U.S. patent application number 15/679599 was filed with the patent office on 2018-03-29 for drug-conjugates, conjugation methods, and uses thereof.
The applicant listed for this patent is Concortis Biosystems, Corp.. Invention is credited to Yufeng Hong, Zhenwei Miao, Tong Zhu.
Application Number | 20180085471 15/679599 |
Document ID | / |
Family ID | 49584226 |
Filed Date | 2018-03-29 |
United States Patent
Application |
20180085471 |
Kind Code |
A1 |
Miao; Zhenwei ; et
al. |
March 29, 2018 |
DRUG-CONJUGATES, CONJUGATION METHODS, AND USES THEREOF
Abstract
In certain aspects, compounds and uses thereof are provided. In
certain aspects, compound-conjugates and uses thereof are
provided.
Inventors: |
Miao; Zhenwei; (San Diego,
CA) ; Hong; Yufeng; (San Diego, CA) ; Zhu;
Tong; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Concortis Biosystems, Corp. |
San Diego |
CA |
US |
|
|
Family ID: |
49584226 |
Appl. No.: |
15/679599 |
Filed: |
August 17, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14401318 |
Nov 14, 2014 |
9801951 |
|
|
PCT/US2013/041028 |
May 14, 2013 |
|
|
|
15679599 |
|
|
|
|
61652512 |
May 29, 2012 |
|
|
|
61648406 |
May 17, 2012 |
|
|
|
61648532 |
May 17, 2012 |
|
|
|
61647300 |
May 15, 2012 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/6889 20170801;
A61P 31/00 20180101; C07K 5/06008 20130101; A61P 43/00 20180101;
C07F 5/025 20130101; A61P 37/02 20180101; C07K 7/06 20130101; A61K
47/6811 20170801; A61K 47/6855 20170801; C07D 277/593 20130101;
A61P 37/04 20180101; C07K 5/1008 20130101; C07D 207/09 20130101;
C07D 207/08 20130101; C07K 5/06034 20130101; A61K 47/6803 20170801;
A61P 3/00 20180101; A61K 47/6849 20170801; A61K 38/07 20130101;
A61P 35/00 20180101; A61P 3/10 20180101; C07D 401/12 20130101; C07K
5/06052 20130101; C07D 403/12 20130101 |
International
Class: |
C07K 5/06 20060101
C07K005/06; C07F 5/02 20060101 C07F005/02; C07D 403/12 20060101
C07D403/12; C07D 401/12 20060101 C07D401/12; C07D 277/593 20060101
C07D277/593; C07D 207/08 20060101 C07D207/08; A61K 38/07 20060101
A61K038/07; C07D 207/09 20060101 C07D207/09 |
Claims
1. A compound having the structure of Formula I: ##STR00221## or a
pharmaceutically acceptable salt thereof, wherein: A is a tubulin
binding moiety; B is an functional moiety; and R.sup.1-R.sup.8 are
each independently selected from the group consisting of H
(hydrogen), optionally substituted C.sub.1-C.sub.8 alkyl,
optionally substituted C.sub.3-C.sub.8 cycloalkyl, optionally
substituted aryl, and optionally substituted heteroaryl, or
optionally R.sup.1 and R.sup.2 together with the nitrogen to which
they are attached are an optionally substituted 5- to 7-membered
ring, or optionally R.sup.1 and R.sup.3 together with the atoms to
which they are attached are an optionally substituted 5- to
7-membered ring, or optionally R.sup.7 and Re together with the
atoms to which they are attached are an optionally substituted 5-
to 7-membered ring, or optionally R.sup.1 is R.sup.1A or R.sup.1B;
R.sup.1A comprises a targeting moiety; R.sup.1B is
-L.sup.1(CH.sub.2).sub.nR.sup.C, -L.sup.1O(CH.sub.2).sub.nR.sup.C
or --(CH.sub.2).sub.nR; R.sup.C is C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 cycloalkyl, aryl, heteroaryl, or heterocyclyl, each
optionally substituted with one or more R.sup.D, or optionally
R.sup.C comprises a targeting moiety; each R.sup.n is independently
selected from the group consisting of --OH, --N.sub.3, halo, cyano,
nitro, --(CH.sub.2).sub.nNR.sup.ER.sup.F,
--(CH.sub.2).sub.nC(.dbd.O)NR.sup.ER.sup.F,
--O(CH.sub.2).sub.nNR.sup.ER.sup.F,
--O(CH.sub.2).sub.nC(.dbd.O)NR.sup.ER.sup.F,
--O(CH.sub.2).sub.mOC(.dbd.O)NR.sup.ER.sup.F,
--NR.sup.GC(.dbd.O)R.sup.H, --NR.sup.GS(O)R.sup.H,
--O(CH.sub.2).sub.mO(CH.sub.2).sub.mR.sup.J,
--O(CH.sub.2).sub.nC(.dbd.O)R.sup.J, --O(CH.sub.2).sub.nR.sup.J,
optionally substituted C.sub.1-C.sub.8 alkyl, optionally
substituted C.sub.3-C.sub.8 cycloalkyl, optionally substituted
aryl, optionally substituted heteroaryl, optionally substituted
heterocyclyl, and optionally substituted --O(C.sub.1-C.sub.8
alkyl); R.sup.E and R.sup.F are each independently selected from
hydrogen,
-[(L.sup.1).sub.s(C(R.sup.2A).sub.2)(NR.sup.2A).sub.s(C(R.sup.2A).sub.2).-
sub.r]-[L.sup.1(C(R.sup.2A).sub.2)(NR.sup.2A).sub.s(C(R.sup.2A).sub.2).sub-
.r].sub.s-(L.sup.1).sub.s-R.sup.J, -[(L.sup.1
(C(R.sup.A).sub.2)(NR.sup.2A).sub.s(C(R.sup.2A).sub.2).sub.r]-(L.sup.1)[(-
C(R.sup.2A).sub.2).sub.rO(C(R.sup.2A).sub.2).sub.r(L.sup.2).sub.s].sub.s-(-
L.sup.1).sub.s-R.sup.J, optionally substituted C.sub.1-8 alkyl,
optionally substituted C.sub.3-8 cycloalkyl, optionally substituted
aryl, optionally substituted heteroaryl, and optionally substituted
heterocyclyl; each R.sup.G is independently hydrogen, optionally
substituted C.sub.1-C.sub.8 alkyl, optionally substituted
C.sub.3-C.sub.8 cycloalkyl, optionally substituted aryl, optionally
substituted heteroaryl, or optionally substituted heterocyclyl;
each R.sup.H is independently hydrogen, optionally substituted
C.sub.1-C.sub.8 alkyl, optionally substituted C.sub.3-C.sub.8
cycloalkyl, optionally substituted aryl, optionally substituted
heteroaryl, optionally substituted heterocyclyl, or
--NR.sup.ER.sup.F; each R is independently selected from the group
consisting of hydrogen, optionally substituted C.sub.1-C.sub.8
alkyl, optionally substituted --O--(C.sub.1-C.sub.8 alkyl),
optionally substituted C.sub.3-C.sub.8 cycloalkyl, optionally
substituted aryl, optionally substituted heteroaryl, optionally
substituted heterocyclyl, --(CH.sub.2).sub.nOR.sup.2B,
--O(CH.sub.2).sub.nOR.sup.2B, --(CH.sub.2).sub.nNR.sup.2BR.sup.2B,
--C(R.sup.2A).sub.2NR.sup.2BR.sup.2B,
--(CH.sub.2).sub.nC(.dbd.O)OR.sup.2B, and --C(.dbd.O)NHR.sup.2B;
each R.sup.2A is independently selected, wherein R.sup.2A is
selected from the group consisting of hydrogen, halo, --OH,
optionally substituted C.sub.1-C.sub.3 alkyl, optionally
substituted --O--(C.sub.1-C.sub.8 alkyl), optionally substituted
C.sub.3-C.sub.8 cycloalkyl, optionally substituted aryl, optionally
substituted heteroaryl, optionally substituted heterocyclyl,
--(CH.sub.2).sub.nOR.sup.2B, --(CH.sub.2).sub.nNR.sup.2CR.sup.2C,
--C(.dbd.O)OR.sup.2B, and --C(.dbd.O)NR.sup.2CR.sup.2C, or
optionally two geminal R.sup.2A and the carbon to which they are
attached form an optionally substituted three- to six-membered
carbocyclic ring; each R.sup.2B is independently selected from the
group consisting of hydrogen, OH, --(CH.sub.2).sub.nC(.dbd.O)OH,
--C(.dbd.O)(C(R.sup.2D).sub.2).sub.nL.sup.3R.sup.2E, optionally
substituted C.sub.1-C.sub.8 alkyl, optionally substituted
C.sub.3-C.sub.8 cycloalkyl, optionally substituted
--O--(C.sub.1-C.sub.8 alkyl), optionally substituted aryl,
optionally substituted heteroaryl, and optionally substituted
heterocyclyl; each R.sup.2c is independently selected from the
group consisting of hydrogen, --OH, optionally substituted
C.sub.1-C.sub.8 alkyl, optionally substituted C.sub.3-C.sub.8
cycloalkyl, optionally substituted --O--(C.sub.1-C.sub.8 alkyl),
optionally substituted aryl, optionally substituted heteroaryl, and
optionally substituted heterocyclyl, or optionally both R.sup.2c
together with the nitrogen to which they are attached are an
optionally substituted heterocyclyl; each R.sup.2D is independently
selected from the group consisting of hydrogen, optionally
substituted C.sub.1-C.sub.8 alkyl, optionally substituted
C.sub.1-C.sub.8 cycloalkyl, optionally substituted
--O--(C.sub.1-C.sub.8 alkyl), optionally substituted aryl,
optionally substituted heteroaryl, and optionally substituted
heterocyclyl; each R.sub.2E is independently selected from the
group consisting of optionally substituted C.sub.1-C.sub.8 alkyl,
optionally substituted C.sub.3-C.sub.8 cycloalkyl, optionally
substituted aryl, optionally substituted heteroaryl, and optionally
substituted heterocyclyl, and --(CH.sub.2).sub.nC(.dbd.O)OR.sup.2F;
each R.sup.2F is independently selected from the group consisting
hydrogen, optionally substituted C1-C8 alkyl, optionally
substituted C3-C8 cycloalkyl, optionally substituted aryl,
optionally substituted heteroaryl, and optionally substituted
heterocyclyl; each L.sup.1 is independently selected from the group
consisting of --C(.dbd.O)--, --S(.dbd.O)--, --C(.dbd.S)--,
--S(.dbd.O).sub.2--, --C(.dbd.O)O--, --C(.dbd.O)NR.sup.2A--,
--S(.dbd.O)NR.sup.2A--, --S(.dbd.O).sub.2NR.sup.2A--,
--C(.dbd.O)NR.sup.2AC(.dbd.O)--, and
--C(CF.sub.3).sub.2NR.sup.2A--; each L.sup.2 is independently
selected from the group consisting of optionally substituted aryl,
optionally substituted heteroaryl, and optionally substituted
heterocyclyl; each L.sup.3 is independently selected from the group
consisting of --C(.dbd.O)--, --S(.dbd.O)--, --C(.dbd.S)--,
--S(.dbd.O).sub.2--, --C(.dbd.O)O--, --C(.dbd.O)NR.sup.2--,
--S(.dbd.O)NR.sup.2A--, --S(.dbd.O).sub.2NR.sup.2A--,
--C(.dbd.O)NR.sup.2AC(.dbd.O)--, and
--C(CF.sub.3).sub.2NR.sup.2A--; each m independently is 1 or 2;
each n independently is 0, 1, 2, 3, 4, 5, or 6; each r
independently is 0, 1, 2, 3, 4, 5, or 6; each s independently is 0
or 1; each z independently is 1 or 2; R.sup.7 is selected from the
group consisting of H (hydrogen), optionally substituted
C.sub.1-C.sub.8 alkyl, optionally substituted C.sub.3-C.sub.8
cycloalkyl, optionally substituted aryl, and optionally substituted
heterocyclyl; R.sup.8 is selected from the group consisting of H
(hydrogen), --(CH.sub.2).sub.nR.sup.C, optionally substituted
C.sub.1-C.sub.8 alkyl, optionally substituted C.sub.3-C.sub.8
cycloalkyl, optionally substituted aryl, and optionally substituted
heterocyclyl; and R is selected from the group consisting of H
(hydrogen), optionally substituted C.sub.1-C.sub.8 alkyl,
optionally substituted C.sub.3-C.sub.8 cycloalkyl, optionally
substituted aryl, and optionally substituted heteroaryl.
2-60. (canceled)
Description
BACKGROUND
[0001] Cytotoxic agents can provide therapeutic benefits in the
treatment of various conditions, including various cancers.
Accordingly, it is desirable to provide cytotoxic agents with
therapeutically useful properties, for example, as chemotherapies
in cancer treatments.
[0002] Clinical uses of cytotoxic agents as chemotherapies usually
develop drug resistances, and thus, drop their therapeutic
efficacies. Accordingly, it is desirable to provide cytotoxic
agents with improved drug resistance profiles. One possibility is
to design cytotoxic compounds with efflux pump resistances. Another
possibility is to design cytotoxic compounds potentially act on
more than one targets.
[0003] Tubulins are a class of targets for anti-mitotic agents as
chemotherapies alone or in combination with other chemotherapies or
as active agent-conjugates. Examples of Tubulin-Binding Agents
include, but are not limited to, the following compounds:
##STR00001## ##STR00002## ##STR00003##
[0004] Many enzymes, for example, proteasome, MMAP, FAP and uPA,
are considered as cancer therapy targets due to their involvements
in cancer proliferations and metastases. Examples of Proteasome
inhibitors include, but are not limited to, the following
compounds:
##STR00004##
[0005] Example of FAP Inhibitors, not Limited to:
##STR00005##
[0006] Examples of uPA Inhibitors, not Limited to:
##STR00006##
[0007] Examples of MMP Inhibitors Disclosed in, not Limited to:
[0008] Compounds disclosed in Bioorganic & Medicinal Chemistry
15 (2007) 2223-2268.
[0009] Compounds disclosed in Cancer Metastasis Rev (2006) 25:
115-136.
SUMMARY
[0010] Some embodiments provide compounds, methods of preparing
compounds, and uses thereof.
[0011] Some embodiments provide compound-conjugates, methods of
preparing compound-conjugates, and uses thereof.
[0012] Some embodiments provide a compound having the structure of
Formula I:
##STR00007##
[0013] Some embodiments provide a compound having the structure of
Formula
##STR00008##
[0014] or a pharmaceutically acceptable salt thereof,
[0015] wherein
[0016] B is a moiety might have contribution to enzyme inhibition
or efflux pump resistance;
[0017] R.sub.1-R.sub.8 are each independently selected from the
group consisting of II (hydrogen), C.sub.1-C.sub.8 alkyl, and
substituted or cyclic C.sub.1-C.sub.8 alkyl, or optionally R.sub.1
and R.sub.2 together with the nitrogen to which they are attached
are a cyclic 5- to 7-membered ring, or optionally R.sub.1 and
R.sub.3 together with the atoms to which they are attached are a
cyclic 5- to 7-membered ring, or optionally R.sub.7 and R together
with the atoms to which they are attached are a cyclic 5- to
7-membered ring;
[0018] Y is CH.sub.2, S, S.dbd.O, C.dbd.O, CHF, CHCN, CHN.sub.3
CH--OH, CH--ONH.sub.2, or CHOR, where R is C.sub.1-C.sub.8 alkyl or
substituted C.sub.1-C.sub.8 alkyl.
[0019] In some embodiments, B may be CN, CHO, CH.sub.2OH,
CH.sub.2F, CH.sub.2CN, CH.sub.2N.sub.3, COOH, CO--N(R)OR,
CO--N(R)CO--R, CO--CO--NHR, CO--N(R)--SO.sub.2R,
(CH.sub.2).sub.pCOOH, (CH.sub.2).sub.p--CH(OH)--COOH,
CO--(CH.sub.2).sub.p--COOH, CH.dbd.CH--COOH, CO--CH.dbd.CH--COOH,
CH.dbd.CH--CONHOH, CH.dbd.CH--CONH--SO.sub.2R,
CO--CH.dbd.CH--CONHOH, B(OH).sub.2, (CH.sub.2).sub.p--B(OH).sub.2,
PO(OH).sub.2, or (CH.sub.2).sub.p--PO(OH).sub.2, --R--COOH,
--R--CO--N(R)OR, --R--CO--NHR, --R--CO--N(R)--SO.sub.2R, where each
occurrence of R is independently selected from H (hydrogen),
C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, substituted
C.sub.1-C.sub.8 alkyl, substituted C.sub.3-C.sub.8 cycloalkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl, and
NR.sup.ER.sup.F, and p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
[0020] In some embodiments, the compound having the structure of
Formula I has the structure of Formula Ib:
##STR00009##
[0021] or a pharmaceutically acceptable salt thereof,
[0022] B is a moiety might have contribution to enzyme inhibition
or efflux pump resistance;
[0023] R.sub.1-R.sub.8 are each independently selected from the
group consisting of HI (hydrogen), C.sub.1-C.sub.8 alkyl, and
substituted or cyclic C.sub.1-C.sub.8 alkyl, or optionally R.sub.1
and R.sub.2 together with the nitrogen to which they are attached
are a cyclic 5- to 7-membered ring, or optionally R.sub.1 and
R.sub.3 together with the atoms to which they are attached are a
cyclic 5- to 7-membered ring, or optionally R.sub.7 and R.sub.8
together with the atoms to which they are attached are a cyclic 5-
to 7-membered ring;
[0024] Z is --F, --SR, --N.sub.3, --NRR, --ONHR, --OAc, or --OR,
where each R is independently H, C.sub.1-C.sub.8 alkyl or
substituted C.sub.1-C.sub.8 alkyl.
[0025] In some embodiments, B may be CN, CHO, CH.sub.2OH,
CH.sub.2F, CH.sub.2CN, CH.sub.2N.sub.3, COOH, CO--NHOH,
CO--CO--NHR, CO--NH--SO.sub.2R, (CH.sub.2).sub.nCOOH,
(CH.sub.2).sub.n--CH(OH)--COOH, CO--(CH.sub.2).sub.n--COOH.
CH.dbd.CH--COOH, CO--CH.dbd.CH--COOH, CH.dbd.CH--CONHOH,
CO--CH.dbd.CH--CONHOH, B(OH).sub.2, (CH.sub.2).sub.n--B(OH).sub.2,
PO(OH).sub.2, or (CH.sub.2).sub.n--PO(OH).sub.2, where
R=C.sub.1-C.sub.8 alkyl or substituted C.sub.1-C.sub.8 alkyl, and n
is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
[0026] Some embodiments provide a compound having the structure of
Formula IIa:
##STR00010##
[0027] or a pharmaceutically acceptable salt thereof,
[0028] wherein X is OR.sup.10, selected from, but not limited to, a
group consisting of at least one hetero atom:
##STR00011##
[0029] wherein, X is SO.sub.2R.sup.10, selected from, but not
limited to, a group consisting of at least one hetero atom:
##STR00012##
[0030] R.sub.1-R.sub.10 are each independently selected from the
group consisting of H (hydrogen), C.sub.1-C.sub.8 alkyl, and
substituted or cyclic C.sub.1-C.sub.8 alkyl, or optionally R.sub.1
and R.sub.3 together with the atoms to which they are attached are
a cyclic 5- to 7-membered ring, or optionally and respectively
R.sub.7, R.sub.8 and R.sub.9 together with the atoms to which they
are attached are a cyclic 5- to 7-membered ring;
[0031] X is a group consisting of at least one heteroatom;
[0032] Y is CH.sub.2, S, S.dbd.O, C.dbd.O, CHF, CHCN, CHN.sub.3
CH--OH, CH--ONH.sub.2, or CHOR, where R is C.sub.1-C.sub.8 alkyl or
substituted C.sub.1-C.sub.8 alkyl.
[0033] In some embodiments, the dual active compound having the
structure of Formula I has the structure of Formula IIb:
##STR00013##
[0034] or a pharmaceutically acceptable salt thereof,
[0035] wherein, n is 0 or 1; X is OR.sup.10, selected from, but not
limited to, a group consisting of at least one hetero atom:
##STR00014##
[0036] wherein, n is 0 or 1; X is SO.sub.2R.sup.10, selected from,
but not limited to, a group consisting of at least one hetero
atom:
##STR00015##
[0037] R.sub.1-R.sub.10 are each independently selected from the
group consisting of H (hydrogen), C.sub.1-C.sub.8 alkyl, and
substituted or cyclic C.sub.1-C.sub.8 alkyl, or optionally R.sub.1
and R.sub.2 together with the nitrogen to which they are attached
are a cyclic 5- to 7-membered ring, or optionally R.sub.1 and
R.sub.3 together with the atoms to which they are attached are a
cyclic 5- to 7-membered ring, or optionally and respectively
R.sub.7, R.sub.8 and R.sub.9 together with the atoms to which they
are attached are a cyclic 5- to 7-membered ring;
[0038] X is a group of at least one heteroatom;
[0039] Z is --F, --SR, --N.sub.3, --NRR, --ONHR, --OAc, or --OR,
where each R is independently H, C.sub.1-C.sub.8 alkyl or
substituted C.sub.1-C.sub.8 alkyl.
[0040] Also provided herein are the compounds described above
conjugated to a targeting moiety with a linker. Also provided
herein are the compounds described above with a linker.
[0041] Some embodiments provide a compound having the structure of
Formula IV:
##STR00016##
[0042] or a pharmaceutically acceptable salt thereof,
[0043] wherein: [0044] R.sup.1-R.sup.8 are each independently
selected from the group consisting of H hydrogen), optionally
substituted C.sub.1-C.sub.8 alkyl, optionally substituted
C.sub.1-C.sub.8 alkoxy, optionally substituted C.sub.3-C.sub.8
cycloalkyl, optionally substituted aryl, and optionally substituted
heteroaryl, or optionally R.sup.1 and R.sup.2 together with the
nitrogen to which they are attached are an optionally substituted
cyclic 5- to 7-membered ring, or optionally R.sup.1 and R.sup.3
together with the atoms to which they are attached are an
optionally substituted cyclic 5- to 7-membered ring, or optionally
R.sup.7 and R.sup.8 together with the atoms to which they are
attached are an optionally substituted cyclic 5- to 7-membered
ring, or optionally R.sup.1 is R.sup.1A or R.sup.1B; [0045]
R.sup.1A comprises a targeting moiety; [0046] R.sup.1B is
-L.sup.1(CH.sub.2).sub.nR.sup.C, -L.sup.1O(CH.sub.2).sub.nR.sup.C
or --(CH.sub.2).sub.nR.sup.C; [0047] R.sup.C is C.sub.1-C.sub.8
alkyl, C.sub.3-C.sub.8 cycloalkyl, aryl, heteroaryl, and
heterocyclyl, each optionally substituted with one or more R.sup.D,
or optionally R.sup.C comprises a targeting moiety; [0048] each
R.sup.D is independently selected from the group consisting of
--OH, --N.sub.3, halo, cyano, nitro,
--(CH.sub.2).sub.nNR.sup.ER.sup.F,
--(CH.sub.2).sub.nC(.dbd.O)NR.sup.ER.sup.F,
--O(CH.sub.2).sub.nNR.sup.ER.sup.F,
--O(CH.sub.2).sub.nC(.dbd.O)NR.sup.ER.sup.F,
--O(CH.sub.2).sub.mOC(.dbd.O)NR.sup.ER.sup.F,
--NR.sup.GC(.dbd.O)R.sup.H, --NR.sup.GS(O).sub.zR.sup.H,
--O(CH.sub.2).sub.mO(CH.sub.2).sub.mR.sup.J,
--O(CH.sub.2).sub.nC(.dbd.O)R.sup.J, --O(CH.sub.2).sub.nR.sup.J,
optionally substituted C.sub.1-C.sub.8 alkyl, optionally
substituted C.sub.3-C.sub.8 cycloalkyl, optionally substituted
aryl, optionally substituted heteroaryl, optionally substituted
heterocyclyl, and optionally substituted --O--(C.sub.1-C.sub.8
alkyl); [0049] each NR.sup.ER.sup.F is independently selected,
wherein R.sup.E and R.sup.F are each independently selected from
hydrogen,
-[(L.sup.1).sub.s(C(R.sup.2A).sub.2).sub.r(NR.sup.2A).sub.s(C(R.sup.2Ah).-
sub.r]-[L.sup.1(C(R.sup.2A).sub.2).sub.r(NR.sup.2A)(C(R.sup.2A).sub.2).sub-
.r]-(L.sup.1).sub.s-R.sup.J,
-[(L.sup.1).sub.s(C(R.sup.2A).sub.2).sub.r(NR.sup.2A).sub.s(C(R.sup.2A).s-
ub.2).sub.r]-(L.sup.1).sub.s[(C(R.sup.2A).sub.2).sub.rO(C(R.sup.2A).sub.2)-
.sub.r(L.sup.2).sub.s].sub.s-(L.sup.1).sub.s-R.sup.J, optionally
substituted C.sub.1-8 alkyl, optionally substituted C.sub.3-8
cycloalkyl, optionally substituted aryl, optionally substituted
heteroaryl, and optionally substituted heterocyclyl; [0050] each
R.sup.G is independently hydrogen, optionally substituted
C.sub.1-C.sub.8 alkyl, optionally substituted C.sub.3-C.sub.8
cycloalkyl, optionally substituted aryl, optionally substituted
heteroaryl, or optionally substituted heterocyclyl; [0051] each
R.sup.H is independently hydrogen, optionally substituted
C.sub.1-C.sub.8 alkyl, optionally substituted C.sub.3-C.sub.8
cycloalkyl, optionally substituted aryl, optionally substituted
heteroaryl, optionally substituted heterocyclyl, or
--NR.sup.ER.sup.F; [0052] each R.sup.J is independently selected
from the group consisting of hydrogen, optionally substituted
C.sub.1-C.sub.8 alkyl, optionally substituted --O--(C.sub.1-C.sub.8
alkyl), optionally substituted C.sub.3-C.sub.8 cycloalkyl,
optionally substituted aryl, optionally substituted heteroaryl,
optionally substituted heterocyclyl, --(CH.sub.2).sub.mOR.sup.2B,
--O(CH.sub.2).sub.mOR.sup.2B, --(CH.sub.2).sub.nNR.sup.2BR.sup.2B,
--C(R.sup.2A).sub.2NR.sup.2BR.sup.2B,
--(CH.sub.2).sub.nC(.dbd.O)OR.sup.2B, and --C(.dbd.O)NHR.sup.2B;
[0053] each R.sup.2A is independently selected, wherein R.sup.2A is
selected from the group consisting of hydrogen, halo, --OH,
optionally substituted C.sub.1-C.sub.8 alkyl, optionally
substituted --O--(C.sub.1-C.sub.8 alkyl), optionally substituted
C.sub.3-C.sub.8 cycloalkyl, optionally substituted aryl, optionally
substituted heteroaryl, optionally substituted heterocyclyl,
--(CH.sub.2).sub.nOR.sup.2B, --(CH.sub.2).sub.nNR.sup.2CR.sup.2C,
--C(.dbd.O)OR.sup.2B, --C(.dbd.O)NR.sup.2CR.sup.2C, or optionally
two geminal R.sup.2A and the carbon to which they are attached are
together an optionally substituted three- to six-membered
carbocyclic ring; [0054] each R.sup.2B is independently selected
from the group consisting of hydrogen, --OH,
--(CH.sub.2).sub.nC(.dbd.O)OH,
C(.dbd.O)(C(R.sup.2D).sub.2).sub.nL.sup.3R.sup.2E, optionally
substituted C.sub.1-C.sub.8 alkyl, optionally substituted
C.sub.3-C.sub.8 cycloalkyl, optionally substituted
--O--(C.sub.1-C.sub.8 alkyl), optionally substituted aryl,
optionally substituted heteroaryl, and optionally substituted
heterocyclyl; [0055] each NR.sup.2CR.sup.2C is independently
selected, wherein each R.sup.2C is independently selected from the
group consisting of hydrogen, --OH, optionally substituted
C.sub.1-C.sub.8 alkyl, optionally substituted C.sub.3-C.sub.8
cycloalkyl, optionally substituted --O--(C.sub.1-C.sub.8 alkyl),
optionally substituted aryl, optionally substituted heteroaryl, and
optionally substituted heterocyclyl, or optionally both R.sup.2C
together with the nitrogen to which they are attached are an
optionally substituted heterocyclyl; [0056] each R.sup.2D is
independently selected from the group consisting of hydrogen,
optionally substituted C.sub.1-C.sub.8 alkyl, optionally
substituted C.sub.3-C.sub.8 cycloalkyl, optionally substituted
--O--(C.sub.1-C.sub.8 alkyl), optionally substituted aryl,
optionally substituted heteroaryl, and optionally substituted
heterocyclyl; [0057] each R.sup.2E is independently selected from
the group consisting of optionally substituted C.sub.1-C.sub.8
alkyl, optionally substituted C.sub.3-C.sub.8 cycloalkyl,
optionally substituted aryl, optionally substituted heteroaryl, and
optionally substituted heterocyclyl, and
--(CH.sub.2).sub.nC(.dbd.O)OR.sup.2F; [0058] each R.sup.2F is
independently selected from the group consisting hydrogen,
optionally substituted C.sub.1-C.sub.8 alkyl, optionally
substituted C.sub.3-C.sub.8 cycloalkyl, optionally substituted
aryl, optionally substituted heteroaryl, and optionally substituted
heterocyclyl; [0059] each L.sup.1 is independently selected from
the group consisting of --C(.dbd.O)--, --S(.dbd.O)--,
--C(.dbd.S)--, --S(.dbd.O).sub.2--, --C(.dbd.O)O--,
--C(.dbd.O)NR.sup.2A--, --S(.dbd.O)NR.sup.2A--,
--S(.dbd.O).sub.2NR.sup.2A--, --C(.dbd.O)NR.sup.2AC(.dbd.O)--, and
--C(CF.sub.3).sub.2NR.sup.2A--; [0060] each L.sup.2 is
independently selected from the group consisting of optionally
substituted aryl, optionally substituted heteroaryl, and optionally
substituted heterocyclyl; [0061] each L.sup.3 is independently
selected from the group consisting of --C(.dbd.O)--, --S(.dbd.O)--,
--C(.dbd.S)--, --S(.dbd.O).sub.2--, --C(.dbd.O)O--,
--C(.dbd.O)NR.sup.2A--, --S(.dbd.O)NR.sup.2A--,
--S(.dbd.O).sub.2NR.sup.2A--, --C(.dbd.O)NR.sup.2AC(.dbd.O)--, and
--C(CF.sub.3).sub.2NR.sup.2A--; [0062] each m independently is 1 or
2; [0063] each n independently is 0, 1, 2, 3, 4, 5, or 6; [0064]
each r independently is 0, 1, 2, 3, 4, 5, or 6; [0065] each s
independently is 0 or 1; and [0066] each z independently is 1 or 2
[0067] R.sup.7 is selected from the group consisting of H
(hydrogen), optionally substituted C.sub.1-C.sub.8 alkyl,
optionally substituted C.sub.3-C.sub.8 cycloalkyl, optionally
substituted aryl, and optionally substituted heterocyclyl; [0068]
R.sup.8 is selected from the group consisting of H (hydrogen),
--(CH.sub.2).sub.nR.sup.C, optionally substituted C.sub.1-C.sub.8
alkyl, optionally substituted C.sub.3-C.sub.8 cycloalkyl,
optionally substituted aryl, and optionally substituted
heterocyclyl; [0069] E is selected from the group consisting
of:
[0069] ##STR00017## [0070] where Y is CH.sub.2, S, S.dbd.O,
C.dbd.O, CHF, CHCN, CHN.sub.3 CH--OH, CH--ONH.sub.2, or
CHOR.sup.14, where R.sup.14 is optionally substituted
C.sub.1-C.sub.8 alkyl, and Z is --F, --SR.sup.14, --N.sub.3,
--NR.sup.14R.sup.14, --ONHR.sup.14, --OAc, or --OR.sup.14, where
each R.sup.14 is independently H (hydrogen), C.sub.1-C.sub.8 alkyl
substituted C.sub.1-C.sub.8 alkyl, or NR.sup.ER.sup.F;
[0071] R.sup.11-R.sup.13 are each independently selected from the
group consisting of H (hydrogen), optionally substituted
C.sub.1-C.sub.8 alkyl, optionally substituted C.sub.1-C.sub.8
alkoxy, optionally substituted C.sub.3-C.sub.8 cycloalkyl,
optionally substituted aryl, and optionally substituted heteroaryl;
and [0072] J is selected from: CN, CHO, CH.sub.2OH, CH.sub.2F,
CH.sub.2CN, CH.sub.2N.sub.3, COOH, CO--N(R.sup.15)OR.sup.15,
CO--N(R.sup.15)CO--R.sup.15, CO--CO--NHR.sup.15,
CO--N(R.sup.15)--SO.sub.2R.sup.15, (CH.sub.2).sub.pCOOH,
(CH.sub.2).sub.p--CH(OH)--COOH, CO--(CH.sub.2).sub.p--COOH,
CH.dbd.CH--COOH, CO--CH.dbd.CH--COOH, CH.dbd.CH--CONHOH,
CH.dbd.CH--CONH--SO.sub.2R, CO--CH.dbd.CH--CONHOH, B(OH).sub.2,
(CH.sub.2).sub.p--B(OH).sub.2, PO(OH).sub.2, or
(CH.sub.2).sub.p--PO(OH).sub.2, --R.sup.15--COOH,
--CO--N(R.sup.15)OR.sup.15, --CO--N(R.sup.15)OR.sup.15,
--R.sup.15--CO--N(R.sup.15)OR.sup.15, --R.sup.15--CO--NHR.sup.15,
--R.sup.15--CO--N(R.sup.15)--SO.sub.2R.sup.15, where each
occurrence of R.sup.15 is independently selected from H (hydrogen),
C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, substituted
C.sub.1-C.sub.8 alkyl, substituted C.sub.3-C.sub.8 cycloalkyl,
aryl, substituted aryl, heteroaryl, substituted heteroaryl and
NR.sup.ER.sup.F, and p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
BRIEF DESCRIPTION OF THE DRAWINGS
[0073] FIGS. 1-10 shows the cytotoxic effects of compounds or
antibody drug conjugates (ADCs) on various cell types. The antibody
used in the antibody drug conjugates is Trastuzumab.
DETAILED DESCRIPTION
[0074] Some embodiments provide a compound.
[0075] In some embodiments, the compound includes a linker.
[0076] In some embodiments, the compound includes a cytotoxic
agent.
[0077] In some embodiments, the compound includes a functional
group that has tubulin-binding properties or tubulin inhibitory
properties.
[0078] In some embodiments, the compound includes a functional
group that has protease inhibitory or efflux pump resistant
properties. For example, the functional group may have proteasome
inhibitory properties.
Definitions
[0079] As used herein, common organic abbreviations are defined as
follows: [0080] Ac Acetyl [0081] aq. Aqueous [0082] BOC or Boc
tert-Butoxycarbonyl [0083] BrOP bromo tris(dimethylamino)
phosphonium hexafluorophosphate [0084] Bu n-Butyl [0085] .degree.
C. Temperature in degrees Centigrade [0086] DCM methylene chloride
[0087] DEPC Diethylcyanophosphonate [0088] DIC
diisopropylcarbodiimide [0089] DIEA Diisopropylethylamine [0090]
DMF N,N-Dimethylformamide [0091] EDC
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide [0092] Et Ethyl
[0093] EtOAc Ethyl acetate [0094] Eq Equivalents [0095] Fmoc
9-Fluorenylmethoxycarbonyl [0096] g Gram(s) [0097] h Hour (hours)
[0098] HATU 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl
uronium hexafluorophosphate [0099] HOAt
1-Hydroxy-7-azabenzotriazole [0100] HOBT N-Hydroxybenzotriazole
[0101] HOSu N-Hydroxysuccinimide [0102] HPLC High-performance
liquid chromatography [0103] LC/MS Liquid chromatography-mass
spectrometry [0104] Me Methyl [0105] MeOH Methanol [0106] MeCN
Acetonitrile [0107] mL Milliliter(s) [0108] MS mass spectrometry
[0109] RP-HPLC reverse phase HPLC [0110] rt room temperature [0111]
t-Bu tert-Butyl [0112] TEA Triethylamine [0113] Tert, t tertiary
[0114] TFA Trifluoracetic acid [0115] THF Tetrahydrofuran [0116]
THP Tetrahydropyranyl [0117] TLC Thin-layer chromatography [0118]
.mu.L Microliter(s)
[0119] The term "pharmaceutically acceptable salt" refers to salts
that retain the biological effectiveness and properties of a
compound and, which are not biologically or otherwise undesirable
for use in a pharmaceutical. In many cases, the compounds disclosed
herein are capable of forming acid and/or base salts by virtue of
the presence of amino and/or carboxyl groups or groups similar
thereto. Pharmaceutically acceptable acid addition salts can be
formed with inorganic acids and organic acids. Inorganic acids from
which salts can be derived include, for example, hydrochloric acid,
hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and
the like. Organic acids from which salts can be derived include,
for example, acetic acid, propionic acid, glycolic acid, pyruvic
acid, oxalic acid, maleic acid, malonic acid, succinic acid,
fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic
acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,
p-toluenesulfonic acid, salicylic acid, and the like.
Pharmaceutically acceptable base addition salts can be formed with
inorganic and organic bases. Inorganic bases from which salts can
be derived include, for example, sodium, potassium, lithium,
ammonium, calcium, magnesium, iron, zinc, copper, manganese,
aluminum, and the like; particularly preferred are the ammonium,
potassium, sodium, calcium and magnesium salts. Organic bases from
which salts can be derived include, for example, primary,
secondary, and tertiary amines, substituted amines including
naturally occurring substituted amines, cyclic amines, basic ion
exchange resins, and the like, specifically such as isopropylamine,
trimethylamine, diethylamine, triethylaminc, tripropylamine, and
ethanolamine. Many such salts are known in the art, as described in
WO 87/05297, Johnston et al., published Sep. 11, 1987 (incorporated
by reference herein in its entirety).
[0120] As used herein, "C.sub.a to C.sub.b" or "C.sub.a-b" in which
"a" and "b" are integers refer to the number of carbon atoms in the
specified group. That is, the group can contain from "a" to "b",
inclusive, carbon atoms. Thus, for example, a "C.sub.1 to C.sub.4
alkyl" or "C.sub.1-4 alkyl" group refers to all alkyl groups having
from 1 to 4 carbons, that is, CH.sub.3--, CH.sub.3CH.sub.2--,
CH.sub.3CH.sub.2CH.sub.2--, (CH).sub.2CH--,
CH.sub.3CH.sub.2CH.sub.2CH.sub.2--, CH.sub.3CH.sub.2CH(CH.sub.3)--
and (CH.sub.3).sub.3C--.
[0121] The term "halogen" or "halo," as used herein, means any one
of the radio-stable atoms of column 7 of the Periodic Table of the
Elements, e.g., fluorine, chlorine, bromine, or iodine, with
fluorine and chlorine being preferred.
[0122] As used herein, "alkyl" refers to a straight or branched
hydrocarbon chain that is fully saturated (i.e., contains no double
or triple bonds). The alkyl group may have 1 to 20 carbon atoms
(whenever it appears herein, a numerical range such as "1 to 20"
refers to each integer in the given range; e.g., "1 to 20 carbon
atoms" means that the alkyl group may consist of 1 carbon atom, 2
carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon
atoms, although the present definition also covers the occurrence
of the term "alkyl" where no numerical range is designated). The
alkyl group may also be a medium size alkyl having 1 to 9 carbon
atoms. The alkyl group could also be a lower alkyl having 1 to 4
carbon atoms. The alkyl group may be designated as "C.sub.1-4
alkyl" or similar designations. By way of example only, "C.sub.1-4
alkyl" indicates that there are one to four carbon atoms in the
alkyl chain, i.e., the alkyl chain is selected from the group
consisting of methyl, ethyl, propyl, iso-propyl, n-butyl,
iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include,
but are in no way limited to, methyl, ethyl, propyl, isopropyl,
butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like.
[0123] As used herein, "alkoxy" refers to the formula --OR wherein
R is an alkyl as is defined above, such as "C.sub.1-9 alkoxy",
including but not limited to methoxy, ethoxy, n-propoxy,
1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, and
tert-butoxy, and the like.
[0124] As used herein, "alkylthio" refers to the formula --SR
wherein R is an alkyl as is defined above, such as "C.sub.1-9
alkylthio" and the like, including but not limited to
methylmercapto, ethylmercapto, n-propylmercapto,
1-methylethylmercapto (isopropylmercapto), n-butylmercapto,
iso-butylmercapto, sec-butylmercapto, tert-butylmercapto, and the
like.
[0125] As used herein, "alkenyl" refers to a straight or branched
hydrocarbon chain containing one or more double bonds. The alkenyl
group may have 2 to 20 carbon atoms, although the present
definition also covers the occurrence of the term "alkenyl" where
no numerical range is designated. The alkenyl group may also be a
medium size alkenyl having 2 to 9 carbon atoms. The alkenyl group
could also be a lower alkenyl having 2 to 4 carbon atoms. The
alkenyl group may be designated as "C.sub.2-4 alkenyl" or similar
designations. By way of example only, "C.sub.2-4 alkenyl" indicates
that there are two to four carbon atoms in the alkenyl chain, i.e.,
the alkenyl chain is selected from the group consisting of ethenyl,
propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl,
buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl,
1-ethyl-ethen-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl,
buta-1,2,-dienyl, and buta-1,2-dien-4-yl. Typical alkenyl groups
include, but are in no way limited to, ethenyl, propenyl, butenyl,
pentenyl, and hexenyl, and the like.
[0126] As used herein, "alkynyl" refers to a straight or branched
hydrocarbon chain containing one or more triple bonds. The alkynyl
group may have 2 to 20 carbon atoms, although the present
definition also covers the occurrence of the term "alkynyl" where
no numerical range is designated. The alkynyl group may also be a
medium size alkynyl having 2 to 9 carbon atoms. The alkynyl group
could also be a lower alkynyl having 2 to 4 carbon atoms. The
alkynyl group may be designated as "C.sub.2-4 alkynyl" or similar
designations. By way of example only, "C.sub.2-4 alkynyl" indicates
that there are two to four carbon atoms in the alkynyl chain, i.e.,
the alkynyl chain is selected from the group consisting of ethynyl,
propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl, and
2-butynyl. Typical alkynyl groups include, but are in no way
limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl, and
the like.
[0127] The term "aromatic" refers to a ring or ring system having a
conjugated pi electron system and includes both carbocyclic
aromatic (e.g., phenyl) and heterocyclic aromatic groups (e.g.,
pyridine). The term includes monocyclic or fused-ring polycyclic
(i.e., rings which share adjacent pairs of atoms) groups provided
that the entire ring system is aromatic.
[0128] As used herein, "aryloxy" and "arylthio" refers to RO-- and
RS--, in which R is an aryl as is defined above, such as
"C.sub.6-10 aryloxy" or "C.sub.6-10 arylthio" and the like,
including but not limited to phenyloxy.
[0129] An "aralkyl" or "arylalkyl" is an aryl group connected, as a
substituent, via an alkylene group, such as "C.sub.7-14 aralkyl"
and the like, including but not limited to benzyl, 2-phenylethyl,
3-phenylpropyl, and naphthylalkyl. In some cases, the alkylene
group is a lower alkylene group (i.e., a C.sub.1-4 alkylene
group).
[0130] As used herein, "heteroaryl" refers to an aromatic ring or
ring system (i.e., two or more fused rings that share two adjacent
atoms) that contain(s) one or more heteroatoms, that is, an element
other than carbon, including but not limited to, nitrogen, oxygen
and sulfur, in the ring backbone. When the heteroaryl is a ring
system, every ring in the system is aromatic. The heteroaryl group
may have 5-18 ring members (i.e., the number of atoms making up the
ring backbone, including carbon atoms and heteroatoms), although
the present definition also covers the occurrence of the term
"heteroaryl" where no numerical range is designated. In some
embodiments, the heteroaryl group has 5 to 10 ring members or 5 to
7 ring members. The heteroaryl group may be designated as "5-7
membered heteroaryl," "5-10 membered heteroaryl," or similar
designations. Examples of heteroaryl rings include, but are not
limited to, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl,
thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl,
triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl,
pyrazinyl, triazinyl, quinolinyl, isoquinlinyl, benzimidazolyl,
benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and
benzothienyl.
[0131] A "heteroaralkyl" or "heteroarylalkyl" is heteroaryl group
connected, as a substituent, via an alkylene group. Examples
include but are not limited to 2-thienylmethyl, 3-thienylmethyl,
furylmethyl, thienylethyl, pyrrolylalkyl, pyridylalkyl,
isoxazollylalkyl, and imidazolylalkyl. In some cases, the alkylene
group is a lower alkylene group (i.e., a C.sub.1-4 alkylene
group).
[0132] As used herein, "carbocyclyl" means a non-aromatic cyclic
ring or ring system containing only carbon atoms in the ring system
backbone. When the carbocyclyl is a ring system, two or more rings
may be joined together in a fused, bridged or spiro-connected
fashion. Carbocyclyls may have any degree of saturation provided
that at least one ring in a ring system is not aromatic. Thus,
carbocyclyls include cycloalkyls, cycloalkenyls, and cycloalkynyls.
The carbocyclyl group may have 3 to 20 carbon atoms, although the
present definition also covers the occurrence of the term
"carbocyclyl" where no numerical range is designated. The
carbocyclyl group may also be a medium size carbocyclyl having 3 to
10 carbon atoms. The carbocyclyl group could also be a carbocyclyl
having 3 to 6 carbon atoms. The carbocyclyl group may be designated
as "C.sub.3-6 carbocyclyl" or similar designations. Examples of
carbocyclyl rings include, but are not limited to, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl,
2,3-dihydro-indene, bicycle[2.2.2]octanyl, adamantyl, and
spiro[4.4]nonanyl.
[0133] A "(carbocyclyl)alkyl" is a carbocyclyl group connected, as
a substituent, via an alkylene group, such as "C.sub.4-10
(carbocyclyl)alkyl" and the like, including but not limited to,
cyclopropylmethyl, cyclobutylmethyl, cyclopropylethyl,
cyclopropylbutyl, cyclobutylethyl, cyclopropylisopropyl,
cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl,
cyclohexylethyl, cycloheptylmethyl, and the like. In some cases,
the alkylene group is a lower alkylene group.
[0134] As used herein, "cycloalkyl" means a fully saturated
carbocyclyl ring or ring system. Examples include cyclopropyl,
cyclobutyl, cyclopentyl, and cyclohexyl.
[0135] As used herein, "cycloalkenyl" means a carbocyclyl ring or
ring system having at least one double bond, wherein no ring in the
ring system is aromatic. An example is cyclohexenyl.
[0136] As used herein, "heterocyclyl" means a non-aromatic cyclic
ring or ring system containing at least one heteroatom in the ring
backbone. Heterocyclyls may be joined together in a fused, bridged
or spiro-connected fashion. Heterocyclyls may have any degree of
saturation provided that at least one ring in the ring system is
not aromatic. The heteroatom(s) may be present in either a
non-aromatic or aromatic ring in the ring system. The heterocyclyl
group may have 3 to 20 ring members (i.e., the number of atoms
making up the ring backbone, including carbon atoms and
heteroatoms), although the present definition also covers the
occurrence of the term "heterocyclyl" where no numerical range is
designated. The heterocyclyl group may also be a medium size
heterocyclyl having 3 to 10 ring members. The heterocyclyl group
could also be a heterocyclyl having 3 to 6 ring members. The
heterocyclyl group may be designated as "3-6 membered heterocyclyl"
or similar designations. In preferred six membered monocyclic
heterocyclyls, the heteroatom(s) are selected from one up to three
of O, N or S, and in preferred five membered monocyclic
heterocyclyls, the heteroatom(s) are selected from one or two
heteroatoms selected from O, N, or S. Examples of heterocyclyl
rings include, but are not limited to, azepinyl, acridinyl,
carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl,
morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl,
piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl,
pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl,
1,3-dioxinyl, 1,3-dioxanyl, 1,4-dioxinyl, 1,4-dioxanyl,
1,3-oxathianyl, 1,4-oxathiinyl, 1,4-oxathianyl, 2H-1,2-oxazinyl,
trioxanyl, hexahydro-1,3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxolanyl,
1,3-dithiolyl, 1,3-dithiolanyl, isoxazolinyl, isoxazolidinyl,
oxazolinyl, oxazolidinyl, oxazolidinonyl, thiazolinyl,
thiazolidinyl, 1,3-oxathiolanyl, indolinyl, isoindolinyl,
tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl,
tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl, thiamorpholinyl,
dihydrobenzofuranyl, benzimidazolidinyl, and
tetrahydroquinoline.
[0137] A "(heterocyclyl)alkyl" is a heterocyclyl group connected,
as a substituent, via an alkylene group. Examples include, but are
not limited to, imidazolinylmethyl and indolinylethyl.
[0138] As used herein, "acyl" refers to --C(.dbd.O)R, wherein R is
hydrogen, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl,
C.sub.3-7 carbocyclyl, C.sub.6-10 aryl, 5-10 membered heteroaryl,
and 5-10 membered heterocyclyl, as defined herein. Non-limiting
examples include formyl, acetyl, propanoyl, benzoyl, and acryl.
[0139] An "O-carboxy" group refers to a "--OC(.dbd.O)R" group in
which R is selected from hydrogen, C.sub.1-6 alkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.3-7 carbocyclyl, C.sub.6-10 aryl,
5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as
defined herein.
[0140] A "C-carboxy" group refers to a "--C(.dbd.O)OR" group in
which R is selected from hydrogen, C.sub.1-6 alkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.3-7 carbocyclyl, C.sub.6-10 aryl,
5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as
defined herein. A non-limiting example includes carboxyl (i.e.,
--C(.dbd.O)OH).
[0141] A "cyano" group refers to a "--CN" group.
[0142] A "cyanato" group refers to an "--OCN" group.
[0143] An "isocyanato" group refers to a "--NCO" group.
[0144] A "thiocyanato" group refers to a "--SCN" group.
[0145] An "isothiocyanato" group refers to an "--NCS" group.
[0146] A "sulfinyl" group refers to an "--S(.dbd.O)R" group in
which R is selected from hydrogen, C.sub.1-6 alkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.3-7 carbocyclyl, C.sub.6-10 aryl,
5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as
defined herein.
[0147] A "sulfonyl" group refers to an "--SO.sub.2R" group in which
R is selected from hydrogen, C.sub.1-6 alklyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, C.sub.3-7 carbocyclyl, C.sub.6-10 aryl, 5-10
membered heteroaryl, and 5-10 membered heterocyclyl, as defined
herein.
[0148] An "S-sulfonamido" group refers to a
"--SO.sub.2NR.sub.AR.sub.B" group in which R.sub.A and R.sub.B are
each independently selected from hydrogen, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-7 carbocyclyl,
C.sub.6-10 aryl, 5-10 membered heteroaryl, and 5-10 membered
heterocyclyl, as defined herein.
[0149] An "N-sulfonamido" group refers to a
"--N(R.sub.A)SO.sub.2R.sub.B" group in which R.sub.A and R.sub.b
are each independently selected from hydrogen, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-7 carbocyclyl,
C.sub.6-10 aryl, 5-10 membered heteroaryl, and 5-10 membered
heterocyclyl, as defined herein.
[0150] An "O-carbamyl" group refers to a
"--OC(.dbd.O)NR.sub.AR.sub.B" group in which R.sub.A and R.sub.B
are each independently selected from hydrogen, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-7 carbocyclyl,
C.sub.6-10 aryl, 5-10 membered heteroaryl, and 5-10 membered
heterocyclyl, as defined herein.
[0151] An "N-carbamyl" group refers to an
"--N(R.sub.A)C(.dbd.O)OR.sub.B" group in which R.sub.A and R.sub.B
are each independently selected from hydrogen, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-7 carbocyclyl,
C.sub.6-10 aryl, 5-10 membered heteroaryl, and 5-10 membered
heterocyclyl, as defined herein.
[0152] An "O-thiocarbamyl" group refers to a
"--OC(.dbd.S)NR.sub.AR.sub.B" group in which R.sub.A and R.sub.B
are each independently selected from hydrogen, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-7 carbocyclyl.
C.sub.6-10 aryl, 5-10 membered heteroaryl, and 5-10 membered
heterocyclyl, as defined herein.
[0153] A "urea" group refers to a
"--N(R.sub.A)C(.dbd.O)NR.sub.AR.sub.B" group in which R.sub.A and
R.sub.B are each independently selected from hydrogen, C.sub.1-6
alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-7 carbocyclyl,
C.sub.6-10 aryl, 5-10 membered heteroaryl, and 5-10 membered
heterocyclyl, as defined herein.
[0154] An "N-thiocarbamyl" group refers to an
"--N(R.sub.A)C(.dbd.S)OR.sub.B" group in which R.sub.A and R.sub.B
are each independently selected from hydrogen, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-7 carbocyclyl,
C.sub.6-10 aryl, 5-10 membered heteroaryl, and 5-10 membered
heterocyclyl, as defined herein.
[0155] A "C-amido" group refers to a "--C(.dbd.O)NR.sub.AR.sub.B"
group in which R.sub.A and R.sub.B are each independently selected
from hydrogen, C.sub.1-6 alkyl, C.sub.2-4 alkenyl, C.sub.2-4
alkynyl, C.sub.3-7 carbocyclyl, C.sub.6-10 aryl, 5-10 membered
heteroaryl, and 5-10 membered heterocyclyl, as defined herein.
[0156] An "N-amido" group refers to a
"--N(R.sub.A)C(.dbd.O)R.sub.B" group in which R.sub.A and R.sub.B
are each independently selected from hydrogen, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-7 carbocyclyl,
C.sub.6-10 aryl, 5-10 membered heteroaryl, and 5-10 membered
heterocyclyl, as defined herein.
[0157] An "amino" group refers to a "--NR.sub.AR.sub.B" group in
which R.sub.A and R.sub.B are each independently selected from
hydrogen, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl,
C.sub.3-7 carbocyclyl, C.sub.6-10 aryl, 5-10 membered heteroaryl,
and 5-10 membered heterocyclyl, as defined herein. A non-limiting
example includes free amino (i.e., --NH.sub.2).
[0158] An "aminoalkyl" group refers to an amino group connected via
an alkylene group.
[0159] An "alkoxyalkyl" group refers to an alkoxy group connected
via an alkylene group, such as a "C.sub.2-8 alkoxyalkyl" and the
like.
[0160] As used herein, a substituted group is derived from the
unsubstituted parent group in which there has been an exchange of
one or more hydrogen atoms for another atom or group. Unless
otherwise indicated, when a group is deemed to be "substituted," it
is meant that the group is substituted with one or more
substitutents independently selected from C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.7
carbocyclyl (optionally substituted with halo, C.sub.1-C.sub.6
alkyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 haloalkyl, and
C.sub.1-C.sub.6 haloalkoxy),
C.sub.3-C.sub.7-carbocyclyl-C.sub.1-C.sub.6-alkyl (optionally
substituted with halo, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkoxy, C.sub.1-C.sub.6 haloalkyl, and C.sub.1-C.sub.6 haloalkoxy),
5-10 membered heterocyclyl (optionally substituted with halo,
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6
haloalkyl, and C.sub.1-C.sub.6 haloalkoxy), 5-10 membered
heterocyclyl-C.sub.1-C.sub.6-alkyl (optionally substituted with
halo, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy,
C.sub.1-C.sub.6 haloalkyl, and C.sub.1-C.sub.6 haloalkoxy), aryl
(optionally substituted with halo, C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 haloalkyl, and
C.sub.1-C.sub.6 haloalkoxy), aryl(C.sub.1-C.sub.6)alkyl (optionally
substituted with halo, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkoxy, C.sub.1-C.sub.6 haloalkyl, and C.sub.1-C.sub.6 haloalkoxy),
5-10 membered heteroaryl (optionally substituted with halo,
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6
haloalkyl, and C.sub.1-C.sub.6 haloalkoxy), 5-10 membered
heteroaryl(C.sub.1-C.sub.6)alkyl (optionally substituted with halo,
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6
haloalkyl, and C.sub.1-C.sub.6 haloalkoxy), halo, cyano, hydroxy,
C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6
alkoxy(C.sub.1-C.sub.6)alkyl (i.e., ether), aryloxy, sulfhydryl
(mercapto), halo(C.sub.1-C.sub.6)alkyl (e.g., --CF.sub.3),
halo(C.sub.1-C.sub.6)alkoxy (e.g., --OCF.sub.3), C.sub.1-C.sub.6
alkylthio, arylthio, amino, amino(C.sub.1-C.sub.6)alkyl, nitro,
O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,
N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, acyl,
cyanato, isocyanato, thiocyanato, isothiocyanato, sulfinyl,
sulfonyl, and oxo (.dbd.O). Wherever a group is described as
"optionally substituted" that group can be substituted with the
above substituents.
[0161] It is to be understood that certain radical naming
conventions can include either a mono-radical or a di-radical,
depending on the context. For example, where a substituent requires
two points of attachment to the rest of the molecule, it is
understood that the substituent is a di-radical. For example, a
substituent identified as alkyl that requires two points of
attachment includes di-radicals such as --CH.sub.2--,
--CH.sub.2CH.sub.2--, --CH.sub.2CH(CH.sub.3)CH.sub.2--, and the
like. Other radical naming conventions clearly indicate that the
radical is a di-radical such as "alkylene" or "alkenylene."
[0162] When two R groups are said to form a ring (e.g., a
carbocyclyl, heterocyclyl, aryl, or heteroaryl ring) "together with
the atom to which they are attached," it is meant that the
collective unit of the atom and the two R groups are the recited
ring. The ring is not otherwise limited by the definition of each R
group when taken individually. For example, when the following
substructure is present:
##STR00018##
[0163] and R.sup.1 and R.sup.2 are defined as selected from the
group consisting of hydrogen and alkyl, or R.sup.1 and R.sup.2
together with the nitrogen to which they are attached form a
heterocyclyl, it is meant that R.sup.1 and R.sup.2 can be selected
from hydrogen or alkyl, or alternatively, the substructure has
structure:
##STR00019##
[0164] where ring E is a heteroaryl ring containing the depicted
nitrogen.
[0165] Similarly, when two "adjacent" R groups are said to form a
ring "together with the atom to which they are attached," it is
meant that the collective unit of the atoms, intervening bonds, and
the two R groups are the recited ring. For example, when the
following substructure is present:
##STR00020##
[0166] and R.sup.1 and R.sup.2 are defined as selected from the
group consisting of hydrogen and alkyl, or R.sup.1 and R.sup.2
together with the atoms to which they are attached form an aryl or
carbocylyl, it is meant that R.sup.1 and R.sup.2 can be selected
from hydrogen or alkyl, or alternatively, the substructure has
structure:
##STR00021##
[0167] where E is an aryl ring or a carbocylyl containing the
depicted double bond.
[0168] Wherever a substituent is depicted as a di-radical (i.e.,
has two points of attachment to the rest of the molecule), it is to
be understood that the substituent can be attached in any
directional configuration unless otherwise indicated. Thus, for
example, a substituent depicted as -AE- or
##STR00022##
includes the substituent being oriented such that the A is attached
at the leftmost attachment point of the molecule as well as the
case in which A is attached at the rightmost attachment point of
the molecule.
[0169] "Subject" as used herein, means a human or a non-human
mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig,
a goat, a non-human primate or a bird, e.g., a chicken, as well as
any other vertebrate or invertebrate.
Compounds
[0170] The skilled artisan will recognize that some structures
described herein may be resonance forms or tautomers of compounds
that may be fairly represented by other chemical structures, even
when kinetically; the artisan recognizes that such structures may
only represent a very small portion of a sample of such
compound(s). Such compounds are considered within the scope of the
structures depicted, though such resonance forms or tautomers are
not represented herein.
[0171] Isotopes may be present in the compounds described. Each
chemical element present in a compound either specifically or
generically described herein may include any isotope of said
element. For example, in a compound specifically or generically
described herein a hydrogen atom may be explicitly disclosed or
understood to be present in the compound and each such hydrogen
atom is any isotope of hydrogen, including but not limited to
hydrogen-1 (protium) and hydrogen-2 (deuterium). Thus, reference
herein to a compound encompasses all potential isotopic forms
unless the context clearly dictates otherwise.
Utilities and Applications
[0172] Some embodiments provide a method of treating a patient in
need thereof comprising administering a compound as disclosed and
described herein to said patient. In some embodiments, the patient
may have cancer, an infection, or an immune system disease. In some
embodiments, the compound may have anti-tumor, antibiotic, or
anti-inflammatory activity.
Structures
[0173] Some embodiments provide a compound having the structure
##STR00023##
[0174] or a pharmaceutically acceptable salt thereof, wherein: A is
a tubulin binding moiety; B is a group might have protease
inhibitory or efflux pump resistant properties; and R.sub.1-R.sub.8
are each independently selected from the group consisting of H
(hydrogen), C.sub.1-C.sub.8 alkyl, substituted or cyclic
C.sub.1-C.sub.8 alkyl, aryl, and substituted aryl, or optionally
R.sub.1 and R.sub.2 together with the nitrogen to which they are
attached are a cyclic 5- to 7-membered ring, or optionally R.sub.1
and R, together with the atoms to which they are attached are a
cyclic 5- to 7-membered ring, or optionally R.sub.7 and R.sub.8
together with the atoms to which they are attached are a cyclic 5-
to 7-membered ring. In some embodiments, the compound may be used
alone as API (active pharmaceutical ingredient) or in a prodrug
form. In some embodiments, the compound may be included in a
conjugate including a linker and another component. In some
embodiments, the compound may be included in a conjugate including
a linker and a targeting moiety, such as antibody, Fab, peptide,
protein ligand, and the like. In some embodiments, the compound may
be included in a conjugate including a linker and a carrier
molecule, such as HSA, lipid, polymers, nanoparticles, and the
like. In some embodiments, the compound may be included in a
conjugate including a linker and a small molecule drug/ligand, such
as folic acid.
[0175] As used herein, the term "tubulin binding moiety" refers to
a structural component of a compound that inhibits tubulin
polymerization under a certain set of conditions. In some
embodiments, the compound may inhibit tubulin polymerization under
in vivo or in vitro conditions. For example, the compound may
inhibit tubulin polymerization in PBS. Examples of compounds that
inhibit tubulin polymerization are described in Peltier, et al.,
"The Total Synthesis of Tubulysin D," J. Am. Chem. Soc., 2006, 128
(50): 16018-16019, and U.S. Publication No.: 2005/0239713 the
disclosures of which are incorporated herein by reference in their
entirety.
[0176] As used herein, the term "functional moiety" refers to a
structural component of a compound that interacts with a biological
moiety or fragment of the biological moiety under a certain set of
conditions. In some embodiments, the functional moiety may interact
with a biological moiety or fragment of the biological moiety under
in vivo or in vitro conditions. For example, the functional moiety
may interact with a biological moiety or fragment of the biological
moiety in PBS. In some embodiments, the functional moiety may
afford a desirable effect in a compound comparison to a compound
that does not include the functional moiety. In some embodiments,
the functional moiety may contribute to enzyme inhibition or efflux
pump resistance in a compound.
[0177] In some embodiments, the cytotoxic compound having the
structure of Formula Ia:
##STR00024##
[0178] or a pharmaceutically acceptable salt thereof,
[0179] wherein,
[0180] B is a moiety might have contribution to enzyme inhibition
or efflux pump resistance;
[0181] R.sub.1-R.sub.8 are each independently selected from the
group consisting of H (hydrogen), C.sub.1-C.sub.8 alkyl, and
substituted or cyclic C.sub.1-C.sub.8 alkyl, or optionally R.sub.1
and R.sub.2 together with the nitrogen to which they are attached
are a cyclic 5- to 7-membered ring, or optionally R.sub.1 and
R.sub.3 together with the atoms to which they are attached are a
cyclic 5- to 7-membered ring, or optionally R.sub.7 and R.sub.8
together with the atoms to which they are attached are a cyclic 5-
to 7-membered ring;
[0182] Y is CH.sub.2, S, S.dbd.O, C.dbd.O, CHF, CHCN, CHN.sub.3
CH--OH, CH--ONH.sub.2, or CHOR, where R is C.sub.1-C.sub.8 alkyl or
substituted C.sub.1-C.sub.8 alkyl.
[0183] In some embodiments, B may be CN, CHO, CH.sub.2OH,
CH.sub.2F, CH.sub.2CN, CH.sub.2N.sub.3, COOH, CO--NHOH,
CO--CO--NHR, CO--NH--SO.sub.2R, (CH.sub.2).sub.nCOOH,
(CH.sub.2).sub.n--CH(OH)--COOH, CO--(CH.sub.2).sub.n--COOH,
CH.dbd.CH--COOH, CO--CH.dbd.CH--COOH, CH.dbd.CH--CONHOH,
CO--CH.dbd.CH--CONHOH, B(OH).sub.2, (CH.sub.2).sub.n--B(OH).sub.2,
PO(OH).sub.2, or (CH.sub.2).sub.n--PO(OH).sub.2, where R is
C.sub.1-C.sub.8 alkyl or substituted C.sub.1-C.sub.8 alkyl, and n
is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
[0184] In some embodiments, the dual active compound having the
structure of Formula I has the structure of Formula Ib:
##STR00025##
[0185] or a pharmaceutically acceptable salt thereof.
[0186] B is a moiety might have contribution to enzyme inhibition
or efflux pump resistance;
[0187] R.sub.1-R.sub.8 are each independently selected from the
group consisting of H (hydrogen), C.sub.1-C.sub.8 alkyl, and
substituted or cyclic C.sub.1-C.sub.8 alkyl, or optionally R.sub.1
and R.sub.2 together with the nitrogen to which they are attached
are a cyclic 5- to 7-membered ring, or optionally R.sub.1 and
R.sub.3 together with the atoms to which they are attached are a
cyclic 5- to 7-membered ring, or optionally R.sub.7 and R.sub.8
together with the atoms to which they are attached are a cyclic 5-
to 7-membered ring;
[0188] Z is --F, --SR, --N.sub.3, --NRR, --ONHR, --OAc, or --OR,
where each R is independently H, C.sub.1-C.sub.8 alkyl or
substituted C.sub.1-C.sub.8 alkyl
[0189] In some embodiments, B may be CN, CHO, CH.sub.2OH,
CH.sub.2F, CH.sub.2CN, CH.sub.2N.sub.3, COOH, CO--NHOH,
CO--CO--NHR, CO--NH--SO.sub.2R, (CH.sub.2).sub.nCOOH,
(CH.sub.2).sub.n--CH(OH)--COOH, CO--(CH.sub.2).sub.n--COOH,
CH.dbd.CH--COOH, CO--CH.dbd.CH--COOH, CH.dbd.CH--CONHOH,
CO--CH.dbd.CH--CONHOH, B(OH).sub.2, (CH.sub.2).sub.n--B(OH).sub.2,
PO(OH).sub.2, or (CH.sub.2).sub.n--PO(OH).sub.2, where
R=C.sub.1-C.sub.8 alkyl or substituted C.sub.1-C.sub.8 alkyl, and n
is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
[0190] Some embodiments provide a compound having the structure of
Formula ha:
##STR00026##
[0191] or a pharmaceutically acceptable salt thereof,
[0192] wherein X is OR.sup.10, selected from, but not limited to, a
group consisting of at least one hetero atom:
##STR00027##
[0193] wherein, X is SO.sub.2R, selected from, but not limited to,
a group consisting of at least one hetero atom:
##STR00028##
[0194] R.sub.1-R.sub.10 are each independently selected from the
group consisting of H (hydrogen), C.sub.1-C.sub.8 alkyl, and
substituted or cyclic C.sub.1-C.sub.8 alkyl, or optionally R.sub.1
and R.sub.3 together with the atoms to which they are attached are
a cyclic 5- to 7-membered ring, or optionally and respectively
R.sub.7, R.sub.8 and R.sub.9 together with the atoms to which they
are attached are a cyclic 5- to 7-membered ring;
[0195] X is a group consisting of at least one heteroatom;
[0196] Y is CH.sub.2, S, S.dbd.O, C.dbd.O, CHF, CHCN, CHN.sub.3
CH--OH, CH--ONH.sub.2, or CHOR, where R is C.sub.1-C.sub.8 alkyl or
substituted C.sub.1-C.sub.8 alkyl.
[0197] In some embodiments, the dual active compound having the
structure of Formula I has the structure of Formula IIb:
##STR00029##
[0198] or a pharmaceutically acceptable salt thereof,
[0199] wherein, n is 0 or 1; X is OR.sup.10, selected from, but not
limited to, a group consisting of at least one hetero atom:
##STR00030##
[0200] wherein, n is 0 or 1; X is SO.sub.2R.sup.10, selected from,
but not limited to, a group consisting of at least one hetero
atom:
##STR00031##
[0201] R.sub.1-R.sub.10 are each independently selected from the
group consisting of H (hydrogen), C.sub.1-C.sub.8 alkyl, and
substituted or cyclic C.sub.1-C.sub.8 alkyl, or optionally R.sub.1
and R.sub.2 together with the nitrogen to which they are attached
are a cyclic 5- to 7-membered ring, or optionally R.sub.1 and
R.sub.3 together with the atoms to which they are attached are a
cyclic 5- to 7-membered ring, or optionally and respectively
R.sub.7, R.sub.8 and R.sub.9 together with the atoms to which they
are attached are a cyclic 5- to 7-membered ring;
[0202] X is a group of at least one heteroatom;
[0203] Z is --F, --SR, --N.sub.3, --NRR, --ONHR, --OAc, or --OR,
where each R is independently H, C.sub.1-C.sub.8 alkyl or
substituted C.sub.1-C.sub.8 alkyl
[0204] Examples of compounds having the structure of Formula Ia
include but not limited to the following:
##STR00032##
[0205] Examples of compounds having the structure of Formula Ia
include the following:
##STR00033## ##STR00034##
[0206] Examples of general compounds having the structure of
Formula Ia include the following:
##STR00035##
[0207] Examples of compounds having the structure of Formula Ia
include the following:
##STR00036## ##STR00037##
[0208] Examples of general compounds having the structure of
Formula Ia include the following:
##STR00038##
[0209] Examples of general compounds having the structure of
Formula Ia include the following:
##STR00039## ##STR00040## ##STR00041##
[0210] Examples of general compounds having the structure of
Formula Ia include the following:
##STR00042##
[0211] Examples of compounds having the structure of Formula Ia
include the following:
##STR00043## ##STR00044##
[0212] Examples of general compounds having the structure of
Formula Ia include the following:
##STR00045##
[0213] Examples of compounds having the structure of Formula Ia
include the following:
##STR00046## ##STR00047##
[0214] Examples of general compounds having the structure of
Formula Ia include the following:
##STR00048##
[0215] Examples of compounds having the structure of Formula Ia
include the following:
##STR00049## ##STR00050##
[0216] Examples of general compounds having the structure of
Formula Ia include the following:
##STR00051##
[0217] Examples of compounds having the structure of Formula Ia
include the following:
##STR00052## ##STR00053##
[0218] Examples of compounds having the structure of Formula Ib
include the following:
##STR00054## ##STR00055##
[0219] Examples of compounds having the structure of Formula Ib
include the following:
##STR00056## ##STR00057## ##STR00058##
[0220] In some embodiments, the compound is conjugated to a
targeting moiety.
[0221] In some embodiments, the targeting moiety includes a
monoclonal antibody (mAB). In some embodiments, the compound
includes a spacer or a multifunctional linker.
[0222] In some embodiments, the spacer connects to the mAB by a
group including a N (nitrogen) atom. In some embodiments, the
multifunctional linker connects to the mAB by a group including a N
(nitrogen) atom. In some embodiments, the spacer or multifunctional
linker may be optionally connected to an auxiliary moiety. In some
embodiments, the auxiliary moiety may be a second targeting moiety
such as mAB and peptide. In some embodiments, the auxiliary moiety
may be a hydrophilic polymer such as polyethylene glycol (PEG), and
the like. In some embodiments, the spacer or multifunctional linker
may include a group including a N (nitrogen) atom. In some
embodiments, the spacer or multifunctional linker may include a
cyclic group including a N (nitrogen) atom.
[0223] In some embodiments, the spacer connects to the mAB by a
sulfide bond. In some embodiments, the multifunctional linker
connects to the mAB by a sulfide bond. In some embodiments, the
spacer or multifunctional linker may be optionally connected to an
auxiliary moiety. In some embodiments, the auxiliary moiety may be
a second targeting moiety such as mAB and peptide. In some
embodiments, the auxiliary moiety may be a hydrophilic polymer such
as polyethylene glycol (PEG), and the like. In some embodiments,
the spacer or multifunctional linker may include a 2- to 5-atom
bridge. In some embodiments, the spacer or multifunctional linker
may include a 4C bridge.
Conjugation Methods, Spacers and Linkers Involved
[0224] Some embodiments provide a method of conjugating of a
targeting moiety through a spacer or a multifunctional linker.
[0225] In some embodiments, the spacer or multifunctional linker
may include a 2- to 5-atom bridge. In some embodiments, the method
includes a single-step or sequential conjugation approach. In some
embodiments, the compound includes a spacer or a multifunctional
linker. In some embodiments, the spacer or multifunctional linker
may include a noncleavable or cleavable unit such as peptides.
[0226] In some embodiments, the spacer or multifunctional linker
may include a group including a N (nitrogen) atom. In some
embodiments, the method includes a single-step or sequential
conjugation approach. In some embodiments, the spacer or
multifunctional linker may include a noncleavable or cleavable unit
such as a peptide.
[0227] Some embodiments provide a compound-conjugate having the
structure of Formula IIa:
##STR00059##
or a pharmaceutically acceptable salt thereof, wherein: [0228]
R.sup.1-R.sup.9 are each independently selected from the group
consisting of H (hydrogen), optionally substituted C.sub.1-C.sub.8
alkyl, optionally substituted C.sub.3-C.sub.8 cycloalkyl,
optionally substituted aryl, and optionally substituted heteroaryl,
or optionally R.sup.1 and R.sup.2 together with the nitrogen to
which they are attached are an optionally substituted cyclic 5- to
7-membered ring, or optionally R.sup.1 and R.sup.3 together with
the atoms to which they are attached are an optionally substituted
cyclic 5- to 7-membered ring, or optionally R.sup.7, R.sup.8 and
R.sup.9 together with the atoms to which they are attached are an
optionally substituted cyclic 5- to 7-membered ring, or optionally
R.sup.1 is R.sup.1A or R.sup.1B; [0229] R.sup.1A comprises a
targeting moiety; [0230] R.sup.1B is
-L.sup.1(CH.sub.2).sub.nR.sup.C, -L.sup.1O(CH.sub.2).sub.nR.sup.C
or --(CH.sub.2).sub.nR.sup.C; [0231] R.sup.C is C.sub.1-C.sub.8
alkyl, C.sub.3-C.sub.8 cycloalkyl, aryl, heteroaryl, and
heterocyclyl, each optionally substituted with one or more R.sup.D,
or optionally R.sup.C comprises a targeting moiety; [0232] each
R.sup.D is independently selected from the group consisting of
--OH, --N.sub.3, halo, cyano, nitro,
--(CH.sub.2).sub.nNR.sup.ER.sup.F,
--(CH.sub.2).sub.nC(.dbd.O)NR.sup.ER.sup.F,
--O(CH.sub.2).sub.nNR.sup.ER.sup.F,
--O(CH.sub.2).sub.nC(.dbd.O)NR.sup.ER.sup.F,
--O(CH.sub.2).sub.mOC(.dbd.O)NR.sup.ER.sup.F,
--NR.sup.GC(.dbd.O)R.sup.H, --NR.sup.GS(O).sub.zR.sup.H,
--O(CH.sub.2).sub.mO(CH.sub.2).sub.mR.sup.J,
--O(CH.sub.2).sub.nC(.dbd.O)R.sup.J, --O(CH.sub.2).sub.mR.sup.J,
optionally substituted C.sub.1-C.sub.8 alkyl, optionally
substituted C.sub.3-C.sub.8 cycloalkyl, optionally substituted
aryl, optionally substituted heteroaryl, optionally substituted
heterocyclyl, and optionally substituted --O--(C.sub.1-C.sub.8
alkyl); [0233] each NR.sup.ER.sup.F is independently selected,
wherein R.sup.E and R.sup.F are each independently selected from
hydrogen,
-[(L.sup.1).sub.s(C(R.sup.2A).sub.2).sub.r(NR.sup.2A).sub.s(C(R.sup.2A).s-
ub.2).sub.r]-[L.sup.1(C(R.sup.2A).sub.2).sub.r(NR.sup.2A).sub.s(C(R.sup.2A-
).sub.2).sub.r]-(L.sup.1).sub.s-R.sup.J,
-[(L.sup.1).sub.s(C(R.sup.2A).sub.2).sub.r(NR.sup.2A).sub.s(C(R.sup.2A).s-
ub.2).sub.r]-(L.sup.1).sub.s[(C(R.sup.2A).sub.2).sub.rO(C(R.sup.2A).sub.2)-
.sub.r(L.sup.2).sub.s].sub.s-(L.sup.1).sub.s-R.sup.J, optionally
substituted C.sub.1-8 alkyl, optionally substituted C.sub.3-8
cycloalkyl, optionally substituted aryl, optionally substituted
heteroaryl, and optionally substituted heterocyclyl; [0234] each
R.sup.G is independently hydrogen, optionally substituted
C.sub.1-C.sub.8 alkyl, optionally substituted C.sub.3-C.sub.8
cycloalkyl, optionally substituted aryl, optionally substituted
heteroaryl, or optionally substituted heterocyclyl; [0235] each
R.sup.H is independently hydrogen, optionally substituted
C.sub.1-C.sub.8 alkyl, optionally substituted C.sub.3-C.sub.8
cycloalkyl, optionally substituted aryl, optionally substituted
heteroaryl, optionally substituted heterocyclyl, or
--NR.sup.ER.sup.F; [0236] each R.sup.J is independently selected
from the group consisting of hydrogen, optionally substituted
C.sub.1-C.sub.8 alkyl, optionally substituted --O--(C.sub.1-C.sub.8
alkyl), optionally substituted C.sub.3-C.sub.8 cycloalkyl,
optionally substituted aryl, optionally substituted heteroaryl,
optionally substituted heterocyclyl, --(CH.sub.2).sub.mOR.sup.2B,
--O(CH.sub.2).sub.mOR.sup.2B, --(CH.sub.2)NR.sup.2BR.sup.2B,
--C(R.sup.2A).sub.2NR.sup.2BR.sup.2B,
--(CH.sub.2)C(.dbd.O)OR.sup.2B, and --C(.dbd.O)NHR.sup.2B; [0237]
each R.sup.2A is independently selected, wherein R.sup.2A is
selected from the group consisting of hydrogen, halo, --OH,
optionally substituted C.sub.1-C.sub.8 alkyl, optionally
substituted --O--(C.sub.1-C.sub.8 alkyl), optionally substituted
C.sub.3-C.sub.8 cycloalkyl, optionally substituted aryl, optionally
substituted heteroaryl, optionally substituted heterocyclyl,
--(CH.sub.2).sub.mOR.sup.2B, --(CH.sub.2)NR.sup.2CR.sup.2C,
--C(O)OR.sup.2B, --C(.dbd.O)NR.sup.2CR.sup.2C, or optionally two
geminal R.sup.2A and the carbon to which they are attached are
together an optionally substituted three- to six-membered
carbocyclic ring; [0238] each R.sup.2B is independently selected
from the group consisting of hydrogen, --OH,
--(CH.sub.2).sub.nC(.dbd.O)OH,
--C(.dbd.O)(C(R.sup.2D).sub.2)).sub.nL.sup.3R.sup.2E, optionally
substituted C.sub.1-C.sub.8 alkyl, optionally substituted
C.sub.3-C.sub.8 cycloalkyl, optionally substituted
--O--(C.sub.1-C.sub.8 alkyl), optionally substituted aryl,
optionally substituted heteroaryl, and optionally substituted
heterocyclyl; [0239] each NR.sup.2CR.sup.2C is independently
selected, wherein each R.sup.2C is independently selected from the
group consisting of hydrogen, --OH, optionally substituted
C.sub.1-C.sub.8 alkyl, optionally substituted C.sub.3-C.sub.8
cycloalkyl, optionally substituted --O--(C.sub.1-C.sub.8 alkyl),
optionally substituted aryl, optionally substituted heteroaryl, and
optionally substituted heterocyclyl, or optionally both R.sup.2C
together with the nitrogen to which they are attached are an
optionally substituted heterocyclyl; [0240] each R.sup.2D is
independently selected from the group consisting of hydrogen,
optionally substituted C.sub.1-C.sub.8 alkyl, optionally
substituted C.sub.3-C.sub.8 cycloalkyl, optionally substituted
--O--(C.sub.1-C.sub.8 alkyl), optionally substituted aryl,
optionally substituted heteroaryl, and optionally substituted
heterocyclyl; [0241] each R.sup.2E is independently selected from
the group consisting of optionally substituted C.sub.1-C.sub.8
alkyl, optionally substituted C.sub.1-C.sub.8 cycloalkyl,
optionally substituted aryl, optionally substituted heteroaryl, and
optionally substituted heterocyclyl, and
--(CH.sub.2).sub.nC(.dbd.O)OR.sup.2F; [0242] each R.sup.2F is
independently selected from the group consisting hydrogen,
optionally substituted C.sub.1-C.sub.8 alkyl, optionally
substituted C.sub.3-C.sub.8 cycloalkyl, optionally substituted
aryl, optionally substituted heteroaryl, and optionally substituted
heterocyclyl; [0243] each L.sup.1 is independently selected from
the group consisting of --C(.dbd.O)--, --S(.dbd.O)--,
--C(.dbd.S)--, --S(.dbd.O).sub.2--, --C(.dbd.O)O--,
--C(.dbd.O)NR.sup.2A--, --S(.dbd.O)NR.sup.2A--,
--S(.dbd.O).sub.2NR.sup.2A--, --C(.dbd.O)NR.sup.2AC(.dbd.O)--, and
--C(CF.sub.3).sub.2NR.sup.2A--; [0244] each L.sup.2 is
independently selected from the group consisting of optionally
substituted aryl, optionally substituted heteroaryl, and optionally
substituted heterocyclyl; [0245] each L.sup.3 is independently
selected from the group consisting of --C(.dbd.O)--, --S(.dbd.O)--,
--C(.dbd.S)--, --S(.dbd.O).sub.2--, --C(.dbd.O)O--,
--C(.dbd.O)NR.sup.2A--, --S(.dbd.O)NR.sup.2A--,
--S(.dbd.O).sub.2NR.sup.2A--, --C(.dbd.O)NR.sup.2AC(.dbd.O)--, and
--C(CF.sub.3).sub.2NR.sup.2A--; [0246] each m independently is 1 or
2; [0247] each n independently is 0, 1, 2, 3, 4, 5, or 6; [0248]
each r independently is 0, 1, 2, 3, 4, 5, or 6; [0249] each s
independently is 0 or 1; and [0250] each z independently is 1 or 2
[0251] R.sup.7 is selected from the group consisting of H
(hydrogen), optionally substituted C.sub.1-C.sub.8 alkyl,
optionally substituted C.sub.3-C.sub.8 cycloalkyl, optionally
substituted aryl, and optionally substituted heterocyclyl; [0252]
R.sup.8 is selected from the group consisting of H (hydrogen),
--(CH.sub.2).sub.nR.sup.C, optionally substituted C.sub.1-C.sub.8
alkyl, optionally substituted C.sub.3-C.sub.8 cycloalkyl,
optionally substituted aryl, and optionally substituted
heterocyclyl; [0253] X is a group consisting of at least one
heteroatom, or selected from groups consisting of --OR.sup.10,
--SO2-R.sup.10, where R.sup.10 is R.sup.C.
[0254] In some embodiments, the active compound having the
structure of Formula I has the structure of Formula IIb:
##STR00060##
[0255] or a pharmaceutically acceptable salt thereof,
[0256] wherein: [0257] R.sup.9 is selected from the group
consisting of H (hydrogen), optionally substituted C.sub.1-C.sub.8
alkyl, optionally substituted C.sub.3-C.sub.8 cycloalkyl,
substituted C.sub.1-C.sub.8 alkyl, substituted C.sub.3-C.sub.8
cycloalkyl, optionally substituted aryl, optionally substituted
heteroaryl, or optionally R.sup.7, R.sup.8 and R.sup.9 together
with the atoms to which they are attached are an optionally
substituted cyclic 5- to 7-membered ring, X is selected from groups
consisting of --OR.sup.10, --SO2-R.sup.10, where R.sup.10 is
R.sup.C; and n is 0 or 1.
[0258] In some embodiments, at least one of R.sup.1, R.sup.10 and X
comprises a targeting moiety. In some embodiments, at least one of
R.sup.1, R.sup.10 and X further comprises a linker. In some
embodiments, at least one of R.sup.1, R.sup.10 and X comprises
--(CH.sub.2).sub.n-- where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
In some embodiments, at least one of R.sup.1, R.sup.10 and X
comprises --(CH.sub.2CH.sub.2O).sub.n-- where n is 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10. In some embodiments, at least one of R.sup.1,
R.sup.10 and X comprises Val-Cit-PAB, Val-Ala-PAB, Phe-Lys-PAB,
D-Val-Leu-Lys, Gly-Gly-Arg, or Ala-Ala-Asn-PAB. In some
embodiments, at least one of R.sup.1, R.sup.10 and X comprises a
peptide, oligosaccharide, --(CH.sub.2).sub.n--,
--(CH.sub.2CH.sub.2O).sub.n--, Val-Cit-PAB, Val-Ala-PAB,
Phe-Lys-PAB, D-Val-Lcu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or
combinations thereof. In some embodiments, the targeting moiety is
a monoclonal antibody (mAB). In some embodiments, the targeting
moiety is an antibody fragment, surrogate, or variant. In some
embodiments, the targeting moiety is a protein ligand. In some
embodiments, the targeting moiety is a protein scaffold. In some
embodiments, the targeting moiety is a peptide. In some
embodiments, the targeting moiety is a small molecule ligand. In
some embodiments, the linker includes a 4-carbon bridge and at
least two sulfur atoms. In some embodiments, the linker includes a
fragment selected from the group consisting of:
##STR00061##
[0259] In some embodiments, at least one of R.sup.1, R.sup.10 and X
comprises:
##STR00062##
wherein: [0260] the A-component is the targeting moiety; the
E-component is an optionally substituted heteroaryl or an
optionally substituted heterocyclyl; L.sup.3 is an optionally
substituted C.sub.1-C.sub.6 alkyl, or L.sup.3 is null, when L.sup.3
is null the sulfur is directly connected to the E-component; and
L.sup.4 is an optionally substituted C.sub.1-C.sub.6 alkyl, or
L.sup.4 is null, when L.sup.4 is null the sulfur is directly
connected to the E-component. In some embodiments, the E-component
includes a fragment selected from the group consisting of:
##STR00063##
[0260] In some embodiments, L.sup.3 is --(CH.sub.2)--; and L.sup.4
is --(CH.sub.2)--. In some embodiments, L.sup.3 is null; and
L.sup.4 is null.
[0261] In some embodiments,
##STR00064##
[0262] In some embodiments, at least one of R.sup.1, R.sup.10 and X
comprises:
##STR00065##
wherein: A is the targeting moiety; X is N (nitrogen) or CH; Y is N
(nitrogen), or CH; m is 0, 1, or 2; L is a linker, or null; and
L.sup.1A is a linker, or null. In some embodiments, L is null. In
some embodiments, L includes --C(.dbd.O)--, --NH--C(.dbd.O)--,
--C(.dbd.O)--O--, --NH--C(.dbd.O)--NH-- or --NH--C(.dbd.O)--O--. In
some embodiments, L is --C(.dbd.O)--, --NH--C(.dbd.O)--,
--C(O)--O--, --NH--C(.dbd.O)--NH-- or --NH--C(.dbd.O)--O--. In some
embodiments, L is --C(.dbd.O)--. In some embodiments,
##STR00066##
[0263] In some embodiments, at least one of R.sup.1, R.sup.10 and X
comprises, consists of, or consists essentially of:
##STR00067##
wherein L.sup.5 may be optionally substituted C.sub.1-C.sub.8
alkyl, optionally substituted C.sub.3-C.sub.8 cycloalkyl,
optionally substituted C.sub.1-C.sub.8 alkoxy, optionally
substituted aryl, optionally substituted heteroaryl, optionally
substituted heterocyclyl, or combination thereof. In some
embodiments, L.sup.5 is an optionally substituted C.sub.1-C.sub.8
alkyl. In some embodiments, L.sup.5 is C.sub.1-C.sub.8 alkyl.
[0264] In some embodiments, at least one of R.sup.1, R.sup.10 and X
comprises, consists of, or consist essentially of:
##STR00068##
wherein A is the targeting moiety, L.sup.5 may be optionally
substituted C.sub.1-C.sub.8 alkyl, optionally substituted
C.sub.3-C.sub.8 cycloalkyl, optionally substituted C.sub.1-C.sub.8
alkoxy, optionally substituted aryl, optionally substituted
heteroaryl, optionally substituted heterocyclyl, or combination
thereof. In some embodiments, L.sup.5 is an optionally substituted
C.sub.1-C.sub.8 alkyl. In some embodiments, L.sup.5 is
C.sub.1-C.sub.8 alkyl.
[0265] In some embodiments, at least one of R.sup.1, R.sup.10 and X
comprises, consists of, or consist essentially of:
##STR00069##
wherein A is the targeting moiety, L.sup.5 may be optionally
substituted C.sub.1-C.sub.8 alkyl, optionally substituted
C.sub.3-C.sub.8 cycloalkyl, optionally substituted C.sub.1-C.sub.8
alkoxy, optionally substituted aryl, optionally substituted
heteroaryl, optionally substituted heterocyclyl, or combination
thereof. In some embodiments, L.sup.5 is an optionally substituted
C.sub.1-C.sub.8 alkyl. In some embodiments, L.sup.5 is
C.sub.1-C.sub.8 alkyl. L.sup.6 may be H, optionally substituted
C.sub.1-C.sub.8 alkyl, optionally substituted C.sub.3-C.sub.8
cycloalkyl, optionally substituted aryl, optionally substituted
heteroaryl, optionally substituted heterocyclyl, or combination
thereof.
[0266] In some embodiments, at least one of R.sup.1, R.sup.10 and X
comprises, consists of, or consist essentially of:
##STR00070##
wherein A is the targeting moiety, L.sup.5 may be optionally
substituted C.sub.1-C.sub.8 alkyl, optionally substituted
C.sub.3-C.sub.8 cycloalkyl, optionally substituted C.sub.3-C.sub.8
cycloalkyl annulated to cyclooctyl ring, optionally substituted
C.sub.1-C.sub.8 alkoxy, optionally substituted aryl, optionally
substituted heteroaryl, optionally substituted heterocyclyl, or
combination thereof. In some embodiments, L.sup.5 is an optionally
substituted C.sub.1-C.sub.8 alkyl. In some embodiments, L.sup.5 is
C.sub.1-C.sub.8 alkyl.
[0267] Some embodiments provide a compound-conjugate having the
structure of Formula V:
##STR00071##
[0268] or a pharmaceutically acceptable salt thereof, wherein:
A.sup.1 may be a targeting moiety; B.sup.1 is an auxiliary moiety
that optionally includes a second targeting moiety, or B.sup.1 is
null; L.sup.1 includes a group including a N (nitrogen) atom or a
group including a 2- to 5-carbon bridge and at least one sulfur
atom; each D is independently selected, where each D includes a
compound; each L.sup.2 is independently a linker, wherein at least
one L.sup.2 links to L.sup.1; and n is 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10. In some embodiments, A.sup.1 may be a monoclonal antibody
(mAB). In some embodiments, A.sup.1 may be an antibody fragment,
surrogate, or variant. In some embodiments, A.sup.1 may be a
protein ligand. In some embodiments, A.sup.1 may be a protein
scaffold. In some embodiments, A.sup.1 may be a peptide. In some
embodiments. A.sup.1 may be RNA or DNA. In some embodiments,
A.sup.1 may be a RNA or DNA fragment. In some embodiments, A.sup.1
may be a small molecule ligand. In some embodiments, B.sup.1 may be
a hydrophilic polymer. In some embodiments, the hydrophilic polymer
may polyethylene glycol (PEG), and the like. In some embodiments,
B.sup.1 may be a biodegradable polymer. In some embodiments, the
biodegradable polymer may be unstructured proteins polyamino acids,
polypeptides polysaccharides and combinations thereof. In some
embodiments, B.sup.1 may be a monoclonal antibody (mAB). In some
embodiments, B.sup.1 may be an antibody fragment, surrogate, or
variant. In some embodiments, B.sup.1 may be a protein ligand. In
some embodiments, B.sup.1 may be a protein scaffold. In some
embodiments, B.sup.1 may be a peptide. In some embodiments, B.sup.1
may be RNA or DNA. In some embodiments, B.sup.1 may be a RNA or DNA
fragment. In some embodiments, B.sup.1 may be a small molecule
ligand. In some embodiments, D may includes a biologically active
compound. In some embodiments, D may includes a core from
tubulin-binder or tubulin-binder derivative. In some embodiments, D
include a core from epothilone A, epothilone B, paclitaxel, or
derivatives thereof. In some embodiments, D includes
##STR00072##
wherein: A is a tubulin binding moiety; B is a protease inhibition
moiety; and R.sub.1-R.sub.8 are each independently selected from
the group consisting of H (hydrogen), C.sub.1-C.sub.8 alkyl,
substituted or cyclic C.sub.1-C.sub.8 alkyl, aryl, and substituted
aryl, or optionally R.sub.1 and R.sub.2 together with the nitrogen
to which they are attached are a cyclic 5- to 7-membered ring, or
optionally R.sub.1 and R.sub.3 together with the atoms to which
they are attached are a cyclic 5- to 7-membered ring, or optionally
R.sub.7 and R.sub.8 together with the atoms to which they are
attached are a cyclic 5- to 7-membered ring. In some embodiments, A
may be
##STR00073##
and Y may be CH.sub.2, S, S.dbd.O, C.dbd.O, CHF, CHCN, CHN.sub.3
CH--OH, CH--ONH.sub.2, or CHOR, where R is C.sub.1-C.sub.8 alkyl or
substituted C.sub.1-C.sub.8 alkyl. In some embodiments, A may
be
##STR00074##
Z may be N (nitrogen), CH, C--OH, C--OR, CSH, CSR, where R is
C.sub.1-C.sub.8 alkyl or substituted C.sub.1-C.sub.8 alkyl; X may
be F, OH, N.sub.3, OMe, or OR, where R is C.sub.1-C.sub.8 alkyl or
substituted C.sub.1-C.sub.8 alkyl; and V may be CH.sub.2, S,
S.dbd.O, C.dbd.O, CHF, CHCN, CHN.sub.3 CH--OH, CH--ONH.sub.2, or
CHOR, where R is C.sub.1-C.sub.8 alkyl or substituted
C.sub.1-C.sub.8 alkyl. In some embodiments, L.sup.2 may include a
spacer or a multifunctional linker. In some embodiments, L.sup.2
may include a spacer and a multifunctional linker. In some
embodiments, L.sup.2 may include a multifunctional linker. In some
embodiments, each L.sup.2 may be a linker, wherein the linker may
be cleavable or non-cleavable under biological conditions. In some
embodiments, the linker may be cleavable by an enzyme. In some
embodiments, L.sup.2 may include Linker. In some embodiments,
L.sup.1 includes a cyclic group including at least one N (nitrogen)
atom. In some embodiments, L.sup.1 includes a cyclic group
including at least two N (nitrogen) atoms. In some embodiments,
L.sup.1 includes a cyclic group including at least one N (nitrogen)
atom and a spacer. In some embodiments, L.sup.1 includes a cyclic
group including at least two N (nitrogen) atoms and a spacer. In
some embodiments, the spacer connects to the mAB by an amide bond.
In some embodiments, the spacer connects to the mAB through an
amine bond. In some embodiments, L.sup.1 includes a 2- to 5-carbon
bridge and at least one sulfur atom. In some embodiments, L.sup.1
includes a 2- to 5-carbon bridge and at least two sulfur atoms. In
some embodiments, L.sup.1 includes a 2- to 5-carbon bridge and a
spacer. In some embodiments, L.sup.1 includes a 2- to 5-carbon
bridge, at least two sulfur atoms and a spacer. In some
embodiments, L.sup.1 may include one or more sulfurs. In some
embodiments, the L.sup.1 may include two or more sulfurs. In some
embodiments, the L.sup.1 may include exactly two sulfurs. In some
embodiments, may include a 4-carbon bridge and/or a spacer. In some
embodiments, L.sup.1 include a 4-carbon bridge or a spacer. In some
embodiments, L.sup.1 may include a 4-carbon bridge and a spacer. In
some embodiments, L.sup.1 includes a 4-carbon bridge and at least
two sulfur atoms. In some embodiments, the spacer connects to the
mAB by a sulfide bond. In some embodiments, the spacer connects to
the mAB through a thioether. In some embodiments, A.sup.1 comprises
at least one modified n-butyl L-.alpha.-amino acid. In some
embodiments, at least one modified L-Lysine residue is from an
L-Lysine residue of a peptide before conjugation. In some
embodiments, at least one nitrogen of L.sup.1 is from an at least
one modified n-butyl L-.alpha.-amino acid of a peptide before
conjugation. In some embodiments, A.sup.1 and L.sup.1 together
comprise at least one modified L-Lysine residue. In some
embodiments, the terminal nitrogen of the side chain of an L-Lysine
residue of a peptide before conjugation is the at least one N
(nitrogen) atom of L.sup.1. In some embodiments, A.sup.1 comprises
the --(CH.sub.2).sub.4-- of the side chain of an L-Lysine residue
of a peptide before conjugation that provides the at least one N
(nitrogen) atom of L.sup.1. In some embodiments, A.sup.1 comprises
a modified n-butyl .alpha.-amino acid residue. In some embodiments,
Linker may be a peptide. In some embodiments, Linker may include an
oligosaccharide. For example, Linker may include chitosan. In some
embodiments, L.sup.2 may include Linker and --(CH.sub.2).sub.n--
where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments,
L.sup.2 may include Linker and --(CH.sub.2CH.sub.2O).sub.n-- where
n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, Linker
may include --(CH.sub.2).sub.n-- where n is 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10. In some embodiments, Linker may include
--(CH.sub.2CH.sub.2O).sub.n-- where n is 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10. In some embodiments, Linker may include Val-Cit-PAB,
Val-Ala-PAB, Phe-Lys-PAB, D-Val-Lcu-Lay, Gly-Gly-Arg,
Ala-Ala-Asn-PAB, or the like. In some embodiments, Linker may
include any combination of peptide, oligosaccharide,
--(CH.sub.2).sub.n--, --(CH.sub.2CH.sub.2O)--, Val-Cit-PAB,
Val-Ala-PAB, Phe-Lys-PAB, D-Val-Leu-Lay, Gly-Gly-Arg,
Ala-Ala-Asn-PAB, and the like. In some embodiments, the spacer may
include a peptide. In some embodiments, the spacer may include an
oligosaccharide. For example, the spacer may include chitosan. In
some embodiments, the spacer may include --(CH.sub.2).sub.n-- where
n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, L.sup.1
may include a component including a 4-carbon bridge and
--(CH.sub.2).sub.n-- where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
In some embodiments, the spacer may include
--(CH.sub.2CH.sub.2O).sub.n-- where n is 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10. In some embodiments, L.sup.1 may include a component
including a 4-carbon bridge and --(CH.sub.2CH.sub.2O).sub.n-- where
n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the
spacer may include Val-Cit-PAB, Val-Ala-PAB, Phe-Lys-PAB,
Ala-Ala-Asn-PAB, or the like. In some embodiments, the spacer may
be any combination of peptide, oligosaccharide,
--(CH.sub.2).sub.n--, --(CH.sub.2CH.sub.2O).sub.n--, Val-Cit-PAB,
Val-Ala-PAB, Phe-Lys-PAB, Ala-Ala-Asn-PAB, and the like. In some
embodiments, L.sup.1 may include,
##STR00075##
but is not limited to,
##STR00076##
and the like. In some embodiments, L.sup.1 may include, but is not
limited to,
##STR00077##
and the like.
[0269] In some embodiments, the compound-conjugates may include one
or more components selected from the group consisting of an amino
acid, an amino acid residue, an amino acid analog, and a modified
amino acid.
[0270] As used herein, the term "peptide" refers to a structure
including one or more components each individually selected from
the group consisting of an amino acid, an amino acid residue, an
amino acid analog, and a modified amino acid. The components are
typically joined to each other through an amide bond.
[0271] As used herein, the term "amino acid" includes naturally
occurring amino acids, a molecule having a nitrogen available for
forming an amide bond and a carboxylic acid, a molecule of the
general formula NH.sub.2--CHR--COOH or the residue within a peptide
bearing the parent amino acid, where "R" is one of a number of
different side chains. "R" can be a substituent found in naturally
occurring amino acids. "R" can also be a substituent referring to
one that is not of the naturally occurring amino acids.
[0272] As used herein, the term "amino acid residue" refers to the
portion of the amino acid which remains after losing a water
molecule when it is joined to another amino acid.
[0273] As used herein, the term "amino acid analog" refers to a
structural derivative of an amino acid parent compound that often
differs from it by a single element.
[0274] As used herein, the term "modified amino acid" refers to an
amino acid bearing an "R" substituent that does not correspond to
one of the twenty genetically coded amino acids.
[0275] As used herein, the abbreviations for the genetically
encoded L-enantiomeric amino acids are conventional and are as
follows: The D-amino acids are designated by lower case, e.g.
D-proline=p, etc.
TABLE-US-00001 TABLE 1 Amino Acids One-Letter Symbol Common
Abbreviation Alanine A Ala Arginine R Arg Asparagine N Asn Aspartic
acid D Asp Cysteine C Cys Glutamine Q Gln Glutamic acid E Glu
Glycine G Gly Histidine H His Isoleucine I Ile Leucine L Leu Lysine
K Lys Phenylalanine F Phe Proline P Pro Serine S Ser Threonine T
Thr Tryptophan W Trp Tyrosine Y Tyr Valine V Val
[0276] Certain amino acid residues in the compound-conjugate can be
replaced with other amino acid residues without significantly
deleteriously affecting, and in many cases even enhancing, the
activity of the peptides. Thus, also contemplated by the preferred
embodiments are altered or mutated forms of the active
agent-conjugate wherein at least one defined amino acid residue in
the structure is substituted with another amino acid residue or
derivative and/or analog thereof. It will be recognized that in
preferred embodiments, the amino acid substitutions are
conservative, i.e., the replacing amino acid residue has physical
and chemical properties that are similar to the amino acid residue
being replaced.
[0277] For purposes of determining conservative amino acid
substitutions, the amino acids can be conveniently classified into
two main categories--hydrophilic and hydrophobic--depending
primarily on the physical-chemical characteristics of the amino
acid side chain. These two main categories can be further
classified into subcategories that more distinctly define the
characteristics of the amino acid side chains. For example, the
class of hydrophilic amino acids can be further subdivided into
acidic, basic and polar amino acids. The class of hydrophobic amino
acids can be further subdivided into nonpolar and aromatic amino
acids. The definitions of the various categories of amino acids are
as follows:
[0278] The term "hydrophilic amino acid" refers to an amino acid
exhibiting a hydrophobicity of less than zero according to the
normalized consensus hydrophobicity scale of Eisenberg et al.,
1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophilic
amino acids include Thr (T), Ser (S), His (H), Glu (E), Asn (N),
Gln (Q), Asp (D), Lys (K) and Arg (R).
[0279] The term "hydrophobic amino acid" refers to an amino acid
exhibiting a hydrophobicity of greater than zero according to the
normalized consensus hydrophobicity scale of Eisenberg, 1984, J.
Mol. Biol. 179:1.25-142. Genetically encoded hydrophobic amino
acids include Pro (P), lie (1), Phe (F), Val (V), Leu (L), Trp (W),
Met (M), Ala (A), Gly (G) and Tyr (Y).
[0280] The term "acidic amino acid" refers to a hydrophilic amino
acid having a side chain pK value of less than 7. Acidic amino
acids typically have negatively charged side chains at
physiological pH due to loss of a hydrogen ion. Genetically encoded
acidic amino acids include Glu (E) and Asp (D).
[0281] The term "basic amino acid" refers to a hydrophilic amino
acid having a side chain pK value of greater than 7. Basic amino
acids typically have positively charged side chains at
physiological pH due to association with hydronium ion. Genetically
encoded basic amino acids include His (H), Arg (R) and Lys (K).
[0282] The term "polar amino acid" refers to a hydrophilic amino
acid having a side chain that is uncharged at physiological pH, but
which has at least one bond in which the pair of electrons shared
in common by two atoms is held more closely by one of the atoms.
Genetically encoded polar amino acids include Asn (N), Gln (Q) Ser
(S) and Thr (T).
[0283] The term "nonpolar amino acid" refers to a hydrophobic amino
acid having a side chain that is uncharged at physiological pH and
which has bonds in which the pair of electrons shared in common by
two atoms is generally held equally by each of the two atoms (i.e.,
the side chain is not polar). Genetically encoded nonpolar amino
acids include Leu (L), Val (V), Ile (I), Met (M), Gly (G) and Ala
(A).
[0284] The term "aromatic amino acid" refers to a hydrophobic amino
acid with a side chain having at least one aromatic or
heteroaromatic ring. In some embodiments, the aromatic or
heteroaromatic ring may contain one or more substituents such as
--OH, --SH, --CN, --F, --Cl, --Br, --I, --NO.sub.2, --NO,
--NH.sub.2, --NHR, --NRR, --C(O)R, --C(O)OH, --C(O)OR,
--C(O)NH.sub.2, --C(O)NHR, --C(O)NRR and the like where each R is
independently (C.sub.1-C.sub.6) alkyl, substituted
(C.sub.1-C.sub.6) alkyl, (C.sub.1-C.sub.6) alkenyl, substituted
(C.sub.1-C.sub.6) alkenyl, (C.sub.1-C.sub.6) alkynyl, substituted
(C.sub.1-C.sub.6) alkynyl, (C.sub.5-C.sub.20) aryl, substituted
(C.sub.5-C.sub.20) aryl, (C.sub.6-C.sub.26) alkaryl, substituted
(C.sub.6-C.sub.26) alkaryl, 5-20 membered heteroaryl, substituted
5-20 membered heteroaryl, 6-26 membered alkheteroaryl or
substituted 6-26 membered alkheteroaryl. Genetically encoded
aromatic amino acids include Phe (F), Tyr (Y) and Trp (W).
[0285] The term "aliphatic amino acid" refers to a hydrophobic
amino acid having an aliphatic hydrocarbon side chain. Genetically
encoded aliphatic amino acids include Ala (A), Val (V), Lcu (L) and
Ile (I).
[0286] The amino acid residue Cys (C) is unusual in that it can
form disulfide bridges with other Cys (C) residues or other
sulfanyl-containing amino acids. The ability of Cys (C) residues
(and other amino acids with --SH containing side chains) to exist
in a peptide in either the reduced free --SH or oxidized
disulfide-bridged form affects whether Cys (C) residues contribute
net hydrophobic or hydrophilic character to a peptide. While Cys
(C) exhibits a hydrophobicity of 0.29 according to the normalized
consensus scale of Eisenberg (Eisenberg, 1984, supra), it is to be
understood that for purposes of the preferred embodiments Cys (C)
is categorized as a polar hydrophilic amino acid, notwithstanding
the general classifications defined above.
[0287] As used herein, the term "targeting moiety" refers to a
structure that binds or associates with a biological moiety or
fragment thereof.
[0288] In some embodiments, the targeting moiety may be a
monoclonal antibody (mAB). In some embodiments, the targeting
moiety may be an antibody fragment, surrogate, or variant. In some
embodiments, the targeting moiety may be a protein ligand. In some
embodiments, the targeting moiety may be a protein scaffold. In
some embodiments, the targeting moiety may be a peptide. In some
embodiments, the targeting moiety may be RNA or DNA. In some
embodiments, the targeting moiety may be a RNA or DNA fragment. In
some embodiments, the targeting moiety may be a small molecule
ligand.
[0289] In some embodiments, the targeting moiety may be an antibody
fragment described in Janthur et al., "Drug Conjugates Such as
Antibody Drug Conjugates (ADCs), Immunotoxins and Immunoliposomes
Challenge Daily Clinical Practice," Int. J. Mol. Sci. 2012, 13,
16020-16045, the disclosure of which is incorporated herein by
reference in its entirety. In some embodiments, the targeting
moiety may be an antibody fragment described in Trail, P A,
"Antibody Drug Conjugates as Cancer Therapeutics," Antibodies 2013,
2, 113-129, the disclosure of which is incorporated herein by
reference in its entirety.
[0290] In some embodiments, the targeting moiety may be
HuM195-Ac-225, HuM195-Bi-213, Anyara (naptumomab estafenatox;
ABR-217620), AS1409, Zevalin (ibritumomab tiuxetan), BIIB015,
BT-062, Neuradiab, CDX-1307, CR011-vcMMAE, Trastuzumab-DM1 (R3502),
Bexxar (tositumomab), IMGN242, IMGN388, IMGN901,
.sup.131I-labetuzumab, IMMU-102 (.sup.90Y-epratuzumab), IMMU-107
(.sup.90Y-clivatuzumab tetraxetan), MDX-1203, CAT-8015, EMD 273063
(hu14.18-IL2), Tucotuzumab celmoleukin (EMD 273066; huKS-IL2),
.sup.188Re-PTI-6D2, Cotara, L19-IL2, Teleukin (F16-IL2), Tenarad
(F16-.sup.131I), L19-.sup.131I, L19-TNF, PSMA-ADC, DI-Leu16-IL2,
SAR3419, SGN-35, or CMC544. In some embodiments, the targeting
moiety may comprise, consist of, or consist essentially of the
antibody portion of HuM195-Ac-225, HuM195-Bi-213, Anyara
(naptumomab estafenatox; ABR-217620), AS1409, Zevalin (ibritumomab
tiuxetan), BIIB015, BT-062, Neuradiab, CDX-1307, CR011-vcMMAE,
Trastuzumab-DM1 (R3502), Bexxar (tositumomab), IMGN242, IMGN388,
IMGN901, .sup.131I-labetuzumab, IMMU-102 (.sup.90Y-epratuzumab),
IMMU-107 (.sup.90Y-clivatuzumab tetraxetan), MDX-1203, CAT-8015,
EMD 273063 (hu14.18-IL2), Tucotuzumab celmoleukin (EMD 273066;
huKS-IL2), .sup.188Re-PTI-6D2, Cotara, L19-IL2, Teleukin (F16-IL2),
Tenarad (F16-.sup.131I), L19-.sup.131I, L19-TNF, PSMA-ADC,
DI-Leu16-IL2, SAR3419, SGN-35, or CMC544.
[0291] In some embodiments, the targeting moiety may be Brentuximab
vedotin, Trastuzumab emtansine, Inotuzumab ozogamicin, Lorvotuzumab
mertansine, Glembatumumab vedotin, SAR3419, Moxetumomab pasudotox,
Moxetumomab pasudotox, AGS-16M8F, AGS-16M8F, BIIB-015, BT-062,
IMGN-388, or IMGN-388.
[0292] In some embodiments, the targeting moiety may comprise,
consist of, or consist essentially of the antibody portion of
Brentuximab vedotin, Trastuzumab emtansine, Inotuzumab ozogamicin,
Lorvotuzumab mertansine, Glembatumumab vedotin, SAR3419,
Moxetumomab pasudotox, Moxetumomab pasudotox, AGS-16M8F, AGS-16M8F,
BIIB-015, BT-062, IMGN-388, or IMGN-388.
[0293] In some embodiments, the targeting moiety may comprise,
consist of, or consist essentially of Brentuximab, Inotuzumab,
Gemtuzumab, Milatuzumab, Trastuzumab, Glembatumomab, Lorvotuzumab,
or Labestuzumab.
[0294] As used herein, the term "linker" refers to a moiety that
connects two or more components to each other.
[0295] In some embodiments, the linker may be a linker disclosed in
Janthur et al., "Drug Conjugates Such as Antibody Drug Conjugates
(ADCs), Immunotoxins and Immunoliposomes Challenge Daily Clinical
Practice," Int. J. Mol. Sci. 2012, 13, 16020-16045. In some
embodiments, the linker may be a linker disclosed in Trail, P A,
"Antibody Drug Conjugates as Cancer Therapeutics," Antibodies 2013,
2, 113-129. In some embodiments, the linker may be a linker
disclosed in U.S. Pat. No. 7,829,531.
[0296] In some embodiments, the linker may comprise, consist of, or
consist essentially of the linker portion of HuM195-Ac-225,
HuM195-Bi-213, Anyara (naptumomab estafenatox; ABR-217620), AS1409,
Zevalin (ibritumomab tiuxetan), BIIB15, BT-062, Neuradiab,
CDX-1307, CR011-vcMMAE, Trastuzumab-DM1 (R3502), Bexxar
(tositumomab), IMGN242, IMGN388, IMGN901, .sup.131I-labetuzumab,
IMMU-102 (.sup.90Y-epratuzumab), IMMU-107 (.sup.90Y-clivatuzumab
tetraxetan), MDX-1203, CAT-8015, EMD 273063 (hu14.18-IL2),
Tucotuzumab celmoleukin (EMD 273066; huKS-IL2), .sup.188Re-PTI-6D2,
Cotara, L19-IL2, Teleukin (F16-IL2), Tenarad (F16-.sup.131I),
L19-.sup.131I, L19-TNF, PSMA-ADC, DI-Lcu16-IL2, SAR3419, SGN-35, or
CMC544.
[0297] In some embodiments, the linker may comprise, consist of, or
consist essentially of the linker portion of Brentuximab vedotin,
Trastuzumab emtansine, Inotuzumab ozogamicin, Lorvotuzumab
mertansine, Glembatumumab vedotin, SAR3419, Moxetumomab pasudotox,
Moxetumomab pasudotox, AGS-16M8F, AGS-16M8F, BIIB-015, BT-062,
IMGN-388, or IMGN-388.
[0298] In some embodiments, the linker may comprise, consist of, or
consist essentially of Valine-citrulline residue, hydrazine,
4-mercaptobutanoyl, 4-(N-succinimidomethyl)cyclohexanecarbonyl
(SMCC), Maleimidocaproyl, Phenylalaninelysine,
6-(3-(thio)propanamido)hexanoyl, 3-mercaptopropanoyl,
4-mercaptopentanoyl, or lysine residue.
[0299] In some embodiments, the linker may comprise, consist of, or
consist essentially of:
##STR00078##
wherein L.sup.5 may be optionally substituted C.sub.1-C.sub.8
alkyl, optionally substituted C.sub.3-C.sub.8 cycloalkyl,
optionally substituted C.sub.1-C.sub.8 alkoxy, optionally
substituted aryl, optionally substituted heteroaryl, optionally
substituted heterocyclyl, or combination thereof. In some
embodiments, L.sup.5 is an optionally substituted C.sub.1-C.sub.8
alkyl. In some embodiments, L.sup.5 is C.sub.1-C.sub.8 alkyl.
[0300] In some embodiments, the linker may comprise, consist of, or
consist essentially of:
##STR00079##
wherein A is the targeting moiety, L.sup.5 may be optionally
substituted C.sub.1-C.sub.8 alkyl, optionally substituted
C.sub.3-C.sub.8 cycloalkyl, optionally substituted C.sub.1-C.sub.8
alkoxy, optionally substituted aryl, optionally substituted
heteroaryl, optionally substituted heterocyclyl, or combination
thereof. In some embodiments, L.sup.5 is an optionally substituted
C.sub.1-C.sub.8 alkyl. In some embodiments, L.sup.5 is
C.sub.1-C.sub.8 alkyl.
[0301] In some embodiments, the linker may comprise, consist of, or
consist essentially of:
##STR00080##
wherein A is the targeting moiety, L.sup.5 may be optionally
substituted C.sub.1-C.sub.8 alkyl, optionally substituted
C.sub.3-C.sub.8 cycloalkyl, optionally substituted C.sub.1-C.sub.8
alkoxy, optionally substituted aryl, optionally substituted
heteroaryl, optionally substituted heterocyclyl, or combination
thereof. In some embodiments, L.sup.5 is an optionally substituted
C.sub.1-C.sub.8 alkyl. In some embodiments, L.sup.5 is
C.sub.1-C.sub.8 alkyl. L.sup.6 may be H, optionally substituted
C.sub.1-C.sub.8 alkyl, optionally substituted C.sub.3-C.sub.8
cycloalkyl, optionally substituted aryl, optionally substituted
heteroaryl, optionally substituted heterocyclyl, or combination
thereof.
[0302] In some embodiments, the linker may comprise, consist of, or
consist essentially of:
##STR00081##
herein A is the targeting moiety, L.sup.5 may be optionally
substituted C.sub.1-C.sub.8 alkyl, optionally substituted
C.sub.3-C.sub.8 cycloalkyl, optionally substituted C.sub.3-C.sub.8
cycloalkyl annulated to cyclooctyl ring, optionally substituted
C.sub.1-C.sub.8 alkoxy, optionally substituted aryl, optionally
substituted heteroaryl, optionally substituted heterocyclyl, or
combination thereof. In some embodiments, L.sup.5 is an optionally
substituted C.sub.1-C.sub.8 alkyl. In some embodiments, L.sup.5 is
C.sub.1-C.sub.8 alkyl.
[0303] In some embodiments, the linker may comprise, consist of, or
consist essentially of:
##STR00082## ##STR00083## ##STR00084##
[0304] As will be appreciated by those of skill in the art, the
above-defined categories are not mutually exclusive. Thus, amino
acids having side chains exhibiting two or more physical-chemical
properties can be included in multiple categories. For example,
amino acid side chains having aromatic moieties that are further
substituted with polar substituents, such as Tyr (Y), may exhibit
both aromatic hydrophobic properties and polar or hydrophilic
properties, and can therefore be included in both the aromatic and
polar categories. The appropriate categorization of any amino acid
will be apparent to those of skill in the art, especially in light
of the detailed disclosure provided herein.
[0305] While the above-defined categories have been exemplified in
terms of the genetically encoded amino acids, the amino acid
substitutions need not be, and in certain embodiments preferably
are not, restricted to the genetically encoded amino acids. In some
embodiments, the active agent-conjugate may contain genetically
non-encoded amino acids. Thus, in addition to the naturally
occurring genetically encoded amino acids, amino acid residues in
the active agent-conjugate may be substituted with naturally
occurring non-encoded amino acids and synthetic amino acids.
[0306] Certain commonly encountered amino acids which provide
useful substitutions for the active agent-conjugates include, but
are not limited to, .beta.-alanine (.beta.-Ala) and other
omega-amino acids such as 3-aminopropionic acid,
2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth;
.alpha.-aminoisobutyric acid (Aib); -aminohexanoic acid (Aha);
.delta.-aminovaleric acid (Ava); N-methylglycine or sarcosine
(MeGly); omithine (Orn); citrulline (Cit); t-butylalanine (t-BuA);
t-butylglycine (t-BuG); N-methylisoleucine (Melle); phenylglycine
(Phg); cyclohexylalanine (Cha); norleucine (Nle); naphthylalanine
(Nal); 4-phenylphenylalanine, 4-chlorophenylalanine (Phe(4-Cl));
2-fluorophenylalanine (Phe(2-F)); 3-fluorophenylalanine (Phe(3-F));
4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen);
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic);
.beta.-2-thienylalanine (Thi); methionine sulfoxide (MSO);
homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric
acid (Dbu); 2,3-diaminobutyric acid (Dab); p-aminophenylalanine
(Phe (pNH.sub.2)); N-methyl valine (MeVal); homocysteine (hCys),
homophenylalanine (hPhe) and homoserine (hSer); hydroxyproline
(Hyp), homoproline (hPro), N-methylated amino acids and peptoids
(N-substituted glycines).
[0307] Other amino acid residues not specifically mentioned herein
can be readily categorized based on their observed physical and
chemical properties in light of the definitions provided
herein.
[0308] The classifications of the genetically encoded and common
non-encoded amino acids according to the categories defined above
are summarized in Table 2, below. It is to be understood that Table
2 is for illustrative purposes only and does not purport to be an
exhaustive list of amino acid residues and derivatives that can be
used to substitute the active agent-conjugate described herein.
TABLE-US-00002 TABLE 2 CLASSIFICATIONS OF COMMONLY ENCOUNTERED
AMINO ACIDS Genetically Classification Encoded Non-Genetically
Encoded Hydrophobic Aromatic F, Y, W Phg, Nal, Thi, Tic, Phe
(4-Cl), Phe (2-F), Phe (3-F), Phe (4-F), hPhe Nonpolar L, V, I, M,
G, t-BuA, t-BuG, MeIle, Nle, MeVal, Cha, A, P McGly, Aib Aliphatic
A, V, L, I b-Ala, Dpr, Aib, Ahx, MeGly, t-BuA, t- BuG, MeIle, Cha,
Nle, MeVal Hydrophilic Acidic D, E Basic H, K, R Dpr, Orn, hArg,
Phe(p-NH.sub.2), Dbu, Dab Polar C, Q, N, S, T Cit, AcLys, MSO,
bAla, hSer Helix-Breaking P, G D-Pro and other D-amino acids (in L-
peptides)
[0309] Other amino acid residues not specifically mentioned herein
can be readily categorized based on their observed physical and
chemical properties in light of the definitions provided
herein.
[0310] While in most instances, the amino acids of the
compound-conjugate will be substituted with L-enantiomeric amino
acids, the substitutions are not limited to L-enantiomeric amino
acids. In some embodiments, the peptides may advantageously be
composed of at least one D-enantiomeric amino acid. Peptides
containing such D-amino acids are thought to be more stable to
degradation in the oral cavity, gut or serum than are peptides
composed exclusively of L-amino acids.
[0311] Examples of compound-conjugates include, but are not limited
to, the following general compounds:
##STR00085##
[0312] R may be H (hydrogen), C.sub.1-C.sub.8 alkyl, substituted or
cyclic C.sub.1-C.sub.8 alkyl
[0313] X may be S (sulfur), CH.sub.2, CH--OH, CH--OR,
CH--ONH.sub.2, or C.dbd.O.
[0314] Examples of compounds include, but are not limited to, the
following general compounds:
##STR00086## ##STR00087##
[0315] Examples of compound-conjugates include, but are not limited
to, the following general compounds:
##STR00088##
[0316] R may be H (hydrogen), C.sub.1-C.sub.8 alkyl, substituted or
cyclic C.sub.1-C.sub.8 alkyl, and the like X may be S (sulfur),
CH.sub.2, CH--OH, CH--OR, CH--ONH.sub.2, C.dbd.O, and the like.
[0317] Examples of compounds include, but are not limited to, the
following compound and general compounds:
##STR00089## ##STR00090##
Pharmaceutical Compositions
[0318] In some embodiments, the compounds disclosed herein are used
in pharmaceutical compositions. The compounds can be used, for
example, in pharmaceutical compositions comprising a
pharmaceutically acceptable carrier prepared for storage and
subsequent administration. Also, embodiments relate to a
pharmaceutically effective amount of the products and compounds
disclosed above in a pharmaceutically acceptable carrier or
diluent. Acceptable carriers or diluents for therapeutic use are
well known in the pharmaceutical art, and are described, for
example, in Remington's Pharmaceutical Sciences, Mack Publishing
Co. (A. R. Gennaro edit. 1985), which is incorporated herein by
reference in its entirety. Preservatives, stabilizers, dyes and
even flavoring agents can be provided in the pharmaceutical
composition. For example, sodium benzoate, ascorbic acid and esters
of p-hydroxybenzoic acid can be added as preservatives. In
addition, antioxidants and suspending agents can be used.
[0319] The compositions can be formulated and used as tablets,
capsules, or elixirs for oral administration; suppositories for
rectal administration; sterile solutions, suspensions for
injectable administration; patches for transdermal administration,
and sub-dermal deposits and the like. Injectables can be prepared
in conventional forms, either as liquid solutions or suspensions,
solid forms suitable for solution or suspension in liquid prior to
injection, or as emulsions. Suitable excipients are, for example,
water, saline, dextrose, mannitol, lactose, lecithin, albumin,
sodium glutamate, cysteine hydrochloride, and the like. In
addition, if desired, the injectable pharmaceutical compositions
may contain minor amounts of nontoxic auxiliary substances, such as
wetting agents, pH buffering agents, and the like. If desired,
absorption enhancing preparations (for example, liposomes), can be
utilized.
[0320] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds can be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or other organic oils such as soybean, grapefruit or almond
oils, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances that increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents that increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0321] Pharmaceutical preparations for oral use can be obtained by
combining the active compounds with solid excipient, optionally
grinding a resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee cores. Suitable excipients are, in particular,
fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol; cellulose preparations such as, for example, maize
starch, wheat starch, rice starch, potato starch, gelatin, gum
tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If
desired, disintegrating agents can be added, such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate. Dragee cores are provided with
suitable coatings. For this purpose, concentrated sugar solutions
can be used, which may optionally contain gum arabic, talc,
polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or
titanium dioxide, lacquer solutions, and suitable organic solvents
or solvent mixtures. Dyestuffs or pigments can be added to the
tablets or dragee coatings for identification or to characterize
different combinations of active compound doses. For this purpose,
concentrated sugar solutions can be used, which may optionally
contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments can be added to the tablets or dragee coatings for
identification or to characterize different combinations of active
compound doses.
[0322] To formulate the compounds of Formulae I and II as an
anti-cancer agent, known surface active agents, excipients,
smoothing agents, suspension agents and pharmaceutically acceptable
film-forming substances and coating assistants, and the like can be
used. Preferably alcohols, esters, sulfated aliphatic alcohols, and
the like can be used as surface active agents; sucrose, glucose,
lactose, starch, crystallized cellulose, mannitol, light anhydrous
silicate, magnesium aluminate, magnesium methasilicate aluminate,
synthetic aluminum silicate, calcium carbonate, sodium acid
carbonate, calcium hydrogen phosphate, calcium carboxymethyl
cellulose, and the like can be used as excipients; magnesium
stearate, talc, hardened oil and the like can be used as smoothing
agents; coconut oil, olive oil, sesame oil, peanut oil, soya can be
used as suspension agents or lubricants; cellulose acetate
phthalate as a derivative of a carbohydrate such as cellulose or
sugar, or methylacetate-methacrylate copolymer as a derivative of
polyvinyl can be used as suspension agents; and plasticizers such
as ester phthalates and the like can be used as suspension agents.
In addition to the foregoing preferred ingredients, sweeteners,
fragrances, colorants, preservatives and the like can be added to
the administered formulation of the compound produced by the method
of the embodiment, particularly when the compound is to be
administered orally.
[0323] When used as an anti-cancer compound, for example, the
compounds of Formulae I and II or compositions including compounds
of Formulae I and II can be administered by either oral or non-oral
pathways. When administered orally, it can be administered in
capsule, tablet, granule, spray, syrup, or other such form. When
administered non-orally, it can be administered as an aqueous
suspension, an oily preparation or the like or as a drip,
suppository, salve, ointment or the like, when administered via
injection, subcutaneously, intraperitoneally, intravenously,
intramuscularly, or the like.
[0324] In one embodiment, the anti-cancer agent can be mixed with
additional substances to enhance their effectiveness.
Methods of Administration
[0325] In an alternative embodiment, the disclosed compounds and
the disclosed pharmaceutical compositions are administered by a
particular method as an anti-cancer, or anti-inflammatory. Such
methods include, among others, (a) administration though oral
pathways, which administration includes administration in capsule,
tablet, granule, spray, syrup, or other such forms; (b)
administration through non-oral pathways, which administration
includes administration as an aqueous suspension, an oily
preparation or the like or as a drip, suppository, salve, ointment
or the like; administration via injection, subcutaneously,
intraperitoneally, intravenously, intramuscularly, intradermally,
or the like; as well as (c) administration topically, (d)
administration rectally, or (e) administration vaginally, as deemed
appropriate by those of skill in the art for bringing the compound
of the present embodiment into contact with living tissue; and (f)
administration via controlled released formulations, depot
formulations, and infusion pump delivery. As further examples of
such modes of administration and as further disclosure of modes of
administration, disclosed herein are various methods for
administration of the disclosed compounds and pharmaceutical
compositions including modes of administration through intraocular,
intranasal, and intraauricular pathways.
[0326] The pharmaceutically effective amount of the compositions
that include the described compounds required as a dose will depend
on the route of administration, the type of animal, including
human, being treated, and the physical characteristics of the
specific animal under consideration. The dose can be tailored to
achieve a desired effect, but will depend on such factors as
weight, diet, concurrent medication and other factors which those
skilled in the medical arts will recognize. In a typical
embodiment, a compound represented by Formulae I and II can be
administered to a patient in need of an anti-cancer agent, until
the need is effectively reduced or preferably removed.
[0327] In practicing the methods of the embodiment, the products or
compositions can be used alone or in combination with one another,
or in combination with other therapeutic or diagnostic agents.
These products can be utilized in vivo, ordinarily in a mammal,
preferably in a human, or in vitro. In employing them in vivo, the
products or compositions can be administered to the mammal in a
variety of ways, including parenterally, intravenously,
subcutaneously, intramuscularly, colonically, rectally, vaginally,
nasally or intraperitoneally, employing a variety of dosage forms.
Such methods may also be applied to testing chemical activity in
vivo.
[0328] As will be readily apparent to one skilled in the art, the
useful in vivo dosage to be administered and the particular mode of
administration will vary depending upon the age, weight and
mammalian species treated, the particular compounds employed, and
the specific use for which these compounds are employed. The
determination of effective dosage levels, that is the dosage levels
necessary to achieve the desired result, can be accomplished by one
skilled in the art using routine pharmacological methods.
Typically, human clinical applications of products are commenced at
lower dosage levels, with dosage level being increased until the
desired effect is achieved. Alternatively, acceptable in vitro
studies can be used to establish useful doses and routes of
administration of the compositions identified by the present
methods using established pharmacological methods.
[0329] In non-human animal studies, applications of potential
products are commenced at higher dosage levels, with dosage being
decreased until the desired effect is no longer achieved or adverse
side effects disappear. The dosage may range broadly, depending
upon the desired affects and the therapeutic indication. Typically,
dosages can be between about 10 mg/kg and 100 mg/kg body weight,
preferably between about 100 mg/kg and 10 mg/kg body weight.
Alternatively dosages can be based and calculated upon the surface
area of the patient, as understood by those of skill in the art.
Administration is preferably oral on a daily or twice daily
basis.
[0330] The exact formulation, route of administration and dosage
can be chosen by the individual physician in view of the patient's
condition. See for example, Fingl et al., in The Pharmacological
Basis of Therapeutics, 1975, which is incorporated herein by
reference in its entirety. It should be noted that the attending
physician would know how to and when to terminate, interrupt, or
adjust administration due to toxicity, or to organ dysfunctions.
Conversely, the attending physician would also know to adjust
treatment to higher levels if the clinical response were not
adequate (precluding toxicity). The magnitude of an administrated
dose in the management of the disorder of interest will vary with
the severity of the condition to be treated and to the route of
administration. The severity of the condition may, for example, be
evaluated, in part, by standard prognostic evaluation methods.
Further, the dose and perhaps dose frequency, will also vary
according to the age, body weight, and response of the individual
patient. A program comparable to that discussed above can be used
in veterinary medicine.
[0331] Depending on the specific conditions being treated, such
agents can be formulated and administered systemically or locally.
A variety of techniques for formulation and administration can be
found in Remington's Pharmaceutical Sciences, 18th Ed., Mack
Publishing Co., Easton, Pa. (1990), which is incorporated herein by
reference in its entirety. Suitable administration routes may
include oral, rectal, transdermal, vaginal, transmucosal, or
intestinal administration; parenteral delivery, including
intramuscular, subcutaneous, intramedullary injections, as well as
intrathecal, direct intraventricular, intravenous, intraperitoneal,
intranasal, or intraocular injections.
[0332] For injection, the agents of the embodiment can be
formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hanks' solution, Ringer's solution, or
physiological saline buffer. For such transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art.
Use of pharmaceutically acceptable carriers to formulate the
compounds herein disclosed for the practice of the embodiment into
dosages suitable for systemic administration is within the scope of
the embodiment. With proper choice of carrier and suitable
manufacturing practice, the compositions disclosed herein, in
particular, those formulated as solutions, can be administered
parenterally, such as by intravenous injection. The compounds can
be formulated readily using pharmaceutically acceptable carriers
well known in the art into dosages suitable for oral
administration. Such carriers enable the compounds of the
embodiment to be formulated as tablets, pills, capsules, liquids,
gels, syrups, slurries, suspensions and the like, for oral
ingestion by a patient to be treated.
[0333] Agents intended to be administered intracellularly can be
administered using techniques well known to those of ordinary skill
in the art. For example, such agents can be encapsulated into
liposomes, then administered as described above. All molecules
present in an aqueous solution at the time of liposome formation
are incorporated into the aqueous interior. The liposomal contents
are both protected from the external micro-environment and, because
liposomes fuse with cell membranes, are efficiently delivered into
the cell cytoplasm. Additionally, due to their hydrophobicity,
small organic molecules can be directly administered
intracellularly.
[0334] Determination of the effective amounts is well within the
capability of those skilled in the art, especially in light of the
detailed disclosure provided herein. In addition to the active
ingredients, these pharmaceutical compositions may contain suitable
pharmaceutically acceptable carriers comprising excipients and
auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically. The
preparations formulated for oral administration can be in the form
of tablets, dragees, capsules, or solutions. The pharmaceutical
compositions can be manufactured in a manner that is itself known,
for example, by means of conventional mixing, dissolving,
granulating, dragee-making, levitating, emulsifying, encapsulating,
entrapping, or lyophilizing processes.
[0335] Compounds disclosed herein can be evaluated for efficacy and
toxicity using known methods. For example, the toxicology of a
particular compound, or of a subset of the compounds, sharing
certain chemical moieties, can be established by determining in
vitro toxicity towards a cell line, such as a mammalian, and
preferably human, cell line. The results of such studies are often
predictive of toxicity in animals, such as mammals, or more
specifically, humans. Alternatively, the toxicity of particular
compounds in an animal model, such as mice, rats, rabbits, dogs or
monkeys, can be determined using known methods. The efficacy of a
particular compound can be established using several art recognized
methods, such as in vitro methods, animal models, or human clinical
trials. Art-recognized in vitro models exist for nearly every class
of condition, including the conditions abated by the compounds
disclosed herein, including cancer, cardiovascular disease, and
various immune dysfunction, and infectious diseases. Similarly,
acceptable animal models can be used to establish efficacy of
chemicals to treat such conditions. When selecting a model to
determine efficacy, the skilled artisan can be guided by the state
of the art to choose an appropriate model, dose, and route of
administration, and regime. Of course, human clinical trials can
also be used to determine the efficacy of a compound in humans.
[0336] As will be understood by one of skill in the art, "need" is
not an absolute term and merely implies that the patient can
benefit from the treatment of the anti-cancer agent in use. By
"patient" what is meant is an organism that can benefit by the use
of an anti-cancer agent.
[0337] "Therapeutically effective amount," "pharmaceutically
effective amount," or similar term, means that amount of drug or
pharmaceutical agent that will result in a biological or medical
response of a cell, tissue, system, animal, or human that is being
sought. In a preferred embodiment, the medical response is one
sought by a researcher, veterinarian, medical doctor, or other
clinician.
[0338] In one embodiment, a described compound, preferably a
compound having any one of Formulas I and II, including those as
described herein, is considered an effective anti-cancer agent if
the compound can influence 10% of the cancer cells, for example. In
a more preferred embodiment, the compound is effective if it can
influence 10 to 50% of the cancer cells. In an even more preferred
embodiment, the compound is effective if it can influence 50-80% of
the cancer cells. In an even more preferred embodiment, the
compound is effective if it can influence 80-95% of the cancer
cells. In an even more preferred embodiment, the compound is
effective if it can influence 95-99% of the cancer cells.
"Influence" is defined by the mechanism of action for each
compound.
EXAMPLES
General Synthetic Procedures
General Procedure A--HATU Mediated Amide Bond Formation
[0339] To an acid (1.1 eq with respect to amine) in anhydrous DMF
was added HATU (1 eq with respect to acid) and DIEA (2 eq with
respect to acid) and the mixture was stirred at room temperature
for 1 minute. The mixture was then added to a solution of amine in
DMF and the reaction mixture was stirred at room temperature till
the completion of the reaction (monitored by LC/MS). The solvent
was removed under reduced pressure and the residue was optionally
purified by reverse phase HPLC to give final pure product.
General Procedure B--DIC/HOAt Mediated Amide Bond Formation
[0340] To a stirred solution of carboxylic acid (1.1 eq), amine and
HOAt (1.1 eq) in anhydrous DMF was added DIC (1.1 eq) and the
reaction mixture was stirred at room temperature. Upon completion
(monitored by LC/MS), the solvent was removed under reduced
pressure and the residue was optionally purified by reverse phase
HPLC to give final pure product.
General Procedure C--Removal of Acid Sensitive Protecting Groups
(Boc, THP, t-Bu) Using HCl/Dioxane
[0341] The acid sensitive protecting groups containing compound was
dissolved in 4N HCl/dioxane and the mixture was stirred at room
temperature for 2 h. The solution was then concentrated under
reduced pressure and the residue was washed twice with cold ether.
Purification was carried out on reverse phase HPLC if
necessary.
General Procedure D--Removal of Fmoc Group
[0342] The Fmoc containing compound was dissolved in 2-5%
piperidine in DMF. The mixture was stirred at room temperature for
1 h. The solvents were removed under reduced pressure. Purification
was carried out on reverse phase HPLC if necessary.
General Procedure E--Reductive Alkylation
[0343] An amine was dissolved in DMF and aldehyde (5 eq) was added,
followed by addition of sodium cyanoborohydride (5 eq). HOAc was
added to adjust the pH of the reaction mixture to 4-5. The mixture
was stirred at room temperature till completion (1-4 h, monitored
by HPLC). Purification was carried out on reverse phase HPLC if
necessary.
General Procedure F--Saponification--Removal of Me/Ft from
Esters
[0344] To a stirred solution of an ester in MeOH was added 1M aq.
solution of LiOH till pH of the mixture was about 13-14 and the
reaction mixture was stirred at room temperature till completion
(.about.16 h, monitored by HPLC). Citric acid (.about.10% aq,) was
added to neutralize the reaction and the solvents were removed
under reduced pressure. The crude product was optionally purified
by RP-HPLC or used directly in the next step.
General Procedure G--Activation of a Hydroxyl/Phenol Group with
Bis(p-Nitrophenyl)Carbonate
[0345] To a stirred solution of an alcohol/phenol in THF/DMF (2/1)
was added bis(p-nitrophenyl) carbonate (3-5 eq), followed by DIEA
(2-4 eq) and the reaction mixture was stirred at room temperature
until most of the starting material was consumed. The progress of
the reaction was monitored by LC/MS. The crude product was
optionally purified by flash column chromatography or by
precipitation and washing.
General Procedure H--Reaction of an Amine with a Cyclic Anhydride
(Glutaric Anhydride or Succinic Anhydride)
[0346] An amine containing compound was dissolved in DMF. Glutaric
anhydride (3 eq) was added, followed by addition of DIEA (4 eq).
The reaction mixture was stirred at room temperature until most of
the starting material was consumed. The progress of the reaction
was monitored by LC/MS. The crude product was purified by RP-HPLC
to yield the pure carboxylic acid.
General Procedure I--Formation of Carbamate with p-Nitrophenyl
Carbonate (e.g. FmocVC-PAB-PNP)
[0347] An amine containing compound was dissolved in DMF and
alkyl/aryl p-nitrophenyl carbonate (1.5 eq) was added, followed by
addition of DIEA (2 eq) and HOBt (cat., 5%). The reaction mixture
was stirred at room temperature until most of the amine was
consumed. The progress of the reaction was monitored by LC/MS. The
crude product was optionally purified by RP-HPLC to yield the pure
carbamate.
General Procedure I--Formation of an Activated Ester (e.g. NHS)
from an Acid
[0348] An acid was dissolved in DCM and DMF was added to aid
dissolution if necessary. N-hydroxysuccinimide (1.5 eq) was added,
followed by EDC.HCl (1.5 eq). The reaction mixture was stirred at
room temperature for 1 h until most of the acid was consumed. The
progress of the reaction was monitored by RP-HPLC. The mixture was
then diluted with DCM and washed successively with citric acid (aq.
10%) and brine. The organic layer was dried and concentrated to
dryness. The crude product was optionally purified by RP-HPLC or
silica gel column chromatography.
General Scheme for Active Agent Conjugates Formation
Conjugation Method A. Conjugation on Lys Residues Via an Activated
Carboxylic Acid
##STR00091##
[0349] Conjugation Method B. Conjugation on Lys Residues Via
Reductive Alkylation with an Dialdehyde
##STR00092##
Conjugation Method C. Conjugation on Individual Cys Side Chain
Employing Maleimide Chemistry
##STR00093##
[0350] Conjugation Method D. Conjugation on Two Cys Side Chains by
Forming a Cyclic Structure
##STR00094##
[0351] Conjugation Method E. Conjugation on Carbonyl
(Ketone/Aldehyde) Bearing Biologics by Formation of an Oxime
Moiety
##STR00095##
[0352] Conjugation Method F. Conjugation on Azide Bearing Biologics
Using Copper-Free Click Chemistry:
##STR00096##
[0353] Experimental Description
Step 1. Drug-Linker Construct Synthesis (-L.sup.2-D)
Methods of Drug-Linker Construct Synthesis, but not Limited to:
[0354] Method 1-1: Linker and Drug Connected Via a Carbamate Bond.
The Following General Procedures were Employed:
General Procedure G and I for Activation and Carbamate Formation
General Procedure C, D, and F for Removal of Protective Groups for
Further Derivatization.
##STR00097## ##STR00098##
[0355] Method 1-2: Linker and Drug Connected Via Reductive
Alkylation (General Procedure E)
##STR00099##
[0356] Method 1-3. Active Molecule Containing a Carboxylic Acid
Moiety Connected to an Alkoxyamino Linker Via Formation of
Hydroxamate (General Procedure A or B), Followed by Removal of
Protective Groups.
##STR00100##
[0358] For active molecules that are hydroxamic acids, the above
method still can be employed since the construct will release
hydroxamic acid under enzymatic cleavage conditions. The reaction
needs to start from its corresponding carboxylic acid.
##STR00101##
Step 2. Introducing Functional Groups to L1-(L2-D)
[0359] Methods to Introduce Functional Groups that Suitable for
Conjugation Reaction, but No Limited to: Method 2-1. Compounds
Bearing Free Amino Group to React with an Cyclic Anhydride to
Introduce Carboxylic Acid (General Procedure H).
##STR00102##
Method 2-2. Compounds Bearing Free Amino Group to React with a
Di-Acid to Introduce Carboxylic Acid (General Procedure B).
##STR00103##
Method 2-3. Removal of Carboxylic Acid Protective Group to Reveal
the Free Carboxylic Acid (General Procedure C, F)
##STR00104##
[0360] Method 2-4. Reductive Alkylation of a Primary Amine with a
Dialdehyde Bearing a Carboxylic Acid Moiety (General Procedure
E)
##STR00105##
[0361] The amine (NH.sub.2-Ahx-Maytansinol) (20 mg) was dissolved
in acetonitrile (2 mL) and 1 mL of NaOAc buffer (100 mM, pH=4.0)
was added. The dialdehyde (0.5 M solution in water, 0.2 mL) was
added, followed by NaCNBH.sub.3 (10 mg). The reaction mixture was
stirred at room temperature for 30 mins and purified directly by
RP-HPLC to give the desired acid (16 mg) as a white solid after
lyophilization. MS found: 790.5 (M+H).sup.+.
[0362] The dialdehyde carboxylic acid was synthesized according to
the following scheme:
##STR00106##
[0363] The ester, 2H-Pyran-4-carboxylic acid,
2-ethoxyl-3,4-dihydro-ethyl ester was synthesized according to a
literature procedure (Chem Communications, (1) 25-26, 1998) was
saponified using General procedure F, followed by treatment with 1N
aq. HCl at room temperature for 1 h. The aq. solution of dialdehyde
was used directly without further purification.
Method 2-5. Introduction of o-Phenylenediamine Moiety
##STR00107##
[0364] The 3-nitro-4-amino benzoic acid was incorporated using
standard amidation reaction (General procedure B). The nitro group
was reduced using sodium dithionite (3 eq) in acetonitrile/water to
give the desired o-phenylene diamine. MS found: 1504.8
(M+H).sup.+.
Step 3. Introducing the Final Functional Groups Prior to
Conjugation
Methods of Introduction of Final Reactive Group Prior to
Conjugation Reaction, but not Limited to:
Method 3-1. Activation of a Carboxylic Acid to its Corresponding
Activated Form
##STR00108##
[0366] G is a leaving group selected from --F, --Cl, --Br, --I,
--N.sub.3, --OR (R=alkyl, aryl, heteroaryl, substituted aryl,
substituted heteroaryl), SR (R=alkyl, aryl heteroaryl, substituted
aryl, substituted heteroaryl), --ON(R.sup.1)R.sup.2, (R.sup.1,
R.sup.2 are each independently selected from --(C.dbd.O)--R, R=H,
alkyl, aryl, heteroaryl, substituted aryl, substituted heteroaryl
or R.sup.1 and R.sup.2 are connected to form a cyclic structure, or
R.sup.1=R.sup.2=(.dbd.C--R), RC(.dbd.O)O--, and RSO.sub.2--O--
(R=alkyl, aryl, heteroaryl, substituted aryl, substituted
heteroaryl).
[0367] The carboxylic acid can be activated using a variety of
methods to afford an activated form. For example, the carboxylic
acid can be activated using the following methods: A) Tetrahedron
61 (2005) 10827-10852; B) Beckwith, A. L. J. In The Chemistry of
Amides; Zabicky, J., Ed.; Synthesis of Amides; Interscience:
London, 1970; pp 105-109; C) Handbook of Reagents for Organic
Synthesis: Activating Agents and Protecting Groups; Pearson, A. J.,
Roush, W. R., Eds.; Wiley: New York, 1999; pp 370-373; D)
Lloyd-Williams, P., Albericio, F., and Giralt, E. (1997). Chemical
approaches to the synthesis of peptide and proteins (Series ed. C.
W. Rees). CRC Press, New York; E) Peptide chemistry: A practical
textbook: By M Bodansky. Springer-Verlag, Heidelberg. 1988; and F)
The practice of peptide synthesis, 2nd ed., by M. Bodansky and A.
Bodansky, Springer-Verlag, New York, each of which is incorporated
herein by reference in its entirety.
Method 3-2. Introducing a maleimido moiety
[0368] a. Via Amidation (General Procedure A, or B, or from an
Activated Ester Bearing a Maleimido Moiety)
##STR00109##
[0369] b. Converting an Existing Amino Group to Maleimide Directly
Using N-Methoxy-Carbonylmaleimide
##STR00110##
[0370] The amine (0.1 mmol) was dissolved in acetonitrile/water
(6/4, v/v, 3 mL). The mixture was cooled in an ice-water bath and
treated with sat. aq. NaHCO.sub.3 (0.5 mL), followed by
N-methoxycarbonylmaleimide (0.12 mmol). The mixture was stirred at
room temperature for 1 h. The pH was adjusted to 6-7 with citric
acid and the solution was concentrated. The residue was purified by
RP-HPLC to yield the desired maleimide as a white powder after
lyophilization (58%). MS found: 1091.2 (M+H).sup.+.
Method 3-3. Formation of Dibromomethyl Quinoxaline
##STR00111##
[0372] The o-phenylenediamino compound (12 mg) was dissolved in
acetonitrile/water. Dibromomethyl diketone (10 mg) was added. The
mixture was stirred at room temperature for 10 min and purified
directly by RP-HPLC to give the desired quinoxaline as a white
powder (12 mg) after lyophilization. MS found 1713.0
(M+H).sup.+.
Method 3-4. Incorporation of Hydroxylamine Moiety
##STR00112##
[0373] Method 3-5. Introduction of Cyclooctyne Moiety
##STR00113##
[0374] Example I. Synthesis of Compound 10
##STR00114## ##STR00115##
[0376] To a solution of compound 1 (23.4 g, 81.53 mmol) in dry EtOH
(200 mL) was added SOCl.sub.2 (100 mL) at 0.degree. C. The mixture
was stirred for overnight and the solvent was removed by
evaporation in vacuo. The residue was immediately used for the next
step without further purification. To a solution of compound 2
(81.53 mmol), compound 3 (50 g, 163.1 mmol) in dry DMF (150 mL) was
added DEPC (15.9 g, 97.8 mmol), TEA (41 g, 0.408 mol) at 0.degree.
C. The mixture was stirred for 2 h at 0.degree. C. Then the mixture
was stirred overnight at room temperature. Solvent was removed by
evaporation in vacuo. The residue was diluted with ethyl
acetate-toluene (2:1, 900 ml) and washed with 1M KHSO.sub.4, water,
sat. NaHCO.sub.3, and brine. The organic layer was dried and
concentrated to give a residue, which was purified by column
(hexanes:ethylacetate:DCM=5:1:1) to give 38 g of compound 4.
[0377] To a solution of Boc-Val-OH (30.6 g, 0.142 mol), compound 5
(from 25 g of compound 4) in DCM (400 mL) was added BrOP (28 g,
70.87 mmol), DIEA (30 g, 0.236 mol) at 0.degree. C. The mixture was
shielded from light and stirred for 0.5 h at 0.degree. C. Then the
mixture was stirred for 48 h at room temperature. The solvent was
removed by evaporation in vacuo. The residue was diluted with ethyl
acetate-toluene (3:1, 900 mL) and washed with 1M KHSO.sub.4, water,
sat. NaHCO.sub.3, and brine. The organic layer was dried and
concentrated to give a residue, which was purified by silica gel
column (hexanes:ethylacetate:DCM=3:1:1) to give 22 g of compound
7.
[0378] To a solution of compound 7 (40 g, 66.7 mmol) in THF (600
mL) was added a mixture of LiOH (14 g, 0.33 mol) in water (300 mL)
below 10.degree. C. The mixture was stirred for 5 days at
25.degree. C. THF was removed by evaporation. The aqueous layer was
washed with Et.sub.2O (200 mL.times.3). The aqueous layer was
acidified to pH 2 with 1N HCl at 0.degree. C., the mixture was
extracted with ethyl acetate and the organic layer was washed with
water and brine. The organic layer was dried and concentrated to
give a residue, which was purified by Prep-HPLC to give 14 g of
compound 8.
[0379] To a solution of compound 8 (3 g) in DCM (100 mL) was added
compound 9 (3 g, prepared according to General procedure J from
Boc-N-Me-Val-OH using EDC and pentafluorophenol). DIEA (2.6 mL) was
added, followed by HOBt (cat. 100 mg) and the reaction mixture was
stirred at room temperature for 16 h. The solvents were removed
under reduced pressure and the residue was purified on a silica gel
column to give compound 10 as a white powder (3.1 g). MS m/z Calcd
for C.sub.35H.sub.64N.sub.4O.sub.9 684.5, Found 707.6
([M+Na].sup.+).
Example II. Preparation of Cytotoxic Compounds--Sulfonamide
Derivatives
TABLE-US-00003 [0380] -L1-(L2-D) MS Synthetic Conj. found ID
Cytotoxic compound (D) method Method [M + H].sup.+ 13 ##STR00116##
1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D,
E, F 835.6 14 ##STR00117## 1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1,
3-2, 3-3, 3-4 A, B, C, D, E, F 809.6 16 ##STR00118## 1-1, 1-2 2-1,
2-2 2-3, 2-4 2-5, 3-1 3-2, 3-3 3-4 A, B, C, D, E, F 801.7 18
##STR00119## 1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4
A, B, C, D, E, F 836.6 30 ##STR00120## 1-1, 1-2, 2-1, 2-2, 2-3,
2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D, E, F 827.5 31 ##STR00121##
1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D,
E, F 807.8 32 ##STR00122## 1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1,
3-2, 3-3, 3-4 A, B, C, D, E, F 793.4 35 ##STR00123## 1-1, 1-2, 1-3,
2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D, E, F 839.6
37 ##STR00124## 1-1, 1-2, 1-3, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2,
3-3, 3-4 A, B, C, D, E, F 865.7 121 ##STR00125## 1-1, 1-2 2-1, 2-2
2-3, 2-4 2-5, 3-1 3-2, 3-3 3-4 A, B, C, D, E, F 838.3 122
##STR00126## 1-1, 1-2 2-1, 2-2 2-3, 2-4 2-5, 3-1 3-2, 3-3 3-4 A, B,
C, D, E, F 864.5 44 ##STR00127## 1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2,
3-4 A, B, C, D, E, F 1011.8 45 ##STR00128## 1-3, 2-1, 2-2, 2-3,
2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 1037.5 123 ##STR00129## 1-1,
1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D, E, F
823.2 124 ##STR00130## 1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2,
3-3, 3-4 A, B, C, D, E, F 837.5 125 ##STR00131## 1-1, 1-2, 2-1,
2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D, E, F 835.7 126
##STR00132## 1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4
A, B, C, D, E, F 871.7 127 ##STR00133## 1-1, 1-2, 2-1, 2-2, 2-3,
2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D, E, F 851.3 128
##STR00134## 1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4
A, B, C, D, E, F 817.5 129 ##STR00135## 1-1, 1-2, 2-1, 2-2, 2-3,
2-4, 2-5, 3-1, 3-2, 3-3, 3-4 A, B, C, D, E, F 817.1 130
##STR00136## 1-1, 1-2, 2-1, 2-2, 2-3, 2-4, 2-5, 3-1, 3-2, 3-3, 3-4
A, B, C, D, E, F 818.4
Example IIa. Synthesis of Compound 13, 14, 16, 18
##STR00137## ##STR00138##
[0382] The amino acid sulfonamide derivatives 11, 15, 17, 19 were
synthesized according to previously reported procedure (ARKIVOC
2004 (xii) 14-22) using Boc protected amino acid and
cyclopropyl/methyl sulfonamide, followed by removal of Boc (General
procedure C)
Example IIa-1. Synthesis of Compound 13
[0383] Compound 13 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between Boc-N-Me-Val-Val-Dil-Dap-OH
(compound 1) and amine II1, followed by removal of Boc (General
procedure C). The final compound was purified by reverse phase HPLC
to give compound 13 as a white powder after lyophilization. MS m/z
Calcd for C.sub.42H.sub.70N.sub.6O.sub.9S 834.5, Found 835.6
([M+H].sup.+).
Example IIa-2. Synthesis of Compound 14
[0384] Compound 14 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between Boc-N-Me-Val-Val-Dil-Dap-OH
(compound 10) and amine 15, followed by removal of Boc (General
procedure C). The final compound was purified by reverse phase HPLC
to give compound 14 as a white powder after lyophilization. MS n/z
Calcd for C.sub.40H.sub.68N.sub.6O.sub.9S 808.5, Found 809.6
([M+H].sup.+).
Example IIa-3. Synthesis of Compound 16
[0385] Compound 16 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between Boc-N-Me-Val-Val-Dil-Dap-OH
(compound 10) and amine 17, followed by removal of Boc (General
procedure C). The final compound was purified by reverse phase HPLC
to give compound 16 as a white powder after lyophilization. MS nm/z
Calcd for C.sub.39H.sub.72N.sub.6O.sub.9S 800.5, Found 801.7
([M+H].sup.+).
Example IIa-4. Synthesis of Compound 18
[0386] Compound 18 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between Boc-N-Me-Val-Val-Dil-Dap-OH
(compound 10) and amine 19, followed by removal of Boc (General
procedure C). The final compound was purified by reverse phase HPLC
to give compound 18 as a white powder after lyophilization. MS m/z
Calcd for C.sub.41H.sub.69N.sub.7O.sub.9S 835.5, Found 836.6
([M+H].sup.+).
Example IIb. Synthesis of Compound 27
##STR00139##
[0388] To a stirred solution of compound 1 (2.9 g, 10 mmol) and
compound 11 (HCl salt, 3 g) in anhydrous DMF (100 mL) was added
HOBt (1.54 g) and EDC.HCl (2 g). DIEA (20 mmol) was added and the
mixture was stirred at room temperature for 16 h. The solvents were
removed under reduced pressure and the residue was dissolved in
ethyl acetate (500 mL) and washed successively with Citric acid
(10%, aq., 200 mL). NaHCO3 (sat. aq., 200 mL) and brine. The
organic layer wad dried and evaporated to dryness to give compound
20 as a syrup which was treated with 6N HCl in iPr-OH (100 mL) for
1 h to give compound 21 after concentration. Compound 21 was used
directly without further purification.
[0389] To a solution of Boc-Val-OH (2.5 g), compound 22 in DCM (150
mL) was added PyBrOP (11 mmol), DIEA (22) at 0.degree. C. The
mixture was shielded from light and stirred for 0.5 h at 0.degree.
C. Then the mixture was stirred for 24 h at room temperature. The
solvent was removed by evaporation in vacuo. The residue was
diluted with ethyl acetate (300 mL) and washed with 1M KHSO.sub.4,
water, sat. NaHCO.sub.3, and brine. The organic layer was dried and
concentrated to give a residue, which was purified by silica gel
column (hexanes: ethyl acetate) to give 3.2 g of compound 23.
[0390] The compound 23 (3 g) was dissolved in DCM (100 mL) and TFA
(30 mL) was added. The mixture was stirred at room temperature for
2 h and concentrated under reduced pressure. The residue was
dissolved in dioxane (200 mL). Sat. aq. NaHCO.sub.3 (80 mL) was
added, followed by Boc anhydride (2.2 g). The mixture was stirred
at room temperature for 4 h and neutralized to pH 3-4 using 1N HCl.
Solvents were removed and the residue was dissolved in ethyl
acetate, which was washed with water and brine. The organic layer
was concentrated and the residue was purified on a silica gel
column to give compound 25 as a syrup.
[0391] Compound 27 was synthesized using the general procedures
described above as following: HATU mediated amide bond formation
(General procedure A) between compound 21 and amine 25, followed by
removal of Boc (General procedure C). The final compound was
purified by reverse phase HPLC to give compound 27 as a white
powder after lyophilization. MS m/z Calcd. for
C.sub.36H.sub.59N.sub.5O.sub.8S 721.4, Found 722.5
([M+H].sup.+).
Example IIc. Synthesis of Compounds 30, 31, and 31
##STR00140##
[0392] Example IIc-1. Synthesis of Compound 30
[0393] Compound 30 was synthesized using the general procedures
described above as following: HATU mediated amide bond formation
(General procedure A) between .beta.-Cl--N-Boc-Ala-OH (compound 28)
and amine 27, followed by removal of Boc (General procedure C). The
final compound was purified by reverse phase HPLC to give compound
30 as a white powder after lyophilization. MS m/z Calcd for
C.sub.39H.sub.63ClN.sub.6O.sub.9S 826.4, Found 827.5
([M+H].sup.+).
Example IIc-2. Synthesis of Compound 31
[0394] Compound 31 was synthesized using the general procedures
described above as following: HATU mediated amide bond formation
(General procedure A) between N-Boc-Aib-OH and amine 27 followed by
removal of Boc (General procedure C). The final compound was
purified by reverse phase HPLC to give compound 31 as a white
powder after lyophilization. MS m/z Calcd. for
C.sub.40H.sub.66N.sub.6O.sub.9S 806.5, Found 807.8
([M+H].sup.+).
Example IIc-3. Synthesis of Compound 32
[0395] Compound 32 was synthesized using the general procedures
described above as following: HATU mediated amide bond formation
(General procedure A) between N-Boc-Sar-OH and amine 27, followed
by removal of Boc (General procedure C). The final compound was
purified by reverse phase HPLC to give compound 32 as a white
powder after lyophilization. MS nm/z Calcd. for
C.sub.39H.sub.64N.sub.6O.sub.9S 792.5, Found 793.4
([M+H].sup.+).
Example IId. Synthesis of Compounds 35 and 37
##STR00141##
[0397] The amino acid sulfonamide derivatives 33 and 36 were
synthesized according to previously reported procedure (WO
2007146695) using Boc protected amino acid and cyclopropyl/methyl
sulfonamide, followed by removal of Boc (General procedure C)
Example IId-1. Synthesis of Compound 35
[0398] Compound 35 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between Boc-N-Me-Val-Val-Dil-Dap-OH
(compound 10) and amine 33, followed by removal of Boc (General
procedure C). The final compound was purified by reverse phase HPLC
to give compound 35 as a white powder after lyophilization. MS m/z
Calcd for C.sub.41H.sub.70N.sub.6O.sub.10S 838.5, Found 839.6
([M+H].sup.-).
Example IId-2. Synthesis of Compound 37
[0399] Compound 37 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between Boc-N-Me-Val-Val-Dil-Dap-OH
(compound 10) and amine 36, followed by removal of Boc (General
procedure C). The final compound was purified by reverse phase HPLC
to give compound 37 as a white powder after lyophilization. MS m/z
Calcd for C.sub.43H.sub.72N.sub.6O.sub.10S 864.5, Found 865.7
([M+H].sup.+).
Example IIe. Synthesis of Compounds 44 and 45
##STR00142##
[0400] Example IIe-1. Synthesis of Compound 44
[0401] The phenol 35 (1 mmol) was treated with 3 eq of
bis(p-nitrophenyl)carbonate to form the activated carbonate 39
(general procedure G). The crude product was used directly without
further purification. 6-Aminohexanoic acid (5 eq) was dissolved in
sat. aq. NaHCO.sub.3 (5 mL) and the solution was added. The
reaction mixture was stirred at room temperature for 16 h. Citric
acid (aq. 10%) was added to acidify the reaction (pH=4-5) and then
diluted with EtOAc (150 mL). Organic layer was dried (over
Na.sub.2SO.sub.4) and concentrated to give the crude product 40
which underwent the following procedures: removal of Boc (General
procedure C), reductive alkylation using HCHO (General procedure
E), DIC/HOAt mediated amide bond formation (General procedure B)
between compound 42 and THP--O--NH.sub.2, followed by removal of
THP (General procedure C, using 4N aq. HCl). The final compound was
purified by reverse phase HPLC to give compound 44 as a white
powder after lyophilization. MS m/z Calcd for
C.sub.4H.sub.82N.sub.80O.sub.3S 1010.6, Found 1011.8
([M+H].sup.+).
Example IIe-2. Synthesis of Compound 45
[0402] The compound 45 was synthesized according to the same
procedures as described for the synthesis of compound 44. The final
compound was purified by reverse phase HPLC to give compound 45 as
a white powder after lyophilization. MS m/z Calcd for
C.sub.50H.sub.84N.sub.8O.sub.13S 1036.6, Found 1037.5
([M+H].sup.+).
Example III Synthesis of Cytotoxic Compounds
TABLE-US-00004 [0403] -L.sup.1-(L.sup.2-D) MS Synthetic Conjugation
found ID Cytotoxic compound (D) method Method [M + H].sup.+ 49
##STR00143## 1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E,
F 712.5 50 ##STR00144## 1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A,
B, C, D, E, F 738.5 52 ##STR00145## 1-3, 2-1, 2-2, 2-3, 2-4, 3-1,
3-2, 3-4 A, B, C, D, E, F 784.7 53 ##STR00146## 1-3, 2-1, 2-2, 2-3,
2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 788.7 54 ##STR00147## 1-3, 2-1,
2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 772.5 137
##STR00148## 1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E,
F 682.4 138 ##STR00149## 1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A,
B, C, D, E, F 698.6
##STR00150##
Example III-1. Synthesis of Compound 49
[0404] Compound 49 was synthesized using the general procedures
described above as following: HATU mediated amide bond formation
(General procedure A) between Boc-N-Me-Val-Val-Dil-Dap-OH (compound
10) and NH.sub.2-Leu-OMe, followed by removal of Boc (General
procedure C), reductive alkylation with HCHO (General procedure E)
and saponification to remove methyl group from ester (General
procedure F). The final compound was purified by reverse phase HPLC
to give compound 49 as a white powder after lyophilization. MS m/z
Calcd for C.sub.37H.sub.69N.sub.5O.sub.8 711.5, Found 712.5
([M+H].sup.+).
Example III-2. Synthesis of Compound 50
[0405] Compound 50 was synthesized using the general procedures
described above as following: HATU mediated amide bond formation
(General procedure A) between Boc-N-Me-Val-Val-Dil-Dap-OH (compound
10) and amine 51, followed by removal of Boc (General procedure C),
reductive alkylation with HCHO (General procedure E) and
saponification to remove methyl group from ester (General procedure
F). The final compound was purified by reverse phase HPLC to give
compound 51 as a white powder after lyophilization. MS m/z Calcd
for C.sub.39H.sub.71N.sub.5O.sub.8 737.5, Found 738.5
([M+H].sup.+).
[0406] Compound 51 was synthesized according to literature
procedures (J. Org. Chem., 2001, 66, 7355-7364)
Example III-3. Synthesis of Compound 52
[0407] Compound 52 was synthesized using the general procedures
described above as following: HATU mediated amide bond formation
(General procedure A) between Boc-N-Me-Val-Val-Dil-Dap-OH (compound
1) and amine 22, followed by removal of Boc and t-Bu (General
procedure C), reductive alkylation with HCHO (General procedure E).
The final compound was purified by reverse phase HPLC to give
compound 52 as a white powder after lyophilization. MS m/z Calcd
for C.sub.41H.sub.66N.sub.5O.sub.8 783.6, Found 784.7
([M+H].sup.+).
Example III-4. Other Compounds Synthesized Using the Same
Procedures as Described for the Synthesis of Compound 49
[0408] For compound 53: MS m/z Calcd for
C.sub.43H.sub.73N.sub.5O.sub.8 787.6, Found 788.7
([M+H].sup.+).
[0409] For compound 54: MS m/z Calcd for
C.sub.42H.sub.69N.sub.5O.sub.8 771.5 Found 772.5 ([M+H].sup.+).
##STR00151##
Example III-5
##STR00152##
[0410] Example III-5a. Synthesis of Compound 137
[0411] The intermediate 136 was synthesized using the general
procedures described above as following (0.1 mmol scale): HATU
mediated amide bond formation (General procedure A) between
Boc-N-Mc-Val-Val-Dil-Dap-OH (compound 1) and amine 134, followed by
removal of Boc (General procedure C). The HCl salt 11 was dissolved
in water (5 mL) and hexane (5 mL) was added. Phenylboronic acid (10
eq) was added and the suspension was stirred vigorously at room
temperature for 1 h. Hexane layer was removed and fresh hexane (5
mL) was added. The mixture was agitated for another 2 h and the
aqueous layer was collect and concentrated. The final compound was
purified by reverse phase HPLC to give compound 12 as a white
powder after lyophilization. MS m/z Calcd for
C.sub.34H.sub.64BN.sub.5O.sub.8 681.5, Found 682.4
([M+H].sup.+).
Example III-5b. Synthesis of Compound 138
[0412] The compound 138 was synthesized employing the same sequence
as described for the preparation of compound 137. (compound 139 as
a starting material). It was obtained as a white powder after
RP-HPLC purification and lyophilization. MS m/z Calcd for
C.sub.35H.sub.68BN.sub.5O.sub.8 697.5, Found 698.6
([M+H].sup.+).
Example IV. Preparation of Cytotoxic Compounds--Hydroxamic Acid
Derivatives
TABLE-US-00005 [0413] -L.sup.1-(L.sup.2-D) MS Synthetic Conjugation
found ID Cytotoxic compound (D) method Method [M + H].sup.+ 56
##STR00153## 1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E,
F 727.6 57 ##STR00154## 1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A,
B, C, D, E, F 761.6 59 ##STR00155## 1-3, 2-1, 2-2, 2-3, 2-4, 3-1,
3-2, 3-4 A, B, C, D, E, F 489.5 58 ##STR00156## 1-3, 2-1, 2-2, 2-3,
2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 743.5 60 ##STR00157## 1-3, 2-1,
2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 753.5 61 ##STR00158##
1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 799.5 73
##STR00159## 1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A, B, C, D, E,
F 719.3 74 ##STR00160## 1-3, 2-1, 2-2, 2-3, 2-4, 3-1, 3-2, 3-4 A,
B, C, D, E, F 759.4 75 ##STR00161## 1-3, 2-1, 2-2, 2-3, 2-4, 3-1,
3-2, 3-4 A, B, C, D, E, F 725.2 76 ##STR00162## 1-3, 2-1, 2-2, 2-3,
2-4, 3-1, 3-2, 3-4 A, B, C, D, E, F 685.7
##STR00163## ##STR00164##
Example IV-1. Synthesis of Compound 56
[0414] Compound 56 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between
Me.sub.2-Val-Val-Dil-Dap-LeuOH (compound 49) and THP--O--NH.sub.2,
followed by removal of THP (General procedure C, using 4N aq. HCl).
The final compound was purified by reverse phase HPLC to give
compound 56 as a white powder after lyophilization. MS m/z Calcd
for C.sub.37H.sub.70N.sub.6O.sub.8 726.5, Found 727.6
([M+H].sup.+).
Example IV-2. Synthesis of Compound 57
[0415] Dimethyl Auristatin F was synthesized from compound 10 and
NH.sub.2-Phe-OMe using the synthetic procedures described above for
the synthesis of compound 49.
[0416] Compound 57 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between dimethyl auristatin F and
THP--O--NH.sub.2, followed by removal of THP (General procedure C,
using 4N aq. HCl). The final compound was purified by reverse phase
HPLC to give compound 57 as a white powder after lyophilization. MS
n/z Calcd for C.sub.40H.sub.68N.sub.6O.sub.8 760.5, Found 761.6
([M+H].sup.+).
Example IV-3. Synthesis of Compound 58
[0417] Compound 58 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between Tubulysin (J. Am. Chem.
Soc., 2006, 128 (50), pp 16018-16019) and THP--O--NH.sub.2,
followed by removal of THP (General procedure C, using 4N aq. HCl).
The final compound was purified by reverse phase HPLC to give
compound 45 as a white powder after lyophilization. MS m/z Calcd
for C.sub.38H.sub.58N.sub.6O.sub.7S 742.4, Found 743.5
([M+H].sup.+).
Example IV-4. Synthesis of Compound 59
[0418] Compound 59 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between HTI-286 (Bioorg Med Chem
Lett. 2004, 14(16):4329-32) and THP--O--NH.sub.2, followed by
removal of THP (General procedure C, using 4N aq. HCl). The final
compound was purified by reverse phase HPLC to give compound 59 as
a white powder after lyophilization. MS m/z Calcd for
C.sub.27H.sub.44N.sub.4O.sub.4 488.3, Found 489.5
([M+H].sup.+).
Example IV-5. Synthesis of Compound 60
[0419] Compound 60 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between compound 50 and
THP--O--NH.sub.2, followed by removal of THP (General procedure C,
using 4N aq. HCl). The final compound was purified by reverse phase
HPLC to give compound 47 as a white powder after lyophilization. MS
m/z Calcd for C.sub.39H.sub.72N.sub.6O.sub.8 752.5, Found 753.5
([M+H].sup.+).
Example IV-6. Synthesis of Compound 61
[0420] Compound 61 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between compound 52 and
THP--O--NH.sub.2, followed by removal of THP (General procedure C,
using 4N aq. HCl). The final compound was purified by reverse phase
HPLC to give compound 61 as a white powder after lyophilization. MS
m/z Calcd for C.sub.41H.sub.78N.sub.6O.sub.9 798.5, Found 799.5
([M+H].sup.+).
Example V. Synthesis of Alkoxyamine Linkers 65, 66, 67, and 68
TABLE-US-00006 [0421] ID Structure 65 ##STR00165## 66 ##STR00166##
67 ##STR00167## 68 ##STR00168##
##STR00169##
Example V-1. Synthesis of Compound 65
[0422] To a stirred solution of Fmoc-VA-PAB (62) (Bioconjugate
Chem., 2002, 13, 855-859) (9 g, 15 mmol) in THF (200 mL) was added
thionyl chloride (18 mmol) dropwise. After the addition was
complete, the reaction mixture was stirred at room temperature for
1 h. TLC analysis (ethyl acetate/hexane, 1/1, v/v) showed the
completion of the reaction. The solvents were removed under reduced
pressure and the residue was washed with hexanes (100 mL) to give
compound 63 as a slightly yellowish solid (8.8 g).
[0423] Compound 63 (6.2 g, 10 mmol) was dissolved in anhydrous DMF
(100 mL). N-Hydroxy-phthalimide (3.2 g, 20 mmol) was added,
followed by solid NaHCO.sub.3 (3.4 g, 40 mmol). The reaction
mixture was stirred at room temperature for 48 h. TLC analysis
showed that most of compound 61 was consumed. The reaction was then
diluted with ethyl acetate (500 mL) and washed successively with
sat. aq. NaHCO.sub.3 (3.times.200 mL) and brine (200 mL). The
organic layer was dried and concentrated to give compound 64 as a
tan solid, which was used directly without further
purification.
[0424] The crude compound 64 from previous step was dissolved in
DMF (100 mL). HOAc (6 mL) was added, followed by hydrazine hydrate
(5 mL). The reaction was stirred at room temperature for 1 h. LC/MS
showed the completion of the reaction. The reaction mixture was
then poured into a beaker containing IL of water under stirring.
The precipitated solid was collected via filtration and washed
twice with water to give compound 65 as a white solid (purity
>85%, can be used directly). Pure compound 63 was obtained after
RP-HPLC purification. MS m/z Calcd for
C.sub.30H.sub.34N.sub.4O.sub.5 530.3, Found 531.4
([M+H].sup.+).
Example V-2. Synthesis of Compound 66
[0425] Compound 66 was synthesized starting from compound
Fmoc-VC-PAB (Bioconjugate Chem., 2002, 13, 855-859) using the
procedures described above for the synthesis of compound 63. MS m/z
Calcd for C.sub.33H.sub.40N.sub.6O.sub.6 616.3, Found 617.5
([M+H].sup.+).
Example V-3. Synthesis of Compound 67
[0426] Compound 64 was synthesized starting from compound
Fmoc-A-PAB (synthesized according to the procedure reported:
Bioconjugate Chem., 2002, 13, 855-859) using the procedures
described above for the synthesis of compound 63. MS m/z Calcd for
C.sub.25H.sub.25N.sub.3O.sub.4 431.2, Found 432.6
([M+H].sup.+).
Example V-4. Synthesis of Compound 68
[0427] Compound 68 was synthesized starting from compound
Fmoc-Ahx-PAB using the procedures described above for the synthesis
of compound 68. MS m/z Calcd for C.sub.28H.sub.31N.sub.3O.sub.4
473.2, Found 474.3 ([M+H].sup.+).
Example VIII. Synthesis of -L.sup.1-(L.sup.2-D)-
TABLE-US-00007 [0428] MS Conj. found ID Structure of
-L.sup.1-(L.sup.2-D) Method [M + H].sup.+ 83 ##STR00170## A 1150.9
84 ##STR00171## A 1237.1 85 ##STR00172## A 1051.9 86 ##STR00173## A
1093.9 88 ##STR00174## A 1091.9 89 ##STR00175## A 1118.0 90
##STR00176## A 1263.1 91 ##STR00177## A 1274.8 93 ##STR00178## A
1278.9 92 ##STR00179## A 1218.9 94 ##STR00180## A 878.4
##STR00181##
Example VIII-1. Synthesis of Compound 83
[0429] Compound 83 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between Auristatin F and compound
65, followed by removal of Fmoc (General procedure D), and reaction
with glutaric anhydride (General procedure H). The final compound
was purified by reverse phase HPLC to give compound 83 as a white
powder after lyophilization. MS m/z Calcd for
C.sub.60H.sub.95N.sub.9O.sub.13 1149.7, Found 1150.9
([M+H].sup.+).
##STR00182##
Example VIII-2 Synthesis of Compound 84
[0430] Compound 84 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between Auristatin F and compound
66, followed by removal of Fmoc (General procedure D), and reaction
with glutaric anhydride (General procedure H). The final compound
was purified by reverse phase HPLC to give compound 84 as a white
powder after lyophilization. MS m/z Calcd for
C.sub.6H.sub.101N.sub.11O.sub.14 1235.8, Found 1237.1
([M+H].sup.+).
Example VIII-3. Synthesis of Compound 85
[0431] Compound 85 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between Auristatin F and compound
67, followed by removal of Fmoc (General procedure D), and reaction
with glutaric anhydride (General procedure H). The final compound
was purified by reverse phase HPLC to give compound 85 as a white
powder after lyophilization. MS n/z Calcd for
C.sub.55H.sub.86N.sub.8O.sub.12 1050.6, Found 1051.9
([M+H].sup.+).
Example VIII-4. Synthesis of Compound 86
[0432] Compound 86 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between Auristatin F and compound
68, followed by removal of Fmoc (General procedure D), and reaction
with glutaric anhydride (General procedure H). The final compound
was purified by reverse phase HPLC to give compound 86 as a white
powder after lyophilization. MS m/z Calcd for
C.sub.58H.sub.92N.sub.8O.sub.12 1092.7, Found 1093.9
([M+H].sup.+).
Example VIII-5. Synthesis of Compound 88
##STR00183##
[0434] Compound 88 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between compound 49 and compound
67, followed by removal of Fmoc (General procedure D), and amide
formation with acid 87 using HATU (General procedure A, 3 eq of
acid 87 and 1 eq of HATU were used). The final compound was
purified by reverse phase HPLC to give compound 88 as a white
powder after lyophilization. MS m/z Calcd for
C.sub.55H.sub.94N.sub.5O.sub.14 1090.7, Found 1091.9
([M+H].sup.+).
Example VIII-6. Synthesis of Compound 89
[0435] Compound 89 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between compound 50 and compound
67, followed by removal of Fmoc (General procedure D), and amide
formation with acid 87 using HATU (General procedure A, 3 eq of
acid 87 and 1 eq of HATU were used). The final compound was
purified by reverse phase HPLC to give compound 89 as a white
powder after lyophilization. MS m/z Calcd for C59H96N8O14 1116.7,
Found 1118.0 ([M+H].sup.+).
Example VIII-7. Synthesis of Compound 90
[0436] Compound 90 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between compound 54 and compound
66, followed by removal of Fmoc (General procedure D), and reaction
with glutaric anhydride (General procedure H). The final compound
was purified by reverse phase HPLC to give compound 90 as a white
powder after lyophilization. MS m/z Calcd for
C.sub.65H.sub.10N.sub.11O.sub.14 1261.8, Found 1263.1
([M+H].sup.+).
##STR00184##
Example VIII-8. Synthesis of Compound 91
[0437] Compound 91 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between compound 52 and compound
66, followed by removal of Fmoc (General procedure D), and reaction
with glutaric anhydride (General procedure H). The final compound
was purified by reverse phase HPLC to give compound 91 as a white
powder after lyophilization. MS m/z Calcd for
C.sub.64H.sub.111N.sub.11O.sub.15 1273.8, Found 1274.8 ([M+H]f.
Example VIII-9. Synthesis of Compound 92
[0438] Compound 92 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between Tubulysin M and compound
66, followed by removal of Fmoc (General procedure D), and reaction
with glutaric anhydride (General procedure H). The final compound
was purified by reverse phase HPLC to give compound 92 as a white
powder after lyophilization. MS m/z Calcd for
C.sub.61H.sub.91N.sub.11O.sub.13S 1217.7, Found 1218.9
([M+H].sup.+).
Example VIII-10. Synthesis of Compound 93
[0439] Compound 93 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between compound 53 and compound
66, followed by removal of Fmoc (General procedure D), and reaction
with glutaric anhydride (General procedure H). The final compound
was purified by reverse phase HPLC to give compound 93 as a white
powder after lyophilization. MS m/z Calcd for
C.sub.66H.sub.107N.sub.11O.sub.14 1277.8, Found 1278.9
([M+H].sup.+).
##STR00185##
Example VIII-11. Synthesis of Compound 94
[0440] Compound 94 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between compound HTI-286 and
compound 66, followed by removal of Fmoc (General procedure D), and
reaction with glutaric anhydride (General procedure H). The final
compound was purified by reverse phase HPLC to give compound 94 as
a white powder after lyophilization. MS min Calcd for
C.sub.47H.sub.71N.sub.71O.sub.9 877.5, Found 878.4
([M+H].sup.+).
Example IX Synthesis of -L1-(L2-D)-
TABLE-US-00008 [0441] MS found Conj. [M + ID Structure of
-L.sup.1-(L.sup.2-D) Method H].sup.+ 97 ##STR00186## A 1354.9 98
##STR00187## A 1243.8 99 ##STR00188## A 995.8 102 ##STR00189## A
1135.6 103 ##STR00190## A 1209.8 104 ##STR00191## A 1486.9 105
##STR00192## A 1512.9 131 ##STR00193## A 1444.0 110 ##STR00194## A
1020.8 113 ##STR00195## A 923.7 114 ##STR00196## A 1034.7 115
##STR00197## A 1325.9 132 ##STR00198## A 976.3
Example IX-1. Synthesis of Compound 97
##STR00199##
[0443] Compound 97 was synthesized using the general procedures
described above as following: Carbamate formation (General
procedure 1) between compound 13 and Fmoc-VC-PAB-PNP, followed by
removal of Fmoc (General procedure D), and reaction with glutaric
anhydride (General procedure H). The final compound was purified by
reverse phase HPLC to give compound 97 as a white powder after
lyophilization. MS nm/z Calcd for
C.sub.66H.sub.103N.sub.11O.sub.17S 1353.7, Found 1354.9
([M+H].sup.+).
Example IX-2. Synthesis of Compound 98
##STR00200##
[0445] Compound 98 was synthesized using the general procedures
described above as following: Carbamate formation (General
procedure I) between compound 4 and Fmoc-A-PAB-PNP, followed by
removal of Fmoc (General procedure D), and amide formation with
acid 87 using HATU (General procedure A, 3 eq of acid 87 and 1 eq
of HATU were used). The final compound was purified by reverse
phase HPLC to give compound 98 as a white powder after
lyophilization. MS m/z Calcd for C.sub.61H.sub.94N.sub.8O.sub.17S
1242.7, Found 1243.8 ([M+H].sup.+).
Example IX-3 Synthesis of Compound 99
##STR00201##
[0447] Compound 99 was synthesized using the general procedures
described above as following: reaction of compound 13 and aldehyde
100 under reductive alkylation conditions (General procedure E) and
Fmoc-A-PAB-PNP, followed by removal of t-Bu ester (General
procedure C). The final compound was purified by reverse phase HPLC
to give compound 99 as a white powder after lyophilization. MS m/z
Calcd for C.sub.49H.sub.82N.sub.6O.sub.13S 994.6, Found 995.8
([M+H].sup.+).
Example IX-4 Synthesis of Compound 102
##STR00202##
[0449] Compound 102 was synthesized using the general procedures
described above as following: Carbamate formation (General
procedure I) between compound 16 and Fmoc-A-PAB-PNP, followed by
removal of Fmoc (General procedure D), and reaction with glutaric
anhydride (General procedure H). The final compound was purified by
reverse phase HPLC to give compound 102 as a white powder after
lyophilization. MS m/z Calcd for C.sub.55H.sub.90N.sub.5O.sub.15S
1134.6, Found 1135.6 ([M+H].sup.+).
Example IX-5 Synthesis of Compound 103
##STR00203##
[0451] Compound 103 was synthesized using the general procedures
described above as following: Carbamate formation (General
procedure I) between compound 16 and Fmoc-A-PAB-PNP, followed by
removal of Fmoc (General procedure D), and amide formation with
acid 87 using HATU (General procedure A, 3 eq of acid 87 and 1 eq
of HATU were used). The final compound was purified by reverse
phase HPLC to give compound 103 as a white powder after
lyophilization. MS m/z Calcd for C.sub.58H.sub.96N.sub.8O.sub.17S
1208.7, Found 1209.8 ([M+H].sup.+).
Example IX-6. Synthesis of Compound 104
##STR00204##
[0453] Compound 104 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between compound 42 and compound
66, followed by removal of Fmoc (General procedure D), and reaction
with glutaric anhydride (General procedure H). The final compound
was purified by reverse phase HPLC to give compound 104 as a white
powder after lyophilization. MS m/z Calcd for
C.sub.1H.sub.115N.sub.13O.sub.19S 1485.8, Found 1486.9
([M+H].sup.+).
Example IX-7. Synthesis of Compound 105
##STR00205##
[0455] Compound 105 was synthesized in the same manner as described
for the synthesis of compound 104. The final compound was purified
by reverse phase HPLC to give compound 105 as a white powder after
lyophilization. MS m/z Calcd for C.sub.73H.sub.117N.sub.13O.sub.19S
1511.8, Found 1512.9 ([M+H].sup.+).
Example IX-8. Synthesis of Compound 110
##STR00206##
[0457] The phenol 106 (1 mmol) was treated with 3 eq of
bis(p-nitrophenyl)carbonate to form the activated carbonate 107
(general procedure G). The crude product was used directly without
further purification. Piperidine 4-carboxylic acid (5 eq) was
dissolved in sat. aq. NaHCO.sub.3 (5 mL) and the solution was
added. The reaction mixture was stirred at room temperature for 8
h. Citric acid (aq. 10%) was added to acidify the reaction (pH=4-5)
and then diluted with EtOAc (150 mL). Organic layer was dried (over
Na.sub.2SO.sub.4) and concentrated to give the crude product 108
which underwent the following procedures: removal of Boc (General
procedure C), and reductive alkylation using HCHO (General
procedure E). The final compound was purified by reverse phase HPLC
to give compound 110 as a white powder after lyophilization. MS m/z
Calcd for C.sub.80H.sub.81N.sub.7O.sub.13S 1019.6, Found 1020.8
([M+H].sup.+).
Example IX-9. Synthesis of Compound 113
##STR00207##
[0459] To a stirred solution of compound 106 (0.2 mmol, 190 mg) in
anhydrous DMF (5 mL) was added t-butyl bromoacetate (0.3 mmol),
followed by solid potassium carbonate (55 mg, 0.4 mmol). The
reaction mixture was stirred at room temperature for 2 h. LC/MS
confirmed that the completion of the reaction. The mixture was
diluted with EtOAc (100 mL) and washed with 10% aq. Citric acid and
brine. The organic layer was dried and concentrated to dryness to
give the crude compound 111, which underwent the following
procedures: removal of Boc and t-Bu (General procedure C), and
reductive alkylation using HCHO (General procedure E). The final
compound was purified by reverse phase HPLC to give compound 113 as
a white powder after lyophilization. MS m/z Calcd for
C.sub.45H.sub.74N.sub.6O.sub.12S, 922.5 Found 923.7
([M+H].sup.+).
Example IX-10. Synthesis of Compound 114
##STR00208##
[0461] Compound 114 was synthesized using the general procedures
described above as following: HATU mediated amide bond formation
(General procedure A) between compound 113 and methyl
isonipecotate, followed by saponification to remove methyl group
from ester (General procedure F). The final compound was purified
by reverse phase HPLC to give compound 114 as a white powder after
lyophilization. MS n/z Calcd for C.sub.51H.sub.83N.sub.7O.sub.13S
1033.6, Found 1034.7 ([M+H].sup.+).
Example IX-11. Synthesis of Compound 115
##STR00209##
[0463] Compound 115 was synthesized using the general procedures
described above as following: DIC/HOAt mediated amide bond
formation (General procedure B) between compound 113 and compound
67, followed by removal of Fmoc (General procedure D), HATU
mediated amidation reaction with acid 116 (General procedure A),
and saponification to remove methyl group from ester (General
procedure F). The final compound was purified by reverse phase HPLC
to give compound 115 as a white powder after lyophilization. MS
nm/z Calcd for C.sub.65H.sub.100N.sub.10O.sub.17S 1324.7, Found
1325.9 ([M+H].sup.+).
Example X. Introduction of the Final Function Group Prior to
Conjugation
TABLE-US-00009 [0464] MS Conjugation found ID Structure Method [M +
H].sup.+ 101 ##STR00210## C 1433.9 118 ##STR00211## C 1091.2 120
##STR00212## D 1829.5 133 ##STR00213## B 1136.6 136 ##STR00214## E
1026.6 139 ##STR00215## F 1184.8
Example X-1. Synthesis of Compound 101
##STR00216##
[0466] Compound 101 was synthesized using the general procedures
described above as following: Carbamate formation (General
procedure 1) between compound 13 and FmocVC-PAB-PNP, followed by
removal of Fmoc (General procedure D), and amidation reaction with
6-maleimidohexanoic acid (General procedure A). The final compound
was purified by reverse phase HPLC to give compound 101 as a white
powder after lyophilization. MS m/z Calcd for
C.sub.71H.sub.108N.sub.12O.sub.17S 1432.8, Found 1433.9
([M+H].sup.+).
Example X-2. Converting an Existing Amino Group to Maleimide
Directly Using N-Methoxy-Carbonylmaleimide
##STR00217##
[0468] The amine (117, 0.1 mmol) was dissolved in
acetonitrile/water (6/4, v/v, 3 mL). The mixture was cooled in an
ice-water bath and treated with sat. aq. NaHCO.sub.3 (0.5 mL),
followed by N-methoxycarbonylmaleimide (0.12 mmol). The mixture was
stirred at room temperature for 1 h. The pH was adjusted to 6-7
with citric acid and the solution was concentrated. The residue was
purified by RP-HPLC to yield the desired maleimide 118 as a white
powder after lyophilization (58%). MS found: 1091.2
(M+H).sup.+.
Example X-3. Formation of Dibromomethyl Quinoxaline
##STR00218##
[0470] The o-phenylenediamino compound 119 (12 mg) was dissolved in
acetonitrile/water (6/4, v/v, 1 mL). NaOAc buffer (100 mM, pH=4.0,
0.3 mL) was added, followed by addition of dibromomethyl diketone
(10 mg). The mixture was stirred at room temperature for 10 min and
purified directly by RP-HPLC to give the desired quinoxaline 120 as
a white powder (12 mg) after lyophilization. MS found 1829.5
(M+H).sup.+.
Example X-4. Synthesis of Compound 136
##STR00219##
[0472] Compound 13 (50 mg) was treated with aldehyde under
reductive alkylation conditions (General procedure E). Without any
purification, hydrazine hydrate (20 .mu.L) was added to the
reaction mixture. After 10 min, the crude mixture was purified by
RP-HPLC to give compound 136 as a white powder (46 mg). MS found
1026.6 (M+H).sup.+.
Example X-5. Synthesis of Compound 139
##STR00220##
[0474] Compound 139 was synthesized from compound 137 and carbonate
138 according to General procedure I. MS found: 1184.8
(M+H).sup.+.
[0475] The Antibody Drug Conjugates depicted in the Figures were
prepared according to Example XI.
Example XI. Preparation of Antibody Drug Conjugate
[0476] To a solution of 0.5-50 mgs/mL of Trastuzumab in buffet at
pH 6.0-9.0 with 0-30% organic solvent, was added 0.1-10 eq of
carboxylic acid activated derivatives of compounds 84, 92, 93, or
98 in a portion wise or continuous flow manner. The reaction was
performed at 0-40.degree. C. for 0.5-50 hours with gentle stirring
or shaking, monitored by HIC-HPLC (Hydrophobic Interaction
Chromatography-HPLC). The resultant crude ADC product underwent
necessary down-stream steps of desalt, buffet changes/formulation,
and optionally, purification, using the state-of-art procedures.
The final ADC product was characterized by HIC-HPLC, SEC, RP-HPLC,
and optionally LC-MS. The average DAR (drug antibody ratio) was
calculated by UV absorption and/or MS spectroscopy.
Example XII. In Vitro Cytotoxicity Experiment
[0477] The cell lines used were SK-BR-3 human breast adenocarcinoma
(HER2 triple positive), HCC1954 human Ductal Carcinoma (HER2 triple
positive), MCF7 human breast adenocarcinoma (HER2 normal), and
MDA-MB-468 human breast adenocarcinoma (HER2 negative). These cells
were available from ATCC. SK-BR-3 cells were grown in McCoy's 5A
medium (Caisson Labs, North Logan, Utah) supplemented with 10%
fetal bovine serum. HCC1954 cells were grown in RPMI-1640 medium
(Caisson Labs, North Logan, Utah) supplemented with 10% fetal
bovine serum. MCF7 and MDA-MB-468 cells were grown in DMEM/F12
medium (Caisson Labs, North Logan, Utah) supplemented with 10%
fetal bovine serum. SK-BR-3, MCF7, and MDA-MB-468 cells were plated
in 96-well plates at approximately 7,500 cells/well, and HCC1954
cells were plated in 96-well plates at approximately 20,000
cells/well. Compounds or the antibody-drug conjugates were added in
duplicates in the same day. After 72 hour incubation at 37.degree.
C., CellTiter-Glo (Promega, Madison, Wis.) were added and cell
viability was determined as describe by the manufacture's protocol.
The percent viability was determined as following:
% Viability=Average Luminescence Value of the duplicates (treated
wells)/Average Luminescence Value of the untreated wells
* * * * *