U.S. patent application number 14/708914 was filed with the patent office on 2015-09-03 for novel binder-drug conjugates (adcs) and use of same.
This patent application is currently assigned to SEATTLE GENETICS, INC.. The applicant listed for this patent is SEATTLE GENETICS, INC.. Invention is credited to Rudolf BEIER, Sandra BORKOWSKI, Sandra BRUDER, Sven GOLFIER, Simone GREVEN, Axel HARRENGA, Iring HEISLER, Hannah JORISSEN, Charlotte C. KOPITZ, Hans-Georg LERCHEN, Lars LINDEN, Christoph MAHLERT, Heike PETRUL, Joachim SCHUHMACHER, Sherif El SHEIKH, Beatrix STELTE-LUDWIG, Karl-Heinz THIERAUCH, Jorg WILLUDA.
Application Number | 20150246136 14/708914 |
Document ID | / |
Family ID | 45976407 |
Filed Date | 2015-09-03 |
United States Patent
Application |
20150246136 |
Kind Code |
A1 |
LERCHEN; Hans-Georg ; et
al. |
September 3, 2015 |
NOVEL BINDER-DRUG CONJUGATES (ADCs) AND USE OF SAME
Abstract
The present patent application relates to novel binder-drug
conjugates (ADCs) of N,N-dialkylauristatins directed against the
target epidermal growth factor receptor (EGFR, gene ID 1956),
effective metabolites of these ADCs, methods for producing these
ADCs, use of these ADCs for treatment and or prevention of diseases
as well as the use of these ADCs to produce pharmaceutical drugs
for treatment and/or prevention of diseases, in particular
hyperproliferative and/or angiogenic diseases such as cancer, for
example. Such treatments may be administered as monotherapy or in
combination with other pharmaceutical drugs or other therapeutic
measures.
Inventors: |
LERCHEN; Hans-Georg;
(Leverkusen, DE) ; LINDEN; Lars; (Duesseldorf,
DE) ; SHEIKH; Sherif El; (Essen, DE) ;
WILLUDA; Jorg; (Glienicke/Nordbahn, DE) ; KOPITZ;
Charlotte C.; (Berlin, DE) ; SCHUHMACHER;
Joachim; (Wuppertal, DE) ; GREVEN; Simone;
(Dormagen, DE) ; MAHLERT; Christoph; (Wuppertal,
DE) ; STELTE-LUDWIG; Beatrix; (Wuelfrath, DE)
; GOLFIER; Sven; (Berlin, DE) ; BEIER; Rudolf;
(Berlin, DE) ; HEISLER; Iring; (Wuppertal, DE)
; HARRENGA; Axel; (Wuppertal, DE) ; THIERAUCH;
Karl-Heinz; (Berlin, DE) ; BRUDER; Sandra;
(Leverkusen, DE) ; PETRUL; Heike; (Berlin, DE)
; JORISSEN; Hannah; (Essen, DE) ; BORKOWSKI;
Sandra; (Hohen Neuendorf, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SEATTLE GENETICS, INC. |
BOTHELL |
WA |
US |
|
|
Assignee: |
SEATTLE GENETICS, INC.
BOTHELL
WA
|
Family ID: |
45976407 |
Appl. No.: |
14/708914 |
Filed: |
May 11, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14113070 |
Jan 17, 2014 |
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PCT/EP2012/057243 |
Apr 20, 2012 |
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14708914 |
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Current U.S.
Class: |
424/181.1 ;
530/391.9; 544/63; 548/467; 548/540 |
Current CPC
Class: |
A61K 31/537 20130101;
A61K 31/5377 20130101; A61K 47/6803 20170801; C07K 5/0207 20130101;
C07K 2317/24 20130101; A61K 31/422 20130101; A61P 43/00 20180101;
C07K 16/2863 20130101; A61K 45/06 20130101; A61K 31/4245 20130101;
A61K 31/40 20130101; A61K 47/6851 20170801; C07K 7/06 20130101;
C07K 2317/565 20130101; C07K 5/06052 20130101; C07K 7/02 20130101;
C07K 2317/76 20130101; C07K 16/30 20130101; A61K 31/4025 20130101;
A61K 31/5355 20130101; A61K 39/3955 20130101; A61K 47/542 20170801;
C07K 5/0606 20130101; C07K 16/2812 20130101; A61K 47/6869 20170801;
C07K 5/06034 20130101; C07K 16/28 20130101; A61P 35/02 20180101;
A61K 47/6889 20170801; C07K 2317/21 20130101; A61K 47/6859
20170801; A61K 31/404 20130101; C07K 16/18 20130101; A61P 9/00
20180101; A61K 31/536 20130101; A61P 35/00 20180101; C07K 5/0205
20130101; C07K 5/06 20130101 |
International
Class: |
A61K 47/48 20060101
A61K047/48; C07K 5/062 20060101 C07K005/062; A61K 45/06 20060101
A61K045/06; C07K 16/28 20060101 C07K016/28; A61K 39/395 20060101
A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 21, 2011 |
EP |
11163467.1 |
Apr 21, 2011 |
EP |
11163470.5 |
Apr 21, 2011 |
EP |
11163472.1 |
Apr 21, 2011 |
EP |
11163474.7 |
Jun 1, 2011 |
EP |
11168556.6 |
Jun 1, 2011 |
EP |
11168557.4 |
Jun 1, 2011 |
EP |
11168558.2 |
Jun 1, 2011 |
EP |
11168559.0 |
Dec 14, 2011 |
EP |
11193609.2 |
Dec 14, 2011 |
EP |
11193618.3 |
Dec 14, 2011 |
EP |
11193621.7 |
Dec 14, 2011 |
EP |
11193623.3 |
Claims
1. Binder-drug ingredient conjugates of general formula (Ia)
##STR01080## in which n is a number from 1 to 50, AK is a binder
the group .sctn.-G-L.sup.1-B-L.sup.2-.sctn..sctn. is a linker,
wherein .sctn. denotes the linkage site to the group AK and
.sctn..sctn. denotes the linkage site to the nitrogen atom, D is a
group of the formula ##STR01081## wherein #.sup.3 denotes the
linkage site to the nitrogen atom, R.sup.1 is hydrogen or methyl,
R.sup.2 is isopropyl, isobutyl, sec-butyl, tert-butyl, phenyl,
benzyl, 1-hydroxyethyl, 4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl,
4-hydroxy-3-aminobenzyl, 1-phenylethyl, diphenylmethyl,
1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl, or R.sup.1 and
R.sup.2 together with the carbon atom to which they are attached
form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR01082## wherein #.sup.4 denotes the linkage site to the
vicinal nitrogen atom, #.sup.5 denotes the linkage site to the
carbonyl group, the ring A with the N--O group contained in it is a
mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR01083## wherein #.sup.6 denotes the linkage site to
the carbonyl group, R.sup.6 is hydrogen, hydroxyl or benzyloxy,
R.sup.3 is hydrogen or methyl, R.sup.4 is isopropyl, isobutyl,
sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl,
4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl,
1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or
1H-indol-3-ylmethyl, or R.sup.3 and R.sup.4 together with the
carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR01084## wherein #.sup.7 denotes the linkage site with the
vicinal nitrogen atom, #.sup.8 denotes the linkage site to the
group T.sup.1, T.sup.1 is a group of the formula
--C(.dbd.O)--OR.sup.7, --C(.dbd.O)--NR.sup.8R.sup.9,
--C(.dbd.O)--NH--NH--R.sup.10 or --CH.sup.2--O--R.sup.11, wherein
R.sup.7 is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, R.sup.8 is hydrogen or methyl, R.sup.9 is
hydrogen, methyl, ethyl, n-propyl or benzyl, or R.sup.8 and R.sup.9
together with the nitrogen atom to which they are bound form a
four- to seven-membered heterocycle, R.sup.10 is benzoyl, R.sup.11
is benzyl, which may be substituted in the phenyl group with
methoxycarbonyl or carboxyl, R.sup.5 is hydrogen, methyl or a group
of the formula ##STR01085## wherein #.sup.9 denotes the linkage
site to --CHC(R.sup.26)-T.sup.2, R.sup.12 is phenyl, which may be
substituted with methoxycarbonyl, carboxyl or a group of the
formula --S(O).sub.2OH, R.sup.13 is phenyl, which may be
substituted with methoxycarbonyl or carboxyl, R.sup.26 is hydrogen
or hydroxyl, T.sup.2 is phenyl, benzyl, 1H-indol-3-yl or
1H-indol-3-ylmethyl, and R.sup.35 is methyl or hydroxyl, as well as
their salts, solvates, and solvates of the salts.
2. Binder-drug conjugates of the general formula (Ia) according to
claim 1, wherein n is a number from 1 to 50, AK is AK.sub.1 or
AK.sub.2 wherein AK.sub.1 is a binder which is bound to the group G
by a sulfur atom of the binder, AK.sub.2 is a binder which is bound
to the group G by a nitrogen atom of the binder, G for the case
when AK=AK.sub.1, is a group of the formula ##STR01086## wherein
#.sup.1 denotes the linkage site to the sulfur atom of the binder,
#.sup.2 denotes the linkage site to the group L.sup.1, or for the
case when AK=AK.sub.2, G is carbonyl, L.sup.1 is a bond, linear
(C.sub.1-C.sub.10)-alkanediyl, or a group of the formula
##STR01087## or ##.sup.1-L.sup.1A-B.sup.1-L.sup.1B-##.sup.2 wherein
m is a number from 2 to 6, ##.sup.1 denotes the linkage site to the
group G, ##.sup.2 denotes the linkage site to the group B, L.sup.1A
is linear (C.sub.2-C.sub.10)-alkanediyl, B.sup.1 is a group of the
formula ##STR01088## wherein ##.sup.5 denotes the linkage site to
the group L.sup.1A, ##.sup.6 denotes the linkage site to the group
L.sup.1B, L.sup.5 is a bond or (C.sub.2-C.sub.4)-alkanediyl,
L.sup.6 is a bond or a group with the formula ##STR01089## wherein
##.sup.7 denotes the linkage site to the carbonyl group, ##.sup.8
denotes the linkage site to L.sup.1B, R.sup.33 is hydrogen,
(C.sub.1-C.sub.4)-alkylcarbonyl, tert-butyloxycarbonyl or
benzyloxycarbonyl, R.sup.34 is hydrogen or methyl, R.sup.29 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, R.sup.30 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, or R.sup.29 and R.sup.30 together with the
atoms to which they are bound form a five- or six-membered
heterocycle, R.sup.31 is hydrogen or (C.sub.1-C.sub.4)-alkyl,
R.sup.32 is hydrogen or (C.sub.1-C.sub.4)-alkyl, or R.sup.31 and
R.sup.32 together with the atoms to which they are bound form a
five- or six-membered heterocycle, L.sup.1B is linear
(C.sub.2-C.sub.10)-alkanediyl, and wherein
(C.sub.1-C.sub.10)-alkanediyl may be substituted with one to four
substituents selected independently of one another from the group
consisting of methyl, hydroxyl and benzyl and wherein two carbon
atoms of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-
relation to one another, including the carbon atoms optionally
situated between them, to form a (C.sub.3-C.sub.6)-cycloalkyl ring
or a phenyl ring, B is a bond or a group of the formula
##STR01090## wherein * denotes the linkage site to L.sup.1, **
denotes the linkage site to L.sup.2, P is O or NH, L.sup.3 is a
bond or (C.sub.2-C.sub.4)-alkanediyl, L.sup.4 is a bond or a group
of the formula ##STR01091## wherein *** denotes the linkage site to
the carbonyl group, **** denotes the linkage site to L.sup.2,
R.sup.25 is hydrogen or methyl, R.sup.28 is hydrogen,
(C.sub.1-C.sub.4)-alkylcarbonyl, tert-butyloxycarbonyl or
benzyloxycarbonyl, Q.sup.1 is a four- to seven-membered
heterocycle, Q.sup.2 is a three- to seven-membered carbocycle or a
four- to seven-membered heterocycle, R.sup.14 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, R.sup.15 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, or R.sup.14 and R.sup.15 together with the
atoms to which they are bound form a five- or six-membered
heterocycle, R.sup.16 is hydrogen or (C.sub.1-C.sub.4)-alkyl,
R.sup.17 is hydrogen or (C.sub.1-C.sub.4)-alkyl, or R.sup.16 and
R.sup.17 together with the atoms to which they are bound form a
five- or six-membered heterocycle, R.sup.18 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, R.sup.19 is hydrogen or the side group of
a natural .alpha.-amino acid or its homologs or isomers, R.sup.20
is hydrogen or (C.sub.1-C.sub.4)-alkyl, or R.sup.19 and R.sup.20
together with the atoms to which they are bound form a pyrrolidinyl
ring, R.sup.21 is hydrogen or (C.sub.1-C.sub.4)-alkyl, R.sup.22 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, or R.sup.21 and R.sup.22
together with the atoms to which they are bound form a three- to
seven-membered carbocycle, R.sup.23 is (C.sub.1-C.sub.4)-alkyl,
R.sup.24 is hydrogen or (C.sub.1-C.sub.4)-alkyl, R.sup.27 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, R.sup.36 is hydrogen,
(C.sub.1-C.sub.4)-alkylcarbonyl, tert-butyloxycarbonyl or
benzyloxycarbonyl, R.sup.37 is hydrogen or methyl, or R.sup.36 and
R.sup.37 together with the atoms to which they are bound form a
pyrrolidine ring, L.sup.2 is linear (C.sub.2-C.sub.10)-alkanediyl
or for a group of the formula ##STR01092## wherein p is a number
from 2 to 6, ##.sup.3 denotes the linkage site to the group B,
##.sup.4 denotes the linkage site to the nitrogen atom, wherein
(C.sub.2-C.sub.10)-alkanediyl may be substituted with 1 to 4
substituents selected independently of one another from the group
consisting of methyl, hydroxyl and benzyl, and wherein two carbon
atoms of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-
relation to one another, including the carbon atoms optionally
present between them to form a (C.sub.3-C.sub.6) cycloalkyl ring or
a phenyl ring, D is a group of the formula ##STR01093## wherein
#.sup.3 denotes the linkage site to the nitrogen atom, R.sup.1 is
hydrogen or methyl, R.sup.2 is isopropyl, isobutyl, sec-butyl,
tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl,
4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl,
diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl, or
R.sup.1 and R.sup.2 together with the carbon atom to which they are
attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula ##STR01094## wherein #.sup.4 denotes the linkage site to
the vicinal nitrogen atom, #.sup.5 denotes the linkage site to the
carbonyl group, the ring A with the N--O grouping contained in it
is a mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR01095## wherein #.sup.6 denotes the linkage site to
the carbonyl group, R.sup.6 is hydrogen, hydroxyl or benzyloxy,
R.sup.3 is hydrogen or methyl, R.sup.4 is isopropyl, isobutyl,
sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl,
4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl,
1-phenylethyl, di-phenylmethyl, 1H-imidazol-4-ylmethyl or
1H-indol-3-ylmethyl, or R.sup.3 and R.sup.4 together with the
carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR01096## wherein #.sup.7 denotes the linkage site to the
vicinal nitrogen atom, #.sup.8 denotes the linkage site to the
group T, T.sup.1 is a group of the formula --C(.dbd.O)--OR.sup.7,
--C(.dbd.O)--NR.sup.8R.sup.9, --C(.dbd.O)--NH--NH--R.sup.10 or
--CH.sub.2--O--R.sup.11, wherein R.sup.7 is hydrogen, methyl,
ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl, R.sup.8 is
hydrogen or methyl, R.sup.9 is hydrogen, methyl, ethyl, n-propyl or
benzyl, or R.sup.8 and R.sup.9 together with the nitrogen atom to
which they are bound form a four- to seven-membered heterocycle,
R.sup.10 is benzoyl, R.sup.11 is benzyl, which may be substituted
in the phenyl group with methoxycarbonyl or carboxyl, R.sup.5 is
hydrogen, methyl or a group of the formula ##STR01097## wherein
#.sup.9 denotes the linkage site to --CHC(R.sup.26)-T.sup.2,
R.sup.12 is phenyl, which may be substituted with methoxycarbonyl,
carboxyl or a group of the formula --S(O).sub.2OH, R.sup.13 is
phenyl, which may be substituted with methoxycarbonyl or carboxyl,
R.sup.26 is hydrogen or hydroxyl, T.sup.2 is phenyl, benzyl,
1H-indol-3-yl or 1H-indol-3-ylmethyl, and R.sup.35 is methyl or
hydroxyl, as well as their salts, solvates, and solvates of the
salts.
3-6. (canceled)
7. Compounds of formula (XXXa) ##STR01098## in which Cys is a
cysteine radical which is bound to a carbon atom of the succinimide
via the sulfur atom of the side chain, L.sup.1 is a bond, linear
(C.sub.1-C.sub.10)-alkanediyl, or a group of the formula
##STR01099## or ##.sup.1-L.sup.1A-B.sup.1-L.sup.1B-##.sup.2 wherein
m is a number from 2 to 6, ##.sup.1 denotes the linkage site to the
group G, ##.sup.2 denotes the linkage site to the group B, L.sup.1A
is linear (C.sub.2-C.sub.10)-alkanediyl, B.sup.1 is a group of the
formula ##STR01100## wherein ##.sup.5 denotes the linkage site to
the group L.sup.1A, ##.sup.6 denotes the linkage site to the group
L.sup.1B, L.sup.5 is a bond or (C.sub.2-C.sub.4)-alkanediyl,
L.sup.6 is a bond, R.sup.29 is hydrogen or (C.sub.1-C.sub.4)-alkyl,
R.sup.30 is hydrogen or (C.sub.1-C.sub.4)-alkyl, or R.sup.29 and
R.sup.30 together with the atoms to which they are bound form a
five- or six-membered heterocycle, R.sup.31 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, R.sup.32 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, or R.sup.31 and R.sup.32 together with the
atoms to which they are bound form a five- or six-membered
heterocycle, L.sup.1B is linear (C.sub.2-C.sub.10)-alkanediyl, and
wherein (C.sub.1-C.sub.10)-alkanediyl may be substituted with one
to four substituents selected independently of one another from the
group consisting of methyl, hydroxyl and benzyl and wherein two
carbon atoms of the alkanediyl chain may be bridged in 1,2-, 1,3-
or 1,4- relation to one another, including the carbon atoms
optionally situated between them, to form a
(C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring, B is a bond or
a group of the formula ##STR01101## wherein * denotes the linkage
site to L.sup.1, ** denotes the linkage site to L.sup.2, P is O or
NH, L.sup.3 is a bond or (C.sub.2-C.sub.4)-alkanediyl, L.sup.4 is a
bond, Q.sup.1 is a four- to seven-membered heterocycle, R.sup.14 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, R.sup.15 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, or R.sup.14 and R.sup.15 together with the
atoms to which they are bound form a five- or six-membered
heterocycle, R.sup.16 is hydrogen or (C.sub.1-C.sub.4)-alkyl,
R.sup.17 is hydrogen or (C.sub.1-C.sub.4)-alkyl, or R.sup.16 and
R.sup.17 together with the atoms to which they are bound form a
five- or six-membered heterocycle, R.sup.27 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, L.sup.2 is linear
(C.sub.2-C.sub.10)-alkanediyl or a group of the formula
##STR01102## wherein p is a number from 2 to 6, ##.sup.3 denotes
the linkage site to the group B, ##.sup.4 denotes the linkage site
to the nitrogen atom, wherein (C.sub.2-C.sub.10)-alkanediyl may be
substituted with 1 to 4 substituents selected independently of one
another from the group consisting of methyl, hydroxyl and benzyl,
and wherein two carbon atoms of the alkanediyl chain may be bridged
in 1,2-, 1,3- or 1,4- relation to one another, including the carbon
atoms optionally present between them to form a (C.sub.3-C.sub.6)
cycloalkyl ring or a phenyl ring, D is a group of the formula
##STR01103## wherein #.sup.3 denotes the linkage site to the
nitrogen atom, R.sup.1 is hydrogen or methyl, R.sup.2 is isopropyl,
isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl,
4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl,
1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or
1H-indol-3-ylmethyl, or R.sup.1 and R.sup.2 together with the
carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR01104## wherein #.sup.4 denotes the linkage site to the
vicinal nitrogen atom, #.sup.5 denotes the linkage site to the
carbonyl group, the ring A with the N--O group contained in it is a
mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR01105## wherein #.sup.6 denotes the linkage site to
the carbonyl group, R.sup.6 is hydrogen, hydroxyl or benzyloxy,
R.sup.3 is hydrogen or methyl, R.sup.4 is isopropyl, isobutyl,
sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl,
4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl,
1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or
1H-indol-3-ylmethyl, or R.sup.3 and R.sup.4 together with the
carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR01106## wherein #.sup.7 denotes the linkage site to the
vicinal nitrogen atom, #.sup.8 denotes the linkage site to the
group T, T.sup.1 is a group of the formula --C(.dbd.O)--OR.sup.7,
--C(.dbd.O)--NR.sup.8R.sup.9, --C(.dbd.O)--NH--NH--R.sup.10 or
--CH.sub.2--O--R.sup.11, wherein R.sup.7 is hydrogen, methyl,
ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl, R.sup.8 is
hydrogen or methyl, R.sup.9 is hydrogen, methyl, ethyl, n-propyl or
benzyl, or R.sup.8 and R.sup.9 together with the nitrogen atom to
which they are bound form a four- to seven-membered heterocycle,
R.sup.10 is benzoyl, R.sup.11 is benzyl, which may be substituted
in the phenyl group with methoxycarbonyl or carboxyl, R.sup.5 is
hydrogen, methyl or a group of the formula ##STR01107## wherein
#.sup.9 denotes the linkage site to --CHC(R.sup.26)-T.sup.2,
R.sup.12 is phenyl, which may be substituted with methoxycarbonyl,
carboxyl or a group of the formula --S(O).sub.2OH, R.sup.13 is
phenyl, which may be substituted with methoxycarbonyl or carboxyl,
R.sup.26 is hydrogen or hydroxyl, T.sup.2 is phenyl, benzyl,
1H-indol-3-yl or 1H-indol-3-ylmethyl, and R.sup.35 is methyl or
hydroxyl, as well as their salts, solvates, and solvates of the
salts.
8. Compounds of the formula (XXXa) according to claim 7, wherein
Cys is a cysteine radical which is bound to a carbon atom of the
succinimide via the sulfur atom of the side chain, L.sup.1 is a
bond, linear (C.sub.2-C.sub.6)-alkanediyl, or a group of formula
##STR01108## or ##.sup.1-L.sup.1A-B.sup.1-L.sup.1B-##.sup.2 wherein
m is a number from 2 to 3, ##.sup.1 denotes the linkage site to the
group G, ##.sup.2 denotes the linkage site to the group B, L.sup.1A
is linear (C.sub.2-C.sub.6)-alkanediyl, B.sup.1 is a group of the
formula ##STR01109## wherein ##.sup.5 denotes the linkage site to
the group L.sup.1A, ##.sup.6 denotes the linkage site to the group
L.sup.1B, L.sup.5 is a bond, L.sup.6 is a bond, R.sup.29 is
hydrogen, R.sup.30 is hydrogen, R.sup.31 is hydrogen or methyl,
R.sup.32 is hydrogen or methyl, L.sup.1B is linear
(C.sub.2-C.sub.6)-alkanediyl, and wherein
(C.sub.2-C.sub.6)-alkanediyl may be substituted with one to two
methyl substituents, B is a bond or a group of the formula
##STR01110## wherein * denotes the linkage site to L.sup.1, **
denotes the linkage site to L.sup.2, L.sup.3 is a bond or
ethane-1,2-diyl, L.sup.4 is a bond, R.sup.14 is hydrogen, R.sup.15
is hydrogen, R.sup.16 is hydrogen or methyl, R.sup.17 is hydrogen
or methyl, or R.sup.16 and R.sup.17 together with the atoms to
which they are bound form a piperazinyl ring, R.sup.23 is methyl,
R.sup.24 is hydrogen or methyl, L.sup.2 is linear
(C.sub.2-C.sub.6)-alkanediyl or a group of the formula ##STR01111##
wherein p is a number from 2 to 3, ##.sup.3 denotes the linkage
site to the group B, ##.sup.4 denotes the linkage site to the
nitrogen atom, D is a group of the formula ##STR01112## wherein
#.sup.3 denotes the linkage site to the nitrogen atom, R.sup.1 is
hydrogen, R.sup.2 is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl,
1-phenylethyl or 1H-indol-3-yl-methyl, or R.sup.1 and R.sup.2
together with the carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR01113## wherein #.sup.4 denotes the linkage site to the
vicinal nitrogen atom, #.sup.5 denotes the linkage site to the
carbonyl group, the ring A with the N--O group contained in it is a
mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR01114## wherein #.sup.6 denotes the linkage site to
the carbonyl group, R.sup.6 is hydrogen, hydroxyl or benzyloxy,
R.sup.3 is hydrogen, R.sup.4 is 1-hydroxyethyl, benzyl,
4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-yl-methyl, or R.sup.3
and R.sup.4 together with the carbon atom to which they are
attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula ##STR01115## wherein #.sup.7 denotes the linkage site to
the vicinal nitrogen atom, #.sup.8 denotes the linkage site to the
group T, T.sup.1 is a group of the formula --C(.dbd.O)--OR.sup.7,
--C(.dbd.O)--NR.sup.8R.sup.9, --C(.dbd.O)--NH--NH--R.sup.10 or
--CH.sub.2--O--R.sup.11, wherein R.sup.7 is hydrogen, methyl,
ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl, R.sup.8 is
hydrogen or methyl, R.sup.9 is hydrogen, methyl, ethyl, n-propyl or
benzyl, or R.sup.8 and R.sup.9 together with the nitrogen atom to
which they are bound form a four- to seven-membered heterocycle,
R.sup.10 is benzoyl, R.sup.11 is benzyl, which may be substituted
in the phenyl group with methoxycarbonyl or carboxyl, R.sup.5 is
hydrogen, methyl or a group of the formula ##STR01116## wherein
#.sup.9 denotes the linkage site to --CHCH.sub.2phenyl, R.sup.12 is
phenyl, which may be substituted with methoxycarbonyl, carboxyl or
a group of the formula --S(O).sub.2OH, R.sup.13 is phenyl, which
may be substituted with methoxycarbonyl or carboxyl, and R.sup.35
is methyl or hydroxyl, as well as their salts, solvates, and
solvates of the salts.
9. Compounds of the formula (XXXa) according to claim 7, wherein
Cys is a cysteine radical which is bound to a carbon atom of the
succinimide via the sulfur atom of the side chain, L.sup.1 is a
bond or linear (C.sub.2-C.sub.6)-alkanediyl, B is a bond or a group
of the formula ##STR01117## wherein * denotes the linkage site to
L.sup.1, ** denotes the linkage site to L.sup.2, L.sup.3 is a bond,
L.sup.4 is a bond, R.sup.16 is hydrogen or methyl, R.sup.17 is
hydrogen or methyl, L.sup.2 is linear (C.sub.2-C.sub.6)-alkanediyl
or a group of the formula ##STR01118## wherein p is a number from 2
to 3, ##.sup.3 denotes the linkage site to the group B, ##.sup.4
denotes the linkage site to the nitrogen atom, D is a group of the
formula ##STR01119## wherein #.sup.3 denotes the linkage site to
the nitrogen atom, R.sup.1 is hydrogen, R.sup.2 is benzyl or
1H-indol-3-ylmethyl, or R.sup.1 and R.sup.2 together with the
carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR01120## wherein #.sup.4 denotes the linkage site to the
vicinal nitrogen atom, #.sup.5 denotes the linkage site to the
carbonyl group, the ring A with the N--O group contained in it is a
mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR01121## wherein #.sup.6 denotes the linkage site to
the carbonyl group, R.sup.3 is hydrogen, R.sup.4 is benzyl or
1H-indol-3-ylmethyl, or R.sup.3 and R.sup.4 together with the
carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR01122## wherein #.sup.7 denotes the linkage site to the
vicinal nitrogen atom, #.sup.8 denotes the linkage site to the
group T.sup.1, T.sup.1 is a group of the formula
--C(.dbd.O)--OR.sup.7 or --C(.dbd.O)--NR.sup.8R.sup.9, wherein
R.sup.7 is hydrogen, R.sup.8 is hydrogen, R.sup.9 is hydrogen, and
R.sup.35 is methyl, as well as their salts, solvates, and solvates
of the salts.
10. Compounds of formula (XXXI) ##STR01123## in which L.sup.1 is a
bond, linear (C.sub.1-C.sub.10)-alkanediyl, or a group of the
formula ##STR01124## or ##.sup.1-L.sup.1A-B.sup.1-L.sup.1B-##.sup.2
wherein m is a number from 2 to 6, ##.sup.1 denotes the linkage
site to the group G, ##.sup.2 denotes the linkage site to the group
B, L.sup.1A is linear (C.sub.2-C.sub.10)-alkanediyl, B.sup.1 is a
group of the formula ##STR01125## wherein ##.sup.5 denotes the
linkage site to the group L.sup.1A, ##.sup.6 denotes the linkage
site to the group L.sup.1B, L.sup.5 is a bond or
(C.sub.2-C.sub.4)-alkanediyl, L.sup.6 is a bond, R.sup.29 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, R.sup.30 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, or R.sup.29 and R.sup.30 together with the
atoms to which they are bound form a five- or six-membered
heterocycle, R.sup.31 is hydrogen or (C.sub.1-C.sub.4)-alkyl,
R.sup.32 is hydrogen or (C.sub.1-C.sub.4)-alkyl, or R.sup.31 and
R.sup.32 together with the atoms to which they are bound form a
five- or six-membered heterocycle, L.sup.1B is linear
(C.sub.2-C.sub.10)-alkanediyl, and wherein
(C.sub.1-C.sub.10)-alkanediyl may be substituted with one to four
substituents selected independently of f-re one another from the
group consisting of methyl, hydroxyl and benzyl and wherein two
carbon atoms of the alkanediyl chain may be bridged in 1,2-, 1,3-
or 1,4- relation to one another, including the carbon atoms
optionally situated between them, to form a
(C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring, B is a bond or
a group of the formula ##STR01126## wherein * denotes the linkage
site to L.sup.1, ** denotes the linkage site to L.sup.2, P is O or
NH, Q.sup.1 is a four- to seven-membered heterocycle, Q.sup.2 is a
three- to seven-membered carbocycle or a four- to seven-membered
heterocycle, R.sup.18 is hydrogen or (C.sub.1-C.sub.4)-alkyl,
R.sup.19 is hydrogen or the side group of a natural .alpha.-amino
acid or its homologs or isomers, R.sup.20 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, or R.sup.19 and R.sup.20 together with the
atoms to which they are bound form a pyrrolidinyl ring, R.sup.21 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, R.sup.22 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, or R.sup.21 and R.sup.22 together with the
atoms to which they are bound form a three- to seven-membered
carbocycle, R.sup.27 is hydrogen or (C.sub.1-C.sub.4)-alkyl,
L.sup.2 is linear (C.sub.2-C.sub.10)-alkanediyl or for a group of
the formula ##STR01127## wherein p is a number from 2 to 6,
##.sup.3 denotes the linkage site to the group B, ##.sup.4 denotes
the linkage site to the nitrogen atom, wherein
(C.sub.2-C.sub.10)-alkanediyl may be substituted with 1 to 4
substituents selected independently of one another from the group
consisting of methyl, hydroxyl and benzyl, and wherein two carbon
atoms of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-
relation to one another, including the carbon atoms optionally
present between them to form a (C.sub.3-C.sub.6) cycloalkyl ring or
a phenyl ring, D is a group of the formula ##STR01128## wherein
#.sup.3 denotes the linkage site to the nitrogen atom, R.sup.1 is
hydrogen or methyl, R.sup.2 is isopropyl, isobutyl, sec-butyl,
tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl,
4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl,
diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl, or
R.sup.1 and R.sup.2 together with the carbon atom to which they are
attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula ##STR01129## wherein #.sup.4 denotes the linkage site to
the vicinal nitrogen atom, #.sup.5 denotes the linkage site to the
carbonyl group, the ring A with the N--O group contained in it is a
mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR01130## wherein #.sup.6 denotes the linkage site to
the carbonyl group, R.sup.6 is hydrogen, hydroxyl or benzyloxy,
R.sup.3 is hydrogen or methyl, R.sup.4 is isopropyl, isobutyl,
sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl,
4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl,
1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or
1H-indol-3-ylmethyl, or R.sup.3 and R.sup.4 together with the
carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR01131## wherein #.sup.7 denotes the linkage site to the
vicinal nitrogen atom, #.sup.8 denotes the linkage site to the
group T.sup.1, T.sup.1 is a group of the formula
--C(.dbd.O)--OR.sup.7, --C(.dbd.O)--NR.sup.8R.sup.9,
--C(.dbd.O)--NH--NH--R.sup.10 or --CH.sub.2--O--R.sup.11, wherein
R.sup.7 is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, R.sup.8 is hydrogen or methyl, R.sup.9 is
hydrogen, methyl, ethyl, n-propyl or benzyl, or R.sup.8 and R.sup.9
together with the nitrogen atom to which they are bound form a
four- to seven-membered heterocycle, R.sup.10 is benzoyl, R.sup.11
is benzyl, which may be substituted in the phenyl group with
methoxycarbonyl or carboxyl, R.sup.5 is hydrogen, methyl or a group
of the formula ##STR01132## wherein #.sup.9 denotes the linkage
site to --CHC(R.sup.26)-T.sup.2, R.sup.12 is phenyl, which may be
substituted with methoxycarbonyl, carboxyl or a group of the
formula --S(O).sub.2OH, R.sup.13 is phenyl, which may be
substituted with methoxycarbonyl or carboxyl, R.sup.26 is hydrogen
or hydroxyl, T.sup.2 is phenyl, benzyl, 1H-indol-3-yl or
1H-indol-3-ylmethyl, and R.sup.35 is methyl or hydroxyl, as well as
their salts, solvates, and solvates of the salts.
11. Compounds of the formula (XXXI) according to claim 10, wherein
L.sup.1 is a bond, linear (C.sub.2-C.sub.6)-alkanediyl or a group
of the formula ##STR01133## wherein m is a number from 2 to 3,
##.sup.1 denotes the linkage site to the group G, ##.sup.2 denotes
the linkage site to the group B, wherein
(C.sub.2-C.sub.6)-alkanediyl may be substituted with one or two
methyl substituents, B is a bond or a group of the formula
##STR01134## wherein * denotes the linkage site to L.sup.1, **
denotes the linkage site to L.sup.2, R.sup.18 is hydrogen, R.sup.19
is hydrogen, methyl, propan-2-yl, 2-methylpropan-1-yl or
1-methylpropan-1-yl, R.sup.20 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, or R.sup.19 and R.sup.20 together with the
atoms to which they are bound form a pyrrolidinyl ring, R.sup.21 is
hydrogen or methyl, R.sup.22 is hydrogen or methyl, or R.sup.21 and
R.sup.22 together with the atoms to which they are bound form a
cyclopropyl ring, R.sup.27 is stands Er hydrogen or methyl, L.sup.2
is linear (C.sub.2-C.sub.6)-alkanediyl or a group of the formula
##STR01135## wherein p is a number from 2 to 3, ##.sup.3 denotes
the linkage site to the group B, ##.sup.4 denotes the linkage site
to the nitrogen atom, wherein (C.sub.2-C.sub.10)-alkanediyl may be
substituted with one or two methyl substituents, and wherein two
carbon atoms of the alkanediyl chain may be bridged in 1,4-
relation to one another, including the carbon atoms optionally
present between them to form a phenyl ring, D is a group of the
formula ##STR01136## wherein #.sup.3 denotes the linkage site to
the nitrogen atom, R.sup.1 is hydrogen, R.sup.2 is 1-hydroxyethyl,
benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-yl-methyl, or
R.sup.1 and R.sup.2 together with the carbon atom to which they are
attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula ##STR01137## wherein #.sup.4 denotes the linkage site to
the vicinal nitrogen atom, #.sup.5 denotes the linkage site to the
carbonyl group, the ring A with the N--O group contained in it is a
mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR01138## wherein #.sup.6 denotes the linkage site to
the carbonyl group, R.sup.6 is hydrogen, hydroxyl or benzyloxy,
R.sup.3 is hydrogen, R.sup.4 is 1-hydroxyethyl, benzyl,
4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-yl-methyl, or R.sup.3
and R.sup.4 together with the carbon atom to which they are
attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula ##STR01139## wherein #.sup.7 denotes the linkage site to
the vicinal nitrogen atom, #.sup.8 denotes the linkage site to the
group T.sup.1, T.sup.1 is a group of the formula
--C(.dbd.O)--OR.sup.7, --C(.dbd.O)--NR.sup.8R.sup.9,
--C(.dbd.O)--NH--NH--R.sup.10 or --CH.sub.2--O--R.sup.11, wherein
R.sup.7 is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, R.sup.8 is hydrogen or methyl, R.sup.9 is
hydrogen, methyl, ethyl, n-propyl or benzyl, or R.sup.8 and R.sup.9
together with the nitrogen atom to which they are bound form a
four- to seven-membered heterocycle, R.sup.10 is benzoyl, R.sup.11
is benzyl, which may be substituted in the phenyl group with
methoxycarbonyl or carboxyl, R.sup.5 is hydrogen, methyl or a group
of the formula ##STR01140## wherein #.sup.9 denotes the linkage
site to --CHCH.sub.2phenyl, R.sup.2 is phenyl, which may be
substituted with methoxycarbonyl, carboxyl or a group of the
formula --S(O).sub.2OH, R.sup.13 is phenyl, which may be
substituted with methoxycarbonyl or carboxyl, and R.sup.35 is
methyl or hydroxyl, as well as their salts, solvates, and solvates
of the salts.
12. Compounds of the formula (XXXI) according to claim 10, wherein
L.sup.1 is a bond, B is a bond, L.sup.2 is linear
(C.sub.2-C.sub.6)-alkanediyl or for a group of the formula
##STR01141## wherein p is a number from 2 to 3, ##.sup.3 denotes
the linkage site to the group B, ##.sup.4 denotes the linkage site
to the nitrogen atom, D is a group of the formula ##STR01142##
wherein #.sup.3 denotes the linkage site to the nitrogen atom,
R.sup.1 is hydrogen, R.sup.2 is benzyl or 1H-indol-3-ylmethyl, or
R.sup.1 and R.sup.2 together with the carbon atom to which they are
attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula ##STR01143## wherein #.sup.4 denotes the linkage site to
the vicinal nitrogen atom, #.sup.5 denotes the linkage site to the
carbonyl group, the ring A with the N--O group contained in it is a
mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR01144## wherein #.sup.6 denotes the linkage site to
the carbonyl group, R.sup.3 is hydrogen, R.sup.4 is benzyl or
1H-indol-3-ylmethyl, or R.sup.3 and R.sup.4 together with the
carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR01145## wherein #.sup.7 denotes the linkage site to the
vicinal nitrogen atom, #.sup.8 denotes the linkage site to the
group T.sup.1, T.sup.1 is a group of the formula
--C(.dbd.O)--OR.sup.7 or --C(.dbd.O)--NR.sup.8R.sup.9, wherein
R.sup.7 is hydrogen, R.sup.8 is hydrogen, R.sup.9 is hydrogen, and
R.sup.35 is methyl, as well as their salts, solvates, and solvates
of the salts.
13. A compound of formula (XXXa) or (XXXI) selected from the group
consisting of:
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)-
hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carbox-
y-2-(1H-indol-3-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin--
1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)-
hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-in-
dol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-
-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methy-
l-L-valinamide,
N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-N-
-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-
-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-
-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate, and
N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-N-
-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-(1H-indol-3-yl)ethyl]-
amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl--
1-oxoheptan-4-yl]-N-methyl-L-valinamide, as well as their salts,
solvates, and solvates of the salts.
14. Binder-drug conjugates of the general formula (Ia) according to
claim 1, wherein R.sup.35 is methyl, thereby providing a
binder-drug conjugate of general formula (I) ##STR01146## in which
n is a number from 1 to 50, AK is a binder, the group
.sctn.-G-L.sup.1-B-L.sup.2-.sctn..sctn. is a linker, wherein .sctn.
denotes the linkage site to the group AK and .sctn..sctn. denotes
the linkage site to the nitrogen atom, D is a group of the formula
##STR01147## wherein #.sup.3 denotes the linkage site to the
nitrogen atom, R.sup.1 is hydrogen, R.sup.2 is 1-hydroxyethyl,
benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl, or R.sup.1 and
R.sup.2 together with the carbon atom to which they are attached
form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR01148## wherein #.sup.4 denotes the linkage site to the
vicinal nitrogen atom, #.sup.5 denotes the linkage site to the
carbonyl group, the ring A with the N--O group contained in it is a
mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR01149## wherein #.sup.6 denotes the linkage site to
the carbonyl group, R.sup.6 is hydrogen, hydroxyl or benzyloxy,
R.sup.3 is hydrogen, R.sup.4 is 1-hydroxyethyl, benzyl,
1-phenylethyl or 1H-indol-3-ylmethyl, or R.sup.3 and R.sup.4
together with the carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR01150## wherein #.sup.7 denotes the linkage site to the
vicinal nitrogen atom, #.sup.8 denotes the linkage site to the
group T.sup.1, T.sup.1 is a group of the formula
--C(.dbd.O)--OR.sup.7, --C(.dbd.O)--NR.sup.8R.sup.9,
--C(.dbd.O)--NH--NH--R.sup.10 or --CH.sub.2--O--R.sup.11, wherein
R.sup.7 is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, R.sup.8 is hydrogen or methyl, R.sup.9 is
hydrogen, methyl, ethyl, n-propyl or benzyl, or R.sup.8 and R.sup.9
together with the nitrogen atom to which they are bound form a
four- to seven-membered heterocycle, R.sup.10 is benzoyl, R.sup.11
is benzyl, which may be substituted in the phenyl group with
methoxycarbonyl or carboxyl, R.sup.5 is hydrogen, methyl or a group
of the formula ##STR01151## wherein #.sup.9 denotes the linkage
site to --CHC(R.sup.26)-T.sup.2, R.sup.12 is phenyl, which may be
substituted with methoxycarbonyl, carboxyl or a group of the
formula --S(O).sub.2OH, R.sup.13 is phenyl, which may be
substituted with methoxycarbonyl or carboxyl, R.sup.26 is hydrogen
or hydroxyl, and T.sup.2 is phenyl, benzyl, 1H-indol-3-yl or
1H-indol-3-ylmethyl, as well as their salts, solvates, and solvates
of the salts.
15. Binder-drug conjugates according to claim 14 of the general
formula (I), in which n is a number from 1 to 50, AK is AK.sub.1 or
AK.sub.2 wherein AK.sub.1 is a binder bound to the group G via a
sulfur atom of the binder, AK.sub.2 is a binder bound to the group
G via a nitrogen atom of the binder, G for the case when
AK=AK.sub.1, is a group of the formula ##STR01152## wherein #.sup.1
denotes the linkage site to the sulfur atom of the binder, #.sup.2
denotes the linkage site to the group L.sup.1, or for the case when
AK=AK.sub.2, G is carbonyl, L.sup.1 is a bond, linear
(C.sub.1-C.sub.10)-alkanediyl or a group of the formula
##STR01153## wherein m is a number from 2 to 6, ##.sup.1 denotes
the linkage site to the group G, ##.sup.2 denotes the linkage site
to the group B, wherein (C.sub.1-C.sub.10)-alkanediyl may be
substituted with 1 to 4 methyl substituents, and wherein two carbon
atoms of the alkanediyl chain in 1,2-, 1,3- or 1,4- relation to one
another may be bridged to form a (C.sub.3-C.sub.6)-cycloalkyl ring
or a phenyl ring including the carbon atoms optionally situated
between them, B is a bond or a group of the formula ##STR01154##
wherein * denotes the linkage site to L.sup.1, ** denotes the
linkage site to L.sup.2, P is O or NH, L.sup.3 is a bond or
(C.sub.2-C.sub.4)-alkanediyl, L.sup.4 is a bond or a group of the
formula ##STR01155## wherein *** denotes the linkage site to the
carbonyl group, **** denotes the linkage site to L.sup.2, R.sup.25
is hydrogen or methyl, Q.sup.1 is a four- to seven-membered
heterocycle, Q.sup.2 is a three- to seven-membered carbocycle or a
four- to seven-membered heterocycle, R.sup.14 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, R.sup.15 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, or R.sup.14 and R.sup.15 together with the
atoms to which they are bound form a five- or six-membered
heterocycle, R.sup.16 is hydrogen or (C.sub.1-C.sub.4)-alkyl,
R.sup.17 is hydrogen or (C.sub.1-C.sub.4)-alkyl, or R.sup.16 and
R.sup.17 together with the atoms to which they are bound form a
five- or six-membered heterocycle, R.sup.18 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, R.sup.19 is hydrogen or the side group of
a natural .alpha.-amino acid or its homologs or isomers, R.sup.20
is hydrogen or (C.sub.1-C.sub.4)-alkyl, or R.sup.19 and R.sup.20
together with the atoms to which they are bound form a pyrrolidinyl
ring, R.sup.21 is hydrogen or (C.sub.1-C.sub.4)-alkyl, R.sup.22 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, or R.sup.21 and R.sup.22
together with the atoms to which they are bound form a three- to
seven-membered carbocycle, R.sup.23 is (C.sub.1-C.sub.4)-alkyl,
R.sup.24 is hydrogen or (C.sub.1-C.sub.4)-alkyl, R.sup.27 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, L.sup.2 is linear
(C.sub.2-C.sub.10)-alkanediyl or a group of the formula
##STR01156## wherein p is a number from 2 to 6, ##.sup.3 denotes
the linkage site to the group B, ##.sup.4 denotes the linkage site
to the nitrogen atom, wherein (C.sub.2-C.sub.10)-alkanediyl may be
substituted with 1 to 4 methyl substituents, and wherein two carbon
atoms of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-
relation to one another, including the carbon atoms optionally
present between them to form a (C.sub.3-C.sub.6) cycloalkyl ring or
a phenyl ring, and D has the meaning as described in claim 14, as
well as their salts, solvates, and solvates of the salts.
16. Binder-drug conjugates according to claim 14 of the general
formula (I), in which n is a number from 1 to 50, AK is AK.sub.1 or
AK.sub.2 wherein AK.sub.1 is an antibody or an antigen-binding
antibody fragment and is bound to the group G via a sulfur atom,
AK.sub.2 is an antibody or an antigen-binding antibody fragment and
is bound to the group G via a nitrogen atom, and n, G, L.sup.1, B,
L.sup.2, and D have the meanings given in claim 14, as well as
their salts, solvates, and solvates of the salts.
17-18. (canceled)
19. Method for production of compounds of formula (I) as defined in
claim 14, the method comprising [A] mixing a solution of binder in
a buffer with a reducing agent selected from the group consisting
of dithiothreitol and tris-(2-carboxyethyl)phosphine hydrochloride,
and then reacting the resulting solution with a compound of formula
(II) ##STR01157## in which D, L.sup.1, B and L.sup.2 each have the
meanings given in claim 14, to form a compound of formula (I-A)
##STR01158## in which n, AK.sub.1, D, L.sup.1, B and L.sup.2 each
have the meanings given in claim 14, or [B] mixing a solution of
binder in a buffer with a reducing agent selected from the group
consisting of dithiothreitol and tris-(2-carboxyethyl)phosphine
hydrochloride, and then reacting the resulting solution with a
compound of formula (III) ##STR01159## in which D, L.sup.1, B and
L.sup.2 each have the meanings given in claim 14, to form a
compound of formula (I-B) ##STR01160## in which n, AK.sub.2, D,
L.sup.1, B and L.sup.2 each have the meanings given in claim
14.
20. (canceled)
21. Compounds of the general formula (XXXa) according to claim 7,
wherein R.sup.35 is methyl, thereby providing a compound of the
general formula (XXX) ##STR01161## in which Cys is a cysteine
radical which is bound to a carbon atom of the succinimide via the
sulfur atom of the side chain, L.sup.1 is a bond, linear
(C.sub.1-C.sub.10)-alkanediyl or a group of the formula
##STR01162## wherein m is a number from 2 to 6, ##.sup.1 denotes
the linkage site to the group G, ##.sup.2 denotes the linkage site
to the group B, wherein (C.sub.1-C.sub.10)-alkanediyl may be
substituted with 1 to 4 methyl substituents, and wherein two carbon
atoms of the alkanediyl chain in 1,2-, 1,3- or 1,4- relation to one
another may be bridged to form a (C.sub.3-C.sub.6)-cycloalkyl ring
or a phenyl ring including the carbon atoms optionally situated
between them, B is a bond or a group of the formula ##STR01163##
wherein * denotes the linkage site to L.sup.1, ** denotes the
linkage site to L.sup.2, P is O or NH, L.sup.3 is a bond or
(C.sub.2-C.sub.4)-alkanediyl, L.sup.4 is a bond or a group of the
formula ##STR01164## wherein *** denotes the linkage site to the
carbonyl group, **** denotes the linkage site to L.sup.2, R.sup.25
is hydrogen or methyl, Q.sup.1 is a three- to seven-membered
carbocycle or a four- to seven-membered azaheterocycle, Q.sup.2 is
a three- to seven-membered carbocycle or a four- to seven-membered
azaheterocycle, R.sup.14 is hydrogen or (C.sub.1-C.sub.4)-alkyl,
R.sup.15 is hydrogen or (C.sub.1-C.sub.4)-alkyl, or R.sup.14 and
R.sup.15 together with the atoms to which they are bound form a
five- or six-membered heterocycle, R.sup.16 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, R.sup.17 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, or R.sup.16 and R.sup.17 together with the
atoms to which they are bound form a five- or six-membered
heterocycle, R.sup.18 is hydrogen or (C.sub.1-C.sub.4)-alkyl,
R.sup.19 is hydrogen or the side group of a natural .alpha.-amino
acid or its homologs or isomers, R.sup.20 is stand for hydrogen or
(C.sub.1-C.sub.4)-alkyl, or R.sup.19 and R.sup.20 together with the
atoms to which they are bound form a pyrrolidinyl ring, R.sup.21 is
for hydrogen or (C.sub.1-C.sub.4)-alkyl, R.sup.22 is stand for
hydrogen or (C.sub.1-C.sub.4)-alkyl, or R.sup.21 and R.sup.22
together with the atoms to which they are bound form a three- to
seven-membered carbocycle, R.sup.23 is (C.sub.1-C.sub.4)-alkyl,
R.sup.24 is hydrogen or (C.sub.1-C.sub.4)-alkyl, R.sup.27 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, L.sup.2 is linear
(C.sub.2-C.sub.10)-alkanediyl or a group of the formula
##STR01165## wherein p is a number from 2 to 6, ##.sup.3 denotes
the linkage site to the group B, ##.sup.4 denotes the linkage site
to the nitrogen atom, wherein (C.sub.2-C.sub.10)-alkanediyl may be
substituted with 1 to 4 methyl substituents, and wherein two carbon
atoms of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-
relation to one another, including the carbon atoms optionally
present between them to form a (C.sub.3-C.sub.6) cycloalkyl ring or
a phenyl ring. D is a group of the formula ##STR01166## wherein
#.sup.3 denotes the linkage site to the nitrogen atom, R.sup.1 is
hydrogen, R.sup.2 is 1-hydroxyethyl, benzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, or R.sup.1 and R.sup.2 together with the
carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR01167## wherein #.sup.4 denotes the linkage site to the
vicinal nitrogen atom, #.sup.5 denotes the linkage site to the
carbonyl group, the ring A with the N--O group contained in it is a
mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR01168## wherein #.sup.6 denotes the linkage site to
the carbonyl group, R.sup.6 is hydrogen, hydroxyl or benzyloxy,
R.sup.3 is hydrogen, R.sup.4 is 1-hydroxyethyl, benzyl,
1-phenylethyl or 1H-indol-3-ylmethyl, or R.sup.3 and R.sup.4
together with the carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR01169## wherein #.sup.7 denotes the linkage site to the
vicinal nitrogen atom, #.sup.8 denotes the linkage site to the
group T.sup.1, T.sup.1 is a group of the formula
--C(.dbd.O)--OR.sup.7, --C(.dbd.O)--NR.sup.8R.sup.9,
--C(.dbd.O)--NH--NH--R.sup.10 or --CH.sub.2--O--R.sup.11, wherein
R.sup.7 is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, R.sup.8 is hydrogen or methyl, R.sup.9 is
hydrogen, methyl, ethyl, n-propyl or benzyl, or R.sup.8 and R.sup.9
together with the nitrogen atom to which they are bound form a
four- to seven-membered heterocycle, R.sup.10 is benzoyl, R.sup.11
is benzyl, which may be substituted in the phenyl group with
methoxycarbonyl or carboxyl, R.sup.5 is hydrogen, methyl or a group
of the formula ##STR01170## wherein #.sup.9 denotes the linkage
site to --CHC(R.sup.26)-T.sup.2, R.sup.12 is phenyl, which may be
substituted with methoxycarbonyl, carboxyl or a group of the
formula --S(O).sub.2OH, R.sup.13 is phenyl, which may be
substituted with methoxycarbonyl or carboxyl, R.sup.26 is hydrogen
or hydroxyl, and T.sup.2 is phenyl, benzyl, 1H-indol-3-yl or
1H-indol-3-ylmethyl, as well as their salts, solvates, and solvates
of the salts.
22. Compounds of the formula (XXX) according to claim 21, wherein
Cys is a cysteine radical which is bound to a carbon atom of the
succinimide via the sulfur atom of the side chain, L.sup.1 is a
bond, linear (C.sub.2-C.sub.6)-alkanediyl or a group of the formula
##STR01171## wherein m is a number from 2 to 6, ##.sup.1 denotes
the linkage site to the group G, ##.sup.2 denotes the linkage site
to the group B, wherein (C.sub.2-C.sub.6)-alkanediyl may be
substituted with one or two methyl substituents, B is a bond or a
group of the formula ##STR01172## wherein * denotes the linkage
site to L.sup.1, ** denotes the linkage site to L.sup.2, L.sup.3 is
a bond or ethane-1,2-diyl, L.sup.4 is a bond, R.sup.14 is hydrogen,
R.sup.15 is hydrogen, R.sup.16 is hydrogen or methyl, R.sup.17 is
hydrogen or methyl, or R.sup.16 and R.sup.17 together with the
atoms to which they are bound form a piperazinyl ring, R.sup.23 is
methyl, R.sup.24 is hydrogen or methyl, L.sup.2 is linear
(C.sub.2-C.sub.6)-alkanediyl or a group of the formula ##STR01173##
wherein p is a number from 2 to 3, ##.sup.3 denotes the linkage
site to the group B, ##.sup.4 denotes the linkage site to the
nitrogen atom, D is a group of the formula ##STR01174## wherein
#.sup.3 denotes the linkage site to the nitrogen atom, R.sup.1 is
hydrogen, R.sup.2 is 1-hydroxyethyl, benzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, or R.sup.1 and R.sup.2 together with the
carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR01175## wherein #.sup.4 denotes the linkage site to the
vicinal nitrogen atom, #.sup.5 denotes the linkage site to the
carbonyl group, the ring A with the N--O group contained in it is a
mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR01176## wherein #.sup.6 denotes the linkage site to
the carbonyl group, R.sup.6 is hydrogen, hydroxyl or benzyloxy,
R.sup.3 is hydrogen, R.sup.4 is benzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, or R.sup.3 and R.sup.4 together with the
carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR01177## wherein #.sup.7 denotes the linkage site to the
vicinal nitrogen atom, #.sup.8 denotes the linkage site to the
group T, T.sup.1 is a group of the formula --C(.dbd.O)--OR.sup.7,
--C(.dbd.O)--NR.sup.8R.sup.9, --C(.dbd.O)--NH--NH--R.sup.10 or
--CH.sub.2--O--R.sup.11, wherein R.sup.7 is hydrogen, methyl,
ethyl, n-propyl, benzyl or adamantylmethyl, R.sup.8 is hydrogen or
methyl, R.sup.9 is hydrogen, methyl, ethyl, n-propyl or benzyl,
R.sup.10 is benzoyl, R.sup.11 is benzyl, which may be substituted
in the phenyl group with methoxycarbonyl or carboxyl, R.sup.5 is
hydrogen or a group of the formula ##STR01178## wherein #.sup.9
denotes the linkage site to --CHC(R.sup.26)phenyl, R.sup.12 is
phenyl, which may be substituted with methoxycarbonyl, carboxyl or
a group of the formula --S(O).sub.2OH, and R.sup.13 is phenyl,
which may be substituted with methoxycarbonyl or carboxyl, as well
as their salts, solvates, and solvates of the salts.
23-44. (canceled)
45. Pharmaceutical drug containing a binder-drug conjugate
according to claim 1, in combination with an inert nontoxic
pharmaceutically suitable excipient.
46. Pharmaceutical drug containing a binder-drug conjugate
according to claim 1, in combination with one or more
anti-hyperproliferative, cytostatic or cytotoxic substances.
47. (canceled)
48. Method for treatment or prevention of hyperproliferative or
angiogenic diseases in humans and animals, the method comprising
administering an effective amount of at least one binder-drug
conjugate according to claim 1.
49. Binder-drug conjugates of the general formula (Ia) according to
claim 1, wherein n is a number from 1 to 20, AK is AK.sub.1 or
AK.sub.2 wherein AK.sub.1 is a binder bound to the group G via a
sulfur atom of the binder, AK.sub.2 is a binder bound to the group
G via a nitrogen atom of the binder, G for the case when
AK=AK.sub.1, is a group of the formula ##STR01179## wherein #.sup.1
denotes the linkage site to the cysteine radical of the binder,
#.sup.2 denotes the linkage site to the group L.sup.1, or for the
case when AK=AK.sub.2, G is carbonyl, L.sup.1 is a bond, linear
(C.sub.2-C.sub.6)-alkanediyl, or a group of the formula
##STR01180## or ##.sup.1-L.sup.1A-B.sup.1-L.sup.1B-##.sup.2 wherein
m is a number from 2 to 6, ##.sup.1 denotes the linkage site to the
group G, ##.sup.2 denotes the linkage site to the group B, L.sup.1A
is linear (C.sub.2-C.sub.6)-alkanediyl, B.sup.1 is a group of the
formula ##STR01181## wherein ##.sup.5 denotes the linkage site to
the group L.sup.1A, ##.sup.6 denotes the linkage site to the group
L.sup.1B, L.sup.5 is a bond, L.sup.6 is a bond or a group with the
formula ##STR01182## wherein ##.sup.7 denotes the linkage site to
the carbonyl group, ##.sup.8 denotes the linkage site to L.sup.1B,
R.sup.33 is hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
R.sup.34 is hydrogen or methyl, R.sup.29 is hydrogen, R.sup.30 is
stands foe hydrogen, R.sup.31 is hydrogen or methyl, R.sup.32 is
hydrogen or methyl, L.sup.1B is linear
(C.sub.2-C.sub.10)-alkanediyl, and wherein
(C.sub.2-C.sub.6)-alkanediyl may be substituted with one to two
methyl substituents, B is a bond or a group of the formula
##STR01183## wherein * denotes the linkage site to L.sup.1, **
denotes the linkage site to L.sup.2, L.sup.3 is a bond or
ethane-1,2-diyl, L.sup.4 is a bond or a group of the formula
##STR01184## wherein *** denotes the linkage site to the carbonyl
group, **** denotes the linkage site to L.sup.2, R.sup.25 is
hydrogen or methyl, R.sup.28 is hydrogen, methylcarbonyl or
tert-butyloxycarbonyl, Q.sup.1 is a four- to seven-membered
heterocycle, R.sup.14 is hydrogen, R.sup.15 is hydrogen, R.sup.16
is hydrogen or methyl, R.sup.17 is hydrogen or methyl, or R.sup.16
and R.sup.17 together with the atoms to which they are bound form a
piperazinyl ring, R.sup.18 is hydrogen, R.sup.19 is hydrogen,
methyl, propan-2-yl, 2-methylpropan-1-yl or 1-methylpropan-1-yl,
R.sup.20 is hydrogen or methyl, or R.sup.19 and R.sup.20 together
with the atoms to which they are bound form a pyrrolidinyl ring,
R.sup.21 is hydrogen or methyl, R.sup.22 is hydrogen or methyl, or
R.sup.21 and R.sup.22 together with the atoms to which they are
bound form a cyclopropyl ring, R.sup.23 is methyl, R.sup.24 is
hydrogen or methyl, R.sup.27 is hydrogen, L.sup.2 is linear
(C.sub.2-C.sub.6)-alkanediyl or a group of the formula ##STR01185##
wherein p is a number from 2 to 6, ##.sup.3 denotes the linkage
site to the group B, ##.sup.4 denotes the linkage site to the
nitrogen atom, wherein (C.sub.2-C.sub.10)-alkanediyl may be
substituted with one or two methyl substituents, D is a group of
the formula ##STR01186## wherein #.sup.3 denotes the linkage site
to the nitrogen atom, R.sup.1 is hydrogen, R.sup.2 is
1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or
1H-indol-3-yl-methyl, or R.sup.1 and R.sup.2 together with the
carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR01187## wherein #.sup.4 denotes the linkage site to the
vicinal nitrogen atom, #.sup.5 denotes the linkage site to the
carbonyl group, the ring A with the N--O group contained in it is a
mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR01188## wherein #.sup.6 denotes the linkage site to
the carbonyl group, R.sup.6 is hydrogen, hydroxyl or benzyloxy,
R.sup.3 is hydrogen, R.sup.4 is 1-hydroxyethyl, benzyl,
4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-yl-methyl, or R.sup.3
and R.sup.4 together with the carbon atom to which they are
attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula ##STR01189## wherein #.sup.7 denotes the linkage site to
the vicinal nitrogen atom, #.sup.8 denotes the linkage site to the
group T.sup.1, T.sup.1 is a group of the formula
--C(.dbd.O)--OR.sup.7, --C(.dbd.O)--NR.sup.8R.sup.9,
--C(.dbd.O)--NH--NH-Ro or --CH.sub.2--O--R.sup.11, wherein R.sup.7
is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, R.sup.8 is hydrogen or methyl, R.sup.9 is
hydrogen, methyl, ethyl, n-propyl or benzyl, or R.sup.8 and R.sup.9
together with the nitrogen atom to which they are bound form a
four- to seven-membered heterocycle, R.sup.10 is benzoyl, R.sup.11
is benzyl, which may be substituted in the phenyl group with
methoxycarbonyl or carboxyl, R.sup.5 is hydrogen, methyl or a group
of the formula ##STR01190## wherein #.sup.9 denotes the linkage
site to --CHC(R.sup.26)-T.sup.2, R.sup.2 is phenyl, which may be
substituted with methoxycarbonyl, carboxyl or a group of the
formula --S(O).sub.2OH, R.sup.13 is phenyl, which may be
substituted with methoxycarbonyl or carboxyl, R.sup.26 is hydrogen
or hydroxyl, T.sup.2 is phenyl, benzyl, 1H-indol-3-yl or
1H-indol-3-ylmethyl, and R.sup.35 is methyl or hydroxyl, as well as
their salts, solvates, and solvates of the salts.
50. Binder-drug conjugates of the general formula (Ia) according to
claim 1, wherein n is a number from 1 to 10, AK is AK.sub.1 or
AK.sub.2 wherein AK.sub.1 is a binder bound to the group G via a
sulfur atom of the binder, AK.sub.2 is a binder bound to the group
G via a nitrogen atom of the binder, G for the case when
AK=AK.sub.1, is a group of the formula ##STR01191## wherein #.sup.1
denotes the linkage site to the cysteine radical of the binder,
#.sup.2 denotes the linkage site to the group L.sup.1, or for the
case when AK=AK.sub.2, G is carbonyl, L.sup.1 is a bond, linear
(C.sub.2-C.sub.6)-alkanediyl, or a group of the formula
##STR01192## wherein m is a number from 2 to 3, ##.sup.1 denotes
the linkage site to the group G, ##.sup.2 denotes the linkage site
to the group B, wherein (C.sub.2-C.sub.6)-alkanediyl may be
substituted with one or two methyl substituents, B is a bond or a
group of the formula ##STR01193## wherein * denotes the linkage
site to L.sup.1, ** denotes the linkage site to L.sup.2, L.sup.3 is
a bond or ethane-1,2-diyl, L.sup.4 is a bond or a group of the
formula ##STR01194## wherein *** denotes the linkage site to the
carbonyl group, **** denotes the linkage site to L.sup.2, R.sup.25
is methyl, R.sup.28 is hydrogen, methylcarbonyl or
tert-butyloxycarbonyl, Q.sup.1 is piperidine-1,4-diyl, R.sup.16 is
hydrogen or methyl, R.sup.17 is hydrogen or methyl, or R.sup.16 and
R.sup.17 together with the atoms to which they are bound form a
piperazinyl ring, R.sup.21 is hydrogen or methyl, R.sup.22 is
hydrogen or methyl, or R.sup.21 and R.sup.22 together with the
atoms to which they are bound form a cyclopropyl ring, R.sup.23 is
methyl, R.sup.24 is hydrogen, L.sup.2 is linear
(C.sub.2-C.sub.6)-alkanediyl or a group of the formula ##STR01195##
wherein p is a number from 2 to 6, ##.sup.3 is linkage site to the
group B, ##.sup.4 is linkage site to the nitrogen atom, D is a
group of the formula ##STR01196## wherein #.sup.3 denotes the
linkage site to the nitrogen atom, R.sup.1 is hydrogen, R.sup.2 is
1-hydroxyethyl, benzyl, 1-hydroxybenzyl, 1-phenylethyl or
1H-indol-3-yl-methyl, or R.sup.1 and R.sup.2 together with the
carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR01197## wherein #.sup.4 denotes the linkage site to the
vicinal nitrogen atom, #.sup.5 denotes the linkage site to the
carbonyl group, the ring A with the N--O group contained in it is a
mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR01198## wherein #.sup.6 denotes the linkage site to
the carbonyl group, R.sup.6 is hydrogen, hydroxyl or benzyloxy,
R.sup.3 is hydrogen, R.sup.4 is benzyl, 1-hydroxybenzyl,
1-phenylethyl or 1H-indol-3-ylmethyl, or R.sup.3 and R.sup.4
together with the carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR01199## wherein #.sup.7 denotes the linkage site to the
vicinal nitrogen atom, #.sup.8 denotes the linkage site to the
group T.sup.1, T.sup.1 is a group of the formula
--C(.dbd.O)--OR.sup.7, --C(.dbd.O)--NR.sup.8R.sup.9 or
--CH.sub.2--O--R.sup.11, wherein R.sup.7 is hydrogen, methyl,
ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl, R.sup.8 is
hydrogen or methyl, R.sup.9 is hydrogen, methyl, ethyl, n-propyl or
benzyl, R.sup.11 is benzyl, which may be substituted in the phenyl
group with methoxycarbonyl or carboxyl, R.sup.5 is hydrogen, methyl
or a group of the formula ##STR01200## wherein #.sup.9 denotes the
linkage site to --CHCH.sub.2phenyl, R.sup.12 is phenyl, which may
be substituted with methoxycarbonyl, carboxyl or a group of the
formula --S(O).sub.2OH, R.sup.13 is phenyl, which may be
substituted with methoxycarbonyl or carboxyl, and R.sup.35 is
methyl or hydroxyl, as well as their salts, solvates, and solvates
of the salts.
51. Method for producing compounds of the general formula (Ia) as
defined in claim 1, the method comprising [A] mixing a solution of
binder in a buffer with a reducing agent selected from the group
consisting of dithiothreitol and tris-(2-carboxyethyl)phosphine
hydrochloride and then reacting the resulting solution with a
compound of formula (IIa) ##STR01201## in which D, L.sup.1, B,
L.sup.2 and R.sup.35 each have the meanings given in claim 1, to
form a compound of formula (Ia-A) ##STR01202## in which n,
AK.sub.1, D, L.sup.1, B, L.sup.2 and R.sup.35 each have the
meanings given in claim 1, or [B] mixing a solution of binder in a
buffer with a reducing agent selected from the group consisting of
dithiothreitol and tris-(2-carboxyethyl)phosphine hydrochloride and
then reacting the resulting solution with a compound of formula
(IIIa) ##STR01203## in which D, L.sup.1, B, L.sup.2 and R.sup.35
each have the meanings given in claim 1, to form a compound of
formula (Ia-B) ##STR01204## in which n, AK.sub.2, D, L.sup.1, B,
L.sup.2 and R.sup.35 each have the meanings given in claim 1.
52. (canceled)
53. Binder-drug conjugates of the general formula (Ia) according to
claim 1, wherein n is a number from 1 to 50, AK is a bond, the
group .sctn.-G-L.sup.1-B-.sctn..sctn. is a linker, wherein .sctn.
denotes the linkage site to the group AK and .sctn..sctn. denotes
the linkage site to the nitrogen atom, L.sup.2 is linear
(C.sub.2-C.sub.10)-alkanediyl or a group of the formula
##STR01205## wherein p is a number from 2 to 6, ##.sup.3 denotes
the linkage site to the group B, ##.sup.4 denotes the linkage site
to the nitrogen atom, wherein (C.sub.2-C.sub.10)-alkanediyl may be
substituted with 1 to 4 substituents selected independently of one
another from the group consisting of methyl, hydroxyl and benzyl,
and wherein two carbon atoms of the alkanediyl chain may be bridged
in 1,2-, 1,3- or 1,4- relation to one another, including the carbon
atoms optionally present between them to form a (C.sub.3-C.sub.6)
cycloalkyl ring or a phenyl ring, D is a group of the formula
##STR01206## wherein #.sup.3 denotes the linkage site to the
nitrogen atom, R.sup.1 is hydrogen or methyl, R.sup.2 is isopropyl,
isobutyl, sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl,
4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl,
1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or
1H-indol-3-ylmethyl, or R.sup.1 and R.sup.2 together with the
carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR01207## wherein #.sup.4 denotes the linkage site to the
vicinal nitrogen atom, #.sup.5 denotes the linkage site to the
carbonyl group, the ring A with the N--O group contained in it is a
mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR01208## wherein #.sup.6 denotes the linkage site to
the carbonyl group, R.sup.6 is hydrogen, hydroxyl or benzyloxy,
R.sup.3 is hydrogen or methyl, R.sup.4 is isopropyl, isobutyl,
sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl,
4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl,
1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or
1H-indol-3-ylmethyl, or R.sup.3 and R.sup.4 together with the
carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR01209## wherein #.sup.7 denotes the linkage site to the
vicinal nitrogen atom, #.sup.8 denotes the linkage site to the
group T.sup.1, T.sup.1 is a group of the formula
--C(.dbd.O)--OR.sup.7, --C(.dbd.O)--NR.sup.8R.sup.9,
--C(.dbd.O)--NH--NH-Ro or --CH.sub.2--O--R.sup.11, wherein R.sup.7
is hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, R.sup.8 is hydrogen or methyl, R.sup.9 is
hydrogen, methyl, ethyl, n-propyl or benzyl, or R.sup.8 and R.sup.9
together with the nitrogen atom to which they are bound form a
four- to seven-membered heterocycle, R.sup.10 is benzoyl, R.sup.11
is benzyl, which may be substituted in the phenyl group with
methoxycarbonyl or carboxyl, R.sup.5 is hydrogen, methyl or a group
of the formula ##STR01210## wherein #.sup.9 denotes the linkage
site to --CHC(R.sup.26)-T.sup.2, R.sup.12 is phenyl, which may be
substituted with methoxycarbonyl, carboxyl or a group of the
formula --S(O).sub.2OH, R.sup.13 is phenyl, which may be
substituted with methoxycarbonyl or carboxyl, R.sup.26 is hydrogen
or hydroxyl, T.sup.2 is phenyl, benzyl, 1H-indol-3-yl or
1H-indol-3-ylmethyl, and R.sup.35 is methyl or hydroxyl, as well as
their salts, solvates, and solvates of the salts.
54. Binder-drug conjugates of the general formula (Ia) according to
claim 1, wherein n is a number from 2 to 8, AK is AK.sub.1 or
AK.sub.2, wherein AK.sub.1 is a binder that is bound to the group G
via a sulfur atom of the cysteine radical of the binder, AK.sub.2
is a binder that is bound to the group G via a nitrogen atom of the
lysine radical of the binder, G for the case when AK=AK.sub.1, is a
group of the formula ##STR01211## wherein #.sup.1 denotes the
linkage site to the cysteine radical of the binder, #.sup.2 denotes
the linkage site to the group L.sup.1, or for the case when
AK=AK.sub.2, G is carbonyl, L.sup.1 is a bond, linear
(C.sub.2-C.sub.6)-alkanediyl, or a group of the formula
##STR01212## wherein m is a number from 2 to 3, ##.sup.1 denotes
the linkage site to the group G, ##.sup.2 denotes the linkage site
to the group B, wherein (C.sub.2-C.sub.6)-alkanediyl may be
substituted with one or two methyl substituents, B is a bond or a
group of the formula ##STR01213## wherein * denotes the linkage
site to L.sup.1, ** denotes the linkage site to L.sup.2, L.sup.3 is
a bond or ethane-1,2-diyl, L.sup.4 is a bond or a group of the
formula ##STR01214## wherein *** denotes the linkage site to the
carbonyl group, **** denotes the linkage site to L.sup.2, R.sup.25
is methyl, R.sup.28 is hydrogen, methylcarbonyl or
tert-butyloxycarbonyl, Q.sup.1 is piperidine-1,4-diyl, R.sup.16 is
hydrogen or methyl, R.sup.17 is hydrogen or methyl, or R.sup.16 and
R.sup.17 together with the atoms to which they are bound form a
piperazinyl ring, R.sup.21 is hydrogen or methyl, R.sup.22 is
hydrogen or methyl, or R.sup.21 and R.sup.22 together with the
atoms to which they are bound form a cyclopropyl ring, R.sup.23 is
methyl, R.sup.24 is hydrogen, L.sup.2 is linear
(C.sub.2-C.sub.6)-alkanediyl or a group of the formula ##STR01215##
wherein p is a number from 2 to 6, ##.sup.3 denotes the linkage
site to the group B, ##.sup.4 denotes the linkage site to the
nitrogen atom, D is a group of the formula ##STR01216## wherein
#.sup.3 denotes the linkage site to the nitrogen atom, R.sup.1 is
hydrogen, R.sup.2 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl, the
ring A with the N--O group contained therein is ##STR01217##
wherein #.sup.6 denotes the linkage site to the carbonyl group,
R.sup.3 is hydrogen, R.sup.4 is 4-hydroxybenzyl or
1H-indol-3-ylmethyl, T.sup.1 is a group of the formula
--C(.dbd.O)--NR.sup.8R.sup.9, wherein R.sup.8 is hydrogen or
methyl, R.sup.9 is hydrogen, methyl or ethyl, and R.sup.35 is
methyl, as well as their salts, solvates, and solvates of the
salts.
55. Binder-drug conjugates of the general formula (Ia) according to
claim 1, wherein n is a number from 2 to 8, AK is AK.sub.1, wherein
AK.sub.1 is binder that is bound to the group G via a sulfur atom
of the cysteine radical of the binder, G is a group of the formula
##STR01218## wherein #.sup.1 denotes the linkage site to the
cysteine radical of the binder, #.sup.2 denotes the linkage site to
the group L.sup.1, L.sup.1 is pentane-1,5-diyl, B is a group of the
formula ##STR01219## wherein * denotes the linkage site to L.sup.1,
** denotes the linkage site to L.sup.2, L.sup.3 is a bond, L.sup.4
is a bond, R.sup.16 is hydrogen, R.sup.17 is hydrogen, L.sup.2 is
propane-1,3-diyl, D is a group of the formula ##STR01220## wherein
#.sup.3 denotes the linkage site to the nitrogen atom, R.sup.1 is
hydrogen, R.sup.2 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl, the
ring A with the N--O group contained therein is ##STR01221##
wherein #.sup.6 denotes the linkage site to the carbonyl group, R
.sup.3 is hydrogen, R.sup.4 is 4-hydroxybenzyl or
1H-indol-3-ylmethyl, T.sup.1 is a group of the formula
--C(.dbd.O)--NR.sup.8R.sup.9, wherein R.sup.8 is hydrogen, R.sup.9
is hydrogen, and R.sup.35 is methyl, as well as their salts,
solvates, and solvates of the salts.
56. Binder-drug conjugates of the general formula (Ia) according to
claim 1, wherein n is a number from 2 to 8, AK is AK.sub.1, wherein
AK.sub.1 is a binder that is bound to the group G via a sulfur atom
of the cysteine radical of the binder, G is a group of the formula
##STR01222## wherein #.sup.1 denotes the linkage site to the
cysteine radical of the binder, #.sup.2 denotes the linkage site to
the group L.sup.1, L .sup.1 is a bond, B is a bond, L.sup.2 is
hexane-1,6-diyl, D is a group of the formula ##STR01223## wherein
#.sup.3 denotes the linkage site to the nitrogen atom, R.sup.1 is
hydrogen, R.sup.2 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl, the
ring A with the N--O group contained therein is ##STR01224##
wherein #.sup.6 denotes the linkage site to the carbonyl group,
R.sup.3 is hydrogen, R.sup.4 is 4-hydroxybenzyl or
1H-indol-3-ylmethyl, T.sup.1 is a group of the formula
--C(.dbd.O)--NR.sup.8R.sup.9, wherein R.sup.8 is hydrogen, and
R.sup.9 is hydrogen, as well as their salts, solvates, and solvates
of the salts.
57. Binder-drug conjugates of the general formula (Ia) according to
claim 1, wherein n is a number from 2 to 8, AK is AK.sub.2, wherein
AK.sub.2 is a binder that is bound to the group G via a nitrogen
atom of the binder, G is carbonyl, L.sup.1 is a bond, B is a bond,
L.sup.2 is pentane-1,5-diyl, D is a group of the formula
##STR01225## wherein #.sup.3 denotes the linkage site to the
nitrogen atom, R.sup.1 is hydrogen, R.sup.2 is 4-hydroxybenzyl or
1H-indol-3-ylmethyl, the ring A with the N--O group contained
therein is ##STR01226## wherein #.sup.6 denotes the linkage site to
the carbonyl group, R.sup.3 is hydrogen, R.sup.4 is 4-hydroxybenzyl
or 1H-indol-3-ylmethyl, T.sup.1 is a group of the formula
--C(.dbd.O)--NR.sup.8R.sup.9, wherein R.sup.8 is hydrogen, R.sup.9
is hydrogen, and R.sup.35 is methyl, as well as their salts,
solvates, and solvates of the salts.
58. Binder-drug conjugates of the general formula (Ia) according to
claim 1, wherein n is a number from 2 to 8, AK is AK.sub.2, wherein
AK.sub.2 is a binder that is bound to the group G via a nitrogen
atom of the lysine radical of the binder, G is carbonyl, L.sup.1 is
a bond, B is a bond, L.sup.2 is a group of the formula ##STR01227##
wherein p is the number 3, ##.sup.3 denotes the linkage site to the
group B, ##.sup.4 denotes the linkage site to the nitrogen atom, D
is a group of the formula ##STR01228## wherein #.sup.3 denotes the
linkage site to the nitrogen atom, R.sup.1 is hydrogen, R.sup.2 is
4-hydroxybenzyl or 1H-indol-3-ylmethyl, the ring A with the N--O
group contained therein is ##STR01229## wherein #.sup.6 denotes the
linkage site to the carbonyl group, R .sup.3 is hydrogen, R.sup.4
is 4-hydroxybenzyl or 1H-indol-3-ylmethyl, T.sup.1 is a group of
the formula --C(.dbd.O)--NR.sup.8R.sup.9, wherein R.sup.8 is
hydrogen, and R.sup.9 is hydrogen, as well as their salts,
solvates, and solvates of the salts.
Description
[0001] The present application relates to novel binder-drug
conjugates (antibody-drug conjugates, ADCs) of
N,N-dialkylauristatins, in particular those directed against the
target epidermal growth factor receptor (EGFR, gene ID 1956),
active metabolites of these ADCs, methods of synthesis of these
ADCs, use of these ADCs for treatment and/or prevention of diseases
and use of these ADCs for production of drugs for treatment and/or
prevention of diseases, in particular hyperproliferative and/or
angiogenic diseases, such as the various forms of cancer, for
example. Such treatments may be administered as monotherapy or in
combination with other drugs or other therapeutic measures.
[0002] Cancer is the result of uncontrolled cell growth of a wide
variety of tissues. In many cases, the cells grow into the existing
tissue (invasive growth) or metastasize to remote organs. Cancer
occurs in a wide variety of organs and the pathology often has a
tissue-specific course. The term cancer is therefore a generic term
that describes a large group of specific diseases of various
organs, tissues and types of cells.
[0003] Early-stage tumors can in some cases be removed by surgical
and radiotherapeutic measures. Metastatic tumors can usually be
treated only palliatively by chemotherapeutic agents. The goal here
is to find the optimum combination of improving the quality of life
and prolonging life.
[0004] Most of the chemotherapeutic agents administered
parenterally today are not distributed to the tumor tissue or tumor
cells in a targeted manner but instead are nonspecifically
distributed throughout the patient's body through systemic
administration, i.e., at sites where exposure to the drug is often
undesirable, such as in healthy cells, tissues and organs, for
example. This may lead to adverse effects or even serious general
toxic effects, which then often severely limit the therapeutically
usable drug dosage range or necessitate complete cessation of the
medication.
[0005] The improved and selective availability of these
chemotherapeutic agents in the tumor cell or the immediate
surrounding tissue and the associated increase in effect, on the
one hand, and minimization of toxic side effects, on the other
hand, have therefore for many years been the focus of work in
developing new chemotherapeutic drugs. There have been numerous
attempts so far to develop efficient methods for introducing drugs
into the target cell. However, it is still a difficult task to
optimize the association between the drug and the intracellular
target and to minimize the intercellular distribution of the drug,
e.g., to neighboring cells.
[0006] Monoclonal antibodies, for example, are suitable for
targeted addressing of tumor tissue and tumor cells. The importance
of such antibodies for clinical treatment of cancer has grown
enormously in recent years based on the efficacy of such agents as
trastuzumab (Herceptin), rituximab (Rituxan), cetuximab (Erbitux)
and bevacizumab (Avastin) which have been approved in the meantime
for treatment of individual specific tumor conditions (see, for
example, G. P. Adams and L. M. Weiner, Nat. Biotechnol. 23,
1147-1157 (2005)). As a result, there has been a significant
increase in interest in so-called immunoconjugates, such as the
aforementioned ADCs, for example, in which an internalizing
antibody directed against a tumor-associated antigen is bound
covalently to a cytotoxic agent by a linking unit ("linker"). After
introducing the ADCs into the tumor cell and then splitting off the
conjugate, either the cytotoxic agent itself or another cytotoxic
metabolite formed from it is then released inside the tumor cell,
where it can manifest its effect directly and selectively. In this
way, the damage to normal tissue can be kept within significantly
narrower limits in comparison with conventional chemotherapy for
cancer (see, for example, J. M. Lambert, Curr. Opin. Pharmacol. 5,
543-549 (2005); A. M. Wu and P. D. Senter, Nat. Biotechnol. 23,
1137-1146 (2005); P. D. Senter, Curr. Opin. Chem. Biol. 13, 235-244
(2009); L. Ducry and B. Stump, Bioconjugate Chem. 21, 5-13
(2010)).
[0007] Instead of antibodies, binders from the field of small drug
molecules may be used as binders to selectively bind to a specific
target, such as, for example, a receptor (see, e.g., E. Ruoslahti
et al., Science, 279, 377-380 (1998); D. Karkan et al., PLoS ONE 3
(6), e2469 (Jun. 25, 2008)). Conjugates of a cytotoxic drug and an
addressing ligand having a defined cleavage site between the ligand
and the drug for release of the drug are also known. One such
"intended breaking point" may consist of a peptide chain, for
example, which can be cleaved selectively at a certain site by a
specific enzyme at the site of action (see, for example, R. A.
Firestone and L. A. Telan, US Patent Application US
2002/0147138).
[0008] Monoclonal antibodies are suitable in particular for
targeted address of tumor tissues and tumor cells, especially those
directed against the target EGFR. The "epidermal growth factor
receptor" (EGFR, gene ID 1956) is a trans-membrane glycoprotein
(170 kDa) belonging to the tyrosine kinase subfamily. Although the
EGF receptor is expressed in many normal cells, it is overexpressed
in many forms of human cancer, including cancer of the large and
small intestine, carcinomas of the head and neck, pancreatic cancer
and gliomas. The extent of this over-expression correlates with a
poor prognosis (Galizia, G. et al., Ann. Surg. Oncol., June 2006,
13(6):823-35).
[0009] Binding of the ligand EGF to the EGF receptor leads to
dimerization of the receptor and activation of the intracellular
kinase domains. These kinase domains undergo autophosphorylation
and thus activate pro-proliferative signal cascades (including
those via mitogen-activated protein kinases (MAPKs) and Akt). These
signal cascades regulate the transcription of genes involved in
cell growth and cell survival, motility and proliferation.
[0010] Signal transduction by the EGF receptor also results in
activation of the wild-type KRAS gene, but the presence of an
activating somatic mutation in the KRAS gene within a cancer cell
leads to dysregulation of the signal pathways and to resistance to
EGFR inhibitory treatments (Allegra et al., J. Clin. Oncol., 20
Apr. 2009, 27(12):2091-6).
[0011] In an ADC approach, an additional antitumor effect can be
achieved by the attached cytotoxic agent in addition to inhibiting
the interaction between ligands and receptor.
[0012] The following publications describe the EGF receptor and
anti-EGFR antibodies in general: WO 00069459 A1, WO 2010145796 A2,
WO 02100348 A2, EP 00979246 B1, EP 00531472 B1, Mendelsohn, J.,
Baselga, J., Oncogene (2000) 19, 6550-6565; M. L. Janmaat and G.
Giaccone, Drugs of Today, Vol. 39, Suppl. C, 2003, pp. 61-80;
Normanno. N., et al., Gene, Jan. 17, 2006, 366(1):2-16, Epidermal
growth factor receptor (EGFR) signaling in cancer.
[0013] Auristatin E (AE) and monomethyl auristatin E (MMAE) are
synthetic analogs of the dolastatins, a special group of linear
pseudopeptides, which were originally isolated from marine sources,
and some of which have a very potent cytotoxic activity with
respect to tumor cells (for an overview, see, for example, G. R.
Pettit, Prog. Chem. Org. Nat. Prod. 70, 1-79 (1997); G. R. Pettit
et al., Anti-Cancer Drug Design 10, 529-544 (1995); G. R. Pettit et
al., Anti-Cancer Drug Design 13, 243-277 (1998)).
##STR00001## [0014] Auristatin E (AE): R=CH.sub.3 [0015]
Monomethylauristatin E (MMAE): R=H
[0016] However, MMAE has the disadvantage of a comparatively high
systemic toxicity. To improve the tumor selectivity, MMAE is used
for targeted tumor therapy in conjunction with enzymatically
cleavable valine-citrulline linkers in the ADC setting in
particular (WO 2005/081711 A2; S. O. Doronima et al., Bioconjugate
Chem. 17, 114-124 (2006)). After proteolytic cleavage, MMAE is
preferably released from the corresponding ADCs
intracellularly.
[0017] However, when used in the form of antibody-drug conjugates
(ADCs), MMAE is not compatible with linking units (linkers) between
the antibody and the drug, which do not have any enzymatically
cleavable intended breaking point (S. O. Doronina et al.,
Bioconjugate Chem. 17, 114-124 (2006)).
[0018] Monomethyl auristatin F (MMAF) is an auristatin derivative
with a C-terminal phenylalanine unit having only a moderate
antiproliferative effect in comparison with MMAE. This can very
likely be attributed to the free carboxyl group, which has a
negative effect on the cell viability of this compound because of
its polarity and charge. In this context, the methyl ester of MMAF
(MMAF-OMe) has been described as a prodrug derivative, which has a
neutral charge and can pass through the cell membrane; it also has
an increased in vitro cytotoxicity, which is greater by several
orders of magnitude in comparison with MMAF with respect to various
carcinoma cell lines (S. O. Doronina et al., Bioconjugate Chem. 17,
114-124 (2006)). It may be assumed that this effect is caused by
the MMAF itself, which is rapidly released by intracellular ester
hydrolysis after the prodrug has been incorporated into the
cells.
##STR00002## [0019] Monomethylauristatin F (MMAF): R=H [0020]
Monomethylauristatin F-methylester (MMAF-OMe): R=CH.sub.3
[0021] However, drug compounds based on simple ester derivatives
are generally at risk of chemical instability due to a nonspecific
ester hydrolysis, which is independent of the intended site of
action, for example, due to esterases present in blood plasma. This
can greatly restrict the usability of such compounds in
treatment.
[0022] Monomethyl auristatin F (MMAF) as well as various esters and
amide derivatives thereof were disclosed in WO 2005/081711 A2.
Additional auristatin analogs having a C-terminal amide-substituted
phenylalanine unit are described in WO 01/18032 A2. MMAF analogs
involving side chain modifications of phenylalanine are claimed in
WO 02/088172 A2 and WO 2007/008603 A1. WO 2007/008848 A2 describes
those in which the carboxyl group of phenylalanine is modified.
Auristatin conjugates linked via the C-terminus were recently
described in WO 2009/117531 A1 (see also S. O. Doronina et al.,
Bioconjugate Chem. 19, 1960-1963 (2008)).
[0023] In addition, auristatin derivatives such as MMAE and MMAF
are also substrates for transporter proteins, which are expressed
by many tumor cells, which can lead to development of resistance to
these drugs.
[0024] The object of the present invention was to provide novel
binder-drug conjugates (ADCs) which, due to the combination of
novel N,N-dialkylauristatin derivatives with suitable novel linkers
and binders, have a very attractive profile of effects with regard
to their specific tumor effect and/or the lower potential of the
metabolites formed intracellularly as a substrate with respect to
transporter proteins, for example, and are therefore suitable for
treatment and/or prevention of hyperproliferative and/or angiogenic
diseases, e.g., cancers.
[0025] The subject matter of the present invention is binder-drug
conjugates of the general formula (Ia)
##STR00003##
[0026] in which [0027] n stands for a number from 1 to 50, [0028]
AK stands for a binder, preferably a chimeric humanized or human
antibody, especially preferably an anti-EGFR antibody, [0029] the
group .sctn.-G-L.sup.1-B-L.sup.2.sctn..sctn. stands for a linker,
[0030] wherein [0031] .sctn. denotes the linkage site to the group
AK and [0032] .sctn..sctn. denotes the linkage site to the nitrogen
atom, [0033] D stands for a group of the formula
[0033] ##STR00004## [0034] wherein [0035] #.sup.3 denotes the
linkage site to the nitrogen atom, [0036] R.sup.1 stands for
hydrogen or methyl, [0037] R.sup.2 stands for isopropyl, isobutyl,
sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl,
4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl,
1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or
1H-indol-3-ylmethyl, [0038] or [0039] R.sup.1 and R.sup.2 together
with the carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0039] ##STR00005## [0040] wherein [0041] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [0042] #.sup.5 denotes
the linkage site to the carbonyl group, [0043] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[0043] ##STR00006## [0044] wherein [0045] #.sup.6 denotes the
linkage site to the carbonyl group, [0046] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [0047] R.sup.3 stands for hydrogen
or methyl, [0048] R.sup.4 stands for isopropyl, isobutyl,
sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl,
4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl,
1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or
1H-indol-3-ylmethyl, [0049] or [0050] R.sup.3 and R.sup.4 together
with the carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0050] ##STR00007## [0051] wherein [0052] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [0053] #.sup.8 denotes
the linkage site to the group T.sup.1, [0054] T.sup.1 stands for a
group of the formula --C(.dbd.O)--OR.sup.7,
--C(.dbd.O)--NR.sup.8R.sup.9, --C(.dbd.O)--NH--NH--R.sup.10 or
--CH.sub.2--O--R.sup.11, [0055] wherein [0056] R.sup.7 stands for
hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, [0057] R.sup.8 stands for hydrogen or methyl,
[0058] R.sup.9 stands for hydrogen, methyl, ethyl, n-propyl or
benzyl, [0059] or [0060] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are bound form a four- to
seven-membered heterocycle, [0061] R.sup.10 stands for benzoyl,
[0062] R.sup.11 stands for benzyl, which may be substituted in the
phenyl group with methoxycarbonyl or carboxyl, [0063] R.sup.5
stands for hydrogen, methyl or a group of the formula
[0063] ##STR00008## [0064] wherein [0065] #.sup.9 denotes the
linkage site to --CHC(R.sup.26)-T.sup.2, [0066] R.sup.12 stands for
phenyl, which may be substituted with methoxycarbonyl, carboxyl or
a group of the formula --S(O).sub.2OH, [0067] R.sup.13 stands for
phenyl, which may be substituted with methoxycarbonyl or carboxyl,
[0068] R.sup.26 stands for hydrogen or hydroxyl, [0069] T.sup.2
stands for phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
[0070] R.sup.35 stands for methyl or hydroxyl, [0071] as well as
their salts and solvates as well as the solvates of the salts.
[0072] Compounds according to the invention include the compounds
of formula (I) and their salts and solvates as well as the solvates
of the salts, the compounds of the formulas given below, covered by
formula (I), and their salts and solvates as well as the solvates
of the salts as well as the compounds covered by formula (I) and
referred to below as exemplary embodiments as well as their salts
and solvates as well as the solvates of the salts inasmuch as the
compounds covered by formula (I) and listed below are not already
the salts and solvates as well as the solvates of the salts.
[0073] The compounds according to the invention may exist in
different stereoisomeric forms depending on their structure, i.e.,
in the form of configurational isomers or optionally also as
conformational isomers (enantiomers and/or diastereomers, including
those in atropisomers). The present invention therefore includes
the enantiomers and diastereomers and their respective mixtures.
The stereoisomerically uniform components can be isolated in a
known way from such mixtures of enantiomers and/or diastereomers.
Chromatographic methods, in particular HPLC chromatography on a
chiral or achiral phase, are preferably used for this purpose.
[0074] If the compounds according to the invention can occur in
tautomeric forms, then the present invention also includes all
tautomeric forms.
[0075] The present invention also includes all suitable isotope
variants of the compounds according to the invention. Isotope
variants of a compound according to the invention are understood
here to refer to a compound, in which at least one atom within the
compound according to the invention is exchanged with another atom
of the same ordinal number but with a different atomic mass than
the atomic mass normally or mainly occurring in nature. Examples of
isotopes that may be incorporated into a compound according to the
invention include those of hydrogen, carbon, nitrogen, oxygen,
phosphorus, sulfur, fluorine, chlorine, bromine and iodine such as
.sup.2H (deuterium), .sup.3H (tritium), .sup.13C, .sup.14C,
.sup.15N, .sup.17O, .sup.18O, .sup.32P, .sup.33P, .sup.33S,
.sup.34S, .sup.35S, .sup.36S, .sup.18F, .sup.36Cl, .sup.82Br,
.sup.123I, .sup.124I, .sup.129I and .sup.131I. Certain isotope
variants of a compound according to the invention, such as in
particular those in which one or more radioactive isotopes are
incorporated, may be beneficial for investigating the mechanism of
action or the distribution of the drug in the body, for example.
Compounds labeled with .sup.3H or .sup.14C isotopes are especially
suitable for this purpose because of their comparative ease of
synthesizing and detection. In addition, the implantation of
isotopes, such as deuterium, for example, may lead to certain
therapeutic advantages as a result of a greater metabolic stability
of the compound, such as prolonging the half-life in the body, for
example, or reducing the required active dose. Therefore, such
modifications of the compounds according to the invention may
optionally also be preferred embodiments of the present invention.
Isotope variants of the compounds according to the invention can be
synthesized by the methods known to those skilled in the art, for
example, according to the methods described below and the
procedures given in the exemplary embodiments by using the
corresponding isotopic modifications of the respective reagents
and/or starting compounds.
[0076] Within the scope of the present invention, the preferred
salts are the physiologically safe salts of the compounds according
to the invention. This also includes salts that are not suitable
for pharmaceutical applications per se but may be used for
isolating or purifying the compounds according to the invention,
for example.
[0077] Physiologically safe salts of the compounds according to the
invention include acid addition salts of mineral acids, carboxylic
acids and sulfonic acids, for example, salts of hydrochloric acid,
hydrobromic acid, sulfuric acid, phosphoric acid, methane sulfonic
acid, ethane sulfonic acid, benzene sulfonic acid, toluene sulfonic
acid, naphthalene disulfonic acid, acetic acid, trifluoroacetic
acid, propionic acid, lactic acid, tartaric acid, malic acid citric
acid, fumaric acid, maleic acid and benzoic acid.
[0078] Physiologically safe salts of the compounds according to the
invention also include the salts of conventional bases such as
preferably and for example, alkali metal salts (e.g., sodium and
potassium salts), alkaline earth salts (e.g., calcium and magnesium
salts) and ammonium salts derived from ammonia or organic amines
with 1 to 16 carbon atoms, such as preferably and for example,
ethylamine, diethylamine, diethylamine, ethyl diisopropylamine,
monoethanolamine, diethanolamine, triethanolamine,
dicyclohexylamine, dimethylaminoethanol, procaine, dibenzoylamine,
N-methylpiperidine, N-methylmorpholine, arginine, lysine and
1,2-ethylene-diamine.
[0079] Within the scope of the invention, the solvates refer to
forms of the compounds according to the invention which form a
complex in the solid or liquid state by coordination with solvent
molecules. Hydrates are a special form of solvates having
coordinated molecules of water. Hydrates are the preferred solvates
within the scope of the present invention.
[0080] Furthermore, the present invention also includes prodrugs of
the compounds according to the invention. The term "prodrugs" here
refers to compounds which may be biologically active or inactive
themselves but are converted to the compounds according to the
invention during their dwell time in the body (for example,
metabolically or hydrolytically).
[0081] Within the scope of the present invention, the substituents
have the following meanings, unless otherwise specified:
[0082] (C.sub.1-C.sub.4)-Alkyl within the scope of the invention
stands for a linear or branched alkyl radical with 1 to 4 carbon
atoms. The following can be mentioned, preferably and for example:
methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,
1-methylpropyl and tert-butyl.
[0083] Alkanediyl within the scope of the invention stands for a
linear am-divalent alkyl radical having the number of carbon atoms
indicated in each case. The following can be mentioned, preferably
and for example: methylene, ethane-1,2-diyl(1,2-ethylene),
propane-1,3-diyl(1,3-propylene), butane-1,4-diyl(1,4-butylene),
pentane-1,5-diyl(1,5-pentylene), hexane-1,6-diyl(1,6-hexylene),
heptane-1,7-diyl(1,7-hexylene), octane-1,8-diyl(1,8-octylene),
nonane-1,9-diyl(1,9-nonylene), decane-1,10-diyl(1,10-decylene).
[0084] (C.sub.3-C.sub.7)-Cycloalkyl and/or three- to seven-membered
carbocycle within the scope of the invention stands for a
monocyclic saturated cycloalkyl group with 3 to 7 carbon atoms. The
following can be mentioned preferably and for example: cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
[0085] The side group of an .alpha.-amino acid in the R.sup.19
meaning includes both the side groups of the naturally occurring
.alpha.-amino acids and the side groups of the homologs and isomers
of these .alpha.-amino acids. The .alpha.-amino acid may be present
in both the L- and D-configurations or as a mixture of these L- and
D-forms. Examples of side groups that can be mentioned include:
methyl (alanine), propan-2-yl (valine), propan-1-yl (norvaline),
2-methylpropan-1-yl (leucine), 1-methylpropan-1-yl (isoleucine),
butan-1-yl (norleucine), tert-butyl (2-tert-butylglycine), phenyl
(2-phenylglycine), benzyl (phenylalanine), p-hydroxybenzyl
(tyrosine), indol-3-ylmethyl (tryptophan), imidazol-4-ylmethyl
(histidine), hydroxymethyl (serine), 2-hydroxyethyl (homoserine),
1-hydroxyethyl (threonine), mercaptomethyl (cysteine),
methylthiomethyl (S-methylcysteine), 2-mercaptoethyl
(homocysteine), 2-methylthioethyl (methionine), carbamoylmethyl
(asparagine), 2-carbamoylethyl (glutamine), carboxymethyl (aspartic
acid), 2-carboxyethyl (glutamic acid), 4-aminobutan-1-yl (lysine),
4-amino-3-hydroxybutan-1-yl (hydroxylysine), 3-aminopropan-1-yl
(ornithine), 2-aminoethyl (2,4-diaminobutyric acid), aminomethyl
(2,3-diaminopropionic acid), 3-guanidinopropan-1-yl (arginine),
3-ureidopropan-1-yl (citrulline). Preferred .alpha.-amino acid side
groups in the meaning of R.sup.19 include methyl (alanine),
propan-2-yl (valine), 2-methylpropan-1-yl (leucine), benzyl
(phenylalanine), imidazole-4-ylmethyl (histidine), hydroxymethyl
(serine), 1-hydroxyethyl (threonine), 4-aminobutan-1-yl (lysine),
3-aminopropan-1-yl (ornithine), 2-aminoethyl (2,4-diaminobutyric
acid), aminomethyl (2,3-diaminopropionic acid),
3-guanidinopropan-1-yl (arginine) The L configuration is preferred
in each case.
[0086] A four- to seven-membered heterocycle within the scope of
the invention stands for a monocyclic saturated heterocycle having
a total of four to seven ring atoms that contain one or two ring
heteroatoms from the series of N, O, S, SO and/or SO.sub.2 and are
linked via a ring carbon atom or optionally a ring nitrogen atom. A
five- to seven-membered heterocycle with one or two ring
heteroatoms from the series N, O and/or S, especially preferably a
five- or six-membered heterocycle with one or two ring heteroatoms
from the series of N and/or O is preferred. Examples include:
azetidinyl, oxetanyl, pyrrolidinyl, pyrazolidinyl,
tetrahydrofuranyl, thiolanyl, piperidinyl, piperazinyl,
tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl,
thiomorpholinyl, hexahydroazepinyl and hexahydro-1,4-diazepinyl.
Preferred examples include pyrrolidinyl, tetrahydrofuranyl,
piperidinyl, piperazinyl, tetrahydropyranyl and morpholinyl.
[0087] In the formula for the group for which A, B, D, G, L.sup.1,
L.sup.2, L.sup.4, R.sup.1, R.sup.2, R.sup.3, R.sup.4 and/or R.sup.5
may stand, the end point of the line at which the symbol #.sup.6,
*, **, #.sup.3, #.sup.1, #.sup.2, ##.sup.1, ##.sup.2, ##.sup.3,
##.sup.4, ***, ****, #.sup.4, #.sup.5, #.sup.6, #.sup.7, #.sup.8
and/or #.sup.9 appears does not stand for a carbon atom or a
CH.sub.2 group but instead is a component of the bond to the
respective atom identified, to which A, B, D, G, L.sup.1, L.sup.2,
L.sup.4, R.sup.1, R.sup.2, R.sup.3, R.sup.4 and/or R.sup.5 is
bound.
[0088] Within the scope of the present invention, it is true that
for all radicals that occur several times, their meanings are
independent of one another. If radicals are substituted in the
compounds according to the invention, then the radicals may be
substituted one or more times, unless otherwise specified.
Substitution with one or two substituents that are the same or
different is preferred. Substitution with one substituent is
especially preferred.
[0089] Within the scope of the present invention, the terms that
are used have the following meanings, unless otherwise
specified:
[0090] The term "linker" is understood in the broadest sense to be
a chemical unit comprising a covalent bond or a row of atoms
covalently linking a binder to a drug. The term "linker" is
preferably understood to be a series of atoms in the sense of the
present invention, which covalently link a binder to a drug. In
addition, linkers may be divalent chemical units, such as
alkyldiyls, aryldiyls, heteroaryldiyls, heterocyclyldiyls,
dicarboxylic acid esters, dicarboxylic acid amides.
[0091] The term "binder" is understood in the broadest sense to be
a molecule, which binds to a target molecule that is present on a
certain target cell population to be addressed by the binder-drug
conjugate. The term "binder" is to be understood in its broadest
interpretation, which also includes, for example, lectins, proteins
that can bind certain sugar chains or phospholipid binding
proteins. Such binders include, for example, high-molecular
proteins (binder proteins), polypeptides or peptides (binder
peptides), nonpeptidic molecules (e.g., aptamers (U.S. Pat. No.
5,270,163; review article by Keefe, A. D. et al., Nat. Rev. Drug
Discov. 2010; 9:537-550) or vitamins) and all other cell-binding
molecules or substances. Binder proteins include, for example,
antibodies and antibody fragments or antibody mimetics such as
affibodies, adnectins, anticalins, DARPins, avimers, nanobodies
(review article by Gebauer, M. et al., Curr. Opinion in Chem. Biol.
2009; 13:245-255; Nuttall, S. D. et al., Curr. Opinion in
Pharmacology, 2008; 8:608-617). Binder peptides include, for
example, ligands of a ligand-receptor pair, e.g., VEGF of the
ligand receptor pair VEGF/KDR, such as transferrin of the
ligand-receptor pair transferrin/transferrin receptor or a
cytokine/cytokine receptor, such as TNF.alpha. of the
ligand-receptor pair TNF.alpha./TNF.alpha. receptor.
[0092] Preferred binders according to the invention include
antibodies (in particular human or humanized monoclonal antibodies)
or antigen binding antibody fragments that bind to EGFR. In the
case of antibodies such as anti-EGFR antibodies, n (i.e., the
number of toxophore molecules per antibody molecule) is preferably
in the range of 1 to 10, especially preferably 2 to 8.
[0093] A "target molecule" is understood in the broadest sense to
be a molecule, which is present in the target cell population and
may be a protein (e.g., a receptor of a growth factor) or a
non-peptidic molecule (e.g., a sugar or phospholipid). It is
preferably a receptor or an antigen.
[0094] The term "extracellular" target molecule describes a target
molecule, which is bound to the cell and is found on the outside of
a cell or part of a target molecule, which is found on the outside
of a cell, i.e., a binder may bind to an intact cell at its
extracellular target molecule. An extracellular target molecule may
be anchored in the cell membrane or may be part of the cell
membrane. Those skilled in the art are familiar with methods for
identifying extracellular target molecules. For proteins this may
take place by determining the transmembrane domain(s) and though
orientation of the protein in the membrane. These specifications
are usually stored in the protein data banks (e.g., SwissProt).
[0095] The term "cancer target molecule" describes a target
molecule, which is present on one or more types of cancer cells in
comparison with noncancer cells of the same type of tissue. The
cancer target molecule is preferably selectively present on one or
more types of cancer cells in comparison with noncancer cells of
the same tissue type, where the term "selective" describes an at
least two-fold enrichment on cancer cells in comparison with
noncancer cells of the same type of tissue (a "selective cancer
target molecule"). Use of cancer target cells allows selective
treatment of cancer cells with the conjugates according to the
invention.
[0096] The binder may be linked to the linker via a bond. Various
possibilities of covalent bonding (conjugation) of organic
molecules to antibodies are known from the literature. The linkage
of the binder may be accomplished by means of a heteroatom of the
binder. Heteroatoms of the binder according to the invention that
may be used for linkage include sulfur (by means of a sulfhydryl
group of the binder in one embodiment), oxygen (by means of a
carboxyl or hydroxyl group of the binder according to the
invention) and nitrogen (by means of a primary or secondary amine
group or amide group of the binder in one embodiment). Conjugation
of the toxophores to the antibodies via one or more sulfur atoms of
cysteine radicals of the antibody and/or via one or more NH groups
of lysine radicals of the antibody is/are preferred according to
the invention. These heteroatoms may be present in the natural
binder or may be introduced through chemical or molecular
biological methods. According to the present invention, the linkage
of the binder to the toxophore only has a low influence on the
binding activity of the binder to the target molecule. In a
preferred embodiment, the linkage has no effect on the binding
activity of the binder to the target molecule.
[0097] The term "antibody" is understood in its broadest sense
according to the present invention and includes immunoglobulin
molecules, for example, intact or modified monoclonal antibodies,
polyclonal antibodies or multispecific antibodies (e.g., bispecific
antibodies). An immunoglobulin molecule preferably comprises a
molecule having four polypeptide chains, two heavy chains (H
chains) and two light chains (L chains), which are typically linked
by disulfide bridges. Each heavy chain comprises one variable
domain of the heavy chain (abbreviated VH) and one constant domain
of the heavy chain. The constant domain of the heavy chain may
comprise, for example, three domains CH1, CH2 and CH3. Each light
chain comprises one variable domain (abbreviated VL) and one
constant domain. The constant domain of the light chain comprises
one domain (abbreviated CL). The VH and VL domains can be further
subdivided into regions of hypervariability, also known as
complementarity determining regions (abbreviated CDR), and regions
of a lower sequence variability ("framework region," abbreviated
FR). Each VH and VL region is typically made up of three CDRs and
up to four FRs, for example, from the amino terminus to the carboxy
terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3,
FR4. An antibody can be obtained from any species suitable for
this, e.g., rabbit, llama, camel, mouse or rat. In one embodiment,
the antibody is of human or murine origin. An antibody may be
human, humanized or chimeric, for example.
[0098] The term "monoclonal" antibody refers to antibodies obtained
from a population of substantially homogeneous antibodies, i.e.,
individual antibodies of the population are identical except for
naturally occurring mutations which may occur in a small number.
Monoclonal antibodies recognize a single antigenic binding site
with a high specificity. The term monoclonal antibody is not based
on a specific synthesis process.
[0099] The term "intact" antibody relates to antibodies comprising
both an antigen binding domain and the constant domain of the light
and heavy chains. The constant domain may be a naturally occurring
domain or a variant thereof in which several amino acid positions
have been altered.
[0100] The term "modified intact" antibody refers to intact
antibodies that have been fused via their amino terminus or carboxy
terminus to another polypeptide or protein that does not originate
from an antibody by means of a covalent bond (for example, a
peptide linkage). In addition, antibodies may also be modified by
inserting reactive cysteines at defined sites to facilitate
coupling to a toxophore (see Junutula et al., Nat. Biotechnol.,
August 2008; 26(8):925-32).
[0101] The term "human" antibody denotes antibodies that can be
obtained from a human or are synthetic human antibodies. A
"synthetic" human antibody is an antibody that can be obtained
entirely or partially by in silico synthesis sequences based on
analysis of human antibody sequences. A human antibody may be coded
by a nucleic acid, for example, isolated from a library of antibody
sequences of human origin. One example of such an antibody is given
by Soderlind et al., Nature Biotech. 2000, 18: 853-856.
[0102] The term "humanized" or "chimeric" antibody describes
antibodies consisting of a human sequence component and a nonhuman
sequence component. In these antibodies, a portion of the sequences
of the human immunoglobulin (recipient) have been replaced by
sequence components of a nonhuman immunoglobulin (donor). The donor
is frequently a murine immunoglobulin. In humanized antibodies,
amino acids of the CDR of the recipient are replaced by amino acids
of the donor. In some cases amino acids of the framework are also
replaced by corresponding amino acids of the donor. In many cases
the humanized antibody contains amino acids not present in the
recipient or donor but inserted during optimization of the
antibody. In chimeric antibodies, variable domains of donor
immunoglobulin are fused to constant regions of a human
antibody.
[0103] The term complementarity determining region (CDR) as used
here refers to the amino acids of a variable antibody domain, which
are necessary for binding to the antigen. A variable region will
typically have three CDR regions, which are identified as CDR1,
CDR2 and CDR3. Each CDR region may comprise amino acids according
to the definition by Kabat and/or amino acids of a hypervariable
loop defined according to Chotia. The definition according to Kabat
includes, for example, the region of approximately amino acid
positions 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) of the
variable light chain and 31-35 (CDR1), 50-65 (CDR2) and 95-102
(CDR3) of the variable heavy chain (Kabat et al., Sequences of
Proteins of Immunological Interest, 5.sup.th ed. Public Health
Service, National Institutes of Health, Bethesda, Md. (1991)). The
definition according to Chotia comprises, for example,
approximately the region of amino acid positions 26-32 (CDR1),
50-52 (CDR2) and 91-96 (CDR3) of the variable light chain and 26-32
(CDR1), 53-55 (CDR2) and 96-101 (CDR3) of the variable heavy chain
(Chotia and Lesk; J. Mol. Biol. 196:901-917 (1987)). In many cases,
a CDR may comprise amino acids from a CDR region as defined by
Kabat and Chiota.
[0104] Antibodies can be divided into several various classes,
depending on the amino acid sequence of the constant domain of the
heavy chain. There are five main classes of intact antibodies: IgA,
IgD, IgE, IgG and IgM, several of which can be divided further into
subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
The constant domain of the heavy chain corresponding to the
different classes are identified as [alpha/.alpha.],
[delta/.delta.], [epsilon/.epsilon.], [gamma/.gamma.] and
[mu/.mu.]. Both the three-dimensional structure and the subunit
structure of antibodies are known.
[0105] The term "functional fragment" or "antigen binding antibody
fragment" of an antibody/-immunoglobulin is defined as a fragment
of an antibody/immunoglobulin (e.g., the variable domains of an
IgG), which still comprises the antigen binding domains of the
antibody/-immunoglobulin. The "antigen binding domain" of an
antibody typically comprises one or more hypervariable regions of
an antibody, e.g., the CDR, CDR2 and/or CDR3 regions. However, the
"framework" region of an antibody may also play a role in binding
the antibody to the antigen. The framework region forms the
framework for the CDRs. The antigen binding domain preferably
comprises at least amino acids 1 through 103 of the variable light
chain and amino acids 5 through 109 of the variable heavy chain,
more preferably amino acids 3 through 107 of the variable light
chain and 4 through 111 of the variable heavy chain, with the
complete variable light and heavy chains being especially
preferred, i.e., amino acids 1 through 109 of the VL and 1 through
113 of the VH (numbering according to WO 97/08320).
[0106] "Functional fragments" or "antigen binding antibody
fragments" of the invention comprise not conclusively Fab, Fab',
F(ab').sub.2 and Fv fragments, diabodies, single domain antibodies
(DAbs), linear antibodies, single chain antibodies (single chain
Fv, abbreviated scFv) and multispecific antibodies, for example,
bi- and tri-specific antibodies formed from antibody fragments
(C.A.K. Borrebaeck, editor (1995), Antibody Engineering
(Breakthroughs in Molecular Biology), Oxford University Press; R.
Kontermann and S. Duebel, editors (2001), Antibody Engineering
(Springer Laboratory Manual), Springer Verlag). Antibodies other
than "multispecific" or "multi-functional" include those with
identical binding sites. Multispecific antibodies may be specific
for different epitopes of an antigen or specific for epitopes of
more than one antigen (see, for example, WO 93/17715; WO 92/08802;
WO 91/00360; WO 92/05793; Tutt et al., 1991, J. Immunol. 147:60-69;
U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920;
5,601,819; or Kostelny et al., 1992, J. Immunol. 148:1547-1553). A
F(ab').sub.2 or Fab molecule may be constructed so that the number
of intermolecular disulfide interactions taking place between the
CH1 and CL domains can be reduced or completely prevented.
[0107] "Functional fragment" or "antigen binding antibody
fragments" may be fused to an additional polypeptide or protein
which does not originate from an antibody by way of their amino
terminus or carboxy terminus by means of a covalent bond (e.g., a
peptide linkage). In addition, antibodies and antigen binding
fragments may be modified so that reactive cysteines are inserted
at defined sites to facilitate coupling to a toxophore (see
Junutula et al., Nat. Biotechnol., August 2008, 26(8):925-32).
[0108] Polyclonal antibodies can be synthesized by methods with
which the average person skilled in the art is familiar. Monoclonal
antibodies can be synthesized by methods with which those skilled
in the art are familiar (Kohler and Milstein, Nature, 256:495-497,
1975). Human and/or humanized monoclonal antibodies can be
synthesized by methods with which the average person skilled in the
art is familiar (Olsson et al., Meth. Enzymol. 92:3-16 and/or
Cabilly et al., U.S. Pat. No. 4,816,567 or Boss et al., U.S. Pat.
No. 4,816,397).
[0109] The average person skilled in the art is familiar with
various methods for synthesis of antibodies and their fragments
such as, for example, by means of transgenic mice (N. Lonberg and
D. Huszar, Int. Rev. Immunol. 1995; 13(1):65-93) or Phage Display
Technologies (Clackson et al., Nature, Aug. 15, 1991,
352(6336):624-628). Antibodies according to the invention can be
obtained from recombinant antibody library consisting of the amino
acid sequences of a plurality of antibodies created from a large
number of healthy volunteer subjects. Antibodies can also be
synthesized by means of known recombinant DNA technologies. The
nucleic acid sequence of an antibody can be obtained by routine
sequencing or is available from publicly accessible data banks.
[0110] An "isolated" antibody or binder has been purified to remove
other constituents of the cell. Contaminating ingredients of a cell
which can interfere with a diagnostic or therapeutic use may be,
for example, enzymes, hormones or other peptidic or nonpeptidic
components of a cell. An antibody or binder that has been purified
to more than 95% by weight, based on the antibody and/or binder
(determined by the Lowry method, UV-Vis spectroscopy or SDS
capillary gel electrophoresis, for example). Furthermore, an
antibody that has been purified to the extent that at least 15
amino acids of the amino terminus or an internal amino acid
sequence can be determined or which has been purified to the point
of homogeneity, where homogeneity is determined by SDS-PAGE under
reducing or nonreducing conditions (detection may be performed by
Coomassie blue staining or preferably by silver staining) may also
be used. However, an antibody is normally synthesized by at least
one purification step.
[0111] The term "specific binding" or "binds specifically" refers
to an antibody or binder that binds to a predetermined
antigen/target molecule. Specific binding of an antibody or binder
typically describes an antibody, i.e., binder having an affinity of
at least 10.sup.-7 M (as the Kd value; i.e., preferably those with
a Kd value of less than 10.sup.-7 M), where the antibody, i.e., the
binder, has an affinity for the predetermined antigen/target
molecule that is at least twice as high as that of a nonspecific
antigen/target molecule (e.g., bovine serum albumin or casein)
which is not the predetermined antigen/target molecule or a closely
related antigen/target molecule.
[0112] Antibodies which are specific against a cancer cell antigen
can be synthesized by the average person skilled in the art using
methods with which he is familiar (such as recombinant expression)
or may be acquired commercially (for example, from Merck KGaA,
Germany). Examples of known commercially available antibodies in
cancer therapy include Erbitux.RTM. (cetuximab, Merck KGaA),
Avastin.RTM. (bevacizumab, Roche) and Herceptin.RTM. (trastuzumab,
Genentech). Trastuzumab is a recombinant humanized monoclonal
antibody of the IgG1.kappa. type which binds the extracellular
domains of human epidermal growth receptor with a high affinity in
a cell-based assay (Kd=5 nM). The antibody is synthesized
recombinantly in CHO cells.
[0113] The compounds of formula (I) constitute a subgroup of the
compounds of formula (Ia).
[0114] The preferred subject matter of the invention is binder-drug
conjugates of the general formula (Ia), wherein
[0115] n stands for a number from 1 to 50,
[0116] AK stands for AK.sub.1 or AK.sub.2 [0117] wherein [0118]
AK.sub.1 stands for a binder (preferably for a chimeric, humanized
or human antibody, especially preferably an anti-EGFR antibody)
which is bound to the group G by a sulfur atom of the binder,
[0119] AK.sub.2 stands for a binder (preferably for a chimeric,
humanized or human antibody, especially preferably an anti-EGFR
antibody) which is bound to the group G by a nitrogen atom of the
binder,
[0120] G for the case when AK=AK.sub.1 stands for a group of the
formula
##STR00009## [0121] wherein [0122] #.sup.1 denotes the linkage site
to the sulfur atom of the binder, [0123] #.sup.2 denotes the
linkage site to the group L.sup.1, [0124] or [0125] for the case
when AK=AK.sub.2, G stands for carbonyl,
[0126] L.sup.1 stands for a bond, linear
(C.sub.1-C.sub.10)-alkanediyl, a group of the formula
##STR00010## [0127] wherein [0128] m stands for a number from 2 to
6, [0129] ##.sup.1 denotes the linkage site to the group G, [0130]
##.sup.2 denotes the linkage site to the group B, [0131] L.sup.1A
stands for linear (C.sub.2-C.sub.10)-alkanediyl, [0132] B.sup.1
stands for a group of the formula
[0132] ##STR00011## [0133] wherein [0134] ##.sup.5 denotes the
linkage site to the group L.sup.1A, [0135] ##.sup.6 denotes the
linkage site to the group L.sup.1B, [0136] L.sup.5 stands for a
bond or (C.sub.2-C.sub.4)-alkanediyl, [0137] L.sup.6 stands for a
bond or a group with the formula
[0137] ##STR00012## [0138] wherein [0139] ##.sup.7 denotes the
linkage site to the carbonyl group, [0140] ##.sup.8 denotes the
linkage site to L.sup.1B, [0141] R.sup.33 stands for hydrogen,
(C.sub.1-C.sub.4)-alkylcarbonyl, tert-butyloxycarbonyl or
benzyloxycarbonyl, [0142] R.sup.34 stands for hydrogen or methyl,
[0143] R.sup.29 stands for hydrogen or (C.sub.1-C.sub.4)-alkyl,
[0144] R.sup.30 stands for hydrogen or (C.sub.1-C.sub.4)-alkyl,
[0145] or [0146] R.sup.29 and R.sup.30 together with the atoms to
which they are bound form a five- or six-membered heterocycle,
[0147] R.sup.31 stands for hydrogen or (C.sub.1-C.sub.4)-alkyl,
[0148] R.sup.32 stands for hydrogen or (C.sub.1-C.sub.4)-alkyl,
[0149] or [0150] R.sup.31 and R.sup.32 together with the atoms to
which they are bound form a five- or six-membered heterocycle,
[0151] L.sup.1B stands for linear (C.sub.2-C.sub.10)-alkanediyl,
[0152] and [0153] wherein (C.sub.1-C.sub.10)-alkanediyl may be
substituted with one to four substituents selected independently
from one another from the group, comprising methyl, hydroxyl and
benzyl, [0154] and [0155] wherein two carbon atoms of the
alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4- relation to
one another, including the carbon atoms optionally situated between
them, to form a (C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl
ring,
[0156] B stands for a bond or a group of the formula
##STR00013## [0157] wherein [0158] denotes the linkage site to
L.sup.1, [0159] ** denotes the linkage site to L.sup.2, [0160] P
stands for O or NH, [0161] L.sup.3 stands for a bond or
(C.sub.2-C.sub.4)-alkanediyl, [0162] L.sup.4 stands for a bond or a
group of the formula
[0162] ##STR00014## [0163] wherein [0164] *** denotes the linkage
site to the carbonyl group, [0165] **** denotes the linkage site to
L.sup.2, [0166] R.sup.25 stands for hydrogen or methyl, [0167]
R.sup.28 stands for hydrogen, (C.sub.1-C.sub.4)-alkylcarbonyl,
tert-butyloxycarbonyl or benzyloxycarbonyl, [0168] Q.sup.1 stands
for a four- to seven-membered heterocycle, [0169] Q.sup.2 stands
for a three- to seven-membered carbocycle or a four- to
seven-membered heterocycle, [0170] R.sup.14 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0171] R.sup.15 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0172] or [0173] R.sup.14 and R.sup.15
together with the atoms to which they are bound form a five- or
six-membered heterocycle, [0174] R.sup.16 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0175] R.sup.17 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0176] or [0177] R.sup.16 and R.sup.17
together with the atoms to which they are bound form a five- or
six-membered heterocycle, [0178] R.sup.18 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0179] R.sup.19 stands for hydrogen or the
side group of .alpha.-amino acid or its homologs or isomers, [0180]
R.sup.20 stands for hydrogen or (C.sub.1-C.sub.4)-alkyl, [0181] or
[0182] R.sup.19 and R.sup.20 together with the atoms to which they
are bound form a pyrrolidinyl ring, [0183] R.sup.21 stands for
hydrogen or (C.sub.1-C.sub.4)-alkyl, [0184] R.sup.22 stands for
hydrogen or (C.sub.1-C.sub.4)-alkyl, [0185] or [0186] R.sup.21 and
R.sup.22 together with the atoms to which they are bound form a
three- to seven-membered carbocycle, [0187] R.sup.23 stands for
(C.sub.1-C.sub.4)-alkyl, [0188] R.sup.24 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0189] R.sup.27 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0190] R.sup.36 stands for hydrogen,
(C.sub.1-C.sub.4)-alkylcarbonyl, tert-butyloxycarbonyl or
benzyloxycarbonyl, [0191] R.sup.37 stands for hydrogen or methyl,
[0192] or [0193] R.sup.36 and R.sup.37 together with the atoms to
which they are bound form a pyrrolidine ring,
[0194] L.sup.2 stands for linear (C.sub.2-C.sub.10)-alkanediyl or
for a group of the formula
##STR00015## [0195] wherein [0196] p stands for a number from 2 to
6, [0197] ##.sup.3 denotes the linkage site to the group B, [0198]
##.sup.4 denotes the linkage site to the nitrogen atom, [0199]
wherein (C.sub.2-C.sub.10)-alkanediyl may be substituted with one
to four substituents, selected independently of one another from
the group comprising methyl, hydroxyl and benzyl, [0200] and [0201]
wherein two carbon atoms of the alkanediyl chain may be bridged in
1,2-, 1,3- or 1,4- relation to one another, including the carbon
atoms optionally present between them, to form a (C.sub.3-C.sub.6)
cycloalkyl ring or a phenyl ring,
[0202] D stands for a group of the formula
##STR00016## [0203] wherein [0204] #.sup.3 denotes the linkage site
to the nitrogen atom, [0205] R.sup.1 stands for hydrogen or methyl,
[0206] R.sup.2 stands for isopropyl, isobutyl, sec-butyl,
tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl,
4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl,
diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
[0207] or [0208] R.sup.1 and R.sup.2 together with the carbon atom
to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0208] ##STR00017## [0209] wherein [0210] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [0211] #.sup.5 denotes
the linkage site to the carbonyl group, [0212] the ring A with the
N--O grouping contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[0212] ##STR00018## [0213] wherein [0214] #.sup.6 denotes the
linkage site to the carbonyl group, [0215] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [0216] R.sup.3 stands for hydrogen
or methyl, [0217] R.sup.4 stands for isopropyl, isobutyl,
sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl,
4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl,
1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or
1H-indol-3-ylmethyl, [0218] or [0219] R.sup.3 and R.sup.4 together
with the carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0219] ##STR00019## [0220] wherein [0221] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [0222] #.sup.8 denotes
the linkage site to the group T.sup.1, [0223] T.sup.1 stands for a
group of the formula --C(.dbd.O)--OR.sup.7,
--C(.dbd.O)--NR.sup.8R.sup.9, --C(.dbd.O)--NH--NH--R.sup.10 or
--CH.sub.2--O--R.sup.11, [0224] wherein [0225] R.sup.7 stands for
hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, [0226] R.sup.8 stands for hydrogen or methyl,
[0227] R.sup.9 stands for hydrogen, methyl, ethyl, n-propyl or
benzyl, [0228] or [0229] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are bound form a four- to
seven-membered heterocycle, [0230] R.sup.10 stands for benzoyl,
[0231] R.sup.11 stands for benzyl, which may be substituted in the
phenyl group with methoxycarbonyl or carboxyl, [0232] R.sup.5
stands for hydrogen, methyl or a group of the formula
[0232] ##STR00020## [0233] wherein [0234] #.sup.9 denotes the
linkage site to --CHC(R.sup.26)-T.sup.2, [0235] R.sup.12 stands for
phenyl, which may be substituted with methoxycarbonyl, carboxyl or
a group of the formula --S(O).sub.2OH, [0236] R.sup.13 stands for
phenyl, which may be substituted with methoxycarbonyl or carboxyl,
[0237] R.sup.26 stands for hydrogen or hydroxyl, [0238] T.sup.2
stands for phenyl, benzyl, 1H-indol-3-yl or
1H-indol-3-ylmethyl,
[0239] R.sup.35 stands for methyl or hydroxyl,
[0240] as well as their salts and solvates as well as the solvates
of the salts.
[0241] Binder-drug conjugates of the general formula (Ia), wherein
[0242] n stands for an integer from 1 to 50, [0243] AK stands for a
binder, preferably a chimeric, humanized or human antibody,
especially preferably an anti-EGFR antibody, [0244] the group
.sctn.-G-L.sup.1-B-.sctn..sctn. for a linker [0245] wherein [0246]
.sctn. denotes the linkage site to the group AK and [0247]
.sctn..sctn. denotes the linkage site to the nitrogen atom, [0248]
L.sup.2 stands for linear (C.sub.2-C.sub.10)-alkanediyl or a group
of the formula
[0248] ##STR00021## [0249] wherein [0250] p stands for a number
from 2 to 6, [0251] ##.sup.3 denotes the linkage site to the group
B, [0252] ##.sup.4 denotes the linkage site to the nitrogen atom,
[0253] wherein (C.sub.2-C.sub.10)-alkanediyl may be substituted
with one to four substituents, independently of one another,
selected from the group comprising methyl, hydroxyl and benzyl,
[0254] and [0255] wherein two carbon atoms of the alkanediyl chain
may be bridged in 1,2-, 1,3- or 1,4- relation to one another to
form a (C.sub.3-C.sub.6) cycloalkyl ring or a phenyl ring,
including the carbon atoms optionally situated between them,
[0256] D stands for a group of the formula
##STR00022## [0257] wherein [0258] #.sup.3 denotes the linkage site
to the nitrogen atom, [0259] R.sup.1 stands for hydrogen or methyl,
[0260] R.sup.2 stands for isopropyl, isobutyl, sec-butyl,
tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl,
4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl,
diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
[0261] or [0262] R.sup.1 and R.sup.2 together with the carbon atom
to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0262] ##STR00023## [0263] wherein [0264] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [0265] #.sup.5 denotes
the linkage site to the carbonyl group, [0266] the ring A with the
N--O group contained therein stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[0266] ##STR00024## [0267] wherein [0268] #.sup.6 denotes the
linkage site to the carbonyl group, [0269] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [0270] R.sup.3 stands for hydrogen
or methyl, [0271] R.sup.4 stands for isopropyl, isobutyl,
sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl,
4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl,
1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or
1H-indol-3-ylmethyl, [0272] or [0273] R.sup.3 and R.sup.4 together
with the carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0273] ##STR00025## [0274] wherein [0275] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [0276] #.sup.8 denotes
the linkage site to the group T.sup.1, [0277] T.sup.1 stands for a
group of the formula --C(.dbd.O)--OR.sup.7,
--C(.dbd.O)--NR.sup.8R.sup.9, --C(.dbd.O)--NH--NH--R.sup.10 or
--CH.sub.2--O--R.sup.11, [0278] wherein [0279] R.sup.7 stands for
hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, [0280] R.sup.8 stands for hydrogen or methyl,
[0281] R.sup.9 stands for hydrogen, methyl, ethyl, n-propyl or
benzyl, [0282] or [0283] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are bound form a four- to
seven-membered heterocycle, [0284] R.sup.10 stands for benzoyl,
[0285] R.sup.11 stands for benzyl, which may be substituted with
methoxycarbonyl or carboxyl in the phenyl group, [0286] R.sup.5
stands for hydrogen, methyl or a group of the formula
[0286] ##STR00026## [0287] wherein [0288] #.sup.9 denotes the
linkage site to --CHC(R.sup.26)-T.sup.2, [0289] R.sup.12 stands for
phenyl, which may be substituted with methoxycarbonyl, carboxyl or
a group of the formula --S(O).sub.2OH, [0290] R.sup.13 stands for
phenyl, which may be substituted with methoxycarbonyl or
carboxyl,
[0291] R.sup.26 stands for hydrogen or hydroxyl,
[0292] T.sup.2 stands for phenyl, benzyl, 1H-indol-3-yl or
1H-indol-3-ylmethyl,
[0293] R.sup.35 stands for methyl or hydroxyl,
[0294] as well as their salts and solvates as well as the solvates
of the salts.
[0295] The preferred subject matter of the present invention
relates to binder-drug conjugates of general formula (Ia) as given
above, wherein
[0296] n stands for a number from 1 to 50,
[0297] AK stands for AK.sub.1 or AK.sub.2 [0298] wherein [0299]
AK.sub.1 stands for a binder (preferably for a chimeric, humanized
or human antibody, especially preferably an anti-EGFR antibody),
which is bound to the group G via a sulfur atom of the binder,
[0300] AK.sub.2 stands for a binder (preferably for a chimeric,
humanized or human antibody, especially preferably an anti-EGFR
antibody) that is bound to the group G via a nitrogen atom of the
binder,
[0301] G for the case when AK=AK.sub.1, stands for a group of the
formula
##STR00027## [0302] wherein [0303] #.sup.1 denotes the linkage site
to the sulfur atom of the binder, [0304] #.sup.2 denotes the
linkage site to the group L.sup.1, [0305] or [0306] for the case
when AK=AK.sub.2, G stands for carbonyl,
[0307] L.sup.1 stands for a bond, linear
(C.sub.1-C.sub.10)-alkanediyl, a group of the formula
##STR00028## [0308] wherein [0309] m stands for a number from 2 to
6, [0310] ##.sup.1 denotes the linkage site to the group G, [0311]
##.sup.2 denotes the linkage site to the group B, [0312] L.sup.1A
stands for linear (C.sub.2-C.sub.10)-alkanediyl, [0313] B.sup.1
stands for a group of the formula
[0313] ##STR00029## [0314] wherein [0315] ##.sup.5 denotes the
linkage site to the group L.sup.1A, [0316] ##.sup.6 denotes the
linkage site to the group L.sup.1B, [0317] L.sup.5 stands for a
bond or (C.sub.2-C.sub.4)-alkanediyl, [0318] L.sup.6 stands for a
bond or a group with the formula
[0318] ##STR00030## [0319] wherein [0320] ##.sup.7 denotes the
linkage site to the carbonyl group, [0321] ##.sup.8 denotes the
linkage site to L.sup.1B, [0322] R.sup.33 stands for hydrogen,
(C.sub.1-C.sub.4)-alkylcarbonyl, tert-butyloxycarbonyl or
benzyloxycarbonyl, [0323] R.sup.34 stands for hydrogen or methyl,
[0324] R.sup.29 stands for hydrogen or (C.sub.1-C.sub.4)-alkyl,
[0325] R.sup.30 stands for hydrogen or (C.sub.1-C.sub.4)-alkyl,
[0326] or [0327] R.sup.29 and R.sup.30 together with the atoms to
which they are bound form a five- or six-membered heterocycle,
[0328] R.sup.31 stands for hydrogen or (C.sub.1-C.sub.4)-alkyl,
[0329] R.sup.32 stands for hydrogen or (C.sub.1-C.sub.4)-alkyl,
[0330] or [0331] R.sup.31 and R.sup.32 together with the atoms to
which they are bound form a five- or six-membered heterocycle,
[0332] L.sup.1B stands for linear (C.sub.2-C.sub.10)-alkanediyl,
[0333] and [0334] wherein (C.sub.1-C.sub.10)-alkanediyl may be
substituted with one to four substituents selected independently of
one another from the group comprising methyl, hydroxyl and benzyl
[0335] and [0336] wherein two carbon atoms of the alkanediyl chain
in 1,2-, 1,3- or 1,4- relation to one another may be bridge to form
a (C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring by inclusion
of the carbon atoms optionally occurring between them,
[0337] B stands for a bond or a group of the formula
##STR00031## [0338] wherein [0339] * denotes the linkage site to
L.sup.1, [0340] ** denotes the linkage site to L.sup.2, [0341] P
stands for O or NH, [0342] L.sup.3 stands for a bond or
(C.sub.2-C.sub.4)-alkanediyl, [0343] L.sup.4 stands for a bond or a
group of the formula
[0343] ##STR00032## [0344] wherein [0345] *** denotes the linkage
site to the carbonyl group, [0346] **** denotes the linkage site to
L.sup.2, [0347] R.sup.25 stands for hydrogen or methyl, [0348]
R.sup.28 stands for hydrogen, (C.sub.1-C.sub.4)-alkylcarbonyl,
tert-butyloxycarbonyl or benzyloxycarbonyl, [0349] Q.sup.1 stands
for a four- to seven-membered heterocycle, [0350] Q.sup.2 stands
for a three- to seven-membered carbocycle or a four- to
seven-membered heterocycle, [0351] R.sup.14 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0352] R.sup.15 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0353] or [0354] R.sup.14 and R.sup.18
together with the atoms to which they are bound may form a five- or
six-membered heterocycle, [0355] R.sup.16 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0356] R.sup.17 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0357] or [0358] R.sup.16 and R.sup.17
together with the atoms to which they are bound form a five- or
six-membered heterocycle, [0359] R.sup.18 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0360] R.sup.19 stands for hydrogen or the
side group of a naturally occurring .alpha.-amino acid or its
homologs or isomers, [0361] R.sup.20 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0362] or [0363] R.sup.19 and R.sup.20
together with the atoms to which they are bound form a pyrrolidinyl
ring, [0364] R.sup.21 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0365] R.sup.22 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0366] or [0367] R.sup.21 and R.sup.22
together with the atoms to which they are bound form a three- to
seven-membered carbocycle, [0368] R.sup.23 stands for
(C.sub.1-C.sub.4)-alkyl, [0369] R.sup.24 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0370] R.sup.27 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0371] R.sup.36 stands for hydrogen,
(C.sub.1-C.sub.4)-alkylcarbonyl, tert-butyloxycarbonyl or
benzyloxycarbonyl, [0372] R.sup.37 stands for hydrogen or methyl,
[0373] or [0374] R.sup.36 and R.sup.37 together with the atoms to
which they are bound form a pyrrolidine ring,
[0375] L.sup.2 stands for linear (C.sub.2-C.sub.10)-alkanediyl or
for a group of the formula
##STR00033## [0376] wherein [0377] p stands for a number from 2 to
6, [0378] ##.sup.3 denotes the linkage site to the group B, [0379]
##.sup.4 denotes the linkage site to the nitrogen atom, [0380]
wherein (C.sub.2-C.sub.10)-alkanediyl may be substituted with 1 to
4 substituents selected independently of one another from the group
comprising methyl, hydroxyl and benzyl, [0381] and [0382] wherein
two carbon atoms of the alkanediyl chain in 1,2-, 1,3- or 1,4-
relation to one another to form a (C.sub.3-C.sub.6) cycloalkyl ring
or a phenyl ring, including the carbon atoms optionally situated
between them,
[0383] D stands for a group of the formula
##STR00034## [0384] wherein [0385] #.sup.3 denotes the linkage site
to the nitrogen atom, [0386] R.sup.1 stands for hydrogen or methyl,
[0387] R.sup.2 stands for isopropyl, isobutyl, sec-butyl,
tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl,
4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl,
diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
[0388] or [0389] R.sup.1 and R.sup.2 together with the carbon atom
to which they are attached may form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0389] ##STR00035## [0390] wherein [0391] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [0392] #.sup.5 denotes
the linkage site to the carbonyl group, [0393] the ring A together
with the N--O group contained therein stands for a monocyclic or
bicyclic, optionally substituted heterocycle of the formula
[0393] ##STR00036## [0394] wherein [0395] #.sup.6 denotes the
linkage site to the carbonyl group, [0396] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [0397] R.sup.3 stands for hydrogen
or methyl, [0398] R.sup.4 stands for isopropyl, isobutyl,
sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl,
4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl,
1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or
1H-indol-3-ylmethyl, [0399] or [0400] R.sup.3 and R.sup.4 together
with the carbon atom to which they are attached forms a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0400] ##STR00037## [0401] wherein [0402] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [0403] #.sup.8 denotes
the linkage site to the group T.sup.1, [0404] T.sup.1 stands for a
group of the formula --C(.dbd.O)--OR.sup.7,
--C(.dbd.O)--NR.sup.8R.sup.9, --C(.dbd.O)--NH--NH--R.sup.10 or
--CH.sub.2--O--R.sup.11, [0405] wherein [0406] R.sup.7 stands for
hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, [0407] R.sup.8 stands for hydrogen or methyl,
[0408] R.sup.9 stands for hydrogen, methyl, ethyl, n-propyl or
benzyl, [0409] or [0410] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are bound form a four- to
seven-membered heterocycle, [0411] R.sup.10 stands for benzoyl,
[0412] R.sup.11 stands for benzyl, which may be substituted in the
phenyl group with methoxycarbonyl or carboxyl, [0413] R.sup.5
stands for hydrogen, methyl or a group of the formula
[0413] ##STR00038## [0414] wherein [0415] #.sup.9 denotes the
linkage site to --CHC(R.sup.26)-T.sup.2, [0416] R.sup.12 stands for
phenyl, which may be substituted with methoxycarbonyl, carboxyl or
a group of the formula --S(O).sub.2OH, [0417] R.sup.13 stands for
phenyl, which may be substituted with methoxycarbonyl or carboxyl,
[0418] R.sup.26 stands for hydrogen or hydroxyl, [0419] T.sup.2
stands for phenyl, benzyl, 1H-indol-3-yl or
1H-indol-3-ylmethyl,
[0420] R.sup.35 stands for methyl or hydroxyl,
[0421] as well as their salts and solvates as well as the solvates
of the salts.
[0422] The preferred subject matter of the invention relates to
binder-drug conjugates of the general formula (Ia), wherein
[0423] n stands for a number from 1 to 20,
[0424] AK stands for AK.sub.1 or AK.sub.2 [0425] wherein [0426]
AK.sub.1 stands for an antibody or an antigen binding antibody
fragment which binds to EGFR and is bound to the group G via the
sulfur atom of a cysteine radical of the binder, [0427] AK.sub.2
stands for an antibody or an antigen binding antibody fragment
which binds to EGFR and is bound to the group G via the NH side
group of a lysine radical of the binder,
[0428] G for the case when AK=AK.sub.1 stands for a group of the
formula
##STR00039## [0429] wherein [0430] #.sup.1 denotes the linkage site
to the cysteine radical of the binder, [0431] #.sup.2 denotes the
linkage site to the group L.sup.1, [0432] or [0433] for the case
when AK=AK.sub.2, G stands for carbonyl,
[0434] L.sup.1 stands for a bond, linear
(C.sub.2-C.sub.6)-alkanediyl, a group of the formula
##STR00040## [0435] wherein [0436] m stands for a number from 2 to
6, [0437] ##.sup.1 denotes the linkage site to the group G, [0438]
##.sup.2 denotes the linkage site to the group B, [0439] L.sup.1A
stands for linear (C.sub.2-C.sub.6)-alkanediyl, [0440] B.sup.1
stands for a group of the formula
[0440] ##STR00041## [0441] wherein [0442] ##.sup.5 denotes the
linkage site to the group L.sup.1A, [0443] ##.sup.6 denotes the
linkage site to the group L.sup.1B, [0444] L.sup.5 stands for a
bond, [0445] L.sup.6 stands for a bond or a group of the
formula
[0445] ##STR00042## [0446] wherein [0447] ##.sup.7 denotes the
linkage site to the carbonyl group, [0448] ##.sup.8 denotes the
linkage site to L.sup.1B, [0449] R.sup.33 for hydrogen,
methylcarbonyl or tert-butyloxycarbonyl, [0450] R.sup.34 stands for
hydrogen or methyl, [0451] R.sup.29 stands for hydrogen, [0452]
R.sup.30 stands for hydrogen, [0453] R.sup.31 stands for hydrogen
or methyl, [0454] R.sup.32 stands for hydrogen or methyl, [0455]
L.sup.1B stands for linear (C.sub.2-C.sub.6)-alkanediyl, [0456] and
[0457] wherein (C.sub.2-C.sub.6)-alkanediyl may be substituted with
one to two methyl substituents,
[0458] B stands for a bond or a group of the formula
##STR00043## [0459] wherein [0460] * denotes the linkage site to
L.sup.1, [0461] ** denotes the linkage site to L.sup.2, [0462]
L.sup.3 stands for a bond or ethane-1,2-diyl, [0463] L.sup.4 stands
for a bond or a group of the formula
[0463] ##STR00044## [0464] wherein [0465] *** denotes the linkage
site to the carbonyl group, [0466] **** denotes the linkage site to
L.sup.2, [0467] R.sup.25 stands for hydrogen or methyl, [0468]
R.sup.28 stands for hydrogen, methylcarbonyl or
tert-butyloxycarbonyl, [0469] Q.sup.1 stands for a four- to
seven-membered heterocycle, [0470] R.sup.14 stands for hydrogen,
[0471] R.sup.15 stands for hydrogen, [0472] R.sup.16 stands for
hydrogen or methyl, [0473] R.sup.17 stands for hydrogen or methyl,
[0474] or [0475] R.sup.16 and R.sup.17 together with the atoms to
which they are bound form a piperazinyl ring, [0476] R.sup.18
stands for hydrogen, [0477] R.sup.19 stands for hydrogen, methyl,
propan-2-yl, 2-methylpropan-1-yl or 1-methyl-propan-1-yl, [0478]
R.sup.20 stands for hydrogen or methyl, [0479] or [0480] R.sup.19
and R.sup.20 together with the atoms to which they are bound form a
pyrrolidinyl ring, [0481] R.sup.21 stands for hydrogen or methyl,
[0482] R.sup.22 stands for hydrogen or methyl, [0483] or [0484]
R.sup.21 and R.sup.22 together with the atoms to which they are
bound form a cyclopropyl ring, [0485] R.sup.23 stands for methyl,
[0486] R.sup.24 stands for hydrogen or methyl, [0487] R.sup.27
stands for hydrogen, [0488] R.sup.36 stands for hydrogen,
methylcarbonyl or tert-butyloxycarbonyl, [0489] R.sup.37 stands for
hydrogen or methyl, [0490] or [0491] R.sup.36 and R.sup.37 together
with the atoms to which they are bound form a pyrrolidine ring,
[0492] L.sup.2 stands for linear (C.sub.2-C.sub.6)-alkanediyl or
for a group of the formula
##STR00045## [0493] wherein [0494] p stands for a number from 2 to
6, [0495] ##.sup.3 denotes the linkage site to the group B, [0496]
##.sup.4 denotes the linkage site to the nitrogen atom, [0497]
wherein (C.sub.2-C.sub.10)-alkanediyl may be substituted with one
or two methyl substituents.
[0498] D stands for a group of the formula
##STR00046## [0499] wherein [0500] #.sup.3 denotes the linkage site
to the nitrogen atom, [0501] R.sup.1 stands for hydrogen, [0502]
R.sup.2 stands for 1-hydroxyethyl, benzyl, 4-hydroxybenzyl,
1-phenylethyl or 1H-indol-3-ylmethyl, [0503] or [0504] R.sup.1 and
R.sup.2 together with the carbon atom to which they are attached
may form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[0504] ##STR00047## [0505] wherein [0506] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [0507] #.sup.5 denotes
the linkage site to the carbonyl group, [0508] the ring A with the
N--O group contained therein stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[0508] ##STR00048## [0509] wherein [0510] #.sup.6 denotes the
linkage site to the carbonyl group, [0511] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [0512] R.sup.3 stands for
hydrogen, [0513] R.sup.4 stands for 1-hydroxyethyl, benzyl,
4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl, [0514] or
[0515] R.sup.3 and R.sup.4 together with the carbon atom to which
they are attached may form a (1S,2R)-2-phenylcyclopropane-1,1-diyl
group of the formula
[0515] ##STR00049## [0516] wherein [0517] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [0518] #.sup.8 denotes
the linkage site to the group T.sup.1, [0519] T.sup.1 stands for a
group of the formula --C(.dbd.O)--OR.sup.7,
--C(.dbd.O)--NR.sup.8R.sup.9, --C(.dbd.O)--NH--NH--R.sup.10 or
--CH.sub.2--O--R.sup.11, [0520] wherein [0521] R.sup.7 stands for
hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, [0522] R.sup.8 stands for hydrogen or methyl,
[0523] R.sup.9 stands for hydrogen, methyl, ethyl, n-propyl or
benzyl, [0524] or [0525] R.sup.8 and R.sup.9 together with the
nitrogen atom which they are attached may form a four- to
seven-membered heterocycle, [0526] R.sup.10 stands for benzoyl,
[0527] R.sup.11 stands for benzyl, which may be substituted with
methoxycarbonyl or carboxyl in the phenyl group, [0528] R.sup.5
stands for hydrogen, methyl or a group of the formula
[0528] ##STR00050## [0529] wherein [0530] #.sup.9 denotes the
linkage site to --CHC(R.sup.26)-T.sup.2, [0531] R.sup.12 stands for
phenyl, which may be substituted with methoxycarbonyl, carboxyl or
a group of the formula --S(O).sub.2OH, [0532] R.sup.13 stands for
phenyl, which may be substituted with methoxycarbonyl or carboxyl,
[0533] R.sup.26 stands for hydrogen or hydroxyl, [0534] T.sup.2
stands for phenyl, benzyl, 1H-indol-3-yl or
1H-indol-3-ylmethyl,
[0535] R.sup.35 stands for methyl or hydroxyl,
[0536] as well as their salts and solvates as well as the solvates
of the salts.
[0537] The preferred subject matter of the present invention
relates to binder-drug conjugates of the general formula (Ia) as
indicated above, wherein
[0538] n stands for a number from 1 to 20,
[0539] AK stands for AK.sub.1 or AK.sub.2 [0540] wherein [0541]
AK.sub.1 stands for an antibody or an antigen binding antibody
fragment which binds to EGFR and is bound to the group G by the
sulfur atom of a cysteine radical of the binder, [0542] AK.sub.2
stands for an antibody or an antigen binding antibody fragment
which binds to EGFR and is bound to the group G by the NH side
group of a lysine radical of the binder,
[0543] G for the case when AK=AK.sub.1 stands for the group of the
formula
##STR00051## [0544] wherein [0545] #.sup.1 denotes the linkage site
to the cysteine radical of the binder, [0546] #.sup.2 denotes the
linkage site to the group L.sup.1, [0547] or [0548] for the case
when AK=AK.sub.2, G stands for carbonyl,
[0549] L.sup.1 stands for a bond, linear
(C.sub.2-C.sub.6)-alkanediyl, a group of the formula
##STR00052## [0550] wherein [0551] m stands for a number from 2 to
6, [0552] ##.sup.1 denotes the linkage site to the group G, [0553]
##.sup.2 denotes the linkage site to the group B, [0554] L.sup.1A
stands for linear (C.sub.2-C.sub.6)-alkanediyl, [0555] B.sup.1
stands for a group with the formula
[0555] ##STR00053## [0556] wherein [0557] ##.sup.5 denotes the
linkage site to the group L.sup.1A, [0558] ##.sup.6 denotes the
linkage site to the group L.sup.1B, [0559] L.sup.5 stands for a
bond, [0560] L.sup.6 stands for a bond or a group of the
formula
[0560] ##STR00054## [0561] wherein [0562] ##.sup.7 denotes the
linkage site to the carbonyl group, [0563] ##.sup.8 denotes the
linkage site to L.sup.1B, [0564] R.sup.33 stands for hydrogen,
methylcarbonyl or tert-butyloxycarbonyl, [0565] R.sup.34 stands for
hydrogen or methyl, [0566] R.sup.29 stands for hydrogen, [0567]
R.sup.30 stands for hydrogen, [0568] R.sup.31 stands for hydrogen
or methyl, [0569] R.sup.32 stands for hydrogen or methyl, [0570]
L.sup.1B stands for linear (C.sub.2-C.sub.6)-alkanediyl, [0571] and
[0572] wherein (C.sub.2-C.sub.6)-alkanediyl may be substituted with
one to two methyl substituents,
[0573] B stands for a bond or a group of the formula
##STR00055## [0574] wherein [0575] * denotes the linkage site to
L.sup.1, [0576] ** denotes the linkage site to L.sup.2, [0577]
L.sup.3 stands for a bond or ethane-1,2-diyl, [0578] L.sup.4 stands
for a bond or a group of the formula
[0578] ##STR00056## [0579] wherein [0580] *** denotes the linkage
site to the carbonyl group, [0581] **** denotes the linkage site to
L.sup.2, [0582] R.sup.25 stands for hydrogen or methyl, [0583]
R.sup.28 stands for hydrogen, methylcarbonyl tert-butyloxycarbonyl,
[0584] Q.sup.1 stands for a four- to seven-membered heterocycle,
[0585] R.sup.14 stands for hydrogen, [0586] R.sup.15 stands for
hydrogen, [0587] R.sup.16 stands for hydrogen or methyl, [0588]
R.sup.17 stands for hydrogen or methyl, [0589] or [0590] R.sup.16
and R.sup.17 together with the atoms to which they are bound form a
piperazinyl ring, [0591] R.sup.18 stands for hydrogen, [0592]
R.sup.19 stands for hydrogen, methyl, propan-2-yl,
2-methylpropan-1-yl or 1-methyl-propan-1-yl, [0593] R.sup.20 stands
for hydrogen or methyl, [0594] or [0595] R.sup.19 and R.sup.20
together with the atoms to which they are bound form a pyrrolidinyl
ring, [0596] R.sup.21 stands for hydrogen or methyl, [0597]
R.sup.22 stands for hydrogen or methyl, [0598] or [0599] R.sup.21
and R.sup.22 together with the atoms to which they are bound form a
cyclopropyl ring, [0600] R.sup.23 stands for methyl, [0601]
R.sup.24 stands for hydrogen or methyl, [0602] R.sup.27 stands for
hydrogen, [0603] R.sup.36 stands for hydrogen,
(C.sub.1-C.sub.4)-alkylcarbonyl, tert-butyloxycarbonyl or
benzyloxycarbonyl, [0604] R.sup.37 stands for hydrogen or
methyl,
[0605] L.sup.2 stands for linear (C.sub.2-C.sub.6)-alkanediyl or
for a group of the formula
##STR00057## [0606] wherein [0607] p stands for a number from 2 to
6, [0608] ##.sup.3 denotes the linkage site to the group B, [0609]
##.sup.4 denotes the linkage site to the nitrogen atom, [0610]
wherein (C.sub.2-C.sub.10)-alkanediyl may be substituted with one
or two methyl substituents,
[0611] D stands for a group of the formula
##STR00058## [0612] wherein [0613] #.sup.3 denotes the linkage site
to the nitrogen atom, [0614] R.sup.1 stands for hydrogen, [0615]
R.sup.2 stands for 1-hydroxyethyl, benzyl, 4-hydroxybenzyl,
1-phenylethyl or 1H-indol-3-ylmethyl, [0616] or [0617] R.sup.1 and
R.sup.2 together with the carbon atom to which they are attached
form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[0617] ##STR00059## [0618] wherein [0619] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [0620] #.sup.5 denotes
the linkage site to the carbonyl group, [0621] the ring A together
with the N--O group contained in it stands for a monocyclic or
bicyclic, optionally substituted heterocycle of the formula
[0621] ##STR00060## [0622] wherein [0623] #.sup.6 denotes the
linkage site to the carbonyl group, [0624] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [0625] R.sup.3 stands for
hydrogen, [0626] R.sup.4 stands for 1-hydroxyethyl, benzyl,
4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl, [0627] or
[0628] R.sup.3 and R.sup.4 together with the carbon atom to which
they are attached may form a (1S,2R)-2-phenylcyclopropane-1,1-diyl
group of the formula
[0628] ##STR00061## [0629] wherein [0630] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [0631] #.sup.8 denotes
the linkage site to the group T.sup.1, [0632] T.sup.1 stands for a
group of the formula --C(.dbd.O)--OR.sup.7,
--C(.dbd.O)--NR.sup.8R.sup.9, --C(.dbd.O)--NH--NH--R.sup.10 or
--CH.sub.2--O--R.sup.11, [0633] wherein [0634] R.sup.7 stands for
hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, [0635] R.sup.8 stands for hydrogen or methyl,
[0636] R.sup.9 stands for hydrogen, methyl, ethyl, n-propyl or
benzyl, [0637] or [0638] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are bound may form a four- to
seven-membered heterocycle, [0639] R.sup.10 stands for benzoyl,
[0640] R.sup.11 stands for benzyl, which may be substituted with
methoxycarbonyl or carboxyl in the phenyl group, [0641] R.sup.5
stands for hydrogen, methyl or a group of the formula
[0641] ##STR00062## [0642] wherein [0643] #.sup.9 denotes the
linkage site to --CHC(R.sup.26)-T.sup.2, [0644] R.sup.12 stands for
phenyl, which may be substituted with methoxycarbonyl, carboxyl or
a group of the formula --S(O).sub.2OH, [0645] R.sup.13 stands for
phenyl, which may be substituted with methoxycarbonyl or carboxyl,
[0646] R.sup.26 stands for hydrogen or hydroxyl, [0647] T.sup.2
stands for phenyl, benzyl, 1H-indol-3-yl or
1H-indol-3-ylmethyl,
[0648] R.sup.35 stands for methyl or hydroxyl,
[0649] as well as their salts and solvates as well as the solvates
of the salts.
[0650] The preferred subject matter of the invention relates to
binder-drug conjugates of the general formula (Ia), wherein
[0651] n stands for a number between 1 and 10,
[0652] AK stands for AK.sub.1 or AK.sub.2 [0653] wherein [0654]
AK.sub.1 stands for cetuximab, pantitumumab or nimutuzumab, which
is bound to the group G via the sulfur atom of a cysteine radical
of the binder, [0655] AK.sub.2 stands for cetuximab, pantitumumab
or nimutuzumab, which is bound to the group G via the NH side group
of a lysine radical of the binder,
[0656] G for the case when AK=AK.sub.1 stands for a group of the
formula
##STR00063## [0657] wherein [0658] #.sup.1 denotes the linkage site
to the cysteine radical of the binder, [0659] #.sup.2 denotes the
linkage site to the group L.sup.1, [0660] or [0661] for the case
when AK=AK.sub.2, G stands for carbonyl,
[0662] L.sup.1 stands for a bond, linear
(C.sub.2-C.sub.6)-alkanediyl, a group of the formula
##STR00064## [0663] wherein [0664] m stands for a number of 2 or 3,
[0665] ##.sup.1 denotes the linkage site to the group G, [0666]
##.sup.2 denotes the linkage site to the group B, [0667] wherein
(C.sub.2-C.sub.6)-alkanediyl may be substituted with one or two
methyl substituents,
[0668] B stands for a bond or a group of the formula
##STR00065## [0669] wherein [0670] * denotes the linkage site to
L.sup.1, [0671] ** denotes the linkage site to L.sup.2, [0672]
L.sup.3 stands for a bond or ethane-1,2-diyl, [0673] L.sup.4 stands
for a bond or a group of the formula
[0673] ##STR00066## [0674] wherein [0675] *** denotes the linkage
site to the carbonyl group, [0676] **** denotes the linkage site to
L.sup.2, [0677] R.sup.25 stands for methyl, [0678] R.sup.28 stands
for hydrogen, methylcarbonyl or tert-butyloxycarbonyl, [0679]
Q.sup.1 stands for piperidine-1,4-diyl, [0680] R.sup.16 stands for
hydrogen or methyl, [0681] R.sup.17 stands for hydrogen or methyl,
[0682] or [0683] R.sup.16 and R.sup.17 together with the atoms to
which they are bound form a piperazinyl ring, [0684] R.sup.21
stands for hydrogen or methyl, [0685] R.sup.22 stands for hydrogen
or methyl, [0686] or [0687] R.sup.21 and R.sup.22 together with the
atoms to which they are bound form a cyclopropyl ring, [0688]
R.sup.23 stands for methyl, [0689] R.sup.24 stands for hydrogen,
[0690] R.sup.36 stands for hydrogen, methylcarbonyl or
tert-butyloxycarbonyl, [0691] R.sup.37 stands for hydrogen or
methyl,
[0692] L.sup.2 stands for linear (C.sub.2-C.sub.6)-alkanediyl or
for a group of the formula
##STR00067## [0693] wherein [0694] p stands for a number from 2 to
6, [0695] ##.sup.3 denotes the linkage site to the group B, [0696]
##.sup.4 denotes the linkage site to the nitrogen atom,
[0697] D stands for a group of the formula
##STR00068## [0698] wherein [0699] #.sup.3 denotes the linkage site
to the nitrogen atom, [0700] R.sup.1 stands for hydrogen, [0701]
R.sup.2 stands for 1-hydroxyethyl, benzyl, 4-hydroxybenzyl,
1-phenylethyl or 1H-indol-3-ylmethyl, [0702] or [0703] R.sup.1 and
R.sup.2 together with the carbon atom to which they are attached
form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[0703] ##STR00069## [0704] wherein [0705] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [0706] #.sup.5 denotes
the linkage site to the carbonyl group, [0707] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[0707] ##STR00070## [0708] wherein [0709] #.sup.6 denotes the
linkage site to the carbonyl group, [0710] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [0711] R.sup.3 stands for
hydrogen, [0712] R.sup.4 stands for benzyl, 4-hydroxybenzyl,
1-phenylethyl or 1H-indol-3-ylmethyl, [0713] or [0714] R.sup.3 and
R.sup.4 together with the carbon atom to which they are attached
form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[0714] ##STR00071## [0715] wherein [0716] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [0717] #.sup.8 denotes
the linkage site to the group T.sup.1, [0718] T.sup.1 stands for a
group of the formula --C(.dbd.O)--OR.sup.7,
--C(.dbd.O)--NR.sup.8R.sup.9 or --CH.sub.2--O--R.sup.11, [0719]
wherein [0720] R.sup.7 stands for hydrogen, methyl, ethyl,
n-propyl, tert-butyl, benzyl or adamantylmethyl, [0721] R.sup.8
stands for hydrogen or methyl, [0722] R.sup.9 stands for hydrogen,
methyl, ethyl, n-propyl or benzyl, [0723] R.sup.11 stands for
benzyl, which may be substituted in the phenyl group with
methoxycarbonyl or carboxyl, [0724] R.sup.5 stands for hydrogen,
methyl or a group of the formula
[0724] ##STR00072## [0725] wherein [0726] #.sup.9 denotes the
linkage site to --CHCH.sub.2-phenyl, [0727] R.sup.12 stands for
phenyl, which may be substituted with methoxycarbonyl, carboxyl or
a group of the formula --S(O).sub.2OH, [0728] R.sup.13 stands for
phenyl, which may be substituted with methoxycarbonyl or
carboxyl,
[0729] R.sup.35 stands for methyl or hydroxyl,
[0730] as well as their salts and solvates as well as the solvates
of the salts.
[0731] The preferred subject matter of the present invention
relates to binder-drug conjugates of the general formula (Ia) as
indicated above, wherein
[0732] n stands for a number from 1 to 10,
[0733] AK stands for AK.sub.1 or AK.sub.2 [0734] wherein [0735]
AK.sub.1 stands for cetuximab, pantitumumab or nimutuzumab, which
is bound to the group G via the sulfur atom of a cysteine radical
of the binder, [0736] AK.sub.2 stands for cetuximab, pantitumumab
or nimutuzumab, which is bound to the group G via the NH side group
of a lysine radical of the binder,
[0737] G for the case when AK=AK.sub.1 stands for a group of the
formula
##STR00073## [0738] wherein [0739] #.sup.1 denotes the linkage site
to the cysteine radical of the binder, [0740] #.sup.2 denotes the
linkage site to the group L.sup.1, [0741] or [0742] for the case
when AK=AK.sub.2, G stands for carbonyl,
[0743] L.sup.1 stands for a bond, linear
(C.sub.2-C.sub.6)-alkanediyl, a group of the formula
##STR00074## [0744] wherein [0745] m stands for a number of 2 or 3,
[0746] ##.sup.1 denotes the linkage site to the group G, [0747]
##.sup.2 denotes the linkage site to the group B, [0748] wherein
(C.sub.2-C.sub.6)-alkanediyl may be substituted with one or two
methyl substituents,
[0749] B stands for a bond or a group of the formula
##STR00075## [0750] wherein [0751] * denotes the linkage site to
L.sup.1, [0752] ** denotes the linkage site to L.sup.2, [0753]
L.sup.3 stands for a bond or ethane-1,2-diyl, [0754] L.sup.4 stands
for a bond or a group of the formula
[0754] ##STR00076## [0755] wherein [0756] *** denotes the linkage
site to the carbonyl group, [0757] **** denotes the linkage site to
L.sup.2, [0758] R.sup.25 stands for methyl, [0759] R.sup.28 stands
for hydrogen, methylcarbonyl or tert-butyloxycarbonyl, [0760]
Q.sup.1 stands for piperidine-1,4-diyl, [0761] R.sup.16 stands for
hydrogen or methyl, [0762] R.sup.17 stands for hydrogen or methyl,
[0763] or [0764] R.sup.16 and R.sup.17 together with the atoms to
which they are bound form a piperazinyl ring, [0765] R.sup.21
stands for hydrogen or methyl, [0766] R.sup.22 stands for hydrogen
or methyl, [0767] or [0768] R.sup.21 and R.sup.22 together with the
atoms to which they are bound form a cyclopropyl ring, [0769]
R.sup.23 stands for methyl, [0770] R.sup.24 stands for
hydrogen,
[0771] L.sup.2 stands for linear (C.sub.2-C.sub.6)-alkanediyl or
for a group of the formula
##STR00077## [0772] wherein [0773] p stands for a number from 2 to
6, [0774] ##.sup.3 denotes the linkage site to the group B, [0775]
##.sup.4 denotes the linkage site to the nitrogen atom,
[0776] D stands for a group of the formula
##STR00078## [0777] wherein [0778] #.sup.3 denotes the linkage site
to the nitrogen atom, [0779] R.sup.1 stands for hydrogen, [0780]
R.sup.2 stands for 1-hydroxyethyl, benzyl, 1-hydroxybenzyl,
1-phenylethyl or 1H-indol-3-ylmethyl, [0781] or [0782] R.sup.1 and
R.sup.2 together with the carbon atom to which they are attached
form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[0782] ##STR00079## [0783] wherein [0784] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [0785] #.sup.5 denotes
the linkage site to the carbonyl group, [0786] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[0786] ##STR00080## [0787] wherein [0788] #.sup.6 denotes the
linkage site to the carbonyl group, [0789] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [0790] R.sup.3 stands for
hydrogen, [0791] R.sup.4 stands for benzyl, 1-hydroxybenzyl,
1-phenylethyl or 1H-indol-3-ylmethyl, [0792] or [0793] R.sup.3 and
R.sup.4 together with the carbon atom to which they are attached
form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[0793] ##STR00081## [0794] wherein [0795] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [0796] #.sup.8 denotes
the linkage site to the group T.sup.1, [0797] T.sup.1 stands for a
group of the formula --C(.dbd.O)--OR.sup.7,
--C(.dbd.O)--NR.sup.8R.sup.9 or --CH.sub.2--O--R.sup.11, [0798]
wherein [0799] R.sup.7 stands for hydrogen, methyl, ethyl,
n-propyl, tert-butyl, benzyl or adamantylmethyl, [0800] R.sup.8
stands for hydrogen or methyl, [0801] R.sup.9 stands for hydrogen,
methyl, ethyl, n-propyl or benzyl, [0802] R.sup.11 stands for
benzyl, which may be substituted in the phenyl group with
methoxycarbonyl or carboxyl, [0803] R.sup.5 stands for hydrogen,
methyl or a group of the formula
[0803] ##STR00082## [0804] wherein [0805] #.sup.9 denotes the
linkage site to --CHCH.sub.2-phenyl, [0806] R.sup.12 stands for
phenyl, which may be substituted with methoxycarbonyl, carboxyl or
a group of the formula --S(O).sub.2OH, [0807] R.sup.13 stands for
phenyl, which may be substituted with methoxycarbonyl or
carboxyl,
[0808] R.sup.35 stands for methyl or hydroxyl,
[0809] as well as their salts and solvates as well as the solvates
of the salts.
[0810] The preferred subject matter of the present invention
relates to binder-drug conjugates of the general formula (Ia) as
indicated above, wherein
[0811] n stands for a number from 1 to 10,
[0812] AK stands for AK.sub.2 [0813] wherein [0814] AK.sub.2 stands
for cetuximab, pantitumumab or nimutuzumab, which is bound to the
group G via the NH side group of a lysine radical of the
binder,
[0815] G stands for carbonyl,
[0816] L.sup.1 stands for a bond,
[0817] B stands for a bond,
[0818] L.sup.2 stands for linear (C.sub.3-C.sub.6)-alkanediyl or
for a group of the formula
##STR00083## [0819] wherein [0820] p stands for a number of 2 or 3,
[0821] ##.sup.3 denotes the linkage site to the group B, [0822]
##.sup.4 denotes the linkage site to the nitrogen atom,
[0823] D stands for a group of the formula
##STR00084## [0824] wherein [0825] #.sup.3 denotes the linkage site
to the nitrogen atom, [0826] R.sup.1 stands for hydrogen, [0827]
R.sup.2 stands for benzyl or 1H-indol-3-ylmethyl, [0828] or [0829]
R.sup.1 and R.sup.2 together with the carbon atom to which they are
attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[0829] ##STR00085## [0830] wherein [0831] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [0832] #.sup.5 denotes
the linkage site to the carbonyl group, [0833] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[0833] ##STR00086## [0834] wherein [0835] #.sup.6 denotes the
linkage site to the carbonyl group, [0836] R.sup.3 stands for
hydrogen, [0837] R.sup.4 stands for benzyl or 1H-indol-3-ylmethyl,
[0838] or [0839] R.sup.3 and R.sup.4 together with the carbon atom
to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0839] ##STR00087## [0840] wherein [0841] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [0842] #.sup.8 denotes
the linkage site to the group T.sup.1, [0843] T.sup.1 stands for a
group of the formula C(.dbd.O)OR.sup.7 or C(.dbd.O)
NR.sup.8R.sup.9, [0844] wherein [0845] R.sup.7 stands for hydrogen,
methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
[0846] R.sup.8 stands for hydrogen, [0847] R.sup.9 stands for
hydrogen or benzyl,
[0848] R.sup.35 stands for methyl,
[0849] as well as their salts and solvates as well as the solvates
of the salts.
[0850] The preferred subject matter of the present invention
relates to binder-drug conjugates of the general formula (Ia) as
indicated above, wherein
[0851] n stands for a number from 1 to 10,
[0852] AK stands for AK.sub.2, [0853] wherein [0854] AK.sub.2
stands for cetuximab, pantitumumab or nimutuzumab, which is bound
to the group G via the NH side group of a lysine radical of the
binder,
[0855] G stands for carbonyl,
[0856] L.sup.1 stands for a bond,
[0857] B stands for a bond,
[0858] L.sup.2 stands for linear (C.sub.3-C.sub.6)-alkanediyl or
for a group of the formula
##STR00088## [0859] wherein [0860] p stands for a number of 2 or 3,
[0861] ##.sup.3 denotes the linkage site to the group B, [0862]
##.sup.4 denotes the linkage site to the nitrogen atom,
[0863] D stands for a group of the formula
##STR00089## [0864] wherein [0865] #.sup.3 denotes the linkage site
to the nitrogen atom, [0866] R.sup.1 stands for hydrogen, [0867]
R.sup.2 stands for benzyl, 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
[0868] or [0869] R.sup.1 and R.sup.2 together with the carbon atom
to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0869] ##STR00090## [0870] wherein [0871] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [0872] #.sup.5 denotes
the linkage site to the carbonyl group, [0873] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[0873] ##STR00091## [0874] wherein [0875] #.sup.6 denotes the
linkage site to the carbonyl group, [0876] R.sup.3 stands for
hydrogen, [0877] R.sup.4 stands for benzyl, 4-hydroxybenzyl or
1H-indol-3-ylmethyl, [0878] or [0879] R.sup.3 and R.sup.4 together
with the carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0879] ##STR00092## [0880] wherein [0881] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [0882] #.sup.8 denotes
the linkage site to the group T.sup.1, [0883] T.sup.1 stands for a
group of the formula --C(.dbd.O)--OR.sup.7 or
--C(.dbd.O)--NR.sup.8R.sup.9, [0884] wherein [0885] R.sup.7 stands
for hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, [0886] R.sup.8 stands for hydrogen, [0887] R.sup.9
stands for hydrogen or benzyl,
[0888] R.sup.35 stands for methyl,
[0889] as well as their salts and solvates as well as the solvates
of the salts.
[0890] The preferred subject matter of the present invention
relates to binder-drug conjugates of the general formula (Ia) as
indicated above, wherein
[0891] n stands for a number from 1 to 10,
[0892] AK stands for AK.sub.1 [0893] wherein [0894] AK.sub.1 stands
for cetuximab, pantitumumab or nimutuzumab, which is bound to the
group G via the sulfur atom of a cysteine radical of the
binder,
[0895] G stands for a group of the formula
##STR00093## [0896] wherein [0897] #.sup.1 denotes the linkage site
to the cysteine radical of the binder, [0898] #.sup.2 denotes the
linkage site to the group L.sup.1,
[0899] L.sup.1 stands for a bond, linear
(C.sub.3-C.sub.5)-alkanediyl or a group of the formula
##STR00094## [0900] wherein [0901] m stands for a number of 2 or 3,
[0902] ##.sup.1 denotes the linkage site to the group G, [0903]
##.sup.2 denotes the linkage site to the group B, [0904] wherein
(C.sub.3-C.sub.5)-alkanediyl may be substituted with one or two
methyl substituents,
[0905] B stands for a bond or a group of the formula
##STR00095## [0906] wherein [0907] * denotes the linkage site to
L.sup.1, [0908] ** denotes the linkage site to L.sup.2, [0909]
L.sup.3 stands for a bond or ethane-1,2-diyl, [0910] L.sup.4 stands
for a bond or a group of the formula
[0910] ##STR00096## [0911] wherein [0912] *** denotes the linkage
site to the carbonyl group, [0913] **** denotes the linkage site to
L.sup.2, [0914] R.sup.25 stands for methyl, [0915] R.sup.28 stands
for hydrogen, methylcarbonyl or tert-butyloxycarbonyl, [0916]
R.sup.16 stands for hydrogen or methyl, [0917] R.sup.17 stands for
hydrogen or methyl, [0918] or [0919] R.sup.16 and R.sup.17 together
with the atoms to which they are bound form a piperazinyl ring,
[0920] L.sup.2 stands for linear (C.sub.3-C.sub.5)-alkanediyl or
for a group of the formula
##STR00097## [0921] wherein [0922] p stands for a number of 2 or 3,
[0923] ##.sup.3 denotes the linkage site to the group B, [0924]
##.sup.4 denotes the linkage site to the nitrogen atom,
[0925] D stands for a group of the formula
##STR00098## [0926] wherein [0927] #.sup.3 denotes the linkage site
to the nitrogen atom, [0928] R.sup.1 stands for hydrogen, [0929]
R.sup.2 stands for benzyl or 1H-indol-3-ylmethyl, [0930] or [0931]
R.sup.1 and R.sup.2 together with the carbon atom to which they are
attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[0931] ##STR00099## [0932] wherein [0933] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [0934] #.sup.5 denotes
the linkage site to the carbonyl group, [0935] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[0935] ##STR00100## [0936] wherein [0937] #.sup.6 denotes the
linkage site to the carbonyl group, [0938] R.sup.3 stands for
hydrogen, [0939] R.sup.4 stands for benzyl or 1H-indol-3-ylmethyl,
[0940] or [0941] R.sup.3 and R.sup.4 together with the carbon atom
to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0941] ##STR00101## [0942] wherein [0943] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [0944] #.sup.8 denotes
the linkage site to the group T.sup.1, [0945] T.sup.1 stands for a
group of the formula C(.dbd.O)OR.sup.7 or C(.dbd.O)
NR.sup.8R.sup.9, [0946] wherein [0947] R.sup.7 stands for hydrogen,
methyl, ethyl, n-propyl, tert-butyl, benzyl or adamantylmethyl,
[0948] R.sup.8 stands for hydrogen, [0949] R.sup.9 stands for
hydrogen or benzyl,
[0950] R.sup.35 stands for methyl,
[0951] as well as their salts and solvates as well as the solvates
of the salts.
[0952] The preferred subject matter of the present invention
relates to binder-drug conjugates of the general formula (Ia) as
indicated above, wherein
[0953] n stands for a number from 1 to 10,
[0954] AK stands for AK.sub.1, [0955] wherein [0956] AK.sub.1
stands for cetuximab, pantitumumab or nimutuzumab, which is bound
to the group G via the sulfur atom of a cysteine radical of the
binder,
[0957] G stands for a group of the formula
##STR00102## [0958] wherein [0959] #.sup.1 denotes the linkage site
to the cysteine radical of the binder, [0960] #.sup.2 denotes the
linkage site to the group L.sup.1,
[0961] L.sup.1 stands for a bond, linear
(C.sub.3-C.sub.5)-alkanediyl or a group of the formula
##STR00103## [0962] wherein [0963] m stands for a number of 2 or 3,
[0964] ##.sup.1 denotes the linkage site to the group G, [0965]
##.sup.2 denotes the linkage site to the group B, [0966] wherein
(C.sub.3-C.sub.5)-alkanediyl may be substituted with one or two
methyl substituents,
[0967] B stands for a bond or a group of the formula
##STR00104## [0968] wherein [0969] * denotes the linkage site to
L.sup.1, [0970] ** denotes the linkage site to L.sup.2, [0971]
L.sup.3 stands for a bond or ethane-1,2-diyl, [0972] L.sup.4 stands
for a bond or a group of the formula
[0972] ##STR00105## [0973] wherein [0974] *** denotes the linkage
site to the carbonyl group, [0975] **** denotes the linkage site to
L.sup.2, [0976] R.sup.25 stands for methyl, [0977] R.sup.28 stands
for hydrogen, methylcarbonyl or tert-butyloxycarbonyl, [0978]
R.sup.16 stands for hydrogen or methyl, [0979] R.sup.17 stands for
hydrogen or methyl, [0980] or [0981] R.sup.16 and R.sup.17 together
with the atoms to which they are bound form a piperazinyl ring,
[0982] L.sup.2 stands for linear (C.sub.3-C.sub.5)-alkanediyl or
for a group of the formula
##STR00106## [0983] wherein [0984] p stands for a number of 2 or 3,
[0985] ##.sup.3 denotes the linkage site to the group B, [0986]
##.sup.4 denotes the linkage site to the nitrogen atom,
[0987] D stands for a group of the formula
##STR00107## [0988] wherein [0989] #.sup.3 denotes the linkage site
to the nitrogen atom, [0990] R.sup.1 stands for hydrogen, [0991]
R.sup.2 stands for benzyl, 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
[0992] or [0993] R.sup.1 and R.sup.2 together with the carbon atom
to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0993] ##STR00108## [0994] wherein [0995] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [0996] #.sup.5 denotes
the linkage site to the carbonyl group, [0997] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[0997] ##STR00109## [0998] wherein [0999] #.sup.6 denotes the
linkage site to the carbonyl group, [1000] R.sup.3 stands for
hydrogen, [1001] R.sup.4 stands for benzyl, 4-hydroxybenzyl or
1H-indol-3-ylmethyl, [1002] or [1003] R.sup.3 and R.sup.4 together
with the carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1003] ##STR00110## [1004] wherein [1005] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [1006] #.sup.8 denotes
the linkage site to the group [1007] T.sup.1 stands for a group of
the formula --C(.dbd.O)--OR.sup.7 or --C(.dbd.O)--NR.sup.8R.sup.9,
[1008] wherein [1009] R.sup.7 stands for hydrogen, methyl, ethyl,
n-propyl, tert-butyl, benzyl or adamantylmethyl, [1010] R.sup.8
stands for hydrogen, [1011] R.sup.9 stands for hydrogen or
benzyl,
[1012] R.sup.35 stands for methyl,
[1013] as well as their salts and solvates as well as the solvates
of the salts.
[1014] Another subject matter of the present invention relates to
compounds of the formula (XXXa)
##STR00111##
[1015] in which [1016] Cys stands for a cysteine radical which is
bound to a carbon atom of the succinimide via the sulfur atom of
the side chain, [1017] L.sup.1 stands for a bond, linear
(C.sub.1-C.sub.10)-alkanediyl, a group of the formula
[1017] ##STR00112## [1018] wherein [1019] m stands for a number
from 2 to 6, [1020] ##.sup.1 denotes the linkage site to the group
G, [1021] ##.sup.2 denotes the linkage site to the group B, [1022]
L.sup.1A stands for linear (C.sub.2-C.sub.10)-alkanediyl, [1023]
B.sup.1 stands for a group of the formula
[1023] ##STR00113## [1024] wherein [1025] ##.sup.5 denotes the
linkage site to the group L.sup.1A, [1026] ##.sup.6 denotes the
linkage site to the group L.sup.1B, [1027] L.sup.5 stands for a
bond or (C.sub.2-C.sub.4)-alkanediyl, [1028] L.sup.6 stands for a
bond, [1029] R.sup.29 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1030] R.sup.30 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1031] or [1032] R.sup.29 and R.sup.30
together with the atoms to which they are bound form a five- or
six-membered heterocycle, [1033] R.sup.31 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1034] R.sup.32 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1035] or [1036] R.sup.31 and R.sup.32
together with the atoms to which they are bound form a five- or
six-membered heterocycle, [1037] L.sup.1B stands for linear
(C.sub.2-C.sub.10)-alkanediyl, [1038] and [1039] wherein
(C.sub.1-C.sub.10)-alkanediyl may be substituted with one to four
substituents selected independently of one another from the group
comprising methyl, hydroxyl and benzyl [1040] and [1041] wherein
two carbon atoms of the alkanediyl chain in 1,2-, 1,3- or 1,4-
relation to one another to form a (C.sub.3-C.sub.6)-cycloalkyl ring
or a phenyl ring by including the carbon atoms optionally situated
between them,
[1042] B stands for a bond or a group of the formula
##STR00114## [1043] wherein [1044] * denotes the linkage site to
L.sup.1, [1045] ** denotes the linkage site to L.sup.2, [1046] P
stands for O or NH, [1047] L.sup.3 stands for a bond or
(C.sub.2-C.sub.4)-alkanediyl, [1048] L.sup.4 stands for a bond,
[1049] Q.sup.1 stands for a four- to seven-membered heterocycle,
[1050] Q.sup.2 stands for a three- to seven-membered carbocycle or
a four- to seven-membered heterocycle, [1051] R.sup.14 stands for
hydrogen or (C.sub.1-C.sub.4)-alkyl, [1052] R.sup.15 stands for
hydrogen or (C.sub.1-C.sub.4)-alkyl, [1053] or [1054] R.sup.14 and
R.sup.15 together with the atoms to which they are bound form a
five- or six-membered heterocycle, [1055] R.sup.16 stands for
hydrogen or (C.sub.1-C.sub.4)-alkyl, [1056] R.sup.17 stands for
hydrogen or (C.sub.1-C.sub.4)-alkyl, [1057] or [1058] R.sup.16 and
R.sup.17 together with the atoms to which they are bound form a
five- or six-membered heterocycle, [1059] R.sup.18 stands for
hydrogen or (C.sub.1-C.sub.4)-alkyl, [1060] R.sup.19 stands for
hydrogen or the side group of a natural .alpha.-amino acid or its
homologs or isomers, [1061] R.sup.20 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1062] or [1063] R.sup.19 and R.sup.20
together with the atoms to which they are bound form a pyrrolidinyl
ring, [1064] R.sup.21 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1065] R.sup.22 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1066] or [1067] R.sup.21 and R.sup.22
together with the atoms to which they are bound form a three- to
seven-membered carbocycle, [1068] R.sup.23 stands for
(C.sub.1-C.sub.4)-alkyl, [1069] R.sup.24 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1070] R.sup.27 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl,
[1071] L.sup.2 stands for linear (C.sub.2-C.sub.10)-alkanediyl or
for a group of the formula
##STR00115## [1072] wherein [1073] p stands for a number from 2 to
6, [1074] ##.sup.3 denotes the linkage site to the group B, [1075]
##.sup.4 denotes the linkage site to the nitrogen atom, [1076]
wherein (C.sub.2-C.sub.10)-alkanediyl may be substituted with 1 to
4 substituents selected independently of one another from the group
comprised of methyl, hydroxyl and benzyl, [1077] and [1078] wherein
two carbon atoms of the alkanediyl chain in 1,2-, 1,3- or 1,4-
relation to one another to form a (C.sub.3-C.sub.6) cycloalkyl ring
or a phenyl ring, including the carbon atoms optionally situated
between them,
[1079] D stands for a group of the formula
##STR00116## [1080] wherein [1081] #.sup.3 denotes the linkage site
to the nitrogen atom, [1082] R.sup.1 stands for hydrogen or methyl,
[1083] R.sup.2 stands for isopropyl, isobutyl, sec-butyl,
tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl,
4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl,
diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
[1084] or [1085] R.sup.1 and R.sup.2 together with the carbon atom
to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1085] ##STR00117## [1086] wherein [1087] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [1088] #.sup.5 denotes
the linkage site to the carbonyl group, [1089] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[1089] ##STR00118## [1090] wherein [1091] #.sup.6 denotes the
linkage site to the carbonyl group, [1092] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [1093] R.sup.3 stands for hydrogen
or methyl, [1094] R.sup.4 stands for isopropyl, isobutyl,
sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl,
4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl,
1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or
1H-indol-3-ylmethyl, [1095] or [1096] R.sup.3 and R.sup.4 together
with the carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1096] ##STR00119## [1097] wherein [1098] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [1099] #.sup.8 denotes
the linkage site to the group [1100] T.sup.1 stands for a group of
the formula --C(.dbd.O)--OR.sup.7, --C(.dbd.O)--NR.sup.8R.sup.9,
--C(.dbd.O)--NH--NH--R.sup.10 or --CH.sub.2--O--R.sup.11, [1101]
wherein [1102] R.sup.7 stands for hydrogen, methyl, ethyl,
n-propyl, tert-butyl, benzyl or adamantylmethyl, [1103] R.sup.8
stands for hydrogen or methyl, [1104] R.sup.9 stands for hydrogen,
methyl, ethyl, n-propyl or benzyl, [1105] or [1106] R.sup.8 and
R.sup.9 together with the nitrogen atom to which they are bound
form a four- to seven-membered heterocycle, [1107] R.sup.10 stands
for benzoyl, [1108] R.sup.11 stands for benzyl, which may be
substituted in the phenyl group with methoxycarbonyl or carboxyl,
[1109] R.sup.5 stands for hydrogen, methyl or a group of the
formula
[1109] ##STR00120## [1110] wherein [1111] #.sup.9 denotes the
linkage site to --CHC(R.sup.26)-T.sup.2, [1112] R.sup.12 stands for
phenyl, which may be substituted with methoxycarbonyl, carboxyl or
a group of the formula --S(O).sub.2OH, [1113] R.sup.13 stands for
phenyl, which may be substituted with methoxycarbonyl or carboxyl,
[1114] R.sup.26 stands for hydrogen or hydroxyl, [1115] T.sup.2
stands for phenyl, benzyl, 1H-indol-3-yl or
1H-indol-3-ylmethyl,
[1116] R.sup.35 stands for methyl or hydroxyl,
[1117] as well as their salts and solvates as well as the solvates
of the salts.
[1118] The preferred subject matter of the present invention is
compounds of formula (XXXa) as indicated above, wherein
##STR00121## [1119] Cys stands for a cysteine radical which is
bound to a carbon atom of the succinimide via the sulfur atom of
the side chain, [1120] L.sup.1 stands for a bond, linear
(C.sub.2-C.sub.6)-alkanediyl, a group of the formula
[1120] ##STR00122## [1121] wherein [1122] m stands for a number of
2 or 3, [1123] ##.sup.1 denotes the linkage site to the group G,
[1124] ##.sup.2 denotes the linkage site to the group B, [1125]
L.sup.1A stands for linear (C.sub.2-C.sub.6)-alkanediyl, [1126]
B.sup.1 stands for a group of the formula
[1126] ##STR00123## [1127] wherein [1128] ##.sup.5 denotes the
linkage site to the group L.sup.1A, [1129] ##.sup.6 denotes the
linkage site to the group L.sup.1B, [1130] L.sup.5 stands for a
bond, [1131] L.sup.6 stands for a bond, [1132] R.sup.29 stands for
hydrogen, [1133] R.sup.30 stands for hydrogen, [1134] R.sup.31
stands for hydrogen or methyl, [1135] R.sup.32 stands for hydrogen
or methyl, [1136] L.sup.1B stands for linear
(C.sub.2-C.sub.6)-alkanediyl, [1137] and [1138] wherein
(C.sub.2-C.sub.6)-alkanediyl may be substituted with one to two
methyl substituents,
[1139] B stands for a bond or a group of the formula
##STR00124## [1140] wherein [1141] * denotes the linkage site to
L.sup.1, [1142] ** denotes the linkage site to L.sup.2, [1143]
L.sup.3 stands for a bond or ethane-1,2-diyl, [1144] L.sup.4 stands
for a bond, [1145] R.sup.14 stands for hydrogen, [1146] R.sup.15
stands for hydrogen, [1147] R.sup.16 stands for hydrogen or methyl,
[1148] R.sup.17 stands for hydrogen or methyl, [1149] or [1150]
R.sup.16 and R.sup.17 together with the atoms to which they are
bound form a piperazinyl ring, [1151] R.sup.23 stands for methyl,
[1152] R.sup.24 stands for hydrogen or methyl,
[1153] L.sup.2 stands for linear (C.sub.2-C.sub.6)-alkanediyl or
for a group of the formula
##STR00125## [1154] wherein [1155] p stands for a number of 2 or 3,
[1156] ##.sup.3 denotes the linkage site to the group B, [1157]
##.sup.4 denotes the linkage site to the nitrogen atom,
[1158] D stands for a group of the formula
##STR00126## [1159] wherein [1160] #.sup.3 denotes the linkage site
to the nitrogen atom, [1161] R.sup.1 stands for hydrogen, [1162]
R.sup.2 stands for 1-hydroxyethyl, benzyl, 4-hydroxybenzyl,
1-phenylethyl or 1H-indol-3-ylmethyl, [1163] or [1164] R.sup.1 and
R.sup.2 together with the carbon atom to which they are attached
form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[1164] ##STR00127## [1165] wherein [1166] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [1167] #.sup.5 denotes
the linkage site to the carbonyl group, [1168] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[1168] ##STR00128## [1169] wherein [1170] #.sup.6 denotes the
linkage site to the carbonyl group, [1171] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [1172] R.sup.3 stands for
hydrogen, [1173] R.sup.4 stands for 1-hydroxyethyl, benzyl,
4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl, [1174] or
[1175] R.sup.3 and R.sup.4 together with the carbon atom to which
they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl
group of the formula
[1175] ##STR00129## [1176] wherein [1177] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [1178] #.sup.8 denotes
the linkage site to the group [1179] T.sup.1 stands for a group of
the formula --C(.dbd.O)--OR.sup.7, --C(.dbd.O)--NR.sup.8R.sup.9,
--C(.dbd.O)--NH--NH--R.sup.10 or --CH.sub.2--O--R.sup.11, [1180]
wherein [1181] R.sup.7 stands for hydrogen, methyl, ethyl,
n-propyl, tert-butyl, benzyl or adamantylmethyl, [1182] R.sup.8
stands for hydrogen or methyl, [1183] R.sup.9 stands for hydrogen,
methyl, ethyl, n-propyl or benzyl, [1184] or [1185] R.sup.8 and
R.sup.9 together with the nitrogen atom to which they are bound
form a four- to seven-membered heterocycle, [1186] R.sup.10 stands
for benzoyl, [1187] R.sup.11 stands for benzyl, which may be
substituted in the phenyl group with methoxycarbonyl or carboxyl,
[1188] R.sup.5 stands for hydrogen, methyl or a group of the
formula
[1188] ##STR00130## [1189] wherein [1190] #.sup.9 denotes the
linkage site to --CHCH.sub.2-phenyl, [1191] R.sup.12 stands for
phenyl, which may be substituted with methoxycarbonyl, carboxyl or
a group of the formula --S(O).sub.2OH, [1192] R.sup.13 stands for
phenyl, which may be substituted with methoxycarbonyl or carboxyl,
[1193] R.sup.35 stands for methyl or hydroxyl, [1194] as well as
their salts and solvates as well as the solvates of the salts.
[1195] The preferred subject matter of the present invention is
compounds of formula (XXXa) as indicated above, wherein [1196] Cys
stands for a cysteine radical which is bound to a carbon atom of
the succinimide via the sulfur atom of the side chain, [1197]
L.sup.1 stands for a bond or linear (C.sub.2-C.sub.6)-alkanediyl,
[1198] B stands for a bond or a group of the formula
[1198] ##STR00131## [1199] wherein [1200] * denotes the linkage
site to L.sup.1, [1201] ** denotes the linkage site to L.sup.2,
[1202] L.sup.3 stands for a bond, [1203] L.sup.4 stands for a bond,
[1204] R.sup.16 stands for hydrogen or methyl, [1205] R.sup.17
stands for hydrogen or methyl,
[1206] L.sup.2 stands for linear (C.sub.2-C.sub.6)-alkanediyl or
for a group of the formula
##STR00132## [1207] wherein [1208] p stands for a number of 2 or 3,
[1209] ##.sup.3 denotes the linkage site to the group B, [1210]
##.sup.4 denotes the linkage site to the nitrogen atom,
[1211] D stands for a group of the formula
##STR00133## [1212] wherein [1213] #.sup.3 denotes the linkage site
to the nitrogen atom, [1214] R.sup.1 stands for hydrogen, [1215]
R.sup.2 stands for benzyl or 1H-indol-3-ylmethyl, [1216] or [1217]
R.sup.1 and R.sup.2 together with the carbon atom to which they are
attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[1217] ##STR00134## [1218] wherein [1219] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [1220] #.sup.5 denotes
the linkage site to the carbonyl group, [1221] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[1221] ##STR00135## [1222] wherein [1223] #.sup.6 denotes the
linkage site to the carbonyl group, [1224] R.sup.3 stands for
hydrogen, [1225] R.sup.4 stands for benzyl or 1H-indol-3-ylmethyl,
[1226] or [1227] R.sup.3 and R.sup.4 together with the carbon atom
to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1227] ##STR00136## [1228] wherein [1229] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [1230] #.sup.8 denotes
the linkage site to the group T.sup.1, [1231] T.sup.1 stands for a
group of the formula C(.dbd.O)OR.sup.7 or C(.dbd.O)
NR.sup.8R.sup.9, [1232] wherein [1233] R.sup.7 stands for hydrogen,
[1234] R.sup.8 stands for hydrogen, [1235] R.sup.9 stands for
hydrogen, [1236] R.sup.35 stands for methyl, [1237] as well as
their salts and solvates as well as the solvates of the salts.
[1238] The preferred subject matter of the present invention is
compounds of formula (XXXa) as indicated above, wherein [1239] Cys
stands for a cysteine radical which is bound to a carbon atom of
the succinimide via the sulfur atom of the side chain, [1240]
L.sup.1 stands for a bond or linear (C.sub.2-C.sub.6)-alkanediyl,
[1241] B stands for a bond or a group of the formula
[1241] ##STR00137## [1242] wherein [1243] * denotes the linkage
site to L.sup.1, [1244] ** denotes the linkage site to L.sup.2,
[1245] L.sup.3 stands for a bond, [1246] L.sup.4 stands for a bond,
[1247] R.sup.16 stands for hydrogen or methyl, [1248] R.sup.17
stands for hydrogen or methyl,
[1249] L.sup.2 stands for linear (C.sub.2-C.sub.6)-alkanediyl or
for a group of the formula
##STR00138## [1250] wherein [1251] p stands for a number of 2 or 3,
[1252] ##.sup.3 denotes the linkage site to the group B, [1253]
##.sup.4 denotes the linkage site to the nitrogen atom,
[1254] D stands for a group of the formula
##STR00139## [1255] wherein [1256] #.sup.3 denotes the linkage site
to the nitrogen atom, [1257] R.sup.1 stands for hydrogen, [1258]
R.sup.2 stands for benzyl, 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
[1259] or [1260] R.sup.1 and R.sup.2 together with the carbon atom
to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1260] ##STR00140## [1261] wherein [1262] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [1263] #.sup.5 denotes
the linkage site to the carbonyl group, [1264] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[1264] ##STR00141## [1265] wherein [1266] #.sup.6 denotes the
linkage site to the carbonyl group, [1267] R.sup.3 stands for
hydrogen, [1268] R.sup.4 stands for benzyl, 4-hydroxybenzyl or
1H-indol-3-ylmethyl, [1269] or [1270] R.sup.3 and R.sup.4 together
with the carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1270] ##STR00142## [1271] wherein [1272] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [1273] #.sup.8 denotes
the linkage site to the group T.sup.1, [1274] T.sup.1 stands for a
group of the formula C(.dbd.O)OR.sup.7 or C(.dbd.O)
NR.sup.8R.sup.9, [1275] wherein [1276] R.sup.7 stands for hydrogen,
[1277] R.sup.8 stands for hydrogen, [1278] R.sup.9 stands for
hydrogen,
[1279] R.sup.35 stands for methyl,
[1280] as well as their salts and solvates as well as the solvates
of the salts.
[1281] Another subject matter of the present invention relates to
compounds of formula (XXXI)
##STR00143##
[1282] in which
[1283] L.sup.1 stands for a bond, linear
(C.sub.1-C.sub.10)-alkanediyl, a group of the formula
##STR00144## [1284] wherein [1285] m stands for a number from 2 to
6, [1286] ##.sup.1 denotes the linkage site to the group G, [1287]
##.sup.2 denotes the linkage site to the group B, [1288] L.sup.1A
stands for linear (C.sub.2-C.sub.10)-alkanediyl, [1289] B.sup.1
stands for a group of the formula
[1289] ##STR00145## [1290] wherein [1291] ##.sup.5 denotes the
linkage site to the group L.sup.1A, [1292] ##.sup.6 denotes the
linkage site to the group L.sup.1B, [1293] L.sup.5 stands for a
bond or (C.sub.2-C.sub.4)-alkanediyl, [1294] L.sup.6 stands for a
bond, [1295] R.sup.29 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1296] R.sup.30 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1297] or [1298] R.sup.29 and R.sup.30
together with the atoms to which they are bound form a five- or
six-membered heterocycle, [1299] R.sup.31 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1300] R.sup.32 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1301] or [1302] R.sup.31 and R.sup.32
together with the atoms to which they are bound form a five- or
six-membered heterocycle, [1303] L.sup.1B stands for linear
(C.sub.2-C.sub.10)-alkanediyl, [1304] and [1305] wherein
(C.sub.1-C.sub.10)-alkanediyl may be substituted with one to four
substituents selected independently from one another from the group
comprising methyl, hydroxyl and benzyl [1306] and [1307] wherein
two carbon atoms of the alkanediyl chain may be bridged in 1,2-,
1,3- or 1,4- relation to one another, including the carbon atoms
optionally situated between them, to form a
(C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring,
[1308] B stands for a bond or a group of the formula
##STR00146## [1309] wherein [1310] * denotes the linkage site to
L.sup.1, [1311] ** denotes the linkage site to L.sup.2, [1312] P
stands for O or NH, [1313] Q.sup.1 stands for a four- to
seven-membered heterocycle, [1314] Q.sup.2 stands for a three- to
seven-membered carbocycle or a four- to seven-membered heterocycle,
[1315] R.sup.18 stands for hydrogen or (C.sub.1-C.sub.4)-alkyl,
[1316] R.sup.19 stands for hydrogen or the side group of a natural
.alpha.-amino acid or its homologs or isomers, [1317] R.sup.20
stands for hydrogen or (C.sub.1-C.sub.4)-alkyl, [1318] or [1319]
R.sup.19 and R.sup.20 together with the atoms to which they are
bound form a pyrrolidinyl ring, [1320] R.sup.21 stands for hydrogen
or (C.sub.1-C.sub.4)-alkyl, [1321] R.sup.22 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1322] or [1323] R.sup.21 and R.sup.22
together with the atoms to which they are bound form a three- to
seven-membered carbocycle, [1324] R.sup.27 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl,
[1325] L.sup.2 stands for linear (C.sub.2-C.sub.10)-alkanediyl or
for a group of the formula
##STR00147## [1326] wherein [1327] p stands for a number from 2 to
6, [1328] ##.sup.3 denotes the linkage site to the group B, [1329]
##.sup.4 denotes the linkage site to the nitrogen atom, [1330]
Wherein (C.sub.2-C.sub.10)-alkanediyl may be substituted with 1 to
4 substituents selected independently of one another from the group
comprising methyl, hydroxyl and benzyl, [1331] and [1332] wherein
two carbon atoms of the alkanediyl chain may be bridged in 1,2-,
1,3- or 1,4- relation to one another, including the carbon atoms
optionally present between them to form a (C.sub.3-C.sub.6)
cycloalkyl ring or a phenyl ring,
[1333] D stands for a group of the formula
##STR00148## [1334] wherein [1335] #.sup.3 denotes the linkage site
to the nitrogen atom, [1336] R.sup.1 stands for hydrogen or methyl,
[1337] R.sup.2 stands for isopropyl, isobutyl, sec-butyl,
tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl,
4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl,
diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
[1338] or [1339] R.sup.1 and R.sup.2 together with the carbon atom
to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1339] ##STR00149## [1340] wherein [1341] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [1342] #.sup.5 denotes
the linkage site to the carbonyl group, [1343] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[1343] ##STR00150## [1344] wherein [1345] #.sup.6 denotes the
linkage site to the carbonyl group, [1346] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [1347] R.sup.3 stands for hydrogen
or methyl, [1348] R.sup.4 stands for isopropyl, isobutyl,
sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl,
4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl,
1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or
1H-indol-3-ylmethyl, [1349] or [1350] R.sup.3 and R.sup.4 together
with the carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1350] ##STR00151## [1351] wherein [1352] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [1353] #.sup.8 denotes
the linkage site to the group T.sup.1, [1354] T.sup.1 stands for a
group of the formula --C(.dbd.O)--OR.sup.7,
--C(.dbd.O)--NR.sup.8R.sup.9, --C(.dbd.O)--NH--NH--R.sup.10 or
--CH.sub.2--O--R.sup.11, [1355] wherein [1356] R.sup.7 stands for
hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, [1357] R.sup.8 stands for hydrogen or methyl,
[1358] R.sup.9 stands for hydrogen, methyl, ethyl, n-propyl or
benzyl, [1359] or [1360] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are bound form a four- to
seven-membered heterocycle, [1361] R.sup.10 stands for benzoyl,
[1362] R.sup.11 stands for benzyl, which may be substituted in the
phenyl group with methoxycarbonyl or carboxyl, [1363] R.sup.5
stands for hydrogen, methyl or a group of the formula
[1363] ##STR00152## [1364] wherein [1365] * denotes the linkage
site to --CHC(R.sup.26)-T.sup.2, [1366] R.sup.12 stands for phenyl,
which may be substituted with methoxycarbonyl, carboxyl or a group
of the formula --S(O).sub.2OH, [1367] R.sup.13 stands for phenyl,
which may be substituted with methoxycarbonyl or carboxyl, [1368]
R.sup.26 stands for hydrogen or hydroxyl, [1369] T.sup.2 stands for
phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl, [1370]
R.sup.35 stands for methyl or hydroxyl, [1371] as well as their
salts and solvates as well as the solvates of the salts.
[1372] The preferred subject matter of the present invention is
compounds of formula (XXXI) as indicated above, wherein
[1373] L.sup.1 stands for a bond, linear
(C.sub.2-C.sub.6)-alkanediyl, a group of the formula
##STR00153## [1374] wherein [1375] m stands for a number of 2 or 3,
[1376] ##.sup.1 denotes the linkage site to the group G, [1377]
##.sup.2 denotes the linkage site to the group B, [1378] wherein
(C.sub.2-C.sub.6)-alkanediyl may be substituted with one or two
methyl substituents,
[1379] B stands for a bond or a group of the formula
##STR00154## [1380] wherein [1381] * denotes the linkage site to
L.sup.1, [1382] ** denotes the linkage site to L.sup.2, [1383]
R.sup.18 stands for hydrogen, [1384] R.sup.19 stands for hydrogen,
methyl, propan-2-yl, 2-methylpropan-1-yl or 1-methyl-propan-1-yl,
[1385] R.sup.20 stands for hydrogen or (C.sub.1-C.sub.4)-alkyl,
[1386] or [1387] R.sup.19 and R.sup.20 together with the atoms to
which they are bound form a pyrrolidinyl ring, [1388] R.sup.21
stands for hydrogen or methyl, [1389] R.sup.22 stands for hydrogen
or methyl, [1390] or [1391] R.sup.21 and R.sup.22 together with the
atoms to which they are bound form a cyclopropyl ring, [1392]
R.sup.27 stands for hydrogen or methyl, L.sup.2 stands for linear
(C.sub.2-C.sub.6)-alkanediyl or for a group of the formula
[1392] ##STR00155## [1393] wherein [1394] p stands for a number of
2 or 3, [1395] ##.sup.3 denotes the linkage site to the group B,
[1396] ##.sup.4 denotes the linkage site to the nitrogen atom,
[1397] wherein (C.sub.2-C.sub.10)-alkanediyl may be substituted
with one or two methyl substituents, [1398] and [1399] wherein two
carbon atoms of the alkanediyl chain may be bridged in 1,4-
relation to one another, including the carbon atoms optionally
present between them to form a phenyl ring,
[1400] D stands for a group of the formula
##STR00156## [1401] wherein [1402] #.sup.3 denotes the linkage site
to the nitrogen atom, [1403] R.sup.1 stands for hydrogen, [1404]
R.sup.2 stands for 1-hydroxyethyl, benzyl, 4-hydroxybenzyl,
1-phenylethyl or 1H-indol-3-ylmethyl, [1405] or [1406] R.sup.1 and
R.sup.2 together with the carbon atom to which they are attached
form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[1406] ##STR00157## [1407] wherein [1408] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [1409] #.sup.5 denotes
the linkage site to the carbonyl group, [1410] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[1410] ##STR00158## [1411] wherein [1412] #.sup.6 denotes the
linkage site to the carbonyl group, [1413] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [1414] R.sup.3 stands for
hydrogen, [1415] R.sup.4 stands for 1-hydroxyethyl, benzyl,
4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl, [1416] or
[1417] R.sup.3 and R.sup.4 together with the carbon atom to which
they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl
group of the formula
[1417] ##STR00159## [1418] wherein [1419] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [1420] #.sup.8 denotes
the linkage site to the group T.sup.1, [1421] T.sup.1 stands for a
group of the formula --C(.dbd.O)--OR.sup.7,
--C(.dbd.O)--NR.sup.8R.sup.9, --C(.dbd.O)--NH--NH--R.sup.10 or
--CH.sub.2--O--R.sup.11, [1422] wherein [1423] R.sup.7 stands for
hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, [1424] R.sup.8 stands for hydrogen or methyl,
[1425] R.sup.9 stands for hydrogen, methyl, ethyl, n-propyl or
benzyl, [1426] or [1427] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are bound form a four- to
seven-membered heterocycle, [1428] R.sup.10 stands for benzoyl,
[1429] R.sup.11 stands for benzyl, which may be substituted in the
phenyl group with methoxycarbonyl or carboxyl, [1430] R.sup.5
stands for hydrogen, methyl or a group of the formula
[1430] ##STR00160## [1431] wherein [1432] #.sup.9 denotes the
linkage site to --CHCH.sub.2-phenyl, [1433] R.sup.12 stands for
phenyl, which may be substituted with methoxycarbonyl, carboxyl or
a group of the formula --S(O).sub.2OH, [1434] R.sup.13 stands for
phenyl, which may be substituted with methoxycarbonyl or carboxyl,
[1435] R.sup.35 stands for methyl or hydroxyl, [1436] as well as
their salts and solvates as well as the solvates of the salts.
[1437] The preferred subject matter of the present invention is
compounds of formula (XXXI) as indicated above, wherein
[1438] L.sup.1 stands for a bond,
[1439] B stands for a bond,
[1440] L.sup.2 stands for linear (C.sub.2-C.sub.6)-alkanediyl or
for a group of the formula
##STR00161## [1441] wherein [1442] p stands for a number of 2 or 3,
[1443] ##.sup.3 denotes the linkage site to the group B, [1444]
##.sup.4 denotes the linkage site to the nitrogen atom,
[1445] D stands for a group of the formula
##STR00162## [1446] wherein [1447] #.sup.3 denotes the linkage site
to the nitrogen atom, [1448] R.sup.1 stands for hydrogen, [1449]
R.sup.2 stands for benzyl or 1H-indol-3-ylmethyl, [1450] or [1451]
R.sup.1 and R.sup.2 together with the carbon atom to which they are
attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[1451] ##STR00163## [1452] wherein [1453] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [1454] #.sup.5 denotes
the linkage site to the carbonyl group, [1455] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[1455] ##STR00164## [1456] wherein [1457] #.sup.6 denotes the
linkage site to the carbonyl group, [1458] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [1459] R.sup.3 stands for
hydrogen, [1460] R.sup.4 stands for benzyl or 1H-indol-3-ylmethyl,
[1461] or [1462] R.sup.3 and R.sup.4 together with the carbon atom
to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1462] ##STR00165## [1463] wherein [1464] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [1465] #.sup.8 denotes
the linkage site to the group T.sup.1, [1466] T.sup.1 stands for a
group of the formula --C(.dbd.O)--OR.sup.7 or
--C(.dbd.O)--NR.sup.8R.sup.9, [1467] wherein [1468] R.sup.7 stands
for hydrogen, [1469] R.sup.8 stands for hydrogen, [1470] R.sup.9
stands for hydrogen, [1471] R.sup.35 stands for methyl, [1472] as
well as their salts and solvates as well as the solvates of the
salts.
[1473] The preferred subject matter of the present invention is
compounds of formula (XXXI) as indicated above, wherein
[1474] L.sup.1 stands for a bond,
[1475] B stands for a bond,
[1476] L.sup.2 stands for linear (C.sub.2-C.sub.6)-alkanediyl or
for a group of the formula
##STR00166## [1477] wherein [1478] p stands for a number of 2 or 3,
[1479] ##.sup.3 denotes the linkage site to the group B, [1480]
##.sup.4 denotes the linkage site to the nitrogen atom,
[1481] D stands for a group of the formula
##STR00167## [1482] wherein [1483] #.sup.3 denotes the linkage site
to the nitrogen atom, [1484] R.sup.1 stands for hydrogen, [1485]
R.sup.2 stands for benzyl, 4-hydroxybenzyl or 1H-indol-3-ylmethyl,
[1486] or [1487] R.sup.1 and R.sup.2 together with the carbon atom
to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1487] ##STR00168## [1488] wherein [1489] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [1490] #.sup.5 denotes
the linkage site to the carbonyl group, [1491] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[1491] ##STR00169## [1492] wherein [1493] #.sup.6 denotes the
linkage site to the carbonyl group, [1494] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [1495] R.sup.3 stands for
hydrogen, [1496] R.sup.4 stands for benzyl, 4-hydroxybenzyl or
1H-indol-3-ylmethyl, [1497] or [1498] R.sup.3 and R.sup.4 together
with the carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1498] ##STR00170## [1499] wherein [1500] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [1501] #.sup.8 denotes
the linkage site to the group T.sup.1, [1502] T.sup.1 stands for a
group of the formula --C(.dbd.O)--OR.sup.7 or
--C(.dbd.O)--NR.sup.8R.sup.9, [1503] wherein [1504] R.sup.7 stands
for hydrogen, [1505] R.sup.8 stands for hydrogen, [1506] R.sup.9
stands for hydrogen,
[1507] R.sup.35 stands for methyl,
[1508] as well as their salts and solvates as well as the solvates
of the salts.
[1509] The preferred subject matter of the present invention
relates to compounds of formula (XXXa) and (XXXI) selected from the
group: [1510]
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)-
hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carbox-
y-2-(1H-indol-3-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin--
1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,
[1511]
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)-
hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-in-
dol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-
-3-oxopropyl]pyrrolidine-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-meth-
yl-L-valinamide, [1512]
N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-N-
-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-
-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-
-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate, [1513]
N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-N-
-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-(1H-indol-3-yl)ethyl]-
amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl--
1-oxoheptan-4-yl]-N-methyl-L-valinamide, [1514] as well as their
salts and solvates as well as the solvates of the salts.
[1515] Another preferred subject matter of the present invention
relates to binder-drug conjugates of the general formula (I)
##STR00171##
[1516] in which [1517] n stands for a number from 1 to 50, [1518]
AK stands for a binder, preferably a chimeric, humanized or human
antibody, especially preferably an anti-EGFR antibody, [1519] the
group .sctn.-G-L1-B-L2-.sctn..sctn. stands for a linker, [1520]
wherein [1521] .sctn. denotes the linkage site to the group AK and
[1522] .sctn..sctn. denotes the linkage site to the nitrogen
atom,
[1523] D stands for a group of the formula
##STR00172## [1524] wherein [1525] #.sup.3 denotes the linkage site
to the nitrogen atom, [1526] R.sup.1 stands for hydrogen, [1527]
R.sup.2 stands for 1-hydroxyethyl, benzyl, 1-phenylethyl,
4-hydroxybenzyl or 1H-indol-3-ylmethyl, [1528] or [1529] R.sup.1
and R.sup.2 together with the carbon atom to which they are
attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[1529] ##STR00173## [1530] wherein [1531] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [1532] #.sup.5 denotes
the linkage site to the carbonyl group, [1533] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[1533] ##STR00174## [1534] wherein [1535] #.sup.6 denotes the
linkage site to the carbonyl group, [1536] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [1537] R.sup.3 stands for
hydrogen, [1538] R.sup.1 stands for 1-hydroxyethyl, benzyl,
1-phenylethyl, 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [1539] or
[1540] R.sup.3 and R.sup.4 together with the carbon atom to which
they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl
group of the formula
[1540] ##STR00175## [1541] wherein [1542] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [1543] #.sup.8 denotes
the linkage site to the group T.sup.1, [1544] T.sup.1 stands for a
group of the formula --C(.dbd.O)--OR.sup.7,
--C(.dbd.O)--NR.sup.3R.sup.9, --C(.dbd.O)--NH--NH--R.sup.10 or
--CH.sub.2--O--R.sup.11, [1545] wherein [1546] R.sup.7 stands for
hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, [1547] R.sup.8 stands for hydrogen or methyl,
[1548] R.sup.9 stands for hydrogen, methyl, ethyl, n-propyl or
benzyl, [1549] or [1550] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are attached form a four- to
seven-membered heterocycle, [1551] R.sup.10 stands for benzoyl,
[1552] R.sup.11 stands for benzyl, which may be substituted in the
phenyl group with methoxycarbonyl or carboxyl, [1553] R.sup.5
stands for hydrogen, methyl or a group of the formula
[1553] ##STR00176## [1554] wherein [1555] #.sup.9 denotes the
linkage site to --CHC(R.sup.26)-T.sup.2, [1556] R.sup.12 stands for
phenyl, which may be substituted with methoxycarbonyl, carboxyl or
a group of the formula --S(O).sub.2OH, [1557] R.sup.13 stands for
phenyl, which may be substituted with methoxycarbonyl or carboxyl,
[1558] R.sup.26 stands for hydrogen or hydroxyl, [1559] T.sup.2
stands for phenyl, benzyl, 1H-indol-3-yl or
1H-indol-3-ylmethyl,
[1560] as well as their salts and solvates as well as the solvates
of the salts.
[1561] The preferred subject matter of the invention relates to
binder-drug conjugates of the general formula (I), wherein
[1562] n stands for a number from 1 to 50,
[1563] AK stands for AK.sub.1 or AK.sub.2 [1564] wherein [1565]
AK.sub.1 denotes a binder which is bound by a nitrogen atom of the
binder to the group G, preferably for a chimeric, humanized or
human antibody, especially preferably an anti-EGFR antibody, [1566]
AK.sub.2 stands for a binder bound to group G by a nitrogen atom of
the binder, preferably standing for a chimeric, humanized or human
antibody, especially preferably an anti-EGFR antibody,
[1567] G for the case when AK=AK.sub.1 stands for a group of the
formula
##STR00177## [1568] wherein [1569] #.sup.1 denotes the linkage site
to the sulfur atom of the binder, [1570] #.sup.2 denotes the
linkage site to the group L.sup.1, [1571] or [1572] for the case
when AK=AK.sub.2, G stands for carbonyl,
[1573] L.sup.1 stands for a bond, linear
(C.sub.1-C.sub.10)-alkanediyl or for a group of the formula
##STR00178## [1574] wherein [1575] m stands for a number from 2 to
6, [1576] ##.sup.1 denotes the linkage site to the group G, [1577]
##.sup.2 denotes the linkage site to the group B, [1578] wherein
(C.sub.1-C.sub.10)-alkanediyl may be substituted with 1 to 4 methyl
substituents, [1579] and [1580] wherein two carbon atoms of the
alkanediyl chain in 1,2-, 1,3- or 1,4- relation to one another to
form a (C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring,
including the carbon atoms optionally situated between them,
[1581] B stands for a bond or a group of the formula
##STR00179## [1582] wherein [1583] * denotes the linkage site to
L.sup.1, [1584] ** denotes the linkage site to L.sup.2, [1585] P
stands for O or NH, [1586] L.sup.3 for a bond or
(C.sub.2-C.sub.4)-alkanediyl, [1587] L.sup.4 stands for a bond or a
group of the formula
[1587] ##STR00180## [1588] wherein [1589] *** denotes the linkage
site to the carbonyl group, [1590] **** denotes the linkage site to
L.sup.2, [1591] R.sup.25 stands for hydrogen or methyl, [1592]
Q.sup.1 stands for a four- to seven-membered heterocycle, [1593]
Q.sup.2 stands for a three- to seven-membered carbocycle or a four-
to seven-membered heterocycle, [1594] R.sup.14 stands for hydrogen
or (C.sub.1-C.sub.4)-alkyl, [1595] R.sup.15 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1596] or [1597] R.sup.14 and R.sup.15
together with the atoms to which they are bound form a five- or
six-membered heterocycle, [1598] R.sup.16 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1599] R.sup.17 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1600] or [1601] R.sup.16 and R.sup.17
together with the atoms to which they are bound form a five- or
six-membered heterocycle, [1602] R.sup.18 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1603] R.sup.19 stands for hydrogen or the
side group of a natural .alpha.-amino acid or its homologs or
isomers, [1604] R.sup.20 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1605] or [1606] R.sup.19 and R.sup.20
together with the atoms to which they are bound form a pyrrolidinyl
ring, [1607] R.sup.21 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1608] R.sup.22 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1609] or [1610] R.sup.21 and R.sup.22
together with the atoms to which they are bound form a three- to
seven-membered carbocycle, [1611] R.sup.23 stands for
(C.sub.1-C.sub.4)-alkyl, [1612] R.sup.24 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1613] R.sup.27 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl,
[1614] L.sup.2 stands for linear (C.sub.2-C.sub.10)-alkanediyl or
for a group of the formula
##STR00181## [1615] wherein [1616] p stands for a number from 2 to
6, [1617] ##.sup.3 denotes the linkage site to the group B, [1618]
##.sup.4 denotes the linkage site to the nitrogen atom, [1619]
wherein (C.sub.2-C.sub.10)-alkanediyl may be substituted with 1 to
4 methyl substituents, [1620] and [1621] wherein two carbon atoms
of the alkanediyl chain in 1,2-, 1,3- or 1,4- relation to one
another to form a (C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl
ring, including the carbon atoms optionally situated between
them,
[1622] D has the meanings given above,
[1623] as well as their salts and solvates as well as the solvates
of the salts.
[1624] The preferred subject matter of the invention is binder-drug
conjugates of the general formula (I)
[1625] in which
[1626] n stands for a number from 1 to 50,
[1627] AK stands for AK.sub.1 or AK.sub.2 [1628] wherein [1629]
AK.sub.1 stands for an antibody or an antigen binding antibody
fragment and is bound to the group G via a sulfur atom, preferably
standing for a chimeric, humanized or human antibody, especially
preferably an anti-EGFR antibody, [1630] AK.sub.2 stands for an
antibody or an antigen binding antibody fragment and is bound to
the group G via a nitrogen atom, preferably standing for a
chimeric, humanized or human antibody, especially preferably an
anti-EGFR antibody,
[1631] G, L.sup.1, B, L.sup.2 and D have the meanings given
above
[1632] as well as their salts and solvates as well as the solvates
of the salts.
[1633] The preferred subject matter of the present invention is
binder-drug conjugates of general formula (I)
[1634] in which
[1635] n stands for a number from 1 to 20,
[1636] AK stands for AK.sub.1 or AK.sub.2 [1637] wherein [1638]
AK.sub.1 stands for an antibody or an antigen binding antibody
fragment which binds to EGFR and is bound to the group G via the
sulfur atom of a cysteine radical of the binder, [1639] AK.sub.2
stands for an antibody or an antigen binding antibody fragment
which binds to EGFR and is bound to the group G via the NH side
group of a lysine radical of the binder,
[1640] G for the case when AK=AK.sub.1 stands for a group of the
formula
##STR00182## [1641] wherein [1642] #.sup.1 denotes the linkage site
to the cysteine radical of the binder, [1643] #.sup.2 denotes the
linkage site to the group L.sup.1, [1644] or [1645] for the case
when AK=AK.sub.2, G stands for carbonyl,
[1646] L.sup.1 stands for a bond, linear
(C.sub.2-C.sub.6)-alkanediyl or for a group of the formula
##STR00183## [1647] wherein [1648] m stands for a number from 2 to
6, [1649] ##.sup.1 denotes the linkage site to the group G, [1650]
##.sup.2 denotes the linkage site to the group B, [1651] wherein
(C.sub.2-C.sub.6)-alkanediyl may be substituted with one or two
methyl substituents,
[1652] B stands for a bond or a group of the formula
##STR00184## [1653] wherein [1654] * denotes the linkage site to
L.sup.1, [1655] ** denotes the linkage site to L.sup.2, [1656] P
stands for O or NH, [1657] L.sup.3 stands for a bond or
ethane-1,2-diyl, [1658] L.sup.4 stands for a bond or a group of the
formula
[1658] ##STR00185## [1659] wherein [1660] *** denotes the linkage
site to the carbonyl group, [1661] **** denotes the linkage site to
L.sup.2, [1662] R.sup.25 stands for methyl, [1663] Q.sup.2 stands
for cyclopentyl or cyclohexyl, [1664] R.sup.16 stands for hydrogen
or methyl, [1665] R.sup.17 stands for hydrogen or methyl, [1666] or
[1667] R.sup.16 and R.sup.17 together with the atoms to which they
are bound form a piperazinyl ring, [1668] R.sup.18 stands for
hydrogen, [1669] R.sup.19 stands for hydrogen, methyl, propan-2-yl,
2-methylpropan-1-yl or 1-methyl-propan-1-yl, [1670] R.sup.20 stands
for hydrogen or methyl, [1671] or [1672] R.sup.19 and R.sup.20
together with the atoms to which they are attached form a
pyrrolidinyl ring,
[1673] L.sup.2 stands for linear (C.sub.2-C.sub.6)-alkanediyl,
[1674] wherein (C.sub.2-C.sub.6)-alkanediyl may be substituted with
one or two methyl substituents,
[1675] D stands for a group of the formula
##STR00186## [1676] wherein [1677] #.sup.3 denotes the linkage site
to the nitrogen atom, [1678] R.sup.1 stands for hydrogen, [1679]
R.sup.2 stands for 1-hydroxyethyl, benzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [1680] or [1681] R.sup.1 and R.sup.2 together
with the carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1681] ##STR00187## [1682] wherein [1683] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [1684] #.sup.5 denotes
the linkage site to the carbonyl group, [1685] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[1685] ##STR00188## [1686] wherein [1687] #.sup.6 denotes the
linkage site to the carbonyl group, [1688] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [1689] R.sup.3 stands for
hydrogen, [1690] R.sup.4 stands for 1-hydroxyethyl, benzyl,
1-phenylethyl or 1H-indol-3-ylmethyl, [1691] or [1692] R.sup.3 and
R.sup.4 together with the carbon atom to which they are attached
form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[1692] ##STR00189## [1693] wherein [1694] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [1695] #.sup.8 denotes
the linkage site to the group T.sup.1, [1696] T.sup.1 stands for a
group of the formula --C(.dbd.O)--OR.sup.7,
--C(.dbd.O)--NR.sup.8R.sup.9, --C(.dbd.O)--NH--NH--R.sup.10 or
--CH.sub.2--O--R.sup.11, [1697] wherein [1698] R.sup.7 stands for
hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, [1699] R.sup.8 stands for hydrogen or methyl,
[1700] R.sup.9 stands for hydrogen, methyl, ethyl, n-propyl or
benzyl, [1701] or [1702] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are attached form a four- to
seven-membered heterocycle, [1703] R.sup.10 stands for benzoyl,
[1704] R.sup.11 stands for benzyl, which may be substituted in the
phenyl group with methoxycarbonyl or carboxyl, [1705] R.sup.5
stands for hydrogen, methyl or a group of the formula
[1705] ##STR00190## [1706] wherein [1707] #.sup.9 denotes the
linkage site to --CHC(R.sup.26)-T.sup.2, [1708] R.sup.12 stands for
phenyl, which may be substituted with methoxycarbonyl, carboxyl or
a group of the formula --S(O).sub.2OH, [1709] R.sup.13 stands for
phenyl, which may be substituted with methoxycarbonyl or carboxyl,
[1710] R.sup.26 stands for hydrogen or hydroxyl, [1711] T.sup.2
stands for phenyl, benzyl, 1H-indol-3-yl or
1H-indol-3-ylmethyl,
[1712] as well as their salts and solvates as well as the solvates
of the salts.
[1713] Especially preferred as the subject matter of the present
invention are binder-drug conjugates of general formula (I)
[1714] in which
[1715] n stands for a number from 1 to 10,
[1716] AK stands for AK.sub.1 or AK.sub.2 [1717] wherein [1718]
AK.sub.1 stands for cetuximab, pantitumumab or nimutuzumab, which
is bound to the group G via the sulfur atom of a cysteine radical
of the binder, [1719] AK.sub.2 stands for cetuximab, pantitumumab
or nimutuzumab, which is bound to the group G via the NH side group
of a lysine radical of the binder,
[1720] G for the case when AK=AK.sub.1 stands for a group of the
formula
##STR00191## [1721] wherein [1722] #.sup.1 denotes the linkage site
to the cysteine radical of the binder, [1723] #.sup.2 denotes the
linkage site to the group L.sup.1, [1724] or [1725] for the case
when AK=AK.sub.2, G stands for carbonyl,
[1726] L.sup.1 stands for a bond, linear
(C.sub.2-C.sub.6)-alkanediyl, a group of the formula
##STR00192## [1727] wherein [1728] m stands for a number of 2 or 3,
[1729] ##.sup.1 denotes the linkage site to the group G, [1730]
##.sup.2 denotes the linkage site to the group B, [1731] wherein
(C.sub.2-C.sub.6)-alkanediyl may be substituted with one or two
methyl substituents,
[1732] B stands for a bond or a group of the formula
##STR00193## [1733] wherein [1734] * denotes the linkage site to
L.sup.1, [1735] ** denotes the linkage site to L.sup.2, [1736]
L.sup.3 stands for a bond or ethane-1,2-diyl, [1737] L.sup.4 stands
for a bond or a group of the formula
[1737] ##STR00194## [1738] wherein [1739] *** denotes the linkage
site to the carbonyl group, [1740] **** denotes the linkage site to
L.sup.2, [1741] R.sup.25 stands for methyl, [1742] R.sup.16 stands
for hydrogen or methyl, [1743] R.sup.17 stands for hydrogen or
methyl, [1744] or [1745] R.sup.16 and R'7 together with the atoms
to which they are bound form a piperazinyl ring,
[1746] L.sup.2 stands for linear (C.sub.2-C.sub.6)-alkanediyl,
[1747] D stands for a group of the formula
##STR00195## [1748] wherein [1749] #.sup.3 denotes the linkage site
to the nitrogen atom, [1750] R.sup.1 stands for hydrogen, [1751]
R.sup.2 stands for 1-hydroxyethyl, benzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [1752] or [1753] R.sup.1 and R.sup.2 together
with the carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1753] ##STR00196## [1754] wherein [1755] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [1756] #.sup.5 denotes
the linkage site to the carbonyl group, [1757] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[1757] ##STR00197## [1758] wherein [1759] #.sup.6 denotes the
linkage site to the carbonyl group, [1760] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [1761] R.sup.3 stands for
hydrogen, [1762] R.sup.4 stands for benzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [1763] or [1764] R.sup.3 and R.sup.4 together
with the carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1764] ##STR00198## [1765] wherein [1766] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [1767] #.sup.8 denotes
the linkage site to the group T, [1768] T.sup.1 stands for a group
of the formula --C(.dbd.O)--OR.sup.7, --C(.dbd.O)--NR.sup.8R.sup.9
or --CH.sub.2--O--R.sup.11, [1769] wherein [1770] R.sup.7 stands
for hydrogen, methyl, ethyl, n-propyl, benzyl or adamantylmethyl,
[1771] R.sup.8 stands for hydrogen or methyl, [1772] R.sup.9 stands
for hydrogen, methyl, ethyl, n-propyl or benzyl, [1773] R.sup.11
stands for benzyl, which may be substituted in the phenyl group
with methoxycarbonyl or carboxyl, [1774] R.sup.5 stands for
hydrogen or a group of the formula
[1774] ##STR00199## [1775] wherein [1776] #.sup.9 denotes the
linkage site to --CHC(R.sup.26)phenyl, [1777] R.sup.12 stands for
phenyl, which may be substituted with methoxycarbonyl, carboxyl or
a group of the formula --S(O).sub.2OH, [1778] R.sup.13 stands for
phenyl, which may be substituted with methoxycarbonyl or
carboxyl,
[1779] as well as their salts and solvates as well as the solvates
of the salts.
[1780] Another subject matter of the present invention relates to
compounds of formula (XXX)
##STR00200##
[1781] in which [1782] Cys stands for a cysteine radical which is
bound to a carbon atom of the succinimide via the sulfur atom of
the side chain, [1783] L.sup.1 stands for a bond, linear
(C.sub.1-C.sub.10)-alkanediyl or for a group of the formula
[1783] ##STR00201## [1784] wherein [1785] m stands for a number
from 2 to 6, [1786] ##.sup.1 denotes the linkage site to the group
G, [1787] ##.sup.2 denotes the linkage site to the group B, [1788]
wherein (C.sub.1-C.sub.10)-alkanediyl may be substituted with 1 to
4 methyl substituents, [1789] and [1790] wherein two carbon atoms
of the alkanediyl chain in 1,2-, 1,3- or 1,4- relation to one
another to form a (C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl
ring, including the carbon atoms optionally situated between
them,
[1791] B stands for a bond or a group of the formula
##STR00202## [1792] wherein [1793] * denotes the linkage site to
L.sup.1, [1794] ** denotes the linkage site to L.sup.2, [1795] P
stands for O or NH, [1796] L.sup.3 for a bond or
(C.sub.2-C.sub.4)-alkanediyl, [1797] L.sup.4 stands for a bond or a
group of the formula
[1797] ##STR00203## [1798] wherein [1799] *** denotes the linkage
site to the carbonyl group, [1800] **** denotes the linkage site to
L.sup.2, [1801] R.sup.25 stands for hydrogen or methyl, [1802]
Q.sup.1 stands for a three- to seven-membered carbocycle or a four-
to seven-membered azaheterocycle, [1803] Q.sup.2 stands for a
three- to seven-membered carbocycle or a four- to seven-membered
azaheterocycle, [1804] R.sup.14 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1805] R.sup.15 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1806] or [1807] R.sup.14 and R.sup.15
together with the atoms to which they are bound form a five- or
six-membered heterocycle, [1808] R.sup.16 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1809] R.sup.17 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1810] or [1811] R.sup.16 and R.sup.17
together with the atoms to which they are bound form a five- or
six-membered heterocycle, [1812] R.sup.18 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1813] R.sup.19 stands for hydrogen or the
side group of a natural .alpha.-amino acid or its homologs or
isomers, [1814] R.sup.20 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1815] or [1816] R.sup.19 and R.sup.20
together with the atoms to which they are bound form a pyrrolidinyl
ring, [1817] R.sup.21 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1818] R.sup.22 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1819] or [1820] R.sup.21 and R.sup.22
together with the atoms to which they are bound form a three- to
seven-membered carbocycle, [1821] R.sup.23 stands for
(C.sub.1-C.sub.4)-alkyl, [1822] R.sup.24 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl,
[1823] L.sup.2 stands for linear (C.sub.2-C.sub.10)-alkanediyl or
for a group of the formula
##STR00204## [1824] wherein [1825] p stands for a number from 2 to
6, [1826] ##.sup.3 denotes the linkage site to the group B, [1827]
##.sup.4 denotes the linkage site to the nitrogen atom, [1828]
wherein (C.sub.2-C.sub.10)-alkanediyl may be substituted with 1 to
4 methyl substituents, [1829] and [1830] wherein two carbon atoms
of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-
relation to one another, including the carbon atoms optionally
present between them to form a (C.sub.3-C.sub.6) cycloalkyl ring or
a phenyl ring,
[1831] D stands for a group of the formula
##STR00205## [1832] wherein [1833] #.sup.3 denotes the linkage site
to the nitrogen atom, [1834] R.sup.1 stands for hydrogen, [1835]
R.sup.2 stands for 1-hydroxyethyl, benzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [1836] or [1837] R.sup.1 and R.sup.2 together
with the carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1837] ##STR00206## [1838] wherein [1839] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [1840] #.sup.5 denotes
the linkage site to the carbonyl group, [1841] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[1841] ##STR00207## [1842] wherein [1843] #.sup.6 denotes the
linkage site to the carbonyl group, [1844] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [1845] R.sup.3 stands for
hydrogen, [1846] R.sup.4 stands for 1-hydroxyethyl, benzyl,
1-phenylethyl or 1H-indol-3-ylmethyl, [1847] or [1848] R.sup.3 and
R.sup.4 together with the carbon atom to which they are attached
form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[1848] ##STR00208## [1849] wherein [1850] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [1851] #.sup.8 denotes
the linkage site to the group T.sup.1, [1852] T.sup.1 stands for a
group of the formula --C(.dbd.O)--OR.sup.7,
--C(.dbd.O)--NR.sup.8R.sup.9, --C(.dbd.O)--NH--NH--R.sup.10 or
--CH.sub.2--O--R.sup.11, [1853] wherein [1854] R.sup.7 stands for
hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, [1855] R.sup.8 stands for hydrogen or methyl,
[1856] R.sup.9 stands for hydrogen, methyl, ethyl, n-propyl or
benzyl, [1857] or [1858] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are bound form a four- to
seven-membered heterocycle, [1859] R.sup.10 stands for benzoyl,
[1860] R.sup.11 stands for benzyl, which may be substituted in the
phenyl group with methoxycarbonyl or carboxyl, [1861] R.sup.5
stands for hydrogen, methyl or a group of the formula
[1861] ##STR00209## [1862] wherein [1863] #.sup.9 denotes the
linkage site to --CHC(R.sup.26)-T.sup.2, [1864] R.sup.12 stands for
phenyl, which may be substituted with methoxycarbonyl, carboxyl or
a group of the formula --S(O).sub.2OH, [1865] R.sup.13 stands for
phenyl, which may be substituted with methoxycarbonyl or carboxyl,
[1866] R.sup.26 stands for hydrogen or hydroxyl, [1867] T.sup.2
stands for phenyl, benzyl, 1H-indol-3-yl or
1H-indol-3-ylmethyl,
[1868] as well as their salts and solvates as well as the solvates
of the salts.
[1869] In addition, compounds of formula (XXX) that are especially
preferred within the scope of the present invention are those in
which [1870] Cys stands for a cysteine radical which is bound to a
carbon atom of the succinimide via the sulfur atom of the side
chain, [1871] L.sup.1 stands for a bond, linear
(C.sub.2-C.sub.6)-alkanediyl, a group of the formula
[1871] ##STR00210## [1872] wherein [1873] m stands for a number
from 2 to 6, [1874] ##.sup.1 denotes the linkage site to the group
G, [1875] ##.sup.2 denotes the linkage site to the group B, [1876]
wherein (C.sub.2-C.sub.6)-alkanediyl may be substituted with one or
two methyl substituents,
[1877] B stands for a bond or a group of the formula
##STR00211## [1878] wherein [1879] * denotes the linkage site to
L.sup.1, [1880] ** denotes the linkage site to L.sup.2, [1881]
L.sup.3 stands for a bond or ethane-1,2-diyl, [1882] L.sup.4 stands
for a bond, [1883] R.sup.14 stands for hydrogen, [1884] R.sup.15
stands for hydrogen, [1885] R.sup.16 stands for hydrogen or methyl,
[1886] R.sup.17 stands for hydrogen or methyl, [1887] or [1888]
R.sup.16 and R.sup.17 together with the atoms to which they are
bound form a piperazinyl ring, [1889] R.sup.23 stands for methyl,
[1890] R.sup.24 stands for hydrogen or methyl,
[1891] L.sup.2 stands for linear (C.sub.2-C.sub.6)-alkanediyl or
for a group of the formula
##STR00212## [1892] wherein [1893] p stands for a number of 2 or 3,
[1894] ##.sup.3 denotes the linkage site to the group B, [1895]
##.sup.4 denotes the linkage site to the nitrogen atom,
[1896] D stands for a group of the formula
##STR00213## [1897] wherein [1898] #.sup.3 denotes the linkage site
to the nitrogen atom, [1899] R.sup.1 stands for hydrogen, [1900]
R.sup.2 stands for 1-hydroxyethyl, benzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [1901] or [1902] R.sup.1 and R.sup.2 together
with the carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1902] ##STR00214## [1903] wherein [1904] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [1905] #.sup.5 denotes
the linkage site to the carbonyl group, [1906] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[1906] ##STR00215## [1907] wherein [1908] #.sup.6 denotes the
linkage site to the carbonyl group, [1909] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [1910] R.sup.3 stands for
hydrogen, [1911] R.sup.4 stands for benzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [1912] or [1913] R.sup.3 and R.sup.4 together
with the carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1913] ##STR00216## [1914] wherein [1915] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [1916] #.sup.8 denotes
the linkage site to the group T, [1917] T.sup.1 stands for a group
of the formula --C(.dbd.O)--OR.sup.7, --C(.dbd.O)--NR.sup.8R.sup.9,
--C(.dbd.O)--NH--NH--R.sup.10 or --CH.sub.2--O--R.sup.11, [1918]
wherein [1919] R.sup.7 stands for hydrogen, methyl ethyl, n-propyl,
benzyl or adamantylmethyl, [1920] R.sup.8 stands for hydrogen or
methyl, [1921] R.sup.9 stands for hydrogen, methyl, ethyl, n-propyl
or benzyl, [1922] R.sup.10 stands for benzoyl, [1923] R.sup.11
stands for benzyl, which may be substituted in the phenyl group
with methoxycarbonyl or carboxyl, [1924] R.sup.5 stands for
hydrogen or a group of the formula
[1924] ##STR00217## [1925] wherein [1926] #.sup.9 denotes the
linkage site to --CHC(R.sup.26)phenyl, [1927] R.sup.12 stands for
phenyl, which may be substituted with methoxycarbonyl, carboxyl or
a group of the formula --S(O).sub.2OH, [1928] R.sup.13 stands for
phenyl, which may be substituted with methoxycarbonyl or
carboxyl,
[1929] as well as their salts and solvates as well as the solvates
of the salts.
[1930] Within the scope of the present invention, compounds of
formula (Ia) are also preferred, in which n=1-20, especially
preferably n=1-10 and most especially preferably n=2-8.
[1931] Within the scope of the present invention, compounds of
formula (Ia) are preferred, in which
[1932] AK stands for AK.sub.1 [1933] wherein [1934] AK.sub.1 stands
for an antibody or an antigen binding antibody fragment which binds
to EGFR and is bound to the group G by the sulfur atom of a
cysteine radical of the binder,
[1935] G stands for a group of the formula
##STR00218## [1936] wherein [1937] #.sup.1 denotes the linkage site
to the cysteine radical of the binder, [1938] #.sup.2 denotes the
linkage site to the group L.sup.1,
[1939] and
[1940] n, L.sup.1, B, L.sup.2, D and R.sup.35 have the meanings
given above
[1941] as well as their salts and solvates as well as the solvates
of the salts.
[1942] Also preferred within the scope of the present invention are
compounds of formula (Ia), wherein
[1943] AK stands for AK.sub.2 [1944] wherein [1945] AK.sub.2 stands
for an antibody or an antigen binding antibody fragment which binds
to EGFR and is bound to the group G via the NH side group of a
lysine radical of the binder,
[1946] G stands for carbonyl,
[1947] and
[1948] n, L.sup.1, B, L.sup.2, D and R.sup.35 have the meanings
given above
[1949] as well as their salts and solvates as well as the solvates
of the salts.
[1950] Also preferred within the scope of the present invention are
compounds of formula (Ia), in which
[1951] AK stands for AK.sub.1 [1952] wherein [1953] AK.sub.1 stands
for cetuximab, pantitumumab or nimutuzumab, which is bound to the
group G via the sulfur atom of a cysteine radical of the
binder,
[1954] G stands for a group of the formula
##STR00219## [1955] wherein [1956] #.sup.1 denotes the linkage site
to the cysteine radical of the binder, [1957] #.sup.2 denotes the
linkage site to the group L.sup.1,
[1958] and
[1959] n, L.sup.1, B, L.sup.2, D and R.sup.35 have the meanings
given above
[1960] as well as their salts and solvates as well as the solvates
of the salts.
[1961] Also preferred within the scope of the present invention are
compounds of formula (Ia), in which
[1962] AK stands for AK.sub.2 [1963] wherein [1964] AK.sub.2 stands
for cetuximab, pantitumumab or nimutuzumab, which is bound to the
group G via the NH side group of a lysine radical of the
binder,
[1965] G stands for carbonyl,
[1966] and
[1967] n, L.sup.1, B, L.sup.2, D and R.sup.35 have the meanings
given above
[1968] as well as their salts and solvates as well as the solvates
of the salts.
[1969] Also preferred within the scope of the present invention are
compounds of general formula (Ia), in which
[1970] AK stands for AK.sub.2 [1971] wherein [1972] AK.sub.2 stands
for cetuximab, pantitumumab or nimutuzumab, which is bound to the
group G via the NH side group of a lysine radical of the
binder,
[1973] G stands for carbonyl,
[1974] L.sup.1 stands for a bond,
[1975] B stands for a bond,
[1976] L.sup.2 stands for linear (C.sub.3-C.sub.6)-alkanediyl or
for a group of the formula
##STR00220## [1977] wherein [1978] p stands for a number of 2 or 3,
[1979] ##.sup.3 denotes the linkage site to the group B, [1980]
##.sup.4 denotes the linkage site to the nitrogen atom,
[1981] n, D and R.sup.35 have the meanings given above
[1982] as well as their salts and solvates as well as the solvates
of the salts.
[1983] Also preferred within the scope of the present invention are
compounds of general formula (Ia), in which
[1984] AK stands for AK.sub.1 [1985] wherein [1986] AK.sub.1 stands
for cetuximab, pantitumumab or nimutuzumab, which is bound to the
group G via the sulfur atom of a cysteine radical of the
binder,
[1987] G stands for a group of the formula
##STR00221## [1988] wherein [1989] #.sup.1 denotes the linkage site
to the cysteine radical of the binder, [1990] #.sup.2 denotes the
linkage site to the group
[1991] L.sup.1 stands for a bond, linear
(C.sub.3-C.sub.5)-alkanediyl, a group of the formula
##STR00222## [1992] wherein [1993] m stands for a number of 2 or 3,
[1994] ##.sup.1 denotes the linkage site to the group G, [1995]
##.sup.2 denotes the linkage site to the group B, [1996] wherein
(C.sub.3-C.sub.5)-alkanediyl may be substituted with one or two
methyl substituents,
[1997] B stands for a bond or a group of the formula
##STR00223## [1998] wherein [1999] * denotes the linkage site to
L.sup.1, [2000] ** denotes the linkage site to L.sup.2, [2001]
L.sup.3 stands for a bond or ethane-1,2-diyl, [2002] L.sup.4 stands
for a bond or a group of the formula
[2002] ##STR00224## [2003] wherein [2004] *** denotes the linkage
site to the carbonyl group, [2005] **** denotes the linkage site to
L.sup.2, [2006] R.sup.25 stands for methyl, [2007] R.sup.28 stands
for hydrogen, methylcarbonyl or tert-butyloxycarbonyl, [2008]
R.sup.16 stands for hydrogen or methyl, [2009] R.sup.17 stands for
hydrogen or methyl, [2010] or [2011] R.sup.16 and R.sup.17 together
with the atoms to which they are bound form a piperazinyl ring,
[2012] L.sup.2 stands for linear (C.sub.3-C.sub.5)-alkanediyl or
for a group of the formula
##STR00225## [2013] wherein [2014] p stands for a number of 2 or 3,
[2015] ##.sup.3 denotes the linkage site to the group B, [2016]
##.sup.4 denotes the linkage site to the nitrogen atom,
[2017] and
[2018] n, D and R.sup.35 have the meanings given above
[2019] as well as their salts and solvates as well as the solvates
of the salts.
[2020] Also preferred within the scope of the present invention are
compounds of formula (Ia), (XXXa) and (XXXI), in which
[2021] L.sup.1 stands for a bond,
[2022] B stands for a bond,
[2023] L.sup.2 stands for linear (C.sub.3-C.sub.6)-alkanediyl or
for a group of the formula
##STR00226## [2024] wherein [2025] p stands for a number of 2 or 3,
[2026] ##.sup.3 denotes the linkage site to the group B, [2027]
##.sup.4 denotes the linkage site to the nitrogen atom,
[2028] and
[2029] n, AK, Cys, G, D and R.sup.35 have the meanings given
above
[2030] as well as their salts and solvates as well as the solvates
of the salts.
[2031] Also preferred within the scope of the present invention are
compounds of formula (Ia), in which
[2032] L.sup.1 stands for linear (C.sub.1-C.sub.10)-alkanediyl or a
group of the formula
##STR00227## [2033] wherein [2034] m stands for a number from 2 to
6, [2035] ##.sup.1 denotes the linkage site to the group G, [2036]
##.sup.2 denotes the linkage site to the group B, [2037] wherein
(C.sub.1-C.sub.10)-alkanediyl may be substituted with 1 to 4
substituents selected independently of one another from the group
methyl, hydroxyl and benzyl,
[2038] B stands for a bond or a group of the formula
##STR00228## [2039] wherein [2040] * denotes the linkage site to
L.sup.1, [2041] ** denotes the linkage site to L.sup.2, [2042]
L.sup.3 for a bond or (C.sub.2-C.sub.4)-alkanediyl, [2043] L.sup.4
stands for a group of the formula
[2043] ##STR00229## [2044] wherein [2045] *** denotes the linkage
site to the carbonyl group, [2046] **** denotes the linkage site to
L.sup.2, [2047] R.sup.25 stands for hydrogen or methyl, [2048]
R.sup.28 stands for hydrogen, (C.sub.1-C.sub.4)-alkylcarbonyl,
tert-butyloxycarbonyl or benzyloxycarbonyl, [2049] Q.sup.1 stands
for a four- to seven-membered heterocycle, [2050] R.sup.16 stands
for hydrogen or (C.sub.1-C.sub.4)-alkyl, [2051] R.sup.17 stands for
hydrogen or (C.sub.1-C.sub.4)-alkyl, [2052] or [2053] R.sup.16 and
R.sup.17 together with the atoms to which they are bound form a
five- or six-membered heterocycle, [2054] R.sup.23 stands for
(C.sub.1-C.sub.4)-alkyl, [2055] R.sup.24 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [2056] R.sup.36 stands for hydrogen,
(C.sub.1-C.sub.4)-alkylcarbonyl, tert-butyloxycarbonyl or
benzyloxycarbonyl, [2057] R.sup.37 stands for hydrogen or methyl,
[2058] or [2059] R.sup.36 and R.sup.37 together with the atoms to
which they are bound form a pyrrolidine ring,
[2060] L.sup.2 stands for linear (C.sub.2-C.sub.10)-alkanediyl or
for a group of the formula
##STR00230## [2061] wherein [2062] p stands for a number from 2 to
6, [2063] ##.sup.3 denotes the linkage site to the group B, [2064]
##.sup.4 denotes the linkage site to the nitrogen atom, [2065]
wherein (C.sub.2-C.sub.10)-alkanediyl may be substituted with 1 to
4 substituents selected independently of one another from the group
comprising methyl, hydroxyl and benzyl,
[2066] and
[2067] n, AK, G, D and R.sup.35 have the meanings given above
[2068] as well as their salts and solvates as well as the solvates
of the salts.
[2069] Also preferred within the scope of the present invention are
compounds of formula (Ia), in which
[2070] L.sup.1 stands for linear (C.sub.2-C.sub.6)-alkanediyl or
for a group of the formula
##STR00231## [2071] wherein [2072] m stands for a number of 2 or 3,
[2073] ##.sup.1 denotes the linkage site to the group G, [2074]
##.sup.2 denotes the linkage site to the group B,
[2075] B stands for a bond or a group of the formula
##STR00232## [2076] wherein [2077] * denotes the linkage site to
L.sup.1, [2078] ** denotes the linkage site to L.sup.2, [2079]
L.sup.3 stands for a bond or ethane-1,2-diyl, [2080] L.sup.4 stands
for a group of the formula
[2080] ##STR00233## [2081] wherein [2082] *** denotes the linkage
site to the carbonyl group, [2083] **** denotes the linkage site to
L.sup.2, [2084] R.sup.25 stands for hydrogen or methyl, [2085]
R.sup.28 stands for hydrogen, methylcarbonyl or
tert-butyloxycarbonyl, [2086] R.sup.16 stands for hydrogen or
methyl, [2087] R.sup.17 stands for hydrogen or methyl, [2088] or
[2089] R.sup.16 and R.sup.17 together with the atoms to which they
are bound form a piperazinyl ring, [2090] R.sup.36 stands for
hydrogen, methylcarbonyl or tert-butyloxycarbonyl, [2091] R.sup.37
stands for hydrogen or methyl, [2092] or [2093] R.sup.36 and
R.sup.37 together with the atoms to which they are bound form a
pyrrolidine ring,
[2094] L.sup.2 stands for linear (C.sub.2-C.sub.6)-alkanediyl or
for a group of the formula
##STR00234## [2095] wherein [2096] p stands for a number of 2 or 3,
[2097] ##.sup.3 denotes the linkage site to the group B, [2098]
##.sup.4 denotes the linkage site to the nitrogen atom,
[2099] and
[2100] n, AK, G, D and R.sup.35 have the meanings given above
[2101] as well as their salts and solvates as well as the solvates
of the salts.
[2102] Also preferred within the scope of the present invention are
compounds of formula (Ia) and (XXXa), in which
[2103] G stands for a group of the formula
##STR00235## [2104] wherein [2105] #.sup.1 denotes the linkage site
to the cysteine radical of the binder, [2106] #.sup.2 denotes the
linkage site to the group L.sup.1,
[2107] L.sup.1 stands for linear (C.sub.3-C.sub.5)-alkanediyl or
for a group of the formula
##STR00236## [2108] wherein [2109] m stands for a number of 2 or 3,
[2110] ##.sup.1 denotes the linkage site to the group G, [2111]
##.sup.2 denotes the linkage site to the group B, [2112] wherein
(C.sub.3-C.sub.5)-alkanediyl may be substituted with one or two
methyl substituents,
[2113] B stands for a bond or a group of the formula
##STR00237## [2114] wherein [2115] * denotes the linkage site to
L.sup.1, [2116] ** denotes the linkage site to L.sup.2, [2117]
L.sup.3 stands for a bond or ethane-1,2-diyl, [2118] L.sup.4 stands
for a bond,
[2119] L.sup.2 for linear (C.sub.3-C.sub.5)-alkanediyl or for a
group of the formula
##STR00238## [2120] wherein [2121] p stands for a number of 2 or 3,
[2122] ##.sup.3 denotes the linkage site to the group B, [2123]
##.sup.4 denotes the linkage site to the nitrogen atom,
[2124] and
[2125] n, AK.sub.1, Cys, D, R.sup.16 and R.sup.17 have the meanings
given above
[2126] as well as their salts and solvates as well as the solvates
of the salts.
[2127] Also preferred within the scope of the present invention are
compounds of formula (Ia) and (XXXa), in which
[2128] B stands for a bond or a group of the formula
##STR00239## [2129] wherein [2130] * denotes the linkage site to
L.sup.1, [2131] ** denotes the linkage site to L.sup.2, [2132]
L.sup.3 stands for a bond or ethane-1,2-diyl, [2133] L.sup.4 stands
for a bond,
[2134] n, AK, Cys, G, L.sup.1, L.sup.2, D, R.sup.16, R.sup.17 and
R.sup.35 have the meanings given above
[2135] as well as their salts and solvates as well as the solvates
of the salts.
[2136] Also preferred within the scope of the present invention are
compounds of formula (Ia), (XXXa) and (XXXI), in which
[2137] L.sup.1 stands for a bond, linear
(C.sub.3-C.sub.5)-alkanediyl, a group of the formula
##STR00240## [2138] wherein [2139] m stands for a number of 2 or 3,
[2140] ##.sup.1 denotes the linkage site to the group G, [2141]
##.sup.2 denotes the linkage site to the group B, [2142] wherein
(C.sub.3-C.sub.5)-alkanediyl may be substituted with one or two
methyl substituents,
[2143] B stands for a bond or a group of the formula
##STR00241## [2144] wherein [2145] * denotes the linkage site to
L.sup.1, [2146] ** denotes the linkage site to L.sup.2, [2147]
L.sup.3 stands for a bond, [2148] L.sup.4 stands for a bond [2149]
R.sup.16 stands for hydrogen, [2150] R.sup.17 stands for
hydrogen,
[2151] L.sup.2 stands for linear (C.sub.3-C.sub.6)-alkanediyl or
for a group of the formula
##STR00242## [2152] wherein [2153] p stands for a number of 2 or 3,
[2154] ##.sup.3 denotes the linkage site to the group B, [2155]
##.sup.4 denotes the linkage site to the nitrogen atom,
[2156] n, AK, Cys, G, D and R.sup.35 have the meanings given
above
[2157] as well as their salts and solvates as well as the solvates
of the salts.
[2158] Also preferred within the scope of the present invention are
compounds of formula (Ia), (XXXa) and (XXXI), in which
[2159] L.sup.1 stands for a bond,
[2160] B stands for a bond,
[2161] L.sup.2 stands for linear (C.sub.3-C.sub.6)-alkanediyl or
for a group of the formula
##STR00243## [2162] wherein [2163] p stands for a number of 2 or 3,
[2164] ##.sup.3 denotes the linkage site to the group B, [2165]
##.sup.4 denotes the linkage site to the nitrogen atom,
[2166] n, AK, Cys, G, D and R.sup.35 have the meanings given
above
[2167] as well as their salts and solvates as well as the solvates
of the salts.
[2168] Also preferred within the scope of the present invention are
compounds of formula (Ia), (XXXa) and (XXXI), in which
[2169] L.sup.1 stands for linear (C.sub.3-C.sub.5)-alkanediyl or
for a group of the formula
##STR00244## [2170] wherein [2171] m stands for a number of 2 or 3,
[2172] ##.sup.1 denotes the linkage site to the group G, [2173]
##.sup.2 denotes the linkage site to the group B, [2174] wherein
(C.sub.3-C.sub.5)-alkanediyl may be substituted with one or two
methyl substituents,
[2175] B stands for a group of the formula
##STR00245## [2176] wherein [2177] * denotes the linkage site to
L.sup.1, [2178] ** denotes the linkage site to L.sup.2, [2179]
L.sup.3 stands for a bond, [2180] L.sup.4 stands for a bond [2181]
R.sup.16 stands for hydrogen, [2182] R.sup.17 stands for
hydrogen,
[2183] L.sup.2 stands for linear (C.sub.3-C.sub.6)-alkanediyl or
for a group of the formula
##STR00246## [2184] wherein [2185] p stands for a number of 2 or 3,
[2186] ##.sup.3 denotes the linkage site to the group B, [2187]
##.sup.4 denotes the linkage site to the nitrogen atom,
[2188] n, AK, Cys, G, D and R.sup.35 have the meanings given
above
[2189] as well as their salts and solvates as well as the solvates
of the salts.
[2190] Also preferred within the scope of the present invention are
compounds of formula (Ia), in which
[2191] n stands for a number from 2 to 8, preferably 2 to 5,
[2192] AK stands for AK.sub.1 or AK.sub.2 [2193] wherein [2194]
AK.sub.1 stands for an antibody or an antigen binding antibody
fragment which binds to C4.4a and is bound to the group G via the
sulfur atom of a cysteine radical of the binder, [2195] AK.sub.2
stands for an antibody or an antigen binding antibody fragment
which binds to C4.4a and is bound to the group G via the NH side
group of a lysine radical of the binder,
[2196] G for the case when AK=AK.sub.1 stands for a group of the
formula
##STR00247## [2197] wherein [2198] #.sup.1 denotes the linkage site
to the cysteine radical of the binder, [2199] #.sup.2 denotes the
linkage site to the group L.sup.1, [2200] or [2201] for the case
when AK=AK.sub.2, G stands for carbonyl,
[2202] L.sup.1 stands for a bond, linear
(C.sub.3-C.sub.5)-alkanediyl, a group of the formula
##STR00248## [2203] wherein [2204] m stands for a number of 2 or 3,
[2205] ##.sup.1 denotes the linkage site to the group G, [2206]
##.sup.2 denotes the linkage site to the group B, [2207] wherein
(C.sub.3-C.sub.5)-alkanediyl may be substituted with one or two
methyl substituents,
[2208] B stands for a bond or a group of the formula
##STR00249## [2209] wherein [2210] * denotes the linkage site to
L.sup.1, [2211] ** denotes the linkage site to L.sup.2, [2212]
L.sup.3 stands for a bond, [2213] L.sup.4 stands for a bond [2214]
R.sup.16 stands for hydrogen, [2215] R.sup.17 stands for
hydrogen,
[2216] L.sup.2 stands for linear (C.sub.3-C.sub.6)-alkanediyl or
for a group of the formula
##STR00250## [2217] wherein [2218] p stands for a number of 2 or 3,
[2219] ##.sup.3 denotes the linkage site to the group B, [2220]
##.sup.4 denotes the linkage site to the nitrogen atom,
[2221] and
[2222] D and R.sup.35 have the meanings given above
[2223] as well as their salts and solvates as well as the solvates
of the salts.
[2224] Also preferred within the scope of the present invention are
compounds of formula (Ia), in which
[2225] n stands for a number from 2 to 8, preferably 2 to 5,
[2226] AK stands for AK.sub.1 or AK.sub.2 [2227] wherein [2228]
AK.sub.1 stands for an antibody or an antigen binding antibody
fragment which binds to C4.4a and is bound to the group G via the
sulfur atom of a cysteine radical of the binder, [2229] AK.sub.2
stands for an antibody or an antigen binding antibody fragment
which binds to C4.4a and is bound to the group G via the NH side
group of a lysine radical of the binder,
[2230] G for the case when AK=AK.sub.1 stands for a group of the
formula
##STR00251## [2231] wherein [2232] #.sup.1 denotes the linkage site
to the cysteine radical of the binder, [2233] #.sup.2 denotes the
linkage site to the group L.sup.1, [2234] or [2235] for the case
when AK=AK.sub.2, G stands for carbonyl,
[2236] L.sup.1 stands for a bond,
[2237] B stands for a bond,
[2238] L.sup.2 stands for linear (C.sub.3-C.sub.6)-alkanediyl or
for a group of the formula
##STR00252## [2239] wherein [2240] p stands for a number of 2 or 3,
[2241] ##.sup.3 denotes the linkage site to the group B, [2242]
##.sup.4 denotes the linkage site to the nitrogen atom,
[2243] D and R.sup.35 have the meanings given above
[2244] as well as their salts and solvates as well as the solvates
of the salts.
[2245] Also preferred within the scope of the present invention are
compounds of formula (Ia), in which
[2246] n stands for a number from 2 to 8, preferably 2 to 5,
[2247] AK stands for AK.sub.1, [2248] wherein [2249] AK.sub.1
stands for an antibody or an antigen binding antibody fragment
which binds to C4.4a and is bound to the group G via the sulfur
atom of a cysteine radical of the binder,
[2250] G stands for a group of the formula
##STR00253## [2251] wherein [2252] #.sup.1 denotes the linkage site
to the cysteine radical of the binder, [2253] #.sup.2 denotes the
linkage site to the group L.sup.1,
[2254] L.sup.1 stands for linear (C.sub.3-C.sub.5)-alkanediyl or
for a group of the formula
##STR00254## [2255] wherein [2256] m stands for a number of 2 or 3,
[2257] ##.sup.1 denotes the linkage site to the group G, [2258]
##.sup.2 denotes the linkage site to the group B, [2259] wherein
(C.sub.3-C.sub.5)-alkanediyl may be substituted with one or two
methyl substituents,
[2260] B stands for a group of the formula
##STR00255## [2261] wherein [2262] * denotes the linkage site to
L.sup.1, [2263] ** denotes the linkage site to L.sup.2, [2264]
L.sup.3 stands for a bond, [2265] L.sup.4 stands for a bond [2266]
R.sup.16 stands for hydrogen, [2267] R.sup.17 stands for
hydrogen,
[2268] L.sup.2 stands for linear (C.sub.3-C.sub.6)-alkanediyl or
for a group of the formula
##STR00256## [2269] wherein [2270] p stands for a number of 2 or 3,
[2271] ##.sup.3 denotes the linkage site to the group B, [2272]
##.sup.4 denotes the linkage site to the nitrogen atom,
[2273] D and R.sup.35 have the meanings given above
[2274] as well as their salts and solvates as well as the solvates
of the salts.
[2275] Also preferred within the scope of the present invention are
compounds of formula (Ia), in which
[2276] L.sup.1 stands for a bond, linear
(C.sub.3-C.sub.5)-alkanediyl, a group of the formula
##STR00257## [2277] wherein [2278] m stands for a number of 2 or 3,
[2279] ##.sup.1 denotes the linkage site to the group G, [2280]
##.sup.2 denotes the linkage site to the group B, [2281] L.sup.1A
stands for linear (C.sub.2-C.sub.6)-alkanediyl, [2282] B.sup.1
stands for a group of the formula
[2282] ##STR00258## [2283] wherein [2284] ##.sup.5 denotes the
linkage site to the group L.sup.1A, [2285] ##.sup.6 denotes the
linkage site to the group L.sup.1B, [2286] L.sup.5 stands for a
bond or ethane-1,2-diyl, [2287] L.sup.6 stands for a bond or a
group of the formula
[2287] ##STR00259## [2288] wherein [2289] ##.sup.7 denotes the
linkage site to the carbonyl group, [2290] ##.sup.8 denotes the
linkage site to L.sup.1B, [2291] R.sup.33 stands for hydrogen,
(C.sub.1-C.sub.4)-alkylcarbonyl or tert-butyloxy-carbonyl, [2292]
R.sup.34 stands for hydrogen or methyl, [2293] R.sup.29 stands for
hydrogen or methyl, [2294] R.sup.30 stands for hydrogen or methyl,
[2295] R.sup.31 stands for hydrogen or methyl, [2296] R.sup.32
stands for hydrogen or methyl, [2297] L.sup.1B stands for linear
(C.sub.3-C.sub.6)-alkanediyl,
[2298] B stands for a bond or a group of the formula
##STR00260## [2299] wherein [2300] * denotes the linkage site to
L.sup.1, [2301] ** denotes the linkage site to L.sup.2, [2302] P
stands for O, [2303] L.sup.3 stands for a bond or ethane-1,2-diyl,
[2304] L.sup.4 stands for a group of the formula
[2304] ##STR00261## [2305] wherein [2306] *** denotes the linkage
site to the carbonyl group, [2307] **** denotes the linkage site to
L.sup.2, [2308] R.sup.25 stands for hydrogen or methyl, [2309]
R.sup.28 stands for hydrogen, (C.sub.1-C.sub.4)-alkylcarbonyl or
tert-butyloxycarbonyl, [2310] Q.sup.1 stands for a four- to
seven-membered heterocycle, [2311] Q.sup.2 stands for a three- to
seven-membered carbocycle or a four- to seven-membered heterocycle,
[2312] R.sup.16 stands for hydrogen or methyl, [2313] R.sup.17
stands for hydrogen or methyl, [2314] R.sup.23 stands for
(C.sub.1-C.sub.4)-alkyl, [2315] R.sup.24 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [2316] R.sup.36 stands for hydrogen,
(C.sub.1-C.sub.4)-alkylcarbonyl or tert-butyloxycarbonyl, [2317]
R.sup.37 stands for hydrogen or methyl, [2318] or [2319] R.sup.36
and R.sup.37 together with the atoms to which they are bound form a
pyrrolidine ring,
[2320] L.sup.2 stands for linear (C.sub.2-C.sub.6)-alkanediyl or
for a group of the formula
##STR00262## [2321] wherein [2322] p stands for a number of 2 or 3,
[2323] ##.sup.3 denotes the linkage site to the group B, [2324]
##.sup.4 denotes the linkage site to the nitrogen atom,
[2325] as well as their salts and solvates as well as the solvates
of the salts.
[2326] Also preferred within the scope of the present invention are
compounds of formula (Ia), in which
[2327] L.sup.1 stands for linear (C.sub.3-C.sub.5)-alkanediyl or
for a group of the formula
##STR00263## [2328] wherein [2329] m stands for a number of 2 or 3,
[2330] ##.sup.1 denotes the linkage site to the group G, [2331]
##.sup.2 denotes the linkage site to the group B,
[2332] B stands for a group of the formula
##STR00264## [2333] wherein [2334] * denotes the linkage site to
L.sup.1, [2335] ** denotes the linkage site to L.sup.2, [2336]
L.sup.3 stands for a bond or ethane-1,2-diyl, [2337] L.sup.4 stands
for a group of the formula
[2337] ##STR00265## [2338] wherein [2339] *** denotes the linkage
site to the carbonyl group, [2340] **** denotes the linkage site to
L.sup.2, [2341] R.sup.25 stands for methyl, [2342] R.sup.28 stands
for hydrogen, methylcarbonyl or tert-butyloxycarbonyl, [2343]
Q.sup.1 stands for piperidine-1,4-diyl, [2344] R.sup.16 stands for
hydrogen or methyl, [2345] R.sup.17 stands for hydrogen or methyl,
[2346] R.sup.23 stands for methyl, [2347] R.sup.24 stands for
hydrogen, [2348] R.sup.36 stands for hydrogen, methylcarbonyl or
tert-butyloxycarbonyl, [2349] R.sup.37 stands for hydrogen or
methyl,
[2350] L.sup.2 stands for linear (C.sub.2-C.sub.6)-alkanediyl or
for a group of the formula
##STR00266## [2351] wherein [2352] p stands for a number of 2 or 3,
[2353] ##.sup.3 denotes the linkage site to the group B, [2354]
##.sup.4 denotes the linkage site to the nitrogen atom,
[2355] as well as their salts and solvates as well as the solvates
of the salts.
[2356] Also preferred within the scope of the present invention are
compounds of formula (Ia), (XXXa) and (XXXI), in which
[2357] D stands for a group of the formula
##STR00267## [2358] wherein [2359] #.sup.3 denotes the linkage site
to the nitrogen atom, [2360] R.sup.1 stands for hydrogen or methyl,
[2361] R.sup.2 stands for isopropyl, isobutyl, sec-butyl,
tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl,
4-hydroxy-3-nitrobenzyl, 4-hydroxy-1-3-aminobenzyl, 1-phenylethyl,
diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
[2362] or [2363] R.sup.1 and R.sup.2 together with the carbon atom
to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[2363] ##STR00268## [2364] wherein [2365] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [2366] #.sup.5 denotes
the linkage site to the carbonyl group, [2367] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[2367] ##STR00269## [2368] wherein [2369] #.sup.6 denotes the
linkage site to the carbonyl group, [2370] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [2371] R.sup.3 stands for hydrogen
or methyl, [2372] R.sup.4 stands for isopropyl, isobutyl,
sec-butyl, tert-butyl, phenyl, benzyl, 1-hydroxyethyl,
4-hydroxybenzyl, 4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl,
1-phenylethyl, diphenylmethyl, 1H-imidazol-4-ylmethyl or
1H-indol-3-ylmethyl, [2373] or [2374] R.sup.3 and R.sup.4 together
with the carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[2374] ##STR00270## [2375] wherein [2376] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [2377] #.sup.8 denotes
the linkage site to the group T.sup.1, [2378] T.sup.1 stands for a
group of the formula C(.dbd.O) OR.sup.7, C(.dbd.O) NR.sup.8R.sup.9,
C(.dbd.O) NH--NH--R.sup.10 or --CH.sub.2--O--R.sup.11, [2379]
wherein [2380] R.sup.7 stands for hydrogen, methyl, ethyl,
n-propyl, tert-butyl, benzyl or adamantylmethyl, [2381] R.sup.8
stands for hydrogen or methyl, [2382] R.sup.9 stands for hydrogen,
methyl, ethyl, n-propyl or benzyl, [2383] or [2384] R.sup.8 and
R.sup.9 together with the nitrogen atom to which they are bound
form a four- to seven-membered heterocycle, [2385] R.sup.10 stands
for benzoyl, [2386] R.sup.11 stands for benzyl, which may be
substituted in the phenyl group with methoxycarbonyl or carboxyl,
[2387] R.sup.5 stands for hydrogen, methyl or a group of the
formula
[2387] ##STR00271## [2388] wherein [2389] #.sup.9 denotes the
linkage site to --CHC(R.sup.26)-T.sup.2, [2390] R.sup.12 stands for
phenyl, which may be substituted with methoxycarbonyl, carboxyl or
a group of the formula --S(O).sub.2OH, [2391] R.sup.13 stands for
phenyl, which may be substituted with methoxycarbonyl or carboxyl,
[2392] R.sup.26 stands for hydrogen, [2393] T.sup.2 stands for
phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
[2394] and
[2395] n, AK, Cys, G, L.sup.1, B, L.sup.2, D and R.sup.35 have the
meanings given above
[2396] as well as their salts and solvates as well as the solvates
of the salts.
[2397] Also preferred within the scope of the present invention are
compounds of formula (Ia), (XXXa) and (XXXI), in which
[2398] D stands for a group of the formula
##STR00272## [2399] wherein [2400] #.sup.3 denotes the linkage site
to the nitrogen atom, [2401] R.sup.1 stands for hydrogen, [2402]
R.sup.2 stands for benzyl, 4-hydroxybenyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [2403] the ring A with the N--O group
contained in it stands for a heterocycle of the formula
[2403] ##STR00273## [2404] wherein [2405] #.sup.6 denotes the
linkage site to the carbonyl group, [2406] R.sup.3 stands for
hydrogen, [2407] R.sup.4 stands for benzyl, 4-hydroxybenzyl,
1-phenylethyl or 1H-indol-3-ylmethyl, [2408] T.sup.1 stands for a
group of the formula --C(.dbd.O)--OR.sup.7 or
--C(.dbd.O)--NR.sup.8R.sup.9, [2409] wherein [2410] R.sup.1 stands
for hydrogen, [2411] R.sup.8 stands for hydrogen, [2412] R.sup.9
stands for hydrogen,
[2413] n, AK, Cys, G, B, L.sup.2, D and R.sup.35 have the meanings
given above
[2414] as well as their salts and solvates as well as the solvates
of the salts.
[2415] Also preferred within the scope of the present invention are
compounds of formula (Ia), (XXXa) and (XXXI), in which
[2416] D stands for a group of the formula
##STR00274## [2417] wherein [2418] #.sup.3 denotes the linkage site
to the nitrogen atom, [2419] R.sup.1 stands for hydrogen, [2420]
R.sup.2 stands for benzyl, 4-hydroxybenzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [2421] the ring A with the N--O group
contained in it stands for a heterocycle of the formula
[2421] ##STR00275## [2422] wherein [2423] #.sup.6 denotes the
linkage site to the carbonyl group, [2424] R.sup.3 stands for
hydrogen, [2425] R.sup.4 stands for benzyl, 4-hydroxybenzyl,
1-phenylethyl or 1H-indol-3-ylmethyl, [2426] T.sup.1 stands for a
group of the formula --C(.dbd.O)--NR.sup.8R.sup.9, [2427] wherein
[2428] R.sup.8 stands for hydrogen, [2429] R.sup.9 stands for
hydrogen,
[2430] n, AK, Cys, G, L.sup.1, B, L.sup.2, D and R.sup.35 have the
meanings given above
[2431] as well as their salts and solvates as well as the solvates
of the salts.
[2432] Also preferred within the scope of the present invention are
compounds of formula (Ia), (XXXa) and (XXXI), in which
[2433] D stands for a group of the formula
##STR00276## [2434] wherein [2435] #.sup.3 denotes the linkage site
to the nitrogen atom, [2436] R.sup.1 stands for hydrogen, [2437]
R.sup.2 stands for 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [2438]
the ring A with the N--O group contained in it stands for a
heterocycle of the formula
[2438] ##STR00277## [2439] wherein [2440] #.sup.6 denotes the
linkage site to the carbonyl group, [2441] R.sup.3 stands for
hydrogen, [2442] R.sup.4 stands for 4-hydroxybenzyl or
1H-indol-3-ylmethyl, [2443] T.sup.1 stands for a group of the
formula --C(.dbd.O)--NR.sup.8R.sup.9, [2444] wherein [2445] R.sup.8
stands for hydrogen, [2446] R.sup.9 stands for hydrogen,
[2447] n, AK, Cys, G, B, L.sup.2, D and R.sup.35 have the meanings
given above
[2448] as well as their salts and solvates as well as the solvates
of the salts.
[2449] Also preferred within the scope of the present invention are
compounds of formula (Ia), (XXXa) and (XXXI), in which
[2450] D stands for a group of the formula
##STR00278## [2451] wherein [2452] #.sup.3 denotes the linkage site
to the nitrogen atom, [2453] R.sup.3 stands for hydrogen, [2454]
R.sup.4 stands for 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [2455]
T.sup.1 stands for a group of the formula --C(.dbd.O)--OR.sup.7 or
--C(.dbd.O)--NR.sup.8R.sup.9, [2456] wherein [2457] R.sup.1 stands
for hydrogen, [2458] R.sup.8 stands for hydrogen, [2459] R.sup.9
stands for hydrogen,
[2460] n, AK, Cys, G, B, L.sup.2, D and R.sup.35 have the meanings
given above
[2461] as well as their salts and solvates as well as the solvates
of the salts.
[2462] Also preferred within the scope of the present invention are
compounds of formula (Ia), (XXXa) and (XXXI), in which
[2463] D stands for a group of the formula
##STR00279## [2464] wherein [2465] #.sup.3 denotes the linkage site
to the nitrogen atom, [2466] R.sup.3 stands for hydrogen, [2467]
R.sup.4 stands for 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [2468]
T.sup.1 stands for a group of the formula
--C(.dbd.O)--NR.sup.8R.sup.9, [2469] wherein [2470] R.sup.8 stands
for hydrogen, [2471] R.sup.9 stands for hydrogen,
[2472] n, AK, Cys, G, B, L.sup.2, D and R.sup.35 have the meanings
given above
[2473] as well as their salts and solvates as well as the solvates
of the salts.
[2474] Also preferred within the scope of the present invention are
compounds of formula (Ia), (XXXa) and (XXXI), in which
[2475] D stands for a group of the formula
##STR00280## [2476] wherein [2477] #.sup.3 denotes the linkage site
to the nitrogen atom, [2478] R.sup.1 stands for hydrogen or methyl,
[2479] R.sup.2 stands for isopropyl, isobutyl, sec-butyl,
tert-butyl, phenyl, benzyl, 1-hydroxyethyl, 4-hydroxybenzyl,
4-hydroxy-3-nitrobenzyl, 4-hydroxy-3-aminobenzyl, 1-phenylethyl,
diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
[2480] or [2481] R.sup.1 and R.sup.2 together with the carbon atom
to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[2481] ##STR00281## [2482] wherein [2483] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [2484] #.sup.5 denotes
the linkage site to the carbonyl group, [2485] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[2485] ##STR00282## [2486] wherein [2487] #.sup.6 denotes the
linkage site to the carbonyl group, [2488] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy,
[2489] and
[2490] n, AK, Cys, G, L.sup.1, B, L.sup.2 and R.sup.35 have the
meanings given above
[2491] as well as their salts and solvates as well as the solvates
of the salts.
[2492] Also preferred within the scope of the present invention are
compounds of formula (Ia), (XXXa) and (XXXI), in which
[2493] D stands for a group of the formula
##STR00283## [2494] wherein [2495] #.sup.3 denotes the linkage site
to the nitrogen atom, [2496] R.sup.1 stands for hydrogen, [2497]
R.sup.2 stands for benzyl, 4-hydroxybenzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [2498] or [2499] R.sup.1 and R.sup.2 together
with the carbon atom to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[2499] ##STR00284## [2500] wherein [2501] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [2502] #.sup.5 denotes
the linkage site to the carbonyl group, [2503] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[2503] ##STR00285## [2504] wherein [2505] #.sup.6 denotes the
linkage site to the carbonyl group,
[2506] and
[2507] n, AK, Cys, G, L.sup.1, B, L.sup.2 and R.sup.35 have the
meanings given above
[2508] as well as their salts and solvates as well as the solvates
of the salts.
[2509] Also preferred within the scope of the present invention are
compounds of formula (Ia), (XXXa) and (XXXI), in which
[2510] R.sup.35 stands for hydroxyl,
[2511] and
[2512] n, AK, Cys, G, L.sup.1, B, L.sup.2, D and R.sup.35 have the
meanings given above
[2513] as well as their salts and solvates as well as the solvates
of the salts.
[2514] Also preferred within the scope of the present invention are
compounds of formula (Ia), (XXXa) and (XXXI), in which
[2515] R.sup.35 stands for methyl,
[2516] and
[2517] n, AK, Cys, G, L.sup.1, B, L.sup.2, D and R.sup.35 have the
meanings given above
[2518] as well as their salts and solvates as well as the solvates
of the salts.
[2519] Also especially preferred within the scope of the present
invention are compounds of formula (XXXa), in which [2520] Cys
stands for a L-cysteine radical which is bound to a carbon atom of
the succinimide via the sulfur atom of the side chain,
[2521] as well as their salts and solvates as well as the solvates
of the salts.
[2522] Also preferred within the scope of the present invention are
compounds of formula (I) and (XXX), in which
[2523] D stands for a group of the formula
##STR00286## [2524] wherein [2525] #.sup.3 denotes the linkage site
to the nitrogen atom, [2526] R.sup.1 stands for hydrogen, [2527]
R.sup.2 stands for benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
[2528] or [2529] R.sup.1 and R.sup.2 together with the carbon atom
to which they are attached form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[2529] ##STR00287## [2530] wherein [2531] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [2532] #.sup.5 denotes
the linkage site to the carbonyl group, [2533] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[2533] ##STR00288## [2534] wherein [2535] #.sup.6 denotes the
linkage site to the carbonyl group, [2536] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [2537] n, AK, Cys, G, L.sup.1,
L.sup.2 and B have the meanings given above
[2538] as well as their salts and solvates as well as the solvates
of the salts.
[2539] Also especially preferred within the scope of the present
invention are compounds of formula (Ia) and (XXXa), in which
[2540] D stands for a group of the formula
##STR00289## [2541] wherein [2542] #.sup.3 denotes the linkage site
to the nitrogen atom, [2543] R.sup.1 stands for hydrogen, [2544]
R.sup.2 stands for benzyl or 1H-indol-3-ylmethyl, [2545] or [2546]
R.sup.1 and R.sup.2 together with the carbon atom to which they are
attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[2546] ##STR00290## [2547] wherein [2548] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [2549] #.sup.5 denotes
the linkage site to the carbonyl group, [2550] the ring A with the
N--O group contained in it stands for a heterocycle of the
formula
[2550] ##STR00291## [2551] wherein [2552] #.sup.6 denotes the
linkage site to the carbonyl group, [2553] n, AK, Cys, G, L.sup.1,
L.sup.2 and B have the meanings given above
[2554] as well as their salts and solvates as well as the solvates
of the salts.
[2555] Another especially preferred subject matter of the present
invention is compounds of formula (I), in which
[2556] D stands for a group of the formula
##STR00292## [2557] wherein [2558] T.sup.1 stands for
--C(.dbd.O)--OH or --C(.dbd.O)--NH.sub.2 and
[2559] n, AK, G, L.sup.1, B, L.sup.2, #.sup.3, R.sup.3 and R.sup.4
have the meanings given above.
[2560] Also preferred within the scope of the present invention are
compounds of formula (I), in which
[2561] n=1-20, especially preferably n=1-10 and most especially
preferably n=2-8.
[2562] Also preferred within the scope of the present invention are
compounds of formula (Ia) and (XXX), in which
[2563] B stands for a bond or a group of the formula
##STR00293## [2564] wherein [2565] * denotes the linkage site to
L.sup.1, [2566] ** denotes the linkage site to L.sup.2, [2567]
L.sup.3 stands for a bond or ethane-1,2-diyl, [2568] L.sup.4 stands
for a bond, [2569] n, AK, Cys, G, L.sup.1, L.sup.2, D, R.sup.16 and
R.sup.47 have the meanings given above
[2570] as well as their salts and solvates as well as the solvates
of the salts.
[2571] Especially preferred within the scope of the present
invention are compounds of formula (I) and (XXX), in which
[2572] B stands for a bond or a group of the formula
##STR00294## [2573] wherein [2574] ** denotes the linkage site to
L.sup.1, [2575] * denotes the linkage site to L.sup.2, [2576]
L.sup.3 and L.sup.4 stands for a bond, [2577] n, AK, Cys, G,
L.sup.1, L.sup.2, D, R.sup.16 and R.sup.47 have the meanings given
above
[2578] as well as their salts and solvates as well as the solvates
of the salts.
[2579] Also preferred within the scope of the present invention are
binder-drug conjugates of general formula (I), in which
[2580] AK stands for AK.sub.1 [2581] wherein [2582] AK.sub.1 stands
for a binder that is bound to the group G via a sulfur atom of the
binder, preferably a chimeric, humanized or human antibody,
especially preferably an anti-EGFR antibody,
[2583] G stands for a group of the formula
##STR00295## [2584] wherein [2585] #.sup.1 denotes the linkage site
to the cysteine radical of the binder, [2586] #.sup.2 denotes the
linkage site to the group L.sup.1,
[2587] L.sup.1 stands for a bond, linear
(C.sub.1-C.sub.10)-alkanediyl or for a group of the formula
##STR00296## [2588] wherein [2589] m stands for a number from 2 to
6, [2590] ##.sup.1 denotes the linkage site to the group G, [2591]
##.sup.2 denotes the linkage site to the group B, [2592] wherein
(C.sub.1-C.sub.10)-alkanediyl may be substituted with 1 to 4 methyl
substituents, [2593] and [2594] wherein two carbon atoms of the
alkanediyl chain in 1,2-, 1,3- or 1,4- relation to one another to
form a (C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring,
including the carbon atoms optionally situated between them,
[2595] B stands for a bond or a group of the formula
##STR00297## [2596] wherein [2597] * denotes the linkage site to
L.sup.1, [2598] ** denotes the linkage site to L.sup.2, [2599]
L.sup.3 for a bond or (C.sub.2-C.sub.4)-alkanediyl, [2600] L.sup.4
stands for a bond or a group of the formula
[2600] ##STR00298## [2601] wherein [2602] *** denotes the linkage
site to the carbonyl group, [2603] **** denotes the linkage site to
L.sup.2, [2604] R.sup.25 stands for hydrogen or methyl, [2605]
Q.sup.1 stands for a four- to seven-membered heterocycle, [2606]
R.sup.14 stands for hydrogen or (C.sub.1-C.sub.4)-alkyl, [2607]
R.sup.15 stands for hydrogen or (C.sub.1-C.sub.4)-alkyl, [2608] or
[2609] R.sup.14 and R.sup.15 together with the atoms to which they
are bound form a five- or six-membered heterocycle, [2610] R.sup.16
stands for hydrogen or (C.sub.1-C.sub.4)-alkyl, [2611] R.sup.17
stands for hydrogen or (C.sub.1-C.sub.4)-alkyl, [2612] or [2613]
R.sup.16 and R.sup.17 together with the atoms to which they are
bound form a five- or six-membered heterocycle,
[2614] L.sup.2 stands for linear (C.sub.2-C.sub.10)-alkanediyl or
for a group of the formula
##STR00299## [2615] wherein [2616] p stands for a number from 2 to
6, [2617] ##.sup.3 denotes the linkage site to the group B, [2618]
##.sup.4 denotes the linkage site to the nitrogen atom, [2619]
wherein (C.sub.2-C.sub.10)-alkanediyl may be substituted with 1 to
4 methyl substituents,
[2620] and [2621] wherein two carbon atoms of the alkanediyl chain
may be bridged in 1,2-, 1,3- or 1,4- relation to one another,
including the carbon atoms optionally present between them to form
a (C.sub.3-C.sub.6) cycloalkyl ring or a phenyl ring,
[2622] as well as their salts and solvates as well as the solvates
of the salts.
[2623] Also preferred within the scope of the present invention are
binder-drug conjugates of general formula (I), in which
[2624] AK stands for AK.sub.2 [2625] wherein [2626] AK.sub.2 stands
for a binder that is bound to the group G via the NH side group of
a lysine radical of the binder,
[2627] G stands for carbonyl,
[2628] L.sup.1 stands for a bond, linear
(C.sub.1-C.sub.10)-alkanediyl, a group of the formula
##STR00300## [2629] wherein [2630] m stands for a number from 2 to
6, [2631] ##.sup.1 denotes the linkage site to the group G, [2632]
##.sup.2 denotes the linkage site to the group B, [2633] wherein
(C.sub.1-C.sub.10)-alkanediyl may be substituted with 1 to 4 methyl
substituents, [2634] and [2635] wherein two carbon atoms of the
alkanediyl chain in 1,2-, 1,3- or 1,4- relation to one another to
form a (C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring,
including the carbon atoms optionally situated between them,
[2636] B stands for a bond or a group of the formula
##STR00301## [2637] wherein [2638] * denotes the linkage site to
L.sup.1, [2639] ** denotes the linkage site to L.sup.2, [2640] P
stands for O or NH, [2641] Q.sup.2 stands for a three- to
seven-membered carbocycle or a four- to seven-membered heterocycle,
[2642] R.sup.18 stands for hydrogen or (C.sub.1-C.sub.4)-alkyl,
[2643] R.sup.19 stands for hydrogen or the side group of a natural
.alpha.-amino acid or its homologs or isomers, [2644] R.sup.20
stands for hydrogen or (C.sub.1-C.sub.4)-alkyl, [2645] or [2646]
R.sup.19 and R.sup.20 together with the atoms to which they are
bound form a pyrrolidinyl ring, [2647] R.sup.21 stands for hydrogen
or (C.sub.1-C.sub.4)-alkyl, [2648] R.sup.22 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl, [2649] or [2650] R.sup.21 and R.sup.22
together with the atoms to which they are bound form a three- to
seven-membered carbocycle, [2651] R.sup.23 stands for
(C.sub.1-C.sub.4)-alkyl, [2652] R.sup.24 stands for hydrogen or
(C.sub.1-C.sub.4)-alkyl,
[2653] L.sup.2 stands for linear (C.sub.2-C.sub.10)-alkanediyl or
for a group of the formula
##STR00302## [2654] wherein [2655] p stands for a number from 2 to
6, [2656] ##.sup.3 denotes the linkage site to the group B, [2657]
##.sup.4 denotes the linkage site to the nitrogen atom, [2658]
wherein (C.sub.2-C.sub.10)-alkanediyl may be substituted with 1 to
4 methyl substituents, [2659] and [2660] wherein two carbon atoms
of the alkanediyl chain may be bridged in 1,2-, 1,3- or 1,4-
relation to one another, including the carbon atoms optionally
present between them to form a (C.sub.3-C.sub.6) cycloalkyl ring or
a phenyl ring,
[2661] as well as their salts and solvates as well as the solvates
of the salts.
[2662] The preferred subject matter of the present invention are
binder-drug conjugates of general formula (Ia) as indicated above,
in which [2663] n stands for a number from 1 to 20, [2664] AK
stands for AK.sub.1 or AK.sub.2 [2665] wherein [2666] AK.sub.1
stands for a binder that is bound to the group G via a sulfur atom
of the binder, preferably a chimeric, humanized or human antibody,
especially preferably an anti-EGFR antibody, [2667] AK.sub.2 stands
for a binder that is bound to the group G via a nitrogen atom of
the binder, preferably a chimeric, humanized or human antibody,
especially preferably an anti-EGFR antibody,
[2668] G for the case when AK=AK.sub.1 stands for a group of the
formula
##STR00303## [2669] wherein [2670] #.sup.1 denotes the linkage site
to the cysteine radical of the binder, [2671] #.sup.2 denotes the
linkage site to the group L.sup.1, [2672] or [2673] for the case
when AK=AK.sub.2, G stands for carbonyl,
[2674] L.sup.1 stands for a bond, linear
(C.sub.2-C.sub.6)-alkanediyl, a group of the formula
##STR00304## [2675] wherein [2676] m stands for a number from 2 to
6, [2677] ##.sup.1 denotes the linkage site to the group G, [2678]
##.sup.2 denotes the linkage site to the group B, [2679] L.sup.1A
stands for linear (C.sub.2-C.sub.6)-alkanediyl, [2680] B.sup.1
stands for a group of the formula
[2680] ##STR00305## [2681] wherein [2682] ##.sup.5 denotes the
linkage site to the group L.sup.1A, [2683] ##.sup.6 denotes the
linkage site to the group L.sup.1B, [2684] L.sup.5 stands for a
bond, [2685] L.sup.6 stands for a bond or a group with the
formula
[2685] ##STR00306## [2686] wherein [2687] ##.sup.7 denotes the
linkage site to the carbonyl group, [2688] ##.sup.8 denotes the
linkage site to L.sup.1B, [2689] R.sup.33 stands for hydrogen,
methylcarbonyl or tert-butyloxycarbonyl, [2690] R.sup.34 stands for
hydrogen or methyl, [2691] R.sup.29 stands for hydrogen, [2692]
R.sup.30 stands for hydrogen, [2693] R.sup.31 stands for hydrogen
or methyl, [2694] R.sup.32 stands for hydrogen or methyl, [2695]
L.sup.1B stands for linear (C.sub.2-C.sub.6)-alkanediyl, [2696] and
[2697] wherein (C.sub.2-C.sub.6)-alkanediyl may be substituted with
one to two methyl substituents,
[2698] B stands for a bond or a group of the formula
##STR00307## [2699] wherein [2700] * denotes the linkage site to
L.sup.1, [2701] ** denotes the linkage site to L.sup.2, [2702]
L.sup.3 stands for a bond or ethane-1,2-diyl, [2703] L.sup.4 stands
for a bond or a group of the formula
[2703] ##STR00308## [2704] wherein [2705] *** denotes the linkage
site to the carbonyl group, [2706] **** denotes the linkage site to
L.sup.2, [2707] R.sup.25 stands for hydrogen or methyl, [2708]
R.sup.28 stands for hydrogen, methylcarbonyl or
tert-butyloxycarbonyl, [2709] Q.sup.1 stands for a four- to
seven-membered heterocycle, [2710] R.sup.14 stands for hydrogen,
[2711] R.sup.15 stands for hydrogen, [2712] R.sup.16 stands for
hydrogen or methyl, [2713] R.sup.17 stands for hydrogen or methyl,
[2714] or [2715] R.sup.16 and R.sup.17 together with the atoms to
which they are bound form a piperazinyl ring, [2716] R.sup.18
stands for hydrogen, [2717] R.sup.19 stands for hydrogen, methyl,
propan-2-yl, 2-methylpropan-1-yl or 1-methyl-propan-1-yl, [2718]
R.sup.20 stands for hydrogen or methyl, [2719] or [2720] R.sup.19
and R.sup.20 together with the atoms to which they are bound form a
pyrrolidinyl ring, [2721] R.sup.21 stands for hydrogen or methyl,
[2722] R.sup.22 stands for hydrogen or methyl, [2723] or [2724]
R.sup.21 and R.sup.22 together with the atoms to which they are
bound form a cyclopropyl ring, [2725] R.sup.23 stands for methyl,
[2726] R.sup.24 stands for hydrogen or methyl, [2727] R.sup.27
stands for hydrogen,
[2728] L.sup.2 stands for linear (C.sub.2-C.sub.6)-alkanediyl or
for a group of the formula
##STR00309## [2729] wherein [2730] p stands for a number from 2 to
6, [2731] ##.sup.3 denotes the linkage site to the group B, [2732]
##.sup.4 denotes the linkage site to the nitrogen atom, [2733]
wherein (C.sub.2-C.sub.10)-alkanediyl may be substituted with one
or two methyl substituents,
[2734] D stands for a group of the formula
##STR00310## [2735] wherein [2736] #.sup.3 denotes the linkage site
to the nitrogen atom, [2737] R.sup.1 stands for hydrogen, [2738]
R.sup.2 stands for 1-hydroxyethyl, benzyl, 4-hydroxybenzyl,
1-phenylethyl or 1H-indol-1-3-ylmethyl, [2739] or [2740] R.sup.1
and R.sup.2 together with the carbon atom to which they are
attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[2740] ##STR00311## [2741] wherein [2742] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [2743] #.sup.5 denotes
the linkage site to the carbonyl group, [2744] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[2744] ##STR00312## [2745] wherein [2746] #.sup.6 denotes the
linkage site to the carbonyl group, [2747] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [2748] R.sup.3 stands for
hydrogen, [2749] R.sup.4 stands for 1-hydroxyethyl, benzyl,
4-hydroxybenzyl, 1-phenylethyl or 1H-indol-1-3-ylmethyl, [2750] or
[2751] R.sup.3 and R.sup.4 together with the carbon atom to which
they are attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl
group of the formula
[2751] ##STR00313## [2752] wherein [2753] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [2754] #.sup.8 denotes
the linkage site to the group [2755] T.sup.1 stands for a group of
the formula --C(.dbd.O)--OR.sup.7, --C(.dbd.O)--NR.sup.8R.sup.9,
--C(.dbd.O)--NH--NH--R.sup.10 or --CH.sub.2--O--R.sup.11, [2756]
wherein [2757] R.sup.7 stands for hydrogen, methyl, ethyl,
n-propyl, tert-butyl, benzyl or adamantylmethyl, [2758] R.sup.8
stands for hydrogen or methyl, [2759] R.sup.9 stands for hydrogen,
methyl, ethyl, n-propyl or benzyl, [2760] or [2761] R.sup.8 and
R.sup.9 together with the nitrogen atom to which they are bound
form a four- to seven-membered heterocycle, [2762] R.sup.10 stands
for benzoyl, [2763] R.sup.11 stands for benzyl, which may be
substituted in the phenyl group with methoxycarbonyl or carboxyl,
[2764] R.sup.5 stands for hydrogen, methyl or a group of the
formula
[2764] ##STR00314## [2765] wherein [2766] #.sup.9 denotes the
linkage site to --CHC(R.sup.26)-T.sup.2, [2767] R.sup.12 stands for
phenyl, which may be substituted with methoxycarbonyl, carboxyl or
a group of the formula --S(O).sub.2OH, [2768] R.sup.13 stands for
phenyl, which may be substituted with methoxycarbonyl or carboxyl,
[2769] R.sup.26 stands for hydrogen or hydroxyl, [2770] T.sup.2
stands for phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
[2771] R.sup.35 stands for methyl or hydroxyl,
[2772] as well as their salts and solvates as well as the solvates
of the salts.
[2773] The preferred subject matter of the present invention is
binder-drug conjugates of general formula (Ia) as indicated above,
in which [2774] n stands for a number from 1 to 10,
[2775] AK stands for AK.sub.1 or AK.sub.2 [2776] wherein [2777]
AK.sub.1 stands for a binder bound to the group G via a sulfur atom
of the binder, [2778] AK.sub.2 stands for a binder bound to the
group G via a nitrogen atom of the binder,
[2779] G for the case when AK=AK.sub.1 stands for a group of the
formula
##STR00315## [2780] wherein [2781] #.sup.1 denotes the linkage site
to the cysteine radical of the binder, [2782] #.sup.2 denotes the
linkage site to the group L.sup.1, [2783] or [2784] for the case
when AK=AK.sub.2, G stands for carbonyl,
[2785] L.sup.1 stands for a bond, linear
(C.sub.2-C.sub.6)-alkanediyl, a group of the formula
##STR00316## [2786] wherein [2787] m stands for a number of 2 or 3,
[2788] ##.sup.1 denotes the linkage site to the group G, [2789]
##.sup.2 denotes the linkage site to the group B, [2790] wherein
(C.sub.2-C.sub.6)-alkanediyl may be substituted with one or two
methyl substituents,
[2791] B stands for a bond or a group of the formula
##STR00317## [2792] wherein [2793] * denotes the linkage site to
L.sup.1, [2794] ** denotes the linkage site to L.sup.2, [2795]
L.sup.3 stands for a bond or ethane-1,2-diyl, [2796] L.sup.4 stands
for a bond or a group of the formula
[2796] ##STR00318## [2797] wherein [2798] *** denotes the linkage
site to the carbonyl group, [2799] **** denotes the linkage site to
L.sup.2, [2800] R.sup.25 stands for methyl, [2801] R.sup.28 stands
for hydrogen, methylcarbonyl or tert-butyloxycarbonyl, [2802]
Q.sup.1 stands for piperidine-1,4-diyl, [2803] R.sup.16 stands for
hydrogen or methyl, [2804] R.sup.17 stands for hydrogen or methyl,
[2805] or [2806] R.sup.16 and R.sup.17 together with the atoms to
which they are bound form a piperazinyl ring, [2807] R.sup.21
stands for hydrogen or methyl, [2808] R.sup.22 stands for hydrogen
or methyl, [2809] or [2810] R.sup.21 and R.sup.22 together with the
atoms to which they are bound form a cyclopropyl ring, [2811]
R.sup.23 stands for methyl, [2812] R.sup.24 stands for
hydrogen,
[2813] L.sup.2 stands for linear (C.sub.2-C.sub.6)-alkanediyl or
for a group of the formula
##STR00319## [2814] wherein [2815] p stands for a number from 2 to
6, [2816] ##.sup.3 denotes the linkage site to the group B, [2817]
##.sup.4 denotes the linkage site to the nitrogen atom,
[2818] D stands for a group of the formula
##STR00320## [2819] wherein [2820] #.sup.3 denotes the linkage site
to the nitrogen atom, [2821] R.sup.1 stands for hydrogen, [2822]
R.sup.2 stands for 1-hydroxyethyl, benzyl, 1-hydroxybenzyl,
1-phenylethyl, or 1H-indol-1-3-ylmethyl, [2823] or [2824] R.sup.1
and R.sup.2 together with the carbon atom to which they are
attached form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[2824] ##STR00321## [2825] wherein [2826] #.sup.4 denotes the
linkage site to the vicinal nitrogen atom, [2827] #.sup.5 denotes
the linkage site to the carbonyl group, [2828] the ring A with the
N--O group contained in it stands for a monocyclic or bicyclic,
optionally substituted heterocycle of the formula
[2828] ##STR00322## [2829] wherein [2830] #.sup.6 denotes the
linkage site to the carbonyl group, [2831] R.sup.6 stands for
hydrogen, hydroxyl or benzyloxy, [2832] R.sup.3 stands for
hydrogen, [2833] R.sup.4 stands for benzyl, 1-hydroxybenzyl,
1-phenylethyl or 1H-indol-3-ylmethyl, [2834] or [2835] R.sup.3 and
R.sup.4 together with the carbon atom to which they are attached
form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[2835] ##STR00323## [2836] wherein [2837] #.sup.7 denotes the
linkage site to the vicinal nitrogen atom, [2838] #.sup.8 denotes
the linkage site to the group T.sup.1, [2839] T.sup.1 stands for a
group of the formula --C(.dbd.O)--OR.sup.7,
--C(.dbd.O)--NR.sup.8R.sup.9 or --CH.sub.2--O--R.sup.11, [2840]
wherein [2841] R.sup.7 stands for hydrogen, methyl, ethyl,
n-propyl, tert-butyl, benzyl or adamantylmethyl, [2842] R.sup.8
stands for hydrogen or methyl, [2843] R.sup.9 stands for hydrogen,
methyl, ethyl, n-propyl or benzyl, [2844] R.sup.11 stands for
benzyl, which may be substituted in the phenyl group with
methoxycarbonyl or carboxyl, [2845] R.sup.5 stands for hydrogen,
methyl or a group of the formula
[2845] ##STR00324## [2846] wherein [2847] #.sup.9 denotes the
linkage site to --CHCH.sub.2-phenyl, [2848] R.sup.12 stands for
phenyl, which may be substituted with methoxycarbonyl, carboxyl or
a group of the formula --S(O).sub.2OH, [2849] R.sup.13 stands for
phenyl, which may be substituted with methoxycarbonyl or carboxyl,
[2850] R.sup.35 stands for methyl or hydroxyl, [2851] as well as
their salts and solvates as well as the solvates of the salts.
[2852] Especially preferred within the scope of the present
invention are binder-drug conjugates of formula (Ia), in which
[2853] n stands for a number from 2 to 8,
[2854] AK stands for AK.sub.1 or AK.sub.2, [2855] wherein [2856]
AK.sub.1 stands for a binder that is bound to the group G via a
sulfur atom of the cysteine radical of the binder, preferably for a
chimeric, humanized or human antibody, especially preferably an
anti-EGFR antibody, [2857] AK.sub.2 stands for a binder that is
bound to the group G via a nitrogen atom of the lysine radical of
the binder, preferably a chimeric, humanized or human antibody,
especially preferably an anti-EGFR antibody,
[2858] G for the case when AK=AK.sub.1 stands for a group of the
formula
##STR00325## [2859] wherein [2860] #.sup.1 denotes the linkage site
to the cysteine radical of the binder, [2861] #.sup.2 denotes the
linkage site to the group L.sup.1, [2862] or [2863] for the case
when AK=AK.sub.2, G stands for carbonyl,
[2864] L.sup.1 stands for a bond, linear
(C.sub.2-C.sub.6)-alkanediyl, a group of the formula
##STR00326## [2865] wherein [2866] m stands for a number of 2 or 3,
[2867] ##.sup.1 denotes the linkage site to the group G, [2868]
##.sup.2 denotes the linkage site to the group B, [2869] wherein
(C.sub.2-C.sub.6)-alkanediyl may be substituted with one or two
methyl substituents,
[2870] B stands for a bond or a group of the formula
##STR00327## [2871] wherein [2872] * denotes the linkage site to
L.sup.1, [2873] ** denotes the linkage site to L.sup.2, [2874]
L.sup.3 stands for a bond or ethane-1,2-diyl, [2875] L.sup.4 stands
for a bond or a group of the formula
[2875] ##STR00328## [2876] wherein [2877] *** denotes the linkage
site to the carbonyl group, [2878] **** denotes the linkage site to
L.sup.2, [2879] R.sup.25 stands for methyl, [2880] R.sup.28 stands
for hydrogen, methylcarbonyl or tert-butyloxycarbonyl, [2881]
Q.sup.1 stands for piperidine-1,4-diyl, [2882] R.sup.16 stands for
hydrogen or methyl, [2883] R.sup.17 stands for hydrogen or methyl,
[2884] or [2885] R.sup.16 and R.sup.17 together with the atoms to
which they are bound form a piperazinyl ring, [2886] R.sup.21
stands for hydrogen or methyl, [2887] R.sup.22 stands for hydrogen
or methyl, [2888] or [2889] R.sup.21 and R.sup.22 together with the
atoms to which they are bound form a cyclopropyl ring, [2890]
R.sup.23 stands for methyl, [2891] R.sup.24 stands for
hydrogen,
[2892] L.sup.2 stands for linear (C.sub.2-C.sub.6)-alkanediyl or
for a group of the formula
##STR00329## [2893] wherein [2894] p stands for a number from 2 to
6, [2895] ##.sup.3 denotes the linkage site to the group B, [2896]
##.sup.4 denotes the linkage site to the nitrogen atom,
[2897] D stands for a group of the formula
##STR00330## [2898] wherein [2899] #.sup.3 denotes the linkage site
to the nitrogen atom, [2900] R.sup.1 stands for hydrogen, [2901]
R.sup.2 stands for 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [2902]
the ring A with the N--O group contained therein stands for
[2902] ##STR00331## [2903] wherein [2904] #.sup.6 denotes the
linkage site to the carbonyl group, [2905] R.sup.3 stands for
hydrogen, [2906] R.sup.4 stands for 4-hydroxybenzyl or
1H-indol-3-ylmethyl, [2907] T.sup.1 stands for a group of the
formula --C(.dbd.O)--NR.sup.8R.sup.9, [2908] R.sup.8 stands for
hydrogen or methyl, [2909] R.sup.9 stands for hydrogen, methyl or
ethyl,
[2910] R.sup.35 stands for methyl,
[2911] as well as their salts and solvates as well as the solvates
of the salts.
[2912] Especially preferred within the scope of the present
invention are binder-drug conjugates of formula (Ia), in which
[2913] n stands for a number from 2 to 8, preferably 2 to 5,
[2914] AK stands for AK.sub.1, [2915] wherein [2916] AK.sub.1
stands for a binder that is bound to the group G via a sulfur atom
of the cysteine radical of the binder, preferably for a chimeric,
humanized or human antibody, especially preferably an anti-EGFR
antibody,
[2917] G stands for a group of the formula
##STR00332## [2918] wherein [2919] #.sup.1 denotes the linkage site
to the cysteine radical of the binder, [2920] #.sup.2 denotes the
linkage site to the group L.sup.1,
[2921] L.sup.1 stands for pentane-1,5-diyl,
[2922] B stands for a group of the formula
##STR00333## [2923] wherein [2924] * denotes the linkage site to
L.sup.1, [2925] ** denotes the linkage site to L.sup.2, [2926]
L.sup.3 stands for a bond, [2927] L.sup.4 stands for a bond, [2928]
R.sup.16 stands for hydrogen, [2929] R.sup.17 stands for
hydrogen,
[2930] L.sup.2 stands for propane-1,3-diyl,
[2931] D stands for a group of the formula
##STR00334## [2932] wherein [2933] #.sup.3 denotes the linkage site
to the nitrogen atom, [2934] R.sup.1 stands for hydrogen, [2935]
R.sup.2 stands for 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [2936]
the ring A with the N--O group contained therein stands for
[2936] ##STR00335## [2937] wherein [2938] #.sup.6 denotes the
linkage site to the carbonyl group, [2939] R.sup.3 stands for
hydrogen, [2940] R.sup.4 stands for 4-hydroxybenzyl or
1H-indol-3-ylmethyl, [2941] T.sup.1 stands for a group of the
formula --C(.dbd.O)--NR.sup.8R.sup.9, [2942] R.sup.8 stands for
hydrogen, [2943] R.sup.9 stands for hydrogen,
[2944] R.sup.35 stands for methyl,
[2945] as well as their salts and solvates as well as the solvates
of the salts.
[2946] Especially preferred within the scope of the present
invention are binder-drug conjugates of formula (Ia), in which
[2947] n stands for a number from 2 to 8, preferably 2 to 5,
[2948] AK stands for AK.sub.1, [2949] wherein [2950] AK.sub.1
stands for a binder that is bound to the group G via a sulfur atom
of the cysteine radical of the binder, preferably for a chimeric,
humanized or human antibody, especially preferably an anti-EGFR
antibody,
[2951] G stands for a group of the formula
##STR00336## [2952] wherein [2953] #.sup.1 denotes the linkage site
to the cysteine radical of the binder, [2954] #.sup.2 denotes the
linkage site to the group L.sup.1,
[2955] L.sup.1 stands for a bond,
[2956] B stands for a bond,
[2957] L.sup.2 stands for hexane-1,6-diyl,
[2958] and D has the meaning given above,
[2959] as well as their salts and solvates as well as the solvates
of the salts.
[2960] Especially preferred within the scope of the present
invention are binder-drug conjugates of formula (Ia), in which
[2961] n stands for a number from 2 to 8, preferably from 2 to
5,
[2962] AK stands for AK.sub.2, [2963] wherein [2964] AK.sub.2
stands for a binder that is bound to the group G via a nitrogen
atom of the binder, preferably a chimeric, humanized or human
antibody, especially preferably an anti-EGFR antibody,
[2965] G stands for carbonyl,
[2966] L.sup.1 stands for a bond,
[2967] B stands for a bond,
[2968] L.sup.2 stands for pentane-1,5-diyl,
[2969] D stands for a group of the formula
##STR00337## [2970] wherein [2971] #.sup.3 denotes the linkage site
to the nitrogen atom, [2972] R.sup.1 stands for hydrogen, [2973]
R.sup.2 stands for 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [2974]
the ring A with the N--O group contained therein stands for
[2974] ##STR00338## [2975] wherein [2976] #.sup.6 denotes the
linkage site to the carbonyl group, [2977] R.sup.3 stands for
hydrogen, [2978] R.sup.4 stands for 4-hydroxybenzyl or
1H-indol-3-ylmethyl, [2979] T.sup.1 stands for a group of the
formula --C(.dbd.O)--NR.sup.8R.sup.9, [2980] R.sup.8 stands for
hydrogen, [2981] R.sup.9 stands for hydrogen,
[2982] R.sup.35 stands for methyl,
[2983] as well as their salts and solvates as well as the solvates
of the salts.
[2984] Especially preferred within the scope of the present
invention are binder-drug conjugates of formula (Ia), in which
[2985] n stands for a number from 2 to 8, preferably 2 to 5,
[2986] AK stands for AK.sub.2, [2987] wherein [2988] AK.sub.2
stands for a binder that is bound to the group G via a nitrogen
atom of the lysine radical of the binder, preferably a chimeric,
humanized or human antibody, especially preferably an anti-EGFR
antibody,
[2989] G stands for carbonyl,
[2990] L.sup.1 stands for a bond,
[2991] B stands for a bond,
[2992] L.sup.2 stands for a group of the formula
##STR00339## [2993] wherein [2994] p stands for the number 3,
[2995] ##.sup.3 denotes the linkage site to the group B, [2996]
##.sup.4 denotes the linkage site to the nitrogen atom,
[2997] and D has the meaning given above,
[2998] as well as their salts and solvates as well as the solvates
of the salts.
[2999] According to the invention, the drug-binder conjugate
preferably comprises the following compounds in particular, where n
stands for a number from 2 to 8, preferably 2 to 8, and AK stands
for a chimeric, human or humanized antibody or an antigen binding
antibody fragment which binds to mesothelin, C4.4a or EGFR:
##STR00340##
[3000] In addition, according to the invention the drug-binder
conjugate is especially preferably selected from the following
compounds:
##STR00341##
[3001] in which
[3002] n stands for a number from 2 to 8, preferably 2 to 5,
[3003] and
[3004] AK.sub.1A, AK.sub.1B, AK.sub.2A, A.sub.K3 and AK.sub.4 stand
for the antibodies indicated.
[3005] AK
[3006] binder-drug conjugate of the following formula Ia
##STR00342##
[3007] wherein
[3008] n stands for a number from 2 to 8;
[3009] AK stands for AK.sub.1 or AK.sub.2 [3010] wherein [3011]
AK.sub.1 stands for a chimeric, human or humanized antibody or an
antigen binding antibody fragment which is bound to mesothelin,
EGFR or C4.4a and is bound to the group G via the sulfur atom of a
cysteine radical of the binder, [3012] AK.sub.2 stands for a
chimeric, human or humanized antibody or an antigen binding
antibody fragment which is bound to mesothelin, EGFR or C4.4a and
is bound to the group G via the NH side group of a lysine radical
of the binder,
[3013] R.sup.35 stands for methyl;
[3014] D stands for a group of the formula
##STR00343##
[3015] wherein
[3016] #.sup.3 denotes the linkage site to the nitrogen atom,
[3017] R.sup.1 stands for hydrogen,
[3018] R.sup.2 stands for 4-hydroxybenzyl or
1H-indol-3-ylmethyl,
[3019] the ring A with the N--O group contained therein stands
for
##STR00344## [3020] wherein [3021] #.sup.6 denotes the linkage site
to the carbonyl group, [3022] R.sup.3 stands for hydrogen, [3023]
R.sup.4 stands for 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [3024]
T.sup.1 stands for a group of the formula
--C(.dbd.O)--NR.sup.8R.sup.9, [3025] R.sup.8 stands for hydrogen or
methyl, [3026] R.sup.9 stands for hydrogen, methyl or ethyl, [3027]
the group .sctn.-G-L1-B-L2-.sctn..sctn. stands for a linker, [3028]
wherein [3029] .sctn. denotes the linkage site to the group AK and
[3030] .sctn..sctn. denotes the linkage site to the nitrogen
atom,
[3031] G for the case when AK=AK.sub.1 stands for a group of the
formula
##STR00345## [3032] wherein [3033] #.sup.1 denotes the linkage site
to the cysteine radical of the binder, [3034] #.sup.2 denotes the
linkage site to the group L.sup.1, [3035] or [3036] for the case
when AK=AK.sub.2, G stands for carbonyl,
[3037] L.sup.1 stands for a bond, linear
(C.sub.2-C.sub.6)-alkanediyl, a group of the formula
##STR00346## [3038] wherein [3039] m stands for a number of 2 or 3,
[3040] ##.sup.1 denotes the linkage site to the group G, [3041]
##.sup.2 denotes the linkage site to the group B, [3042] wherein
(C.sub.2-C.sub.6)-alkanediyl may be substituted with one or two
methyl substituents,
[3043] B stands for a bond or a group of the formula
##STR00347## [3044] wherein [3045] * denotes the linkage site to
L.sup.1, [3046] ** denotes the linkage site to L.sup.2, [3047]
L.sup.3 stands for a bond or ethane-1,2-diyl, [3048] L.sup.4 stands
for a bond or a group of the formula
[3048] ##STR00348## [3049] wherein [3050] *** denotes the linkage
site to the carbonyl group, [3051] **** denotes the linkage site to
L.sup.2, [3052] R.sup.25 stands for methyl, [3053] R.sup.28 stands
for hydrogen, methylcarbonyl or tert-butyloxycarbonyl, [3054]
Q.sup.1 stands for piperidine-1,4-diyl, [3055] R.sup.16 stands for
hydrogen or methyl, [3056] R.sup.17 stands for hydrogen or methyl,
[3057] or [3058] R.sup.16 and R.sup.17 together with the atoms to
which they are bound form a piperazinyl ring, [3059] R.sup.21
stands for hydrogen or methyl, [3060] R.sup.22 stands for hydrogen
or methyl, [3061] or [3062] R.sup.21 and R.sup.22 together with the
atoms to which they are bound form a cyclopropyl ring, [3063]
R.sup.23 stands for methyl, [3064] R.sup.24 stands for
hydrogen,
[3065] L.sup.2 stands for linear (C.sub.2-C.sub.6)-alkanediyl or
for a group of the formula
##STR00349## [3066] wherein [3067] p stands for a number from 2 to
6, [3068] ##.sup.3 denotes the linkage site to the group B, [3069]
##.sup.4 denotes the linkage site to the nitrogen atom,
[3070] as well as their salts and solvates as well as the solvates
of the salts.
[3071] Especially preferred are conjugates of the following
formula,
##STR00350##
[3072] wherein
[3073] n stands for a number from 2 to 8, preferably 2 to 5;
[3074] AK stands for a human or humanized antibody or an antigen
binding antibody fragment which is bound to mesothelin, EGFR or
C4.4a and is bound to the group G via the sulfur atom of a cysteine
radical of the binder,
[3075] X.sub.1 stands for NH.sub.2 or
##STR00351##
[3076] and
[3077] X.sub.2 stands for 4-hydroxybenzyl or
1H-indol-3-ylmethyl.
[3078] When the toxophore is bound to a cysteine radical of the
antibody, the linker
##STR00352##
[3079] may be replaced by the following linker, for example:
##STR00353##
[3080] When the toxophore is bound to an NH group of the lysine
radical of the antibody, the linker may be replaced by the
following:
##STR00354##
##STR00355##
[3081] According to the invention the drug-binder conjugate is
especially comprised of the following compounds, where n stands for
a number from 2 to 8, preferably 2 to 5, and AK stands for a
chimeric, human or humanized antibody or an antigen binding
antibody fragment which binds to mesothelin, EGFR or C4.4a:
##STR00356##
[3082] In these formulas, AK.sub.1F, AK.sub.1B and AK.sub.2B may be
replaced by other chimeric, human or humanized anti-C4.4a
antibodies, anti-EGFR antibodies or anti-mesothelin antibodies.
[3083] The definitions of radicals given in the respective
combinations and/or preferred combinations of radicals in detail
can also be replaced by definitions of radicals of other
combinations independently of the respective combinations of
radicals given.
[3084] Combinations of two or more of the aforementioned preferred
ranges are most especially preferred.
[3085] An additional subject matter of the present invention is a
method for synthesis of the compounds of formula (Ia) according to
the invention, which is characterized in that a solution of the
binder in PBS buffer [3086] [A] is mixed with a suitable reducing
agent such as, for example, dithiothreitol or
tris-(2-carboxyethyl)phosphine hydrochloride and then is reacted
with a compound of formula (IIa)
[3086] ##STR00357## [3087] in which D, L.sup.1, B, L.sup.2 and
R.sup.35 each have the meanings given above, [3088] to form a
compound of formula (I-A)
[3088] ##STR00358## [3089] in which n, AK.sub.1, D, L.sup.1, B,
L.sup.2 and R.sup.35 each have the meanings given above,
[3090] or [3091] [B] reacting it with a compound of formula
(IIIa)
[3091] ##STR00359## [3092] in which D, L.sup.1, B, L.sup.2 and
R.sup.35 each have the meanings given above, [3093] to form a
compound of formula (Ia-B)
[3093] ##STR00360## [3094] in which n, AK.sub.2, D, L.sup.1, B,
L.sup.2 and R.sup.35 each have the meanings given above.
[3095] An additional subject matter of the present invention is a
method for synthesis of the compounds of formula (I) according to
the invention, which is characterized in that a solution of the
binder in PBS buffer [3096] [A] is mixed with a suitable reducing
agent such as, for example, dithiothreitol or
tris-(2-carboxyethyl)phosphine hydrochloride and then is reacted
with a compound of formula (II)
[3096] ##STR00361## [3097] in which D, L.sup.1, B and L.sup.2 each
have the meanings given above, [3098] to form a compound of formula
(I-A)
[3098] ##STR00362## [3099] in which n, AK.sub.1, D, L.sup.1, B and
L.sup.2 each have the meanings given above,
[3100] or [3101] [B] reacting it with a compound of formula
(III)
[3101] ##STR00363## [3102] in which D, L.sup.1, B and L.sup.2 each
have the meanings given above, [3103] to form a compound of formula
(I-B)
[3103] ##STR00364## [3104] in which n, AK.sub.2, D, L.sup.1, B and
L.sup.2 each have the meanings given above.
[3105] Cysteine Coupling:
[3106] Partial reduction of the antibody and subsequent conjugation
of the (partially) reduced antibody with a compound of formula (II)
and/or (IIa) takes place according to methods with which those
skilled in the art are familiar; see, for example, Ducry et. al.,
Bioconj. Chem. 2010, 21, 5 and references therein, Klussman et.
al., Bioconj. Chem. 2004, 15(4), 765-773. The mild reduction of the
antibody by adding 2-6 equivalence of TCEP to the antibody present
in a suitable buffer solution, preferably phosphate buffer, and
stirring for 30-180 minutes at temperatures between 15.degree. C.
and 40.degree. C., preferably at room temperature. Next the
conjugation is performed by adding a solution of a compound of
formula (II) and/or (IIa) in DMSO, acetonitrile or DMF to the
solution of the (partially) reduced antibody in PBS buffer and then
reacting them at a temperature of 0.degree. C. to +40.degree. C.,
in particular from +10.degree. C. to +30.degree. C. for a period of
30 minutes to 6 hours, in particular one to two hours.
[3107] Lysine Coupling:
[3108] First the compounds of formula (III) and/or (IIa) or
comparable activated carboxyl components are synthesized by
traditional methods of peptide chemistry. These compounds are then
dissolved in inert solvents such as DMSO or DMF and added to the
antibody, which is preferably present in phosphate buffer at a
neutral pH. The solution is stirred for 1-16 hours at a temperature
between 15.degree. C. and 40.degree. C., preferably at RT.
[3109] The synthesis processes described above are then illustrated
as an example on the basis of the following schemes (schemes 1 and
2):
##STR00365##
[3110] [a): 1. AK, TCEP, PBS buffer, RT; 2. Addition of maleimide
derivative in DMSO, RT].
##STR00366##
[3111] [a): AK, PBS buffer, RT mixed with activated carboxyl
derivative of the linker-drug components].
[3112] The compounds of formula (II) in which L.sup.1 and B stand
for a bond can be synthesized by reductive amination of a compound
of formula (IV)
##STR00367##
[3113] in which D has the meaning given above
[3114] in an inert solvent with a compound of formula (V)
##STR00368##
[3115] in which [3116] L.sup.2A has the meaning of L.sup.2 as
defined above but is shortened in the alkyl chain length by one
carbon atom, [3117] PG.sup.1 stands for an amino protective group
such as, for example, (9H-fluorene-9-ylmethoxy) carbonyl,
tert-butoxycarbonyl or benzyloxycarbonyl,
[3118] to form a compound of formula (VI)
##STR00369##
[3119] in which D, L.sup.2 and PG.sup.1 have the meanings given
above,
[3120] splitting off the protective group PG.sup.1 from this
compound by methods with which those skilled in the art are
familiar and then reacting the deprotected compound in an inert
solvent in the presence of a suitable base with
methyl-2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate to form a
compound of formula (II-A)
##STR00370##
[3121] in which D and L.sup.2 each have the meanings given
above.
[3122] The compounds of formula (II) in which B stands for a group
of the formula (B.sup.1)
##STR00371##
[3123] in which *, **, R.sup.14 and R.sup.15 each have the meanings
given above,
[3124] can be synthesized by splitting off the protective group
PG.sup.1 from a compound of formula (VI) by methods with which
those skilled in the art are familiar and then reacting the
deprotected compound in an inert solvent in the presence of a
suitable base with a compound of formula (VII)
##STR00372##
[3125] in which L.sup.1 has the meaning given above
[3126] to form a compound of formula (II-B)
##STR00373##
[3127] in which D, L.sup.1 and L.sup.2 each have the meanings given
above.
[3128] The compounds of formula (II) in which B stands for a group
of the formula (B.sup.2)
##STR00374##
[3129] in which *, **, L.sup.3, R.sup.16 and R.sup.17 each have the
meanings given above,
[3130] can be synthesized by reductive amination of a compound of
formula (IV) in an inert solvent with a compound of formula
(VIII)
##STR00375##
[3131] in which [3132] L.sup.2A has the meaning given above for
L.sup.2 but the alkyl chain length has been shortened by one carbon
atom,
[3133] to form a compound of formula (IX)
##STR00376##
[3134] in which D and L.sup.2 have the meanings given above
[3135] and this compound is reacted in an inert solvent in the
presence of a suitable coupling reagent and a suitable base with a
compound of formula (X)
##STR00377##
[3136] in which L.sup.1 and L.sup.3 each have the meanings given
above,
[3137] to form a compound of formula (II-C)
##STR00378##
[3138] in which D, L.sup.1, L.sup.2 and L.sup.3 each have the
meanings given above.
[3139] A compound of formula (II), in which B stands for a group of
the formula (B.sup.3)
##STR00379##
[3140] in which *, **, L.sup.3, R.sup.16 and R.sup.17 each have the
meanings given above and
[3141] L.sup.4A stands for a group of the formula
##STR00380## [3142] wherein [3143] *** denotes the linkage site to
the carbonyl group, [3144] **** denotes the linkage site to
L.sup.2, [3145] R.sup.25 stands for hydrogen or methyl,
[3146] can be synthesized by reacting a compound a compound of
formula (IX) in an inert solvent in the presence of a suitable base
and a suitable coupling reagent with a compound of formula (XI-A)
or (XI-B)
##STR00381##
[3147] in which R.sup.25 and PG.sup.1 each have the meanings given
above and
[3148] PG.sup.2 stands for a suitable carboxyl protective group, in
particular benzyl,
[3149] to form a compound (XII-A) and/or (XII-B)
##STR00382##
[3150] in which D, PG.sup.1, PG.sup.2 and L.sup.2 have the meanings
given above,
[3151] then splitting off the protective group PG.sup.2 from this
compound using methods known to those skilled in the art and
reacting the deprotected compound in an inert solvent in the
presence of a suitable coupling reagent and a suitable base with a
compound of formula (X), and then splitting off the protective
group PG.sup.1 by methods with which those skilled in the art are
familiar to form a compound of formula (II-D-A) and/or (II-D-B)
##STR00383##
[3152] in which D, L.sup.1, L.sup.2 and L.sup.3 have the meanings
given above.
[3153] A compound of formula (II) in which B stands for a group of
the formula (B.sup.4)
##STR00384##
[3154] in which *, ** each have the meanings given above and
[3155] Q.sup.1A stands for an N-linked four- to seven-membered
heterocycle,
[3156] can be synthesized by reacting a compound of formula (IX) in
an inert solvent in the presence of a suitable base and a suitable
coupling reagent with a compound of formula (XXI)
##STR00385##
[3157] in which PG.sup.1 and Q.sup.1A each have the meanings given
above,
[3158] to form a compound of formula (XXII)
##STR00386##
[3159] in which PG.sup.1, Q.sup.1A, D and L.sup.2 have the meanings
given above
[3160] then splitting off the protective group PG.sup.1 from this
compound by methods with which those skilled in the art are
familiar and then reacting the deprotected compound in an inert
solvent in the presence of a suitable coupling reagent and a
suitable base with a compound of formula (XXIII)
##STR00387##
[3161] in which L.sup.1 has the meaning given above
[3162] to form a compound of formula (II-D)
##STR00388##
[3163] in which Q.sup.1A, D, L.sup.1 and L.sup.2 have the meanings
given above.
[3164] The compounds of formula (III), in which L.sup.1 and B stand
for a bond can be synthesized by reacting a compound of formula
(IX) with N-hydroxysuccinimide in an inert solvent in the presence
of a suitable coupling regent and a suitable base to form a
compound of formula (III-A):
##STR00389##
[3165] in which D and L.sup.2 each have the meanings given
above.
[3166] The compounds of formula (III), in which L.sup.1 stands for
a bond and B stands for a group of the formula (B.sup.5A)
##STR00390##
[3167] in which *, ** and P each have the meanings given above
and
[3168] Q.sup.2A stands for a three- to seven-membered
carbocycle,
[3169] can be synthesized by reacting a compound of formula (IX) in
an inert solvent in the presence of a suitable coupling reagent and
a suitable base with a compound of formula (XIII)
##STR00391##
[3170] in which P, Q.sup.2A and PG.sup.2 each have the meanings
given above,
[3171] to form a compound of formula (XIV)
##STR00392##
[3172] in which D, P, Q.sup.2A, L.sup.2 and PG.sup.2 each have the
meanings given above,
[3173] splitting off the protective group PG.sup.2 from this
compound by methods with which those skilled in the art are
familiar and then reacting the deprotected compound in an inert
solvent in the presence of a suitable base with
N-hydroxysuccinimide to form a compound of formula (III-B)
##STR00393##
[3174] in which D, P, Q.sup.2A and L.sup.2 each have the meanings
given above.
[3175] The compounds of formula (III), in which L.sup.1 stands for
a bond and B stands for a group of the formula (B.sup.6)
##STR00394##
[3176] in which *, **, R.sup.18, R.sup.19 and R.sup.20 each have
the meanings given above,
[3177] can be synthesized by reacting a compound of formula (IX) in
an inert solvent in the presence of a suitable coupling reagent and
a suitable base with a compound of formula (XV)
##STR00395##
[3178] in which R.sup.18, R.sup.19, R.sup.20 and PG.sup.2 each have
the meanings given above,
[3179] to form a compound of formula (XVI)
##STR00396##
[3180] in which D, R.sup.18, R.sup.19, R.sup.20, L.sup.2 and
PG.sup.2 each have the meanings given above,
[3181] then splitting off the protective group PG.sup.2 from this
compound by methods with which those skilled in the art are
familiar and then reacting the deprotected compound in an inert
solvent in the presence of a suitable coupling reagent and a
suitable base with N-hydroxysuccinimide to form a compound of
formula (III-C)
##STR00397##
[3182] in which D, R.sup.18, R.sup.19, R.sup.20 and L.sup.2 each
have the meanings given above.
[3183] The compounds of formula (III), in which L.sup.1 stands for
a bond and B stands for a group of formula (B.sup.7)
##STR00398##
[3184] in which *, **, R.sup.21 and R.sup.22 each have the meanings
given above,
[3185] may be synthesized by splitting off the protective group
PG.sup.1 from a compound of formula (VI) by methods with which
those skilled in the art are familiar, and then reacting the
resulting deprotected compound in an inert solvent in the presence
of a suitable base with a compound of formula (XVII)
##STR00399##
[3186] in which R.sup.21 and R.sup.22 each have the meanings given
above,
[3187] to form a compound of formula (III-D)
##STR00400##
[3188] in which D, R.sup.21, R.sup.22 and L.sup.2 each have the
meanings given above.
[3189] The compounds of formula (III) in which B stands for a group
of the formula (B.sup.8)
##STR00401##
[3190] in which *, **, R.sup.23 and R.sup.24 each have the meanings
given above,
[3191] can by synthesized by reacting a compound of formula (IX) in
an inert solvent in the presence of a suitable coupling reagent and
a suitable base with a compound of formula (XVIII)
##STR00402##
[3192] in which R.sup.23, R.sup.24 and PG.sup.1 each have the
meanings given above
[3193] to form a compound of formula (XIX)
##STR00403##
[3194] in which D, R.sup.23, R.sup.24, L.sup.2 and PG.sup.1 each
have the meanings given above,
[3195] splitting off the protective group PG.sup.1 from this
compound by methods with which those skilled in the art are
familiar and then reacting the deprotected compound in an inert
solvent in the presence of a suitable coupling reagent and a
suitable base with a compound of formula (XX)
##STR00404##
[3196] in which
[3197] L.sup.1A stands for linear (C.sub.1-C.sub.10)-alkanediyl or
for a group of the formula
##STR00405## [3198] wherein [3199] m stands for a number from 2 to
6, [3200] ##.sup.1 denotes the linkage site to the group G, [3201]
##.sup.2 denotes the linkage site to the group B, [3202] wherein
(C.sub.1-C.sub.10)-alkanediyl may be substituted with 1 to 4 methyl
substituents, [3203] and [3204] wherein two carbon atoms of the
alkanediyl chain in 1,2-, 1,3- or 1,4- relation to one another may
be bridged to form a (C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl
ring including the carbon atoms optionally situated between
them,
[3205] to form a compound of formula (III-E)
##STR00406##
[3206] in which D, R.sup.23, R.sup.24, L.sup.1A and L.sup.2 each
have the meanings given above.
[3207] The compounds of formula (III), in which B stands for a
group of the formula (B.sup.5B)
##STR00407##
[3208] in which * and ** each have the meanings given above and
[3209] Q.sup.2B stands for a N-linked four- to seven-membered
heterocycle,
[3210] can be synthesized by reacting a compound of formula (IX) in
an inert solvent in the presence of a suitable base and a suitable
coupling reagent with a compound of formula (XXIV
##STR00408##
[3211] in which PG.sup.1 and Q.sup.2B each have the meanings given
above,
[3212] to form a compound of formula (XXV)
##STR00409##
[3213] in which PG.sup.1, Q.sup.2B, D and L.sup.2 have the meanings
given above,
[3214] splitting off the protective group PG.sup.1 from this
compound by methods with which those skilled in the art are
familiar
[3215] and then reacting the deprotected compound in an inert
solvent in the presence of a suitable base with a compound of
formula (XX) to yield a compound of formula (III-F)
##STR00410##
[3216] in which Q.sup.2B, D, L.sup.1A and L.sup.2 have the meanings
given above.
[3217] The reactions (IV)+(V).fwdarw.(VI) and
(IV)+(VIII).fwdarw.(IX) take place in the usual solvents that are
typically used for reductive amination and are inert under the
reaction conditions, optionally in the presence of an acid and/or a
water extracting agent as the catalyst. Such solvents include, for
example, alcohols such as methanol, ethanol, n-propanol,
isopropanol, n-butanol or tert-butanol, ethers such as
tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane or
bis-(2-methoxyethyl)ether or other solvents such as
dichloromethane, 1,2-dichloroethane, N,N-dimethyl formamide and
water. It is also possible to use mixtures of these solvents. The
preferred solvent is a 1,4-dioxane/water mixture that is used with
the addition of acetic acid or dilute hydrochloric acid as the
catalyst.
[3218] Complex borohydrides in particular are suitable reducing
agents for this reaction, such as, for example, sodium borohydride,
sodium cyanoborohydride, sodium triacetoxyborohydride,
tetra-n-butylammonium borohydride or borane-pyridine complex.
Sodium cyanoborohydride or borane pyridine complex is preferably
used.
[3219] The reactions (IV)+(V).fwdarw.(VI) and
(IV)+(VIII).fwdarw.(IX) usually take place in a temperature range
from 0.degree. C. to +120.degree. C., preferably at +50.degree. C.
to +100.degree. C. The reactions may be performed at normal,
elevated or reduced pressure (e.g., from 0.5 to 5 bar). It is
customary to work under normal pressure.
[3220] The coupling reactions described above
(IX)+(X).fwdarw.(II-C), (XII-A) and/or (XII-B)+(X).fwdarw.(II-D-A)
and/or (II-D-B), (IX)+(XIII).fwdarw.(XIV), (IX)+(XV).fwdarw.(XVI)
and (X)+(XOH).fwdarw.(II-D) (amide formed from the respective amine
and carboxylic acid components) are performed according to the
standard methods of peptide chemistry (see, for example, M.
Bodanszky, Principles of Peptide Synthesis, Springer Verlag,
Berlin, 1993; M. Bodanszky and A. Bodanszky, The Practice of
Peptide Synthesis, Springer Verlag, Berlin, 1984; H.-D. Jakubke and
H. Jeschkeit, Aminosauren, Peptide, Proteine [Amino Acids,
Peptides, Proteins], Verlag Chemie, Weinheim, 1982).
[3221] Inert solvents for these coupling reactions include, for
example, ethers like diethyl ether, diisopropyl ether,
tert-butylmethyl ether, tetrahydrofuran, 1,4-dioxane,
1,2-dimethoxyethane or bis-(2-methoxyethyl)ether, hydrocarbons such
as benzene, toluene, xylene, pentane, hexane, heptane, cyclohexane
or petroleum fractions, halohydrocarbons such as dichloromethane,
trichloromethane, tetrachloromethane, 1,2-dichloroethane,
trichloroethylene or chlorobenzene or dipolar aprotic solvents such
as acetone, methyl ethyl ketone, acetonitrile, ethyl acetate,
pyridine, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF),
N,N-dimethylacetamide (DMA), N,N'-dimethylpropylene urea (DMPU) or
N-methylpyrrolidinone (NMP). It is likewise possible to use
mixtures of such solvents. N,N-Dimethylformamide is preferred.
[3222] Suitable activation/condensation agents for these coupling
reactions include, for example, carbodiimides such as N,N'-diethyl,
N,N'-dipropyl, N,N'-diisopropyl, N,N'-dicyclohexyl-carbodiimide
(DCC) or N-(3-dimethylaminoisopropyl)-N-ethylcarbodiimide
hydrochloride (EC), phosgene derivatives such as
N,N'-carbonyldiimidazole (CDI) or isobutyl chloroformate,
1,2-oxazolium compounds such as 2-ethyl-5-phenyl-1,2-oxazolium
3-sulfate or 2-tert-butyl-5-methylisoxazolium perchlorate,
acylamino compounds such as
2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, phosphorus
compounds such as propane phosphonic acid anhydride,
cyanophosphonic acid diethyl ester,
bis-(2-oxo-3-oxazolidinyl)phosphoryl chloride,
benzotriazole-1-yloxy-tris-(dimethylamino)phosphonium
hexafluorophosphate or
benzotriazol-1-yloxytris-(pyrrolidino)phosphonium
hexafluorophosphate (PyBOP), or uronium compounds such as
O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TBTU), O-(benzo-triazol 1-yl)
N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU),
2-(2-oxo-1-(2H)-pyridyl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TPTU),
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU) or
O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TCTU), optionally in combination with additional
excipients such as 1-hydroxybenzotriazole (HOBt) or
N-hydroxysuccinimide (HOSu) as well as bases such as alkali
carbonates, e.g., sodium or potassium carbonate or tertiary amine
bases such as triethylamine, N-methylmorpholine,
N-methylpiperidine, N,N-diisopropylethylamine, pyridine or
4-N,N-dimethylaminopyridine.
[3223] Within the context of the present invention, the preferred
activation/condensation agents for such coupling reactions include
N-(3-dimethylaminoisopropyl)-N'-ethylcarbodiimide hydrochloride
(EDC) in combination with 1-hydroxybenzotriazole (HOBt) and
N,N-diisopropylethylamine or O-(7-azabenzotriazol-1-yl)
N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) likewise in
combination with N,N-diisopropylethylamine.
[3224] The coupling reactions (IX)+(X).fwdarw.(II-C), (XII-A)
and/or (XII-B)+(X).fwdarw.(II-D-A) and/or (II-D-B),
(IX)+(XIII).fwdarw.(XIV), (IX)+(XV).fwdarw.(XVI) and
(XXII)+(XXIII).fwdarw.(II-D) are usually performed in a temperature
range from -20.degree. C. to +60.degree. C., preferably at
0.degree. C. to +40.degree. C. The reactions may be performed under
normal, elevated or reduced pressure (e.g., from 0.5 to 5 bar). It
is customary to work under normal pressure.
[3225] The ester-forming reactions (IX)+(XVIII).fwdarw.(XII) and
(IX)+(XI-A) and/or (XI-B).fwdarw.(XII-A) and/or (XII-B),
(IX)+(XXIV).fwdarw.(XXV) as well as (IX)+(XXI).fwdarw.(XXII) take
place like the amide coupling reactions described above. These
reactions preferably take place in dichloromethane using
N-(3-dimethylaminoisopropyl)-N-ethylcarbodiimide hydrochloride
(EDC) and 4-dimethylaminopyridine at a temperature of +50.degree.
C. to 100.degree. C. under normal pressure.
[3226] The functional groups optionally present in the
compounds--such as amino, hydroxyl and carboxyl groups in
particular--may also be present in a temporarily protected form in
the process steps described above, if this is expedient or
necessary. Such protective groups are introduced and removed
according to conventional methods known in peptide chemistry (see,
for example, T.W. Greene and P.G.M. Wuts, Protective Groups in
Organic Synthesis, Wiley, New York, 1999; M. Bodanszky and A.
Bodanszky, The Practice of Peptide Synthesis, Springer Verlag,
Berlin, 1984). In the presence of multiple protected groups, their
re-release may optionally be performed simultaneously in a one-pot
reaction or also in separate reaction steps.
[3227] The preferred amino protective groups PG.sup.1 include
tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Z) or
(9H-fluorene-9-ylmethoxy)carbonyl (Fmoc); tert-butyl or benzyl is
preferably used as the protective group PG.sup.2 for a hydroxyl or
carboxyl function. A tert-butyl or tert-butoxycarbonyl group is
usually split off by treatment with a strong acid such as
hydrochloric acid, hydrobromic acid or trifluoroacetic acid in an
inert solvent such as diethyl ether, 1,4-dioxane, dichloromethane
or acetic acid. This reaction may optionally also take place
without the addition of an inert solvent. In the case of benzyl or
benzyloxycarbonyl as the protective group, such a protective group
is preferably removed by hydrogenolysis in the presence of a
suitable palladium catalyst such as, for example, palladium on
activated carbon. The (9H-fluorene-9-ylmethoxy)carbonyl group is
generally split off with the help of a secondary amine base such as
diethylamine or piperidine.
[3228] The reaction (VI).fwdarw.(II-A) takes place in a solvent
that is inert under the reaction conditions, such as, for example,
ethers, e.g., tetrahydrofuran, 1,4-dioxane, 1,2-dimetoxyethane or
bis-(2-methoxyethyl)ether, alcohols such as methanol, ethanol,
isopropanol, n-butanol or tert-butanol or dipolar aprotic solvents
such as acetone, methyl ethyl ketone, acetonitrile, ethyl acetate,
pyridine, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF),
N,N-dimethylacetamide (DMA), N,N'-dimethylpropylene urea (DMPU) or
N-methylpyrrolidinone (NMP) or water. It is likewise possible to
use mixtures of such solvents. A mixture of 1,4-dioxane and water
is preferably used.
[3229] Suitable bases for the reaction (VI).fwdarw.(II-A) include,
for example, alkali carbonates such as potassium carbonate, sodium
carbonate or lithium carbonate, alkali bicarbonates such as sodium
or potassium bicarbonate or alkali alcoholates such as sodium
methanolate, sodium ethanolate or potassium tert-butylate. Sodium
bicarbonate is preferred.
[3230] The reaction (VI).fwdarw.(II-A) takes place in a temperature
range from 0.degree. C. to +50.degree. C., preferably at
+10.degree. C. to +30.degree. C. The reaction may be performed
under normal, elevated or reduced pressure (e.g., from 0.5 to 5
bar). It is customary to work under normal pressure.
[3231] The reaction (VI)+(VII).fwdarw.(II-B) takes place in a
solvent that is inert under the reaction conditions such as, for
example, ethers, e.g., tetrahydrofuran, 1,4-dioxane,
1,2-dimethoxyethane or bis-(2-methoxyethyl) ether, alcohols such as
methanol, ethanol, isopropanol, n-butanol or tert-butanol or
dipolar aprotic solvents like acetone, methyl ethyl ketone,
acetonitrile, ethyl acetate, pyridine, dimethyl sulfoxide (DMSO),
N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA),
N,N'-dimethylpropylene urea (DMPU) or N-methylpyrrolidinone (NMP)
or water. It is also possible to use mixtures of such solvents. DMF
is preferred.
[3232] Suitable bases for the reaction (VI)+(VII).fwdarw.(II-B)
include, for example, tertiary amine bases such as triethylamine,
N-methylmorpholine, N-methylpiperidine, N,N-diisopropylethylamine,
pyridine or 4-N,N-dimethylaminopyridine. N,N-Diisopropylethylamine
is preferred.
[3233] The reaction (VI)+(VII).fwdarw.(II-B) takes place in a
temperature range from 0.degree. C. to +50.degree. C., preferably
at +10.degree. C. to +30.degree. C. The reaction may take place
under normal, elevated or reduced pressure (e.g., from 0.5 to 5
bar). It is customary to work under normal pressure.
[3234] The reactions (IX).fwdarw.(III-A), (XIV).fwdarw.(III-B) and
(XVI).fwdarw.(III-C) as well as (VI)+(XVII).fwdarw.(III-D),
(XIX)+(XX).fwdarw.(III-E) and (XXV)+(XX).fwdarw.(III-F) take place
in a solvent that is inert under the reaction conditions. Suitable
solvents include, for example, ethers such as diethyl ether,
diisopropyl ether, tert-butylmethyl ether, tetrahydrofuran,
1,4-dioxane, 1,2-dimethoxyethane or bis-(2-methoxyethyl)ether,
hydrocarbons such as benzene, toluene, xylene, pentane, hexane,
heptane, cyclohexane or petroleum fractions, halohydrocarbons such
as dichloromethane, trichloromethane, tetrachloromethane,
1,2-dichloroethane, trichloroethylene or chlorobenzene or dipolar
aprotic solvents such as acetone, methyl ethyl ketone,
acetonitrile, ethyl acetate, pyridine, dimethyl sulfoxide (DMSO),
N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA),
N,N'-dimethylpropylene urea (DMPU) or N-methylpyrrolidinone (NMP).
It is likewise possible to use mixtures of such solvents.
N,N-Dimethylformamide is preferred.
[3235] Suitable bases for these reactions include, for example,
tertiary amines like triethylamine, N-methylmorpholine,
N-methylpiperidine, N,N-diisopropylethylamine, pyridine or
4-N,N-di-methylaminopyridine. N,N-Diisopropylethylamine is
preferred, optionally with the addition of
4-N,N-dimethylaminopyridine.
[3236] The reactions (IX).fwdarw.(III-A), (XIV).fwdarw.(III-B) and
(XVI).fwdarw.(III-C) as well as (VI)+(XVII).fwdarw.(III-D) and
(XIX)+(XX).fwdarw.(III-E) take place in a temperature range from
0.degree. C. to +50.degree. C., preferably at +10.degree. C. to
+30.degree. C. The reaction may be carried out under normal,
elevated or reduced pressure (e.g., from 0.5 to 5 bar). It is
customary to work under normal pressure.
[3237] The compounds of formulas (II), (III), (I-A) and/or (I-B)
are subsets of the compounds of formulas (IIa), (IIIa), (Ia-A)
and/or (Ia-B), where R.sup.35 stands for methyl. Compounds (IIa)
and (Ma) are synthesized as in the synthesis of the compound of
formula (II) and (III) as described above.
[3238] The methods described above are illustrated by the following
synthesis schemes (schemes 3 through 13, 18) as examples:
##STR00411##
##STR00412##
##STR00413##
##STR00414##
##STR00415##
##STR00416##
##STR00417##
##STR00418##
##STR00419##
##STR00420##
##STR00421##
##STR00422##
[3239] The compounds of formula (IV) may be synthesized from
commercially available amino acid building blocks or those known
from the literature (see, for example, Pettit et al., Synthesis
1996, 719; Shioiri et al., Tetrahedron Lett. 1991, 32, 931; Shioiri
et al., Tetrahedron 1993, 49, 1913; Koga et al., Tetrahedron Lett.
1991, 32, 2395; Vidal et al., Tetrahedron 2004, 60, 9715; Poncet et
al., Tetrahedron 1994, 50, 5345. Pettit et al., J. Org. Chem. 1994,
59, 1796) as with the processes known from the literature, by using
the standard methods of peptide chemistry and as described in the
present experimental part. The following synthesis schemes (schemes
14 through 16) illustrate this synthesis process as an example.
##STR00423##
##STR00424##
##STR00425##
[3240] The compounds of formulas (XI), (XIII), (XV), (XVII) and
(XXI), including where applicable chiral or diastereomeric forms
thereof are commercially available or have been described as such
in the literature or can be synthesized by methods like those
published in the literature in a manner that would be self-evident
to those skilled in the art. Several detailed publications and
specifications in the literature regarding the synthesis of the
starting materials can also be found in the experimental part in
the section for synthesis of the starting compounds and
intermediates.
[3241] The compounds of formulas (V), (VII), (VIII), (X), (XVIII),
(XX) and (XXIII) including where appropriate chiral or
diastereomeric forms thereof are known in the literature or they
can be synthesized by methods like those described in the
literature in a manner obvious to those skilled in the art.
Numerous detailed specifications as well as references from the
literature regarding the synthesis of the starting materials can be
found in the experimental part in the section on synthesis of the
starting compounds and intermediates.
[3242] Alternatively individually steps of the synthesis sequence
may be performed in a different order. This procedure is
illustrated in the following synthesis schemes (schemes 17, 19 and
20) as an example.
##STR00426## ##STR00427##
##STR00428##
##STR00429## ##STR00430##
[3243] In one embodiment, the binder is bound to a target molecule
that is present on a cancer cell. In a preferred embodiment, the
binder binds to a cancer target molecule.
[3244] In another preferred embodiment, the target molecule is a
selected cancer target molecule.
[3245] In an especially preferred embodiment, the target molecule
is a protein.
[3246] In one embodiment, the target molecule is an extracellular
target molecule. In a preferred embodiment, the extracellular
target molecule is a protein.
[3247] Cancer target molecules are known to those skilled in the
art. Examples of these are given below.
[3248] Examples of cancer target molecules include:
[3249] (1) EGF receptor (NCBI reference sequence
NP.sub.--005219.2)
[3250] Sequence (1210 amino acids): [3251]
>gi|29725609|ref|NP.sub.--005219.2| epidermal growth factor
receptor isoform a precursor [Homo sapiens]
TABLE-US-00001 [3251]
MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFL
SLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVER
IPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGA
VRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPN
GSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRE
SDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKK
CPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIG
EFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQEL
DILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVV
SLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKI
ISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKC
NLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDG
PHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCP
TNGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQER
ELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEG
EKVKIPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTS
TVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRL
VHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMA
LESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGE
RLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLV
IQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFESSPST
SRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALT
EDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQD
PHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDF
FPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA
[3252] The extracellular domain is underlined for emphasis.
[3253] (2) Mesothelin (SwissProt reference Q13421-3)
[3254] Sequence (622 amino acids): [3255]
>sp|Q13421-3|MSLN_HUMAN isoform 2 of mesothelin OS=Homo sapiens
GN=MSLN
TABLE-US-00002 [3255]
MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLD
GVLANPPNISSLSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLST
EQLRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTRFFSRITKA
NVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALGGLACDLPGRF
VAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTM
DALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFR
REVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIP
FTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSL
ETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYL
CSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGS
EYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEV
QKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLV
LDLSMQEALSGTPCLLGPGPVLTVLALLLASTLA
[3256] where mesothelin is coded by amino acids 296-598. Amino
acids 37-286 code for "megakaryocyte potentiating factor."
Mesothelin is anchored in the cell membrane by a GPI anchor and is
localized extracellularly.
[3257] (3) Carboanhydrase IX (SwissProt reference Q16790) [3258]
Sequence (459 amino acids): [3259] >sp|Q16790|CAH9_HUMAN
Carbonic anhydrase 9 OS=Homo sapiens GN=CA9 PE=1 SV=2
TABLE-US-00003 [3259]
MAPLCPSPWLPLLIPAPAPGLTVQLLLSLLLLVPVHPQRLPRMQEDSPLG GGSSGEDDPL
GEEDLPSEEDSPREEDPPGEEDLPGEEDLPGEEDLPEVKPKSEEEGSLKL EDLPTVEAPG
DPQEPQNNAHRDKEGDDQSHWRYGGDPPWPRVSPACAGRFQSPVDIRPQL AAFCPALRPL
ELLGFQLPPLPELRLRNNGHSVQLTLPPGLEMALGPGREYRALQLHLHWG AAGRPGSEHT
VEGHRFPAEIHVVHLSTAFARVDEALGRPGGLAVLAAFLEEGPEENSAYE QLLSRLEEIA
EEGSETQVPGLDISALLPSDFSRYFQYEGSLTTPPCAQGVIWTVFNQTVM LSAKQLHTLS
DTLWGPGDSRLQLNFRATQPLNGRVIEASFPAGVDSSPRAAEPVQLNSCL AAGDILALVF
GLLFAVTSVAFLVQMRRQHRRGTKGGVSYRPAEVAETGA
[3260] The extracellular domain is underlined for emphasis.
[3261] (4) C4.4a (NCBI reference sequence NP.sub.--055215.2;
synonym LYPD3)
[3262] Sequence (346 amino acids): [3263]
>gi|93004088|ref|NP.sub.--055215.2| ly6/PLAUR domain-containing
protein 3 precursor [Homo sapiens]
TABLE-US-00004 [3263]
MDPARKAGAQAMIWTAGWLLLLLLRGGAQALECYSCVQKADDGCSPNKMK
TVKCAPGVDVCTEAVGAVETIHGQFSLAVRGCGSGLPGKNDRGLDLHGLL
AFIQLQQCAQDRCNAKLNLTSRALDPAGNESAYPPNGVECYSCVGLSREA
CQGTSPPVVSCYNASDHVYKGCFDGNVTLTAANVTVSLPVRGCVQDEFCT
RDGVTGPGFTLSGSCCQGSRCNSDLRNKTYFSPRIPPLVRLPPPEPTTVA
STTSVTTSTSAPVRPTSTTKPMPAPTSQTPRQGVEHEASRDEEPRLTGGA
AGHQDRSNSGQYPAKGGPQQPHNKGCVAPTAGLAALLLAVAAGVLL
[3264] The matured extracellular domain is underlined for emphasis
(SEQ ID NO: 1).
[3265] (5) CD52 (NCBI reference sequence NP.sub.--001794.2) [3266]
>gi|68342030|ref|NP.sub.--001794.2| CAMPATH-1 antigen precursor
[Homo sapiens]
TABLE-US-00005 [3266]
MKRFLFLLLTISLLVMVQIQTGLSGQNDTSQTSSPSASSNISGGIFLFFV ANAIIHLFCFS
[3267] (6) HER2 (NCBI reference sequence NP.sub.--004439.2) [3268]
>gi|54792096|ref|NP.sub.--004439.2| receptor tyrosine-protein
kinase erbB-2 isoform a [Homo sapiens]
TABLE-US-00006 [3268]
MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLY
QGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLR
IVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILK
GGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCK
GSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHS
DCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACP
YNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHL
REVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVF
ETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGI
SWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRP
EDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGL
PREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARC
PSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASP
LTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPL
TPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPV
AIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQL
MPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARN
VLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFT
HQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTID
VYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPL
DSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSS
STRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQS
LPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPP
SPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQ
GGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLG LDVPV
[3269] (7) CD20 (NCBI reference sequence NP.sub.--068769.2) [3270]
>gi|23110987|ref|NP.sub.--068769.2| B-lymphocyte antigen CD20
[Homo sapiens]
TABLE-US-00007 [3270]
MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESK
TLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSL
LAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKME
SLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIF
AFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLT
ETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP
[3271] (8) The lymphocyte activating antigen CD30 (SwissProt ID
P28908) [3272] >gi|68348711|ref|NP.sub.--001234.2| tumor
necrosis factor receptor superfamily member 8 isoform 1 precursor
[Homo sapiens]
TABLE-US-00008 [3272]
MRVLLAALGLLFLGALRAFPQDRPFEDTCHGNPSHYYDKAVRRCCYRCPM
GLFPTQQCPQRPTDCRKQCEPDYYLDEADRCTACVTCSRDDLVEKTPCAW
NSSRVCECRPGMFCSTSAVNSCARCFFHSVCPAGMIVKFPGTAQKNTVCE
PASPGVSPACASPENCKEPSSGTIPQAKPTPVSPATSSASTMPVRGGTRL
AQEAASKLTRAPDSPSSVGRPSSDPGLSPTQPCPEGSGDCRKQCEPDYYL
DEAGRCTACVSCSRDDLVEKTPCAWNSSRTCECRPGMICATSATNSRARC
VPYPICAAETVTKPQDMAEKDTTFEAPPLGTQPDCNPTPENGEAPASTSP
TQSLLVDSQASKTLPIPTSAPVALSSTGKPVLDAGPVLFWVILVLVVVVG
SSAFLLCHRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSG
ASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDL
PEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEE
ELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK
[3273] (9) The lymphocyte adhesion molecule CD22 (SwissProt ID
P20273) [3274] >gi|157168355|ref|NP.sub.--001762.2| B-cell
receptor CD22 isoform 1 precursor [Homo sapiens]
TABLE-US-00009 [3274]
MHLLGPWLLLLVLEYLAFSDSSKWVFEHPETLYAWEGACVWIPCTYRALD
GDLESFILFHNPEYNKNTSKFDGTRLYESTKDGKVPSEQKRVQFLGDKNK
NCTLSIHPVHLNDSGQLGLRMESKTEKWMERIHLNVSERPFPPHIQLPPE
IQESQEVTLTCLLNFSCYGYPIQLQWLLEGVPMRQAAVTSTSLTIKSVFT
RSELKFSPQWSHHGKIVTCQLQDADGKFLSNDTVQLNVKHTPKLEIKVTP
SDAIVREGDSVTMTCEVSSSNPEYTTVSWLKDGTSLKKQNTFTLNLREVT
KDQSGKYCCQVSNDVGPGRSEEVFLQVQYAPEPSTVQILHSPAVEGSQVE
FLCMSLANPLPTNYTWYHNGKEMQGRTEEKVHIPKILPWHAGTYSCVAEN
ILGTGQRGPGAELDVQYPPKKVTTVIQNPMPIREGDTVTLSCNYNSSNPS
VTRYEWKPHGAWEEPSLGVLKIQNVGWDNTTIACAACNSWCSWASPVALN
VQYAPRDVRVRKIKPLSEIHSGNSVSLQCDFSSSHPKEVQFFWEKNGRLL
GKESQLNFDSISPEDAGSYSCWVNNSIGQTASKAWTLEVLYAPRRLRVSM
SPGDQVMEGKSATLTCESDANPPVSHYTWFDWNNQSLPYHSQKLRLEPVK
VQHSGAYWCQGTNSVGKGRSPLSTLTVYYSPETIGRRVAVGLGSCLAILI
LAICGLKLQRRWKRTQSQQGLQENSSGQSFFVRNKKVRRAPLSEGPHSLG
CYNPMMEDGISYTTLRFPEMNIPRTGDAESSEMQRPPPDCDDTVTYSALH
KRQVGDYENVIPDFPEDEGIHYSELIQFGVGERPQAQENVDYVILKH
[3275] (10) The myeloid cell surface antigen CD33 (SwissProt ID
P20138) [3276] >gi|130979981|ref|NP.sub.--001763.3| myeloid cell
surface antigen CD33 isoform 1 precursor [Homo sapiens]
TABLE-US-00010 [3276]
MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYY
DKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNN
CSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIP
GTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIIT
PRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGK
QETRAGVVHGAIGGAGVTALLALCLCLIFFFVKTHRRKAARTAVGRNDTH
PTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNP
SKDTSTEYSEVRTQ
[3277] (11) The transmembrane glycoprotein NMB (SwissProt ID
Q14956) [3278] >gi|52694752|ref|NP.sub.--001005340.1|
transmembrane glycoprotein NMB isoform a precursor [Homo
sapiens]
TABLE-US-00011 [3278]
MECLYYFLGFLLLAARLPLDAAKRFHDVLGNERPSAYMREHNQLNGWSSD
ENDWNEKLYPVWKRGDMRWKNSWKGGRVQAVLTSDSPALVGSNITFAVNL
IFPRCQKEDANGNIVYEKNCRNEAGLSADPYVYNWTAWSEDSDGENGTGQ
SHHNVFPDGKPFPHHPGWRRWNFIYVFHTLGQYFQKLGRCSVRVSVNTAN
VTLGPQLMEVTVYRRHGRAYVPIAQVKDVYVVTDQIPVFVTMFQKNDRNS
SDETFLKDLPIMFDVLIHDPSHFLNYSTINYKWSFGDNTGLFVSTNHTVN
HTYVLNGTFSLNLTVKAAAPGPCPPPPPPPRPSKPTPSLATTLKSYDSNT
PGPAGDNPLELSRIPDENCQINRYGHFQATITIVEGILEVNIIQMTDVLM
PVPWPESSLIDFVVTCQGSIPTEVCTIISDPTCEITQNTVCSPVDVDEMC
LLTVRRTFNGSGTYCVNLTLGDDTSLALTSTLISVPDRDPASPLRMANSA
LISVGCLAIFVTVISLLVYKKHKEYNPIENSPGNVVRSKGLSVFLNRAKA
VFFPGNQEKDPLLKNQEFKGVS
[3279] (12) The adhesion molecule CD56 (SwissProt ID P13591) [3280]
>gi|94420689|ref|NP.sub.--000606.3| neural cell adhesion
molecule 1 isoform 1 [Homo sapiens]
TABLE-US-00012 [3280]
MLQTKDLIWTLFFLGTAVSLQVDIVPSQGEISVGESKFFLCQVAGDAKDK
DISWFSPNGEKLTPNQQRISVVWNDDSSSTLTIYNANIDDAGIYKCVVTG
EDGSESEATVNVKIFQKLMFKNAPTPQEFREGEDAVIVCDVVSSLPPTII
WKHKGRDVILKKDVRFIVLSNNYLQIRGIKKTDEGTYRCEGRILARGEIN
FKDIQVIVNVPPTIQARQNIVNATANLGQSVTLVCDAEGFPEPTMSWTKD
GEQIEQEEDDEKYIFSDDSSQLTIKKVDKNDEAEYICIAENKAGEQDATI
HLKVFAKPKITYVENQTAMELEEQVTLTCEASGDPIPSITWRTSTRNISS
EEKTLDGHMVVRSHARVSSLTLKSIQYTDAGEYICTASNTIGQDSQSMYL
EVQYAPKLQGPVAVYTWEGN
QVNITCEVFAYPSATISWFRDGQLLPSSNYSNIKIYNTPSASYLEVTPDS
ENDFGNYNCTAVNRIGQESLEFILVQADTPSSPSIDQVEPYSSTAQVQFD
EPEATGGVPILKYKAEWRAVGEEVWHSKWYDAKEASMEGIVTIVGLKPET
TYAVRLAALNGKGLGEISAASEFKTQPVQGEPSAPKLEGQMGEDGNSIKV
NLIKQDDGGSPIRHYLVRYRALSSEWKPEIRLPSGSDHVMLKSLDWNAEY
EVYVVAENQQGKSKAAHFVFRTSAQPTAIPANGSPTSGLSTGAIVGILIV
IFVLLLVVVDITCYFLNKCGLFMCIAVNLCGKAGPGAKGKDMEEGKAAFS
KDESKEPIVEVRTEEERTPNHDGGKHTEPNETTPLTEPEKGPVEAKPECQ
ETETKPAPAEVKTVPNDATQTKENESKA
[3281] (13) The surface molecule CD70 (SwissProt ID P32970) [3282]
>gi|4507605|ref|NP.sub.--001243.1| CD70 antigen [Homo
sapiens]
TABLE-US-00013 [3282]
MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPL
ESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHR
DGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQG
CTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP
[3283] (14) The surface molecule CD74 (SwissProt ID P04233) [3284]
>gi|10835071|ref|NP.sub.--004346.1| HLA class II
histocompatibility antigen gamma chain isoform b [Homo sapiens]
TABLE-US-00014 [3284]
MHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALY
TGFSILVTLLLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPKP
PKPVSKMRMATPLLMQALPMGALPQGPMQNATKYGNMTEDHVMHLLQNAD
PLKVYPPLKGSFPENLRHLKNTMETIDWKVFESWMHHWLLFEMSRHSLEQ
KPTDAPPKESLELEDPSSGLGVTKQDLGPVPM
[3285] (15) The B-lymphocyte antigen CD19 (SwissProt ID P15391)
[3286] >gi|296010921|ref|NP.sub.--001171569.1| B-lymphocyte
antigen CD19 isoform 1 precursor [Homo sapiens]
TABLE-US-00015 [3286]
MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQL
TWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPG
PPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGK
LMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSC
GVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPR
ATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYL
IFCLCSLVGILHLQRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYGN
VLSLPTPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQADGALGSRSPPGVG
PEEEEGEGYEEPDSEEDSEFYENDSNLGQDQLSQDGSGYENPEDEPLGPE
DEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLAGSQS
YEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWGGGGR MGTWSTR
[3287] (16) The surface protein mucin 1 (SwissProt ID P15941)
[3288] >gi|65301117|ref|NP.sub.--002447.4| mucin-1 isoform 1
precursor [Homo sapiens]
TABLE-US-00016 [3288]
MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGEKETSATQRSSVPSSTE
KNALSTGVSFFFLSFHISNLQFNSSLEDPSTDYYQELQRDISEMFLQIYK
QGGFLGLSNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRY
NLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALA
VCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKV
SAGNGGSSLSYTNPAVAATSANL
[3289] (17) The surface protein CD138 (SwissProt ID P18827) [3290]
>gi|29568086|ref|NP.sub.--002988.3| syndecan-1 precursor [Homo
sapiens]
TABLE-US-00017 [3290]
MRRAALWLWLCALALSLQPALPQIVATNLPPEDQDGSGDDSDNFSGSGAG
ALQDITLSQQTPSTWKDTQLLTAIPTSPEPTGLEATAASTSTLPAGEGPK
EGEAVVLPEVEPGLTAREQEATPRPRETTQLPTTHQASTTTATTAQEPAT
SHPHRDMQPGHHETSTPAGPSQADLHTPHTEDGGPSATERAAEDGASSQL
PAAEGSGEQDFTFETSGENTAVVAVEPDRRNQSPVDQGATGASQGLLDRK
EVLGGVIAGGLVGLIFAVCLVGFMLYRMKKKDEGSYSLEEPKQANGGAYQ KPTKQEEFYA
[3291] (18) Integrin alphaV (GenBank Accession No.
NP.sub.--002201.1) [3292] >gi|4504763|ref|NP.sub.--002201.1|
integrin alpha-V isoform 1 precursor [Homo sapiens]
TABLE-US-00018 [3292]
MAFPPRRRLRLGPRGLPLLLSGLLLPLCRAFNLDVDSPAEYSGPEGSYFG
FAVDFFVPSASSRMFLLVGAPKANTTQPGIVEGGQVLKCDWSSTRRCQPI
EFDATGNRDYAKDDPLEFKSHQWFGASVRSKQDKILACAPLYHWRTEMKQ
EREPVGTCFLQDGTKTVEYAPCRSQDIDADGQGFCQGGFSIDFTKADRVL
LGGPGSFYWQGQLISDQVAEIVSKYDPNVYSIKYNNQLATRTAQAIFDDS
YLGYSVAVGDFNGDGIDDFVSGVPRAARTLGMVYIYDGKNMSSLYNFTGE
QMAAYFGFSVAATDINGDDYADVFIGAPLFMDRGSDGKLQEVGQVSVSLQ
RASGDFQTTKLNGFEVFARFGSAIAPLGDLDQDGFNDIAIAAPYGGEDKK
GIVYIFNGRSTGLNAVPSQILEGQWAARSMPPSFGYSMKGATDIDKNGYP
DLIVGAFGVDRAILYRARPVITVNAGLEVYPSILNQDNKTCSLPGTALKV
SCFNVRFCLKADGKGVLPRKLNFQVELLLDKLKQKGAIRRALFLYSRSPS
HSKNMTISRGGLMQCEELIAYLRDESEFRDKLTPITIFMEYRLDYRTAAD
TTGLQPILNQFTPANISRQAHILLDCGEDNVCKPKLEVSVDSDQKKIYIG
DDNPLTLIVKAQNQGEGAYEAELIVSIPLQADFIGVVRNNEALARLSCAF
KTENQTRQVVCDLGNPMKAGTQLLAGLRFSVHQQSEMDTSVKFDLQIQSS
NLFDKVSPVVSHKVDLAVLAAVEIRGVSSPDHIFLPIPNWEHKENPETEE
DVGPVVQHIYELRNNGPSSFSKAMLHLQWPYKYNNNTLLYILHYDIDGPM
NCTSDMEINPLRIKISSLQTTEKNDTVAGQGERDHLITKRDLALSEGDIH
TLGCGVAQCLKIVCQVGRLDRGKSAILYVKSLLWTETFMNKENQNHSYSL
KSSASFNVIEFPYKNLPIEDITNSTLVTTNVTWGIQPAPMPVPVWVIILA
VLAGLLLLAVLVFVMYRMGFFKRVRPPQEEQEREQLQPHENGEGNSET
[3293] (19) The teratocarcinoma-derived growth factor 1 protein
TDGF1 (GenBank Accession No.: NP.sub.--003203.1) [3294]
>gi|4507425|ref|NP.sub.--003203.1| teratocarcinoma-derived
growth factor 1 isoform 1 precursor [Homo sapiens]
TABLE-US-00019 [3294]
MDCRKMARFSYSVIWIMAISKVFELGLVAGLGHQEFARPSRGYLAFRDDS
IWPQEEPAIRPRSSQRVPPMGIQHSKELNRTCCLNGGTCMLGSFCACPPS
FYGRNCEHDVRKENCGSVPHDTWLPKKCSLCKCWHGQLRCFPQAFLPGCD
GLVMDEHLVASRTPELPPSARTTTFMLVGICLSIQSYY
[3295] (20) The prostate-specific membrane antigen PSMA (SwissProt
ID: Q04609) [3296] >gi|4758398|ref|NP.sub.--004467.1| glutamate
carboxypeptidase 2 isoform 1 [Homo sapiens]
TABLE-US-00020 [3296]
MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEAT
NITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQW
KEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPG
YENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKI
VIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPG
GGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYY
DAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTN
EVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVR
SFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYI
NADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKK
SPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYP
LYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDY
AVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERL
QDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKY
AGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA
[3297] (21) Tyrosine protein kinase EPHA2 (SwissProt ID: P29317)
[3298] >gi|32967311|ref|NP.sub.--004422.2| ephrin type-A
receptor 2 precursor [Homo sapiens]
TABLE-US-00021 [3298]
MELQAARACFALLWGCALAAAAAAQGKEVVLLDFAAAGGELGWLTHPYGK
GWDLMQNIMNDMPIYMYSVCNVMSGDQDNWLRTNWVYRGEAERIFIELKF
TVRDCNSFPGGASSCKETFNLYYAESDLDYGTNFQKRLFTKIDTIAPDEI
TVSSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRVYYKK
CPELLQGLAHFPETIAGSDAPSLATVAGTCVDHAVVPPGGEEPRMHCAVD
GEWLVPIGQCLCQAGYEKVEDACQACSPGFFKFEASESPCLECPEHTLPS
PEGATSCECEEGFFRAPQDPASMPCTRPPSAPHYLTAVGMGAKVELRWTP
PQDSGGREDIVYSVTCEQCWPESGECGPCEASVRYSEPPHGLTRTSVTVS
DLEPHMNYTFTVEARNGVSGLVTSRSFRTASVSINQTEPPKVRLEGRSTT
SLSVSWSIPPPQQSRVWKYEVTYRKKGDSNSYNVRRTEGFSVTLDDLAPD
TTYLVQVQALTQEGQGAGSKVHEFQTLSPEGSGNLAVIGGVAVGVVLLLV
LAGVGFFIHRRRKNQRARQSPEDVYFSKSEQLKPLKTYVDPHTYEDPNQA
VLKFTTEIHPSCVTRQKVIGAGEFGEVYKGMLKTSSGKKEVPVAIKTLKA
GYTEKQRVDFLGEAGIMGQFSHHNIIRLEGVISKYKPMMIITEYMENGAL
DKFLREKDGEFSVLQLVGMLRGIAAGMKYLANMNYVHRDLAARNILVNSN
LVCKVSDFGLSRVLEDDPEATYTTSGGKIPIRWTAPEAISYRKFTSASDV
WSFGIVMWEVMTYGERPYWELSNHEVMKAINDGFRLPTPMDCPSAIYQLM
MQCWQQERARRPKFADIVSILDKLIRAPDSLKTLADFDPRVSIRLPSTSG
SEGVPFRTVSEWLESIKMQQYTEHFMAAGYTAIEKVVQMTNDDIKRIGVR
LPGHQKRIAYSLLGLKDQVNTVGIPI
[3299] (22) The surface protein SLC44A4 (GenBank Accession No.
NP.sub.--001171515) [3300]
>gi|295849282|ref|NP.sub.--001171515.1| choline transporter-like
protein 4 isoform 2 [Homo sapiens]
TABLE-US-00022 [3300]
MGGKQRDEDDEAYGKPVKYDPSFRGPIKNRSCTDVICCVLFLLFILGYIV
VGIVAWLYGDPRQVLYPRNSTGAYCGMGENKDKPYLLYFNIFSCILSSNI
ISVAENGLQCPTPQTVITSLQQELCPSFLLPSAPALGRCFPWTNVTPPAL
PGITNDTTIQQGISGLIDSLNARDISVKIFEDFAQSWYWILVALGVALVL
SLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISQ
LGFTTNLSAYQSVQETWLAALIVLAVLEAILLLMLIFLRQRIRIAIALLK
EASKAVGQMMSTMFYPLVTFVLLLICIAYWAMTALYLATSGQPQYVLWAS
NISSPGCEKVPINTSCNPTAHLVNSSCPGLMCVFQGYSSKGLIQRSVFNL
QIYGVLGLFWTLNWVLALGQCVLAGAFASFYWAFHKPQDIPTFPLISAFI
RTLRYHTGSLAFGALILTLVQIARVILEYIDHKLRGVQNPVARCIMCCFK
CCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSAKNAFMLLMRNIVRVVVLD
KVTDLLLFFGKLLVVGGVGVLSFFFFSGRIPGLGKDFKSPHLNYYWLPIM
TSILGAYVIASGFFSVFGMCVDTLFLCFLEDLERNNGSLDRPYYMSKSLL
KILGKKNEAPPDNKKRKK
[3301] (23) The surface protein BMPR1B (SwissProt: 000238)
[3302] (24) The transport protein SLC7A5 (SwissProt: Q01650)
[3303] (25) The epithelial antigen of the prostate STEAP1
(SwissProt: Q9UHE8)
[3304] (26) The ovarian carcinoma antigen MUC16 (SwissProt:
Q8WXI7)
[3305] (27) The transport protein SLC34A2 (SwissProt: 095436)
[3306] (28) The surface protein SEMA5b (SwissProt: Q9P283)
[3307] (29) The surface protein LYPD1 (SwissProt: Q8N2G4)
[3308] (30) The endothelin receptor type B EDNRB (SwissProt:
P24530)
[3309] (31) The ring finger protein RNF43 (SwissProt: Q68DV7)
[3310] (32) The prostate carcinoma associated protein STEAP2
(SwissProt: Q8NFT2)
[3311] (33) The cation channel TRPM4 (SwissProt: Q8TD43)
[3312] (34) The complement receptor CD21 (SwissProt: P20023)
[3313] (35) The B-cell antigen receptor complex associated protein
CD79b (SwissProt: P40259)
[3314] (36) The cell adhesion antigen CEACAM6 (SwissProt:
P40199)
[3315] (37) The dipeptidase DPEP1 (SwissProt: P16444)
[3316] (38) The interleukin receptor IL20Ralpha (SwissProt:
Q9UHF4)
[3317] (39) The proteoglycan BCAN (SwissProt: Q96GW7)
[3318] (40) The ephrine receptor EPHB2 (SwissProt: P29323)
[3319] (41) The prostatic stem cell associated protein PSCA
(GenBank Accession No. NP.sub.--005663.2)
[3320] (42) The surface protein LHFPL3 (SwissProt: Q86UP9)
[3321] (43) The receptor protein TNFRSF13C (SwissProt: Q96RJ3)
[3322] (44) The B-cell antigen receptor complex associated protein
CD79a (SwissProt: P11912)
[3323] (45) The receptor protein CXCRS (SwissProt: P32302)
[3324] (46) The ion channel P2X5 (SwissProt: Q93086)
[3325] (47) The lymphocyte antigen CD180 (SwissProt: Q99467)
[3326] (48) The receptor protein FCRL1 (SwissProt: Q96LA6)
[3327] (49) The receptor protein FCRLS (SwissProt: Q96RD9)
[3328] (50) The MHC class II molecule Ia antigen HLA-DOB (GenBank
Accession No: NP.sub.--002111.1)
[3329] (51) The T-cell protein VTCN1 (SwissProt: Q7Z7D3).
[3330] (52) Single-pass type-I membrane protein "Programmed cell
death 1 ligand 1"
[3331] (synonyms: CD274, B7H1, PDCD1L1, PDCD1LG1, PDL1) (SwissProt:
Q9NZQ7)--both are isoforms [3332] >sp|Q9NZQ7|PD1L1_HUMAN
Programmed cell death 1 ligand 1 OS=Homo sapiens GN=CD274 PE=1
SV=1
TABLE-US-00023 [3332]
MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDL
AALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQ
ITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSE
HELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRIN
TTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLC
LGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET
[3333] (53) Single-pass type I membrane protein "ICOSLG"
(synonyms:B7H2, B7RP1, ICOSL, KIAA0653, CD275)--(SwissProt:
O75144), both are isoforms [3334] >sp|O75144|ICOSL_HUMAN ICOS
ligand OS=Homo sapiens GN=ICOSLG PE=1 SV=2
TABLE-US-00024 [3334]
MRLGSPGLLFLLFSSLRADTQEKEVRAMVGSDVELSCACPEGSRFDLNDV
YVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRL
FNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPS
QDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVV
SVLRIARTPSVNIGCCIENVLLQQNLTVGSQTGNDIGERDKITENPVSTG
EKNAATWSILAVLCLLVVVAVAIGWVCRDRCLQHSYAGAWAVSPETELTG HV
[3335] (54) Tyrosine kinase "Fibroblast growth factor receptor 3"
(FGFR-3, EC=2.7.10.1, CD333, JTK4), (SwissProt: P22607)--four
isoforms (alternative splicing) [3336] >sp|P22607|FGFR3_HUMAN
Fibroblast growth factor receptor 3 OS=Homo sapiens GN=FGFR3 PE=1
SV=1
TABLE-US-00025 [3336]
MGAPACALALCVAVAIVAGASSESLGTEQRVVGRAAEVPGPEPGQQEQLV
FGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNAS
HEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGA
PYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHR
IGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSP
HRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGP
DGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHH
SAWLVVLPAEEELVEADEAGSVYAGILSYGVGFFLFILVVAAVTLCRLRS
PPKKGLGSPTVHKISRFPLKRQVSLESNASMSSNTPLVRIARLSSGEGPT
LANVSELELPADPKWELSRARLTLGKPLGEGCFGQVVMAEAIGIDKDRAA
KPVTVAVKMLKDDATDKDLSDLVSEMEMMKMIGKHKNIINLLGACTQGGP
LYVLVEYAAKGNLREFLRARRPPGLDYSFDTCKPPEEQLTFKDLVSCAYQ
VARGMEYLASQKCIHRDLAARNVLVTEDNVMKIADFGLARDVHNLDYYKK
TTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLLWEIFTLGGSPYPGIPV
EELFKLLKEGHRMDKPANCTHDLYMIMRECWHAAPSQRPTFKQLVEDLDR
VLTVTSTDEYLDLSAPFEQYSPGGQDTPSSSSSGDDSVFAHDLLPPAPPS SGGSRT
[3337] (55) Single-pass type-I membrane protein "TYRP1" (CAS2,
TYRP, TYRRP, DHICA oxidase, 5,6-dihydroxyindole-2-carboxylic acid
oxidase, catalase B, glycoprotein 75, melanoma antigen gp75,
tyrosinase-related protein 1), (SwissProt: P17643) [3338]
>sp|P17643|TYRP1_HUMAN 5,6-dihydroxyindole-2-carboxylic acid
oxidase OS=Homo sapiens GN=TYRP1 PE=1 SV=2
TABLE-US-00026 [3338]
MSAPKLLSLGCIFFPLLLFQQARAQFPRQCATVEALRSGMCCPDLSPVSG
PGTDRCGSSSGRGRCEAVTADSRPHSPQYPHDGRDDREVWPLRFFNRTCH
CNGNFSGHNCGTCRPGWRGAACDQRVLIVRRNLLDLSKEEKNHFVRALDM
AKRTTHPLFVIATRRSEEILGPDGNTPQFENISIYNYFVWTHYYSVKKTF
LGVGQESFGEVDFSHEGPAFLTWHRYHLLRLEKDMQEMLQEPSFSLPYWN
FATGKNVCDICTDDLMGSRSNFDSTLISPNSVFSQWRVVCDSLEDYDTLG
TLCNSTEDGPIRRNPAGNVARPMVQRLPEPQDVAQCLEVGLFDTPPFYSN
STNSFRNTVEGYSDPTGKYDPAVRSLHNLAHLFLNGTGGQTHLSPNDPIF
VLLHTFTDAVFDEWLRRYNADISTFPLENAPIGHNRQYNMVPFWPPVTNT
EMFVTAPDNLGYTYEIQWPSREFSVPEIIAIAVVGALLLVALIFGTASYL
IRARRSMDEANQPLLTDQYQCYAEEYEKLQNPNQSVV
[3339] (56) Cell membrane protein, cleaved into secreted glypican-3
(GPC3, OCI5, GTR2-2, intestinal protein OCI-5, MXR7), (SwissProt:
P51654) [3340] >sp|P51654|GPC3_HUMAN Glypican-3 OS=Homo sapiens
GN=GPC3 PE=1 SV=1
TABLE-US-00027 [3340]
MAGTVRTACLVVAMLLSLDFPGQAQPPPPPPDATCHQVRSFFQRLQPGLK
WVPETPVPGSDLQVCLPKGPTCCSRKMEEKYQLTARLNMEQLLQSASMEL
KFLIIQNAAVFQEAFEIVVRHAKNYTNAMFKNNYPSLTPQAFEFVGEFFT
DVSLYILGSDINVDDMVNELFDSLFPVIYTQLMNPGLPDSALDINECLRG
ARRDLKVFGNFPKLIMTQVSKSLQVTRIFLQALNLGIEVINTTDHLKFSK
DCGRMLTRMWYCSYCQGLMMVKPCGGYCNVVMQGCMAGVVEIDKYWREYI
LSLEELVNGMYRIYDMENVLLGLFSTIHDSIQYVQKNAGKLTTTIGKLCA
HSQQRQYRSAYYPEDLFIDKKVLKVAHVEHEETLSSRRRELIQKLKSFIS
FYSALPGYICSHSPVAENDTLCWNGQELVERYSQKAARNGMKNQFNLHEL
KMKGPEPVVSQIIDKLKHINQLLRTMSMPKGRVLDKNLDEEGFESGDCGD
DEDECIGGSGDGMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEIS
TFHNLGNVHSPLKLLTSMAISVVCFFFLVH
[3341] In a preferred subject matter of the invention, the cancer
target molecule is selected from the group consisting of the cancer
target molecules (1) though (56).
[3342] In another preferred subject matter of the invention, the
binder binds to an extracellular cancer target molecule, which is
selected from the group consisting of the cancer target molecules
(1) through (56).
[3343] In another preferred subject matter of the invention, the
binder binds specifically to an extracellular cancer target
molecule, which is selected from the group consisting of the cancer
target molecules (1) through (56).
[3344] In an especially preferred subject matter of the invention,
the cancer target molecule is selected from the group consisting of
EGF receptor (NP.sub.--005219.2), mesothelin (Q13421-3), C4.4a
(NP.sub.--055215.2), carboanhydrase IX (CA IX; Q16790,
NP.sub.--001207.2), HER2, glypican-3, TYRP1, fibroblast growth
factor receptor 3, single-pass type I membrane protein ICOSLG and
programmed cell death 1 ligand 1.
[3345] In another especially preferred subject matter of the
invention, the binder binds to an extracellular cancer target
molecule, which is selected from the group consisting of EGF
receptor (NP.sub.--005219.2), mesothelin (Q13421-3), C4.4a
(NP.sub.--055215.2), carboanhydrase IX (CA IX; Q16790,
NP.sub.--001207.2), HER2, glypican-3, TYRP1, fibroblast growth
factor receptor 3, single-pass type I membrane protein ICOSLG and
programmed cell death 1 ligand 1.
[3346] In one preferred embodiment, the binder is internalized by
the target cell after binding to its extracellular target molecule
on the target cell by the binding. The result of this is that the
binder-drug conjugate which may be an immunoconjugate or an ADC, is
absorbed by the target cell.
[3347] In one embodiment, the binder is a binding protein. In a
preferred embodiment, the binder is an antibody, an antigen-binding
antibody fragment, a multispecific antibody or an antibody
mimetic.
[3348] Preferred antibody mimetics include affibodies, adnectins,
anticalins, DARPins, avimers or nanobodies. Preferred multispecific
antibodies include bi-specific and tri-specific antibodies.
[3349] In a preferred embodiment, the binder is an antibody or an
antigen-binding antibody fragment; more preferably it is an
isolated antibody or an isolated antigen-binding antibody
fragment.
[3350] Preferred antigen binding antibody fragments include Fab,
Fab', F(ab').sub.2 and Fv fragments, diabodies, Dabs, linear
antibodies and scFv, Fab, diabodies and scFv are especially
preferred.
[3351] In an especially preferred embodiment, the binder is an
antibody. Especially preferred are monoclonal antibodies or
antigen-binding antibody fragments thereof. Additionally especially
preferred are human, humanized or chimeric antibodies or
antigen-binding antibody fragments thereof.
[3352] Antibodies or antigen-binding antibody fragments that bind
cancer target molecules can be synthesized by the average person
skilled in the art using known methods, for example, recombinant
synthesis or recombinant expression. Binders for cancer target
molecules can be purchased commercially or can be synthesized by an
average person skilled in the art by using known methods, e.g.,
chemical synthesis or recombinant expression. Additional methods of
synthesis of antibodies or antigen-binding antibody fragments are
described in WO 2007070538 (see page 22 "Antibodies"). Those
skilled in the art are familiar with methods such as the so-called
phage display technique which creates libraries (e.g., Morphosys
HuCAL Gold) and can be used to discover antibodies or
antigen-binding antibody fragments (see WO 200707058, pages 24
ff.,
[3353] Example 1 on page 70 and Example 2 on page 72). Additional
methods of synthesis of antibodies using DNA libraries from B cells
are described on page 26 of WO 2007070538, for example. Methods of
humanizing antibodies are described on pages 30-32 of WO 2007070538
and in detail in Queen et al., Proc. Natl. Acad. Sci. USA
86:10029-10033, 1989 or in WO 90/0786. In addition, those skilled
in the art are familiar with methods of recombinant expression of
proteins in general and in specific by antibodies (see, e.g., in
Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods
in Enzymology, vol. 152, Academic Press, Inc.; Sambrook, et al.,
Molecular Cloning: A Laboratory Manual, 2.sup.nd edition, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989,
vol. 1-3; Current Protocols in Molecular Biology, F. M. Ausabel et
al. (eds.), Current Protocols, Green Publishing Associates, Inc.,
John Wiley & Sons, Inc.; Harlow et al., Monoclonal Antibodies:
A Laboratory Manual, Cold Spring Harbor Laboratory Press, 19881,
Paul (ed.); Fundamental Immunology, (Lippincott Williams &
Wilkins, 1998; and Harlow, et al., Using Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, 1998. Those skilled in
the art are familiar with the corresponding vectors, promoters and
signal peptides, which are necessary for expression of a
protein/antibody. Conventional methods are also described on pages
41-45 of WO 2007070538. Methods of synthesis of an IgG1 antibody
are described in WO 2007070538, e.g., on pages 74 ff. of Example 6;
these methods, described on page 80 of WO 2007070538, for example,
make it possible to internalize an antibody after binding it to its
antigen. Similarly, those skilled in the art can utilize the
methods described in WO 2007070538 for synthesis of carboanhydrase
IX (Mn) antibodies to synthesize antibodies having other target
molecule specificities.
[3354] Especially preferred binders according to the invention are
antibodies in particular human or humanized antibodies. The
antibodies preferably have an affinity of at least 10.sup.-7 M (as
a Kd value; i.e., preferably those with smaller Kd values than
10.sup.-7 M), preferably of at least 10.sup.-8M, especially
preferably in the range of 10.sup.-9 M to 10.sup.-11 M. These Kd
values can be determined, for example, by surface plasmon resonance
spectroscopy.
[3355] The antibody-drug conjugates according to the invention also
have affinities in these ranges. Through conjugation of the active
ingredients, the affinity is preferably not influenced
significantly (the affinity is usually reduced by less than one
order of magnitude, e.g., max. from 10.sup.-8M to 10.sup.-7 M).
[3356] The antibodies used according to the invention are also
preferably characterized by a high selectivity. Selectivity is high
when the antibody according to the invention has a better affinity
for the target protein than for another independent antigen, e.g.,
human serum albumin, said affinity being better by a factor of 2, a
factor of 5, a factor of 10 or in particular preferably a factor of
100 (the affinity can be determined, for example, by surface
plasmon resonance spectroscopy).
[3357] Furthermore, the antibodies used according to the invention
are preferably cross-reactive. To facilitate preclinical trials,
e.g., toxicological studies or efficacy studies (e.g., in xenograft
mice) and to be able to interpret them better, it is advantageous
if the antibody to be used according to the invention not only
binds the human target protein but also binds the species target
protein in the species used for these studies. In one embodiment,
the antibody used according to the invention, which is
cross-reactive with the antibody used according to the invention
but is also cross-reactive with the human target protein of at
least one additional species. For toxicological studies and
efficacy studies, species of rodent, dog and non-human primate
families are especially preferred. Preferred rodent species include
the mouse and the rat. Preferred non-human primates include Rhesus
monkeys, chimpanzees and long-tailed macaques.
[3358] In one embodiment, the antibody used according to the
invention is also cross-reactive with the target protein of at
least one additional species in addition to being cross-reactive
with the human target protein, said additional species being
selected from the group of species consisting of the mouse, the rat
and the long-tailed macaque (Macaca fascicularis). Antibodies that
are used according to the invention and are cross-reactive at least
with the mouse target protein in addition to being cross-reactive
with the human target protein are preferred in particular.
Cross-reactive antibodies whose affinity for the target protein of
the additional non-human species does not differ from the affinity
for the human target protein by more than a factor of 50, in
particular not more than a factor of 10 are preferred.
[3359] EGFR Antibodies
[3360] Examples of antibodies that bind the cancer target molecule
EGFR include cetuximab (INN No. 7906), panitumumab (INN No. 8499)
and nimotuzumab (INN No. 8545). Cetuximab (Drug Bank Accession No.
DB00002) is a chimeric anti-EGFR1 antibody that is produced in
SP2/0 mouse myeloma cells and is distributed by ImClone Systems
Inc., Merck KGaA/Bristol Myers Squibb Co. Cetuximab is indicated
for treatment of metastatic EGFR-expressing colorectal carcinoma
with the wild-type K-Ras gene. It has an affinity of 10.sup.-10
M.
[3361] Sequence:
[3362] Cetuximab light chain (kappa):
TABLE-US-00028 DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKY
ASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGA
GTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
[3363] Cetuximab heavy chain:
TABLE-US-00029 QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGV
IWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALT
YYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[3364] Panitumumab (INN No. 8499) (Drug Bank Accession No. DB01269)
is a recombinant monoclonal human IgG2 antibody that binds
specifically to human EGF receptor 1 and is distributed by
Abgenix/Amgen. Panitumumab originates from the immunization of
transgenic mice (XenoMouse). These mice are capable of producing
human immunoglobulins (light and heavy chains). A special B-cell
clone that produces antibodies to EGFR was selected and was
immortalized with CHO cells (Chinese hamster ovary cells). These
cells are now being used for the production of a 100% human
antibody. Panitumumab is indicated for the treatment of
EGFR-expressing, metastatic colorectal carcinoma, which is
refractory to chemotherapeutic treatment with fluoropyrimidine,
oxaliplatin and irinotecan. It has an affinity of 10.sup.-11 M.
[3365] Sequence:
[3366] Panitumumab light chain (kappa):
TABLE-US-00030 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYD
ASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGG
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
[3367] Panitumumab heavy chain:
TABLE-US-00031 QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWI
GHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRD
RVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTY
TCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLM
ISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV
VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
[3368] Nimotuzumab (INN No. 8545) (EP 00586002, EP 00712863) is a
humanized monoclonal IgG1 antibody that binds specifically to the
human EGF receptor 1 and is distributed by YM BioSciences Inc.
(Mississauga, Canada). It is produced in non-secreting NSO cells
(mammalian cell line). Nimotuzumab has been approved for treatment
of head and neck tumors, highly malignant astrocytomas and
glioblastoma multiforme (not in EU or US) and pancreatic cancer
(orphan drug, EMA). It has an affinity of 10.sup.-8 M.
[3369] Additional embodiments of EGFR antibodies include: [3370]
Zalutumumab/2F8/HuMax-EGFr, Genmab Co. A/S (WO 02100348, WO
2004-056847, INN No. 8605) [3371] Necitumumab/11F8,
ImClone/IMC-11F8, ImClone Systems Inc. (Eli Lilly & Co.) (WO
2005090407, (EP 01735348-A1, US 20070264253-A1, U.S. Ser. No.
07/598,350, WO 2005-090407 A1), INN No. 9083) [3372]
Matuzumab/anti-EGFR mAb, Merck KGaA/anti-EGFR mAb, Takeda/EMD
72000/EMD-6200/EMD-72000 and EMD-55900/mAb 425/monoclonal antibody
425, Merck KGaA/Takeda (WO 09215683, INN No. 8103 (matuzumab))
[3373]
RG-7160/GA-201/GA201/R-7160/R7160/RG7160/RO-4858696/RO-5083945/RO4-
858696/RO5083945, Glycart Biotechnology AG (Roche Holding AG) (WO
2010-112413 A1, WO 2010115554) [3374] GT-MAB 5.2-GEX/CetuGEX,
Glycotope GmbH (WO 2008028686 A2, EP 01900750 A1, EP 01911766 A1,
EP 02073842 A2, US 20100028947 A1) [3375] ISU-101, Isu Abxis Inc.
(ISU Chemical Co. Ltd.)/Scancell (patent: WO 2008-004834 A1) [3376]
ABT-806/mAb 806/ch-806/anti-EGFR monoclonal antibody 806, Ludwig
Institute for Cancer Research/Abbott/Life Science Pharmaceuticals
(WO02092771, WO2005-081854 and WO-2009023265) [3377] SYM-004
(consists of two chimeric IgG1 antibodies (992 and 1024)),
Symphogen A/S (WO 2010022736 A2) [3378] MR1-1/MR1-1KDEL, WAX Corp
(Teva Pharmaceutical Industries Ltd.) (Duke University), (patent:
WO 2001062931 A2) [3379] Antibodies to the deletion mutant,
EGFRvIII, Amgen/Abgenix (WO 2005010151, U.S. Ser. No. 07/628,986)
[3380] SC-100, Scancell Ltd. (WO-2001088138-A1) [3381] MDX-447/EMD
82633/BAB-447/H 447/MAb, EGFR, Medarex/Merck KGaA, Bristol-Myers
Squibb (US)/Merck KGaA (DE)/Takeda (JP), (WO 09105871, WO 09215683)
[3382] Anti-EGFR mAb, Xencor (WO 2005056606) [3383]
DXL-1218/anti-EGFR monoclonal antibody (cancer), InNexus, InNexus
Biotechnology Inc., pharmaceutical projects PH048638
[3384] In a preferred embodiment, the anti-EGFR antibodies are
selected from the group consisting of cetuximab, panitumumab,
nimotuzumab, zalutumumab, necitumumab, matuzumab, RG-716, GT-MAB
5.2-GEX, ISU-101, ABT-806, SYM-004, MR1-1, SC-100, MDX-447 and
DXL-1218.
[3385] In an especially preferred embodiment, the anti-EGFR
antibodies are selected from the group consisting of cetuximab,
panitumumab, nimotuzumab, zalutumumab, necitumumab and
matuzumab.
[3386] Those skilled in the art will be familiar with methods with
which additional antibodies having a similar or better affinity
and/or specificity for the target molecule can be synthesized from
the CDR regions of the aforementioned antibodies by sequence
variations.
[3387] In another embodiment, the anti-EGFR antibodies or
antigen-binding antibody fragments are selected from the group
consisting of
[3388] antibodies or antigen-binding antibody fragments comprising
the three CDR regions of the light chain and the three CDR regions
of the heavy chain of one of the following antibodies: cetuximab,
panitumumab, nimotuzumab, zalutumumab, necitumumab, matuzumab,
RG-716, GT-MAB 5.2-GEX, ISU-101, ABT-806, SYM-004, MR1-1, SC-100,
MDX-447, and DXL-1218.
[3389] In another preferred embodiment, the anti-EGFR antibodies or
antigen-binding antibody fragments are selected from the group
consisting of
[3390] antibodies or antigen-binding antibody fragments comprising
the three CDR regions of the light chain and the three CDR regions
of the heavy chain of one of the following antibodies: cetuximab,
panitumumab, nimotuzumab, zalutumumab, necitumumab, matuzumab.
[3391] Carboanhydrase IX Antibodies
[3392] Especially preferred binders according to the invention
include anti-CAIX antibodies, in particular human or humanized
anti-CAIX antibodies. The antibodies preferably have an affinity of
at least 10.sup.-7 M (as the Kd value, i.e., preferably those with
Kd values of less than 10.sup.-7 M), preferably of at least
10.sup.-8 M, especially preferably in the range of 10.sup.-9 M to
10.sup.-11 M. The Kd values can be determined, for example, by
surface plasmon resonance spectroscopy.
[3393] The antibody-drug conjugates according to the invention also
have affinities in these ranges. The affinity is preferably not
influenced significantly by conjugation of the active ingredients
(the affinity is usually reduced less than one order of magnitude,
i.e., from max. 10.sup.-8 M to 10.sup.-7 M, for example).
[3394] The antibodies used according to the invention are also
characterized preferably by a high selectivity. A high selectivity
occurs when the antibodies according to the invention have a better
affinity for the target protein by a factor of at least 2, a factor
of 5, a factor of 10 or especially preferably a factor of 100 than
the affinity for another independent antigen, e.g., human serum
albumin (the affinity can be determined, for example, by surface
plasmon resonance spectroscopy).
[3395] Furthermore, the antibodies used according to the invention
are preferably cross-reactive. To facilitate preclinical trials,
e.g., toxicological or efficacy studies (e.g., in xenograft mice),
and to be better able to interpret them, it is advantageous if the
antibodies used according to the invention bind not only the human
target protein but also bind the species target protein in the
species used for the studies. In one embodiment, the antibody used
according to the invention is cross-reactive with the target
protein of at least one species in addition to the human target
protein. For toxicological studies and efficacy studies, the
preferred species for use are those of the rodent, dog and
non-human primate families. Preferred rodent species include the
mouse and the rate. Preferred non-human primates include Rhesus
monkeys, chimpanzees and long-tailed macaques.
[3396] In one embodiment, the antibody used according to the
invention is cross-reactive with the target protein of at least one
additional species selected from the group of species consisting of
mouse, rat and long-tailed macaque (Macaca fascicularis) in
addition to being cross-reactive with the human target protein.
Especially preferred are antibodies that can be used according to
the invention and are at least cross-reactive with the mouse target
protein in addition to being cross-reactive with the human target
protein. The preferred cross-reactive antibodies are those whose
affinity for the target protein of the additional non-human species
does not differ from the affinity for the human target protein by a
factor of more than 50, in particular no more than a factor of 10.
Anti-CAIX antibodies include those described, for example, in WO
2007/070538 A2. These antibodies may be used according to the
invention.
[3397] Examples of antibodies that bind the cancer target molecule
carboanhydrase IX are described in WO 2007/070538 A2 (e.g., claims
1-16).
[3398] In a preferred embodiment, the anti-carboanhydrase IX
antibodies or antigen-binding antibody fragments are selected from
the group consisting of anti-carboanhydrase IX antibodies or
antigen-binding antibody fragments 3ee9 (claim 4 (a) in WO
2007070538 A2), 3ef2 (claim 4 (b) in WO 2007070538 A2), 1e4 (claim
4 (c) in WO 2007070538 A2), 3a4 (claim 4 (d) in WO 2007070538 A2),
3ab4 (claim 4 (e) in WO 2007070538 A2), 3ah10 (claim 4 (f) in WO
2007070538 A2), 3bb2 (claim 4 (g) in WO 2007070538 A2), 1aa1 (claim
4 (h) in WO 2007070538 A2), 5a6 (claim 4 (i) in WO 2007070538 A2)
and 5aa3 (claim 4 (j) in WO 2007070538 A2).
[3399] In a preferred embodiment, the anti-carboanhydrase IX
antibodies or antigen-binding antibody fragments are selected from
the group consisting of:
[3400] anti-carboanhydrase IX antibodies or antigen-binding antigen
fragments thereof, which comprise the sequences of the three CDR
regions of the light chain and the sequences of the three CDR
regions of the heavy chain of the antibody 3ee9 (from WO 2007070538
A2),
[3401] anti-carboanhydrase IX antibodies or antigen-binding antigen
fragments thereof, which comprise the sequences of the three CDR
regions of the light chain and the sequences of the three CDR
regions of the heavy chain of the antibody 3ef2 (from WO 2007070538
A2),
[3402] anti-carboanhydrase IX antibodies or antigen-binding antigen
fragments thereof, which comprise the sequences of the three CDR
regions of the light chain and the sequences of the three CDR
regions of the heavy chain of the antibody 1e4 (from WO 2007070538
A2),
[3403] anti-carboanhydrase IX antibodies or antigen-binding antigen
fragments thereof, which comprise the sequences of the three CDR
regions of the light chain and the sequences of the three CDR
regions of the heavy chain of the antibody 3a4 (from WO 2007070538
A2),
[3404] anti-carboanhydrase IX antibodies or antigen-binding antigen
fragments thereof, which comprise the sequences of the three CDR
regions of the light chain and the sequences of the three CDR
regions of the heavy chain of the antibody 3ab4 (from WO 2007070538
A2),
[3405] anti-carboanhydrase IX antibodies or antigen-binding antigen
fragments thereof, which comprise the sequences of the three CDR
regions of the light chain and the sequences of the three CDR
regions of the heavy chain of the antibody 3ah10 (from WO
2007070538 A2),
[3406] anti-carboanhydrase IX antibodies or antigen-binding antigen
fragments thereof, which comprise the sequences of the three CDR
regions of the light chain and the sequences of the three CDR
regions of the heavy chain of the antibody 3bb2 (from WO 2007070538
A2),
[3407] anti-carboanhydrase IX antibodies or antigen-binding antigen
fragments thereof, which comprise the sequences of the three CDR
regions of the light chain and the sequences of the three CDR
regions of the heavy chain of the antibody 1aa1 (from WO 2007070538
A2),
[3408] anti-carboanhydrase IX antibodies or antigen-binding antigen
fragments thereof, which comprise the sequences of the three CDR
regions of the light chain and the sequences of the three CDR
regions of the heavy chain of the antibody 5a6 (from WO 2007070538
A2) and
[3409] anti-carboanhydrase IX antibodies or antigen-binding antigen
fragments thereof, which comprise the sequences of the three CDR
regions of the light chain and the sequences of the three CDR
regions of the heavy chain of the antibody 5aa3 (from WO 2007070538
A2).
[3410] The given sequences of the CDR regions are shown in FIGS.
2a-2c, pages 128-130 in WO 2007070538 A2.
[3411] In a preferred embodiment, the anti-carboanhydrase IX
antibodies or antigen-binding antibody fragments are selected from
the group consisting of:
[3412] an antibody or antigen-binding fragment consisting of the
amino acid sequence of the variable light and variable heavy chains
of the antibody 3ee9, as defined in WO 2007070538 A2 in FIG. 4b on
page 137,
[3413] an antibody or antigen-binding fragment consisting of the
amino acid sequence of the variable light and variable heavy chains
of the antibody 3ef2, as defined in WO 2007070538 A2 in FIG. 4c on
page 138 and/or in FIG. 4b on page 137,
[3414] an antibody or antigen-binding fragment consisting of the
amino acid sequence of the variable light and variable heavy chains
of the antibody 1e4, as defined in WO 2007070538 A2 in FIG. 4a on
page 136,
[3415] an antibody or antigen-binding fragment consisting of the
amino acid sequence of the variable light and variable heavy chains
of the antibody 3a4, as defined in WO 2007070538 A2 in FIG. 4a on
page 136,
[3416] an antibody or antigen-binding fragment consisting of the
amino acid sequence of the variable light and variable heavy chains
of the antibody 3ab4, as defined in WO 2007070538 A2 in FIG. 4a on
page 136,
[3417] an antibody or antigen-binding fragment consisting of the
amino acid sequence of the variable light and variable heavy chains
of the antibody 3ah10, as defined in WO 2007070538 A2 in FIG. 4a on
page 136,
[3418] an antibody or antigen-binding fragment consisting of the
amino acid sequence of the variable light and variable heavy chains
of the antibody 3bb2, as defined in WO 2007070538 A2 in FIG. 4b on
page 137,
[3419] an antibody or antigen-binding fragment consisting of the
amino acid sequence of the variable light and variable heavy chains
of the antibody 1aa1, as defined in WO 2007070538 A2 in FIG. 4a on
page 136,
[3420] an antibody or antigen-binding fragment consisting of the
amino acid sequence of the variable light and variable heavy chains
of the antibody 5a6, as defined in WO 2007070538 A2 in FIG. 4b on
page 137, and
[3421] an antibody or antigen-binding fragment consisting of the
amino acid sequence of the variable light and variable heavy chains
of the antibody 5aa3, as defined in WO 2007070538 A2 in FIG. 4b on
page 137.
[3422] In an especially preferred embodiment, the
anti-carboanhydrase IX antibody is the antibody 3ee9 from WO
2007070538 A2.
[3423] In an especially preferred embodiment, the
anti-carboanhydrase IX antibody or he antigen-binding antibody
fragment comprises the amino acid sequences of the CDR regions of
the variable heavy chain of the antibody 3ee9 (VH3-CDR1:
GFTFSSYGMS; VH3-CDR2: GISSLGSTTYYADSVKG; VH3-CDR3: TGSPGTFMHGDH,
see FIG. 2a, page 128 in WO 2007070538 A2) and the amino acid
sequences of the CDR regions of the variable light chain of the
antibody 3ee9 (VLk1-CDR1: RASQDINNYLS; VLk1-CDR2: YGASNLQS;
VLk1-CDR3: QQYYGRPT, see FIG. 2b, page 129 in WO 2007070538
A2).
[3424] In an especially preferred embodiment, the
anti-carboanhydrase IX antibody or the antigen-binding antibody
fragment comprises the amino acid sequences of the variable heavy
chain of the antibody 3ee9
[3425] (VH3:ELVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVSGISSLGST
TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTGSPGTFMHHGDHWGQ GTLVTVSS,
see FIG. 4b, page 137 in WO 2007070538 A2) and the amino acid
sequences of the variable light chain of the antibody 3ee9
[3426]
(VLk1:DIQMTQSPSSLSASVGDRVTITCRaSQDINNYLSWYQQKPGKAPKLLIYGASNLQS
GVPSRFSGSGSGTDFTLTISLQPEDFAVYYCQQYYGRPTTFGQGTKVEIKRT, see FIG. 4b,
page 137 in WO 2007070538 A2).
[3427] In a preferred embodiment, the anti-carboanhydrase IX
antibody 3ee9 is a IgG antibody.
[3428] In an especially preferred embodiment, the
anti-carboanhydrase IX antibody 3ee9 is an IgG1 antibody
(3ee9-IgG1),
[3429] wherein the amino acid sequence of the heavy chain comprises
the following sequence:
TABLE-US-00032 QVELVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVSG
ISSLGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTG
SPGTFMHGDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K
[3430] and the amino acid sequence of the light chain comprises the
following sequence:
TABLE-US-00033 DIQMTQSPSSLSASVGDRVTITCRASQDINNYLSWYQQKPGKAPKLLIYG
ASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYYGRPTTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
[3431] anti-carboanhydrase IX antibody 3ee9-IgG1:
[3432] Another aspect of the present invention is supplying the
anti-carboanhydrase IX antibody 3ee9-IgG1.
[3433] C4.4a Antibody:
[3434] Especially preferred binders according to the invention are
anti-C4.4a antibodies, in particular human or humanized anti-C4.4a
antibodies. These antibodies have an affinity of preferably at
least 10.sup.-7 M (as Kd value, i.e., preferably those with Kd
values of less than 10.sup.-7 M), especially at least 10.sup.-8 M,
most especially preferably in the range of 10.sup.-9 M to
10.sup.-11 M. The Kd values can be determined by surface plasmon
resonance spectroscopy, for example.
[3435] The antibody-drug conjugates according to the invention also
have affinities in these ranges. Through conjugation of the active
ingredients, the affinity is preferably not influenced
significantly (the affinity is usually reduced by less than one
order of magnitude, e.g., max. from 10.sup.-8M to 10.sup.-7M).
[3436] The antibodies used according to the invention are also
preferably characterized by a high selectivity. A high selectivity
occurs when the antibody according to the invention has a better
affinity for the target protein than for another independent
antigen, e.g., human serum albumin by a factor of at least 2,
preferably by a factor of 5 or in particular preferably a factor of
10 (the affinity can be determined, for example, by surface plasmon
resonance spectroscopy).
[3437] Furthermore, the antibodies to be used according to the
invention are preferably cross-reactive. To facilitate preclinical
trials, e.g., toxicological studies or efficacy studies (e.g., in
xenograft mice) and to be able to interpret them better, it is
advantageous if the antibody to be used according to the invention
not only binds the human target protein but also binds the species
target protein in the species used for the studies. In one
embodiment, the antibody used according to the invention is
additionally cross-reactive with the target protein of at least one
other species in addition to the human target protein. For
toxicological studies and efficacy studies, species of the rodent,
dog and non-human primate families are preferably used. Preferred
rodent species include the mouse and the rat. Preferred non-human
primates include Rhesus monkeys, chimpanzees and long-tailed
macaques.
[3438] In one embodiment, the antibody used according to the
invention is also cross-reactive with the target protein of at
least one other species in addition to being cross-reactive with
the human target protein, said additional species being selected
from the group of species consisting of the mouse, rat and
long-tailed macaque (Macaca fascicularis). Especially preferred
antibodies for use according to the invention include those that
are cross-reactive with at least the mouse target protein in
addition to being cross-reactive with the human target protein. The
preferred cross-reactive antibodies are those whose affinity for
the target protein of the additional non-human species does not
differ from the affinity for the human target protein by a factor
of more than 50, in particular more than 10.
[3439] Anti-C4.4a antibodies are described in WO 01/23553 or WO
2011070088, for example. These antibodies may be used according to
the present invention.
[3440] Examples of C4.4a antibodies and antigen-binding fragments
are described below. The sequences of the antibodies are given in
Table 1, where each row shows the respective CDR amino acid
sequences of the variable light chain and/or of the variable heavy
chain of the antibody listed in column 1. This table also shows the
amino acid sequences of the variable light chain and of the
variable heavy chain and also the amino acid sequence of the
respective antibody listed lists in column 1.
[3441] In one embodiment, the anti-C4.4a antibodies or the
antigen-binding antibody fragments bind to the S1 domain S1 (amino
acid positions 1-85 of SEQ ID NO: 1) of C4.4a.
[3442] In one embodiment, the anti-C4.4a antibodies or the
antigen-binding antibody fragments have cross-reactivity with human
C4.4a (SEQ ID NO: 1) and with murine C4.4a (SEQ ID NO: 2).
[3443] In one exemplary embodiment, the anti-C4.4a antibodies or
the antigen-binding antibody fragments thereof are internalized by
the cell after binding to a C4.4a-expressing cell.
[3444] In another embodiment, the anti-C4.4a antibodies or the
antigen-binding antibody fragments compete with the antibody
M31-B01 and/or with the antibody M20-D02-S-A for binding to C4.4a.
Antibodies M31-B01 and M20-D02-S-A compete for binding to C4.4a.
Antibodies B01-1 to B01-12 were synthesized by affinity maturation
from M31-B01 and compete with M31-B01 for binding to C4.4a. The
antibodies D02-1 through D02-13 were synthesized by affinity
maturation from M20-D02-S-A and compete with M20-D02-S-A for
binding to C4.4a.
[3445] In another embodiment, the anti-4.4a antibodies or the
antigen-binding antibody fragments comprise at least one, two or
three of the CDR amino acid sequences listed in Table 1 or Table
2.
[3446] In another embodiment, the anti-4.4a antibodies or the
antigen-binding antibody fragments comprise at least one, two or
three CDR amino acid sequences of an antibody listed in Table 1 or
Table 2.
[3447] In another embodiment, the anti-4.4a antibodies or the
antigen-binding antibody fragments comprise at least one, two or
three CDR amino acid sequences of the variable light chain and at
least one, two or three CDR amino acid sequences of the variable
heavy chain of an antibody listed in Table 1 or Table 2.
[3448] In another embodiment, the anti-4.4a antibodies or the
antigen-binding antibody fragments, which are at least 50%, 60%,
70%, 80%, 90% or 95% identical to the CDR amino acid sequences of
the variable light chain and are identical with the CDR amino acid
sequences of the variable heavy chain comprise an antibody as
listed in Table 1 or Table 2.
[3449] In another embodiment, the CDR sequences of the anti-C4.4a
antibodies or of the antigen-binding fragments comprise:
[3450] CDR sequences of the heavy chain, which conform to CDR
sequences SEQ ID NO: 297 (CDR H1), SEQ ID NO: 298 (CDR H2) and SEQ
ID NO: 299 (CDR H3), and CDR sequences of the light chain, which
conform to CDR sequences SEQ ID NO: 300 (CDR L1), SEQ ID NO: 22
(CDR L2) and SEQ ID NO: 301 (CDR L3), or
[3451] CDR sequences of the heavy chain, which conform to CDR
sequences SEQ ID NO: 302 (CDR H1), SEQ ID NO: 303 (CDR H2) and SEQ
ID NO: 304 (CDR H3) and CDR sequences of the light chain, which
conform to CDR sequences SEQ ID NO: 305 (CDR L1), SEQ ID NO: 306
(CDR L2) and SEQ ID NO: 307 (CDR L3).
[3452] In another embodiment, the anti-C4.4a antibodies or
antigen-binding antibody fragments which are at least 50%, 60%,
70%, 80%, 90% or 95% identical to the variable light chain and to
the variable heavy chain comprise an antibody as listed in Table 1
or Table 2.
[3453] In another embodiment, the anti-C4.4a antibodies or
antigen-binding antibody fragments comprise the three CDR amino
acid sequences of the variable light chain and the three CDR amino
acid sequences of the variable heavy chain as listed in Table 1 or
Table 2.
[3454] In another embodiment, the anti-C4.4a antibodies or
antigen-binding antibody fragments comprise a variable light chain
and/or a variable heavy chain of an antibody as listed in Table 1
or Table 2.
[3455] In another embodiment, the anti-C4.4a antibodies or
antigen-binding antibody fragments comprise the variable light
chain and the variable heavy chain of an antibody as listed in
Table 1 or Table 2.
[3456] In a preferred embodiment, the C4.4a antibodies and the
antigen-binding antibody fragments are selected from the group
consisting of
[3457] antibody comprising the CDR sequences of the variable heavy
chain represented by SEQ ID NO: 75-77 and which reflects the CDR
sequences of the variable light chain, as represented by sequence
SEQ ID NOS: 78-80 (B01-10),
[3458] antibody comprising the CDR sequences of the variable heavy
chain represented by the sequences SEQ ID NOS: 5, 9 and 13 and
comprising the CDR sequences of the variable light chain
represented by the sequences SEQ ID NOS: 17, 21 and 25
(M31-B01),
[3459] antibody comprising the CDR sequences of the variable heavy
chain represented by the sequences SEQ ID NOS: 6, 10 and 14 and
comprising the CDR sequences of the variable light chain
represented by the sequences SEQ ID NOS: 18, 22 and 26
(M20-D02-S-A),
[3460] antibody comprising the CDR sequences of the variable heavy
chain represented by the sequences SEQ ID NOS: 7, 11 and 15 and
comprising the CDR sequences of the variable light chain
represented by the sequences SEQ ID NOS: 19, 23 and 27
(M60-G03),
[3461] antibody comprising the CDR sequences of the variable heavy
chain represented by the sequences SEQ ID NOS: 8, 12 and 16 and
comprising the CDR sequences of the variable light chain
represented by the sequences SEQ ID NOS: 20, 24 and 28
(36-H02),
[3462] antibody comprising the CDR sequences of the variable heavy
chain represented by the sequences SEQ ID NOS: 45-47 and comprising
the CDR sequences of the variable light chain represented by the
sequences SEQ ID NOS: 48-50 (B01-3),
[3463] antibody comprising the CDR sequences of the variable heavy
chain represented by the sequences SEQ ID NOS: 55-57 and comprising
the CDR sequences of the variable light chain represented by the
sequences SEQ ID NOS: 58-60 (B01-5),
[3464] antibody comprising the CDR sequences of the variable heavy
chain represented by the sequences SEQ ID NOS: 65-67 and comprising
the CDR sequences of the variable light chain represented by the
sequences SEQ ID NOS: 68-70 (B01-7),
[3465] antibody comprising the CDR sequences of the variable heavy
chain represented by the sequences SEQ ID NOS: 85-87 and comprising
the CDR sequences of the variable light chain represented by the
sequences SEQ ID NOS: 88-90 (B01-12),
[3466] antibody comprising the CDR sequences of the variable heavy
chain represented by the sequences SEQ ID NOS: 95-97 and comprising
the CDR sequences of the variable light chain represented by the
sequences SEQ ID NOS: 98-100 (D02-4),
[3467] antibody comprising the CDR sequences of the variable heavy
chain represented by the sequences SEQ ID NOS: 105-107 and
comprising the CDR sequences of the variable light chain
represented by the sequences SEQ ID NOS: 108-110 (D02-6),
[3468] antibody comprising the CDR sequences of the variable heavy
chain represented by the sequences SEQ ID NOS: 115-117 and
comprising the CDR sequences of the variable light chain
represented by the sequences SEQ ID NOS: 118-120 (D02-7),
[3469] antibody comprising the CDR sequences of the variable heavy
chain represented by the sequences SEQ ID NOS: 125-127 and
comprising the CDR sequences of the variable light chain
represented by the sequences SEQ ID NOS: 128-130 (D02-11) and
[3470] antibody comprising the CDR sequences of the variable heavy
chain represented by the sequences SEQ ID NOS: 135-137 and
comprising the CDR sequences of the variable light chain
represented by the sequences SEQ ID NOS: 138-140 (D02-13).
[3471] In a preferred embodiment, the C4.4a antibodies and the
antigen-binding antibody fragments are selected from the group
consisting of antibodies comprising the amino acid sequence of the
variable heavy chain represented by the sequence SEQ ID NO: 81 and
comprising the amino acid sequence of the variable light chain
represented by the sequence SEQ ID NO: 82 (B01-7), antibodies
comprising the amino acid sequence of the variable heavy chain
represented by the sequence SEQ ID NOS: 33 and comprising the amino
acid sequence of the variable light chain represented by the
sequence SEQ ID NO: 29 (M31-B01), antibodies comprising the amino
acid sequence of the variable heavy chain represented by the
sequence SEQ ID NO: 34 and comprising the amino acid sequence of
the variable light chain represented by the sequence SEQ ID NO: 30
(M20-D02 S-A), antibodies comprising the amino acid sequence of the
variable heavy chain represented by the sequence SEQ ID NO: 35 and
comprising the amino acid sequence of the variable light chain
represented by the sequence SEQ ID NO: 31 (M60-G03), antibodies
comprising the amino acid sequence of the variable heavy chain
represented by the sequence SEQ ID NO: 36 and comprising the amino
acid sequence of the variable light chain represented by the
sequence SEQ ID NO: 32 (M36-H02), antibodies comprising the amino
acid sequence of the variable heavy chain represented by the
sequence SEQ ID NO: 51 and comprising the amino acid sequence of
the variable light chain represented by the sequence SEQ ID NO: 52
(B01-3), antibodies comprising the amino acid sequence of the
variable heavy chain represented by the sequence SEQ ID NO: 61 and
comprising the amino acid sequence of the variable light chain
represented by the sequence SEQ ID NO: 62 (B01-5), antibodies
comprising the amino acid sequence of the variable heavy chain
represented by the sequence SEQ ID NO: 71 and comprising the amino
acid sequence of the variable light chain represented by the
sequence SEQ ID NO: 72 (B01-7), antibodies comprising the amino
acid sequence of the variable heavy chain represented by the
sequence SEQ ID NO: 91 and comprising the amino acid sequence of
the variable light chain represented by the sequence SEQ ID NO: 92
(B01-12), antibodies comprising the amino acid sequence of the
variable heavy chain represented by the sequence SEQ ID NO: 101 and
comprising the amino acid sequence of the variable light chain
represented by the sequence SEQ ID NO: 102 (D02-4), antibodies
comprising the amino acid sequence of the variable heavy chain
represented by the sequence SEQ ID NO: 111 and comprising the amino
acid sequence of the variable light chain represented by the
sequence SEQ ID NO: 112 (D02-6), antibodies comprising the amino
acid sequence of the variable heavy chain represented by the
sequence SEQ ID NO: 121 and comprising the amino acid sequence of
the variable light chain represented by the sequence SEQ ID NO: 122
(D02-7), antibodies comprising the amino acid sequence of the
variable heavy chain represented by the sequence SEQ ID NO: 131 and
comprising the amino acid sequence of the variable light chain
represented by the sequence SEQ ID NO: 132 (D02-11) and antibodies
comprising the amino acid sequence of the variable heavy chain
represented by the sequence SEQ ID NO: 141 and comprising the amino
acid sequence of the variable light chain represented by the
sequence SEQ ID NO: 142 (D02-13).
[3472] In another embodiment, the anti-C4.4a antibodies comprise
the light chain and the heavy chain of an antibody as listed in
Table 2.
[3473] In a preferred embodiment, the anti-C4.4a antibodies
comprise the light chain and the heavy chain of an antibody as
listed in Table 2.
[3474] In an especially preferred embodiment, the C4.4a antibody is
selected from the group consisting of:
[3475] an antibody comprising the amino acid sequence of the light
chain represented by SEQ ID NO: 346 and comprising the amino acid
sequence of the heavy chain represented by SEQ ID NO: 347
(M31-B01),
[3476] an antibody comprising the amino acid sequence of the light
chain represented by SEQ ID NO: 352 and comprising the amino acid
sequence of the heavy chain represented by SEQ ID NO: 353
(B01-3),
[3477] an antibody comprising the amino acid sequence of the light
chain represented by SEQ ID NO: 364 and comprising the amino acid
sequence of the heavy chain represented by SEQ ID NO: 365 (B01-10)
and
[3478] an antibody comprising the amino acid sequence of the light
chain represented by SEQ ID NO: 382 and comprising the amino acid
sequence of the heavy chain represented by SEQ ID NO: 383
(D02-6).
TABLE-US-00034 TABLE 1 Sequences of the C4.4a antibodies SEQ ID SEQ
ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: SEQ ID NO:
NO: NO: NO: NO: NO: NO: NO: NO: VH VL Antibody HCDR1 HCDR2 HCDR3
LCDR1 LCDR2 LCDR3 VH protein VL protein nucleotide nucleotide
M31-B01 5 9 13 17 21 25 33 29 41 37 M20-D02 6 10 14 18 22 26 34 30
42 38 S-A M60-G03 7 11 15 19 23 27 35 31 43 39 M36-H02 8 12 16 20
24 28 36 32 44 40 B01-3 45 46 47 48 49 50 51 52 53 54 B01-5 55 56
57 58 59 60 61 62 63 64 B01-7 65 66 67 68 69 70 71 72 73 74 B01-10
75 76 77 78 79 80 81 82 83 84 B01-12 85 86 87 88 89 90 91 92 93 94
D02-4 95 96 97 98 99 100 101 102 103 104 D02-6 105 106 107 108 109
110 111 112 113 114 D02-7 115 116 117 118 119 120 121 122 123 124
D02-11 125 126 127 128 129 130 131 132 133 134 D02-13 135 136 137
138 139 140 141 142 143 144 B01-nn1 145 146 147 148 149 150 151 152
308 309 B01-nn2 153 154 155 156 157 158 159 160 310 311 B01-nn3 161
162 163 164 165 166 167 168 312 313 B01-nn4 169 170 171 172 173 174
175 176 314 315 B01-nn5 177 178 179 180 181 182 183 184 316 317
B01-2 185 186 187 188 189 190 191 192 318 319 B01-4 193 194 195 196
197 198 199 200 320 321 B01-6 201 202 203 204 205 206 207 208 322
323 B01-8 209 210 211 212 213 214 215 216 324 325 B01-9 217 218 219
220 221 222 223 224 326 327 B01-11 225 226 227 228 229 230 231 232
328 329 B01-12 233 234 235 236 237 238 239 240 330 331 D02-og1 241
242 243 244 245 246 247 248 332 333 D02-5 249 250 251 252 253 254
255 256 334 335 D02-8 257 258 259 260 261 262 263 264 336 337 D02-9
265 266 267 268 269 270 271 272 338 339 D02-10 273 274 275 276 277
278 279 280 340 341 D02-11 281 282 283 284 285 286 287 288 342 343
D02-12 289 290 291 292 293 294 295 296 344 345
TABLE-US-00035 TABLE 2 Sequences of the light and heavy chains of
the C4.4a antibodies Light chain Heavy chain Antibody SEQ ID NO:
SEQ ID NO: M31-B01 346 347 B01-1 348 349 B01-2 350 351 B01-3 352
353 B01-4 354 355 B01-5 356 357 B01-6 358 359 B01-7 360 361 B01-8
362 363 B01-10 364 365 B01-11 366 367 B01-12 368 369 M20-D02 S-A
370 371 D02-1 372 373 D02-2 374 375 D02-3 376 377 D02-4 378 379
D02-5 380 381 D02-6 382 383 D02-7 384 385 D02-8 386 387 D02-9 388
389 D02-10 390 391 D02-11 392 393 D02-12 394 395 D02-13 396 397
[3479] Anti-C4.4a Antibody IgG:
[3480] Another aspect of the present invention is providing an
anti-C4.4a IgG1 antibody comprising the amino acid sequence of the
light chain and the heavy chain of an antibody as listed in Table
2.
[3481] One example of an antibody that binds the cancer target
molecule HER2 is trastuzumab (Genentech). Trastuzumab is a
humanized antibody used for treatment of breast cancer, among other
things. An example of an antibody that binds the cancer target
molecule CD20 is rituximab (Genentech). Rituximab (CAS No.
174722-31-7) is a chimeric antibody used for treating non-Hodgkin's
lymphoma. One example of an antibody that binds the cancer target
molecule CD52 is alemtuzumab (Genzyme). Alemtuzumab (CAS No.
216503-57-0) is a humanized antibody that is used for treatment of
chronic lymphatic leukemia.
[3482] Mesothelin Antibody
[3483] According to the invention an especially preferred binders
are anti-mesothelin antibodies, in particular human or humanized
anti-mesothelin antibodies. The antibodies preferably have an
affinity of at least 10.sup.-7 M (as Kd value, i.e., preferably
those with Kd values less than 10.sup.-7 M), preferably of at least
10.sup.-8 M, especially preferably in the range from 10.sup.-9 M to
10.sup.-11 M. The Kd values can be determined by surface plasmon
resonance spectroscopy.
[3484] The antibody-drug conjugates according to the invention also
have affinities in these ranges. Through conjugation of the active
ingredients, the affinity is preferably not influenced
significantly (the affinity is usually reduced by less than one
order of magnitude, e.g., max. from 10.sup.-8M to 10.sup.-7M).
[3485] The antibodies used according to the invention are also
preferably characterized by a high selectivity. A high selectivity
occurs when the antibody according to the invention has a better
affinity for the target protein than for another independent
antigen, e.g., human serum albumin by a factor of at least 2,
preferably by a factor of 5 or in particular preferably a factor of
10 (the affinity can be determined, for example, by surface plasmon
resonance spectroscopy).
[3486] Furthermore, the antibodies used according to the invention
are preferably cross-reactive. To facilitate preclinical trials,
e.g., toxicological studies or efficacy studies (e.g., in xenograft
mice) and to be able to interpret them better, it is advantageous
if the antibody to be used according to the invention not only
binds the human target protein but also binds the species target
protein in the species used for these studies. In one embodiment,
the antibody used according to the invention which is
cross-reactive with the antibody used according to the invention
but is also cross-reactive with the human target protein of at
least one additional species. For toxicological studies and
efficacy studies, species of the rodent, dog and non-human primate
families are especially preferred. Preferred rodent species include
the mouse and the rat. Preferred non-human primates include Rhesus
monkeys, chimpanzees and long-tailed macaques.
[3487] In one embodiment, the antibody used according to the
invention is also cross-reactive with the target protein of at
least one additional species in addition to being cross-reactive
with the human target protein, said additional species being
selected from the group of species consisting of the mouse, the rat
and the long-tailed macaque (Macaca fascicularis). Preferred
antibodies in particular are those that are used according to the
invention and are cross-reactive with at least the mouse target
protein in addition to being cross-reactive with the human target
protein. The preferred cross-reactive antibodies are those whose
affinity for the target protein of the additional non-human species
does not differ from the affinity for the human target protein by
more than a factor of 50, in particular not more than a factor of
10.
[3488] The antibodies used according to the invention are
additionally preferably characterized by invariant binding to
mesothelin. Invariant binding is characterized, for example, in
that the antibody used according to the invention binds to an
epitope of mesothelin, which cannot be masked by another
extracellular protein. Such an additional extracellular protein is,
for example, the ovarian cancer antigen 125 protein (CA125). The
antibodies used are preferably characterized in that their binding
to mesothelin is not blocked by CA125.
[3489] Anti-mesothelin antibodies are described, for example, in WO
2009/068204. These antibodies may be used according to the
invention.
[3490] Another aspect of the present invention is providing a novel
anti-mesothelin antibody (MF-Ta) whose amino acid sequence
comprises the CDR sequences of the variable heavy chain represented
by the sequences SEQ ID NO:398 (HCDR1), SEQ ID NO:399 (HCDR2) and
SEQ ID NO:400 (HCDR3) and the CDR sequences of the variable light
chain represented by the sequences SEQ ID NO:401 (LCDR1), SEQ ID
NO:402 (LCDR2) and SEQ ID NO:403 (LCDR3).
[3491] In a preferred embodiment, the amino acid sequence of the
anti-mesothelin antibody MF-Ta or the antigen-binding antibody
fragment comprises a sequence of the variable heavy chain
represented by the sequences SEQ ID NO: 404 and the sequence of the
variable light chain represented by the sequence SEQ ID NO: 405. In
a preferred embodiment, the amino acid sequence of the
anti-mesothelin antibody MF-Ta or the antigen-binding antibody
fragment comprises the sequence of the variable heavy chain, which
is coded by the nucleic acid sequence SEQ ID NO: 406 and the
sequence of the variable light chain, which is coded by the nucleic
acid sequence SEQ ID NO: 407.
[3492] In an especially preferred embodiment, the amino acid
sequence of the anti-mesothelin antibody MF-Ta comprises the
sequence of the heavy chain represented by the sequences SEQ ID NO:
408 and the sequence of the light chain represented by the sequence
SEQ ID NO: 409.
[3493] In an especially preferred embodiment, the amino acid
sequence of the anti-mesothelin antibody MF-Ta comprises the
sequence of the heavy chain, which is coded by the nucleic acid
sequence SEQ ID NO: 410 and the sequence of the light chain, which
is coded by the nucleic acid sequence SEQ ID NO: 411.
[3494] Additional examples of antibodies that bind the cancer
target molecule mesothelin are familiar to those skilled in the art
and are described in WO 2009/068204, for example, and can be used
for the binder-drug conjugates according to the invention.
[3495] In one embodiment of the binder-drug conjugates, the binder
is an anti-mesothelin antibody or an antigen-binding antibody
fragment wherein the antibody binds to mesothelin and has invariant
binding.
[3496] In one embodiment, the binder-drug conjugate comprises an
anti-mesothelin antibody or an antigen-binding antibody fragment
comprises the amino acid sequences of the three CDR regions of the
light chain and the amino acid sequences of the three CDR regions
of the heavy chain of an antibody as described in WO 2009/068204 A1
(Table 7, pages 61-63).
[3497] In a preferred embodiment, the mesothelin antibodies or the
antigen-binding antibody fragments are selected from the group
consisting of:
[3498] anti-mesothelin antibodies or antigen-binding antibody
fragments which comprise the sequences of the three CDR regions of
the light chain and the sequences of the three CDR regions of the
heavy chain of the antibody MF-Ta,
[3499] anti-mesothelin antibodies or antigen-binding antigen
fragments thereof, which comprise the sequences of the three CDR
regions of the light chain and the sequences of the three CDR
regions of the heavy chain of the antibody MF-J (WO 2009068204 A1;
Table 7, page 61),
[3500] anti-mesothelin antibodies or antigen-binding antigen
fragments thereof, which comprise the sequences of the three CDR
regions of the light chain and the sequences of the three CDR
regions of the heavy chain of the antibody MOR06640 (WO 2009/068204
A1; Table 7, page 61),
[3501] anti-mesothelin antibodies or antigen-binding antigen
fragments thereof, which comprise the sequences of the three CDR
regions of the light chain and the sequences of the three CDR
regions of the heavy chain of the antibody MF-226 (WO 2009/068204
A1; Table 7, page 61), and
[3502] anti-mesothelin antibodies or antigen-binding antigen
fragments thereof, which comprise the sequences of the three CDR
regions of the light chain and the sequences of the three CDR
regions of the heavy chain of the antibody MOR06626 (WO
2009/068204-A1; Table 7, page 61).
[3503] In an especially preferred embodiment the mesothelin
antibodies or antigen-binding antibody fragments are selected from
the group
[3504] anti-mesothelin antibodies or antigen-binding antibody
fragments thereof comprising the sequence of the variable light
chain and the sequence of the variable heavy chain of the antibody
MF-Ta,
[3505] anti-mesothelin antibodies or antigen-binding antibody
fragments thereof comprising the sequence of the variable light
chain and the sequence of the variable heavy chain of the antibody
MF-J (WO 2009/068204 A1; Table 7, page 61),
[3506] anti-mesothelin antibodies or antigen-binding antibody
fragments thereof comprising the sequence of the variable light
chain and the sequence of the variable heavy chain of the antibody
MOR06640 (WO 2009/068204 A1; Table 7, page 61),
[3507] anti-mesothelin antibodies or antigen-binding antibody
fragments thereof comprising the sequence of the variable light
chain and the sequence of the variable heavy chain of the antibody
MF-226 (WO 2009/068204 A1; Table 7, page 61), and
[3508] anti-mesothelin antibodies or antigen-binding antibody
fragments thereof comprising the sequence of the variable light
chain and the sequence of the variable heavy chain of the antibody
MOR06626 (WO 2009/068204 A1; Table 7, page 61).
[3509] Additional Antibodies:
[3510] Trastuzumab (Genentech) is an example of antibody that binds
the cancer target molecule HER2. Trastuzumab is a humanized
antibody used for treatment of breast cancer among other things.
Rituximab (Genentech) is an example of an antibody that binds the
cancer target molecule CD20. Rituximab (CAS No. 174722-31-7) is a
chimeric antibody used for treatment of non-Hodgkin's lymphoma.
Alemtuzumab (Genzyme) is an example of an antibody that binds the
cancer target molecule CD52. Alemtuzumab (CAS No. 216503-57-0) is a
humanized antibody that is used for treatment of chronic lymphatic
leukemia.
[3511] Additional examples of antibodies that bind to HER2 in
addition to trastuzumab (INN No. 7637, CAS No. 180288-69-1) and
pertuzumab (CAS No.: 380610-27-5) also include antibodies such as
those proposed in WO 2009123894 A2, WO2008140603 A2 or WO
2011044368 A2. One example of anti-HER2 conjugate is trastuzumab
emtansine (INN No. 9295).
[3512] Examples of antibodies that bind the cancer target molecule
CD30 and can be used for treatment of cancer, e.g., Hodgkin's
lymphoma include brentuximab, iratumumab and antibodies as
disclosed in WO 2008092117, WO 2008036688 or WO 2006089232. An
example of an anti-CD30 conjugate is brentuximab vedotin (INN No.
9144).
[3513] Examples of antibodies that bind the cancer target molecule
CD22 and can be used for treatment of cancer, e.g., lymphoma
include inotuzumab or epratuzumab. Examples of anti-CD22 conjugates
include inotuzumab ozagamycin (INN No. 8574) or anti-CD22-MMAE and
anti-CD22-MC-MMAE (CAS No.: 139504-50-0 and/or 474645-27-7).
[3514] Examples of antibodies that bind the cancer target molecule
CD33 and can be used for treatment of cancer, e.g., leukemia
include gemtuzumab or lintuzumab (INN No. 7580). One example of an
anti-CD33 conjugate is gemtuzumab ozagamycin.
[3515] One example of an antibody that binds the cancer target
molecule NMB and can be used for treatment of cancer, e.g.,
melanoma or breast cancer is glembatumumab (INN No. 9199). One
example of an anti-NMB conjugate is glembatumumab vedotin (CAS No.:
474645-27-7).
[3516] One example of an antibody that binds the cancer target
molecule CD56 and can be used for treatment of cancer, e.g.,
multiple myeloma, small-cell lung carcinoma, MCC or ovarian
carcinoma is lorvotuzumab. One example of an anti-CD56 conjugate is
lorvotuzumab mertansine (CAS No.: 139504-50-0).
[3517] Examples of antibodies that bind the cancer target molecule
CD70 and can be used for treatment of cancer, e.g., non-Hodgkin's
lymphoma or renal cell cancer are disclosed in WO 2007038637 A2 or
WO 2008070593 A2. One example of an anti-CD70 conjugate is SGN-75
(CD70 MMAF)
[3518] One example of an antibody that binds the cancer target
molecule CD74 and can be used for treatment of cancer, e.g.,
multiple myeloma, is milatuzumab. One example of an anti-CD74
conjugate is milatuzumab doxorubicin (CAS No.: 23214-92-8).
[3519] One example of an antibody that binds the cancer target
molecule CD19 and can be used for treatment of cancer, e.g.,
non-Hodgkin's lymphoma is disclosed in WO 2008031056 A2. Additional
antibodies and examples of an anti-CD19 conjugate (SAR3419) are
disclosed in WO 2008047242 A2.
[3520] Examples of antibodies that bind the cancer target molecule
Mucin-1 and can be used for treatment of cancer, e.g.,
non-Hodgkin's lymphoma include clivatuzumab or the antibodies
disclosed in WO 2003106495 A2, WO 2008028686 A2. Examples of
anti-mucin conjugates are disclosed in WO 2005009369 A2.
[3521] Examples of antibodies that bind the cancer target molecule
CD138 and conjugate thereof that can be used for treatment of
cancer, e.g., multiple myeloma are disclosed in WO 2009080829 A1,
WO 2009080830 A1.
[3522] Examples of antibodies that bind the cancer target molecule
integrin alphaV and can be used for treatment of cancer, e.g.,
melanoma, sarcoma or carcinoma include intetumumab (CAS No.:
725735-28-4), abciximab (CAS No.: 143653-53-6), etaracizumab (CAS
No.: 892553-42-3) or the antibodies disclosed in U.S. Pat. No.
7,465,449 B2, EP 719859 A1, WO 2002012501 A1 or WO 2006062779 A2.
Examples of anti-integrin alphaV conjugates include intetumumab DM4
and additional ADCs disclosed in WO 2007024536 A2.
[3523] Examples of antibodies that bind the cancer target molecule
TDGF1 and can be used for treatment of cancer include the
antibodies disclosed in WO 2002077033 A1, U.S. Pat. No. 7,318,924,
WO 2003083041 A2 and WO 2002088170 A2. Examples of anti-TDGF1
conjugates are disclosed in WO2002088170-A2.
[3524] Examples of antibodies that bind the cancer target molecule
and can be used for treatment of cancer, e.g., prostatic carcinoma
are the antibodies disclosed in WO 199735615 A1, WO 199947554 A1
and WO 2001009192 A1. Examples of anti-PSMA conjugates are
disclosed in WO 2009026274 A1.
[3525] Examples of antibodies that bind the cancer target molecule
EPHA2 and can be used for producing a conjugate and for treatment
of cancer are disclosed in WO 2004091375 A2.
[3526] Examples of antibodies that bind the cancer target molecule
SLC44A4 and can be used for producing a conjugate and for treatment
of cancer, e.g., pancreatic or prostatic carcinoma, are disclosed
in WO 2009033094 A2 and US 20090175796 A1.
[3527] One example of an antibody that binds the cancer target
molecule HLA-DOB is the antibody Lym-1 (CAS No.: 301344-99-0),
which can be used for treatment of cancer, e.g., non-Hodgkin's
lymphoma. Examples of anti-HLA-DOB conjugates are disclosed, for
example, in WO 2005081711 A2.
[3528] Examples of antibodies that bind the cancer target molecule
VTCN1 and can be used for producing a conjugate and for treatment
of cancer, e.g., ovarian cancer, pancreatic, lung cancer or breast
cancer are disclosed in WO 2006074418 A2.
[3529] An example of an antibody that can be used to bind the
cancer target molecule PDL1 is the antibody 3G10 from patent WO
2007005874 A2. The antibody 3G10 may be used, for example, in the
human IgG1 format and as the anti-PDL1 used in the exemplary
embodiments where the antibody comprises the following
sequences;
[3530] Light chain:
TABLE-US-00036 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLVWYQQKPGQAPRLLIYD
ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
[3531] Heavy chain:
TABLE-US-00037 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYGFSWVRQAPGQGLEWMGW
ITAYNGNTNYAQKLQGRVTMTTDTSTSTVYMELRSLRSDDTAVYYCARDY
FYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT
LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
[3532] An example of an antibody that binds the cancer target
molecule ICOSLG is the antibody 16H (SEQ ID NOS: 70 and 45) from WO
2007011941 A2. Anti-ICOSLG antibody 16H may be used in the human
IgG1 format and as the anti-ICOSLG used in the exemplary
embodiments comprising the following sequences:
[3533] Light chain:
TABLE-US-00038 DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWYQQKPEKAPKSLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDSYPRTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
[3534] Heavy chain:
TABLE-US-00039 EVQLVESGGGLVQPGGSLRLSCAGSGFTFSSYWMSWVRQAPGKGLEWVAY
IKQDGNEKYYVDSVKGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCAREG
ILWFGDLPTFWGQGILVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
[3535] One example of an antibody that binds the target molecule
FGFR3 is the antibody 15D8 (SEQ ID NO: 74 for the heavy chain and
SEQ ID NO: 76 for the light chain) from WO 2010002862 A2.
Anti-FGFR3 antibody 15D8 may be used in the human IgG1 format, for
example, and as the anti-FGFR3 used in the exemplary embodiment,
this antibody comprises the following sequences:
[3536] Light chain:
TABLE-US-00040 DIQLTQSPSSLSASVGDRVTITCSASSSVSYMYWFQQKPGKAPKPLIYLT
SYLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSYPLTFGGG
TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
SSPVTKSFNRGEC
[3537] Heavy chain:
TABLE-US-00041 EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYNMYWVRQMPGKGLEWMGY
IDPYNGGTSYNQKFKGKATLTVDKSISTAYLQWSSLKASDTAMYYCAREG
GNYEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
[3538] One example of an antibody that binds the target cancer
molecule 5,6-dihydroxyindole-2-carboxylic acid oxidase (TYRP1) is
the antibody 20D7S (SEQ ID NOS: 30 and 32) from patent WO
2009114585 A1. In the exemplary embodiments, anti-TYRP1 antibody
20D7S may be used in human IgG1 format, for example, and when used
as the anti-TYRP1, the antibody comprises the following
sequences:
[3539] Light chain
TABLE-US-00042 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD
ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWLMYTFG
QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK
VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ
GLSSPVTKSFNRGEC
[3540] Heavy chain:
TABLE-US-00043 QVQLVQSGSELKKPGASVKISCKASGYTFTSYAMNWVRQAPGQGLESMGW
INTNTGNPTYAQGFTGRFVFSMDTSVSTAYLQISSLKAEDTAIYYCAPRY
SSSWYLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
[3541] One example of an antibody that binds the cancer target
molecule glypican-3 is the antibody that is known from the patent
U.S. Ser. No. 07/776,329 B2 and comprises the amino acid sequences
SEQ ID NOS: 84 and 92 (human mouse chimera). The anti-glypican-3
antibody described above may be used in human IgG1 format, for
example, and as the anti-glypican-3 used in the exemplary
embodiments, this antibody has the following sequences:
[3542] Light chain:
TABLE-US-00044 DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQ
LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVP
PTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC
[3543] Heavy chain:
TABLE-US-00045 QVQLVESGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGA
LDPKTGDTAYSQKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARFY
SYTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE
PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM
ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
[3544] The compounds according to the invention have valuable
pharmacological properties and can be used to prevent and treat
diseases in humans and animals.
[3545] The binder-drug conjugates (ADCs) of formula (Ia) according
to the invention have a high and specific cytotoxic activity with
respect to tumor cells which can be demonstrated on the basis of
assays performed in the present experimental part (C-1 to C-7e).
This high and specific cytotoxic activity of the binder-drug
conjugates (ADCs) of formula (Ia) according to the invention is
achieved by the suitable combination of the novel
N,N-dialkylauristatin derivatives and binders with linkers which
have both an enzymatic, hydrolytic or reductively cleavable
intended breaking point for release of the toxophore as well as
those not having any such intended breaking point. The effect on
the tumor cell is delineated very specifically by using stable
linkers in particular, which do not have any intended breaking
points that can be cleaved enzymatically, hydrolytically or
reductively to release the toxophore and which still remain
entirely or partially intact after receiving the ADC into the tumor
cell and after complete intracellular enzymatic degradation of the
antibody. The compatibility of ADCs with stable linkers
presupposes, among things, that the metabolites formed
intracellularly will be formed with enough efficiency to reach
their target and be able to manifest their antiproliferative effect
on the target there in adequate potency without first being removed
from the tumor cell by transporter proteins. The metabolites formed
intracellularly after incorporation of the compounds of formula
(Ia) according to the invention have a reduced potential as a
substrate with respect to transporter proteins, so that a
redistribution to the systemic circulation and thus the triggering
of potential adverse effects are suppressed by the toxophore
itself. In addition the basic character at the amino terminus of
the monomethylauristatin peptide is preserved by the novel N-alkyl
binding. In particular with the binder-drug conjugates (ADCs) of
formula (Ia) according to the invention, the total charge of the
antibody remains constant regardless of the number of
toxophore-linker charges.
[3546] The compatibility of the ADCs with a stable linker chemistry
and the respective target in conjunction with metabolites that
represent a substrate for transporter proteins to a slight extent
offers an enlarged therapeutic window.
[3547] Because of this profile of properties, the compounds
according to the invention are therefore suitable to a particular
extent for treatment of hyperproliferative diseases in humans and
infants in general. These compounds may on the one hand block,
inhibit, reduce or decrease cell proliferation and cell division on
the one hand while on the other hand potentiating the
apoptosis.
[3548] The hyperproliferative diseases for treatment of which the
compounds according to the invention may be used include in
particular the group of cancer and tumor diseases. These are
understood to include in particular the following diseases within
the scope of the present invention without being limited to these:
breast cancer and breast tumors (ductile and lobular forms, also in
situ), respiratory tract tumors (small cell and non-small-cell
carcinomas, bronchial carcinoma), brain tumors (e.g., of the brain
stem and the hypothalamus, astrocytoma, medulloblastoma, ependymoma
and neuroectodermal and pineal tumors), tumors of the digestive
tract (esophagus, stomach, gallbladder, small intestine, large
intestine, rectum), liver tumors (including hepatocellular
carcinoma, cholangiocarcinoma and mixed hepatocellular
cholangiocarcinoma), tumors of the head and neck area (larynx,
hypopharynx, nasopharynx, oropharynx, lips and oral cavity), skin
tumors (squamous epithelial carcinoma, Kaposi's sarcoma, malignant
melanoma, Merkel cell skin cancer and non-melanoma type skin
cancer), tumors of the soft tissues (including soft tissue
sarcomas, malignant fibrous histiocytoma, lymphosarcoma and
rhabdomyosarcoma), tumors of the eyes (including intraocular
melanoma and retinoblastoma), tumors of the endocrine and exocrine
glands (e.g., thyroid and parathyroid glands, pancreatic gland and
esophageal gland), tumors of the urinary tract (bladder, penis,
kidney, renal pelvis and urethral tumors) as well as tumors of the
reproductive organs (endometrium, cervical, ovarian, vaginal,
vulval and uterine carcinomas in the woman and prostatic and
testicular carcinomas in males). These also include proliferative
blood diseases in solid form and as circulating blood cells such as
lymphomas, leukemias and myeloproliferative diseases, e.g., acute
myeloid leukemia, acute lymphoblastic, chronic lymphocytic
leukemia, chronic myelogenous leukemia and hairy cell leukemia as
well as AIDS-related lymphomas, Hodgkin's lymphomas, non-Hodgkin's
lymphomas, cutaneous T-cell lymphomas, Burkitt's lymphomas and
lymphomas of the central nervous system.
[3549] Preferred Hyperproliferative Diseases for Anti-CA9
Binder-Drug Conjugates
[3550] Hyperproliferative diseases for the treatment of which the
compounds according to the invention may preferably be used include
CA9 overexpressing tumors, breast cancers and breast tumors
(ductile and lobular forms, also in situ); respiratory tract tumors
(small cell and non-small-cell carcinomas, bronchial carcinomas),
of which preferably non-small-cell lung carcinoma; brain tumors
(e.g., of the brain stem and of the hypothalamus, astrocytoma,
medulloblastoma, ependymoma and/or neuroectodermal and pineal
tumors); tumors of the digestive organs (esophagus, stomach,
gallbladder, small intestine, large intestine, rectum), of which
those that are especially preferred are stomach and intestinal
tumors; liver tumors (including hepatocellular carcinoma,
cholangiocarcinoma and mixed hepatocellular cholangiocarcinoma);
tumors of the head and neck area (larynx, hypopharynx, nasopharynx,
oropharynx, lips, oral cavity, tongue and esophagus); tumors of the
urinary tract (bladder, penis, kidney, renal pelvis and urethral
tumors), of which the tumors of the kidneys and bladder are
especially preferred; and/or tumors of the reproductive organs
(endometrial, cervical, ovarian, vaginal, vulval and uterine
carcinomas of the woman and/or prostatic and testicular carcinomas
in the man), of which cervical and uterine carcinomas are
especially preferred.
[3551] Preferred Hyperproliferative Diseases for Anti-EGFR
Binder-Drug Conjugates
[3552] Hyperproliferative diseases for the treatment of which the
compounds according to the invention may preferably be used include
EGFR overexpressing tumors, respiratory tract tumors (e.g., small
cell and non-small-cell carcinoma, bronchial carcinoma), of which
non-small-cell lung carcinoma is especially preferred; tumors of
the digestive tract (e.g., esophagus, stomach, gallbladder, small
intestine, large intestine, rectum), of which the intestinal tumors
are especially preferred; tumors of the endocrine and exocrine
glands (e.g., thyroid and parathyroid glands, pancreatic gland and
salivary gland), of which the pancreas is preferred; tumors of the
head and neck area (e.g., larynx, hypopharynx, nasopharynx,
oropharynx, lips, oral cavity, tongue and esophagus); and/or
gliomas.
[3553] Preferred Hyperproliferative Diseases for Anti-Mesothelin
Binder-Drug Conjugates
[3554] Hyperproliferative diseases for treatment of which the
compounds according to the invention are preferably used include
mesothelin overexpressing tumors, tumors of the reproductive organs
(endometrial, cervical, ovarian, vaginal, vulva and uterine
carcinomas in the woman and/or prostatic and testicular carcinomas
in the man) of which ovarian carcinomas are preferred; tumors of
the endocrine and exocrine glands (e.g., thyroid and parathyroid
glands, pancreatic gland and salivary gland), of which the pancreas
is preferred; respiratory tract tumors (e.g., small cell and
non-small-cell carcinomas, bronchial carcinoma), of which non-small
lung cancer is preferred; and/or mesotheliomas.
[3555] Preferred Hyperproliferative Diseases for Anti-C4.4a
Binder-Drug Conjugates
[3556] Hyperproliferative diseases for treatment of which the
compounds according to the invention are preferably used include
C4.4a hyperexpressing tumors, squamous epithelial cell carcinomas
(e.g., of the cervix, vulva, vagina, the anal canal, endometrium,
fallopian tube, penis, scrotum, esophagus, breast, bladder, bile
duct, endometrium, uterus and ovaries); breast cancer and breast
tumors (e.g., ductal and lobular forms, also in situ); respiratory
tract tumors (e.g., small-cell and non-small-cell carcinomas,
bronchial carcinoma), of which the non-small-cell lung cancer is
preferred along with squamous epithelial cell and adenocarcinomas
of the lungs; tumors of the head and neck area (e.g., larynx,
hypopharynx, nasopharynx, oropharynx, lips, oral cavity, tongue and
esophagus, squamous epithelial cell carcinomas of the head and neck
area); tumors of the urinary tract (bladder, penis, kidney, renal
pelvis and urethral tumors, squamous epithelial cell carcinomas of
the bladder), of which tumors of the kidney and bladder are
especially preferred; skin tumors (squamous epithelial cell
carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin
cancer and non-melanoma-type skin cancer), of which melanomas are
especially preferred; tumors of the endocrine and exocrine glands
(e.g., thyroid and parathyroid glands, pancreatic gland and
salivary gland), of which the pancreas is preferred; tumors of the
digestive organs (e.g., esophagus, stomach, gallbladder, small
intestine, large intestine, rectum), of which colorectal carcinomas
are especially preferred; and/or tumors of the reproductive organs
(endometrial, cervical, ovarian, vaginal, vulval and uterine
carcinomas in the woman and/or prostatic and testicular carcinomas
in the man), of which uterine carcinomas are especially
preferred.
[3557] These human diseases that have been described extensively
can also occur with a comparable etiology in other mammals and can
be treated there using the compounds according to the present
invention.
[3558] The terms "treatment" or "to treat" is used in the
conventional sense within the scope of this invention and refers to
the care, treatment and consultation of a patient with the goal of
combatting, reducing, diminishing or ameliorating a disease or
health deviation and improving the quality of life which is
impaired by this disease such as, for example, in a cancer.
[3559] An additional subject matter of the present invention is
thus the use of the compounds according to the invention for
treatment and/or prevention of diseases, in particular the diseases
cited above.
[3560] An additional subject matter of the present invention is the
use of the compounds according to the invention for producing a
pharmaceutical drug for treatment and/or prevention of diseases, in
particular the diseases cited above.
[3561] An additional subject matter of the present invention is the
use of the compounds according to the invention in a method for
treatment and/or prevention of diseases, in particular the diseases
cited above.
[3562] An additional subject matter of the present invention is a
method for treatment and/or prevention of diseases, in particular
the diseases cited above, using an effective amount of at least one
of the compounds according to the invention.
[3563] The inventor-drug conjugate is preferably used for treating
cancer in a patient, where the cancer cells of the patient to be
treated express the target (preferably EGFR, CA9, mesothelin or
C4.4a), preferably having a greater expression of this target than
in non-tumorous tissue.
[3564] A method for identifying patients who will response
advantageously to an anti-target binder-drug conjugate for
treatment of cancer includes determining the target expression in
the patient's cancer cells. In one embodiment, the target
expression is determined by target gene expression analysis. Those
skilled in the art are familiar with methods for gene expression
analysis such as RNA detection, quantitative or qualitative
polymerase chain reaction or fluorescence in situ hybridization
(FISH). In another preferred embodiment, the target expression is
determined by means of immunohistochemistry using an anti-target
antibody. Immunohistochemistry is preferably performed on tissue
fixed in formaldehyde. The antibody used for the
immunohistochemistry is the same antibody also used in the
conjugate. The antibody used for the immunohistochemistry is a
second antibody that recognizes the target protein/target,
preferably specifically.
[3565] The compounds according to the invention may be used alone
or, if necessary, in combination with one or more other
pharmacologically active substances as long as this combination
does not lead to adverse and unacceptable effects. Another subject
matter of the present invention therefore relates to pharmaceutical
drugs containing at least one of the compounds according to the
invention and one or more additional active ingredients, in
particular for treating and/or preventing the diseases listed
above.
[3566] For example, the compounds according to the invention may be
combined with known anti-hyperproliferative, cytostatic or
cytotoxic for treatment of cancer. Suitable combination active
ingredients and drugs that can be listed as examples include:
[3567] aldesleukin, alendronic acid, alfaferone, alitretinoin,
allopurinol, aloprim, aloxi, altretamine, amino glutethimide,
amifostine, amrubicin, amsacrine, anastrozol, anzmet, aranesp,
arglabin, arsentrioxide, aromasine, 5-azacytidine, azathioprine,
BCG or tice-BCG, bestatin, betamethasone acetate, betamethasone
sodium phosphate, bexarotene, bleomycin sulfate, broxuridine,
bortezomib, busulfane, calcitonine, campath, capecitabine,
carboplatin, casodex, cefesone, celmoleukin, cerubidine,
chlorambucil, cisplatin, cladribine, clodronic acid,
cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunoxome,
decadrone, decadrone phosphate, delestrogen, denileukin diftitox,
depo medrol, desloreline, dexrazoxane, diethylstilbestrol,
diflucan, docetaxel, doxifluridine, doxorubicin, dronabinol,
DW-166HC, eligard, elitek, ellence, emend, epirubicin, epoetin
alfa, epogen, eptaplatin, ergamisole, estrace, estradiol,
estramustine sodium phosphate, ethinyl estradiol, ethyol, etidronic
acid, etopophos, etoposide, fadrozole, farstone, filgrastim,
finasteride, fligrastim, floxuridine, fluconazole, fludarabin,
5-fluorodeoxyuridine monophosphate, 5-fluorouracil (5-FU),
fluoxymesterone, flutamide, formestane, fosteabine, fotemustine,
fulvestrant, gammagard, gemcitabine, gemtuzumab, gleevec, gliadel,
gosereline, granisetrone hydrochloride, histreline, hycamtine,
hydrocortone, erythrohydroxynonyladenine, hydroxyurea, ibritumomab
tiuxetan, idarubicin, ifosfamide, interferon-alpha,
interferon-alpha-2, interferon-alpha-2a, interferon-alpha-2P,
interferon-alpha-n1, interferon-alpha-n3, interferon-beta,
interferon-gamma-la, interleukin-2, intron A, iressa, irinotecan,
kytril, lentinane sulfate, letrozole, leucovorine, leuprolide,
leuprolide acetate, levamisole, levofolic acid calcium salt,
levothroid, levoxyl, lomustine, lonidamine, marinol,
mechlorethamine, mecobalamine, medroxyprogesterone acetate,
megestrole acetate, melphalane, menest, 6-mercaptopurine, mesna,
methotrexate, metvix, miltefosine, minocycline, mitomycin C,
mitotane, mitoxantrone, modrenal, myocet, nedaplatin, neulasta,
neumega, neupogen, nilutamide, nolvadex, NSC-631570, OCT-43,
octreotide, ondansetrone hydrochloride, orapred, oxaliplatin,
paclitaxel, pediapred, pegaspargase, pegasys, pentostatin,
picibanil, pilocarpine hydrochloride, pirarubicin, plicamycin,
porfimer sodium, prednimustin, prednisolone, prednisone, premarin,
procarbazine, procrit, raltitrexed, rebif, rhenium 186 etidronate,
rituximab, roferone A, romurtide, salagen, sandostatin,
sargramostim, semustine, sizofiran, sobuzoxane, solu-medrol,
streptozocine, strontium-89 cehlorid, synthroid, tamoxifen,
tamsulosine, tasonermine, tastolactone, taxoter, teceleukin,
temozolomide, teniposide, testosterone propionate, testred,
thioguanine, thiotepa, thyrotropin, tiludronic acid, topotecan,
toremifen, tositumomab, tastuzumab, teosulfane, tretinoin, trexall,
trimethylmelamine, trimetrexate, triptoreline acetate, triptoreline
pamoate, uft, uridine, valrubicin, vesnarinone, vinblastine,
vincristine, vindesine, vinorelbine, virulizine, zinecard,
zinostatin stimalamer, zofran; ABI-007, acolbifene, actimmune,
affinitak, aminopterine, arzoxifene, asoprisnil, atamestane,
atrasentane, avastin, BAY 43-9006 (sorafenib), CCI-779, CDC-501,
celebrex, cetuximab, crisnatol, cyproterone acetate, decitabin,
DN-101, doxorubicin MTC, dSLIM, dutasteride, edotecarin,
eflornithine, exatecane, fenretinide, histamine dihydrochloride,
histreline hydrogel implant, holmium-166-DOTMP, ibandronic acid,
interferon-gamma, intron PEG, ixabepilone, keyhole limpet
hemocyanine, L-651582, lanreotide, lasofoxifen, libra, lonafarnib,
miproxifen, minodronate, MS-209, liposomales MTP-PE, MX-6,
nafareline, nemorubicin, neovastat, nolatrexed, oblimersen,
onko-TCS, osidem, paclitaxel polyglutamate, pamidronate disodium,
PN-401, QS-21, quazepam, Rr-1549, raloxifene, ranpirnase,
13-cis-retinic acid, satraplatin, seocalcitol, T-138067, tarceva,
taxoprexine, thymosine-alpha-1, tiazofurine, tipifarnib,
tirapazamine, TLK-286, toremifene, transmid 107R, valspodar,
vapreotide, vatalanib, verteporfin, vinflunine, Z-100, zoledronic
acid as well as combinations thereof.
[3568] In a preferred embodiment, the compounds according to the
present invention may be combined with antihyperproliferative
agents, which may include the following, for example, although this
list is not conclusive:
[3569] aminoglutethimide, L-asparaginase, azathioprine,
5-azacytidine, bleomycin, busulfan, carboplatin, carmustin,
chlorambucil, cisplatin, colaspase, cyclophosphamide, cytarabine,
dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol,
2',2'-difluorodeoxycytidine, docetaxel, doxorubicin (adriamycin),
epirubicin, epothilone and seine derivate,
erythrohydroxynonyladenine, ethinyl estradiol, etoposide,
fludarabine phosphate, 5-fluorodeoxyuridine, 5-fluordeoxyuridine
monophosphate, 5-fluoruracil, fluoxymesterone, flutamide,
hexamethyl melamine, hydroxyurea, hydroxyprogesterone caproate,
idarubicin, ifosfamide, interferon, irinotecan, leucovorin,
lomustine, mechlorethamine, medroxyprogesterone acetate, megestrol
acetate, melphalan, 6-mercaptopurine, mesna, methotrexate,
mitomycin C, mitotane, mitoxantrone, paclitaxel, pentostatin,
n-phosphonoacetyl-L-aspartate (PALA), plicamycin, prednisolone,
prednisone, procarbazine, raloxifene, semustine, streptozocine,
tamoxifen, teniposide, testosterone propionate, thioguanine,
thiotepa, topotecan, trimethylmelamine, uridine, vinblastine,
vincristine, vindesine and vinorelbine.
[3570] In one very promising aspect, the compounds according to the
invention can also be combined with biological therapeutic agents
such as antibodies (e.g., Avastin, Rituxan, Erbitux, Herceptin).
The compounds according to the invention may also achieve positive
effects in combination with treatments directed against
angiogenesis, for example, with Avastin, axitinib, recentin,
regorafenib, sorafenib or sunitinib. Combinations with inhibitors
of the proteasome and of mTOR as well as combinations with
antihormones and steroidal metabolic enzyme inhibitors are also
especially suitable because of their favorable profile of side
effects.
[3571] In general, the following goals can be pursued with the
combination of compounds of the present invention with other active
cytostatic or cytotoxic agents: [3572] an improved efficacy in
retarding the growth of a tumor, in reducing its size or even
completely eliminating it in comparison with a treatment with a
single active ingredient; [3573] the possibility of using the
chemotherapeutic agents that are used in a lower dosage than in
monotherapy; [3574] the possibility of a tolerable therapy with few
adverse effects in comparison with an individual administration;
[3575] the possibility of treatment of a broader spectrum of
tumors; [3576] achieving a higher response rate to the treatment;
[3577] a longer survival time for the patients in comparison with
today's standard therapy.
[3578] In addition, the compounds according to the invention may
also be used in combination with radiation therapy and/or a
surgical intervention.
[3579] An additional subject matter of the present invention is
pharmaceutical drugs that contain at least one compound according
to the invention, usually together with one or more inert nontoxic
pharmaceutically suitable excipients as well as their use for the
aforementioned purposes.
[3580] The compounds according to the invention may act
systemically and/or locally. For this purpose they are applied in a
suitable way such as, for example, orally or parenterally. The
compounds according to the invention may act systemically and/or
locally. For this purpose they are applied in a suitable way such
as, for example, parenterally, possibly by inhalation or as an
implant and/or stent.
[3581] For these application methods, the compounds according to
the invention may be administered in suitable dosage forms.
[3582] Parenteral administration may be performed in order to
bypass a resorption step (e.g., intravenously, intra-arterially,
intracardially, intraspinally or intralumbarly) or with the
inclusion of resorption (e.g., intramuscularly, subcutaneously,
intracutaneously, percutaneously or intraperitoneally). Suitable
forms of administration for parenteral administration include
infusion and injection preparations in the form of solutions,
suspensions, emulsions or lyophilisates.
[3583] Parenteral administration in particular intravenous
administration is preferred.
[3584] i.v. Solution:
[3585] The compounds according to the invention may be converted to
the dosage forms indicated. This may be done in the known way by
"mixing with" and/or "dissolving in" inert nontoxic
pharmaceutically suitable excipients (e.g., buffer substances,
stabilizers, solubilizer, preservatives). These may include, for
example: amino acids (glycine, histidine, methionine, arginine,
lysine, leucine, isoleucine, threonine, glutamic acid,
phenylalanine and others), sugars and related substances (glucose,
saccharose, mannitol, trehalose, sucrose, mannose, lactose,
sorbitol), glycerol, sodium, potassium, ammonium and calcium salts
(e.g., NaCl, KCl or Na.sub.2HPO.sub.4 and many more),
acetate/acetic acid buffer systems, phosphate buffer systems,
citric acid and citrate buffer systems, trometamol (TRIS and TRIS
salts), polysorbates (e.g., polysorbate 80 and polysorbate 20),
poloxamers (e.g., poloxamer 188 and poloxamer 171), macrogols (PEG
derivatives, e.g., 3350), Triton X-100, EDTA salts, glutathione,
albumins (e.g., human), urea, benzyl alcohol, phenol, chlorocresol,
metacresol, benzalkonium chloride and many others.
[3586] It has proven advantageous in general to administer doses of
approx. 0.001 to 1 mg/kg, preferably approx. 0.01 to 0.5 mg/kg body
weight to achieve effective results in parenteral
administration.
[3587] Nevertheless, it may be necessary under some circumstances
to deviate from the stated amounts, namely depending on the body
weight, the method of administration, the individual response to
the active ingredient, the type of preparation and the point in
time or interval at which the application occurs. Thus, in many
cases, it may be sufficient to use less than the aforementioned
minimum amount, whereas in other cases the aforementioned upper
limit must be exceeded. In the case of administration of larger
amounts, it may be advisable to distribute the larger amount among
several individual doses throughout the day.
[3588] The following examples are presented to illustrate the
invention, although the invention is not limited to these
examples.
[3589] The percentage amounts specified in the following tests and
examples are percent by weight (wt %), unless otherwise indicated;
parts refer to parts by weight. Solvent ratios, dilution ratios and
concentration specifications for liquid-liquid solutions are each
based on volume.
A. EXAMPLES
Abbreviations and Acronyms
[3590] A431NS human tumour cell line [3591] A549 human tumour cell
line [3592] ABCB1 ATP-binding cassette sub-family B member 1
(synonym for P-gp and MDR1) [3593] abs. absolute [3594] ADC
antibody-drug-conjugate [3595] Ac acetyl [3596] aq. aqueous,
aqueous solution [3597] ATP adenosine triphosphate [3598] BCRP
breast cancer resistance protein, an efflux transporter [3599] Boc
tert-butoxycarbonyl [3600] br. broad (in NMR) [3601] Ex. example
[3602] ca. circa, approximately [3603] CAIX carboanhydrase IX
[3604] CI chemical ionization (in MS) [3605] d doublet (in NMR)
[3606] d day(s) [3607] TLC thin-layer chromatography [3608] DCI
direct chemical ionization (in MS) [3609] dd doublet of a doublet
(in NMR) [3610] DMAP 4-N,N-dimethylaminopyridine [3611] DME
1,2-dimethoxyethane [3612] DMEM Dulbecco's modified eagle medium
(standardized nutrient medium for the cell culture) [3613] DMF
N,N-dimethylformamide [3614] DMSO dimethyl sulphoxide [3615] DPBS,
D-PBS, PBS Dulbecco's phosphate-buffered saline solution
PBS=DPBS=D-PBS, pH 7.4, from Sigma, No. D8537 [3616] Composition:
[3617] 0.2 g KCl [3618] 0.2 g KH.sub.2PO.sub.4 (anhydrous) [3619]
8.0 g NaCl [3620] 1.15 g Na.sub.2HPO.sub.4 (anhydrous) [3621] make
up to 1 l with H.sub.2O [3622] dt doublet of a triplet (in NMR)
[3623] DTT DL-dithiothreitol [3624] EDC
N'-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride [3625]
EGFR epidermal growth factor receptor [3626] EI electron impact
ionization (in MS) [3627] ELISA enzyme-linked immunosorbent assay
[3628] eq. equivalent(s) [3629] ESI electrospray ionization (in MS)
[3630] ESI-MicroTofq ESI-MicroTofq (name of the mass spectrometer,
with Tof=time of flight and q=quadrupole) [3631] FCS foetal calf
serum [3632] Fmoc (9H-fluoren-9-ylmethoxy)carbonyl [3633] sat.
Saturated [3634] GTP guanosine 5'-triphosphate [3635] h hour(s)
[3636] HATU O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate [3637] HCT-116 human tumour cell line [3638]
HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulphonic acid [3639]
HOAc acetic acid [3640] HOBt 1-hydroxy-1H-benzotriazole hydrate
[3641] HOSu N-hydroxysuccinimide [3642] HPLC high-pressure,
high-performance liquid chromatography [3643] HT29 human tumour
cell line [3644] IC.sub.50 half-maximum inhibitory concentration
[3645] i.m. intramuscular, administration into the muscle [3646]
i.v. intravenous, administration into the vein [3647] conc.
Concentrated [3648] LC-MS liquid chromatography-coupled mass
spectrometry [3649] LLC-PK1 cells Lewis lung carcinoma pork kidney
cell line [3650] L-MDR human MDR1 transfected LLC-PK1 cells [3651]
m multiplet (in NMR) [3652] MDR1 multidrug resistence protein 1
[3653] min minute(s) [3654] MS mass spectrometry [3655] MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide
[3656] NCI-H292 human tumour cell line [3657] NCI-H520 human tumour
cell line [3658] NMM N-methylmorpholine [3659] NMP
N-methyl-2-pyrrolidinone [3660] NMR nuclear magnetic resonance
spectrometry [3661] NMRI mouse strain, originating from Naval
Medical Research Institute (NMRI) [3662] Nude mice experimental
animals [3663] NSCLC non-small cell lung cancer (non-small cell
bronchial carcinoma) [3664] PBS phosphate-buffered saline solution
[3665] Pd/C palladium on activated carbon [3666] P-gp
P-glycoprotein, a transporter protein [3667] PNGaseF enzyme for
sugar elimination [3668] quant. quantitative (for yield) [3669]
quart quartet (in NMR) [3670] quint quintet (in NMR) [3671] R.sub.f
retention index (for TLC) [3672] RT room temperature [3673] R.sub.t
retention time (for HPLC) [3674] singlet (in NMR) [3675] s.c.
subcutaneous, administration beneath the skin [3676] SCC-4 human
tumour cell line [3677] SCC-9 human tumour cell line [3678] SCID
mice experimental mice with a severe combined immunodeficiency
[3679] t triplet (in NMR) [3680] tert tertiary [3681] TFA
trifluoroacetic acid [3682] THF tetrahydrofuran [3683] UV
ultraviolet spectrometry [3684] v/v volume to volume ratio (of a
solution) [3685] Z benzyloxycarbonyl
[3686] HPLC and LC-MS methods:
[3687] Method 1 (LC-MS):
[3688] Instrument: Waters Acquity SQD UPLC System; column: Waters
Acquity UPLC HSS T3 1.8.mu. 50 mm.times.1 mm; eluent A: 1 l
water+0.25 ml 99% strength formic acid, eluent B: 1 l
acetonitrile+0.25 ml 99% strength formic acid; gradient: 0.0 min
90% A.fwdarw.1.2 min 5% A.fwdarw.2.0 min 5% A; flow rate: 0.40
ml/min; oven: 50.degree. C.; UV detection: 210-400 nm.
[3689] Method 2 (LC-MS):
[3690] Instrument: Micromass QuattroPremier with Waters UPLC
Acquity; column: Thermo Hypersil GOLD 1.9.mu. 50 mm.times.1 mm;
eluent A: 1 l water+0.5 ml 50% strength formic acid, eluent B: 1 l
acetonitrile+0.5 ml 50% strength formic acid; gradient: 0.0 min 90%
A.fwdarw.0.1 min 90% A.fwdarw.1.5 min 10% A.fwdarw.2.2 min 10% A;
flow rate: 0.33 ml/min; oven: 50.degree. C.; UV detection: 210
nm.
[3691] Method 3 (LC-MS):
[3692] Instrument: Micromass Quattro Micro MS with HPLC Agilent
Series 1100; column: Thermo Hypersil GOLD 3.mu. 20 mm.times.4 mm;
eluent A: 1 l water+0.5 ml 50% strength formic acid, eluent B: 1 l
acetonitrile+0.5 ml 50% strength formic acid; gradient: 0.0 min
100% A.fwdarw.3.0 min 10% A.fwdarw.4.0 min 10% A.fwdarw.4.01 min
100% A (flow rate 2.5 ml/min).fwdarw.5.00 min 100% A; oven:
50.degree. C.; flow rate: 2 ml/min; UV detection: 210 nm.
[3693] Method 4 (LC-MS):
[3694] MS instrument: Micromass ZQ; HPLC instrument: HP 1100
Series; UV DAD; column: Phenomenex Gemini 3.mu. 30 mm.times.3.00
mm; eluent A: 1 l water+0.5 ml 50% strength formic acid, eluent B:
1 l acetonitrile+0.5 ml 50% strength formic acid; gradient: 0.0 min
90% A.fwdarw.2.5 min 30% A.fwdarw.3.0 min 5% A.fwdarw.4.5 min 5% A;
flow rate: 0.0 min 1 ml/min 2.5 min/3.0 min/4.5 min 2 ml/min; oven:
50.degree. C.; UV detection: 210 nm.
[3695] Method 5 (HPLC):
[3696] Instrument: HP 1090 Series II; column: Merck Chromolith
Speed ROD RP-18e, 50 mm.times.4.6 mm; preliminary column: Merck
Chromolith Guard Cartridge Kit RP-18e, 5 mm.times.4.6 mm; injection
volume: 5 .mu.l; eluent A: 70% HClO.sub.4 in water (4 ml/litre),
eluent B: acetonitrile; gradient: 0.00 min 20% B.fwdarw.0.50 min
20% B.fwdarw.3.00 min 90% B.fwdarw.3.50 min 90% B.fwdarw.3.51 min
20% B.fwdarw.4.00 min 20% B; flow rate: 5 ml/min; column
temperature: 40.degree. C.
[3697] Method 6 (HPLC):
[3698] Instrument: Waters 2695 with DAD 996; column: Merck
Chromolith SpeedROD RP-18e, 50 mm.times.4.6 mm; Ord. No.:
1.51450.0001, preliminary column: Merck Chromolith Guard Cartridge
Kit RP-18e, 5 mm.times.4.6 mm; Ord. No.: 1.51470.0001, eluent A:
70% HC1O.sub.4 in water (4 ml/litre), eluent B: acetonitrile;
gradient: 0.00 min 5% B.fwdarw.0.50 min 5% B.fwdarw.3.00 min 95%
B.fwdarw.4.00 min 95% B; flow rate: 5 ml/min.
[3699] Method 7 (LC-MS):
[3700] MS instrument: Waters ZQ; HPLC instrument: Agilent 1100
Series; UV DAD; column: Thermo Hypersil GOLD 3.mu. 20 mm.times.4
mm; eluent A: 1 l water+0.5 ml 50% strength formic acid, eluent B:
1 l acetonitrile+0.5 ml 50% strength formic acid; gradient: 0.0 min
100% A.fwdarw.3.0 min 10% A.fwdarw.4.0 min 10% A.fwdarw.4.1 min
100% A (flow rate 2.5 ml/min); oven: 55.degree. C.; flow rate: 2
ml/min; UV detection: 210 nm.
[3701] Method 8 (LC-MS):
[3702] MS instrument: Waters ZQ; HPLC instrument: Agilent 1100
Series; UV DAD; column: Thermo Hypersil GOLD 3.mu. 20 mm.times.4
mm; eluent A: 1 l water+0.5 ml 50% strength formic acid, eluent B:
1 l acetonitrile+0.5 ml 50% strength formic acid; gradient: 0.0 min
100% A.fwdarw.2.0 min 60% A.fwdarw.2.3 min 40% A.fwdarw.3.0 min 20%
A.fwdarw.4.0 min 10% A.fwdarw.4.2 min 100% A (flow rate 2.5
ml/min); oven: 55.degree. C.; flow rate: 2 ml/min; UV detection:
210 nm.
[3703] Method 9 (LC-MS):
[3704] Instrument: Waters Acquity SQD UPLC System; column: Waters
Acquity UPLC HSS T3 1.8.mu. 50 mm.times.1 mm; eluent A: 1 l
water+0.25 ml 99% strength formic acid, eluent B: 1 l
acetonitrile+0.25 ml 99% strength formic acid; gradient: 0.0 min
95% A.fwdarw.6.0 min 5% A.fwdarw.7.5 min 5% A; oven: 50.degree. C.;
flow rate: 0.35 ml/min; UV detection: 210-400 nm.
[3705] Method 10 (HPLC):
[3706] Instrument: Agilent 1200 Series; column: Agilent Eclipse
XDB-C18 5.mu. 4.6 mm.times.150 mm; preliminary column: Phenomenex
KrudKatcher Disposable Pre-Column; injection volume: 5 .mu.l;
eluent A: 1 l water+0.01% trifluoroacetic acid; eluent B: 1 l
acetonitrile+0.01% trifluoroacetic acid; gradient: 0.00 min 10%
B.fwdarw.1.00 min 10% B.fwdarw.1.50 min 90% B.fwdarw.5.5 min 10% B;
flow rate: 2 ml/min; column temperature: 30.degree. C.
[3707] For all reactants or reagents whose preparation is not
explicitly described below, they were obtained commercially from
generally available sources. For all other reactants or reacents
whose preparation is likewise not described below, and which were
not available commercially or were obtained from sources which are
not generally available, a reference is given to the published
literature in which their preparation is described.
[3708] Method 11 (LC-MS):
[3709] Instrument: Waters ACQUITY SQD UPLC System; column: Waters
Acquity UPLC HSS T3 1.8.mu. 30.times.2 mm; eluent A: 1 l water+0.25
ml 99% strength formic acid, eluent B: 1 l acetonitrile+0.25 ml 99%
strength formic acid; gradient: 0.0 min 90% A.fwdarw.1.2 min 5%
A.fwdarw.2.0 min 5% A oven: 50.degree. C.; flow rate: 0.60 ml/min;
UV detection: 208-400 nm.
[3710] Method 12 (HPLC):
[3711] Instrument: Agilent 1200 Series with column oven and DAD;
column: Merck Chromolith SpeedROD RP-18e, 50 mm.times.4.6 mm; Ord.
No.: 1.51450.0001; preliminary column: Merck Chromolith Guard
Cartridge Kit RP-18e, 5 mm.times.4.6 mm; Ord. No.: 1.51470.0001;
eluent A: 70% HC1O.sub.4 in water (4 ml/litre), eluent B:
acetonitrile; gradient: 0.00 min 5% B.fwdarw.0.50 min 5%
B.fwdarw.3.00 min 95% B.fwdarw.4.00 min 95% B; flow rate: 5 ml/min;
column temperature: 30.degree. C.
[3712] Method 13 (LC-MS):
[3713] MS instrument: Waters (Micromass) Quattro Micro; Instrument
HPLC: Agilent 1100 Series; column: YMC-Triart C18 3.mu. 50.times.3
mm; eluent A: 1 l water+0.01 mol ammonium carbonate, eluent B: 1 l
acetonitrile; gradient: 0.0 min 100% A.fwdarw.2.75 min 5%
A.fwdarw.4.5 min 5% A; oven: 40.degree. C.; flow rate: 1.25 ml/min;
UV detection: 210 nm.
Starting Compounds and Intermediates
Starting Compound 1
(2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methyl-
propanoic acid (Boc-dolaproine)
##STR00431##
[3715] The title compound can be prepared in various ways according
to literature methods; see, for example, Pettit et al., Synthesis
1996, 719; Shioiri et al., Tetrahedron Lett. 1991, 32, 931; Shioiri
et al., Tetrahedron 1993, 49, 1913; Koga et al., Tetrahedron Lett.
1991, 32, 2395; Vidal et al., Tetrahedron 2004, 60, 9715; Poncet et
al., Tetrahedron 1994, 50, 5345. It was prepared either as the free
acid or as a 1:1 salt with dicyclohexylamine.
Starting Compound 2a
tert-butyl (3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanoate
hydrochloride (dolaisoleucine-OtBu.times.HCl)
##STR00432##
[3717] The title compound can be prepared in various ways according
to literature methods; see, for example, Pettit et al., J. Org.
Chem. 1994, 59, 1796; Koga et al., Tetrahedron Lett. 1991, 32,
2395; Shioiri et al., Tetrahedron Lett. 1991, 32, 931; Shioiri et
al., Tetrahedron 1993, 49, 1913.
Starting Compound 2b
tert-butyl-(3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanoate
(dolaisoleucine-O.sup.tBu)
##STR00433##
[3719] The compound was prepared in analogy to starting compound
2a, except that the hydrogenation was performed without addition of
1N hydrochloric acid.
Starting Compound 3
N.sup..alpha.-(tert-butoxycarbonyl)-N-hydroxy-L-phenylalaninamide
##STR00434##
[3721] The title compound was prepared by the literature method (A.
Ritter et al., J. Org. Chem. 1994, 59, 4602).
[3722] Yield: 750 mg (75% of theory)
[3723] LC-MS (Method 3): R.sub.t=1.67 min; MS (ESIpos): m/z=281
(M+H).sup.+.
Starting Compound 4
1,2-oxazolidine hydrochloride
##STR00435##
[3725] The title compound can be prepared by literature methods
(see, for example, H. King, J. Chem. Soc. 1942, 432); it is also
commercially available.
Starting Compound 5
1,2-oxazinane hydrochloride
##STR00436##
[3727] The title compound can be prepared by literature methods
(see, for example, H. King, J. Chem. Soc. 1942, 432).
Starting Compound 6
2-oxa-3-azabicyclo[2.2.2]oct-5-ene
##STR00437##
[3729] The title compound can be prepared in Boc-protected form by
the literature method (see, for example, C. Johnson et al.,
Tetrahedron Lett. 1998, 39, 2059); the deprotection was effected in
a customary manner by treatment with trifluoroacetic acid and
subsequent neutralization.
[3730] Yield: 149 mg (89% of theory)
Starting Compound 7
tert-butyl (1S,2R)-1-(hydroxycarbamoyl)-2-phenylcyclopropyl
carbamate
##STR00438##
[3732] The title compound was prepared by a literature method (A.
Ritter et al., J. Org. Chem. 1994, 59, 4602) proceeding from
commercially available
(1S,2R)-1-[(tert-butoxycarbonyl)amino]-2-phenylcyclopropanecarb-
oxylic acid (C. Cativiela et al., Chirality 1999, 11, 583).
[3733] Yield: 339 mg (59% of theory)
[3734] LC-MS (Method 1): R.sub.t=0.82 min; MS (ESIpos): m/z=293
(M+H).sup.+.
Intermediate 1
tert-butyl-(3R,4S,5S)-4-[{N-[(benzyloxy)carbonyl]-L-valyl}(methyl)amino]-3-
-methoxy-5-methyl heptanoate
##STR00439##
[3736] 10.65 g (41.058 mmol) of tert-butyl
(3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)-heptanoate (starting
compound 2b) were taken up in 250 ml of dichloromethane, and the
solution was cooled to -10.degree. C. Then, while stirring, 10.317
g (41.058 mmol) of N-[(benzyloxy)carbonyl]-L-valine, 16.866 g
(61.586 mmol) of 2-bromo-1-ethylpyridinium tetrafluoroborate (BEP)
and 28.6 ml of N,N-diisopropylethylamine were added, and the
mixture was subsequently stirred at RT for 20 h. The reaction
mixture was then diluted with dichloromethane and shaken twice with
saturated sodium chloride solution, dried over sodium sulphate,
filtered and concentrated. The residue was purified by flash
chromatography on silica gel with 4:1 petroleum ether/ethyl acetate
as the eluent. The corresponding fractions were concentrated, and
the residue was dried under high vacuum overnight. 10.22 g (51% of
theory) of the title compound were obtained as a yellowish oil.
[3737] HPLC (Method 5): R.sub.t=2.3 min;
[3738] LC-MS (Method 2): R.sub.t=1.59 min; MS (ESIpos): m/z=493
(M+H).sup.+.
Intermediate 2
tert-butyl
(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(L-valyl)amino]heptanoat-
e
##STR00440##
[3740] 500 mg (1 mmol) of tert-butyl
(3R,4S,5S)-4-[{N-[(benzyloxy)carbonyl]-L-valyl}(methyl)amino]-3-methoxy-5-
-methylheptanoate (intermediate 1) were dissolved in 50 ml of
methanol and, after addition of 100 mg of 10% palladium on
activated carbon, hydrogenated under standard hydrogen pressure at
RT for 1 h. The catalyst was then filtered off, and the solvent was
removed under reduced pressure. This gave 370 mg (quant.) of the
title compound as a virtually colourless oil.
[3741] HPLC (Method 5): R.sub.t=1.59 min;
[3742] LC-MS (Method 1): R.sub.t=0.74 min; MS (ESIpos): m/z=359
(M+H).sup.+.
Intermediate 3
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-tert-
-butoxy-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00441##
[3744] 4.64 g (13.13 mmol) of
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valine were
dissolved in 20 ml of DMF and admixed successively with 4.28 g
(11.94 mmol) of tert-butyl
(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(L-valyl)amino]heptanoate
(Intermediate 2), 2.75 g (14.33 mmol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 2.2
g (14.33 mmol) of 1-hydroxy-1H-benzotriazole hydrate. The mixture
was stirred at RT overnight. The reaction mixture was then poured
into a mixture of semisaturated aqueous ammonium chloride solution
and ethyl acetate. The organic phase was removed, washed
successively with saturated sodium hydrogencarbonate solution and
saturated sodium chloride solution, dried over magnesium sulphate,
filtered and concentrated. The residue was used directly in the
next stage, without further purification.
[3745] Yield: 9.1 g (quant., 60% purity)
[3746] HPLC (Method 5): R.sub.t=2.7 min;
[3747] LC-MS (Method 2): R.sub.t=1.99 min; MS (ESIpos): m/z=694
(M+H).sup.+.
Intermediate 4
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carb-
oxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide
##STR00442##
[3749] 9.1 g of the crude product
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-ter-
t-butoxy-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
(Intermediate 3) were taken up in 56.6 ml of dichloromethane, 56.6
ml of trifluoroacetic acid were added, and the mixture was stirred
at RT for 2 h. Subsequently, the reaction mixture was concentrated
in vacuo and the remaining residue was purified by flash
chromatography, using dichloromethane, 3:1 dichloromethane/ethyl
acetate and 15:5:0.5 dichloromethane/ethyl acetate/methanol as
eluent. After purification of the corresponding fractions and
concentration, 5.8 g (86% of theory) of the title compound were
obtained as a colourless foam.
[3750] HPLC (Method 5): R.sub.t=2.2 min;
[3751] LC-MS (Method 1): R.sub.t=1.3 min; MS (ESIpos): m/z=638
(M+H).sup.+.
Intermediate 5
tert-butyl (2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl
carbamate
##STR00443##
[3753] 500 mg (1.9 mmol) of N-(tert-butoxycarbonyl)-L-phenylalanine
were dissolved in 10 ml of DMF and admixed successively with 466 mg
(3.8 mmol) of 1,2-oxazinane hydrochloride (Starting Compound 5),
433 mg (2.3 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride, 382 mg (2.8 mmol) of 1-hydroxy-1H-benzotriazole
hydrate and 731 mg (5.7 mmol) of N,N-diisopropylethylamine. The
mixture was stirred at RT overnight. The reaction mixture was then
poured into a mixture of semisaturated aqueous ammonium chloride
solution and ethyl acetate. The organic phase was removed, washed
successively with saturated sodium hydrogencarbonate solution and
saturated sodium chloride solution, dried over magnesium sulphate,
filtered and concentrated. 620 mg (98% of theory) of the title
compound were obtained.
[3754] HPLC (Method 5): R.sub.t=1.8 min;
[3755] LC-MS (Method 2): R.sub.t=1.62 min; MS (ESIpos): m/z=235
(M-C.sub.4H.sub.8--CO.sub.2+H).sup.+.
Intermediate 6
(2S)-2-amino-1-(1,2-oxazinan-2-yl)-3-phenylpropan-1-one
trifluoroacetate
##STR00444##
[3757] 620 mg (1.85 mmol) of ten-butyl
(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl carbamate
(Intermediate 5) were taken up in 5 ml of dichloromethane, 10 ml of
trifluoroacetic acid were added and the mixture was stirred at RT
for 30 min. Subsequently, the reaction mixture was concentrated in
vacuo, and the remaining residue was lyophilized from
water/acetonitrile. In this way, 779 mg (91% of theory) of the
title compound were obtained as a colourless foam.
[3758] HPLC (Method 5): R.sub.t=0.45 min;
[3759] LC-MS (Method 3): R.sub.t=1.09 min; MS (ESIpos): m/z=235
(M+H).sup.+.
Intermediate 7
(2R,3R)-3-methoxy-2-methyl-N-[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpro-
pan-2-yl]-3-[(2S)-pyrrolidin-2-yl]propanamide trifluoroacetate
##STR00445##
[3761] 360 mg (1.25 mmol) of
(2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methy-
lpropanoic acid (Starting Compound 1) were taken up in 10 ml of DMF
and admixed successively with 579.2 mg (1.25 mmol) of
(2S)-2-amino-1-(1,2-oxazinan-2-yl)-3-phenylpropan-1-one
trifluoroacetate (Intermediate 6), 714.5 mg (1.88 mmol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) and 655 .mu.l of
N,N-diisopropylethylamine. The mixture was stirred at RT for 16 h.
The reaction mixture was then concentrated, and the residue was
taken up in ethyl acetate and extracted by shaking first with 5%
aqueous citric acid solution, then with 5% aqueous sodium
hydrogencarbonate solution and subsequently with saturated sodium
chloride solution. The organic phase was concentrated, and the
residue was purified by flash chromatography on silica gel with
16:4 dichloromethane/methanol as the eluent. The corresponding
fractions were combined and the solvent was removed under in vacuo.
After the residue had been dried under high vacuum, 503.5 mg (74%
of theory) of the Boc-protected intermediate tert-butyl
(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-
-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidine-1-carboxylate
were obtained.
[3762] HPLC (Method 12): R.sub.t=2.0 min;
[3763] LC-MS (Method 1): R.sub.t=1.12 min; MS (ESIpos): m/z=504
(M+H).sup.+.
[3764] 503 mg (1 mmol) of this intermediate were taken up in 20 ml
of dichloromethane, 10 ml of trifluoroacetic acid were added, and
the mixture was stirred at RT for 30 min. Subsequently, the
reaction mixture was concentrated in vacuo and redistilled with
dichloromethane. The remaining residue was precipitated from ethyl
acetate with n-pentane, and the solvent was decanted off. The
residue thus obtained was dissolved in water and extracted by
shaking with ethyl acetate, and the aqueous phase was subsequently
lyophilized. In this way, 462 mg (89% of theory) of the title
compound were obtained as a colourless foam.
[3765] HPLC (Method 12): R.sub.t=1.53 min;
[3766] LC-MS (Method 11): R.sub.t=0.57 min; MS (ESIpos): m/z=404
(M+H).sup.+.
Intermediate 8
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methoxy-
-4-methylhexan-3-yl]-N-methyl-L-valinamide
##STR00446##
[3768] 51 mg (0.08 mmol) of
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-car-
boxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide
(Intermediate 4) were dissolved in 10 ml of DMF, and 0.5 ml of
piperidine was added. After stirring at RT for 10 min, the reaction
mixture was concentrated in vacuo, and the residue was stirred with
diethyl ether. The insoluble constituents were filtered off and
washed repeatedly with diethyl ether. Then the filter residue was
taken up in 5 ml of dioxane/water, and the solution was adjusted to
pH 11 with 1 N sodium hydroxide solution. Under ultrasound
treatment, a total of 349 mg (1.6 mmol) of di-tert-butyl
dicarbonate were added in several portions, in the course of which
the pH of the solution was kept at 11. After the reaction had
ended, the dioxane was evaporated off and the aqueous solution was
adjusted to a pH of 2-3 with citric acid. The mixture was extracted
twice, with 50 ml each time of ethyl acetate. The organic phases
were combined, dried over magnesium sulphate and concentrated under
reduced pressure. The residue was taken up in diethyl ether and the
of the title compound was precipitated with pentane. The solvent
was removed by decantation. The residue was digested several times
more with pentane and finally dried under high vacuum. 40 mg (97%
of theory) of the title compound were thus obtained.
[3769] HPLC (Method 6): R.sub.t=2.2 min;
[3770] LC-MS (Method 2): R.sub.t=1.32 min; MS (ESIpos): m/z=516
(M+H).sup.+.
Intermediate 9
tert-butyl-(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan--
2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidine-1-carboxy-
late
##STR00447##
[3772] The title compound was prepared in analogy to the synthesis
of Intermediates 5, 6 and 7 over three stages by coupling of
commercially available
(1S,2R)-1-[(tert-butoxycarbonyl)amino]-2-phenylcyclopropanecarb-
oxylic acid with 1,2-oxazinane hydrochloride (Starting Compound 5),
subsequent deprotection with trifluoroacetic acid and coupling with
Starting Compound 1. The end product was purified by preparative
HPLC.
[3773] HPLC (Method 5): R.sub.t=2.12 min;
[3774] LC-MS (Method 2): R.sub.t=1.25 min; MS (ESIpos): m/z=516
(M+H).sup.+.
Intermediate 10
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S-
)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-
-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00448##
[3776] 315 mg (0.494 mmol) of
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-car-
boxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide
(Intermediate 4) were dissolved in 12 ml of DMF and admixed with
104 mg (0.543 mmol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 83
mg (0.543 mmol) of 1-hydroxy-1H-benzotriazole hydrate, and the
mixture was stirred at RT for 90 min. Subsequently, 112 .mu.l of
N,N-diisopropylethylamine and 149 mg (0.494 mmol) of
(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoic acid
trifluoroacetate, which had been prepared beforehand from Starting
Compound 1 by elimination of the Boc protecting group by means of
trifluoroacetic acid, were added. The mixture was stirred at RT for
2 h and then concentrated under high vacuum. The remaining residue
was purified twice by preparative HPLC. 140 mg (35% of theory) of
the title compound were obtained in the form of a colourless
foam.
[3777] HPLC (Method 5): R.sub.t=2.40 min;
[3778] LC-MS (Method 1): R.sub.t=1.38 min; MS (ESIpos): m/z=807
(M+H).sup.+.
Intermediate 11
N-[(benzyloxy)carbonyl]-N-methyl-L-threonyl-N-[(2R,3S,4S)-1-carboxy-2-meth-
oxy-4-methylhexan-3-yl]-N-methyl-L-valinamide
##STR00449##
[3780] First, N-[(benzyloxy)carbonyl]-N-methyl-L-threonine was
released from 237 mg (0.887 mmol) of its dicyclohexylamine salt
thereof by taking it up in ethyl acetate and extractive shaking
with 5% aqueous sulphuric acid. The organic phase was dried over
magnesium sulphate, filtered and concentrated. The residue was
taken up in 16 ml of DMF and admixed successively with 365 mg (1
mmol) of tert-butyl
(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl(L-valyl)amino]heptanoate
(Intermediate 2), 185 mg (0.967 mmol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 148
mg (0.967 mmol) of 1-hydroxy-1H-benzotriazole hydrate. The mixture
was stirred at RT for 2 h. The reaction mixture was then poured
into a mixture of semisaturated aqueous ammonium chloride solution
and ethyl acetate. The organic phase was removed, washed
successively with saturated sodium hydrogencarbonate solution and
saturated sodium chloride solution, dried over magnesium sulphate,
filtered and concentrated. The residue was purified by preparative
HPLC. 283 mg (53% of theory) of the tert-butyl ester intermediate
N-[(benzyloxy)carbonyl]-N-methyl-L-threonyl-N-[(3R,4S,5S)-1-tert-butoxy-3-
-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide were thus
obtained.
[3781] HPLC (Method 5): R.sub.t=2.17 min.
[3782] 283 mg (0.466 mmol) of this intermediate were taken up in 5
ml of dichloromethane, 5 ml of anhydrous trifluoroacetic acid were
added, and the mixture was stirred at RT for 2 h. Subsequently, the
reaction mixture was concentrated under high vacuum and the
remaining residue was purified by means of preparative HPLC. This
gave 156 mg (61% of theory) of the title compound as a colourless
foam.
[3783] HPLC (Method 5): R.sub.t=1.50 min;
[3784] LC-MS (Method 2): R.sub.t=1.09 min; MS (ESIpos): m/z=552
(M+H).sup.+.
Intermediate 12
Benzyl-N-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoyl}-L--
phenylalaninate trifluoroacetate
##STR00450##
[3786] In the first step, Starting Compound 1 was released from 600
mg (1.28 mmol) of the corresponding dicyclohexylammonium salt by
dissolving the salt in 100 ml of ethyl acetate and extractive
shaking, first with 50 ml of 0.5% sulphuric acid and then with
saturated sodium chloride solution. Then the organic phase was
dried over magnesium sulphate, filtered, concentrated and reacted
immediately with benzyl L-phenylalaninate in analogy to the
synthesis of Intermediate 7, and then deprotected.
[3787] Yield: 650 mg (94% over 2 stages)
[3788] HPLC (Method 6): R.sub.t=1.76 min;
[3789] LC-MS (Method 2): R.sub.t=1.68 min; MS (ESIpos): m/z=425
(M+H).sup.+.
Intermediate 13
Benzyl-(.beta.S)--N-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]pr-
opanoyl}-.beta.-methyl-L-phenylalaninate trifluoroacetate
##STR00451##
[3791] First,
(2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methy-
lpropanoic acid was released from 351 mg (0.75 mmol) of the
dicyclohexylamine salt (Starting Compound 1) by taking it up in
ethyl acetate and extractive shaking with aqueous 5% potassium
hydrogensulphate solution. The organic phase was dried over
magnesium sulphate, filtered and concentrated. The residue was
taken up in 10 ml of DMF and admixed successively with 373 mg (0.75
mmol) of benzyl-(.beta.S)-.beta.-methyl-L-phenylalaninate
trifluoroacetate [prepared from commercially available
(.beta.S)--N-(tert-butoxycarbonyl)-.beta.-methyl-L-phenylalanine by
EDC/DMAP-mediated esterification with benzyl alcohol and subsequent
cleaving of the Boc protecting group with trifluoroacetic acid],
428 mg (1.125 mmol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) and 392 .mu.l of
N,N-diisopropylethylamine. The mixture was stirred at RT for 20 h.
The reaction mixture was then poured onto a mixture of
semisaturated aqueous ammonium chloride solution and ethyl acetate.
The organic phase was removed, washed successively with saturated
sodium hydrogencarbonate solution and saturated sodium chloride
solution, and subsequently concentrated. The residue was purified
by means of preparative HPLC. This gave 230 mg (57% of theory) of
the Boc-protected intermediate
benzyl-(.beta.S)--N-{(2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2--
yl]-3-methoxy-2-methylpropanoyl}-.beta.-methyl-L-phenylalaninate.
[3792] HPLC (Method 6): R.sub.t=2.3 min;
[3793] LC-MS (Method 1): R.sub.t=1.36 min; MS (ESIpos): m/z=539
(M+H).sup.+.
[3794] 230 mg (0.42 mmol) of this intermediate were taken up in 5
ml of dichloromethane, 5 ml of trifluoroacetic acid were added, and
the mixture was stirred at RT for 30 min. Subsequently, the
reaction mixture was concentrated in vacuo. The remaining residue
was the reaction mixture dried further in vacuo and then
lyophilized from acetonitrile/water. In this way, 230 mg (quant.)
of the title compound were obtained.
[3795] HPLC (Method 6): R.sub.t=1.6 min.
Intermediate 14
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-me-
thyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxo-
propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate
##STR00452##
[3797] 143 mg (0.223 mmol) of
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-car-
boxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide
(Intermediate 4) were taken up in 15 ml of DMF and admixed
successively with 141 mg (0.22 mmol) of
(2R,3R)-3-methoxy-2-methyl-N-[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-
-phenylpropan-2-yl]-3-[(2S)-pyrrolidin-2-yl]propanamide
trifluoroacetate (Intermediate 7), 102 mg (0.27 mmol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) and 128 .mu.l (0.74 mmol) of
N,N-diisopropylethylamine. The mixture was stirred at RT for 3 h.
The reaction mixture was then poured into a mixture of
semisaturated aqueous ammonium chloride solution and ethyl acetate.
The organic phase was removed, washed successively with saturated
sodium hydrogencarbonate solution and saturated sodium chloride
solution, dried over magnesium sulphate, filtered and concentrated.
This gave 275 mg (quant.) of the Fmoc-protected intermediate
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-3-met-
hoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)--
1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-o-
xoheptan-4-yl]-N-methyl-L-valinamide.
[3798] HPLC (Method 5): R.sub.t=2.73 min;
[3799] LC-MS (Method 4): R.sub.t=3.19 min; MS (ESIpos): m/z=1023
(M+H).sup.+.
[3800] 46 mg (0.045 mmol) of this intermediate were dissolved in 4
ml of DMF. After 1 ml of piperidine had been added, the reaction
mixture was stirred at RT for 1 h. Subsequently, the reaction
mixture was concentrated in vacuo and the residue was purified by
means of preparative HPLC (eluent: acetonitrile+0.01%
TFA/water+0.01% TFA). 22 mg (54% of theory) of the title compound
were obtained as a colourless foam.
[3801] HPLC (Method 5): R.sub.t=1.68 min;
[3802] LC-MS (Method 2): R.sub.t=1.03 min; MS (ESIpos): m/z=801
(M+H).sup.+
[3803] .sup.1H NMR (600 MHz, DMSO-d.sub.6): .delta.=8.8 (m, 2H),
8.7 (m, 1H), 8.42 and 8.15 (2d, 1H), 7.3-7.1 (m, 5H), 5.12 and 4.95
(2m, 1H), 4.70 and 4.62 (2m, 1H), 4.62 and 4.50 (2t, 1H), 4.1-3.9
(m, 3H), 3.85 (m, 1H), 3.75-3.6 (m, 2H), 3.23, 3.18, 3.17, 3.14,
3.02 and 2.96 (6s, 9H), 3.1-2.9 and 2.75 (2m, 2H), 2.46 (m, 3H),
2.4-2.1 (m, 2H), 2.05 (br. m, 2H), 1.85-1.55 (br. m, 6H), 1.5-1.2
(br. m, 3H), 1.1-0.8 (m, 18H), 0.75 (t, 3H) [further signals hidden
under H.sub.2O peak].
Intermediate 15
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-me-
thyl-3-{[(2S,3S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylbutan-2-yl]amino}-3-o-
xopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate
##STR00453##
[3805] 126 mg (0.198 mmol) of
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-car-
boxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide
(Intermediate 4) were taken up in 10 ml of DMF and admixed
successively with 105 mg (0.198 mmol) of
(2R,3R)-3-methoxy-2-methyl-N-[(2S,3S)-1-(1,2-oxazinan-2-yl)-1-ox-
o-3-phenylbutan-2-yl]-3-[(2S)-pyrrolidin-2-yl]propanamide
trifluoroacetate (Intermediate 17), 41.6 mg (0.217 mmol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 33 mg
(0.217 mmol) of 1-hydroxy-1H-benzotriazole hydrate and 79 .mu.l
(0.454 mmol) of N,N-diisopropylethylamine. The mixture was stirred
at RT overnight. The reaction mixture was then poured into a
mixture of semisaturated aqueous ammonium chloride solution and
ethyl acetate. The organic phase was removed, washed successively
with saturated sodium hydrogencarbonate solution and saturated
sodium chloride solution, dried over magnesium sulphate, filtered
and concentrated. This gave 220 mg (quant.) of the Fmoc-protected
intermediate
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-3-met-
hoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S,3S)-1-(1,2-oxazinan-2-y-
l)-1-oxo-3-phenylbutan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-
-oxoheptan-4-yl]-N-methyl-L-valinamide.
[3806] HPLC (Method 5): R.sub.t=2.77 min;
[3807] LC-MS (Method 1): R.sub.t=1.5 min; MS (ESIpos): m/z=1037
(M+H).sup.+.
[3808] 220 mg (0.212 mmol) of this intermediate were dissolved in 5
ml of DMF. After 1 ml of piperidine had been added, the reaction
mixture was stirred at RT for 1 h. Subsequently, the reaction
mixture was concentrated under reduced pressure and the residue was
purified by means of preparative HPLC (eluent: acetonitrile+0.01%
TFA/water+0.01% TFA). 91 mg (46% of theory) of the title compound
were obtained as a colourless foam.
[3809] HPLC (Method 5): R.sub.t=1.71 min;
[3810] LC-MS (Method 1): R.sub.t=0.9 min; MS (ESIpos): m/z=815
(M+H).sup.+
[3811] .sup.1H NMR (600 MHz, DMSO-d.sub.6): .delta.=8.87 and 8.80
(2d, 2H), 8.75 (m, 1H), 8.40 and 7.98 (2d, 1H), 7.3-7.1 (m, 5H),
5.45 and 5.2 (2t, 1H), 4.78 and 4.62 (2m, 1H), 4.73 and 4.58 (2t,
1H), 4.2-4.0 (m, 3H), 3.7-3.6 (m, 1H), 3.35, 3.20, 3.18, 3.14, 3.12
and 3.00 (6s, 9H), 3.1 and 2.95 (2m, 2H), 2.46 (m, 3H), 2.4-2.0 (m,
4H), 1.9-1.6 (m, 4H), 1.6-1.2 (m, 5H), 1.1-0.75 (m, 21H), 0.80 (t,
3H) [further signals hidden under H.sub.2O peak].
Intermediate 16
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-me-
thyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}--
3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinam-
ide trifluoroacetate
##STR00454##
[3813] 617 mg (1.2 mmol) of tert-butyl
(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbon-
yl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidine-1-carboxylate
(Intermediate 24) were taken up in 44 ml of dichloromethane, 4.4 ml
of trifluoroacetic acid were added, and the mixture was stirred at
RT for 30 min. Subsequently, the reaction mixture was concentrated
in vacuo and the remaining residue was lyophilized from
dioxane/water. 702 mg (quant.) of the deprotected compound
(2R,3R)-3-methoxy-2-methyl-N-[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phe-
nylcyclopropyl]-3-[(2S)-pyrrolidin-2-yl]propanamide
trifluoroacetate were obtained as a crude product, which was used
in the following stage without further purification.
[3814] 470 mg (0.74 mmol) of
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-car-
boxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide
(Intermediate 4) were taken up in 57 ml of DMF and admixed
successively with 390 mg (approx. 0.74 mmol) of the above-obtained
(2R,3R)-3-methoxy-2-methyl-N-[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phe-
nylcyclopropyl]-3-[(2S)-pyrrolidin-2-yl]propanamide
trifluoroacetate, 336 mg (0.88 mmol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) and 423 .mu.l (2.4 mmol) of
N,N-diisopropylethylamine. The mixture was stirred at RT for 2 h.
The reaction mixture was then poured into a mixture of
semisaturated aqueous ammonium chloride solution and ethyl acetate.
The organic phase was removed, washed successively with saturated
sodium hydrogencarbonate solution and saturated sodium chloride
solution, dried over sodium sulphate, filtered and concentrated.
The residue was purified by preparative HPLC. This gave 453 mg (59%
of theory) of the Fmoc-protected intermediate
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-3-met-
hoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-y-
lcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methy-
l-1-oxoheptan-4-yl]-N-methyl-L-valinamide.
[3815] HPLC (Method 5): R.sub.t=2.58 min;
[3816] LC-MS (Method 1): R.sub.t=3.10 min; MS (ESIpos): m/z=1035
(M+H).sup.+.
[3817] 453 mg (0.438 mmol) of this intermediate were dissolved in
24 ml of DMF. After 2.4 ml of piperidine had been added, the
reaction mixture was stirred at RT for 30 min. Subsequently, the
reaction mixture was concentrated under reduced pressure and the
residue was purified by preparative HPLC (eluent: acetonitrile/0.1%
TFA in water). 260 mg (64% of theory) of the title compound were
obtained as a colourless foam.
[3818] HPLC (Method 5): R.sub.t=1.64 min;
[3819] LC-MS (Method 1): R.sub.t=0.86 min; MS (ESIpos): m/z=813
(M+H).sup.+
[3820] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=8.8 (m, 2H),
8.65 (m, 2H), 7.3-7.1 (m, 5H), 4.8-4.05 (m, 2H), 4.0 and 3.82 (2m,
2H), 3.8-3.5 (m, 8H), 3.32, 3.29, 3.20, 3.19, 3.12 and 3.00 (6s,
9H), 2.65 (t, 1H), 2.5-2.45 (m, 3H), 2.4-1.3 (m, 15H), 1.15-0.85
(m, 18H), 0.8 and 0.75 (2d, 3H) [further signals hidden under
H.sub.2O peak].
Intermediate 17
N-benzyl-N-methyl-L-phenylalaninamide trifluoroacetate
##STR00455##
[3822] 1000 mg (3.77 mmol) of
N-(tert-butoxycarbonyl)-L-phenylalanine were dissolved in 10 ml of
DMF and admixed with 457 mg (3.77 mmol) of N-methylbenzylamine,
2150 mg (5.65 mmol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and 657 .mu.l of N,N-diisopropylethylamine. The
reaction mixture was stirred at RT for 30 min and then concentrated
in vacuo. The residue was taken up in dichloromethane and extracted
by shaking three times with water. The organic phase was dried over
magnesium sulphate and concentrated. The residue was purified by
flash chromatography on silica gel with 3:1 petroleum ether/ethyl
acetate as the eluent. The product fractions were concentrated, and
the residue was dried under high vacuum. This gave 1110 mg (75% of
theory) of the Boc-protected intermediate
N-benzyl-N.sup..alpha.-(tert-butoxycarbonyl)-N-methyl-L-phenylalaninamide-
.
[3823] HPLC (Method 6): R.sub.t=2.1 min;
[3824] LC-MS (Method 1): R.sub.t=1.14 min; MS (ESIpos): m/z=369
(M+H).sup.+.
[3825] 1108 mg (3,007 mmol) of this intermediate were taken up in
30 ml of dichloromethane, 10 ml of trifluoroacetic acid were added,
and the mixture was stirred at RT for 30 min. Subsequently, the
reaction mixture was concentrated in vacuo, the remaining residue
was stirred with dichloromethane, and the solvent was distilled
off. The residue was stirred twice more with pentane, the solvent
was decanted off again each time and the of the title compound was
finally dried under high vacuum. 1075 mg (93% of theory) of the
title compound were thus obtained as a resin.
[3826] HPLC (Method 6): R.sub.t=1.6 min;
[3827] LC-MS (Method 1): R.sub.t=0.6 min; MS (ESIpos): m/z=269
(M+H).sup.+.
Intermediate 18
N-benzyl-N.sup..alpha.-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl-
]propanoyl}-N-methyl-L-phenylalaninamide trifluoroacetate
##STR00456##
[3829] First,
(2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methy-
lpropanoic acid (Starting Compound 1) was released from 141 mg
(0.491 mmol) of its dicyclohexylamine salt by taking it up in ethyl
acetate and extractive shaking with 5% aqueous sulphuric acid. The
organic phase was dried over magnesium sulphate, filtered and
concentrated. The residue was taken up in 10 ml of DMF and 187.6 mg
(0.49 mmol) of N-benzyl-N-methyl-L-phenylalaninamide
trifluoroacetate (Intermediate 9), 190.3 mg (1.47 mmol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) and 256 .mu.l of
N,N-diisopropylethylamine were added. The mixture was stirred at RT
for 1 h. The reaction mixture was then concentrated, the residue
was taken up in ethyl acetate, and the solution was subsequently
extracted by shaking successively with saturated ammonium chloride
solution, saturated sodium hydrogencarbonate solution and water.
The organic phase was dried over magnesium sulphate and
concentrated. The residue was purified by flash chromatography on
silica gel with 30:1 acetonitrile/water as the eluent. The product
fractions were concentrated and the residue was dried under high
vacuum. This gave 168 mg (64% of theory) of the Boc-protected
intermediate tert-butyl
(2S)-2-[(1R,2R)-3-({(2S)-1-[benzyl(methyl)amino]-1-oxo-3-phenylpropan-2-y-
l}amino)-1-methoxy-2-methyl-3-oxopropyl]pyrrolidine-1-carboxylate.
[3830] HPLC (Method 6): R.sub.t=2.2 min;
[3831] LC-MS (Method 2): R.sub.t=1.22 min; MS (ESIpos): m/z=538
(M+H).sup.+.
[3832] 168 mg (0.312 mmol) of this intermediate were taken up in 15
ml of dichloromethane, 3 ml of trifluoroacetic acid were added, and
the mixture was stirred at RT for 30 min. Subsequently, the
reaction mixture was concentrated in vacuo. The remaining residue
was stirred first with dichloromethane, then with diethyl ether,
and the solvent was distilled off again each time. After drying
under high vacuum, 170 mg (99% of theory) of the title compound
were obtained as a resin.
[3833] HPLC (Method 6): R.sub.t=1.7 min;
[3834] LC-MS (Method 1): R.sub.t=0.73 min; MS (ESIpos): m/z=438
(M+H).sup.+.
Intermediate 19
Methyl-N-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoyl}-L--
phenylalaninate trifluoroacetate
##STR00457##
[3836] The title compound was prepared in analogy to the synthesis
of Intermediate 18, proceeding from
(2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methy-
lpropanoic acid (Starting Compound 1), which was released from the
dicyclohexylamine salt, and methyl L-phenylalaninate
hydrochloride.
[3837] HPLC (Method 5): R.sub.t=0.6 min;
[3838] LC-MS (Method 3): R.sub.t=1.17 min; MS (ESIpos): m/z=349
(M+H).sup.+.
Intermediate 20
Benzyl-N-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoyl}-L--
tryptophanate trifluoroacetate
##STR00458##
[3840] The title compound was prepared in analogy to the synthesis
of Intermediate 18, proceeding from
(2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methy-
lpropanoic acid (Starting Compound 1), which was released from the
dicyclohexylamine salt, and benzyl L-tryptophanate.
[3841] HPLC (Method 6): R.sub.t=2.0 min;
[3842] LC-MS (Method 1): R.sub.t=0.8 min; MS (ESIpos): m/z=464
(M+H).sup.+.
Intermediate 21
Benzyl-(1S,2R)-1-({(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]prop-
anoyl}amino)-2-phenylcyclopropanecarboxylate trifluoroacetate
##STR00459##
[3844] The title compound was prepared in analogy to the synthesis
of Intermediate 18, proceeding from
(2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methy-
lpropanoic acid (Starting Compound 1), which was released from the
dicyclohexylamine salt, and benzyl
(1S,2R)-1-amino-2-phenylcyclopropanecarboxylate.
Benzyl-(1S,2R)-1-amino-2-phenylcyclopropane-carboxylate had been
prepared beforehand by standard methods, by esterifying
commercially available
(1S,2R)-1-[(tert-butoxycarbonyl)amino]-2-phenylcyclopropanecarboxylic
acid with benzyl alcohol and subsequent Boc cleaving with
trifluoroacetic acid.
[3845] HPLC (Method 5): R.sub.t=1.5 min;
[3846] LC-MS (Method 2): R.sub.t=0.93 min; MS (ESIpos): m/z=437
(M+H).sup.+.
Intermediate 22
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N'-methylhexanehydrazide
trifluoroacetate
##STR00460##
[3848] 100 mg (473 .mu.mol) of
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid were
dissolved in 71 .mu.l of DMF and then admixed with 139 mg (947
.mu.mol) of tert-butyl-1-methylhydrazinecarboxylate, 182 mg (947
.mu.mol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride and 145 mg (947 .mu.mol) of
1-hydroxy-1H-benzotriazole hydrate. The mixture was stirred at RT
overnight and then concentrated in vacuo. The remaining residue was
purified by means of preparative HPLC. After lyophilization from
dioxane/water, 129 mg (80% of theory) of the protected intermediate
were obtained as a colourless foam.
[3849] Subsequently, the 129 mg (380 .mu.mol) were deblocked with 2
ml of trifluoroacetic acid in 8 ml of dichloromethane. After
stirring at RT for 1 h, the reaction mixture was concentrated in
vacuo. The residue was lyophilized from acetonitrile/water, which
left 125 mg (83% of theory) of the title compound as a colourless
foam.
[3850] LC-MS (Method 1): R.sub.t=0.38 min; MS (ESIpos): m/z=240
(M+H).sup.+
Intermediate 23
N-(2-aminoethyl)-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylbutanami-
de trifluoroacetate
##STR00461##
[3852] First, 35 mg (164 .mu.mol) of
tert-butyl-[2-(methylamino)ethyl]carbamate-hydrochloride-trifluoroacetate-
, 30 mg (164 .mu.mol) of
4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid, 75 mg (197
.mu.mol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium-hexafluorophospha-
te and 57 .mu.l of N,N-diisopropylethylamine were combined in 5 ml
of DMF and stirred at RT overnight. Subsequently, the solvent was
removed in vacuo, and the remaining residue was purified by means
of preparative HPLC. The corresponding fractions were concentrated
and, by lyophilization from dioxane/water, 35 mg (63% of theory) of
the protected intermediate were obtained.
[3853] HPLC (Method 12): R.sub.t=1.6 min;
[3854] LC-MS (Method 1): R.sub.t=0.71 min; MS (ESIpos): m/z=340
(M+H).sup.+. Subsequently, the entire amount of the protected
intermediate was deblocked with 1 ml of trifluoroacetic acid in 5
ml of dichloromethane to obtain 28 mg (77% of theory) of the title
compound.
[3855] LC-MS (Method 3): R.sub.t=0.75 min; MS (ESIpos): m/z=240
(M+H).sup.+.
Intermediate 24
4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-[2-(methylamino)ethyl]butanamid-
e trifluoroacetate
##STR00462##
[3857] First, 35 mg (164 .mu.mol) of
tert-butyl-(2-aminoethyl)methylcarbamate hydrochloride
trifluoroacetate, 30 mg (164 .mu.mol) of
4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid, 75 mg (197
.mu.mol) of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and 57 .mu.l of N,N-diisopropylethylamine were
combined in 5 ml of DMF and stirred at RT for 30 min. Subsequently,
the solvent was removed in vacuo, and the remaining residue was
purified by means of preparative HPLC. The corresponding fractions
were concentrated and, by lyophilization from dioxane/water, 51 mg
(91% of theory) of the protected intermediate were obtained.
[3858] HPLC (Method 12): R.sub.t=1.6 min;
[3859] LC-MS (Method 1): R.sub.t=0.77 min; MS (ESIpos): m/z=340
(M+H).sup.+.
[3860] Subsequently, the entire amount was deprotected with 1 ml of
trifluoroacetic acid in 5 ml of dichloromethane to obtain 45 mg
(69% of theory) of the title compound.
[3861] LC-MS (Method 1): R.sub.t=0.19 min; MS (ESIpos): m/z=240
(M+H).sup.+.
Intermediate 25
Benzyl-(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoate
trifluoroacetate
##STR00463##
[3863] First,
(2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methy-
lpropanoic acid was released from 1.82 g (3.88 mmol) of its
dicyclohexylamine salt by taking it up in 150 ml of ethyl acetate
and extractive shaking with 100 ml of 0.5% sulphuric acid. The
organic phase was dried over magnesium sulphate, filtered and
concentrated. The residue was taken up in 10 ml of dioxane and 10
ml of water, 1517 mg (4.66 mmol) of caesium carbonate were added,
and the mixture was treated in an ultrasound bath for 5 min, then
concentrated under in vacuo and redistilled once with DMF. The
residue was then taken up in 15 ml of dichloromethane, and 1990 mg
(11.64 mmol) of benzyl bromide were added to this. The mixture was
treated in an ultrasound bath for 15 min and then concentrated in
vacuo. The residue was partitioned between ethyl acetate and water,
the organic phase was removed and extracted by shaking with
saturated sodium chloride solution and then concentrated. The
residue was then purified by preparative HPLC. This gave 1170 mg
(80% of theory) of the Boc-protected intermediate.
[3864] Subsequently, these 1170 mg were deprotected immediately
with 5 ml of trifluoroacetic acid in 15 ml of dichloromethane.
After stirring at RT for 15 min, the reaction mixture was
concentrated in vacuo. The residue was lyophilized from dioxane.
After drying under high vacuum, there remained 1333 mg (84% of
theory) of the title compound as a yellow oil.
[3865] HPLC (Method 6): R.sub.t=1.5 min;
[3866] LC-MS (Method 1): R.sub.t=0.59 min; MS (ESIpos): m/z=278
(M+H).sup.+.
Intermediate 26
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)--
2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan--
4-yl]-N-methyl-L-valinamide
##STR00464##
[3868] 1200 mg (2.33 mmol) of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methox-
y-4-methylhexan-3-yl]-N-methyl-L-valinamide (Intermediate 5) were
combined with 910.8 mg (2.33 mmol) of benzyl
(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoate
trifluoroacetate (Intermediate 14), 1327 mg (3.49 mmol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and 2027 .mu.l of N,N-diisopropylethylamine in
50 ml of DMF, and the mixture was stirred at RT for 5 min.
Thereafter, the solvent was removed in vacuo. The remaining residue
was taken up in ethyl acetate and extracted by shaking it
successively with 5% aqueous citric acid solution and saturated
sodium hydrogencarbonate solution. The organic phase was removed
and concentrated. The residue was purified by means of preparative
HPLC. The product fractions were combined and concentrated, and the
residue was dried under high vacuum. This gave 1000 mg (55% of
theory) of the benzyl ester intermediate
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-3-(benzyloxy)-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-
-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide as a resin.
[3869] LC-MS (Method 1): R.sub.t=1.56 min; MS (ESIpos): m/z=775
(M+H).sup.+.
[3870] The entire amount of this intermediate obtained was taken up
in 25 ml of a mixture of methanol and dichloromethane (20:1), and
the benzyl ester group was removed by hydrogenation under standard
hydrogen pressure with 10% palladium on activated carbon as a
catalyst. After stirring at RT for 30 min, the catalyst was
filtered off and the filtrate was concentrated in vacuo. This gave
803 mg (91% of theory) of the title compound as a white solid.
[3871] HPLC (Method 6): R.sub.t=2.1 min;
[3872] LC-MS (Method 1): R.sub.t=1.24 min; MS (ESIpos): m/z=685
(M+H).sup.+.
Intermediate 27
(1S,2R)-1-amino-2-phenyl-N-propylcyclopropanecarboxamide
trifluoroacetate
##STR00465##
[3874] The title compound was prepared by coupling commercially
available
(1S,2R)-1-[(tert-butoxycarbonyl)amino]-2-phenylcyclopropanecarboxylic
acid with n-propylamine in the presence of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) and subsequent Boc cleaving with
trifluoroacetic acid (yield: 85% of theory over both stages).
[3875] HPLC (Method 6): R.sub.t=1.2 min;
[3876] LC-MS (Method 1): R.sub.t=0.52 min; MS (ESIpos): m/z=219
(M+H).sup.+.
Intermediate 28
Ethyl-(1S,2R)-1-amino-2-phenylcyclopropanecarboxylate
trifluoroacetate
##STR00466##
[3878] The title compound was prepared according to standard
methods by esterifying commercially available
(1S,2R)-1-[(tert-butoxycarbonyl)amino]-2-phenylcyclopropanecarboxylic
acid with ethanol and subsequent Boc cleaving with trifluoroacetic
acid.
[3879] LC-MS (Method 1): R.sub.t=0.50 min; MS (ESIpos): m/z=206
(M+H).sup.+.
Intermediate 29
4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethylbutanoic
acid
##STR00467##
[3881] To a solution of 1.39 g (8.95 mmol) of
N-methoxycarbonylmaleimide in 44 ml of saturated sodium
hydrogencarbonate solution were added, at 0.degree. C., 1.5 g (8.95
mmol) of 4-amino-2,2-dimethylbutyric acid, and the mixture was
stirred for 40 min. Subsequently, the cooling bath was removed, and
the reaction mixture was stirred for 1 h more. While cooling with
ice, the reaction mixture was then adjusted to pH 3 by adding
sulphuric acid, then extracted with ethyl acetate. The combined
organic phases were dried over magnesium sulphate and concentrated.
1.17 g (79% purity, 49% of theory) of the title compound were
obtained.
[3882] LC-MS (Method 1): R.sub.t=0.64 min; m/z=212 (M+H).sup.+.
Intermediate 30
tert-butyl
2-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethylbutanoy-
l]hydrazinecarboxylate
##STR00468##
[3884] To a solution of 50 mg (237 .mu.mol) of
4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethylbutanoic acid
in 2 ml of THF were added, at 0.degree. C., first 26 .mu.l (237
.mu.mol) of 4-methylmorpholine and then 31 .mu.l (237 .mu.mol) of
isobutyl chloroformate. After removing the cooling bath and
stirring at RT for a further 15 min, 31.3 mg (237 .mu.mol) of
tert-butyloxycarbonyl hydrazide were added. The reaction mixture
was stirred overnight and then concentrated. The residue was
purified by preparative HPLC. 50.8 mg (66% of theory) of the title
compound were obtained.
[3885] LC-MS (Method 1): R.sub.t=0.71 min; m/z=324 (M-H).sup.-.
Intermediate 31
4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethylbutanehydrazide
trifluoroacetate
##STR00469##
[3887] 50 mg (154 mmol) of tert-butyl
2-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethylbutanoyl]hydrazin-
ecarboxylate were dissolved in 2 ml of dichloromethane, and 0.4 ml
of trifluoroacetic acid was added. The reaction mixture was stirred
at RT for 30 min and then concentrated. 55.2 mg (93% purity, 99% of
theory) of the title compound were obtained.
[3888] LC-MS (Method 1): R.sub.t=0.36 min; m/z=226 (M+H).sup.+.
Intermediate 32
Adamantan-1-ylmethyl N-(tert-butoxycarbonyl)-L-phenylalaninate
##STR00470##
[3890] To a solution of 500 mg (1.89 mmol) of N-Boc-L-phenylalanine
in 25 ml of dichloromethane were added, at RT, 1192 mg (6.2 mmol)
of EDC, 578 .mu.l (4.1 mmol) of triethylamine, 345 mg (2.8 mmol) of
DMAP and 345 mg (2.1 mmol) of 1-adamantylmethanol. The reaction
mixture was stirred overnight, then diluted with 50 ml of
dichloromethane, and was successively washed with 10% aqueous
citric acid solution, water and saturated sodium chloride solution.
The organic phase was dried over magnesium sulphate, then
concentrated, and the residue was purified by preparative HPLC. 769
mg (90% of theory) of the title compound were obtained.
[3891] LC-MS (Method 2): R.sub.t=1.84 min; m/z=414 (M+H).sup.+.
Intermediate 33
Adamantan-1-ylmethyl L-phenylalaninate hydrochloride
##STR00471##
[3893] 769 mg (1.86 mmol) of adamantan-1-ylmethyl
N-(tert-butoxycarbonyl)-L-phenylalaninate (Intermediate 13) were
dissolved in 25 ml of a 4 N solution of hydrogen chloride in
dioxane and stirred at RT for 1 h. Subsequently, the reaction
mixture was concentrated, and the residue was dried in vacuo. 619
mg (95% of theory) of the title compound were obtained.
[3894] LC-MS (Method 1): R.sub.t=0.82 min; m/z=314 (M+H).sup.+.
Intermediate 34
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)--
3-{[(2S)-1-(adamantan-1-ylmethoxy)-1-oxo-3-phenylpropan-2-yl]amino}-1-meth-
oxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-
-yl]-N-methyl-L-valinamide
##STR00472##
[3896] To a solution of 20 mg (29 .mu.mol) of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide in 1 ml of DMF were added, at RT, 15.3
.mu.l (88 .mu.mol) of N,N-diisopropylethylamine, 6.7 mg (44
.mu.mol) of HOBt and 6.7 mg (35 .mu.mol) of EDC, and the mixture
was stirred for 30 min. Subsequently, 10.1 mg (32 .mu.mol) of
adamantan-1-yl L-phenylalaninate hydrochloride were added. After
stirring overnight, the reaction mixture was separated directly
into its components via preparative HPLC. 27.5 mg (93% of theory)
of the title compound were obtained.
[3897] LC-MS (Method 1): R.sub.t=1.70 min; m/z=980 (M+H).sup.+.
Intermediate 35
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(adamantan-1--
ylmethoxy)-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl-
]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinami-
de trifluoroacetate
##STR00473##
[3899] 27.5 mg (28 .mu.mol) of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-3-{[(2S)-1-(adamantan-1-ylmethoxy)-1-oxo-3-phenylpropan-2-yl]amino}-1-met-
hoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan--
4-yl]-N-methyl-L-valinamide were dissolved in 1.8 ml of
dichloromethane, and 361 .mu.l of TFA were added. The reaction
mixture was stirred for 30 min and then concentrated. The residue
was taken up in water and lyophilized. 22.7 mg (81% of theory) of
the title compound were obtained.
[3900] LC-MS (Method 1): R.sub.t=1.14 min; m/z=880 (M+H).sup.+.
Intermediate 36
tert-butyl (2S)-1-(benzyloxy)-3-phenylpropan-2-yl carbamate
##STR00474##
[3902] Under an argon atmosphere, 500 mg (1.99 mmol) of
N-Boc-L-phenylalaninol were dissolved in 5 ml of DMF and cooled to
0.degree. C. Subsequently, 159 mg (3.98 mmol) of a 60% suspension
of sodium hydride in paraffin oil were added. The reaction mixture
was stirred until the evolution of gas had ended, and then 260
.mu.l (2.19 mmol) of benzyl bromide were added. The cooling bath
was removed, and the reaction mixture was stirred at RT for 2 h.
Thereafter, the reaction mixture was concentrated, the residue was
taken up in ice water, and the mixture was extracted with
dichloromethane. The organic phase was washed with saturated sodium
chloride solution, dried over magnesium sulphate and concentrated.
The residue was purified by means of preparative HPLC. 226 mg (33%
of theory) of the title compound were obtained.
[3903] LC-MS (Method 1): R.sub.t=1.28 min; m/z=342 (M+H).sup.+.
Intermediate 37
(2S)-1-(benzyloxy)-3-phenylpropan-2-amine hydrochloride
##STR00475##
[3905] 220 mg (644 .mu.mol) of tert-butyl
(2S)-1-(benzyloxy)-3-phenylpropan-2-yl carbamate were dissolved in
11 ml of a 4 N solution of hydrogen chloride in dioxane and stirred
at RT for 1 h. Then the reaction mixture was concentrated, and the
residue was dried in vacuo. 138 mg (77% of theory) of the title
compound were obtained.
[3906] LC-MS (Method 1): R.sub.t=0.65 min; m/z=242 (M+H).sup.+.
Intermediate 38
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)--
3-{[(2S)-1-(benzyloxy)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxo-
propyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-va-
linamide
##STR00476##
[3908] To a solution of 20 mg (29 .mu.mol) of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide in 1 ml of DMF were added, at RT, 15.3
.mu.l (88 .mu.mol) of N,N-diisopropylethylamine, 6.7 mg (44
.mu.mol) of HOBt and 6.7 mg (35 .mu.mol) of EDC, and the mixture
was stirred for 30 min. Subsequently, 7.8 mg (32 .mu.mol) of
(2S)-1-(benzyloxy)-3-phenylpropan-2-amine hydrochloride were added.
After stirring overnight, the reaction mixture was separated
directly into its components via preparative HPLC. 26 mg (98% of
theory) of the title compound were obtained.
[3909] LC-MS (Method 1): Rt=1.51 min; m/z=909 (M+H)+.
Intermediate 39
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzyloxy)-3-
-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}--
3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate
##STR00477##
[3911] 26 mg (29 .mu.mol) of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-3-{[(2S)-1-(benzyloxy)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-ox-
opropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-v-
alinamide were dissolved in 1.8 ml of dichloromethane, and 370
.mu.l of TFA were added. The reaction mixture was stirred at RT for
30 min and then concentrated. The residue was taken up in water and
lyophilized. 26.4 mg (quant.) of the title compound were
obtained.
[3912] LC-MS (Method 1): R.sub.t=0.97 min; m/z=809 (M+H).sup.+.
Intermediate 40
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1--
phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-
-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00478##
[3914] 50 mg (70 .mu.mol) of Intermediate 26 and 11 mg (70 .mu.mol)
of (1S,2R)-2-amino-1-phenylpropan-1-ol in 10 ml of DMF were admixed
with 42 mg (0.11 .mu.mol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and 25 .mu.l of N,N-diisopropylethylamine, and
the reaction mixture was stirred at RT for 5 min. This was followed
by concentration and purification of the residue by means of
preparative HPLC. After combining the corresponding fractions,
concentrating and drying under high vacuum, 49 mg (81%) of the
protected intermediate were obtained. Subsequently, the Boc group
was cleaved using known conditions with trifluoroacetic acid in
dichloromethane. Concentration was followed by the purification of
the title compound via preparative HPLC, and 26 mg (52%) of the
title compound were obtained.
[3915] HPLC (Method 12): R.sub.t=1.65 min;
[3916] LC-MS (Method 1): R.sub.t=0.77 min; MS (ESIpos): m/z=718
(M+H).sup.+.
Intermediate 41
3-{2-[2-(2-aminoethoxyl)ethoxy]ethoxy}propanoic acid
trifluoroacetate
##STR00479##
[3918] 150 mg (541 .mu.mol) of tert-butyl
3-{2-[2-(2-aminoethoxyl)ethoxy]ethoxy}propanoate were dissolved in
3 ml of dichloromethane, 1.5 ml of trifluoroacetic acid were added,
and the reaction mixture was stirred at RT for 1 h, then
concentrated. 181 mg (100% of theory) of the title compound were
obtained.
[3919] MS (EI): m/z 222 (M+H).sup.+
Intermediate 42
3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy)prop-
anoic acid
##STR00480##
[3921] 186 mg (555 .mu.mol) of
3-{2-[2-(2-aminoethoxyl)ethoxy]ethoxy}propanoic acid
trifluoroacetate were dissolved in 2.6 ml of saturated sodium
hydrogencarbonate solution and admixed at 0.degree. C. with 86 mg
(555 .mu.mol) of N-methoxycarbonylmaleimide. The reaction mixture
was stirred at 0.degree. C. for 40 min and at RT for 1 h, then
cooled again to 0.degree. C., adjusted to pH 3 with sulphuric acid
and extracted 3.times. with 25 ml of ethyl acetate. The combined
organic phases were dried over magnesium sulphate and concentrated.
126 mg (75% of theory) of the title compound were obtained.
[3922] LC-MS (Method 1): R.sub.t=0.53 min; m/z=302 (M+H).sup.+.
Intermediate 43
tert-butyl-15-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-oxo-7,10,13-trioxa--
2,3-diazapentadecan-1-oate
##STR00481##
[3924] 125 mg (417 .mu.mol) of
3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy)
propanoic acid were dissolved at 0.degree. C. in 2.1 ml of THF and
admixed with 46 .mu.l (417 mmol) of 4-methylmorpholine and 54.5
.mu.l (417 .mu.mol) of isobutyl chloroformate. The ice bath was
removed, and the reaction mixture was stirred at RT for 30 min.
Subsequently, at 0.degree. C., 55 mg (417 .mu.mol) of
tert-butyloxycarbonyl hydrazide were added. The reaction mixture
was warmed to RT overnight, concentrated and purified via
preparative HPLC.
[3925] 60 mg (33% of theory) of the title compound were
obtained.
[3926] LC-MS (Method 1): R.sub.t=0.66 min; m/z=416 (M+H).sup.+.
Intermediate 44
3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy)prop-
anehydrazide trifluoroacetate
##STR00482##
[3928] 60 mg (145 .mu.mol) of
tert-butyl-15-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4-oxo-7,10,13-trioxa-
-2,3-diazapentadecan-1-oate were dissolved in 1 ml of
dichloromethane, and 0.2 ml of trifluoroacetic acid was added. The
reaction mixture was stirred at RT for 30 min and then
concentrated.
[3929] 62 mg (100% of theory) of the title compound were
obtained.
[3930] LC-MS (Method 1): R.sub.t=0.35 min; m/z=316 (M+H).sup.+.
Intermediate 45
Benzyl-(1S,2R)-1-amino-2-phenylcyclopropanecarboxylate
trifluoroacetate
##STR00483##
[3932] The title compound was prepared according to standard
methods by esterifying commercially available
(1S,2R)-1-[(tert-butoxycarbonyl)amino]-2-phenylcyclopropanecarboxylic
acid with benzyl alcohol and subsequent Boc cleaving with
trifluoroacetic acid.
[3933] LC-MS (Method 1): R.sub.t=0.72 min; MS (ESIpos): m/z=268
(M+H).sup.+.
Intermediate 46
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)--
3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyr-
rolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00484##
[3935] 383 mg (0.743 mmol) of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methox-
y-4-methylhexan-3-yl]-N-methyl-L-valinamide (Intermediate 8) were
combined with 485 mg (0.743 mmol) of
benzyl-N-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoyl}-L-
-phenylalaninate trifluoroacetate (Intermediate 12), 424 mg (1.114
mmol) of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and 388 .mu.l of N,N-diisopropylethylamine in
15 ml of DMF, and the mixture was stirred at RT for 10 min.
Subsequently, the solvent was removed in vacuo. The remaining
residue was taken up in ethyl acetate and extracted by shaking
successively with 5% aqueous citric acid solution and saturated
sodium hydrogencarbonate solution. The organic phase was removed
and concentrated, and the residue was purified by means of
preparative HPLC. The product fractions were combined and
concentrated, and the residue was dried under high vacuum. 335 mg
(48% of theory) of the benzyl ester intermediate were obtained as a
foam.
[3936] LC-MS (Method 1): R.sub.t=1.49 min; MS (ESIpos): m/z=922
(M+H).sup.+.
[3937] 100 mg (0.11 mmol) of this intermediate were taken up in 15
ml of methanol and the benzyl ester group was removed by
hydrogenation under standard hydrogen pressure with 10% palladium
on activated carbon as a catalyst. After stirring at RT for 1 h,
the catalyst was filtered off and the filtrate was concentrated in
vacuo. After lyophilization from dioxane, 85 mg (94% of theory) of
the title compound were obtained as a solid.
[3938] HPLC (Method 12): R.sub.t=2.4 min;
[3939] LC-MS (Method 1): R.sub.t=1.24 min; MS (ESIpos): m/z=832
(M+H).sup.+.
Intermediate 47
N-benzyl-L-tryptophanamide trifluoroacetate
##STR00485##
[3941] 202 mg (0.5 mmol) of 2,5-dioxopyrrolidin-1-yl
N-(tert-butoxycarbonyl)-L-tryptophanate and 45 mg (0.42 mmol) of
benzylamine were dissolved in 10 ml of DMF, and 110 .mu.l (630
.mu.mol) of N,N-diisopropylethylamine were added. The reaction
mixture was stirred at RT for 3 h. Subsequently, the reaction
mixture was concentrated in vacuo and the residue was purified by
flash chromatography on silica gel (eluent: 20:0.5:0.05
dichloromethane/methanol/17% aq. ammonia). The corresponding
fractions were combined and concentrated. The resulting residue was
digested with diethyl ether and then dried under high vacuum.
Subsequently, this residue was taken up in 10 ml of
dichloromethane, and 3 ml of anhydrous trifluoroacetic acid were
added. After stirring at RT for 45 minutes, the mixture was
concentrated, and the residue was purified via preparative HPLC.
After drying in vacuo, 117 mg (57% of theory over both stages) of
the title compound were obtained.
[3942] HPLC (Method 12): R.sub.t=1.6 min;
[3943] LC-MS (Method 1): R.sub.t=0.66 min; MS (ESIpos): m/z=294
(M+H).sup.+.
Intermediate 48
(1S,2R)-1-amino-2-phenylcyclopropanecarboxamide
trifluoroacetate
##STR00486##
[3945] 50 mg (180 .mu.mol) of commercially available
(1S,2R)-1-[(tert-butoxycarbonyl)amino]-2-phenylcyclopropanecarboxylic
acid were dissolved in 5 ml of DMF, 94 .mu.l (541 .mu.mol) of
N,N-diisopropylethylamine, 31 mg (270 .mu.mol) of
N-hydroxysuccinimide and 41.5 mg (216 .mu.mol) of EDC were added,
and then the mixture was stirred at RT overnight. The reaction
mixture was then concentrated, the residue was taken up in dioxane,
71 mg (901 .mu.mol) of ammonium hydrogencarbonate were added, and
the reaction mixture was then left to stand at RT for 3 days. The
reaction mixture was then diluted with a 1:1 mixture of ethyl
acetate and water. The organic phase was removed, dried over
magnesium sulphate and concentrated. The resulting residue was
subsequently taken up in 3 ml of dichloromethane, and 3 ml of
anhydrous trifluoroacetic acid were added. Stirring at RT for 1 h
was followed by concentration. The residue was stirred with
pentane, suctioned off and lyophilized from dioxane. In this way,
32 mg (62% of theory over both stages) of the title compound were
obtained.
[3946] HPLC (Method 6): R.sub.t=0.38 min;
[3947] LC-MS (Method 1): R.sub.t=0.20 min; MS (ESIpos): m/z=177
(M+H).sup.+.
Intermediate 49
N.sup..alpha.-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoy-
l}-L-tryptophanamide trifluoroacetate
##STR00487##
[3949] The title compound was prepared in analogy to the synthesis
of Intermediate 13 from Starting Compound 1 and L-tryptophanamide
hydrochloride.
[3950] HPLC (Method 5): R.sub.t=1.4 min;
[3951] LC-MS (Method 1): R.sub.t=0.92 min; MS (ESIpos): m/z=473
(M+H).sup.+.
Intermediate 50
4-nitrophenyl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl
carbamate
##STR00488##
[3953] 813 mg (3.1 mmol) of triphenylphosphine were dissolved in 25
ml of THF and cooled to -70.degree. C. under argon. After the
dropwise addition of 627 mg (3.1 mmol) of diisopropyl
azodicarboxylate, the mixture was stirred for 5 min. Subsequently,
500 mg (3.1 mmol) of tert-butyl-(2-aminoethyl) carbamate dissolved
in 5 ml of THF were added dropwise, and the reaction mixture was
stirred at -70.degree. C. for another 5 min. Then 136.6 mg (1.55
mmol) of 2,2-dimethyl-1-propanol dissolved in 1 ml of THF and 301
mg (3.1 mmol) of maleimide were added, the reaction mixture was
stirred at -70.degree. C. for another 10 min, and then the mixture
was warmed to RT. After stirring at RT for another 16 h, the
solvent was removed in vacuo, and the residue was purified by means
of preparative HPLC. This gave 463 mg (62%) of the protected
intermediate.
[3954] After removing the Boc protecting group under standard
conditions, 652 mg of 1-(2-aminoethyl)-1H-pyrrole-2,5-dione were
obtained as trifluoroacetate.
[3955] 112.9 mg (543 .mu.mol) of nitrophenyl chloroformate were
dissolved in 30 ml of THF and, after the addition of 100 mg (271.6
.mu.mol) of 1-(2-aminoethyl)-1H-pyrrole-2,5-dione trifluoroacetate,
the mixture was stirred at RT for 30 min. The mixture was filtered,
and the filtrate was concentrated to dryness and then slurried with
diethyl ether. After suctioning off and drying, 60 mg (95% of
theory) of the title compound were obtained.
[3956] HPLC (Method 5): Rt=0.65 min;
[3957] LC-MS (Method 1): Rt=0.74 min; MS (ESIpos): m/z=306
(M+H)+.
Intermediate 51
(1S)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethanamine
trifluoroacetate
##STR00489##
[3959] 200 mg (0.75 mmol) of
N-(tert-butoxycarbonyl)-L-phenylalanine were initially provided at
0.degree. C. in 5.5 ml of dichloromethane, and 128 mg (0.79 mmol)
of 1,1'-carbonyldiimidazole were added to this. After 30 min, 103
mg (0.75 mmol) of benzoyl hydrazide were added. After a further 45
min at 0.degree. C., 500 mg (1.5 mmol) of carbon tetrabromide and
395 mg (1.5 mmol) of triphenylphosphine were finally added. The
reaction mixture was stirred first at 0.degree. C. for 2 h and then
at RT overnight. The mixture was subsequently concentrated on a
rotary evaporator, and the residue was dried under high vacuum. The
crude product thus obtained was purified by means of preparative
HPLC. 217 mg (78% of theory) of the Boc-protected intermediate
tert-butyl-[(1S)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl
carbamate were obtained.
[3960] LC-MS (Method 12): R.sub.t=1.15 min; MS (ESIpos): m/z=366
(M+H).sup.+
[3961] 217 mg (0.59 mmol) of this intermediate were taken up in 3
ml of dichloromethane, 0.6 ml of trifluoroacetic acid were added,
and the mixture was stirred at RT for 30 min. Subsequently, the
reaction mixture was concentrated in vacuo. The remaining residue
was the reaction mixture and was dried further in vacuo, then
lyophilized from dioxane. In this way, 214 mg (90% of theory) of
the title compound were obtained.
[3962] LC-MS (Method 11): R.sub.t=0.62 min; MS (ESIpos): m/z=266
(M+H).sup.+
Intermediate 52
(1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethanamine
trifluoroacetate
##STR00490##
[3964] 200 mg (0.75 mmol) of
N-(tert-butoxycarbonyl)-D-phenylalanine were initially provided at
0.degree. C. in 5.5 ml of dichloromethane, and 128.3 mg (0.79 mmol)
of 1,1'-carbonyldiimidazole were added to this. After 30 min, 103
mg (0.75 mmol) of benzoyl hydrazide were added. After another 45
min at 0.degree. C., 500 mg (1.5 mmol) of carbon tetrabromide and
395 mg (1.5 mmol) of triphenylphosphine were finally added. The
reaction mixture was stirred first at 0.degree. C. for 2 h and then
at RT overnight. The mixture was subsequently concentrated on a
rotary evaporator, and the residue was dried under high vacuum. The
crude product thus obtained was purified by means of preparative
HPLC. 219 mg (80% of theory) of the Boc-protected intermediate
tert-butyl (1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl
carbamate were obtained.
[3965] LC-MS (Method 2): R.sub.t=1.36 min; MS (ESIpos): m/z=366
(M+H).sup.+
[3966] 219 mg (0.6 mmol) of this intermediate were taken up in 3 ml
of dichloromethane, 0.6 ml of trifluoroacetic acid were added, and
the mixture was stirred at RT for 30 min. Subsequently, the
reaction mixture was concentrated in vacuo. The remaining residue
was the reaction mixture and was dried further in vacuo, then
lyophilized from dioxane. In this way, 196 mg (86% of theory) of
the title compound were obtained as a solid.
[3967] HPLC (Method 10): R.sub.t=2.41 min
Intermediate 53
(2S)-1-(benzylsulphonyl)-3-phenylpropan-2-amine
##STR00491##
[3969] 200 mg (1.13 mmol) of (4S)-4-benzyl-1,3-oxazolidin-2-one
were initially provided in 3 ml of tert-butanol, and 280 mg (2.26
mmol) of benzyl mercaptan were added to this. The mixture was
subsequently heated under reflux for 2 days. Thereafter, the
reaction mixture was concentrated on a rotary evaporator, and the
resulting intermediate
(2S)-1-(benzylsulphanyl)-3-phenylpropan-2-amine was directly
converted further, without workup.
[3970] HPLC (Method 10): R.sub.t=2.63 min
[3971] LC-MS (Method 1): R.sub.t=0.67 min; MS (ESIpos): m/z=258
(M+H).sup.+
[3972] The crude intermediate obtained above was dissolved in a
solution of 2 ml of 30% hydrogen peroxide and 5 ml of formic acid,
and the mixture was stirred at RT for 12 h. Then the reaction
mixture was added to saturated sodium sulphate solution and
extracted three times with ethyl acetate. The organic phase was
dried over magnesium sulphate and concentrated in vacuo. The
obtained crude product was purified by means of preparative HPLC.
343 mg (61% of theory) of the title compound were thus
obtained.
[3973] HPLC (Method 10): R.sub.t=2.40 min;
[3974] LC-MS (Method 12): R.sub.t=0.65 min; MS (ESIpos): m/z=290
(M+H).sup.+
Intermediate 54
(2S,3E)-1,4-diphenylbut-3-en-2-amine
##STR00492##
[3976] 552.7 mg (9.85 mmol) of potassium hydroxide were dissolved
in methanol, adsorbed onto 1.1 g of neutral aluminium oxide and
then dried under high vacuum. To a solution of 240 mg (0.82 mmol)
of (2S)-1-(benzylsulphonyl)-3-phenylpropan-2-amine and 1.56 g of
the potassium hydroxide on aluminium oxide thus prepared in 6.2 ml
of n-butanol were added dropwise, at 5-10.degree. C., 307 .mu.l
(3.3 mmol) of dibromodifluoromethane. The reaction mixture was
stirred at RT for 2 h, then filtered through Celite, and the
residue was washed thoroughly with dichloromethane afterwards. The
filtrate was concentrated, and the resulting residue was dried in
vacuo. The crude product thus obtained was purified by means of
preparative HPLC. 98 mg (35% of theory) of the title compound were
obtained with an E/Z diastereomer ratio of 4:1.
[3977] HPLC (Method 10): R.sub.t=2.46 min;
[3978] LC-MS (Method 12): R.sub.t=0.75 min; MS (ESIpos): m/z=224
(M+H).sup.+
[3979] The E/Z diastereomer mixture obtained above was dissolved in
2 ml of ethanol and 0.2 ml of N,N-diisopropylethylamine and
separated by means of HPLC on chiral phase [column: Daicel
Chiralpak AD-H, 5 .mu.m, 250 mm.times.20 mm, eluent:
hexane/(ethanol+0.2% diethylamine) 50:50 v/v; UV detection: 220 nm;
temperature: 30.degree. C.]. The appropriate fractions were
concentrated on a rotary evaporator, and the residue was dried in
vacuo. 45 mg of the title compound were obtained.
[3980] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. [ppm]=2.62-2.83
(m, 2H) 3.52-3.71 (m, 1H) 6.18-6.30 (m, 1H) 6.34-6.46 (m, 1H)
6.98-7.57 (m, 10H) [further signals hidden under solvent
peaks].
Intermediate 55
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-me-
thyl-3-oxo-3-{[(1S)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]amino}-
propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate
##STR00493##
[3982] 20 mg (29 .mu.mol) of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide were dissolved in 1 ml of DMF, 13.3 mg
(35 .mu.mol) of HATU and 15.3 .mu.l (88 .mu.mol) of
N,N-diisopropylethylamine were added, and the mixture was stirred
at RT for 30 min. Subsequently, 12.2 mg (32 .mu.mol) of
(1S)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethanamine
trifluoroacetate were added. The reaction mixture was stirred at RT
overnight and then separated by means of preparative HPLC. This
gave 22 mg (81% of theory) of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)--
2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(5-phenyl-1,3,4-ox-
adiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl-
]-N-methyl-L-valinamide.
[3983] LC-MS (Method 12): R.sub.t=1.45 min; MS (ESIpos): m/z=933
(M+H).sup.+
[3984] By subsequently cleaving the BOC protecting group with
trifluoroacetic acid, 22.4 mg (98% of theory) of the title compound
were then obtained.
[3985] LC-MS (Method 11): R.sub.t=0.85 min; MS (ESIpos): m/z=833
(M+H).sup.+
Intermediate 56
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-me-
thyl-3-oxo-3-{[(1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]amino}-
propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate
##STR00494##
[3987]
N-(tert-Butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1--
{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1R)-2-phenyl-1-(5-phenyl-1,-
3,4-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxohepta-
n-4-yl]-N-methyl-L-valinamide was prepared in analogy to the
synthesis of Intermediate 55, by reacting 20 mg (29 .mu.mol) of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide with 12.2 mg (32 .mu.mol) of
(1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethanamine
trifluoroacetate.
[3988] Yield: 17 mg (64% of theory)
[3989] HPLC (Method 10): R.sub.t=3.74 min;
[3990] LC-MS (Method 1): R.sub.t=1.45 min; MS (ESIpos): m/z=933
(M+H).sup.+
[3991] By subsequently cleaving the BOC protecting group with
trifluoroacetic acid, 17.1 mg (99% of theory) of the title compound
were thus obtained.
[3992] HPLC (Method 10): R.sub.t=2.55 min;
[3993] LC-MS (Method 11): R.sub.t=0.85 min; MS (ESIpos): m/z=833
(M+H).sup.+
Intermediate 57
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylsulpho-
nyl)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin--
1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate
##STR00495##
[3995]
N-(tert-Butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(-
1R,2R)-3-{[(2S)-1-(benzylsulphonyl)-3-phenylpropan-2-yl]amino}-1-methoxy-2-
-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]--
N-methyl-L-valinamide was prepared in analogy to the synthesis of
Intermediate 55, by reacting 20 mg (29 .mu.mol) of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide with 9.3 mg (20 .mu.mol) of
(2S)-1-(benzylsulphonyl)-3-phenylpropan-2-amine.
[3996] Yield: 19.2 mg (68% of theory)
[3997] HPLC (Method 10): R.sub.t=3.5 min;
[3998] LC-MS (Method 12): R.sub.t=1.41 min; MS (ESIpos): m/z=957
(M+H).sup.+
[3999] By subsequently cleaving the BOC protecting group with
trifluoroacetic acid, 19.3 mg (99% of theory) of the title compound
were thus obtained.
[4000] HPLC (Method 10): R.sub.t=2.52 min;
[4001] LC-MS (Method 1): R.sub.t=0.86 min; MS (ESIpos): m/z=857
(M+H).sup.+
Intermediate 58
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3E)-1,4-diphenyl-
but-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-met-
hoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate
##STR00496##
[4003]
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(-
1R,2R)-3-{[(2S,3E)-1,4-diphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3-o-
xopropyl]pyrrolidin-1-yl}-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-va-
linamide was prepared in analogy to the synthesis of Intermediate
55, by reacting 20 mg (29 .mu.mol)
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(R,2R)--
2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan--
4-yl]-N-methyl-L-valinamide with 7.1 mg (32 .mu.mol) of
(2S,3E)-1,4-diphenylbut-3-en-2-amine.
[4004] Yield: 15.1 mg (58% of theory)
[4005] HPLC (Method 10): R.sub.t=4.2 min;
[4006] LC-MS (Method 12): R.sub.t=1.51 min; MS (ESIpos): m/z=891
(M+H).sup.+
[4007] By subsequently cleaving the BOC protecting group with
trifluoroacetic acid, 15.7 mg (99% of theory) of the title compound
were then obtained.
[4008] HPLC (Method 10): R.sub.t=2.62 min;
[4009] LC-MS (Method 12): R.sub.t=0.97 min; MS (ESIpos): m/z=791
(M+H).sup.+
Intermediate 61
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1-
R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-pheny-
lcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl-
]-N-methyl-L-valinamide
##STR00497##
[4011] 50 mg (0.054 mmol) of
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-m-
ethyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-
-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valina-
mide trifluoroacetate (Intermediate 16) were dissolved in 8 ml of
dioxane/water, and 70 ml (0.108 mmol) of a 15% solution of
4-oxobutanoic acid in water were added. The reaction mixture was
subsequently stirred at 100.degree. C. for 1 h. After cooling to
RT, 3.7 mg (0.059 mmol) of sodium cyanoborohydride were added, and
the mixture was adjusted to a pH of 3 by adding about 300 .mu.l of
0.1 N hydrochloric acid. The reaction mixture was then stirred at
100.degree. C. for another 2 h. After cooling, another 70 ml (0.108
mmol) of the 15% 4-oxobutanoic acid solution were added, and the
reaction mixture was stirred again at 100.degree. C. for 1 h. Then
a further 3.7 mg (0.059 mmol) of sodium cyanoborohydride were
added, and about 300 .mu.l of 0.1 N hydrochloric acid were
subsequently used to readjust the pH to 3. The reaction mixture was
then stirred at 100.degree. C. for another 2 h. For a conversion
that was still incomplete, this procedure was repeated for a third
time. The reaction mixture was finally concentrated, and the
residue was purified by means of preparative HPLC. In this way, 32
mg (65% of theory) of the title compound were obtained in the form
of a colourless foam.
[4012] HPLC (Method 5): R.sub.t=1.64 min;
[4013] LC-MS (Method 9): R.sub.t=4.76 min; MS (ESIpos): m/z=899
(M+H).sup.+
[4014] .sup.1H NMR (500 MHz, DMSO-d.sub.6): .delta.=8.95 and 8.8
(2m, 1H), 8.88 and 8.65 (2s, 1H), 7.4-7.1 (m, 5H), 5.0, 4.78, 4.65
and 4.55 (4m, 2H), 4.1-3.7 (m, 5H), 3.32, 3.29, 3.20, 3.12, 3.1 and
3.0 (6s, 9H), 2.75 (m, 2H), 2.63 (t, 1H), 2.4-2.2 (m, 4H), 2.1-1.2
(m, 12H), 1.2-0.8 (m, 16H), 0.75 (m, 3H) [further signals hidden
under H.sub.2O and DMSO peaks].
Intermediate 62
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1-
R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylprop-
an-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-m-
ethyl-L-valinamide
##STR00498##
[4016] The title compound was prepared in analogy to the synthesis
of Intermediate 61, by reacting 50 mg of
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-m-
ethyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-ox-
opropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate (Intermediate 14) with 4-oxobutanoic acid.
[4017] Yield: 34 mg (70% of theory)
[4018] HPLC (Method 5): R.sub.t=1.64 min;
[4019] LC-MS (Method 9): R.sub.t=4.77 min; MS (ESIpos): m/z=887
(M+H).sup.+.
Intermediate 63
N-(4-carboxybenzyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1-
R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-pheny-
lcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl-
]-N-methyl-L-valinamide
##STR00499##
[4021] The title compound was prepared in analogy to the synthesis
of Intermediate 61 by reacting 15 mg of
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-m-
ethyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-
-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valina-
mide trifluoroacetate (Intermediate 16) with 4-formylbenzoic
acid.
[4022] Yield: 7.5 mg (48% of theory)
[4023] HPLC (Method 5): R.sub.t=1.75 min;
[4024] LC-MS (Method 1): R.sub.t=0.97 min; MS (ESIpos): m/z=947
(M+H).sup.+.
Intermediate 64
N-(5-carboxypentyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1-
R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-pheny-
lcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl-
]-N-methyl-L-valinamide
##STR00500##
[4026] 10 mg (0.011 mmol) of
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-m-
ethyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-
-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valina-
mide trifluoroacetate (Intermediate 16) were dissolved in 2 ml of
dioxane/water, and 2.8 mg (0.022 mmol) of 6-oxohexanoic acid were
added. The reaction mixture was subsequently stirred at 100.degree.
C. for 1 h.
[4027] After cooling to RT, 0.75 mg (0.012 mmol) of sodium
cyanoborohydride were added, and the mixture was adjusted to a pH
of 3 by adding 0.1 N hydrochloric acid. The reaction mixture was
then stirred at 100.degree. C. for another hour. After cooling,
another 2.8 mg (0.022 mmol) of 6-oxohexanoic acid were added, and
the reaction mixture was again stirred at 100.degree. C. for 1 h. A
further 0.75 mg (0.012 mmol) of sodium cyanoborohydride were added,
and 0.1 N hydrochloric acid was subsequently used to readjust the
pH to 3. The reaction mixture was then stirred at 100.degree. C.
for another 1 h. This procedure was then repeated for a third time.
The reaction mixture was finally concentrated, and the crude
product was purified by means of preparative HPLC. This gave 6.4 mg
(64% of theory) of the title compound in the form of a colourless
foam.
[4028] HPLC (Method 5): R.sub.t=1.68 min;
[4029] LC-MS (Method 9): R.sub.t=4.86 min; MS (ESIpos): m/z=927
(M+H).sup.+.
Intermediate 65
N-(2-aminoethyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2-
R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan--
2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-meth-
yl-L-valinamide bistrifluoroacetate
##STR00501##
[4031] The title compound was prepared by reacting 68 mg of
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-m-
ethyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-ox-
opropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate (Intermediate 14) with
tert-butyl-(2-oxoethyl)carbamate and subsequent cleaving of the Boc
protecting group with trifluoroacetic acid.
[4032] Yield: 49 mg (62% of theory over two stages)
[4033] HPLC (Method 5): R.sub.t=1.58 min;
[4034] LC-MS (Method 2): R.sub.t=1.05 min; MS (ESIpos): m/z=844
(M+H).sup.+
[4035] .sup.1H NMR (600 MHz, DMSO-do): .delta.=8.25 (m, 1H), 8.45
and 8.15 (2d, 1H), 7.65-7.55 (m, 3H), 7.23-7.1 (m, 5H), 5.12 and
4.95 (2m, 1H), 4.72 and 4.62 (2m, 1H), 4.6 and 4.52 (2t, 1H),
4.2-3.8 (m, 4H), 3.7 (d, 1H), 3.23, 3.20, 3.19, 3.18, 3.03 and 2.98
(6s, 9H), 3.0-2.7 (m, 6H), 2.4-1.2 (m, 15H), 1.05, 1.0, 0.88 and
0.82 (4d, 6H), 0.92 (m, 6H), 0.73 (m, 6H) [further signals hidden
under H.sub.2O peak].
Intermediate 66
N-(3-aminopropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,-
2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylc-
yclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]--
N-methyl-L-valinamide
##STR00502##
[4037] The title compound was prepared in analogy to the synthesis
of Intermediate 65 by reacting 25 mg (0.027 mmol) of
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-m-
ethyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-
-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valina-
mide trifluoroacetate (Intermediate 16) with
benzyl-(3-oxopropyl)carbamate and subsequent hydrogenolytic
cleaving of the Z protecting group (with 10% palladium on charcoal
as a catalyst, in ethanol as a solvent).
[4038] Yield: 11 mg (41% of theory over two stages)
[4039] HPLC (Method 5): R.sub.t=1.53 min;
[4040] LC-MS (Method 1): R.sub.t=0.72 min; MS (ESIpos): m/z=870
(M+H).sup.+.
Intermediate 67
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
(2S)-1-(adamantan-1-ylmethoxy)-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy--
2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-
-N-methyl-L-valinamide
##STR00503##
[4042] 26 mg (26 .mu.mol) of
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(adamantan-1-
-ylmethoxy)-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropy-
l]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinam-
ide trifluoroacetate and 33.9 .mu.l of a 15% aqueous
succinaldehydic acid solution (53 .mu.mol) were dissolved in 957
.mu.l of a 1:1-dioxane/water mixture and heated to 100.degree. C.
for 1 h. After brief cooling, 1.81 mg (29 .mu.mol) of sodium
cyanoborohydride were added. The reaction mixture was adjusted to
pH 3 by adding 0.1 N hydrochloric acid, and the mixture was heated
to 100.degree. C. for another 2 h. After again adding the same
amounts of succinaldehydic acid solution, sodium cyanoborohydride
and hydrochloric acid, the mixture was heated once again to
100.degree. C. for 2 h. The reaction mixture was then directly
separated into its components by means of preparative HPLC. 18.5 mg
(73% of theory) of the title compound were thus obtained.
[4043] LC-MS (Method 1): R.sub.t=1.17 min; m/z=967 (M+H).
Intermediate 68
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
(2S)-1-(benzyloxy)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxoprop-
yl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valina-
mide
##STR00504##
[4045] 24 mg (26 .mu.mol) of
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzyloxy)--
3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-
-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate and 33.7 .mu.l of a 15% aqueous succinaldehydic
acid solution (52 .mu.mol) were dissolved in 953 .mu.l of a
1:1-dioxane/water mixture and heated to 100.degree. C. for 1 h.
After brief cooling, 1.80 mg (29 .mu.mol) of sodium
cyanoborohydride were added. The reaction mixture was adjusted to
pH 3 by adding 0.1 N hydrochloric acid and the mixture was heated
to 100.degree. C. for another 2 h. After adding the same amounts of
succinaldehydic acid solution, sodium cyanoborohydride and
hydrochloric acid again, the mixture was heated once again to
100.degree. C. for 2 h. The reaction mixture was then directly
separated into its components by means of preparative HPLC. 15.2 mg
(65% of theory) of the title compound were thus obtained.
[4046] LC-MS (Method 1): R.sub.t=1.01 min; m/z=895 (M+H).sup.+
Intermediate 69
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
(2S)-1-(benzyloxy)-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-o-
xopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L--
valinamide
##STR00505##
[4048] 53 mg (84 .mu.mol) of
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-car-
boxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide
(Intermediate 4) and 45 mg (84 .mu.mol) of benzyl
N-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoyl}-L-phenyl-
alaninate trifluoroacetate (Intermediate 12) were taken up in 2 ml
of DMF, 19 .mu.l of N,N-diisopropylethylamine, 14 mg (92 .mu.mol)
of HOBt and 17.6 mg (92 .mu.mol) of EDC were added, and then the
mixture was stirred at RT overnight. Subsequently, the reaction
mixture was concentrated and the residue was purified by means of
preparative HPLC. This gave 59 mg (68% of theory) of the
Fmoc-protected intermediate
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2-
S)-2-[(1R,2R)-3-{[(2S)-1-(benzyloxy)-1-oxo-3-phenylpropan-2-yl]amino}-1-me-
thoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide.
[4049] LC-MS (Method 1): R.sub.t=1.55 min; m/z=1044
(M+H).sup.+.
[4050] 57 mg (0.055 mmol) of this intermediate were treated with
1.2 ml of piperidine in 5 ml of DMF to cleave the Fmoc protecting
group. After concentration and purification by means of preparative
HPLC, 39 mg (76% of theory) of the free amine intermediate
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzyloxy)--
1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-
-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
were obtained as the trifluoroacetate.
[4051] HPLC (Method 5): R.sub.t=1.9 min;
[4052] LC-MS (Method 1): R.sub.t=1.01 min; m/z=822 (M+H).sup.+.
[4053] 37 mg (0.045 mmol) of this intermediate were dissolved in 5
ml of dioxane/water and, in analogy to the preparation of the
compound in Intermediate 66, reacted with a 15% aqueous solution of
4-oxobutanoic acid in the presence of sodium cyanoborohydride. 16
mg (39% of theory) of the title compound were obtained in the form
of a colourless foam.
[4054] HPLC (Method 6): R.sub.t=2.1 min;
[4055] LC-MS (Method 1): R.sub.t=1.01 min; MS (ESIpos): m/z=908
(M+H).sup.+.
Intermediate 70
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
(2S,3S)-1-(benzyloxy)-1-oxo-3-phenylbutan-2-yl]amino}-1-methoxy-2-methyl-3-
-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl--
L-valinamide
##STR00506##
[4057] First, in analogy to the synthesis described in Intermediate
14, proceeding from Intermediates 4 and 26, the amine compound
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3S)-1-(benzylox-
y)-1-oxo-3-phenylbutan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolid-
in-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
was prepared.
[4058] 30 mg (0.032 mmol) of this compound were dissolved in 6 ml
of dioxane/water, and 41 .mu.l (0.063 mmol) of a 15% aqueous
solution of 4-oxobutanoic acid were added. The reaction mixture was
subsequently stirred at 100.degree. C. for 1 h. After cooling to
RT, 2.2 mg (0.035 mmol) of sodium cyanoborohydride were added, and
the mixture was adjusted to a pH of 3 by adding about 300 .mu.l of
0.1 N hydrochloric acid. The reaction mixture was then stirred at
100.degree. C. for another 2 h. After cooling, another 41 .mu.l
(0.063 mmol) of the 15% 4-oxobutanoic acid solution were added, and
the reaction mixture was again stirred at 100.degree. C. for 1 h.
Then a further 2.2 mg (0.035 mmol) of sodium cyanoborohydride were
added, and about 300 .mu.l of 0.1 N hydrochloric acid were
subsequently used to adjust the pH back to 3. The reaction mixture
was then stirred at 100.degree. C. for another 2 h. In the event of
the conversion still being incomplete, this procedure was repeated
for a third time. The reaction mixture was finally concentrated and
the crude product was purified by means of preparative HPLC. This
gave 24 mg (82% of theory) of the title compound in the form of a
colourless foam.
[4059] HPLC (Method 5): R.sub.t=1.9 min;
[4060] LC-MS (Method 9): R.sub.t=5.15 min; MS (ESIpos): m/z=922
(M+H).sup.+.
Intermediate 71
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1-
R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-meth-
yl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-vali-
namide
##STR00507##
[4062] First, in analogy to the synthesis described in Intermediate
14, proceeding from Intermediates 4 and 39, the amine compound
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{-
[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrr-
olidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide was
prepared. 7 mg (0.009 mmol) of this compound were then used, in
analogy to the preparation of Intermediate 61, by reaction with
4-oxobutanoic acid in the presence of sodium cyanoborohydride, to
obtain 2 mg (22% of theory) of the title compound in the form of a
colourless foam.
[4063] HPLC (Method 6): R.sub.t=1.9 min;
[4064] LC-MS (Method 2): R.sub.t=1.06 min; MS (ESIpos): m/z=832
(M+H).sup.+.
Intermediate 72
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
(2S)-1-(benzyloxy)-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-m-
ethyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N--
methyl-L-valinamide
##STR00508##
[4066] 212 mg (411 .mu.mol) of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(2R,3S,4S)-1-carboxy-2-methox-
y-4-methylhexan-3-yl]-N-methyl-L-valinamide (Intermediate 8) and
237 mg (411 .mu.mol) of
benzyl-N-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoyl}-L-
-tryptophanate trifluoroacetate (Intermediate 20) were taken up in
30 ml of DMF, and 188 mg (493 .mu.mol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and 215 .mu.l N,N-diisopropylethylamine were
added. The reaction mixture was stirred at RT for 20 h, then
concentrated in vacuo, and the residue was purified by means of
preparative HPLC. The product fractions were combined and
concentrated, and the residue was dried under high vacuum. This
gave 315 mg (80% of theory) of the Boc-protected intermediate
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-3-{[(2S)-1-(benzyloxy)-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methox-
y-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-y-
l]-N-methyl-L-valinamide as a colourless foam.
[4067] LC-MS (Method 1): R.sub.t=1.45 min; m/z=961 (M+H).sup.+.
[4068] 50 mg (52 .mu.mol) of this intermediate were treated with 1
ml of trifluoroacetic acid in 9 ml of dichloromethane to cleave the
Boc protecting group. After concentration and purification by means
of preparative HPLC, 29 mg (57% of theory) of the free amine
intermediate
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzyloxy)--
3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]p-
yrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
were obtained as trifluoroacetate.
[4069] LC-MS (Method 1): R.sub.t=0.99 min; m/z=861 (M+H).sup.+.
[4070] 29 mg (0.03 mmol) of this intermediate were dissolved in 6
ml of dioxane/water, and 39 .mu.l (0.059 mmol) of a 15% aqueous
solution of 4-oxobutanoic acid were added. The reaction mixture was
subsequently stirred at 100.degree. C. for 1 h. After cooling to
RT, 2 mg (0.033 mmol) of sodium cyanoborohydride were added, and
the mixture was adjusted to a pH of 3 by adding about 300 .mu.l of
0.1 N hydrochloric acid. The reaction mixture was then stirred at
100.degree. C. for a further 2 h. After cooling, another 39 .mu.l
(0.059 mmol) of the 15% 4-oxobutanoic acid solution were added, and
the reaction mixture was again stirred at 100.degree. C. for 1 h.
Then a further 2 mg (0.033 mmol) of sodium cyanoborohydride were
added, and about 300 .mu.l of 0.1 N hydrochloric acid were
subsequently used to adjust the pH back to 3. The mixture was then
stirred at 100.degree. C. for another 2 h. Thereafter, the reaction
mixture was poured onto a 1:1 mixture of semisaturated aqueous
ammonium chloride solution and ethyl acetate. The organic phase was
removed, washed with saturated sodium chloride solution, dried over
sodium sulphate and concentrated. The residue was freeze-dried from
water/acetonitrile. This gave 27 mg (94% of theory) of the title
compound in the form of a colourless foam.
[4071] HPLC (Method 5): R.sub.t=2.2 min;
[4072] LC-MS (Method 9): R.sub.t=5.04 min; MS (ESIpos): m/z=947
(M+H).sup.+.
Intermediate 73
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-({-
(2S)-1-[benzyl(methyl)amino]-1-oxo-3-phenylpropan-2-yl}amino)-1-methoxy-2--
methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-
-methyl-L-valinamide
##STR00509##
[4074] First, in analogy to the synthesis described in Intermediate
14, proceeding from Intermediates 4 and 38, the amine compound
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-({(2S)-1-[benzyl(meth-
yl)amino]-1-oxo-3-phenylpropan-2-yl}amino)-1-methoxy-2-methyl-3-oxopropyl]-
pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamid-
e was prepared. 25 mg (0.026 mmol) of this compound were then used,
in analogy to the preparation of Intermediate 61, by reaction with
4-oxobutanoic acid in the presence of sodium cyanoborohydride, to
obtain 13 mg (54% of theory) of the title compound in the form of a
colourless foam.
[4075] HPLC (Method 12): R.sub.t=2.2 min;
[4076] LC-MS (Method 9): R.sub.t=5.01 min; MS (ESIpos): m/z=921
(M+H).sup.+.
Intermediate 74
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-({-
(1S,2R)-1-[(benzyloxy)carbonyl]-2-phenylcyclopropyl}amino)-1-methoxy-2-met-
hyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-me-
thyl-L-valinamide
##STR00510##
[4078] 50 mg (73 .mu.mol) of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide (Intermediate 26) and 28 mg (73
.mu.mol) of benzyl (1S,2R)-1-amino-2-phenylcyclopropanecarboxylate
trifluoroacetate (Intermediate 45) were taken up in 5 ml of DMF,
and 42 mg (110 .mu.mol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and 38 .mu.l of N,N-diisopropylethylamine were
added. The reaction mixture was stirred at RT for 5 h, then
concentrated in vacuo, and the residue was purified by means of
preparative HPLC. The product fractions were combined and
concentrated. After lyophilization from dioxane/water, 35 mg (51%
of theory) of the Boc-protected intermediate
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-3-({(1S,2R)-1-[(benzyloxy)carbonyl]-2-phenylcyclopropyl}amino)-1-methoxy--
2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-
-N-methyl-L-valinamide were obtained as a colourless foam.
[4079] LC-MS (Method 1): R.sub.t=1.52 min; m/z=934 (M+H).sup.+.
[4080] 35 mg of this intermediate were treated with 1 ml of
trifluoroacetic acid in 5 ml of dichloromethane to cleave the Boc
protecting group. After concentration and lyophilization from
dioxane/water, 34 mg (97% of theory) of the free amine intermediate
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-({(1S,2R)-1-[(benzylo-
xy)carbonyl]-2-phenylcyclopropyl}amino)-1-methoxy-2-methyl-3-oxopropyl]pyr-
rolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
were obtained as the trifluoroacetate.
[4081] LC-MS (Method 1): R.sub.t=0.91 min; m/z=834 (M+H).sup.+.
[4082] 11 mg (0.011 mmol) of this intermediate were then used, in
analogy to the preparation of Intermediate 66, by reaction with
4-oxobutanoic acid in the presence of sodium cyanoborohydride, to
obtain 2.5 mg (24% of theory) of the title compound in the form of
a colourless foam.
[4083] HPLC (Method 12): R.sub.t=2.2 min;
[4084] LC-MS (Method 9): R.sub.t=5.1 min; MS (ESIpos): m/z=920
(M+H).sup.+.
Intermediate 75
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1-
R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S,2R)-2-phenyl-1-(propylcarbamoyl)cyc-
lopropyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-
-L-valinamide
##STR00511##
[4086] First, in analogy to the synthesis described in Intermediate
74, by coupling of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide (Intermediate 26) and
(1S,2R)-1-amino-2-phenyl-N-propylcyclopropanecarboxamide
trifluoroacetate (Intermediate 27) in the presence of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and subsequent cleaving of the Boc protecting
group by means of trifluoroacetic acid, the amine compound
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-m-
ethyl-3-oxo-3-{[(1S,2R)-2-phenyl-1-(propylcarbamoyl)cyclopropyl]amino}prop-
yl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
was prepared as trifluoroacetate. 14 mg (0.016 mmol) of this
compound were then used, in analogy to the preparation of
Intermediate 61, by reaction with 4-oxobutanoic acid in the
presence of sodium cyanoborohydride, to obtain 11.3 mg (83% of
theory) of the title compound.
[4087] HPLC (Method 6): R.sub.t=1.9 min;
[4088] LC-MS (Method 2): R.sub.t=1.27 min; MS (ESIpos): m/z=871
(M+H).sup.+.
Intermediate 76
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
(1S,2R)-1-(ethoxycarbonyl)-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-
-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl--
L-valinamide
##STR00512##
[4090] First, by coupling of Intermediate 46
(N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R-
)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxohepta-
n-4-yl]-N-methyl-L-valinamide) with Intermediate 48 (ethyl
(1S,2R)-1-amino-2-phenylcyclopropanecarboxylate trifluoroacetate)
in the presence of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and subsequent Boc cleaving, the starting
compound
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-(ethoxyca-
rbonyl)-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolid-
in-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate was prepared. 70 mg (0.079 mmol) of this starting
material were then used, by reaction with 4-oxobutanoic acid, in
analogy to Intermediate 61, to obtain 46 mg (68% of theory) of the
title compound.
[4091] HPLC (Method 6): R.sub.t=1.9 min;
[4092] LC-MS (Method 2): R.sub.t=1.28 min; MS (ESIpos): m/z=858
(M+H).sup.+
Intermediate 77
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
(2S)-1-amino-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxoprop-
yl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valina-
mide
##STR00513##
[4094] First, in analogy to the synthesis described in Intermediate
75, by coupling of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide (Intermediate 26) and
L-phenylalaninamide hydrochloride in the presence of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and subsequent cleaving of the Boc protecting
group by means of trifluoroacetic acid, the amine compound
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino-1-oxo--
3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-
-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide was
prepared as the trifluoroacetate. 47 mg (0.049 mmol) of this
compound were then used, in analogy to the preparation of
Intermediate 61, by reaction with 4-oxobutanoic acid in the
presence of sodium cyanoborohydride, to obtain 39 mg (96% of
theory) of the title compound.
[4095] HPLC (Method 6): R.sub.t=1.7 min;
[4096] LC-MS (Method 9): R.sub.t=4.44 min; MS (ESIpos): m/z=817
(M+H).sup.+
[4097] .sup.1H NMR (500 MHz, DMSO-d.sub.6): .delta.=8.95 and 8.8
(2m, 1H), 8.25 and 8.0 (2d, 1H), 7.45, 7.35 and 7.0 (3s, broad,
2H), 7.3-7.1 (m, 5H), 4.8-4.4 (2m, 3H), 3.95 (m, 1H), 3.82 (m, 1H),
3.72 (d, 1H), 3.22, 3.18, 3.15, 3.05 and 3.00 (5s, 9H), 2.85-2.7
(m, 4H), 2.45-1.6 (m, 12H), 1.5-1.2 (m, 3H), 1.1-0.7 (m, 21H)
[further signals hidden under solvent peaks].
Intermediate 78
N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2-
R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan--
2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-meth-
yl-L-valinamide
##STR00514##
[4099] This compound was prepared in analogy to Intermediate 66
over 2 stages, proceeding from 20 mg (16 .mu.mol) of the compound
from Intermediate 14 and benzyl-(6-oxohexyl)carbamate, and the
hydrogenation was performed in methanol as the solvent.
[4100] Yield: 7.6 mg (55% of theory over 2 stages)
[4101] HPLC (Method 6): R.sub.t=1.8 min;
[4102] LC-MS (Method 1): R.sub.t=0.7 min; MS (ESIpos): m/z=901
(M+H).sup.+.
Intermediate 79
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
(2S)-1-(benzylamino)-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-
-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl--
L-valinamide
##STR00515##
[4104] 36 mg (43 .mu.mol) of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]py-
rrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
(Intermediate 46) and 4.6 mg (43 .mu.mol) of benzylamine were taken
up in 5 ml of DMF, 7.5 .mu.l (88 .mu.mol) of
N,N-diisopropylethylamine, 10 mg (65 .mu.mol) of HOBt and 10 mg (52
.mu.mol) of EDC were added, and then the mixture was stirred at RT
overnight. Subsequently, the reaction mixture was concentrated and
the residue was purified by means of preparative HPLC. 29 mg (73%
of theory) of the Boc-protected intermediate
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-3-{[(2S)-1-(benzylamino)-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-met-
hyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-me-
thyl-L-valinamide were obtained.
[4105] LC-MS (Method 1): R.sub.t=1.43 min; m/z=921 (M+H).sup.+.
[4106] 29 mg of this intermediate were treated with 1 ml of
trifluoroacetic acid in 6 ml of dichloromethane to cleave the Boc
protecting group. After concentration and lyophilization from
dioxane/water, 30 mg (quant.) of the free amine intermediate
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylamino-
)-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolid-
in-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
were obtained as the trifluoroacetate.
[4107] LC-MS (Method 1): R.sub.t=0.95 min; m/z=821 (M+H).sup.+.
[4108] 17 mg (0.018 mmol) of this intermediate were then used, in
analogy to the preparation of Intermediate 61, by reaction with
4-oxobutanoic acid in the presence of sodium cyanoborohydride, to
obtain 13 mg (80% of theory) of the title compound in the form of a
colourless foam.
[4109] HPLC (Method 5): R.sub.t=1.7 min;
[4110] LC-MS (Method 9): R.sub.t=4.97 min; MS (ESIpos): m/z=907
(M+H).sup.+.
Intermediate 80
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
(2S)-1-(benzylamino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-
-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]--
N-methyl-L-valinamide
##STR00516##
[4112] First, in analogy to the synthesis described in Intermediate
74, by coupling of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide (Intermediate 26) and
N-benzyl-L-tryptophanamide trifluoroacetate (Intermediate 47) in
the presence of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and subsequent cleaving of the Boc protecting
group by means of trifluoroacetic acid, the amine compound
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylamino-
)-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl-
]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinami-
de was prepared as the trifluoroacetate. 10 mg (0.01 mmol) of this
compound were then used, in analogy to the preparation of
Intermediate 61, by reaction with 4-oxobutanoic acid in the
presence of sodium cyanoborohydride, to obtain 2.5 mg (26% of
theory) of the title compound.
[4113] HPLC (Method 5): R.sub.t=1.7 min;
[4114] LC-MS (Method 2): R.sub.t=1.13 min; MS (ESIpos): m/z=946
(M+H).sup.+.
Intermediate 81
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
(1S,2R)-1-carbamoyl-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopro-
pyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valin-
amide
##STR00517##
[4116] First, in analogy to the synthesis described in Intermediate
74, by coupling of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide (Intermediate 26) and
(1S,2R)-1-amino-2-phenylcyclopropanecarboxamide trifluoroacetate
(Intermediate 48) in the presence of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and subsequent cleaving of the Boc protecting
group by means of trifluoroacetic acid, the amine compound
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-carbamoyl-
-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl-
}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide was
prepared as the trifluoroacetate. 14 mg (0.0163 mmol) of this
compound were then used, in analogy to the preparation of
Intermediate 61, by reaction with 4-oxobutanoic acid in the
presence of sodium cyanoborohydride, to obtain 8 mg (57% of theory)
of the title compound.
[4117] HPLC (Method 5): R.sub.t=1.6 min;
[4118] LC-MS (Method 9): R.sub.t=4.64 min; MS (ESIpos): m/z=829
(M+H).sup.+.
Intermediate 82
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
(2S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl--
3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-
-L-valinamide
##STR00518##
[4120] First, in analogy to the synthesis described in Intermediate
69, by coupling of
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-car-
boxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide
(Intermediate 4) and
N.sup..alpha.-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]pro-
panoyl}-L-tryptophanamide trifluoroacetate (Intermediate 49) in the
presence of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and subsequent cleaving of the Fmoc protecting
group by means of piperidine, the amine compound
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino-3-(1H--
indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrroli-
din-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
was prepared as the trifluoroacetate. 78 mg (0.088 mmol) of this
compound were then used, in analogy to the preparation of
Intermediate 61, by reaction with 4-oxobutanoic acid in the
presence of sodium cyanoborohydride, to obtain 68 mg (90% of
theory) of the title compound.
[4121] HPLC (Method 5): R.sub.t=1.8 min;
[4122] LC-MS (Method 9): R.sub.t=4.49 min; MS (ESIpos): m/z=856
(M+H).sup.+.
Intermediate 83
N-(5-carboxypentyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
(2S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl--
3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-
-L-valinamide
##STR00519##
[4124] This compound was prepared in analogy to the compound in
Intermediate 82, proceeding from 20 mg (26 .mu.mol) of
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino-3-(1H--
indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrroli-
din-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate, by reaction with 4-oxobutanoic acid in the
presence of sodium cyanoborohydride.
[4125] Yield: 5 mg (25% of theory)
[4126] HPLC (Method 5): R.sub.t=1.6 min;
[4127] LC-MS (Method 11): R.sub.t=0.72 min; MS (ESIpos): m/z=884
(M+H).sup.+.
Intermediate 84
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1-
R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(morpholin-4-yl)-1-oxo-3-phenylpropan--
2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-meth-
yl-L-valinamide
##STR00520##
[4129] First, in analogy to the synthesis described in Intermediate
79, by coupling of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]py-
rrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
(Intermediate 46) and morpholine in the presence of EDC and HOBT
and subsequent cleaving of the Boc protecting group by means of
trifluoroacetic acid, the amine compound
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-m-
ethyl-3-{[(2S)-1-(morpholin-4-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopr-
opyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
was prepared as the trifluoroacetate. 30 mg (0.033 mmol) of this
compound were then used, in analogy to the preparation of
Intermediate 61, by reaction with 4-oxobutanoic acid in the
presence of sodium cyanoborohydride, to obtain 22 mg (76% of
theory) of the title compound.
[4130] HPLC (Method 5): R.sub.t=1.6 min;
[4131] LC-MS (Method 9): R.sub.t=4.58 min; MS (ESIpos): m/z=887
(M+H).sup.+.
Intermediate 85
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
(2S,3R)-1-(benzylamino)-3-hydroxy-1-oxobutan-2-yl]amino}-1-methoxy-2-methy-
l-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-meth-
yl-L-valinamide
##STR00521##
[4133] First, in analogy to the synthesis described in Intermediate
79, by coupling of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]py-
rrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
(Intermediate 46) and N-benzyl-L-threoninamide trifluoroacetate in
the presence of HATU and subsequent cleaving of the Boc protecting
group by means of trifluoroacetic acid, the amine compound
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3R)-1-(benzylam-
ino)-3-hydroxy-1-oxobutan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrro-
lidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
was prepared as the trifluoroacetate. 21 mg (0.024 mmol) of this
compound were then used, in analogy to the preparation of
Intermediate 61, by reaction with 4-oxobutanoic acid in the
presence of sodium cyanoborohydride, to obtain 20 mg (97% of
theory) of the title compound.
[4134] HPLC (Method 5): R.sub.t=1.54 min;
[4135] LC-MS (Method 9): R.sub.t=4.49 min; MS (ESIpos): m/z=861
(M+H).sup.+.
Intermediate 86
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
(2S)-1-tert-butoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-o-
xopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L--
valinamide
##STR00522##
[4137] First, in analogy to the synthesis described in Intermediate
74, by coupling of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide (Intermediate 26) and
tert-butyl-L-phenylalaninate hydrochloride in the presence of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and subsequent cleavingt of the Boc protecting
group by means of trifluoroacetic acid to obtain the tert-butyl
ester (stirring with trifluoroacetic acid in dichloromethane for 40
minutes), the amine compound
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-ter-
t-butoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]p-
yrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
was prepared as the trifluoroacetate. 22 mg (0.02 mmol) of this
compound were then used, in analogy to the preparation of
Intermediate 61, by reaction with 4-oxobutanoic acid in the
presence of sodium cyanoborohydride, to obtain 16 mg (94% of
theory) of the title compound.
[4138] HPLC (Method 5): R.sub.t=2.0 min;
[4139] LC-MS (Method 9): R.sub.t=5.05 min; MS (ESIpos): m/z=874
(M+H).sup.+.
Intermediate 87
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
(2S)-1-tert-butoxy-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-m-
ethyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N--
methyl-L-valinamide
##STR00523##
[4141] This compound was prepared in analogy to the synthesis
described in Intermediate 86, proceeding from 230 mg (336 .mu.mol)
of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide (Intermediate 26) and
tert-butyl-L-tryptophanate hydrochloride over 3 stages.
[4142] Yield: 95 mg (31% of theory over 3 stages)
[4143] HPLC (Method 5): R.sub.t=2.0 min;
[4144] LC-MS (Method 9): R.sub.t=5.05 min; MS (ESIpos): m/z=913
(M+H).sup.+.
Intermediate 88
N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S-
)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-o-
xopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L--
valinamide
##STR00524##
[4146] First, in analogy to the syntheses described in Intermediate
69, by coupling of
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-car-
boxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide
(Intermediate 4) and
N.sup..alpha.-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]pro-
panoyl}-L-tryptophanamide trifluoroacetate (Intermediate 49) in the
presence of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and subsequent cleaving of the Fmoc protecting
group by means of piperidine, the amine compound
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino-3-(1H--
indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrroli-
din-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
was prepared as the trifluoroacetate. 30 mg (0.03 mmol) of this
compound were then used, in analogy to the preparation of the
compound of Intermediate 61, by reaction with
benzyl-(6-oxohexyl)carbamate, which had been obtained beforehand by
oxidation of benzyl-(6-hydroxyhexyl)carbamate, in the presence of
sodium cyanoborohydride, to obtain 17 mg (45% of theory) of the
Z-protected compound. Subsequently, hydrogenolysis in methanol over
10% palladium/activated carbon yielded the title compound.
[4147] Yield: 14 mg (95% of theory)
[4148] HPLC (Method 5): R.sub.t=1.5 min;
[4149] LC-MS (Method 1): R.sub.t=0.73 min; MS (ESIpos): m/z=869
(M+H).sup.+.
Intermediate 89
N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S-
)-1-tert-butoxy-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-meth-
yl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-met-
hyl-L-valinamide
##STR00525##
[4151] First, in analogy to the synthesis described in Intermediate
86, by coupling of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide (Intermediate 26) and
tert-butyl-L-tryptophanate hydrochloride in the presence of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and subsequent cleaving of the Boc protecting
group by means of trifluoroacetic acid to obtain the tert-butyl
ester (stirring with 1:10 trifluoroacetic acid/dichloromethane for
30 min), the amine compound
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-ter-
t-butoxy-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-ox-
opropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-v-
alinamide was prepared as the trifluoroacetate. 71 mg (0.075 mmol)
of this compound were then used, in analogy to the preparation of
the compound of Intermediate 61, by reaction with
benzyl-(6-oxohexyl)carbamate, which had been obtained beforehand by
oxidation of benzyl-(6-hydroxyhexyl)carbamate, in the presence of
sodium cyanoborohydride, to obtain 35 mg (44% of theory) of the
Z-protected compound. Subsequently, hydrogenolysis in methanol over
10% palladium/activated carbon yielded the title compound.
[4152] Yield: 30 mg (98% of theory)
[4153] HPLC (Method 5): Rt=1.9 min;
[4154] LC-MS (Method 1): Rt=0.77 min; MS (ESIpos): m/z=926
(M+H)+.
Intermediate 90
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
2-(1H-indol-3-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1--
yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00526##
[4156] First, in analogy to the synthesis described in Intermediate
74, by coupling of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide (Intermediate 26) and
2-(1H-indol-3-yl)ethanamine in the presence of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and subsequent cleaving of the Boc protecting
group by means of trifluoroacetic acid, the amine compound
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(1H-indol-3-yl)et-
hyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-met-
hyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide was prepared as the
trifluoroacetate. 100 mg (0.119 mmol) of this compound were then
used, in analogy to the preparation of Intermediate 61, by reaction
with 4-oxobutanoic acid in the presence of sodium cyanoborohydride,
to obtain 50 mg (49% of theory) of the title compound. The title
compound was purified here by flash chromatography on silica gel
with dichloromethane/methanol/17% ammonia as the eluent, in the
course of which the mixing ratio was switched from initially
15/2/02 to 15/4/0.5.
[4157] HPLC (Method 6): R.sub.t=1.8 min;
[4158] LC-MS (Method 1): R.sub.t=0.87 min; MS (ESIpos): m/z=813
(M+H).sup.+.
Intermediate 91
N-(3-carboxypropyl)-N-methyl-L-valyl-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1-
R,2R)-1-methoxy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-
-yl]-5-methyl-1-oxoheptan-4-yl}-N-methyl-L-valinamide
##STR00527##
[4160] First, in analogy to the synthesis described in Intermediate
74, by coupling of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide (Intermediate 26) and phenylethylamine
in the presence of
O-(7-azabenzotriazol-1-yl)-N,N,N'N'-tetramethyluronium
hexafluorophosphate and subsequent cleaving of the Boc protecting
group by means of trifluoroacetic acid, the amine compound
N-methyl-L-valyl-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1-methoxy-2-m-
ethyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl]-5-methyl-1-oxo-
heptan-4-yl}-N-methyl-L-valinamide was prepared as the
trifluoroacetate. 57 mg (0.071 mmol) of this compound were then
used, in analogy to the preparation of Intermediate 61, by reaction
with 4-oxobutanoic acid in the presence of sodium cyanoborohydride,
to obtain 44 mg (80% of theory) of the title compound. The title
compound can also be purified here by flash chromatography on
silica gel with dichloromethane/methanol/17% ammonia as the eluent
(15/2/02->15/4/0.5).
[4161] HPLC (Method 5): R.sub.t=1.7 min;
[4162] LC-MS (Method 9): R.sub.t=4.64 min; MS (ESIpos): m/z=774
(M+H).sup.+.
Intermediate 92
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropy-
l]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinam-
ide
##STR00528##
[4164] 100 mg (0.139 mmol) of
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-
-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}--
3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
(Intermediate 40) were used, in analogy to the preparation of
Intermediate 61, by reaction with 4-oxobutanoic acid in the
presence of sodium cyanoborohydride, to obtain 94 mg (84% of
theory) of the title compound.
[4165] The title compound was purified by preparative HPLC.
[4166] HPLC (Method 5): R.sub.t=1.5 min;
[4167] LC-MS (Method 9): R.sub.t=4.46 min; MS (ESIpos): m/z=804
(M+H).sup.+.
Intermediate 93
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1-
R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(5-phenyl-1,3,4-oxadiaz-
ol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-m-
ethyl-L-valinamide
##STR00529##
[4169] 22.4 mg (24 .mu.mol) of
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-m-
ethyl-3-oxo-3-{[(1S)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]amino-
}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate were dissolved in 1.4 ml of dioxane/water and,
analogously to the preparation of Intermediate 61, reacted with 15%
aqueous solution of 4-oxobutanoic acid in the presence of sodium
cyanoborohydride. After lyophilization from dioxane, 8.2 mg (38% of
theory) of the title compound were obtained in the form of a white
solid.
[4170] HPLC (Method 10): R.sub.t=2.54 min
[4171] LC-MS (Method 12): R.sub.t=0.94 min; MS (ESIpos): m/z=919
(M+H).sup.+
Intermediate 94
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1-
R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiaz-
ol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-m-
ethyl-L-valinamide
##STR00530##
[4173] 17.1 mg (18 .mu.mol) of
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-m-
ethyl-3-oxo-3-{[(1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]amino-
}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate were dissolved in 1.1 ml of dioxane/water and,
analogously to the preparation of Intermediate 61, reacted with 15%
aqueous solution of 4-oxobutanoic acid in the presence of sodium
cyanoborohydride. After lyophilization from dioxane, 14.8 mg (89%
of theory) of the title compound were obtained in the form of a
white solid.
[4174] HPLC (Method 10): R.sub.t=2.54 min;
[4175] LC-MS (Method 12): R.sub.t=0.92 min; MS (ESIpos): m/z=919
(M+H).sup.+
Intermediate 95
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
(2S)-1-(benzylsulphonyl)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-o-
xopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L--
valinamide
##STR00531##
[4177] 19.3 mg (20 .mu.mol)
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylsulph-
onyl)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-
-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate were dissolved in 1.2 ml of dioxane/water and,
analogously to the preparation of Intermediate 61, reacted with 15%
aqueous solution of 4-oxobutanoic acid in the presence of sodium
cyanoborohydride. After lyophilization from dioxane, 8.6 mg (45% of
theory) of the title compound were obtained in the form of a
solid.
[4178] LC-MS (Method 11): R.sub.t=0.85 min; MS (ESIpos): m/z=943
(M+H).sup.+
Intermediate 96
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
(2S,3E)-1,4-diphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]py-
rrolidin-1-yl}-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00532##
[4180] 15.5 mg (10 .mu.mol) of
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3E)-1,4-dipheny-
lbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-meth-
oxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate were dissolved in 1.0 ml of dioxane/water and,
analogously to the preparation of Intermediate 61, reacted with 15%
aqueous solution of 4-oxobutanoic acid in the presence of sodium
cyanoborohydride. After lyophilization from dioxane, 10.3 mg (68%
of theory) of the title compound were obtained in the form of a
white solid.
[4181] HPLC (Method 10): R.sub.t=2.59 min;
[4182] LC-MS (Method 11): R.sub.t=0.94 min; MS (ESIpos): m/z=877
(M+H).sup.+
Intermediate 97
N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2-
R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcy-
clopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-
-methyl-L-valinamide
##STR00533##
[4184] The title compound was prepared in analogy to the synthesis
of Intermediate 66, by reaction of 200 mg (0.108 mmol) of
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-m-
ethyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-
-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valina-
mide trifluoroacetate (Intermediate 16) with
benzyl-(6-oxohexyl)carbamate and subsequent hydrogenolytic cleaving
of the Z protecting group (with 5% palladium on charcoal as a
catalyst, in methanol as a solvent).
[4185] Yield: 69 mg (65% of theory over two stages)
[4186] HPLC (Method 5): R.sub.t=1.7 min;
[4187] LC-MS (Method 1): R.sub.t=0.76 min; MS (ESIpos): m/z=912
(M+H).sup.+.
Intermediate 98
N-(5-carboxypentyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
(2S)-1-(benzylamino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-
-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]--
N-methyl-L-valinamide
##STR00534##
[4189] This compound was prepared in analogy to the synthesis
described in Intermediate 80. The purification was effected by
preparative HPLC.
[4190] Yield: 40 mg (29% of theory over 3 stages)
[4191] HPLC (Method 5): R.sub.t=1.9 min;
[4192] LC-MS (Method 1): R.sub.t=0.92 min; MS (ESIpos): m/z=974
(M+H).sup.+.
Intermediate 99
(2S)-2-amino-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)propan-1-one
trifluoroacetate
##STR00535##
[4194] 324 mg (0.81 mmol) of 2,5-dioxopyrrolidin-1-yl
N-(tert-butoxycarbonyl)-L-tryptophanate were dissolved in 20 ml of
DMF, and 200 mg (1.62 mmol) of 1,2-oxazinane hydrochloride
(Starting Compound 5) and 850 .mu.l of N,N-diisopropylethylamine
were added. The reaction mixture was stirred at 50.degree. C.
overnight and then concentrated under in vacuo. The residue was
taken up in dichloromethane and extracted with water. The organic
phase was dried over magnesium sulphate and concentrated. The
residue was purified by flash chromatography on silica gel with 4:1
dichloromethane/ethyl acetate as the eluent. The product fractions
were concentrated, and the residue was dried under high vacuum.
This gave 147.5 mg (48% of theory) of the Boc-protected
intermediate.
[4195] HPLC (Method 12): R.sub.t=1.9 min;
[4196] LC-MS (Method 1): R.sub.t=1.03 min; MS (ESIpos): m/z=374
(M+H).sup.+.
[4197] Using 166 mg (444.5 .mu.mol) of this intermediate, under
standard conditions with 3 ml of trifluoroacetic acid in 20 ml of
dichloromethane, the Boc protecting group was cleaved and, after
HPLC purification, 155 mg (86% of theory) of the title compound
were obtained.
[4198] HPLC (Method 12): R.sub.t=1.43 min;
[4199] LC-MS (Method 11): R.sub.t=0.56 min; MS (ESIpos): m/z=274
(M+H).sup.+.
Intermediate 100
N-(6-{[(benzyloxy)carbonyl]amino}hexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{-
(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxoprop-
an-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-
-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00536##
[4201] 177 mg (260 .mu.mol) of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide (Intermediate 26) and 100 mg (260
.mu.mol) of
(2S)-2-amino-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)propan-1-one
trifluoroacetate (Intermediate 99) were taken up in 15 ml of DMF,
and 118 mg (310 .mu.mol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and 140 .mu.l of N,N-diisopropylethylamine were
added. The reaction mixture was stirred at RT for 30 min, then
concentrated in vacuo, and the residue was purified by means of
preparative HPLC. The product fractions were combined and
concentrated. After lyophilization from dioxane, 170 mg (68% of
theory) of the Boc-protected intermediate were obtained.
[4202] LC-MS (Method 1): R.sub.t=1.36 min; m/z=940 (M+H).sup.+.
[4203] 170 mg of this intermediate were treated with 3 ml of
trifluoroacetic acid in 30 ml of dichloromethane for 30 min for
cleaving the Boc protecting group. Then the reaction mixture was
concentrated in vacuo, and the residue was purified by means of
preparative HPLC to obtain 155 mg (86% of theory) of the
deprotected
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3--
yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxo-
propyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-va-
linamide intermediate.
[4204] HPLC (Method 12): R.sub.t=1.85 min;
[4205] LC-MS (Method 1): R.sub.t=0.86 min; MS (ESIpos): m/z=840
(M+H).sup.+.
[4206] 50 mg (0.052 mmol) of this intermediate were then used, in
analogy to the preparation of Intermediate 97, with
benzyl-(6-oxohexyl)carbamate in the presence of sodium
cyanoborohydride and subsequent hydrogenolytic cleaving of the Z
protecting group (with 5% palladium on charcoal as a catalyst, in
methanol as a solvent), to prepare the title compound.
[4207] Yield: 21 mg (37% of theory)
[4208] HPLC (Method 12): R.sub.t=2.1 min;
[4209] LC-MS (Method 1): R.sub.t=1.02 min; MS (ESIpos): m/z=1073
(M+H).sup.+.
Intermediate 101
N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S-
)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methox-
y-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-y-
l]-N-methyl-L-valinamide
##STR00537##
[4211] 26.7 mg (24.87 .mu.mol) of Intermediate 100 were dissolved
in 10 ml of methanol and hydrogenated over palladium/activated
carbon (5%) under standard hydrogen pressure for 30 min. The
catalyst was filtered off and the solvent was evaporated off in
vacuo. After the residue had been dried under high vacuum, 22.5 mg
(96% of theory) of the title compound were obtained.
[4212] HPLC (Method 5): R.sub.t=1.7 min;
[4213] LC-MS (Method 1): R.sub.t=0.76 min; MS (ESIpos): m/z=939
(M+H).sup.+.
Intermediate 102
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-metho-
xy-2-methyl-3-{[(2S)-1-(morpholin-4-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-
-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinami-
de
##STR00538##
[4215] This compound was prepared in analogy to the synthesis
described in Intermediate 157 from
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(morpholin-4-yl)-1-oxo-3-phenylpropan-
-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-met-
hyl-L-valinamide and commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide.
[4216] Yield: 8 mg (71% of theory)
[4217] HPLC (Method 12): R.sub.t=1.9 min;
[4218] LC-MS (Method 1): R.sub.t=0.87 min; MS (ESIpos): m/z=1094
(M+H).sup.+.
Intermediate 103
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3R)-1-(be-
nzylamino)-3-hydroxy-1-oxobutan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl-
]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinami-
de
##STR00539##
[4220] This compound was prepared in analogy to the synthesis
described in Intermediate 157 from
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{-
[(2S,3R)-1-(benzylamino)-3-hydroxy-1-oxobutan-2-yl]amino}-1-methoxy-2-meth-
yl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-met-
hyl-L-valinamide and commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide.
[4221] Yield: 3 mg (22% of theory)
[4222] HPLC (Method 5): R.sub.t=1.6 min;
[4223] LC-MS (Method 1): R.sub.t=0.78 min; MS (ESIpos): m/z=1069
(M+H).sup.+.
Intermediate 104
N-{4-[(trans-4-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}cyclohexyl)amino]--
4-oxobutyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-a-
mino-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopro-
pyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valin-
amide
##STR00540##
[4225] First, benzyl trans-4-aminocyclohexanecarboxylate
trifluoroacetate was prepared from
trans-4-aminocyclohexanecarboxylic acid by introducing the Boc
protecting group, then introducing the benzyl ester protecting
group and subsequently cleaving the Boc protecting group by
conventional peptide chemistry methods.
[4226] 15 mg (18 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{-
[(2S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-
-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methy-
l-L-valinamide were then dissolved in 5 ml of dimethylformamide and
subsequently admixed with 13 mg (35 .mu.mol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate, 9 .mu.l of N,N-diisopropylethylamine and with
15 mg (44 .mu.mol) of benzyl trans-4-aminocyclohexanecarboxylate
trifluoroacetate. The mixture was stirred at RT for 1 h and then
concentrated in vacuo. The remaining residue was purified by means
of preparative HPLC. The corresponding fractions were combined and
the solvent was evaporated off in vacuo. After the residue had been
dried under high vacuum, 14.7 mg (78% of theory) of the protected
intermediate were obtained as a colourless foam.
[4227] HPLC (Method 6): R.sub.t=2.0 min;
[4228] LC-MS (Method 1): R.sub.t=0.95 min; MS (ESIpos): m/z=1072
(M+H).sup.+.
[4229] From this protected intermediate, the benzyl ester was first
removed by hydrogenolytic means, and the free carboxyl component
was obtained in quantitative yield. 14 mg (14 .mu.mol; 1 equiv.) of
the deprotected compound were taken up in 5 ml of DMF and admixed
with 3.3 mg (29 .mu.mol; 2.1 equiv.) of N-hydroxysuccinimide in the
presence of 4.1 mg (21 .mu.mol; 1.5 equiv.) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 7.5
.mu.l (44 .mu.mol; 3.1 equiv.) of N,N-diisopropylethylamine and 0.9
mg (7 .mu.mol; 0.5 equiv.) of 4-dimethylaminopyridine, and the
mixture was stirred at RT overnight. Then another 10 equiv. of
N-hydroxysuccinimide, 5 equiv. of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 5
equiv. of N,N-diisopropylethylamine and 0.5 equiv. of
4-dimethylaminopyridine were added, and the reaction mixture was
treated in an ultrasound bath for 5 h. Subsequently, the solvent
was evaporated off, the residue was purified by means of
preparative HPLC and the corresponding fractions were combined and
concentrated. After lyophilization of the residue from dioxane, 9.7
mg (62% of theory) of the title compound were obtained as a
colourless foam.
[4230] HPLC (Method 6): R.sub.t=1.8 min;
[4231] LC-MS (Method 11): R.sub.t=0.77 min; MS (ESIpos): m/z=1078
(M+H).sup.+.
Intermediate 105
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carbox-
y-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-m-
ethoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00541##
[4233] This compound was prepared in analogy to the synthesis
described in Intermediate 157, proceeding from
4-{[(2S)-1-{[(2S)-1-{[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-tert-butox-
y-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolid-
in-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-methylbutan--
2-yl]amino}-3-methyl-1-oxobutan-2-yl](methyl)amino}butanoic acid
and commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The ester
intermediate was obtained in 42% yield. In a second step, 6 mg (6
.mu.mol) of this intermediate were cleaved with trifluoroacetic
acid the tert-butyl ester. After HPLC purification, 3.4 mg (48% of
theory) of the title compound were obtained.
[4234] HPLC (Method 5): R.sub.t=1.66 min;
[4235] LC-MS (Method 2): R.sub.t=1.04 min; MS (ESIpos): m/z=1025
(M+H).sup.+.
Intermediate 106
N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,-
4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-
-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-met-
hyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00542##
[4237] 14 mg (16 mol) of
N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2-
S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3--
oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-
-valinamide (Intermediate 88) were taken up in 750 .mu.l of dioxane
and admixed with 1.5 ml of saturated sodium hydrogencarbonate
solution and then with 3.2 mg (21 .mu.mol) of methyl
2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate. The reaction
mixture was stirred at RT for 1 h and then concentrated in vacuo.
The remaining residue was purified by means of preparative HPLC.
After lyophilization, 5.5 mg (36% of theory) of the title compound
were obtained.
[4238] HPLC (Method 5): RC=1.7 in;
[4239] LC-MS (Method 1): R=0.84 min; MS (ESIpos): m/z=949
(M+H).sup.+.
Intermediate 107
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[2-(1H-indol-3-
-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-
-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00543##
[4241] 38 mg (47 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{-
[2-(1H-indol-3-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-
-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
were dissolved in 37 ml of DMF and then admixed with 71 mg (187
.mu.mol) of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate, 33 .mu.l of N,N-diisopropylethylamine and with
37 mg (140 .mu.mol) of commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The
mixture was stirred at RT for 1 h. This was followed by
concentration under high vacuum and purification of the remaining
residue by means of preparative HPLC. Thus, 12.2 mg (26% of theory)
of the title compound were obtained as a colourless foam.
[4242] HPLC (Method 5): R.sub.t=1.6 min;
[4243] LC-MS (Method 1): R.sub.t=0.85 min; MS (ESIpos): m/z=1020
(M+H).sup.+.
Intermediate 108
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-{(3R,4S,5S)-3-methoxy-1-[(2S)-2-{(1R,2R)-1-metho-
xy-2-methyl-3-oxo-3-[(2-phenylethyl)amino]propyl}pyrrolidin-1-yl]-5-methyl-
-1-oxoheptan-4-yl}-N-methyl-L-valinamide
##STR00544##
[4245] The compound was prepared in analogy to Intermediate
107.
[4246] Yield: 2.5 mg (30% of theory)
[4247] HPLC (Method 12): R.sub.t=1.9 min;
[4248] LC-MS (Method 1): R.sub.t=0.9 min; MS (ESIpos): m/z=981
(M+H).sup.+.
Intermediate 109
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hyd-
roxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin--
1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00545##
[4250] The compound was prepared in analogy to Intermediate 107
from the compound in Intermediate 92.
[4251] Yield: 35 mg (65% of theory)
[4252] HPLC (Method 5): R.sub.t=1.9 min;
[4253] LC-MS (Method 11): R.sub.t=0.76 min; MS (ESIpos): m/z=1011
(M+H).sup.+.
Intermediate 110
N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-N-[(3R,4-
S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2--
yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-meth-
yl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00546##
[4255] This compound was prepared in analogy to Intermediate 147
from the compound in Intermediate 83.
[4256] Yield: 2.4 mg (24% of theory)
[4257] HPLC (Method 6): R.sub.t=1.8 min;
[4258] LC-MS (Method 1): R.sub.t=0.87 min; MS (ESIpos): m/z=981
(M+H).sup.+.
Intermediate 111
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-1-methylhydrazi-
no}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-
-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-ox-
opropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-v-
alinamide
##STR00547##
[4260] This compound was prepared in analogy to Intermediate 140
from Intermediate 82 and Intermediate 22.
[4261] Yield: 6.5 mg (51% of theory)
[4262] HPLC (Method 6): R.sub.t=1.8 min;
[4263] LC-MS (Method 1): R.sub.t=4.71 min; MS (ESIpos): m/z=1077
(M+H).sup.+.
Intermediate 112
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-car-
bamoyl-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidi-
n-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00548##
[4265] This compound was prepared in analogy to Intermediate 157
from the compound in Intermediate 81.
[4266] Yield: 5.7 mg (57% of theory)
[4267] HPLC (Method 5): R.sub.t=1.6 min;
[4268] LC-MS (Method 1): R.sub.t=0.87 min; MS (ESIpos): m/z=1036
(M+H).sup.+.
Intermediate 113
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carbox-
y-2-(1H-indol-3-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin--
1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00549##
[4270] 95 mg (104 .mu.mol) of
4-{[(2S)-1-{[(2S)-1-{[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-tert-butox-
y-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl-
]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl](methyl)amino}-3-met-
hylbutan-2-yl]amino}-3-methyl-1-oxobutan-2-yl](methyl)amino}butanoic
acid were dissolved in DMF and then admixed with 79.5 mg (209
.mu.mol) of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate, 73 .mu.l of N,N-diisopropylethylamine and with
68 mg (261 .mu.mol) of commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The
mixture was stirred at RT for 2 h. This was followed by
concentration under high vacuum and purification of the remaining
residue by means of preparative HPLC. Thus, 104 mg (89% of theory)
of the tert-butyl ester of the title compound were obtained as a
colourless foam.
[4271] HPLC (Method 5): R.sub.t=2.0 min;
[4272] LC-MS (Method 1): R.sub.t=0.93 min; MS (ESIpos): m/z=1121
(M+H).sup.+.
[4273] The intermediate was taken up in 33.4 ml of dichloromethane,
17 ml of trifluoroacetic acid were added, and the mixture was
stirred at RT for 1 h. Subsequently, the reaction mixture was
concentrated under reduced pressure and the residue was purified by
preparative HPLC.
[4274] Thus, 61 mg (62% of theory) of the title compound were
obtained as a colourless foam.
[4275] HPLC (Method 5): R.sub.t=1.7 min;
[4276] LC-MS (Method 1): R.sub.t=0.86 min; MS (ESIpos): m/z=1064
(M+H).sup.+.
Intermediate 114
N-[6-({[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]carbamoyl}amino)hexy-
l]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino-3-(1-
H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrro-
lidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00550##
[4278] 5 mg (5 .mu.mol) of
N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2-
S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3--
oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-
-valinamide were taken up in 885 .mu.l of DMF and admixed with 5.3
mg (8 .mu.mol) of
4-nitrophenyl-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]carbamate
and 2.8 .mu.l of N,N-diisopropylethylamine. The reaction mixture
was stirred at RT for 2 h and then concentrated to dryness. The
residue was purified by means of preparative HPLC.
[4279] Yield: 0.58 mg (11% of theory) of a colourless foam
[4280] HPLC (Method 5): R.sub.t=1.6 min;
[4281] LC-MS (Method 1): R.sub.t=0.83 min; MS (ESIpos): m/z=1035
(M+H).sup.+.
Intermediate 115
N-{4-[(2,5-dioxopyrrolidin-1-yl)oxy]-4-oxobutyl}-N-methyl-L-valyl-N-[(3R,4-
S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-o-
xazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1--
yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00551##
[4283] This compound was prepared in analogy to the compound in
Intermediate 147, starting from 8 mg (9 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phen-
ylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-y-
l]-N-methyl-L-valinamide. After concentration, the activated ester
was purified by means of preparative HPLC and, after removal of the
solvent in vacuo, reacted immediately with the antibody.
[4284] Yield: 3 mg (27% of theory) (hydrolysis-sensitive)
[4285] HPLC (Method 5): R.sub.t=1.7 min;
[4286] LC-MS (Method 1): R.sub.t=0.87 min; MS (ESIpos): m/z=996
(M+H).sup.+.
Intermediate 116
N-{4-[(2,5-dioxopyrrolidin-1-yl)oxy]-4-oxobutyl}-N-methyl-L-valyl-N-[(3R,4-
S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxaz-
inan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-
-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00552##
[4288] This compound was prepared in analogy to the compound in
Intermediate 147, starting from 5 mg (6 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpro-
pan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N--
methyl-L-valinamide. After concentration, the activated ester was
purified by means of preparative HPLC and, after removal of the
solvent in vacuo, reacted immediately with the antibody.
[4289] Yield: 3.2 mg (43% of theory) (hydrolysis-sensitive)
[4290] HPLC (Method 5): R.sub.t=1.7 min;
[4291] LC-MS (Method 1): R.sub.t=0.92 min; MS (ESIpos): m/z=984
(M+H).sup.+.
Intermediate 117
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-tert-b-
utoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrr-
olidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00553##
[4293] This compound was prepared in analogy to Intermediate 157
from the compound in Intermediate 86.
[4294] Yield: 7 mg (42% of theory)
[4295] HPLC (Method 5): R.sub.t=1.6 min;
[4296] LC-MS (Method 1): R.sub.t=0.94 min; MS (ESIpos): m/z=1081
(M+H).sup.+.
Intermediate 118
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2R)-1-(benzy-
loxy)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-
-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00554##
[4298] The target compound was prepared in analogy to Intermediate
157 from 7 mg (7.8 .mu.mol) of the compound in Intermediate 68.
Yield: 6.3 mg (53% of theory)
[4299] LC-MS (Method 1): R.sub.t=1.00 min; MS (ESIpos): m/z=1102
(M+H).sup.+.
Intermediate 119
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-metho-
xy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl-
]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valin-
amide
##STR00555##
[4301] 7.4 mg (8.1 mmol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(5-phenyl-1,3,4-oxadia-
zol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N--
methyl-L-valinamide and 6.3 mg (24.2 mmol) of
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide
hydrochloride were coupled and worked up in analogy to Intermediate
157. 1.6 mg (13% of theory) of the title compound were obtained as
a solid.
[4302] LC-MS (Method 11): R.sub.t=0.89 min; MS (ESIpos): m/z=1126
(M+H)
Intermediate 120
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-metho-
xy-2-methyl-3-oxo-3-{[(1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl-
]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valin-
amide
##STR00556##
[4304] 12.8 mg (13.9 mmol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadia-
zol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N--
methyl-L-valinamide and 10.9 mg (41.8 mmol) of
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide
hydrochloride were coupled and worked up in analogy to Intermediate
157. 10.8 mg (59% of theory) of the title compound were obtained as
a solid.
[4305] LC-MS (Method 11): R.sub.t=0.90 min; MS (ESIpos): m/z=1126
(M+H)
Intermediate 121
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzy-
lsulphonyl)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrr-
olidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00557##
[4307] 7.4 mg (7.9 mmol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{-
[(2S)-1-(benzylsulphonyl)-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3--
oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-
-valinamide and 6.2 mg (23.5 mmol) of
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide
hydrochloride were coupled and worked up in analogy to Intermediate
157. 6.9 mg (74% of theory) of the title compound were obtained as
a solid.
[4308] LC-MS (Method 11): R.sub.t=0.87 min; MS (ESIpos): m/z=1150
(M+H).sup.+
Intermediate 122
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3E)-1,4-d-
iphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl-
}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00558##
[4310] 8 mg (9.1 mmol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{-
[(2S,3E)-1,4-diphenylbut-3-en-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]p-
yrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
and 7.2 mg (27.4 mmol) of
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide
hydrochloride were coupled and worked up in analogy to Intermediate
157. 8.2 mg (82% of theory) of the title compound were obtained as
a white solid.
[4311] LC-MS (Method 11): R.sub.t=0.95 min; MS (ESIpos): m/z=1083
(M+H)
Intermediate 123
N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,-
4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-tert-butoxy-3-(1H-indol-3-yl)-1-oxopr-
opan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-
-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00559##
[4313] 30 mg (30 .mu.mol) of Intermediate 89 were taken up in 2 ml
of 1,4-dioxane and admixed with 4 ml of saturated sodium
hydrogencarbonate solution and then with 7.5 mg (50 .mu.mol) of
methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate. The reaction
mixture was stirred at RT for 1 h and then concentrated in vacuo.
The remaining residue was purified by means of preparative HPLC.
After lyophilization, 24 mg (74% of theory) of the title compound
were obtained.
[4314] HPLC (Method 5): R.sub.t=2.2 min;
[4315] LC-MS (Method 1): R.sub.t=1.01 min; MS (ESIpos): m/z=1006
(M+H).sup.+.
Intermediate 124
N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,-
4S,5S)-1-{(2S)-2-[(1R,2R)-3-[(1S)-1-carboxy-2-(1H-indol-3-yl)ethyl]amino-1-
-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxohep-
tan-4-yl]-N-methyl-L-valinamide
##STR00560##
[4317] 22 mg (20 .mu.mol) of Intermediate 123 were reacted with 4
ml of trifluoroacetic acid in 8 ml of dichloromethane at RT for 1
h. Thereafter, the reaction mixture was concentrated in vacuo. The
remaining residue was purified by means of preparative HPLC. After
lyophilization, 11 mg (54% of theory) of the title compound were
obtained.
[4318] HPLC (Method 5): R.sub.t=1.8 min;
[4319] LC-MS (Method 11): R.sub.t=0.85 min; MS (ESIpos): m/z=950
(M+H).sup.+.
Intermediate 125
N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,-
4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-
-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-
-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00561##
[4321] 22.5 mg (20 .mu.mol) of Intermediate 101 were taken up in 2
ml of 1:1 dioxane/water and then admixed with 5.6 mg (40 .mu.mol)
of methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate and with
0.25 ml of saturated sodium hydrogencarbonate solution. The
reaction mixture was stirred at RT for 30 min. Then another 0.25 ml
of the saturated sodium hydrogencarbonate solution were added, and
the reaction mixture was stirred at RT for another 15 min and then
concentrated in vacuo. The remaining residue was purified by means
of preparative HPLC. After lyophilization, 12.8 mg (50% of theory)
of the title compound were obtained as a colourless foam.
[4322] HPLC (Method 5): R.sub.t=1.9 min;
[4323] LC-MS (Method 1): R.sub.t=0.95 min; MS (ESIpos): m/z=1019
(M+H).sup.+.
Intermediate 126
N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,-
4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2--
oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-
-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00562##
[4325] 64 mg (70 .mu.mol) of
N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,-
2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylc-
yclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]--
N-methyl-L-valinamide (Intermediate 97) were taken up in 3 ml of
1:1 dioxane/water, then adjusted to pH 9 with 4 ml of saturated
sodium hydrogencarbonate solution and subsequently admixed with
16.3 mg (110 .mu.mol) of methyl
2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate. The reaction
mixture was stirred at RT for 1 h and then concentrated in vacuo.
Then another 8 mg (55 .mu.mol) of methyl
2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate were added, and the
reaction mixture was adjusted again to pH 9 and stirred at RT for
another hour. This was followed by concentration and purification
of the remaining residue by means of preparative HPLC. At first, 31
mg of an as yet uncyclized intermediate were obtained. 27 mg of
this intermediate were taken up again in 2 ml of 1:1 dioxane/water
and then admixed with 250 .mu.l of saturated sodium
hydrogencarbonate solution. After stirring at RT for 2 hours, the
reaction mixture was concentrated, and the residue was purified by
means of preparative HPLC. After lyophilization, 20 mg (29% of
theory) of the title compound were obtained.
[4326] HPLC (Method 5): R.sub.t=1.96 min;
[4327] LC-MS (Method 1): R.sub.t=0.97 min; MS (ESIpos): m/z=992
(M+H).sup.+.
Intermediate 127
N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-N-[(3R,4-
S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzylamino)-3-(1H-indol-3-yl)-1-oxop-
ropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methox-
y-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00563##
[4329] 17 mg (18 .mu.mol) of
N-(5-carboxypentyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{-
[(2S)-1-(benzylamino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy--
2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-
-N-methyl-L-valinamide (Intermediate 98) were dissolved in 2.8 ml
of dichloromethane and admixed with 20 mg (174 mmol) of
1-hydroxypyrrolidine-2,5-dione and then admixed with 10 mg (52
.mu.mol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride and 0.21 mg (0.17 .mu.mol) of DMAP. After stirring at
RT for 4 h, the reaction mixture was concentrated in vacuo. The
remaining residue was purified by means of preparative HPLC. After
lyophilization, 8.2 mg (43% of theory) of the title compound were
obtained.
[4330] HPLC (Method 5): R.sub.t=2.0 min;
[4331] LC-MS (Method 1): R.sub.t=0.98 min; MS (ESIpos): m/z=1071
(M+H).sup.+.
Intermediate 128
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-metho-
xy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-yl]amino-
}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valin-
amide
##STR00564##
[4333] 5 mg (5.6 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpro-
pan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N--
methyl-L-valinamide were dissolved in 845 .mu.l of DMF and then
admixed with 3.2 mg (17 .mu.mol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 2.6 mg
(17 .mu.mol) of 1-hydroxy-1H-benzotriazole hydrate, 1.96 .mu.l of
N,N-diisopropylethylamine and with 5.9 mg (22.5 .mu.mol) of
commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The
mixture was stirred at RT overnight and then concentrated under
high vacuum. The remaining residue was purified by means of
preparative HPLC. Thus, 2.2 mg (36% of theory) of the title
compound were obtained as a colourless foam.
[4334] HPLC (Method 5): R.sub.t=1.7 min;
[4335] LC-MS (Method 1): R.sub.t=0.88 min; MS (ESIpos): m/z=1094
(M+H).sup.+.
Intermediate 129
N-(6-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-6-oxo-
hexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-metho-
xy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]-
amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L--
valinamide
##STR00565##
[4337] 4 mg (4.3 .mu.mol) of
N-(5-carboxypentyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phen-
ylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-y-
l]-N-methyl-L-valinamide were dissolved in 646 .mu.l of DMF and
then admixed with 2.5 mg (13 .mu.mol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 2.0 mg
(13 .mu.mol) of 1-hydroxy-1H-benzotriazole hydrate, 2.25 .mu.l of
N,N-diisopropylethylamine and with 4.5 mg (17 .mu.mol) of
commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The
mixture was stirred at RT for 3 h and then concentrated under high
vacuum. The remaining residue was purified by means of preparative
HPLC. Thus, 1.9 mg (39% of theory) of the title compound were
obtained as a colourless foam.
[4338] HPLC (Method 5): R.sub.t=1.7 min;
[4339] LC-MS (Method 9): R.sub.t=4.9 min; MS (ESIpos): m/z=1134
(M+H).
Intermediate 130
N-(4-{[(2R)-1-({5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}amino)pro-
pan-2-yl]oxy}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-
-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-
-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxohepta-
n-4-yl]-N-methyl-L-valinamide
##STR00566##
[4341] 10.5 mg (11.7 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phen-
ylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-y-
l]-N-methyl-L-valinamide were dissolved in 3.7 ml of
dichloromethane and then admixed with 6.7 mg (35 .mu.mol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 0.7 mg
(5.8 .mu.mol) of 4-dimethylaminopyridine and with 8.2 mg (47
.mu.mol) of commercially available
tert-butyl-[(2R)-2-hydroxypropyl]carbamate. The mixture was stirred
at RT overnight and then concentrated under high vacuum. The
remaining residue was purified by means of preparative HPLC. Thus,
7.5 mg (61% of theory) of the Boc-protected intermediate were
obtained as a colourless foam.
[4342] HPLC (Method 5): R.sub.t=2.0 min;
[4343] LC-MS (Method 1): R.sub.t=1.03 min; MS (ESIpos): m/z=1056
(M+H).sup.+.
[4344] Subsequently, the Boc protecting group was cleaved with
trifluoroacetic acid. 4.9 mg (0.005 mmol) of the deprotected crude
product were then, without further purification, taken up in 1.8 ml
of dichloromethane and admixed with 3.7 mg (0.011 mmol) of
1,1'-[(1,5-dioxopentane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dione,
2.4 .mu.l (0.014 mmol) of N,N-diisopropylethylamine and 0.6 mg (5
.mu.mol) of 4-dimethylaminopyridine. The mixture was stirred at RT
for 2 h and then concentrated under high vacuum. The remaining
residue was purified by means of preparative HPLC. Thus, 0.77 mg
(15% of theory) of the title compound were obtained as a colourless
foam.
[4345] HPLC (Method 5): R.sub.t=1.8 min;
[4346] LC-MS (Method 1): R.sub.t=0.93 min; MS (ESIpos): m/z=1167
(M+H).sup.+.
Intermediate 131
N-{4-[(1-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}piperidin-4-yl)-
oxy]-4-oxobutyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2-
R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcy-
clopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-
-methyl-L-valinamide
##STR00567##
[4348] 10 mg (11 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phen-
ylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-y-
l]-N-methyl-L-valinamide were dissolved in 2 ml of dichloromethane
and then admixed with 4.3 mg (22 .mu.mol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 0.88
mg (6 .mu.mol) of 4-dimethylaminopyridine and with 5.2 mg (22
.mu.mol) of commercially available benzyl
4-hydroxypiperidine-1-carboxylate. The mixture was stirred at RT
overnight and then concentrated under high vacuum. The remaining
residue was purified by means of preparative HPLC. Thus, 5 mg (40%
of theory) of the Z-protected intermediate were obtained as a
colourless foam.
[4349] HPLC (Method 5): R.sub.t=2.1 min;
[4350] LC-MS (Method 1): R.sub.t=1.04 min; MS (ESIpos): m/z=1116
(M+H).sup.+.
[4351] Subsequently, the Z protecting group was cleaved by
hydrogenolytic means in ethanol over palladium/activated carbon.
4.6 mg (0.005 mmol) of the deprotected crude product were then,
without further purification, taken up in 1.8 ml of dichloromethane
and admixed with 3.8 mg (0.012 mmol) of
1,1'-[(1,5-dioxopentane-1,5-diyl)bis(oxy)]dipyrrolidine-2,5-dion-
e, 0.8 .mu.l (0.005 mmol) of N,N-diisopropylethylamine and 0.6 mg
(5 .mu.mol) of 4-dimethylaminopyridine. The mixture was stirred at
RT overnight and then concentrated under high vacuum. The remaining
residue was purified by means of preparative HPLC. Thus, 0.96 mg
(16% of theory) of the title compound were obtained as a colourless
foam.
[4352] HPLC (Method 5): R.sub.t=1.8 min;
[4353] LC-MS (Method 1): R.sub.t=0.94 min; MS (ESIpos): m/z=1193
(M+H).sup.+.
Intermediate 132
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazinyl}-4-ox-
obutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(R,2R)-1-metho-
xy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]-
amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L--
valinamide
##STR00568##
[4355] 15 mg (16.7 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phen-
ylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-y-
l]-N-methyl-L-valinamide were dissolved in 2500 .mu.l of DMF and
then admixed with 9.6 mg (50 .mu.mol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 7.6 mg
(50 .mu.mol) of 1-hydroxy-1H-benzotriazole hydrate, 5.8 .mu.l of
N,N-diisopropylethylamine and with 17.4 mg (67 .mu.mol) of
commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The
mixture was stirred at RT overnight and then concentrated under
high vacuum. The remaining residue was purified by means of
preparative HPLC. Thus, 11.2 mg (52% of theory) of the title
compound were obtained as a colourless foam.
[4356] HPLC (Method 5): R.sub.t=1.7 min;
[4357] LC-MS (Method 2): R.sub.t=1.09 min; MS (ESIpos): m/z=1106
(M+H).sup.+.
Intermediate 133
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazinyl}-4-ox-
obutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3S)-1-(b-
enzyloxy)-1-oxo-3-phenylbutan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]p-
yrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00569##
[4359] 5.8 mg (6.3 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{-
[(2S,3S)-1-(benzyloxy)-1-oxo-3-phenylbutan-2-yl]amino}-1-methoxy-2-methyl--
3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-
-L-valinamide were dissolved in 943 .mu.l of DMF and then admixed
with 3.6 mg (19 .mu.mol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 2.9 mg
(19 .mu.mol) of 1-hydroxy-1H-benzotriazole hydrate, 2.2 .mu.l of
N,N-diisopropylethylamine and with 6.6 mg (25 .mu.mol) of
commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The
mixture was stirred at RT overnight and then concentrated under
high vacuum. The remaining residue was purified by means of
preparative HPLC. Thus, 4.5 mg (64% of theory) of the title
compound were obtained as a colourless foam.
[4360] HPLC (Method 5): R.sub.t=2.0 min;
[4361] LC-MS (Method 1): R.sub.t=1.03 min; MS (ESIpos): m/z=1129
(M+H).sup.+.
Intermediate 134
N-[3-({[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]carbamoyl}amino)prop-
yl]-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy--
2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]ami-
no}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-val-
inamide
##STR00570##
[4363] First, 4-nitrophenyl
2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl carbamate was
prepared under standard conditions, starting from commercially
available 1-(2-aminoethyl)-1H-pyrrole-2,5-dione trifluoroacetate
and 4-nitrophenyl chlorocarbonate.
[4364] 5 mg (6 .mu.mol) of
N-(3-aminopropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R-
,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenyl-
cyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-
-N-methyl-L-valinamide were dissolved in 1000 .mu.l of DMF and then
admixed with 2 .mu.l of N,N-diisopropylethylamine and with 2.2 mg
(9 .mu.mol) of
4-nitrophenyl-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]carbamate.
The mixture was stirred at RT for 1 h and then concentrated under
high vacuum. The remaining residue was purified by means of
preparative HPLC. Thus, 1.6 mg (23% of theory) of the title
compound were obtained as a colourless foam.
[4365] HPLC (Method 5): R.sub.t=1.7 min;
[4366] LC-MS (Method 2): R.sub.t=1.09 min; MS (ESIpos): m/z=1036
(M+H).sup.+.
Intermediate 135
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzy-
loxy)-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrr-
olidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00571##
[4368] 10 mg (11 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{-
[(2S)-1-(benzyloxy)-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3--
oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-
-valinamide were dissolved in 4000 .mu.l of DMF and then admixed
with 6.3 mg (33 .mu.mol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4.5 mg
(33 .mu.mol) of 1-hydroxy-1H-benzotriazole hydrate, 5.7 .mu.l of
N,N-diisopropylethylamine and with 11.5 mg (44 .mu.mol) of
commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The
mixture was stirred at RT overnight and then concentrated under
high vacuum. The remaining residue was purified by means of
preparative HPLC. Thus, 2.6 mg (14% of theory) of the title
compound were obtained as a colourless foam.
[4369] HPLC (Method 6): R.sub.t=2.1 min;
[4370] LC-MS (Method 1): R.sub.t=1.01 min; MS (ESIpos): m/z=1115
(M+H).sup.+.
Intermediate 136
N-(4-{4-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl]piperazin-1-yl}--
4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1--
methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopr-
opyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-meth-
yl-L-valinamide
##STR00572##
[4372] First,
1-[4-oxo-4-(piperazin-1-yl)butyl]-1H-pyrrole-2,5-dione
trifluoroacetate was prepared under standard conditions, starting
from tert-butyl piperazine-1-carboxylate and
4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid over 2
stages.
[4373] 5 mg (5.6 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phen-
ylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-y-
l]-N-methyl-L-valinamide were dissolved in 1000 .mu.l of DMF and
then admixed with 2.1 mg (11 .mu.mol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1.7 mg
(11 .mu.mol) of 1-hydroxy-1H-benzotriazole hydrate, 2 .mu.l of
N,N-diisopropylethylamine and with 3.5 mg (5.6 .mu.mol) of
1-[4-oxo-4-(piperazin-1-yl)butyl]-1H-pyrrole-2,5-dione
trifluoroacetate. The mixture was stirred at RT overnight. Then 2.1
mg (5.6 .mu.mol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate were added, and the reaction mixture was
stirred at RT for another 3 h. Subsequently, the solvent was
removed in vacuo, and the remaining residue was purified by means
of preparative HPLC. The corresponding fractions were concentrated
and, by lyophilization from water, 0.6 mg (10% of theory) of the
title compound were obtained as a colourless foam.
[4374] HPLC (Method 6): R.sub.t=1.9 min;
[4375] LC-MS (Method 1): R.sub.t=0.9 min; MS (ESIpos): m/z=1132
(M+H).sup.+.
Intermediate 137
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-1-methylhydrazi-
no}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R-
)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2-
-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methy-
l-L-valinamide
##STR00573##
[4377] First,
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanehydrazide
trifluoroacetate was prepared under standard conditions, starting
from commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid and
tert-butyl 1-methylhydrazinecarboxylate over 2 stages.
[4378] 6.9 mg (8 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpro-
pan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N--
methyl-L-valinamide were dissolved in 2540 .mu.l of DMF and then
admixed with 3.6 mg (9 .mu.mol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate, 3 .mu.l of N,N-diisopropylethylamine and with
4.1 mg (12 .mu.mol) of
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanehydrazide
trifluoroacetate. The mixture was stirred at RT overnight.
Subsequently, the solvent was removed in vacuo, and the remaining
residue was purified by means of preparative HPLC. Thus, 3.9 mg
(45% of theory) of the title compound were obtained as a colourless
foam.
[4379] HPLC (Method 5): R.sub.t=1.8 min;
[4380] LC-MS (Method 1): R.sub.t=0.93 min; MS (ESIpos): m/z=1108
(M+H).sup.+.
Intermediate 138
N-{4-[(2-{[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl](methyl)amino}-
ethyl)(methyl)
amino]-4-oxobutyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1-
R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-pheny-
lcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl-
]-N-methyl-L-valinamide
##STR00574##
[4382] Starting from
tert-butylmethyl[2-(methylamino)ethyl]carbamate and
4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid, over 2
stages,
4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methyl-N-[2-(methylamino)ethyl-
]butanamide trifluoroacetate was prepared first.
[4383] 6.6 mg (7.3 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phen-
ylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-y-
l]-N-methyl-L-valinamide were dissolved in 2000 .mu.l of DMF and
then admixed with 5.6 mg (14.7 .mu.mol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate, 2.6 .mu.l of N,N-diisopropylethylamine and
with 4.1 mg (9 .mu.mol) of
4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methyl-N-[2-(methylamino)ethyl-
]butanamide trifluoroacetate. After stirring at RT for 3 h, the
same amounts of HATU and N,N-diisopropylethylamine were added once
more, and the reaction mixture was then stirred at RT overnight.
Subsequently, the solvent was removed in vacuo, and the remaining
residue was purified by means of preparative HPLC. Thus, 4 mg (44%
of theory) of the title compound were obtained as a colourless
foam.
[4384] HPLC (Method 6): R.sub.t=2.0 min;
[4385] LC-MS (Method 1): R.sub.t=0.91 min; MS (ESIpos): m/z=1134
(M+H).sup.+.
Intermediate 139
(2R,3S)-3-amino-4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hyd-
razino}-4-oxobutan-2-yl
(3R,4S,7S,10S)-4-[(2S)-butan-2-yl]-7,10-diisopropyl-3-(2-{(2S)-2-[(1R,2R)-
-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpropan-2--
yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-5,11-dimethyl-6,9-dioxo-
-2-oxa-5,8,11-triazapentadecan-15-oate
##STR00575##
[4387] 13 mg (14.7 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpro-
pan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N--
methyl-L-valinamide were dissolved in 10 ml of dichloromethane and
then admixed with 8.4 mg (44 .mu.mol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 5.4 mg
(44 .mu.mol) of 4-dimethylaminopyridine and with 9 mg (29.3
.mu.mol) of commercially available benzyl
N-(tert-butoxycarbonyl)-L-threoninate. The mixture was stirred at
RT for 5 h. Subsequently, the reaction mixture was extracted twice
by shaking with water, and the organic phase was dried over sodium
sulphate and concentrated in vacuo. The remaining residue was
purified by means of preparative HPLC. After lyophilization from
dioxane/water, 14 mg (81% of theory) of the protected intermediate
were obtained as a colourless foam.
[4388] HPLC (Method 12): R.sub.t=2.3 min;
[4389] LC-MS (Method 1): R.sub.t=1.13 min; MS (ESIpos): m/z=1178
(M+H).sup.+.
[4390] Subsequently, the Z protecting group was cleaved by
hydrogenolytic means in methanol over 10% palladium/activated
carbon. 9.5 mg (0.0087 mmol) of the deprotected crude product were
then, without further purification, taken up in 5 ml of DMF and
admixed with 5 mg (26.2 .mu.mol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4 mg
(26.2 .mu.mol) of 1-hydroxy-1H-benzotriazole hydrate, 54.6 .mu.l of
N,N-diisopropylethylamine and with 9.1 mg (34.9 .mu.mol) of
commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The
mixture was stirred at RT for 1 h and then concentrated under high
vacuum. The remaining residue was purified by means of preparative
HPLC. After lyophilization from dioxane, 9.5 mg (84% of theory) of
the Boc-protected intermediate were obtained.
[4391] HPLC (Method 12): R.sub.t=2.1 min;
[4392] LC-MS (Method 1): R.sub.t=0.97 min; MS (ESIpos): m/z=1295
(M+H).sup.+.
[4393] Subsequently, 9.5 mg (7.3 .mu.mol) were deprotected with 0.5
ml of trifluoroacetic acid in 2 ml of dichloromethane of the
Boc-protected intermediate and, after lyophilization from dioxane,
9 mg (82% of theory) of the title compound were obtained as a
colourless foam.
[4394] HPLC (Method 12): R.sub.t=2.1 min;
[4395] LC-MS (Method 1): R.sub.t=0.84 min; MS (ESIpos): m/z=1195
(M+H).sup.+.
Intermediate 140
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-1-methylhydrazi-
no}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R-
)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyc-
lopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N--
methyl-L-valinamide
##STR00576##
[4397] 4.1 mg (12 .mu.mol) of
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N'-methylhexanehydrazide
trifluoroacetate (Intermediate 22) were dissolved together with 6.9
mg (8 .mu.mol) of the compound from Intermediate 61 in 2.5 ml of
DMF and then admixed with 3.5 mg (9 .mu.mol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and 3 .mu.l of N,N-diisopropylethylamine. The
mixture was stirred at RT overnight and then concentrated under
high vacuum. The remaining residue was purified by means of
preparative HPLC. After lyophilization from dioxane, 2.6 mg (30% of
theory) of the title compound were obtained.
[4398] HPLC (Method 5): R.sub.t=1.8 min;
[4399] LC-MS (Method 1): R.sub.t=0.90 and 0.91 min; MS (ESIpos):
m/z=1120 (M+H).sup.+.
Intermediate 141
N-[4-({1-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl]piperidin-4-yl}-
oxy)-4-oxobutyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2-
R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcy-
clopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-
-methyl-L-valinamide
##STR00577##
[4401] 44 mg (49 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phen-
ylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-y-
l]-N-methyl-L-valinamide were dissolved in 2 ml of dichloromethane
and then admixed with 18.8 mg (98 .mu.mol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 3.8 mg
(24 .mu.mol) of 4-dimethylaminopyridine and with 23 mg (98 .mu.mol)
of commercially available benzyl 4-hydroxypiperidine-1-carboxylate.
The mixture was stirred at RT overnight and then concentrated under
high vacuum. The remaining residue was purified by means of
preparative HPLC. Thus, 22 mg (40% of theory) of the Z-protected
intermediate were obtained as a colourless foam.
[4402] HPLC (Method 5): R.sub.t=2.1 min;
[4403] LC-MS (Method 1): R.sub.t=1.04 min; MS (ESIpos): m/z=1116
(M+H).sup.+.
[4404] Subsequently, the Z protecting group was cleaved by
hydrogenolytic means in ethanol over palladium/activated
carbon.
[4405] 19 mg (19 .mu.mol) of the deprotected crude product were
then, without further purification, taken up in 4 ml of DMF and
admixed with 7 mg (39 .mu.mol) of
4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid, 11 mg (29
.mu.mol) of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and 5 .mu.l of N,N-diisopropylethylamine. The
mixture was stirred at RT for 1 h and then concentrated under high
vacuum. The remaining residue was purified by means of preparative
HPLC. After lyophilization from dioxane, 7.5 mg (34% of theory) of
the title compound were obtained.
[4406] HPLC (Method 5): R.sub.t=1.8 min;
[4407] LC-MS (Method 1): R.sub.t=0.94 min; MS (ESIpos): m/z=1147
(M+H).sup.+.
Intermediate 142
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzy-
loxy)-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopr-
opyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-vali-
namide
##STR00578##
[4409] 9 mg (9.5 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{-
[(2S)-1-(benzyloxy)-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2--
methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-
-methyl-L-valinamide (Intermediate 72) were dissolved in 1000 .mu.l
of DMF and then admixed with 10 mg (38 .mu.mol) of commercially
available 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide,
7.2 mg (19 .mu.mol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and 8 .mu.l of N,N-diisopropylethylamine, and
the reaction mixture was stirred at RT for 1 h. Subsequently, the
solvent was removed in vacuo and the remaining residue was purified
by means of preparative HPLC. The corresponding fractions were
concentrated and, by lyophilization, 6.4 mg (58% of theory) of the
title compound were obtained as a colourless foam.
[4410] HPLC (Method 5): R.sub.t=1.9 min;
[4411] LC-MS (Method 1): R.sub.t=0.99 min; MS (ESIpos): m/z=1154
(M+H).sup.+.
Intermediate 143
N-(4-{2-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethylbutanoyl]hyd-
razino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1-
R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-pheny-
lcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl-
]-N-methyl-L-valinamide
##STR00579##
[4413] 6 mg (6.7 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phen-
ylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-y-
l]-N-methyl-L-valinamide (Intermediate 61) were reacted with 3 mg
(8.7 .mu.mol) of
4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethylbutanehydrazide
trifluoroacetate in analogy to Intermediate 142 to yield 2 mg (27%
of theory) of the title compound.
[4414] HPLC (Method 12): R.sub.t=2.1 min;
[4415] LC-MS (Method 3): R.sub.t=1.92 min; MS (ESIpos): m/z=1106
(M+H).sup.+.
Intermediate 144
N-(4-{2-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethylbutanoyl]hyd-
razino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1-
R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylprop-
an-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-m-
ethyl-L-valinamide
##STR00580##
[4417] To a solution of 5 mg (5.6 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpro-
pan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N--
methyl-L-valinamide in 1 ml of DMF were added 7.65 mg (22.5
.mu.mol) of
4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethylbutanehydrazide
trifluoroacetate, 3.2 mg (16.9 .mu.mol) of EDC, 1.96 .mu.l (11.3
.mu.mol) of diisopropylethylamine and 2.6 mg (16.9 .mu.mol) of
HOBT. The reaction mixture was stirred at RT for 3 h. Subsequently,
a further 0.95 mg (2.8 .mu.mol) of
4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2,2-dimethylbutanehydrazide
trifluoroacetate were added. After stirring overnight, the reaction
mixture was concentrated and purified by means of preparative HPLC.
3.5 mg (85% purity, 48% of theory) of the title compound were
obtained.
[4418] LC-MS (Method 3): R.sub.t=1.86 min; m/z=1094
(M+H).sup.+.
Intermediate 145
N-[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl]-N-methyl-L-valyl-N-[(3R-
,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-
-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin--
1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00581##
[4420] 12 mg (14 .mu.mol) of
N-(3-aminopropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R-
,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenyl-
cyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-
-N-methyl-L-valinamide (Intermediate 66) were taken up in 750 .mu.l
of dioxane and admixed with 1.5 ml of saturated sodium
hydrogencarbonate solution and then with 3.2 mg (21 .mu.mol) of
methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate. The reaction
mixture was stirred at RT for 1 h and then concentrated under
reduced pressure. The remaining residue was purified by means of
preparative HPLC. After lyophilization, 4.2 mg (32% of theory) of
the title compound were obtained.
[4421] HPLC (Method 5): R.sub.t=1.7 min;
[4422] LC-MS (Method 1): R.sub.t=0.94 min; MS (ESIpos): m/z=950
(M+H).sup.+.
Intermediate 146
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-({(2S)-1-[benzy-
l(methyl)amino]-1-oxo-3-phenylpropan-2-yl}amino)-1-methoxy-2-methyl-3-oxop-
ropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-val-
inamide
##STR00582##
[4424] 9 mg (9.8 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-(-
{(2S)-1-[benzyl(methyl)amino]-1-oxo-3-phenylpropan-2-yl}amino)-1-methoxy-2-
-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]--
N-methyl-L-valinamide (Intermediate 73) were reacted in analogy to
Intermediate 133 with 10 mg (39 .mu.mol) of
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide to yield
1.8 mg (15% of theory) of the title compound.
[4425] HPLC (Method 12): R.sub.t=2.2 min;
[4426] LC-MS (Method 9): R.sub.t=5.11 min; MS (ESIpos): m/z=1128
(M+H).sup.+.
Intermediate 147
N-{4-[(2,5-dioxopyrrolidin-1-yl)oxy]-4-oxobutyl}-N-methyl-L-valyl-N-[(3R,4-
S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S,3S)-1-(benzyloxy)-1-oxo-3-phenylbutan-2-y-
l]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methy-
l-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00583##
[4428] 16 mg (17 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{-
[(2S,3S)-1-(benzyloxy)-1-oxo-3-phenylbutan-2-yl]amino}-1-methoxy-2-methyl--
3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-
-L-valinamide (Intermediate 70) were dissolved in 2 ml of
dichloromethane and admixed with 2.6 mg (23 mmol) of
1-hydroxypyrrolidine-2,5-dione and then with 4 mg (21 .mu.mol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. After
stirring at RT for 2 h, the same amounts of
1-hydroxypyrrolidine-2,5-dione and
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were
added once again. Then stirring at RT overnight, the reaction
mixture was concentrated in vacuo. The remaining residue was
purified by means of preparative HPLC. After lyophilization, 10 mg
(56% of theory) of the title compound were obtained.
[4429] HPLC (Method 5): R.sub.t=2.0 min;
Intermediate 148
N-{4-[(2-{[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl](methyl)amino}-
ethyl)amino]-4-oxobutyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)--
2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2--
phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide
##STR00584##
[4431] 6 mg (7 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phen-
ylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-y-
l]-N-methyl-L-valinamide (Intermediate 61) were combined with 2.8
mg (8 .mu.mol) of
N-(2-aminoethyl)-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylbutanam-
ide trifluoroacetate, 10.1 mg (27 .mu.mol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and 5 .mu.l of N,N-diisopropylethylamine in 2
ml of DMF and stirred at RT overnight. Then another 5 mg (23.5
.mu.mol) of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and 3 .mu.l of N,N-diisopropylethylamine were
added. After stirring at RT for a further 5 h, the solvent was
removed in vacuo, and the remaining residue was purified by means
of preparative HPLC. The corresponding fractions were concentrated
and, by lyophilization from dioxane, 1.3 mg (15% of theory) of the
title compound were obtained.
[4432] HPLC (Method 12): R.sub.t=2.1 min;
[4433] LC-MS (Method 2): R.sub.t=1.21 min; MS (ESIpos): m/z=1120
(M+H).sup.+.
Intermediate 149
N-{4-[(2-{[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl]amino}ethyl)(m-
ethyl)amino]-4-oxobutyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)--
2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2--
phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide
##STR00585##
[4435] 6 mg (7 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phen-
ylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-y-
l]-N-methyl-L-valinamide (Intermediate 61) were combined with 3.1
mg (9 .mu.mol) of
4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-[2-(methylamino)ethyl]butanami-
de trifluoroacetate, 10.1 mg (27 .mu.mol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and 5 .mu.l of N,N-diisopropylethylamine in 2
ml of DMF, and the mixture was stirred at RT for 4 h. Then the
solvent was removed in vacuo, and the remaining residue was
purified by means of preparative HPLC. The corresponding fractions
were concentrated and, by lyophilization from dioxane, 1 mg (13.4%
of theory) of the title compound were obtained.
[4436] HPLC (Method 12): R.sub.t=2.1 min;
[4437] LC-MS (Method 1): R.sub.t=0.89 min; MS (ESIpos): m/z=1121
(M+H).sup.+.
Intermediate 150
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-metho-
xy-2-methyl-3-oxo-3-{[(1S,2R)-2-phenyl-1-(propylcarbamoyl)cyclopropyl]amin-
o}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00586##
[4439] 7.9 mg (9 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S,2R)-2-phenyl-1-(propylcarbamoyl)cy-
clopropyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methy-
l-L-valinamide were dissolved in 3 ml of DMF and then admixed with
10.4 mg (54 .mu.mol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 8.3 mg
(54 .mu.mol) of 1-hydroxy-1H-benzotriazole hydrate, 9 .mu.l of
N,N-diisopropylethylamine and with 9.5 mg (36 .mu.mol) of
commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The
mixture was stirred at RT overnight and then concentrated under
high vacuum. The remaining residue was purified by means of
preparative HPLC. Thus, 4.3 mg (22% of theory) of the title
compound were obtained as a colourless foam.
[4440] HPLC (Method 6): R.sub.t=1.9 min;
[4441] LC-MS (Method 9): R.sub.t=4.93 min; MS (ESIpos): m/z=1078
(M+H).sup.+.
Intermediate 151
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-car-
bamoyl-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidi-
n-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00587##
[4443] The compound was prepared in analogy to Intermediate 150
starting from the compound in Intermediate 81.
[4444] HPLC (Method 5): R.sub.t=1.7 min;
[4445] LC-MS (Method 1): R.sub.t=0.87 min; MS (ESIpos): m/z=1036
(M+H).sup.+.
Intermediate 152
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-(et-
hoxycarbonyl)-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopropyl]py-
rrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00588##
[4447] 10 mg (12 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{-
[(1S,2R)-1-(ethoxycarbonyl)-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl--
3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-
-L-valinamide were dissolved in 3 ml of DMF and then admixed with
8.9 mg (23 .mu.mol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate, 10 .mu.l of N,N-diisopropylethylamine and with
12 mg (47 .mu.mol) of commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The
mixture was stirred at RT for 1 h. This was followed by
concentration under high vacuum and purification of the remaining
residue by means of preparative HPLC. Thus, 5.8 mg (37% of theory)
of the title compound were obtained as a colourless foam.
[4448] HPLC (Method 6): R.sub.t=2.0 min;
[4449] LC-MS (Method 9): R.sub.t=4.99 min; MS (ESIpos): m/z=1066
(M+H).sup.+.
Intermediate 153
N-[1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-12,15-dioxo-3,6,9-trioxa-13,14-
-diazaoctadecan-18-yl]-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2--
[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylp-
ropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]--
N-methyl-L-valinamide
##STR00589##
[4451] To a solution of 5 mg (5.6 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylpro-
pan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N--
methyl-L-valinamide in 1 ml of DMF were added 9.7 mg (22.5 .mu.mol)
of
3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy)prop-
anehydrazide trifluoroacetate, 3.2 mg (16.9 .mu.mol) of EDC, 1.96
N1 (11.3 .mu.mol) of N,N-diisopropylethylamine and 2.6 mg (16.9
.mu.mol) of HOBT. The reaction mixture was stirred at RT for 3 h.
Subsequently, another 1.2 mg (2.8 .mu.mol) of
3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy)pro-
panehydrazide trifluoroacetate were added. The reaction mixture was
stirred at RT overnight and then purified by means of preparative
HPLC.
[4452] 3.6 mg (51% of theory) of the title compound were
obtained.
[4453] LC-MS (Method 1): R.sub.t=0.90 min; m/z=1185
(M+H).sup.+.
Intermediate 154
(2R,3S)-3-amino-4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hyd-
razino}-4-oxobutan-2-yl-(3R,4S,7S,10S)-4-[(2S)-butan-2-yl]-7,10-diisopropy-
l-3-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-y-
lcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-2-oxoet-
hyl)-5,11-dimethyl-6,9-dioxo-2-oxa-5,8,11-triazapentadecan-15-oate
##STR00590##
[4455] 15 mg (17 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-{[(1S)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylc-
yclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]--
N-methyl-L-valinamide were dissolved in 10 ml of dichloromethane
and then admixed with 12.8 mg (67 .mu.mol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 10 mg
(83 .mu.mol) of 4-dimethylaminopyridine and with 10.3 mg (33
.mu.mol) of commercially available benzyl
N-(tert-butoxycarbonyl)-L-threoninate. The mixture was heated to
reflux for 4 h. Then the same amounts of coupling reagent and
4-dimethylaminopyridine were added again, and the reaction mixture
was heated overnight with reflux. Subsequently, the reaction
mixture was diluted with dichloromethane and extracted by shaking
once with water, the organic phase was removed and concentrated
under high vacuum. The remaining residue was purified by means of
preparative HPLC. Thus, 7.7 mg (37% of theory) of the protected
intermediate were obtained as a colourless foam.
[4456] HPLC (Method 12): R.sub.t=2.5 min;
[4457] LC-MS (Method 1): R.sub.t=1.13 min; MS (ESIpos): m/z=1190
(M+H).sup.+.
[4458] Subsequently, the benzyl ester protecting group was removed
by hydrogenation under standard hydrogen pressure in methanol over
10% palladium/activated carbon, and the acid thus obtained, as
described in Intermediate 151, was coupled to
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. In a last
step, the Boc protecting group was detached with trifluoroacetic
acid. The remaining residue was purified by means of preparative
HPLC. Thus, 0.22 mg (2.5% of theory over 3 stages) of the title
compound was obtained as a colourless foam.
[4459] HPLC (Method 12): R.sub.t=2.0 min;
[4460] LC-MS (Method 1): R.sub.t=0.81 min; MS (ESIpos): m/z=1207
(M+H).sup.+.
Intermediate 155
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino--
1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-
-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00591##
[4462] This compound was prepared in analogy to the synthesis
described in Intermediate 152 from
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{-
[(2S)-1-amino-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopro-
pyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valin-
amide and commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide.
[4463] HPLC (Method 5): R.sub.t=1.6 min;
[4464] LC-MS (Method 1): R.sub.t=0.82 min; MS (ESIpos): m/z=1024
(M+H)+.
Intermediate 156
N-(3-{[(1-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}cyclopropyl)carbonyl]am-
ino}propyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1--
methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopr-
opyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-meth-
yl-L-valinamide
##STR00592##
[4466] This compound was prepared in analogy to the synthesis
described in the last stage of Intermediate 131 from
N-(3-aminopropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R-
,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenyl-
cyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-
-N-methyl-L-valinamide and
1,1'-[cyclopropane-1,1-diylbis(carbonyloxy)]dipyrrolidine-2,5-dione,
which had been obtained from the corresponding dicarboxylic acid
beforehand.
[4467] HPLC (Method 12): R.sub.t=2.0 min;
[4468] LC-MS (Method 1): R.sub.t=0.92 min; MS (ESIpos): m/z=1080
(M+H).sup.+.
Intermediate 157
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino--
3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]p-
yrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00593##
[4470] 15 mg (18 .mu.mol) of
(N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3--
{[(2S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methy-
l-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-meth-
yl-L-valinamide were dissolved in 3.8 ml of DMF and then admixed
with 27 mg (70 .mu.mol) of O-(7
azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate, 12 .mu.l of N,N-diisopropylethylamine and with
14 mg (53 .mu.mol) of commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide. The
reaction mixture was stirred at RT for 1 h. This was followed by
concentration under high vacuum and purification of the remaining
residue by means of preparative HPLC. Thus, 6.2 mg (33% of theory)
of the title compound were obtained as a colourless foam.
[4471] HPLC (Method 5): R.sub.t=1.6 min;
[4472] LC-MS (Method 1): R.sub.t=0.85 min; MS (ESIpos): m/z=1063
(M+H).sup.+.
[4473] .sup.1H-NMR (500 MHz, DMSO-d.sub.6, characteristic signals):
.delta.=10.8 (d, 1H), 9.8-9.7 (m, 2H), 9.6 and 9.4 (2m, 1H), 8.9,
8.88, 8.78 and 8.75 (4d, 1H), 8.08 and 7.85 (2d, 1H), 7.6-6.9 (m,
9H), 4.7-4.4 (m, 3H), 3.4 (t, 2H), 3.23, 3.2, 3.18, 3.0, and 2.99
(5s, 9H), 2.8 (m, 3H), 2.1 (t, 2H), 1.06 and 1.01 (2d, 3H),
0.95-0.8 (m, 15H), 0.8-0.75 (dd, 3H).
Intermediate 158
N-[4-({(2R)-1-[(2,5-dioxopyrrolidin-1-yl)oxy]-4-methyl-1-oxopentan-2-yl}am-
ino)-4-oxobutyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S-
)-1-(benzylamino)-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-ox-
opropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-v-
alinamide
##STR00594##
[4475] 13 mg (14.7 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{-
[(2S)-1-(benzylamino)-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl--
3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-
-L-valinamide were dissolved in 4 ml of dimethylformamide and then
admixed with 9.4 mg (25 .mu.mol) of
0-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate, 6 .mu.l of N,N-diisopropylethylamine and with
7 mg (31 .mu.mol) of commercially available tert-butyl-D-leucinate
hydrochloride. The mixture was stirred at RT for 5 h and then
concentrated in vacuo. The remaining residue was purified by means
of preparative HPLC. After lyophilization from dioxane/water, 6.5
mg (49% of theory) of the protected intermediate were obtained as a
colourless foam.
[4476] HPLC (Method 5): R.sub.t=2.2 min;
[4477] LC-MS (Method 1): R.sub.t=1.21 min; MS (ESIpos): m/z=1076
(M+H).sup.+.
[4478] Trifluoroacetic acid in dichloromethane was first used to
cleave the Boc protecting group from this protected intermediate,
yielding 6.2 mg (99% of theory) of the deprotected compound. 5.2 mg
(5 .mu.mol) of this intermediate were taken up in 1.5 ml of
dichloromethane and reacted with 0.8 mg (7 .mu.mol) of
N-hydroxysuccinimide, in the presence of 1.2 mg (6 .mu.mol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and
0.16 mg (1 .mu.mol) of 4-dimethylaminopyridine. After stirring at
RT for 2 h, the reaction mixture was concentrated and purified by
means of preparative HPLC. 1.3 mg of the title compound were
obtained, some of which was hydrolysed into an educt.
Intermediate 159
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzy-
lamino)-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]py-
rrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00595##
[4480] This compound was prepared in analogy to the synthesis
described in Intermediate 157 from
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{-
[(2S)-1-(benzylamino)-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl--
3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-
-L-valinamide and commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide.
[4481] Yield: 6 mg (53% of theory)
[4482] HPLC (Method 5): R.sub.t=1.9 min;
[4483] LC-MS (Method 1): R.sub.t=0.94 min; MS (ESIpos): m/z=1114
(M+H).sup.+.
Intermediate 160
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzy-
lamino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxo-
propyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-va-
linamide
##STR00596##
[4485] This compound was prepared in analogy to the synthesis
described in Intermediate 157 from 20 mg (21 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{-
[(2S)-1-(benzylamino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy--
2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-
-N-methyl-L-valinamide and commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide.
[4486] Yield: 13 mg (52% of theory)
[4487] HPLC (Method 5): R.sub.t=1.9 min;
[4488] LC-MS (Method 1): R.sub.t=0.92 min; MS (ESIpos): m/z=1153
(M+H).sup.+.
Intermediate 161
N-(6-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-6-oxo-
hexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino--
3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]p-
yrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00597##
[4490] This compound was prepared in analogy to the synthesis
described in Intermediate 157 from
N-(5-carboxypentyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{-
[(2S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-
-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methy-
l-L-valinamide and commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide.
[4491] Yield: 0.8 mg (16% of theory)
[4492] HPLC (Method 5): R.sub.t=1.6 min;
[4493] LC-MS (Method 1): R.sub.t=0.78 min; MS (ESIpos): m/z=1092
(M+H).sup.+.
Intermediate 162
N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-N-[(3R,4-
S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-o-
xazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1--
yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00598##
[4495] 18 mg (20 .mu.mol) of
N-(5-carboxypentyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phen-
ylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-y-
l]-N-methyl-L-valinamide (Intermediate 64) were dissolved in 3.2 ml
of dichloromethane and admixed with 22 mg (190 mmol) of
1-hydroxypyrrolidine-2,5-dione and then with 11 mg (60 .mu.mol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and
0.24 mg (0.17 .mu.mol) of DMAP. After stirring at RT for 2 h,
another 22 mg (190 mmol) of 1-hydroxypyrrolidine-2,5-dione, 11 mg
(60 .mu.mol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride and 0.24 mg (0.17 .mu.mol) of DMAP were added, and
the reaction mixture was stirred at RT for another hour. This was
followed by concentration in vacuo. The remaining residue was
purified by means of preparative HPLC. After lyophilization, 8.2 mg
(41% of theory) of the title compound were obtained.
[4496] HPLC (Method 5): R.sub.t=2.0 min;
[4497] LC-MS (Method 11): R.sub.t=0.9 min; MS (ESIpos): m/z=1024
(M+H).sup.+.
Intermediate 163
[(1S,2R)-1-amino-2-phenylcyclopropyl](1,4-dihydro-3H-2,3-benzoxazin-3-yl)m-
ethanone trifluoroacetate
##STR00599##
[4499] First, starting with 265 mg (0.82 mmol) of tert-butyl
(1S,2R)-1-(hydroxycarbamoyl)-2-phenylcyclopropyl carbamate
(Starting Compound 7), and by reaction with
1,2-bis(bromomethyl)benzene analogously to a literature method (see
H. King, J. Chem. Soc. 1942, 432), the Boc-protected intermediate
tert-butyl-[(1S,2R)-1-(1,4-dihydro-3H-2,3-benzoxazin-3-ylcarbonyl)-2-phen-
ylcyclopropyl]carbamate was prepared.
[4500] Yield: 108 mg (34% of theory)
[4501] LC-MS (Method 2): R.sub.t=1.3 min; MS (ESIpos): m/z=395
(M+H).sup.+.
[4502] 108 mg (0.27 mmol) of this intermediate were taken up in 3.7
ml of dichloromethane, 1.8 ml of trifluoroacetic acid were added,
and the mixture was stirred at RT for 15 min. This was followed by
concentration in vacuo and lyophilization of the remaining residue
from dioxane. 112 mg of the title compound were obtained in
quantitative yield as a colourless foam.
[4503] LC-MS (Method 1): R.sub.t=0.7 min; MS (ESIpos): m/z=295
(M+H).sup.+.
Intermediate 164
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-(1,4-dihyd-
ro-3H-2,3-benzoxazin-3-ylcarbonyl)-2-phenylcyclopropyl]amino}-1-methoxy-2--
methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-
-methyl-L-valinamide trifluoroacetate
##STR00600##
[4505] 166 mg (0.196 mmol) of
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2-
S)-2-[(1R,2R)-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methy-
l-1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 10) were
taken up in 40 ml of DMF and admixed successively with 80 mg (0.196
mmol) of
[(1S,2R)-1-amino-2-phenylcyclopropyl](1,4-dihydro-3H-2,3-benzoxazin-3-yl)-
methanone trifluoroacetate (Intermediate 163), 112 mg (0.294 mmol)
of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) and 682 .mu.l (3.9 mmol) of
N,N-diisopropylethylamine. The mixture was subsequently stirred at
RT overnight. The reaction mixture was then concentrated in vacuo,
the residue was taken up in ethyl acetate, and the solution was
washed with saturated aqueous sodium chloride solution. The organic
phase was dried over magnesium sulphate, filtered and concentrated.
The residue was finally purified by means of preparative HPLC. In
this way, 19 mg (9% of theory) of the Fmoc-protected intermediate
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2-
S)-2-[(1R,2R)-3-{[(1S,2R)-1-(1,4-dihydro-3H-2,3-benzoxazin-3-ylcarbonyl)-2-
-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}--
3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide were
obtained.
[4506] HPLC (Method 5): R.sub.t=1.68 min;
[4507] LC-MS (Method 1): R.sub.t=1.51 min; MS (ESIpos): m/z=1083
(M+H).sup.+.
[4508] 19 mg (0.015 mmol) of this intermediate were dissolved in 4
ml of DMF. After adding 817 .mu.l of piperidine, the reaction
mixture was stirred at RT for 5 min. This was followed by
concentration in vacuo, and the residue was first digested with
diethyl ether and then purified by means of preparative HPLC
(eluent: acetonitrile+0.1% TFA/0.1% aq. TFA). The corresponding
fractions were combined, the solvent was removed in vacuo, and then
the residue was lyophilized from dioxane/water. 12 mg (92% of
theory) of the title compound were obtained as a colourless
foam.
[4509] HPLC (Method 6): R.sub.t=2.0 min;
[4510] LC-MS (Method 1): R.sub.t=0.94 min; MS (ESIpos): m/z=861
(M+H).sup.+.
Intermediate 165
N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S-
,2R)-1-(1,4-dihydro-3H-2,3-benzoxazin-3-ylcarbonyl)-2-phenylcyclopropyl]am-
ino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1--
oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00601##
[4512] 20 mg (0.021 mmol) of Intermediate 164 were used, in analogy
to the preparation of Intermediate 97, together with
benzyl-(6-oxohexyl)carbamate in the presence of sodium
cyanoborohydride and with subsequent hydrogenolytic cleaving of the
Z protecting group (using 5% palladium on carbon as catalyst, in
methanol as a solvent), to prepare the title compound.
[4513] Yield: 4.5 mg (23% of theory over 2 stages)
[4514] HPLC (Method 12): R.sub.t=1.9 min;
[4515] LC-MS (Method 1): R.sub.t=0.9 min; MS (ESIpos): m/z=960
(M+H).sup.+.
Intermediate 166
N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,-
4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-(1,4-dihydro-3H-2,3-benzoxazin-3-y-
lcarbonyl)-2-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrro-
lidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00602##
[4517] 4.4 mg (4.5 .mu.mol) of Intermediate 165 were taken up in 1
ml of 1:1 dioxane/water and then admixed with 1 mg (6.8 .mu.mol) of
methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate and with 50
.mu.l of saturated aqueous sodium hydrogencarbonate solution. The
reaction mixture was stirred at RT for 30 min. Then another 50
.mu.l of the saturated aqueous sodium hydrogencarbonate solution
were added, and the reaction mixture was stirred at RT for a
further 15 min and then concentrated in vacuo. The remaining
residue was purified by means of preparative HPLC. After
lyophilization, 1 mg (21% of theory) of the title compound were
obtained as a colourless foam.
[4518] HPLC (Method 12): R.sub.t=2.1 min;
[4519] LC-MS (Method 1): R.sub.t=1.08 min; MS (ESIpos): m/z=1040
(M+H).sup.+.
Intermediate 167
Benzyl 3-{2-[2-(2-oxoethoxyl)ethoxy]ethoxy}propanoate
##STR00603##
[4521] The title compound was prepared from 6 g (21.55 mmol) of
commercially available
3-{2-[2-(2-hydroxyethoxyl)ethoxy]ethoxy}propanoic acid under
standard conditions, first by esterification with benzyl chloride
and caesium carbonate and subsequent oxidation with sulphur
trioxide-pyridine complex.
[4522] Yield: 611 mg (10% of theory over 2 stages)
[4523] LC-MS (Method 2): R.sub.t=1.69 min; MS (ESIpos): m/z=311
(M+H).sup.+.
Intermediate 168
N-(2-{2-[2-(2-carboxyethoxyl)ethoxy]ethoxy}ethyl)-N-methyl-L-valyl-N-[(3R,-
4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-
-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-met-
hyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00604##
[4525] First, in analogy to the synthesis described in Intermediate
69, by coupling of
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-car-
boxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide
(Intermediate 4) and
N.sup..alpha.-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]pro-
panoyl}-L-tryptophanamide trifluoroacetate (Intermediate 49) in the
presence of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and subsequent cleaving of the Fmoc protecting
group by means of piperidine, the amine compound
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino-3-(1H--
indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrroli-
din-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
was prepared as the trifluoroacetate.
[4526] 25 mg (0.028 mmol) of this compound and 17.5 mg (0.06 mmol)
of Intermediate 167 were combined in 2 ml of methanol and admixed
with 12.6 mg (0.14 mmol) of borane-pyridine complex and 2.5 ml of
acetic acid. The reaction mixture was stirred at RT overnight.
Then, the same amounts of borane-pyridine complex and acetic acid
were added once more, and the reaction mixture was stirred at RT
for another 24 h. This was followed by concentration in vacuo, and
the residue was purified by means of preparative HPLC. After
concentration of the corresponding fractions and lyophilization
from 1:1 dioxane/water, 26.5 mg (88% of theory) of the Z-protected
title compound were obtained.
[4527] HPLC (Method 12): R.sub.t=2.04 min;
[4528] LC-MS (Method 1): R.sub.t=0.97 min; MS (ESIpos): m/z=1064
(M+H).sup.+.
[4529] 25 mg (0.024 mmol) of this intermediate were taken up in 10
ml of methanol and hydrogenated over 10% palladium on activated
carbon under standard hydrogen pressure at RT for 45 min. The
catalyst was then filtered off, and the solvent was removed in
vacuo. After lyophilization from dioxane, 19.7 mg (85% of theory)
of the title compound were obtained.
[4530] HPLC (Method 12): R.sub.t=1.8 min;
[4531] LC-MS (Method 1): R.sub.t=0.83 min; MS (ESIpos): m/z=974
(M+H).sup.+.
Intermediate 169
N-{2-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropoxy}ethoxy)ethoxy]e-
thyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino-3-
-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]py-
rrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00605##
[4533] 10 mg (10 .mu.mol) of Intermediate 168 were dissolved in 3
ml of DMF and admixed with 3.5 mg (30 mmol) of
1-hydroxypyrrolidine-2,5-dione and then with 2.4 mg (10 .mu.mol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 5
.mu.l of N,N-diisopropylethylamine. After stirring at RT for 20 h,
8 mg (0.02 mmol) of HATU were added, and the reaction mixture was
stirred once again at RT overnight and then concentrated in vacuo.
The remaining residue was purified by means of preparative HPLC.
After lyophilization from dioxane, 8.6 mg (64% of theory) of the
title compound were obtained.
[4534] HPLC (Method 12): Rt=1.9 min;
[4535] LC-MS (Method 11): Rt=0.81 min; MS (ESIpos): m/z=1071
(M+H)+.
Intermediate 170
N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2-
R)-1-methoxy-2-methyl-3-{[(2S,3S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylbuta-
n-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-me-
thyl-L-valinamide
##STR00606##
[4537] This compound was prepared in analogy to Intermediate 101
over 2 stages, starting from 26 mg (0.028 mmol) of Intermediate
15.
[4538] Yield: 16.7 mg (63% of theory over 2 stages)
[4539] HPLC (Method 12): R.sub.t=1.9 min;
[4540] LC-MS (Method 1): R.sub.t=0.81 min; MS (ESIpos): m/z=914
(M+H).sup.+.
Intermediate 171
N-(6-{[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl]amino}hexyl)-N-met-
hyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl--
3-{[(2S,3S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylbutan-2-yl]amino}-3-oxopro-
pyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00607##
[4542] 6.7 mg (7.3 .mu.mol) of the compound from Intermediate 170
and 3 mg (14.7 .mu.mol) of commercially available
4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid were taken up
in 2 ml of DMF and admixed with 5.6 mg (14.7 .mu.mol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) and 2 .mu.l of
N,N-diisopropylethylamine. The mixture was stirred at RT for 30
min. The reaction mixture was concentrated, and the residue was
purified by means of preparative HPLC. The corresponding fractions
were combined, the solvent was removed in vacuo, and then the
residue was lyophilized from dioxane. Thus, 4.5 mg (56% of theory)
of the title compound were obtained.
[4543] HPLC (Method 12): R.sub.t=2.0 min;
[4544] LC-MS (Method 1): R.sub.t=1.12 min; MS (ESIpos): m/z=1079
(M+H).sup.+.
Intermediate 172
Benzyl 2-{2-[2-(2-oxoethoxyl)ethoxy]ethoxy}ethyl)carbamate
##STR00608##
[4546] The title compound was prepared from commercially available
2-{2-[2-(2-aminoethoxyl)ethoxy]ethoxy}ethanol under standard
conditions by first introducing the Z protecting group and then
oxidizing with sulphur trioxide-pyridine complex.
[4547] HPLC (Method 12): R.sub.t=1.4 min;
[4548] LC-MS (Method 11): R.sub.t=0.65 min; MS (ESIpos): m/z=326
(M+H).sup.+.
Intermediate 173
Benzyl {2-[2-(2-oxoethoxyl)ethoxy]ethyl}carbamate
##STR00609##
[4550] The title compound was prepared in analogy to Intermediate
172 from commercially available 2-[2-(2-aminoethoxyl)ethoxy]ethanol
under standard conditions by first introducing the Z protecting
group and then oxidizing with sulphur trioxide-pyridine
complex.
[4551] HPLC (Method 12): R.sub.t=1.3 min;
[4552] LC-MS (Method 11): R.sub.t=0.68 min; MS (ESIpos): m/z=282
(M+H).sup.+.
Intermediate 174
N-(2-{2-[2-(2-aminoethoxyl)ethoxy]ethoxy}ethyl)-N-methyl-L-valyl-N-[(3R,4S-
,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-ox-
azinan-2-ylcarbonyl)-2-phenyl
cyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl-
]-N-methyl-L-valinamide
##STR00610##
[4554] 47 mg (0.05 mmol) of Intermediate 16 were reductively
aminated in analogy to the preparation of Intermediate 167 with
benzyl-(2-{2-[2-(2-oxoethoxyl)ethoxy]ethoxy}ethyl)carbamate in the
presence of borane-pyridine complex. Subsequently, the Z protecting
group was removed by hydrogenolytic means with 5% palladium on
carbon as a catalyst and in methanol as a solvent, and 38 mg (66%
of theory over 2 stages) of the title compound were prepared.
[4555] HPLC (Method 5): R.sub.t=1.7 min;
[4556] LC-MS (Method 1): R.sub.t=0.8 min; MS (ESIpos): m/z=988
(M+H).sup.+.
Intermediate 175
N-[2-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy)e-
thyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methox-
y-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]a-
mino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-v-
alinamide
##STR00611##
[4558] The preparation was done in analogy zu Intermediate 166
starting from 34 mg (0.03 mmol) of Intermediate 174.
[4559] Yield: 8.3 mg (23% of theory)
[4560] HPLC (Method 5): R.sub.t=1.9 min;
[4561] LC-MS (Method 1): R.sub.t=0.97 min; MS (ESIpos): m/z=1068
(M+H).
Intermediate 176
N-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethyl)-N-me-
thyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino-3-(1H-indol-
-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-
-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00612##
[4563] The preparation was done in analogy to Intermediates 174 and
175 starting with the reductive amination of Intermediate 16 with
Intermediate 173, subsequent deprotection and formation of the
maleimide.
[4564] HPLC (Method 12): R.sub.t=1.8 min;
[4565] LC-MS (Method 11): R.sub.t=0.8 min; MS (ESIpos): m/z=981
(M+H).sup.+.
Intermediate 177
N-[2-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy)e-
thyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino-3-
-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]py-
rrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00613##
[4567] The preparation was done in analogy to Intermediates 174 and
175 starting with the reductive amination of Intermediate 16 with
Intermediate 172, subsequent deprotection and formation of the
maleimide.
[4568] HPLC (Method 12): R.sub.t=1.9 min;
[4569] LC-MS (Method 1): R.sub.t=0.86 min; MS (ESIpos): m/z=1025
(M+H).sup.+.
Intermediate 178
N-{4-[(2,5-dioxopyrrolidin-1-yl)oxy]-4-oxobutyl}-N-methyl-L-valyl-N-[(3R,4-
S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2--
yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-meth-
yl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00614##
[4571] The preparation was done in analogy to Intermediate 162
starting from 6 mg of Intermediate 82.
[4572] LC-MS (Method 1): R.sub.t=0.82 min; MS (ESIpos): m/z=953
(M+H).
Intermediate 179
4-[(1E,3S)-3-amino-4-phenylbut-1-en-1-yl]benzenesulphonic acid
trifluoroacetate
##STR00615##
[4574] A mixture of 13.6 mg (0.06 mmol) of palladium(II) acetate,
469 mg (1.46 mmol) of potassium 4-iodobenzenesulphonate, 300 mg
(1.21 mmol) of (S)-tert-butyl-1-phenylbut-3-en-2-yl-carbamate, 16.5
mg (0.12 mmol) of phenylurea and 167.6 mg (1.21 mmol) of potassium
carbonate in 7.5 ml of DMF was heated in a microwave for 15 min to
160.degree. C. The crude product was subsequently purified directly
by means of preparative HPLC. This yielded 312 mg of a mixture of
31% of the BOC-protected compound and 69% of the free amine.
[4575] This mixture was subsequently taken up in 30 ml of
dichloromethane, admixed with 1 ml of trifluoroacetic acid and
stirred at RT for 20 h. After concentrating in vacuo, the residue
was stirred in with diethyl ether, and the precipitate that formed
was suctioned off and washed with diethyl ether. This yielded 200
mg (62% of theory) of the title compound.
[4576] LC-MS (Method 11): R.sub.t=0.44 min; MS (ESIpos): m/z=304
(M+H).sup.+.
Intermediate 180
4-[(3R)-3-amino-4-phenylbutyl]benzenesulphonic acid
##STR00616##
[4578] 100 mg (0.25 mmol) of
4-[(1E,3S)-3-amino-4-phenylbut-1-en-1-yl]benzenesulphonic acid
trifluoroacetate were suspended in 10 ml of acetic acid and a few
drops of DMF and water, admixed with 70 mg (0.07 mmol) of palladium
on carbon (10%) and hydrogenated at hydrogen pressure 2.2 bar for
24 h. The solution was filtered and the filtrate purified by means
of preparative HPLC.
[4579] 29 mg (76% purity, 21% of theory) of product were
obtained.
[4580] LC-MS (Method 1): R.sub.t=0.46 min; MS (ESIpos): m/z=306
(M+H).sup.+.
Intermediate 181
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-
-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S,3E)-1-phenyl-4-(4-sulphophenyl)-
but-3-en-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-m-
ethyl-L-valinamide
##STR00617##
[4582] To a solution of 90 mg (0.13 mmol) of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide in 4 ml of DMF were added 60 mg (0.16
mmol) of HATU and 69 .mu.l of (0.39 mmol) Hunig's base. The
reaction mixture was stirred at RT for 30 min and then admixed with
60 mg (0.15 mmol) 60.3 mg (0.13 mmol) of
4-[(1E,3S)-3-amino-4-phenylbut-1-en-1-yl]benzenesulphonic acid
trifluoroacetate. After stirring overnight, the reaction mixture
was purified by means of preparative HPLC. This yielded 127 mg of a
44:56 mixture of the title compound and of the already deprotected
amine.
[4583] LC-MS (Method 1): R.sub.t=1.21 min; MS (ESIpos): m/z=971
(M+H)+; R.sub.t=0.84 min; MS (ESIpos): m/z=871 (M+H).sup.+ for the
deprotected compound.
Intermediate 182
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-me-
thyl-3-oxo-3-{[(2S,3E)-1-phenyl-4-(4-sulphophenyl)but-3-en-2-yl]amino}prop-
yl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate
##STR00618##
[4585] 90 mg of Intermediate 180 were dissolved in 4.6 ml of
dichloromethane, and 0.92 ml of trifluoroacetic acid were added.
The reaction mixture was stirred at RT for 30 min and then
concentrated. The obtained crude product was purified by means of
preparative HPLC.
[4586] 91 mg (98% of theory) of the target compound were
obtained.
[4587] LC-MS (Method 1): R.sub.t=0.85 min; MS (ESIpos): m/z=871
(M+H).sup.+
Intermediate 183
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1-
R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S,3E)-1-phenyl-4-(4-sulphophenyl)but--
3-en-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methy-
l-L-valinamide
##STR00619##
[4589] 16.7 .mu.l (0.03 mmol) of a 15% aqueous succinaldehyde
solution were initially provided in 943 .mu.l of methanol and
admixed with 17 mg (0.02 mmol) of
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-m-
ethyl-3-oxo-3-{[(2S,3E)-1-phenyl-4-(4-sulphophenyl)but-3-en-2-yl]amino}pro-
pyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate (Intermediate 181) and 1.1 .mu.l (0.02 mmol) of
acetic acid. The reaction mixture was stirred for 5 min at RT, and
then 2.9 .mu.l (0.02 mmol) of borane-pyridine complex were added.
After 1 h, a further 2 equivalents each of succinaldehyde, acetic
acid and borane-pyridine complex were added, and the mixture was
stirred at RT for 20 h. The reaction mixture was then purified by
means of preparative HPLC.
[4590] This yielded 20 mg (83% purity, 80% of theory) of the title
compound.
[4591] LC-MS (Method 1): R.sub.t=0.87 min; MS (ESIpos): m/z=957
(M+H).sup.+
Intermediate 184
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-metho-
xy-2-methyl-3-oxo-3-{[(2S,3E)-1-phenyl-4-(4-sulphophenyl)but-3-en-2-yl]ami-
no}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamid-
e
##STR00620##
[4593] 8 mg (7.5 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S,3E)-1-phenyl-4-(4-sulphophenyl)but-
-3-en-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-meth-
yl-L-valinamide, 2.8 mg (8.2 .mu.mol) of
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide
trifluoroacetate, 3.4 mg (9 .mu.mol) of HATU and 3.9 .mu.l of
Hunig's base were stirred in 0.77 ml of DMF at RT for 20 h.
Subsequently, the reaction mixture was purified by means of
preparative HPLC.
[4594] 3 mg (31% of theory) of the title compound were
obtained.
[4595] LC-MS (Method 1): R.sub.t=0.90 min; MS (ESIpos): m/z=1164
(M+H).sup.+
Intermediate 185
N-{4-[(2,5-dioxopyrrolidin-1-yl)oxy]-4-oxobutyl}-N-methyl-L-valyl-N-[(3R,4-
S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S,3E)-1--
phenyl-4-(4-sulphophenyl)but-3-en-2-yl]amino}propyl]pyrrolidin-1-yl}-5-met-
hyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00621##
[4597] To a solution of 8 mg (7.5 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S,3E)-1-phenyl-4-(4-sulphophenyl)but-
-3-en-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-meth-
yl-L-valinamide in 2 ml of DMF were added 8.6 mg (74.8 .mu.mol) of
N-hydroxysuccinimide, 8.5 mg (22.4 .mu.mol) of EDCI and 0.1 mg
(0.75 .mu.mol) of DMAP. The reaction mixture was stirred at RT for
20 h. Subsequently, 1.3 .mu.l (7.5 .mu.mol) of Hanig's base were
added, and the mixture was stirred for another 1 h. The reaction
mixture was then purified by means of preparative HPLC. 2.6 mg (72%
purity, 21% of theory) of the title compound were obtained.
[4598] LC-MS (Method 1): R.sub.t=0.89 min; MS (ESIpos): m/z=1054
(M+H).sup.+
Intermediate 186
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-
-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2R)-1-phenyl-4-(4-sulphophenyl)but-
an-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl--
L-valinamide
##STR00622##
[4600] To a solution of 43 mg (0.06 mmol) of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide in 1.9 ml of DMF were added 29 mg
(0.07 mmol) of HATU and 33 .mu.l (0.19 mmol) of Htinig's base. The
reaction mixture was stirred at RT for 30 min and then admixed with
29 mg (0.07 mmol) of 4-[(3R)-3-amino-4-phenylbutyl]benzenesulphonic
acid trifluoroacetate. After stirring overnight, the reaction
mixture was purified by means of preparative HPLC. This yielded 58
mg of a 45:55 mixture of the title compound and of the already
deprotected amine.
[4601] LC-MS (Method 1): R.sub.t=1.09 min; MS (ESIpos): m/z=973
(M+H)+; R.sub.t=0.87 min; MS (ESIpos): m/z=873 (M+H).sup.+ for the
deprotected compound.
Intermediate 187
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-me-
thyl-3-oxo-3-{[(2R)-1-phenyl-4-(4-sulphophenyl)butan-2-yl]amino}propyl]pyr-
rolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate
##STR00623##
[4603] 58 mg of Intermediate 186 were dissolved in 4.1 ml of
dichloromethane, 0.41 ml of trifluoroacetic acid were added, and
the mixture was stirred at RT for 30 min. After concentration in
vacuo, the crude product was purified by means of preparative
HPLC.
[4604] 50 mg (90% purity, 85% of theory) of the title compound were
obtained.
[4605] LC-MS (Method 1): R.sub.t=0.87 min; MS (ESIpos): m/z=873
(M+H).sup.+
Intermediate 188
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1-
R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2R)-1-phenyl-4-(4-sulphophenyl)butan-2-
-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-va-
linamide
##STR00624##
[4607] 171 .mu.l (0.26 mmol) of a 15% aqueous succinaldehyde
solution were initially provided in 2.5 ml of methanol and admixed
with 50 mg (0.05 mmol) of
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-me-
thoxy-2-methyl-3-oxo-3-{[(2R)-1-phenyl-4-(4-sulphophenyl)butan-2-yl]amino}-
propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate and 11.6 .mu.l (0.2 mmol) of acetic acid. The
reaction mixture was stirred for 5 min at RT, and then 30 .mu.l
(0.24 mmol) of borane-pyridine complex were added. After stirring
for 24 hours, another equivalent of borane-pyridine complex was
added, and the mixture was stirred for another 2 h. The reaction
mixture was then purified by means of preparative HPLC.
[4608] 40 mg (90% purity, 66% of theory) of the title compound were
obtained.
[4609] LC-MS (Method 1): R.sub.t=0.91 min; MS (ESIpos): m/z=959
(M+H).sup.+
Intermediate 189
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-metho-
xy-2-methyl-3-oxo-3-{[(2R)-1-phenyl-4-(4-sulphophenyl)butan-2-yl]amino}pro-
pyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00625##
[4611] 10 mg (9.3 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2R)-1-phenyl-4-(4-sulphophenyl)butan--
2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-v-
alinamide, 3.5 mg (10.3 .mu.mol) of
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide
trifluoroacetate, 4.3 mg (11.2 .mu.mol) of HATU and 4.9 .mu.l (28
.mu.mol) of Hunig's base were stirred in 1 ml of DMF at RT for 20
h. Subsequently, the reaction mixture was purified by means of
preparative HPLC. 4.2 mg (92% purity, 33% of theory) of the title
compound were obtained.
[4612] LC-MS (Method 1): R.sub.t=0.91 min; MS (ESIpos): m/z=1166
(M+H).sup.+
Intermediate 190
N-{4-[(2,5-dioxopyrrolidin-1-yl)oxy]-4-oxobutyl}-N-methyl-L-valyl-N-[(3R,4-
S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2R)-1-phe-
nyl-4-(4-sulphophenyl)butan-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1--
oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00626##
[4614] To a solution of 10 mg (9.3 mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(-
1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2R)-1-phenyl-4-(4-sulphophenyl)butan--
2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-v-
alinamide in 2.5 ml of DMF were added 10.7 mg (93 .mu.mol) of
N-hydroxysuccinimide, 10.6 mg (28 .mu.mol) of EDCI and 0.12 mg (0.9
.mu.mol) of DMAP. The reaction mixture was stirred at RT for 20 h
and then purified by means of preparative HPLC.
[4615] 3.8 mg (72% purity, 25% of theory) of the title compound
were obtained.
[4616] LC-MS (Method 1): R.sub.t=0.90 min; MS (ESIpos): m/z=1055
(M+H).sup.+
Intermediate 191
(2R,3R)--N-[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]-
-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanamide
trifluoroacetate
##STR00627##
[4618] The title compound was prepared in analogy to the synthesis
of Intermediate 7 over two stages from Starting Compound 1 and
(2S)-2-amino-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)propan-1-one
trifluoroacetate (Intermediate 99).
[4619] Yield over 2 stages: 62 mg (67% of theory)
[4620] HPLC (Method 6): R.sub.t=1.65 min;
[4621] LC-MS (Method 1): R.sub.t=0.7 min; MS (ESIpos): m/z=443
(M+H).sup.+.
Intermediate 192
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-y-
l)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxop-
ropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-val-
inamide
##STR00628##
[4623] 1015 mg (1.59 mmol) of
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-car-
boxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide
(Intermediate 4) were taken up in 50 ml of DMF, admixed with 654 mg
(2.39 mmol) of 2-bromo-1-ethylpyridinium tetrafluoroborate (BEP)
and 2.8 ml of N,N-diisopropylethylamine, then stirred at RT for 10
min. Then 1083 mg (1.75 mmol) of
(2R,3R)--N-[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl-
]-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanamide
trifluoroacetate (Intermediate 191) were added, and then the
mixture was treated in an ultrasound bath at RT for 30 min. The
reaction mixture was then concentrated in vacuo, and the residue
was taken up in 300 ml of ethyl acetate. The organic phase was
washed successively with 5% aqueous citric acid solution and 5%
aqueous sodium hydrogencarbonate solution, dried over magnesium
sulphate, filtered and concentrated. The crude product thus
obtained (1684 mg) was, without further purification, taken up in
20 ml of acetonitrile, 2 ml of piperidine were added tho this, and
the reaction mixture was then stirred at RT for 10 min. Then the
mixture was concentrated in vacuo, and the residue was admixed with
diethyl ether. The solvent was again concentrated by evaporation,
and the residue was purified by flash chromatography on silica gel
(eluent: 15:1:0.1->15:2:0.2 dichloromethane/methanol/17% aqueous
ammonia solution). The corresponding fractions were combined, the
solvent was removed in vacuo, and the residue was lyophilized from
acetonitrile/water. Thus, 895 mg (67% over 2 stages) of the title
compound were obtained.
[4624] HPLC (Method 12): R.sub.t=1.8 min;
[4625] LC-MS (Method 1): R.sub.t=0.84 min; MS (ESIpos): m/z=840
(M+H).sup.+.
[4626] .sup.1H NMR (500 MHz, DMSO-d.sub.6): .delta.=10.8 (d, 1H),
8.3 and 8.05 (2d, 1H), 8.0 (d, 1H), 7.5 (m, 1H), 7.3 (m, 1H), 7.15
and 7.08 (2s, 1H) 7.05-6.9 (m, 2H), 5.12 and 4.95 (2m, 1H), 4.65
(m, 1H), 4.55 (m, 1H), 4.1-3.8 (m, 4H), 3.75 (d, 1H), 3.23, 3.18,
3.17, 3.12, 2.95 and 2.88 (6s, 9H), 3.1-3.0 and 2.85 (2m, 2H), 2.65
(d, 1H), 2.4-2.2 (m, 3H), 2.15 (m, 3H), 1.95 (br. m, 2H), 1.85-0.8
(br. m, 11H), 1.08 and 1.04 (2d, 3H), 0.9-0.75 (m, 15H), 0.75-0.65
(dd, 3H) [further signals hidden under H.sub.2O peak].
Intermediate 193
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-met-
hoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan--
4-yl]-N-methyl-L-valinamide
##STR00629##
[4628] 50 mg (0.052 mmol) of
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3--
yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxo-
propyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-va-
linamide (Intermediate 192) and 204 .mu.l einer of a 15% aqueous
solution of 4-oxobutanoic acid were combined in 2 ml of methanol
and admixed with 23.4 mg (0.252 mmol) of borane-pyridine complex
and 6 .mu.l of acetic acid. The reaction mixture was stirred at RT
overnight. This was followed by concentration in vacuo, and the
residue was purified by means of preparative HPLC. After
concentration of the corresponding fractions, 38 mg (78% of theory)
of the title compound were obtained.
[4629] HPLC (Method 5): R.sub.t=1.7 min;
[4630] LC-MS (Method 9): R.sub.t=4.7 min; MS (ESIpos): m/z=926
(M+H).sup.+.
Intermediate 194
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-in-
dol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-
-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methy-
l-L-valinamide
##STR00630##
[4632] This compound was prepared in analogy to the synthesis
described in Intermediate 157 from 10 mg (11 .mu.mol) of
N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{-
[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-me-
thoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide and commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide.
[4633] Yield: 4.4 mg (35% of theory)
[4634] HPLC (Method 5): R.sub.t=1.8 min;
[4635] LC-MS (Method 1): R.sub.t=0.90 min; MS (ESIpos): m/z=1133
(M+H).sup.+.
Intermediate 195
N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,-
4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S,3S)-1-(1,2--
oxazinan-2-yl)-1-oxo-3-phenylbutan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl-
}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00631##
[4637] This compound was prepared in analogy to Intermediate 166
starting from 9 mg (0.010 mmol) of Intermediate 170.
[4638] Yield: 1.1 mg (10% of theory)
[4639] HPLC (Method 12): R.sub.t=2.0 min;
[4640] LC-MS (Method 1): R.sub.t=0.99 min; MS (ESIpos): m/z=994
(M+H).sup.+.
Intermediate 196
(2S)-2-amino-1-(2-oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl)-3-phenylpropan-1-o-
ne trifluoroacetate
##STR00632##
[4642] 41 mg (0.37 mmol) of 2,5-dioxopyrrolidin-1-yl
N-(tert-butoxycarbonyl)-L-phenylalaninate were taken up in 10 ml of
DMF and admixed with 149 mg (0.41 mmol) of
2-oxa-3-azabicyclo[2.2.2]oct-5-ene (Starting Compound 6) and 72
.mu.l (0.41 mmol) of N,N-diisopropylethylamine. The mixture was
stirred at RT for 1 h. The solvent was then removed in vacuo, and
the residue was taken up in ethyl acetate and extracted by shaking
with 5% aqueous citric acid solution and then with 5% aqueous
sodium hydrogencarbonate solution. The organic phase was
concentrated, and the residue was purified by flash chromatography
on silica gel with 10:1 toluene/ethanol as the eluent. The
corresponding fractions were combined, and the solvent was removed
in vacuo. After the residue had been dried under high vacuum, 69 mg
(47% of theory) of the Boc-protected intermediate
tert-butyl-[(2S)-1-(2-oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl)-1-oxo-3-pheny-
lpropan-2-yl]carbamate were thus obtained as a diastereomer
mixture.
[4643] LC-MS (Method 1): R.sub.t=1.1 min; MS (ESIpos): m/z=359
(M+H).sup.+.
[4644] 64 mg (0.18 mmol) of this intermediate were taken up in 10
ml of dichloromethane, 1 ml of trifluoroacetic acid was added, and
the mixture was stirred at RT for 30 min. This was followed by
concentration in vacuo and lyophilization of the remaining residue
from water/dioxane. In this way, 66 mg (quant.) of the title
compound were obtained as a foam.
[4645] HPLC (Method 6): R.sub.t=1.45 min;
[4646] LC-MS (Method 3): R.sub.t=1.12 min; MS (ESIpos): m/z=259
(M+H).sup.+.
Intermediate 197
(2R,3R)-3-methoxy-2-methyl-N-[(2S)-1-(2-oxa-3-azabicyclo[2.2.2]oct-5-en-3--
yl)-1-oxo-3-phenylpropan-2-yl]-3-[(2S)-pyrrolidin-2-yl]propanamide
trifluoroacetate
##STR00633##
[4648] First,
(2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methy-
lpropanoic acid (Starting Compound 1) was released from 83 mg (0.18
mmol) of its dicyclohexylamine salt by taking it up in ethyl
acetate and extractive shaking with 5% aqueous potassium
hydrogensulphate solution. The organic phase was dried over
magnesium sulphate, filtered and concentrated. The residue was
taken up in 10 ml of DMF and admixed successively with 66 mg (0.18
mmol) of
(2S)-2-amino-1-(2-oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl)-3-phenylpropan-1--
one trifluoroacetate (Intermediate 196), 101 mg (0.266 mmol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) and 93 .mu.l (0.53 mmol) of
N,N-diisopropylethylamine. The mixture was stirred at RT for 30
min. The reaction mixture was then concentrated, and the residue
was purified by means of preparative HPLC. This yielded 52 mg (56%
of theory) of the Boc-protected intermediate
tert-butyl-(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(2-oxa-3-azabicy-
clo[2.2.2]oct-5-en-3-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrr-
olidine-1-carboxylate.
[4649] HPLC (Method 6): R.sub.t=2.13 min;
[4650] LC-MS (Method 1): R.sub.t=1.13 min; MS (ESIpos): m/z=528
(M+H).sup.+.
[4651] 52 mg (0.1 mmol) of this intermediate were taken up in 10 ml
of dichloromethane, 1 ml of trifluoroacetic acid was added, and the
mixture was stirred at RT for 20 min. This was followed by
concentration in vacuo and stirring of the remaining residue with
20 ml of diethyl ether. After 10 min, the mixture was filtered, and
the filter residue was dried under high vacuum. In this way, 39 mg
(72% of theory) of the title compound were obtained.
[4652] HPLC (Method 6): R.sub.t=1.62 min;
[4653] LC-MS (Method 1): R.sub.t=0.68 min; MS (ESIpos): m/z=428
(M+H).sup.+.
Intermediate 198
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-me-
thyl-3-{[(2S)-1-(2-oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl)-1-oxo-3-phenylpro-
pan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N--
methyl-L-valinamide trifluoroacetate
##STR00634##
[4655] 44.5 mg (0.071 mmol) of
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-car-
boxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide
(Intermediate 4) were taken up in 10 ml of DMF and admixed
successively with 38.6 mg (0.071 mmol) of
(2R,3R)-3-methoxy-2-methyl-N-[(2S)-1-(2-oxa-3-azabicyclo[2.2.2]oct-5-en-3-
-yl)-1-oxo-3-phenylpropan-2-yl]-3-[(2S)-pyrrolidin-2-yl]propanamide
trifluoroacetate (Intermediate 197), 32.5 mg (0.086 mmol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) and 41 .mu.l (0.235 mmol) of
N,N-diisopropylethylamine. The mixture was stirred at RT for 1 h.
The reaction mixture was then concentrated in vacuo, and the
residue was taken up in ethyl acetate. The organic phase was washed
successively with 5% aqueous citric acid solution and 5% aqueous
sodium hydrogencarbonate solution, dried over magnesium sulphate,
filtered and concentrated. This yielded 73 mg (98% of theory) of
the Fmoc-protected intermediate
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-3-met-
hoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S)-1-(2-oxa-3-azabicyclo[-
2.2.2]oct-5-en-3-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolid-
in-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide.
[4656] HPLC (Method 6): R.sub.t=2.78 min;
[4657] LC-MS (Method 3): R.sub.t=2.96 min; MS (ESIpos): m/z=1047
(M+H).sup.+.
[4658] 73 mg (0.071 mmol) of this intermediate were dissolved in 5
ml of DMF. After adding 0.5 ml of piperidine, the reaction mixture
was stirred at RT for 10 min. This was followed by concentration in
vacuo, and the residue was digested repeatedly with diethyl ether.
After the diethyl ether had been decanted off, the residue was
purified by means of preparative HPLC (eluent: acetonitrile/0.1%
aq. TFA). 16 mg (26% of theory) of the title compound were obtained
as a foam.
[4659] HPLC (Method 6): R.sub.t=1.94 min;
[4660] LC-MS (Method 3): R.sub.t=1.71 min; MS (ESIpos): m/z=825
(M+H).sup.+
[4661] .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta.=8.9-8.6 (m,
3H), 8.4, 8.3, 8.1 and 8.0 (4d, 1H), 7.3-7.1 (m, 5H), 6.7-6.5 (m,
2H), 5.2-4.8 (m, 3H), 4.75-4.55 (m, 3H), 4.05-3.95 (m, 1H), 3.7-3.4
(m, 4H), 3.22, 3.17, 3.15, 3.05, 3.02 and 2.95 (6s, 9H), 3.0 and
2.7 (2 br. m, 2H), 2.46 (m, 3H), 2.4-1.2 (br. m, 13H), 1.1-0.85 (m,
18H), 0.75 (m, 3H) [further signals hidden under H.sub.2O
peak].
Intermediate 199
N-(4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]hydrazino}-4-oxo-
butyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-metho-
xy-2-methyl-3-{[(2S)-1-(2-oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl)-1-oxo-3-ph-
enylpropan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-
-yl]-N-methyl-L-valinamide
##STR00635##
[4663] The title compound was prepared in analogy to Intermediates
193 and 194 starting from 23 mg (24 .mu.mol) of
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-m-
ethyl-3-{[(2S)-1-(2-oxa-3-azabicyclo[2.2.2]oct-5-en-3-yl)-1-oxo-3-phenylpr-
opan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-
-methyl-L-valinamide trifluoroacetate (Intermediate 198).
[4664] HPLC (Method 12): Rt=1.9 min;
[4665] LC-MS (Method 2): Rt=2.1 min; MS (ESIpos): m/z=1118
(M+H)+.
Intermediate 200
N-[2-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy)e-
thyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-ind-
ol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl--
3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-
-L-valinamide
##STR00636##
[4667] The preparation was done in analogy to Intermediates 174 and
175 starting with the reductive alkylation of Intermediate 192 with
Intermediate 172, subsequent deprotection and formation of the
maleimide.
[4668] HPLC (Method 12): Rt=1.9 min;
[4669] LC-MS (Method 1): Rt=0.86 min; MS (ESIpos): m/z=1025
(M+H)+.
Intermediate 201
N-{6-[(bromoacetyl)amino]hexyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[-
(1R,2R)-3-[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]a-
mino-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1--
oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00637##
[4671] 22 mg (0.023 mmol) of
N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2-
S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-metho-
xy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4--
yl]-N-methyl-L-valinamide (Intermediate 101) were dissolved in 9.5
ml of THF and admixed at 0.degree. C. with 4.2 .mu.l of
triethylamine. A solution of bromoacetyl chloride in THF was added
dropwise, and the reaction mixture was stirred at 0.degree. C. for
30 min. The reaction mixture was concentrated and the residue was
purified by means of preparative HPLC. Thus, 6.9 mg (26% of theory)
of the title compound were obtained as a foam.
[4672] HPLC (Method 5): R.sub.t=1.8 min;
[4673] LC-MS (Method 11): R.sub.t=0.9 min; MS (ESIpos): m/z=1059
and 1061 (M+H).sup.+.
Intermediate 202
N-{2-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropoxy}ethoxy)ethoxy]e-
thyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-ind-
ol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl--
3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-
-L-valinamide
##STR00638##
[4675] The preparation was at first done in analogy to Intermediate
168 starting with the reductive alkylation of Intermediate 192 with
Intermediate 167 and subsequent hydrogenolytic cleavage of the
benzyl ester of
N-(2-{2-[2-(2-carboxyethoxyl)ethoxy]ethoxy}ethyl)-N-methyl-L-val-
yl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxaz-
inan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolid-
in-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide.
[4676] 13 mg (10 .mu.mol) of this intermediate were dissolved in 5
ml of DMF and admixed with 2.1 mg (20 mmol) of
1-hydroxypyrrolidine-2,5-dione, 6.5 .mu.l of
N,N-diisopropylethylamine and 7.1 mg (0.02 mmol) of HATU. The
reaction mixture was stirred at RT overnight and then concentrated
in vacuo. The remaining residue was purified by means of
preparative HPLC. After lyophilization from acetonitrile/water, 9.2
mg (62% of theory) of the title compound were obtained.
[4677] HPLC (Method 12): R.sub.t=2.0 min;
[4678] LC-MS (Method 2): R.sub.t=2.1 min; MS (ESIpos): m/z=1141
(M+H).sup.+.
Intermediate 203
tert-butyl-(6-hydrazino-6-oxohexyl)carbamate
##STR00639##
[4680] This compound was prepared by standard peptide chemistry
methods by coupling of 6-[(tert-butoxycarbonyl)amino]hexanoic acid
with benzyl hydrazinecarboxylate in the presence of EDCI and HOBT
and subsequent hydrogenolytic cleavage of the benzyloxycarbonyl
protecting group.
[4681] LC-MS (Method 11): R.sub.t=0.59 min; MS (ESIpos): m/z=246
(M+H).sup.+.
Intermediate 204
N-{4-[2-(6-aminohexanoyl)hydrazino]-4-oxobutyl}-N-methyl-L-valyl-N-[(3R,4S-
,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-y-
l]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methy-
l-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate
##STR00640##
[4683] 146 mg (50 .mu.mol) of
(N-(3-carboxypropyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3--
{[(2S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methy-
l-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-meth-
yl-L-valinamide were dissolved in 5 ml of DMF and then admixed with
30.6 mg (80 .mu.mol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate, 19 .mu.l of N,N-diisopropylethylamine and with
22.4 mg (60 .mu.mol) of
tert-butyl-(6-hydrazino-6-oxohexyl)carbamate. The reaction mixture
was stirred at RT for 1.5 h. This was followed by concentration
under high vacuum and purification of the remaining residue by
means of preparative HPLC. Thus, 43 mg (68% of theory) of the
protected intermediate were obtained, which were then taken up in
10 ml of dichloromethane and deprotected with 1 ml of
trifluoroacetic acid. The reaction mixture was concentrated, and
the residue was stirred in with dichloromethane, and the solvent
was removed again in vacuo. Thus, 45 mg (68% of theory over 2
stages) of the title compound were obtained.
[4684] HPLC (Method 12): R.sub.t=1.6 min;
[4685] LC-MS (Method 11): R.sub.t=0.66 min; MS (ESIpos): m/z=983
(M+H).sup.+.
Intermediate 205
N-(4-{2-[6-({[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]carbamoyl}amin-
o)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-
-[(1R,2R)-3-{[(2S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-met-
hoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan--
4-yl]-N-methyl-L-valinamide
##STR00641##
[4687] This compound was prepared in analogy to Intermediate 114
starting from Intermediates 50 and 204.
[4688] Yield: 4 mg (78% of theory)
[4689] HPLC (Method 12): R.sub.t=1.7 min;
[4690] LC-MS (Method 11): R.sub.t=0.73 min; MS (ESIpos): m/z=1149
(M+H).sup.+.
Intermediate 206
N-(6-{[3-({3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropyl}disulphanyl)propa-
noyl]amino}hexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2-
S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-metho-
xy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4--
yl]-N-methyl-L-valinamide
##STR00642##
[4692] 8 mg (10 .mu.mol) of Intermediate 101 were dissolved in 2 ml
of DMF and admixed with 8.6 mg (20 .mu.mol) of
1,1'-{disulphanediylbis[(1-oxopropane-3,1-diyl)oxy]}dipyrrolidine-2,5-dio-
ne and 3.7 .mu.l of N,N-diisopropylethylamine. The reaction mixture
was stirred at RT for 2 h, and then the solvent was evaporated off
in vacuo, and the residue was purified by means of preparative
HPLC. 7.2 mg (68% of theory) of the title compound were
obtained.
[4693] HPLC (Method 5): R.sub.t=1.9 min;
[4694] LC-MS (Method 11): R.sub.t=0.94 min; MS (ESIpos): m/z=615
[1/2 (M+2H.sup.+]
Intermediate 207
(1S,2R)-1-amino-2-phenylcyclopropanecarboxylic acid
trifluoroacetate
##STR00643##
[4696] The title compound was obtained in quantitative yield by
deprotecting 210 mg (0.76 mmol) of commercially available
(1S,2R)-1-[(tert-butoxycarbonyl)amino]-2-phenylcyclopropanecarboxylic
acid with trifluoroacetic acid.
[4697] LC-MS (Method 1): R.sub.t=0.23 min; MS (ESIpos): m/z=178
(M+H).sup.+.
Intermediate 208
9H-fluoren-9-ylmethyl-(6-oxohexyl)carbamate
##STR00644##
[4699] The title compound was prepared from 1 g (2.95 mmol) of
commercially available
9H-fluoren-9-ylmethyl-(6-hydroxyhexyl)carbamate under standard
conditions, by oxidation with sulphur trioxide-pyridine complex.
840 mg (85% of theory) of the title compound were obtained.
[4700] HPLC (Method 12): R.sub.t=2.0 min;
[4701] LC-MS (Method 1): R.sub.t=1.1 min; MS (ESIpos): m/z=338
(M+H).sup.+.
Intermediate 209
N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,-
4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-carboxy-2-phenylcyclopropyl]amino}-
-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoh-
eptan-4-yl]-N-methyl-L-valinamide
##STR00645##
[4703] First prepared was, in analogy to the synthesis described in
Intermediate 75, by coupling of
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide (Intermediate 26) and
(1S,2R)-1-amino-2-phenylcyclopropanecarboxylic acid
trifluoroacetate (Intermediate 207) in the presence of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and the subsequent cleaving of the Boc
protecting group by means of trifluoroacetic acid, the amine
compound
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-carboxy-2-
-phenylcyclopropyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}--
3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide as the
trifluoroacetate.
[4704] To 22 mg (0.026 mmol) of this compound in 10 ml of methanol
were then added 17 mg (0.05 mmol) of
9H-fluoren-9-ylmethyl-(6-oxohexyl)carbamate (Intermediate 208) and
2.3 mg of acetic acid, and also 11.4 mg (0.12 mmol) of
borane-pyridine complex. The reaction mixture was stirred at RT
overnight. Then the same amounts of borane-pyridine complex and
acetic acid, and also 8 mg of
fluoren-9-ylmethyl-(6-oxohexyl)carbamate were added once again, and
the reaction mixture was stirred at RT for a further 24 h. This was
followed by concentration in vacuo, and the residue was purified by
means of preparative HPLC. After concentration of the corresponding
fractions, the product was used immediately in the next stage.
[4705] 33 mg of the still contaminated intermediate were taken up
in 5 ml of DMF, and 1 ml of piperidine was added. After stirring at
RT for 15 min, the reaction mixture was concentrated, and the
obtained residue was purified by means of preparative HPLC. Thus,
11 mg (55% of theory over 2 stages) of the aminocarboxylic acid
intermediate were obtained.
[4706] HPLC (Method 12): R.sub.t=1.7 min;
[4707] LC-MS (Method 11): R.sub.t=0.7 min; MS (ESIpos): m/z=843
(M+H).sup.+.
[4708] 6 mg (7.12 .mu.mol) of this intermediate were taken up in 1
ml of dioxane and then admixed with 6.6 mg (42.7 .mu.mol) of methyl
2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate and with 5 .mu.l of
saturated aqueous sodium hydrogencarbonate solution. The reaction
mixture was stirred at RT for 1 h. Then another 3 portions each of
50 .mu.l of the saturated aqueous sodium hydrogencarbonate solution
were added, and the reaction mixture was stirred at RT for another
30 min. Then the reaction mixture was acidified to pH 2 with
trifluoroacetic acid and subsequently concentrated in vacuo. The
remaining residue was purified by means of preparative HPLC. After
lyophilization from acetonitrile/water, 4 mg (60% of theory) of the
title compound were obtained as a foam.
[4709] HPLC (Method 12): R.sub.t=1.9 min;
[4710] LC-MS (Method 11): R.sub.t=0.88 min; MS (ESIpos): m/z=923
(M+H).sup.+.
Intermediate 210
N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-N-[(3R,4-
S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)--
1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3--
methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00646##
[4712] First, 6-oxohexanoic acid was prepared by a literature
method (J. Org. Chem. 58, 1993, 2196-2200).
[4713] 80 mg (0.08 mmol) of
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3--
yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxo-
propyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-va-
linamide (Intermediate 192) and 65.4 mg (0.5 mmol) of 6-oxohexanoic
acid were combined in 9 ml of methanol and admixed with 10 .mu.l of
acetic acid and 37.4 mg (0.4 mmol) of borane-pyridine complex. The
reaction mixture was stirred at RT overnight. This was followed by
concentration in vacuo, and the residue was taken up in 1:1
acetonitrile/water and adjusted to pH 2 with trifluoroacetic acid.
The reaction mixture was concentrated again, and the residue was
purified by means of preparative HPLC. After concentration of the
corresponding fractions, 70 mg (86% of theory) of
N-(5-carboxypentyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{-
[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-me-
thoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide were obtained as the
trifluoroacetate.
[4714] HPLC (Method 12): R.sub.t=1.9 min;
[4715] LC-MS (Method 1): R.sub.t=0.87 min; MS (ESIpos): m/z=955
(M+H).sup.+.
[4716] .sup.1H NMR (500 MHz, DMSO-d.sub.6, characteristic signals):
.delta.=12.0 (br. M, 1H), 10.8 (s, 1H), 9.4 (m, 1H), 8.9 and 8.8
(2d, 1H), 8.3 and 8.02 (2d, 1H), 7.5 (m, 1H), 7.3 (m, 1H), 7.15 and
7.1 (2s, 1H) 7.05-6.9 (m, 2H), 5.12 and 4.95 (2m, 1H), 4.7-4.5 (m,
2H), 4.1-3.8 (m, 4H), 3.75 (d, 1H), 3.25, 3.2, 3.18, 3.13, 2.98 and
2.88 (6s, 9H), 2.8 (m, 3H), 1.08 and 1.04 (2d, 3H), 0.95-0.8 (m,
15H), 0.8-0.65 (dd, 3H).
[4717] 22 mg (23 .mu.mol) of this intermediate were dissolved in
1.8 ml of dichloromethane and admixed with 13.2 mg (70 .mu.mol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 26.5
mg (230 .mu.mol) of 1-hydroxypyrrolidine-2,5-dione and 0.28 mg (2
.mu.mol) of dimethylaminopyridine, and the reaction mixture was
stirred at RT for 2 h. Subsequently, the reaction mixture was
concentrated in vacuo and the remaining residue was purified by
means of preparative HPLC. After lyophilization from
acetonitrile/water, 21.3 mg (88% of theory) of the title compound
were obtained.
[4718] HPLC (Method 12): R.sub.t=1.9 min;
[4719] LC-MS (Method 1): R.sub.t=0.94 min; MS (ESIpos): m/z=1052
(M+H).sup.+.
Intermediate 211
N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-N-[(3R,4-
S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S,3S)-1-(1,2-o-
xazinan-2-yl)-1-oxo-3-phenylbutan-2-yl]amino}-3-oxopropyl]pyrrolidin-1-yl}-
-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00647##
[4721] 15 mg (20 .mu.mol) of
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-m-
ethyl-3-{[(2S,3S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylbutan-2-yl]amino}-3--
oxopropyl]pyrrolidin-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate (Intermediate 15) were reductively alkylated with
6-oxohexanoic acid, in analogy to Intermediate 210.
[4722] Yield: 9.2 mg (61% of theory)
[4723] HPLC (Method 12): R.sub.t=1.9 min;
[4724] LC-MS (Method 1): R.sub.t=0.87 min; MS (ESIpos): m/z=929
(M+H).sup.+.
[4725] 9 mg (10 .mu.mol) of this intermediate were dissolved in 3
ml of DMF and admixed with 5.6 mg (48 .mu.mol) of
1-hydroxypyrrolidine-2,5-dione, 5 .mu.l of
N,N-diisopropylethylamine and 5.5 mg (0.015 mmol) of HATU, and the
reaction mixture was treated in an ultrasound bath for 6 h. In the
course of this, 5.5 mg of HATU were re-added every hour.
Subsequently, the reaction mixture was concentrated in vacuo, and
the residue was taken up in acetonitrile/water and adjusted to pH 2
with trifluoroacetic acid. After concentrating again in vacuo, the
remaining residue was purified by means of preparative HPLC. After
lyophilization from acetonitrile/water, 5.8 mg (57% of theory) of
the title compound were obtained.
[4726] HPLC (Method 12): R.sub.t=2.0 min;
[4727] LC-MS (Method 1): R.sub.t=0.95 min; MS (ESIpos): m/z=1027
(M+H)+.
Intermediate 212
N-{2-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropoxy}ethoxy)ethoxy]e-
thyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methox-
y-2-methyl-3-{[(2S,3S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylbutan-2-yl]amin-
o}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-vali-
namide
##STR00648##
[4729] The preparation was at first done in analogy to Intermediate
168 starting with the reductive alkylation of Intermediate 15 with
Intermediate 167 and subsequent hydrogenolytic cleavage of the
benzyl ester of
N-(2-{2-[2-(2-carboxyethoxyl)ethoxy]ethoxy}ethyl)-N-methyl-L-val-
yl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(2S,3-
S)-1-(1,2-oxazinan-2-yl)-1-oxo-3-phenylbutan-2-yl]amino}-3-oxopropyl]pyrro-
lidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide.
[4730] 8.4 mg (8 .mu.mol) of this intermediate were dissolved in 3
ml of DMF and admixed with 9.5 mg (80 .mu.mol) of
1-hydroxypyrrolidine-2,5-dione, 10 .mu.l of
N,N-diisopropylethylamine and 9.4 mg (25 .mu.mol) of HATU, and the
reaction mixture was stirred at RT overnight and then concentrated
in vacuo. Subsequently, the reaction mixture was concentrated in
vacuo, and the residue was taken up in acetonitrile/water and
adjusted to pH 2 with trifluoroacetic acid. After concentrating
again in vacuo, the remaining residue was purified by means of
preparative HPLC. After lyophilization from acetonitrile/water, 4
mg (32% of theory) of the title compound were obtained.
[4731] HPLC (Method 12): R.sub.t=2.0 min;
[4732] LC-MS (Method 1): R.sub.t=0.96 min; MS (ESIpos): m/z=1117
(M+H).sup.+.
Intermediate 213
N-{6-[(trans-4-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}cyclohexyl)amino]--
6-oxohexyl}-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(-
1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-m-
ethyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N--
methyl-L-valinamide
##STR00649##
[4734] This compound was prepared in analogy to Intermediate 104
starting from
N-(5-carboxypentyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R-
)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-
-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoh-
eptan-4-yl]-N-methyl-L-valinamide, the synthesis of which was
described under Intermediate 210. 9.3 mg of the title compound (37%
of theory over 3 stages) were obtained.
[4735] HPLC (Method 12): R.sub.t=1.9 min;
[4736] LC-MS (Method 1): R.sub.t=0.9 min; MS (ESIpos): m/z=1177
(M+H).
Intermediate 214
N-{4-[(2,5-dioxopyrrolidin-1-yl)oxy]-4-oxobutyl}-N-methyl-L-valyl-N-[(3R,4-
S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}--
1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxohe-
ptan-4-yl]-N-methyl-L-valinamide
##STR00650##
[4738] This compound was prepared in analogy to Intermediate 210 by
conversion of Intermediate 92 to the active ester.
[4739] HPLC (Method 5): R.sub.t=1.6 min;
[4740] LC-MS (Method 11): R.sub.t=0.82 min; MS (ESIpos): m/z=901
(M+H).sup.+.
Intermediate 215
N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-N-[(3R,4-
S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}--
1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxohe-
ptan-4-yl]-N-methyl-L-valinamide
##STR00651##
[4742] First, from Intermediate 40, in analogy to Intermediate 183
with borane-pyridine complex, was prepared
N-(5-carboxypentyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{-
[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxoprop-
yl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valina-
mide. From this compound, in analogy to Intermediate 210, the
active ester was then generated. 34 mg (36% of theory over 2
stages) of the title compound were obtained.
[4743] HPLC (Method 5): R.sub.t=1.6 min;
[4744] LC-MS (Method 1): R.sub.t=0.85 min; MS (ESIpos): m/z=930
(M+H).sup.+.
Intermediate 216
N-(4-{[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}benzyl)-N-methyl-L-valyl-N-[-
(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-
-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-y-
l}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00652##
[4746] First, in analogy to the preparation of Intermediate 183,
Intermediate 192 was reacted with 4-formylbenzoic acid with
borane-pyridine complex to yield
N-(4-carboxybenzyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{-
[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-me-
thoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide. This compound was then used, in
analogy to Intermediate 210, to generate 11 mg (68% of theory) of
the title compound.
[4747] HPLC (Method 5): R.sub.t=1.8 min;
[4748] LC-MS (Method 1): R.sub.t=1.13 min; MS (ESIpos): m/z=1072
(M+H).sup.+.
Intermediate 217
N-(5-carboxypentyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
(2S)-1-(benzyloxy)-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-o-
xopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L--
valinamide
##STR00653##
[4750] 53 mg (84 .mu.mol) of
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(2R,3S,4S)-1-car-
boxy-2-methoxy-4-methylhexan-3-yl]-N-methyl-L-valinamide
(Intermediate 4) and 45 mg (84 .mu.mol) of
benzyl-N-{(2R,3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoyl}-L-
-phenylalaninate trifluoroacetate (Intermediate 12) were taken up
in 2 ml of DMF, 19 .mu.l of N,N-diisopropylethylamine, 14 mg (92
.mu.mol) of HOBt and 17.6 mg (92 .mu.mol) of EDC were added, and
then the mixture was stirred at RT overnight. Subsequently, the
reaction mixture was concentrated and the residue was purified by
means of preparative HPLC. This yielded 59 mg (68% of theory) of
the Fmoc-protected intermediate
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2-
S)-2-[(1R,2R)-3-{[(2S)-1-(benzyloxy)-1-oxo-3-phenylpropan-2-yl]amino}-1-me-
thoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide.
[4751] LC-MS (Method 1): R.sub.t=1.55 min; m/z=1044
(M+H).sup.+.
[4752] 57 mg (0.055 mmol) of this intermediate were treated with
1.2 ml of piperidine in 5 ml of DMF to cleave the Fmoc protecting
group. After concentration and purification by means of preparative
HPLC, 39 mg (76% of theory) of the free amine intermediate
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzyloxy)--
1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-
-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
were obtained as the trifluoroacetate.
[4753] HPLC (Method 5): R.sub.t=1.9 min;
[4754] LC-MS (Method 1): R.sub.t=1.01 min; m/z=822 (M+H).sup.+.
[4755] 60 mg (0.06 mmol) of this intermediate were reacted, in
analogy to Intermediate 210, with 6-oxohexanoic acid in the
presence of borane-pyridine complex. 45 mg (75% of theory) of the
title compound were obtained as a foam.
[4756] HPLC (Method 5): R.sub.t=1.9 min;
[4757] LC-MS (Method 1): R.sub.t=0.97 min; MS (ESIpos): m/z=9936
(M+H).sup.+.
Intermediate 218
N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-N-[(3R,4-
S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzyloxy)-1-oxo-3-phenylpropan-2-yl]-
amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl--
1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00654##
[4759] This compound was prepared by conversion of 42 mg (0.05
mmol) of Intermediate 217 to the active ester.
[4760] Yield: 26 mg (54%)
[4761] HPLC (Method 5): R.sub.t=2.1 min;
[4762] LC-MS (Method 1): R.sub.t=1.01 min; MS (ESIpos): m/z=1034
(M+H).sup.+.
Intermediate 219
N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-N-[(3R,4-
S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-
-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl-
]-N-methyl-L-valinamide
##STR00655##
[4764] 20 mg (0.02 mol) of the compound from Intermediate 218 were
taken up in 2.4 ml of methanol and hydrogenated over 5% palladium
on activated carbon under standard hydrogen pressure at RT for 30
min. The catalyst was then filtered off, and the solvent was
removed in vacuo. The residue was lyophilized from 1:1
acetonitrile/water. This yielded 14 mg (92% of theory) of the title
compound as a colourless foam.
[4765] HPLC (Method 5): R.sub.t=1.7 min;
[4766] LC-MS (Method 1): R.sub.t=0.86 min; MS (ESIpos): m/z=944
(M+H).sup.+.
Intermediate 220
N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazina-
n-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin--
1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00656##
[4768] 0.5 g (1.01 mmol) of Intermediate 1 were admixed in 10 ml of
dichloromethane with 1 ml of trifluoroacetic acid. After treatment
in an ultrasound bath for 30 min, the batch was concentrated and
redistilled first with DCM and then with diethyl ether, then dried
under high vacuum. The oily residue was used without further
purification in the next stage.
[4769] 500 mg of this intermediate were dissolved in 20 ml of DMF
and admixed with 466 mg (3.8 mmol) of Intermediate 191, 382 mg
(1.01 mmol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) and 440 .mu.l (2.5 mmol) of
N,N-diisopropylethylamine. The mixture was stirred at RT for 1 h
and then concentrated. The residue was taken up in dichloromethane
and extracted by shaking first twice with 5% aqueous citric acid
solution and then with saturated aqueous sodium hydrogencarbonate
solution. The organic phase was concentrated, and the residue was
purified by flash chromatography on silica gel with 95:5
dichloromethane/methanol as the eluent. The corresponding fractions
were combined, and the solvent was removed in vacuo. After the
residue had been dried under high vacuum, 562 mg (65% of theory
over both stages) of the Z-protected intermediate were
obtained.
[4770] 562 mg (0.57 mmol) of this intermediate were taken up in 50
ml of methanol and hydrogenated with 155 mg of 10% palladium on
activated carbon under standard hydrogen pressure at RT for 20 min.
The catalyst was then filtered off, and the solvent was removed in
vacuo. The residue was purified by means of preparative HPLC. The
corresponding fractions were combined, the solvent was evaporated
in vacuo, and the residue was lyophilized from dioxane. This
yielded 361 mg (87% of theory) of the title compound as a foam.
[4771] HPLC (Method 5): double peak with Rt=1.75 and 1.86 min;
[4772] LC-MS (Method 1): double peak at Rt=0.84 min and 0.91 min
with the same mass; MS (ESIpos): m/z=944 (M+H).sup.+.
Intermediate 221
N-{(2S)-2-[(tert-butoxycarbonyl)amino]-3-phenylpropyl}-N-methyl-L-valine
##STR00657##
[4774] 100 mg (0.76 mmol) of commercially available
N-methyl-L-valine and 285 mg (1.14 mmol) of commercially available
tert-butyl (2S)-1-oxo-3-phenylpropan-2-yl carbamate were combined
in 22 ml of methanol and admixed with 340 mg (3.66 mmol) of
borane-pyridine complex and 70 .mu.l of acetic acid. The reaction
mixture was stirred at RT overnight. This was followed by
concentration in vacuo, and the residue was purified by flash
chromatography on silica gel with dichloromethane/methanol/17%
aqueous ammonia solution as the eluent. After concentration of the
corresponding fractions and lyophilization from 1:1 dioxane/water,
259 mg (93% of theory) of the title compound were obtained.
[4775] HPLC (Method 12): R.sub.t=1.6 min;
[4776] LC-MS (Method 11): R.sub.t=0.76 min; MS (ESIpos): m/z=365
(M+H).sup.+.
Intermediate 222
N-[(2S)-2-amino-3-phenylpropyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[-
(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]-
amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl--
1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate
##STR00658##
[4778] 40 mg (0.11 mmol) of
N-{(2S)-2-[(tert-butoxycarbonyl)amino]-3-phenylpropyl}-N-methyl-L-valine
(Intermediate 221) were dissolved in 5 ml of DMF and admixed with
80 mg (0.11 mmol) of
N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazin-
an-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-
-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
(Intermediate 220), 50 mg (0.13 mmol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) and 57 .mu.l (2.5 mmol) of
N,N-diisopropylethylamine. The mixture was stirred at RT for 1 h
and then concentrated. The residue was taken up in ethyl acetate
and washed first with 5% aqueous citric acid solution and then with
water. The organic phase was concentrated, and the residue was
purified by means of preparative HPLC. The corresponding fractions
were combined, and the solvent was removed in vacuo. After
lyophilization from dioxane, 60 mg (50% of theory) of the protected
intermediate were obtained.
[4779] HPLC (Method 12): R.sub.t=2.2 min;
[4780] LC-MS (Method 1): R.sub.t=1.17 min; MS (ESIpos): m/z=1073
(M+H).sup.+.
[4781] 60 mg (0.05 mmol) of this intermediate were taken up in 10
ml of dichloromethane, 2 ml of trifluoroacetic acid were added, and
the reaction mixture was stirred at RT for 1.5 h. Subsequently, the
reaction mixture was concentrated in vacuo, and the remaining
residue was purified by means of preparative HPLC. The
corresponding fractions were combined, the solvent was removed in
vacuo, and the residue was lyophilized from dioxane/water. In this
way, 25 mg (42% of theory) of the title compound were obtained as a
foam.
[4782] HPLC (Method 12): R.sub.t=1.9 min;
[4783] LC-MS (Method 1): R.sub.t=0.95 min; MS (ESIpos): m/z=974
(M+H).sup.+.
Intermediate 223
N-[(2S)-2-({[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]carbamoyl}amino-
)-3-phenylpropyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2-
S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-metho-
xy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4--
yl]-N-methyl-L-valinamide
##STR00659##
[4785] The preparation was done in analogy to Intermediate 134
starting from 5 mg (4.6 .mu.mol) of Intermediate 222. 3.4 mg (65%
of theory) of the title compound were obtained.
[4786] HPLC (Method 12): R.sub.t=2.0 min;
[4787] LC-MS (Method 1): R.sub.t=0.99 min; MS (ESIpos): m/z=1140
(M+H).sup.+.
Intermediate 224
N-[(2S)-2-({[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]carbamoyl}amino-
)propyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H--
indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-meth-
yl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-met-
hyl-L-valinamide
##STR00660##
[4789] The preparation was done in analogy to the synthesis of
Intermediate 223.
[4790] HPLC (Method 12): R.sub.t=1.9 min;
[4791] LC-MS (Method 1): R.sub.t=0.92 min; MS (ESIpos): m/z=1064
(M+H).sup.+.
Intermediate 225
N-(2-aminoethyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S-
)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methox-
y-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-y-
l]-N-methyl-L-valinamide trifluoroacetate
##STR00661##
[4793] 100 mg (0.76 mmol) of commercially available
N-methyl-L-valine and 182 mg (1.14 mmol) of commercially available
tert-butyl 2-oxoethyl carbamate were combined in 20 ml of methanol
and admixed with 340 mg (3.66 mmol) of borane-pyridine complex and
65 .mu.l of acetic acid. The reaction mixture was stirred at RT
overnight. This was followed by concentration under reduced
pressure, and the residue was purified by flash chromatography on
silica gel with dichloromethane/methanol/17% aqueous ammonia
solution (15/4/0.5) as the eluent. After concentration of the
corresponding fractions and lyophilization from 1:1 dioxane/water,
190 mg in 39% purity (35% of theory) of the intermediate were
obtained, which were converted further without further
purification.
[4794] 50 mg (0.07 mmol) of this intermediate were dissolved in 10
ml of DMF and admixed with 52 mg (0.07 mmol) of
N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazin-
an-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-
-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
(Intermediate 220), 32 mg (0.09 mmol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) and 37 .mu.l (0.2 mmol) of
N,N-diisopropylethylamine. The mixture was stirred at RT overnight
and then concentrated. The residue was taken up in ethyl acetate
and extracted by shaking first with 5% aqueous citric acid solution
and then with water. The organic phase was concentrated and the
residue was purified by means of preparative HPLC. The
corresponding fractions were combined, and the solvent was removed
in vacuo. After lyophilization from dioxane, 53 mg (76% of theory)
of the protected intermediate were obtained.
[4795] HPLC (Method 12): R.sub.t=2.0 min;
[4796] LC-MS (Method 1): R.sub.t=1.02 min; MS (ESIpos): m/z=984
(M+H).sup.+.
[4797] 53 mg (0.05 mmol) of this intermediate were taken up in 10
ml of dichloromethane, 2 ml of trifluoroacetic acid were added, and
the reaction mixture was stirred at RT for 30 min. Subsequently,
the reaction mixture was concentrated in vacuo and the remaining
residue was purified by means of preparative HPLC. The
corresponding fractions were combined, the solvent was removed in
vacuo, and the residue was lyophilized from dioxane/water. In this
way, 21 mg (40% of theory) of the title compound were obtained with
65% purity.
[4798] HPLC (Method 12): R.sub.t=1.7 min;
[4799] LC-MS (Method 1): R.sub.t=0.87 min; MS (ESIpos): m/z=884
(M+H).sup.+.
Intermediate 226
N-[2-({[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]carbamoyl}amino)ethy-
l]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol--
3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-o-
xopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L--
valinamide
##STR00662##
[4801] The preparation was done starting from Intermediate 225 in
analogy to the synthesis of Intermediate 134. 11.6 mg (59% of
theory) of the title compound were obtained.
[4802] HPLC (Method 12): R.sub.t=1.9 min;
[4803] LC-MS (Method 1): R.sub.t=0.90 min; MS (ESIpos): m/z=1050
(M+H).sup.+.
Intermediate 227
N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-N-[(3R,4-
S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(benzyloxy)-3-(1H-indol-3-yl)-1-oxopro-
pan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy--
5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00663##
[4805] This compound was prepared analogously to Intermediate 218
by conversion to the active ester.
[4806] Yield: 18 mg (51% of theory)
[4807] HPLC (Method 5): R.sub.t=2.1 min;
[4808] LC-MS (Method 1): R.sub.t=0.98 min; MS (ESIpos): m/z=1073
(M+H).sup.+.
Intermediate 228
(2R,3S)-3-[(tert-butoxycarbonyl)amino]-4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-p-
yrrol-1-yl)hexanoyl]hydrazino}-4-oxobutan-2-yl(3R,4S,7S,10S)-4-[(2S)-butan-
-2-yl]-7,10-diisopropyl-3-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2-
R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]py-
rrolidin-1-yl}-2-oxoethyl)-5,11-dimethyl-6,9-dioxo-2-oxa-5,8,11-triazapent-
adecan-15-oate
##STR00664##
[4810] The title compound was prepared by coupling the
Boc-protected intermediate obtained from the synthesis of
Intermediate 154 with commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide.
[4811] HPLC (Method 12): R.sub.t=2.1 min;
[4812] LC-MS (Method 1): R.sub.t=0.97 min; MS (ESIpos): m/z=1308
(M+H).sup.+.
Intermediate 229
(2R,3S)-3-acetamido-4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl-
]hydrazino}-4-oxobutan-2-yl
(3R,4S,7S,10S)-4-[(2S)-butan-2-yl]-7,10-diisopropyl-3-(2-{(2S)-2-[(1R,2R)-
-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarbonyl)-2-phenylcycl-
opropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-5,11-dimethyl-6,9--
dioxo-2-oxa-5,8,11-triazapentadecan-15-oate
##STR00665##
[4814] The title compound was prepared from 7.5 mg (2.5 .mu.mol) of
Intermediate 154 by acetylation with 2.3 .mu.l of acetic anhydride
in 1 ml of DMF in the presence of 0.4 .mu.l of
N,N-diisopropylethylamine.
[4815] Yield: 1.4 mg (40% of theory)
[4816] HPLC (Method 12): R.sub.t=1.9 min;
[4817] LC-MS (Method 1): R.sub.t=0.86 min; MS (ESIpos): m/z=1250
(M+H).sup.+.
Intermediate 230
(2R,3S)-3-[(tert-butoxycarbonyl)amino]-4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-p-
yrrol-1-yl)hexanoyl]hydrazino}-4-oxobutan-2-yl
(3R,4S,7S,10S)-4-[(2S)-butan-2-yl]-3-(2-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-i-
ndol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methy-
l-3-oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl--
6,9-dioxo-2-oxa-5,8,11-triazapentadecan-15-oate
##STR00666##
[4819] This compound was prepared in analogy to Intermediate 228
starting from Intermediate 193. 16 mg (30% of theory over 3 stages)
of the title compound were obtained.
[4820] HPLC (Method 12): R.sub.t=2.0 min;
[4821] LC-MS (Method 1): R.sub.t=1.02 min; MS (ESIpos): m/z=1335
(M+H).sup.+.
Intermediate 231
(2R,3S)-3-acetamido-4-{2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl-
]hydrazino}-4-oxobutan-2-yl
(3R,4S,7S,10S)-4-[(2S)-butan-2-yl]-3-(2-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-i-
ndol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methy-
l-3-oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl--
6,9-dioxo-2-oxa-5,8,11-triazapentadecan-15-oate
##STR00667##
[4823] This compound was prepared from 8 mg (6 .mu.mol) of
Intermediate 230, first by deprotection with trifluoroacetic acid
and subsequent acetylation with acetic anhydride in DMF in the
presence of N,N-diisopropylethylamine. 2 mg (37% of theory over 2
stages) of the title compound were obtained.
[4824] HPLC (Method 12): R.sub.t=1.9 min;
[4825] LC-MS (Method 1): R.sub.t=0.88 min; MS (ESIpos): m/z=1277
(M+H).sup.+.
Intermediate 232
Benzyl-N-[(4-nitrophenoxy)carbonyl]-beta-alaninate
##STR00668##
[4827] 200 mg (0.57 mmol) of commercially available
4-methylbenzenesulphonic acid benzyl beta-alaninate and 229 mg
(1.14 mmol) of 4-nitrophenyl chlorocarbonate were taken up in 15 ml
of tetrahydrofuran, and the reaction mixture was then heated to
reflux for 30 min. Subsequently, the reaction mixture was
concentrated in vacuo, and the residue was purified by means of
preparative HPLC. After concentration of the corresponding
fractions and drying of the residue under high vacuum, 86 mg (44%
of theory) of the title compound were obtained.
[4828] HPLC (Method 12): R.sub.t=1.8 min;
[4829] LC-MS (Method 1): R.sub.t=1.07 min; MS (ESIpos): m/z=345
(M+H).sup.+.
Intermediate 233
N-{2-[({3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropyl}carbamoyl)amino]ethy-
l}-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol--
3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-o-
xopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L--
valinamide
##STR00669##
[4831] 13 mg (10 .mu.mol) of Intermediate 225 and 6.7 mg (20
.mu.mol) of Intermediate 232 were dissolved in 3 ml of DMF, and
then 7 .mu.l of N,N-diisopropylethylamine were added. The mixture
was stirred at RT overnight and then concentrated under high
vacuum. The remaining residue was purified by means of preparative
HPLC. After concentration of the corresponding fractions and drying
of the residue under high vacuum, 5.4 mg (38% of theory) of the
protected intermediate were obtained.
[4832] HPLC (Method 5): R.sub.t=2.1 min;
[4833] LC-MS (Method 1): R.sub.t=0.6 in; MS (ESIpos): m/z=1089
(M+H).sup.+.
[4834] 5.4 mg (5 .mu.mol) of this intermediate were dissolved in 5
ml of methanol and, after adding 2 mg of 10% palladium on activated
carbon, hydrogenated under standard hydrogen pressure at RT for 20
min. The catalyst was then filtered off, and the solvent was
removed in vacuo. After drying of he residue under high vacuum, 5
mg (quant.) of the acid intermediate were obtained.
[4835] HPLC (Method 12): R.sub.t=1.8 min;
[4836] LC-MS (Method 1): R.sub.t=0.84 min; MS (ESIpos): m/z=999
(M+H).sup.+.
[4837] 5 mg (10 .mu.mol) of this intermediate were dissolved in 1
ml of DMF and admixed with 5.8 mg (50 mmol) of
1-hydroxypyrrolidine-2,5-dione and then with 2.6 .mu.l of
N,N-diisopropylethylamine and 3.8 mg (10 .mu.mol) of HATU. After
stirring at RT for 20 h, the reaction mixture was concentrated in
vacuo. The remaining residue was purified by means of preparative
HPLC. After lyophilization from 1:1 dioxane/water, 1.1 mg (20% of
theory) of the title compound were obtained.
[4838] HPLC (Method 12): R.sub.t=1.9 min;
[4839] LC-MS (Method 1): R.sub.t=0.87 min; MS (ESIpos): m/z=1096
(M+H).sup.+.
Intermediate 234
N-(6-{[(benzyloxy)carbonyl]amino}hexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-m-
ethoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(5--
phenyl-1,3,4-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-
-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00670##
[4841] 25 mg (30 .mu.mol) of
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-m-
ethyl-3-oxo-3-{[(1S)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]amino-
}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
(Intermediate 55) and 45 mg (180 .mu.mol) of
benzyl-(6-oxohexyl)carbamate were taken up in 3 ml of methanol and
acidified with acetic acid. At room temperature, 15 .mu.l (144
.mu.mol; 9.4M) of borane-pyridine complex were subsequently added.
The batch was subsequently stirred at RT for 24 h, and acetic acid
and 15 .mu.l (144 .mu.mol; 9.4M) of borane-pyridine complex were
added again after 8 h. The reaction mixture was subsequently
adjusted to pH 2 with TFA and purified by means of preparative
HPLC. The product fractions were combined and concentrated, and the
residue was dried under high vacuum. This gave 15 mg (46% of
theory) of the title compound as a foam.
[4842] LC-MS (Method 1): R.sub.t=1.03 min; m/z=1066
(M+H).sup.+.
Intermediate 235
N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2-
R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol--
2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-meth-
yl-L-valinamide
##STR00671##
[4844] 15 mg (14 .mu.mol) of
N-(6-{[(benzyloxy)carbonyl]amino}hexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3--
methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(5-
-phenyl-1,3,4-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl--
1-oxoheptan-4-yl]-N-methyl-L-valinamide (Intermediate 234) were
taken up in 3 ml of methanol, and 1.8 mg of palladium on carbon
(5%) were added. The reaction mixture was subsequently hydrogenated
under standard hydrogen pressure at RT for 2 h. The catalyst was
then filtered off, and the solvent was removed in vacuo. The
residue was lyophilized from 1:1 acetonitrile/water. 11 mg (86% of
theory) of the title compound were obtained as a foam.
[4845] LC-MS (Method 1): R.sub.t=0.81 min; m/z=932 (M+H).sup.+.
Intermediate 236
N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,-
4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-ph-
enyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}--
5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00672##
[4847] 11 mg (12 .mu.mol) of
N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,-
2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-
-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-met-
hyl-L-valinamide (Intermediate 235) were taken up in 500 .mu.l of
1:1 dioxane/water and admixed with 253 .mu.l of 1M aqueous sodium
hydrogencarbonate solution and then with 2.8 mg (18 .mu.mol) of
methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate. The reaction
mixture was stirred at RT for 30 min and then acidified with
trifluoroacetic acid. The reaction mixture was purified by means of
preparative HPLC. After lyophilization, 0.8 mg (7% of theory) of
the title compound was obtained.
[4848] LC-MS (Method 1): R.sub.t=1.01 min; m/z=1012
(M+H).sup.+.
Intermediate 237
N-(5-carboxypentyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1-
R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(5-phenyl-1,3,4-oxadiaz-
ol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-m-
ethyl-L-valinamide
##STR00673##
[4850] 25 mg (30 .mu.mol) of
N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-m-
ethyl-3-oxo-3-{[(1S)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]amino-
}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
(Intermediate 55) and 23 mg (180 .mu.mol) of 6-oxohexanoic acid
were taken up in 3 ml of methanol and acidified with acetic acid.
At room temperature, 15 .mu.l (144 .mu.mol; 9.4M) of
borane-pyridine complex were subsequently added. The reaction
mixture was subsequently stirred at RT for 20 h, and acetic acid
and 15 .mu.l (144 .mu.mol; 9.4M) of borane-pyridine complex were
added again after 8 h. The reaction mixture was subsequently
adjusted to pH 2 with trifluoroacetic acid and purified by means of
preparative HPLC. The product fractions were combined and
concentrated, and the residue was lyophilized. 21 mg (74% of
theory) of the title compound were thus obtained as a foam.
[4851] LC-MS (Method 1): R.sub.t=0.91 min; m/z=947 (M+H).sup.+.
Intermediate 238
N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-N-[(3R,4-
S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phe-
nyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-
-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00674##
[4853] 21 mg (22 .mu.mol) of Intermediate 237 were dissolved in 1
ml of DMF and admixed with 38 mg (333 .mu.mol) of
1-hydroxypyrrolidine-2,5-dione and then with 2.4 mg (10 .mu.mol) of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) and 19 .mu.l of
N,N-diisopropylethylamine. After stirring at RT for 2 h, the
reaction mixture was purified by means of preparative HPLC. After
lyophilization from dioxane, 22 mg (96% of theory) of the title
compound were obtained.
[4854] LC-MS (Method 1): R.sub.t=0.95 min; m/z=1044
(M+H).sup.+.
Intermediate 239
N-methyl-L-threonyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol--
3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-o-
xopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L--
valinamide trifluoroacetate
##STR00675##
[4856] First, N-[(benzyloxy)carbonyl]-N-methyl-L-threonine was
released from 237 mg (0.887 mmol) of its dicyclohexylamine salt by
taking it up in ethyl acetate and extractive shaking with 5%
aqueous sulphuric acid. The organic phase was dried over magnesium
sulphate, filtered and concentrated. 14.7 mg (0.055 mmol) of
N-[(benzyloxy)carbonyl]-N-methyl-L-threonine were taken up in 3 ml
of DMF and admixed successively with 40 mg (0.055 mmol) of
Intermediate 220, 12.7 mg (0.066 mmol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 10
mg (0.066 mmol) of 1-hydroxy-1H-benzotriazole hydrate. The mixture
was subsequently stirred at RT for 2 h. The solvent was then
removed in vacuo, and the residue purified by means of preparative
HPLC. 29 mg (54% of theory) of the Z-protected intermediate were
thus obtained.
[4857] LC-MS (Method 1): R.sub.t=1.15 min; MS (ESIpos): m/z=976
(M+H).sup.+.
[4858] 29 mg (0.003 mmol) of this intermediate were dissolved in 5
ml of methanol and hydrogenated over 5 mg of 5% palladium/carbon at
RT and standard pressure for 1 h. The catalyst was subsequently
filtered off and the solvent evaporated. The remaining residue was
purified by means of preparative HPLC. 17 mg (54% of theory) of the
title compound were obtained.
[4859] LC-MS (Method 1): R.sub.t=0.77 min; MS (ESIpos): m/z=842
(M+H).sup.+.
Intermediate 240
N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-threonyl-N-[(3-
R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan-2-y-
l)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-
-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00676##
[4861] This compound was prepared in analogy to Intermediate 210
from 15.6 mg (0.016 mmol) of Intermediate 239. 10.8 mg (67% of
theory over 2 stages) of the title compound were obtained.
[4862] HPLC (Method 5): R.sub.t=1.7 min;
[4863] LC-MS (Method 1): R.sub.t=0.85 min; MS (ESIpos): m/z=1053
(M+H).sup.+.
Intermediate 241
N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(4-hydroxyphe-
nyl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-ox-
opropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-v-
alinamide trifluoroacetate
##STR00677##
[4865] First, in analogy to Intermediate 5, trifluoroacetic
acid-(2S)-2-amino-3-(4-hydroxyphenyl)-1-(1,2-oxazinan-2-yl)propan-1-one
(1:1) was prepared. This component was then used to obtain the
title compound, in analogy to the synthesis described in
Intermediate 75, by coupling with
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide (Intermediate 26) in the presence of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and subsequent cleaving of the Boc protecting
group by means of trifluoroacetic acid.
[4866] HPLC (Method 12): R.sub.t=1.7 min;
[4867] LC-MS (Method 1): R.sub.t=0.85 min; MS (ESIpos): m/z=817
(M+H).sup.+.
Intermediate 242
N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-N-[(3R,4-
S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(4-hydroxyphenyl)-1-(1,2-oxazinan-2-yl-
)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}--
3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00678##
[4869] 50 mg (0.05 mmol) of Intermediate 241 were reacted, in
analogy to Intermediate 210, with 6-oxohexanoic acid in the
presence of borane-pyridine complex. Subsequently, 22.5 mg (0.02
mmol) of the obtained acid were converted to the activated ester.
13.5 mg (36% of theory over 2 stages) of the title compound were
obtained.
[4870] HPLC (Method 12): R.sub.t=1.8 in;
[4871] LC-MS (Method 1): R.sub.t=0.86 min; MS (ESIpos): m/z=1028
(M+H).sup.+.
Intermediate 243
N-(6-aminohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S-
)-3-(4-hydroxyphenyl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-meth-
oxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1l-oxoheptan--
4-yl]-N-methyl-L-valinamide
##STR00679##
[4873] The preparation was done, in analogy to Intermediate 78, by
reductive alkylation of Intermediate 241 with
benzyl-(6-oxohexyl)carbamate and borane-pyridine complex and
subsequent hydrogenation in methanol as the solvent.
[4874] Yield: 17.5 mg (34% of theory over 2 stages)
[4875] HPLC (Method 12): R.sub.t=1.7 min;
[4876] LC-MS (Method 1): R.sub.t=0.63 min; MS (ESIpos): m/z=916
(M+H).sup.+.
Intermediate 244
N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl]-N-methyl-L-valyl-N-[(3R,-
4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(4-hydroxyphenyl)-1-(1,2-oxazinan-2-y-
l)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-
-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00680##
[4878] The preparation was done in analogy to Intermediate 166
starting from Intermediate 243.
[4879] Yield: 1.3 mg (12% of theory)
[4880] HPLC (Method 12): R.sub.t=1.9 min;
[4881] LC-MS (Method 1): R.sub.t=0.89 min; MS (ESIpos): m/z=996
(M+H).sup.+.
Intermediate 245
2,5-dioxopyrrolidin-1-yl
O-[(3R,4S,7S,10S)-4-[(2S)-butan-2-yl]-3-(2-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1-
H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-me-
thyl-3-oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-7,10-diisopropyl-5,11-dimeth-
yl-6,9,15-trioxo-2-oxa-5,8,11-triazapentadecan-15-yl]-Nert-butoxycarbonyl)-
-L-threonyl-beta-alaninate
##STR00681##
[4883] First, Intermediate 193, as described for Intermediate 154,
was reacted with benzyl N-(tert-butoxycarbonyl)-L-threoninate, and
then the benzyl ester was removed by hydrogenolytic means. 30 mg
(0.027 mmol) of the thus obtained
N-[4-({(1S,2R)-1-[(tert-butoxycarbonyl)amino]-1-carboxypropan-2-yl}oxy)-4-
-oxobutyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1-
H-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-me-
thyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-m-
ethyl-L-valinamide were then coupled with 4-methylbenzenesulphonic
acid benzyl-beta-alaninate in the presence of HATU, and the benzyl
ester was removed again by hydrogenolysis (yield: 24 mg (71% of
theory over 2 stages)). Finally, 10 mg (0.008 mmol) of the obtained
acid were converted to the activated ester. After HPLC
purification, 2.7 mg (23% of theory) of the title compound were
obtained.
[4884] HPLC (Method 5): R.sub.t=1.9 min;
[4885] LC-MS (Method 1): R.sub.t=1.01 min; MS (ESIpos): m/z=1295
(M+H).sup.+
Intermediate 246a
(2S)-2-amino-1-(4-hydroxy-1,2-oxazolidin-2-yl)-3-(1H-indol-3-yl)propan-1-o-
ne trifluoroacetate (Diastereomer 1)
##STR00682##
[4887] 1.6 g (3.982 mmol) of 2,5-dioxopyrrolidin-1-yl
N-(tert-butoxycarbonyl)-L-tryptophanate were dissolved in 15 ml of
DMF and admixed with 500 mg (3.982 mmol) of 1,2-oxazolidin-4-ol and
100 .mu.l of N,N-diisopropylethylamine. The reaction mixture was
stirred at RT overnight. Then another 100 .mu.l of
N,N-diisopropylethylamine were added, and the mixture was first
treated in an ultrasound bath for 5 h, then stirred at RT overnight
and subsequently concentrated in vacuo. The remaining residue was
taken up in ethyl acetate and extracted first twice with 5% aqueous
citric acid solution, then with saturated aqueous sodium
hydrogencarbonate solution and finally with water. The organic
phase was concentrated and the residue separated into the
diastereomers by means of flash chromatography on silica gel with
95:5 dichloromethane/methanol as the eluent. The corresponding
fractions of both diastereomers were combined and the solvent was
removed in vacuo. After drying of the residues under high vacuum,
272 mg (18% of theory) of Diastereomer 1 (R.sub.f=0.18 (95:5
dichloromethane/methanol) and 236 mg (16% of theory) of
Diastereomer 2 (R.sub.f=0.13 (95:5 dichloromethane/methanol) as
wells as 333 mg (22% of theory) of a mixed fraction of the
Boc-protected intermediates were obtained.
[4888] 5 ml of trifluoroacetic acid in 20 ml of dichloromethane
were used under standard conditions for cleaving the Boc protecting
group from 272 mg (725 .mu.mol) of Diastereomer 1 of this
intermediate and, after lyophilization from dioxane/water, 290 mg
(quant) of the title compound were obtained in 75% purity and used
without further purification in the next stage.
[4889] HPLC (Method 12): R.sub.t=1.1 min;
[4890] LC-MS (Method 13): R.sub.t=1.80 min; MS (ESIpos): m/z=276
(M+H).sup.+
Intermediate 246b
(2S)-2-amino-1-(4-hydroxy-1,2-oxazolidin-2-yl)-3-(1H-indol-3-yl)propan-1-o-
ne trifluoroacetate (Diastereomer 2)
##STR00683##
[4892] 5 ml of trifluoroacetic acid in 20 ml of dichloromethane
were used under standard conditions for cleaving the Boc protecting
group from 236 mg (630 .mu.mol) of Diastereomer 2 of the
intermediate described in 246a and, after concentration, stirring
with diethyl ether and drying of the residue under high vacuum, 214
mg (76%) of the title compound were obtained.
[4893] LC-MS (Method 13): R.sub.t=1.84 min; MS (ESIpos): m/z=276
(M+H).sup.+
Intermediate 247a
N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-N-[(3R,4-
S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(4-hydroxy-1,2-oxazolidin-2-yl)-3-(1H--
indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrroli-
din-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
(Diastereomer 1)
##STR00684##
[4895] To synthesize this compound, the coupling of Intermediates
26 and 246a with subsequent cleaving of the Boc protecting group
was first performed as described for Intermediate 74. Subsequently,
the alkylation with 6-oxohexanoic acid in the presence of
borane-pyridine complex and subsequent conversion of the acid to
the active ester were performed, as described for Intermediate 210.
The title compound was purified by means of preparative HPLC.
[4896] HPLC (Method 12): R.sub.t=1.8 min;
[4897] LC-MS (Method 1): R.sub.t=0.86 min; MS (ESIpos): m/z=1053
(M+H).sup.+
Intermediate 247b
N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-N-methyl-L-valyl-N-[(3R,4-
S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-(4-hydroxy-1,2-oxazolidin-2-yl)-3-(1H--
indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrroli-
din-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
(Diastereomer 2)
##STR00685##
[4899] To synthesize this compound, the coupling of Intermediates
26 and 246b with subsequent cleaving of the Boc protecting group
was first performed as described for Intermediate 74. Subsequently,
the alkylation with 6-oxohexanoic acid in the presence of
borane-pyridine complex and subsequent conversion of the acid to
the active ester were performed, as described for Intermediate 210.
The title compound was purified by means of preparative HPLC.
[4900] HPLC (Method 12): R.sub.t=1.8 min;
[4901] LC-MS (Method 1): R.sub.t=0.86 min; MS (ESIpos): m/z=1053
(M+H).sup.+
Intermediate 248
N-(5-carboxypentyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[-
(2S)-1-tert-butoxy-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino}-1-methoxy-2-
-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]--
N-methyl-L-valinamide
##STR00686##
[4903] First, in analogy to the synthesis described in Intermediate
86, the amine compound tert-butyl
N-[(2R,3R)-3-methoxy-3-{(2S)-1-[(3R,4S,5S)-3-methoxy-5-methyl-4-(methyl
{(2S)-3-methyl-2-[(N-methyl-L-valyl)amino]butyl}amino)heptanoyl]pyrrolidi-
n-2-yl}-2-methylpropanoyl]-L-tyrosinate was prepared as the
trifluoroacetate by coupling
N-(tert-butoxycarbonyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-
-2-carboxy-1-methoxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-4-yl]-N-methyl-L-valinamide (Intermediate 26) and
tert-butyl-L-tyrosinate in the presence of
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate and subsequent cleaving of the Boc protecting
group by means of trifluoroacetic acid to obtain the tert-butyl
ester (stirring with trifluoroacetic acid in dichloromethane for 40
min). 38 mg (0.04 mmol) of this compound were then used to obtain
31 mg (99% of theory) of the title compound, in analogy to the
preparation of Intermediate 210, by reaction with 6-oxohexanoic
acid in the presence of borane-pyridine complex.
[4904] HPLC (Method 12): R.sub.t=1.8 min;
[4905] LC-MS (Method 1): R.sub.t=0.88 min; MS (ESIpos): m/z=918
(M+H).sup.+.
Exemplary Embodiments
Anti-EGFR1 Antibodies Used
Cetuximab (INN No. 7906)
[4906] Additional names: IMC-225, C225, EMR-62202, BMS-564717, Fab
C225
[4907] Cetuximab (Drug Bank Accession No. DB00002) is a chimeric
anti-EGFR1-antibody that is produced in SP2/0 mouse myeloma cells
and is distributed by ImClone Systems Inc./Merck KGaA/Bristol-Myers
Squibb Co.
[4908] Cetuximab is indicated for treatment of metastatic
EGFR-expressing colorectal carcinoma with the wild-type K-Ras gene.
It has an affinity of 10.sup.-10 M.
[4909] Sequence:
[4910] Light chain (kappa):
TABLE-US-00046 DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKY
ASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGA
GTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
[4911] Heavy chain:
TABLE-US-00047 QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGV
IWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALT
YYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[4912] Panitumumab (INN No. 8499)
[4913] Panitumumab (additional names: ABX-EGF, E7.6.3) (Drug Bank
Accession No. DB01269) is a recombinant monoclonal human IgG2
antibody that binds specifically to the human EGF receptor 1 and is
distributed by Abgenix/Amgen.
[4914] Panitumumab originates from the immunization of transgenic
mice (XenoMouse). These mice are capable of producing human
immunoglobulins (light chains and heavy chains). A special B cell
clone was selected which produces antibodies to EGFR and it was
immortalized with CHO cells (Chinese hamster ovary cells). These
cells are now used for the production of a 100% human antibody.
[4915] Panitumumab is indicated for treatment of an EGFR-expressing
metastatic colorectal carcinoma that is refractory to a
chemotherapeutic treatment with fluoropyrimidine, oxaliplatin and
irinotecan. It has an affinity of 10.sup.-11 M.
[4916] Sequence:
[4917] Light chain (kappa):
TABLE-US-00048 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYD
ASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGG
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
[4918] Heavy chain:
TABLE-US-00049 QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWI
GHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRD
RVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTY
TCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLM
ISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV
VSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
[4919] Nimotuzumab (INN No. 8545)
[4920] Nimotuzumab (additional names: TheraCIM-h-R3; h-R3;
Theraloc; BioMAb; BIOMAb-EGFR; Vecthix; KI-0501) (patents EP
00586002, EP 00712863) is a humanized monoclonal IgG1 antibody that
binds specifically to human EGF receptor 1 and is produced by YM
BioSciences Inc. (Mississauga, Canada). It is produced in
non-secreting NSO cells (mammalian line).
[4921] Nimotuzumab is approved for treatment of head and neck
tumors, highly malignant astrocytomas and glioblastoma multiform
(not in the EU and US) and pancreatic cancer (orphan drug, EMA). It
has an affinity of 10.sup.-8 M.
B. PRODUCTION OF ADCS
[4922] The intermediates described above can be linked to the
anti-EGF receptor antibodies cetuximab, nimotuzumab or panitumumab,
for example, as well as additional antibodies listed below, and
such linkages may optionally be via cysteine or lysine side chains
of the antibody protein according to the methods described
below.
[4923] B-1.1 Workup of the EGFR Antibodies Before Conjugation
[4924] Erbitux commercial product (Erbitux.RTM. 5 mg/mL infusion
solution 100 mL, PZN 0493540, N1, 500 mg, Merck), Vectibix
commercial product (Vectibix.RTM. 20 mg/mL concentrate for
preparing an infusion solution, one puncturable vial (N1) 100 mg,
20 mL, PZN 6078606, Amgen) or CIMAher commercial product
(CIMAher.RTM. 50 mg AMP 4.times.10 mL, imported from Cuba, YM
BioSciences Inc. (Mississauga, Canada) were obtained commercially
from a pharmacy.
[4925] To remove the polysorbate 80 contained in the formulation,
it was bound to protein A (MabSelectSure) and rinsed with 15%
isopropanol. After elution with acidic acetate buffer, the mixture
was rebuffered after gel filtration on D-PBS, and the resulting
material was coupled to the respective toxophores.
[4926] B-1.2 General Procedure for Expression of Antibodies in
Mammalian Cells
[4927] The antibodies, e.g., anti-PDL1 or other antibodies to the
various targets are produced in mammalian cell culture by
transfecting HEK293 6E cells transiently with a suitable CMV
promoter-based expression plasmid. The light and heavy chains of
the antibodies were cloned either together in a single-vector
system or separately in a two-vector system. The cell culture
standard was up to 1.5 L in an agitated flask or 10 L in the "Wave
Bag." The expression occurred at 37.degree. C. for 5-6 days in F17
medium (Invitrogen) supplemented with tryptone TN1 (Organotechnie)
with 1% "FCS ultra-low IgG" (Invitrogen) and 0.5 mM valproic acid.
The expression yields were between 7 and 310 mg/L.
[4928] B-1.3 General Method for Purifying Antibodies from Cell
Supernatants
[4929] The antibodies, e.g., PDL1 or other antibodies to the
various targets were obtained from the cell culture supernatants.
The cell culture supernatants were clarified by centrifugation of
cells. Then the supernatant was purified by affinity chromatography
on a MabSelectSure (GE Healthcare) chromatography column. The
column was therefore equilibrated in DPBS, pH 7.4 (Sigma/Aldrich),
the cell supernatant was applied and the column was washed with
approx. 10 column volumes of DPBS, pH 7.4, +500 mM sodium chloride.
The antibodies were eluted in 50 mM sodium acetate, pH 3.5, +500 mM
sodium chloride and then purified further by gel filtration
chromatography on a Superdex 200 column (GE Healthcare) in DPBS, pH
7.4.
[4930] B-1.4 General Method for Coupling to Cysteine Side
Chains
[4931] The following antibodies were used in the coupling
reaction:
[4932] Anti-EGFR1 Antibodies:
[4933] cetuximab
[4934] nimotuzumab
[4935] panitumumab
[4936] Other Antibodies:
[4937] anti-PDL1
[4938] anti-ICOSLG
[4939] anti-FGFR3
[4940] herceptin
[4941] anti-TYRP1
[4942] anti-glypican-3
[4943] To a solution of the corresponding antibody in PBS buffer in
the concentration range between 1 mg/mL and 10 mg/mL, 3 eq. of
tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) dissolved in
PBS buffer were added and stirred for one hour at RT. Then,
depending on the desired load, between two and ten equivalents of
the maleimide precursor compound to be coupled or the halide
precursor compound (intermediates 102, 103, 105-109, 111-114,
117-126, 128, 129, 132-146, 148-155, 157, 159-161, 166, 171,
175-177, 184, 189, 194-195, 199-201, 205, 209, 223-224, 226,
228-231, 236 and 244) were added as a solution in DMSO. The amount
of DMSO should not exceed 10% of the total volume. The batch was
stirred for 60-120 minutes at RT and then applied to PD10 columns
(Sephadex.RTM. G-25, GE Healthcare) equilibrated in PBS and then
eluted with PBS buffer. If necessary, the concentration was
increased further by ultracentrifugation.
[4944] Unless otherwise indicated, 5 mg of the corresponding
antibody was generally used in PBS buffer for reduction and the
following coupling. After purification on the PD10 column, the
solutions of the corresponding ADC in 3.5 mL PBS buffer were each
obtained. The protein concentration indicated in each case was then
determined for these solutions. In addition, the load of the
antibody (drug/mAb ratio) was determined by the methods described
below.
[4945] According to this method, the immunoconjugates synthesized
in Examples 1-34, 36-37, 39-41, 43-44, 52-53, 55, 338-339, 341-344,
349, 351-352, 354, 356-358 and 374 were prepared.
[4946] In the structural formulas presented, AK.sub.1A-AK.sub.1J
have the following meanings:
[4947] AK.sub.1A=cetuximab (partially reduced)-S.sctn..sup.1
[4948] AK.sub.1B=nimotuzumab (partially reduced)-S.sctn..sup.1
[4949] AK.sub.1C=panitumumab (partially reduced)-S.sctn..sup.1
[4950] AK.sub.1D=anti-PDL1 (partially reduced)-S.sctn..sup.1
[4951] AK.sub.1E=anti-ICOSLG (partially reduced)-S.sctn..sup.1
[4952] AK.sub.1F=anti-FGFR3 (partially reduced)-S.sctn..sup.1
[4953] AK.sub.1G=herceptin (partially reduced)-S.sctn..sup.1
[4954] AK.sub.1H=anti-TYRP1 (partially reduced)-S.sctn..sup.1
[4955] AK.sub.1J=anti-glypican-3 (partially
reduced)-S.sctn..sup.1
[4956] wherein
[4957] .sctn..sup.1 denotes the linkage to the succinimide
group
[4958] and
[4959] S stands for the sulfur atom of a cysteine radical of the
partially reduced antibody.
[4960] B-1.5 General Method for Coupling to Lysine Side Chains
[4961] The following antibodies were used in the coupling
reactions:
[4962] Anti-EGFR1 Antibodies:
[4963] cetuximab
[4964] nimotuzumab
[4965] panitumumab
[4966] Other Antibodies:
[4967] anti-PDL1
[4968] anti-ICOSLG
[4969] anti-FGFR3
[4970] herceptin
[4971] anti-TYRP1 hIgG1-kapp
[4972] anti-glypican-3
[4973] Between 2 and 5 eq. of the precursor compound to be coupled
from the intermediates 104, 110, 115, 116, 127, 130, 131, 147, 156,
158, 162, 169, 178, 185, 190, 202, 206, 210-216, 218, 219, 227,
233, 238, 240, 242, 245, 247a and 247b were added as a solution in
DMSO to a solution of the corresponding antibody in PBS buffer in
the concentration range between 1 mg/mL and 10 mg/mL, depending on
the desired load. After stirring for 30 minutes at RT, the same
amount of precursor compound in DMSO was again added. The amount of
DMSO should not exceed 10% of the total volume. After stirring for
30 minutes more at RT the batch was poured over PD10 columns
(Sephadex.RTM. G-25) and then eluted with PBS buffer. Another
concentration step was optionally performed by ultrafiltration. If
necessary, for better separation of low-molecular components, the
concentration step by ultrafiltration was repeated after diluting
again with PBS buffer.
[4974] Unless otherwise indicated, 5 mg of the corresponding
antibody in PBS buffer was generally used for coupling. After
purification on the PD10 column, solutions of the corresponding
ADCs in 3.5 mL PBS buffer were obtained. For these solutions, the
respective protein concentrations then given were determined, and
the antibody load (drug/mAb ratio) was determined according to the
methods described below.
[4975] According to this method, the immunoconjugates described in
Examples 35, 38, 42, 54, 337, 340, 345-348, 350, 353, 355, 359,
363, 375 and 376 were prepared.
[4976] In the structural formulas shown here, AK.sub.2A, AK.sub.2B,
AK.sub.2C, AK.sub.2D, AK.sub.2E, AK.sub.2F, AK.sub.2G, AK.sub.2H
and AK.sub.2J have the following meanings:
[4977] AK.sub.2A=cetuximab-NH.sctn..sup.2
[4978] AK.sub.2B=nimotuzumab-NH.sctn..sup.2
[4979] AK.sub.2C=panitumumab-NH.sctn..sup.2
[4980] AK.sub.2D=anti-PDL1-NH.sctn..sup.2
[4981] AK.sub.1E=anti-ICOSLG-NH.sctn..sup.2
[4982] AK.sub.2F=anti-FGFR3-NH.sctn..sup.2
[4983] AK.sub.2G=herceptin-NH.sctn..sup.2
[4984] AK.sub.2H=anti-TYRP1-NH.sctn..sup.2
[4985] AK.sub.2J=anti-glypican-3-NH.sctn..sup.2
[4986] wherein
[4987] .sctn..sup.2 denotes the linkage to the carbonyl group
[4988] and
[4989] NH stands for the side chain amino group of a lysine radical
of the antibody.
[4990] B-1.6a General Method for Preparing Cysteine Adducts
[4991] 10 .mu.mol of the maleimide precursor compounds described
above was placed in 3 mL DMF and mixed with 2.1 mg (20 .mu.mol)
L-cysteine. The batch was stirred for 2 hours at RT, then
concentrated in vacuo and next purified by preparative HPLC.
[4992] In the structural formulas shown here, Cys has the following
meaning:
##STR00687##
[4993] wherein
[4994] .sctn..sup.3 denotes the linkage to the linker toxophore
unit.
[4995] B-1.6b General Method for Preparing Ivsine Adducts:
[4996] 10 .mu.mol of the active ester precursor compounds described
above was placed in 5 mL DMF and mixed with .alpha.-amino-protected
L-lysine in the presence of 30 .mu.mol N,N-diisopropylethylamine.
The reaction mixture was stirred for 2 hours at RT and then
concentrated in vacuo and next was purified by preparative HPLC.
Then the protective group was removed by known methods.
[4997] Further Purification and Characterization of the Conjugates
According to the Invention
[4998] After the reaction was successful, the reaction mixture was
concentrated in some cases by ultracentrifugation, for example, and
then was desalinated and purified by chromatography using a
Sephadex.RTM. G-25 column, for example. Elution was performed using
phosphate-buffered saline solution (PBS), for example. Then the
solution was sterile filtered and frozen. Alternatively, the
conjugate may be lyophilized.
[4999] B-1.7 Determining the Toxophore Load
[5000] The toxophore load was determined as follows on the
resulting solutions of the conjugates in PBS buffer as described in
the exemplary embodiments:
[5001] The toxophore load of lysine-linked ADCs was determined by
mass spectrometric determination of the molecular weights of the
individual conjugated species. The antibody conjugates were first
deglycosylated by PNGaseF, the sample was acidified and next, after
HPLC separation, the sample was analyzed by mass spectrometry using
ESI MicroTof.sub.Q (Bruker Daltonik). All the spectra over the
signal in the TIC (total ion chromatogram) were added up and the
molecular weights of the various conjugate species were calculated
on the basis of MaxEnt deconvolution. After signal integration of
the various species, the DAR (drug/antibody ratio) was
calculated.
[5002] For protein identification, after deglycosylation and/or
denaturing, in addition to determination of the molecular weight,
tryptic digestion was performed, confirming the identity of the
protein on the basis of the tryptic peptides identified after
denaturing, reduction and derivatization.
[5003] The toxophore load of cysteine-linked conjugates was
determined by reversed-phase chromatography of the reduced and
dentured ADCs. Guanidinium hydrochloride (GuHCl, 28.6 mg) and a
solution of DL-dithiothreitol (DTT, 500 mM, 3 .mu.L) were added to
the ADC solution (1 mg/mL, 50 .mu.L). The mixture was incubated for
one hour at 55.degree. C. and then analyzed by HPLC.
[5004] HPLC analysis was performed on an adjuvant 1260 HPLC system
with detection at 220 nm, using a Polymer Laboratories PLRP-S
polymeric reversed-phase column (catalog number PL1912-3802)
(2.1.times.150 mm, 8 .mu.m particle size, 1000 .ANG.) at a flow
rate of 1 mL/min with the following gradient: 0 min, 25% B; 3 min,
25% B; 28 min, 50% B. Eluent A consisted of 0.05% trifluoroacetic
acid (TFA) in water, and eluent B consisted of 0.05%
trifluoroacetic acid in acetonitrile.
[5005] The peaks detected were assigned based on a comparison of
the retention times with the light chain (L0) and the heavy chain
(H0) of the unconjugated antibody. Peaks detected exclusively in
the conjugated sample were assigned to the light chain with one
toxophore (L1) and to the heavy chains with one, two and three
toxophores (H1, H2, H3).
[5006] The average toxophore load of the antibody was calculated
from the peak areas determined by integration as the sum of the
integration results of all peaks times 2, weighted by the number of
toxophores, divided by the total of the integration results of all
peaks with simple weighting. In isolated cases, it may happen that
the toxophore load cannot be determined accurately due to
co-elution of some peaks.
[5007] B-1.8 Testing the Antigen Binding of the ADC
[5008] The ability of the binder to bind to the target molecule was
tested after successful coupling. Those skilled in the art are
familiar with a variety of methods for doing so; for example, the
affinity of the conjugate can be tested by means of ELISA
technology or surface plasmon resonance analysis (BIAcore.TM.
measurements). The skilled person can measure the conjugate
concentration using conventional methods, e.g., protein assay for
antibody conjugates (see also Doronina et al., Nature Biotechnol.
2003; 21:778-784 and Polson et al., Blood 2007, 1102:616-623).
[5009] B2 Producing Antibody-Drug Conjugates (ADCs)
[5010] The intermediates described above were linked to the
anti-mesothelin antibody MF-Ta, for example, with the linkage
optionally taking place via the cysteine or lysine side chains of
the antibody protein using the methods described below. The
anti-mesothelin antibody MF-Ta was produced by methods like those
described in WO 2009/068204 A1. The antibody MF-Ta was expressed in
eukaryotic CHO cells (stable cell line) and purified by protein A
and gel filtration before being subjected to conjugation in DPBS
buffer.
[5011] B-2.1 General Working Procedure 1 (Coupling Via
Cysteine):
[5012] To a solution of the corresponding antibody in PBS buffer in
the concentration range between 1 mg/mL and 10 mg/mL, 3 eq. of
tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) dissolved in
PBS buffer were added and stirred for one hour at RT. Next,
depending on the desired load, between 2 and 10 eq. of the
maleimide precursor compound or the halide precursor compound to be
coupled (intermediates 128, 129, 132-146, 148-155, 157, 159-161,
171, 175-177, 184, 189, 194-195, 199-201, 205, 209, 223-224, 226,
228-231, 236 and 244) were added as a solution in DMSO. The amount
of DMSO should not exceed 10% of the total volume. The reaction
mixture was stirred for 60-120 minutes at RT and then applied to
PD10 columns (Sephadex.RTM. G-25) and eluted with PBS buffer. The
solution was then optionally concentrated by ultrafiltration.
Concentration by ultrafiltration was repeated, if necessary, after
diluting again with PBS buffer to achieve a better separation of
low-molecular components.
[5013] Unless otherwise indicated, 5 mg of the corresponding
antibody in PBS buffer was generally used for reduction and the
subsequent coupling. After purification over the PD10 column,
solutions of the corresponding ADCs in 3.5 mL PBS buffer were thus
obtained. Then the respective protein concentration was determined
for each of these solutions. In addition, the antibody load
(drug/mAb ratio) was determined by the methods described in B4.
[5014] The immunoconjugates synthesized in Examples 56, 57, 60-74,
76-83, 85, 86, 88-92, 94-101, 103, 106-112, 114, 115, 126, 128-131,
133-135, 137-139, 141-142, 151, 153-154, 366 and 377 were prepared
by this method.
[5015] In the structural formulas given, AK.sub.3 has the
meaning
AK.sub.3=MF-Ta(partially reduced)-S.sctn..sup.1, [5016] wherein
[5017] .sctn..sup.1 denotes the linkage to the succinimide group,
[5018] MF-Ta (partially reduced) stands for the partially reduced
MF-Ta antibody (heavy chain SEQ ID NO: 408 and light chain SEQ ID
NO: 409), [5019] and [5020] S stands for the sulfur atom of a
cysteine radical of the partially reduced antibody.
[5021] B-2.2 General Working Procedure 2 (Coupling Via Lysine Side
Chains):
[5022] Between 2 and 5 eq. of the precursor compound to be coupled
(intermediates 104, 110, 115, 116, 127, 130, 131, 147, 156, 158,
162, 169, 178, 185, 190, 202, 206, 210-216, 218, 219, 227, 233,
238, 240, 242, 245, 247a and 247b) were added as a solution in DMSO
to a solution of the corresponding antibody in PBS buffer in the
concentration range between 1 mg/mL and 10 mg/mL, depending on the
desired load. After stirring for 30 minutes at RT, the same amount
of precursor compound in DMSO was added. The amount of DMSO should
not exceed 10% of the total volume. After stirring for 30 minutes
more at RT, the batch was poured over PD10 columns (Sephadex.RTM.
G-25) and eluted with PBS buffer. Further concentration by
ultrafiltration was optionally also performed. Concentration was
repeated by ultrafiltration, if necessary, after diluting again
with PBS buffer to improve the separation of low-molecular
components.
[5023] Unless otherwise indicated, 5 mg of the corresponding
antibody in PBS buffer was generally used for coupling. After
purification over the PD10 column, solutions of the corresponding
ADC in 3.5 mL PBS buffer were obtained. Then the protein
concentration indicated was determined for these solutions and the
load of the antibody (drug/mAb ratio) was determined by the methods
described under B4.
[5024] Following this method, the immunoconjugates synthesized in
Examples 58, 59, 75, 84, 87, 93, 102, 104, 105, 113, 116, 127, 132,
136, 140, 143-150, 152, 367-369 and 378-380 were prepared.
[5025] In the structural formulas given, AK.sub.4 has the
meaning
AK.sub.4=MF-Ta-NH.sctn..sup.2,
[5026] wherein
[5027] .sctn..sup.2 denotes the linkage to the carbonyl group
[5028] MF-Ta stands for the unreduced MF-Ta antibody (heavy chain
SEQ ID NO: 408 and light chain SEQ ID NO: 409)
[5029] and
[5030] NH stands for the side chain amino group of a lysine radical
of the antibody.
[5031] B-2.3a General Method for Synthesis of Cysteine Adducts:
[5032] 10 .mu.mol of the maleimide precursor compounds described
above was dissolved in 3 mL DMF and mixed with 2.1 mg (10 .mu.mol)
L-cysteine. The reaction mixture was stirred for 2 hours at RT,
then concentrated in vacuo and purified by preparative HPLC.
[5033] Cys in the structural formulas given has the meaning
##STR00688##
[5034] wherein
[5035] .sctn..sup.3 denotes the linkage to the linker toxophore
unit.
[5036] B-2.3b General Method for Preparing Lysine Adducts:
[5037] 10 .mu.mol of the active ester precursor compounds described
above was placed in 5 mL DMF and mixed with .alpha.-amino-protected
L-lysine in the presence of 30 .mu.mol N,N-diisopropylethylamine.
The reaction mixture was stirred for 2 hours at RT and then
concentrated in vacuo and next was purified by preparative HPLC.
Then the protective group was removed by known methods.
[5038] Further Purification and Characterization of the Conjugates
According to the Invention
[5039] After a successful reaction, the reaction mixture was
concentrated by ultracentrifugation, for example, in some cases and
then was desalinated and purified by chromatography using a
Sephadex.RTM. G-25 column, for example. Elution was performed using
phosphate-buffered saline solution (PBS), for example. Then the
solution was sterile filtered and frozen. Alternatively, the
conjugate may be lyophilized.
[5040] B-2.4 Determining the Toxophore Load
[5041] The toxophore load was determined as follows on the
resulting solutions of the conjugates in PBS buffer as described in
the exemplary embodiments:
[5042] The toxophore load of lysine-linked ADCs was determined by
mass spectrometric determination of the molecular weights of the
individual conjugated species. First, the antibody conjugates were
deglycosylated by PNGaseF, then the sample was acidified; next,
after HPLC separation, the sample was analyzed by mass spectrometry
using ESI MicroTof.sub.Q (Bruker Daltonik). All the spectra over
the signal in the TIC (total ion chromatogram) were added up and
the molecular weights of the various conjugate species were
calculated based on MaxEnt deconvolution. The DAR (drug/antibody
ratio) was calculated after signal integration of the various
species.
[5043] For protein identification, after deglycosylation and/or
denaturing, tryptic digestion was performed, in addition to
determination of the molecular weight, the identity of the protein
being confirmed on the basis of the tryptic peptides identified
after denaturing, reduction and derivatization.
[5044] The toxophore load of cysteine-linked conjugates was
determined by reversed-phase chromatography of the reduced and
dentured ADCs. Guanidinium hydrochloride (GuHCl, 28.6 mg) and a
solution of DL-dithiothreitol (DTT, 500 mM, 3 .mu.L) were added to
the ADC solution (1 mg/mL, 50 .mu.L). The mixture was then
incubated for one hour at 55.degree. C. and analyzed by HPLC.
[5045] The HPLC analysis was performed on an adjuvant 1260 HPLC
system with detection at 220 nm, using a Polymer Laboratories
PLRP-S polymeric reversed-phase column (catalog no. PL1912-3802)
(2.1.times.150 mm, 8 .mu.m particle size, 1000 .ANG.) at a flow
rate of 1 mL/min with the following gradient: 0 min, 25% B; 3 min,
25% B; 28 min, 50% B. Eluent A was 0.05% trifluoroacetic acid (TFA)
in water, and eluent B was 0.05% trifluoroacetic acid in
acetonitrile.
[5046] The peaks detected were assigned based on a comparison of
the retention times with the light chain (L0) and the heavy chain
(H0) of the unconjugated antibody. Peaks detected exclusively in
the conjugated sample were assigned to the light chain with one
toxophore (L1) and to the heavy chains with one, two and three
toxophores (H1, H2, H3).
[5047] The average toxophore load of the antibody was calculated
from the peak areas determined by integration as two times the sum
of the integration results of all peaks, weighted by the number of
toxophores, divided by the total of the integration results of all
peaks with simple weighting. In isolated cases, it may happen that
the toxophore load cannot be determined accurately due to
co-elution of some peaks.
[5048] B-2.5 Testing the Antigen Binding of the ADC
[5049] The ability of the binder to bind to the target molecule was
tested after successful coupling. Those skilled in the art are
familiar with a variety of methods for doing so; for example, the
affinity of the conjugate can be tested by ELISA technology or
surface plasmon resonance analysis (BIAcore.TM. measurements). The
skilled person can measure the conjugate concentration using
conventional methods, e.g., by protein determination for antibody
conjugates (see also Doronina et al., Nature Biotechnol. 2003;
21:778-784 and Polson et al., Blood 2007, 1102:616-623).
[5050] B3 Synthesis of Antibody-Drug Conjugates (ADCs)
[5051] B-3.1 General Method for Generating Anti-C4.4a
Antibodies
[5052] The anti-C4.4a antibodies described by the sequences
according to Table 1 and Table 2 were generated by screening of a
phage display library for recombinant human C4.4a SEQ ID NO: 1 and
murine C4.4a SEQ ID NO: 2 and for C4.4a-expressing cells. The
antibodies thereby obtained were reformatted into the human IgG1
format and used for the exemplary embodiments described here.
[5053] B-3.2 General Method for Expression of Anti-C4.4a Antibodies
in Mammalian Cells
[5054] The antibodies, e.g., M31-B01 (light chain SEQ ID NO: 346
and heavy chain SEQ ID NO: 347) or other antibodies according to
Table 2, were produced in mammalian cell culture. To do so, HEK293
6E cells were transfected transiently using a suitable CMV
promoter-based expression plasmid. The heavy and light chains of
the antibodies were cloned either together in a single-vector
system or separately in a two-vector system. This cell culture
standard was up to 1.5 L in a shaken flask or 10 L in a Wave Bag.
The cells were expressed for 5-6 days at 37.degree. C. in F17
medium (Invitrogen) supplemented with tryptone TN1 (Organotechnie)
with 1% FCS ultralow IgG (Invitrogen) and 0.5 mM valproic acid.
Expression yields were between 100 mg/L and 600 mg/L.
[5055] B-3.3 General Method for Purification of Antibodies from
Cell Supernatants
[5056] The antibodies, e.g., M31-B01 (light chain SEQ ID NO: 346
and heavy chain SEQ ID NO: 347) or additional antibodies according
to Table 2 were obtained from the cell culture supernatants. The
cell supernatants were clarified of cells by centrifugation. Then
the cell supernatant was purified by affinity chromatography on a
MabSelectSure (GE Healthcare) chromatography column. To do so, the
column was equilibrated in DPBS, pH 7.4 (Sigma/Aldrich), the cell
supernatant was applied and the column was washed with approx. 10
column volumes of DPBS, pH 7.4, +500 mM NaCl. The antibodies were
eluted in 50 mM sodium acetate, pH 3.5, +500 mM NaCl and then
purified further by gel filtration chromatography on a Superdex 200
column (GE Healthcare) in DPBS, pH 7.4.
[5057] B-3.4 General Method for Coupling to Cysteine Side
Chains
[5058] The following antibodies were used in the coupling
reactions:
[5059] anti-C4.4a M31-B01
[5060] anti-C4.4a B01-3
[5061] anti-C4.4a B01-10
[5062] anti-C4.4a B01-7
[5063] anti-C4.4a D02-4
[5064] anti-C4.4a D02-6
[5065] anti-C4.4a D02-7
[5066] Three equivalents of tris-(2-carboxyethyl)phosphine
hydrochloride (TCEP) dissolved in PBS buffer were added to a
solution of the corresponding antibody in PBS buffer in the
concentration range between 1 mg/mL and 10 mg/mL and stirred for
one hour at RT. Next, between 2 and 10 eq. of the maleimide
precursor compound from intermediates 128, 129, 132-146, 148-155,
157, 159-161, 166, 171, 175-177, 184, 188, 190, 194-195, 199-201,
205, 209, 223-224, 226, 228-231, 236 and 244 to be coupled,
depending on the desired load, were added as a solution in DMSO.
The amount of DMSO should not exceed 10% of the total volume. The
batch was stirred for 60-120 minutes at RT and then applied to PD10
columns (Sephadex.RTM. G-25, GE Healthcare) equilibrated in PBS and
eluted with PBS buffer. If necessary, further concentration was
performed by ultracentrifugation.
[5067] Unless otherwise indicated, 5 mg of the corresponding
antibody in PBS buffer was generally used for reduction and for the
subsequent coupling. After purification on the PD10 column,
solutions of the corresponding ADC in 3.5 mL PBS buffer were
obtained. The protein concentration indicated was then determined
for each of these solutions. In addition, the load of the antibody
(drug/mAb ratio) was determined according to the methods described
below.
[5068] The immunoconjugates prepared in Examples 163-165, 167-192,
194-198, 200-221, 223-228, 230-232, 242, 244-247, 249, 250,
254-257, 259-260, 269, 271-275, 371 and 385 were produced by this
method.
[5069] In the structural formulas shown, AK.sub.5A through
AK.sub.5G have the meanings given below:
[5070] AK.sub.5A=anti-C4.4a antibody M31-B01 (partially
reduced)-S.sctn..sup.1
[5071] AK.sub.5B=anti-C4.4a antibody B01-3 (partially
reduced)-S.sctn..sup.1
[5072] AK.sub.5C=anti-C4.4a antibody B01-10 (partially
reduced)-S.sctn..sup.1
[5073] AK.sub.5D=anti-C4.4a antibody B01-7 (partially
reduced)-S.sctn..sup.1
[5074] AK.sub.5E=anti-C4.4a antibody D02-4 (partially
reduced)-S.sctn..sup.1
[5075] AK.sub.5F=anti-C4.4a antibody D02-6 (partially
reduced)-S.sctn..sup.1
[5076] AK.sub.5G=anti-C4.4a antibody D02-7 (partially
reduced)-S.sctn..sup.1
[5077] wherein
[5078] .sctn..sup.1 denotes the linkage to the succinimide
group
[5079] and
[5080] S stands for the sulfur atom of a cysteine radical of the
partially reduced antibody.
[5081] B-3.5 General Method for Coupling to Lysine Side Chains:
[5082] The following antibodies were used in the coupling
reactions:
[5083] anti-C4.4a antibody M31-B01
[5084] anti-C4.4a antibody B01-3
[5085] To a solution of the corresponding antibody in PBS buffer in
the concentration range between 1 mg/mL and 10 mg/mL, between 2 and
5 eq. of the precursor compound to be coupled, depending on the
desired load, from the intermediates 104, 110, 115, 116, 127, 130,
131, 147, 156, 158, 162, 169, 178, 185, 190, 202, 206, 210-216,
218-219, 227, 233, 238, 240, 242, 245, 247a and 247b were added as
a solution in DMSO. After stirring for 30 minutes at RT, the same
amount of precursor compound in DMSO was added again. In doing so,
the amount of DMSO should not exceed 10% of the total volume. After
stirring for 30 minutes more at RT, the batch was applied to PD10
columns (Sephadex.RTM. G-25) and eluted with PBS buffer. The batch
was optionally concentrated further by ultrafiltration. For better
separation of low-molecular components, the ultrafiltration
concentration step was repeated after diluting with PBS buffer
again, if necessary.
[5086] Usually, unless otherwise indicated, 5 mg of the
corresponding antibody in PBS buffer was used for coupling. After
purification on the PD10 column, solutions of the corresponding ADC
in 3.5 mL PBS buffer were thus obtained. Then the specific protein
concentration indicated was determined for these solutions, and the
antibody load (drug/mAb ratio) was determined according to the
methods described below.
[5087] The immunoconjugates synthesized in Examples 166, 193, 199,
222, 229, 243, 248, 251-253, 258, 261-268, 270, 276, 370, 372-373
and 386-388 were prepared by this method.
[5088] In the structural formulas given, AK.sub.6A and AK.sub.6B
have the following meanings
[5089] AK.sub.6A=anti-C4.4a antibody M31-B01-NH.sctn..sup.2
[5090] AK.sub.6B=anti-C4.4a antibody B01-3-NH.sctn..sup.2
[5091] wherein
[5092] .sctn..sup.2 denotes the linkage to the carbonyl group
[5093] and
[5094] NH stands for the side chain amino group of a lysine radical
of the antibody.
[5095] B-3.6 General Method for Synthesis of Cysteine Adducts:
[5096] 10 .mu.mol of the maleimide precursor compounds described
above was placed in 3 mL DMF and mixed with 2.1 mg (10 .mu.mol)
L-cysteine. The reaction mixture was stirred for 2 hours at RT,
then concentrated in vacuo and purified by preparative HPLC.
[5097] Cys in the structural formulas given has the meaning
##STR00689##
[5098] wherein
[5099] .sctn..sup.3 denotes the linkage to the linker toxophore
unit.
[5100] B-3.6 General Method 2.3a for Synthesis of Cvsteine
Adducts:
[5101] 10 .mu.mol of the maleimide precursor compounds described
above was placed in 3 mL DMF and mixed with 2.1 mg (10 .mu.mol)
L-cysteine. The reaction mixture was stirred for 2 hours at RT,
then concentrated in vacuo and purified by preparative HPLC.
[5102] Cys in the structural formulas given has the meaning
##STR00690##
[5103] wherein
[5104] .sctn..sup.3 denotes the linkage to the linker toxophore
unit.
[5105] Further Purification and Characterization of the Conjugates
According to the Invention
[5106] After successful reaction, the reaction mixture was
concentrated further in some cases by ultracentrifugation, for
example, and then was desalinated and purified by chromatography,
for example, using a Sephadex.RTM. G-25 column. Then elution was
performed using phosphate-buffered saline solution (PBS), for
example. Then the solution was sterile filtered and frozen.
Alternatively, the conjugate may be lyophilized.
[5107] B-3.7 Determining the Toxophore Load
[5108] The toxophore load was determined as follows on the
resulting solutions of the conjugates in PBS buffer, as described
in the exemplary embodiments:
[5109] The toxophore load of lysine-linked ADCs was determined by
mass spectrometric determination of the molecular weights of the
individual conjugated species. The antibody conjugates were first
deglycosylated by PNGaseF, the sample was acidified and then, after
HPLC separation, the sample was analyzed by mass spectrometry using
ESI-MicroTof.sub.Q (Bruker Daltonik). All the spectra over the
signal in the TIC (total ion chromatogram) were added up and the
molecular weights of the various conjugate species were calculated
based on MaxEnt deconvolution. The DAR (drug/antibody ratio) was
then calculated after signal integration of the various
species.
[5110] For protein identification, after deglycosylation and/or
denaturing, in addition to determination of the molecular weight,
tryptic digestion was performed, confirming the identity of the
protein on the basis of the tryptic peptides identified after
denaturing, reduction and derivatization.
[5111] The toxophore load of cysteine-linked conjugates was
determined by reversed-phase chromatography of the reduced and
dentured ADCs. Guanidinium hydrochloride (GuHCl, 28.6 mg) and a
solution of DL-dithiothreitol (DTT, 500 mM, 3 .mu.L) were added to
the ADC solution (1 mg/mL, 50 .mu.L). The mixture was then
incubated for one hour at 55.degree. C. and then analyzed by
HPLC.
[5112] The HPLC analysis was performed on an adjuvant 1260 HPLC
system with detection at 220 nm, using a Polymer Laboratories
PLRP-S polymeric reversed-phase column (catalog number PL1912-3802)
(2.1.times.150 mm, 8 m particle size, 1000 .ANG.) at a flow rate of
1 mL/min with the following gradient: 0 min, 25% B; 3 min, 25% B;
28 min, 50% B. Eluent A consisted of 0.05% trifluoroacetic acid
(TFA) in water; eluent B consisted of 0.05% trifluoroacetic acid in
acetonitrile.
[5113] The peaks detected were assigned on the basis of a
comparison of the retention times with the light chain (L0) and the
heavy chain (H0) of the unconjugated antibody. Peaks detected
exclusively in the conjugated sample were assigned to the light
chain with one toxophore (L1) and to the heavy chains with one, two
and three toxophores (H1, H2, H3). The average toxophore load of
the antibody was calculated from the peak areas determined by
integration as two times the sum of the integration results of all
peaks, weighted by the number of toxophores, dividing by the total
of the integration results of all peaks with simple weighting. In
isolated cases, it may happen that the toxophore load cannot be
determined accurately due to co-elution of some peaks.
[5114] B-3.8 Testing the Antigen Binding of the ADC
[5115] The ability of the binder to bind to the target molecule was
tested after successful coupling. Those skilled in the art are
familiar with a variety of methods for doing so. For example, the
affinity of the conjugate can be tested by means of ELISA
technology or surface plasmon resonance analysis (BIAcore.TM.
measurements). The skilled person can measure the conjugate
concentration using conventional methods, e.g., by protein
determination for antibody conjugates (see also Doronina et al.,
Nature Biotechnol. 2003; 21:778-784 and Polson et al., Blood 2007,
1102:616-623).
[5116] B4 Producing Antibody-Drue Coniugates (ADCs)
[5117] The intermediates described above were linked to the
anti-CA9 antibody (3ee9), for example, with the linkage taking
place optionally by way of the cysteine or lysine side chains of
the antibody protein using the methods described below.
[5118] B-4.1 General Method for Generating Anti-CA9 Antibodies
[5119] The antibodies to CA9, e.g., the antibody 3ee9, were
obtained by panning the HuCAL GOLD phage display library for
recombinant antigen. The Fab antibody fragments thereby isolated
were recloned to the IgG format (WO 2007/070538 A2).
[5120] B-4.2 General Method for Expression of Anti-CA9 Antibodies
in Mammalian Cells and for Purification
[5121] The anti-CA9 IgG antibodies, e.g., 3ee9, were expressed by
transient transfection of HEK 293 and purified from their cell
supernatants by methods familiar to those skilled in the art. These
methods are described in WO 2007/070538 A2.
[5122] B-4.3 General Method for Coupling to Cysteine Side
Chains
[5123] To a solution of the corresponding anti-CA9 antibody, e.g.,
3ee9, which may be present PBS buffer or in Tris buffer in the
concentration range between 1 mg/mL and 10 mg/mL, for example,
three equivalents of tris-(2-carboxyethyl)phosphine hydrochloride
(TCEP) dissolved in PBS buffer were added and stirred for one hour
at RT. Then, depending on the desired load, between 2 and 10 eq. of
the maleimide precursor compound or the halide precursor compound
from intermediates 102, 103, 105-109, 111-114, 117-126, 128, 129,
132-146, 148-155, 157, 159-161, 166, 171, 175-177, 184, 189,
194-195, 199-201, 205, 209, 223-224, 226, 228-231, 236 and 244 to
be coupled were added as a solution in DMSO. The amount of DMSO
should not exceed 10% of the total volume. The batch was stirred
for 60-120 minutes at RT, then applied to PD10 columns
(Sephadex.RTM. G-25, GE Healthcare) and eluted with PBS buffer. A
further concentration was optionally performed by
ultracentrifugation. If necessary, for better separation of
low-molecular components, the concentration step by ultrafiltration
was repeated after diluting again with PBS buffer.
[5124] Unless otherwise indicated, 5 mg of the corresponding
antibody was generally used in PBS buffer for reduction and
subsequent coupling. After purification on the PD10 column,
solutions of the corresponding ADC in 3.5 mL PBS buffer were
obtained. Then the respective protein concentration given was
determined for each of these solutions. In addition, the antibody
load (drug/mAb ratio) was determined by the methods described
below.
[5125] According to this method the immunoconjugates synthesized in
Examples 280-289, 291-302, 304-305, 313, 315-318, 320-321, 324-325,
327-328 and 330-331 were prepared.
[5126] In the structural formulas shown, AK.sub.1 has the
meaning
AK.sub.7=3ee9(partially reduced)-S.sctn..sup.1, [5127] wherein
[5128] .sctn..sup.1 denotes the linkage to the succinimide group
[5129] 3ee9 (partially reduced) stands for the partially reduced
anti-CA9 3ee9 antibody, [5130] and [5131] S stands for the sulfur
atom of a cysteine radical of the partially reduced antibody.
[5132] B-4.4 General Method for Coupling to Lysine Side Chains
[5133] To a solution of the corresponding anti-CA antibody 3ee9 in
PBS buffer in the concentration range between 1 mg/mL and 10 mg/mL,
between two and five equivalents, depending on the desired load, of
the precursor compound of the intermediates 104, 110, 115, 116,
127, 130, 131, 147, 156, 158, 162, 169, 178, 185, 190, 202, 206,
210-216, 218, 219, 227, 233, 238, 240, 242, 245, 247a and 247b to
be coupled were added as a solution in DMSO. After stirring for 30
minutes at RT, the same amount of precursor compound in DMSO was
added again. The amount of DMSO should not exceed 10% of the total
volume. After stirring for 30 minutes more at RT, the batch was
applied to PD10 columns (Sephadex.RTM. G-25) and eluted with PBS
buffer. A further concentration step by ultrafiltration was
optionally performed. Concentration by ultrafiltration was
repeated, if necessary, after diluting with PBS buffer again for
better separation of the low-molecular components.
[5134] Unless otherwise indicated, 5 mg of the corresponding
antibody in PBS buffer was generally used for coupling. After
purification on the PD10 column, solutions of the corresponding ADC
in 3.5 mL PBS buffer were obtained. Then the respective protein
concentration indicated was determined for each of these solutions,
and the antibody load (drug/mAb ratio) by the methods described
below.
[5135] According to this method the immunoconjugates synthesized in
Examples 290, 303, 306, 314, 319, 322-323, 326, 329, 332-333 and
384 were prepared.
[5136] In the structural formulas given, AK.sub.2 has the
meaning
AK.sub.8=anti-CA9-NH.sctn..sup.2
[5137] wherein
[5138] .sctn..sup.2 denotes the linkage to the carbonyl group
[5139] anti-CA9 stands for the unreduced CA9 antibody 3ee9,
[5140] and
[5141] NH stands for the side chain amino group of a lysine radical
of the antibody.
[5142] B-4.5a General Method for Synthesis of Cysteine Adducts
[5143] 10 .mu.mol of the maleimide precursor compounds described
above was placed in 3 mL DMF and mixed with 2.1 mg (20 .mu.mol)
L-cysteine. The batch was stirred for 2 hours at RT, then
concentrated in vacuo and purified by preparative HPLC.
[5144] Cys in the structural formulas given has the following
meaning:
##STR00691##
[5145] wherein
[5146] .sctn..sup.3 denotes the linkage to the linker toxophore
unit.
[5147] B-4.5b General Method for Synthesis of Ivsine Adducts:
[5148] 10 .mu.mol of the active ester precursor compounds described
above was placed in 5 mL DMF and mixed with .alpha.-amino-protected
L-lysine in the presence of 30 .mu.mol N,N-diisopropylethylamine.
The reaction mixture was stirred for 2 hours at RT and then
concentrated in vacuo and next was purified by preparative HPLC.
Then the protective group was removed by known methods.
[5149] Further Purification and Characterization of the Conjugates
According to the Invention
[5150] After successful reaction, in some cases the reaction
mixture was concentrated by ultracentrifugation, for example, and
then was desalinated and purified by chromatography for example,
using a Sephadex.RTM. G-25 column. Elution was performed using
phosphate-buffered saline solution (PBS), for example. Then the
solution was sterile filtered and frozen. Alternatively, the
conjugate may be lyophilized.
[5151] B-4.6 Determining the Toxophore Load
[5152] The toxophore load was determined as follows on the
resulting solutions of the conjugates in PBS buffer as described in
the exemplary embodiments:
[5153] The toxophore load of lysine-linked ADCs was determined by
mass spectrometric determination of the molecular weights of the
individual conjugated species. The antibody conjugates were first
deglycosylated by PNGaseF, the sample was acidified and then, after
HPLC separation, the sample was analyzed by mass spectrometry using
ESI MicroTof.sub.Q (Bruker Daltonik). All the spectra over the
signal in the TIC (total ion chromatogram) were added up and the
molecular weights of the various conjugate species were calculated
on the basis of MaxEnt deconvolution. After signal integration of
the various species, the DAR (drug/antibody ratio) was
calculated.
[5154] For protein identification, after deglycosylation and/or
denaturing, tryptic digestion was performed in addition to
determination of the molecular weight, with the identity of the
protein being confirmed on the basis of the tryptic peptides
identified after denaturing, reduction and derivatization.
[5155] The toxophore load of cysteine-linked conjugates was
determined by reversed-phase chromatography of the reduced and
dentured ADCs. Guanidinium hydrochloride (GuHCl, 28.6 mg) and a
solution of DL-dithiothreitol (DTT, 500 mM, 3 .mu.L) were added to
the ADC solution (1 mg/mL, 50 .mu.L). The mixture was then
incubated for one hour at 55.degree. C. and then analyzed by
HPLC.
[5156] HPLC analysis was performed on an adjuvant 1260 HPLC system
with detection at 220 nm, using a Polymer Laboratories PLRP-S
polymeric reversed-phase column (catalog number PL1912-3802)
(2.1.times.150 mm, 8 .mu.m particle size, 1000 .ANG.) at a flow
rate of 1 mL/min with the following gradient: 0 min, 25% B; 3 min,
25% B; 28 min, 50% B. Eluent A was 0.05% trifluoroacetic acid (TFA)
in water, and eluent B was 0.05% trifluoroacetic acid in
acetonitrile.
[5157] The peaks detected were assigned based on a comparison of
the retention times with the light chain (L0) and the heavy chain
(H0) of the unconjugated antibody. Peaks detected exclusively in
the conjugated sample were assigned to the light chain with one
toxophore (L1) and to the heavy chains with one, two and three
toxophores (H1, H2, H3).
[5158] The average toxophore load of the antibody was calculated
from the peak areas determined by integration as two times the sum
of the integration results of all peaks, weighted by the number of
toxophores, divided by the total of the integration results of all
peaks with simple weighting. In isolated cases it may occur that
the toxophore load cannot be determined accurately due to
co-elution of some peaks.
[5159] B-4.7 Testing the Antigen Binding of the ADC
[5160] The ability of the binder to bind to the target molecule was
tested after successful coupling. Those skilled in the art are
familiar with a variety of methods for doing so; for example, the
affinity of the conjugate can be tested by means of ELISA
technology or surface plasmon resonance analysis (BIAcore.TM.
measurements). The skilled person can measure the conjugate
concentration using conventional methods, for example, by protein
determination for antibody conjugates (see also Doronina et al.,
Nature Biotechnol. 2003; 21:778-784 and Polson et al., Blood 2007,
1102:616-623).
EMBODIMENTS
Example 1
##STR00692##
[5162] Protein concentration: 1.27 mg/ml
[5163] Drug/mAb ratio: 1.5
Example 2
##STR00693##
[5165] Protein concentration: 0.64 mg/ml
[5166] Drug/mAb ratio: 3.3
Example 3
##STR00694##
[5168] Protein concentration: 0.61 mg/ml
[5169] Drug/mAb ratio: 5.1
Example 4
##STR00695##
[5171] Protein concentration: 0.61 mg/ml
[5172] Drug/mAb ratio: not detectable
Example 5
##STR00696##
[5174] Protein concentration: 1.37 mg/ml
[5175] Drug/mAb ratio: 1.9
Example 6
##STR00697##
[5177] Protein concentration: 1.12 mg/ml
[5178] Drug/mAb ratio: 1.3
Example 7
##STR00698##
[5180] 270 mg cetuximab in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation
[5181] Protein concentration: 10.46 mg/ml
[5182] Drug/mAb ratio: 2.8
Example 8
##STR00699##
[5184] Protein concentration: 1.86 mg/ml
[5185] Drug/mAb ratio: 2.4
Example 9
##STR00700##
[5187] Protein concentration: 1.39 mg/ml
[5188] Drug/mAb ratio: not detectable
Example 10
##STR00701##
[5190] Protein concentration: 0.66 mg/ml
[5191] Drug/mAb ratio: 2.4
Example 11
##STR00702##
[5193] Protein concentration: 0.66 mg/ml
[5194] Drug/mAb ratio: not detectable
Example 12
##STR00703##
[5196] Protein concentration: 0.9 mg/ml
[5197] Drug/mAb ratio: 1.1
Example 13
##STR00704##
[5199] Protein concentration: 1.52 mg/ml
[5200] Drug/mAb ratio: not detectable
Example 14
##STR00705##
[5202] Protein concentration: 1.44 mg/ml
[5203] Drug/mAb ratio: 3.2
Example 16
##STR00706##
[5205] Protein concentration: 1.23 mg/ml
[5206] Drug/mAb ratio: 2.7
Example 16
##STR00707##
[5208] Protein concentration: 1.27 mg/mi
[5209] Drug/mAb ratio: 1.3
Example 17
##STR00708##
[5211] Protein concentration: 1.61 mg/ml
[5212] Drug/mAb ratio: 4.7
Example 18
##STR00709##
[5214] Protein concentration: 1.24 mg/ml
[5215] Drug/mAb ratio: 2.4
Example 19
##STR00710##
[5217] Protein concentration: 1.49 mg/ml
[5218] Drug/mAb ratio: 1.9
Example 20
##STR00711##
[5220] Protein concentration: 1.49 mg/ml
[5221] Drug/mAb ratio: 2.0
Example 21
##STR00712##
[5223] Protein concentration: 1.46 mg/ml
[5224] Drug/mAb ratio: >0.9
Example 22
##STR00713##
[5226] Protein concentration: 1.28 mg/ml
[5227] Drug/mAb ratio: not detectable
Example 23
##STR00714##
[5229] Protein concentration: 1.33 mg/ml
[5230] Drug/mAb ratio: 1.8
Example 24
##STR00715##
[5232] Protein concentration: 1.39 mg/ml
[5233] Drug/mAb ratio: >0.8
Example 25
##STR00716##
[5235] Protein concentration: 1.26 mg/ml
[5236] Drug/mAb ratio: not detectable
Example 26
##STR00717##
[5238] Protein concentration: 1.51 mg/ml
[5239] Drug/mAb ratio: 1.8
Example 27
##STR00718##
[5241] Protein concentration: 1.6 mg/ml
[5242] Drug/mAb ratio: 1.8
Example 28
##STR00719##
[5244] Protein concentration: 1.21 mg/ml
[5245] Drug/mAb ratio: not detectable
Example 29
##STR00720##
[5247] Protein concentration: 1.53 mg/ml
[5248] Drug/mAb ratio: 2.7
Example 30
##STR00721##
[5250] Protein concentration: 1.4 mg/ml
[5251] Drug/mAb ratio: not detectable
Example 31
##STR00722##
[5253] Protein concentration: 1.66 mg/ml
[5254] Drug/mAb ratio: 2.2
Example 32
##STR00723##
[5256] Protein concentration: 1.21 mg/ml
[5257] Drug/mAb ratio: 2.2
Example 33
##STR00724##
[5259] Protein concentration: 1.46 mg/ml
[5260] Drug/mAb ratio: 2
Example 34
##STR00725##
[5262] Protein concentration: 1.2 mg/ml
[5263] Drug/mAb ratio: not detectable
Example 35
##STR00726##
[5265] Protein concentration: 1.66 mg/ml
[5266] Drug/mAb ratio: not detectable
Example 36
##STR00727##
[5268] Protein concentration: 1.48 mg/ml
[5269] Drug/mAb ratio: 2.2
Example 37
##STR00728##
[5271] Protein concentration: 1.45 mg/ml
[5272] Drug/mAb ratio: 2.7
Example 38
##STR00729##
[5274] Protein concentration: 1.5 mg/ml
[5275] Drug/mAb ratio: 0.15
Example 39
##STR00730##
[5277] Protein concentration: 1.5 mg/ml
[5278] Drug/mAb ratio: 2.1
Example 40
##STR00731##
[5280] Protein concentration: 1.54 mg/ml
[5281] Drug/mAb ratio: >0.9
Example 41
##STR00732##
[5283] Protein concentration: 1.39 mg/ml
[5284] Drug/mAb ratio: not detectable
Example 42
##STR00733##
[5286] Protein concentration: 1.52 mg/ml
[5287] Drug/mAb ratio: 1.5
Example 43
##STR00734##
[5289] Protein concentration: 1.44 mg/ml
[5290] Drug/mAb ratio: 2.6
Example 44
##STR00735##
[5292] Protein concentration: 1.45 mg/ml
[5293] Drug/mAb ratio: 1.9
Examples for Cysteine Adducts
Example 45
N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin--
1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S-
)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-(1H-indol-3-yl)ethyl]amino}-1-methoxy-2--
methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-
-methyl-L-valinamide
##STR00736##
[5295] 10 mg (10 .mu.mol) of Intermediate 113 were taken up in 5.2
ml DMF and mixed with 2.28 mg (20 .mu.mol) L-cysteine. The batch
was stirred for 2 hours at RT, then concentrated in vacuo and next
purified by means of preparative HPLC. The yield was 6 mg (54% of
theory) of the title compound.
[5296] HPLC (Method 5): R.sub.t=1.5 min;
[5297] LC-MS (Method 1): R.sub.t=0.77 min; MS (ESIpos): m/z=1185
(M+H).sup.+.
Example 46
N-(4-{(2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-
-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-met-
hoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-y-
lcarbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methy-
l-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00737##
[5299] 9 mg (8.3 .mu.mol) of Intermediate 132 were taken up in 4 ml
DMF and mixed with 3 mg (24.4 .mu.mol) L-cysteine. The batch was
stirred overnight at RT, then concentrated in vacuo and next
purified by means of preparative HPLC. The yield was 6.8 mg (68% of
theory) of the title compound.
[5300] HPLC (Method 12): R.sub.t=1.8 min;
[5301] LC-MS (Method 1): R.sub.t=0.78 min; MS (ESIpos): m/z=1227
(M+H).sup.+.
Example 47
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)h-
exyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino-3-
-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]py-
rrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00738##
[5303] 10 mg (10 .mu.mol) of Intermediate 106 were taken up in 5.8
ml DMF and mixed with 2.5 mg (20 .mu.mol) L-cysteine. The batch was
stirred for 2 hours at RT, then concentrated in vacuo and next
purified by means of preparative HPLC. The yield was 5.2 mg (46% of
theory) of the title compound.
[5304] HPLC (Method 5): R.sub.t=1.5 min;
[5305] LC-MS (Method 11): R.sub.t=0.71 min; MS (ESIpos): m/z=1070
(M+H).sup.+.
Example 48
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)h-
exyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-
-2-(1H-indol-3-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-
-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00739##
[5307] 10 mg (10 .mu.mol) of Intermediate 124 were taken up in 4 ml
DMF and mixed with 2.5 mg (20 .mu.mol) L-cysteine. The batch was
stirred for 2 hours at RT, then concentrated in vacuo and next
purified by means of preparative HPLC. The yield was 7.2 mg (64% of
theory) of the title compound.
[5308] HPLC (Method 5): R.sub.t=1.6 min;
[5309] LC-MS (Method 1): R.sub.t=0.8 min; MS (ESIpos): m/z=1071
(M+H).sup.+.
Example 49
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)h-
exyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-ind-
ol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl--
3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-
-L-valinamide
##STR00740##
[5311] 10 mg (10 .mu.mol) of Intermediate 125 were taken up in 4 ml
DMF and mixed with 2.4 mg (20 .mu.mol) L-cysteine. The batch was
stirred for 2 hours at RT, then concentrated in vacuo and next
purified by means of preparative HPLC. The yield was 7.7 mg (69% of
theory) of the title compound.
[5312] HPLC (Method 5): R.sub.t=1.7 min;
[5313] LC-MS (Method 2): R.sub.t=1.91 min; MS (ESIpos): m/z=1140
(M+H).sup.+.
Example 50
N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin--
1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S-
)-2-[(1R,2R)-3-{[(2S)-1-(benzylamino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl]a-
mino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-
-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00741##
[5315] 10 mg (10 .mu.mol) of Intermediate 160 were taken up in 3 ml
DMF and mixed with 2.1 mg (20 .mu.mol) L-cysteine. The batch was
stirred for 2 hours at RT, then concentrated in vacuo and next
purified by means of preparative HPLC. The yield was 8.1 mg (73% of
theory) of the title compound.
[5316] HPLC (Method 5): R.sub.t=1.7 min;
[5317] LC-MS (Method 1): R.sub.t=0.86 min; MS (ESIpos): m/z=1274
(M+H).sup.+.
Example 51
N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin--
1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S-
)-2-[(1R,2R)-3-{[(2S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1--
methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxohept-
an-4-yl]-N-methyl-L-valinamide
##STR00742##
[5319] 10 mg (10 .mu.mol) of Intermediate 157 were taken up in 5.2
ml DMF and mixed with 2.28 mg (20 .mu.mol) L-cysteine. The batch
was stirred for 2 hours at RT, then concentrated in vacuo and next
purified by means of preparative HPLC. The yield was 5.8 mg (48% of
theory) of the title compound.
[5320] HPLC (Method 5): R.sub.t=1.45 min;
[5321] LC-MS (Method 1): R.sub.t=0.74 min; MS (ESIpos): m/z=1184
(M+H).sup.+.
Example 52
##STR00743##
[5323] 5 mg cetuximab in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[5324] Protein concentration: 1.73 mg/ml
[5325] Drug/mAb ratio: 2.8
Example 53
##STR00744##
[5327] 5 mg cetuximab in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[5328] Protein concentration: 0.86 mg/ml
[5329] Drug/mAb ratio: 4.9
Example 54
##STR00745##
[5331] 5 mg cetuximab in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[5332] Protein concentration: 1.64 mg/ml
[5333] Drug/mAb ratio: 0.7
Example 55
##STR00746##
[5335] 5 mg cetuximab in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[5336] Protein concentration: 1.43 mg/ml
[5337] Drug/mAb ratio: 3.2
Example 56
##STR00747##
[5339] Protein concentration: 0.96 mg/ml
[5340] Drug/mAb ratio: 3.1
Example 57
##STR00748##
[5342] Protein concentration: 0.44 mg/ml
[5343] Drug/mAb ratio: 4.6
Example 58
##STR00749##
[5345] Protein concentration: 1.09 mg/ml
[5346] Drug/mAb ratio: 2.1
Example 59
##STR00750##
[5348] Protein concentration: 0.87 mg/ml
[5349] Drug/mAb ratio: 3.8
Example 60
##STR00751##
[5351] Protein concentration: 0.45 mg/ml
[5352] Drug/mAb ratio: 6.5
Example 61
##STR00752##
[5354] Protein concentration: 0.15 mg/ml
[5355] Drug/mAb ratio: 3.1
Example 62
##STR00753##
[5357] Protein concentration: 0.94 mg/ml
[5358] Drug/mAb ratio: 2.8
Example 63
##STR00754##
[5360] Protein concentration: 0.45 mg/ml
[5361] Drug/mAb ratio: 0.9
Example 64
##STR00755##
[5363] Protein concentration: 0.51 mg/ml
[5364] Drug/mAb ratio: 6.6
Example 65
##STR00756##
[5366] Protein concentration: 0.47 mg/ml
[5367] Drug/mAb ratio: 4.2
Example 66
##STR00757##
[5369] Protein concentration: 0.45 mg/ml
[5370] Drug/mAb ratio: 5.9
Example 67
##STR00758##
[5372] Protein concentration: 0.47 mg/ml
[5373] Drug/mAb ratio: 3.3
Example 68
##STR00759##
[5375] Protein concentration: 0.53 mg/ml
[5376] Drug/mAb ratio: 2.8
Example 69
##STR00760##
[5378] Protein concentration: 0.92 mg/ml
[5379] Drug/mAb ratio: 3.5
Example 70
##STR00761##
[5381] Protein concentration: 0.09 mg/ml
[5382] Drug/mAb ratio: nd
Example 71
##STR00762##
[5384] Protein concentration: 0.62 mg/ml
[5385] Drug/mAb ratio: 1.8
Example 72
##STR00763##
[5387] Protein concentration: 0.55 mg/ml
[5388] Drug/mAb ratio: 3.8
Example 73
##STR00764##
[5390] Protein concentration: 0.54 mg/ml
[5391] Drug/mAb ratio: 4.4
Example 74
##STR00765##
[5393] Protein concentration: 0.56 mg/ml
[5394] Drug/mAb ratio: 4.0
Example 75
##STR00766##
[5396] Protein concentration: 1.1 mg/ml
[5397] Drug/mAb ratio: 0.3
Example 76
##STR00767##
[5399] Protein concentration: 0.61 mg/ml
[5400] Drug/mAb ratio: 0.9
Example 77
##STR00768##
[5402] Protein concentration: 0.57 mg/ml
[5403] Drug/mAb ratio: 1.2
Example 78
##STR00769##
[5405] 100 mg MF-Ta in PBS were presently used for the coupling
and, after purification on a Sephadex column, the reaction mixture
was concentrated by ultracentrifugation.
[5406] Protein concentration: 11.2 mg/ml
[5407] Drug/mAb ratio: 3.4
Example 79
##STR00770##
[5409] Protein concentration: 1.56 mg/ml
[5410] Drug/mAb ratio: 2.8
Example 80
##STR00771##
[5412] Protein concentration: 0.60 mg/ml
[5413] Drug/mAb ratio: 2.4
Example 81
##STR00772##
[5415] Protein concentraion: 0.584 mg/ml
[5416] Drug/mAb ratio: 2.6
Example 82
##STR00773##
[5418] Protein concentration: 0.39 mg/ml
[5419] Drug/mAb ratio: 0.8
Example 83
##STR00774##
[5421] 100 mg MF-Ta in PBS were presently used for the coupling
and, after purification on a Sephadex column, the reaction mixture
was concentrated by ultracentrifugation.
[5422] Protein concentration: 13.2 mg/ml
[5423] Drug/mAb ratio: 4.6
Example 84
##STR00775##
[5425] Protein concentration: 0.98 ml
[5426] Drug/mAb ratio: 1.1
Example 85
##STR00776##
[5428] Protein concentration: 0.55 mg/ml
[5429] Drug/mAb ratio: not detectable
Example 86
##STR00777##
[5431] 40 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation.
[5432] Protein concentration: 10.6 mg/ml
[5433] Drug/mAb ratio: 4.1
Example 87
##STR00778##
[5435] Protein concentration: 0.96 mg/ml
[5436] Drug/mAb ratio: 0.4
Example 88
##STR00779##
[5438] 70 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation.
[5439] Protein concentration: 12.7 mg/ml
[5440] Drug/mAb ratio: 3.6
Example 89
##STR00780##
[5442] Protein concentration: 1.1 mg/ml
[5443] Drug/mAb ratio: 2.7
Example 90
##STR00781##
[5445] Protein concentration: 1.24 mg/ml
[5446] Drug/mAb ratio: 2.6
Example 91
##STR00782##
[5448] Protein concentration: 0.99 mg/ml
[5449] Drug/mAb ratio: 2.3
Example 92
##STR00783##
[5451] Protein concentration: 1.22 mg/ml
[5452] Drug/mAb ratio: 3.3
Example 93
##STR00784##
[5454] Protein concentration: 1.34 mg/ml
[5455] Drug/mAb ratio: 1.2
Example 94
##STR00785##
[5457] Protein concentration: 1.28 mg/ml
[5458] Drug/mAb ratio: 3.2
Example 95
##STR00786##
[5460] 70 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation.
[5461] Protein concentration: 10.9 mg/ml
[5462] Drug/mAb ratio: 5.1
Example 96
##STR00787##
[5464] 100 mg MF-Ta in PBS were presently used for the coupling
and, after purification on a Sephadex column, the reaction mixture
was concentrated by ultracentrifugation.
[5465] Protein concentration: 10.3 mg/ml
[5466] Drug/mAb ratio: 4.3
Example 97
##STR00788##
[5468] Protein concentration: 1.08 mg/ml
[5469] Drug/mAb ratio: 2.8
Example 98
##STR00789##
[5471] Protein concentration: 1.24 mg/ml
[5472] Drug/mAb ratio: 2.8
Example 99
##STR00790##
[5474] Protein concentration: 1.28 mg/mi
[5475] Drug/mAb ratio: 3.8
Example 100
##STR00791##
[5477] Protein concentration: 1.07 mg/ml
[5478] Drug/mAb ratio: 3.0
Example 101
##STR00792##
[5480] Protein concentration: 1.35 mg/ml
[5481] Drug/mAb ratio: 4.0
Example 102
##STR00793##
[5483] 100 mg MF-Ta in PBS were presently used for the coupling
and, after purification on a Sephadex column, the reaction mixture
was concentrated by ultracentrifugation.
[5484] Protein concentration: 12.2 mg/ml
[5485] Drug/mAb ratio: 5.6
Example 103
##STR00794##
[5487] Protein concentration: 1.32 mg/ml
[5488] Drug/mAb ratio: 3.2
Example 104
##STR00795##
[5490] Protein concentration: 1.01 mg/ml
[5491] Drug/mAb ratio: 0.9
Example 105
##STR00796##
[5493] Protein concentration: 1.03 mg/ml
[5494] Drug/mAb ratio: 0.3
Example 106
##STR00797##
[5496] Protein concentration: 0.62 mg/ml
[5497] Drug/mAb ratio: 3.1
[5498] This ADC was concentrated by Vivaspin centrifugation and
diluted again, followed by another concentration and dilution.
Example 107
##STR00798##
[5500] Protein concentration: 1.26 mg/ml
[5501] Drug/mAb ratio: not detectable
Example 108
##STR00799##
[5503] Protein concentration: 1.55 mg/ml
[5504] Drug/mAb ratio: not detectable
Example 109
##STR00800##
[5506] Protein concentration: 1.23 mg/ml
[5507] Drug/mAb ratio: 3.5
Example 110
##STR00801##
[5509] Protein concentration: 1.44 mg/ml
[5510] Drug/mAb ratio: 4.1
Example 111
##STR00802##
##STR00803##
[5512] 5 mg MF-T in PBS were presently used for the coupling and,
after purification on a Sephadex column, the batch was concentrated
by ultracentrifugation and diluted again.
[5513] Protein concentration: 0.77 mg/ml
[5514] Drug/mAb ratio: >1.5 (not exactly detectable)
Example 113
##STR00804##
[5516] Protein concentration: 1.3 mg/ml
[5517] Drug/mAb ratio: 2.0
Example 114
##STR00805##
[5519] 500 mg MF-Ta in PBS were presently used for the coupling
and, after purification on a Sephadex column, the reaction mixture
was concentrated by ultracentrifugation.
[5520] Protein concentration: 11.2 mg/ml
[5521] Drug/mAb ratio: 3.7
Example 115
##STR00806##
[5523] 100 mg MF-Ta in PBS were presently used for the coupling
and, after purification on a Sephadex column, the reaction mixture
was concentrated by ultracentrifugation.
[5524] Protein concentration: 11.4 mg/ml
[5525] Drug/mAb ratio: 3.9
Example 116
##STR00807##
[5527] 60 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation.
[5528] Protein concentration: 10.5 mg/ml
[5529] Drug/mAb ratio: 4.4
Example 117
N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin--
1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S-
)-2-[(1R,2R)-3-{[(2S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1--
methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxohept-
an-4-yl]-N-methyl-L-valinamide
##STR00808##
[5531] 10 mg (10 .mu.mol) of Intermediate 157 were taken up in 5.2
ml DMF and mixed with 2.28 mg (20 .mu.mol) L-cysteine. The reaction
mixture was stirred for 2 hours at RT, then concentrated in vacuo
and next purified by means of preparative HPLC. The yield was 5.8
mg (48% of theory) of the title compound.
[5532] HPLC (Method 5): R.sub.t=1.45 min;
[5533] LC-MS (Method 1): R.sub.t=0.74 min; MS (ESIpos): m/z=1184
(M+H).sup.+.
Example 118
N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin--
1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S-
)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-(1H-indol-3-yl)ethyl]amino}-1-methoxy-2--
methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-
-methyl-L-valinamide
##STR00809##
[5535] 10 mg (10 .mu.mol) of Intermediate 113 were taken up in 5.2
ml DMF and mixed with 2.28 mg (20 .mu.mol) L-cysteine. The reaction
mixture was stirred for 2 hours at RT, then concentrated in vacuo
and next purified by means of preparative HPLC. The yield was 6 mg
(54% of theory) of the title compound.
[5536] HPLC (Method 5): R.sub.t=1.5 min;
[5537] LC-MS (Method 1): R.sub.t=0.77 min; MS (ESIpos): m/z=1185
(M+H).sup.+.
Example 119
N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin--
1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-meth-
oxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-yl-
carbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-
-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00810##
[5539] 9 mg (8.3 .mu.mol) of Intermediate 132 were taken up in 4 ml
DMF and mixed with 3 mg (24.4 .mu.mol) L-cysteine. The reaction
mixture was stirred overnight at RT, then concentrated in vacuo and
next purified by means of preparative HPLC. The yield was 6.8 mg
(68% of theory) of the title compound.
[5540] HPLC (Method 12): R.sub.t=1.8 min;
[5541] LC-MS (Method 1): R.sub.t=0.78 min; MS (ESIpos): m/z=1227
(M+H).sup.+.
Example 120
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)h-
exyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino-3-
-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]py-
rrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00811##
[5543] 10 mg (10 .mu.mol) of Intermediate 106 were taken up in 5.8
ml DMF and mixed with 2.5 mg (20 .mu.mol) L-cysteine. The reaction
mixture was stirred for 2 hours at RT, then concentrated in vacuo
and next purified by means of preparative HPLC. The yield was 5.2
mg (46% of theory) of the title compound.
[5544] HPLC (Method 5): R.sub.t=1.5 min;
[5545] LC-MS (Method 11): R.sub.t=0.71 min; MS (ESIpos): m/z=1070
(M+H).
Example 121
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)h-
exyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-
-2-(1H-indol-3-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-
-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00812##
[5547] 10 mg (10 .mu.mol) of Intermediate 124 were taken up in 4 ml
DMF and mixed with 2.5 mg (20 .mu.mol) L-cysteine. The reaction
mixture was stirred for 2 hours at RT, then concentrated in vacuo
and next purified by means of preparative HPLC. The yield was 7.2
mg (64% of theory) of the title compound.
[5548] HPLC (Method 5): R.sub.t=1.6 min;
[5549] LC-MS (Method 1): R.sub.t=0.8 min; MS (ESIpos): m/z=1071
(M+H).sup.+.
Example 122
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)h-
exyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-ind-
ol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl--
3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-
-L-valinamide
##STR00813##
[5551] 10 mg (10 .mu.mol) of Intermediate 125 were taken up in 4 ml
DMF and mixed with 2.4 mg (20 .mu.mol) L-cysteine. The reaction
mixture was stirred for 2 hours at RT, then concentrated in vacuo
and next purified by means of preparative HPLC. The yield was 7.7
mg (69% of theory) of the title compound.
[5552] HPLC (Method 5): R.sub.t=1.7 min;
[5553] LC-MS (Method 2): R.sub.t=1.91 min; MS (ESIpos): m/z=1140
(M+H).sup.+.
Example 123
N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin--
1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S-
)-2-[(1R,2R)-3-{[(2S)-1-(benzylamino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl]a-
mino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-
-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00814##
[5555] 10 mg (10 .mu.mol) of Intermediate 160 were taken up in 3 ml
DMF and mixed with 2.1 mg (20 .mu.mol) L-cysteinet. The reaction
mixture was stirred for 2 hours at RT, then concentrated in vacuo
and next purified by means of preparative HPLC. The yield was 8.1
mg (73% of theory) of the title compound.
[5556] HPLC (Method 5): R.sub.t=1.7 min;
[5557] LC-MS (Method 1): R.sub.t=0.86 min; MS (ESIpos): m/z=1274
(M+H).sup.+.
Example 124
N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin--
1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S-
)-2-[(1R,2R)-3-{[(2S)-1-(benzylamino)-1-oxo-3-phenylpropan-2-yl]amino}-1-m-
ethoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxohepta-
n-4-yl]-N-methyl-L-valinamide
##STR00815##
[5559] 3.5 mg (3 .mu.mol) of Intermediate 159 were taken up in 1 ml
DMF and mixed with 0.76 mg (6 .mu.mol) L-cysteine. The reaction
mixture was stirred for 2 hours at RT, then concentrated in vacuo
and next purified by means of preparative HPLC. The yield was 2.6
mg (65% of theory) of the title compound.
[5560] HPLC (Method 5): R.sub.t=1.75 min;
[5561] LC-MS (Method 1): R.sub.t=0.85 min; MS (ESIpos): m/z=1235
(M+H).sup.+.
Example 125
N-(6-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin--
1-yl)hexanoyl]hydrazino}-6-oxohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-meth-
oxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-yl-
carbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-
-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00816##
[5563] 3.6 mg (3 .mu.mol) of Intermediate 129 were taken up in 1 ml
DMF and mixed with 0.77 mg (6 .mu.mol) L-cysteine. The reaction
mixture was stirred for 2 hours at RT, then concentrated in vacuo
and next purified by means of preparative HPLC. The yield was 1.55
mg (39% of theory) of the title compound.
[5564] HPLC (Method 5): R.sub.t=1.6 min;
[5565] LC-MS (Method 1): R.sub.t=0.87 min; MS (ESIpos): m/z=1255
(M+H).sup.+.
Example 126
##STR00817##
[5567] 5 mg MF-T[a] in PBS were presently used for the coupling
and, after purification on a Sephadex column, the reaction mixture
was concentrated by ultracentrifugation and diluted again.
[5568] Protein concentration: 0.9 mg/ml
[5569] Drug/mAb ratio: 1
Example 127
##STR00818##
[5571] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5572] Protein concentration: 1.86 mg/ml
[5573] Drug/mAb ratio: 2.9
Example 128
##STR00819##
[5575] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5576] Protein concentration: 1.05 mg/ml
[5577] Drug/mAb ratio: 4.4
Example 129
##STR00820##
[5579] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5580] Protein concentration: 1.13 mg/ml
[5581] Drug/mAb ratio: 2.8
Example 130
##STR00821##
[5583] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5584] Protein concentration: 1.41 mg/ml
[5585] Drug/mAb ratio: 3.9
Example 131
##STR00822##
[5587] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5588] Protein concentration: 1.38 mg/ml
[5589] Drug/mAb ratio: 4.3
Example 132
##STR00823##
[5591] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5592] Protein concentration: 1.32 mg/ml
[5593] Drug/mAb ratio: 1
Example 133
##STR00824##
[5595] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5596] Protein concentration: 1.14 mg/ml
[5597] Drug/mAb ratio: 5.3
Example 134
##STR00825##
[5599] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5600] Protein concentration: 1.25 mg/ml
[5601] Drug/mAb ratio: 4.8
Example 135
##STR00826##
[5603] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5604] Protein concentration: 1.12 mg/ml
[5605] Drug/mAb ratio: 1.7
Example 136
##STR00827##
[5607] 150 mg MF-Ta in PBS were presently used for the coupling
and, after purification on a Sephadex column, the reaction mixture
was concentrated by ultracentrifugation, diluted again with PBS and
concentrated again.
[5608] Protein concentration: 12.2 mg/ml
[5609] Drug/mAb ratio: 4.1
Example 137
##STR00828##
[5611] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5612] Protein concentration: 0.86 mg/ml
[5613] Drug/mAb ratio: 3.4
Example 138
##STR00829##
[5615] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5616] Protein concentration: 1.43 mg/ml
[5617] Drug/mAb ratio: 3.7
Example 139
##STR00830##
[5619] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5620] Protein concentration: 0.8 mg/ml
[5621] Drug/mAb ratio: 0.7
Example 140
##STR00831##
[5623] 50 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation, diluted again with PBS and
reconcentrated.
[5624] Protein concentration: 9.5 mg/ml
[5625] Drug/mAb ratio: 2.9
Example 141
##STR00832##
[5627] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5628] Protein concentration: 1.52 mg/ml
[5629] Drug/mAb ratio: 3.2
Example 142
##STR00833##
[5631] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5632] Protein concentration: 1.25 mg/ml
[5633] Drug/mAb ratio: 4.6
Example 143
##STR00834##
[5635] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5636] Protein concentration: 1.47 mg/ml
[5637] Drug/mAb ratio: 1.6
Example 144
##STR00835##
[5639] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5640] Protein concentration: 0.99 mg/ml
[5641] Drug/mAb ratio: 5.5
Example 145
##STR00836##
[5643] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5644] Protein concentration: 1.02 mg/ml
[5645] Drug/mAb ratio: 4.0
Example 146
##STR00837##
[5647] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5648] Protein concentration: 1.63 mg/ml
[5649] Drug/mAb ratio: 3.8
Example 147
##STR00838##
[5651] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5652] Protein concentration: 1.27 mg/ml
[5653] Drug/mAb ratio: 3.0
Example 148
##STR00839##
[5655] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5656] Protein concentration: 1.58 mg/ml
[5657] Drug/mAb ratio: 0.6
Example 149
##STR00840##
[5659] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5660] Protein concentration: 1.31 mg/ml
[5661] Drug/mAb ratio: 6.6
Example 150
##STR00841##
[5663] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5664] Protein concentration: 1.75 mg/ml
[5665] Drug/mAb ratio: 1.8
Example 151
##STR00842##
[5667] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5668] Protein concentration: 1.44 mg/ml
[5669] Drug/mAb ratio: 2.5
Example 152
##STR00843##
[5671] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5672] Protein concentration: 1.96 mg/ml
[5673] Drug/mAb ratio: 5.6
Example 153
##STR00844##
[5675] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5676] Protein concentration: 1.58 mg/ml
[5677] Drug/mAb ratio: 4.2
Example 154
##STR00845##
[5679] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5680] Protein concentration: 1.48 mg/ml
[5681] Drug/mAb ratio: 4.6
Example 155
##STR00846##
[5683] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5684] Protein concentration: 1.5 mg/ml
[5685] Drug/mAb ratio: 3.1
Example 156
##STR00847##
[5687] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5688] Protein concentration: mg/ml
[5689] Drug/mAb ratio:
Example 157
##STR00848##
[5691] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5692] Protein concentration: 1.62 mg/ml
[5693] Drug/mAb ratio: 2.2
Example 158
##STR00849##
[5695] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5696] Protein concentration: 1.37 mg/ml
[5697] Drug/mAb ratio: 2.8
Example 159
##STR00850##
[5699] 5 mg MF-Ta in PBS were presently used for the coupling and,
after purification on a Sephadex column, the reaction mixture was
concentrated by ultracentrifugation and diluted again.
[5700] Protein concentration: 1.43 mg/ml
[5701] Drug/mAb ratio: 4.0
Example 160**
N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-N--
[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan--
2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1--
yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate
##STR00851##
[5703] 15.5 mg (15 .mu.mol) of Intermediate 210 were taken up in 5
ml DMF and mixed with 4.4 mg (18 .mu.mol)
N.sup.2-(tert.-butoxycarbonyl)-L-lysine and 7.7 .mu.L (44 .mu.mol)
N,N-diisopropylethylamine. The reaction mixture was stirred
overnight at RT, then concentrated in vacuo. The residue was
purified by means of preparative HPLC. The yield was 14 mg (81% of
theory) of the protected intermediate of the title compound, which
were then taken up in 1 ml dichloromethane and deprotected with 1
ml trifluoroascetic acid. The reaction mixture was concentrated
and, after lyophilization of the residue from acetonitrile/wasser
1:1, 15 mg (97% of theory) of the title compound were obtained.
[5704] HPLC (Method 12): R.sub.t=1.8 min;
[5705] LC-MS (Method 1): R.sub.t=0.79 min; MS (ESIpos): m/z=1083
(M+H).sup.+.
Example 161
N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-N--
[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-(1H-indol-3-yl)ethyl]a-
mino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-
-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00852##
[5707] 40 mg (40 .mu.mol) of Intermediate 226 were taken up in 5 ml
DMF and mixed with 11.5 mg (40 .mu.mol)
N.sup.2-[(benzyloxy)carbonyl]-L-lysine and 13 .mu.l (80 .mu.mol)
N,N-diisopropylethylamine. The reaction mixture was stirred
overnight at RT, then concentrated in vacuo and next purified by
means of preparative HPLC. The yield was 32.5 mg (70% of theory) of
the protected intermediate of the title compound.
[5708] These 32.5 mg of the intermediate were dissolved in 10 ml
methanol and, after adding 2 mg 10% palladium on activated carbon,
hydrogenated for 30 min at RT under standard hydrogen pressure. The
catalyst was then filtered off and the solvent removed in vacuo.
Following lyophilization of the residue from dioxane/water 1:1, 26
mg (99% of theory) of the title compound were obtained.
[5709] HPLC (Method 12): R.sub.t=1.7 min;
[5710] LC-MS (Method 1): R.sub.t=0.76 min; MS (ESIpos): m/z=1014
(M+H).sup.+.
Example 162
N-[(18S)-18-amino-18-carboxy-12-oxo-3,6,9-trioxa-13-azaoctadec-1-yl]-N-met-
hyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1--
(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl-
]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinami-
de trifluoroacetate
##STR00853##
[5712] 3.5 mg (3 .mu.mol) of Intermediate 202 were taken up in 2 ml
DMF and mixed with 0.8 mg (3 .mu.mol)
N.sup.2-(tert-butoxycarbonyl)-L-lysine and 1.6 .mu.l (10 .mu.mol)
N,N-diisopropylethylamine. The reaction mixture was stirred
overnight at RT and then concentrated in vacuo. The residue was
taken up in acetonitrile/water 1:1, adjusted to pH 2 with
trifluoroascetic acid and then purified by means of preparative
HPLC. The yield was 1 mg (25% of theory) of the protected
intermediate of the title compound that was then taken up in 500
.mu.l dichloromethane and deprotected with 500 .mu.l
trifluoroascetic acid. The reaction mixture was concentrated and,
following lyophilization of the residue from acetonitrile/water
1:1, 1 mg (89% of theory) of the title compound was obtained.
[5713] HPLC (Method 12): R.sub.t=1.9 min;
[5714] LC-MS (Method 1): R.sub.t=0.82 min; MS (ESIpos): m/z=1173
(M+H).sup.+.
Example 163
##STR00854##
[5716] 70 mg anti-C4.4a M31-B01 in DPBS pH 7.4 were presently used
for the coupling and, after purification on a Sephadex column, the
batch was concentrated by ultracentrifugation.
[5717] Protein concentration: 12.2 mg/ml
[5718] Drug/mAb ratio: 1.5
Example 164
##STR00855##
[5720] Protein concentration: 0.87 mg/ml
[5721] Drug/mAb ratio: 5.8
##STR00856##
Example 165
[5722] Protein concentration: 1.16 mg/ml
[5723] Drug/mAb ratio: 3.1
Example 166
##STR00857##
[5725] Protein concentration: 1.24 mg/ml
[5726] Drug/mAb ratio: 1.6
Example 167
##STR00858##
[5728] Protein concentration: 0.88 mg/ml
[5729] Drug/mAb ratio: 6.9
Example 168
##STR00859##
[5731] Protein concentration: 1.2 mg/ml
[5732] Drug/mAb ratio: 2.8
Example 169
##STR00860##
[5734] Protein concentration: 0.9 mg/ml
[5735] Drug/mAb ratio: 3.9
Example 170
##STR00861##
[5737] Protein concentration: 0.52 mg/ml
[5738] Drug/mAb ratio: 1.6
Example 171
##STR00862##
[5740] Protein concentration: 0.47 mg/ml
[5741] Drug/mAb ratio: 6.6
Example 172
##STR00863##
[5743] Protein concentration: 0.77 mg/ml
[5744] Drug/mAb ratio: 6.9
Example 173
##STR00864##
[5746] Protein concentration: 0.47 mg/ml
[5747] Drug/mAb ratio: 4.0
Example 174
##STR00865##
[5749] Protein concentration: 1.46 mg/ml
[5750] Drug/mAb ratio: 2.5
Example 175
##STR00866##
[5752] Protein concentration: 0.45 mg/ml
[5753] Drug/mAb ratio: 3.3
Example 176
##STR00867##
[5755] Protein concentration: 0.98 mg/ml
[5756] Drug/mAb ratio: 3.6
Example 177
##STR00868##
[5758] 70 mg anti-C4.4a M31-B01 in DPBS pH 7.4 were presently used
for the coupling and, after purification on a Sephadex column, the
batch was concentrated by ultracentrifugation.
[5759] Protein concentration: 9.42 mg/ml
[5760] Drug/mAb ratio: 4.1
Example 178
##STR00869##
[5762] Protein concentration: 0.65 mg/ml
[5763] Drug/mAb ratio: 1.8
Example 179
##STR00870##
[5765] Protein concentration: 1.07 mg/ml
[5766] Drug/mAb ratio: not detectable
Example 180
##STR00871##
[5768] Protein concentration: 0.47 mg/ml
[5769] Drug/mAb ratio: 4.4
Example 181
##STR00872##
[5771] Protein concentration: 0.43 mg/ml
[5772] Drug/mAb ratio: 4.8
Example 182
##STR00873##
[5774] Protein concentration: 1.01 mg/ml
[5775] Drug/mAb ratio: 2.6
Example 183
##STR00874##
[5777] Protein concentration: 0.53 mg/ml
[5778] Drug/mAb ratio: 0.6
Example 184
##STR00875##
[5780] Protein concentration: 0.55 mg/ml
[5781] Drug/mAb ratio: 1.3
Example 185
##STR00876##
[5783] Protein concentration: 0.65 mg/ml
[5784] Drug/mAb ratio: 1.1
Example 186
##STR00877##
[5786] Protein concentration: 1.04
[5787] Drug/mAb ratio: 3.5
Example 187
##STR00878##
[5789] Protein concentration: 0.62 mg/ml
[5790] Drug/mAb ratio: 2.4
Example 188
##STR00879##
[5792] 90 mg anti-C4.4a M31-B01 in DPBS pH 7.4 were presently used
for the coupling and, after purification on a Sephadex column, the
batch was concentrated by ultracentrifugation.
[5793] Protein concentration: 11.2 mg/ml
[5794] Drug/mAb-Ratio: 2.3
Example 189
##STR00880##
[5796] Protein concentration: 1.11 mg/ml
[5797] Drug/mAb-Ratio: 2.4
Example 190
##STR00881##
[5799] 70 mg anti-C4.4a M31-B01 in DPBS pH 7.4 were presently used
for the coupling and, after purification on a Sephadex column, the
batch was concentrated by ultracentrifugation.
[5800] Protein concentration: 10.7 mg/ml
[5801] Drug/mAb ratio: 2.2
Example 191
##STR00882##
[5803] Protein concentration: 0.87 mg/ml
[5804] Drug/mAb ratio: 1.8
Example 192
##STR00883##
[5806] Protein concentration: 1.3 mg/ml
[5807] Drug/mAb ratio: 2.1
Example 193
##STR00884##
[5809] Protein concentration: 1.3 mg/ml
[5810] Drug/mAb ratio: 0.3
Example 194
##STR00885##
[5812] 70 mg anti-C4.4a M31-B01 in DPBS pH 7.4 were presently used
for the coupling and, after purification on a Sephadex column, the
batch was concentrated by ultracentrifugation.
[5813] Protein concentration: 12.0 mg/ml
[5814] Drug/mAb ratio: 3.2
Example 195
##STR00886##
[5816] 90 mg anti-C4.4a M31-B01 in DPBS pH 7.4 were presently used
for the coupling and, after purification on a Sephadex column, the
batch was concentrated by ultracentrifugation.
[5817] Protein concentration: 10.2 mg/ml
[5818] Drug/mAb ratio: 4.3
Example 196
##STR00887##
[5820] Protein concentration: 1.37 mg/ml
[5821] Drug/mAb ratio: 2.6
Example 197
##STR00888##
[5823] Protein concentration: 1.14 mg/ml
[5824] Drug/mAb ratio: 2.0
Example 198
##STR00889##
[5826] Protein concentration: 1.07 mg/ml
[5827] Drug/mAb ratio: 3.5
Example 199
##STR00890##
[5829] Protein concentration: 1.14 mg/ml
[5830] Drug/mAb ratio: 1.9
Example 200
##STR00891##
[5832] Protein concentration: 1.22 mg/ml
[5833] Drug/mAb ratio: 3.3
Example 201
##STR00892##
[5835] Protein concentration: 1.3 mg/ml
[5836] Drug/mAb ratio: 3.2
Example 202
##STR00893##
[5838] Protein concentration: 1.23 mg/ml
[5839] Drug/mAb ratio: 3.3
Example 203
##STR00894##
[5841] Protein concentration: 1.64 mg/ml
[5842] Drug/mAb ratio: 1.8
Example 204
##STR00895##
[5844] Protein concentration: 1.07 mg/ml
[5845] Drug/mAb ratio: 3.1
Example 205
##STR00896##
[5847] Protein concentration: 1.14 mg/ml
[5848] Drug/mAb ratio: 2.3
Example 206
##STR00897##
[5850] Protein concentration: 1.23 mg/ml
[5851] Drug/mAb ratio: 3.4
Example 207
##STR00898##
[5853] Protein concentration: 1.22 mg/ml
[5854] Drug/mAb ratio: 2.5
Example 208
##STR00899##
[5856] Protein concentration: 1.22 mg/ml
[5857] Drug/mAb ratio: 2.4
Example 209
##STR00900##
[5859] Protein concentration: 1.32 mg/ml
[5860] Drug/mAb ratio: not detectable
Example 210
##STR00901##
[5862] Protein concentration: 1.44 mg/ml
[5863] Drug/mAb ratio: 2.3
Example 211
##STR00902##
[5865] 250 mg anti-C4.4a B01-10 in DPBS pH 7.4 were presently used
for the coupling and, after purification on a Sephadex column, the
batch was concentrated by ultracentrifugation.
[5866] Protein concentration: 12.8 mg/ml
[5867] Drug/mAb ratio: 5.2
Example 212
##STR00903##
[5869] Protein concentration: 0.9 mg/ml
[5870] Drug/mAb ratio: 2
Example 213
##STR00904##
[5872] 250 mg anti-C4.4a B01-3 in DPBS pH 7.4 were presently used
for the coupling and, after purification on a Sephadex column, the
batch was concentrated by ultracentrifugation.
[5873] Protein concentration: 8.0 mg/ml
[5874] Drug/mAb ratio: 4.5
Example 214
##STR00905##
[5876] 250 mg anti-C4.4a B01-10 in DPBS pH 7.4 were presently used
for the coupling and, after purification on a Sephadex column, the
batch was concentrated by ultracentrifugation.
[5877] Protein concentration: 12.3 mg/ml
[5878] Drug/mAb ratio: 5.2
Example 215
##STR00906##
[5880] 250 mg anti-C4.4a B01-10 in DPBS pH 7.4 were presently used
for the coupling and, after purification on a Sephadex column, the
batch was concentrated by ultracentrifugation.
[5881] Protein concentration: 10.2 mg/ml
[5882] Drug/mAb ratio: 4.4
Example 216
##STR00907##
[5884] 50 mg anti-C4.4a B01-3 in DPBS pH 7.4 were presently used
for the coupling and, after purification on a Sephadex column, the
batch was concentrated by ultracentrifugation.
[5885] Protein concentration: 11.5 mg/ml
[5886] Drug/mAb ratio: 5.2
Example 217
##STR00908##
[5888] 250 mg anti-C4.4a D02-6 in DPBS pH 7.4 were presently used
for the coupling and, after purification on a Sephadex column, the
batch was concentrated by ultracentrifugation.
[5889] Protein concentration: 13 mg/ml
[5890] Drug/mAb ratio: 5.2
Example 218
##STR00909##
[5892] 250 mg anti-C4.4a B01-3 in DPBS pH 7.4 were presently used
for the coupling and, after purification on a Sephadex column, the
batch was concentrated by ultracentrifugation.
[5893] Protein concentration: 10.3 mg/ml
[5894] Drug/mAb ratio: 4.9
Example 219
##STR00910##
[5896] Protein concentration: 0.88 mg/ml
[5897] Drug/mAb ratio: 3.2
Example 220
##STR00911##
[5899] Protein concentration: 1.18 mg/ml
[5900] Drug/mAb ratio: 3.4
Example 221
##STR00912##
[5902] Protein concentration: 1.23 mg/ml
[5903] Drug/mAb ratio: 3.0
Example 222
##STR00913##
[5905] Protein concentration: 1.3 mg/ml
[5906] Drug/mAb ratio: 3.3
Example 223
##STR00914##
[5908] Protein concentration: 1.11 mg/ml
[5909] Drug/mAb ratio: not detectable
Example 224
##STR00915##
[5911] Protein concentration: 1.25 mg/ml
[5912] Drug/mAb ratio: 2.4
Example 225
##STR00916##
[5914] Protein concentration: 0.88 mg/ml
[5915] Drug/mAb ratio: 5.0
Example 226
##STR00917##
[5917] Protein concentration: 1.23 mg/ml
[5918] Drug/mAb ratio: 3.3
Example 227
##STR00918##
[5920] Protein concentration: 0.93 mg/ml
[5921] Drug/mAb ratio: 1.8
Example 228
##STR00919##
[5923] Protein concentration: 0.85 mg/ml
[5924] Drug/mAb ratio: 5.3
Example 229
##STR00920##
[5926] Protein concentration: 1.51 mg/ml
[5927] Drug/mAb ratio: 1.4
Example 230
##STR00921##
[5929] 150 mg anti-C4.4a B01-3 in DPBS pH 7.4 were presently used
for the coupling and, after purification on a Sephadex column, the
batch was concentrated by ultracentrifugation.
[5930] Protein concentration: 11.0 mg/ml
[5931] Drug/mAb ratio: 4.5
Example 231
##STR00922##
[5933] Protein concentration: 1.2 mg/ml
[5934] Drug/mAb ratio: 3.3
Example 232
##STR00923##
[5936] Protein concentration: 1.25 mg/ml
[5937] Drug/mAb ratio: 3.1
Example 233
N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin--
1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S-
)-2-[(1R,2R)-3-{[(2S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1--
methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxohept-
an-4-yl]-N-methyl-L-valinamide
##STR00924##
[5939] 10 mg (10 .mu.mol) of Intermediate 157 were taken up in 5.2
ml DMF and mixed with 2.28 mg (20 .mu.mol) L-cysteine. The reaction
mixture was stirred for 2 hours at RT, then concentrated in vacuo
and next purified by means of preparative HPLC. The yield was 5.8
mg (48% of theory) of the title compound.
[5940] HPLC (Method 5): R.sub.t=1.45 min;
[5941] LC-MS (Method 1): R.sub.t=0.74 min; MS (ESIpos): m/z=1184
(M+H).sup.+.
Example 234
N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin--
1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S-
)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-(1H-indol-3-yl)ethyl]amino}-1-methoxy-2--
methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-
-methyl-L-valinamide
##STR00925##
[5943] 10 mg (10 .mu.mol) of Intermediate 113 were taken up in 5.2
ml DMF and mixed with 2.28 mg (20 .mu.mol) L-cysteine. The reaction
mixture was stirred for 2 hours at RT, then concentrated in vacuo
and next purified by means of preparative HPLC. The yield was 6 mg
(54% of theory) of the title compound.
[5944] HPLC (Method 5): R.sub.t=1.5 min;
[5945] LC-MS (Method 1): R.sub.t=0.77 min; MS (ESIpos): m/z=1185
(M+H).sup.+.
Example 235
N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin--
1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-meth-
oxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-yl-
carbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-
-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00926##
[5947] 9 mg (8.3 .mu.mol) of Intermediate 132 were taken up in 4 ml
DMF and mixed with 3 mg (24.4 .mu.mol) L-cysteine. The reaction
mixture was stirred overnight at RT, then concentrated in vacuo and
next purified by means of preparative HPLC. The yield was 6.8 mg
(68% of theory) of the title compound.
[5948] HPLC (Method 12): R.sub.t=1.8 min;
[5949] LC-MS (Method 1): R.sub.t=0.78 min; MS (ESIpos): m/z=1227
(M+H).sup.+.
Example 236
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)h-
exyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino-3-
-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]py-
rrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00927##
[5951] 10 mg (10 .mu.mol) of Intermediate 106 were taken up in 5.8
ml DMF and mixed with 2.5 mg (20 .mu.mol) L-cysteine. The reaction
mixture was stirred for 2 hours at RT, then concentrated in vacuo
and next purified by means of preparative HPLC. The yield was 5.2
mg (46% of theory) of the title compound.
[5952] HPLC (Method 5): R.sub.t=1.5 min;
[5953] LC-MS (Method 11): R.sub.t=0.71 min; MS (ESIpos): m/z=1070
(M+H).sup.+.
Example 237
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)h-
exyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-
-2-(1H-indol-3-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-
-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00928##
[5955] 10 mg (10 .mu.mol) of Intermediate 124 was taken up in 4 ml
DMF and mixed with 2.5 mg (20 .mu.mol) L-cysteine. The reaction
mixture was stirred for 2 hours at RT, then concentrated in vacuo
and next purified by means of preparative HPLC. The yield was 7.2
mg (64% of theory) of the title compound.
[5956] HPLC (Method 5): R.sub.t=1.6 min;
[5957] LC-MS (Method 1): R.sub.t=0.8 min; MS (ESIpos): m/z=1071
(M+H).sup.+.
Example 238
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)h-
exyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-ind-
ol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl--
3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-
-L-valinamide
##STR00929##
[5959] 10 mg (10 .mu.mol) of Intermediate 125 were taken up in 4 ml
DMF and mixed with 2.4 mg (20 .mu.mol) L-cysteine. The reaction
mixture was stirred for 2 hours at RT, then concentrated in vacuo
and next purified by means of preparative HPLC. The yield was 7.7
mg (69% of theory) of the title compound.
[5960] HPLC (Method 5): R.sub.t=1.7 min;
[5961] LC-MS (Method 2): R.sub.t=1.91 min; MS (ESIpos): m/z=1140
(M+H).sup.+.
Example 239
N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin--
1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S-
)-2-[(1R,2R)-3-{[(2S)-1-(benzylamino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl]a-
mino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-
-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00930##
[5963] 10 mg (10 .mu.mol) of intermediate 160 were taken up in 3 ml
DMF and mixed with 2.1 mg (20 .mu.mol) L-cysteine. The reaction
mixture was stirred for 2 hours at RT, then concentrated in vacuo
and next purified by means of preparative HPLC. The yield was 8.1
mg (73% of theory) of the title compound.
[5964] HPLC (Method 5): R.sub.t=1.7 min;
[5965] LC-MS (Method 1): R.sub.t=0.86 min; MS (ESIpos): m/z=1274
(M+H).sup.+.
Example 240
N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin--
1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S-
)-2-[(1R,2R)-3-{[(2S)-1-(benzylamino)-1-oxo-3-phenylpropan-2-yl]amino}-1-m-
ethoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxohepta-
n-4-yl]-N-methyl-L-valinamide
##STR00931##
[5967] 3.5 mg (3 .mu.mol) of Intermediate 159 were taken up in 1 ml
DMF and mixed with 0.76 mg (6 .mu.mol) L-cysteine. The reaction
mixture was stirred for 2 hours at RT, then concentrated in vacuo
and next purified by means of preparative HPLC. The yield was 2.6
mg (65% of theory) of the title compound.
[5968] HPLC (Method 5): R.sub.t=1.75 min;
[5969] LC-MS (Method 1): R.sub.t=0.85 min; MS (ESIpos): m/z=1235
(M+H).sup.+.
Example 241
N-(6-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin--
1-yl)hexanoyl]hydrazino}-6-oxohexyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-meth-
oxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-yl-
carbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-
-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00932##
[5971] 3.6 mg (3 .mu.mol) of Intermediate 129 were taken up in 1 ml
DMF and mixed with 0.77 mg (6 .mu.mol) L-cysteine. The reaction
mixture was stirred for 2 hours at RT, then concentrated in vacuo
and next purified by means of preparative HPLC. The yield was 1.55
mg (39% of theory) of the title compound.
[5972] HPLC (Method 5): R.sub.t=1.6 min;
[5973] LC-MS (Method 1): R.sub.t=0.87 min; MS (ESIpos): m/z=1255
(M+H).sup.+.
Example 242
##STR00933##
[5975] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the reaction
mixture was concentrated by ultracentrifugation and diluted
again.
[5976] Protein concentration: 0.83 mg/ml
[5977] Drug/mAb ratio: 1.6
Example 243
##STR00934##
[5979] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the reaction
mixture was concentrated by ultracentrifugation and diluted
again.
[5980] Protein concentration: 1.59 mg/ml
[5981] Drug/mAb ratio: 3.1
[5982] Drug/mAb ratio: 2.9
Example 244
##STR00935##
[5984] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the reaction
mixture was concentrated by ultracentrifugation and diluted
again.
[5985] Protein concentration: 1.25 mg/ml
[5986] Drug/mAb ratio: 4.0
Example 245
##STR00936##
[5988] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the reaction
mixture was concentrated by ultracentrifugation and diluted
again.
[5989] Protein concentration: 1.27 mg/ml
[5990] Drug/mAb ratio: 3.6
Example 246
##STR00937##
[5992] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the reaction
mixture was concentrated by ultracentrifugation and diluted
again.
[5993] Protein concentration: 1.54 mg/ml
[5994] Drug/mAb ratio: 4.7
Example 247
##STR00938##
[5996] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the reaction
mixture was concentrated by ultracentrifugation and diluted
again.
[5997] Protein concentration: 1.73 mg/ml
[5998] Drug/mAb ratio: 4.7
Example 248
##STR00939##
[6000] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the reaction
mixture was concentrated by ultracentrifugation and diluted
again.
[6001] Protein concentration: 1.66 mg/ml
[6002] Drug/mAb ratio: 1.3
Example 249
##STR00940##
[6004] Protein concentration: 2.11 mg/ml
[6005] Drug/mAb ratio: 5.5
Example 250
##STR00941##
[6007] Protein concentration: 1.53 mg/ml
[6008] Drug/mAb ratio: 3.4
Example 251
##STR00942##
[6010] Protein concentration: 1.5 mg/ml
[6011] Drug/mAb ratio: 0.2
Example 252
##STR00943##
[6013] Protein concentration: 1.32 mg/ml
[6014] Drug/mAb ratio: 0.1
Example 253
##STR00944##
[6016] 80 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation, diluted again with PBS and
reconcentrated.
[6017] Protein concentration: 10.3 mg/ml
[6018] Drug/mAb ratio: 3.1
Example 254
##STR00945##
[6020] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6021] Protein concentration: 1.09 mg/ml
[6022] Drug/mAb ratio: 1.8
Example 255
##STR00946##
[6024] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6025] Protein concentration: 1.52 mg/ml
[6026] Drug/mAb ratio: 4.2
Example 256
##STR00947##
[6028] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6029] Protein concentration: 1.1 mg/ml
[6030] Drug/mAb ratio: 3.3
Example 257
##STR00948##
[6032] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6033] Protein concentration: 1.43 mg/ml
[6034] Drug/mAb ratio: 4.8
Example 258
##STR00949##
[6036] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugationm diluted again with PBS and
reconcentrated.
[6037] Protein concentration: 1.36 mg/ml
[6038] Drug/mAb ratio: 4.6
Example 259
##STR00950##
[6040] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6041] Protein concentration: 1.33 mg/ml
[6042] Drug/mAb ratio: 4.0
Example 260
##STR00951##
[6044] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6045] Protein concentration: 1.33 mg/ml
[6046] Drug/mAb ratio: 4.6
Example 261
##STR00952##
[6048] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6049] Protein concentration: 1.47 mg/ml
[6050] Drug/mAb ratio: 1.6
Example 262
##STR00953##
[6052] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6053] Protein concentration: 1.49 mg/ml
[6054] Drug/mAb ratio: 4.5
Example 263
##STR00954##
[6056] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6057] Protein concentration: 1.29 mg/ml
[6058] Drug/mAb ratio: 3.3
Example 264
##STR00955##
[6060] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6061] Protein concentration: 1.74 mg/ml
[6062] Drug/mAb ratio: 3.5
Example 265
##STR00956##
[6064] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6065] Protein concentration: 1.09 mg/ml
[6066] Drug/mAb ratio: 3.2
Example 266
##STR00957##
[6068] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6069] Protein concentration: 1.63 mg/ml
[6070] Drug/mAb ratio: 0.2
Example 267
##STR00958##
[6072] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6073] Protein concentration: 1.41 mg/ml
[6074] Drug/mAb ratio: 7.6
Example 268
##STR00959##
[6076] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6077] Protein concentration: 2.0 mg/ml
[6078] Drug/mAb ratio: 1.6
Example 269
##STR00960##
[6080] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6081] Protein concentration: 1.67 mg/ml
[6082] Drug/mAb ratio: 2.8
Example 270
##STR00961##
[6084] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6085] Protein concentration: 1.91 mg/ml
[6086] Drug/mAb ratio: 5.3
Example 271
##STR00962##
[6088] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6089] Protein concentration: 1.82 mg/ml
[6090] Drug/mAb ratio: 4.6
##STR00963##
Example 272
[6091] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6092] Protein concentration: 1.9 mg/ml
[6093] Drug/mAb ratio: 4.2
Example 273
##STR00964##
[6095] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6096] Protein concentration: 1.89 mg/ml
[6097] Drug/mAb-Ratio: 2.7
Example 274
##STR00965##
[6099] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6100] Protein concentration: 1.73 mg/ml
[6101] Drug/mAb-Ratio: 2.3
Example 275
##STR00966##
[6103] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6104] Protein concentration: 1.71 mg/ml
[6105] Drug/mAb-Ratio: 3.3
Example 276
##STR00967##
[6107] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6108] Protein concentration: 1.47 mg/ml
[6109] Drug/mAb ratio: 3.9
Example 277
N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-N--
[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan--
2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1--
yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate
##STR00968##
[6111] 15.5 mg (15 .mu.mol) of Intermediate 210 were taken up in 5
ml DMF and mixed with 4.4 mg (18 .mu.mol)
N.sup.2-(tert.-butoxycarbonyl)-L-lysine as well as 7.7 .mu.L (44
.mu.mol) N,N-diisopropylethylamine. The reaction mixture was
stirred overnight at RT and then concentrated in vacuo. Next, the
residue was purified by means of preparative HPLC. The yield was 14
mg (81% of theory) of the protected intermediate of the title
compound that was subsequently taken up in 1 ml dichloromethane and
deprotected with 1 ml trifluoroascetic acid. The batch was
concentrated and, following lyophilization of the residue from
acetonitrile/water (1:1), 15 mg (97% of theory) of the title
compound were obtained.
[6112] HPLC (Method 12): R.sub.t=1.8 min;
[6113] LC-MS (Method 1): R.sub.t=0.79 min; MS (ESIpos): m/z=1083
(M+H).sup.+.
Example 278
N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-N--
[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-(1H-indol-3-yl)ethyl]a-
mino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-
-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00969##
[6115] 40 mg (40 .mu.mol) of Intermediate 227 were taken up in 5 ml
DMF and mixed with 11.5 mg (40 .mu.mol)
N.sup.2-[(benzyloxy)carbonyl]-L-lysine as well as 13 .mu.l (80
.mu.mol) N,N-diisopropylethylamine. The reaction mixture was
stirred overnight at RT, then concentrated in vacuo and next
purified by means of preparative HPLC. The yield was 32.5 mg (70%
of theory) of the protected intermediate of the title compound.
[6116] These 32.5 mg of the intermediate were dissolved in 10 ml
methanol and, after adding 2 mg 10% palladium on activated carbon,
hydrogenated for 30 min at RT under standard hydrogen pressure. The
catalyst was then filtered off and the solvent removed in vacuo.
Following lyophilization of the residue from dioxane/water 1:1, 26
mg (99% of theory) of the title compound were obtained.
[6117] HPLC (Method 12): R.sub.t=1.7 min;
[6118] LC-MS (Method 1): R.sub.t=0.76 min; MS (ESIpos): m/z=1014
(M+H).sup.+.
Example 279
N-[(18S)-18-amino-18-carboxy-12-oxo-3,6,9-trioxa-13-azaoctadec-1-yl]-N-met-
hyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1--
(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl-
]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinami-
de trifluoroacetate
##STR00970##
[6120] 3.5 mg (3 .mu.mol) of Intermediate 202 were taken up in 2 ml
DMF and mixed with 0.8 mg (3 .mu.mol) N.sup.2-(tert.
butoxycarbonyl)-L-lysine as well as 1.6 .mu.l (10 .mu.mol)
N,N-diisopropylethylamine. The reaction mixture was stirred
overnight at RT and then concentrated in vacuo. Next, the residue
was taken up in acetonitrile/water (1:1), adjusted to pH 2 with
trifluoroascetic acid, and purified by means of preparative HPLC.
The yield was 1 mg (25% of theory) of the protected intermediate of
the title compound that was subsequently taken up in 500 .mu.l
dichloromethane and deprotected with 500 .mu.l trifluoroascetic
acid. The batch was concentrated and, following lyophilization of
the residue from acetonitrile/water (1:1), 1 mg (89% of theory) of
the title compound was obtained.
[6121] HPLC (Method 12): R.sub.t=1.9 min;
[6122] LC-MS (Method 1): R.sub.t=0.82 min; MS (ESIpos): m/z=1173
(M+H).sup.+.
Example 280
##STR00971##
[6124] Protein concentration: 0.9 mg/ml
[6125] Drug/mAb ratio: 2.8
Example 281
##STR00972##
[6127] Protein concentration: 1.08 mg/ml
[6128] Drug/mAb ratio: 1.1
Example 282
##STR00973##
[6130] Protein concentration: 0.98 mg/ml
[6131] Drug/mAb ratio: 2.4
Example 283
##STR00974##
[6133] Protein concentration: 1.23 mg/ml
[6134] Drug/mAb ratio: 4.6
Example 284
##STR00975##
[6136] 100 mg anti-CA9 were presently used for the coupling and,
after purification on a Sephadex column, the batch was concentrated
by ultracentrifugation. The solution was then diluted again,
reconcentrated, and the process was repeated one more time.
[6137] Protein concentration: 9.2 mg/ml
[6138] Drug/mAb ratio: 3.2
Example 285
##STR00976##
[6140] Protein concentration: 1.21 mg/ml
[6141] Drug/mAb ratio: not detectable
Example 286
##STR00977##
[6143] Protein concentration: 1.26 mg/ml
[6144] Drug/mAb ratio: 4.2
Example 287
##STR00978##
[6146] Protein concentration: 1.01 mg/ml
[6147] Drug/mAb ratio: 3.0
Example 288
##STR00979##
[6149] Protein concentration: 1.28 mg/ml
[6150] Drug/mAb ratio: 2.3
Example 289
##STR00980##
[6152] Protein concentration: 1.12 mg/ml
[6153] Drug/mAb ratio: 2.6
Example 290
##STR00981##
[6155] Protein concentration: 1.4 mg/ml
[6156] Drug/mAb ratio: 3.3
Example 291
##STR00982##
[6158] Protein concentration: 1.3 mg/ml
[6159] Drug/mAb ratio: 2.5
Example 292
##STR00983##
[6161] Protein concentration: 1.27 mg/ml
[6162] Drug/mAb ratio: 2.6
Example 293
##STR00984##
[6164] Protein concentration: 1.55 mg/ml
[6165] Drug/mAb ratio: not detectable
Example 294
##STR00985##
[6167] 100 mg anti-CA9 were presently used for the coupling and,
after purification on a Sephadex column, the batch was concentrated
by ultracentrifugation.
[6168] Protein concentration: 11.8 mg/ml
[6169] Drug/mAb ratio: 4.4
Example 295
##STR00986##
[6171] 100 mg anti-CA9 were presently used for the coupling and,
after purification on a Sephadex column, the batch was concentrated
by ultracentrifugation.
[6172] Protein concentration: 12.29 mg/ml
[6173] Drug/mAb ratio: 3.8
Example 296
##STR00987##
[6175] Protein concentration: 1.1 mg/ml
[6176] Drug/mAb ratio: 1.6
Example 297
##STR00988##
[6178] Protein concentration: 1.00 mg/ml
[6179] Drug/mAb ratio: 1.9
Example 298
##STR00989##
[6181] 100 mg anti-CA9 were presently used for the coupling and,
after purification on a Sephadex column, the batch was concentrated
by ultracentrifugation.
[6182] Protein concentration: 11.5 mg/ml
[6183] Drug/mAb ratio: 4.9
Example 299
##STR00990##
[6185] Protein concentration: 0.98 mg/ml
[6186] Drug/mAb ratio: 2.5
Example 300
##STR00991##
[6188] Protein concentration: 0.99 mg/ml
[6189] Drug/mAb ratio: 2.0
Example 301
##STR00992##
[6191] Protein concentration: 0.87 mg/ml
[6192] Drug/mAb ratio: 2.1
Example 302
##STR00993##
[6193] Example 303
[6194] 100 mg anti-CA9 were presently used for the coupling and,
after purification on a Sephadex column, the batch was concentrated
by ultracentrifugation.
[6195] Protein concentration: 12.2 mg/ml
[6196] Drug/mAb ratio: 4.6
Example 303
##STR00994##
[6198] Protein concentration: 1.58 mg/ml
[6199] Drug/mAb ratio: 1.9
Example 304
##STR00995##
[6201] 70 mg anti-CA9 were presently used for the coupling and,
after purification on a Sephadex column, the batch was concentrated
by ultracentrifugation.
[6202] Protein concentration: 11.5 mg/ml
[6203] Drug/mAb ratio: 3.9
Example 305
##STR00996##
[6205] 60 mg anti-CA9 were presently used for the coupling and,
after purification on a Sephadex column, the batch was concentrated
by ultracentrifugation.
[6206] Protein concentration: 11.6 mg/ml
[6207] Drug/mAb ratio: 3.9
Example 306
##STR00997##
[6209] 60 mg anti-CA9 were presently used for the coupling and,
after purification on a Sephadex column, the batch was concentrated
by ultracentrifugation.
[6210] Protein concentration: 10 mg/ml
Example 307
N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin--
1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S-
)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-(1H-indol-3-yl)ethyl]amino}-1-methoxy-2--
methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-
-methyl-L-valinamide
##STR00998##
[6212] 10 mg (10 .mu.mol) of Intermediate 113 were taken up in 5.2
ml DMF and mixed with 2.28 mg (20 .mu.mol) L-cysteine. The batch
was stirred for 2 hours at RT, then concentrated in vacuo and next
purified by means of preparative HPLC. The yield was 6 mg (54% of
theory) of the title compound.
[6213] HPLC (Method 5): R.sub.t=1.5 min;
[6214] LC-MS (Method 1): R.sub.t=0.77 min; MS (ESIpos): m/z=1185
(M+H).sup.+.
Example 308
N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin--
1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-3-meth-
oxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-yl-
carbonyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-
-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00999##
[6216] 9 mg (8.3 .mu.mol) of Intermediate 132 were taken up in 4 ml
DMF and mixed with 3 mg (24.4 .mu.mol) L-cysteine. The batch was
stirred overnight at RT, then concentrated in vacuo and next
purified by means of preparative HPLC. The yield was 6.8 mg (68% of
theory) of the title compound.
[6217] HPLC (Method 12): R.sub.t=1.8 min;
[6218] LC-MS (Method 1): R.sub.t=0.78 min; MS (ESIpos): m/z=1227
(M+H).sup.+.
Example 309
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)h-
exyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-amino-3-
-(1H-indol-3-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]py-
rrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR01000##
[6220] 10 mg (10 .mu.mol) of Intermediate 106 were taken up in 5.8
ml DMF and mixed with 2.5 mg (20 .mu.mol) L-cysteine. The batch was
stirred for 2 hours at RT, then concentrated in vacuo and next
purified by means of preparative HPLC. The yield was 5.2 mg (46% of
theory) of the title compound.
[6221] HPLC (Method 5): R.sub.t=1.5 min;
[6222] LC-MS (Method 11): R.sub.t=0.71 min; MS (ESIpos): m/z=1070
(M+H).sup.+.
Example 310
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)h-
exyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-
-2-(1H-indol-3-yl)ethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-
-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR01001##
[6224] 10 mg (10 .mu.mol) of Intermediate 124 were taken up in 4 ml
DMF and mixed with 2.5 mg (20 .mu.mol) L-cysteine. The batch was
stirred for 2 hours at RT, then concentrated in vacuo and next
purified by means of preparative HPLC. The yield was 7.2 mg (64% of
theory) of the title compound.
[6225] HPLC (Method 5): R.sub.t=1.6 min;
[6226] LC-MS (Method 1): R.sub.t=0.8 min; MS (ESIpos): m/z=1071
(M+H).sup.+.
Example 311
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin-1-yl)h-
exyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-ind-
ol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl--
3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-
-L-valinamide
##STR01002##
[6228] 10 mg (10 .mu.mol) of Intermediate 125 were taken up in 4 ml
DMF and mixed with 2.4 mg (20 .mu.mol) L-cysteine. The batch was
stirred for 2 hours at RT, then concentrated in vacuo and next
purified by means of preparative HPLC. The yield was 7.7 mg (69% of
theory) of the title compound.
[6229] HPLC (Method 5): R.sub.t=1.7 min;
[6230] LC-MS (Method 2): R.sub.t=1.91 min; MS (ESIpos): m/z=1140
(M+H).sup.+.
Example 312
N-(4-{2-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulfanyl}-2,5-dioxopyrrolidin--
1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S-
)-2-[(1R,2R)-3-{[(2S)-1-(benzylamino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl]a-
mino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-
-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR01003##
[6232] 10 mg (10 .mu.mol) of Intermediate 160 was taken up in 3 ml
DMF and mixed with 2.1 mg (20 .mu.mol) L-cystein. The batch was
stirred for 2 hours at RT, then concentrated in vacuo and next
purified by means of preparative HPLC. The yield was 8.1 mg (73% of
theory) of the title compound.
[6233] HPLC (Method 5): R.sub.t=1.7 min;
[6234] LC-MS (Method 1): R.sub.t=0.86 min; MS (ESIpos): m/z=1274
(M+H)+.
Example 313
##STR01004##
[6236] 5 mg anti-CA9 in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6237] Protein concentration: 1.23 mg/ml
[6238] Drug/mAb ratio: .about.1-1.5
Example 314
##STR01005##
[6240] 5 mg anti-CA9 in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6241] Protein concentration: 1.98 mg/ml
[6242] Drug/mAb ratio: 2.8
Example 315
##STR01006##
[6244] 5 mg anti-CA9 in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6245] Protein concentration: 1.0 mg/ml
[6246] Drug/mAb ratio: 3
Example 316
##STR01007##
[6248] 5 mg anti-CA9 in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6249] Protein concentration: 1.59 mg/ml
[6250] Drug/mAb ratio: 3.1
Example 317
##STR01008##
[6252] 5 mg anti-CA9 in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6253] Protein concentration: 1.75 mg/ml
[6254] Drug/mAb ratio: 3.3
Example 318
##STR01009##
[6256] 5 mg anti-CA9 in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6257] Protein concentration: 1.54 mg/ml
[6258] Drug/mAb ratio: 3.5
Example 319
##STR01010##
[6260] 5 mg anti-CA9 in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6261] Protein concentration: 2 mg/ml
[6262] Drug/mAb ratio: 1.1
Example 320
##STR01011##
[6264] Protein concentration: 1.66 mg/ml
[6265] Drug/mAb ratio: 4.9
Example 321
##STR01012##
[6267] Protein concentration: 1.7 mg/ml
[6268] Drug/mAb ratio: 3.0
Example 322
##STR01013##
[6270] Protein concentraion: 1.08 mg/ml
[6271] Drug/mAb ratio: 1.9
Example 323
##STR01014##
[6273] 5 mg anti-CA9 in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6274] Protein concentration: 1.57 mg/ml
[6275] Drug/mAb ratio: 2.9
Example 324
##STR01015##
[6277] 5 mg anti-CA9 in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6278] Protein concentration: 1.7 mg/ml
[6279] Drug/mAb ratio: 1.4
Example 325
##STR01016##
[6281] 5 mg anti-CA9 in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6282] Protein concentration: 1.53 mg/ml
[6283] Drug/mAb ratio: 3.6
Example 326
##STR01017##
[6285] 5 mg anti-CA9 in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again with PBS.
[6286] Protein concentration: 1.77 mg/ml
[6287] Drug/mAb ratio: 6.1
Example 327
##STR01018##
[6289] 5 mg anti-CA9 in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6290] Protein concentration: 1.14 mg/ml
[6291] Drug/mAb ratio: 2.5
Example 328
##STR01019##
[6293] 5 mg anti-CA9 in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6294] Protein concentration: 1.57 mg/ml
[6295] Drug/mAb ratio: 3.8
Example 329
##STR01020##
[6297] 5 mg anti-CA9 in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6298] Protein concentration: 1.72 mg/ml
[6299] Drug/mAb ratio: 3.9
Example 330
##STR01021##
[6301] 5 mg anti-CA9 in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6302] Protein concentration: 1.56 mg/ml
[6303] Drug/mAb ratio: 2.9
Example 331
##STR01022##
[6305] 5 mg anti-CA9 in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6306] Protein concentration: 1.81 mg/ml
[6307] Drug/mAb ratio: 3.5
Example 332
##STR01023##
[6309] 5 mg anti-CA9 in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6310] Protein concentration: 1.54 mg/ml
[6311] Drug/mAb ratio: 1.3
Example 333
##STR01024##
[6313] 5 mg anti-CA9 in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6314] Protein concentration: 1.72 mg/ml
[6315] Drug/mAb ratio: 4.0
Example 334
N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-N--
[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan--
2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1--
yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate
##STR01025##
[6317] 15.5 mg (15 .mu.mol) of Intermediate 210 were taken up in 5
ml DMF and mixed with 4.4 mg (18 .mu.mol)
N.sup.2-(tert.-butoxycarbonyl)-L-lysine as well as 7.7 .mu.l (44
.mu.mol) N,N-diisopropylethylamine. The reaction mixture was
stirred overnight at RT and then concentrated in vacuo. Next, the
residue was purified by means of preparative HPLC. 14 mg (81% of
theory) of the protected intermediate of the title compound were
obtained, which were then taken up in 1 ml dichloromethane and
deprotected with 1 ml trifluoroascetic acid. The reaction mixture
was concentrated and, after lyophilization of the residue from
acetonitrile/water (1:1), 15 mg (97% of theory) of the title
compound were obtained.
[6318] HPLC (Method 12): R.sub.t=1.8 min;
[6319] LC-MS (Method 1): R.sub.t=0.79 min; MS (ESIpos): m/z=1083
(M+H).sup.+.
Example 335
N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-N--
[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-(1H-indol-3-yl)ethyl]a-
mino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-
-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR01026##
[6321] 40 mg (40 .mu.mol) of Intermediate 227 were taken up in 5 ml
DMF and mixed with 11.5 mg (40 .mu.mol)
N.sup.2-[(benzyloxy)carbonyl]-L-lysine as well as 13 .mu.l (80
.mu.mol) N,N-diisopropylethylamine. The reaction mixture was
stirred overnight at RT, then concentrated in vacuo and next
purified by means of preparative HPLC. The yield was 32.5 mg (70%
of theory) of the protected intermediate of the title compound.
[6322] 32.5 mg of this intermediate was dissolved in 10 ml methanol
and, after adding 2 mg 10% palladium on activated carbon,
hydrogenated for 30 min at RT under standard hydrogen pressure. The
catalyst was then filtered off and the solvent removed in vacuo.
After lyophilization of the residue from dioxane/water (1:1), the
yield was 26 mg (99% of theory) of the title compound.
[6323] HPLC (Method 12): R.sub.t=1.7 min;
[6324] LC-MS (Method 1): R.sub.t=0.76 min; MS (ESIpos): m/z=1014
(M+H).sup.+.
Example 336
N-[(18S)-18-amino-18-carboxy-12-oxo-3,6,9-trioxa-13-azaoctadec-1-yl]-N-met-
hyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1--
(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl-
]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinami-
de trifluoroacetate
##STR01027##
[6326] 3.5 mg (3 .mu.mol) of Intermediate 202 were taken up in 2 ml
DMF and mixed with 0.8 mg (3 .mu.mol)
N.sup.2-(tert.-butoxycarbonyl)-L-lysine as well as 1.6 .mu.l (10
.mu.mol) N,N-diisopropylethylamine.
[6327] The reaction mixture was stirred overnight at RT and then
concentrated in vacuo. Next, the residue was taken up in
acetonitrile/water (1:1), adjusted to pH 2 with trifluoroascetic
acid and then purified by means of preparative HPLC. 1 mg (25% of
theory) of the protected intermediate of the title compound was
obtained, which was subsequently taken up in 500 .mu.l
dichloromethane and deprotected with 500 .mu.l trifluoroascetic
acid. The reaction mixture was concentrated and, after
lyophilization of the residue from acetonitrile/water (1:1), the
yield was 1 mg (89% of theory) of the title compound.
[6328] HPLC (Method 12): R.sub.t=1.9 min;
[6329] LC-MS (Method 1): R.sub.t=0.82 min; MS (ESIpos): m/z=1173
(M+H).sup.+.
Example 337
##STR01028##
[6331] 5 mg cetuximab in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again with PBS.
[6332] Protein concentration: 1.57 mg/ml
[6333] Drug/mAb ratio: 4.6
Example 338
##STR01029##
[6335] 5 mg cetuximab were presently used for the coupling and,
following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again with PBS.
[6336] Protein concentration: 1.48 mg/ml
[6337] Drug/mAb ratio: 3.4
Example 339
##STR01030##
[6339] 5 mg cetuximab in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6340] Protein concentration: 1.21 mg/ml
[6341] Drug/mAb ratio: 2.4
Example 340
##STR01031##
[6343] 5 mg cetuximab in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again with PBS.
[6344] Protein concentration: 1.75 mg/ml
[6345] Drug/mAb ratio: 3.4
Example 341
##STR01032##
[6347] 5 mg cetuximab in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again with PBS.
[6348] Protein concentration: 1.69 mg/ml
[6349] Drug/mAb ratio: 2.9
Example 342
##STR01033##
[6351] 5 mg cetuximab were presently used for the coupling and,
following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again with PBS.
[6352] Protein concentration: 1.27 mg/ml
[6353] Drug/mAb ratio: 2.9
Example 343
##STR01034##
[6355] 5 mg panitumuab in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again with PBS.
[6356] Protein concentration: 1.27 mg/ml
[6357] Drug/mAb ratio: not detectable
Example 344
##STR01035##
[6359] 5 mg cetuximab were presently used for the coupling and,
following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again with PBS.
[6360] Protein concentration: 1.55 mg/ml
[6361] Drug/mAb ratio: 3.1
Example 345
##STR01036##
[6363] 5 mg cetuximab in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6364] Protein concentration: 1.67 mg/ml
[6365] Drug/mAb ratio: 3.5
Example 346
##STR01037##
[6367] 5 mg cetuximab in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again with PBS.
[6368] Protein concentration: 1.44 mg/ml
[6369] Drug/mAb ratio: 2.5
Example 347
##STR01038##
[6371] 5 mg cetuximab in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6372] Protein concentration: 1.73 mg/ml
[6373] Drug/mAb ratio: 1.2
Example 348
##STR01039##
[6375] 2 mg anti-PDL1 in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again with PBS.
[6376] Protein concentration: 1.04 mg/ml
[6377] Drug/mAb ratio: 4.8
Example 349
##STR01040##
[6379] 3 mg anti-PDL1 in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6380] Protein concentration: 1.7 mg/ml
[6381] Drug/mAb ratio: 2.3
Example 350
##STR01041##
[6383] 2 mg anti-ICOSLG in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again with PBS.
[6384] Protein concentration: 1.77 mg/ml
[6385] Drug/mAb ratio: 3.7
Example 351
##STR01042##
[6387] 4 mg anti-FGFR3 in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again with PBS.
[6388] Protein concentration: 1.41 mg/ml
[6389] Drug/mAb ratio: 1.8
Example 352
##STR01043##
[6391] 3 mg herceptin in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6392] Protein concentration: 1.49 mg/ml
[6393] Drug/mAb ratio: 2.3
Example 353
##STR01044##
[6395] 5 mg herceptin in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6396] Protein concentration: 1.62 mg/ml
[6397] Drug/mAb ratio: 5.0
Example 354
##STR01045##
[6399] 5 mg herceptin in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6400] Protein concentration: 1.63 mg/ml
[6401] Drug/mAb ratio: 2.4
Example 355
##STR01046##
[6403] 5 mg herceptin in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6404] Protein concentration: 1.65 mg/ml
[6405] Drug/mAb ratio: 3.9
Example 356
##STR01047##
[6407] 5 mg herceptin in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6408] Protein concentration: 1.78 mg/ml
[6409] Drug/mAb ratio: 3.1
Example 357
##STR01048##
[6411] 5 mg herceptin in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6412] Protein concentration: 1.72 mg/ml
[6413] Drug/mAb ratio: 3.2
Example 358
##STR01049##
[6415] 5 mg herceptin in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6416] Proteinkonzentration: 1.95 mg/ml
[6417] Drug/mAb-Ratio: .about.3.7
Example 359
##STR01050##
[6419] 5 mg herceptin in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again with PBS.
[6420] Protein concentration: 1.79 mg/ml
[6421] Drug/mAb ratio: 9.1
Example 360
N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-N--
[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazinan--
2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1--
yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate
##STR01051##
[6423] 15.5 mg (15 .mu.mol) of Intermediate 210 were taken up in 5
ml DMF and mixed with 4.4 mg (18 .mu.mol)
N.sup.2-(tert.-butoxycarbonyl)-L-lysine as well as 7.7 .mu.l (44
.mu.mol) N,N-diisopropylethylamine. The reaction mixture was
stirred overnight at RT, then concentrated in vacuo. The residue
was purified by means of preparative HPLC. 14 mg (81% of theory) of
the protected intermediate of the title compound were obtained,
which were then taken up in 1 ml dichloromethane and deprotected
with 1 ml trifluoroascetic acid. The batch was concentrated and,
after lyophilization of the residue from acetonitrile/water (1:1),
the yield was 15 mg (97% of theory) of the title compound.
[6424] HPLC (Method 12): R.sub.t=1.8 min;
[6425] LC-MS (Method 1): R.sub.t=0.79 min; MS (ESIpos): m/z=1083
(M+H).sup.+.
Example 361
N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-N--
[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-(1H-indol-3-yl)ethyl]a-
mino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-
-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR01052##
[6427] 40 mg (40 .mu.mol) of Intermediate 227 were taken up in 5 ml
DMF and mixed with 11.5 mg (40 .mu.mol)
N.sup.2-[(benzyloxy)carbonyl]-L-lysine as well as 13 .mu.l (80
.mu.mol) N,N-diisopropylethylamine. The reaction mixture was
stirred overnight at RT, then concentrated in vacuo and next
purified by means of preparative HPLC. The yield was 32.5 mg (70%
of theory) of the protected intermediate of the title compound.
[6428] 32.5 mg of this intermediate were taken up in 10 ml methanol
and, after adding 2 mg 10% palladium on activated carbon,
hydrogenated at RT under hydrogen normal pressure. The catalyst was
then filtered off and the solvent removed in vacuo. After
lyophilization of the residue from dioxane/water (1:1), the yield
was 26 mg (99% of theory) of the title compound.
[6429] HPLC (Method 12): R.sub.t=1.7 min;
[6430] LC-MS (Method 1): R.sub.t=0.76 min; MS (ESIpos): m/z=1014
(M+H).sup.+.
Example 362
N-[(18S)-18-amino-18-carboxy-12-oxo-3,6,9-trioxa-13-azaoctadec-1-yl]-N-met-
hyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1--
(1,2-oxazinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl-
]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinami-
de trifluoroacetate
##STR01053##
[6432] 3.5 mg (3 .mu.mol) of Intermediate 202 were taken up in 2 ml
DMF and mixed with 0.8 mg (3 .mu.mol)
N.sup.2-(tert.-butoxycarbonyl)-L-lysine as well as 1.6 .mu.l (10
.mu.mol) N,N-diisopropylethylamine. The reaction mixture was
stirred overnight at RT, then concentrated in vacuo. The residue
was taken up in acetonitrile/water (1:1), adjusted to pH 2 with
trifluoroascetic acid, then purified by means of preparative HPLC.
The yield was 1 mg (25% of theory) of the protected intermediate of
the title compound that was subsequently taken up in 500 .mu.l
dichloromethane and deprotected with 500 .mu.l trifluoroascetic
acid. The batch was concentrated and, after lyophilization of the
residue from acetonitrile/water (1:1), the yield was 1 mg (89% of
theory) of the title compound.
[6433] HPLC (Method 12): R.sub.t=1.9 min;
[6434] LC-MS (Method 1): R.sub.t=0.82 min; MS (ESIpos): m/z=1173
(M+H).sup.+.
Example 363
##STR01054##
[6436] 2.2 mg anti-TYRP1 in PBS were presently used for the
coupling and, following purification on a Sephadex column, the
batch was concentrated by ultracentrifugation and diluted
again.
[6437] Protein concentration: 1.14 mg/ml
[6438] Drug/mAb ratio: 4.1
Example 364
##STR01055##
[6440] 3 mg anti-glypican-3 were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again with PBS.
[6441] Protein concentration: 1.17 mg/ml
[6442] Drug/mAb ratio: 3.0
Example 365
##STR01056##
[6444] 3 mg anti-glypican-3 in PBS were presently used for the
coupling and, following purification on a Sephadex column, the
batch was concentrated by ultracentrifugation and diluted again
with PBS.
[6445] Protein concentration: 1.25 mg/ml
[6446] Drug/mAb ratio: 2.9
Example 366
##STR01057##
[6448] 5 mg MF-Ta in PBS were presently used for the coupling and,
following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6449] Protein concentration: 0.81 mg/ml
[6450] Drug/mAb ratio: 2.5
Example 367
##STR01058##
[6452] 5 mg MF-Ta in PBS were presently used for the coupling and,
following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6453] Protein concentration: 1.06 mg/ml
[6454] Drug/mAb ratio: 1.8
Example 368
##STR01059##
[6456] 5 mg MF-Ta in PBS were presently used for the coupling and,
following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again with PBS.
[6457] Protein concentration: 1.36 mg/ml
[6458] Drug/mAb ratio: 7.2
Example 369
##STR01060##
[6460] 5 mg MF-Ta in PBS were presently used for the coupling and,
following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again with PBS.
[6461] Protein concentration: 1.57 mg/ml
[6462] Drug/mAb ratio: 2.9
Example 370
##STR01061##
[6464] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, following purification on a Sephadex column, the
batch was concentrated by ultracentrifugation and diluted
again.
[6465] Protein concentration: 0.89 mg/ml
[6466] Drug/mAb ratio: 1.8
Example 371
##STR01062##
[6468] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, following purification on a Sephadex column, the
batch was concentrated by ultracentrifugation and diluted
again.
[6469] Protein concentration: 0.57 mg/ml
[6470] Drug/mAb ratio: 1.5
Example 372
##STR01063##
[6472] 5 mg anti-C4.4a B01-3 were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again with PBS.
[6473] Protein concentration: 1.39 mg/ml
[6474] Drug/mAb ratio: 7.1
Example 373
##STR01064##
[6476] 5 mg anti-C4.4a B01-3 were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again with PBS.
[6477] Protein concentration: 1.54 mg/ml
[6478] Drug/mAb ratio: 2.4
Example 374
##STR01065##
[6480] 5 mg cetuximab in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6481] Protein concentration: 1.55 mg/ml
[6482] Drug/mAb-Ratio: 1.8
Example 375
##STR01066##
[6484] 5 mg cetuximab were presently used for the coupling and,
following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again with PBS.
[6485] Protein concentration: 1.36 mg/ml
[6486] Drug/mAb ratio: 1.9
Example 376
##STR01067##
[6488] 5 mg cetuximab in PBS were presently used for the coupling
and, following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6489] Protein concentration: 1.73 mg/ml
[6490] Drug/mAb-Ratio: 3.7
Example 377
##STR01068##
[6492] 5 mg MF-Ta in PBS were presently used for the coupling and,
following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6493] Protein concentration: 1.44 mg/ml
[6494] Drug/mAb ratio: 2.5
Example 378
##STR01069##
[6496] 5 mg MF-Ta in PBS were presently used for the coupling and,
following purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again.
[6497] Protein concentration: 1.74 mg/ml
[6498] Drug/mAb-Ratio: 3.6
Example 379
Diastereomer 1
##STR01070##
[6500] Intermediate 247a and 5 mg MF-Ta in PBS were presently used
for the coupling and, following purification on a Sephadex column,
the batch was concentrated by ultracentrifugation and diluted again
with PBS.
[6501] Protein concentration: 1.57 mg/ml
[6502] Drug/mAb ratio: 4.2
Example 380
Diastereomer 2
##STR01071##
[6504] Intermediate 247a and 5 mg MF-Ta in PBS were presently used
for the coupling and, following purification on a Sephadex column,
the batch was concentrated by ultracentrifugation and diluted again
with PBS.
[6505] Protein concentration: 1.42 mg/ml
[6506] Drug/mAb ratio: 4.0
Example 381
N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-threonyl-
-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazin-
an-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-
-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate
##STR01072##
[6508] 8.6 mg (8 .mu.mol) of Intermediate 240 were taken up in 5 ml
DMF and mixed with 4.0 mg (16 .mu.mol)
N.sup.2-(tert.-butoxycarbonyl)-L-lysine as well as 2 .mu.l (16
.mu.mol) N,N-diisopropylethylamine. The reaction mixture was
stirred for 4 hours at RT, then mixed again with the same
quantities of N.sup.2-(tert.-butoxycarbonyl)-L-lysine and
N,N-diisopropylethylamine and stirred overnight at RT. The batch
was then concentrated in vacuo. Next, the residue was purified by
means of preparative HPLC. The yield was 7 mg (72% of theory) of
the protected intermediate of the title compound that was then
taken up in 1 ml of dichloromethane and deprotected with 0.5 ml
trifluoroascetic acid. The batch was concentrated and the residue
purified by means of preparative HPLC. After drying under high
vacuum, the yield was 3.3 mg (47% of theory) of the title
compound.
[6509] HPLC (Method 5): R.sub.t=1.5 min;
[6510] LC-MS (Method 1): R.sub.t=0.8 min; MS (ESIpos): m/z=1084
(M+H).sup.+.
Example 382
N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-N--
[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(4-hydroxyphenyl)-1-(1,2-oxazina-
n-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin--
1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate
##STR01073##
[6512] 8 mg (8 .mu.mol) of Intermediate 242 were taken up in 3 ml
DMF and mixed with 2.9 mg (12 .mu.mol)
N.sup.2-(tert.-butoxycarbonyl)-L-lysine as well as 2.7 .mu.l (16
.mu.mol) N,N-diisopropylethylamine. The reaction mixture was
stirred overnight at RT, then mixed again with the same quantities
of N.sup.2-(tert.-butoxycarbonyl)-L-lysine and
N,N-diisopropylethylamine, then stirred for another 4 hours at RT.
The batch was then concentrated in vacuo. Next, the residue was
purified by means of preparative HPLC. Following lyophilization
from acetonitrile/water, the yield was 6.5 mg (72% of theory) of
the protected intermediate of the title compound that was then
taken up in 5 ml dichloromethane and deprotected with 0.75 ml
trifluoroascetic acid. The batch was concentrated and, following
lyophilization of the residue from dioxane/water, the yield was 5
mg (76% of theory) of the title compound.
[6513] HPLC (Method 12): R.sub.t=1.7 min;
[6514] LC-MS (Method 1): R.sub.t=0.69 min; MS (ESIpos): m/z=1059
(M+H).sup.+.
Example 383
N-(6-{[(5S)-5-amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-valyl-N--
[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-(4-hydroxyphenyl)ethyl-
]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-
-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate
##STR01074##
[6516] 38 mg (41 .mu.mol) of Intermediate 248 were first converted
to the N-hydroxysuccinimide ester. 72 mg of the obtained raw
product were taken up in 5 ml DMF and mixed with 24 mg (100
.mu.mol) N.sup.2-(tert.-butoxycarbonyl)-L-lysine as well as 23
.mu.l N,N-diisopropylethylamine. The reaction mixture was stirred
overnight at RT, then mixed again with 16 mg
N.sup.2-(tert.-butoxycarbonyl)-L-lysine and 12 .mu.l
N,N-diisopropylethylamine and finally treated for another 2 hours
in an ultrasonic bath. The batch was then concentrated in vacuo and
the residue purified by means of preparative HPLC. After
lyophilization from acetonitrile/water, the yield was 20 mg (50% of
theory) of the protected intermediate of the title compound.
[6517] 15 mg (12 .mu.mol) of this intermediate were then taken up
in 3 ml dichloromethane and mixed with 1 ml trifluoroascetic acid.
After stirring for 40 min at RT, further 1.5 ml trifluoroascetic
acid were added, and the batch was treated for 1 h in an ultrasonic
bath. The batch was then concentrated and, after lyophilization of
the residue from dioxane/water, the yield was 13 mg (90% of theory)
of the title compound.
[6518] HPLC (Method 12): R.sub.t=1.5 min;
[6519] LC-MS (Method 1): R.sub.t=0.68 min; MS (ESIpos): m/z=990
(M+H).sup.+.
Example 384
##STR01075##
[6521] 5 mg anti-CA9 in PBS were presently used for the coupling
and, after purification on a Sephadex column, the batch was
concentrated by ultracentrifugation and diluted again with PBS.
[6522] Protein concentration: 1.4 mg/ml
[6523] Drug/mAb ratio: 3.0
Example 385
##STR01076##
[6525] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6526] Protein concentration: 1.48 mg/ml
[6527] Drug/mAb ratio: 2.4
Example 386
##STR01077##
[6529] 5 mg anti-C4.4a B01-3 in PBS were presently used for the
coupling and, after purification on a Sephadex column, the batch
was concentrated by ultracentrifugation and diluted again.
[6530] Proteinkonzentration: 1.43 mg/ml
[6531] Drug/mAb-Ratio: 3.6
Example 387 Diastereomer 1
##STR01078##
[6533] Intermediate 247a and 5 mg anti-C4.4a B01-3 in PBS were
presently used for the coupling and, after purification on a
Sephadex column, the batch was concentrated by ultracentrifugation
and diluted again with PBS.
[6534] Protein concentration: 1.45 mg/ml
[6535] Drug/mAb ratio: 3.8
Example 388 Diastereomer 2
##STR01079##
[6537] Intermediate 247a and 5 mg anti-C4.4a B01-3 in PBS were
presently used for the coupling and, after purification on a
Sephadex column, the batch was concentrated by ultracentrifugation
and diluted again with PBS.
[6538] Protein concentration: 1.42 mg/ml
[6539] Drug/mAb ratio: 4.0
C. EVALUATION OF BIOLOGICAL EFFICACY
[6540] The biological activity of the compound according to the
invention can be demonstrated by in vitro and in vivo tests, such
as those with which those skilled in the art are familiar.
[6541] The biological effect of the compounds according to the
invention was revealed in the assays described below:
[6542] C-1.1 In Vitro Cell Proliferation Test
[6543] Human EGFR-expressing tumors cells are used to test the
efficacy of anti-EGFR ADCs. The cells may be, for example, NCI-H292
or A431 with a high expression. Cells with a low EGFR expression,
such as HT29 or cells with practically no EGFR expression such as
NCI-H520 are used as the controls for the EGFR-dependent
cytotoxicity.
[6544] Description of the Experiment
[6545] Day 1: The cells are plated out in the medium in 100
.mu.L/well in a 96-well plate (Perkin Elmer, white, catalog
6005680). Cells for determination of the time zero are plated out
in a parallel plate. All plates are incubated overnight at
37.degree. C.
[6546] Day 2: A three-fold dilution series of the test substances
in medium is prepared and 100 .mu.L of the three-fold dilutions is
pipetted into each well in the plates. The plates are incubated for
96 hours at 37.degree. C. in an incubator. The time zero plate is
measured: 100 .mu.L/well CTG solution (Promega Cell Titer Glo
solution (catalog nos. G755B and G756B)) are pipetted into the
corresponding wells and incubated for 2 min on a shaker for +10
minutes in the dark. Next the luminescence is measured using a
VICTOR V instrument (Perkin Elmer).
[6547] Day 6: Measurement of all other batches: 100 .mu.L/well CTG
solution (Promega Cell Titer Glo solution (catalog nos. G755B and
G756B)) is pipetted into the corresponding wells and incubated for
2 min on a shaker for +10 minutes in the dark. Then the
luminescence is measured using a VICTOR V instrument (Perkin
Elmer).
[6548] The luminescence is used as a marker for the number of
viable cells.
[6549] The measured value of the time zero plate is equated with
zero, and the measured value of the cells incubated only in medium
without active ingredient is equated with 100%. The result is a
sigmoidal dose-effect curve from which the IC.sub.50 can be
determined (GraphPad Prism software).
[6550] A431: 2500 cells/well, medium: DMEM Hams, Biochrom,
#FG4815+10% FCS
[6551] NCI-H292: 2500 cells/well, medium: RPMI1640; Biochrom,
#FG1215+10% FCS
[6552] HT29L 2500 cells/well, medium: DMEM Hams; Biochrom,
#FG4815+10% FCS
[6553] Substances that inhibit cell proliferation at
<1.times.10.sup.-7 M are classified as effective.
[6554] Substances that inhibit cell proliferation at
<1.times.10.sup.-9 M are classified as especially effective.
[6555] Table 3 below lists the IC.sub.50 values.sup.1) of
representative exemplary embodiments from this assay.
TABLE-US-00050 TABLE 3 IC.sub.50 (nM) IC.sub.50 (nM) IC.sub.50 (nM)
Example A431 NCI-H292 HT29 1 0.03 <0.03 <0.03 2 1.50 1.47 3
0.28 0.12 4 0.19 0.17 5 <0.03 <0.03 <0.03 6 2.79 0.79 6.22
7 0.91 0.06 10.40 10 0.08 <0.03 2.46 11 2.91 0.36 46.70 12 0.39
<0.03 0.27 13 1.59 0.49 2.94 38 0.60 0.24 1.06 .sup.1)The
efficacy data given are based on the ADC batches described in
concrete terms here and may deviate in other batches having a
different drug/antibody ratio.
[6556] C-1.2 Protocol of Proliferation Assay with Short Substance
Incubation (Pulse Assay)
[6557] The protocol is performed as described above but the
substance is removed by suction after 4 hours of incubation with
the test substances and is replaced by fresh medium. The analysis
is performed as described above after a total of 96 hours.
[6558] Table 4 below lists the IC.sub.50 values.sup.1,2) of
representative exemplary embodiments from this assay.
TABLE-US-00051 TABLE 4 Example Antibody A431 NCI H292 7 Cetuximab
0.99 0.01 8 Nimotuzumab >200 34.4 9 Panitumumab 90.5 26.4 10
Cetuximab 0.0526 0.0884 14 Cetuximab 0.258 0.248 15 Cetuximab 13.4
3.5 16 Cetuximab 0.943 0.689 17 Cetuximab 0.285 0.133 18 Cetuximab
17.4 3.63 19 Cetuximab 0.89 20 Cetuximab 31.9 42.3 21 Nimotuzumab
54.6 120 22 Panitumumab 44.3 51.8 23 Cetuximab 88.1 124 24
Nimotuzumab 111 >200 25 Panitumumab 53 77 26 Cetuximab 10.6 3.02
27 Nimotuzumab 11.4 20.4 28 Panitumumab 5.78 8.6 29 Cetuximab 0.37
0.03 30 Panitumumab 23.2 3.34 31 Nimotuzumab 13.71 4.24 32
Cetuximab 50.8 20.4 33 Nimotuzumab 49 42 34 Panitumumab 51.1 32.2
35 Cetuximab 0.716 0.125 36 Cetuximab 0.357 0.0589 37 Cetuximab
0.52 0.17 38 Cetuximab 7.21 39 Cetuximab 1.49 0.03 40 Nimotuzumab
36 2.38 41 Panitumumab 110 6.91 42 Cetuximab 2.84 0.0949 43
Cetuximab 2.44 0.587 44 Cetuximab 0.456 0.571 52 Cetuximab >200
0.961 53 Cetuximab 0.253 0.133 54 Cetuximab 166 1.83 55 Cetuximab
1.47 0.11 337 Cetuximab 0.175 0.0746 338 Cetuximab 1.79 0.152 339
Cetuximab 2.57 0.194 340 Cetuximab 0.225 0.101 341 Cetuximab 2.25
0.232 342 Cetuximab 24.5 0.267 343 Panitumumab >200 20.3
.sup.1)The efficacy data given are based on the ADC batches
described in concrete terms here and may deviate in other batches
having a different drug/antibody ratio. .sup.2)This shows the
averages of two experiments (A431) or three (NCI-H292)
experiments.
[6559] C-1.3 Determination of the Influence on Tubulin
Polymerization
[6560] Cancer cells are degenerate cells which often lead to
development of a tumor due to an increased cell division.
Microtubules form the spindle fibers of the spindle apparatus and
are an essential component of the cell cycle. Regulated synthesis
and degradation of microtubules permits an accurate classification
of chromosomes on daughter cells and constitutes a continuous
dynamic process. A disturbance in the dynamics leads to a faulty
cell division and ultimately leads to cell death. However, the
increased cell division of cancer cells also makes them especially
sensitive to spindle fiber toxins which are a fixed component of
chemotherapy. Spindle fiber toxins such as paclitaxel or epothilone
lead to a greatly increased polymerization rate of the
microtubules, whereas the vinca alkaloids or monomethyl auristatin
E (MMAE) lead to a greatly reduced polymerization rate of the
microtubules. Those cases involve a sensitive disturbance in the
essential dynamics of the cell cycle. The compounds tested within
the scope of the present invention result in a reduced
polymerization rate of the microtubules.
[6561] The "Fluorescence-Based Microtubule Polymerization Assay
Kit" from Cytoskeleton (Denver, Colo., USA, order no. BKO11) was
used to investigate tubulin polymerization. In this assay, GTP is
added to unpolymerized tubulin, so that polymerization can take
place spontaneously. The assay is based on binding of the
fluorophore 4',6'-diamidino-2-phenylindole (DAPI) to tubulin. Free
and bond DAPI can be differentiated on the basis of their different
emission spectra. Tubulin polymerization can be tracked according
to the increase in fluorescence of bound DAPI fluorophores, because
DAPI has a much higher affinity for polymerized tubulin in
comparison with unpolymerized tubulin.
[6562] To perform this assay, the test substances dissolved in DMSO
were diluted from their initial concentration of 10 mM to 1 .mu.M
in water. In addition to the buffer controls,
polymerization-increasing paclitaxel was also included as an assay
control, and on the other hand, polymerization-inhibiting
vinblastine was also included. For the measurement 96-well plates
with a half bottom area were used, tracking the kinetics of tubulin
polymerization for one hour at 37.degree. C. in a fluorimeter. The
excitation wavelength was 355 nm, and the emission was tracked at
460 nm. For the range of the linear increase within the first 10
minutes, the change in fluorescence per minute (.DELTA.F/min) which
represents the polymerization rate of the microtubules was
calculated. The potency of the test substances was quantified on
the basis of the respective reduction in polymerization rate.
[6563] The value of the inhibition of MMAF at a concentration of 1
.mu.M is set at 100%.
[6564] Table 5 shows the data for inhibition of tubulin
polymerization of representative exemplary embodiments.
TABLE-US-00052 TABLE 5 Toxophore/ Concentration Percentage
inhibition of Example (.mu.M) tubulin polymerization MMAF 1 100
MMAF 10 34 MMAF 100 0 360 1 45 360 10 1 361 1 80 361 10 14 362 1 60
362 10 0 233 1 88 233 10 25 234 1 109 234 10 27 235 1 120 236 1 117
236 10 64 237 1 107 237 10 25 238 1 121 238 10 35 239 1 111 239 10
45 240 1 110 381 1 102 381 10 31 382 1 88 382 10 21 383 1 90 383 10
17
[6565] The toxophore MMAF and the exemplary embodiments inhibit
tubulin polymerization in a concentration-dependent. Tubulin
polymerization is completely inhibited at 100 .mu.M MMAF. Example
115 inhibits the tubulin polymerization rate at 1 .mu.M to 45% of
the value measured at 1 .mu.M MMAF.
[6566] C-1.4 Efficacy Test In Vivo
[6567] The efficacy of the conjugates according to the invention
was tested in vivo, e.g., by means of xenograft models. Those
skilled in the art are familiar with state-of-the-art methods for
testing the efficacy of a conjugate according to the invention
(see, for example, WO 2005081711; Polson et al., Cancer Res. Mar.
15, 2009, 69(6):2358-64). For example, a tumor cell line that
expresses the target molecule of the binder would be implanted in
rodents (e.g., mice). Then a conjugate according to the invention
or a control antibody or an isotonic saline solution would be
administered to the implant animals. The substance would be
administered either one or more times. After an incubation time of
several days, the tumor size would be determined in comparison with
animals treated with conjugate and with the control group.
[6568] C-1.4a Growth Inhibition/Regression of Experimental Tumors
in the Mouse
[6569] Human tumor cells that express the antigen for ADC are
injected subcutaneously into the flanks of immunosuppressed mice
for inoculation, for example, nude mice or SCID mice. Of the cell
culture, 1-10 million cells are isolated, centrifuged and
resuspended with Medium or Medium/Matrigel. The cell suspension is
then injected subcutaneously into the mice.
[6570] A tumor will then grow within a few days. The treatment
begins after establishment of the tumor but at a tumor size of 20
mm.sup.2. To investigate the effect of larger tumors, the treatment
may be started only when the tumor size reaches 50-100
mm.sup.2.
[6571] Treatment with ADCs is performed via the intravenous route
into the caudal vein of the mouse. The ADC is administered in a
volume of 5 mL/kg.
[6572] The treatment regime depends on the pharmacokinetics of the
antibody. The standard treatment consists of treatment three times
every fourth day. However, the treatment may also be continued
further or a second cycle with three days of treatment may also
follow at a later point in time.
[6573] Eight animals are used per treatment group as the standard.
This number may be increased if especially great fluctuations in
tumor growth or according to treatment are to be expected. In
addition to the groups receiving the active substances, one group
as the control group is treated only with the buffer according to
the same scheme.
[6574] In the case of this experiment, the area of the tumor is
measured regularly using a caliper in two dimensions
(length/width). The area of the tumor is determined by
length.times.width.
[6575] At the end of the experiment, the tumors are excised and
weighed. The comparison of the average tumor weights of the
treatment group with the control group is reported as T/C.
[6576] C-1.4b Efficacy in the BxPC3 Pancreatic Carcinoma Tumor
Model
[6577] Two million BxPC3 cells are injected subcutaneously into the
flanks of female NMRI nude mice for inoculation.
[6578] In a tumor cell of 40 mm.sup.2 on day 15, the treatment is
initiated with an intravenous dose of 10 mg/kg (days 15, 19, 22).
Following the treatment, the tumor growth is tracked until day 77.
The animals in the control group had to be sacrificed on day 50 for
veterinary reasons because of the large tumors.
[6579] A naked anti-EGFR antibody shows a delayed tumor growth at
approx. 14 days.
[6580] Animals treated with the anti-EGFR ADCs (Example 7 and
Example 10) did not exhibit any further tumor growth until the end
of the experiment on day 77.
[6581] C-1.4c Efficacy in the NCI-H292 NSCLC Tumor Model
[6582] Five million NCI-H292 cells were injected subcutaneously
into the flanks of female NMRI nude mice for inoculation.
[6583] At a tumor size of 100 mm.sup.2 on day 15, the treatment was
initiated with an intravenous dose of 3 mg/kg (days 15, 19, 23).
The tumor growth was tracked until day 27 after the treatment. At
the end of the experiment, the tumors were excised and weighed. The
experiment was terminated on day 27 because the animals in the
control group had to be euthanized due to the large tumors. The
animals treated with the naked anti-EGFR antibody exhibited
inhibited tumor growth. The animals treated with the anti-EGFR ADCs
show regression of the tumor. After an incubation time of several
days, the tumor size was determined in comparison of
conjugate-treated animals with the control group (T/C). The animals
treated with the conjugate had tumors of a smaller size.
[6584] The T/C ratio for the ADCs is between 0.05 (Example 10) and
0.1 (Example 7), whereas that with the naked anti-EGFR antibody is
0.3.
[6585] C-2.1 In Vitro Cell Proliferation Tests
[6586] The cytotoxic effect of the conjugates according to the
invention was tested in an in vitro cell proliferation test by
incubating a mammal cell that expresses the target molecule of the
binder either endogenously or recombinantly with the conjugate
according to the invention. After an incubation time of several
hours to several days, cell proliferation was determined on the
basis of the cell count in comparison with controls to which no
conjugated was added. The unconjugated toxophore alone may be added
as additional controls and/or cells that do not express the target
molecule of the binder may be used. The cell count was determined
by methods with which those skilled in the art are familiar, for
example, by counting or by using a test kit which allows a
determination of the cell count based on a measurement of ATP
(e.g., ATPlite.TM., Perkin Elmer). The IC.sub.50 value of the
conjugates according to the invention was determined in this way.
The selectivity of the conjugate could be determined by comparing
the IC.sub.50 value of the conjugate in measurements on cells
carrying the target molecule of the binder and cells not carrying
that molecule.
[6587] C-2.2 Determination of the Antiproliferative Effect of
Anti-Mesothelin ADC on the Human Colon Carcinoma Cell Line HT29
[6588] A defined cell count of the human colon carcinoma cell line
HT29 wt (2500 c/well, wild type) was sown in a 96-well MTP in whole
medium (10% FCS RPMI) and incubated overnight at 37.degree. C., 5%
carbon dioxide. In parallel with that transfected HT29 cells with
stable mesothelin expression were plated out in a 96-well MTP in
whole medium and incubated overnight (2500 c/well, 37.degree. C.,
5% carbon dioxide).
[6589] After 18 hours, the inoculation medium was replaced by fresh
medium with 10% FCS. The treatment was initiated with the addition
of the compounds according to the invention. The transfected cells
and the HT29 wt cells were treated identically here.
[6590] Of the substances to be tested, dose-effect curves were
determined in a concentration range from 10.sup.-5 M to 10.sup.14 M
(1:10 dilution series).
[6591] Incubation times of 48-96 hours were selected.
[6592] Proliferation was determined with the help of the MTT assay
(ATCC, Manassas, Va., USA, catalog no. 30-1010K). After the
selected time had elapsed, the HT29 wt cells were incubated for 4
hours with MTT before lysis of the cells was performed overnight by
adding detergent.
[6593] The dye thus formed was detected at 570 nm.
[6594] The 100% value was defined as the proliferation not with
test substance but otherwise identically treated cells. The data
obtained from this test represents triple determinations and at
least two independent experiments were conducted.
[6595] Table 6 below lists the IC.sub.50 values.sup.1) of
representative exemplary embodiments of this assay:
TABLE-US-00053 TABLE 6 HT29 meso.sup.+ HT29 wt Example IC.sub.50
(nM) IC.sub.50 (nM) 56 0.17 5.3 57 0.4 162 58 5 22 59 1 13 60 0.06
8 60 0.16 5.4 61 0.019 0.27 62 20 >300 63 0.03 1 64 0.11 1.4 65
0.14 3.3 66 1.5 8.6 67 0.015 11 68 2 9 69 0.07 20 70 0.02 2 71 0.6
11 72 0.05 8 73 25 221 74 9 >50 75 0.2 0.29 76 0.35 3 77 0.8 3.6
78 1.1 6 79 0.5 5 80 4.4 14 81 0.02 1.4 82 24 >1000 83 0.3 31 84
6 14 85 0.2 2 86 0.45 >500 87 0.9 3 88 0.01 0.2 89 0.06 3.1 90
2.6 60 91 0.35 1.1 92 38 167 93 52 89 94 0.6 >500 95 0.013 4.9
96 25 >500 97 0.2 0.2 98 1.7 6.3 99 0.27 1.6 100 2 44 101 3 500
102 7.2 294 103 10 32 104 1.8 0.9 105 0.6 1.4 106 0.8 15 107 3 2
108 0.74 9.2 109 0.09 1.1 110 0.002 0.28 111 0.7 162 112 0.018 4
113 0.4 2.5 114 0.6 4.1 115 0.06 10 129 5.3 57 130 0.13 672 131
0.09 769 132 3.3 >1000 133 2.1 219 134 0.03 199 135 0.25 317 136
1.6 3000 137 0.04 10 138 0.3 >1000 139 70 >1000 140 6.8
>1000 141 43 >1000 142 0.27 554 143 0.3 54 144 2.7 74 145 7.4
135 146 13 >1000 147 5.6 18 148 425 >1000 149 3.1 33 150
<0.1 0.15 151 >1000 >1000 152 4.1 >1000 153 30 >1000
154 2.2 >1000 155 0.5 >500 156 4.6 >500 157 0.09 839 158
0.18 602 159 177 >500 366 1.3 16 367 0.06 10 368 3.1 >500 369
0.02 14 377 1.6 >500 378 0.04 >500 379 7 >500 380 15
>500 .sup.1)Efficacy data listed here is based on the exemplary
embodiments described in the present experimental section with the
drug/mAb ratios indicated. The values may deviate with different
drug/mAb ratios.
[6596] C-2.3 Pharmacokinetics in the HT29 Tumor Model with
Mesothelin-Transfected HT29 Cells and Non-Transfected Cells
[6597] After i.v. administration of 16 mg/kg from Example 60, the
plasma and tumor concentrations of Example 60 were measured along
with potentially occurring metabolites such as those of Example
119, for example. The area under the curve (AUC) of the compound
from Example 119 in the plasma of animals with
mesothelin-transfected tumors was approx. 0.50 mgh/L; in the tumor,
the exposure of the compound from Example 119 was approx. 400 times
higher (AUC=203 mgh/L). In the animals with non-transfected tumors,
the exposure in the plasma in Example 119 was identical to the
exposure in the plasma of animals with transfected tumors. However,
the AUC in the tumor of the non-transfected animals was approx.
eight times lower than in the transfected animals. This is
indicative of a definite targeting effect in the tumor in the
presence of the antigen.
[6598] Analysis for Quantitation of the Compound from Example
119
[6599] The measurement of the compound from Example 119 in the
plasma and the tumor was performed after precipitation of the
proteins with methanol by high pressure liquid chromatography
(HPLC) coupled to a tandem mass spectrometer (MS).
[6600] For workup of 100 .mu.L plasma, it was mixed with 400 .mu.L
methanol and 10 .mu.L internal standard (ISTD, 50 ng/mL methanol)
and agitated for 10 seconds. After centrifuging for 5 minutes at
16,000 g, 250 .mu.L supernatant was transferred to an autosampler
vial, topped off with 250 .mu.L ammonium acetate buffer (AAC, 10
mM, pH 6.8) and agitated again.
[6601] In workup of a tumor, it was mixed with a four-fold amount
of methanol. The sample was pulverized for 6 minutes at 30 beats
per minute in the Tissuelyser II (Quiagen) and then centrifuged for
5 minutes at 16,000 g; 50 .mu.L of the supernatant was transferred
to an autosampler vial and topped off with 50 .mu.L ammonium
acetate buffer (10 mM, pH 6.8) and 5 L ISTD. After agitating again,
the tumor sample was ready for measurement.
[6602] Finally, the measurement of both matrix samples was
performed on the HPLC coupled atmospheric pressure
ionization/tandem mass spectrometer by means of turbo ion spray
interface (TISP) on an API4000 instrument from the company SCIEX.
The following m/z transitions were measured:
[6603] Example 119 (quantifier) 614.652.fwdarw.570.9
[6604] Example 119 (qualifier 1) 614.652.fwdarw.555.0
[6605] Example 119 (qualifier 2) 614.652.fwdarw.500.4
[6606] Internal standard (ISTD) 726.665.fwdarw.694.5
[6607] HPLC/LC MSMS (TISP) analysis was performed under the
following gradient conditions on an HP1100 pump (Agilent) using the
Gemini column (5 .mu.L C18 110 A, 50.times.3 mm, Phenomenex); flow
rate 0.4 mL/min; gradient: 0.0 min to 1.0 min 10% acetonitrile/90%
AAC, 1.0 min to 3.0 min 10% acetonitrile/90% AAC.fwdarw.50%
acetonitrile/50% AAC, 3.0 min to 5.5 min 50% acetonitrile/50% AAC,
5.5 min to 5.6 min 50% acetonitrile/50% AAC.fwdarw.10%
acetonitrile/90% AAC, 5.6 min to 6.0 min 10% acetonitrile/90%
AAC.
[6608] For the calibration, plasma samples with concentrations of
0.5-2000 .mu.g/L were mixed. The limit of quantitation (LOQ) was 2
.mu.g/L. The linear range was from 2 to 1000 .mu.g/L.
[6609] For calibration of the tumor samples, the supernatants of
untreated tumors with concentrations of 0.5 to 200 .mu.g/L were
mixed. The limit of quantitation was 5 .mu.g/L. The linear range
was from 5 to 200 .mu.g/L.
[6610] Quality controls for validity testing contained 5 and 50
.mu.g/L, with an additional 500 .mu.g/L in the plasma. The
concentrations determined on these samples deviated by as much as
20% from the ideal value (data not included).
[6611] C-2.4 Efficacy Test In Vivo
[6612] The efficacy of the conjugates according to the invention
was tested in vivo by using xenograft models, for example. Those
skilled in the art are familiar with the state-of-the-art methods
for testing the efficacy of a conjugate according to the invention
(see, for example, WO 2005081711; Polson et al., Cancer Res. Mar.
15, 2009, 69(6):2358-64). For example, a tumor cell line that
expresses the target molecule of the binder would be implanted in
rodents (e.g., mice). Then a conjugate according to the invention
or a control antibody or an isotonic saline solution would be
administered to the implant animals. The substance would be
administered one or more times. After an incubation time of several
days, the tumor size would be determined in comparison with animals
treated with the conjugate and with the control group. The size of
the tumors was smaller in the animals treated with the
conjugate.
[6613] C-2.4a Testing Anti-Mesothelin ADCs in Experimental Tumors
in the Mouse
[6614] Human mesothelin-expressing tumor cells are injected
subcutaneously into the flanks of immunosuppressed mice, for
example, nude or SCID mice; 1-10 million cells are isolated from
the cell culture, centrifuged and resuspended in 100 .mu.L Medium
or 1:1 Medium/Matrigel. The cell suspension is then injected
subcutaneously into the mice.
[6615] A tumor will then grow within a few days. The treatment
begins after establishment of the tumor but at a tumor size of 20
mm.sup.2. To investigate the effect of larger tumors, the treatment
may be started only when the tumor size reaches 50-100
mm.sup.2.
[6616] Treatment with ADCs is performed via the intravenous route
into the caudal vein of the mouse. The ADC is dissolved in PBS and
administered in a volume of 5 mL/kg.
[6617] The treatment regime depends on the pharmacokinetics of the
antibody. The standard treatment consists of treatment three times
every fourth day. However, the treatment may also be continued
further or a second cycle with three days of treatment may also
follow at a later point in time.
[6618] Eight animals are used per treatment group as the standard.
This number may be increased if especially great fluctuations in
tumor growth or according to treatment are to be expected. In
addition to the groups receiving the active substances, one group
as the control group is treated only with the buffer according to
the same scheme.
[6619] In this experiment, the area of the tumor is measured
regularly in two dimensions (length/width) using a caliper. The
area of the tumor is determined by length.times.width.
[6620] At the end of the experiment, the tumors are excised and
weighed. The comparison of the average tumor weights of the
treatment group (T) with the control group (C) is reported as
T/C.
[6621] C-2.4b Efficacy in the HT29 Colon Carcinoma Tumor Model with
Mesothelin-Transfected HT29 Cells
[6622] One million HT29 cells (stable transfection with mesothelin)
were injected subcutaneously into the flanks of NMRI nude mice for
inoculation. At a tumor size of 20-30 mm.sup.2 on day 6,
intravenous treatment begins (days 6, 10, 14). Following the
treatment, the tumor growth was tracked until day 48.
[6623] C-2.4c Efficacy in the Ovcar3 Ovarian Carcinoma Tumor
Model
[6624] Seven million Ovcar3 cells were injected subcutaneously into
the flanks of NMRI nude mice for inoculation.
[6625] At a tumor size of 25-30 mm.sup.2 on day 31, the intravenous
treatment was initiated in the dosage range of 5-30 mg/kg (days 31,
35, 39). Following the treatment, the tumor growth was tracked
until day 94. At the end of the experiment, the tumors were excised
and weighed.
[6626] C-3.1 Analysis of the Cytotoxic Effect of the ADCs Directed
Against C4.4a
[6627] The cytotoxic effect of the anti-C4.4a ADCs was analyzed on
various cell lines: [6628] A549 (CCL-185, ATCC) transfected with
the sequence for the complete C4.4a receptor [6629] A549, mock
transfected [6630] A549 wild type (DSMZ, lot 11) [6631] NCI-H292,
endogenous C4.4a-expressing lung cancer cell line CRL-1848, ATCC)
[6632] SCC-4 endogenous C4.4a-expressing squamous epithelial cell
carcinoma cell line (CRL-1624, ATCC) [6633] SCC-9 endogenous
C4.4a-expressing squamous epithelial cell carcinoma cell line
(CRL-1629, ATCC) [6634] HCT-116 endogenous C4.4a-expressing colon
carcinoma cell line (CCL-247, ATCC) [6635] HCT-116/VM46, HCT-116
transfected with VM46 [6636] A431NS (CRL-2592, ATCC)
[6637] The cells were cultured according to the standard method as
stipulated in the American Tissue Type Collection (ATCC) for the
respective cell lines. For this procedure, the cells were isolated
with a solution of trypsin (0.05%) and EDTA (0.02%) in PBS
(Biochrom AG #L2143), pelletized, resuspended in culture medium,
counted and sown in a 94-hole culture plate with a white bottom
(Costar #3610) (2500 cells in 100 .mu.L/well) and then incubated at
37.degree. C. and 5% carbon dioxide in an incubator. After 24
hours, the antibody-drug conjugates were added to the cells in 100
.mu.L culture medium in concentrations of 10.sup.-7 M to 10.sup.-11
M (double values) and incubated at 37.degree. C. and 5% carbon
dioxide in an incubator. After 72 hours, the cell viability was
determined by the Cell Titer Glow Luminescent Cell Viability Assay
(Promega #G7573 and #G7571). To do so, 100 .mu.L of substrate was
added per cell batch, then the plates were covered with aluminum
foil, agitated for 2 minutes with the plate agitator at 180 rpm,
left to stand for 8 minutes on the laboratory bench and then
measured using the Victor X2 instrument (Perkin Elmer). The
substrate detects the ATP content in the viable cells, thus
generating a luminescence signal, the height of which is directly
proportional to the vitality of the cells. The IC.sub.50 is
calculated from the measured data using the Graph Pad Prism
laboratory software.
[6638] Table 7 below shows the IC.sub.50 value.sup.1) of
representative exemplary embodiments of this assay:
TABLE-US-00054 TABLE 7 IC.sub.50 (nM) IC.sub.50 (nM) A549: A549
Example C4.4a Mock 163 0.081 11.15 164 0.7 50 165 0.47 4.75 166 0.6
100 167 0.4 20 0.2 26 0.1 17 168 0.53 4.50 169 0.39 32 170 0.01
0.15 171 0.43 10 172 0.01 25 173 4 >100 174 0.58 6.36 175 0.7
14.9 176 0.1 65.5 177 0.030 9.53 178 3.8 21 179 0.62 4.19 180 0.4
>100 181 1.2 66.1 182 0.46 4.20 183 4.5 12.7 184 5 16 185 0.4
0.7 186 0.3 23 187 5.4 53 188 0.052 11.27 189 0.65 6.70 190 0.062
>100 191 0.02 2.5 192 0.1 71 193 0.32 9 194 0.035 6.19 195 0.037
-30 196 197 0.3 20 198 0.08 >100 199 0.1 kH 200 0.03 50 201 0.04
1.5 202 0.6 50 203 0.4 2 204 0.1 14 205 0.062 6.33 206 0.044 6.93
207 0.058 4.01 208 0.062 7.74 209 0.066 9.11 210 0.061 6.78 211
0.076 100 212 0.02 0.02 213 0.044 44 214 0.04 45 215 0.046 26 216
0.074 >100 217 0.053 >100 218 0.037 60 219 0.3 1 220 0.04
>100 221 0.1 >100 222 0.04 >100 223 0.44 6.8 224 0.09 50
225 0.1 0.4 226 0.04 0.52 227 0.03 0.04 228 0.03 0.04 229 0.08 26
230 0.02 >100 231 0.17 0.27 212 0.06 7 242 3.0 >100 243 0.045
>100 244 0.06 >100 245 0.27 >100 246 0.13 >100 247 0.14
>100 248 0.17 >100 249 0.28 >100 250 1.1 >100 251 1.3
>100 252 >100 >100 253 0.15 kH 254 0.29 >100 255 0.04
>100 256 0.035 100 257 0.036 >100 258 0.018 >100 259 0.062
>100 260 0.06 >100 261 0.1 80 262 0.1 kH 263 0.3 kH 264 0.1
kH 265 0.2 30 266 3 kH 267 0.03 50 268 0.05 20 269 >100 kH 270
0.03 >100 271 1 >100 272 0.2 kH 373 0.03 7 385 0.04 >100
386 0.02 >100 387 0.04 >100 388 0.04 >100 .sup.1) Efficacy
data listed here is based on the exemplary embodiments described in
the present experimental section with the drug/mAb ratios
indicated. The values may deviate with different drug/mAb
ratios.
[6639] C-3.3 In Vitro Tests for Determining Cell Permeability
[6640] The cell permeability of a substance can be investigated by
means of in vitro testing in a flux assay using Caco2 cells (M. D.
Troutman and D. R. Thakker, Pharm. Res. 20(8):1210-1224 (2003)). To
do so, the cells are cultured on 24-hole filter plates for 15-16
days. To determine the permeation, the respective test substance in
a HEPES buffer was applied to the cells either apically (A) or
basally (B) and incubated for 2 hours. After 0 h and 2 h, samples
were taken from the cis and trans compartments. The samples were
separated by HPLC (Agilent 1200, Biblingen, Germany) using
reverse-phase columns. The HPLC system was linked to a triple
quadrupole mass spectrometer API 4000 (Applied Biosystems Applera,
Darmstadt, Germany) via a turbo ion spray interface. Permeability
was evaluated on the basis of a P.sub.app value which was
calculated by means of the formula published by Schwab et al. (D.
Schwab et al., J. Med. Chem. 46, 1716-1725 (2003)). A substance was
classified as having active transport when the ratio of P.sub.app
(B-A) to P.sub.app (A-B) was >2 or <0.5.
[6641] The permeability of B to A (P.sub.app (B-A)) is of crucial
importance for toxophores but are released intracellularly: the
lower this permeability, the longer is the dwell time of the
substance in the cell after intracellular release and thus also the
time available for interaction with the biochemical target (here
tubulin).
[6642] The following table shows permeability data of
representative exemplary embodiments of this assay:
TABLE-US-00055 TABLE 8 Exemplary P.sub.app (B-A) embodiment (nm/s)
233 2 234 1.6 235 2.5 236 5 237 1 239 7 277 2 278 1
[6643] In comparison with this monomethyl auristatin E (MMAE) and
monomethyl auristatin F (MMAF) have a P.sub.app (B-A) value of 73
nm/s in this test.
[6644] C-3.4 In Vitro Tests for Determining the Substrate
Properties for P-glycoprotein (P-gp)
[6645] Many tumor cells express transporter proteins for drugs,
which is often associated with the development of a resistance to
cytostatics. Substances that are not substrates of such transporter
proteins such as P-glycoprotein (P-gp) or BCRP, for example, might
thus have an improved profile of effect.
[6646] The substrate properties of a substance for P-gp (ABCB1) are
determined by means of flux assay using LLC-PK1 cells that
overexpress P-gp (L-MDR1 cells) (A. H. Schinkel et al., J. Clin.
Invest. 96, 1698-1705 (1995)). To do so, LLC-PK1 cells or L-MDR1
cells were cultured on 96-hole filter plates for 3-4 days. To
determine the permeation, the respective test substance was applied
to the cells and incubated for 2 hours, either alone or in the
presence of an inhibitor (e.g., ivermectin or verapamil) in a HEPES
buffer either apically (A) or basally (B). After 0 h and 2 h,
samples were taken from the cis and trans compartments. The samples
were separated by HPLC using reverse-phase columns. The HPLC system
was coupled to a triple quadrupole mass spectrometer API 3000
(Applied Biosystems Applera, Darmstadt, Germany) by way of a turbo
ion spray interface. The permeability was evaluated on the basis of
a P.sub.app value which was calculated by using the formula
published by Schwab et al. (D. Schwab et al., J. Med. Chem. 46,
1716-1725 (2003)). A substance was classified as being a P-gp
substrate if the efflux ratio P.sub.app (B-A) to P.sub.app (A-B)
was >2.
[6647] The efflux ratios in L-MDR1 and LLC-PK1 cells or the efflux
ratio in the presence or absence of an inhibitor can be compared as
additional criteria for evaluating the P-gp substrate properties.
The respective substance is considered to be a P-gp substrate if
these values differ by more than a factor of 2.
TABLE-US-00056 TABLE 9 Exemplary P.sub.app (B-A) embodiment (nm/s)
233 3 234 3.6 235 2.1 236 3.6 237 4 239 2 277 6 278 4
[6648] Again in this assay the permeability of the examples of B to
A cited here is low, i.e., the dwell time of the toxophores in the
cells is long.
[6649] C-3.5 Efficacy Test In Vivo
[6650] The efficacy of the conjugates according to the invention
was tested in vivo, for example, by means of xenograft models.
Those skilled in the art are familiar with the state-of-the-art
methods for testing the efficacy of a conjugate according to the
invention (see, for example, WO 2005081711; Polson et al., Cancer
Res. Mar. 15, 2009, 69(6):2358-64). For example, a tumor cell line
that expresses the target molecule of the binder would be implanted
in rodents (e.g., mice). Then a conjugate according to the
invention or a control antibody or an isotonic saline solution
would be administered to the implant animals. The substance would
be administered either one or more times. The tumor growth was
determined using a caliper twice a week. After an incubation time
of several days, the tumor size would be determined in comparison
with animals treated with conjugate and with the control group.
[6651] C-3.5a Testing of ADCs in Experimental Tumors in the
Mouse
[6652] Human tumor cells that express the antigen for ADC are
injected subcutaneously into the flanks of immunosuppressed mice
for inoculation, for example, nude mice or SCID mice. Of the cell
culture, 1-10 million cells are isolated, centrifuged and
resuspended with 100 .mu.L medium or 50% medium/50% Matrigel. The
cell suspension is then injected subcutaneously into the mice.
[6653] A tumor will then grow within a few days. The treatment
begins after establishment of the tumor but at a tumor size of 25
mm.sup.2.
[6654] Treatment with ADCs is performed via the intravenous route
into the caudal vein of the mouse. The ADC is dissolved in PBS and
administered in a volume of 10 mL/kg.
[6655] The treatment regime depends on the pharmacokinetics of the
antibody. The standard treatment consists of treatment three times
every fourth day. However, the treatment may also be continued
further or a second cycle with three days of treatment may also
follow at a later point in time.
[6656] Eight animals are used per treatment group as the standard.
This number may be increased if especially great fluctuations in
tumor growth or according to treatment are to be expected. In
addition to the groups receiving the active substances, one group
as the control group is treated only with the buffer according to
the same scheme.
[6657] In the case of this experiment, the area of the tumor is
measured regularly using a caliper in two dimensions
(length/width). The area of the tumor is determined by
length.times.width.
[6658] At the end of the experiment, the tumors are excised and
weighed. The quotient of the average tumor weights of the treatment
group (T) and the control group (C) is given as T/C. If the control
group and treatment group are ended at different points in time,
the T/C value is calculated on the basis of the tumor areas of the
last joint measurement of all treatment groups and control
groups.
[6659] One million SCC-4 cells are injected subcutaneously into the
flanks of female NMRI nude mice for inoculation.
[6660] The intravenous treatment with the ADCs is started when the
average tumor size has reached 30-35 mm.sup.2. When the control
groups have reached the maximum allowed size, the test is
terminated and the tumors are excised and weighed. All the C-4.4a
targeting ADCs tested were found to have inhibited tumor growth in
a dose dependent ratio. In a dose of 30 mg/kg all the tested ADCs
achieved a T/C ratio of <0.1 (Example 216, Example 211, Example
215, Example 213). A significant antitumor effect was achieved in
comparison with the controls for all the tested ADCs down to a dose
of 15 mg/kg.
[6661] One million NCI-H292 cells are injected subcutaneously into
the flanks of female NMRI nude mice for inoculation.
[6662] The intravenous treatment with the ADCs was initiated at an
average tumor size of 30-35 mm.sup.2. The control groups and
treatment groups were terminated whenever the maximum allowed tumor
size was reached. Therefore, differences in the subsequent growth
of tumors after the end of treatment can contribute toward a
further characterization of the ADCs. The tumor areas at the last
joint measurement time were therefore used to determine the
antitumor effect in comparison with the controls (T/C). In the
NCI-H292 mouse model used in this experiment, it is demonstrated
that all the tested ADCs were able to reduce tumor growth in a dose
dependent manner in comparison with the control. For Example 216, a
significant antitumor effect was achieved down to a dose of 1.9
mg/kg; for Example 211, this was achieved down to a dose of 3.75
mg/kg. The minimal T/C values achieved in this model include a T/C
of 0.16 at 30 mg/kg for Example 216; a T/C of 0.17 at 30 mg/kg for
Example 211; a T/C of 0.16 at 30 mg/kg for Example 215, and a T/C
of 0.17 at 15 mg/kg for Example 213.
[6663] C-4. Pharmacokinetics in the A549 Tumor Model with
C4.4a-Transfected and Non-Transfected A549 Cells
[6664] After i.v. administration of 7-30 mg/kg of various ADCs, the
plasma and tumor concentrations of ADC and any potential
metabolites that might occur were measured and the pharmacokinetic
parameters such as the clearance (CL), area under the curve (AUC)
and half-life (t.sub.1/2) were calculated.
[6665] Analysis for Quantitation of the Metabolites Occurring
Potentially
[6666] After precipitation of the proteins with methanol, the
compounds in the plasma and tumor were measured by high pressure
liquid chromatography (HPLC) coupled to a tandem mass spectrometer
(MS).
[6667] For workup of 100 .mu.L plasma, it was mixed with 400 .mu.L
methanol and 10 .mu.L internal standard (ISTD, 50 ng/mL in
methanol) and shaken for 10 seconds. After centrifuging for 5
minutes at 16,000 g, 250 .mu.L supernatant was transferred to an
autosampler vial, topped off with 250 .mu.L ammonium acetate buffer
(AAC, 10 mM, pH 6.8) and shaken again.
[6668] In workup of a tumor, it was mixed with a four-fold amount
of methanol. The sample was pulverized for 6 minutes at 30 beats
per minute in the Tissuelyser II (Quiagen) and then centrifuged for
5 minutes at 16,000 g; 50 .mu.L of the supernatant was transferred
to an autosampler vial and topped off with 50 .mu.L ammonium
acetate buffer (10 mM, pH 6.8) and 5 .mu.L ISTD. The tumor sample
was ready for measurement.
[6669] Finally, the two matrix samples were measured on the
atmospheric pressure ionization/tandem mass spectrometer by means
of turbo ion spray interface (TISP) on an API4000 instrument from
the company SCIEX with the mass spectrometer linked to an HPLC.
[6670] HPLC/LC-MSMS (TISP) analysis was performed on an HP1100 pump
(Agilent) with a Gemini column (5 .mu.m C18 110 A, 50.times.3 mm,
Phenomenex).
[6671] For calibration, plasma samples with concentrations of
0.5-2000 .mu.g/L were mixed. The limit of quantitation (LOQ) was 2
.mu.g/L. The linear range extended from 2 to 1000 .mu.g/L.
[6672] For calibration of the tumor samples, the supernatant of
untreated tumors with concentrations of 0.5-200 .mu.g/L was mixed.
The limit of quantitation was 5 .mu.g/L. The linear range extended
from 5 to 200 .mu.g/L.
[6673] Quality controls for validity testing contained 5 and 50
.mu.g/L plus 500 .mu.g/L in the plasma. The concentrations
determined on these samples deviated as much as 20% from the ideal
value (data not included).
[6674] C-4.1 In Vitro Cell Proliferation Tests
[6675] The cytotoxic effect of the conjugates according to the
invention was tested in an in vitro cell proliferation test by
incubating a mammalian cell that expresses the target molecule of
the binder either endogenously or recombinantly with the conjugate
according to the invention. After an incubation time of several
hours to several days, cell proliferation was determined on the
basis of the cell count in comparison with controls to which no
conjugated was added. The unconjugated toxophore alone may be added
as additional controls and/or cells that do not express the target
molecule of the binder may be used. The cell count was determined
by methods with which those skilled in the art are familiar, for
example, by counting or by using a test kit which allows a
determination of the cell count based on a measurement of ATP
(e.g., ATPlite.TM., Perkin Elmer). The IC.sub.50 value of the
conjugates according to the invention was determined in this way.
The selectivity of the conjugate was determined by comparing the
IC.sub.50 value of the conjugate in measurements on cells carrying
the target molecule of the binder and cells not carrying that
molecule.
[6676] C-4.2 In Vitro Cytotoxicity on the Cell Lines HT29, DLD-1
and SNU-5
[6677] For testing the CA9 selective, cytotoxic effect on tumor
cells that are endogenously CAIX-positive or CAIX-negative, the
human colon carcinoma cell lines HT29 (CA9-positive) and DLD-1
(CA9-negative) as well as the gastric carcinoma cell line SNU-5
(CA9-positive) were used. A defined cell count of the cell line
HT29 (5000 c/well) was sown in a 96-well MTP for luminescent in
whole medium (DMEM/HAM's F12, 10% FCS heat inactivated) and
incubated overnight at 37.degree. C., 5% CO.sub.2. A similar
procedure was followed with the SNU-5 cell line, but the medium
here was ISCOVE's+10% FCS (heat inactivated). The antigen-negative
cell line DLD-1 was plated out in parallel in a 96-well MTP for
luminescence in whole medium (RPMI 1640, 10% FCS, heat inactivated)
and incubated overnight (5000 c/well, 37.degree. C., 5%
CO.sub.2).
[6678] After 24 hours, the substances to be tested were
concentrated three times in RPMI/5% FCS and prepared. The treatment
began with the addition of the substances to be tested and/or the
ADC to the cells. The HT29, SNU-5 and DLD-1 cells were treated
identically.
[6679] Of the substances to be tested, dose-effect curves were
determined in a concentration range of 3.times.10.sup.-7 M to
10.sup.-12M.
[6680] Incubation times of 2 to 96 hours were selected.
[6681] Detection of proliferation was performed with the help of
the Cell Titer Glo Luminescent Cell Viability Assay (PROMEGA
catalog no. #G7571). After the selected incubation time had
elapsed, the Cell Titer Glo reagent was incubated with the cells
for 20 minutes and then the measurement of the luminescence was
performed with the luminescence reader VICTOR Light (Perkin
Elmer).
[6682] The proliferation not with test substance but with otherwise
identically treated cells was defined as the 100% value. The data
obtained from this test represents triple determinations and at
least two independent experiments were performed.
[6683] The following table lists the IC.sub.50 values of
representative exemplary embodiments from this assay:
TABLE-US-00057 TABLE 10 EC.sub.50 (nM) EC.sub.50 (nM) EC.sub.50
(nM) Example HT29 DLD-1 SNU-5 280 2.9 58 n.d. 281 1.7 23 n.d. 282
<0.01 <0.01 n.d. 283 0.3 20 n.d. 284 0.7 39 3 284 1.6 5.8 2.2
285 0.5 11.1 n.d. 286 0.03 6.6 n.d. 287 >30 >68 n.d. 288 39
72 n.d. 289 49 >300 n.d. 290 115 7938 84 291 27 107 n.d. 292 223
74 n.d. 293 0.1 0.6 n.d. 294 13.6 5.5 21.4 295 >100 >100 63
296 0.4 16.4 n.d. 297 <0.01 <0.01 n.d. 298 2.5 31 7.8 299 4.1
32.3 n.d. 300 5 41 n.d. 301 >30 >300 n.d. 302 >30 >300
n.d. 303 2.2 18.3 3.3 304 0.05 8 0.6 305 0.9 23 2.2 306 66 >100
33
[6684] C-4.3 Determining the Antiproliferative Effect of Anti-CAIX
ADC on the Human Pancreatic Carcinoma Cell Line MIAPaCa 2 and Colon
Carcinoma Cell Line HT29
[6685] A defined cell count of the human pancreatic carcinoma cell
line MIAPaCa 2 (2500 c/well, wild type) was sown in a 96-well MTP
in whole medium (DMEM, 10% FCS, 2.5% equine serum) and incubated
overnight at 37.degree. C., 5% CO.sub.2. Transfected MIAPaCa 2
cells (MIAPaCaMSL) with stable CAIX expression were plated out in a
96-well MTP in whole medium and incubated overnight (2500 c/well,
37.degree. C., 5% CO.sub.2).
[6686] To test the cytotoxic effect on cells that endogenously
express CAIX, the colon carcinoma cell line HT29 was used. The
cells (2500 c/well, wild type) were also sown in a 96-well MTP and
incubated overnight in whole medium (RPMI, 10% FCS).
[6687] After 18 hours, the inoculation medium was replaced by fresh
medium with serum. Treatment was initiated by adding the substances
to be tested and/or the ADC. The transfected cells and MIAPaCa2
cells as well as the HT29 cells were treated identically.
[6688] Of the substances to be tested, dose-effect curves in a
concentration range of 10.sup.-5M to 10.sup.-14 M (1:10 dilution
series) were determined.
[6689] Incubation times of 48-96 hours were selected.
[6690] Proliferation was detected with the help of the MTT assay
(ATCC, Manassas, Va., USA, catalog no. 30-1010K). After the end of
the selected incubation time, the MTT reagent was incubated for 4
hours with the cells before lysis of the cells was performed
overnight by adding the detergent.
[6691] The dye that was formed was detected at 570 nm.
[6692] Proliferation not with test substance but otherwise
identically treated cells was defined as the 100% value. Data
obtained from this test represents triple determinations and at
least two independent experiments were conducted in each case.
[6693] The following table shows the IC.sub.50 values of
representative exemplary embodiments from this assay:
TABLE-US-00058 TABLE 11 MIAPaCa 2/CA9+ MIAPaCa 2 wt Example
IC.sub.50 (nM) IC.sub.50 (nM) 280 0.4 8 281 1.2 4.1 282 0.05 0.5
283 0.4 2.1 284 0.9 5 285 0.6 1.8 286 0.8 2.7 287 69 >1000 288
12 42 289 78 >1000 290 1.9 357 291 22 n.d. 292 35 1686 293
<0.1 <0.1 294 1 7 295 25 1400 296 0.6 0.9 297 0.02 0.4 298
1.1 9 299 0.8 3.3 300 0.3 1.1 301 22 34 302 3.9 39 303 0.009 0.015
304 0.019 0.03 305 2.6 3.7 306 21 25 313 3.8 44 314 150 >1000
315 3.2 23 320 19 78 321 220 1142 322 172 >1000 323 238 >500
324 11 15 325 137 >1000 326 2 >500 327 19 26
[6694] C-4.4 Efficacy Test In Vivo
[6695] The efficacy of the conjugates according to the invention
was tested in vivo, for example, by means of xenograft models.
Those skilled in the art are familiar with the state-of-the-art
methods for testing the efficacy of a conjugate according to the
invention (see, for example, WO 2005081711; Polson et al., Cancer
Res. Mar. 15, 2009, 69(6):2358-64). For example, a tumor cell line
that expresses the target molecule of the binder would be implanted
in rodents (e.g., mice). Then a conjugate according to the
invention or a control antibody or an isotonic saline solution
would be administered to the implant animals. The substance would
be administered either one or more times. After an incubation time
of several days, the tumor size would be determined in comparison
with animals treated with conjugate and with the control group.
[6696] C-4.4a Testing the Efficacy of Anti-CA9 ADCs in Experimental
Tumors in the Mouse
[6697] Human CA9-expressing tumor cells are injected s.c. into the
flanks of immunosuppressed mice, for example, nude or SCID mice;
1-10 million cells are isolated from the cell culture, centrifuged
and resuspended with 100 .mu.L Medium or Medium/Matrigel. The cell
suspension is then injected subcutaneously into the mice.
[6698] A tumor will then grow within a few days. The treatment
begins after establishment of the tumor but at a tumor size of 20
mm.sup.2. To investigate the effect of larger tumors, the treatment
may be started only when the tumor size reaches 50-100
mm.sup.2.
[6699] Treatment with ADCs is performed via the intravenous route
into the caudal vein of the mouse. The ADC is dissolved in PBS and
administered in a volume of 5 mL/kg.
[6700] The treatment regime depends on the pharmacokinetics of the
antibody. The standard treatment consists of treatment three times
every fourth day. However, the treatment may also be continued
further or a second cycle with three days of treatment may also
follow at a later point in time.
[6701] Eight animals are used per treatment group as the standard.
This number may be increased if especially great fluctuations in
tumor growth or according to treatment are to be expected. In
addition to the groups receiving the active substances, one group
as the control group is treated only with the buffer according to
the same scheme.
[6702] In the case of this experiment, the area of the tumor is
measured regularly using a caliper in two dimensions
(length/width).
[6703] At the end of the experiment, the tumors are excised and
weighed. The quotient of the average tumor weights of the treatment
group (T) and the control group (C) is given as T/C. If the control
group and treatment group are terminated on different days, the
tumor area at the last measurement point at which all the groups
were still in the experiment was used to calculate the T/C.
[6704] C-4.4b Efficacy in SNU-5 Gastric Carcinoma Tumor Model
[6705] Three million SNU-5 cells were injected subcutaneously into
the flanks of nodSCID mice for inoculation.
[6706] At a tumor size of 30-40 mm.sup.2 on day 15, the treatment
is initiated by intravenous injection of doses in the range between
5 and 30 mg/kg (days 15, 19, 23). Following the treatment the tumor
growth of all groups was tracked. The control groups were ended
when the tumors had reached the maximum allowed tumor size. Either
all the groups were terminated together at this point in time, the
tumors were excised and weighed and the T/C value was formed based
on the tumor weight, or the treatment groups were observed with
regard to further growth of the tumors. In the latter case, the T/C
value was calculated based on the tumor area at the last joint
measurement time. The animals treated with the anti-CA9 ADCs showed
inhibited tumor growth.
[6707] C-4.4c HT29 Model
[6708] Female athymic nude mice carrying the nu/nu gene were used
for the human xenograft study of mice. These inbred mice (NMRI
background) were obtained from Taconic, Denmark with a body weight
between 18 and 21 g. Human HT29 colon carcinoma cells were cultured
in the recommended medium with 10% fetal calf serum (FCS) in
accordance with the ATCC protocol. The cells were harvested for
transplantation in the subconfluent state (80% confluence). On day
0, tumors were initiated by subcutaneous (s.c.) injection of
1.times.10.sup.6 HT29 cells in 50% Matrigel/50% culture medium
(without FCS) in the mice. The transplantation volume was 100
.mu.L, the transplantation site was the left flank. Tumor growth
was determined by measuring the area of the tumor (calculation:
longest diameter.times.length of the perpendicular to that
diameter), measured by caliper. According to the statement of
object, the tumors were used up to a predefined size of 20-30 or
40-50 mm.sup.2. At this point in time, the animals were randomized
and assigned to the individual test groups--control groups and
treatment groups. The treatment with ADCs was administered every
fourth day for a total of three times (Q4D.times.3). The form of
the treatment was an intravenous (i.v.) injection into the caudal
vein. The treatment of each animal was based on the individual body
weight and the treatment volumes were 5-10 mL/kg body weight. The
area of the tumor and the animal's weight were determined twice a
week, monitoring the body weight as a measure of the
treatment-associated toxicity. Animals were euthanized when signs
of toxicity developed or when the tumor reached the size of 150
mm.sup.2 or when tumors became necrotic. At the time of termination
of a group, the animals were euthanized, the tumors were excised
and the respective tumor wet weight was determined. The response to
treatment was calculated as the ratio of treatment to controls
based on the tumor area or the final tumor weight where
appropriate.
D. EXEMPLARY EMBODIMENTS FOR PHARMACEUTICAL COMPOSITIONS
[6709] The compounds according to the invention can be converted to
pharmaceutical preparations by the following procedure:
[6710] i.v. Solution:
[6711] The compound according to the invention is dissolved in a
concentration below the saturation solubility in a physiologically
safe solvent (e.g., isotonic saline solution, D-PBS or a
formulation with glycine and sodium chloride in citrate puffer with
the addition of polysorbate 80). The solution is sterile-filtered
and bottled in sterile and pyrogen-free injection containers.
[6712] i.v. Solution
[6713] The compounds according to the invention can be converted to
the dosage forms indicated. This may be done in a known way by
"mixing with" or "dissolving in" inert nontoxic pharmaceutical
suitable excipients (e.g., buffer substances, stabilizers,
solubilizers, preservatives). The following may be included for
example: amino acids (glycine, histidine, methionine, arginine,
lysine, leucine, isoleucine, threonine, glutamic acid,
phenylalanine and others), sugars and related substances (glucose,
saccharose, mannitol, trehalose, sucrose, mannose, lactose,
sorbitol), glycerol, sodium, potassium, ammonium and calcium salts
(e.g., sodium chloride, potassium chloride or disodium hydrogen
phosphate and many more) acetate/acetic acid buffer systems,
phosphate buffer systems, citric acid and citrate buffer systems
trometamol (TRIS and TRIS salts), polysorbates (e.g., polysorbate
80 and polysorbate 20), poloxamers (e.g., poloxamer 188 and
poloxamer 171), macrogols (PEG-derivatives, e.g., 3350), Triton
X-100, EDTA salts. glutathione, albumins (e.g., human), urea,
benzyl alcohol, phenol, chlorocresol, metacresol, benzalkonium
chloride and many others.
[6714] Lyophilisate for Later Conversion to an i.v., s.c. or i.m.
Solution:
[6715] Alternatively, the compounds according to the invention may
be converted to a stable lyophilisate (possibly with the help of
the excipients listed above) and reconstituted with a suitable
solvent (e.g., water for injection, isotonic, saline solution)
before administration and then administered.
Sequence CWU 1
1
4111278PRTHomo Sapiens 1Leu Glu Cys Tyr Ser Cys Val Gln Lys Ala Asp
Asp Gly Cys Ser Pro 1 5 10 15 Asn Lys Met Lys Thr Val Lys Cys Ala
Pro Gly Val Asp Val Cys Thr 20 25 30 Glu Ala Val Gly Ala Val Glu
Thr Ile His Gly Gln Phe Ser Leu Ala 35 40 45 Val Arg Gly Cys Gly
Ser Gly Leu Pro Gly Lys Asn Asp Arg Gly Leu 50 55 60 Asp Leu His
Gly Leu Leu Ala Phe Ile Gln Leu Gln Gln Cys Ala Gln 65 70 75 80 Asp
Arg Cys Asn Ala Lys Leu Asn Leu Thr Ser Arg Ala Leu Asp Pro 85 90
95 Ala Gly Asn Glu Ser Ala Tyr Pro Pro Asn Gly Val Glu Cys Tyr Ser
100 105 110 Cys Val Gly Leu Ser Arg Glu Ala Cys Gln Gly Thr Ser Pro
Pro Val 115 120 125 Val Ser Cys Tyr Asn Ala Ser Asp His Val Tyr Lys
Gly Cys Phe Asp 130 135 140 Gly Asn Val Thr Leu Thr Ala Ala Asn Val
Thr Val Ser Leu Pro Val 145 150 155 160 Arg Gly Cys Val Gln Asp Glu
Phe Cys Thr Arg Asp Gly Val Thr Gly 165 170 175 Pro Gly Phe Thr Leu
Ser Gly Ser Cys Cys Gln Gly Ser Arg Cys Asn 180 185 190 Ser Asp Leu
Arg Asn Lys Thr Tyr Phe Ser Pro Arg Ile Pro Pro Leu 195 200 205 Val
Arg Leu Pro Pro Pro Glu Pro Thr Thr Val Ala Ser Thr Thr Ser 210 215
220 Val Thr Thr Ser Thr Ser Ala Pro Val Arg Pro Thr Ser Thr Thr Lys
225 230 235 240 Pro Met Pro Ala Pro Thr Ser Gln Thr Pro Arg Gln Gly
Val Glu His 245 250 255 Glu Ala Ser Arg Asp Glu Glu Pro Arg Leu Thr
Gly Gly Ala Ala Gly 260 265 270 His Gln Asp Arg Ser Asn 275 2
293PRTMus musculus 2Leu Glu Cys Tyr Ser Cys Val Gln Lys Ala Asp Asp
Gly Cys Ser Pro 1 5 10 15 His Arg Met Lys Thr Val Lys Cys Gly Pro
Gly Val Asp Val Cys Thr 20 25 30 Glu Ala Val Gly Ala Val Glu Thr
Ile His Gly Gln Phe Ser Val Ala 35 40 45 Val Arg Gly Cys Gly Ser
Gly Ile Pro Gly Lys Asn Asp Arg Gly Leu 50 55 60 Asp Leu His Gly
Leu Leu Ala Phe Phe Gln Leu Gln Gln Cys Ser Glu 65 70 75 80 Asp Arg
Cys Asn Ala Lys Leu Asn Leu Thr Leu Arg Gly Leu Asn Pro 85 90 95
Ala Gly Asn Glu Ser Ala Tyr Glu Pro Asn Gly Ala Glu Cys Tyr Ser 100
105 110 Cys Val Gly Leu Ser Arg Glu Lys Cys Gln Gly Ser Met Pro Pro
Val 115 120 125 Val Asn Cys Tyr Asn Ala Ser Gly Arg Val Tyr Lys Gly
Cys Phe Asp 130 135 140 Gly Asn Val Thr Leu Thr Ala Ala Asn Val Thr
Val Ser Leu Pro Val 145 150 155 160 Arg Gly Cys Val Gln Asp Glu Thr
Cys Thr Arg Asp Gly Val Thr Gly 165 170 175 Pro Gly Phe Thr Leu Ser
Gly Ser Cys Cys Gln Gly Pro Arg Cys Asn 180 185 190 Ala Asp Leu Arg
Asn Lys Thr Tyr Phe Ser Pro Arg Ile Pro Pro Leu 195 200 205 Val Leu
Leu Pro Pro Pro Thr Thr Ala Ala Pro Ser Thr Arg Ala Gln 210 215 220
Asn Ser Ser Ser Thr Thr Ser Thr Ala Ala Pro Thr Thr Thr Thr Ser 225
230 235 240 Ile Ile Lys Pro Thr Thr Ala Gln Ala Ser His Thr Ser Pro
His Glu 245 250 255 Met Asp Leu Glu Val Ile Gln Glu Glu Gly Ala Ser
Leu Ser Gly Gly 260 265 270 Ala Ala Gly His Gly Gly Thr Ala Gly His
Gly Gly Ala Ala Gly His 275 280 285 Gln Asp Arg Ser Asn 290
3346PRTHomo Sapiens 3Met Asp Pro Ala Arg Lys Ala Gly Ala Gln Ala
Met Ile Trp Thr Ala 1 5 10 15 Gly Trp Leu Leu Leu Leu Leu Leu Arg
Gly Gly Ala Gln Ala Leu Glu 20 25 30 Cys Tyr Ser Cys Val Gln Lys
Ala Asp Asp Gly Cys Ser Pro Asn Lys 35 40 45 Met Lys Thr Val Lys
Cys Ala Pro Gly Val Asp Val Cys Thr Glu Ala 50 55 60 Val Gly Ala
Val Glu Thr Ile His Gly Gln Phe Ser Leu Ala Val Arg 65 70 75 80 Gly
Cys Gly Ser Gly Leu Pro Gly Lys Asn Asp Arg Gly Leu Asp Leu 85 90
95 His Gly Leu Leu Ala Phe Ile Gln Leu Gln Gln Cys Ala Gln Asp Arg
100 105 110 Cys Asn Ala Lys Leu Asn Leu Thr Ser Arg Ala Leu Asp Pro
Ala Gly 115 120 125 Asn Glu Ser Ala Tyr Pro Pro Asn Gly Val Glu Cys
Tyr Ser Cys Val 130 135 140 Gly Leu Ser Arg Glu Ala Cys Gln Gly Thr
Ser Pro Pro Val Val Ser 145 150 155 160 Cys Tyr Asn Ala Ser Asp His
Val Tyr Lys Gly Cys Phe Asp Gly Asn 165 170 175 Val Thr Leu Thr Ala
Ala Asn Val Thr Val Ser Leu Pro Val Arg Gly 180 185 190 Cys Val Gln
Asp Glu Phe Cys Thr Arg Asp Gly Val Thr Gly Pro Gly 195 200 205 Phe
Thr Leu Ser Gly Ser Cys Cys Gln Gly Ser Arg Cys Asn Ser Asp 210 215
220 Leu Arg Asn Lys Thr Tyr Phe Ser Pro Arg Ile Pro Pro Leu Val Arg
225 230 235 240 Leu Pro Pro Pro Glu Pro Thr Thr Val Ala Ser Thr Thr
Ser Val Thr 245 250 255 Thr Ser Thr Ser Ala Pro Val Arg Pro Thr Ser
Thr Thr Lys Pro Met 260 265 270 Pro Ala Pro Thr Ser Gln Thr Pro Arg
Gln Gly Val Glu His Glu Ala 275 280 285 Ser Arg Asp Glu Glu Pro Arg
Leu Thr Gly Gly Ala Ala Gly His Gln 290 295 300 Asp Arg Ser Asn Ser
Gly Gln Tyr Pro Ala Lys Gly Gly Pro Gln Gln 305 310 315 320 Pro His
Asn Lys Gly Cys Val Ala Pro Thr Ala Gly Leu Ala Ala Leu 325 330 335
Leu Leu Ala Val Ala Ala Gly Val Leu Leu 340 345 4363PRTMus musculus
4Met Asp Ala Ala Arg Arg Gly Asp Thr Gln Pro Val Met Trp Thr Thr 1
5 10 15 Gly Trp Leu Leu Leu Leu Pro Leu Leu Leu Cys Glu Gly Ala Gln
Ala 20 25 30 Leu Glu Cys Tyr Ser Cys Val Gln Lys Ala Asp Asp Gly
Cys Ser Pro 35 40 45 His Arg Met Lys Thr Val Lys Cys Gly Pro Gly
Val Asp Val Cys Thr 50 55 60 Glu Ala Val Gly Ala Val Glu Thr Ile
His Gly Gln Phe Ser Val Ala 65 70 75 80 Val Arg Gly Cys Gly Ser Gly
Ile Pro Gly Lys Asn Asp Arg Gly Leu 85 90 95 Asp Leu His Gly Leu
Leu Ala Phe Phe Gln Leu Gln Gln Cys Ser Glu 100 105 110 Asp Arg Cys
Asn Ala Lys Leu Asn Leu Thr Leu Arg Gly Leu Asn Pro 115 120 125 Ala
Gly Asn Glu Ser Ala Tyr Glu Pro Asn Gly Ala Glu Cys Tyr Ser 130 135
140 Cys Val Gly Leu Ser Arg Glu Lys Cys Gln Gly Ser Met Pro Pro Val
145 150 155 160 Val Asn Cys Tyr Asn Ala Ser Gly Arg Val Tyr Lys Gly
Cys Phe Asp 165 170 175 Gly Asn Val Thr Leu Thr Ala Ala Asn Val Thr
Val Ser Leu Pro Val 180 185 190 Arg Gly Cys Val Gln Asp Glu Thr Cys
Thr Arg Asp Gly Val Thr Gly 195 200 205 Pro Gly Phe Thr Leu Ser Gly
Ser Cys Cys Gln Gly Pro Arg Cys Asn 210 215 220 Ala Asp Leu Arg Asn
Lys Thr Tyr Phe Ser Pro Arg Ile Pro Pro Leu 225 230 235 240 Val Leu
Leu Pro Pro Pro Thr Thr Ala Ala Pro Ser Thr Arg Ala Gln 245 250 255
Asn Ser Ser Ser Thr Thr Ser Thr Ala Ala Pro Thr Thr Thr Thr Ser 260
265 270 Ile Ile Lys Pro Thr Thr Ala Gln Ala Ser His Thr Ser Pro His
Glu 275 280 285 Met Asp Leu Glu Val Ile Gln Glu Glu Gly Ala Ser Leu
Ser Gly Gly 290 295 300 Ala Ala Gly His Gly Gly Thr Ala Gly His Gly
Gly Ala Ala Gly His 305 310 315 320 Gln Asp Arg Ser Asn Met Glu Lys
Tyr Pro Gly Lys Gly Gly Ala Gln 325 330 335 Ile Pro Ala Lys Gly Gly
Ser Gly Thr Leu Gly Ser Trp Leu Ser Ala 340 345 350 Val Leu Leu Thr
Val Val Ala Gly Ala Met Leu 355 360 59PRTArtificial SequenceC4.4a
binder 5Phe Ser Asn Ala Trp Met Ser Trp Val 1 5 69PRTArtificial
SequenceC4.4a binder 6Phe Ser Asp Tyr Gln Met Thr Trp Ile 1 5
79PRTArtificial SequenceC4.4a binder 7Phe Gly His Tyr Tyr Met Phe
Trp Ile 1 5 88PRTArtificial SequenceC4.4a binder 8Phe Ser Ser Asn
Tyr Met Ser Trp 1 5 920PRTArtificial SequenceC4.4a binder 9Val Ser
Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser 1 5 10 15
Val Lys Gly Arg 20 1020PRTArtificial SequenceC4.4a binder 10Val Ser
Gly Val Ser Trp Asn Gly Ala Arg Thr His Tyr Ala Asp Ser 1 5 10 15
Val Lys Gly Arg 20 1120PRTArtificial SequenceC4.4a binder 11Val Ser
Ala Ile Ser Gly Ser Gly Tyr Ser Thr His Tyr Ala Asp Ser 1 5 10 15
Val Lys Gly Arg 20 1220PRTArtificial SequenceC4.4a binder 12Val Ser
Ala Ile Ser Ser Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser 1 5 10 15
Val Lys Gly Arg 20 1310PRTArtificial SequenceC4.4a binder 13Ala Arg
Glu Gly Leu Trp Ala Phe Asp Tyr 1 5 10 1415PRTArtificial
SequenceC4.4a binder 14Ala Lys Gly Asp Tyr Leu Val Tyr Ser Ala Tyr
Tyr Phe Asp Ser 1 5 10 15 1513PRTArtificial SequenceC4.4a binder
15Ala Arg Leu Pro Tyr Gly Ser Gln Ser Gly Val Asp Tyr 1 5 10
1617PRTArtificial SequenceC4.4a binder 16Ala Arg Glu Ser Gly Gly
Ser Gly Pro Asn Tyr Tyr Tyr Gly Met Asp 1 5 10 15 Val
1714PRTArtificial SequenceC4.4a binder 17Thr Gly Ser Ser Ser Asn
Ile Gly Ala Gly Tyr Val Val His 1 5 10 1813PRTArtificial
SequenceC4.4a binder 18Ser Gly Ser Ser Ser Asn Val Gly Ser Asn Pro
Val Asn 1 5 10 1914PRTArtificial SequenceC4.4a binder 19Thr Gly Ser
Ser Ser Asn Ile Gly Ala Gly Tyr Val Val His 1 5 10
2014PRTArtificial SequenceC4.4a binder 20Thr Gly Ser Ser Ser Asn
Ile Gly Ala Gly Tyr Val Val His 1 5 10 217PRTArtificial
SequenceC4.4a binder 21Asp Asn Asn Lys Arg Pro Ser 1 5
227PRTArtificial SequenceC4.4a binder 22Arg Asn Asn Gln Arg Pro Ser
1 5 237PRTArtificial SequenceC4.4a binder 23Ser Asn Asn Gln Arg Pro
Ser 1 5 247PRTArtificial SequenceC4.4a binder 24Ser Asn Asn Gln Arg
Pro Ser 1 5 2512PRTArtificial SequenceC4.4a binder 25Cys Ala Ala
Trp Asp Asp Arg Leu Asn Gly Pro Val 1 5 10 2612PRTArtificial
SequenceC4.4a binder 26Cys Ala Ala Trp Asp Asp Arg Leu Asn Gly Trp
Val 1 5 10 2710PRTArtificial SequenceC4.4a binder 27Cys Gln Ser Tyr
Asp Ser Ser His Val Leu 1 5 10 2812PRTArtificial SequenceC4.4a
binder 28Cys Gln Ser Tyr Asp Arg Ser Leu Arg Gly Trp Val 1 5 10
29113PRTArtificial SequenceC4.4a binder 29Gln Ser Val Leu Thr Gln
Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile
Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Val
Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45
Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50
55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly
Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp
Asp Asp Arg 85 90 95 Leu Asn Gly Pro Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu Gly 100 105 110 Gln 30112PRTArtificial
SequenceC4.4a binder 30Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser
Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser
Ser Ser Asn Val Gly Ser Asn 20 25 30 Pro Val Asn Trp Tyr Gln Gln
Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn
Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys
Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser
Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Arg Leu 85 90
95 Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln
100 105 110 31111PRTArtificial SequenceC4.4a binder 31Gln Ser Val
Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg
Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25
30 Tyr Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
35 40 45 Leu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp
Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala
Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys
Gln Ser Tyr Asp Ser Ser 85 90 95 His Val Leu Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu Gly Gln 100 105 110 32113PRTArtificial
SequenceC4.4a binder 32Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser
Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser
Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Val Val His Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Ser Asn
Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg Ser 85 90
95 Leu Arg Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110 Gln 33117PRTArtificial SequenceC4.4a binder 33Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20
25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Leu Trp Ala
Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100
105 110 Val Thr Val Thr Ser 115 34122PRTArtificial SequenceC4.4a
binder 34Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Asp Tyr 20 25 30 Gln Met Thr Trp Ile Arg Gln Thr Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Val Ser Trp Asn Gly Ala
Arg Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys
Gly Asp Tyr Leu Val Tyr Ser Ala Tyr Tyr Phe Asp Ser Trp 100 105 110
Gly Gln Gly Thr Leu Val Thr Val Thr Ser 115 120 35120PRTArtificial
SequenceC4.4a binder 35Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Gly His Tyr 20 25 30 Tyr Met Phe Trp Ile Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly
Ser Gly Tyr Ser Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Leu Pro Tyr Gly Ser Gln Ser Gly Val Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Leu Val Thr Val Thr Ser 115 120
36124PRTArtificial SequenceC4.4a binder 36Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Tyr Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ala Ile Ser Ser Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Glu Ser Gly Gly Ser Gly Pro Asn Tyr
Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Leu Val Thr
Val Thr Ser 115 120 37339DNAArtificial SequenceC4.4a binder
37cagtctgtgc tgactcagcc accctcagcg tctgggaccc ctgggcagag ggtcaccatc
60tcctgcactg ggagcagctc caacattggg gcgggttatg ttgtacattg gtatcagcag
120ctcccaggaa cggcccccaa actcctcatc tatgacaata ataagcgacc
ctcaggggtc 180cctgaccgat tctctggctc caagtctggc acctcagcct
ccctggccat cagtgggctc 240cggtccgagg atgaggctga ttattactgt
gcagcatggg atgacaggct gaatggtccg 300gtgttcggcg gaggaaccaa
gttaaccgtc ctaggtcag 33938336DNAArtificial SequenceC4.4a binder
38cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc
60tcttgttctg gaagcagctc caacgtcggg agtaatcctg taaactggta tcagcagctc
120ccaggaacgg cccccaaact cctcatctat aggaataatc agcggccctc
aggggtccct 180gaccgattct ctggctccaa gtctggcacc tcagcctccc
tggccatcag tgggctccgg 240tccgaggatg aggctgatta ttactgtgca
gcatgggatg acaggctgaa tggttgggtg 300ttcggcggag gaaccaagct
gacggtccta ggtcag 33639333DNAArtificial SequenceC4.4a binder
39cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc
60tcctgcactg ggagcagctc caacattggg gcgggttatg ttgtacattg gtatcagcag
120ctcccaggaa cggcccccaa actcctcatc tatagtaata atcagcggcc
ctcaggagtc 180cctgaccgat tctctggctc caagtctggc acctcagcct
ccctggccat cagtgggctc 240cggtccgagg atgaggctga ttattactgc
cagtcctatg acagcagcca tgttttattc 300ggcggaggaa ccaagctgac
ggtcctaggt cag 33340339DNAArtificial SequenceC4.4a binder
40cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc
60tcctgcactg ggagcagctc caacattggg gcgggttatg ttgtacattg gtatcagcag
120ctcccaggaa cggcccccaa actcctcatc tatagtaata atcagcggcc
ctcaggggtc 180cctgaccgat tctctggctc caagtctggc acctcagcct
ccctggccat cagtgggctc 240cggtccgagg atgaggctga ttattactgc
cagtcctatg acagaagcct gcgtggttgg 300gtgttcggcg gaggaaccaa
gctgacggtc ctaggtcag 33941351DNAArtificial SequenceC4.4a binder
41gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc
60tcctgtgcag cctctggatt caccttcagt aacgcctgga tgagctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtttcatac attagtagta gtggtagtac
catatactac 180gcagactctg tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
actgccgtgt attactgtgc gagagaaggg 300ttatgggcct ttgactactg
gggccagggt accctggtca ccgtgactag t 35142366DNAArtificial
SequenceC4.4a binder 42gaggtgcagc tgttggagtc tgggggaggc ttggtacagc
ctggggggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt gactatcaga
tgacctggat ccgccagact 120ccagggaagg ggctggagtg ggtatcgggt
gttagttgga atggcgctag gacgcactat 180gcagactctg tgaagggccg
attcaccatc tccagagaca attccaagaa cacgctgtat 240ctacaaatga
acagcctgag agccgaggac actgccgtgt attactgtgc gaagggcgac
300tacctggttt actccgcata ctactttgac tcctggggcc agggtaccct
ggtcaccgtg 360actagt 36643360DNAArtificial SequenceC4.4a binder
43gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc
60tcctgtgcag cctctggatt caccttcggt cactactata tgttctggat ccgtcaggct
120ccagggaagg ggctggagtg ggtctcagct attagtggta gtggttatag
cacacactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
actgccgtgt attactgtgc gagactgcca 300tatggttcgc agagtggcgt
tgactactgg ggccagggta ccctggtcac cgtgactagt 36044372DNAArtificial
SequenceC4.4a binder 44gaggtgcagc tgttggagtc tgggggaggc ttggtacagc
ctggggggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt agcaactaca
tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcagct
attagtagta gtggtagtag cacatactac 180gcagactccg tgaagggccg
gttcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga
acagcctgag agccgaggac actgccgtgt attactgtgc gagagaatct
300ggtgggagcg gaccgaacta ctactacggt atggacgtct ggggccaagg
taccctggtc 360accgtgacta gt 372459PRTArtificial SequenceC4.4a
binder 45Phe Ser Asn Ala Trp Met Ser Trp Val 1 5 4620PRTArtificial
SequenceC4.4a binder 46Val Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile
Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Gly Arg 20 4710PRTArtificial
SequenceC4.4a binder 47Ala Arg Glu Gly Leu Trp Ala Phe Asp Tyr 1 5
10 4814PRTArtificial SequenceC4.4a binder 48Thr Gly Ser Ser Ser Asn
Ile Gly Ala Gly Tyr Val Val His 1 5 10 497PRTArtificial
SequenceC4.4a binder 49Asp Asn Asn Lys Arg Pro Ser 1 5
5012PRTArtificial SequenceC4.4a binder 50Cys Ala Ala Trp Asp Asp
Arg Leu Asn Gly Pro Val 1 5 10 51117PRTArtificial SequenceC4.4a
binder 51Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser
Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Gly Leu Trp Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 52113PRTArtificial SequenceC4.4a binder
52Glu Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1
5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala
Gly 20 25 30 Tyr Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala
Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly
Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala
Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp
Tyr Tyr Cys Ala Ala Trp Asp Asp Arg 85 90 95 Leu Asn Gly Pro Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln
53351DNAArtificial SequenceC4.4a binder 53gaggtgcagc tgctggaaag
cggcggagga ctggtgcagc ctggaggcag cctgagactg 60tcttgcgccg ccagcggctt
caccttcagc aacgcctgga tgagctgggt ccgacaggct 120cctggcaagg
gcctggaatg ggtgtcctac atcagcagca gcggcagcac catctactac
180gccgacagcg tgaagggccg gttcaccatc agccgggaca acagcaagaa
caccctgtac 240ctgcagatga acagcctgcg ggccgaggac accgccgtgt
actactgcgc cagagaaggc 300ctgtgggcct tcgactactg gggccagggc
accctggtca ccgtgtctag c 35154339DNAArtificial SequenceC4.4a binder
54gagagcgtgc tgacccagcc tcctagcgtg tccggcgctc ctggccagag agtgaccatc
60agctgcaccg gcagcagcag caacatcgga gccggctacg tggtgcactg gtatcagcag
120ctgcccggca ccgcccccaa gctgctgatc tacgacaaca acaagcggcc
tagcggcgtg 180cccgacagat tcagcggcag caagagcggc accagcgcca
gcctggccat cagcggcctg 240agaagcgagg acgaggccga ctactactgc
gccgcctggg acgacagact gaacggccct 300gtgttcggcg gaggcaccaa
gctgaccgtg ctgggacag 339559PRTArtificial SequenceC4.4a binder 55Phe
Ser Asn Ala Trp Met Ser Trp Val 1 5 5620PRTArtificial SequenceC4.4a
binder 56Val Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala
Asp Ser 1 5 10 15 Val Lys Gly Arg 20 5710PRTArtificial
SequenceC4.4a binder 57Ala Arg Glu Gly Leu Trp Ala Phe Asp Tyr 1 5
10 5814PRTArtificial SequenceC4.4a binder 58Thr Gly Ser Ser Ser Asn
Ile Gly Ala Gly Tyr Val Val His 1 5 10 597PRTArtificial
SequenceC4.4a binder 59Asp Asn Asn Lys Arg Pro Ser 1 5
6012PRTArtificial SequenceC4.4a binder 60Cys Ala Ala Tyr Asp Asp
Ser Leu Ser Gly Pro Val 1 5 10 61117PRTArtificial SequenceC4.4a
binder 61Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser
Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Gly Leu Trp Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 62113PRTArtificial SequenceC4.4a binder
62Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1
5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala
Gly 20 25 30 Tyr Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala
Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly
Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala
Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp
Tyr Tyr Cys Ala Ala Tyr Asp Asp Ser 85 90 95 Leu Ser Gly Pro Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln
63351DNAArtificial SequenceC4.4a binder 63gaggtgcagc tgctggaatc
cggcggaggc ctggtgcagc ctggcggatc tctgagactg 60tcctgcgccg ccagcggctt
taccttctcc aacgcctgga tgtcctgggt ccgacaggcc 120cctggcaagg
gactggaatg ggtgtcctac atctcctcct ccggctccac catctactac
180gccgactccg tgaagggccg gttcaccatc tcccgggaca actccaagaa
caccctgtac 240ctgcagatga actccctgcg ggccgaggac accgccgtgt
actactgcgc ccgagagggc 300ctgtgggcct tcgattattg gggccagggc
accctggtca ccgtcagctc a 35164339DNAArtificial SequenceC4.4a binder
64cagtccgtgc tgacccagcc cccttctgtg tctggcgccc ctggccagag agtgaccatc
60tcttgcaccg gctcctccag caacatcggc gctggctacg tggtgcactg gtatcagcag
120ctgcccggca ccgcccccaa gctgctgatc tacgacaaca acaagcggcc
ctccggcgtg 180cccgacagat tctccggctc caagtccggc acctccgcct
ccctggccat ctccggcctg 240agatctgagg acgaggccga ctactactgc
gccgcctacg acgactccct gtccggccct 300gtgttcggcg gaggcacaaa
gttaaccgtg ctgggccag 339659PRTArtificial SequenceC4.4a binder 65Phe
Ser Asn Ala Trp Met Ser Trp Val 1 5 6620PRTArtificial SequenceC4.4a
binder 66Val Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala
Asp Ser 1 5 10 15 Val Lys Gly Arg 20 6710PRTArtificial
SequenceC4.4a binder 67Ala Arg Glu Gly Leu Trp Ala Phe Asp Tyr 1 5
10 6814PRTArtificial SequenceC4.4a binder 68Thr Gly Ser Ser Ser Asn
Ile Gly Ala Gly Tyr Val Val His 1 5 10 697PRTArtificial
SequenceC4.4a binder 69Asp Asn Asn Lys Arg Pro Ser 1 5
7012PRTArtificial SequenceC4.4a binder 70Cys Ala Ala Phe Asp Asp
Ser Leu Asn Gly Pro Val 1 5 10 71117PRTArtificial SequenceC4.4a
binder 71Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser
Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Gly Leu Trp Ala Phe Asp Lys Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 72113PRTArtificial SequenceC4.4a binder
72Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1
5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala
Gly 20 25 30 Tyr Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala
Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly
Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala
Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp
Tyr Tyr Cys Ala Ala Phe Asp Asp Arg 85 90 95 Leu Asn Gly Pro Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln
73351DNAArtificial SequenceC4.4a binder 73gaggtgcagc tgctggaatc
cggcggaggc ctggtgcagc ctggcggatc tctgagactg 60tcctgcgccg cctccggctt
taccttctcc aacgcctgga tgtcctgggt ccgacaggct 120cctggcaagg
gcctggaatg ggtgtcctac atctcctcct ccggctccac catctactac
180gccgactccg tgaagggccg gttcaccatc tcccgggaca actccaagaa
caccctgtac 240ctgcagatga actccctgcg ggccgaggac accgccgtgt
actactgcgc ccgagagggc 300ctgtgggcct tcgataagtg gggccagggc
accctggtca ccgtcagctc a 35174339DNAArtificial SequenceC4.4a binder
74cagtccgtgc tgacccagcc tccttccgcc tctggcaccc ctggccagag agtgaccatc
60tcctgcaccg gctcctccag caacatcggc gctggctacg tggtgcactg gtatcagcag
120ctgcccggca ccgcccccaa gctgctgatc tacgacaaca acaagcggcc
ctccggcgtg 180cccgacagat tctccggctc caagtccggc acctccgcct
ccctggccat ctccggcctg 240agatctgagg acgaggccga ctactactgc
gccgccttcg acgaccggct gaacggccct 300gtgttcggcg gaggcacaaa
gttaaccgtg ctgggccag 339759PRTArtificial SequenceC4.4a binder 75Phe
Ser Ser Ala Trp Met Ser Trp Val 1 5 7620PRTArtificial SequenceC4.4a
binder 76Val Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala
Asp Ser 1 5 10 15 Val Lys Gly Arg 20 7710PRTArtificial
SequenceC4.4a binder 77Ala Arg Glu Gly Leu Trp Ala Phe Asp Tyr 1 5
10 7814PRTArtificial SequenceC4.4a binder 78Thr Gly Ser Ser Ser Asn
Ile Gly Ala Gly Tyr Val Val His 1 5 10 797PRTArtificial
SequenceC4.4a binder 79Asp Asn Asn Lys Arg Pro Ser 1 5
8012PRTArtificial SequenceC4.4a binder 80Cys Ala Ala Tyr Asp Asp
Ser Leu Ser Gly Pro Val 1 5 10 81117PRTArtificial SequenceC4.4a
binder 81Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser
Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Gly Leu Trp Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 82113PRTArtificial SequenceC4.4a binder
82Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1
5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala
Gly 20 25 30 Tyr Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala
Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly
Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala
Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp
Tyr Tyr Cys Ala Ala Tyr Asp Asp Ser 85 90 95 Leu Ser Gly Pro Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln
83351DNAArtificial SequenceC4.4a binder 83gaggtgcagc tgctggaatc
cggcggaggc ctggtgcagc ctggcggatc tctgagactg 60tcctgcgccg ccagcggctt
taccttctcc agcgcctgga tgtcctgggt ccgacaggcc 120cctggcaagg
gactggaatg ggtgtcctac atctcctcct ccggctccac catctactac
180gccgactccg tgaagggccg gttcaccatc tcccgggaca actccaagaa
caccctgtac 240ctgcagatga actccctgcg ggccgaggac accgccgtgt
actactgcgc ccgagagggc 300ctgtgggcct tcgattattg gggccagggc
accctggtca ccgtcagctc a 35184339DNAArtificial SequenceC4.4a binder
84cagtccgtgc tgacccagcc cccttctgtg tctggcgccc ctggccagag agtgaccatc
60tcttgcaccg gctcctccag caacatcggc gctggctacg tggtgcactg gtatcagcag
120ctgcccggca ccgcccccaa gctgctgatc tacgacaaca acaagcggcc
ctccggcgtg 180cccgacagat tctccggctc caagtccggc acctccgcct
ccctggccat ctccggcctg 240agatctgagg acgaggccga ctactactgc
gccgcctacg acgactccct gtccggccct 300gtgttcggcg gaggcacaaa
gttaaccgtg ctgggccag 339859PRTArtificial SequenceC4.4a binder 85Phe
Ser Ser Ala Trp Met Ser Trp Val 1 5 8620PRTArtificial SequenceC4.4a
binder 86Val Ser Tyr Ile Ser Ser Ser Gly Ser Ser Thr Tyr Tyr Ala
Asp Ser 1 5 10 15 Val Lys Gly Arg 20 8710PRTArtificial
SequenceC4.4a binder 87Ala Arg Glu Gly Leu Trp Ala Phe Asp Lys 1 5
10 8814PRTArtificial SequenceC4.4a binder 88Ser Gly Ser Ser Ser Asn
Ile Gly Ala Gly Tyr Val Val His 1 5 10 897PRTArtificial
SequenceC4.4a binder 89Asp Asn Asn Gln Arg Pro Ser 1 5
9012PRTArtificial SequenceC4.4a binder 90Cys Ala Ala Phe Asp Asp
Arg Leu Ser Gly Pro Val 1 5 10 91117PRTArtificial SequenceC4.4a
binder 91Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Gly Leu Trp Ala Phe Asp Lys Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 92113PRTArtificial SequenceC4.4a binder
92Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1
5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ala
Gly 20 25 30 Tyr Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala
Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn Asn Gln Arg Pro Ser Gly
Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala
Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp
Tyr Tyr Cys Ala Ala Phe Asp Asp Arg 85 90 95 Leu Ser Gly Pro Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln
93351DNAArtificial SequenceC4.4a binder 93gaggtgcagc tgctggaaag
cggcggaggc ctggtgcagc ctggcggaag cctgagactg 60agctgtgccg ccagcggctt
caccttcagc agcgcctgga tgagctgggt ccgacaggcc 120cctggcaagg
gcctggaatg ggtgtcctac atcagcagca gcggcagcag cacctactac
180gccgacagcg tgaagggccg gttcaccatc agccgggaca acagcaagaa
caccctgtac 240ctgcagatga acagcctgcg ggccgaggac accgccgtgt
actactgcgc cagagaaggc 300ctgtgggcct tcgataagtg gggccagggc
accctggtca ccgtcagctc a 35194339DNAArtificial SequenceC4.4a binder
94cagagcgtgc tgacccagcc tcctagcgcc tctggcaccc ctggccagag agtgaccatc
60agctgcagcg gcagcagcag caacatcgga gccggctacg tggtgcactg gtatcagcag
120ctgcccggca ccgcccccaa gctgctgatc tacgacaaca accagcggcc
cagcggcgtg 180cccgacagat tttccggcag caagagcggc accagcgcca
gcctggccat cagcggcctg 240agaagcgagg acgaggccga ctactactgc
gccgccttcg acgacagact gagcggccct 300gtgttcggcg gaggcacaaa
gttaaccgtg ctgggccag 339959PRTArtificial SequenceC4.4a binder 95Phe
Ser Asp Tyr Gln Met Thr Trp Ile 1 5 9620PRTArtificial SequenceC4.4a
binder 96Val Ser Gly Val Ser Trp Asn Gly Ala Arg Thr His Tyr Ala
Asp Ser 1 5 10 15 Val Lys Gly Arg 20 9715PRTArtificial
SequenceC4.4a binder 97Ala Lys Gly Asp Tyr Leu Val Tyr Ser Ala Tyr
Tyr Phe Asp Ser 1 5 10 15 9813PRTArtificial SequenceC4.4a binder
98Ser Gly Ser Ser Ser Asn Val Gly Ser Asn Pro Val Asn 1 5 10
997PRTArtificial SequenceC4.4a binder 99Arg Asn Asn Gln Arg Pro Ser
1 5 10012PRTArtificial SequenceC4.4a binder 100Cys Ala Ala Trp Asp
Asp Arg Leu Asn Gly Trp Val 1 5 10 101122PRTArtificial
SequenceC4.4a binder 101Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gln Met Thr Trp Ile Arg Gln
Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp
Asn Gly Gly Ser Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Lys Gly Asp Tyr Leu Val Tyr Ser Ala Tyr Tyr Phe Asp Ser Trp
100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
102112PRTArtificial SequenceC4.4a binder 102Glu Ser Val Leu Thr Gln
Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile
Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Pro Val
Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45
Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50
55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu
Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp
Asp Arg Leu 85 90 95 Asn Gly Trp Gly Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly Gln 100 105 110 103366DNAArtificial SequenceC4.4a
binder 103gaggtgcagc tgctggaaag cggcggagga ctggtgcagc ctggaggcag
cctgagactg 60tcttgcgccg ccagcggctt caccttcagc gactaccaga tgacctggat
ccgacagacc 120cctggcaagg gcctggaatg ggtgtccggc atcagctgga
acggaggcag cacccactac 180gccgacagcg tgaagggccg gttcaccatc
agccgggaca acagcaagaa caccctgtac 240ctgcagatga acagcctgcg
ggccgaggac accgccgtgt actactgcgc caagggcgac 300tacctggtgt
acagcgccta ctacttcgac agctggggcc agggcaccct ggtcaccgtg 360tctagc
366104336DNAArtificial SequenceC4.4a binder 104gagagcgtgc
tgacccagcc tcctagcgcc tctggcaccc ctggccagag agtgaccatc 60agctgctctg
gcagcagcag caacatcgga agcaaccccg tgaactggta tcagcagctg
120cccggcaccg cccccaagct gctgatctac cggaacaacc agcggcctag
cggcgtgccc 180gacagattca gcggcagcaa gagcggcacc agcgccagcc
tggccatcag cggcctgaga 240agcgaggacg aggccgacta ctactgcgcc
gcctgggacg acagactgaa cggctggggc 300ttcggcggag gcaccaagct
gaccgtgctg ggacag 3361059PRTArtificial SequenceC4.4a binder 105Phe
Ser Asp Tyr Gln Met Thr Trp Ile 1 5 10620PRTArtificial
SequenceC4.4a binder 106Val Ser Gly Ile Ser Trp Asn Gly Gly Ser Thr
His Tyr Ala Asp Ser 1 5 10 15 Val Lys Gly Arg 20 10715PRTArtificial
SequenceC4.4a binder 107Ala Lys Gly Asp Tyr Leu Val Tyr Ser Ser Tyr
Tyr Phe Lys Ser 1 5 10 15 10813PRTArtificial SequenceC4.4a binder
108Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Pro Val Asn 1 5 10
1097PRTArtificial SequenceC4.4a binder 109Arg Asn Asn Gln Arg Pro
Ser 1 5 11012PRTArtificial SequenceC4.4a binder 110Cys Ala Ala Trp
Asp Asp Arg Leu Ser Gly Trp Ala 1 5 10 111122PRTArtificial
SequenceC4.4a binder 111Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gln Met Thr Trp Ile Arg Gln
Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp
Asn Gly Gly Ser Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Lys Gly Asp Tyr Leu Val Tyr Ser Ser Tyr Tyr Phe Lys Ser Trp
100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
112112PRTArtificial SequenceC4.4a binder 112Gln Ser Val Leu Thr Gln
Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile
Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Pro Val
Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45
Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50
55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu
Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp
Asp Arg Leu 85 90 95 Ser Gly Trp Ala Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly Gln 100 105 110 113366DNAArtificial SequenceC4.4a
binder 113gaggtgcagc tgctggaatc cggcggaggc ctggtgcagc ctggcggatc
tctgagactg 60tcctgcgccg cctccggctt caccttctcc gactaccaga tgacctggat
cagacagacc 120cccggcaagg gcctggaatg ggtgtccggc atctcctgga
acggcggctc cacccactac 180gccgactctg tgaagggccg gttcaccatc
tcccgggaca actccaagaa caccctgtac 240ctgcagatga actccctgcg
ggccgaggac accgccgtgt actactgcgc caagggcgac 300tacctggtgt
actcctccta ctacttcaag tcctggggcc agggcaccct ggtcaccgtc 360agctca
366114336DNAArtificial SequenceC4.4a binder 114cagtccgtgc
tgacccagcc tccttccgcc tctggcaccc ctggccagag agtgaccatc 60tcctgctccg
gctcctcctc caacatcggc tccaaccccg tgaactggta tcagcagctg
120cccggcaccg cccccaagct gctgatctac cggaacaacc agcggccctc
cggcgtgccc 180gacagattct ccggctccaa gtccggcacc tccgcctccc
tggccatctc cggcctgaga 240tctgaggacg aggccgacta ctactgcgcc
gcctgggacg accggctgtc tggctgggct 300tttggcggcg gaacaaagtt
aaccgtgctg ggccag 3361159PRTArtificial SequenceC4.4a binder 115Phe
Ser Asp Tyr Gln Met Thr Trp Ile 1 5 11620PRTArtificial
SequenceC4.4a binder 116Val Ser Gly Ile Ser Trp Asn Gly Gly Ser Thr
His Tyr Ala Asp Ser 1 5 10 15 Val Lys Gly Arg 20 11715PRTArtificial
SequenceC4.4a binder 117Ala Lys Gly Asp Tyr Leu Val Tyr Lys Ser Tyr
Tyr Phe Lys Ser 1 5 10 15 11813PRTArtificial SequenceC4.4a binder
118Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Pro Val Asn 1 5 10
1197PRTArtificial SequenceC4.4a binder 119Arg Asn Asn Gln Arg Pro
Ser 1 5 12012PRTArtificial SequenceC4.4a binder 120Cys Ala Ala Trp
Asp Asp Ser Leu Ser Gly Trp Ala 1 5 10 121122PRTArtificial
SequenceC4.4a binder 121Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gln Met Thr Trp Ile Arg Gln
Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp
Asn Gly Gly Ser Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Lys Gly Asp Tyr Leu Val Tyr Lys Ser Tyr Tyr Phe Lys Ser Trp
100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
122112PRTArtificial SequenceC4.4a binder 122Gln Ser Val Leu Thr Gln
Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile
Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Pro Val
Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45
Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50
55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu
Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp
Asp Ser Leu 85 90 95 Ser Gly Trp Ala Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly Gln 100 105 110 123366DNAArtificial SequenceC4.4a
binder 123gaggtgcagc tgctggaatc cggcggaggc ctggtgcagc ctggcggatc
tctgagactg 60tcctgcgccg cctccggctt caccttctcc gactaccaga tgacctggat
cagacagacc 120cccggcaagg gcctggaatg
ggtgtccggc atctcctgga acggcggctc cacccactac 180gccgactctg
tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgtac
240ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgcgc
caagggcgac 300tacctggtgt acaagtccta ctacttcaag tcctggggcc
agggcaccct ggtcaccgtc 360agctca 366124336DNAArtificial
SequenceC4.4a binder 124cagtccgtgc tgacccagcc tccttccgcc tctggcaccc
ctggccagag agtgaccatc 60tcctgctccg gctcctcctc caacatcggc tccaaccccg
tgaactggta tcagcagctg 120cccggcaccg cccccaagct gctgatctac
cggaacaacc agcggccctc cggcgtgccc 180gacagattct ccggctccaa
gtccggcacc tccgcctccc tggccatctc cggcctgaga 240tctgaggacg
aggccgacta ctactgcgcc gcctgggacg actccctgtc tggctgggct
300tttggcggcg gaacaaagtt aaccgtgctg ggccag 3361259PRTArtificial
SequenceC4.4a binder 125Phe Ser Asp Tyr Gln Met Thr Trp Ile 1 5
12620PRTArtificial SequenceC4.4a binder 126Val Ser Gly Ile Ser Trp
Asn Gly Gly Ser Thr His Tyr Ala Asp Ser 1 5 10 15 Val Lys Gly Arg
20 12715PRTArtificial SequenceC4.4a binder 127Ala Lys Gly Asp Tyr
Leu Val Tyr Ser Ser Tyr Tyr Phe Lys Ser 1 5 10 15
12813PRTArtificial SequenceC4.4a binder 128Ser Gly Ser Ser Ser Asn
Ile Gly Ser Asn Pro Val Asn 1 5 10 1297PRTArtificial SequenceC4.4a
binder 129Arg Asn Asn Gln Arg Pro Ser 1 5 13012PRTArtificial
SequenceC4.4a binder 130Cys Ala Ala Trp Asp Asp Arg Leu Ser Gly Trp
Gly 1 5 10 131122PRTArtificial SequenceC4.4a binder 131Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25
30 Gln Met Thr Trp Ile Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Gly Ile Ser Trp Asn Gly Gly Ser Thr His Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Asp Tyr Leu Val Tyr
Ser Ser Tyr Tyr Phe Lys Ser Trp 100 105 110 Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120 132112PRTArtificial SequenceC4.4a binder
132Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly
Ser Asn 20 25 30 Pro Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala
Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly
Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala
Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp
Tyr Tyr Cys Ala Ala Trp Asp Asp Arg Leu 85 90 95 Ser Gly Trp Gly
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110
133366DNAArtificial SequenceC4.4a binder 133gaggtgcagc tgctggaatc
cggcggaggc ctggtgcagc ctggcggatc tctgagactg 60tcctgcgccg cctccggctt
caccttctcc gactaccaga tgacctggat cagacagacc 120cccggcaagg
gcctggaatg ggtgtccggc atctcctgga acggcggctc cacccactac
180gccgactctg tgaagggccg gttcaccatc tcccgggaca actccaagaa
caccctgtac 240ctgcagatga actccctgcg ggccgaggac accgccgtgt
actactgcgc caagggcgac 300tacctggtgt actcctccta ctacttcaag
tcctggggcc agggcaccct ggtcaccgtc 360agctca 366134336DNAArtificial
SequenceC4.4a binder 134cagtccgtgc tgacccagcc tccttccgcc tctggcaccc
ctggccagag agtgaccatc 60tcctgctccg gctcctcctc caacatcggc tccaaccccg
tgaactggta tcagcagctg 120cccggcaccg cccccaagct gctgatctac
cggaacaacc agcggccctc cggcgtgccc 180gacagattct ccggctccaa
gtccggcacc tccgcctccc tggccatctc cggcctgaga 240tctgaggacg
aggccgacta ctactgcgcc gcctgggacg accggctgtc tggctgggga
300tttggcggcg gaacaaagtt aaccgtgctg ggccag 3361359PRTArtificial
SequenceC4.4a binder 135Phe Ser Ser Tyr Gln Met Thr Trp Ile 1 5
13620PRTArtificial SequenceC4.4a binder 136Val Ser Gly Ile Ser Trp
Asn Gly Gly Ser Thr His Tyr Ala Asp Ser 1 5 10 15 Val Lys Gly Arg
20 13715PRTArtificial SequenceC4.4a binder 137Ala Lys Gly Asp Tyr
Leu Val Tyr Lys Ser Tyr Tyr Phe Lys Ser 1 5 10 15
13813PRTArtificial SequenceC4.4a binder 138Ser Gly Ser Ser Ser Asn
Ile Gly Ser Asn Pro Val Asn 1 5 10 1397PRTArtificial SequenceC4.4a
binder 139Arg Asn Asn Gln Arg Pro Ser 1 5 14012PRTArtificial
SequenceC4.4a binder 140Cys Ala Ala Trp Asp Asp Ser Leu Ser Gly Trp
Ala 1 5 10 141122PRTArtificial SequenceC4.4a binder 141Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30 Gln Met Thr Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Gly Ile Ser Trp Asn Gly Gly Ser Thr His Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Asp Tyr Leu Val Tyr
Lys Ser Tyr Tyr Phe Lys Ser Trp 100 105 110 Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120 142112PRTArtificial SequenceC4.4a binder
142Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly
Ser Asn 20 25 30 Pro Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala
Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly
Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala
Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp
Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Ser Gly Trp Ala
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110
143366DNAArtificial SequenceC4.4a binder 143gaggtgcagc tgctggaaag
cggcggaggc ctggtgcagc ctggcggaag cctgagactg 60agctgtgccg ccagcggctt
caccttcagc agctaccaga tgacctggat cagacaggcc 120cctggcaagg
gcctggaatg ggtgtccggc atcagctgga acggcggcag cacccactac
180gccgacagcg tgaagggccg gttcaccatc agccgggaca acagcaagaa
caccctgtac 240ctgcagatga acagcctgcg ggccgaggac accgccgtgt
actactgcgc caagggcgac 300tacctggtgt acaagagcta ctacttcaag
agctggggcc agggcacact ggtcaccgtc 360agctca 366144336DNAArtificial
SequenceC4.4a binder 144cagagcgtgc tgacccagcc tcctagcgcc tctggcaccc
ctggccagag agtgaccatc 60agctgcagcg gcagcagcag caacatcggc agcaaccccg
tgaactggta tcagcagctg 120cccggcaccg cccccaagct gctgatctac
cggaacaacc agcggcccag cggcgtgccc 180gacagatttt ccggcagcaa
gagcggcacc agcgccagcc tggccatcag cggcctgaga 240agcgaggacg
aggccgacta ctactgcgcc gcctgggacg atagcctgag cggctgggcc
300tttggcggcg gaacaaagtt aaccgtgctg ggccag 3361459PRTArtificial
SequenceC4.4a binder 145Phe Ser Asn Ala Trp Met Ser Trp Val 1 5
14620PRTArtificial SequenceC4.4a binder 146Val Ser Tyr Ile Ser Ser
Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Gly Arg
20 14710PRTArtificial SequenceC4.4a binder 147Ala Arg Glu Gly Leu
Trp Ala Phe Asp Lys 1 5 10 14814PRTArtificial SequenceC4.4a binder
148Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Val Val His 1 5 10
1497PRTArtificial SequenceC4.4a binder 149Asp Asn Asn Lys Arg Pro
Ser 1 5 15012PRTArtificial SequenceC4.4a binder 150Cys Ala Ala Tyr
Asp Asp Ser Leu Lys Gly Pro Val 1 5 10 151117PRTArtificial
SequenceC4.4a binder 151Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser
Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Glu Gly Leu Trp Ala Phe Asp Lys Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Thr Ser 115 152113PRTArtificial
SequenceC4.4a binder 152Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser
Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser
Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Val Val His Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn
Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Tyr Asp Asp Ser 85 90
95 Leu Lys Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110 Gln 1539PRTArtificial SequenceC4.4a binder 153Phe Ser
Asn Ala Trp Met Ser Trp Val 1 5 15420PRTArtificial SequenceC4.4a
binder 154Val Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala
Asp Ser 1 5 10 15 Val Lys Gly Arg 20 15510PRTArtificial
SequenceC4.4a binder 155Ala Arg Glu Gly Leu Trp Ala Phe Asp Lys 1 5
10 15614PRTArtificial SequenceC4.4a binder 156Thr Gly Ser Ser Ser
Asn Ile Gly Ala Gly Tyr Val Val His 1 5 10 1577PRTArtificial
SequenceC4.4a binder 157Asp Asn Asn Lys Arg Pro Ser 1 5
15812PRTArtificial SequenceC4.4a binder 158Cys Ala Ala Phe Asp Asp
Ser Leu Asn Gly Pro Val 1 5 10 159117PRTArtificial SequenceC4.4a
binder 159Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser
Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Gly Leu Trp Ala Phe Asp Lys Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Thr Ser 115 160113PRTArtificial SequenceC4.4a binder
160Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln
1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly
Ala Gly 20 25 30 Tyr Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr
Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser
Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser
Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala
Asp Tyr Tyr Cys Ala Ala Phe Asp Asp Ser 85 90 95 Leu Asn Gly Pro
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln
1619PRTArtificial SequenceC4.4a binder 161Phe Ser Asn Ala Trp Met
Ser Trp Val 1 5 16220PRTArtificial SequenceC4.4a binder 162Val Ser
Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser 1 5 10 15
Val Lys Gly Arg 20 16310PRTArtificial SequenceC4.4a binder 163Ala
Arg Glu Gly Leu Trp Ala Phe Asp Lys 1 5 10 16414PRTArtificial
SequenceC4.4a binder 164Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr
Val Val His 1 5 10 1657PRTArtificial SequenceC4.4a binder 165Asp
Asn Asn Lys Arg Pro Ser 1 5 16612PRTArtificial SequenceC4.4a binder
166Cys Ala Ala Tyr Asp Asp Ser Leu Ser Gly Pro Val 1 5 10
167117PRTArtificial SequenceC4.4a binder 167Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Leu Trp Ala Phe Asp Lys Trp
Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Thr Ser 115
168113PRTArtificial SequenceC4.4a binder 168Gln Ser Val Leu Thr Gln
Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile
Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Val
Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45
Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50
55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly
Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Phe
Asp Asp Ser 85 90 95 Leu Asn Gly Pro Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu Gly 100 105 110 Gln 1699PRTArtificial SequenceC4.4a
binder 169Phe Ser Ser Ala Trp Met Ser Trp Val 1 5
17020PRTArtificial SequenceC4.4a binder 170Val Ser Tyr Ile Ser Ser
Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Gly Arg
20 17110PRTArtificial SequenceC4.4a binder 171Ala Arg Glu Gly Leu
Trp Ala Phe Asp Trp 1 5 10 17214PRTArtificial SequenceC4.4a binder
172Ser Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Val Val His 1 5 10
1737PRTArtificial SequenceC4.4a binder 173Asp Asn Asn Gln Arg Pro
Ser 1 5 17412PRTArtificial SequenceC4.4a binder 174Cys Ala Ala Tyr
Asp Asp Ser Leu Ser Gly Pro Val 1 5 10 175117PRTArtificial
SequenceC4.4a binder 175Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Ala 20 25 30 Trp Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser
Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Leu Trp Ala Phe Asp Trp Trp
Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Thr Ser 115
176113PRTArtificial SequenceC4.4a binder 176Gln Ser Val Leu Thr Gln
Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile
Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Val
Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45
Leu Ile Tyr Asp Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe 50
55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly
Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Tyr
Asp Asp Ser 85 90 95 Leu Ser Gly Pro Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu Gly 100 105 110 Gln 1779PRTArtificial SequenceC4.4a
binder 177Phe Ser Ser Ala Trp Met Ser Trp Val 1 5
17820PRTArtificial SequenceC4.4a binder 178Val Ser Tyr Ile Ser Ser
Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Gly Arg
20 17910PRTArtificial SequenceC4.4a binder 179Ala Arg Glu Gly Leu
Trp Ala Phe Asp Asn 1 5 10 18014PRTArtificial SequenceC4.4a binder
180Ser Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Val Val His 1 5 10
1817PRTArtificial SequenceC4.4a binder 181Asp Asn Asn Gln Arg Pro
Ser 1 5 18212PRTArtificial SequenceC4.4a binder 182Cys Ala Ala Tyr
Asp Asp Ser Leu Asn Gly Pro Val 1 5 10 183117PRTArtificial
SequenceC4.4a binder 183Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser
Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Glu Gly Leu Trp Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Thr Ser 115 184113PRTArtificial
SequenceC4.4a binder 184Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser
Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser
Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Val Val His Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn
Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Tyr Asp Asp Ser 85 90
95 Leu Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110 Gln 1859PRTArtificial SequenceC4.4a binder 185Phe Ser
Ser Ala Trp Met Ser Trp Val 1 5 18620PRTArtificial SequenceC4.4a
binder 186Val Ser Tyr Ile Ser Ser Ser Gly Ser Ser Thr Tyr Tyr Ala
Asp Ser 1 5 10 15 Val Lys Gly Arg 20 18710PRTArtificial
SequenceC4.4a binder 187Ala Arg Glu Gly Leu Trp Ala Phe Asp Tyr 1 5
10 18814PRTArtificial SequenceC4.4a binder 188Ser Gly Ser Ser Ser
Asn Ile Gly Ala Gly Tyr Val Val His 1 5 10 1897PRTArtificial
SequenceC4.4a binder 189Asp Asn Asn Gln Arg Pro Ser 1 5
19012PRTArtificial SequenceC4.4a binder 190Cys Ala Ala Trp Asp Asp
Arg Leu Asn Gly Pro Val 1 5 10 191117PRTArtificial SequenceC4.4a
binder 191Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Gly Leu Trp Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 192113PRTArtificial SequenceC4.4a binder
192Glu Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly
Ala Gly 20 25 30 Tyr Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr
Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn Asn Gln Arg Pro Ser
Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser
Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala
Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Arg 85 90 95 Leu Asn Gly Pro
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln
19311PRTArtificial SequenceC4.4a binder 193Phe Thr Phe Ser Asn Ala
Trp Met Ser Trp Val 1 5 10 19420PRTArtificial SequenceC4.4a binder
194Val Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser
1 5 10 15 Val Lys Gly Arg 20 19510PRTArtificial SequenceC4.4a
binder 195Ala Arg Glu Gly Leu Trp Ala Phe Asp Tyr 1 5 10
19614PRTArtificial SequenceC4.4a binder 196Thr Gly Ser Ser Ser Asn
Ile Gly Ala Gly Tyr Val Val His 1 5 10 1977PRTArtificial
SequenceC4.4a binder 197Asp Asn Asn Lys Arg Pro Ser 1 5
19812PRTArtificial SequenceC4.4a binder 198Cys Ala Ala Tyr Asp Asp
Arg Leu Asn Gly Pro Val 1 5 10 199117PRTArtificial SequenceC4.4a
binder 199Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser
Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Gly Leu Trp Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 200113PRTArtificial SequenceC4.4a binder
200Glu Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln
1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly
Ala Gly 20 25 30 Tyr Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr
Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser
Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser
Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala
Asp Tyr Tyr Cys Ala Ala Tyr Asp Asp Arg 85 90 95 Leu Asn Gly Pro
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln
20111PRTArtificial SequenceC4.4a binder 201Phe Thr Phe Ser Ser Ala
Trp Met Ser Trp Val 1 5 10 20220PRTArtificial SequenceC4.4a binder
202Val Ser Tyr Ile Ser Ser Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser
1 5 10 15 Val Lys Gly Arg 20 20310PRTArtificial SequenceC4.4a
binder 203Ala Arg Glu Gly Leu Trp Ala Phe Asp Gly 1 5 10
20414PRTArtificial SequenceC4.4a binder 204Ser Gly Ser Ser Ser Asn
Ile Gly Ala Gly Tyr Val Val His 1 5 10 2057PRTArtificial
SequenceC4.4a binder 205Asp Asn Asn Gln Arg Pro Ser 1 5
20612PRTArtificial SequenceC4.4a binder 206Cys Ala Ala Tyr Asp Asp
Ser Leu Asn Arg Pro Val 1 5 10 207117PRTArtificial SequenceC4.4a
binder 207Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Gly Leu Trp Ala Phe Asp Gly Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 208113PRTArtificial SequenceC4.4a binder
208Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly
Ala Gly 20 25 30 Tyr Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr
Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn Asn Gln Arg Pro Ser
Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser
Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala
Asp Tyr Tyr Cys Ala Ala Tyr Asp Asp Ser 85 90 95 Leu Asn Arg Pro
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln
20911PRTArtificial SequenceC4.4a binder 209Phe Thr Phe Ser Asn Ala
Trp Met Ser Trp Val 1 5 10 21020PRTArtificial SequenceC4.4a binder
210Val Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser
1 5 10 15 Val Lys Gly Arg 20 21110PRTArtificial SequenceC4.4a
binder 211Ala Arg Glu Gly Leu Trp Ala Phe Asp Tyr 1 5 10
21214PRTArtificial SequenceC4.4a binder 212Thr Gly Ser Ser Ser Asn
Ile Gly Ala Gly Tyr Val Val His 1 5 10 2137PRTArtificial
SequenceC4.4a binder 213Asp Asn Asn Lys Arg Pro Ser 1 5
21412PRTArtificial SequenceC4.4a binder 214Cys Ala Ala Phe Asp Asp
Ser Leu Asn Gly Pro Val 1 5 10 215117PRTArtificial SequenceC4.4a
binder 215Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser
Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Gly Leu Trp Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 216113PRTArtificial SequenceC4.4a binder
216Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln
1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly
Ala Gly 20 25 30 Tyr Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr
Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser
Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser
Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala
Asp Tyr Tyr Cys Ala Ala Phe Asp Asp Ser 85 90 95 Leu Asn Gly Pro
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln
2179PRTArtificial SequenceC4.4a binder 217Phe Ser Ser Ala Trp Met
Ser Trp Val 1 5 21820PRTArtificial SequenceC4.4a binder 218Val Ser
Tyr Ile Ser Ser Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser 1 5 10 15
Val Lys Gly Arg 20 21910PRTArtificial SequenceC4.4a binder 219Ala
Arg Glu Gly Leu Trp Ala Phe Asp Lys 1 5 10 22014PRTArtificial
SequenceC4.4a binder 220Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr
Val Val His 1 5 10 2217PRTArtificial SequenceC4.4a binder 221Asp
Asn Asn Lys Arg Pro Ser 1 5 22212PRTArtificial SequenceC4.4a binder
222Cys Ala Ala Phe Asp Asp Ser Leu Asn Gly Pro Val 1 5 10
223117PRTArtificial SequenceC4.4a binder 223Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ala 20 25 30 Trp Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Tyr Ile Ser Ser Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Leu Trp Ala Phe Asp Lys Trp
Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Thr Ser 115
224113PRTArtificial SequenceC4.4a binder 224Gln Ser Val Leu Thr Gln
Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile
Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Val
Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45
Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50
55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly
Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Phe
Asp Asp Ser 85 90 95 Leu Asn Gly Pro Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu Gly 100 105 110 Gln 22511PRTArtificial
SequenceC4.4a binder 225Phe Thr Phe Ser Ser Ala Trp Met Ser Trp Val
1 5 10 22620PRTArtificial SequenceC4.4a binder 226Val Ser Tyr Ile
Ser Ser Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser 1 5
10 15 Val Lys Gly Arg 20 22710PRTArtificial SequenceC4.4a binder
227Ala Arg Glu Gly Leu Trp Ala Phe Asp Lys 1 5 10
22814PRTArtificial SequenceC4.4a binder 228Ser Gly Ser Ser Ser Asn
Ile Gly Ala Gly Tyr Val Val His 1 5 10 2297PRTArtificial
SequenceC4.4a binder 229Asp Asn Asn Gln Arg Pro Ser 1 5
23012PRTArtificial SequenceC4.4a binder 230Cys Ala Ala Phe Asp Asp
Ser Leu Asn Gly Pro Val 1 5 10 231117PRTArtificial SequenceC4.4a
binder 231Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Gly Leu Trp Ala Phe Asp Lys Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 232113PRTArtificial SequenceC4.4a binder
232Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly
Ala Gly 20 25 30 Tyr Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr
Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn Asn Gln Arg Pro Ser
Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser
Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala
Asp Tyr Tyr Cys Ala Ala Phe Asp Asp Ser 85 90 95 Leu Asn Gly Pro
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln
23311PRTArtificial SequenceC4.4a binder 233Phe Thr Phe Ser Ser Ala
Trp Met Ser Trp Val 1 5 10 23420PRTArtificial SequenceC4.4a binder
234Val Ser Tyr Ile Ser Ser Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser
1 5 10 15 Val Lys Gly Arg 20 23510PRTArtificial SequenceC4.4a
binder 235Ala Arg Glu Gly Leu Trp Ala Phe Asp Lys 1 5 10
23614PRTArtificial SequenceC4.4a binder 236Ser Gly Ser Ser Ser Asn
Ile Gly Ala Gly Tyr Val Val His 1 5 10 2377PRTArtificial
SequenceC4.4a binder 237Asp Asn Asn Gln Arg Pro Ser 1 5
23812PRTArtificial SequenceC4.4a binder 238Cys Ala Ala Phe Asp Asp
Arg Leu Ser Gly Pro Val 1 5 10 239117PRTArtificial SequenceC4.4a
binder 239Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Gly Leu Trp Ala Phe Asp Lys Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 240113PRTArtificial SequenceC4.4a binder
240Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly
Ala Gly 20 25 30 Tyr Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr
Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn Asn Gln Arg Pro Ser
Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser
Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala
Asp Tyr Tyr Cys Ala Ala Phe Asp Asp Arg 85 90 95 Leu Ser Gly Pro
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln
2419PRTArtificial SequenceC4.4a binder 241Phe Ser Asp Tyr Gln Met
Thr Trp Ile 1 5 24220PRTArtificial SequenceC4.4a binder 242Val Ser
Gly Val Ser Trp Asn Gly Ala Arg Thr His Tyr Ala Asp Ser 1 5 10 15
Val Lys Gly Arg 20 24315PRTArtificial SequenceC4.4a binder 243Ala
Lys Gly Asp Tyr Leu Val Tyr Ser Ser Tyr Tyr Phe Lys Ser 1 5 10 15
24413PRTArtificial SequenceC4.4a binder 244Ser Gly Ser Ser Ser Asn
Val Gly Ser Asn Pro Val Asn 1 5 10 2457PRTArtificial SequenceC4.4a
binder 245Arg Asn Asn Gln Arg Pro Ser 1 5 24613PRTArtificial
SequenceC4.4a binder 246Cys Ala Ala Trp Asp Asp Arg Leu Asn Gly Trp
Thr Gly 1 5 10 247122PRTArtificial SequenceC4.4a binder 247Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20
25 30 Gln Met Thr Trp Ile Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ser Gly Val Ser Trp Asn Gly Ala Arg Thr His Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Asp Tyr Leu Val
Tyr Ser Ser Tyr Tyr Phe Lys Ser Trp 100 105 110 Gly Gln Gly Thr Leu
Val Thr Val Thr Ser 115 120 248112PRTArtificial SequenceC4.4a
binder 248Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro
Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn
Val Gly Ser Asn 20 25 30 Pro Val Asn Trp Tyr Gln Gln Leu Pro Gly
Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro
Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr
Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu
Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Arg Leu 85 90 95 Asn Gly
Trp Gly Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110
24911PRTArtificial SequenceC4.4a binder 249Phe Thr Phe Ser Ser Tyr
Gln Met Thr Trp Ile 1 5 10 25020PRTArtificial SequenceC4.4a binder
250Val Ser Gly Ile Ser Trp Asn Gly Gly Ser Thr His Tyr Ala Asp Ser
1 5 10 15 Val Lys Gly Arg 20 25115PRTArtificial SequenceC4.4a
binder 251Ala Lys Gly Asp Tyr Leu Val Tyr Ser Ala Tyr Tyr Phe Asp
Ser 1 5 10 15 25213PRTArtificial SequenceC4.4a binder 252Ser Gly
Ser Ser Ser Asn Ile Gly Ser Asn Pro Val Asn 1 5 10
2537PRTArtificial SequenceC4.4a binder 253Arg Asn Asn Gln Arg Pro
Ser 1 5 25412PRTArtificial SequenceC4.4a binder 254Cys Ala Ala Trp
Asp Asp Arg Leu Asn Gly Trp Gly 1 5 10 255122PRTArtificial
SequenceC4.4a binder 255Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gln Met Thr Trp Ile Arg Gln
Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp
Asn Gly Gly Ser Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Lys Gly Asp Tyr Leu Val Tyr Ser Ala Tyr Tyr Phe Asp Ser Trp
100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
256112PRTArtificial SequenceC4.4a binder 256Glu Ser Val Leu Thr Gln
Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile
Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Pro Val
Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45
Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50
55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu
Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp
Asp Arg Leu 85 90 95 Asn Gly Trp Gly Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly Gln 100 105 110 25711PRTArtificial SequenceC4.4a
binder 257Phe Thr Phe Ser Asp Tyr Gln Met Thr Trp Ile 1 5 10
25820PRTArtificial SequenceC4.4a binder 258Val Ser Gly Ile Ser Trp
Asn Gly Gly Ser Thr His Tyr Ala Asp Ser 1 5 10 15 Val Lys Gly Arg
20 25915PRTArtificial SequenceC4.4a binder 259Ala Lys Gly Asp Tyr
Leu Val Tyr Ser Ser Tyr Tyr Phe Lys Tyr 1 5 10 15
26013PRTArtificial SequenceC4.4a binder 260Ser Gly Ser Ser Ser Asn
Ile Gly Ser Asn Pro Val Asn 1 5 10 2617PRTArtificial SequenceC4.4a
binder 261Arg Asn Asn Gln Arg Pro Ser 1 5 26212PRTArtificial
SequenceC4.4a binder 262Cys Ala Ala Trp Asp Asp Arg Leu Asn Gly Trp
Ala 1 5 10 263122PRTArtificial SequenceC4.4a binder 263Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25
30 Gln Met Thr Trp Ile Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Gly Ile Ser Trp Asn Gly Gly Ser Thr His Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Asp Tyr Leu Val Tyr
Ser Ser Tyr Tyr Phe Lys Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120 264112PRTArtificial SequenceC4.4a binder
264Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly
Ser Asn 20 25 30 Pro Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala
Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly
Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala
Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp
Tyr Tyr Cys Ala Ala Trp Asp Asp Arg Leu 85 90 95 Asn Gly Trp Ala
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110
26511PRTArtificial SequenceC4.4a binder 265Phe Thr Phe Ser Asp Tyr
Gln Met Thr Trp Ile 1 5 10 26620PRTArtificial SequenceC4.4a binder
266Val Ser Gly Ile Ser Trp Asn Gly Gly Ser Thr His Tyr Ala Asp Ser
1 5 10 15 Val Lys Gly Arg 20 26715PRTArtificial SequenceC4.4a
binder 267Ala Lys Gly Asp Tyr Leu Val Tyr Ser Ser Tyr Tyr Phe Lys
Ser 1 5 10 15 26813PRTArtificial SequenceC4.4a binder 268Ser Gly
Ser Ser Ser Asn Ile Gly Ser Asn Pro Val Asn 1 5 10
2697PRTArtificial SequenceC4.4a binder 269Arg Asn Asn Gln Arg Pro
Ser 1 5 27012PRTArtificial SequenceC4.4a binder 270Cys Ala Ala Trp
Asp Asp Ser Leu Asn Gly Trp Gly 1 5 10 271122PRTArtificial
SequenceC4.4a binder 271Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gln Met Thr Trp Ile Arg Gln
Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp
Asn Gly Gly Ser Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Lys Gly Asp Tyr Leu Val Tyr Ser Ser Tyr Tyr Phe Lys Ser Trp
100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
272112PRTArtificial SequenceC4.4a binder 272Gln Ser Val Leu Thr Gln
Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile
Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Pro Val
Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45
Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50
55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu
Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp
Asp Ser Leu 85 90 95 Asn Gly Trp Gly Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly Gln 100 105 110 27311PRTArtificial SequenceC4.4a
binder 273Phe Thr Phe Ser Asp Tyr Gln Met Thr Trp Ile 1 5 10
27420PRTArtificial SequenceC4.4a binder 274Val Ser Gly Ile Ser Trp
Asn Gly Gly Ser Thr His Tyr Ala Asp Ser 1 5 10 15 Val Lys Gly Arg
20 27515PRTArtificial SequenceC4.4a binder 275Ala Lys Gly Asp Tyr
Leu Val Tyr Lys Ser Tyr Tyr Phe Lys Ser 1 5 10 15
27613PRTArtificial SequenceC4.4a binder 276Ser Gly Ser Ser Ser Asn
Ile Gly Ser Asn Pro Val Asn 1 5 10 2777PRTArtificial SequenceC4.4a
binder 277Arg Asn Asn Gln Arg Pro Ser 1 5 27812PRTArtificial
SequenceC4.4a binder 278Cys Ala Ala Trp Asp Asp Arg Leu Ser Gly Trp
Gly 1 5 10 279122PRTArtificial SequenceC4.4a binder 279Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25
30 Gln Met Thr Trp Ile Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Gly Ile Ser Trp Asn Gly Gly Ser Thr His Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Lys Gly Asp Tyr Leu Val Tyr Lys Ser Tyr Tyr Phe Lys
Ser Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
280112PRTArtificial SequenceC4.4a binder 280Gln Ser Val Leu Thr Gln
Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile
Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Pro Val
Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45
Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50
55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu
Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp
Asp Arg Leu 85 90 95 Ser Gly Trp Gly Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly Gln 100 105 110 28111PRTArtificial SequenceC4.4a
binder 281Phe Thr Phe Ser Asp Tyr Gln Met Thr Trp Ile 1 5 10
28220PRTArtificial SequenceC4.4a binder 282Val Ser Gly Ile Ser Trp
Asn Gly Gly Ser Thr His Tyr Ala Asp Ser 1 5 10 15 Val Lys Gly Arg
20 28315PRTArtificial SequenceC4.4a binder 283Ala Lys Gly Asp Tyr
Leu Val Tyr Ser Ser Tyr Tyr Phe Lys Ser 1 5 10 15
28413PRTArtificial SequenceC4.4a binder 284Ser Gly Ser Ser Ser Asn
Ile Gly Ser Asn Pro Val Asn 1 5 10 2857PRTArtificial SequenceC4.4a
binder 285Arg Asn Asn Gln Arg Pro Ser 1 5 28612PRTArtificial
SequenceC4.4a binder 286Cys Ala Ala Trp Asp Asp Arg Leu Ser Gly Trp
Gly 1 5 10 287122PRTArtificial SequenceC4.4a binder 287Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25
30 Gln Met Thr Trp Ile Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Gly Ile Ser Trp Asn Gly Gly Ser Thr His Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Asp Tyr Leu Val Tyr
Ser Ser Tyr Tyr Phe Lys Ser Trp 100 105 110 Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120 288112PRTArtificial SequenceC4.4a binder
288Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly
Ser Asn 20 25 30 Pro Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala
Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly
Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala
Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp
Tyr Tyr Cys Ala Ala Trp Asp Asp Arg Leu 85 90 95 Ser Gly Trp Gly
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110
28911PRTArtificial SequenceC4.4a binder 289Phe Thr Phe Ser Asp Tyr
Gln Met Thr Trp Ile 1 5 10 29020PRTArtificial SequenceC4.4a binder
290Val Ser Gly Ile Ser Trp Asn Gly Gly Ser Thr His Tyr Ala Asp Ser
1 5 10 15 Val Lys Gly Arg 20 29115PRTArtificial SequenceC4.4a
binder 291Ala Lys Gly Asp Tyr Leu Val Tyr Lys Ser Tyr Tyr Phe Lys
Ser 1 5 10 15 29213PRTArtificial SequenceC4.4a binder 292Ser Gly
Ser Ser Ser Asn Ile Gly Ser Asn Pro Val Asn 1 5 10
2937PRTArtificial SequenceC4.4a binder 293Arg Asn Asn Gln Arg Pro
Ser 1 5 29412PRTArtificial SequenceC4.4a binder 294Cys Ala Ala Trp
Asp Asp Ser Leu Asn Gly Trp Gly 1 5 10 295122PRTArtificial
SequenceC4.4a binder 295Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gln Met Thr Trp Ile Arg Gln
Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp
Asn Gly Gly Ser Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Lys Gly Asp Tyr Leu Val Tyr Lys Ser Tyr Tyr Phe Lys Ser Trp
100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
296112PRTArtificial SequenceC4.4a binder 296Gln Ser Val Leu Thr Gln
Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile
Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Pro Val
Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45
Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50
55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu
Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp
Asp Ser Leu 85 90 95 Asn Gly Trp Gly Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly Gln 100 105 110 2979PRTArtificial Sequenceconsensus
sequence for M20 D02 S-A derived CDR H1 297Phe Ser Xaa Tyr Gln Met
Thr Trp Ile 1 5 29820PRTArtificial Sequenceconsensus sequence for
M20 D02 S-A derived CDRH2 298Val Ser Gly Xaa Ser Trp Asn Gly Xaa
Xaa Thr His Tyr Ala Asp Ser 1 5 10 15 Val Lys Gly Arg 20
29915PRTArtificial Sequenceconsensus for M20 D02 S-A derived CDRH3
299Ala Lys Gly Asp Tyr Leu Val Tyr Xaa Xaa Tyr Tyr Phe Xaa Xaa 1 5
10 15 30013PRTArtificial Sequenceconsensus seqeunce derived from
M20 D02 S-A CDRL1 300Ser Gly Ser Ser Ser Asn Xaa Gly Ser Asn Pro
Val Asn 1 5 10 30112PRTArtificial Sequenceconsensus sequence
derived from M20 D02 S-A derived CDR L3 301Cys Ala Xaa Trp Xaa Asp
Xaa Leu Xaa Gly Trp Xaa 1 5 10 3029PRTArtificial Sequenceconsensus
sequence derived from M31 B01 CDR H1 302Phe Ser Xaa Xaa Trp Met Ser
Trp Val 1 5 30320PRTArtificial Sequenceconsensus sequence derived
from M31 B01 CDR H2 303Val Ser Tyr Ile Ser Ser Ser Gly Ser Xaa Xaa
Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Gly Arg 20 30410PRTArtificial
Sequenceconsensus sequence derived from M31 B01 CDR H3 304Ala Arg
Glu Gly Leu Trp Ala Phe Asp Xaa 1 5 10 30514PRTArtificial
Sequenceconsensus sequence derived from M31 B01 CDR L1 305Xaa Gly
Ser Ser Ser Asn Ile Gly Ala Gly Tyr Val Val His 1 5 10
3067PRTArtificial Sequenceconsensus sequence derived from M31 B01
CDR L2 306Asp Asn Asn Xaa Arg Pro Ser 1 5 30712PRTArtificial
Sequenceconsensus sequence derived from M31 B01 CDR L3 307Cys Ala
Ala Xaa Asp Asp Xaa Leu Xaa Xaa Xaa Val 1 5 10 308351DNAArtificial
SequenceC4.4a binder 308gaggtgcagc tgctggagag cggggggggg ctggtgcagc
cgggggggag cctgcgcctg 60agctgcgcgg cgagcgggtt tacctttagc aacgcgtgga
tgagctgggt gcgccaggcg 120ccggggaaag ggctggagtg ggtgagctat
attagcagca gcgggagcac catttattat 180gcggatagcg tgaaagggcg
ctttaccatt agccgcgata acagcaaaaa caccctgtat 240ctgcagatga
acagcctgcg cgcggaggat accgcggtgt attattgcgc gcgcgagggg
300ctgtgggcgt ttgataaatg ggggcagggg accctggtga ccgtgaccag c
351309339DNAArtificial SequenceC4.4a binder 309cagagcgtgc
tgacccagcc gccgagcgcg agcgggaccc cggggcagcg cgtgaccatt 60agctgcaccg
ggagcagcag caacattggg gcggggtatg tggtgcattg gtatcagcag
120ctgccgggga ccgcgccgaa actgctgatt tatgataaca acaaacgccc
gagcggggtg 180ccggatcgct ttagcgggag caaaagcggg accagcgcga
gcctggcgat tagcgggctg 240cgcagcgagg atgaggcgga ttattattgc
gcggcgtatg atgatagcct gaaagggccg 300gtgtttgggg gggggaccaa
actgaccgtg ctggggcag 339310351DNAArtificial SequenceC4.4a binder
310gaggtgcagc tgctggagag cggggggggg ctggtgcagc cgggggggag
cctgcgcctg 60agctgcgcgg cgagcgggtt tacctttagc aacgcgtgga tgagctgggt
gcgccaggcg 120ccggggaaag ggctggagtg ggtgagctat attagcagca
gcgggagcac catttattat 180gcggatagcg tgaaagggcg ctttaccatt
agccgcgata acagcaaaaa caccctgtat 240ctgcagatga acagcctgcg
cgcggaggat accgcggtgt attattgcgc gcgcgagggg 300ctgtgggcgt
ttgataaatg ggggcagggg accctggtga ccgtgaccag c
351311339DNAArtificial SequenceC4.4a binder 311cagagcgtgc
tgacccagcc gccgagcgtg agcggggcgc cggggcagcg cgtgaccatt 60agctgcaccg
ggagcagcag caacattggg gcggggtatg tggtgcattg gtatcagcag
120ctgccgggga ccgcgccgaa actgctgatt tatgataaca acaaacgccc
gagcggggtg 180ccggatcgct ttagcgggag caaaagcggg accagcgcga
gcctggcgat tagcgggctg 240cgcagcgagg atgaggcgga ttattattgc
gcggcgtttg atgatagcct gaacgggccg 300gtgtttgggg gggggaccaa
actgaccgtg ctggggcag 339312351DNAArtificial SequenceC4.4a binder
312gaggtgcagc tgctggagag cggggggggg ctggtgcagc cgggggggag
cctgcgcctg 60agctgcgcgg cgagcgggtt tacctttagc aacgcgtgga tgagctgggt
gcgccaggcg 120ccggggaaag ggctggagtg ggtgagctat attagcagca
gcgggagcac catttattat 180gcggatagcg tgaaagggcg ctttaccatt
agccgcgata acagcaaaaa caccctgtat 240ctgcagatga acagcctgcg
cgcggaggat accgcggtgt attattgcgc gcgcgagggg 300ctgtgggcgt
ttgataaatg ggggcagggg accctggtga ccgtgaccag c
351313339DNAArtificial SequenceC4.4a binder 313cagagcgtgc
tgacccagcc gccgagcgtg agcggggcgc cggggcagcg cgtgaccatt 60agctgcaccg
ggagcagcag caacattggg gcggggtatg tggtgcattg gtatcagcag
120ctgccgggga ccgcgccgaa actgctgatt tatgataaca acaaacgccc
gagcggggtg 180ccggatcgct ttagcgggag caaaagcggg accagcgcga
gcctggcgat tagcgggctg 240cgcagcgagg atgaggcgga ttattattgc
gcggcgtttg atgatagcct gaacgggccg 300gtgtttgggg gggggaccaa
actgaccgtg ctggggcag 339314351DNAArtificial SequenceC4.4a binder
314gaggtgcagc tgctggagag cggggggggg ctggtgcagc cgggggggag
cctgcgcctg 60agctgcgcgg cgagcgggtt tacctttagc agcgcgtgga tgagctgggt
gcgccaggcg 120ccggggaaag ggctggagtg ggtgagctat attagcagca
gcgggagcag cacctattat 180gcggatagcg tgaaagggcg ctttaccatt
agccgcgata acagcaaaaa caccctgtat 240ctgcagatga acagcctgcg
cgcggaggat accgcggtgt attattgcgc gcgcgagggg 300ctgtgggcgt
ttgattggtg ggggcagggg accctggtga ccgtgaccag c
351315339DNAArtificial SequenceC4.4a binder 315cagagcgtgc
tgacccagcc gccgagcgcg agcgggaccc cggggcagcg cgtgaccatt 60agctgcagcg
ggagcagcag caacattggg gcggggtatg tggtgcattg gtatcagcag
120ctgccgggga ccgcgccgaa actgctgatt tatgataaca accagcgccc
gagcggggtg 180ccggatcgct ttagcgggag caaaagcggg accagcgcga
gcctggcgat tagcgggctg 240cgcagcgagg atgaggcgga ttattattgc
gcggcgtatg atgatagcct gagcgggccg 300gtgtttgggg gggggaccaa
actgaccgtg ctggggcag 339316351DNAArtificial SequenceC4.4a binder
316gaggtgcagc tgctggagag cggggggggg ctggtgcagc cgggggggag
cctgcgcctg 60agctgcgcgg cgagcgggtt tacctttagc aacgcgtgga tgagctgggt
gcgccaggcg 120ccggggaaag ggctggagtg ggtgagctat attagcagca
gcgggagcac catttattat 180gcggatagcg tgaaagggcg ctttaccatt
agccgcgata acagcaaaaa caccctgtat 240ctgcagatga acagcctgcg
cgcggaggat accgcggtgt attattgcgc gcgcgagggg 300ctgtgggcgt
ttgattattg ggggcagggg accctggtga ccgtgaccag c
351317339DNAArtificial SequenceC4.4a binder 317cagagcgtgc
tgacccagcc gccgagcgcg agcgggaccc cggggcagcg cgtgaccatt 60agctgcagcg
ggagcagcag caacattggg gcggggtatg tggtgcattg gtatcagcag
120ctgccgggga ccgcgccgaa actgctgatt tatgataaca accagcgccc
gagcggggtg 180ccggatcgct ttagcgggag caaaagcggg accagcgcga
gcctggcgat tagcgggctg 240cgcagcgagg atgaggcgga ttattattgc
gcggcgtatg atgatagcct gaacgggccg 300gtgtttgggg gggggaccaa
actgaccgtg ctggggcag 339318351DNAArtificial SequenceC4.4a binder
318gaggtgcagc tgctggagag cggggggggg ctggtgcagc cgggggggag
cctgcgcctg 60agctgcgcgg cgagcgggtt tacctttagc agcgcgtgga tgagctgggt
gcgccaggcg 120ccggggaaag ggctggagtg ggtgagctat attagcagca
gcgggagcag cacctattat 180gcggatagcg tgaaagggcg ctttaccatt
agccgcgata acagcaaaaa caccctgtat 240ctgcagatga acagcctgcg
cgcggaggat accgcggtgt attattgcgc gcgcgagggg 300ctgtgggcgt
ttgattattg ggggcagggg accctggtga ccgtgagcag c
351319339DNAArtificial SequenceC4.4a binder 319gagagcgtgc
tgacccagcc gccgagcgcg agcgggaccc cggggcagcg cgtgaccatt 60agctgcagcg
ggagcagcag caacattggg gcggggtatg tggtgcattg gtatcagcag
120ctgccgggga ccgcgccgaa actgctgatt tatgataaca accagcgccc
gagcggggtg 180ccggatcgct ttagcgggag caaaagcggg accagcgcga
gcctggcgat tagcgggctg 240cgcagcgagg atgaggcgga ttattattgc
gcggcgtggg atgatcgcct gaacgggccg 300gtgtttgggg gggggaccaa
actgaccgtg ctggggcag 339320351DNAArtificial SequenceC4.4a binder
320gaggtgcagc tgctggagag cggggggggg ctggtgcagc cgggggggag
cctgcgcctg 60agctgcgcgg cgagcgggtt tacctttagc aacgcgtgga tgagctgggt
gcgccaggcg 120ccggggaaag ggctggagtg ggtgagctat attagcagca
gcgggagcac catttattat 180gcggatagcg tgaaagggcg ctttaccatt
agccgcgata acagcaaaaa caccctgtat 240ctgcagatga acagcctgcg
cgcggaggat accgcggtgt attattgcgc gcgcgagggg 300ctgtgggcgt
ttgattattg ggggcagggg accctggtga ccgtgagcag c
351321339DNAArtificial SequenceC4.4a binder 321gagagcgtgc
tgacccagcc gccgagcgtg agcggggcgc cggggcagcg cgtgaccatt 60agctgcaccg
ggagcagcag caacattggg gcggggtatg tggtgcattg gtatcagcag
120ctgccgggga ccgcgccgaa actgctgatt tatgataaca acaaacgccc
gagcggggtg 180ccggatcgct ttagcgggag caaaagcggg accagcgcga
gcctggcgat tagcgggctg 240cgcagcgagg atgaggcgga ttattattgc
gcggcgtatg atgatcgcct gaacgggccg 300gtgtttgggg gggggaccaa
actgaccgtg ctggggcag 339322351DNAArtificial SequenceC4.4a binder
322gaggtgcagc tgctggagag cggggggggg ctggtgcagc cgggggggag
cctgcgcctg 60agctgcgcgg cgagcgggtt tacctttagc agcgcgtgga tgagctgggt
gcgccaggcg 120ccggggaaag ggctggagtg ggtgagctat attagcagca
gcgggagcag cacctattat 180gcggatagcg tgaaagggcg ctttaccatt
agccgcgata acagcaaaaa caccctgtat 240ctgcagatga acagcctgcg
cgcggaggat accgcggtgt attattgcgc gcgcgagggg 300ctgtgggcgt
ttgatgggtg ggggcagggg accctggtga ccgtgagcag c
351323339DNAArtificial SequenceC4.4a binder 323cagagcgtgc
tgacccagcc gccgagcgcg agcgggaccc cggggcagcg cgtgaccatt 60agctgcagcg
ggagcagcag caacattggg gcggggtatg tggtgcattg gtatcagcag
120ctgccgggga ccgcgccgaa actgctgatt tatgataaca accagcgccc
gagcggggtg 180ccggatcgct ttagcgggag caaaagcggg accagcgcga
gcctggcgat tagcgggctg 240cgcagcgagg atgaggcgga ttattattgc
gcggcgtatg atgatagcct gaaccgcccg 300gtgtttgggg gggggaccaa
actgaccgtg ctggggcag 339324351DNAArtificial SequenceC4.4a binder
324gaggtgcagc tgctggagag cggggggggg ctggtgcagc cgggggggag
cctgcgcctg 60agctgcgcgg cgagcgggtt tacctttagc aacgcgtgga tgagctgggt
gcgccaggcg 120ccggggaaag ggctggagtg ggtgagctat attagcagca
gcgggagcac catttattat 180gcggatagcg tgaaagggcg ctttaccatt
agccgcgata acagcaaaaa caccctgtat 240ctgcagatga acagcctgcg
cgcggaggat accgcggtgt attattgcgc gcgcgagggg 300ctgtgggcgt
ttgattattg ggggcagggg accctggtga ccgtgagcag c
351325339DNAArtificial SequenceC4.4a binder 325cagagcgtgc
tgacccagcc gccgagcgtg agcggggcgc cggggcagcg cgtgaccatt 60agctgcaccg
ggagcagcag caacattggg gcggggtatg tggtgcattg gtatcagcag
120ctgccgggga ccgcgccgaa actgctgatt tatgataaca acaaacgccc
gagcggggtg 180ccggatcgct ttagcgggag caaaagcggg accagcgcga
gcctggcgat tagcgggctg 240cgcagcgagg atgaggcgga ttattattgc
gcggcgtttg atgatagcct gaacgggccg 300gtgtttgggg gggggaccaa
actgaccgtg ctggggcag 339326351DNAArtificial SequenceC4.4a binder
326gaggtgcagc tgctggagag cggggggggg ctggtgcagc cgggggggag
cctgcgcctg 60agctgcgcgg cgagcgggtt tacctttagc agcgcgtgga tgagctgggt
gcgccaggcg 120ccggggaaag ggctggagtg ggtgagctat attagcagca
gcgggagcag cacctattat 180gcggatagcg tgaaagggcg ctttaccatt
agccgcgata acagcaaaaa caccctgtat 240ctgcagatga acagcctgcg
cgcggaggat accgcggtgt attattgcgc gcgcgagggg 300ctgtgggcgt
ttgataaatg ggggcagggg accctggtga ccgtgaccag c
351327339DNAArtificial SequenceC4.4a binder 327cagagcgtgc
tgacccagcc
gccgagcgcg agcgggaccc cggggcagcg cgtgaccatt 60agctgcaccg ggagcagcag
caacattggg gcggggtatg tggtgcattg gtatcagcag 120ctgccgggga
ccgcgccgaa actgctgatt tatgataaca acaaacgccc gagcggggtg
180ccggatcgct ttagcgggag caaaagcggg accagcgcga gcctggcgat
tagcgggctg 240cgcagcgagg atgaggcgga ttattattgc gcggcgtttg
atgatagcct gaacgggccg 300gtgtttgggg gggggaccaa actgaccgtg ctggggcag
339328351DNAArtificial SequenceC4.4a binder 328gaggtgcagc
tgctggagag cggggggggg ctggtgcagc cgggggggag cctgcgcctg 60agctgcgcgg
cgagcgggtt tacctttagc agcgcgtgga tgagctgggt gcgccaggcg
120ccggggaaag ggctggagtg ggtgagctat attagcagca gcgggagcag
cacctattat 180gcggatagcg tgaaagggcg ctttaccatt agccgcgata
acagcaaaaa caccctgtat 240ctgcagatga acagcctgcg cgcggaggat
accgcggtgt attattgcgc gcgcgagggg 300ctgtgggcgt ttgataaatg
ggggcagggg accctggtga ccgtgagcag c 351329339DNAArtificial
SequenceC4.4a 329cagagcgtgc tgacccagcc gccgagcgcg agcgggaccc
cggggcagcg cgtgaccatt 60agctgcagcg ggagcagcag caacattggg gcggggtatg
tggtgcattg gtatcagcag 120ctgccgggga ccgcgccgaa actgctgatt
tatgataaca accagcgccc gagcggggtg 180ccggatcgct ttagcgggag
caaaagcggg accagcgcga gcctggcgat tagcgggctg 240cgcagcgagg
atgaggcgga ttattattgc gcggcgtttg atgatagcct gaacgggccg
300gtgtttgggg gggggaccaa actgaccgtg ctggggcag
339330351DNAArtificial SequenceC4.4a binder 330gaggtgcagc
tgctggagag cggggggggg ctggtgcagc cgggggggag cctgcgcctg 60agctgcgcgg
cgagcgggtt tacctttagc agcgcgtgga tgagctgggt gcgccaggcg
120ccggggaaag ggctggagtg ggtgagctat attagcagca gcgggagcag
cacctattat 180gcggatagcg tgaaagggcg ctttaccatt agccgcgata
acagcaaaaa caccctgtat 240ctgcagatga acagcctgcg cgcggaggat
accgcggtgt attattgcgc gcgcgagggg 300ctgtgggcgt ttgataaatg
ggggcagggg accctggtga ccgtgagcag c 351331339DNAArtificial
SequenceC4.4a binder 331cagagcgtgc tgacccagcc gccgagcgcg agcgggaccc
cggggcagcg cgtgaccatt 60agctgcagcg ggagcagcag caacattggg gcggggtatg
tggtgcattg gtatcagcag 120ctgccgggga ccgcgccgaa actgctgatt
tatgataaca accagcgccc gagcggggtg 180ccggatcgct ttagcgggag
caaaagcggg accagcgcga gcctggcgat tagcgggctg 240cgcagcgagg
atgaggcgga ttattattgc gcggcgtttg atgatcgcct gagcgggccg
300gtgtttgggg gggggaccaa actgaccgtg ctggggcag
339332366DNAArtificial SequenceC4.4a binder 332gaggtgcagc
tgctggagag cggggggggg ctggtgcagc cgggggggag cctgcgcctg 60agctgcgcgg
cgagcgggtt tacctttagc gattatcaga tgacctggat tcgccagacc
120ccggggaaag ggctggagtg ggtgagcggg gtgagctgga acggggcgcg
cacccattat 180gcggatagcg tgaaagggcg ctttaccatt agccgcgata
acagcaaaaa caccctgtat 240ctgcagatga acagcctgcg cgcggaggat
accgcggtgt attattgcgc gaaaggggat 300tatctggtgt atagcagcta
ttattttaaa agctgggggc aggggaccct ggtgaccgtg 360accagc
366333336DNAArtificial SequenceC4.4a binder 333cagagcgtgc
tgacccagcc gccgagcgcg agcgggaccc cggggcagcg cgtgaccatt 60agctgcagcg
ggagcagcag caacgtgggg agcaacccgg tgaactggta tcagcagctg
120ccggggaccg cgccgaaact gctgatttat cgcaacaacc agcgcccgag
cggggtgccg 180gatcgcttta gcgggagcaa aagcgggacc agcgcgagcc
tggcgattag cgggctgcgc 240agcgaggatg aggcggatta ttattgcgcg
gcgtgggatg atcgcctgaa cgggtggggg 300tttggggggg ggaccaaact
gaccgtgctg gggcag 336334363DNAArtificial SequenceC4.4a binder
334gagcagctgc tggagagcgg gggggggctg gtgcagccgg gggggagcct
gcgcctgagc 60tgcgcggcga gcgggtttac ctttagcagc tatcagatga cctggattcg
ccagaccccg 120gggaaagggc tggagtgggt gagcgggatt agctggaacg
gggggagcac ccattatgcg 180gatagcgtga aagggcgctt taccattagc
cgcgataaca gcaaaaacac cctgtatctg 240cagatgaaca gcctgcgcgc
ggaggatacc gcggtgtatt attgcgcgaa aggggattat 300ctggtgtata
gcgcgtatta ttttgatagc tgggggcagg ggaccctggt gaccgtgagc 360agc
363335333DNAArtificial SequenceC4.4a binder 335caggtgctga
cccagccgcc gagcgcgagc gggaccccgg ggcagcgcgt gaccattagc 60tgcagcggga
gcagcagcaa cattgggagc aacccggtga actggtatca gcagctgccg
120gggaccgcgc cgaaactgct gatttatcgc aacaaccagc gcccgagcgg
ggtgccggat 180cgctttagcg ggagcaaaag cgggaccagc gcgagcctgg
cgattagcgg gctgcgcagc 240gaggatgagg cggattatta ttgcgcggcg
tgggatgatc gcctgaacgg gtgggggttt 300ggggggggga ccaaactgac
cgtgctgggg cag 333336366DNAArtificial SequenceC4.4a binder
336gaggtgcagc tgctggagag cggggggggg ctggtgcagc cgggggggag
cctgcgcctg 60agctgcgcgg cgagcgggtt tacctttagc gattatcaga tgacctggat
tcgccagacc 120ccggggaaag ggctggagtg ggtgagcggg attagctgga
acggggggag cacccattat 180gcggatagcg tgaaagggcg ctttaccatt
agccgcgata acagcaaaaa caccctgtat 240ctgcagatga acagcctgcg
cgcggaggat accgcggtgt attattgcgc gaaaggggat 300tatctggtgt
atagcagcta ttattttaaa tattgggggc aggggaccct ggtgaccgtg 360agcagc
366337336DNAArtificial SequenceC4.4a binder 337cagagcgtgc
tgacccagcc gccgagcgcg agcgggaccc cggggcagcg cgtgaccatt 60agctgcagcg
ggagcagcag caacattggg agcaacccgg tgaactggta tcagcagctg
120ccggggaccg cgccgaaact gctgatttat cgcaacaacc agcgcccgag
cggggtgccg 180gatcgcttta gcgggagcaa aagcgggacc agcgcgagcc
tggcgattag cgggctgcgc 240agcgaggatg aggcggatta ttattgcgcg
gcgtgggatg atcgcctgaa cgggtgggcg 300tttggggggg ggaccaaact
gaccgtgctg gggcag 336338366DNAArtificial SequenceC4.4a binder
338gaggtgcagc tgctggagag cggggggggg ctggtgcagc cgggggggag
cctgcgcctg 60agctgcgcgg cgagcgggtt tacctttagc gattatcaga tgacctggat
tcgccagacc 120ccggggaaag ggctggagtg ggtgagcggg attagctgga
acggggggag cacccattat 180gcggatagcg tgaaagggcg ctttaccatt
agccgcgata acagcaaaaa caccctgtat 240ctgcagatga acagcctgcg
cgcggaggat accgcggtgt attattgcgc gaaaggggat 300tatctggtgt
atagcagcta ttattttaaa agctgggggc aggggaccct ggtgaccgtg 360agcagc
366339336DNAArtificial SequenceC4.4a binder 339cagagcgtgc
tgacccagcc gccgagcgcg agcgggaccc cggggcagcg cgtgaccatt 60agctgcagcg
ggagcagcag caacattggg agcaacccgg tgaactggta tcagcagctg
120ccggggaccg cgccgaaact gctgatttat cgcaacaacc agcgcccgag
cggggtgccg 180gatcgcttta gcgggagcaa aagcgggacc agcgcgagcc
tggcgattag cgggctgcgc 240agcgaggatg aggcggatta ttattgcgcg
gcgtgggatg atagcctgaa cgggtggggg 300tttggggggg ggaccaaact
gaccgtgctg gggcag 336340366DNAArtificial SequenceC4.4a binder
340gaggtgcagc tgctggagag cggggggggg ctggtgcagc cgggggggag
cctgcgcctg 60agctgcgcgg cgagcgggtt tacctttagc gattatcaga tgacctggat
tcgccagacc 120ccggggaaag ggctggagtg ggtgagcggg attagctgga
acggggggag cacccattat 180gcggatagcg tgaaagggcg ctttaccatt
agccgcgata acagcaaaaa caccctgtat 240ctgcagatga acagcctgcg
cgcggaggat accgcggtgt attattgcgc gaaaggggat 300tatctggtgt
ataaaagcta ttattttaaa agctgggggc aggggaccct ggtgaccgtg 360agcagc
366341336DNAArtificial SequenceC4.4a binder 341cagagcgtgc
tgacccagcc gccgagcgcg agcgggaccc cggggcagcg cgtgaccatt 60agctgcagcg
ggagcagcag caacattggg agcaacccgg tgaactggta tcagcagctg
120ccggggaccg cgccgaaact gctgatttat cgcaacaacc agcgcccgag
cggggtgccg 180gatcgcttta gcgggagcaa aagcgggacc agcgcgagcc
tggcgattag cgggctgcgc 240agcgaggatg aggcggatta ttattgcgcg
gcgtgggatg atcgcctgag cgggtggggg 300tttggggggg ggaccaaact
gaccgtgctg gggcag 336342366DNAArtificial SequenceC4.4a binder
342gaggtgcagc tgctggagag cggggggggg ctggtgcagc cgggggggag
cctgcgcctg 60agctgcgcgg cgagcgggtt tacctttagc gattatcaga tgacctggat
tcgccagacc 120ccggggaaag ggctggagtg ggtgagcggg attagctgga
acggggggag cacccattat 180gcggatagcg tgaaagggcg ctttaccatt
agccgcgata acagcaaaaa caccctgtat 240ctgcagatga acagcctgcg
cgcggaggat accgcggtgt attattgcgc gaaaggggat 300tatctggtgt
atagcagcta ttattttaaa agctgggggc aggggaccct ggtgaccgtg 360agcagc
366343336DNAArtificial SequenceC4.4a binder 343cagagcgtgc
tgacccagcc gccgagcgcg agcgggaccc cggggcagcg cgtgaccatt 60agctgcagcg
ggagcagcag caacattggg agcaacccgg tgaactggta tcagcagctg
120ccggggaccg cgccgaaact gctgatttat cgcaacaacc agcgcccgag
cggggtgccg 180gatcgcttta gcgggagcaa aagcgggacc agcgcgagcc
tggcgattag cgggctgcgc 240agcgaggatg aggcggatta ttattgcgcg
gcgtgggatg atcgcctgag cgggtggggg 300tttggggggg ggaccaaact
gaccgtgctg gggcag 336344366DNAArtificial SequenceC4.4a binder
344gaggtgcagc tgctggagag cggggggggg ctggtgcagc cgggggggag
cctgcgcctg 60agctgcgcgg cgagcgggtt tacctttagc gattatcaga tgacctggat
tcgccagacc 120ccggggaaag ggctggagtg ggtgagcggg attagctgga
acggggggag cacccattat 180gcggatagcg tgaaagggcg ctttaccatt
agccgcgata acagcaaaaa caccctgtat 240ctgcagatga acagcctgcg
cgcggaggat accgcggtgt attattgcgc gaaaggggat 300tatctggtgt
ataaaagcta ttattttaaa agctgggggc aggggaccct ggtgaccgtg 360agcagc
366345336DNAArtificial SequenceC4.4a binder 345cagagcgtgc
tgacccagcc gccgagcgcg agcgggaccc cggggcagcg cgtgaccatt 60agctgcagcg
ggagcagcag caacattggg agcaacccgg tgaactggta tcagcagctg
120ccggggaccg cgccgaaact gctgatttat cgcaacaacc agcgcccgag
cggggtgccg 180gatcgcttta gcgggagcaa aagcgggacc agcgcgagcc
tggcgattag cgggctgcgc 240agcgaggatg aggcggatta ttattgcgcg
gcgtgggatg atagcctgaa cgggtggggg 300tttggggggg ggaccaaact
gaccgtgctg gggcag 336346217PRTHomo Sapiens 346Asp Ile Val Leu Thr
Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr
Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr
Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40
45 Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe
50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser
Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala
Trp Asp Asp Arg 85 90 95 Leu Asn Gly Pro Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val
Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys
Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala
Val Thr Val Ala Trp Lys Gly Asp Ser Ser Pro Val 145 150 155 160 Lys
Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 165 170
175 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
180 185 190 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr
Val Glu 195 200 205 Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215
347447PRTHomo Sapiens 347Gln Val Glu Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser
Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Glu Gly Leu Trp Ala Phe Asp Tyr Trp Gly Gln Gly Thr
Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210
215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330
335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445
348217PRTHomo Sapiens 348Glu Ser Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly
Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Val Val His Trp Tyr
Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp
Asn Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80
Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Arg 85
90 95 Leu Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro
Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys
Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Val Thr Val Ala Trp
Lys Ala Asp Ser Ser Pro Val 145 150 155 160 Lys Ala Gly Val Glu Thr
Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 165 170 175 Tyr Ala Ala Ser
Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180 185 190 His Arg
Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 195 200 205
Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215 349446PRTHomo Sapiens
349Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile
Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly
Leu Trp Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130
135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210
215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330
335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445
350217PRTHomo Sapiens 350Glu Ser Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Val Val His Trp Tyr
Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp
Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80
Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Arg 85
90 95 Leu Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro
Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys
Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Val Thr Val Ala Trp
Lys Ala Asp Ser Ser Pro Val 145 150 155 160 Lys Ala Gly Val Glu Thr
Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 165 170 175 Tyr Ala Ala Ser
Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180 185 190 His Arg
Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 195 200 205
Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215 351446PRTHomo Sapiens
351Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Ser Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Ser Thr
Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly
Leu Trp Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130
135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250
255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly 435 440 445 352217PRTHomo Sapiens 352Glu Ser
Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15
Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20
25 30 Tyr Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys
Leu 35 40 45 Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Val Pro
Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu
Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr
Cys Ala Ala Trp Asp Asp Arg 85 90 95 Leu Asn Gly Pro Val Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Ala
Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln
Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr
Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 145 150
155 160 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
Lys 165 170 175 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln
Trp Lys Ser 180 185 190 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu
Gly Ser Thr Val Glu 195 200 205 Lys Thr Val Ala Pro Thr Glu Cys Ser
210 215 353446PRTHomo Sapiens 353Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr
Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Leu Trp Ala Phe Asp Tyr Trp Gly
Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185
190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310
315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445
354217PRTHomo Sapiens 354Glu Ser Val Leu Thr Gln Pro Pro Ser Val
Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly
Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Val Val His Trp Tyr
Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp
Asn Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80
Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Tyr Asp Asp Arg 85
90 95 Leu Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro
Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys
Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Val Thr Val Ala Trp
Lys Ala Asp Ser Ser Pro Val 145 150 155 160 Lys Ala Gly Val Glu Thr
Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 165 170 175 Tyr Ala Ala Ser
Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180 185 190 His Arg
Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 195 200 205
Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215 355446PRTHomo Sapiens
355Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile
Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly
Leu Trp Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130
135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250
255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly 435 440 445 356217PRTHomo Sapiens 356Gln Ser
Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15
Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20
25 30 Tyr Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala
Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly
Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala
Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala Asp
Tyr Tyr Cys Ala Ala Tyr Asp Asp Ser 85 90 95 Leu Ser Gly Pro Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln Pro Lys
Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125 Glu
Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130 135
140 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val
145 150 155 160 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser
Asn Asn Lys 165 170 175 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro
Glu Gln Trp Lys Ser 180 185 190 His Arg Ser Tyr Ser Cys Gln Val Thr
His Glu Gly Ser Thr Val Glu 195 200 205 Lys Thr Val Ala Pro Thr Glu
Cys Ser 210 215 357446PRTHomo Sapiens 357Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Leu Trp Ala Phe Asp Tyr Trp
Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180
185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305
310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425
430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445 358217PRTHomo Sapiens 358Gln Ser Val Leu Thr Gln Pro Pro Ser
Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser
Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Val Val His Trp
Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr
Asp Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70
75 80 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Tyr Asp Asp
Ser 85 90 95 Leu Asn Arg Pro Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe
Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr Leu
Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Val Thr Val
Ala Trp Lys Ala Asp Ser Ser Pro Val 145 150 155 160 Lys Ala Gly Val
Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 165 170 175 Tyr Ala
Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180 185 190
His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 195
200 205 Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215 359446PRTHomo
Sapiens 359Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Gly Leu Trp Ala Phe Asp Gly Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115
120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235
240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360
365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 435 440 445 360217PRTHomo Sapiens
360Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly
Ala Gly 20 25 30 Tyr Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr
Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser
Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser
Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala
Asp Tyr Tyr Cys Ala Ala Phe Asp Asp Arg 85 90 95 Leu Asn Gly Pro
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln Pro
Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130
135 140 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
Val 145 150 155 160 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln
Ser Asn Asn Lys 165 170 175 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr
Pro Glu Gln Trp Lys Ser 180 185 190 His Arg Ser Tyr Ser Cys Gln Val
Thr His Glu Gly Ser Thr Val Glu 195 200 205 Lys Thr Val Ala Pro Thr
Glu Cys Ser 210 215 361446PRTHomo Sapiens 361Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Leu Trp Ala Phe Asp Lys
Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170
175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295
300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420
425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435
440 445 362217PRTHomo Sapiens 362Gln Ser Val Leu Thr Gln Pro Pro
Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys
Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Val Val His
Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile
Tyr Asp Asn Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60
Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65
70 75 80 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Phe Asp
Asp Ser 85 90 95 Leu Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu
Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr
Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Val Thr
Val Ala Trp Lys Ala Asp Ser Ser Pro Val 145 150 155 160 Lys Ala Gly
Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 165 170 175 Tyr
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180 185
190 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205 Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215
363446PRTHomo Sapiens 363Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser
Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Glu Gly Leu Trp Ala Phe Asp Tyr Trp Gly Gln Gly Thr
Leu 100 105
110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230
235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355
360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 364217PRTHomo Sapiens
364Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln
1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly
Ala Gly 20 25 30 Tyr Val Val His Trp Tyr Gln Gln Leu Pro Gly Thr
Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser
Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser
Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Arg Ser Glu Asp Glu Ala
Asp Tyr Tyr Cys Ala Ala Tyr Asp Asp Ser 85 90 95 Leu Ser Gly Pro
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln Pro
Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130
135 140 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
Val 145 150 155 160 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln
Ser Asn Asn Lys 165 170 175 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr
Pro Glu Gln Trp Lys Ser 180 185 190 His Arg Ser Tyr Ser Cys Gln Val
Thr His Glu Gly Ser Thr Val Glu 195 200 205 Lys Thr Val Ala Pro Thr
Glu Cys Ser 210 215 365446PRTHomo Sapiens 365Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ala 20 25 30 Trp
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Leu Trp Ala Phe Asp Tyr
Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170
175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295
300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420
425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435
440 445 366217PRTHomo Sapiens 366Gln Ser Val Leu Thr Gln Pro Pro
Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys
Ser Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Val Val His
Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile
Tyr Asp Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60
Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65
70 75 80 Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Phe Asp
Asp Ser 85 90 95 Leu Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu
Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr
Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Val Thr
Val Ala Trp Lys Ala Asp Ser Ser Pro Val 145 150 155 160 Lys Ala Gly
Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 165 170 175 Tyr
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180 185
190 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205 Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215
367446PRTHomo Sapiens 367Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Ala 20 25 30 Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser
Ser Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Glu Gly Leu Trp Ala Phe Asp Lys Trp Gly Gln Gly Thr
Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210
215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330
335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445
368217PRTHomo Sapiens 368Gln Ser Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Val Val His Trp Tyr
Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp
Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80
Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Phe Asp Asp Arg 85
90 95 Leu Ser Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro
Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys
Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Val Thr Val Ala Trp
Lys Ala Asp Ser Ser Pro Val 145 150 155 160 Lys Ala Gly Val Glu Thr
Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 165 170 175 Tyr Ala Ala Ser
Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180 185 190 His Arg
Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 195 200 205
Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215 369446PRTHomo Sapiens
369Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Ser Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Ser Thr
Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly
Leu Trp Ala Phe Asp Lys Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130
135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250
255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 370216PRTHomo Sapiens
370Asp Ile Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Val Gly
Ser Asn 20 25 30 Pro Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala
Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly
Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala
Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp
Tyr Tyr Cys Ala Ala Trp Asp Asp Arg Leu 85 90 95 Asn Gly Trp Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys
Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125
Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130
135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Gly Asp Ser Ser Pro Val
Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser
Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro
Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr
His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu
Cys Ser 210 215 371452PRTHomo Sapiens 371Gln Val Glu Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gln Met
Thr Trp Ile Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Gly Val Ser Trp Asn Gly Ala Arg Thr His Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Lys Gly Asp Tyr Leu Val Tyr Ser Ala Tyr
Tyr Phe Asp Ser Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180
185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305
310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425
430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445 Ser Pro Gly Lys 450 372216PRTHomo Sapiens 372Glu Ser
Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Val Gly Ser Asn 20
25 30 Pro Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp
Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala
Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys
Ala Ala Trp Asp Asp Arg Leu 85 90 95 Asn Gly Trp Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro
Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala
Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro
Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150
155 160 Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp
Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly
Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210
215 373451PRTHomo Sapiens 373Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gln Met Thr Trp Ile
Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Val
Ser Trp Asn Gly Ala Arg Thr His Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Lys Gly Asp Tyr Leu Val Tyr Ser Ala Tyr Tyr Phe
Asp Ser Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195
200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315
320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440
445 Ser Pro Gly 450 374216PRTHomo Sapiens 374Glu Ser Val Leu Thr
Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr
Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Pro
Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly
Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp
Asp Asp Arg Leu 85 90 95 Asn Gly Trp Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr
Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala
Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val
Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala
Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170
175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His
180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215
375451PRTHomo Sapiens 375Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gln Met Thr Trp Ile Arg
Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser
Trp Asn Gly Gly Ser Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys Gly Asp Tyr Leu Val Tyr Ser Ala Tyr Tyr Phe Asp Ser
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210
215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330
335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser
Pro Gly 450 376216PRTHomo Sapiens 376Glu Ser Val Leu Thr Gln Pro
Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser
Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Pro Val Asn
Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile
Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55
60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp
Arg Leu 85 90 95 Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe
Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu
Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val
Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val
Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala
Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185
190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215 377451PRTHomo
Sapiens 377Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gln Met Thr Trp Ile Arg
Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser
Trp Asn Gly Gly Ser Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys Gly Asp Tyr Leu Val Tyr Ser Ala Tyr Tyr Phe Asp Ser
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210
215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330
335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser
Pro Gly 450 378216PRTHomo Sapiens 378Glu Ser Val Leu Thr Gln Pro
Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser
Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Pro Val Asn
Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile
Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55
60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp
Arg Leu 85 90 95 Asn Gly Trp Gly Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe
Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu
Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val
Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val
Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala
Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185
190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215 379451PRTHomo
Sapiens 379Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Asp Tyr 20 25 30 Gln Met Thr Trp Ile Arg Gln Thr Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Gly Gly
Ser Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys
Gly Asp Tyr Leu Val Tyr Ser Ala Tyr Tyr Phe Asp Ser Trp 100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115
120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235
240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360
365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly 450
380216PRTHomo Sapiens 380Glu Ser Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Pro Val Asn Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn
Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Arg Leu 85
90 95 Asn Gly Trp Gly Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser
Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu
Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys
Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr
Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser
Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser
Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205
Thr Val Ala Pro Thr Glu Cys Ser 210 215 381451PRTHomo Sapiens
381Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Ser Tyr 20 25 30 Gln Met Thr Trp Ile Arg Gln Thr Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Gly Gly Ser Thr
His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Asp
Tyr Leu Val Tyr Ser Ala Tyr Tyr Phe Asp Ser Trp 100 105 110 Gly Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130
135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250
255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375
380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly 450
382216PRTHomo Sapiens 382Gln Ser Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Pro Val Asn Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn
Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Arg Leu 85
90 95 Ser Gly Trp Ala Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser
Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu
Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys
Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr
Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser
Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser
Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205
Thr Val Ala Pro Thr Glu Cys Ser 210 215 383451PRTHomo Sapiens
383Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Asp Tyr 20 25 30 Gln Met Thr Trp Ile Arg Gln Thr Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Gly Gly Ser Thr
His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Asp
Tyr Leu Val Tyr Ser Ser Tyr Tyr Phe Lys Ser Trp 100 105 110 Gly Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130
135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250
255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 290 295 300
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305
310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425
430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445 Ser Pro Gly 450 384216PRTHomo Sapiens 384Gln Ser Val
Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg
Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25
30 Pro Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg
Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile
Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala
Ala Trp Asp Asp Ser Leu 85 90 95 Ser Gly Trp Ala Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser
Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn
Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly
Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155
160 Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr
165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys
Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser
Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215
385451PRTHomo Sapiens 385Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gln Met Thr Trp Ile Arg
Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser
Trp Asn Gly Gly Ser Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys Gly Asp Tyr Leu Val Tyr Lys Ser Tyr Tyr Phe Lys Ser
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210
215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330
335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser
Pro Gly 450 386216PRTHomo Sapiens 386Gln Ser Val Leu Thr Gln Pro
Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser
Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Pro Val Asn
Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile
Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55
60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp
Arg Leu 85 90 95 Asn Gly Trp Ala Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe
Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu
Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val
Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val
Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala
Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185
190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215 387451PRTHomo
Sapiens 387Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Asp Tyr 20 25 30 Gln Met Thr Trp Ile Arg Gln Thr Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Gly Gly
Ser Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys
Gly Asp Tyr Leu Val Tyr Ser Ser Tyr Tyr Phe Lys Tyr Trp 100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115
120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235
240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360
365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly 450
388216PRTHomo Sapiens 388Gln Ser Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Pro Val Asn Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn
Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85
90 95 Asn Gly Trp Gly Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser
Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu
Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys
Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr
Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser
Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser
Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205
Thr Val Ala Pro Thr Glu Cys Ser 210 215 389451PRTHomo Sapiens
389Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Asp Tyr 20 25 30 Gln Met Thr Trp Ile Arg Gln Thr Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Gly Gly Ser Thr
His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Asp
Tyr Leu Val Tyr Ser Ser Tyr Tyr Phe Lys Ser Trp 100 105 110 Gly Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130
135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250
255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375
380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly 450
390216PRTHomo Sapiens 390Gln Ser Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Pro Val Asn Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn
Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Arg Leu 85
90 95 Ser Gly Trp Gly Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser
Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu
Ile Ser Asp
Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser
Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Thr Pro Ser
Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser
Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys
Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala
Pro Thr Glu Cys Ser 210 215 391451PRTHomo Sapiens 391Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25
30 Gln Met Thr Trp Ile Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Gly Ile Ser Trp Asn Gly Gly Ser Thr His Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Asp Tyr Leu Val Tyr
Lys Ser Tyr Tyr Phe Lys Ser Trp 100 105 110 Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155
160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280
285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405
410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu 435 440 445 Ser Pro Gly 450 392216PRTHomo Sapiens
392Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly
Ser Asn 20 25 30 Pro Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala
Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly
Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala
Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp
Tyr Tyr Cys Ala Ala Trp Asp Asp Arg Leu 85 90 95 Ser Gly Trp Gly
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys
Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125
Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130
135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val
Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser
Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro
Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr
His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu
Cys Ser 210 215 393451PRTHomo Sapiens 393Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gln Met
Thr Trp Ile Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Gly Ile Ser Trp Asn Gly Gly Ser Thr His Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Lys Gly Asp Tyr Leu Val Tyr Ser Ser Tyr
Tyr Phe Lys Ser Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180
185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305
310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425
430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445 Ser Pro Gly 450 394216PRTHomo Sapiens 394Gln Ser Val
Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg
Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25
30 Pro Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg
Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile
Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala
Ala Trp Asp Asp Ser Leu 85 90 95 Asn Gly Trp Gly Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser
Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn
Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly
Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155
160 Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr
165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys
Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser
Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215
395451PRTHomo Sapiens 395Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gln Met Thr Trp Ile Arg
Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser
Trp Asn Gly Gly Ser Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys Gly Asp Tyr Leu Val Tyr Lys Ser Tyr Tyr Phe Lys Ser
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210
215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330
335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser
Pro Gly 450 396216PRTHomo Sapiens 396Gln Ser Val Leu Thr Gln Pro
Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser
Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Pro Val Asn
Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile
Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55
60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp
Ser Leu 85 90 95 Ser Gly Trp Ala Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe
Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu
Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val
Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val
Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala
Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185
190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys
195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215 397451PRTHomo
Sapiens 397Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30 Gln Met Thr Trp Ile Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Gly Gly
Ser Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys
Gly Asp Tyr Leu Val Tyr Lys Ser Tyr Tyr Phe Lys Ser Trp 100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115
120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305
310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425
430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445 Ser Pro Gly 450 39810PRTHomo sapiens 398Gly Tyr Ser Phe
Thr Ser Tyr Trp Ile Gly 1 5 10 39920PRTHomo sapiens 399Trp Met Gly
Ile Ile Asp Pro Gly Asp Ser Arg Thr Arg Tyr Ser Pro 1 5 10 15 Ser
Phe Gln Gly 20 40011PRTHomo sapiens 400Gly Gln Leu Tyr Gly Gly Thr
Tyr Met Asp Gly 1 5 10 40114PRTHomo sapiens 401Thr Gly Thr Ser Ser
Asp Ile Gly Gly Tyr Asn Ser Val Ser 1 5 10 40211PRTHomo sapiens
402Leu Met Ile Tyr Gly Val Asn Asn Arg Pro Ser 1 5 10 40310PRTHomo
sapiens 403Ser Ser Tyr Asp Ile Glu Ser Ala Thr Pro 1 5 10
404120PRTHomo sapiens 404Gln Val Glu Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly
Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile Gly Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asp
Pro Gly Asp Ser Arg Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly
Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80
Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85
90 95 Ala Arg Gly Gln Leu Tyr Gly Gly Thr Tyr Met Asp Gly Trp Gly
Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
405113PRTHomo sapiens 405Asp Ile Ala Leu Thr Gln Pro Ala Ser Val
Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly
Thr Ser Ser Asp Ile Gly Gly Tyr 20 25 30 Asn Ser Val Ser Trp Tyr
Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Gly
Val Asn Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Asp Ile Glu 85
90 95 Ser Ala Thr Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
Gly 100 105 110 Gln 406360DNAHomo sapiens 406caggtggaat tggttcagag
cggcgcggaa gtgaaaaaac cgggcgaaag cctgaaaatt 60agctgcaaag gttccggata
ttcctttact tcttattgga ttggttgggt gcgccaggcc 120cctgggaagg
gtctcgagtg gatgggcatt atcgatccgg gtgatagccg tacccgttat
180tctccgagct ttcagggcca ggtgaccatt agcgcggata aaagcattag
caccgcgtat 240cttcaatgga gcagcctgaa agcgagcgat acggccatgt
attattgcgc gcgtggtcag 300ctttatggtg gtacttatat ggatggttgg
ggccaaggca ccctggtgac ggttagctca 360407339DNAHomo sapiens
407gatatcgcac tgacccagcc agcttcagtg agcggctcac caggtcagag
cattaccatc 60tcgtgtacgg gtactagcag cgatattggt ggttataatt ctgtgtcttg
gtaccagcag 120catcccggga aggcgccgaa acttatgatt tatggtgtta
ataatcgtcc ctcaggcgtg 180agcaaccgtt ttagcggatc caaaagcggc
aacaccgcga gcctgaccat tagcggcctg 240caagcggaag acgaagcgga
ttattattgc tcttcttatg atattgagtc tgctactcct 300gtgtttggcg
gcggcacgaa gttaaccgtc ctaggtcag 339408450PRTHomo sapiens 408Gln Val
Glu Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20
25 30 Trp Ile Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Met 35 40 45 Gly Ile Ile Asp Pro Gly Asp Ser Arg Thr Arg Tyr Ser
Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser
Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser
Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gly Gln Leu Tyr Gly
Gly Thr Tyr Met Asp Gly Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150
155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275
280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395
400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro 435 440 445 Gly Lys 450 409217PRTHomo sapiens
409Asp Ile Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln
1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Ile Gly
Gly Tyr 20 25 30 Asn Ser Val Ser Trp Tyr Gln Gln His Pro Gly Lys
Ala Pro Lys Leu 35 40 45 Met Ile Tyr Gly Val Asn Asn Arg Pro Ser
Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr
Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala
Asp Tyr Tyr Cys Ser Ser Tyr Asp Ile Glu 85 90 95 Ser Ala Thr Pro
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln Pro
Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125
Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130
135 140 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Gly Asp Ser Ser Pro
Val 145 150 155 160 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln
Ser Asn Asn Lys 165 170 175 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr
Pro Glu Gln Trp Lys Ser 180 185 190 His Arg Ser Tyr Ser Cys Gln Val
Thr His Glu Gly Ser Thr Val Glu 195 200 205 Lys Thr Val Ala Pro Thr
Glu Cys Ser 210 215 4101353DNAHomo sapiens 410caggtggaat tggttcagag
cggcgcggaa gtgaaaaaac cgggcgaaag cctgaaaatt 60agctgcaaag gttccggata
ttcctttact tcttattgga ttggttgggt gcgccaggcc 120cctgggaagg
gtctcgagtg gatgggcatt atcgatccgg gtgatagccg tacccgttat
180tctccgagct ttcagggcca ggtgaccatt agcgcggata aaagcattag
caccgcgtat 240cttcaatgga gcagcctgaa agcgagcgat acggccatgt
attattgcgc gcgtggtcag 300ctttatggtg gtacttatat ggatggttgg
ggccaaggca ccctggtgac ggttagctca 360gcctccacca agggtccatc
ggtcttcccc ctggcaccct cctccaagag cacctctggg 420ggcacagcgg
ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg
480tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct
acagtcctca 540ggactctact ccctcagcag cgtggtgacc gtgccctcca
gcagcttggg cacccagacc 600tacatctgca acgtgaatca caagcccagc
aacaccaagg tggacaagaa agttgagccc 660aaatcttgtg acaaaactca
cacatgccca ccgtgcccag cacctgaact cctgggggga 720ccgtcagtct
tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
780gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa
gttcaactgg 840tacgtggacg gcgtggaggt gcataatgcc aagacaaagc
cgcgggagga gcagtacaac 900agcacgtacc gggtggtcag cgtcctcacc
gtcctgcacc aggactggct gaatggcaag 960gagtacaagt gcaaggtctc
caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020aaagccaaag
ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag
1080ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc
cagcgacatc 1140gccgtggagt gggagagcaa tgggcagccg gagaacaact
acaagaccac gcctcccgtg 1200ctggactccg acggctcctt cttcctctac
agcaagctca ccgtggacaa gagcaggtgg 1260cagcagggga acgtcttctc
atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320cagaagagcc
tctccctgtc tccgggtaaa tga 1353411654DNAHomo sapiens 411gatatcgcac
tgacccagcc agcttcagtg agcggctcac caggtcagag cattaccatc 60tcgtgtacgg
gtactagcag cgatattggt ggttataatt ctgtgtcttg gtaccagcag
120catcccggga aggcgccgaa acttatgatt tatggtgtta ataatcgtcc
ctcaggcgtg 180agcaaccgtt ttagcggatc caaaagcggc aacaccgcga
gcctgaccat tagcggcctg 240caagcggaag acgaagcgga ttattattgc
tcttcttatg atattgagtc tgctactcct 300gtgtttggcg gcggcacgaa
gttaaccgtc ctaggtcagc ccaaggctgc cccctcggtc 360actctgttcc
cgccctcctc tgaggagctt caagccaaca aggccacact ggtgtgtctc
420ataagtgact tctacccggg agccgtgaca gtggcctgga agggagatag
cagccccgtc 480aaggcgggag tggagaccac cacaccctcc aaacaaagca
acaacaagta cgcggccagc 540agctatctga gcctgacgcc tgagcagtgg
aagtcccaca gaagctacag ctgccaggtc 600acgcatgaag ggagcaccgt
ggagaagaca gtggccccta cagaatgttc atag 654
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