U.S. patent application number 15/461569 was filed with the patent office on 2018-05-31 for novel conjugates of cc-1065 analogs and bifunctional linkers.
This patent application is currently assigned to SYNTARGA BV. The applicant listed for this patent is SYNTARGA BV. Invention is credited to Patrick Henry BEUSKER, Rudy Gerardus Elisabeth Coumans, Franciscus Marinus Hendrikus De Groot, Ronald Christiaan Elgersma, Johannes Albertus Frederikus Joosten, Wiro Michael Petrus Bernardus Menge, Henri Johannes Spijker.
Application Number | 20180147289 15/461569 |
Document ID | / |
Family ID | 44343209 |
Filed Date | 2018-05-31 |
United States Patent
Application |
20180147289 |
Kind Code |
A9 |
BEUSKER; Patrick Henry ; et
al. |
May 31, 2018 |
Novel Conjugates of CC-1065 Analogs and Bifunctional Linkers
Abstract
This invention relates to novel analogs of the DNA-alkylating
agent CC-1065 and to their conjugates. Furthermore this invention
concerns intermediates for the preparation of said agents and
conjugates. The conjugates are designed to release their (multiple)
payload after one or more activation steps and/or at a rate and
time span controlled by the conjugate in order to selectively
deliver and/or controllably release one or more of said DNA
alkylating agents. The agents, conjugates, and intermediates can be
used to treat an illness that is characterized by undesired (cell)
proliferation. As an example, the agents and the conjugates of this
invention may be used to treat a tumor.
Inventors: |
BEUSKER; Patrick Henry;
(Nijmegen, NL) ; Coumans; Rudy Gerardus Elisabeth;
(Nijmegen, NL) ; Elgersma; Ronald Christiaan;
(Nijmegen, NL) ; Menge; Wiro Michael Petrus
Bernardus; (Nijmegen, NL) ; Joosten; Johannes
Albertus Frederikus; (Nijmegen, NL) ; Spijker; Henri
Johannes; (Nijmegen, NL) ; De Groot; Franciscus
Marinus Hendrikus; (Nijmegen, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SYNTARGA BV |
Nijmegen |
|
NL |
|
|
Assignee: |
SYNTARGA BV
Nijmegen
NL
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20170333567 A1 |
November 23, 2017 |
|
|
Family ID: |
44343209 |
Appl. No.: |
15/461569 |
Filed: |
March 17, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13642847 |
Nov 27, 2012 |
9629924 |
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PCT/NL11/50278 |
Apr 21, 2011 |
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15461569 |
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61421824 |
Dec 10, 2010 |
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61326437 |
Apr 21, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/04 20180101;
A61K 47/65 20170801; A61K 47/60 20170801; C07D 471/04 20130101;
A61P 35/00 20180101; A61K 47/643 20170801; A61K 47/545 20170801;
A61K 39/44 20130101; A61K 38/385 20130101; A61K 47/64 20170801;
A61K 31/437 20130101; A61K 47/646 20170801; A61K 47/6851 20170801;
A61K 47/6803 20170801 |
International
Class: |
A61K 47/64 20060101
A61K047/64; C07D 471/04 20060101 C07D471/04; A61K 39/44 20060101
A61K039/44; A61K 38/38 20060101 A61K038/38; A61K 47/68 20060101
A61K047/68; A61K 31/437 20060101 A61K031/437 |
Claims
1. A compound of formula (III): ##STR00654## or a pharmaceutically
acceptable salt, hydrate, or solvate thereof, wherein V.sup.2 is
either absent or a functional moiety, wherein said functional
moiety is other than an antibody, an antibody fragment or a
derivative thereof; each L.sup.2 is independently absent or a
linking group linking V.sup.2 to L; each L is independently absent
or a linking group linking L.sup.2 to one or more V.sup.1 and/or Y;
each V.sup.1 is independently absent or a conditionally-cleavable
or conditionally-transfoiniable moiety, which can be cleaved or
transformed by a chemical, photochemical, physical, biological, or
enzymatic process; each Y is independently absent or a
self-eliminating spacer system which is comprised of 1 or more
self-elimination spacers and is linked to V.sup.1, optionally L,
and one or more Z; each p and q are numbers representing a degree
of branching and are each independently a positive integer; z is a
positive integer equal to or smaller than the total number of
attachment sites for Z; each Z is independently a compound of
formula (I), (II), (I'), or (II'): ##STR00655## or a
pharmaceutically acceptable salt, hydrate, or solvate thereof,
wherein DB is a DNA-binding moiety and is selected from the group
consisting of ##STR00656## R.sup.1 is a leaving group; R.sup.2,
R.sup.2', R.sup.3, R.sup.3', R.sup.4, R.sup.4', R.sup.12, and
R.sup.19 are independently selected from the group consisting of H,
OH, SH, NH.sub.2, N.sub.3, NO.sub.2, NO, CF.sub.3, CN,
C(O)NH.sub.2, C(O)H, C(O)OH, halogen, R.sup.a, SR.sup.a,
S(O)R.sup.a, S(O).sub.2R.sup.a, S(O)OR.sup.a, S(O).sub.2OR.sup.a,
OS(O)R.sup.a, OS(O).sub.2R.sup.a, OS(O)OR.sup.a,
OS(O).sub.2OR.sup.a, OR.sup.a, NHR.sup.a, N(R.sup.a)R.sup.b,
.sup.+N(R.sup.a)(R.sup.b)R.sup.c, P(O)(OR.sup.a)(OR.sup.b),
OP(O)(OR.sup.a)(OR.sup.b), SiR.sup.aR.sup.bR.sup.c, C(O)R.sup.a,
C(O)OR.sup.a, C(O)N(R.sup.a)R.sup.b, OC(O)R.sup.a, OC(O)OR.sup.a,
OC(O)N(R.sup.a)R.sup.b, N(R.sup.a)C(O)R.sup.b,
N(R.sup.a)C(O)OR.sup.b, and N(R.sup.a)C(O)N(R.sup.b)R.sup.c,
wherein R.sup.a, R.sup.b, and R.sup.c are independently selected
from H and optionally substituted C.sub.1-3 alkyl or C.sub.1-3
heteroalkyl, or R.sup.3+R.sup.3' and/or R.sup.4+R.sup.4' are
independently selected from .dbd.O, .dbd.S, .dbd.NOR.sup.18,
.dbd.C(R.sup.18)R.sup.18', and .dbd.NR.sup.18, R.sup.18 and
R.sup.18' being independently selected from H and optionally
substituted C.sub.1-3 alkyl, two or more of R.sup.2, R.sup.2',
R.sup.3, R.sup.3', R.sup.4, R.sup.4', and R.sup.12 optionally being
joined by one or more bonds to form one or more optionally
substituted carbocycles and/or heterocycles; X.sup.2 is selected
from the group consisting of O, C(R.sup.14)(R.sup.14'), and
NR.sup.14', wherein R.sup.14 and R.sup.14' have the same meaning as
defined for R.sup.7 and are independently selected, or R.sup.14'
and R.sup.7' are absent resulting in a double bond between the
atoms designated to bear R.sup.7' and R.sup.14'; R.sup.5, R.sup.5',
R.sup.6, R.sup.6', R.sup.7, and R.sup.7' are independently selected
from the group consisting of H, OH, SH, NH.sub.2, N.sub.3,
NO.sub.2, NO, CF.sub.3, CN, C(O)NH.sub.2, C(O)H, C(O)OH, halogen,
R.sup.e, SR.sup.e, S(O)R.sup.e, S(O).sub.2R.sup.e, S(O)OR.sup.e,
S(O).sub.2OR.sup.e, OS(O)R.sup.e, OS(O).sub.2R.sup.e,
OS(O)OR.sup.e, OS(O).sub.2OR.sup.e, OR.sup.e, NHR.sup.e,
N(R.sup.e)R.sup.f, .sup.+N(R.sup.e)(R.sup.f)R.sup.g,
P(O)(OR.sup.e)(OR.sup.f), OP(O)(OR.sup.e)(OR.sup.f),
SiR.sup.eR.sup.fR.sup.g, C(O)R.sup.e, C(O)OR.sup.e,
C(O)N(R.sup.e)R.sup.f, OC(O)R.sup.e, OC(O)OR.sup.e,
OC(O)N(R.sup.e)R.sup.f, N(R.sup.e)C(O)R.sup.f,
N(R.sup.e)C(O)OR.sup.f, N(R.sup.e)C(O)N(R.sup.f)R.sup.g, and a
water-soluble group, wherein R.sup.e, R.sup.f, and R.sup.g are
independently selected from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-15 alkyl, C.sub.1-15 heteroalkyl, C.sub.3-15 cycloalkyl,
C.sub.1-15 heterocycloalkyl, C.sub.5-15 aryl, or C.sub.1-15
heteroaryl, wherein ee is selected from 1 to 1000, X.sup.13 is
selected from O, S, and NR.sup.f1, and R.sup.f1 and R.sup.e1 are
independently selected from H and C.sub.1-3 alkyl, one or more of
the optional substituents in R.sup.e, R.sup.f, and/or R.sup.g
optionally being a water-soluble group, two or more of R.sup.e,
R.sup.f, and R.sup.g optionally being joined by one or more bonds
to form one or more optionally substituted carbocycles and/or
heterocycles, or R.sup.5+R.sup.5' and/or R.sup.6+R.sup.6' and/or
R.sup.7+R.sup.7' are independently selected from .dbd.O, .dbd.S,
.dbd.NOR.sup.e3, .dbd.C(R.sup.e3)R.sup.e4, and .dbd.NR.sup.e3,
R.sup.e3 and R.sup.e4 being independently selected from H and
optionally substituted C.sub.1-3 alkyl, or R.sup.5'+R.sup.6' and/or
R.sup.6'+R.sup.7' and/or R.sup.7'+R.sup.14' are absent, resulting
in a double bond between the atoms designated to bear R.sup.5' and
R.sup.6', and/or R.sup.6' and R.sup.7', and/or R.sup.7' and
R.sup.14', respectively, two or more of R.sup.5, R.sup.5', R.sup.6,
R.sup.6', R.sup.7, R.sup.7', R.sup.14, and R.sup.14' optionally
being joined by one or more bonds to form one or more optionally
substituted carbocycles and/or heterocycles; X.sup.1 is selected
from the group consisting of O, S, and NR.sup.13, wherein R.sup.13
is selected from H and optionally substituted C.sub.1-8 alkyl or
C.sub.1-8 heteroalkyl and not joined with any other substituent;
X.sup.3 is selected from the group consisting of O, S,
C(R.sup.15)R.sup.15',
--C(R.sup.15)(R.sup.15')--C(R.sup.15')(R.sup.15''')--,
--N(R.sup.15)--N(R.sup.15')--,
--C(R.sup.15)(R.sup.15')--N(R.sup.15'')--,
--N(R.sup.15'')--C(R.sup.15)(R.sup.15')--,
--C(R.sup.15)(R.sup.15')--O--, --O--C(R.sup.15)(R.sup.15')--,
--C(R.sup.15)(R.sup.15')--S--, --S--C(R.sup.15)(R.sup.15')--,
--C(R.sup.15).dbd.C(R.sup.15')--,
.dbd.C(R.sup.15)--C(R.sup.15').dbd., --N.dbd.C(R.sup.15')--,
.dbd.N--C(R.sup.15').dbd., --C(R.sup.15).dbd.N--,
.dbd.C(R.sup.15)--N.dbd., --N.dbd.N--, .dbd.N--N.dbd., CR.sup.15,
N, and NR.sup.15, or in DB1 and DB2 --X.sup.3-- represents
--X.sup.3a and X.sup.3b--, wherein X.sup.3a is connected to
X.sup.34, a double bond is present between X.sup.34 and X.sup.4,
and X.sup.3b is connected to X.sup.11, wherein X.sup.3a is
independently selected from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-8 alkyl, or C.sub.1-8 heteroalkyl and not joined with any
other substituent; X.sup.4 is selected from the group consisting of
O, S, C(R.sup.16)R.sup.16', NR.sup.16, N, and CR.sup.16; X.sup.5 is
selected from the group consisting of O, S, C(R.sup.17)R.sup.17',
NOR.sup.17, and NR.sup.17, wherein R.sup.17 and R.sup.17' are
independently selected from H and optionally substituted C.sub.1-8
alkyl or C.sub.1-8 heteroalkyl and not joined with any other
substituent; X.sup.6 is selected from the group consisting of
CR.sup.11, CR.sup.11(R.sup.11'), N, NR.sup.11, O, and S; X.sup.7 is
selected from the group consisting of CR.sup.8, CR.sup.8(R.sup.8'),
N, NR.sup.8, O, and S; X.sup.8 is selected from the group
consisting of CR.sup.9, CR.sup.9(R.sup.9'), N, NR.sup.9, O, and S;
X.sup.9 is selected from the group consisting of CR.sup.10,
CR.sup.10(R.sup.10'), N, NR.sup.10, O, and S; X.sup.10 is selected
from the group consisting of CR.sup.20, CR.sup.20(R.sup.20'), N,
NR.sup.20, O, and S; X.sup.11 is selected from the group consisting
of C, CR.sup.21, and N, or X.sup.11--X.sup.3b is selected from the
group consisting of CR.sup.21, CR.sup.21(R.sup.21'), N, NR.sup.21,
O, and S; X.sup.12 is selected from the group consisting of C,
CR.sup.22, and N; X.sup.6*, X.sup.7*, X.sup.8*, X.sup.9*,
X.sup.10*, and X.sup.11* have the same meaning as defined for
X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, and X.sup.11,
respectively, and are independently selected; X.sup.34 is selected
from the group consisting of C, CR.sup.23, and N; the ring B atom
of X.sup.11* in DB6 and DB7 is connected to a ring atom of ring A
such that ring A and ring B in DB6 and DB7 are directly connected
via a single bond; means that the indicated bond may be a single
bond or a non-cumulated, optionally delocalized, double bond;
R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10, R.sup.10',
R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15'', R.sup.15''',
R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21, R.sup.21',
R.sup.22, and R.sup.23 are each independently selected from the
group consisting of H, OH, SH, NH.sub.2, N.sub.3, NO.sub.2, NO,
CF.sub.3, CN, C(O)NH.sub.2, C(O)H, C(O)OH, halogen, R.sup.h,
SR.sup.h, S(O)R.sup.h, S(O).sub.2R.sup.h, S(O)OR.sup.h,
S(O).sub.2OR.sup.h, OS(O)R.sup.h, OS(O).sub.2R.sup.h,
OS(O)OR.sup.h, OS(O).sub.2OR.sup.h, OR.sup.h, NHR.sup.h,
N(R.sup.h)R.sup.i, .sup.+N(R.sup.h)(R.sup.i)R.sup.j,
P(O)(OR.sup.h)(OR.sup.i), OP(O)(OR.sup.h)(OR.sup.i),
SiR.sup.hR.sup.iR.sup.j, C(O)R.sup.h, C(O)OR.sup.h,
C(O)N(R.sup.h)R.sup.i, OC(O)R.sup.h, OC(O)OR.sup.h,
OC(O)N(R.sup.h)R.sup.i, N(R.sup.h)C(O)R.sup.i,
N(R.sup.h)C(O)OR.sup.i, N(R.sup.h)C(O)N(R.sup.i)R.sup.j, and a
water-soluble group, wherein R.sup.h, R.sup.i, and R.sup.j are
independently selected from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-15 alkyl, C.sub.1-15 heteroalkyl, C.sub.3-15 cycloalkyl,
C.sub.1-15 heterocycloalkyl, C.sub.5-15 aryl, or C.sub.1-15
heteroaryl, one or more of the optional substituents in R.sup.h,
R.sup.i, and/or R.sup.j optionally being a water-soluble group, two
or more of R.sup.h, R.sup.i, and R.sup.j optionally being joined by
one or more bonds to form one or more optionally substituted
carbocycles and/or heterocycles, or R.sup.8+R.sup.8' and/or
R.sup.9+R.sup.9' and/or R.sup.10+R.sup.10' and/or
R.sup.11+R.sup.11' and/or R.sup.15+R.sup.15' and/or
R.sup.15''+R.sup.15''' and/or R.sup.16+R.sup.16' and/or
R.sup.20+R.sup.20' and/or R.sup.21+R.sup.21' are independently
selected from .dbd.O, .dbd.S, .dbd.NOR.sup.h1,
.dbd.C(R.sup.h1)R.sup.h2, and .dbd.NR.sup.h1, R.sup.h1 and R.sup.h2
being independently selected from H and optionally substituted
C.sub.1-3 alkyl, two or more of R.sup.8, R.sup.8', R.sup.9,
R.sup.9', R.sup.10, R.sup.10', R.sup.11, R.sup.11', R.sup.15,
R.sup.15', R.sup.15'', R.sup.15''', R.sup.16, R.sup.16', R.sup.20,
R.sup.20', R.sup.21, R.sup.21', R.sup.22, and R.sup.23 optionally
being joined by one or more bonds to form one or more optionally
substituted carbocycles and/or heterocycles; R.sup.8b and R.sup.9b
are independently selected and have the same meaning as R.sup.8,
except that they may not be joined with any other substituent; one
of R.sup.4 and R.sup.4' and one of R.sup.16 and R.sup.16' may
optionally be joined by one or more bonds to form one or more
optionally substituted carbocycles and/or heterocycles; one of
R.sup.2, R.sup.2', R.sup.3, and R.sup.3' and one of R.sup.5 and
R.sup.5' may optionally be joined by one or more bonds to form one
or more optionally substituted carbocycles and/or heterocycles; a
and b are independently selected from 0 and 1; the DB moiety does
not comprise a DA1, DA2, DA1', or DA2' moiety; ring B in DB1 is a
heterocycle; if X.sup.3 in DB1 represents --X.sup.3a and X.sup.3b--
and ring B is aromatic, then two vicinal substituents on said ring
B are joined to form an optionally substituted carbocycle or
heterocycle fused to said ring B; if X.sup.3 in DB2 represents
--X.sup.3a and X.sup.3b-- and ring B is aromatic, then two vicinal
substituents on said ring B are joined to form an optionally
substituted heterocycle fused to said ring B, an optionally
substituted non-aromatic carbocycle fused to said ring B, or a
substituted aromatic carbocycle which is fused to said ring B and
to which at least one substituent is attached that contains a
hydroxy group, a primary amino group, or a secondary amino group,
the primary or secondary amine not being a ring atom in an aromatic
ring system nor being part of an amide; if ring A in DB2 is a
6-membered aromatic ring, then substituents on ring B are not
joined to form a ring fused to ring B; two vicinal substituents on
ring A in DB8 are joined to form an optionally substituted
carbocycle or heterocycle fused to said ring A to form a bicyclic
moiety to which no further rings are fused; ring A in DB9 together
with any rings fused to said ring A contains at least two ring
heteroatoms; and one or more of X.sup.1, R.sup.5, R.sup.5',
R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14, R.sup.14', R.sup.8,
R.sup.8', R.sup.9, R.sup.9', R.sup.10, R.sup.10', R.sup.11,
R.sup.11', R.sup.15, R.sup.15', R.sup.15'', R.sup.15''', R.sup.16,
R.sup.16', R.sup.20, R.sup.20', R.sup.21, R.sup.21', R.sup.22, and
R.sup.23 may optionally in addition be substituted by or be a
substituent of formula (V): ##STR00657## wherein each V.sup.2',
L.sup.2', L', V.sup.1', Y', Z', p', q', and z' has the same meaning
as defined for V.sup.2, L.sup.2, L, V.sup.1, Y, Z, p, q, and z,
respectively, and is independently selected, the one or more
substituents of formula (V) being independently connected via Y' to
one or more of X.sup.1, R.sup.5, R.sup.5', R.sup.6, R.sup.6',
R.sup.7, R.sup.7', R.sup.14, R.sup.14', R.sup.8, R.sup.8', R.sup.9,
R.sup.9', R.sup.10, R.sup.10', R.sup.11, R.sup.11', R.sup.15,
R.sup.15', R.sup.15'', R.sup.15''', R.sup.16, R.sup.16', R.sup.20,
R.sup.20', R.sup.21, R.sup.21', R.sup.22, R.sup.23, and/or to one
or more atoms bearing these R substituents; each Z is independently
connected to Y through either X.sup.1, an atom in R.sup.5,
R.sup.5', R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14,
R.sup.14', R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10,
R.sup.10', R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15'',
R.sup.15''', R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21,
R.sup.21', R.sup.22, R.sup.23, or an atom bearing any of these R
substituents; and at least V.sup.2 or a V.sup.1 is present.
2. The compound according to claim 1 wherein DB is DB1.
3. The compound according to claim 2, wherein DB is selected from
the group consisting of ##STR00658##
4. The compound according to claim 2, wherein DB is selected from
the group consisting of ##STR00659## ##STR00660## ##STR00661##
5. The compound according to claim 1, wherein Z is ##STR00662## or
an isomer of one of these, or a mixture of isomers.
6. The compound according to claim 1, wherein Z is ##STR00663##
##STR00664## ##STR00665## ##STR00666## ##STR00667## or an isomer of
one of these, or a mixture of isomers.
7. The compound according to claim 1, wherein X.sup.1 is O and Y is
connected to X.sup.1 via an .omega.-amino aminocarbonyl cyclization
spacer being part of Y.
8. The compound according to claim 7, wherein the .omega.-amino
aminocarbonyl cyclization spacer being part of Y is selected from
the group consisting of ##STR00668## ##STR00669## ##STR00670##
##STR00671## wherein R.sup.115 and R.sup.116 are independently
selected from the group consisting of H, methyl, ethyl, isopropyl,
tert-butyl, phenyl,
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
##STR00672## ##STR00673## ##STR00674## wherein dd is selected from
0 to 10, dd' is selected from 0 and 1, each R.sup.123 is
independently selected from H and methyl, ee is selected from 1 to
1000, X.sup.13 is selected from the group consisting of O, S, and
NR.sup.f1, and R.sup.f1 and R.sup.e1 are independently selected
from the group consisting of H and C.sub.1-3 alkyl, and wherein
R.sup.117, R.sup.118, R.sup.119, R.sup.120, R.sup.121, and
R.sup.122 are independently selected from the group consisting of
H, OH, SH, NH.sub.2, N.sub.3, NO.sub.2, NO, CF.sub.3, CN,
C(O)NH.sub.2, C(O)H, C(O)OH, halogen, R.sup.z, SR.sup.z,
S(O)R.sup.z, S(O).sub.2R.sup.z, S(O)OR.sup.Z, S(O).sub.2OR.sup.z,
OS(O)R.sup.z, OS(O).sub.2R.sup.z, OS(O)OR.sup.z,
OS(O).sub.2OR.sup.z, OR.sup.z, NHR.sup.z, N(R.sup.z)R.sup.z1,
.sup.+N(R.sup.z)(R.sup.z1)R.sup.z2, P(O)(OR.sup.z)(OR.sup.z1),
OP(O)(OR.sup.z)(OR.sup.z1), C(O)R.sup.z, C(O)OR.sup.z,
C(O)N(R.sup.z1)R.sup.z, OC(O)R.sup.z, OC(O)OR.sup.z,
OC(O)N(R.sup.z)R.sup.z1, N(R.sup.z1)C(O)R.sup.z,
N(R.sup.z1)C(O)OR.sup.z, and N(R.sup.z1)C(O)N(R.sup.z2)R.sup.z,
wherein R.sup.z, R.sup.z1, and R.sup.z2 are independently selected
from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-20 alkyl, C.sub.1-20 heteroalkyl, C.sub.3-20 cycloalkyl,
C.sub.1-20 heterocycloalkyl, C.sub.5-20 aryl, or C.sub.1-20
heteroaryl, wherein ee is selected from 1 to 1000, X.sup.13 is
selected from the group consisting of O, S, and NR.sup.f1, and
R.sup.f1 and R.sup.e1 are independently selected from H and
C.sub.1-3 alkyl.
9. The compound according to claim 8, wherein the .omega.-amino
aminocarbonyl cyclization spacer being part of Y is
##STR00675##
10. The compound according to claim 1, wherein the spacer system Y
is ##STR00676## wherein A is selected from the group consisting of
##STR00677##
11. The compound according to claim 1, wherein V.sup.1 contains a
substrate that can be cleaved by a proteolytic enzyme, plasmin, a
cathepsin, cathepsin B, .beta.-glucuronidase, a galactosidase,
prostate-specific antigen (PSA), urokinase-type plasminogen
activator (u-PA), a member of the family of matrix
metalloproteinases, or an enzyme localized by means of directed
enzyme prodrug therapy, or wherein V.sup.1 contains a moiety that
can be cleaved or transformed through reduction under hypoxic
conditions, through reduction by a nitroreductase, or through
oxidation.
12. The compound according to claim 1, wherein the chain of atoms
of L linking L.sup.2 to V.sup.1 consists of less than 19 atoms.
13. The compound according to claim 1, wherein L is selected from
the group consisting of ##STR00678##
14. The compound according to claim 1, wherein L.sup.2 is
##STR00679##
15-17. (canceled)
18. A compound of formula (IV): ##STR00680## or a pharmaceutically
acceptable salt, hydrate, or solvate thereof, wherein RM is a
reactive moiety, L is independently absent or a linking group
linking RM to one or more V.sup.1 and/or Y; each V.sup.1 is
independently absent or a conditionally-cleavable or
conditionally-transformable moiety, which can be cleaved or
transformed by a chemical, photochemical, physical, biological, or
enzymatic process; each Y is independently absent or a
self-eliminating spacer system which is comprised of 1 or more
self-elimination spacers and is linked to V.sup.1, optionally L,
and one or more Z; p is a number representing a degree of branching
and is a positive integer; z is a positive integer equal to or
smaller than the total number of attachment sites for Z; each Z is
independently a compound of formula (I), (II), (I'), or (II'):
##STR00681## or a pharmaceutically acceptable salt, hydrate, or
solvate thereof, wherein DB is a DNA-binding moiety and is selected
from the group consisting of ##STR00682## R.sup.1 is a leaving
group; R.sup.2, R.sup.2', R.sup.3, R.sup.3', R.sup.4, R.sup.4',
R.sup.12, and R.sup.19 are independently selected from the group
consisting of H, OH, SH, NH.sub.2, N.sub.3, NO.sub.2, NO, CF.sub.3,
CN, C(O)NH.sub.2, C(O)H, C(O)OH, halogen, R.sup.a, SR.sup.a,
S(O)R.sup.a, S(O).sub.2R.sup.a, S(O)OR.sup.a, S(O).sub.2OR.sup.a,
OS(O)R.sup.a, OS(O).sub.2R.sup.a, OS(O)OR.sup.a,
OS(O).sub.2OR.sup.a, OR.sup.a, NHR.sup.a, N(R.sup.a)R.sup.b,
.sup.+N(R.sup.a)(R.sup.b)R.sup.c, P(O)(OR.sup.a)(OR.sup.b),
OP(O)(OR.sup.a)(OR.sup.b), SiR.sup.aR.sup.bR.sup.c, C(O)R.sup.a,
C(O)OR.sup.a, C(O)N(R.sup.a)R.sup.b, OC(O)R.sup.a, OC(O)OR.sup.a,
OC(O)N(R.sup.a)R.sup.b, N(R.sup.a)C(O)R.sup.b,
N(R.sup.a)C(O)OR.sup.b, and N(R.sup.a)C(O)N(R.sup.b)R.sup.c,
wherein R.sup.a, R.sup.b, and R.sup.c are independently selected
from H and optionally substituted C.sub.1-3 alkyl or C.sub.1-3
heteroalkyl, or R.sup.3+R.sup.3' and/or R.sup.4+R.sup.4' are
independently selected from .dbd.O, .dbd.S, .dbd.NOR.sup.18,
.dbd.C(R.sup.18)R.sup.18', and .dbd.NR.sup.18, R.sup.18 and
R.sup.18' being independently selected from H and optionally
substituted C.sub.1-3 alkyl, two or more of R.sup.2, R.sup.2',
R.sup.3, R.sup.3', R.sup.4, R.sup.4', and R.sup.12 optionally being
joined by one or more bonds to form one or more optionally
substituted carbocycles and/or heterocycles; X.sup.2 is selected
from the group consisting of O, C(R.sup.14)(R.sup.14') and
NR.sup.14', wherein R.sup.14 and R.sup.14' have the same meaning as
defined for R.sup.7 and are independently selected, or R.sup.14'
and R.sup.7' are absent resulting in a double bond between the
atoms designated to bear R.sup.7' and R.sup.14'; R.sup.5, R.sup.5',
R.sup.6, R.sup.6', R.sup.7, and R.sup.7' are independently selected
from the group consisting of H, OH, SH, NH.sub.2, H.sub.3,
NO.sub.2, NO, CF.sub.3, CN, C(O)NH.sub.2, C(O)H, C(O)OH, halogen,
R.sup.e, SR.sup.e, S(O)R.sup.e, S(O).sub.2R.sup.e, S(O)OR.sup.e,
S(O).sub.2OR.sup.e, OS(O)R.sup.e, OS(O).sub.2R.sup.e,
OS(O)OR.sup.e, OS(O).sub.2OR.sup.e, OR.sup.e, NHR.sup.e,
N(R.sup.e)R.sup.f, .sup.+N(R.sup.e)(R.sup.f)R.sup.g,
P(O)(OR.sup.e)(OR.sup.f), OP(O)(OR.sup.e)(OR.sup.f),
SiR.sup.eR.sup.fR.sup.g, C(O)R.sup.e, C(O)OR.sup.e,
C(O)N(R.sup.e)R.sup.f, OC(O)R.sup.e, OC(O)OR.sup.e,
OC(O)N(R.sup.e)R.sup.f, N(R.sup.e)C(O)R.sup.f,
N(R.sup.e)C(O)OR.sup.f, N(R.sup.e)C(O)N(R.sup.f)R.sup.g, and a
water-soluble group, wherein R.sup.e, R.sup.f, and R.sup.g are
independently selected from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-15 alkyl, C.sub.1-15 heteroalkyl, C.sub.1-15 cycloalkyl,
C.sub.1-15 heterocycloalkyl, C.sub.5-15 aryl, or C.sub.1-15
heteroaryl, wherein ee is selected from 1 to 1000, X.sup.13 is
selected from O, S, and NR.sup.f1, and R.sup.f1 and R.sup.e1 are
independently selected from H and C.sub.1-3 alkyl, one or more of
the optional substituents in R.sup.e, R.sup.f, and/or R.sup.g
optionally being a water-soluble group, two or more of R.sup.e,
R.sup.f, and R.sup.g optionally being joined by one or more bonds
to form one or more optionally substituted carbocycles and/or
heterocycles, or R.sup.5+R.sup.5' and/or R.sup.6+R.sup.6' and/or
R.sup.7+R.sup.7' are independently selected from .dbd.O, .dbd.S,
.dbd.NOR.sup.e3, .dbd.C(R.sup.e3)R.sup.e4, and .dbd.NR.sup.e3,
R.sup.e3 and R.sup.e4 being independently selected from H and
optionally substituted C.sub.1-3 alkyl, or R.sup.5'+R.sup.6' and/or
R.sup.6'+R.sup.7' and/or R.sup.7'+R.sup.14' are absent, resulting
in a double bond between the atoms designated to bear R.sup.5' and
R.sup.6', and/or R.sup.6' and R.sup.7', and/or R.sup.7' and
R.sup.14', respectively, two or more of R.sup.5, R.sup.5', R.sup.6,
R.sup.6', R.sup.7, R.sup.7', R.sup.14, and R.sup.14' optionally
being joined by one or more bonds to form one or more optionally
substituted carbocycles and/or heterocycles; X.sup.1 is selected
from the group consisting of O, S, and NR.sup.13, wherein R.sup.13
is selected from H and optionally substituted C.sub.1-8 alkyl or
C.sub.1-8 heteroalkyl and not joined with any other substituent;
X.sup.3 is selected from the group consisting of O, S,
C(R.sup.15)R.sup.15',
--C(R.sup.15)(R.sup.15')--C(R.sup.15'')(R.sup.15''')--,
--N(R.sup.15)--N(R.sup.15')--,
--C(R.sup.15)(R.sup.15')--N(R.sup.15'')--,
--N(R.sup.15'')--C(R.sup.15)(R.sup.15')--,
--C(R.sup.15)(R.sup.15')--O--, --O--C(R.sup.15)(R.sup.15')--,
--C(R.sup.15)(R.sup.15')--S--, --S--C(R.sup.15)(R.sup.15')--,
--C(R.sup.15).dbd.C(R.sup.15')--,
.dbd.C(R.sup.15)--C(R.sup.15').dbd., --N.dbd.C(R.sup.15')--,
.dbd.N--C(R.sup.15').dbd., --C(R.sup.15).dbd.N--,
.dbd.C(R.sup.15)--N.dbd., --N.dbd.N--, .dbd.N--N.dbd., CR.sup.15,
N, and NR.sup.15, or in DB1 and DB2 --X.sup.3-- represents
--X.sup.3a and X.sup.3b--, wherein X.sup.3a is connected to
X.sup.34, a double bond is present between X.sup.34 and X.sup.4,
and X.sup.3b is connected to X.sup.11, wherein X.sup.3a is
independently selected from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-8 alkyl, or C.sub.1-8 heteroalkyl and not joined with any
other substituent; X.sup.4 is selected from the group consisting of
O, S, C(R.sup.16)R.sup.16', NR.sup.16, N, and CR.sup.16; X.sup.5 is
selected from the group consisting of O, S, C(R.sup.17)R.sup.17',
NOR.sup.17, and NR.sup.17, wherein R.sup.17 and R.sup.17' are
independently selected from H and optionally substituted C.sub.1-8
alkyl or C.sub.1-8 heteroalkyl and not joined with any other
substituent; X.sup.6 is selected from the group consisting of
CR.sup.11, CR.sup.11(R.sup.11'), N, NR.sup.11, O, and S; X.sup.7 is
selected from the group consisting of CR.sup.8, CR.sup.8(R.sup.8'),
N, NR.sup.8, O, and S; X.sup.8 is selected from the group
consisting of CR.sup.9, CR.sup.9(R.sup.9'), N, NR.sup.9, O, and S;
X.sup.9 is selected from the group consisting of CR.sup.10,
CR.sup.10(R.sup.10'), N, NR.sup.10, O, and S; X.sup.10 is selected
from the group consisting of CR.sup.20, CR.sup.20(R.sup.20'), N,
NR.sup.20, O, and S; X.sup.11 is selected from the group consisting
of C, CR.sup.21, and N, or X.sup.11-X.sup.3b is selected from the
group consisting of CR.sup.21, CR.sup.21(R.sup.21'), N, NR.sup.21,
O, and S; X.sup.12 is selected from the group consisting of C,
CR.sup.22, and N; X.sup.6*, X.sup.7*, X.sup.8*, X.sup.9*,
X.sup.10*, and X.sup.11* have the same meaning as defined for
X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, and X.sup.11,
respectively, and are independently selected; X.sup.34 is selected
from the group consisting of C, CR.sup.23, and N; the ring B atom
of X.sup.11* in DB6 and DB7 is connected to a ring atom of ring A
such that ring A and ring B in DB6 and DB7 are directly connected
via a single bond; means that the indicated bond may be a single
bond or a non-cumulated, optionally delocalized, double bond;
R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10, R.sup.10',
R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15'', R.sup.15''',
R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21, R.sup.21',
R.sup.22, and R.sup.23 are each independently selected from the
group consisting of H, OH, SH, NH.sub.2, N.sub.3, NO.sub.2, NO,
CF.sub.3, CN, C(O)NH.sub.2, C(O)H, C(O)OH, halogen, R.sup.h,
SR.sup.h, S(O)R.sup.h, S(O).sub.2R.sup.h, S(O)OR.sup.h,
S(O).sub.2OR.sup.h, OS(O)R.sup.h, OS(O).sub.2R.sup.h,
OS(O)OR.sup.h, OS(O).sub.2OR.sup.h, OR.sup.h, NHR.sup.h,
N(R.sup.h)R.sup.i, .sup.+N(R.sup.h)(R.sup.i)R.sup.j,
P(O)(OR.sup.h)(OR.sup.i), OP(O)(OR.sup.h)(OR.sup.i),
SiR.sup.hR.sup.iR.sup.j, C(O)R.sup.h, C(O)OR.sup.h,
C(O)N(R.sup.h)R.sup.i, OC(O)R.sup.h, OC(O)OR.sup.h,
OC(O)N(R.sup.h)R.sup.i, N(R.sup.h)C(O)R.sup.i,
N(R.sup.h)C(O)OR.sup.i, N(R.sup.h)C(O)N(R.sup.i)R.sup.j, and a
water-soluble group, wherein R.sup.h, R.sup.i, and R.sup.j are
independently selected from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-15 alkyl, C.sub.1-15 heteroalkyl, C.sub.3-15 cycloalkyl,
C.sub.1-15 heterocycloalkyl, C.sub.5-15 aryl, or C.sub.1-15
heteroaryl, one or more of the optional substituents in R.sup.h,
R.sup.i, and/or R.sup.j optionally being a water-soluble group, two
or more of R.sup.h, R.sup.i, and R.sup.j optionally being joined by
one or more bonds to form one or more optionally substituted
carbocycles and/or heterocycles, or R.sup.8+R.sup.8' and/or
R.sup.9+R.sup.9' and/or R.sup.10+R.sup.10' and/or
R.sup.11+R.sup.11' and/or R.sup.15+R.sup.15' and/or
R.sup.15''+R.sup.15''' and/or R.sup.16+R.sup.16' and/or
R.sup.20+R.sup.20' and/or R.sup.21+R.sup.21' are independently
selected from .dbd.O, .dbd.S, .dbd.NOR.sup.h1,
.dbd.C(R.sup.h1)R.sup.h2, and .dbd.NR.sup.h1, R.sup.h1 and R.sup.h2
being independently selected from H and optionally substituted
C.sub.1-3 alkyl, two or more of R.sup.8, R.sup.8', R.sup.9,
R.sup.9', R.sup.10, R.sup.10', R.sup.11, R.sup.11', R.sup.15,
R.sup.15', R.sup.15'', R.sup.15''', R.sup.16, R.sup.16', R.sup.20,
R.sup.20', R.sup.21, R.sup.21', R.sup.22, and R.sup.23 optionally
being joined by one or more bonds to form one or more optionally
substituted carbocycles and/or heterocycles; R.sup.8b and R.sup.9b
are independently selected and have the same meaning as R.sup.8,
except that they may not be joined with any other substituent; one
of R.sup.4 and R.sup.4' and one of R.sup.16 and R.sup.16' may
optionally be joined by one or more bonds to form one or more
optionally substituted carbocycles and/or heterocycles; one of
R.sup.2, R.sup.2', R.sup.3, and R.sup.3' and one of R.sup.5 and
R.sup.5' may optionally be joined by one or more bonds to form one
or more optionally substituted carbocycles and/or heterocycles; a
and b are independently selected from 0 and 1; the DB moiety does
not comprise a DA1, DA2, DA1', or DA2' moiety; ring B in DB1 is a
heterocycle; if X.sup.3 in DB1 represents --X.sup.3a and X.sup.3b--
and ring B is aromatic, then two vicinal substituents on said ring
B are joined to form an optionally substituted carbocycle or
heterocycle fused to said ring B; if X.sup.3 in DB2 represents
--X.sup.3a and X.sup.3b-- and ring B is aromatic, then two vicinal
substituents on said ring B are joined to form an optionally
substituted heterocycle fused to said ring B, an optionally
substituted non-aromatic carbocycle fused to said ring B, or a
substituted aromatic carbocycle which is fused to said ring B and
to which at least one substituent is attached that contains a
hydroxy group, a primary amino group, or a secondary amino group,
the primary or secondary amine not being a ring atom in an aromatic
ring system nor being part of an amide; if ring A in DB2 is a
6-membered aromatic ring, then substituents on ring B are not
joined to form a ring fused to ring B; two vicinal substituents on
ring A in DB8 are joined to form an optionally substituted
carbocycle or heterocycle fused to said ring A to form a bicyclic
moiety to which no further rings are fused; ring A in DB9 together
with any rings fused to said ring A contains at least two ring
heteroatoms; and one or more of X.sup.1, R.sup.5, R.sup.5',
R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14, R.sup.14', R.sup.8,
R.sup.8', R.sup.9, R.sup.9', R.sup.10, R.sup.10', R.sup.11,
R.sup.11', R.sup.15, R.sup.15', R.sup.15'', R.sup.15''', R.sup.16,
R.sup.16', R.sup.20, R.sup.20', R.sup.21, R.sup.21', R.sup.22, and
R.sup.23 may optionally in addition be substituted by or be a
substituent of formula (V): ##STR00683## wherein each V.sup.2' is
absent or a functional moiety, each L.sup.2' is independently
absent or a linking group linking V.sup.2' to L', q' is a number
representing a degree of branching and is a positive integer, L',
V.sup.1', Y', Z', p', and z' has the same meaning as defined for L,
V.sup.1, Y, Z, p, and z, respectively, and is independently
selected, the one or more substituents of foimula (V) being
independently connected via Y' to one or more of X.sup.1, R.sup.5,
R.sup.5', R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14,
R.sup.14', R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10,
R.sup.10', R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15'',
R.sup.15''', R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21,
R.sup.21', R.sup.22, R.sup.23, and/or to one or more atoms bearing
these R substituents; each Z is independently connected to Y
through either X.sup.1, an atom in R.sup.5, R.sup.5', R.sup.6,
R.sup.6', R.sup.7, R.sup.7', R.sup.14, R.sup.14', R.sup.8,
R.sup.8', R.sup.9, R.sup.9', R.sup.10, R.sup.10', R.sup.11,
R.sup.11', R.sup.15, R.sup.15', R.sup.15'', R.sup.15''', R.sup.16,
R.sup.16', R.sup.20, R.sup.20', R.sup.21, R.sup.21', R.sup.22,
R.sup.23, or an atom bearing any of these R substituents, V.sup.1,
Y, and Z may contain protecting groups, and the one or more
V.sup.2'-L.sup.2' moieties optionally present in Z as defined
hereinabove may optionally and independently be RM' instead, which
is a reactive moiety, and wherein, if there is more than 1 reactive
moiety in (IV), some or all reactive moieties are the same or
different.
19. The compound according to claim 18 wherein the reactive moiety
RM is ##STR00684## wherein X.sup.35 is selected from the group
consisting of halide, hydroxy, OC(O)R.sup.dd, and OC(O)OR.sup.dd,
or C(O)--X.sup.35 is an active ester, X.sup.36 is selected from the
group consisting of halide, mesyloxy, triflyloxy, and tosyloxy, and
R.sup.dd is selected from the group consisting of optionally
substituted C.sub.1-10 alkyl, C.sub.1-l0 heteroalkyl, C.sub.3-10
cycloalkyl, C.sub.1-10 heterocycloalkyl, C.sub.5-10 aryl, and
C.sub.1-10 heteroaryl.
20. The compound according to claim 18, which is ##STR00685## or an
isomer, or a mixture of isomers, wherein R.sup.5a is selected from
H, methyl and methoxy, AZ is ##STR00686## ##STR00687## ##STR00688##
##STR00689## ##STR00690## ##STR00691## V.sup.1 is selected from the
group consisting of valylcitrulline, valyllysine,
phenylalanyllysine, alanylphenylalanyllysine, and
D-alanylphenylalanyllysine, CL is ##STR00692## and L is selected
from the group consisting of ##STR00693##
21. A compound of formula (I) or (II): ##STR00694## or a
pharmaceutically acceptable salt, hydrate, or solvate thereof,
wherein DB is a DNA-binding moiety of formula: ##STR00695## R.sup.1
is a leaving group; R.sup.2, R.sup.2', R.sup.3, R.sup.3', R.sup.4,
R.sup.4', R.sup.12, and R.sup.19 are independently selected from
the group consisting of H, OH, SH, NH.sub.2, N.sub.3, NO.sub.2, NO,
CF.sub.3, CN, C(O)NH.sub.2, C(O)H, C(O)OH, halogen, R.sup.a,
SR.sup.a, S(O)R.sup.a, S(O).sub.2R.sup.a, S(O)OR.sup.a,
S(O).sub.2OR.sup.a, OS(O)R.sup.a, OS(O).sub.2R.sup.a,
OS(O)OR.sup.a, OS(O).sub.2OR.sup.a, OR.sup.a, NHR.sup.a,
N(R.sup.a)R.sup.b, .sup.+N(R.sup.a)(R.sup.b)R.sup.c,
P(O)(OR.sup.a)(OR.sup.b), OP(O)(OR.sup.a)(OR.sup.b),
SiR.sup.aR.sup.bR.sup.c, C(O)R.sup.a, C(O)OR.sup.a,
C(O)N(R.sup.a)R.sup.b, OC(O)R.sup.a, OC(O)OR.sup.a,
OC(O)N(R.sup.a)R.sup.b, N(R.sup.a)C(O)R.sup.b,
N(R.sup.a)C(O)OR.sup.b, and N(R.sup.a)C(O)N(R.sup.b)R.sup.c,
wherein R.sup.a, R.sup.b, and R.sup.c are independently selected
from H and optionally substituted C.sub.1-3 alkyl or C.sub.1-3
heteroalkyl, or R.sup.3+R.sup.3' and/or R.sup.4+R.sup.4' are
independently selected from .dbd.O, .dbd.S, .dbd.NOR.sup.18,
.dbd.C(R.sup.18)R.sup.18', and .dbd.NR.sup.18, R.sup.18 and
R.sup.18' being independently selected from H and optionally
substituted C.sub.1-3 alkyl, two or more of R.sup.2, R.sup.2',
R.sup.3, R.sup.3', R.sup.4, R.sup.4', and R.sup.12 optionally being
joined by one or more bonds to form one or more optionally
substituted carbocycles and/or heterocycles; X.sup.2 is selected
from the group consisting of O, C(R.sup.14)(R.sup.14'), and
NR.sup.14', wherein R.sup.14 and R.sup.14' have the same meaning as
defined for R.sup.7 and are independently selected, or R.sup.14'
and R.sup.7' are absent resulting in a double bond between the
atoms designated to bear R.sup.7' and R.sup.14'; R.sup.5, R.sup.5',
R.sup.6, R.sup.6', R.sup.7, and R.sup.7' are independently selected
from the group consisting of H, OH, SH, NH.sub.2, N.sub.3,
NO.sub.2, NO, CF.sub.3, CN, C(O)NH.sub.2, C(O)H, C(O)OH, halogen,
R.sup.e, SR.sup.e, S(O)R.sup.e, S(O).sub.2R.sup.e, S(O)OR.sup.e,
S(O).sub.2OR.sup.e, OS(O)R.sup.e, OS(O).sub.2R.sup.e,
OS(O)OR.sup.e, OS(O).sub.2OR.sup.e, OR.sup.e, NHR.sup.e,
N(R.sup.e)R.sup.f, .sup.+N(R.sup.e)(R.sup.f)R.sup.g,
P(O)(OR.sup.e)(OR.sup.f), OP(O)(OR.sup.e)(OR.sup.f),
SiR.sup.eR.sup.fR.sup.g, C(O)R.sup.e, C(O)OR.sup.e,
C(O)N(R.sup.e)R.sup.f, OC(O)R.sup.e, OC(O)OR.sup.e,
OC(O)N(R.sup.e)R.sup.f, N(R.sup.e)C(O)R.sup.f,
N(R.sup.e)C(O)OR.sup.f, N(R.sup.e)C(O)N(R.sup.f)R.sup.g, and a
water-soluble group, wherein R.sup.e, R.sup.f, and R.sup.g are
independently selected from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-15 alkyl, C.sub.1-15 heteroalkyl, C.sub.3-15 cycloalkyl,
C.sub.1-15 heterocycloalkyl, C.sub.5-15 aryl, or C.sub.1-15
heteroaryl, wherein ee is selected from 1 to 1000, X.sup.13 is
selected from O, S, and NR.sup.f1, and R.sup.f1 and R.sup.e1 are
independently selected from H and C.sub.1-3 alkyl, one or more of
the optional substituents in R.sup.e, R.sup.f, and/or R.sup.g
optionally being a water-soluble group, two or more of R.sup.e,
R.sup.f, and R.sup.g optionally being joined by one or more bonds
to form one or more optionally substituted carbocycles and/or
heterocycles, or R.sup.5+R.sup.5' and/or R.sup.6+R.sup.6' and/or
R.sup.7+R.sup.7' are independently selected from .dbd.O, .dbd.S,
.dbd.NOR.sup.e3, .dbd.C(R.sup.e3)R.sup.e4, and .dbd.NR.sup.e3,
R.sup.e3 and R.sup.e4 being independently selected from H and
optionally substituted C.sub.1-3 alkyl, or R.sup.5'+R.sup.6' and/or
R.sup.6'+R.sup.7' and/or R.sup.7'+R.sup.14' are absent, resulting
in a double bond between the atoms designated to bear R.sup.5' and
R.sup.6', and/or R.sup.6' and R.sup.7', and/or R.sup.7' and
R.sup.14', respectively, two or more of R.sup.5, R.sup.5', R.sup.6,
R.sup.6', R.sup.7, R.sup.7', R.sup.14, and R.sup.14' optionally
being joined by one or more bonds to form one or more optionally
substituted carbocycles and/or heterocycles; X.sup.1 is selected
from O, S, and NR.sup.13, wherein R.sup.13 is selected from H and
optionally substituted C.sub.1-8 alkyl or C.sub.1-8 heteroalkyl and
not joined with any other substituent; R.sup.8, R.sup.9, R.sup.10,
R.sup.11, R.sup.15, and R.sup.16 are each independently selected
from the group consisting of H, OH, SH, NH.sub.2, N.sub.3,
NO.sub.2, NO, CF.sub.3, CN, C(O)NH.sub.2, C(O)H, C(O)OH, halogen,
R.sup.h, SR.sup.h, S(O)R.sup.h, S(O).sub.2R.sup.h, S(O)OR.sup.h,
S(O).sub.2OR.sup.h, OS(O)R.sup.h, OS(O).sub.2R.sup.h,
OS(O)OR.sup.h, OS(O).sub.2OR.sup.h, OR.sup.h, NHR.sup.h,
N(R.sup.h)R.sup.i, .sup.+N(R.sup.h)(R.sup.i)R.sup.j,
P(O)(OR.sup.h)(OR.sup.i), OP(O)(OR.sup.h)(OR.sup.i),
SiR.sup.hR.sup.iR.sup.j, C(O)R.sup.h, C(O)OR.sup.h,
C(O)N(R.sup.h)R.sup.i, OC(O)R.sup.h, OC(O)OR.sup.h,
OC(O)N(R.sup.h)R.sup.i, N(R.sup.h)C(O)R.sup.i,
N(R.sup.h)C(O)OR.sup.i, N(R.sup.h)C(O)N(R.sup.i)R.sup.j, and a
water-soluble group, wherein R.sup.h, R.sup.i, and R.sup.j are
independently selected from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-15 alkyl, C.sub.1-15 heteroalkyl, C.sub.3-15 cycloalkyl,
C.sub.1-15 heterocycloalkyl, C.sub.5-15 aryl, or C.sub.1-15
heteroaryl, one or more of the optional substituents in R.sup.h,
R.sup.i, and/or R.sup.j optionally being a water-soluble group, two
or more of R.sup.h, R.sup.i, and R.sup.j optionally being joined by
one or more bonds to form one or more optionally substituted
carbocycles and/or heterocycles, two or more of R.sup.8, R.sup.9,
R.sup.10, R.sup.11, R.sup.15, and R.sup.16 optionally being joined
by one or more bonds to form one or more optionally substituted
carbocycles and/or heterocycles; one of R.sup.4 and R.sup.4' and
R.sup.16 may optionally be joined by one or more bonds to form one
or more optionally substituted carbocycles and/or heterocycles; one
of R.sup.2, R.sup.2', R.sup.3, and R.sup.3' and one of R.sup.5 and
R.sup.5' may optionally be joined by one or more bonds to form one
or more optionally substituted carbocycles and/or heterocycles; and
a and b are independently selected from 0 and 1.
22-23. (canceled)
24. A pharmaceutical composition comprising a compound according to
claim 1 and a pharmaceutically acceptable carrier.
25. A process for preparing a pharmaceutical composition comprising
the step of mixing a compound according to claim 1 with a
pharmaceutically acceptable carrier.
26. A method of treating or preventing a tumor in a mammal, wherein
the method comprises the administration of a pharmaceutical
composition according to claim 24 to the mammal in a
therapeutically effective dose.
27. A pharmaceutical composition comprising a compound according to
claim 18 and a pharmaceutically acceptable carrier.
28. A method of treating or preventing a tumor in a mammal, wherein
the method comprises the administration of a pharmaceutical
composition according to claim 27 to the mammal in a
therapeutically effective dose.
29. A pharmaceutical composition comprising a compound according to
claim 21 and a pharmaceutically acceptable carrier.
30. A method of treating or preventing a tumor in a mammal, wherein
the method comprises the administration of a pharmaceutical
composition according to claim 29 to the mammal in a
therapeutically effective dose.
31. A method of treating or preventing a tumor in a mammal, wherein
the method comprises the administration of a pharmaceutical
composition comprising a compound of formula (III): ##STR00696## or
a pharmaceutically acceptable salt, hydrate, or solvate thereof,
and a pharmaceutically acceptable carrier, wherein V.sup.2 is
antibody or an antibody fragment or a derivative thereof; each
L.sup.2 is independently absent or a linking group linking V.sup.2
to L; each L is independently absent or a linking group linking
L.sup.2 to one or more V.sup.1 and/or Y; each V.sup.1 is
independently absent or a conditionally-cleavable or
conditionally-transformable moiety, which can be cleaved or
transformed by a chemical, photochemical, physical, biological, or
enzymatic process; each Y is independently absent or a
self-eliminating spacer system which is comprised of 1 or more
self-elimination spacers and is linked to V.sup.1, optionally L,
and one or more Z; each p and q are numbers representing a degree
of branching and are each independently a positive integer; z is a
positive integer equal to or smaller than the total number of
attachment sites for Z; each Z is independently a compound of
formula (I), (II), (I'), or (II'): ##STR00697## or a
pharmaceutically acceptable salt, hydrate, or solvate thereof,
wherein DB is a DNA-binding moiety and is selected from the group
consisting of ##STR00698## R.sup.1 is a leaving group; R.sup.2,
R.sup.2', R.sup.3, R.sup.3', R.sup.4, R.sup.4', R.sup.12, and
R.sup.19 are independently selected from the group consisting of H,
OH, SH, NH.sub.2, N.sub.3, NO.sub.2, NO, CF.sub.3, CN,
C(O)NH.sub.2, C(O)H, C(O)OH, halogen, R.sup.a, SR.sup.a,
S(O)R.sup.a, S(O).sub.2R.sup.a, S(O)OR.sup.a, S(O).sub.2OR.sup.a,
OS(O)R.sup.a, OS(O).sub.2R.sup.a, OS(O)OR.sup.a,
OS(O).sub.2OR.sup.a, OR.sup.a, NHR.sup.a, N(R.sup.a)R.sup.b,
.sup.+N(R.sup.a)(R.sup.b)R.sup.c, P(O)(OR.sup.a)(OR.sup.b),
OP(O)(OR.sup.a)(OR.sup.b), SiR.sup.aR.sup.bR.sup.c, C(O)R.sup.a,
C(O)OR.sup.a, C(O)N(R.sup.a)R.sup.b, OC(O)R.sup.a, OC(O)OR.sup.a,
OC(O)N(R.sup.a)R.sup.b, N(R.sup.a)C(O)R.sup.b,
N(R.sup.a)C(O)OR.sup.b, and N(R.sup.a)C(O)N(R.sup.b)R.sup.c,
wherein R.sup.a, R.sup.b, and R.sup.c are independently selected
from H and optionally substituted C.sub.1-3 alkyl or C.sub.1-3
heteroalkyl, or R.sup.3+R.sup.3' and/or R.sup.4+R.sup.4' are
independently selected from .dbd.O, .dbd.S, .dbd.NOR.sup.18,
.dbd.C(R.sup.18)R.sup.18', and .dbd.NR.sup.18, R.sup.18 and
R.sup.18' being independently selected from H and optionally
substituted C.sub.1-3 alkyl, two or more of R.sup.2, R.sup.2',
R.sup.3, R.sup.3', R.sup.4, R.sup.4', and R.sup.12 optionally being
joined by one or more bonds to form one or more optionally
substituted carbocycles and/or heterocycles; X.sup.2 is selected
from the group consisting of O, C(R.sup.14)(R.sup.14'), and
NR.sup.14', wherein R.sup.14 and R.sup.14' have the same meaning as
defined for R.sup.7 and are independently selected, or R.sup.14'
and R.sup.7' are absent resulting in a double bond between the
atoms designated to bear R.sup.7' and R.sup.14'; R.sup.5, R.sup.5',
R.sup.6, R.sup.6', R.sup.7, and R.sup.7' are independently selected
from the group consisting of H, OH, SH, NH.sub.2, N.sub.3,
NO.sub.2, NO, CF.sub.3, CN, C(O)NH.sub.2, C(O)H, C(O)OH, halogen,
R.sup.e, SR.sup.e, S(O)R.sup.e, S(O).sub.2R.sup.e, S(O)OR.sup.e,
S(O).sub.2OR.sup.e, OS(O)R.sup.e, OS(O).sub.2R.sup.e,
OS(O)OR.sup.e, OS(O).sub.2OR.sup.e, OR.sup.e, NHR.sup.e,
N(R.sup.e)R.sup.f, .sup.+N(R.sup.e)(R.sup.f)R.sup.g,
P(O)(OR.sup.e)(OR.sup.f), OP(O)(OR.sup.e)(OR.sup.f),
SiR.sup.eR.sup.fR.sup.g, C(O)R.sup.e, C(O)OR.sup.e,
C(O)N(R.sup.e)R.sup.f, OC(O)R.sup.e, OC(O)OR.sup.e,
OC(O)N(R.sup.e)R.sup.f, N(R.sup.c)C(O)R.sup.f,
N(R.sup.e)C(O)OR.sup.f, N(R.sup.e)C(O)N(R.sup.f)R.sup.g, and a
water-soluble group, wherein R.sup.e, R.sup.f, and R.sup.g are
independently selected from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-15 alkyl, C.sub.1-15 heteroalkyl, C.sub.3-15 cycloalkyl,
C.sub.1-15 heterocycloalkyl, C.sub.5-15 aryl, or C.sub.1-15
heteroaryl, wherein ee is selected from 1 to 1000, X.sup.13 is
selected from O, S, and NR.sup.f1, and R.sup.fl and R.sup.e1 are
independently selected from H and C.sub.1-3 alkyl, one or more of
the optional substituents in R.sup.e, R.sup.f, and/or R.sup.g
optionally being a water-soluble group, two or more of R.sup.e,
R.sup.f, and R.sup.g optionally being joined by one or more bonds
to form one or more optionally substituted carbocycles and/or
heterocycles, or R.sup.5+R.sup.5' and/or R.sup.6+R.sup.6' and/or
R.sup.7+R.sup.7' are independently selected from .dbd.O, .dbd.S,
.dbd.NOR.sup.e3, .dbd.C(R.sup.e3)R.sup.e4, and .dbd.NR.sup.e3,
R.sup.e3 and R.sup.e4 being independently selected from H and
optionally substituted C.sub.1-3 alkyl, or R.sup.5'+R.sup.6' and/or
R.sup.6'+R.sup.7' and/or R.sup.7'+R.sup.14' are absent, resulting
in a double bond between the atoms designated to bear R.sup.5' and
R.sup.6', and/or R.sup.6' and R.sup.7', and/or R.sup.7' and
R.sup.14', respectively, two or more of R.sup.5, R.sup.5', R.sup.6,
R.sup.6', R.sup.7, R.sup.7', R.sup.14, and R.sup.14' optionally
being joined by one or more bonds to form one or more optionally
substituted carbocycles and/or heterocycles; X.sup.1 is selected
from the group consisting of O, S, and NR.sup.13, wherein R.sup.13
is selected from H and optionally substituted C.sub.1-8 alkyl or
C.sub.1-8 heteroalkyl and not joined with any other substituent;
X.sup.3 is selected from the group consisting of O, S,
C(R.sup.15)R.sup.15',
--C(R.sup.15)(R.sup.15')--C(R.sup.15'')(R.sup.15''')--,
--N(R.sup.15)--N(R.sup.15')--,
--C(R.sup.15)(R.sup.15')--N(R.sup.15'')--,
--N(R.sup.15'')--C(R.sup.15)(R.sup.15')--,
--C(R.sup.15)(R.sup.15')--O--, --O--C(R.sup.15)(R.sup.15')--,
--C(R.sup.15)(R.sup.15')--S--, --S--C(R.sup.15)(R.sup.15')--,
--C(R.sup.15).dbd.C(R.sup.15')--,
.dbd.C(R.sup.15)--C(R.sup.15').dbd., --N.dbd.C(R.sup.15')--,
.dbd.N--C(R.sup.15').dbd., --C(R.sup.15).dbd.N--,
.dbd.C(R.sup.15)--N.dbd., --N.dbd.N--, .dbd.N--N.dbd., CR.sup.15,
N, and NR.sup.15, or in DB1 and DB2 --X.sup.3-- represents
--X.sup.3a and X.sup.3b--, wherein X.sup.3a is connected to
X.sup.34, a double bond is present between X.sup.34 and X.sup.4,
and X.sup.3b is connected to X.sup.11, wherein X.sup.3a is
independently selected from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-8 alkyl, or C.sub.1-8 heteroalkyl and not joined with any
other substituent; X.sup.4 is selected from the group consisting of
O, S, C(R.sup.16)R.sup.16', NR.sup.16, N, and CR.sup.16; X.sup.5 is
selected from the group consisting of O, S, C(R.sup.17)R.sup.17',
NOR.sup.17, and NR.sup.17, wherein R.sup.17 and R.sup.17' are
independently selected from H and optionally substituted C.sub.1-8
alkyl or C.sub.1-8 heteroalkyl and not joined with any other
substituent; X.sup.6 is selected from the group consisting of
CR.sup.11, CR.sup.11(R.sup.11'), N, NR.sup.11, O, and S; X.sup.7 is
selected from the group consisting of CR.sup.8, CR.sup.8(R.sup.8'),
N, NR.sup.8, O, and S; X.sup.8 is selected from the group
consisting of CR.sup.9, CR.sup.9(R.sup.9'), N, NR.sup.9, O, and S;
X.sup.9 is selected from the group consisting of CR.sup.10,
CR.sup.10(R.sup.10'), N, NR.sup.10, O, and S; X.sup.10 is selected
from the group consisting of CR.sup.20, CR.sup.20(R.sup.20'), N,
NR.sup.20, O, and S; X.sup.11 is selected from the group consisting
of C, CR.sup.21, and N, or X.sup.11-X.sup.3b is selected from the
group consisting of CR.sup.21, CR.sup.21(R.sup.21'), N, NR.sup.21,
O, and S; X.sup.12 is selected from the group consisting of C,
CR.sup.22, and N; X.sup.6*, X.sup.7*, X.sup.8*, X.sup.9*,
X.sup.10*, and X.sup.11* have the same meaning as defined for
X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, and X.sup.11,
respectively, and are independently selected; X.sup.34 is selected
from the group consisting of C, CR.sup.23, and N; the ring B atom
of X.sup.11* in DB6 and DB7 is connected to a ring atom of ring A
such that ring A and ring B in DB6 and DB7 are directly connected
via a single bond; means that the indicated bond may be a single
bond or a non-cumulated, optionally delocalized, double bond;
R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10, R.sup.10',
R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15'', R.sup.15''',
R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21, R.sup.21',
R.sup.22, and R.sup.23 are each independently selected from the
group consisting of H, OH, SH, NH.sub.2, N.sub.3, NO.sub.2, NO,
CF.sub.3, CN, C(O)NH.sub.2, C(O)H, C(O)OH, halogen, R.sup.h,
SR.sup.h, S(O)R.sup.h, S(O).sub.2R.sup.h, S(O)OR.sup.h,
S(O).sub.2OR.sup.h, OS(O)R.sup.h, OS(O).sub.2R.sup.h,
OS(O)OR.sup.h, OS(O).sub.2OR.sup.h, OR.sup.h, NHR.sup.h,
N(R.sup.h)R.sup.i, .sup.+N(R.sup.h)(R.sup.iV)R.sup.j,
P(O)(OR.sup.h)(OR.sup.i), OP(O)(OR.sup.h)(OR.sup.i),
SiR.sup.hR.sup.iR.sup.j, C(O)R.sup.h, C(O)OR.sup.h,
C(O)N(R.sup.h)R.sup.i, OC(O)R.sup.h, OC(O)OR.sup.h,
OC(O)N(R.sup.h)R.sup.i, N(R.sup.h)C(O)R.sup.i,
N(R.sup.h)C(O)OR.sup.i, N(R.sup.h)C(O)N(R.sup.i)R.sup.j, and a
water-soluble group, wherein R.sup.h, R.sup.i, and R.sup.j are
independently selected from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-15 alkyl, C.sub.1-15 hetero alkyl, C.sub.3-15 cycloalkyl,
C.sub.1-15 heterocycloalkyl, C.sub.5-15 aryl, or C.sub.1-15
heteroaryl, one or more of the optional substituents in R.sup.h,
R.sup.i, and/or R.sup.j optionally being a water-soluble group, two
or more of R.sup.h, R.sup.i, and R.sup.j optionally being joined by
one or more bonds to form one or more optionally substituted
carbocycles and/or heterocycles, or R.sup.8+R.sup.8' and/or
R.sup.9+R.sup.9' and/or R.sup.10+R.sup.10' and/or
R.sup.11+R.sup.11' and/or R.sup.15+R.sup.15' and/or
R.sup.15''+R.sup.15''' and/or R.sup.16+R.sup.16' and/or
R.sup.20+R.sup.20' and/or R.sup.21+R.sup.21' are independently
selected from .dbd.O, .dbd.S, .dbd.NOR.sup.h1,
.dbd.C(R.sup.h1)R.sup.h2, and .dbd.NR.sup.h1, R.sup.h1 and R.sup.h2
being independently selected from H and optionally substituted
C.sub.1-3 alkyl, two or more of R.sup.8, R.sup.8', R.sup.9,
R.sup.9', R.sup.10, R.sup.10', R.sup.11, R.sup.11', R.sup.15,
R.sup.15', R.sup.15'', R.sup.15''', R.sup.16, R.sup.16', R.sup.20,
R.sup.20', R.sup.21, R.sup.21', R.sup.22, and R.sup.23 optionally
being joined by one or more bonds to form one or more optionally
substituted carbocycles and/or heterocycles; R.sup.8b and R.sup.9b
are independently selected and have the same meaning as R.sup.8,
except that they may not be joined with any other substituent; one
of R.sup.4 and R.sup.4' and one of R.sup.16 and R.sup.16' may
optionally be joined by one or more bonds to form one or more
optionally substituted carbocycles and/or heterocycles; one of
R.sup.2, R.sup.2', R.sup.3, and R.sup.3' and one of R.sup.5 and
R.sup.5' may optionally be joined by one or more bonds to form one
or more optionally substituted carbocycles and/or heterocycles; a
and b are independently selected from 0 and 1; the DB moiety does
not comprise a DA1, DA2, DA1', or DA2' moiety; ring B in DB1 is a
heterocycle; if X.sup.3 in DB1 represents --X.sup.3a and X.sup.3b--
and ring B is aromatic, then two vicinal substituents on said ring
B are joined to form an optionally substituted carbocycle or
heterocycle fused to said ring B; if X.sup.3 in DB2 represents
--X.sup.3a and X.sup.3b-- and ring B is aromatic, then two vicinal
substituents on said ring B are joined to form an optionally
substituted heterocycle fused to said ring B, an optionally
substituted non-aromatic carbocycle fused to said ring B, or a
substituted aromatic carbocycle which is fused to said ring B and
to which at least one substituent is attached that contains a
hydroxy group, a primary amino group, or a secondary amino group,
the primary or secondary amine not being a ring atom in an aromatic
ring system nor being part of an amide; if ring A in DB2 is a
6-membered aromatic ring, then substituents on ring B are not
joined to form a ring fused to ring B; two vicinal substituents on
ring A in DB8 are joined to form an optionally substituted
carbocycle or heterocycle fused to said ring A to form a bicyclic
moiety to which no further rings are fused; ring A in DB9 together
with any rings fused to said ring A contains at least two ring
heteroatoms; and one or more of X.sup.1, R.sup.5, R.sup.5',
R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14, R.sup.14', R.sup.8,
R.sup.8', R.sup.9, R.sup.9', R.sup.10, R.sup.10', R.sup.11,
R.sup.11', R.sup.15, R.sup.15', R.sup.15'', R.sup.15''', R.sup.16,
R.sup.16', R.sup.20, R.sup.20', R.sup.21, R.sup.21', R.sup.22, and
R.sup.23 may optionally in addition be substituted by or be a
substituent of formula (V): ##STR00699## wherein each V.sup.2',
L.sup.2', L', V.sup.1', Y', Z', q', and z' has the same meaning as
defined for V.sup.2, L.sup.2, L, V.sup.1, Y, Z, p, q, and z,
respectively, and is independently selected, the one or more
substituents of formula (V) being independently connected via Y' to
one or more of X.sup.1, R.sup.5, R.sup.5', R.sup.6, R.sup.6',
R.sup.7, R.sup.7', R.sup.14, R.sup.14', R.sup.8, R.sup.8', R.sup.9,
R.sup.9', R.sup.10, R.sup.10', R.sup.11, R.sup.11', R.sup.15,
R.sup.15', R.sup.15'', R.sup.15''', R.sup.16, R.sup.16', R.sup.20,
R.sup.20', R.sup.21, R.sup.21', R.sup.22, R.sup.23, and/or to one
or more atoms bearing these R substituents; each Z is independently
connected to Y through either X.sup.1, an atom in R.sup.5,
R.sup.5', R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14,
R.sup.14', R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10,
R.sup.10', R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15'',
R.sup.15''', R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21,
R.sup.21', R.sup.22, R.sup.23, or an atom bearing any of these R
substituents; and at least V.sup.2 or a V.sup.1 is present.
32. The method according to claim 31, wherein the compound has the
folumla ##STR00700## or an isomer, or a mixture of isomers, wherein
R.sup.5 is selected from the group consisting of H, methyl and
methoxy, R.sup.6, R.sup.7, and R.sup.14 are H, DB is DB1, V.sup.1
is selected from the group consisting of valylcitrulline,
valyllysine, phenylalanyllysine, alanylphenylalanyllysine, and
D-alanylphenylalanyllysine, f is 1 or 2, CL is selected from
##STR00701## L is selected from the group consisting of
##STR00702## q ranges from 1 to 4, and Ab is an antibody or a
fragment or derivative thereof.
33. The method according to claim 31, wherein the V.sup.2 moiety is
selected from the group consisting of an anti-CD19 antibody, an
anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody,
an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD74
antibody, an anti-CD138 antibody, an anti-CLL-1 antibody, an
anti-5T4 antibody, an anti-CD303 antibody, an anti-Tag 72 antibody,
an anti-Lewis A like carbohydrate antibody, an anti-EphB3 antibody,
an anti-HMW-MAA antibody, an anti-CD38 antibody, an anti-Cripto
antibody, an anti-EphA2 antibody, an anti-GPNMB antibody, an
anti-integrin antibody, an anti-MN antibody, an anti-Her2 antibody,
and an anti-PSMA antibody, or an epitope-binding functional
fragment or a derivative thereof.
34. A method of treating or preventing a tumor in a mammal, wherein
the method comprises the administration of a pharmaceutical
composition comprising a compound having the formula ##STR00703##
wherein R is (CH.sub.2CH.sub.2O).sub.2H,
(CH.sub.2).sub.3C(O)NH.sub.2, (CH.sub.2).sub.4C(NH.sub.2)COOH,
(CH.sub.2).sub.3COOH, (CH.sub.2).sub.3NHC(O)NH.sub.2, or
(CH.sub.2CH.sub.2O).sub.3H; q ranges from 1 to 4; and Ab is
selected from trastuzumab or an epitope-binding functional fragment
of trastuzumab, and a pharmaceutically acceptable carrier.
Description
FIELD OF THE INVENTION
[0001] This invention relates to novel analogs of the
DNA-alkylating agent CC-1065 and to their conjugates. Furthermore
this invention concerns intermediates for the preparation of said
agents and conjugates. The conjugates are designed to release their
(multiple) payload after one or more activation steps and/or at a
rate and time span controlled by the conjugate in order to
selectively deliver and/or controllably release one or more of said
DNA-alkylating agents. The agents, conjugates, and intermediates
can be used to treat an illness that is characterized by undesired
(cell) proliferation. As an example, the agents and the conjugates
of this invention may be used to treat a tumor.
BACKGROUND OF THE INVENTION
[0002] The duocarmycins, first isolated from a culture broth of
Streptomyces species, are members of a family of antitumor
antibiotics that also includes CC-1065. These extremely potent
agents allegedly derive their biological activity from an ability
to sequence-selectively alkylate DNA at the N3 of adenine in the
minor groove, which initiates a cascade of events that terminates
in an apoptotic cell death mechanism..sup.1
[0003] Although CC-1065 has shown very potent cytotoxicity, it
could not be used in the clinic because of serious delayed
hepatotoxicity..sup.2 This observation led to the development of
synthetic analogs of CC-1065 (see for CC-1065 derivatives for
example Aristoff et al., J. Org. Chem. 1992, 57, 6234; Boger et
al., Bioorg. Med. Chem. Lett. 1996, 6, 2207; Boger et al., Chem.
Rev. 1997, 97, 787; Milbank et al., J. Med. Chem. 1999, 42, 649;
Atwell et al., J. Med. Chem. 1999, 42, 3400; Wang et al., J. Med.
Chem. 2000, 43, 1541; Boger et al., Bioorg. Med. Chem. Lett. 2001,
11, 2021; Parrish et al., Bioorg. Med. Chem. 2003, 11, 3815;
Daniell et al., Bioorg. Med. Chem. Lett. 2005, 15, 177; Tichenor et
al., J. Am. Chem. Soc. 2006, 128, 15683; Purnell et al., Bioorg.
Med. Chem. 2006, 16, 5677; Bando and Sugiyama, Acc. Chem. Res.
2006, 39, 935; Tichenor et al., Nat. Prod. Rep. 2008, 25, 220;
MacMillan et al., J. Am. Chem. Soc. 2009, 131, 1187; Tietze et al.,
Anti-Cancer Agents Med. Chem. 2009, 9, 304; Gauss et al.,
Tetrahedron 2009, 65, 6591; Robertson et al., Bioorg. Med. Chem.
Lett. 2010, 20, 2722; Boyle et al., Bioorg. Med. Chem. Lett. 2010,
20, 1854; Chavda et al., Bioorg. Med. Chem. 2010, 18, 5016; EP
0154445; WO 88/04659; WO 90/02746; WO 97/12862; WO 97/32850; WO
97/45411; WO 98/52925; WO 99/19298; WO 01/83482; WO 02/067937; WO
02/067930; WO 02/068412; WO 03/022806; WO 2004/101767; WO
2006/043839; and WO 2007/051081), which generally showed to have
similar cytotoxicity, but reduced hepatotoxicity. Still, however,
these derivatives lack sufficient selectivity for tumor cells, as
the selectivity of these agents--and cytotoxic agents in
general--is for a certain part based on the difference in the rate
of proliferation of tumor cells and normal cells, and therefore
they also affect healthy cells that show a relatively high
proliferation rate. This typically leads to severe side effects.
Drug concentrations that would completely eradicate the tumor
cannot be reached because of dose-limiting side effects such as
gastrointestinal tract and bone marrow toxicity. In addition,
tumors can develop resistance against anticancer agents after
prolonged treatment. In modern drug development, targeting of
cytotoxic drugs to the tumor site can therefore be considered one
of the primary goals.
[0004] One promising approach to obtain increased selectivity for
tumor cells or tumor tissue is to exploit the existence of
tumor-associated antigens, receptors, and other receptive moieties,
which can serve as a target. Such a target may be upregulated or to
some degree be specifically present in tumor tissue or in closely
associated tissue, such as neovascular tissue, with respect to
other tissues in order to achieve efficient targeting. Many targets
have been identified and validated and several methods to identify
and validate targets have been developed..sup.3 By coupling a
ligand, e.g. an antibody or antibody fragment, for such a
tumor-associated antigen, receptor, or other receptive moiety to a
therapeutic agent, this agent can be selectively targeted to tumor
tissue.
[0005] Another promising approach to obtain selectivity for tumor
cells or tumor tissue is to exploit the existence of
tumor-associated enzymes. An enzyme that is mainly localized at the
tumor site can convert a pharmacologically inactive prodrug, which
consists of an enzyme substrate directly or indirectly linked to
the toxic drug, to the corresponding drug in the vicinity of or
inside the tumor. Via this concept a high concentration of toxic
anticancer agent can be selectively generated at the tumor site.
All tumor cells may be killed if the dose is sufficiently high,
which may decrease development of drug-resistant tumor cells.
[0006] Enzymes can also be transported to the vicinity of or inside
target cells or target tissue via for example antibody-directed
enzyme prodrug therapy (ADEPT).sup.4, polymer-directed enzyme
prodrug therapy (PDEPT) or macromolecular-directed enzyme prodrug
therapy (MDEPT).sup.5, virus-directed enzyme prodrug therapy
(VDEPT).sup.6, or gene-directed enzyme prodrug therapy
(GDEPT).sup.7. With ADEPT, for example, a non-toxic prodrug is
selectively converted into a cytotoxic compound at the surface of
target cells by an antibody-enzyme conjugate that has been
pretargeted to the surface of those cells.
[0007] Yet another promising approach to obtain selectivity for
tumor cells or tumor tissue is to exploit the enhanced permeability
and retention (EPR) effect. Through this EPR effect, macromolecules
passively accumulate in solid tumors as a consequence of the
disorganized pathology of angiogenic tumor vasculature with its
discontinuous endothelium, leading to hyperpermeability to large
macromolecules, and the lack of effective tumor lymphatic
drainage..sup.8 By coupling a therapeutic agent directly or
indirectly to a macromolecule, said agent can be selectively
targeted to tumor tissue.
[0008] Besides efficient targeting, other important criteria for
the successful application of targeted conjugates of cytotoxic
agents in tumor therapy are that the one or more agents are
released efficiently from the conjugate at the tumor site and that
the conjugate is non-cytotoxic or only very weakly cytotoxic,
whereas the cytotoxic agent itself exhibits highly potent
cytotoxicity. Ideally, this leads to the generation of cytotoxic
molecules only at the tumor site, which results in a greatly
increased therapeutic index with respect to the untargeted
cytotoxic agent. Another important criterion for a successful
targeted conjugate is that the conjugate must have suitable
pharmacological properties, such as sufficient stability in the
circulation, low aggregation tendency, and good water solubility.
Appropriate water-solubility and hydrophilicity of the drug and/or
the linker may contribute to improved pharmacological
properties.
[0009] Several conjugates of CC-1065 and derivatives have been
described (see for conjugates of CC-1065 derivatives for example
Suzawa et al., Bioorg. Med. Chem. 2000, 8, 2175; Jeffrey et al., J.
Med. Chem. 2005, 48, 1344; Wang et al., Bioorg. Med. Chem. 2006,
14, 7854; Tietze et al., Chem. Eur. J. 2007, 13, 4396; Tietze et
al., Chem. Eur. J. 2008, 14, 2811; Tietze et al., Chem Med Chem
2008, 3, 1946; Li et al., Tetrahedron Lett. 2009, 50, 2932; Tietze
et al., Angew. Chem. Int. Ed. 2010, 49, 7336; WO 91/16324; WO
94/04535; WO 95/31971; U.S. Pat. No. 5,475,092; U.S. Pat. No.
5,585,499; U.S. Pat. No. 5,646,298; WO 97/07097; WO 97/44000; U.S.
Pat. No. 5,739,350; WO 98/11101; WO 98/25898; U.S. Pat. No.
5,843,937; U.S. Pat. No. 5,846,545; WO 02/059122; WO 02/30894; WO
03/086318; WO 2005/103040; WO 2005/112919; WO 2006/002895; WO
2006/110476; WO 2007/038658; WO 2007/059404; WO 2008/083312; WO
2008/103693; WO 2009/026274; WO 2009/064908; WO 2009/073533; WO
2009/073524; WO 2009/073546; WO 2009/134977; and US 2009/0162372).
In these conjugates, one or more of the favorable properties
discussed above may be non-optimal.
[0010] Accordingly, there is still a clear need for conjugates of
CC-1065 derivatives that show a high therapeutic window, contain
CC-1065 derivatives that have potent cytotoxicity and favorable
pharmacological properties, and release the CC-1065 derivatives
efficiently.
SUMMARY OF THE INVENTION
[0011] The present invention fulfils the above-mentioned need with
a compound of formula (I) or (II):
##STR00001##
[0012] or a pharmaceutically acceptable salt, hydrate, or solvate
thereof, wherein
[0013] DB is a DNA-binding moiety and is selected from the group
consisting of
##STR00002##
[0014] R.sup.1 is a leaving group;
[0015] R.sup.2, R.sup.2', R.sup.3, R.sup.3', R.sup.4, R.sup.4',
R.sup.12, and R.sup.19 are independently selected from H, OH, SH,
NH.sub.2, N.sub.3, NO.sub.2, NO, CF.sub.3, CN, C(O)NH.sub.2, C(O)H,
C(O)OH, halogen, R.sup.a, SR.sup.a, S(O)R.sup.a, S(O).sub.2R.sup.a,
S(O)OR.sup.a, S(O).sub.2OR.sup.a, OS(O)R.sup.a, OS(O).sub.2R.sup.a,
OS(O)OR.sup.a, OS(O).sub.2OR.sup.a, OR.sup.a, NHR.sup.a,
N(R.sup.a)R.sup.b, .sup.+N(R.sup.a)(R.sup.b)R.sup.c,
P(O)(OR.sup.a)(OR.sup.b), OP(O)(OR.sup.a)(OR.sup.b),
SiR.sup.aR.sup.bR.sup.c, C(O)R.sup.a, C(O)OR.sup.a,
C(O)N(R.sup.a)R.sup.b, OC(O)R.sup.a, OC(O)OR.sup.a,
OC(O)N(R.sup.a)R.sup.b, N(R.sup.a)C(O)R.sup.b,
N(R.sup.a)C(O)OR.sup.b, and N(R.sup.a)C(O)N(R.sup.b)R.sup.e,
wherein [0016] R.sup.a, R.sup.b, and R.sup.c are independently
selected from H and optionally substituted C.sub.1-3 alkyl or
C.sub.1-3 heteroalkyl,
[0017] or R.sup.3+R.sup.3' and/or R.sup.4+R.sup.4' are
independently selected from .dbd.O, .dbd.S, .dbd.NOR.sup.18,
=C(R.sup.18)R.sup.18', and .dbd.NR.sup.18, R.sup.18 and R.sup.18'
being independently selected from H and optionally substituted
C.sub.1-3 alkyl, two or more of R.sup.2, R.sup.2', R.sup.3,
R.sup.3', R.sup.4, R.sup.4', and R.sup.12 optionally being joined
by one or more bonds to form one or more optionally substituted
carbocycles and/or heterocycles;
[0018] X.sup.2 is selected from O, C(R.sup.14)(R.sup.14'), and
NR.sup.14', wherein R.sup.14 and R.sup.14' have the same meaning as
defined for R.sup.7 and are independently selected, or R.sup.14'
and R.sup.7' are absent resulting in a double bond between the
atoms designated to bear R.sup.7' and R.sup.14';
[0019] R.sup.5, R.sup.5', R.sup.6, R.sup.6', R.sup.7, and R.sup.7'
are independently selected from H, OH, SH, NH.sub.2, N.sub.3,
NO.sub.2, NO, CF.sub.3, CN, C(O)NH.sub.2, C(O)H, C(O)OH, halogen,
R.sup.e, SR.sup.e, S(O)R.sup.e, S(O).sub.2R.sup.e, S(O)OR.sup.e,
S(O).sub.2OR.sup.e, OS(O)R.sup.e, OS(O).sub.2R.sup.e,
OS(O)OR.sup.e, OS(O).sub.2OR.sup.e, OR.sup.e, NHR.sup.e,
N(R.sup.e)R.sup.f, .sup.+N(R.sup.e)(R.sup.f)R.sup.g,
P(O)(OR.sup.e)(OR.sup.f), OP(O)(OR.sup.e)(OR.sup.f),
SiR.sup.eR.sup.fR.sup.g, C(O)R.sup.e, C(O)OR.sup.e,
C(O)N(R.sup.e)R.sup.f, OC(O)R.sup.e, OC(O)OR.sup.e,
OC(O)N(R.sup.e)R.sup.f, N(R.sup.e)C(O)R.sup.f,
N(R.sup.e)C(O)OR.sup.f, N(R.sup.e)C(O)N(R.sup.f)R.sup.g, and a
water-soluble group, wherein [0020] R.sup.e, R.sup.f, and R.sup.g
are independently selected from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1 ,
C.sub.1-15 alkyl, C.sub.1-15 heteroalkyl, C.sub.3-15 cycloalkyl,
C.sub.1-15 heterocycloalkyl, C.sub.5-15 aryl, or C.sub.1-15
heteroaryl, wherein ee is selected from 1 to 1000, X.sup.13 is
selected from O, S, and NR.sup.f1, and R.sup.f1 and R.sup.e1 are
independently selected from H and C.sub.1-3 alkyl, one or more of
the optional substituents in R.sup.e, R.sup.f, and/or R.sup.g
optionally being a water-soluble group, two or more of R.sup.e,
R.sup.f, and R.sup.g optionally being joined by one or more bonds
to form one or more optionally substituted carbocycles and/or
heterocycles,
[0021] or R.sup.5+R.sup.5' and/or R.sup.6+R.sup.6' and/or
R.sup.7+R.sup.7' are independently selected from .dbd.O, .dbd.S,
.dbd.NOR.sup.e3, .dbd.C(R.sup.e3)R.sup.e4, and .dbd.NR.sup.e3,
R.sup.e3 and R.sup.e4 being independently selected from H and
optionally substituted C.sub.1-3 alkyl, or R.sup.5'+R.sup.6' and/or
R.sup.6'+R.sup.7' and/or R.sup.7'+R.sup.14' are absent, resulting
in a double bond between the atoms designated to bear R.sup.5' and
R.sup.6', and/or R.sup.6' and R.sup.7', and/or R.sup.7' and
R.sup.14', respectively, two or more of R.sup.5, R.sup.5', R.sup.6,
R.sup.6', R.sup.7, R.sup.7', R.sup.14, and R.sup.14' optionally
being joined by one or more bonds to form one or more optionally
substituted carbocycles and/or heterocycles;
[0022] X.sup.1 is selected from O, S, and NR.sup.13, wherein
R.sup.13 is selected from H and optionally substituted C.sub.1-8
alkyl or C.sub.1-8 heteroalkyl and not joined with any other
substituent;
[0023] X.sup.3 is selected from O, S, C(R.sup.15)R.sup.15',
--C(R.sup.15)(R.sup.15')--C(R.sup.15'')(R.sup.15''')--,
--N(R.sup.15)--N(R.sup.15')--,
--C(R.sup.15)(R.sup.15')--N(R.sup.15'')--,
--N(R.sup.15'')--C(R.sup.15)(R.sup.15')',
--C(R.sup.15)(R.sup.15')--O--, --O--C(R.sup.15)(R.sup.15')--,
--C(R.sup.15)(R.sup.15')--S--, --S--C(R.sup.15)(R.sup.15')--,
--C(R.sup.15).dbd.C(R.sup.15')--,
.dbd.C(R.sup.15)--C(R.sup.15').dbd., --N.dbd.C(R.sup.15')--,
.dbd.N--C(R.sup.15').dbd., --C(R.sup.15).dbd.N--,
.dbd.C(R.sup.15)--N.dbd., --N.dbd.N--, .dbd.N--N.dbd., CR.sup.15,
N, and NR.sup.15, or in DB1 and DB2 --X.sup.3-- represents
--X.sup.3a and X.sup.3b--, wherein X.sup.3a is connected to
X.sup.34, a double bond is present between X.sup.34 and X.sup.4,
and X.sup.3b is connected to X.sup.11, wherein X.sup.3a is
independently selected from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-8 alkyl, or C.sub.1-8 heteroalkyl and not joined with any
other substituent;
[0024] X.sup.4 is selected from O, S, C(R.sup.16)R.sup.16',
NR.sup.16, N, and CR.sup.16;
[0025] X.sup.5 is selected from O, S, C(R.sup.17)R.sup.17',
NOR.sup.17 , and NR.sup.17, wherein R.sup.17 and R.sup.17' are
independently selected from H and optionally substituted C.sub.1-8
alkyl or C.sub.1-8 heteroalkyl and not joined with any other
substituent;
[0026] X.sup.6 is selected from CR.sup.11, CR.sup.11(R.sup.11') N,
NR.sup.11, O, and S;
[0027] X.sup.7 is selected from CR.sup.8, CR.sup.8(R.sup.8'), N,
NR.sup.B, O, and S;
[0028] X.sup.8 is selected from CR.sup.9, CR.sup.9(R.sup.9'), N,
NR.sup.9, O, and S;
[0029] X.sup.9 is selected from CR.sup.10, CR.sup.10(R.sup.10'), N,
NR.sup.10, O, and S;
[0030] X.sup.10 is selected from CR.sup.20, CR.sup.20(R.sup.20'),
N, NR.sup.20, O, and S;
[0031] X.sup.11 is selected from C, CR.sup.21, and N, or
X.sup.11--X.sup.3b is selected from CR.sup.21,
CR.sup.21(R.sup.21'), N, NR.sup.21, O, and S;
[0032] X.sup.12 is selected from C, CR.sup.22, and N;
[0033] X.sup.6*, X.sup.7*, X.sup.8*, X.sup.9*, X.sup.10*, and
X.sup.11* have the same meaning as defined for X.sup.6, X.sup.7,
X.sup.8, X.sup.9, X.sup.10, and X.sup.11, respectively, and are
independently selected;
[0034] X.sup.34 is selected from C, CR.sup.23, and N;
[0035] the ring B atom of X.sup.11* in DB6 and DB7 is connected to
a ring atom of ring A such that ring A and ring B in DB6 and DB7
are directly connected via a single bond;
[0036] means that the indicated bond may be a single bond or a
non-cumulated, optionally delocalized, double bond;
[0037] R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10, R.sup.10',
R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15'', R.sup.15''',
R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21, R.sup.21',
R.sup.22, and R.sup.23 are each independently selected from H, OH,
SH, NH.sub.2, N.sub.3, NO.sub.2, NO, CF.sub.3, CN, C(O)NH.sub.2,
C(O)H, C(O)OH, halogen, R.sup.h, SR.sup.h, S(O)R.sup.h,
S(O).sub.2R.sup.h, S(O)OR.sup.h, S(O).sub.2OR.sup.h, OS(O)R.sup.h,
OS(O).sub.2R.sup.h, OS(O)OR.sup.h, OS(O).sub.2OR.sup.h, OR.sup.h,
NHR.sup.h, N(R.sup.h)R.sup.i, .sup.+N(R.sup.h)(R.sup.i)R.sup.j,
P(O)(OR.sup.h)(OR.sup.i), OP(O)(OR.sup.h)(OR.sup.i),
SiR.sup.hR.sup.iR.sup.j, C(O)R.sup.h, C(O)OR.sup.h,
C(O)N(R.sup.h)R.sup.i, OC(O)R.sup.h, OC(O)OR.sup.h,
OC(O)N(R.sup.h)R.sup.i, N(R.sup.h)C(O)R.sup.i,
N(R.sup.h)C(O)OR.sup.i, N(R.sup.h)C(O)N(R.sup.i)R.sup.j, and a
water-soluble group, wherein [0038] R.sup.h, R.sup.i, and R.sup.j
are independently selected from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-15 alkyl, C.sub.1-15 heteroalkyl, C.sub.3-15 cycloalkyl,
C.sub.1-15 heterocycloalkyl, C.sub.5-15 aryl, or C.sub.1-15
heteroaryl, one or more of the optional substituents in R.sup.h,
R.sup.i, and/or R.sup.j optionally being a water-soluble group, two
or more of R.sup.h, R.sup.i, and R.sup.j optionally being joined by
one or more bonds to form one or more optionally substituted
carbocycles and/or heterocycles,
[0039] or R.sup.8+R.sup.8' and/or R.sup.9+R.sup.9' and/or
R.sup.10+R.sup.10' and/or R.sup.11+R.sup.11' and/or
R.sup.15+R.sup.15' and/or R.sup.15''+R.sup.15''' and/or
R.sup.16+R.sup.16' and/or R.sup.20+R.sup.20' and/or
R.sup.21+R.sup.21' are independently selected from .dbd.O, .dbd.S,
.dbd.NOR.sup.h1, .dbd.C(R.sup.h1)R.sup.h2, and .dbd.NR.sup.h1,
R.sup.h1 and R.sup.h2 being independently selected from H and
optionally substituted C.sub.1-3 alkyl, two or more of R.sup.8,
R.sup.8', R.sup.9, R.sup.9', R.sup.10, R.sup.10', R.sup.11,
R.sup.11', R.sup.15, R.sup.15', R.sup.15'', R.sup.15''', R.sup.16,
R.sup.16', R.sup.20, R.sup.20', R.sup.21, R.sup.21', R.sup.22, and
R.sup.23 optionally being joined by one or more bonds to form one
or more optionally substituted carbocycles and/or heterocycles;
[0040] R.sup.8b and R.sup.9b are independently selected and have
the same meaning as R.sup.8, except that they may not be joined
with any other substituent;
[0041] one of R.sup.4 and R.sup.4' and one of R.sup.16 and
R.sup.16' may optionally be joined by one or more bonds to form one
or more optionally substituted carbocycles and/or heterocycles;
[0042] one of R.sup.2, R.sup.2', R.sup.3, and R.sup.3' and one of
R.sup.5 and R.sup.5' may optionally be joined by one or more bonds
to form one or more optionally substituted carbocycles and/or
heterocycles; and
[0043] a and b are independently selected from 0 and 1.
[0044] In a further aspect, this invention relates to a compound of
formula (I') or (II'):
##STR00003##
[0045] which is formed through rearrangement of and concomitant
elimination of H-R.sup.1 from the corresponding compounds of
formulae (I) and (II), which are seco compounds (see FIG. 1). Said
cyclopropyl ring-containing analogs are believed to be active
species, allegedly being formed from compounds of formulae (I) and
(II) in vivo via said rearrangement.
[0046] In a more specific embodiment, this invention relates to a
compound of formula (I) or (II) as described hereinabove, wherein
[0047] a) the DB moiety does not comprise a DA1, DA2, DA1', or DA2'
moiety; and [0048] b) ring B in DB1 is a heterocycle; and [0049] c)
if X.sup.3 in DB1 represents --X.sup.3a and X.sup.3b-- and ring B
is aromatic, then two vicinal substituents on said ring B are
joined to form an optionally substituted carbocycle or heterocycle
fused to said ring B; and [0050] d) if X.sup.3 in DB2 represents
--X.sup.3a and X.sup.3b-- and ring B is aromatic, then two vicinal
substituents on said ring B are joined to form an optionally
substituted heterocycle fused to said ring B, an optionally
substituted non-aromatic carbocycle fused to said ring B, or a
substituted aromatic carbocycle which is fused to said ring B and
to which at least one substituent is attached that contains a
hydroxy group, a primary amino group, or a secondary amino group,
the primary or secondary amine not being a ring atom in an aromatic
ring system nor being part of an amide; and [0051] e) if ring A in
DB2 is a 6-membered aromatic ring, then substituents on ring B are
not joined to form a ring fused to ring B; and [0052] f) two
vicinal substituents on ring A in DB8 are joined to form an
optionally substituted carbocycle or heterocycle fused to said ring
A to form a bicyclic moiety to which no further rings are fused;
and [0053] g) ring A in DB9 together with any rings fused to said
ring A contains at least two ring heteroatoms.
[0054] In a further embodiment, this invention relates to a
compound of formula (I) or (II) as described hereinabove, wherein
at least one of the substituents R.sup.1, R.sup.5, R.sup.5',
R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14, R.sup.14', R.sup.8,
R.sup.8', R.sup.9, R.sup.9', R.sup.10, R.sup.10', R.sup.11,
R.sup.11', R.sup.15, R.sup.15', R.sup.15'', R.sup.15''', R.sup.16,
R.sup.16', R.sup.20, R.sup.20', R.sup.21, R.sup.21', R.sup.22, and
R.sup.23 contains a
X.sup.14(CH.sub.2CH.sub.2O).sub.ffCH.sub.2CH.sub.2X.sup.14 moiety,
wherein ff is selected from 1 to 1000 and each X.sup.14 is
independently selected from
##STR00004##
[0055] that is connected to the attachment site of said substituent
either via a direct bond or via a moiety, being part of said same
substituent, that does not comprise a disulfide, a hydrazone, a
hydrazide, an ester, a natural amino acid, or a peptide containing
at least one natural amino acid, and wherein if ring B in DB1 is an
all-carbon ring, X.sup.3 is O or NR.sup.15, X.sup.4 is CH, X.sup.34
is C, there is only one
X.sup.14(CH.sub.2CH.sub.2O).sub.ffCH.sub.2CH.sub.2X.sup.14 moiety
present in said compound of formula (I) or (II) and said moiety is
part of R.sup.6, R.sup.7, R.sup.8, R.sup.10, or R.sup.15, then b=1
and ff is .gtoreq.5.
[0056] A compound of formula (I) or (II) or a conjugate thereof in
which ff is larger than 1000 is encompassed by this invention.
[0057] In a further embodiment, this invention relates to a
compound of formula (I) or (II) as described hereinabove, wherein
at least one of the substituents R.sup.1, R.sup.5, R.sup.5',
R.sup.6, R.sup.6', R.sup.7, R.sup.7', R14.sub., R.sup.14,
R.sup.14', R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10,
R.sup.10', R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15'',
R.sup.15''', R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21,
R.sup.21', R.sup.22, and R.sup.23 contains a triazole moiety.
[0058] It is to be understood that if --X.sup.3-- represents
--X.sup.3a and X.sup.3b-- in moieties DB1 and DB2 these moieties
are actually represented by the following structures:
##STR00005##
[0059] In another aspect, the present invention relates to a
conjugate of a compound of formula (I), (II), (I'), or (II').
[0060] In yet another aspect, this invention relates to a compound
of formula (III):
##STR00006##
[0061] or a pharmaceutically acceptable salt, hydrate, or solvate
thereof, wherein V.sup.2 is either absent or a functional
moiety;
[0062] each L.sup.2 is independently absent or a linking group
linking V.sup.2 to L;
[0063] each L is independently absent or a linking group linking
L.sup.2 to one or more V.sup.1 and/or Y;
[0064] each V.sup.1 is independently absent or a
conditionally-cleavable or conditionally-transformable moiety,
which can be cleaved or transformed by a chemical, photochemical,
physical, biological, or enzymatic process;
[0065] each Y is independently absent or a self-eliminating spacer
system which is comprised of 1 or more self-elimination spacers and
is linked to V.sup.1, optionally L, and one or more Z;
[0066] each p and q are numbers representing a degree of branching
and are each independently a positive integer;
[0067] z is a positive integer equal to or smaller than the total
number of attachment sites for Z;
[0068] each Z is independently a compound of formula (I), (II),
(I'), or (II') as defined hereinabove wherein one or more of
X.sup.1, R.sup.5, R.sup.5', R.sup.6, R.sup.6', R.sup.7, R.sup.7',
R.sup.14, R.sup.14', R.sup.8, R.sup.8', R.sup.9, R.sup.9',
R.sup.10, R.sup.10', R.sup.11, R.sup.11', R.sup.15, R.sup.15',
R.sup.15'', R.sup.15''', R.sup.16, R.sup.16', R.sup.20, R.sup.20',
R.sup.21, R.sup.21', R.sup.22, and R.sup.23 may optionally in
addition be substituted by or be a substituent of formula (V):
##STR00007##
[0069] wherein each V.sup.2', L.sup.2', L', V.sup.1', Y', Z', p',
q', and z' has the same meaning as defined for V.sup.2, L.sup.2, L,
V.sup.1, Y, Z, p, q, and z, respectively, and is independently
selected, the one or more substituents of formula (V) being
independently connected via Y' to one or more of X.sup.1, R.sup.5,
R.sup.5', R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14,
R.sup.14', R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10,
R.sup.10', R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15'',
R.sup.15''', R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21,
R.sup.21', R.sup.22, R.sup.23, and/or to one or more atoms bearing
these R substituents;
[0070] each Z is independently connected to Y through either
X.sup.1, an atom in R.sup.5, R.sup.5', R.sup.6, R.sup.6', R.sup.7,
R.sup.7', R.sup.14, R.sup.14', R.sup.8, R.sup.8', R.sup.9,
R.sup.9', R.sup.10, R.sup.10', R.sup.11, R.sup.11', R.sup.15,
R.sup.15', R.sup.15'', R.sup.15''', R.sup.16, R.sup.16', R.sup.20,
R.sup.20', R.sup.21, R.sup.21', R.sup.22, R.sup.23, or an atom
bearing any of these R substituents; and
[0071] at least V.sup.2 or a V.sup.1 is present.
[0072] It is noted that in a compound of formula (III), V.sup.2 or
a V.sup.1 needs to be present. However, in the one or more moieties
of formula (V) that are optionally present in Z, each V.sup.2' and
V.sup.1' may be independently selected to be absent or present.
[0073] In a further aspect, this invention relates to a compound of
formula (III), wherein
[0074] V.sup.2 is present and selected to be a targeting moiety and
there is at least one group of formula (V) that contains a V.sup.1'
moiety and either comprises a V.sup.2', L.sup.2', or L' moiety that
contains a
X.sup.14(CH.sub.2CH.sub.2O).sub.ggCH.sub.2CH.sub.2X.sup.14 moiety,
wherein gg is selected from 3 to 1000 and each X.sup.14 is
independently selected from
##STR00008##
[0075] or said same group of formula (V) comprises at least 2
X.sup.14CH.sub.2CH.sub.2OCH.sub.2CH.sub.2X.sup.14 moieties, in
which each X.sup.14 is independently selected.
[0076] It is noted that the separate X.sup.14 moieties in the
--CH.sub.2CH.sub.2X.sup.14 moieties that may be present in a
compound of formula (III) are independently selected.
[0077] It is further noted that z does not represent a degree of
polymerization; hence z does not indicate that a number of moieties
Z or RM2 are connected to one another.
[0078] It is further noted that if Y or Y' is connected to an atom
of Z or RM2 bearing a specific R substituent instead of to this R
substituent itself, this in fact means that this R substituent is
absent if this is necessary to meet valency rules.
[0079] It is further noted that if X.sup.14 in for example
--CH.sub.2CH.sub.2X.sup.14 represents
##STR00009##
then --CH.sub.2CH.sub.2X.sup.14 should be read as
--CH.sub.2CHX.sup.14.
[0080] The present invention also relates to a compound of formula
(IV):
##STR00010##
[0081] or a pharmaceutically acceptable salt, hydrate, or solvate
thereof, wherein
[0082] RM is a reactive moiety and L, V.sup.1, Y, Z, p, and z are
as defined hereinabove, except that L is now linking RM to one or
more V.sup.1 and/or Y, and V.sup.1, Y, and Z may contain protecting
groups, and the one or more V.sup.2'-L.sup.2' moieties optionally
present in Z as defined hereinabove may optionally and
independently be RM' instead, which is a reactive moiety, and
wherein, if there is more than 1 reactive moiety in (IV), some or
all reactive moieties are the same or different. These linker-agent
conjugates of formula (IV) may or may not be considered
intermediates for compounds of formula (III).
[0083] In a further aspect, the present invention relates to a
compound of formula (IV), wherein RM is a reactive moiety selected
from carbamoyl halide, acyl halide, active ester, anhydride,
.alpha.-haloacetyl, .alpha.-haloacetamide, maleimide, isocyanate,
isothiocyanate, disulfide, thiol, hydrazine, hydrazide, sulfonyl
chloride, aldehyde, methyl ketone, vinyl sulfone, halomethyl, and
methyl sulfonate, and wherein at least one group of formula (V),
being part of Z, contains a V.sup.1' moiety and either comprises a
V.sup.2', L.sup.2', or L' moiety that contains a
X.sup.14(CH.sub.2CH.sub.2O).sub.ggCH.sub.2CH.sub.2X.sup.14 moiety,
wherein gg is selected from 3 to 1000 and each X.sup.14 is
independently selected from
##STR00011##
[0084] or said same group of formula (V) comprises at least 2
X.sup.14CH.sub.2CH.sub.2OCH.sub.2CH.sub.2X.sup.14 moieties, in
which each X.sup.14 is independently selected. These linker-agent
conjugates of formula (IV) may or may not be considered
intermediates for compounds of formula (III).
[0085] In yet a further aspect, this invention relates to novel
bifunctional linkers that contain a cleavage site, a
self-elimination spacer system and two reactive moieties, one of
which can be reacted with a therapeutic or diagnostic moiety, e.g.
a compound of formula (II or (II), and the other of which can be
reacted with a functional moiety, such as a targeting moiety. These
bifunctional linkers can be used to prepare conjugates of formulae
(III) and (IV) of this invention or similar compounds with
different therapeutic or diagnostic moieties.
[0086] More specifically, this invention relates to a compound of
formula (VIII):
##STR00012##
[0087] or a pharmaceutically acceptable salt, hydrate, or solvate
thereof, wherein
[0088] L, V.sup.1, Y, RM, p, and z are as defined for a compound of
formula (IV), and RM2 is a reactive moiety or a leaving group. RM
and each RM2 are independently selected. These bifunctional linkers
of formula (VIII) may or may not be considered intermediates for
compounds of formula (III) and (IV).
[0089] This invention relates to enantiomerically pure and/or
diastereomerically pure compounds of formulae (I), (II), (III),
(IV), and (VIII) as well as to enantiomeric and/or diastereomeric
mixtures of compounds of formulae (I), (II), (III), (IV), and
(VIII). This invention relates to pure compounds of formulae (I),
(II), (III), (IV), and (VIII) as well as to mixtures of isomers of
compounds of formulae (I), (II), (III), (IV), and (VIII).
[0090] Compounds of formulae (I) and (II) represent duocarmycin
derivatives that preferably have heteroatoms or polar groups at
selected positions in the DNA-binding moiety or in substituents on
the DNA-binding or DNA-alkylating moiety. Compounds of formula
(III), which are conjugates of compounds of formulae (I) and (II),
were unexpectedly found to be more efficacious in vivo and to have
improved properties, such as increased polarity and optimized drug
release, compared to similar compounds from the prior art.
[0091] In one embodiment, the current invention relates to a
conjugate of a compound of formula (I) or (II) according to one of
the above embodiments and to derivatives thereof. Such a conjugate
contains one or more promoieties. Advantageously, such a conjugate
has a sufficient stability in the circulation, but is efficiently
and selectively activated to release the compound of formula (I) or
(II) at the target site, leading to a suitable therapeutic window.
The length and nature of the linker between functional moiety and
the compound of formula (I) or (II) proved to be an important
contributor. In one aspect of this invention, the linker has a
reduced linker length with respect to linker lengths in similar
conjugates from the prior art, which leads to improved efficacy. In
another aspect, the linker contains a self-elimination spacer
system with improved properties, which leads for example to an
optimized self-elimination rate, optimized drug release and/or
increased polarity. In yet another aspect, the linker between
functional moiety and the compound of formula (I) or (II) contains
one or more groups designed to improve the pharmacokinetic
properties of the conjugate. These groups may be present in L
and/or Y and/or in any of the other moieties making up a compound
of formula (III).
[0092] Premature release of the parent agent, i.e., the compound of
formula (I) or (II), in the circulation may not be desirable, but a
relatively fast deactivation of the released compound might reduce
toxic side effects in this case. Deactivation may be tuned by
choosing the appropriate DNA-alkylating and DNA-binding moiety.
Deactivation may occur by several mechanisms, including enzymatic
or hydrolytic cleavage of the DNA-alkylating unit from the
DNA-binding unit.
[0093] Compounds of formulae (I) and (II) are suited for
application in drug delivery purposes, including drug targeting and
controlled release applications, using compounds of formulae (III)
and (IV).
BRIEF DESCRIPTION OF DRAWINGS
[0094] FIG. 1. Rearrangement of a seco compound to a
cyclopropyl-containing compound.
[0095] FIG. 2. Graphical illustration of cyclization rates for a
series of cyclization spacer-duocarmycin compounds at 25.degree. C.
and pH 7.4 (upper line) and at 37.degree. C. and pH 5 (lower
line).
[0096] FIG. 3. Human plasma stability for a series of
HSA-conjugated linker-agent conjugates.
[0097] FIGS. 4A-4C. Single dose efficacy study with
Trastuzumab-based antibody-drug conjugates (ADCs) in female nu/nu
mice bearing an N87 xenograft described in Example 15: FIG. 4A
shows the average tumor burden for each group, FIG. 4B shows the
average body weight change for each group, and FIG. 4C shows the
survival percentage in each group.
[0098] FIG. 5. Cleavage of N-acetylcysteine-quenched linker-agent
conjugates by cathepsin B.
DESCRIPTION OF THE INVENTION
[0099] The following detailed description is provided so that the
invention may be more fully understood.
[0100] Definitions
[0101] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art.
[0102] The term "antibody", as used herein, refers to a full-length
immunoglobulin molecule, an immunologically active portion of a
full-length immunoglobulin molecule, or a derivative of a
full-length immunoglobulin molecule or an active portion thereof,
i.e., a molecule that contains an antigen-binding site that
immunospecifically binds an antigen of a target of interest or part
thereof, such targets including, but not limited to, tumor cells. A
fragment or derivative of a full-length immunoglobulin molecule
therefore immunospecifically binds the same antigen as said
full-length immunoglobulin molecule. The immunoglobulin can be of
any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), class (e.g.,
IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or subclass. The
immunoglobulin, or a derivative or active portion thereof, can be
derived from any species, e.g., human, rodent (e.g., mouse, rat, or
hamster), donkey, sheep, rabbit, goat, guinea pig, camelid, horse,
cow, or chicken, but preferably, it is of human, murine, or rabbit
origin, or it is derived from more than one species. Antibodies
useful in the invention include, but are not limited to,
monoclonal, polyclonal, bispecific, multispecific, human,
humanized, chimeric, and engineered antibodies, single chain
antibodies, Fv fragments, Fd fragments, Fab fragments, F(ab')
fragments, F(ab').sub.2 fragments, dAb fragments, fragments
produced by a Fab expression library, anti-idiotypic antibodies,
isolated CDRs, and epitope-binding fragments of any of the above
that immunospecifically bind to an antigen-of-interest.
[0103] The term "leaving group" refers to a group that can be
substituted by another group in a substitution reaction. Such
leaving groups are well-known in the art, and examples include, but
are not limited to, a halide (fluoride, chloride, bromide, and
iodide), azide, a sulfonate (e.g., an optionally substituted
C.sub.1-6 alkanesulfonate, such as methanesulfonate,
trifluoromethanesulfonate, and trifluoroethanesulfonate, or an
optionally substituted benzenesulfonate, such as p-toluenesulfonate
and nosylate), imidazole, a cyclic imide thione,
succinimide-N-oxide, phtalimide-N-oxide, p-nitrophenoxide,
o-nitrophenoxide, pentafluorophenoxide, tetrafluorophenoxide,
1,3,5-trichlorophenoxide, 1,3,5-trifluorophenoxide, a carboxylate,
an aminocarboxylate (carbamate), and an alkoxycarboxylate
(carbonate). For substitutions at saturated carbon, halides and
sulfonates are preferred leaving groups. For substitutions at a
carbonyl carbon a halide, succinimide-N-oxide, p-nitrophenoxide,
pentafluorophenoxide, tetrafluorophenoxide, a carboxylate, or an
alkoxycarboxylate (carbonate) may for example be used as a leaving
group. The term "leaving group" also refers to a group that is
eliminated as a consequence of an elimination reaction, e.g., an
electronic cascade reaction or a spirocyclization reaction. In this
instance, a halide, a sulfonate, azide, an aminocarboxylate
(carbamate) or an alkoxycarboxylate (carbonate) may for example be
used as a leaving group. Therefore, an agent or a derivative
thereof released from a conjugate through a (multiple)
self-elimination is defined as a leaving group according to this
definition.
[0104] The term "active ester" refers to a functional group in
which the alkoxy group of the ester moiety is a good leaving group.
Examples of such alkoxy groups include, but are not limited to,
succinimide-N-oxide, p-nitrophenoxide, pentafluorophenoxide,
tetrafluorophenoxide, 1-hydroxybenzotriazole, and
1-hydroxy-7-azabenzotriazole, and groups with comparable leaving
capability. Unsubstituted alkyl-based alkoxy groups such as
methoxy, ethoxy, isopropoxy, and t-butoxy do not qualify as good
leaving groups and methyl, ethyl, isopropyl, and t-butyl esters are
therefore not considered to be active esters.
[0105] The term "reactive moiety" herein refers to a functional
group that can react with a second functional group under
relatively mild conditions and without the need of prior
functionalization of the reactive moiety. The reaction between the
reactive moiety and said second functional group will only require
the application of some heat, pressure, a catalyst, acid, and/or
base. Examples of reactive moieties include, but are not limited
to, carbamoyl halide, acyl halide, active ester, anhydride,
.alpha.-haloacetyl, .alpha.-haloacetamide, maleimide, isocyanate,
isothiocyanate, disulfide, thiol, hydrazine, hydrazide, sulfonyl
chloride, aldehyde, methyl ketone, vinyl sulfone, halomethyl, and
methyl sulfonate.
[0106] The term "promoiety" refers to a moiety that is coupled to a
compound of formula (I) or (II) to modify its properties and that
is to be (partly) removed in vivo from said compound of formula (I)
or (II).
[0107] The term "water-soluble group" refers to a functional group
that is well solvated in aqueous environments and that imparts
improved water solubility to the compound to which it is attached.
Examples of water-soluble groups include, but are not limited to,
polyalcohols, straight chain or cyclic saccharides, primary,
secondary, tertiary, or quaternary amines and polyamines, sulfate
groups, sulfonate groups, sulfinate groups, carboxylate groups,
phosphate groups, phosphonate groups, phosphinate groups, ascorbate
groups, glycols, including polyethylene glycols, and polyethers.
Preferred water-soluble groups are primary, secondary, tertiary,
and quaternary amines, carboxylates, phosphonates, phosphates,
sulfonates, sulfates,
--(CH.sub.2CH.sub.2O).sub.yyCH.sub.2CH.sub.2X.sup.17R.sup.yy,
--(CH.sub.2CH.sub.2O).sub.yyCH.sub.2CH.sub.2X.sup.17--,
--X.sup.17(CH.sub.2CH.sub.2O).sub.yyCH.sub.2CH.sub.2--, glycol,
oligoethylene glycol, and polyethylene glycol, wherein yy is
selected from 1 to 1000, X.sup.17 is selected from O, S, and
NR.sup.zz, and R.sup.zz and R.sup.yy are independently selected
from H and C.sub.1-3 alkyl.
[0108] The term "substituted", when used as an adjective to
"alkyl", "heteroalkyl", "cycloalkyl", "heterocycloalkyl", "aryl",
"heteroaryl", or the like, indicates that said "alkyl",
"heteroalkyl", "cycloalkyl", "heterocycloalkyl", "aryl",
"heteroaryl", or similar group contains one or more substituents
(introduced by substitution for hydrogen). Exemplary substituents
include, but are not limited to, OH, .dbd.O, .dbd.S, .dbd.NR.sup.k,
.dbd.N--OR.sup.k, SH, NH.sub.2, NO.sub.2, NO, N.sub.3, CF.sub.3,
CN, OCN, SCN, NCO, NCS, C(O)NH.sub.2, C(O)H, C(O)OH, halogen,
R.sup.k, SR.sup.k, S(O)R.sup.k, S(O)OR.sup.k, S(O).sub.2R.sup.k,
S(O).sub.2OR.sup.k, OS(O)R.sup.k, OS(O)OR.sup.k,
OS(O).sub.2R.sup.k, OS(O).sub.2OR.sup.k, S(O)N(R.sup.k)R.sup.1,
OS(O)N(R.sup.k)R.sup.1, S(O).sub.2N(R.sup.k)R.sup.1,
OS(O).sub.2N(R.sup.k)R.sup.1, OP(O)(OR.sup.k)(OR.sup.1),
P(O)(OR.sup.k)(OR.sup.1), OR.sup.k, NHR.sup.k, N(R.sup.k)R.sup.l,
.sup.+N(R.sup.k)(R.sup.l)R.sup.m, Si(R.sup.k)(R.sup.l)(R.sup.m),
C(O)R.sup.k, C(O)OR.sup.k, C(O)N(R.sup.k)R.sup.l, OC(O)R.sup.k,
OC(O)OR.sup.k, OC(O)N(R.sup.k)R.sup.l, N(R.sup.k)C(O)R.sup.l,
N(R.sup.k)C(O)OR.sup.1, N(R.sup.k)C(O)N(R.sup.l)R.sup.m, a
water-soluble group, and the thio derivatives of these
substituents, and protonated, charged, and deprotonated forms of
any of these substituents, wherein R.sup.k, R.sup.l, and R.sup.m
are independently selected from H and optionally substituted
--(CH.sub.2CH.sub.2O).sub.yyCH.sub.2CH.sub.2X.sup.17R.sup.yy,
C.sub.1-15 alkyl, C.sub.1-15 heteroalkyl, C.sub.3-15 cycloalkyl,
C.sub.1-15 heterocycloalkyl, C.sub.5-15 aryl, or C.sub.1-15
heteroaryl, or a combination thereof, wherein yy is selected from 1
to 1000, X.sup.17 is independently selected from O, S, and
NR.sup.zz, and R.sup.zz and R.sup.yy are independently selected
from H and C.sub.1-3 alkyl, two or more of R.sup.k, R.sup.l, and
R.sup.m optionally being joined by one or more bonds to form one or
more optionally substituted carbocycles and/or heterocycles. When
there is more than one substituent, each substituent is
independently selected. Two or more substituents may be connected
to each other by replacement of one or more hydrogen atoms on each
of the substituents by one or more connecting bonds, which may be
single, double, or triple bonds, or, if resonance structures are
possible, the bond order of said bonds may be different in two or
more of these resonance structures. Two substituents may thus be
joined under formation of one or more rings.
[0109] When substituents may be "joined by one or more bonds to
form one or more optionally substituted carbocycles and/or
heterocycles", this means that the substituents may be connected to
each other through replacement of one or more hydrogen atoms on
each of the substituents by one or more connecting bonds.
[0110] The term "aryl" as used herein refers to a carbocyclic
aromatic substituent comprising 5 to 24 ring carbon atoms, which
may be charged or uncharged and which may consist of one ring or
two or more rings fused together. Examples of aryl groups include,
but are not limited to, phenyl, naphthyl, and anthracenyl.
[0111] The term "heteroaryl" as used herein refers to a
heterocyclic aromatic substituent comprising 1 to 24 ring carbon
atoms and at least one ring heteroatom, e.g., oxygen, nitrogen,
sulfur, silicon, or phosphorus, wherein nitrogen and sulfur may
optionally be oxidized and nitrogen may optionally be quaternized,
which may consist of one ring or two or more rings fused together.
Heteroatoms may be directly connected to each other. Examples of
heteroaryl groups include, but are not limited to, pyridinyl,
pyrimidyl, furanyl, pyrrolyl, triazolyl, pyrazolyl, pyrazinyl,
oxazolyl, isoxazolyl, thiazolyl, imidazolyl, thienyl, indolyl,
benzofuranyl, benzimidazolyl, benzothiazolyl, purinyl, indazolyl,
benzotriazolyl, benzisoxazolyl, quinoxalinyl, isoquinolyl, and
quinolyl. In one embodiment, a heteroaryl group comprises 1 to 4
heteroatoms. It should be noted that "C.sub.1 heteroaryl group"
denotes that there is only one carbon present in the ring system of
the heteroaromatic group (carbon atoms in optional substituents are
thus not counted). An example of such a heteroaromatic group is a
tetrazolyl group.
[0112] "Aryl" and "heteroaryl" groups also encompass ring systems
in which one or more non-aromatic rings are fused to an aryl or
heteroaryl ring or ring system.
[0113] The term "alkyl" as used herein refers to a straight chain
or branched, saturated or unsaturated hydrocarbyl substituent.
Examples of alkyl groups include, but are not limited to, methyl,
ethyl, propyl, butyl, pentyl, hexyl, octyl, decyl, isopropyl,
sec-butyl, isobutyl, tent-butyl, isopentyl, 2-methylbutyl, vinyl,
allyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,
and 1-butynyl.
[0114] The term "heteroalkyl" as used herein refers to a straight
chain or branched, saturated or unsaturated hydrocarbyl substituent
in which at least one carbon atom is replaced by a heteroatom,
e.g., by oxygen, nitrogen, sulfur, silicon, or phosphorus, wherein
nitrogen and sulfur may optionally be oxidized and nitrogen may
optionally be quaternized. Heteroatoms may be directly connected to
each other. Examples include, but are not limited to, methoxy,
ethoxy, propoxy, isopropoxy, n-butyloxy, tert-butyloxy,
methyloxymethyl, ethyloxymethyl, methyloxyethyl, ethyloxyethyl,
methylaminomethyl, dimethylaminomethyl, methylaminoethyl,
dimethylaminoethyl, methylthiomethyl, ethylthiomethyl,
ethylthioethyl, and methylthioethyl.
[0115] The term "cycloalkyl" as used herein refers to a saturated
or unsaturated non-aromatic cyclic hydrocarbyl substituent, which
may consist of one ring or two or more rings fused together.
Examples include, but are not limited to, cyclopropyl, cyclobutyl,
cyclopentyl, cyclopentenyl, cyclopentadienyl, cyclohexyl,
cyclohexenyl, 1,3-cyclohexadienyl, decalinyl, and
1,4-cyclohexadienyl.
[0116] The term "heterocycloalkyl" as used herein refers to a
saturated or unsaturated non-aromatic cyclic hydrocarbyl
substituent, which may consist of one ring or two or more rings
fused together, wherein at least one carbon in one of the rings is
replaced by a heteroatom, e.g., by oxygen, nitrogen, sulfur,
silicon, or phosphorus, wherein nitrogen and sulfur may optionally
be oxidized and nitrogen may optionally be quaternized. Heteroatoms
may be directly connected to each other. Examples include, but are
not limited to, tetrahydrofuranyl, pyrrolidinyl, piperidinyl,
1,4-dioxanyl, decahydroquinolinyl, piperazinyl, oxazolidinyl, and
morpholinyl. It should be noted that "C.sub.1 heterocycloalkyl
group" denotes that there is only one carbon present in the ring
system of the heterocycloalkane (carbon atoms in optional
substituents are thus not counted). An example of such a group is a
dioxiranyl group.
[0117] The number of carbon atoms that an "alkyl", "heteroalkyl",
"cycloalkyl", "heterocycloalkyl", "aryl", "heteroaryl", and the
like, may contain is indicated by a designation preceding said
terms, i.e., C.sub.1-10 alkyl means that said alkyl may contain
from one to ten carbons (carbon atoms in optional substituents
attached to this alkyl are not counted).
[0118] The term "carbocycle" herein refers to a saturated or
unsaturated cycloalkane or arene moiety, wherein the terms
"cycloalkane" and "arene" are defined as parent moieties of the
"cycloalkyl" and "aryl" substituents, respectively, as defined
hereinabove.
[0119] The term "heterocycle" herein refers to a saturated or
unsaturated heterocycloalkane or heteroarene moiety, wherein the
terms "heterocycloalkane" and "heteroarene" are defined as parent
moieties of the "heterocycloalkyl" and "heteroaryl" substituents,
respectively, as defined hereinabove.
[0120] The extension "-ylene" as opposed to "-yl" in for example
"alkylene" as opposed to "alkyl" indicates that said for example
"alkylene" is a divalent (or multivalent) moiety connected to one
or more other moieties via at least one or more double bonds or two
or more single bonds, as opposed to being a monovalent group
connected to one moiety via one single bond in said for example
"alkyl". The term "alkylene" therefore refers to a straight chain
or branched, saturated or unsaturated hydrocarbylene moiety; the
term "heteroalkylene" as used herein refers to a straight chain or
branched, saturated or unsaturated hydrocarbylene moiety in which
at least one carbon is replaced by a heteroatom; the term "arylene"
as used herein refers to a carbocyclic aromatic moiety, which may
consist of one ring or two or more rings fused together; the term
"heteroarylene" as used herein refers to a carbocyclic aromatic
moiety, which may consist of one ring or two or more rings fused
together, wherein at least one carbon in one of the rings is
replaced by a heteroatom; the term "cycloalkylene" as used herein
refers to a saturated or unsaturated non-aromatic cyclic
hydrocarbylene moiety, which may consist of one ring or two or more
rings fused together; the term "heterocycloalkylene" as used herein
refers to a saturated or unsaturated non-aromatic cyclic
hydrocarbylene moiety, which may consist of one ring or two or more
rings fused together, wherein at least one carbon in one of the
rings is replaced by a heteroatom. Exemplary divalent moieties
include those examples given for the monovalent groups hereinabove
in which one hydrogen atom is removed.
[0121] The prefix "poly" in "polyalkylene", "polyheteroalkylene",
"polyarylene", "polyheteroarylene", polycycloalkylene",
"polyheterocycloalkylene", and the like, indicates that two or more
of such "-ylene" moieties, e.g., alkylene moieties, are joined
together to form a branched or unbranched multivalent moiety
containing two or more attachment sites for adjacent moieties.
Similarly, the prefix "oligo" in for example oligoethylene glycol
indicates that two or more ethylene glycol moieties are joined
together to form a branched or unbranched multivalent moiety. The
difference between the prefixes "oligo" and "poly" is that the
prefix "oligo" is most frequently used to denote a relatively small
number of repeating units, while the prefix "poly" usually refers
to a relatively large number of repeating units.
[0122] Certain compounds of the invention possess chiral centers
and/or double bonds, and/or may have tautomers or atropisomers; the
tautomeric, enantiomeric, diastereomeric, atropisomeric, and
geometric mixtures of two or more isomers, in any composition, as
well as the individual isomers (including tautomers and
atropisomers) are encompassed within the scope of the present
invention. Whenever the term "isomer" is used, it refers to an
atropisomeric, tautomeric, enantiomeric, diastereomeric, and/or
geometric isomer or to a mixture of two or more of these isomers,
unless the context dictates otherwise.
[0123] The term "peptidomimetic" refers to a group or moiety that
has a structure that is different from the general chemical
structure of an amino acid or peptide, but functions in a manner
similar to a naturally occurring amino acid or peptide. Therefore,
a peptidomimetic is an amino acid mimic or peptide mimic.
[0124] The term "unnatural amino acid" is intended to represent the
D stereoisomer of a naturally occurring amino acid.
[0125] The term "bond" herein refers to a covalent connection
between two atoms and may refer to a single bond, a double bond, or
a triple bond, or, if resonance structures are possible, the bond
order of said bond may be different in two or more of these
resonance structures. For example, if the bond is part of an
aromatic ring, the bond may be a single bond in one resonance
structure and a double bond in another resonance structure. If it
is stated that a "double bond" or "triple bond" is present between
two atoms, this double, or triple bond may be localized, but it may
also be that this double or triple bond is delocalized, which means
that only in one or some resonance structures a double or triple
bond is indeed present between the two atoms, whereas the bond
order may be different in one or more other resonance structures.
At the same time, bonds marked as single bond in one resonance
structure, may be double bonds in another resonance structure.
[0126] Compounds of the invention may also contain unnatural
proportions of atomic isotopes at one or more atoms that constitute
such compounds. All isotopic variations of compounds of this
invention, whether radioactive or not, are intended to be
encompassed within the scope of this invention.
[0127] The phrase "pharmaceutically active salt" as used herein
refers to a pharmaceutically acceptable organic or inorganic salt
of a compound of the invention. For compounds containing one or
more basic groups, e.g., an amine group, acid addition salts can be
formed. For compounds containing one or more acidic groups, e.g., a
carboxylic acid group, base addition salts can be formed. For
compounds containing both acidic and basic groups, zwitterions may
in addition be obtained as salts. When the compound of the
invention comprises more than one charged atom or group, there may
be multiple (distinct) counterions.
[0128] The phrase "pharmaceutically acceptable solvate" refers to
an association of one or more solvent molecules with a compound of
the invention. Examples of solvents that form pharmaceutically
acceptable solvates include, but are not limited to, water,
isopropyl alcohol, ethanol, methanol, DMSO, ethyl acetate, and
acetic acid. When referring to water as a solvate, the term
"hydrate" can be used.
[0129] The term "conjugate" hereinbelow refers to a compound of
formula (III) or to a conjugate of a compound of formula (I) or
(II) or a derivative thereof, unless the context dictates
otherwise. The term "linker-agent conjugate" hereinbelow refers to
a compound of formula (IV), unless the context dictates otherwise.
The term "agent" hereinbelow refers to a compound of formula (I),
(II), (I'), or (II'), unless the context dictates otherwise.
[0130] The term "bifunctional linker" hereinbelow refers to a
compound of formula (VIII), unless the context dictates otherwise.
The term "linker" generally refers to the moiety linking V.sup.2 to
Z in a compound of formula (III) or the promoiety connected to Z in
a compound of formula (IV).
[0131] The term "core" or "core structure" of a moiety, for example
the DNA-binding or DNA-alkylating moiety, refers to the structure
that remains when all R substituents are removed from the formula
representing said moiety.
[0132] The term "targeting moiety" refers to any moiety that
specifically binds or reactively associates or complexes with a
moiety specifically or in relative excess present at or near the
target site, on, in, or near the target cell, or in (the proximity
of) the target tissue or organ, e.g., a receptor, a receptor
complex, substrate, antigenic determinant, or other receptive
moiety, or that can target the conjugate to the target site via
other mechanisms by virtue of its nature, e.g., through the EPR
effect. Examples of a targeting moiety include, but are not limited
to, an aptamer, an antibody or antibody fragment or derivative, a
polymer, a dendrimer, a lectin, a biologic response modifier, an
enzyme, a vitamin, a growth factor, a steroid, a sugar residue, an
oligosaccharide residue, a carrier protein, and a hormone, or any
combination thereof
[0133] The phrase "moiety that improves the pharmacological
properties of the compound" refers to a moiety that changes the
pharmacological properties (e.g., pharmacodynamic, pharmacokinetic,
physicochemical, and biopharmaceutic properties) of a compound of
this invention in such a way that a better therapeutic effect can
be obtained. The moiety can for example increase the water
solubility, increase the circulation time, increase the therapeutic
index, or reduce immunogenicity.
[0134] The term "linking group" refers to a structural element of a
compound that links one structural element of said compound to one
or more other structural elements of said same compound.
[0135] The phrase "a number representing degree of branching" is
used to denote that the subscript number next to a closing bracket
represents how many units of the moiety within the brackets are
each directly attached to the moiety immediately to the left of the
corresponding opening bracket. For example, A-(B).sub.b with b
being a number representing a degree of branching means that b
units B are all directly attached to A. This means that when b is
2, the formula reduces to B-A-B.
[0136] The phrase "a number representing degree of polymerization"
is used to denote that the subscript number next to a closing
bracket represents how many units of the moiety within the brackets
are connected to each other. For example, A-(B).sub.b with b being
a number representing a degree of polymerization means that when b
is 2, the formula reduces to A-B-B.
[0137] The term "single-release spacer" refers to a
self-elimination spacer that can release one moiety upon
self-immolation.
[0138] The term "multiple-release spacer" refers to a
self-elimination spacer that can release two or more moieties upon
(repetitive) self-immolation.
[0139] The term "electronic cascade spacer" refers to a
self-elimination spacer, either branched or unbranched, which may
self-eliminate through one or more 1,2+2n electronic cascade
eliminations (n.gtoreq.1).
[0140] The term ".omega.-amino aminocarbonyl cyclization spacer"
refers to a self-elimination spacer that may eliminate through a
cyclization process under formation of a cyclic ureum
derivative.
[0141] The term "spacer system" refers to a single self-eliminating
spacer moiety or to two or more of the same or different
self-eliminating spacer moieties coupled together. A spacer system
may be branched or unbranched and contain one or more attachment
sites for Z as well as V.sup.1 and optionally L.
[0142] In this document and in its claims, the verbs "to comprise",
"to have", "to contain" and their conjugations are used in their
non-limiting sense to mean that items that are "comprised", "had",
or "contained" are included, but items non-specifically mentioned
are not excluded. In addition, reference to an element by the
indefinite article "a" or "an" does not exclude the possibility
that more than one of the element is present, unless the context
clearly requires that there be one and only one of the elements.
The indefinite article "a" or "an" thus usually means "at least
one".
[0143] This specification includes many embodiments. It is noted
that embodiments that are not specifically mentioned and that
result from combination of two or more of said mentioned
embodiments are also encompassed by this invention.
[0144] In the generic structures throughout this description and in
the claims letters are used to define structural elements. Some of
these letters can be mistaken to represent an atom, such as C, N,
O, P, K, B, F, S, U, V, W, I, and Y. To avoid confusion whenever
these letters do not represent an atom they are given in bold
typeface.
[0145] When there are one or more adjectives and/or adjective
phrases to a noun that is a) the first in a list of nouns or b)
anywhere in the middle of a list of nouns and said noun and
adjectives together are preceded by the word "and" or "or", the
adjectives do not only bear on said noun, but on all following
nouns separately, unless the context dictates otherwise. This for
example means that the phrase "optionally substituted C.sub.1-4
alkyl, C.sub.1-4 heteroalkyl, C.sub.3-7 cycloalkyl, or C.sub.1-7
heterocycloalkyl" should be read as "optionally substituted
C.sub.1-4 alkyl, optionally substituted C.sub.1-4 heteroalkyl,
optionally substituted C.sub.3-7 cycloalkyl, or optionally
substituted C.sub.1-7 heterocycloalkyl" and that the phrase
"C.sub.1-4 alkyl, C.sub.1-4 heteroalkyl, and optionally substituted
C.sub.3-7 cycloalkyl, C.sub.5-8 aryl, or C.sub.1-7
heterocycloalkyl" should be read as "C.sub.1-4 alkyl, C.sub.1-4
heteroalkyl, and optionally substituted C.sub.3-7 cycloalkyl,
optionally substituted C.sub.5-8 aryl, or optionally substituted
C.sub.1-7 heterocycloalkyl".
[0146] Throughout this description and in the claims molecular
structures or parts thereof are drawn. As usual in such drawings
bonds between atoms are represented by lines, in some cases, to
indicate stereochemistry, by wavy, bold, or broken or wedged lines.
A bond represented by a wavy line indicates unspecified
stereochemistry at the chiral center to which it is connected; a
structure or substructure with one or more of such wavy bonds in
fact represents a set of (sub)structures in which each chiral
center can either have the R or S configuration. Usually a line
ending in space (a "loose" end), i.e., at one end not having
another line or specific atom connected to it, represents a
CH.sub.3 group. This is correct for the drawings representing the
compounds of this invention. For those structures representing a
structural element of the compounds of this invention a line ending
in space may indicate the position of attachment of another
structural element of the compound. This has been indicated with a
wavy line perpendicular to and crossing the "loose" line.
[0147] Furthermore, the structures or parts thereof have been
drawn, under the assumption that the structures are read from left
to right, meaning that for example in the drawings of compounds of
formula (III) V.sup.2 (if present) is located on the left side and
Z is located on the right side of such structures or parts thereof,
unless the context implies otherwise.
[0148] The following abbreviations are used herein and have the
indicated definitions: Ac: acetyl; Bn: benzyl; Boc:
tert-butyloxycarbonyl; CBI:
1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one; Cbz:
carbobenzyloxy; Cit: citrulline; DCC:
N,N'-dicyclohexylcarbodiimide; DCE: 1,2-dichloroethane; DCM:
dichloromethane; DMAP: 4-dimethylaminopyridine; DMF:
N,N-dimethylformamide; DMSO: dimethylsulfoxide; DiPEA:
N,N-diisopropylethylamine; EEDQ:
2-ethoxy-l-ethoxycarbonyl-1,2-dihydroquinoline; ESI: electrospray
ionization; Fmoc: fluorenylmethyloxycarbonyl; HOBt:
1-hydroxy-benzotriazole; HOSu: hydroxysuccinimide; HSA: human serum
albumin; LC/MS: liquid chromatography-mass spectrometry; MOMCl:
methyl chloromethyl ether; PABA: p-aminobenzyl alcohol; PNPCl:
p-nitrophenyl chloroformate; RT: room temperature; SEC:
size-exclusion chromatography; TCEP: tris(2-carboxyethyl)phosphine;
TFA: trifluoroacetic acid; THF: tetrahydrofuran; Val: valine.
[0149] Agents, Linker-Agent Conjugates, Conjugates, and
Bifunctional Linkers
[0150] This invention relates to novel analogs of the
DNA-alkylating agent CC-1065. The agents of the present invention
are deemed to be used to treat an illness that is characterized by
undesired (cell) proliferation. For example, an agent of this
invention can be used to treat a tumor, cancer, an autoimmune
disease, or an infectious disease.
[0151] The conjugates of the present invention are in one aspect
deemed to be applicable to target agents of formulae (I) and (II)
to a specific target site where the conjugate can be converted into
one or more agents or be induced to be converted into one or more
of said agents. This invention can furthermore find application in
(non-specific) controlled release of one or more of said agents
from a conjugate, with the aim of for example enhancing
physicochemical, biopharmaceutic, pharmacodynamic, and/or
pharmacokinetic properties.
[0152] Compounds of formulae (I) and (II) represent duocarmycin
derivatives that preferably have heteroatoms or polar groups at
selected positions in the DNA-binding moiety or in substituents on
the DNA-binding or DNA-alkylating moiety. Compounds of formula
(III), which are conjugates of compounds of formulae (I) and (II),
were unexpectedly found to be more efficacious in vivo and to have
improved properties, such as increased polarity and optimized drug
release, compared to similar compounds from the prior art.
[0153] In one embodiment, the current invention relates to a
conjugate of a compound of formula (I) or (II) according to one of
the above embodiments and to derivatives thereof. Such a conjugate
contains one or more promoieties. In a more specific embodiment,
such a conjugate has a sufficient stability in the circulation, but
is efficiently and selectively activated to release the compound of
formula (I) or (II) at the target site, leading to a suitable
therapeutic window. The length and nature of the linker between
functional moiety and the compound of formula (I) or (II) proved to
be an important contributor. In one aspect of this invention, the
linker has a reduced linker length with respect to linker lengths
in similar conjugates from the prior art, which leads to improved
efficacy. In another aspect, the linker contains a self-elimination
spacer system with improved properties, which leads for example to
an optimized self-elimination rate, optimized drug release and/or
increased polarity. In yet another aspect, the linker between
functional moiety and the compound of formula (I) or (II) contains
one or more groups designed to improve the pharmacokinetic
properties of the conjugate. These groups may be present in L
and/or Y and/or in any of the other moieties making up a compound
of formula (III). Pharmacokinetic properties affected may for
example include water solubility, multi-drug resistance, plasma
stability, proteolytic lability, absorption, distribution,
metabolism, excretion, and internalization. Some of these
properties may not only affect in vivo behavior, but also in vitro
behavior and behavior during the preparation of a compound of
formula (III). For example, increased water solubility of a
compound of formula (IV) may favorably affect conjugation of such a
compound to a functional moiety in aqueous medium.
[0154] Premature release of the parent agent, i.e., the compound of
formula (I) or (II), in the circulation may not be desirable, but a
relatively fast deactivation of the released compound might reduce
toxic side effects in this case. Deactivation may be tuned by
choosing the appropriate DNA-alkylating and DNA-binding moiety.
Deactivation may occur by several mechanisms, including enzymatic
or hydrolytic cleavage of the DNA-alkylating unit from the
DNA-binding unit.
[0155] Compounds of formulae (I) and (II) are suited for
application in drug delivery purposes, including drug targeting and
controlled release applications, using compounds of formulae (III)
and (IV).
[0156] In a further aspect, this invention relates to novel
bifunctional linkers that contain a cleavage site, a
self-elimination spacer system and two reactive moieties, one of
which can be reacted with a therapeutic or diagnostic moiety and
the other of which can be reacted with a functional moiety, such as
a targeting moiety. These bifunctional linkers contain the novel
linker elements of this invention and can be used to prepare
conjugates of formulae (III) and (IV) of this invention or similar
compounds with different therapeutic or diagnostic moieties.
[0157] Agents
[0158] In one aspect, the present invention provides a compound of
formula (I) or (II):
##STR00013##
[0159] or a pharmaceutically acceptable salt, hydrate, or solvate
thereof, wherein
[0160] DB is a DNA-binding moiety and is selected from the group
consisting of
##STR00014##
[0161] R.sup.1 is a leaving group;
[0162] R.sup.2, R.sup.2', R.sup.3, R.sup.3', R.sup.4, R.sup.4',
R.sup.12 and R.sup.19 are independently selected from H, OH, SH,
NH.sub.2, N.sub.3, NO.sub.2, NO, CF.sub.3, CN, C(O)NH.sub.2, C(O)H,
C(O)OH, halogen, R.sup.a, SR.sup.a, S(O)R.sup.a, S(O).sub.2R.sup.a,
S(O)OR.sup.a, S(O).sub.2OR.sup.a, OS(O)R.sup.a, OS(O).sub.2R.sup.a,
OS(O)OR.sup.a, OS(O).sub.2OR.sup.a, OR.sup.a, NHR.sup.a,
N(R.sup.a)R.sup.b, .sup.+N(R.sup.a)(R.sup.b)R.sup.c,
P(O)(OR.sup.a)(OR.sup.b), OP(O)(OR.sup.a)(OR.sup.b),
SiR.sup.aR.sup.bR.sup.c, C(O)R.sup.a, C(O)OR.sup.a,
C(O)N(R.sup.a)R.sup.b, OC(O)R.sup.a, OC(O)OR.sup.a,
OC(O)N(R.sup.a)R.sup.b, N(R.sup.a)C(O)R.sup.b,
N(R.sup.a)C(O)OR.sup.b, and N(R.sup.a)C(O)N(R.sup.b)R.sup.c,
wherein [0163] R.sup.a, R.sup.b, and R.sup.c are independently
selected from H and optionally substituted C.sub.1-3 alkyl or
C.sub.1-3 heteroalkyl,
[0164] or R.sup.3+R.sup.3' and/or R.sup.4+R.sup.4' are
independently selected from .dbd.O, .dbd.S, .dbd.NOR.sup.18,
.dbd.C(R.sup.18)R.sup.18', and .dbd.NR.sup.18, R.sup.18 and
R.sup.18 being independently selected from H and optionally
substituted C.sub.1-3 alkyl, two or more of R.sup.2, R.sup.2',
R.sup.3, R.sup.3', R.sup.4, R.sup.4', and R.sup.12 optionally being
joined by one or more bonds to form one or more optionally
substituted carbocycles and/or heterocycles;
[0165] X.sup.2 is selected from O, C(R.sup.14)(R.sup.14'), and
NR.sup.14', wherein R.sup.14 and R.sup.14' have the same meaning as
defined for R.sup.7 and are independently selected, or R.sup.14'
and R.sup.7' are absent resulting in a double bond between the
atoms designated to bear R.sup.7' and R.sup.14';
[0166] R.sup.5, R.sup.5', R.sup.6, R.sup.6', R.sup.7, and R.sup.7'
are independently selected from H, OH, SH, NH.sub.2, N.sub.3,
NO.sub.2, NO, CF.sub.3, CN, C(O)NH.sub.2, C(O)H, C(O)OH, halogen,
R.sup.e, SR.sup.e, S(O)R.sup.e, S(O).sub.2R.sup.e, S(O)OR.sup.e,
S(O).sub.2OR.sup.e, OS(O)R.sup.e, OS(O).sub.2R.sup.e,
OS(O)OR.sup.e, OS(O).sub.2OR.sup.e, OR.sup.e, NHR.sup.e,
N(R.sup.e)R.sup.f, .sup.+N(R.sup.e)(R.sup.f)R.sup.g,
P(O)(OR.sup.e)(OR.sup.f), OP(O)(OR.sup.e)(OR.sup.f),
SiR.sup.eR.sup.fR.sup.g, C(O)R.sup.e, C(O)OR.sup.e,
C(O)N(R.sup.e)R.sup.f, OC(O)R.sup.e, OC(O)OR.sup.e,
OC(O)N(R.sup.e)R.sup.f, N(R.sup.e)C(O)R.sup.f,
N(R.sup.e)C(O)OR.sup.f, N(R.sup.e)C(O)N(R.sup.f)R.sup.g, and a
water-soluble group, wherein [0167] R.sup.e, R.sup.f, and R.sup.g
are independently selected from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-15 alkyl, C.sub.1-15 heteroalkyl, C.sub.3-15 cycloalkyl,
C.sub.1-15 heterocycloalkyl, C.sub.5-15 aryl, or C.sub.1-15
heteroaryl, wherein ee is selected from 1 to 1000, X.sup.13 is
selected from O, S, and NR.sup.fl, and R.sup.f1 and R.sup.e1 are
independently selected from H and C.sub.1-3 alkyl, one or more of
the optional substituents in R.sup.e, R.sup.f, and/or R.sup.g
optionally being a water-soluble group, two or more of R.sup.e,
R.sup.f, and R.sup.g optionally being joined by one or more bonds
to form one or more optionally substituted carbocycles and/or
heterocycles,
[0168] or R.sup.5+R.sup.5' and/or R.sup.6+R.sup.6' and/or
R.sup.7+R.sup.7' are independently selected from .dbd.O, .dbd.S,
.dbd.NOR.sup.e3, .dbd.C(R.sup.e3)R.sup.e4, and .dbd.NR.sup.e3,
R.sup.e3 and R.sup.e4 being independently selected from H and
optionally substituted C.sub.1-3 alkyl, or R.sup.5'+R.sup.6' and/or
R.sup.6'+R.sup.7' and/or R.sup.7'+R.sup.14' are absent, resulting
in a double bond between the atoms designated to bear R.sup.5' and
R.sup.6', and/or R.sup.6' and R.sup.7', and/or R.sup.7' and
R.sup.14', respectively, two or more of R.sup.5, R.sup.5', R.sup.6,
R.sup.6', R.sup.7, R.sup.7', R.sup.14, and R.sup.14' optionally
being joined by one or more bonds to form one or more optionally
substituted carbocycles and/or heterocycles;
[0169] X.sup.1 is selected from O, S, and NR.sup.13, wherein
R.sup.13 is selected from H and optionally substituted C.sub.1-8
alkyl or C.sub.1-8 heteroalkyl and not joined with any other
substituent;
[0170] X.sup.3 is selected from O, S, C(R.sup.15)R.sup.15',
--C(R.sup.15)(R.sup.15')--C(R.sup.15'')(R.sup.15''')--,
--N(R.sup.15)--N(R.sup.15')--,
--C(R.sup.15)(R.sup.15')--N(R.sup.15'')--,
--N(R.sup.15'')--C(R.sup.15)(R.sup.15')--,
--C(R.sup.15)(R.sup.15')--O--, --O--C(R.sup.15)(R.sup.15')--,
--C(R.sup.15)(R.sup.15')--S--, --S--C(R.sup.15)(R.sup.15')--,
--C(R.sup.15).dbd.C(R.sup.15')--,
.dbd.C(R.sup.15)--C(R.sup.15').dbd., --N.dbd.C(R.sup.15')--,
.dbd.N--C(R.sup.15').dbd., --C(R.sup.15).dbd.N--,
.dbd.C(R.sup.15)--N.dbd., --N.dbd.N--, .dbd.N--N.dbd., CR.sup.15,
N, and NR.sup.15, or in DB1 and DB2 -X.sup.3-- represents
--X.sup.3a and X.sup.3b--, wherein X.sup.3a is connected to
X.sup.34, a double bond is present between X.sup.34 and X.sup.4,
and X.sup.3b is connected to X.sup.11, wherein X.sup.3a is
independently selected from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-8 alkyl, or C.sub.1-8 heteroalkyl and not joined with any
other substituent;
[0171] X.sup.4 is selected from O, S, C(R.sup.16)R.sup.16',
NR.sup.16, N, and CR.sup.16;
[0172] X.sup.5 is selected from O, S, C(R.sup.17)R.sup.17',
NOR.sup.17, and NR.sup.17, wherein R.sup.17 and R.sup.17' are
independently selected from H and optionally substituted C.sub.1-8
alkyl or C.sub.1-8 heteroalkyl and not joined with any other
substituent;
[0173] X.sup.6 is selected from CR.sup.11, CR.sup.11(R.sup.11'), N,
NR.sup.11, O, and S;
[0174] X.sup.7 is selected from CR.sup.8, CR.sup.8(R.sup.8'), N,
NR.sup.8, O, and S;
[0175] X.sup.8 is selected from CR.sup.9, CR.sup.9(R.sup.9'), N,
NR.sup.9, O, and S;
[0176] X.sup.9 is selected from CR.sup.10, CR.sup.10(R.sup.10'), N,
NR.sup.10, O, and S;
[0177] X.sup.10 is selected from CR.sup.20, CR.sup.20(R.sup.20'),
N, NR.sup.20, O, and S;
[0178] X.sup.11 is selected from C, CR.sup.21, and N, or
X.sup.11-X.sup.3b is selected from CR.sup.21, CR.sup.21(R.sup.21'),
N, NR.sup.21, O, and S;
[0179] X.sup.12 is selected from C, CR.sup.22, and N;
[0180] X.sup.6*, X.sup.7*, X.sup.8*, X.sup.9*, X.sup.10*, and
X.sup.11* have the same meaning as defined for X.sup.6, X.sup.7,
X.sup.8, X.sup.9, X.sup.10, and
[0181] X.sup.11, respectively, and are independently selected;
[0182] X.sup.34 is selected from C, CR.sup.23, and N;
[0183] the ring B atom of X.sup.11* in DB6 and DB7 is connected to
a ring atom of ring A such that ring A and ring B in DB6 and DB7
are directly connected via a single bond;
[0184] means that the indicated bond may be a single bond or a
non-cumulated, optionally delocalized, double bond;
[0185] R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10, R.sup.10',
R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15'', R.sup.15''',
R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21, R.sup.21',
R.sup.22, and R.sup.23 are each independently selected from H, OH,
SH, NH.sub.2, N.sub.3, NO.sub.2, NO, CF.sub.3, CN, C(O)NH.sub.2,
C(O)H, C(O)OH, halogen, R.sup.h, SR.sup.h, S(O)R.sup.h,
S(O).sub.2R.sup.h, S(O)OR.sup.h, S(O).sub.2OR.sup.h, OS(O)R.sup.h,
OS(O).sub.2R.sup.h, OS(O)OR.sup.h, OS(O).sub.2OR.sup.h, OR.sup.h,
NHR.sup.h, N(R.sup.h)R.sup.i, .sup.+N(R.sup.h)(R.sup.i)R.sup.j,
P(O)(OR.sup.h)(OR.sup.j), OP(O)(OR.sup.h)(OR.sup.i),
SiR.sup.hR.sup.iR.sup.j, C(O)R.sup.h, C(O)OR.sup.h,
C(O)N(R.sup.h)R.sup.i, OC(O)R.sup.h, OC(O)OR.sup.h,
OC(O)N(R.sup.h)R.sup.i, N(R.sup.h)C(O)R.sup.i,
N(R.sup.h)C(O)OR.sup.i, N(R.sup.h)C(O)N(R.sup.i)R.sup.j, and a
water-soluble group, wherein [0186] R.sup.h, R.sup.i, and R.sup.j
are independently selected from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-15 alkyl, C.sub.1-15 heteroalkyl, C.sub.3-15 cycloalkyl,
C.sub.1-15 heterocycloalkyl, C.sub.5-15 aryl, or C.sub.1-15
heteroaryl, one or more of the optional substituents in R.sup.h,
R.sup.i, and/or R.sup.j optionally being a water-soluble group, two
or more of R.sup.h, R.sup.i, and R.sup.j optionally being joined by
one or more bonds to form one or more optionally substituted
carbocycles and/or heterocycles,
[0187] or R.sup.8+R.sup.8' and/or R.sup.9+R.sup.9' and/or
R.sup.10+R.sup.10' and/or R.sup.11+R.sup.11' and/or
R.sup.15+R.sup.15' and/or R.sup.15'+R.sup.15''' and/or
R.sup.16+R.sup.16' and/or R.sup.20+R.sup.20' and/or
R.sup.21+R.sup.21' are independently selected from .dbd.O, .dbd.S,
.dbd.NOR.sup.h1, .dbd.C(R.sup.h1)R.sup.h2, and .dbd.NR.sup.h1,
R.sup.h1 and R.sup.h2 being independently selected from H and
optionally substituted C.sub.1-3 alkyl, two or more of R.sup.8,
R.sup.8', R.sup.9, R.sup.9', R.sup.10, R.sup.10', R.sup.11,
R.sup.11', R.sup.15, R.sup.15', R.sup.15'', R.sup.''', R.sup.16,
R.sup.16', R.sup.20, R.sup.20', R.sup.21, R.sup.21', R.sup.22, and
R.sup.23 optionally being joined by one or more bonds to form one
or more optionally substituted carbocycles and/or heterocycles;
[0188] R.sup.8b and R.sup.9b are independently selected and have
the same meaning as R.sup.8, except that they may not be joined
with any other substituent;
[0189] one of R.sup.4 and R.sup.4' and one of R.sup.16 and
R.sup.16' may optionally be joined by one or more bonds to form one
or more optionally substituted carbocycles and/or heterocycles;
[0190] one of R.sup.2, R.sup.2', R.sup.3, and R.sup.3' and one of
R.sup.5 and R.sup.5' may optionally be joined by one or more bonds
to form one or more optionally substituted carbocycles and/or
heterocycles; and
[0191] a and b are independently selected from 0 and 1.
[0192] In a further aspect, this invention relates to a compound of
formula (I') or (II'):
##STR00015##
[0193] or a pharmaceutically acceptable salt, hydrate, or solvate
thereof, wherein all substituents have the same meaning as
described for compounds of formulae (I) and (II). Compounds of
formulae (I) and (II) are alleged to be converted to (I') and
(II'), respectively, in vivo with concomitant elimination of
H--R.sup.1.
[0194] Therefore, this invention relates to a compound of formula
(I') or (II'), said compound comprising a cyclopropyl group, which
can be formed through rearrangement of and concomitant elimination
of H--R' from a compound of formula (I) or (II). All embodiments
for a compound of formula (I) or (II) or a moiety thereof also hold
for a compound of formula (I') or (II') or a moiety thereof, unless
the context dictates otherwise.
[0195] In a more specific embodiment, this invention relates to a
compound of formula (I) or (II) as described hereinabove, wherein
[0196] a) the DB moiety does not comprise a DA1, DA2, DA1', or DA2'
moiety; and [0197] b) ring B in DB1 is a heterocycle; and [0198] c)
if X.sup.3 in DB1 represents --X.sup.3a and X.sup.3b-- and ring B
is aromatic, then two vicinal substituents on said ring B are
joined to form an optionally substituted carbocycle or heterocycle
fused to said ring B; and [0199] d) if X.sup.3 in DB2 represents
--X.sup.3a and X.sup.3b-- and ring B is aromatic, then two vicinal
substituents on said ring B are joined to form an optionally
substituted heterocycle fused to said ring B, an optionally
substituted non-aromatic carbocycle fused to said ring B, or a
substituted aromatic carbocycle which is fused to said ring B and
to which at least one substituent is attached that contains a
hydroxy group, a primary amino group, or a secondary amino group,
the primary or secondary amine not being a ring atom in an aromatic
ring system nor being part of an amide; and [0200] e) if ring A in
DB2 is a 6-membered aromatic ring, then substituents on ring B are
not joined to form a ring fused to ring B; and [0201] f) two
vicinal substituents on ring A in DB8 are joined to form an
optionally substituted carbocycle or heterocycle fused to said ring
A to form a bicyclic moiety to which no further rings are fused;
and [0202] g) ring A in DB9 together with any rings fused to said
ring A contains at least two ring heteroatoms.
[0203] In a further more specific embodiment, this invention
relates to a compound of formula (I) or (II) as described
hereinabove, wherein at least one of the substituents R.sup.1,
R.sup.5, R.sup.5', R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14,
R.sup.14', R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10,
R.sup.10', R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15'',
R.sup.15''', R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21,
R.sup.21', R.sup.22, and R.sup.23 contains a
X.sup.14(CH.sub.2CH.sub.2O).sub.ffCH.sub.2CH.sub.2X.sup.14 moiety,
wherein ff is selected from 1 to 1000 and each X.sup.14 is
independently selected from
##STR00016##
[0204] that is connected to the attachment site of said substituent
either via a direct bond or via a moiety, being part of said same
substituent, that does not comprise a disulfide, a hydrazone, a
hydrazide, an ester, a natural amino acid, or a peptide containing
at least one natural amino acid, and wherein if ring B in DB1 is an
all-carbon ring, X.sup.3 is O or NR.sup.15, X.sup.4 is CH, X.sup.34
is C, there is only one
X.sup.14(CH.sub.2CH.sub.2O).sub.ffCH.sub.2CH.sub.2X.sup.14 moiety
present in said compound of formula (I) or (II) and said moiety is
part of R.sup.6, R.sup.7, R.sup.8, R.sup.10, or R.sup.15, then b=1
and ff is .gtoreq.5.
[0205] A compound of formula (I) or (II) or a conjugate thereof in
which ff is larger than 1000 is encompassed by this invention.
[0206] In a further more specific embodiment, this invention
relates to a compound of formula (I) or (II) as described
hereinabove, wherein at least one of the substituents R.sup.1,
R.sup.5, R.sup.5', R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14,
R.sup.14', R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10,
R.sup.10', R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15'',
R.sup.15''', R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21,
R.sup.21', R.sup.22, and R.sup.23, contains a triazole moiety.
[0207] It should be understood that in this entire document, when
referring to a compound of formula (I) or (II), this includes
reference to a compound of formula (I') or (II'), respectively,
unless structural parts of (I) and (II) not present in (I') and
(II') are concerned or the context dictates otherwise. Similarly,
when referring to a structural part (fragment), linker-agent
conjugate, or conjugate derived from a compound of formula (I) or
(II), this includes reference to a similar structural part
(fragment), linker-agent conjugate, or conjugate derived from a
compound of formula (I') or (II'), respectively, unless structural
parts of (I) and (II) not present in (I') and (II') are concerned
or the context dictates otherwise.
[0208] It should also be understood that when reference is made to
a compound of formula (I) or (II) or a fragment, derivative, or
conjugate thereof and the scope of R.sup.2' or R.sup.12 is
specified, this specification only affects a compound of formula
(I) as R.sup.2' and R.sup.12 are absent in a compound of formula
(II). Therefore, wherever it reads "R.sup.2'" or "R.sup.12" in this
document, one could read "R.sup.2' (if present)" or "R.sup.12 (if
present)", respectively. This holds as well for (other)
substituents that may be present or absent in compounds of formulae
(I) and (II) and their fragments, linker-agent conjugates, and
conjugates.
[0209] It should further be understood that this invention relates
to enantiomerically pure and/or diastereomerically pure compounds
of formulae (I) and (II) as well as to enantiomeric and/or
diastereomeric mixtures of compounds of formulae (I) and (II).
[0210] Considerations about substituent effects and the effects of
linkers, DNA-alkylating units and DNA-binding units in compounds of
formulae (I) and (II), their cyclopropyl-containing analogs, and
their conjugates and linker-agent conjugates given in this document
are presented without consenting to a specific mechanism of action
for compounds of formulae (I) and (II), their
cyclopropyl-containing analogs, and their linker-agent conjugates
and conjugates.
[0211] Compounds of formula (I) and (II) can be considered to be
built up of a DNA-binding unit (DB1-DB9) and a DNA-alkylating unit
(DA1, DA2, DA1', or DA2'), as indicated in the figures
hereinabove.
[0212] The DNA-alkylating unit of compounds of formulae (I) and
(II) is considered to contain the site of alkylation. Alkylation of
DNA may occur through attack of DNA on the carbon bearing R.sup.1
in a compound of formula (I) or (II) or on that same carbon in the
cyclopropyl-containing analog of said compound.
[0213] The DNA-binding unit of compounds of formulae (I) and (II)
is considered to assist in efficient binding of these compounds to
DNA. It may be coupled to the DNA-alkylating moiety via, for
instance, an amide bond. Therefore in one embodiment, X.sup.5 is
O.
[0214] In one embodiment, this invention relates to a compound of
formula (I). In another embodiment, this invention relates to a
compound of formula (II).
[0215] R.sup.1 in a compound of formula (I) or (II) is a leaving
group.
[0216] In one embodiment, the leaving group R.sup.1 is selected
from halogen, azide (N.sub.3), carboxylate [OC(O)R.sup.n],
carbonate [OC(O)OR.sup.n], carbamate [OC(O)N(R.sup.n)R.sup.n1],
.sup.+N(R.sup.n)(R.sup.n1)R.sup.n2, S(O).sub.2R.sup.o, and
OS(O).sub.2R.sup.o, wherein R.sup.n, R.sup.n1, R.sup.n2, and
R.sup.o are independently selected from H and optionally
substituted C.sub.1-10 alkyl, C.sub.1-10 heteroalkyl, C.sub.5-10
aryl, or C.sub.1-10 heteroaryl. An optional substituent may be an
oligoethylene glycol or a polyethylene glycol moiety. When the
R.sup.1 group comprises an oligoethylene glycol or polyethylene
glycol moiety, i.e., a
X.sup.14(CH.sub.2CH.sub.2O).sub.ffCH.sub.2CH.sub.2X.sup.14 moiety,
a compound of formula (I) or (II) or its conjugate may show
improved physicochemical, biopharmaceutical, pharmacodynamic,
and/or pharmacokinetic properties, which, as indicated hereinabove,
may also be valid for the presence of oligoethylene glycol or
polyethylene glycol moieties at other positions in a compound of
formula (I) or (II). In addition, however, the relatively large
size of the R.sup.1 substituent may reduce non-specific alkylation
of a compound of formula (I) or (II) or its conjugate. Furthermore,
the R.sup.1 group will be eliminated when the compound of formula
(I) or (II) rearranges to a compound of formula (I') or (II'). This
means that the oligoethylene glycol or polyethylene glycol moiety
may not have a negative effect on the cytotoxic potential of the
compound of formula (I) or (II).
[0217] In one embodiment, R.sup.l is selected from halogen and
OS(O).sub.2R.sup.o. In another embodiment, the leaving group
R.sup.l in a compound of formula (I) or (II) is a halogen. In
another embodiment, R.sup.1 is selected from chloro (Cl), bromo
(Br), and iodo (I). In yet another embodiment, R.sup.1 is chloro
(Cl). In yet another embodiment, R.sup.1 is bromo (Br). In yet
another embodiment, R.sup.1 is OS(O).sub.2R.sup.o. In yet another
embodiment, R.sup.1 is OS(O).sub.2R.sup.o and R.sup.o contains a
X.sup.14(CH.sub.2CH.sub.2O).sub.ffCH.sub.2CH.sub.2X.sup.14 moiety.
In yet another embodiment, R.sup.1 is selected from
OS(O).sub.2CF.sub.3, OS(O).sub.2C.sub.6H.sub.4CH.sub.3, and
OS(O).sub.2CH.sub.3.
[0218] By varying the leaving group R.sup.1, one may tune the
alkylating activity of the seco agents and affect the
transformation rate of a seco agent to a cyclopropyl-containing
agent of formula (I') or (II'). If the leaving capability of
R.sup.1 is too good, this may cause the seco agent to become an
aspecific alkylating agent, which may decrease the cytotoxicity
quotient and therapeutic index of conjugates of compounds of
formulae (I) and (II) as the agent may for example be able to
alkylate while still being bound in the conjugate. On the other
hand, if R.sup.1 is too bad a leaving group, the seco agent may not
close to form a cyclopropyl-containing agent, believed to be the
active species, which may reduce its cytotoxicity and the
cytotoxicity quotient. Therefore, in one embodiment, the
Swain-Scott parameter s of the alkylating site is larger than 0.3.
In other embodiments, the Swain-Scott parameters is larger than 0.5
or 0.7 or 1.0.
[0219] The size of R.sup.1 may affect the non-DNA alkylation rate
of a compound of formula (I) or (II) or a conjugate thereof. If
R.sup.1 is a relatively bulky group, aspecific alkylation may be
reduced as the carbon bearing R.sup.1 is somewhat shielded.
[0220] Another means to tune the alkylating activity of the seco
agents and their cyclopropyl-containing derivatives may be to
somewhat shield the carbon to which the leaving group R.sup.1 is
attached or on which nucleophilic attack can occur by choosing at
least one of R.sup.2, R.sup.2', R.sup.3, R.sup.3', R.sup.4,
R.sup.4', R.sup.5, R.sup.5', R.sup.6, R.sup.6', R.sup.12, R.sup.16,
and R.sup.16' present to be other than hydrogen. Shielding of said
carbon may reduce aspecific alkylation by compounds of formulae (I)
and (II), their cyclopropyl-containing analogs, and their
conjugates. Although introduction of steric hindrance may also
affect the DNA alkylation rate, it may be reasonable to assume that
aspecific alkylation may be affected relatively more than DNA
alkylation as the latter occurs presumably after the agent is
ideally positioned for nucleophilic attack being bound to the DNA
minor groove. The carbon bearing R.sup.1 in a compound of formula
(II), being a secondary carbon atom (when R.sup.2 is H), is already
somewhat shielded in comparison to the carbon bearing R.sup.1 in a
compound of formula (I) when R.sup.2 and R.sup.2' are both H. In
this respect, a compound of formula (II) may be compared to a
compound of formula (I) in which R.sup.2' is other than hydrogen.
Further shielding may however be accomplished by choosing one or
more of R.sup.2, R.sup.3, R.sup.3', R.sup.4, R.sup.4', R.sup.5,
R.sup.5', R.sup.6, R.sup.6', R.sup.16, and R.sup.16' present to be
other than hydrogen.
[0221] In one embodiment, R.sup.2 and R.sup.2' are both hydrogen.
In another embodiment, R.sup.2' is hydrogen and R.sup.2 is not
hydrogen. In another embodiment, R.sup.2 is selected from N.sub.3,
NO.sub.2, NO, CF.sub.3, CN, C(O)NH.sub.2, C(O)H, C(O)OH, halogen,
R.sup.a, SR.sup.a, S(O)R.sup.a, S(O).sub.2R.sup.a, S(O)OR.sup.a,
S(O).sub.2OR.sup.a, OS(O)R.sup.a, OS(O).sub.2R.sup.a,
OS(O)OR.sup.a, OS(O).sub.2OR.sup.a, OR.sup.a, N(R.sup.a)R.sup.b,
.sup.+N(R.sup.a)(R.sup.b)R.sup.c, P(O)(OR.sup.a)(OR.sup.b),
OP(O)(OR.sup.a)(OR.sup.b), SiR.sup.aR.sup.bR.sup.c, C(O)R.sup.a,
C(O)OR.sup.a, C(O)N(R.sup.a)R.sup.b, OC(O)R.sup.a, OC(O)OR.sup.a,
OC(O)N(R.sup.a)R.sup.b, N(R.sup.a)C(O)R.sup.b,
N(R.sup.a)C(O)OR.sup.b, and N(R.sup.a)C(O)N(R.sup.b)R.sup.c,
wherein R.sup.a, R.sup.b, and R.sup.c are independently selected
from H and optionally substituted C.sub.1-3 alkyl or C.sub.1-3
heteroalkyl.
[0222] In one embodiment, R.sup.2 is selected from optionally
substituted C.sub.1-3 alkyl and C.sub.1-3 heteroalkyl. In another
embodiment, R.sup.2 is optionally substituted C.sub.1-3 alkyl. In
another embodiment, R.sup.2 is selected from methyl, ethyl, propyl,
and isopropyl. In another embodiment, R.sup.2 is methyl.
[0223] In yet another embodiment, R.sup.2 and R.sup.2' are both
other than hydrogen. In one embodiment, both R.sup.2 and R.sup.2'
are methyl.
[0224] Alternatively, or simultaneously, steric shielding of the
carbon bearing R.sup.1 may be introduced by choosing one or more of
R.sup.3, R.sup.3', R.sup.4, R.sup.4', R.sup.12, R.sup.16, and
R.sup.16' present to be other than hydrogen. In one embodiment,
R.sup.3, R.sup.3', R.sup.4, and R.sup.4' are each H. In another
embodiment, R.sup.3 and R.sup.3' are both H. In another embodiment,
R.sup.4 and R.sup.4' are both H. In another embodiment, one of
R.sup.3 and R.sup.3' is C.sub.1-3 alkyl while the other is H. In
another embodiment, one of R.sup.4 and R.sup.4' is C.sub.1-3 alkyl
while the other is H. In another embodiment, one of R.sup.3 and
R.sup.3' is C.sub.1-3 alkyl and one of R.sup.4 and R.sup.4' is
C.sub.1-3 alkyl while the others are H. In another embodiment, both
R.sup.3 and R.sup.3' are independently C.sub.1-3 alkyl. In another
embodiment, both R.sup.4 and R.sup.4' are independently C.sub.1-3
alkyl. In another embodiment, one of R.sup.3, R.sup.3', R.sup.4,
and R.sup.4' is methyl. In another embodiment, one of R.sup.4 and
R.sup.4' is methyl. In yet another embodiment, both R.sup.4 and
R.sup.4' are methyl. In yet other embodiments, one or both of
R.sup.4 and R.sup.4' are fluoro.
[0225] In one embodiment, R.sup.12 is H. In another embodiment,
R.sup.12 is C.sub.1-3 alkyl. In yet other embodiments, R.sup.12 is
methyl or ethyl. In yet another embodiment, R.sup.12 equals
C(R.sup.2')(R.sup.2)R.sup.1, which means that the carbon bearing
R.sup.12 bears two identical groups.
[0226] In another embodiment, R.sup.16 and R.sup.16' are both H. In
another embodiment, R.sup.16 is H. In other embodiments, R.sup.16
is fluoro (F) or methyl or ethyl.
[0227] The alkylating activity of a compound of formula (I) or (II)
or its cyclopropyl-containing analog may also be affected by the
nature of X.sup.1. The nature of X.sup.1 may affect the rate at
which and the conditions under which the seco agents ring close to
the cyclopropyl analogs and/or the rate at which the cyclopropyl
ring is opened by nucleophilic attack (by DNA), and thus affect the
alkylation behavior. In one embodiment, X.sup.1 is O. In another
embodiment, X.sup.1 is NR.sup.13.
[0228] The substituents R.sup.5, R.sup.5', R.sup.6, R.sup.6',
R.sup.7, R.sup.7', and X.sup.2 as well as the size of the ring
connected to the left-hand side of the ring bearing X.sup.1 may for
example, each independently or two or more taken together, affect
the pharmacological properties of the agent, e.g., affect the water
solubility, affect the aggregation behavior, affect the DNA
alkylation process, and/or affect the DNA binding strength.
Furthermore, especially R.sup.5 and R.sup.5', and to some degree
R.sup.6 and R.sup.6' as well, may also affect the degree of
shielding of the carbon on which nucleophilic attack should
occur.
[0229] R.sup.5 and R.sup.5' may both be H, or R.sup.5 may be H
while R.sup.5' is absent. In another embodiment, at least one of
R.sup.5 and R.sup.5' is not hydrogen nor absent. In another
embodiment, R.sup.5 is not hydrogen.
[0230] In one embodiment, R.sup.5 is selected from OH, SH,
NH.sub.2, N.sub.3, NO.sub.2, NO, CF.sub.3, CN, C(O)NH.sub.2, C(O)H,
C(O)OH, halogen, R.sup.e2, SR.sup.e2, S(O)R.sup.e2,
S(O).sup.2R.sup.e2, S(O)OR.sup.e2, S(O).sub.2OR.sup.e2,
OS(O)R.sup.e2, OS(O).sub.2R.sup.e2, OS(O)OR.sup.e2,
OS(O).sub.2OR.sup.e2, OR.sup.e2, NHR.sup.e2, N(R.sup.e2)R.sup.f2,
.sup.+N(R.sup.e2)(R.sup.f2)R.sup.g2, P(O)(OR.sup.e2)(OR.sup.f2),
OP(O)(OR.sup.e2)(OR.sup.f2), SiR.sup.e2R.sup.f2R.sup.g2,
C(O)R.sup.e2, C(O)OR.sup.e2, C(O)N(R.sup.e2)R.sup.f2,
OC(O)R.sup.e2, OC(O)OR.sup.e2, OC(O)N(R.sup.e2)R.sup.f2,
N(R.sup.e2)C(O)R.sup.f2, N(R.sup.e2)C(O)OR.sup.f2, and
N(R.sup.e2)C(O)N(R.sup.f2)R.sup.g2, wherein R.sup.e2, R.sup.f2, and
R.sup.g2 are independently selected from H and optionally
substituted C.sub.1-3 alkyl, C.sub.1-3 heteroalkyl, C.sub.3
cycloalkyl, or C.sub.1-3 heterocycloalkyl, two or more of R.sup.e2,
R.sup.f2, and R.sup.g2 optionally being joined by one or more bonds
to form one or more optionally substituted carbocycles and/or
heterocycles.
[0231] In another embodiment, R.sup.5 is selected from OH, SH,
NH.sub.2, N.sub.3, NO.sub.2, NO, CF.sub.3, CN, C(O)NH.sub.2, C(O)H,
C(O)OH, halogen, R.sup.e2, SR.sup.e2, S(O)R.sup.e2,
S(O).sub.2R.sup.e2, S(O)OR.sup.e2, S(O).sub.2OR.sup.e2,
OS(O)R.sup.e2, OS(O).sub.2R.sup.e2, OS(O)OR.sup.e2,
OS(O).sub.2OR.sup.e2, OR.sup.e2, NHR.sup.e2, N(R.sup.e2)R.sup.f2,
.sup.+N(R.sup.e2)(R.sup.f2)R.sup.g2, P(O)(OR.sup.e2)(OR.sup.f2),
OP(O)(OR.sup.e2)(OR.sup.f2), SiR.sup.e2R.sup.f2R.sup.g2,
C(O)R.sup.e2, C(O)OR.sup.e2, C(O)N(R.sup.e2)R.sup.f2,
OC(O)R.sup.e2, OC(O)OR.sup.e2, OC(O)N(R.sup.e2)R.sup.f2,
N(R.sup.e2)C(O)R.sup.f2, N(R.sup.e2)C(O)OR.sup.f2, and
N(R.sup.e2)C(O)N(R.sup.f2)R.sup.g2, wherein R.sup.e2, R.sup.f2, and
R.sup.g2 are independently selected from H and optionally
substituted C.sub.1-3 alkyl, C.sub.1-3 heteroalkyl, C.sub.3
cycloalkyl, or C.sub.1-3 heterocycloalkyl, two or more of R.sup.e2,
R.sup.f2, and R.sup.g2 optionally being joined by one or more bonds
to form one or more optionally substituted carbocycles and/or
heterocycles, provided that R.sup.e2 is not H when R.sup.5 is
R.sup.e2.
[0232] In another embodiment, R.sup.5 is selected from nitro,
halogen, amino, cyano, hydroxy, and optionally substituted
C.sub.1-3 alkylamino, di(C .sub.1-3 alkyl)amino, C.sub.1-3
alkylcarbonylamino, C.sub.1-3 alkoxycarbonylamino, C.sub.1-3
alkylaminocarbonylamino, C.sub.1-3 alkyloxy, C.sub.1-3
alkylcarbonyloxy, C.sub.1-3 alkoxycarbonyloxy, C.sub.1-3
alkylaminocarbonyloxy, or C.sub.1-3 alkyl. In yet another
embodiment, R.sup.5 is optionally substituted linear C.sub.1-3
alkyl. In another embodiment, R.sup.5 is unsubstituted linear
C.sub.1-3 alkyl. In another embodiment, R.sup.5 is selected from
methyl, ethyl, propyl, isopropyl, nitro, CF.sub.3, F, Cl, Br,
cyano, methoxy, ethoxy, propoxy, isopropoxy, amino (NH.sub.2),
methylamino, formyl, hydroxymethyl, and dimethylamino. In another
embodiment, R.sup.5 is methyl, ethyl, methoxy, or ethoxy. In
another embodiment, R.sup.5 is methyl. In other embodiments,
R.sup.5 is ethyl or methoxy or ethoxy.
[0233] R.sup.6 and R.sup.6' may both be hydrogen, or R.sup.6 may be
hydrogen while R.sup.6' is absent. In another embodiment, at least
one of R.sup.6 and R.sup.6' is not hydrogen nor absent. In another
embodiment, R.sup.6 is not hydrogen.
[0234] R.sup.5 and R.sup.6 may be joined to form, together with the
two carbon atoms to which they are attached, an optionally
substituted 5- or 6-membered ring. This ring may for example be a
dihydropyrrole, dihydrofuran, cyclopentene, 1,3-dioxolene,
pyrrolidine, tetrahydrofuran, cyclopentane, or 1,3-dioxolane
moiety.
[0235] The substituents R.sup.16 and R.sup.16' may affect the
degree of shielding of the carbon on which nucleophilic attack can
occur as well. In one embodiment X.sup.4 is CR.sup.16. In a further
embodiment, R.sup.16 is hydrogen. In yet another embodiment,
R.sup.16 is C.sub.1-3 alkyl or C.sub.1-3 heteroalkyl. In another
embodiment, R.sup.16 is methyl or ethyl. In yet another embodiment,
R.sup.16 is methyl. In yet another embodiment, R.sup.16 is
fluoro.
[0236] R.sup.14 and R.sup.14' may affect the degree of shielding of
X.sup.1, or when a compound of formula (I) or (II) is part of a
conjugate or linker-agent conjugate in which the compound of
formula (I) or (II) is connected via X.sup.1, they may affect the
degree of shielding of the linkage between the compound of formula
(I) or (II) and the promoiety. To increase the stability of this
linkage, R.sup.14 may be selected to be other than hydrogen. In one
embodiment, R.sup.14 is hydrogen. In another embodiment, R.sup.14
is methyl. In yet other embodiments, R.sup.14 is chloro or ethyl or
isopropyl. In yet another embodiment, R.sup.5 and R.sup.14 are the
same and not hydrogen. For example, both R.sup.5 and R.sup.14 may
be methyl.
[0237] In one embodiment, R.sup.2, R.sup.2', R.sup.3, R.sup.3',
R.sup.4, R.sup.4', R.sup.5, R.sup.5', R.sup.6, R.sup.6', R.sup.12,
R.sup.16, and R.sup.16' present are each hydrogen. In another
embodiment R.sup.2, R.sup.2', R.sup.3, R.sup.3', R.sup.4, R.sup.4',
R.sup.5', R.sup.6, R.sup.6', R.sup.12, R.sup.16, and R.sup.16'
present are each hydrogen. In yet another embodiment, R.sup.2,
R.sup.2', R.sup.3, R.sup.3', R.sup.4, R.sup.4', R.sup.5, R.sup.5',
R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.12, R.sup.14,
R.sup.14', R.sup.16, R.sup.16', and R.sup.19 present are each
hydrogen. In yet another embodiment, R.sup.2, R.sup.2', R.sup.3,
R.sup.3', R.sup.4, R.sup.4', R.sup.5', R.sup.6, R.sup.6', R.sup.7,
R.sup.7', R.sup.12, R.sup.14, R.sup.14', R.sup.16, R.sup.16', and
R.sup.19 present are each hydrogen.
[0238] Although the alkylation rate and efficiency of compounds of
formulae (I) and (II) may optionally be tuned in several ways, in
one aspect of this invention, this may be achieved by introducing
steric shielding choosing for a compound of formula (I) one or more
of R.sup.2, R.sup.2', R.sup.3, R.sup.3', R.sup.4, R.sup.4',
R.sup.5, R.sup.5', R.sup.6, R.sup.6', R.sup.12, R.sup.16, and
R.sup.16' present to be other than hydrogen and for a compound of
formula (II) one or more of R.sup.2, R.sup.3, R.sup.3', R.sup.4,
R.sup.4', R.sup.5, R.sup.5', R.sup.6, R.sup.6', R.sup.16, and
R.sup.16' present to be other than hydrogen. Substituents should
not cause too much steric hindrance, however, especially when more
than one of these substituents is other than hydrogen, as this
might adversely affect DNA alkylation. Furthermore, it may provide
for less efficient binding in the DNA minor groove and may pose
synthetic difficulties.
[0239] In one aspect of this invention, at least one of R.sup.1,
R.sup.5, R.sup.5', R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14,
R.sup.14', R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10,
R.sup.10', R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15''',
R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21, R.sup.21',
R.sup.22, and R.sup.23 contains a
X.sup.14(CH.sub.2CH.sub.2O).sub.ffCH.sub.2CH.sub.2X.sup.14 moiety,
wherein ff is selected from 1 to 1000 and each X.sup.14 is
independently selected from
##STR00017##
[0240] This moiety must be connected to the core of the
DNA-alkylating moiety or DNA-binding moiety via a direct bond or
via a linking unit that is part of said same R group and that does
not comprise a disulfide, a hydrazone, a hydrazide, an ester, a
natural amino acid, or a peptide containing at least one natural
amino acid. Said linking unit should preferably be cleaved less
than 20%, more preferably less than 10%, and most preferably less
than 5% in 24 hours upon administration of a compound of formula
(I) or (II) in vivo.
[0241] The
X.sup.14(CH.sub.2CH.sub.2O).sub.ffCH.sub.2CH.sub.2X.sup.14 moiety
may for example be selected to be
##STR00018## ##STR00019## ##STR00020## ##STR00021##
[0242] wherein ff is selected from 1 to 1000. In more specific
embodiments, ff is selected from 1 to 100 or from 1 to 10. In other
embodiments, ff is selected to be 1 or 2 or 3 or 4. In another
embodiment, ff is 3 or 4.
[0243] The oligoethylene glycol or polyethylene glycol moiety or
derivative thereof is connected via a linking unit to the core
structure of a compound of formula (I) or (II). Such a linking unit
may be a single bond, in which case the oligoethylene glycol or
polyethylene glycol or derivative thereof is connected to the core
structure via for example an amine, ether, or sulfide bond.
Alternatively, the oligoethylene glycol or polyethylene glycol
moiety or derivative thereof may be connected to the core structure
via for example a carbamate, a carbonate, an amide, an alkyl, a
heteroalkyl, an aryl, or a heteroaryl moiety, or a combination of
any of these. In one embodiment, at least one of R.sup.1, R.sup.5,
R.sup.5', R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14,
R.sup.14', R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10,
R.sup.10', R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15'',
R.sup.15''', R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21,
R.sup.21', R.sup.22, and R.sup.23 is selected from
##STR00022## ##STR00023## ##STR00024##
[0244] wherein hh is selected from 1 to 1000, X.sup.15 is selected
from S and NR.sup.32, each X.sup.16 is independently selected from
O, S, and NR.sup.34, R.sup.30 is independently selected from H and
optionally substituted C.sub.1-10 alkyl, C.sub.1-10 heteroalkyl,
C.sub.3-10 cycloalkyl, C.sub.1-10 heterocycloalkyl, C.sub.5-10
aryl, or C.sub.1-10 heteroaryl, R.sup.32, R.sup.33, and R.sup.34
are independently selected from H and C.sub.1-3 alkyl, and R.sup.31
has the same meaning as defined for R.sup.7. R.sup.30 may for
example be selected from H, methyl, ethyl, methoxymethyl,
p-aminobenzoyl, and p-aminoanilinocarbonyl.
[0245] In a further embodiment, at least one of R.sup.1, R.sup.5,
R.sup.5', R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14,
R.sup.14', R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10,
R.sup.10', R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15'',
R.sup.15''', R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21,
R.sup.21', R.sup.22, and R.sup.23 is selected from
##STR00025## ##STR00026## ##STR00027## ##STR00028##
[0246] In another embodiment, R.sup.1 is selected from
##STR00029##
[0247] In one embodiment, at least one of R.sup.1, R.sup.5,
R.sup.5', R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14,
R.sup.14', R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10,
R.sup.10', R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15'',
R.sup.15''', R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21,
R.sup.21', R.sup.22, and R.sup.23 contains a
X.sup.14(CH.sub.2CH.sub.2O).sub.ffCH.sub.2CH.sub.2X.sup.14 moiety.
In another embodiment, at least one of R.sup.5, R.sup.6, R.sup.7,
and R.sup.14 contains a
X.sup.14(CH.sub.2CH.sub.2O).sub.ffCH.sub.2CH.sub.2X.sup.14 moiety.
In yet another embodiment, at least one of R.sup.6 and R.sup.7
contains a
X.sup.14(CH.sub.2CH.sub.2O).sub.ffCH.sub.2CH.sub.2X.sup.14 moiety.
In yet another embodiment, at least one of R.sup.8, R.sup.8',
R.sup.9, R.sup.9', R.sup.10, R.sup.10', R.sup.11, R.sup.11',
R.sup.15, R.sup.15', R.sup.15'', R.sup.15''', R.sup.16, R.sup.16',
R.sup.20, R.sup.20', R.sup.21, R.sup.21', R.sup.22, and R.sup.23
contains a
X.sup.14(CH.sub.2CH.sub.2O).sub.ffCH.sub.2CH.sub.2X.sup.14 moiety.
In yet another embodiment, at least one of R.sup.8, R.sup.9,
R.sup.10, R.sup.11, R.sup.20, R.sup.21, and R.sup.22 contains a
X.sup.14(CH.sub.2CH.sub.2O).sub.ffCH.sub.2CH.sub.2X.sup.14 moiety.
In yet another embodiment, at least one of R.sup.8 and R.sup.9
contains a
X.sup.14(CH.sub.2CH.sub.2O).sub.ffCH.sub.2CH.sub.2X.sup.14 moiety.
In yet another embodiment, at least R.sup.1 contains a
X.sup.14(CH.sub.2CH.sub.2O).sub.ffCH.sub.2CH.sub.2X.sup.14
moiety.
[0248] A compound of formula (I) or (II) may also contain 2 or more
X.sup.14(CH.sub.2CH.sub.2O).sub.ffCH.sub.2CH.sub.2X.sup.14
moieties. In one embodiment, a compound of formula (I) or (II)
contains 2
X.sup.14(CH.sub.2CH.sub.2O).sub.ffCH.sub.2CH.sub.2X.sup.14
moieties. In another embodiment, a compound of formula (I) or (II)
contains 2
X.sup.14(CH.sub.2CH.sub.2O).sub.ffCH.sub.2CH.sub.2X.sup.14 moieties
that are part of 2 separate R groups. It may be beneficial to put
the two or more
X.sup.14(CH.sub.2CH.sub.2O).sub.ffCH.sub.2CH.sub.2X.sup.14 moieties
at distant positions in the compound of formula (I) or (II) as this
may shield the relatively hydrophobic core more efficiently.
[0249] Compounds of formulae (I) and (II) may contain one or more
oligoethylene glycol or polyethylene glycol moieties or derivatives
thereof. Such a moiety may improve the water solubility and
aggregation behavior of a compound of formula (I) or (II) and may
cause increased activity against multidrug-resistant targets due to
increased polarity. If a compound of formula (I) or (II) with such
a moiety is incorporated in a conjugate, it may be that the
oligoethylene glycol or polyethylene glycol moiety is located in
between the promoiety and the remainder of the compound of formula
(I) or (II) or that it is located at a position somewhat opposite
to the attachment site of the promoiety, thus placing the remainder
of the compound of formula (I) or (II) in between the promoiety and
the oligoethylene glycol or polyethylene glycol moiety. The latter
may be more beneficial for the water solubility of the conjugates.
Improved water solubility of compounds of formulae (I) and (II) and
their conjugates may lead to improved yields and purity of the
conjugates during synthesis, for example due to reduced aggregate
formation. Furthermore, a reduced tendency for aggregation and a
higher purity of the conjugate may for example lead to fewer side
effects after administration of the conjugate. In addition, the
presence of one or more oligoethylene glycol and/or polyethylene
glycol moieties in a conjugate may reduce excretion of the
conjugate via the kidneys or liver, which increases the circulation
time in the body.
[0250] In another aspect of this invention, compounds of formula
(I) and (II) may contain one or more triazole rings. Incorporation
of a 1,2,3-triazole ring may provide for a synthetic advantage as
the two moieties that eventually may become attached to the
1,2,3-triazole ring may be attached to each other via said triazole
ring using a mild and efficient cycloaddition reaction between an
alkyne and azide moiety. Because the conditions for this
cycloaddition reaction are very mild and are compatible with almost
all functional groups, the reaction can be performed in one of the
last steps of the synthetic route towards a compound of formula (I)
or (II), its linker-agent conjugate, or conjugate, thus allowing
for easy generation of series of compounds of formula (I) and (II)
and their conjugates for SAR (structure-activity relationship)
studies.
[0251] Preferably, the triazole moiety is located in such a way
within the DNA-alkylating unit or DNA-binding unit that it can
contribute to the binding of the compound to DNA. Additional
DNA-binding moieties, such as indole or benzofuran moieties, that
are connected to the DNA-binding or DNA-alkylating unit may
increase the potency of the compound, allegedly through enhanced
DNA binding. These additional aromatic moieties may however have a
detrimental effect on pharmacological properties, such as water
solubility. A triazole, being an aromatic group, may also enhance
binding to DNA and thus increase cytotoxic potency of the compound,
but as it is more polar than other aromatic moieties such as a
phenyl ring, negative effects on pharmacological properties may be
less pronounced.
[0252] In one embodiment, this invention relates to a compound of
formula (I) or (II) wherein at least one of R.sup.1, R.sup.5,
R.sup.5', R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14,
R.sup.14', R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10,
R.sup.10', R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15'',
R.sup.15''', R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21,
R.sup.21', R.sup.22, and R.sup.23 contains a triazole moiety.
[0253] In another embodiment, at least one of R.sup.8, R.sup.8',
R.sup.9, R.sup.9', R.sup.10, R.sup.10', R.sup.11, R.sup.11',
R.sup.15, R.sup.15', R.sup.15'', R.sup.15''', R.sup.16, R.sup.16',
R.sup.20, R.sup.20', R.sup.21, R.sup.21', R.sup.22, and R.sup.23
contains a triazole moiety. In another embodiment, at least one of
R.sup.8, R.sup.9, and R.sup.10 contains a triazole moiety. In
another embodiment, at least one of R.sup.8 and R.sup.9 contains a
triazole moiety. In yet another embodiment, at least R.sup.8
contains a triazole moiety.
[0254] In another embodiment, at least one of R.sup.5, R.sup.5',
R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14, and R.sup.14'
contains a triazole moiety. In another embodiment, at least one of
R.sup.6, R.sup.6', R.sup.7, and R.sup.7' contains a triazole
moiety. In yet another embodiment, R.sup.1 contains a triazole
moiety.
[0255] For an optimum DNA-binding effect, the triazole moiety may
be connected via a linker that keeps the triazole moiety in
conjugation with or in close proximity to the core of the
DNA-binding or DNA-alkylating unit. The linker may for example be a
single bond, --N(R.sup.35)C(O)--, --C(O)N(R.sup.35)--, --C(O)--,
--C(R.sup.35)(R.sup.36)--, --C(R.sup.35).dbd.C(R.sup.36)--, --O--,
--S--, or --N(R.sup.35)--, wherein R.sup.35 and R.sup.36 are
selected from H and optionally substituted C.sub.1-4 alkyl or
C.sub.1-4 heteroalkyl, or be any other optionally substituted small
linker that does not have more than 4 connecting atoms (e.g., the
--N(R.sup.20)C(O)-- moiety has two connecting atoms: N and C) in
between the core of the DNA-binding unit or DNA-alkylating unit and
the triazole ring.
[0256] The triazole ring may be a 1,2,3-triazole or a
1,2,4-triazole. In one embodiment, the triazole ring is a
1,2,3-triazole. In another embodiment, the triazole is a
1,2,4-triazole. A 1,2,3-triazole ring may be 4,5-, 1,5-, or
1,4-disubstituted. If the 1,2,3-triazole ring is 1,4-substituted,
this means that the substituent that contains the 1,2,3-triazole
ring has an extended form. If the 1,2,3-triazole ring is 4,5- or
1,5-substituted, the 1,2,3-triazole ring in fact forms a kind of
turn and puts the two substituents on the triazole in close
proximity to each other. The triazole ring may also be located at
the end of the substituent, in which case the triazole ring is only
monosubstituted. Substitution may in this case occur at N-1 or C-4.
A 1,2,4-triazole may be 1,3-, 1,5-, or 3,5-disubstituted. A
substituent that contains a 1,3- or 3,5-disubstituted
1,2,4-triazole has an extended form, whereas in a 1,5-disubstituted
1,2,4-triazole both substituents on the triazole are in close
proximity to each other. The triazole ring may also be
trisubstituted.
[0257] In one aspect, at least one of R.sup.1, R.sup.5, R.sup.5',
R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14, R.sup.14', R.sup.8,
R.sup.8', R.sup.9, R.sup.9', R.sup.10, R.sup.10', R.sup.11,
R.sup.11', R.sup.15, R.sup.15', R.sup.15'', R.sup.15''', R.sup.16,
R.sup.16', R.sup.20, R.sup.20', R.sup.21, R.sup.21', R.sup.22, and
R.sup.23 in a compound of formula (I) or (II) is
##STR00030##
[0258] wherein X.sup.18 and X.sup.19 are selected from O, S,
NR.sup.25, H.sub.2, and C(R.sup.25)R.sup.26, wherein R.sup.25 and
R.sup.26 are selected from H and optionally substituted C.sub.1-3
alkyl or C.sub.1-3 heteroalkyl, and R.sup.24 has the same meaning
as R.sup.8 and is independently selected.
[0259] R.sup.24 may for example be selected from H and
##STR00031##
[0260] wherein jj, jj', jj'', and jj''' are independently selected
from 0 to 8, X.sup.74 is selected from
##STR00032##
[0261] each tt, tt', and tt'' is independently selected from 0 and
1, each X.sup.21 and X.sup.22 is independently selected from O, S,
NR.sup.67, H.sub.2, and C(R.sup.67)R.sup.68, wherein R.sup.67 and
R.sup.68 are independently selected from H and optionally
substituted C.sub.1-3 alkyl or C.sub.1-3 heteroalkyl, and R.sup.66
is selected from H, COOH, CO.sub.2Me, OH, OMe, NR.sup.69R.sup.70,
NR.sup.69C(O)CH.sub.3, SH, SMe,
##STR00033## ##STR00034##
[0262] wherein X.sup.23 is selected from halide, hydroxy,
OC(O)R.sup.bb, and OC(O)OR.sup.bb, or C(O)--X.sup.23 is an active
ester, X.sup.24 is selected from halide, mesyloxy, triflyloxy, and
tosyloxy, R.sup.bb is selected from optionally substituted
C.sub.1-10 alkyl, C.sub.1-10 heteroalkyl, C.sub.3-10 cycloalkyl,
C.sub.1-10 heterocycloalkyl, C.sub.5-10 aryl, and C.sub.1-10
heteroaryl, and R.sup.69, R.sup.70, and R.sup.71 are independently
selected from methyl and H.
[0263] In other embodiments, at least one of R.sup.5, R.sup.6,
R.sup.7, and R.sup.14, or at least one of R.sup.8, R.sup.9,
R.sup.10 and R.sup.11, or at least one of R.sup.6 and R.sup.7, or
at least one of R.sup.8 and R.sup.9, or at least R.sup.8, or at
least R.sup.6, or at least R.sup.7 in a compound of formula (I) or
(II) is
##STR00035##
[0264] wherein R.sup.24, X.sup.18, and X.sup.19 are as defined
hereinabove.
[0265] In some embodiments at least one of R.sup.1, R.sup.5,
R.sup.5', R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14,
R.sup.14', R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10,
R.sup.10', R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15'',
R.sup.15''', R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21,
R.sup.21', R.sup.22, and R.sup.23, or at least one of R.sup.8,
R.sup.9, R.sup.10 and R.sup.11, or at least one of R.sup.8 and
R.sup.9, or at least R.sup.8, or at least one of R.sup.5, R.sup.6,
R.sup.7, and R.sup.14, or at least one of R.sup.6 and R.sup.7 in a
compound of formula (I) or (II) is selected from
##STR00036## ##STR00037##
[0266] wherein R.sup.37, R.sup.38, R.sup.39, and R.sup.40 are
independently selected from H and methyl.
[0267] In other embodiments, at least one of R.sup.1, R.sup.5,
R.sup.5', R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14,
R.sup.14', R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10,
R.sup.10', R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15'',
R.sup.15''', R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21,
R.sup.21', R.sup.22, and R.sup.23, or at least one of R.sup.8,
R.sup.9, R.sup.10, and R.sup.11, or at least one of R.sup.8 and
R.sup.9, or at least R.sup.8, or at least one of R.sup.5, R.sup.6,
R.sup.7, and R.sup.14, or at least one of R.sup.6 and R.sup.7 in a
compound of formula (I) or (II) is selected from
##STR00038## ##STR00039##
[0268] wherein R.sup.38, R.sup.39, and R.sup.40 are independently
selected from H and methyl.
[0269] In other embodiments, at least one of R.sup.1, R.sup.5,
R.sup.5', R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14,
R.sup.14', R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10,
R.sup.10', R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15'',
R.sup.15''', R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21,
R.sup.21', R.sup.22, and R.sup.23, or at least one of R.sup.8,
R.sup.9, R.sup.10, and R.sup.11, or at least one of R.sup.8 and
R.sup.9, or at least R.sup.8, or at least one of R.sup.5, R.sup.6,
R.sup.7, and R.sup.14, or at least one of R.sup.6 and R.sup.7 in a
compound of formula (I) or (II) is selected from
##STR00040## ##STR00041##
[0270] wherein R.sup.38, R.sup.39, and R.sup.40 are independently
selected from H and methyl.
[0271] In other embodiments, at least one of R.sup.1, R.sup.5,
R.sup.5', R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14,
R.sup.14', R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10,
R.sup.10', R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15'',
R.sup.15''', R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21,
R.sup.21', R.sup.22, and R.sup.23, or at least one of R.sup.8,
R.sup.9, R.sup.10, and R.sup.11, or at least one of R.sup.8 and
R.sup.9, or at least R.sup.8, or at least one of R.sup.5, R.sup.6,
R.sup.7, and R.sup.14, or at least one of R.sup.6 and R.sup.7 in a
compound of formula (I) or (II) is selected from
##STR00042## ##STR00043##
[0272] wherein R.sup.38, R.sup.39, and R.sup.40 are independently
selected from H and methyl.
[0273] In one aspect, compounds of formulae (I) and (II) are
represented by compounds of formulae (Ib) and (IIb),
respectively:
##STR00044##
[0274] In one embodiment, X.sup.2 in (Ib) or (IIb) is N.
[0275] In another embodiment, X.sup.2 in (Ib) or (IIb) is
CR.sup.14.
[0276] In a further embodiment, X.sup.2 in (Ib) is CR.sup.14 and a
is 0.
[0277] In another embodiment, X.sup.2 in (Ib) or (IIb) is CH.
[0278] In yet another embodiment, R.sup.5 in (Ib) or (IIb) is
selected from nitro, halogen, amino, cyano, hydroxy, and optionally
substituted C.sub.1-3 alkylamino, di(C.sub.1-3 alkyl)amino,
C.sub.1-3 alkylcarbonylamino, C.sub.1-3 alkoxycarbonylamino,
C.sub.1-3 alkylaminocarbonylamino, C.sub.1-3 alkyloxy, C.sub.1-3
alkylcarbonyloxy, C.sub.1-3 alkylaminocarbonyloxy, or C.sub.1-3
alkyl. In yet another embodiment, R.sup.5 in (Ib) or (IIb) is
optionally substituted linear C.sub.1-3 alkyl. In another
embodiment, R.sup.5 in (Ib) or (IIb) is unsubstituted linear
C.sub.1-3 alkyl. In another embodiment, R.sup.5 in (Ib) or (IIb) is
methyl. In other embodiments, R.sup.5 in (Ib) or (IIb) is ethyl or
methoxy or ethoxy.
[0279] In yet another aspect, compounds of formulae (I) and (II)
are represented by compounds of formulae (Ic) and (IIc),
respectively:
##STR00045##
[0280] In one embodiment, X.sup.2 in (Ic) or (IIc) is NH.
[0281] In yet another aspect, compounds of formulae (I) and (II)
are represented by compounds of formulae (Id) and (IId),
respectively:
##STR00046##
[0282] In one embodiment, X.sup.2 in (Id) or (IId) is NH.
[0283] In another embodiment, compounds of formulae (I) and (II)
are represented by (Ia) and (IIa), respectively:
[0284] DA1-DB (Ia) DA2-DB (IIa)
[0285] wherein DA1 is
##STR00047##
[0286] or an isomer or a mixture of isomers thereof
[0287] In other embodiments, compounds of formulae (I) and (II) are
represented by (Ia) and (IIa), respectively:
[0288] DA1-DB (Ia) DA2-DB (IIa)
[0289] wherein DA1 is
##STR00048## ##STR00049## ##STR00050## ##STR00051## ##STR00052##
##STR00053## ##STR00054## ##STR00055##
[0290] or an isomer of one of these, or a mixture of isomers.
[0291] In other embodiments, compounds of formulae (I) and (II) are
represented by (Ia) and (IIa), respectively:
[0292] DA1-DB (Ia) DA2-DB (IIa)
[0293] wherein DA1 is
##STR00056## ##STR00057##
[0294] or an isomer of one of these, or a mixture of isomers.
[0295] In yet other embodiments, compounds of formulae (I) and (II)
are represented by (Ia) and (IIa), respectively:
[0296] DA1-DB (Ia) DA2-DB (IIa)
[0297] wherein DA1 is
##STR00058## ##STR00059##
[0298] wherein R.sup.54 is selected from H and optionally
substituted C.sub.1-3 alkyl (e.g., methyl or trifluoromethyl),
R.sup.55 is selected from H, methyl, ethyl, and methoxy, X.sup.25
and X.sup.26 are independently selected from O, S, CH.sub.2, and
NR.sup.51, and R.sup.51, R.sup.52, and R.sup.53 are independently
selected from H, C.sub.1-3 alkyl and
##STR00060##
[0299] wherein ii, ii', ii'', and ii''' are independently selected
from 0 to 8, X.sup.74 is selected from
##STR00061##
[0300] each ss, ss', and ss'' is independently selected from 0 and
1, each X.sup.25 and X.sup.26 is independently selected from O, S,
NR.sup.56, H.sub.2, and C(R.sup.56)R.sup.57, wherein R.sup.56 and
R.sup.57 are independently selected from H and optionally
substituted C.sub.1-3 alkyl or C.sub.1-3 heteroalkyl, and R.sup.58
is selected from H, COOH, CO.sub.2Me, OH, OMe, NR.sup.59R.sup.60,
NR.sup.59C(O)CH.sub.3, SH, SMe,
##STR00062## ##STR00063##
wherein X.sup.27 is selected from halide, hydroxy, OC(O)R.sup.aa,
and OC(O)OR.sup.aa, or C(O)--X.sup.23 is an active ester, X.sup.24
is selected from halide, mesyloxy, triflyloxy, and tosyloxy,
R.sup.aa is selected from optionally substituted C.sub.1-10 alkyl,
C.sub.1-10 heteroalkyl, C.sub.3-10 cycloalkyl, C.sub.1-10
heterocycloalkyl, C.sub.5-10 aryl, and C.sub.1-10 heteroaryl, and
R.sup.59, R.sup.60, and R.sup.61 are independently selected from
methyl and H, or an isomer of one of these, or a mixture of
isomers.
[0301] In another embodiment, a compound of formula (I) or (II)
is
##STR00064##
[0302] or an isomer thereof, or a mixture of isomers.
[0303] In another embodiment, a compound of formula (I) or (II)
is
##STR00065##
[0304] or an isomer thereof, or a mixture of isomers.
[0305] In other embodiments, a compound of formula (I) or (II)
is
##STR00066##
[0306] or an isomer of one of these, or a mixture of isomers.
[0307] In one embodiment, in a compound of formula (I) or (II),
b=1. In another embodiment, b=0. In another embodiment, a=0. In yet
another embodiment, a=1. In yet another embodiment, a=0 and
b=1.
[0308] Increased water solubility of a compound of formula (I) or
(II) may not only be achieved through the introduction of
water-soluble or polar groups, such as a triazole group or an
oligoethylene glycol or polyethylene glycol moiety or a combination
thereof, but may also be achieved through substitution of carbon
ring atoms by heteroatoms, for example in the DNA-binding unit.
Improved water solubility of compounds of formulae (I) and (II) and
their conjugates may lead to improved yields and purity of the
conjugates during synthesis, for example due to reduced aggregate
formation. Furthermore, a reduced tendency for aggregation and a
higher purity of the conjugate may for example lead to fewer side
effects after administration of the conjugate.
[0309] Increased metabolic degradation, e.g., in the liver, may for
example be achieved through the introduction of groups in the
DNA-binding units that can be oxidized with relative ease, for
example acetylene and alkene moieties. Oxidation of toxic compounds
is one of the mechanisms by which a mammal may detoxify such
compounds. If compounds of this invention are taken up in the
liver, efficient detoxification may for example circumvent liver
toxicity as a side effect.
[0310] The stability of the linkage between the DNA-binding unit
and DNA-alkylating unit, for example in the circulation, may be
tuned by modifications in the DNA-binding unit. It may be favorable
to have the DNA-alkylating unit and DNA-binding unit linked via an
essentially non-cleavable bond. Especially when the compound of
formula (I) or (II) is administered as the active ingredient,
stability in the circulation may be desired. However, when a
compound of formula (I) or (II) is administered as a part of a
conjugate, it may be favorable to have a bond between the
DNA-alkylating unit and the DNA-binding unit that becomes less
stable in the circulation as soon as the compound of formula (I) or
(II) is prematurely released from the conjugate. This may reduce
toxic side effects as a consequence of premature release of the
agent. The DNA-binding units of this invention may cause a compound
of formula (I) or (II) or its conjugate or linker-agent conjugate
to have a more stable linkage between DNA-binding and
DNA-alkylating unit in the circulation than a compound of formula
(I') or (II'), which may be formed from a compound of formula (I)
or (II) as soon as X.sup.1 is unprotected.
[0311] Extension of the .pi.-conjugated system in the DNA-binding
moiety may increase the binding affinity of the DNA binder for DNA.
The .pi. system may be extended by the introduction of additional
aromatic rings and/or conjugated double and/or triple bonds.
[0312] Promoieties may be connected to the DNA-binding units if a
suitable functional group is present. This may for example be a
hydroxyl group or a primary or secondary amino group. Coupling of a
promoiety to the DNA-binding unit in addition to or instead of to
the alkylating unit, e.g., to X.sup.1, may provide advantages. For
example, the presence of two promoieties may increase
target-selective delivery and/or activation and/or reduce the
amount of free agent in non-targeted areas, thereby reducing side
effects and increasing the therapeutic index.
[0313] The DNA-binding unit DB in a compound of formula (I) or (II)
is selected from structures DB1-DB9:
##STR00067##
[0314] In one embodiment, the DNA-binding unit comprises at least
two aromatic rings of which at least one contains at least one ring
atom that is a heteroatom or the DNA-binding unit comprises at
least a bicyclic aromatic system in which at least one ring atom is
a heteroatom. In another embodiment, the DNA-binding unit comprises
at least two aromatic rings and both contain at least one ring atom
that is a heteroatom or the DNA-binding unit comprises at least a
bicyclic aromatic system in which at least two ring atoms are a
heteroatom.
[0315] In one aspect of this invention, a compound of formula (I)
or (II) has a DNA-binding unit of formula DB1. This moiety
comprises structures that at least contain a 6-membered ring B that
is connected to the DNA-alkylating unit via a fused 5- or
6-membered ring A or vinyl group. The optional heteroatom in said
ring B may provide for improved water solubility with respect to
DNA binder analogs having an all-carbon ring. In one embodiment,
ring B in unit DB1 contains a heteroatom.
[0316] Preferably, ring B is aromatic. It may for example be a
phenyl, pyridine, pyrimidine, pyridazine, pyrazine, 1,3,5-triazine,
1,2,3,5-tetrazine, 1,2,3,4-tetrazine, pentazine, phosphinine,
1,3-diphosphinine, or 1,3-azaphosphinine moiety. Alternatively,
this ring may be non-aromatic and either be unsaturated or
completely saturated.
[0317] A compound of formula (I) or (II) wherein ring B is
connected to the DNA-alkylating unit via a vinyl group may contain
a handle that allows for detoxification by means of for example
oxidation or hydration of the double bond.
[0318] The moiety DB1 may for example be
##STR00068## ##STR00069## ##STR00070## ##STR00071##
[0319] Moiety DB1 may for example also be
##STR00072##
[0320] In another embodiment, the moiety DB1 may be
##STR00073##
[0321] In another embodiment, the moiety DB1 may be
##STR00074##
[0322] In another embodiment, the moiety DB1 may for example be
##STR00075## ##STR00076##
[0323] wherein R.sup.9a has the same meaning as defined for R.sup.9
and is independently selected.
[0324] In a further embodiment, the moiety DB1 may be selected
from
##STR00077##
[0325] In one embodiment, the moiety DB1 is
##STR00078##
[0326] In another embodiment, the moiety DB1 is
##STR00079##
[0327] In yet another embodiment, the moiety DB1 is
##STR00080##
[0328] The moiety DB1 may for example also be
##STR00081##
[0329] In the exemplary structures of DB1, R.sup.8, R.sup.8'
R.sup.9, R.sup.9', R.sup.9a, R.sup.10, R.sup.10', R.sup.11,
R.sup.11', R.sup.15, R.sup.16, and R.sup.21 may for example each
independently be selected to be H, be or contain another moiety
selected from structures DB1-DB9 or a derivative thereof, or be
##STR00082## ##STR00083## ##STR00084## ##STR00085## ##STR00086##
##STR00087## ##STR00088##
[0330] wherein R.sup.62, R.sup.63, R.sup.64, and R.sup.65 are
independently selected from H, C.sub.1-3 alkyl, and
##STR00089##
[0331] wherein jj, jj', jj'', and jj''' are independently selected
from 0 to 8, X.sup.74 is selected from
##STR00090##
[0332] each tt, tt', and tt'' is independently selected from 0 and
1, each X.sup.21 and X.sup.22 is independently selected from O, S,
NR.sup.67, H.sub.2, and C(R.sup.67)R.sup.68, wherein R.sup.67 and
R.sup.68 are independently selected from H and optionally
substituted C.sub.1-3 alkyl or C.sub.1-3 heteroalkyl, and R.sup.66
is selected from H, COOH, CO.sub.2Me, OH, OMe, NR.sup.69R.sup.70,
NR.sup.69C(O)CH.sub.3, SH, SMe,
##STR00091## ##STR00092##
[0333] wherein X.sup.23 is selected from halide, hydroxy,
OC(O)R.sup.bb, and OC(O)OR.sup.bb, or C(O)--X.sup.23 is an active
ester, X.sup.24 is selected from halide, mesyloxy, triflyloxy, and
tosyloxy, R.sup.bb is selected from optionally substituted
C.sub.1-10 alkyl, C.sub.1-10 heteroalkyl, C.sub.3-10 cycloalkyl,
C.sub.1-10 heterocycloalkyl, C.sub.5-10 aryl, and C.sub.1-10
heteroaryl, and R.sup.69, R.sup.70, and R.sup.71 are independently
selected from methyl and H.
[0334] In a further embodiment, the moiety DB1 may for example
be
##STR00093## ##STR00094## ##STR00095## ##STR00096## ##STR00097##
##STR00098## ##STR00099## ##STR00100## ##STR00101##
##STR00102##
[0335] In a further embodiment, the moiety DB1 may be selected
from
##STR00103## ##STR00104## ##STR00105## ##STR00106##
[0336] In a further embodiment, the moiety DB1 may be selected
from
##STR00107## ##STR00108## ##STR00109##
[0337] In a further embodiment, the moiety DB1 may be selected
from
##STR00110## ##STR00111## ##STR00112##
[0338] In another embodiment, the moiety DB1 may for example be
##STR00113## ##STR00114## ##STR00115## ##STR00116##
[0339] In another aspect of this invention, a compound of formula
(I) or (II) has a DNA-binding unit of formula DB2. This moiety
comprises structures that at least contain a 5-membered ring B that
is connected to the DNA-alkylating unit via a fused 5- or
6-membered ring A or a vinyl group. Especially in the latter case,
ring B may be fused to another heterocyclic or carbocyclic aromatic
or non-aromatic ring in order to have an improved DNA-binding
affinity. For reasons of increased water solubility, the fused ring
may be a heterocycle, or a carbocycle substituted with relatively
polar groups that at the same time may provide handles for coupling
to promoieties. A DNA binder in which three or more rings are fused
together to form an aromatic multicyclic system may be less
favorable as this may increase the hydrophobicity and/or the
aggregation tendency of the DNA-binder and therefore increase the
hydrophobicity and/or the aggregation tendency of a compound of
formula (I) or (II) and its conjugates. This may be especially true
for multicyclic aromatic systems in which none or only one of the
ring atoms is a heteroatom.
[0340] DNA binder DB2 may comprise an aromatic core structure.
Alternatively, one or more rings may be non-aromatic and be either
unsaturated or completely saturated.
[0341] A compound of formula (I) or (II) wherein ring B is
connected to the DNA-alkylating unit via a vinyl group may contain
a handle that allows for detoxification by means of for example
oxidation or hydration of the double bond.
[0342] The moiety DB2 may for example be
##STR00117## ##STR00118## ##STR00119##
[0343] wherein R.sup.8a, R.sup.9a, R.sup.10a, and R.sup.11a have
the same meaning as defined for R.sup.8, R.sup.9, R.sup.10, and
R.sup.11, respectively, and are independently selected.
[0344] In a more specific embodiment, the moiety DB2 may for
example be
##STR00120## ##STR00121## ##STR00122##
[0345] wherein R.sup.72 and R.sup.73 are independently selected
from H and methyl.
[0346] In the exemplary structures of DB2, R.sup.8, R.sup.8a,
R.sup.9a, R.sup.10, R.sup.10a, R.sup.11, R.sup.11a, R.sup.15,
R.sup.16, and R.sup.21 may for example each independently be
selected to be H, be or contain another moiety selected from
structures DB1-DB9 or a derivative thereof, or be
##STR00123## ##STR00124## ##STR00125## ##STR00126## ##STR00127##
##STR00128## ##STR00129##
[0347] wherein R.sup.62, R.sup.63, R.sup.64, and R.sup.65 are
independently selected from H, C.sub.1-3 alkyl, and
##STR00130##
[0348] wherein jj, jj', jj'', and jj''' are independently selected
from 0 to 8, X.sup.74 is selected from
##STR00131##
[0349] each tt, tt', and tt'' is independently selected from 0 and
1, each X.sup.21 and X.sup.22 is independently selected from O, S,
NR.sup.67, H.sub.2, and C(R.sup.67)R.sup.68, wherein R.sup.67 and
R.sup.68 are independently selected from H and optionally
substituted C.sub.1-3 alkyl or C.sub.1-3 heteroalkyl, and R.sup.66
is selected from H, COOH, CO.sub.2Me, OH, OMe, NR.sup.69R.sup.70,
NR.sup.69C(O)CH.sub.3, SH, SMe,
##STR00132## ##STR00133##
[0350] wherein X.sup.23 is selected from halide, hydroxy,
OC(O)R.sup.bb, and OC(O)OR.sup.bb, or C(O)--X.sup.23 is an active
ester, X.sup.24 is selected from halide, mesyloxy, triflyloxy, and
tosyloxy, R.sup.bb is selected from optionally substituted
C.sub.1-10 alkyl, C.sub.1-10 heteroalkyl, C.sub.3-10 cycloalkyl,
C.sub.1-10 heterocycloalkyl, C.sub.5-10 aryl, and C.sub.1-10
heteroaryl, and R.sup.69, R.sup.79, and R.sup.71 are independently
selected from methyl and H.
[0351] In a further embodiment, the moiety DB2 may for example
be
##STR00134## ##STR00135## ##STR00136## ##STR00137## ##STR00138##
##STR00139## ##STR00140## ##STR00141## ##STR00142## ##STR00143##
##STR00144## ##STR00145## ##STR00146## ##STR00147## ##STR00148##
##STR00149##
[0352] In another aspect of this invention, a compound of formula
(I) or (II) has a DNA-binding unit of formula DB3 or DB4. These two
moieties comprise structures that are built up of an acetylene
moiety coupled to a 5- or 6-membered ring. This ring may be
aromatic or non-aromatic. In the latter case, it may be either
unsaturated or completely saturated. Furthermore, the 5- or
6-membered ring may be fused to one or more other rings to form an
aromatic or non-aromatic ring system. Such a ring system is
preferably flat as this may increase the DNA binding affinity.
Either polar substituents or heteroatoms in the ring may provide
for increased water solubility and may favorably affect the
pharmacological properties of a compound of formula (I) or (II).
The presence of an acetylene moiety in DNA-binding units DB3 and
DB4 may provide for a handle that allows detoxification by means of
for example oxidation or hydration.
[0353] The moiety DB3 may for example be
##STR00150##
[0354] The moiety DB4 may for example be
##STR00151##
[0355] In a more specific embodiment, the moiety DB3 may for
example be
##STR00152##
[0356] In another more specific embodiment, the moiety DB4 may for
example be
##STR00153##
[0357] wherein R.sup.72 is selected from H and methyl.
[0358] In the exemplary structures of DB3 and DB4, R.sup.8,
R.sup.9, R.sup.10, R.sup.11, and R.sup.20 may for example each
independently be selected to be H, be or contain another moiety
selected from structures DB1-DB9 or a derivative thereof, or be
##STR00154## ##STR00155## ##STR00156## ##STR00157## ##STR00158##
##STR00159## ##STR00160## ##STR00161##
[0359] wherein R.sup.62, R.sup.63, R.sup.64, and R.sup.65 are
independently selected from H, C.sub.1-3 alkyl, and
##STR00162##
[0360] wherein jj, jj', jj'', and jj''' are independently selected
from 0 to 8, X.sup.74 is selected from
##STR00163##
[0361] each tt, tt', and tt'' is independently selected from 0 and
1, each X.sup.23 and X.sup.22 is independently selected from O, S,
NR.sup.67, H.sub.2, and C(R.sup.67)R.sup.68, wherein R.sup.67 and
R.sup.68 are independently selected from H and optionally
substituted C.sub.1-3 alkyl or C.sub.1-3 heteroalkyl, and R.sup.66
is selected from H, COOH, CO.sub.2Me, OH, OMe, NR.sup.69R.sup.70,
NR.sup.69C(O)CH.sub.3, SH, SMe,
##STR00164## ##STR00165##
[0362] wherein X.sup.23 is selected from halide, hydroxy,
OC(O)R.sup.bb, and OC(O)OR.sup.bb, or C(O)--X.sup.23 is an active
ester, X.sup.24 is selected from halide, mesyloxy, triflyloxy, and
tosyloxy, R.sup.bb is selected from optionally substituted
C.sub.1-10 alkyl, C.sub.1-10 heteroalkyl, C.sub.3-10 cycloalkyl,
C.sub.1-10 heterocycloalkyl, C.sub.5-10 aryl, and C.sub.1-10
heteroaryl, and R.sup.69, R.sup.70, and R.sup.71 are independently
selected from methyl and H.
[0363] In a further embodiment, the moiety DB3 may for example
be
##STR00166## ##STR00167##
[0364] In yet a further embodiment, the moiety DB4 may for example
be
##STR00168##
[0365] In another aspect of this invention, a compound of formula
(I) or (II) has a DNA-binding unit of formula DB5. This moiety
comprises structures that are built up of a 5-membered or
6-membered ring coupled to an optionally substituted vinyl moiety.
The 5-membered or 6-membered ring may be aromatic or non-aromatic.
In the latter case it may be either unsaturated or completely
saturated. Polar substituents or heteroatoms in the ring and/or
polar substituents on the vinyl group may provide for increased
water solubility and favorably affect the pharmacological
properties of a compound of formula (I) or (II). Aromatic
substituents on the ring or vinyl moiety may increase the binding
affinity. The presence of a vinyl moiety in DNA-binding unit DB5
may provide for a handle that allows detoxification by means of for
example oxidation or hydration.
[0366] The moiety DB5 may for example be
##STR00169##
[0367] In the exemplary structures of DB5, R.sup.8b, R.sup.9b, and
R.sup.15 may for example each independently be selected to be H, be
or contain another moiety selected from structures DB1-DB9 or a
derivative thereof, or be
##STR00170## ##STR00171## ##STR00172## ##STR00173## ##STR00174##
##STR00175## ##STR00176## ##STR00177##
[0368] wherein R.sup.62, R.sup.63, R.sup.64, and R.sup.65 are
independently selected from H, C.sub.1-3 alkyl, and
##STR00178##
[0369] wherein jj, jj', jj'', and jj''' are independently selected
from 0 to 8, X.sup.74 is selected from
##STR00179##
[0370] each tt, tt', and tt'' is independently selected from 0 and
1, each X.sup.21 and X.sup.22 is independently selected from O, S,
NR.sup.67, H.sub.2, and C(R.sup.67)R.sup.68, wherein R.sup.67 and
R.sup.68 are independently selected from H and optionally
substituted C.sub.1-3 alkyl or C.sub.1-3 heteroalkyl, and R.sup.66
is selected from H, COOH, CO.sub.2Me, OH, OMe, NR.sup.69R.sup.70,
NR.sup.69C(O)CH.sub.3, SH, SMe,
##STR00180## ##STR00181##
[0371] wherein X.sup.23 is selected from halide, hydroxy,
OC(O)R.sup.bb, and OC(O)OR.sup.bb, or C(O)--X.sup.23 is an active
ester, X.sup.24 is selected from halide, mesyloxy, triflyloxy, and
tosyloxy, R.sup.bb is selected from optionally substituted
C.sub.1-10 alkyl, C.sub.1-10 heteroalkyl, C.sub.3-10 cycloalkyl,
C.sub.1-10 heterocycloalkyl, C.sub.5-10 aryl, and C.sub.1-10
heteroaryl, and R.sup.69, R.sup.70, and R.sup.71 are independently
selected from methyl and H.
[0372] In a further embodiment, the moiety DB5 may for example
be
##STR00182##
[0373] In another aspect of this invention, a compound of formula
(I) or (II) has a DNA-binding unit of formula DB6 or DB7. These two
moieties comprise structures that are built up of two 5- or
6-membered rings that are connected together via a direct single
bond. These rings may each independently be aromatic or
non-aromatic. In the latter case, they may be either unsaturated or
completely saturated. Furthermore, ring B may be fused to one or
more other rings to form an aromatic or non-aromatic ring system,
which is preferably flat. This may increase the DNA binding
affinity. Either polar substituents or heteroatoms in one or more
of the rings may provide for increased water solubility and may
favorably affect the pharmacological properties of a compound of
formula (I) or (II).
[0374] The moiety DB6 may for example be
##STR00183## ##STR00184##
[0375] The moiety DB7 may for example be
##STR00185## ##STR00186## ##STR00187## ##STR00188##
[0376] wherein R.sup.8a, R.sup.9a, R.sup.10a, and R.sup.11a have
the same meaning as defined for R.sup.8, R.sup.9, R.sup.10, and
R.sup.11, respectively, and are independently selected.
[0377] In a more specific embodiment, moiety DB6 may for example
be
##STR00189## ##STR00190##
[0378] In another more specific embodiment, moiety DB7 may for
example be
##STR00191## ##STR00192##
[0379] In the exemplary structures of DB6 and DB7, R.sup.8,
R.sup.8a, R.sup.9, R.sup.9a, R.sup.10, R.sup.10a, R.sup.11,
R.sup.11a, R.sup.15, and R.sup.20 may for example each
independently be selected to be H, be or contain another moiety
selected from structures DB1-DB9 or a derivative thereof, or be
##STR00193## ##STR00194## ##STR00195## ##STR00196## ##STR00197##
##STR00198## ##STR00199## ##STR00200##
[0380] wherein R.sup.62, R.sup.63, R.sup.64, and R.sup.65 are
independently selected from H, C.sub.1-3 alkyl, and
##STR00201##
[0381] wherein jj, jj', jj'', and jj''' are independently selected
from 0 to 8, X.sup.74 is selected from
##STR00202##
[0382] each tt, tt', and tt'' is independently selected from 0 and
1, each X.sup.21 and X.sup.22 is independently selected from O, S,
NR.sup.67, H.sub.2, and C(R.sup.67)R.sup.68, wherein R.sup.67 and
R.sup.68 are independently selected from H and optionally
substituted C.sub.1-3 alkyl or C.sub.1-3 heteroalkyl, and R.sup.66
is selected from H, COOH, CO.sub.2Me, OH, OMe, NR.sup.69R.sup.70,
NR.sup.69C(O)CH.sub.3, SH, SMe,
##STR00203## ##STR00204##
[0383] wherein X.sup.23 is selected from halide, hydroxy,
OC(O)R.sup.bb, and OC(O)OR.sup.bb, or C(O)--X.sup.23 is an active
ester, X.sup.24 is selected from halide, mesyloxy, triflyloxy, and
tosyloxy, R.sup.bb is selected from optionally substituted
C.sub.1-10 alkyl, C.sub.1-10 heteroalkyl, C.sub.3-10 cycloalkyl,
C.sub.1-10 heterocycloalkyl, C.sub.5-10 aryl, and C.sub.1-10
heteroaryl, and R.sup.69, R.sup.79, and R.sup.71 are independently
selected from methyl and H.
[0384] In a further embodiment, moiety DB6 may for example be
##STR00205## ##STR00206## ##STR00207##
[0385] In another further embodiment, moiety DB7 may for example
be
##STR00208## ##STR00209##
[0386] In another aspect of this invention, a compound of formula
(I) or (II) has a DNA-binding unit of formula DB8. This moiety
comprises structures that are built up of a monocyclic or
multicyclic ring system coupled to the DNA-alkylating unit via a
methylene unit. Preferably, the DB8 moiety comprises a bicyclic
ring system. The ring system may be aromatic or non-aromatic. In
the latter case it may be either unsaturated or completely
saturated. Either polar substituents or heteroatoms in one or more
of the rings may provide for increased water solubility and may
favorably affect the pharmacological properties of a compound of
formula (I) or (II).
[0387] The moiety DB8 may for example be
##STR00210##
[0388] wherein R.sup.8a, R.sup.9a, R.sup.10a, and R.sup.11a have
the same meaning as defined for R.sup.8, R.sup.9, R.sup.10, and
R.sup.11, respectively, and are independently selected.
[0389] In the exemplary structures of DB8, R.sup.8a, R.sup.9a,
R.sup.10a, R.sup.11a, R.sup.15, R.sup.15', and R.sup.16 may for
example each independently be selected to be H, be or contain
another moiety selected from structures DB1-DB9 or a derivative
thereof, or be
##STR00211## ##STR00212## ##STR00213## ##STR00214## ##STR00215##
##STR00216## ##STR00217## ##STR00218##
[0390] wherein R.sup.62, R.sup.63, R.sup.64, and R.sup.65 are
independently selected from H, C.sub.1-3 alkyl, and
##STR00219##
[0391] wherein jj, jj', jj'', and jj''' are independently selected
from 0 to 8, X.sup.74 is selected from
##STR00220##
[0392] each tt, tt', and tt'' is independently selected from 0 and
1, each X.sup.21 and X.sup.22 is independently selected from O, S,
NR.sup.67, H.sub.2, and C(R.sup.67)R.sup.68, wherein R.sup.67 and
R.sup.68 are independently selected from H and optionally
substituted C.sub.1-3 alkyl or C.sub.1-3 heteroalkyl, and R.sup.66
is selected from H, COOH, CO.sub.2Me, OH, OMe, NR.sup.69R.sup.70,
NR.sup.69C(O)CH.sub.3, SH, SMe,
##STR00221## ##STR00222##
[0393] wherein X.sup.23 is selected from halide, hydroxy,
OC(O)R.sup.bb, and OC(O)OR.sup.bb, or C(O)--X.sup.23 is an active
ester, X.sup.24 is selected from halide, mesyloxy, triflyloxy, and
tosyloxy, R.sup.bb is selected from optionally substituted
C.sub.1-10 alkyl, C.sub.1-10 heteroalkyl, C.sub.3-10 cycloalkyl,
C.sub.1-10 heterocycloalkyl, C.sub.5-10 aryl, and C.sub.1-10
heteroaryl, and R.sup.69, R.sup.70, and R.sup.71 are independently
selected from methyl and H.
[0394] In a further embodiment, moiety DB8 may for example be
##STR00223##
[0395] In another aspect of this invention, a compound of formula
(I) or (II) has a DNA-binding unit of formula DB9. This moiety
comprises structures that are built up of a 5-membered ring that is
directly connected to the nitrogen atom of the DNA-alkylating unit
via a single bond. The 5-membered ring may be connected or fused to
one or more other rings to form a multicyclic ring system, which is
preferably flat. This may increase the DNA binding affinity. The
ring system may be aromatic or non-aromatic. In the latter case it
may be either unsaturated or completely saturated. Either polar
substituents or heteroatoms in one or more of the rings may provide
for increased water solubility and may favorably affect the
pharmacological properties of a compound of formula (I) or (II). In
one embodiment, the DB9 moiety contains at least two ring
heteroatoms.
[0396] The moiety DB9 may for example be
##STR00224##
[0397] wherein R.sup.8a, R.sup.9a, R.sup.10a, and R.sup.11a have
the same meaning as defined for R.sup.8, R.sup.9, R.sup.10, and
R.sup.11, respectively, and are independently selected.
[0398] In the exemplary structures of DB9, R.sup.8a, R.sup.9a,
R.sup.10a, R.sup.11a, and R.sup.9 may for example each
independently be selected to be H, be or contain another moiety
selected from structures DB1-DB9 or a derivative thereof, or be
##STR00225## ##STR00226## ##STR00227## ##STR00228## ##STR00229##
##STR00230## ##STR00231## ##STR00232##
[0399] wherein R.sup.62, R.sup.63, R.sup.64, and R.sup.65 are
independently selected from H, C.sub.1-3 alkyl, and
##STR00233##
[0400] wherein jj, jj', jj'', and jj''' are independently selected
from 0 to 8, X.sup.74 is selected from
##STR00234##
[0401] each tt, tt', and tt'' is independently selected from 0 and
1, each X.sup.21 and X.sup.22 is independently selected from O, S,
NR.sup.67, H.sub.2, and C(R.sup.67)R.sup.68, wherein R.sup.67 and
R.sup.68 are independently selected from H and optionally
substituted C.sub.1-3 alkyl or C.sub.1-3 heteroalkyl, and R.sup.66
is selected from H, COOH, CO.sub.2Me, OH, OMe, NR.sup.69R.sup.70,
NR.sup.69C(O)CH.sub.3, SH, SMe,
##STR00235## ##STR00236##
[0402] wherein X.sup.23 is selected from halide, hydroxy,
OC(O)R.sup.bb, and OC(O)OR.sup.bb, or C(O)--X.sup.23 is an active
ester, X.sup.24 is selected from halide, mesyloxy, triflyloxy, and
tosyloxy, R.sup.bb is selected from optionally substituted
C.sub.1-10 alkyl, C.sub.1-10 heteroalkyl, C.sub.3-10 cycloalkyl,
C.sub.1-10 heterocycloalkyl, C.sub.5-10 aryl, and C.sub.1-10
heteroaryl, and R.sup.69, R.sup.70, and R.sup.71 are independently
selected from methyl and H.
[0403] In a further embodiment, moiety DB9 may for example be
##STR00237##
[0404] In one embodiment of this invention, the DB unit is DB1. In
another embodiment, the DB unit is DB2. In yet another embodiment,
the DB unit is DB3. In yet another embodiment, the DB unit is DB4.
In yet another embodiment, the DB unit is DB5. In yet another
embodiment, the DB unit is DB6. In yet another embodiment, the DB
unit is DB7. In yet another embodiment, the DB unit is DB8. In yet
another embodiment, the DB unit is DB9. In another embodiment, the
DB unit is selected from DB1, DB2, DB3, DB4, DB5, DB6, and DB7. In
another embodiment, the DB unit is selected from DB1, DB2, DB5,
DB6, and DB7. In a further embodiment, DB is selected from DB1,
DB2, DB6, and DB7. In yet a further embodiment, DB is selected from
DB1 and DB2. In yet a further embodiment, DB is selected from DB6
and DB7.
[0405] In one embodiment, R.sup.5, R.sup.5', R.sup.6, R.sup.6',
R.sup.7, and R.sup.7' are independently selected from H, OH, SH,
NH.sub.2, N.sub.3, NO.sub.2, NO, CF.sub.3, CN, C(O)NH.sub.2, C(O)H,
C(O)OH, halogen, R.sup.e, SR.sup.e, S(O)R.sup.e, S(O).sub.2R.sup.e,
S(O)OR.sup.e, S(O).sub.2OR.sup.e, OS(O)R.sup.e, OS(O).sub.2R.sup.e,
OS(O)OR.sup.e, OS(O).sub.2OR.sup.e, OR.sup.e, NHR.sup.e,
N(R.sup.e)R.sup.f, .sup.+N(R.sup.e)(R.sup.f)R.sup.g,
P(O)(OR.sup.e)(OR.sup.f), OP(O)(OR.sup.e)(OR.sup.f),
SiR.sup.eR.sup.fR.sup.g, C(O)R.sup.e, C(O)OR.sup.e,
C(O)N(R.sup.e)R.sup.f, OC(O)R.sup.e, OC(O)OR.sup.e,
OC(O)N(R.sup.e)R.sup.f, N(R.sup.e)C(O)R.sup.f,
N(R.sup.e)C(O)OR.sup.f, and N(R.sup.e)C(O)N(R.sup.f)R.sup.g,
wherein R.sup.e, R.sup.f, and R.sup.g are independently selected
from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-15 alkyl, C.sub.1-15 heteroalkyl, C.sub.3-15 cycloalkyl,
C.sub.1-15 heterocycloalkyl, C.sub.5-15 aryl, or C.sub.1-15
heteroaryl, wherein ee is selected from 1 to 1000, X.sup.13 is
selected from O, S, and NR.sup.f1, and R.sup.f1 and R.sup.e1 are
independently selected from H and C.sub.1-3 alkyl, two or more of
R.sup.e, R.sup.f, and R.sup.g optionally being joined by one or
more bonds to form one or more optionally substituted carbocycles
and/or heterocycles, or R.sup.5+R.sup.5' and/or R.sup.6+R.sup.6'
and/or R.sup.7+R.sup.7' are independently selected from .dbd.O,
.dbd.S, .dbd.NOR.sup.e3, .dbd.C (R.sup.e3)R.sup.e4, and
.dbd.NR.sup.e3, R.sup.e3 and R.sup.e4 being independently selected
from H and optionally substituted C.sub.1-3 alkyl, or
R.sup.5'+R.sup.6' and/or R.sup.6'+R.sup.7' and/or
R.sup.7'+R.sup.14' are absent, resulting in a double bond between
the atoms designated to bear R.sup.5' and R.sup.6', and/or R.sup.6'
and R.sup.7', and/or R.sup.7'and R.sup.14', respectively, two or
more of R.sup.5, R.sup.5', R.sup.6, R.sup.6', R.sup.7, R.sup.7',
R.sup.14, and R.sup.14' optionally being joined by one or more
bonds to form one or more optionally substituted carbocycles and/or
heterocycles.
[0406] In another embodiment R.sup.8, R.sup.8', R.sup.9, R.sup.9',
R.sup.10, R.sup.10', R.sup.11, R.sup.11', R.sup.15, R.sup.15',
R.sup.15'', R.sup.15''', R.sup.16, R.sup.16', R.sup.20, R.sup.20',
R.sup.21, R.sup.21', R.sup.22, and R.sup.23 are each independently
selected from H, OH, SH, NH.sub.2, N.sub.3, NO.sub.2, NO, CF.sub.3,
CN, C(O)NH.sub.2, C(O)H, C(O)OH, halogen, R.sup.h, SR.sup.h,
S(O)R.sup.h, S(O).sub.2R.sup.h, S(O)OR.sup.h, S(O).sub.2OR.sup.h,
OS(O)R.sup.h, OS(O).sub.2R.sup.h, OS(O)OR.sup.h,
OS(O).sub.2OR.sup.h, OR.sup.h, NHR.sup.h, N(R.sup.h)R.sup.i,
.sup.+N(R.sup.h)(R.sup.i)R.sup.j, P(O)(OR.sup.h)(OR.sup.i),
OP(O)(OR.sup.h)(OR.sup.i), SiR.sup.hR.sup.iR.sup.j, C(O)R.sup.h,
C(O)OR.sup.h, C(O)N(R.sup.h)R.sup.i, OC(O)R.sup.h, OC(O)OR.sup.h,
OC(O)N(R.sup.h)R.sup.i, N(R.sup.h)C(O)R.sup.i,
N(R.sup.h)C(O)OR.sup.i, and N(R.sup.h)C(O)N(R.sup.i)R.sup.j,
wherein R.sup.h, R.sup.i, and R.sup.j are independently selected
from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-15 alkyl, C.sub.1-15 heteroalkyl, C.sub.3-15 cycloalkyl,
C.sub.1-15 heterocycloalkyl, C.sub.5-15 aryl, or C.sub.1-15
heteroaryl, two or more of R.sup.h, R.sup.i, and R.sup.j optionally
being joined by one or more bonds to form one or more optionally
substituted carbocycles and/or heterocycles, or R.sup.8+R.sup.8'
and/or R.sup.9+R.sup.9' and/or R.sup.10+R.sup.10' and/or
R.sup.11+R.sup.11' and/or R.sup.15+R.sup.15' and/or
R.sup.15''+R.sup.15''' and/or R.sup.16+R.sup.16' and/or
R.sup.20+R.sup.20' and/or R.sup.21+R.sup.21' are independently
selected from .dbd.O, .dbd.S, .dbd.NOR.sup.h1,
.dbd.C(R.sup.h1)R.sup.h2, and .dbd.NR.sup.h1, R.sup.h1 and R.sup.h1
being independently selected from H and optionally substituted
C.sub.1-3 alkyl, two or more of R.sup.8, R.sup.8', R.sup.9,
R.sup.9', R.sup.10, R.sup.10', R.sup.11, R.sup.11', R.sup.15,
R.sup.15', R.sup.15'', R.sup.15''', R.sup.16, R.sup.16', R.sup.20,
R.sup.20', R.sup.21, R.sup.21', R.sup.22, and R.sup.23 optionally
being joined by one or more bonds to form one or more optionally
substituted carbocycles and/or heterocycles.
[0407] In another embodiment, X.sup.3 is not represented by
--X.sup.3a and X.sup.3b--.
[0408] In a further embodiment, if DB is DB2 in a compound of
formula (I) or (II), then X.sup.1 is O.
[0409] In a further embodiment, if DB is DB2 in a compound of
formula (I) or (II) and X.sup.3 is represented by --X.sup.3a and
X.sup.3b--, then X.sup.1 is O.
[0410] Any of the substituents present on any of the rings in DB1,
DB2, DB3, DB4, DB5, DB6, DB7, DB8, and DB9 may be or comprise
another DB1, DB2, DB3, DB4, DB5, DB6, DB7, DB8, or DB9 moiety or
any other DNA-binding moiety. Such another DB moiety or DNA-binding
moiety may be connected to the first DB moiety via for example an
amide or ketone linkage.
[0411] In one embodiment, at least one ring in the DNA-binding
moiety is aromatic. In another embodiment, at least one ring system
is aromatic. In yet another embodiment, all rings in the
DNA-binding moiety are aromatic or form an aromatic ring system. In
yet another embodiment, the DNA-binding moiety contains at least a
bicyclic aromatic moiety.
[0412] Substituents R.sup.1 to R.sup.23 may assist in improving the
pharmacological properties of a compound of formula (I) or (II) or
its conjugate, for example, its water solubility. This may for
example be achieved by selecting one or more of the substituents
R.sup.1, R.sup.5, R.sup.5', R.sup.6, R.sup.6', R.sup.7, R.sup.7',
R.sup.14, R.sup.14', R.sup.8, R.sup.8', R.sup.9, R.sup.9',
R.sup.10, R.sup.10', R.sup.11, R.sup.11', R.sup.15, R.sup.15',
R.sup.15'', R.sup.15''', R.sup.16, R.sup.20, R.sup.20', R.sup.21,
R.sup.21', R.sup.22, and R.sup.23 to comprise or be an
oligoethylene glycol or polyethylene glycol moiety or a triazole
moiety. Alternatively or simultaneously, one or more of the
substituents may comprise or be a water-soluble group. The presence
of a water-soluble group may not only result in enhanced water
solubility, but may also prevent a compound of formula (I) or (II)
from crossing a biological barrier, especially when it is an apolar
barrier, such as a cell membrane. This may be advantageous,
especially when a compound of formula (I) or (II) is delivered into
a targeted cell through conjugation to a targeting moiety before it
is released from the conjugate as the compound of formula (I) or
(II) will be unable to leave the cell. Even active transport via
for example the P-glycoprotein pump may be (partially) impaired.
When a compound of formula (I) or (II) is prematurely released from
the conjugate, e.g., in the circulation, it may be unable or only
moderately able to enter (non-targeted) cells aspecifically as its
membrane translocation capabilities may be impaired by the
water-soluble group. This may lead to increased selectivity and
therefore to fewer side effects. In addition, at least in some
instances, for example when the water-soluble group is positively
charged under physiological conditions, the water-soluble group may
also improve the binding affinity for DNA by means of favorable
electrostatic interactions with the negatively charged phosphate
groups.
[0413] A water-soluble group is a group that imparts increased
solubility on a compound of formula (I) or (II) and/or a conjugate
thereof. In one embodiment, water solubility of a compound of this
invention carrying a water-soluble group is increased by more than
100% compared to the compound lacking said water-soluble group. In
other embodiments, water solubility of a compound of this invention
carrying a water-soluble group is increased by more than 75% or 50%
or 25% or 10% compared to the compound lacking said water-soluble
group. The water-soluble group may also contribute to prevent or
reduce aggregation of compounds of this invention or to reduce side
effects. Examples of water-soluble groups include, but are not
limited to, --NH.sub.2, --NH--, --NHR.sup.s, --NR.sup.s--,
--N(R.sup.s)(R.sup.t), --.sup.+N(R.sup.s)(R.sup.t)--,
--.sup.+N(R.sup.s)(R.sup.t)(R.sup.u), --COOH, --OP(O)(OH).sub.2,
--OP(O)(OH)O--, --OP(O)(OR.sup.s)O--, --OP(O)(OH)OR.sup.s,
--OP(O)(OR.sup.s)OR.sup.t, --P(O)(OH).sub.2, --P(O)(OH)O--,
--P(O)(OR.sup.s)OH, --P(O)(OR.sup.s)O--,
--P(O)(OR.sup.s)(OR.sup.t), --OS(O).sub.2OH, --OS(O).sub.2O--,
--OS(O).sub.2OR.sup.s, --S(O).sub.2OH, --S(O).sub.2O--,
--S(O).sub.2OR.sup.s, --OS(O)OH, --OS(O)O--, --OS(O)OR.sup.s,
--S(O)OH, --S(O)O--, --OS(O)--, --S(O)OR.sup.s, --OS(O).sub.2--,
--OS(O).sub.2R.sup.s, --S(O).sub.2--, --S(O).sub.2R.sup.s,
--OS(O)R.sup.s, --S(O)--, --S(O)R.sup.s,
--(OCH.sub.2CH.sub.2).sub.vOH, --(OCH.sub.2CH.sub.2).sub.vO--,
--(OCH.sub.2CH.sub.2).sub.vOR.sup.s, a sugar moiety, a mono-, di-,
or oligosaccharide moiety, and an oligopeptide moiety, or a
protonated or deprotonated form thereof and further any combination
thereof, wherein R.sup.s, R.sup.t, and R.sup.u are independently
selected from H and optionally substituted C.sub.1-3 alkyl, two or
more of R.sup.s, R.sup.t, and R.sup.u optionally being joined by
one or more bonds to form one or more carbocycles and/or
heterocycles, and v' is an integer selected from 2 to 1000. The
water-soluble group may be at any position within a substituent or
may constitute the whole substituent. The water-soluble group may
for example be located at any interior position, be part of the
main chain, be part of a ring structure, be a functional group
pending to the main chain or a ring, or be placed at the position
at which the substituent is attached to the remainder of the
agent.
[0414] In one embodiment at least one of R.sup.1, R.sup.5,
R.sup.5', R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14,
R.sup.14', R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10,
R.sup.10', R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15'',
R.sup.15''', R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21,
R.sup.21', R.sup.22, and R.sup.23 contains a water-soluble
group.
[0415] In another embodiment, at least one of R.sup.6, R.sup.7,
R.sup.14, R.sup.8, R.sup.9, and R.sup.10 contains a water-soluble
group.
[0416] In yet other embodiments, R.sup.8 or R.sup.9 or R.sup.10 or
R.sup.6 or R.sup.7 or R.sup.14 contains a water-soluble group.
[0417] In one embodiment, the water-soluble group is a carboxylic
acid group.
[0418] In another embodiment, the water-soluble group is an amino
group.
[0419] In further embodiments, the water-soluble group is a primary
or secondary or tertiary or quaternary amino (ammonium) group. In
other embodiments, the water-soluble group is a primary or
secondary or tertiary or quaternary aliphatic amino (ammonium)
group.
[0420] In other embodiments, the water-soluble group is a
phosphonate group or a phosphate group or a sulfonate group or a
sulfate group or a glycol group or an oligoethylene glycol group or
an polyethylene glycol group.
[0421] A compound of formula (I) or (II) may not have a reactive
moiety incorporated in its structure. On the other hand, as becomes
clear from the above, a reactive moiety may be present in its
structure that allows for reaction of a compound of formula (I) or
(II) with another moiety. For example, a compound of formula (I) or
(II) may be reacted with a targeting moiety or a linker-targeting
moiety construct, e.g., an antibody or an antibody fragment, or an
antibody-linker construct or an antibody fragment-linker construct,
to prepare a targeting moiety-agent conjugate in one or more steps,
which may or may not be a conjugate of formula (III). Such a
targeting moiety-agent conjugate may either be cleavable or
non-cleavable. The formation of a targeting moiety-agent conjugate
may not only be carried out through chemical synthesis, but may
also occur in situ, i.e., upon administration of a compound of
formula (I) or (II) in vivo. The compound of formula (I) or (II)
may for example bind to endogenous proteins, e.g., albumin, upon
administration.
[0422] In one embodiment, a compound of formula (I) or (II) is
##STR00238##
[0423] or an isomer thereof, or a mixture of isomers.
[0424] In a further embodiment, a compound of formula (I) or (II)
is
##STR00239##
[0425] or an isomer of one of these, or a mixture of isomers.
[0426] In a further embodiment, a compound of formula (I) or (II)
is
##STR00240##
[0427] or an isomer of one of these, or a mixture of isomers.
[0428] In yet a further embodiment, a compound of formula (I) or
(II) is
##STR00241## ##STR00242## ##STR00243## ##STR00244##
##STR00245##
[0429] or an isomer of one of these, or a mixture of isomers.
[0430] In yet a further embodiment, a compound of formula (I) or
(II) is
##STR00246## ##STR00247## ##STR00248##
[0431] or an isomer of one of these, or a mixture of isomers.
[0432] In yet a further embodiment, a compound of formula (I) or
(II) is
##STR00249## ##STR00250## ##STR00251## ##STR00252##
[0433] or an isomer of one of these, or a mixture of isomers.
[0434] In yet a further embodiment, a compound of formula (I) or
(II) is
##STR00253## ##STR00254## ##STR00255## ##STR00256##
[0435] or an isomer of one of these, or a mixture of isomers.
[0436] In yet a further embodiment, a compound of formula (I) or
(II) is
##STR00257## ##STR00258## ##STR00259##
[0437] or an isomer of one of these, or a mixture of isomers.
[0438] In yet a further embodiment, a compound of formula (I) or
(II) is
##STR00260## ##STR00261## ##STR00262## ##STR00263##
[0439] or an isomer of one of these, or a mixture of isomers.
[0440] In yet a further embodiment, a compound of formula (I) or
(II) is
##STR00264## ##STR00265## ##STR00266## ##STR00267##
[0441] or an isomer of one of these, or a mixture of isomers.
[0442] Conjugates, Linker-Agent Conjugates and Bifunctional
Linkers
[0443] In another aspect, this invention relates to a conjugate of
a compound of formula (I) or (II) that can be converted in vivo in
one or more steps to a compound of formula (I) or (II),
respectively. The conjugate may also be converted to a derivative
of a compound of formula (I) or (II) in which a part of the
promoiety attached to a compound of formula (I) or (II) in the
conjugate remains attached to the compound of formula (I) or (II)
after in vivo conversion. An alternative way of looking at this is
that the remaining moiety of the linker is part of the compound of
formula (I) or (II).
[0444] These conjugates may favorably affect the pharmacological
properties and other characteristics of a compound of formula (I)
or (II). In one embodiment, this invention relates to a conjugate
comprising a compound of formula (I) or (II) conjugated to at least
one promoiety. In another embodiment, this invention relates to a
conjugate comprising a compound of formula (I) or (II) conjugated
to a promoiety.
[0445] In a further embodiment, this invention relates to a
compound of formula (III):
##STR00268##
[0446] or a pharmaceutically acceptable salt, hydrate, or solvate
thereof, wherein
[0447] V.sup.2 is either absent or a functional moiety;
[0448] each L.sup.2 is independently absent or a linking group
linking V.sup.2 to L;
[0449] each L is independently absent or a linking group linking
L.sup.2 to one or more V.sup.1 and/or Y;
[0450] each V.sup.1 is independently absent or a
conditionally-cleavable or conditionally-transformable moiety,
which can be cleaved or transformed by a chemical, photochemical,
physical, biological, or enzymatic process;
[0451] each Y is independently absent or a self-eliminating spacer
system which is comprised of 1 or more self-elimination spacers and
is linked to V.sup.1, optionally L, and one or more Z;
[0452] each p and q are numbers representing a degree of branching
and are each independently a positive integer;
[0453] z is a positive integer equal to or smaller than the total
number of attachment sites for Z;
[0454] each Z is independently a compound of formula (I), (II),
(I'), or (II') as defined hereinabove wherein one or more of
X.sup.1, R.sup.5, R.sup.5', R.sup.6, R.sup.6', R.sup.7, R.sup.7',
R.sup.14, R.sup.14', R.sup.8, R.sup.8', R.sup.9, R.sup.9',
R.sup.10, R.sup.10', R.sup.11, R.sup.11', R.sup.15, R.sup.15',
R.sup.15'', R.sup.15''', R.sup.16, R.sup.16', R.sup.20, R.sup.20',
R.sup.21, R.sup.21', R.sup.22, and R.sup.23 may optionally in
addition be substituted by or be a substituent of formula (V):
##STR00269##
[0455] wherein each V.sup.2', L.sup.2', L', V.sup.1', Z', p', q',
and z' has the same meaning as defined for V.sup.2, L.sup.2, L,
V.sup.1, Y, Z, p, q, and z, respectively, and is independently
selected, the one or more substituents of formula (V) being
independently connected via Y' to one or more of X.sup.1, R.sup.5,
R.sup.5', R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14,
R.sup.14', R.sup.8, R.sup.8', R.sup.9, R.sup.9', R.sup.10,
R.sup.10', R.sup.11, R.sup.11', R.sup.15, R.sup.15', R.sup.15'',
R.sup.15''', R.sup.16, R.sup.16', R.sup.20, R.sup.20', R.sup.21,
R.sup.21', R.sup.22, R.sup.23, and/or to one or more atoms bearing
these R substituents;
[0456] each Z is independently connected to Y through either
X.sup.1, an atom in R.sup.5, R.sup.5', R.sup.6, R.sup.6', R.sup.7,
R.sup.7', R.sup.14, R.sup.14', R.sup.8, R.sup.8', R.sup.9,
R.sup.9', R.sup.10, R.sup.10', R.sup.11, R.sup.11', R.sup.15,
R.sup.15', R.sup.15'', R.sup.15''', R.sup.16, R.sup.16', R.sup.20,
R.sup.20', R.sup.21, R.sup.21', R.sup.22, R.sup.23, or an atom
bearing any of these R substituents; and
[0457] at least V.sup.2 or a V.sup.1 is present.
[0458] In a further aspect, this invention relates to a compound of
formula (III), wherein
[0459] V.sup.2 is present and selected to be a targeting moiety and
there is at least one group of formula (V) that contains a V.sup.1'
moiety and either comprises a V.sup.2', L.sup.2', or L' moiety that
contains a
X.sup.14(CH.sub.2CH.sub.2O).sub.ggCH.sub.2CH.sub.2X.sup.14 moiety,
wherein gg is selected from 3 to 1000 and each X.sup.14 is
independently selected from
##STR00270##
[0460] or said same group of formula (V) comprises at least 2
X.sup.14CH.sub.2CH.sub.2OCH.sub.2CH.sub.2X.sup.14 moieties, in
which each X.sup.14 is independently selected.
[0461] In a further aspect, this invention relates to a compound of
formula (III) that contains at least one
X.sup.14(CH.sub.2CH.sub.2O).sub.ffCH.sub.2CH.sub.2X.sup.14 moiety,
wherein ff is selected from 1 to 1000 and each X.sup.14 is
independently selected from
##STR00271##
[0462] It should be understood from formula (III) that L can be
connected to V.sup.1 and/or to Y. If L is connected to Y, this
means that both V.sup.1 and L, as well as one or more Z, are
connected to Y. If L is connected to V.sup.1, this means that
V.sup.1 and one or more Z are connected to Y. L may also be
connected to both V.sup.1 and Y at the same time. If Y is absent, L
is connected to V.sup.1 or, if V.sup.1 is absent, L is directly
connected to Z.
[0463] The V.sup.2(-L.sup.2-L-(V.sup.1--Y)).sub.p).sub.q(Z).sub.z-1
and one or more
V.sup.2'(-L.sup.2'-L'(-(V.sup.1'--Y')).sub.p').sub.q')(Z').sub.z'-1
moieties, wherein L(-(V.sup.1--Y)).sub.p indicates that L can be
connected to V.sup.1 and/or to Y, connected to Z are herein
referred to as promoieties.
[0464] The present invention also relates to a compound of formula
(IV):
##STR00272##
[0465] or a pharmaceutically acceptable salt, hydrate, or solvate
thereof, wherein
[0466] RM is a reactive moiety and L, V.sup.1, Y, Z, p, and z are
as defined hereinabove, except that L is now linking RM to one or
more V.sup.1 and/or Y, and V.sup.1, Y, and Z may contain protecting
groups, and the one or more V.sup.2'-L.sup.2' moieties optionally
present in Z as defined hereinabove may optionally and
independently be RM' instead, which is a reactive moiety, and
wherein, if there is more than 1 reactive moiety in (IV), some or
all reactive moieties are the same or different. These linker-agent
conjugates of formula (IV) may or may not be considered
intermediates for compounds of formula (III). In a compound of
formula (IV), RM must be present while V.sup.1 may be either
present or absent.
[0467] In a further aspect, the present invention relates to a
compound of formula (IV), wherein RM is a reactive moiety selected
from carbamoyl halide [--N(R)C(O)X], acyl halide [--C(O)X], active
ester [--C(O)OR], anhydride [--C(O)OC(O)OR], .alpha.-haloacetyl
[.alpha.C(O)CH.sub.2X], .alpha.-haloacetamide
[--N(R)C(O)CH.sub.2X], maleimide, isocyanate [--N.dbd.C.dbd.O],
isothiocyanate [--N.dbd.C.dbd.S], disulfide [--S--SR], thiol
[--SH], hydrazine [--NH.sub.2NH.sub.2], hydrazide
[--C(O)NH.sub.2NH.sub.2], sulfonyl chloride [--S(O).sub.2Cl],
aldehyde [--C(O)H], methyl ketone [--C(O)CH.sub.3], vinyl sulfone
[--S(O).sub.2--CH.dbd.CH.sub.2], halomethyl [--CH.sub.2Cl], and
methyl sulfonate [--CH.sub.2OS(O).sub.2R], and wherein at least one
group of formula (V), being part of Z, contains a moiety and either
comprises a V.sup.2', L.sup.2', or L' moiety that contains a
X.sup.14(CH.sub.2CH.sub.2O).sub.ggCH.sub.2CH.sub.2X.sup.14 moiety,
wherein gg is selected from 3 to 1000 and each X.sup.14 is
independently selected from
##STR00273##
[0468] or said same group of formula (V) comprises at least 2
X.sup.14CH.sub.2CH.sub.2OCH.sub.2CH.sub.2X.sup.14 moieties, in
which each X.sup.14 is independently selected. These linker-agent
conjugates of formula (IV) may or may not be considered
intermediates for compounds of formula (III). In such a compound of
formula (IV), RM must be present.
[0469] In another embodiment, a compound of formula (IV) contains
an RM moiety selected from carbamoyl halide [--N(R)C(O)X], acyl
halide [--C(O)X], active ester [--C(O)OR], anhydride
[--C(O)OC(O)OR], .alpha.-haloacetyl [--C(O)CH.sub.2X],
.alpha.-haloacetamide [--N(R)C(O)CH.sub.2X], maleimide, isocyanate
[--N.dbd.C.dbd.O], isothiocyanate [--N.dbd.C.dbd.S], disulfide
[--S--SR], thiol [--SH], hydrazine [--NH.sub.2NH.sub.2], hydrazide
[--C(O)NH.sub.2NH.sub.2], sulfonyl chloride [--S(O).sub.2Cl],
aldehyde [--C(O)H], methyl ketone [--C(O)CH.sub.3], vinyl sulfone
[--S(O).sub.2--CH.dbd.CH.sub.2], halomethyl [--CH.sub.2Cl], and
methyl sulfonate [--CH.sub.2OS(O).sub.2R].
[0470] The RM-L(-(V.sup.1--Y)).sub.p(Z).sub.z-1 and one or more
RM'-L'(-(V.sup.1'--Y')).sub.p'(Z').sub.z'-1 moieties, wherein
L(-(V.sup.1--Y)).sub.p indicates that L can be connected to V.sup.1
and/or to Y, connected to Z are herein referred to as
promoieties.
[0471] In yet a further aspect, this invention relates to novel
bifunctional linkers that contain a cleavage site, a
self-elimination spacer system and two reactive moieties, one of
which can be reacted with a therapeutic or diagnostic moiety, e.g.
a compound of formula (II or (II), and the other of which can be
reacted with a functional moiety, such as a targeting moiety. These
bifunctional linkers can be used to prepare conjugates of formulae
(III) and (IV) of this invention or similar compounds with
different therapeutic or diagnostic moieties.
[0472] More specifically, this invention relates to a compound of
formula (VIII):
##STR00274##
[0473] or a pharmaceutically acceptable salt, hydrate, or solvate
thereof, wherein
[0474] L, V.sup.1, Y, RM, p, and z are as defined for a compound of
formula (IV), and RM2 is a reactive moiety or a leaving group. RM
and each RM2 are independently selected. These bifunctional linkers
of formula (VIII) may or may not be considered intermediates for
compounds of formula (III) and (IV). Furthermore, these compounds
may be considered to be intermediates for conjugates and
linker-agent conjugates similar to compounds of formula (III) and
(IV) in which the Z moiety is a therapeutic or diagnostic moiety
different from a compound of formula (I), (II), (I'), or (II'), or
a promoiety-containing derivative thereof. Therefore, in another
aspect, this invention relates to a conjugate of formula (III) in
which the one or more Z moieties are independently a therapeutic or
diagnostic moiety. In yet another aspect, this invention relates to
a linker-agent conjugate of formula (IV) in which the one or more Z
moieties are independently a therapeutic or diagnostic moiety.
[0475] It is noted that the separate X.sup.14 moieties in the
--CH.sub.2CH.sub.2X.sup.14 moieties that may be present in a
compound of formula (III), (IV), or (VIII) are independently
selected.
[0476] It is also noted that z does not represent a degree of
polymerization; hence z does not indicate that a number of moieties
Z or RM2 are connected to one another.
[0477] It is further noted that if Y or Y' is connected to an atom
of Z or RM2 bearing a specific R substituent instead of to this R
substituent itself, this in fact means that this R substituent is
absent if this is necessary to meet valency rules.
[0478] It is further noted that if X.sup.14 in for example
--CH.sub.2CH.sub.2X.sup.14 represents
##STR00275##
then --CH.sub.2CH.sub.2X.sup.14 should be read as
--CH.sub.2CHX.sup.14.
[0479] It is noted that if a compound of this invention contains an
oligoethylene glycol or polyethylene glycol moiety the actual
number of ethylene glycol units may vary from molecule to molecule
and the variable designating the number of units actually
represents the average number of units. Although the average number
of ethylene glycol units is generally limited to 1000 in the
definitions of compounds of this invention, compounds with a larger
average number of ethylene glycol units are also encompassed by
this invention.
[0480] It should be understood that this invention relates to
enantiomerically pure and/or diastereomerically pure compounds of
formulae (III), (IV), and (VIII) as well as to enantiomeric and/or
diastereomeric mixtures of compounds of formulae (III), (IV), and
(VIII).
[0481] When a compound of formula (III) or (IV) contains attachment
sites in Y for Z that are not coupled to Z, for instance as a
consequence of an incomplete coupling reaction with Z during
synthesis, these attachment sites are considered to be attached to
H, OH, a reactive moiety (e.g., RM2), or a leaving group (e.g.,
RM2) instead. If all of said attachment sites are connected to Z,
then z equals the number of said attachment sites; otherwise, z is
lower. Compounds of this invention may exist as a mixture, wherein
each component of the mixture has a different z value. For example,
the compound may exist as a mixture of two separate compounds, one
compound wherein z is 4 and another compound wherein z is 3.
Furthermore, for a given z, the compound may exist as a mixture of
(constitutional) isomers as Z may be connected to distinct (sets
of) attachment sites.
[0482] For reasons of clarity, when referring to the connections of
one first moiety to other moieties within formula (III), (IV), or
(VIII), in general only those said other moieties are mentioned
that are directly attached to said first moiety in formula (III),
(IV), or (VIII). It should be understood that if one of said other
moieties is not present, said first moiety is actually connected to
the moiety first in line that is present, unless explicitly stated
otherwise. For example, if it is stated that "V.sup.1 is cleaved
from Y", this phrase actually means "V.sup.1 is cleaved from Y, or
from Z if Y is absent" and should be read as "V.sup.1 is cleaved
from Z" when reference is made to a compound lacking Y.
[0483] In a compound of formula (III) or (IV), Z may be conjugated
to a promoiety through its water-soluble group, e.g., an
oligoethylene glycol or polyethylene glycol moiety. In this way,
the water-soluble group may contribute less to the water solubility
of the compound of formula (III) or (IV), but may contribute again
to the water solubility of Z upon removal of said promoiety.
[0484] In this document, whenever V.sup.2, L.sup.2, L, V.sup.1, Y,
Z, RM, p, q, or z is mentioned, it should be understood that the
same can apply for each V.sup.2', L.sup.2', L', V.sup.1', Y', Z',
RM', p', q', or z', respectively, unless the context dictates
otherwise.
[0485] The V.sup.1 Moiety
[0486] In a compound of formula (III), (IV), or (VIII), the V.sup.1
moiety is a group that is conditionally cleavable or transformable.
In other words, it is designed to be transformed and/or cleaved
from Y by a chemical, photochemical, physical, biological, or
enzymatic process upon being brought in or under a certain
condition. This condition may for example be bringing a compound of
the invention in an aqueous environment, which leads to hydrolysis
of or bringing a compound of the invention in an environment that
contains an enzyme that recognizes and cleaves V.sup.1, or bringing
a compound of the invention under reducing conditions, which leads
to reduction and/or removal of V.sup.1, or bringing a compound of
the invention under oxidizing conditions, which leads to oxidation
and/or removal of V.sup.1, or bringing a compound of the invention
in contact with radiation, e.g., UV light, which leads to
transformation and/or cleavage, or bringing a compound of the
invention in contact with heat, which leads to transformation
and/or cleavage, or bringing a compound of the invention under
reduced pressure, which leads to transformation, e.g., a
retrocycloaddition, and/or cleavage, or bringing a compound of the
invention under elevated or high pressure, which leads to
transformation and/or cleavage. This condition may be met after
administering a compound of this invention to an animal, e.g., a
mammal, for example a human: the condition may be met when the
compound localizes to for example a specific organ, tissue, cell,
subcellular target, or bacterial, viral, or microbial target, for
example by the presence of internal factors (e.g., target-specific
enzymes or hypoxia) or application of external factors (e.g.,
radiation, magnetic fields) or the condition may already be met
directly upon administration (e.g., ubiquitous enzymes in the
circulation).
[0487] Cleavage of V.sup.1 means that the bond between V.sup.1 and
Y is broken. Transformation of V.sup.1 means that V.sup.1 is
converted into a different moiety and this event may directly or
indirectly lead to self-cleavage of V.sup.1 from Y. Alternatively,
transformation of V.sup.1 may lead to formation of a V.sup.1--Y
moiety which is a self-immolative linker. In this case, Y only
becomes self-immolative after transformation of V.sup.1. The
transformed V.sup.1 moiety actually becomes (partially) part of Y.
For example, oxidation of V.sup.1 being a hydrogen atom to a
hydroxyl group may lead to formation of a para- or
ortho-hydroxybenzyloxycarbonyl V.sup.1--Y moiety that
self-eliminates. As another example, reduction of V.sup.1 being a
nitro group may lead to formation of a para- or
ortho-aminobenzyloxycarbonyl V.sup.1--Y moiety that
self-eliminates.
[0488] Alternatively again, V.sup.1 may be absent. In this
instance, the promoiety is intended to be non-removable from Z and
the whole promoiety or a part thereof (in case of degradation of a
compound of formula (III) or (IV) at one or more other sites in the
molecule) will stay connected to the one or more moieties Z. One
alternative way to look at this is that the part of the promoiety
that remains attached to the moiety Z is in fact a part of moiety
Z.
[0489] A compound of this invention may contain more than one
V.sup.1 moiety per promoiety (p and/or q>1). These V.sup.1
moieties may or may not be the same and may or may not require the
same conditions for transformation and/or cleavage.
[0490] The rate at which V.sup.1 is transformed and/or cleaved may
be dependent on the other moieties in a compound of formula (III).
For example, use of a shorter L and/or Y moiety may bring the
functional moiety and/or Z closer to the cleavage site, which may
lead to a reduced rate of transformation and/or cleavage. (Bulky)
substituents on the L and/or Y moiety may lead to a reduced rate of
transformation and/or cleavage of V.sup.1 as well, especially when
they are situated close to the site of transformation/cleavage.
Other effects, such as hydrogen bonding, neighboring group, and
electronic effects, may play a role as well.
[0491] In one aspect of this invention, a conjugate is used to
target one or more moieties Z to target cells. In this instance, a
V.sup.1 moiety may for example contain a substrate molecule that is
cleaved by an enzyme present in the vicinity of the target cells or
inside the target cells, for example tumor cells. V.sup.1 can for
example contain a substrate that is cleaved by an enzyme present at
elevated levels in the vicinity of or inside the target cells as
compared to other parts of the body, or by an enzyme that is
present only in the vicinity of or inside the target cells.
[0492] It is important to recognize that if target site specificity
is achieved solely based upon the selective transformation and/or
cleavage of said V.sup.1 at the target site, the condition causing
the cleavage should preferably, at least to a certain degree, be
target site-specific, whereas the presence of another
target-specific moiety in the compound of the invention, for
instance in a V.sup.2 moiety, weakens or takes away this
requirement. For example, when V.sup.2 causes selective
internalization into a target cell, an enzyme also present in other
cells may transform and/or cleave V.sup.1. However, cleavage should
preferably not occur at a site distant from the target site.
Therefore, the conjugate should not be exposed to enzymes or
conditions that can cause cleavage of V.sup.1 at sites other than
the target site. In one embodiment, transformation and/or cleavage
of V.sup.1 occur intracellularly. In another embodiment,
transformation and/or cleavage of V.sup.1 occur extracellularly. In
another embodiment, transformation and/or cleavage of V.sup.1 occur
by a ubiquitous intracellular enzyme. In another embodiment,
transformation and/or cleavage of V.sup.1 occur by a ubiquitous
extracellular enzyme.
[0493] In one embodiment, V.sup.1 contains a single amino acid, a
di-, tri-, tetra-, or oligopeptide, or a peptidomimetic, which
consists of an amino acid or amino acid sequence or mimetic thereof
recognized and cleavable by a proteolytic enzyme, for example
plasmin, a cathepsin, cathepsin B, prostate-specific antigen (PSA),
urokinase-type plasminogen activator (u-PA), or a member of the
family of matrix metalloproteinases, present in the vicinity of or
inside the target cells, for example tumor cells. In one
embodiment, V.sup.1 is a peptide. In another embodiment, V.sup.1 is
a single amino acid. In another embodiment, V.sup.1 is a dipeptide.
In another embodiment, V.sup.1 is a tripeptide. In another
embodiment, V.sup.1 is a tetrapeptide. In yet another embodiment,
V.sup.1 is a peptidomimetic. A peptidomimetic may be an amino acid
mimic or peptide mimic. An amino acid mimic may for example be a
derivative of a natural amino acid in which the amino group has
been replaced by a hydroxy group or a triazole group, in which the
.alpha.-amino group of the amino acid is alkylated, or in which the
side chain is connected to the .alpha.-amino group instead of to
the a-carbon. A peptide mimic may be a peptide that contains one or
more of such amino acid mimics.
[0494] In another embodiment, V.sup.1 contains a .beta.-glucuronide
that is recognized by .beta.-glucuronidase present in the vicinity
of or inside tumor cells.
[0495] In one embodiment, V.sup.1 contains a substrate for an
enzyme.
[0496] In another embodiment, V.sup.1 is a substrate for an
enzyme.
[0497] In one embodiment, V.sup.1 contains a substrate for an
extracellular enzyme.
[0498] In another embodiment, V.sup.1 contains a substrate for an
intracellular enzyme.
[0499] In yet another embodiment, V.sup.l contains a substrate for
a lysosomal enzyme.
[0500] In yet another embodiment, V.sup.1 contains a substrate for
the serine protease plasmin.
[0501] In yet another embodiment, V.sup.1 contains a substrate for
one or more of the cathepsins, for example cathepsin B.
[0502] In yet another embodiment, V.sup.1 contains a substrate for
a galactosidase.
[0503] In yet another embodiment, V.sup.1 contains a substrate for
quinone reductase NQO1.
[0504] In yet another embodiment, V.sup.1 contains a hydrazide,
hydrazone or imine moiety that is to be hydrolyzed
intracellularly.
[0505] In yet another embodiment, V.sup.1 contains a disulfide
moiety that is to be cleaved intracellularly.
[0506] When V.sup.1 is cleaved extracellularly, the one or more Z
moieties may be released extracellularly. This may provide the
advantage that these Z moieties are not only able to affect the
cell(s) directly surrounding the site of activation (e.g.,
target-positive cells), but also cells somewhat further away from
the site of activation (e.g., target-negative cells) due to
diffusion (bystander effect), provided that the Z moieties are able
to penetrate the cell membrane.
[0507] An enzyme to cleave V.sup.1 can also be transported to the
vicinity of or inside target cells or target tissue via for example
antibody-directed enzyme prodrug therapy (ADEPT), polymer-directed
enzyme prodrug therapy (PDEPT), macromolecular-directed enzyme
prodrug therapy (MDEPT), virus-directed enzyme prodrug therapy
(VDEPT), or gene-directed enzyme prodrug therapy (GDEPT). In these
approaches, the enzyme that needs to cleave V.sup.1 is transported
to or induced to be produced at the target site before
administration of the prodrug, e.g., a compound of formula (III) or
(IV). In one embodiment, transformation and/or cleavage of V.sup.1
occur through an enzyme linked to an antibody using the ADEPT
approach.
[0508] In again another embodiment, V.sup.1 contains a moiety, for
example a nitrobenzyl moiety that can be transformed and/or cleaved
by reduction under hypoxic conditions or by reduction by a
nitroreductase. After reduction of the nitro group and cleavage of
the resulting moiety via self-elimination, self-elimination of the
spacer system Y, if present, leads to release of one or more
moieties Z.
[0509] In one embodiment, the invention relates to a conjugate
wherein V.sup.1 is a single amino acid, a dipeptide, a tripeptide,
a tetrapeptide, or an oligopeptide moiety comprised of natural L
amino acids, unnatural D amino acids, or synthetic amino acids, or
a peptidomimetic, or any combination thereof. In another
embodiment, the invention relates to a compound wherein V.sup.1
comprises a tripeptide. The tripeptide may be linked via its
C-terminus to Y. In one embodiment, the C-terminal amino acid
residue of the tripeptide is selected from alanine, arginine,
citrulline, and lysine, the middle amino acid residue of the
tripeptide is selected from alanine, valine, leucine, isoleucine,
methionine, phenylalanine, cyclohexylglycine, tryptophan, and
proline, and the N-terminal amino acid residue of the tripeptide is
selected from any natural or unnatural amino acid.
[0510] In another embodiment, the invention relates to a compound
wherein V.sup.1 comprises a dipeptide. The dipeptide may be linked
via its C-terminus to Y. In one embodiment, the C-terminal amino
acid residue of the dipeptide is selected from alanine, arginine,
citrulline, and lysine, and the N-terminal amino acid residue of
the dipeptide is selected from any natural or unnatural amino
acid.
[0511] In yet another embodiment, the invention relates to a
compound wherein V.sup.1 comprises a single amino acid. The amino
acid may be linked via its carboxyl group to Y. In one embodiment,
the amino acid is selected from alanine, arginine, citrulline, and
lysine.
[0512] In one embodiment, when the .alpha.-amino group of the
N-terminal amino acid of V.sup.1 is not coupled to L, this amino
acid may be functionalized with a suitable blocking group coupled
to the .alpha.-amino group or may be an unnatural amino acid such
that undesired premature degradation of V.sup.1 by for example
ubiquitous enzymes, e.g., exopeptidases, is prevented. Such a
blocking group may be any group that prevents or considerably
retards premature degradation of V.sup.1. Examples of such a
blocking group include a D-amino acid, an acetyl group, a
tert-butyloxycarbonyl group, and an oligoethylene or polyethylene
glycol.
[0513] In a further embodiment, V.sup.1 is selected from
D-alanylphenylalanyllysine, D-valylleucyllysine,
D-alanylleucyllysine, D-valylphenylalanyllysine,
D-valyltryptophanyllysine, D-alanyltryptophanyllysine,
alanylphenylalanyllysine, valylleucyllysine, alanylleucyllysine,
valylphenylalanyllysine, valyltryptophanyllysine,
alanyltryptophanyllysine, D-alanylphenylalanylcitrulline,
D-valylleucylcitrulline, D-alanylleucylcitrulline,
D-valylphenylalanylcitrulline, D-valyltryptophanylcitrulline,
D-alanyltryptophanylcitrulline, alanylphenylalanylcitrulline,
valylleucylcitrulline, alanylleucylcitrulline,
valylphenylalanylcitrulline, valyltryptophanylcitrulline, and
alanyltryptophanylcitrulline.
[0514] In yet another embodiment, V.sup.1 is selected from
phenylalanyllysine, valyllysine, valylalanine,
D-phenylalanylphenylalanyllysine, phenylalanylphenylalanyllysine,
glycylphenylalanyllysine, alanyllysine, valylcitrulline,
N-methylvalylcitrulline, phenylalanylcitrulline,
isoleucylcitrulline, tryptophanyllysine, tryptophanylcitrulline,
phenylalanylarginine, phenylalanylalanine,
glycylphenylalanylleucylglycine, alanylleucylalanylleucine,
alanylarginylarginine, phenylalanyl-N.sup.9-tosylarginine,
phenylalanyl-N.sup.9-nitroarginine, leucyllysine, leucylcitrulline,
and phenylalanyl-O-benzoylthreonine.
[0515] In a further embodiment, V.sup.1 is selected from
phenylalanyllysine, valyllysine, and valylcitrulline.
[0516] In yet further embodiments, V.sup.1 is phenylalanyllysine or
valyllysine or valylcitrulline.
[0517] Therefore, in one embodiment this invention relates to a
compound wherein V.sup.1 contains a substrate that can be cleaved
by a proteolytic enzyme, plasmin, a cathepsin, cathepsin B,
.beta.-glucuronidase, a galactosidase, prostate-specific antigen
(PSA), urokinase-type plasminogen activator (u-PA), a member of the
family of matrix metalloproteinases, or an enzyme localized by
means of directed enzyme prodrug therapy, such as ADEPT, VDEPT,
MDEPT, GDEPT, or PDEPT, or wherein V.sup.1 contains a moiety that
can be cleaved or transformed through reduction under hypoxic
conditions, through reduction by a nitroreductase, or through
oxidation.
[0518] In another aspect of this invention, a conjugate of this
invention is used primarily to improve the pharmacological
properties of Z. When a promoiety does not need to be selectively
removed at a target site, V.sup.1 of said promoiety may for example
be or contain a group that is cleaved by ubiquitous enzymes, e.g.,
esterases that are present in the circulation or intracellular
enzymes, such as for example proteases and phosphatases, by
pH-controlled intramolecular cyclization, or by acid-catalyzed,
base-catalyzed, or non-catalyzed hydrolysis, or V.sup.1 may for
example be or contain a disulfide or form a disulfide with a
neighboring moiety. V.sup.1 may therefore, optionally together with
the connecting atom(s) of L and/or Y, for example form a carbonate,
carbamate, ureum, ester, amide, imine, hydrazone, hydrazide, oxime,
disulfide, acetal, or ketal group that can be cleaved in vivo. This
means that V.sup.1, optionally together with the connecting atom(s)
of L and/or Y, can for example also represent --OC(O)--, --C(O)O--,
--OC(O)O--, --N(R.sup.v)C(O)--, --C(O)N(R.sup.v)--,
--N(R.sup.v)C(O)O--, --OC(O)N(R.sup.v)--,
--N(R.sup.v)C(O)N(R.sup.w)--, --C(O)--, --OC(R.sup.v)(R.sup.w)--,
--C(R.sup.v)(R.sup.w)O--, --OC(R.sup.v)(R.sup.w)O--,
--C(R.sup.v)(R.sup.w)--, --S--, --S--S--, --C.dbd., .dbd.C--,
--N.dbd., .dbd.N--, --C.dbd.N--, --N.dbd.C--, --O--N.dbd.,
.dbd.N--O--, --C.dbd.N--O--, --O--N.dbd.C--, --N(R.sup.v)--N.dbd.,
.dbd.N--N(R.sup.v)--, --N(R.sup.v)--N.dbd.C--, or
--C.dbd.N--N(R.sup.v)--, wherein R.sup.v and R.sup.w are
independently selected from H and optionally substituted C.sub.1-10
alkyl, C.sub.1-10 heteroalkyl, C.sub.1-10 heteroaryl, C.sub.3-10
cycloalkyl, C.sub.1-10 heterocycloalkyl, or C.sub.5-10 aryl,
R.sup.v and R.sup.w optionally being joined by one or more bonds to
form one or more optionally substituted carbocycles and/or
heterocycles.
[0519] V.sup.1 may therefore for example be or contain, optionally
together with the connecting atom(s) of L and/or Y, a peptide, an
amino acid, a peptidomimetic, a disulfide, a monosaccharide or
disaccharide or a derivative thereof, a nitroaromatic moiety, an
imine, a hydrazide, or a hydrazone moiety.
[0520] If V.sup.1 or V.sup.1--Y represents a whole promoiety or L
is connected to Y and not to V.sup.1, V.sup.1 may for example also
be selected from a mono-, di-, or oligosaccharide,
R.sup.p--[O(R.sup.p'O)P(O)].sub.pp--, R.sup.p--C(O)--,
R.sup.p--OC(O)--, and R.sup.p--N(R.sup.p')C(O)--, wherein pp is
selected from 1 to 3 and each R.sup.p and R.sup.p' is independently
selected from H and optionally substituted C.sub.1-15 alkyl,
C.sub.1-15 heteroalkyl, C.sub.3-15 cycloalkyl, C.sub.1-15
heterocycloalkyl, C.sub.5-15 aryl, or C.sub.1-15 heteroaryl,
R.sup.p and R.sup.p' optionally being joined by one or more bonds
to form one or more optionally substituted carbocycles and/or
heterocycles.
[0521] In one embodiment, V.sup.1 is selected from phosphono,
phenylaminocarbonyl, 4-(piperidin-1-yl)piperidin-1-ylcarbonyl,
piperazin-1-ylcarbonyl, piperidin-1-ylcarbonyl,
pyrrolidin-1-ylcarbonyl, and 4-methylpiperazin-1-ylcarbonyl.
[0522] V.sup.1 itself may contribute to favorable pharmacological
properties of the conjugate, for example through the presence of
polar functional groups in V.sup.1.
[0523] If a conjugate of this invention contains more than 1
promoiety, one of these promoieties may be used to target the
conjugate to a target site (targeting promoiety), whereas another
promoiety is used to improve the pharmacological properties. In
this instance, the V.sup.1 moiety in the targeting promoiety is
preferably cleaved at the target site, for example through a target
site-specific process such as an enzymatic cleavage by an enzyme
predominantly present at the target site or through a more generic
intracellular process which can only occur after target
cell-selective internalization of the conjugate, whereas the
promoiety that helps to improve the pharmacological properties may
be cleaved either at the target site or systemically, for example
by ubiquitous enzymes.
[0524] It should be noted that V.sup.1, either in the form of an
amino acid, a di-, tri-, tetra-, or oligopeptide, or in any other
form, may contain protecting groups. Compounds of the invention
comprising such a protected V.sup.1 may not release any Z moiety
when put under conditions that will transform and/or cleave the
corresponding unprotected V.sup.1. However, when said compounds are
deprotected, such compounds will release one or more Z moieties
when put under the appropriate conditions. Compounds comprising
such a protected V.sup.1 also fall under the scope of this
invention. In one aspect the above can be envisioned for compounds
of formula (IV). Suitable protecting groups for functional groups,
in particular for amino acids, are well-known to the organic
chemist and may for example be found in T. W. Greene, Protective
Groups in Organic Synthesis, John Wiley & Sons, New York, 1981.
In another aspect, the protecting group or protecting moiety may be
present in a compound of formula (III) and this compound may be
administered as such. This means that deprotection should occur in
vivo before release of any Z can occur through transformation
and/or cleavage of V.sup.1. In vivo deprotection of the protected
V.sup.1 moiety in a compound of formula (III) may occur by, for
example, hydrolysis or enzymatic conversion. This deprotection may
occur at the target site or aspecifically.
[0525] Compounds of formulae (III) and (IV) can be designed to
eventually release a compound of formula (I) or (II), or a compound
of formula (I') or (II'), after transformation and/or cleavage of
the one or more V.sup.1 and V.sup.1' moieties. Release of a
compound of formula (I) or (II), a compound of formula (I') or
(II'), or a derivative thereof (for example due to only partial
degradation of the promoiety) from a conjugate of this invention
via another mechanism is however not excluded from this
invention.
[0526] In another aspect of this invention, a compound of formula
(III) represents an intermediate for the preparation of a compound
of formula (I) or (II) or another compound of formula (III). In
this instance, for example, V.sup.2, L.sup.2, L, and Y are absent,
p, q, and z all are 1, and the V.sup.1 moiety may be a protecting
group. There may or may not be one or more
V.sup.2'(-L.sup.2'-L'(-(V.sup.1'--Y')).sub.p').sub.q'(Z').sub.z'-1
moieties present, in which V.sup.2', L.sup.2', L', and Y' may or
may not be absent, and p', q', and z' all may or may not be 1. In
one embodiment, a compound of formula (III) is a compound of
formula (I) or (II) to which a V.sup.1 moiety is attached. In
another embodiment, a compound of formula (III) is a compound of
formula (I) or (II) to which a V.sup.1 moiety and a
V.sup.2'(-L.sup.2'-L'(-(V.sup.1'--Y').sub.p').sub.q'(Z').sub.z'-1
moiety are attached. In yet another embodiment, a compound of
formula (III) is a compound of formula (I) or (II) to which a
V.sup.1 moiety and a V.sup.1' moiety are attached.
[0527] In one embodiment, V.sup.1 is not a protecting group.
[0528] In another embodiment, V.sup.2, L.sup.2, L, and Y are
absent, and p, q, and z all are 1.
[0529] In a further embodiment, V.sup.1 is a chemically removable
group.
[0530] In yet a further embodiment, V.sup.1 is a chemically
removable group connected to Z via X.sup.1.
[0531] In yet another further embodiment, V.sup.1 is a benzyl group
connected to Z via X.sup.1.
[0532] In another embodiment, V.sup.1 is
tert-butoxycarbonyl(methylamino)ethyl(methylamino)carbonyl.
[0533] In another embodiment, V.sup.1 is
4-(tert-butoxycarbonyl)piperazine-1-carbonyl.
[0534] In one embodiment, V.sup.1 is connected to L via more than
one functional group on V.sup.1.
[0535] In another embodiment, V.sup.1 is connected to L via one
functional group on V.sup.1.
[0536] In another embodiment, V.sup.1 is connected to L via a
functional group in the side chain of one of the natural or
unnatural amino acids of V.sup.1.
[0537] In another embodiment, the N-terminal amino acid of V.sup.1
is connected via its a amino group to L.
[0538] In another embodiment, V.sup.1 is absent.
[0539] The Self-Eliminating Spacer System Y
[0540] The self-elimination spacer system Y, if present, links
V.sup.1 and optionally L to one or more moieties Z, or RM2 in case
of a compound of formula (VIII).
[0541] A self-elimination spacer system Y may be incorporated in a
conjugate of this invention for example to improve the properties
of Z or the conjugate in general, to provide for suitable coupling
chemistries, and/or to create space between V.sup.1 and Z.
[0542] A compound of this invention may contain more than one
spacer system Y per promoiety. These moieties Y may or may not be
the same.
[0543] After cleavage or transformation of V.sup.1, the left-hand
side of Y may become unblocked or a V.sup.1--Y self-elimination
moiety may be formed, which results in eventual release of one or
more moieties Z. The self-elimination spacer systems may for
example be those described in WO 02/083180 and WO 2004/043493,
which are incorporated herein by reference in their entirety, the
ones described in this section, as well as other self-elimination
spacers known to a person skilled in the art.
[0544] Moiety Y may, in addition to providing suitable coupling
chemistries and creation of space between the cleavage site and Z,
help improve the pharmacological properties of the conjugate. For
example, the presence of a water-soluble moiety or substituent,
such as a polyethylene glycol moiety or a substituent that is
charged, at least partly, at physiological pH may contribute to the
water solubility and/or increase the storage stability and/or
plasma stability of the conjugate.
[0545] In one aspect the invention is related to compounds wherein
Y is
[0546] (W--).sub.w(X-).sub.x(A-).sub.s,
[0547] wherein
[0548] W and X are each a single-release 1,2+2n electronic cascade
spacer (n.gtoreq.1), being the same or different;
[0549] A is an .omega.-amino aminocarbonyl cyclization spacer that
forms a cyclic ureum derivative upon cyclization;
[0550] s is 0 or 1;
[0551] w and x are numbers representing degree of polymerization
and are independently an integer from 0 (included) to 5
(included).
[0552] In one embodiment, w+x is 0, 1, or 2. In another embodiment,
s is 0. In yet another embodiment, s is 1. In yet another
embodiment, w+x is 1 or 2 and s is 1. In yet another embodiment,
w+x is 1 and s is 1. In yet another embodiment, w is 1, x is 0, and
s is 1.
[0553] According to a further embodiment of this invention, the
1,2+2n electronic cascade spacers W and X are independently a
moiety having the formula:
##STR00276##
[0554] wherein
[0555] Q' is selected from --R.sup.110C.dbd.CR.sup.111--, S, O,
NR.sup.111, --R.sup.111C.dbd.N--, and --N.dbd.CR.sup.111--;
[0556] B is selected from NR.sup.112, O, and S;
[0557] P is C(R.sup.108)(R.sup.109)Q;
[0558] R.sup.106, R.sup.107, B, and
(T-).sub.t(T'-).sub.t'(T''-).sub.t''P are connected to C.sup.a,
C.sup.b, C.sup.c, and C.sup.d in such a way that B and
(T-).sub.t(T'-).sub.t'(T''-).sub.t''P are connected to two adjacent
carbon atoms or to C.sup.a and C.sup.d, respectively;
[0559] Q is absent or --O--C(O)--;
[0560] t, t', and t'' are numbers representing degree of
polymerization and are independently an integer from 0 (included)
to 5 (included);
[0561] T, T', and T'' are independently selected from moieties
having the formula:
##STR00277##
[0562] R.sup.106, R.sup.107, R.sup.108, R.sup.109, R.sup.110,
R.sup.111, R.sup.112, R.sup.113, and R.sup.114 are independently
selected from H, OH, SH, NH.sub.2, N.sub.3, NO.sub.2, NO, CF.sub.3,
CN, C(O)NH.sub.2, C(O)H, C(O)OH, halogen, R.sup.y, SR.sup.y,
S(O)R.sup.y, S(O).sub.2R.sup.y, S(O)OR.sup.y, S(O).sub.2OR.sup.y,
OS(O)R.sup.y, OS(O).sub.2R.sup.y, OS(O)OR.sup.y,
OS(O).sub.2OR.sup.y, OR.sup.y, NHR.sup.y, N(R.sup.y)R.sup.y1,
.sup.+N(R.sup.y)(R.sup.y1)R.sup.y2, P(O)(OR.sup.y)(OR.sup.y1),
OP(O)(OR.sup.y)(OR.sup.y1), C(O)R.sup.y, C(O)OR.sup.y,
C(O)N(R.sup.y1)R.sup.y, OC(O)R.sup.y, OC(O)OR.sup.y,
OC(O)N(R.sup.y)R.sup.y1, N(R.sup.y1)C(O)R.sup.y,
N(R.sup.y1)C(O)OR.sup.y, and N(R.sup.y1)C(O)N(R.sup.y2)R.sup.y,
wherein R.sup.y, R.sup.y1, and R.sup.y2 are independently selected
from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-20 alkyl, C.sub.1-20 heteroalkyl, C.sub.3-20 cycloalkyl,
C.sub.1-20 heterocycloalkyl, C.sub.5-20 aryl, or C.sub.1-20
heteroaryl, wherein ee is selected from 1 to 1000, X.sup.13 is
selected from O, S, and NR.sup.f1, and R.sup.f1 and R.sup.e1 are
independently selected from H and C.sub.1-3 alkyl, two or more of
R.sup.y, R.sup.y1, and R.sup.y2 optionally being joined by one or
more bonds to form one or more optionally substituted carbocycles
and/or heterocycles, two or more of the substituents R.sup.106,
R.sup.107, R.sup.108, R.sup.109, R.sup.110, R.sup.111, R.sup.112,
R.sup.113, and R.sup.114 optionally being joined by one or more
bonds to form one or more optionally substituted carbocycles and/or
heterocycles.
[0563] In the formulae above, Q may be O--C(O), but it may also be
absent. For example, a compound with a benzyl ether linkage between
self-elimination spacer and the group that leaves, the oxycarbonyl
moiety thus being absent (Q is absent), has been reported to
undergo self-elimination.sup.9.
[0564] In one embodiment, t, t' and t'' are 0.
[0565] Substituents R.sup.108 and R.sup.109 can be used to tune the
degree of shielding of the linkage to the moiety attached to Q
(e.g., A or Z). They may also be used to tune the rate at which the
1,2+2n electronic cascade spacers W and X self-eliminate. In one
embodiment, both R.sup.108 and R.sup.109 are H. In another
embodiment, R.sup.108 is not H. In yet another embodiment, both
R.sup.108 and R.sup.109 are not H.
[0566] Substituents R.sup.106, R.sup.107, R.sup.110, and R.sup.111
can be used to tune the degree of shielding of the linkage between
V.sup.1 and Y and may thus be used to tune the cleavage rate of a
compound of formula (III) or (IV). Furthermore, these substituents
can be used to introduce additional water solubility into a
compound of this invention. In some embodiments, the 1,2+2n
electronic cascade spacer W or X is
##STR00278##
[0567] wherein bb and cc are independently an integer from 0 to 10
and R.sup.123 and R.sup.124 are independently selected from H and
methyl.
[0568] In another embodiment, the 1,2+2n electronic cascade spacer
W or X is
##STR00279##
[0569] According to a further embodiment of this invention, the
w-amino aminocarbonyl cyclization elimination spacer A is a moiety
having the formula:
##STR00280##
[0570] wherein
[0571] u is an integer of 0 or 1;
[0572] R.sup.115, R.sup.116, R.sup.117, R.sup.118, R.sup.119,
R.sup.120, R.sup.121, and R.sup.122 are independently selected from
H, OH, SH, NH.sub.2, N.sub.3, NO.sub.2, NO, CF.sub.3, CN,
C(O)NH.sub.2, C(O)H, C(O)OH, halogen, R.sup.z, SR.sup.z,
S(O)R.sup.z, S(O).sub.2R.sup.z, S(O)OR.sup.z, S(O).sub.2OR.sup.z,
OS(O)R.sup.z, OS(O).sub.2R.sup.z, OS(O)OR.sup.z,
OS(O).sub.2OR.sup.z, OR.sup.z, NHR.sup.z, N(R.sup.z)R.sup.z1,
.sup.+N(R.sup.z)(R.sup.z1)R.sup.z2, P(O)(OR.sup.z)(OR.sup.z1),
OP(O)(OR.sup.z)(OR.sup.z1), C(O)R.sup.z, C(O)OR.sup.z,
C(O)N(R.sup.z1)R.sup.z, OC(O)R.sup.z, OC(O)OR.sup.z,
OC(O)N(R.sup.z)R.sup.z1, N(R.sup.z1)C(O)R.sup.z,
N(R.sup.z1)C(O)OR.sup.z, and N(R.sup.z1)C(O)N(R.sup.z2)R.sup.z,
wherein R.sup.z, R.sup.z1, and R.sup.z2 are independently selected
from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-20 alkyl, C.sub.1-20 heteroalkyl, C.sub.3-20 cycloalkyl,
C.sub.1-20 heterocycloalkyl, C.sub.5-20 aryl, or C.sub.1-20
heteroaryl, wherein ee is selected from 1 to 1000, X.sup.13 is
selected from O, S, and NR.sup.f1, and R.sup.f1 and R.sup.e1 are
independently selected from H and C.sub.1-3 alkyl, two or more of
R.sup.z, R.sup.z1, and R.sup.z2 optionally being joined by one or
more bonds to form one or more optionally substituted carbocycles
and/or heterocycles, two or more of the substituents R.sup.115,
R.sup.116, R.sup.117, R.sup.118, R.sup.119, R.sup.120, R.sup.121,
and R.sup.122 optionally being joined by one or more bonds to form
one or more optionally substituted carbocycles and/or
heterocycles.
[0573] R.sup.115 and R.sup.116 are substituent groups that are
located on the nitrogen atoms that are part of the connecting
linkages to the moieties adjacent to the co-amino aminocarbonyl
cyclization elimination spacer in a compound of formula (III),
(IV), or (VIII). The nitrogen bearing R.sup.116 is connected to Z
or RM2 via a carbonyl moiety. The nitrogen atom bearing R.sup.115
is connected to W, X, V.sup.1, or L. The size and nature of
substituents R.sup.115 and R.sup.116 may, in addition to affecting
pharmacological properties of a compound of formula (III), (IV), or
(VIII) in general, especially affect the stability of said
connecting linkages. For example, choosing R.sup.116 to be a small
substituent may in general cause the linkage to Z to be less stable
to hydrolysis or enzymatic degradation than when R.sup.116 is a
more bulky substituent (e.g., a substituent that is branched at the
carbon .alpha. or .beta. to the nitrogen atom). The nature of the
R.sup.116 substituent, e.g., whether polar or apolar or whether or
not charged at physiological pH, may affect the stability of the
linkage to Z as well, because the substituent may affect the
substrate properties of the connecting linkage for specific enzymes
or retard or increase the rate of hydrolysis. If the connecting
linkage is susceptible to enzymes that are ubiquitously present,
for example enzymes present in the circulation, this may cause
premature degradation of the conjugate. The same holds for
substituent R.sup.115 with regard to the connecting linkage to W,
X, V.sup.1, or L. Similarly, the substituents on the carbons
directly attached to the nitrogen atoms (R.sup.117, R.sup.118,
R.sup.119, R.sup.121, and R.sup.122) may contribute to the
stability of the connecting linkages.
[0574] Substituents R.sup.115 and R.sup.116 may also affect the
cyclization rate of the co-amino aminocarbonyl cyclization
elimination spacer A. Bulky substituents may hinder cyclization and
thus generally reduce the cyclization rate. Small substituents may
therefore in general be favorable for fast cyclization rates.
However, other properties of the substituent, such as
electronegativity, polarity, and availability of hydrogen bond
donor and/or acceptor groups, may also affect the cyclization rate.
Furthermore, if only one of R.sup.115 and R.sup.116 is a bulky
substituent, the cyclization rate may be enhanced with respect to
cyclization spacers containing two non-bulky substituents, possibly
because of B-strain. The cyclization rate may also be affected by
the other substituents on the co-amino aminocarbonyl cyclization
elimination spacer. Properties of these substituents such as
polarity, electronegativity, and hydrogen bonding capabilities, may
have an effect on the cyclization rate. Furthermore, the presence
of geminal substituents on one of the carbons in between the two
nitrogen atoms may enhance the cyclization rate as well
(Thorpe-Ingold effect). The joinder of substituents to form one or
more rings may also have an effect on the cyclization rate. In this
respect, every modification that may bring the two nitrogen atoms
closer together or that reduces entropy may have an accelerating
effect on the cyclization rate.
[0575] Effects on the cyclization rate may be pH dependent and the
order of the cyclization rate of two .omega.-amino aminocarbonyl
cyclization elimination spacers may be reversed going from for
example pH 7 to pH 5.
[0576] Although it may be beneficial in some cases for a conjugate
of this invention to have an .omega.-amino aminocarbonyl
cyclization elimination spacer with a fast cyclization rate, thus
releasing the active drug shortly after cleavage of V.sup.1, in
other cases it may be more beneficial to have an .omega.-amino
aminocarbonyl cyclization elimination spacer that has a slow
cyclization rate or a cyclization rate that lies between two
predetermined limits or that preferably cyclizes at a fast rate
only within certain pH limits. This may cause the cyclization
spacer-drug intermediate to survive for a certain amount of time as
a prodrug that will release the drug slowly or after a certain
condition has been met.
[0577] In one embodiment, R.sup.115 and R.sup.116 are independently
selected from R.sup.z, wherein R.sup.z is selected from H and
optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-20 alkyl, C.sub.1-20 heteroalkyl, C.sub.3-20 cycloalkyl,
C.sub.1-20 heterocycloalkyl, C.sub.5-20 aryl, and C.sub.1-20
heteroaryl, wherein ee is selected from 1 to 1000, X.sup.13 is
selected from O, S, and NR.sup.f1, and R.sup.f1 and R.sup.e1 are
independently selected from H and C.sub.1-3 alkyl.
[0578] In another embodiment, both R.sup.115 and R.sup.116 are not
H. In another embodiment, both R.sup.115 and R.sup.116 are not
methyl. In yet another embodiment, both R.sup.115 and R.sup.116 are
not H and at least one of R.sup.115 and R.sup.116 is not methyl. In
yet another embodiment, both R.sup.115 and R.sup.116 are not H and
R.sup.116 is not methyl. In yet another embodiment, at least one of
R.sup.115 and R.sup.116 is not H or methyl. Such cyclization
spacers incorporated in a compound of formula (III) or (IV) have
been shown to have improved properties with respect to cyclization
spacers in which R.sup.115 and R.sup.116 are both selected from
only H and methyl. For example, the rate of self-elimination, the
rate of drug release, conjugate stability, and/or conjugate
polarity may be improved.
[0579] In one embodiment, R.sup.115 and R.sup.116are independently
selected from optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-6 alkyl, C.sub.1-6 heteroalkyl, C.sub.3-6 cycloalkyl,
C.sub.1-6 heterocycloalkyl, C.sub.5-6 aryl, and C.sub.1-6
heteroaryl, wherein ee is selected from 1 to 9, X.sup.13 is
selected from O, S, and NR.sup.f1, and R.sup.f1 and R.sup.e1 are
independently selected from H and C.sub.1-3 alkyl.
[0580] In another embodiment, R.sup.115 and R.sup.116 are
independently selected from H, methyl, ethyl, isopropyl,
tert-butyl, phenyl,
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
##STR00281## ##STR00282##
[0581] wherein dd is selected from 0 to 10, dd' is selected from 0
and 1, each R.sup.123 is independently selected from H and methyl,
ee is selected from 1 to 1000, X.sup.13 is selected from O, S, and
NR.sup.f1, and R.sup.f1 and R.sup.e1 are independently selected
from H and C.sub.1-3 alkyl.
[0582] In further embodiments, R.sup.115 and R.sup.116 are
independently selected from H, methyl, ethyl, isopropyl,
tert-butyl, phenyl,
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
##STR00283## ##STR00284##
[0583] wherein dd' is selected from 0 and 1, each R.sup.123 is
independently selected from H and methyl, X.sup.13 is selected from
O, S, and NR.sup.f1, R.sup.f1 and R.sup.e1 are independently
selected from H and C.sub.1-3 alkyl, dd is 1 or 2 or 3 or 4 or 5,
and ee is 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8.
[0584] In another embodiment, R.sup.115 is methyl and R.sup.116 is
selected from H, methyl, ethyl, isopropyl, tert-butyl, phenyl,
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
##STR00285## ##STR00286##
[0585] wherein dd is selected from 0 to 10, dd' is selected from 0
and 1, each R.sup.123 is independently selected from H and methyl,
X.sup.13 is selected from O, S, and NR.sup.f1, and R.sup.f1 and
R.sup.e1 are independently selected from H and C.sub.1-3 alkyl, and
ee is selected from 1 to 1000.
[0586] In another embodiment, R.sup.115 is methyl and R.sup.116 is
selected from ethyl, isopropyl, tert-butyl, phenyl,
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
##STR00287## ##STR00288##
[0587] wherein dd is selected from 0 to 10, dd' is selected from 0
and 1, each R.sup.123 is independently selected from H and methyl,
X.sup.13 is selected from O, S, and NR.sup.f1, and R.sup.f1 and
R.sup.e1 are independently selected from H and C.sub.1-3 alkyl, and
ee is selected from 1 to 1000.
[0588] In yet another embodiment, R.sup.116 is methyl and R.sup.115
is selected from H, methyl, ethyl, isopropyl, tert-butyl, phenyl,
(CH.sub.2CH.sub.2O)CH.sub.2CH.sub.2X.sup.13R.sup.e1,
##STR00289## ##STR00290##
[0589] wherein dd is selected from 0 to 10, dd' is selected from 0
and 1, each R.sup.123 is independently selected from H and methyl,
X.sup.13 is selected from O, S, and NR.sup.f1, and R.sup.f1 and
R.sup.e1 are independently selected from H and C.sub.1-3 alkyl, and
ee is selected from 1 to 1000.
[0590] In yet a further embodiment, R.sup.115 and R.sup.116are
independently selected from H, methyl, ethyl, isopropyl,
tert-butyl, phenyl,
##STR00291## ##STR00292## ##STR00293##
[0591] In yet a further embodiment, R.sup.115 and R.sup.116 are
independently selected from H, methyl, ethyl, isopropyl,
tert-butyl, phenyl,
##STR00294## ##STR00295## ##STR00296##
[0592] In yet a further embodiment, R.sup.115 is methyl and
R.sup.116 is selected from H, methyl, ethyl, isopropyl, tert-butyl,
phenyl,
##STR00297## ##STR00298## ##STR00299## ##STR00300##
[0593] In yet a further embodiment, R.sup.116 is methyl and
R.sup.115 is selected from H, methyl, ethyl, isopropyl, tert-butyl,
phenyl,
##STR00301## ##STR00302## ##STR00303## ##STR00304##
##STR00305##
[0594] In yet another embodiment, R.sup.115 or R.sup.116 is joined
with one of R.sup.117, R.sup.118, R.sup.119, R.sup.120, R.sup.121,
and R.sup.122 to form a saturated or unsaturated ring. In a further
embodiment, R.sup.115 or R.sup.116 is joined with one of R.sup.117,
R.sup.118, R.sup.119, R.sup.120, R.sup.121, and R.sup.122 to form a
saturated or unsaturated 5- or 6-membered ring. In yet a further
embodiment, R.sup.115 is joined with one of R.sup.117, R.sup.118,
R.sup.119, R.sup.120, R.sup.121, and R.sup.122 to form a saturated
or unsaturated 5- or 6-membered ring. In yet a further embodiment,
R.sup.116 is joined with one of R.sup.117, R.sup.118, R.sup.119,
R.sup.120, R.sup.121, and R.sup.122 to form a saturated or
unsaturated 5- or 6-membered ring.
[0595] In another embodiment, two of R.sup.117, R.sup.118,
R.sup.119, R.sup.120, R.sup.121, and R.sup.122 are joined to form a
saturated or unsaturated ring. In yet another embodiment, two of
R.sup.117, R.sup.118, R.sup.119, R.sup.120, R.sup.121, and
R.sup.122 not being connected to the same carbon atom are joined to
form a saturated or unsaturated 5- or 6-membered ring.
[0596] In one embodiment, the cyclization spacer A is selected
from
##STR00306##
[0597] In another embodiment, the cyclization spacer A is selected
from
##STR00307##
[0598] In a further embodiment, the cyclization spacer A is
selected from
##STR00308##
[0599] In a further embodiment, the cyclization spacer A is
##STR00309##
[0600] In a further embodiment, the cyclization spacer A is
selected from
##STR00310## ##STR00311## ##STR00312## ##STR00313##
[0601] wherein R.sup.115, R.sup.116, R.sup.117, R.sup.118,
R.sup.119, R.sup.120, R.sup.121, and R.sup.122 are independently
selected from H, OH, SH, NH.sub.2, N.sub.3, NO.sub.2, NO, CF.sub.3,
CN, C(O)NH.sub.2, C(O)H, C(O)OH, halogen, R.sup.z, SR.sup.z,
S(O)R.sup.z, S(O).sub.2R.sup.z, S(O)OR.sup.z, S(O).sub.2OR.sup.z,
OS(O)R.sup.z, OS(O).sub.2R.sup.z, OS(O)OR.sup.z,
OS(O).sub.2OR.sup.z, OR.sup.z, NHR.sup.z, N(R.sup.z)R.sup.z1,
.sup.+N(R.sup.z)(R.sup.z1)R.sup.z2, P(O)(OR.sup.z)(OR.sup.z1),
OP(O)(OR.sup.z)(OR.sup.z1), C(O)R.sup.z, C(O)OR.sup.z,
C(O)N(R.sup.z1)R.sup.z, OC(O)R.sup.z, OC(O)OR.sup.z,
OC(O)N(R.sup.z)R.sup.z1, N(R.sup.z1)C(O)R.sup.z,
N(R.sup.z1)C(O)OR.sup.z, and N(R.sup.z1)C(O)N(R.sup.z2)R.sup.z,
wherein R.sup.z, R.sup.z1, and R.sup.z2 are independently selected
from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-20 alkyl, C.sub.1-20 heteroalkyl, C.sub.3-20 cycloalkyl,
C.sub.1-20 heterocycloalkyl, C.sub.5-20 aryl, or C.sub.1-20
heteroaryl, wherein ee is selected from 1 to 1000, X.sup.13 is
selected from O, S, and NR.sup.f1, and R.sup.f1 and R.sup.e1 are
independently selected from H and C.sub.1-3 alkyl, two or more of
R.sup.z, R.sup.z1, and R.sup.z2 optionally being joined by one or
more bonds to form one or more optionally substituted carbocycles
and/or heterocycles, two or more of the substituents R.sup.115,
R.sup.116, R.sup.117, R.sup.118, R.sup.119, R.sup.120, R.sup.121,
and R.sup.122 optionally being joined by one or more bonds to form
one or more optionally substituted carbocycles and/or
heterocycles.
[0602] In yet a further embodiment, the cyclization spacer A is
selected from
##STR00314## ##STR00315## ##STR00316## ##STR00317##
[0603] wherein R.sup.115 and R.sup.116 are independently selected
from H, methyl, ethyl, isopropyl, tert-butyl, phenyl,
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
##STR00318## ##STR00319##
[0604] wherein dd is selected from 0 to 10, dd' is selected from 0
and 1, each R.sup.123 is independently selected from H and methyl,
ee is selected from 1 to 1000, X.sup.13 is selected from O, S, and
NR.sup.f1, and R.sup.f1 and R.sup.e1 are independently selected
from H and C.sub.1-3 alkyl,
[0605] and wherein R.sup.117, R.sup.118, R.sup.119, R.sup.120,
R.sup.121, and R.sup.122 are independently selected from H, OH, SH,
NH.sub.2, N.sub.3, NO.sub.2, NO, CF.sub.3, CN, C(O)NH.sub.2, C(O)H,
C(O)OH, halogen, R.sup.z, SR.sup.z, S(O)R.sup.z, S(O).sub.2R.sup.z,
S(O)OR.sup.z, S(O).sub.2OR.sup.z, OS(O)R.sup.z, OS(O).sub.2R.sup.z,
OS(O)OR.sup.z, OS(O).sub.2OR.sup.z, OR.sup.z, NHR.sup.z,
N(R.sup.z)R.sup.z1, .sup.+N(R.sup.z)(R.sup.z1)R.sup.z2,
P(O)(OR.sup.z)(OR.sup.z1), OP(O)(OR.sup.z)(OR.sup.z1), C(O)R.sup.z,
C(O)OR.sup.z, C(O)N(R.sup.z1)R.sup.z, OC(O)R.sup.z, OC(O)OR.sup.z,
OC(O)N(R.sup.z)R.sup.z1, N(R.sup.z1)C(O)R.sup.z,
N(R.sup.z1)C(O)OR.sup.z, and N(R.sup.z1)C(O)N(R.sup.z2)R.sup.z,
wherein R.sup.z, R.sup.z1, and R.sup.z2 are independently selected
from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-20 alkyl, C.sub.1-20 heteroalkyl, C.sub.3-20 cycloalkyl,
C.sub.1-20 heterocycloalkyl, C.sub.5-20 aryl, or C.sub.1-20
heteroaryl, wherein ee is selected from 1 to 1000, X.sup.13 is
selected from O, S, and NR.sup.f1, and R.sup.f1 and R.sup.e1 are
independently selected from H and C.sub.1-3 alkyl.
[0606] In yet a further embodiment, the cyclization spacer A is
selected from
##STR00320## ##STR00321## ##STR00322## ##STR00323##
[0607] wherein R.sup.115 and R.sup.116 are independently selected
from H, methyl, ethyl, isopropyl, tert-butyl, phenyl,
##STR00324## ##STR00325## ##STR00326##
[0608] and wherein R.sup.117, R.sup.118, R.sup.119, R.sup.120,
R.sup.121, and R.sup.122 are independently selected from H, OH, SH,
NH.sub.2, N.sub.3, NO.sub.2, NO, CF.sub.3, CN, C(O)NH.sub.2, C(O)H,
C(O)OH, halogen, R.sup.z, SR.sup.z, S(O)R.sup.z, S(O).sub.2R.sup.z,
S(O)OR.sup.z, S(O).sub.2OR.sup.z, OS(O)R.sup.z, OS(O).sub.2R.sup.z,
OS(O)OR.sup.z, OS(O).sub.2OR.sup.z, OR.sup.z, NHR.sup.z,
N(R.sup.z)R.sup.z1, .sub.+N(R.sup.z)(R.sup.z1)R.sup.z2,
P(O)(OR.sup.z)(OR.sup.z1), OP(O)(OR.sup.z)(OR.sup.z1), C(O)R.sup.z,
C(O)OR.sup.z, C(O)N(R.sup.z1)R.sup.z, OC(O)R.sup.z, OC(O)OR.sup.z,
OC(O)N(R.sup.z)R.sup.z1, N(R.sup.z1)C(O)R.sup.z,
N(R.sup.z1)C(O)OR.sup.z, and N(R.sup.z1)C(O)N(R.sup.z2)R.sup.z,
wherein R.sup.z, R.sup.z1, and R.sup.z2 are independently selected
from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-20 alkyl, C.sub.1-20 heteroalkyl, C.sub.3-20 cycloalkyl,
C.sub.1-20 heterocycloalkyl, C.sub.5-20 aryl, or C.sub.1-20
heteroaryl, wherein ee is selected from 1 to 1000, X.sup.13 is
selected from O, S, and NR.sup.f1, and R.sup.f1 and R.sup.e1 are
independently selected from H and C.sub.1-3 alkyl.
[0609] In another embodiment, the cyclization spacer A is
##STR00327##
[0610] wherein R.sup.115 and R.sup.116 are independently selected
from H, methyl, ethyl, isopropyl, tert-butyl, phenyl,
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
##STR00328## ##STR00329##
[0611] wherein dd is selected from 0 to 10, dd' is selected from 0
and 1, each R.sup.123 is independently selected from H and methyl,
ee is selected from 1 to 1000, X.sup.13 is selected from O, S, and
NR.sup.f1, and R.sup.f1 and R.sup.e1 are independently selected
from H and C.sub.1-3 alkyl.
[0612] In another embodiment, the cyclization spacer A is
##STR00330##
[0613] wherein R.sup.115 and R.sup.116 are independently selected
from H, methyl, ethyl, isopropyl, tert-butyl, phenyl,
##STR00331## ##STR00332## ##STR00333## ##STR00334##
##STR00335##
[0614] In another embodiment, the cyclization spacer A is
##STR00336##
[0615] wherein R.sup.116 is selected from methyl, ethyl, isopropyl,
tert-butyl, phenyl,
##STR00337## ##STR00338## ##STR00339##
[0616] In another embodiment, the cyclization spacer A is
##STR00340##
[0617] wherein R.sup.116 is selected from ethyl, isopropyl,
tert-butyl, phenyl,
##STR00341## ##STR00342## ##STR00343##
[0618] In another embodiment, the cyclization spacer A is selected
from
##STR00344## ##STR00345## ##STR00346## ##STR00347## ##STR00348##
##STR00349## ##STR00350## ##STR00351## ##STR00352## ##STR00353##
##STR00354## ##STR00355##
[0619] In another embodiment, cyclization linker A is selected
from
##STR00356## ##STR00357##
[0620] In other embodiments, the cyclization linker A is
##STR00358##
[0621] In other embodiments, the cyclization linker A is
##STR00359##
[0622] In one embodiment, Y is absent.
[0623] In another embodiment, this invention relates to a compound
of formula (III) or (IV) wherein X.sup.1 is O and Y is connected to
X.sup.1 via an co-amino aminocarbonyl cyclization spacer being part
of Y.
[0624] In one embodiment, the spacer system Y is selected from
##STR00360##
[0625] In another embodiment, the spacer system Y is
##STR00361##
[0626] In another embodiment, the spacer system Y is
##STR00362##
[0627] In a further embodiment, the spacer system Y is
##STR00363##
[0628] wherein A is selected from
##STR00364## ##STR00365## ##STR00366##
[0629] wherein R.sup.115 and R.sup.116 are independently selected
from H, methyl, ethyl, isopropyl, tert-butyl, phenyl,
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
##STR00367## ##STR00368## ##STR00369##
[0630] wherein dd is selected from 0 to 10, dd' is selected from 0
and 1, each R.sup.123 is independently selected from H and methyl,
ee is selected from 1 to 1000, X.sup.13 is selected from O, S, and
NR.sup.f1, and R.sup.f1 and R.sup.e1 are independently selected
from H and C.sub.1-3 alkyl,
[0631] and wherein R.sup.117, R.sup.118, R.sup.119, R.sup.120,
R.sup.121, and R.sup.122 are independently selected from H, OH, SH,
NH.sub.2, N.sub.3, NO.sub.2, NO, CF.sub.3, CN, C(O)NH.sub.2, C(O)H,
C(O)OH, halogen, R.sup.z, SR.sup.z, S(O)R.sup.z, S(O).sub.2R.sup.z,
S(O)OR.sup.z, S(O).sub.2OR.sup.z, OS(O)R.sup.z, OS(O).sub.2R.sup.z,
OS(O)OR.sup.z, OS(O).sub.2OR.sup.z, OR.sup.z, NHR.sup.z,
N(R.sup.z)R.sup.z1, .sup.+N(R.sup.z)(R.sup.z1)R.sup.z2,
P(O)(OR.sup.z)(OR.sup.z1), OP(O)(OR.sup.z)(OR.sup.z1), C(O)R.sup.z,
C(O)OR.sup.z, C(O)N(R.sup.z1)R.sup.z, OC(O)R.sup.z, OC(O)OR.sup.z,
OC(O)N(R.sup.z)R.sup.z1, N(R.sup.z1)C(O)R.sup.z,
N(R.sup.z1)C(O)OR.sup.z, and N(R.sup.z1)C(O)N(R.sup.z2)R.sup.z,
wherein R.sup.z, R.sup.z1, and R.sup.z2 are independently selected
from H and optionally substituted
(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
C.sub.1-20 alkyl, C.sub.1-20 heteroalkyl, C.sub.3-20 cycloalkyl,
C.sub.1-20 heterocycloalkyl, C.sub.5-20 aryl, or C.sub.1-20
heteroaryl, wherein ee is selected from 1 to 1000, X.sup.13 is
selected from O, S, and NR.sup.f1, and R.sup.f1 and R.sup.e1 are
independently selected from H and C.sub.1-3 alkyl.
[0632] In a further embodiment, the spacer system Y is
##STR00370##
wherein A is selected from
##STR00371##
[0633] wherein R.sup.116 is selected from methyl, ethyl, isopropyl,
tert-butyl, phenyl,
##STR00372## ##STR00373## ##STR00374## ##STR00375##
##STR00376##
[0634] In a further embodiment, the spacer system Y is
##STR00377##
wherein A is selected from
##STR00378##
[0635] wherein R.sup.116 is selected from ethyl, isopropyl,
tert-butyl, phenyl,
##STR00379## ##STR00380## ##STR00381## ##STR00382##
##STR00383##
[0636] In a further embodiment, the spacer system Y is
##STR00384##
[0637] wherein A is selected from
##STR00385##
[0638] In another embodiment, at least one of R.sup.115 and
R.sup.116 is not H or methyl when the two nitrogen atoms of the
.omega.-amino aminocarbonyl cyclization spacer are connected via an
unsubstituted ethylene bridge.
[0639] Other examples of self-eliminating spacers include, but are
not limited to, other spacers that can undergo cyclization.sup.10,
such as optionally substituted 4-aminobutyric acid amides,
appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring
systems, 2-aminophenylpropionic acid amides, and "trimethyl-lock"
cyclization spacers.sup.11. A glycine spacer in which an
amine-containing leaving group is connected at the a-position is
another useful spacer for the compounds of the
invention..sup.12
[0640] In a conjugate of this invention, a spacer system Y may be
connected to more than one V.sup.1 moiety. In this case,
transformation and/or cleavage of one of these V.sup.1 moieties may
trigger the release of one or more Z moieties. When V.sup.1
moieties that are transformed or cleaved under different conditions
are connected to the same Y, release of one or more Z moieties may
occur when a conjugate of this invention is brought under one of
several distinct conditions if Y can undergo self-elimination in
multiple ways. Alternatively, a spacer system Y may be used that
requires to be triggered twice or even more times in order to
self-eliminate. An example of such a self-elimination spacer is a
bicine spacer..sup.13 When such a spacer is used in combination
with different, selectively cleavable V.sup.1 moieties connected to
said spacer, selectivity of release of Z may be increased as two
different conditions must be met before Z is released.
[0641] The Linking Group L
[0642] The linking group L links one or more V.sup.1 and/or Y
moieties to L.sup.2 or RM. Synthesis may be more straightforward
when L is connected to V.sup.1 instead of Y and the compound may be
less prone to premature degradation as V.sup.1 may be more
shielded. Connection of L to Y may have the advantage that V.sup.1
may be transformed and/or cleaved with more ease. Other reasons to
connect L to Y may for example be that (part of) Y remains bound to
L upon cleavage of V.sup.1, which prevents the release of reactive
small molecules, and that the compound may display improved
pharmacological properties, solubility, or aggregation behavior. L
may be absent, which means that V.sup.1 or Y is directly connected
to either L.sup.2 or RM. In another aspect, however, L is a linking
group that functionally links or spaces the one or more V.sup.1
and/or Y moieties and the L.sup.2 or RM moiety. In a compound of
formula (IV), spacing may make the reactive moiety RM more
accessible to the reaction partner, for example when the functional
moiety V.sup.2 is being coupled. In a compound of formula (III),
spacing may provide for a better accessibility of V.sup.1, because
V.sup.2 is further away, which, especially in the case of enzymatic
cleavage or transformation of V.sup.1, may improve the rate at
which V.sup.1 is transformed and/or cleaved. However, it was found
that a relatively short L moiety may improve in vivo efficacy of a
compound of formula (III) compared to similar compounds with a
relatively long L moiety, provided that V.sup.1 can still be
transformed and/or cleaved at the target site.
[0643] The linking group L must contain suitable functional groups
at both of its ends to provide for selective coupling with the one
or more V.sup.1 and/or Y moieties and L.sup.2 or RM.
[0644] The linking group L may be a water-soluble moiety or contain
one or more water-soluble moieties or water-soluble functional
groups, such that L contributes to the water solubility of a
compound of formula (III) or (IV). L may also be a moiety or
contain one or more moieties that reduce(s) aggregation of a
compound of formula (III) or (IV), which may or may not be a
moiety/moieties that also increase(s) the water solubility of a
compound of formula (III) or (IV). Furthermore, L may also contain
or be a moiety that causes a compound of formula (III) or (IV) to
be less susceptible to an immune response or increased efflux from
cells by multidrug resistance-associated transporter proteins. The
L moiety may contain an oligoethylene glycol or polyethylene glycol
moiety or a derivative thereof. This moiety may for example improve
the water solubility, reduce aggregation, reduce the immune
response, and/or reduce the efflux from cells of a compound of
formula (III) or (IV). L may for example contain a
--(CH.sub.2CH.sub.2O).sub.ffCH.sub.2CH.sub.2X.sup.13-- or
--(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1
moiety, wherein ff is selected from 1 to 1000, X.sup.13 is selected
from O, S, and NR.sup.f1, and R.sup.f1 and R.sup.e1 are
independently selected from H and C.sub.1-3 alkyl. In one
embodiment, such a moiety is part of a substituent attached to the
main chain of L that connects RM or L.sup.2 to V.sup.1 and not
present in the main chain of L itself in order to keep the length
of the main chain relatively short. The L moiety may also contain
polar groups and/or groups that are charged, at least partly, at
physiological pH in order to improve the pharmacological properties
of a compound of formula (III) or (IV).
[0645] In one aspect, the L moiety is a linear, branched, or
dendritic moiety, so that it can be connected to one or more
V.sup.1 and/or Y moieties. Branching can occur via one or more
cyclic structures or at one or more branching atoms that may for
example be carbon, nitrogen, silicon, or phosphorus.
[0646] The number of branches in L that are connected to V.sup.1
and/or Y does not necessarily equal the total number of branches as
in the coupling reaction with V.sup.1 and/or Y not all branches may
be coupled to V.sup.1 and/or Y moieties due to incomplete chemical
conversion. This means that L may contain branches that are not
coupled to V.sup.1 or Y, but instead end in for example a
functional group, H, OH, or a leaving group.
[0647] Therefore, when L is branched, compounds of this invention
may exist as a mixture, wherein each component of the mixture has a
different p value. For example, the compound may exist as a mixture
of two separate compounds, one compound wherein p is 2 and another
compound wherein p is 3. Furthermore, for a given p, the compound
may exist as a mixture of (constitutional) isomers as V.sup.1
and/or Y may be connected to distinct (sets of) branches on L.
[0648] In one embodiment, L is absent.
[0649] In another embodiment, L is a linear linker.
[0650] In another embodiment, the chain of atoms linking L.sup.2 or
RM to V.sup.1 consists of less than 19 atoms.
[0651] In another embodiment, the chain of atoms linking L.sup.2 or
RM to V.sup.1 consists of less than 15 atoms.
[0652] In another embodiment, the chain of atoms linking L.sup.2 or
RM to V.sup.1 consists of less than 10 atoms.
[0653] In another embodiment, the chain of atoms linking L.sup.2 or
RM to V.sup.1 consists of less than 5 atoms.
[0654] In another embodiment, L is a linear linker containing a
1,2,3-triazole moiety. Such a linker may be built up through a
cycloaddition reaction between a molecule containing an azide group
and one containing an acetylene group.
[0655] In another embodiment, L is a linear linker containing a
water-soluble group. In another embodiment, L is a linear linker
that comprises an oligoethylene glycol or polyethylene glycol or a
derivative thereof. In a further embodiment, L is a linear linker
that contains a functional group that is charged, at least partly,
at physiological pH.
[0656] In another embodiment, L is a branched linker.
[0657] In another embodiment, L is a dendritic linker. The
dendritic structure may for example be built up through
cycloaddition reactions between molecules containing one or more
azide groups and ones containing one or more acetylene groups.
[0658] In one embodiment, p is 1.
[0659] In other embodiments, p is 2 or 3 or 4 or 6 or 8 or 9.
[0660] In another embodiment, L is represented by the formula:
##STR00386##
[0661] wherein
[0662] X.sup.101 and X102 are each independently O, NR.sup.131, or
S;
[0663] Each X.sup.103 and X.sup.104 is independently O, NR.sup.132,
or S;
[0664] Each xa, xb, xc, and xd is independently 0 or 1;
[0665] kk is a number representing a degree of branching and is an
integer selected from 1 (included) to 128 (included);
[0666] ll is a number representing a degree of branching and is an
integer selected from 0 (included) to 127 (included);
[0667] kk+ll.ltoreq.128;
[0668] Each DD is independently H, OH, or a leaving group;
[0669] R.sup.130 is either a dendritic, branched, or unbranched
multivalent moiety and selected from optionally substituted
alkylene, oligoalkylene, or polyalkylene, and optionally
substituted heteroalkylene, oligoheteroalkylene, or
polyheteroalkylene, and optionally substituted arylene,
oligoarylene, or polyarylene, and optionally substituted
heteroarylene, oligoheteroarylene, or polyheteroarylene, and
optionally substituted cycloalkylene, oligocycloalkylene, or
polycycloalkylene, and optionally substituted heterocycloalkylene,
oligoheterocycloalkylene, or polyheterocycloalkylene, and
--(CH.sub.2CH.sub.2O).sub.v--,
-alkylene-(CH.sub.2CH.sub.2O).sub.v--,
--(CH.sub.2CH.sub.2O).sub.v-alkylene-,
-alkylene-(CH.sub.2CH.sub.2O).sub.v-alkylene-,
-heteroalkylene-(CH.sub.2CH.sub.2O).sub.v--,
--(CH.sub.2CH.sub.2O).sub.v-heteroalkylene-,
-heteroalkylene-(CH.sub.2CH.sub.2O).sub.v-alkylene-,
-heteroalkylene-(CH.sub.2CH.sub.2O).sub.v-heteroalkylene-,
-alkylene-(CH.sub.2CH.sub.2O).sub.v-heteroalkylene-,
X.sup.14(CH.sub.2CH.sub.2O).sub.ffCH.sub.2CH.sub.2X.sup.14, a
dendritic structure, a sugar residue, and an oligopeptide, or any
combination of two or more of the above, wherein optional
substituents may for example be selected from C.sub.1-8 alkyl,
C.sub.1-8 heteroalkyl, C.sub.5-8 aryl, C.sub.1-8 heteroaryl,
X.sup.14(CH.sub.2CH.sub.2O).sub.eeCH.sub.2CH.sub.2X.sup.13R.sup.e1,
--(CHOR.sup.133').sub.vv--R.sup.133, a sugar residue, and charged
substituents selected from SO.sub.3.sup.-, OPO.sub.3.sup.2-,
PO.sub.3.sup.2-, CO.sub.2.sup.-, and
NR.sup.133R.sup.133'R.sup.133'', or from any combination
thereof;
[0670] R.sup.131, R.sup.132, R.sup.133, R.sup.133', and R.sup.133''
are independently selected from H, C.sub.1-8 alkyl, and C.sub.1-8
heteroalkyl;
[0671] X.sup.13 is selected from O, S, and NR.sup.f1;
[0672] R.sup.f1 and R.sup.e1 are independently selected from H and
C.sub.1-3 alkyl;
[0673] ee and ff are independently selected from 1 to 1000;
[0674] Each X.sup.14 is independently selected from
##STR00387##
[0675] v is selected from 1 (included) to 1000 (included);
[0676] vv is selected from 1 to 10.
[0677] In another embodiment, L is selected from
##STR00388## ##STR00389## ##STR00390## ##STR00391## ##STR00392##
##STR00393##
[0678] In yet another embodiment, L is selected from
##STR00394##
[0679] In yet another embodiment, L is selected from
##STR00395##
[0680] In yet another embodiment, L contains a moiety that may for
example be
##STR00396## ##STR00397## ##STR00398## ##STR00399##
[0681] wherein ff is selected from 1 to 1000. In other embodiments,
ff is selected from 3 to 1000 or 500 or 100 or 50 or 10. In other
embodiments, ff is selected to be 1 or 2 or 3 or 4 or 5.
[0682] In yet another embodiment, L is selected from
##STR00400##
[0683] wherein rr, rr', rr'', and rr''' each independently range
from 0 to 8, X.sup.74 is selected from
##STR00401##
[0684] each X.sup.40 and X.sup.41 is independently selected from O,
S, and NR.sup.135, wherein R.sup.135 is selected from H and
C.sub.1-3 alkyl, and each uu, uu', and uu'' is independently
selected from 0 and 1.
[0685] In another embodiment, L is selected from
##STR00402##
[0686] wherein X.sup.70, X.sup.71, X.sup.72, and X.sup.73 are
independently selected from O, S, and NR.sup.82, X.sup.74 is
selected from
##STR00403##
[0687] d is selected from 0 to 8, e is 0 or 1, gg', gg'', gg''',
gg'''', and gg* are independently selected from 0 to 1000, and
R.sup.81 and R.sup.82 are independently selected from H and
optionally substituted C.sub.1-3 alkyl. In other embodiments, gg',
gg'', gg''', gg'''', and gg* are independently selected from 3 to
1000 or 500 or 100 or 50 or 10.
[0688] In vet another embodiment, L is not
##STR00404##
[0689] The linkage between L and V.sup.1 or Y may for example be an
amide, a ureum, a carbonate, or a carbamate linkage. In one
embodiment, the linkage between L and V.sup.1 is a ureum,
carbonate, or carbamate linkage. Alternatively, when V.sup.1 is a
peptide in which the N-terminal amino acid is an amino acid mimic
that carries an .alpha.-azido group instead of an .alpha.-amino
group, the linkage between L and V.sup.1 may be a triazole group
formed through reaction of an acetylene group being part of L and
the .alpha.-azido group of V.sup.1.
[0690] The Reactive Moiety RM and the Linking Group L.sup.2
[0691] The reactive moiety RM in a compound of formula (IV) is
connected to the linking group L and is able to react with a
suitable functional group on a reaction partner.
[0692] In one embodiment of this invention, the reactive moiety RM
is designed to react with a functional group on the moiety V.sup.2,
which results in formation of a compound of formula (III). In this
reaction, the moiety RM is transformed into the moiety L.sup.2. In
another embodiment, the reactive moiety RM is designed to react
with a complementary moiety in situ, e.g., in vivo, for example
with serum albumin, to give a compound that may or may not be a
compound of formula (III).
[0693] In one aspect of this invention, the reactive moiety RM
contains an electrophilic group that reacts with a nucleophilic
group on the reaction partner, for example V.sup.2, e.g., a thiol
group, an amino group, or a hydroxy group.
[0694] In another aspect of this invention, the reactive moiety RM
contains a nucleophilic group that reacts with an electrophilic
group on the reaction partner, for example V.sup.2, e.g., an
aldehyde group.
[0695] In another aspect of the invention, the reactive moiety RM
contains a cycloaddition partner moiety, e.g., an alkene, a diene,
a 1,3-dipole, or a 1,3-dipolarophile, that reacts with a suitable
complementary cycloaddition partner moiety on the reaction partner,
for example V.sup.2, e.g., a diene, an alkene, a 1,3-dipolarophile,
or a 1,3-dipole.
[0696] In another aspect of the invention, the reactive moiety RM
contains a group that can be coupled with a suitable complementary
group on the reaction partner, for example V.sup.2, under
metal-catalyzed, biocatalyzed, or enzyme-catalyzed conditions,
e.g., palladium-catalyzed conditions.
[0697] In one aspect of the invention, the reactive moiety RM is,
without limitation,
##STR00405##
[0698] wherein
[0699] X.sup.35 is selected from halide, hydroxy, OC(O)R.sup.dd,
and OC(O)OR.sup.dd, or C(O)--X.sup.35 is an active ester, X.sup.36
is selected from halide, mesyloxy, triflyloxy, and tosyloxy, and
R.sup.dd is selected from optionally substituted C.sub.1-10 alkyl,
C.sub.1-10 heteroalkyl, C.sub.3-10 cycloalkyl, heterocycloalkyl,
C.sub.5-10 aryl, and C.sub.1-10 heteroaryl.
[0700] In one embodiment, the moiety RM is selected from
##STR00406##
[0701] which makes it able to react with a thiol group on the
reaction partner, for example moiety V.sup.2.
[0702] In another embodiment, the moiety RM is
##STR00407##
[0703] which makes it able to react with a thiol group on the
reaction partner, for example moiety V.sup.2.
[0704] In another embodiment, the moiety RM is
##STR00408##
[0705] wherein X.sup.36 is Br, which makes it able to react with a
thiol group on the reaction partner, for example moiety
V.sup.2.
[0706] In another embodiment, the moiety RM is selected from
##STR00409##
[0707] which makes it able to react with an amino group, e.g., a
primary or secondary amino group, on the reaction partner, for
example moiety V.sup.2.
[0708] In another embodiment, the moiety RM is selected from
##STR00410##
[0709] which makes it able to react with an aldehyde group on the
reaction partner, for example moiety V.sup.2.
[0710] The linking group L.sup.2 in a compound of formula (III)
represents the remainder of RM when the reactive moiety RM has
reacted with V.sup.2. This group then links the moiety V.sup.2 with
L. The group that remains may be a bond, meaning that L.sup.2 is
absent. Typically, however, L.sup.2 is a linking group. When a
compound of formula (III) is formed other than via a compound of
formula (IV), L.sup.2 does not represent the remainder of RM, but
may represent a similar or the same moiety and in addition be
selected from for example optionally substituted C.sub.1-10
alkylene, C.sub.1-10 heteroalkylene, C.sub.3-10 cycloalkylene,
C.sub.1-10 heterocycloalkylene, C.sub.5-10 arylene, and C.sub.1-10
heteroarylene. The L.sup.2 moiety may optionally comprise a
X.sup.14(CH.sub.2CH.sub.2O).sub.ggCH.sub.2CH.sub.2X.sup.14
moiety.
[0711] In one embodiment, the moiety L.sup.2 is absent.
[0712] In another embodiment, the moiety L.sup.2 is, without
limitation,
##STR00411##
[0713] In a further embodiment, the moiety L.sup.2 is
##STR00412##
[0714] In yet a further embodiment, the moiety L.sup.2 is
##STR00413##
[0715] The RM2 Moiety
[0716] RM2 may be a reactive moiety or a leaving group.
[0717] If RM2 is a reactive moiety, it is preferably different from
the RM reactive moiety. In this way, reactions with the
bifunctional linker may be carried out selectively and individually
with each of the reactive moieties. When RM2 is a reactive moiety,
this means that after reaction with a therapeutic or diagnostic
moiety or a promoiety-containing derivative thereof, the RM2 moiety
(or what remains of it after reaction) may become part of said
therapeutic or diagnostic moiety or a promoiety-containing
derivative thereof optionally together with any part of Y which may
not self-eliminate because of a limited or absent leaving
capability of therapeutic or diagnostic moiety-conjugated RM2.
[0718] In one embodiment, the RM2 may be connected to Y and be
##STR00414##
[0719] wherein
[0720] X.sup.35 is selected from halide, hydroxy, OC(O)R.sup.dd,
and OC(O)OR.sup.dd, or C(O)--X.sup.35 is an active ester, X.sup.36
is selected from halide, mesyloxy, triflyloxy, and tosyloxy,
X.sup.95 is either absent or selected from O, S, NR.sup.95,
C.sub.1-3 alkyl, and C.sub.1-3 heteroalkyl, R.sup.dd is selected
from optionally substituted C.sub.1-10 alkyl, C.sub.1-10
heteroalkyl, C.sub.3-10 cycloalkyl, C.sub.1-10 heterocycloalkyl,
C.sub.5-10 aryl, and C.sub.1-10 heteroaryl, and R.sup.95 is
selected from H and C.sub.1-3 alkyl.
[0721] RM2 may also be a leaving group. In this case, RM2 is
replaced by the therapeutic or diagnostic moiety or a
promoiety-containing derivative thereof in a reaction of a compound
of formula (VIII) with such a therapeutic or diagnostic moiety or a
derivative thereof and this therapeutic or diagnostic moiety or a
derivative thereof becomes directly attached to Y.
[0722] In one embodiment, RM2 is selected from a halide (fluoride,
chloride, bromide, and iodide), azide, a sulfonate (e.g., an
optionally substituted C.sub.1-6 alkanesulfonate, such as
methanesulfonate, trifluoromethanesulfonate, and
trifluoroethanesulfonate, or an optionally substituted
benzenesulfonate, such as p-toluenesulfonate and nosylate),
imidazole, a cyclic imide thione, succinimide-N-oxide,
phtalimide-N-oxide, p-nitrophenoxide, o-nitrophenoxide,
pentafluorophenoxide, tetrafluorophenoxide,
1,3,5-trichlorophenoxide, 1,3,5-trifluorophenoxide, a carboxylate,
an aminocarboxylate (carbamate), an alkoxycarboxylate (carbonate),
and an alkoxy group that together with the carbonyl group of Y can
be referred to as an active ester group. Such alkoxy groups
include, but are not limited to, succinimide-N-oxide,
p-nitrophenoxide, pentafluorophenoxide, tetrafluorophenoxide,
1-hydroxybenzotriazole, and 1-hydroxy-7-azabenzotriazole, and
groups with comparable leaving capability.
[0723] The Moiety V.sup.2
[0724] The moiety V.sup.2 is a functional moiety, which means that
it adds additional functionality to a compound of the
invention.
[0725] In one embodiment, V.sup.2 is a targeting moiety. In another
embodiment, the V.sup.2 moiety is a moiety that improves the
pharmacological properties of a compound of the invention. In yet
another embodiment, the V.sup.2 moiety is a moiety that causes
accumulation of a compound of the invention at a target site. In
yet another embodiment, the V.sup.2 moiety is a moiety that
improves the aqueous solubility of a compound of the invention. In
yet another embodiment, the V.sup.2 moiety is a moiety that
increases the hydrophobicity of a compound of the invention. In yet
another embodiment, the V.sup.2 moiety is a moiety that reduces
extravasation of a compound of the invention. In yet another
embodiment, the V.sup.2 moiety is a moiety that reduces excretion
of a compound of the invention. In yet another embodiment, the
V.sup.2 moiety is a moiety that reduces the immunogenicity of a
compound of the invention. In yet another embodiment, the V.sup.2
moiety is a moiety that enhances the circulation time of a compound
of the invention. In yet another embodiment, the V.sup.2 moiety is
a moiety that enhances the ability of a compound of the invention
to cross a biological barrier, e.g., a membrane, cell wall, or the
blood-brain barrier. In yet another embodiment, the V.sup.2 moiety
is a moiety that enhances the ability of a compound of the
invention to internalize. In yet another embodiment, the V.sup.2
moiety is a moiety that enables a compound of the invention to
internalize. In yet another embodiment, the V.sup.2 moiety is a
moiety that causes the compounds of the invention to aggregate. In
yet another embodiment, the V.sup.2 moiety is a moiety that reduces
aggregation of a compound of the invention. In yet another
embodiment, the V.sup.2 moiety is a moiety that causes a compound
of the invention to form micelles or liposomes. In yet another
embodiment, the V.sup.2 moiety is a moiety that causes complexation
of a compound of the invention to another molecule, e.g., a
biomolecule. In yet another embodiment, the V.sup.2 moiety is a
polynucleotide moiety that complexes with a complementary
nucleotide sequence, for example RNA or DNA. In yet another
embodiment, the V.sup.2 moiety is a moiety that causes a compound
of the invention to bind, associate, interact, or complex to
another moiety, for example a (functionalized) surface or solid
support.
[0726] In another embodiment, V.sup.2 exhibits two or more
different functions. The V.sup.2 moiety may for example be a
targeting moiety and at the same time improve the pharmacological
properties, including water solubility.
[0727] In one aspect of the invention, the moiety V.sup.2 includes
within its scope any unit that binds or reactively associates or
complexes with a receptor, a receptor complex, antigen, or other
moiety associated with a given target cell population. V.sup.2 can
be any molecule that binds to, complexes with, or reacts with a
moiety of a cell population sought to be therapeutically or
otherwise biologically modified. The V.sup.2 moiety acts to deliver
the one or more moieties Z to the particular target cell population
with which V.sup.2 reacts or to which V.sup.2 binds. Such V.sup.2
moieties include, but are not limited to, aptamers, full-length
antibodies and antibody fragments and derivatives thereof, lectins,
biologic response modifiers, enzymes, vitamins, growth factors,
steroids, nutrients, sugar residues, oligosaccharide residues,
hormones, and any derivatives thereof, or any combination of any of
these. Upon binding, reactively associating, or complexing, the
compounds of the invention may or may not be internalized. If
internalization occurs, transformation and/or cleavage of V.sup.1
preferably occur inside the target cell.
[0728] Useful non-immunoreactive protein, polypeptide, or peptide
V.sup.2 moieties include, but are not limited to, transferrin,
epidermal growth factors ("EGF"), bombesin, gastrin and its
derivatives, gastrin-releasing peptide, platelet-derived growth
factor, IL-2, IL-6, transforming growth factors ("TGF"), such as
TGF-a and TGF-P, tumor growth factors, vaccinia growth factor
("VGF"), insulin and insulin-like growth factors I and II, lectins,
and apoprotein from low density lipoprotein.
[0729] Useful polyclonal antibody V.sup.2 moieties are
heterogeneous populations of antibody molecules. Various procedures
well-known in the art may be used for the production of polyclonal
antibodies to an antigen-of-interest.
[0730] Useful monoclonal antibody V.sup.2 moieties are homogeneous
populations of antibodies to a particular antigen (e.g., a cancer
cell antigen). A monoclonal antibody (mAb) to an
antigen-of-interest can be prepared by using any technique known in
the art which provides for the production of monoclonal antibody
molecules.
[0731] Useful monoclonal antibody V.sup.2 moieties include, but are
not limited to, human monoclonal antibodies, humanized monoclonal
antibodies, or chimeric human-mouse (or other species) monoclonal
antibodies. Monoclonal antibodies may be made by any of numerous
techniques known in the art.
[0732] The V.sup.2 moiety can also be a bispecific antibody.
Methods for making bispecific antibodies are known in the art.
[0733] The V.sup.2 moiety can be a functionally active fragment,
derivative, or analog of an antibody that immunospecifically binds
to an antigen on a target cell, e.g., a cancer cell antigen. In
this regard, "functionally active" means that the fragment,
derivative, or analog is able to elicit anti-anti-idiotype
antibodies that recognize the same antigen that the antibody from
which the fragment, derivative, or analog is derived,
recognizes.
[0734] Other useful V.sup.2 moieties comprise fragments of
antibodies including, but not limited to, F(ab').sub.2 fragments,
which contain the variable region, the light chain constant region,
and the CH1 domain of the heavy chain, which can be produced by
pepsin digestion of the antibody molecule, and Fab fragments, which
can be generated by reducing the disulfide bridges of the
F(ab').sub.2 fragments. Other useful V.sup.2 moieties are heavy
chain and light chain dimers of antibodies, or any minimal fragment
thereof such as Fvs or single chain antibodies (SCAs), domain
antibodies, anticalins, affibodies, nanobodies, and any other
molecules with the same, similar, or comparable specificity as the
parent antibody.
[0735] Additionally, recombinant antibodies, such as chimeric and
humanized monoclonal antibodies, comprising both human and
non-human portions, which can be made using standard recombinant
DNA techniques, are useful V.sup.2 moieties. A chimeric antibody is
a molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine monoclonal and a human immunoglobulin constant region.
Humanized antibodies are antibody molecules from non-human species
having one or more complementarity determining regions (CDRs) from
the non-human species and a framework region from a human
immunoglobulin molecule.
[0736] Completely human antibodies are particularly desirable as
V.sup.2 moieties. Such antibodies can for example be produced using
transgenic mice that are incapable of expressing endogenous
immunoglobulin heavy and light chains genes, but which can express
human heavy and light chain genes.
[0737] In other embodiments, the V.sup.2 moiety is a fusion protein
of an antibody, or a functionally active fragment or derivative
thereof, for example one in which the antibody is fused via a
covalent bond (e.g., a peptide bond) at either the N-terminus or
the C-terminus to an amino acid sequence of another protein (or
portion thereof, preferably at least a 10, 20, or 50 amino acid
portion of the protein) that is not the antibody. Preferably, the
antibody or fragment thereof is covalently linked to the other
protein at the N-terminus of the constant domain.
[0738] The V.sup.2 moiety antibodies include analogs and
derivatives that are modified, i.e., by the covalent attachment of
any type of molecule as long as such covalent attachment permits
the antibody to retain its antigen-binding immunospecificity. For
example, but not by way of limitation, derivatives and analogs of
antibodies include those that have been further modified, e.g., by
glycosylation, acetylation, pegylation, disulfide reduction,
phosphylation, amidation, derivatization by known protecting or
blocking groups, proteolytic cleavage, linkage to another protein,
etc. Additionally, the analog or derivative can contain one or more
unnatural amino acids.
[0739] The V.sup.2 moiety antibodies include antibodies having
modifications (e.g., substitutions (for example cysteine to serine
or serine to cysteine), deletions, or additions). In particular,
they include antibodies having modifications in amino acid residues
identified as involved in the interaction between the Fc domain and
the FcRn receptor. Modifications may also be introduced to be able
to couple the antibody to linker-agent conjugates at specific
positions on the antibody.
[0740] In a specific embodiment, an antibody immunospecific for a
cancer or tumor antigen is used as a V.sup.2 moiety in accordance
with the compounds, compositions, and methods of the invention.
[0741] Antibodies immunospecific for a cancer cell antigen can be
obtained commercially or produced by any method known to one of
skill in the art, such as chemical synthesis or recombinant
expression techniques. The nucleotide sequences encoding antibodies
immunospecific for a cancer cell antigen can be obtained, e.g.,
from the GenBank database or a database like it, a commercial or
other source, literature publications, or by routine cloning and
sequencing.
[0742] Examples of antibodies available for the treatment of cancer
that may be useful for incorporation into conjugates of this
invention as a V.sup.2 moiety include, but are not limited to,
HERCEPTIN (trastuzumab), which is a humanized anti-HER2 monoclonal
antibody for the treatment of patients with metastatic breast
cancer; RITUXAN (rituximab), which is a chimeric anti-CD20
monoclonal antibody for the treatment of patients with
non-Hodgkin's lymphoma; OvaRex (oregovomab), which is a murine
antibody for the treatment of ovarian cancer; Panorex
(edrecolomab), which is a murine IgG.sub.2a antibody for the
treatment of colorectal cancer; IMC-BEC2 (mitumomab), which is a
murine IgG antibody for the treatment of lung cancer; IMC-C225
(erbitux), which is a chimeric IgG antibody for the treatment of
head and neck cancer; Vitaxin, which is a humanized antibody for
the treatment of sarcoma; Campath I/H (alemtuzumab), which is a
humanized IgG.sub.1 antibody for the treatment of chronic
lymphocytic leukemia (CLL); SGN-70, which is a humanized anti-CD70
antibody for the treatment of hematologic malignancies; Smart MI95,
which is a humanized IgG antibody for the treatment of acute
myeloid leukemia (AML); J591, which is a humanized antibody against
prostate specific membrane antigen; LymphoCide (epratuzumab), which
is a humanized IgG antibody for the treatment of non-Hodgkin's
lymphoma; SGN-33, which is a humanized anti-CD33 antibody for the
treatment of acute myeloid leukemia; Smart ID 10, which is a
humanized antibody for the treatment of non-Hodgkin's lymphoma;
Oncolym, which is a murine antibody for the treatment of
non-Hodgkin's lymphoma; Allomune, which is a humanized anti-CD2 mAb
for the treatment of Hodgkin's disease or non-Hodgkin's lymphoma;
Avastin (bevacizumab), which is a humanized anti-VEGF antibody for
the treatment of lung and colorectal cancers; SGN-40, which is a
humanized anti-CD40 antibody for the treatment of multiple myeloma;
SGN-30, which is a chimeric anti-CD30 antibody for the treatment of
Hodgkin's disease; CEAcide, which is a humanized anti-CEA antibody
for the treatment of colorectal cancer; IMC-1C11, which is an
anti-KDR chimeric antibody for the treatment of colorectal cancer,
lung cancers, and melanoma; and Cetuximab, which is an anti-EGFR
chimeric antibody for the treatment of epidermal growth factor
positive cancers.
[0743] Other antibodies that may be useful for incorporation into
conjugates of this invention as a V.sup.2 moiety include, but are
not limited to, antibodies against the following antigens: CA125,
CA9, CA6, CA15-3, CA19-9, L6, Lewis Y, Lewis X, Lewis A, alpha
fetoprotein, CA 242, placental alkaline phosphatase, prostate
specific antigen (PSA), prostate specific membrane antigen (PSMA),
prostatic acid phosphatase, epidermal growth factor receptors,
interleukin receptors, integrins, insulin-like growth factor
receptors, CanAg, DAF, PEM, IRTA-2, IRTA-4, AFP, HER2, EGFR,
VEGFR1, VEGFR2, MAGE-1, LUCA1, LUCA2, MAGE-2, MAGE-3, MAGE-4, ED-B,
MADCAM, CEACAMS, MCP-1, Cripto, TAT226, VLA-4, C3B,
anti-transferrin receptor, endosialin, E-selectin, GCC, GP-75,
Syndecan-1, GPNMB, ROBO4, STEAP-1, CMET, EGP-1, Kim-1, Tim-1, Eph
receptor tyrosine kinases, HMW-MAA, TMEFF2, PSCA, CLL-1,
TNF-.alpha., FAP-.alpha., IFN-.alpha., EphA2, EphB2, EphB3, EphB4,
EGFL-7, DLL-4, RS7, 4-1BB, TENB2, FLT3, p97, FGF19, FGFR2,
glypican-3, P53, L53, RON, MN, GFR-.alpha.3, FDF03, TSLPR,
MUC1-KLH, Tag 72, MUC18, B7H4, PTK7, RG-1, MUC16, CSAP, PDGF, PSMA,
5T4, EpCAM, SGA-1M, SGA-56M, SGA-72M, IGF1R, CCR2, CCRS, CTLA4,
CLCA-1, ELAM1, DRS, CEA, CXCR-4, GD2, gp100, GD3 ganglioside, L243,
HMGB1, GPC-3, MART1, IL-2 receptor, CD2, CD3, CD303, CD4, CD20,
CD43, CD44, CD30, CD55, CD151, CD154, CD19, CD21, CD23, CD79, CD52,
CD25, CD45, CD46, CD56, CD59, CD7, CD137, CD138, CD74, CD133, CD80,
CD63, CD64, CD66, CD140b, CD32, CD33, CD37, CD22, CD27, Apo-2,
ERBB4, HLA-DR, HLA-DR10, human chorionic gonadotropin, CD38, CD40,
CD70, mucin, P21, a cancer stem cell-specific receptor, MPG, and
Neu oncogene product. Many other internalizing or non-internalizing
antibodies that bind to tumor-associated antigens can be used in
this invention as a V.sup.2 moiety, some of which have been
reviewed.sup.14.
[0744] In one embodiment, the anti-Her2 antibody trastuzumab is
selected as the V.sup.2 moiety. In another embodiment, an
epitope-binding functional fragment or derivative of trastuzumab is
selected as the V.sup.2 moiety. In yet another embodiment, an
anti-Her2 antibody or a functional fragment or derivative thereof
is selected as the V.sup.2 moiety. In yet another embodiment, an
anti-Her2 antibody or functional fragment or derivative thereof
that has improved properties with respect to trastuzumab is
selected as the V.sup.2 moiety; improved properties may for example
be increased binding, longer circulation half-life, increased
internalization rate, higher binding specificity for tumor tissue
compared to non-tumor tissue, and/or reduced immunogenicity.
[0745] In yet another embodiment, the anti-PSMA antibody J591 is
selected as the V.sup.2 moiety. In another embodiment, an
epitope-binding functional fragment or derivative of J591 is
selected as the V.sup.2 moiety. In yet another embodiment, an
anti-PSMA antibody or a functional fragment or derivative thereof
is selected as the V.sup.2 moiety. In yet another embodiment, an
anti-PSMA antibody or functional fragment or derivative thereof
that has improved properties with respect to J591 is selected as
the V.sup.2 moiety; improved properties may for example be
increased binding, longer circulation half-life, increased
internalization rate, higher binding specificity for tumor tissue
compared to non-tumor tissue, and/or reduced immunogenicity.
[0746] In yet other embodiments, an anti-CD19 antibody or an
anti-CD22 antibody or an anti-CD30 antibody or an anti-CD33
antibody or an anti-CD56 antibody or an anti-CD70 antibody or an
anti-CD74 antibody or an anti-CD138 antibody or an anti-CLL-1
antibody or an anti-5T4 antibody or an anti-CD303 antibody or an
anti-Tag 72 antibody or an anti-Lewis A like carbohydrate antibody
or an anti-EphB3 antibody or an anti-HMW-MAA antibody or an
anti-CD38 antibody or an anti-Cripto antibody or an anti-EphA2
antibody or an anti-GPNMB antibody or an anti-integrin antibody or
an anti-MN antibody is selected as the V.sup.2 moiety. In yet other
embodiments, an epitope-binding functional fragment or derivative
of an anti-CD19 antibody or an anti-CD22 antibody or an anti-CD30
antibody or an anti-CD33 antibody or an anti-CD56 antibody or an
anti-CD70 antibody or an anti-CD74 antibody or an anti-CD138
antibody or an anti-CLL-1 antibody or an anti-5T4 antibody or an
anti-CD303 antibody or an anti-Tag 72 antibody or an anti-Lewis A
like carbohydrate antibody or an anti-EphB3 antibody or an
anti-HMW-MAA antibody or an anti-CD38 antibody or an anti-Cripto
antibody or an anti-EphA2 antibody or an anti-GPNMB antibody or an
anti-integrin antibody or an anti-MN antibody is selected as the
V.sup.2 moiety. Therefore, in one embodiment, the V.sup.2 moiety
may be selected from an anti-CD19 antibody, an anti-CD22 antibody,
an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD56
antibody, an anti-CD70 antibody, an anti-CD74 antibody, an
anti-CD138 antibody, an anti-CLL-1 antibody, an anti-5T4 antibody,
an anti-CD303 antibody, an anti-Tag 72 antibody, an anti-Lewis A
like carbohydrate antibody, an anti-EphB3 antibody, an anti-HMW-MAA
antibody, an anti-CD38 antibody, an anti-Cripto antibody, an
anti-EphA2 antibody, an anti-GPNMB antibody, an anti-integrin
antibody, an anti-MN antibody, an anti-Her2 antibody, and an
anti-PSMA antibody, or from an epitope-binding functional fragment
or derivative of any of these.
[0747] In some embodiments, the antibody is an anti-nuclear
antibody or an antibody that can bind to a receptor or receptor
complex expressed on a target cell. The receptor or receptor
complex can comprise an immunoglobulin gene superfamily member, an
integrin, a chemokine receptor, a TNF receptor superfamily member,
a cytokine receptor, a major histocompatibility protein, a
complement control protein, or a lectin.
[0748] In another specific embodiment, an antibody immunospecific
for an antigen associated with an autoimmune disease is used as a
V.sup.2 moiety in accordance with the compounds, compositions, and
methods of the invention. In another specific embodiment, an
antibody immunospecific for a viral or microbial antigen is used as
a V.sup.2 moiety in accordance with the compounds, compositions,
and methods of the invention. As used herein, the term "viral
antigen" includes, but is not limited to, any viral peptide,
polypeptide, protein, saccharide, polysaccharide, or lipid that is
capable of eliciting an immune response. As used herein, the term
"microbial antigen" includes, but is not limited to, any microbial
peptide, polypeptide, protein, saccharide, polysaccharide, or lipid
that is capable of eliciting an immune response.
[0749] New antibodies are continually being discovered and
developed, and the present invention provides that these new
antibodies may also be incorporated into a compound of this
invention.
[0750] V.sup.2 can react with the reactive moiety RM via for
example a heteroatom on V.sup.2. Heteroatoms that may be present on
V.sup.2 include, without limitation, sulfur (in one embodiment,
from a sulfhydryl group), oxygen (in one embodiment, from a
carboxyl or hydroxyl group), and nitrogen (in one embodiment, from
a primary or secondary amino group). V.sup.2 may also react via for
example a carbon atom (in one embodiment, from a carbonyl group).
These atoms can be present on V.sup.2 in V.sup.2's natural state,
for example a naturally occurring antibody, or can be introduced
into V.sup.2 via (chemical) modification.
[0751] Free sulfhydryl groups can be generated in an antibody or
antibody fragment by reduction of the antibody (fragment) with a
reducing agent such as dithiothreitol (DTT) or
tris(2-carboxyethyl)phosphine (TCEP). In this way, modified
antibodies can be obtained that can have from 1 to about 20
sulfhydryl groups, but typically between about 1 and about 9
sulfhydryl groups.
[0752] Alternatively, V.sup.2 can have one or more carbohydrate
groups that can be chemically modified to contain one or more
sulfhydryl groups. As another alternative, sulfhydryl groups can be
generated by reaction of amino groups, for example from lysine
moieties, on V.sup.2 with 2-iminothiolane (Traut's reagent),
N-succinimidyl S-acetylthioacetate (SATA), or another
sulfhydryl-generating reagent. Such a reagent may also be used to
introduce additional functionality. For example, a
sulfhydryl-generating reagent may be used that not only introduces
a sulfhydryl group onto V.sup.2, but at the same time introduces a
water-soluble moiety such as an oligoethylene glycol or
polyethylene glycol. Such a group may be present as a substituent
in the reagent (instead of being part of the main chain) in order
to keep the sulfhydryl group close to the V.sup.2 moiety. The
presence of such a water-soluble moiety may eventually improve the
pharmacological properties of a compound of formula (III).
[0753] In one embodiment, the V.sup.2 moiety is a receptor-binding
moiety.
[0754] In another embodiment, the V.sup.2 moiety is an antibody or
an antibody fragment or a derivative thereof.
[0755] In another embodiment, the V.sup.2 moiety is a monoclonal
antibody or a fragment or derivative thereof. In one embodiment,
V.sup.2 has one or more sulfhydryl groups and V.sup.2 reacts with
one or more RM moieties of one or more compounds of formula (IV)
via one or more of these sulfhydryl groups' sulfur atoms to form a
compound of formula (III) in which one or more compounds of formula
(IV) have thus been incorporated.
[0756] In yet another embodiment, V.sup.2 contains one or more
disulfide bonds that can be chemically reduced to sulfhydryl groups
(two for each disulfide bond), which can then be reacted with one
or more reactive moieties RM to form a compound of formula
(III).
[0757] In another embodiment, V.sup.2 contains about 1 to about 3
sulfhydryl groups, which can be reacted with one or more reactive
moieties RM to form a compound of formula (III).
[0758] In another embodiment, V.sup.2 contains about 2 sulfhydryl
groups, which can be reacted with one or more reactive moieties RM
to form a compound of formula (III).
[0759] In another embodiment, V.sup.2 contains about 3 to about 5
sulfhydryl groups, which can be reacted with one or more reactive
moieties RM to form a compound of formula (III).
[0760] In another embodiment, V.sup.2 contains about 4 sulfhydryl
groups, which can be reacted with one or more reactive moieties RM
to form a compound of formula (III).
[0761] In another embodiment, V.sup.2 contains about 7 to about 9
sulfhydryl groups, which can be reacted with one or more reactive
moieties RM to form a compound of formula (III).
[0762] In another embodiment, V.sup.2 contains about 8 sulfhydryl
groups, which can be reacted with one or more reactive moieties RM
to form a compound of formula (III).
[0763] In another embodiment, V.sup.2 can have one or more
carbohydrate groups that can be chemically modified to have one or
more sulfhydryl groups. V.sup.2 reacts with RM moieties via these
one or more sulfhydryl groups' sulfur atoms to form a compound of
formula (III).
[0764] In another embodiment, V.sup.2 can have one or more lysine
groups that can be chemically modified to have one or more
sulfhydryl groups, which can be reacted with one or more reactive
moieties RM to form a compound of formula (III).
[0765] Reactive moieties that can react with a sulfhydryl group
include, but are not limited to, carbamoyl halide, acyl halide,
a-haloacetamide, halomethyl ketone, vinyl sulfone, maleimide, and
2-disulfanylpyridine.
[0766] In yet another embodiment, V.sup.2 can have one or more
carbohydrate groups that can be oxidized to provide one or more
aldehyde groups. The corresponding aldehyde(s) can then react with
one or more reactive moieties RM to form a compound of formula
(III). Reactive moieties that can react with an aldehyde group on
V.sup.2 include, but are not limited to, hydrazine, hydrazide,
amine, and hydroxylamine.
[0767] In yet another embodiment, V.sup.2 can have one or more
amino groups, e.g., from lysine residues, which can be reacted with
one or more reactive moieties RM to form a compound of formula
(III). Reactive moieties that can react with an amino group
include, but are not limited to, carbamoyl halide,
.alpha.-haloacetamide, acyl halide, aldehyde, sulfonyl chloride,
alkyl halide, alkyl sulfonate, isocyanate, and isothiocyanate.
[0768] A conjugate of formula (III) may exist as a mixture, wherein
each component of the mixture has a different q value. For example,
the compound may exist as a mixture of two separate compounds, one
compound wherein q is 2 and another compound wherein q is 3. As
another example, a compound may exist as a mixture of 5 separate
compounds, in which q is 1, 2, 3, 4, and 5, respectively. As yet
another example, a compound may exist as a mixture of more than 5
separate compounds. Such mixtures might further be "contaminated"
with unconjugated V.sup.2. When analyzing the compound of formula
(III) it is understood that q may be the (rounded) average number
of L.sup.2-L(-(V.sup.1--Y)).sub.p(Z).sub.z/q units per V.sup.2
moiety. Furthermore, for a given q, the compound may exist as a
mixture of (constitutional) isomers as the q
L.sup.2-L(-(V.sup.1--Y)).sub.p(Z).sub.z/q moieties may be connected
to distinct (sets of) functional groups on V.sup.2. It should be
noted that the number of Z moieties in each unit only equals z/q if
all units are the same and/or contain the same number of Z
moieties.
[0769] In one embodiment, the V.sup.2 moiety is connected to
L.sup.2 via a sulfur atom of V.sup.2.
[0770] In another embodiment, the V.sup.2 moiety is connected to
L.sup.2 via a sulfur atom and q ranges from about 1 to about
20.
[0771] In another embodiment, the V.sup.2 moiety is connected to
L.sup.2 via a sulfur atom and q ranges from about 1 to about 9.
[0772] In another embodiment, the V.sup.2 moiety is connected to
L.sup.2 via a sulfur atom and q ranges from about 1 to about 3.
[0773] In another embodiment, the V.sup.2 moiety is connected to
L.sup.2 via a sulfur atom and q is about 1.
[0774] In another embodiment, the V.sup.2 moiety is connected to
L.sup.2 via a sulfur atom and q is about 2.
[0775] In another embodiment, the V.sup.2 moiety is connected to
L.sup.2 via a sulfur atom and q ranges from about 3 to about 5.
[0776] In another embodiment, the V.sup.2 moiety is connected to
L.sup.2 via a sulfur atom and q is about 4.
[0777] In another embodiment, the V.sup.2 moiety is connected to
L.sup.2 via a sulfur atom and q ranges from about 7 to about 9.
[0778] In another embodiment, the V.sup.2 moiety is connected to
L.sup.2 via a sulfur atom and q is about 8.
[0779] In one embodiment, a compound of formula (III) exists as a
mixture of separate compounds.
[0780] In one embodiment, a compound of formula (III) exists as a
mixture of separate compounds wherein q for three compounds is 1,
2, and 3, respectively.
[0781] In one embodiment, a compound of formula (III) exists as a
mixture of separate compounds wherein q for three compounds is 3,
4, and 5, respectively.
[0782] In one embodiment, a compound of formula (III) exists as a
mixture of separate compounds wherein q for three compounds is 5,
6, and 7, respectively.
[0783] In one embodiment, a compound of formula (III) exists as a
mixture of separate compounds wherein q for three compounds is 7,
8, and 9, respectively.
[0784] In another embodiment, the V.sup.2 moiety is connected to
L.sup.2 via a nitrogen atom of V.sup.2.
[0785] In yet another embodiment, the V.sup.2 moiety is connected
to L.sup.2 via a carbon atom of V.sup.2.
[0786] In another aspect of this invention, the V.sup.2 moiety
includes any unit that causes accumulation of compounds of the
invention at the target site or in the vicinity thereof by a
mechanism other than binding or reactively associating or
complexing with a receptor, antigen, or other receptive moiety
associated with a given target site, e.g., a target cell
population. One way to achieve this is for example to use a large
macromolecule as a V.sup.2 moiety, which targets to solid tumor
tissue through the enhanced permeability and retention (EPR)
effect. Ringsdorf reported use of polymers to target antitumor
agents to tumors..sup.15 Through this EPR effect, macromolecules
passively accumulate in solid tumors as a consequence of the
disorganized pathology of angiogenic tumor vasculature with its
discontinuous endothelium, leading to hyperpermeability to large
macromolecules, and the lack of effective tumor lymphatic
drainage.
[0787] The V.sup.2 moiety may for example be a branched or
unbranched polymer, such as for example poly
[N-(2-hydroxypropyl)methacrylamide] (HPMA), hydroxy ethyl starch
(HES), poly(2-hydroxyethyl methacrylate) (HEMA), polyglutamic acid
or poly-L-glutamic acid (PG), carboxymethyldextran (CMDex), a
polyacetal, chitosan, a polypeptide, an oligoethylene glycol or
polyethylene glycol (PEG), or a copolymer, such as an HPMA
copolymer, an HPMA-methacrylic acid copolymer, a HEMA-methacrylic
acid copolymer, a CMDex copolymer, a .beta.-cyclodextrin copolymer,
a PEG copolymer, or a poly(lactic-co-glycolic) acid
copolymer..sup.16 In this document both polymer and copolymer are
referred to as polymer.
[0788] The polymer may be connected to L.sup.2 via any suitable
functional group, which can be located at one or both ends of the
polymer, meaning that in the conjugate q ranges from 1 to 2, or
alternatively, the functional groups may (also) be located on
groups pendant on the polymer such that L.sup.2 is (also) connected
to the polymer via these pendant groups with q typically ranging
from 1 to about 1000. Optionally, the polymer may also contain an
additional targeting group that can bind or reactively associate or
complex with a receptive moiety, e.g., an antibody or antibody
derivative, bonded to the polymer either via a pendant group or end
group, such that improved targeting to the target site is
achieved.
[0789] Alternatively, the V.sup.2 moiety may be a dendrimer or a
protein or protein fragment, e.g., serum albumin, which has no
targeting properties except for its ability to accumulate at the
target site because of its size or molecular weight.
[0790] In one embodiment, the V.sup.2 moiety contains a
polymer.
[0791] In another embodiment, the V.sup.2 moiety is a polymer.
[0792] In another embodiment, the V.sup.2 moiety is a polymer and q
ranges from 1 to about 1000.
[0793] In other embodiments, the V.sup.2 moiety is a polymer and q
ranges from 1 to about 500 or 400 or 300 or 200 or 100 or less than
100.
[0794] In another embodiment, the V.sup.2 moiety is a polymer and q
ranges from 1 to 2.
[0795] In another embodiment, the V.sup.2 moiety is a polymer and q
is 1.
[0796] In a specific embodiment, the V.sup.2 moiety is an
oligoethylene glycol or a polyethylene glycol or a derivative
thereof.
[0797] In another embodiment, the V.sup.2 moiety is a dendrimer, a
protein, or a protein fragment.
[0798] In another embodiment, V.sup.2 is absent.
[0799] In another embodiment, the V.sup.2 moiety is a moiety that
is able to transport the conjugate across a biological barrier,
e.g., a cell membrane, either with or without prior binding,
associating, or complexing with a receptor or receptor complex. In
one embodiment, the V.sup.2 moiety is a Tat peptide or a
derivative, fragment, or analog thereof, or a moiety that has
similar transmembrane delivery properties. In another embodiment,
the V.sup.2 moiety is a protein or protein fragment, an antibody or
an antibody fragment, a receptor-binding or peptide vector moiety,
or a polymeric or dendritic moiety, or any combination thereof, to
which is attached a Tat peptide or a derivative, fragment, or
analog thereof, or a moiety that has similar transmembrane delivery
properties.
[0800] Thus, in one aspect of the invention, the moiety V.sup.2 is
a targeting moiety and is selected from the group consisting of a
protein or protein fragment, an antibody or an antibody fragment, a
receptor-binding or peptide vector moiety, and a polymeric or
dendritic moiety, and any combination or derivative thereof.
[0801] In another aspect of the invention, the V.sup.2 moiety is a
moiety that improves the pharmacological properties of a conjugate
of the invention. For example, the moiety V.sup.2 can be chosen
such that the water solubility of the conjugate is (further)
improved. This can be achieved by choosing V.sup.2 to be a
hydrophilic moiety. Alternatively, the V.sup.2 moiety can be used
for example to increase the residence time of the compound in the
circulation, to reduce extravasation and/or excretion, to reduce
aggregation, and/or to reduce the immunogenicity of the compound.
This may for example be achieved by choosing V.sup.2 to be or
contain a polyethylene glycol or oligoethylene glycol or derivative
thereof. When the moiety V.sup.2 is a moiety that improves the
pharmacological properties of a compound of the invention and
V.sup.1 is a moiety that can be cleaved or transformed
aspecifically and there are no V.sup.1' and V.sup.2' moieties, the
compound solely serves to improve the (pharmacological) properties
of the one or more Z moieties.
[0802] In one embodiment, V.sup.2 is a moiety that improves the
pharmacological properties and V.sup.1 is a moiety that can be
cleaved or transformed specifically.
[0803] In another embodiment, V.sup.2 is an oligoethylene glycol or
a polyethylene glycol or a derivative thereof and V.sup.1 is a
moiety that can be cleaved or transformed specifically.
[0804] In another embodiment, V.sup.2 is a moiety that improves the
pharmacological properties and V.sup.1 is a moiety that can be
cleaved or transformed aspecifically.
[0805] In another embodiment, V.sup.2 is an oligoethylene glycol or
a polyethylene glycol or a derivative thereof and V.sup.1 is a
moiety that can be cleaved or transformed aspecifically.
[0806] In another embodiment, V.sup.2 is an oligoethylene glycol or
a polyethylene glycol or a derivative thereof and V.sup.1 is a
moiety that can be cleaved by ubiquitous enzymes.
[0807] In another embodiment, V.sup.2 is an oligoethylene glycol or
a polyethylene glycol or a derivative thereof and V.sup.1 is a
hydrolyzable moiety.
[0808] In another embodiment, V.sup.2 contains a
X.sup.14(CH.sub.2CH.sub.2O).sub.ggCH.sub.2CH.sub.2X.sup.14
moiety.
[0809] In one aspect of this invention, the V.sup.2 moiety is
represented by formula (VI):
##STR00415##
[0810] wherein V.sup.2*, L.sup.2*, L*, V.sup.1*, Y*, p*, q*, and z*
have the same meaning as V.sup.2, L.sup.2, L, Y, p, q, and z,
respectively, as defined in this document and are selected
independently, except that Y* is connected to L.sup.2. It should be
noted that z* actually equals q, assuming that all Y* are indeed
connected to L.sup.2. When a compound of formula (III) contains a
V.sup.2 moiety represented by formula (VI), the one or more L.sup.2
moieties are thus connected to Y.
[0811] Use of a V.sup.2 moiety of formula (VI) in a conjugate of
formula (III) implicates that two conditionally-cleavable or
conditionally-transformable moieties may be present in between the
functional moiety V.sup.2* and Z, and therefore two separate
cleavages/transformations may be required to release Z. The
requirement that two different conditions need to have been met--in
consecutive order--before one or more Z are released might
favorably affect the properties of the conjugate. For instance, it
may increase the targeting efficiency and therapeutic index of the
conjugate. The two transformations/cleavages may occur at different
extracellular/intracellular locations. The moiety to be removed by
the second cleavage or as a consequence of the second
transformation may for example be used to help transport Z from a
first extracellular or intracellular location (where the first
cleavage has occurred) to a second extracellular or intracellular
location, or to stabilize Z until it is closer to its target, or to
(temporarily) increase the water solubility of Z. In order to
increase the targeting efficiency and/or therapeutic index using
this concept, the second transformation and/or cleavage should only
occur after the first transformation and/or cleavage have occurred.
If the second transformation and/or cleavage can also occur before
the first transformation and/or cleavage have occurred, an improved
targeting efficiency and/or an improved therapeutic index due to
this concept seems unlikely.
[0812] It will be apparent that a V.sup.2 moiety of formula (VI) or
a promoiety containing such a V.sup.2 cannot only be useful in
conjugates of a compound of formula (I) or (II), but may be used in
similar conjugates of other therapeutic agents, diagnostic
moieties, and the like.
[0813] A compound of formula (III) containing a V.sup.2 moiety of
formula (VI) may be prepared from a compound of formula (III)
containing a V.sup.2 moiety of formula (VII):
##STR00416##
[0814] wherein RM* has the same meaning as RM and is selected
independently.
[0815] It should be understood that in this document, whenever
V.sup.2, L.sup.2, L, Y, RM, p, q, or z is mentioned, the same can
apply for each V.sup.2*, L.sup.2*, L*, V.sup.1*, Y*, RM*, p*, q*,
or z*, respectively, unless the context dictates otherwise.
[0816] It should be understood that the functional moiety V.sup.2
can have several functional properties combined. For example,
V.sup.2 can be a moiety that improves the pharmacological
properties of a compound of this invention and at the same time be
or contain a targeting moiety.
[0817] Conjugates of this invention may contain one or more
promoieties. These promoieties may be the same or different. The
presence of two or more promoieties may favorably affect the
properties of the conjugate. For instance, it may improve the water
solubility and/or increase the targeting efficiency of the
conjugate. Furthermore, if in a targeted conjugate there are two
promoieties and the promoiety required for targeting is prematurely
cleaved from Z, for example in the circulation, the second
promoiety attenuates the cytotoxicity of Z.
[0818] In one embodiment, when there are two or more promoieties,
said promoieties are different from each other. The two or more
different promoieties may have different functions and may be
removed under different conditions and at different
extracellular/intracellular locations.
[0819] In one embodiment, there is one promoiety linked to Z. In
another embodiment, there is one promoiety linked to Z via X.sup.1.
In another embodiment, there are two promoieties linked to Z. In
another embodiment, there are two promoieties linked to Z, of which
one is connected via X.sup.1. In another embodiment, there are two
promoieties linked to Z, of which one is connected via X.sup.1 and
the other to the DNA-alkylating unit. In another embodiment, there
are two promoieties linked to Z, of which one is connected via
X.sup.1 and the other to the DNA-binding unit. In another
embodiment, there are two promoieties linked to Z, of which one is
connected to the DNA-binding unit and the other to the
DNA-alkylating unit. In yet another embodiment, there are three
promoieties linked to Z. In yet another embodiment, there are three
promoieties linked to Z, of which one is connected via X.sup.l.
[0820] In another aspect, this invention relates to conjugates and
linker-agent conjugates similar to compounds of formulae (III) and
(IV) in which the Z moiety is a therapeutic or diagnostic moiety
different from a compound of formula (I), (II), (I'), or (II'), or
a promoiety-containing derivative thereof. Therapeutic moieties may
for example be selected from anthracyclines (e.g., daunorubicin,
doxorubicin), antimetabolites (e.g., methotrexate, cytarabine,
6-mercaptopurine), calicheamycins, dolastatins, auristatins,
tubulysins, epothilones, taxoids (e.g., paclitaxel, docetaxel),
maytansinoids, mitomycins, other alkylating agents (e.g.,
melphalan, carmustine, chlorambucil, cyclophosphamide), and other
tubulin-binding agents (e.g., vincristine, vinblastine). All
embodiments for compounds of formulae (III) and (IV) also apply to
conjugates and linker-agent conjugates similar to compounds of
formulae (III) and (IV) in which the Z moiety is a therapeutic or
diagnostic moiety different from a compound of formula (I), (II),
(I'), or (II'), or a promoiety-containing derivative thereof,
unless the context dictates otherwise.
[0821] In one aspect of this invention, a compound of formula (III)
comprises at least 2 promoieties. The first promoiety contains at
least a targeting moiety and the second comprises at least a
X.sup.14(CH.sub.2CH.sub.2O).sub.ggCH.sub.2CH.sub.2X.sup.14 moiety
or 2 X.sup.14CH.sub.2CH.sub.2OCH.sub.2CH.sub.2X.sup.14 moieties,
and V.sup.1* of said same second promoiety is present. Similarly, a
compound of formula (IV) may comprise at least 2 promoieties. The
first promoiety contains at least a reactive moiety RM and the
second comprises at least a
X.sup.14(CH.sub.2CH.sub.2O).sub.ggCH.sub.2CH.sub.2X.sup.14 moiety
or 2 X.sup.14CH.sub.2CH.sub.2OCH.sub.2CH.sub.2X.sup.14 moieties,
and V.sup.1* of said same second promoiety is present. Said second
promoieties of compounds of formulae (III) and (IV) may for example
be represented by
##STR00417##
[0822] In one embodiment, said second promoiety is selected
from
##STR00418## ##STR00419##
[0823] wherein X.sup.70, X.sup.71, X.sup.72, and X.sup.73 are
independently selected from O, S, and NR.sup.82, X.sup.74 is
selected from
##STR00420##
[0824] d is selected from 0 to 8, e is 0 or 1, gg', gg'', gg''',
gg'''', and gg* are independently selected from 0 to 1000, and
R.sup.81 and R.sup.82 are independently selected from H and
optionally substituted C.sub.1-3 alkyl. In other embodiments, gg',
gg'', gg''', gg'''', and gg* are independently selected from 3 to
1000 or 500 or 100 or 50 or 10.
[0825] In another embodiment, said second promoiety is selected
from
##STR00421## ##STR00422## ##STR00423##
[0826] wherein gg' is selected from 0 to 1000. In other
embodiments, gg' is selected from 3 to 1000 or 500 or 100 or 50 or
10.
[0827] In a further embodiment, said second promoiety is selected
from
##STR00424##
[0828] wherein gg' is selected from 0 to 1000, AS is
##STR00425##
[0829] wherein A is
##STR00426##
[0830] R.sup.116 is selected from methyl, ethyl, isopropyl,
tert-butyl, phenyl,
##STR00427## ##STR00428## ##STR00429## ##STR00430##
[0831] f is 0, 1, or 2, g is 0 or 1, and PM is an amino acid or a
peptide coupled with its N-terminus to L'.
[0832] In other embodiments, gg' is selected from 3 to 1000 or 500
or 100 or 50 or 10 or 5.
[0833] In one embodiment, (III) is represented by a compound of
formula (III-1) or (III-2):
##STR00431##
[0834] In more specific embodiments, the DB unit in a compound of
formula (III-1) or (III-2) is DB1 or DB2 or DB3 or DB4 or DB5 or
DB6 or DB7 or DB8 or DB9.
[0835] In another embodiment, a compound of formula (III) is
represented by a compound of formula (III-3a) or (III-4a), wherein
the DNA-binding moiety is DB1:
##STR00432##
[0836] wherein Y' is connected to an atom being part of X.sup.3,
X.sup.34, X.sup.4, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.11, or
X.sup.12.
[0837] In another embodiment, a compound of formula (III) is
represented by a compound of formula (III-3b) or (III-4b), wherein
the DNA-binding moiety is DB2:
##STR00433##
[0838] wherein Y' is connected to an atom being part of X.sup.3,
X.sup.34, X.sup.4, X.sup.6, X.sup.7, X.sup.9, X.sup.11, or
X.sup.12.
[0839] In another embodiment, a compound of formula (III) is
represented by a compound of formula (III-3c) or (III-4c), wherein
the DNA-binding moiety is DB3:
##STR00434##
[0840] wherein Y' is connected to an atom being part of X.sup.6,
X.sup.7, X.sup.8, X.sup.9, X.sup.10, or X.sup.11.
[0841] In another embodiment, a compound of formula (III) is
represented by a compound of formula (III-3d) or (III-4d), wherein
the DNA-binding moiety is DB4:
##STR00435##
[0842] wherein Y' is connected to an atom being part of X.sup.6,
X.sup.7, X.sup.8, X.sup.9, or X.sup.11.
[0843] In another embodiment, a compound of formula (III) is
represented by a compound of formula (III-3e) or (III-4e), wherein
the DNA-binding moiety is DB5:
##STR00436##
[0844] wherein Y' is connected to an atom being part of R.sup.8b,
R.sup.9b, X.sup.3, X.sup.34, X.sup.4, X.sup.7, or X.sup.11.
[0845] In another embodiment, a compound of formula (III) is a
compound of formula (III-3f) or (III-4f), wherein the DNA-binding
moiety is DB6:
##STR00437##
[0846] In another embodiment, a compound of formula (III) is a
compound of formula (III-3g) or (III-4g), wherein the DNA-binding
moiety is DB7:
##STR00438##
[0847] wherein Y' is connected to an atom being part of X.sup.3,
X.sup.34, X.sup.4, X.sup.6*, X.sup.7, X.sup.7*, X.sup.8, X.sup.8*,
X.sup.9*, or X.sup.11*.
[0848] In another embodiment, a compound of formula (III) is a
compound of formula (III-3h) or (III-4h), wherein the DNA-binding
moiety is DB8:
##STR00439##
[0849] wherein Y' is connected to an atom being part of X.sup.3,
X.sup.34, X.sup.4, X.sup.7, or X.sup.8.
[0850] In another embodiment, a compound of formula (III) is
represented by a compound of formula (III-3i) or (III-4i), wherein
the DNA-binding moiety is DB9:
##STR00440##
[0851] wherein Y' is connected to an atom being part of X.sup.6,
X.sup.7, X.sup.8, X.sup.9, or X.sup.11.
[0852] This invention further relates to compounds of formulae
(III-3j)-(III-3r) and (III-4j)-(III-4r), which are identical to
compounds of formulae (III-3a)-(III-3i) and (III-4a)-(III-4i),
respectively, except that the two promoieties have switched places,
Y now being connected to an atom in the DNA-binding unit and Y'
being connected to X.sup.1.
[0853] It is noted that if in any of compounds of formulae
(III-3a)-(III-3i) and (III-4a)-(III-4i) Y' is connected to a ring
atom being part of ring A or ring B instead of to an atom in an R
substituent connected to said ring atom, this in fact means that
such an R substituent is absent if this is necessary to meet
valency rules. The same holds for Y in compounds of formulae
(III-3j)-(III-3r) and (III-4j)-(III-4r).
[0854] In another embodiment, a compound of formula (III) is
represented by a compound of formula (III-5) or (III-6):
##STR00441##
[0855] wherein Y' is connected to an atom being part of R.sup.5,
R.sup.5', R.sup.6, R.sup.6', R.sup.7, R.sup.7', R.sup.14,
R.sup.14', X.sup.2 or to any of the atoms bearing these R
substituents.
[0856] In more specific embodiments, the DB unit in a compound of
formula (III-5) or (III-6) is DB1 or DB2 or DB3 or DB4 or DB5 or
DB6 or DB7 or DB8 or DB9.
[0857] In a further embodiment, a compound of formula (III) is
represented by compounds of formulae (III-7) and (III-8), which are
identical to compounds (III-5) and (III-6), respectively, except
that the two promoieties have switched places, Y now being
connected to an atom in the DNA-alkylating unit and Y' being
connected to X.sup.1.
[0858] In more specific embodiments, the DB unit in a compound of
formula (III-7) or (III-8) is DB1 or DB2 or DB3 or DB4 or DB5 or
DB6 or DB7 or DB8 or DB9.
[0859] When Y' in compounds of formulae (III-5) and (III-6) is
connected to a ring atom instead of to an atom in an R substituent
connected to said ring atom, this in fact means that such an R
substituent is absent if this is necessary to meet valency rules.
The same holds for Y in compounds of formulae (III-7) and
(III-8).
[0860] Similar embodiments can be envisioned for compounds of
formula (IV) by replacing V.sup.2-L.sup.2 and/or V.sup.2'-L.sup.2'
by RM and/or RM', respectively, and removing the parentheses with
subscript q and/or q'. Therefore, compounds of formulae (IV-1),
(IV-2), (IV-3a)-(IV-3r), (IV-4a)-(IV-4r), (IV-5), (IV-6), (IV-7),
and (IV-8) are represented by the structures of compounds of
formulae (III-1), (III-2), (III-3a)-(III-3r), (III-4a)-(III-4r),
(III-5), (III-6), (III-7), and (III-8), respectively, in which at
least one of V.sup.2-L.sup.2 and V.sup.2'-L.sup.2' is replaced by
RM and RM', respectively.
[0861] In one embodiment, the
V.sup.2'(-L.sup.2'-L'(-(V.sup.1'--Y')).sub.p').sub.q'(Z').sub.z'-1
moiety in any of compounds of formulae (III-3a)-(III-3r),
(III-4a)-(III-4r), (III-5), (III-6), (III-7), and (III-8) or an
analogous compound thereof based on a compound of formula (IV) is
represented by
##STR00442##
[0862] In another embodiment, the
V.sup.2'(-L.sup.2'-L'(-(V.sup.1'-Y')).sub.p').sub.q'(Z').sub.z'-1
moiety in any of compounds of formulae (III-3a)-(III-3r),
(III-4a)-(III-4r), (III-5), (III-6), (III-7), and (III-8) or an
analogous compound thereof based on a compound of formula (IV) is
represented by
##STR00443##
[0863] In one embodiment, p is an integer from 1 (included) to 128
(included). In another embodiment, q is an integer from 1
(included) to 1000 (included). In other embodiments, p is an
integer from 1 (included) to 64 (included) or 32 (included) or 16
(included) or 8 (included) or 4 (included) or 2 (included), or p is
1. In other embodiments, q is an integer from 1 (included) to 500
(included) or 400 (included) or 300 (included) or 200 (included) or
100 (included) or 16 (included) or 8 (included) or 6 (included) or
4 (included) or 2 (included), or q is 1. In another embodiment, q
is selected from 1 to 4.
[0864] In one embodiment, if more than 1 promoiety is connected to
a first Z and in one of the promoieties there is more than one
attachment site for Z moieties, then the other ones of said
promoieties connected to said first Z each contain a single
attachment site for a Z moiety.
[0865] In one embodiment, a compound of formula (III) is
represented by
##STR00444##
[0866] In one embodiment, p in a compound of formula (IIIa) is
1.
[0867] In another embodiment, in a compound of formula (IIIa) p is
1 and z equals q, which reduces formula (IIIa) to:
##STR00445##
[0868] In another embodiment, a compound of formula (IIIa) is
represented by
##STR00446##
[0869] or by an isomer, or by a mixture of isomers, wherein
R.sup.5, R.sup.6, R.sup.7, R.sup.14, and DB are as previously
defined, V.sup.1 is selected from valylcitrulline, valylly sine,
phenylalanylly sine, alanylphenylalanyllysine, and
D-alanylphenylalanyllysine, f is 1 or 2, CL is selected from
##STR00447## ##STR00448##
[0870] L is selected from
##STR00449##
[0871] q ranges from 1 to 20, rr, a', rr'', and rr''' each
independently range from 0 to 8, X.sup.74 is selected from
##STR00450##
[0872] each X.sup.40 and X.sup.41 is independently selected from O,
S, and NR.sup.135, wherein R.sup.135 is selected from H and
C.sub.1-3 alkyl, each uu, uu', and uu'' is independently selected
from 0 and 1, and Ab is an antibody or a fragment or derivative
thereof.
[0873] In yet another embodiment, a compound of formula (IIIa) is
represented by
##STR00451##
[0874] or by an isomer, or by a mixture of isomers, wherein
R.sup.5, R.sup.6, R.sup.7, R.sup.14, and DB are as previously
defined, V.sup.1 is selected from valylcitrulline, valyllysine,
phenylalanyllysine, alanylphenylalanyllysine, and
D-alanylphenylalanyllysine, f is 1 or 2, CL is
##STR00452##
[0875] R.sup.116 is selected from methyl, ethyl, isopropyl,
tert-butyl, phenyl,
##STR00453## ##STR00454## ##STR00455##
[0876] L is selected from
##STR00456##
[0877] q ranges from 1 to 20, rr, rr', rr'', and a''' each
independently range from 0 to 8, X.sup.74 is selected from
##STR00457##
[0878] each X.sup.40 and X.sup.41 is independently selected from O,
S, and NR.sup.135, wherein R.sup.135 is selected from H and
C.sub.1-3 alkyl, each uu, uu', and uu'' is independently selected
from 0 and 1, and Ab is an antibody or a fragment or derivative
thereof.
[0879] In yet another embodiment, a compound of formula (IIIa) is
represented by
##STR00458##
[0880] or by an isomer, or by a mixture of isomers, wherein
R.sup.5, R.sup.6, R.sup.7, R.sup.14, and DB are as previously
defined, V.sup.1 is selected from valylcitrulline, valyllysine,
phenylalanyllysine, alanylphenylalanyllysine, and
D-alanylphenylalanyllysine, f is 1 or 2, CL is selected from
##STR00459## ##STR00460##
[0881] L is selected from
##STR00461##
[0882] q ranges from 1 to 20, and Ab is an antibody or a fragment
or derivative thereof.
[0883] In yet another embodiment, a compound of formula (IIIa) is
represented by
##STR00462##
[0884] or by an isomer, or by a mixture of isomers, wherein R.sup.5
is selected from H, methyl and methoxy, R.sup.6, R.sup.7, and
R.sup.H are H, DB is DB1, V.sup.1 is selected from valylcitrulline,
valyllysine, phenylalanyllysine, alanylphenylalanyllysine, and
D-alanylphenylalanyllysine, f is 1 or 2, CL is selected from
##STR00463##
[0885] L is selected from
##STR00464##
[0886] q ranges from 1 to 4, and Ab is an antibody or a fragment or
derivative thereof.
[0887] In another embodiment, a compound of formula (IIIa) is
represented by
##STR00465##
[0888] or by an isomer, or by a mixture of isomers, wherein
R.sup.5, R.sup.6, R.sup.7, R.sup.14, and DB are as previously
defined, CL and CL' are independently selected from
##STR00466## ##STR00467##
[0889] V.sup.1 and V.sup.1' are independently selected from
valylcitrulline, valyllysine, phenylalanyllysine,
alanylphenylalanyllysine, and D-alanylphenylalanyllysine, f is 1 or
2, f' is 0, 1, or 2, g' is 0 or 1, the CL' group--or the
p-aminobenzyloxycarbonyl group if g' is 0, or the V.sup.1' group if
f is 0 as well--is connected to an atom in DB, L is selected
from
##STR00468##
[0890] q ranges from 1 to 20, rr, rr', rr'', and rr''' each
independently range from 0 to 8, X.sup.74 is selected from
##STR00469##
[0891] each X.sup.40 and X.sup.41 is independently selected from O,
S, and NR.sup.135, wherein R.sup.135 is selected from H and
C.sub.1-3 alkyl, each uu, uu', and uu'' is independently selected
from 0 and 1, Ab is an antibody or a fragment or derivative
thereof, and L' is selected from
##STR00470## ##STR00471##
[0892] wherein gg' is selected from 0 to 1000.
[0893] In another embodiment, a compound of formula (IIIa) is
represented by
##STR00472##
[0894] or by an isomer, or by a mixture of isomers, wherein
R.sup.5, R.sup.6, R.sup.7, R.sup.14, and DB are as previously
defined, CL and CL' are independently selected from
##STR00473## ##STR00474##
[0895] V.sup.1 and V.sup.1' are independently selected from
valylcitrulline, valyllysine, phenylalanyllysine,
alanylphenylalanyllysine, and D-alanylphenylalanyllysine, f is 0,
1, or 2, f' is 1, or 2, g is 0 or 1, the CL group--or the
p-aminobenzyloxycarbonyl group if g is 0, or the V.sup.1 group if f
is 0 as well--is connected to an atom in DB, L is selected from
##STR00475##
[0896] q ranges from 1 to 20, rr, rr', rr'', and rr''' each
independently range from 0 to 8, X.sup.74 is selected from
##STR00476##
[0897] each X.sup.40 and X.sup.41 is independently selected from O,
S, and NR.sup.135, wherein R.sup.135 is selected from H and
C.sub.1-3 alkyl, each uu, uu', and uu'' is independently selected
from 0 and 1, Ab is an antibody or a fragment or derivative
thereof, and L' is selected from
##STR00477## ##STR00478##
[0898] wherein gg' is selected from 0 to 1000.
[0899] In another embodiment, a compound of formula (III) is
represented by
##STR00479##
[0900] In one embodiment, p* in a compound of formula (IIIa*) is
1.
[0901] In another embodiment, in a compound of formula (IIIa*) p*
is 1 and z* equals q*.
[0902] In another embodiment, in a compound of formula (IIIa*) p*
is 1 and z* as well as z equal q*, which reduces formula (IIIa*)
to:
##STR00480##
[0903] In another embodiment, a compound of formula (III) is
represented by
##STR00481##
[0904] In one embodiment, p in a compound of formula (IIIb) is
1.
[0905] In another embodiment, p in a compound of formula (IIIb) is
1 and z equals q, which reduces formula (IIIb) to:
##STR00482##
[0906] In another embodiment, a compound of formula (III) is
represented by
##STR00483##
[0907] In one embodiment, p* in a compound of formula (IIIc) is
1.
[0908] In another embodiment, in a compound of formula (IIIc) p* is
1 and z* equals q*.
[0909] In yet another embodiment, in a compound of formula (IIIc)
p* is 1 and z* as well as z equal q*, which reduces formula (IIIc)
to:
##STR00484##
[0910] In another embodiment, V.sup.l in a compound of formula
(IIIc) is an enzyme-cleavable substrate. In a further embodiment,
V.sup.1 can be cleaved by an intracellular enzyme. In another
embodiment, V.sup.1 is an optionally substituted
N,N-dialkylaminocarbonyl group wherein the two alkyl groups may be
the same or different and optionally be connected to each other to
form an optionally substituted heterocycle. In yet another
embodiment, V.sup.1 is piperazinocarbonyl. Such a V.sup.1 group may
be cleaved enzymatically, for example by carboxylesterases.
[0911] In another embodiment, a compound of formula (IIIc) is
represented by
##STR00485##
[0912] or by an isomer, or by a mixture of isomers, wherein
R.sup.5, R.sup.6, R.sup.7, R.sup.14, and DB are as previously
defined, V.sup.1* is selected from valylcitrulline, valyllysine,
phenylalanyllysine, alanylphenylalanyllysine, and
D-alanylphenylalanyllysine, is 1 or 2, L* is selected from
##STR00486##
[0913] q* ranges from 1 to 20, rr, rr', rr'', and rr''' each
independently range from 0 to 8, X.sup.74 is selected from
##STR00487##
[0914] each X.sup.40 and X.sup.41 is independently selected from O,
S, and NR.sup.135, wherein R.sup.135 is selected from H and
C.sub.1-3 alkyl, each uu, uu', and uu'' is independently selected
from 0 and 1, and Ab is an antibody or a fragment or derivative
thereof.
[0915] In yet another embodiment, a compound of formula (IIIc) is
represented by
##STR00488##
[0916] or by an isomer, or by a mixture of isomers, wherein
R.sup.5, R.sup.6, R.sup.7, R.sup.14, and DB are as previously
defined, V.sup.1* is selected from valylcitrulline, valyllysine,
phenylalanyllysine, alanylphenylalanyllysine, and
D-alanylphenylalanyllysine, is 1 or 2, L* is selected from
##STR00489##
[0917] q* ranges from 1 to 20, and Ab is an antibody or a fragment
or derivative thereof.
[0918] In yet another embodiment, a compound of formula (III) is
represented by
[0919] V.sup.1-Z (IIId)
[0920] In one embodiment, a compound of formula (IIId) is
represented by
##STR00490##
[0921] or by an isomer, or by a mixture of isomers, wherein
R.sup.5, R.sup.6, R.sup.7, R.sup.14, and DB are as previously
defined, CL' is selected from
##STR00491## ##STR00492##
[0922] f' is 0, 1, or 2, g' is 0 or 1, V.sup.1' is selected from
valylcitrulline, valyllysine, phenylalanyllysine,
alanylphenylalanyllysine, and D-alanylphenylalanyllysine or is
absent, the CL' group--or the p-aminobenzyloxycarbonyl group if g'
is 0, or the group if f' is 0 as well, or the L' group if the
V.sup.1' group is absent as well--is connected to an atom in DB, L'
is selected from
##STR00493##
[0923] q' ranges from 1 to 20, rr, rr', rr'', and rr''' each
independently range from 0 to 8, X.sup.74 is selected from
##STR00494##
[0924] each X.sup.40 and X.sup.41 is independently selected from O,
S, and NR.sup.135, wherein R.sup.135 is selected from H and
C.sub.1-3 alkyl, each uu, uu', and uu'' is independently selected
from 0 and 1, Ab is an antibody or a fragment or derivative
thereof, and V.sup.1 is selected from a mono-, di-, or
oligosaccharide or a reduced, oxidized, or protected derivative
thereof and
##STR00495##
[0925] wherein R.sup.141, R.sup.142, and R.sup.143 are
independently selected from H and optionally substituted C.sub.1-8
alkyl, C.sub.1-8 heteroalkyl, C.sub.3-8 cycloalkyl, C.sub.1-8
heterocycloalkyl, C.sub.5-8 aryl, or C.sub.1-8 heteroaryl.
[0926] In another embodiment, a compound of formula (IIId) is
represented by
##STR00496##
[0927] or by an isomer, or by a mixture of isomers, wherein
R.sup.5, R.sup.6, R.sup.7, R.sup.14, and DB are as previously
defined, CL' is
##STR00497##
[0928] R.sup.116 is selected from methyl, ethyl, isopropyl,
tert-butyl, phenyl,
##STR00498## ##STR00499## ##STR00500## ##STR00501##
[0929] f' is 0, 1, or 2, g' is 0 or 1, V.sup.1' is selected from
valylcitrulline, valyllysine, phenylalanyllysine,
alanylphenylalanyllysine, and D-alanylphenylalanyllysine or is
absent, the CL' group or the p-aminobenzyloxycarbonyl group if g'
is 0, or the V.sup.1' group if f' is 0 as well, or the L' group if
the V.sup.1' group is absent as well--is connected to an atom in
DB, L' is selected from
##STR00502##
[0930] q' ranges from 1 to 20, rr, rr', rr'', and rr''' each
independently range from 0 to 8, X.sup.74 is selected from
##STR00503##
[0931] each X.sup.40 and X.sup.41 is independently selected from O,
S, and NR.sup.135, wherein R.sup.135 is selected from H and
C.sub.1-3 alkyl, each uu, uu', and uu'' is independently selected
from 0 and 1, Ab is an antibody or a fragment or derivative
thereof, and V.sup.1 is selected from a mono-, di-, or
oligosaccharide or a reduced, oxidized, or protected derivative
thereof and
##STR00504##
[0932] wherein R.sup.141, R.sup.142, and R.sup.143 are
independently selected from H and optionally substituted C.sub.1-8
alkyl, C.sub.1-8 heteroalkyl, C.sub.3-8 cycloalkyl, C.sub.1-8
heterocycloalkyl, C.sub.5-8 aryl, or C.sub.1-8 heteroaryl.
[0933] In another embodiment, a compound of formula (IIId) is
represented by
##STR00505##
[0934] or by an isomer, or by a mixture of isomers, wherein
R.sup.5, R.sup.6, R.sup.7, R.sup.14, and DB are as previously
defined, CL' is selected from
##STR00506## ##STR00507##
[0935] f' is 1 or 2, V.sup.1' is selected from valylcitrulline,
valyllysine, phenylalanyllysine, alanylphenylalanyllysine, and
D-alanylphenylalanyllysine, L' is selected from
##STR00508##
[0936] q' ranges from 1 to 20, rr, rr', rr'', and rr''' each
independently range from 0 to 8, each X.sup.40 and X.sup.41 is
independently selected from O, S, and NR.sup.135, wherein R.sup.135
is selected from H and C.sub.1-3 alkyl, each uu, uu', and uu'' is
independently selected from 0 and 1, Ab is an antibody or a
fragment or derivative thereof, and V.sup.1 is coupled to an atom
of DB and is selected from a mono-, di-, or oligosaccharide or a
reduced. oxidized. or protected derivative thereof and
##STR00509##
[0937] wherein R.sup.141, R.sup.142, and R.sup.143 are
independently selected from H and optionally substituted C.sub.1-8
alkyl, C.sub.1-8 heteroalkyl, C.sub.3-8 cycloalkyl, C.sub.1-8
heterocycloalkyl, C.sub.5-8 aryl, or C.sub.1-8 heteroaryl.
[0938] In another embodiment, a compound of formula (IIId) is
represented by
##STR00510##
[0939] or by an isomer, or by a mixture of isomers, wherein
R.sup.5, R.sup.6, R.sup.7, R.sup.14, and DB are as previously
defined, CL' is
##STR00511##
[0940] R.sup.116 is selected from methyl, ethyl, isopropyl,
tert-butyl, phenyl,
##STR00512## ##STR00513## ##STR00514## ##STR00515##
[0941] f' is 1 or 2, V.sup.1' is selected from valylcitrulline,
valyllysine, phenylalanyllysine, alanylphenylalanyllysine, and
D-alanylphenylalanyllysine, L' is selected from
##STR00516##
[0942] q' ranges from 1 to 20, rr, rr', rr'', and rr''' each
independently range from 0 to 8, each X.sup.40 and X.sup.41 is
independently selected from O, S, and NR.sup.135, wherein R.sup.135
is selected from H and C.sub.1-3 alkyl, each uu, uu', and uu'' is
independently selected from 0 and 1, Ab is an antibody or a
fragment or derivative thereof, and V.sup.1 is coupled to an atom
of DB and is selected from a mono-, di-, or oligosaccharide or a
reduced, oxidized, or protected derivative thereof and
##STR00517##
[0943] wherein R.sup.141, R.sup.142, and R.sup.143 are
independently selected from H and optionally substituted C.sub.1-8
alkyl, C.sub.1-8 heteroalkyl, C.sub.3-8 cycloalkyl, C.sub.1-8
heterocycloalkyl, C.sub.5-8 aryl, or C.sub.1-8 heteroaryl.
[0944] In yet another embodiment, a compound of formula (III) is
represented by V.sup.2-L.sup.2-L-Z (IIIe).
[0945] Similar embodiments can be envisioned for compounds of
formula (IV) by replacing V.sup.2-L.sup.2 and/or V.sup.2'-L.sup.2'
by RM and/or RM', respectively, and removing the parentheses with
subscript q and/or q', or by eliminating Ab-SH from the structures.
Therefore, compounds of formulae (IVa)-(IVe) are represented by the
structures of compounds of formulae (IIIa)-(IIIe), respectively, in
which V.sup.2-L.sup.2 or V.sup.2'-L.sup.2' is replaced by RM or
RM', respectively, and parentheses with subscript q or q' are
removed.
[0946] In one embodiment, the DB moiety of a Z in a compound of
formula (III) is DB1.
[0947] In other embodiments, the DB moiety of a Z in a compound of
formula (III) is DB2 or DB3 or DB4 or DB5 or DB6 or DB7 or DB8 or
DB9.
[0948] In another embodiment, the DB moiety of a Z in a compound of
formula (IV) is DB1.
[0949] In other embodiments, the DB moiety of a Z in a compound of
formula (IV) is DB2 or DB3 or DB4 or DB5 or DB6 or DB7 or DB8 or
DB9.
[0950] In yet another embodiment, the DB moiety of a Z in a
compound of formula (III) is
##STR00518##
[0951] In yet another embodiment, the DB moiety of a Z in a
compound of formula (IV) is
##STR00519##
[0952] In one embodiment, a compound of formula (IV) is represented
by
##STR00520##
[0953] or by an isomer, or by a mixture of isomers, wherein
R.sup.5a is selected from H, methyl and methoxy, AZ is
##STR00521## ##STR00522## ##STR00523## ##STR00524## ##STR00525##
##STR00526## ##STR00527##
[0954] V.sup.1 is selected from valylcitrulline, valyllysine,
phenylalanyllysine, alanylphenylalanyllysine, and
D-alanylphenylalanyllysine, CL is
##STR00528##
[0955] R.sup.116 is selected from methyl, ethyl, isopropyl,
tert-butyl, phenyl,
##STR00529## ##STR00530## ##STR00531## ##STR00532##
[0956] and L is selected from
##STR00533##
[0957] In another embodiment, a compound of formula (IV) is
represented by
##STR00534##
[0958] or by an isomer, or by a mixture of isomers, wherein
R.sup.5a is selected from H, methyl and methoxy, AZ is
##STR00535## ##STR00536## ##STR00537## ##STR00538## ##STR00539##
##STR00540## ##STR00541##
[0959] V.sup.1 is selected from valylcitrulline, valyllysine,
phenylalanyllysine, alanylphenylalanyllysine, and
D-alanylphenylalanyllysine, CL is
##STR00542##
[0960] and L is selected from
##STR00543##
[0961] In another embodiment, a compound of formula (III) is
represented by
##STR00544##
[0962] or by an isomer, or by a mixture of isomers, wherein
R.sup.5a is selected from H, methyl and methoxy, AZ is
##STR00545## ##STR00546## ##STR00547## ##STR00548## ##STR00549##
##STR00550## ##STR00551##
[0963] V.sup.1 is selected from valylcitrulline, valyllysine,
phenylalanyllysine, alanylphenylalanyllysine, and
D-alanylphenylalanyllysine, CL is
##STR00552##
[0964] R.sup.116 is selected from methyl, ethyl, isopropyl,
tert-butyl, phenyl,
##STR00553## ##STR00554## ##STR00555## ##STR00556##
[0965] a ranes from 1 to 8, Ab is an antibody or a fragment or
derivative thereof, and L is selected from
##STR00557##
[0966] In yet another embodiment, a compound of formula (III) is
represented by
##STR00558##
[0967] or by an isomer, or by a mixture of isomers, wherein
R.sup.5a is selected from H, methyl and methoxy, AZ is
##STR00559## ##STR00560## ##STR00561## ##STR00562## ##STR00563##
##STR00564## ##STR00565##
[0968] V.sup.1 is selected from valylcitrulline, valyllysine,
phenylalanyllysine, alanylphenylalanyllysine, and
D-alanylphenylalanyllysine, CL is
##STR00566##
[0969] L is selected from
##STR00567##
[0970] q ranges from 1 to 4, and Ab is an antibody or a fragment or
derivative thereof.
[0971] A series of compounds (III-AZ) wherein R.sup.5a is methyl,
AZ is
##STR00568##
[0972] L is
##STR00569##
[0973] Ab is Trastuzumab, V.sup.1 is valylcitrulline, and CL is
##STR00570##
[0974] were prepared according to the procedure described in
Example 15 and evaluated against the corresponding reference
compound in which CL is
##STR00571##
[0975] for amount of aggregate present after the conjugation step.
The evaluated compounds had relative aggregate amounts of 33%, 15%,
8%, 58%, and 12%, respectively, compared to that of the reference
compound (100%), clearly showing the advantage of the present
cyclization spacers.
[0976] In one embodiment, a compound of formula (IV) is selected
from
##STR00572##
[0977] In another embodiment, a compound of formula (IV) is
selected from
##STR00573##
[0978] In another embodiment, a compound of formula (IV) is
selected from
##STR00574##
[0979] In another embodiment, a compound of formula (IV) is
selected from
##STR00575##
[0980] In another embodiment, a compound of formula (IV) is
selected from
##STR00576##
[0981] In another embodiment, a compound of formula (IV) is
selected from
##STR00577## ##STR00578##
[0982] In another embodiment, a compound of formula (IV) is
selected from
##STR00579## ##STR00580##
[0983] In another embodiment, a compound of formula (IV) is
selected from
##STR00581## ##STR00582##
[0984] In another embodiment, a compound of formula (IV) is
selected from
##STR00583## ##STR00584##
[0985] In another embodiment, a compound of formula (IV) is
selected from
##STR00585## ##STR00586##
[0986] In another embodiment, a compound of formula (IV) is
selected from
##STR00587## ##STR00588##
[0987] In another embodiment, a compound of formula (IV) is
selected from
##STR00589##
[0988] In another embodiment, a compound of formula (IV) is
selected from
##STR00590## ##STR00591##
[0989] In another embodiment, a compound of formula (IV) is
selected from
##STR00592##
[0990] In another embodiment, a compound of formula (IV) is
selected from
##STR00593## ##STR00594##
[0991] In another embodiment, a compound of formula (IV) is
selected from
##STR00595## ##STR00596##
[0992] In another embodiment, a compound of formula (IV) is
selected from
##STR00597##
[0993] In another embodiment, a compound of formula (IV) is
selected from
##STR00598## ##STR00599##
[0994] In another embodiment, a compound of formula (IV) is
selected from
##STR00600## ##STR00601##
[0995] In another embodiment, a compound of formula (IV) is
selected from
##STR00602##
[0996] In another embodiment, a compound of formula (IV) is
selected from
##STR00603## ##STR00604##
[0997] In another embodiment, a compound of formula (IV) is
selected from
##STR00605## ##STR00606##
[0998] In another embodiment, a compound of formula (IV) is
selected from
##STR00607## ##STR00608##
[0999] In another embodiment, a compound of formula (IV) is
selected from
##STR00609## ##STR00610##
[1000] In another embodiment, a compound of formula (IV) is
selected from
##STR00611## ##STR00612##
[1001] In another embodiment, a compound of formula (IV) is
selected from
##STR00613## ##STR00614##
[1002] In another embodiment, a compound of formula (IV) is
selected from
##STR00615## ##STR00616##
[1003] In another embodiment, a compound of formula (IV) is
selected from
##STR00617## ##STR00618##
[1004] In another embodiment, a compound of formula (IV) is
selected from
##STR00619## ##STR00620##
[1005] In another embodiment, a compound of formula (IV) is
selected from
##STR00621## ##STR00622##
[1006] In another embodiment, a compound of formula (IV) is
selected from
##STR00623## ##STR00624##
[1007] Synthesis of Compounds of the Invention
[1008] Compounds of formulae (I)-(IV) and (VIII) can be
conveniently prepared in a way for some part analogous to compounds
reported in WO 01/83448, WO 02/083180, WO 2004/043493, WO
2007/018431, WO 2007/089149, WO 2009/017394, and WO2010/062171.
Further representative synthetic protocols can be found in the
Examples.
[1009] In one embodiment, a compound of formula (I) or (II) is used
to prepare a compound of formula (III). In another embodiment, a
compound of formula (I) or (II) is used to prepare a compound of
formula (IV). In another embodiment, a compound of formula (IV) is
used to prepare a compound of formula (III). In another embodiment,
a compound of formula (III) wherein V.sup.1 is a protecting group
is used to prepare another compound of formula (III) wherein
V.sup.1 is an in vivo cleavable/transformable moiety. In yet
another embodiment, a compound of formula (VIII) is used to prepare
a compound of formula (IV). In yet another embodiment, a compound
of formula (VIII) is used to prepare a compound of formula (III),
optionally via a compound of formula (IV).
[1010] Uses, Methods, and Compositions
[1011] In one aspect, this invention relates to use of a compound
of formula (I) or (II) for the preparation of a compound of formula
(III).
[1012] In another aspect, this invention relates to use of a
compound of formula (IV) for the preparation of a compound of
formula (III).
[1013] In yet another aspect, this invention relates to use of a
compound of formula (I) or (II) for the preparation of a compound
of formula (IV).
[1014] In yet another aspect, this invention relates to use of a
compound of formula (VIII) for the preparation of a compound of
formula (IV).
[1015] In yet another aspect, this invention relates to use of a
compound of formula (VIII) for the preparation of a compound of
formula (III), optionally via a compound of formula (III).
[1016] In yet another aspect, this invention relates to use of a
compound of formula (VIII) for the preparation of conjugates and
linker-agent conjugates similar to compounds of formulae (III) and
(IV) in which the Z moiety is a therapeutic or diagnostic moiety
different from a compound of formula (I), (II), (I'), or (II'), or
a promoiety-containing derivative thereof.
[1017] In yet another aspect, this invention relates to use of a
compound of formula (III) wherein V.sup.1 is a protecting group for
the preparation of another compound of formula (III) wherein
V.sup.1 is an in vivo cleavable/transformable moiety.
[1018] In yet another aspect, the invention relates to the use of
any of the compounds defined hereinabove for the manufacture of a
pharmaceutical composition for the treatment of a mammal being in
need thereof. In one embodiment, the invention relates to the use
of any of the compounds defined hereinabove for the manufacture of
a pharmaceutical composition for the treatment of a tumor in a
mammal. In another embodiment, the invention relates to the use of
any of the compounds defined hereinabove for the manufacture of a
pharmaceutical composition for the prevention of a tumor in a
mammal. The invention can also be worded as any of the compounds
defined hereinabove for use in the treatment of a mammal being in
need thereof, or for use in the treatment of a tumor in a mammal or
for use in the prevention of a tumor in a mammal.
[1019] The invention also relates to any of the compounds defined
hereinabove as a medicament or an active component or active
substance in a medicament.
[1020] In a further aspect, the invention relates to a process for
preparing a pharmaceutical composition containing a compound as
defined hereinabove, to provide a solid or a liquid formulation for
administration orally, topically, or by injection. Such a method or
process at least comprises the step of mixing the compound with a
pharmaceutically acceptable carrier.
[1021] In one embodiment, a compound of the invention is used to
treat or prevent an illness characterized by undesired
proliferation. In another embodiment, a compound of the invention
is used to treat or prevent an illness characterized by undesired
cell proliferation. In another embodiment, a compound of the
invention is used to treat a tumor. In another embodiment, a
compound of the invention is used to prevent a tumor. In yet
another embodiment, a compound of the invention is used to treat or
prevent an inflammatory disease. In yet another embodiment, a
compound of the invention is used to treat or prevent an autoimmune
disease. In yet another embodiment, a compound of the invention is
used to treat or prevent a bacterial, viral, or microbial
infection.
[1022] In a further embodiment, this invention relates to a method
of treating a mammal having an illness characterized by undesired
(cell) proliferation with a compound of this invention. In another
embodiment, this invention relates to a method of treating a mammal
carrying a tumor with a compound of this invention. In yet another
embodiment, this invention relates to a method of treating a mammal
having an inflammatory disease with a compound of this invention.
In yet another embodiment, this invention relates to a method of
treating a mammal having an autoimmune disease with a compound of
this invention. In yet another embodiment, this invention relates
to a method of treating a mammal having a bacterial, viral, or
microbial infection with a compound of this invention.
[1023] In a further embodiment, the invention relates to a method
of treating a mammal being in need thereof, whereby the method
comprises the administration of a pharmaceutical composition
comprising a compound of this invention to the mammal in a
therapeutically effective dose.
[1024] In one embodiment, the invention relates to a method of
treating or preventing a tumor in a mammal, whereby the method
comprises the administration of a pharmaceutical composition
comprising a compound of this invention to the mammal in a
therapeutically effective dose. In another embodiment, the
invention relates to a method of treating a tumor in a mammal,
whereby the method comprises the administration of a pharmaceutical
composition comprising a compound of this invention to the mammal
in a therapeutically effective dose. In yet another embodiment, the
invention relates to a method of treating a tumor in a human,
whereby the method comprises the administration of a pharmaceutical
composition comprising a compound of this invention to the human in
a therapeutically effective dose.
[1025] In another embodiment, the invention relates to a method of
treating or preventing an inflammatory disease in a mammal, whereby
the method comprises the administration of a pharmaceutical
composition comprising a compound of this invention to the mammal
in a therapeutically effective dose.
[1026] In another embodiment, the invention relates to a method of
treating or preventing an autoimmune disease in a mammal, whereby
the method comprises the administration of a pharmaceutical
composition comprising a compound of this invention to the mammal
in a therapeutically effective dose.
[1027] In another embodiment, the invention relates to a method of
treating or preventing a bacterial, viral, or microbial infection
in a mammal, whereby the method comprises the administration of a
pharmaceutical composition comprising a compound of this invention
to the mammal in a therapeutically effective dose.
[1028] The invention also relates to pharmaceutical compositions
comprising the compounds of the invention as defined hereinabove. A
compound of the invention may be administered in purified form
together with a pharmaceutical carrier as a pharmaceutical
composition. The preferred form depends on the intended mode of
administration and therapeutic application. The pharmaceutical
carrier can be any compatible, nontoxic substance suitable to
deliver the compounds of the invention to the patient.
Pharmaceutically acceptable carriers are well known in the art and
include, for example, aqueous solutions such as (sterile) water or
physiologically buffered saline or other solvents or vehicles such
as glycols, glycerol, oils such as olive oil or injectable organic
esters, alcohol, fats, waxes, and inert solids. A pharmaceutically
acceptable carrier may further contain physiologically acceptable
compounds that act for example to stabilize or to increase the
absorption of the compounds of the invention. Such physiologically
acceptable compounds include, for example, carbohydrates, such as
glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid
or glutathione, chelating agents, low molecular weight proteins, or
other stabilizers or excipients. One skilled in the art would know
that the choice of a pharmaceutically acceptable carrier, including
a physiologically acceptable compound, depends, for example, on the
route of administration of the composition. Pharmaceutically
acceptable adjuvants, buffering agents, dispersing agents, and the
like, may also be incorporated into the pharmaceutical
compositions.
[1029] For oral administration, the active ingredient can be
administered in solid dosage forms, such as capsules, tablets, and
powders, or in liquid dosage forms, such as elixirs, syrups, and
suspensions.
[1030] Active component(s) can be encapsulated in gelatin capsules
together with inactive ingredients and powdered carriers, such as
glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose
derivatives, magnesium stearate, stearic acid, sodium saccharin,
talcum, magnesium carbonate, and the like. Examples of additional
inactive ingredients that may be added to provide desirable color,
taste, stability, buffering capacity, dispersion, or other known
desirable features are red iron oxide, silica gel, sodium lauryl
sulfate, titanium dioxide, edible white ink, and the like. Similar
diluents can be used to make compressed tablets. Both tablets and
capsules can be manufactured as sustained release products to
provide for continuous release of medication over a period of
hours. Compressed tablets can be sugar-coated or film-coated to
mask any unpleasant taste and protect the tablet from the
atmosphere, or enteric-coated for selective disintegration in the
gastrointestinal tract. Liquid dosage forms for oral administration
can contain coloring and flavoring to increase patient
acceptance.
[1031] The compounds of the invention are however preferably
administered parenterally. Preparations of the compounds of the
invention for parenteral administration must be sterile.
Sterilization is readily accomplished by filtration through sterile
filtration membranes, optionally prior to or following
lyophilization and reconstitution. The parenteral route for
administration of compounds of the invention is in accord with
known methods, e.g. injection or infusion by intravenous,
intraperitoneal, intramuscular, intraarterial, or intralesional
routes. The compounds of the invention may be administered
continuously by infusion or by bolus injection. A typical
composition for intravenous infusion could be made up to contain
100 to 500 ml of sterile 0.9% NaCl or 5% glucose optionally
supplemented with a 20% albumin solution and 1 mg to 10 g of the
compound of the invention, depending on the particular type of
compound of the invention and its required dosing regime. Methods
for preparing parenterally administrable compositions are well
known in the art and described in more detail in various sources,
including, for example, Remington's Pharmaceutical
Science.sup.17.
[1032] A compound of the invention may also be used in combination
therapy, in which a compound of this invention is used in
combination with one or more other therapeutic agents. Combination
of two or more therapeutics may favorably affect treatment outcome.
The agents may be administered either sequentially or
concomitantly. Therefore, in one embodiment this invention relates
to use of a compound of this invention or a pharmaceutical
composition comprising a compound of this invention in combination
therapy.
[1033] The invention is further exemplified by the following
examples. These examples are for illustrative purposes only and are
not intended to limit the scope of the invention.
EXAMPLES
Example 1
Synthesis of Boc-Protected Cyclization Spacers
##STR00625##
[1035] Route A
[1036] Synthesis of compound 1: 7.5 ml (85 mmol) oxalyl chloride
was dissolved in 200 ml DCM and cooled to T<-60.degree. C. and
12.1 ml (171 mmol) DMSO in 10 ml DCM was added dropwise
(T<-60.degree. C.) and stirred an additional 10 min. 10.0 g (57
mmol) N-Boc-N-methylaminoethanol in 40 ml DCM were added dropwise
(T<-60.degree. C.) and stirred an additional 10 min. 40 ml (285
mmol) Et.sub.3N was added dropwise followed by 50 ml DCM
(T<-60.degree. C.) and stirred for 30 min. The reaction mixture
was warmed to 0.degree. C. and washed with 3.times.100 ml water,
100 ml 0.5 M KHSO.sub.4, 75 ml brine, dried with MgSO.sub.4 and
concentrated in vacuo. The product was purified by column
chromatography (SiO.sub.2, DCM/ethyl acetate, 1:0 to 9:1) to give
7.36 g (74%) of compound 1. .sup.1H-NMR (300 MHz, CDCl.sub.3):
.delta.=1.42/1.46 (s, 9H, Boc), 2.93/2.96 (s, 3H, Me), 3.90/4.01
(s, 2H, CH.sub.2), 9.60 (s, 1H, CHO). Z/E isomers.
[1037] General procedure reductive amination: 1 mmol R-amine and 1
mmol compound 1 were stirred in 10 ml MeOH for 4 hrs. The reaction
mixture was cooled in ice and 2 mmol sodium borohydride was added
in portions and the mixture stirred at RT overnight. The mixture
was concentrated and purified by column chromatography (SiO.sub.2,
DCM/MeOH, 1:0 to 1:1) to give
N-Boc-N-Methyl-N'-R-diaminoethane.
[1038] Compound 4a, R.dbd.(CH.sub.2CH.sub.2O).sub.2H: 40% yield,
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=1.46 (9H, s, Boc),
2.75-2.87 (4H, m, 2.times.CH.sub.2), 2.88 (3H, s, NMe), 3.35 (2H,
t, J=6.6 Hz, CH.sub.2), 3.56-3.63 (4H, m, 2.times.CH.sub.2, CH),
3.70-3.75 (2H, m, CH.sub.2, CH); MS (ESI) m/z=263.5 (M+H+).
[1039] Compound 4b, R.dbd.(CH.sub.2).sub.3COOMe: 61% yield, .sup.1H
NMR (300 MHz, CDCl.sub.3): .delta.=1.46 (9H, s, Boc), 1.83 (2H, m,
CH.sub.2), 2.39 (2H, t, J=7.4 Hz, CH.sub.2) 2.64-2.86 (3H, m,
CH/CH.sub.2), 2.88 (3H, s, NMe), 2.99 (1H, m, CH), 3.30-3.49 (2H,
m, CH.sub.2), 3.67 (3H, s, CH.sub.3); MS (ESI) m/z=275.5
(M+H.sup.+).
##STR00626##
[1040] Compound 4c: .sup.1H-NMR (300 MHz, DMSO-d.sub.6):
.delta.=1.15 (3H, d, J=6.9 Hz, CH.sub.3 Ala), 1.38 (9H, s, Boc),
2.57-2.67 (1H, m, .alpha.-H), 2.76 (3H, s, NCH.sub.3), 3.08-3.36
(4H, m, CH.sub.2-CH.sub.2), 3.63 (3H, s, OCH.sub.3); MS (ESI):
m/z=261.3 (M+H.sup.+).
##STR00627##
[1041] Compound 4d: .sup.1H-NMR (300 MHz, DMSO-d.sub.6):
.delta.=1.12 (3H, d, J=6.9 Hz, CH.sub.3 Ala), 1.39 (9H, s, Boc),
2.54-2.61 (1H, m, .alpha.-H), 2.77 (3H, s, NCH.sub.3), 3.10-3.51
(13H, m, 6.times.CH.sub.2+OH), 7.90 (1H, s, NH amide); MS (ESI):
m/z=334.4 (M+H.sup.+).
##STR00628##
[1042] Compound 4e: 27% yield, .sup.1H-NMR (300 MHz, CDCl.sub.3):
.delta.=1.44 (9H, s, Boc), 2.72-2.85 (4H, m, 2.times.CH.sub.2),
2.81 (3H, s, MeN), 3.31 (2H, m, NCH.sub.2), 3.5-3.7 (32H, m,
15.times.CH.sub.2+OH+NH); MS (ESI): m/z=527.4 (M+H.sup.+).
##STR00629##
[1043] Compound 4f: 30% yield, .sup.1H-NMR (300 MHz, CDCl.sub.3):
.delta.=1.44 (9H, s, Boc), 2.65-2.79 (4H, m, 2.times.CH.sub.2),
2.80 (3H, s, MeN), 3.26 (2H, m, NCH.sub.2), 3.5-3.7 (48H, m,
23.times.CH.sub.2+OH+NH); MS (ESI): m/z 703.5 (M+H.sup.+).
##STR00630##
[1044] Compound 4g: .sup.1H-NMR (300 MHz, DMSO-d.sub.6):
.delta.=1.14 (5H, m, 2 CH and CH.sub.3), 1.35 (9H, s, Boc), 1.47
(4H, m, 4 CH), 1.74-2.38 (4H, m, 4 CH), 2.59 (2H, m, CH.sub.2),
2.75 (3H, s, NCH.sub.3), 3.16 (2H, m, CH.sub.2), 3.99 (2H, m,
CH.sub.2); MS (ESI): m/z =329.4 (M+H.sup.+).
##STR00631##
[1045] Compound 4h: 63% yield, .sup.1H-NMR (300 MHz, CDCl.sub.3):
.delta.=1.46 (9H, s, Boc), 1.69 (2H, m, CH.sub.2), 2.24 (6H, s,
Me.sub.2), 2.36 (2H, t, CH.sub.2), 2.52 (1H, m, NH), 2.72 (2H, t,
CH.sub.2), 2.80 (2H, t, CH.sub.2), 2.88 (3H, s, MeN), 3.37 (2H, m,
CH.sub.2); MS (ESI) m/z=260.2 (M+H.sup.+).
##STR00632##
[1046] Compound 4i: .sup.1H-NMR (300 MHz, DMSO-d.sub.6):
.delta.=1.30-1.36 (4H, m, 2 CH.sub.2 Lys), 1.35 (18H, 2 s, 2 Boc),
1.53 (2H, m, CH.sub.2 Lys), 2.46 (2H, m, CH.sub.2 Lys), 2.56 (2H,
m, CH.sub.2), 2.75 (3H, s, NCH.sub.3), 3.13 (3H, m, CH.sub.2 and
.alpha.-H Lys), 3.58 (3H, s, OCH.sub.3); MS (ESI): m/z=481.4
(M+H.sup.+).
##STR00633##
[1047] Compound 4j:.sup.1H-NMR (300 MHz, DMSO-d.sub.6):
.delta.=1.36 (31H, m, 1 tBu, 2 Boc and 2 CH.sub.2 Lys), 1.53 (2H,
m, CH.sub.2 Lys), 2.46 (2H, m, CH.sub.2 Lys), 2.56 (2H, t,
CH.sub.2), 2.75 (3H, s, NCH.sub.3), 3.14 (2H, t, CH.sub.2) 3.64
(1H, m, a-H Lys), 7.01 (d, 1H, NH); MS (ESI): m/z =460.3
(M+H.sup.+).
##STR00634##
[1048] Compound 4k:.sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=1.15
(2H, m), 1.30 (2H, m), 1.94 (4H, m), 2.46 (1H, m), 2.76 (2H, t,
J=6.8 Hz), 2.87 (3H, s), 3.30 (2H, t, J=6.8 Hz), 3.60 (1H, m); MS
(ESI): m/z=273.3 (M+H.sup.+).
[1049] Route B
[1050] General procedure alkylation reaction: To a solution of 1
mmol R-amine and 1 mmol mesylate in dry THF (1.5 mL) was added
K.sub.2CO.sub.3 (1.5 mmol) and the mixture was stirred at
60.degree. C. overnight. The mixture was cooled to RT, concentrated
and purified by column chromatography (SiO.sub.2, DCM/MeOH, 1:0 to
1:1) to give the N-Boc-N-Methyl-N'-R-diaminoethane.
[1051] Compound 5a, R=iso-propyl:.sup.1H NMR (300 MHz, CDCl.sub.3),
.delta. (ppm): 1.05 (6H, d, 2.times.CH.sub.3), 1.46 (9H, s, Boc),
2.75 (2H, t, J=6.6 Hz, CH.sub.2), 2.82 (1H, t, J=6.6 Hz, CH), 2.87
(3H, s, NMe), 3.31 (2H, t, J=6.6 Hz, CH.sub.2); MS (ESI) m/z=217.2
(M+H.sup.+).
##STR00635##
[1052] Compound 5b: Starting from N-Boc-prolinol.sup.18 compound 5b
was prepared according to route B. MS (ESI) m/z=215.2
(M+H.sup.+).
##STR00636##
[1053] Compound 5c: .sup.1H-NMR (300 MHz, DMSO-d.sub.6):
.delta.=1.39 (9H, s, Boc), 2.73-2.85 (7H, m,
NCH.sub.3+2.times.CH.sub.2), 3.24-3.30 (2H, m, CH.sub.2), 3.39-3.55
(11H, m, 5.times.CH.sub.2+OH).
Example 2
Synthesis of Compound 2
##STR00637##
[1055] 2.28 g (10 mmol) benzyl phenylcarbonate was dissolved in 20
ml ethanol and 1.03 ml (10 mmol) 1,2-diamino-1-methylpropane was
added dropwise and stirred at RT overnight. The mixture was diluted
with 25 ml water and acidified with 1M HCl until pH<3 and
extracted with DCM. The aqueous phase was basified with 4M NaOH and
extracted with DCM. The extract was dried with MgSO.sub.4 and
concentrated in vacuo. This gave 1.93 g (87%) monoprotected
diamine. This material was dissolved in 25 ml dioxane, 2.028 g
(10.5 mmol) Boc.sub.2O and 0.12 g (1 mmol) DMAP were added and the
reaction was stirred at RT overnight. The mixture was concentrated
in vacuo and purified by column chromatography (SiO.sub.2,
ether/heptane, 1:0 to 7:3) to give 1.13 g (40%) of diprotected
diamine. The diprotected diamine was dissolved in 10 ml dry DMF,
1.1 ml (17.5 mmol) iodomethane was added and the reaction mixture
cooled in ice. 0.50 g (10.5 mmol) sodium hydride (60% in oil) was
added in portions and stirred in ice for 2 hrs. The mixture was
warmed to RT, quenched with 10 ml saturated NH.sub.4Cl and 50 ml
water, extracted with ethyl acetate, dried with MgSO.sub.4 and
concentrated in vacuo. The product was purified by column
chromatography (SiO.sub.2, DCM/ethyl acetate, 1:0 to 50:1) to give
0.358 g (29%) of Cbz-protected compound 2. .sup.1H-NMR (300 MHz,
CDCl.sub.3): .delta.=1.29 (s, 3H, Me), 1.35 (s, 3H, Me), 1.46 (s,
9H, Boc), 2.82/2.86 (s, 3H, Me, Z/E), 2.93 (s, 3H, Me), 3.72 (s,
2H, CH.sub.2N), 5.12 (s, 2H, benzyl), 7.35 (m, 5H, Phe). This
material was dissolved in 20 ml methanol, 0.04 g Pd/C was added,
the mixture was stirred under hydrogen for 3 hrs, filtered, and the
filtrate concentrated. This gave 0.21 g (100%) of compound 2. MS
(ESI): m/z=217.2 (M+H.sup.+).
Example 3
Synthesis of Compound 3
##STR00638##
[1057] 2.15 g (18.8 mmol) cis-1,2-diaminocyclohexane was dissolved
in 20 ml ethyl formate and refluxed overnight. The suspension was
cooled, filtered and dried under vacuum to give 2.59 g (80%) of
product. The solid was added in portions to a cooled mixture of 2.1
g (53 mmol) lithium aluminumhydride in 50 ml THF and the mixture
was stirred at RT for lhr and then refluxed overnight. The clear
solution was cooled in ice and quenched by dropwise addition of 2.1
ml water followed by 15 ml 20% NaOH. The suspension was briefly
heated until the salts were white. The mixture was cooled to RT and
filtered. The residue was washed with 2 portions of 50 ml THF. The
filtrate was concentrated and dissolved in 50 ml DCM and washed
with 20 ml 4M NaOH. The aqueous phase was extracted with 25 ml DCM,
dried with MgSO.sub.4 and concentrated in vacuo to give 1.94 g
(89%) of product. The crude product was dissolved in 50 ml DCM and
the mixture cooled to -40.degree. C. and 2.95 g (13.5 mmol)
Boc.sub.2O in 10 ml DCM was added dropwise. The mixture was slowly
warmed to RT and washed with 20 ml 2M NaOH, dried with MgSO.sub.4,
concentrated in vacuo and the crude product purified by column
chromatography (SiO.sub.2, DCM/MeOH, 1:0 to 9:1) to give 2.09 g
(63%) of compound 3. .sup.1H-NMR (300 MHz, CDCl.sub.3):
.delta.=1.38 (m, 6H, cyclohex), 1.46 (s, 9H, Boc), 1.83 (m, 3H,
cyclohex+NH), 2.35 (s, 3H, NMe), 2.89 (s, 3H, NMe), 2.95 (m, 1H,
CHN), 3.86 (m, 1H, CHN); MS (ESI): m/z=243.2 (M+H.sup.+).
Example 4
Synthesis of Compound 6
##STR00639##
[1059] To a solution of 2.55 mmol N-Boc-prolinal in dry THF (5 mL)
under an Argon atmosphere was added 0.7 mL glacial acetic acid and
272 mg 2-(2-aminoethoxy)ethanol (2.58 mmol). The mixture was
stirred overnight at room temperature. 2.59 mmol sodium borohydride
was added portion wise, and the mixture was stirred for another 4
hrs after which water was added. The mixture was washed with ethyl
acetate, the aqueous layer basified up to pH 10 using
Na.sub.2CO.sub.3 and extracted with ethyl acetate (9.times.). The
combined organic layers were washed with brine, dried with
MgSO.sub.4, and concentrated in vacuo to give 335 mg (46%) crude 6,
which was used without further purification. MS (ESI): m/z=289
(M+H.sup.+).
Example 5
Synthesis of Compounds 7 and 8
##STR00640##
[1061] 0.17 g (1.5 mmol) N-methylpiperid-4-one and 0.178 g (1.0
mmol) N-Boc-N-methyl-1,2-diaminoethane were reacted with 0.42 g
(2.0 mmol) sodium triacetoxyborohydride in 5 ml DCE at RT
overnight. The reaction was quenched with Me0H and the mixture
extracted with DCM and brine (basified to pH>12), dried with
MgSO.sub.4 and concentrated in vacuo to give 0.31 g (100%) of 7 as
an oil. .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta.=.sup.1H-NMR (300
MHz, CDCl.sub.3): .delta.=1.38 (2, m, CH.sub.2), 1.45 (9H, s, Boc),
1.76 (1H, br s, NH), 1.85 (2H, m, CH.sub.2), 2.00 (2H, m,
CH.sub.2), 2.27 (3H, s, MeN), 2.46 (1H, m, CH), 2.76 (4H, m,
2.times.CH.sub.2), 2.86 (3H, s, MeN), 3.30 (2H, t, CH.sub.2); MS
(ESI) m/z=272.2 (M+H.sup.+).
##STR00641##
[1062] Compound 8 was prepared similarly to compound 7 using 0.75 g
(4.0 mmol) N-hydroxyethoxyethyl-4-piperidine. The crude product was
purified by column chromatography (SiO.sub.2, DCM/MeOH, 1:0 to
95:5) to give 0.83 g (66%) of 8 as an oil. .sup.1H-NMR (300 MHz,
CDCl.sub.3): .delta.=1.46 (9H, s, Boc), 1.89 (2H, m, CH.sub.2),
1.91 (2H, m, CH.sub.2), 2.50 (3H, m, CH+OH+NH), 2.57 (2H, t,
CH.sub.2), 2.77 (2H, t, CH.sub.2), 2.87 (3H, s, MeN), 2.94 (2H, m,
CH.sub.2), 3.30 (2H, t, CH.sub.2), 3.64 (6H, m, 3.times.CH.sub.2O);
MS (ESI) m/z=346.3 (M+H.sup.+).
Example 6
Synthesis of Compound 9
##STR00642##
[1064] 0.18 g (1.0 mmol)N-Boc-N-methyl-1,2-diaminoethane and 0.15 g
(1.0 mmol) 4-formylbenzoic acid were reacted according to general
procedure A of example 1 to give 0.34 g (90%) 9 (HCl salt).
.sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta.=2.89 (3H, s, MeN),
3.11 (2H, m, CH.sub.2N), 3.57 (2H, t, CH.sub.2N), 4.25 (2H, m,
CH.sub.2N), 7.33 (2H, d, ArH), 8.02 (2H, d, ArH), 9.03 (2H, br s,
NH.sub.2+), 13.03 (1H, s, CO.sub.2H); MS (ESI) m/z=309.2
(M+H.sup.+).
Example 7
Synthesis of Compound 11
##STR00643##
[1066] 1.84 g (7.5 mmol) N-Cbz-1,3-diamine hydrochloride and 3 ml
Et.sub.3N were dissolved in 50 ml DCM, cooled in ice and 0.94 ml
phenyl chloroformate dissolved in 5 ml DCM was added dropwise. The
mixture was stirred at 0.degree. C. for 2 hrs and concentrated in
vacuo. The residue was dissolved in 50 ml DCM, washed with 25 ml
0.5 M KHSO.sub.4, dried with MgSO.sub.4 and concentrated in vacuo.
The residue was dissolved in 25 ml EtOH, 4 ml concentrated ammonia
was added and the mixture was stirred for 3 hrs. The mixture was
concentrated in vacuo and the residue dissolved in 25 ml
CHCl.sub.3, washed with 10 ml 4 M NaOH and brine, dried with
MgSO.sub.4 and concentrated in vacuo to give 1.9 g of intermediate.
This material was dissolved in 50 ml MeOH, 0.1 g Pd/C was added and
the reaction mixture stirred under hydrogen for 5 hrs. The mixture
was filtered and the filtrate concentrated in vacuo to give 0.87 g
(95%) of compound 10. 0.26 g (1.7 mmol) of this amine was reacted
according to general procedure A to give 0.17 g (36%) of 11 as an
oil. .sup.1H-NMR (300 MHz, DMSO-d.sub.6): .delta.=1.39 (9H, s,
Boc), 1.45 (2H, m, CH.sub.2), 2.48 (2H, t, CH.sub.2), 2.58 (2H, t,
CH.sub.2), 2.78 (3H, s, MeN), 2.97 (2H, m, CH.sub.2), 3.18 (2H, t,
CH.sub.2), 5.35 (2H, s, NH.sub.2), 5.87 (1H, t, NH); MS (ESI)
m/z=275.2 (M+H.sup.+).
Example 8
Synthesis of Compound 12
##STR00644##
[1068] 0.98 g (4.0 mmol) N-Cbz-1,3-diamine hydrochloride and 3 ml
Et.sub.3N were dissolved in 15 ml DCM and added dropwise to 1.65 ml
(12 mmol) diethyl oxalate in 15 ml ethanol. The mixture was stirred
at RT overnight, concentrated in vacuo and the residue purified by
column chromatography (SiO.sub.2, DCM/MeOH, 1:0 to 95:5) to give
the mono-oxalamide. This material was dissolved in 15 ml ethanol
and 5 ml concentrated ammonia was added and the mixture was stirred
at RT overnight. The mixture was diluted with 50 ml MeOH and 100 mg
Pd/C was added. The mixture was stirred under hydrogen atmosphere
for 3 hrs. 10 ml water was added, the mixture was filtered over
Celite and the filtrate concentrated in vacuo to give 0.57 g (94%)
of white solid. This was reacted according to general procedure A
of Example 1 to give 0.45 g (38%) of 12 as a white solid.
.sup.1H-NMR (300 MHz, CDCl.sub.3): .delta.=1.45 (9H, s, Boc), 1.80
(2H, m, CH.sub.2), 2.80 (4H, m, 2.times.CH.sub.2), 2.89 (3H, s,
MeN), 3.39 (4H, m, 2.times.CH.sub.2), 6.85 (1H, s, NH), 7.40 (1H,
s, NH), 8.39 (1H, t, NH); MS (ESI) m/z=303.2 (M+H.sup.+).
Example 9
Synthesis of Compounds 14a-b and 15a-b
##STR00645##
[1070] Compound 14c: Compound 13c (0.51 mmol) was dissolved in THF
(12 ml); LiOH (1.02 mmol) and water (1 mL) were added and the
reaction mixture was stirred for 4 h. Then, the mixture was
acidified with aqueous HCl (1M, 1.5 ml), concentrated, concentrated
and the residue purified by column chromatography (SiO.sub.2,
DCM/MeOH, 1:0 to 9:1) to yield compound 14c (0.41 mmol)
[1071] Compound 15a: Compound 14a (0.11 mmol) was dissolved in
dioxane (2 ml); pyridine (0.07 mmol) and Boc.sub.2O (0.14 mmol)
were added followed by NH.sub.4HCO.sub.3 (0.14 mmol). The resulting
mixture was stirred for 8 hrs. Afterwards, the mixture was
concentrated and purified by column chromatography (SiO.sub.2,
DCM/MeOH, 1:0 to 9:1) to yield compound 15a (0.06 mmol).
Example 10
Synthesis of Linker-Agent Conjugates
##STR00646## ##STR00647##
[1073] General procedure for the coupling of the cyclization spacer
to the activated protected drug (step 2): 0.2 mmol activated
protected drug, 0.6 mmol of mono-protected spacer, and 0.02 mmol
HOBt were dissolved in 2 ml DMF, 1 mmol Et.sub.3N was added and the
mixture was heated at 50.degree. C. for 2 hrs. The mixture was
concentrated in vacuo and the crude product purified by column
chromatography (SiO.sub.2, DCM/MeOH) to give the cyclization
spacer-drug.
[1074] General procedure for coupling of the linker to the
cyclization spacer-drug intermediate (steps 3 and 4): 0.1 mmol
cyclization spacer-drug was suspended in 6 ml CHCl.sub.3, the
reaction mixture was cooled in ice and 2 ml of TFA was added and
the mixture stirred for 3 hrs. The mixture was then concentrated in
vacuo. The residue was dissolved in 4 ml DMF, the solution cooled
in ice and 0.13 mmol activated linker (16 or 17) and 1 mmol
Et.sub.3N were added. The mixture was stirred for 2 hrs,
concentrated in vacuo and the residue purified by column
chromatography (SiO.sub.2, DCM/MeOH, 1:0 to 8:2) to an oil which
was further purified by preparative reversed phase HPLC
(acetonitrile/water+0.1% TFA) and freeze-dried to give construct 18
as a pale yellow solid.
[1075] Compound 18b, R=Me: .sup.1H-NMR (400 MHz, DMSO-d.sub.6):
.delta.=0.84 (6H, dd, J=6.4 Hz, 2.times.CH.sub.3 Val), 1.32-1.47
(2H, m, CH.sub.2 Cit), 1.52-1.71 (2H, m, CH.sub.2 Cit), 1.91-1.99
(1H, m, .beta.-H Val), 2.81-3.03 (11H, m,
3.times.CH.sub.3+NCH.sub.2), 4.36-4.43 (1H, m, H-2), 4.45-4.51 (1H,
m, .alpha.-H), 4.63-4.41 (1H, m, H-2), 4.97-5.08 (3H, m, CH.sub.2
PABA+H-1), 5.97 (1H, br s, NH), 6.90 (2H, d, J=8.8 Hz, H-3''), 7.01
(2H, s, CH.dbd.CH), 7.14-7.59 (7H, m, ArH+2.times.NH), 7.67-7.80
(3H, m, ArH), 7.91 (2H, d, J=8.8 Hz, H-T'), 8.06 (1H, br s, H-6),
8.33 (H, br s, H-4), 8.81 (1H, s, H-3'), 9.55 (1H, s, H-4'), 9.99
(1H, s, NH), 10.20 (1H, s, NH), 10.28 (1H, s, OH); MS (ESI):
m/z=1257.3 (M+H.sup.+).
[1076] Compound 18c, R=Me: .sup.1H NMR (400 MHz, DMSO-d.sub.6):
.delta.=0.84 (6H, dd, Val), 1.28-1.73 (4H, m, CH.sub.2CH.sub.2Cit),
1.95 (1H, m, .beta.HVal), 2.85 (3H, s, MeAr), 2.94 (3H, s, MeN),
2.87-3.05 (4H, m, 2.times.CH.sub.2NMe), 4.05 (2H, t, CH.sub.2O),
4.40 (1H, m, H-1), 4.48 (1H, s, CH), 4.67 (1H, m, H-2), 5.05 (3H,
m, CH.sub.2O, H-2), 5.40 (2H, br s, NH.sub.2), 5.96 (1H, br s, NH),
6.90 (2H, d, H-3''), 7.01 (2H, s, CH.dbd.CH), 7.2-7.7 (10H, m,
ArH+NH), 7.91 (2H, d, H-T'), 8.05 (1H, s, H-6), 8.33 (1H, br s,
H-4), 8.77 (1H, s, H-3'), 9.52 (1H, s, H-4'), 10.0 (1H, s, NH),
10.19 (1H, s, NH), 10.25 (1H, s, OH); MS (ESI): m/z=1345.7
(M+H.sup.+).
[1077] Compound 18b, R.dbd.(CH.sub.2CH.sub.2O).sub.2H: .sup.1H NMR
(400 MHz, DMSO-d.sub.6), .delta. (ppm): .delta.=0.84 (6H, dd, Val),
1.28-1.73 (4H, m, CH.sub.2CH.sub.2Cit), 1.96 (1H, m, .beta.HVal),
2.85 (3H, s, MeAr), 2.95 (3H, s, MeN), 2.87-3.05 (4H, m,
2.times.CH.sub.2NMe), 4.00 (2H, t, CH.sub.2O), 4.35 (1H, m, H-1),
4.48 (1H, m, CH), 4.67 (1H, m, H-2), 4.93-5.11 (4H, m, CH.sub.2O,
H-2, CH), 5.40 (2H, br s, NH.sub.2), 5.97 (1H, br s, NH), 6.91 (2H,
d, H-3''), 7.01 (2H, s, CH.dbd.CH), 7.2-7.7 (10H, m, ArH+NH), 7.91
(2H, d, H-2''), 8.06 (1H, d, H-6), 8.33 (1H, br s, H-4), 8.80 (1H,
s, H-3'), 9.54 (1H, s, H-4'), 10.01 (1H, s, NH), 10.20 (1H, s, NH),
10.27 (1H, s, OH); MS (ESI): m/z=1331.7 (M+H.sup.+).
[1078] Compound 18c, R.dbd.(CH.sub.2).sub.4C(NH.sub.2)COOH:
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=0.84 (6H, dd, J=6.4
Hz, 2.times.CH.sub.3 Val), 1.29-1.98 (11H, m, 3.times.CH.sub.2
Lys+2.times.CH.sub.2 Cit+.beta.-H Val), 2.82-3.04 (8H, m,
2.times.CH.sub.3+NCH.sub.2), 4.35-4.42 (1H, m, H-1), 4.45-4.51 (1H,
m, .alpha.-H), 4.63-4.70 (1H, m, H-2), 4.95-5.07 (2H, m, CH.sub.2
PABA), 5.09-5.16 (1H, m, H-2), 5.99 (1H, br s, NH), 6.90 (2H, d,
J=8.4 Hz, H-3''), 7.01 (2H, s, CH.dbd.CH), 7.16-7.77 (11H, m,
ArH+3.times.NH), 7.90 (2H, d, J=8.4 Hz, H-2''), 8.05-8.09 (1H, m,
H-6), 8.19 (3H, br s, NH.sub.2 Lys+OH), 8.31-8.37 (1H, m, H-4),
8.75 (1H, br s, H-3'), 9.53 (1H, br s, H-4'), 10.00 (1H, br s, NH),
10.16-10.26 (2H, m, NH+OH); MS (ESI): m/z =1460.7 (M+H.sup.+).
[1079] Compound 18d, R=(CH.sub.2CH.sub.2O).sub.2H: .sup.1H-NMR (400
MHz, DMSO-d.sub.6): .delta.=0.84 (6H, dd, Val, J=6.8), 1.30-1.46
(2H, m, CH.sub.2 Cit), 1.52-1.70 (2H, m, CH.sub.2 Cit), 1.91-1.99
(1H, m, .beta.-H Val), 2.80-3.05 (10H, m,
ArCH.sub.3+NCH.sub.3+2.times.NCH2), 3.97-4.06 (5H, m,
2.times.CH.sub.2O+H-2), 4.20 (2H, d, J=5.6 Hz, NCH.sub.2-triazole),
4.36-4.43 (1H, m, H-1), 4.44-4.52 (3H, m,
CH.sub.2-triazole+.alpha.-H), 4.63-4.71 (1H, m, H-2), 4.94-5.09
(3H, m, CH.sub.2 PABA+H-2), 5.98 (1H, br s, NH), 6.90 (2H, d, J=8.8
Hz, H-3''), 6.99 (2H, s, CH.dbd.CH), 7.16-7.81 (11H, m, ArH+NH),
7.87 (1H, s, triazole-H), 7.91 (2H, d, J=8.8 Hz, H-2''), 8.05-8.09
(1H, m, H-6), 8.33 (H, m, H-4), 8.83 (1H, s, H-3'), 9.55 (1H, s,
H-4'), 10-00 (1H, s, NH), 10.20 (1H, s, NH), 10.28 (1H, s, OH); MS
(ESI): m/z=1543.7 (M+H.sup.+).
[1080] Compound 18c, R.dbd.(CH.sub.2CH.sub.2O).sub.2H: .sup.1H NMR
(400 MHz, DMSO-d.sub.6), .delta.=0.84 (6H, dd, Val), 1.28-1.73 (4H,
m, CH.sub.2CH.sub.2Cit), 1.96 (1H, m, .beta.HVal), 2.85 (3H, s,
MeAr), 2.94 (3H, s, MeN), 2.87-3.05 (4H, m, 2.times.CH.sub.2NMe),
4.05 (2H, t, CH.sub.2O), 4.40 (1H, m, H-1), 4.48 (1H, m, CH), 4.67
(1H, m, H-2), 4.93-5.16 (4H, m, CH.sub.2O, CH, H-2), 5.40 (2H, br
s, NH.sub.2), 5.96 (1H, br s, NH), 6.90 (2H, d, H-3''), 7.01 (2H,
s, CH.dbd.CH), 7.2-7.8 (11H, m, ArH+3.times.NH), 7.90 (2H, d,
H-T'), 8.05 (1H, d, H-6), 8.32 (1H, br s, H-4), 8.76 (1H, s, H-3'),
9.51 (1H, s, H-4'), 10.01 (1H, s, NH), 10.19 (1H, s, NH), 10.24
(1H, s, OH); MS (ESI): m/z=1419.7 (M+H.sup.+).
[1081] Compound 18a, R.dbd.(CH.sub.2CH.sub.2O).sub.2H: .sup.1H NMR
(400 MHz, DMSO-d.sub.6): .delta.=0.81 (6H, dd, J=6.4 Hz,
2.times.CH.sub.3), 1.30-1.47 (2H, m, CH.sub.2 Cit), 1.53-1.71 (2H,
m, CH.sub.2 Cit), 1.89-1.99 (1H, m, .beta.-H Val), 2.82-3.05 (8H,
m, 2.times.CH.sub.3+NCH.sub.2), 3.96-4.02 (1H, m), 4.15-4.23 (1H,
m), 4.33-4.40 (1H, m, H-1), 4.44-4.52 (1H, m, .alpha.-H), 4.63-4.71
(1H, m, H-2), 4.94-5.12 (3H, m, CH.sub.2+H-2), 5.96 (1H, br s, NH),
6.90 (2H, d, J=8.4 Hz, H-3''), 7.01 (2H, s, CH.dbd.CH), 7.10-7.54
(7H, m, 5 ArH+2.times.NH), 7.66-7.81 (3H, m, ArH), 7.91 (2H, d,
J=8.8 Hz, H-2''), 8.00-8.05 (1H, m, H-6), 8.29-8.37 (1H, m, H-4),
8.79 (1H, br s, H-3'), 9.54 (1H, br s, H-4'), 9.91-10.00 (1H, m,
NH), 10.20 (1H, br s, OH), 10.27 (1H, s, NH); MS (ESI): m/z=1287.7
(M+H.sup.+).
[1082] Compound 18b, R.dbd.(CH.sub.2).sub.3C(O)NH.sub.2:
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=0.83 (6H, dd, J=6.4
Hz, 2.times.CH.sub.3 Val), 1.25-2.15 (9H, m, 2.times.CH.sub.2
butyramide+2.times.CH.sub.2 Cit+.beta.-H Val), 2.82-3.04 (8H, m,
2.times.CH.sub.3+NCH.sub.2), 3.18-3.98 (15H, m,
2.times.H-10+6.times.CH.sub.2+.alpha.-H), 4.34-4.42 (1H, m, H-1),
4.46-4.54 (1H, m, .alpha.-H), 4.63-4.71 (1H, m, H-2), 4.93-5.07
(3H, m, CH.sub.2 PABA+H-2), 5.98 (1H, br s, NH), 6.76 (1H, br s,
NH), 6.91 (2H, d, J=8.8 Hz, H-3''), 7.00 (2H, s, CH=CH), 7.13-7.58
(8H, m, ArH+2.times.NH), 7.65-7.83 (3H, m, ArH), 7.91 (2H, d, J=8.4
Hz, H-2''), 8.04-8.09 (1H, m, H-6), 8.28-8.37 (1H, m, H-4), 8.89
(1H, br s, H-3'), 9.60 (1H, br s, H-4'), 9.93-10.02 (1H, m, NH),
10.34 (1H, br s, NH); MS (ESI): m/z=1328.3 (M+H.sup.+).
[1083] Compound 18b, R.dbd.(CH.sub.2).sub.4C(NH.sub.2)COOH:
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=0.83 (6H, dd, J=6.4
Hz, 2.times.CH.sub.3 Val), 1.30-1.98 (11H, m, 3.times.CH.sub.2
Lys+2.times.CH.sub.2 Cit+.beta.-H Val), 2.82-3.04 (8H, m,
2.times.CH.sub.3+NCH.sub.2), 4.33-4.42 (1H, m, H-1), 4.45-4.51 (1H,
m, .alpha.-H), 4.62-4.70 (1H, m, H-2), 4.95-5.07 (2H, m, CH.sub.2
PABA), 5.10-5.16 (1H, m, H-2), 5.98 (1H, br s, NH), 6.90 (2H, d,
J=8.8 Hz, H-3''), 7.01 (2H, s, CH.dbd.CH), 7.13-7.77 (11H, m,
ArH+3.times.NH), 7.90 (2H, d, J=8.8 Hz, H-2''), 8.05-8.09 (1H, m,
H-6), 8.19 (3H, br s, NH.sub.2 Lys+OH), 8.31-8.37 (1H, m, H-4),
8.73 (1H, br s, H-3'), 9.51 (1H, br s, H-4'), 10.00 (1H, br s, NH),
10.17-10.25 (2H, m, NH+OH); MS (ESI): m/z=1372.6 (M+H.sup.+).
[1084] Compound 18b, R.dbd.(CH.sub.2).sub.3COOH: .sup.1H NMR (400
MHz, DMSO-d.sub.6), .delta.=0.83 (6H, dd, Val), 1.28-1.73 (4H, m,
CH.sub.2CH.sub.2Cit), 1.74-2.02 (2H, m, CH.sub.2), 1.95 (1H, m,
.beta.HVal), 2.19-2.39 (2H, m, CH.sub.2), 2.85 (3H, s, MeAr), 2.94
(3H, s, MeN), 2.87-3.05 (4H, m, 2.times.CH.sub.2NMe), 4.00 (2H, m,
CH.sub.2O), 4.40 (1H, m, H-1), 4.48 (1H, m, CH), 4.67 (1H, m, H-2),
4.93-5.18 (4H, m, CH.sub.2O, CH, H-2), 5.40 (2H, br s, NH.sub.2),
5.96 (1H, br s, NH), 6.90 (2H, d, H-3''), 7.01 (2H, s, CH.dbd.CH),
7.2-7.8 (10H, m, ArH+NH), 7.91 (2H, d, H-'), 8.06 (1H, d, H-6),
8.33 (1H, br s, H-4), 8.76 (1H, s, H-3'), 9.51 (1H, s, H-4'), 10.01
(1H, s, NH), 10.19 (1H, s, NH), 10.24 (1H, s, OH); MS (ESI):
m/z=1329.8 (M+H.sup.+).
[1085] Compound 18c, R.dbd.(CH.sub.2).sub.3COOH: .sup.1H NMR (400
MHz, DMSO-d.sub.6), .delta.=0.84 (6H, dd, Val), 1.30-1.73 (4H, m,
CH.sub.2CH.sub.2Cit), 1.74-2.02 (2H, m, CH.sub.2), 1.95 (1H, m,
.beta.HVal), 2.19-2.39 (2H, m, CH.sub.2), 2.85 (3H, s, MeAr), 2.94
(3H, s, MeN), 2.87-3.05 (4H, m, 2.times.CH.sub.2NMe), 4.05 (2H, m,
CH.sub.2O), 4.40 (1H, m, H-1), 4.48 (1H, m, CH), 4.67 (1H, m, H-2),
4.95-5.16 (4H, m, CH.sub.2O, CH, H-2), 5.40 (2H, br s, NH.sub.2),
5.96 (1H, br s, NH), 6.90 (2H, d, H-3''), 7.01 (2H, s, CH.dbd.CH),
7.2-7.8 (9H, m, ArH+NH), 7.91 (2H, d, H-2''), 8.05 (1H, d, H-6),
8.34 (1H, br s, H-4), 8.77 (1H, s, H-3'), 9.52 (1H, s, H-4'), 10.01
(1H, s, NH), 10.19 (1H, s, NH), 10.24 (1H, s, OH); MS (ESI):
m/z=1417.9 (M+H.sup.+).
[1086] Compound 18b, R.dbd.(CH.sub.2).sub.3NHC(O)NH.sub.2:
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=0.79 (6H, dd, Val),
1.25-1.8 (6H, m, 3.times.CH.sub.2), 1.91 (1H, m, .beta.HVal), 2.80
(3H, s, MeAr), 2.90 (3H, s, MeN), 2.95 (4H, m, 2.times.CH.sub.2N),
4.34 (1H, m, H-1), 4.61 (1H, m, H-2), 4.94 (1H, m, H-2), 4.99 (2H,
s, CH.sub.2O), 5.32 (4H, br s, 2.times.NH.sub.2), 5.91 (1H, br s,
NH), 5.98 (1H, br s, NH), 6.86 (2H, d, H-3''), 6.95 (2H, s,
CH.dbd.CH), 7.2-7.7 (10H, m, ArH+2.times.NH), 7.86 (2H, d, H-2''),
8.02 (1H, d, H-6), 8.28 (1H, br s, H-4), 8.82 (1H, s, H-3'), 9.54
(1H, s, H-4'), 9.95 (1H, s, NH), 10.15 (1H, s, NH), 10.27 (1H, s,
OH); MS (ESI): m/z=1343.7 (M+H.sup.+).
[1087] Compound 18c, R.dbd.(CH.sub.2).sub.3NHC(O)NH.sub.2:
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=0.79 (6H, dd, Val),
1.25-1.8 (6H, m, 3*CH.sub.2), 1.91 (1H, m, .beta.HVal), 2.80 (3H,
s, MeAr), 2.90 (3H, s, MeN), 2.95 (4H, m, 2.times.CH.sub.2N), 4.34
(1H, m, CH), 4.45 (1H, m, CH), 4.61 (1H, m, H-2), 4.94 (1H, m,
H-2), 4.99 (2H, s, CH.sub.2O), 5.32 (4H, br s, 2.times.NH.sub.2),
5.91 (1H, br s, NH), 5.98 (1H, br s, NH), 6.86 (2H, d, H-3''), 6.95
(2H, s, CH.dbd.CH), 7.2-7.7 (10H, m, ArH+2.times.NH), 7.86 (2H, d,
H-2''), 8.02 (1H, d, H-6), 8.28 (H, br s, H-4), 8.82 (1H, s, H-3'),
9.54 (1H, s, H-4'), 9.95 (1H, s, NH), 10.15 (1H, s, NH), 10.27 (1H,
s, OH); MS (ESI): m/z =1431.5 (M+H.sup.+).
[1088] Compound 18d, R.dbd.(CH.sub.2).sub.3NHC(O)NH.sub.2:
.sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=0.83 (6H, dd, Val),
1.25-1.8 (6H, m, 3.times.CH.sub.2), 1.95 (1H, m, .beta.HVal), 2.85
(3H, s, MeAr), 2.94 (3H, s, MeN), 2.95 (4H, m, 2.times.CH.sub.2N),
4.20 (2H, d, NCH.sub.2), 4.39 (1H, m, CH), 4.48 (3H, m,
CH+CH.sub.2), 4.67 (1H, m, H-2), 5.02 (3H, m, H-2, CH.sub.2O), 5.41
(4H, br s, 2.times.NH.sub.2), 5.97 (2H, br s, 2.times.NH), 6.91
(2H, d, H-3''), 6.99 (2H, s, CH.dbd.CH), 7.2-7.7 (11H, m,
ArH+3.times.NH), 7.76 (1H, s, triazole-H), 7.88 (2H, d, H-2''),
8.08 (1H, d, H-6), 8.33 (H, br s, H-4), 8.84 (1H, s, H-3'), 9.57
(1H, s, H-4'), 10.00 (1H, s, NH), 10.20 (1H, s, NH), 10.30 (1H, s,
OH); MS (ESI): m/z=1556.2 (M+H.sup.+).
[1089] Compound 18b, R.dbd.(CH.sub.2CH.sub.2O).sub.3H: .sup.1H-NMR
(400 MHz, DMSO): .delta.=0.83 (6H, dd, J=6.5 Hz, J=15.8 Hz, Val),
1.3-1.7 (4H, m), 1.96 (1H, m), 2.90 (8H, m), 3.00-4.00 (6H, m),
4.44 (2H, m), 4.67 (1H, m), 6.69 (1H, s), 5.04 (2H, d, J=9.8 Hz),
5.40 (1H, br s), 5.97 (1H, s, NH), 6.9 (2H, d, J=8.3 Hz), 7.00 (2H,
s, CH.dbd.CH), 7.13-7.61 (7H, m), 7.75 (3H, m) 7.91 (2H, d, J=8.3
Hz), 8.06 (1H, m), 8.33 (1H, s), 8.83 (1H, s), 9.56 (1H, s), 9.97
(1H, s), 10.20 (1H, s), 10.30 (1H, s); MS (ESI): m/z=1376
(M+H.sup.+).
##STR00648##
[1090] Compound 22: Compound 22 was prepared analogously starting
with the corresponding activated drug. .sup.1H-NMR (400 MHz,
DMSO-d.sub.6): .delta.=0.84 (6H, dd, J=6.4 Hz, 2.times.CH.sub.3
Val), 1.30-1.47 (2H, m, CH.sub.2 Cit), 1.52-1.71 (2H, m, CH.sub.2
Cit), 1.91-1.99 (1H, m, .beta.-H Val), 2.82-3.03 (8H, m,
2.times.CH.sub.3+NCH.sub.2), 4.18-4.22 (2H, m, CH.sub.2O),
4.35-4.43 (1H, m, H-1), 4.46-4.52 (1H, m, .alpha.-H), 4.63-4.71
(1H, m, H-2), 4.94-5.08 (3H, m, CH.sub.2 PABA+H-2), 5.97 (1H, br s,
NH), 7.01 (2H, s, CH.dbd.CH), 7.09-7.20 (4H, m, H-3''+2 ArH),
7.22-7.58 (5H, m, 3 ArH+2.times.NH), 7.69-7.82 (3H, m, ArH), 8.00
(2H, d, J=8.8 Hz, H-2''), 8.03-8.08 (1H, br s, H-6), 8.28-8.37 (1H,
m, H-4), 8.83 (1H, br s, H-3'), 9.57 (1H, br s, H-4'), 9.93-10.02
(1H, m, NH), 10.39 (1H, s, NH); MS (ESI): m/z=1509.6
(M+H.sup.+).
##STR00649##
[1091] Compound 23: Compound 23 was prepared analogously starting
with the corresponding activated drug. .sup.1H-NMR (400 MHz, DMSO):
.delta.=0.80 (6H, dd, J=6.5 Hz, J=15.9 Hz, 2.times.CH.sub.3),
1.3-1.7 (4H, m), 1.93 (1H, m), 2.9 (11H, m), 3.00-4.50 (10H, m),
5.03 (3H, m), 5.40 (1H, br s), 5.94 (1H, s, NH), 6.98 (2H, s,
CH.dbd.CH), 7.10-7.85 (11H, m), 8.03 (3H, m), 8.32 (1H, m) 8.76
(1H, s), 9.50 (1H, s), 9.78 (1H, s), 9.98 (1H, br s), 10.51 (1H, br
s); MS (ESI): m/z=1457 (M+H.sup.+).
##STR00650##
[1092] Compound 26: .sup.1H-NMR (400 MHz, DMSO-d.sub.6):
.delta.=0.83 (6H, dd, J=6.8 Hz, 2.times.CH.sub.3), 1.30-1.47 (2H,
m, CH.sub.2 Cit), 1.54-1.71 (2H, m, CH.sub.2 Cit), 1.90-2.00 (1H,
m, .beta.-H Val), 2.82-3.03 (10H, m,
ArCH.sub.3+NCH.sub.3+2.times.NCH2), 3.19 (3H, s, OCH.sub.3),
3.97-4.03 (2H, m, NCH.sub.2-triazole), 4.37-4.52 (2H, m,
H-1+.alpha.-H), 4.64-4.69 (3H, m, H-2+CH.sub.2), 4.94-5.13 (3H, m,
CH.sub.2 PABA+H-2), 5.96 (1H, br s, NH), 7.01 (2H, s, CH.dbd.CH),
7.14-7.58 (7H, m, 5 ArH+3.times.NH), 7.72-7.84 (3H, m, ArH),
8.04-8.07 (1H, m, H-6), 8.29-8.38 (H, m, H-4), 8.74-8.80 (2H, s,
H-3'+triazole-H), 9.53 (1H, s, H-4'), 10-00 (1H, s, NH), 10.85 (1H,
s, NH); MS (ESI): m/z=1496.9 (M+H.sup.+).
Example 11
Synthesis of Compound 19
##STR00651##
[1094] 0.21 g (0.85 mmol)
N-Boc-N-methyl-N'-methyl-1,2-diamino-1-methylpropane, 0.45 g (0.64
mmol) 20 and 8 mg HOBt were dissolved in 5 ml DMF. 0.75 ml DiPEA
was added and the reaction mixture stirred at RT overnight. The
mixture was concentrated in vacuo and purified by column
chromatography (SiO.sub.2, DCM/MeOH, 1:0 to 20:1) to give 0.50 g
(100%) of intermediate. This material was suspended in 4 ml
CHCl.sub.3 and cooled in ice. 2 ml TFA was added and the reaction
mixture was stirred for 2 hrs at 0.degree. C. and then concentrated
in vacuo. To this material in 6 ml DMF, 0.11 g (0.15 mmol)
activated MOM-protected drug, 2 mg HOBt and 0.2 ml (1.2 mmol)
Et.sub.3N were added and the mixture was heated at 50.degree. C.
for 3 hrs. The mixture was concentrated in vacuo and purified by
column chromatography (SiO.sub.2, DCM/MeOH, 1:0 to 75:15) to give
0.14 g (72%) material. This was suspended in 6 ml CHCl.sub.3 cooled
in ice. 0.5 ml of TFA was added and the mixture was stirred for 2
hrs and then concentrated in vacuo. This material and 0.045 g (0.17
mmol) maleimide-alkyne linker were dissolved in 2 ml DMF. 0.6 mL of
a solution of 0.025 g (0.1 mmol) CuSO.sub.4, 0.022 g (0.11 mmol)
1,10-phenanthroline and 0.022 g (0.11 mmol) sodium ascorbate in 1
ml acetonitrile/water (1:2) was added to the reaction mixture and
the mixture was stirred for 2.5 hrs. The mixture was acidified with
acetic acid and concentrated in vacuo. The crude product was
purified by column chromatography (SiO.sub.2, DCM/MeOH, 1:0 to 8:2)
to give 0.026 g (16%) material which was further purified by
preparative reversed phase HPLC and freeze dried to give 0.015 g of
19. .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=0.79 (6H, dd,
Val), 1.34 (3H, br s, gem-Me.sub.2), 1.38 (3H, br s, gem-Me.sub.2),
1.3-1.7 (4H, m, CH.sub.2CH.sub.2Cit), 1.90 (1H, m, .beta.HVal),
2.80 (3H, s, MeAr), 2.91 (3H, s, MeN). 3.11 (3H, br s, MeN), 3.95
(2H, t, CH.sub.2O), 3.98 (2H, t, CH.sub.2O), 4.15 (2H, d,
NCH.sub.2-triazole), 4.30 (1H, m, H-1), 4.43 (2H, t,
CH.sub.2-triazole), 4.61 (1H, m, H-2), 5.00 (2H, s, CH.sub.2O),
5.04 (1H, m, H-2), 5.37 (2H, br s, NH.sub.2), 5.92 (1H, br s, NH),
6.85 (2H, d, H-3''), 6.94 (2H, s, CH.dbd.CH), 7.2-7.7 (11H, m,
ArH+3.times.NH), 7.81 (1H, s, triazole-H), 7.84 (2H, d, H-T'), 8.03
(1H, d, H-6), 8.27 (H, br s, H-4), 8.73 (1H, s, H-3'), 9.48 (1H, s,
H-4'), 9.98 (1H, s, NH), 10.15 (1H, s, NH), 10.21 (1H, s, OH); MS
(ESI): m/z=1497.6 (M+H.sup.+).
##STR00652##
[1095] Compound 21: The same procedure was followed as for compound
19. .sup.1H-NMR (400 MHz, DMSO-d.sub.6): .delta.=0.83 (6H, dd,
Val), 1.3-2.1 (3 Hm, cyclohexyl+CH.sub.2CH.sub.2Cit+.beta.HVal),
2.84 (3H, s, MeAr), 3.01 (8H, m, 2.times.MeNCH), 3.98 (4H, m,
2.times.CH.sub.2O), 4.20 (2H, d, NCH.sub.2-triazole), 4.36 (1H, m,
H-1), 4.48 (2H, t, CH.sub.2-triazole), 4.65 (1H, m, H-2), 5.04 (1H,
m, H-2), 5.06 (2H, s, CH.sub.2O), 5.39 (2H, br s, NH.sub.2), 5.96
(1H, br s, NH), 6.90 (2H, d, H-3''), 6.99 (2H, s, CH.dbd.CH),
7.2-7.7 (11H, m, ArH+3.times.NH), 7.87 (1H, s, triazole-H), 7.91
(2H, d, H-2''), 8.06 (1H, d, H-6), 8.36 (H, br s, H-4), 8.80 (1H,
s, H-3'), 9.55 (1H, s, H-4'), 9.97 (1H, s, NH), 10.20 (1H, s, NH),
10.27 (1H, s, OH); MS (ESI): m/z=1523.6 (M+H.sup.+).
Example 12
Synthesis of Maleimide-Peptide Linkers
##STR00653##
[1097] Compound 25a: .sup.1H-NMR (300 MHz, DMSO-d.sub.6):
.delta.=0.83 (6H, dd, J=6.9 Hz, 2.times.CH.sub.3 Val), 1.30-1.51
(2H, m, CH.sub.2 Cit), 1.53-1.76 (2H, m, CH.sub.2 Cit), 1.90-2.01
(1H, m, (3-H Val), 2.89-3.08 (2H, m, CH.sub.2 Cit), 3.62 (2H, t,
J=5.4 Hz, CH.sub.2), 3.83-3.88 (1H, m, .alpha.-H), 3.97-4.05 (1H,
m), 4.16-4.24 (1H, m), 4.35-4.43 (1H, m, .alpha.-H), 5.25 (2H, s,
CH.sub.2), 5.41 (2H, s, NH.sub.2), 5.95-5.99 (1H, m, NH), 7.02 (2H,
s, HC.dbd.CH), 7.11-7.14 (1H, m, NH), 7.40 (2H, d, J=8.7 Hz,
Ar--H), 7.54-7.59 (2H, m, Ar--H), 7.64 (2H, d, J=8.4 Hz, Ar--H),
8.03-8.06 (1H, m, NH), 8.29-8.33 (2H, m, Ar--H), 10.08 (1H, s, NH);
MS (ESI): m/z=712.5 (M+H.sup.+).
[1098] Compound 25b: .sup.1H-NMR (300 MHz, DMSO-d.sub.6):
.delta.=0.85 (6H, dd, J=6.9 Hz, 2.times.CH.sub.3 Val), 1.32-1.50
(2H, m, CH.sub.2 Cit), 1.54-1.76 (2H, m, CH.sub.2 Cit), 1.91-2.02
(1H, m, .beta.-H Val), 2.90-3.09 (2H, m, CH.sub.2), 3.49-3.60 (6H,
m, CH.sub.2), 3.87-3.92 (1H, m, .alpha.-H), 3.97-4.06 (2H, m,
CH.sub.2), 4.37-4.46 (1H, m, .alpha.-H), 5.24 (2H, s, CH.sub.2),
5.41 (2H, s, NH.sub.2), 5.95-6.00 (1H, m, NH), 7.01 (2H, s,
HC.dbd.CH), 7.15-7.18 (1H, m, NH), 7.41 (2H, d, J=8.7 Hz, Ar--H),
7.54-7.59 (2H, m, Ar--H), 7.64 (2H, d, J=8.4 Hz, Ar--H), 8.07-8.10
(1H, m, NH), 8.29-8.33 (2H, m, Ar--H), 10.11 (1H, s, NH); MS (ESI):
m/z=756.5 (M+H.sup.+).
[1099] Compound 25c: .sup.1H-NMR (300 MHz, CDCl.sub.3/CD.sub.3OD)
.delta.=0.98 (6H, dd, J=6.9 Hz, J=14.4 Hz, CH.sub.3), 1.59 (2H, m),
1.76 (1H, m), 1.94 (1H, m), 2.13 (1H, m), 3.08-3.28 (2H, m)
3.54-3.76 (10H, m, CH.sub.2O), 4.00 (1H, d, J=6.3 Hz), 4.20 (2H,
m), 4.57 (1H, m), 5.27 (2H, s, OCH.sub.2Ar), 6.78 (2H, s,
CH.dbd.CH), 7.42 (4H, m, ArH), 7.65 (2H, d, J=8.5 Hz), 8.30 (2H, d,
J=9.2 Hz).
[1100] Compound 25d: .sup.1H-NMR (400 MHz, CDCl.sub.3/CD.sub.3OD)
.delta.=0.98 (6H, dd, J=6.9 Hz, J=14.4 Hz, CH.sub.3), 1.59 (2H, m),
1.76 (1H, m), 1.94 (1H, m), 2.13 (1H, m), 3.08-3.28 (2H, m)
3.57-3.76 (14H, m, CH.sub.2O), 4.00 (1H, m), 4.22 (2H, m), 4.57
(1H, m), 5.26 (2H, s, OCH.sub.2Ar), 6.78 (2H, s, CH.dbd.CH), 7.42
(4H, m, ArH), 7.66 (2H, d, J=8.3 Hz), 8.29 (2H, d, J=9.3 Hz).
Example 13
Determination of the Cyclization Rate of Cyclization
Spacer-Duocarmycin Compounds
[1101] Cyclization spacer-duocarmycin compounds in which the
cyclization spacer is coupled to the hydroxy group of the
DNA-alkylator were prepared from the corresponding Boc-protected
derivatives such as and similar to compound 14a by treatment with
TFA in DCM for 10 minutes, followed by concentration. For
measurement of the cyclization rate at pH 7.4, the cyclization
spacer-duocarmycin compound was dissolved in a mixture of 100 mM
phosphate buffer pH 7.4 and acetonitrile (60/40) at 25.degree. C.
and disappearance of starting material/appearance of free drug was
followed over time by LC/MS (direct injection of reaction mixture).
For measurement of the cyclization rate at pH 5, the cyclization
spacer-duocarmycin compound was dissolved in a mixture of 100 mM
sodium acetate buffer pH 5 and acetonitrile (60/40) at 37.degree.
C. and disappearance of starting material/appearance of free drug
was followed over time by LC/MS (direct injection of reaction
mixture). Cyclization rates were calculated from the LC/MS data.
FIG. 2 shows some representative data. All cyclization
spacer-duocarmycin compounds depicted in FIG. 2 contain the same
duocarmycin compound. CM-Drugl contains a cyclization spacer used
in the prior art. All other cyclization spacer-duocarmycin
compounds contain cyclization spacers selected from the ones
described in Examples 1-9. The results presented in FIG. 2 indicate
that the cyclization spacers of the present invention can be used
to modulate the cyclization rate.
Example 14
Human Plasma Stability of HSA-Conjugated Linker-Agent
Conjugates
[1102] Linker-agent conjugate containing a maleimide group was
dissolved in DMSO and added to sodium heparine-stabilized human
plasma at 37.degree. C. such that the concentration of linker-agent
conjugate was 7.5 .mu.M and percentage DMSO was 2.5%. Linker-agent
conjugate reacted in situ with human serum albumin within 5
minutes, as indicated by LC/MS. Formation of free drug was followed
over time by LC/MS. Plasma samples were treated with acetonitrile
and centrifuged before analysis. FIG. 3 presents the human plasma
stability data for a representative selection of linker-agent
conjugates of the present invention. All linker-agent conjugates
contain the same duocarmycin analog and linkers are coupled to the
DNA alkylator moiety, except for LD13, in which the linker is bound
to the DNA binder moiety. LDS, LD12, and LD13 contain linkers that
have been used in the prior art and that contain a relatively long
linker as in compound 18d. LD10, LD25, LD26, LD28 and LD29 contain
similar relatively short linkers as in compound 18b and have
different cyclization spacers. LD10 and LD12 only differ in L
moiety. The results in FIG. 3 demonstrate that conjugates with a
relatively short linker have a high human plasma stability, which
generally exceeds that of corresponding conjugates with a
relatively long linker. Plasma stability is furthermore
demonstrated to be affected by the cyclization spacer.
Example 15
single Dose Efficacy Study in Female nu/nu Mice Bearing an N87
Xenograft
[1103] Preparation of ADCs: Trastuzumab (50 mg, 10 mg/mL) was
reduced using 1.1 molar equivalents of TCEP by incubating at
20.degree. C. for 90 minutes to generate 2 free thiols per mAb. The
incorporation ratio was confirmed via the Ellman's assay.
Linker-agent conjugate dissolved in DMSO was added to the reduced
antibody solution dropwise at a ratio of 1.3 molar equivalents per
free thiol such that the final concentration of DMSO was 10%. After
mixing for a further 50 minutes at 20.degree. C., the reaction was
quenched by the addition of one molar equivalent of
N-acetylcysteine per linker-agent conjugate. After quenching, the
conjugate was desalted into PBS pH 7.4 buffer and then purified
using a 5 ml r-Protein A column, with the product collected off
this column being desalted into presentation buffer (same buffer as
in commercially available Herceptin). The product was filtered to
0.2 .mu.m and characterized for aggregate (SEC), drug-to-antibody
ratio (UV, 280 nm vs 335 nm), and free linker-agent conjugate
(LC/MS). ADCs used in the efficacy study described below contain a
representative selection of linker-agent conjugates of the present
invention and have an average drug-to-antibody ratio of
approximately 2.
[1104] Efficacy study: 7-8 week old female nu/nu mice were
implanted with N87 tumor fragments subcutaneously. Treatment
started 19 days after implant when the mean estimated tumor mass
for all groups was 130 mg. ADC groups contained 6 animals per
group; control groups contained 4 animals per group. Animals were
treated with a single dose of 12 mg ADC/kg (0.2 mL/20 g for
vehicle; 12 mg mAb/kg for trastuzumab) on day 0. Animals were
euthanized when the tumor reached a mass of 1,000 mg. FIG. 4A shows
the average tumor burden for each group. A line stops after a first
animal in the corresponding group has been euthanized due to tumor
burden. FIG. 4B shows the average body weight change for each
group. FIG. 4C shows the survival percentage in each group. ADC5 is
based on a linker-agent that contains a relatively long linker as
in 18d, whereas the ADCs based on the other linker-agents contain a
relatively short linker as in 18b. ADC30 contains a different drug
than the other ADCs, but has the same linker as ADC28. ADC5 and
ADC28 only differ in L moiety. The results in FIGS. 4A-4C
demonstrate that conjugates that contain a relatively short linker
have a better efficacy than the corresponding conjugate with a
relatively long linker. Both the nature of the linker and the
nature of the drug were demonstrated to have an effect on efficacy
as well.
Example 16
Cleavage of Quenched Linker-Agent Conjugates by Cathepsin B
[1105] Linker-agent conjugate was dissolved in acetonitrile/water
and 5 equivalents of N-acetylcysteine were added. The reaction
mixture was incubated for 1 h at RT and subsequently freeze-dried
to give the quenched linker-agent conjugate. Quenched linker-agent
conjugate dissolved in DMSO was added to a 5 .mu.g/mL solution of
cathepsin B (>200 U/mg) in sodium acetate buffer pH 5 at
37.degree. C. The final concentration of quenched linker-agent
conjugate was 130 .mu.M. Disappearance of starting material was
monitored over time by LC/MS. FIG. 5 presents the cathepsin B
cleavage data for a representative selection of quenched
linker-agent conjugates of the present invention. LD5, LD12, and
LD13 are based on linker-agents from the prior art and contain
linkers that are relatively long as in 18d, whereas the linkers in
the other quenched linker-agent conjugates are as in 18b. The said
other quenched linker-agent conjugates primarily differ in
cyclization spacer. The results from FIG. 5 demonstrate that all
quenched linker-agent conjugates are cleaved efficiently by
cathepsin B.
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* * * * *