U.S. patent application number 13/274412 was filed with the patent office on 2012-05-24 for carboline derivatives useful in the treatment of cancer and other diseases.
This patent application is currently assigned to PTC Therapeutics, Inc.. Invention is credited to Liangxian Cao, Thomas Davis, Samit Hirawat, Langdon Miller, Marla L. Weetall.
Application Number | 20120129841 13/274412 |
Document ID | / |
Family ID | 39582067 |
Filed Date | 2012-05-24 |
United States Patent
Application |
20120129841 |
Kind Code |
A1 |
Cao; Liangxian ; et
al. |
May 24, 2012 |
CARBOLINE DERIVATIVES USEFUL IN THE TREATMENT OF CANCER AND OTHER
DISEASES
Abstract
In accordance with the present invention, compounds that inhibit
the expression of VEGF post-transcriptionally have been identified,
and compositions, and methods for the administration and use of
those compounds. provided. In one aspect of the invention,
compounds useful in the inhibition of VEGF production, in the
treatment of solid tumor cancer, and in reducing serum, plasma,
and/or tumor VEGF levels, are provided. In another aspect of the
invention, methods are provided for the inhibition of VEGF
production, the treatment of cancer, and the reduction of plasma
and/or tumor VEGF levels, using the compounds of the invention.
Inventors: |
Cao; Liangxian; (Parlin,
NJ) ; Hirawat; Samit; (South Plainfield, NJ) ;
Miller; Langdon; (Lebanon, NJ) ; Davis; Thomas;
(South Orange, NJ) ; Weetall; Marla L.;
(Morristown, NJ) |
Assignee: |
PTC Therapeutics, Inc.
|
Family ID: |
39582067 |
Appl. No.: |
13/274412 |
Filed: |
October 17, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11735069 |
Apr 13, 2007 |
8076352 |
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13274412 |
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11107783 |
Apr 18, 2005 |
7767689 |
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11735069 |
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11079420 |
Mar 15, 2005 |
7601840 |
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11107783 |
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60552725 |
Mar 15, 2004 |
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Current U.S.
Class: |
514/218 ;
514/228.2; 514/232.8; 514/253.03; 514/255.05; 514/275; 514/292 |
Current CPC
Class: |
A61K 31/5377 20130101;
A61K 31/444 20130101; A61P 27/02 20180101; A61K 31/437 20130101;
A61P 35/00 20180101; A61K 31/4353 20130101 |
Class at
Publication: |
514/218 ;
514/292; 514/275; 514/253.03; 514/232.8; 514/228.2; 514/255.05 |
International
Class: |
A61K 31/551 20060101
A61K031/551; A61K 31/506 20060101 A61K031/506; A61K 31/497 20060101
A61K031/497; A61K 31/5377 20060101 A61K031/5377; A61K 31/541
20060101 A61K031/541; A61P 35/00 20060101 A61P035/00; A61K 31/437
20060101 A61K031/437; A61K 31/496 20060101 A61K031/496 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 15, 2005 |
US |
PCT/US2005/008481 |
Apr 17, 2006 |
US |
PCT/US2006/014547 |
Claims
1.-18. (canceled)
19. A method for inhibiting pathologically induced VEGF expression
comprising administering a therapeutically effective amount of a
compound of Formula (I), (II), or (III): ##STR00385## or a
pharmaceutically acceptable salt, racemate or stereoisomer of said
compound, to a subject in need thereof; wherein X is hydrogen; a
C.sub.1 to C.sub.6 alkyl, optionally substituted with one or more
halogens; a hydroxyl group; a halogen; a C.sub.1 to C.sub.5 alkoxy,
optionally substituted with a C.sub.6 to C.sub.10 aryl group; A is
CH or N; B is CH or N, with the proviso that at least one of A or B
is N, and that when A is N, B is CH; R.sub.1 is a hydroxyl group; a
C.sub.1 to C.sub.8 alkyl group, optionally substituted with an
alkylthio group, a 5 to 10 membered heteroaryl, a C.sub.6 to
C.sub.10 aryl group optionally substituted with at least one
independently selected R.sub.o group; a C.sub.2 to C.sub.8 alkenyl
group; a C.sub.2 to C.sub.8 alkynyl group; a 3 to 12 membered
heterocycle group, wherein the heterocycle group is optionally
substituted with at least one independently selected halogen, oxo,
amino, alkylamino, acetamino, thio, or alkylthio group; a 5 to 12
membered heteroaryl group, wherein the heteroaryl group is
optionally substituted with at least one independently selected
halogen, oxo, amino, alkylamino, acetamino, thio, or alkylthio
group; or a C.sub.6 to C.sub.10 aryl group, optionally substituted
with at least one independently selected R.sub.o group; R.sub.o is
a halogen; a cyano; a nitro; a sulfonyl, wherein the sulfonyl is
optionally substituted with a C.sub.1 to C.sub.6 alkyl or a 3 to 10
membered heterocycle; an amino group, wherein the amino group is
optionally substituted with a C.sub.1 to C.sub.6 alkyl,
--C(O)--R.sub.b, --C(O)O--R.sub.b, a sulfonyl, an alkylsulfonyl, a
3 to 10 membered heterocycle group optionally substituted with a
--C(O)O--R.sub.n; --C(O)--NH--R.sub.b; a 5 to 6 membered
heterocycle; a 5 to 6 membered heteroaryl; a C.sub.1 to C.sub.6
alkyl group, wherein the alkyl group is optionally substituted with
at least one independently selected hydroxyl, halogen, amino, or 3
to 12 membered heterocycle group, wherein the amino group and
heterocycle group are optionally substituted with at least one
independently selected C.sub.1 to C.sub.4 alkyl group, which
C.sub.1 to C.sub.4 alkyl group is optionally substituted with at
least one independently selected C.sub.1 to C.sub.4 alkoxy group,
amino group, alkylamino group, or 5 to 10 membered heterocycle
group; a --C(O)--R.sub.n group; or an --OR.sub.a group; R.sub.a is
hydrogen; C.sub.2 to C.sub.8 alkenyl; a --C(O)O--R.sub.b group; a
--C(O)--NH--R.sub.b; a C.sub.1 to C.sub.8 alkyl, wherein the alkyl
group is optionally substituted with at least one independently
selected hydroxyl, halogen, C.sub.1 to C.sub.4 alkoxy, amino,
alkylamino, acetamide, --C(O)--R.sub.b, --C(O)O--R.sub.b, C.sub.6
to C.sub.10 aryl, 3 to 12 membered heterocycle, or 5 to 12
heteroaryl group, further wherein the alkylamino is optionally
substituted with a hydroxyl, a C.sub.1 to C.sub.4 alkoxy, or a 5 to
12 membered heteroaryl optionally substituted with a C.sub.1 to
C.sub.4 alkyl, further wherein the acetamide is optionally
substituted with a C.sub.1 to C.sub.4 alkoxy, sulfonyl, or
alkylsulfonyl, and further wherein the heterocycle group is
optionally substituted with a C.sub.1 to C.sub.4 alkyl optionally
substituted with a hydroxyl group, --C(O)--R.sub.n,
--C(O)O--R.sub.n, or an oxo group; R.sub.b is hydroxyl; an amino;
an alkylamino, wherein the alkylamino is optionally substituted
with a hydroxyl, an amino, an alkylamino, a C.sub.1 to C.sub.4
alkoxy, a 3 to 12 membered heterocycle optionally substituted with
at least one independently selected C.sub.1 to C.sub.6 alkyl, oxo,
--C(O)O--R.sub.n, or a 5 to 12 membered heteroaryl optionally
substituted with a C.sub.1 to C.sub.4 alkyl; a C.sub.1 to C.sub.4
alkoxy; a C.sub.2 to C.sub.8 alkenyl; a C.sub.2 to C.sub.8 alkynyl;
a C.sub.6 to C.sub.10 aryl, wherein the aryl is optionally
substituted with at least one independently selected halogen or
C.sub.1 to C.sub.4 alkoxy; a 5 to 12 membered heteroaryl; 3 to 12
membered heterocycle group, wherein the heterocycle is optionally
substituted with at least one independently selected acetamide,
--C(O)O--R.sub.n, 5 to 6 membered heterocycle, or C.sub.1 to
C.sub.6 alkyl optionally substituted with a hydroxyl, C.sub.1 to
C.sub.4 alkoxy, amino group, or alkylamino group; or a C.sub.1 to
C.sub.8 alkyl, wherein the alkyl is optionally substituted with at
least one independently selected C.sub.1 to C.sub.4 alkoxy, C.sub.6
to C.sub.10 aryl, amino, or 3 to 12 membered heterocycle group,
wherein the amino and heterocycle groups are optionally substituted
with at least one independently selected C.sub.1 to C.sub.6 alkyl,
oxo, or --C(O)O--R.sub.n group; R.sub.2 is a hydrogen; a hydroxyl;
a 5 to 10 membered heteroaryl group; a C.sub.1 to C.sub.8 alkyl
group, wherein the alkyl group is optionally substituted with a
hydroxyl, a C.sub.1 to C.sub.4 alkoxy, a 3 to 10 membered
heterocycle, a 5 to 10 membered heteroaryl, or C.sub.6 to C.sub.10
aryl group; a --C(O)--R.sub.c group; a --C(O)O--R.sub.d group; a
--C(O)--N(R.sub.dR.sub.d) group; a --C(S)--N(R.sub.dR.sub.d) group;
a --C(S)--O--R.sub.e group; a --S(O.sub.2)--R.sub.e group; a
--C(NR.sub.e)--S--R.sub.e group; or a --C(S)--S--R.sub.f group;
R.sub.c is hydrogen; an amino, wherein the amino is optionally
substituted with at least one independently selected C.sub.1 to
C.sub.6 alkyl or C.sub.6 to C.sub.10 aryl group; a C.sub.6 to
C.sub.10 aryl, wherein the aryl is optionally substituted with at
least one independently selected halogen, haloalkyl, hydroxyl,
C.sub.1 to C.sub.4 alkoxy, or C.sub.1 to C.sub.6 alkyl group;
--C(O)--R.sub.n; a 5 to 6 membered heterocycle, wherein the
heterocycle is optionally substituted with a --C(O)--R.sub.n group;
a 5 to 6 membered heteroaryl; a thiazoleamino group; a C.sub.1 to
C.sub.8 alkyl group, wherein the alkyl group is optionally
substituted with at least one independently selected halogen, a
C.sub.1 to C.sub.4 alkoxy, a phenyloxy, a C.sub.6 to C.sub.10 aryl,
--C(O)--R.sub.n, --O--C(O)--R.sub.n, hydroxyl, or amino group,
optionally substituted with a --C(O)O--R.sub.n group; R.sub.d is
independently hydrogen; a C.sub.2 to C.sub.8 alkenyl group; a
C.sub.2 to C.sub.8 alkynyl group; a C.sub.6 to C.sub.10 aryl group,
wherein the aryl is optionally substituted with at least one
independently selected halogen, nitro, C.sub.1 to C.sub.6 alkyl,
--C(O)O--R.sub.e, or --OR.sub.e; or a C.sub.1 to C.sub.8 alkyl
group, wherein the alkyl group is optionally substituted with at
least one independently selected halogen, C.sub.1 to C.sub.4 alkyl,
C.sub.1 to C.sub.4 alkoxy, phenyloxy, C.sub.6 to C.sub.10 aryl, 5
to 6 membered heteroaryl, --C(O)--R.sub.n, --O--C(O)--R.sub.n, or
hydroxyl group, wherein the C.sub.6 to C.sub.10 aryl group is
optionally substituted with at least one independently selected
halogen or haloalkyl group; R.sub.e is a hydrogen; a C.sub.1 to
C.sub.6 alkyl group, wherein the alkyl group is optionally
substituted with at least one independently selected halogen or
alkoxy group; or a C.sub.6 to C.sub.10 aryl group, wherein the aryl
group is optionally substituted with at least one independently
selected halogen or alkoxy group; R.sub.f is a C.sub.1 to C.sub.6
alkyl group, optionally substituted with at least one independently
selected halogen, hydroxyl, C.sub.1 to C.sub.4 alkoxy, cyano,
C.sub.6 to C.sub.10 aryl, or --C(O)--R.sub.n group, wherein the
alkoxy group may be optionally substituted with at least one
C.sub.1 to C.sub.4 alkoxy group and the aryl group may be
optionally substituted with at least one independently selected
halogen, hydroxyl, C.sub.1 to C.sub.4 alkoxy, cyano, or C.sub.1 to
C.sub.6 alkyl group; R.sub.n is a hydroxyl, C.sub.1 to C.sub.4
alkoxy, amino, or C.sub.1 to C.sub.6 alkyl group; R.sub.3 is
hydrogen or --C(O)--R.sub.g; R.sub.g is a hydroxyl group; an amino
group, wherein the amino is optionally substituted with a C.sub.6
to C.sub.10 cycloalkyl group or a 5 to 10 membered heteroaryl
group; or a 5 to 10 membered heterocycle group, wherein the
heterocycle group is optionally substituted with a-C(O)--R.sub.n
group; and wherein said stereoisomer of said compound has a chiral
carbon at the point of attachment of R.sub.1; with the proviso that
the compound of Formula (I) is other than a compound, wherein
R.sub.1 is phenyl, A is N, B is CH, and R.sub.2 is
--C(O)--O-phenyl, wherein the phenyl is substituted or
unsubstituted; and wherein the administered compound inhibits
pathologically induced VEGF expression as shown by an ELISA in HeLa
cells or in a HT1080 solid tumor grown in a nude mouse, inhibits
HT1080 solid tumor growth in a nude mouse or inhibits angiogenesis
in a HT1080 solid tumor grown in a nude mouse.
20. The method of claim 19, wherein said compound is a compound of
any of Formula (I-d), (I-e), (I-f), (I-g), (I-h), or (I-i):
##STR00386## wherein all other variables are as previously defined,
and wherein said stereoisomer of said compound has a chiral carbon
at the point of attachment of R.sub.1.
21. A method for inhibiting pathologically induced VEGF expression
comprising administering a therapeutically effective amount of a
compound selected from the group consisting of: ##STR00387##
##STR00388## ##STR00389## ##STR00390## ##STR00391## ##STR00392##
##STR00393## ##STR00394## ##STR00395## ##STR00396## ##STR00397##
##STR00398## ##STR00399## ##STR00400## ##STR00401## ##STR00402##
##STR00403## ##STR00404## ##STR00405## ##STR00406## ##STR00407##
##STR00408## ##STR00409## ##STR00410## ##STR00411## ##STR00412##
##STR00413## ##STR00414## ##STR00415## ##STR00416## ##STR00417##
##STR00418## ##STR00419## ##STR00420## ##STR00421## ##STR00422##
##STR00423## ##STR00424## ##STR00425## ##STR00426## ##STR00427##
##STR00428## ##STR00429## ##STR00430## ##STR00431## ##STR00432##
##STR00433## ##STR00434## ##STR00435## ##STR00436## ##STR00437##
##STR00438## ##STR00439## ##STR00440## ##STR00441## ##STR00442##
##STR00443## ##STR00444## ##STR00445## ##STR00446## ##STR00447##
##STR00448## ##STR00449## ##STR00450## ##STR00451## ##STR00452##
##STR00453## ##STR00454## ##STR00455## ##STR00456## ##STR00457##
##STR00458## ##STR00459## ##STR00460## ##STR00461## ##STR00462##
##STR00463## ##STR00464## ##STR00465## ##STR00466## ##STR00467##
##STR00468## ##STR00469## ##STR00470## ##STR00471## ##STR00472##
##STR00473## ##STR00474## ##STR00475## ##STR00476## ##STR00477##
##STR00478## ##STR00479## ##STR00480## ##STR00481## ##STR00482##
##STR00483## ##STR00484## ##STR00485## ##STR00486## ##STR00487##
##STR00488## ##STR00489## ##STR00490## ##STR00491## ##STR00492##
##STR00493## ##STR00494## ##STR00495## ##STR00496## ##STR00497##
##STR00498## ##STR00499## ##STR00500## ##STR00501## ##STR00502##
##STR00503## ##STR00504## ##STR00505## ##STR00506## ##STR00507##
##STR00508## ##STR00509## ##STR00510## ##STR00511## ##STR00512##
##STR00513## ##STR00514## ##STR00515## ##STR00516## ##STR00517##
##STR00518## ##STR00519## ##STR00520## ##STR00521## ##STR00522##
##STR00523## ##STR00524## ##STR00525## ##STR00526## ##STR00527##
##STR00528## ##STR00529## ##STR00530## ##STR00531## ##STR00532##
##STR00533## ##STR00534## ##STR00535## ##STR00536## ##STR00537##
##STR00538## ##STR00539## ##STR00540## ##STR00541## ##STR00542##
##STR00543## ##STR00544## ##STR00545## ##STR00546## ##STR00547##
##STR00548## ##STR00549## ##STR00550## ##STR00551## ##STR00552##
##STR00553## ##STR00554## ##STR00555## ##STR00556## ##STR00557##
##STR00558## ##STR00559## ##STR00560## or a pharmaceutically
acceptable salt, racemate or stereoisomer thereof, wherein said
stereoisomer of said compound has a chiral carbon at the point of
attachment of the phenyl ring directly attached to the tricyclic
core.
22. The method of any one of claims 19 to 21, wherein said
stereoisomer of said compound is an (S) isomer at said chiral
carbon.
23. The method of any one of claims 19 to 21, wherein inhibiting
pathologically induced VEGF expression treats a solid tumor
cancer.
24. The method of any one of claims 19 to 21, wherein the compound
has an EC.sub.50 of less than 50 .mu.M for inhibiting
hypoxia-induced VEGF expression in cultured HeLa cells.
25. The method of claim 19, wherein the compound inhibits
pathologically induced VEGF production in a HT1080 solid tumor
grown in a nude mouse.
26. The method of claim 19, wherein the compound inhibits HT1080
solid tumor growth in a nude mouse.
27. The method of claim 19, wherein the compound inhibits
angiogenesis in a HT1080 solid tumor grown in a nude mouse.
28. The method of claim 19, wherein the compound inhibits
pathologically induced VEGF production as shown by an ELISA in HeLa
cells.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. application Ser.
No. 11/735,069, filed Apr. 13, 2007, which is a
continuation-in-part of U.S. application Ser. No. 11/107,783, filed
Apr. 18, 2005, which is a continuation-in-part of U.S. application
Ser. No. 11/079,420, filed Mar. 15, 2005, and having the title
"Carboline Derivatives Useful in the Inhibition of Angiogenesis,"
which claims the benefit of and priority to U.S. Provisional
Application No. 60/552,725, filed Mar. 15, 2004, which applications
are incorporated herein by reference; this application also claims
priority to International Application No. PCT/US2005/008481, filed
Mar. 15, 2005, and PCT/US2006/014547, filed on Apr. 17, 2006, which
application is incorporated herein by reference
FIELD OF THE INVENTION
[0002] The present invention relates to compounds for inhibiting
the expression of VEGF post-transcriptionally or for inhibiting
angiogenesis, and compositions, methods, and kits for the
administration or use of such compounds.
BACKGROUND OF THE INVENTION
[0003] Aberrant angiogenesis plays a critical role in the
pathogenesis of numerous diseases, including malignant, ischemic,
inflammatory and immune disorders (Carmeliet, Nat. Med.,
9(6):653-60 (2003), Ferrara, Semin. Oncol., 29(6 Suppl 16):10-4
(2002)). The best-known of these disorders are cancer, exudative
macular degeneration and diabetic retinopathy (DR), the last two of
which are leading cause of blindness in the United States (Witmer
et al., Prog. Retin Eye Res., 22(1):1-29 (2003), Clark et al., Nat.
Rev. Drug Discovery, 2:448-459 (2003)). During the last decade our
understanding of the molecular basis of angiogenesis has grown
considerably. Numerous cytokines and growth factors that stimulate
angiogenesis, such as VEGF, FGF-2, PDGF, IGF-1, TGF, TNF-.alpha.,
G-CSF have been identified (Ferrara et al., Nat. Med.,
5(12):1359-64 (1999), Kerbel et al., Nat. Rev. Cancer, 2(10):727-39
(2002), Rofstad et al., Cancer Res., 60(17):4932-8 (2000)). Among
these growth factors, Vascular Endothelial Growth Factor (VEGF)
plays a central role in angiogenesis (Ferrara, Semin. Oncol., 29(6
Suppl 16):10-4 (2002)).
[0004] VEGF, also known as VEGF-A, was initially identified for its
ability to induce vascular permeability and to promote vascular
endothelial cell proliferation (Leung et al., Science,
246:1306-1309 (1989), Plouet et al., EMBO J., 8:3801-3806 (1989),
Connolly et al., J. Biol. Chem., 264:20017-20024 (1989)). VEGF is
encoded by a single gene that gives rise to four isoforms by
alternative splicing (Tischer et al., J. Biol. Chem.,
266:11947-11954 (1991)). All four isoforms share the same unusually
long and GC rich 5'-UTR, as well as a 3'-UTR that includes multiple
RNA stability determinants. The receptors VEGFR-2 (also known as
KDR or Flk-1) and VEGFR-1 (previously known as Flt1) recognize the
dimeric form of VEGF (Ortega et al., Front. Biosci., 4:D141-52
(1999), Sato et al., Annals of New York Academy of Science,
902:201-207, (2000)). The highly specific VEGFR-2 receptor is
expressed on endothelial cells. VEGF binding to the VEGFR-2
receptor activates the receptor's tyrosine kinase activity, leading
to endothelial cell proliferation, differentiation and primitive
vessel formation (Shalaby et al., Nature, 376:62-66, (1995)).
VEGFR-1 inhibits endothelial cell growth either by acting as a
decoy or by suppressing signaling pathways through VEGFR-2 (Fong et
al., Nature, 376:66-70 (1995)).
[0005] Over 30 years ago, it was proposed that inhibition of tumor
angiogenesis could be an effective approach for the treatment of
cancer (Folkman, N. Engl. J. Med., 285(21):1182-6 (1971)). VEGF and
its receptor have been demonstrated to have a central role in tumor
angiogenesis, especially in the early stages of tumor growth
(Hanahan et al., Cell, 86:353-364, 1996)). Indeed, increased levels
of VEGF expression have been correlated with microvessel density in
primary tumor tissues (Gasparini et al., J. Natl. Cancer Inst.,
89:139-147 (1997)). Moreover, increased levels of the VEGF
transcript are found in virtually all of the common solid tumors
(Ferrara et al., Endocr. Rev., 18:4-25, 1997)). In general,
tumor-bearing patients have higher levels of VEGF compared to those
in tumor-free individuals, and high VEGF levels in serum/plasma are
associated with poor prognosis (Dirix et al., Br. J. Cancer,
76:238-243 (1997)). Consistent with the role of VEGF in tumor
angiogenesis, VEGF null embryonic stem cells showed a dramatically
reduced ability to form tumors in nude mice (Carmeliet et al.,
Nature, 380:435-439 (1996)). Direct evidence for the involvement of
VEGF in tumorgenesis was demonstrated by using specific antibodies
against VEGF in human xenografts implanted in nude mice (Kim et
al., Nature, 362:841-844 (1993), Hichlin et al., Drug Discovery
Today, 6:517-528 (2001)). In these studies, the inhibition of tumor
growth correlated positively with decreased vessel formation in the
antibody-treated tumors. Subsequent experiments using the soluble
receptors substantiated the importance of VEGF activity in tumor
growth (Lin et al., Cell Growth Differ., 9(1):49-58 (1998)), and
demonstrated that inactivation of VEGF by specific antibody
treatment directly resulted in a nearly complete suppression of
tumor-associated neovascularization (Borgstrom et al., Prostate,
35:1-10 (1998), Yuan et al. Proc. Natl. Acad. Sci. USA,
93:14765-14770 (1996)).
[0006] In exudative macular degeneration and diabetic retinopathy,
pre-clinical experiments and clinical trials have demonstrated that
over production of VEGF is critical for aberrant retinal or
choroidal neovascularization (reviewed in Witmer et al., Prog.
Retin Eye Res., 22(1):1-29 (2003)). Evidence has been obtained that
intra-ocular VEGF levels are strongly correlated with active
retinal/choroidal neovascularization (CNV) in patients with
diseases such as diabetic retinopathy and wet form macular
degeneration (Funatsu et al., Am. J. Ophthalmol., 133(4):537-43
(2002), Lip et al., Ophthalmology, 108(4):705-10 (2001)). In
addition, studies using transgenic mice demonstrated that
overexpression of VEGF in retinal pigment epithelial cells or
photoreceptor cells results in choroidal or retinal
neovasucularization (Schwesinger et al., Am. J. Pathol.,
158(3):1161-72 (2001), Ohno-Matsui et al., Am. J. Pathol.,
160(2):711-9 (2002)). In recent studies neutralizing antibodies,
soluble receptor, receptor antagonists, or siRNA have proven
efficacious in reducing VEGF-mediated blood vessel formation in
animal models and in the clinic. (Eyetech Study Group, 22(2):143-52
(2002), Krzystolik et al., Arch. Ophthalmol., 120(3):338-46 (2002),
Shen et al., Lab Invest., 82(2):167-82 (2002), Honda et al., Gene
Ther., 7(11):978-85 (2000), Saishin et al., J. Cell Physiol.,
195(2):241-8 (2003)).
[0007] VEGF expression is regulated by a number of factors and
agents including cytokines, growth factors, steroid hormones and
chemicals, and mutations that modulate the activity of oncogenes
such as ras or the tumor suppressor gene VHL (Maxwell et al.,
Nature, 399:271-275 (1999), Rak et al., Cancer Res., 60:490-498
(2000)). Nevertheless, hypoxia is the most significant physiologic
signal for regulating VEGF expression. Hypoxia results in enhanced
VEGF expression by increasing both the transcription rate and
stability of the VEGF transcript (Ikeda et al., J. Biol. Chem.
270:19761-19766 (1995), Stein et al., Mol. Cell. Biol. 18:3112-3119
(1998), Levy et al., J. Biol. Chem. 271:2746-2753 (1996)).
Hypoxia-inducible factor 1.alpha. (HIF-1.alpha.) is a transcription
factor that increases VEGF gene expression in cells undergoing
hypoxia by binding to the hypoxia response element (HRE) located in
the VEGF promoter (Liu et al., Circ. Res., 77:638-643 (1995),
Semenza, Annu. Rev. Cell. Dev. Biol., 5:551-578 (1999)). Both the
stability and translation efficiency of the VEGF transcript is
influenced by sequences in the 5'- and 3'-untranslated regions
(UTRs). The 5'-UTR contains an internal ribosomal entry site (IRES)
and mediates cap-independent translation initiation while the
3'-UTR harbors multiple AU-rich (AUR) stability determinants that
have been previously shown to regulate turnover of VEGF mRNA. In
addition, the translation initiation of the VEGF transcript is
uniquely regulated. Under hypoxic conditions, translation of most
cellular transcripts mediated by cap-dependent translation
initiation process is greatly impaired (Kraggerud et al.,
Anticancer Res., 15:683-686 (1995)). Initiation of translation of
the VEGF mRNA, however, is unique under hypoxic conditions in that
it is mediated via an internal ribosome entry site (IRES) within
the VEGF 5'UTR (Stein et al., Mol. Cell. Biol. 18:3112-3119 (1998),
Levy et al., J. Biol. Chem. 271:2746-2753 (1996), Huez et al., Mol.
Cell. Biol., 18:6178-6190 (1998), Akiri et al., Oncogene,
17:227-236 (1998)). Thus, this form of post-transciptional
regulation permits cells to produce large amounts of VEGF protein
to support either further tumor growth or aberrant
neovascularization in ocular diseases under hypoxic conditions. The
stability of VEGF mRNA is also greatly enhanced as a consequence of
the binding of factors to elements in the 3'-UTR (Goldberg et al.,
J. Biol. Cell. J. Biol. Chem., 277(16):13635-40 (2002)).
[0008] There is a large body of experimental evidence indicating
that tumor growth can be inhibited by the prevention of
neovascularization (Lin et al., Cell Growth Differ., 9(1):49-58
(1998), Zhu et al., Invest. New Drugs, 17:195-212 (1999)). Tumor
vessels are generally immature and constantly undergo remodeling
(Carmeliet, Nat. Med., 9(6):653-60 (2003), Carmeliet et al.,
Nature, 407:249-257 (2000)). Active and aberrant angiogenesis is
the result of a disruption in the normal balance of proangiogenic
and anti-angiogenic factors, including various cytokines, growth
factors and steroid hormones. Despite the complexity of the
regulation of tumor angiogenesis, accumulated evidence indicates
that targeting a single proangiogenic factor might be sufficient to
inhibit tumor angiogenesis and suppress tumor growth (Kim et al.,
Nature, 362:841-844 (1993), Millauer et al., Nature, 367:576-579
(1994), Fong et al., Cancer Res., 59:99-106 (1999)). Among many
angiogenesis targets, VEGF and its receptor are most attractive
(Carmeliet, Nat. Med., 9(6):653-60 (2003), Ortega et al., Front.
Biosci., 4:D141-52 (1999)). As noted above, treatment with a
monoclonal antibody specifically targeting VEGF inhibited the
growth of tumors in human xenografts implanted in nude mice.
Subsequently, various approaches designed to inactivate VEGF
signaling have been tested in tumor models and have proven to be
highly effective in a broad range of tumor cell lines including
carcinomas, sarcomas and gliomas (Ferrara et al., Endocr. Rev.,
18:4-25, 1997), Kim et al., Nature, 362:841-844 (1993), Millauer et
al., Nature, 367:576-579 (1994), Fong et al., Cancer Res.,
59:99-106 (1999), Geng et al., Cancer Res., 61:2413-2419 (2001)).
In addition, inhibition of VEGF by anti-VEGF antibody did not
result in significant side effects in fully developed rodents or
primates (Ryan et al, Toxicol. Pathol., 27:78-86 (1999), Ferrara et
al., Nat. Med., 4:336-340 (1998)). Taken together, these results
indicate that VEGF is a valid target for the development of tumor
therapy. Indeed, a number of clinical trials are underway using
VEGF inhibitors (Matter, Drug Discovery Today, 6:1005-1024 (2001),
Hichlin et al., Drug Discovery Today, 6:517-528 (2001)).
[0009] Although several pro-angiogenic factors are implicated in
the pathology of exudative age-related macular degeneration, VEGF
appears to be the most critical in the pathogenesis and development
of this disease (Witmer et al., Prog. Retin Eye Res., 22(1):1-29
(2003), Holash et al., Science, 284:1994-1998 (1999)). Data from
preclinical experiments and clinical trials have demonstrated that
blockade of VEGF alone is sufficient to alleviate or stabilize
disease progression (Eyetech Study Group, 22(2):143-52 (2002),
Krzystolik et al., Arch. Ophthalmol., 120(3):338-46 (2002), Shen et
al., Lab Invest., 82(2):167-82 (2002), Honda et al., Gene Ther.,
7(11):978-85 (2000), Saishin et al., J. Cell Physiol., 195(2):241-8
(2003)). For example, inhibition of VEGFR signaling by a specific
tyrosine kinase inhibitor is sufficient to completely prevent
retinal neovascularization in a murine retinopathy of prematurity
model (Ozaki H, Seo M S, Ozaki et al., Am. J. Pathol.,
156(2):697-707 (2000)). Furthermore, it has recently been
demonstrated that small interfering RNAs (siRNA) directed against
murine VEGF significantly inhibited ocular neovascularization after
laser photocoagulation in a mouse model (Reich et al., Mol. Vis.
30; 9:210-6 (2003)). These results indicate that selective
inhibition of VEGF expression is achievable and offers validation
of this approach for the treatment of ocular neovascular diseases
such as exudative macular degeneration and diabetic
retinopathy.
[0010] Three approaches have been used to inhibit VEGF activity,
including (1) neutralization of VEGF activity by using a specific
antibody, soluble VEGF receptor or aptamer oligos against the
VEGF/VEGFR interaction (Kim et al., Nature, 362:841-844 (1993), Lin
et al., Cell Growth Differ., 9(1):49-58 (1998), Borgstrom et al.,
Prostate, 35:1-10 (1998), Zhu et al., Invest. New Drugs, 17:195-212
(1999), Millauer et al., Nature, 367:576-579 (1994), Asano et al.,
Jpn. J. Cancer Res., 90(1):93-100 (1999), Brekken et al., Cancer
Res., 60(18):5117-24 (2000)); (2) inhibition of VEGFR mediated
signal transduction by specific small molecule tyrosine kinase
inhibitors (Fong et al., Cancer Res., 59:99-106 (1999), Wedge et
al., Cancer Res., 60(4):970-5 (2000), Laird et al., Cancer Res.,
60(15):4152-60 (2000)); and (3) inhibition of VEGF/VEGFR expression
by using antisense, siRNA or ribozyme (Reich et al., Mol. Vis. 30;
9:210-6 (2003), Parry et al., Nucleic Acids Res., 27:2569-2577
(1999), Ellis et al., Surgery, 120:871-878 (1996), Filleur et al.,
Cancer Res., 63(14):3919-22 (2003)). Although all of these
approaches show significant inhibition of angiogenesis in vivo,
they all possess significant limitations. For example, therapeutic
proteins (antibody and soluble receptors) or oligos (antisense,
siRNA and ribozyme) are large molecules with poor permeability that
usually require parenteral administration and are costly to
produce. For treatment of chronic ocular neovascularization,
multiple injections may be impractical due to potential
complications such as retinal detachment and procedure related
infection. Moreover, tyrosine kinase inhibitors have the potential
for limited specificity. VEGF is constitutively expressed at a low
level in normal eyes and other tissues and thus it may be harmful
to completely suppress VEGF function by administration of antibody
or tyrosine kinase inhibitors systemically, especially for patients
with AMD and RD many of whom are also hypertensive (Giles et al.,
Cancer, 97(8):1920-8 (2003), Sugimoto et al., J. Biol. Chem.,
278(15):12605-8 (2003), Bergsland et al., American Society of
Clinical Oncology 36.sup.th Annual Meeting, 20-23 May, 2000, New
Orleans, La., USA, Abstract 939), DeVore et al.,American Society of
Clinical Oncology 36.sup.th Annual Meeting, 20-23 May, 2000, New
Orleans, La., USA, Abstract 1896).
[0011] Thus, there remains a need to develop, characterize and
optimize lead molecules for the development of novel
anti-angiogenesis drugs. Accordingly, it is an object of the
present invention to provide such compounds.
[0012] All documents referred to herein are incorporated by
reference into the present application as though fully set forth
herein.
SUMMARY OF THE INVENTION
[0013] The present invention relates to compounds for inhibiting
the expression of VEGF post-transcriptionally or for inhibiting
angiogenesis, and compositions, methods, and kits for the
administration or use of such compounds.
[0014] In accordance with the present invention, compounds that
inhibit the expression of VEGF post-transcriptionally have been
identified, and methods for their use provided.
[0015] In one aspect of the invention, compounds of Formulas (I),
(II) and (III), including Formulas (I-a) to (1-1), are provided
which are useful in the inhibition of VEGF production, in the
inhibition of angiogenesis, and/or in the treatment of cancer,
diabetic retinopathy or exudative macular degeneration.
[0016] In another aspect of the invention, methods are provided for
the inhibition of VEGF production, the inhibition of angiogenesis,
and/or the treatment of cancer, cystic fibrosis, muscular
dystrophy, diabetic retinopathy, rheumatoid arthritis, psoriasis,
atherosclerosis, chronic inflammation, other chronic
inflammation-related diseases and disorders, obesity, or exudative
macular degeneration using the compounds described herein.
[0017] In one embodiment, the invention is directed to methods for
inhibiting VEGF production comprising administering a
VEGF-expression inhibiting amount of at least one compound of the
invention to a subject in need thereof.
[0018] In another embodiment, methods for inhibiting angiogenesis
are provided comprising administering an anti-angiogenic amount of
at least one compound of the invention to a subject in need
thereof.
[0019] The present invention also provides methods for treating a
solid tumor cancer comprising administering a therapeutically
effective amount of a compound of Formula (I), or a
pharmaceutically acceptable salt, hydrate, solvate, clathrate,
polymorph, racemate or stereoisomer of said compound, to a subject
in need thereof.
[0020] The present invention also provides methods for treating a
Ewing's sarcoma or a Wilms tumor comprising administering a
therapeutically effective amount of a compound of Formula (I), or a
pharmaceutically acceptable salt, hydrate, solvate, clathrate,
polymorph, racemate or stereoisomer of said compound, to a subject
in need thereof.
[0021] The present invention also provides methods for treating a
neuroblastoma comprising administering a therapeutically effective
amount of a compound of Formula (I), or a pharmaceutically
acceptable salt, hydrate, solvate, clathrate, polymorph, racemate
or stereoisomer of said compound, to a subject in need thereof.
[0022] The present invention further provides methods of treating a
malignant melanoma comprising administering a therapeutically
effective amount of a compound of Formula (I), or a
pharmaceutically acceptable salt, hydrate, solvate, clathrate,
polymorph, racemate or stereoisomer of said compound, to a subject
in need thereof.
[0023] The present invention yet further provides methods for
treating cervical cancer comprising administering a therapeutically
effective amount of a compound of Formula (I), or a
pharmaceutically acceptable salt, hydrate, solvate, clathrate,
polymorph, racemate or stereoisomer of said compound, to a subject
in need thereof.
[0024] The present invention also provides methods for treating
lung cancer comprising administering a therapeutically effective
amount of a compound of Formula (I), or a pharmaceutically
acceptable salt, hydrate, solvate, clathrate, polymorph, racemate
or stereoisomer of said compound, to a subject in need thereof.
[0025] The present invention also provides methods for treating
colon cancer comprising administering a therapeutically effective
amount of a compound of Formula (I), or a pharmaceutically
acceptable salt, hydrate, solvate, clathrate, polymorph, racemate
or stereoisomer of said compound, to a subject in need thereof.
[0026] The present invention further provides methods for treating
a solid tumor cancer by post-transcriptionally modifying VEGF
expression comprising administering a therapeutically effective
amount of a compound of Formula (I), or a pharmaceutically
acceptable salt, hydrate, solvate, clathrate, polymorph, racemate
or stereoisomer of said compound, to a subject in need thereof.
[0027] The present invention yet further provides methods for
treating a solid tumor cancer by slowing tumorigenesis at a
pre-vascular stage comprising administering a therapeutically
effective amount of a compound of Formula (I), or a
pharmaceutically acceptable salt, hydrate, solvate, clathrate,
polymorph, racemate or stereoisomer of said compound, to a subject
in need thereof.
[0028] The present invention also provides methods for treating a
solid tumor cancer by reducing tumor VEGF levels comprising
administering a therapeutically effective amount of a compound of
Formula (I), or a pharmaceutically acceptable salt, hydrate,
solvate, clathrate, polymorph, racemate or stereoisomer of said
compound, to a subject in need thereof.
[0029] The present invention further provides methods for treating
a solid tumor cancer by reducing perivascularly sequestered VEGF
comprising administering a therapeutically effective amount of a
compound of Formula (I), or a pharmaceutically acceptable salt,
hydrate, solvate, clathrate, polymorph, racemate or stereoisomer of
said compound, to a subject in need thereof.
[0030] The present invention also provides methods of inhibiting
VEGF mRNA translation comprising administering a therapeutically
effective amount of a compound of Formula (I), or a
pharmaceutically acceptable salt, hydrate, solvate, clathrate,
polymorph, racemate or stereoisomer of said compound, to a subject
in need thereof.
[0031] The present invention further provides methods of slowing
tumorigenesis at a pre-vascular stage comprising administering a
therapeutically effective amount of a compound of Formula (I), or a
pharmaceutically acceptable salt, hydrate, solvate, clathrate,
polymorph, racemate or stereoisomer of said compound, to a subject
in need thereof.
[0032] The present invention yet further provides methods of
reducing tumor VEGF levels comprising administering a
therapeutically effective amount of a compound of Formula (I), or a
pharmaceutically acceptable salt, hydrate, solvate, clathrate,
polymorph, racemate or stereoisomer of said compound, to a subject
in need thereof.
[0033] The present invention also provides methods of reducing
plasma and serum VEGF levels comprising administering a
therapeutically effective amount of a compound of Formula (I), or a
pharmaceutically acceptable salt, hydrate, solvate, clathrate,
polymorph, racemate or stereoisomer of said compound, to a subject
in need thereof.
[0034] The present invention further provides methods of reducing
perivascularly sequestered VEGF comprising administering a
therapeutically effective amount of a compound of Formula (I), or a
pharmaceutically acceptable salt, hydrate, solvate, clathrate,
polymorph, racemate or stereoisomer of said compound, to a subject
in need thereof.
[0035] The present invention yet further provides methods of
treating a solid tumor cancer comprising measuring serum or plasma
levels of VEGF, tumor levels of VEGF, or both, and administering a
therapeutically effective amount of a compound of Formula (I), or a
pharmaceutically acceptable salt, hydrate, solvate, clathrate,
polymorph, racemate or stereoisomer of said compound, to a subject
in need thereof.
[0036] The present invention also provides methods of diagnosing
solid tumor cancers comprising measuring serum or plasma levels of
VEGF.
[0037] The present invention further provides methods of diagnosing
solid tumor cancers comprising measuring tumor levels of VEGF.
[0038] The present invention yet further provides methods of
treating a solid tumor cancer comprising administering a
therapeutically effective amount of a compound of Formula (I), or a
pharmaceutically acceptable salt, hydrate, solvate, clathrate,
polymorph, racemate or stereoisomer thereof, together with one or
more additional agents useful for treating cancer to a subject in
need thereof.
[0039] These and other aspects of the invention will be more
clearly understood with reference to the following preferred
embodiments and detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 illustrates inhibition of lung cancer tumor growth by
a certain compound #10.
[0041] FIG. 2 illustrates inhibition of Ewing's sarcoma and
neuroblastoma tumor growth by compound #10.
[0042] FIG. 3 illustrates inhibition of fibrosarcoma tumor growth
by compound #10.
[0043] FIG. 4 illustrates inhibition of malignant melanoma tumor
growth by compound #10.
[0044] FIG. 5 illustrates inhibition of VEGF expression in a
fibrosarcoma tumor by compound #10.
[0045] FIG. 6 illustrates inhibition of colon cancer tumor growth
by compound #10, either alone or in combination with 5-FU
(fluorouracil).
[0046] FIG. 7 illustrates inhibition of cervical cancer tumor
growth by compound #10, either alone or in combination with Taxol
(paclitaxel).
[0047] FIG. 8 illustrates inhibition of tumor growth by compound
#10, either alone or in combination with Avastin (bevacizumab, an
antibody to VEGF).
[0048] FIG. 9 illustrates inhibition of tumor and plasma VEGF
expression by compound #10, either alone or in combination with
Avastin.
[0049] FIG. 10 illustrates inhibition of angiogenesis by compound
#10.
[0050] FIG. 11 illustrates that compound #10 does not affect VEGF
mRNA levels under hypoxic conditions.
[0051] FIG. 12 illustrates that the activity of phosphdiesterase 5
(PDE-5) is not affected by certain compounds.
[0052] FIG. 13 illustrates the effect of compound #10 on the
production of VEGF by cultured keratinocytes exposed to normoxic
and hypoxic conditions.
[0053] FIG. 14 illustrates the mean (average) plasma concentrations
of compound #10 at several times following administration of a
single dose of compound #10 to normal healthy subjects. The error
bars show the standard deviation.
[0054] FIG. 15 illustrates the mean plasma concentrations of VEGF
(FIG. 15, panel A) and the change in VEGF relative to baseline
measurements (FIG. 15, panel B) over time following a single dose
0.0 mg/ml (placebo) or 3.0 mg/kg of compound #10.
[0055] FIG. 16 illustrates the mean plasma concentrations of
compound #10 at day one and seven of a seven day dosing study of
normal healthy subjects at three different doses administered twice
per day (0.3, 0.6 or 1.2 mg/kg). The error bars show the standard
deviation.
[0056] FIG. 17 illustrates the mean plasma and serum concentrations
of VEGF relative to placebo treated subjects at several time points
following administration of doses of compound #10. Groups of
subjects are treated with twice daily (BID) with 0.3 mg/kg, 0.6
mg/kg, or 1.2 mg/kg of the compound. For assessment of effects on
VEGF levels with administration three times daily (TID) compound
was administered at 1.6 mg/kg (4.8 mg/kg/day total).
DETAILED DESCRIPTION OF THE INVENTION
[0057] Aberrant up-regulation of Vascular Endothelial Growth Factor
(VEGF), a key factor for angiogenesis, is an important contributor
to the pathogenesis of disease states such as cancer, diabetic
retinopathy, rheumatoid arthritis, psoriasis, atherosclerosis,
chronic inflammation, other chronic inflammation-related diseases
and disorders, obesity, or exudative macular degeneration. In
accordance with the present invention, compounds that inhibit the
expression of VEGF post-transcriptionally have been identified, and
methods for their use provided. The compounds of the invention have
nanomolar to sub-nanomolar activity for the inhibition of VEGF
expression.
A. Compounds of the Invention
[0058] In one aspect of the invention, compounds are provided which
are useful in the inhibition of VEGF production, in the inhibition
of angiogenesis, and/or in the treatment of cancer, diabetic
retinopathy or exudative macular degeneration. In certain
embodiments, the compounds of the invention specifically inhibit
VEGF production, while in other embodiments, the compounds of the
invention inhibit VEGF expression as well as that of other
angiogenesis factors such as FGF-2. In this regard, pan-angiogenic
inhibitor may be preferred in methods of inhibiting tumor growth,
while VEGF specific inhibitors may be preferred for the treatment
of ocular neovascular disorders (Eyetech Study Group, 22(2):143-52
(2002)).
[0059] The compounds of the invention generally include one or more
chiral centers, and as such may exist as racemic mixtures (R/S) or
as enantiomerically pure compositions. The compounds may exist as
(R) or (S) isomers (when one chiral center is present) in
enantiomerically pure compositions. In a preferred embodiment, the
compounds of the invention are the (S) isomers and may exist as
enantiomerically pure compositions comprising only the (S) isomer.
As one of skill will recognize, when more than one chiral center is
present, the compounds of the invention may exist as (R,R), (R,S),
(S,R), (S,S), etc. isomer. Preferred compounds included (S,S) and
(S,R) isomers.
[0060] As used herein, "enantiomerically pure" refers to
compositions consisting substantially of a single isomer,
preferably consisting of greater than or equal to 90%, 92%, 95%,
98%, 99%, or equal to 100% of a single isomer.
[0061] As used herein, a "racemic mixture" is any mixture of
isometric forms that are not "enantiomerically pure," including,
without limitation, about 50/50, about 60/40, and about 70/30
mixtures.
[0062] Preferred compounds of the present invention useful in the
inhibition of VEGF production include those of Formula (I) as shown
below.
##STR00001##
wherein,
[0063] X is hydrogen; a C.sub.1 to C.sub.6 alkyl, optionally
substituted with one or more halogens; a hydroxyl group; a halogen;
a C.sub.1 to C.sub.5 alkoxy, optionally substituted with a C.sub.6
to C.sub.10 aryl group;
[0064] A is C or N;
[0065] B is C or N, with the proviso that at least one of A or B is
N, and that when A is N, B is C;
[0066] R.sub.1 is a hydroxyl group; a C.sub.1 to C.sub.8 alkyl
group, optionally substituted with an alkylthio group, a 5 to 10
membered heteroaryl, a C.sub.6 to C.sub.10 aryl group optionally
substituted with at least one independently selected R.sub.o group;
a C.sub.2 to C.sub.8 alkyenyl group; a C.sub.2 to C.sub.8 alkynyl
group; a 3 to 12 membered heterocycle group, wherein the
heterocycle group is optionally substituted with at least one
independently selected halogen, oxo, amino, alkylamino, acetamino,
thio, or alkylthio group; a 5 to 12 membered heteroaryl group,
wherein the heteroaryl group is optionally substituted with at
least one independently selected halogen, oxo, amino, alkylamino,
acetamino, thio, or alkylthio group; or a C.sub.6 to C.sub.10 aryl
group, optionally substituted with at least one independently
selected R.sub.o group;
[0067] R.sub.o is a halogen; a cyano; a nitro; a sulfonyl, wherein
the sulfonyl is optionally substituted with a C.sub.1 to C.sub.6
alkyl or a 3 to 10 membered heterocycle; an amino group, wherein
the amino group is optionally substituted with a C.sub.1 to C.sub.6
alkyl, --C(O)--R.sub.b, --C(O)O--R.sub.b, a sulfonyl, an
alkylsulfonyl, a 3 to 10 membered heterocycle group optionally
substituted with a --C(O)O--R.sub.n; --C(O)--NH--R.sub.b; a 5 to 6
membered heterocycle; a 5 to 6 membered heteroaryl; a C.sub.1 to
C.sub.6 alkyl group, wherein the alkyl group is optionally
substituted with at least one independently selected hydroxyl,
halogen, amino, or 3 to 12 membered heterocycle group,
[0068] wherein the amino group and heterocycle group are optionally
substituted with at least one independently selected C.sub.1 to
C.sub.4 alkyl group, which C.sub.1 to C.sub.4 alkyl group is
optionally substituted with at least one independently selected
C.sub.1 to C.sub.4 alkoxy group, amino group, alkylamino group, or
5 to 10 membered heterocycle group; a --C(O)--R.sub.n group; or an
--OR.sub.hd a group;
[0069] R.sub.a is hydrogen; C.sub.2 to C.sub.8 alkenyl; a
--C(O)O--R.sub.b group; a --C(O)--NH--R.sub.b; a C.sub.1 to C.sub.8
alkyl, wherein the alkyl group is optionally substituted with at
least one independently selected hydroxyl, halogen, C.sub.1 to
C.sub.4 alkoxy, amino, alkylamino, acetamide, --C(O)--R.sub.b,
--C(O)O--R.sub.b, C.sub.6 to C.sub.10 aryl, 3 to 12 membered
heterocycle, or 5 to 12 heteroaryl group, further wherein the
alkylamino is optionally substituted with a hydroxyl, a C.sub.1 to
C.sub.4 alkoxy, or a 5 to 12 membered heteroaryl optionally
substituted with a C.sub.1 to C.sub.4 alkyl, further wherein the
acetamide is optionally substituted with a C.sub.1 to C.sub.4
alkoxy, sulfonyl, or alkylsulfonyl, further wherein and the
heterocycle group is optionally substituted with a C.sub.1 to
C.sub.4 alkyl optionally substituted with a hydroxyl group,
--C(O)--R.sub.n, --C(O)O--R.sub.n, or an oxo group;
[0070] R.sub.b is hydroxyl; an amino; an alkylamino, wherein the
alkylamino is optionally substituted with a hydroxyl, an amino, an
alkylamino, a C.sub.1 to C.sub.4 alkoxy, a 3 to 12 membered
heterocycle optionally substituted with at least one independently
selected C.sub.1 to C.sub.6 alkyl, oxo, --C(O)O--R.sub.n, or a 5 to
12 membered heteroaryl optionally substituted with a C.sub.1 to
C.sub.4 alkyl; a C.sub.1 to C.sub.4 alkoxy; a C.sub.2 to C.sub.8
alkenyl; a C.sub.2 to C.sub.8 alkynyl; a C.sub.6 to C.sub.10 aryl,
wherein the aryl is optionally substituted with at least one
independently selected halogen or C.sub.1 to C.sub.4 alkoxy; a 5 to
12 membered heteroaryl; 3 to 12 membered heterocycle group, wherein
the heterocycle is optionally substituted with at least one
independently selected acetamide, --C(O)O--R.sub.n, 5 to 6 membered
heterocycle, or C.sub.1 to C.sub.6 alkyl optionally substituted
with a hydroxyl, C.sub.1 to C.sub.4 alkoxy, amino group, or
alkylamino group; or a C.sub.1 to C.sub.8 alkyl, wherein the alkyl
is optionally substituted with at least one independently selected
C.sub.1 to C.sub.4 alkoxy, C.sub.6 to C.sub.10 aryl, amino, or 3 to
12 membered heterocycle group, wherein the amino and heterocycle
groups are optionally substituted with at least one independently
selected C.sub.1 to C.sub.6 alkyl, oxo, or --C(O)O--R.sub.n
group;
[0071] R.sub.2 is a hydrogen; a hydroxyl; a 5 to 10 membered
heteroaryl group; a C.sub.1 to C.sub.8 alkyl group, wherein the
alkyl group is optionally substituted with a hydroxyl, a C.sub.1 to
C.sub.4 alkoxy, a 3 to 10 membered heterocycle, a 5 to 10 membered
heteroaryl, or C.sub.6 to C.sub.10 aryl group; a --C(O)--R.sub.c
group; a --C(O)O--R.sub.d group; a --C(O)--N(R.sub.dR.sub.d) group;
a --C(S)--N(R.sub.dR.sub.d) group; a --C(S)--O--R.sub.e group; a
--S(O.sub.2)--R.sub.e group; a --C(NR.sub.E)--S--R.sub.e group; or
a --C(S)--S--R.sub.f group;
[0072] R.sub.c is hydrogen; an amino, wherein the amino is
optionally substituted with at least one independently selected
C.sub.1 to C.sub.6 alkyl or C.sub.6 to C.sub.10 aryl group; a
C.sub.6 to C.sub.10 aryl, wherein the aryl is optionally
substituted with at least one independently selected halogen,
haloalkyl, hydroxyl, C.sub.1 to C.sub.4 alkoxy, or C.sub.1 to
C.sub.6 alkyl group; --C(O)--R.sub.n; a 5 to 6 membered
heterocycle, wherein the heterocycle is optionally substituted with
a --C(O)--R.sub.n group; a 5 to 6 membered heteroaryl; a
thiazoleamino group; a C.sub.1 to C.sub.8 alkyl group, wherein the
alkyl group is optionally substituted with at least one
independently selected halogen, a C.sub.1 to C.sub.4 alkoxy, a
phenyloxy, a C.sub.6 to C.sub.10 aryl, --C(O)--R.sub.n,
--O--C(O)--R.sub.n, hydroxyl, or amino group, optionally
substituted with a --C(O)O--R.sub.n group;
[0073] R.sub.d is independently hydrogen; a C.sub.2 to C.sub.8
alkenyl group; a C.sub.2 to C.sub.8 alkynyl group; a C.sub.6 to
C.sub.10 aryl group, wherein the aryl is optionally substituted
with at least one independently selected halogen, nitro, C.sub.1 to
C.sub.6 alkyl, --C(O)O--R.sub.e, or --OR.sub.e; or a C.sub.1 to
C.sub.8 alkyl group, wherein the alkyl group is optionally
substituted with at least one independently selected halogen,
C.sub.1 to C.sub.4 alkyl, C.sub.1 to C.sub.4 alkoxy, phenyloxy,
C.sub.6 to C.sub.10 aryl, 5 to 6 membered heteroaryl,
--C(O)--R.sub.n, --O--C(O)--R.sub.n, or hydroxyl group, wherein the
C.sub.6 to C.sub.10 aryl group is optionally substituted with at
least one independently selected halogen or haloalkyl group;
[0074] R.sub.e is a hydrogen; a C.sub.1 to C.sub.6 alkyl group,
wherein the alkyl group is optionally substituted with at least one
independently selected halogen or alkoxy group; or a C.sub.6 to
C.sub.10 aryl group, wherein the aryl group is optionally
substituted with at least one independently selected halogen or
alkoxy group;
[0075] R.sub.f is a C.sub.1 to C.sub.6 alkyl group, optionally
substituted with at least one independently selected halogen,
hydroxyl, C.sub.1 to C.sub.4 alkoxy, cyano, C.sub.6 to C.sub.10
aryl, or --C(O)--R.sub.n group, wherein the alkoxy group may be
optionally substituted with at least one C.sub.1 to C.sub.4 alkoxy
group and the aryl group may be optionally substituted with at
least one independently selected halogen, hydroxyl, C.sub.1 to
C.sub.4 alkoxy, cyano, or C.sub.1 to C.sub.6 alkyl group;
[0076] R.sub.n is a hydroxyl, C.sub.1 to C.sub.4 alkoxy, amino, or
C.sub.1 to C.sub.6 alkyl group;
[0077] R.sub.3 is hydrogen or --C(O)--R.sub.g;
[0078] R.sub.g is a hydroxyl group; an amino group, wherein the
amino is optionally substituted with a C.sub.6 to C.sub.10
cycloalkyl group or a 5 to 10 membered heteroaryl group; or a 5 to
10 membered heterocycle group, wherein the heterocycle group is
optionally substituted with a --C(O)--R.sub.n group; and
[0079] n is 0, 1, 2, or 3.
[0080] As will be evident to one of skill in the art, the compounds
of Formula (I) comprise at least one stereocenter (e.g., at the
R.sub.1 substituent), and may exist as a racemic mixture or as an
enantiomerically pure composition. In a preferred embodiment, the
compounds of Formula (I) are the (S) isomer, in an enantiomerically
pure composition.
[0081] As used herein, the term "alkyl" generally refers to
saturated hydrocarbyl radicals of straight, branched or cyclic
configuration including methyl, ethyl, n-propyl, isopropyl,
n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl,
cyclohexyl, n-heptyl, octyl, n-octyl, and the like. In some
embodiments, alkyl substituents may be include C.sub.1 to C.sub.8,
C.sub.1 to C.sub.6, or C.sub.1 to C.sub.4 alkyl groups. The alkyl
group may be optionally substituted with one or more halogen or
alkoxy groups. For instance, the alkyl group may be a haloalkyl,
dihaloalkyl, or trihaloalkyl.
[0082] As used herein, "alkenyl" generally refers to linear,
branched or cyclic alkene radicals having one or more carbon-carbon
double bonds, such as C.sub.2 to C.sub.8 and C.sub.2 to C.sub.6
alkenyl groups, including 3-propenyl.
[0083] As used herein, "alkynyl" generally refers to linear,
branched or cyclic alkyne radicals having one or more carbon-carbon
triple bonds, such as C.sub.2 to C.sub.8 and C.sub.2 to C.sub.6
alkynyl groups, including hex-3-yne.
[0084] As used herein, "aryl" refers to a carbocyclic aromatic ring
structure. Included in the scope of aryl groups are aromatic rings
having from five to twenty carbon atoms. Aryl ring structures
include compounds having one or more ring structures, such as
mono-, bi-, or tricyclic compounds. Examples of aryl groups that
include phenyl, tolyl, anthracenyl, fluorenyl, indenyl, azulenyl,
phenanthrenyl (i.e., phenanthrene), and napthyl (i.e., napthalene)
ring structures. In certain embodiments, the aryl group may be
optionally substituted.
[0085] As used herein, "heteroaryl" refers to cyclic aromatic ring
structures in which one or more atoms in the ring, the
heteroatom(s), is an element other than carbon. Heteroatoms are
typically O, S or N atoms. Included within the scope of heteroaryl,
and independently selectable, are O, N, and S heteroaryl ring
structures. The ring structure may include compounds having one or
more ring structures, such as mono-, bi-, or tricyclic compounds.
In some embodiments, the heteroaryl groups may be selected from
heteroaryl groups that contain one or more heteroatoms, two or more
heteroatoms, three or more heteroatoms, or four or more
heteroatoms. Heteroaryl ring structures may be selected from those
that contain five or more atoms, six or more atoms, or eight or
more atoms. Examples of heteroaryl ring structures include:
acridine, benzimidazole, benzoxazole, benzodioxole, benzofuran,
dihydro-chromen-4-only, 1,3-diazine, 1,2-diazine, 1,2-diazole,
1,4-diazanaphthalene, furan, furazan, imidazole, indole, isoxazole,
isoquinoline, isothiazole, isoindolyl, oxazole, purine, pyridazine,
pyrazole, pyridine, pyrazine, pyrimidine, pyrrole, quinoline,
quinoxaline, thiazole, thiophene, 1,3,5-triazine, 1,2,4-triazine,
1,2,3-triazine, tetrazole and quinazoline. In certain embodiments,
the heteroaryl may be optionally substituted.
[0086] As used herein, "heterocycle" refers to cyclic ring
structures in which one or more atoms in the ring, the
heteroatom(s), is an element other than carbon. Heteroatoms are
typically O, S or N atoms. Included within the scope of
heterocycle, and independently selectable, are O, N, and S
heterocycle ring structures. The ring structure may include
compounds having one or more ring structures, such as mono-, bi-,
or tricyclic compounds. In some embodiments, the heterocycle groups
may be selected from heterocycle groups that contain one or more
heteroatoms, two or more heteroatoms, three or more heteroatoms, or
four or more heteroatoms. Example of heterocycle groups include
morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl,
piperazinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl,
tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl,
tetrahydroprimidinyl, tetrahydrothiophenyl or tetrahydrothiopyranyl
and the like. In certain embodiments, the heterocycle may
optionally be substituted.
[0087] As used herein, "alkanoyl" generally refers to a group with
the structure --C(O)--R. In certain embodiments, R may be a
hydrogen, an alkyl, an 4-morpholinyl group, or a thiazoleamino
group.
[0088] As used herein, "alkoxy" generally refers to a group with
the structure --O--R. In certain embodiments, R may be an alkyl
group, such as a C.sub.1 to C.sub.5 alkyl group.
[0089] For the purposes of this invention, halo substituents may be
independently selected from the halogens such as fluorine,
chlorine, bromine, iodine, and astatine.
[0090] In certain preferred embodiments, X may be hydrogen,
methoxy, hydroxyl, benzoxy, or a halogen, preferably bromide or
chloride. In other embodiments, X may preferably be a C.sub.1 to
C.sub.4 alkyl or a haloalkyl.
[0091] R.sub.1 may preferably be a C.sub.6 to C.sub.8 aryl group,
optionally substituted with at least one R.sub.o group. R.sub.0 may
then preferably be methoxy, benzoxy, a C.sub.1 to C.sub.6 alkyl, a
5 to 6 membered heteroaryl (such as furyl or imidazole), cyano,
nitro, tri-fluoro methyl, or a halogen, more preferably methoxy,
benzoxy, iso-butyl or a halogen, and more preferably methoxy,
iso-butyl, bromide or chloride. Alternatively, R.sub.1 may be a 5
to 10 membered heteroaryl or 3 to 12 membered heterocycle, such as
a pyridinyl group, a thiophene group, a furyl group, a tetrahydro
furyl group, and a thiazole group dihydro-chromen-4-onyl group, a
1H-isoindolyl group, or a benzodioxole group.
[0092] R.sub.2 may preferably be a --CH.sub.2-furyl group, a
pyrimidyl group, or a --C(O)O--R.sub.d group. R.sub.d may
preferably then be a C.sub.1 to C.sub.6 alkyl, optionally
substituted with at least one halogen; or a C.sub.5 to C.sub.6
aryl, optionally substituted with at least one methyl, methoxy, or
halogen.
[0093] Preferred R.sub.1 substituents also include the following,
where the * indicates the bond of attachment to the carboline
scaffold molecule.
##STR00002## ##STR00003## ##STR00004##
[0094] Other preferred R.sub.1 substituents include the following,
where the * indicates the bond of attachment to the carboline
scaffold molecule.
##STR00005## ##STR00006## ##STR00007## ##STR00008## ##STR00009##
##STR00010## ##STR00011## ##STR00012## ##STR00013## ##STR00014##
##STR00015## ##STR00016## ##STR00017## ##STR00018## ##STR00019##
##STR00020##
[0095] Preferred R.sub.2 substituents also include the following,
where the * indicates the bond of attachment to the carboline
scaffold molecule.
##STR00021## ##STR00022##
[0096] Other preferred R.sub.2 substituents include the following,
where the * indicates the bond of attachment to the carboline
scaffold molecule.
##STR00023## ##STR00024## ##STR00025## ##STR00026##
[0097] Preferred R.sub.3 substituents include the following, where
the * indicates the bond of attachment to the carboline scaffold
molecule.
##STR00027##
[0098] A preferred class of compounds within Formula (I) include
those compounds of Formula (I-a) as shown below.
##STR00028##
[0099] wherein X, R.sub.1 and R.sub.2 are defined as described with
regard to Formula (I) and the preferred embodiments described
above.
[0100] Another preferred class of compounds within Formula (I)
include those compounds of Formula (I-b) as shown below.
##STR00029##
wherein:
[0101] X is a halogen;
[0102] R.sub.2 is as described above with regard to Formula
(I);
[0103] R.sub.0 is as described above with regard to Formula
(I);
[0104] m is 0, 1, 2, or 3; and
[0105] n is 0, 1, 2, or 3.
[0106] Other preferred classes of compounds within Formula (I)
include the following.
##STR00030##
[0107] It is understood that substituents X and R.sub.1, R.sub.c,
R.sub.d, and R.sub.e of the compounds of Formulas (I-c) to (I-i)
are defined as in Formula (I).
[0108] In other embodiments, preferred compounds of the present
invention useful in the inhibition of VEGF production and treating
cancer and other disorders include those of Formulas (I-i) through
(1-1), as shown below. In the embodiments of Formulas (I-j) through
(I-1), substituents X, R.sub.1, R.sub.2, R.sub.3, etc. are defined
as in Formula (I), as well as Formulas (I-a) to (I-i).
##STR00031##
[0109] Also included within the scope of the invention are
pharmaceutically acceptable salts, hydrates, solvates, calthrates,
polymorphs, racemates and stereoisomers of the compounds described
herein.
[0110] In another aspect of the invention, preferred compounds of
the present invention useful in the inhibition of VEGF production,
and treating cancer and other disorders include those of Formula
(I-1) as shown below.
##STR00032##
wherein,
[0111] X is hydrogen; a hydroxyl group; a halogen; a
C.sub.1-C.sub.4 alkyl; a C.sub.1 to C.sub.5 alkoxy, optionally
substituted with a C.sub.6 to C.sub.8 aryl group;
[0112] R.sub.1 is a hydroxyl group; a C.sub.1 to C.sub.8 alkyl
group, optionally substituted with a C.sub.6 to C.sub.8 aryl group,
wherein the C.sub.6 to C.sub.8 aryl group is optionally substituted
with at least one R.sub.o group; a heterocycle group; a heteroaryl
group; and a C.sub.6 to C.sub.8 aryl group, optionally substituted
with at least one R.sub.0 group;
[0113] R.sub.0 is a halogen; a C.sub.1 to C.sub.6 alkyl, optionally
substituted with one or more halogen groups; a cyano group; a nitro
group; an amino group; an aminoalkyl group; an acetamide group; an
imidazole group; or OR.sub.a;
[0114] R.sub.a is hydrogen; a C.sub.1 to C.sub.6 alkyl, optionally
substituted with a heterocycle group or a C.sub.6 to C.sub.8 aryl
group; or a --C(O)O--R.sub.b;
[0115] R.sub.b is C.sub.1 to C.sub.4 alkyl group;
[0116] R.sub.2 is a hydrogen; a hydroxyl; a heteroaryl group; a
C.sub.1 to C.sub.8 alkyl group, optionally substituted with an
alkoxy, hydroxyl, heteroaryl, or C.sub.6 to C.sub.8 aryl group; a
--C(O)--R.sub.c group; a --C(O)O--R.sub.d group; a
--C(O)NH--R.sub.d group; a --C(S)NH--R.sub.d group; a
--S(O.sub.2)--R.sub.e group; or (1S)-isopropyl-carbamic acid
tert-butyl ester;
[0117] R.sub.c is hydrogen; a 4-morpholinyl group; a thiazoleamino
group; a piperazinyl group, optionally substituted with a
--C(O)CH.sub.3 group; a C.sub.1 to C.sub.6 alkyl group, optionally
substituted with a halogen, an alkoxy, or hydroxyl group;
[0118] R.sub.d is hydrogen; a benzyl group; a C.sub.1 to C.sub.8
alkyl group, optionally substituted with a halogen or an alkoxy
group; a C.sub.6 to C.sub.8 aryl group, optionally substituted with
at least one halogen, C.sub.1 to C.sub.5 alkyl, --C(O)OR.sub.e, or
OR.sub.e;
[0119] R.sub.e is a hydrogen; a C.sub.1 to C.sub.6 alkyl group,
optionally substituted with at least one halogen or alkoxy group;
or a C.sub.6 to C.sub.8 aryl group; and
[0120] n is 0, 1, 2, or 3.
[0121] In another embodiment, compounds of Formulas (II), (III) and
(IV) are provided, which are useful for inhibiting VEGF production,
and treating cancer and other disorders.
##STR00033##
[0122] Wherein X, R.sub.1, R.sub.2, R.sub.o and R.sub.d are defined
as described above with regard with Formula (I).
[0123] For the purposes of this invention, where one or more
functionalities encompassing X R.sub.1, R.sub.2, R.sub.0, R.sub.a,
R.sub.b, R.sub.c, R.sub.d, and R.sub.e, are incorporated into a
molecule of Formulas (I), (II), and (III), including Formulas (I-a)
to (I-k), each of the functionalities appearing at any location
within the disclosed may be independently selected, and as
appropriate, independently substituted. Further, where a more
generic substituent is set forth for any position in the molecules
of the present invention, it is understood that the generic
substituent may be replaced with more specific substituents, and
the resulting molecules are within the scope of the molecules of
the present invention.
[0124] Preferred compounds of the invention include the
following.
##STR00034## ##STR00035## ##STR00036## ##STR00037## ##STR00038##
##STR00039## ##STR00040## ##STR00041## ##STR00042## ##STR00043##
##STR00044## ##STR00045## ##STR00046## ##STR00047## ##STR00048##
##STR00049## ##STR00050## ##STR00051## ##STR00052## ##STR00053##
##STR00054## ##STR00055## ##STR00056## ##STR00057## ##STR00058##
##STR00059## ##STR00060## ##STR00061## ##STR00062## ##STR00063##
##STR00064## ##STR00065## ##STR00066## ##STR00067## ##STR00068##
##STR00069## ##STR00070## ##STR00071## ##STR00072## ##STR00073##
##STR00074## ##STR00075## ##STR00076## ##STR00077## ##STR00078##
##STR00079## ##STR00080## ##STR00081## ##STR00082## ##STR00083##
##STR00084## ##STR00085## ##STR00086## ##STR00087## ##STR00088##
##STR00089## ##STR00090## ##STR00091## ##STR00092## ##STR00093##
##STR00094## ##STR00095## ##STR00096## ##STR00097## ##STR00098##
##STR00099## ##STR00100## ##STR00101## ##STR00102## ##STR00103##
##STR00104## ##STR00105## ##STR00106## ##STR00107## ##STR00108##
##STR00109## ##STR00110## ##STR00111## ##STR00112## ##STR00113##
##STR00114## ##STR00115## ##STR00116## ##STR00117## ##STR00118##
##STR00119## ##STR00120## ##STR00121## ##STR00122## ##STR00123##
##STR00124## ##STR00125## ##STR00126## ##STR00127## ##STR00128##
##STR00129## ##STR00130## ##STR00131## ##STR00132## ##STR00133##
##STR00134## ##STR00135## ##STR00136## ##STR00137## ##STR00138##
##STR00139## ##STR00140## ##STR00141## ##STR00142## ##STR00143##
##STR00144## ##STR00145## ##STR00146## ##STR00147## ##STR00148##
##STR00149## ##STR00150## ##STR00151## ##STR00152## ##STR00153##
##STR00154## ##STR00155## ##STR00156## ##STR00157## ##STR00158##
##STR00159## ##STR00160## ##STR00161## ##STR00162## ##STR00163##
##STR00164## ##STR00165## ##STR00166## ##STR00167## ##STR00168##
##STR00169## ##STR00170## ##STR00171## ##STR00172## ##STR00173##
##STR00174## ##STR00175## ##STR00176## ##STR00177## ##STR00178##
##STR00179## ##STR00180## ##STR00181## ##STR00182## ##STR00183##
##STR00184## ##STR00185## ##STR00186## ##STR00187## ##STR00188##
##STR00189## ##STR00190## ##STR00191## ##STR00192## ##STR00193##
##STR00194## ##STR00195## ##STR00196## ##STR00197## ##STR00198##
##STR00199## ##STR00200## ##STR00201## ##STR00202## ##STR00203##
##STR00204## ##STR00205## ##STR00206## ##STR00207## ##STR00208##
##STR00209## ##STR00210## ##STR00211## ##STR00212## ##STR00213##
##STR00214## ##STR00215## ##STR00216## ##STR00217## ##STR00218##
##STR00219## ##STR00220## ##STR00221## ##STR00222## ##STR00223##
##STR00224## ##STR00225## ##STR00226## ##STR00227## ##STR00228##
##STR00229## ##STR00230## ##STR00231## ##STR00232## ##STR00233##
##STR00234## ##STR00235## ##STR00236## ##STR00237## ##STR00238##
##STR00239## ##STR00240## ##STR00241## ##STR00242## ##STR00243##
##STR00244## ##STR00245## ##STR00246## ##STR00247## ##STR00248##
##STR00249## ##STR00250## ##STR00251## ##STR00252## ##STR00253##
##STR00254## ##STR00255## ##STR00256## ##STR00257## ##STR00258##
##STR00259## ##STR00260## ##STR00261## ##STR00262## ##STR00263##
##STR00264## ##STR00265## ##STR00266##
[0125] In certain embodiments, preferred compounds include those
with an EU.sub.50 in the VEGF ELISA assay described in Example 5 of
less than about 2 uM, more preferably between about 2 uM and about
0.04 uM (200 nM to 40 nM); more preferably from about 0.04 uM to
about 0.008 uM to (40 nM to 8 nM); and more preferably less than
about 0.008 uM (<8 nM). Particularly preferred compounds are
Compound Nos: 2, 4, 5, 7, 8, 10, 11, 12, 17, 23, 25, 81, 102, 112,
140, 328, 329, 330, 331, 332, 355, 816, 817, 818, 823, 824, 825,
830, 831, 832, 837, 838, 841, 842, 843, and regioisomers thereof.
In one embodiment, the preferred compounds of the invention form a
racemic mixture, and in another embodiment the compounds of the
invention are the (R), (S), (R,R), (S,S), (R,S), (S,R) isomer, in
an enantiomerically pure composition. More preferably, the
compounds of the invention are the (S) isomers, in an
enantiomerically pure composition.
[0126] The above compounds are listed only to provide examples that
may be used in the methods of the invention. Based upon the instant
disclosure, the skilled artisan would recognize other compounds
intended to be included within the scope of the presently claimed
invention that would be useful in the methods recited herein.
B. Preparation of Compounds of the Invention
[0127] Compounds of the invention may be produced in any manner
known in the art. By way of example, compounds of the invention may
be prepared according to the following general schemes. More
specifically, Scheme I may be used to make compounds of Formula I.
Scheme Ia can be used when in conjunction with Scheme I when
R.sub.2 is a --CH.sub.2-furanyl group. Alternatively, for
asymmetric synthesis when R.sub.2 is hydrogen or hydroxyl, Scheme
Ib may be used.
##STR00267##
##STR00268##
##STR00269##
[0128] Scheme II can be used to prepare compounds of Formula
I-h.
##STR00270##
[0129] Schemes IIIa or IIIb can be used to prepare compounds of
Formula I-i.
##STR00271##
##STR00272##
[0130] In a preferred embodiment, compounds of the invention may be
resolved to enantiomerically pure compositions using any method
known in art. By way of example, compounds of the invention may be
resolved by direct crystallization of enantiomer mixtures, by
diastereomer salt formation of enantiomers, by the formation of
diasteriomers and separation, or by enzymatic resolution.
[0131] In a preferred embodiment, compounds of the invention may be
resolved through crystallization using, e.g.,
N-acetyl-L-phenylalanine to obtain the (S) isomer, or
N-acetyl-D-phenylalanine to obtain the (R) isomer, in a manner
similar to that illustrated in Scheme IV.
##STR00273##
[0132] In certain embodiments, exemplary methods of Scheme I for
preparing preferred compounds of Formula I involve the formation of
free amine Pictet-Spengler reaction products/intermediates, as
described below in Procedure-I.
##STR00274##
[0133] In one embodiment, Procedure-I may involve adding a desired
Aldehyde (II) to a suspension of 5-substituted tryptamine. HCl (I)
in 0.1N sulfuric acid. The solution may then be stirred at about
110.degree. C.-120.degree. C. in a closed reaction vessel until the
reaction is sufficient to complete, e.g., for about 15 minutes to
about 20 hours. After completion of the reaction, the reaction
mixture may be cooled to room temperature and the precipitated salt
may be filtered. The filtered residue may then be washed with
ether, EtOAc or a mixture of DCM and DMF and dried to give the
product (III) as acid salt. Alternatively, a desired Aldehyde (II)
may be added to a suspension of 5-substituted tryptamine.HCl (I) in
acetic acid and refluxed until the reaction is sufficiently
complete, e.g., for about 15 minutes to about 20 hours. After
completion of the reaction, the reaction mixture may be cooled to
room temperature and the acid salt may be filtered. The filtered
residue may then be washed with acetic acid followed by DCM and
dried to give the product (III) as acid salt. The free amine (III)
may be obtained by extraction with EtOAc and washing with aqueous
ammonium hydroxide or 1M aq. sodium hydroxide.
[0134] The free amine, or its salt, may then be used to form other
preferred compounds of Formula I, such as carbamate analogs
(Formula I-c, Procedure-II), amide analogs, including N-acetyl
analogs (Formula I-c, Procedure-Ma and Procedure-Mb), urea and
thiourea analogs (Formula I-e and I-f, Procedure-IV and Procedure-V
respectively), sulfoxide analogs (Formula I-g, Procedure-VI), and
pyrimidine analogs (Procedure-VII).
[0135] More particularly, Procedure-II may be used to synthesize
carbamate analogs of free amines (III), or their salts.
##STR00275##
[0136] In accordance with Procedure-II, diisopropylethylamine
(DIEA) may be added to the free amine (III), or its acid salt in
dichloromethane (DCM), followed by slow addition of substituted
chloroformate. The reaction mixture may be stirred at room
temperature for about 1 to 20 hours. The solvent may then be
evaporated and the crude product may either be purified by HPLC or
silica gel column chromatography.
[0137] Procedure-Ma may be used to synthesize amide analogs of free
amine (III), or their salts.
##STR00276##
[0138] In accordance with Procedure-IIIa, a 15 min pre-stirred
mixture of an R.sub.2-acid and diisopropyl carbodiimide (DIC) may
be added to the free amine (III), or its acid salt in DCM and DIEA.
The reaction mixture may be stirring for about 1 h. The solvents
may then be evaporated and the crude product purified by HPLC.
[0139] Alternatively, Procedure-Mb may be used to synthesize
N-acetyl analogs of free amines (III), or their salts.
##STR00277##
[0140] In accordance with Procedure-IIIb, pyridine may be added to
the free amine (III), or its acid salt in DCM, followed by acetic
anhydride. The reaction mixture may be stirred at room temperature
for about 8 to 20 hours. The solvents may then be evaporated and
the crude product was purified by HPLC.
[0141] Procedure-IV may be used to synthesize urea analogs of free
amines (III), or their salts.
##STR00278##
[0142] In accordance with Procedure-IV, DIEA and R.sub.2-isocyanate
may be added to the free amine (III), or its acid salt in DCM. The
reaction mixture may be refluxed for about 1.5 h. The solvents may
then be evaporated and the crude product purified by HPLC.
[0143] Procedure-V may be used to synthesize thiourea analogs of
free amines (III), or their salts.
##STR00279##
[0144] In accordance with Procedure-V, DIEA and
R.sub.2-isothiocyanate may be added to the free amine (III), or its
acid salt in DCM. The reaction mixture may be refluxed for about 12
h. The solvents may then be evaporated and the crude product
purified by HPLC.
[0145] Procedure-VI may be used to synthesize sulfonyl analogs of
free amines (III), or their salts.
##STR00280##
[0146] In accordance with Procedure-VI, DIEA and
R.sub.2-sulfonylchloride may be added to the free amine (III), or
its acid salt in DCM. The reaction mixture may be stirred at room
temperature for about 12 h. The solvents may then be evaporated and
the crude product purified by HPLC.
[0147] Procedure-VII may be used to synthesize pyrimidine analogs
of free amines (III), or their salts.
##STR00281##
[0148] In accordance with Procedure-VII, triethylamine and
2-bromopyrimidine in N,N-dimethylformamide (DMF) may be added to
the free amine (III), or its acid salt in DCM. The reaction mixture
may be heated to about 120.degree. C. for about 12 h. The solvents
may then be evaporated and the crude product purified by HPLC.
[0149] These and other reaction methodologies may be useful in
preparing the compounds of the invention, as recognized by one of
skill in the art. Various modifications to the above schemes and
procedures will be apparent to one of skill in the art, and the
invention is not limited specifically by the method of preparing
the compounds of the invention.
C. Methods of the Invention
[0150] In another aspect of the invention, methods are provided for
the inhibition of VEGF production, the inhibition of angiogenesis,
and/or the treatment of cancer, diabetic retinopathy, rheumatoid
arthritis, psoriasis, atherosclerosis, chronic inflammation, other
chronic inflammation-related diseases and disorders, obesity, or
exudative macular degeneration using the compounds described
herein.
[0151] In one embodiment, the invention is directed to methods for
inhibiting VEGF production comprising administering a
VEGF-expression inhibiting amount of at least one compound of the
invention to a subject in need thereof.
[0152] In another embodiment, methods for inhibiting angiogenesis
are provided comprising administering an anti-angiogenic amount of
at least one compound of the invention to a subject in need
thereof.
[0153] In yet another embodiment, methods for treating cancer,
diabetic retinopathy, rheumatoid arthritis, psoriasis,
atherosclerosis, chronic inflammation, other chronic
inflammation-related diseases and disorders, obesity, or exudative
macular degeneration are provided comprising administering a
therapeutically effective amount of at least one compound of the
invention to a subject in need thereof.
[0154] In yet a further embodiment, the cancers which can be
treated by administering a therapeutically effective amount of at
least one compound of the invention to a subject in need thereof
include solid tumor cancers. Solid tumor cancers that can be
treated by the present invention include solid tumor carcinomas and
solid tumor sarcomas. Solid tumor carcinomas include, but are not
limited to, pediatric solid tumors, such as Ewing's sarcoma or
Wilms tumor and neuroblastoma, and carcinomas of the epidermis,
such as malignant melanomas, as well as lung cancers, cervical
cancers, colon cancers and renal cancers. Solid tumor sarcomas
include, but are not limited to, fibrosarcomas. The methods of
treating cancer can further include the administration of one or
more additional agents useful for treating cancer.
[0155] In yet another embodiment of the invention, methods for
treating a solid tumor cancer by slowing tumorigenesis at a
pre-vascular stage are provided, comprising administering a
therapeutically effective amount of at least one compound of the
invention to a subject in need thereof, either alone or together
with one or more additional agents useful for treating cancer.
[0156] In another embodiment of the invention, methods for treating
a solid tumor cancer by inhibiting VEGF mRNA translation are
provided, comprising administering a therapeutically effective
amount of at least one compound of the invention to a subject in
need thereof, either alone or together with one or more additional
agents useful for treating cancer.
[0157] In yet another embodiment of the invention methods for
treating a solid tumor cancer by reducing tumor VEGF levels are
provided, comprising administering a therapeutically effective
amount of at least one compound of the invention to a subject in
need thereof, either alone or together with one or more additional
agents useful for treating cancer.
[0158] In yet a further embodiment of the invention, methods for
treating a solid tumor cancer by reducing perivascularly
sequestered or intratumoral VEGF are provided, comprising
administering a therapeutically effective amount of at least one
compound of the invention to a subject in need thereof, either
alone or together with one or more additional agents useful for
treating cancer. In this aspect, reduced perivascularly sequestered
VEGF is an in situ comparison of perivascular VEGF in tumors
treated with the compound of the invention and tumors not treated
with the compound of the invention. In a preferred aspect, reduced
perivascularly sequestered VEGF is compared with levels of
perivascular VEGF in tumors treated with antibodies to VEGF.
[0159] Without intending to be limited by theory, it is believed
that the methods of the present invention act through a combination
of mechanisms that modulate the activity of VEGF. In this
embodiment of the invention, methods for inhibiting VEGF mRNA
translation are provided, comprising administering a
therapeutically effective amount of at least one compound of the
invention to a subject in need thereof. In a preferred aspect, VEGF
mRNA translation is inhibited by greater than 10%, 25%, 50%, 75%,
80%, or 90% compared with an untreated tumor or cell.
[0160] In another embodiment of the invention, methods for slowing
tumorigenesis at a pre-vascular stage are provided, comprising
administering a therapeutically effective amount of at least one
compound of the invention to a subject in need thereof, either
alone or together with one or more additional cancer agents. The
pre-vascular stage of tumorigenesis is clinically known as
"carcinoma in situ" and tumors at this stage are characterized by
their reliance on nearby blood vessels for oxygen and diffusion of
nutrients, due to the tumors absence of its own vascular
infrastructure. So, by slowing tumorigenesis at a pre-vascular
stage, one is preventing or slowing the development of a vascular
infrastructure in the tumor. In this embodiment of the invention,
whether tumorigenesis has been slowed at the pre-vascular stage is
determined by identifying to what extent the tumor has developed a
vascular infrastructure. In a preferred aspect, treated tumor
growth is prevented or slowed, as compared to the untreated tumors,
by 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%.
[0161] In yet a further embodiment of the invention, methods for
reducing perivascularly sequestered or intratumoral VEGF are
provided, comprising administering a therapeutically effective
amount of at least one compound of the invention to a subject in
need thereof.
[0162] In yet an additional embodiment of the invention, methods of
diagnosing cancer by measuring tumor plasma, and/or serum levels of
VEGF are provided. Tumor levels of VEGF can be measured using
biopsy tissue, where plasma or serum VEGF levels can be measured by
taking blood. In humans, different tumors secrete different levels
of VEGF. Standard ELISA procedures can be used to measure the
amount of VEGF in the tumor, serum or plasma. See, for example,
Verheul, H. M. W. et al. (2000) Platelet and coagulation activation
with vascular endothelial growth factor generation in soft tissue
sarcomas. Clin. Cancer Res. 6:166. For tumors that do not secrete
large amounts of VEGF into the plasma, the tumor VEGF concentration
can be determined to diagnose the tumor progression. For tumors
that do secrete large amount of VEGF into the plasma, plasma VEGF
concentration can be determined to diagnose the tumor progression.
After most known cancer treatments, VEGF levels are not affected,
and therefore the plasma or tumor levels of VEGF do not predict
efficacy of the treatment (i.e., progression of the cancer).
Compounds of the present invention can affect VEGF levels, both in
the plasma and tumor, and therefore measuring VEGF levels is an
accurate way to monitor and/or predict the progression of the
cancer (i.e., the efficacy of the treatment) when the methods of
the present invention are used for treating cancer.
[0163] In yet another embodiment of the invention, methods for
reducing tumor or plasma VEGF levels are provided, comprising
administering a therapeutically effective amount of at least one
compound of the invention to a subject in need thereof. In this
embodiment, VEGF levels can be measured in a tumor not treated with
the compounds of the present invention and the VEGF levels compared
to the VEGF levels measured in a tumor treated with the compounds
of the present invention, thereby showing that by treatment of
tumors with the compounds of the present invention VEGF levels are
reduced.
[0164] In yet another embodiment of the invention, methods for
treating a solid tumor cancer are provided, comprising (a)
measuring one or more of serum VEGF levels, plasma VEGF levels, or
tumor VEGF levels, and administering a therapeutically effective
amount of at least one compound of the invention to a subject in
need thereof. In an embodiment, VEGF concentration is measured to
determine whether treatment with a compound of the present
invention should be undertaken. In this aspect, treatment with a
compound of the present invention is preferred and more effective
as the VEGF levels increase.
[0165] In yet a further embodiment of the invention, methods for
treating a solid tumor cancer are provided, comprising
administering a therapeutically effective amount of at least one
compound of the invention to a subject in need thereof, together
with one or more additional cancer agents.
[0166] In preferred embodiments, the methods of the invention
comprise administering a therapeutically effective amount of at
least one compound of the invention, wherein the compound is an (S)
isomer.
[0167] According to the methods of the invention, the compound(s)
may be administered to the subject via any drug delivery route
known in the art. Specific exemplary administration routes include
oral, ocular, rectal, buccal, topical, nasal, ophthalmic,
subcutaneous, intramuscular, intravenous (bolus and infusion),
intracerebral, transdermal, and pulmonary.
[0168] The terms "VEGF-inhibiting amount", "anti-angiogenic
amount", and "therapeutically effective amount", as used herein,
refer to an amount of a pharmaceutical agent to treat, ameliorate,
or prevent the identified disease or condition, or to exhibit a
detectable therapeutic or inhibitory affect. The affect can be
detected by, for example, the assays disclosed in the following
examples. The precise effective amount for a subject will depend
upon the subject's body weight, size, and health; the nature and
extent of the condition; and the therapeutic or combination of
therapeutics selected for administration. Therapeutically effective
amounts for a given situation can be determined by routine
experimentation that is within the skill and judgment of the
clinician.
[0169] For any compound, the therapeutically effective amount can
be estimated initially either in cell culture assays, e.g., of
neoplastic cells, or in animal models, usually rats, mice, rabbits,
dogs, or pigs. The animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans. Therapeutic/prophylactic efficacy and
toxicity may be determined by standard pharmaceutical procedures in
cell cultures or experimental animals, e.g., ED.sub.50 (the dose
therapeutically effective in 50% of the population) and LD.sub.50
(the dose lethal to 50% of the population). The dose ratio between
therapeutic and toxic effects is the therapeutic index, and it can
be expressed as the ratio, ED.sub.50/LD.sub.50. Pharmaceutical
compositions that exhibit large therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies may
be used in formulating a range of dosage for human use. The dosage
contained in such compositions is preferably within a range of
circulating concentrations that include an ED.sub.50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed, sensitivity of the patient, and the route
of administration.
[0170] More specifically, the concentration-biological effect
relationships observed with regard to the compound(s) of the
present invention indicate an initial target plasma concentration
ranging from approximately 0.1 .mu.g/mL to approximately 100
.mu.g/mL, preferably from approximately 0.5 .mu.g/mL to
approximately 50 .mu.g/mL, more preferably from approximately 0.5
.mu.g/mL to approximately 10 .mu.g/mL. To achieve such plasma
concentrations, the compounds of the invention may be administered
at doses that vary from 0.1 .mu.g to 100,000 mg/day, depending upon
the route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature and is generally
available to practitioners in the art. In general the dose will be
in the range of about 1 mg/day to about 10 g/day, or about 0.100 mg
to about 3 g/day, or about 300 mg to about 3 g/day, or about 500 mg
to about 2 g/day, in single, divided, or continuous doses for a
patient or subject weighing between about 40 to about 100 kg (which
dose may be adjusted for patients or subjects above or below this
weight range, particularly children under 40 kg).
[0171] The dose administered to achieve an effective target plasma
concentration may also be administered based upon the weight of the
subject or patient. Doses administered on a weight basis may be in
the range of about 0.01 mg/kg/day to about 100 mg/kg/day, or about
0.015 mg/kg/day to about 50 mg/kg/day, or about 0.02 mg/kg/day to
about 10 mg/kg/day, or about 0.025 mg/kg/day to about 7.5
mg/kg/day, or about 0.03 mg/kg/day to about 5 mg/kg/day. In one
embodiment, the dose may be from about 0.3 to about 0.48 mg/kg/day.
In another embodiment, where daily doses are adjusted based upon
the weight of the subject or patient, compounds of the invention
may be formulated for delivery at about 0.02, 0.025, 0.03, 0.05,
0.06, 0.075, 0.08, 0.09. 0.10, 0.20, 0.25, 0.30, 0.50, 0.60, 0.75,
0.80, 0.90, 1.0, 1.10, 1.20, 1.25, 1.50, 1.75, 2.0, 5.0, 10, 25,
50, 75, or 100 mg/kg/day. Daily doses adjusted based upon the
weight of the subject or patient may be administered as a single,
divided, or continuous dose. In embodiments where a dose of
compound is given more than once per day, it may be administered
twice, thrice, or more per day.
[0172] Compounds of the invention may be advantageously
administered over one or more days without causing significant side
effects. Compounds of the invention administered over one or more
days may reduce VEGF production induced by pathological events such
as hypoxia or tumor transformation without causing a statistically
significant reduction in normal plasma VEGF concentrations and
associated problems. In some embodiments, the compounds maybe
administered without reducing plasma VEGF below the lower limit of
the normal physiological range for plasma VEGF. In other
embodiments, the compounds maybe administered without reducing
plasma VEGF below the lower limit of the normal physiological range
for plasma VEGF of healthy adult mammals, or healthy adult human
subjects. For the purpose of this invention, the lower limit of
VEGF's normal physiological range in plasma may be about 10 pg/ml,
or about 15 pg/ml or about 20 pg/ml or about 25 pg/ml in healthy
mammalian or human subjects.
[0173] In one aspect, compounds of the invention are generally well
tolerated by subjects. For example, the compounds can be
administered by the oral route with no occurrences, or only mild to
moderate occurrences, of nausea, productive cough, constipation,
diarrhea, eye pruritus, headache, back pain, or insomnia in one or
more subjects. For the purposes of this disclosure, mild or
moderate occurrences may be described as those occurrences that
would not prevent individual subjects or groups of subjects from
continuing to receive the compounds.
[0174] Compounds of the invention may be advantageously
administered over one or more days without raising safety concerns
based upon hERG assays, Novascreen.RTM. assays, phosphatase and
kinase panel assessments Similarly, no meaningful cytotoxicity is
observed in lung fibroblast, skin fibroblast or bone marrow
progenitor cell cytotoxicity testing. In some embodiments,
compounds of the invention do not display mutagenecity in the Ames
assay, chromosomal aberrations, polyploidy or endoreduplication in
CHO cell assays, or clastogenic effects in rat micronucleus assays.
In yet other embodiments, no negative cardiopulmonary effects are
observed with single doses up to 140 mg/kg, no negative
neuorological effects are observed with single doses up to 200
mg/kg, and no significant toxicity is observed at dose of 120 mg/kg
QD or at doses of 60 mg/kg BID for 7 days.
[0175] Compounds of the invention may be advantageously
administered without one or more negative effects observed with
other compounds having VEGF inhibitory activity, such as anti VEGF
antibodies (e.g., bevacizumab) and other compounds inhibiting VEGFR
tyrosine kinase activity. Recognized side effects of bevacizumab
include hypertension (see, e.g., Gordon et al., J. Clin. Oncol.
19(3) 843-850 (2001)), proteinuria (id.), and thromboembolism.
Other inhibitors of VEGFR, (e.g., PTK787, sunitinib, and ZD6474)
also induce a number of additional off-target effects including:
light-headedness, ataxia, headache, nausea, vomiting, diarrhea,
rash, subungual hemorrhage, myelosuppression, fatigue,
hypothyroidism, QT interval prolongation or heart failure. Such
effects appear to be due to nonspecific inhibition of tyrosine
kinase receptors other than the VEGFR.
[0176] In some embodiments, the compounds of the invention may
advantageously be administered without causing a substantial
incidence of either proteinuria or hypertension. In other
embodiments, the compounds of the invention may advantageously be
administered without causing a substantial incidence of an increase
in the grade of proteinuria. In other embodiments, the compounds of
the invention may advantageously be administered without causing a
substantial increase in blood pressure.
[0177] In some embodiments, a substantial incidence of proteinuria,
an increase in the grade of proteinuria, or hypertension is the
occurrence of those side effects in greater than about 20% of the
subjects or patients treated. In other embodiments, a substantial
incidence of either proteinuria or hypertension is the occurrence
of either of those side effects in greater than 15% of the subjects
or patients treated. In still other embodiments, a substantial
incidence of either proteinuria or hypertension is the occurrence
of those side effects in greater than 10%, or 5% or 2% or 1% of the
subjects or patients treated. Compounds of the invention may cause
minor transient changes in heart rate, blood pressure, respiratory
rate, and core body temperature. Such changes may remain within
normal limits and may be observed at dosages of 30 mg/kg or
greater.
[0178] For the purposes of this invention, a patient or subject is
considered to have hypertension when their blood pressure is
greater than 150 mm Hg/100 mm Hg (systolic/diastolic). For the
purpose of this invention, hypertension is defined as an elevation
of blood pressure of 20 mm Hg (diastolic), or blood pressure above
150 mm Hg/100 mm Hg (systolic/diastolic), for more than 24 hours
during which time one or more compounds of the invention are
present at or above a therapeutically effective concentration in
the plasma of a subject.
[0179] For the purposes of this invention, proteinuria is defined
as an elevation in the amount of protein found in the urine of a
patient or subject outside of the normal range during the period a
compound of the invention is present at or above a therapeutically
effective concentration in the plasma of a subject. For example,
proteinuria may be found when there is more than 0.15 grams of
protein present in a 24 hour urine sample. Grade 1 proteinuria may
be found when the amount of protein in a 24 hour urine sample is
from 0.15 g to 1 g of protein in a 24 hour urine sample, Grade 2
proteinuria may be found when the amount of protein in a 24 hour
urine sample is greater 1 g but less than 3.5 grams of protein in a
24 hour urine sample. Grade 3 proteinuria may be found when the
amount of protein in a 24 hour urine sample is greater than 3.5 g
of protein in a 24 hour urine sample. Grade 4 proteinuria equates
to nephrotic syndrome. An elevation in the amount of protein found
in the urine of a patient or subject outside of the normal range
may also be found based upon dipstick measurements. A dipstick
measurement of "1+" equates to grade 1 proteinuria, a measure of 2+
or 3+ equates to grade 2 proteinuria, and a dipstick measure of 4+
equates to grade 3 proteinuria.
[0180] For the purposes of this invention a patient or subject may
be considered to have a risk of having a stroke when they have
hypertension, and particularly when they have hypertension and a
prior medical history of one or more strokes.
[0181] In some embodiments, the compounds of the invention may be
administered without causing a substantial incidence of grade 1
proteinuria as measured by 24 hour urine analysis or by dipstick
analysis of a urine sample. In other embodiments, compounds of the
invention may be administered without causing a substantial
incidence of grade 2 proteinuria as measured by 24 hour urine
analysis or by dipstick analysis of a urine sample. In yet other
embodiments, compounds of the invention may be administered without
causing an increase in a the proteinuria status (e.g., the grade of
proteinuria) of a patient from grade 1 proteinuria to grade 2
proteinuria, or from grade 2 proteinuria to grade 3 proteinuria, as
measured by 24 hour urine analysis or by dipstick analysis of a
urine sample.
[0182] In one embodiment the compounds of the invention are
administered to patients having solid tumors that have one or more
of high blood pressure (hypertension), proteinuria, or risk of
having a stroke.
[0183] The exact dosage will be determined by the practitioner, in
light of factors related to the subject that requires treatment.
Dosage and administration are adjusted to provide sufficient levels
of the active agent(s) or to maintain the desired effect. Factors
which may be taken into account include the severity of the disease
state, general health of the subject, age, weight, and gender of
the subject, diet, time and frequency of administration, drug
combination(s), reaction sensitivities, and tolerance/response to
therapy. Long-acting pharmaceutical compositions may be
administered every 3 to 4 days, every week, or once every two weeks
depending on half-life and clearance rate of the particular
formulation.
D. Metabolites of the Compounds of the Invention
[0184] Also falling within the scope of the present invention are
the in vivo metabolic products of the compounds described herein.
Such products may result for example from the oxidation, reduction,
hydrolysis, imidation, esterification and the like of the
administered compound, primarily due to enzymatic processes.
Accordingly, the invention includes compounds produced by a process
comprising contacting a compound of this invention with a mammalian
tissue or a mammal for a period of time sufficient to yield a
metabolic product thereof. Such products typically are identified
by preparing a radio-labeled (e.g. C.sup.14 or H.sup.3) compound of
the invention, administering it in a detectable dose (e.g., greater
than about 0.5 mg/kg) to a mammal such as rat, mouse, guinea pig,
monkey, or to man, allowing sufficient time for metabolism to occur
(typically about 30 seconds to 30 hours), and isolating its
conversion products from urine, blood or other biological samples.
These products are easily isolated since they are labeled (others
are isolated by the use of antibodies capable of binding epitopes
surviving in the metabolite). The metabolite structures are
determined in conventional fashion, e.g., by MS or NMR analysis. In
general, analysis of metabolites may be done in the same way as
conventional drug metabolism studies well-known to those skilled in
the art. The conversion products, so long as they are not otherwise
found in vivo, are useful in diagnostic assays for therapeutic
dosing of the compounds of the invention even if they possess no
biological activity of their own.
E. Pharmaceutical Compositions of the Invention
[0185] While it is possible for the compounds of the present
invention to be administered neat, it may be preferable to
formulate the compounds as pharmaceutical compositions. As such, in
yet another aspect of the invention, pharmaceutical compositions
useful in the methods of the invention are provided. The
pharmaceutical compositions of the invention may be formulated with
pharmaceutically acceptable excipients such as carriers, solvents,
stabilizers, adjuvants, diluents, etc., depending upon the
particular mode of administration and dosage form. The
pharmaceutical compositions should generally be formulated to
achieve a physiologically compatible pH, and may range from a pH of
about 3 to a pH of about 11, preferably about pH 3 to about pH 7,
depending on the formulation and route of administration. In
alternative embodiments, it may be preferred that the pH is
adjusted to a range from about pH 5.0 to about pH 8.0.
[0186] More particularly, the pharmaceutical compositions of the
invention comprise a therapeutically or prophylactically effective
amount of at least one compound of the present invention, together
with one or more pharmaceutically acceptable excipients.
Optionally, the pharmaceutical compositions of the invention may
comprise a combination of compounds of the present invention, or
may include a second active ingredient useful in the treatment of
cancer, diabetic retinopathy, or exudative macular
degeneration.
[0187] Formulations of the present invention, e.g., for parenteral
or oral administration, are most typically solids, liquid
solutions, emulsions or suspensions, while inhalable formulations
for pulmonary administration are generally liquids or powders, with
powder formulations being generally preferred. A preferred
pharmaceutical composition of the invention may also be formulated
as a lyophilized solid that is reconstituted with a physiologically
compatible solvent prior to administration. Alternative
pharmaceutical compositions of the invention may be formulated as
syrups, creams, ointments, tablets, and the like.
[0188] The term "pharmaceutically acceptable excipient" refers to
an excipient for administration of a pharmaceutical agent, such as
the compounds of the present invention. The term refers to any
pharmaceutical excipient that may be administered without undue
toxicity. Pharmaceutically acceptable excipients are determined in
part by the particular composition being administered, as well as
by the particular method used to administer the composition.
Accordingly, there exists a wide variety of suitable formulations
of pharmaceutical compositions of the present invention (see, e.g.,
Remington's Pharmaceutical Sciences).
[0189] Suitable excipients may be carrier molecules that include
large, slowly metabolized macromolecules such as proteins,
polysaccharides, polylactic acids, polyglycolic acids, polymeric
amino acids, amino acid copolymers, and inactive virus particles.
Other exemplary excipients include antioxidants such as ascorbic
acid; chelating agents such as EDTA; carbohydrates such as dextrin,
hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid;
liquids such as oils, water, saline, glycerol and ethanol; wetting
or emulsifying agents; pH buffering substances; and the like.
Liposomes are also included within the definition of
pharmaceutically acceptable excipients.
[0190] The pharmaceutical compositions of the invention may be
formulated in any form suitable for the intended method of
administration. When intended for oral use for example, tablets,
troches, lozenges, aqueous or oil suspensions, non-aqueous
solutions, dispersible powders or granules (including micronized
particles or nanoparticles), emulsions, hard or soft capsules,
syrups or elixirs may be prepared. Compositions intended for oral
use may be prepared according to any method known to the art for
the manufacture of pharmaceutical compositions, and such
compositions may contain one or more agents including sweetening
agents, flavoring agents, coloring agents and preserving agents, in
order to provide a palatable preparation.
[0191] Pharmaceutically acceptable excipients particularly suitable
for use in conjunction with tablets include, for example, inert
diluents, such as celluloses, calcium or sodium carbonate, lactose,
calcium or sodium phosphate; disintegrating agents, such as
croscarmellose sodium, cross-linked povidone, maize starch, or
alginic acid; binding agents, such as povidone, starch, gelatin or
acacia; and lubricating agents, such as magnesium stearate, stearic
acid or talc. Tablets may be uncoated or may be coated by known
techniques including microencapsulation to delay disintegration and
adsorption in the gastrointestinal tract and thereby provide a
sustained action over a longer period. For example, a time delay
material such as glyceryl monostearate or glyceryl distearate alone
or with a wax may be employed.
[0192] Formulations for oral use may be also presented as hard
gelatin capsules where the active ingredient is mixed with an inert
solid diluent, for example celluloses, lactose, calcium phosphate
or kaolin, or as soft gelatin capsules wherein the active
ingredient is mixed with non-aqueous or oil medium, such as
glycerin, propylene glycol, polyethylene glycol, peanut oil, liquid
paraffin or olive oil.
[0193] In another embodiment, pharmaceutical compositions of the
invention may be formulated as suspensions comprising a compound of
the present invention in admixture with at least one
pharmaceutically acceptable excipient suitable for the manufacture
of a suspension. In yet another embodiment, pharmaceutical
compositions of the invention may be formulated as dispersible
powders and granules suitable for preparation of a suspension by
the addition of suitable excipients.
[0194] Excipients suitable for use in connection with suspensions
include suspending agents, such as sodium carboxymethylcellulose,
methylcellulose, hydroxypropyl methylcelluose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth, gum acacia, dispersing or
wetting agents such as a naturally occurring phosphatide (e.g.,
lecithin), a condensation product of an alkylene oxide with a fatty
acid (e.g., polyoxyethylene stearate), a condensation product of
ethylene oxide with a long chain aliphatic alcohol (e.g.,
heptadecaethyleneoxycethanol), a condensation product of ethylene
oxide with a partial ester derived from a fatty acid and a hexitol
anhydride (e.g., polyoxyethylene sorbitan monooleate); and
thickening agents, such as carbomer, beeswax, hard paraffin or
cetyl alcohol. The suspensions may also contain one or more
preservatives such as acetic acid, methyl and/or n-propyl
p-hydroxy-benzoate; one or more coloring agents; one or more
flavoring agents; and one or more sweetening agents such as sucrose
or saccharin.
[0195] The pharmaceutical compositions of the invention may also be
in the form of oil-in-water emulsions. The oily phase may be a
vegetable oil, such as olive oil or arachis oil, a mineral oil,
such as liquid paraffin, or a mixture of these. Suitable
emulsifying agents include naturally-occurring gums, such as gum
acacia and gum tragacanth; naturally occurring phosphatides, such
as soybean lecithin, esters or partial esters derived from fatty
acids; hexitol anhydrides, such as sorbitan monooleate; and
condensation products of these partial esters with ethylene oxide,
such as polyoxyethylene sorbitan monooleate. The emulsion may also
contain sweetening and flavoring agents. Syrups and elixirs may be
formulated with sweetening agents, such as glycerol, sorbitol or
sucrose. Such formulations may also contain a demulcent, a
preservative, a flavoring or a coloring agent.
[0196] Additionally, the pharmaceutical compositions of the
invention may be in the form of a sterile injectable preparation,
such as a sterile injectable aqueous emulsion or oleaginous
suspension. This emulsion or suspension may be formulated according
to the known art using those suitable dispersing or wetting agents
and suspending agents which have been mentioned above. The sterile
injectable preparation may also be a sterile injectable solution or
suspension in a non-toxic parenterally acceptable diluent or
solvent, such as a solution in 1,2-propane-diol. The sterile
injectable preparation may also be prepared as a lyophilized
powder. Among the acceptable vehicles and solvents that may be
employed are water, Ringer's solution, and isotonic sodium chloride
solution. In addition, sterile fixed oils may be employed as a
solvent or suspending medium. For this purpose any bland fixed oil
may be employed including synthetic mono- or diglycerides. In
addition, fatty acids such as oleic acid may likewise be used in
the preparation of injectables.
[0197] Generally, the compounds of the present invention useful in
the methods of the present invention are substantially insoluble in
water and are sparingly soluble in most pharmaceutically acceptable
protic solvents and in vegetable oils. However, the compounds are
generally soluble in medium chain fatty acids (e.g., caprylic and
capric acids) or triglycerides and have high solubility in
propylene glycol esters of medium chain fatty acids. Also
contemplated in the invention are compounds which have been
modified by substitutions or additions of chemical or biochemical
moieties which make them more suitable for delivery (e.g., increase
solubility, bioactivity, palatability, decrease adverse reactions,
etc.), for example by esterification, glycosylation, PEGylation,
etc.
[0198] In a preferred embodiment, the compounds of the present
invention may be formulated for oral administration in a
lipid-based formulation suitable for low solubility compounds.
Lipid-based formulations can generally enhance the oral
bioavailability of such compounds. As such, a preferred
pharmaceutical composition of the invention comprises a
therapeutically or prophylactically effective amount of a compound
of the present invention, together with at least one
pharmaceutically acceptable excipient selected from the group
consisting of: medium chain fatty acids or propylene glycol esters
thereof (e.g., propylene glycol esters of edible fatty acids such
as caprylic and capric fatty acids) and pharmaceutically acceptable
surfactants such as polyoxyl 40 hydrogenated castor oil.
[0199] In an alternative preferred embodiment, cyclodextrins may be
added as aqueous solubility enhancers. Preferred cyclodextrins
include hydroxypropyl, hydroxyethyl, glucosyl, maltosyl and
maltotriosyl derivatives of .alpha.-, .beta.-, and
.gamma.-cyclodextrin. A particularly preferred cyclodextrin
solubility enhancer is hydroxypropyl-.beta.-cyclodextrin (HPBC),
which may be added to any of the above-described compositions to
further improve the aqueous solubility characteristics of the
compounds of the present invention. In one embodiment, the
composition comprises 0.1% to 20%
hydroxypropyl-.beta.-cyclodextrin, more preferably 1% to 15%
hydroxypropyl-.beta.-cyclodextrin, and even more preferably from
2.5% to 10% hydroxypropyl-.beta.-cyclodextrin. The amount of
solubility enhancer employed will depend on the amount of the
compound of the present invention in the composition.
F. Combination Therapy
[0200] It is also possible to combine any compound of the present
invention with one or more other active ingredients or agents
useful in the treatment of cancer, including compounds, in a
unitary dosage form, or in separate dosage forms intended for
simultaneous or sequential administration to a patient in need of
treatment. When administered sequentially, the combination may be
administered in two or more administrations. In an alternative
embodiment, it is possible to administer one or more compounds of
the present invention and one or more additional active ingredients
by different routes.
[0201] The skilled artisan will recognize that a variety of active
ingredients may be administered in combination with the compounds
of the present invention that may act to augment or synergistically
enhance the VEGF-inhibiting and/or anti-angiogenesis activity of
the compounds of the invention.
[0202] More specifically, for methods involving the treatment of
cancer, agents known in the art to be useful for treating cancer
are provided. Such agents include, but are not limited to,
radiation therapy, agents that cause DNA damage, agents that reduce
the concentration or effect of a growth factor, agents that inhibit
angiogenesis, paclitaxel, fluorouracil, CPT-11, a tyrosine kinase
inhibitor, a COX-2 inhibitor, thalidomide, gemcitabine, squalamine,
endostatin, angiostatin, AE-941, lenalidomide, medi-522,
2-methoxyestradiol, carboxyamidotriazole, combretastatin A4
phosphate, SU6668, SU11248, BMS-275291, COL-3, cilengitide,
IMC-1121B, vatalanib, LY317615, VEGF Trap, ZD6474, halofuginone,
hydrobromide, celecoxib, interferon alpha, interleukin-12, and
antibodies capable of binding VEGF or a VEGF receptor, such as
bevacizumab. VEGF receptors include VEGF receptor 1, VEGF receptor
2, and VEGF receptor 3, and the neuropilins (e.g., neurophilin-1
(np-1) and neurorphilin-2 (np-2)). In another embodiment, the
compounds of the present invention are used in combination with an
agent that blocks the activity of a VEGF receptor. In yet another
embodiment, the compounds of the present invention can be used in
combination with agents that can block the VEGF signaling pathway.
Treatment only with a factor that can block VEGF signaling may
cause an increase in VEGF concentration. In such a case, including
a compound of the present invention in the treatment protocol can
prevent the subsequent increase in VEGF levels. Similarly, use of
the compounds of the present invention in combination with an
antibody is highly preferred. Antibodies are relatively large and
may not cross tight barriers, allowing secreted VEGF to remain in
areas such as the perivascular space. Post-transcriptional control
of VEGF expression can prevent the tumor from retaining as much
VEGF in the perivascular space, in the extracellular matrix, or in
other spaces and vessels that have a physical barrier to
antibodies.
[0203] According to the methods of the invention, the combination
of active ingredients may be: (1) co-formulated and administered or
delivered simultaneously in a combined formulation; (2) delivered
by alternation or in parallel as separate formulations; or (3) by
any other combination therapy regimen known in the art. When
delivered in alternation therapy, the methods of the invention may
comprise administering or delivering the active ingredients
sequentially, e.g., in separate solution, emulsion, suspension,
tablets, pills or capsules, or by different injections in separate
syringes. In general, during alternation therapy, an effective
dosage of each active ingredient is administered sequentially,
i.e., serially, whereas in simultaneous therapy, effective dosages
of two or more active ingredients are administered together.
Various sequences of intermittent combination therapy may also be
used.
[0204] To assist in understanding the present invention, the
following Examples are included. The experiments relating to this
invention should not, of course, be construed as specifically
limiting the invention and such variations of the invention, now
known or later developed, which would be within the purview of one
skilled in the art are considered to fall within the scope of the
invention as described herein and hereinafter claimed.
EXAMPLES
[0205] The present invention is described in more detail with
reference to the following non-limiting examples, which are offered
to more fully illustrate the invention, but are not to be construed
as limiting the scope thereof. The examples illustrate the
preparation of certain compounds of the invention, and the testing
of these compounds in vitro and/or in vivo. Those of skill in the
art will understand that the techniques described in these examples
represent techniques described by the inventors to function well in
the practice of the invention, and as such constitute preferred
modes for the practice thereof. However, it should be appreciated
that those of skill in the art should in light of the present
disclosure, appreciate that many changes can be made in the
specific methods that are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Preparation of Compounds of the Invention
[0206] Using the schemes and procedures described above in Section
B, one may prepare certain compounds of the invention as follows.
Other preferred compounds of the invention, such as those in Table
5 below, may be similarly prepared.
Example 1A
Compounds of Formula I, Scheme I
[0207] Certain compounds of Formula I may be prepared according to
Scheme I using free amine products/intermediates, or their salts
prepared in accordance with Procedure I. By way of example, certain
free amines (III), or their salts are prepared using Procedure I.
Table 4 illustrates certain free amines (III) or their salts,
Intermediates 1-11.
TABLE-US-00001 TABLE 4 (III) ##STR00282## Intermediate R--of Free
Amine (III) R.sub.1--of Free Amine (III) 1 Cl 4-OMe 2 Cl
2,3-difluoro 3 Cl 4-Cl 4 Cl 4-CN 5 Cl 4-F 6 Cl 4-iPr 7 Br 4-Cl 8 Br
4-Me 9 Br 4-iPr 10 Br 3-Cl 11 Br 4-OMe 12 Cl
4-(2-morpholine-4-yl-ethoxy)
[0208] Intermediate-1:
[0209] This intermediate is prepared using Procedure-I with
5-chlorotryptamine.HCl (5.8 g, 25 mmol), p-anisaldehyde (6.13 mL,
50 mmol) and 0.1N sulfuric acid (60 mL) to give the title compound
as an acid salt (6.1 g, 59%). ES-MS: 313 (M+H).sup.+.
Alternatively, this intermediate is prepared using Procedure-1B
with 5-chlorotryptamine.HCl (20 g, 86.5 mmol), p-anisaldehyde (15.9
mL, 130 mmol) and acetic acid (250 mL) to give the title compound
as an acid salt (25.8 g, 79%). ES-MS: 313 (M+H).sup.+.
[0210] Intermediate-2:
[0211] This intermediate is prepared using Procedure-I with
5-chlorotryptamine.HCl (116 mg, 0.5 mmol), 2,3-difluoro
benzaldehyde (109 .mu.L, 1 mmol) and 0.1N sulfuric acid (2 mL) to
give the title compound as an acid salt (158 mg, 75%). ES-MS: 319
(M+H).sup.+
[0212] Intermediate-3:
[0213] This intermediate is prepared using Procedure-I with
5-chlorotryptamine.HCl (462 mg, 2 mmol), 4-chloro benzaldehyde (562
mg, 4 mmol) and 0.1N sulfuric acid (8 mL) to give the title
compound as an acid salt (825 mg, 99%). ES-MS: 317 (M+H).sup.+
[0214] Intermediate-4:
[0215] This intermediate is prepared using Procedure-I with
5-chlorotryptamine.HCl (462 mg, 2 mmol), 4-cyano benzaldehyde (525
mg, 4 mmol) and 0.1N sulfuric acid (8 mL) to give the title
compound as an acid salt (810 mg, 100%). ES-MS: 308 (M+H).sup.+
[0216] Intermediate-5:
[0217] This intermediate is prepared using Procedure-I with
5-chlorotryptamine.HCl (374 mg, 1.5 mmol), 4-fluoro benzaldehyde
(322 .mu.L, 3 mmol) and 0.1N sulfuric acid (4 mL) to give the title
compound as an acid salt (250 mg, 42%). ES-MS: 301 (M+H).sup.+
[0218] Intermediate-6:
[0219] This intermediate is prepared using Procedure-I with
5-chlorotryptamine.HCl (1.15 g, 5 mmol), 4-isopropyl benzaldehyde
(1.516 mL, 10 mmol) and 0.1N sulfuric acid (12 mL) to give the
title compound as an acid salt (628 mg, 30%). ES-MS: 325
(M+H).sup.+
[0220] Intermediate-7:
[0221] This intermediate is prepared using Procedure-I with
5-bromotryptamine.HCl (551 mg, 2 mmol), 4-chloro benzaldehyde (562
mg, 4 mmol) and 0.1N sulfuric acid (8 mL) to give the title
compound as an acid salt (330 mg, 36%). ES-MS: 363 (M+H).sup.+
[0222] Intermediate-8:
[0223] This intermediate is prepared using Procedure-I with
5-bromotryptamine.HCl (551 mg, 2 mmol), p-tolualdehyde (471 .mu.L,
4 mmol) and 0.1N sulfuric acid (8 mL) to give the title compound as
hydrogen sulfate salt (257 mg, 29%). ES-MS: 341 (M+H).sup.+.
Alternatively, this intermediate is prepared using Procedure-1B
with 5-bromotryptamine.HCl (10 g, 36.3 mmol), p-tolualdehyde (6.41
mL, 54.5 mmol) and acetic acid (120 mL) to give the title compound
as acetate salt (14.5 g, 100%). ES-MS: 341 (M+H).sup.+
[0224] Intermediate-9 (Compound 112): This product/intermediate is
prepared using Procedure-I with 5-bromotryptamine.HCl (551 mg, 2
mmol), 4-isopropyl benzaldehyde (606 .mu.L, 4 mmol) and 0.1N
sulfuric acid (8 mL) to give the title compound as hydrogen sulfate
salt (329 mg, 35%). ES-MS: 369 (M+H).sup.+. Alternatively, this
intermediate is prepared using Procedure-1B with
5-bromotryptamine.HCl (10 g, 36.3 mmol), 4-isopropyl benzaldehyde
(8.24 mL, 54.5 mmol) and acetic acid (120 mL) to give the title
compound as acetate salt (13 g, 77%). ES-MS: 369 (M+H).sup.+
[0225] Intermediate-10:
[0226] This intermediate is prepared using Procedure-I with
5-bromotryptamine.HCl (551 mg, 2 mmol), 3-chloro benzaldehyde (453
.mu.L, 4 mmol) and 0.1N sulfuric acid (8 mL) to give the title
compound as an acid salt (662 mg, 72%). ES-MS: 361 (M+H).sup.+
[0227] Intermediate-11:
[0228] This intermediate is prepared using Procedure-I with
5-bromotryptamine.HCl (551 mg, 2 mmol), p-anisaldehyde (491 .mu.L,
4 mmol) and 0.1N sulfuric acid (8 mL) to give the title compound as
an acid salt (611 mg, 67%). ES-MS: 357 (M+H).sup.+
[0229] Intermediate-12:
[0230] The 4-(2-Morpholin-4-yl-ethoxy)-benzaldehyde reaction
intermediate is prepared by combining 4-hydroxybenzaldehyde (1.2 g,
10.0 mmol), 4-(2-chloroethyl)-morpholine hydrochloride (2.0 g, 11.0
mmol), potassium carbonate (4.1 g, 30.0 mmol), and potassium iodide
(170 mg, 1 mmol) in 100 ml of acetone and heating to reflux with
stirring. After all the 4-hydroxybenzaldehyde is consumed (48 hours
by LC/MS), the solids are filtered and the solvent is removed in
vacuo. The yield is 4.1 g.
[0231] Then Intermediate 12 is prepared in accordance with
Procedure-IB. Thus, 5-Chlorotryptamine hydrochloride (231 mg, 1.0
mmol) is combined with 4-(2-Morpholin-4-yl-ethoxy)-benzaldehyde
(565 mg, .about.1.2 mmol) in 3 mL of glacial acetic acid. The
suspension is heated to about 120.degree. C. for 10 minutes with
constant cooling and a max power of 300 W using the CEM Explorer
microwave system. Acetonitrile (2 mL) is added to the cooled
reaction mixture, and the solid is filtered and washed with 1 mL of
acetonitrile to produce the acetic acid salt of Intermediate 12
(6-Chloro-1-[4-(2-morpholin-4-yl-ethoxy)-phenyl]-2,3,4,9-tetrahydro-1H-.b-
eta.-carboline) (179 mg, 34%). Intermediates 1-12 may then be used
to prepare compounds of the invention according to Procedures II
through VII as follows.
[0232] Compound 2:
[0233] This product is prepared by Procedure-II using the
Intermediate-1 (3 g, 9.6 mmol), ethyl chloroformate (1.37 mL, 14.4
mmol) and DIEA (2.5 mL, 14.4 mmol) in dichloromethane (70 mL) to
give the title compound as white powder (1.56 g, 42%). ES-MS: 385
(M+H).sup.+.
[0234] Compound 4:
[0235] This product is prepared by Procedure-II using the
Intermediate-7 (72 mg, 0.2 mmol), ethyl chloroformate (29 .mu.L,
0.3 mmol) and DIEA (52 .mu.L, 0.3 mmol) in dichloromethane (2 mL)
to give the title compound as white powder (37 mg, 43%). ES-MS: 435
(M+H).sup.+.
[0236] Compound 5:
[0237] This product is prepared by the Procedure-II using the
Intermediate-2 (50 mg, 0.16 mmol), ethyl chloroformate (23 .mu.L,
0.24 mmol) and DIEA (42 .mu.L, 0.24 mmol) in dichloromethane (2 mL)
to give the title compound as white powder (25 mg, 41%). ES-MS: 391
(M+H).sup.+.
[0238] Compound 7:
[0239] This product is prepared by the Procedure-II using the
Intermediate-9 (74 mg, 0.2 mmol), ethyl chloroformate (29 .mu.L,
0.3 mmol) and DIEA (52 .mu.L, 0.3 mmol) in dichloromethane (2 mL)
to give the title compound as white powder (34 mg, 38%). ES-MS: 441
(M+H).sup.+.
[0240] Compound 8:
[0241] This product is prepared by the Procedure-II using the
Intermediate-8 (72 mg, 0.2 mmol), ethyl chloroformate (29 .mu.L,
0.3 mmol) and DIEA (52 .mu.L, 0.3 mmol) in dichloromethane (2 mL)
to give the title compound as white powder (39 mg, 47%). ES-MS: 413
(M+H).sup.+.
[0242] Compound 10:
[0243] This product is prepared by the Procedure-II using the
Intermediate-1 acetate (10.5 g, 28.2 mmol), 4-chlorophenyl
chloroformate (4.74 mL, 33.8 mmol) and DIEA (9.8 mL, 56.4 mmol) in
dichloromethane (300 mL) to give the title compound as white powder
(10.2 g, 78%). ES-MS: 467 (M+H).sup.+.
[0244] Compound 11:
[0245] This product is prepared by the Procedure-II using the
Intermediate-3 (63 mg, 0.2 mmol), ethyl chloroformate (29 .mu.L,
0.3 mmol) and DIEA (52 .mu.L, 0.3 mmol) in dichloromethane (2 mL)
to give the title compound as white powder (31 mg, 40%). ES-MS: 389
(M+H).sup.+.
[0246] Compound 12:
[0247] This product is prepared by the Procedure-II using the
Intermediate-4 (31 mg, 0.1 mmol), 2-chloroethyl chloroformate (16
.mu.L, 0.15 mmol) and DIEA (26 .mu.L, 0.15 mmol) in dichloromethane
(2 mL) to give the title compound as white powder (22 mg, 53%).
ES-MS: 414 (M+H).sup.+.
[0248] Compound 17:
[0249] This product is prepared by the Procedure-II using the
Intermediate-1 (47 mg, 0.15 mmol), 4-methylphenyl chloroformate (33
.mu.L, 0.23 mmol) and DIEA (39 .mu.L, 0.23 mmol) in dichloromethane
(2 mL) to give the title compound as white powder (34 mg, 51%).
ES-MS: 447 (M+H).sup.+.
[0250] Compound 23:
[0251] This product is prepared by the Procedure-II using the
Intermediate-5 (30 mg, 0.1 mmol), ethyl chloroformate (14 .mu.L,
0.15 mmol) and DIEA (26 .mu.L, 0.15 mmol) in dichloromethane (2 mL)
to give the title compound as white powder (21 mg, 56%). ES-MS: 373
(M+H).sup.+.
[0252] Compound 25:
[0253] This product is prepared by the Procedure-VII using the
Intermediate-9 (74 mg, 0.2 mmol), 2-bromopyrimidine (48 mg, 0.3
mmol) and triethylamine (42 .mu.L, 0.3 mmol) in DMF (2 mL) to give
the title compound (42 mg, 47%). ES-MS: 447 (M+H).sup.+.
[0254] Compound 102:
[0255] This product is prepared by the Procedure-Mb using the
Intermediate-9 (74 mg, 0.2 mmol), acetic anhydride (47 .mu.L, 0.5
mmol) and pyridine (41 .mu.L, 0.5 mmol) in dichloromethane (2 mL)
to give the title compound as white powder (31 mg, 38%). ES-MS: 411
(M+H).sup.+.
[0256] Compound 140:
[0257] This product is prepared by the Procedure-IV using the
Intermediate-10 (72 mg, 0.2 mmol), cyclohexyl isocyanate (26 .mu.L,
0.2 mmol) and DIEA (37 .mu.L, 0.21 mmol) in dichloromethane (2 mL)
to give the title compound as white powder (51 mg, 53%). ES-MS: 486
(M+H).sup.+.
[0258] Compound 166:
[0259] This product is prepared by the Procedure-Ma using its free
amine intermediate (141 mg, 0.5 mmol), Boc-L-Alanine (105 mg, 0.6
mmol), DIC (94 .mu.L, 0.6 mmol), DIEA (105 .mu.L, 0.6 mmol) and
dichloromethane (4 mL) to give the title compound (105 mg, 46%).
ES-MS: 420 (M+H).sup.+.
[0260] Compound 225:
[0261] This product is prepared by the Procedure-VI using its free
amine intermediate (78 mg, 0.2 mmol), methyl sulfonylchloride (16
.mu.L, 0.2 mmol) and DIEA (37 .mu.L, 0.21 mmol) and dichloromethane
(2 mL) to give the title compound (32 mg, 34%). ES-MS: 461
(M+H).sup.+.
[0262] Compound 242:
[0263] This product is prepared by the Procedure-V using its free
amine intermediate (59 mg, 0.2 mmol), cyclohexyl isothiocyanate (29
.mu.L, 0.2 mmol), DIEA (35 .mu.L, 0.2 mmol) and dichloromethane (4
mL) to give the title compound (52 mg, 60%). ES-MS: 438
(M+H).sup.+.
[0264] Compound 279:
[0265] This product is prepared by generating Intermediate 12
(6-Chloro-1-[4-(2-morpholin-4-yl-ethoxy)-phenyl]-2,3,4,9-tetrahydro-1H-.b-
eta.-carboline) using Procedure-I. Intermediate 12 is then used to
generate Compound 279
(6-Chloro-1-[4-(2-morpholin-4-yl-ethoxy)-phenyl]-1,3,4,9-tetrahydro-b-car-
boline-2-carboxylic acid ethyl ester) using Procedure-II.
[0266] In accordance with Procedure-II, Intermediate 12 (82 mg,
0.20 mmol), ethyl chloroformate (24 mg, 214, 0.22 mmol), and
diisopropylethylamine (175 .mu.L, 1.00 mmol) are dissolved in
methylene chloride (2 mL) and stirred at room temperature for 15
minutes to form Compound 279. The solvent is removed under a stream
of nitrogen. The crude mixture is purified by preparative reversed
phase HPLC on a C-18 column using a gradient of acetonitrile in
water buffered with 0.2% trifluoroacetic acid (TFA). The TFA salt
of Compound 279 (3.7 mg, 3%) is isolated as a yellow solid. The
same procedure may be applied for other carbamate formation
reactions according to Procedure-II.
[0267] Compound 320:
[0268] This product/intermediate is prepared using Procedure-I with
5-benzyloxy tryptamine.HCl (100 mg, 0.33 mmol),
pyridine-3-carboxaldehyde (62 .mu.L, 0.66 mmol) and 0.1N sulfuric
acid (2 mL) to give the title compound as dihydrogen sulfate salt
(64 mg, 55%). ES-MS: 356 (M+H).sup.+
[0269] Compound 329:
[0270] This product is prepared by the Procedure-VII using the
Intermediate-11 (71 mg, 0.2 mmol), 2-bromopyrimidine (48 mg, 0.3
mmol) and triethylamine (42 .mu.L, 0.3 mmol) in DMF (2 mL) to give
the title compound (41 mg, 49%). ES-MS: 434 (M+H).sup.+.
[0271] Compound 330:
[0272] This product is prepared by the Procedure-II using the
Intermediate-6 (65 mg, 0.2 mmol), 2-fluoroethyl chloroformate (38
.mu.L, 0.3 mmol) and DIEA (70 .mu.L, 0.4 mmol) in dichloromethane
(2 mL) to give the title compound as white powder (34 mg, 41%).
ES-MS: 415 (M+H).sup.+.
[0273] Compound 332:
[0274] This product is prepared by the Procedure-II using the
Intermediate-7 (36 mg, 0.1 mmol), 4-methoxyphenyl chloroformate (22
.mu.L, 0.15 mmol) and DIEA (26 .mu.L, 0.15 mmol) in dichloromethane
(2 mL) to give the title compound as white powder (41 mg, 81%).
ES-MS: 511 (M+H).sup.+.
Example 1B
Certain Starting Materials, Scheme Ia
[0275] Scheme Ia can be used when in conjunction with Scheme I
(above) to generate starting materials when R.sub.2 is a
--CH.sub.2-furanyl group, as follows.
##STR00283##
[0276] 2-furaldehyde (0.05 mL, 1.1 eq) is added to a solution of
5-chlorotryptamine (114 mg, 0.586 mmol) in 2 mL of MeOH. The
reaction mixture is stirred at room temperature for about 1 hour.
NaBH.sub.4 (110 mg, 5 eq) is added slowly. The reaction mixture is
stirred at room temperature for about 30 min. MeOH is evaporated
and the residue is partitioned between water and methylene
chloride. The organic layer is separated and dried over
K.sub.2CO.sub.3. The collected organic layer is concentrated to
give 134.9 mg of viscous oil (84%).
Example 1C
Compounds of Formula I, Scheme Ib
[0277] Alternatively, certain compounds of Formula I may be
prepared according to Scheme Ib as follows.
##STR00284##
[0278] A suspension of reaction material A (8.05 g, 35.9 mmol) and
CH.sub.3COONH.sub.4 (4.15 g, 1.5 eq) in 60 mL of CH.sub.3NO.sub.2
is refluxed in oil bath at about 110.degree. C. After about 30
minutes, the reaction mixture is cooled with ice-bath. The
precipitated solid is filtered and washed with water (3.times.100
mL), followed by hexane (2.times.50 mL) to give crude indole
product B. The collected solid is dried under vacuum at about
40.degree. C. for about 30 min to give 6.97 g of brown solid
(73%).
##STR00285##
[0279] A solution of indole product B (12.32 g, 46.1 mmol) in THF
(130 mL) is then treated with a solution of tetrabutylammonium
borohydride (11.9 g, 1 eq) in 75 mL of THF slowly for about 60
minutes at about -5.degree. C. The reaction is stirred at room
temperature for about 1 hour and diluted with dichloromethane (200
mL). The organic layer is washed with water twice and brine. The
combined organic layers are dried and evaporated under vacuum. The
residue is purified on silica gel to give 10.28 g of solid C
(83%).
##STR00286##
[0280] Ammonium chloride (9.9 mL of aqueous solution (100 mg/mL), 2
eq) and Zn (725 mg, 1.2 eq) are then added to a solution of indole
product C (2.49 g, 9.24 mmol) in 161 mL of THF. The reaction
mixture is stirred at room temperature for about 10 min and Zn (725
mg, 1.2 eq) is then added. After about 30 min, additional Zn (967
mg, 1.6 eq) is added and stirred for about 2 hours, followed by the
addition of further Zn (845 mg, 1.4 eq). After stirring at room
temperature for about 15 min, Zn is filtered off and the residue is
concentrated and dissolved in THF. The resulting solution is then
treated with p-chlorobenzaldehyde (0.7 eq) and stirred at room
temperature for about 15 hours. The reaction mixture is
concentrated under vacuum and purified on silica gel to give 953.5
mg of the desired nitrone product D.
##STR00287##
[0281] (+)-DIP-Cl (6.93 mL, 2 eq, 85.8 mg/mL in CH.sub.2Cl.sub.2)
is then added to a solution of nitrone product D (350 mg, 0.93
mmol) in 60 mL of dichloromethane. The reaction mixture is stirred
at about -78.degree. C. for about 10 days and quenched with a
mixture of 10% NaHCO.sub.3 (7 mL) and 10 mL of water. The aqueous
layer is extracted with dichloromethane three times. Combined
organic layers are concentrated and purified on silica gel to give
the desired hydroxylamine product E (>98% ee).
##STR00288##
[0282] Water (11.5 mL), NH.sub.4Cl (2.5 mL, 5 eq) and Zn (908 mg,
15 eq) are then added to a solution of hydroxylamine product E
(0.927 mmol) in THF (28 mL). The reaction mixture is stirred at
room temperature for about 1 day. Additional THF (10 mL),
NH.sub.4Cl (5 mL, 10 eq) and Zn (1.8 g, 30 eq) are then added and
stirred for about another 21 hours. Again, THF (10 mL), NH4Cl (5
mL, 10 eq) and Zn (1.8 g, 30 eq) are added and stirred for about
another 20 hours. The reaction mixture is then filtered through
celite and washed with MC. The collected dichloromethane layer is
washed with water and brine. The organic layer is dried and
concentrated to give a boron complex of beta-carboline. This
product is dissolved in 20 mL of THF. This solution is loaded into
prepacked cation exchange resin (preconditioned with MeOH and THF)
and washed with THF. The combined THF solution is concentrated to
give 390 mg of free amine. The solid is then washed with ether and
hexane consecutively to yield 130 mg of the enantiomerically pure
compound F.
Example 1D
Compounds of Formula I, Scheme II
[0283] Compounds of Formula I-h may be prepared according to Scheme
II as follows.
##STR00289##
[0284] p-anisaldehyde (2.16 g, 15.9 mmol, 1.93 mL) is added to a
suspension of 5-Bromotryptophan A (3 g, 10.6 mmol) in 100 mL of
Acetic acid at room temperature. The reaction mixture is then
heated to reflux at about 125.degree. C. in silicon oil bath and
maintained at that temperature for about 3 hours 20 minutes. The
resultant solution is concentrated under vacuum. The residue is
triturated with dichloromethane, diethyl ether and hexane to yield
a powdery brown solid. The acetic salts of the intermediate product
B is collected and washed with hexane three times.
[0285] The intermediate product B is suspended (70 mg, 0.174 mmol)
in 2 mL of dichloromethane, and triethylamine (52.8 mg, 0.522
mmol), 5-methyl-2-aminothiazole (37.6 mg, 0.26 mmol) and PyBOP
(135.8 mg, 0.26 mmol) is added to the suspension. The reaction
mixture is stirred at room temperature for about 6 hour and
quenched with sat. NaHCO.sub.3 solution. The aqueous layer is
extracted with dichloromethane. The combined organic layers are
dried over K.sub.2CO.sub.3 and concentrated. Purification on silica
gel with 40% ethyl acetate in hexane yields 8.1 mg of the desired
amide C. LCMS [MH.sup.+]498, Rt=2.54.
Example 1E
Compounds of Formula I, Scheme III
[0286] Compounds of Formula I-i may be prepared according to Scheme
III as follows.
##STR00290##
[0287] Tryptophan A (1.0 g, 5.0 mmol) and 3-methoxybenzaldehyde
(670 .mu.L, 5.5 mmol) are suspended/dissolved in acetonitrile (100
mL) and concentrated sulfuric acid (100 .mu.L) is added. The
reaction is heated to reflux until all the aldehyde was consumed
(overnight). The solvent was removed in vacuo and the residue was
dissolved in 5 mL of ethanol. The product was precipitated out with
ether, filtered, and washed with 10 mL of ether. The desired
.beta.-carboline product/intermediate B
(1-(3-Methoxy-phenyl)-2,3,4,9-tetrahydro-1H-.beta.-carboline-3-carboxylic
acid) is isolated as a beige solid (1.2 g, 76%). LC/MS RT=2.33 min.
M/Z+323, 100%.
[0288] The .beta.-carboline product/intermediate B (200 mg, 0.62
mmol) is then dissolved in 5 mL of dry THF and cooled to about
0.degree. C. Lithium aluminum hydride (LAH) solution (1.2 mL, 1.0M
in ether, 1.2 mmol) is added to the cooled reaction mixture under
nitrogen. After the addition is complete (about 10 minutes), the
reaction is allowed to warm to room temperature for about 4 hours.
The reaction mixture is then cooled back to 0.degree. C., and
saturated sodium sulfate solution (750 .mu.L) is added and the
mixture stirred for about 5 minutes at 0.degree. C. The reaction
mixture is then filtered and washed with THF (100 mL). The solvent
is removed in vacuo, and the crude product purified by preparative
HPLC. The product C
([1-(3-Methoxy-phenyl)-2,3,4,9-tetrahydro-1H-b-carbolin-3-yl]-methanol)
is isolated as a white solid (106 mg, 55%). LC/MS RT=2.25 min.
M/Z+309, 100%.
Example 1F
Chemical Resolution of Compounds of the Invention
[0289] Compounds of the invention may optionally be chemical
resolved to enantiomerically pure compositions, preferably
enantiomerically pure (S) isomer compositions as follows.
##STR00291##
[0290] The racemic amine A (18.21 g, 58.2 mmol) is mixed with
N-acetyl-L-phenylalanine (12.05 g, 58.2 mmol) in EtOH (1.28 L) and
refluxed to get a clear solution. The solution is then allowed to
cool to room temperature. After overnight standing, the
precipitated solid is filtered and washed with EtOH (200 mL) to
give the salt B (16.4 g). The salt B is taken in EtOAc (500 mL) and
washed with aqueous 1N NaOH (300 mL.times.2) or NH.sub.4OH (200
mL.times.2), dried and evaporated to give the S-isomer of the free
amine C (7.4 g). The R-isomer is prepared by similar procedure
using N-acetyl-D-phenylalanine
Example 1G
Further Exemplary Compounds of the Invention
[0291] By way of further non-limiting example, the following
compounds (Table 5) may be prepared by similar methodology to that
described above, as will be recognized by one of skill in the
art.
TABLE-US-00002 TABLE 5 Mass Reten. Spec Time Compound NMR (LCMS)
(min) ##STR00292## (CDCl3, 400 MHz), .delta. 8.16 (s, 1H), 7.48 (s,
1H), 7.22 (d, J = 8.8 Hz, 1H), 7.19 (d, J = 8.8 Hz, 2H), 7.13 (d, J
= 8.8 Hz, 1H), 6.94 (s, 1H), 6.80 (d, J = 8.8 Hz, 2H), 3.92-3.91
(m, 1H), 3.86 (t, J = 7.2 Hz, 2H), 3.77 (s, 3H), 3.46-3.39 (m, 1H),
3.11-3.09 (m, 1H), 2.91-2.83 (m, 3H) 402.8 4.37 ##STR00293##
(CDCl3, 400 MHz), .delta. 8.29 (s, 1H), 7.47-7.09 (m, 10H), 6.98
(s, 1H), 6.77 (d, J = 8.8 Hz, 2H), 3.93 (dd, J = 13.6 Hz and 4.8
Hz, 1H), 3.82-3.80 (m, 2H), 3.77 (s, 3H), 3.38-3.30 (m, 1H),
2.69-2.65 (m, 1H), 2.53-2.45 (m, 1H) 430.9 4.79 ##STR00294##
(CDCl3, 400 MHz), .delta. 8.21 (s, 1H), 7.46 (s, 1H), 7.22 (d, J =
8.4 Hz, 1H), 7.17 (d, J = 8.4 Hz, 2H), 7.12 (dd, J = 8.4 Hz and 2.0
Hz), 6.92 (s, 1H), 6.77 (d, J = 8.4 Hz, 2H), 3.94 (dd, J = 13.2 Hz
and 4.4 Hz, 1H), 3.76 (s, 3H), 3.65 (s, 3H), 3.43-3.35 (m, 1H),
2.87-2.62 (m, 6H) 427.0 4.06 ##STR00295## (CDCl3, 400 MHz), .delta.
8.23, 8.12 (s, 1H), 7.48, 7.42 (d, J = 1.6 Hz, 1.2 Hz, 1H),
7.22-7.10 (m, 4H), 6.94, 6.88 (s, 1H), 6.79 (d, J = 8.8 Hz, 2H),
5.48-5.45 (m, 1H), 3.96-3.80 (m, 1H), 3.77 (s, 3H), 3.47-3.36 (m,
1H), 3.08-2.77 (m, 2H), 2.14, 2.09 (s, 3H), 1.48, 1.41 (d, J = 6.8
Hz, 6.4 Hz, 3H) 427.0 3.99 ##STR00296## (CDCl3, 400 MHz), .delta.
7.87 (s, 1H), 7.51 (s, 1H), 7.47 (dd, J = 6.8 Hz and 1.6 Hz, 1H),
7.30-7.15 (m, 6H), 6.98 (b, 1H), 6.76 (d, J = 8.8 Hz, 2H), 3.80 (s,
3H), 3.77-3.74 (m, 1H), 3.49-3.39 (m, 1H), 2.93-2.82 (m, 2H) 469.0
5.27 ##STR00297## (CDCl3, 400 MHz), .delta. 8.07 (dd, J = 7.6 Hz
and 1.2 Hz, 1H), 7.74 (s, 1H), 7.45-7.32 (m, 4H), 7.18 (d, J = 8.4
Hz, 1H), 7.12 (dd, J = 8.8 Hz and 2.0 Hz, 1H), 7.07 (d, J = 8.4 Hz,
2H), 6.76 (d, J = 8.8 Hz, 2H), 6.35 (s, 1H), 3.97 (dd, J = 14.8 Hz
and 5.2 Hz, 1H), 3.77 (s, 3H), 3.49-3.41 (m, 1H), 2.67 (dd, J =
15.6 Hz and 3.2 Hz, 1H), 2.57-2.53 (m, 1H) 486.9 4.96 ##STR00298##
(CDCl3, 400 MHz), .delta. 7.95 (s, 1H), 7.48 (s, 1H), 7.30 (d, J =
8.4 Hz, 2H), 7.23 (d, J = 8.8 Hz, 1H), 7.16 (dd, J = 8.8 Hz and 1.6
Hz, 1H), 7.05 (b, 3H), 6.86 (d, J = 8.4 Hz, 2H), 3.80 (s, 3H), 3.61
(dd, J = 13.6 Hz and 5.2 Hz, 1H), 3.52-3.44 (m, 1H), 2.91-2.88 (m,
1H), 2.78 (dd, J = 15.2 Hz and 3.2 Hz, 1H) 470.8 5.01 ##STR00299##
(CDCl3, 400 MHz), .delta. 8.09 (s, 1H), 7.45 (s, 1H), 7.21-7.17 (m,
4H), 7.12 (d, J = 8.8 Hz, 1H), 6.98 (s, 1H), 6.91 (d, J = 4 Hz,
1H), 6.80 (s, 1H), 6.79 (d, J = 8.4 Hz, 2H), 3.99 (s, 2H), 3.96 (d,
J = 4.4 Hz, 1H), 3.77 (s, 3H), 3.43-3.38 (m, 1H), 2.77-2.63 (m, 2H)
436.9 4.66 ##STR00300## (CDCl3, 400 MHz), .delta. 8.19, 8.16 (s,
1H), 7.48, 8.42 (s, 1H), 7.24-7.09 (m, 6H), 6.94 (t, J = 7.8 Hz,
2H), 6.85 (t, J = 8.2 Hz, 2H), 6.77 (d, J = 8.4 Hz, 1H), 6.72 (d, J
= 8.4 Hz, 1H), 5.09-4.98 (m, 1H), 4.39-4.17 (m, 1H), 3.77, 3.75 (s,
3H), 3.41-3.28 (m, 1H), 3.02-2.65 (m, 2H), 1.61-1.59 (m, 3H) 461
4.92 ##STR00301## (CDCl3, 400 MHz), .delta. 8.39 (s, 1H), 7.48 (s,
1H), 7.23 (d, J = 8.4 Hz, 1H), 7.19 (d, J = 8.4 Hz, 2H), 7.13 (dd,
J = 8.8 Hz and 1.6 Hz, 1H), 6.89 (s, 1H), 6.77 (d, J = 8.4 Hz, 2H),
4.17 (q, J = 12.8 Hz, 2H), 3.88 (d, J = 10 Hz, 1H), 3.75 (s, 3H),
3.41 (s, 3H), 3.38-3.34 (m, 1H), 2.95-2.81 (m, 2H) 385 3.79
##STR00302## (CD3OD, 400 MHz), .delta. 7.48-7.46 (m, 4H), 7.35 (b,
1H), 7.23 (d, J = 8.8 Hz, 1H), 7.07 (dd, J = 8.8 Hz and 2.0 Hz,
1H), 6.46 (b, 1H), 4.35-4.14 (m, 5H), 3.52-3.47 (m, 2H), 3.22-3.19
(m, 7H), 2.98-2.93 (m, 3H), 2.89 (s, 6H), 2.67-2.63 (m, 5H),
2.06-1.96 (m, 2H), 1.31 (t, J = 7.2 Hz, 3H) 538.3 4.29 ##STR00303##
(DMSO, 400 MHz), .delta. 11.00 (s, 1H), 8.47 (s, 2H), 7.67 (s, 1H),
7.26 (d, J = 8.4 Hz, 1H), 7.19 (dd, J = 8.8 Hz and 2.0 Hz, 1H),
6.26 (b, 1H), 4.25 (b, 1H), 4.11 (t, J = 6.8 Hz, 2H), 3.22-3.17 (m,
1H), 2.86-2.81 (m, 1H), 2.77-2.66 (m, 1H), 2.50 (b, 3H), 1.21 (t, J
= 6.8 Hz, 3H) 447.1 6.55 ##STR00304## (CD3OD, 400 MHz), .delta.
8.43-8.41 (m, 4H), 7.63 (d, J = 1.2 Hz, 1H), 7.22 (d, J = 8.8 Hz,
1H), 7.19 (dd, J = 8.4 Hz and 1.6 Hz, 1H), 7.04 (s, 1H), 6.67 (t, J
= 4.8 Hz, 1H), 5.01 (dd, J = 14.0 Hz and 3.6 Hz, 1H), 3.29-3.26 (m,
1H), 3.21 (s, 6H), 2.91-2.86 (m, 2H) 450.1 5.48 ##STR00305## (DMSO,
400 MHz), .delta. 11.15, 11.05 (b, 1H), 7.53 (d, J = 1.6 Hz, 1H),
7.29 (d, J = 8.8 Hz, 1H), 7.20-7.18 (m, 6H), 7.06 (dd, J = 8.8 Hz
and 2 Hz, 1H), 6.93 (d, J = 7.2 Hz, 2H), 6.45-6.37 (m, 1H), 4.30
(b, 1H), 3.72 (s, 3H), 3.18 (b, 1H), 2.82 (b, 2H) 451.3 3.99
##STR00306## (CD3OD, 400 MHz), .delta. 10.98 (b, 1H), 7.49 (d, J =
2.0 Hz, 1H), 7.34-7.30 (m, 5H), 7.25-7.21 (m, 1H), 7.13 (dd, J =
8.8 Hz and 2.0 Hz, 1H), 4.81-4.79 (m, 1H), 3.82-3.76 (m, 1H),
3.54-3.49 (m, 1H), 3.11-3.07 (m, 2H), 2.91-2.87 (m, 2H), 2.59-2.55
(m, 1H), 2.24-2.20 (m, 1H) 311.1 4.39 ##STR00307## (CD3OD, 400
MHz), .delta. 7.61 (s, 1H), 7.46 (d, J = 8.0 Hz, 2H), 7.38 (d, J =
8.0 Hz, 2H), 7.19 (s, 2H), 6.47 (s, 1H), 4.32-4.19 (m, 5H), 3.62
(t, J = 3.9 Hz, 2H), 3.42 (s, 1H), 3.19-3.10 (m, 3H), 2.29-2.76 (m,
2H), 1.30 (s, 3H) 486.6 3.45 ##STR00308## (CD3OD, 400 MHz), .delta.
7.63 (s, 1H), 7.49 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H),
7.19 (s, 2H), 6.49 (b, 1H), 4.34-4.19 (m, 4H), 3.60 (b, 4H),
3.29-3.17 (m, 6H), 2.89-2.75 (m, 2H), 1.36 (t, J = 7.2 Hz, 3H),
1.30 (b, 3H) 539.2 3.11 ##STR00309## (CDCl3, 400 MHz), .delta. 8.56
(b, 1H), 8.40 (b, 2H), 7.68 (s, 1H), 7.28 (d, J = 2.0 Hz, 1H), 7.14
(d, J = 8.4 Hz, 1H), 7.00 (d, J = 9.2 Hz, 2H), 6.80 (d, J = 8.4 Hz,
2H), 6.48-6.38 (m, 1H), 4.55-4.52 (m, 1H), 3.81-3.74 (m, 4H), 3.24
(s, 6H), 3.00-2.91 (m, 1H), 2.88-2.84 (m, 1H) 522.2 5.05
##STR00310## (DMSO, 400 MHz), .delta. 11.00 (s, 1H), 8.14 (s, 2H),
7.64 (s, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.18 (d, J = 8.8 Hz, 1H),
6.14 (s, 1H), 4.23 (b, 1H), 4.11-4.08 (m, 2H), 3.14-3.10 (m, 1H),
3.08 (s, 6H), 2.81-2.77 (m, 1H), 2.70-2.66 (m, 1H), 1.21 (t, J =
6.8 Hz, 3H) 444.3 3.95 ##STR00311## (CD3OD, 400 MHz), .delta. 7.79
(d, J = 8.4 Hz, 2H), 7.63 (s, 1H), 7.37 (d, J = 8.4 Hz, 2H), 7.20
(s, 2H), 6.51 (b, 1H), 4.32-4.22 (m, 3H), 3.54 (s, 3H), 3.36 (s,
2H), 3.30 (s, 2H), 3.21-3.11 (m, 1H), 2.90-2.77 (m, 2H), 1.32 (s,
3H) 500.1 4.35 ##STR00312## (CDCl3, 400 MHz), .delta. 7.98, 7.81
(s, 1H), 7.42 (s, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.11 (d, J = 8.4
Hz, 1H), 5.40-5.23 (m, 3H), 4.55-4.35 (m, 1H), 4.20-4.11 (m, 2H),
3.24-3.13 (m, 1H), 2.79-2.63 (m, 2H), 2.22 (d, J = 6.8 Hz, 2H),
2.08 (b, 2H), 1.89-1.81 (m, 2H), 1.30 (b, 3H), 0.97 (b, 3H) 361.2
5.95 ##STR00313## (CD3OD, 400 MHz), .delta. 7.47 (d, J = 1.6 Hz,
1H), 7.43 (d, J = 7.6 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.24 (d, J
= 8.8 Hz, 1H), 7.06 (dd, J = 8.4 Hz and 1.6 Hz, 1H), 6.49 (b, 1H),
4.35-4.21 (m, 3H), 3.83 (s, 4H), 3.19-3.10 (m, 1H), 2.90-2.79 (m,
2H), 1.57 (b, 6H), 1.32 (s, 3H) 482.1 5.11 ##STR00314## (CDCl3, 400
MHz), .delta. 8.48-8.09 (m, 1H), 7.44-7.42 (m, 1H), 7.24 (t, J = 9
Hz, 1H), 7.11-7.09 (m, 1H), 5.59-5.40 (m, 1H), 4.54-4.34 (m, 1H),
4.21-4.18 (m, 2H), 3.23-3.13 (m, 1H), 2.87-2.81 (m, 2H), 2.76-263
(m, 1H), 2.17 (s, 3H), 2.12-1.90 (m, 2H), 1.42-1.24 (m, 6H) 367.1
2.92 ##STR00315## (CD3OD, 400 MHz), .delta. 8.62 (d, J = 4.4 Hz,
2H), 8.59 (s, 2H), 7.84 (s, 1H), 7.43-7.39 (m, 2H), 7.24 (s, 1H),
6.88 (t, J = 8.0 Hz, 1H), 5.24-5.20 (m, 1H), 3.47-3.44 (m, 1H),
3.16 (s, 3H), 3.11-3.05 (m, 2H) 436.2 5.25 ##STR00316## (CDCl3, 400
MHz), .delta. 8.12 (s, 1H), 7.45 (s, 1H), 7.26 (d, J = 8 Hz, 2H),
7.18 (d, J = 8.8 Hz, 2H), 7.14-7.12 (m, 4H), 6.97 (s, 1H), 6.78 (d,
J = 8.8 Hz, 2H), 3.89 (dd, J = 14 Hz and 1.2 Hz, 1H), 3.80-3.78 (m,
5H), 3.41-3.33 (m, 1H), 2.73 (dd, J = 15.2 Hz and 3.2 Hz, 1H),
2.64-2.60 (m, 1H) 464.9 5.11 ##STR00317## (CD3OD, 400 MHz), .delta.
7.78 (d, J = 8.0 Hz, 2H), 7.47 (d, J = 1.6 Hz, 1H), 7.37 (d, J =
8.0 Hz, 2H), 7.24 (d, J = 8.4 Hz, 1H), 7.06 (dd, J = 8.8 Hz and 1.6
Hz, 1H), 6.49 (b, 1H), 4.31-4.05 (m, 8H), 3.20-3.11 (m, 1H),
3.00-2.77 (m, 4H), 1.94-1.90 (m, 2H), 1.54-1.45 (m, 2H), 1.31 (b,
3H), 1.25 (t, J = 7.2 Hz, 3H) 553.1 6.13 ##STR00318## (CD3OD, 400
MHz), .delta. 7.80 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 16 Hz, 1H),
7.38 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.8 Hz, 1H), 7.07 (dd, J =
8.4 Hz and 1.6 Hz, 1H), 6.49 (b, 1H), 4.31-4.21 (m, 4H), 4.06 (t, J
= 8.4 Hz, 1H), 3.74 (t, J = 8.0 Hz, 1H), 3.51 (d, J = 5.2 Hz, 2H),
3.21-3.11 (m, 1H), 2.90-2.79 (m, 2H), 2.26 (s, 1H), 1.39 (s, 3H),
1.32 (s, 6H) 454.3 5.98 ##STR00319## (CDCl3, 400 MHz), .delta. 8.29
(b, 1H), 7.64 (d, J = 8.0 Hz, 2H), 7.61 (d, J = 7.2 Hz, 2H),
7.50-7.45 (m, 5H), 7.39 (d, J = 7.6 Hz, 1H), 7.33 (d, J = 7.6 Hz,
1H), 7.19 (d, J = 8.8 Hz, 1H), 7.14 (dd, J = 8.4 Hz and 1.6 Hz,
1H), 7.08 (s, 1H), 6.84 (d, J = 8 Hz, 2H), 3.87 (d, J = 9.2 Hz,
1H), 3.79 (s, 3H), 3.45-3.40 (m, 1H), 2.96-2.94 (m, 1H), 2.80-2.76
(m, 1H) 493.0 5.71 ##STR00320## (CD3OD, 400 MHz), .delta. 7.63 (s,
1H), 7.48 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 7.20 (s,
2H), 6.49 (b, 1H), 4.33-4.22 (b, 3H), 3.89 (t, J = 5.2 Hz, 2H),
3.50 (b, 4H), 3.21-3.11 (m, 2H), 2.91-2.78 (m, 2H), 1.31 (s, 3H)
555.2 3.14 ##STR00321## (CD3OD, 400 MHz), .delta. 7.47 (d, J = 2.0
Hz, 1H), 7.39 (s, 4H), 7.23 (d, J = 8.8 Hz, 1H), 7.06 (dd, J = 8.4
Hz and 2.0 Hz, 1H), 6.49 (b, 1H), 4.35-4.21 (m, 3H), 3.75 (b, 2H),
3.53 (t, J = 5.4 Hz, 2H), 3.44 (b, 2H), 3.26-3.30 (m, 4H),
3.22-3.13 (m, 1H), 2.89-2.78 (m, 2H), 2.60 (t, J = 5.4 Hz, 4H),
2.46 (b, 2H), 1.32 (s, 3H) 525.2 5.07 ##STR00322## (CDCl3, 400
MHz), .delta. 7.80, 7.75 (s, 1H), 7.43, 7.41 (s, 1H), 7.21 (d, J =
8.4 Hz, 1H), 7.10 (d, J = 8.0 Hz, 1H), 5.43, 5.27 (d, J = 7.2 Hz,
1H), 4.51-4.30 (m, 1H), 4.21-4.10 (m, 2H), 3.18 (q, J = 12.8 Hz,
1H), 2.82-2.76 (m, 1H), 2.64-2.61 (m, 1H), 1.82-1.76 (m, 2H),
1.55-1.53 (m, 1H), 1.29-1.24 (m, 3H), 1.08 (b, 3H), 0.98 (d, J =
6.8 Hz, 3H) 335.3 5.52 ##STR00323## (CD3OD, 400 MHz), .delta. 7.47
(d, J = 2.0 Hz, 1H), 7.39 (s, 4H), 7.23 (d, J = 8.8 Hz, 1H), 7.05
(dd, J = 8.4 Hz and 2.0 Hz, 1H), 6.49 (b, 1H), 4.32-4.20 (m, 3H),
3.76 (b, 2H), 3.46 (b, 2H), 3.21-3.13 (m, 1H), 2.90-2.78 (m, 2H),
2.54 (b, 2H), 2.49-2.43 (m, 4H), 1.32 (b, 3H), 1.10 (t, J = 7.2 Hz,
3H) 495.3 4.68 ##STR00324## ((CD3OD, 400 MHz), .delta. 7.61 (s,
1H), 7.44 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 7.20-7.16
(m, 2H), 6.45 (b, 1H), 4.28-4.14 (m, 3H), 4.11 (s, 2H), 3.47 (s,
4H), 3.26 (s, 4H), 3.19-3.12 (m, 1H), 2.91 (s, 3H), 2.88-2.79 (m,
2H), 1.30 (s, 3H) 511.2 4.99 ##STR00325## (CD3OD, 400 MHz), .delta.
7.48 (d, J = 1.6 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.12 (dd, J =
8.8 Hz and 2.0 Hz, 1H), 4.68 (s, 1H), 3.77-3.72 (m, 1H), 3.47-3.44
(m, 1H), 3.10-3.03 (m, 2H), 2.65-2.61 (m, 1H), 1.25 (d, J = 7.2 Hz,
3H), 0.96 (d, J = 7.2 Hz, 3H) 249.1 3.67 ##STR00326## CD3OD, 400
MHz), .delta. 7.63 (s, 1H), 7.48 (d, J = 8.0 Hz, 2H), 7.42 (d, J =
8.0 Hz, 2H), 7.20 (s, 2H), 6.49 (b, 1H), 4.32-4.21 (m, 3H), 3.50
(b, 4H), 3.21-3.15 (m, 3H), 2.92 (s, 3H), 2.90-2.73 (m, 2H), 1.32
(s, 3H) 525.1 3.25 ##STR00327## (CD3OD, 400 MHz), .delta. 7.78 (d,
J = 8.0 Hz, 2H), 7.63 (s, 1H), 7.37 (d, J = 8.4 Hz, 2H), 7.20 (s,
2H), 6.49 (b, 1H), 4.31-4.22 (m, 3H), 3.19-3.11 (m, 1H), 2.90 (s,
3H), 2.86-2.77 (m, 2H), 1.32 (s, 3H) 456.1 4.26 ##STR00328##
(CD3OD, 400 MHz), .delta. 7.48 (d, J = 2 Hz, 1H), 7.41-7.36 (m,
4H), 7.23 (d, J = 8.4 Hz, 1H), 7.06 (dd, J = 8.8 Hz and 2.0 Hz,
1H), 6.49 (b, 1H), 4.35-4.21 (m, 3H), 3.64 (b, 2H), 3.45 (b, 2H),
3.20-3.11 (m, 1H), 2.92-2.78 (m, 2H), 2.68 (b, 2H), 2.55 (b, 2H),
1.92-1.80 (m, 4H), 1.66-1.62 (m, 1H), 1.32-1.22 (m, 8H) 549.3 5.29
##STR00329## (CD3OD, 400 MHz), .delta. 7.63 (s, 1H), 7.41 (d, J =
8.4 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.19 (s, 2H), 6.49 (b, 1H),
4.35-4.22 (m, 3H), 3.22-3.13 (m, 1H), 3.08 (s, 3H), 2.98 (s, 3H),
2.89-2.77 (m, 2H), 1.32 (s, 3H) 470.1 4.46 ##STR00330## (CD3OD, 400
MHz), .delta. 7.63 (s, 1H), 7.48 (d, J = 7.2 Hz, 2H), 7.40 (d, J =
8.0 Hz, 2H), 7.20 (s, 2H), 6.49 (b, 1H), 4.35-4.22 (m, 4H),
3.82-3.50 (m, 6H), 3.45 (b, 1H), 3.21-3.11 (m, 1H), 3.00-2.78 (m,
5H), 2.25-2.15 (m, 2H), 1.32 (s, 3H) 539.2 3.02 ##STR00331##
(CDCl3, 400 MHz), .delta. 8.06 7.98 (s, 1H), 7.50, 7.49 (s, 1H),
7.22 (d, J = 6.0 Hz, 1H), 7.21 (d, J = 6.4 Hz, 2H), 7.15 (dd, J =
8.8 Hz and 1.6 Hz, 1H), 6.81 (d, J = 8.4 Hz, 2H), 6.77 (s, 1H),
3.91 (s, 3H), 3.77 (s, 3H), 3.72 (d, J = 5.2 Hz, 1H), 3.51-3.43 (m,
1H), 3.02-2.96 (m, 1H), 2.86-2.81 (m, 1H) 398.9 4.18 ##STR00332##
(CDCl3, 400 MHz), .delta. 7.77, 7.70 (s, 1H), 7.42, 7.39 (s, 1H),
7.20 (dd, J = 8.4 and 1.6 Hz, 1H), 7.09 (d, J = 8.0 Hz, 1H),
5.52-5.36 (m, 1H), 4.44-4.17 (m, 3H), 3.28-3.20 (m, 1H), 2.88-2.77
(m, 1H), 2.60 (d, J = 15.2 Hz, 1H), 2.05-1.88 (m, 1H), 1.58-1.54
(m, 1H), 1.30-1.26 (m, 3H), 1.04 (d, J = 2 Hz, 9H) 349.1 6.03
##STR00333## (CD3OD, 400 MHz), .delta. 7.85 (d, J = 8.0 Hz, 2H),
7.64 (s, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.20 (s, 2H), 6.52 (b, 1H),
4.33-4.22 (b, 3H), 4.07 (b, 2H), 3.77 (t, J = 5.6 Hz, 4H), 3.65 (b,
2H), 3.39 (t, J = 5.6 Hz, 2H), 3.21-3.11 (m, 3H), 2.91-2.78 (m,
2H), 1.32 (s, 3H) 555.2 3.34 ##STR00334## (CD3OD, 400 MHz), .delta.
7.81 (d, J = 8.4 Hz, 2H), 7.63 (s, 1H), 7.37 (d, J = 8.0 Hz, 2H),
7.20 (s, 2H), 6.51 (b, 1H), 4.32-4.22
(m, 3H), 3.69 (t, J = 5.8 Hz, 2H), 3.48 (t, J = 5.6 Hz, 2H),
3.21-3.11 (m, 1H), 2.90-2.77 (m, 2H), 1.32 (s, 3H) 486.1 3.80
##STR00335## (CD3OD, 400 MHz), .delta. 7.47 (s, 1H), 7.41-7.38 (m,
4H), 7.23 (d, J = 8.8 Hz, 1H), 7.06 (dd, J = 8.8 Hz and 1.6 Hz,
1H), 6.49 (b, 1H), 4.35-4.21 (m, 3H), 3.73-3.62 (m, 6H), 3.44 (b,
2H), 3.19-3.10 (m, 1H), 2.91-2.78 (m, 2H), 1.32 (b, 3H) 468 5.52
##STR00336## (DMSO, 400 MHz), .delta. 11.19 (b, 1H), 8.49 (b, 1H),
7.81 (d, J = 8.0 Hz, 2H), 7.51 (d, J = 1.6 Hz, 1H), 7.30 (d, J =
8.4 Hz, 2H), 7.29 (d, J = 14.0 Hz, 1H), 7.07 (dd, J = 8.4 Hz and
1.6 Hz, 1H), 6.39 (b, 1H), 4.21-4.16 (m, 3H), 3.93 (t, J = 6.4 Hz,
1H), 3.74 (q, J = 6.8 Hz, 1H), 3.59 (q, J = 6.8 Hz, 1H), 3.28 (s,
2H), 3.08-3.01 (m, 1H), 2.81-2.70 (m, 2H), 1.91-1.79 (m, 3H),
1.59-1.52 (m, 1H), 1.21 (s, 3H) 482.2 5.74 ##STR00337## (CD3OD, 400
MHz), .delta. 11.05 (s, 1H), 8.09 (s, 2H), 7.64 (s, 1H), 7.32 (b,
1H), 7.24 (d, J = 8.4 Hz, 1H), 7.17 (dd, J = 8.8 Hz and 2.0 Hz,
1H), 6.24 (s, 1H), 4.22 (b, 1H), 4.12-4.09 (m, 2H), 3.15-3.09 (m,
1H), 2.83-2.65 (m, 5H), 1.21 (t, J = 6.8 Hz, 3H) 430.2 3.65
##STR00338## (CD3OD, 400 MHz), .delta. 7.49 (d, J = 1.6 Hz, 1H),
7.34 (d, J = 8.8 Hz, 1H), 7.13 (dd, J = 8.8 Hz and 2.0 Hz, 1H),
3.77-3.72 (m, 1H), 3.52-3.45 (m, 1H), 3.15-3.01 (m, 2H), 2.80-2.74
(m, 2H), 2.60-2.52 (m, 1H), 2.27-2.20 (m, 4H) 281.0 3.84
##STR00339## (CDCl3, 400 MHz), .delta. 8.35 (b, 1H), 7.51 (s, 1H),
7.32-7.26 (m, 4H), 7.20 (d, J = 8.4 Hz, 1H), 7.13 (dd, J = 8.8 Hz
and 2.4 Hz, 1H), 6.39 (b, 1H), 4.25-4.21 (m, 2H), 3.80 (b, 2H),
3.47 (b, 2H), 3.16-3.10 (m, 1H), 2.96-2.88 (m, 3H), 2.79-2.75 (m,
1H), 2.54-2.36 (m, 6H), 1.32 (s, 3H) 481.4 4.81 ##STR00340## (DMSO,
400 MHz), .delta. 10.86 (s, 1H), 8.17 (s, 1H), 8.03 (d, J = 7.6 Hz,
1H), 7.81 (t, J = 8.0 Hz, 1H), 7.65 (d, J = 8.4 Hz, 1H), 7.50 (b,
2H), 7.26 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 8.8 Hz, 1H), 6.24 (s,
1H), 4.35 (b, 1H), 4.09-4.05 (m, 2H), 3.61-3.49 (m, 1H), 2.78-2.65
(m, 2H), 1.45 (t, J = 6.8 Hz, 3H) 423.3 5.15 ##STR00341## (CD3OD,
400 MHz), .delta. 8.33 (s, 2H), 7.67 (s, 1H), 7.23 (s, 2H), 7.05
(d, J = 8.4 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 6.54-6.38 (m, 1H),
4.52 (b, 1H), 3.78 (s, 3H), 3.36-3.34 (m, 1H), 2.99 (s, 3H),
2.92-2.88 (m, 2H) 508.2 5.72 ##STR00342## (CDCl3, 400 MHz), .delta.
7.88-7.77 (m, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.23 (d, J = 8.8 Hz,
1H), 7.11 (d, J = 8.8 Hz 1H), 5.70-7.68 (m, 2H), 5.19-4.97 (m, 1H),
4.60-4.38 (m, 1H), 4.19-4.07 (m, 2H), 2.82-2.80 (m, 1H), 2.68-2.64
(m, 1H), 2.29-1.84 (m, 6H), 1.55-1.46 (m, 1H), 1.36-1.24 (m, 3H)
359.1 5.65 ##STR00343## (CD3OD, 400 MHz), .delta. 7.84 (d, J = 8.0
Hz, 2H), 7.63 (s, 1H), 7.38 (d, J = 8.0 Hz, 2H), 7.20 (s, 2H), 6.49
(b, 1H), 4.31-4.22 (m, 3H), 3.19-3.11 (m, 1H), 2.89-2.77 (m, 2H),
1.32 (s, 3H) 442.0 4.06 ##STR00344## (CD3OD, 400 MHz), .delta. 8.44
(s, 2H), 7.67 (d, J = 2.0 Hz, 1H), 7.44 (d, J = 8.8 Hz, 1H), 7.28
(dd, J = 8.8 Hz and 2.0 Hz, 1H), 6.52 (s, 1H), 4.58-4.55 (m, 1H),
4.43-4.40 (m, 2H), 3.41-3.31 (m, 1H), 3.15 (s, 3H), 3.03-3.01 (m,
2H), 1.32 (b, 3H) 386.3 5.32 ##STR00345## (CDCl3, 400 MHz), .delta.
7.66 (d, J = 24.8 Hz, 1H), 7.39-6.89 (m, 8H), 5.44-5.02 (m, 1H),
4.49-4.10 (m, 3H), 3.23-2.94 (m, 2H), 2.38-2.74 (m, 1H), 2.64-2.58
(m, 1H), 2.26-1.98 (m, 2H), 1.47-1.26 (m, 6H) 397.1 5.97
##STR00346## (CD3OD, 400 MHz), .delta. 7.80 (d, J = 8.4 Hz, 2H),
7.47 (d, J = 1.6 Hz, 1H), 7.38 (d, J = 8.0 Hz, 2H), 7.24 (d, J =
8.8 Hz, 1H), 7.07 (dd, J = 8.0 Hz and 1.6 Hz, 1H), 6.49 (b, 1H),
4.35-4.21 (m, 3H), 3.69 (t, J = 4.6 Hz, 4H), 3.53 (t, J = 6.8 Hz,
2H), 3.19-3.10 (m, 1H), 2.90-2.78 (m, 2H), 2.59 (t, J = 6.6 Hz,
4H), 2.53 (s, 2H), 1.32 (s, 3H) 511.4 5.05 ##STR00347## (CDCl3, 400
MHz), .delta. 8.09, 7.83 (s, 1H), 7.42 (s, 1H), 7.21 (d, J = 8.4
Hz, 1H), 7.09 (dd, J = 8.4 Hz and 1.2 Hz, 1H), 5.33-5.21 (m, 1H),
4.50-4.34 (m, 1H), 4.21-4.10 (m, 2H), 3.19-3.17 (m, 1H), 2.77-2.74
(m, 1H), 2.67-2.61 (m, 1H), 1.81 (s, 2H), 1.52 (s, 2H), 1.29-1.23
(m, 3H), 0.96 (s, 3H) 321.4 5.19 ##STR00348## (CDCl3, 400 MHz),
.delta. 7.73-7.52 (m, 1H), 7.47 (s, 1H), 7.42-7.18 (m, 6H), 7.09
(dd, J = 8.8 Hz and 2.0 Hz, 1H), 5.41-5.26 (m, 1H), 4.56-4.32 (m,
1H), 4.23-4.10 (m, 2H), 3.21 (b, 1H), 2.85-2.72 (m, 3H), 2.65 (d, J
= 14.2 Hz, 1H), 2.23-2.10 (m, 2H), 1.38 (b, 3H) 383.1 5.75
##STR00349## (CD3OD, 400 MHz), .delta. 7.80 (d, J = 8.4 Hz, 2H),
7.47 (d, J = 1.6 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2H), 7.24 (d, J =
8.8 Hz, 1H), 7.06 (dd, J = 8.8 Hz and 2.0 Hz, 1H), 6.50 (b, 1H),
4.32-4.21 (m, 3H), 3.47 (t, J = 7.2 Hz, 2H), 3.38-3.34 (m, 4H),
3.19-3.10 (m, 1H), 2.89-2.78 (m, 2H), 2.39 (t, J = 8.4 Hz, 2H),
2.09-2.00 (m, 2H), 1.86-1.80 (m, 2H), 1.32 (b, 3H) 523.1 5.69
##STR00350## (CDCl3, 400 MHz), .delta. 7.81 (s, 1H), 7.51 (d, J =
6.8 Hz, 1H), 7.29 (dd, J = 12.0 Hz and 2.8 Hz, 1H), 7.21 (d, J =
8.4 Hz, 1H), 7.13 (dd, J = 8.4 Hz and 2.0 Hz, 1H), 7.12-7.08 (m,
1H), 7.07 (s, 1H), 6.50 (b, 1H), 4.49-4.21 (m, 3H), 3.17-3.09 (m,
1H), 2.91-2.85 (m, 1H), 2.77-2.73 (m, 1H), 1.39 (s, 3H) 361.1 5.12
##STR00351## (CD3OD, 400 MHz), .delta. 7.47 (d, J = 2.0 Hz, 1H),
7.46-7.37 (m, 4H), 7.23 (d, J = 8.4 Hz, 1H), 7.06 (dd, J = 8.8 Hz
and 2.0 Hz, 1H), 6.49 (b, 1H), 4.35-4.21 (m, 3H), 3.77-3.69 (m,
2H), 3.55-3.45 (m, 2H), 3.20-3.11 (m, 1H), 2.90-2.78 (m, 3H),
2.67-2.55 (m, 3H), 2.39-2.31 (m, 3H), 2.01-1.95 (m, 1H), 1.82-1.79
(m, 1H), 1.32 (s, 3H) 495.3 4.67 ##STR00352## (CDCl3, 400 MHz),
.delta. 7.92, 7.82 (s, 1H), 7.42 (s, 1H), 7.22 (dd, J = 8.4 Hz and
1.2 Hz, 1H), 7.10 (d, J = 8.8 Hz, 1H), 5.31, 5.19 (s, 1H), 4.52,
4.32 (d, J = 10.8 Hz, 1H), 4.20-4.12 (m, 2H), 3.19-3.12 (m, 1H),
2.81-2.62 (m, 2H), 1.81 (d, J = 6.8 Hz, 2H), 1.48-22 (m, 12H), 0.88
(s, 3H) 363.5 6.34 ##STR00353## (CD3OD, 400 MHz), .delta. 7.63 (s,
1H), 7.50 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 7.20 (s,
2H), 6.49 (b, 1H), 4.30-4.20 (m, 3H), 3.89 (s, 2H), 3.45 (b, 2H),
3.20-3.10 (m, 1H), 3.03-3.01 (m, 9H), 2.91-2.80 (m, 2H), 1.32 (s,
3H) 527.1 3.16 ##STR00354## (CD3OD, 400 MHz), .delta. 8.27 (s, 2H),
7.52 (d, J = 2 Hz, 1H), 7.27 (d, J = 8.8 Hz, 1H), 7.17 (d, J = 8.8
Hz, 1H), 7.10 (dd, J = 8.8 Hz and 2.4 Hz, 1H), 7.05 (d, J = 8.8 Hz,
2H), 6.95 (d, J = 9.2 Hz, 2H), 6.92 (s, 1H), 6.58-6.38 (m, 1H),
4.52 (b, 1H), 3.80 (s, 1H), 3.79 (s, 3H), 3.31-3.30 (m, 1H), 2.95
(s, 3H), 2.92-2.88 (m, 1H) 464.2 5.86 ##STR00355## (CD3OD, 400
MHz), .delta. 8.49, 8.29 (d, J = 4.4 Hz, 2.8 Hz, 1H), 7.82, 7.70
(t, J = 2.0 Hz, 1H), 7.46 (s, 1H), 7.38-7.23 (m, 5H), 7.15 (d, J =
7.6 Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H), 6.98 (d, J = 6.8 Hz, 1H),
6.46 (b, 1H), 4.35-4.21 (m, 3H), 3.88 (t, J = 7.0 Hz, 1H),
3.71-3.67 (m, 1H), 3.20-3.11 (m, 3H), 3.01-2.80 (m, 4H), 1.32 (s,
3H) 517.6 5.03 ##STR00356## (DMSO, 400 MHz), .delta. 11.15 (s, 1H),
7.51 (d, J = 2.0 Hz, 1H), 7.42 (t, J = 7.6 Hz, 1H), 7.35 (d, J =
7.6 Hz, 1H), 7.30 (d, J = 8.8 Hz, 2H), 7.16 (s, 1H), 7.06 (dd, J =
8.8 Hz and 2.0 Hz, 1H), 6.36 (b, 1H), 4.18-4.10 (m, 3H), 3.09-3.00
(m, 1H), 2.91-2.64 (m, 8H), 1.21 (t, J = 6.6 Hz, 3H) 426.2 4.29
##STR00357## (CD3OD, 400 MHz), .delta. 7.81 (d, J = 8.4 Hz, 2H),
7.47 (d, J = 1.6 Hz, 1H), 7.39 (d, J = 8.4 Hz, 2H), 7.24 (d, J =
8.4 Hz, 1H), 7.07 (dd, J = 8.4 Hz and 2.0 Hz, 1H), 6.50 (b, 1H),
4.35-4.29 (m, 3H), 3.70-3.60 (m, 1H), 3.51-3.47 (m, 2H), 3.37-3.29
(m, 1H), 3.19-3.11 (m, 2H), 2.92 (s, 3H), 2.88-2.78 (m, 2H),
2.51-2.41 (m, 1H), 2.29-2.20 (m, 1H), 2.17-2.00 (m, 2H), 1.89-1.78
(m, 2H), 1.32 (s, 3H) 509.4 4.99 ##STR00358## (CDCl3, 400 MHz),
.delta. 7.91, 7.72 (s, 1H), 7.50-7.43 (s, 1H), 7.22-7.06 (m, 6H),
5.28-5.19 (m, 1H), 4.64-4.45 (m, 1H), 4.20 (b, 2H), 3.27-3.10 (m,
2H), 2.91-2.72 (m, 2H), 2.70-2.66 (m, 1H), 2.49-2.28 (m, 2H),
1.38-1.24 (m, 9H), 1.01, 0.96 (d, J = 6.8 Hz, 3H) 439.0 6.11
##STR00359## (DMSO, 400 MHz), .delta. 11.10 (s, 1H), 8.42 (s, 1H),
7.75 (d, J = 7.2 Hz, 1H), 7.67 (s, 1H), 7.51 (d, J = 1.6 Hz, 1H),
7.43 (t, J = 7.2 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.29 (d, J =
8.4 Hz, 1H), 7.06 (dd, J = 8.8 Hz and 2.4 Hz, 1H), 6.39 (b, 1H),
4.13-4.09 (m, 3H), 3.10-3.04 (m, 1H), 2.81-2.72 (m, 5H), 1.21 (s,
3H) 412.1 4.13 ##STR00360## (CD3OD, 400 MHz), .delta. 7.53-7.46 (m,
4H), 7.29 (b, 1H), 7.25 (d, J = 8.8 Hz, 1H), 7.08 (dd, J = 8.8 Hz
and 2.0 Hz, 1H), 6.49 (b, 1H), 4.34-4.23 (m, 3H), 3.53-3.42 (m,
2H), 3.18-3.12 (m, 5H), 2.91-2.74 (m, 3H), 1.32 (t, J = 7.2 Hz, 6H)
4.95.3 3.46 ##STR00361## (CD3OD, 400 MHz), .delta. 7.63 (s, 1H),
7.51 (d, J = 8.0 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 7.19 (d, J =
1.2 Hz, 2H), 6.46 (b, 1H), 4.31 (s, 2H), 4.23-4.20 (m, 3H),
3.62-3.50 (m, 4H), 3.19-3.11 (m, 1H), 2.92 (s, 6H), 2.87-2.81 (m,
2H), 2.76 (s, 3H), 1.31 (s, 3H) 513.2 4.43 ##STR00362## (CD3OD, 400
MHz), .delta. 7.47 (d, J = 2 Hz, 1H), 7.46-7.37 (m, 4H), 7.24 (d, J
= 8.8 Hz, 1H), 7.07 (d, J = 8.8 Hz and 2.0 Hz, 1H), 6.49 (b, 1H),
4.74 (b, 1H), 4.35-4.21 (m, 3H), 3.85 (b, 1H), 3.64 (b, 2H),
3.45-3.37 (m, 1H), 3.19-3.12 (m, 4H), 2.91-2.80 (m, 3H), 2.28-2.00
(m, 6H), 2.12-2.05 (m, 2H), 1.61 (b, 2H), 1.32 (s, 3H) 535.3 4.94
##STR00363## (CDCl3, 400 MHz), .delta. 7.89-7.69 (m, 1H), 7.43 (b,
1H), 7.33-7.30 (m, 2H), 7.20-7.06 (m, 4H), 5.29-5.19 (m, 1H),
4.64-4.45 (m, 1H), 4.20 (b, 2H), 3.27-3.10 (m, 2H), 2.91-2.72 (m,
2H), 2.70-2.66 (m, 1H), 2.50 (b, 2H), 2.29 (b, 1H), 1.32-1.31 (m,
12H), 1.02, 0.90 (d, J = 6.8 Hz, 3H) 453.0 6.30 ##STR00364##
(CD3OD, 400 MHz), .delta. 7.52-7.45 (m, 4H), 7.31 (b, 1H), 7.25 (d,
J = 8.4 Hz, 1H), 7.08 (dd, J = 8.4 Hz and 2.0 Hz, 1H), 6.48 (b,
1H), 4.34-4.23 (m, 3H), 3.45 (b, 3H), 3.23-3.13 (m, 4H), 2.92-2.80
(m, 5H), 1.32 (s, 3H) 481.3 3.43 ##STR00365## (CD3OD, 400 MHz),
.delta. 7.48 (d, J = 1.6 Hz, 1H), 7.43 (d, J = 8.4 Hz, 2H), 7.40
(d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.4 Hz, 1H), 7.07 (dd, J = 8.4 Hz
and 2.0 Hz, 1H), 6.50 (b, 1H), 4.35-4.29 (m, 3H), 3.90 (b, 1H),
3.52-3.47 (m, 3H), 3.20-3.16 (m, 2H), 3.01 (t, J = 12.0 Hz, 2H),
2.91-2.79 (m, 3H), 2.20 (b, 1H), 2.00-1.97 (m, 3H), 1.82-1.71 (m,
6H), 1.56-1.48 (m, 1H), 1.32 (b, 3H) 549.6 5.21 ##STR00366## (DMSO,
400 MHz), .delta. 11.39 (s, 1H), 9.80 (b, 1H), 9.40 (b, 1H), 7.52
(d, J = 1.6 Hz, 1H), 7.48 (s, 1H), 7.37-7.31 (m, 4H), 7.25-7.19 (m,
1H), 7.00 (dd, J = 8.8 Hz and 2 Hz, 1H), 4.76 (d, J = 5.6 Hz, 1H),
3.61-3.53 (m, 1H), 3.25-3.20 (m, 1H), 2.94-2.92 (m, 2H), 2.13-1.97
(m, 1H), 1.35, 1.24 (d, J = 6.8 Hz, 3H) 325.3 4.75 ##STR00367##
(CD3OD, 400 MHz), .delta. 8.99 (s, 1H), 7.80 (d, J = 8.0 Hz, 2H),
7.71 (d, J = 1.2 Hz, 1H), 7.57 (s, 1H), 7.47 (d, J = 1.6 Hz, 1H),
7.39 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.4 Hz, 1H), 7.07 (d, J =
8.0 Hz, 1H), 6.51 (b, 1H), 4.32 (t, J = 4.8 Hz, 3H), 4.23-4.21 (m,
2H), 3.43 (t, J = 6.4 Hz, 2H), 3.20-3.11 (m, 1H), 2.91-2.78 (m,
2H), 2.23-2.17 (m, 2H), 1.32 (b, 3H) 506.2 4.96 ##STR00368##
(CD3OD, 400 MHz), .delta. 7.79 (d, J = 8.4 Hz, 2H), 7.48 (s, 1H),
7.38 (d, J = 8.0 Hz, 2H), 7.25 (d, J = 8.8 Hz, 1H), 7.07 (dd, J =
8.4 Hz and 2.0 Hz, 1H), 6.51 (b, 1H), 4.35-4.21 (m, 3H), 3.67 (t, J
= 4.6 Hz, 4H), 3.41 (q, J = 4.8 Hz, 2H), 3.20-3.11 (m, 1H),
2.91-2.79 (m, 2H), 2.62 (s, 1H), 2.46-2.42 (m, 5H), 1.83-1.79 (m,
2H), 1.32 (s, 3H) 525.2 4.76 ##STR00369## (CD3OD, 400 MHz), .delta.
7.62 (s, 1H), 7.49 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.4 Hz, 2H),
7.19 (s, 2H), 6.48 (s, 1H), 4.27-4.18 (m, 5H), 3.87 (t, J = 4.6 Hz,
4H), 3.47 (t, J = 6.8 Hz, 2H), 3.34-3.30 (m, 2H), 3.16-3.12 (m,
5H), 2.89-2.75 (m, 2H), 1.30 (s, 3H) 541.2 3.51 ##STR00370##
(CD3OD, 400 MHz), .delta. 7.60 (s, 1H), 7.51 (d, J = 8.0 Hz, 2H),
7.40 (d, J = 8.0 Hz, 2H), 7.21-7.16 (m, 2H), 6.46 (b, 1H), 4.41 (s,
2H), 4.28-4.19 (m, 3H), 3.79-3.74 (m, 4H), 3.51-3.49 (m, 4H),
3.19-3.11 (m, 1H), 2.95 (s, 3H), 2.88-2.75 (m, 2H), 2.30 (s, 2H),
1.30 (s, 3H) 525.2 4.42 ##STR00371## (CD3OD, 400 MHz), .delta. 7.84
(d, J = 8.0 Hz, 2H), 7.47 (d, J = 2.0 Hz, 1H), 7.37 (d, J = 8.4 Hz,
2H), 7.24 (d, J = 8.4 Hz, 1H), 7.06 (dd, J = 8.4 Hz and 2.0 Hz,
1H), 6.49 (b, 1H), 4.35-4.16 (m, 3H), 3.21-3.10 (m, 1H), 2.90-2.71
(m, 2H), 1.32 (b, 3H) 398.1 3.95 ##STR00372## (CDCl3, 400 MHz),
.delta. 7.92-7.77 (m, 1H), 7.42-7.39 (m, 8H), 7.26-7.21 (m, 1H),
7.10 (d, J = 8.4 Hz, 1H), 5.16-4.97 (m, 1H), 4.56-4.36 (m, 1H),
4.19-4.11 (m, 2H), 3.27-3.19 (m, 1H), 2.78-2.63 (m, 2H), 1.90 (d, J
= 5.6 Hz, 1H), 1.74 (b, 1H), 1.49-1.26 (m, 4H), 1.10-0.91 (m, 6H)
335.2 5.45 ##STR00373## (CD3OD, 400 MHz), .delta. 7.82 (s, 1H),
7.80 (s, 1H), 7.55-7.48 (m, 3H), 7.23 (d, J = 8.4 Hz, 1H), 7.07
(dd, J = 8.4 Hz and 2.0 Hz, 1H), 6.49 (b, 1H), 4.33-4.21 (m, 3H),
4.05 (b, 2H), 3.5-3.73 (m, 4H), 3.61 (b, 2H), 3.37 (t, J = 5.8 Hz,
2H), 3.25-3.17 (m, 3H), 2.92-2.80 (m, 2H), 1.32 (s, 3H) 511.3 3.56
##STR00374## (CDCl3, 400 MHz), .delta. 8.01, 7.91 (s, 1H), 7.43 (s,
1H), 7.23 (d, J = 8.4 Hz, 1H), 7.11 (d, J = 7.2 Hz, 1H), 6.71 (d, J
= 7.6 Hz, 1H), 6.63 (s, 1H), 6.57 (d, J = 7.6 Hz, 1H), 5.92 (s,
2H), 5.18-5.07 (m, 1H), 4.63-4.41 (m, 1H), 4.30-4.11 (m, 2H),
3.36-3.31 (m, 1H), 2.91-2.83 (m, 2H), 2.70-2.61 (m, 1H), 2.38-2.15
(m, 2H), 1.38-1.30 (m, 3H), 1.09-1.01 (m, 3H) 440.9 5.75
##STR00375## (CD3OD, 400 MHz), .delta. 7.76 (s, 1H), 7.75 (s, 1H),
7.52-7.43 (m, 2H), 7.23 (d, J = 8.4 Hz, 1H), 7.06 (d, J = 7.6 Hz,
1H), 6.47 (b, 1H), 4.30-4.21 (m, 3H), 3.52 (s, 4H), 3.33 (s, 3H),
3.26-3.18 (m, 1H), 2.91-2.80 (m, 2H), 1.32 (s, 3H) 456.1 4.21
##STR00376## (CD3OD, 400 MHz), .delta. 7.48 (s, 1H), 7.46 (d, J =
8.8 Hz, 2H), 7.40 (d, J = 7.6 Hz, 2H), 7.24 (d, J = 8.4 Hz, 1H),
7.07 (d, J = 8.0 Hz, 1H), 6.49 (b, 1H), 4.35-4.21 (m, 3H),
3.64-3.61 (m, 2H), 3.20-3.11 (m, 3H), 3.01 (s, 3H), 2.93 (s, 5H),
2.89-2.78 (m, 3H), 2.12-2.05 (m, 2H), 1.32 (s, 3H) 497.2 4.69
##STR00377## (CDCl3, 400 MHz), .delta. 8.17, 8.00 (s, 1H), 7.50 (s,
1H), 7.23-7.13 (m, 4H), 6.97, 6.92 (s, 1H), 6.80 (d, J = 8.4 Hz,
2H), 4.43,
4.34 (t, J = 7.0 Hz, 1H), 4.04-3.98 (m, 1H), 3.77 (s, 3H),
3.47-3.41 (m, 1H), 3.25-2.81 (m, 2H), 2.23-2.06 (m, 2H), 1.02 (t, J
= 6.2 Hz, 3H) 460.8 4.96 ##STR00378## (DMSO, 300 MHz), .delta. 7.63
(s, 1H), 7.49 (d, J = 6.3 Hz, 2H), 7.42 (d, J = 6.0 Hz, 2H), 7.20
(s, 2H), 6.49 (s, 1H), 4.32-4.21 (m, 3H), 3.85 (b, 4H), 3.39-3.30
(m, 3H), 3.26-3.15 (m, 5H), 2.92-2.73 (m, 9H), 2.26-2.20 (m, 2H),
1.31 (s, 3H) 596.3 4.45 ##STR00379## (CD3OD, 400 MHz), .delta. 7.52
(d, J = 8.4 Hz, 2H), 7.47 (s, 1H), 7.39-7.36 (m, 2H), 7.24 (d, J =
8.8 Hz, 1H), 7.06 (dd, J = 8.4 Hz and 1.6 Hz, 1H), 6.49 (b, 1H),
4.45-4.23 (m, 4H), 3.84-3.45 (m, 4H), 3.20-3.12 (m, 1H), 2.91-2.78
(m, 2H), 2.25-2.10 (m, 1H), 1.98-1.89 (m, 4H), 1.32 (s, 3H) 509.2
5.18 ##STR00380## (CD3OD, 400 MHz), .delta. 7.52-7.45 (m, 4H), 7.32
(b, 1H), 7.25 (d, J = 8.4 Hz, 1H), 7.08 (dd, J = 8.4 Hz and 1.6 Hz,
1H), 6.49 (b, 1H), 4.34-4.23 (m, 4H), 3.69 (s, 3H), 3.31-3.30 (m,
8H), 3.21-3.12 (m, 3H), 2.91-2.74 (m, 2H), 1.32 (s, 3H) 525.3 3.52
##STR00381## (CD3OD, 400 MHz), .delta. 7.51-7.48 (m, 3H), 7.40 (d,
J = 8.0 Hz, 2H), 7.24 (d, J = 8.8 Hz, 1H), 7.07 (dd, J = 8.4 Hz and
1.2 Hz, 1H), 6.49 (b, 1H), 4.35-4.21 (m, 3H), 3.89 (b, 2H), 3.45
(b, 2H), 3.19-3.10 (m, 1H), 3.05-3.01 (m, 9H), 2.91-2.78 (m, 2H),
1.32 (b, 3H) 483.1 4.96 ##STR00382## (CD3OD, 400 MHz), .delta. 7.47
(d, J = 1.6 Hz 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.12 (dd, J = 8.4 Hz
and J = 2.0 Hz, 1H), 4.87 (s, 1H), 3.75-3.72 (m, 1H), 3.50-3.47 (m,
1H), 3.09-3.03 (m, 2H), 2.22 (dd, J = 15.6 Hz and J = 2.4 Hz, 1H),
1.84 (dd, J = 15.6 Hz and 8.4 Hz, 1H), 1.17 (s, 9H) 276.9 4.00
##STR00383## (CD3OD, 400 MHz), .delta. 7.48 (d, J = 1.6 Hz, 1H),
7.41-7.32 (m, 3H), 7.23 (d, J = 8.4 Hz, 2H), 7.07 (dd, J = 8.4 Hz
and 2.0 Hz, 1H), 6.46 (b, 1H), 4.32-4.17 (m, 3H), 3.80 (s, 2H),
3.67 (t, J = 5.0 Hz, 2H), 3.39 (s, 3H), 3.30-15 (m, 6H), 2.88-2.83
(m, 6H), 1.32 (s, 3H) 511.4 4.71 ##STR00384## (DMSO, 400 MHz),
.delta. 11.39 (d, J = 2.8 Hz, 1H), 9.75 (s, 1H), 9.34 (s, 1H), 7.53
(s, 1H), 7.36 (dd, J = 8.4 Hz and 4.0 Hz, 1H), 7.10 (dd, J = 8.8 Hz
and 2.0 Hz, 1H), 4.82-4.71 (m, 1H), 3.62-3.56 (m, 1H), 3.14 (b,
1H), 3.00-2.83 (m, 2H), 2.35-2.23 (m, 1H), 2.18-1.82 (m, 4H), 1.34
(q, J = 6.4 Hz, 3H) 295.0 4.14
Example 2
Assay to Evaluate Affect on Hypoxia-Inducible Endogenous VEGF
Expression
[0292] The ability of the compounds of the invention to modulate
hypoxia-inducible endogenous VEGF expression may be analyzed as
follows. VEGF protein levels may be monitored by an ELISA assay
(R&D Systems). Briefly, HeLa cells may be cultured for 24-48
hours under hypoxic conditions (1% O.sub.2, 5% CO.sub.2, balanced
with nitrogen) in the presence or absence of a compound of the
invention. The conditioned media may then be assayed by ELISA, and
the concentration of VEGF calculated from the standard ELISA curve
of each assay.
[0293] A dose-response analysis may be performed using the ELISA
assay and conditions described above. The conditions for the
dose-response ELISA are analogous to those described above. A
series of, e.g., seven different concentrations may be analyzed. In
parallel, a dose-response cytotoxicity assay may be performed using
Cell Titer Glo (Promega) under the same conditions as the ELISA to
ensure that the inhibition of VEGF expression was not due to the
cytotoxicity. Dose-response curves may be plotted using percentage
inhibition versus concentration of the compound, and EC.sub.50 and
CC.sub.50 values may be generated for each compound with the
maximal inhibition set as 100% and the minimal inhibition as 0%.
Preferred compounds of the invention will have an EC.sub.50 of less
than 50, preferably less than 10, more preferably less than 2, even
more preferably less than 0.5, and even more preferably less than
0.01.
[0294] The EC.sub.50 for a series of preferred compounds of the
invention is provided in Table 6.
TABLE-US-00003 TABLE 6 Com- LCMS LCMS Retention ELISA pound [M + H]
Time (min) EC50 .mu.M 1 391.20 3.67 **** 2 385.28 4.01 ***** 3
479.18 4.35 ***** 4 435.23 4.28 ***** 5 391.28 4.05 ***** 6 425.28
4.07 ***** 7 443.28 4.61 ***** # 8 415.26 4.25 ***** 9 431.25 4.07
***** # 10 467.15 4.51 ***** 11 389.24 4.24 ***** 12 414.31 3.94
***** 13 411.24 4.89 ***** 14 397.22 4.57 ***** 15 457.3 4.24 *****
16 435.19 4.47 ***** 17 447.14 4.44 ***** 18 431.14 4.55 ***** 19
437.26 4.54 ***** 20 389.24 4.22 ***** 21 391.28 4.04 ***** 22
425.28 4.11 ***** 23 373.23 4.04 ***** 24 411.24 4.8 ***** 25
449.23 4.03 ***** 26 437.15 4.52 ***** 27 399.25 4.11 ***** 28
399.19 4.2 ***** 29 435.09 4.14 ***** 30 413.22 4.42 ***** 31
423.17 4.32 ***** 32 467.25 4.26 ***** 33 457.15 4.29 ***** 34
383.19 4.42 ***** 35 425.28 4.14 ***** 36 383.2 4.37 ***** 37 423.3
4.24 ***** 38 355.24 4.07 ***** 39 391.28 4.12 ***** 40 403.15 4.45
***** 41 449.11 4.59 ***** 42 383.19 4.44 ***** 43 371.31 3.89
***** 44 479.18 4.35 ***** 45 394.16 4.09 ***** 46 421.19 4.22 ****
47 449.07 4.54 **** 48 403.32 4.2 **** 49 403.15 4.51 **** 50
405.18 3.81 **** 51 373.23 4.11 **** 52 355.3 4.07 **** 53 375.26
3.92 **** 54 435.23 4.3 **** 55 425.27 4.26 **** 56 414.14 4.19
**** 57 399.19 4.2 **** 58 469.22 4.32 **** 59 444.12 4.12 **** 60
433.17 4.27 **** 61 419.28 4.04 **** 62 409.14 4.22 **** 63 435.09
4.16 **** 64 435.12 4.27 **** 65 387.2 3.95 **** 66 414.17 4.24
**** 67 429.3 4.47 **** 68 359.19 3.89 **** 69 449.08 4.55 **** 70
375.25 4.19 **** 71 394.16 4.12 **** 72 403.15 4.49 **** 73 381.09
3.59 **** # 74 400.15 4.05 **** 75 387.22 4.29 **** 76 449.26 4.3
**** 77 391.28 4.19 **** 78 435.12 4.24 **** 79 437.19 4.49 **** 80
437.2 3.84 **** 81 375.03 3.57 **** 82 391.28 4.05 **** 83 425.28
4.16 **** 84 359.22 3.95 **** 85 437.15 4.44 **** 86 399.19 4.22
**** 87 403.15 4.44 **** 88 399.19 4.17 **** 89 434.07 4.04 **** 90
387.23 4.26 **** 91 369.27 4.17 **** 92 377.29 4.04 **** 93 435.23
4.29 **** 94 369.17 4.24 **** 95 449.06 4.51 **** 96 341.27 3.89
**** 97 387.19 4.2 **** 98 405.18 3.79 **** 99 469.22 4.29 **** 100
461.32 4.61 **** 101 369.17 4.26 **** 102 413.28 4.02 **** 103
407.1 4.05 **** 104 375.27 4.11 **** 105 387.21 4.19 **** 106
373.18 4.04 **** 107 385.28 4.02 **** 108 359.16 3.92 **** 109
369.34 4.16 **** 110 374.24 3.07 **** 111 386.19 3.89 **** 112
369.27 2.63 **** 113 399.13 4.01 **** 114 389.3 4.05 **** 115
435.13 4.14 **** 116 407.16 4.09 **** 117 419.28 4.05 **** 118
366.29 3.79 **** 119 521.19 4.16 **** 120 380.31 3.92 **** 121
403.32 4.27 **** 122 383.31 4.37 **** 123 319.2 2.19 **** 124
351.14 2.53 *** 125 409.3 4.14 *** 126 423.3 3.95 *** 127 371.31
3.9 *** 128 371.31 3.62 *** 129 449.13 3.81 *** 130 401.23 3.56 ***
131 385.22 3.74 *** 132 363.06 2.31 *** 133 385.15 3.86 *** 134
377.3 4.04 *** 135 397.15 2.42 *** 136 443.33 4.11 *** 137 361.07
2.53 *** 138 345.07 3.15 *** 139 400.27 4.01 *** 140 488.23 4.36
*** 141 425.21 4.37 *** 142 462.15 4.11 *** 143 369.23 3.74 *** 144
415.33 3.84 *** 145 361.3 4.39 *** 146 400.21 3.81 *** 147 438.21
3.97 *** 148 469.01 4.42 *** 149 425.25 4.24 *** 150 504.2 4.68 ***
151 397.01 2.44 *** 152 369.21 3.59 *** 153 372.21 2.36 *** 154
377.29 3.97 *** 155 363.11 2.32 *** 156 341.21 2.46 *** 157 407.14
1.78 *** 158 428.11 3.85 *** 159 351.13 2.47 *** 160 450.15 3.95
*** 161 363.05 2.32 *** 162 325.26 2.66 *** 163 319.2 2.24 *** 164
462.19 3.87 *** 165 371.31 3.65 *** 166 354.28 (-Boc) 3.95 *** 167
432.16 3.87 *** 168 351.08 2.4 *** 169 385.35 4.09 *** 170 351.07
2.51 *** 171 363.09 2.68 ** 172 384.21 3.52 ** 173 319.2 2.24 **
174 N/A 2.38 ** 175 443.33 4.09 ** 176 417.30 2.77 ** 177 398.17
3.67 ** 178 363.11 2.31 ** 179 450.14 3.89 ** 180 421.19 2.65 **
181 363.15 2.46 ** 182 419.14 4.14 ** 183 389.29 4.14 ** 184 431.27
4.1 ** 185 328.02 2.41 ** 186 462.19 3.81 ** 187 443.28 3.99 ** 188
446.19 3.81 ** 189 405.19 3.8 ** 190 317.16 2.7 ** 191 369.23 3.89
** 192 495.28 4.89 ** 193 297.2 2.53 ** 194 319.21 2.19 ** 195
494.25 2.79 ** 196 419.22 4.09 ** 197 317.16 2.41 ** 198 317.08
2.53 ** 199 448.24 3.95 ** 200 363.09 2.45 ** 201 365.09 2.36 **
202 464.2 4.32 ** 203 301.18 2.27 ** 204 429.23 3.57 ** 205 301.15
2.27 ** 206 476.3 4.33 ** 207 395.17 2.55 ** 208 367.36 2.72 ** 209
353.33 3.97 ** 210 313.21 2.33 ** 211 415.26 4.07 ** 212 389.2 2.88
** 213 407.1 2.46 ** 214 357.07 2.48 ** 215 319.23 2.24 ** 216
283.1 2.41 ** 217 418.17 3.62 ** 218 435.23 3.77 ** 220 308.23 2.37
** 221 460.29 4.05 ** 222 365.11 2.52 ** 223 441.02 2.6 ** 224
341.27 2.6 ** 225 467.25 4.18 ** 226 369.34 4.01 ** 227 327.16 2.26
** 228 369.34 2.64 ** 229 373.29 4.04 * 230 401.23 3.2 * 231 313.12
2.43 * 232 433.25 2.73 * 233 430.38 (-Boc) 4.34 * 234 351.17 2.4 *
235 351.25 3.79 * 236 379.35 2.74 * 237 439.11 4.41 * 238 479.24
3.77 * 239 328.16 2.35 * 240 307.27 3.87 * 241 523.19 3.7 * 242
438.27 4.14 * 243 323.20 3.49 * 244 512 2.27 * 245 485 2.62 * 246
498 2.54 *
247 471 2.36 * 248 283.23 2.24 * 249 339.17 3.07 * 250 355.30 3.57
* 251 297.26 2.26 * 252 341.21 2.44 * 253 301.27 2.29 * 254 301.25
2.27 * 255 281.31 2.2 * 256 345.2 2.26 * 257 335.21 2.34 * 258
459.27 3.72 * 259 479.24 3.52 * 260 287.26 2.36 * 261 287.26 2.56 *
262 380.24 3.92 * 263 503.50 3.20 * 264 369.36 2.52 * 265 355.26
2.54 * 266 355.26 2.42 * 267 370.22 3.61 * 268 355.26 2.42 * 269
355.27 2.37 * 270 370.23 3.19 * 271 369.34 2.62 * 272 374.31 2.90 *
273 492.25 2.76 * 274 451.30 3.17 * 275 374.31 2.61 * 276 374.31
2.72 * 277 349.28 1.5 * 278 457.28 4.11 * 279 ***** 280 407.10 3.92
* 281 508.15 4.74 * 282 507.08 4.42 * 283 422.32 3.86 * 284 373.29
4.01 * 285 385.24 2.25 * 286 297.2 2.52 * 287 289.22 2.48 * 288
461.26 2.57 * 289 380.29 3.82 * 290 396.27 3.60 * 291 299.17 2.43 *
292 385.18 2.6 * 293 413.22 3.8 * 294 340.25 2.27 * 295 404.34 3.84
* 296 299.17 2.23 * 297 326.24 2.4 * 298 235.13 2.18 * 299 351.16
2.62 * 300 401 2.57 * 301 313.21 2.35 * 302 398.28 3.74 * 303
355.22 2.58 * 304 440.32 4.09 * 305 341.08 2.48 * 306 364.3 3.65 *
307 350.32 3.35 * 308 432.27 3.92 * 309 474.26 3.02 **** 310 289.03
2.35 * 311 345.19 2.58 * 312 420.28 4.12 * 313 279.28 2.18 * 314
293.24 2.20 * 315 297.26 2.17 * 316 472.26 3.85 * 317 428.25 3.95 *
318 309 2.25 * 319 284.09 2.1 * 320 356.21 2.37 * 321 279.2 2.1 *
322 279.2 1.76 * 323 309.23 1.82 * 324 280.19 1.76 * 325 279.2 1.76
* 326 263.17 1.93 * 327 343.18 2.33 * 328 ~0.005 4.16 * 329 0.0036
4.26 * 330 0.0047 4.24 * 331 ~0.010 2.94 * # 332 ~0.010 4 * 333
410.27 3.64 ** 334 426.24 3.39 * 335 466.23 4.64 *** 336 438.31
4.31 ** 337 454.24 4.63 *** 338 474.32 4.33 ** 339 412.3 3.83 * 340
446.33 4.49 * 341 447.26 4.25 *** 342 371.31 3.88 *** 343 371.31
3.61 * 344 459.31 4.91 **** 345 383.35 4.44 **** 346 587 4.04 ****
347 451.16 3.93 ***** 348 479.28 4.13 ***** 349 481.21 3.74 ****
350 462.17 3.66 ***** 351 471.17 3.93 **** 352 403.29 3.98 **** 353
497.16 3.94 ***** 354 525.2 4.19 ***** 355 511.21 3.81 ***** 356
490.3 3.93 ** 357 534.23 3.93 *** 358 433.2 3.45 *** 359 511.25
3.64 *** 360 516 3.82 **** 361 474.26 3.02 **** 362 427 4.2 *****
363 412.4 1.80 * 364 484.3 2.49 ***** 365 457.3 4.06 *** 366 553.3
4.42 * 367 402.8 4.37 **** 368 430.9 4.79 ** 369 427.0 4.06 ** 370
427.0 3.99 ***** 371 469.0 5.27 *** 372 486.9 4.96 * 373 470.8 5.01
*** 374 436.9 4.66 *** 375 461 4.92 ** 376 385 3.79 ** 377 n/d n/d
* 378 n/d n/d * 379 n/d n/d * 380 n/d n/d * 381 n/d n/d * 382 n/d
n/d * 383 417.2 4.93 ***** 384 403.22 4.65 ***** 385 509.51 2.57
**** 386 465.26 2.52 ***** 387 465.26 2.52 ***** 388 495.4 3.94
***** 389 538.3 4.29 ***** 390 480.5 3.23 ***** 391 562.55 3.63
***** 392 443.4 3.88 ***** 393 447.1 6.55 ***** 394 450.1 5.48
***** 395 481.32 3.51 ***** 396 411.3 3.99 ***** 397 535.3 4.29
***** 398 481.3 4.23 ***** 399 429.3 3.81 ***** 400 493.3 4.43
***** 401 451.3 3.99 ***** 402 494.4 3.71 ***** 403 479.3 4.23
***** 404 473.6 3.78 ***** 405 551.17 4.58 ***** 406 425.4 4.13
***** 407 457.4 4.04 ***** 408 425.4 4.09 ***** 409 477.4 4.18
***** 410 451.3 3.99 ***** 411 443.4 3.86 ***** 412 473.4 4.23
***** 413 459.3 4.16 ***** 414 439.4 4.31 ***** 415 637.64 2.82
***** 416 311.1 4.39 ***** 417 562.47 4.15 ***** 418 511.3 4.13
***** 419 491.4 3.98 ***** 420 486.6 3.45 ***** 421 553.30 4.05
***** 422 359.29 4.17 ***** 423 447.4 3.56 ***** 424 594.2 [M - H]
4.58 ***** 425 539.2 3.11 ***** 426 535.27 4.29 ***** 427 554.3
4.45 ***** 428 563.55 4.64 ***** 429 564.42 2.77 ***** 430 431.3
3.41 ***** 431 522.2 5.05 ***** 432 489.4 4.14 ***** 433 578.44
2.82 ***** 434 467.18 4.11 ***** 435 444.3 3.95 ***** 436 477.4
3.93 ***** 437 543.4 3.92 ***** 438 500.1 4.35 ***** 439 361.2 5.95
***** 440 536.43 3.95 ***** 441 482.1 5.11 **** 442 367.1 2.92 ****
443 436.2 5.25 **** 444 455.28 3.73 **** 445 478 3.67 **** 446
383.3 4.10 **** 447 464.9 5.11 **** 448 501.27 3.65 **** 449 482.24
2.62 **** 450 587 4.04 **** 451 644.3 [M - H] 4.80 **** 452 439.3
3.56 **** 453 553.1 6.13 **** 454 579.3 2.75 **** 455 583 3.84 ****
456 474.3 2.44 **** 457 455 3.4 **** 458 456.3 2.51 **** 459 470.3
2.61 **** 460 509.30 4.16 **** 461 454.3 5.98 **** 462 580.56 2.85
**** 463 495.44 4.13 **** 464 493.0 5.71 **** 465 507.4 3.98 ****
466 555.2 3.14 **** 467 524.2 4.02 **** 468 582.2 2.81 **** 469
525.2 5.07 **** 470 554.3 3.90 **** 471 620.18 3.85 **** 472 335.3
5.52 **** 473 495.3 4.68 *** 474 511.2 4.99 *** 475 483 3.87 ***
476 400 3.45 *** 477 249.1 3.67 *** 478 525.1 3.25 *** 479 538.3
2.76 *** 480 456.1 4.26 *** 481 549.3 5.29 *** 482 522.3 3.95 ***
483 470.1 4.46 *** 484 539.2 3.02 *** 485 398.9 4.18 *** 486 349.1
6.03 *** 487 505 3.66 *** 488 555.2 3.34 *** 489 538.3 4.15 *** 490
486.1 3.80 *** 491 537.31 2.64 *** 492 468 5.52 *** 493 504.3 2.68
*** 494 482.2 5.74 *** 495 403.3 4.16 *** 496 430.2 3.65 *** 497
281.0 3.84 ***
498 481.4 4.81 *** 499 423.3 5.15 *** 500 506.29 3.85 *** 501 534.3
2.68 *** 502 518.3 2.76 *** 503 508.2 5.72 *** 504 359.1 5.65 ***
505 442.0 4.06 *** 506 386.3 5.32 *** 507 450 3.19 *** 508 397.1
5.97 *** 509 511.4 5.05 *** 510 321.4 5.19 *** 511 383.1 5.75 ***
512 523.1 5.69 *** 513 361.1 5.12 *** 514 495.3 4.67 *** 515 363.5
6.34 ** 516 527.1 3.16 ** 517 464.2 5.86 ** 518 517.6 5.03 ** 519
527.2 3.88 ** 520 426.2 4.29 ** 521 509.4 4.99 ** 522 383.3 4.10 **
523 439.0 6.11 ** 524 412.1 4.13 ** 525 4.95.3 3.46 ** 526 513.2
4.43 ** 527 535.3 4.94 ** 528 453.0 6.30 ** 529 481.3 3.43 ** 530
466.28 3.21 ** 531 549.6 5.21 ** 532 325.3 4.75 ** 533 506.2 4.96
** 534 525.2 4.76 ** 535 541.2 3.51 ** 536 482.29 3.29 ** 537 476.3
2.51 ** 538 516.37 3.49 ** 539 337.3 [M - H] 2.14 ** 540 428.28
3.43 ** 541 525.2 4.42 ** 542 398.1 3.95 ** 543 466.34 3.29 ** 544
723.58 3.92 ***** 545 466.31 3.28 ** 546 426.3 2.26 ** 547 335.2
5.45 ** 548 516.37 3.46 ** 549 414 2.89 ** 550 496 4.58 ** 551
544.5 2.78 ** 552 511.3 3.56 ** 553 440.9 5.75 ** 554 482.32 3.41
** 555 372 2.89 ** 556 456.1 4.21 ** 557 538.4 3.71 ** 558 497.2
4.69 ** 559 460.8 4.96 ** 560 596.3 4.45 * 561 509.2 5.18 * 562
525.3 3.52 * 563 483.1 4.96 * 564 432 2.18 * 565 276.9 4.00 * 566
384.4 1.73 * 567 511.4 4.71 * 568 295.0 4.14 * 569 480.21 3.50
***** 570 549.22 4.59 ***** 571 497.13 3.50 ** 572 525.29 4.14
***** 573 341.34 2.14 **** 574 427.37 2.23 * 575 437.33 3.16 ** 576
575.43 3.71 *** 577 453.28 3.34 *** 578 610.45 3.94 *** 579 481.32
3.51 ***** 580 495.29 3.64 ***** 581 465.43 3.64 * 582 516.34 3.31
* 583 512.26 3.39 *** 584 466.37 3.34 *** 585 516.33 3.46 *** 586
387.27 2.13 ***** 587 467.29 3.66 *** 588 455.26 3.69 *** 589 471.3
3.83 *** 590 495.31 3.64 **** 591 541.35 3.73 ***** 592 523.42 3.58
***** 593 541.38 3.69 **** 594 505.38 3.83 *** 595 431.21 4.01 ****
596 431.24 3.99 ***** 597 445.24 4.19 ***** 598 459.24 4.36 *****
599 513.17 4.19 **** 600 479.23 3.99 ***** 601 504.21 3.79 **** 602
493.2 4.18 **** 603 513.16 4.19 **** 604 446.18 2.86 * 605 503.23
3.84 ***** 606 461.19 3.46 *** 607 442.25 3.46 *** 608 489.2 3.72
*** 609 433.27 3.98 ** 610 n/d n/d **** 611 n/d n/d ** 612 491.23
3.56 *** 613 513.14 4.18 **** 614 463 3.88 ** 615 381 3.48 *** 616
540 4.17 ** 617 621.57 4.13 **** 618 493.6 2.63 ***** 619 521.6
2.80 ***** 620 445.5 3.23 **** 621 459.5 3.40 ***** 622 459.5 3.38
***** 623 473.5 3.57 ***** 624 479.5 3.28 **** 625 507.6 3.53 *****
626 493.6 3.48 **** 627 511.6 3.53 ***** 628 527.4 3.62 *** 629
527.5 3.72 ***** 630 573.5 3.75 ***** 631 507.6 3.65 ***** 632
538.6 3.53 **** 633 443.5 3.32 ***** 634 457.6 3.30 ***** 635 523.6
3.47 **** 636 463.6 3.12 ***** 637 621.62 2.77 ***** 638 580.56
2.80 ***** 639 496.54 3.28 ***** 640 552.64 2.48 **** 641 445.55
4.13 ***** 642 381.49 3.97 ***** 643 397.47 3.95 ***** 644 395.45
3.78 ***** 645 521.15 4.17 ***** 646 531.11 4.58 **** 647 505.18
4.7 ***** 648 437.19 4.15 **** 649 477.21 4.1 ***** 650 487.18 4.3
**** 651 548.3 2.53 **** 652 419.23 4.15 **** 653 449.24 4.12 ****
654 433.26 4.3 ***** 655 453.19 4.33 **** 656 444.17 4.02 ***** 657
464.22 4.08 ***** 658 461.6 4.30 ***** 659 489.7 4.78 ***** 660
543.7 4.92 ***** 661 459.5 3.63 ***** 662 471.5 3.87 ***** 663
491.6 3.63 ***** 664 507.6 3.80 ***** 665 485.6 3.85 **** 666 485.6
3.83 ***** 667 486.6 3.95 ***** 668 503.6 3.58 ***** 669 521.6 3.88
***** 670 521.6 4.02 ***** 671 501.6 4.13 ***** 672 501.6 4.10
***** 673 539.6 4.02 674 555.6 4.13 **** 675 555.6 4.22 **** 676
535.6 4.05 **** 677 535.6 4.15 **** 678 551.6 3.98 *** 679 487.6
3.93 **** 680 599.5 4.27 ***** 681 566.6 4.02 **** 682 496.5 2.13
** 683 486.5 2.03 *** 684 484.6 2.67 *** 685 514.6 2.15 *** 686
512.6 2.12 **** 687 510.6 2.13 *** 688 525.6 1.85 *** 689 494.5
3.12 *** 690 524.6 2.32 *** 691 514.6 2.23 *** 692 512.6 2.35 ***
693 542.6 2.35 **** 694 540.6 2.27 **** 695 538.6 2.35 **** 696
553.6 2.07 *** 697 522.6 3.95 ***** 698 578.5 2.43 **** 699 568.5
2.35 **** 700 566.6 2.45 **** 701 596.6 2.47 **** 702 594.6 2.43
**** 703 592.6 2.48 **** 704 607.6 2.20 *** 705 575.5 2.47 **** 706
576.5 3.58 ***** 707 477.51 2.77 ***** 708 491.53 2.73 ***** 709
503.55 2.68 ***** 710 495.45 4.42 ***** 711 475.51 4.62 ***** 712
513.50 4.42 ***** 713 529.46 4.62 **** 714 509.51 4.43 ***** 715
482.46 4.28 ***** 716 457.47 4.05 **** 717 459.59 4.33 ***** 718
491.5 4.10 ***** 719 527.5 4.47 ***** 720 489.5 4.75 ***** 721
517.5 4.26 ***** 722 519.5 3.84 ***** 723 555.4 4.09 (non polar)
***** 724 541.54 2.90 ***** 725 478.47 3.58 ***** 726 516.5 2.67 **
727 526.5 2.78 **** 728 544.5 2.80 *** 729 542.5 2.72 ***** 730
540.5 2.83 **** 731 555.6 2.43 *** 732 580.6 2.40 *** 733 523.5
2.78 ***** 734 524.5 3.40 ***** 735 552.5 2.98 ***** 736 562.5 3.15
***** 737 580.6 3.17 **** 738 578.5 3.02 ***** 739 576.6 3.17 *****
740 591.6 2.75 *** 741 616.5 2.62 *** 742 559.5 3.13 ***** 743
560.5 3.83 ***** 744 514.6 2.80 ***** 745 524.6 2.92 ***** 746
512.5 2.93 ***** 747 542.6 2.93 ***** 748 540.5 2.85 *****
749 538.6 2.93 ***** 750 553.6 2.55 ***** 751 521.5 2.92 **** 752
522.5 3.87 ***** 753 542.6 2.98 **** 754 552.6 n/d ***** 755 540.6
3.17 **** 756 570.6 3.17 **** 757 568.6 3.07 ***** 758 566.6 3.17
*** 759 581.6 2.78 *** 760 549.6 3.13 ***** 761 550.5 4.17 *****
762 544.5 2.68 **** 763 554.5 2.77 ***** 764 542.6 2.78 **** 765
572.5 2.75 **** 766 570.6 2.70 ***** 767 568.6 2.82 **** 768 583.6
2.47 **** 769 608.6 2.38 *** 770 551.5 2.73 ***** 771 552.5 3.65
***** 772 580.5 3.03 ***** 773 590.6 3.12 ***** 774 578.5 3.12 ****
775 608.6 3.05 ***** 776 606.5 3.05 ***** 777 604.6 3.12 ***** 778
619.6 2.77 ***** 779 644.5 2.63 *** 780 587.5 3.10 ***** 781 588.5
4.05 ***** 782 596.5 3.10 ***** 783 606.5 3.18 ***** 784 594.5 3.27
***** 785 624.5 3.22 ***** 786 622.5 3.12 ***** 787 620.5 3.20
***** 788 635.6 2.85 **** 789 660.5 2.68 *** 790 603.5 3.22 *****
791 604.5 4.25 ***** 792 480.50 2.98 ***** 793 494.50 2.97 **** 794
494.50 2.97 *** 795 496.48 2.97 **** 796 563.50 2.41 **** 797
522.48 2.50 ***** 798 538.48 2.92 ***** 799 535.49 2.35 *** 800
503.40 2.52 **** 801 504.43 3.42 ***** 802 504.42 3.37 ***** 803
579.48 2.42 **** 804 538.48 2.43 ***** 805 584.50 2.52 ***** 806
554.40 2.47 ***** 807 540.47 2.50 ***** 808 551.48 2.33 **** 809
516.45 2.47 ***** 810 520.40 3.21 ***** 811 520.40 3.12 ***** 812
466.4 3.27 ***** 813 466.4 3.18 ***** 814 465.4 2.38 ***** 815
465.4 3.45 ***** # 816 497.4 2.70 ***** # 817 511.4 2.62 ***** #
818 491.4 2.43 **** 819 494.4 3.53 ***** 820 494.4 3.47 **** 821
493.4 2.55 **** 822 493.4 3.73 ***** # 823 525.4 2.95 ***** # 824
539.4 2.83 ***** # 825 519.4 2.58 * 826 496.4 3.07 *** 827 496.4
2.98 **** 828 495.4 2.32 *** 829 495.4 3.28 *** # 830 527.4 2.53
***** # 831 541.4 2.50 ***** # 832 521.4 2.35 833 532.4 3.50 ***
834 532.4 3.42 **** 835 531.4 2.57 *** 836 531.4 3.67 **** # 837
563.4 2.93 ***** # 838 577.4 2.82 ***** 839 548.3 3.63 **** 840
548.3 3.58 **** # 841 579.3 3.08 ***** # 842 593.3 2.95 ***** # 843
573.4 2.75 ***** 844 451.91 3.58 *** 845 648.48 4.45 *** 846 526.45
2.57 *** 847 568.37 3.40 **** 848 585.30 3.57 ***** 849 604.37 3.52
**** 850 540.39 2.60 *** 851 495.06 4.37 ***** 852 539.08 4.17
***** 853 549.09 4.38 ***** 854 523.17 4.73 ***** 855 455.19 4.15
**** 856 495.18 4.10 ***** 857 505.16 4.30 ***** 858 566.3 2.57
***** 859 437.22 4.15 ***** 860 467.2 4.13 ***** 861 451.12 4.10
**** 862 471.17 4.32 ***** 863 514.55 4.38 ***** 864 462.28 4.00
**** 865 482.13 4.08 **** 866 447.37 4.04 ***** 867 577.43 2.85
**** 868 477.14 4.37 ***** 869 504.53 3.62 ***** 870 493.55 2.80
***** 871 489.54 2.72 ***** 872 493.55 2.80 ***** 873 503.54 2.73
***** 874 479.2 2.74 ***** 875 425.52 4.27 ***** 876 492.52 3.57
***** 877 489.54 2.72 ***** 878 508.55 3.82 ***** 879 507.55 2.90
***** 880 459.49 4.24 ***** 881 471.45 4.22 ***** 882 542.51 3.87
***** 883 494.50 3.67 ***** 884 544.27 2.79 ***** 885 490.54 3.54
***** 886 494.57 3.68 ***** 887 521.62 2.93 ***** 888 558.54 3.70
***** 889 545.55 2.93 ***** 890 490.49 3.48 ***** 891 528.49 3.69
***** 892 546.50 3.75 ***** 893 461.49 4.36 ***** 894 580.47 2.72
***** 895 491.51 2.77 ***** 896 576.49 4.00 ***** 897 504.51 3.52
***** 898 457.53 4.25 ***** 899 481.37 4.17 ***** 900 541.55 3.00
***** 901 575.54 2.98 ***** 902 471.49 4.12 ***** 903 621.39 2.72
***** 904 596.54 2.85 ***** 905 542.54 3.78 ***** 906 489.53 4.82
***** 907 514.47 3.54 ***** 908 582.43 2.79 ***** 909 514.21 2.75
***** 910 539.45 3.97 ***** 911 527.54 2.88 ***** 912 530.53 2.67
***** 913 626.6 2.88 ***** 914 514.55 2.60 ***** 915 509.56 4.63
***** 916 626.40 2.82 ***** 917 561.46 2.95 ***** 918 642.56 2.85
***** 919 543.45 4.82 ***** 920 557.57 2.87 ***** 921 527.39 4.52
***** 922 561.53 2.85 ***** 923 612.51 2.92 ***** 924 498.20 2.71
***** 925 596.54 2.88 ***** 926 5.62 3.85 ***** 927 540.65 4.25
***** 928 510.52 3.10 ***** 929 506.46 2.95 ***** 930 500.48 2.83
***** 931 467.39 4.17 ***** 932 548.49 3.17 ***** 933 596.37 2.79
***** 934 561.53 2.95 ***** 935 496.54 3.37 ***** 936 582.6 2.83
***** 937 555.61 2.55 ***** 938 582.53 2.85 ***** 939 560.63 2.68
***** 940 541.43 2.45 ***** 941 562.55 3.63 ***** 942 623.35 2.73
**** 943 499 2.72 **** 944 525.56 4.36 **** 945 509.43 4.73 ****
946 566.53 2.77 **** 947 510 2.44 **** 948 482.47 2.88 **** 949
524.55 3.22 **** 950 506.46 2.87 **** 951 544.53 3.27 **** 952
530.53 3.12 **** 953 552.46 2.90 **** 954 403 4.11 **** 955 397 3.9
**** 956 484.55 2.42 **** 957 495.52 2.62 **** 958 542.36 3.84 ****
959 496.24 2.81 **** 960 639.57 2.70 **** 961 593.52 2.64 **** 962
516.59 2.65 **** 963 593.61 2.72 **** 964 598.55 2.83 **** 965
544.53 3.15 **** 966 564.45 3.32 **** 967 491.57 4.00 **** 968
512.51 2.73 **** 969 492.46 2.90 **** 970 609.54 2.72 **** 971
468.46 2.78 **** 972 496.47 3.02 **** 973 578.47 3.80 **** 974
528.34 3.79 *** 975 431.5 3.10 *** 976 564.46 3.23 *** 977 568.53
2.85 *** 978 578.45 3.30 *** 979 470.55 2.45 *** 980 527.61 2.50
*** 981 560.51 3.12 *** 982 425.60 3.78 *** 983 375.37 2.27 *** 984
5.06.19 3.97 ** 985 407.31 1.82 * 986 531.56 2.17 * 987 497.1 4.4
***** 988 605.62 2.52 ***** 989 564.61 2.55 ***** 990 610.62 2.67
***** 991 580.58 2.60 *** 992 566.61 2.60 *** 993 577.61 2.45 *****
994 545.54 2.57 ***** 995 546.57 3.53 ***** 996 578.46 3.71 ***** #
(S) Isomer prepared and tested. Wherein:
1 star, >1 uM (1000 nM) 2 stars, 0.2 to 1 uM (200 nM to 1000 nM)
3 stars, 0.04 uM to 0.2 uM (40 nM to 200 nM) 4 stars, 0.008 uM to
0.04 uM (8 nM to 40 nM) 5 stars, <0.008 uM (<8 nM)
Example 3
Compounds of the Invention Inhibit VEGF Expression and Tumor
Growth, and Inhibit angiogenesis, in an In Vivo Tumor Growth PD
Model
[0295] Compounds of the invention also show activity in the
following pharmacodynamic model that assesses tumor VEGF levels.
Briefly, HT1080 cells (a human fibrosarcoma cell line) may be
implanted subcutaneously in nude mice. After seven days, mice may
be administrated compounds orally at a desired dosage range, e.g.,
200 mg/kg/day, for seven days. The tumors may then be excised from
mice, weighed and homogenized in Tris-HCl buffer containing
proteinase inhibitors. Moulder et al., Cancer Res. 61(24):8887-95
(2001). Tumor VEGF levels are subsequently measured using a human
VEGF ELISA kit (R&D System). Protein concentrations of the
homogenates are measured with a Bio-Rad Protein assay kit and tumor
VEGF levels are normalized to the protein concentrations.
[0296] Preferred compounds of the invention, when used for one week
on a 100 mm.sup.3 tumor, will generally inhibit tumor growth by at
least 50%, as compared to the vehicle-treated control groups. In
similarly conducted experiments, compounds of the invention are
shown to reduce lung cancer tumor growth (at a dosage of 3 mg/kg,
twice a day, or 30 mg/kg, twice a day) (FIG. 1), Ewing's sarcoma
and neuroblastoma tumor growth (at a dosage of 30 mg/kg, once a
day) (FIG. 2), fibrosarcoma tumor growth (at a dosage of 10 mg/kg
3.times./week) (FIG. 3), and human skin malignant melanoma (at a
dosage of 10 mg/kg 5.times./week) (FIG. 4) as compared to vehicle.
Furthermore, the compounds of the invention are shown to reduce
VEGF plasma and tumor levels in fibrosarcoma tumors as compared to
vehicle (at a dosage of 10 mg/kg 3.times./week) (FIG. 5). The
compounds of the invention also inhibit tumor growth when
administered together with other cancer agents, such as 5-FU
(fluorouracil) in a colon cancer tumor (FIG. 6), taxol (paclitaxel)
in a human cervical carcinoma (FIG. 7), and Avastin (bevacizumab)
(FIG. 8). Furthermore, as shown in FIG. 9, the compounds of the
invention also reduce tumor and plasma VEGF levels when
administered together with another cancer agent (such as
Avastin).
[0297] The tumors excised from the mice can also be used in
immunochemistry experiments to determine levels of angiogenesis. As
seen in FIG. 10, when antibodies to Collagen IV are used,
angiogenesis is shown to be inhibited by the administration of a
compound of the invention in Ewing's sarcoma (SK-NEP-1) and
neuroblastoma (SY5Y and NGP) tumors. SK-NEP-1 cells were previously
believed to be a useful Wilms tumor model. More recent genotyping,
however, suggest that the cells, which may have characteristics of
both transformed cell/tumor type, are useful as a Ewing's sarcoma
model.
Example 4
Compounds of the Invention do not Affect VEGF mRNA Levels
[0298] Human solid tumor cells are treated with various
concentrations of a compound of the present invention or a control
under hypoxic conditions. Actinomycin D blocks transcription and is
used as a control. Total RNA is isolated using the RNeasy kit
(Qiagen, Inc., Valencia, Calif., USA) or an acid-guanidinium
thiocyanate-phenol-chloroform based method (See Chomczynski, P. and
Sacchi, N., Anal. Biochem. 162: 156 (1987), hereby incorporated by
reference).
[0299] Concentration of total RNA can be determined using the
RiboGreen.RTM. fluorescent dye (Molecular Probes, Inc., Eugene,
Oreg., USA) with the VersaFluor Fluorometer System (BioRad,
Hercules, Calif., USA). Quality and integrity of total RNA can be
assessed on 1% formaldehyde-agarose gels. First-strand cDNA was
synthesized using 1 .mu.g of total RNA (DNase-treated) in a 20
.mu.A reverse transcriptase reaction mixture (Leclerc G J, Barredo
J C, Clin. Cancer Res. 2001, 7:942-951, which is hereby
incorporated by reference). A region of the VEGF mRNA is amplified
using PCR primers. The cDNA amplified fragment (approximately 300
basepairs) is cloned into a vector such as the pCR2.1-TOPO vector
(Invitrogen, Carlsbad, Calif., USA) to generate a plasmid. Serial
ten-fold dilutions (104 to 109 molecules) of this plasmid are used
as a reference molecule for standard curve calculation. All
Real-Time PCR reactions are performed in a 25 .mu.A mixture
containing 1/20 volume of cDNA preparation (1 .mu.A), 1.times.SYBR
Green buffer (PE Applied Biosystems, Foster City, Calif., USA), 4
mM MgCl2, 0.2 .mu.M of each VEGF primer used above, 0.2 mM dNTPs
mix and 0.025 Unit of AmpliTaq Gold.RTM. thermostable DNA
polymerase (Applied Biosystems, Foster City, Calif., USA).
Real-Time quantitations are performed using the BIO-RAD iCycler iQ
system (BioRad, Hercules, Calif., USA). The fluorescence threshold
value can be calculated using the iCycle iQ system software. Using
similar methodologies, the data shown in FIG. 11 was obtained, and
shows that a compound of the present invention does not affect VEGF
mRNA levels.
Example 5
Compounds of the Invention do not Affect the Activity of PDE5
[0300] The compounds of the invention are tested to assess their
affect on phosphodiesterase 5 (PDE5) activity. The affect on PDE5
activity is determined using the High-Efficiency Fluorescence
Polarization Assay (HEFP) kit from Molecular Devices. The HEFP
assay measures the activity of PDE-5 by using fluorescein-labeled
derivatives of cGMP as a substrate. When hydrolyzed by PDE-5,
fluorescein-labeled cGMP derivatives are able to bind to a binding
reagent. The cGMP substrate:binding reagent complex results in a
highly polarized fluorescent state.
[0301] FIG. 12 shows the results of compounds 10, 7, and 25 of the
invention on PDE-5 activity. After combining recombinant PDE5
(CalBioChem) and the cGMP substrate, the mixture is incubated at
room temperature for 45 minutes in the presence or absence of
compounds or a positive control (Tadalafil). The reaction is
stopped upon addition of the binding reagent. Fluorescence
polarization is determined on a Viewlux using a setting recommended
by the manufacturer. As is evident from FIG. 12, compounds 10, 7,
and 25 of the invention do not inhibit the activity of PDE-5 in
comparison to the positive control.
Example 6
Effects on VEGF Production Induced by Hypoxia in Keratinocytes
[0302] Compounds of the invention reduce the hypoxia induced
production of VEGF from keratinocytes. In the absence of one or
more compounds of the invention, media from hypoxic keratinocytes
cell culture has substantial concentrations of VEGF concentration
(mean 1413, pg/mL). Treatment with a compound of the invention
induces a dose-dependent reduction in VEGF concentrations in the
mediaproduction, resulting in a maximal 57% decrease in the VEGF
concentration (to a mean of 606 pg/mL). By contrast, media from
normoxic cells has a relatively low low concentrations of VEGF
(mean 242 pg/mL) in the absence of a compound of the invention and
showed only a 21% decrease in the concentration of VEGF found in
the keratinocyte culture media concentrations (to a mean of 192
pg/mL) in the presence of a compound of the invention. No
cytotoxicity was observed at any concentration tested.
[0303] All VEGF measurements are conducted by ELISA and the data is
presented as the mean+/- the standard error (SE). See FIG. 13.
Example 7
Plasma Concentrations of VEGF and Compounds of the Invention in
Healthy Human Subjects Following a Single Dose of Compound #10
[0304] Groups of normal healthy human volunteers (six per group)
are administered a single oral dose (0.03, 0.10, 0.30, 1.00, or
3.00 mg/kg) of a compound of the invention. Two volunteers receive
placebo. At the indicated times after administration samples of
venous blood are withdrawn from the volunteers and the plasma
concentration of the compound in each sample is determined using
liquid chromatography and tandem mass spectroscopy (LC-MS/MS). Mean
plasma concentrations of compound are plotted versus time along
with the standard deviation of the values. See FIG. 14. Mean plasma
concentrations of VEGF (FIG. 15, panel A) and the change in VEGF
relative to baseline measurements (FIG. 15, panel B) are plotted
versus time along with the standard error of the values for the 0.0
and 3.0 mg/kg groups.
Example 8
Plasma Concentrations of VEGF and Compounds of the Invention in
Healthy Human Subjects with Dosing Twice Daily Over Multiple
Days
[0305] Groups of normal healthy human volunteers (8 per group, 3
male and 3 female receiving drug, 1 placebo per gender at each dose
level, 24 individuals total) are administered oral doses (0.3,
0.60, or 1.20 mg/kg) of a compound of the invention twice daily for
seven days. Vital signs (including pulse and blood pressure) and
samples for urinalysis are collected at baseline and repeatedly
during the study. Samples of venous blood are withdrawn from the
volunteers prior to administration of the first dose. Samples of
venous blood are also withdrawn at multiple times on the first and
seventh day, before the morning and evening doses on days two
through six of the study, then at 6, 12, 24 and 36 hours after
administration of the last dose of the compound, and once on days
14 and 21 of the study. The plasma concentration of the compound in
each sample is determined using liquid chromatography and tandem
mass spectroscopy (LC-MS/MS). While side effects including
headache, dizziness, nausea, vomiting and stomach discomfort are
observed, no serious, dose limiting, or definitive drug-related
events, such as the development of hypertension or proteinuria are
observed. All side effects are reversible and there is no
correlation of any side effect with dose. Mean plasma
concentrations of compound are plotted versus time along with the
standard deviation of the values for study days 1 and 7. See FIG.
16. The mean plasma and serum VEGF levels, along with the standard
deviations, on days 1 and 7 are plotted vs. time at each dose
level. See FIG. 17. Vital sign and urinalysis data indicate the
compounds of the invention do not give rise to hypertension or
proteinuria (data not shown).
Example 9
Plasma Concentrations Over Multiple Days with Dosing Thrice
Daily
[0306] Groups of normal healthy human volunteers (eight per group)
are administered oral doses of 1.6 mg/kg of a compound of the
invention three times daily for seven days. Samples of venous blood
are withdrawn from the volunteers prior to administration of the
first dose. Samples of venous blood are withdrawn at multiple times
on the first and seventh day, before the morning and evening doses
on days two through six of the study, at 6, 12, 24, and 36 hours
after administration of the last dose of the compound and once on
days 14 and 21 of the study. The plasma and/or serum concentration
of the compound in each sample is determined using liquid
chromatography and tandem mass spectroscopy (LC-MS/MS). While side
effects including headache, dizziness, nausea, vomiting and stomach
discomfort are observed, no serious, dose limiting, or definitive
drug-related events, such as the development of hypertension or
proteinuria are observed. In addition, no bleeding, clotting,
hypertension or proteinuria are observed. All side effects are
reversible and there is no correlation of any side effect with
dose. Mean plasma compound concentrations are plotted versus time
along with the standard deviation of the values. See FIG. 17. The
mean plasma and serum VEGF levels, along with the standard
deviations, on days 1 and 7 are plotted vs. time are plotted. See
FIG. 17, the lines labeled 1.6 mg/kg TID (n=6).
[0307] The discussion above provides illustrative features and
embodiments of the present invention, but the invention is not
limited to the particular features and embodiments disclosed. Those
skilled in the relevant arts will readily appreciate that
variations to the disclosed features and embodiments may be made
without departing from the spirit and scope of the present
invention. For example, one or more of the disclosed features or
embodiments may be combined with one or more other features or
embodiments.
[0308] All publications and patent applications cited herein are
incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually
indicated to be incorporated by reference.
* * * * *