U.S. patent application number 14/269577 was filed with the patent office on 2015-01-29 for novel binder-drug conjugates (adcs) and use thereof.
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, Sherif El Sheikh, Sven Golfier, Simone Greven, Axel Harrenga, Iring Heisler, Hannah Jorissen, Charlotte C. Kopitz, Hans-Georg LERCHEN, Lars Linden, Christoph Mahlert, Heike Petrul, Joachim Schuhmacher, Beatrix Stelte-Ludwig, Karl-Heinz Thierauch, Jorg Willuda.
Application Number | 20150030618 14/269577 |
Document ID | / |
Family ID | 45976407 |
Filed Date | 2015-01-29 |
United States Patent
Application |
20150030618 |
Kind Code |
A1 |
LERCHEN; Hans-Georg ; et
al. |
January 29, 2015 |
NOVEL BINDER-DRUG CONJUGATES (ADCS) AND USE THEREOF
Abstract
The present application relates to new binder-drug conjugates
(ADCs) of N,N-dialkylauristatins that are directed against the
target C4.4a, to active metabolites of these ADCs, to processes for
preparing these ADCs, to the use of these ADCs for treating and/or
preventing illnesses, and also to the use of these ADCs for
producing medicaments for treating and/or preventing illnesses,
more particularly hyperproliferative and/or angiogenic diseases
such as, for example, cancer diseases. Such treatments may be
practised as a monotherapy or else in combination with other
medicaments or further therapeutic measures.
Inventors: |
LERCHEN; Hans-Georg;
(Leverkusen, DE) ; Linden; Lars; (Dusseldorf,
DE) ; El Sheikh; Sherif; (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; (Wulfrath, 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.: |
14/269577 |
Filed: |
May 5, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13656681 |
Oct 20, 2012 |
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14269577 |
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PCT/EP2012/057247 |
Apr 20, 2012 |
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13656681 |
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PCT/EP2012/057249 |
Apr 20, 2012 |
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PCT/EP2012/057247 |
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PCT/EP2012/057245 |
Apr 20, 2012 |
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PCT/EP2012/057249 |
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PCT/EP2012/057243 |
Apr 20, 2012 |
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PCT/EP2012/057245 |
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Current U.S.
Class: |
424/181.1 ;
530/391.9; 544/141; 544/63; 548/143; 548/243; 548/468; 548/518 |
Current CPC
Class: |
A61K 31/536 20130101;
C07K 7/06 20130101; C07K 16/30 20130101; A61P 9/00 20180101; A61K
31/4025 20130101; A61K 31/4245 20130101; A61P 35/02 20180101; A61K
47/6869 20170801; A61K 31/40 20130101; C07K 16/18 20130101; C07K
2317/76 20130101; C07K 7/02 20130101; A61K 47/6859 20170801; A61K
31/537 20130101; C07K 16/2863 20130101; A61K 31/5377 20130101; C07K
5/0205 20130101; C07K 2317/24 20130101; A61K 39/3955 20130101; C07K
5/06034 20130101; A61K 45/06 20130101; A61K 31/5355 20130101; A61K
31/404 20130101; A61K 47/6803 20170801; A61K 47/542 20170801; C07K
2317/21 20130101; A61K 31/422 20130101; C07K 5/0606 20130101; C07K
16/28 20130101; C07K 2317/565 20130101; A61K 47/6851 20170801; C07K
5/06 20130101; C07K 5/06052 20130101; A61K 47/6889 20170801; A61P
43/00 20180101; A61P 35/00 20180101; C07K 5/0207 20130101; C07K
16/2812 20130101 |
Class at
Publication: |
424/181.1 ;
544/141; 548/518; 548/468; 544/63; 548/143; 548/243; 530/391.9 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 31/5355 20060101 A61K031/5355; A61K 31/4025
20060101 A61K031/4025; A61K 31/404 20060101 A61K031/404; A61K 45/06
20060101 A61K045/06; A61K 31/4245 20060101 A61K031/4245; A61K
31/536 20060101 A61K031/536; A61K 31/537 20060101 A61K031/537; A61K
31/422 20060101 A61K031/422; A61K 47/48 20060101 A61K047/48; A61K
31/5377 20060101 A61K031/5377 |
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 |
11163474.1 |
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. (canceled)
2. Binder-drug conjugates of the general formula (Ia) ##STR00820##
in which n is a number from 1 to 50, AK is AK.sub.1 or AK.sub.2
where AK.sub.1 is a binder which is bonded via a sulphur atom of
the binder to the group G, AK.sub.2 is a binder which is bonded via
a nitrogen atom of the binder to the group G, G when AK=AK.sub.1,
is a group of the formula ##STR00821## where #.sup.1 marks the
linkage site with the sulphur atom of the binder, #.sup.2 marks the
linkage site with the group L.sup.1, or when AK=AK.sub.2, is
carbonyl, L.sup.1 is a bond, linear (C.sub.1-C.sub.10)-alkanediyl,
or a group of the formula ##STR00822## where m is a number from 2
to 6, ##.sup.1 marks the linkage site with the group G, ##.sup.2
marks the linkage site with the group B, L.sup.1A is linear
(C.sub.2-C.sub.10)-alkanediyl, B.sup.1 is a group of the formula
##STR00823## in which ##.sup.5 marks the linkage site with the
group L.sup.1A, ##.sup.6 marks the linkage site with 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 of the formula ##STR00824## in which
##.sup.7 marks the linkage site with the carbonyl group, ##.sup.8
marks the linkage site with 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 bonded form a 5- or 6-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 bonded form a 5- or
6-membered heterocycle, L.sup.1B is linear
(C.sub.2-C.sub.10)-alkanediyl, and where
(C.sub.1-C.sub.10)-alkanediyl may be substituted by 1 to 4
substituents selected independently of one another from the group
consisting of methyl, hydroxy and benzyl, and where two carbon
atoms of the alkanediyl chain in 1,2, 1, 3 or 1,4 relation to one
another, with inclusion of any carbon atoms situated between them,
may be bridged 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 ##STR00825##
where * marks the linkage site with L.sup.1, ** marks the linkage
site with 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 ##STR00826## in which *** marks the linkage site with the
carbonyl group, **** marks the linkage site with 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 4- to 7-membered heterocycle,
Q.sup.2 is a 3- to 7-membered carbocycle or a 4- to 7-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 bonded form a 5-
or 6-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 bonded form a 5- or 6-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 of
its homologues 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 bonded 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 bonded form a 3- to 7-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
benzyl-oxycarbonyl, R.sup.37 is hydrogen or methyl, or R.sup.36 and
R.sup.37 together with the atoms to which they are bonded form a
pyrrolidine ring, L.sup.2 is linear (C.sub.2-C.sub.10)-alkanediyl
or is a group of the formula ##STR00827## where p is a number from
2 to 6, ##.sup.3 marks the linkage site with the group B, ##.sup.4
marks the linkage site with the nitrogen atom, where
(C.sub.2-C.sub.10)-alkanediyl may be substituted by 1 to 4
substituents selected independently of one another from the group
consisting of methyl, hydroxy and benzyl, and where two carbon
atoms of the alkanediyl chain in 1,2-, 1,3- or 1,4 relation to one
another, with inclusion of any carbon atoms situated between them,
may be bridged to form a (C.sub.3-C.sub.6)-cycloalkyl ring or a
phenyl ring, D is a group of the formula ##STR00828## in which
#.sup.3 marks the linkage site with 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
bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula ##STR00829## in which #.sup.4 marks the linkage site with
the adjacent nitrogen atom, #.sup.5 marks the linkage site with the
carbonyl group, the ring A with the N--O moiety present therein is
a mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR00830## in which #.sup.6 marks the linkage site with
the carbonyl group, R.sup.6 is hydrogen, hydroxy 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 bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR00831## in which #.sup.7 marks the linkage site with the
adjacent nitrogen atom, #.sup.8 marks the linkage site with 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, in which
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 bonded form a 4-
to 7-membered heterocycle, R.sup.10 is benzoyl, R.sup.11 is benzyl,
which may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, R.sup.5 is hydrogen, methyl or a group of the formula
##STR00832## in which #.sup.9 marks the linkage site with
--CHC(R.sup.26)-T.sup.2, R.sup.12 is phenyl which may be
substituted by methoxycarbonyl, carboxyl or a group of the formula
--S(O).sub.2OH, R.sup.13 is phenyl which may be substituted by
methoxycarbonyl or carboxyl, R.sup.26 is hydrogen or hydroxy,
T.sup.2 is phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
R.sup.35 is methyl or hydroxy, and also their salts, solvates and
solvates of the salts.
3. Binder-drug conjugates of the general formula (Ia) according to
claim 2, in which n is a number from 1 to 20, AK is AK.sub.1 or
AK.sub.2 where AK.sub.1 is an antibody or an antigen-binding
antibody fragment which binds to C4.4a and is bonded via the
sulphur atom of a cysteine residue of the binder to the group G,
AK.sub.2 is an antibody or an antigen-binding antibody fragment
which binds to C4.4a and is bonded via the NH side group of a
lysine residue of the binder to the group G, G when AK=AK.sub.1, is
a group of the formula ##STR00833## in which #.sup.1 marks the
linkage site with the cysteine residue of the binder, #.sup.2 marks
the linkage site with the group L.sup.1, or when AK=AK.sub.2, is
carbonyl, L.sup.1 is a bond, linear (C.sub.2-C.sub.6)-alkanediyl, a
group of the formula ##STR00834## where m is a number from 2 to 6,
##.sup.1 marks the linkage site with the group G, ##.sup.2 marks
the linkage site with the group B, L.sup.1A is linear
(C.sub.2-C.sub.6)-alkanediyl, B.sup.1 is a group of the formula
##STR00835## in which ##.sup.5 marks the linkage site with the
group L.sup.1A, ##.sup.6 marks the linkage site with the group
L.sup.1B, L.sup.5 is a bond, L.sup.6 is a bond or a group of the
formula ##STR00836## in which ##.sup.7 marks the linkage site with
the carbonyl group, ##.sup.8 marks the linkage site with 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
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 where
(C.sub.2-C.sub.6)-alkanediyl may be substituted by 1 or 2 methyl
substituents, B is a bond or a group of the formula ##STR00837##
where * marks the linkage site with L.sup.1, ** marks the linkage
site with 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 ##STR00838## in which *** marks
the linkage site with the carbonyl group, **** marks the linkage
site with L.sup.2, R.sup.25 is hydrogen or methyl, R.sup.28 is
hydrogen, methylcarbonyl or tert-butyloxycarbonyl, Q.sup.1 is a 4-
to 7-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 bonded 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 bonded 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 bonded form a cyclopropyl ring, R.sup.23 is
methyl, R.sup.24 is hydrogen or methyl, R.sup.27 is hydrogen,
R.sup.36 is hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
R.sup.37 is hydrogen or methyl, or R.sup.36 and R.sup.37 together
with the atoms to which they are bonded form a pyrrolidine ring,
L.sup.2 is linear (C.sub.2-C.sub.6)-alkanediyl or is a group of the
formula ##STR00839## where p is a number from 2 to 6, ##.sup.3
marks the linkage site with the group B, ##.sup.4 marks the linkage
site with the nitrogen atom, where (C.sub.2-C.sub.10)-alkanediyl
may be substituted by 1 or 2 methyl substituents, D is a group of
the formula ##STR00840## where #.sup.3 marks the linkage site with
the nitrogen atom, R.sup.1 is hydrogen, R.sup.2 is 1-hydroxyethyl,
benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl, or
R.sup.1 and R.sup.2 together with the carbon atom to which they are
bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula ##STR00841## in which #.sup.4 marks the linkage site with
the adjacent nitrogen atom, #.sup.5 marks the linkage site with the
carbonyl group, the ring A with the N--O moiety present therein is
a mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR00842## in which #.sup.6 marks the linkage site with
the carbonyl group, R.sup.6 is hydrogen, hydroxy or benzyloxy,
R.sup.3 is hydrogen, R.sup.4 is 1-hydroxyethyl, benzyl,
4-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 bonded
form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR00843## in which #.sup.7 marks the linkage site with the
adjacent nitrogen atom, #.sup.8 marks the linkage site with 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, in which
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 bonded form a 4-
to 7-membered heterocycle, R.sup.10 is benzoyl, R.sup.11 is benzyl,
which may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, R.sup.5 is hydrogen, methyl or a group of the formula
##STR00844## in which #.sup.9 marks the linkage site with
--CHC(R.sup.26)-T.sup.2, R.sup.12 is phenyl which may be
substituted by methoxycarbonyl, carboxyl or a group of the formula
--S(O).sub.2OH, R.sup.13 is phenyl which may be substituted by
methoxycarbonyl or carboxyl, R.sup.26 is hydrogen or hydroxy,
T.sup.2 is phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
R.sup.35 is methyl or hydroxy, and also their salts, solvates and
solvates of the salts.
4-6. (canceled)
7. Compounds of the formula (XXXa) ##STR00845## in which Cys is a
cysteine residue which is bonded via the sulphur atom of the side
chain to a carbon atom of the succinimide, L.sup.1 is a bond,
linear (C.sub.1-C.sub.10)-alkanediyl, or a group of the formula
##STR00846## where m is a number from 2 to 6, ##.sup.1 marks the
linkage site with the group G, ##.sup.2 marks the linkage site with
the group B, L.sup.1A is linear (C.sub.2-C.sub.10)-alkanediyl,
B.sup.1 is a group of the formula ##STR00847## in which ##.sup.5
marks the linkage site with the group L.sup.1A, ##.sup.6 marks the
linkage site with 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 bonded form a 5- or 6-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 bonded form a 5- or
6-membered heterocycle, L.sup.1B is linear
(C.sub.2-C.sub.10)-alkanediyl, and where
(C.sub.1-C.sub.10)-alkanediyl may be substituted by 1 to 4
substituents selected independently of one another from the group
consisting of methyl, hydroxy and benzyl, and where two carbon
atoms of the alkanediyl chain in 1,2, 1, 3 or 1,4 relation to one
another, with inclusion of any carbon atoms situated between them,
may be bridged 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 ##STR00848##
where * marks the linkage site with L.sup.1, ** marks the linkage
site with 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 4- to
7-membered heterocycle, Q.sup.2 is a 3- to 7-membered carbocycle or
a 4- to 7-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 bonded form a 5- or 6-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 bonded form a 5- or
6-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 of its homologues 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 bonded 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 bonded form a 3-
to 7-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 is a group of the formula
##STR00849## where p is a number from 2 to 6, ##.sup.3 marks the
linkage site with the group B, ##.sup.4 marks the linkage site with
the nitrogen atom, where (C.sub.2-C.sub.10)-alkanediyl may be
substituted by 1 to 4 substituents selected independently of one
another from the group consisting of methyl, hydroxy and benzyl,
and where two carbon atoms of the alkanediyl chain in 1,2-, 1,3- or
1,4 relation to one another, with inclusion of any carbon atoms
situated between them, may be bridged to form a
(C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring, D is a group of
the formula ##STR00850## where #.sup.3 marks the linkage site with
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 bonded form
a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR00851## in which #.sup.4 marks the linkage site with the
adjacent nitrogen atom, #.sup.5 marks the linkage site with the
carbonyl group, the ring A with the N--O moiety present therein is
a mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR00852## in which #.sup.6 marks the linkage site with
the carbonyl group, R.sup.6 is hydrogen, hydroxy 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 bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR00853## in which #.sup.7 marks the linkage site with the
adjacent nitrogen atom, #.sup.8 marks the linkage site with 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, in which
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 bonded form a 4-
to 7-membered heterocycle, R.sup.10 is benzoyl, R.sup.11 is benzyl,
which may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, R.sup.5 is hydrogen, methyl or a group of the formula
##STR00854## in which #.sup.9 marks the linkage site with
--CHC(R.sup.26)-T.sup.2, R.sup.12 is phenyl which may be
substituted by methoxycarbonyl, carboxyl or a group of the formula
--S(O).sub.2OH, R.sup.13 is phenyl which may be substituted by
methoxycarbonyl or carboxyl, R.sup.26 is hydrogen or hydroxy,
T.sup.2 is phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
R.sup.35 is methyl or hydroxy, and also their salts, solvates and
solvates of the salts.
8. Compounds of the formula (XXXa) according to claim 7, in which
Cys is a cysteine residue which is bonded via the sulphur atom of
the side chain to via a carbon atom of the succinimide, L.sup.1 is
a bond, linear (C.sub.2-C.sub.6)-alkanediyl, or a group of the
formula ##STR00855## where m is a number 2 or 3, ##.sup.1 marks the
linkage site with the group G, ##.sup.2 marks the linkage site with
the group B, L.sup.1A is linear (C.sub.2-C.sub.6)-alkanediyl,
B.sup.1 is a group of the formula ##STR00856## in which ##.sup.5
marks the linkage site with the group L.sup.A, ##.sup.6 marks the
linkage site with 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 where
(C.sub.2-C.sub.6)-alkanediyl may be substituted by 1 or 2 methyl
substituents, B is a bond or a group of the formula ##STR00857##
where * marks the linkage site with L.sup.1, ** marks the linkage
site with 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 bonded form
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 is a
group of the formula ##STR00858## where p is a number 2 or 3,
##.sup.3 marks the linkage site with the group B, ##.sup.4 marks
the linkage site with the nitrogen atom, D is a group of the
formula ##STR00859## where #.sup.3 marks the linkage site with the
nitrogen atom, R.sup.1 is hydrogen, R.sup.2 is 1-hydroxyethyl,
benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl, or
R.sup.1 and R.sup.2 together with the carbon atom to which they are
bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula ##STR00860## in which #.sup.4 marks the linkage site with
the adjacent nitrogen atom, #.sup.5 marks the linkage site with the
carbonyl group, the ring A with the N--O moiety present therein is
a mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR00861## in which #.sup.6 marks the linkage site with
the carbonyl group, R.sup.6 is hydrogen, hydroxy or benzyloxy,
R.sup.3 is hydrogen, R.sup.4 is 1-hydroxyethyl, benzyl,
4-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 bonded
form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR00862## in which #.sup.7 marks the linkage site with the
adjacent nitrogen atom, #.sup.8 marks the linkage site with 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, in which
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 bonded form a 4-
to 7-membered heterocycle, R.sup.10 is benzoyl, R.sup.11 is benzyl
which may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, R.sup.5 is hydrogen, methyl or a group of the formula
##STR00863## in which #.sup.9 marks the linkage site to
--CHCH.sub.2-phenyl, R.sup.12 is phenyl which may be substituted by
methoxycarbonyl, carboxyl or a group of the formula --S(O).sub.2OH,
R.sup.13 is phenyl which may be substituted by methoxycarbonyl or
carboxyl, R.sup.35 is methyl or hydroxy, and also their salts,
solvates and solvates of the salts.
9. Compounds of the formula (XXXa) according to claim 7, in which
Cys is a cysteine residue which is bonded via the sulphur atom of
the side chain to a carbon atom of the succinimide, 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 ##STR00864## where * marks the linkage site with
L.sup.1, ** marks the linkage site with 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 is a group of the formula ##STR00865## where p is a number 2 or
3, ##.sup.3 marks the linkage site with the group B, ##.sup.4 marks
the linkage site with the nitrogen atom, D is a group of the
formula ##STR00866## where #.sup.3 marks the linkage site with 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 bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR00867## in which #.sup.4 marks the linkage site with the
adjacent nitrogen atom, #.sup.5 marks the linkage site with the
carbonyl group, the ring A with the N--O moiety present therein is
a mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR00868## in which #.sup.6 marks the linkage site with
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 bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR00869## in which #.sup.7 marks the linkage site with the
adjacent nitrogen atom, #.sup.8 marks the linkage site with 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, in which
R.sup.7 is hydrogen, R.sup.8 is hydrogen, R.sup.9 is hydrogen,
R.sup.35 is methyl, and also their salts, solvates and solvates of
the salts.
10. Compounds of the formula (XXXI) ##STR00870## in which L.sup.1
is a bond, linear (C.sub.1-C.sub.10)-alkanediyl, or a group of the
formula ##STR00871## where m is a number from 2 to 6, ##.sup.1
marks the linkage site with the group G, ##.sup.2 marks the linkage
site with the group B, L.sup.1A is linear
(C.sub.2-C.sub.10)-alkanediyl, B.sup.1 is a group of the formula
##STR00872## in which ##.sup.5 marks the linkage site with the
group L.sup.1A, ##.sup.6 marks the linkage site with 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 bonded form a 5-
or 6-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 bonded form a 5- or 6-membered heterocycle,
L.sup.1B is linear (C.sub.2-C.sub.10)-alkanediyl, and where
(C.sub.1-C.sub.10)-alkanediyl may be substituted by 1 to 4
substituents selected independently of one another from the group
consisting of methyl, hydroxy and benzyl, and where two carbon
atoms of the alkanediyl chain in 1,2, 1, 3 or 1,4 relation to one
another, with inclusion of any carbon atoms situated between them,
may be bridged 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 ##STR00873##
where * marks the linkage site with L.sup.1, ** marks the linkage
site with L.sup.2, P is O or NH, Q.sup.1 is a 4- to 7-membered
heterocycle, Q.sup.2 is a 3- to 7-membered carbocycle or a 4- to
7-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 of its homologues 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 bonded 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 bonded form a 3-
to 7-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 is a group of the formula
##STR00874## where p is a number from 2 to 6, ##.sup.3 marks the
linkage site with the group B, ##.sup.4 marks the linkage site with
the nitrogen atom, where (C.sub.2-C.sub.10)-alkanediyl may be
substituted by 1 to 4 substituents selected independently of one
another from the group consisting of methyl, hydroxy and benzyl,
and where two carbon atoms of the alkanediyl chain in 1,2, 1, 3 or
1,4 relation to one another, with inclusion of any carbon atoms
situated between them, may be bridged to form a
(C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring, D is a group of
the formula ##STR00875## in which #.sup.3 marks the linkage site
with 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 bonded form
a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR00876## in which #.sup.4 marks the linkage site with the
adjacent nitrogen atom, #.sup.5 marks the linkage site with the
carbonyl group, the ring A with the N--O moiety present therein is
a mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR00877## in which #.sup.6 marks the linkage site with
the carbonyl group, R.sup.6 is hydrogen, hydroxy 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 bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR00878## in which #.sup.7 marks the linkage site with the
adjacent nitrogen atom, #.sup.8 marks the linkage site with 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, in which
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 bonded form a 4-
to 7-membered heterocycle, R.sup.10 is benzoyl, R.sup.11 is benzyl,
which may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, R.sup.5 is hydrogen, methyl or a group of the formula
##STR00879## #.sup.9 marks the linkage site with
--CHC(R.sup.26)-T.sup.2, R.sup.12 is phenyl which may be
substituted by methoxycarbonyl, carboxyl or a group of the formula
--S(O).sub.2OH, R.sup.13 is phenyl which may be substituted by
methoxycarbonyl or carboxyl, R.sup.26 is hydrogen or hydroxy,
T.sup.2 is phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
R.sup.35 is methyl or hydroxy, and also their salts, solvates and
solvates of the salts.
11. Compounds of the formula (XXXI) according to claim 10, in which
L.sup.1 is a bond, linear (C.sub.2-C.sub.6)-alkanediyl or a group
of the formula ##STR00880## where m is a number 2 or 3, ##.sup.1
marks the linkage site with the group G, ##.sup.2 marks the linkage
site with the group B, where (C.sub.2-C.sub.6)-alkanediyl may be
substituted by 1 or 2 methyl substituents, B is a bond or a group
of the formula ##STR00881## where * marks the linkage site with
L.sup.1, ** marks the linkage site with L.sup.2, R.sup.18 is
hydrogen, R.sup.19 is 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 bonded 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 bonded form
a cyclopropyl ring, R.sup.27 is hydrogen or methyl, L.sup.2 is
linear (C.sub.2-C.sub.6)-alkanediyl or is a group of the formula
##STR00882## where p is a number 2 or 3, ##.sup.3 marks the linkage
site with the group B, ##.sup.4 marks the linkage site with the
nitrogen atom, where (C.sub.2-C.sub.10)-alkanediyl may be
substituted by 1 or 2 methyl substituents, and where two carbon
atoms of the alkanediyl chain in 1,4 relation to one another, with
inclusion of any carbon atoms situated between them, may be bridged
to form a phenyl ring, D is a group of the formula ##STR00883## in
which #.sup.3 marks the linkage site with the nitrogen atom,
R.sup.1 is hydrogen, R.sup.2 is 1-hydroxyethyl, benzyl,
4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl, or R.sup.1
and R.sup.2 together with the carbon atom to which they are bonded
form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR00884## in which #.sup.4 marks the linkage site with the
adjacent nitrogen atom, #.sup.5 marks the linkage site with the
carbonyl group, the ring A with the N--O moiety present therein is
a mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR00885## in which #.sup.6 marks the linkage site with
the carbonyl group, R.sup.6 is hydrogen, hydroxy or benzyloxy,
R.sup.3 is hydrogen, R.sup.4 is 1-hydroxyethyl, benzyl,
4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl, or R.sup.3
and R.sup.4 together with the carbon atoms to which they are bonded
form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR00886## in which #.sup.7 marks the linkage site with the
adjacent nitrogen atom, #.sup.8 marks the linkage site with 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, in which
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 bonded form a 4-
to 7-membered heterocycle, R.sup.10 is benzoyl, R.sup.11 is benzyl,
which may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, R.sup.5 is hydrogen, methyl or a group of the formula
##STR00887## in which #.sup.9 marks the linkage site with
--CHCH.sub.2-phenyl, R.sup.12 is phenyl which may be substituted by
methoxycarbonyl, carboxyl or a group of the formula --S(O).sub.2OH,
R.sup.13 is phenyl which may be substituted by methoxycarbonyl or
carboxyl, R.sup.35 is methyl or hydroxy, and also their salts,
solvates and solvates of the salts.
12. Compounds of the formula (XXXI) according to claim 10, in which
L.sup.1 is a bond, B is a bond, L.sup.2 is linear
(C.sub.2-C.sub.6)-alkanediyl or is a group of the formula
##STR00888## where p is a number 2 or 3, ##.sup.3 marks the linkage
site with the group B, ##.sup.4 marks the linkage site with the
nitrogen atom, D is a group of the formula ##STR00889## where
#.sup.3 marks the linkage site with 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 bonded form
a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR00890## in which #.sup.4 marks the linkage site with the
adjacent nitrogen atom, #.sup.5 marks the linkage site with the
carbonyl group, the ring A with the N--O moiety present therein is
a mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR00891## in which #.sup.6 marks the linkage site with
the carbonyl group, R.sup.6 is hydrogen, hydroxy or benzyloxy,
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
bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula ##STR00892## in which #.sup.7 marks the linkage site with
the adjacent nitrogen atom, #.sup.8 marks the linkage site with 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, in which
R.sup.7 is hydrogen, R.sup.8 is hydrogen, R.sup.9 is hydrogen,
R.sup.35 is methyl, and also their salts, solvates and solvates of
the salts.
13. Compounds of the formulae (XXXa) ##STR00893## selected from the
group consisting of:
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulphanyl}-2,5-dioxopyrrolidin-1-yl-
)hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carbo-
xy-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]sulphanyl}-2,5-dioxopyrrolidin-1-yl-
)hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(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}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-meth-
yl-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,
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, and also their salts,
solvates and solvates of the salts.
14. (canceled)
15. Binder-drug conjugates of the general formula (Ia) according to
claim 2, in which n is a number from 1 to 50, AK is AK.sub.1 or
AK.sub.2 where AK.sub.1 is a binder which is bonded via a sulphur
atom of the binder to the group G, AK.sub.2 is a binder which is
bonded via a nitrogen atom of the binder to the group G, G when
AK=AK.sub.1, is a group of the formula ##STR00894## where #.sup.1
marks the linkage site with the sulphur atom of the binder, #.sup.2
marks the linkage site with the group L.sup.1, or when AK=AK.sub.2,
is carbonyl, L.sup.1 is a bond, linear
(C.sub.1-C.sub.10)-alkanediyl or is a group of the formula
##STR00895## where m is a number from 2 to 6, ##.sup.1 marks the
linkage site with the group G, ##.sup.2 marks the linkage site with
the group B, where (C.sub.1-C.sub.10)-alkanediyl may be substituted
by 1 to 4 methyl substituents, and where two carbon atoms of the
alkanediyl chain in 1,2, 1, 3 or 1,4 relation to one another, with
inclusion of any carbon atoms situated between them, may be bridged
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 ##STR00896## where * marks the
linkage site with L.sup.1, ** marks the linkage site with 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 ##STR00897## in which
*** marks the linkage site with the carbonyl group, **** marks the
linkage site with L.sup.2, R.sup.25 is hydrogen or methyl, Q.sup.1
is a 4- to 7-membered heterocycle, Q.sup.2 is a 3- to 7-membered
carbocycle or a 4- to 7-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 bonded form a 5- or 6-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 bonded form a 5- or
6-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 of its homologues 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 bonded 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 bonded form a 3-
to 7-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 is a group of the formula
##STR00898## where p is a number from 2 to 6, ##.sup.3 marks the
linkage site with the group B, ##.sup.4 marks the linkage site with
the nitrogen atom, where (C.sub.2-C.sub.10)-alkanediyl may be
substituted by 1 to 4 methyl substituents, and where two carbon
atoms of the alkanediyl chain in 1,2, 1, 3 or 1,4 relation to one
another, with inclusion of any carbon atoms situated between them,
may be bridged to form a (C.sub.3-C.sub.6)-cycloalkyl ring or a
phenyl ring, D is a group of the formula ##STR00899## where #.sup.3
marks the linkage site with 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 bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR00900## in which #.sup.4 marks the linkage site with the
adjacent nitrogen atom, #.sup.5 marks the linkage site with the
carbonyl group, the ring A with the N--O moiety present therein is
a mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR00901## in which #.sup.6 marks the linkage site with
the carbonyl group, R.sup.6 is hydrogen, hydroxy or benzyloxy,
R.sup.3 is hydrogen, R.sup.4 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 bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR00902## in which #.sup.7 marks the linkage site with the
adjacent nitrogen atom, #.sup.8 marks the linkage site with 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 in which
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 bonded form a 4-
to 7-membered heterocycle, R.sup.10 is benzoyl, R.sup.11 is benzyl,
which may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, R.sup.5 is hydrogen, methyl or a group of the formula
##STR00903## in which #.sup.9 marks the linkage site with
--CHC(R.sup.26)-T.sup.2, R.sup.12 is phenyl which may be
substituted by methoxycarbonyl, carboxyl or a group of the formula
--S(O).sub.2OH, R.sup.13 is phenyl which may be substituted by
methoxycarbonyl or carboxyl, R.sup.26 is hydrogen or hydroxy,
T.sup.2 is phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
and R.sup.35 is methyl, and also their salts, solvates and solvates
of the salts.
16. (canceled)
17. Binder-drug conjugates of the general formula (Ia) according to
claim 15, in which n is a number from 1 to 20, AK is AK.sub.1 or
AK.sub.2 where AK.sub.1 is an antibody or an antigen-binding
antibody fragment which binds to C4.4a and is bonded via the
sulphur atom of a cysteine residue of the binder to the group G,
AK.sub.2 is an antibody or an antigen-binding antibody fragment
which binds to C4.4a and is bonded via the NH side group of a
lysine residue of the binder to the group G, G when AK=AK.sub.1, is
a group of the formula ##STR00904## in which #.sup.1 marks the
linkage site with the cysteine residue of the binder, #.sup.2 marks
the linkage site with the group L.sup.1, or when AK=AK.sub.2, is
carbonyl, L.sup.1 is a bond, linear (C.sub.2-C.sub.6)-alkanediyl or
is a group of the formula ##STR00905## where m is a number from 2
to 6, ##.sup.1 marks the linkage site with the group G, ##.sup.2
marks the linkage site with the group B, where
(C.sub.2-C.sub.6)-alkanediyl may be substituted by 1 or 2 methyl
substituents, B is a bond or a group of the formula ##STR00906##
where * marks the linkage site with L.sup.1, ** marks the linkage
site with 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 ##STR00907## in which *** marks
the linkage site with the carbonyl group, **** marks the linkage
site with L.sup.2, R.sup.25 is methyl, Q.sup.1 is a 4- to
6-membered carbocycle or piperidine-1,4-diyl, 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 bonded 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 bonded 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 bonded form a cyclopropyl
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 is a group of the formula
##STR00908## where p is a number from 2 to 6, ##.sup.3 marks the
linkage site with the group B, ##.sup.4 marks the linkage site with
the nitrogen atom, where (C.sub.2-C.sub.6)-alkanediyl may be
substituted by 1 or 2 methyl substituents, D is a group of the
formula ##STR00909## where #.sup.3 marks the linkage site with 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 bonded form
a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR00910## in which #.sup.4 marks the linkage site with the
adjacent nitrogen atom, #.sup.5 marks the linkage site with the
carbonyl group, the ring A with the N--O moiety present therein is
a mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR00911## in which #.sup.6 marks the linkage site with
the carbonyl group, R.sup.6 is hydrogen, hydroxy 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 bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR00912## in which #.sup.7 marks the linkage site with the
adjacent nitrogen atom, #.sup.8 marks the linkage site with 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, in which
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 bonded form a 4-
to 7-membered heterocycle, R.sup.10 is benzoyl, R.sup.11 is benzyl,
which may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, R.sup.5 is hydrogen, methyl or a group of the formula
##STR00913## in which #.sup.9 marks the linkage site with
--CHC(R.sup.26)-T.sup.2, R.sup.12 is phenyl which may be
substituted by methoxycarbonyl, carboxyl or a group of the formula
--S(O).sub.2OH, R.sup.13 is phenyl which may be substituted by
methoxycarbonyl or carboxyl, R.sup.26 is hydrogen or hydroxy,
T.sup.2 is phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
and also their salts, solvates and solvates of the salts.
18. (canceled)
19. Process for preparing the compounds of the general formula (Ia)
according to claim 15, characterized in that a solution of the
binder in a buffer [A] is admixed with a suitable reducing agent,
such as, for example, dithiothreitol or
tris(2-carboxyethyl)phosphine hydrochloride, and is subsequently
reacted with a compound of the formula (II) ##STR00914## in which
D, L.sup.1, B and L.sup.2 each have the definitions indicated in
claim 15, to give a compound of the formula (I-A) ##STR00915## in
which n, AK.sub.1, D, L.sup.1, B and L.sup.2 each have the
definitions indicated in claim 15, or [B] is reacted with a
compound of the formula (III) ##STR00916## in which D, L.sup.1, B
and L.sup.2 each have the definitions indicated in claim 15, to
give a compound of the formula (I-B) ##STR00917## in which n,
AK.sub.2, D, L.sup.1, B and L.sup.2 each have the definitions
indicated in claim 15.
20. (canceled)
21. Compounds of the formula (XXX) ##STR00918## in which Cys is a
cysteine residue which is bonded via the sulphur atom of the side
chain to a carbon atom of the succinimide, L.sup.1 is a bond,
linear (C.sub.1-C.sub.10)-alkanediyl or is a group of the formula
##STR00919## where m is a number from 2 to 6, ##.sup.1 marks the
linkage site with the group G, ##.sup.2 marks the linkage site with
the group B, where (C.sub.1-C.sub.10)-alkanediyl may be substituted
by 1 to 4 methyl substituents, and where two carbon atoms of the
alkanediyl chain in 1,2, 1, 3 or 1,4 relation to one another, with
inclusion of any carbon atoms situated between them, may be bridged
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 ##STR00920## where * marks the
linkage site with L.sup.1, ** marks the linkage site with 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 ##STR00921## in which
*** marks the linkage site with the carbonyl group, **** marks the
linkage site with L.sup.2, R.sup.25 is hydrogen or methyl, Q.sup.1
is a 3- to 7-membered carbocycle or a 4- to 7-membered aza
heterocycle, Q.sup.2 is a 3- to 7-membered carbocycle or a 4- to
7-membered aza 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 bonded form a 5- or 6-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 bonded form a 5- or
6-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 of its homologues 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 bonded 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 bonded form a 3-
to 7-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 is a group of the formula
##STR00922## where p is a number from 2 to 6, ##.sup.3 marks the
linkage site with the group B, ##.sup.4 marks the linkage site with
the nitrogen atom, where (C.sub.2-C.sub.10)-alkanediyl may be
substituted by 1 to 4 methyl substituents, and where two carbon
atoms of the alkanediyl chain in 1,2, 1, 3 or 1,4 relation to one
another, with inclusion of any carbon atoms situated between them,
may be bridged to form a (C.sub.3-C.sub.6)-cycloalkyl ring or a
phenyl ring, D is a group of the formula ##STR00923## in which
#.sup.3 marks the linkage site with 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 bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR00924## in which #.sup.4 marks the linkage site with the
adjacent nitrogen atom, #.sup.5 marks the linkage site with the
carbonyl group, the ring A with the N--O moiety present therein is
a mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR00925## in which #.sup.6 marks the linkage site with
the carbonyl group, R.sup.6 is hydrogen, hydroxy 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 bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR00926## in which #.sup.7 marks the linkage site with the
adjacent nitrogen atom, #.sup.8 marks the linkage site with 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, in which
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 bonded form a 4-
to 7-membered heterocycle, R.sup.10 is benzoyl, R.sup.11 is benzyl,
which may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, R.sup.5 is hydrogen, methyl or a group of the formula
##STR00927## in which #.sup.9 marks the linkage site with
--CHC(R.sup.26)-T.sup.2, R.sup.12 is phenyl which may be
substituted by methoxycarbonyl, carboxyl or a group of the formula
--S(O).sub.2OH, R.sup.13 is phenyl which may be substituted by
methoxycarbonyl or carboxyl, R.sup.26 is hydrogen or hydroxy,
T.sup.2 is phenyl, benzyl, 1H-indol-3-yl or 1H-indol-3-ylmethyl,
and also their salts, solvates and solvates of the salts.
22. Compounds of the formula (XXX) according to claim 21, in which
Cys is a cysteine residue which is bonded via the sulphur atom of
the side chain to a carbon atom of the succinimide, L.sup.1 is a
bond, linear (C.sub.2-C.sub.6)-alkanediyl or is a group of the
formula ##STR00928## where m is a number from 2 to 6, ##.sup.1
marks the linkage site with the group G, ##.sup.2 marks the linkage
site with the group B, where (C.sub.2-C.sub.6)-alkanediyl may be
substituted by 1 or 2 methyl substituents, B is a bond or a group
of the formula ##STR00929## where * marks the linkage site with
L.sup.1, ** marks the linkage site with 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 bonded 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 is a group of the formula
##STR00930## where p is a number 2 or 3, ##.sup.3 marks the linkage
site with the group B, ##.sup.4 marks the linkage site with the
nitrogen atom, D is a group of the formula ##STR00931## in which
#.sup.3 marks the linkage site with 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 bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR00932## in which #.sup.4 marks the linkage site with the
adjacent nitrogen atom, #.sup.5 marks the linkage site with the
carbonyl group, the ring A with the N--O moiety present therein is
a mono- or bicyclic, optionally substituted heterocycle of the
formula ##STR00933## in which #.sup.6 marks the linkage site with
the carbonyl group, R.sup.6 is hydrogen, hydroxy 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 bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
##STR00934## in which #.sup.7 marks the linkage site with the
adjacent nitrogen atom, #.sup.8 marks the linkage site with 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, in which
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 by
methoxycarbonyl or carboxyl, R.sup.5 is hydrogen or a group of the
formula ##STR00935## in which #.sup.9 marks the linkage site with
--CHC(R.sup.26)phenyl, R.sup.12 is phenyl which may be substituted
by methoxycarbonyl, carboxyl or a group of the formula
--S(O).sub.2OH, R.sup.13 is phenyl which may be substituted by
methoxycarbonyl or carboxyl, and also their salts, solvates and
solvates of the salts.
23. Binder-drug conjugate according to claim 2, where the binder
binds to a cancer target molecule
24-28. (canceled)
29. Binder-drug conjugate according to claim 2, where the binder is
an antibody or an antigen-binding antibody fragment thereof or an
antibody mimetic.
30. Binder-drug conjugate according to claim 29, where the antibody
is a monoclonal antibody.
31. (canceled)
32. Binder-drug conjugate according to claim 29, where the antibody
is an intact or a modified intact antibody and wherein the antibody
is a human, humanized or chimeric antibody.
33. Binder-drug conjugate according to claim 29, where the antibody
is an antibody of the IgG class.
34-49. (canceled)
50. Process for preparing the compounds of the general formula (Ia)
according to claim 2, characterized in that a solution of the
binder in a buffer [A] is admixed with a suitable reducing agent,
such as, for example, dithiothreitol or
tris(2-carboxyethyl)phosphine hydrochloride, and is subsequently
reacted with a compound of the formula (IIa) ##STR00936## in which
D, L.sup.1, B, L.sup.2 and R.sup.35 each have the definitions
indicated in claim 2, to give a compound of the formula (Ia-A)
##STR00937## in which n, AK.sub.1, D, L.sup.1, B, L.sup.2 and
R.sup.35 each have the definitions indicated in claim 2, or [B] is
reacted with a compound of the formula (IIIa) ##STR00938## in which
D, L.sup.1, B, L.sup.2 and R.sup.35 each have the definitions
indicated in claim 2, to give a compound of the formula (Ia-B)
##STR00939## in which n, AK.sub.2, D, L.sup.1, B, L.sup.2 and
R.sup.35 each have the definitions indicated in claim 2.
51. Binder-drug conjugate according to claim 2 selected from the
following compounds: ##STR00940## ##STR00941## in which n is a
number 2 to 8, and AK.sub.1 is a binder which is bonded via a
cysteine residue to the toxophor-linker unit, AK.sub.2 is a binder
which is bonded via a lysine residue to the toxophor-linker
unit.
52. (canceled)
53. A medicament comprising the binder-drug conjugate of claim 2 in
combination with a pharmaceutically acceptable excipient.
54. The medicament of claim 53, further comprising one or more
anti-hyperproliferative, cytostatic, or cytotoxic substances.
55. A method for the treatment of cancer in a human or animal
subject comprising administering to said subject the binder-drug
conjugate of claim 2.
56. A method for the treatment of cancer in a human or animal
subject comprising administering to said subject the medicament of
claim 54.
Description
[0001] This application is a continuation-in-part of International
Application PCT/EP/2012/057245, filed Apr. 20, 2012, which claims
priority to EP11163472.1, filed Apr. 21, 2012, EP11168559.0, filed
Jun. 1, 2011, and EP11193609.2, filed Dec. 14, 2011. This
application is also a continuation-in-part of International
Application PCT/EP/2012/057243, filed Apr. 20, 2012, which claims
priority to EP11163470.5, filed June Apr. 21, 2011, EP11168558.2,
filed Jun. 1, 2011, and EP11193618.3, filed Dec. 14, 2011. This
application is also a continuation-in-part of International
Application PCT/EP/2012/057247, filed Apr. 20, 2012, which claims
priority to EP11163467.1, filed June Apr. 21, 2011, EP11168557.4,
filed Jun. 1, 2011, and EP11193621.7, filed Dec. 14, 2011. This
application is also a continuation-in-part of International
Application PCT/EP/2012/057249, filed Apr. 20, 2012, which claims
priority to EP11163474.7, filed June Apr. 21, 2011, EP11168556.6,
filed Jun. 1, 2011, and EP11193623.3, filed Dec. 14, 2011. Each of
the applications is incorporated by reference herein in its
entirety for all purposes.
[0002] The present application relates to new binder-drug
conjugates (ADCs) of N,N-dialkylauristatins that are directed
against the target C4.4a, to active metabolites of these ADCs, to
processes for preparing these ADCs, to the use of these ADCs for
treating and/or preventing illnesses, and also to the use of these
ADCs for producing medicaments for treating and/or preventing
illnesses, more particularly hyperproliferative and/or angiogenic
diseases such as, for example, cancer diseases. Such treatments may
be practised as a monotherapy or else in combination with other
medicaments or further therapeutic measures.
[0003] Cancer diseases are the consequence of uncontrolled cell
growth in a wide variety of tissues. In many cases the new cells
penetrate existing tissue (invasive growth), or they metastase into
remote organs. Cancer diseases occur in a wide variety of organs,
and the illnesses often progress in a tissue-specific manner. The
designation "cancer disease" as a generic term therefore describes
a large group of defined diseases of different organs, tissues and
cell types.
[0004] Early-stage tumours may be able to be removed by surgical
and radiotherapeutic measures. Metastasized tumours can generally
only be given palliative therapy by means of chemotherapeutic
agents. The objective in that case is to achieve the optimum
combination of improving quality of life and prolonging remaining
lifetime.
[0005] The majority of the chemotherapeutic agents which are
presently administered parenterally are often not target-directed
at the tumour tissue or the tumour cells, but instead, as a result
of their systemic administration, are distributed non-specifically
within the body, hence including at locations at which exposure to
the drug is undesirable, such as in healthy cells, tissues and
organs, for example. This may lead to unwanted side-effects and
even to serious effects of general toxicity, which then often
greatly limit the therapeutically useful dose range of the drug, or
necessitate complete cessation of medication.
[0006] The improved and selective availability of these
chemotherapeutic agents in the tumour cell or the immediately
surrounding tissue, and the associated boost in effect, on the one
hand, and minimization of toxic side-effects, on the other hand,
have therefore been a focal point for a number of years in the
development of new chemotherapeutic agents. Many attempts have been
made to date to develop efficient methods of introducing the drug
into the target cell. Optimizing the association between drug and
intracellular target and minimizing the intercellular distribution
of drug, to adjacent cells, for example, nevertheless continue to
constitute a difficult problem.
[0007] Monoclonal antibodies, for example, are suitable for the
target-directed addressing of tumour tissue and tumour cells. The
significance of such antibodies for the clinical treatment of
cancer diseases has seen a considerable general increase in recent
years, based on the activity of such agents as trastuzumab
(Herceptin), rituximab (Rituxan), cetuximab (Erbitux) and
bevacizumab (Avastin), which have since been approved for the
therapy of individual, specific tumour diseases [see e.g. G. P.
Adams and L. M. Weiner, Nat. Biotechnol. 23, 1147-1157 (2005)].
Consequently there has also been a marked increase in interest in
so-called immunoconjugates such as, for example, the aforementioned
ADCs, in which an internalizing antibody directed against a
tumour-associated antigen is joined covalently via a linking unit
("linker") to a cytotoxic agent. Following introduction of the ADC
into the tumour cell and subsequent cleavage of the conjugate,
either the cytotoxic agent itself or another metabolite with
cytotoxic activity, formed from the cytotoxic agent, is released
within the tumour cell, where it is able to develop its effect
directly and selectively. In this way it would be possible to keep
the damage to normal tissue within significantly closer limits in
comparison to a conventional chemotherapy of the cancer disease
[see e.g. 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)].
[0008] Instead of antibodies, it is also possible for binders from
the small-molecule drug sphere to be used as binders which bind
selectively to a specific target location ("target"), such as to a
receptor, for example [see e.g. E. Ruoslahti et al., Science 279,
377-380 (1998); D. Karkan et al., PLoS ONE 3 (6), e2469 (Jun. 25,
2008)]. Also known are conjugates of cytotoxic drug and addressing
ligand that exhibit a defined cleavage point between ligand and
drug for the release of the drug. A "predetermined break point" of
this kind may exist, for example, within a peptide chain which can
be cleaved selectively at a particular site by a specific enzyme at
the location of action [see e.g. R. A. Firestone and L. A. Telan,
US Patent Application US 2002/0147138].
[0009] Especially suitable for the target-directed addressing of
tumour tissue and tumour cells are monoclonal antibodies directed
against the antigen C4.4a. C4.4a (gene: LYPD3) was first described
as a metastasis-associated, cell surface protein in rat pancreas
tumour cells (Rosel M. et al., Oncogene 1998, 17(15):1989-2002).
Human C4.4a was isolated from its placental cDNA library (Wurfel,
J. et. al. Gene 2001, 262:35-41). C4.4a exhibits structural
homology with the uPA receptor and contains two LY6 domains, which
exhibit the typical three-finger folding pattern and are linked via
9 disulphide bridges (Jacobsen B. & Ploug M., Current Medicinal
Chemistry 2008, 15:2559-2573). C4.4a is anchored in the cell via
glycophosphatidylinositol (GPI). The protein is highly glycosylated
and contains numerous N- and O-glycosylation sites. C4.4a exhibits
strong expression in tumour cells of lung cancer, large bowel
cancer, breast cancer, ovarian cancer, pancreatic cancer, kidney
cancer, head-and-neck tumours and melanomas. RNA analyses have
shown C4.4a expression in .about.50% of primary pulmonary tumours
and .about.75% of lung cancer metastases, although expression in
healthy lung tissue was not detectable (Wurfel J. et. al., Gene
2001, 262:35-41). C4.4a can be used as a prognostic marker in
non-small-cell lung cancer--a high level of C4.4a expression
correlates with a poor prognosis (Hansen L. et al., Lung Cancer
2007, 58:260-266). The same is true for large bowel cancer. C4.4a
is cleaved off from the surface of the tumour cell and can be used
as a prognostic serum marker (K. Konishi et al., Cancer Science
2010). A detailed expression analysis of melanomas has shown that
C4.4a is expressed in .about.60% of primary malignant melanomas and
in 100% of lymph-node and skin metastases (Seiter S. et al., J
Invest Dermatol. 2001, 116(2):344-347). Upregulation of C4.4a gene
expression is observed in breast cancer tissue as compared with
adjacent normal tissues (Fletcher G. C., Br. J. Cancer 2003,
88(4):579-585). C4.4a is an ideal target protein for a tumour
therapy, since C4.4a expression in healthy tissues is confined to
skin keratinocytes and oesophageal endothelial cells, and also to
placenta cells (Wurfel J. et. al., Gene 2001, 262:35-41).
WO01/23553 describes the use of a C4.4a inhibitor (e.g. an
anti-C4.4a antibody) which in a cancer therapy is able to inhibit
C4.4a expression or activity.
[0010] The precise function of C4.4a is unknown. In the course of
wound healing, it is upregulated in migrating keratinocytes (Hansen
L. et al., Biochem J. 2004, 380:845-857). It is thought that C4.4a
plays a part in tumour cell invasion, presumably through
interaction with the extracellular matrix (Rosel M. et al.,
Oncogene 1998, 17(15):1989-2002; Paret C. et al., British Journal
of Cancer 2007, 97:1146-1156). Potential ligands are laminin 1 and
5, and also galectin 3 (Paret C., Int. J. Cancer 2005,
115:724-733).
[0011] Auristatin E (AE) and monomethylauristatin E (MMAE) are
synthetic analogues of the dolastatins, a specific group of linear
pseudopeptides which were originally isolated from marine sources
and which have in some cases very potent cytotoxic activity with
respect to tumour cells [for a review see e.g. 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##
[0012] MMAE, however, possesses the disadvantage of a comparatively
high systemic toxicity. For improving tumour selectivity, MMAE is
used more particularly in conjunction with enzymatically cleavable
valine-citrulline linkers in the ADC setting for more targeted
tumour therapy [WO 2005/081711-A2; S. O. Doronina et al.,
Bioconjugate Chem. 17, 114-124 (2006)]. Following proteolytic
cleavage, MMAE is released preferably intracellularly from
corresponding ADCs.
[0013] When employed in the form of antibody-drug conjugates
(ADCs), however, MMAE is not compatible with linking units
(linkers) between antibody and drug that do not have an
enzymatically cleavable predetermined break point [S. O. Doronina
et al., Bioconjugate Chem. 17, 114-124 (2006)].
[0014] Monomethylauristatin F (MMAF) is an auristatin derivative
having a C-terminal phenylalanine unit which exhibits only moderate
antiproliferative activity in comparison to MMAE. This fact is very
probably attributable to the free carboxyl group, whose polarity
and charge adversely affect the capacity of this compound to access
cells. In this connection, the methyl ester of MMAF (MMAF-OMe) has
been described, as a neutral-charged prodrug derivative with cell
access capability, which, in comparison to MMAF, has an in vitro
cytotoxicity for various carcinoma cell lines that is increased by
a number of orders of magnitude [S. O. Doronina et al.,
Bioconjugate Chem. 17, 114-124 (2006)]. It can be assumed that this
effect is brought about by MMAF itself, which, following uptake of
the prodrug into the cells, is rapidly released by intracellular
ester hydrolysis.
##STR00002##
[0015] However, drug compounds based on simple ester derivatives
are generally subject to the risk of chemical instability on
account of non-specific ester hydrolysis, independent of the
intended site of action, by means, for example, of esterases that
are present in the blood plasma; this non-specific hydrolysis may
significantly restrict the usefulness of such compounds in
therapy.
[0016] Monomethylauristatin F (MMAF) and also various ester
derivatives and amide derivatives thereof have been disclosed in WO
2005/081711-A2. Further auristatin analogues with a C-terminal,
amidically substituted phenylalanine unit are described in WO
01/18032-A2. WO 02/088172-A2 and WO 2007/008603-A1 claim MMAF
analogues which relate to side-chain modifications of the
phenylalanine, while WO 2007/008848-A2 claims those in which the
carboxyl group of the phenylalanine has been modified. Auristatin
conjugates linked via the C-terminus have been recently described
in WO 2009/117531-A1 [see also S. O. Doronina et al., Bioconjugate
Chem. 19, 1960-1963 (2008)].
[0017] Furthermore, auristatin derivatives such as MMAE and MMAF
are also substrates for transporter proteins which are expressed by
many tumour cells, and this may lead to the development of
resistance to these drugs.
[0018] The problem addressed with the present invention was that of
providing new binder-drug conjugates (ADCs) which, through
combination of new N,N-dialkylauristatin derivatives with
innovative, suitable linkers and binder, exhibit a very attractive
activity profile, such as, for example, in terms of their specific
tumour effect and/or the reduced potential of the metabolites
formed intracellularly to be a substrate with respect to
transporter proteins, and which are therefore suitable for the
treatment and/or prophylaxis of hyperproliferative and/or
angiogenic diseases, such as cancer diseases, for example.
[0019] The present invention provides binder-drug conjugates of the
general formula (Ia)
##STR00003## [0020] in which [0021] n is a number from 1 to 50,
[0022] AK is a binder, [0023] the group
.sctn.-G-L.sup.1-B-L.sup.2-.sctn..sctn. is a linker, [0024] where
[0025] marks the linkage site with the group AK and [0026]
.sctn..sctn. marks the linkage site with the nitrogen atom, [0027]
D is a group of the formula
[0027] ##STR00004## [0028] where [0029] #.sup.3 marks the linkage
site with the nitrogen atom, [0030] R.sup.1 is hydrogen or methyl,
[0031] 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,
[0032] or [0033] R.sup.1 and R.sup.2 together with the carbon atom
to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0033] ##STR00005## [0034] in which [0035] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [0036] #.sup.5 marks
the linkage site with the carbonyl group, [0037] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[0037] ##STR00006## [0038] in which [0039] #.sup.6 marks the
linkage site with the carbonyl group, [0040] R.sup.6 is hydrogen,
hydroxy or benzyloxy, [0041] R.sup.3 is hydrogen or methyl, [0042]
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, [0043] or [0044]
R.sup.3 and R.sup.4 together with the carbon atom to which they are
bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[0044] ##STR00007## [0045] in which [0046] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [0047] #.sup.8 marks
the linkage site with the group T.sup.1, [0048] 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, [0049] in
which [0050] R.sup.7 is hydrogen, methyl, ethyl, n-propyl,
tert-butyl, benzyl or adamantylmethyl, [0051] R.sup.8 is hydrogen
or methyl, [0052] R.sup.9 is hydrogen, methyl, ethyl, n-propyl or
benzyl, [0053] or [0054] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are bonded form a 4- to 7-membered
heterocycle, [0055] R.sup.10 is benzoyl, [0056] R.sup.11 is benzyl,
which may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, [0057] R.sup.5 is hydrogen, methyl or a group of the
formula
[0057] ##STR00008## [0058] in which [0059] #.sup.9 marks the
linkage site with --CHC(R.sup.26)-T.sup.2, [0060] R.sup.12 is
phenyl which may be substituted by methoxycarbonyl, carboxyl or a
group of the formula --S(O).sub.2OH, [0061] R.sup.13 is phenyl
which may be substituted by methoxycarbonyl or carboxyl, [0062]
R.sup.26 is hydrogen or hydroxy, [0063] T.sup.2 is phenyl, benzyl,
1H-indol-3-yl or 1H-indol-3-ylmethyl, [0064] R.sup.35 is methyl or
hydroxy, [0065] and also their salts, solvates and solvates of the
salts.
[0066] Compounds of the invention are the compounds of the formula
(Ia) and (I) and their salts, solvates and solvates of the salts,
the compounds of the formulae identified below and encompassed by
formula (Ia) and (I), and their salts, solvates and solvates of the
salts, and also the compounds identified below as working examples
and encompassed by formula (Ia) and (I), and their salts, solvates
and solvates of the salts, to the extent that the compounds
identified below and encompassed by formula (Ia) and (I) are not
already salts, solvates and solvates of the salts.
[0067] Depending on their structure, the compounds of the invention
may exist in different stereoisomeric forms, i.e. in the form of
configurational isomers or else where appropriate as conformational
isomers (enantiomers and/or diastereoisomers, including those in
the case of atropisomers). The present invention therefore
encompasses the enantiomers and diastereomers and their respective
mixtures. The stereoisomerically homogeneous constituents can be
isolated from such mixtures of enantiomers and/or diastereomers in
a known way; for this purpose it is preferred to use
chromatographic processes, more particularly HPLC chromatography on
an achiral or chiral phase.
[0068] Where the compounds of the invention can occur in tautomeric
forms, the present invention encompasses all of the tautomeric
forms.
[0069] The present invention also encompasses all suitable isotopic
variants of the compounds of the invention. An isotopic variant of
a compound of the invention is understood here to mean a compound
in which at least one atom within the compound of the invention has
been exchanged for another atom of the same atomic number but with
a different atomic mass from the atomic mass which occurs commonly
or predominantly in nature. Examples of isotopes which can be
incorporated into an inventive compound are those of hydrogen,
carbon, nitrogen, oxygen, phosphorus, sulphur, 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. Particular isotope variants of a compound of the
invention, such as more particularly those in which one or more
radioactive isotopes are incorporated, may be of benefit, for
example, for investigating the mechanism of action or the
distribution of drug in the body; owing to the comparative ease of
preparation and detectability, compounds labelled with .sup.3H or
.sup.14C isotopes are especially suitable for these purposes.
Furthermore, the incorporation of isotopes, such as of deuterium,
for example, may lead to certain therapeutic advantages as a
consequence of greater metabolic stability of the compound, such as
an extension to the half-life in the body or a reduction in the
active dose required, for example; such modifications of the
compounds of the invention may therefore, where appropriate, also
constitute a preferred embodiment of the present invention.
Isotopic variants of the compounds of the invention can be prepared
by the processes known to the skilled person, as for example in
accordance with the methods described later on below and the
procedures reproduced in the working examples, by using
corresponding isotopic modifications of the respective reagents
and/or starting compounds.
[0070] Preferred salts in the context of the present invention are
physiologically acceptable salts of the compounds of the invention.
Also encompassed are salts which although themselves not suitable
for pharmaceutical applications may nevertheless be used, for
example, for isolating or purifying the compounds of the
invention.
[0071] Physiologically acceptable salts of the compounds of the
invention encompass acid addition salts of mineral acids,
carboxylic acids and sulphonic acids, examples being salts of
hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric
acid, methanesulphonic acid, ethanesulphonic acid, benzenesulphonic
acid, toluenesulphonic acid, naphthalenedisulphonic acid, acetic
acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric
acid, malic acid, citric acid, fumaric acid, maleic acid and
benzoic acid.
[0072] Physiologically acceptable salts of the compounds of the
invention also encompass salts of customary bases, such as, by way
of example and preferably, alkali metal salts (e.g. sodium and
potassium salts), alkaline earth metal salts (e.g. calcium and
magnesium salts) and ammonium salts, derived from ammonia or
organic amines having 1 to 16 C atoms, such as, by way of example
and preferably, ethylamine, diethylamine, triethylamine,
ethyldiisopropylamine, monoethanolamine, diethanolamine,
triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine,
dibenzylamine, N-methylpiperidine, N-methylmorpholine, arginine,
lysine and 1,2-ethylenediamine.
[0073] Solvates in the context of the invention are those forms of
the compounds of the invention that form a complex in the solid or
liquid state through coordination with solvent molecules. Hydrates
are one specific form of solvates, in which the coordination takes
place with water. Preferred solvates in the context of the present
invention are hydrates.
[0074] Furthermore, the present invention also encompasses prodrugs
of the compounds of the invention. The term "prodrugs" here
identifies compounds which may themselves be biologically active or
inactive but are converted during their residence in the body into
compounds of the invention (by metabolism or hydrolysis, for
example).
[0075] In the context of the present invention the definitions of
the substituents, unless otherwise specified, are as follows:
[0076] (C.sub.1-C.sub.4)-Alkyl in the context of the invention is a
linear or branched alkyl radical having 1 to 4 carbon atoms. By way
of example and with preference, the following may be mentioned:
methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,
1-methylpropyl and tert-butyl.
[0077] Alkanediyl in the context of the invention is a linear,
.alpha.,.omega.-divalent alkyl radical having the particular number
of carbon atoms indicated. By way of example and of preference, the
following may be mentioned: 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).
[0078] (C.sub.3-C.sub.7)-Cycloalkyl and 3- to 7-membered carbocycle
respectively in the context of the invention is a monocyclic,
saturated cycloalkyl group having 3 to 7 carbon atoms. By way of
example and of preference, the following may be mentioned:
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and
cycloheptyl.
[0079] The side group of an .alpha.-amino acid in the definition of
R.sup.19 encompasses not only the side groups of the naturally
occurring .alpha.-amino acids but also the side groups of
homologues and isomers of these .alpha.-amino acids. The
.alpha.-amino acid here may be in the L or D configuration or else
may be present as a mixture of the L and D forms. Examples that may
be given of side groups are as follows: 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 definition of R.sup.19 are methyl (alanine),
propan-2-yl (valine), 2-methylpropan-1-yl (leucine), benzyl
(phenylalanine), imidazol-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.
[0080] A 4- to 7-membered heterocycle in the context of the
invention is a monocyclic, saturated heterocycle having a total of
4 to 7 ring atoms, which contains one or two ring heteroatoms from
the series N, O, S, SO and/or SO.sub.2 and is linked via a ring
carbon atom or optionally a ring nitrogen atom. Preference is given
to a 5- to 7-membered heterocycle having one or two ring
heteroatoms from the series N, O and/or S, more preferably a 5- or
6-membered heterocycle having one or two ring heteroatoms from the
series N and/or O. By way of example, the following may be
mentioned: azetidinyl, oxetanyl, pyrrolidinyl, pyrazolidinyl,
tetrahydrofuranyl, thiolanyl, piperidinyl, piperazinyl,
tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl,
thiomorpholinyl, hexahydroazepinyl and hexahydro-1,4-diazepinyl.
Preference is given to pyrrolidinyl, tetra-hydrofuranyl,
piperidinyl, piperazinyl, tetrahydropyranyl and morpholinyl.
[0081] In the formula of the group which may be represented by 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 R.sup.5, respectively, 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 or #.sup.9 is located is not a
carbon atom or a CH.sub.2 group, but instead is part of the bond to
the atom designated in each case, to which the 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 or R.sup.5 is
bonded.
[0082] In the context of the present invention, all radicals which
occur multiply have their definition independently of one another.
If radicals in the compounds of the invention are substituted, the
radicals, unless otherwise specified, may be substituted one or
more times. Substitution by one or by two identical or different
substituent(s) is preferred. Particularly preferred is substitution
by one substituent.
[0083] In the context of the present invention the terms used,
unless otherwise specified, have the following definitions:
[0084] The term "linker" is understood in the broadest sense as a
chemical unit which comprises a covalent bond or a series of atoms
that links a binder covalently to a drug. The term "linker" is
understood preferably as a series of atoms in the sense of the
present invention that links a binder covalently to a drug.
Furthermore, linkers may be represented, for example, by divalent
chemical units, such as alkyldiyls, aryldiyls, heteroaryldiyls,
heterocyclyldiyls, dicarbonyl acid esters, dicarbonyl acid
amides.
[0085] The term "binder" is understood in the broadest sense as a
molecule which binds to a target molecule which is present on a
particular target cell population to be addressed with the
binder-drug conjugate. The term "binder" should be understood in
its broadest interpretation and encompasses, for example, lectins,
proteins which are able to bind particular sugar chains, or
phospholipid-binding proteins. Such binders comprise, for example,
high molecular mass proteins (binding proteins), polypeptides or
peptides (binding peptides), non-peptidic (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. Binding proteins are, for
example, antibodies and antibody fragments or antibody mimetics
such as, for example, affibodies, adnectins, anticalins, DARPins,
avimers, nanobodies (review articles 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). Binding peptides
are, for example, ligands of a ligand-receptor pair, such as VEGF
in the ligand-receptor pair VEGF/KDR, such as transferrin of the
ligand-receptor pair transferrin/transferrin receptor, or
cytokines/cytokine receptor, such as TNFalpha in the ligand
receptor pair TNFalpha/TNFalpha receptor.
[0086] Preferred binders in accordance with the invention are (more
particularly human, monoclonal) antibodies or antigen-binding
antibody fragments which bind to C4.4a. In the case of anti-C4.4a
antibodies, n, in other words the number of toxophore molecules per
antibody molecule, is preferably in the range from 1 to 10, more
preferably 2 to 8.
[0087] 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). Preferably it
is a receptor or an antigen.
[0088] The term "extracellular" target molecule describes a target
molecule which is attached to the cell and which is located on the
outside of a cell or the part of a target molecule which is located
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. The skilled person knows of methods for identifying
extracellular target molecules. For proteins this may be done via
determination of the transmembrane domain(s) and the orientation of
the protein in the membrane. This data is generally recorded in
protein databases (e.g. SwissProt).
[0089] The term "cancer target molecule" describes a target
molecule which is multiply present on one or more cancer cell types
in comparison to non-cancer cells of the same tissue type. The
cancer target molecule is preferably present selectively on one or
more cancer cell types in comparison to non-cancer cells of the
same tissue type, with "selectively" describing an at least twofold
accumulation on cancer cells in comparison to non-cancer cells of
the same tissue type (a "selective cancer target molecule"). The
use of cancer target molecules allows selective therapy of cancer
cells with the conjugates of the invention.
[0090] The binder may be linked via a bond to the linker. Known
from the literature are various possibilities of covalent coupling
(conjugation) of organic molecules to antibody. The linking of the
binder may take place by means of a heteroatom of the binder.
Inventive heteroatoms of the binder that may be used for linking
are sulphur (in one embodiment via a sulphhydryl group of the
binder), oxygen (in accordance with the invention by means of a
carboxyl or hydroxy group of the binder) and nitrogen (in one
embodiment via a primary or secondary amine group or amide group of
the binder). Preferred in accordance with the invention is the
conjugation of the toxophores to the antibody via one or more
sulphur atoms of cysteine residues of the antibody and/or via one
or more NH groups of lysine residues of the antibody. These
heteroatoms may be present in the natural binder or may be
introduced by means of methods of chemistry or molecular biology.
In accordance with the invention, the linking of the binder to the
toxophore has little influence over the binding activity of the
binder to the target molecule. In a preferred embodiment the
linking has no influence on the binding activity of the binder to
the target molecule.
[0091] The term "antibody" is understood in accordance with the
present invention in its broadest sense and encompasses
immunoglobulin molecules, examples being 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
linked typically by disulphide bridges. Each heavy chain comprises
a variable domain of the heavy chain (abbreviated to VH) and a
constant domain of the heavy chain. The constant domain of the
heavy chain may encompass, for example, three domains CH1, CH2 and
CH3. Each light chain comprises a variable domain (abbreviated to
VL) and a constant domain. The constant domain of the light chain
comprises one domain (abbreviated to CL). The VH and VL domains may
be further subdivided into regions having hypervariability, also
called complementarity-determining regions (abbreviated to CDR),
and regions having a low sequence variability ("framework region",
abbreviated to FR). Each VH and VL region is typically composed of
three CDRs and up to four FRs. For example, in the following order
from the amino terminus to the carboxy terminus: FR1, CDR1, FR2,
CDR2, FR3, CDR3, FR4. An antibody may be obtained from any species
suitable for the antibody, such as, for example, rabbit, lama,
camel, mouse or rat. In one embodiment the antibody is of human or
murine origin. An antibody may for example be human, humanized or
chimeric.
[0092] The term "monoclonal" antibody identifies antibodies which
have been 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 small numbers. Monoclonal antibodies recognize a single
antigenic binding site with a high specificity. The term
"monoclonal antibody" does not refer to a particular production
method.
[0093] The term "intact" antibody refers to antibodies which
comprise not only an antigen-binding domain but also the constant
domain of the light and heavy chain. The constant domain may be a
naturally occurring domain, or a variant thereof in which one or
more amino acid positions have been altered.
[0094] The term "modified intact" antibody refers to intact
antibodies which have been fused with another polypeptide or
protein, not originating from an antibody, via the amino terminus
or carboxyl terminus thereof, by means of a covalent bond (e.g. a
peptide linkage). Furthermore, antibodies may be modified by
introducing reactive cysteines at defined locations, in order to
facilitate coupling to a toxophore (see Junutula et al. Nat.
Biotechnol. 2008 August; 26(8):925-32).
[0095] The term "human" antibody identifies antibodies which can be
obtained from a human being or are synthetic human antibodies. A
"synthetic" human antibody is an antibody which in parts or as a
whole is obtainable from synthetic sequences in silico which are
based on the analysis of human antibody sequences. A human antibody
may be encoded, for example, by a nucleic acid which has been
isolated from a library of antibody sequences which are of human
origin. One example of such antibodies can be found in Soderlind et
al., Nature Biotech. 2000, 18:853-856.
[0096] The term "humanized" or "chimeric" antibody describes
antibodies which consist of a non-human and of a human sequence
component. In these antibodies, part of the sequences of the human
immunoglobulin (recipient) is replaced by sequence components of a
non-human immunoglobulin (donor). In many cases the donor is a
murine immunoglobulin. With humanized antibodies, amino acids of
the CDR in the recipient are replaced by amino acids of the donor.
In some cases, amino acids of the framework as well are replaced by
corresponding amino acids of the donor. In some cases the humanized
antibody contains amino acids which were present neither in the
recipient nor in the donor and which were inserted during the
optimization of the antibody. In the case of chimeric antibodies,
for example, the variable domains of the donor immunoglobulin, or
else the entire Fab fraction, in other words VL-CL and VH+CH1, are
fused with the constant regions of a human antibody.
[0097] The term complementarity-determining region (CDR) as used
here refers to those amino acids in a variable antibody domain that
are necessary for binding to the antigen. Every variable region
typically has three CDR regions, identified as CDR1, CDR2 and CDR3.
Each CDR region may comprise amino acids according to the
definition of Kabat and/or amino acids of a hypervariable loop,
defined according to Chotia. The definition according to Kabat
encompasses, for example, the region of approximately amino acid
position 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 Immulological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)). The
definition according to Chotia encompasses, for example, the region
of approximately amino acid position 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 Chothia and
Lesk; J Mol Biol 196: 901-917 (1987)). In some cases a CDR may
comprise amino acids from one CDR region as defined by Kabat and
Chotia.
[0098] Depending on the amino acid sequence of the constant domain
of the heavy chain, antibodies may be divided into different
classes. There are five main classes of intact antibodies: IgA,
IgD, IgE, IgG and IgM, and a number of them may be broken down into
further subclasses (isotypes), e.g. IgG1, IgG2, IgG3, IgG4, IgA1
and IgA2. The constant domains of the heavy chain that correspond
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.
[0099] The term "functional fragment" or "antigen-binding antibody
fragments" of a antibody/immunoglobulin is defined as a fragment of
an antibody/immunoglobulin (e.g. the variable domains of an IgG)
which further encompasses the antigen binding domains of the
antibody/immunoglobulin. The "antigen binding domain" of an
antibody typically encompasses one or more hypervariable regions of
an antibody, e.g. the CDR1, CDR2 and/or CDR3 region. However, the
"framework" or "scaffold" region of an antibody may also play a
part with regard to the binding of the antibody to the antigen. The
framework region forms the scaffold for the CDRs. The
antigen-binding domain preferably encompasses at least amino acids
4 to 103 of the variable light chain and amino acid 5 to 109 of the
variable heavy chain, more preferably amino acid 3 to 107 of the
variable light chain and 4 to 111 of the variable heavy chain,
particular preference being given to the complete variable light
and heavy chains, i.e. amino acid 1-109 of the VL and 1 to 113 of
the VH (numbering according to WO97/08320).
[0100] "Functional fragments" or "antigen-binding antibody
fragments" of the invention encompass, non-conclusively, Fab, Fab',
F(ab').sub.2 and Fv fragments, diabodies, Single Domain Antibodies
(DAbs), linear antibodies, individual chains of antibodies
(single-chain Fv, abbreviated to ScFv); and multispecific
antibodies, such as bi and tri-specific antibodies, for example,
formed from antibody fragments C. A. K Borrebaeck, editor (1995)
Antibody Engineering (Breakthroughs in Molecular Biology), Oxford
University Press; R. Kontermann & S. Duebel, editors (2001)
Antibody Engineering (Springer Laboratory Manual), Springer
Verlag). Antibodies other than "multispecific" or "multifunctional"
antibodies are those having identical binding sites. Multispecific
antibodies may be specific for different epitopes of an antigen or
may be specific for epitopes of more than one antigen (see, for
example WO93/17715; 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). An F(ab').sub.2 or Fab molecule
may be constructed such that the number of intermolecular
disulphide interactions occurring between the Ch1 and the CL
domains can be reduced or else completely prevented.
[0101] "Functional fragments" or "antigen-binding antibody
fragments" may be fused with another polypeptide or protein, not
originating from an antibody, via the amino terminus or carboxyl
terminus thereof, by means of a covalent bond (e.g. a peptide
linkage). Furthermore, antibodies and antigen-binding fragments may
be modified by introducing reactive cysteines at defined locations,
in order to facilitate coupling to a toxophore (see Junutula et al.
Nat. Biotechnol. 2008 August; 26(8):925-32).
[0102] Polyclonal antibodies can be prepared by methods known to a
person of ordinary skill in the art. Monoclonal antibodies may be
prepared by methods known to a person of ordinary skill in the art
(Kohler and Milstein, Nature, 256, 495-497, 1975). Human and
humanized monoclonal antibodies may be prepared by methods known to
a person of ordinary skill in the art (Olsson et al., Meth Enzymol.
92, 3-16 or Cabilly et al U.S. Pat. No. 4,816,567 or Boss et al
U.S. Pat. No. 4,816,397).
[0103] A person of ordinary skill in the art is aware of diverse
methods for preparing human antibodies and fragments thereof, 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. 1991 Aug. 15;
352(6336):624-8). Antibodies of the invention may be obtained from
recombinant antibody libraries consisting for example of the amino
acid sequences of a multiplicity of antibodies compiled from a
large number of healthy volunteers. Antibodies may also be produced
by means of known recombinant DNA technologies. The nucleic acid
sequence of an antibody can be obtained by routine sequencing or is
available from publically accessible databases.
[0104] An "isolated" antibody or binder has been purified to remove
other constituents of the cell. Contaminating constituents of a
cell which may interfere with a diagnostic or therapeutic use are,
for example, enzymes, hormones, or other peptidic or non-peptidic
constituents of the cell. A preferred antibody or binder is one
which has been purified to an extent of more than 95%, relative to
the antibody or binder (determined for example by Lowry method,
UV-Vis spectroscopy or by SDS capillary gel electrophoresis), the
purification thereof being such that it is possible to determine at
least 15 amino acids of the amino terminus or of an internal amino
acid sequence, or which has been purified to homogeneity, the
homogeneity being determined by SDS-PAGE under reducing or
non-reducing conditions (detection may be determined by means of
Coomassie Blau staining or preferably by silver coloration).
However, an antibody is normally prepared by one or more
purification steps.
[0105] The term "specific binding" or "binds specifically" refers
to an antibody or binder which binds to a predetermined
antigen/target molecule. Specific binding of an antibody or binder
typically describes an antibody or binder having an affinity of at
least 10.sup.-7 M (as Kd value; i.e. preferably those with smaller
Kd values than 10.sup.-7 M), with the antibody or binder having an
at least two times higher affinity for the predetermined
antigen/target molecule than for a non-specific 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.
[0106] Antibodies which are specific against a cancer cell antigen
can be prepared by a person of ordinary skill in the art by means
of methods with which he or she is familiar (such as recombinant
expression, for example) or may be acquired commercially (as for
example from Merck KGaA, Germany). Examples of known commercially
available antibodies in cancer therapy are 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 in a cell-based
assay (Kd=5 nM) binds the extracellular domains of the human
epidermal growth receptor with high affinity. The antibody is
produced recombinantly in CHO cells.
[0107] The compounds of the formula (I) represent a subgroup of the
compounds of the formula (Ia).
[0108] A preferred subject of the invention are binder-drug
conjugates of the general formula (Ia) in which D is
##STR00009##
wherein the asterisks marks the linkage site with the nitrogen
atom; and the remainder of the variables are as defined.
[0109] A preferred subject of the invention are binder-drug
conjugates of the general formula (Ia) in which [0110] n is a
number from 1 to 50, [0111] AK is AK.sub.1 or AK.sub.2 [0112] where
[0113] AK.sub.1 is a binder (preferably an antibody or
antigen-binding antibody fragment (e.g., an anti-C4.4a antibody or
anti-C4.4a antigen-binding antibody fragment)) which is bonded via
a sulphur atom of the binder to the group G, [0114] AK.sub.2 is a
binder (preferably an antibody or antigen-binding antibody fragment
(e.g., an anti-C4.4a antibody or anti-C4.4a antigen-binding
antibody fragment)) which is bonded via a nitrogen atom of the
binder to the group G, [0115] G when AK=AK.sub.1, is a group of the
formula
[0115] ##STR00010## [0116] where [0117] #.sup.1 marks the linkage
site with the sulphur atom of the binder, [0118] #.sup.2 marks the
linkage site with the group L.sup.1, [0119] or [0120] when
AK=AK.sub.2, is carbonyl, [0121] L.sup.1 is a bond, linear
(C.sub.1-C.sub.10)-alkanediyl, a group of the formula
[0121] ##STR00011## [0122] where [0123] m is a number from 2 to 6,
[0124] ##.sup.1 marks the linkage site with the group G, [0125]
##.sup.2 marks the linkage site with the group B, [0126] L.sub.1A
is linear (C.sub.2-C.sub.10)-alkanediyl, [0127] B.sup.1 is a group
of the formula
[0127] ##STR00012## [0128] in which [0129] ##.sup.5 marks the
linkage site with the group L.sup.1A, [0130] ##.sup.6 marks the
linkage site with the group L.sup.1B, [0131] L.sup.5 is a bond or
(C.sub.2-C.sub.4)-alkanediyl, [0132] L.sup.6 is a bond or a group
of the formula
[0132] ##STR00013## [0133] in which [0134] ##.sup.7 marks the
linkage site with the carbonyl group, [0135] ##.sup.8 marks the
linkage site with L.sup.1B, [0136] R.sup.33 is hydrogen,
(C.sub.1-C.sub.4)-alkylcarbonyl, tert-butyloxycarbonyl or
benzyloxycarbonyl, [0137] R.sup.34 is hydrogen or methyl, [0138]
R.sup.29 is hydrogen or (C.sub.1-C.sub.4)-alkyl, [0139] R.sup.30 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, [0140] or [0141] R.sup.29 and
R.sup.30 together with the atoms to which they are bonded form a 5-
or 6-membered heterocycle, [0142] R.sup.31 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0143] R.sup.32 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0144] or [0145] R.sup.31 and R.sup.32
together with the atoms to which they are bonded form a 5- or
6-membered heterocycle, [0146] L.sup.1B is linear
(C.sub.2-C.sub.10)-alkanediyl, [0147] and [0148] where
(C.sub.1-C.sub.10)-alkanediyl may be substituted by 1 to 4
substituents selected independently of one another from the group
consisting of methyl, hydroxy and benzyl, [0149] and [0150] where
two carbon atoms of the alkanediyl chain in 1,2, 1, 3 or 1,4
relation to one another, with inclusion of any carbon atoms
situated between them, may be bridged to form a
(C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring, [0151] B is a
bond or a group of the formula
[0151] ##STR00014## [0152] where [0153] * marks the linkage site
with L.sup.1, [0154] ** marks the linkage site with L.sup.2, [0155]
P is O or NH, [0156] L.sup.3 is a bond or
(C.sub.2-C.sub.4)-alkanediyl, [0157] L.sup.4 is a bond or a group
of the formula
[0157] ##STR00015## [0158] in which [0159] *** marks the linkage
site with the carbonyl group, [0160] **** marks the linkage site
with L.sup.2, [0161] R.sup.25 is hydrogen or methyl, [0162]
R.sup.28 is hydrogen, (C.sub.1-C.sub.4)-alkylcarbonyl,
tert-butyloxycarbonyl or benzyloxycarbonyl, [0163] Q.sup.1 is a 4-
to 7-membered heterocycle, [0164] Q.sup.2 is a 3- to 7-membered or
a 4- to 7-membered heterocyle, [0165] R.sup.14 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0166] R.sup.15 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0167] or [0168] R.sup.14 and R.sup.15
together with the atoms to which they are bonded form a 5- or
6-membered heterocycle, [0169] R.sup.16 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0170] R.sup.17 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0171] or [0172] R.sup.16 and R.sup.17
together with the atoms to which they are bonded form a 5- or
6-membered heterocycle, [0173] R.sup.18 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0174] R.sup.19 is hydrogen or the side
group of a natural .alpha.-amino acid or of its homologues or
isomers, [0175] R.sup.20 is hydrogen or (C.sub.1-C.sub.4)-alkyl,
[0176] or [0177] R.sup.19 and R.sup.20 together with the atoms to
which they are bonded form a pyrrolidinyl ring, [0178] R.sup.21 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, [0179] R.sup.22 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0180] or [0181] R.sup.21 and R.sup.22
together with the atoms to which they are bonded form a 3- to
7-membered carbocycle, [0182] R.sup.23 is (C.sub.1-C.sub.4)-alkyl,
[0183] R.sup.24 is hydrogen or (C.sub.1-C.sub.4)-alkyl, [0184]
R.sup.27 is hydrogen or (C.sub.1-C.sub.4)-alkyl, [0185] R.sup.36 is
hydrogen, (C.sub.1-C.sub.4)-alkylcarbonyl, tert-butyloxycarbonyl or
benzyloxycarbonyl, [0186] R.sup.37 is hydrogen or methyl, [0187] or
[0188] R.sup.36 and R.sup.37 together with the atoms to which they
are bonded form a pyrrolidine ring, [0189] L.sup.2 is linear
(C.sub.2-C.sub.10)-alkanediyl or is a group of the formula
[0189] ##STR00016## [0190] where [0191] p is a number from 2 to 6,
[0192] ##.sup.3 marks the linkage site with the group B, [0193]
##.sup.4 marks the linkage site with the nitrogen atom, [0194]
where (C.sub.2-C.sub.10)-alkanediyl may be substituted by 1 to 4
substituents selected independently of one another from the group
consisting of methyl, hydroxy and benzyl, [0195] and [0196] where
two carbon atoms of the alkanediyl chain in 1,2, 1, 3 or 1,4
relation to one another, with inclusion of any carbon atoms
situated between them, may be bridged to form a
(C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring, [0197] D is a
group of the formula
[0197] ##STR00017## [0198] in which [0199] #.sup.3 marks the
linkage site with the nitrogen atom, [0200] R.sup.1 is hydrogen or
methyl, [0201] 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-phenyl-ethyl,
diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
[0202] or [0203] R.sup.1 and R.sup.2 together with the carbon atom
to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0203] ##STR00018## [0204] in which [0205] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [0206] #.sup.5 marks
the linkage site with the carbonyl group, [0207] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[0207] ##STR00019## [0208] in which [0209] #.sup.6 marks the
linkage site with the carbonyl group, [0210] R.sup.6 is hydrogen,
hydroxy or benzyloxy, [0211] R.sup.3 is hydrogen or methyl, [0212]
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-phenyl-ethyl, diphenylmethyl,
1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl, [0213] or [0214]
R.sup.3 and R.sup.4 together with the carbon atom to which they are
bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[0214] ##STR00020## [0215] in which [0216] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [0217] #.sup.8 marks
the linkage site with the group T.sup.1, [0218] 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, [0219] in
which [0220] R.sup.7 is hydrogen, methyl, ethyl, n-propyl,
tert-butyl, benzyl or adamantylmethyl, [0221] R.sup.8 is hydrogen
or methyl, [0222] R.sup.9 is hydrogen, methyl, ethyl, n-propyl or
benzyl, [0223] or [0224] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are bonded form a 4- to 7-membered
heterocycle, [0225] R.sup.10 is benzoyl, [0226] R.sup.11 is benzyl,
which may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, [0227] R.sup.5 is hydrogen, methyl or a group of the
formula
[0227] ##STR00021## [0228] in which [0229] #.sup.9 marks the
linkage site with --CHC(R.sup.26)-T.sup.2, [0230] R.sup.12 is
phenyl which may be substituted by methoxycarbonyl, carboxyl or a
group of the formula --S(O).sub.2OH, [0231] R.sup.13 is phenyl
which may be substituted by methoxycarbonyl or carboxyl, [0232]
R.sup.26 is hydrogen or hydroxy, [0233] T.sup.2 is phenyl, benzyl,
1H-indol-3-yl or 1H-indol-3-ylmethyl, [0234] R.sup.35 is methyl or
hydroxy, and also their salts, solvates and solvates of the
salts.
[0235] A preferred subject of the present invention are binder-drug
conjugates of the general formula (Ia) as indicated above, in which
[0236] n is a number from 1 to 50, [0237] AK is AK.sub.1 or
AK.sub.2 [0238] where [0239] AK.sub.1 is a binder (preferably an
antibody or antigen-binding antibody fragment (e.g., an anti-C4.4a
antibody or anti-C4.4a antigen-binding antibody fragment)) which is
bonded via a sulphur atom of the binder to the group G, [0240]
AK.sub.2 is a binder (preferably an antibody or antigen-binding
antibody fragment (e.g., an anti-C4.4a antibody or anti-C4.4a
antigen-binding antibody fragment)) which is bonded via a nitrogen
atom of the binder to the group G, [0241] G when AK=AK.sub.1, is a
group of the formula
[0241] ##STR00022## [0242] in which [0243] #.sup.1 marks the
linkage site with the sulphur atom of the binder, [0244] #.sup.2
marks the linkage site with the group L.sup.1, [0245] or [0246]
when AK=AK.sub.2, is carbonyl, [0247] L.sup.1 is a bond, linear
(C.sub.1-C.sub.10)-alkanediyl, a group of the formula
[0247] ##STR00023## [0248] where [0249] m is a number from 2 to 6,
[0250] ##.sup.1 marks the linkage site with the group G, [0251]
##.sup.2 marks the linkage site with the group B, [0252] L.sup.1A
is linear (C.sub.2-C.sub.10)-alkanediyl, [0253] B.sup.1 is a group
of the formula
[0253] ##STR00024## [0254] in which [0255] ##.sup.5 marks the
linkage site with the group L.sup.1A, [0256] ##.sup.6 marks the
linkage site with the group L.sup.1B, [0257] L.sup.5 is a bond or
(C.sub.2-C.sub.4)-alkanediyl, [0258] L.sup.6 is a bond or a group
of the formula
[0258] ##STR00025## [0259] in which [0260] ##.sup.7 marks the
linkage site with the carbonyl group, [0261] ##.sup.8 marks the
linkage site with L.sup.1B, [0262] R.sup.33 is hydrogen
(C.sub.1-C.sub.4)-alkylcarbonyl, tert-butyloxycarbonyl or
benzyloxycarbonyl, [0263] R.sup.34 is hydrogen or methyl, [0264]
R.sup.29 is hydrogen or (C.sub.1-C.sub.4)-alkyl, [0265] R.sup.30 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, [0266] or [0267] R.sup.29 and
R.sup.30 together with the atoms to which they are bonded form a 5-
or 6-membered heterocycle, [0268] R.sup.31 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0269] R.sup.32 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0270] or [0271] R.sup.31 and R.sup.32
together with the atoms to which they are bonded form a 5- or
6-membered heterocycle, [0272] L.sup.1B is linear
(C.sub.2-C.sub.10)-alkanediyl, [0273] and [0274] where
(C.sub.1-C.sub.10)-alkanediyl may be substituted by 1 to 4
substituents selected independently of one another from the group
consisting of methyl, hydroxy and benzyl, [0275] and [0276] where
two carbon atoms of the alkanediyl chain in 1,2, 1, 3 or 1,4
relation to one another, with inclusion of any carbon atoms
situated between them, may be bridged to form a
(C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring, [0277] B is a
bond or a group of the formula
[0277] ##STR00026## [0278] where [0279] * marks the linkage site
with L.sup.1, [0280] ** marks the linkage site with L.sup.2, [0281]
P is O or NH, [0282] L.sup.3 is a bond or
(C.sub.2-C.sub.4)-alkanediyl, [0283] L.sup.4 is a bond or a group
of the formula
[0283] ##STR00027## [0284] in which [0285] * * * marks the linkage
site with the carbonyl group, [0286] **** marks the linkage site
with L.sup.2, [0287] R.sup.25 is hydrogen or methyl, [0288]
R.sup.28 is hydrogen, (C.sub.1-C.sub.4)-alkylcarbonyl,
tert-butyloxycarbonyl or benzyloxycarbonyl, [0289] Q.sup.1 is a 4-
to 7-membered heterocycle, [0290] Q.sup.2 is a 3- to 7-membered
carbocycle or a 4- to 7-membered heterocycle, [0291] R.sup.14 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, [0292] R.sup.15 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0293] or [0294] R.sup.14 and R.sup.15
together with the atoms to which they are bonded form a 5- or
6-membered heterocycle, [0295] R.sup.16 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0296] R.sup.17 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0297] or [0298] R.sup.16 and R.sup.17
together with the atoms to which they are bonded form a 5- or
6-membered heterocycle, [0299] R.sup.18 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0300] R.sup.19 is hydrogen or the side
group of a natural .alpha.-amino acid or of its homologues or
isomers, [0301] R.sup.20 is hydrogen or (C.sub.1-C.sub.4)-alkyl,
[0302] or [0303] R.sup.19 and R.sup.20 together with the atoms to
which they are bonded form a pyrrolidinyl ring, [0304] R.sup.21 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, [0305] R.sup.22 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0306] or [0307] R.sup.21 and R.sup.22
together with the atoms to which they are bonded form a 3- to
7-membered carbocycle, [0308] R.sup.23 is (C.sub.1-C.sub.4)-alkyl,
[0309] R.sup.24 is hydrogen or (C.sub.1-C.sub.4)-alkyl, [0310]
R.sup.27 is hydrogen or (C.sub.1-C.sub.4)-alkyl, [0311] R.sup.36 is
hydrogen, (C.sub.1-C.sub.4)-alkylcarbonyl, tert-butyloxycarbonyl or
benzyloxycarbonyl, [0312] R.sup.37 is hydrogen or methyl, [0313] or
[0314] R.sup.36 and R.sup.37 together with the atoms to which they
are bonded form a pyrrolidine ring, [0315] L.sup.2 is linear
(C.sub.2-C.sub.10)-alkanediyl or is a group of the formula
[0315] ##STR00028## [0316] where [0317] p is a number from 2 to 6,
[0318] ##.sup.3 marks the linkage site with the group B, [0319]
##.sup.4 marks the linkage site with the nitrogen atom, [0320]
where (C.sub.2-C.sub.10)-alkanediyl may be substituted by 1 to 4
substituents selected independently of one another from the group
consisting of methyl, hydroxy and benzyl, [0321] and [0322] where
two carbon atoms of the alkanediyl chain in 1,2, 1, 3 or 1,4
relation to one another, with inclusion of any carbon atoms
situated between them, may be bridged to form a
(C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring, [0323] D is a
group of the formula
[0323] ##STR00029## [0324] in which [0325] #.sup.3 marks the
linkage site with the nitrogen atom, [0326] R.sup.1 is hydrogen or
methyl, [0327] 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-phenyl-ethyl,
diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
[0328] or [0329] R.sup.1 and R.sup.2 together with the carbon atom
to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0329] ##STR00030## [0330] in which [0331] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [0332] #.sup.5 marks
the linkage site with the carbonyl group, [0333] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[0333] ##STR00031## [0334] in which [0335] #.sup.6 marks the
linkage site with the carbonyl group, [0336] R.sup.6 is hydrogen,
hydroxy or benzyloxy, [0337] R.sup.3 is hydrogen or methyl, [0338]
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-phenyl-ethyl, diphenylmethyl,
1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl, [0339] or [0340]
R.sup.3 and R.sup.4 together with the carbon atom to which they are
bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[0340] ##STR00032## [0341] in which [0342] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [0343] #.sup.8 marks
the linkage site with the group T.sup.1, [0344] 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, [0345] in
which [0346] R.sup.7 is hydrogen, methyl, ethyl, n-propyl,
tert-butyl, benzyl or adamantylmethyl, [0347] R.sup.8 is hydrogen
or methyl, [0348] R.sup.9 is hydrogen, methyl, ethyl, n-propyl or
benzyl, [0349] or [0350] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are bonded form a 4- to 7-membered
heterocycle, [0351] R.sup.10 is benzoyl, [0352] R.sup.11 is benzyl,
which may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, [0353] R.sup.5 is hydrogen, methyl or a group of the
formula
[0353] ##STR00033## [0354] in which [0355] #.sup.9 marks the
linkage site with --CHC(R.sup.26)-T.sup.2, [0356] R.sup.12 is
phenyl which may be substituted by methoxycarbonyl, carboxyl or a
group of the formula --S(O).sub.2OH, [0357] R.sup.13 is phenyl
which may be substituted by methoxycarbonyl or carboxyl, [0358]
R.sup.26 is hydrogen or hydroxy, [0359] T.sup.2 is phenyl, benzyl,
1H-indol-3-yl or 1H-indol-3-ylmethyl, [0360] R.sup.35 is methyl or
hydroxy, and also their salts, solvates and solvates of the
salts.
[0361] Preferred subject of the invention are binder-drug
conjugates of the general formula (Ia), in which [0362] n is a
number from 1 to 20, [0363] AK is AK.sub.1 or AK.sub.2 [0364] Where
[0365] AK.sub.1 is an antibody or an antigen-binding antibody
fragment and is bonded via the sulphur atom of a cysteine residue
of the binder to the group G (e.g., an antibody or an
antigen-binding antibody fragment which binds to C4.4a and is
bonded via the sulphur atom of a cysteine residue of the binder to
the group G), [0366] AK.sub.2 is an antibody or an antigen-binding
antibody fragment and is bonded via the NH side group of a lysine
residue of the binder to the group G (e.g., an antibody or an
antigen-binding antibody fragment which binds to C4.4a and is
bonded via the NH side group of a lysine residue of the binder to
the group G), [0367] G when AK=AK.sub.1, is a group of the
formula
[0367] ##STR00034## [0368] in which [0369] #.sup.1 marks the
linkage site with the cysteine residue of the binder, [0370]
#.sup.2 marks the linkage site with the group L.sup.1, [0371] or
[0372] when AK=AK.sub.2, is carbonyl, [0373] L.sup.1 is a bond,
linear (C.sub.2-C.sub.6)-alkanediyl, a group of the formula
[0373] ##STR00035## [0374] where [0375] m is a number from 2 to 6,
[0376] ##.sup.1 marks the linkage site with the group G, [0377]
##.sup.2 marks the linkage site with the group B, [0378] L.sup.1A
is linear (C.sub.2-C.sub.6)-alkanediyl, [0379] B.sup.1 is a group
of the formula
[0379] ##STR00036## [0380] in which [0381] ##.sup.5 marks the
linkage site with the group L.sup.1A, [0382] ##.sup.6 marks the
linkage site with the group L.sup.1B, [0383] L.sup.5 is a bond,
[0384] L.sup.6 is a bond or a group of the formula
[0384] ##STR00037## [0385] in which [0386] ##.sup.7 marks the
linkage site with the carbonyl group, [0387] ##.sup.8 marks the
linkage site with L.sup.1B, [0388] R.sup.33 is hydrogen,
methylcarbonyl or tert-butyloxycarbonyl, [0389] R.sup.34 is
hydrogen or methyl, [0390] R.sup.29 is hydrogen, [0391] R.sup.36 is
hydrogen, [0392] R.sup.31 is hydrogen or methyl, [0393] R.sup.32 is
hydrogen or methyl, [0394] L.sup.1B is linear
(C.sub.2-C.sub.6)-alkanediyl, [0395] and [0396] where
(C.sub.2-C.sub.6)-alkanediyl may be substituted by 1 or 2 methyl
substituents, [0397] B is a bond or a group of the formula
[0397] ##STR00038## [0398] where [0399] * marks the linkage site
with L.sup.1, [0400] ** marks the linkage site with L.sup.2, [0401]
L.sup.3 is a bond or ethane-1,2-diyl, [0402] L.sup.4 is a bond or a
group of the formula
[0402] ##STR00039## [0403] in which [0404] *** marks the linkage
site with the carbonyl group, [0405] **** marks the linkage site
with L.sup.2, [0406] R.sup.25 is hydrogen or methyl, [0407]
R.sup.28 is hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
[0408] Q.sup.1 is a 4- to 7-membered heterocycle, [0409] R.sup.14
is hydrogen, [0410] R.sup.15 is hydrogen, [0411] R.sup.16 is
hydrogen or methyl, [0412] R.sup.17 is hydrogen or methyl, [0413]
or [0414] R.sup.16 and R.sup.17 together with the atoms to which
they are bonded form a piperazinyl ring, [0415] R.sup.18 is
hydrogen, [0416] R.sup.19 is hydrogen, methyl, propan-2-yl,
2-methylpropan-1-yl or 1-methylpropan-1-yl, [0417] R.sup.20 is
hydrogen or methyl, [0418] or [0419] R.sup.19 and R.sup.20 together
with the atoms to which they are bonded form a pyrrolidinyl ring,
[0420] R.sup.21 is hydrogen or methyl, [0421] R.sup.22 is hydrogen
or methyl, [0422] or [0423] R.sup.21 and R.sup.22 together with the
atoms to which they are bonded form a cyclopropyl ring, [0424]
R.sup.23 is methyl, [0425] R.sup.24 is hydrogen or methyl, [0426]
R.sup.27 is hydrogen, [0427] R.sup.36 is hydrogen, methylcarbonyl
or tert-butyloxycarbonyl, [0428] R.sup.37 is hydrogen or methyl,
[0429] or [0430] R.sup.36 and R.sup.37 together with the atoms to
which they are bonded form a pyrrolidine ring, [0431] L.sup.2 is
linear (C.sub.2-C.sub.6)-alkanediyl or is a group of the
formula
[0431] ##STR00040## [0432] where [0433] p is a number from 2 to 6,
[0434] ##.sup.3 marks the linkage site with the group B, [0435]
##.sup.4 marks the linkage site with the nitrogen atom, [0436]
where (C.sub.2-C.sub.10)-alkanediyl may be substituted by 1 or 2
methyl substituents, [0437] D is a group of the formula
[0437] ##STR00041## [0438] where [0439] #.sup.3 marks the linkage
site with the nitrogen atom, [0440] R.sup.1 is hydrogen, [0441]
R.sup.2 is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl
or 1H-indol-3-ylmethyl, [0442] or [0443] R.sup.1 and R.sup.2
together with the carbon atom to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0443] ##STR00042## [0444] in which [0445] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [0446] #.sup.5 marks
the linkage site with the carbonyl group, [0447] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[0447] ##STR00043## [0448] in which [0449] #.sup.6 marks the
linkage site with the carbonyl group, [0450] R.sup.6 is hydrogen,
hydroxy or benzyloxy, [0451] R.sup.3 is hydrogen, [0452] R.sup.4 is
1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [0453] or [0454] R.sup.3 and R.sup.4 together
with the carbon atom to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0454] ##STR00044## [0455] in which [0456] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [0457] #.sup.8 marks
the linkage site with the group T.sup.1, [0458] 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, [0459] in
which [0460] R.sup.7 is hydrogen, methyl, ethyl, n-propyl,
tert-butyl, benzyl or adamantylmethyl, [0461] R.sup.8 is hydrogen
or methyl, [0462] R.sup.9 is hydrogen, methyl, ethyl, n-propyl or
benzyl, [0463] or [0464] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are bonded form a 4- to 7-membered
heterocycle, [0465] R.sup.10 is benzoyl, [0466] R.sup.11 is benzyl,
which may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, [0467] R.sup.5 is hydrogen, methyl or a group of the
formula
[0467] ##STR00045## [0468] in which [0469] #.sup.9 marks the
linkage site with --CHC(R.sup.26)-T.sup.2, [0470] R.sup.12 is
phenyl which may be substituted by methoxycarbonyl, carboxyl or a
group of the formula --S(O).sub.2OH, [0471] R.sup.13 is phenyl
which may be substituted by methoxycarbonyl or carboxyl, [0472]
R.sup.26 is hydrogen or hydroxy, [0473] T.sup.2 is phenyl, benzyl,
1H-indol-3-yl or 1H-indol-3-ylmethyl, [0474] R.sup.35 is methyl or
hydroxy, and also their salts, solvates and solvates of the
salts.
[0475] Preferred subject matter of the present invention are
binder-drug conjugates of the general formula (Ia) as indicated
above, in which [0476] n is a number from 1 to 20, [0477] AK is
AK.sub.1 or AK.sub.2 [0478] where [0479] AK.sub.1 is an antibody or
an antigen-binding antibody fragment and is bonded via the sulphur
atom of a cysteine residue of the binder to the group G (e.g., an
antibody or an antigen-binding antibody fragment which binds to
C4.4a and is bonded via the sulphur atom of a cysteine residue of
the binder to the group G), [0480] AK.sub.2 is an antibody or an
antigen-binding antibody fragment and is bonded via the NH side
group of a lysine residue of the binder to the group G (e.g., an
antibody or an antigen-binding antibody fragment which binds to
C4.4a and is bonded via the NH side group of a lysine residue of
the binder to the group G) [0481] G when AK=AK.sub.1, is a group of
the formula
[0481] ##STR00046## [0482] in which [0483] #.sup.1 marks the
linkage site with the cysteine residue of the binder, [0484]
#.sup.2 marks the linkage site with the group L.sup.1, [0485] or
[0486] when AK=AK.sub.2, is carbonyl, [0487] L.sup.1 is a bond,
linear (C.sub.2-C.sub.6)-alkanediyl, a group of the formula
[0487] ##STR00047## [0488] where [0489] m is a number from 2 to 6,
[0490] ##.sup.1 marks the linkage site with the group G, [0491]
##.sup.2 marks the linkage site with the group B, [0492] L.sup.1A
is linear (C.sub.2-C.sub.6)-alkanediyl, [0493] B.sup.1 is a group
of the formula
[0493] ##STR00048## [0494] in which [0495] ##.sup.5 marks the
linkage site with the group L.sup.1A, [0496] ##.sup.6 marks the
linkage site with the group L.sup.1B, [0497] L.sup.5 is a bond,
[0498] L.sup.6 is a bond or a group of the formula
[0498] ##STR00049## [0499] in which [0500] ##.sup.7 marks the
linkage site with the carbonyl group, [0501] ##.sup.8 marks the
linkage site with L.sup.1B, [0502] R.sup.33 is hydrogen,
methylcarbonyl or tert-butyloxycarbonyl, [0503] R.sup.34 is
hydrogen or methyl, [0504] R.sup.29 is hydrogen, [0505] R.sup.30 is
hydrogen, [0506] R.sup.31 is hydrogen or methyl, [0507] R.sup.32 is
hydrogen or methyl, [0508] L.sup.1B is linear
(C.sub.2-C.sub.6)-alkanediyl, [0509] and [0510] where
(C.sub.2-C.sub.6)-alkanediyl may be substituted by 1 or 2 methyl
substituents, [0511] B is a bond or a group of the formula
[0511] ##STR00050## [0512] where [0513] * marks the linkage site
with L.sup.1, [0514] ** marks the linkage site with L.sup.2, [0515]
L.sup.3 is a bond or ethane-1,2-diyl, [0516] L.sup.4 is a bond or a
group of the formula
[0516] ##STR00051## [0517] in which [0518] *** marks the linkage
site with the carbonyl group, [0519] **** marks the linkage site
with L.sup.2, [0520] R.sup.25 is hydrogen or methyl, [0521]
R.sup.28 is hydrogen, methylcarbonyl or tert-butyloxycarbonyl,
[0522] Q.sup.1 is a 4- to 7-membered heterocycle, [0523] R.sup.14
is hydrogen, [0524] R.sup.15 is hydrogen, [0525] R.sup.16 is
hydrogen or methyl, [0526] R.sup.17 is hydrogen or methyl, [0527]
or [0528] R.sup.16 and R.sup.17 together with the atoms to which
they are bonded form a piperazinyl ring, [0529] R.sup.18 is
hydrogen, [0530] R.sup.19 is hydrogen, methyl, propan-2-yl,
2-methylpropan-1-yl or 1-methylpropan-1-yl, [0531] R.sup.20 is
hydrogen or methyl, [0532] or [0533] R.sup.19 and R.sup.20 together
with the atoms to which they are bonded form a pyrrolidinyl ring,
[0534] R.sup.21 is hydrogen or methyl, [0535] R.sup.22 is hydrogen
or methyl, [0536] or [0537] R.sup.21 and R.sup.22 together with the
atoms to which they are bonded form a cyclopropyl ring, [0538]
R.sup.23 is methyl, [0539] R.sup.24 is hydrogen or methyl, [0540]
R.sup.27 is hydrogen, [0541] R.sup.36 is hydrogen,
(C.sub.1-C.sub.4)-alkylcarbonyl, tert-butyloxycarbonyl, or
benzyloxycarbonyl, [0542] R.sup.37 is hydrogen or methyl, [0543]
L.sup.2 is linear (C.sub.2-C.sub.6)-alkanediyl or is a group of the
formula
[0543] ##STR00052## [0544] where [0545] p is a number from 2 to 6,
[0546] ##.sup.3 marks the linkage site with the group B, [0547]
##.sup.4 marks the linkage site with the nitrogen atom, [0548]
where (C.sub.2-C.sub.10)-alkanediyl may be substituted by 1 or 2
methyl substituents, [0549] D is a group of the formula
[0549] ##STR00053## [0550] where [0551] #.sup.3 marks the linkage
site with the nitrogen atom, [0552] R.sup.1 is hydrogen, [0553]
R.sup.2 is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl
or 1H-indol-3-ylmethyl, [0554] or [0555] R.sup.1 and R.sup.2
together with the carbon atom to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0555] ##STR00054## [0556] in which [0557] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [0558] #.sup.5 marks
the linkage site with the carbonyl group, [0559] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[0559] ##STR00055## [0560] in which [0561] #.sup.6 marks the
linkage site with the carbonyl group, [0562] R.sup.6 is hydrogen,
hydroxy or benzyloxy, [0563] R.sup.3 is hydrogen, [0564] R.sup.4 is
1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [0565] or [0566] R.sup.3 and R.sup.4 together
with the carbon atom to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0566] ##STR00056## [0567] in which [0568] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [0569] #.sup.8 marks
the linkage site with the group T.sup.1, [0570] 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, [0571] in
which [0572] R.sup.7 is hydrogen, methyl, ethyl, n-propyl,
tert-butyl, benzyl or adamantylmethyl, [0573] R.sup.8 is hydrogen
or methyl, [0574] R.sup.9 is hydrogen, methyl, ethyl, n-propyl or
benzyl, [0575] or [0576] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are bonded form a 4- to 7-membered
heterocycle, [0577] R.sup.10 is benzoyl, [0578] R.sup.11 is benzyl,
which may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, [0579] R.sup.5 is hydrogen, methyl or a group of the
formula
[0579] ##STR00057## [0580] in which [0581] #.sup.9 marks the
linkage site with --CHC(R.sup.26)-T.sup.2, [0582] R.sup.12 is
phenyl which may be substituted by methoxycarbonyl, carboxyl or a
group of the formula --S(O).sub.2OH, [0583] R.sup.13 is phenyl
which may be substituted by methoxycarbonyl or carboxyl, [0584]
R.sup.26 is hydrogen or hydroxy, [0585] T.sup.2 is phenyl, benzyl,
1H-indol-3-yl or 1H-indol-3-ylmethyl, [0586] R.sup.35 is methyl or
hydroxy, and also their salts, solvates and solvates of the
salts.
[0587] Preferred subject matter of the invention are binder-drug
conjugates of the general formula (Ia), in which [0588] n is a
number from 1 to 10, [0589] AK is AK.sub.1 or AK.sub.2 [0590] where
[0591] AK.sub.1 is an antibody or antigen-binding antibody fragment
(e.g., an antibody which comprises the six CDR sequences of the
antibody B01-3, B01-10, M31-B01 or D02-6, the variable light and
variable heavy chain of the antibody B01-3, B01-10, M31-B01 or
D02-6 or the light and heavy chain of the antibody B01-3, B01-10,
M31-B01 or D02-6), and is bonded via the sulphur atom of a cysteine
residue of the binder to the group G, [0592] AK.sub.2 is an
antibody or antigen-binding antibody fragment (e.g., an antibody
which comprises the six CDR sequences of the antibody B01-3,
B01-10, M31-B01 or D02-6, the variable light and variable heavy
chain of the antibody B01-3, B01-10, M31-B01 or D02-6 or the light
and heavy chain of the antibody B01-3, B01-10, M31-B01 or D02-6),
and is bonded via the NH side group of a lysine residue of the
binder to the group G, [0593] G when AK=AK.sub.1, is a group of the
formula
[0593] ##STR00058## [0594] in which [0595] #.sup.1 marks the
linkage site with the cysteine residue of the binder, [0596]
#.sup.2 marks the linkage site with the group L.sup.1, [0597] or
[0598] when AK=AK.sub.2, is carbonyl, [0599] L.sup.1 is a bond,
linear (C.sub.2-C.sub.6)-alkanediyl, a group of the formula
[0599] ##STR00059## [0600] where [0601] m is a number 2 or 3,
[0602] ##.sup.1 marks the linkage site with the group G, [0603]
##.sup.2 marks the linkage site with the group B, [0604] where
(C.sub.2-C.sub.6)-alkanediyl may be substituted by 1 or 2 methyl
substituents, [0605] B is a bond or a group of the formula
[0605] ##STR00060## [0606] where [0607] * marks the linkage site
with L.sup.1, [0608] ** marks the linkage site with L.sup.2, [0609]
L.sup.3 is a bond or ethane-1,2-diyl, [0610] L.sup.4 is a bond or a
group of the formula
[0610] ##STR00061## [0611] in which [0612] * * * marks the linkage
site with the carbonyl group, [0613] **** marks the linkage site
with L.sup.2, [0614] R.sup.25 is methyl, [0615] R.sup.28 is
hydrogen, methylcarbonyl or tert-butyloxycarbonyl, [0616] Q.sup.1
is piperidine-1,4-diyl, [0617] R.sup.16 is hydrogen or methyl,
[0618] R.sup.17 is hydrogen or methyl, [0619] or [0620] R.sup.16
and R.sup.17 together with the atoms to which they are bonded form
a piperazinyl ring, [0621] R.sup.21 is hydrogen or methyl, [0622]
R.sup.22 is hydrogen or methyl, [0623] or [0624] R.sup.21 and
R.sup.22 together with the atoms to which they are bonded form a
cyclopropyl ring, [0625] R.sup.23 is methyl, [0626] R.sup.24 is
hydrogen, [0627] R.sup.36 is hydrogen, methylcarbonyl or
tert-butyloxycarbonyl, [0628] R.sup.37 is hydrogen or methyl,
[0629] L.sup.2 is linear (C.sub.2-C.sub.6)-alkanediyl or is a group
of the formula
[0629] ##STR00062## [0630] where [0631] p is a number from 2 to 6,
[0632] ##.sup.3 marks the linkage site with the group B, [0633]
##.sup.4 marks the linkage site with the nitrogen atom, [0634] D is
a group of the formula
[0634] ##STR00063## [0635] where [0636] #.sup.3 marks the linkage
site with the nitrogen atom, [0637] R.sup.1 is hydrogen, [0638]
R.sup.2 is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl
or 1H-indol-3-ylmethyl, [0639] or [0640] R.sup.1 and R.sup.2
together with the carbon atom to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0640] ##STR00064## [0641] in which [0642] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [0643] #.sup.5 marks
the linkage site with the carbonyl group, [0644] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[0644] ##STR00065## [0645] in which [0646] #.sup.6 marks the
linkage site with the carbonyl group, [0647] R.sup.6 is hydrogen,
hydroxy or benzyloxy, [0648] R.sup.3 is hydrogen, [0649] R.sup.4 is
benzyl, 4-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
[0650] or [0651] R.sup.3 and R.sup.4 together with the carbon atom
to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0651] ##STR00066## [0652] in which [0653] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [0654] #.sup.8 marks
the linkage site with the group T.sup.1, [0655] 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, [0656] in which [0657] R.sup.7 is
hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, [0658] R.sup.8 is hydrogen or methyl, [0659]
R.sup.9 is hydrogen, methyl, ethyl, n-propyl or benzyl, [0660]
R.sup.11 is benzyl, which may be substituted in the phenyl group by
methoxycarbonyl or carboxyl, [0661] R.sup.5 is hydrogen, methyl or
a group of the formula
[0661] ##STR00067## [0662] in which [0663] #.sup.9 marks the
linkage site with --CHCH.sub.2-phenyl, [0664] R.sup.12 is phenyl
which may be substituted by methoxycarbonyl, carboxyl or a group of
the formula --S(O).sub.2OH, [0665] R.sup.13 is phenyl which may be
substituted by methoxycarbonyl or carboxyl, [0666] R.sup.35 is
methyl or hydroxy, and also their salts, solvates and solvates of
the salts.
[0667] Preferred subject matter of the present invention are
binder-drug conjugates of the general formula (Ia), as indicated
above, in which [0668] n is a number from 1 to 10, [0669] AK is
AK.sub.1 or AK.sub.2 [0670] where [0671] AK.sub.1 is an antibody or
antigen-binding antibody fragment (e.g., an antibody which
comprises the six CDR sequences of the antibody B01-3, B01-10,
M31-B01 or D02-6, the variable light and variable heavy chain of
the antibody B01-3, B01-10, M31-B01 or D02-6 or the light and heavy
chain of the antibody B01-3, B01-10, M31-B01 or D02-6), and is
bonded via the sulphur atom of a cysteine residue of the binder to
the group G, [0672] AK.sub.2 is an antibody or antigen-binding
antibody fragment (e.g., an antibody which comprises the six CDR
sequences of the antibody B01-3, B01-10, M31-B01 or D02-6, the
variable light and variable heavy chain of the antibody B01-3,
B01-10, M31-B01 or D02-6 or the light and heavy chain of the
antibody B01-3, B01-10, M31-B01 or D02-6), and is bonded via the NH
side group of a lysine residue of the binder to the group G, [0673]
G when AK=AK.sub.1, is a group of the formula
[0673] ##STR00068## [0674] in which [0675] #.sup.1 marks the
linkage site with the cysteine residue of the binder, [0676]
#.sup.2 marks the linkage site with the group L.sup.1, [0677] or
[0678] when AK=AK.sub.2, is carbonyl, [0679] L.sup.11 is a bond,
linear (C.sub.2-C.sub.6)-alkanediyl, a group of the formula
[0679] ##STR00069## [0680] where [0681] m is a number 2 or 3,
[0682] ##.sup.1 marks the linkage site with the group G, [0683]
##.sup.2 marks the linkage site with the group B, [0684] where
(C.sub.2-C.sub.6)-alkanediyl may be substituted by 1 or 2 methyl
substituents, [0685] B is a bond or a group of the formula
[0685] ##STR00070## [0686] where [0687] * marks the linkage site
with L.sup.1, [0688] ** marks the linkage site with L.sup.2, [0689]
L.sup.3 is a bond or ethane-1,2-diyl, [0690] L.sup.4 is a bond or a
group of the formula
[0690] ##STR00071## [0691] in which [0692] * * * marks the linkage
site with the carbonyl group, [0693] **** marks the linkage site
with L.sup.2, [0694] R.sup.25 is methyl, [0695] R.sup.28 is
hydrogen, methylcarbonyl or tert-butyloxycarbonyl, [0696] Q.sup.1
is piperidine-1,4-diyl, [0697] R.sup.16 is hydrogen or methyl,
[0698] R.sup.17 is hydrogen or methyl, [0699] or [0700] R.sup.16
and R.sup.17 together with the atoms to which they are bonded form
a piperazinyl ring, [0701] R.sup.21 is hydrogen or methyl, [0702]
R.sup.22 is hydrogen or methyl, [0703] or [0704] R.sup.21 and
R.sup.22 together with the atoms to which they are bonded form a
cyclopropyl ring, [0705] R.sup.23 is methyl, [0706] R.sup.24 is
hydrogen, [0707] L.sup.2 is linear (C.sub.2-C.sub.6)-alkanediyl or
is a group of the formula
[0707] ##STR00072## [0708] where [0709] p is a number from 2 to 6,
[0710] ##.sup.3 marks the linkage site with the group B, [0711]
##.sup.4 marks the linkage site with the nitrogen atom, [0712] D is
a group of the formula
[0712] ##STR00073## [0713] where [0714] #.sup.3 marks the linkage
site with the nitrogen atom, [0715] R.sup.1 is hydrogen, [0716]
R.sup.2 is 1-hydroxyethyl, benzyl, 1-hydroxybenzyl, 1-phenylethyl
or 1H-indol-3-ylmethyl, [0717] or [0718] R.sup.1 and R.sup.2
together with the carbon atom to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0718] ##STR00074## [0719] in which [0720] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [0721] #.sup.5 marks
the linkage site with the carbonyl group, [0722] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[0722] ##STR00075## [0723] in which [0724] #.sup.6 marks the
linkage site with the carbonyl group, [0725] R.sup.6 is hydrogen,
hydroxy or benzyloxy, [0726] R.sup.3 is hydrogen, [0727] R.sup.4 is
benzyl, 1-hydroxybenzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
[0728] or [0729] R.sup.3 and R.sup.4 together with the carbon atom
to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[0729] ##STR00076## [0730] in which [0731] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [0732] #.sup.8 marks
the linkage site with the group T.sup.1, [0733] 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, [0734] in which [0735] R.sup.7 is
hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, [0736] R.sup.8 is hydrogen or methyl, [0737]
R.sup.9 is hydrogen, methyl, ethyl, n-propyl or benzyl, [0738]
R.sup.11 is benzyl, which may be substituted in the phenyl group by
methoxycarbonyl or carboxyl, [0739] R.sup.5 is hydrogen, methyl or
a group of the formula
[0739] ##STR00077## [0740] in which [0741] #.sup.9 marks the
linkage site with --CHCH.sub.2-phenyl, [0742] R.sup.12 is phenyl
which may be substituted by methoxycarbonyl, carboxyl or a group of
the formula --S(O).sub.2OH, [0743] R.sup.13 s phenyl which may be
substituted by methoxycarbonyl or carboxyl, [0744] R.sup.35 is
methyl or hydroxy, [0745] and also their salts, solvates and
solvates of the salts.
[0746] Preferred subject matter of the present invention are
binder-drug conjugates of the general formula (Ia) as indicated
above, in which [0747] n is a number from 1 to 10, [0748] AK is
AK.sub.2, [0749] where [0750] AK.sub.2 is an antibody or
antigen-binding antibody fragment (e.g., an antibody which
comprises the six CDR sequences of the antibody B01-3, B01-10,
M31-B01 or D02-6, the variable light and variable heavy chain of
the antibody B01-3, B01-10, M31-B01 or D02-6 or the light and heavy
chain of the antibody B01-3, B01-10, M31-B01 or D02-6), and is
bonded via the NH side group of a lysine residue of the binder to
the group G, [0751] G is carbonyl, [0752] L.sup.1 is a bond, [0753]
B is a bond, [0754] L.sup.2 is linear (C.sub.3-C.sub.6)-alkanediyl
or is a group of the formula
[0754] ##STR00078## [0755] where [0756] p is a number 2 or 3,
[0757] ##.sup.3 marks the linkage site with the group B, [0758]
##.sup.4 marks the linkage site with the nitrogen atom, [0759] D is
a group of the formula
[0759] ##STR00079## [0760] where [0761] #.sup.3 marks the linkage
site with the nitrogen atom, [0762] R.sup.1 is hydrogen, [0763]
R.sup.2 is benzyl or 1H-indol-3-ylmethyl, [0764] or [0765] R.sup.1
and R.sup.2 together with the carbon atom to which they are bonded
form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[0765] ##STR00080## [0766] in which [0767] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [0768] #.sup.5 marks
the linkage site with the carbonyl group, [0769] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[0769] ##STR00081## [0770] in which [0771] #.sup.6 marks the
linkage site with the carbonyl group, [0772] R.sup.3 is hydrogen,
[0773] R.sup.4 is benzyl or 1H-indol-3-ylmethyl, [0774] or [0775]
R.sup.3 and R.sup.4 together with the carbon atom to which they are
bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[0775] ##STR00082## [0776] in which [0777] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [0778] #.sup.8 marks
the linkage site with the group T.sup.1, [0779] T.sup.1 is a group
of the formula --C(.dbd.O)--OR.sup.7 or
--C(.dbd.O)--NR.sup.8R.sup.9 [0780] in which [0781] R.sup.7 is
hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, [0782] R.sup.8 is hydrogen, [0783] R.sup.9 is
hydrogen or benzyl, [0784] R.sup.35 is methyl, [0785] and also
their salts, solvates and solvates of the salts.
[0786] Preferred subject matter of the present invention are
binder-drug conjugates of the general formula (Ia) as indicated
above, in which [0787] n is a number from 1 to 10, [0788] AK is
AK.sub.2, [0789] where [0790] AK.sub.2 is an antibody or
antigen-binding antibody fragment (e.g., an antibody which
comprises the six CDR sequences of the antibody B01-3, B01-10,
M31-B01 or D02-6, the variable light and variable heavy chain of
the antibody B01-3, B01-10, M31-B01 or D02-6 or the light and heavy
chain of the antibody B01-3, B01-10, M31-B01 or D02-6), and is
bonded via the NH side group of a lysine residue of the binder to
the group G, [0791] G is carbonyl, [0792] L.sup.1 is a bond, [0793]
B is a bond, [0794] L.sup.2 is linear (C.sub.3-C.sub.6)-alkanediyl
or is a group of the formula
[0794] ##STR00083## [0795] where [0796] p is a number 2 or 3,
[0797] ##.sup.3 marks the linkage site with the group B, [0798]
##.sup.4 marks the linkage site with the nitrogen atom, [0799] D is
a group of the formula
[0799] ##STR00084## [0800] where [0801] #.sup.3 marks the linkage
site with the nitrogen atom, [0802] R.sup.1 is hydrogen, [0803]
R.sup.2 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [0804] or [0805]
R.sup.1 and R.sup.2 together with the carbon atom to which they are
bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[0805] ##STR00085## [0806] in which [0807] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [0808] #.sup.5 marks
the linkage site with the carbonyl group, [0809] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[0809] ##STR00086## [0810] in which [0811] #.sup.6 marks the
linkage site with the carbonyl group, [0812] R.sup.3 is hydrogen,
[0813] R.sup.4 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [0814] or
[0815] R.sup.3 and R.sup.4 together with the carbon atom to which
they are bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group
of the formula
[0815] ##STR00087## [0816] in which [0817] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [0818] #.sup.8 marks
the linkage site with the group T.sup.1, [0819] T.sup.1 is a group
of the formula --C(.dbd.O)--OR.sup.7 or
--C(.dbd.O)--NR.sup.8R.sup.9 [0820] in which [0821] R.sup.7 is
hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, [0822] R.sup.8 is hydrogen, [0823] R.sup.9 is
hydrogen or benzyl, [0824] R.sup.35 is methyl, [0825] and also
their salts, solvates and solvates of the salts.
[0826] Preferred subject matter of the present invention are
binder-drug conjugates of the general formula (Ia) as indicated
above, in which [0827] n is a number from 1 to 10, [0828] AK is
AK.sub.1, [0829] where [0830] AK.sub.1 is an antibody or
antigen-binding antibody fragment (e.g., an antibody which
comprises the six CDR sequences of the antibody B01-3, B01-10,
M31-B01 or D02-6, the variable light and variable heavy chain of
the antibody B01-3, B01-10, M31-B01 or D02-6 or the light and heavy
chain of the antibody B01-3, B01-10, M31-B01 or D02-6), and is
bonded via the sulphur atom of a cysteine residue of the binder to
the group G, [0831] G is a group of the formula
[0831] ##STR00088## [0832] where [0833] #.sup.1 marks the linkage
site with the cysteine residue of the binder, [0834] #.sup.2 marks
the linkage site with the group L.sup.1, [0835] L.sup.1 is a bond,
linear (C.sub.3-C.sub.5)-alkanediyl or a group of the formula
[0835] ##STR00089## [0836] where [0837] m is a number 2 or 3,
[0838] ##.sup.1 marks the linkage site with the group G, [0839]
##.sup.2 marks the linkage site with the group B, [0840] where
(C.sub.3-C.sub.5)-alkanediyl may be substituted by 1 or 2 methyl
substituents, [0841] B is a bond or a group of the formula
[0841] ##STR00090## [0842] where [0843] * marks the linkage site
with L.sup.1, [0844] ** marks the linkage site with L.sup.2, [0845]
L.sup.3 is a bond or ethane-1,2-diyl, [0846] L.sup.4 is a bond or a
group of the formula
[0846] ##STR00091## [0847] in which [0848] *** marks the linkage
site with the carbonyl group, [0849] **** marks the linkage site
with L.sup.2, [0850] R.sup.25 is methyl, [0851] R.sup.28 is
hydrogen, methylcarbonyl or tert-butyloxycarbonyl, [0852] R.sup.16
is hydrogen or methyl, [0853] R.sup.17 is hydrogen or methyl,
[0854] or [0855] R.sup.16 and R.sup.17 together with the atoms to
which they are bonded form a piperazinyl ring, [0856] L.sup.2 is
linear (C.sub.3-C.sub.5)-alkanediyl or is a group of the
formula
[0856] ##STR00092## [0857] where [0858] p is a number 2 or 3,
[0859] ##.sup.3 marks the linkage site with the group B, [0860]
##.sup.4 marks the linkage site with the nitrogen atom, [0861] D is
a group of the formula
[0861] ##STR00093## [0862] where [0863] #.sup.3 marks the linkage
site with the nitrogen atom, [0864] R.sup.1 is hydrogen, [0865]
R.sup.2 is benzyl or 1H-indol-3-ylmethyl, [0866] or [0867] R.sup.1
and R.sup.2 together with the carbon atom to which they are bonded
form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[0867] ##STR00094## [0868] in which [0869] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [0870] #.sup.5 marks
the linkage site with the carbonyl group, [0871] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[0871] ##STR00095## [0872] in which [0873] #.sup.6 marks the
linkage site with the carbonyl group, [0874] R.sup.3 is hydrogen,
[0875] R.sup.4 is benzyl or 1H-indol-3-ylmethyl, [0876] or [0877]
R.sup.3 and R.sup.4 together with the carbon atom to which they are
bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[0877] ##STR00096## [0878] in which [0879] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [0880] #.sup.8 marks
the linkage site with the group T.sup.1, [0881] T.sup.1 is a group
of the formula --C(.dbd.O)--OR.sup.7 or
--C(.dbd.O)--NR.sup.8R.sup.9, [0882] in which [0883] R.sup.7 is
hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, [0884] R.sup.8 is hydrogen, [0885] R.sup.9 is
hydrogen or benzyl, [0886] R.sup.35 is methyl, and also their
salts, solvates and solvates of the salts.
[0887] Preferred subject matter of the present invention are
binder-drug conjugates of the general formula (Ia) as indicated
above, in which [0888] n is a number from 1 to 10, [0889] AK is
AK.sub.1, [0890] where [0891] AK.sub.1 is an antibody or
antigen-binding antibody fragment (e.g., an antibody which
comprises the six CDR sequences of the antibody B01-3, B01-10,
M31-B01 or D02-6, the variable light and variable heavy chain of
the antibody B01-3, B01-10, M31-B01 or D02-6 or the light and heavy
chain of the antibody B01-3, B01-10, M31-B01 or D02-6), and is
bonded via the sulphur atom of a cysteine residue of the binder to
the group G, [0892] G is a group of the formula
[0892] ##STR00097## [0893] where [0894] #.sup.1 marks the linkage
site with the cysteine residue of the binder, [0895] #.sup.2 marks
the linkage site with the group L.sup.1, [0896] L.sup.1 is a bond,
linear (C.sub.3-C.sub.5)-alkanediyl or a group of the formula
[0896] ##STR00098## [0897] where [0898] m is a number 2 or 3,
[0899] ##.sup.1 marks the linkage site with the group G, [0900]
##.sup.2 marks the linkage site with the group B, [0901] where
(C.sub.3-C.sub.5)-alkanediyl may be substituted by 1 or 2 methyl
substituents, [0902] B is a bond or a group of the formula
[0902] ##STR00099## [0903] where [0904] * marks the linkage site
with L.sup.1 [0905] ** marks the linkage site with L.sup.2, [0906]
L.sup.3 is a bond or ethane-1,2-diyl, [0907] L.sup.4 is a bond or a
group of the formula
[0907] ##STR00100## [0908] in which [0909] *** marks the linkage
site with the carbonyl group, [0910] **** marks the linkage site
with L.sup.2, [0911] R.sup.25 is methyl, [0912] R.sup.28 is
hydrogen, methylcarbonyl or tert-butyloxycarbonyl, [0913] R.sup.16
is hydrogen or methyl, [0914] R.sup.17 is hydrogen or methyl,
[0915] or [0916] R.sup.16 and R.sup.17 together with the atoms to
which they are bonded form a piperazinyl ring, [0917] L.sup.2 is
linear (C.sub.3-C.sub.5)-alkanediyl or is a group of the
formula
[0917] ##STR00101## [0918] where [0919] p is a number 2 or 3,
[0920] ##.sup.3 marks the linkage site with the group B, [0921]
##.sup.4 marks the linkage site with the nitrogen atom, [0922] D is
a group of the formula
[0922] ##STR00102## [0923] where [0924] #.sup.3 marks the linkage
site with the nitrogen atom, [0925] R.sup.1 is hydrogen, [0926]
R.sup.2 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [0927] or [0928]
R.sup.1 and R.sup.2 together with the carbon atom to which they are
bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[0928] ##STR00103## [0929] in which [0930] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [0931] #.sup.5 marks
the linkage site with the carbonyl group, [0932] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[0932] ##STR00104## [0933] in which [0934] #.sup.6 marks the
linkage site with the carbonyl group, [0935] R.sup.3 is hydrogen,
[0936] R.sup.4 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [0937] or
[0938] R.sup.3 and R.sup.4 together with the carbon atom to which
they are bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group
of the formula
[0938] ##STR00105## [0939] in which [0940] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [0941] #.sup.8 marks
the linkage site with the group T.sup.1, [0942] T.sup.1 is a group
of the formula --C(.dbd.O)--OR.sup.7 or
--C(.dbd.O)--NR.sup.8R.sup.9, [0943] in which [0944] R.sup.7 is
hydrogen, methyl, ethyl, n-propyl, tert-butyl, benzyl or
adamantylmethyl, [0945] R.sup.8 is hydrogen, [0946] R.sup.9 is
hydrogen or benzyl, [0947] R.sup.35 is methyl, and also their
salts, solvates and solvates of the salts.
[0948] Additionally provided by the present invention are compounds
of the formula (XXXa)
##STR00106## [0949] in which [0950] Cys is a cysteine residue which
is bonded via the sulphur atom of the side chain to a carbon atom
of the succinimide, [0951] L.sup.1 is a bond, linear
(C.sub.1-C.sub.10)-alkanediyl, a group of the formula
[0951] ##STR00107## [0952] where [0953] m is a number from 2 to 6,
[0954] ##.sup.1 marks the linkage site with the group G, [0955]
##.sup.2 marks the linkage site with the group B, [0956] L.sup.1A
is linear (C.sub.2-C.sub.10)-alkanediyl, [0957] B.sup.1 is a group
of the formula
[0957] ##STR00108## [0958] in which [0959] ##.sup.5 marks the
linkage site with the group L.sup.1A, [0960] ##.sup.6 marks the
linkage site with the group L.sup.1B, [0961] L.sup.5 is a bond or
(C.sub.2-C.sub.4)-alkanediyl, [0962] L.sup.6 is a bond, [0963]
R.sup.29 is hydrogen or (C.sub.1-C.sub.4)-alkyl, [0964] R.sup.30 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, [0965] or [0966] R.sup.29 and
R.sup.30 together with the atoms to which they are bonded form a 5-
or 6-membered heterocycle, [0967] R.sup.31 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0968] R.sup.32 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0969] or [0970] R.sup.31 and R.sup.32
together with the atoms to which they are bonded form a 5- or
6-membered heterocycle, [0971] L.sup.1B is linear
(C.sub.2-C.sub.10)-alkanediyl, [0972] and [0973] where
(C.sub.1-C.sub.10)-alkanediyl may be substituted by 1 to 4
substituents selected independently of one another from the group
consisting of methyl, hydroxy and benzyl, [0974] and [0975] where
two carbon atoms of the alkanediyl chain in 1,2, 1, 3 or 1,4
relation to one another, with inclusion of any carbon atoms
situated between them, may be bridged to form a
(C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring, [0976] B is a
bond or a group of the formula
[0976] ##STR00109## [0977] where [0978] * marks the linkage site
with L.sup.1, [0979] ** marks the linkage site with L.sup.2, [0980]
P is O or NH, [0981] L.sup.3 is a bond or
(C.sub.2-C.sub.4)-alkanediyl, [0982] L.sup.4 is a bond, [0983]
Q.sup.1 is a 4- to 7-membered heterocycle, [0984] Q.sup.2 is a 3-
to 7-membered carbocycle or a 4- to 7-membered heterocycle, [0985]
R.sup.14 is hydrogen or (C.sub.1-C.sub.4)-alkyl, [0986] R.sup.15 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, [0987] or [0988] R.sup.14 and
R.sup.15 together with the atoms to which they are bonded form a 5-
or 6-membered heterocycle, [0989] R.sup.16 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0990] R.sup.17 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0991] or [0992] R.sup.16 and R.sup.17
together with the atoms to which they are bonded form a 5- or
6-membered heterocycle, [0993] R.sup.18 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [0994] R.sup.19 is hydrogen or the side
group of a natural .alpha.-amino acid or of its homologues or
isomers, [0995] R.sup.20 is hydrogen or (C.sub.1-C.sub.4)-alkyl,
[0996] or [0997] R.sup.19 and R.sup.20 together with the atoms to
which they are bonded form a pyrrolidinyl ring, [0998] R.sup.21 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, [0999] R.sup.22 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1000] or [1001] R.sup.21 and R.sup.22
together with the atoms to which they are bonded form a 3- to
7-membered carbocycle, [1002] R.sup.23 is (C.sub.1-C.sub.4)-alkyl,
[1003] R.sup.24 is hydrogen or (C.sub.1-C.sub.4)-alkyl, [1004]
R.sup.27 is hydrogen or (C.sub.1-C.sub.4)-alkyl, [1005] L.sup.2 is
linear (C.sub.2-C.sub.10)-alkanediyl or is a group of the
formula
[1005] ##STR00110## [1006] where [1007] p is a number from 2 to 6,
[1008] ##.sup.3 marks the linkage site with the group B, [1009]
##.sup.4 marks the linkage site with the nitrogen atom, [1010]
where (C.sub.2-C.sub.10)-alkanediyl may be substituted by 1 to 4
substituents selected independently of one another from the group
consisting of methyl, hydroxy and benzyl, [1011] and [1012] where
two carbon atoms of the alkanediyl chain in 1,2, 1, 3 or 1,4
relation to one another, with inclusion of any carbon atoms
situated between them, may be bridged to form a
(C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring, [1013] D is a
group of the formula
[1013] ##STR00111## [1014] where [1015] #.sup.3 marks the linkage
site with the nitrogen atom, [1016] R.sup.1 is hydrogen or methyl,
[1017] 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-phenyl-ethyl,
diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
[1018] or [1019] R.sup.1 and R.sup.2 together with the carbon atom
to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1019] ##STR00112## [1020] in which [1021] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [1022] #.sup.5 marks
the linkage site with the carbonyl group, [1023] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[1023] ##STR00113## [1024] in which [1025] #.sup.6 marks the
linkage site with the carbonyl group, [1026] R.sup.6 is hydrogen,
hydroxy or benzyloxy, [1027] R.sup.3 is hydrogen or methyl, [1028]
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-phenyl-ethyl, diphenylmethyl,
1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl, [1029] or [1030]
R.sup.3 and R.sup.4 together with the carbon atom to which they are
bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[1030] ##STR00114## [1031] in which [1032] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [1033] #.sup.8 marks
the linkage site with the group T.sup.1, [1034] 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, [1035] in
which [1036] R.sup.7 is hydrogen, methyl, ethyl, n-propyl,
tert-butyl, benzyl or adamantylmethyl, [1037] R.sup.8 is hydrogen
or methyl, [1038] R.sup.9 is hydrogen, methyl, ethyl, n-propyl or
benzyl, [1039] or [1040] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are bonded form a 4- to 7-membered
heterocycle, [1041] R.sup.10 is benzoyl, [1042] R.sup.11 is benzyl,
which may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, [1043] R.sup.5 is hydrogen, methyl or a group of the
formula
[1043] ##STR00115## [1044] in which [1045] #.sup.9 marks the
linkage site with --CHC(R.sup.26)-T.sup.2, [1046] R.sup.12 is
phenyl which may be substituted by methoxycarbonyl, carboxyl or a
group of the formula --S(O).sub.2OH, [1047] R.sup.13 is phenyl
which may be substituted by methoxycarbonyl or carboxyl, [1048]
R.sup.26 is hydrogen or hydroxy, [1049] T.sup.2 is phenyl, benzyl,
1H-indol-3-yl or 1H-indol-3-ylmethyl, [1050] R.sup.35 is methyl or
hydroxy, and also their salts, solvates and solvates of the
salts.
[1051] Preferred subject matter of the present invention are
compounds of the formula (XXXa) as indicated above, in which [1052]
Cys is a cysteine residue which is bonded via the sulphur atom of
the side chain via a carbon atom of the succinimide, [1053] L.sup.1
is a bond, linear (C.sub.2-C.sub.6)-alkanediyl, a group of the
formula
[1053] ##STR00116## [1054] where [1055] m is a number 2 or 3,
[1056] ##.sup.1 marks the linkage site with the group G, [1057]
##.sup.2 marks the linkage site with the group B, [1058] L.sup.1A
is linear (C.sub.2-C.sub.6)-alkanediyl, [1059] B.sup.1 is a group
of the formula
[1059] ##STR00117## [1060] in which [1061] ##.sup.5 marks the
linkage site with the group L.sup.1A, [1062] ##.sup.6 marks the
linkage site with the group L.sup.1B, [1063] L.sup.5 is a bond,
[1064] L.sup.6 is a bond, [1065] R.sup.29 is hydrogen, [1066]
R.sup.30 is hydrogen, [1067] R.sup.31 is hydrogen or methyl, [1068]
R.sup.32 is hydrogen or methyl, [1069] L.sup.1B is linear
(C.sub.2-C.sub.6)-alkanediyl, [1070] and [1071] where
(C.sub.2-C.sub.6)-alkanediyl may be substituted by 1 or 2 methyl
substituents, [1072] B is a bond or a group of the formula
[1072] ##STR00118## [1073] where [1074] * marks the linkage site
with L.sup.1, [1075] ** marks the linkage site with L.sup.2, [1076]
L.sup.3 is a bond or ethane-1,2-diyl, [1077] L.sup.4 is a bond,
[1078] R.sup.14 is hydrogen, [1079] R.sup.15 is hydrogen, [1080]
R.sup.16 is hydrogen or methyl, [1081] R.sup.17 is hydrogen or
methyl, [1082] or [1083] R.sup.16 and R.sup.17 together with the
atoms to which they are bonded form piperazinyl ring, [1084]
R.sup.23 is methyl, [1085] R.sup.24 is hydrogen or methyl, [1086]
L.sup.2 is linear (C.sub.2-C.sub.6)-alkanediyl or is a group of the
formula
[1086] ##STR00119## [1087] where [1088] p is a number 2 or 3,
[1089] ##.sup.3 marks the linkage site with the group B, [1090]
##.sup.4 marks the linkage site with the nitrogen atom, [1091] D is
a group of the formula
[1091] ##STR00120## [1092] where [1093] #.sup.3 marks the linkage
site with the nitrogen atom, [1094] R.sup.1 is hydrogen, [1095]
R.sup.2 is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl
or 1H-indol-3-ylmethyl, [1096] or [1097] R.sup.1 and R.sup.2
together with the carbon atom to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1097] ##STR00121## [1098] in which [1099] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [1100] #.sup.5 marks
the linkage site with the carbonyl group, [1101] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[1101] ##STR00122## [1102] in which [1103] #.sup.6 marks the
linkage site with the carbonyl group, [1104] R.sup.6 is hydrogen,
hydroxy or benzyloxy, [1105] R.sup.3 is hydrogen, [1106] R.sup.4 is
1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [1107] or [1108] R.sup.3 and R.sup.4 together
with the carbon atom to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1108] ##STR00123## [1109] in which [1110] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [1111] #.sup.8 marks
the linkage site with the group T.sup.1, [1112] 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, [1113] in
which [1114] R.sup.7 is hydrogen, methyl, ethyl, n-propyl,
tert-butyl, benzyl or adamantylmethyl, [1115] R.sup.8 is hydrogen
or methyl, [1116] R.sup.9 is hydrogen, methyl, ethyl, n-propyl or
benzyl, [1117] or [1118] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are bonded form a 4- to 7-membered
heterocycle, [1119] R.sup.10 is benzoyl, [1120] R.sup.11 s benzyl
which may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, [1121] R.sup.5 is hydrogen, methyl or a group of the
formula
[1121] ##STR00124## [1122] in which [1123] #.sup.9 marks the
linkage site with --CHCH.sub.2-phenyl, [1124] R.sup.12 is phenyl
which may be substituted by methoxycarbonyl, carboxyl or a group of
the formula --S(O).sub.2OH, [1125] R.sup.13 is phenyl which may be
substituted by methoxycarbonyl or carboxyl, [1126] R.sup.35 is
methyl or hydroxy, and also their salts, solvates and solvates of
the salts.
[1127] Preferred subject matter of the present invention are
compounds of the formula (XXXa) as indicated above, in which [1128]
Cys is a cysteine residue which is bonded via the sulphur atom of
the side chain via a carbon atom of the succinimide, [1129] L.sup.1
is a bond or linear (C.sub.2-C.sub.6)-alkanediyl, [1130] B is a
bond or a group of the formula
[1130] ##STR00125## [1131] where [1132] * marks the linkage site
with L.sup.1, [1133] ** marks the linkage site with L.sup.2,
[1134] L.sup.3 is a bond,
[1135] L.sup.4 is a bond, [1136] R.sup.16 is hydrogen or
methyl,
[1137] R.sup.17 is hydrogen or methyl, [1138] L.sup.2 is linear
(C.sub.2-C.sub.6)-alkanediyl or is a group of the formula
[1138] ##STR00126## [1139] where [1140] p is a number 2 or 3,
[1141] ##.sup.3 marks the linkage site with the group B, [1142]
##.sup.4 marks the linkage site with the nitrogen atom, [1143] D is
a group of the formula
[1143] ##STR00127## [1144] where [1145] #.sup.3 marks the linkage
site with the nitrogen atom, [1146] R.sup.1 is hydrogen, [1147]
R.sup.2 is benzyl or 1H-indol-3-ylmethyl, [1148] or [1149] R.sup.1
and R.sup.2 together with the carbon atom to which they are bonded
form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[1149] ##STR00128## [1150] in which [1151] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [1152] #.sup.5 marks
the linkage site with the carbonyl group, [1153] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[1153] ##STR00129## [1154] in which [1155] #.sup.6 marks the
linkage site with the carbonyl group, [1156] R.sup.3 is hydrogen,
[1157] R.sup.4 is benzyl or 1H-indol-3-ylmethyl, [1158] or [1159]
R.sup.3 and R.sup.4 together with the carbon atom to which they are
bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[1159] ##STR00130## [1160] in which [1161] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [1162] #.sup.8 marks
the linkage site with the group T.sup.1, [1163] T.sup.1 is a group
of the formula --C(.dbd.O)--OR.sup.7 or
--C(.dbd.O)--NR.sup.8R.sup.9, [1164] in which [1165] R.sup.7 is
hydrogen, [1166] R.sup.8 is hydrogen, [1167] R.sup.9 is hydrogen,
[1168] R.sup.35 is methyl, and also their salts, solvates and
solvates of the salts.
[1169] Preferred subject matter of the present invention are
compounds of the formula (XXXa) as indicated above, in which [1170]
Cys is a cysteine residue which is bonded via the sulphur atom of
the side chain via a carbon atom of the succinimide, [1171] L.sup.1
is a bond or linear (C.sub.2-C.sub.6)-alkanediyl, [1172] B is a
bond or a group of the formula
[1172] ##STR00131## [1173] where [1174] * marks the linkage site
with L.sup.1, [1175] ** marks the linkage site with L.sup.2, [1176]
L.sup.3 is a bond, [1177] L.sup.4 is a bond, [1178] R.sup.16 is
hydrogen or methyl, [1179] R.sup.17 is hydrogen or methyl, [1180]
L.sup.2 is linear (C.sub.2-C.sub.6)-alkanediyl or is a group of the
formula
[1180] ##STR00132## [1181] where [1182] p is a number 2 or 3,
[1183] ##.sup.3 marks the linkage site with the group B, [1184]
##.sup.4 marks the linkage site with the nitrogen atom, [1185] D is
a group of the formula
[1185] ##STR00133## [1186] where [1187] #.sup.3 marks the linkage
site with the nitrogen atom, [1188] R.sup.1 is hydrogen, [1189]
R.sup.2 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [1190] or [1191]
R.sup.1 and R.sup.2 together with the carbon atom to which they are
bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[1191] ##STR00134## [1192] in which [1193] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [1194] #.sup.5 marks
the linkage site with the carbonyl group, [1195] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[1195] ##STR00135## [1196] in which [1197] #.sup.6 marks the
linkage site with the carbonyl group, [1198] R.sup.3 is hydrogen,
[1199] R.sup.4 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [1200] or
[1201] R.sup.3 and R.sup.4 together with the carbon atom to which
they are bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group
of the formula
[1201] ##STR00136## [1202] in which [1203] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [1204] #.sup.8 marks
the linkage site with the group T.sup.1, [1205] T.sup.1 is a group
of the formula --C(.dbd.O)--OR.sup.7 or
--C(.dbd.O)--NR.sup.8R.sup.9, [1206] in which [1207] R.sup.7 is
hydrogen, [1208] R.sup.8 is hydrogen, [1209] R.sup.9 is hydrogen,
[1210] R.sup.35 is methyl, and also their salts, solvates and
solvates of the salts.
[1211] The present invention additionally provides compounds of the
formula (XXXI)
##STR00137## [1212] in which [1213] L.sup.1 is a bond, linear
(C.sub.1-C.sub.10)-alkanediyl, a group of the formula
[1213] ##STR00138## [1214] where [1215] m is a number from 2 to 6,
[1216] ##.sup.1 marks the linkage site with the group G, [1217]
##.sup.2 marks the linkage site with the group B, [1218] L.sup.1A
is linear (C.sub.2-C.sub.10)-alkanediyl, [1219] B.sup.1 is a group
of the formula
[1219] ##STR00139## [1220] in which [1221] ##.sup.5 marks the
linkage site with the group L.sup.1A, [1222] ##.sup.6 marks the
linkage site with the group L.sup.1B, [1223] L.sup.5 is a bond or
(C.sub.2-C.sub.4)-alkanediyl, [1224] L.sup.6 is a bond, [1225]
R.sup.29 is hydrogen or (C.sub.1-C.sub.4)-alkyl, [1226] R.sup.30 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, [1227] or [1228] R.sup.29 and
R.sup.30 together with the atoms to which they are bonded form a 5-
or 6-membered heterocycle, [1229] R.sup.31 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1230] R.sup.32 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1231] or [1232] R.sup.31 and R.sup.32
together with the atoms to which they are bonded form a 5- or
6-membered heterocycle, [1233] L.sup.1B is linear
(C.sub.2-C.sub.10)-alkanediyl, [1234] and [1235] where
(C.sub.1-C.sub.10)-alkanediyl may be substituted by 1 to 4
substituents selected independently of one another from the group
consisting of methyl, hydroxy and benzyl, [1236] and [1237] where
two carbon atoms of the alkanediyl chain in 1,2, 1, 3 or 1,4
relation to one another, with inclusion of any carbon atoms
situated between them, may be bridged to form a
(C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring, [1238] B is a
bond or a group of the formula
[1238] ##STR00140## [1239] where [1240] * marks the linkage site
with L.sup.1, [1241] ** marks the linkage site with L.sup.2, [1242]
P is O or NH, [1243] Q.sup.1 is a 4- to 7-membered heterocycle,
[1244] Q.sup.2 is a 3- to 7-membered carbocycle or a 4- to
7-membered heterocycle, [1245] R.sup.18 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1246] R.sup.19 is hydrogen or the side
group of a natural .alpha.-amino acid or of its homologues or
isomers, [1247] R.sup.20 is hydrogen or (C.sub.1-C.sub.4)-alkyl,
[1248] or [1249] R.sup.19 and R.sup.20 together with the atoms to
which they are bonded form a pyrrolidinyl ring, [1250] R.sup.21 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, [1251] R.sup.22 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1252] or [1253] R.sup.21 and R.sup.22
together with the atoms to which they are bonded form a 3- to
7-membered carbocycle, [1254] R.sup.27 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1255] L.sup.2 is linear
(C.sub.2-C.sub.10)-alkanediyl or is a group of the formula
[1255] ##STR00141## [1256] where [1257] p is a number from 2 to 6,
[1258] ##.sup.3 marks the linkage site with the group B, [1259]
##.sup.4 marks the linkage site with the nitrogen atom, [1260]
where (C.sub.2-C.sub.10)-alkanediyl may be substituted by 1 to 4
substituents selected independently of one another from the group
consisting of methyl, hydroxy and benzyl, [1261] and [1262] where
two carbon atoms of the alkanediyl chain in 1,2, 1, 3 or 1,4
relation to one another, with inclusion of any carbon atoms
situated between them, may be bridged to form a
(C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring, [1263] D is a
group of the formula
[1263] ##STR00142## [1264] in which [1265] #.sup.3 marks the
linkage site with the nitrogen atom, [1266] R.sup.1 is hydrogen or
methyl, [1267] 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-phenyl-ethyl,
diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
[1268] or [1269] R.sup.1 and R.sup.2 together with the carbon atom
to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1269] ##STR00143## [1270] in which [1271] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [1272] #.sup.5 marks
the linkage site with the carbonyl group, [1273] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[1273] ##STR00144## [1274] in which [1275] #.sup.6 marks the
linkage site with the carbonyl group, [1276] R.sup.6 is hydrogen,
hydroxy or benzyloxy, [1277] R.sup.3 is hydrogen or methyl, [1278]
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-phenyl-ethyl, diphenylmethyl,
1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl, [1279] or [1280]
R.sup.3 and R.sup.4 together with the carbon atom to which they are
bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[1280] ##STR00145## [1281] in which [1282] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [1283] #.sup.8 marks
the linkage site with the group T.sup.1, [1284] 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, [1285] in
which [1286] R.sup.7 is hydrogen, methyl, ethyl, n-propyl,
tert-butyl, benzyl or adamantylmethyl, [1287] R.sup.8 is hydrogen
or methyl, [1288] R.sup.9 is hydrogen, methyl, ethyl, n-propyl or
benzyl, [1289] or [1290] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are bonded form a 4- to 7-membered
heterocycle, [1291] R.sup.10 is benzoyl, [1292] R.sup.11 is benzyl,
which may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, [1293] R.sup.5 is hydrogen, methyl or a group of the
formula
[1293] ##STR00146## [1294] in which [1295] #.sup.9 marks the
linkage site with --CHC(R.sup.26)-T.sup.2, [1296] R.sup.12 is
phenyl which may be substituted by methoxycarbonyl, carboxyl or a
group of the formula --S(O).sub.2OH, [1297] R.sup.13 is phenyl
which may be substituted by methoxycarbonyl or carboxyl, [1298]
R.sup.26 is hydrogen or hydroxy, [1299] T.sup.2 is phenyl, benzyl,
1H-indol-3-yl or 1H-indol-3-ylmethyl, [1300] R.sup.35 is methyl or
hydroxy, and also their salts, solvates and solvates of the
salts.
[1301] Preferred subject matter of the present invention are
compounds of the formula (XXXI) as indicated above, in which [1302]
L.sup.1 is a bond, linear (C.sub.2-C.sub.6)-alkanediyl or a group
of the formula
[1302] ##STR00147## [1303] where [1304] m is a number 2 or 3,
[1305] ##.sup.1 marks the linkage site with the group G, [1306]
##.sup.2 marks the linkage site with the group B, [1307] where
(C.sub.2-C.sub.6)-alkanediyl may be substituted by 1 or 2 methyl
substituents, [1308] B is a bond or a group of the formula
[1308] ##STR00148## [1309] where [1310] * marks the linkage site
with L.sup.1, [1311] ** marks the linkage site with L.sup.2, [1312]
R.sup.18 is hydrogen, [1313] R.sup.19 is methyl, propan-2-yl,
2-methylpropan-1-yl or 1-methylpropan-1-yl, [1314] R.sup.20 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, [1315] or [1316] R.sup.19 and
R.sup.20 together with the atoms to which they are bonded form a
pyrrolidinyl ring, [1317] R.sup.21 is hydrogen or methyl, [1318]
R.sup.22 is hydrogen or methyl, [1319] or [1320] R.sup.21 and
R.sup.22 together with the atoms to which they are bonded form a
cyclopropyl ring, [1321] R.sup.27 is hydrogen or methyl, [1322]
L.sup.2 is linear (C.sub.2-C.sub.6)-alkanediyl or is a group of the
formula
[1322] ##STR00149## [1323] where [1324] p is a number 2 or 3,
[1325] ##.sup.3 marks the linkage site with the group B, [1326]
##.sup.4 marks the linkage site with the nitrogen atom, [1327]
where (C.sub.2-C.sub.10)-alkanediyl may be substituted by 1 or 2
methyl substituents, [1328] and [1329] where two carbon atoms of
the alkanediyl chain in 1,4 relation to one another, with inclusion
of any carbon atoms situated between them, may be bridged to form a
phenyl ring, [1330] D is a group of the formula
[1330] ##STR00150## [1331] in which [1332] #.sup.3 marks the
linkage site with the nitrogen atom, [1333] R.sup.1 is hydrogen,
[1334] R.sup.2 is 1-hydroxyethyl, benzyl, 4-hydroxybenzyl,
1-phenylethyl or 1H-indol-3-ylmethyl, [1335] or [1336] R.sup.1 and
R.sup.2 together with the carbon atom to which they are bonded form
a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1336] ##STR00151## [1337] in which [1338] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [1339] #.sup.5 marks
the linkage site with the carbonyl group, [1340] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[1340] ##STR00152## [1341] in which [1342] #.sup.6 marks the
linkage site with the carbonyl group, [1343] R.sup.6 is hydrogen,
hydroxy or benzyloxy, [1344] R.sup.3 is hydrogen, [1345] R.sup.4 is
1-hydroxyethyl, benzyl, 4-hydroxybenzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [1346] or [1347] R.sup.3 and R.sup.4 together
with the carbon atom to which they are bonded form a
(1S,2R)-2-phenylcyclopropan-1,1-diyl group of the formula
[1347] ##STR00153## [1348] in which [1349] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [1350] #.sup.8 marks
the linkage site with the group T.sup.1, [1351] 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, [1352] in
which [1353] R.sup.7 is hydrogen, methyl, ethyl, n-propyl,
tert-butyl, benzyl or adamantylmethyl, [1354] R.sup.8 is hydrogen
or methyl, [1355] R.sup.9 is hydrogen, methyl, ethyl, n-propyl or
benzyl, [1356] or [1357] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are bonded form a 4- to 7-membered
heterocycle, [1358] R.sup.10 is benzoyl, [1359] R.sup.11 is benzyl,
which may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, [1360] R.sup.5 is hydrogen, methyl or a group of the
formula
[1360] ##STR00154## [1361] in which [1362] #.sup.9 marks the
linkage site with --CHCH.sub.2-phenyl, [1363] R.sup.12 is phenyl
which may be substituted by methoxycarbonyl, carboxyl or a group of
the formula --S(O).sub.2OH, [1364] R.sup.13 is phenyl which may be
substituted by methoxycarbonyl or carboxyl, [1365] R.sup.35 is
methyl or hydroxy, and also their salts, solvates and solvates of
the salts.
[1366] Preferred subject matter of the present invention are
compounds of the formula (XXXI) as indicated above, in which [1367]
L.sup.1 is a bond, [1368] B is a bond, [1369] L.sup.2 is linear
(C.sub.2-C.sub.6)-alkanediyl or is a group of the formula
[1369] ##STR00155## [1370] where [1371] is a number 2 or 3, [1372]
##.sup.3 marks the linkage site with the group B, [1373] ##.sup.4
marks the linkage site with the nitrogen atom, [1374] D is the
group of the formula
[1374] ##STR00156## [1375] where [1376] #.sup.3 marks the linkage
site with the nitrogen atom, [1377] R.sup.1 is hydrogen, [1378]
R.sup.2 is benzyl or 1H-indol-3-ylmethyl, [1379] or [1380] R.sup.1
and R.sup.2 together with the carbon atom to which they are bonded
form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[1380] ##STR00157## [1381] in which [1382] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [1383] #.sup.5 marks
the linkage site with the carbonyl group, [1384] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[1384] ##STR00158## [1385] in which [1386] #.sup.6 marks the
linkage site with the carbonyl group, [1387] R.sup.6 is hydrogen,
hydroxy or benzyloxy, [1388] R.sup.3 is hydrogen, [1389] R.sup.4 is
benzyl or 1H-indol-3-ylmethyl, [1390] or [1391] R.sup.3 and R.sup.4
together with the carbon atom to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1391] ##STR00159## [1392] in which [1393] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [1394] #.sup.8 marks
the linkage site with the group T.sup.1, [1395] T.sup.1 is a group
of the formula --C(.dbd.O)--OR.sup.7 or
--C(.dbd.O)--NR.sup.8R.sup.9, [1396] in which [1397] R.sup.7 is
hydrogen, [1398] R.sup.8 is hydrogen, [1399] R.sup.9 is hydrogen,
[1400] R.sup.35 is methyl, and also their salts, solvates and
solvates of the salts.
[1401] Preferred subject matter of the present invention are
compounds of the formula (XXXI) as indicated above, in which [1402]
L.sup.1 is a bond, [1403] B is a bond, [1404] L.sup.2 is linear
(C.sub.2-C.sub.6)-alkanediyl or is a group of the formula
[1404] ##STR00160## [1405] where [1406] p is a number 2 or 3,
[1407] ##.sup.3 marks the linkage site with the group B, [1408]
##.sup.4 marks the linkage site with the nitrogen atom, [1409] D is
a group of the formula
[1409] ##STR00161## [1410] where [1411] #.sup.3 marks the linkage
site with the nitrogen atom, [1412] R.sup.1 is hydrogen, [1413]
R.sup.2 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [1414] or [1415]
R.sup.1 and R.sup.2 together with the carbon atom to which they are
bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[1415] ##STR00162## [1416] in which [1417] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [1418] #.sup.5 marks
the linkage site with the carbonyl group, [1419] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[1419] ##STR00163## [1420] in which [1421] #.sup.6 marks the
linkage site with the carbonyl group, [1422] R.sup.6 is hydrogen,
hydroxy or benzyloxy, [1423] R.sup.3 is hydrogen, [1424] R.sup.4 is
4-hydroxybenzyl or 1H-indol-3-ylmethyl, [1425] or [1426] R.sup.3
and R.sup.4 together with the carbon atom to which they are bonded
form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[1426] ##STR00164## [1427] in which [1428] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [1429] #.sup.8 marks
the linkage site with the group T.sup.1, [1430] T.sup.1 is a group
of the formula --C(.dbd.O)--OR.sup.7 or
--C(.dbd.O)--NR.sup.8R.sup.9, [1431] in which [1432] R.sup.7 s
hydrogen, [1433] R.sup.8 is hydrogen, [1434] R.sup.9 is hydrogen,
[1435] R.sup.35 is methyl, and also their salts, solvates and
solvates of the salts.
[1436] Preferred subject matter of the present invention are
compounds of the formulae (XXXa) and (XXXI) selected from the
following group: [1437]
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulphanyl}-2,5-dioxopyrrolidin-1-yl-
)hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carbo-
xy-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,
[1438]
N-[6-(3-{[(2R)-2-amino-2-carboxyethyl]sulphanyl}-2,5-dioxopyrrolidin-1-yl-
)hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(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}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-meth-
yl-L-valinamide, [1439]
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, [1440]
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, and also their salts,
solvates and solvates of the salts.
[1441] The present invention additionally provides binder-drug
conjugates of the general formula (I)
##STR00165## [1442] in which [1443] n is a number from 1 to 50,
[1444] AK is a binder, [1445] the group
.sctn.-G-L.sup.1-B-L.sup.2-.sctn..sctn. is a linker, [1446] where
[1447] .sctn. marks the linkage site with the group AK and [1448]
.sctn..sctn. marks the linkage site with the nitrogen atom, [1449]
D is a group of the formula
[1449] ##STR00166## [1450] where [1451] #.sup.3 marks the linkage
site with the nitrogen atom, [1452] R.sup.1 is hydrogen, [1453]
R.sup.2 is 1-hydroxyethyl, benzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [1454] or [1455] R.sup.1 and R.sup.2 together
with the carbon atom to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1455] ##STR00167## [1456] in which [1457] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [1458] #.sup.5 marks
the linkage site with the carbonyl group, [1459] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[1459] ##STR00168## [1460] in which [1461] #.sup.6 marks the
linkage site with the carbonyl group, [1462] R.sup.6 is hydrogen,
hydroxy or benzyloxy, [1463] R.sup.3 is hydrogen, [1464] R.sup.4
1-hydroxyethyl, benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
[1465] or [1466] R.sup.3 and R.sup.4 together with the carbon atom
to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1466] ##STR00169## [1467] in which [1468] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [1469] #.sup.8 marks
the linkage site with the group T.sup.1, [1470] 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, [1471] in
which [1472] R.sup.7 is hydrogen, methyl, ethyl, n-propyl,
tert-butyl, benzyl or adamantylmethyl, [1473] R.sup.8 is hydrogen
or methyl, [1474] R.sup.9 is hydrogen, methyl, ethyl, n-propyl or
benzyl, [1475] or [1476] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are bonded form a 4- to 7-membered
heterocycle, [1477] R.sup.10 is benzoyl, [1478] R.sup.11 is benzyl,
which may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, [1479] R.sup.5 is hydrogen, methyl or a group of the
formula
[1479] ##STR00170## [1480] in which [1481] #.sup.9 marks the
linkage site with --CHC(R.sup.26)-T.sup.2, [1482] R.sup.12 is
phenyl which may be substituted by methoxycarbonyl, carboxyl or a
group of the formula --S(O).sub.2OH, [1483] R.sup.13 is phenyl
which may be substituted by methoxycarbonyl or carboxyl, [1484]
R.sup.26 is hydrogen or hydroxy, [1485] T.sup.2 is phenyl, benzyl,
1H-indol-3-yl or 1H-indol-3-ylmethyl, and also their salts,
solvates and solvates of the salts.
[1486] Preferred subject matter of the invention are binder-drug
conjugates of the general formula (I), in which [1487] n is a
number from 1 to 50, [1488] AK is AK.sub.1 or AK.sub.2 [1489] where
[1490] AK.sub.1 is a binder which is bonded via a sulphur atom of
the binder to the group G, [1491] AK.sub.2 is a binder which is
bonded via a nitrogen atom of the binder to the group G, [1492] G
when AK=AK.sub.1, is a group of the formula
[1492] ##STR00171## [1493] where [1494] #.sup.1 marks the linkage
site with the sulphur atom of the binder, [1495] #.sup.2 mark ks
the linkage site with the group L.sup.1, [1496] or [1497] when
AK=AK.sub.2, is carbonyl, [1498] L.sup.1 is a bond, linear
(C.sub.1-C.sub.10)-alkanediyl or is a group of the formula
[1498] ##STR00172## [1499] where [1500] m is a number from 2 to 6,
[1501] ##.sup.1 marks the linkage site with the group G, [1502]
##.sup.2 marks the linkage site with the group B, [1503] where
(C.sub.1-C.sub.10)-alkanediyl may be substituted by 1 to 4 methyl
substituents, [1504] and [1505] where two carbon atoms of the
alkanediyl chain in 1,2, 1, 3 or 1,4 relation to one another, with
inclusion of any carbon atoms situated between them, may be bridged
to form a (C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring,
[1506] B is a bond or a group of the formula
[1506] ##STR00173## [1507] where [1508] * marks the linkage site
with L.sup.1, [1509] ** marks the linkage site with L.sup.2, [1510]
P is O or NH, [1511] L.sup.3 is a bond or
(C.sub.2-C.sub.4)-alkanediyl, [1512] L.sup.4 is a bond or a group
of the formula
[1512] ##STR00174## [1513] in which [1514] *** marks the linkage
site with the carbonyl group, [1515] **** marks the linkage site
with L.sup.2, [1516] R.sup.25 is hydrogen or methyl, [1517] Q.sup.1
is a 4- to 7-membered heterocycle, [1518] Q.sup.2 is a 3- to
7-membered carbocycle or a 4- to 7-membered heterocycle, [1519]
R.sup.14 is hydrogen or (C.sub.1-C.sub.4)-alkyl, [1520] R.sup.15 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, [1521] or [1522] R.sup.14 and
R.sup.15 together with the atoms to which they are bonded form a 5-
or 6-membered heterocycle, [1523] R.sup.16 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1524] R.sup.17 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1525] or [1526] R.sup.16 and R.sup.17
together with the atoms to which they are bonded form a 5- or
6-membered heterocycle, [1527] R.sup.18 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1528] R.sup.19 is hydrogen or the side
group of a natural .alpha.-amino acid or of its homologues or
isomers, [1529] R.sup.20 is hydrogen or (C.sub.1-C.sub.4)-alkyl,
[1530] or [1531] R.sup.19 and R.sup.20 together with the atoms to
which they are bonded form a pyrrolidinyl ring, [1532] R.sup.21 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, [1533] R.sup.22 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1534] or [1535] R.sup.21 and R.sup.22
together with the atoms to which they are bonded form a 3- to
7-membered carbocycle, [1536] R.sup.23 is (C.sub.1-C.sub.4)-alkyl,
[1537] R.sup.24 is hydrogen or (C.sub.1-C.sub.4)-alkyl, [1538]
R.sup.27 is hydrogen or (C.sub.1-C.sub.4)-alkyl, [1539] L.sup.2 is
linear (C.sub.2-C.sub.10)-alkanediyl or is a group of the
formula
[1539] ##STR00175## [1540] where [1541] p is a number from 2 to 6,
[1542] ##.sup.3 marks the linkage site with the group B, [1543]
##.sup.4 marks the linkage site with the nitrogen atom, [1544]
where (C.sub.2-C.sub.10)-alkanediyl may be substituted by 1 to 4
methyl substituents, [1545] and [1546] where two carbon atoms of
the alkanediyl chain in 1,2, 1, 3 or 1,4 relation to one another,
with inclusion of any carbon atoms situated between them, may be
bridged to form a (C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl
ring, [1547] D has the definitions indicated above, and also their
salts, solvates and solvates of the salts.
[1548] Preferred subject matter of the invention are binder-drug
conjugates of the general formula (I), [1549] in which [1550] n is
a number from 1 to 50, [1551] AK is AK.sub.1 or AK.sub.2 [1552]
where [1553] AK.sub.1 is an antibody or an antigen-binding antibody
fragment and are bonded via a sulphur atom to the group G, [1554]
AK.sub.2 is an antibody or an antigen-binding antibody fragment and
are bonded via a nitrogen atom to the group G, [1555] G, L.sup.1,
B, L.sup.2 and D have the definitions indicated above, and also
their salts, solvates and solvates of the salts.
[1556] Preferred subject matter of the present invention are
binder-drug conjugates of the general formula (I), in which [1557]
n is a number from 1 to 20, [1558] AK is AK.sub.1 or AK.sub.2
[1559] where [1560] AK.sub.1 is an antibody or antigen-binding
antibody fragment (e.g., an antibody or an antigen-binding antibody
fragment which binds to C4.4a) and is bonded via the sulphur atom
of a cysteine residue of the binder to the group G, [1561] AK.sub.2
is an antibody or antigen-binding antibody fragment (e.g., an
antibody or an antigen-binding antibody fragment which binds to
C4.4a) and is bonded via the NH side group of a lysine residue of
the binder to the group G, [1562] G when AK=AK.sub.1, is a group of
the formula
[1562] ##STR00176## [1563] in which [1564] #.sup.1 marks the
linkage site with the cysteine residue of the binder, [1565]
#.sup.2 marks the linkage site with the group L.sup.1, [1566] or
[1567] when AK=AK.sub.2, is carbonyl, [1568] L.sup.1 is a bond,
linear (C.sub.2-C.sub.6)-alkanediyl or is a group of the
formula
[1568] ##STR00177## [1569] where [1570] m is a number from 2 to 6,
[1571] ##.sup.1 marks the linkage site with the group G, [1572]
##.sup.2 marks the linkage site with the group B, [1573] where
(C.sub.2-C.sub.6)-alkanediyl may be substituted by 1 or 2 methyl
substituents, [1574] B is a bond or a group of the formula
[1574] ##STR00178## [1575] where [1576] * marks the linkage site
with L.sup.1, [1577] ** marks the linkage site with L.sup.2, [1578]
P is O or NH, [1579] L.sup.3 is a bond or ethane-1,2-diyl, [1580]
L.sup.4 is a bond or a group of the formula
[1580] ##STR00179## [1581] in which [1582] *** marks the linkage
site with the carbonyl group, [1583] **** marks the linkage site
with L.sup.2, [1584] R.sup.25 is methyl, [1585] Q.sup.1 is a 4- to
6-membered carbocycle or piperidine-1,4-diyl, [1586] Q.sup.2 is
cyclopentyl or cyclohexyl, [1587] R.sup.14 is hydrogen, [1588]
R.sup.15 is hydrogen, [1589] R.sup.16 is hydrogen or methyl, [1590]
R.sup.17 is hydrogen or methyl, [1591] or [1592] R.sup.16 and
R.sup.17 together with the atoms to which they are bonded form a
piperazinyl ring, [1593] R.sup.18 is hydrogen, [1594] R.sup.19 is
hydrogen, methyl, propan-2-yl, 2-methylpropan-1-yl or
1-methylpropan-1-yl, [1595] R.sup.20 is hydrogen or methyl, [1596]
or [1597] R.sup.19 and R.sup.20 together with the atoms to which
they are bonded form a pyrrolidinyl ring, [1598] L.sup.2 is linear
(C.sub.2-C.sub.6)-alkanediyl or is a group of the formula
[1598] ##STR00180## [1599] where [1600] p is a number from 2 to 6,
[1601] ##.sup.3 marks the linkage site with the group B, [1602]
##.sup.4 marks the linkage site with the nitrogen atom, [1603]
where (C.sub.2-C.sub.6)-alkanediyl may be substituted by 1 or 2
methyl substituents, [1604] D is a group of the formula
[1604] ##STR00181## [1605] where [1606] #.sup.3 marks the linkage
site with the nitrogen atom, [1607] R.sup.1 is hydrogen, [1608]
R.sup.2 is 1-hydroxyethyl, benzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [1609] or [1610] R.sup.1 and R.sup.2 together
with the carbon atom to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1610] ##STR00182## [1611] in which [1612] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [1613] #.sup.5 marks
the linkage site with the carbonyl group, [1614] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[1614] ##STR00183## [1615] in which [1616] #.sup.6 marks the
linkage site with the carbonyl group, [1617] R.sup.6 is hydrogen,
hydroxy or benzyloxy, [1618] R.sup.3 is hydrogen, [1619] R.sup.4 is
1-hydroxyethyl, benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
[1620] or [1621] R.sup.3 and R.sup.4 together with the carbon atom
to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1621] ##STR00184## [1622] in which [1623] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [1624] #.sup.8 marks
the linkage site with the group T.sup.1, [1625] 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, [1626] in
which [1627] R.sup.7 is hydrogen, methyl, ethyl, n-propyl,
tert-butyl, benzyl or adamantylmethyl, [1628] R.sup.8 is hydrogen
or methyl, [1629] R.sup.9 is hydrogen, methyl, ethyl, n-propyl or
benzyl, [1630] or [1631] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are bonded form a 4- to 7-membered
heterocycle, [1632] R.sup.10 is benzoyl, [1633] R.sup.11 is benzyl,
which may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, [1634] R.sup.5 is hydrogen, methyl or a group of the
formula
[1634] ##STR00185## [1635] in which [1636] #.sup.9 marks the
linkage site with --CHC(R.sup.26)-T.sup.2, [1637] R.sup.12 is
phenyl which may be substituted by methoxycarbonyl, carboxyl or a
group of the formula --S(O).sub.2OH, [1638] R.sup.13 is phenyl
which may be substituted by methoxycarbonyl or carboxyl, [1639]
R.sup.26 is hydrogen or hydroxy, [1640] T.sup.2 is phenyl, benzyl,
1H-indol-3-yl or 1H-indol-3-ylmethyl, and also their salts,
solvates and solvates of the salts.
[1641] Particularly preferred subject matter of the present
invention are binder-drug conjugates of the general formula (I), in
which [1642] n is a number from 1 to 10, [1643] AK is AK.sub.1 or
AK.sub.2 [1644] where [1645] AK.sub.1 is an antibody or
antigen-binding antibody fragment (e.g., an antibody which
comprises the six CDR sequences of an antibody listed in Table 2,
the variable light and variable heavy chain of an antibody listed
in Table 2 or the light and heavy chain of an antibody listed in
Table 2), and is bonded via the sulphur atom of a cysteine residue
of the binder to the group G, [1646] AK.sub.2 is an antibody or
antigen-binding antibody fragment (e.g., an antibody which
comprises the six CDR sequences of an antibody listed in Table 2,
the variable light and variable heavy chain of an antibody listed
in Table 2 or the light and heavy chain of an antibody listed in
Table 2), and is bonded via the NH side group of a lysine residue
of the binder to the group G, [1647] G when AK=AK.sub.1, is a group
of the formula
[1647] ##STR00186## [1648] in which [1649] #.sup.1 marks the
linkage site with the cysteine residue of the binder, [1650]
#.sup.2 marks the linkage site with the group L.sup.1, [1651] or
[1652] when AK=AK.sub.2, is carbonyl, [1653] L.sup.1 is a bond,
linear (C.sub.2-C.sub.6)-alkanediyl or is a group of the
formula
[1653] ##STR00187## [1654] where [1655] m is a number 2 or 3,
[1656] ##.sup.1 marks the linkage site with the group G, [1657]
##.sup.2 marks the linkage site with the group B, [1658] where
(C.sub.2-C.sub.6)-alkanediyl may be substituted by 1 or 2 methyl
substituents, [1659] B is a bond or a group of the formula
[1659] ##STR00188## [1660] where [1661] * marks the linkage site
with L.sup.1, [1662] ** marks the linkage site with L.sup.2, [1663]
L.sup.3 is a bond or ethane-1,2-diyl, [1664] L.sup.4 is a bond or a
group of the formula
[1664] ##STR00189## [1665] where [1666] *** marks the linkage site
with the carbonyl group, [1667] **** marks the linkage site with
L.sup.2, [1668] R.sup.25 is methyl, [1669] R.sup.16 is hydrogen or
methyl, [1670] R.sup.17 is hydrogen or methyl, [1671] or [1672]
R.sup.16 and R.sup.17 together with the atoms to which they are
bonded form a piperazinyl ring, [1673] L.sup.2 is linear
(C.sub.2-C.sub.6)-alkanediyl, [1674] D is a group of the
formula
[1674] ##STR00190## [1675] where [1676] #.sup.3 marks the linkage
site with the nitrogen atom, [1677] R.sup.1 is hydrogen, [1678]
R.sup.2 is 1-hydroxyethyl, benzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [1679] or [1680] R.sup.1 and R.sup.2 together
with the carbon atom to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1680] ##STR00191## [1681] in which [1682] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [1683] #.sup.5 marks
the linkage site with the carbonyl group, [1684] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[1684] ##STR00192## [1685] in which [1686] #.sup.6 marks the
linkage site with the carbonyl group, [1687] R.sup.6 is hydrogen,
hydroxy or benzyloxy, [1688] R.sup.3 is hydrogen, [1689] R.sup.4 is
benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl, [1690] or [1691]
R.sup.3 and R.sup.4 together with the carbon atom to which they are
bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[1691] ##STR00193## [1692] in which [1693] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [1694] #.sup.8 marks
the linkage site with the group T.sup.1, [1695] 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, [1696] in which [1697] R.sup.7 is
hydrogen, methyl, ethyl, n-propyl, benzyl or adamantylmethyl,
[1698] R.sup.8 is hydrogen or methyl, [1699] R.sup.9 is hydrogen,
methyl, ethyl, n-propyl or benzyl, [1700] R.sup.11 is benzyl, which
may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, [1701] R.sup.5 is hydrogen or a group of the formula
[1701] ##STR00194## [1702] in which [1703] #.sup.9 marks the
linkage site with --CHC(R.sup.26)phenyl, [1704] R.sup.12 is phenyl
which may be substituted by methoxycarbonyl, carboxyl or a group of
the formula --S(O).sub.2OH, [1705] R.sup.13 is phenyl which may be
substituted by methoxycarbonyl or carboxyl, and also their salts,
solvates and solvates of the salts.
[1706] Particularly preferred subject matter of the present
invention are binder-drug conjugates of the general formula (I), in
which [1707] n is a number from 1 to 10, [1708] AK is AK.sub.1 or
AK.sub.2 [1709] where [1710] AK.sub.1 is an antibody or
antigen-binding antibody fragment (e.g., an antibody which
comprises the six CDR sequences of the antibody B01-3, B01-10 or
D02-6, the variable light and variable heavy chain of the antibody
B01-3, B01-10 or D02-6 or the light and heavy chain of the antibody
B01-3, B01-10 or D02-6), and is bonded via the sulphur atom of a
cysteine residue of the binder to the group G, [1711] AK.sub.2 is
an antibody or antigen-binding antibody fragment (e.g., an antibody
which comprises the six CDR sequences of the antibody B01-3,
B01-10, M31-B01 or D02-6, the variable light and variable heavy
chain of the antibody B01-3, B01-10, M31-B01 or D02-6 or the light
and heavy chain of the antibody B01-3, B01-10, M31-B01 or D02-6),
and is bonded via the NH side group of a lysine residue of the
binder to the group G, [1712] G when AK=AK.sub.1, is a group of the
formula
[1712] ##STR00195## [1713] in which [1714] #.sup.1 marks the
linkage site with the cysteine residue of the binder, [1715]
#.sup.2 marks the linkage site with the group L.sup.1, [1716] or
[1717] when AK=AK.sub.2, is carbonyl, [1718] L.sup.1 is a bond,
linear (C.sub.2-C.sub.6)-alkanediyl or is a group of the
formula
[1718] ##STR00196## [1719] where [1720] m is a number 2 or 3,
[1721] ##.sup.1 marks the linkage site with the group G, [1722]
##.sup.2 marks the linkage site with the group B, [1723] where
(C.sub.2-C.sub.6)-alkanediyl may be substituted by 1 or 2 methyl
substituents, [1724] B is a bond or a group of the formula
[1724] ##STR00197## [1725] where [1726] * marks the linkage site
with L.sup.1, [1727] ** marks the linkage site with L.sup.2, [1728]
L.sup.3 is a bond or ethane-1,2-diyl, [1729] L.sup.4 is a bond or a
group of the formula
[1729] ##STR00198## [1730] in which [1731] *** marks the linkage
site with the carbonyl group, [1732] **** marks the linkage site
with L.sup.2, [1733] R.sup.25 is methyl, [1734] R.sup.16 is
hydrogen or methyl, [1735] R.sup.17 is hydrogen or methyl, [1736]
or [1737] R.sup.16 and R.sup.17 together with the atoms to which
they are bonded form a piperazinyl ring, [1738] L.sup.2 is linear
(C.sub.2-C.sub.6)-alkanediyl, [1739] D is a group of the
formula
[1739] ##STR00199## [1740] where [1741] #.sup.3 marks the linkage
site with the nitrogen atom, [1742] R.sup.1 is hydrogen, [1743]
R.sup.2 is 1-hydroxyethyl, benzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [1744] or [1745] R.sup.1 and R.sup.2 together
with the carbon atom to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1745] ##STR00200## [1746] in which [1747] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [1748] #.sup.5 marks
the linkage site with the carbonyl group, [1749] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[1749] ##STR00201## [1750] in which [1751] #.sup.6 marks the
linkage site with the carbonyl group, [1752] R.sup.6 is hydrogen,
hydroxy or benzyloxy, [1753] R.sup.3 is hydrogen, [1754] R.sup.4 is
benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl, [1755] or [1756]
R.sup.3 and R.sup.4 together with the carbon atom to which they are
bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[1756] ##STR00202## [1757] in which [1758] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [1759] #.sup.8 marks
the linkage site with the group T, [1760] 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, [1761] in which [1762] R.sup.7 is
hydrogen, methyl, ethyl, n-propyl, benzyl or adamantylmethyl,
[1763] R.sup.8 is hydrogen or methyl, [1764] R.sup.9 is hydrogen,
methyl, ethyl, n-propyl or benzyl, [1765] R.sup.11 is benzyl, which
may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, [1766] R.sup.5 is hydrogen or a group of the formula
[1766] ##STR00203## [1767] in which [1768] #.sup.9 marks the
linkage site with --CHC(R.sup.26)phenyl, [1769] R.sup.12 is phenyl
which may be substituted by methoxycarbonyl, carboxyl or a group of
the formula --S(O).sub.2OH, [1770] R.sup.13 is phenyl which may be
substituted by methoxycarbonyl or carboxyl, and also their salts,
solvates and solvates of the salts.
[1771] The present invention additionally provides compounds of the
formula (XXX)
##STR00204## [1772] in which [1773] Cys is a cysteine residue which
is bonded via the sulphur atom of the side chain to a carbon atom
of the succinimide. [1774] L.sup.1 is a bond, linear
(C.sub.1-C.sub.10)-alkanediyl or is a group of the formula
[1774] ##STR00205## [1775] in which [1776] m is a number from 2 to
6, [1777] ##.sup.1 marks the linkage site with the group G, [1778]
##.sup.2 marks the linkage site with the group B, [1779] where
(C.sub.1-C.sub.10)-alkanediyl may be substituted by 1 to 4 methyl
substituents, [1780] and [1781] where two carbon atoms of the
alkanediyl chain in 1,2, 1, 3 or 1,4 relation to one another, with
inclusion of any carbon atoms situated between them, may be bridged
to form a (C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring,
[1782] B is a bond or a group of the formula
[1782] ##STR00206## [1783] where [1784] * marks the linkage site
with L.sup.1, [1785] ** marks the linkage site with L.sup.2, [1786]
P is O or NH, [1787] L.sup.3 is a bond or
(C.sub.2-C.sub.4)-alkanediyl, [1788] L.sup.4 is a bond or a group
of the formula
[1788] ##STR00207## [1789] in which [1790] *** marks the linkage
site with the carbonyl group, [1791] **** marks the linkage site
with L.sup.2, [1792] R.sup.25 is hydrogen or methyl [1793] Q.sup.1
is a 3- to 7-membered carbocycle or a 4- to 7-membered aza
heterocycle, [1794] Q.sup.2 is a 3- to 7-membered carbocycle or a
4- to 7-membered aza heterocycle, [1795] R.sup.14 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1796] R.sup.15 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1797] or [1798] R.sup.14 and R.sup.15
together with the atoms to which they are bonded form a 5- or
6-membered heterocycle, [1799] R.sup.16 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1800] R.sup.17 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1801] or [1802] R.sup.16 and R.sup.17
together with the atoms to which they are bonded form a 5- or
6-membered heterocycle, [1803] R.sup.18 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1804] R.sup.19 is hydrogen or the side
group of a natural .alpha.-amino acid or of its homologues or
isomers, [1805] R.sup.20 is hydrogen or (C.sub.1-C.sub.4)-alkyl,
[1806] or [1807] R.sup.19 and R.sup.20 together with the atoms to
which they are bonded form a pyrrolidinyl ring, [1808] R.sup.21 is
hydrogen or (C.sub.1-C.sub.4)-alkyl, [1809] R.sup.22 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [1810] or [1811] R.sup.21 and R.sup.22
together with the atoms to which they are bonded form a 3- to
7-membered carbocycle, [1812] R.sup.23 is (C.sub.1-C.sub.4)-alkyl,
[1813] R.sup.24 is hydrogen or (C.sub.1-C.sub.4)-alkyl, [1814]
R.sup.27 is hydrogen or (C.sub.1-C.sub.4)-alkyl, [1815] L.sup.2 is
linear (C.sub.2-C.sub.10)-alkanediyl or is a group of the
formula
[1815] ##STR00208## [1816] where [1817] p is a number from 2 to 6,
[1818] ##.sup.3 marks the linkage site with the group B, [1819]
##.sup.4 marks the linkage site with the nitrogen atom, [1820]
where (C.sub.2-C.sub.10)-alkanediyl may be substituted by 1 to 4
substituents, [1821] and [1822] where two carbon atoms of the
alkanediyl chain in 1,2, 1, 3 or 1,4 relation to one another, with
inclusion of any carbon atoms situated between them, may be bridged
to form a (C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring,
[1823] D is a group of the formula
[1823] ##STR00209## [1824] in which [1825] #.sup.3 marks the
linkage site with the nitrogen atom, [1826] R.sup.1 is hydrogen,
[1827] R.sup.2 is 1-hydroxyethyl, benzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [1828] or [1829] R.sup.1 and R.sup.2 together
with the carbon atom to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1829] ##STR00210## [1830] in which [1831] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [1832] #.sup.5 marks
the linkage site with the carbonyl group, [1833] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[1833] ##STR00211## [1834] in which [1835] #.sup.6 marks the
linkage site with the carbonyl group, [1836] R.sup.6 is hydrogen,
hydroxy or benzyloxy, [1837] R.sup.3 is hydrogen, [1838] R.sup.4 is
1-hydroxyethyl, benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl,
[1839] or [1840] R.sup.3 and R.sup.4 together with the carbon atom
to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1840] ##STR00212## [1841] in which [1842] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [1843] #.sup.8 marks
the linkage site with the group T.sup.1, [1844] 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, [1845] in
which [1846] R.sup.7 is hydrogen, methyl, ethyl, n-propyl,
tert-butyl, benzyl or adamantylmethyl, [1847] R.sup.8 is hydrogen
or methyl, [1848] R.sup.9 is hydrogen, methyl, ethyl, n-propyl or
benzyl, [1849] or [1850] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are bonded form a 4- to 7-membered
heterocycle, [1851] R.sup.10 is benzoyl, [1852] R.sup.11 is benzyl,
which may be substituted in the phenyl group by methoxycarbonyl or
carboxyl, [1853] R.sup.5 is hydrogen, methyl or a group of the
formula
[1853] ##STR00213## [1854] in which [1855] #.sup.9 marks the
linkage site with --CHC(R.sup.26)-T.sup.2, [1856] R.sup.12 is
phenyl which may be substituted by methoxycarbonyl, carboxyl or a
group of the formula --S(O).sub.2OH, [1857] R.sup.13 is phenyl
which may be substituted by methoxycarbonyl or carboxyl, [1858]
R.sup.26 is hydrogen or hydroxy, [1859] T.sup.2 is phenyl, benzyl,
1H-indol-3-yl or 1H-indol-3-ylmethyl, and also their salts,
solvates and solvates of the salts.
[1860] Particularly preferred in the context of the present
invention in addition are also compounds of the formula (XXX), in
which [1861] Cys is a cysteine residue which is bonded via the
sulphur atom of the side chain via a carbon atom of the
succinimide, [1862] L.sup.1 is a bond, linear
(C.sub.2-C.sub.6)-alkanediyl or is a group of the formula
[1862] ##STR00214## [1863] in which [1864] m is a number from 2 to
6, [1865] ##.sup.1 marks the linkage site with the group G, [1866]
##.sup.2 marks the linkage site with the group B, [1867] where
(C.sub.2-C.sub.6)-alkanediyl may be substituted by 1 or 2 methyl
substituents, [1868] B is a bond or a group of the formula
[1868] ##STR00215## [1869] where [1870] * marks the linkage site
with L.sup.1, [1871] ** marks the linkage site with L.sup.2, [1872]
L.sup.3 is a bond or ethane-1,2-diyl, [1873] L.sup.4 is a bond,
[1874] R.sup.14 is hydrogen, [1875] R.sup.15 is hydrogen, [1876]
R.sup.16 is hydrogen or methyl, [1877] R.sup.17 is hydrogen or
methyl, [1878] or [1879] R.sup.16 and R.sup.17 together with the
atoms to which they are bonded form a piperazinyl ring, [1880]
R.sup.23 is methyl, [1881] R.sup.24 is hydrogen or methyl, [1882]
L.sup.2 is linear (C.sub.2-C.sub.6)-alkanediyl or is a group of the
formula
[1882] ##STR00216## [1883] where [1884] p is a number 2 or 3,
[1885] ##.sup.3 marks the linkage site with the group B, [1886]
##.sup.4 marks the linkage site with the nitrogen atom, [1887] D is
a group of the formula
[1887] ##STR00217## [1888] in which [1889] #.sup.3 marks the
linkage site with the nitrogen atom, [1890] R.sup.1 is hydrogen,
[1891] R.sup.2 is 1-hydroxyethyl, benzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [1892] or [1893] R.sup.1 and R.sup.2 together
with the carbon atom to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[1893] ##STR00218## [1894] in which [1895] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [1896] #.sup.5 marks
the linkage site with the carbonyl group, [1897] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[1897] ##STR00219## [1898] in which [1899] #.sup.6 marks the
linkage site with the carbonyl group, [1900] R.sup.6 is hydrogen,
hydroxy or benzyloxy, [1901] R.sup.3 is hydrogen, [1902] R.sup.4 is
benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl, [1903] or [1904]
R.sup.3 and R.sup.4 together with the carbon atom to which they are
bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[1904] ##STR00220## [1905] in which [1906] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [1907] #.sup.8 marks
the linkage site with the group T, [1908] 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, [1909] in
which [1910] R.sup.7 is hydrogen, methyl, ethyl, n-propyl, benzyl
or adamantylmethyl, [1911] R.sup.8 is hydrogen or methyl, [1912]
R.sup.9 is hydrogen, methyl, ethyl, n-propyl or benzyl, [1913]
R.sup.10 is benzoyl, [1914] R.sup.11 is benzyl, which may be
substituted in the phenyl group by methoxycarbonyl or carboxyl,
[1915] R.sup.5 is hydrogen or a group of the formula
[1915] ##STR00221## [1916] in which [1917] #.sup.9 marks the
linkage site with --CHC(R.sup.26)phenyl, [1918] R.sup.12 is phenyl
which may be substituted by methoxycarbonyl, carboxyl or a group of
the formula --S(O).sub.2OH, [1919] R.sup.13 is phenyl which may be
substituted by methoxycarbonyl or carboxyl, and also their salts,
solvates and solvates of the salts.
[1920] Preferred in the context of the present invention are also
compounds of the formula (Ia), in which n=1-20, more preferably
n=1-10 and very preferably n=2-8.
[1921] Preferred in the context of the present invention are also
compounds of the formula (Ia), in which [1922] AK is AK.sub.1
[1923] where [1924] AK.sub.1 is an antibody or antigen-binding
antibody fragment (e.g., an antibody or an antigen-binding antibody
fragment which binds to C4.4a) and is bonded via the sulphur atom
of a cysteine residue of the binder to the group G, [1925] G is a
group of the formula
[1925] ##STR00222## [1926] where [1927] #.sup.1 marks the linkage
site with the cysteine residue of the binder, [1928] #.sup.2 marks
the linkage site with the group L.sup.1, and [1929] n, L.sup.1, B,
L.sup.2, D and R.sup.35 have the definitions indicated above, and
also their salts, solvates and solvates of the salts.
[1930] Preferred in the context of the present invention are also
compounds of the formula (Ia), in which [1931] AK is AK.sub.2
[1932] where [1933] AK.sub.2 is an antibody or antigen-binding
antibody fragment (e.g., an antibody or an antigen-binding antibody
fragment which binds to C4.4a) and is bonded via the NH side group
of a lysine residue of the binder to the group G, [1934] G is
carbonyl, and [1935] n, L.sup.1, B, L.sup.2, D and R.sup.35 have
the definitions indicated above, and also their salts, solvates and
solvates of the salts.
[1936] Preference in the context of the present invention is also
given to compounds of the formula (Ia), in which [1937] AK is
AK.sub.1 [1938] where [1939] AK.sub.1 is an antibody or
antigen-binding antibody fragment (e.g., an antibody which
comprises the six CDR sequences of the antibody B01-3, B01-10 or
D02-6, the variable light and variable heavy chain of the antibody
B01-3, B01-10 or D02-6 or the light and heavy chain of the antibody
B01-3, B01-10 or D02-6), and which is attached via the sulphur atom
of a cysteine residue of the binder to the group G, [1940] G is a
group of the formula
[1940] ##STR00223## [1941] where [1942] #.sup.1 marks the linkage
site with the cysteine residue of the binder, [1943] #.sup.2 marks
the linkage site with the group L.sup.1, and [1944] n, L.sup.1, B,
L.sup.2, D and R.sup.35 have the definitions indicated above, and
also their salts, solvates and solvates of the salts.
[1945] Preference in the context of the present invention is also
given to compounds of the formula (Ia), in which [1946] AK is
AK.sub.2 [1947] where [1948] AK.sub.2 is an antibody or
antigen-binding antibody fragment (e.g., an antibody which
comprises the six CDR sequences of the antibody B01-3, B01-10,
M31-B01 or D02-6, the variable light and variable heavy chain of
the antibody B01-3, B01-10, M31-B01 or D02-6 or the light and heavy
chain of the antibody B01-3, B01-10, M31-B01 or D02-6), and which
is bonded via the NH side group of a lysine residue of the binder
to the group G, [1949] G is carbonyl, and [1950] n, L.sup.1, B,
L.sup.2, D and R.sup.35 have the definitions indicated above, and
also their salts, solvates and solvates of the salts.
[1951] Preference in the context of the present invention is also
given to compounds of the general formula (Ia), in which [1952] AK
is AK.sub.2 [1953] where [1954] AK.sub.2 is an antibody or
antigen-binding antibody fragment (e.g., an antibody which
comprises the six CDR sequences of the antibody B01-3, B01-10,
M31-B01 or D02-6, the variable light and variable heavy chain of
the antibody B01-3, B01-10, M31-B01 or D02-6 or the light and heavy
chain of the antibody B01-3, B01-10, M31-B01 or D02-6), and which
is bonded via the NH side group of a lysine residue of the binder
to the group G, [1955] G is carbonyl, [1956] L.sup.1 is a bond,
[1957] B is a bond, [1958] L.sup.2 is linear
(C.sub.3-C.sub.6)-alkanediyl or is a group of the formula
[1958] ##STR00224## [1959] where [1960] p is a number 2 or 3,
[1961] ##.sup.3 marks the linkage site with the group B, [1962]
##.sup.4 marks the linkage site with the nitrogen atom, [1963] n, D
and R.sup.35 have the definitions indicated above, and also their
salts, solvates and solvates of the salts.
[1964] Preference in the context of the present invention is also
given to compounds of the general formula (Ia), in which [1965] AK
is AK.sub.1 [1966] where [1967] AK.sub.1 is an antibody or
antigen-binding antibody fragment (e.g., an antibody which
comprises the six CDR sequences of the antibody B01-3, B01-10 or
D02-6, the variable light and variable heavy chain of the antibody
B01-3, B01-10 or D02-6 or the light and heavy chain of the antibody
B01-3, B01-10 or D02-6), and which is attached via the sulphur atom
of a cysteine residue of the binder to the group G, [1968] G is a
group of the formula
[1968] ##STR00225## [1969] where [1970] #.sup.1 marks the linkage
site with the cysteine residue of the binder, [1971] #.sup.2 marks
the linkage site with the group L.sup.1, [1972] L.sup.1 is a bond,
linear (C.sub.3-C.sub.5)-alkanediyl or a group of the formula
[1972] ##STR00226## [1973] where [1974] m is a number 2 or 3,
[1975] ##.sup.1 marks the linkage site with the group G, [1976]
##.sup.2 marks the linkage site with the group B, [1977] where
(C.sub.3-C.sub.5)-alkanediyl may be substituted by 1 or 2 methyl
substituents, [1978] B is a bond or a group of the formula
[1978] ##STR00227## [1979] where [1980] * marks the linkage site
with L.sup.1, [1981] ** marks the linkage site with L.sup.2, [1982]
L.sup.3 is a bond or ethane-1,2-diyl, [1983] L.sup.4 is a bond or a
group of the formula
[1983] ##STR00228## [1984] in which [1985] *** marks the linkage
site with the carbonyl group, [1986] **** marks the linkage site
with L.sup.2, [1987] R.sup.25 is methyl, [1988] R.sup.28 is
hydrogen, methylcarbonyl or tert-butyloxycarbonyl, [1989] R.sup.16
is hydrogen or methyl, [1990] R.sup.17 is hydrogen or methyl,
[1991] or [1992] R.sup.16 and R.sup.17 together with the atoms to
which they are bonded form a piperazinyl ring, [1993] L.sup.2 is
linear (C.sub.3-C.sub.5)-alkanediyl or is a group of the
formula
[1993] ##STR00229## [1994] where [1995] p is a number 2 or 3,
[1996] ##.sup.3 marks the linkage site with the group B, [1997]
##.sup.4 marks the linkage site with the nitrogen atom, and [1998]
n, D and R.sup.35 have the definitions indicated above, and also
their salts, solvates and solvates of the salts.
[1999] Preferred in the context of the present invention are also
compounds of the formula (Ia), (XXXa) and (XXXI), in which [2000]
L.sup.1 is a bond, [2001] B is a bond, [2002] L.sup.2 is linear
(C.sub.3-C.sub.6)-alkanediyl or is a group of the formula
[2002] ##STR00230## [2003] where [2004] p is a number 2 or 3,
[2005] ##.sup.3 marks the linkage site with the group B, [2006]
##.sup.4 marks the linkage site with the nitrogen atom, and [2007]
n, AK, Cys, G, D and R.sup.35 have the definitions indicated above,
and also their salts, solvates and solvates of the salts.
[2008] Preferred in the context of the present invention are also
compounds of the formula (Ia), in which [2009] L.sup.1 is linear
(C.sub.1-C.sub.10)-alkanediyl or a group of the formula
[2009] ##STR00231## [2010] where [2011] m is a number from 2 to 6,
[2012] ##.sup.1 marks the linkage site with the group G, [2013]
##.sup.2 marks the linkage site with the group B, [2014] where
(C.sub.1-C.sub.10)-alkanediyl may be substituted by 1 to 4
substituents selected independently of one another from the group
consisting of methyl, hydroxyl and benzyl, [2015] B is a bond or a
group of the formula
[2015] ##STR00232## [2016] where [2017] * marks the linkage site
with L.sup.1, [2018] ** marks the linkage site with L.sup.2, [2019]
L.sup.3 is a bond or (C.sub.2-C.sub.4)-alkanediyl, [2020] L.sup.4
is a group of the formula
[2020] ##STR00233## [2021] in which [2022] *** marks the linkage
site with the carbonyl group, [2023] **** marks the linkage site
with L.sup.2, [2024] R.sup.25 is hydrogen or methyl, [2025]
R.sup.28 is hydrogen, (C.sub.1-C.sub.4)-alkylcarbonyl,
tert-butyloxycarbonyl or benzyloxycarbonyl, [2026] Q.sup.1 is a 4-
to 7-membered heterocycle, [2027] R.sup.16 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [2028] R.sup.17 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [2029] or [2030] R.sup.16 and R.sup.17
together with the atoms to which they are bonded form a 5- or
6-membered heterocycle, [2031] R.sup.23 is (C.sub.1-C.sub.4)-alkyl,
[2032] R.sup.24 is hydrogen or (C.sub.1-C.sub.4)-alkyl, [2033]
R.sup.36 is hydrogen, (C.sub.1-C.sub.4)-alkylcarbonyl,
tert-butyloxycarbonyl or benzyloxycarbonyl, [2034] R.sup.37 is
hydrogen or methyl, [2035] or [2036] R.sup.36 and R.sup.37 together
with the atoms to which they are bonded form a pyrrolidine ring,
[2037] L.sup.2 is linear (C.sub.2-C.sub.10)-alkanediyl or is a
group of the formula
[2037] ##STR00234## [2038] where [2039] p is a number from 2 to 6,
[2040] ##.sup.3 marks the linkage site with the group B, [2041]
##.sup.4 marks the linkage site with the nitrogen atom, [2042]
where (C.sub.2-C.sub.10)-alkanediyl may be substituted by 1 to 4
substituents selected independently of one another from the group
consisting of methyl, hydroxyl and benzyl, [2043] and [2044] n, AK,
G, D and R.sup.35 have the definitions indicated above, [2045] and
also their salts, solvates and solvates of the salts.
[2046] Preferred in the context of the present invention are also
compounds of the formula (Ia), in which [2047] L.sup.1 is linear
(C.sub.2-C.sub.6)-alkanediyl or a group of the formula
[2047] ##STR00235## [2048] where [2049] m is a number 2 or 3,
[2050] ##.sup.1 marks the linkage site with the group G, [2051]
##.sup.2 marks the linkage site with the group B, [2052] B is a
bond or a group of the formula
[2052] ##STR00236## [2053] where [2054] * marks the linkage site
with L.sup.1, [2055] ** marks the linkage site with L.sup.2, [2056]
L.sup.3 is a bond or ethane-1,2-diyl, [2057] L.sup.4 is a group of
the formula
[2057] ##STR00237## [2058] where [2059] *** marks the linkage site
with the carbonyl group, [2060] **** marks the linkage site with
L.sup.2, [2061] R.sup.25 is hydrogen or methyl, [2062] R.sup.28 is
hydrogen, methylcarbonyl or tert-butyloxycarbonyl, [2063] R.sup.16
is hydrogen or methyl, [2064] R.sup.17 is hydrogen or methyl,
[2065] or [2066] R.sup.16 and R.sup.17 together with the atoms to
which they are bonded form a piperazinyl ring, [2067] R.sup.36 is
hydrogen, methylcarbonyl or tert-butyloxycarbonyl, [2068] R.sup.37
is hydrogen or methyl, [2069] or [2070] R.sup.36 and R.sup.37
together with the atoms to which they are bonded form a pyrrolidine
ring, [2071] L.sup.2 is linear (C.sub.2-C.sub.6)-alkanediyl or is a
group of the formula
[2071] ##STR00238## [2072] where [2073] p is a number 2 or 3,
[2074] ##.sup.3 marks the linkage site with the group B, [2075]
##.sup.4 marks the linkage site with the nitrogen atom, and [2076]
n, AK, G, D and R.sup.35 have the definitions indicated above, and
also their salts, solvates and solvates of the salts.
[2077] Preferred in the context of the present invention are also
compounds of the formula (Ia) and (XXXa), in which [2078] G is a
group of the formula
[2078] ##STR00239## [2079] in which [2080] #.sup.1 marks the
linkage site with the cysteine residue of the binder, [2081]
#.sup.2 marks the linkage site with the group L.sup.1, [2082]
L.sup.1 is linear (C.sub.3-C.sub.5)-alkanediyl or a group of the
formula
[2082] ##STR00240## [2083] in which [2084] m is a number 2 or 3,
[2085] ##.sup.1 marks the linkage site with the group G, [2086]
##.sup.2 marks the linkage site with the group B, [2087] where
(C.sub.3-C.sub.5)-alkanediyl may be substituted by 1 or 2 methyl
substituents, [2088] B is a bond or a group of the formula
[2088] ##STR00241## [2089] where [2090] * marks the linkage site
with L.sup.1, [2091] ** marks the linkage site with L.sup.2, [2092]
L.sup.3 is a bond or ethane-1,2-diyl, [2093] L.sup.4 is a bond,
[2094] L.sup.2 is linear (C.sub.3-C.sub.5)-alkanediyl or is a group
of the formula
[2094] ##STR00242## [2095] where [2096] p is a number 2 or 3,
[2097] ##.sup.3 marks the linkage site with the group B, [2098]
##.sup.4 marks the linkage site with the nitrogen atom, and [2099]
n, AK.sub.1, Cys, D, R.sup.16 and R.sup.17 have the definitions
indicated above, and also their salts, solvates and solvates of the
salts.
[2100] Preferred in the context of the present invention are also
compounds of the formula (Ia) and (XXXa), in which [2101] B is a
bond or a group of the formula
[2101] ##STR00243## [2102] where [2103] * marks the linkage site
with L.sup.1, [2104] ** marks the linkage site with L.sup.2, [2105]
L.sup.3 is a bond or ethane-1,2-diyl, [2106] L.sup.4 is a bond,
[2107] n, AK, Cys, G, L.sup.1, L.sup.2, D, R.sup.16, R.sup.17 and
R.sup.35 have the definitions indicated above, and also their
salts, solvates and solvates of the salts.
[2108] Preferred in the context of the present invention are also
compounds of the formula (Ia), (XXXa) and (XXXI), in which [2109]
L.sup.1 is a bond, linear (C.sub.3-C.sub.5)-alkanediyl or a group
of the formula
[2109] ##STR00244## [2110] where [2111] m is a number 2 or 3,
[2112] ##.sup.1 marks the linkage site with the group G, [2113]
##.sup.2 marks the linkage site with the group B, [2114] where
(C.sub.3-C.sub.5)-alkanediyl may be substituted by 1 or 2 methyl
substituents, [2115] B is a bond or a group of the formula
[2115] ##STR00245## [2116] where [2117] * marks the linkage site
with L.sup.1, [2118] ** marks the linkage site with L.sup.2, [2119]
L.sup.3 is a bond, [2120] L.sup.4 is a bond [2121] R.sup.16 is
hydrogen, [2122] R.sup.17 is hydrogen, [2123] L.sup.2 is linear
(C.sub.3-C.sub.6)-alkanediyl or is a group of the formula
[2123] ##STR00246## [2124] where [2125] p is a number 2 or 3,
[2126] ##.sup.3 marks the linkage site with the group B, [2127]
##.sup.4 marks the linkage site with the nitrogen atom, [2128] n,
AK, Cys, G, D and R.sup.35 have the definitions indicated above,
and also their salts, solvates and solvates of the salts.
[2129] Preferred in the context of the present invention are also
compounds of the formula (Ia), (XXXa) and (XXXI), in which [2130]
L.sup.1 is a bond, [2131] B is a bond, [2132] L.sup.2 is linear
(C.sub.3-C.sub.6)-alkanediyl or is a group of the formula
[2132] ##STR00247## [2133] where [2134] p is a number 2 or 3,
[2135] ##.sup.3 marks the linkage site with the group B, [2136]
##.sup.4 marks the linkage site with the nitrogen atom, [2137] n,
AK, Cys, G, D and R.sup.35 have the definitions indicated above,
and also their salts, solvates and solvates of the salts.
[2138] Preferred in the context of the present invention are also
compounds of the formula (Ia), (XXXa) and (XXXI), in which [2139]
L.sup.1 is linear (C.sub.3-C.sub.5)-alkanediyl or a group of the
formula
[2139] ##STR00248## [2140] where [2141] m is a number 2 or 3,
[2142] ##.sup.1 marks the linkage site with the group G, [2143]
##.sup.2 marks the linkage site with the group B, [2144] where
(C.sub.3-C.sub.5)-alkanediyl may be substituted by 1 or 2 methyl
substituents, [2145] B is a group of the formula
[2145] ##STR00249## [2146] where [2147] * marks the linkage site
with L.sup.1, [2148] ** marks the linkage site with L.sup.2, [2149]
L.sup.3 is a bond, [2150] L.sup.4 is a bond, [2151] R.sup.16 is
hydrogen, [2152] R.sup.17 is hydrogen, [2153] L.sup.2 is linear
(C.sub.3-C.sub.6)-alkanediyl or is a group of the formula
[2153] ##STR00250## [2154] where [2155] p is a number 2 or 3,
[2156] ##.sup.3 marks the linkage site with the group B, [2157]
##.sup.4 marks the linkage site with the nitrogen atom, [2158] n,
AK, Cys, G, D and R.sup.35 have the definitions indicated above,
and also their salts, solvates and solvates of the salts.
[2159] Preferred in the context of the present invention are also
compounds of the formula (Ia), in which [2160] n is a number from 2
to 8, preferably 2 to 5, [2161] AK is AK.sub.1 or AK.sub.2 [2162]
where [2163] AK.sub.1 is an antibody or antigen-binding antibody
fragment (e.g., an antibody or an antigen-binding antibody fragment
which binds to C4.4a) and is bonded via the sulphur atom of a
cysteine residue of the binder to the group G, [2164] AK.sub.2 is
an antibody or antigen-binding antibody fragment (e.g., is an
antibody or an antigen-binding antibody fragment which binds to
C4.4a) and is bonded via the NH side group of a lysine residue of
the binder to the group G, [2165] G when AK=AK.sub.1, is a group of
the formula
[2165] ##STR00251## [2166] in which [2167] #.sup.1 marks the
linkage site with the cysteine residue of the binder, [2168]
#.sup.2 marks the linkage site with the group L.sup.1, [2169] or
[2170] when AK=AK.sub.2, is carbonyl, [2171] L.sup.1 is a bond,
linear (C.sub.3-C.sub.5)-alkanediyl or a group of the formula
[2171] ##STR00252## [2172] where [2173] m is a number 2 or 3,
[2174] ##.sup.1 marks the linkage site with the group G, [2175]
##.sup.2 marks the linkage site with the group B, [2176] where
(C.sub.3-C.sub.5)-alkanediyl may be substituted by 1 or 2 methyl
substituents, [2177] B is a bond or a group of the formula
[2177] ##STR00253## [2178] where [2179] * marks the linkage site
with L.sup.1, [2180] ** marks the linkage site with L.sup.2, [2181]
L.sup.3 is a bond, [2182] L.sup.4 is a bond, [2183] R.sup.16 is
hydrogen, [2184] R.sup.17 is hydrogen, [2185] L.sup.2 is linear
(C.sub.3-C.sub.6)-alkanediyl or is a group of the formula
[2185] ##STR00254## [2186] where [2187] p is a number 2 or 3,
[2188] ##.sup.3 marks the linkage site with the group B, [2189]
##.sup.4 marks the linkage site with the nitrogen atom, and [2190]
D and R.sup.35 have the definitions indicated above, and also their
salts, solvates and solvates of the salts.
[2191] Preferred in the context of the present invention are also
compounds of the formula (Ia), in which [2192] n is a number from 2
to 8, preferably 2 to 5, [2193] AK is AK.sub.1 or AK.sub.2, [2194]
where [2195] AK.sub.1 is an antibody or antigen-binding antibody
fragment (e.g., an antibody or an antigen-binding antibody fragment
which binds to C4.4a) and is bonded via the sulphur atom of a
cysteine residue of the binder to the group G, [2196] AK.sub.2 is
an antibody or antigen-binding antibody fragment (e.g., an antibody
or an antigen-binding antibody fragment which binds to C4.4a) and
is bonded via the NH side group of a lysine residue of the binder
to the group G, [2197] G when AK=AK.sub.1, is a group of the
formula
[2197] ##STR00255## [2198] in which [2199] #.sup.1 marks the
linkage site with the cysteine residue of the binder, [2200]
#.sup.2 marks the linkage site with the group L.sup.1, [2201] or
[2202] when AK=AK.sub.2, is carbonyl, [2203] L.sup.1 is a bond,
[2204] B is a bond, [2205] L.sup.2 is linear
(C.sub.3-C.sub.6)-alkanediyl or is a group of the formula
[2205] ##STR00256## [2206] where [2207] p is a number 2 or 3,
[2208] ##.sup.3 marks the linkage site with the group B, [2209]
##.sup.4 marks the linkage site with the nitrogen atom, [2210] D
and R.sup.35 have the definitions indicated above, and also their
salts, solvates and solvates of the salts.
[2211] Preferred in the context of the present invention are also
compounds of the formula (Ia), in which [2212] n is a number from 2
to 8, preferably 2 to 5, [2213] AK is AK.sub.1, [2214] where [2215]
AK.sub.1 is an antibody or antigen-binding antibody fragment (e.g.,
an antibody or an antigen-binding antibody fragment which binds to
C4.4a) and is bonded via the sulphur atom of a cysteine residue of
the binder to the group G, [2216] G is a group of the formula
[2216] ##STR00257## [2217] where [2218] #.sup.1 marks the linkage
site with the cysteine residue of the binder, [2219] #.sup.2 marks
the linkage site with the group L.sup.1, [2220] L.sup.1 is linear
(C.sub.3-C.sub.5)-alkanediyl or a group of the formula
[2220] ##STR00258## [2221] where [2222] m is a number 2 or 3,
[2223] ##.sup.1 marks the linkage site with the group G, [2224]
##.sup.2 marks the linkage site with the group B, [2225] where
(C.sub.3-C.sub.5)-alkanediyl may be substituted by 1 or 2 methyl
substituents, [2226] B is a group of the formula
[2226] ##STR00259## [2227] where [2228] * marks the linkage site
with L.sup.1, [2229] ** marks the linkage site with L.sup.2, [2230]
L.sup.3 is a bond, [2231] L.sup.4 is a bond, [2232] R.sup.16 is
hydrogen, [2233] R.sup.17 is hydrogen, [2234] L.sup.2 is linear
(C.sub.3-C.sub.6)-alkanediyl or is a group of the formula
[2234] ##STR00260## [2235] where [2236] p is a number 2 or 3,
[2237] ##.sup.3 marks the linkage site with the group B, [2238]
##.sup.4 marks the linkage site with the nitrogen atom, [2239] D
and R.sup.35 have the definitions indicated above, and also their
salts, solvates and solvates of the salts.
[2240] Preferred in the context of the present invention are also
compounds of the formula (Ia), in which [2241] L.sup.1 is a bond,
linear (C.sub.3-C.sub.5)-alkanediyl, a group of the formula
[2241] ##STR00261## [2242] where [2243] m is a number 2 or 3,
[2244] ##.sup.1 marks the linkage site with the group G, [2245]
##.sup.2 marks the linkage site with the group B, [2246] L.sup.1A
is linear (C.sub.3-C.sub.6)-alkanediyl, [2247] B.sup.1 is a group
of the formula
[2247] ##STR00262## [2248] in which [2249] ##.sup.5 marks the
linkage site with the group L.sup.1A, [2250] ##.sup.6 marks the
linkage site with the group L.sup.1B, [2251] L.sup.5 is a bond or
ethane-1,2-diyl, [2252] L.sup.6 is a bond or a group of the
formula
[2252] ##STR00263## [2253] in which [2254] ##.sup.7 marks the
linkage site with the carbonyl group, [2255] ##.sup.8 marks the
linkage site with L.sup.1B, [2256] R.sup.33 is hydrogen,
(C.sub.1-C.sub.4)-alkylcarbonyl or tert-butyloxycarbonyl, [2257]
R.sup.34 is hydrogen or methyl, [2258] R.sup.29 is hydrogen or
methyl, [2259] R.sup.30 is hydrogen or methyl, [2260] R.sup.31 is
hydrogen or methyl, [2261] R.sup.32 is hydrogen or methyl, [2262]
L.sup.1B is linear (C.sub.3-C.sub.6)-alkanediyl, [2263] B is a bond
or a group of the formula
[2263] ##STR00264## [2264] where [2265] marks the linkage site with
L.sup.1, [2266] ** marks the linkage site with L.sup.2, [2267] P is
O, [2268] L.sup.3 is a bond or ethane-1,2-diyl, [2269] L.sup.4 is a
group of the formula
[2269] ##STR00265## [2270] in which [2271] *** marks the linkage
site with the carbonyl group, [2272] **** marks the linkage site
with L.sup.2, [2273] R.sup.25 is hydrogen or methyl, [2274]
R.sup.28 is hydrogen, (C.sub.1-C.sub.4)-alkylcarbonyl or
tert-butyloxycarbonyl, [2275] Q.sup.1 is a 4- to 7-membered
heterocycle, [2276] Q.sup.2 is a 3- to 7-membered carbocycle or a
4- to 7-membered heterocycle, [2277] R.sup.16 is hydrogen or
methyl, [2278] R.sup.17 is hydrogen or methyl, [2279] R.sup.23 is
(C.sub.1-C.sub.4)-alkyl, [2280] R.sup.24 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [2281] R.sup.36 is hydrogen,
(C.sub.1-C.sub.4)-alkylcarbonyl or tert-butyloxycarbonyl, [2282]
R.sup.37 is hydrogen or methyl, [2283] or [2284] R.sup.36 and
R.sup.37 together with the atoms to which they are bonded form a
pyrrolidine ring, [2285] L.sup.2 is linear
(C.sub.2-C.sub.6)-alkanediyl or is a group of the formula
[2285] ##STR00266## [2286] where [2287] p is a number 2 or 3,
[2288] ##.sup.3 marks the linkage site with the group B, [2289]
##.sup.4 marks the linkage site with the nitrogen atom, and also
their salts, solvates and solvates of the salts.
[2290] Preferred in the context of the present invention are also
compounds of the formula (Ia), in which [2291] L.sup.1 is linear
(C.sub.3-C.sub.5)-alkanediyl or a group of the formula
[2291] ##STR00267## [2292] where [2293] m is a number 2 or 3,
[2294] ##.sup.1 marks the linkage site with the group G, [2295]
##.sup.2 marks the linkage site with the group B, [2296] B is a
group of the formula
[2296] ##STR00268## [2297] where [2298] * marks the linkage site
with L.sup.1, [2299] ** marks the linkage site with L.sup.2, [2300]
L.sup.3 is a bond or ethane-1,2-diyl, [2301] L.sup.4 is a group of
the formula
[2301] ##STR00269## [2302] in which [2303] *** marks the linkage
site with the carbonyl group, [2304] **** marks the linkage site
with L.sup.2, [2305] R.sup.25 is methyl, [2306] R.sup.28 is
hydrogen, methylcarbonyl or tert-butyloxycarbonyl, [2307] Q.sup.6
is piperidine-1,4-diyl, [2308] R.sup.16 is hydrogen or methyl,
[2309] R.sup.17 is hydrogen or methyl, [2310] R.sup.23 is methyl,
[2311] R.sup.24 is hydrogen, [2312] R.sup.36 is hydrogen,
methylcarbonyl or tert-butyloxycarbonyl, [2313] R.sup.37 is
hydrogen or methyl, [2314] L.sup.2 is linear
(C.sub.2-C.sub.6)-alkanediyl or is a group of the formula
[2314] ##STR00270## [2315] where [2316] p is a number 2 or 3,
[2317] ##.sup.3 marks the linkage site with the group B, [2318]
##.sup.4 marks the linkage site with the nitrogen atom, and also
their salts, solvates and solvates of the salts.
[2319] Preferred in the context of the present invention are also
compounds of the formula (Ia), (XXXa) and (XXXI), in which [2320] D
is a group of the formula
[2320] ##STR00271## [2321] where [2322] #.sup.3 marks the linkage
site with the nitrogen atom, [2323] R.sup.1 is hydrogen or methyl,
[2324] 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-phenyl-ethyl,
diphenylmethyl, 1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl,
[2325] or [2326] R.sup.1 and R.sup.2 together with the carbon atom
to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[2326] ##STR00272## [2327] in which [2328] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [2329] #.sup.5 marks
the linkage site with the carbonyl group, [2330] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[2330] ##STR00273## [2331] in which [2332] #.sup.6 marks the
linkage site with the carbonyl group, [2333] R.sup.6 is hydrogen,
hydroxy or benzyloxy, [2334] R.sup.3 is hydrogen or methyl, [2335]
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-phenyl-ethyl, diphenylmethyl,
1H-imidazol-4-ylmethyl or 1H-indol-3-ylmethyl, [2336] or [2337]
R.sup.3 and R.sup.4 together with the carbon atom to which they are
bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[2337] ##STR00274## [2338] in which [2339] #.sup.7 marks the
linkage site with the adjacent nitrogen atom, [2340] #.sup.8 marks
the linkage site with the group T.sup.1, [2341] 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, [2342] in
which [2343] R.sup.7 is hydrogen, methyl, ethyl, n-propyl,
tert-butyl, benzyl or adamantylmethyl, [2344] R.sup.8 is hydrogen
or methyl, [2345] R.sup.9 is hydrogen, methyl, ethyl, n-propyl or
benzyl, [2346] or [2347] R.sup.8 and R.sup.9 together with the
nitrogen atom to which they are bonded form a 4- to 7-membered
heterocycle, [2348] R.sup.10 is benzoyl, [2349] R.sup.11 is benzyl,
which may be substituted in the phenyl group by methoxycarbonyl or
carboxyl [2350] R.sup.5 is hydrogen, methyl or a group of the
formula
[2350] ##STR00275## [2351] in which [2352] #.sup.9 marks the
linkage site with --CHC(R.sup.26)-T.sup.2, [2353] R.sup.12 is
phenyl which may be substituted by methoxycarbonyl, carboxyl or a
group of the formula --S(O).sub.2OH, [2354] R.sup.13 is phenyl
which may be substituted by methoxycarbonyl or carboxyl, [2355]
R.sup.26 is hydrogen, [2356] T.sup.2 is phenyl, benzyl,
1H-indol-3-yl or 1H-indol-3-ylmethyl, and [2357] n, AK, Cys, G,
L.sup.1, B, L.sup.2, D and R.sup.35 have the definitions indicated
above, and also their salts, solvates and solvates of the
salts.
[2358] Preferred in the context of the present invention are also
compounds of the formula (Ia), (XXXa) and (XXXI), in which [2359] D
is a group of the formula
[2359] ##STR00276## [2360] where [2361] #.sup.3 marks the linkage
site with the nitrogen atom, [2362] R.sup.1 is hydrogen, [2363]
R.sup.2 is benzyl, 4-hydroxybenzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [2364] the ring A with the N--O moiety present
therein is a heterocycle of formula
[2364] ##STR00277## [2365] in which [2366] #.sup.6 marks the
linkage site with the carbonyl group, [2367] R.sup.3 is hydrogen,
[2368] R.sup.4 is benzyl, 4-hydroxybenzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [2369] T.sup.1 is a group of the formula
--C(.dbd.O)--OR.sup.7 or --C(.dbd.O)--NR.sup.8R.sup.9, [2370] in
which [2371] R.sup.7 is hydrogen, [2372] R.sup.8 is hydrogen,
[2373] R.sup.9 is hydrogen, [2374] n, AK, Cys, G, L.sup.1, B,
L.sup.2, D and R.sup.35 have the definitions indicated above, and
also their salts, solvates and solvates of the salts.
[2375] Preferred in the context of the present invention are also
compounds of the formula (Ia), (XXXa) and (XXXI), in which [2376] D
is a group of the formula
[2376] ##STR00278## [2377] where [2378] #.sup.3 marks the linkage
site with the nitrogen atom, [2379] R.sup.1 is hydrogen, [2380]
R.sup.2 is benzyl, 4-hydroxybenzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [2381] the ring A with the N--O moiety present
therein is a heterocycle of the formula
[2381] ##STR00279## [2382] in which [2383] #.sup.6 marks the
linkage site with the carbonyl group, [2384] R.sup.3 is hydrogen,
[2385] R.sup.4 is benzyl, 4-hydroxybenzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [2386] T.sup.1 is a group of the formula
--C(.dbd.O)--NR.sup.8R.sup.9, [2387] in which [2388] R.sup.8 is
hydrogen, [2389] R.sup.9 is hydrogen, [2390] n, AK, Cys, G,
L.sup.1, B, L.sup.2, D and R.sup.35 have the definitions indicated
above, and also their salts, solvates and solvates of the
salts.
[2391] Preferred in the context of the present invention are also
compounds of the formula (Ia), (XXXa) and (XXXI), in which [2392] D
is a group of the formula
[2392] ##STR00280## [2393] where [2394] #.sup.3 marks the linkage
site with the nitrogen atom, [2395] R.sup.1 is hydrogen, [2396]
R.sup.2 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [2397] the ring
A with the N--O moiety present therein is a heterocycle of the
formula
[2397] ##STR00281## [2398] in which [2399] #.sup.6 marks the
linkage site with the carbonyl group, [2400] R.sup.3 is hydrogen,
[2401] R.sup.4 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [2402]
T.sup.1 is a group of the formula --C(.dbd.O)--NR.sup.8R.sup.9,
[2403] in which [2404] R.sup.8 is hydrogen, [2405] R.sup.9 is
hydrogen, [2406] n, AK, Cys, G, L.sup.1, B, L.sup.2, D and R.sup.35
have the definitions indicated above, and also their salts,
solvates and solvates of the salts.
[2407] Preferred in the context of the present invention are also
compounds of the formula (Ia), (XXXa) and (XXXI), in which [2408] D
is a group of the formula
[2408] ##STR00282## [2409] where [2410] #.sup.3 marks the linkage
site with the nitrogen atom, [2411] R.sup.3 is hydrogen, [2412]
R.sup.4 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [2413] T.sup.1
is a group of the formula --C(.dbd.O)--OR.sup.7 or
--C(.dbd.O)--NR.sup.8R.sup.9, [2414] in which [2415] R.sup.7 is
hydrogen, [2416] R.sup.8 is hydrogen, [2417] R.sup.9 is hydrogen,
[2418] n, AK, Cys, G, L.sup.1, B, L.sup.2, D and R.sup.35 have the
definitions indicated above, and also their salts, solvates and
solvates of the salts.
[2419] Preferred in the context of the present invention are also
compounds of the formula (Ia), (XXXa) and (XXXI), in which [2420] D
is a group of the formula
[2420] ##STR00283## [2421] where [2422] #.sup.3 marks the linkage
site with the nitrogen atom, [2423] R.sup.3 is hydrogen, [2424]
R.sup.4 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [2425] T.sup.1
is a group of the formula --C(.dbd.O)--NR.sup.8R.sup.9, [2426] in
which [2427] R.sup.8 is hydrogen, [2428] R.sup.9 is hydrogen,
[2429] n, AK, Cys, G, L.sup.1, B, L.sup.2, D and R.sup.35 have the
definitions indicated above, and also their salts, solvates and
solvates of the salts.
[2430] Preferred in the context of the present invention are also
compounds of the formula (Ia), (XXXa) and (XXXI), in which [2431] D
is a group of the formula
[2431] ##STR00284## [2432] where [2433] #.sup.3 marks the linkage
site with the nitrogen atom, [2434] R.sup.1 is hydrogen or methyl,
[2435] 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,
[2436] or [2437] R.sup.1 and R.sup.2 together with the carbon atom
to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[2437] ##STR00285## [2438] in which [2439] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [2440] #.sup.5 marks
the linkage site with the carbonyl group, [2441] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[2441] ##STR00286## [2442] in which [2443] #.sup.6 marks the
linkage site with the carbonyl group, [2444] R.sup.6 is hydrogen,
hydroxy or benzyloxy, and [2445] n, AK, Cys, G, L.sup.1, B, L.sup.2
and R.sup.35 have the definitions indicated above, and also their
salts, solvates and solvates of the salts.
[2446] Preferred in the context of the present invention are also
compounds of the formula (Ia), (XXXa) and (XXXI), in which [2447] D
is a group of the formula
[2447] ##STR00287## [2448] where [2449] #.sup.3 marks the linkage
site with the nitrogen atom, [2450] R.sup.1 is hydrogen, [2451]
R.sup.2 is benzyl, 4-hydroxybenzyl, 1-phenylethyl or
1H-indol-3-ylmethyl, [2452] or [2453] R.sup.1 and R.sup.2 together
with the carbon atom to which they are bonded form a
(1S,2R)-2-phenylcyclopropane-1,1-diyl group of the formula
[2453] ##STR00288## [2454] in which [2455] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [2456] #.sup.5 marks
the linkage site with the carbonyl group, [2457] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[2457] ##STR00289## [2458] in which [2459] #.sup.6 marks the
linkage site with the carbonyl group, and [2460] n, AK, Cys, G,
L.sup.1, B, L.sup.2 and R.sup.35 have the definitions indicated
above, and also their salts, solvates and solvates of the
salts.
[2461] Preferred in the context of the present invention are also
compounds of the formula (Ia), (XXXa) and (XXXI), in which [2462]
R.sup.35 is hydroxyl, and [2463] n, AK, Cys, G, L.sup.1, B,
L.sup.2, D and R.sup.35 have the definitions indicated above, and
also their salts, solvates and solvates of the salts.
[2464] Preferred in the context of the present invention are also
compounds of the formula (Ia), (XXXa) and (XXXI), in which [2465]
R.sup.35 is methyl, and [2466] n, AK, Cys, G, L.sup.1, B, L.sup.2,
D and R.sup.35 have the definitions indicated above, and also their
salts, solvates and solvates of the salts.
[2467] Particularly preferred in the context of the present
invention are, furthermore, also compounds of the formula (XXXa),
in which [2468] Cys is an L-cysteine residue which is bonded via
the sulphur atom of the side chain via a carbon atom of the
succinimide, and also their salts, solvates and solvates of the
salts.
[2469] Preferred in the context of the present invention are also
compounds of the formula (I) and (XXX), in which [2470] D is a
group of the formula
[2470] ##STR00290## [2471] where [2472] #.sup.3 marks the linkage
site with the nitrogen atom, [2473] R.sup.1 is hydrogen, [2474]
R.sup.2 is benzyl, 1-phenylethyl or 1H-indol-3-ylmethyl, [2475] or
[2476] R.sup.1 and R.sup.2 together with the carbon atom to which
they are bonded form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group
of the formula
[2476] ##STR00291## [2477] in which [2478] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [2479] #.sup.5 marks
the linkage site with the carbonyl group, [2480] the ring A with
the N--O moiety present therein is a mono- or bicyclic, optionally
substituted heterocycle of the formula
[2480] ##STR00292## [2481] in which [2482] #.sup.6 marks the
linkage site with the carbonyl group, [2483] R.sup.6 is hydrogen,
hydroxy or benzyloxy, [2484] n, AK, Cys, G, L.sup.1, L.sup.2 and B
have the definitions indicated above, and also their salts,
solvates and solvates of the salts.
[2485] Particularly preferred in the context of the present
invention are also compounds of the formula (I) and (XXX), in which
[2486] D is a group of the formula
[2486] ##STR00293## [2487] where [2488] #.sup.3 marks the linkage
site with the nitrogen atom, [2489] R.sup.1 is hydrogen, [2490]
R.sup.2 is benzyl or 1H-indol-3-ylmethyl, [2491] or [2492] R.sup.1
and R.sup.2 together with the carbon atom to which they are bonded
form a (1S,2R)-2-phenylcyclopropane-1,1-diyl group of the
formula
[2492] ##STR00294## [2493] in which [2494] #.sup.4 marks the
linkage site with the adjacent nitrogen atom, [2495] #.sup.5 marks
the linkage site with the carbonyl group, [2496] the ring A with
the N--O moiety present therein is a heterocycle of the formula
[2496] ##STR00295## [2497] in which [2498] #.sup.6 marks the
linkage site with the carbonyl group, [2499] n, AK, Cys, G,
L.sup.1, L.sup.2 and B have the definitions indicated above, and
also their salts, solvates and solvates of the salts.
[2500] A further particularly preferred subject of the present
invention are compounds of the formula (I), in which [2501] D is a
group of the formula
[2501] ##STR00296## [2502] where [2503] T.sup.1 is --C(.dbd.O)--OH
or --C(.dbd.O)--NH.sub.2 and [2504] n, AK, G, L.sup.1, B, L.sup.2,
#.sup.3, R.sup.3 and R.sup.4 have the definitions indicated
above.
[2505] Preferred in the context of the present invention are also
compounds of the formula (I), in which [2506] n=1-20, more
preferably n=1-10 and very preferably n=2-8.
[2507] Preferred in the context of the present invention are also
compounds of the formula (I) and (XXX), in which [2508] B is a bond
or a group of the formula
[2508] ##STR00297## [2509] where [2510] * marks the linkage site
with L.sup.1, [2511] ** marks the linkage site with L.sup.2, [2512]
L.sup.3 is a bond or ethane-1,2-diyl, [2513] L.sup.4 is a bond,
[2514] n, AK, Cys, G, L.sup.1, L.sup.2, D, R.sup.16 and R.sup.17
have the definitions indicated above, and also their salts,
solvates and solvates of the salts.
[2515] Particularly preferred in the context of the present
invention are also compounds of the formula (I) and (XXX), in which
[2516] B is a bond or a group of the formula
[2516] ##STR00298## [2517] where [2518] * marks the linkage site
with L.sup.1, [2519] ** marks the linkage site with L.sup.2, [2520]
L.sup.3 and L.sup.4 is a bond, [2521] n, AK, Cys, G, L.sup.1,
L.sup.2, D, R.sup.16 and R.sup.17 have the definitions indicated
above, and also their salts, solvates and solvates of the
salts.
[2522] Preferred in the context of the present invention are also
binder-drug conjugates of the general formula (I), in which [2523]
AK is AK.sub.1, [2524] where [2525] AK.sub.1 is a binder which is
bonded via the sulphur atom of a cysteine residue of the binder to
the group G, [2526] G is a group of the formula
[2526] ##STR00299## [2527] where [2528] #.sup.1 marks the linkage
site with the cysteine residue of the binder, [2529] #.sup.2 marks
the linkage site with the group L.sup.1, [2530] L.sup.1 is a bond,
linear (C.sub.1-C.sub.10)-alkanediyl or is a group of the
formula
[2530] ##STR00300## [2531] where [2532] m is a number from 2 to 6,
[2533] ##.sup.1 marks the linkage site with the group G, [2534]
##.sup.2 marks the linkage site with the group B, [2535] where
(C.sub.1-C.sub.10)-alkanediyl may be substituted by 1 to 4 methyl
substituents, [2536] and [2537] where two carbon atoms of the
alkanediyl chain in 1,2, 1, 3 or 1,4 relation to one another, with
inclusion of any carbon atoms situated between them, may be bridged
to form a (C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring,
[2538] B is a bond or a group of the formula
[2538] ##STR00301## [2539] where [2540] * marks the linkage site
with L.sup.1, [2541] ** marks the linkage site with L.sup.2, [2542]
L.sup.3 is a bond or (C.sub.2-C.sub.4)-alkanediyl, [2543] L.sup.4
is a bond or a group of the formula
[2543] ##STR00302## [2544] in which [2545] *** marks the linkage
site with the carbonyl group, [2546] **** marks the linkage site
with L.sup.2, [2547] R.sup.25 is hydrogen or methyl, [2548] Q.sup.1
is a 4- to 7-membered heterocycle, [2549] R.sup.14 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [2550] R.sup.15 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [2551] or [2552] R.sup.14 and R.sup.15
together with the atoms to which they are bonded form a 5- or
6-membered heterocycle, [2553] R.sup.16 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [2554] R.sup.17 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [2555] or [2556] R.sup.16 and R.sup.17
together with the atoms to which they are bonded form a 5- or
6-membered heterocycle, [2557] L.sup.2 is linear
(C.sub.2-C.sub.10)-alkanediyl or is a group of the formula
[2557] ##STR00303## [2558] where [2559] p is a number from 2 to
6,
[2560] ##.sup.3 marks the linkage site with the group B, [2561]
##.sup.4 marks the linkage site with the nitrogen atom, [2562]
where (C.sub.2-C.sub.10)-alkanediyl may be substituted by 1 to 4
methyl substituents, [2563] and [2564] where two carbon atoms of
the alkanediyl chain in 1,2, 1, 3 or 1,4 relation to one another,
with inclusion of any carbon atoms situated between them, may be
bridged to form a (C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl
ring, and also their salts, solvates and solvates of the salts.
[2565] Preferred in the context of the present invention are also
binder-drug conjugates of the general formula (I), in which [2566]
AK is AK.sub.2, [2567] where [2568] AK.sub.2 is a binder which is
bonded via the NH side group of a lysine residue of the binder to
the group G, [2569] G is carbonyl, [2570] L.sup.1 is a bond, linear
(C.sub.1-C.sub.10)-alkanediyl or is a group of the formula
[2570] ##STR00304## [2571] where [2572] m is a number from 2 to 6,
[2573] ##.sup.1 marks the linkage site with the group G, [2574]
##.sup.2 marks the linkage site with the group B, [2575] where
(C.sub.1-C.sub.10)-alkanediyl may be substituted by 1 to 4 methyl
substituents, [2576] and [2577] where two carbon atoms of the
alkanediyl chain in 1,2, 1, 3 or 1,4 relation to one another, with
inclusion of any carbon atoms situated between them, may be bridged
to form a (C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring,
[2578] B is a bond or a group of the formula
[2578] ##STR00305## [2579] where [2580] * marks the linkage site
with L.sup.1, [2581] ** marks the linkage site with L.sup.2, [2582]
P is O or NH, [2583] Q.sup.2 is a 3- to 7-membered carbocycle or a
4- to 7-membered heterocycle, [2584] R.sup.18 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [2585] R.sup.19 is hydrogen or the side
group of a natural .alpha.-amino acid or of its homologues or
isomers, [2586] R.sup.20 is hydrogen or (C.sub.1-C.sub.4)-alkyl,
[2587] or [2588] R.sup.19 and R.sup.20 together with the atoms to
which they are bonded form a pyrrolidinyl ring, [2589] R.sup.21 s
hydrogen or (C.sub.1-C.sub.4)-alkyl, [2590] R.sup.22 is hydrogen or
(C.sub.1-C.sub.4)-alkyl, [2591] or [2592] R.sup.21 and R.sup.22
together with the atoms to which they are bonded form a 3- to
7-membered carbocycle, [2593] R.sup.23 is (C.sub.1-C.sub.4)-alkyl,
[2594] R.sup.24 is hydrogen or (C.sub.1-C.sub.4)-alkyl, [2595]
L.sup.2 is linear (C.sub.2-C.sub.10)-alkanediyl or is a group of
the formula
[2595] ##STR00306## [2596] where [2597] p is a number from 2 to 6,
[2598] ##.sup.3 marks the linkage site with the group B, [2599]
##.sup.4 marks the linkage site with the nitrogen atom, [2600]
where (C.sub.2-C.sub.10)-alkanediyl may be substituted by 1 to 4
methyl substituents, [2601] and [2602] where two carbon atoms of
the alkanediyl chain in 1,2, 1, 3 or 1,4 relation to one another,
with inclusion of any carbon atoms situated between them, may be
bridged to form a (C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl
ring, and also their salts, solvates and solvates of the salts.
[2603] Particularly preferred in the context of the present
invention are binder-drug conjugates of the formula (Ia), in which
[2604] n is a number from 2 to 8, [2605] AK is AK.sub.1 or
AK.sub.2, [2606] where [2607] AK.sub.1 is an antibody or
antigen-binding antibody fragment (e.g., a human or humanized
antibody or an antigen-binding antibody fragment which binds to
C4.4a) and is bonded via the sulphur atom of a cysteine residue of
the binder to the group G, [2608] AK.sub.2 is an antibody or
antigen-binding antibody fragment (e.g., a human or humanized
antibody or an antigen-binding antibody fragment which binds to
C4.4a) and is bonded via the NH side group of a lysine residue of
the binder to the group G, [2609] G when AK=AK.sub.1, is a group of
the formula
[2609] ##STR00307## [2610] in which [2611] #.sup.1 marks the
linkage site with the cysteine residue of the binder, [2612]
#.sup.2 marks the linkage site with the group L.sup.1, [2613] or
[2614] when AK=AK.sub.2, is carbonyl, [2615] L.sup.1 is a bond,
linear (C.sub.2-C.sub.6)-alkanediyl or is a group of the
formula
[2615] ##STR00308## [2616] where [2617] m is a number 2 or 3,
[2618] ##.sup.1 marks the linkage site with the group G, [2619]
##.sup.2 marks the linkage site with the group B, [2620] where
(C.sub.2-C.sub.6)-alkanediyl may be substituted by 1 or 2 methyl
substituents, [2621] B is a bond or a group of the formula
[2621] ##STR00309## [2622] where [2623] * marks the linkage site
with L.sup.1, [2624] ** marks the linkage site with L.sup.2, [2625]
L.sup.3 is a bond or ethane-1,2-diyl, [2626] L.sup.4 is a bond or a
group of the formula
[2626] ##STR00310## [2627] in which [2628] *** marks the linkage
site with the carbonyl group, [2629] **** marks the linkage site
with L.sup.2, [2630] R.sup.25 is methyl, [2631] R.sup.28 is
hydrogen, methylcarbonyl or tert-butyloxycarbonyl, [2632] Q.sup.1
is piperidine-1,4-diyl, [2633] R.sup.16 is hydrogen or methyl,
[2634] R.sup.17 is hydrogen or methyl, [2635] or [2636] R.sup.16
and R.sup.17 together with the atoms to which they are bonded form
a piperazinyl ring, [2637] R.sup.21 is hydrogen or methyl, [2638]
R.sup.22 is hydrogen or methyl, [2639] or [2640] R.sup.21 and
R.sup.22 together with the atoms to which they are bonded form a
cyclopropyl ring, [2641] R.sup.23 is methyl, [2642] R.sup.24 is
hydrogen, [2643] L.sup.2 is linear (C.sub.2-C.sub.6)-alkanediyl or
is a group of the formula
[2643] ##STR00311## [2644] where [2645] p is a number from 2 to 6,
[2646] ##.sup.3 marks the linkage site with the group B, [2647]
##.sup.4 marks the linkage site with the nitrogen atom, [2648] D is
a group of the formula
[2648] ##STR00312## [2649] where [2650] #.sup.3 marks the linkage
site with the nitrogen atom, [2651] R.sup.1 is hydrogen, [2652]
R.sup.2 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [2653] the ring
A with the N--O moiety present therein is
[2653] ##STR00313## [2654] in which [2655] #.sup.6 marks the
linkage site with the carbonyl group, [2656] R.sup.3 is hydrogen,
[2657] R.sup.4 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [2658]
T.sup.1 is a group of the formula --C(.dbd.O)--NR.sup.8R.sup.9,
[2659] R.sup.8 is hydrogen or methyl, [2660] R.sup.9 is hydrogen,
methyl, or ethyl, [2661] R.sup.35 is methyl, and also their salts,
solvates and solvates of the salts.
[2662] Particularly preferred in the context of the present
invention are binder-drug conjugates of the formula (Ia), in which
[2663] n is a number from 2 to 8, preferably 2 to 5, [2664] AK is
AK.sub.1, [2665] where [2666] AK.sub.1 is an antibody or
antigen-binding antibody fragment (e.g., a human or humanized
antibody or an antigen-binding antibody fragment which binds to
C4.4a) and is bonded via the sulphur atom of a cysteine residue of
the binder to the group G, [2667] G is a group of the formula
[2667] ##STR00314## [2668] where [2669] #.sup.1 marks the linkage
site with the cysteine residue of the binder, [2670] #.sup.2 marks
the linkage site with the group L.sup.1, [2671] L.sup.1 is
pentane-1,5-diyl, [2672] B is a group of the formula
[2672] ##STR00315## [2673] where [2674] * marks the linkage site
with L.sup.1, [2675] ** marks the linkage site with L.sup.2, [2676]
L.sup.3 is a bond, [2677] L.sup.4 is a bond, [2678] R.sup.16 is
hydrogen, [2679] R.sup.17 is hydrogen, [2680] L.sup.2 is
propane-1,3-diyl, [2681] D is a group of the formula
[2681] ##STR00316## [2682] where [2683] #.sup.3 marks the linkage
site with the nitrogen atom, [2684] R.sup.1 is hydrogen, [2685]
R.sup.2 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [2686] the ring
A with the N--O moiety present therein is
[2686] ##STR00317## [2687] in which [2688] #.sup.6 marks the
linkage site with the carbonyl group, [2689] R.sup.3 is hydrogen,
[2690] R.sup.4 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [2691]
T.sup.1 is a group of the formula --C(.dbd.O)--NR.sup.8R.sup.9,
[2692] R.sup.8 is hydrogen, [2693] R.sup.9 is hydrogen, [2694]
R.sup.35 is methyl, and also their salts, solvates and solvates of
the salts.
[2695] Particularly preferred in the context of the present
invention are binder-drug conjugates of the formula (Ia), in which
[2696] n is a number from 2 to 8, preferably 2 to 5, [2697] AK is
AK.sub.1, [2698] where [2699] AK.sub.1 is an antibody or
antigen-binding antibody fragment (e.g., a human or humanized
antibody or an antigen-binding antibody fragment which binds to
C4.4a) and is bonded via the sulphur atom of a cysteine residue of
the binder to the group G, [2700] G is a group of the formula
[2700] ##STR00318## [2701] where [2702] #.sup.1 marks the linkage
site with the cysteine residue of the binder, [2703] #.sup.2 marks
the linkage site with the group L.sup.1, [2704] L.sup.1 is a bond,
[2705] B is a bond, [2706] L.sup.2 is hexane-1,6-diyl, and D has
the definition indicated above, and also their salts, solvates and
solvates of the salts.
[2707] Particularly preferred in the context of the present
invention are binder-drug conjugates of the formula (Ia), in which
[2708] n is a number from 2 to 8, preferably 2 to 5, [2709] AK is
AK.sub.2, [2710] where [2711] AK.sub.2 is an antibody or
antigen-binding antibody fragment (e.g., a human or humanized
antibody or an antigen-binding antibody fragment which binds to
C4.4a) and is bonded via the NH side group of a lysine residue of
the binder to the group G, [2712] G is carbonyl, [2713] L.sup.1 is
a bond, [2714] B is a bond, [2715] L.sup.2 is pentane-1,5-diyl,
[2716] D is a group of the formula
[2716] ##STR00319## [2717] where [2718] * marks the linkage site
with the nitrogen atom, [2719] R.sup.1 is hydrogen, [2720] R.sup.2
is 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [2721] the ring A with
the N--O moiety present therein is
[2721] ##STR00320## [2722] in which [2723] #.sup.6 marks the
linkage site with the carbonyl group, [2724] R.sup.3 is hydrogen,
[2725] R.sup.4 is 4-hydroxybenzyl or 1H-indol-3-ylmethyl, [2726]
T.sup.1 is a group of the formula --C(.dbd.O)--NR.sup.8R.sup.9,
[2727] R.sup.8 is hydrogen, [2728] R.sup.9 is hydrogen, [2729]
R.sup.35 is methyl, and also their salts, solvates and solvates of
the salts.
[2730] Particularly preferred in the context of the present
invention are binder-drug conjugates of the formula (Ia), in which
[2731] n is a number from 2 to 8, preferably 2 to 5, [2732] AK is
AK.sub.2, [2733] where [2734] AK.sub.2 is an antibody or
antigen-binding antibody fragment (e.g., a human or humanized
antibody or an antigen-binding antibody fragment which binds to
C4.4a) and is bonded via the NH side group of a lysine residue of
the binder to the group G, [2735] G is carbonyl, [2736] L.sup.1 is
a bond, [2737] B is a bond, [2738] L.sup.2 is a group of the
formula
[2738] ##STR00321## [2739] where [2740] p is the number 3, [2741]
##.sup.3 marks the linkage site with the group B, [2742] ##.sup.4
marks the linkage site with the nitrogen atom, and D has the
meaning indicated above, and also their salts, solvates and
solvates of the salts.
[2743] Particularly preferred in the context of the present
invention are binder-drug conjugates of the formula (Ia), in
which
[2744] A preferred subject of the invention are binder-drug
conjugates of the general formula (Ia) in which D is
##STR00322##
wherein the asterisks marks the linkage site with the nitrogen
atom; and the linker .sctn.-G-L.sup.1-B-L.sup.2-.sctn..sctn. and
the remainder of the variables are as defined in any of the
embodiments provided herein; and also their salts, solvates and
solvates of the salts.
[2745] A preferred subject of the invention are binder-drug
conjugates of the general formula (Ia) in which D is as shown in
any of the exemplary drug-binder conjugates provided herein; and
the linker .sctn.-G-L.sup.1-B-L.sup.2-.sctn..sctn. and the
remainder of the variables are as defined in any of the embodiments
provided herein; and also their salts, solvates and solvates of the
salts.
[2746] Exemplary drug-binder conjugates are as follows:
##STR00323## ##STR00324## ##STR00325## ##STR00326## ##STR00327##
##STR00328## ##STR00329##
and also their salts, solvates and solvates of the salts, wherein
AK (e.g., AK.sub.2A, AK.sub.4, AK.sub.3, AK.sub.6B, AK.sub.8, or
AK.sub.2G) is an antibody or antigen-binding antibody fragment
(preferably a human or humanized monoclonal antibody or
antigen-binding fragment thereof) and is, in some embodiments,
bonded via a nitrogen atom of the antibody or antigen binding
fragment thereof to the drug-linker and n is from 1 to 20,
preferably 1 to 10.
[2747] Exemplary drug conjugate are also as follows:
##STR00330## ##STR00331## ##STR00332## ##STR00333## ##STR00334##
##STR00335## ##STR00336## ##STR00337## ##STR00338## ##STR00339##
##STR00340## ##STR00341## ##STR00342## ##STR00343## ##STR00344##
##STR00345## ##STR00346## ##STR00347## ##STR00348##
##STR00349##
and also their salts, solvates and solvates of the salts, wherein
AK (e.g., AK.sub.1A, AK.sub.3, AK.sub.4, AK.sub.5A, AK.sub.5B,
AK.sub.S, AK.sub.1F, or AK.sub.1G) is an antibody or
antigen-binding antibody fragment (preferably a human or humanized
monoclonal antibody or antigen-binding fragment thereof) and is, in
some embodiments, bonded via the sulphur atom of a cysteine residue
of to the antibody or antigen binding fragment thereof to the
drug-linker, and n is from 1 to 20, preferably 1 to 10.
[2748] Particularly preferred in the context of the present
invention are also drug-binder conjugates selected from the
following compounds:
##STR00350## ##STR00351##
where in each case [2749] n is a number from 2 to 8, preferably 2
to 5, [2750] AK.sub.1 is a human or humanized antibody or an
antigen-binding antibody fragment (e.g., a human or humanized
antibody or an antigen-binding antibody fragment which binds to
C4.4a) and is bonded via the sulphur atom of a cysteine residue of
the binder to the group G, and [2751] AK.sub.2 is a human or
humanized antibody or an antigen-binding antibody fragment (e.g., a
human or humanized antibody or an antigen-binding antibody fragment
which binds to C4.4a) and is bonded via the NH side group of a
lysine residue of the binder to the group G.
[2752] More particularly preferred in the context of the present
invention are binder-drug conjugates selected from the following
compounds:
##STR00352## ##STR00353##
where in each case [2753] n is a number from 2 to 8, preferably 2
to 5, and [2754] AK is a human or humanized antibody or an
antigen-binding antibody fragment (e.g., a human or humanized
antibody or an antigen-binding antibody fragment which binds to
C4.4a).
[2755] In all of these formulae, the antibody may be any one of the
antibodies described herein.
[2756] More particularly preferred in the context of the present
invention are binder-drug conjugates selected from the following
compounds:
##STR00354## ##STR00355##
in which [2757] n is a number 2 to 8, preferably 2 to 5, and [2758]
AK.sub.1 is a human or humanized antibody or an antigen-binding
antibody fragment (e.g., a human or humanized antibody or an
antigen-binding antibody fragment which binds to C4.4a) and is
bonded via a cysteine residue to the toxophor-linker unit, [2759]
AK.sub.2 is a human or humanized antibody or an antigen-binding
antibody fragment (e.g., a human or humanized antibody or an
antigen-binding antibody fragment which binds to C4.4a and is
bonded via a lysine residue to the toxophor-linker unit).
[2760] More particularly preferred in the context of the present
invention are binder-drug conjugates selected from the following
compounds:
##STR00356## ##STR00357## ##STR00358##
in which [2761] n is a number 2 to 8, preferably 2 to 5, and [2762]
AK.sub.1B and AK.sub.2B is B01-3.
[2763] Exemplary cysteine adducts are as follows:
##STR00359## ##STR00360##
[2764] The definitions of radicals that are indicated individually
in the respective combinations and preferred combinations of
radicals are also replaced arbitrarily by radical definitions of
other combinations, independently of the respective combinations of
radicals that are indicated.
[2765] Especially preferred are combinations of two or more of the
abovementioned preference ranges.
[2766] Further provided by the invention is a process for preparing
the compounds of the invention of the formula (Ia), characterized
in that a solution of the binder (preferably in buffer such as, for
example, PBS buffer) [2767] [A] is admixed with a suitable reducing
agent, such as, for example, dithiothreitol or
tris(2-carboxyethyl)phosphine hydrochloride, and is subsequently
reacted with a compound of the formula (IIa)
[2767] ##STR00361## [2768] in which D, L.sup.1, B, L.sup.2 and
R.sup.35 each have the definitions indicated above, [2769] to give
a compound of the formula (I-A)
[2769] ##STR00362## [2770] in which n, AK.sub.2, D, L.sup.1, B,
L.sup.2 and R.sup.35 each have the definitions indicated above, or
[2771] [B] is reacted with a compound of formula (IIIa)
[2771] ##STR00363## [2772] in which D, L.sup.1, B, L.sup.2 and
R.sup.35 each have the definitions indicated above, [2773] to give
a compound of the formula (Ia-B)
[2773] ##STR00364## [2774] in which n, AK.sub.2, D, L.sup.1, B,
L.sup.2 and R.sup.35 each have the definitions indicated above.
[2775] Further provided by the invention is a process for preparing
the compounds of the invention of the formula (I), characterized in
that a solution of the binder in PBS buffer [2776] [A] is admixed
with a suitable reducing agent, such as, for example,
dithiothreitol or tris(2-carboxyethyl)phosphine hydrochloride, and
is subsequently reacted with a compound of the formula (II)
[2776] ##STR00365## [2777] in which D, L.sup.1, B and L.sup.2 each
have the definitions indicated above, [2778] to give a compound of
the formula (I-A)
[2778] ##STR00366## [2779] in which n, AK.sub.1, D, L.sup.1, B and
L.sup.2 each have the definitions indicated above, or [2780] [B] is
reacted with a compound of the formula (III)
[2780] ##STR00367## [2781] in which D, L.sup.1, B and L.sup.2 each
have the definitions indicated above, [2782] to give a compound of
the formula (I-B)
[2782] ##STR00368## [2783] in which n, AK.sub.2, D, L.sup.1, B and
L.sup.2 each have the definitions indicated above.
[2784] Cysteine Coupling:
[2785] The partial reduction of the antibody and also the
subsequent conjugation of the (partially) reduced antibody with a
compound of the formula (II) or (IIa) takes place in accordance
with the methods known to the skilled person, see e.g. Ducry et.
al., Bioconj. Chem. 2010, 21, 5 and references herein, Klussman et.
al., Bioconj. Chem. 2004, 15(4), 765-773. The mild reduction of the
antibody is accomplished preferably by addition of 2-6 equivalents
of TCEP to the antibody, which is present in a suitable buffer
solution, preferably phosphate buffer, and by stirring for 30-180
minutes at temperatures between 15 and 40.degree. C., preferably at
RT. This is followed by the conjugation, by addition of a solution
of a compound of the formula (II) or (IIa) in DMSO, acetonitrile or
DMF to the solution of the (partially) reduced antibody in PBS
buffer, and subsequent reaction at a temperature of 0.degree. C. to
+40.degree. C., more particularly of +10.degree. C. to +30.degree.
C., for a period of 30 minutes to 6 hours, more particularly 1 to 2
hours.
Lysine Coupling:
[2786] First of all the compounds of the formula (III) or (IIa) or
comparable activated carboxyl components are prepared by
conventional methods of peptide chemistry. They are then taken up
in inert solvents such as DMSO or DMF, for example, and added to
the antibody, which is preferably present in phosphate buffer at a
neutral pH. The solution is stirred for 1-16 h at a temperature
between 15 and 40.degree. C., preferably RT.
[2787] The preparation processes described above are elucidated by
way of example using the schemes below (Scheme 1 and 2):
##STR00369##
##STR00370##
[2788] The compounds of the formula (II) in which L.sup.1 and B are
a bond can be prepared by subjecting a compound of the formula
(IV)
##STR00371##
in which D has the definition indicated above, to reductive
amination in an inert solvent with a compound of the formula
(V)
##STR00372##
in which [2789] L.sup.2A has the above-defined definition of
L.sup.2, but is shortened by one carbon atom in the alkyl chain
length, [2790] PG.sup.1 is an amino-protective group such as, for
example, (9H-fluoren-9-ylmethoxy)carbonyl, tert-butoxycarbonyl or
benzyloxycarbonyl, to give a compound of the formula (VI)
##STR00373##
[2790] in which D, L.sup.2 and PG.sup.1 have the definition
indicated above, eliminating the protective group PG.sup.1 from
this compound by methods known to the skilled person, and 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 give a compound
of the formula (II-A)
##STR00374##
in which D and L.sup.2 each have the definitions indicated
above.
[2791] The compounds of the formula (II) in which B is a group of
the formula (B.sup.1)
##STR00375##
in which *, **, R.sup.14 and R.sup.15 each have the conditions
indicated above, can be prepared by eliminating the protective
group PG.sup.1 from a compound of the formula (VI) by methods known
to the skilled person, and reacting the deprotected compound in an
inert solvent in the presence of a suitable base with a compound of
the formula (VII)
##STR00376##
in which L.sup.1 has the definition indicated above, to give a
compound of the formula (II-B)
##STR00377##
in which D, L.sup.1 and L.sup.2 each have the definitions indicated
above.
[2792] The compounds of the formula (II) in which B is a group of
the formula (B.sup.2)
##STR00378##
in which *, **, L.sup.3, R.sup.16 and R.sup.17 each have the
conditions indicated above can be prepared by subjecting a compound
of the formula (IV) to reductive amination in an inert solvent with
a compound of the formula (VIII)
##STR00379##
in which [2793] L.sup.2A has the above-defined definition of
L.sup.2, but is shortened by one carbon atom in the alkyl chain
length, to give a compound of the formula (IX)
##STR00380##
[2793] in which D and L.sup.2 have the definitions indicated above,
and reacting this compound in an inert solvent in the presence of a
suitable coupling reagent and a suitable base with a compound of
the formula (X)
##STR00381##
in which L.sup.1 and L.sup.3 each have the definitions indicated
above, to give a compound of the formula (II-C)
##STR00382##
in which D, L.sup.1, L.sup.2 and L.sup.3 each have the definitions
indicated above.
[2794] Compound of the formula (II), in which B is a group of the
formula (B.sup.3)
##STR00383##
in which *, **, L.sup.3, R.sup.16 and R.sup.17 each have the
conditions indicated above and [2795] L.sup.4A is a group of the
formula
[2795] ##STR00384## [2796] in which [2797] *** marks the linkage
site with the carbonyl group, [2798] **** marks the linkage site
with L.sup.2, [2799] R.sup.25 is hydrogen or methyl, can be
prepared by reacting a compound of the formula (IX) in an inert
solvent in the presence of a suitable base and a suitable coupling
reagent with a compound of the formula (XI-A) or (XI-B)
##STR00385##
[2799] in which R.sup.25 and PG.sup.1 each have the definitions
indicated above and PG.sup.2 is a suitable carboxyl-protective
group, more particularly benzyl, to give a compound (XII-A) or
(XII-B)
##STR00386##
in which D, PG.sup.1, PG.sup.2 and L.sup.2 have the definitions
indicated above, eliminating the protective group PG.sup.2 from
this compound subsequently, by methods known to the skilled person,
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 the formula (X), and finally, eliminating the
protective group PG1 from this compound, by methods known to the
skilled person, to give a compound of the formula (II-D-A) or
(II-D-B)
##STR00387##
in which D, L.sup.1, L.sup.2 and L.sup.3 have the definitions
indicated above.
[2800] Compound of the formula (II), in which B is a group of the
formula (B.sup.4)
##STR00388##
in which *, ** each have the conditions indicated above and [2801]
Q.sup.1A is an N-linked 4- to 7-membered heterocycle, can be
prepared by reacting a compound of the formula (IX) in an inert
solvent in the presence of a suitable base and a suitable coupling
reagent with a compound of the formula (XXI)
##STR00389##
[2801] in which PG.sup.1 and Q.sup.1A each have the definitions
indicated above, to give a compound of the formula (XXII)
##STR00390##
in which PG.sup.1, Q.sup.1A, D and L.sup.2 have the definitions
indicated above, eliminating the protective group PG.sup.1 from
this compound, by methods known to the skilled person, and
subsequently reacting the deprotected compound in an inert solvent
in the presence of a suitable coupling reagent and a suitable base
with a compound of the formula (XXIII)
##STR00391##
in which L.sup.1 has the definition indicated above, to give a
compound of the formula (II-D)
##STR00392##
in which Q.sup.1A, D, L.sup.1 and L.sup.2 have the definitions
indicated above.
[2802] The compounds of the formula (III), in which L.sup.1 and B
are a bond can be prepared by reacting a compound of the formula
(IX) in an inert solvent in the presence of a suitable coupling
reagent and a suitable base with N-hydroxysuccinimide to give a
compound of the formula (III-A)
##STR00393##
in which D and L.sup.2 each have the definitions indicated
above.
[2803] The compounds of the formula (III), in which L.sup.1 is a
bond and B is a group of the formula (B.sup.5A)
##STR00394##
in which *, ** and P each have the definitions indicated above and
[2804] Q.sup.2A is a 3- to 7-membered carbocycle, can be prepared
by reacting a compound of the formula (IX) in an inert solvent in
the presence of a suitable coupling reagent and a suitable base
with a compound of the formula (XIII)
##STR00395##
[2804] in which P, Q.sup.2A and PG.sup.2 each have the definitions
indicated above, to give a compound of the formula (XIV)
##STR00396##
in which D, P, Q.sup.2A, L.sup.2 and PG.sup.2 each have the
definitions indicated above, eliminating the protective group
PG.sup.2 from this compound by methods known to the skilled person,
and subsequently reacting the deprotected compound in an inert
solvent in the presence of a suitable base with
N-hydroxysuccinimide to give a compound of the formula (III-B)
##STR00397##
in which D, P, Q.sup.2A and L.sup.2 each have the definitions
indicated above.
[2805] The compounds of the formula (III), in which L.sup.1 is a
bond and B is a group of the formula (B.sup.6)
##STR00398##
in which *, **, R.sup.18, R.sup.19 and R.sup.20 each have the
definitions indicated above, can be prepared by reacting a compound
of the formula (IX) in an inert solvent in the presence of a
suitable coupling reagent and a suitable base with a compound of
the formula (XV)
##STR00399##
in which R.sup.18, R.sup.19, R.sup.20 and PG.sup.2 each have the
definitions indicated above, to give a compound of the formula
(XVI)
##STR00400##
in which D, R.sup.18, R.sup.19, R.sup.20, L.sup.2 and PG.sup.2 each
have the definitions indicated above, eliminating the protective
group PG.sup.2 from this compound by methods known to the skilled
person, and subsequently reacting the deprotected compound in an
inert solvent in the presence of a suitable coupling reagent and a
suitable base with N-hydroxysuccinimide to give a compound of the
formula (III-C)
##STR00401##
in which D, R.sup.18, R.sup.19, R.sup.20 and L.sup.2 each have the
definitions indicated above.
[2806] The compounds of the formula (III), in which L.sup.1 is a
bond and B is a group of the formula (B.sup.7)
##STR00402##
in which *, **, R.sup.21 and R.sup.22 each have the definitions
indicated above, can be prepared by eliminating the protective
group PG.sup.1 from a compound of the formula (VI) by methods known
to the skilled person, and reacting the resultant deprotected
compound in an inert solvent in the presence of a suitable base
with a compound of the formula (XVII)
##STR00403##
in which R.sup.21 and R.sup.22 each have the definitions indicated
above, to give a compound of the formula (III-D)
##STR00404##
in which D, R.sup.21, R.sup.22 and L.sup.2 each have the
definitions indicated above.
[2807] The compounds of the formula (III), in which B is a group of
the formula (B.sup.8)
##STR00405##
in which *, **, R.sup.23 and R.sup.24 each have the definitions
indicated above, can be prepared by reacting a compound of the
formula (IX) in an inert solvent in the presence of a suitable
coupling reagent and a suitable base with a compound of the formula
(XVIII)
##STR00406##
in which R.sup.23, R.sup.24 and PG.sup.1 each have the definitions
indicated above, to give a compound of the formula (XIX)
##STR00407##
in which D, R.sup.23, R.sup.24, L.sup.2 and PG.sup.1 each have the
definitions indicated above, eliminating the protective group
PG.sup.1 from this compound by methods known to the skilled person,
and subsequently reacting the deprotected compound in an inert
solvent in the presence of a suitable coupling reagent and a
suitable base with a compound of the formula (XX)
##STR00408##
in which [2808] L.sup.1A is linear (C.sub.1-C.sub.10)-alkanediyl or
is a group of the formula
[2808] ##STR00409## [2809] where [2810] m is a number from 2 to 6,
[2811] ##.sup.1 marks the linkage site with the group G, [2812]
##.sup.2 marks the linkage site with the group B, [2813] where
(C.sub.1-C.sub.10)-alkanediyl may be substituted by 1 to 4 methyl
substituents, [2814] and [2815] where two carbon atoms of the
alkanediyl chain in 1,2, 1, 3 or 1,4 relation to one another, with
inclusion of any carbon atoms situated between them, may be bridged
to form a (C.sub.3-C.sub.6)-cycloalkyl ring or a phenyl ring, to
give a compound of the formula (III-E)
##STR00410##
[2815] in which D, R.sup.23, R.sup.24, L.sup.1A and L.sup.2 each
have the definitions indicated above.
[2816] The compounds of the formula (III), in which B is a group of
the formula (B.sup.5B)
##STR00411##
in which * and ** each have the definitions indicated above and
[2817] Q.sup.2B is an N-linked 4- to 7-membered heterocycle, can be
prepared by reacting a compound of the formula (IX) in an inert
solvent in the presence of a suitable base and a suitable coupling
reagent with a compound of the formula (XXIV)
##STR00412##
[2817] in which PG.sup.1 and Q.sup.2B each have the definitions
indicated above, to give a compound of the formula (XXV)
##STR00413##
in which PG.sup.1, Q.sup.2B, D and L.sup.2 have the definitions
indicated above, eliminating the protective group PG.sup.1 from
this compound by methods known to the skilled person, and
subsequently converting the deprotected compound in an inert
solvent in the presence of a suitable base with a compound of the
formula (XX) into a compound of the formula (III-F)
##STR00414##
in which Q.sup.2B, D, L.sup.1A and L.sup.2 have the definitions
indicated above.
[2818] The reactions (IV)+(V).fwdarw.(VI) and
(IV)+(VIII).fwdarw.(IX) take place in the solvents which are
customary for a reductive amination and are inert under the
reaction conditions, optionally in the presence of an acid and/or
of a water-removing agent as 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-dimethylformamide or else
water. It is also possible to use mixtures of these solvents. As
solvent it is preferred to use a 1,4-dioxane/water mixture, with
addition of acetic acid or dilute hydrochloric acid as
catalyst.
[2819] Reducing agents suitable for this reaction are, in
particular, complex borohydrides, such as, for example, sodium
borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride,
tetra-n-butylammonium borohydride or borane-pyridine complex. It is
preferred to use sodium cyanoborohydride or borane-pyridine
complex.
[2820] The reactions (IV)+(V).fwdarw.(VI) and
(IV)+(VIII).fwdarw.(IX) take place in general 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 carried out
under atmospheric, increased or reduced pressure (e.g. from 0.5 to
5 bar); it is usual to operate at atmospheric pressure.
[2821] The above-described coupling reactions
(IX)+(X).fwdarw.(II-C), (XII-A) or (XII-B)+(X).fwdarw.(II-D-A) or
(II-D-B), (IX)+(XIII).fwdarw.(XIV), (IX)+(XV).fwdarw.(XVI) and
(XXII)+(XXIII).fwdarw.(II-D) (amide formation from amine component
and carboxylic acid component respectively) are carried out by
standard methods of peptide chemistry [see e.g. 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, Verlag Chemie, Weinheim, 1982].
[2822] Examples of inert solvents for these coupling reactions are
ethers such as diethyl ether, diisopropyl ether, tert-butyl methyl
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, halogenated hydrocarbons 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 sulphoxide (DMSO), N,N-dimethylformamide (DMF),
N,N-dimethylacetamide (DMA), N,N'-dimethylpropyleneurea (DMPU) or
N-methylpyrrolidinone (NMP). It is also possible to use mixtures of
such solvents. Preference is given to using
N,N-dimethylformamide.
[2823] Examples of suitable activating/condensing agents for these
couplings include carbodiimides such as N,N'-diethyl-,
N,N'-dipropyl-, N,N'-diisopropyl-, N,N'-dicyclohexylcarbodiimide
(DCC) or N-(3-dimethylaminoisopropyl)-N'-ethylcarbodiimide
hydrochloride (EDC), 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-sulphate or 2-tert-butyl-5-methylisoxazolium perchlorate,
acylamino compounds such as
2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, phosphorus
compounds such as propanephosphonic anhydride, diethyl
cyanophosphonate, bis(2-oxo-3-oxazolidinyl)phosphoryl chloride,
benzotriazol-1-yloxytris(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-(benzotriazol-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)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) or
O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium-tetrafluorobo-
rate (TCTU), optionally in combination with further auxiliaries
such as 1-hydroxybenzotriazole (HOBt) or N-hydroxysuccinimide
(HOSu), and also, as bases, alkali metal 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.
[2824] In the context of the present invention, as
activating/condensing agents for such coupling reactions, it is
preferred to use 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 conjunction with
N,N-diisopropylethylamine.
[2825] The coupling reactions (IX)+(X).fwdarw.(II-C), (XII-A) or
(XII-B)+(X).fwdarw.(II-D-A) or (II-D-B), (IX)+(XIII).fwdarw.(XIV),
(IX)+(XV).fwdarw.(XVI) and (XXII)+(XXIII).fwdarw.(II-D) are carried
out in general in a temperature range from -20.degree. C. to
+60.degree. C., preferably at 0.degree. C. to +40.degree. C. The
reactions may take place under atmospheric, at increased or at
reduced pressure (e.g. from 0.5 to 5 bar); it is usual to operate
under atmospheric pressure.
[2826] The esterifications (IX)+(XVIII).fwdarw.(XII) and
(IX)+(XI-A) or (XI-B).fwdarw.(XII-A) or (XI-B),
(IX)+(XXIV).fwdarw.(XXV) and also (IX)+(XXI).fwdarw.(XXII) take
place in analogy to the above-described amide coupling reactions.
These reactions take place preferably in dichloromethane, using
N-(3-dimethylaminoisopropyl)-N'-ethylcarbodiimide hydrochloride
(EDC) and 4-dimethylamino-pyridine at a temperature of +50.degree.
C. to 100.degree. C. under atmospheric pressure.
[2827] 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
during the above-described process steps, if useful or necessary.
In these cases, such protective groups are introduced and removed
in accordance with customary methods known from 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]. Where two or more protected groups
are present, they can be liberated again optionally simultaneously
in a one-pot reaction, or else liberated again in separate reaction
steps.
[2828] As an amino-protective group PG.sup.1 it is preferred to use
tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Z) or
(9H-fluoren-9-ylmethoxy)carbonyl (Fmoc); for a hydroxyl or carboxyl
function it is preferred to use tert-butyl or benzyl as protective
group PG.sup.2. The elimination of a tert-butyl or
tert-butoxycarbonyl group is typically accomplished by treatment
with a strong acid, such as hydrogen chloride, hydrogen bromide or
trifluoroacetic acid, in an inert solvent such as diethyl ether,
1,4-dioxane, dichloromethane or acetic acid; this reaction may
optionally also be carried out without addition of an inert
solvent. In the case of benzyl or benzyloxycarbonyl as protective
group, this group is removed preferably by hydrogenolysis in the
presence of a suitable palladium catalyst, such as palladium on
activated carbon, for example. The (9H-fluoren-9-ylmethoxy)carbonyl
group is generally eliminated using a secondary amine base such as
diethylamine or piperidine.
[2829] The reaction (VI).fwdarw.(II-A) takes place in a solvent
which is inert under the reaction conditions, such as, for example,
ethers such as 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
such as acetone, methyl ethyl ketone, acetonitrile, ethyl acetate,
pyridine, dimethyl sulphoxide (DMSO), N,N-dimethylformamide (DMF),
N,N-dimethylacetamide (DMA), N,N'-dimethylpropyleneurea (DMPU) or
N-methylpyrrolidinone (NMP) or water. It is also possible to use
mixtures of such solvents. Preference is given to using a mixture
of 1,4-dioxane and water.
[2830] Suitable bases for the reaction (VI).fwdarw.(II-A) are, for
example, alkali metal carbonates such as potassium carbonate,
sodium carbonate or lithium carbonate, alkali metal
hydrogencarbonates such as sodium or potassium hydrogencarbonate or
alkali metal alkoxides such as sodium methoxide, sodium ethoxide or
potassium tert-butoxide. It is preferred to use sodium
hydrogencarbonate.
[2831] 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 take place under
atmospheric, under elevated or under reduced pressure (e.g. from
0.5 to 5 bar); it is usual to operate under atmospheric
pressure.
[2832] The reaction (VI)+(VII).fwdarw.(II-B) takes place in a
solvent which is inert under the reaction conditions, such as, for
example, ethers such as 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 such as acetone, methyl ethyl ketone,
acetonitrile, ethyl acetate, pyridine, dimethyl sulphoxide (DMSO),
N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA),
N,N'-dimethylpropyleneurea (DMPU) or N-methylpyrrolidinone (NMP) or
water. It is also possible to use mixtures of such solvents.
Preference is given to using DMF.
[2833] Suitable bases for the reaction (VI)+(VII).fwdarw.(II-B)
are, for example, tertiary amine bases such as triethylamine,
N-methylmorpholine, N-methylpiperidine, N,N-diisopropylethylamine,
pyridine or 4-N,N-dimethylaminopyridine. Preference is given to
using N,N-diisopropylethylamine.
[2834] 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 atmospheric, under elevated or under reduced pressure (e.g.
from 0.5 to 5 bar); it is usual to operate under atmospheric
pressure.
[2835] The reactions (IX).fwdarw.(III-A), (XIV).fwdarw.(III-B) and
(XVI).fwdarw.(III-C) and also (VI)+(XVII).fwdarw.(III-D),
(XIX)+(XX).fwdarw.(III-E) and (XXV)+(XX).fwdarw.(III-F) take place
in a solvent which is inert under the reaction conditions. Examples
of suitable solvents are ethers such as diethyl ether, diisopropyl
ether, tert-butyl methyl 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, halogenated hydrocarbons 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 sulphoxide (DMSO),
N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA),
N,N'-dimethylpropyleneurea (DMPU) or N-methylpyrrolidinone (NMP).
It is also possible to use mixtures of such solvents. Preference is
given to using N,N-dimethylformamide.
[2836] Suitable bases for these reactions are, for example,
tertiary amines such as triethylamine, N-methylmorpholine,
N-methylpiperidine, N,N-diisopropylethylamine, pyridine or
4-N,N-dimethylaminopyridine. Preference is given to using
N,N-diisopropylethylamine, optionally with addition of
4-N,N-dimethylaminopyridine.
[2837] The reactions (IX).fwdarw.(III-A), (XIV).fwdarw.(III-B) and
(XVI).fwdarw.(III-C) and also (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 take place under atmospheric, under
elevated or under reduced pressure (e.g. from 0.5 to 5 bar); it is
usual to operate under atmospheric pressure.
[2838] The compounds of the formulae (II), (III), (1-A) and (1-B)
are sub-quantities of the compounds of the formulae (IIa), (IIIa),
(Ia-A) and (Ia-B), respectively, where R.sup.35 is methyl. The
preparation of the compounds (IIa) and (IIIa) takes place in
analogy to the preparation of the compound of the formulae (II) and
(III) as described above.
[2839] The above-described processes are illustrated by way of
example by the following synthesis schemes (Scheme 3 to 13,
18):
##STR00415##
##STR00416##
##STR00417##
##STR00418##
##STR00419##
##STR00420##
##STR00421##
##STR00422##
##STR00423##
##STR00424##
##STR00425##
##STR00426##
[2840] The compounds of the formula (IV) can be prepared 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) in analogy to processes known from the literature, in
accordance with customary methods of peptide chemistry, and as
described in the present experimental section. The synthesis
schemes below (Scheme 14 to 16) illustrate the preparation by way
of example.
##STR00427##
##STR00428##
##STR00429##
[2841] The compounds of the formulae (XI), (XIII), (XV), (XVII) and
(XXI), including, where appropriate, chiral or diastereomeric forms
thereof, are available commercially or are described as such in the
literature, or they can be prepared by routes that are obvious to
the skilled person, in analogy to methods published in the
literature. Numerous comprehensive instructions and also literature
information on the preparation of the starting materials are also
given in the experimental section, in the section relating to the
preparation of the starting compounds and intermediates.
[2842] The compounds of the formulae (V), (VII), (VIII), (X),
(XVIII), (XX) and (XXIII), including, where appropriate, chiral or
diastereomeric forms thereof, are known from the literature, or can
be prepared by routes which are obvious to the skilled person, in
analogy to methods published in the literature. Numerous
comprehensive instructions and also literature information on the
preparation of the starting materials are also given in the
experimental section, in the section relating to the preparation of
the starting compounds and intermediates.
[2843] Alternatively, individual steps of the preparation sequence
may be carried out in a different order. This approach is
illustrated by way of example in the synthesis schemes below
(Scheme 17, 19 and 20).
##STR00430## ##STR00431##
##STR00432##
##STR00433## ##STR00434##
[2844] In one embodiment the binder binds to a target molecule
which is present on a cancer cell. In one preferred embodiment the
binder binds to a cancer target molecule.
[2845] In another preferred embodiment the target molecule is a
selective cancer target molecule.
[2846] In one particularly preferred embodiment the target molecule
is a protein.
[2847] In one embodiment the target molecule is an extracellular
target molecule. In one preferred embodiment the extracellular
target molecule is a protein.
[2848] Cancer target molecules are known to the skilled person.
Examples thereof are listed below.
[2849] Examples of cancer target molecules are as follows:
(1) EGF receptor (NCBI reference sequence NP.sub.--005219.2)
[2850] Sequence (1210 amino acids):
TABLE-US-00001 >gi|29725609|ref|NP_005219.2| epidermal growth
factor receptor isoform a precursor [Homo sapiens]
MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFL
SLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVER
IPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGA
VRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPN
GSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRE
SDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKK
CPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIG
EFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQEL
DILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVV
SLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKI
ISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKC
NLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDG
PHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCP
TNGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQER
ELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEG
EKVKIPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTS
TVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRL
VHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMA
LESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGE
RLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLV
IQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPST
SRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALT
EDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQD
PHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDF
FPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA
[2851] The extracellular domain is marked by underlining.
(2) Mesothelin (SwissProt reference Q13421-3)
[2852] Sequence (622 amino acids):
TABLE-US-00002 >sp|Q13421-3|MSLN_HUMAN Isoform 2 of Mesothelin
OS = Homo sapiens GN = MSLN
MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPL
DGVLANPPNISSLSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKL
STEQLRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTRFFSRI
TKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALGGLACDL
PGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTW
SVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTI
LRPRFRREVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQM
DRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDI
RKWNVTSLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDT
LTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKA
RLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRT
DAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGL
GLQGGIPNGYLVLDLSMQEALSGTPCLLGPGPVLTVLALLLASTLA
[2853] Mesothelin is encoded 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.
(3) Carboanhydrase IX (SwissProt reference Q16790)
[2854] Sequence (459 amino acids):
TABLE-US-00003 >sp|Q16790|CAH9_HUMAN Carbonic anhydrase 9 OS =
Homo sapiens GN = CA9 PE = 1 SV = 2
MAPLCPSPWLPLLIPAPAPGLTVQLLLSLLLLVPVHPQRLPRMQEDSP
LGGGSSGEDDPLGEEDLPSEEDSPREEDPPGEEDLPGEEDLPGEEDLP
EVKPKSEEEGSLKLEDLPTVEAPGDPOEPONNAHRDKEGDDOSHWRYG
GDPPWPRVSPACAGRFOSPVDIRPOLAAFCPALRPLELLGFQLPPLPE
LRLRNNGHSVQLTLPPGLEMALGPGREYRALQLHLHWGAAGRPGSEHT
VEGHRFPAEIHVVHLSTAFARVDEALGRPGGLAVLAAFLEEGPEENSA
YEOLLSRLEEIAEEGSETQVPGLDISALLPSDFSRYFOYEGSLTTPPC
AQGVIWTVFNOTVMLSAKOLHTLSDTLWGPGDSRLOLNFRATOPLNGR
VIEASFPAGVDSSPRAAEPVOLNSCLAAGDILALVFGLLFAVTSVAFL
VQMRRQHRRGTKGGVSYRPAEVAETGA
[2855] The extracellular domain is marked by underlining.
(4) C4.4a (NCBI reference sequence NP.sub.--055215.2; Synonym
LYPD3)
[2856] Sequence (346 amino acids):
TABLE-US-00004 >gi|93004088|ref|NP_055215.2| ly6/PLAUR domain-
containing protein 3 precursor [Homo sapiens]
MDPARKAGAQAMIWTAGWLLLLLLRGGAQALECYSCVQKADDGCSPNKM
KTVKCAPGVDVCTEAVGAVETIHGQFSLAVRGCGSGLPGKNDRGLDLHG
LLAFIQLQQCAQDRCNAKLULTSRALDPAGNESAYPPNGVECYSCVGLS
REACQGTSPPVVSCYNASDHVYKGCFDGNVTLTAANVTVSLPVRGCVQD
EFCTRDGVTGPGFTLSGSCCQGSRCNSDLRNKTYFSPRIPPLVRLPPPE
PTTVASTTSVTTSTSAPVRPTSTTKPMPAPTSQTPRQGVEHEASRDEEP
RLTGGAAGHQDRSNSGQYPAKGGPQQPHNKGCVAPTAGLAALLLAVAAG VLL
[2857] The matured, extracellular domain is marked by underlining
(SEQ ID NO:1).
(5) CD52 (NCBI reference sequence NP.sub.--001794.2)
TABLE-US-00005 >gi|68342030|ref|NP_001794.2| CAMPATH-1 antigen
precursor [Homo sapiens]
MKRFLFLLLTISLLVMVQIQTGLSGQNDTSQTSSPSASSNISGGIFLF FVANAIIHLFCFS
(6) Her2 (NCBI reference sequence NP.sub.--004439.2)
TABLE-US-00006 >gi|54792096|ref|NP_004439.2| receptor tyrosine-
protein kinase erbB-2 isoform a [Homo sapiens]
MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHL
YQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQR
LRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTE
ILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCS
PMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCT
GPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGAS
CVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCY
GLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPL
QPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAY
SLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPH
QALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQE
CVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACA
HYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDL
DDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYT
MRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVY
KGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSR
LLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGM
SYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGG
KVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREI
PDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMA
RDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQG
FFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPS
EGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGY
VAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPG
KNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQ
DPPERGAPPSTFKGTPTAENPEYLGLDVPV
(7) CD20 (NCBI reference sequence NP.sub.--068769.2)
TABLE-US-00007 >gi|23110987|ref|NP_068769.2| B-lymphocyte
antigen CD20 [Homo sapiens]
MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMR
ESKTLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMY
IISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNI
KISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQS
LFLGILSVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEK
KEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEP PQDQESSPIENDSSP
(8) The lymphocyte-activating antigen CD30 (SwissProt ID
P28908)
TABLE-US-00008 >gi|68348711|ref|NP_001234.2| tumor necrosis
factor receptor superfamily member 8 isoform 1 precursor [Homo
sapiens] MRVLLAALGLLFLGALRAFPQDRPFEDTCHGNPSHYYDKAVRRCCYRCP
MGLFPTQQCPQRPTDCRKQCEPDYYLDEADRCTACVTCSRDDLVEKTPC
AWNSSRVCECRPGMFCSTSAVNSCARCFFHSVCPAGMIVKFPGTAQKNT
VCEPASPGVSPACASPENCKEPSSGTIPQAKPTPVSPATSSASTMPVRG
GTRLAQEAASKLTRAPDSPSSVGRPSSDPGLSPTQPCPEGSGDCRKQCE
PDYYLDEAGRCTACVSCSRDDLVEKTPCAWNSSRTCECRPGMICATSAT
NSRARCVPYPICAAETVTKPQDMAEKDTTFEAPPLGTQPDCNPTPENGE
APASTSPTQSLLVDSQASKTLPIPTSAPVALSSTGKPVLDAGPVLFWVI
LVLVVVVGSSAFLLCHRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPR
RSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASP
AGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRG
LAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPL PTAASGK
(9) The lymphocyte adhesion molecule CD22 (SwissProt ID P20273)
TABLE-US-00009 >gi|157168355|ref|NP_001762.2| B-cell receptor
CD22 isoform 1 precursor [Homo sapiens]
MHLLGPWLLLLVLEYLAFSDSSKWVFEHPETLYAWEGACVWIPCTYRAL
DGDLESFILFHNPEYNKNTSKFDGTRLYESTKDGKVPSEQKRVQFLGDK
NKNCTLSIHPVHLNDSGQLGLRMESKTEKWMERIHLNVSERPFPPHIQL
PPEIQESQEVTLTCLLNFSCYGYPIQLQWLLEGVPMRQAAVTSTSLTIK
SVFTRSELKFSPQWSHHGKIVTCQLQDADGKFLSNDTVQLNVKHTPKLE
IKVTPSDAIVREGDSVTMTCEVSSSNPEYTTVSWLKDGTSLKKQNTFTL
NLREVTKDQSGKYCCQVSNDVGPGRSEEVFLQVQYAPEPSTVQILHSPA
VEGSQVEFLCMSLANPLPTNYTWYHNGKEMQGRTEEKVHIPKILPWHAG
TYSCVAENILGTGQRGPGAELDVQYPPKKVTTVIQNPMPIREGDTVTLS
CNYNSSNPSVTRYEWKPHGAWEEPSLGVLKIQNVGWDNTTIACAACNSW
CSWASPVALNVQYAPRDVRVRKIKPLSEIHSGNSVSLQCDFSSSHPKEV
QFFWEKNGRLLGKESQLNFDSISPEDAGSYSCWVNNSIGQTASKAWTLE
VLYAPRRLRVSMSPGDQVMEGKSATLTCESDANPPVSHYTWFDWNNQSL
PYHSQKLRLEPVKVQHSGAYWCQGTNSVGKGRSPLSTLTVYYSPETIGR
RVAVGLGSCLAILILAICGLKLQRRWKRTQSQQGLQENSSGQSFFVRNK
KVRRAPLSEGPHSLGCYNPMMEDGISYTTLRFPEMNIPRTGDAESSEMQ
RPPPDCDDTVTYSALHKRQVGDYENVIPDFPEDEGIHYSELIQFGVGER
PQAQENVDYVILKH
(10) The myloid cell surface antigen CD33 (SwissProt ID P20138)
TABLE-US-00010 >gi|130979981|ref|NP_001763.3| myeloid cell
surface antigen CD33 isoform 1 precursor [Homo sapiens]
MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPY
YDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSR
NNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKI
LIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSV
LIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPG
DGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAV
GRNDTHPTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASL
NFHGMNPSKDTSTEYSEVRTQ
(11) The transmembrane glycoprotein NMB (SwissProt ID Q14956)
TABLE-US-00011 >gi|52694752|ref|NP_001005340.1|transmembrane
glycoprotein NMB isoform a precursor [Homo sapiens]
MECLYYFLGFLLLAARLPLDAAKRFHDVLGNERPSAYMREHNQLNGWS
SDENDWNEKLYPVWKRGDMRWKNSWKGGRVQAVLTSDSPALVGSNITF
AVNLIFPRCQKEDANGNIVYEKNCRNEAGLSADPYVYNWTAWSEDSDG
ENGTGQSHHNVFPDGKPFPHHPGWRRWNFIYVFHTLGQYFQKLGRCSV
RVSVNTANVTLGPQLMEVTVYRRHGRAYVPIAQVKDVYVVTDQIPVFV
TMFQKNDRNSSDETFLKDLPIMFDVLIHDPSHFLNYSTINYKWSFGDN
TGLFVSTNHTVNHTYVLNGTFSLNLTVKAAAPGPCPPPPPPPRPSKPT
PSLATTLKSYDSNTPGPAGDNPLELSRIPDENCQINRYGHFQATITIV
EGILEVNIIQMTDVLMPVPWPESSLIDFVVTCQGSIPTEVCTIISDPT
CEITQNTVCSPVDVDEMCLLTVRRTFNGSGTYCVNLTLGDDTSLALTS
TLISVPDRDPASPLRMANSALISVGCLAIFVTVISLLVYKKHKEYNPI
ENSPGNVVRSKGLSVFLNRAKAVFFPGNQEKDPLLKNQEFKGVS
(12) The adhesion molecule CD56 (SwissProt ID P13591)
TABLE-US-00012 >gi|94420689|ref|NP_000606.3|neural cell adhesion
molecule 1 isoform 1 [Homo sapiens]
MLQTKDLIWTLFFLGTAVSLQVDIVPSQGEISVGESKFFLCQVAGDAKD
KDISWFSPNGEKLTPNQQRISVVWNDDSSSTLTIYNANIDDAGIYKCVV
TGEDGSESEATVNVKIFQKLMFKNAPTPQEFREGEDAVIVCDVVSSLPP
TIIWKHKGRDVILKKDVRFIVLSNNYLQIRGIKKTDEGTYRCEGRILAR
GEINFKDIQVIVNVPPTIQARQNIVNATANLGQSVTLVCDAEGFPEPTM
SWTKDGEQIEQEEDDEKYIFSDDSSQLTIKKVDKNDEAEYICIAENKAG
EQDATIHLKVFAKPKITYVENQTAMELEEQVTLTCEASGDPIPSITWRT
STRNISSEEKTLDGHMVVRSHARVSSLTLKSIQYTDAGEYICTASNTIG
QDSQSMYLEVQYAPKLQGPVAVYTWEGNQVNITCEVFAYPSATISWFRD
GQLLPSSNYSNIKIYNTPSASYLEVTPDSENDFGNYNCTAVNRIGQESL
EFILVQADTPSSPSIDQVEPYSSTAQVQFDEPEATGGVPILKYKAEWRA
VGEEVWHSKWYDAKEASMEGIVTIVGLKPETTYAVRLAALNGKGLGEIS
AASEFKTQPVQGEPSAPKLEGQMGEDGNSIKVNLIKQDDGGSPIRHYLV
RYRALSSEWKPEIRLPSGSDHVMLKSLDWNAEYEVYVVAENQQGKSKAA
HFVFRTSAQPTAIPANGSPTSGLSTGAIVGILIVIFVLLLVVVDITCYF
LNKCGLFMCIAVNLCGKAGPGAKGKDMEEGKAAFSKDESKEPIVEVRTE
EERTPNHDGGKHTEPNETTPLTEPEKGPVEAKPECQETETKPAPAEVKT
VPNDATQTKENESKA
(13) The surface molecule CD70 (SwissProt ID P32970)
TABLE-US-00013 >gi|4507605|ref|NP_001243.1|CD70 antigen [Homo
sapiens] MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLP
LESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRI
HRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSF
HQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFFGVQWVRP
(14) The surface molecule CD74 (SwissProt ID P04233)
TABLE-US-00014 >gi|10835071|ref|NP_004346.1| HLA class II
histocompatibility antigen gamma chain isoform b [Homo sapiens]
MHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPESKCSRGA
LYTGFSILVTLLLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMK
LPKPPKPVSKMRMATPLLMQALPMGALPQGPMQNATKYGNMTEDHVMH
LLQNADPLKVYPPLKGSFPENLRHLKNTMETIDWKVFESWMHHWLLFE
MSRHSLEQKPTDAPPKESLELEDPSSGLGVTKQDLGPVPM
(15) The B-lymphocyte antigen CD19 (SwissProt ID P15391)
TABLE-US-00015 >gi|296010921|ref|NP_001171569.1| B-lymphocyte
antigen CD19 isoform 1 precursor [Homo sapiens]
MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPT
QQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGF
YLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSS
EGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDL
TMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRP
ARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWH
WLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKRKRMTDP
TRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQRWAAGLGGTAP
SYGNPSSDVQADGALGSRSPPGVGPEEEEGEGYEEPDSEEDSEFYEN
DSNLGQDQLSQDGSGYENPEDEPLGPEDEDSFSNAESYENEDEELTQ
PVARTMDFLSPHGSAWDPSREATSLAGSQSYEDMRGILYAAPQLRSI
RGQPGPNHEEDADSYENMDNPDGPDPAWGGGGRMGTWSTR
(16) The surface protein mucin-1 (SwissProt ID P15941)
TABLE-US-00016 >gi|65301117|ref|NP_002447.4|mucin-1 isoform 1
precursor [Homo sapiens]
MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGEKETSATQRSSVPS
STEKNALSTGVSFFFLSFHISNLQFNSSLEDPSTDYYQELQRDISEM
FLQIYKQGGFLGLSNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQ
YKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVL
VALAIVYLIALAVCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGR
YVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAATSANL
(17) The surface protein CD138 (SwissProt ID P18827)
TABLE-US-00017 >gi|29568086|ref|NP_002988.3| syndecan-1
precursor [Homo sapiens]
MRRAALWLWLCALALSLQPALPQIVATNLPPEDQDGSGDDSDNFSGSG
AGALQDITLSQQTPSTWKDTQLLTAIPTSPEPTGLEATAASTSTLPAG
EGPKEGEAVVLPEVEPGLTAREQEATPRPRETTQLPTTHQASTTTATT
AQEPATSHPHRDMQPGHHETSTPAGPSQADLHTPHTEDGGPSATERAA
EDGASSQLPAAEGSGEQDFTFETSGENTAVVAVEPDRRNQSPVDQGAT
GASQGLLDRKEVLGGVIAGGLVGLIFAVCLVGFMLYRMKKKDEGSYSL
EEPKQANGGAYQKPTKQEEFYA
(18) The integrin alphaV (Genbank Accession No.:
NP.sub.--002201.1)
TABLE-US-00018 >gi|4504763|ref|NP_002201.1|integrin alpha-V
isoform 1 precursor [Homo sapiens]
MAFPPRRRLRLGPRGLPLLLSGLLLPLCRAFNLDVDSPAEYSGPEGSY
FGFAVDFFVPSASSRMFLLVGAPKANTTQPGIVEGGQVLKCDWSSTRR
CQPIEFDATGNRDYAKDDPLEFKSHQWFGASVRSKQDKILACAPLYHW
RTEMKQEREPVGTCFLQDGTKTVEYAPCRSQDIDADGQGFCQGGFSID
FTKADRVLLGGPGSFYWQGQLISDQVAEIVSKYDPNVYSIKYNNQLAT
RTAQAIFDDSYLGYSVAVGDFNGDGIDDFVSGVPRAARTLGMVYIYDG
KNMSSLYNFTGEQMAAYFGFSVAATDINGDDYADVFIGAPLFMDRGSD
GKLQEVGQVSVSLQRASGDFQTTKLNGFEVFARFGSAIAPLGDLDQDG
FNDIAIAAPYGGEDKKGIVYIFNGRSTGLNAVPSQILEGQWAARSMPP
SFGYSMKGATDIDKNGYPDLIVGAFGVDRAILYRARPVITVNAGLEVY
PSILNQDNKTCSLPGTALKVSCFNVRFCLKADGKGVLPRKLNFQVELL
LDKLKQKGAIRRALFLYSRSPSHSKNMTISRGGLMQCEELIAYLRDES
EFRDKLTPITIFMEYRLDYRTAADTTGLQPILNQFTPANISRQAHILL
DCGEDNVCKPKLEVSVDSDQKKIYIGDDNPLTLIVKAQNQGEGAYEAE
LIVSIPLQADFIGVVRNNEALARLSCAFKTENQTRQVVCDLGNPMKAG
TQLLAGLRFSVHQQSEMDTSVKFDLQIQSSNLFDKVSPVVSHKVDLAV
LAAVEIRGVSSPDHIFLPIPNWEHKENPETEEDVGPVVQHIYELRNNG
PSSFSKAMLHLQWPYKYNNNTLLYILHYDIDGPMNCTSDMEINPLRIK
ISSLQTTEKNDTVAGQGERDHLITKRDLALSEGDIHTLGCGVAQCLKI
VCQVGRLDRGKSAILYVKSLLWTETFMNKENQNHSYSLKSSASFNVIE
FPYKNLPIEDITNSTLVTINVIWGIQPAPMPVPVWVIILAVLAGLLLL
AVLVFVMYRMGFFKRVRPPQEEQEREQLQPHENGEGNSET
(19) The teratocarcinoma-derived growth factor 1 protein TDGF1
(Genbank Accession No.: NP.sub.--003203.1)
TABLE-US-00019 >gi|4507425|ref|NP_003203.1| teratocarcinoma-
derived growth factor 1 isoform 1 precursor [Homo sapiens]
MDCRKMARFSYSVIWIMAISKVFELGLVAGLGHQEFARPSRGYLAFRD
DSIWPQEEPAIRPRSSQRVPPMGIQHSKELNRTCCLNGGTCMLGSFCA
CPPSFYGRNCEHDVRKENCGSVPHDTWLPKKCSLCKCWHGQLRCFPQA
FLPGCDGLVMDEHLVASRTPELPPSARTTTFMLVGICLSIQSYY
(20) The prostate-specific membrane antigen PSMA (Swiss Prot ID:
Q04609)
TABLE-US-00020 >gi|4758398|ref|NP_004467.1| glutamate carboxy-
peptidase 2 isoform 1 [Homo sapiens]
MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNE
ATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQI
QSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLF
EPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDM
KINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVK
SYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVG
LPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGN
FSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFG
GIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGST
EWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKE
LKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRL
GIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTV
AQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKT
YSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLE
RAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVD
PSKAWGEVKRQIYVAAFTVQAAAETLSEVA
(21) The tyrosine protein kinase EPHA2 (Swiss Prot ID: P29317)
TABLE-US-00021 >gi|32967311|ref|NP_004422.2|ephrin type-A
receptor 2 precursor [Homo sapiens]
MELQAARACFALLWGCALAAAAAAQGKEVVLLDFAAAGGELGWLTHPYG
KGWDLMQNIMNDMPIYMYSVCNVMSGDQDNWLRTNWVYRGEAERIFIEL
KFTVRDCNSFPGGASSCKETFNLYYAESDLDYGTNFQKRLFTKIDTIAP
DEITVSSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRV
YYKKCPELLQGLAHFPETIAGSDAPSLATVAGTCVDHAVVPPGGEEPRM
HCAVDGEWLVPIGQCLCQAGYEKVEDACQACSPGFFKFEASESPCLECP
EHTLPSPEGATSCECEEGFFRAPQDPASMPCTRPPSAPHYLTAVGMGAK
VELRWTPPQDSGGREDIVYSVTCEQCWPESGECGPCEASVRYSEPPHGL
TRTSVTVSDLEPHMNYTFTVEARNGVSGLVTSRSFRTASVSINQTEPPK
VRLEGRSTTSLSVSWSIPPPQQSRVWKYEVTYRKKGDSNSYNVRRTEGF
SVTLDDLAPDTTYLVQVQALTQEGQGAGSKVHEFQTLSPEGSGNLAVIG
GVAVGVVLLLVLAGVGFFIHRRRKNQRARQSPEDVYFSKSEQLKPLKTY
VDPHTYEDPNQAVLKFTTEIHPSCVTRQKVIGAGEFGEVYKGMLKTSSG
KKEVPVAIKTLKAGYTEKQRVDFLGEAGIMGQFSHHNIIRLEGVISKYK
PMMIITEYMENGALDKFLREKDGEFSVLQLVGMLRGIAAGMKYLANMNY
VHRDLAARNILVNSNLVCKVSDFGLSRVLEDDPEATYTTSGGKIPIRWT
APEAISYRKFTSASDVWSFGIVMWEVMTYGERPYWELSNHEVMKAINDG
FRLPTPMDCPSAIYQLMMQCWQQERARRPKFADIVSILDKLIRAPDSLK
TLADFDPRVSIRLPSTSGSEGVPFRTVSEWLESIKMQQYTEHFMAAGYT
AIEKVVQMTNDDIKRIGVRLPGHQKRIAYSLLGLKDQVNTVGIPI
(22) The surface protein SLC44A4 (Genbank Accession No:
NP.sub.--001171515)
TABLE-US-00022 >gi|295849282|ref|NP_001171515.1|choline
transporter-like protein 4 isoform 2 [Homo sapiens]
MGGKQRDEDDEAYGKPVKYDPSFRGPIKNRSCTDVICCVLFLLFILGY
IVVGIVAWLYGDPRQVLYPRNSTGAYCGMGENKDKPYLLYFNIFSCIL
SSNIISVAENGLQCPTPQTVITSLQQELCPSFLLPSAPALGRCFPWIN
VIPPALPGITNDTTIQQGISGLIDSLNARDISVKIFEDFAQSWYWILV
ALGVALVLSLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRV
LRDKGASISQLGFTTNLSAYQSVQETWLAALIVLAVLEAILLLMLIFL
RQRIRIAIALLKEASKAVGQMMSTMFYPLVTFVLLLICIAYWAMTALY
LATSGQPQYVLWASNISSPGCEKVPINTSCNPTAHLVNSSCPGLMCVF
QGYSSKGLIQRSVFNLQIYGVLGLFWTLNWVLALGQCVLAGAFASFYW
AFHKPQDIPTFPLISAFIRTLRYHTGSLAFGALILTLVQIARVILEYI
DHKLRGVQNPVARCIMCCFKCCLWCLEKFIKFLNRNAYIMIAIYGKNF
CVSAKNAFMLLMRNIVRVVVLDKVIDLLLFFGKLLVVGGVGVLSFFFF
SGRIPGLGKDFKSPHLNYYWLPIMTSILGAYVIASGFFSVFGMCVDTL
FLCFLEDLERNNGSLDRPYYMSKSLLKILGKKNEAPPDNKKRKK
(23) The surface protein BMPR1B (SwissProt: O00238) (24) The
transport protein SLC7A5 (SwissProt: Q01650) (25) The epithelial
prostate antigen STEAP1 (SwissProt: Q9UHE8) (26) The ovarian
carcinoma antigen MUC16 (SwissProt: Q8WXI7) (27) The transport
protein SLC34A2 (SwissProt: O95436) (28) The surface protein SEMA5b
(SwissProt: Q9P283) (29) The surface protein LYPD1 (SwissProt:
Q8N2G4) (30) The endothelin receptor type B EDNRB (SwissProt:
P24530) (31) The ring finger protein RNF43 (SwissProt: Q68DV7) (32)
The prostate carcinoma-associated protein STEAP2 (SwissProt:
Q8NFT2) (33) The cation channel TRPM4 (SwissProt: Q8TD43) (34) The
complement receptor CD21 (SwissProt: P20023) (35) The B-cell
antigen receptor complex-associated protein CD79b (SwissProt:
P40259) (36) The cell adhesion antigen CEACAM6 (SwissProt: P40199)
(37) The dipeptidase DPEP1 (SwissProt: P16444) (38) The interleukin
receptor IL20Ralpha (SwissProt: Q9UHF4) (39) The proteoglycan BCAN
(SwissProt: Q96GW7) (40) The ephrin receptor EPHB2 (SwissProt:
P29323) (41) The prostate stem cell-associated protein PSCA
(Genbank Accession No: NP.sub.--005663.2) (42) The surface protein
LHFPL3 (SwissProt: Q86UP9) (43) The receptor protein TNFRSF13C
(SwissProt: Q96RJ3) (44) The B-cell antigen receptor
complex-associated protein CD79a (SwissProt: P11912) (45) The
receptor protein CXCR5 (SwissProt: P32302) (46) The ion channel
P2X5 (SwissProt: Q93086) (47) The lymphocyte antigen CD180
(SwissProt: Q99467) (48) The receptor protein FCRL1 (SwissProt:
Q96LA6) (49) The receptor protein FCRL5 (SwissProt: Q96RD9) (50)
The MHC class II molecule Ia antigen HLA-DOB (Genbank Accession No:
NP.sub.--002111.1) (51) The T-cell protein VTCN1 (SwissProt:
Q7Z7D3). (52) The Lewis Y antigen (53) The Lewix X antigen
[2858] In one preferred subject of the invention the cancer target
molecule is selected from the group consisting of the cancer target
molecules (1)-(51). In one preferred subject of the invention the
cancer target molecule is selected from the group consisting of the
cancer target molecules (1)-(53).
[2859] In another particularly preferred subject 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)-(51).
[2860] In another particularly preferred subject 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)-(51).
[2861] In one particularly preferred subject 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) and carboanhydrase IX (CA IX;
NP.sub.--001207.2), more particularly C4.4a
(NP.sub.--055215.2).
[2862] In another particularly preferred subject 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) and carboanhydrase IX (CA IX; Q16790)), more
particularly C4.4a (NP.sub.--055215.2).
[2863] In another particularly preferred subject of the invention
the binder binds specifically 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) and carboanhydrase IX (CA IX; Q16790)), more
particularly C4.4a (NP.sub.--055215.2).
[2864] In one preferred embodiment the binder, after binding to its
extracellular target molecule on the target cell, is internalized
by the target cell as a result of the binding. The effect of this
is that the binder-drug conjugate, which may be an immunoconjugate
or an ADC, is taken up by the target cell.
[2865] In one embodiment the binder is a binding protein. In one
preferred embodiment the binder is an antibody, an antigen-binding
antibody fragment, a multispecific antibody or an antibody
mimetic.
[2866] Preferred antibody mimetics are affibodies, adnectins,
anticalins, DARPins, avimers, or nanobodies. Preferred
multispecific antibodies are bispecific and trispecific
antibodies.
[2867] In one preferred embodiment the binder is an antibody or an
antigen-binding antibody fragment, more preferably an isolated
antibody or an isolated antigen-binding antibody fragment.
[2868] Preferred antigen-binding antibody fragments are Fab, Fab',
F(ab').sub.2 and Fv fragments, diabodies, DAbs, linear antibodies
and scFv. Particularly preferred are Fab, diabodies and scFv.
[2869] In one particularly preferred embodiment the binder is an
antibody. Particularly preferred are monoclonal antibodies or
antigen-binding antibody fragments thereof. Further particularly
preferred are human, humanized or chimeric antibodies or
antigen-binding antibody fragments thereof.
[2870] Antibodies or antigen-binding antibody fragments which bind
cancer target molecules may be prepared by a person of ordinary
skill in the art using known processes, such as, for example,
chemical synthesis or recombinant expression. Binders for cancer
target molecules may be acquired commercially or may be prepared by
a person of ordinary skill in the art using known processes, such
as, for example, chemical synthesis or recombinant expression.
Further processes for preparing antibodies or antigen-binding
antibody fragments are described in WO 2007/070538 (see page 22
"Antibodies"). The skilled person knows how processes such as phage
display libraries (e.g. Morphosys HuCAL Gold) can be compiled and
used for discovering antibodies or antigen-binding antibody
fragments (see WO 2007/070538, page 24 ff and Example 1 on page 70,
Example 2 on page 72). Further processes for preparing antibodies
that use DNA libraries from B-cells are described for example on
page 26 (WO 2007/070538). Processes for humanizing antibodies are
described on page 30-32 of WO2007070538 and in detail in Queen, et
al., Pros. Natl. Acad. Sci. USA 86:10029-10033, 1989 or in WO
90/0786. Furthermore, processes for the recombinant expression of
proteins in general and of antibodies in particular are known to
the skilled person (see, for example, 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, (Second Edition, Cold Spring Harbor Laboratory
Press; Cold Spring Harbor, N.Y.; 1989) Vol. 1-3); Current Protocols
in Molecular Biolony, (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)). The skilled person knows
the corresponding vectors, promoters and signal peptides which are
necessary for the expression of a protein/antibody. Commonplace
processes are also described in WO 2007/070538 on pages 41-45.
Processes for preparing an IgG1 antibody are described for example
in WO 2007/070538 in Example 6 on page 74 ff. Processes which allow
the determination of the internalization of an antibody after
binding to its antigen are known to the skilled person and are
described for example in WO 2007/070538 on page 80. The skilled
person is able to use the processes described in WO 2007/070538
that have been used for preparing carboanhydrase IX (Mn) antibodies
in analogy for the preparation of antibodies with different target
molecule specificity.
EGFR Antibodies
[2871] Examples of antibodies which bind the cancer target
molecules EGFR are cetuximab (INN number 7906), panitumumab (INN
number 8499) and nimotuzumab (INN number 8545). Cetuximab (Drug
Bank Accession Number DB00002) is a chimeric anti-EGFR1 antibody
which is produced in SP2/0 mouse myeloma cells and is sold by
ImClone Systems Inc/Merck KgaA/Bristol-Myers Squibb Co. Cetuximab
is indicated for the treatment of metastasizing, EGFR expressing,
colorectal carcinoma with wild type K-Ras gene. It has an affinity
of 10.sup.-10 M.
Sequence:
Cetuximab Light Chain (Kappa):
TABLE-US-00023 [2872]
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLL
KYASESISGIPSRFSGSGSGTDFILSINSVESEDIADYYCQQNNNWP
TTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR
EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
KVYACEVTHQGLSSPVTKSFNRGEC
Cetuximab Heavy Chain:
TABLE-US-00024 [2873]
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLE
WLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAI
YYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP
CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[2874] Panitumumab (INN number 8499) (Drug Bank Accession Number
DB01269) is a recombinant monoclonal human IgG2 antibody which
binds specifically to the human EGF receptor 1 and is sold by
Abgenix/Amgen. Panitumumab originates from the immunization of
transgenic mice (XenoMouse). These mice are capable of producing
human immunoglobulin (light and heavy chains). A specific B-cell
clone was selected which produces antibodies against EGFR, and this
clone was immortalized with CHO cells (Chinese hamster ovary
cells). These cells are now used for the production of a 100% human
antibody. Panitumumab is indicated for the treatment of
EGFR-expressing, metastasizing colorectal carcinoma, which is
resistant to chemotherapeutic treatment with fluoropyrimidine,
oxaliplatin and irinotecan. It has an affinity of 10-11M.
Sequence:
Panitumumab Light Chain (Kappa):
TABLE-US-00025 [2875]
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLL
IYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHL
PLAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP
REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKSFNRGEC
Panitumumab Heavy Chain:
TABLE-US-00026 [2876]
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKG
LEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADT
AIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTS
ESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPA
PPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWY
VDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS
NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPG
[2877] Nimotuzumab (INN number 8545) (EP 00586002, EP 00712863) is
a humanized monoclonal IgG1 antibody which binds specifically to
the human EGF receptor 1 and is sold by YM BioScienecs Inc.
(Mississauga Canada). It is produced in non-secreting NSO cells
(mammalian cell line). Nimotuzumab is approved for the treatment of
head-and-neck tumours, highly malignant astrocytoma and
glioblastoma multiforms (not in EU and US) and pancreatic carcinoma
(Orphan drug, EMA). It has an affinity of 10.sup.-8M.
[2878] Further embodiments of EGFR antibodies are as follows:
[2879] Zalutumumab/2F8/HuMax-EGFr, from Genmab A/S (WO 02/100348,
WO 2004/056847, INN number 8605) [2880] Necitumumab/11F8,
ImClone/IMC-11F8, from ImClone Systems Inc. [Eli Lilly & Co]
(WO 2005/090407 (EP 01735348-A1, US 2007/0264253-A1, U.S. Pat. No.
7,598,350, WO 2005/090407-A1), INN number 9083) [2881]
Matuzumab/anti-EGFR MAb, Merck KGaA/anti-EGFR MAb, Takeda/EMD
72000/EMD-6200/EMD-72000 and EMD-55900/MAb 425/monoclonal antibody
425, from Merck KGaA/Takeda (WO 92/15683, INN number 8103
(Matuzumab)) [2882]
RG-7160/GA-201/GA201/R-7160/R7160/RG7160/RO-4858696/RO-5083945/RO4858696/-
RO5083945, from Glycart Biotechnology AG (Roche Holding AG) (WO
2010/112413-A1, WO 2010/115554) [2883] GT-MAB 5.2-GEX/CetuGEX, from
Glycotope GmbH (WO 2008/028686-A2 (EP 01900750-A1, EP 01911766-A1,
EP 02073842-A2, US 2010/0028947-A1) [2884] ISU-101, from Isu Abxis
Inc (ISU Chemical Co Ltd)/Scancell (WO 2008/004834-A1) [2885]
ABT-806/mAb-806/ch-806/anti-EGFR monoc. antibody 806, from Ludwig
Institute for Cancer Research/Abbott/Life Science Pharmaceuticals
(WO 02/092771, WO 2005/081854 and WO 2009/023265) [2886] SYM-004
(consists of two chimeric IgG1 antibodies (992 and 1024)), from
Symphogen A/S (WO 2010/022736-A2) [2887] MR1-1/MR1-1 KDEL, from WAX
Corp (Teva Pharmaceutical Industries Ltd) (Duke University),
(Patent: WO2001/062931-A2) [2888] Antibody against the deletion
mutant, EGFRvIII, from Amgen/Abgenix (WO 2005/010151, U.S. Pat. No.
7,628,986) [2889] SC-100, from Scancell Ltd (WO 01/088138-A1)
[2890] MDX-447/EMD 82633/BAB-447/H 447/MAb, EGFR, Medarex/Merck
KgaA, from Bristol-Myers Squibb (US)/Merck KGaA (DE)/Takeda (JP),
(WO 91/05871, WO 92/15683) [2891] Anti-EGFR-Mab, from Xencor (WO
2005/056606) [2892] DXL-1218/anti-EGFR monoclonal antibody
(cancer), InNexus, from InNexus Biotechnology Inc., Pharmaprojects
PH048638
[2893] In one 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.
[2894] In one particularly preferred embodiment the anti-EGFR
antibodies are selected from the group consisting of cetuximab,
panitumumab, nimotuzumab, zalutumumab, necitumumab and
matuzumab.
[2895] The skilled person knows of processes which can be used to
prepare further antibodies, from the CDR regions of the
abovementioned antibodies by means of sequence variations, these
further antibodies having a similar or better affinity and/or
specificity for the target molecule.
[2896] In a further embodiment, the anti-EGFR antibodies or
antigen-binding antibody fragments are selected from the group
consisting of
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.
[2897] In another embodiment the anti-EGFR antibodies or
antigen-binding antibody fragments are selected from the group
consisting of
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.
Carboanhydrase IX Antibodies
[2898] Examples of antibodies which bind the cancer target molecule
carbonahydrase IX are described in WO 2007/070538-A2 (e.g. Claims
1-16).
[2899] In one 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
2007/070538-A2), 3ef2 (Claim 4 (b) in WO2007/070538-A2), 1e4 (Claim
4 (c) in WO 2007/070538-A2), 3a4 (Claim 4 (d) in WO
2007/070538-A2), 3ab4 (Claim 4 (e) in WO 2007/070538-A2), 3ah10
(Claim 4 (f) in WO 2007/070538-A2), 3bb2 (Claim 4 (g) in WO
2007/070538-A2), 1aa1 (Claim 4 (h) in WO 2007/070538-A2), 5a6
(Claim 4 (i) in WO 2007/070538-A2) and 5aa3 (Claim 4 (j) in WO
2007/070538-A2).
[2900] In one 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 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
2007/070538-A2), anti-carboanhydrase IX antibodies or
antigen-binding antibody 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 2007/070538-A2), anti-carboanhydrase IX
antibodies or antigen-binding antibody 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 2007/070538-A2), anti-carboanhydrase IX
antibodies or antigen-binding antibody 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 2007/070538-A2), anti-carboanhydrase IX
antibodies or antigen-binding antibody 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 2007/070538-A2), anti-carboanhydrase IX
antibodies or antigen-binding antibody 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 2007/070538-A2), anti-carboanhydrase IX
antibodies or antigen-binding antibody 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 2007/070538-A2), anti-carboanhydrase IX
antibodies or antigen-binding antibody 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 2007/070538-A2), anti-carboanhydrase IX
antibodies or antigen-binding antibody 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 2007/070538-A2), and anti-carboanhydrase
IX antibodies or antigen-binding antibody 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 2007/070538-A2).
[2901] The here-indicated sequences of the CDR regions are
disclosed in FIGS. 2a-2c, page 128-130 in WO 2007/070538-A2.
[2902] In one preferred embodiment the anti-carboanhydrase IX
antibodies or antigen-binding antibody fragments are selected from
the group consisting of:
an antibody or antigen-binding fragment which comprises the amino
acid sequence of the variable light and variable heavy chains of
the antibody 3ee9, as indicated in WO 2007/070538-A2 in FIG. 4b on
page 137, an antibody or antigen-binding fragment which comprises
the amino acid sequence of the variable light and variable heavy
chains of the antibody 3ef2, as indicated in WO 2007/070538-A2 in
FIG. 4c on page 138 and in FIG. 4b on page 137, an antibody or
antigen-binding fragment which comprises the amino acid sequence of
the variable light and variable heavy chains of the antibody 1e4,
as indicated in WO 2007/070538-A2 in FIG. 4a on page 136, an
antibody or antigen-binding fragment which comprises the amino acid
sequence of the variable light and variable heavy chains of the
antibody 3a4, as indicated in WO 2007/070538-A2 in FIG. 4a on page
136, an antibody or antigen-binding fragment which comprises the
amino acid sequence of the variable light and variable heavy chains
of the antibody 3ab4, as indicated in WO 2007/070538-A2 in FIG. 4a
on page 136, an antibody or antigen-binding fragment which
comprises the amino acid sequence of the variable light and
variable heavy chains of the antibody 3ah10, as indicated in WO
2007/070538-A2 in FIG. 4a on page 136, an antibody or
antigen-binding fragment which comprises the amino acid sequence of
the variable light and variable heavy chains of the antibody 3bb2,
as indicated in WO 2007/070538-A2 in FIG. 4b on page 137, an
antibody or antigen-binding fragment which comprises the amino acid
sequence of the variable light and variable heavy chains of the
antibody 1aa1l, as indicated in WO 2007/070538-A2 in FIG. 4a on
page 136, an antibody or antigen-binding fragment which comprises
the amino acid sequence of the variable light and variable heavy
chains of the antibody 5a6, as indicated in WO 2007/070538-A2 in
FIG. 4b on page 137, and an antibody or antigen-binding fragment
which comprises the amino acid sequence of the variable light and
variable heavy chains of the antibody 5aa3, as indicated in WO
2007/070538-A2 in FIG. 4b on page 137.
[2903] In one particularly preferred embodiment the
anti-carboanhydrase IX antibody is antibody 3ee9 from WO
2007/070538-A2.
[2904] In one particularly preferred embodiment the
anti-carboanhydrase IX antibody or the 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 WO2007070538-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
2007/070538-A2).
[2905] In one particularly preferred embodiment the
anti-carboanhydrase IX antibody or the antigen-binding antibody
fragment comprises the amino acid sequences of a variable heavy
chain of the antibody 3ee9
(VH3:ELVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVSGISSLGSTT
YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTGSPGTFMHGDHWGQGTL VTVSS,
see FIG. 4b, page 137 in WO 2007070538-A2) and the amino acid
sequences of the variable light chain of the antibody 3ee9
(VLk1:DIQMTQSPSSLSASVGDRVTITCRaSQDINNYLSWYQQKPGKAPKLLIYGASNLQSG
VPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYYGRPTTFGQGTKVEIKRT, see FIG. 4b,
page 137 in WO 2007070538-A2).
[2906] In one preferred embodiment the anti-carboanhydrase IX
antibody 3ee9 is an IgG antibody.
[2907] In one particularly preferred embodiment the
anti-carboanhydrase IX antibody 3ee9 is an IgG1 antibody
(3ee9-IgG1),
where the amino acid sequence of the heavy chain comprises the
following sequence:
TABLE-US-00027 QVELVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEW
VSGISSLGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY
YCARTGSPGTFMHGDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGK
and the amino acid sequences of the light chain comprises the
following sequence:
TABLE-US-00028 DIQMTQSPSSLSASVGDRVTITCRASQDINNYLSWYQQKPGKAPKLLI
YGASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYYGRPT
TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA
KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
ACEVTHQGLSSPVTKSFNRGEC
anti-carboanhydrase IX antibody 3ee9-IgG1:
[2908] A further aspect of the present invention is the provision
of the anti-carboanhydrase IX antibody 3ee9-IgG1.
C4.4a Antibodies:
[2909] Binders particularly preferred in accordance with the
invention are anti-C4.4a antibodies, more particularly human or
humanized anti-C4.4a antibodies. The antibodies preferably have an
affinity of at least 10.sup.-7 M (as Kd value; in other words
preferably those with smaller Kd values than 10.sup.-7 M),
preferably of at least 10.sup.-8 M, more preferably in the range
from 10.sup.-9 M to 10.sup.-11 M. The Kd values may be determined,
for example, by means of surface plasmon resonance
spectroscopy.
[2910] The antibody-drug conjugates of the invention likewise
exhibit affinities in these ranges. The affinity is preferably not
substantially affected by the conjugation of the drugs (in general,
the affinity is reduced by less than one order of magnitude, in
other words, for example, at most from 10.sup.-8 M to 10.sup.-7
M).
[2911] The antibodies used in accordance with the invention are
also notable preferably for a high selectivity. A high selectivity
exists when the antibody of the invention exhibits an affinity for
the target protein which is better by a factor of at least 2,
preferably by a factor of 5 or more preferably by a factor of 10,
than for an independent other antigen, e.g. human serum albumin
(the affinity may be determined, for example, by means of surface
plasmon resonance spectroscopy).
[2912] Furthermore, the antibodies of the invention that are used
are preferably cross-reactive. In order to be able to facilitate
and better interpret preclinical studies, for example toxicological
or activity studies (e.g. in xenograft mice), it is advantageous if
the antibody used in accordance with 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 in accordance with the invention, in addition to the human
target protein, is cross-reactive to the target protein of at least
one further species. For toxicological and activity studies it is
preferred to use species of the families of rodents, dogs and
non-human primates. Preferred rodent species are mouse and rat.
Preferred non-human primates are rhesus monkeys, chimpanzees and
long-tailed macaques.
[2913] In one embodiment the antibody used in accordance with the
invention, in addition to the human target protein, is
cross-reactive to the target protein of at least one further
species selected from the group of species consisting of mouse, rat
and long-tailed macaque (Macaca fascicularis). Especially preferred
are antibodies used in accordance with the invention which in
addition to the human target protein are at least cross-reactive to
the mouse target protein. Preference is given to cross-reactive
antibodies whose affinity for the target protein of the further
non-human species differs by a factor of not more than 50, more
particularly by a factor of not more than ten, from the affinity
for the human target protein.
[2914] Anti-C4.4a antibodies are described for example in WO
01/23553 or WO 2011070088. These antibodies can be used in
accordance with the invention.
[2915] Examples of C4.4a antibodies and antigen-binding fragments
are described below. The sequences of the antibodies are indicated
in Table 1, with each line reproducing the respective CDR amino
acid sequences of the variable light chain and of the variable
heavy chain, respectively of the antibody listed in column 1. The
amino acid sequences of the variable light chain and of the
variable heavy chain, and the nucleic acid sequence of the antibody
indicated in column 1 in each case, are also indicated.
[2916] In one embodiment the anti-C4.4a antibodies or
antigen-binding antibody fragments bind to the Si domain S1 (amino
acid position 1-85 of SEQ ID NO: 1) of C4.4a.
[2917] In one embodiment the anti-C4.4a antibodies or
antigen-binding antibody fragments are cross-reactive with human
C4.4a (SEQ ID NO:1) and with murine C4.4a (SEQ ID NO:2).
[2918] In one embodiment the anti-C4.4a antibodies or
antigen-binding antibody fragments thereof, after binding to a cell
which expresses C4.4a, are internalized by the cell.
[2919] In another embodiment the anti-C4.4a antibodies or
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.
The antibodies B01-1 to B01-12 were prepared from M31-B01 by means
of affinity maturation and compete with M31-B01 for binding to
C4.4a. The antibodies D02-1 to D02-13 were prepared from
M20-D02-S-A by means of affinity maturation and compete with
M20-D02-S-A for binding to C4.4a.
[2920] In a further embodiment the anti-C4.4a antibodies or
antigen-binding antibody fragments comprise at least one, two or
three of the CDR amino acid sequences given in Table 1 or Table
2.
[2921] In another embodiment the anti-C4.4a antibodies or
antigen-binding antibody fragments comprise at least one, two or
three CDR amino acid sequences of an antibody given in Table 1 or
Table 2.
[2922] In a further embodiment the anti-C4.4a antibodies or
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 given in Table 1 or Table 2.
[2923] In another embodiment the anti-C4.4a antibodies or
antigen-binding antibody fragments comprise which are at least 50%,
60%, 70%, 80%, 90% or 95% identical with the CDR amino acid
sequences of the variable light chain and with the CDR amino acid
sequences of the variable heavy chain, of an antibody given in
Table 1 or Table 2.
[2924] In another embodiment the CDR sequences of the anti-C4.4a
antibodies or antigen-binding antibody fragments comprise
[2925] CDR sequences of the heavy chain which conform to the 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 the CDR sequences SEQ ID NO: 300 (CDR L1), SEQ ID NO: 22
(CDR L2) and SEQ ID NO: 301 (CDR L3), or
[2926] CDR sequences of the heavy chain which conform to the 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 the CDR sequences SEQ ID NO: 305 (CDR L1), SEQ ID NO:
306 (CDR L2) and SEQ ID NO: 307 (CDR L3).
[2927] In another embodiment the anti-C4.4a antibodies or
antigen-binding antibody fragments comprise which are at least 50%,
60%, 70%, 80%, 90% or 95% identical with the variable light chain
and with the variable heavy chain, of an antibody given in Table 1
or Table 2.
[2928] 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 of an antibody given in
Table 1 or Table 2.
[2929] 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 given in Table 1 or
Table 2.
[2930] 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 given in Table 1
or Table 2.
[2931] In one preferred embodiment the C4.4a antibodies and the
antigen-binding antibody fragments are selected from the group
consisting of
antibody which comprises the CDR sequences of the variable heavy
chain represented by the sequences SEQ ID NO: 75-77 and which
comprises the CDR sequences of the variable light chain represented
by the sequences SEQ ID NO: 78-80 (B01-10), antibody which
comprises the CDR sequences of the variable heavy chain represented
by the sequences SEQ ID NO: 5, 9 and 13 and which comprises the CDR
sequences of the variable light chain represented by the sequences
SEQ ID NO: 17, 21 and 25 (M31-B01), antibody which comprises the
CDR sequences of the variable heavy chain represented by the
sequences SEQ ID NO: 6, 10 and 14 an which comprises the CDR
sequences of the variable light chain represented by the sequences
SEQ ID NO: 18, 22 and 26 (M20-D02-S-A), antibody which comprises
the CDR sequences of the variable heavy chain represented by the
sequences SEQ ID NO: 7, 11 and 15 and which comprises the CDR
sequences of the variable light chain represented by the sequences
SEQ ID NO: 19, 23 and 27 (M60-G03), antibody which comprises the
CDR sequences of the variable heavy chain represented by the
sequences SEQ ID NO: 8, 12 and 16 and which comprise the CDR
sequences of the variable light chain represented by the sequences
SEQ ID NO: 20, 24 and 28 (36-H02), antibody which comprises the CDR
sequences of the variable heavy chain represented by the sequences
SEQ ID NO: 45-47 and which comprises the CDR sequences of the
variable light chain represented by the sequences SEQ ID NO: 48-50
(B01-3), antibody which comprises the CDR sequences of the variable
heavy chain represented by the sequences SEQ ID NO: 55-57 and which
comprises the CDR sequences of the variable light chain represented
by the sequences SEQ ID NO: 58-60 (B01-5), antibody which comprises
the CDR sequences of the variable heavy chain represented by the
sequences SEQ ID NO: 65-67 and which comprises the CDR sequences of
variable light chain represented by the sequences SEQ ID NO: 68-70
(B01-7), antibody which comprises the CDR sequences of the variable
heavy chain represented by the sequences SEQ ID NO: 85-87 and which
comprises the CDR sequences of the variable light chain represented
by the sequences SEQ ID NO: 88-90 (B01-12), antibody which
comprises the CDR sequences of the variable heavy chain represented
by the sequences SEQ ID NO: 95-97 and which comprises the CDR
sequences of the variable light chain represented by the sequences
SEQ ID NO: 98-100 (D02-4), antibody which comprises the CDR
sequences of the variable heavy chain represented by the sequences
SEQ ID NO: 105-107 and which comprises CDR sequences of the
variable light chain represented by the sequences SEQ ID NO:
108-110 (D02-6), antibody which comprises the CDR sequences of the
variable heavy chain represented by the sequences SEQ ID NO:
115-117 and which comprises the CDR sequences of the variable light
chain represented by the sequences SEQ ID NO: 118-120 (D02-7),
antibody which comprises the CDR sequences of the variable heavy
chain represented by the sequences SEQ ID NO: 125-127 and which
comprises the CDR sequences of the variable light chain represented
by the sequences SEQ ID NO: 128-130 (D02-11), and antibody which
comprises the CDR sequences of the variable heavy chain represented
by the sequences SEQ ID NO: 135-137 which comprises the CDR
sequences of the variable light chain represented by the sequences
SEQ ID NO: 138-140 (D02-13).
[2932] In one preferred embodiment the C4.4a antibodies and the
antigen-binding antibody fragments are selected from the group
consisting of
antibodies which comprise the amino acid sequence of the variable
heavy chain represented by the sequence SEQ ID NO: 81 and which
comprise the amino acid sequence of the variable light chain
represented by the sequence SEQ ID NO: 82 (B01-7), antibodies which
comprise the amino acid sequence of the variable heavy chain
represented by the sequence SEQ ID NO: 33 and which comprise the
amino acid sequence of the variable light chain represented by the
sequence SEQ ID NO: 29 (M31-B01), antibodies which comprise the
amino acid sequence of the variable heavy chain represented by the
sequence SEQ ID NO: 34 and which comprise the amino acid sequence
of the variable light chain represented by the sequence SEQ ID NO:
30 (M20-D02 S-A), antibodies which comprise the amino acid sequence
of the variable heavy chain represented by the sequence SEQ ID NO:
35 and which comprise the amino acid sequence of the variable light
chain represented by the sequence SEQ ID NO: 31 (M60-G03),
antibodies which comprise the amino acid sequence of the variable
heavy chain represented by the sequence SEQ ID NO: 36 and which
comprise the amino acid sequence of the variable light chain
represented by the sequence SEQ ID NO: 32 (M36-H02), antibodies
which comprise the amino acid sequence of the variable heavy chain
represented by the sequence SEQ ID NO: 51 and which comprise the
amino acid sequence of the variable light chain represented by the
sequence SEQ ID NO: 52 (B01-3), antibodies which comprise the amino
acid sequence of the variable heavy chain represented by the
sequence SEQ ID NO: 61 and which comprise the amino acid sequence
of the variable light chain represented by the sequence SEQ ID NO:
62 (B01-5), antibodies which comprise the amino acid sequence of
the variable heavy chain represented by the sequence SEQ ID NO: 71
and which comprise the amino acid sequence of the variable light
chain represented by the sequence SEQ ID NO: 72 (B01-7) antibodies
which comprise the amino acid sequence of the variable heavy chain
represented by the sequence SEQ ID NO: 91 and which comprise the
amino acid sequence of the variable light chain represented by the
sequence SEQ ID NO: 92 (B01-12), antibodies which comprise the
amino acid sequence of the variable heavy chain represented by the
sequence SEQ ID NO: 101 and which comprise the amino acid sequence
of the variable light chain represented by the sequence SEQ ID NO:
102 (D02-4), antibodies which comprise the amino acid sequence of
the variable heavy chain represented by the sequence SEQ ID NO: 111
and which comprise the amino acid sequence of the variable light
chain represented by the sequence SEQ ID NO: 112 (D02-6),
antibodies which comprise the amino acid sequence of the variable
heavy chain represented by the sequence SEQ ID NO: 121 and which
comprise the amino acid sequence of the variable light chain
represented by the sequence SEQ ID NO: 122 (D02-7), antibodies
which comprise the amino acid sequence of the variable heavy chain
represented by the sequence SEQ ID NO: 131 and which comprise the
amino acid sequence of the variable light chain represented by the
sequence SEQ ID NO: 132 (D02-11), and antibodies which comprise the
amino acid sequence of the variable heavy chain represented by the
sequence SEQ ID NO: 141 and which comprise the amino acid sequence
of the variable light chain represented by the sequence SEQ ID NO:
142 (D02-13).
[2933] In another embodiment the anti-C4.4a antibodies comprise the
light chain and the heavy chain of an antibody given in Table
2.
[2934] In one preferred embodiment the anti-C4.4a antibodies
comprise the light chain and the heavy chain of an antibody given
in Table 2.
[2935] In one particularly preferred embodiment the C4.4a antibody
is selected from the group consisting of
antibody which comprises the amino acid sequence of the light chain
represented by SEQ ID NO: 346 and which comprises the amino acid
sequence of the heavy chain represented by SEQ ID NO: 347
(M31-B01), antibody which comprises the amino acid sequence of the
light chain represented by SEQ ID NO: 352 and which comprises the
amino acid sequence of the heavy chain represented by SEQ ID NO:
353 (B01-3), antibody which comprises the amino acid sequence of
the light chain represented by SEQ ID NO: 364 and which comprises
the amino acid sequence of the heavy chain represented by SEQ ID
NO: 365 (B01-10), and antibody which comprises the amino acid
sequence of the light chain represented by SEQ ID NO: 382 and which
comprises the amino acid sequence of the heavy chain represented by
SEQ ID NO: 383 (D02-6).
TABLE-US-00029 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ
ID SEQ ID SEQ ID NO: NO: NO: NO: NO: NO: NO: VH NO: VL NO: VH NO:
VL Antibody HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 Protein Protein
Nucleotide Nucleotide M31-B01 5 9 13 17 21 25 33 29 41 37 M20-D02
S-A 6 10 14 18 22 26 34 30 42 38 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-ogl 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-00030 TABLE 2 Sequences of the light and heavy chain 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
Anti-C4.4a Antibody IgG:
[2936] A further aspect of the present invention is the provision
of an anti-C4.4a IgG1 antibody which comprises the amino acid
sequence of the light chain and of the heavy chain of an antibody
given in Table 2.
Mesothelin Antibody
[2937] A further aspect of the present invention is the provision
of a new 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).
[2938] In one preferred embodiment the amino acid sequence of the
anti-mesothelin antibody MF-Ta or antigen-binding antibody
fragments comprises the 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
one preferred embodiment the amino acid sequence of the
anti-mesothelin antibody MF-Ta or antigen-binding antibody
fragments comprises the sequence of the variable heavy chain which
is encoded by the nucleic acid sequence SEQ ID NO:406, and the
sequence of the variable light chain which is encoded by the
nucleic acid sequence SEQ ID NO:407.
[2939] In one particularly 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.
[2940] In one particularly preferred embodiment the amino acid
sequence of the anti-mesothelin antibody MF-Ta comprises the
sequence of the heavy chain which is encoded with a nucleic acid
sequence SEQ ID NO:410, and the sequence of the light chain with is
encoded with a nucleic acid sequence SEQ ID NO: 411.
[2941] Further examples of antibodies which bind the cancer target
molecule mesothelin are known to the skilled person and are
described for example in WO 2009/068204 and can be used for the
binder-drug conjugates of the invention.
[2942] In one embodiment of the binder-drug conjugates, the binder
is an anti-mesothelin antibody or antigen-binding antibody
fragment, where the antibody binds to mesothelin and exhibits
invariant binding.
[2943] In one embodiment of the binder-drug conjugates, an
anti-mesothelin antibody or 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 described in WOv2009/068204-A1
(Table 7; page 61-63).
[2944] In one preferred embodiment the mesothelin antibodies or
antigen-binding antibody fragments are selected from the group
consisting of
anti-mesothelin antibodies or antigen-binding antibody 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-Ta, anti-mesothelin antibodies or
antigen-binding antibody 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 (WO2009068204-A1; Table 7; page 61), anti-mesothelin
antibodies or antigen-binding antibody 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),
anti-mesothelin antibodies or antigen-binding antibody 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 anti-mesothelin antibodies or antigen-binding antibody
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).
[2945] In one particularly preferred embodiment the mesothelin
antibodies or antigen-binding antibody fragments are selected from
the group consisting of
anti-mesothelin antibodies or antigen-binding antibody fragments
thereof which comprise the sequence of the variable light chain and
the sequence of the variable heavy chain of the antibody MF-Ta,
anti-mesothelin antibodies or antigen-binding antibody fragments
thereof which comprise 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), anti-mesothelin antibodies or
antigen-binding antibody fragments thereof which comprise 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), anti-mesothelin antibodies or antigen-binding
antibody fragments thereof which comprise 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),
anti-mesothelin antibodies or antigen-binding antibody fragments
thereof which comprise 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).
Further Antibodies:
[2946] An example of an antibody which binds the cancer target
molecule Her2 is trastuzumab (Genentech). Trastuzumab is a
humanized antibody which is used for the treatment inter alia of
breast cancer. One example of an antibody which binds the cancer
target molecule CD20 is rituximab (Genentech). Rituximab (CAS
number: 174722-31-7) is a chimeric antibody which is used for the
treatment of non-Hodgkin's lymphoma. One example of an antibody
which binds the cancer target molecule CD52 is alemtuzumab
(Genzyme). Alemtuzumab (CAS number: 216503-57-0) is a humanized
antibody which is used for the treatment of chronic lymphatic
leukaemia.
[2947] Other examples of antibodies which bind to HER2, besides
trastuzumab (INN 7637, CAS No: RN: 180288-69-1) and pertuzumab (Cas
No: 380610-27-5), are antibodies as disclosed in WO 2009/123894-A2,
WO 200/8140603-A2, or in WO 2011/044368-A2. An example of an
anti-HER2 conjugate is trastuzumab-emtansine (INN No. 9295).
[2948] Examples of antibodies which bind the cancer target molecule
CD30 and can be used for the treatment of cancer, e.g. Hodgkin's
lymphoma, are brentuximab, iratumumab and antibodies as disclosed
in WO 2008/092117, WO 2008/036688 or WO 2006/089232. An example of
an anti-CD30 conjugate is brentuximab vedotine (INN No. 9144).
[2949] Examples of antibodies which bind the cancer target molecule
CD22 and can be used for the treatment of cancer, e.g. lymphoma,
are inotuzumab or epratuzumab. Examples of anti-CD22 conjugates are
inotuzumab ozagamycin (INN No. 8574), or anti-CD22-MMAE and
anti-CD22-MC-MMAE (CAS RN: 139504-50-0 and 474645-27-7).
[2950] Examples of antibodies which bind the cancer target molecule
CD33 and can be used for the treatment of cancer, e.g. leukaemia,
are gemtuzumab or lintuzumab (INN 7580). An example of an anti-CD33
conjugate is gemtuzumab-ozagamycin.
[2951] An example of an antibody which binds the cancer target
molecule NMB and can be used for the treatment of cancer, e.g.
melanoma or breast cancer, is glembatumumab (INN 9199). An example
of an anti-NMB conjugate is glembatumumab vedotine (CAS RN:
474645-27-7).
[2952] An example of an antibody which binds the cancer target
molecule CD56 and can be used for the treatment of cancer, e.g.
multiple myeloma, small-cell carcinoma of the lung, MCC or ovarian
carcinoma, is lorvotuzumab. An example of an anti-CD56 conjugate is
lorvotuzumab mertansine (CAS RN: 139504-50-0).
[2953] Examples of antibodies which bind the cancer target molecule
CD70 and can be used for the treatment of cancer, e.g.
non-Hodgkin's lymphoma or kidney cell cancer, are disclosed in WO
2007/038637-A2 or WO 2008/070593-A2. An example of an anti-CD70
conjugate is SGN-75 (CD70 MMAF).
[2954] An example of an antibody which binds the cancer target
molecule CD74 and can be used for the treatment of cancer, e.g.
multiple myeloma, is milatuzumab. An example of an anti-CD74
conjugate is milatuzumab-doxorubicin (CAS RN: 23214-92-8).
[2955] An example of an antibody which binds the cancer target
molecule CD19 and can be used for the treatment of cancer, e.g.
non-Hodgkin's lymphoma, is disclosed in WO 2008/031056-A2. Further
antibodies and examples of an anti-CD19 conjugate (SAR3419) are
disclosed in WO 2008/047242-A2.
[2956] Examples of antibodies which bind the cancer target molecule
mucin-1 and can be used for the treatment of cancer, e.g.
non-Hodgkin's lymphoma, are clivatuzumab or the antibodies
disclosed in WO 2003/106495-A2, WO 2008/028686-A2. Examples of
anti-mucin conjugates are disclosed in WO 2005/009369-A2.
[2957] Examples of antibodies which bind the cancer target molecule
CD138 and conjugates thereof which can be used for the treatment of
cancer, e.g. multiple myeloma, are disclosed in WO 2009/080829-A1,
WO 2009/080830-A1.
[2958] Examples of antibodies which bind the cancer target molecule
integrin alphaV and can be used for the treatment of cancer, e.g.
melanoma, sarcoma or carcinoma, are intetumumab (Cas RN:
725735-28-4), abciximab (Cas-RN: 143653-53-6), etaracizumab
(Cas-RN: 892553-42-3) or the antibodies disclosed in U.S. Pat. No.
7,465,449, EP 19859-A1, WO 2002/012501-A1 or WO 2006/062779-A2.
Examples of anti-integrin alphaV conjugates are intetumumab-DM4 and
other ADCs disclosed in WO 2007/024536-A2.
[2959] Examples of antibodies which bind the cancer target molecule
TDGF1 and can be used for the treatment of cancer are the
antibodies disclosed in WO 02/077033-A1, U.S. Pat. No. 7,318,924,
WO 2003/083041-A2 and WO 2002/088170-A2. Examples of anti-TDGF1
conjugates are disclosed in WO 2002/088170-A2.
[2960] Examples of antibodies which bind the cancer target molecule
PSMA and can be used for the treatment of cancer, e.g. prostate
carcinoma, are the antibodies disclosed in WO 97/35616-A1, WO
99/47554-A1, and WO 01/009192-A1. Examples of anti-PSMA conjugates
are disclosed in WO 2009/026274-A1.
[2961] Examples of antibodies which bind the cancer target molecule
EPHA2, can be used for preparing a conjugate and can be used for
the treatment of cancer are disclosed in WO 2004/091375-A2.
[2962] Examples of antibodies which bind the cancer target molecule
SLC44A4, can be used for preparing a conjugate and can be used for
the treatment of cancer, e.g. pancreatic or prostate carcinoma, are
disclosed in WO 2009/033094-A2 and US 2009/0175796-A1.
[2963] An example of an antibody which binds the cancer target
molecule HLA-DOB is the antibody lym-1 (Cas-RN: 301344-99-0), which
can be used for the treatment of cancer, e.g. non-Hodgkin's
lymphoma. Examples of anti-HLA-DOB conjugates are disclosed for
example in WO 2005/081711-A2.
[2964] Examples of antibodies which bind the cancer target molecule
VTCN1, can be used for preparing a conjugate and can be used for
the treatment of cancer, e.g. ovarian carcinoma, pancreatic, lung
or breast cancer, are disclosed in WO 2006/074418-A2.
[2965] The compounds of the invention possess valuable
pharmacological properties and can be used for the prevention and
treatment of diseases in humans and animals.
[2966] The binder-drug conjugates (ADCs) of the invention, of the
formula (Ia), exhibit a high and specific cytotoxic activity with
regard to tumour cells, as may be shown on the basis of the assays
set out in the present experimental section (C-1. to C-6.). This
high and specific cytotoxic activity on the part of the binder-drug
conjugates (ADCs) of the invention, of the formula (Ia), is
achieved through the appropriate combination of the new
N,N-dialkylauristatin derivative and binder with linkers which
exhibit not only an enzymatically, hydrolytically or reductively
cleavable predetermined break point, for the release of the
toxophores, but also no such predetermined break point. More
particularly, through the use of stable linkers which have no
enzymatically, hydrolytically or reductively cleavable
predetermined break point for the release of the toxophores, and
which, following uptake of the ADCs into the tumour cell and
following complete intracellular, enzymatic breakdown of the
antibody, still remain wholly or partly intact, the activity is
confined very specifically to the tumour cell. Compatibility
between ADCs and stable linkers presupposes, among other things,
that the metabolites formed intracellularly can be formed with
sufficient efficacy, are able to reach their target and are able
there to develop their anti-proliferative activity on the target
with sufficient potency, without being carried out of the tumour
cell again beforehand by transporter proteins. The metabolites
formed intracellularly after the compounds of the formula (Ia) of
the invention have been taken up exhibit a reduced potential as a
substrate with respect to transporter proteins, thereby suppressing
their redistribution into the systemic circulation and hence the
triggering of potential side effects by the toxophore itself.
[2967] The compatibility of the ADCs with a stabile linker
chemistry and with the target in question, in conjunction with
metabolites which represent a substrate for transporter proteins to
a relatively low degree, offers an enlarged therapeutic window.
[2968] More particularly, the binder-drug conjugates of the
invention, of the formula (Ia), exhibit a high and specific
cytotoxic activity with respect to tumour cells which express
C4.4a. The activity with respect to tumour cells which do not
express C4.4a is significantly weaker at the same time.
[2969] On the basis of this profile of properties, the compounds of
the invention are therefore suitable to a particular degree for the
treatment of hyperproliferative diseases in humans and in mammals
generally. The compounds are able on the one hand to inhibit,
block, reduce or lower cell proliferation and cell division, and on
the other hand to increase apoptosis.
[2970] The hyperproliferative diseases for the treatment of which
the compounds of the invention can be employed include in
particular the group of cancer and tumour diseases. In the context
of the present invention, these are understood as meaning, in
particular, the following diseases, but without being limited to
them: mammary carcinomas and mammary tumours (ductal and lobular
forms, also in situ), tumours of the respiratory tract
(parvicellular and non-parvicellular carcinoma, bronchial
carcinoma), cerebral tumours (e.g. of the brain stem and of the
hypothalamus, astrocytoma, medulloblastoma, ependymoma and
neuro-ectodermal and pineal tumours), tumours of the digestive
organs (oesophagus, stomach, gall bladder, small intestine, large
intestine, rectum), liver tumours (including hepatocellular
carcinoma, cholangiocellular carcinoma and mixed hepatocellular and
cholangiocellular carcinoma), tumours of the head and neck region
(larynx, hypopharynx, nasopharynx, oropharynx, lips and oral
cavity), skin tumours (squamous epithelial carcinoma, Kaposi
sarcoma, malignant melanoma, Merkel cell skin cancer and
non-melanomatous skin cancer), tumours of soft tissue (including
soft tissue sarcomas, osteosarcomas, malignant fibrous
histiocytomas, lymphosarcomas and rhabdomyosarcomas), tumours of
the eyes (including intraocular melanoma and retinoblastoma),
tumours of the endocrine and exocrine glands (e.g. thyroid and
parathyroid glands, pancreas and salivary gland), tumours of the
urinary tract (tumours of the bladder, penis, kidney, renal pelvis
and ureter) and tumours of the reproductive organs (carcinomas of
the endometrium, cervix, ovary, vagina, vulva and uterus in women
and carcinomas of the prostate and testicles in men). These also
include proliferative blood diseases in solid form and as
circulating blood cells, such as lymphomas, leukaemias and
myeloproliferative diseases, e.g. acute myeloid, acute
lymphoblastic, chronic lymphocytic, chronic myelogenic and hair
cell leukaemia, and also AIDS-correlated lymphomas, Hodgkin's
lymphomas, non-Hodgkin's lymphomas, cutaneous T-cell lymphomas,
Burkitt's lymphomas and lymphomas in the central nervous
system.
Preferred Hyperproliferative Diseases for Anti-CA9 Binder-Drug
Conjugates
[2971] Hyperproliferative diseases for the treatment of which the
compounds of the invention can be preferably employed are
CA9-overexpressing tumours, mammary carcinomas and mammary tumours
(e.g. ductal and lobular forms, also in situ); tumours of the
respiratory tract (e.g. parvicellular and non-parvicellular
carcinoma, bronchial carcinoma), including preferably
non-parvicellular carcinoma of the lung; cerebral tumours (e.g. of
the brain stem and of the hypothalamus, astrocytoma,
medulloblastoma, ependymoma and/or neuro-ectodermal and pineal
tumours); tumours of the digestive organs (oesophagus, stomach,
gall bladder, small intestine, large intestine, rectum), including
more preferably stomach tumours and intestinal tumours; liver
tumours (including hepatocellular carcinoma, cholangiocellular
carcinoma and mixed hepatocellular and cholangiocellular
carcinoma); tumours of the head and neck region (e.g. larynx,
hypopharynx, nasopharynx, oropharynx, lips, oral cavity, tongue and
oesophagus); tumours of the urinary tract (tumours of the bladder,
penis, kidney, renal pelvis and ureter), including more preferably
tumours of the kidneys and of the bladder; and/or tumours of the
reproductive organs (carcinomas of the endometrium, cervix, ovary,
vagina, vulva and uterus in women and/or carcinomas of the prostate
and testicles in men), including more preferably carcinomas of the
cervix and uterus.
Preferred Hyperproliferative Diseases for Anti-EGFR Binder-Drug
Conjugates
[2972] Hyperproliferative diseases for the treatment of which the
compounds of the invention can be preferably employed are
EGFR-overexpressing tumours, respiratory tract tumours (e.g.
parvicellular and non-pavicellular carcinomas, bronchial
carcinoma), including preferably non-parvicellular carcinoma of the
lung; tumours of the digestive organs (e.g. oesophagus, stomach,
gall bladder, small intestine, large intestine, rectum), including
especially intestinal tumours; tumours of the endocrine and
exocrine glands (e.g. thyroid and parathyroid glands, pancreas and
salivary gland), including preferably pancreas; tumours of the head
and neck region (e.g. larynx, hypopharynx, nasopharynx, oropharynx,
lips, oral cavity, tongue and oesophagus); and/or gliomas.
Preferred Hyperproliferative Diseases for Anti-Mesothelin
Binder-Drug Conjugates
[2973] Hyperproliferative diseases for the treatment of which the
compounds of the invention can be preferably employed are
mesothelin-overexpressing tumours, tumours of the reproductive
organs (carcinomas of the endometrium, cervix, ovary, vagina, vulva
and uterus in women and/or carcinomas of the prostate and testicles
in men), including preferably ovarian carcinomas; tumours of the
endocrine and exocrine glands (e.g. thyroid and parathyroid glands,
pancreas and salivary gland), including preferably pancreas;
respiratory tract tumours (e.g. parvicellular and non-parvicellular
carcinoma, bronchial carcinoma), including preferably
non-parvicellular carcinoma of the lung; and/or mesotheliomas.
Preferred Hyperproliferative Diseases for Anti-C4.4a Binder-Drug
Conjugates
[2974] Hyperproliferative diseases for the treatment of which the
compounds of the invention can be preferably employed are
C4.4a-overexpressing tumours, squamous epithelial carcinomas (e.g.
of the cervix, vulva, vagina, of the anal duct, endometrium,
fallopian tube, penis, scrotum, of the oesophagus, breast, of the
bladder, of the bile duct, endometrium, uterus and ovary); mammary
carcinomas and mammary tumours (e.g. ductal and lobular forms, also
in situ); tumours of the respiratory tract (e.g. parvicellular and
non-parvicellular carcinoma, bronchial carcinoma), including
preferably non-parvicellular carcinoma of the lung, squamous
epithelial carcinoma and adenocarcinoma of the lung; tumours of the
head and neck region (e.g. larynx, hypopharynx, nasopharynx,
oropharynx, lips, oral cavity, tongue and oesophagus, squamous
epithelial carcinomas of the head and neck region); tumours of the
urinary tract (tumours of the bladder, penis, kidney, renal pelvis
and ureter, squamous epithelial carcinomas of the bladder),
including more preferably tumours of the kidneys and of the
bladder; skin tumours (squamous epithelial carcinoma, Kaposi
sarcoma, malignant melanoma, Merkel cell skin cancer and
non-melanomatous skin cancer), including more preferably melanomas;
tumours of the endocrine and exocrine glands (e.g. thyroid and
parathyroid glands, pancreas and salivary gland), including
preferably pancreas; tumours of the digestive organs (e.g.
oesophagus, stomach, gall bladder, small intestine, large
intestine, rectum), including especially colorectal carcinomas;
and/or tumours of the reproductive organs (carcinomas of the
endometrium, cervix, ovary, vagina, vulva and uterus in women
and/or carcinomas of the prostate and testicles in men), including
more preferably uterine carcinomas.
[2975] These well-described diseases in humans can also occur with
a comparable aetiology in other mammals and can be treated there
with the compounds of the present invention.
[2976] In the context of this invention the term "treatment" or
"treat" is used in the conventional sense and means attending to,
caring for and nursing a patient with the aim of combating,
reducing, attenuating or alleviating an illness or health
abnormality and improving the living conditions impaired by this
illness, such as, for example, with a cancer disease.
[2977] The present invention furthermore provides the use of the
compounds of the invention for the treatment and/or prevention of
diseases, in particular the abovementioned diseases.
[2978] The present invention furthermore provides the use of the
compounds of the invention for the preparation of a medicament for
the treatment and/or prevention of diseases, in particular the
abovementioned diseases.
[2979] The present invention furthermore provides the use of the
compounds of the invention in a method for the treatment and/or
prevention of diseases, in particular the abovementioned
diseases.
[2980] The present invention furthermore provides a method for the
treatment and/or prevention of diseases, in particular the
abovementioned diseases, using an effective amount of at least one
of the compounds of the invention.
[2981] The anti-C4.4a binder-drug conjugate of the invention is
used preferably for treating cancer in a patient, where the cancer
cells of the patient that are to be treated have C4.4a expression.
Treatment is administered more preferably to patients whose C4.4a
expression in cancer cells is higher than in healthy cells.
[2982] One method of identifying patients who respond
advantageously to an anti-C4.4a binder-drug conjugate for the
treatment of cancer involves determining the C4.4a expression in
cancer cells of the patient. In one embodiment the C4.4a expression
is determined by C4.4a gene expression analysis. The skilled person
knows of methods for gene expression analysis such as, for example,
RNA detection, quantitative or qualitative polymerase chain
reaction or fluorescence in situ hybridization (FISH). In another
preferred embodiment the C4.4a expression is determined by means of
immunohistochemistry with an anti-C4.4a antibody. The
immunohistochemistry is carried out preferably on
formaldehyde-fixed tissue. The antibody for use in the
immunohistochemistry is the same antibody which is also used in the
conjugate. The antibody for use in the immunohistochemistry is a
second antibody which--preferably specifically--recognizes the
C4.4a target protein.
[2983] The compounds according to the invention can be employed by
themselves or, if required, in combination with one or more other
pharmacologically active substances, as long as this combination
does not lead to undesirable and unacceptable side effects. The
present invention furthermore therefore provides medicaments
comprising at least one of the compounds of the invention and one
or more further drugs, in particular for the treatment and/or
prevention of the abovementioned diseases.
[2984] For example, the compounds of the present invention can be
combined with known antihyperproliferative, cytostatic or cytotoxic
substances for the treatment of cancer diseases. Suitable drugs in
the combination which may be mentioned by way of example are as
follows:
aldesleukin, alendronic acid, alfaferone, alitretinoin,
allopurinol, aloprim, aloxi, altretamine, aminoglutethimide,
amifostine, amrubicin, amsacrine, anastrozole, anzmet, aranesp,
arglabin, arsenic trioxide, aromasin, 5-azacytidine, azathioprine,
BCG or tice-BCG, bestatin, betamethasone acetate, betamethasone
sodium phosphate, bexarotene, bleomycin sulphate, broxuridine,
bortezomib, busulfan, calcitonin, campath, capecitabine,
carboplatin, casodex, cefesone, celmoleukin, cerubidin,
chlorambucil, cisplatin, cladribin, clodronic acid,
cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunoxome,
decadron, decadron phosphate, delestrogen, denileukin diftitox,
depomedrol, deslorelin, dexrazoxane, diethylstilbestrol, diflucan,
docetaxel, doxifluridine, doxorubicin, dronabinol, DW-166HC,
eligard, elitek, ellence, emend, epirubicin, epoetin-alfa, epogen,
eptaplatin, ergamisol, estrace, estradiol, estramustine sodium
phosphate, ethinylestradiol, 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,
goserelin, granisetron hydrochloride, histrelin, hycamtin,
hydrocortone, erythro-hydroxynonyladenine, hydroxyurea, ibritumomab
tiuxetan, idarubicin, ifosfamide, interferon-alpha,
interferon-alpha-2, interferon-alpha-2.alpha.,
interferon-alpha-2.beta., interferon-alpha-n1, interferon-alpha-n3,
interferon-beta, interferon-gamma-1.alpha., interleukin-2, intron
A, iressa, irinotecan, kytril, lentinan sulphate, letrozole,
leucovorin, leuprolide, leuprolide acetate, levamisole, levofolic
acid calcium salt, levothroid, levoxyl, lomustine, lonidamine,
marinol, mechlorethamine, mecobalamin, medroxyprogesterone acetate,
megestrol acetate, melphalan, 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, ondansetron hydrochloride, orapred, oxaliplatin,
paclitaxel, pediapred, pegaspargase, pegasys, pentostatin,
picibanil, pilocarpine hydrochloride, pirarubicin, plicamycin,
porfimer sodium, prednimustine, prednisolone, prednisone, premarin,
procarbazine, procrit, raltitrexed, rebif, rhenium-186 etidronate,
rituximab, roferon-A, romurtide, salagen, sandostatin,
sargramostim, semustine, sizofuran, sobuzoxane, solu-medrol,
streptozocin, strontium-89 chloride, synthroid, tamoxifen,
tamsulosin, tasonermin, tastolactone, taxoter, teceleukin,
temozolomide, teniposide, testosterone propionate, testred,
thioguanine, thiotepa, thyrotropin, tiludronic acid, topotecan,
toremifen, tositumomab, tastuzumab, teosulfan, tretinoin, trexall,
trimethylmelamine, trimetrexate, triptorelin acetate, triptorelin
pamoate, UFT, uridine, valrubicin, vesnarinone, vinblastine,
vincristine, vindesine, vinorelbine, virulizin, zinecard,
zinostatin-stimalamer, zofran; ABI-007, acolbifen, actimmune,
affinitak, aminopterin, arzoxifen, asoprisnil, atamestane,
atrasentan, avastin, BAY 43-9006 (sorafenib), CCI-779, CDC-501,
celebrex, cetuximab, crisnatol, cyproterone acetate, decitabine,
DN-101, doxorubicin-MTC, dSLIM, dutasteride, edotecarin,
eflornithine, exatecan, fenretinide, histamine dihydrochloride,
histrelin 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, liposomal MTP-PE, MX-6, nafarelin,
nemorubicin, neovastat, nolatrexed, oblimersen, onko-TCS, osidem,
paclitaxel polyglutamate, pamidronate disodium, PN-401, QS-21,
quazepam, R-1549, raloxifen, ranpirnas, 13-cis-retic acid,
satraplatin, seocalcitol, T-138067, tarceva, taxoprexin,
thymosin-alpha-1, tiazofurin, tipifarnib, tirapazamine, TLK-286,
toremifen, transMID-107R, valspodar, vapreotide, vatalanib,
verteporfin, vinflunin, Z-100, zoledronic acid and combinations of
these.
[2985] In a preferred embodiment, the compounds of the present
invention can be combined with antihyperproliferative agents, which
can be, by way of example--without this list being conclusive as
follows:
aminoglutethimide, L-asparaginase, azathioprine, 5-azacytidine,
bleomycin, busulfan, carboplatin, carmustine, chlorambucil,
cisplatin, colaspase, cyclophosphamide, cytarabine, dacarbazine,
dactinomycin, daunorubicin, diethylstilbestrol,
2',2'-difluorodeoxycytidine, docetaxel, doxorubicin (adriamycin),
epirubicin, epothilone and its derivatives,
erythro-hydroxynonyladenin, ethinyl-estradiol, etoposide,
fludarabin phosphate, 5-fluorodeoxyuridine, 5-fluorodeoxyuridine
mono-phosphate, 5-fluorouracil, fluoxymesterone, flutamide,
hexamethylmelamine, 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, raloxifen, semustine, streptozocin,
tamoxifen, teniposide, testosterone propionate, thioguanine,
thiotepa, topotecan, trimethylmelamine, uridine, vinblastine,
vincristine, vindesine and vinorelbine.
[2986] The compounds of the invention can also be combined in a
very promising manner with biological therapeutics such as
antibodies (e.g. avastin, rituxan, erbitux, herceptin). The
compounds of the invention can also achieve positive effects in
combination with therapies directed against angiogenesis, such as,
for example, with avastin, axitinib, recentin, regorafenib,
sorafenib or sunitinib. Combinations with inhibitors of the
proteasome and of mTOR and also with antihormones and steroidal
metabolic enzyme inhibitors are likewise particularly suitable
because of their favourable profile of side effects.
[2987] Generally, the following aims can be pursued with the
combination of compounds of the present invention with other agents
having a cytostatic or cytotoxic action: [2988] an improved
activity in slowing down the growth of a tumour, in reducing its
size or even in its complete elimination compared with treatment
with an individual drug; [2989] the possibility of employing the
chemotherapeutics used in a lower dosage than in monotherapy;
[2990] the possibility of a more tolerable therapy with few side
effects compared with individual administration; [2991] the
possibility of treatment of a broader spectrum of tumour diseases;
[2992] the achievement of a higher rate of response to the therapy;
[2993] a longer survival time of the patient compared with
present-day standard therapy.
[2994] The compounds according to the invention can moreover also
be employed in combination with radiotherapy and/or surgical
intervention.
[2995] The present invention furthermore provides medicaments which
comprise at least one compound of the invention, conventionally
together with one or more inert, non-toxic, pharmaceutically
suitable excipients, and the use thereof for the abovementioned
purposes.
[2996] The compounds of the invention can act systemically and/or
locally. They can be administered in a suitable manner for this
purpose, such as for example orally, parenterally, pulmonally,
nasally, sublingually, lingually, buccally, rectally, dermally,
transdermally, conjunctivally, optically or as an implant or
stent.
[2997] The compounds of the invention can be administered in
suitable administration forms for these administration routes.
[2998] Administration forms which function according to the prior
art, release the compounds of the invention rapidly and/or in a
modified manner and contain the compounds of the invention in
crystalline and/or amorphized and/or dissolved form are suitable
for oral administration, such as e.g. tablets (non-coated or coated
tablets, for example with coatings which are resistant to gastric
juice or dissolve in a delayed manner or are insoluble and control
the release of the compound of the invention), films/oblates or
tablets, which disintegrate rapidly in the oral cavity,
films/lyophilizates, capsules (for example hard or soft gelatine
capsules), film-coated tablets, granules, pellets, powders,
emulsions, suspensions, aerosols or solutions.
[2999] Parenteral administration can be effected with bypassing of
an absorption step (e.g. intravenously, intraarterially,
intracardially, intraspinally or intralumbally) or with inclusion
of an absorption (e.g. intramuscularly, subcutaneously,
intracutaneously, percutaneously or intraperitoneally).
Administration forms which are suitable for parenteral
administration include injection and infusion formulations in the
form of solutions, suspensions, emulsions, lyophilizates or sterile
powders.
[3000] For the other administration routes e.g. inhalation
medicament forms (including powder inhalers, nebulizers), nasal
drops, solutions or sprays, tablets, films/oblates or capsules for
lingual, sublingual or buccal administration, suppositories, ear or
eye preparations, vaginal capsules, aqueous suspensions (lotions,
shaking mixtures), lipophilic suspensions, ointments, creams,
transdermal therapeutic systems (e.g. patches), milk, pastes,
foams, sprinkling powders, implants or stents are suitable.
[3001] Oral and parenteral administration are preferred, in
particular oral and intravenous administration.
[3002] The compounds of the invention can be converted into the
administration forms mentioned. This can be effected in a manner
known per se by mixing with inert, non-toxic, pharmaceutically
suitable excipients. These excipients include inter alia carrier
substances (for example microcrystalline cellulose, lactose,
mannitol), solvents (e.g. liquid polyethylene glycols), emulsifiers
and dispersing or wetting agents (for example sodium dodecyl
sulphate, polyoxysorbitan oleate), binders (for example
polyvinylpyrrolidone), synthetic and natural polymers (for example
albumin), stabilizers (e.g. antioxidants, such as, for example,
ascorbic acid), colorants (e.g. inorganic pigments, such as, for
example, iron oxides) and taste and/or odour correctants.
[3003] In general, it has proved advantageous in the case of
parenteral administration to administer amounts of from about 0.001
to 1 mg/kg, preferably about 0.01 to 0.5 mg/kg of body weight to
achieve effective results. In the case of oral administration the
dosage is about 0.01 to 100 mg/kg, preferably about 0.01 to 20
mg/kg and very particularly preferably 0.1 to 10 mg/kg of body
weight.
[3004] Nevertheless it may be necessary to deviate from the amounts
mentioned, and in particular depending on the body weight,
administration route, individual behaviour towards the active
compound, nature of the formulation and point of time or interval
at which administration takes place. Thus in some cases it may be
sufficient to manage with less than the above-mentioned minimum
amount, while in other cases the upper limit mentioned must be
exceeded. In the case where relatively large amounts are
administered, it may be advisable to distribute these into several
individual doses over the day.
[3005] The following working examples illustrate the invention. The
invention is not limited to the examples.
[3006] The percentage figures in the following tests and examples
are percentages by weight, unless stated otherwise; parts are parts
by weight. Solvent ratios, dilution ratios and concentration data
of liquid/liquid solutions in each case relate to the volume.
A. EXAMPLES
Abbreviations and Acronyms
[3007] A431NS human tumour cell line [3008] A549 human tumour cell
line [3009] ABCB1 ATP-binding cassette sub-family B member 1
(synonym for P-gp and MDR1) [3010] abs. absolute [3011] ADC
antibody-drug-conjugate [3012] Ac acetyl [3013] aq. aqueous,
aqueous solution [3014] ATP adenosine triphosphate [3015] BCRP
breast cancer resistance protein, an efflux transporter [3016] Boc
tert-butoxycarbonyl [3017] br. broad (in NMR) [3018] Ex. example
[3019] ca. circa, approximately [3020] CAIX carboanhydrase IX
[3021] CI chemical ionization (in MS) [3022] d doublet (in NMR)
[3023] d day(s) [3024] TLC thin-layer chromatography [3025] DCI
direct chemical ionization (in MS) [3026] dd doublet of a doublet
(in NMR) [3027] DMAP 4-N,N-dimethylaminopyridine [3028] DME
1,2-dimethoxyethane [3029] DMEM Dulbecco's modified eagle medium
(standardized nutrient medium for cell culture) [3030] DMF
N,N-dimethylformamide [3031] DMSO dimethyl sulphoxide [3032] DPBS,
D-PBS, PBS Dulbecco's phosphate-buffered saline solution [3033]
PBS=DPBS=D-PBS, pH 7.4, from Sigma, No. D8537 [3034] Composition:
[3035] 0.2 g KCl [3036] 0.2 g KH.sub.2PO.sub.4 (anhydrous) [3037]
8.0 g NaCl [3038] 1.15 g Na.sub.2HPO.sub.4 (anhydrous) [3039] make
up to 1 l with H.sub.2O [3040] dt doublet of a triplet (in NMR)
[3041] DTT DL-dithiothreitol [3042] EDC
N'-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride [3043]
EGFR epidermal growth factor receptor [3044] EI electron impact
ionization (in MS) [3045] ELISA enzyme-linked immunosorbent assay
[3046] eq. equivalent(s) [3047] ESI electrospray ionization (in MS)
[3048] ESI-MicroTofq ESI-MicroTofq (name of the mass spectrometer,
with Tof=time of flight and q=quadrupole) [3049] FCS foetal calf
serum [3050] Fmoc (9H-fluoren-9-ylmethoxy)carbonyl [3051] sat.
Saturated [3052] GTP guanosine 5'-triphosphate [3053] h hour(s)
[3054] HATU O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate [3055] HCT-116 human tumour cell line [3056]
HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulphonic acid [3057]
HOAc acetic acid [3058] HOBt 1-hydroxy-1H-benzotriazole hydrate
[3059] HOSu N-hydroxysuccinimide [3060] HPLC high-pressure,
high-performance liquid chromatography [3061] HT29 human tumour
cell line [3062] IC.sub.50 half-maximum inhibitory concentration
[3063] i.m. intramuscular, administration into the muscle [3064]
i.v. intravenous, administration into the vein [3065] conc.
Concentrated [3066] LC-MS liquid chromatography-coupled mass
spectrometry [3067] LLC-PK1 cells Lewis lung carcinoma pork kidney
cell line [3068] L-MDR human MDR1 transfected LLC-PK1 cells [3069]
m multiplet (in NMR) [3070] MDR1 multidrug resistance protein 1
[3071] min minute(s) [3072] MS mass spectrometry [3073] MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide
[3074] NCI-H292 human tumour cell line [3075] NCI-H520 human tumour
cell line [3076] NMM N-methylmorpholine [3077] NMP
N-methyl-2-pyrrolidinone [3078] NMR nuclear magnetic resonance
spectrometry [3079] NMRI mouse strain, originating from Naval
Medical Research Institute (NMRI) [3080] Nude mice experimental
animals [3081] NSCLC non-small cell lung cancer (non-parvicellular
bronchial carcinoma) [3082] PBS phosphate-buffered saline solution
[3083] Pd/C palladium on activated carbon [3084] P-gp
P-glycoprotein, a transporter protein [3085] PNGaseF enzyme for
sugar elimination [3086] quant. quantitative (for yield) [3087]
quart quartet (in NMR) [3088] quint quintet (in NMR) [3089] R.sub.f
retention index (for TLC) [3090] RT room temperature [3091] R.sub.t
retention time (for HPLC) [3092] singlet (in NMR) [3093] s.c.
subcutaneous, administration beneath the skin [3094] SCC-4 human
tumour cell line [3095] SCC-9 human tumour cell line [3096] SCID
mice experimental mice with a severe combined immunodeficiency
[3097] t triplet (in NMR) [3098] tert tertiary [3099] TFA
trifluoroacetic acid [3100] THF tetrahydrofuran [3101] UV
ultraviolet spectrometry [3102] v/v volume to volume ratio (of a
solution) [3103] Z benzyloxycarbonyl
HPLC and LC-MS Methods:
Method 1 (LC-MS):
[3104] 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.
Method 2 (LC-MS):
[3105] 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.
Method 3 (LC-MS):
[3106] 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.
Method 4 (LC-MS):
[3107] 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.fwdarw.2.5 min/3.0 min/4.5 min 2
ml/min; oven: 50.degree. C.; UV detection: 210 nm.
Method 5 (HPLC):
[3108] Instrument: HP 1090 Series II; column: Merck Chromolith
SpeedROD 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.
Method 6 (HPLC):
[3109] 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% HClO.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.
Method 7 (LC-MS):
[3110] 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.
Method 8 (LC-MS):
[3111] 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.fwdarw.(flow rate 2.5
ml/min); oven: 55.degree. C.; flow rate: 2 ml/min; UV detection:
210 nm.
Method 9 (LC-MS):
[3112] 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.
Method 10 (HPLC):
[3113] 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.
[3114] 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.
Method 11 (LC-MS):
[3115] 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.
Method 12 (HPLC):
[3116] 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% HClO.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.
Method 13 (LC-MS):
[3117] 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)
##STR00435##
[3119] 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)
##STR00436##
[3121] 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)
##STR00437##
[3123] 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.alpha.-(tert-butoxycarbonyl)-N-hydroxy-L-phenylalaninamide
##STR00438##
[3125] The title compound was prepared by the literature method (A.
Ritter et al., J. Org. Chem. 1994, 59, 4602).
[3126] Yield: 750 mg (75% of theory)
[3127] 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
##STR00439##
[3129] 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
##STR00440##
[3131] 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
##STR00441##
[3133] 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.
[3134] Yield: 149 mg (89% of theory)
Starting Compound 7
tert-butyl (1S,2R)-1-(hydroxycarbamoyl)-2-phenylcyclopropyl
carbamate
##STR00442##
[3136] 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).
[3137] Yield: 339 mg (59% of theory)
[3138] 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-methylheptanoate
##STR00443##
[3140] 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.
[3141] HPLC (Method 5): R.sub.t=2.3 min;
[3142] 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
##STR00444##
[3144] 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.
[3145] HPLC (Method 5): R.sub.t=1.59 min;
[3146] 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
##STR00445##
[3148] 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.
[3149] Yield: 9.1 g (quant., 60% purity)
[3150] HPLC (Method 5): R.sub.t=2.7 min;
[3151] 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
##STR00446##
[3153] 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
under reduced pressure 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.
[3154] HPLC (Method 5): R.sub.t=2.2 min;
[3155] 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
##STR00447##
[3157] 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.
[3158] HPLC (Method 5): R.sub.t=1.8 min;
[3159] 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
##STR00448##
[3161] 620 mg (1.85 mmol) of tert-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
under reduced pressure 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.
[3162] HPLC (Method 5): R.sub.t=0.45 min;
[3163] 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
##STR00449##
[3165] 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 reduced
pressure. 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.
[3166] HPLC (Method 12): R.sub.t=2.0 min;
[3167] LC-MS (Method 1): R.sub.t=1.12 min; MS (ESIpos): m/z=504
(M+H).sup.+.
[3168] 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 under reduced pressure 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.
[3169] HPLC (Method 12): R.sub.t=1.53 min;
[3170] 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
##STR00450##
[3172] 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 under reduced pressure 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.
[3173] HPLC (Method 6): R.sub.t=2.2 min;
[3174] 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
##STR00451##
[3176] 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.
[3177] HPLC (Method 5): R.sub.t=2.12 min;
[3178] 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
##STR00452##
[3180] 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.
[3181] HPLC (Method 5): R.sub.t=2.40 min;
[3182] 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
##STR00453##
[3184] 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.
[3185] HPLC (Method 5): R.sub.t=2.17 min.
[3186] 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.
[3187] HPLC (Method 5): R.sub.t=1.50 min;
[3188] 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
##STR00454##
[3190] 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.
[3191] Yield: 650 mg (94% over 2 stages)
[3192] HPLC (Method 6): R.sub.t=1.76 min;
[3193] 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
##STR00455##
[3195] 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
detachment 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-m-
ethoxy-2-methylpropanoyl}-.beta.-methyl-L-phenylalaninate.
[3196] HPLC (Method 6): R.sub.t=2.3 min;
[3197] LC-MS (Method 1): R.sub.t=1.36 min; MS (ESIpos): m/z=539
(M+H).sup.+.
[3198] 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 under reduced pressure. The
remaining residue was the reaction mixture dried further under
reduced pressure and then lyophilized from acetonitrile/water. In
this way, 230 mg (quant.) of the title compound were obtained.
[3199] 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
##STR00456##
[3201] 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.
[3202] HPLC (Method 5): R.sub.t=2.73 min;
[3203] LC-MS (Method 4): R.sub.t=3.19 min; MS (ESIpos): m/z=1023
(M+H).sup.+.
[3204] 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 under reduced pressure 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.
[3205] HPLC (Method 5): R.sub.t=1.68 min;
[3206] LC-MS (Method 2): R.sub.t=1.03 min; MS (ESIpos): m/z=801
(M+H).sup.+
[3207] .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
##STR00457##
[3209] 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
[3210] HPLC (Method 5): R.sub.t=2.77 min;
[3211] LC-MS (Method 1): R.sub.t=1.5 min; MS (ESIpos): m/z=1037
(M+H).sup.+.
[3212] 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.
[3213] HPLC (Method 5): R.sub.t=1.71 min;
[3214] LC-MS (Method 1): R.sub.t=0.9 min; MS (ESIpos): m/z=815
(M+H).sup.+
[3215] .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
##STR00458##
[3217] 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
under reduced pressure 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.
[3218] 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.
[3219] HPLC (Method 5): R.sub.t=2.58 min;
[3220] LC-MS (Method 1): R.sub.t=3.10 min; MS (ESIpos): m/z=1035
(M+H).sup.+.
[3221] 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.
[3222] HPLC (Method 5): R.sub.t=1.64 min;
[3223] LC-MS (Method 1): R.sub.t=0.86 min; MS (ESIpos): m/z=813
(M+H).sup.+
[3224] .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
##STR00459##
[3226] 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
under reduced pressure. 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-
.
[3227] HPLC (Method 6): R.sub.t=2.1 min;
[3228] LC-MS (Method 1): R.sub.t=1.14 min; MS (ESIpos): m/z=369
(M+H).sup.+.
[3229] 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 under reduced pressure, 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.
[3230] HPLC (Method 6): R.sub.t=1.6 min;
[3231] 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
##STR00460##
[3233] 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.
[3234] HPLC (Method 6): R.sub.t=2.2 min;
[3235] LC-MS (Method 2): R.sub.t=1.22 min; MS (ESIpos): m/z=538
(M+H).sup.+.
[3236] 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 under reduced pressure. 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.
[3237] HPLC (Method 6): R.sub.t=1.7 min;
[3238] 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
##STR00461##
[3240] 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.
[3241] HPLC (Method 5): R.sub.t=0.6 min;
[3242] 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
##STR00462##
[3244] 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.
[3245] HPLC (Method 6): R.sub.t=2.0 min;
[3246] 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
##STR00463##
[3248] 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-phenylcyclopropanecarboxylate 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 detachment with
trifluoroacetic acid.
[3249] HPLC (Method 5): R.sub.t=1.5 min;
[3250] 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
##STR00464##
[3252] 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 under reduced pressure. 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.
[3253] 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 under
reduced pressure. The residue was lyophilized from
acetonitrile/water, which left 125 mg (83% of theory) of the title
compound as a colourless foam.
[3254] 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
##STR00465##
[3256] 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
hexafluorophosphate 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 under reduced pressure 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.
[3257] HPLC (Method 12): R.sub.t=1.6 min;
[3258] LC-MS (Method 1): R.sub.t=0.71 min; MS (ESIpos): m/z=340
(M+H).sup.+.
[3259] 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.
[3260] 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
##STR00466##
[3262] First, 35 mg (164 .mu.mol) of tert-butyl
(2-aminoethyl)methyl 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
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 under reduced pressure 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.
[3263] HPLC (Method 12): R.sub.t=1.6 min;
[3264] LC-MS (Method 1): R.sub.t=0.77 min; MS (ESIpos): m/z=340
(M+H).sup.+.
[3265] 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.
[3266] 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
##STR00467##
[3268] First,
(2R,3R)-3-[(2S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl]-3-methoxy-2-methy-
lpropanoic acid was released from 1.82 g (388 mmol) of its
dicyclohexylamine salt by taking it up in 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 and
concentrated under reduced pressure 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. The mixture was
treated in an ultrasound bath for 15 min and then concentrated
under reduced pressure. The residue was partitioned between ethyl
acetate and water, and 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.
[3269] 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 under reduced pressure. 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.
[3270] HPLC (Method 6): R.sub.t=1.5 min;
[3271] 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
##STR00468##
[3273] 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 under reduced pressure. 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. 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.
[3274] LC-MS (Method 1): R.sub.t=1.56 min; MS (ESIpos): m/z=775
(M+H).sup.+.
[3275] 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 under reduced
pressure. This gave 803 mg (91% of theory) of the title compound as
a white solid.
[3276] HPLC (Method 6): R.sub.t=2.1 min;
[3277] 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
##STR00469##
[3279] 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 detachment with
trifluoroacetic acid (yield: 85% of theory over both stages).
[3280] HPLC (Method 6): R.sub.t=1.2 min;
[3281] 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
##STR00470##
[3283] The title compound was prepared by standard methods, by
esterifying commercially available
(1S,2R)-1-[(tert-butoxycarbonyl)amino]-2-phenylcyclopropanecarboxylic
acid with ethanol and subsequent Boc detachment with
trifluoroacetic acid.
[3284] 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
##STR00471##
[3286] 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 a further 1 h. While cooling
with ice, the reaction mixture was then adjusted to pH 3 by adding
sulphuric acid, and 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.
[3287] 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
##STR00472##
[3289] 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.
[3290] 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
##STR00473##
[3292] 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.
[3293] 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
##STR00474##
[3295] 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.
[3296] LC-MS (Method 2): R.sub.t=1.84 min; m/z=414 (M+H).sup.+.
Intermediate 33
adamantan-1-ylmethyl L-phenylalaninate hydrochloride
##STR00475##
[3298] 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 under reduced
pressure. 619 mg (95% of theory) of the title compound were
obtained.
[3299] 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
##STR00476##
[3301] 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.
[3302] 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
##STR00477##
[3304] 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.
[3305] 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
##STR00478##
[3307] Under an argon atmosphere, 500 mg (1.99 mmol) of
N-Boc-L-phenylalaminol 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.
[3308] 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
##STR00479##
[3310] 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 under reduced pressure. 138 mg (77% of theory) of
the title compound were obtained.
[3311] 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
##STR00480##
[3313] 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.
[3314] LC-MS (Method 1): R.sub.t=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
##STR00481##
[3316] 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.
[3317] 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
##STR00482##
[3319] 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 detached by known conditions with trifluoroacetic acid in
dichloromethane. Concentration was followed by the purification of
the title compound by preparative HPLC, and 26 mg (52%) of the
title compound were obtained.
[3320] HPLC (Method 12): R.sub.t=1.65 min;
[3321] LC-MS (Method 1): R.sub.t=0.77 min; MS (ESIpos): m/z=718
(M+H).sup.+.
Intermediate 41
3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propanoic acid
trifluoroacetate
##STR00483##
[3323] 150 mg (541 .mu.mol) of tert-butyl
3-{2-[2-(2-aminoethoxy)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.
[3324] 181 mg (100% of theory) of the title compound were
obtained.
[3325] 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
##STR00484##
[3327] 186 mg (555 .mu.mol) of
3-{2-[2-(2-aminoethoxy)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.
[3328] 126 mg (75% of theory) of the title compound were
obtained.
[3329] 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
##STR00485##
[3331] 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 by
preparative HPLC.
[3332] 60 mg (33% of theory) of the title compound were
obtained.
[3333] 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
##STR00486##
[3335] 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-diazap-
entadecan-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.
[3336] 62 mg (100% of theory) of the title compound were
obtained.
[3337] 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
##STR00487##
[3339] The title compound was prepared by standard methods, by
esterifying commercially available
(1S,2R)-1-[(tert-butoxycarbonyl)amino]-2-phenylcyclopropanecarboxylic
acid with benzyl alcohol and subsequent Boc detachment with
trifluoroacetic acid.
[3340] 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
##STR00488##
[3342] 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-phenyl-
alaninate 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 under reduced pressure. 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.
[3343] LC-MS (Method 1): R.sub.t=1.49 min; MS (ESIpos): m/z=922
(M+H).sup.+.
[3344] 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
under reduced pressure. After lyophilization from dioxane, 85 mg
(94% of theory) of the title compound were obtained as a solid.
[3345] HPLC (Method 12): R.sub.t=2.4 min;
[3346] 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
##STR00489##
[3348] 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 under reduced pressure 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 by preparative HPLC.
After drying under high vacuum, 117 mg (57% of theory over both
stages) of the title compound were obtained.
[3349] HPLC (Method 12): R.sub.t=1.6 min;
[3350] 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
##STR00490##
[3352] 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, filtered off with suction and lyophilized from dioxane. In
this way, 32 mg (62% of theory over both stages) of the title
compound were obtained.
[3353] HPLC (Method 6): R.sub.t=0.38 min;
[3354] 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
##STR00491##
[3356] The title compound was prepared in analogy to the synthesis
of Intermediate 13 from Starting Compound 1 and L-tryptophanamide
hydrochloride.
[3357] HPLC (Method 5): R.sub.t=1.4 min;
[3358] 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
##STR00492##
[3360] 813 mg (3.1 mmol) of triphenylphosphine were dissolved in 25
ml of THF and cooled to -70.degree. C. under argon. After 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 a further 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 a further 10 min and then the mixture was warmed
to RT. After stirring at RT for a further 16 h, the solvent was
removed under reduced pressure and the residue was purified by
means of preparative HPLC. This gave 463 mg (62%) of the protected
intermediate.
[3361] After removing the Boc protecting group under standard
conditions, 652 mg of 1-(2-aminoethyl)-1H-pyrrole-2,5-dione were
obtained as the trifluoroacetate.
[3362] 112.9 mg (543 .mu.mol) of nitrophenyl chloroformate were
dissolved in 30 ml of THF and, after 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 filtration with suction and drying, 60 mg (95%
of theory) of the title compound were obtained.
[3363] HPLC (Method 5): R.sub.t=0.65 min;
[3364] LC-MS (Method 1): R.sub.t=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
##STR00493##
[3366] 200 mg (0.75 mmol) of
N-(tert-butoxycarbonyl)-L-phenylalanine were initially charged at
0.degree. C. in 5.5 ml of dichloromethane, and 128 mg (0.79 mmol)
of 1,1'-carbonyldiimidazole were added. 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.
[3367] LC-MS (Method 12): R.sub.t=1.15 min; MS (ESIpos): m/z=366
(M+H).sup.+
[3368] 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 under reduced pressure. The
remaining residue was the reaction mixture dried further under
reduced pressure and then lyophilized from dioxane. In this way,
214 mg (90% of theory) of the title compound were obtained.
[3369] 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
##STR00494##
[3371] 200 mg (0.75 mmol) of
N-(tert-butoxycarbonyl)-D-phenylalanine were initially charged at
0.degree. C. in 5.5 ml of dichloromethane, and 128.3 mg (0.79 mmol)
of 1,1'-carbonyldiimidazole were added. 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.
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.
[3372] LC-MS (Method 2): R.sub.t=1.36 min; MS (ESIpos): m/z=366
(M+H).sup.+
[3373] 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 under reduced pressure. The
remaining residue was the reaction mixture dried further under
reduced pressure and then lyophilized from dioxane. In this way,
196 mg (86% of theory) of the title compound were obtained as a
solid.
[3374] HPLC (Method 10): R.sub.t=2.41 min
Intermediate 53
(2S)-1-(benzylsulphonyl)-3-phenylpropan-2-amine
##STR00495##
[3376] 200 mg (1.13 mmol) of (4S)-4-benzyl-1,3-oxazolidin-2-one
were initially charged in 3 ml of tert-butanol, and 280 mg (2.26
mmol) of benzyl mercaptan were added. The mixture was subsequently
heated under reflux for 2 days. Thereafter, the reaction mixture
was concentrated on a rotary evaporator and the resulting
(2S)-1-(benzylsulphanyl)-3-phenylpropan-2-amine intermediate was
converted further directly, without workup.
[3377] HPLC (Method 10): R.sub.t=2.63 min
[3378] LC-MS (Method 1): R.sub.t=0.67 min; MS (ESIpos): m/z=258
(M+H).sup.+
[3379] 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 under reduced
pressure. The crude product obtained was purified by means of
preparative HPLC. 343 mg (61% of theory) of the title compound were
thus obtained.
[3380] HPLC (Method 10): R.sub.t=2.40 min;
[3381] 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
##STR00496##
[3383] 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. The filtrate
was concentrated and the resulting residue was dried under reduced
pressure. 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.
[3384] HPLC (Method 10): R.sub.t=2.46 min;
[3385] LC-MS (Method 12): R.sub.t=0.75 min; MS (ESIpos): m/z=224
(M+H).sup.+
[3386] 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
under reduced pressure. 45 mg of the title compound were
obtained.
[3387] .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
##STR00497##
[3389] 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 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.
[3390] LC-MS (Method 12): R.sub.t=1.45 min; MS (ESIpos): m/z=933
(M+H).sup.+
[3391] By subsequently detaching the BOC protecting group with
trifluoroacetic acid, 22.4 mg (98% of theory) of the title compound
were then obtained.
[3392] 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
##STR00498##
[3394]
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 reaction 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 with 12.2 mg (32 .mu.mol) of
(1R)-2-phenyl-1-(5-phenyl-1,3,4-oxadiazol-2-yl)ethanamine
trifluoroacetate.
[3395] Yield: 17 mg (64% of theory)
[3396] HPLC (Method 10): R.sub.t=3.74 min;
[3397] LC-MS (Method 1): R.sub.t=1.45 min; MS (ESIpos): m/z=933
(M+H).sup.+
[3398] By subsequently detaching the BOC protecting group with
trifluoroacetic acid, 17.1 mg (99% of theory) of the title compound
were then obtained.
[3399] HPLC (Method 10): R.sub.t=2.55 min;
[3400] 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
##STR00499##
[3402]
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 reaction 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 with 9.3 mg (20 .mu.mol) of
(2S)-1-(benzylsulphonyl)-3-phenylpropan-2-amine
[3403] Yield: 19.2 mg (68% of theory)
[3404] HPLC (Method 10): R.sub.t=3.5 min;
[3405] LC-MS (Method 12): R.sub.t=1.41 min; MS (ESIpos): m/z=957
(M+H).sup.+
[3406] By subsequently detaching the BOC protecting group with
trifluoroacetic acid, 19.3 mg (99% of theory) of the title compound
were then obtained.
[3407] HPLC (Method 10): R.sub.t=2.52 min;
[3408] 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
##STR00500##
[3410]
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]-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L--
valinamide was prepared in analogy to the synthesis of Intermediate
55, by reaction of 20 mg (29 .mu.mol)
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 7.1 mg (32 .mu.mol) of
(2S,3E)-1,4-diphenylbut-3-en-2-amine.
[3411] Yield: 15.1 mg (58% of theory)
[3412] HPLC (Method 10): R.sub.t=4.2 min;
[3413] LC-MS (Method 12): R.sub.t=1.51 min; MS (ESIpos): m/z=891
(M+H).sup.+
[3414] By subsequently detaching the BOC protecting group with
trifluoroacetic acid, 15.7 mg (99% of theory) of the title compound
were then obtained.
[3415] HPLC (Method 10): R.sub.t=2.62 min;
[3416] 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
##STR00501##
[3418] 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 a further 2 h. After cooling, another 70 ml
(0.108 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 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 adjust the pH back to 3. The reaction mixture
was then stirred at 100.degree. C. for another 2 h. In the event of
conversion still being 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.
[3419] HPLC (Method 5): R.sub.t=1.64 min;
[3420] LC-MS (Method 9): R.sub.t=4.76 min; MS (ESIpos): m/z=899
(M+H).sup.+
[3421] .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
##STR00502##
[3423] The title compound was prepared in analogy to the synthesis
of Intermediate 61, by reaction of 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.
[3424] Yield: 34 mg (70% of theory)
[3425] HPLC (Method 5): R.sub.t=1.64 min;
[3426] 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
##STR00503##
[3428] The title compound was prepared in analogy to the synthesis
of Intermediate 61, by reaction of 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.
[3429] Yield: 7.5 mg (48% of theory)
[3430] HPLC (Method 5): R.sub.t=1.75 min;
[3431] 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
##STR00504##
[3433] 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. After cooling to RT, 0.75 mg (0.012 mmol) of sodium
cyanoborohydride was 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 a further 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 was added
and 0.1 N hydrochloric acid was subsequently used to adjust the pH
back 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.
[3434] HPLC (Method 5): R.sub.t=1.68 min;
[3435] 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
##STR00505##
[3437] The title compound was prepared by reaction of 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 detachment of the Boc protecting group
with trifluoroacetic acid.
[3438] Yield: 49 mg (62% of theory over two stages)
[3439] HPLC (Method 5): R.sub.t=1.58 min;
[3440] LC-MS (Method 2): R.sub.t=1.05 min; MS (ESIpos): m/z=844
(M+H).sup.+
[3441] .sup.1H NMR (600 MHz, DMSO-d.sub.6): .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
##STR00506##
[3443] The title compound was prepared in analogy to the synthesis
of Intermediate 65, by reaction of 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 detachment of the Z
protecting group (with 10% palladium on charcoal as a catalyst, in
ethanol as a solvent).
[3444] Yield: 11 mg (41% of theory over two stages)
[3445] HPLC (Method 5): R.sub.t=1.53 min;
[3446] 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
##STR00507##
[3448] 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 a further 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 separated
directly into its components by means of preparative HPLC. 18.5 mg
(73% of theory) of the title compound were thus obtained.
[3449] LC-MS (Method 1): R.sub.t=1.17 min; m/z=967 (M+H).sup.+.
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
##STR00508##
[3451] 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 a further 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 separated
directly into its components by means of preparative HPLC. 15.2 mg
(65% of theory) of the title compound were thus obtained.
[3452] 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
##STR00509##
[3454] 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
[3455] LC-MS (Method 1): R.sub.t=1.55 min; m/z=1044
(M+H).sup.+.
[3456] 57 mg (0.055 mmol) of this intermediate were treated with
1.2 ml of piperidine in 5 ml of DMF to detach 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.
[3457] HPLC (Method 5): R.sub.t=1.9 min;
[3458] LC-MS (Method 1): R.sub.t=1.01 min; m/z=822 (M+H).sup.+.
[3459] 37 mg (0.045 mmol) of this intermediate were dissolved in 5
ml of dioxane/water and, analogously 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.
[3460] HPLC (Method 6): R.sub.t=2.1 min;
[3461] 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
##STR00510##
[3463] 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.
[3464] 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 a further 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
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.
[3465] HPLC (Method 5): R.sub.t=1.9 min;
[3466] 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-[(1R-
,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methy-
l-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valin-
amide
##STR00511##
[3468] 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.
[3469] HPLC (Method 6): R.sub.t=1.9 min;
[3470] 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
##STR00512##
[3472] 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 under reduced pressure, 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.
[3473] LC-MS (Method 1): R.sub.t=1.45 min; m/z=961
(M.alpha.H).sup.+.
[3474] 50 mg (52 .mu.mol) of this intermediate were treated with 1
ml of trifluoroacetic acid in 9 ml of dichloromethane to detach 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 the trifluoroacetate.
[3475] LC-MS (Method 1): R.sub.t=0.99 min; m/z=861 (M+H).sup.+.
[3476] 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.
[3477] HPLC (Method 5): R.sub.t=2.2 min;
[3478] 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
##STR00513##
[3480] 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.
[3481] HPLC (Method 12): R.sub.t=2.2 min;
[3482] 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
##STR00514##
[3484] 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 under reduced pressure, 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.
[3485] LC-MS (Method 1): R.sub.t=1.52 min; m/z=934 (M+H).sup.+.
[3486] 35 mg of this intermediate were treated with 1 ml of
trifluoroacetic acid in 5 ml of dichloromethane to detach 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.
[3487] LC-MS (Method 1): R.sub.t=0.91 min; m/z=834 (M+H).sup.+.
[3488] 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.
[3489] HPLC (Method 12): R.sub.t=2.2 min;
[3490] 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
##STR00515##
[3492] 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 detachment 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 the 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.
[3493] HPLC (Method 6): R.sub.t=1.9 min;
[3494] 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
##STR00516##
[3496] 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 detachment, 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.
[3497] HPLC (Method 6): R.sub.t=1.9 min;
[3498] 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
##STR00517##
[3500] 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 detachment 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.
[3501] HPLC (Method 6): R.sub.t=1.7 min;
[3502] LC-MS (Method 9): R.sub.t=4.44 min; MS (ESIpos): m/z=817
(M+H).sup.+
[3503] .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
##STR00518##
[3505] 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.
[3506] Yield: 7.6 mg (55% of theory over 2 stages)
[3507] HPLC (Method 6): R.sub.t=1.8 min;
[3508] 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
##STR00519##
[3510] 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.
[3511] LC-MS (Method 1): R.sub.t=1.43 min; m/z=921 (M+H).sup.+.
[3512] 29 mg of this intermediate were treated with 1 ml of
trifluoroacetic acid in 6 ml of dichloromethane to detach 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.
[3513] LC-MS (Method 1): R.sub.t=0.95 min; m/z=821 (M+H).sup.+.
[3514] 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.
[3515] HPLC (Method 5): R.sub.t=1.7 min;
[3516] 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
##STR00520##
[3518] 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 detachment 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.
[3519] HPLC (Method 5): R.sub.t=1.7 min;
[3520] 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
##STR00521##
[3522] 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 detachment 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.
[3523] HPLC (Method 5): R.sub.t=1.6 min;
[3524] 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
##STR00522##
[3526] 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 detachment 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.
[3527] HPLC (Method 5): R.sub.t=1.8 min;
[3528] 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
##STR00523##
[3530] 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 prepared.
[3531] Yield: 5 mg (25% of theory)
[3532] HPLC (Method 5): R.sub.t=1.6 min;
[3533] 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
##STR00524##
[3535] 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 detachment 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.
[3536] HPLC (Method 5): R.sub.t=1.6 min;
[3537] 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
##STR00525##
[3539] 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 detachment 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.
[3540] HPLC (Method 5): R.sub.t=1.54 min;
[3541] 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
##STR00526##
[3543] 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 detachment 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.
[3544] HPLC (Method 5): R.sub.t=2.0 min;
[3545] 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
##STR00527##
[3547] 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.
[3548] Yield: 95 mg (31% of theory over 3 stages)
[3549] HPLC (Method 5): R.sub.t=2.0 min;
[3550] 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
##STR00528##
[3552] 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 detachment 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 afforded the title compound.
[3553] Yield: 14 mg (95% of theory)
[3554] HPLC (Method 5): R.sub.t=1.5 min;
[3555] 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
##STR00529##
[3557] 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 detachment 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 afforded the title compound.
[3558] Yield: 30 mg (98% of theory)
[3559] HPLC (Method 5): R.sub.t=1.9 min;
[3560] LC-MS (Method 1): R.sub.t=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
##STR00530##
[3562] 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 detachment 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.
[3563] HPLC (Method 6): R.sub.t=1.8 min;
[3564] 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
##STR00531##
[3566] 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 detachment 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).
[3567] HPLC (Method 5): R.sub.t=1.7 min;
[3568] 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
##STR00532##
[3570] 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. The title compound was purified by
preparative HPLC.
[3571] HPLC (Method 5): R.sub.t=1.5 min;
[3572] 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
##STR00533##
[3574] 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.
[3575] HPLC (Method 10): R.sub.t=2.54 min
[3576] 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
##STR00534##
[3578] 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.
[3579] HPLC (Method 10): R.sub.t=2.54 min;
[3580] 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
##STR00535##
[3582] 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.
[3583] 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}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00536##
[3585] 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-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 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.
[3586] HPLC (Method 10): R.sub.t=2.59 min;
[3587] 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
##STR00537##
[3589] 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 detachment of the Z
protecting group (with 5% palladium on charcoal as a catalyst, in
methanol as a solvent).
[3590] Yield: 69 mg (65% of theory over two stages)
[3591] HPLC (Method 5): R.sub.t=1.7 min;
[3592] 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
##STR00538##
[3594] This compound was prepared in analogy to the synthesis
described in Intermediate 80. The purification was effected by
preparative HPLC.
[3595] Yield: 40 mg (29% of theory over 3 stages)
[3596] HPLC (Method 5): R.sub.t=1.9 min;
[3597] 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
##STR00539##
[3599] 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 reduced pressure. 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.
[3600] HPLC (Method 12): R.sub.t=1.9 min;
[3601] LC-MS (Method 1): R.sub.t=1.03 min; MS (ESIpos): m/z=374
(M+H).sup.+.
[3602] 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 detached and, after
HPLC purification, 155 mg (86% of theory) of the title compound
were obtained.
[3603] HPLC (Method 12): R.sub.t=1.43 min;
[3604] 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
##STR00540##
[3606] 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 under reduced pressure, 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.
[3607] LC-MS (Method 1): R.sub.t=1.36 min; m/z=940 (M+H).sup.+.
[3608] 170 mg of this intermediate were treated with 3 ml of
trifluoroacetic acid in 30 ml of dichloromethane for 30 min to
detach the Boc protecting group. Then the reaction mixture was
concentrated under reduced pressure 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.
[3609] HPLC (Method 12): R.sub.t=1.85 min;
[3610] LC-MS (Method 1): R.sub.t=0.86 min; MS (ESIpos): m/z=840
(M+H).sup.+.
[3611] 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 detachment of the Z protecting group
(with 5% palladium on charcoal as a catalyst, in methanol as a
solvent), prepared to prepare the title compound.
[3612] Yield: 21 mg (37% of theory)
[3613] HPLC (Method 12): R.sub.t=2.1 min;
[3614] 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
##STR00541##
[3616] 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 under
reduced pressure. After the residue had been dried under high
vacuum, 22.5 mg (96% of theory) of the title compound were
obtained.
[3617] HPLC (Method 5): R.sub.t=1.7 min;
[3618] 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
##STR00542##
[3620] 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.
[3621] Yield: 8 mg (71% of theory)
[3622] HPLC (Method 12): R.sub.t=1.9 min;
[3623] 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
##STR00543##
[3625] 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.
[3626] Yield: 3 mg (22% of theory)
[3627] HPLC (Method 5): R.sub.t=1.6 min;
[3628] 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
##STR00544##
[3630] 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 detaching the Boc protecting group by
conventional peptide chemistry methods.
[3631] 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 under reduced pressure. The remaining residue was
purified by means of preparative HPLC. The corresponding fractions
were combined and the solvent was evaporated off under reduced
pressure. 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.
[3632] HPLC (Method 6): R.sub.t=2.0 min;
[3633] LC-MS (Method 1): R.sub.t=0.95 min; MS (ESIpos): m/z=1072
(M+H).sup.+.
[3634] 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.
[3635] HPLC (Method 6): R.sub.t=1.8 min;
[3636] 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
##STR00545##
[3638] 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.
[3639] HPLC (Method 5): R.sub.t=1.66 min;
[3640] 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-meth-
yl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00546##
[3642] 14 mg (16 .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 (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 under
reduced pressure. The remaining residue was purified by means of
preparative HPLC. After lyophilization, 5.5 mg (36% of theory) of
the title compound were obtained.
[3643] HPLC (Method 5): R.sub.t=1.7 min;
[3644] LC-MS (Method 1): R.sub.t=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
##STR00547##
[3646] 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.
[3647] HPLC (Method 5): R.sub.t=1.6 min;
[3648] 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
##STR00548##
[3650] The compound was prepared in analogy to Intermediate
107.
[3651] Yield: 2.5 mg (30% of theory)
[3652] HPLC (Method 12): R.sub.t=1.9 min;
[3653] 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
##STR00549##
[3655] The compound was prepared in analogy to Intermediate 107
from the compound in Intermediate 92.
[3656] Yield: 35 mg (65% of theory)
[3657] HPLC (Method 5): R.sub.t=1.9 min;
[3658] 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
##STR00550##
[3660] This compound was prepared in analogy to Intermediate 147
from the compound in Intermediate 83.
[3661] Yield: 2.4 mg (24% of theory)
[3662] HPLC (Method 6): R.sub.t=1.8 min;
[3663] 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
##STR00551##
[3665] This compound was prepared in analogy to Intermediate 140
from Intermediate 82 and Intermediate 22.
[3666] Yield: 6.5 mg (51% of theory)
[3667] HPLC (Method 6): R.sub.t=1.8 min;
[3668] 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
##STR00552##
[3670] This compound was prepared in analogy to Intermediate 157
from the compound in Intermediate 81.
[3671] Yield: 5.7 mg (57% of theory)
[3672] HPLC (Method 5): R.sub.t=1.6 min;
[3673] 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
##STR00553##
[3675] 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.
[3676] HPLC (Method 5): R.sub.t=2.0 min;
[3677] LC-MS (Method 1): R.sub.t=0.93 min; MS (ESIpos): m/z=1121
(M+H).sup.+.
[3678] 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.
[3679] Thus, 61 mg (62% of theory) of the title compound were
obtained as a colourless foam.
[3680] HPLC (Method 5): R.sub.t=1.7 min;
[3681] 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
##STR00554##
[3683] 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.
[3684] Yield: 0.58 mg (11% of theory) of a colourless foam
[3685] HPLC (Method 5): R.sub.t=1.6 min;
[3686] 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
##STR00555##
[3688] This compound was prepared in analogy to the compound in
Intermediate 147, proceeding 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 under reduced pressure, reacted immediately with the
antibody.
[3689] Yield: 3 mg (27% of theory) (hydrolysis-sensitive)
[3690] HPLC (Method 5): R.sub.t=1.7 min;
[3691] 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
##STR00556##
[3693] This compound was prepared in analogy to the compound in
Intermediate 147, proceeding 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 under reduced pressure, reacted immediately with the
antibody.
[3694] Yield: 3.2 mg (43% of theory) (hydrolysis-sensitive)
[3695] HPLC (Method 5): R.sub.t=1.7 min;
[3696] 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
##STR00557##
[3698] This compound was prepared in analogy to Intermediate 157
from the compound in Intermediate 86.
[3699] Yield: 7 mg (42% of theory)
[3700] HPLC (Method 5): R.sub.t=1.6 min;
[3701] 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
##STR00558##
[3703] The target compound was prepared analogously to Intermediate
157 from 7 mg (7.8 .mu.mol) of the compound in Intermediate 68.
Yield: 6.3 mg (53% of theory)
[3704] 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
##STR00559##
[3706] 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.
[3707] LC-MS (Method 11): R.sub.t=0.89 min; MS (ESIpos): m/z=1126
(M+H).sup.+
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
##STR00560##
[3709] 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.
[3710] LC-MS (Method 11): R.sub.t=0.90 min; MS (ESIpos): m/z=1126
(M+H).sup.+
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
##STR00561##
[3712] 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.
[3713] 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
##STR00562##
[3715] 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.
[3716] LC-MS (Method 11): R.sub.t=0.95 min; MS (ESIpos): m/z=1083
(M+H).sup.+
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
##STR00563##
[3718] 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 under
reduced pressure. The remaining residue was purified by means of
preparative HPLC. After lyophilization, 24 mg (74% of theory) of
the title compound were obtained.
[3719] HPLC (Method 5): R.sub.t=2.2 min;
[3720] 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-oxoh-
eptan-4-yl]-N-methyl-L-valinamide
##STR00564##
[3722] 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 under reduced
pressure. The remaining residue was purified by means of
preparative HPLC. After lyophilization, 11 mg (54% of theory) of
the title compound were obtained.
[3723] HPLC (Method 5): R.sub.t=1.8 min;
[3724] 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
##STR00565##
[3726] 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 was added and
the reaction mixture was stirred at RT for a further 15 min and
then concentrated under reduced pressure. 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.
[3727] HPLC (Method 5): R.sub.t=1.9 min;
[3728] 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
##STR00566##
[3730] 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 under
reduced pressure. 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 a
further 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.
[3731] HPLC (Method 5): R.sub.t=1.96 min;
[3732] LC-MS (Method 1): R.sub.t=0.97 min; MS (ESIpos): m/z=992
(M+H).sup.+.
Intermediate 127
[3733]
N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-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
##STR00567##
[3734] 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 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 under reduced pressure. The
remaining residue was purified by means of preparative HPLC. After
lyophilization, 8.2 mg (43% of theory) of the title compound were
obtained.
[3735] HPLC (Method 5): R.sub.t=2.0 min;
[3736] 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
##STR00568##
[3738] 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.
[3739] HPLC (Method 5): R.sub.t=1.7 min;
[3740] 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
##STR00569##
[3742] 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.
[3743] HPLC (Method 5): R.sub.t=1.7 min;
[3744] LC-MS (Method 9): R.sub.t=4.9 min; MS (ESIpos): m/z=1134
(M+H).sup.+.
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
##STR00570##
[3746] 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.
[3747] HPLC (Method 5): R.sub.t=2.0 min;
[3748] LC-MS (Method 1): R.sub.t=1.03 min; MS (ESIpos): m/z=1056
(M+H).sup.+.
[3749] Subsequently, the Boc protecting group was detached 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.
[3750] HPLC (Method 5): R.sub.t=1.8 min;
[3751] 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
##STR00571##
[3753] 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.
[3754] HPLC (Method 5): R.sub.t=2.1 min;
[3755] LC-MS (Method 1): R.sub.t=1.04 min; MS (ESIpos): m/z=1116
(M+H).sup.+.
[3756] Subsequently, the Z protecting group was detached 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.
[3757] HPLC (Method 5): R.sub.t=1.8 min;
[3758] 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-[(1R,2R)-1-meth-
oxy-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
##STR00572##
[3760] 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.
[3761] HPLC (Method 5): R.sub.t=1.7 min;
[3762] 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
##STR00573##
[3764] 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.
[3765] HPLC (Method 5): R.sub.t=2.0 min;
[3766] 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
##STR00574##
[3768] First, 4-nitrophenyl
2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl carbamate was
prepared under standard conditions, proceeding from commercially
available 1-(2-aminoethyl)-1H-pyrrole-2,5-dione trifluoroacetate
and 4-nitrophenyl chlorocarbonate.
[3769] 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.
[3770] HPLC (Method 5): R.sub.t=1.7 min;
[3771] 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
##STR00575##
[3773] 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.
[3774] HPLC (Method 6): R.sub.t=2.1 min;
[3775] 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
##STR00576##
[3777] First,
1-[4-oxo-4-(piperazin-1-yl)butyl]-1H-pyrrole-2,5-dione
trifluoroacetate was prepared under standard conditions, proceeding
from tert-butyl piperazine-1-carboxylate and
4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoic acid over 2
stages.
[3778] 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-pheny-
lcyclopropyl]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 2.1 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 a further 3 h. Subsequently, the solvent was removed
under reduced pressure 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 was obtained as a colourless
foam.
[3779] HPLC (Method 6): R.sub.t=1.9 min;
[3780] 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
##STR00577##
[3782] First,
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N'-methylhexanehydrazide
trifluoroacetate was prepared under standard conditions, proceeding
from commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid and
tert-butyl 1-methylhydrazinecarboxylate over 2 stages.
[3783] 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 under reduced pressure 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.
[3784] HPLC (Method 5): R.sub.t=1.8 min;
[3785] 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-[(1R,2R)-1-methoxy-2-methyl-3-{[(1S,2R)-1-(1,2-oxazinan-2-ylcarb-
onyl)-2-phenylcyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-o-
xoheptan-4-yl]-N-methyl-L-valinamide
##STR00578##
[3787] Proceeding 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 first prepared by.
[3788] 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
again, and the reaction mixture was then stirred at RT overnight.
Subsequently, the solvent was removed under reduced pressure 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.
[3789] HPLC (Method 6): R.sub.t=2.0 min;
[3790] 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
##STR00579##
[3792] 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 twice extracted
by shaking with water and the organic phase was dried over sodium
sulphate and concentrated under reduced pressure. 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.
[3793] HPLC (Method 12): R.sub.t=2.3 min;
[3794] LC-MS (Method 1): R.sub.t=1.13 min; MS (ESIpos): m/z=1178
(M+H).sup.+.
[3795] Subsequently, the Z protecting group was detached 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 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.
[3796] HPLC (Method 12): R.sub.t=2.1 min;
[3797] LC-MS (Method 1): R.sub.t=0.97 min; MS (ESIpos): m/z=1295
(M+H).sup.+.
[3798] 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.
[3799] HPLC (Method 12): R.sub.t=2.1 min;
[3800] 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
##STR00580##
[3802] 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.
[3803] HPLC (Method 5): R.sub.t=1.8 min;
[3804] 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
##STR00581##
[3806] 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.
[3807] HPLC (Method 5): R.sub.t=2.1 min;
[3808] LC-MS (Method 1): R.sub.t=1.04 min; MS (ESIpos): m/z=1116
(M+H).sup.+.
[3809] Subsequently, the Z protecting group was detached by
hydrogenolytic means in ethanol over palladium/activated
carbon.
[3810] 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.
[3811] HPLC (Method 5): R.sub.t=1.8 min;
[3812] 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
##STR00582##
[3814] 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 under reduced pressure 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.
[3815] HPLC (Method 5): R.sub.t=1.9 min;
[3816] 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
##STR00583##
[3818] 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 give 2 mg (27%
of theory) of the title compound.
[3819] HPLC (Method 12): R.sub.t=2.1 min;
[3820] 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
##STR00584##
[3822] 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 was added. After stirring overnight, the reaction
mixture was concentrated and purified by preparative HPLC. 3.5 mg
(85% purity, 48% of theory) of the title compound were
obtained.
[3823] LC-MS (Method 3): R.sub.t=1.86 min; m/z=1094
(M+H).sup.+.
Intermediate 145
[3824]
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-phenyl
cyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl-
]-N-methyl-L-valinamide
##STR00585##
[3825] 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.
[3826] HPLC (Method 5): R.sub.t=1.7 min;
[3827] 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
##STR00586##
[3829] 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 give 1.8
mg (15% of theory) of the title compound.
[3830] HPLC (Method 12): R.sub.t=2.2 min;
[3831] 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
##STR00587##
[3833] 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 under reduced pressure. The remaining
residue was purified by means of preparative HPLC. After
lyophilization, 10 mg (56% of theory) of the title compound were
obtained.
[3834] 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
##STR00588##
[3836] 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 under reduced pressure 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.
[3837] HPLC (Method 12): R.sub.t=2.1 min;
[3838] 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
##STR00589##
[3840] 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 under reduced pressure 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.
[3841] HPLC (Method 12): R.sub.t=2.1 min;
[3842] 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
##STR00590##
[3844] 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.
[3845] HPLC (Method 6): R.sub.t=1.9 min;
[3846] 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
##STR00591##
[3848] The compound was prepared analogously to Intermediate 150,
proceeding from the compound in Intermediate 81.
[3849] HPLC (Method 5): R.sub.t=1.7 min;
[3850] 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
##STR00592##
[3852] 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.
[3853] HPLC (Method 6): R.sub.t=2.0 min;
[3854] 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
##STR00593##
[3856] 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-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy)pro-
panehydrazide trifluoroacetate, 3.2 mg (16.9 .mu.mol) of EDC, 1.96
.mu.l (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, a further 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 preparative HPLC.
[3857] 3.6 mg (51% of theory) of the title compound were
obtained.
[3858] 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-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
##STR00594##
[3860] 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-yl)-2-phenylcycloprop-
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 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 under reflux overnight. Subsequently, the reaction
mixture was diluted with dichloromethane and extracted by shaking
once with water, and 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.
[3861] HPLC (Method 12): R.sub.t=2.5 min;
[3862] LC-MS (Method 1): R.sub.t=1.13 min; MS (ESIpos): m/z=1190
(M+H).sup.+.
[3863] 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 joined 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.
[3864] 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.
[3865] HPLC (Method 12): R.sub.t=2.0 min;
[3866] 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
##STR00595##
[3868] 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.
[3869] HPLC (Method 5): R.sub.t=1.6 min;
[3870] LC-MS (Method 1): R.sub.t=0.82 min; MS (ESIpos): m/z=1024
(M+H).sup.+.
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
##STR00596##
[3872] 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.
[3873] HPLC (Method 12): R.sub.t=2.0 min;
[3874] 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
##STR00597##
[3876] 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.
[3877] HPLC (Method 5): R.sub.t=1.6 min;
[3878] LC-MS (Method 1): R.sub.t=0.85 min; MS (ESIpos): m/z=1063
(M+H).sup.+.
[3879] .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
##STR00598##
[3881] 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
O-(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 under reduced pressure. 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.
[3882] HPLC (Method 5): R.sub.t=2.2 min;
[3883] LC-MS (Method 1): R.sub.t=1.21 min; MS (ESIpos): m/z=1076
(M+H).sup.+.
[3884] Trifluoroacetic acid in dichloromethane was first used to
detach the Boc protecting group from this protected intermediate,
giving 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 to the reactant.
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
##STR00599##
[3886] 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.
[3887] Yield: 6 mg (53% of theory)
[3888] HPLC (Method 5): R.sub.t=1.9 min;
[3889] 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
##STR00600##
[3891] 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.
[3892] Yield: 13 mg (52% of theory)
[3893] HPLC (Method 5): R.sub.t=1.9 min;
[3894] 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
##STR00601##
[3896] 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.
[3897] Yield: 0.8 mg (16% of theory)
[3898] HPLC (Method 5): R.sub.t=1.6 min;
[3899] 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
##STR00602##
[3901] 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 mol) of DMAP were added and the
reaction mixture was stirred at RT for a further hour. This was
followed by concentration under reduced pressure. The remaining
residue was purified by means of preparative HPLC. After
lyophilization, 8.2 mg (41% of theory) of the title compound were
obtained.
[3902] HPLC (Method 5): R.sub.t=2.0 min;
[3903] 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
##STR00603##
[3905] First, proceeding from 265 mg (0.82 mmol) of tert-butyl
(1S,2R)-1-(hydroxycarbamoyl)-2-phenylcyclopropyl carbamate
(Starting Compound 7), by reaction with
1,2-bis(bromomethyl)benzene, analogously to a literature method
(see H. King, J. Chem. Soc. 1942, 432), the Boc-protected
tert-butyl
(1S,2R)-1-(1,4-dihydro-3H-2,3-benzoxazin-3-ylcarbonyl)-2-phenylcyclopropy-
l carbamate intermediate was prepared.
[3906] Yield: 108 mg (34% of theory)
[3907] LC-MS (Method 2): R.sub.t=1.3 min; MS (ESIpos): m/z=395
(M+H).sup.+.
[3908] 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 under reduced pressure and lyophilization of the
remaining residue from dioxane. 112 mg of the title compound were
obtained in quantitative yield as a colourless foam.
[3909] 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
##STR00604##
[3911] 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 under
reduced pressure, 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
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.
[3912] HPLC (Method 5): R.sub.t=1.68 min;
[3913] LC-MS (Method 1): R.sub.t=1.51 min; MS (ESIpos): m/z=1083
(M+H).sup.+.
[3914] 19 mg (0.015 mmol) of this intermediate were dissolved in 4
ml of DMF. After 817 .mu.l of piperidine had been added, the
reaction mixture was stirred at RT for 5 min. This was followed by
concentration under reduced pressure, 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
under reduced pressure and then the residue was lyophilized from
dioxane/water. 12 mg (92% of theory) of the title compound were
obtained as a colourless foam.
[3915] HPLC (Method 6): R.sub.t=2.0 min;
[3916] 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
##STR00605##
[3918] 20 mg (0.021 mmol) of Intermediate 164 were used, in analogy
to the preparation of Intermediate 97, with benzyl 6-oxohexyl
carbamate in the presence of sodium cyanoborohydride and subsequent
hydrogenolytic detachment of the Z protecting group (with 5%
palladium on charcoal as a catalyst, in methanol as a solvent), to
prepare the title compound.
[3919] Yield: 4.5 mg (23% of theory over 2 stages)
[3920] HPLC (Method 12): R.sub.t=1.9 min;
[3921] 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
##STR00606##
[3923] 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 under reduced pressure. 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.
[3924] HPLC (Method 12): R.sub.t=2.1 min;
[3925] 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-oxoethoxy)ethoxy]ethoxy}propanoate
##STR00607##
[3927] The title compound was prepared from 6 g (21.55 mmol) of
commercially available
3-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}propanoic acid under
standard conditions, first by esterification with benzyl chloride
and caesium carbonate and subsequent oxidation with sulphur
trioxide-pyridine complex.
[3928] Yield: 611 mg (10% of theory over 2 stages)
[3929] 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-carboxyethoxy)ethoxy]ethoxy}ethyl)-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
##STR00608##
[3931] 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 detachment 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.
[3932] 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 again and the reaction mixture was stirred at RT for a
further 24 h. This was followed by concentration under reduced
pressure, 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.
[3933] HPLC (Method 12): R.sub.t=2.04 min;
[3934] LC-MS (Method 1): R.sub.t=0.97 min; MS (ESIpos): m/z=1064
(M+H).sup.+.
[3935] 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 under
reduced pressure. After lyophilization from dioxane, 19.7 mg (85%
of theory) of the title compound were obtained.
[3936] HPLC (Method 12): R.sub.t=1.8 min;
[3937] 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
##STR00609##
[3939] 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 under
reduced pressure. 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.
[3940] HPLC (Method 12): Rt=1.9 min;
[3941] 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
##STR00610##
[3943] This compound was prepared in analogy to Intermediate 101
over 2 stages, proceeding from 26 mg (0.028 mmol) of Intermediate
15.
[3944] Yield: 16.7 mg (63% of theory over 2 stages)
[3945] HPLC (Method 12): R.sub.t=1.9 min;
[3946] 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-yflbutanoyl]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
##STR00611##
[3948] 6.7 mg (7.3 .mu.mol) of the compound formed 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 under reduced pressure and
then the residue was lyophilized from dioxane. Thus, 4.5 mg (56% of
theory) of the title compound were obtained.
[3949] HPLC (Method 12): R.sub.t=2.0 min;
[3950] 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-oxoethoxy)ethoxy]ethoxy}ethyl carbamate
##STR00612##
[3952] The title compound was prepared from commercially available
2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethanol under standard
conditions, by first introducing the Z protecting group and then
oxidizing with sulphur trioxide-pyridine complex.
[3953] HPLC (Method 12): R.sub.t=1.4 min;
[3954] LC-MS (Method 11): R.sub.t=0.65 min; MS (ESIpos): m/z=326
(M+H).sup.+.
Intermediate 173
benzyl {2-[2-(2-oxoethoxy)ethoxy]ethyl carbamate
##STR00613##
[3956] The title compound was prepared analogously to Intermediate
172 from commercially available 2-[2-(2-aminoethoxy)ethoxy]ethanol
under standard conditions, by first introducing the Z protecting
group and then oxidizing with sulphur trioxide-pyridine
complex.
[3957] HPLC (Method 12): R.sub.t=1.3 min;
[3958] 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-aminoethoxy)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-oxa-
zinan-2-ylcarbonyl)-2-phenyl
cyclopropyl]amino}-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl-
]-N-methyl-L-valinamide
##STR00614##
[3960] 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-oxoethoxy)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
charcoal as a catalyst and in methanol as a solvent, and 38 mg (66%
of theory over 2 stages) of the title compound were prepared.
[3961] HPLC (Method 5): R.sub.t=1.7 min;
[3962] 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
##STR00615##
[3964] The preparation was effected in analogy zu Intermediate 166,
proceeding from 34 mg (0.03 mmol) of Intermediate 174.
[3965] Yield: 8.3 mg (23% of theory)
[3966] HPLC (Method 5): R.sub.t=1.9 min;
[3967] LC-MS (Method 1): R.sub.t=0.97 min; MS (ESIpos): m/z=1068
(M+H).sup.+.
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
##STR00616##
[3969] The preparation was effected in analogy to Intermediates 174
and 175, commencing with the reductive amination of Intermediate 16
with Intermediate 173, subsequent deprotection and formation of the
maleimide.
[3970] HPLC (Method 12): R.sub.t=1.8 min;
[3971] 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
##STR00617##
[3973] The preparation was effected in analogy to Intermediates 174
and 175, commencing with the reductive amination of Intermediate 16
with Intermediate 172, subsequent deprotection and formation of the
maleimide.
[3974] HPLC (Method 12): R.sub.t=1.9 min;
[3975] LC-MS (Method 1): R.sub.t=0.86 min; MS (ESIpos): m/z=1025
(M.alpha.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
##STR00618##
[3977] The preparation was effected in analogy to Intermediates
162, proceeding from 6 mg of Intermediate 82.
[3978] LC-MS (Method 1): R.sub.t=0.82 min; MS (ESIpos): m/z=953
(M+H).sup.+.
Intermediate 179
4-[(1E,3S)-3-amino-4-phenylbut-1-en-1-yl]benzenesulphonic acid
trifluoroacetate
##STR00619##
[3980] 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 to 160.degree. C. in a
microwave for 15 min. The crude product was subsequently purified
directly by preparative HPLC. This gave 312 mg of a mixture of 31%
of the BOC-protected compound and 69% of the free amine.
[3981] 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 under reduced pressure,
the residue was stirred with diethyl ether, and the precipitate
formed was filtered off with suction and washed with diethyl ether.
This gave 200 mg (62% of theory) of the title compound.
[3982] 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
##STR00620##
[3984] 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 charcoal (10%) and hydrogenated at hydrogen pressure 2.2 bar for
24 h. The solution was filtered and the filtrate purified by prep.
HPLC.
[3985] 29 mg (76% purity, 21% of theory) of product were
obtained.
[3986] 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
##STR00621##
[3988] 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 prep. HPLC. This gave 127 mg of a 44:56 mixture of
the title compound and of the already deprotected amine.
[3989] LC-MS (Method 1): R.sub.t=1.21 min; MS (ESIpos): m/z=971
(M+H).sup.+; 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
##STR00622##
[3991] 90 mg of Intermediate 180 were dissolved in 4.6 ml of
dichloromethane, and 0.92 ml of trifluoroacetic acid was added. The
reaction mixture was stirred at RT for 30 min and then
concentrated. The crude product obtained was purified by prep.
HPLC.
[3992] 91 mg (98% of theory) of the target compound were
obtained.
[3993] 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
##STR00623##
[3995] 16.7 .mu.l (0.03 mmol) of a 15% aqueous succinaldehyde
solution were initially charged 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
prep. HPLC.
[3996] This gave 20 mg (83% purity, 80% of theory) of the title
compound.
[3997] 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
##STR00624##
[3999] 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 prep. HPLC.
[4000] 3 mg (31% of theory) of the title compound were
obtained.
[4001] 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
##STR00625##
[4003] 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 Hunig's base were
added and the mixture was stirred for 1 h. The reaction mixture was
then purified by prep. HPLC. 2.6 mg (72% purity, 21% of theory) of
the title compound were obtained.
[4004] 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
##STR00626##
[4006] 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 Hunig'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 prep. HPLC. This gave 58 mg of a 45:55
mixture of the title compound and of the already deprotected
amine.
[4007] LC-MS (Method 1): R.sub.t=1.09 min; MS (ESIpos): m/z=973
(M+H).sup.+; 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-sulphophenyllbutan-2-yl]amino}propyl]pyr-
rolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate
##STR00627##
[4009] 58 mg of Intermediate 186 were dissolved in 4.1 ml of
dichloromethane, 0.41 ml of trifluoroacetic acid was added and the
mixture was stirred at RT for 30 min. After concentration under
reduced pressure, the crude product was purified by prep. HPLC.
[4010] 50 mg (90% purity, 85% of theory) of the title compound were
obtained.
[4011] 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-oxohentan-4-yl]-N-methyl-L-va-
linamide
##STR00628##
[4013] 171 .mu.l (0.26 mmol) of a 15% aqueous succinaldehyde
solution were initially charged 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, a further equivalent of borane-pyridine complex was
added and the mixture was stirred for a further 2 h. The reaction
mixture was then purified by prep. HPLC.
[4014] 40 mg (90% purity, 66% of theory) of the title compound were
obtained.
[4015] 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-sulphophenyllbutan-2-yl]amino}pro-
pyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00629##
[4017] 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 prep.
HPLC.
[4018] 4.2 mg (92% purity, 33% of theory) of the title compound
were obtained.
[4019] 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-sulphophenyllbutan-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1--
oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00630##
[4021] To a solution of 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 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 prep. HPLC.
[4022] 3.8 mg (72% purity, 25% of theory) of the title compound
were obtained.
[4023] 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
##STR00631##
[4025] 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).
[4026] Yield over 2 stages: 62 mg (67% of theory)
[4027] HPLC (Method 6): R.sub.t=1.65 min;
[4028] 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
##STR00632##
[4030] 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, and 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 under reduced pressure 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), without further purification, was taken up in
20 ml of acetonitrile, 2 ml of piperidine were added and the
reaction mixture was then stirred at RT for 10 min. Then the
mixture was concentrated under reduced pressure and the residue was
admixed with diethyl ether. The solvent was concentrated by
evaporation again 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
under reduced pressure and the residue was lyophilized from
acetonitrile/water. Thus, 895 mg (67% over 2 stages) of the title
compound were obtained.
[4031] HPLC (Method 12): R.sub.t=1.8 min;
[4032] LC-MS (Method 1): R.sub.t=0.84 min; MS (ESIpos): m/z=840
(M+H).sup.+.
[4033] .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
##STR00633##
[4035] 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 under reduced
pressure, 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.
[4036] HPLC (Method 5): R.sub.t=1.7 min;
[4037] 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
##STR00634##
[4039] 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.
[4040] Yield: 4.4 mg (35% of theory)
[4041] HPLC (Method 5): R.sub.t=1.8 min;
[4042] 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
##STR00635##
[4044] This compound was prepared in analogy to Intermediate 166,
proceeding from 9 mg (0.010 mmol) of Intermediate 170.
[4045] Yield: 1.1 mg (10% of theory)
[4046] HPLC (Method 12): R.sub.t=2.0 min;
[4047] 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
##STR00636##
[4049] 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 removed under reduced
pressure, 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 under reduced pressure. 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-phenylpropan-2-yl
carbamate were thus obtained as a diastereomer mixture.
[4050] LC-MS (Method 1): R.sub.t=1.1 min; MS (ESIpos): m/z=359
(M+H).sup.+.
[4051] 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 under reduced pressure and lyophilization of the
remaining residue from water/dioxane. In this way, 66 mg (quant.)
of the title compound were obtained as a foam.
[4052] HPLC (Method 6): R.sub.t=1.45 min;
[4053] 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
##STR00637##
[4055] 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 preparative HPLC. This gave 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-azabicyclo[2.2.2]oc-
t-5-en-3-yl)-1-oxo-3-phenylpropan-2-yl]amino}-3-oxopropyl]pyrrolidine-1-ca-
rboxylate.
[4056] HPLC (Method 6): R.sub.t=2.13 min;
[4057] LC-MS (Method 1): R.sub.t=1.13 min; MS (ESIpos): m/z=528
(M+H).sup.+.
[4058] 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 under reduced pressure 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.
[4059] HPLC (Method 6): R.sub.t=1.62 min;
[4060] 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
##STR00638##
[4062] 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 under reduced pressure
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 gave 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
[4063] HPLC (Method 6): R.sub.t=2.78 min;
[4064] LC-MS (Method 3): R.sub.t=2.96 min; MS (ESIpos): m/z=1047
(M+H).sup.+.
[4065] 73 mg (0.071 mmol) of this intermediate were dissolved in 5
ml of DMF. After 0.5 ml of piperidine had been added, the reaction
mixture was stirred at RT for 10 min. This was followed by
concentration under reduced pressure, and the residue was digested
repeatedly with diethyl ether. After the diethyl ether had been
decanted off, the residue was purified by preparative HPLC (eluent:
acetonitrile/0.1% aq. TFA). 16 mg (26% of theory) of the title
compound were obtained as a foam.
[4066] HPLC (Method 6): R.sub.t=1.94 min;
[4067] LC-MS (Method 3): R.sub.t=1.71 min; MS (ESIpos): m/z=825
(M+H).sup.+
[4068] .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
##STR00639##
[4070] The title compound was prepared in analogy to Intermediates
193 and 194, proceeding 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).
[4071] HPLC (Method 12): Rt=1.9 min;
[4072] LC-MS (Method 2): Rt=2.1 min; MS (ESIpos): m/z=1118
(M+H).sup.+.
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
##STR00640##
[4074] The preparation was effected in analogy to Intermediates 174
and 175, commencing with the reductive alkylation of Intermediate
192 with Intermediate 172, subsequent deprotection and formation of
the maleimide.
[4075] HPLC (Method 12): Rt=1.9 min;
[4076] LC-MS (Method 1): Rt=0.86 min; MS (ESIpos): m/z=1025
(M+H).sup.+.
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]-
amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl--
1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00641##
[4078] 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 preparative HPLC. Thus, 6.9 mg (26% of theory) of the
title compound were obtained as a foam.
[4079] HPLC (Method 5): R.sub.t=1.8 min;
[4080] 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
##STR00642##
[4082] The preparation was at first effected in analogy to
Intermediate 168, commencing with the reductive alkylation of
Intermediate 192 with Intermediate 167 and subsequent
hydrogenolytic cleavage of the benzyl ester of
N-(2-{2-[2-(2-carboxyethoxy)ethoxy]ethoxy}ethyl)-N-methyl-L-valy-
l-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-oxazi-
nan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidi-
n-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide.
[4083] 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
under reduced pressure. 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.
[4084] HPLC (Method 12): R.sub.t=2.0 min;
[4085] 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
##STR00643##
[4087] 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.
[4088] 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
##STR00644##
[4090] 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 with dichloromethane, and the solvent
was removed again under reduced pressure. Thus, 45 mg (68% of
theory over 2 stages) of the title compound were obtained.
[4091] HPLC (Method 12): R.sub.t=1.6 min;
[4092] 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
##STR00645##
[4094] This compound was prepared in analogy to Intermediate 114,
proceeding from Intermediates 50 and 204.
[4095] Yield: 4 mg (78% of theory)
[4096] HPLC (Method 12): R.sub.t=1.7 min;
[4097] 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
##STR00646##
[4099] 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
under reduced pressure and the residue was purified by preparative
HPLC. 7.2 mg (68% of theory) of the title compound were
obtained.
[4100] HPLC (Method 5): R.sub.t=1.9 min;
[4101] 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
##STR00647##
[4103] 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.
[4104] 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
##STR00648##
[4106] 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.
[4107] HPLC (Method 12): R.sub.t=2.0 min;
[4108] 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
##STR00649##
[4110] 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
(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 subsequent detachment 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 was
prepared as the trifluoroacetate.
[4111] 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 under reduced pressure, 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.
[4112] 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
resulting residue was purified by preparative HPLC. Thus, 11 mg
(55% of theory over 2 stages) of the aminocarboxylic acid
intermediate were obtained.
[4113] HPLC (Method 12): R.sub.t=1.7 min;
[4114] LC-MS (Method 11): R.sub.t=0.7 min; MS (ESIpos): m/z=843
(M+H).sup.+.
[4115] 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 a further
30 min. Then the reaction mixture was acidified to pH 2 with
trifluoroacetic acid and subsequently concentrated under reduced
pressure. 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.
[4116] HPLC (Method 12): R.sub.t=1.9 min;
[4117] 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
##STR00650##
[4119] First, 6-oxohexanoic acid was prepared by a literature
method (J. Org. Chem. 58, 1993, 2196-2200).
[4120] 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 under reduced pressure, 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.
[4121] HPLC (Method 12): R.sub.t=1.9 min;
[4122] LC-MS (Method 1): R.sub.t=0.87 min; MS (ESIpos): m/z=955
(M+H).sup.+.
[4123] .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).
[4124] 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 under reduced pressure 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.
[4125] HPLC (Method 12): R.sub.t=1.9 min;
[4126] 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
##STR00651##
[4128] 15 mg (20 .mu.mmol) 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.
[4129] Yield: 9.2 mg (61% of theory)
[4130] HPLC (Method 12): R.sub.t=1.9 min;
[4131] LC-MS (Method 1): R.sub.t=0.87 min; MS (ESIpos): m/z=929
(M+H).sup.+.
[4132] 9 mg (10 .mu.mol) of this intermediate were dissolved in 3
ml of DMF and admixed with 5.6 mg (48 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 added every hour. Subsequently,
the reaction mixture was concentrated under reduced pressure, and
the residue was taken up in acetonitrile/water and adjusted to pH 2
with trifluoroacetic acid. After concentrating again under reduced
pressure, 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.
[4133] HPLC (Method 12): R.sub.t=2.0 min;
[4134] LC-MS (Method 1): R.sub.t=0.95 min; MS (ESIpos): m/z=1027
(M+H).sup.+.
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
##STR00652##
[4136] The preparation was at first effected in analogy to
Intermediate 168, commencing with the reductive alkylation of
Intermediate 15 with Intermediate 167 and subsequent hydrogenolytic
cleavage of the benzyl ester of
N-(2-{2-[2-(2-carboxyethoxy)ethoxy]ethoxy}ethyl)-N-methyl-L-valy-
l-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]pyrrol-
idin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide.
[4137] 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
under reduced pressure. Subsequently, the reaction mixture was
concentrated under reduced pressure, and the residue was taken up
in acetonitrile/water and adjusted to pH 2 with trifluoroacetic
acid. After concentrating again under reduced pressure, 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.
[4138] HPLC (Method 12): R.sub.t=2.0 min;
[4139] 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
##STR00653##
[4141] This compound was prepared in analogy to Intermediate 104,
proceeding 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-me-
thoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-
-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.
[4142] HPLC (Method 12): R.sub.t=1.9 min;
[4143] LC-MS (Method 1): R.sub.t=0.9 min; MS (ESIpos): m/z=1177
(M+H).sup.+.
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
##STR00654##
[4145] This compound was prepared in analogy to Intermediate 210,
by conversion of Intermediate 92 to the active ester.
[4146] HPLC (Method 5): R.sub.t=1.6 min;
[4147] 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
##STR00655##
[4149] First, Intermediate 40, in analogy to Intermediate 183, was
used with borane-pyridine complex to prepare
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.
[4150] HPLC (Method 5): R.sub.t=1.6 min;
[4151] 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
##STR00656##
[4153] First, in analogy to the preparation of Intermediate 183,
Intermediate 192 was reacted with 4-formylbenzoic acid with
borane-pyridine complex to give
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.
[4154] HPLC (Method 5): R.sub.t=1.8 min;
[4155] 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
##STR00657##
[4157] 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
[4158] LC-MS (Method 1): R.sub.t=1.55 min; m/z=1044
(M+H).sup.+.
[4159] 57 mg (0.055 mmol) of this intermediate were treated with
1.2 ml of piperidine in 5 ml of DMF to detach 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.
[4160] HPLC (Method 5): R.sub.t=1.9 min;
[4161] LC-MS (Method 1): R.sub.t=1.01 min; m/z=822 (M+H).sup.+.
[4162] 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.
[4163] HPLC (Method 5): R.sub.t=1.9 min;
[4164] 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
##STR00658##
[4166] This compound was prepared by conversion of 42 mg (0.05
mmol) of Intermediate 217 to the active ester.
[4167] Yield: 26 mg (54%)
[4168] HPLC (Method 5): R.sub.t=2.1 min;
[4169] 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
##STR00659##
[4171] 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
under reduced pressure. The residue was lyophilized from 1:1
acetonitrile/water. This gave 14 mg (92% of theory) of the title
compound as a colourless foam.
[4172] HPLC (Method 5): R.sub.t=1.7 min;
[4173] 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
##STR00660##
[4175] 0.5 g (1.01 mmol) of Intermediate 1 in 10 ml of
dichloromethane were admixed with 1 ml of trifluoroacetic acid.
After treatment in an ultrasound bath for 30 min, the mixture was
concentrated and redistilled first with DCM and then with diethyl
ether, and dried under high vacuum. The oily residue was used in
the next stage, without further purification.
[4176] 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 under reduced pressure.
After the residue had been dried under high vacuum, 562 mg (65% of
theory over both stages) of the Z-protected intermediate were
obtained.
[4177] 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
under reduced pressure. The residue was purified by preparative
HPLC. The corresponding fractions were combined, the solvent was
evaporated off under reduced pressure and the residue was
lyophilized from dioxane. This gave 361 mg (87% of theory) of the
title compound as a foam.
[4178] HPLC (Method 5): double peak with Rt=1.75 and 1.86 min;
[4179] 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
##STR00661##
[4181] 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 under reduced pressure, 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.
[4182] HPLC (Method 12): R.sub.t=1.6 min;
[4183] 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]a-
mino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-
-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetate
##STR00662##
[4185] 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 under reduced pressure.
After lyophilization from dioxane, 60 mg (50% of theory) of the
protected intermediate were obtained.
[4186] HPLC (Method 12): R.sub.t=2.2 min;
[4187] LC-MS (Method 1): R.sub.t=1.17 min; MS (ESIpos): m/z=1073
(M+H).sup.+.
[4188] 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 under reduced pressure and the
remaining residue was purified by means of preparative HPLC. The
corresponding fractions were combined, the solvent was removed
under reduced pressure 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.
[4189] HPLC (Method 12): R.sub.t=1.9 min;
[4190] 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}amin-
o)-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-meth-
oxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-
-yl]-N-methyl-L-valinamide
##STR00663##
[4192] The preparation was effected in analogy to Intermediate 134,
proceeding from 5 mg (4.6 .mu.mol) of Intermediate 222. 3.4 mg (65%
of theory) of the title compound were obtained.
[4193] HPLC (Method 12): R.sub.t=2.0 min;
[4194] 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
##STR00664##
[4196] The preparation was effected in analogy to the synthesis of
Intermediate 223.
[4197] HPLC (Method 12): R.sub.t=1.9 min;
[4198] 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
##STR00665##
[4200] 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.
[4201] 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
under reduced pressure. After lyophilization from dioxane, 53 mg
(76% of theory) of the protected intermediate were obtained.
[4202] HPLC (Method 12): R.sub.t=2.0 min;
[4203] LC-MS (Method 1): R.sub.t=1.02 min; MS (ESIpos): m/z=984
(M+H).sup.+.
[4204] 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 under reduced pressure and
the remaining residue was purified by means of preparative HPLC.
The corresponding fractions were combined, the solvent was removed
under reduced pressure and the residue was lyophilized from
dioxane/water. In this way, 21 mg (40% of theory) of the title
compound were obtained in 65% purity.
[4205] HPLC (Method 12): R.sub.t=1.7 min;
[4206] 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
##STR00666##
[4208] The preparation was effected proceeding from Intermediate
225, in analogy to the synthesis of Intermediate 134. 11.6 mg (59%
of theory) of the title compound were obtained.
[4209] HPLC (Method 12): R.sub.t=1.9 min;
[4210] 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
##STR00667##
[4212] This compound was prepared analogously to Intermediate 218,
by conversion to the active ester.
[4213] Yield: 18 mg (51% of theory)
[4214] HPLC (Method 5): R.sub.t=2.1 min;
[4215] 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
##STR00668##
[4217] The title compound was prepared by coupling the
Boc-protected intermediate obtained in the synthesis of
Intermediate 154 with commercially available
6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanehydrazide.
[4218] HPLC (Method 12): R.sub.t=2.1 min;
[4219] 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
##STR00669##
[4221] 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
[4222] Yield: 1.4 mg (40% of theory)
[4223] HPLC (Method 12): R.sub.t=1.9 min;
[4224] 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
##STR00670##
[4226] This compound was prepared in analogy to Intermediate 228,
proceeding from Intermediate 193. 16 mg (30% of theory over 3
stages) of the title compound were obtained.
[4227] HPLC (Method 12): R.sub.t=2.0 min;
[4228] 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-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-2-y-
l]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-oxoethyl)-7,10--
diisopropyl-5,11-dimethyl-6,9-dioxo-2-oxa-5,8,11-triazapentadecan-15-oate
##STR00671##
[4230] 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.
[4231] HPLC (Method 12): R.sub.t=1.9 min;
[4232] 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
##STR00672##
[4234] 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 under reduced pressure 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.
[4235] HPLC (Method 12): R.sub.t=1.8 min;
[4236] 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
##STR00673##
[4238] 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.
[4239] HPLC (Method 5): R.sub.t=2.1 min;
[4240] LC-MS (Method 1): R.sub.t=0.6 in; MS (ESIpos): m/z=1089
(M+H).sup.+.
[4241] 5.4 mg (5 .mu.mol) of this intermediate were dissolved in 5
ml of methanol and, after addition of 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 under reduced pressure. After the residue had been
dried under high vacuum, 5 mg (quant.) of the acid intermediate
were obtained.
[4242] HPLC (Method 12): R.sub.t=1.8 min;
[4243] LC-MS (Method 1): R.sub.t=0.84 min; MS (ESIpos): m/z=999
(M+H).sup.+.
[4244] 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
under reduced pressure. 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.
[4245] HPLC (Method 12): R.sub.t=1.9 min;
[4246] 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
##STR00674##
[4248] 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 mixture 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.
[4249] 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
##STR00675##
[4251] 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 charcoal
(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 under reduced
pressure. The residue was lyophilized from 1:1 acetonitrile/water.
11 mg (86% of theory) of the title compound were obtained as a
foam.
[4252] 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
##STR00676##
[4254] 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.
[4255] 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
##STR00677##
[4257] 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.
[4258] 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
##STR00678##
[4260] 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.
[4261] 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
##STR00679##
[4263] 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 under reduced pressure and the residue purified by
preparative HPLC. 29 mg (54% of theory) of the Z-protected
intermediate were thus obtained.
[4264] LC-MS (Method 1): R.sub.t=1.15 min; MS (ESIpos): m/z=976
(M+H).sup.+.
[4265] 29 mg (0.003 mmol) of this intermediate were dissolved in 5
ml of methanol and hydrogenated over 5 mg of 5% palladium/charcoal
at RT and standard pressure for 1 h. The catalyst was subsequently
filtered off and the solvent was evaporated off. The remaining
residue was purified by preparative HPLC. 17 mg (54% of theory) of
the title compound were obtained.
[4266] 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
##STR00680##
[4268] 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.
[4269] HPLC (Method 5): R.sub.t=1.7 min;
[4270] 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
##STR00681##
[4272] 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 reagent was then used, 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 detachment of the Boc protecting
group by means of trifluoroacetic acid, to prepare the title
compound.
[4273] HPLC (Method 12): R.sub.t=1.7 min;
[4274] 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
##STR00682##
[4276] 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 resulting acid were converted to the activated ester.
13.5 mg (36% of theory over 2 stages) of the title compound were
obtained.
[4277] HPLC (Method 12): R.sub.t=1.8 min;
[4278] 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-1-oxoheptan-4-
-yl]-N-methyl-L-valinamide
##STR00683##
[4280] The preparation was effected 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.
[4281] Yield: 17.5 mg (34% of theory over 2 stages)
[4282] HPLC (Method 12): R.sub.t=1.7 min;
[4283] 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
##STR00684##
[4285] The preparation was effected in analogy to Intermediate 166,
proceeding from Intermediate 243.
[4286] Yield: 1.3 mg (12% of theory)
[4287] HPLC (Method 12): R.sub.t=1.9 min;
[4288] 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]-N-(tert-butoxycarbon-
yl)-L-threonyl-beta-alaninate
##STR00685##
[4290] 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 hydrogenolysis. 30 mg (0.027
mmol) of the
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-(1H-
-indol-3-yl)-1-(1,2-oxazinan-2-yl)-1-oxopropan-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 thus obtained 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 resulting acid were converted to the activated ester.
After HPLC purification, 2.7 mg (23% of theory) of the title
compound were obtained.
[4291] HPLC (Method 5): R.sub.t=1.9 min;
[4292] 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)
##STR00686##
[4294] 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 under reduced pressure.
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 was
separated into the diastereomers by 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 under reduced pressure. After the residues had
been dried 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), and also 333 mg (22% of theory) of a
mixed fraction of the Boc-protected intermediates were
obtained.
[4295] Under standard conditions, 5 ml of trifluoroacetic acid in
20 ml of dichloromethane were used to detach 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
in the next stage without further purification.
[4296] HPLC (Method 12): R.sub.t=1.1 min;
[4297] 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)
##STR00687##
[4299] Under standard conditions, 5 ml of trifluoroacetic acid in
20 ml of dichloromethane were used to detach 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.
[4300] 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)
##STR00688##
[4302] To synthesize this compound, the coupling of Intermediates
26 and 246a with subsequent detachment 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 preparative HPLC.
[4303] HPLC (Method 12): R.sub.t=1.8 min;
[4304] 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)
##STR00689##
[4306] To synthesize this compound, the coupling of Intermediates
26 and 246b with subsequent detachment 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 preparative HPLC.
[4307] HPLC (Method 12): R.sub.t=1.8 min;
[4308] 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
##STR00690##
[4310] First, in analogy to the synthesis described in Intermediate
86, 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 detachment 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), 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]pyrrolidin-2-
-yl}-2-methylpropanoyl]-L-tyrosinate was prepared as the
trifluoroacetate. 38 mg (0.04 mmol) of this compound were then
used, in analogy to the preparation of Intermediate 210, by
reaction with 6-oxohexanoic acid in the presence of borane-pyridine
complex, to obtain 31 mg (99% of theory) of the title compound.
[4311] HPLC (Method 12): R.sub.t=1.8 min;
[4312] LC-MS (Method 1): R.sub.t=0.88 min; MS (ESIpos): m/z=918
(M+H).sup.+.
B: PREPARATION OF ANTIBODY-DRUG CONJUGATES (ADCs)
B-1. General Process for Generating Anti-C4.4a Antibodies
[4313] The anti-C4.4a antibodies described by the sequences of
Table 1 and Table 2 were generated by screening a phage display
library for recombinant human C4.4a SEQ ID NO: 1 and murine C4.4a
SEQ ID NO: 2 and for cells expressing C4.4a. The antibodies
obtained in this way were reformatted to the human IgG1 format and
used for the working examples described here.
B-2. General Process for Expressing Anti-C4.4a Antibodies in
Mammalian Cells
[4314] The antibodies, for example M31-B01 (light chain SEQ ID NO:
346 and heavy chain SEQ ID NO: 347) or further antibodies of Table
2 were produced in a mammalian cell culture. For this purpose,
HEK293 6E cells were transiently transfected with a suitable CMV
promotor based expression plasmid. The heavy and light chains of
the antibodies were cloned either together into a one-vector
system, or separately into a two-vector system. The cell culture
scale was either up to 1.5 L in a shake flask or 10 L in a
"wave-bag". Expression took place 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 100 and 600
mg/l.
B-3. General Process for Purifying Antibodies from Cell
Supernatants
[4315] The antibodies, for example M31-B01 (light chain SEQ ID NO:
346 and heavy chain SEQ ID NO: 347) or further antibodies of Table
2 were obtained from the cell culture supernatants. The cell
supernatants were clarified by centrifugation to remove cells.
Subsequently the cell supernatant was purified by affinity
chromatography on a MabSelect Sure (GE Healthcare) chromatography
column. For this purpose the column was equilibrated in DPBS pH 7.4
(Sigma/Aldrich), the cell supernatant was applied, and the column
was washed with about 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 subsequently purified further by
gel filtration chromatography on a Superdex 200 column (GE
Healthcare) in DPBS pH 7.4.
B-4. General Process for Coupling to Cysteine Side Chains
[4316] The antibodies used in the coupling reactions were as
follows:
anti-C4.4a M31-B01 anti-C4.4a B01-3 anti-C4.4a B01-10 anti-C4.4a
B01-7 anti-C4.4a D02-4 anti-C4.4a D02-6 anti-C4.4a D02-7
[4317] Added to a solution of the corresponding antibody in PBS
buffer in the concentration range between 1 mg/ml and 15 mg/ml were
3 equivalents of tris(2-carboxyethyl)phosphine hydro-chloride
(TCEP), in solution in PBS buffer, and the mixture was stirred at
RT for 1 hour. Subsequently, depending on the desired loading,
between 2 and 10 equivalents of the maleimide precursor compound or
halide precursor compound for coupling (Intermediate 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 here ought not to exceed 10% of the
overall volume. The batch was stirred at RT for 60-120 minutes and
then applied to PD 10 columns (Sephadex.RTM. G-25, GE Healthcare)
equilibrated with PBS, and eluted with PBS buffer. Optionally a
concentration procedure was carried out additionally by means of
ultracentrifugation. If necessary, for more effective removal of
low molecular mass constituents, the concentration by
ultrafiltration was repeated after re-dilution with PBS buffer.
[4318] Normally, unless otherwise indicated, 5 mg of the
corresponding antibody in PBS buffer were used for the reduction
and the subsequent coupling. Following purification via the PD10
column, this gave, in each case, solutions of the corresponding ADC
in 3.5 ml of PBS buffer. The particular protein concentration
indicated was then determined for these solutions. Furthermore, the
loading of the antibody (drug/mAb ratio) was determined in
accordance with the methods described below.
[4319] This process was used to prepare the immunoconjugates
represented in Examples 1-3, 5-30, 32-36, 38-59, 61-66, 68-70, 80,
82-85, 87, 88, 92-95, 97, 98, 107, 109-114, 119 and 122.
[4320] In the structural formulae illustrated, the definition of
AK.sub.1A-AK.sub.1G is as follows
AK.sub.1A=anti-C4.4a antibody M31-B01 (partially
reduced)--S.sctn..sup.1 AK.sub.1B=anti-C4.4a antibody B01-3
(partially reduced)--S.sctn..sup.1 AK.sub.1c=anti-C4.4a antibody
B01-10 (partially reduced)--S.sctn..sup.1 AK.sub.1D=anti-C4.4a
antibody B01-7 (partially reduced)--S.sctn..sup.1
AK.sub.1E=anti-C4.4a antibody D02-4 (partially
reduced)--S.sctn..sup.1 AK.sub.1F=anti-C4.4a antibody D02-6
(partially reduced)--S.sctn..sup.1 AK.sub.1G=anti-C4.4a antibody
D02-7 (partially reduced)--S.sctn..sup.1 where .sctn..sup.1 denotes
the link with the succinimide group, and S stands for the sulphur
atom of a cysteine residue of the partially reduced antibody.
B-5. General Process for Coupling to Lysine Side Chains
[4321] The antibodies used in the coupling reactions were as
follows:
anti-C4.4a antibody M31-B01 anti-C4.4a antibody B01-3
[4322] Added to a solution of the corresponding antibody in PBS
buffer in the concentration range between 1 mg/ml and 15 mg/ml
were, depending on the desired loading, between 2 and 5 equivalents
of the precursor compound for coupling (Intermediate 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)) as a solution in DMSO. After 30 minutes of stirring at RT,
the same amount of precursor compound in DMSO was added again.
Alternatively it was possible to add 4-10 equivalents of the
precursor compound for coupling, in one go. The amount of DMSO here
ought not to exceed 10% of the overall volume. After a further 30
minutes of stirring at RT, the batch was applied to PD 10 columns
(Sephadex.RTM. G-25, GE Healthcare) equilibrated with PBS, and
eluted with PBS buffer. Optionally a concentration procedure was
carried out additionally by means of ultracentrifugation. If
necessary, for more effective removal of low molecular mass
constituents, the concentration by ultrafiltration was repeated
after re-dilution with PBS buffer.
[4323] Normally, unless otherwise indicated, 5 mg of the
corresponding antibody in PBS buffer were used for the coupling.
Following purification via the PD10 column, this gave, in each
case, solutions of the corresponding ADC in 3.5 ml of PBS buffer.
The particular protein concentration indicated was then determined
for these solutions and the loading of the antibody (drug/mAb
ratio) was determined in accordance with the methods described
below.
[4324] This process was used to prepare the immunoconjugates
represented in Examples 4, 31, 37, 60, 67, 81, 86, 89-91, 96,
99-106, 108, 118, 120, 121 and 123-125.
[4325] In the structural formulae illustrated, the definition of
AK.sub.2A and AK.sub.2B is as follows
AK.sub.2A=anti-C4.4a antibody M31-B01-NH.sctn..sup.2
AK.sub.2B=anti-C4.4a antibody B01-3-NH.sctn..sup.2 where
.sctn..sup.2 denotes the link with the carbonyl group, and NH
stands for the side chain amino group of a lysine residue of the
antibody.
B-6. General Process for Preparing Cysteine Adducts:
[4326] 10 .mu.mol of the above-described maleimide precursor
compounds were taken up in 3 ml of DMF and admixed with 2.1 mg (20
.mu.mol) of L-cysteine. The reaction mixture was stirred at RT for
2 hours, then concentrated under reduced pressure and subsequently
purified by preparative HPLC.
[4327] In the structural formulae illustrated, the definition of
Cys is as follows
##STR00691##
where .sctn..sup.3 denotes the link with the linker-toxophore
unit.
Further Purification and Characterization of the Conjugates of the
Invention
[4328] After reaction had taken place, in certain cases the
reaction mixture was concentrated, by ultrafiltration, for example,
and then desalted and purified by means of chromatography, for
example using a Sephadex.RTM. G-25. Elution took place with, for
example, phosphate-buffered saline (PBS). The solution was
subsequently subjected to sterile filtration and freezing. An
alternative option is to lyophilize the conjugate.
B-7. Determination of the Toxophore Loading
[4329] The toxophore loading of the resultant solutions of the
conjugates described in the working examples, in PBS buffer, was
determined as follows:
[4330] The toxophore loading of lysine-linked ADCs was determined
by mass-spectrometric determination of the molecular weights of the
individual conjugate species. In this case, to start with, the
antibody conjugates were deglycosylated by means of PNGaseF, and
the sample was acidified and, following HPLC separation, was
analysed by mass spectrometry using an ESI-MicroTofQ (Bruker
Daltonik). All of the spectra were added via the signal in the TIC
(Total Ion Chromatogram), and the molecular weight of the various
conjugate species was calculated on the basis of MaxEnt
Deconvolution. Following signal integration of the different
species, the DAR (Drug/Antibody Ratio) was then calculated.
[4331] For protein identification, in addition to the molecular
weight determination, a tryptic digestion was carried out after
deglycosylation and/or denaturing, and this digestion, after
denaturing, reduction and derivatization, confirmed the identity of
the protein on the basis of the tryptic peptides detected.
[4332] The toxophore loading of cysteine-linked conjugates was
determined via reversed-phase chromatography of the reduced and
denatured ADC. The ADC solution (1 mg/mL, 50 .mu.L) was admixed
with guanidinium hydrochloride (GuHCl) (28.6 mg) and with a
solution of DL-dithiothreitol (DTT) (500 mM, 3 .mu.L). The mixture
was incubated at 55.degree. C. for an hour and analysed by
HPLC.
[4333] The HPLC analysis was carried out on an Agilent 1260 HPLC
System with detection at 220 nm. The column used was a Polymer
Laboratories PLRP-S Polymeric Reversed Phase column (catalogue
number PL1912-3802) (2.1.times.150 mm, 8 .mu.m particle size, 1000
.ANG.) with a flow rate of 1 mL/min, using 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 of 0.05%
trifluoroacetic acid in acetonitrile.
[4334] The peaks detected were assigned by retention time
comparison with the light chain (L0) and the heavy chain (H0) of
the unconjugated antibody. Peaks which were detected exclusively in
the conjugated sample were assigned to the light chain, with a
toxophore (L1), and to the heavy chains, with one, two and three
toxophores (H1, H2, H3).
[4335] The average loading of the antibody with toxophores was
calculated as follows: first of all, the light-chain loading was
calculated from the peak areas--determined by integration--of the
peaks L0 and L1 belonging to the light chains, as the sum of the
toxophore number weighted integration results of L0 and L1, divided
by the sum of the singularly weighted integration results of L0 and
L1. In the same way, the heavy-chain loading was calculated from
the peak areas--determined by integration--of the peaks H0, H1, H2
and H3, belonging to the heavy chains, as the sum of the toxophore
number weighted integration results of H0, H1, H2 and H3, divided
by the sum of the singularly weighted integration results of H0,
H1, H2 and H3. The DAR is given by the light-chain loading and the
heavy-chain loading, as the twofold sum of light-chain loading and
heavy-chain loading. The factor 2 takes account of the fact that an
antibody consists of two light chains and two heavy chains. In
certain individual cases it may be impossible exactly to determine
the toxophore loading, owing to co-elutions of certain peaks.
B-8. Testing of the Antigen Binding of the ADC
[4336] The binding capacity of the binder to the target molecule
was tested after coupling had taken place. The skilled worker knows
of diverse methods for achieving this--for example, the affinity of
the conjugate can be tested by means of ELISA technology or surface
plasmon resonance analysis (BIAcore.TM. measurements). The
conjugate concentration can be measured by the skilled person using
common methods--for example, for antibody conjugates, by means of
protein determination (see also Doronina et al.; Nature Biotechnol.
2003; 21:778-784 and Polson et al., Blood 2007; 1102:616-623).
WORKING EXAMPLES
Immunoconjugates
Example 1
##STR00692##
[4338] In this case coupling was carried out using 70 mg of
anti-C4.4a M31-B01 in DPBS pH 7.4 and following the Sephadex
purification the batch was concentrated by ultracentifugation.
Protein concentration: 12.2 mg/ml
Drug/mAb Ratio: 1.5
Example 2
##STR00693##
[4339] Protein concentration: 0.87 mg/ml
Drug/mAb Ratio: 5.8
Example 3
##STR00694##
[4340] Protein concentration: 1.16 mg/ml
Drug/mAb Ratio: 3.1
Example 4
##STR00695##
[4341] Protein concentration: 1.24 mg/ml
Drug/mAb Ratio: 1.6
Example 5
##STR00696##
[4342] Protein concentration: 0.88 mg/ml
Drug/mAb Ratio: 6.9
Example 6
##STR00697##
[4343] Protein concentration: 1.2 mg/ml
Drug/mAb Ratio: 2.8
Example 7
##STR00698##
[4344] Protein concentration: 0.9 mg/ml
Drug/mAb Ratio: 3.9
Example 8
##STR00699##
[4345] Protein concentration: 0.52 mg/ml
Drug/mAb Ratio: 1.6
Example 9
##STR00700##
[4346] Protein concentration: 0.47 mg/ml
Drug/mAb Ratio: 6.6
Example 10
##STR00701##
[4347] Protein concentration: 0.77 mg/ml
Drug/mAb Ratio: 6.9
Example 11
##STR00702##
[4348] Protein concentration: 0.47 mg/ml
Drug/mAb Ratio: 4.0
Example 12
##STR00703##
[4349] Protein concentration: 1.46 mg/ml
Drug/mAb Ratio: 2.5
Example 13
##STR00704##
[4350] Protein concentration: 0.45 mg/ml
Drug/mAb Ratio: 3.3
Example 14
##STR00705##
[4351] Protein concentration: 0.98 mg/ml
Drug/mAb Ratio: 3.6
Example 15
##STR00706##
[4353] Coupling here was carried out using 70 mg of anti-C4.4a
M31-B01 in DPBS pH 7.4, and following the Sephadex purification the
batch was concentrated by ultracentifugation.
Protein concentration: 9.42 mg/ml
Drug/mAb Ratio: 4.1
Example 16
##STR00707##
[4354] Protein concentration: 0.65 mg/ml
Drug/mAb Ratio: 1.8
Example 17
##STR00708##
[4355] Protein concentration: 1.07 mg/ml Drug/mAb Ratio: not
determinable
Example 18
##STR00709##
[4356] Protein concentration: 0.47 mg/ml
Drug/mAb Ratio: 4.4
Example 19
##STR00710##
[4357] Protein concentration: 0.43 mg/ml
Drug/mAb Ratio: 4.8
Example 20
##STR00711##
[4358] Protein concentration: 1.01 mg/ml
Drug/mAb Ratio: 2.6
Example 21
##STR00712##
[4359] Protein concentration: 0.53 mg/ml
Drug/mAb Ratio: 0.6
Example 22
##STR00713##
[4360] Protein concentration: 0.55 mg/ml
Drug/mAb Ratio: 1.3
Example 23
##STR00714##
[4361] Protein concentration: 0.65 mg/ml
Drug/mAb Ratio: 1.1
Example 24
##STR00715##
[4362] Protein concentration: 1.04
Drug/mAb Ratio: 3.5
Example 25
##STR00716##
[4363] Protein concentration: 0.62 mg/ml
Drug/mAb Ratio: 2.4
Example 26
##STR00717##
[4365] Coupling here was carried out using 90 mg of anti-C4.4a
M31-B01 in DPBS pH 7.4 and following the Sephadex purification the
batch was concentrated by ultracentifugation.
Protein concentration: 11.2 mg/ml
Drug/mAb-Ratio: 2.3
Example 27
##STR00718##
[4366] Protein concentration: 1.11 mg/ml
Drug/mAb-Ratio: 2.4
Example 28
##STR00719##
[4368] Coupling here was carried out using 70 mg of anti-C4.4a
M31-B01 in DPBS pH 7.4 and following the Sephadex purification the
batch was concentrated by ultracentifugation.
Protein concentration: 10.7 mg/ml
Drug/mAb Ratio: 2.2
Example 29
##STR00720##
[4369] Protein concentration: 0.87 mg/ml
Drug/mAb Ratio: 1.8
Example 30
##STR00721##
[4370] Protein concentration: 1.3 mg/ml
Drug/mAb Ratio: 2.1
Example 31
##STR00722##
[4371] Protein concentration: 1.3 mg/ml
Drug/mAb Ratio: 0.3
Example 32
##STR00723##
[4373] Coupling here was carried out using 70 mg of anti-C4.4a
M31-B01 in DPBS pH 7.4 and following the Sephadex purification the
batch was concentrated by ultracentifugation.
Protein concentration: 12.0 mg/ml
Drug/mAb Ratio: 3.2
Example 33
##STR00724##
[4375] Coupling here was carried out using 90 mg of anti-C4.4a
M31-B01 in DPBS pH 7.4 and following the Sephadex purification the
batch was concentrated by ultracentifugation.
Protein concentration: 10.2 mg/ml
Drug/mAb Ratio: 4.3
Example 34
##STR00725##
[4376] Protein concentration: 1.37 mg/ml
Drug/mAb Ratio: 2.6
Example 35
##STR00726##
[4377] Protein concentration: 1.14 mg/ml
Drug/mAb Ratio: 2.0
Example 36
##STR00727##
[4378] Protein concentration: 1.07 mg/ml
Drug/mAb Ratio: 3.5
Example 37
##STR00728##
[4379] Protein concentration: 1.14 mg/ml
Drug/mAb Ratio: 1.9
Example 38
##STR00729##
[4380] Protein concentration: 1.22 mg/ml
Drug/mAb Ratio: 3.3
Example 39
##STR00730##
[4381] Protein concentration: 1.3 mg/ml
Drug/mAb Ratio: 3.2
Example 40
##STR00731##
[4382] Protein concentration: 1.23 mg/ml
Drug/mAb Ratio: 3.3
Example 41
##STR00732##
[4383] Protein concentration: 1.64 mg/ml
Drug/mAb Ratio: 1.8
Example 42
##STR00733##
[4384] Protein concentration: 1.07 mg/ml
Drug/mAb Ratio: 3.1
Example 43
##STR00734##
[4385] Protein concentration: 1.14 mg/ml
Drug/mAb Ratio: 2.3
Example 44
##STR00735##
[4386] Protein concentration: 1.23 mg/ml
Drug/mAb Ratio: 3.4
Example 45
##STR00736##
[4387] Protein concentration: 1.22 mg/ml
Drug/mAb Ratio: 2.5
Example 46
##STR00737##
[4388] Protein concentration: 1.22 mg/ml
Drug/mAb Ratio: 2.4
Example 47
##STR00738##
[4389] Protein concentration: 1.32 mg/ml Drug/mAb Ratio: not
determinable
Example 48
##STR00739##
[4390] Protein concentration: 1.44 mg/ml
Drug/mAb Ratio: 2.3
Example 49
##STR00740##
[4392] Coupling here was carried out using 250 mg of anti-C4.4a
B01-10 in DPBS pH 7.4 and following the Sephadex purification the
batch was concentrated by ultracentifugation.
Protein concentration: 12.8 mg/ml
Drug/mAb Ratio: 5.2
Example 50
##STR00741##
[4393] Protein concentration: 0.9 mg/ml
Drug/mAb Ratio: 2
Example 51
##STR00742##
[4395] Coupling here was carried out using 250 mg of anti-C4.4a
B01-3 in DPBS PH 7.4 and following the Sephadex purification the
batch was concentrated by ultracentifugation.
Protein concentration: 8.0 mg/ml
Drug/mAb Ratio: 4.5
Example 52
##STR00743##
[4397] Coupling here was carried out using 250 mg of anti-C4.4a
B01-10 in DPBS pH 7.4 and following the Sephadex purification the
batch was concentrated by ultracentifugation.
Protein concentration: 12.3 mg/ml
Drug/mAb Ratio: 5.2
Example 53
##STR00744##
[4399] Coupling here was carried out using 250 mg of anti-C4.4a
B01-10 in DPBS pH 7.4 and following the Sephadex purification the
batch was concentrated by ultracentifugation.
Protein concentration: 10.2 mg/ml
Drug/mAb Ratio: 4.4
Example 54
##STR00745##
[4401] Coupling here was carried out using 50 mg of anti-C4.4a
B01-3 in DPBS pH 7.4 and following the Sephadex purification the
batch was concentrated by ultracentifugation.
Protein concentration: 11.5 mg/ml
Drug/mAb Ratio: 5.2
Example 55
##STR00746##
[4403] Coupling here was carried out using 250 mg of anti-C4.4a
D02-6 in DPBS pH 7.4 and following the Sephadex purification the
batch was concentrated by ultracentifugation.
Protein concentration: 13 mg/ml
Drug/mAb Ratio: 5.2
Example 56
##STR00747##
[4405] Coupling here was carried out using 250 mg of anti-C4.4a
B01-3 in DPBS pH 7.4 and following the Sephadex purification the
batch was concentrated by ultracentifugation.
Protein concentration: 10.3 mg/ml
Drug/mAb Ratio: 4.9
Example 57
##STR00748##
[4406] Protein concentration: 0.88 mg/ml
Drug/mAb Ratio: 3.2
Example 58
##STR00749##
[4407] Protein concentration: 1.18 mg/ml
Drug/mAb Ratio: 3.4
Example 59
##STR00750##
[4408] Protein concentration: 1.23 mg/ml
Drug/mAb Ratio: 3.0
Example 60
##STR00751##
[4409] Protein concentration: 1.3 mg/ml
Drug/mAb Ratio: 3.3
Example 61
##STR00752##
[4410] Protein concentration: 1.11 mg/ml Drug/mAb Ratio: not
determinable
Example 62
##STR00753##
[4411] Protein concentration: 1.25 mg/ml
Drug/mAb Ratio: 2.4
Example 63
##STR00754##
[4412] Protein concentration: 0.88 mg/ml
Drug/mAb Ratio: 5.0
Example 64
##STR00755##
[4413] Protein concentration: 1.23 mg/ml
Drug/mAb Ratio: 3.3
Example 65
##STR00756##
[4414] Protein concentration: 0.93 mg/ml
Drug/mAb Ratio: 1.8
Example 66
##STR00757##
[4415] Protein concentration: 0.85 mg/ml
Drug/mAb Ratio: 5.3
Example 67
##STR00758##
[4416] Protein concentration: 1.51 mg/ml
Drug/mAb Ratio: 1.4
Example 68
##STR00759##
[4418] Coupling here was carried out using 150 mg of anti-C4.4a
B01-3 in DPBS PH 7.4 and following the Sephadex purification the
batch was concentrated by ultracentifugation.
Protein concentration: 11.0 mg/ml
Drug/mAb Ratio: 4.5
Example 69
##STR00760##
[4419] Protein concentration: 1.2 mg/ml
Drug/mAb Ratio: 3.3
Example 70
##STR00761##
[4420] Protein concentration: 1.25 mg/ml
Drug/mAb Ratio: 3.1
Example 71
N-(4-{2-[6-(3-{[(2R)-2-Amino-2-carboxyethyl]sulphanyl}-2,5-dioxopyrrolidin-
-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2-
S)-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-oxohep-
tan-4-yl]-N-methyl-L-valinamide
##STR00762##
[4422] 10 mg (10 .mu.mol) of Intermediate 157 were taken up in 5.2
ml of DMF and admixed with 2.28 mg (20 .mu.mol) of L-cysteine. The
reaction mixture was stirred at RT for 2 hours, then concentrated
under reduced pressure and subsequently purified by preparative
HPLC. This gave 5.8 mg (48% of theory) of the title compound.
[4423] HPLC (Method 5): R.sub.t=1.45 min;
[4424] LC-MS (Method 1): R.sub.t=0.74 min; MS (ESIpos): m/z=1184
(M+H).sup.+.
Example 72
N-(4-{2-[6-(3-{[(2R)-2-Amino-2-carboxyethyl]sulphanyl}-2,5-dioxopyrrolidin-
-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2-
S)-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
##STR00763##
[4426] 10 mg (10 .mu.mol) of Intermediate 113 were taken up in 5.2
ml of DMF and admixed with 2.28 mg (20 .mu.mol) of L-cysteine. The
reaction mixture was stirred at RT for 2 hours, then concentrated
under reduced pressure and subsequently purified by preparative
HPLC. This gave 6 mg (54% of theory) of the title compound.
[4427] HPLC (Method 5): R.sub.t=1.5 min;
[4428] LC-MS (Method 1): R.sub.t=0.77 min; MS (ESIpos): m/z=1185
(M+H).sup.+.
Example 73
N-(4-{2-[6-(3-{[(2R)-2-Amino-2-carboxyethyl]sulphanyl}-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
##STR00764##
[4430] 9 mg (8.3 .mu.mol) of Intermediate 132 were taken up in 4 ml
of DMF and admixed with 3 mg (24.4 .mu.mol) of L-cysteine. The
reaction mixture was stirred at RT overnight, then concentrated
under reduced pressure and subsequently purified by preparative
HPLC. This gave 6.8 mg (68% of theory) of the title compound.
[4431] HPLC (Method 12): R.sub.t=1.8 min;
[4432] LC-MS (Method 1): R.sub.t=0.78 min; MS (ESIpos): m/z=1227
(M+H).sup.+.
Example 74
N-[6-(3-{[(2R)-2-Amino-2-carboxyethyl]sulphanyl}-2,5-dioxopyrrolidin-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]p-
yrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
##STR00765##
[4434] 10 mg (10 .mu.mol) of Intermediate 106 were taken up in 5.8
ml of DMF and admixed with 2.5 mg (20 .mu.mol) of L-cysteine. The
reaction mixture was stirred at RT for 2 hours, then concentrated
under reduced pressure and subsequently purified by preparative
HPLC. This gave 5.2 mg (46% of theory of the title compound.
[4435] HPLC (Method 5): R.sub.t=1.5 min;
[4436] LC-MS (Method 11): R.sub.t=0.71 min; MS (ESIpos): m/z=1070
(M+H).sup.+.
Example 75
N-[6-(3-{[(2R)-2-Amino-2-carboxyethyl]sulphanyl}-2,5-dioxopyrrolidin-1-yl)
hexyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carbo-
xy-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
##STR00766##
[4438] 10 mg (10 .mu.mol) of Intermediate 124 were taken up in 4 ml
of DMF and admixed with 2.5 mg (20 .mu.mol) of L-cysteine. The
reaction mixture was stirred at RT for 2 hours, then concentrated
under reduced pressure and subsequently purified by preparative
HPLC. This gave 7.2 mg (64% of theory of the title compound.
[4439] HPLC (Method 5): R.sub.t=1.6 min;
[4440] LC-MS (Method 1): R.sub.t=0.8 min; MS (ESIpos): m/z=1071
(M+H).sup.+.
Example 76
N-[6-(3-{[(2R)-2-Amino-2-carboxyethyl]sulphanyl}-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
##STR00767##
[4442] 10 mg (10 .mu.mol) of Intermediate 125 were taken up in 4 ml
of DMF and admixed with 2.4 mg (20 .mu.mol) of L-cysteine. The
reaction mixture was stirred at RT for 2 hours, then concentrated
under reduced pressure and subsequently purified by preparative
HPLC. This gave 7.7 mg (69% of theory of the title compound.
[4443] HPLC (Method 5): R.sub.t=1.7 min;
[4444] LC-MS (Method 2): R.sub.t=1.91 min; MS (ESIpos): m/z=1140
(M+H).sup.+.
Example 77
N-(4-{2-[6-(3-{[(2R)-2-Amino-2-carboxyethyl]sulphanyl}-2,5-dioxopyrrolidin-
-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2-
S)-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
##STR00768##
[4446] 10 mg (10 .mu.mol) of Intermediate 160 were taken up in 3 ml
of DMF and admixed with 2.1 mg (20 .mu.mol) of L-cysteine. The
reaction mixture was stirred at RT for 2 hours, then concentrated
under reduced pressure and subsequently purified by preparative
HPLC. This gave 8.1 mg (73% of theory of the title compound.
[4447] HPLC (Method 5): R.sub.t=1.7 min;
[4448] LC-MS (Method 1): R.sub.t=0.86 min; MS (ESIpos): m/z=1274
(M+H).sup.+.
Example 78
N-(4-{2-[6-(3-{[(2R)-2-Amino-2-carboxyethyl]sulphanyl}-2,5-dioxopyrrolidin-
-1-yl)hexanoyl]hydrazino}-4-oxobutyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2-
S)-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-oxohept-
an-4-yl]-N-methyl-L-valinamide
##STR00769##
[4450] 3.5 mg (3 .mu.mol) of Intermediate 159 were taken up in 1 ml
of DMF and admixed with 0.76 mg (6 .mu.mol) of L-cysteine. The
reaction mixture was stirred at RT for 2 hours, then concentrated
under reduced pressure and subsequently purified by preparative
HPLC. This gave 2.6 mg (65% of theory of the title compound.
[4451] HPLC (Method 5): R.sub.t=1.75 min;
[4452] LC-MS (Method 1): R.sub.t=0.85 min; MS (ESIpos): m/z=1235
(M+H).sup.+.
Example 79
N-(6-{2-[6-(3-{[(2R)-2-Amino-2-carboxyethyl]sulphanyl}-2,5-dioxopyrrolidin-
-1-yl)hexanoyl]hydrazino}-6-oxohexyl)-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
##STR00770##
[4454] 3.6 mg (3 .mu.mol) of Intermediate 129 were taken up in 1 ml
of DMF and admixed with 0.77 mg (6 .mu.mol) of L-cysteine. The
reaction mixture was stirred at RT for 2 hours, then concentrated
under reduced pressure and subsequently purified by preparative
HPLC. This gave 1.55 mg (39% of theory of the title compound.
[4455] HPLC (Method 5): R.sub.t=1.6 min;
[4456] LC-MS (Method 1): R.sub.t=0.87 min; MS (ESIpos): m/z=1255
(M+H).sup.+.
Example 80
##STR00771##
[4458] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 0.83 mg/ml
Drug/mAb Ratio: 1.6
Example 81
##STR00772##
[4460] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.59 mg/ml
Drug/mAb Ratio: 3.1
Drug/mAb Ratio: 2.9
Example 82
##STR00773##
[4462] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.25 mg/ml
Drug/mAb Ratio: 4.0
Example 83
##STR00774##
[4464] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.27 mg/ml
Drug/mAb Ratio: 3.6
Example 84
##STR00775##
[4466] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.54 mg/ml
Drug/mAb Ratio: 4.7
Example 85
##STR00776##
[4468] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.73 mg/ml
Drug/mAb Ratio: 4.7
Example 86
##STR00777##
[4470] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.66 mg/ml
Drug/mAb Ratio: 1.3
Example 87
##STR00778##
[4471] Protein concentration: 2.11 mg/ml
Drug/mAb Ratio: 5.5
Example 88
##STR00779##
[4472] Protein concentration: 1.53 mg/ml
Drug/mAb Ratio: 3.4
Example 89
##STR00780##
[4473] Protein concentration: 1.5 mg/ml
Drug/mAb Ratio: 0.2
Example 90
##STR00781##
[4474] Protein concentration: 1.32 mg/ml
Drug/mAb Ratio: 0.1
Example 91
##STR00782##
[4476] Coupling here was carried out using 80 mg of anti-C4.4a
B01-3 in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation, re-diluted with PBS and
concentrated again.
Protein concentration: 10.3 mg/ml
Drug/mAb Ratio: 3.1
Example 92
##STR00783##
[4478] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.09 mg/ml
Drug/mAb Ratio: 1.8
Example 93
##STR00784##
[4480] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.52 mg/ml
Drug/mAb Ratio: 4.2
Example 94
##STR00785##
[4482] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.1 mg/ml
Drug/mAb Ratio: 3.3
Example 95
##STR00786##
[4484] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.43 mg/ml
Drug/mAb Ratio: 4.8
Example 96
##STR00787##
[4486] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation, re-diluted with PBS and
concentrated again.
Protein concentration: 1.36 mg/ml
Drug/mAb Ratio: 4.6
Example 97
##STR00788##
[4488] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.33 mg/ml
Drug/mAb Ratio: 4.0
Example 98
##STR00789##
[4490] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.33 mg/ml
Drug/mAb Ratio: 4.6
Example 99
##STR00790##
[4492] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.47 mg/ml
Drug/mAb Ratio: 1.6
Example 100
##STR00791##
[4494] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.49 mg/ml
Drug/mAb Ratio: 4.5
Example 101
##STR00792##
[4496] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.29 mg/ml
Drug/mAb Ratio: 3.3
Example 102
##STR00793##
[4498] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.74 mg/ml
Drug/mAb Ratio: 3.5
Example 103
##STR00794##
[4500] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.09 mg/ml
Drug/mAb Ratio: 3.2
Example 104
##STR00795##
[4502] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.63 mg/ml
Drug/mAb Ratio: 0.2
Example 105
##STR00796##
[4504] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.41 mg/ml
Drug/mAb Ratio: 7.6
Example 106
##STR00797##
[4506] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 2.0 mg/ml
Drug/mAb Ratio: 1.6
Example 107
##STR00798##
[4508] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.67 mg/ml
Drug/mAb Ratio: 2.8
Example 108
##STR00799##
[4510] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.91 mg/ml
Drug/mAb Ratio: 5.3
Example 109
##STR00800##
[4512] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.82 mg/ml
Drug/mAb Ratio: 4.6
Example 110
##STR00801##
[4514] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.9 mg/ml
Drug/mAb Ratio: 4.2
Example 111
##STR00802##
[4516] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.89 mg/ml
Drug/mAb-Ratio: 2.7
Example 112
##STR00803##
[4518] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.73 mg/ml
Drug/mAb-Ratio: 2.3
Example 113
##STR00804##
[4520] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.71 mg/ml
Drug/mAb-Ratio: 3.3
Example 114
##STR00805##
[4522] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.47 mg/ml
Drug/mAb Ratio: 3.9
Example 115
[4523]
N-(6-{[(5S)-5-Amino-5-carboxypentyl]amino}-6-oxohexyl)-N-methyl-L-v-
alyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-3-(1H-indol-3-yl)-1-(1,2-ox-
azinan-2-yl)-1-oxopropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrol-
idin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide
trifluoroacetate
##STR00806##
[4524] 15.5 mg (15 .mu.mol) of Intermediate 210 were taken up in 5
ml of DMF and admixed with 4.4 mg (18 .mu.mol) of
N.sup.2-(tert-butoxycarbonyl)-L-lysine and also 7.7 .mu.L (44
.mu.mol of N,N-diisopropylethylamine. The reaction mixture was
stirred at RT overnight and then concentrated under reduced
pressure. The residue was subsequently purified by preparative
HPLC. This gave 14 mg (81% of theory) of the protected intermediate
of the title compound, which was subsequently taken up in 1 ml of
dichloromethane and deprotected with 1 ml of trifluoroacetic 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.
[4525] HPLC (Method 12): R.sub.t=1.8 min;
[4526] LC-MS (Method 1): R.sub.t=0.79 min; MS (ESIpos): m/z=1083
(M+H).sup.+.
Example 116
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
##STR00807##
[4528] 40 mg (40 .mu.mol) of Intermediate 227 were taken up in 5 ml
of DMF and admixed with 11.5 mg (40 .mu.mol) of
N.sup.2-[(benzyloxy)carbonyl]-L-lysine and also 13 .mu.L (80
.mu.mol) of N,N-diisopropylethylamine. The reaction mixture was
stirred at RT overnight, then concentrated under reduced pressure
and subsequently purified by preparative HPLC. This gave 32.5 mg
(70% of theory) of the protected intermediate of the title
compound.
[4529] 32.5 mg of this intermediate were dissolved in 10 ml of
methanol and, following addition of 2 mg of 10% palladium on
activated carbon, were hydrogenated under standard hydrogen
pressure at RT for 30 minutes. The catalyst was then removed by
filtration and the solvent was removed under reduced pressure.
Lyophilization of the residue from dioxane/water 1:1 gave 26 mg
(99% of theory) of the title compound.
[4530] HPLC (Method 12): R.sub.t=1.7 min;
[4531] LC-MS (Method 1): R.sub.t=0.76 min; MS (ESIpos): m/z=1014
(M+H).sup.+.
Example 117
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
##STR00808##
[4533] 3.5 mg (3 .mu.mol) of Intermediate 202 were taken up in 2 ml
of DMF and admixed with 0.8 mg (3 .mu.mol) of
N.sup.2-(tert-butoxycarbonyl)-L-lysine and also 1.6 .mu.L (10
.mu.mol) of N,N-diisopropylethylamine. The reaction mixture was
stirred at RT overnight and then concentrated under reduced
pressure. The residue was taken up in acetonitrile/water: (1:1),
brought to a pH of 2 with trifluoroacetic acid and then purified by
preparative HPLC. This gave 1 mg (25% of theory) of the protected
intermediate of the title compound, which was subsequently taken up
in 500 .mu.l of dichloromethane and deprotected with 500 .mu.l of
trifluoroacetic acid. The batch was concentrated and, following
lypophilization of the residue from acetonitrile/water (1:1), 1 mg
(89% of theory) of the title compound was obtained.
[4534] HPLC (Method 12): R.sub.t=1.9 min;
[4535] LC-MS (Method 1): R.sub.t=0.82 min; MS (ESIpos): m/z=1173
(M+H).sup.+.
Example 118
##STR00809##
[4537] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS, and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 0.89 mg/ml
Drug/mAb Ratio: 1.8
Example 119
##STR00810##
[4539] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
and the batch, following Sephadex purification, was concentrated by
ultracentrifugation and re-diluted.
Protein concentration: 0.57 mg/ml
Drug/mAb Ratio: 1.5
Example 120
##STR00811##
[4541] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
and the reaction mixture, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted with PBS.
Protein concentration: 1.39 mg/ml
Drug/mAb Ratio: 7.1
Example 121
##STR00812##
[4543] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
and the reaction mixture, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted with PBS.
Protein concentration: 1.54 mg/ml
Drug/mAb Ratio: 2.4
Example 122
##STR00813##
[4545] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS, and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.48 mg/ml
Drug/mAb Ratio: 2.4
Example 123
##STR00814##
[4547] Coupling here was carried out using 5 mg of anti-C4.4a B01-3
in PBS, and the batch, following Sephadex purification, was
concentrated by ultracentrifugation and re-diluted.
Protein concentration: 1.43 mg/ml
Drug/mAb-Ratio: 3.6
Example 124
Diastereomer 1
##STR00815##
[4549] Coupling here was carried out using Intermediate 247a and 5
mg of anti-C4.4a B01-3 in PBS, and the batch, following Sephadex
purification, was concentrated by ultracentrifugation and
re-diluted with PBS.
Protein concentration: 1.45 mg/ml
Drug/mAb Ratio: 3.8
Example 125
Diastereomer 2
##STR00816##
[4551] Coupling here was carried out using Intermediate 247a and 5
mg of anti-C4.4a B01-3 in PBS, and the batch, following Sephadex
purification, was concentrated by ultracentrifugation and
re-diluted with PBS.
Protein concentration: 1.42 mg/ml
Drug/mAb Ratio: 4.0
Example 126
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
##STR00817##
[4553] 8.6 mg (8 .mu.mol) of Intermediate 240 were taken up in 5 ml
of DMF and admixed with 4.0 mg (16 .mu.mol) of
N.sup.2-(tert-butoxycarbonyl)-L-lysine and also 2 .mu.L (16
.mu.mol) of N,N-diisopropylethylamine. The reaction mixture was
stirred at RT for 4 hours, then admixed again with the same amounts
of N.sup.2-(tert-butoxycarbonyl)-L-lysine and
N,N-diisopropylethylamine, and stirred at RT overnight. The
reaction mixture was subsequently concentrated under reduced
pressure. The residue was then purified by preparative HPLC. This
gave 7 mg (72% of theory) of the protected intermediate of the
title compound, which was subsequently taken up in 1 ml of
dichloromethane and deprotected with 0.5 ml of trifluoroacetic
acid. The reaction mixture was concentrated and the residue was
purified by preparative HPLC. Drying under a high vacuum gave 3.3
mg (47% of theory) of the title compound.
[4554] HPLC (Method 5): R.sub.t=1.5 min;
[4555] LC-MS (Method 1): R.sub.t=0.8 min; MS (ESIpos): m/z=1084
(M+H).sup.+.
Example 127
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
##STR00818##
[4557] 8 mg (8 .mu.mol) of Intermediate 242 were taken up in 3 ml
of DMF and admixed with 2.9 mg (12 .mu.mol) of
N.sup.2-(tert-butoxycarbonyl)-L-lysine and also 2.7 .mu.L (16
.mu.mol) of N,N-diisopropylethylamine. The reaction mixture was
stirred at RT overnight, then admixed again with the same amounts
of N.sup.2-(tert-butoxycarbonyl)-L-lysine and
N,N-diisopropylethylamine, and stirred at RT for a further 4 hours.
The reaction mixture was subsequently concentrated under reduced
pressure. The residue was then purified by preparative HPLC.
Lyophilization from acetonitrile/water gave 6.5 mg (72% of theory)
of the protected intermediate of the title compound, which was
subsequently taken up in 5 ml of dichloromethane and deprotected
with 0.75 ml of trifluoroacetic acid. The batch was concentrated,
and lyophilization of the residue from dioxane/water gave 5 mg (76%
of theory) of the title compound.
[4558] HPLC (Method 12): R.sub.t=1.7 min;
[4559] LC-MS (Method 1): R.sub.t=0.69 min; MS (ESIpos): m/z=1059
(M+H).sup.+.
Example 128
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
##STR00819##
[4561] 38 mg (41 .mu.mol) of Intermediate 248 were first converted
into the N-hydroxysuccinimide ester. 72 mg of the crude product
obtained were taken up in 5 ml of DMF and admixed with 24 mg (100
.mu.mol) of N.sup.2-(tert-butoxycarbonyl)-L-lysine and 23 .mu.L of
N,N-diisopropylethylamine. The reaction mixture was stirred at RT
overnight, and then admixed again with 16 mg of
N.sup.2-(tert-butoxycarbonyl)-L-lysine and 12 .mu.L of
N,N-diisopropylethylamine, and subsequently treated in an
ultrasound bath for a further 2 hours. The batch was then
concentrated under reduced pressure and the residue was purified by
preparative HPLC. Lyophilization from acetonitrile/water gave 20 mg
(50% of theory) of the protected intermediate of the title
compound.
[4562] 15 mg (12 .mu.mol) of this intermediate were subsequently
taken up in 3 ml of dichloromethane and admixed with 1 ml of
trifluoroacetic acid. After 40 minutes of stirring at RT, a further
1.5 ml of trifluoroacetic acid were added and the batch was treated
in an ultrasound bath for 1 hour. Thereafter the reaction mixture
was concentrated, and lyophilization of the residue from
dioxane/water gave 13 mg (90% of theory) of the title compound.
[4563] HPLC (Method 12): R.sub.t=1.5 min;
[4564] LC-MS (Method 1): R.sub.t=0.68 min; MS (ESIpos): m/z=990
(M+H).sup.+.
C: EVALUATION OF BIOLOGICAL ACTIVITY
[4565] The biological effect of the compounds of the invention was
demonstrated in the assays described below
C-1. Analysis of the Cytotoxic Effect of the ADCs Directed Against
C4.4a
[4566] The cytotoxic effect of the anti-C4.4a ADCs is analysed in
different cell lines: [4567] A549 (CCL-185, ATCC), transfected with
the sequence for the complete C4.4a receptor, [4568] A549, Mock
transfected [4569] A549 Wildtype (DSMZ, lot 11) [4570] NCI-H292,
endogenously C4.4a expressing lung cancer cell line (CRL-1848,
ATCC) [4571] SCC-4 endogenously C4.4a expressing squamous
epithelial carcinoma cell line (CRL-1624, ATCC) [4572] SCC-9
endogenously C4.4a expressing squamous epithelial carcinoma cell
line (CRL-1629, ATCC) [4573] HCT-116 endogenously C4.4a expressing
colonic carcinoma cell line (CCL-247, ATCC) [4574] HCT-116/VM46,
HCT-116 transfected with VM46 [4575] A431NS(CRL-2592, ATCC)
[4576] The cells are cultivated by a standard method, as indicated
in the American Tissue Type Collection (ATCC) for the respective
cell lines. For the procedure, the cells are detached using a
solution of trypsin (0.05%) and EDTA (0.02%) in PBS (Biochrom AG
#L2143), pelletized, resuspended in culture medium, counted and
seeded out into a 96-well culture plate with a white base (Costar
#3610) (2500 cells in 100 .mu.l/well) and incubated in an incubator
at 37.degree. C. with 5% carbon dioxide. After 24 hours, the
antibody-drug conjugates in 100 .mu.l of culture medium are applied
to the cells at concentrations of 10.sup.-7M to 10.sup.-11 M
(duplicate values), and were incubated in the incubator at
37.degree. C. with 5% carbon dioxide. After 72 hours, cell
viability is determined using the Cell Titer Glow Luminescent Cell
Viability Assay (Promega #G7573 and #G7571). For this purpose, 100
.mu.l of the substrate are added per cell batch, and the plates are
subsequently covered with aluminium foil, shaken at 180 rpm in a
plate shaker for 2 minutes, left to stand on the laboratory bench
for 8 minutes, and then measured using a Victor X2 (Perkin Elmer).
The substrate detects the ATP content of the living cells,
producing a luminescence signal whose extent is directly
proportional to the vitality of the cells. The data measured is
used for calculating the IC.sub.50 using the Graph Pad Prism
Laboratory software.
[4577] Table 3 lists the IC.sub.50 values.sup.1) of representative
working examples from this assay:
TABLE-US-00031 TABLE 3 IC.sub.50 IC.sub.50 [nM] [nM] A549: A549
Example C4.4a Mock 1 0.081 11.15 2 0.7 50 3 0.47 4.75 4 0.6 100 5
0.4 20 0.2 26 0.1 17 6 0.53 4.50 7 0.39 32 8 0.01 0.15 9 0.43 10 10
0.01 25 11 4 >100 12 0.58 6.36 13 0.7 14.9 14 0.1 65.5 15 0.030
9.53 16 3.8 21 17 0.62 4.19 18 0.4 >100 19 1.2 66.1 20 0.46 4.20
21 4.5 12.7 22 5 16 23 0.4 0.7 24 0.3 23 25 5.4 53 26 0.052 11.27
27 0.65 6.70 28 0.062 >100 29 0.02 2.5 30 0.1 71 31 0.32 9 32
0.035 6.19 33 0.037 ~30 34 35 0.3 20 36 0.08 >100 37 0.1 kH 38
0.03 50 39 0.04 1.5 40 0.6 50 41 0.4 2 42 0.1 14 43 0.062 6.33 44
0.044 6.93 45 0.058 4.01 46 0.062 7.74 47 0.066 9.11 48 0.061 6.78
49 0.076 100 50 0.02 0.02 51 0.044 44 52 0.04 45 53 0.046 26 54
0.074 >100 55 0.053 >100 56 0.037 60 57 0.3 1 58 0.04 >100
59 0.1 >100 60 0.04 >100 61 0.44 6.8 62 0.09 50 63 0.1 0.4 64
0.04 0.52 65 0.03 0.04 66 0.03 0.04 67 0.08 26 68 0.02 >100 69
0.17 0.27 70 0.06 7 80 3.0 >100 81 0.045 >100 82 0.06 >100
83 0.27 >100 84 0.13 >100 85 0.14 >100 86 0.17 >100 87
0.28 >100 88 1.1 >100 89 1.3 >100 90 >100 >100 91
0.15 kH 92 0.29 >100 93 0.04 >100 94 0.035 100 95 0.036
>100 96 0.018 >100 97 0.062 >100 98 0.06 >100 99 0.1 80
100 0.1 kH 101 0.3 kH 102 0.1 kH 103 0.2 30 104 3 kH 105 0.03 50
106 0.05 20 107 >100 kH 108 0.03 >100 109 1 >100 110 0.2
kH 112 0.27 >100 113 3 >100 114 0.05 >100 118 0.29 20 119
0.32 15 120 0.07 >100 121 0.03 7 122 0.04 >100 123 0.02
>100 124 0.04 >100 125 0.04 >100 .sup.1) The activity data
reported relate to the working examples described in the present
experimental section, with the drug/mAB ratios indicated. The
values may possibly deviate for different drug/mAB ratios.
C-2. Determination of the Effect on Tubulin Polymerization
[4578] Cancer cells are denatured cells which frequently lead to
the formation of tumours also as a result of increased cell
division. Microtubuli form the spindle fibres of the spindle
apparatus and are an essential constituent of the cell cycle. The
regulated construction and breakdown of microtubuli allows the
precise division of the chromosomes among the daughter cells, and
constitutes a continuously dynamic process. Disruption to this
dynamic process results in incorrect cell division and ultimately
in cell death. The increased cell division of cancer cells,
however, also makes them particularly sensitive towards spindle
fibre poisons, which constitute a fixed constituent of
chemotherapy. Spindle fibre poisons such as paclitaxel or
epothilone lead to a sharply increased polymerization rate of the
microtubuli, while vinca alkaloids or else monomethylauristatin E
(MMAE) lead to a sharply reduced polymerization rate of the
microtubuli. In both cases, the necessary dynamism of the cell
cycle is critically disrupted. The compounds investigated in the
context of the present invention result in a reduced polymerization
rate of the microtubuli. Tubulin polymerization was investigated
using the "Fluorescence-based Microtubule Polymerisation Assay Kit"
from Cytoskeleton (Denver, Colo., USA; order number: BK011). With
this assay, GTP is added to unpolymerized tubulin, allowing
polymerization to take place spontaneously. The assay is based on
the binding of the fluorophore 4',6-diamidino-2-phenylindole (DAPI)
to tubulin. Free and bound DAPI can be differentiated on the basis
of different emission spectra. Since DAPI exhibits a significantly
high affinity for polymerized tubulin in comparison to
non-polymerized tubulin, the tubulin polymerization can be followed
via the increase in the fluorescence of bound DAPI
fluorophores.
[4579] For the implementation of this assay, the compounds of the
invention, in solution in DMSO, were diluted from their initial
concentration of 10 mM to 1 .mu.M in water. In addition to the
buffer control, paclitaxel, with a polymerization-increasing
effect, and vinblastin, with a polymerization inhibiting effect,
were run additionally as assay controls. Measurement was carried
out using 96-well plates with a half base area. The kinetics of the
tubulin polymerization were monitored in a Fluorimeter at
37.degree. C. for 1 hour. The excitation wavelength was 355 nm, and
emission was monitored at 460 nm. For the region of linear increase
within the first 10 minutes, a calculation was made of the change
in fluorescence per minute (AF/min), which represents the
polymerization rate of the microtubuli. The potency of the test
substances was quantified on the basis of their respective
reduction of the polymerization rate.
[4580] The value for the inhibition of MMAF at a concentration of 1
.mu.M is set as being 100%.
[4581] Table 4 gives data for the inhibition of tubulin
polymerization by representative working examples.
TABLE-US-00032 TABLE 4 Concentration Tubulin polymerization in the
of presence of toxophore in [%]. Working toxophore Tubulin
polymerization rate at example [.mu.M] 1 .mu.M MMAF set at 100%
MMAF 1 100 MMAF 10 34 MMAF 100 0 115 1 45 115 10 1 116 1 80 116 10
14 117 1 60 117 10 0 71 1 88 71 10 25 72 1 109 72 10 27 73 1 120 74
1 117 74 10 64 75 1 107 75 10 25 76 1 121 76 10 35 77 1 111 77 10
45 78 1 110 117 1 78 117 10 24 126 1 102 126 10 31 127 1 88 127 10
21 128 1 90 128 10 17
[4582] The MMAF toxophore and the working examples inhibit tubulin
polymerization as a function of their concentration. At 100 .mu.M
MMAF, the tubulin polymerization is inhibited completely. Working
Example 115 inhibits the tubulin polymerization rate at 1 .mu.M to
45% of the value measured for 1 .mu.M MMAF.
C-3. In Vitro Tests for Determining Cell Permeability
[4583] The cell permeability of a substance can be investigated by
means of in vitro testing in a flux assay using Caco-2 cells [M. D.
Troutman and D. R. Thakker, Pharm. Res. 20 (8), 1210-1224 (2003)].
For this purpose, the cells were cultured for 15-16 days on 24-well
filter plates. For the determination of permeation, the respective
working example was applied in a HEPES buffer to the cells either
apically (A) or basally (B) and incubated for 2 hours. After 0
hours and after 2 hours, samples were taken from the cis and trans
compartments. The samples were separated by HPLC (Agilent 1200,
Boblingen, Germany) using reverse phase columns. The HPLC system
was coupled via a Turbo Ion Spray Interface to a Triple Quadropol
mass spectrometer API 4000 (Applied Biosystems Applera, Darmstadt,
Germany). The permeability was evaluated on the basis of a
P.sub.app value, which was calculated using the formula published
by Schwab et al. [D. Schwab et al., J. Med. Chem. 46, 1716-1725
(2003)].
[4584] Of critical importance for toxophores which are released
intracellularly is the permeability from B to A [P.sub.app (B-A)]:
the lower this permeability, the longer the residence time of the
working example in the cell following intracellular release, and
hence also the longer the time available for interaction with the
biochemical target (in this case: tubulin).
[4585] Table 5 below sets out permeability data for representative
working examples from this assay:
TABLE-US-00033 TABLE 5 Working P.sub.app (B-A) example [nm/s] 71 2
72 1.6 73 2.5 74 5 75 1 77 7 115 2 116 1 126 1.8 127 1.5
[4586] The working examples exhibit a low permeability from B to A
[P.sub.app (B-A) and therefore have a long residence time in the
CaCo-2 cells. In comparison, monomethylauristatin E (MMAE) and
monomethylauristatin F (MMAF) in this test exhibit a P.sub.app
(B-A) value of 73 nm/s, and therefore have a significantly shorter
residence time in the Caco-2 cells.
C-4. In Vitro Tests for Determining the Substrate Properties for
P-Glycoprotein (P-gp)
[4587] Many tumour cells express transporter proteins for drugs,
and this frequently accompanies the development of resistance
towards cytostatics. Substances which are not substrates of such
transporter proteins, such as P-glycoprotein (P-gp) or BCRP, for
example, could therefore exhibit an improved activity profile.
[4588] The substrate properties of a substance for P-gp (ABCB1)
were determined by means of a flux assay using LLC-PK1 cells which
overexpress P-gp (L-MDR1 cells) [A. H. Schinkel et al., J. Clin.
Invest. 96, 1698-1705 (1995)]. For this purpose, the LLC-PK1 cells
or L-MDR1 cells were cultured on 96-well filter plates for 3-4
days. For determination of the permeation, the respective test
substance, alone or in the presence of an inhibitor (such as
Ivermectin or Verapamil, for example), was applied in a HEPES
buffer to the cells either apically (A) or basally (B) and
incubated for 2 hours. After 0 hours and after 2 hours, samples
were taken from the cis and trans compartments. The samples were
separated by HPLC using reverse phase columns. The HPLC system was
coupled via a Turbo Ion Spray Interface to a Triple Quadropol mass
spectrometer API 3000 (Applied Biosystems Applera, Darmstadt,
Germany). The permeability was evaluated on the basis of a
P.sub.app value which was calculated using the formula published by
Schwab et al. [D. Schwab et al., J. Med. Chem. 46, 1716-1725
(2003)].
[4589] Of critical importance for toxophores which are released
intracellularly is the permeability from B to A [P.sub.app (B-A)]:
the lower this permeability, the longer the residence time of the
working example in the cell following intracellular release, and
hence also the longer the time available for interaction with the
biochemical target (in this case: tubulin).
[4590] Table 6 below lists permeability data for representative
working examples from this assay, which was carried out in L-MDR1
cells:
TABLE-US-00034 TABLE 6 Working P.sub.app (B-A) example [nm/s] 71 3
72 3.6 73 2.1 74 3.6 75 4 77 2 115 6 116 4
[4591] The working examples exhibit a low permeability from B to A
[P.sub.app (B-A) and therefore have a long residence time in the
L-MDR1 cells.
C-5. Activity Test in Vivo
[4592] The activity of the conjugates of the invention was tested
in vivo by means, for example, of xenograft models. The skilled
person knows of methods in the prior art for testing the activity
of a conjugate of the invention (see, for example, WO 2005/081711;
Polson et al., Cancer Res. 2009 Mar. 15; 69(6):2358-64). For this
purpose, for example, rodents (e.g. mice) were implanted with a
tumour cell line which expresses the target molecule of the binder.
These tumour-bearing rodents were subsequently administered either
a conjugate of the invention or a control antibody conjugate, or
isotonic salt solution. Administration took place singularly or
more often. Tumour growth was determined twice weekly with the aid
of a sliding calliper. After tumour growth for several weeks, the
tumour size of conjugate-treated animals was compared with that of
the control group. The conjugate-treated animals showed a
significantly smaller tumour size.
C-5a. Testing of ADCs in Experimental Tumours in the Mouse
[4593] The predictive force of mice xenograft tumour models,
relative to the clinical situation in the case of immunotoxin
therapies, is often limited, on the one hand by the deficient
cross-reactivity of the therapeutic antibodies with the murine
species, and on the other hand by the incidence of anti-drug
antibodies (ADAs) in the human body on administration of murine or
chimeric antibodies. In order to utilize the full potential of the
specific C4.4a expression for cancer therapy, for an
immunoconjugate approach, for example, there is a need for human
antibodies which are of high affinity, are selective and exhibit
species cross-reactivity, of the kind employed preferably in
accordance with the invention. With such antibodies, mice xenograft
tumour models yield meaningful findings relative to the clinical
situation.
[4594] Human tumour cells which express C4.4a are inoculated
subcutaneously into the flank of immunosuppressed mice, such as
nude mice or SCID mice. 1-10 million cells are detached from the
cell culture, centrifuged and resuspended with 100 .mu.l of medium
or 50% medium/50% Matrigel. The cell suspension is injected beneath
the skin of the mouse.
[4595] Within a few days, a tumour grows. Treatment begins no
earlier than after tumour establishment with a tumour size of 25
mm.sup.2.
[4596] Treatment with ADCs takes place by the intravenous route
into the caudal vein of the mouse. The ADC is dissolved in PBS and
is administered with a volume of 10 ml/kg.
[4597] The treatment scheme is governed by the pharmacokinetics of
the antibody. As a standard, the treatment takes place three times
following every fourth day. Treatment, however, may also be
continued further, or a second cycle with three days of treatment
may follow at a later point in time.
[4598] As a standard basis, 8 animals are used per treatment group.
This number may be higher if particularly strong fluctuations in
tumour growth or after treatment are anticipated. As well as the
groups which receive the active substances, one group, as a control
group, is treated only with the buffer, in accordance with the same
scheme.
[4599] In the course of the experiment, the area of the tumour is
measured regularly using a sliding calliper in two dimensions
(length/width).
[4600] At the end of the experiment, the tumours are removed and
weighed. The ratio of the average tumour weights for the therapy
group (T) to the control group (C) is expressed as T/C. Where
control groups and treatment groups are ended at different times,
the T/C value is calculated on the basis of the tumour areas of the
last joint measurement of all the treatment groups and control
groups.
[4601] 1 million SCC-4 cells are inoculated subcutaneously into the
flank of female NMRI nude mice.
[4602] Intravenous treatment with the ADCs is commenced at an
average tumour size of 30-35 mm.sup.2. When the control groups have
reached the maximum allowed size, the experiment is ended and the
tumours are removed and weighed. All of the ADCs tested that target
C4.4a have inhibited tumour growth in a dose-dependent manner. At a
dose of 30 mg/kg, Example 54, Example 49, Example 51 and Example 53
each reached a T/C of <0.1. Significant anti-tumour activity in
comparison to the control was achieved for Examples 49, 52, 53, 54
and 56 at a dose of down to 15 mg/kg, achieving T/C values of
.ltoreq.0.29.
[4603] 1 million NCI-H292 cells were inoculated subcutaneously into
the flank of female NMRI nude mice.
[4604] Intravenous treatment with the ADCs is commenced at an
average tumour size of 30-35 mm.sup.2. Control groups and treatment
groups are each ended when the maximum allowable tumour size is
reached. In this way, differences in the further growth of tumours
after the end of treatment may contribute to further
characterization of the ADCs. Consequently, the tumour areas at the
last joint point in time of measurement was employed for
determining the anti-tumour activity in comparison to the control
(T/C). In the NCI-H292 mouse model used, it is shown that all of
the ADCs tested reduce tumour growth dose-dependently in comparison
to the control. A significant anti-tumour effect was obtained for
Example 54 at a dose of down to 1.9 mg/kg, and for Example 49 at a
dose of down to 3.75 mg/kg. The minimum T/C values obtained in this
model are a T/C of 0.16 at 30 mg/kg for Example 54, a T/C of 0.17
at 30 mg/kg for Example 49, a T/C of 0.16 at 30 mg/kg for Example
53, a T/C of 0.17 at 15 mg/kg for Example 51, and a T/C of 0.19 at
3.75 mg/kg for Example 70. On comparative administration of the
ADCs with a constant dose of 7.5 mg/kg, it was possible to achieve
a T/C of 0.20 with each of Examples 49 and 54, a T/C of 0.27 with
Example 51, a T/C of 0.22 with Example 52, a T/C of 0.23 with
Example 53, a T/C of 0.24 with Example 55, a T/C of 0.21 with
Example 56 and a T/C of 0.17 with Example 70.
C-6. Pharmacokinetics in the A549 Tumour Model with
C4.4a-Transfected and Non-Transfected A549 Cells
[4605] Following intravenous administration of 7-30 mg/kg of
various ADCs, the plasma concentrations and tumour concentrations
of ADC and also of potential metabolites were measured, and the
pharmacokinetic parameters such as clearance (CL), area under the
curve (AUC) and half-life (t.sub.1/2) were calculated.
Analysis for Quantifying the Potential Metabolites
[4606] The measurement of the compounds in plasma and tumour took
place following precipitation of the proteins with methanol, by
means of high-pressure liquid chromatography (HPLC) coupled to a
tandem mass spectrometer (MS).
[4607] For the processing of 100 .mu.L of plasma, it was admixed
with 400 .mu.L of methanol and 10 .mu.L of 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 of supernatant were
transferred to an autosampler vial, which was made up with 250
.mu.L of ammonium acetate buffer (AAC, 10 mM, pH 6.8) and shaken
again.
[4608] For the processing of a tumour, it was admixed with 4 times
the amount of methanol. In a Tissuelyser II (Quiagen), the sample
was comminuted at 30 impacts per minute for 6 minutes and then
centrifuged off at 16 000 g for 5 minutes. 50 .mu.L of the
supernatant were transferred to an autosampler vial and made up
with 50 .mu.L of ammonium acetate buffer (10 mM, pH 6.8) and with 5
.mu.L of ISTD. After again being shaken, the tumour sample was
ready for measurement.
[4609] The measurement of both matrix samples took place, lastly,
on the HPLC-coupled, atmospheric pressure ionization/tandem mass
spectrometer by means of a Turbo Ion Spray Interface (TISP) on an
API4000 instrument from SCIEX.
[4610] HPLC/LC-MSMS (TISP) analysis ran on an HP1100 pump (Agilent)
with a Gemini column (5 .mu.m C18 110 A, 50.times.3 mm,
Phenomenex).
[4611] For calibration, plasma samples were admixed with
concentrations of 0.5-2000 .mu.g/L. The detection limit (LOQ) was
about 2 .mu.g/L. The linear range extended from 2 to 1000
.mu.g/L
[4612] For the calibration of the tumour samples, the supernatant
of untreated tumours was admixed with concentrations of 0.5-200
.mu.g/L. The detection limit was 5 .mu.g/L. The linear range
extended from to 200 .mu.g/L.
[4613] Quality controls for validity testing contained 5 and 50
.mu.g/L, with an additional 500 .mu.g/L in plasma. The
concentrations found for these samples deviated by up to 20% from
the intended value (data not attached).
D. WORKING EXAMPLES FOR PHARMACEUTICAL COMPOSITIONS
[4614] The compounds of the invention can be converted as follows
into pharmaceutical preparations:
i.v. Solution:
[4615] The compound of the invention is dissolved at a
concentration below the saturation solubility in a physiologically
tolerated solvent (e.g. isotonic saline solution, D-PBS, or a
formulation with glycine and sodium chloride in citrate buffer with
addition of polysorbate 80). The solution is subjected to sterile
filtration and dispensed into sterile and pyrogen-free injection
containers.
i.v. Solution:
[4616] The compounds of the invention can be converted into the
administration forms cited. This can be accomplished in a known way
by "mixing with" or "dissolving in" inert, non-toxic,
pharmaceutically suitable excipients (e.g. buffer substances,
stabilizers, solubilizers, preservatives). The following, for
example, may be present: amino acids (glycine, histidine,
methionine, arginine, lysine, leucine, isoleucine, threonine,
glutamic acid, phenylalanine and others), sugars and related
compounds (glucose, saccharose, mannitol, trehalose, sucrose,
mannose, lactose, sorbitol), glycerol, sodium salts, potassium,
ammonium salts and calcium salts (e.g. sodium chloride, potassium
chloride or disodiumhydrogenphosphate and many others),
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.
Lyophilizate for Subsequent Conversion into an i.v., s.c. or i.m.
Solution:
[4617] Alternatively the compounds of the invention may be
converted into a stable lyophilizate (possibly with the aid of
abovementioned excipients) and, before being administered,
reconstituted with a suitable solvent (e.g. injection-grade water,
isotonic saline solution) and 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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