U.S. patent application number 17/087597 was filed with the patent office on 2021-08-26 for hemiasterlin derivatives for conjugation and therapy.
The applicant listed for this patent is SUTRO BIOPHARMA, INC. Invention is credited to Krishna Bajjuri, Toni KLINE, Qun Yin.
Application Number | 20210261611 17/087597 |
Document ID | / |
Family ID | 1000005497173 |
Filed Date | 2021-08-26 |
United States Patent
Application |
20210261611 |
Kind Code |
A1 |
KLINE; Toni ; et
al. |
August 26, 2021 |
HEMIASTERLIN DERIVATIVES FOR CONJUGATION AND THERAPY
Abstract
Provided herein are hemiasterlin derivatives, conjugates
thereof, compositions comprising the derivatives or conjugates
thereof, methods of producing the derivatives and conjugates
thereof, and methods of using the derivatives, conjugates, and
compositions for the treatment of cell proliferation. The
derivatives, conjugates, and compositions are useful in methods of
treatment and prevention of cell proliferation and cancer, methods
of detection of cell proliferation and cancer, and methods of
diagnosis of cell proliferation and cancer. In an embodiment, the
hemiasterlin derivatives are according to Formula 1000:
##STR00001## or a pharmaceutically acceptable salt, solvate, or
tautomer thereof, wherein Ar, L, W.sup.1, W.sup.4, W.sup.5, SG, and
R are as described herein.
Inventors: |
KLINE; Toni; (San Francisco,
CA) ; Yin; Qun; (Palo Alto, CA) ; Bajjuri;
Krishna; (Fremont, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SUTRO BIOPHARMA, INC, |
South San Francisco |
CA |
US |
|
|
Family ID: |
1000005497173 |
Appl. No.: |
17/087597 |
Filed: |
November 2, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15011388 |
Jan 29, 2016 |
10844090 |
|
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17087597 |
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62110390 |
Jan 30, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/28 20130101;
C07K 9/003 20130101; C07K 16/30 20130101; C07K 5/0205 20130101;
C07K 5/0808 20130101; A61K 47/6855 20170801; A61K 38/00 20130101;
C07K 5/06078 20130101; C07K 16/40 20130101; C07K 2317/76 20130101;
A61K 47/6817 20170801 |
International
Class: |
C07K 5/083 20060101
C07K005/083; C07K 5/02 20060101 C07K005/02; A61K 47/68 20060101
A61K047/68; C07K 9/00 20060101 C07K009/00; C07K 16/28 20060101
C07K016/28; C07K 16/30 20060101 C07K016/30; C07K 16/40 20060101
C07K016/40 |
Claims
1. A compound according to Formula 1000: ##STR00217## or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein: Ar is a divalent six membered,
substituted or unsubstituted, monocyclic aryl; divalent five- or
six-membered, substituted or unsubstituted, monocyclic heteroaryl;
divalent nine- or ten-membered, substituted or unsubstituted,
monocyclic aryl; or a divalent eight-, nine- or ten-membered,
substituted or unsubstituted, fused bicyclic aryl or heteroaryl; L
is absent or --CH.sub.2--; X is ##STR00218## W.sup.1, W.sup.2,
W.sup.3, W.sup.4, and W.sup.5 are each independently a single bond,
absent, or a divalent attaching group; EG is absent or an
eliminator group; RT in the backbone of Formula 1000, is a release
trigger group or is not present or RT, when bonded to EG, is a
release trigger group or is hydrogen; RT.sup.1 is a release trigger
group, or a cleavable linker, or RT.sup.1 is absent; HP is a single
bond, absent, or a divalent hydrophilic group; HP.sup.1 is a single
bond, absent, a divalent hydrophilic group, or ##STR00219## where
R.sup.HP is a monovalent hydrophilic group; SG is a single bond,
absent, or a divalent spacer group; and R is hydrogen or a terminal
conjugating group; or, in the alternative, W.sup.1, W.sup.2,
W.sup.3, W.sup.4, W.sup.5, EG, each RT, and HP are all absent, SG
is a single bond, and R is --H.
2-33. (canceled)
34. A conjugate comprising the compound of claim 1, or a
pharmaceutically acceptable salt, solvate, or tautomer thereof,
linked to a second compound.
35. The conjugate of claim 34 according to Formula (E1):
##STR00220## or a pharmaceutically acceptable salt, solvate,
stereoisomer, or tautomer thereof, wherein: COMP is a residue of a
second compound; Ar is a divalent six membered, substituted or
unsubstituted, monocyclic aryl; divalent five- or six-membered,
substituted or unsubstituted, monocyclic heteroaryl; divalent nine-
or ten-membered, substituted or unsubstituted, monocyclic aryl; or
a divalent eight-, nine- or ten-membered, substituted or
unsubstituted, fused bicyclic aryl or heteroaryl; L is absent or
--CH.sub.2--; X is ##STR00221## W.sup.1, W.sup.2, W.sup.3, W.sup.4,
and W.sup.5 are each independently a single bond, absent, or a
divalent attaching group; EG is absent or an eliminator group; each
RT is a release trigger group, in the backbone of Formula 1000, is
a release trigger group or is not present or RT, when bonded to EG,
is a release trigger group or is hydrogen, wherein each RT is
optional; RT.sup.4 is a release trigger group, or a cleavable
linker, or RT.sup.4 is absent; HP is a single bond, absent, or a
divalent hydrophilic group; HP.sup.1 is a single bond, absent, a
divalent hydrophilic group, or ##STR00222## where R.sup.HP is a
monovalent hydrophilic group; SG is a single bond, absent, or a
divalent spacer group; and R' is a divalent residue of a terminal
conjugating group.
36-65. (canceled)
66. A pharmaceutical composition comprising the conjugate of claim
35 or a pharmaceutically acceptable salt, solvate, or tautomer
thereof; and a pharmaceutically acceptable excipient, carrier, or
diluent.
67. (canceled)
68. A method of inhibiting tubulin polymerization in a subject in
need thereof comprising administering an effective amount of the
conjugate of claim 35 or a pharmaceutically acceptable salt,
solvate, or tautomer thereof to the subject.
69. A method of treating cell proliferation or cancer in a subject
in need thereof comprising administering an effective amount of the
conjugate of claim 35 or a pharmaceutically acceptable salt,
solvate, or tautomer thereof to the subject.
70. (canceled)
71. A method of producing a conjugate, comprising contacting the
compound of claim 1 with a second compound under conditions
suitable for conjugating the compound of claim 1 with the second
compound; wherein the second compound comprises an alkyne, strained
alkene, tetrazine, thiol, maleimide, carbonyl, oxyamine, or
azide.
72-90. (canceled)
91. A conjugate produced by the method of claim 71, wherein the
first compound is linked to a second compound via a linker; or a
pharmaceutically acceptable salt, solvate, or tautomer thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 15/011,388, filed on Jan. 29, 2016, which
claims priority to, and the benefit of, U.S. Provisional
Application No. 62/110,390, filed on Jan. 30, 2015, which are
incorporated herein by reference in their entirety.
FIELD
[0002] Provided herein are hemiasterlin derivatives, conjugates
thereof, compositions comprising the derivatives or conjugates
thereof, methods of producing the derivatives and conjugates
thereof, and methods of using the derivatives, conjugates, and
compositions for the treatment of cell proliferation. The
derivatives, conjugates, and compositions are useful in methods of
treatment and prevention of cell proliferation and cancer, methods
of detection of cell proliferation and cancer, and methods of
diagnosis of cell proliferation and cancer.
BACKGROUND
[0003] Hemiasterlins are a class of tripeptides modified from the
original natural product hemiasterlin. Hemiasterlin is isolated
from marine sponges Cymbastela sp., Hemiasterella minor,
Siphonochalina sp., and Auletta sp. (Talpir et al., Tetrahedron
Letters, vol. 35, no. 25, pp. 4453-4456, 1994).
##STR00002##
[0004] Hemiasterlins are pseudopeptides which are inhibitors of
tubulin polymerization, sharing an antimitotic mechanism of action
with dolastatins and cryptophycins. Noncompetitive binding at the
vinblastine site on tubulin has been demonstrated. Hemiasterlins
are in general poor permeability glycoprotein (pGP) substrates,
rendering them effective against tumors that overexpress pGP as a
resistance mechanism. (Loganzo et al., Cancer Research, vol 63, pp.
1838-1845, 15 Apr. 2003).
[0005] Extensive modification of natural hemiasterlin demonstrated
key features contributing to the nanomolar activity of this class
against a wide variety of tumor cell lines. Two derivatives, E7974,
an N-terminal piperidine derivative developed at Eisai, and
HTI-286, an N-terminal phenyl developed at Wyeth, entered Phase I
trials. Encouraging results were presented in 2007 for E7974.
(Madajewicz et al., "A phase I trial of E7974 administered on days
1 and 15 of a 28-day cycle in patients with solid malignancies,"
presented at American Society of Clinical Oncology Annual Meeting;
Jun. 1-5, 2007; Chicago, Ill.; and Zojwalla et al., "A phase I
trial of E7974 administered on days 1, 8, and 15 of a 28-day cycle
in patients with solid malignancies," presented at American Society
of Clinical Oncology Annual Meeting; Jun. 1-5, 2007; Chicago, Ill.;
both summarized in Rocha-Lima et al., Cancer, Sep. 1, 2012 pp.
4262-4270). However, no results have been reported for HTI-286 to
date.
##STR00003##
[0006] In addition, conjugation of HTI-286 at the C-terminus to a
gastrin decapeptide VLALAEEEAYGWNleDF-NH.sub.2 for tumor targeting
is described in Tarsova et al., United States patent application
publication number US 2005/0171014 A1. Reported activity, however,
was very weak.
SUMMARY
[0007] Provided herein are hemiasterlin derivatives, conjugates
thereof, compositions comprising the derivatives or conjugates
thereof, methods of producing the derivatives and conjugates
thereof, and methods of using the derivatives, conjugates, and
compositions for the treatment of cell proliferation. The
derivatives, conjugates, and compositions are useful in methods of
treatment and prevention of cell proliferation and cancer, methods
of detection of cell proliferation and cancer, and methods of
diagnosis of cell proliferation and cancer.
[0008] In one aspect, provided herein is a compound according to
Formula 1000:
##STR00004##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein:
[0009] Ar is a divalent five- or six-membered, substituted or
unsubstituted, monocyclic aryl or heteroaryl ring or a divalent
eight-, nine- or ten-membered, substituted or unsubstituted, fused
bicyclic aryl or heteroaryl ring;
[0010] L is absent or --CH.sub.2--;
[0011] X is
##STR00005##
[0012] W.sup.1, W.sup.2, W.sup.3, W.sup.4, and W.sup.5 are each
independently a single bond, absent, or a divalent attaching
group;
[0013] EG is absent or an eliminator group;
[0014] each RT is a release trigger group, in the backbone of
Formula 1000 or bonded to EG, wherein each RT is optional;
[0015] RT.sup.1 is a release trigger group, or a cleavable linker,
or RT.sup.1 is absent;
[0016] HP is a single bond, absent, or a divalent hydrophilic
group;
[0017] HP.sup.1 is a single bond, absent, a divalent hydrophilic
group, or
##STR00006##
where R.sup.HP is a monovalent hydrophilic group;
[0018] SG is a single bond, absent, or a divalent spacer group;
and
[0019] R is hydrogen, a terminal conjugating group, or a divalent
residue of a terminal conjugating group;
or, in the alternative, W.sup.1, W.sup.2, W.sup.3, W.sup.4,
W.sup.5, EG, RT, HP, SG, and R combine to form --H.
[0020] In one aspect, provided herein is a conjugate comprising a
compound described herein (e.g., a compound according to any of
Formulas 1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2,
101-111b, or 1-8b) linked to a second compound.
[0021] In an aspect, provided herein is a pharmaceutical
composition comprising:
[0022] a compound (e.g., a compound according to any of Formulas
1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2, 101-111b, or
1-8b) or conjugate (e.g., a conjugate according to any of Formulas
C1-C17b, E1, F1-F17b, and G1-G17b) as described herein; and
[0023] a pharmaceutically acceptable excipient, carrier, or
diluent.
[0024] In an aspect, provided herein is a method of inhibiting
tubulin polymerization in a subject in need thereof comprising
administering an effective amount of a compound (e.g., a compound
according to any of Formulas 1000-1000b, 1001-1001b, 1002-1002b,
and I-XIXb-2, 101-111b, or 1-8b), conjugate (e.g., a conjugate
according to any of Formulas C1-C17b, E1, F1-F17b, and G1-G17b), or
composition comprising a compound or conjugate, as described
herein, to the subject.
[0025] In an aspect, provided herein is a method of treating cell
proliferation or cancer in a subject in need thereof comprising
administering an effective amount of a compound (e.g., a compound
according to any of Formulas 1000-1000b, 1001-1001b, 1002-1002b,
and I-XIXb-2, 101-111b, or 1-8b), conjugate (e.g., a conjugate
according to any of Formulas C1-C17b, E1, F1-F17b, and G1-G17b), or
composition comprising a compound or conjugate, as described
herein, to the subject.
[0026] In an aspect, provided herein is a method of producing a
conjugate, comprising contacting a compound described herein (e.g.,
a compound according to any of Formulas 1000-1000b, 1001-1001b,
1002-1002b, and I-XIXb-2 or 101-111b) with a second compound under
conditions suitable for conjugating the second compound with the
compound described herein (e.g., a compound according to any of
Formulas 1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2 or
101-111b); wherein the second compound comprises a modified amino
acid comprising an alkyne, strained alkene, tetrazine, thiol,
maleimide, carbonyl, oxyamine, or azide.
BRIEF DESCRIPTION OF THE FIGURES
[0027] FIG. 1 provides results of a cell killing assay described in
detail herein. Racemic [R/S,S,S] Compound 1 is evaluated as a
conjugate on the trastuzumab heavy chain at F404 and also on the
light chain at S7. All other conjugates in FIG. 1 are on the
trastuzumab heavy chain at F404. The relative cell viability of
SKBR3 cells is plotted against concentration for trastuzumab
(exes), trastuzumab F404 [S,S,S] Compound 1 conjugate (filled
squares), trastuzumab F404 racemic [R/S,S,S] Compound 1 conjugate
(split squares), trastuzumab S7 racemic [R/S,S,S] Compound 1
conjugate (split circles), and a trastuzumab auristatin (MMAF)
conjugate (open triangles).
[0028] FIG. 2a provides results of a cell killing assay described
in detail herein. In FIG. 2a relative cell viability is plotted
against concentration of [S,S,S] Compound 1 (filled squares) and
[R,S,S] Compound 1 (open squares) for SKBR3 cells in panel (a),
MDA-MB-453 cells in panel (b), and MDA-MB-468 cells in panel
(c).
[0029] FIG. 2b provides results of a cell killing assay described
in detail herein. In FIG. 2b relative cell viability is plotted
against concentration of [S,S,S] Compound 1 (filled squares) and
[R,S,S] Compound 1 (open squares) for HTC116 cells in panel (a),
HT29 cells in panel (b), and SKCO1 cells in panel (c).
[0030] FIG. 2c provides results of a cell killing assay described
in detail herein. In FIG. 2c relative cell viability is plotted
against concentration of [S,S,S] Compound 1 (filled squares) and
[R,S,S] Compound 1 (open squares) for MDA-MB-435 cells in panel
(a), SUDHL6 cells in panel (b), and OMP2 cells in panel (c).
DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0031] Provided herein are compounds (e.g., according to any of
Formulas 1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2,
101-111b, or 1-8b), conjugates thereof (e.g., according to any of
Formulas C1-C17b, E1, F1-F17b, and G1-G17b), compositions
comprising the compounds or conjugates thereof, methods of
producing the compounds and conjugates thereof, and methods of
using the compounds, conjugates, and compositions. The compounds,
conjugates, and compositions are useful in methods of treatment and
prevention of cell proliferation and cancer, methods of detection
of cell proliferation and cancer, and methods of diagnosis of cell
proliferation and cancer.
Definitions
[0032] When referring to the compounds provided herein, the
following terms have the following meanings unless indicated
otherwise. Unless defined otherwise, all technical and scientific
terms used herein have the same meaning as is commonly understood
by one of ordinary skill in the art. In the event that there is a
plurality of definitions for a term herein, those in this section
prevail unless stated otherwise. Unless specified otherwise, where
a term is defined as being unsubstituted or substituted, the groups
in the list of substituents are unsubstituted. For example, an
alkyl group can be substituted with a cycloalkyl group and the
cycloalkyl group is not further substituted.
[0033] The term "alkyl," as used herein, unless otherwise
specified, refers to a saturated straight or branched hydrocarbon
which can be substituted with halo groups. In certain embodiments,
the alkyl group is a primary, secondary, or tertiary hydrocarbon.
In certain embodiments, the alkyl group includes one to ten carbon
atoms, i.e., C.sub.1 to C.sub.10 alkyl. In certain embodiments, the
alkyl group is, for example, methyl, CF.sub.3, CCl.sub.3,
CFCl.sub.2, CF.sub.2C1, ethyl, CH.sub.2CF.sub.3, CF.sub.2CF.sub.3,
propyl, isopropyl, butyl, isobutyl, secbutyl, t-butyl, pentyl,
isopentyl, neopentyl, hexyl, isohexyl, 3-methylpentyl,
2,2-dimethylbutyl, or 2,3-dimethylbutyl. The term includes both
substituted and unsubstituted alkyl groups, including halogenated
alkyl groups. In certain embodiments, the alkyl group is a
fluorinated alkyl group. In certain embodiments, the alkyl group
can be substituted with at least one (in another example with 1, 2,
3, 4, or 5) halogen (fluoro, chloro, bromo or iodo), oxo, epoxy,
hydroxyl, alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl,
cycloalkyl, aralkyl, sulfanyl, alkylsulfanyl, cycloalkylsulfanyl,
arylsulfanyl, alkylsulfonyl, cycloalkylsulfonyl, arylsulfonyl,
aminocarbonyl, carbamoyl, sulfonamido, amino (as defined herein,
e.g. alkylamino, dialkylamino, arylamino, etc.), alkoxy, aryloxy,
nitro, cyano, sulfonic acid, sulfate, sulfonate, phosphonic acid,
phosphate, or phosphonate, either unprotected, or protected as
necessary, as known to those skilled in the art, for example, as
taught in Greene, et al., Protective Groups in Organic Synthesis,
John Wiley and Sons, Second Edition, 1991, hereby incorporated by
reference.
[0034] The term "lower alkyl," as used herein, and unless otherwise
specified, refers to a saturated straight or branched hydrocarbon
having one to six carbon atoms, i.e., C.sub.1 to C.sub.6 alkyl. In
certain embodiments, the lower alkyl group is a primary, secondary,
or tertiary hydrocarbon. The term includes both substituted and
unsubstituted moieties.
[0035] The term "upper alkyl," as used herein, and unless otherwise
specified, refers to a saturated straight or branched hydrocarbon
having seven to thirty carbon atoms, i.e., C.sub.7 to C.sub.30
alkyl. In certain embodiments, the upper alkyl group is a primary,
secondary, or tertiary hydrocarbon. The term includes both
substituted and unsubstituted moieties.
[0036] The term "alkylcarbonyl" refers to the group --C(O)(alkyl)
where alkyl is as defined herein.
[0037] The term "alkylsulfanyl" refers to the group --S(alkyl)
where alkyl is as defined herein.
[0038] The term "carboxylene" refers to a --C(O)O-- or --OC(O)--
group.
[0039] The term "cycloalkylsulfanyl" refers to the group
--S(cycloalkyl) where cycloalkyl is as defined herein.
[0040] The term "arylsulfanyl" refers to the group --S(aryl) where
aryl is as defined herein.
[0041] The term "alkylsulfonyl" refers to the group
--S(O).sub.2(alkyl) where alkyl is as defined herein.
[0042] The term "cycloalkylsulfonyl" refers to the group
--S(O).sub.2(cycloalkyl) where cycloalkyl is as defined herein.
[0043] The term "arylsulfonyl" refers to the group
--S(O).sub.2(aryl) where aryl is as defined herein.
[0044] The term "cycloalkyl," as used herein, unless otherwise
specified, refers to a saturated monocyclic or polycyclic
hydrocarbon. In certain embodiments, cycloalkyl includes fused,
bridged, and spiro ring systems. In certain embodiments, the
cycloalkyl group includes three to ten carbon atoms, i.e., C.sub.3
to C.sub.10 cycloalkyl. In some embodiments, the cycloalkyl has
from 3 to 15 (C.sub.3-15), from 3 to 10 (C.sub.3-10), or from 3 to
7 (C.sub.3-7) carbon atoms. In certain embodiments, the cycloalkyl
group is, for example, cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, cyclohexylmethyl, cycloheptyl, bicyclo[2.1.1]hexyl,
bicyclo[2.2.1]heptyl, decalinyl or adamantyl. The term includes
both substituted and unsubstituted cycloalkyl groups, including
halogenated cycloalkyl groups. In certain embodiments, the
cycloalkyl group is a fluorinated cycloalkyl group. In certain
embodiments, the cycloalkyl group can be substituted with at least
one (in another example with 1, 2, 3, 4, or 5) halogen (fluoro,
chloro, bromo or iodo), oxo, epoxy, hydroxyl, alkylcarbonyl,
cycloalkylcarbonyl, arylcarbonyl, sulfanyl, alkylsulfanyl,
cycloalkylsulfanyl, arylsulfanyl, alkylsulfonyl,
cycloalkylsulfonyl, arylsulfonyl, aminocarbonyl, carbamoyl,
sulfonamido, amino (as defined herein, e.g. alkylamino,
dialkylamino, arylamino, etc.), alkoxy, aryloxy, nitro, cyano,
sulfonic acid, sulfate, sulfonate, phosphonic acid, phosphate, or
phosphonate, either unprotected, or protected as necessary.
[0045] The term "cycloalkylalkyl" refers to an alkyl group as
defined herein substituted with at least one (in some embodiments,
one or two) cycloalkyl groups as defined herein.
[0046] The term "cycloalkylcarbonyl" refers to the group
--C(O)(cycloalkyl) where cycloalkyl is as defined herein.
[0047] "Alkylene" refers to divalent saturated aliphatic
hydrocarbon groups, including those having from one to eleven
carbon atoms which can be straight-chained or branched. In certain
embodiments, the alkylene group contains 1 to 10 carbon atoms. The
term includes both substituted and unsubstituted moieties. In
certain embodiments, alkylene is, for example, methylene
(--CH.sub.2--), ethylene (--CH.sub.2CH.sub.2--), the propylene
isomers (e.g., --CH.sub.2CH.sub.2CH.sub.2-- and
--CH(CH.sub.3)CH.sub.2--) and the like. The term includes
halogenated alkylene groups. In certain embodiments, the alkylene
group is a fluorinated alkylene group. In certain embodiments, the
alkylene group can be substituted with at least one (in another
example with 1, 2, 3, 4, or 5) halogen (fluoro, chloro, bromo or
iodo), oxo, epoxy, hydroxyl, alkylcarbonyl, cycloalkylcarbonyl,
arylcarbonyl, sulfanyl, alkylsulfanyl, cycloalkylsulfanyl,
arylsulfanyl, alkylsulfonyl, cycloalkylsulfonyl, arylsulfonyl,
aminocarbonyl, carbamoyl, sulfonamido, amino (as defined herein,
e.g. alkylamino, dialkylamino, arylamino, etc.), alkylaryl, alkoxy,
aryloxy, nitro, cyano, sulfonic acid, sulfate, sulfonate,
phosphonic acid, phosphate, and phosphonate, either unprotected, or
protected as necessary.
[0048] "Alkenyl" refers to monovalent olefinically unsaturated
hydrocarbon groups, in certain embodiments, having up to about 11
carbon atoms, including from 2 to 8 carbon atoms, or from 2 to 6
carbon atoms, which can be straight-chained or branched and having
at least 1, including from 1 to 2, site of olefinic unsaturation.
The term includes both substituted and unsubstituted moieties. In
certain embodiments, alkenyl is, for example, ethenyl (i.e., vinyl,
or --CH.dbd.CH.sub.2), n-propenyl (--CH.sub.2CH.dbd.CH.sub.2),
isopropenyl (--C(CH.sub.3)=CH.sub.2), and the like. The term
includes halogenated alkenyl groups. In certain embodiments, the
alkenyl group is a fluorinated alkenyl group. In certain
embodiments, the alkenyl group can be substituted with at least one
(in another example with 1, 2, 3, 4, or 5) halogen (fluoro, chloro,
bromo or iodo), oxo, epoxy, hydroxyl, alkylcarbonyl,
cycloalkylcarbonyl, arylcarbonyl, sulfanyl, alkylsulfanyl,
cycloalkylsulfanyl, arylsulfanyl, alkylsulfonyl,
cycloalkylsulfonyl, arylsulfonyl, aminocarbonyl, carbamoyl,
sulfonamido, amino (as defined herein, e.g. alkylamino,
dialkylamino, arylamino, etc.), alkoxy, aryloxy, nitro, cyano,
sulfonic acid, sulfate, sulfonate, phosphonic acid, phosphate, or
phosphonate, either unprotected, or protected as necessary.
[0049] The term "cycloalkenyl," as used herein, unless otherwise
specified, refers to an unsaturated (but not aromatic) cyclic
hydrocarbon. In certain embodiments, cycloalkenyl refers to mono-
or multicyclic ring systems that include at least one double bond.
In certain embodiments, cycloalkyl includes fused, bridged, and
spiro ring systems. In certain embodiments, the cycloalkyl group
includes at least three carbon atoms, including three to ten carbon
atoms, i.e., C.sub.3 to C.sub.10 cycloalkyl. In some embodiments,
the cycloalkenyl has from 3 to 10 (C.sub.3-10), or from 4 to 7
(C.sub.4-7) carbon atoms. The term includes both substituted and
unsubstituted cycloalkenyl groups, including halogenated
cycloalkenyl groups. In certain embodiments, the cycloalkenyl group
is a fluorinated cycloalkenyl group. In certain embodiments, the
cycloalkenyl group can be substituted with at least one (in another
example with 1, 2, 3, 4, or 5) halogen (fluoro, chloro, bromo or
iodo), oxo, epoxy, hydroxyl, alkylcarbonyl, cycloalkylcarbonyl,
arylcarbonyl, sulfanyl, alkylsulfanyl, cycloalkylsulfanyl,
arylsulfanyl, alkylsulfonyl, cycloalkylsulfonyl, arylsulfonyl,
aminocarbonyl, carbamoyl, sulfonamido, amino (as defined herein,
e.g. alkylamino, dialkylamino, arylamino, etc.), alkoxy, aryloxy,
nitro, cyano, sulfonic acid, sulfate, sulfonate, phosphonic acid,
phosphate, or phosphonate, either unprotected, or protected as
necessary.
[0050] "Alkenylene" refers to divalent olefinically unsaturated
hydrocarbon groups, in certain embodiments, having up to about 11
carbon atoms or from 2 to 6 carbon atoms which can be
straight-chained or branched and having at least 1 or from 1 to 2
sites of olefinic unsaturation. In certain embodiments, alkenylene
is, for example, ethenylene (--CH.dbd.CH--), the propenylene
isomers (e.g., --CH.dbd.CHCH.sub.2-- and --C(CH.sub.3)=CH-- and
--CH.dbd.C(CH.sub.3)--) and the like. The term includes both
substituted and unsubstituted alkenylene groups, including
halogenated alkenylene groups. In certain embodiments, the
alkenylene group is a fluorinated alkenylene group. Non-limiting
examples of moieties with which the alkenylene group can be
substituted with at least one (in another example with 1, 2, 3, 4,
or 5) halogen (fluoro, chloro, bromo or iodo), oxo, epoxy,
hydroxyl, alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl,
sulfanyl, alkylsulfanyl, cycloalkylsulfanyl, arylsulfanyl,
alkylsulfonyl, cycloalkylsulfonyl, arylsulfonyl, aminocarbonyl,
carbamoyl, sulfonamido, amino (as defined herein, e.g. alkylamino,
dialkylamino, arylamino, etc.), alkoxy, aryloxy, nitro, cyano,
sulfonic acid, sulfate, sulfonate, phosphonic acid, phosphate, or
phosphonate, either unprotected, or protected as necessary.
[0051] "Alkynyl" refers to acetylenically unsaturated hydrocarbon
groups, in certain embodiments, having up to about 11 carbon atoms
or from 2 to 6 carbon atoms which can be straight-chained or
branched and having at least 1 or from 1 to 2 sites of alkynyl
unsaturation. In certain embodiments, alkynyl is, for example,
acetylenic, ethynyl (--C.ident.CH), propargyl
(--CH.sub.2C.ident.CH), and the like. The term includes both
substituted and unsubstituted alkynyl groups, including halogenated
alkynyl groups. In certain embodiments, the alkynyl group is a
fluorinated alkynyl group. In certain embodiments, the alkynyl
group can be substituted with at least one (in another example with
1, 2, 3, 4, or 5) halogen (fluoro, chloro, bromo or iodo), oxo,
epoxy, hydroxyl, alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl,
sulfanyl, alkylsulfanyl, cycloalkylsulfanyl, arylsulfanyl,
alkylsulfonyl, cycloalkylsulfonyl, arylsulfonyl, aminocarbonyl,
carbamoyl, sulfonamido, amino (as defined herein, e.g. alkylamino,
dialkylamino, arylamino, etc.), alkoxy, aryloxy, nitro, cyano,
sulfonic acid, sulfate, sulfonate, phosphonic acid, phosphate, or
phosphonate, either unprotected, or protected as necessary.
[0052] The term "aryl," as used herein, and unless otherwise
specified, refers to a monovalent six- to fourteen-membered, mono-,
bi-, or tri-carbocyclic ring, wherein the monocyclic ring is
aromatic and at least one of the rings in the bicyclic and
tricyclic ring is aromatic. The aryl group can be bonded to the
rest of the molecule through any carbon in the ring system. In an
embodiment, an aryl group is a C6-C12 aryl group. In an embodiment,
an aryl group is phenyl, indanyl, or naphthyl. The term includes
both substituted and unsubstituted moieties. In certain
embodiments, an aryl group can be substituted with one or more (for
example 1, 2, 3, 4, or 5) moieties independently selected from the
group halogen (fluoro, chloro, bromo or iodo), alkyl, haloalkyl,
hydroxyl, alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl,
sulfanyl, alkylsulfanyl, cycloalkylsulfanyl, arylsulfanyl,
alkylsulfonyl, cycloalkylsulfonyl, arylsulfonyl, aminocarbonyl,
carbamoyl, sulfonamido, amino (as defined herein, e.g. alkylamino,
dialkylamino, arylamino, etc.), alkoxy, aryloxy, nitro, cyano,
sulfonic acid, sulfate, sulfonate, phosphonic acid, phosphate, and
phosphonate, either unprotected, or protected as necessary, as
known to those skilled in the art, for example, as taught in
Greene, et al., Protective Groups in Organic Synthesis, John Wiley
and Sons, Second Edition, 1991.
[0053] The term "arylcarbonyl" refers to the group --C(O)(aryl)
where aryl is as defined herein.
[0054] The term "aryloxy" refers to the group --OR' where R' is
aryl, as defined herein.
[0055] The term "aryloxyalkyl" refers to an alkyl group as defined
herein substituted with at least one (in some embodiments one or
two) aryloxy groups as defined herein.
[0056] "Alkoxy" and "alkoxyl" refer to the group --OR' where R' is
alkyl or cycloalkyl as defined herein. In certain embodiments,
alkoxy and alkoxyl groups include, by way of example, methoxy,
ethoxy, n-propoxy, isopropoxy, cyclopropoxy, n-butoxy, tert-butoxy,
sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the
like.
[0057] The term "alkoxyalkyl" refers to an alkyl group substituted
with at least one (in another embodiment, one or two) alkoxy groups
as defined herein.
[0058] "Alkoxycarbonyl" refers to a radical --C(O)-alkoxy where
alkoxy is as defined herein.
[0059] "Alkoxycarbonylalkyl" refers to an alkyl group substituted
with at least one, in another example 1 or 2, alkoxycarbonyl
groups, as defined herein.
[0060] "Amino" refers to the group --NR.sup.1'R.sup.2' or
--NR.sup.1'--, wherein R.sup.1' and R.sup.2' are independently
hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
heterocyclic, aryl, or heteroaryl, each of which is as defined
herein. In an embodiment, "Amino" is --NH.sub.2 or --NH--.
[0061] "Carboxyl" or "carboxy" refers to the radical --C(O)OH.
[0062] The term "alkylamino" or "acylamino" refers to an amino
group that has one alkyl or aryl substituent, respectively, e.g.
--NHCH.sub.3, and --NH(phenyl). In certain embodiments, the alkyl
substituent is lower alkyl. In another embodiment, the alkyl or
lower alkyl is unsubstituted.
[0063] The term "dialkylamino" refers to an amino group that has
two alkyl substituents, e.g. --N(CH.sub.3).sub.2. In certain
embodiments, the alkyl substituent is lower alkyl. In another
embodiment, the alkyl or lower alkyl is unsubstituted.
[0064] The term "diarylamino" refers to an amino group that has two
aryl substituents.
[0065] "Halogen" or "halo" refers to chloro, bromo, fluoro, or
iodo.
[0066] "Thioalkoxy" refers to the group --SR' where R' is alkyl or
cycloalkyl.
[0067] The term "heterocyclo" or "heterocyclic" refers to a
monovalent monocyclic non-aromatic ring system and/or multicyclic
ring system that contains at least one non-aromatic ring, wherein
one or more of the non-aromatic ring atoms are heteroatoms
independently selected from O, S, or N and the remaining ring atoms
are carbon atoms and where the multicyclic ring system further
comprises a carbocyclic or heterocyclic, aromatic or nonaromatic
ring. In certain embodiments, the heterocyclo or heterocyclic group
has from 3 to 20, from 3 to 15, from 3 to 10, from 3 to 8, from 4
to 7, or from 5 to 6 ring atoms. Heterocyclo groups are bonded to
the rest of the molecule through a non-aromatic ring. In certain
embodiments, the heterocyclo is a monocyclic, bicyclic, tricyclic,
or tetracyclic ring system, which may include a fused, spirocyclic,
or bridged ring system, and in which the nitrogen or sulfur atoms
may be optionally oxidized, the nitrogen atoms may be optionally
quaternized, and some rings may be partially or fully saturated, or
aromatic. The heterocyclo may be attached to the main structure at
any heteroatom or carbon atom of the non-aromatic ring which
results in the creation of a stable compound. In certain
embodiments, heterocyclic is azepinyl, benzodioxanyl,
benzodioxolyl, benzofuranonyl, benzopyranonyl, benzopyranyl,
benzotetrahydrofuranyl, benzotetrahydrothienyl, benzothiopyranyl,
benzoxazinyl, .beta.-carbolinyl, chromanyl, chromonyl, cinnolinyl,
coumarinyl, decahydroisoquinolinyl, dihydrobenzisothiazinyl,
dihydrobenzisoxazinyl, dihydrofuryl, dihydroisoindolyl,
dihydropyranyl, dihydropyrazolyl, dihydropyrazinyl,
dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dioxolanyl,
1,4-dithianyl, furanonyl, imidazolidinyl, imidazolinyl, indolinyl,
isobenzotetrahydrofuranyl, isobenzotetrahydrothienyl, isochromanyl,
isocoumarinyl, isoindolinyl, isothiazolidinyl, isoxazolidinyl,
morpholinyl, octahydroindolyl, octahydroisoindolyl, oxazolidinonyl,
oxazolidinyl, oxiranyl, piperazinyl, piperidinyl, 4-piperidonyl,
pyrazolidinyl, pyrazolinyl, pyrrolidinyl, pyrrolinyl,
quinuclidinyl, tetrahydrofuryl, tetrahydroisoquinolinyl,
tetrahydropyranyl, tetrahydrothienyl, thiamorpholinyl,
thiazolidinyl, tetrahydroquinolinyl, and 1,3,5-trithianyl. In
certain embodiments, heterocyclic may also be optionally
substituted as described herein. In certain embodiments, the
heterocyclic group can be substituted with at least one (in another
example with 1, 2, 3, 4, or 5) halogen (fluoro, chloro, bromo or
iodo), oxo, epoxy, hydroxyl, alkylcarbonyl, cycloalkylcarbonyl,
arylcarbonyl, alkoxycarbonyl, alkoxycarbonylalkyl, sulfanyl,
alkylsulfanyl, cycloalkylsulfanyl, arylsulfanyl, alkylsulfonyl,
cycloalkylsulfonyl, arylsulfonyl, aminocarbonyl, carbamoyl,
sulfonamido, amino (as defined herein, e.g., alkylamino,
dialkylamino, arylamino, etc.), alkoxy, aryloxy, nitro, cyano,
sulfonic acid, sulfate, sulfonate, phosphonic acid, phosphate, or
phosphonate, either unprotected, or protected as necessary.
[0068] The term "heteroaryl" refers to a monovalent monocyclic
aromatic group and/or multicyclic group that contains at least one
aromatic ring, wherein the monocyclic ring contains one or more
heteroatoms independently selected from O, S and N in the ring and
where the multicyclic ring system comprises at least one aromatic
ring and further comprises a carbocyclic or heterocyclic, aromatic
or nonaromatic ring and where one or more of the ring atoms in the
multicyclic ring system is a heteroatom independently selected from
O, S and N. Heteroaryl groups are bonded to the rest of the
molecule through an aromatic ring. Each ring of a heteroaryl group
can contain one or two O atoms, one or two S atoms, and/or one to
four N atoms, provided that the total number of heteroatoms in each
ring is four or less and each ring contains at least one carbon
atom. In certain embodiments, the heteroaryl has from 5 to 20, from
5 to 15, or from 5 to 10 ring atoms. In certain embodiments,
monocyclic heteroaryl groups include, but are not limited to,
furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl,
oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl,
pyrimidinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl,
tetrazolyl, triazinyl and triazolyl. In certain embodiments,
bicyclic heteroaryl groups include, but are not limited to,
benzofuranyl, benzimidazolyl, benzoisoxazolyl, benzopyranyl,
benzothiadiazolyl, benzothiazolyl, benzothienyl, benzotriazolyl,
benzoxazolyl, furopyridyl, imidazopyridinyl, imidazothiazolyl,
indolizinyl, indolyl, indazolyl, isobenzofuranyl, isobenzothienyl,
isoindolyl, isoquinolinyl, isothiazolyl, naphthyridinyl,
oxazolopyridinyl, phthalazinyl, pteridinyl, purinyl, pyridopyridyl,
pyrrolopyridyl, quinolinyl, quinoxalinyl, quinazolinyl,
thiadiazolopyrimidyl, and thienopyridyl. In certain embodiments,
tricyclic heteroaryl groups include, but are not limited to,
acridinyl, benzindolyl, carbazolyl, dibenzofuranyl, perimidinyl,
phenanthrolinyl, phenanthridinyl, phenarsazinyl, phenazinyl,
phenothiazinyl, phenoxazinyl and xanthenyl. In certain embodiments,
the heteroaryl group can be substituted with at least one (in
another example with 1, 2, 3, 4, or 5) group halogen (fluoro,
chloro, bromo or iodo), alkyl, haloalkyl, hydroxyl, alkylcarbonyl,
cycloalkylcarbonyl, arylcarbonyl, sulfanyl, alkylsulfanyl,
cycloalkylsulfanyl, arylsulfanyl, alkylsulfonyl,
cycloalkylsulfonyl, arylsulfonyl, aminocarbonyl, carbamoyl,
sulfonamido, amino (as defined herein, e.g. alkylamino,
dialkylamino, arylamino, etc.), alkoxy, aryloxy, nitro, cyano,
sulfonic acid, sulfate, sulfonate, phosphonic acid, phosphate, and
phosphonate, either unprotected, or protected as necessary.
[0069] The term "alkylaryl" refers to an aryl group with an alkyl
substituent, wherein aryl and alkyl are as defined herein. The term
"aralkyl" or "arylalkyl" refers to an alkyl group with an aryl
substituent, wherein aryl and alkyl are as defined herein.
[0070] The term "phenylene," as used herein, and unless otherwise
specified, refers to a divalent phenyl group and includes both
substituted and unsubstituted moieties. In certain embodiments,
phenylene group can be substituted with one or more (for example 1,
2, 3, 4, or 5) moieties independently selected from the group
halogen (fluoro, chloro, bromo or iodo), alkyl, haloalkyl,
hydroxyl, alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl,
sulfanyl, alkylsulfanyl, cycloalkylsulfanyl, arylsulfanyl,
alkylsulfonyl, cycloalkylsulfonyl, arylsulfonyl, aminocarbonyl,
carbamoyl, sulfonamido, amino (as defined herein, e.g. alkylamino,
dialkylamino, arylamino, etc.), alkoxy, aryloxy, nitro, cyano,
sulfonic acid, sulfate, sulfonate, phosphonic acid, phosphate, and
phosphonate, either unprotected, or protected as necessary, as
known to those skilled in the art, for example, as taught in
Greene, et al., Protective Groups in Organic Synthesis, John Wiley
and Sons, Second Edition, 1991. When phenylene is used in the
context of an EG group, the phenylene is substituted with 1, 2, 3,
or 4 R.sup.EG groups, as defined herein, and/or with 1 or 2-O-[RT]
groups, and/or with 1 or 2 --CH.sub.2OC(O)[RT] groups, where RT is
as defined herein.
[0071] The term "protecting group" as used herein and unless
otherwise defined refers to a group that is added to an oxygen,
nitrogen or phosphorus atom to prevent its further reaction or for
other purposes. A wide variety of oxygen and nitrogen protecting
groups are known to those skilled in the art of organic
synthesis.
[0072] "Pharmaceutically acceptable salt" refers to any salt of a
compound provided herein which retains its biological properties
and which is not toxic or otherwise undesirable for pharmaceutical
use. Such salts may be derived from a variety of organic and
inorganic counter-ions well known in the art. Such salts include,
but are not limited to: (1) acid addition salts formed with organic
or inorganic acids such as hydrochloric, hydrobromic, sulfuric,
nitric, phosphoric, sulfamic, acetic, trifluoroacetic,
trichloroacetic, propionic, hexanoic, cyclopentylpropionic,
glycolic, glutaric, pyruvic, lactic, malonic, succinic, sorbic,
ascorbic, malic, maleic, fumaric, tartaric, citric, benzoic,
3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic, phthalic,
lauric, methanesulfonic, ethanesulfonic, 1,2-ethane-disulfonic,
2-hydroxyethanesulfonic, benzenesulfonic, 4-chlorobenzenesulfonic,
2-naphthalenesulfonic, 4-toluenesulfonic, camphoric,
camphorsulfonic, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic,
glucoheptonic, 3-phenylpropionic, trimethylacetic,
tert-butylacetic, lauryl sulfuric, gluconic, benzoic, glutamic,
hydroxynaphthoic, salicylic, stearic, cyclohexylsulfamic, quinic,
muconic acid and the like acids; or (2) base addition salts formed
when an acidic proton present in the parent compound either (a) is
replaced by a metal ion, e.g., an alkali metal ion, an alkaline
earth ion or an aluminum ion, or alkali metal or alkaline earth
metal hydroxides, such as sodium, potassium, calcium, magnesium,
aluminum, lithium, zinc, and barium hydroxide, ammonia or (b)
coordinates with an organic base, such as aliphatic, alicyclic, or
aromatic organic amines, such as ammonia, methylamine,
dimethylamine, diethylamine, picoline, ethanolamine,
diethanolamine, triethanolamine, ethylenediamine, lysine, arginine,
ornithine, choline, N,N'-dibenzylethylene-diamine, chloroprocaine,
diethanolamine, procaine, N-benzylphenethylamine, N-methylglucamine
piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium
hydroxide, and the like.
[0073] Pharmaceutically acceptable salts further include, by way of
example only and without limitation, sodium, potassium, calcium,
magnesium, ammonium, tetraalkylammonium and the like, and when the
compound contains a basic functionality, salts of non-toxic organic
or inorganic acids, such as hydrohalides, e.g. hydrochloride and
hydrobromide, sulfate, phosphate, sulfamate, nitrate, acetate,
trifluoroacetate, trichloroacetate, propionate, hexanoate,
cyclopentylpropionate, glycolate, glutarate, pyruvate, lactate,
malonate, succinate, sorbate, ascorbate, malate, maleate, fumarate,
tartarate, citrate, benzoate, 3-(4-hydroxybenzoyl)benzoate,
picrate, cinnamate, mandelate, phthalate, laurate, methanesulfonate
(mesylate), ethanesulfonate, 1,2-ethane-disulfonate,
2-hydroxyethanesulfonate, benzenesulfonate (besylate),
4-chlorobenzenesulfonate, 2-naphthalenesulfonate,
4-toluenesulfonate, camphorate, camphorsulfonate,
4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate, glucoheptonate,
3-phenylpropionate, trimethylacetate, tert-butyl acetate, lauryl
sulfate, gluconate, benzoate, glutamate, hydroxynaphthoate,
salicylate, stearate, cyclohexylsulfamate, quinate, muconate and
the like.
[0074] The term "acyl" refers to a group of the formula --C(O)R',
wherein R' is alkyl (including lower alkyl); cycloalkyl;
cycloalkylalkyl; cycloalkenyl; aryl; arylalkyl (including benzyl);
substituted alkyl (including lower alkyl and for example
alkoxyalkyl and aryloxyalkyl); heterocyclo; heterocycloalkyl;
heteroaryl; and heteroarylalkyl; where the cycloalkyl,
cycloalkenyl, aryl, heterocyclo, and heteroaryl may be substituted.
In certain embodiments, aryl groups in the acyl or esters comprise
a phenyl group. In certain embodiments, acyl groups include, for
example, acetyl, trifluoroacetyl, methylacetyl, cyclpropylacetyl,
propionyl, butyryl, hexanoyl, heptanoyl, octanoyl, neo-heptanoyl,
phenylacetyl, 2-acetoxy-2-phenylacetyl, diphenylacetyl,
.alpha.-methoxy-.alpha.-trifluoromethyl-phenylacetyl, bromoacetyl,
2-nitro-benzeneacetyl, 4-chloro-benzeneacetyl,
2-chloro-2,2-diphenylacetyl, 2-chloro-2-phenylacetyl,
trimethylacetyl, chlorodifluoroacetyl, perfluoroacetyl,
fluoroacetyl, bromodifluoroacetyl, methoxyacetyl,
2-thiopheneacetyl, chlorosulfonylacetyl, 3-methoxyphenylacetyl,
phenoxyacetyl, tert-butylacetyl, trichloroacetyl,
monochloro-acetyl, dichloroacetyl, 7H-dodecafluoro-heptanoyl,
perfluoro-heptanoyl, 7H-dodeca-fluoroheptanoyl,
7-chlorododecafluoro-heptanoyl, 7-chloro-dodecafluoro-heptanoyl,
7H-dodecafluoroheptanoyl, 7H-dodeca-fluoroheptanoyl,
nona-fluoro-3,6-dioxa-heptanoyl, nonafluoro-3,6-dioxaheptanoyl,
perfluoroheptanoyl, methoxybenzoyl, methyl
3-amino-5-phenylthiophene-2-carboxyl,
3,6-dichloro-2-methoxy-benzoyl,
4-(1,1,2,2-tetrafluoro-ethoxy)-benzoyl, 2-bromo-propionyl,
omega-aminocapryl, decanoyl, n-pentadecanoyl, stearyl,
3-cyclopentyl-propionyl, 1-benzene-carboxyl, O-acetylmandelyl,
pivaloyl acetyl, 1-adamantane-carboxyl, cyclohexane-carboxyl,
2,6-pyridinedicarboxyl, cyclopropane-carboxyl,
cyclobutane-carboxyl, perfluorocyclohexyl carboxyl,
4-methylbenzoyl, chloromethyl isoxazolyl carbonyl,
perfluorocyclohexyl carboxyl, crotonyl,
1-methyl-1H-indazole-3-carbonyl, 2-propenylcarbonyl, isovaleryl,
1-pyrrolidinecarbonyl, and 4-phenylbenzoyl.
[0075] The term "amino acid" refers to naturally occurring and
synthetic .alpha., .beta., .gamma., or .delta. amino acids, and
includes but is not limited to, amino acids found in proteins, i.e.
glycine, alanine, valine, leucine, isoleucine, methionine,
phenylalanine, tryptophan, proline, serine, threonine, cysteine,
tyrosine, asparagine, glutamine, aspartate, glutamate, lysine,
arginine and histidine. In certain embodiments, the amino acid is
in the L-configuration. In certain embodiments, the amino acid is
in the D-configuration. In certain embodiments, the amino acid is
provided as a substituent of a compound described herein, wherein
the amino acid is a residue selected from alanyl, valinyl,
leucinyl, isoleuccinyl, prolinyl, phenylalaninyl, tryptophanyl,
methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl,
asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl,
histidinyl, .beta.-alanyl, .beta.-valinyl, .beta.-leucinyl,
.beta.-isoleuccinyl, .beta.-prolinyl, .beta.-phenylalaninyl,
.beta.-tryptophanyl, .beta.-methioninyl, .beta.-glycinyl,
.beta.-serinyl, .beta.-threoninyl, .beta.-cysteinyl,
.beta.-tyrosinyl, .beta.-asparaginyl, .beta.-glutaminyl,
.beta.-aspartoyl, .beta.-glutaroyl, .beta.-lysinyl,
.beta.-argininyl, or .beta.-histidinyl.
[0076] The term "amino acid derivative" refers to a group derivable
from a naturally or non-naturally occurring amino acid, as
described and exemplified herein. Amino acid derivatives are
apparent to those of skill in the art and include, but are not
limited to, ester, amino alcohol, amino aldehyde, amino lactone,
and N-methyl derivatives of naturally and non-naturally occurring
amino acids. In an embodiment, a compound described herein
comprises an amino acid derivative, wherein the amino acid
derivative is --NR.sup.X-G(S.sub.C)--C(O)-Q.sup.1-, wherein Q.sup.1
is --S--, --NR.sup.Y--, or --O--, R.sup.Y is hydrogen or alkyl,
S.sub.C is a side chain of a naturally occurring or non-naturally
occurring amino acid, G is C.sub.1-C.sub.2 alkylene, and R.sup.X is
hydrogen or R.sup.X and S.sub.C, together with the atoms to which
they are attached, combine to form a five-membered heterocyclic
ring. In an embodiment, an amino acid derivative is provided as a
substituent of a compound described herein, wherein the substituent
is --O--C(O)-G(S.sub.C)--NH-Q.sup.2-, wherein Q.sup.2 is a single
bond or --O--, S.sub.C is a side chain of a naturally occurring or
non-naturally occurring amino acid and G is C.sub.1-C.sub.2
alkylene. In certain embodiments, Q.sup.2 and S.sub.C, together
with the atoms to which they are attached, combine to form a
five-membered heterocyclic ring. In certain embodiments, G is
C.sub.1 alkylene and S.sub.C is hydrogen, alkyl, arylalkyl,
heterocycloalkyl, carboxylalkyl, heteroarylalkyl, aminoalkyl,
hydroxylalkyl, aminoiminoaminoalkyl, aminocarbonylalkyl,
sulfanylalkyl, carbamoylalkyl, alkylsulfanylalkyl, or
hydroxylarylalkyl. In an embodiment, an amino acid derivative is
provided as a substituent of a compound described herein, wherein
the amino acid derivative is in the D-configuration. In an
embodiment, an amino acid derivative is provided as a substituent
of a compound described herein, wherein the amino acid derivative
is in the L-configuration.
[0077] The term "alkylheterocyclo" refers to a heterocyclo group
with an alkyl substituent. The term "heterocycloalkyl" refers to an
alkyl group with a heterocyclo substituent.
[0078] As used herein, the term "carboxylalkyl" refers to an alkyl
substituted with at least 1, in another example 1 or 2, carboxy,
where alkyl is as described herein.
[0079] The term "alkylheteroaryl" refers to a heteroaryl group with
an alkyl substituent. The term "heteroarylalkyl" refers to an alkyl
group with a heteroaryl substituent.
[0080] As used herein, the term "aminoalkyl" refers to an alkyl
group substituted with at least 1, in another example 1 or 2, amino
substituent(s), where alkyl and amino are as described herein.
[0081] As used herein, the terms "hydroxylalkyl" and "hydroxyalkyl"
refer to an alkyl group substituted with at least 1, in another
example 1 or 2, hydroxyl, where alkyl is as described herein.
[0082] As used herein, the term "aminoiminoaminoalkyl" refers to an
alkyl substituted with at least 1, in another example 1 or 2,
-amino-C(NH)-amino, where alkyl and amino are as described
herein.
[0083] The term aminocarbonyl refers to the group --C(O)(amino)
where amino is as defined herein.
[0084] As used herein, the term "aminocarbonylalkyl" refers to an
alkyl substituted with at least 1, in another example 1 or 2,
--C(O)-amino, where alkyl and amino are as described herein.
[0085] As used herein, the term "sulfanylalkyl" refers to an alkyl
substituted with at least 1, in another example 1 or 2, --SH, where
alkyl is as described herein.
[0086] The term "carbamoyl" refers to a --NRC(OR', where R is
hydrogen or alkyl and R' is alkyl, cycloalkyl, heterocyclo,
heteroaryl, or aryl, as defined herein.
[0087] As used herein, the term "carbamoylalkyl" refers to an alkyl
substituted with at least 1, in another example 1 or 2, carbamoyl
groups, as defined herein.
[0088] As used herein, the term "alkylsulfanylalkyl" refers to an
alkyl substituted with at least 1, in another example 1 or 2, --S--
alkyl, where alkyl is as described herein.
[0089] As used herein, the term "hydroxylarylalkyl" refers to the
group -alkyl-aryl-OH, where alkyl and aryl are as described
herein.
[0090] The term "sulfonic acid" refers to the group
--S(O).sub.2OH.
[0091] The term "sulfate" refers to the group --OS(O).sub.2OR where
R is alkyl or arylalkyl.
[0092] The term "sulfonate" refers to the group --S(O).sub.2OR
where R is alkyl or arylalkyl.
[0093] The term "sulfonamido" refers to the group --S(O).sub.2NRR'
where R is hydrogen or alkyl and R' is alkyl, cycloalkyl,
heterocyclo, heteroaryl, or aryl, as defined herein.
[0094] The term "phosphate" refers to the group --OP(O)(OR).sub.2
where each R is independently alkyl or arylalkyl.
[0095] The term "phosphonic acid" refers to --P(O)(OH).sub.2.
[0096] The term "phosphonate" refers to the group --P(O)(OR).sub.2
where each R is independently alkyl or arylalkyl.
[0097] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. That is, a description directed to a polypeptide applies
equally to a description of a peptide and a description of a
protein, and vice versa. The terms apply to naturally occurring
amino acid polymers as well as amino acid polymers in which one or
more amino acid residues is a modified amino acid. Additionally,
such "polypeptides," "peptides" and "proteins" include amino acid
chains of any length, including full length proteins, wherein the
amino acid residues are linked by covalent peptide bonds.
[0098] Many of the compounds and conjugates described herein have
chiral centers. The present disclosure encompasses each
stereoisomer of each compound or conjugate with each possible
stereochemistry at each chiral center. Certain compounds are
identified by stereochemical notation that is known to those of
skill. In particular embodiments, stereochemistry is identified
with R and S notation for each chiral center, from left to right as
depicted in formula 1000, 1001, 1002, and (I), etc. or formula
(C1), F1, and G1, etc. For instance, the notation [R,S,S] indicates
R, S and S stereochemistry at the chiral centers of formula (I)
from left to right, beginning with the methylamino substituent
position and ending with the isopropyl substituent position.
Similarly, the notation [S,S,S] indicates S, S and S
stereochemistry at the chiral centers of formula (I) from left to
right. Further, the notation racemic [R/S,S,S] indicates a mixture
of [R,S,S] and [S,S,S] compounds. For other compounds and
conjugates herein, the notation can be applied to corresponding
structures.
[0099] The term "substantially free of" or "substantially in the
absence of," when used in connection with an article (including,
but not limited to, a compound, a salt thereof, a solvate thereof,
a solid form thereof, and the like), refers to the article that
includes at least 85% or 90% by weight, in certain embodiments,
95%, 98%, 99%, or 100% by weight, of the designated article. For
example, the term "substantially free of" or "substantially in the
absence of" with respect to a composition can refer to a
composition that includes at least 85% or 90% by weight, in certain
embodiments, 95%, 98%, 99%, or 100% by weight, of a designated
stereoisomer of a compound. In certain embodiments, in the methods
and compounds provided herein, the compounds are substantially free
of undesignated stereoisomers or other compounds. For another
example, the term "substantially free of" or "substantially in the
absence of" with respect to a solid form can refer to a solid form
that includes at least 85% or 90% by weight, in certain
embodiments, 95%, 98%, 99%, or 100% by weight, of the designated
solid form. In certain embodiments, in the methods and compounds
provided herein, the solid form is substantially free of other
solid forms.
[0100] Similarly, the term "isolated" with respect to a composition
refers to a composition that includes at least 85%, 90%, 95%, 98%,
or 99% to 100% by weight, of a designated compound, the remainder
comprising other chemical species or stereoisomers. Similarly, the
term "isolated" with respect to a solid form of a compound refers
to a solid that includes at least 85%, 90%, 95%, 98%, or 99% to
100% by weight, of such solid form of the compound, the remainder
comprising other solid forms of the compound, other compounds,
solvents, and/or other impurities.
[0101] "Solvate" refers to a compound provided herein or a salt
thereof, that further includes a stoichiometric or
non-stoichiometric amount of solvent bound by non-covalent
intermolecular forces. Where the solvent is water, the solvate is a
hydrate.
[0102] "Isotopic composition" refers to the amount of each isotope
present for a given atom, and "natural isotopic composition" refers
to the naturally occurring isotopic composition or abundance for a
given atom. Atoms containing their natural isotopic composition may
also be referred to herein as "non-enriched" atoms. Unless
otherwise designated, the atoms of the compounds recited herein are
meant to represent any stable isotope of that atom. For example,
unless otherwise stated, when a position is designated specifically
as "H" or "hydrogen," the position is understood to have hydrogen
at its natural isotopic composition.
[0103] "Isotopic enrichment" refers to the percentage of
incorporation of an amount of a specific isotope at a given atom in
a molecule in the place of that atom's natural isotopic abundance.
For example, deuterium enrichment of 1% at a given position means
that 1% of the molecules in a given sample contain deuterium at the
specified position. Because the naturally occurring distribution of
deuterium is about 0.0156%, deuterium enrichment at any position in
a compound synthesized using non-enriched starting materials is
about 0.0156%. The isotopic enrichment of the compounds provided
herein can be determined using conventional analytical methods
known to one of ordinary skill in the art, including mass
spectrometry and nuclear magnetic resonance spectroscopy.
[0104] "Isotopically enriched" refers to an atom having an isotopic
composition other than the natural isotopic composition of that
atom. "Isotopically enriched" may also refer to a compound
containing at least one atom having an isotopic composition other
than the natural isotopic composition of that atom.
[0105] As used herein, "alkyl," "cycloalkyl," "alkenyl,"
"cycloalkenyl," "alkynyl," "aryl," "alkoxy," "alkoxycarbonyl,"
"amino," "carboxyl," "alkylamino," "acylamino," "thioalkyoxy,"
"heterocyclyl," "heteroaryl," "alkylheterocyclyl,"
"alkylheteroaryl," "acyl," "aralkyl," "alkaryl," "purine,"
"pyrimidine," "carboxyl" and "amino acid" groups optionally
comprise deuterium at one or more positions where hydrogen atoms
are present, and wherein the deuterium composition of the atom or
atoms is other than the natural isotopic composition.
[0106] Also as used herein, "alkyl," "cycloalkyl," "alkenyl,"
"cycloalkenyl," "alkynyl," "aryl," "alkoxy," "alkoxycarbonyl,"
"carboxyl," "alkylamino," "acylamino," "thioalkyoxy,"
"heterocyclyl," "heteroaryl," "alkylheterocyclyl,"
"alkylheteroaryl," "acyl," "aralkyl," "alkaryl," "purine,"
"pyrimidine," "carboxyl" and "amino acid" groups optionally
comprise carbon-13 at an amount other than the natural isotopic
composition.
[0107] As used herein, EC.sub.50 refers to a dosage, concentration
or amount of a particular test compound that elicits a
dose-dependent response at 50% of maximal expression of a
particular response that is induced, provoked or potentiated by the
particular test compound.
[0108] As used herein, the IC.sub.50 refers to an amount,
concentration or dosage of a particular test compound that achieves
a 50% inhibition of a maximal response in an assay that measures
such response.
[0109] "Cancer" refers to cellular-proliferative disease states,
including but not limited to: Cardiac: sarcoma (angiosarcoma,
fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma,
fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma
(squamous cell, undifferentiated small cell, undifferentiated large
cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial
adenoma, sarcoma, lymphoma, chondromatous hanlartoma,
inesothelioma; Gastrointestinal: colon (colon carcinoma, colon
adenocarcinoma, colorectal adenocarcinoma), esophagus (squamous
cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach
(carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal
adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid
tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid
tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma,
neurofibroma, fibroma), large bowel (adenocarcinoma, tubular
adenoma, villous adenoma, hamartoma, leiomyoma); Genitourinary
tract: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma],
lymphoma, leukemia), bladder and urethra (squamous cell carcinoma,
transitional cell carcinoma, adenocarcinoma), prostate
(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal
carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial
cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma);
Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma,
hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma;
Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant
fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant
lymphoma (reticulum cell sarcoma), multiple myeloma, malignant
giant cell tumor chordoma, osteochronfroma (osteocartilaginous
exostoses), benign chondroma, chondroblastoma, chondromyxofibroma,
osteoid osteoma and giant cell tumors; Nervous system: skull
(osteoma, hemangioma, granuloma, xanthoma, osteitis deformans),
meninges (meningioma, meningiosarcoma, gliomatosis), brain
(astrocytoma, medulloblastoma, glioma, ependymoma, germinoma
[pineal oma], glioblastoma multiform, oligodendroglioma,
schwannoma, retinoblastoma, congenital tumors), spinal cord
neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus
(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor
cervical dysplasia), ovaries (ovarian carcinoma [serous
cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified
carcinoma], platinum-resistant ovarian, granulosa-thecal cell
tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant
teratoma, ovarian adenocarcinoma), vulva (squamous cell carcinoma,
intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),
vagina (clear cell carcinoma, squamous cell carcinoma, botryoid
sarcoma (embryonal rhabdomyosarcoma], fallopian tubes (carcinoma);
Hematologic: blood (myeloid leukemia [acute and chronic], acute
lymphoblastic leukemia, chronic lymphocytic leukemia,
myeloproliferative diseases, multiple myeloma, myelodysplastic
syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant
lymphoma]; Skin: melanoma, malignant melanoma, basal cell
carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles
dysplastic nevi, lipoma, angioma, dermatofibroma, keloids,
psoriasis; Adrenal Glands: neuroblastoma; a lymphoma; large cell
lymphoma; diffuse mixed histiocytic and lymphocytic lymphoma;
follicular B cell lymphoma; and breast (breast cancer which
overexpresses Her2, triple-negative breast cancer). Thus, the term
"cancerous cell" as provided herein, includes a cell afflicted by
any one of the above-identified conditions. As used herein, the
terms "subject" and "patient" are used interchangeably herein. The
terms "subject" and "subjects" refer to an animal, such as a mammal
including a non-primate (e.g., a cow, pig, horse, cat, dog, rat,
and mouse) and a primate (e.g., a monkey, a chimpanzee, and a
human), and for example, a human. In certain embodiments, the
subject is refractory or non-responsive to current treatments for
cell proliferation and/or cancer. In another embodiment, the
subject is a farm animal (e.g., a horse, a cow, a pig, etc.) or a
pet (e.g., a dog or a cat). In certain embodiments, the subject is
a human.
[0110] As used herein, the terms "therapeutic agent" and
"therapeutic agents" refer to any agent(s) which can be used in the
treatment or prevention of a disease or condition, or one or more
symptoms thereof. In certain embodiments, the term "therapeutic
agent" includes a compound provided herein. In certain embodiments,
a therapeutic agent is an agent which is known to be useful for, or
has been or is currently being used for the treatment or prevention
of a or condition, or one or more symptoms thereof.
[0111] "Therapeutically effective amount" refers to an amount of a
compound or composition that, when administered to a subject for
treating a disease or condition, is sufficient to effect such
treatment for the disease or condition. A "therapeutically
effective amount" can vary depending on, inter alia, the compound,
the disease or condition and its severity, and the age, weight,
etc., of the subject to be treated.
[0112] "Treating" or "treatment" of any disease or condition
refers, in certain embodiments, to ameliorating a disease or
condition that exists in a subject. In another embodiment,
"treating" or "treatment" includes ameliorating at least one
physical parameter, which may be indiscernible by the subject. In
yet another embodiment, "treating" or "treatment" includes
modulating the disease or condition, either physically (e.g.,
stabilization of a discernible symptom) or physiologically (e.g.,
stabilization of a physical parameter) or both. In yet another
embodiment, "treating" or "treatment" includes delaying the onset
of the disease or condition.
[0113] As used herein, the terms "prophylactic agent" and
"prophylactic agents" as used refer to any agent(s) which can be
used in the prevention of a disease or condition, or one or more
symptoms thereof. In certain embodiments, the term "prophylactic
agent" includes a compound provided herein. In certain other
embodiments, the term "prophylactic agent" does not refer a
compound provided herein. For example, a prophylactic agent is an
agent which is known to be useful for, or has been or is currently
being used to prevent or impede the onset, development, progression
and/or severity of a disease or condition.
[0114] As used herein, the phrase "prophylactically effective
amount" refers to the amount of a therapy (e.g., prophylactic
agent) which is sufficient to result in the prevention or reduction
of the development, recurrence or onset of one or more symptoms
associated with a disease or condition, or to enhance or improve
the prophylactic effect(s) of another therapy (e.g., another
prophylactic agent).
[0115] The term "antibody" refers to any macromolecule that would
be recognized as an antibody by those of skill in the art.
Antibodies share common properties including binding and at least
one polypeptide chain that is substantially identical to a
polypeptide chain that can be encoded by any of the immunoglobulin
genes recognized by those of skill in the art. The immunoglobulin
genes include, but are not limited to, the .kappa., .lamda.,
.alpha., .gamma. (IgG1, IgG2, IgG3, and IgG4), .delta., .epsilon.
and .mu. constant region genes, as well as the immunoglobulin
variable region genes. The term includes full-length antibodies and
antibody fragments recognized by those of skill in the art, and
variants thereof.
[0116] The term "antibody fragment" refers to any form of an
antibody other than the full-length form. Antibody fragments herein
include antibodies that are smaller components that exist within
full-length antibodies, and antibodies that have been engineered.
Antibody fragments include but are not limited to Fv, Fc, Fab, and
(Fab').sub.2, single chain Fv (scFv), diabodies, triabodies,
tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3,
combinations of CDR's, variable regions, framework regions,
constant regions, and the like (Maynard & Georgiou, 2000, Annu.
Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol.
9:395-402).
[0117] The term "immunoglobulin (Ig)" refers to a protein
consisting of one or more polypeptides substantially encoded by one
of the immunoglobulin genes, or a protein substantially identical
thereto in amino acid sequence. Immunoglobulins include but are not
limited to antibodies. Immunoglobulins may have a number of
structural forms, including but not limited to full-length
antibodies, antibody fragments, and individual immunoglobulin
domains including but not limited to V.sub.H, C.gamma.1, C.gamma.2,
C.gamma.3, V.sub.L, and C.sub.L.
[0118] The term "immunoglobulin (Ig) domain" refers to a protein
domain consisting of a polypeptide substantially encoded by an
immunoglobulin gene. Ig domains include but are not limited to
V.sub.H, C.gamma.1, C.gamma.2, C.gamma.3, V.sub.L, and C.sub.L.
[0119] The term "variable region" of an antibody refers to a
polypeptide or polypeptides composed of the V.sub.H immunoglobulin
domain, the V.sub.L immunoglobulin domains, or the V.sub.H and
V.sub.L immunoglobulin domains. Variable region may refer to this
or these polypeptides in isolation, as an Fv fragment, as a scFv
fragment, as this region in the context of a larger antibody
fragment, or as this region in the context of a full-length
antibody or an alternative, non-antibody scaffold molecule.
[0120] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are responsible for the binding specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed through the variable domains
of antibodies. It is concentrated in three segments called
Complementarity Determining Regions (CDRs) both in the light chain
and the heavy chain variable domains. The more highly conserved
portions of the variable domains are called the framework regions
(FR). The variable domains of native heavy and light chains each
comprise four FR regions, largely adopting a .beta.-sheet
configuration, connected by three or four CDRs, which form loops
connecting, and in some cases forming part of, the .beta.-sheet
structure. The CDRs in each chain are held together in close
proximity by the FR regions and, with the CDRs from the other
chain, contribute to the formation of the antigen binding site of
antibodies (see Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)).
[0121] The constant domains are not typically involved directly in
binding an antibody to an antigen, but exhibit various effector
functions. Depending on the amino acid sequence of the constant
region of their heavy chains, antibodies or immunoglobulins can be
assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these
may be further divided into subclasses (isotypes), e.g. IgG1, IgG2,
IgG3, and IgG4; IgA1 and IgA2. The heavy chain constant regions
that correspond to the different classes of immunoglobulins are
called .alpha., .delta., .epsilon., .gamma., and .mu.,
respectively. Of the various human immunoglobulin classes, only
human IgG1, IgG2, IgG3 and IgM are known to activate
complement.
[0122] The term "conjugate" refers to any compound that can be
formed by conjugating a compound described herein to a second
compound. The second compound can be a small molecule or a
macromolecule. In some embodiments, the second compound is a
bioactive molecule including, but not limited to, a protein, a
peptide, a nucleic active or a hybrid thereof. In some embodiments,
the second compound is a polymer such as polyethylene glycol. In
some embodiments, the second compound is a therapeutic agent,
including a commercially available drug. In some embodiments, the
second compound is a label that can recognize and bind to specific
targets, such as a molecular payload that is harmful to target
cells or a label useful for detection or diagnosis. In some
embodiments, the compound described herein is connected to the
second compound via a linker. In some embodiments, the compound
described herein is directly connected to the second compound
without a linker. In another embodiment the second compound is a
small molecule; a macromolecule; bioactive molecule including, but
not limited to, a protein, a peptide, a nucleic active or a hybrid
thereof; a polymer such as polyethylene glycol; a therapeutic
agent, including a commercially available drug; or a label that can
recognize and bind to specific targets, such as a molecular payload
that is harmful to target cells or a label useful for detection or
diagnosis. In another embodiment, the second compound comprises a
modified amino acid comprising an alkyne, strained alkene,
tetrazine, thiol, maleimide, carbonyl, oxyamine, or azide.
[0123] The term "variant protein sequence" refers to a protein
sequence that has one or more residues that differ in amino acid
identity from another similar protein sequence. Said similar
protein sequence may be the natural wild type protein sequence, or
another variant of the wild type sequence. Variants include
proteins that have one or more amino acid insertions, deletions or
substitutions. Variants also include proteins that have one or more
post-translationally modified amino acids.
[0124] The term "parent antibody" refers to an antibody known to
those of skill in the art that is modified according to the
description provided herein. The modification can be physical,
i.e., chemically or biochemically replacing or modifying one or
more amino acids of the parent antibody to yield an antibody within
the scope of the present description. The modification can also be
conceptual, i.e., using the sequence of one or more polypeptide
chains of the parent antibody to design an antibody comprising one
or more site-specific modified amino acids according to the present
description. Parent antibodies can be naturally occurring
antibodies or antibodies designed or developed in a laboratory.
Parent antibodies can also be artificial or engineered antibodies,
e.g., chimeric or humanized antibodies.
[0125] The term "conservatively modified variant" refers to a
protein that differs from a related protein by conservative
substitutions in amino acid sequence. One of skill will recognize
that individual substitutions, deletions or additions to a peptide,
polypeptide, or protein sequence which alters, adds or deletes a
single amino acid or a small percentage of amino acids in the
encoded sequence is a "conservatively modified variant" where the
alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles.
[0126] The following eight groups each contain amino acids that are
conservative substitutions for one another:
1) Alanine (A), Glycine (G);
[0127] 2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cysteine (C), Methionine (M).
[0128] See, e.g., Creighton, Proteins: Structures and Molecular
Properties, W H Freeman & Co.; 2nd edition (December 1993).
[0129] The terms "identical" or "identity," in the context of two
or more polypeptide sequences, refer to two or more sequences or
subsequences that are the same. Sequences are "substantially
identical" if they have a percentage of amino acid residues or
nucleotides that are the same (i.e., about 60% identity, optionally
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,
or about 95% identity over a specified region), when compared and
aligned for maximum correspondence over a comparison window, or
designated region as measured using one of the following sequence
comparison algorithms or by manual alignment and visual inspection.
The identity can exist over a region that is at least about 50
amino acids or nucleotides in length, or over a region that is
75-100 amino acids or nucleotides in length, or, where not
specified, across the entire sequence or a polypeptide. In the case
of antibodies, identity can be measured outside the variable CDRs.
Optimal alignment of sequences for comparison can be conducted,
including but not limited to, by the local homology algorithm of
Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology
alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol.
48:443, by the search for similarity method of Pearson and Lipman
(1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.); or by manual
alignment and visual inspection (see, e.g., Ausubel et al., Current
Protocols in Molecular Biology (1995 supplement)).
[0130] Examples of algorithms that are suitable for determining
percent sequence identity and sequence similarity include the BLAST
and BLAST 2.0 algorithms, which are described in Altschul et al.
(1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J.
Mol. Biol. 215:403-410, respectively. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information. The BLAST algorithm parameters W, T, and
X determine the sensitivity and speed of the alignment. The BLASTN
program (for nucleotide sequences) uses as defaults a wordlength
(W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of
both strands. For amino acid sequences, the BLASTP program uses as
defaults a wordlength of 3, and expectation (E) of 10, and the
BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc.
Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation
(E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST
algorithm is typically performed with the "low complexity" filter
turned off. In some embodiments, the BLAST algorithm is typically
performed with the "low complexity" filter turned on.
[0131] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin and
Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, in another
embodiment less than about 0.01, and in another embodiment less
than about 0.001.
[0132] The term "amino acid" refers to naturally occurring and
non-naturally occurring amino acids, as well as amino acids such as
proline, amino acid analogs and amino acid mimetics that function
in a manner similar to naturally occurring amino acids.
[0133] Naturally encoded amino acids are the proteinogenic amino
acids known to those of skill in the art. They include the 20
common amino acids (alanine, arginine, asparagine, aspartic acid,
cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,
leucine, lysine, methionine, phenylalanine, proline, serine,
threonine, tryptophan, tyrosine, and valine) and the less common
pyrrolysine and selenocysteine. Naturally encoded amino acids
include post-translational variants of the 22 naturally occurring
amino acids such as prenylated amino acids, isoprenylated amino
acids, myrisoylated amino acids, palmitoylated amino acids,
N-linked glycosylated amino acids, O-linked glycosylated amino
acids, phosphorylated amino acids and acylated amino acids.
[0134] The term "modified amino acid" refers to an amino acid that
is not a proteinogenic amino acid, or a post-translationally
modified variant thereof. In particular, the term refers to an
amino acid that is not one of the 20 common amino acids or
pyrrolysine or selenocysteine, or post-translationally modified
variants thereof.
[0135] The term "strained alkene" refers to a molecule comprising
an alkene moiety that is capable of reacting with tetrazine in a
tetrazine ligation. Exemplary tetrazine ligations are described in
Blackman et al., 2008, J. Am. Chem. Soc. 130:13518-13519. Examples
include trans-cyclooctenes and norbornenes. Useful compounds
include, but are not limited to, trans-cyclooctene,
(E)-cyclooct-4-enol, (E)-cyclooct-4-enyl 2,5-dioxo-1-pyrrolidinyl
carbonate, 5-norbornene-2-acetic acid succinimidyl ester, and
5-norbornene-2-endo-acetic acid.
[0136] The term "tetrazine" refers to a compound or group
comprising the following structure:
##STR00007##
wherein R.sup.201 is lower alkyl. For example, R.sup.201 can be
methyl, ethyl, or propyl. In certain aspects, R.sup.201 is
methyl.
Compounds
[0137] In certain embodiments, the compound is not of formula
(101), (101a), or (101b) and the conjugate does not comprise the
compound of formula (101), (101a), or (101b). In certain
embodiments, the compound is not of formula (101a), and the
conjugate does not comprise the compound of formula (101a). In
certain embodiments where X is
##STR00008##
the compound is not the compound of formula (101), (101a), or
(101b) and the conjugate does not comprise the compound of formula
(101), (101a), or (101b). In certain embodiments where X is
##STR00009##
the compound is not the compound of formula (101a), and the
conjugate does not comprise the compound of formula (101a). In
certain embodiments, the compound is not of formula (101), (101a),
or (101b). In certain embodiments, the compound is not of formula
(101a). In certain embodiments where X is
##STR00010##
the compound is not the compound of formula (101), (101a), or
(101b). In certain embodiments where X is
##STR00011##
the compound is not the compound of formula (101a). In certain
embodiments, the conjugate does not comprise the compound of
formula (101), (101a), or (101b). In certain embodiments, the
conjugate does not comprise the compound of formula (101a). In
certain embodiments where X is
##STR00012##
the conjugate does not comprise the compound of formula (101),
(101a), or (101b). In certain embodiments where X is
##STR00013##
the conjugate does not comprise the compound of formula (101a).
[0138] When a range of formula are used, for example I-XIXb-2, each
formula within that range is included and is as if it were
explicitly listed, including where the roman numeral is followed
by, for example, "a," "-1," etc. For example, I-XIXb-2 includes Va,
XIV, and XIXa-1, etc.
[0139] In an embodiment, provided herein is a compound according to
Formula I:
##STR00014##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein:
[0140] Ar is a divalent five- or six-membered, substituted or
unsubstituted, monocyclic aryl or heteroaryl ring or a divalent
eight-, nine- or ten-membered, substituted or unsubstituted, fused
bicyclic aryl or heteroaryl ring;
[0141] L is absent or --CH.sub.2--;
[0142] W.sup.1, W.sup.2, W.sup.3, W.sup.4, and W.sup.5 are each
independently a single bond, absent, or a divalent attaching
group;
[0143] EG is an eliminator group;
[0144] each RT is a release trigger group, in the backbone of
Formula (I) or bonded to EG, wherein one RT is optional;
[0145] HP is a single bond, absent, or a divalent hydrophilic
group;
[0146] SG is a single bond, absent, or a divalent spacer group;
and
[0147] R is hydrogen, a terminal conjugating group, or a divalent
residue of a terminal conjugating group;
[0148] or, in the alternative, W.sup.1, W.sup.2, W.sup.3, W.sup.4,
W.sup.5, EG, RT, HP, SG, and R combine to form --H.
[0149] In one embodiment, provided herein is a compound of Formula
1000 according to 1001:
##STR00015##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein:
[0150] Ar is a divalent five- or six-membered, substituted or
unsubstituted, monocyclic aryl or heteroaryl ring or a divalent
eight-, nine- or ten-membered, substituted or unsubstituted, fused
bicyclic aryl or heteroaryl ring;
[0151] L is absent or --CH.sub.2--;
[0152] W.sup.1, W.sup.2, W.sup.3, W.sup.4, and W.sup.5 are each
independently a single bond, absent, or a divalent attaching
group;
[0153] EG is absent or an eliminator group;
[0154] RT.sup.1 is a release trigger group or a cleavable
linker;
[0155] RT is a release trigger group bonded to EG; and wherein RT
is optional;
[0156] HP.sup.1 is single bond, absent, a divalent hydrophilic
group, or
##STR00016##
where R.sup.SG is a monovalent hydrophilic group;
[0157] SG is a single bond, absent, or a divalent spacer group;
and
[0158] R is hydrogen, a terminal conjugating group, or a divalent
residue of a terminal conjugating group.
[0159] In one embodiment, provided herein is a compound of Formula
1000 according to 1002:
##STR00017##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein:
[0160] Ar is a divalent five- or six-membered, substituted or
unsubstituted, monocyclic aryl or heteroaryl ring or a divalent
eight-, nine- or ten-membered, substituted or unsubstituted, fused
bicyclic aryl or heteroaryl ring;
[0161] L is absent or --CH.sub.2--;
[0162] W.sup.1, W.sup.2, W.sup.3, W.sup.4, and W.sup.5 are each
independently a single bond, absent, or a divalent attaching
group;
[0163] EG is absent or an eliminator group;
[0164] RT.sup.1 is a release trigger group or a cleavable
linker;
[0165] RT is a release trigger group bonded to EG; and wherein RT
is optional;
[0166] HP.sup.1 is single bond, absent, a divalent hydrophilic
group, or
##STR00018##
where R.sup.SG is a monovalent hydrophilic group;
[0167] SG is a single bond, absent, or a divalent spacer group;
and
[0168] R is hydrogen, a terminal conjugating group, or a divalent
residue of a terminal conjugating group.
[0169] In certain embodiments, a conjugating group can be used to
conjugate a modified Hemiasterlin as described herein (e.g.,
according to any of Formulas 1000-1000b, 1001-1001b, 1002-1002b,
and I-XIXb-2, 101-111b, or 1-8b) to any molecular entity capable of
reacting with the conjugating group to form the conjugate. In
certain embodiments, the conjugating group is designated R herein.
The conjugating group can be directly or indirectly linked to the
modified Hemiasterlin as described herein (e.g., according to any
of Formulas 1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2,
101-111b, or 1-8b) via one or more attaching groups, eliminator
groups, release trigger groups, hydrophobic groups, and/or spacer
groups.
[0170] Attaching Groups
[0171] Attaching groups facilitate incorporation of eliminator
groups, release trigger groups, hydrophobic groups, spacer groups,
and/or conjugating groups into a compound, such as a modified
Hemiasterlin as described herein (e.g., according to any of
Formulas 1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2,
101-111b, or 1-8b). Useful attaching groups are known to, and are
apparent to, those of skill in the art. Examples of useful
attaching groups are provided herein. In certain embodiments,
attaching groups are designated W.sup.1, W.sup.2, W.sup.3, W.sup.4,
or W.sup.5. In certain embodiments, an attaching group can comprise
a divalent ester, divalent ether, divalent amide, divalent amine,
alkylene, arylene, sulfide, disulfide, --C(O)--, or a combination
thereof. In certain embodiments an attaching group can comprise
--C(O)--, --O--, --C(O)O--, --OC(O)--, --C(O)NH--, --C(O)NH-alkyl-,
--OC(O)NH--, --S.sub.C(O)NH--, --NH--, --N(alkyl)-,
--N(R)-alkylene-N(R)-- (where each R is independently H or alkyl),
--N(CH.sub.3)CH.sub.2CH.sub.2N(CH.sub.3)--, --CH.sub.2--,
--CH.sub.2CH.sub.2--, --CH(CH.sub.3)--, --C(CH.sub.3).sub.2--,
--CH.sub.2CH.sub.2CH.sub.2--, phenylene,
--NHCH.sub.2CH.sub.2C(O)--, --C(O)CH.sub.2CH.sub.2NH--, --S--,
--S--S--, --OCH.sub.2CH.sub.2O--, or the reverse (e.g. --NHC(O)--)
thereof, or a combination thereof.
[0172] Eliminator Groups
[0173] Eliminator groups facilitate separation of a biologically
active portion of a compound or conjugate described herein from the
remainder of the compound or conjugate in vivo and/or in vitro.
Eliminator groups can also facilitate separation of a biologically
active portion of a compound or conjugate described herein in
conjunction with a release trigger group. For example, the
eliminator group and the release trigger group can react in a
Releasing Reaction to release a biologically active portion of a
compound or conjugate described herein from the compound or
conjugate in vivo and/or in vitro. Upon initiation of the releasing
reaction by the release trigger, the eliminator group cleaves the
biologically active moiety, or a prodrug form of the biologically
active moiety, and forms a stable, non-toxic entity that has no
further effect on the activity of the biologically active
moiety.
[0174] In certain embodiments, the eliminator group is designated
EG herein. Useful eliminator groups include those described herein.
In certain embodiments, the eliminator group comprises a phenylene,
a --C(O)--, an amino, or a combination thereof. In certain
embodiments, the eliminator group is:
##STR00019##
wherein each R.sup.EG is independently selected from the group
consisting of hydrogen, alkyl, biphenyl, --CF.sub.3, --NO.sub.2,
--CN, fluoro, bromo, chloro, alkoxyl, alkylamino, dialkylamino,
alkyl-C(O)O--, alkylamino-C(O)-- and dialkylamino-C(O)--. In the
second and third structures, those of skill will recognize that EG
is bonded to an RT that is not within the backbone of the e.g.
formula 1000, or (I), as indicated in the above description of
formula 1000 and (I). In some embodiments, each R.sup.EG is
independently selected from the group consisting of hydrogen,
alkyl, biphenyl, --CF.sub.3, alkoxyl, alkylamino, dialkylamino,
alkyl-C(O)O--, alkylamino-C(O)-- and dialkylamino-C(O)--. In
further embodiments, each R.sup.EG is independently selected from
the group consisting of hydrogen, --NO.sub.2, --CN, fluoro, bromo,
and chloro.
[0175] Release Trigger Groups and Cleavable Linkers
[0176] In certain embodiments, release trigger groups facilitate
separation of a biologically active portion of a compound or
conjugate described herein from the remainder of the compound or
conjugate in vivo and/or in vitro. In certain embodiments, release
trigger groups can also facilitate separation of a biologically
active portion of a compound or conjugate described herein in
conjunction with an eliminator group. In some embodiments, the
eliminator group and the release trigger group can react in a
Releasing Reaction to release a biologically active portion of a
compound or conjugate described herein from the compound or
conjugate in vivo and/or in vitro. In certain embodiments, the
release trigger can act through a biologically-driven reaction with
high tumor:nontumor specificity, such as the proteolytic action of
an enzyme overexpressed in a tumor environment.
[0177] In certain embodiments, the release trigger group is
designated RT herein. In certain embodiments, RT is divalent and
bonded within the backbone of formula (I) or 1000. In other
embodiments, RT is monovalent and bonded to EG as depicted above.
Useful release trigger groups include those described herein. In
certain embodiments, the release trigger group comprises a residue
of a natural or non-natural amino acid or residue of a sugar ring.
In certain embodiments, the release trigger group comprises a
residue of a natural or non-natural amino acid or residue of a
sugar ring.
[0178] In some embodiments, the release trigger group is derived
from a linker precursor selected from the group consisting of
dipeptides, tripeptides, tetrapeptides, and pentapeptides, each of
which comprises one citrulline. Exemplary dipeptides include, but
are not limited to, valine-citrulline (vc or val-cit), and
N-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides
include, but are not limited to, glycine-valine-citrulline
(gly-val-cit). In some embodiments, the release trigger group is
derived from a linker precursor selected from the group consisting
of valine-citrulline, N-methyl-valine-citrulline, and
glycine-valine-citrulline, In certain embodiments, the release
trigger group is:
##STR00020##
Those of skill will recognize that the first structure is divalent
and can be bonded within the backbone of formula (I) or 1000, and
that the second structure is monovalent and can be bonded to EG as
depicted in formula (I) and 1000 above.
[0179] Cleavable linkers facilitate separation of a biologically
active portion of a compound or conjugate described herein from the
remainder of the compound or conjugate in vivo and/or in vitro. In
certain embodiments, the release trigger can act through a
biologically-driven reaction with high tumor:nontumor specificity,
such as the proteolytic action of an enzyme overexpressed in a
tumor environment. In certain embodiments, the cleavable linker is
designated RT.sup.1 herein. Useful cleavable linkers include those
described herein. In some embodiments, the cleavable linker is
derived from a linker precursor selected from the group consisting
of dipeptides, tripeptides, tetrapeptides, and pentapeptides. In
such embodiments, the linker can be cleaved by a protease.
Exemplary dipeptides include, but are not limited to,
valine-alanine (VA or Val-Ala); valine-glutamic acid (Val-Glu);
alanine-phenylalanine (AF or Ala-Phe); phenylalanine-lysine (FK or
Phe-Lys); and phenylalanine-homolysine (Phe-homoLys). Exemplary
tripeptides include, but are not limited to glycine-glycine-glycine
(Gly-Gly-Gly). In certain embodiments, the cleavable linker is
derived from a linker precursor selected from the group consisting
of dipeptides and tripeptides. In certain embodiments, the
cleavable linker is derived from a dipeptide. In certain
embodiments, the cleavable linker is derived from a tripeptide. In
certain embodiments the cleavable linker is derived from a linker
precursor derived from valine-alanine, valine-glutamic acid,
phenylalanine-homolysine, phenylalanine-lysine,
phenylalanine-homolysine, or glycine-glycine-glycine.
[0180] In certain embodiments the cleavable linker is derived from
a linker precursor selected from the group consisting of
dipeptides, tripeptides, tetrapeptides, and pentapeptides; or
is
##STR00021##
or is
##STR00022##
where aa is a natural or non-natural amino acid residue; or is
##STR00023##
where the
##STR00024##
ring is a 4-7 membered heterocyclic ring comprising 3-6 carbon
atoms. In certain embodiments the cleavable linker is derived from
a linker precursor selected from the group consisting of dipeptides
and tripeptides; or is
##STR00025##
or is
##STR00026##
where aa is a natural or non-natural amino acid residue; or is
##STR00027##
where the
##STR00028##
ring is a 4-7 membered heterocyclic ring comprising 3-6 carbon
atoms.
[0181] In certain embodiments the cleavable linker is derived from
a linker precursor selected from valine-alanine, valine-glutamic
acid, alanine-phenylalanine; phenylalanine-lysine;
phenylalanine-homolysine; and glycine-glycine-glycine
(Gly-Gly-Gly); or is
##STR00029##
or is
##STR00030##
where aa is a natural or non-natural amino acid residue; or is
##STR00031##
where the
##STR00032##
ring is a 4-7 membered heterocyclic ring comprising 3-6 carbon
atoms.
[0182] In certain embodiments the cleavable linker is
##STR00033##
Hydrophilic Groups
[0183] Hydrophilic groups facilitate increasing the hydrophilicity
of the compounds described herein. It is believed that increased
hydrophilicity allows for greater solubility in aqueous solutions,
such as aqueous solutions found in biological systems. Hydrophilic
groups can also function as spacer groups or substituents, which
are described in further detail herein.
[0184] In certain embodiments, the hydrophilic group is designated
HP and HP.sup.1 herein. Useful hydrophilic groups include those
described herein. In certain embodiments, the HP hydrophilic group
is a divalent poly(ethylene glycol). In certain embodiments, the HP
hydrophilic group is a divalent poly(ethylene glycol) according to
the formula:
##STR00034##
wherein m is an integer from 1 to 12, optionally 1 to 4, optionally
2 to 4. In certain embodiments, the HP.sup.1 hydrophilic group is a
divalent hydrophilic group or a
##STR00035##
where R.sup.SG is a monovalent hydrophilic group. In certain
embodiments, R.sup.SG is a monovalent poly(ethylene glycol). In
certain embodiments, R.sup.SG is a monovalent poly(ethylene glycol)
according to the formula:
##STR00036##
wherein R is --H or --CH.sub.3 and m is an integer from 1 to 12,
optionally 1 to 4, optionally 2 to 4. In certain embodiments,
R.sup.SG is a monovalent poly(ethylene glycol) according to the
formula:
##STR00037##
wherein R is --H or --CH.sub.3 and m is an integer from 1 to 12,
optionally 1 to 4, optionally 2 to 4; R.sup.SG or
is--C1-C6-alkylene-S(O).sub.3.sup.-. In certain embodiments,
R.sup.SG is a monovalent poly(ethylene glycol) according to the
formula:
##STR00038##
wherein R is --H or --CH.sub.3 and m is 2 to 4; or is
--CH.sub.2CH.sub.2--S(O).sub.3.sup.-. In certain embodiments,
R.sup.SG is a monovalent poly(ethylene glycol) according to the
formula:
##STR00039##
[0185] or R.sup.SG is --CH.sub.2CH.sub.2--S(O).sub.3.sup.-. In
certain embodiments, R.sup.SG is
--C.sub.1-C.sub.6-alkylene-S(O).sub.3.sup.-. In certain
embodiments, R.sup.SG is --CH.sub.2CH.sub.2--S(O).sub.3.sup.-.
[0186] Spacer Groups
[0187] Spacer groups facilitate spacing of the conjugating group
from the other groups of the compounds described herein. This
spacing can lead to more efficient conjugation of the compounds
described herein to a second compound. The spacer group can also
stabilize the conjugating group.
[0188] In certain embodiments, the spacer group is designated SG
herein. Useful spacer groups include those described herein. In
certain embodiments, the spacer group is:
##STR00040##
In certain embodiments, SG, W.sup.4, and the HP or HP.sup.1 group
combine to form a divalent poly(ethylene glycol) according to the
formula:
##STR00041##
wherein m is an integer from 1 to 12, optionally 1 to 4, optionally
2 to 4.
[0189] Conjugating Groups and Residues Thereof
[0190] Conjugating groups facilitate conjugation of the compounds
described herein to a second compound, such as a targeting moiety.
In certain embodiments, the conjugating group is designated R
herein. Conjugating groups can react via any suitable reaction
mechanism known to those of skill in the art. In certain
embodiments, a conjugating group reacts through a [3+2]
alkyne-azide cycloaddition reaction, inverse-electron demand
Diels-Alder ligation reaction, thiol-electrophile reaction, or
carbonyl-oxyamine reaction, as described in detail herein. In
certain embodiments, the conjugating group comprises an alkyne,
strained alkene, tetrazine, thiol, para-acetyl-phenylalanine
residue, oxyamine, maleimide, or azide. In certain embodiments, the
conjugating group is:
##STR00042##
--N.sub.3, or --SH; wherein R.sup.201 is lower alkyl. In an
embodiment, R.sup.201 is methyl, ethyl, or propyl. In an
embodiment, R.sup.201 is methyl.
[0191] After conjugation, a divalent residue of the conjugating
group is formed and is bonded to the residue of a second compound.
The structure of the divalent residue is determined by the type of
conjugation reaction employed to form the conjugate.
[0192] In certain embodiments when a conjugate is formed through a
[3+2] alkyne-azide cycloaddition reaction, the divalent residue of
the conjugating group comprises a triazole ring or fused cyclic
group comprising a triazole ring. In certain embodiment when a
conjugate is formed through a [3+2] alkyne-azide cycloaddition
reaction, the divalent residue of the conjugating group is:
##STR00043##
[0193] In certain embodiments when a conjugate is formed through a
tetrazine inverse electron demand Diels-Alder ligation reaction,
the divalent residue of the conjugating group comprises a fused
bicyclic ring having at least two adjacent nitrogen atoms in the
ring. In certain embodiments when a conjugate is formed through a
tetrazine inverse electron demand Diels-Alder ligation reaction,
the divalent residue of the conjugating group is:
##STR00044##
[0194] In certain embodiments when a conjugate is formed through a
thiol-maleimide reaction, the divalent residue of the conjugating
group comprises succinimidylene and a sulfur linkage. In certain
embodiments when a conjugate is formed through a thiol-maleimide
reaction, the divalent residue of the conjugating group is:
##STR00045##
[0195] In certain embodiments when a conjugate is formed through a
carbonyl-oxyamine reaction, the divalent residue of the conjugating
group comprises a divalent residue of a non-natural amino acid. In
certain embodiments when a conjugate is formed through a
carbonyl-oxyamine reaction, the divalent residue of the conjugating
group is:
##STR00046##
In certain embodiments when a conjugate is formed through a
carbonyl-oxyamine reaction, the divalent residue of the conjugating
group comprises an oxime linkage. In certain embodiments when a
conjugate is formed through a carbonyl-oxyamine reaction, the
divalent residue of the conjugating group is:
##STR00047##
[0196] In an embodiment, provided herein is a compound according to
any of Formulas 1000-1002b, I-Ib, or X-XIXb-2, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein Ar is a divalent five- or six-membered,
substituted or unsubstituted, monocyclic aryl or heteroaryl ring.
In an embodiment, provided herein is a compound according to any of
Formulas XVIb1000-1002b, Mb, or X-XIXb-2, or a pharmaceutically
acceptable salt, solvate, stereoisomer, or tautomer thereof;
wherein Ar is a divalent six-membered, substituted or
unsubstituted, monocyclic aryl or heteroaryl ring. In an
embodiment, provided herein is a compound according to any of
Formulas XVIb1000-1002b, I-Ib, or X-XIXb-2, or a pharmaceutically
acceptable salt, solvate, stereoisomer, or tautomer thereof;
wherein Ar is a divalent eight-, nine- or ten-membered, substituted
or unsubstituted, fused bicyclic aryl or heteroaryl ring. In an
embodiment, provided herein is a compound according to any of
Formulas XVIb1000-1002b, I-Ib, or X-XIXb-2, or a pharmaceutically
acceptable salt, solvate, stereoisomer, or tautomer thereof;
wherein Ar is a divalent nine-membered, substituted or
unsubstituted, fused bicyclic heteroaryl ring. In an embodiment,
provided herein is a compound according to any of Formulas
XVIb1000-1002b, I-Ib, or X-XIXb-2, or a pharmaceutically acceptable
salt, solvate, stereoisomer, or tautomer thereof; wherein Ar is
phenylene or indolylene, each of which is unsubstituted or
substituted. In an embodiment, provided herein is a compound
according to any of Formulas XVIb1000-1002b, I-Ib, or X-XIXb-2, or
a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein Ar is any of the following:
##STR00048##
[0197] In an embodiment, provided herein is a compound according to
any of Formulas XVIb1000-1002b, I-Ib, or X-XIXb-2, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein L is absent. In an embodiment, provided
herein is a compound according to any of Formulas XVIb1000-1002b,
I-Ib, or X-XIXb-2, or a pharmaceutically acceptable salt, solvate,
stereoisomer, or tautomer thereof; wherein L is --CH.sub.2--.
[0198] In an embodiment, provided herein is a compound according to
any of Formulas 1000-1002b and I-XIXb-2 in which the group "EG" is
present in the formula, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof; wherein EG comprises
phenylene, carboxylene, amino, or a combination thereof. In an
embodiment, provided herein is a compound according to any of
Formulas I 1000-1002b and I-XIXb-2 in which the group "EG" is
present in the formula, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof; wherein EG is:
##STR00049##
wherein each R.sup.EG is independently selected from the group
consisting of hydrogen, alkyl, biphenyl, --CF.sub.3, --NO.sub.2,
--CN, fluoro, bromo, chloro, alkoxyl, alkylamino, dialkylamino,
alkyl-C(O)O--, alkylamino-C(O)-- and dialkylaminoC(O)--. In the
second and third structures, those of skill will recognize that EG
is bonded to an RT that is not within the backbone of formula 1000
or (I) as indicated in the above description of formula 1000 and
(I). In some embodiments, each R.sup.EG is independently selected
from the group consisting of hydrogen, alkyl, biphenyl, --CF.sub.3,
alkoxyl, alkylamino, dialkylamino, alkyl-C(O)O--, alkylamino-C(O)--
and dialkylaminoC(O)--. In further embodiments, each R.sup.EG is
independently selected from the group consisting of hydrogen,
--NO.sub.2, --CN, fluoro, bromo, and chloro.
[0199] In an embodiment, provided herein is a compound according to
any of Formulas 1000-1002b and I-XIXb-2 in which the group "RT" is
present in the formula, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof; wherein RT comprises a
residue of a natural or non-natural amino acid or a residue of a
sugar. In an embodiment, provided herein is a compound according to
any of Formulas I1000-1002b and I-XIXb-2 in which the group "RT" is
present in the formula, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof; wherein RT is:
##STR00050##
Those of skill will recognize that the first structure is divalent
and can be bonded within the backbone of formula 1000 or (I), and
that the second structure is monovalent and can be bonded to EG as
depicted in formula 1000 and (I) above.
[0200] In an embodiment, provided herein is a compound according to
any of Formulas 1000-1002b and I-XIXb-2 in which the group "HP" is
present in the formula, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof; wherein HP comprises
poly(ethylene glycol). In an embodiment, provided herein is a
compound according to any of Formulas 1000-1002b and I-XIXb-2 in
which the group "HP" is present in the formula, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein HP is:
##STR00051##
wherein m is an integer from 1 to 12.
[0201] In an embodiment, provided herein is a compound according to
any of Formulas 1000-1002b and I-XIXb-2 in which the group "SG" is
present in the formula, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof; wherein SG com
comprises C.sub.1-C.sub.10 alkylene, C.sub.4-C.sub.6 alkylene,
--C(O)--, or combination thereof. In an embodiment, provided herein
is a compound according to any of Formulas 1000-1002b and I-XIXb-2
in which the group "SG" is present in the formula, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein SG is:
##STR00052##
[0202] In an embodiment, provided herein is a compound according to
any of Formulas 1000-1002b and I-XIXb-2 in which the group
"W.sup.1," "W.sup.2," "W.sup.3," "W.sup.4," and/or "W.sup.5" is
present in the formula, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof, wherein W.sup.1,
W.sup.2, W.sup.3, W.sup.4, and W.sup.5 are each independently a
single bond, absent, or comprise a divalent ketone, divalent ester,
divalent ether, divalent amide, divalent amine, alkylene, arylene,
sulfide, disulfide, --C(O)--, or a combination thereof. In an
embodiment, provided herein is a compound according to any of
Formulas 1000-1002b and I-XIXb-2 in which the group "W.sup.1,"
"W.sup.2," "W.sup.3," "W.sup.4," and/or "W.sup.5" is present in the
formula, or a pharmaceutically acceptable salt, solvate,
stereoisomer, or tautomer thereof; wherein W.sup.1, W.sup.2,
W.sup.3, W.sup.4, and W.sup.5 are each independently a single bond,
absent, or comprise --C(O)--, --O--, --C(O)NH--, --C(O)NH-alkyl-,
--OC(O)NH--, --S.sub.C(O)NH--, --NH--, --NH- alkyl-,
--N(CH.sub.3)CH.sub.2CH.sub.2N(CH.sub.3)--, --S--, --S--S--,
--OCH.sub.2CH.sub.2O--, or a combination thereof.
[0203] In an embodiment, provided herein is a compound according to
any of Formulas 1000-1002b and I-XIXb-2 in which the group "R" is
present in the formula, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof; wherein R is a
conjugating group or a residue of a conjugating group. In an
embodiment, provided herein is a compound according to any of
Formulas 1000-1002b and I-XIXb-2 in which the group "R" is present
in the formula, or a pharmaceutically acceptable salt, solvate,
stereoisomer, or tautomer thereof; wherein R comprises an alkyne,
strained alkene, tetrazine, thiol, para-acetyl-phenylalanine
residue, oxyamine, maleimide, carbonyl alkyl halide, aryl sulfide,
or azide. In an embodiment, provided herein is a compound according
to any of Formulas 1000-1002b and I-XIXb-2 in which the group "R"
is present in the formula, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof; wherein R is:
##STR00053##
--N.sub.3, or --SH; wherein R.sup.201 is lower alkyl. In an
embodiment, provided herein is a compound according to any of
Formulas 1000-1002b and I-XIXb-2 in which the group "R" is present
in the formula, or a pharmaceutically acceptable salt, solvate,
stereoisomer, or tautomer thereof; wherein R is:
##STR00054##
and R.sup.201 is methyl, ethyl, or propyl. In an embodiment,
provided herein is a compound according to any of Formulas
1000-1002b and I-XIXb-2 in which the group "R" is present in the
formula, or a pharmaceutically acceptable salt, solvate,
stereoisomer, or tautomer thereof; wherein R is:
##STR00055##
and R.sup.201 is methyl.
[0204] In an embodiment, provided herein is a compound according to
any of Formulas I-IXb, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof; wherein W.sup.1,
W.sup.2, W.sup.3, W.sup.4, W.sup.5, EG, RT, HP, SG, and R combine
to form --H.
[0205] In an embodiment, provided herein is a compound according to
Formula 1000a or Formula 1000b:
##STR00056##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein X, EG, RT, HP, SG, W.sup.1, W.sup.2,
W.sup.3, W.sup.4, W.sup.5, R, L, and Ar are as described in the
context of Formula 1000 and/or any of the embodiments described
herein.
[0206] In an embodiment, provided herein is a compound according to
Formula 1a or Formula Ib:
##STR00057##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein EG, RT, HP, SG, W.sup.1, W.sup.2,
W.sup.3, W.sup.4, W.sup.5, R, L, and Ar are as described in the
context of Formula I and/or any of the embodiments described
herein.
[0207] In an embodiment, provided herein is a compound according to
Formula 1001a or Formula 1001b:
##STR00058##
[0208] or a pharmaceutically acceptable salt, solvate,
stereoisomer, or tautomer thereof; wherein EG, RT.sup.1, HP.sup.1,
SG, W.sup.1, W.sup.2, W.sup.3, W.sup.4, W.sup.5, R, L, and Ar are
as described in the context of Formula 1001 and/or any of the
embodiments described herein.
[0209] In an embodiment, provided herein is a compound according to
Formula 1002a or Formula 1002b:
##STR00059##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein EG, RT.sup.1, HP.sup.1, SG, W.sup.1,
W.sup.2, W.sup.3, W.sup.4, W.sup.5, R, L, and Ar are as described
in the context of Formula 1002 and/or any of the embodiments
described herein.
[0210] In an embodiment, provided herein is a compound according to
any of Formulas II-IX-1:
##STR00060## ##STR00061## ##STR00062## ##STR00063##
##STR00064##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein EG, RT, HP, RT.sup.1, HP.sup.1, SG,
W.sup.1, W.sup.2, W.sup.3, W.sup.4, W.sup.5, and R are as described
in the context of Formula 1000, I, 1001, and/or any of the
embodiments described herein.
[0211] In an embodiment, provided herein is a compound according to
any of Formulas IIa-IXa-1:
##STR00065## ##STR00066## ##STR00067## ##STR00068##
##STR00069##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein EG, RT, HP, RT.sup.1, HP.sup.1, SG,
W.sup.1, W.sup.2, W.sup.3, W.sup.4, W.sup.5, and R are as described
in the context of Formula 1000, I, 1001, and/or any of the
embodiments described herein.
[0212] In an embodiment, provided herein is a compound according to
any of Formulas IIb-IXb-1:
##STR00070## ##STR00071## ##STR00072## ##STR00073##
##STR00074##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein EG, RT, HP, RT.sup.1, HP.sup.1, SG,
W.sup.1, W.sup.2, W.sup.3, W.sup.4, W.sup.5, and R are as described
in the context of Formula 1000, I, 1001, and/or any of the
embodiments described herein.
[0213] In an embodiment, provided herein is a compound according to
Formula X:
##STR00075##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein L and Ar are as described in the context
of Formula I.
[0214] In an embodiment, provided herein is a compound according to
Formula Xa or Xb:
##STR00076##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein L and Ar are as described in the context
of Formula I.
[0215] In an embodiment, provided herein is a compound according to
any of Formulas XI-XVI-1:
##STR00077## ##STR00078## ##STR00079## ##STR00080##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein EG, RT, HP, RT.sup.1, HP.sup.1, SG,
W.sup.1, W.sup.2, W.sup.3, W.sup.4, W.sup.5, R, L, and Ar are as
described in the context of Formula 1000, I, 1001, and/or any of
the embodiments described herein.
[0216] In an embodiment, provided herein is a compound according to
any of Formulas XIa-XVIa-1:
##STR00081## ##STR00082## ##STR00083## ##STR00084##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein EG, RT, HP, RT.sup.1, HP.sup.1, SG,
W.sup.1, W.sup.2, W.sup.3, W.sup.4, W.sup.5, R, L, and Ar are as
described in the context of Formula 1000, I, 1001, and/or any of
the embodiments described herein.
[0217] In an embodiment, provided herein is a compound according to
any of Formulas XIb-XIVb-1:
##STR00085## ##STR00086## ##STR00087##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein EG, RT, HP, RT.sup.1, HP.sup.1, SG,
W.sup.1, W.sup.2, W.sup.3, W.sup.4, W.sup.5, R, L, and Ar are as
described in the context of Formula 1000, I, 1001, and/or any of
the embodiments described herein.
[0218] In one embodiment, the compound of Formula 1000 or 1001 is
that where X is
##STR00088##
HP.sup.1 is
##STR00089##
[0219] and RT.sup.1 is a release trigger group, or a cleavable
linker; or HP.sup.1 is absent and RT.sup.1 is a cleavable linker;
and all other groups are as defined for Formula 1000, 1001, and/or
any embodiments described herein. In one embodiment, the compound
of Formula 1000 or 1001 is that where X is
##STR00090##
HP.sup.1 is
##STR00091##
[0220] and RT.sup.1 is a release trigger group, or a cleavable
linker; or HP.sup.1 is absent and RT.sup.1 is a cleavable linker;
EG is
##STR00092##
[0221] and all other groups are as defined for Formula 1000, 1001,
and/or any embodiments described herein. In one embodiment, the
compound of Formula 1000 or 1001 is that where X is
##STR00093##
HP.sup.1 is
##STR00094##
[0222] and RT.sup.1 is a release trigger group, or a cleavable
linker; or HP.sup.1 is absent and RT.sup.1 is a cleavable linker;
EG is
##STR00095##
W.sup.1-W.sup.5 are absent; and all other groups are as defined for
Formula 1000, 1001, and/or any embodiments described herein. In
certain embodiments, L is absent.
[0223] In one embodiment, provided herein is a compound according
to Formula 1000:
##STR00096##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein:
[0224] Ar is a substituted or unsubstituted indolylene or
substituted or unsubstituted phenylene ring;
[0225] L is absent or --CH.sub.2--;
[0226] X is
##STR00097##
W.sup.1, W.sup.2, W.sup.3, W.sup.4, and W.sup.5 are each
independently a single bond, absent, --C(O)--, --O--, --C(O)O--,
--OC(O)--, --C(O)NH--, --C(O)NH-alkyl-, --OC(O)NH--,
--S.sub.C(O)NH--, --NH--, --N(alkyl)-, --N(R)-alkylene-N(R)--
(where each R is independently H or alkyl),
--N(CH.sub.3)CH.sub.2CH.sub.2N(CH.sub.3)--, --CH.sub.2--,
--CH.sub.2CH.sub.2--, --CH(CH.sub.3)--, --C(CH.sub.3).sub.2--,
--CH.sub.2CH.sub.2CH.sub.2--, phenylene,
--NHCH.sub.2CH.sub.2C(O)--, --C(O)CH.sub.2CH.sub.2NH--, --S--,
--S--S--, --OCH.sub.2CH.sub.2O--, or the reverse thereof;
[0227] EG is absent, or EG is selected from
##STR00098##
wherein each R.sup.EG is independently selected from the group
consisting of hydrogen, alkyl, biphenyl, --CF.sub.3, --NO.sub.2,
--CN, fluoro, bromo, chloro, alkoxyl, alkylamino, dialkylamino,
alkyl-C(O)O--, alkylamino-C(O)-- and dialkylamino-C(O)--;
[0228] RT when in the backbone is
##STR00099##
and RT when bonded to an EG group is
##STR00100##
wherein each RT is optional;
[0229] RT.sup.1 is absent,
##STR00101##
valine-alanine, valine-glutamic acid, alanine-phenylalanine;
phenylalanine-lysine; phenylalanine-homolysine; and
glycine-glycine-glycine (gly-gly-gly),
##STR00102##
where aa is a natural or non-natural amino acid residue, or
##STR00103##
where the
##STR00104##
ring is a 4-7 membered heterocyclic ring comprising 3-6 carbon
atoms;
[0230] HP is absent or
##STR00105##
wherein m is an integer from 1 to 12;
[0231] HP.sup.1 is absent or
##STR00106##
where R.sup.HP is
##STR00107##
wherein R is --H or --CH.sub.3 and m is an integer from 1 to 12 or
R.sup.HP is -alkylene-S(O).sub.3.sup.-.
[0232] SG is absent,
##STR00108##
and
[0233] R is a terminal conjugating group;
or, in the alternative, W.sup.1, W.sup.2, W.sup.3, W.sup.4,
W.sup.5, EG, RT, HP, SG, and R combine to form --H.
[0234] In some embodiments, the compound is that where X is
--HP.sup.1--RT.sup.1-EG-, --HP.sup.1--RT.sup.1-where RT.sup.1 is a
release trigger group, --HP.sup.1--RT.sup.1-where RT.sup.1 is a
cleavable linker, --HP.sup.1--RT.sup.1-where RT.sup.1 is a release
trigger group, -RT-, -RT-EG-, or -EG(RT)-; and all other groups are
as defined in any of the Formula and/or embodiments described
herein. In some embodiments, the compound is that where X is
--HP.sup.1--RT.sup.1-EG-, --HP.sup.1--RT.sup.1-where RT.sup.1 is a
release trigger group, --HP.sup.1--RT.sup.1-where RT.sup.1 is a
cleavable linker, --HP.sup.1--RT.sup.1-where RT.sup.1 is a release
trigger group, -RT-, -RT-EG-, or -EG(RT)-; the release trigger
group facilitates separation of a biologically active portion of a
compound or conjugate in conjunction with an eliminator group; and
all other groups are as defined in any of the Formula and/or
embodiments described herein. In some embodiments, the compound is
that where X is --HP.sup.1--RT.sup.1-where RT.sup.1 is a release
trigger group, --HP.sup.1--RT.sup.1-where RT.sup.1 is a cleavable
linker, --HP.sup.1--RT.sup.1-where RT.sup.1 is a release trigger
group, -RT-, -RT-EG-, RT.sup.1-EG-, or -EG(RT)-; W.sup.4, W.sup.5,
and L are independently a single bond or absent; the release
trigger group facilitates separation of a biologically active
portion of a compound or conjugate in conjunction with an
eliminator group; and all other groups are as defined in any of the
Formula and/or embodiments described herein. In some embodiments,
the compound is that where X is --HP.sup.1--RT.sup.1-EG-,
--HP.sup.1--RT.sup.1-where RT.sup.1 is a release trigger group,
--HP.sup.1--RT.sup.1-where RT.sup.1 is a cleavable linker,
--HP.sup.1--RT.sup.1-where RT.sup.1 is a release trigger group,
-RT-, -RT-EG-, or -EG(RT)-; W.sup.1, W.sup.4, W.sup.5, and L are
independently a single bond or absent; SG is
##STR00109##
the release trigger group facilitates separation of a biologically
active portion of a compound or conjugate in conjunction with an
eliminator group; and all other groups are as defined in any of the
Formula and/or embodiments described herein.
[0235] In an embodiment, provided herein is a compound according to
any of Formulas 101-108 or 1-8:
##STR00110## ##STR00111## ##STR00112## ##STR00113##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof.
[0236] In an embodiment, provided herein is a compound according to
any of Formulas 101a-108a or 1a-8a:
##STR00114## ##STR00115## ##STR00116## ##STR00117##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof.
[0237] In an embodiment, provided herein is a compound according to
any of Formulas 101b-108b or 1-8b:
##STR00118## ##STR00119## ##STR00120## ##STR00121##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof.
Conjugates
[0238] The compounds described herein (e.g., a compound according
to any of Formulas 1000-1000b, 1001-1001b, 1002-1002b, and
I-XIXb-2, 101-111b, or 1-8b, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof) can be reacted with a
second compound (e.g., a polypeptide or antibody) to form a
conjugate. The second compound can be any compound known to be
useful for conjugation to the compounds described herein (e.g., a
compound according to any of Formulas 1000-1000b, 1001-1001b,
1002-1002b, and I-XIXb-2, 101-111b, or 1-8b, or a pharmaceutically
acceptable salt, solvate, stereoisomer, or tautomer thereof).
Useful second compounds include polypeptides and antibodies.
[0239] Therefore, in an aspect, provided herein is a conjugate
comprising a compound described herein (e.g., a compound according
to any of Formulas 1000-1000b, 1001-1001b, 1002-1002b, and
I-XIXb-2, 101-111b, or 1-8b, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof) linked to a second
compound.
[0240] In an embodiment, the conjugate is according to the
following Formula E1:
##STR00122##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein:
[0241] Ar is a divalent five- or six-membered, substituted or
unsubstituted, monocyclic aryl or heteroaryl ring or a divalent
eight-, nine- or ten-membered, substituted or unsubstituted, fused
bicyclic aryl or heteroaryl ring;
[0242] L is absent or --CH.sub.2--;
[0243] X is
##STR00123##
[0244] W.sup.1, W.sup.2, W.sup.3, W.sup.4, and W.sup.5 are each
independently a single bond, absent, or a divalent attaching
group;
[0245] EG is absent or an eliminator group;
[0246] each RT is a release trigger group, in the backbone of
Formula 1000 or bonded to EG, wherein each RT is optional;
[0247] RT.sup.1 is a release trigger group, or a cleavable linker,
or RT.sup.1 is absent;
[0248] HP is a single bond, absent, or a divalent hydrophilic
group;
[0249] HP.sup.1 is a single bond, absent, a divalent hydrophilic
group, or
##STR00124##
where R.sup.HP is a monovalent hydrophilic group;
[0250] SG is a single bond, absent, or a divalent spacer group;
and
[0251] R is a divalent residue of a terminal conjugating group;
or, in the alternative, W.sup.1, W.sup.2, W.sup.3, W.sup.4,
W.sup.5, EG, RT, HP, SG, and R combine to form --H.
[0252] In an embodiment, the conjugate is according to the
following Formula C1:
##STR00125##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein:
[0253] COMP is a residue of a second compound;
[0254] Ar is a divalent five- or six-membered, substituted or
unsubstituted, monocyclic aryl or heteroaryl ring or a divalent
eight-, nine- or ten-membered, substituted or unsubstituted, fused
bicyclic aryl or heteroaryl ring;
[0255] L is absent or --CH.sub.2--;
[0256] W.sup.1, W.sup.2, W.sup.3, W.sup.4, and W.sup.5 are each
independently a single bond, absent, or a divalent attaching
group;
[0257] EG is an eliminator group;
[0258] each RT is a release trigger group, and one RT is
optional;
[0259] HP is a single bond, absent, or a divalent hydrophilic
group;
[0260] SG is a single bond, absent, or a divalent spacer group;
and
[0261] R is a divalent residue of a terminal conjugating group.
[0262] In one embodiment, provided herein is a conjugate according
to Formula (F1) or (G1):
##STR00126##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein:
[0263] COMP is a residue of a second compound;
[0264] Ar is a divalent five- or six-membered, substituted or
unsubstituted, monocyclic aryl or heteroaryl ring or a divalent
eight-, nine- or ten-membered, substituted or unsubstituted, fused
bicyclic aryl or heteroaryl ring;
[0265] L is absent or --CH.sub.2--;
[0266] W.sup.1, W.sup.2, W.sup.3, W.sup.4, and W.sup.5 are each
independently a single bond, absent, or a divalent attaching
group;
[0267] EG is absent or an eliminator group;
[0268] RT.sup.1 is a release trigger group or a cleavable linker;
RT is a release trigger group bonded to EG; and wherein RT and
RT.sup.1 are optional;
[0269] HP.sup.1 is single bond, absent, a divalent hydrophilic
group, or
##STR00127##
where R.sup.SG is a monovalent hydrophilic group;
[0270] SG is a single bond, absent, or a divalent spacer group;
and
[0271] R is a divalent residue of a terminal conjugating group.
[0272] In an embodiment, provided herein is a conjugate according
to any of Formulas C1-C17b, E1, F1-F17b, and G1-G17b or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein Ar is a divalent five- or six-membered,
substituted or unsubstituted, monocyclic aryl or heteroaryl ring.
In an embodiment, provided herein is a conjugate according to any
of Formulas C1-C17b, E1, F1-F17b, and G1-G17b, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein Ar is a divalent six-membered,
substituted or unsubstituted, monocyclic aryl or heteroaryl ring.
In an embodiment, provided herein is a conjugate according to any
of Formulas C1-C17b, E1, F1-F17b, and G1-G17b, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein Ar is a divalent eight-, nine- or
ten-membered, substituted or unsubstituted, fused bicyclic aryl or
heteroaryl ring. In an embodiment, provided herein is a conjugate
according to any of Formulas C1-C17b, E1, F1-F17b, and G1-G17b, or
a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein Ar is a divalent eight-, nine-membered,
substituted or unsubstituted, fused bicyclic heteroaryl ring. In an
embodiment, provided herein is a conjugate according to any of
Formulas C1-C17b, E1, F1-F17b, and G1-G17b, or a pharmaceutically
acceptable salt, solvate, stereoisomer, or tautomer thereof;
wherein Ar is phenylene or indolylene, each of which is
unsubstituted or substituted. In an embodiment, provided herein is
a conjugate according to any of Formulas C1-C17b, E1, F1-F17b, and
G1-G17b, or a pharmaceutically acceptable salt, solvate,
stereoisomer, or tautomer thereof; wherein Ar is any of the
following:
##STR00128##
[0273] In an embodiment, provided herein is a conjugate according
to any of Formulas C1-C17b, E1, F1-F17b, and G1-G17b, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein L is absent. In an embodiment, provided
herein is a conjugate according to any of Formulas C1-C17b, E1,
F1-F17b, and G1-G17b, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof; wherein L is
--CH.sub.2--.
[0274] In an embodiment, provided herein is a conjugate according
to any of Formulas C1-C17b, E1, F1-F17b, and G1-G17b or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein EG comprises phenylene, carboxylene,
amine, or a combination thereof. In an embodiment, provided herein
is a conjugate according to any of Formulas C1-C17b, E1, F1-F17b,
and G1-G17b, or a pharmaceutically acceptable salt, solvate,
stereoisomer, or tautomer thereof; wherein EG is:
##STR00129##
wherein each R.sup.EG is independently selected from the group
consisting of hydrogen, alkyl, biphenyl, --CF.sub.3, --NO.sub.2,
--CN, fluoro, bromo, chloro, alkoxyl, alkylamino, dialkylamino,
alkyl-C(O)O--, alkylamino-C(O)-- and dialkylaminoC(O)--. In the
second and third structures, those of skill will recognize that EG
is bonded to an RT that is not within the backbone of formula (I)
as indicated in the above description of formula (I). In some
embodiments, each R.sup.EG is independently selected from the group
consisting of hydrogen, alkyl, biphenyl, --CF.sub.3, alkoxyl,
alkylamino, dialkylamino, alkyl-C(O)O--, alkylamino-C(O)-- and
dialkylaminoC(O)--. In further embodiments, each R.sup.EG is
independently selected from the group consisting of hydrogen,
--NO.sub.2, --CN, fluoro, bromo, and chloro.
[0275] In an embodiment, provided herein is a conjugate according
to any of Formulas C1-C17b, E1, F1-F17b, and G1-G17b, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein RT comprises a residue of a natural or
non-natural amino acid or a residue of a sugar. In an embodiment,
provided herein is a conjugate according to any of Formulas
C1-C17b, E1, F1-F17b, and G1-G17b, or a pharmaceutically acceptable
salt, solvate, stereoisomer, or tautomer thereof; wherein RT
is:
##STR00130##
Those of skill will recognize that the first structure is divalent
and can be bonded within the backbone of formula 1000 or (I), and
that the second structure is monovalent and can be bonded to EG as
depicted in formula (I) and 1000 above.
[0276] In an embodiment, provided herein is a conjugate according
to any of Formulas C1-C17b, E1, F1-F17b, and G1-G17b, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein HP comprises poly(ethylene glycol). In an
embodiment, provided herein is a conjugate according to any of
Formulas C1-C17b, E1, F1-F17b, and G1-G17b, or a pharmaceutically
acceptable salt, solvate, stereoisomer, or tautomer thereof;
wherein HP is:
##STR00131##
wherein m is an integer from 1 to 12.
[0277] In an embodiment, provided herein is a conjugate according
to any of Formulas C1-C17b, E1, F1-F17b, and G1-G17b, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein SG comprises C1-C.sub.10 alkylene, C4-C6
alkylene, --C(O)--, or combination thereof. In an embodiment,
provided herein is a conjugate according to any of Formulas
C1-C17b, E1, F1-F17b, and G1-G17b, or a pharmaceutically acceptable
salt, solvate, stereoisomer, or tautomer thereof; wherein SG
is:
##STR00132##
[0278] In an embodiment, provided herein is a conjugate according
to any of Formulas C1-C17b, E1, F1-F17b, and G1-G17b, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein W.sup.1, W.sup.2, W.sup.3, W.sup.4, and
W.sup.5 are each independently a single bond, absent, or comprise a
divalent ketone, divalent ester, divalent ether, divalent amide,
divalent amine, alkylene, arylene, sulfide, disulfide, --C(O)--, or
a combination thereof. In an embodiment, provided herein is a
conjugate according to any of Formulas C1-C17b, E1, F1-F17b, and
G1-G17b, or a pharmaceutically acceptable salt, solvate,
stereoisomer, or tautomer thereof; wherein W.sup.1, W.sup.2,
W.sup.3, W.sup.4, and W.sup.5 are each independently a single bond,
absent, or comprise --C(O)--, --O--, --C(O)NH--, --C(O)NH-alkyl-,
--OC(O)NH--, --S.sub.C(O)NH--, --NH--, --NH-alkyl-,
--N(CH.sub.3)CH.sub.2CH.sub.2N(CH.sub.3)--, --S--, --S--S--,
--OCH.sub.2CH.sub.2O--, or a combination thereof.
[0279] In an embodiment, provided herein is a conjugate according
to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein R comprises a triazole ring. In an
embodiment, provided herein is a conjugate according to any of
Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceutically
acceptable salt, solvate, stereoisomer, or tautomer thereof;
wherein R is a triazole ring or fused cyclic group comprising a
triazole ring. In an embodiment, provided herein is a conjugate
according to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or
a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein R is:
##STR00133##
[0280] In an embodiment, provided herein is a conjugate according
to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein R comprises a fused bicyclic ring having
at least two adjacent nitrogen atoms in the ring. In an embodiment,
provided herein is a conjugate according to any of Formulas
C1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceutically acceptable
salt, solvate, stereoisomer, or tautomer thereof; wherein R is:
##STR00134##
[0281] In an embodiment, provided herein is a conjugate according
to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein R comprises a sulfur linkage. In an
embodiment, provided herein is a conjugate according to any of
Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceutically
acceptable salt, solvate, stereoisomer, or tautomer thereof;
wherein R is:
##STR00135##
[0282] In an embodiment, provided herein is a conjugate according
to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein R comprises a divalent residue of a
non-natural amino acid. In an embodiment, provided herein is a
conjugate according to any of Formulas C1-C13b, E1, F1-F13b, and
G1-G13b, or a pharmaceutically acceptable salt, solvate,
stereoisomer, or tautomer thereof; wherein R is:
##STR00136##
In an embodiment, provided herein is a conjugate according to any
of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein comprises an oxime linkage. In an
embodiment, provided herein is a conjugate according to any of
Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceutically
acceptable salt, solvate, stereoisomer, or tautomer thereof;
wherein R is:
##STR00137##
[0283] In an embodiment, provided herein is a conjugate according
to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein R is:
##STR00138##
[0284] In an embodiment, provided herein is a compound according to
any of Formulas C1-C17b, E1, F1-F17b, and G1-G17b, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein COMP is a residue of any compound known
to be useful for conjugation to the modified Hemiasterlin compounds
described herein (e.g., a compound according to any of Formulas
1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2, 101-111b, or
1-8b, or a pharmaceutically acceptable salt, solvate, stereoisomer,
or tautomer thereof). In an embodiment, provided herein is a
compound according to any of Formulas C1-C17b, E1, F1-F17b, and
G1-G17b, or a pharmaceutically acceptable salt, solvate,
stereoisomer, or tautomer thereof, wherein COMP is a residue of a
polypeptide, antibody, or antibody chain. In an embodiment,
provided herein is a compound according to any of Formulas C1-C17b,
E1, F1-F17b, and G1-G17b, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof; wherein COMP is a
residue of a polypeptide. In an embodiment, provided herein is a
compound according to any of Formulas C1-C17b, E1, F1-F17b, and
G1-G17b, or a pharmaceutically acceptable salt, solvate,
stereoisomer, or tautomer thereof; wherein COMP is a residue of an
antibody. In an embodiment, provided herein is a compound according
to any of Formulas C1-C17b, E1, F1-F17b, and G1-G17b, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof; wherein COMP is a residue of an antibody
chain.
[0285] In an aspect, provided herein is a polypeptide conjugate
comprising a compound described herein (e.g., a compound according
to any of Formulas 1000-1000b, 1001-1001b, 1002-1002b, and
I-XIXb-2, 101-111b, or 1-8b, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof) linked to a
polypeptide, wherein the polypeptide conjugate is according to any
of Formulas C1-C17b, E1, F1-F17b, and G1-G17b, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein COMP is a residue of the polypeptide. In
an embodiment, provided herein is a polypeptide conjugate according
to any of Formulas C1-C15b, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof, wherein: COMP is a
residue of the polypeptide; and R comprises a triazole ring or
fused cyclic group comprising a triazole ring. In an embodiment,
provided herein is a polypeptide conjugate according to any of
Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceutically
acceptable salt, solvate, stereoisomer, or tautomer thereof,
wherein: COMP is a residue of the polypeptide; and R is:
##STR00139##
[0286] In an embodiment, provided herein is a polypeptide conjugate
according to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or
a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein: COMP is a residue of the polypeptide;
and R comprises a fused bicyclic ring, wherein the fused bicyclic
ring has at least two adjacent nitrogen atoms in the ring. In an
embodiment, provided herein is a polypeptide conjugate according to
any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein: COMP is a residue of the polypeptide;
and R is:
##STR00140##
[0287] In an embodiment, provided herein is a polypeptide conjugate
according to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or
a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein: COMP is a residue of the polypeptide;
and R comprises a sulfur linkage. In an embodiment, provided herein
is a polypeptide conjugate according to any of Formulas C1-C13b,
E1, F1-F13b, and G1-G13b, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof, wherein: COMP is a
residue of the polypeptide; and R is:
##STR00141##
[0288] In an embodiment, provided herein is a polypeptide conjugate
according to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or
a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein: COMP is a residue of the polypeptide;
and R comprises a divalent residue of a non-natural amino acid. In
an embodiment, provided herein is a polypeptide conjugate according
to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein: COMP is a residue of the polypeptide;
and R is:
##STR00142##
[0289] In an embodiment, provided herein is a polypeptide conjugate
according to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or
a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein: COMP is a residue of the polypeptide;
and R comprises an oxime linkage. In an embodiment, provided herein
is a polypeptide conjugate according to any of Formulas C1-C13b,
E1, F1-F13b, and G1-G13b, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof, wherein: COMP is a
residue of the polypeptide; and R is:
##STR00143##
[0290] In an aspect, provided herein is an antibody conjugate
comprising a compound described herein (e.g., a compound according
to any of Formulas 1000-1000b, 1001-1001b, 1002-1002b, and
I-XIXb-2, 101-111b, or 1-8b, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof) linked to an antibody
according to any of Formulas C1-C17b, E1, F1-F17b, and G1-G17b, or
a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein COMP is a residue of the antibody. In an
embodiment, provided herein is an antibody conjugate according to
any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein: COMP is a residue of the antibody; and R
comprises a triazole ring or fused cyclic group comprising a
triazole ring. In an embodiment, provided herein is an antibody
conjugate according to any of Formulas C1-C13b, E1, F1-F13b, and
G1-G13b, or a pharmaceutically acceptable salt, solvate,
stereoisomer, or tautomer thereof, wherein: COMP is a residue of
the antibody; and R is:
##STR00144##
[0291] In an embodiment, provided herein is an antibody conjugate
according to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or
a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein: COMP is a residue of the antibody; and R
comprises a fused bicyclic ring, wherein the fused bicyclic ring
has at least two adjacent nitrogen atoms in the ring. In an
embodiment, provided herein is an antibody conjugate according to
any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein: COMP is a residue of the antibody; and R
is:
##STR00145##
[0292] In an embodiment, provided herein is an antibody conjugate
according to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or
a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein: COMP is a residue of the antibody; and R
comprises a sulfur linkage. In an embodiment, provided herein is an
antibody conjugate according to any of Formulas C1-C13b, E1,
F1-F13b, and G1-G13b, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof, wherein: COMP is a
residue of the antibody; and R is:
##STR00146##
[0293] In an embodiment, provided herein is an antibody conjugate
according to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or
a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein: COMP is a residue of the antibody; and R
comprises a divalent residue of a non-natural amino acid. In an
embodiment, provided herein is an antibody conjugate according to
any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or a
pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein: COMP is a residue of the antibody; and R
is:
##STR00147##
[0294] In an embodiment, provided herein is an antibody conjugate
according to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or
a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein: COMP is a residue of the antibody; and R
comprises an oxime linkage. In an embodiment, provided herein is an
antibody conjugate according to any of Formulas C1-C13b, E1,
F1-F13b, and G1-G13b, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof, wherein: COMP is a
residue of the antibody; and R is:
##STR00148##
[0295] In an aspect, provided herein is an antibody chain conjugate
comprising a compound described herein (e.g., a compound according
to any of Formulas 1000-1000b, 1001-1001b, 1002-1002b, and
I-XIXb-2, 101-111b, or 1-8b, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof) linked to an antibody
chain according to any of Formulas C1-C17b, E1, F1-F17b, and
G1-G17b, or a pharmaceutically acceptable salt, solvate,
stereoisomer, or tautomer thereof, wherein COMP is a residue of the
antibody chain. In an embodiment, provided herein is an antibody
chain conjugate according to any of Formulas C1-C13b, E1, F1-F13b,
and G1-G13b, or a pharmaceutically acceptable salt, solvate,
stereoisomer, or tautomer thereof, wherein: COMP is a residue of
the antibody chain; and R comprises a triazole ring or fused cyclic
group comprising a triazole ring. In an embodiment, provided herein
is an antibody chain conjugate according to any of Formulas
C1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceutically acceptable
salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is
a residue of the antibody chain; and R is:
##STR00149##
[0296] In an embodiment, provided herein is an antibody chain
conjugate according to any of Formulas C1-C13b, E1, F1-F13b, and
G1-G13b, or a pharmaceutically acceptable salt, solvate,
stereoisomer, or tautomer thereof, wherein: COMP is a residue of
the antibody chain; and R comprises a fused bicyclic ring, wherein
the fused bicyclic ring has at least two adjacent nitrogen atoms in
the ring. In an embodiment, provided herein is an antibody chain
conjugate according to any of Formulas C1-C13b, E1, F1-F13b, and
G1-G13b, or a pharmaceutically acceptable salt, solvate,
stereoisomer, or tautomer thereof, wherein: COMP is a residue of
the antibody chain; and R is:
##STR00150##
[0297] In an embodiment, provided herein is an antibody chain
conjugate according to any of Formulas C1-C13b, E1, F1-F13b, and
G1-G13b, or a pharmaceutically acceptable salt, solvate,
stereoisomer, or tautomer thereof, wherein: COMP is a residue of
the antibody chain; and R comprises a sulfur linkage. In an
embodiment, provided herein is an antibody chain conjugate
according to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or
a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein: COMP is a residue of the antibody chain;
and R is:
##STR00151##
[0298] In an embodiment, provided herein is an antibody chain
conjugate according to any of Formulas C1-C13b, E1, F1-F13b, and
G1-G13b, or a pharmaceutically acceptable salt, solvate,
stereoisomer, or tautomer thereof, wherein: COMP is a residue of
the antibody chain; and R comprises a divalent residue of a
non-natural amino acid. In an embodiment, provided herein is an
antibody chain conjugate according to any of Formulas C1-C13b, E1,
F1-F13b, and G1-G13b, or a pharmaceutically acceptable salt,
solvate, stereoisomer, or tautomer thereof, wherein: COMP is a
residue of the antibody chain; and R is:
##STR00152##
[0299] In an embodiment, provided herein is an antibody chain
conjugate according to any of Formulas C1-C13b, E1, F1-F13b, and
G1-G13b, or a pharmaceutically acceptable salt, solvate,
stereoisomer, or tautomer thereof, wherein: COMP is a residue of
the antibody chain; and R comprises an oxime linkage. In an
embodiment, provided herein is an antibody chain conjugate
according to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or
a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein: COMP is a residue of the antibody chain;
and R is:
##STR00153##
[0300] In an embodiment, provided herein is a conjugate according
to Formula C1a or Formula C1b:
##STR00154##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein COMP, R, SG, HP, RT, EG, W.sup.1,
W.sup.2, W.sup.3, W.sup.4, W.sup.5, L, and Ar are a described in
the context of Formulas C1 and I-XVIb.
[0301] In an embodiment, provided herein is a conjugate according
to the following Formula:
##STR00155##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein all other groups are as defined in any of
the Formulas and/or embodiments described herein.
[0302] In an embodiment, provided herein is a compound according to
any of Formulas C2-C9:
##STR00156## ##STR00157##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein COMP, R, SG, HP, RT, EG, W.sup.1,
W.sup.2, W.sup.3, W.sup.4, W.sup.5, L, and Ar are a described in
the context of Formulas C1 and I-XVIb.
[0303] In an embodiment, provided herein is a compound according to
any of the following Formula:
##STR00158## ##STR00159## ##STR00160##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein all other groups are a described in the
context of any of the Formulas or embodiments described herein.
[0304] In an embodiment, provided herein is a compound according to
any of Formulas C2a-C9a:
##STR00161## ##STR00162##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein COMP, R, SG, HP, RT, EG, W.sup.1,
W.sup.2, W.sup.3, W.sup.4, W.sup.5, L, and Ar are a described in
the context of Formulas C1 and I-XVIb.
[0305] In an embodiment, provided herein is a compound according to
any of the following Formula:
##STR00163## ##STR00164## ##STR00165##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein all other groups are a described in the
context of any of the Formulas or embodiments described herein.
[0306] In an embodiment, provided herein is a compound according to
any of Formulas C2b-C9b:
##STR00166## ##STR00167##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein COMP, R, SG, HP, RT, EG, W.sup.1,
W.sup.2, W.sup.3, W.sup.4, W.sup.5, L, and Ar are a described in
the context of Formulas C1 and I-XVIb.
[0307] In an embodiment, provided herein is a compound according to
any of the following Formula:
##STR00168## ##STR00169## ##STR00170## ##STR00171##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein all other groups are a described in the
context of any of the Formulas or embodiments described herein.
[0308] In an embodiment, provided herein is a compound according to
any of Formulas C10-C13:
##STR00172##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein COMP, R, SG, HP, RT, EG, W.sup.1,
W.sup.2, W.sup.3, W.sup.4, W.sup.5, L, and Ar are a described in
the context of Formulas C1 and I-XVIb.
[0309] In an embodiment, provided herein is a compound according to
any of the following Formula:
##STR00173##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein all other groups are as defined in any of
the Formulas or embodiments herein.
[0310] In an embodiment, provided herein is a compound according to
any of Formulas C10a-C13a:
##STR00174##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein COMP, R, SG, HP, RT, EG, W.sup.1,
W.sup.2, W.sup.3, W.sup.4, W.sup.5, L, and Ar are a described in
the context of Formulas C1 and I-XVIb.
[0311] In an embodiment, provided herein is a compound according to
any of the following Formula:
##STR00175##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein all other groups are as defined in any of
the Formulas or embodiments herein.
[0312] In an embodiment, provided herein is a compound according to
any of Formulas C10b-C13b:
##STR00176##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein COMP, R, SG, HP, RT, EG, W.sup.1,
W.sup.2, W.sup.3, W.sup.4, W.sup.5, L, and Ar are a described in
the context of Formulas C1 and I-XVIb.
[0313] In an embodiment, provided herein is a compound according to
any of the following Formula:
##STR00177##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein all other groups are as defined in any of
the Formulas or embodiments herein.
[0314] In an embodiment, provided herein is a compound according to
any of Formula C14-C17:
##STR00178##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein COMP, SG, HP, RT, EG, W.sup.1, W.sup.2,
W.sup.3, W.sup.4, W.sup.5, L, and Ar are a described in the context
of Formulas C1 and I-XVIb.
[0315] In an embodiment, provided herein is a compound according to
any of the following Formula:
##STR00179## ##STR00180##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein all other groups are as defined in any of
the Formulas or embodiments herein.
[0316] In an embodiment, provided herein is a compound according to
any of Formula C14a-C17a:
##STR00181##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein COMP, SG, HP, RT, EG, W.sup.1, W.sup.2,
W.sup.3, W.sup.4, W.sup.5, L, and Ar are a described in the context
of Formulas C1 and I-XVIb.
[0317] In an embodiment, provided herein is a compound according to
any of the following Formula:
##STR00182## ##STR00183##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein all other groups are as defined in any of
the Formulas or embodiments herein.
[0318] In an embodiment, provided herein is a compound according to
any of Formula C14b-C17b:
##STR00184##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein COMP, SG, HP, RT, EG, W.sup.1, W.sup.2,
W.sup.3, W.sup.4, W.sup.5, L, and Ar are a described in the context
of Formulas C1 and I-XVIb.
[0319] In an embodiment, provided herein is a compound according to
any of the following Formula:
##STR00185## ##STR00186##
or a pharmaceutically acceptable salt, solvate, stereoisomer, or
tautomer thereof, wherein all other groups are as defined in any of
the Formulas or embodiments herein.
[0320] In an aspect, provided herein is a method of producing a
conjugate (e.g., according to any of Formulas C1-C17b, E1, F1-F17b,
and G1-G17b), comprising contacting a compound described herein
(e.g., a compound according to any of Formulas I-XVIIIb, 101-111b,
or 1-8b) with a second compound under conditions suitable for
conjugating the compound described herein with the second compound;
wherein the second compound comprises a modified amino acid
comprising an alkyne, strained alkene, tetrazine, thiol, maleimide,
carbonyl, oxyamine, or azide. In an embodiment, the second compound
is a polypeptide. In an embodiment, the second compound is an
antibody.
Conjugation Reactions
[3+2] Alkyne-Azide Cycloaddition Reaction
##STR00187##
[0322] Advantageously, the compounds described herein comprising a
terminal conjugating alkyne group or an azide group (e.g., a
compound according to any of Formulas I-IXb, XI-XVIIb, and
101-111b) facilitate selective and efficient reactions with a
second compound comprising a complementary azide group or alkyne
group. It is believed the azide and alkyne groups react in a
1,3-dipolar cycloaddition reaction to form a 1,2,3-triazolylene
moiety which links the compound described herein comprising an
alkyne group or an azide group to the second compound. This
reaction between an azide and alkyne to form a triazole is
generally known to those in the art as a Huisgen cycloaddition
reaction or a [3+2] alkyne-azide cycloaddition reaction.
[0323] The unique reactivity of azide and alkyne functional groups
makes them useful for the selective modification of polypeptides
and other biological molecules. Organic azides, particularly
aliphatic azides, and alkynes are generally stable toward common
reactive chemical conditions. In particular, both the azide and the
alkyne functional groups are inert toward the side chains of the 20
common amino acids found in naturally-occurring polypeptides. It is
believed that, when brought into close proximity, the
"spring-loaded" nature of the azide and alkyne groups is revealed
and they react selectively and efficiently via a [3+2] alkyne-azide
cycloaddition reaction to generate the corresponding triazole. See,
e.g., Chin J., et al., Science 301:964-7 (2003); Wang, Q., et al.,
J. Am. Chem. Soc. 125, 3192-3193 (2003); Chin, J. W., et al., J.
Am. Chem. Soc. 124:9026-9027 (2002).
[0324] Because the [3+2] alkyne-azide cycloaddition reaction
involves a selective cycloaddition reaction [see, e.g., Padwa, A.,
in COMPREHENSIVE ORGANIC SYNTHESIS, Vol. 4, (ed. Trost, B. M.,
1991), pp. 1069-1109; Huisgen, R. in 1,3-DIPOLAR CYCLOADDITION
CHEMISTRY, (ed. Padwa, A., 1984), pp. 1-176] rather than a
nucleophilic substitution, the incorporation of non-naturally
encoded amino acids bearing azide and alkyne-containing side chains
permits the resultant polypeptides to be modified selectively at
the position of the non-naturally encoded amino acid. Cycloaddition
reactions involving azide or alkyne-containing compounds can be
carried out at room temperature under aqueous conditions by the
addition of Cu(II) (including but not limited to, in the form of a
catalytic amount of CuSO.sub.4) in the presence of a reducing agent
for reducing Cu(II) to Cu(I), in situ, in catalytic amount. See,
e.g., Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003);
Tornoe, C. W., et al., J. Org. Chem. 67:3057-3064 (2002);
Rostovtsev, et al., Angew. Chem. Int. Ed. 41:2596-2599 (2002).
Exemplary reducing agents include, but not limited to, ascorbate,
metallic copper, quinine, hydroquinone, vitamin K, glutathione,
cysteine, Fe.sup.2+, Co.sup.2+, and an applied electric
potential.
Inverse Electron Demand Ligation Reaction
##STR00188##
[0326] Advantageously, the compounds comprising a terminal
tetrazine or strained alkene group provided herein facilitate
selective and efficient reactions with a second compound comprising
a strained alkene or tetrazine group. It is believed that the
tetrazine and strained alkene react in an inverse-demand
Diels-Alder reaction followed by a retro-Diels-Alder reaction which
links the compounds comprising a terminal tetrazine or strained
alkene group provided herein to the second compound. The reaction
is believed to be specific, with little to no cross-reactivity with
functional groups that occur on biomolecules. The reaction may be
carried out under mild conditions, for example at room temperature
and without a catalyst. This reaction between a tetrazine and a
strained alkene is generally known to those in the art as a
tetrazine ligation reaction.
Thiol Reactions
##STR00189##
[0328] Advantageously, the compounds comprising a terminal thiol
group or suitable electrophilic or disulfide-forming group provided
herein facilitate selective and efficient reactions with a second
compound comprising a complementary electrophilic or
disulfide-forming group or thiol group. These reactions are
believed to be selective with little to no cross-reactivity with
functional groups that occur on biomolecules. In another
embodiment, the thiol reaction does not include reaction of a
maleimide group.
Carbonyl-Oxyamine Reaction
##STR00190##
[0330] Advantageously, the compounds comprising a terminal carbonyl
or oxyamine group provided herein facilitate selective and
efficient reactions with a second compound comprising an oxyamine
or carbonyl group. It is believed that the carbonyl and oxyamine
react to form an oxime linkage. The reaction is believed to be
specific, with little to no cross-reactivity with functional groups
that occur on biomolecules.
Other Reactions
[0331] Other suitable conjugation reactions are described in the
literature. See, for example, Lang, K. and Chin, J. 2014,
Bioorthogonal Reactions for Labeling Proteins, ACS Chem Biol 9,
16-20; Paterson, D. M. et al. 2014, Finding the Right
(Bioorthogonal) Chemistry, ACS Chem Biol 9, 592-605; King, M. and
Wagner, A. 2014, Developments in the Field of Bioorthogonal Bond
Forming Reactions --Past and Present Trends, Bioconjugate Chem.,
2014, 25 (5), pp 825-839; and Ramil, C. P. and Lin, Q., 2013,
Bioorthogonal chemistry: strategies and recent developments, Chem
Commun 49, 11007-11022.
Releasing Reactions
[0332] Releasing Reactions are reactions that act to release a
biologically active portion of a compound or conjugate described
herein from the compound or conjugate in vivo and/or in vitro. In
certain embodiments, the released biologically active portion is a
compound according to any of Formulas 1-8b, or a pharmaceutically
acceptable salt, solvate, stereoisomer, or tautomer thereof. One
example of a releasing reaction is an intramolecular reaction
between an eliminator group and a release trigger group of a
compound or conjugate described herein to release a biologically
active portion of a compound or conjugate described herein. The
eliminator group may itself devolve into two reactive components,
as exemplified in these reactions where X-- is a drug having a
heteroatom N or O for linkage. Exemplary Releasing Reactions are
depicted in the schemes below:
##STR00191##
Compositions
[0333] The compounds and conjugates described herein can be
formulated into compositions using methods available in the art and
those disclosed herein. Any of the compounds and conjugates
described herein can be provided in an appropriate pharmaceutical
composition and be administered by a suitable route of
administration.
[0334] In an aspect, provided herein is a pharmaceutical
composition comprising:
[0335] a compound (e.g., a compound according to any of Formulas
1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2, 101-111b, or
1-8b) or conjugate (e.g., a conjugate according to any of Formulas
C1-C17b, E1, F1-F17b, and G1-G17b) as described herein; and
[0336] a pharmaceutically acceptable excipient, carrier, or
diluent.
[0337] In certain embodiments, the pharmaceutical compositions
provided herein further comprise a pharmaceutically acceptable
carrier. The carrier can be a diluent, excipient, or vehicle with
which the pharmaceutical composition is administered. Such
pharmaceutical carriers can be sterile liquids, such as water and
oils, including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. Saline solutions and aqueous dextrose and glycerol
solutions can also be employed as liquid carriers, particularly for
injectable solutions. Suitable pharmaceutical excipients include
starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,
chalk, silica gel, sodium stearate, glycerol monostearate, talc,
sodium chloride, dried skim milk, glycerol, propylene, glycol,
water, ethanol and the like. The pharmaceutical composition, if
desired, can also contain minor amounts of wetting or emulsifying
agents, or pH buffering agents. The pharmaceutical compositions can
take the form of solutions, suspensions, emulsions, tablets, pills,
capsules, powders, sustained-release formulations and the like.
Oral formulations can include standard carriers such as
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
Examples of suitable pharmaceutical carriers are described in E. W.
Martin, 1990, Remington's Pharmaceutical Sciences, Mack Publishing
Co.
[0338] In some embodiments, the pharmaceutical composition is
provided in a form suitable for administration to a human subject.
In some embodiments, the pharmaceutical composition will contain a
prophylactically or therapeutically effective amount of the
polypeptide together with a suitable amount of carrier so as to
provide the form for proper administration to the patient. The
formulation should suit the mode of administration.
[0339] In some embodiments, the pharmaceutical composition is
provided in a form suitable for intravenous administration.
Typically, compositions suitable for intravenous administration are
solutions in sterile isotonic aqueous buffer. Where necessary, the
composition may also include a solubilizing agent and a local
anesthetic such as lignocaine to ease pain at the site of the
injection. Such compositions, however, may be administered by a
route other than intravenous administration.
[0340] In particular embodiments, the pharmaceutical composition is
suitable for subcutaneous administration. In particular
embodiments, the pharmaceutical composition is suitable for
intramuscular administration.
[0341] Components of the pharmaceutical composition can be supplied
either separately or mixed together in unit dosage form, for
example, as a dry lyophilized powder or water free concentrate.
Where the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ample of sterile water for injection or saline can be
provided so that the ingredients may be mixed prior to
administration.
[0342] In some embodiments, the pharmaceutical composition is
supplied as a dry sterilized lyophilized powder that is capable of
being reconstituted to the appropriate concentration for
administration to a subject. In some embodiments, polypeptides are
supplied as a water free concentrate. In some embodiments, the
polypeptide is supplied as a dry sterile lyophilized powder at a
unit dosage of at least 0.5 mg, at least 1 mg, at least 2 mg, at
least 3 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least
25 mg, at least 30 mg, at least 35 mg, at least 45 mg, at least 50
mg, at least 60 mg, or at least 75 mg.
[0343] In another embodiment, the pharmaceutical composition is
supplied in liquid form. In some embodiments, the pharmaceutical
composition is provided in liquid form and is substantially free of
surfactants and/or inorganic salts. In some embodiments, the
polypeptide is supplied as in liquid form at a unit dosage of at
least 0.1 mg/ml, at least 0.5 mg/ml, at least 1 mg/ml, at least 2.5
mg/ml, at least 3 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at
least 10 mg/ml, at least 15 mg/ml, at least 25 mg/ml, at least 30
mg/ml, or at least 60 mg/ml.
[0344] In some embodiments, the pharmaceutical composition is
formulated as a salt form. Pharmaceutically acceptable salts
include those formed with anions such as those derived from
hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and
those formed with cations such as those derived from sodium,
potassium, ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0345] In therapeutic use, the practitioner will determine the
posology most appropriate according to a preventive or curative
treatment and according to the age, weight, stage of the disease
and other factors specific to the subject to be treated. In certain
embodiments, doses are from about 1 to about 1000 mg per day for an
adult, or from about 5 to about 250 mg per day or from about 10 to
50 mg per day for an adult. In certain embodiments, doses are from
about 5 to about 400 mg per day or 25 to 200 mg per day per adult.
In certain embodiments, dose rates of from about 50 to about 500 mg
per day are also contemplated.
Methods of Use for Therapy or Prophylaxis
[0346] Certain compounds, conjugates, polypeptides, and antibodies
provided herein can be used for the treatment or prevention of any
disease or condition deemed suitable to the practitioner of skill
in the art. Generally, a method of treatment or prevention
encompasses the administration of a therapeutically or
prophylactically effective amount of a compound, conjugate,
polypeptide, antibody, or pharmaceutical composition comprising the
same to a subject in need thereof to treat or prevent the disease
or condition.
[0347] In an aspect, provided herein is a method of inhibiting
tubulin polymerization in a subject in need thereof comprising
administering an effective amount of a compound (e.g., a compound
according to any of Formulas 1000-1000b, 1001-1001b, 1002-1002b,
and I-XIXb-2, 101-111b, or 1-8b), conjugate (e.g., a conjugate
according to any of Formulas C1-C17b, E1, F1-F17b, and G1-G17b), or
composition comprising the compound or conjugate, as described
herein, to the subject.
[0348] In an aspect, provided herein is a method of treating cell
proliferation or cancer in a subject in need thereof comprising
administering an effective amount of a compound (e.g., a compound
according to any of Formulas 1000-1000b, 1001-1001b, 1002-1002b,
and I-XIXb-2, 101-111b, or 1-8b), conjugate (e.g., a conjugate
according to any of Formulas C1-C17b, E1, F1-F17b, and G1-G17b), or
composition comprising the compound or conjugate, as described
herein, to the subject.
[0349] A therapeutically effective amount of the compound,
conjugate, polypeptide, antibody, or pharmaceutical composition
comprising the same is an amount that is effective to reduce the
severity, the duration and/or the symptoms of a particular disease
or condition. The amount of the compound, conjugate, polypeptide,
antibody, or pharmaceutical composition comprising the same that
will be therapeutically effective in the prevention, management,
treatment and/or amelioration of a particular disease can be
determined by standard clinical techniques. The precise amount of
the compound, conjugate, polypeptide, antibody, or pharmaceutical
composition comprising the same to be administered with depend, in
part, on the route of administration, the seriousness of the
particular disease or condition, and should be decided according to
the judgment of the practitioner and each subject's
circumstances.
[0350] In some embodiments, the effective amount of the compound,
conjugate, polypeptide, antibody, or pharmaceutical composition
comprising the same provided herein is between about 0.025 mg/kg
and about 1000 mg/kg body weight of a human subject. In certain
embodiments, the compound, conjugate, polypeptide, antibody, or
pharmaceutical composition comprising the same is administered to a
human subject at an amount of about 1000 mg/kg body weight or less,
about 950 mg/kg body weight or less, about 900 mg/kg body weight or
less, about 850 mg/kg body weight or less, about 800 mg/kg body
weight or less, about 750 mg/kg body weight or less, about 700
mg/kg body weight or less, about 650 mg/kg body weight or less,
about 600 mg/kg body weight or less, about 550 mg/kg body weight or
less, about 500 mg/kg body weight or less, about 450 mg/kg body
weight or less, about 400 mg/kg body weight or less, about 350
mg/kg body weight or less, about 300 mg/kg body weight or less,
about 250 mg/kg body weight or less, about 200 mg/kg body weight or
less, about 150 mg/kg body weight or less, about 100 mg/kg body
weight or less, about 95 mg/kg body weight or less, about 90 mg/kg
body weight or less, about 85 mg/kg body weight or less, about 80
mg/kg body weight or less, about 75 mg/kg body weight or less,
about 70 mg/kg body weight or less, or about 65 mg/kg body weight
or less.
[0351] In some embodiments, the effective amount of compound,
conjugate, polypeptide, antibody, or pharmaceutical composition
comprising the same provided herein is between about 0.025 mg/kg
and about 60 mg/kg body weight of a human subject. In some
embodiments, the effective amount of a compound, conjugate,
polypeptide, antibody, or pharmaceutical composition comprising the
same of the pharmaceutical composition provided herein is about
0.025 mg/kg or less, about 0.05 mg/kg or less, about 0.10 mg/kg or
less, about 0.20 mg/kg or less, about 0.40 mg/kg or less, about
0.80 mg/kg or less, about 1.0 mg/kg or less, about 1.5 mg/kg or
less, about 3 mg/kg or less, about 5 mg/kg or less, about 10 mg/kg
or less, about 15 mg/kg or less, about 20 mg/kg or less, about 25
mg/kg or less, about 30 mg/kg or less, about 35 mg/kg or less,
about 40 mg/kg or less, about 45 mg/kg or less, about 50 mg/kg or
about 60 mg/kg or less.
[0352] The pharmaceutical composition of the method can be
administered using any method known to those skilled in the art.
For example, the pharmaceutical composition can be administered
intramuscularly, intradermally, intraperitoneally, intravenously,
subcutaneously administration, or any combination thereof. In some
embodiments, the pharmaceutical composition is administered
subcutaneously. In some embodiments, the composition is
administered intravenously. In some embodiments, the composition is
administered intramuscularly.
[0353] Cancers which can be treated using a compound, conjugate,
polypeptide, antibody, or pharmaceutical composition disclosed
herein include cancers where Her2 is overexpressed, CD7 is
overexpressed, Her2 is not overexpressed, and CD7 is not
overexpressed, In some embodiments, the cancer is small cell lung
cancer, non-small cell lung cancer, ovarian cancer,
platinum-resistant ovarian cancer, ovarian adenocarcinoma,
endometrial cancer, breast cancer, breast cancer which
overexpresses Her2, triple-negative breast cancer, a lymphoma,
large cell lymphoma, diffuse mixed histiocytic and lymphocytic
lymphoma, follicular B cell lymphoma, colon cancer, colon
carcinoma, colon adenocarcinoma, colorectal adenocarcinoma,
melanoma, prostate, or multiple myeloma. In certain embodiments,
the cancer is breast cancer, lung cancer, ovarian cancer,
endometrial cancer, prostate cancer. colon cancer, colorectal
cancer, melanoma, prostate cancer, or multiple myeloma.
Assay Methods
[0354] Compounds, conjugates, polypeptides, antibodies, and
pharmaceutical composition comprising the same described herein can
be assayed for their expected activity, or for a new activity,
according to any assay apparent to those of skill in the art. The
compound, conjugate, polypeptide, antibody, or pharmaceutical
composition comprising the same can be assayed for activity in a
functional assay or by quantitating the amount of protein present
in a non-functional assay, e.g. immunostaining, ELISA, quantitation
on Coomasie or silver stained gel, etc., and determining the ratio
of biologically active protein to total protein.
[0355] The amount of protein produced in a translation reaction can
be measured in various fashions. One method relies on the
availability of an assay which measures the activity of the
particular protein being translated. An example of an assay for
measuring protein activity is a luciferase assay system, or
chloramphenical acetyl transferase assay system. These assays
measure the amount of functionally active protein produced from the
translation reaction. Activity assays will not measure full length
protein that is inactive due to improper protein folding or lack of
other post translational modifications necessary for protein
activity.
[0356] Another method of measuring the amount of protein produced
in coupled in vitro transcription and translation reactions is to
perform the reactions using a known quantity of radiolabeled amino
acid such as .sup.35S-methionine, .sup.3H-leucine or
.sup.14C-leucine and subsequently measuring the amount of
radiolabeled amino acid incorporated into the newly translated
protein. Incorporation assays will measure the amount of
radiolabeled amino acids in all proteins produced in an in vitro
translation reaction including truncated protein products. The
radiolabeled protein may be further separated on a protein gel, and
by autoradiography confirmed that the product is the proper size
and that secondary protein products have not been produced.
Preparation of Modified Hemiasterlin Compounds
[0357] The compounds provided herein can be prepared, isolated or
obtained by any method apparent to those of skill in the art.
Compounds provided herein can be prepared according to the General
Preparation Scheme provided herein. Reaction conditions, steps and
reactants not provided in the General Preparation Scheme would be
apparent to, and known by, those skilled in the art in light of the
Examples provided herein.
##STR00192##
[0358] In the General Preparation Scheme R, SG, HP, RT, EG,
W.sup.1, W.sup.2, W.sup.3, W.sup.4, W.sup.5, L, and Ar are a
described in the context of Formulas C1 and 1000-1000b, 1001-1001b,
1002-1002b, and I-XIXb-2.
EXAMPLES
[0359] As used herein, the symbols and conventions used in these
processes, schemes and examples, regardless of whether a particular
abbreviation is specifically defined, are consistent with those
used in the contemporary scientific literature, for example, the
Journal of Biological Chemistry and/or the Journal of the American
Chemical Society.
[0360] For all of the following examples, standard work-up and
purification methods known to those skilled in the art can be
utilized. Unless otherwise indicated, all temperatures are
expressed in .degree. C. (degrees Celsius). All methods are
conducted at room temperature ("rt" or "r.t."), unless otherwise
noted.
Example 1a
Synthesis of Compound 1 (Two Diastereomers)
##STR00193##
##STR00194## ##STR00195##
[0361] Preparation of Compound B2
[0362] To a mixed solvent of dichloromethane (100 mL) and 2N HCl
(78 mL, 156 mmol) at -5.degree. C. was added cold bleach (contain
6% NaOCl, 108 mL, 87 mmol) in portions. The mixture was stirred at
0.degree. C. (inside temperature) for 5 min. Sodium
2-mercaptobezothioazole (B, 5 g, 26 mmol) was then added into the
mixture in multi-portions. The mixture stirred at -5 to -10.degree.
C. for 20 min. The organic layer (B1, major is BtsCl) was collected
and mixed with L-valinol (3.2 g, 31.2 mmol) and triethyl amine (8.7
mL, 121 mmol) in dichloromethane at r.t. The mixture allowed
stirring at r.t. for 1 h. Solvent was removed and product was
purified by silica gel column (Hexanes:Ethyl acetate=1:1) to give
product B2 (3.1 g, 40%, two steps) as white solid.
[0363] LC-MS (ESI): 301 (M+1).
[0364] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.08 (dd, J=2.1
and 7.2 Hz, 1H), 7.96 (dd, J=1.8 and 6.9 Hz, 1H), 7.58 (m, 2H),
5.46 (brs, 1H), 3.67 (d, J=4.5 Hz, 2H), 3.54 (brs, 1H), 3.23 (brs,
1H), 1.93 (m, 1H), 0.97 (d, J=6.9 Hz, 6H).
Preparation of Compound B3
[0365] To a solution of B2 (3 g, 10 mmol, 1.0 eq) in
dimethylformamide (50 mL) was added potassium carbonate (2.77 g, 20
mmol, 2.0 eq) and iodomethane (1.25 mL, 20 mmol, 2.0 eq) at rt. The
mixture was heated to 35.degree. C., 4 h. The solvent was removed
and the residue was worked up with ethyl acetate and water
(3.times.), dried with Na.sub.2SO.sub.4 and concentrated to give
product B3 (3.14 g, 100%) as white solid.
[0366] LC-MS (ESI): 315 (M+1).
[0367] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.09 (dd, J=1.6
and 7.5 Hz, 1H), 7.95 (dd, J=1.8 and 6.9 Hz, 1H), 7.58 (m, 2H),
4.25 (brs, 1H), 2.90 (s, 3H), 1.93 (m, 1H), 1.02 (dd, J=2.1 and 6.6
Hz, 6H).
Preparation of Compound B4
[0368] To a mixed solvent of dichloromethane (50 mL) and DMSO (1.56
mL, 22 mmol, 2.2 eq) at -78.degree. C. was added oxalyl chloride
(1.05 mL, 12 mmol, 1.2 eq) slowly under nitrogen and stirred at
this temperature for 30 min. B3 (3.14 g, 10 mmol, 1.0 eq) in 20 mL
of dichloromethane was then added into this reaction mixture at
-78.degree. C. under nitrogen. The reaction mixture allowed
stirring at -78.degree. C. for 2 h. Triethylamine (7 mL, 50 mmol, 5
eq) was then added into the reaction and stirred at -78.degree. C.
for 30 min. and continued to warm up to 0.degree. C. for another 30
min. The reaction mixture was poured into an ice-water and
extracted with DCM (3.times.). The organic layer was washed with
half saturated ammonium chloride (2.times.) solution, brine and
dried with sodium sulfate. It was concentrated at low temperature
(below 30.degree. C.) to give product B4 (3.0 g, 96%) as white
solid.
[0369] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 9.69 (s, 1H), 8.17
(dd, J=1.5 and 8.1 Hz, 1H), 7.95 (dd, J=2.1 and 6.9 Hz, 1H), 7.58
(m, 2H), 4.30 (d, J=10.2 Hz, 1H), 3.01 (s, 3H), 2.21 (m, 1H), 1.15
(d, J=6.6 Hz, 3H), 0.98 (d, J=6.6 Hz, 3H).
Preparation of Compound B5
[0370] Product B4 (3 g, 9.58 mmol, 1.0 eq) and
[(1-ethoxycarbonyl)ethylidene]Ph.sub.3P (6.95 g, 19.2 mmol, 2 eq)
were dissolved in anhydrous tetrahydrofuran (60 mL) and was heated
to reflux, 3 h. The reaction was cooled to r.t. and poured into ice
water. Product was extracted with ethyl acetate (3.times.). The
organic layer was washed with brine, dried with sodium sulfate and
then concentrated to give crude product. It was further purified by
silica gel column (Hexanes:Ethyl acetate=8:2) to give product B5
(2.9 g, 82%).
[0371] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.09 (dd, J=1.2
and 7.2 Hz, 1H), 7.93 (dd, J=1.8 and 8.1 Hz, 1H), 7.53 (m, 2H),
6.39 (dd, J=1.6 and 10.5 Hz, 1H), 4.41 (t, J=10.5 Hz, 1H), 3.87 (q,
J=7.2 Hz, 2H), 3.08 (s, 3H), 1.85 (s, 3H), 1.02-1.08 (m, 6H), 0.83
(d, J=6.9 Hz, 3H).
Preparation of Compound B6
[0372] To a solution of product B5 (2.9 g, 7.31 mmol, 1.0 eq) in
dimethylformamide (30 mL) was added potassium carbonate (4.04 g,
29.2 mmol, 4.0 eq) and thiophenol (2.25 mL, 21.9 mmol, 3.0 eq). The
reaction stirred at r.t. for 1 h. It was then worked up with
diethyl ether and water (3.times.). The ether layer was extracted
with 1% HCl, the aqueous was washed with ether. The aqueous layer
was neutralized with sodium bicarbonate to pH 8 and extracted with
dichloromethane (3.times.). The organic layer was dried with sodium
sulfate and concentrated to give pure product B6 (1.2 g, 84%) as
yellow oil.
[0373] LC-MS (ESI): 200 (M+1).
[0374] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.48 (dd, J=1.2
and 10.2 Hz, 1H), 4.18 (q, J=7.2 Hz, 1H), 3.06 (q, J=6.3 Hz, 2H),
2.30 (s, 3H), 1.86 (d, J=1.8 Hz, 2H), 1.72 (m, 1H), 1.28 (t, J=7.2
Hz, 3H), 0.93 (d, J=6.9 Hz, 3H), 0.87 (d, J=6.9 Hz, 3H).
Preparation of Compounds Bts-Leu-C1 and B7
[0375] This synthesis is fully described in Vedejs and
Kongkittingam, "A Total Synthesis of (-)-Hemiasterlin Using N-Bts
Methodology," J. Org. Chem. 2001, 66(22), 7355-7364. A summary is
provided below.
[0376] To a solution of Bts-Leu (2.4 g, 7.3 mmol, 1.0 eq) in
anhydrous dichloromethane (30 mL) at 0.degree. C. was added thionyl
chloride (1.6 mL, 21.9 mmol, 3.0 eq) under nitrogen. The reaction
mixture was refluxed at 42.degree. C. for 2 h. It was concentrated
and co-evaporated with toluene to give Bt-Leu-C1 as a crude solid
and was used in the next step reaction without further
purification.
[0377] To a solution of product B6 (1.2 g, 6.02 mmol) in a mixed
solvent of dichloromethane and water (1:1, 40 mL) at 0.degree. C.
was added a solution of sodium carbonate (1.28 g, 12.04 mmol, 2.0
eq) and sodium bicarbonate (1.32 g, 15.7 mmol. 3.2 eq) under
nitrogen. The fresh made Bts-Leu-C1 (from above) in dichloromethane
(10 mL) was added into this reaction with syringe. The mixture
stirred at 0-5.degree. C. for 1 h. Product B7 was extracted with
dichloromethane and water (3.times.), dried with sodium sulfate and
concentrated to give crude product B7, which was purified by silica
gel column (Hexanes:ethyl acetate=1:1) to give product B7 (1.8 g,
59%) as white solid. LC-MS (ESI): 510 (M+1).
[0378] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.11 (dd, J=1.5
Hz, 8.7 Hz, 1H), 7.93 (dd, J=1.2 Hz, 8.7 Hz, 1H), 7.58 (m, 2H),
6.52 (dd, J=1.2 Hz, 9.9 Hz, 1H), 6.10 (d, J=8.7 Hz, 1H), 4.85 (t,
J=10.2 Hz, 1H), 4.47 (d, J=8.7 Hz, 1H), 4.16 (m, 2H), 2.94 (s, 3H),
1.82 (d, J=1.2 Hz, 2H), 1.27 (m, 3H), 0.98 (s, 6H), 0.63 (d, J=6.6
Hz, 3H), -0.12 (d, J=6.6 Hz, 3H).
Preparation of Compound B8
[0379] To a solution of B7 (200 mg, 0.392 mmol, 1.0 eq) in DMF (2
mL) was added potassium carbonate (217 mg, 1.57 mmol, 4.0 eq) and
thiophenol (121 .mu.L, 1.18 mmol, 3.0 eq) under nitrogen at r.t.
The reaction mixture was stirred at rt. for 4 h and LC-MS showed
the reaction completed. The reaction was worked up with water and
ether and 10% hydrochloric acid (as the literature described) and
pure B8 (100 mg, 82%) obtained. LC-MS (ESI): 313 (M+1). .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 7.99 (s, 1H), 6.63 (dd, J=1.2 Hz, 9.9
Hz, 1H), 5.15 (t, J=9.9 Hz, 1H), 4.19 (m, 2H), 3.45 (s, 1H),
2.86-2.94 (m, 6H), 1.89 (m, 3H), 1.70 (s, bro, 2H), 1.28 (t, J=5.7
Hz, 3H), -0.86-1.01 (m, 12H).
##STR00196##
Preparation of Compound A1
[0380] A mixture of 3-bromobenzaldehyde (25.0 g, 135 mmol, 1.0 eq),
N-acetyl glycine (15.8 g, 135 mmol, 1.0 eq) and sodium acetate
(10.6 g, 135 mmol, 1.0 eq) were suspended in acetic anhydride (40
mL) and heated with stirring to reflux under N.sub.2 for 5 hr. The
resulting solution solidified upon cooling to room temperature and
was quenched with ice-cold water and filtered. The solids were
washed twice more with water, air dried for 4 h, then further dried
in vacuo to give compound A1 (31 g, 86%).
Preparation of Compound A2
[0381] Oxazolone A1 (31 g, 117 mmol, 1.0 eq) in 1.0 N NaOH (175 mL,
175 mmol, 1.5 eq) was stirred at 85.degree. C. until a translucent
reddish solution was obtained. The reaction was cooled down to room
temperature and acidified to pH 1.0 with 5 N HCl to precipitate a
brown solid. Concentrated HCl (30 mL) was added to the flask, and
the reaction solution diluted to about 500 mL. A reflux was
maintained for another 5 hr. The solids were collected by
filtration and washed with water twice, and dried under high vacuum
to deliver the crude material A2 (23 g, 81%) which was used without
further purification.
Preparation of Compound A3
[0382] Pyruvic acid A2 (23 g, 94.7 mmol, 1.0 eq) was dissolved in
THF (100 mL) and cooled to 0.degree. C. Methyl iodide (36 g, 256
mmol, 2.7 eq) followed by 5 N NaOH (80 mL) were slowly added, and
the reaction brought to reflux overnight. The volatiles were
stripped off and the residual aqueous solution was extracted with
ethyl acetate, and acidified with 10% HCl at 0.degree. C. to pH 1.
The resulting aqueous layer was extracted with ethyl acetate
(2.times.). The combined organics were washed with brine, dried
over sodium sulfate, and purified by column chromatography
(EtOAc/hexanes 1:1) to yield pure compound A3 (11 g, 43%).
Preparation of Compound A4
[0383] A 2 N solution of methylamine (14.4 mL, 28.8 mmol, 2.0 eq)
was added into a solution of the keto-acid A3 (11 g, 40.6 mmol, 1.0
eq) in THF (100 mL) at room temperature and stirred for 4 hr. An 8
N solution of pyridine-borane complex (5 mL, 40.6 mmol, 1.0 eq) was
added, and the mixture heated to 55.degree. C. for 3 hr. The
reaction was quenched with methanol, concentrated, and diluted with
THF (50 mL) to form a white precipitate. The white solid
precipitate was filtered and dried on vacuum to give compound A4 (5
g, 61%).
Preparation of Compound A5
[0384] To a solution of compound A4 (1.0 g, 3.5 mmol, 1.0 eq) and
(Boc).sub.2O (1.15 g, 5.24 mmol, 1.5 eq) in THF and water (1:1, 20
mL) was added sodium hydroxide (280 mg, 6.99 mmol, 2.0 eq). The
mixture was heated at 60.degree. C. for 5 h. The reaction mixture
was cooled and concentrated. The residual aqueous solution was
acidified with 10% HCl at 0.degree. C. to pH 1, and extracted with
ethyl acetate (3.times.). The combined organic layers were dried
over sodium sulfate, and purified with flash column chromatography
to give compound A5 (420 mg, 31%).
Preparation of Compound A6
[0385] To compound A5 (1.58 g, 4.07 mmol, 1 eq) in toluene (15 mL)
in a sealed tube was added ammonium hydroxide (2.7 mL, 40.7 mmol,
10 eq) and copper powder (39 mg, 0.61 mmol, 0.15 eq). The tube was
heated to 100.degree. C. overnight and concentrated to give a
residue, which was diluted with aqueous NaHCO.sub.3 and n-butanol.
The aqueous layer was extracted with n-butanol. The organic layers
were concentrated, and purified by silica gel column
(DCM:MeOH:Et3N=9:1:1) to give compound A6 (680 mg, 52%).
Preparation of Compound A7
[0386] To a solution of compound A6 (1.42 g, 3.36 mmol, 1 eq) in
THF (10 mL) was added Alloc-OSu (1.34 g, 6.72 mmol, 2 eq) and
triethylamine (1.4 mL, 10.1 mmol, 3 eq). The mixture was stirred at
rt overnight. The solvent was removed and the residue was purified
by flash column chromatography (DCM:MeOH=9:1) to give compound A7
(1.01 g, 74%).
##STR00197##
Preparation of Compound A8
[0387] To a solution of compound A7 (41 mg, 0.1 mmol, 1 eq) in dry
DCM (1.5 mL) was added B8 (31 mg, 0.1 mmol, 1 eq) and PyBOP (57.2
mg, 0.11 mmol, 1.1 eq). The mixture was cooled down to 0.degree.
C., and DMA (49 .mu.L, 0.3 mmol, 3 eq) was added. The reaction was
stirred at rt overnight, and diluted with DCM and washed with
water. The aqueous was further extracted with DCM (2.times.). The
organic layers were combined, and dried over sodium sulfate,
concentrated to dryness to give a crude product. It was purified by
pre-HPLC to give A8 (10 mg, 14%) as a mixture of two
diastereoisomers (60:40).
##STR00198##
Preparation of Compound A9
[0388] To a solution of compound A8 (150 mg, 0.21 mmol, 1.0 eq) and
Pd(PPh.sub.3).sub.4 (12.4 mg, 0.011 mmol, 0.05 eq) in THF (10 mL)
was added tri-n-butyl-tin hydride (113 .mu.L, 0.43 mmol, 2.0 eq).
The mixture was degassed and backfilled with nitrogen (3.times.).
The reaction was stirred at rt for 6 h. The solvent was removed,
and the crude product was purified by silica gel column
(DCM:MeOH=9:1) to give A9 (78 mg, 60%) as a mixture of two
isomers.
##STR00199##
[0389] To a solution of compound A9 (28 mg, 0.046 mmol, 1 eq) in
MeOH (1 mL) was added LiOH (10 mg, 0.23 mmol, 5 eq) in water (0.5
mL). The mixture was stirred at rt overnight. The product was
purified by prep-HPLC to give A12 (23 mg, 85%).
[0390] To a solution of A12 (11 mg, 0.0187 mmol, 1 eq) in DCM (1
mL) was added 10% TFA in DCM (1 mL). The mixture stirred at rt for
4 h. Solvent was removed and the crude product 1 was purified by
preparative RP-HPLC twice to give two isomers 1a (0.8 mg), and 1b
(1 mg).
Example 1b
Synthesis of Compound 101 (Two Diastereomers)
##STR00200##
[0392] Linkers synthesized from the aryl amine Compound 1 give rise
to cleavable Compound 101 which releases the novel aniline parent
compounds as a diastereomeric pair.
##STR00201##
Preparation of Compound A10
[0393] To an argon-flushed solution of A9 (27 mg, 0.04 mmol) in 1
mL CH.sub.2Cl.sub.2 was added 15% w/v phosgene in toluene (0.6 mL,
0.06 mmol). The reaction mixture was heated to 50.degree. C. in a
sealed tube for 4 h, cooled to ambient temperature, and the
volatiles removed in vacuo. To the residue was added a vacuum-dried
solution of Fmoc-valine-citruline-p-aminobenzyl alcohol (26 mg,
0.04 mmol) in 1 mL DMF. The reaction mixture was stirred at
45.degree. C. under argon for 6 h, then at ambient temperature for
24 hr. After removal of all volatiles in vacuo the residue was
purified on silica gel (90:10 CH.sub.2Cl.sub.2:MeOH eluent) to give
10 mg (0.008 mmol) A10 as a white solid.
Preparation of Compound A11
[0394] To a solution of A10 in CH.sub.2Cl.sub.2 (1 mL) was added
piperidine (0.1 mL) and the reaction mixture was stirred at ambient
temperature for 1 hr. After removal of all volatiles in vacuo, to
the residue was added DBCO-succinyl N-hydroxysuccinimidyl ester
(3.6 mg, 0.009 mmol), DMF (1 mL), and diisopropylethylamine (0.004
mL, 0.02 mmol). The reaction mixture was stirred at ambient
temperature for 24 hr. After removal of all volatiles in vacuo the
residue was purified on silica gel (90:10 CH.sub.2Cl.sub.2:MeOH
eluent) to give 7 mg (0.005 mmol) A11.
Preparation of Compound 101
[0395] Compound A11 (7 mg, 0.005 mmol) was dissolved in 3:1:1
THF:MeOH:H.sub.2O (1 mL) and the solution cooled to 0.degree. C.
Solid LiOH.H.sub.2O (1.7 mg, 0.4 mmol) was added and the reaction
mixture stirred at ambient temperature overnight. A few microliters
of glacial acetic acid were added, the volatiles removed in vacuo,
and the free acid 101 was purified by reverse phase-high
performance liquid chromatography (RP-HPLC) using an Ultro 120 (7
.mu.m), 150.times.20 mm ID column (water-acetonitrile (10 mm
NH.sub.4OAc) solvent system, gradient mode from 10% ACN to 100% ACN
in 50 min, 15 ml/min). LC-MS (ESI): 1282.6 (M+1), 1182.4
(M-Boc+1).
[0396] The N-protected acid of A11 (5 mg, 0.004) was dissolved in
CH.sub.2Cl.sub.2 (1 mL) and the solution was cooled to 0.degree. C.
To this was added a 0.2 M solution of HCO.sub.2H in
CH.sub.2Cl.sub.2 (0.039 mL) and the reaction mixture allowed to
stir at ambient temperature overnight. After the volatiles were
removed in vacuo, the free amino acid was purified by reverse
phase-high performance liquid chromatography (RP-HPLC) using Ultro
120 (7 .mu.m), 150.times.20 mm ID column (water-acetonitrile (10 mm
NH.sub.4OAc) solvent system, gradient mode from 10% ACN to 100% ACN
in 50 min, 15 ml/min) to give 3 mg (0.0025 mmol, 65%) compound 101
as white solid.
Example 1c
Chiral Synthesis of Compound A9a
##STR00202##
[0398] Linkers synthesized from the aryl amine Compound 1 give rise
to cleavable Compound 101 which releases the novel aniline parent
compounds as a diastereomeric pair.
##STR00203##
Preparation of Compound D12
[0399] 3,3-Dimethylacrylic acid, (97%, 15.8 g, 157.9 mmol),
AlCl.sub.3 (22 g, 164.9 mmol) and DCM (100 mL) were placed in a
one-neck round-bottomed flask under an argon atmosphere.
Bromobenzene (31 g, 197.4 mmol) was added producing vigorous
bubbling. Upon completion of the bubbling, the reaction mixture was
stirred in an oil bath at 65.degree. C. for 1 h and 30 min and
overnight at rt under N.sub.2 atm. Reaction was poured in
HCl:H.sub.2O (1:1) 200 mL slowly, EtOAc (300 mL) was added and the
organic phase was separated, organics were washed with brine, dried
over Na.sub.2SO.sub.4, and concentrated. The crude mixture .sup.1H
NMR data showed mixture of m,p-regioisomers. Crude material was
crystallized from hexane to give pure
3-(3-bromophenyl)-3-methylbutanoicacid (meta isomer) (14 g, 54.7
mmol, 42%) as brown crystals.
[0400] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.52-7.50 (m, 1H),
7.37-7.35 (m, 1H), 7.35-7.30 (m, 1H), 7.23-7.20 (m, 1H), 2.66 (s,
2H), 1.47 (s, 6H).
Preparation of Compound D13
[0401] 3-(3-bromophenyl)-3-methylbutanoicacid (Compound D12, 7.7 g,
29.94 mmol) was dissolved in 170 ml of THF and cooled to
-20.degree. C. Triethylamine (8.3 ml, 59.89 mmol) and
trimethylacetyl chloride (3.7 ml, 29.94 mmol) were added to the
reaction flask producing a white precipitate. The resulting mixture
was stirred at -20.degree. C. for 1 h under N.sub.2 atm, after
which LiCl (1.27 g, 29.94 mmol) and
(4S)-(-)-4-isopropyl-2oxazolidinone (3.87 g, 29.94 mmol) were added
sequentially and the resulting reaction mixture was stirred at
-20.degree. C. for 2 h and overnight at rt under N.sub.2 atm. Water
was added and the reaction mixture was extracted with EtOAc
(2.times.100 mL). The combined organic extracts were dried over
magnesium sulfate and concentrated in vacuo. The product was
purified by flash column chromatography (silica gel, hexane:EtOAc,
4:1) affording compound D13 as a clear, colorless oil in 87% yield
(9.5 g, 25.79 mmol).
[0402] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.52-7.50 (m, 1H),
7.36-7.31 (m, 2H), 7.21-7.18 (m, 1H), 4.25-4.21 (m, 1H, H-4),
4.17-4.09 (m, 2H), 3.42-3.31 (m, 2H, H-10), 2.22-2.10 (m, 1H), 1.50
(s, 3H), 1.49 (s, 3H), 0.86 (d, 3H, J=6.80 Hz), 0.77 (d, 3H, J=6.80
Hz)
Preparation of Compound D14
[0403] Oxazolidinone compound D13 (8.4 g, 22.8 mmol) was dissolved
in THF (100 ml) under an argon atmosphere, and cooled to
-78.degree. C. Potassium bis(trimethylsilyl)amide (25.1 ml, 1 M in
THF, 25.1 mmol) was added and the resulting solution was stirred at
-78.degree. C. for 1 h and 20 min. A solution of
2,4,6-triisopropylbenzenesulfonyl azide (9.2 g, 29.64 mmol) in THF
(40 ml) at -78.degree. C. was added via cannula and after 5 min,
the reaction mixture was treated with glacial acetic acid (6.3 ml,
104.9 mmol), warmed to 40.degree. C., and stirred for an additional
10 h at rt. Brine (270 ml) and Water (35 ml) were added to the
light yellow mixture and the aqueous phase was extracted with
(2.times.500 ml) diethyl ether. The combined organic extracts were
washed with a saturated sodium hydrogen carbonate solution
(2.times.110 ml), dried with magnesium sulfate, and concentrated in
vacuo. The product was purified by column chromatography (3:7
EtOAc-hexanes), affording azide compound D14 as a colorless oil
(8.1 g, 19.84 mmol) in 87% yield.
[0404] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.55-7.52 (m, 1H),
7.41-7.39 (m, 1H), 7.23-7.20 (m, 2H), 5.67 (s, 1H), 4.21-4.07 (m,
3H), 3.61 (t, 1H, J=8.3 Hz), 2.37-2.25 (m, 1H, H-6), 1.56 (s, 3H),
1.54 (s, 3H), 0.89 (d, 3H, J=6.8 Hz), 0.85 (d, 3H, J=7.2 Hz).
Preparation of Compound D15
[0405] SnCl.sub.2 (5.5 g, 29.32 mmol) was dissolved in
1,4-Dioxane:H.sub.2O (2:1) 75 mL and the resulted colorless clear
solution was cooled to 0.degree. C., to which compound D14 (4 g,
9.77 mmol) dissolved in 20 mL of dioxane was added, and the
reaction mixture was stirred at rt overnight. Reaction was cooled
back to 0.degree. C., NaHCO.sub.3 (4.1 g, 48.86 mmol) and
Boc.sub.2O (6.4 g, 29.31 mmol) were added sequentially, and the
reaction was stirred 1 day at rt under N.sub.2 atm. Solvent was
removed under reduced pressure, extracted with EtOAc (2.times.300
mL) and the organics layer was washed with brine, dried over
Na.sub.2SO.sub.4, concentrated, and purified by column
chromatography (3:7 EtOAc-hexanes), affording compound D15 as a
colorless oil (4.1 g, 8.48 mmol) in 87.2% yield. .sup.1H NMR (400
MHz, CDCl.sub.3) .delta. 7.45 (bs, 1H), 7.35-7.31 (m, 1H), 7.32-729
(m, 1H), 6.17 (d, 1H, J=9.6 Hz), 5.15 (bs, 1H, NH), 3.89-3.80 (m,
2H), 3.52 (t, 1H, J=8.3 Hz), 2.33-2.21 (m, 1H), 1.41 (s, 3H), 1.39
(s, 9H), 1.38 (s, 3H), 0.80 (d, 3H, J=7.2 Hz), 0.78 (d, 3H, J=6.8
Hz)
Preparation of Compound D16
[0406] Oxazolidinone Compound D15 (4.1 g, 8.48 mmol) was dissolved
in a mixture of 4:1 THF:H.sub.2O (50 mL). The solution was cooled
to 0.degree. C. Hydrogen peroxide (2.7 ml, 30% aqueous, 25.44 mmol)
and lithium hydroxide (610 mg, 25.44 mmol) were then added to the
oxazolidinone solution and stirred at room temperature overnight.
The excess peroxide was quenched by the slow addition of sodium
hydrogen sulfite and stirring was continued for 1 hr. The mixture
was diluted with EtOAc (50 mL) and H.sub.2O (100 mL), the aqueous
phase was separated and acidified with 1.0 M HCl at 0.degree. C.,
and extracted with ethyl acetate (2.times.200 ml). The organic
layer was washed with brine, dried over magnesium sulfate, and
concentrated in vacuo to give clear colorless oil (2.9 g, 7.83
mmol, 95%) pure enough to use in the next step without further
purification. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.32-7.31
(m, 1H), 7.17-7.10 (m, 2H), 7.00-6.97 (m, 1H), 4.83 (d, 1H, J=8.8
Hz), 4.41 (d, 1H, J=8.8 Hz), 1.45 (s, 6H), 1.38 (s, 9H).
Preparation of Compound A5a
[0407] Under an argon atmosphere, sodium hydride (60%, 830 mg,
22.64 mmol), a catalytic amount of tetrabutylammonium iodide,
followed by methyl iodide (2.0 ml, 32 mmol) were added to a
vigorously stirred solution of acid compound D16 (1.2 g, 3.23 mmol)
in 50 ml dry THF. The resulting suspension was stirred 1 day at
room temperature. The excess sodium hydride was quenched by
cautious addition of ice cold water and the mixture was acidified
by drop wise addition of 1.0 M HCl to pH 3 at 0.degree. C. The
acidic mixture was extracted with ethyl acetate (3.times.100 mL),
the combined organic layer was washed with brine, dried over
magnesium sulfate, and concentrated in vacuo. Purification of acid
Compound A5a was performed by silica gel column chromatography (1:2
EtOAc-hexanes with 1% acetic acid) resulting in a 77% yield (0.74
g, 1.93 mmol, 60%) of a clear colorless oil as mixture of rotamers.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.50-7.49 (m, 1H),
7.37-7.27 (m, 2H), 7.13-7.07 (m, 1H), 5.15 (s, 0.65 H, H-2), 4.95
(s, 0.35H, H-2), 2.51 (s, 1H, H-6), 2.27 (s, 2H, H-6), 1.57 (s,
3H), 1.53-1.38 (m, 12H).
Preparation of Compound A6a
[0408] Compound A5a (600 mg, 1.56 mmol, 1 eq) in toluene (10 mL) in
a sealed tube was added ammonium hydroxide (3 mL, 15.6 mmol, 10 eq)
and copper powder (20 mg, 0.23 mmol, 0.15 eq). The tube was heated
to 100.degree. C. overnight and was cooled to rt, sealed tube cap
was carefully released, concentrated to give a residue, which was
diluted with aqueous NaHCO.sub.3 and n-butanol. The aqueous layer
was extracted with n-butanol. The organic layers were concentrated,
and purified by silica gel column (DCM:MeOH:Et3N=9:1:1) to give
compound A6a (300 mg, 0.930 mmol, 60%). LC-MS (ESI): 323.4 (M+1),
223.5 (M-Boc+1).
Preparation of Compound A7A
[0409] To a solution of compound A6a (300 mg, 0.930 mmol, 1 eq) in
THF (7 mL) was added Alloc-OSu (199.1 mg, 1.86 mmol, 2 eq) and
triethylamine (0.51 mL, 3.72 mmol, 4 eq). The mixture was stirred
overnight at room temperature under N.sub.2 atm. The solvent was
removed and the residue was purified by flash column chromatography
(DCM:MeOH=9:1) to give compound A7A (284 mg, 0.70 mmol, 75%). LC-MS
(ESI): 407.4 (M+1), 307.6 (M-Boc+1).
Preparation of Compound A8A
[0410] To a solution of compound A7A (220 mg, 0.54 mmol, 1 eq) in
dry DCM (5 mL) was added B8 (202 mg, 0.65 mmol, 1.2 eq). The
mixture was cooled to 0.degree. C., and DIEA (49 .mu.L, 0.3 mmol, 3
eq) and PyBop (338 mg, 0.65 mmol, 1.2 eq) were added sequentially.
The reaction was stirred at rt overnight under N.sub.2 atm, and
diluted with DCM and washed with water. The aqueous was further
extracted with DCM (2.times.50 mL). The organic layers were
combined, dried over sodium sulfate, and concentrated to dryness to
give a crude product. This crude product was purified by flash
column chromatography (EtOAc:Hexane=1:1) to give compound A8A (200
mg, 0.28 mmol, 53%). LC-MS (ESI): 701.4 (M+1), 601.6 (M-Boc+1).
Preparation of Compound A9a
[0411] To a solution of compound A8A (170 mg, 0.24 mmol, 1.0 eq)
and Pd(PPh.sub.3).sub.4 (28 mg, 0.024 mmol, 0.1 eq) in DCM (10 mL)
was added tri-n-butyl-tin hydride (78 .mu.L, 0.29 mmol, 1.2 eq).
The reaction mixture was degassed and backfilled with nitrogen. The
reaction was stirred for 3-4 h at rt under N.sub.2 atm. The solvent
was removed, and the crude product was purified by silica gel
column (EtOAc:Hexane=1:1) to give A9a (130 mg, 87%) as a clear oil.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.11-7.07 (m, 1H),
6.97-6.70 (m, 2H), 6.61-6.48 (m, 2H), 6.03-5.97 (m, 1H), 5.12-4.89
(m, 1H), 4.63-4.49 (m, 1H), 4.21-4.01 (m, 2H), 2.93 (s, 3H), 2.83
(m, 3H), 1.83-1.81 (bs, 4H), 1.42 (s, 12H), 1.27-1.17 (m, 6H),
0.87-0.0.81 (m, 6H), 0.78-0.63 (m, 6H), 0.63 (s, 6H). LC-MS (ESI):
617.6 (M+1).
Example 1d
Chiral Synthesis of Compound 101a (Single Diastereomer)
##STR00204##
[0413] Compound 101a is produced from compound A9a according to
Scheme 8.
##STR00205##
Preparation of Compound A10a
[0414] To an argon-flushed solution of Aga (27 mg, 0.04 mmol) in 1
mL CH.sub.2Cl.sub.2 is added 15% w/v phosgene in toluene (0.6 mL,
0.06 mmol). The reaction mixture is heated to 50.degree. C. in a
sealed tube for 4 h, cooled to ambient temperature, and the
volatiles removed in vacuo. To the residue is added a vacuum-dried
solution of Fmoc-valine-citruline-p-aminobenzyl alcohol (26 mg,
0.04 mmol) in 1 mL DMF. The reaction mixture is stirred at
45.degree. C. under argon for 6 h, then at ambient temperature for
24 hr. After removal of all volatiles in vacuo the residue is
purified on silica gel (90:10 CH.sub.2Cl.sub.2:MeOH eluent) to give
A10a.
Preparation of compound A11a
[0415] To a solution of A10a in CH.sub.2Cl.sub.2 (1 mL) is added
piperidine (0.1 mL) and the reaction mixture is stirred at ambient
temperature for 1 hr. After removal of all volatiles in vacuo, to
the residue is added DBCO-succinyl N-hydroxysuccinimidyl ester (3.6
mg, 0.009 mmol), DMF (1 mL), and diisopropylethylamine (0.004 mL,
0.02 mmol). The reaction mixture is stirred at ambient temperature
for 24 hr. After removal of all volatiles in vacuo the residue is
purified on silica gel (90:10 CH.sub.2Cl.sub.2:MeOH eluent) to give
A11a.
Preparation of compound 101a
[0416] Compound A11a (7 mg, 0.005 mmol) is dissolved in 3:1:1
THF:MeOH:H.sub.2O (1 mL) and the solution cooled to 0.degree. C.
Solid LiOH.H.sub.2O (1.7 mg, 0.4 mmol) is added and the reaction
mixture stirred at ambient temperature overnight. A few microliters
of glacial acetic acid are added, the volatiles removed in vacuo,
and the free acid 101a is purified by reverse phase-high
performance liquid chromatography (RP-HPLC) using an Ultro 120 (7
.mu.m), 150.times.20 mm ID column (water-acetonitrile (10 mm
NH.sub.4OAc) solvent system, gradient mode from 10% ACN to 100% ACN
in 50 min, 15 ml/min).
[0417] The N-protected acid of A11a (5 mg, 0.004) is dissolved in
CH.sub.2Cl.sub.2 (1 mL) and the solution is cooled to 0.degree. C.
To this is added a 0.2 M solution of HCO.sub.2H in CH.sub.2Cl.sub.2
(0.039 mL) and the reaction mixture is allowed to stir at ambient
temperature overnight. After the volatiles are removed in vacuo,
the free amino acid is purified by reverse phase-high performance
liquid chromatography (RP-HPLC) using Ultro 120 (7 .mu.m),
150.times.20 mm ID column (water-acetonitrile (10 mm NH.sub.4OAc)
solvent system, gradient mode from 10% ACN to 100% ACN in 50 min,
15 ml/min) to give compound 101a.
Example 1e
Synthesis of Compound (110a)
##STR00206##
[0419] Compound (110a) is prepared according to Scheme 9.
##STR00207##
[0420] Preparation of compound A14: Compound A14 was prepared from
compound A13 (using procedures similar to those described for
compound A8a) by the method described for compound A9a. Yield: 379
mg (45%) as a white foam. MS (ESI) m/z 603 (M+H).sup.+; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta.: 6.96-7.12 (m, 1H), 6.91 (brd, J=7.5
Hz, 1H), 6.83 (brs, 1H), 6.66-6.80 (m, 1H), 6.51-6.65 (m, 1H),
6.41-6.50 (m, 1H), 5.95 (brd, J=8.8 Hz, 1H), 5.33 (s, 1H),
4.94-5.11 (m, 1H), 4.92 (brs, 1H), 4.58 (brd, J=9.5 Hz, 1H), 4.48
(brd, J=8.5 Hz, 1H), 3.72 (brs, 1H), 3.67 (brs, 3H), 2.73-2.96 (m,
7H), 1.75-1.98 (m, 5H), 1.72 (br s, 1H), 1.49 (brs, 2H), 1.18-1.45
(m, 15H), 0.60-0.86 (m, 17H); .sup.13C NMR (400 MHz, CDCl.sub.3)
.delta.: 171.2, 169.9, 168.2, 168.1, 157.1, 148.8, 146.9, 139.2,
139.0, 132.5, 132.2, 129.7, 116.6, 113.4, 79.8, 65.4, 55.9, 55.2,
52.0, 42.5, 42.5, 34.7, 34.3, 33.9, 31.0, 31.0, 30.2, 30.1, 28.3,
28.3, 26.5, 26.3, 26.2, 19.5, 19.4, 18.8, 18.5, 13.9, 13.8.
[0421] Preparation of compound A15: To an argon-flushed, ice-cooled
solution of A14 (63 mg, 0.1 mmol) in CH.sub.2Cl.sub.2 (0.8 mL) was
added trifluoroacetic acid (0.2 mL, 2.6 mmol). The ice bath was
removed, and the reaction was stirred at ambient temperature for 1
h. The volatiles were removed in vacuo to give A15 as a pale yellow
glass that was used directly in the subsequent reaction. MS (ESI)
m/z 503 (M+H).sup.+; .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
10.36 (br, 3H), 7.19-8.14 (m, 4H), 6.57 (brd, J=7.5 Hz, 1H),
5.02-4.45 (m, 3H), 3.68 (s, 3H), 2.97 (s, 3H), 2.38-2.48 (m, 3H),
1.74-1.80 s (m, 3H), 1.18-1.38 (m, 6H), 0.80-0.95 (m, 17H);
.sup.13C NMR (400 MHz, CDCl.sub.3) .delta.: 170.9, 168.2, 161.5,
145.3, 139.2, 138.0, 131.5, 126.8, 122.3, 117.2, 86.9, 52.1, 40.9,
40.5, 35.3, 34.4, 33.6, 31.4, 29.7, 28.2, 28.3, 27.5, 26.5, 26.4,
26.3, 26.2, 19.1, 187, 18.4, 13.8.
[0422] Preparation of compound A16: Compound A16 was prepared from
compound A15 by the general method described for compound A10.
Yield: 52 mg (50%) as a white foam. MS (ESI) m/z 1044 (M+H).sup.+;
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 9.10 (br, 1H), 7.84 (brd,
J=7.8 Hz, 1H), 7.62 (d, J=7.6 Hz, 2H), 7.42-7.31 (m, 7H), 7.11-7.23
(m, 7H), 6.94-6.98 (m, 1H), 6.56 (d, J=8.0 Hz, 1H), 5.94-5.96 (m,
1H), 4.85-5.1 (m, 3H), 4.61-4.80 (m, 2H), 4.15 (t, J=7.3 Hz, 1H),
4.25 (t, J=7.0 Hz, 1H), 4.15 (t, J=7.1 Hz, 1H), 4.05 (t, J=7.0 Hz,
1H), 3.67 (s, 3H), 3.60 (d J=3.6 Hz, 1H), 2.89-3.05 (m, 5H),
1.80=2.09 (m, 10H), 1.28-1.36 (m, 10H), 0.66-0.99 (m, 17H);
.sup.13C NMR (400 MHz, CDCl.sub.3) .delta.: 172.4, 172.0, 171.5,
170.9, 168.2, 156.7, 153.9, 147.9, 143.7, 143.6, 141.3, 138.9,
132.3, 129.2, 127.7, 127.0, 125.0, 120.67.1, 66.0, 54.7, 52.0,
49.6, 47.1, 41.2, 35.6, 35.1, 31.1, 31.0, 29.8, 29.2, 26.7, 21.7,
19.5, 19.3, 19.2, 19.0, 18.9, 18.1, 18.0, 13.8.
[0423] Preparation of compound A17: To an argon-flushed solution of
A16 (52 mg, 0.05 mmol) in DMF (0.4 mL) was added N,N-diethylamine
(0.2 mL, 5 mmol), and the reaction was stirred at ambient
temperature 2 h. The volatiles were removed in vacuo, and the
residue purified on silica gel (Biotage Isolera) using a gradient
of 2-100% methanol in chloroform to yield 23 mg free amine as a
white solid. This was further purified on RP-HPLC (.times.mm C18
5.quadrature..quadrature..quadrature. using a linear gradient of 10
to 90% B in A over 20 minutes (A=10 mM NH.sub.4OAc in water; B=10
mM NH.sub.4OAc in CH.sub.3CN) and detected at 254 and 280 nM to
give A17 Yield 9.5 mg, 24%). MS (ESI) m/z 822 (M+H).sup.+; .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 9.11 (s, 1H), 7.73 (br, 1H), 7.81
(d, J=9.5 Hz, 1H), 7.48 (d, J=9.0 Hz, 2H), 7.01-7.3 (m, 4H), 6.57
(dd, J=1.4, 9.5 Hz, 1H), 5.07 (s, 2H), 5.04 (d, J=1.1 Hz, 1H), 4.72
(d, J=9.8 Hz, 1H), 4.6 (t, J=7.0 Hz, 1H), 3.6 (s, 3H), 3.63 (s,
1H), 3.24 (br, 1H), 3.05 (s, 1H), 3.01, (d J=6.3 Hz, 1H), 2.95 (s,
3H), 2.08 (br, 2H), 2.01 (s, 3H), 1.98 (s, 3H), 1.80-1.84 (m, 1H),
1.40 (d, J=6.2 Hz, 4H), 1.31 (s, 6H), 0.92-0.93 (m, 13H), 0.79 (d,
J=6.6 Hz, 8H), 0.71 (d, J=6.6 Hz, 4H)
[0424] Preparation of (110a): Under an argon atmosphere, compound
A17 (9.8 mg, 0.012 mmol) was stirred with
dibenzocyclooctynyladipoyl N-hydroxysuccinimidyl ester (Broadpharm
22447, 7.7 mg, 0.018 mmol) in DMF (0.150 mL). To this was added N,
N-diisopropylethylamine (0.006 mL, 0.036 mmol) and the reaction was
stirred at ambient temperature for 3 h. The volatiles were removed
in vacuo, and the residue partially purified on silica gel using a
gradient of 2 to 10% methanol in chloroform. The product-containing
fractions were concentrated in vacuo to a residue MS m/z 1137.4.
This residue (10.6 mg, 0.009 mmol), under an argon atmosphere, was
dissolved in 3:1:1 THF:methanol:water (0.5 mL), cooled in an
ice-bath, and treated with LiOH.H.sub.2O (2.6 mg, 0.063 mmol). The
reaction mixture was allowed to equilibrate to ambient temperature
overnight. Volatiles were removed in vacuo and the residue purified
by RP-HPLC as above to yield, after lyophilization, (110a), 2.1 mg,
0.002 mmol, as a flocculent white solid. MS (ESI) m/z 1123
(M+H).sup.+; .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.: 10.02
(brd, J=3.8 Hz, 1H), 9.67 (brs, 1H), 8.25 (brd, J=6.8 Hz, 1H), 7.88
(brd, J=8.8 Hz, 1H), 7.79 (brd, J=8.3 Hz, 1H), 7.54-7.70 (m, 4H),
7.29-7.54 (m, 9H), 7.06-7.27 (m, 3H), 6.40-6.50 (m, 1H), 5.10 (s,
2H), 5.05 (brd, J=14.1 Hz, 1H), 4.91 (br t, J=10.2 Hz, 1H), 4.77
(brd, J=9.5 Hz, 1H), 4.25-4.49 (m, 2H), 4.06-4.24 (m, 2H),
3.60-2.99 (m, broad water envelope), 2.99 (m, 3H), 2.69-2.82 (m,
3H), 2.56-2.49 (m, DMSO envelope), 2.42 (brs, 1H), 2.12-2.29 (m,
3H), 1.90-2.11 (m, 6H), 1.73-1.90 (m, 6H), 1.15-0.76 (m, 18H), 0.72
(brd, J=6.3 Hz, 6H).
Example 1f
Synthesis of Compound (111a)
##STR00208##
[0426] Compound (111a) was prepared according to Scheme 10.
##STR00209##
Preparation of Compound A19: ethyl
(S,E)-4-((R)-2-((S)-3-(3-aminophenyl)-3-methyl-2-(methylamino)butanamido)-
-N, 3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoate
[0427] An oven-dried 50 mL single-necked round-bottomed flask
equipped with a teflon-coated magnetic stir bar was charged with
compound A18 (200 mg, 0.32 mmol, 1.0 eq) dry CH.sub.2Cl.sub.2 (3
mL) and the clear solution was cooled to 0.degree. C. with an ice
bath, to this 1 mL of Trifluoroacetic acid was added. The reaction
mixture was allowed to stir 4 h at room temperature. After which
LC-MS showed completion of the reaction. The solvent was removed
under reduced pressure, and the crude material was lyophilized for
16 h to give compound A19 (167 mg, 100%) as an off-white solid.
LC-MS (ESI): 517.5 (M+1).
Preparation of compound A20:
ethyl(S,E)-4-((S)-2-((R)-3-(3-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)c-
arbonyl)amino)-3-methylbutanamido)propanamido)phenyl)-3-methyl-2-(methylam-
ino)butanamido)-N,
3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoate
[0428] To an oven-dried 50 mL single-necked round-bottomed flask
equipped with a teflon-coated magnetic stir bar is charged with
compound A19 (70 mg, 0.135 mmol, 1 eq), Fmoc-Valine-Alanine-OH (67
mg, 0.162 mmol, 1.2 eq) and 1 mL of anhydrous
N,N-Dimethylformamide. The resulted clear solution was cooled to
0.degree. C. with an ice bath, N,N-Diisopropylethylamine (72 .mu.L,
0.405 mmol, 3 eq),
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate (HATU) (62 mg, 0.162 mmol, 1.2 eq) were
sequentially added to the reaction. The reaction mixture was
allowed to stir at room temperature overnight under N.sub.2
atmosphere. LC-MS showed completion of the reaction. The reaction
was quenched by the addition of saturated NH.sub.4Cl (10 mL) and
then extracted with CH.sub.2Cl.sub.2 (2.times.100 mL). The organic
layer was washed with saturated brine (50 mL), dried over anhydrous
Na.sub.2SO.sub.4, filtered and then concentrated to dryness at
reduced pressure. The crude product was purified by preparative
reverse phase-high performance liquid chromatography using an Ultro
120 (7 .mu.m) C18Q, 150.times.20 mmID column. Solvent system used
Solvent A: water containing 10 mm NH.sub.4OAc; Solvent B:
acetonitrile containing 10 mm NH.sub.4OAc., Gradient mode from 10%
Solvent B to 90% solvent B, over 20 minutes, 10 mL/min), pure
fractions were collected and lyophilized to give the compound A20
(86 mg, 0.095 mmol, 70%) as a white solid. LC-MS (ESI): 909.5
(M+1).
Preparation of Compound A21
[0429] An oven-dried 25 mL single-necked round-bottomed flask
equipped with a teflon-coated magnetic stir bar was charged with
compound A20 (80 mg, 0.088 mmol), dry CH.sub.2Cl.sub.2 (2 mL), to
this clear solution was added piperidine (0.5 mL) and the reaction
mixture was stirred at ambient temperature for 1 h under N.sub.2
atm. LC-MS showed completion of the reaction, all volatiles were
removed under reduced pressure. The crude free amine was purified
by preparative reverse phase-high performance liquid chromatography
using an Ultro 120 (7 .mu.m) C18Q, 150.times.20 mmID column.
Solvent system used Solvent A: water containing 10 mm NH.sub.4OAc;
Solvent B: acetonitrile containing 10 mm NH.sub.4OAc., Gradient
mode from 10% Solvent B to 90% solvent B, over 20 minutes, 10
mL/min), pure fractions were collected and lyophilized to give the
free amine A20a (51 mg, 0.074 mmol, 85%) as a white solid. LC-MS
(ESI): 688 (M+1).
[0430] An oven-dried 25 mL single-necked round-bottomed flask
equipped with a teflon-coated magnetic stir bar was charged with
the free amine A20a (51 mg, 0.074 mmol). DBCO adipinyl
N-hydroxysuccinimidyl ester (41 mg, 0.096 mmol), anhydrous
N,N-Dimethylformamide (0.5 mL), and N,N-Diisopropylethylamine (40
.mu.L, 0.22 mmol) were sequentially added. The reaction mixture was
flushed with Argon and stirred at ambient temperature for 3 hours
under N.sub.2 atm. LC-MS showed completion of the reaction. After
removal of all volatiles in vacuo, the residue was purified by
reverse phase-high performance liquid chromatography using a Ultro
120 (7 .mu.m) C18Q, 150.times.20 mm ID column Solvent system used
Solvent A: water containing 10 mm NH.sub.4OAc; Solvent B:
acetonitrile containing 10 mm NH.sub.4OAc., Gradient mode from 10%
B Solvent to 90% solvent B, over 20 minutes, 10 mL/min), pure
fractions were collected and lyophilized to give compound A21a (52
mg, 0.052 mmol, 70%) as a white powder. LC-MS (ESI): 1003.8
(M+1).
Preparation of Compound (111a)
[0431] An oven-dried 25 mL single-necked round-bottomed flask
equipped with a teflon-coated magnetic stir bar is charged with
compound A21 (50 mg, 0.05 mmoles, 1 equiv.), THF:MeOH:H.sub.2O
(3:1:1) (1 mL). The clear solution was cooled to 0.degree. C. with
an ice bath. Solid LiOH.H.sub.2O (16 mg, 0.349 mmol, 7 eq) was
added and the reaction was allowed to stir at room temperature
under N.sub.2 atm for 7 h, after which LC-MS showed completion of
the reaction, the volatiles were removed in vacuo, and the crude
material was purified by reverse phase-high performance liquid
chromatography using Ultro 120 (7 .mu.m) C18Q, 150.times.20 mmID
column, Solvent system used Solvent A: water containing 10 mm
NH.sub.4OAc; Solvent B: acetonitrile containing 10 mm NH.sub.4OAc.,
Gradient elution mode from 10% Solvent B to 90% solvent B, over 20
minutes, 10 mL/min), pure fractions were collected and lyophilized
to give compound (111a) (34 mg, 0.034 mmol, 70%) as a white powder.
LC-MS (ESI): 974.5 (M+1). .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 9.78-9.63 (bd, 1H), 8.34-8.10 (m, 1H), 7.82-7.68 (2H),
7.58-7.35 (m, 8H), 7.34-7.18 (m, 4H), 7.16-7.08 (m, 4H), 6.53-6.52
(m, 1H) 4.98-4.93 (m, 1H), 4.85 (m, 1H), 4.71 (brd, 1H), 4.36-4.26
(m, 1.5H), 4.09-4.04 (m, 1H), 3.67-3.47 (m, 2H), 3.12 (brd, 1H),
2.91-2.87 (m, 3H), 2.13-2.02 (m, 2H), 1.96-1.78 (m, 9H), 1.74-1.61
(m, 5H), 1.31-1.03 (m, 16H), 0.90-0.67 (m, 15H).
Example 1g
Synthesis of Compound (109a)
##STR00210##
[0433] Compound (109a) was prepared according to Scheme 11a and
11b.
##STR00211##
[0434] Preparation of A22: A22 was prepared by a straightforward
adaptation of literature methods. (See, for example, Florent et al
1998, J Med Chem 41, 3572; and Jeffrey et al 2006, Biocong Chem 17,
831). LC-MS: T.sub.R 9.21 min. m/z 763 (M+H).sup.+.
##STR00212##
Preparation of compound A23:
(2S,3R,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanam-
ido)-4-((((3-((R)-4-(((S)-1-(((S,E)-6-ethoxy-2,5-dimethyl-6-oxohex-4-en-3--
yl)(methyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-methyl-3-(methylam-
ino)-4-oxobutan-2-yl)phenyl)carbamoyl)oxy)methyl)-6-methylphenoxy)-6-(meth-
oxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate
[0435] To an oven-dried 25 mL pressure vessel equipped with a
teflon-coated magnetic stir bar is charged with compound A19 (25
mg, 0.048 mmol, 1 eq) and 1 mL of anhydrous CH.sub.2Cl.sub.2, to
this was added 15% w/v phosgene in toluene (0.7 mL) at rt. The
reaction mixture was flushed with Argon and it was stirred in a
sealed vessel for 17 h at rt, then concentrated under reduced
pressure to remove volatiles, and dried under high vacuum for at
least 2 hours. To this residue was added compound A22 (44 mg, 0.058
mmol, 1.2 eq,). This mixture was dissolved in 1 mL of anhydrous
N,N-Dimethylformamide. The reaction mixture was stirred at
45.degree. C. for 2 h, then at ambient temperature for 12 h. After
removal of all volatiles in vacuo the residue was purified by
reverse phase-high performance liquid chromatography using Ultro
120 (7 .mu.m) C18Q, 150.times.20 mmID column, Solvent system used
Solvent A: water containing 10 mm NH.sub.4OAc; Solvent B:
acetonitrile containing 10 mm NH.sub.4OAc., Gradient elution mode
from 10% Solvent B to 90% solvent B, over 20 minutes, 10 mL/min),
pure fractions were collected and lyophilized to give compound A23
(25 mg, 0.019 mmol, 70%) as a white powder. LC-MS (ESI): 1305.8
(M+1).
Preparation of Compound (109a)
[0436] An oven-dried 25 mL single-necked round-bottomed flask
equipped with a teflon-coated magnetic stir bar was charged with
compound A23 (23 mg, 0.018 mmol), dry CH.sub.2Cl.sub.2 (0.5 mL), to
this clear solution was added piperidine (0.2 mL) and the reaction
mixture was stirred at ambient temperature for 1 h under N.sub.2
atm, all volatiles were removed under reduced pressure. The crude
product was dissolved in THF:MeOH:H.sub.2O (3:1:1) (1 mL). The
clear solution was cooled to 0.degree. C. with an ice bath. Solid
LiOH.H.sub.2O (8 mg, 0.176 mmol, 10 eq) was added and the reaction
was allowed to stir at room temperature under N.sub.2 atm for 7 h,
after which LC-MS showed completion of the reaction, the volatiles
were removed in vacuo, and the crude material was purified by
reverse phase-high performance liquid chromatography using Ultro
120 (7 .mu.m) C18Q, 150.times.20 mmID column, Solvent system used
Solvent A: water containing 10 mm NH.sub.4OAc; Solvent B:
acetonitrile containing 10 mm NH.sub.4OAc., Gradient elution mode
from 10% Solvent B to 90% solvent B, over 20 minutes, 10 mL/min),
pure fractions were collected and lyophilized to give compound A23a
(8 mg, 0.009 mmol, 50%) as a white powder. LC-MS (ESI): 915.7
(M+H), 897.7 (M-H.sub.2O+H)
[0437] An oven-dried 10 mL single-necked round-bottomed flask
equipped with a teflon-coated magnetic stir bar was charged with
the free amine A23a (8 mg, 0.009 mmol). DBCO adipinyl
N-hydroxysuccinimidyl ester (5 mg, 0.011 mmol), anhydrous
N,N-Dimethylformamide (0.3 mL), and N,N-Diisopropylethylamine (10
.mu.L, 0.033 mmol) were sequentially added. The reaction mixture
was flushed with Argon and stirred at ambient temperature for 3
hours under N.sub.2 atm. LC-MS showed completion of the reaction.
After removal of all volatiles in vacuo, the residue was purified
by reverse phase-high performance liquid chromatography using a
Ultro 120 (7 .mu.m) C18Q, 150.times.20 mmID column Solvent system
used Solvent A: water containing 10 mm NH.sub.4OAc; Solvent B:
acetonitrile containing 10 mm NH.sub.4OAc., Gradient mode from 10%
B Solvent to 90% solvent B, over 20 minutes, 10 mL/min), pure
fractions were collected and lyophilized to give compound (109a) (5
mg, 0.004 mmol, 50%) as a white powder. LC-MS (ESI): 1230.8 (M+H),
1212.8 (M-H.sub.2O+H). .sup.1H NMR (400 MHz, DMSO-d6) .delta.
9.61-9.55 (m, 2H), 7.85 (bs, 2H), 7.80 (bd, 1H), 7.61-6.97 (m,
15H), 6.64-6.50 (m, 2H), 6.02 (bs, 1H), 5.48 (bs, 1H), 5.17-4.89
(m, 6H), 4.85-4.76 (bt, 1H), 4.71-4.62 (m, 1H), 4.01 (bs, 1H),
3.69-3.66 (m, 3H), 3.10 (bs, 2H), 2.89 (bs, 3H), 2.29-2.26 (m,
19H), 2.13-2.00 (m, H), 1.96-1.56 (m, 18H), 1.80-1.02 (m, 14H),
0.89 (bs, 10H), 0.70 (d, 3H), 0.66 (d, 3H).
Example 1h
Synthesis of Compound (111a)
[0438] Compound (111a) was prepared according to Schemes 12 and
13.
##STR00213##
Preparation of Compound A8a: ethyl
(6S,9S,12S,E)-6-(2-(3-(((allyloxy)carbonyl)amino)phenyl)propan-2-yl)-9-(t-
ert-butyl)-12-isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-3-oxa-5,8,11-
-triazapentadec-13-en-15-oate
[0439] An oven-dried 100 mL single-necked round-bottomed flask
equipped with a teflon-coated magnetic stir bar is charged with
compound Ala (1.1 g, 2.70 mmoles, 1 equiv, prepared internally),
dry CH.sub.2Cl.sub.2 (10 mL) and compound B8 (1.00 g, 3.24 mmoles,
1.2 equiv., prepared internally) in dry CH.sub.2Cl.sub.2 (10 mL).
The resulted clear solution was cooled to 0.degree. C. with an ice
bath, N,N-Diisopropylethylamine (1.5 mL, 8.1 mmoles, 3 equiv.,) and
(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
(PyBOP, 1.7 g, 3.24 mmol, 1.2 equiv.,) were sequentially added to
the cooled solution. The reaction mixture was allowed to stir at
room temperature overnight under N.sub.2 atmosphere. LC-MS showed
completion of the reaction. The reaction was quenched by the
dropwise addition of saturated NH.sub.4Cl (10 mL) and then
extracted with CH.sub.2Cl.sub.2 (2.times.200 mL). The organic layer
was washed with saturated brine (50 mL), dried over anhydrous
Na.sub.2SO.sub.4, filtered and then concentrated to dryness at
reduced pressure. The crude product was purified by flash silica
column chromatography on a Teledyne ISCO system (40 g silica flash
column, gradient: Hexane to 30% EtOAc/Hexane) to give compound A8a
(1.2 g, 1.71 mmol, 63%) as a colorless viscous oil which slowly
solidifies to give an off-white solid upon standing. LC-MS (ESI):
701.5 (M+1).
Preparation of Compound A9a: Ethyl
(6R,9S,12S,E)-6-(2-(3-aminophenyl)propan-2-yl)-9-(tert-butyl)-12-isopropy-
l-2,2,5,11,14-pentamethyl-4,7,10-trioxo-3-oxa-5,8,11-triazapentadec-13-en--
15-oate
[0440] An oven-dried 100 mL single-necked round-bottomed flask
equipped with a teflon-coated magnetic stir bar is charged with
compound A8a (1.2 g, 1.71 mmols, 1.0 equiv, prepared internally),
dry CH.sub.2Cl.sub.2 (10 mL). To this clear solution,
tetrakis(triphenylphosphine)palladium(O) (0.98 g, 0.856 mmoles, 0.5
equiv.,) and tri-n-butyl-tin hydride (0.55 mL, 2.05 mmoles, 1.2
equiv.,) were sequentially added at room temperature under N.sub.2
atmosphere. Upon completion of addition, the reaction mixture was
flushed with argon and then stirred at room temperature under a
N.sub.2 atmosphere. LC-MS & TLC (1:1 EtOAc/Hexane) showed the
reaction was complete in 4 hours. The solvent was removed under
reduced pressure and the resulting crude material was purified by
flash column chromatography on a Teledyne ISCO system (40 g silica
flash column, gradient: Hexane to 50% EtOAc/Hexane) to give
compound A9a (0.95 g, 1.54 mmole, yield: 90%) as a foamy off-white
solid. LC-MS (ESI): 617.3 (M+1).
##STR00214##
Preparation of Compound A19: ethyl
(S,E)-4-((R)-2-((S)-3-(3-aminophenyl)-3-methyl-2-(methylamino)butanamido)-
-N, 3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoate
[0441] An oven-dried 50 mL single-necked round-bottomed flask
equipped with a teflon-coated magnetic stir bar was charged with
compound A9a (200 mg, 0.32 mmol, 1.0 eq) dry CH.sub.2Cl.sub.2 (3
mL) and the clear solution was cooled to 0.degree. C. with an ice
bath, to this 1 mL of Trifluoroacetic acid was added. The reaction
mixture was allowed to stir 4 h at room temperature. After which
LC-MS showed completion of the reaction. The solvent was removed
under reduced pressure, and the crude material was lyophilized for
16 h to give compound A19 (167 mg, 100%) as an off-white solid.
LC-MS (ESI): 517.5 (M+1).
Preparation of compound A18: ethyl
(S,E)-4-((S)-2-((R)-3-(3-((((4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)-
carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbony-
l)amino)phenyl)-3-methyl-2-(methylamino)butanamido)-N,
3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoate
[0442] To an oven-dried 50 mL pressure vessel equipped with a
teflon-coated magnetic stir bar is charged with compound A19 (167
mg, 0.323 mmol, 1 eq) (**Note: compound A19 was dried under
lyophilizer pump for 15 h prior to use) and 4 mL of anhydrous
CH.sub.2Cl.sub.2 to this was added 15% w/v phosgene in toluene (5
mL) at rt. The reaction mixture was flushed with Argon and it was
stirred in a sealed vessel for 17 h at rt, then concentrated under
reduced pressure to remove volatiles, and dried under high vacuum
for at least 2 hours. To this residue was added Fmoc
valine-citruline-p-aminobenzyl alcohol (253 mg, 0.42 mmol, 1.3 eq).
This mixture was dissolved in 3 mL of anhydrous
N,N-Dimethylformamide. The reaction mixture was stirred at
45.degree. C. for 2 h, then at ambient temperature for 12 h. After
removal of all volatiles in vacuo the residue was purified by flash
column chromatography on a Teledyne ISCO (24 g silica flash column,
gradient: CH.sub.2Cl.sub.2 to 12% MeOH/CH.sub.2Cl.sub.2) to give
compound A18 (247 mg, 0.215 mmol, 67%) as an off-white solid. LC-MS
(ESI): 1144.7 (M+1).
Preparation of Compound A25
[0443] An oven-dried 50 mL single-necked round-bottomed flask
equipped with a teflon-coated magnetic stir bar was charged with
compound A18 (240 mg, 0.209 mmol), dry CH.sub.2Cl.sub.2 (5 mL), to
this clear solution was added piperidine (1.5 mL) and the reaction
mixture was stirred at ambient temperature for 1 h under N.sub.2
atm. LC-MS showed completion of the reaction, all volatiles were
removed under reduced pressure. The crude free amine was purified
by preparative reverse phase-high performance liquid chromatography
using an Ultro 120 (7 .mu.m) C18Q, 150.times.20 mmID column.
Solvent system used Solvent A: water containing 10 mm NH.sub.4OAc;
Solvent B: acetonitrile containing 10 mm NH.sub.4OAc., Gradient
mode from 10% Solvent B to 90% solvent B, over 20 minutes, 10
mL/min), pure fractions were collected and lyophilized to give the
free amine A24 (164 mg, 0.177 mmol, 85%) as a white solid.
[0444] An oven-dried 50 mL single-necked round-bottomed flask
equipped with a teflon-coated magnetic stir bar was charged with
the free amine A24 (164 mg, 0.177 mmol). DBCO succinyl
N-hydroxysuccinimidyl ester (126 mg, 0.313 mmol), anhydrous
N,N-Dimethylformamide (3 mL), and N,N-Diisopropylethylamine (110
.mu.L, 0.627 mmol) were sequentially added. The reaction mixture
was flushed with Argon and stirred at ambient temperature for 3
hours under N.sub.2 atm. LC-MS showed completion of the reaction.
After removal of all volatiles in vacuo, the residue was purified
by reverse phase-high performance liquid chromatography using a
Ultro 120 (7 .mu.m) C18Q, 150.times.20 mmID column Solvent system
used Solvent A: water containing 10 mm NH.sub.4OAc; Solvent B:
acetonitrile containing 10 mm NH.sub.4OAc., Gradient mode from 10%
B Solvent to 90% solvent B, over 20 minutes, 10 mL/min), pure
fractions were collected and lyophilized to give compound A25 (177
mg, 0.146 mmol, 70%) as a white powder. LC-MS (ESI): 1209.6
(M+1).
[0445] An oven-dried 25 mL single-necked round-bottomed flask
equipped with a teflon-coated magnetic stir bar is charged with
compound A25 (177 mg, 0.146 mmoles, 1 equiv.), THF:MeOH:H.sub.2O
(3:1:1) (5 mL). The clear solution was cooled to 0.degree. C. with
an ice bath. Solid LiOH.H.sub.2O (46 mg, 1.022 mmol, 7 eq) was
added and the reaction was allowed to stir at room temperature
under N.sub.2 atm for 7 h, after which LC-MS showed completion of
the reaction, the volatiles were removed in vacuo, and the crude
material was purified by reverse phase-high performance liquid
chromatography using Ultro 120 (7 .mu.m) C18Q, 150.times.20 mmID
column, Solvent system used Solvent A: water containing 10 mm
NH.sub.4OAc; Solvent B: acetonitrile containing 10 mm NH.sub.4OAc.,
Gradient elution mode from 10% Solvent B to 90% solvent B, over 20
minutes, 10 mL/min), pure fractions were collected and lyophilized
to give compound (101a) (138 mg, 0.117 mmol, 80%) as a white
powder. LC-MS (ESI): 1181.5 (M+1).
Example 1i
[0446] Antibodies were expressed in a Xpress CF.TM. reaction using
procedures know to one of skill in the art. (See, for example, Cai
et al. Biotechnol. 2015, 31(3), 823; and Zimmerman et al.
Bioconjugate Chem. 2014, 25, 351.) The cell free extract for this
work were created from an OmpT sensitive RF1 attenuated E. coli
strain engineered to overexpress E. coli DsbC and FkpA as well as
an orthogonal tRNA containing the CUA anti-codon for decoding the
Amber Stop Codon. Extract was treated with 75 .mu.M iodoacetamide
for 45 min at RT (20.degree. C.) and added to a premix containing
all other components, except for IgG heavy and light chain DNA. The
final concentration in the protein synthesis reaction was 30% (v/v)
cell extract, 2 mM para-azidomethylphenylalanine (pAMF) (RSP Amino
Acids), 5 uM engineered pAMF-specific amino-acyl tRNA synthetase
(FRS variant), 2 mM GSSG, 8 mM magnesium glutamate, 10 mM ammonium
glutamate, 130 mM potassium glutamate, 35 mM sodium pyruvate, 1.2
mM AMP, 0.86 mM each of GMP, UMP, and CMP, 2 mM amino acids (except
0.5 mM for Tyrosine and Phenylalanine), 4 mM sodium oxalate, 1 mM
putrescine, 1.5 mM spermidine, 15 mM potassium phosphate, 100 nM T7
RNAP, 1 .mu.g/mL antiCD74 light chain DNA, and 4 .mu.g/mL antiCD74
heavy chain DNA. Site directed mutagenesis was used to introduce an
amber stop codon (TAG) into the nucleotide sequence to encode for
the pAMF non-natural amino acid at positions S7 and F404 (light and
heavy chains respectively, kabat numbering). Cell free reactions
were initiated by addition of plasmid DNA and incubated at
30.degree. C. for 16 h in 100.times.10 mm petri dishes containing
10 mL.
[0447] The cell free reactions were clarified by centrifugation at
10,000 rpm's for 30 minutes. The clarified supernatant was applied
to Protein A MabSelect SuRe (GE Healthcare) with standard wash and
low pH elution. Impurities such as aggregates were removed via
preparative SEC (Sepax SRT-10C) equilibrated in 50 mM sodium
phosphate, 200 mM arginine, pH 6.5. Final formulation of the sample
was done in Dulbecco's Phosphate Buffered Saline
(1.times.DPBS).
[0448] Purified IgGs containing pAMF were conjugated to a cytotoxic
test compound using copper-free click chemistry with strained
cyclooctyne reagent (SpAAC, strain-promoted alkyne azide
cycloadition). In brief, test compounds were dissolved in DMSO to a
final concentration of 5 mM. Each compound was added to 1 mg/mL
purified protein in PBS at a drug-linker to antibody molar ratio of
12 to 1. The reaction mixture was incubated at RT (20.degree. C.)
for 17 hours. Excess free drug was removed by Zeba plate (Thermo
Scientific) equilibrated in PBS. DAR analysis was done by MALDI-TOF
(Bruker AutoFlex Speed). The conjugated protein was reduced for 10
min at 37.degree. C. with 10 mM TCEP in water and diluted to a
final concentration of 50 .mu.g/mL in 30% acetonitrile, 0.1%
trifluoroacetic acid. Samples were combined 1:1 with S-DHB MALDI
matrix (50 mg/mL in 50% acetonitrile, 0.1% trifluoroacetic acid)
and 1 .mu.L was applied to the MALDI target and dried under vacuum.
Each MALDI spectra was accumulated for 5000 shots at full laser
power in linear mode and the final DAR analysis was calculated by
comparing the relative peak intensity for conjugated and
unconjugated species.
Example 1j
[0449] Conjugates of Compound 1 with trastuzumab were prepared as
described below.
[0450] Compound 101 or 101a was dissolved in DMSO to a
concentration of 5 mM. The solution was added to purified C225 HC
C-term antibody in PBS buffer to a final compound concentration of
200 .mu.M and a final antibody concentration of 3 mg/mL (20 .mu.M)
for a 10:1 molar ratio of compound:antibody. The mixture was
incubated at ambient temperature (25.degree. C.) for 16 h. The
excess compound was removed using zeba plates (Thermo Scientific)
equilibrated in 1.times.PBS.
[0451] This procedure was used to conjugate compounds 101 and 101a
to trastuzumab HC at F404 and to trastuzumab LC at S7.
[0452] To make trastuzumab containing a reactive azide group for
conjugation, DNA encoding the molecule's, heavy and light chains
were cloned into pUG expression vector. A TAG codon was inserted at
the indicated positions by overlapping PCR. Stop codon TAA was used
to terminate translation.
[0453] To express protein, cell-free extracts were thawed to room
temperature and incubated with 50 .mu.M iodoacetamide for 30 min.
Cell-free reactions were run at 30 C for up to 16 h containing 30%
(v/v) iodoacetamide-treated extract with 8 mM magnesium glutamate,
10 mM ammonium glutamate, 130 mM potassium glutamate, 35 mM sodium
pyruvate, 1.2 mM AMP, 0.86 mM each of GMP, UMP, and CMP, 2 mM amino
acids for all 18 amino acids except tyrosine and phenylalanine
which were added at 0.5 mM, 4 mM sodium oxalate, 1 mM putrescine,
1.5 mM spermidine, 15 mM potassium phosphate, 100 nM T7 RNAP, 2 mM
oxidized (GSSG) glutathione, 2 mM pAzidoMethylPhenylanine (pAMF),
2.5 .mu.M amber suppressor tRNA synthetase. The concentrations of
heavy chain TAG variant plasmid and wild type light chain plasmid
were 7.5 ug/mL and 2.5 ug/mL respectively.
[0454] The antibodies containing non natural amino acids were
purified by MabSelect and polished by Capto adhere and stored in
PBS buffer before use.
[0455] The anti-CD74 cell free reactions were clarified by
centrifugation at 10,000 rpm's for 30 minutes. The clarified
supernatant was applied to Protein A MabSelect SuRe (GE Healthcare)
with standard wash and low pH elution. Impurities such as
aggregates were removed via preparative SEC (Sepax SRT-10C)
equilibrated in 50 mM sodium phosphate, 200 mM arginine, pH 6.5.
Final formulation of the sample was done in Dulbecco's Phosphate
Buffered Saline (1.times.DPBS).
Example 1k
Production of Anti-CD74 Antibodies with Non-Natural Amino Acids
[0456] Antibodies were expressed in an Xpress CF.TM. reaction as
described previously with the following modifications. The cell
free extract for this work were created from an OmpT sensitive RF1
attenuated E. coli strain engineered to overexpress E. coli DsbC
and FkpA as well as an orthogonal tRNA containing the CUA
anti-codon for decoding the Amber Stop Codon. Extract was treated
with 75 .mu.M iodoacetamide for 45 min at RT (20.degree. C.) and
added to a premix containing all other components, except for IgG
heavy and light chain DNA. The final concentration in the protein
synthesis reaction was 30% (v/v) cell extract, 2 mM
para-azidomethylphenylalanine (pAMF) (RSP Amino Acids), 5 uM
engineered pAMF-specific amino-acyl tRNA synthetase (FRS variant),
2 mM GSSG, 8 mM magnesium glutamate, 10 mM ammonium glutamate, 130
mM potassium glutamate, 35 mM sodium pyruvate, 1.2 mM AMP, 0.86 mM
each of GMP, UMP, and CMP, 2 mM amino acids (except 0.5 mM for
Tyrosine and Phenylalanine), 4 mM sodium oxalate, 1 mM putrescine,
1.5 mM spermidine, 15 mM potassium phosphate, 100 nM T7 RNAP, 1
.mu.g/mL antiCD74 light chain DNA, and 4 .mu.g/mL antiCD74 heavy
chain DNA. Site directed mutagenesis was used to introduce an amber
stop codon (TAG) into the nucleotide sequence to encode for the
pAMF non-natural amino acid at positions S7 and F404 (light and
heavy chains respectively, kabat numbering). Cell free reactions
were initiated by addition of plasmid DNA and incubated at
30.degree. C. for 16 h in 100.times.10 mm petri dishes containing
10 mL.
[0457] The anti-CD74 cell free reactions were clarified by
centrifugation at 10,000 rpm's for 30 minutes. The clarified
supernatant was applied to Protein A MabSelect SuRe (GE Healthcare)
with standard wash and low pH elution. Impurities such as
aggregates were removed via preparative SEC (Sepax SRT-10C)
equilibrated in 50 mM sodium phosphate, 200 mM arginine, pH 6.5.
Final formulation of the sample was done in Dulbecco's Phosphate
Buffered Saline (1.times.DPBS).
[0458] Antibodies prepared having non-natural amino acids at
positions heavy chain residues 404, 241, and 222, according to the
EU number scheme, and at light chain residue 7, according to the
Kabat or Chothia numbering scheme. One antibody comprised residue
(56), above, at position 404, and four antibodies comprised residue
(30), above, at each of positions 404, 241, 222 (heavy chain) and 7
(light chain). Each antibody was expressed at a total yield of at
least 400 mg/L as shown in FIG. 2A, and intact IgG were detected by
SDS-PAGE as shown in FIG. 2B.
Production of Antibody-PEG.sub.4-Maytansine Conjugate
[0459] Purified anti-CD74 IgG containing modified amino acid
residue 30 (i.e. para-azido-methyl-L-phenylalanine, or pAMF) at EU
position 404 in its heavy chains was obtained according to Example
2. The anti-CD74 IgG was conjugated to a hemiasterlin, using a
strained cyclooctyne reagent to yield Conjugate A.
[0460] In brief, DBCO-val-cit-pAB-hemiasterlin according to the
following:
##STR00215##
was dissolved in DMSO to a final concentration of 5 mM. The
compound was added to 1 mg/mL purified protein in PBS at a drug to
antibody molar ratio of 12 to 1. The reaction mixture was incubated
at RT (20.degree. C.) for 17 hours. Excess free drug was removed by
Zeba plate (Thermo Scientific) equilibrated in PBS.
[0461] DAR analysis was done by MALDI-TOF (Bruker AutoFlex Speed).
The conjugated protein was reduced for 10 min at 37.degree. C. with
10 mM TCEP in water and diluted to a final concentration of 50
.mu.g/mL in 30% acetonitrile, 0.1% trifluoroacetic acid. Samples
were combined 1:1 with S-DHB MALDI matrix (50 mg/mL in 50%
acetonitrile, 0.1% trifluoroacetic acid) and 1 uL was applied to
the MALDI target and dried under vacuum. Each MALDI spectra was
accumulated for 5000 shots at full laser power in linear mode and
the final DAR analysis was calculated by comparing the relative
peak heights for conjugated and unconjugated masses for both the
heavy and light chains.
[0462] By peak intensity, MALDI-TOF showed a drug to antibody ratio
(DAR) of 1.88. Conjugate A, as two regioisomers:
##STR00216##
Example 2a
Tumor Cell Line Assay
[0463] Summary
[0464] The two diastereomers of Compound 1, [S,S,S] and [R,S,S]
were assayed against breast cancer cell lines expressing Her2, CD74
expressing and non-expressing cell lines, and CD30 expressing and
non-expressing cell lines. Her2 expressing cell lines included
SKBR3, MDA-MB-453, and MDA-MB-468 which are respectively high-,
medium-, and low-Her2 expressing. CD74 expressing and
non-expressing cell lines included SU-DHL6 and OPM2, which are
respectively CD74 expressing and non-expressing. CD30 expressing
and non-expressing cell lines included L540 and Raji cells, which
are respectively CD30 expressing and non-expressing.
[0465] [S,S,S] Compound 1 was found to be 20-fold more potent
(average IC.sub.50 ca 1 nM) against a panel of these tumor cell
lines when compared to [R,S,S] Compound 1.
[0466] Methods
[0467] Cytotoxicity effects of test compounds were evaluated with a
cell proliferation assay. Adherent cancer cell lines (SKBR3,
MDA-MB435, MDA-MB-468, HCT116, HT29, Skcol, and MDA-MB-453) were
obtained from ATCC and maintained in high glucose DMEM/F12 (50/50)
medium (Cellgro-Mediatech; Manassas, Va.) supplemented with 10%
heat-inactivated fetal bovine serum (Hyclone; Thermo Scientific;
Waltham, Mass.), 2 mM glutamax (Invitrogen; Carlsbad, Calif.) and
1.times. Pencillin/streptomycin (Cellgro-Mediatech; Manassas, Va.).
Suspension cell lines (SU-DHL-6 and OPM-2) were obtained from ATCC
and maintained in high glucose RPMI medium (Cellgro-Mediatech;
Manassas, Va.) supplemented with 20% heat-inactivated fetal bovine
serum (Hyclone; Thermo Scientific; Waltham, Mass.), 2 mM glutamax
(Invitrogen; Carlsbad, Calif.) and 1.times. Pencillin/streptomycin
(Cellgro-Mediatech; Manassas, Va.).
[0468] For adherent cells, a total of 1000 cells in a volume of 40
.mu.L were seeded in a 96-well half area flat bottom white
polystyrene plate the day before the assay. For suspension cells, a
total of 20000 cells in a volume of 40 .mu.I, were seeded in a
96-well half area flat bottom white polystyrene plate on the day of
assay.
[0469] Testing compounds were formulated at 2.times. concentration
in culture medium and filtered through MultiScreen HTS 96-Well
Filter Plates (Millipore; Billerica, Mass.). Filter sterilized
compounds were serial diluted in culture medium and 40 .mu.L of the
compounds were added into treatment wells. For adherent cells,
plates were cultured at 37.degree. C. in a CO.sub.2 incubator for
96 hours. For suspension cells, the incubation time was 72 hours.
For cell viability measurements, 80 .mu.L of Cell Titer-Glo.RTM.
reagent (Promega Corp.; Madison, Wis.) was added into each well,
and plates processed as per product instructions.
[0470] Relative luminescence was measured on an ENVISION.RTM. plate
reader (Perkin-Elmer; Waltham, Mass.). Relative luminescence
readings were converted to percent viability using untreated cells
as controls. Data was fitted with non-linear regression analysis,
using log(inhibitor) vs. response, variable slope, 4 parameter fit
equation using GraphPad Prism (GraphPad v 5.00, Software; San
Diego, Calif.). Data was expressed as percent relative cell
viability vs. dose of compounds in nM. Cell killing IC50 calculated
by Prism was used to evaluate the potency of each compound on each
cell line.
[0471] Results
[0472] For all the cancer cell lines tested, [S,S,S] Compound 1
(cell killing IC50 ranged from 0.74 nM to 9.18 nM) was found to be
10 to 20-fold more potent when compared to [R,S,S] Compound 1 (cell
killing IC50 ranged from 12.21 nM to 91.53 nM).
[0473] Results for trastuzumab conjugates are provided in FIG. 1
and Table 1. Results for hemiasterlin derivatives [S,S,S] and
[R,S,S] Compound 1, are provided in FIGS. 2a-c and Table 2.
TABLE-US-00001 TABLE 1 Tumor Cell Line Assay with Trastuzumab
Conjugates IC50 Span Test Conjugate (nM) (%) Trastuzumab 10.0 66
Trastuzumab F404 [S,S,S] 0.2 91 Compound 1, Conjugate Trastuzumab
heavy chain F404 0.4 87 racemic [R/S,S,S] Compound 1 conjugate
Trastuzumab light chain S7 0.4 86 racemic [R/S,S,S] Compound 1
conjugate Trastuzumab heavy chain F404 0.1 88 MMAF Conjugate
TABLE-US-00002 TABLE 2 Tumor Cell Line Assay with Hemiasterlin
Derivatives Cell Killing IC50 (nM) [S,S,S] Origin Cell Line Markers
Compound 1 Breast SKBR3 Her2 High 1.42 19.97 MDA-MB-453 Her2 Medium
1.80 27.63 MDA-MB-468 Her2 Low 0.74 16.71 Colon HCT116 EpCAM High
3.18 62.18 HT29 EpCAM Medium 9.18 91.53 Skco1 EpCAM Low 3.59 46.88
Melanoma MDA-MB-435 -- 2.29 33.74 Multiple OPM-2 CD74 Negative 2.42
14.35 Myeloma Lymphoma SU-DHL-6 CD74 Positive 1.42 12.21
Example 2b
Tumor Cell Line Assay
[0474] Cytotoxicity effects of test compounds on target positive
and target negative cells were measured with a cell proliferation
assay. Tumor cell lines were obtained from American Type Culture
Collection (ATCC) and maintained in Ham's F-12: high glucose DMEM
(50:50) glucose medium supplemented with 10% heat-inactivated fetal
bovine serum, 1% Penicillin/Streptomcin and 2 mmol/L L-glutamax.
Target positive and negative cells (a total of 625 cells per well)
were seeded in a volume of 25 .mu.L in a 384-well flat bottom white
polystyrene plate. The cells were allowed to adhere overnight at
37.degree. C. in a CO.sub.2 incubator. ADC variants were formulated
at 2.times. concentration in DMEM/F12 medium and filtered through
MultiScreen HTS 96-Well Filter Plates. Filter sterilized ADCs were
serial diluted (1:3) and 25 .mu.I, of diluted samples were added
into each treatment wells. Plates were then cultured at 37.degree.
C. in a CO.sub.2 incubator for 120 hrs. For cell viability
measurement, 30 .mu.L of Cell Titer-Glo.RTM. reagent (Promega Corp)
was added into each well, and plates processed as per product
instructions. Relative luminescence was measured on an
ENVISION.RTM. plate reader (Perkin-Elmer; Waltham, Mass.). Relative
luminescence readings were converted to % viability using untreated
cells as controls. Data was fitted with non-linear regression
analysis, using log(inhibitor) vs. response, variable slope, 4
parameter fit equation using GraphPad Prism (GraphPad v 5.00,
Software; San Diego, Calif.). Data was expressed as % relative cell
viability vs. dose of ADC in nM.
Results
TABLE-US-00003 [0475] TABLE 3 Tumor Cell Line Assay with
Hemiasterlin Derivatives Target Positive Cell Target Negative Cell
Compound IC50 (nM) Span (%) IC50 (nM) Span (%) 1a 2.2 =90 6.8 =88
101a 222 93 2011 110 110a 26 93 337 90 109a 661 92 2440 95 111a 65
92 717 91
TABLE-US-00004 TABLE 4 Tumor Cell Line Assay with Antibody
Conjugates Target Positive Cell Target Negative Cell Conjugate IC50
(nM) Span (%) IC50 (nM) Span (%) Antibody 0.016 74 IA IA
HC-Y180/F404-110a Antibody 0.039 77 IA IA HC-Y180/F404-109a
Antibody IA IA IA IA HC-Y180/F404-111a Antibody 0.11 73 IA IA
HC-Y180/F404-101a IA means not active as tested.
Example 2c
Cell Binding and Cell Killing
[0476] Conjugate A was evaluated for the ability to bind and kill
cells expressing CD74 by the methods below. Cell lines tested
included B-lymphoma, multiple myeloma, and leukemia cells. Controls
included unconjugated anti-CD74 antibody.
Cell Binding Assay
[0477] Cell lines were maintained in RPMI, high glucose
(Cellgro-Mediatech; Manassas, Va.) supplemented with 20%
heat-inactivated fetal bovine serum (Hyclone; Thermo Scientific;
Waltham, Mass.), 2 mM glutamax (Invitrogen; Carlsbad, Calif.) and
1.times. Pencillin/streptomycin (Cellgro-Mediatech; Manassas, Va.).
Cells were harvested and re-suspended in FACS buffer (DPBS buffer
supplemented with 1% bovine serum albumin). A total of 200,000
cells per well were incubated on ice with serial dilutions of
anti-CD74 lead SP7919 without conjugation for 60 minutes. Cells
were washed twice with ice-cold FACS buffer and incubated with 5
ug/ml Alexa 647 labeled donkey anti-human IgG antibody (Jackson
Immune-Research) on ice for another 60 mins. Unstained cells and
cells stained with secondary antibody alone were used as controls.
Samples were then washed twice using FACS buffer and analyzed using
a BD FACS Canto system. Mean fluorescence intensities were fitted
using non-linear regression analysis with one site specific binding
equation on GraphPad Prism. Data was expressed as geometric mean
fluorescent intensity vs. antibody concentration in nM.
Cell Killing Assay
[0478] Cytotoxicity effects of the free drug linkers and conjugates
were measured with a cell proliferation assay. A total of 12500
cells in a volume of 25 .mu.l were seeded in a 384-well flat bottom
white polystyrene plate on the day of assay. Free drug-linkers and
conjugates were formulated at 2.times. starting concentration (1000
nM for free drug linkers and 100 nM for ADCs) in RPMI medium and
filtered through MultiScreen FITS 96-Well Filter Plates
(Millipore). Filter sterilized conjugated leads were serial diluted
(1:3) under sterile conditions and added into treatment wells.
Plates were cultured at 37.degree. C. in a CO.sub.2 incubator for
72 hrs. For cell viability measurement, 30 .mu.l of Cell
Titer-Glo.RTM. reagent (Promega Corp.) was added into each well,
and plates processed as per product instructions. Relative
luminescence was measured on an ENVISION.RTM. plate reader
(Perkin-Elmer; Waltham, Mass.). Relative luminescence readings were
converted to % viability using untreated cells as controls. Data
was fitted with non-linear regression analysis, using
log(inhibitor) vs. response, variable slope, 4 parameter fit
equation using GraphPad Prism. Data was expressed as % relative
cell viability vs. dose of free drug-linker or conjugate in nM.
[0479] Conjugate A was evaluated for the ability to bind and kill
cells expressing CD74. Cell lines tested included B-lymphoma,
multiple myeloma, and leukemia cells. Controls included
unconjugated anti-CD74 antibody. The results are summarized in the
following table:
TABLE-US-00005 Cell Binding Cell Killing Activity anti-CD74
Conjugate A Kd IC50 Span Disease Cell Lines Tested Bmax (nM) (nM)
(%) B-Lymphoma RPMI-6666 (HL) 3879 2.3 0.9 84 SU-DHL-6 (NHL) 1565
2.0 0.3 97 Multiple ARD (MM) 190 2.6 26.0 74 Myeloma ARP (MM) 7.6
87 RPMI-8226 (MM) 119 3.6 17.0 43 OPM-2 (MM) NB NB NK NK Leukemia
BDCM (AML) 3059 4.5 1.1 89 SUP-B15 (ALL) 680 3.5 2.8 68 JVM-13
(CLL) 447 2.5 0.9 54 K562 (CML) NB NB NK NK
[0480] While the claimed subject matter has been described in terms
of various embodiments, the skilled artisan will appreciate that
various modifications, substitutions, omissions, and changes may be
made without departing from the spirit thereof. Accordingly, it is
intended that the scope of the claimed subject matter is limited
solely by the scope of the following claims, including equivalents
thereof.
* * * * *