U.S. patent application number 11/928101 was filed with the patent office on 2009-01-08 for chemical compounds.
Invention is credited to Jerry Leroy Adams, Jiri Kasparec, Domingos J. Silva.
Application Number | 20090012309 11/928101 |
Document ID | / |
Family ID | 27765915 |
Filed Date | 2009-01-08 |
United States Patent
Application |
20090012309 |
Kind Code |
A1 |
Adams; Jerry Leroy ; et
al. |
January 8, 2009 |
CHEMICAL COMPOUNDS
Abstract
Pyrazole derivatives are described herein. The described
invention also includes methods of making such pyrazole derivatives
as well as methods of using the same in the treatment of
diseases.
Inventors: |
Adams; Jerry Leroy; (King of
Prussia, PA) ; Kasparec; Jiri; (King of Prussia,
PA) ; Silva; Domingos J.; (Collegeville, PA) |
Correspondence
Address: |
GLAXOSMITHKLINE;CORPORATE INTELLECTUAL PROPERTY, MAI B482
FIVE MOORE DR., PO BOX 13398
RESEARCH TRIANGLE PARK
NC
27709-3398
US
|
Family ID: |
27765915 |
Appl. No.: |
11/928101 |
Filed: |
October 30, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10490921 |
Mar 26, 2004 |
7307097 |
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PCT/US02/30542 |
Sep 25, 2002 |
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11928101 |
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60325397 |
Sep 27, 2001 |
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Current U.S.
Class: |
548/371.7 |
Current CPC
Class: |
A61P 29/00 20180101;
C07D 405/12 20130101; A61P 3/10 20180101; A61P 17/02 20180101; A61P
25/28 20180101; A61P 35/04 20180101; A61P 35/00 20180101; A61P 9/00
20180101; A61P 19/02 20180101; A61P 1/16 20180101; A61P 9/10
20180101; A61P 9/14 20180101; C07D 233/90 20130101; A61P 27/02
20180101; A61P 17/06 20180101; A61P 43/00 20180101; A61P 13/12
20180101; A61P 37/06 20180101 |
Class at
Publication: |
548/371.7 |
International
Class: |
C07D 231/06 20060101
C07D231/06 |
Claims
1. A compound of Formula (I): ##STR00005## wherein: A is
C.sub.3-C.sub.7 cycloalkyl, aryl or heteroaryl, wherein said
C.sub.3-C.sub.7 cycloalkyl, aryl and heteroaryl is substituted with
at least one independently selected R.sup.2 group, and optionally
further substituted; D is hydrogen; R is independently selected
from C.sub.1-C.sub.6 alkylene, C.sub.2-C.sub.6 alkenylene, or
C.sub.2-C.sub.6 alkynylene, which alkylene, alkenylene, and
alkynylene may optionally be substituted; R.sup.1 is independently
selected from hydrogen, C.sub.1-C.sub.6 alkyl, aryl, --RR.sup.3,
--C(O)OR.sup.4, --C(O)NR.sup.5R.sup.6, and --C(O)R.sup.4, which
alkyl and aryl may optionally be substituted; R.sup.2 is
independently selected from --NR'''C(O)NR'''R'''',
--NR'''--C(.dbd.N--CN)--NR'''R'''' or the structure: ##STR00006##
R.sup.3 is independently selected from hydrogen, halo,
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl, C.sub.3-C.sub.7
cycloalkyl, C.sub.1-C.sub.6 alkoxy, C.sub.3-C.sub.7 cycloalkoxy,
C.sub.1-C.sub.6 haloalkoxy, aryl, aralkyl, aryloxy, heteroaryl,
heterocyclyl, --CN, --NHC(O)R, --NHC(S)R.sup.4, --NR.sup.5R.sup.6,
--RNR.sup.5R.sup.6, --SR.sup.4, --S(O)R.sup.4, --S(O).sub.2R.sup.4,
--R.sup.C(O)OR.sup.4, --C(O)OR.sup.4, --C(O)R.sup.4,
--C(O)NR.sup.5R.sup.6, --NHS(O).sub.2R.sup.4,
--S(O).sub.2NR.sup.5R.sup.6, or --NHC(.dbd.NH)R.sup.4, which alkyl,
haloalkyl, cycloalkyl, alkoxy, cycloalkoxy, haloalkoxy, aryl,
aralkyl, aryloxy, heteroaryl and heterocyclyl may optionally be
substituted; R.sup.4 is independently selected from hydrogen,
C.sub.1-C.sub.6 alkyl, aryl, heteroaryl, heterocyclyl, --RR.sup.3,
--NR'''R'''', or --NR'NR'''R'''', which alkyl, aryl, heteroaryl,
and heterocyclyl may optionally be substituted; R.sup.5 is
independently selected from hydrogen, C.sub.1-C.sub.6 alkyl,
C.sub.3-C.sub.7 cycloalkyl, cyanoalkyl, --R'R'', aryl, aralkyl,
heteroaryl, --NHC(O)OR''', --R'NHC(O)OR''', --R'NHC(O)NR'''R'''',
or --R'C(O)OR''', which alkyl, cycloalkyl, cyanoalkyl, aryl,
aralkyl, and heteroaryl may optionally be substituted; R.sup.6 is
independently selected from hydrogen, C.sub.1-C.sub.6 alkyl,
C.sub.3-C.sub.7 cycloalkyl, cyanoalkyl, --R'R'', aryl, aralkyl,
heteroaryl, --C(O)OR''', or --R'C(O)NR'''R''', which alkyl,
cycloalkyl, cyanoalkyl, aryl, aralkyl, and heteroaryl may
optionally be substituted; R' is independently selected from
optionally substituted C.sub.1-C.sub.4 alkylene; R'' is
independently selected from optionally substituted heteroalkyl or
NR'''R''''; R''' is independently selected from hydrogen,
C.sub.1-C.sub.6 alkyl, aryl, aralkyl, heteroaryl, or
C.sub.3-C.sub.7 cycloalkyl, which alkyl, aryl, aralkyl, heteroaryl,
and cycloalkyl may optionally be substituted; and R'''' is
independently selected from hydrogen, C.sub.1-C.sub.6 alkyl, aryl,
heteroaryl, or C.sub.3-C.sub.7 cycloalkyl, which alkyl, aryl,
heteroaryl, and cycloalkyl may optionally be substituted; or a
salt, solvate, or physiologically functional derivative
thereof.
2. A compound as claimed in claim 1, wherein A, said
C.sub.3-C.sub.7 cycloalkyl, aryl or heteroaryl is substituted with
one R.sup.2 group and optionally substituted with one or more
groups other than R.sup.2; or a salt, solvate, or physiologically
functional derivative thereof.
3. A compound as claimed in claim 1, wherein A is aryl or
heteroaryl, substituted with at least one independently selected
R.sup.2 group and optionally further substituted; or a salt,
solvate, or physiologically functional derivative thereof.
4. A compound as claimed in claim 1, wherein R.sup.2 is
--NR'''C(O)NR'''R''''.
5. A compound as claimed in claim 1, wherein R''' is hydrogen.
6. A compound as claimed in claim 1, wherein R'''' is aryl or
heteroaryl, either optionally substituted.
7. A compound as claimed in claim 1, wherein R.sup.1 is H or
optionally substituted C.sub.1-C.sub.6 alkyl; or a salt, solvate,
or physiologically functional derivative thereof.
8. A compound as claimed in claim 1, which is:
-Amino-3-{4-[3-2-(fluoro-5-trifluoromethyl-phenyl)-ureido]-phenyl}-1H-pyr-
azole-4-carboxylic acid amide; or a salt, solvate, or
physiologically functional derivative thereof.
9. A compound as claimed in claim 1, wherein A is phenyl
substituted with at least one independently selected R.sup.2 group
and optionally further substituted; or a salt, solvate, or
physiologically functional derivative thereof.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to pyrazole derivatives,
compositions and medicaments containing the same, as well as
processes for the preparation and use of such compounds,
compositions and medicaments. Such pyrazole derivatives are useful
in the treatment of diseases associated with inappropriate
angiogenesis.
[0002] The process of angiogenesis is the development of new blood
vessels, generally capillaries, from pre-existing vasculature.
Angiogenesis is defined as involving: (i) activation of endothelial
cells; (ii) increased vascular permeability; (iii) subsequent
dissolution of the basement membrane and extravisation of plasma
components leading to formation of a provisional fibrin gel
extracellular matrix; (iv) proliferation and mobilization of
endothelial cells; (v) reorganization of mobilized endothelial
cells to form functional capillaries; (vi) capillary loop
formation; and (vii) deposition of basement membrane and
recruitment of perivascular cells to newly formed vessels. Normal
angiogenesis is activated during tissue growth, from embryonic
development through maturity, and then enters a period of relative
quiescence during adulthood. Normal angiogenesis is also activated
during wound healing, and at certain stages of the female
reproductive cycle. Inappropriate angiogenesis has been associated
with several disease states including various retinopathies;
ischemic disease; atherosclerosis; chronic inflammatory disorders;
and cancer. The role of angiogenesis in disease states is
discussed, for instance, in Fan et al., Trends in Pharmacol. Sci.
16: 54-66; Shawver et al., DDT Vol. 2, No. 2 February 1997;
Folkmann, 1995, Nature Medicine 1: 27-31; Colville-Nash and Scott,
Ann. Rheum. Dis., 51, 919, 1992; Brooks et al., Cell, 79, 1157,
1994; Kahlon et al., Can. J. Cardiol. 8, 60, 1992; Folkman, Cancer
Biol, 3, 65, 1992; Denekamp, Br. J. Rad. 66, 181, 1993; Fidler and
Ellis, Cell, 79, 185, 1994; O'Reilly et al., Cell, 79, 315, 1994;
Ingber et al., Nature, 348, 555, 1990; Friedlander et al., Science,
270, 1500, 1995; Peacock et al., J. Exp. Med. 175, 1135, 1992;
Peacock et al., Cell. Immun. 160, 178, 1995; and Taraboletti et
al., J. Natl. Cancer Inst. 87, 293, 1995.
[0003] In cancer the growth of solid tumors has been shown to be
angiogenesis dependent. (See Folkmann, J., J. Nat'l. Cancer Inst.,
1990, 82, 4-6.) Consequently, the targeting of pro-angiogenic
pathways is a strategy being widely pursued in order to provide new
therapeutics in these areas of great, unmet medical need. The role
of tyrosine kinases involved in angiogenesis and in the
vascularization of solid tumors has drawn interest. Until recently
most interest in this area has focused on growth factors such as
vascular endothelial growth factor (VEGF) and its receptors termed
vascular endothelial growth factor receptor(s) (VEGFR). VEGF, a
polypeptide, is mitogenic for endothelial cells in vitro and
stimulates angiogenic responses in vivo. VEGF has also been linked
to inappropriate angiogenesis (Pinedo, H. M. et al. The Oncologist,
Vol. 5, No. 90001, 1-2, Apr. 2000). VEGFR(s) are protein tyrosine
kinases (PTKs). PTKs catalyze the phosphorylation of specific
tyrosyl residues in proteins involved in the regulation of cell
growth and differentiation. (A. F. Wilks, Progress in Growth Factor
Research, 1990, 2, 97-111; S. A. Courtneidge, Dev. Supp. 1, 1993,
57-64; J. A. Cooper, Semin. Cell Biol., 1994, 5(6), 377-387; R. F.
Paulson, Semin. Immunol., 1995, 7(4), 267-277; A. C. Chan, Curr.
Opin. Immunol., 1996, 8(3), 394-401).
[0004] Three PTK receptors for VEGF have been identified: VEGFR-1
(Flt-1); VEGFR-2 (Flk-1 or KDR) and VEGFR-3 (Flt-4). These
receptors are involved in angiogenesis and participate in signal
transduction (Mustonen, T. et al. J. Cell Biol. 1995, 129:
895-898). Of particular interest is VEGFR-2, which is a
transmembrane receptor PTK expressed primarily in endothelial
cells. Activation of VEGFR-2 by VEGF is a critical step in the
signal transduction pathway that initiates tumor angiogenesis. VEGF
expression may be constitutive to tumor cells and can also be
upregulated in response to certain stimuli. One such stimuli is
hypoxia, where VEGF expression is upregulated in both tumor and
associated host tissues. The VEGF ligand activates VEGFR-2 by
binding with its extracellular VEGF binding site. This leads to
receptor dimerization of VEGFRs and autophosphorylation of tyrosine
residues at the intracellular kinase domain of VEGFR-2. The kinase
domain operates to transfer a phosphate from ATP to the tyrosine
residues, thus providing binding sites for signaling proteins
downstream of VEGFR-2 leading ultimately to initiation of
angiogenesis (McMahon, G., The Oncologist, Vol. 5, No. 90001, 3-10,
Apr. 2000).
[0005] Angiopoieten 1 (Ang1), a ligand for the endothelium-specific
receptor tyrosine kinase TIE-2 is a novel angiogenic factor (Davis
et al., Cell, 1996, 87: 1161-1169; Partanen et al., Mol. Cell.
Biol., 12: 1698-1707 (1992); U.S. Pat. Nos. 5,521,073; 5,879,672;
5,877,020; and 6,030,831). The acronym TIE represents "tyrosine
kinase containing Ig and EGF homology domains". TIE is used to
identify a class of receptor tyrosine kinases, which are
exclusively expressed in vascular endothelial cells and early
hemopoietic cells. Typically, TIE receptor kinases are
characterized by the presence of an EGF-like domain and an
immunoglobulin (IG) like domain, which consists of extracellular
folding units, stabilized by intra-chain disulfide bonds (Partanen
et al., Curr. Topics Microbiol. Immunol., 1999, 237: 159-172).
Unlike VEGF, which functions during the early stages of vascular
development, Ang1 and its receptor TIE-2 function in the later
stages of vascular development, i.e., during vascular remodeling
(remodeling refers to formation of a vascular lumen) and maturation
(Yancopoulos et al., Cell, 1998, 93: 661-664; Peters, K. G., Circ.
Res., 1998, 83(3): 342-3; Suri et al., Cell 87, 1996:
1171-1180).
[0006] Consequently, inhibition of TIE-2 would be expected to serve
to disrupt remodeling and maturation of new vasculature initiated
by angiogenesis thereby disrupting the angiogenic process.
Furthermore, inhibition at the kinase domain binding site of
VEGFR-2 would block phosphorylation of tyrosine residues and serve
to disrupt initiation of angiogenesis. Presumably then, inhibition
of TIE-2 and/or VEGFR-2 should prevent tumor angiogenesis and serve
to retard or eradicate tumor growth. Accordingly, a treatment for
cancer or other disorder associated with inappropriate angiogenesis
could be provided.
[0007] The present inventors have discovered novel pyrazole
compounds, which are inhibitors of one or more of TIE-2 kinase
activity, VEGFR-2 kinase activity, and VEGFR-3 kinase activity,
e.g., one or both TIE-2 kinase and VEGFR-2 kinase activity. Such
pyrazole derivatives are useful in the treatment of disorders
mediated by at least one of inappropriate TIE-2 kinase, VEGFR-2
kinase, and VEGFR-3 kinase activity (which may include cancer
and/or diseases afflicting mammals which is characterized by
cellular proliferation in the area of disorders associated with
neo-vascularization and/or vascular permeability), and/or disorders
characterized by inappropriate angiogenesis; and/or for treating
cancer and/or a disease afflicting afflicting mammals which is
characterized by cellular proliferation in the area of disorders
associated with neo-vascularization and/or vascular
permeability.
BRIEF SUMMARY OF THE INVENTION
[0008] In one aspect of the present invention, there is provided a
compound of Formula (I):
##STR00001##
wherein:
[0009] A is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.7 cycloalkyl, aryl or
heteroaryl, wherein said C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.7 cycloalkyl, aryl
and heteroaryl is substituted with at least one independently
selected R.sup.2 group and optionally further substituted;
[0010] D is hydrogen; C.sub.1-C.sub.6 alkyl; aryl; heteroaryl;
heterocyclyl; --RR.sup.3; --C(O)OR.sup.4; C(O)NR.sup.5R.sup.6; or
--C(O)R.sup.4; wherein the alkyl, aryl, heteroaryl, and
heterocyclyl are optionally substituted (including aryl or
heteroaryl substituted with at least one independently selected
R.sup.3 group and optionally further substituted);
[0011] R is independently selected from C.sub.1-C.sub.6 alkylene,
C.sub.2-C.sub.6 alkenylene, or C.sub.2-C.sub.6 alkynylene; wherein
the alkylene, alkenylene and alkynylene are optionally
substituted;
[0012] R.sup.1 is independently selected from hydrogen;
C.sub.1-C.sub.6 alkyl; aryl; heteroaryl; heterocyclyl; --RR.sup.3;
--C(O)OR.sup.4; --C(O)NR.sup.5R.sup.6; and --C(O)R.sup.4; wherein
the alkyl, aryl, heteroaryl, and heterocyclyl are optionally
substituted (including aryl or heteroaryl substituted with at least
one independently selected R.sup.3 group and optionally further
substituted);
[0013] R.sup.2 is independently selected from
--NR'''C(O)NR'''R'''', --NR'''--C(.dbd.N--CN)--NR'''R'''' or the
structure:
##STR00002##
[0014] R.sup.3 is independently selected from hydrogen, halo,
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 haloalkyl, C.sub.3-C.sub.7
cycloalkyl, C.sub.1-C.sub.6 alkoxy, C.sub.3-C.sub.7 cycloalkoxy,
C.sub.1-C.sub.6 haloalkoxy, aryl, aralkyl, aryloxy, heteroaryl,
heterocyclyl, --CN, --NHC(O)R.sup.4, --NHC(S)R.sup.4,
--NR.sup.5R.sup.6, --RNR.sup.5R.sup.6, --SR.sup.4, --S(O)R.sup.4,
--S(O).sub.2R.sup.4, --RC(O)OR.sup.4, --C(O)OR.sup.4,
--C(O)R.sup.4, --C(O)NR.sup.5R.sup.6--NHS(O).sub.2R.sup.4,
--S(O).sub.2NR.sup.5R.sup.6, or --NHC(.dbd.NH)R.sup.4; wherein the
alkyl, haloalkyl, cycloalkyl, alkoxy, cycloalkoxy, haloalkoxy,
aryl, aralkyl, aryloxy, heteroaryl, and heterocyclyl are optionally
substituted;
[0015] R.sup.4 is independently selected from hydrogen,
C.sub.1-C.sub.6 alkyl, aryl, heteroaryl, heterocyclyl, --RR.sup.3,
--NR'''R'''', or --NR'NR'''R''''; wherein the alkyl, aryl,
heteroaryl, and heterocyclyl are optionally substituted;
[0016] R.sup.5 is independently selected from hydrogen,
C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.7 cycloalkyl, cyanoalkyl,
--R'R'', aryl, aralkyl, heteroaryl, --NHC(O)OR''', --R'NHC(O)OR''',
--R'NHC(O)NR'''R'''', or --R'C(O)OR'''; wherein the alkyl,
cycloalkyl, cyanoalkyl, aryl, aralkyl, and heteroaryl are
optionally substituted;
[0017] R.sup.6 is independently selected from hydrogen,
C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.7 cycloalkyl, cyanoalkyl,
--R'R'', aryl, aralkyl, heteroaryl, --C(O)OR''', or
--R'C(O)NR'''R'''; wherein the alkyl, cycloalkyl, cyanoalkyl, aryl,
aralkyl, and heteroaryl are optionally substituted;
[0018] R' is independently selected from optionally substituted
C.sub.1-C.sub.4 alkylene (especially C.sub.1-C.sub.3 and
C.sub.2-C.sub.3 alkylene);
[0019] R'' is independently selected from optionally substituted
heteroalkyl or NR'''R'''';
[0020] R''' is independently selected from hydrogen,
C.sub.1-C.sub.6 alkyl, aryl, aralkyl, heteroaryl, or
C.sub.3-C.sub.7 cycloalkyl; wherein the alkyl, aryl, aralkyl,
heteroaryl and cycloalkyl are optionally substituted; and
[0021] R'''' is independently selected from hydrogen,
C.sub.1-C.sub.6 alkyl, aryl, heteroaryl, or C.sub.3-C.sub.7
cycloalkyl; wherein the alkyl, aryl, heteroaryl, and cycloalkyl are
optionally substituted; or a salt, solvate, or physiologically
functional derivative thereof.
[0022] In a second aspect of the present invention, there is
provided a compound of Formula (I) wherein said A, said
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkenyl, C.sub.1-C.sub.6
alkynyl, C.sub.3-C.sub.7 cycloalkyl, aryl or heteroaryl is
substituted with one R.sup.2 group and optionally substituted with
one or more groups other than R.sup.2.
[0023] In a third aspect of the present invention, there is
provided a compound of Formula (I) wherein A is aryl or heteroaryl
(preferably phenyl), substituted with at least one independently
selected R.sup.2 group and optionally further substituted, and D,
R, R.sup.1, R.sup.2R.sup.3, R.sup.4, R.sup.5, R.sup.6, R', R'',
R''' and R'''' are as defined above; or a salt, solvate, or
physiologically functional derivative thereof. In a particular
embodiment. A is substituted with one R.sup.2 group and optionally
substituted with one or more groups other than R.sup.2.
[0024] In a fourth aspect of the present invention, there is
provided a compound of Formula (I) wherein A is aryl or heteroaryl
(preferably phenyl), substituted with at least one independently
selected R.sup.2 group and optionally further substituted, R.sup.2
is --NR'''C(O)NR'''R'''', and D, R, R.sup.1, R.sup.3, R.sup.4,
R.sup.5, R.sup.6, R', R'', R''' and R'''' are as defined above; or
a salt, solvate, or physiologically functional derivative thereof.
In a particular embodiment, A is substituted with one R.sup.2 group
and optionally substituted with one or more groups other than
R.sup.2.
[0025] In a fifth aspect of the present invention, there is
provided a compound of Formula (I) wherein R.sup.1 is independently
H or optionally substituted C.sub.1-C.sub.6 alkyl, and A, D, R,
R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R', R'', R'''
and R'''' are as defined above, or a salt, solvate, or
physiologically functional derivative thereof. In such compounds, A
is preferably aryl or heteroaryl (especially phenyl), substituted
with at least one R.sup.2 group and optionally further substituted,
and more preferably wherein R.sup.2 is --NR'''C(O)NR'''R''''. In a
particular embodiment, A is substituted with one R.sup.2 group and
optionally substituted with one or more groups other than
R.sup.2.
[0026] In a sixth aspect of the present invention, there is
provided a pharmaceutical composition comprising a therapeutically
effective amount of a compound of formula (I), or a salt, solvate,
or a physiologically functional derivative thereof and one or more
of pharmaceutically acceptable carriers, diluents and
excipients.
[0027] In a seventh aspect of the present invention, there is
provided a method of treating a disorder in a mammal, said disorder
being mediated by at least one of inappropriate TIE-2, VEGFR-2 and
VEGFR-3, especially at least one of TIE-2 and VEGFR-2 activity,
comprising: administering to said mammal a therapeutically
effective amount of a compound of formula (I) or a salt, solvate or
a physiologically functional derivative thereof.
[0028] In an eighth aspect of the present invention, there is
provided a compound of formula (I), or a salt, solvate, or a
physiologically functional derivative thereof for use in
therapy.
[0029] In a ninth aspect of the present invention, there is
provided the use of a compound of formula (I), or a salt, solvate,
or a physiologically functional derivative thereof in the
preparation of a medicament for use in the treatment of a disorder
mediated by at least one of inappropriate TIE-2, VEGFR-2 and
VEGFR-3, activity, especially at least one of inappropriate TIE-2
and VEGFR-2 activity.
[0030] In a tenth aspect of the present invention, there is
provided a method of treating a disorder in a mammal, said disorder
being mediated by at least one of inappropriate TIE-2, VEGFR-2, and
VEGFR-3, especially at least one of inappropriate TIE-2 and
VEGFR-2, activity, comprising: administering to said mammal
therapeutically effective amounts of (i) a compound of formula (I),
or a salt, solvate or physiologically functional derivative thereof
and (ii) an agent to inhibit growth factor receptor function.
[0031] In an eleventh aspect of the present invention, there is
provided a method of treating a disorder in a mammal, said disorder
being characterized by inappropriate angiogenesis, comprising:
administering to said mammal a therapeutically effective amount of
a compound of formula (I), or a salt, solvate or physiologically
functional derivative thereof.
DETAILED DESCRIPTION
[0032] All documents cited or referred to herein, including issued
patents, published and unpublished patent applications, and other
publications are hereby incorporated herein by reference as though
fully set forth.
[0033] As used herein, the term "effective amount" means that
amount of a drug or pharmaceutical agent that will elicit the
biological or medical response of a tissue, system, animal or human
that is being sought, for instance, by a researcher or clinician.
Furthermore, the term "therapeutically effective amount" means any
amount which, as compared to a corresponding subject who has not
received such amount, results in improved treatment, healing,
prevention, or amelioration of a disease, disorder, or side effect,
or a decrease in the rate of advancement of a disease or disorder.
The term also includes within its scope amounts effective to
enhance normal physiological function.
[0034] As used herein, the numbering of the
5-amino-4-carboxamido-pyrazole scaffold in formula (I) is assigned
as shown in the structure following.
##STR00003##
[0035] As used herein, the term "alkyl" refers to a straight or
branched chain hydrocarbon radical having from one to twelve carbon
atoms, optionally substituted with substituents selected from the
group which includes C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
hydroxyalkyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6
alkylsulfanyl, C.sub.1-C.sub.6 alkylsulfenyl, C.sub.1-C.sub.6
alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted
by alkyl or aryl, carboxy, carbamoyl optionally substituted by
alkyl or aryl, aryl, aryloxy, heteroaryl, aminosulfonyl optionally
substituted by alkyl or aryl, nitro, cyano, halogen, or
C.sub.1-C.sub.6 perfluoroalkyl, multiple degrees of substitution
being allowed. Examples of "alkyl" as used herein include, but are
not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl,
isobutyl, t-butyl, n-pentyl, isopentyl, and the like.
[0036] As used herein, the term "C.sub.1-C.sub.6 alkyl" refers to
an alkyl group as defined above containing at least 1, and at most
6, carbon atoms. Examples of branched or straight chained
"C.sub.1-C.sub.6 alkyl" groups useful in the present invention
include, but are not limited to, methyl, ethyl, n-propyl,
isopropyl, isobutyl, n-butyl, t-butyl, n-pentyl, and isopentyl.
[0037] As used herein, the term "alkylene" refers to a straight or
branched chain divalent hydrocarbon radical having from one to ten
carbon atoms, optionally substituted with substituents selected
from the group which includes C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 alkylsulfanyl,
C.sub.1-C.sub.6 alkylsulfenyl, C.sub.1-C.sub.6 alkylsulfonyl, oxo,
hydroxy, mercapto, amino optionally substituted by alkyl or aryl,
carboxy, carbamoyl optionally substituted by alkyl or aryl,
aminosulfonyl optionally substituted by alkyl or aryl, nitro,
cyano, halogen and C.sub.1-C.sub.6 perfluoroalkyl, multiple degrees
of substitution being allowed. Examples of "alkylene" as used
herein include, but are not limited to, methylene, ethylene,
n-propylene, n-butylene, and the like.
[0038] As used herein, the terms "C.sub.1-C.sub.6 alkylene" and
"C.sub.1-C.sub.3 alkylene" refer to an alkylene group, as defined
above, which contains at least 1, and, respectively, at most 6 or
at most 3, carbon atoms. Examples of "C.sub.1-C.sub.3 alkylene"
groups useful in the present invention include, but are not limited
to, methylene, ethylene, and n-propylene.
[0039] As used herein, the term "alkenyl" refers to a straight or
branched chain hydrocarbon radical having from two to ten carbons
and at least one carbon-carbon double bond, optionally substituted
with substituents selected from the group which includes
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6
alkylsulfanyl, C.sub.1-C.sub.6 alkylsulfenyl, C.sub.1-C.sub.6
alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted
by alkyl or aryl, carboxy, carbamoyl optionally substituted by
alkyl or aryl, aminosulfonyl optionally substituted by alkyl or
aryl, nitro, cyano, halogen and C.sub.1-C.sub.6 perfluoroalkyl,
multiple degrees of substitution being allowed. Examples of
"alkenyl" as used herein include ethenyl, propenyl, 1-butenyl,
2-butenyl, and isobutenyl.
[0040] As used herein, the term "C.sub.2-C.sub.6 alkenyl" refers to
an alkenyl group as defined above containing at least 2, and at
most 6, carbon atoms. Examples of "C.sub.2-C.sub.6 alkenyl" groups
useful in the present invention include, but are not limited to,
ethenyl, propenyl, 1-butenyl, 2-butenyl, and isobutenyl
[0041] As used herein, the term "alkenylene" refers to a straight
or branched chain divalent hydrocarbon radical having from two to
ten carbon atoms and one or more carbon-carbon double bonds,
optionally substituted with substituents selected from the group
which includes C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy,
C.sub.1-C.sub.6 alkylsulfanyl, C.sub.1-C.sub.6 alkylsulfenyl,
C.sub.1-C.sub.6 alkylsulfonyl, oxo, hydroxy, mercapto, amino
optionally substituted by alkyl or aryl, carboxy, carbamoyl
optionally substituted by alkyl or aryl, aminosulfonyl optionally
substituted by alkyl or aryl, nitro, cyano, halogen and
C.sub.1-C.sub.6 perfluoroalkyl, multiple degrees of substitution
being allowed. Examples of "alkenylene" as used herein include, but
are not limited to, ethene-1,2-diyl, propene-1,3-diyl,
methylene-1,1-diyl, and the like.
[0042] As used herein, the terms "C.sub.2-C.sub.6 alkenylene" and
"C.sub.2-C.sub.3 alkenylene" refer to an alkenylene group as
defined above containing at least 2, and, respectively, at most 6
or at most 3 carbon atoms. Examples of "C.sub.2-C.sub.3 alkenylene"
groups useful in the present invention include, but are not limited
to, ethene-1,2-diyl, propene-1,3-diyl, methylene-1,1-diyl, and the
like.
[0043] As used herein, the term "alkynyl" refers to a straight or
branched chain hydrocarbon radical having from two to ten carbons
and at least one carbon-carbon triple bond, optionally substituted
with substituents selected from the group which includes
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6
alkylsulfanyl, C.sub.1-C.sub.6 alkylsulfenyl, C.sub.1-C.sub.6
alkylsulfonyl, oxo, aryl, hydroxy, mercapto, amino optionally
substituted by alkyl or aryl, carboxy, carbamoyl optionally
substituted by alkyl or aryl, aminosulfonyl optionally substituted
by alkyl or aryl, nitro, cyano, halogen and C.sub.1-C.sub.6
perfluoroalkyl, multiple degrees of substitution being allowed.
Examples of "alkynyl" as used herein, include but are not limited
to acetylenyl, 1-propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, and
1-hexynyl.
[0044] As used herein, the term "alkynylene" refers to a straight
or branched chain divalent hydrocarbon radical having from two to
ten carbon atoms and one or more carbon-carbon triple bonds,
optionally substituted with substituents selected from the group
which includes C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy,
C.sub.1-C.sub.6 alkylsulfanyl, C.sub.1-C.sub.6 alkylsulfenyl,
C.sub.1-C.sub.6 alkylsulfonyl, oxo, hydroxy, mercapto, amino
optionally substituted by alkyl or aryl, carboxy, carbamoyl
optionally substituted by alkyl or aryl, aminosulfonyl optionally
substituted by alkyl or aryl, nitro, cyano, halogen and
C.sub.1-C.sub.6 perfluoroalkyl, multiple degrees of substitution
being allowed. Examples of "alkynylene" as used herein include, but
are not limited to, ethyne-1,2-diyl, propyne-1,3-diyl, and the
like.
[0045] As used herein, the terms "C.sub.2-C.sub.6 alkynylene" and
"C.sub.2-C.sub.3 alkynylene" refers to an alkynylene group as
defined above containing at least 2, and, respectively, at most 6
carbon atoms or at most 3 carbon atoms. Examples of
"C.sub.2-C.sub.3 alkynylene" groups useful in the present invention
include, but are not limited to, ethyne-1,2-diyl, propyne-1,3-diyl,
and the like.
[0046] As used herein, the term "halogen" refers to fluorine (F),
chlorine (Cl), bromine (Br), or iodine (I) and the term "halo"
refers to the halogen radicals fluoro, chloro, bromo, and iodo.
[0047] As used herein, the term "C.sub.1-C.sub.6 haloalkyl" refers
to an alkyl group as defined above containing at least 1, and at
most 6, carbon atoms substituted with at least one halo group, halo
being as defined herein. Examples of branched or straight chained
"C.sub.1-C.sub.6 haloalkyl" groups useful in the present invention
include, but are not limited to, methyl, ethyl, propyl, isopropyl,
isobutyl and n-butyl substituted independently with one or more
halos, e.g., fluoro, chloro, bromo and iodo, e.g.,
trifluoromethyl.
[0048] As used herein, the term "C.sub.3-C.sub.7 cycloalkyl" refers
to a non-aromatic cyclic hydrocarbon radical having from three to
seven carbon atoms and which optionally includes a C.sub.1-C.sub.6
alkyl linker through which it may be attached, and which is
optionally substituted with substituents selected from the group
which includes C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy,
C.sub.1-C.sub.6 alkylsulfanyl, C.sub.1-C.sub.6 alkylsulfenyl,
C.sub.1-C.sub.6 alkylsulfonyl, oxo, hydroxy, mercapto, amino
optionally substituted by alkyl or aryl, carboxy, carbamoyl
optionally substituted by alkyl or aryl, aminosulfonyl optionally
substituted by alkyl or aryl, nitro, cyano, halogen,
C.sub.1-C.sub.6 perfluoroalkyl, multiple degrees of substitution
being allowed. The C.sub.1-C.sub.6 alkyl group is as defined above.
Exemplary "C.sub.3-C.sub.7 cycloalkyl" groups include, but are not
limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and
cycloheptyl.
[0049] As used herein, the term "C.sub.3-C.sub.7 cycloalkylene"
refers to a divalent non-aromatic cyclic hydrocarbon radical having
from three to seven carbon atoms, optionally substituted with
substituents selected from the group which includes C.sub.1-C.sub.6
alkyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 alkylsulfanyl,
C.sub.1-C.sub.6 alkylsulfenyl, C.sub.1-C.sub.6 alkylsulfonyl, oxo,
hydroxy, mercapto, amino optionally substituted by alkyl or aryl,
carboxy, carbamoyl optionally substituted by alkyl or aryl,
aminosulfonyl optionally substituted by alkyl or aryl, nitro,
cyano, halogen, C.sub.1-C.sub.6 perfluoroalkyl, multiple degrees of
substitution being allowed. Examples of "cycloalkylene" as used
herein include, but are not limited to, cyclopropyl-1,1-diyl,
cyclopropyl-1,2-diyl, cyclobutyl-1,2-diyl, cyclopentyl-1,3-diyl,
cyclohexyl-1,4-diyl, cycloheptyl-1,4-diyl, or cyclooctyl-1,5-diyl,
and the like.
[0050] As used herein, the term "heterocyclic" or the term
"heterocyclyl" refers to a three to twelve-membered ring containing
one or more heteroatomic substitutions selected from S, SO,
SO.sub.2, O, N, or N-oxide, optionally substituted with
substituents selected from the group which includes C.sub.1-C.sub.6
alkyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 alkylsulfanyl,
C.sub.1-C.sub.6 alkylsulfenyl, C.sub.1-C.sub.6 alkylsulfonyl, oxo,
hydroxy, mercapto, amino optionally substituted by alkyl or aryl,
carboxy, carbamoyl optionally substituted by alkyl or aryl,
aminosulfonyl optionally substituted by alkyl or aryl, nitro,
cyano, halogen, or C.sub.1-C.sub.6 perfluoroalkyl, multiple degrees
of substitution being allowed. Such a ring can be saturated or have
one or more degrees of saturation. Such a ring may be optionally
fused to one or more other optionally substituted, "heterocyclic"
ring(s), aryl rings (including benzene rings), heteroaryl rings, or
cycloalkyl ring(s). Examples of "heterocyclic" moieties include,
but are not limited to, tetrahydrofuran, pyran, 1,4-dioxane,
1,3-dioxane, piperidine, pyrrolidine, morpholine,
tetrahydrothiopyran, tetrahydrothiophene, and the like.
[0051] As used herein, the term "heterocyclylene" refers to an
unsaturated three to twelve-membered ring diradical containing one
or more heteroatoms selected from S, SO, SO.sub.2, O, N, or
N-oxide, optionally substituted with substituents selected from the
group which includes C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy,
C.sub.1-C.sub.6 alkylsulfanyl, C.sub.1-C.sub.6 alkylsulfenyl,
C.sub.1-C.sub.6 alkylsulfonyl, oxo, hydroxy, mercapto, amino
optionally substituted by alkyl or aryl, carboxy, carbamoyl
optionally substituted by alkyl or aryl, aminosulfonyl optionally
substituted by alkyl or aryl, nitro, cyano, halogen and
C.sub.1-C.sub.6 perfluoroalkyl, multiple degrees of substitution
being allowed. Such a ring has one or more degrees of unsaturation.
Such a ring may be optionally fused to one or more optionally
substituted aryl rings (including benzene rings), heterocyclic
rings, heteroaryl rings, or cycloalkyl rings. Examples of
"heterocyclylene" include, but are not limited to,
tetrahydrofuran-2,5-diyl, morpholine-2,3-diyl, pyran-2,4-diyl,
1,4-dioxane-2,3-diyl, 1,3-dioxane-2,4-diyl, piperidine-2,4-diyl,
piperidine-1,4-diyl, pyrrolidine-1,3-diyl, morpholine-2,4-diyl, and
the like.
[0052] As used herein, the term "aryl" refers to an optionally
substituted benzene ring or to an optionally substituted benzene
ring fused to one or more optionally substituted benzene rings to
form a ring system. Exemplary optional substituents include
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6
haloalkyl, C.sub.1-C.sub.6 haloalkoxy, C.sub.1-C.sub.6
alkylsulfanyl, C.sub.1-C.sub.6 alkylsulfenyl, C.sub.1-C.sub.6
alkylsulfonyl, C.sub.1-C.sub.6 alkylsulfonylamino,
arylsulfonylamino, alkylcarboxy, alkylcarboxamide, oxo, hydroxy,
mercapto, amino optionally substituted by alkyl or acyl, carboxy,
tetrazolyl, carbamoyl optionally substituted by alkyl, aryl, or
heteroaryl, aminosulfonyl optionally substituted by alkyl, acyl,
aroyl, aroylamino, heteroaroyl, acyloxy, aroyloxy, heteroaroyloxy,
alkoxycarbonyl, nitro, cyano, halogen, heteroaryl, heterocyclyl,
aryl optionally substituted with aryl, halogen, C.sub.1-C.sub.6
alkyl, C.sub.1-C.sub.6 haloalkyl, or C.sub.1-C.sub.6 alkylsulfonyl,
ureido, arylurea, alkylurea, cycloalkylurea, alkylthiourea,
aryloxy, or aralkoxy, multiple degrees of substitution being
allowed. Such a ring or ring system may be optionally fused to one
or more optionally substituted aryl rings (including benzene rings)
or cycloalkyl rings. Examples of "aryl" groups include, but are not
limited to, phenyl, 2-naphthyl, tetrahydronaphthyl, 1-naphthyl,
biphenyl, indanyl, anthracyl, phenanthryl, or napthyl, as well as
substituted derivatives thereof.
[0053] As used herein, the term "arylene" refers to an optionally
substituted benzene ring diradical or to a benzene ring system
diradical containing an optionally substituted benzene ring fused
to one or more optionally substituted benzene rings. Exemplary
optional substituents are selected from the group which includes
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6
alkylsulfanyl, C.sub.1-C.sub.6 alkylsulfenyl, C.sub.1-C.sub.6
alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted
by alkyl or aryl, carboxy, tetrazolyl, carbamoyl optionally
substituted by alkyl or aryl, aminosulfonyl optionally substituted
by alkyl or aryl, acyl, aroyl, heteroaroyl, acyloxy, aroyloxy,
heteroaroyloxy, alkoxycarbonyl, nitro, cyano, halogen,
C.sub.1-C.sub.6 perfluoroalkyl, heteroaryl and aryl, multiple
degrees of substitution being allowed. Such a ring or ring system
may be optionally fused to one or more optionally substituted aryl
rings (including benzene rings), or cycloalkyl rings. Examples of
"arylene" include, but are not limited to, benzene-1,4-diyl,
naphthalene-1,8-diyl, anthracene-1,4-diyl, and the like.
[0054] As used herein, the term "aralkyl" refers to an aryl or
heteroaryl group, as defined herein, attached through a
C.sub.1-C.sub.3 alkylene linker, wherein the C.sub.1-C.sub.3
alkylene is as defined herein. Examples of "aralkyl" include, but
are not limited to, benzyl, phenylpropyl, 2-pyridylmethyl,
3-isoxazolylmethyl, 3-(1-methyl-5-t-butyl-pyrazyl)methyl,
3-isoxazolylmethyl, and 2-imidazoyly ethyl.
[0055] As used herein, the term "heteroaryl" refers to an
optionally substituted monocyclic five to seven membered aromatic
ring containing one or more heteroatomic substitutions selected
from S, SO, SO.sub.2, O, N, or N-oxide, or to such an aromatic ring
fused to one or more, optionally substituted, such heteroaryl
rings, aryl rings (including benzene rings), heterocyclic rings, or
cycloalkyl rings (e.g., a bicyclic or tricyclic ring system).
Examples of optional substituents are selected from the group which
includes C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy,
C.sub.1-C.sub.6 alkylsulfanyl, C.sub.1-C.sub.6 alkylsulfenyl,
C.sub.1-C.sub.6 alkylsulfonyl, oxo, hydroxy, mercapto, amino
optionally substituted by alkyl or aryl, carboxy, tetrazolyl,
carbamoyl optionally substituted by alkyl or aryl, aminosulfonyl
optionally substituted by alkyl, acyl, aroyl, heteroaroyl, acyloxy,
aroyloxy, heteroaroyloxy, alkoxycarbonyl, nitro, cyano, halogen,
C.sub.1-C.sub.6 perfluoroalkyl, heteroaryl, or aryl, multiple
degrees of substitution being allowed. Examples of "heteroaryl"
groups used herein include, but are not limited to, furanyl,
thiophenyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,
thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, oxo-pyridyl,
thiadiazolyl, isothiazolyl, pyridyl, pyridazyl, pyrazinyl,
pyrimidyl, quinolinyl, isoquinolinyl, benzofuranyl,
benzothiophenyl, indolyl, indazolyl, and substituted versions
thereof.
[0056] As used herein, the term "heteroarylene" refers to a five-
to seven-membered monocyclic aromatic diradical, or to a polycyclic
aromatic diradical, containing one or more heteroatomic
substitutions selected from S, SO, SO.sub.2, O, N, or N-oxide,
optionally substituted with substituents selected from the group
which includes C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy,
C.sub.1-C.sub.6 alkylsulfanyl, C.sub.1-C.sub.6 alkylsulfenyl,
C.sub.1-C.sub.6 alkylsulfonyl, oxo, hydroxy, mercapto, amino
optionally substituted by alkyl or aryl, carboxy, tetrazolyl,
carbamoyl optionally substituted by alkyl or aryl, aminosulfonyl
optionally substituted by alkyl or aryl, acyl, aroyl, heteroaroyl,
acyloxy, aroyloxy, heteroaroyloxy, alkoxycarbonyl, nitro, cyano,
halogen, C.sub.1-C.sub.6 perfluoroalkyl, heteroaryl, or aryl,
multiple degrees of substitution being allowed. For polycyclic
aromatic system diradicals, one or more of the rings may contain
one or more heteroatoms, and one or more of the rings may be aryl,
heterocyclic, heteroaryl, or cycloalkyl. Examples of
"heteroarylene" used herein are furan-2,5-diyl, thiophene-2,4-diyl,
1,3,4-oxadiazole-2,5-diyl, 1,3,4-thiadiazole-2,5-diyl,
1,3-thiazole-2,4-diyl, 1,3-thiazole-2,5-diyl, pyridine-2,4-diyl,
pyridine-2,3-diyl, pyridine-2,5-diyl, pyrimidine-2,4-diyl,
quinoline-2,3-diyl, and the like.
[0057] As used herein, the term "alkoxy" refers to the group
R.sub.aO--, where R is alkyl as defined above and the term
"C.sub.1-C.sub.6 alkoxy" refers to an alkoxy group as defined
herein wherein the alkyl moiety contains at least 1, and at most 6,
carbon atoms. Exemplary C.sub.1-C.sub.6 alkoxy groups useful in the
present invention include, but are not limited to, methoxy, ethoxy,
n-propoxy, isopropoxy, n-butoxy, and t-butoxy.
[0058] As used herein the term "aralkoxy" refers to the group
R.sub.bR.sub.aO--, where R.sub.a is alkyl and R.sub.b is aryl as
defined above.
[0059] As used herein the term "aryloxy" refers to the group
R.sub.aO--, where R.sub.a is aryl as defined above.
[0060] As used herein the term "ureido" refers to the group
--NHC(O)NH.sub.2.
[0061] As used herein, the term "arylurea" refers to the group
--NHC(O)NHR.sub.a wherein R.sub.a is aryl as defined above.
[0062] As used herein, the term "arylthiourea" refers to the group
--NHC(S)NHR.sub.a wherein R.sub.a is aryl as defined above.
[0063] As used herein, the term "alkylurea" refers to the group
--NHC(O)NHR.sub.a wherein R.sub.a is alkyl as defined above.
[0064] As used herein, the term "cycloalkylurea" refers to the
group --NHC(O)NHR.sub.a wherein R.sub.a is cycloalkyl as defined
above.
[0065] As used herein, the term "C.sub.3-C.sub.7 cycloalkoxy"
refers to the group R.sub.aO--, where R.sub.a is C.sub.3-C.sub.7
cycloalkyl as defined above. Exemplary C.sub.3-C.sub.7 cycloalkoxy
groups useful in the present invention include, but are not limited
to cyclopropoxy, cyclobutoxy, and cyclopentoxy.
[0066] As used herein, the term "haloalkoxy" refers to the group
R.sub.aO--, where R.sub.a is haloalkyl as defined above and the
term "C.sub.1-C.sub.6 haloalkoxy" refers to a haloalkoxy group as
defined herein wherein the haloalkyl moiety contains at least 1,
and at most 6, carbon atoms. Exemplary C.sub.1-C.sub.6 haloalkoxy
groups useful in the present invention include, but is not limited
to, trifluoromethoxy.
[0067] As used herein, the term "alkylsulfanyl" refers to the group
R.sub.aS--, where R.sub.a is alkyl as defined above and the term
"C.sub.1-C.sub.6 alkylsulfanyl" refers to an alkylsulfanyl group as
defined herein wherein the alkyl moiety contains at least 1, and at
most 6, carbon atoms.
[0068] As used herein, the term "haloalkylsulfanyl" refers to the
group R.sub.aS--, where R.sub.a is haloalkyl as defined above and
the term "C.sub.1-C.sub.6 haloalkylsulfanyl" refers to a
haloalkylsulfanyl group as defined herein wherein the alkyl moiety
contains at least 1, and at most 6, carbon atoms.
[0069] As used herein, the term "alkylsulfenyl" refers to the group
R.sub.aS(O)--, where R.sub.a is alkyl as defined above and the term
"C.sub.1-C.sub.6 alkylsulfenyl" refers to an alkylsulfenyl group as
defined herein wherein the alkyl moiety contains at least 1, and at
most 6, carbon atoms.
[0070] As used herein, the term "alkylsulfonyl" refers to the group
R.sub.aS(O).sub.2--, where R.sub.a is alkyl as defined above and
the term "C.sub.1-C.sub.6 alkylsulfonyl" refers to an alkylsulfonyl
group as defined herein wherein the alkyl moiety contains at least
1, and at most 6, carbon atoms.
[0071] As used herein, the term "alkylsulfonylamino" refers to the
group --NHS(O).sub.2R.sub.a wherein R.sub.a is alkyl as defined
above and the term "C.sub.1-C.sub.6 alkylsulfonylamino" refers to
an alkylsulfonylamino group as defined herein wherein the alkyl
moiety contains at least 1, and at most 6, carbon atoms.
[0072] As used herein, the term "arylsulfonylamino" refers to the
group --NHS(O).sub.2R.sub.a wherein R.sub.a is aryl as defined
above.
[0073] As used herein, the term "alkylcarboxyamide" refers to the
group --NHC(O)R.sub.a wherein R.sub.a is alkyl, amino, or amino
substituted with alkyl, aryl or heteroaryl as described above.
[0074] As used herein the term "alkylcarboxy" refers to the group
--C(O)R.sub.a wherein R.sub.a is alkyl as described above.
[0075] As used herein, the term "oxo" refers to the group
.dbd.O.
[0076] As used herein, the term "mercapto" refers to the group
--SH.
[0077] As used herein, the term "carboxy" refers to the group
--C(O)OH.
[0078] As used herein, the term "cyano" refers to the group
--CN.
[0079] As used herein the term "cyanoalkyl" refers to the group
--R.sub.aCN, wherein R.sub.a is alkyl as defined above. Exemplary
"cyanoalkyl" groups useful in the present invention include, but
are not limited to, cyanomethyl, cyanoethyl, and
cyanoisopropyl.
[0080] As used herein, the term "aminosulfonyl" refers to the group
--S(O).sub.2NH.sub.2.
[0081] As used herein, the term "carbamoyl" refers to the group
--C(O)NH.sub.2.
[0082] As used herein, the term "sulfanyl" shall refer to the group
--S--.
[0083] As used herein, the term "sulfenyl" shall refer to the group
--S(O)--.
[0084] As used herein, the term "sulfonyl" shall refer to the group
--S(O).sub.2-- or --SO.sub.2--.
[0085] As used herein, the term "acyl" refers to the group
R.sub.aC(O)--, where R.sub.a is alkyl, cycloalkyl, or heterocyclyl
as defined herein.
[0086] As used herein, the term "aroyl" refers to the group
R.sub.aC(O)--, where R.sub.a is aryl as defined herein.
[0087] As used herein, the term "aroylamino" refers to the group
R.sub.aC(O)NH--, where R.sub.a is aryl as defined herein.
[0088] As used herein, the term "heteroaroyl" refers to the group
R.sub.aC(O)--, where R.sub.a is heteroaryl as defined herein.
[0089] As used herein, the term "alkoxycarbonyl" refers to the
group R.sub.aOC(O)--, where R.sub.a is alkyl as defined herein.
[0090] As used herein, the term "acyloxy" refers to the group
R.sub.aC(O)O--, where R.sub.a is alkyl, cycloalkyl, or heterocyclyl
as defined herein.
[0091] As used herein, the term "aroyloxy" refers to the group
R.sub.aC(O)O--, where R.sub.a is aryl as defined herein.
[0092] As used herein, the term "heteroaroyloxy" refers to the
group R.sub.aC(O)O--, where R.sub.a is heteroaryl as defined
herein.
[0093] As used herein, the term "optionally" means that the
subsequently described event(s) may or may not occur, and includes
both event(s) which occur, and events that do not occur.
[0094] As used herein, the term "physiologically functional
derivative" refers to any pharmaceutically acceptable derivative of
a compound of the present invention, for example, an ester or an
amide, which upon administration to a mammal is capable of
providing (directly or indirectly) a compound of the present
invention or an active metabolite thereof. Such derivatives are
clear to those skilled in the art, without undue experimentation,
and with reference to the teaching of Burger's Medicinal Chemistry
And Drug Discovery, 5.sup.th Edition, Vol 1: Principles and
Practice, which is incorporated herein by reference to the extent
that it teaches physiologically functional derivatives.
[0095] As used herein, the term "solvate" refers to a complex of
variable stoichiometry formed by a solute (in this invention, a
compound of formula (I) or a salt or physiologically functional
derivative thereof) and a solvent. Such solvents for the purpose of
the invention may not interfere with the biological activity of the
solute. Examples of suitable solvents include, but are not limited
to, water, methanol, ethanol and acetic acid. Preferably the
solvent used is a pharmaceutically acceptable solvent. Examples of
suitable pharmaceutically acceptable solvents include, without
limitation, water, ethanol and acetic acid. Most preferably the
solvent used is water.
[0096] As used herein, the term "substituted" refers to
substitution with the named substituent or substituents, multiple
degrees of substitution being allowed unless otherwise stated.
[0097] Certain of the compounds described herein contain one or
more chiral atoms, or may otherwise be capable of existing as two
enantiomers. The compounds of this invention include mixtures of
enantiomers as well as purified enantiomers or enantiomerically
enriched mixtures. Also included within the scope of the invention
are the individual isomers of the compounds represented by formula
(I) above as well as any wholly or partially equilibrated mixtures
thereof. The present invention also covers the individual isomers
of the compounds represented by the formulas above as mixtures with
isomers thereof in which one or more chiral centers are inverted.
Also, it is understood that all tautomers and mixtures of tautomers
of the compounds of formula (I) are included within the scope of
the compounds of formula (I).
[0098] It is to be understood that reference to compounds of
formula (I) herein refers to all compounds within the scope of
formula (I) as defined above with respect to A, D, R, R.sup.1,
R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R', R'',
R''', or R'''' unless specifically limited otherwise.
[0099] Specific examples of compounds of the present invention
include the following: [0100]
5-Amino-1-tert-butyl-3-{4-[3-(2-fluoro-5-trifluoromethyl-phenyl)-ureido]--
phenyl}-1H-pyrazole-4-carboxylic acid amide [0101]
5-Amino-3-{4-[3-(2-fluoro-5-trifluoromethyl-phenyl)-ureido]-phenyl}-1-met-
hyl-1H-pyrazole-4-carboxylic acid amide [0102]
5-Amino-3-{4-[3-(2-chloro-5-trifluoromethyl-phenyl)-ureido]-phenyl}-1-met-
hyl-1H-pyrazole-4-carboxylic acid amide [0103]
5-Amino-3-{4-[3-(bis-3,5-trifluoromethyl-phenyl)-ureido]-phenyl}-1-methyl-
-1H-pyrazole-4-carboxylic acid amide [0104]
5-Amino-1-methyl-3-[4-(3-naphthalen-2-yl-ureido)-phenyl]-1H-pyrazole-4-ca-
rboxylic acid amide [0105]
5-Amino-3-{4-[3-(3-chloro-phenyl)-ureido]-phenyl}-1-methyl-1H-pyrazole-4--
carboxylic acid amide [0106]
5-Amino-3-{4-[3-(2,5-dichloro-phenyl)-ureido]-phenyl}-1-methyl-1H-pyrazol-
e-4-carboxylic acid amide [0107]
5-Amino-1-methyl-3-[4-(3-naphthalen-1-yl-ureido)-phenyl]-1H-pyrazole-4-ca-
rboxylic acid amide [0108]
5-Amino-1-methyl-3-{4-[3-(3-trifluoromethyl-phenyl)-ureido]-phenyl}-1H-py-
razole-4-carboxylic acid amide [0109]
5-Amino-3-[4-(3-biphenyl-4-yl-ureido)-phenyl]-1-methyl-1H-pyrazole-4-carb-
oxylic acid amide [0110]
5-Amino-3-{4-[3-(2,6-difluoro-phenyl)-ureido]-phenyl}-1-methyl-1H-pyrazol-
e-4-carboxylic acid amide [0111]
5-Amino-3-[4-(3-benzo[1,3]dioxol-5-yl-ureido)-phenyl]-1-methyl-1H-pyrazol-
e-4-carboxylic acid amide [0112]
5-Amino-1-methyl-3-[4-(3-o-tolyl-ureido)-phenyl]-1H-pyrazole-4-carboxylic
acid amide [0113]
5-Amino-1-methyl-3-[4-(3-m-tolyl-ureido)-phenyl]-1H-pyrazole-4-carboxylic
acid amide [0114]
5-Amino-3-{4-[3-(2,5-difluoro-phenyl)-ureido]-phenyl}-1-methyl-1H-pyrazol-
e-4-carboxylic acid amide [0115]
5-Amino-3-{4-[3-(2,4-dimethoxy-phenyl)-ureido]-phenyl}-1-methyl-1H-pyrazo-
le-4-carboxylic acid amide [0116]
5-Amino-1-methyl-3-{4-[3-(4-phenoxy-phenyl)-ureido]-phenyl}-1H-pyrazole-4-
-carboxylic acid amide [0117]
5-Amino-1-tert-butyl-3-{4-[3-(2-chloro-5-trifluoromethyl-phenyl)-ureido]--
phenyl}-1H-pyrazole-4-carboxylic acid amide [0118]
5-Amino-3-{4-[3-(bis-3,5-trifluoromethyl-phenyl)-ureido]-phenyl}-1-tert-b-
utyl-1H-pyrazole-4-carboxylic acid amide [0119]
5-Amino-1-tert-butyl-3-[4-(3-naphthalen-1-yl-ureido)-phenyl]-1H-pyrazole--
4-carboxylic acid amide [0120]
5-Amino-1-tert-butyl-3-{4-[3-(3-chloro-phenyl)-ureido]-phenyl}-1H-pyrazol-
e-4-carboxylic acid amide [0121]
5-Amino-1-tert-butyl-3-{4-[3-(2,5-dichloro-phenyl)-ureido]-phenyl}-1H-pyr-
azole-4-carboxylic acid amide [0122]
5-Amino-1-tert-butyl-3-[4-(3-naphthalen-2-yl-ureido)-phenyl]-1H-pyrazole--
4-carboxylic acid amide [0123]
5-Amino-1-tert-butyl-3-{4-[3-(3-trifluoromethyl-phenyl)-ureido]-phenyl}-1-
H-pyrazole-4-carboxylic acid amide [0124]
5-Amino-3-[4-(3-biphenyl-4-yl-ureido)-phenyl]-1-tert-butyl-1H-pyrazole-4--
carboxylic acid amide [0125]
5-Amino-1-tert-butyl-3-{4-[3-(2,6-difluoro-phenyl)-ureido]-phenyl}-1H-pyr-
azole-4-carboxylic acid amide [0126]
5-Amino-3-[4-(3-benzo[1,3]dioxol-5-yl-ureido)-phenyl]-1-tert-butyl-1H-pyr-
azole-4-carboxylic acid amide [0127]
5-Amino-1-tert-butyl-3-[4-(3-o-tolyl-ureido)-phenyl]-1H-pyrazole-4-carbox-
ylic acid amide [0128]
5-Amino-1-tert-butyl-3-[4-(3-m-tolyl-ureido)-phenyl]-1H-pyrazole-4-carbox-
ylic acid amide [0129]
5-Amino-1-tert-butyl-3-{4-[3-(2,5-difluoro-phenyl)-ureido]-phenyl}-1H-pyr-
azole-4-carboxylic acid amide [0130]
5-Amino-1-tert-butyl-3-{4-[3-(2,4-dimethoxyo-phenyl)-ureido]-phenyl}-1H-p-
yrazole-4-carboxylic acid amide [0131]
5-Amino-1-tert-butyl-3-{4-[3-(4-phenoxy-phenyl)-ureido]-phenyl}-1H-pyrazo-
le-4-carboxylic acid amide [0132]
5-Amino-3-{4-[3-2-(fluoro-5-trifluoromethyl-phenyl)-ureido]-phenyl}-1H-py-
razole-4-carboxylic acid amide
N,N-Dimethylamino-{4-[3-(fluoro-trifluoromethyl-phenyl)-ureido]-phenyl}-m-
ethyl-1H-pyrazole-4-carboxylic acid amide
5-Amino-1-tert-butyl-3-{4-[3-(5-tert-butyl-isoxazol-3-yl)-ureido]-phenyl}-
-1H-pyrazole-4-carboxylic acid amide or a salt, solvate, or
physiologically functional derivative thereof.
[0133] Typically, the salts of the present invention are
pharmaceutically acceptable salts. Salts encompassed within the
term "pharmaceutically acceptable salts" refer to non-toxic salts
of the compounds of this invention. Salts of the compounds of the
present invention may comprise acid addition salts derived from a
nitrogen on a substituent in the compound of formula (I).
Representative salts include the following salts: acetate,
benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate,
borate, bromide, calcium edetate, camsylate, carbonate, chloride,
clavulanate, citrate, dihydrochloride, edetate, edisylate,
estolate, esylate, fumarate, gluceptate, gluconate, glutamate,
glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,
hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate,
lactobionate, laurate, malate, maleate, mandelate, mesylate,
methylbromide, methylnitrate, methylsulfate, monopotassium maleate,
mucate, napsylate, nitrate, N-methylglucamine, oxalate, pamoate
(embonate), palmitate, pantothenate, phosphate/diphosphate,
polygalacturonate, potassium, salicylate, sodium, stearate,
subacetate, succinate, tannate, tartrate, teoclate, tosylate,
triethiodide, trimethylammonium and valerate. Other salts, which
are not pharmaceutically acceptable, may be useful in the
preparation of compounds of this invention and these form a further
aspect of the invention.
[0134] While it is possible that, for use in therapy,
therapeutically effective amounts of a compound of formula (I), as
well as salts, solvates and physiological functional derivatives
thereof, may be administered as the raw chemical, it is possible to
present the active ingredient as a pharmaceutical composition.
Accordingly, the invention further provides pharmaceutical
compositions, which include therapeutically effective amounts of
compounds of the formula (I) and/or salts, solvates and/or
physiological functional derivatives thereof, and one or more
pharmaceutically acceptable carriers, diluents, or excipients. The
compounds of the formula (I) and salts, solvates and physiological
functional derivatives thereof, are as described above. The
carrier(s), diluent(s) or excipient(s) must be acceptable in the
sense of being compatible with the other ingredients of the
formulation and not deleterious to the recipient thereof. In
accordance with another aspect of the invention there is also
provided a process for the preparation of a pharmaceutical
formulation including admixing a compound of the formula (I), or
salts, solvates and physiological functional derivatives thereof,
with one or more pharmaceutically acceptable carriers, diluents or
excipients.
[0135] Pharmaceutical formulations may be presented in unit dose
forms containing a predetermined amount of active ingredient per
unit dose. Such a unit may contain, for example, 0.5 mg to 1 g,
preferably 1 mg to 700 mg, more preferably 5 mg to 100 mg of a
compound of the formula (I), depending on the condition being
treated, the route of administration and the age, weight and
condition of the patient, or pharmaceutical formulations may be
presented in unit dose forms containing a predetermined amount of
active ingredient per unit dose. Preferred unit dosage formulations
are those containing a daily dose or sub-dose, as herein above
recited, or an appropriate fraction thereof, of an active
ingredient. Furthermore, such pharmaceutical formulations may be
prepared by any of the methods well known in the pharmacy art.
[0136] Pharmaceutical formulations may be adapted for
administration by any appropriate route, for example by the oral
(including buccal or sublingual), rectal, nasal, topical (including
buccal, sublingual or transdermal), vaginal or parenteral
(including subcutaneous, intramuscular, intravenous or intradermal)
route. Such formulations may be prepared by any method known in the
art of pharmacy, for example by bringing into association the
active ingredient with the carrier(s) or excipient(s).
[0137] For example, pharmaceutical formulations adapted for oral
administration may be presented as discrete units such as capsules
or tablets; powders or granules; solutions or suspensions in
aqueous or non-aqueous liquids; edible foams or whips; or
oil-in-water liquid emulsions or water-in-oil liquid emulsions.
[0138] For instance, for oral administration in the form of a
tablet or capsule, the active drug component can be combined with
an oral, non-toxic pharmaceutically acceptable inert carrier such
as ethanol, glycerol, water and the like. Powders are prepared by
comminuting the compound to a suitable fine size and mixing with a
similarly comminuted pharmaceutical carrier such as an edible
carbohydrate, as, for example, starch or mannitol. Flavoring,
preservative, dispersing and coloring agent can also be
present.
[0139] Capsules are made by preparing a powder mixture, as
described above, and filling formed gelatin sheaths. Glidants and
lubricants such as colloidal silica, talc, magnesium stearate,
calcium stearate or solid polyethylene glycol can be added to the
powder mixture before the filling operation. A disintegrating or
solubilizing agent such as agar-agar, calcium carbonate or sodium
carbonate can also be added to improve the availability of the
medicament when the capsule is ingested.
[0140] Moreover, when desired or necessary, suitable binders,
lubricants, disintegrating agents and coloring agents can also be
incorporated into the mixture. Suitable binders include starch,
gelatin, natural sugars such as glucose or beta-lactose, corn
sweeteners, natural and synthetic gums such as acacia, tragacanth
or sodium alginate, carboxymethylcellulose, polyethylene glycol,
waxes and the like. Lubricants used in these dosage forms include
sodium oleate, sodium stearate, magnesium stearate, sodium
benzoate, sodium acetate, sodium chloride and the like.
Disintegrators include, without limitation, starch, methyl
cellulose, agar, bentonite, xanthan gum and the like. Tablets are
formulated, for example, by preparing a powder mixture, granulating
or slugging, adding a lubricant and disintegrant and pressing into
tablets. A powder mixture is prepared by mixing the compound,
suitably comminuted, with a diluent or base as described above, and
optionally, with a binder such as carboxymethylcellulose, an
aliginate, gelatin, or polyvinyl pyrrolidone, a solution retardant
such as paraffin, a resorption accelerator such as a quaternary
salt and/or an absorption agent such as bentonite, kaolin or
dicalcium phosphate. The powder mixture can be granulated by
wetting with a binder such as syrup, starch paste, acadia mucilage
or solutions of cellulosic or polymeric materials and forcing
through a screen. As an alternative to granulating, the powder
mixture can be run through the tablet machine and the result is
imperfectly formed slugs broken into granules. The granules can be
lubricated to prevent sticking to the tablet forming dies by means
of the addition of stearic acid, a stearate salt, talc or mineral
oil. The lubricated mixture is then compressed into tablets. The
compounds of the present invention can also be combined with a free
flowing inert carrier and compressed into tablets directly without
going through the granulating or slugging steps. A clear or opaque
protective coating comprising a sealing coat of shellac, a coating
of sugar or polymeric material and a polish coating of wax can be
provided. Dyestuffs can be added to these coatings to distinguish
different unit dosages.
[0141] Oral fluids such as solution, syrups and elixirs can be
prepared in dosage unit form so that a given quantity contains a
predetermined amount of the compound. Syrups can be prepared by
dissolving the compound in a suitably flavored aqueous solution,
while elixirs are prepared through the use of a non-toxic alcoholic
vehicle. Suspensions can be formulated by dispersing the compound
in a non-toxic vehicle. Solubilizers and emulsifiers such as
ethoxylated isostearyl alcohols and polyoxy ethylene sorbitol
ethers, preservatives, flavor additive such as peppermint oil or
natural sweeteners or saccharin or other artificial sweeteners, and
the like can also be added.
[0142] Where appropriate, dosage unit formulations for oral
administration can be microencapsulated. The formulation can also
be prepared to prolong or sustain the release as for example by
coating or embedding particulate material in polymers, wax or the
like.
[0143] The compounds of formula (I), and salts, solvates and
physiological functional derivatives thereof, can also be
administered in the form of liposome delivery systems, such as
small unilamellar vesicles, large unilamellar vesicles and
multilamellar vesicles. Liposomes can be formed from a variety of
phospholipids, such as cholesterol, stearylamine or
phosphatidylcholines.
[0144] The compounds of formula (I) and salts, solvates and
physiological functional derivatives thereof may also be delivered
by the use of monoclonal antibodies as individual carriers to which
the compound molecules are coupled. The compounds may also be
coupled with soluble polymers as targetable drug carriers. Such
polymers can include polyvinylpyrrolidone, pyran copolymer,
polyhydroxypropylmethacrylamide-phenol,
polyhydroxyethylaspartamidephenol, or polyethyleneoxidepolylysine
substituted with palmitoyl residues. Furthermore, the compounds may
be coupled to a class of biodegradable polymers useful in achieving
controlled release of a drug, for example, polylactic acid,
polepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters,
polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked
or amphipathic block copolymers of hydrogels.
[0145] Pharmaceutical formulations adapted for transdermal
administration may be presented as discrete patches intended to
remain in intimate contact with the epidermis of the recipient for
a prolonged period of time. For example, the active ingredient may
be delivered from the patch by iontophoresis as generally described
in Pharmaceutical Research, 1986, 3(6):318.
[0146] Pharmaceutical formulations adapted for topical
administration may be formulated as ointments, creams, suspensions,
lotions, powders, solutions, pastes, gels, sprays, aerosols or
oils.
[0147] For treatments of the eye or other external tissues, for
example mouth and skin, the formulations are preferably applied as
a topical ointment or cream. When formulated in an ointment, the
active ingredient may be employed with either a paraffinic or a
water-miscible ointment base. Alternatively, the active ingredient
may be formulated in a cream with an oil-in-water cream base or a
water-in-oil base.
[0148] Pharmaceutical formulations adapted for topical
administrations to the eye include eye drops wherein the active
ingredient is dissolved or suspended in a suitable carrier,
especially an aqueous solvent.
[0149] Pharmaceutical formulations adapted for topical
administration in the mouth include lozenges, pastilles and mouth
washes.
[0150] Pharmaceutical formulations adapted for rectal
administration may be presented as suppositories or as enemas.
[0151] Pharmaceutical formulations adapted for nasal administration
wherein the carrier is a solid include a coarse powder having a
particle size for example in the range 20 to 500 microns which is
administered in the manner in which snuff is taken, i.e. by rapid
inhalation through the nasal passage from a container of the powder
held close up to the nose. Suitable formulations wherein the
carrier is a liquid, for administration as a nasal spray or as
nasal drops, include aqueous or oil solutions of the active
ingredient.
[0152] Pharmaceutical formulations adapted for administration by
inhalation include fine particle dusts or mists, which may be
generated by means of various types of metered, dose pressurised
aerosols, nebulizers or insufflators.
[0153] Pharmaceutical formulations adapted for vaginal
administration may be presented as pessaries, tampons, creams,
gels, pastes, foams or spray formulations.
[0154] Pharmaceutical formulations adapted for parenteral
administration include aqueous and non-aqueous sterile injection
solutions which may contain anti-oxidants, buffers, bacteriostats
and solutes which render the formulation isotonic with the blood of
the intended recipient; and aqueous and non-aqueous sterile
suspensions which may include suspending agents and thickening
agents. The formulations may be presented in unit-dose or
multi-dose containers, for example sealed ampoules and vials, and
may be stored in a freeze-dried (lyophilized) condition requiring
only the addition of the sterile liquid carrier, for example water
for injections, immediately prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets.
[0155] It should be understood that in addition to the ingredients
particularly mentioned above, the formulations may include other
agents conventional in the art having regard to the type of
formulation in question, for example those suitable for oral
administration may include flavouring agents.
[0156] A therapeutically effective amount of a compound of the
present invention will depend upon a number of factors including,
for example, the age and weight of the animal, the precise
condition requiring treatment and its severity, the nature of the
formulation, and the route of administration, and will ultimately
be at the discretion of the attendant physician or veterinarian.
However, an effective amount of a compound of formula (I) for the
treatment of neoplastic growth, for example colon or breast
carcinoma, will generally be in the range of 0.1 to 100 mg/kg body
weight of recipient (mammal) per day and more usually in the range
of 1 to 10 mg/kg body weight per day. Thus, for a 70 kg adult
mammal, the actual amount per day would usually be from 70 to 700
mg and this amount may be given in a single dose per day or more
usually in a number (such as two, three, four, five or six) of
sub-doses per day such that the total daily dose is the same. An
effective amount of a salt or solvate, or physiologically
functional derivative thereof, may be determined as a proportion of
the effective amount of the compound of formula (I) per se. It is
envisaged that similar dosages would be appropriate for treatment
of the other conditions referred to herein.
[0157] The compounds of the present invention and their salts and
solvates, and physiologically functional derivatives thereof, may
be employed alone or in combination with other therapeutic agents
for the treatment of the conditions mentioned herein. In
particular, in anti-cancer therapy, combination with other
chemotherapeutic, hormonal or antibody agents is envisaged as well
as combination with surgical therapy and/or radiotherapy.
Combination therapies according to the present invention thus
comprise the administration of at least one compound of formula (I)
or a pharmaceutically acceptable salt or solvate thereof, or a
physiologically functional derivative thereof, and the use of at
least one other cancer treatment method. Preferably, combination
therapies according to the present invention comprise the
administration of at least one compound of formula (I) or a
pharmaceutically acceptable salt or solvate thereof, or a
physiologically functional derivative thereof, and at least one
other pharmaceutically active agent, preferably an anti-neoplastic
agent. The compound(s) of formula (I) and the other
pharmaceutically active agent(s) may be administered together or
separately and, when administered separately this may occur
simultaneously or sequentially in any order. The amounts of the
compound(s) of formula (I) and the other pharmaceutically active
agent(s) and the relative timings of administration will be
selected in order to achieve the desired combined therapeutic
effect.
[0158] The compounds of the Formula (I) or salts, solvates, or
physiologically functional derivatives thereof and at least one
additional cancer treatment therapy may be employed in combination
concomitantly or sequentially in any therapeutically appropriate
combination with such other anti-cancer therapies. In one
embodiment, the other anti-cancer therapy is at least one
additional chemotherapeutic therapy including administration of at
least one anti-neoplastic agent. The administration in combination
of a compound of formula (I) or salts, solvates, or physiologically
functional derivatives thereof with other anti-neoplastic agents
may be in combination in accordance with the invention by
administration concomitantly in (1) a unitary pharmaceutical
composition including both compounds or (2) separate pharmaceutical
compositions each including one of the compounds. Alternatively,
the combination may be administered separately in a sequential
manner wherein one anti-neoplastic agent is administered first and
the other second or vice versa. Such sequential administration may
be close in time or remote in time.
[0159] Anti-neoplastic agents may induce anti-neoplastic effects in
a cell-cycle specific manner, i.e., are phase specific and act at a
specific phase of the cell cycle, or bind DNA and act in a non
cell-cycle specific manner, i.e., are non-cell cycle specific and
operate by other mechanisms.
[0160] Anti-neoplastic agents useful in combination with the
compounds and salts, solvates or physiologically functional
derivatives thereof of formula (I) include the following:
[0161] (1) cell cycle specific anti-neoplastic agents include, but
are not limited to, diterpenoids such as paclitaxel and its analog
docetaxel; vinca alkaloids such as vinblastine, vincristine,
vindesine, and vinorelbine; epipodophyllotoxins such as etoposide
and teniposide; fluoropyrimidines such as 5-fluorouracil and
fluorodeoxyuridine; antimetabolites such as allopurinol,
fludurabine, methotrexate, cladrabine, cytarabine, mercaptopurine
and thioguanine; and camptothecins such as 9-amino camptothecin,
irinotecan, topotecan, CPT-11 and the various optical forms of
7-(4-methylpiperazino-methylene)-10,11-ethylenedioxy-20-camptothecin;
[0162] (2) cytotoxic chemotherapeutic agents including, but not
limited to, alkylating agents such as melphalan, chlorambucil,
cyclophosphamide, mechlorethamine, hexamethylmelamine, busulfan,
carmustine, lomustine, and dacarbazine; anti-tumour antibiotics
such as doxorubicin, daunomycin, epirubicin, idarubicin,
mitomycin-C, dacttinomycin and mithramycin; and platinum
coordination complexes such as cisplatin, carboplatin, and
oxaliplatin; and
[0163] (3) other chemotherapeutic agents including, but not limited
to, anti-estrogens such as tamoxifen, toremifene, raloxifene,
droloxifene and iodoxyfene; progestrogens such as megestrol
acetate; aromatase inhibitors such as anastrozole, letrazole,
vorazole, and exemestane; antiandrogens such as flutamide,
nilutamide, bicalutamide, and cyproterone acetate; LHRH agonists
and antagagonists such as goserelin acetate and luprolide,
testosterone 5.alpha.-dihydroreductase inhibitors such as
finasteride; metalloproteinase inhibitors such as marimastat;
antiprogestogens; urokinase plasminogen activator receptor function
inhibitors; growth factor function inhibitors such as inhibitors of
the functions of hepatocyte growth factor; erb-B2, erb-B4,
epidermal growth factor receptor (EGFR), platelet derived growth
factor receptor (PDGFR), vascular endothelial growth factor
receptor (VEGFR, and TIE-2 (other than those VEGFR and TIE-2
inhibitors described in the present invention); and other tyrosine
kinase inhibitors such as inhibitors of CDK2 and CDK4
inhibitors.
[0164] The compounds of formula (I) and salts, solvates and
physiological functional derivatives thereof, are active as
inhibitors of at least one of the protein kinases TIE-2, VEGFR-2
and VEGFR-3.
[0165] The present invention thus also provides compounds of
formula (I) and pharmaceutically acceptable salts or solvates
thereof, or physiologically functional derivatives thereof, for use
in medical therapy, and particularly in the treatment of disorders
mediated by at least one of inappropriate TIE-2, VEGFR-2, and
VEGFR-3 kinase activity.
[0166] The inappropriate TIE-2, VEGFR-2, and/or VEGFR-3 activity
referred to herein is any TIE-2. VEGFR-2, and/or VEGFR-3 activity
that deviates from the normal TIE-2, VEGFR-2, and/or VEGFR-3
activity expected in a particular mammalian subject. Inappropriate
TIE-2. VEGFR-2 and/or VEGFR-3 activity may take the form of, for
instance, an abnormal increase in activity, or an aberration in the
timing and or control of TIE-2, VEGFR-2, and/or VEGFR-3 activity.
Such inappropriate activity may result then, for example, from
overexpression or mutation of the protein kinase leading to
inappropriate or uncontrolled activation. Furthermore, it is also
understood that unwanted TIE-2, VEGFR-2, and/or VEGFR-3 activity
may reside in an abnormal source, such as a malignancy. That is,
the level of TIE-2, VEGFR-2 and/or VEGFR-3 activity does not have
to be abnormal to be considered inappropriate, rather the activity
derives from an abnormal source.
[0167] In a like manner, the inappropriate angiogenesis referred to
herein is any angiogenic activity that deviates from the normal
angiogenic activity expected in a particular mammalian subject.
Inappropriate angiogenesis may take the form of, for instance, an
abnormal increase in activity, or an aberration in the timing and
or control of angiogenic activity. Such inappropriate activity may
result then, for example, from overexpression or mutation of a
protein kinase leading to inappropriate or uncontrolled activation.
Furthermore, it is also understood that unwanted angiogenic
activity may reside in an abnormal source, such as a malignancy.
That is, the level of angiogenic activity does not have to be
abnormal to be considered inappropriate, rather the activity
derives from an abnormal source.
[0168] The present invention is directed to methods of regulating,
modulating, or inhibiting TIE-2, VEGFR-2 and/or VEGFR-3, especially
TIE-2 and/or VEGFR-2, for the prevention and/or treatment of
disorders related to inappropriate TIE-2, VEGFR-2 and/or VEGFR-3
activity, respectively.
[0169] In particular, the compounds of the present invention are
useful in the treatment of susceptible forms of cancer, including
tumor growth and metastatis. Furthermore, the compounds of the
present invention can be used to provide additive or synergistic
effects with certain existing cancer chemotherapies, and/or be used
to restore effectiveness of certain existing cancer chemotherapies
and radiation.
[0170] The compounds of the present invention are also useful in
the treatment of one or more diseases afflicting mammals which are
characterized by cellular proliferation in the area of disorders
associated with neo-vascularization and/or vascular permeability
including blood vessel proliferative disorders including arthritis
and restenosis; fibrotic disorders including hepatic cirrhosis and
atherosclerosis; mesangial cell proliferative disorders including
glomerulonephritis, diabetic nephropathy, malignant
nephrosclerosis, thrombotic microangiopathy syndromes, organ
transplant rejection and glomerulopathies; metabolic disorders
including psoriasis, diabetes mellitus, chronic wound healing,
inflammation and neurodegenerative diseases; also diabetic
retinopathy; macular degeneration; other diseases characterized by
ocular neovascularization; and diseases characterized by
hemangiomas.
[0171] A further aspect of the invention provides a method of
treatment of a mammal suffering from a disorder mediated by at
least one of inappropriate TIE-2, VEGFR-2, and VEGFR-3 activity,
which includes administering to said subject an effective amount of
a compound of formula (I) or a pharmaceutically acceptable salt,
solvate, or a physiologically functional derivative thereof. In a
preferred embodiment, the disorder is cancer, e.g., malignant
tumors. This aspect of the invention also provides such a method
wherein the disorder is a disease afflicting mammals which are
characterized by cellular proliferation in the area of disorders
associated with neovascularization and/or vascular permeability,
including those disclosed herein.
[0172] A further aspect of the invention provides a method of
treatment of a mammal suffering from cancer which includes
administering to said subject an effective amount of a compound of
formula (I) or a pharmaceutically acceptable salt or solvate
thereof, or a physiologically functional derivative thereof.
[0173] A further aspect of the present invention provides the use
of a compound of formula (I), or a pharmaceutically acceptable salt
or solvate thereof, or a physiologically functional derivative
thereof, in the preparation of a medicament for the treatment of a
disorder characterized by at least one of inappropriate TIE-2,
VEGFR-2, and VEGFR-3 activity. In a preferred embodiment, the
disorder is cancer, malignant tumors. This aspect of the invention
also provides such a use wherein the disorder is a disease
afflicting mammals which are characterized by cellular
proliferation in the area of disorders associated with
neovascularization and/or vascular permeability, including those
disclosed herein.
[0174] A further aspect of the present invention provides the use
of a compound of formula (I), or a pharmaceutically acceptable salt
or solvate thereof, or a physiologically functional derivative
thereof, in the preparation of a medicament for the treatment of
cancer, e.g., malignant tumors.
[0175] The mammal requiring treatment with a compound of the
present invention is typically a human being.
[0176] In another embodiment, therapeutically effective amounts of
(a) the compounds of formula (I) or salts, solvates or
physiologically derived derivatives thereof, and (b) agents which
inhibit growth factor receptor function may be administered in
combination to a mammal for treatment of a disorder mediated by at
least one of inappropriate TIE-2, VEGFR-2 and VEGFR-3 activity, for
instance in the treatment of cancer, e.g., malignant tumors. Such
growth factor receptors include, for example, EGFR, PDGFR, erbB2,
erbB4, VEGFR, and/or TIE-2. Growth factor receptors and agents that
inhibit growth factor receptor function are described, for
instance, in Kath, John C., Exp. Opin. Ther. Patents (2000) 10(6):
803-818 and in Shawver et al. DDT Vol 2, No. 2 Feb. 1997.
[0177] The compounds of the formula (I) or salts, solvates, or
physiologically functional derivatives thereof and the agent for
inhibiting growth factor receptor function may be employed in
combination concomitantly or sequentially in any therapeutically
appropriate combination. The combination may be employed in
combination in accordance with the invention by administration
concomitantly in (1) a unitary pharmaceutical composition including
both compounds, or (2) separate pharmaceutical compositions each
including one of the compounds. Alternatively, the combination may
be administered separately in a sequential manner wherein one is
administered first and the other second or vice versa. Such
sequential administration may be close in time or remote in
time.
[0178] In another aspect of the present invention, there is
provided a method of treating a disorder in a mammal, said disorder
being mediated by inappropriate angiogenesis, including:
administering to said mammal a therapeutically effective amount of
a compound of formula (I), or a salt, solvate or physiologically
functional derivative thereof. In one embodiment, the inappropriate
angiogenic activity is due to at least one of inappropriate
VEGFR-1, VEGFR-2, VEGFR-3, and TIE-2 activity. In another
embodiment, the inappropriate angiogenesis is due to at least one
of inappropriate VEGFR-2, VEGFR-3, and TIE-2 activity. In another
embodiment, the inappropriate angiogenesis is due to at least one
of inappropriate VEGFR-2 and TIE-2 activity. In a further
embodiment, the method further includes administering a
therapeutically effective amount of a VEGFR-2 inhibitor along with
the compounds of formula (I) or salts, solvates or physiologically
functional derivatives thereof. Preferably the disorder is cancer,
e.g., malignant tumors. This aspect of the invention also provides
such methods wherein the disorder is a disease afflicting mammals
which are characterized by cellular proliferation in the area of
disorders associated with neovascularization and/or vascular
permeability, including those disclosed herein.
[0179] In another aspect of the present invention, there is
provided the use of a compound of formula (I), or a salt, solvate
or physiologically functional derivative thereof in the preparation
of a medicament for use in treating a disorder in a mammal, said
disorder being characterized by inappropriate angiogenesis. In one
embodiment, the inappropriate angiogenic activity is due to at
least one of inappropriate VEGFR-1, VEGFR-2, VEGFR-3 and TIE-2
activity. In another embodiment, the inappropriate angiogenesis is
due to at least one of inappropriate VEGFR-2, VEGFR-3 and TIE-2
activity. In another embodiment, the inappropriate angiogenesis is
due to at least one of inappropriate VEGFR-2 and TIE-2 activity. In
a further embodiment, the use further includes use of a VEGFR-2
inhibitor to prepare said medicament. Preferably the disorder is
cancer, e.g., malignant tumors. This aspect of the invention also
provides such uses wherein the disorder is a disease afflicting
mammals which are characterized by cellular proliferation in the
area of disorders associated with neovascularization and/or
vascular permeability, including those disclosed herein.
[0180] The combination of a compound of formula (I) or salts,
solvates, or physiologically functional derivatives thereof with a
VEGFR-2 inhibitor may be employed in combination in accordance with
the invention by administration concomitantly in (1) a unitary
pharmaceutical composition including both compounds, or
[0181] (2) separate pharmaceutical compositions each including one
of the compounds. Alternatively, the combination may be
administered separately in a sequential manner wherein one is
administered first and the other second or vice versa. Such
sequential administration may be close in time or remote in
time.
[0182] The compounds of this invention may be made by a variety of
methods, including standard chemistry. Any previously defined
variable will continue to have the previously defined meaning
unless otherwise indicated. Illustrative general synthetic methods
are set out below and the preparation of specific compounds of the
invention is described in the Examples.
[0183] Compounds of general formula (I) may be prepared by methods
known in the art of organic synthesis as set forth in part by the
following synthesis schemes. In all of the schemes described below,
it is well understood that protecting groups for sensitive or
reactive groups are employed where necessary in accordance with
general principles of chemistry. Protecting groups are manipulated
according to standard methods of organic synthesis (T. W. Green and
P. G. M. Wuts (1991) Protecting Groups in Organic Synthesis, John
Wiley & Sons). These groups are removed at a convenient stage
of the compound synthesis using methods that are readily apparent
to those skilled in the art. The selection of processes as well as
the reaction conditions and order of their execution shall be
consistent with the preparation of compounds of Formula (I). Those
skilled in the art will recognize if a stereocenter exists in
compounds of Formula (I). Accordingly, the present invention
includes both possible stereoisomers and includes not only racemic
compounds but the individual enantiomers as well. When a compound
is desired as a single enantiomer, it may be obtained by
stereospecific synthesis or by resolution of the final product or
any convenient intermediate. Resolution of the final product, an
intermediate, or a starting material may be effected by any
suitable method known in the art. See, for example, Stereochemistry
of Organic Compounds by E. L. Eliel, S. H. Wilen, and L. N. Mander
(Wiley-Interscience, 1994).
[0184] Compounds of Formula (I) can be prepared according to the
synthetic sequences illustrated in Scheme 1, which shows general
routes for the synthesis of the targeted
5-amino-4-carboxamido-pyrazoles. Specific details of synthetic
routes according to Scheme 1 are shown in the Examples following
which include preparation of 5-amino-4-carboxamido-pyrazole
compound examples having Tie-2, VEGFR-2 and/or VEGFR-3 inhibitory
activity.
[0185] The effective synthesis of 5-amino-4-carboxamido pyrazoles
is achieved by the route shown in Scheme 1, using the method
described by Hanefeld et al. (J. Chem. Soc., Perkin Trans. 1
(1996), 1545). This synthesis requires an appropriately substituted
acid chloride (i), which is here exemplified with, but should not
be seen as limited to, a phenyl-based acid chloride. This acid
chloride contains a protected or disguised amino group "N" such as,
but not limited to, the following functionalities: nitro,
N-carbamoyl or a N-acyl amino. The acyl chloride (i) is reacted
with malononitrile in the presence of sodium hydride to generate
the bis-nitrile (ii), which upon treatment with dimethyl sulfate
yields the corresponding vinyl ether (iii). Treatment of (iii) with
a monosubstituted hydrazine affords the pyrazole (iv), which is
reacted with an alkylating or acylating agent to yield the desired
N-alkyl or N-acyl analogue (v). N-alkyl derivatives could also be
obtained by treating the 2-amino pyrazole with the appropriate
aldehyde or ketone in the presence of a reducing agent. Hydrolysis
of nitrile (v) under conditions described by Cacchiet et al.
(Synthesis (1980), 243-4) yields the amide (vi). Unmasking of the
amino functionality produces the intermediate (vii) and appropriate
derivatization affords the desired final product (viii).
##STR00004##
EXAMPLES
[0186] Certain embodiments of the present invention will now be
illustrated by way of example only. The physical data given for the
compounds exemplified is consistent with the assigned structure of
those compounds.
[0187] As used herein the symbols and conventions used in these
processes, schemes and examples are consistent with those used in
the contemporary scientific literature, for example, the Journal of
the American Chemical Society or the Journal of Biological
Chemistry. Standard single-letter or three-letter abbreviations are
generally used to designate amino acid residues, which are assumed
to be in the L-configuration unless otherwise noted. Unless
otherwise noted, all starting materials were obtained from
commercial suppliers and used without further purification.
Specifically, the following abbreviations may be used in the
examples and throughout the specification: [0188] g (grams); mg
(milligrams); [0189] L (liters); mL (milliliters); [0190] .mu.L
(microliters); psi (pounds per square inch); [0191] M (molar); mM
(millimolar); [0192] i. v. (intravenous); Hz (Hertz); [0193] MHz
(megahertz); mol (moles); [0194] mmol (millimoles); rt (room
temperature); [0195] min (minutes); h (hours); [0196] mp (melting
point); TLC (thin layer chromatography); [0197] T.sub.r (retention
time); RP (reverse phase); [0198] MeOH (methanol); i-PrOH
(isopropanol); [0199] TEA (triethylamine); TFA (trifluoroacetic
acid); [0200] TFAA (trifluoroacetic anhydride); THF
(tetrahydrofuran); [0201] DMSO (dimethylsulfoxide); AcOEt (ethyl
acetate); [0202] DME (1,2-dimethoxyethane); DCM (dichloromethane);
[0203] DCE (dichloroethane); DMF (N,N-dimethylformamide); [0204]
DMPU (N,N'-dimethylpropyleneurea); atm (atmosphere); [0205] CDI
(1,1-carbonyldiimidazole); TMS (trimethylsilyl); [0206] IBCF
(isobutyl chloroformate); TBS (t-butyldimethylsilyl); [0207] HOAc
(acetic acid); BSA (bovine serum albumin) [0208] HOSu
(N-hydroxysuccinimide); HRP (horseradish peroxidase); [0209] HOBT
(1-hydroxybenzotriazole); [0210] mCPBA (meta-chloroperbenzoic acid;
[0211] EDC (ethylcarbodiimide hydrochloride); [0212] BOC
(tert-butyloxycarbonyl); [0213] FMOC (9-fluorenylmethoxycarbonyl);
[0214] DCC (dicyclohexylcarbodiimide); [0215] CBZ
(benzyloxycarbonyl); [0216] Ac (acetyl); [0217] TMSE
(2-(trimethylsilyl)ethyl); [0218] TIPS (triisopropylsilyl); [0219]
DMAP (4-dimethylaminopyridine); [0220] ATP (adenosine
triphosphate); [0221] DMEM (Dulbecco's modified Eagle medium);
[0222] HPLC (high pressure liquid chromatography); [0223] BOP
(bis(2-oxo-3-oxazolidinyl)phosphinic chloride); [0224] TBAF
(tetra-n-butylammonium fluoride); [0225] HBTU
(O-Benzotriazole-1-yl-N,N,N',N'-tetramethyluronium
hexafluorophosphate); [0226] HEPES (4-(2-hydroxyethyl)-1-piperazine
ethane sulfonic acid); [0227] DPPA (diphenylphosphoryl azide);
[0228] fHN03 (fumed HNO.sub.3); and [0229] EDTA
(ethylenediaminetetraacetic acid).
[0230] All references to ether are to diethyl ether; brine refers
to a saturated aqueous solution of NaCl. Unless otherwise
indicated, all temperatures are expressed in .degree. C. (degrees
Centigrade). All reactions are conducted under an inert atmosphere
at room temperature unless otherwise noted.
[0231] .sup.1H NMR (hereinafter alternatively "NMR") spectra were
recorded on a Varian VXR-300, a Varian Unity-300, a Varian
Unity-400 instrument, a Brucker AVANCE-400, a Bruker AM 400
spectrometer or a General Electric QE-300. Chemical shifts are
expressed in parts per million (ppm, 6 units). Coupling constants
are in units of hertz (Hz). Splitting patterns describe apparent
multiplicities and are designated as s (singlet), d (doublet), t
(triplet), q (quartet), quint (quintet), m (multiplet), br
(broad).
[0232] Mass spectra were run on an open access LC-MS system using
electrospray ionization. LC conditions: 4.5% to 90% CH.sub.3CN
(0.02% TFA) in 3.2 min with a 0.4 min hold and 1.4 min
re-equilibration; detection by MS, UV at 214 nm, and a light
scattering detector (ELS). Column: 1.times.40 mm Aquasil (C18).
[0233] For preparative (prep) hplc; ca 50 mg of the final products
were injected in 500 uL of DMSO onto a 50.times.20 mm I. D. YMC
CombiPrep ODS-A column at 20 mL/min with a 10 min gradient from 10%
CH.sub.3CN (0.1% TFA) to 90% CH.sub.3CN (0.1% TFA) in H.sub.2O
(0.1% TFA) and a 2 min hold. Flash chromatography was run over
Merck Silica gel 60 (230-400 mesh).
[0234] Infrared (1R) spectra were obtained on a Nicolet 510 FT-IR
spectrometer using a 1-mm NaCl cell. Most of the reactions were
monitored by thin-layer chromatography on 0.25 mm E. Merck silica
gel plates (60F-254), visualized with UV light, 5% ethanolic
phosphomolybdic acid or p-anisaldehyde solution.
Preparation 1
[0235]
5-Amino-1-tert-butyl-3-(4-nitro-phenyl)-1H-pyrazole-4-carbonitrile
To a suspension of sodium hydride (4.80 g, 0.2 mol, 2 eq, 60%
dispersion in mineral oil) in tetrahydrofuran (100 mL) was slowly
added malonodinitrile (6.61 g, 0.1 mol). 4-Nitrobenzoyl chloride
(18.8 g, 0.1 mol, 1 eq) was then added in portions while keeping
the reaction temperature below 10.degree. C. The reaction mixture
was stirred for 1 hour at room temperature. Dimethyl sulfate (15.1
g, 0.12 mol, 1.2 eq) was then added and the reaction mixture was
refluxed for 2 hours. Triethylamine (25.3 g, 0.25 mol, 2.5 eq),
followed by tert-butyl hydrazine hydrochloride (12.4 g, 10 mmol),
was added and the reaction mixture was heated under reflux for an
additional hour. The solvent was then evaporated and 50 mL of
ethanol were added. The reaction mixture was heated under reflux,
and water (250 mL) was added while keeping the temperature above
50.degree. C. The reaction mixture was then cooled to 10.degree. C.
and
5-amino-1-tert-butyl-3-(4-nitro-phenyl)-1H-pyrazole-4-carbonitrile
was isolated by filtration. .sup.1H-NMR (DMSO-d.sub.6): ppm 1.59
(s, 9H), 6.55 (br s, 2H), 8.05 (d, 2H, J=7.2 Hz), 8.33 (d, 2H,
J=7.2 Hz). LC MS (m/e)=285.8 (MH+).
Preparation 2
[0236]
5-Amino-1-methyl-3-(4-nitro-phenyl)-1H-pyrazole-4-carbonitrile
Prepared as described above in Preparation 1 starting from
4-nitrobenzoyl chloride and methylhydrazine to give the title
compound
5-amino-1-methyl-3-(4-nitro-phenyl)-1H-pyrazole-4-carbonitrile.
.sup.1H-NMR (DMSO-d.sub.6): ppm 3.64 (s, 3H), 6.86 (br s, 2H), 8.03
(d, 2H, J=8.8 Hz), 8.33 (d, 2H, J=8.8 Hz). LC MS (m/e)=244.2
(MH+).
Preparation 3
[0237]
5-Amino-1-tert-butyl-3-(4-nitro-phenyl)-1H-pyrazole-4-carboxylic
acid amide To a solution of
5-amino-1-tert-butyl-3-(4-nitro-phenyl)-1H-pyrazole-4-carbonitrile
(2.85 g, 10 mmol) in 40 mL of tetrahydrofuran were added a 30%
aqueous solution of hydrogen peroxide (80 mL), a 1 M solution of
sodium hydroxide (20 mL) and 3 g of tetrabutylammonium
hydrogensulfate. The reaction mixture was stirred for 48 hours at
room temperature and extracted with ethyl acetate (2.times.200 mL).
The organic layers were combined, washed with brine, dried over
solid MgSO.sub.4, filtered and evaporated. The yellow residue was
purified by normal phase silica column chromatography (100% ethyl
acetate) to give 750 mg of pure
5-amino-1-tert-butyl-3-(4-nitro-phenyl)-1H-pyrazole-4-carboxylic
acid amide. .sup.1H-NMR (DMSO-d.sub.6): ppm 1.54 (s, 9H), 6.11 (br
s, 2H), 7.81 (d, 2H, J=7.6 Hz), 8.22 (d, 2H, J=7.6 Hz). LC MS
(m/e)=304.2 (MH+).
Preparation 4
[0238] 5-Amino-1-methyl-3-(4-nitro-phenyl)-1H-pyrazole-4-carboxylic
acid amide Prepared as described above in Preparation 3 starting
from 5-amino-1-methyl-3-(4-nitro-phenyl)-1H-pyrazole-4-carbonitrile
to give the title compound
5-amino-1-methyl-3-(4-nitro-phenyl)-1H-pyrazole-4-carboxylic acid
amide. .sup.1H-NMR (DMSO-d.sub.6): ppm 3.61 (s, 3H), 6.19 (br s,
2H), 7.78 (d, 2H, J=8.4 Hz), 8.25 (d, 2H, J=8.4 Hz). LC MS
(m/e)=262.0 (MH+).
Preparation 5
5-Amino-3-(4-amino-phenyl)-1-tert-butyl-1H-pyrazole-4-carboxylic
acid amide
[0239] To a solution of
5-amino-1-tert-butyl-3-(4-nitro-phenyl)-1H-pyrazole-4-carboxylic
acid amide (304 mg, 1 mmol) in 6 mL of 6 N HCl was added tin powder
(304 mg), and the reaction mixture was stirred for 1 hour at room
temperature. Upon completion of reaction (as judged by TLC
analysis), the reaction mixture was filtered and basified with 1 M
sodium hydroxide (to pH.about.10-12). Pure
5-amino-3-(4-amino-phenyl)-1-tert-butyl-3-1H-pyrazole-4-carboxylic
acid amide was isolated by filtration. .sup.1H-NMR (DMSO-d.sub.6):
ppm 1.53 (s, 9H), 5.22 (br s, 3H), 6.32 (br s, 2H), 6.51 (d, 2H,
J=7.9 Hz), 6.88 (br s, 1H), 7.01 (d, 2H, J=7.9 Hz). LC MS
(m/e)=274.2 (MH+).
Preparation 6
5-Amino-3-(4-amino-phenyl)-1-methyl-1H-pyrazole-4-carboxylic acid
amide
[0240] Prepared as described above in Preparation 5 starting from
5-amino-1-methyl-3-(4-nitro-phenyl)-1H-pyrazole-4-carboxylic acid
amide to give the title compound
5-amino-3-(4-aminophenyl)-1-methyl-1H-pyrazole-4-carboxylic acid
amide. .sup.1H-NMR (DMSO-d.sub.6): ppm 3.61 (s, 3H), 5.31 (br s,
3H), 6.27 (br s, 2H), 6.61 (d, 2H, J=8.4 Hz), 7.09 (d, 2H, J=8.4
Hz). LC MS (m/e)=231.2 (MH+).
Preparation 7
N,N-Dimethylamino-methyl-(4-nitro-phenyl)-1H-pyrazole-4-carbonitrile
[0241] To a solution of
5-amino-1-methyl-3-(4-nitro-phenyl)-1H-pyrazole-4-carbonitrile (2
g, 8.23 mmol) in anhydrous THF (30 mL) was added sodium hydride
(360 mg, 60% suspension in mineral oil, 9 mmol, 1.1 eq) and the
reaction mixture was allowed to stir for 20 minutes at room
temperature. Iodomethane (0.56 mL, 9 mmol, 1.1 eq) was added and
the reaction mixture was monitored by TLC (ethyl acetate as mobile
phase). After 1 hour, a ratio of 1:1:1 mixture (starting material
R.sub.f=0.51, monomethylated product R.sub.f=0.64 and
bis-methylated product R.sub.f=0.88) was observed. Saturated
solution of ammonium chloride (10 mL) was added followed by 50 mL
of ethyl acetate. Layers were separated, organic washed with brine,
dried over MgSO.sub.4, filtered and evaporated to give a yellow
oil, which was purified by column chromatography (10%
dichloromethane: ethyl acetate) to give 100 mg (8% yield) of pure
N,N-dimethylamino-methyl-(4-nitro-phenyl)-1H-pyrazole-4-carbonitrile.
.sup.1H-NMR (CDCl.sub.3): ppm 3.02 (s, 6H), 3.88 (s, 3H), 8.11 (d,
2H, J=9.0 Hz), 8.31 (d, 2H, J=9.0 Hz).
Preparation 8
N,N-dimethylamino-methyl-(4-nitro-phenyl)-1H-pyrazole-4-carboxylic
acid amide
[0242] Prepared as described above in Preparation 3 starting from
N,N-dimethylamino-methyl-(4-nitro-phenyl)-1H-pyrazole-4-carbonitrile
to give the title compound
N,N-dimethylamino-methyl-(4-nitro-phenyl)-1H-pyrazole-4-carboxylic
acid amide. LC MS (m/e)=289.8 (MH+). Rt=1.38 min.
Example 1
5-Amino-1-tert-butyl-3-[4-[3-(2-fluoro-5-trifluoromethyl-phenyl)-ureido]-p-
henyl]-1H-pyrazole-4-carboxylic acid amide
[0243] To a solution of
5-amino-3-(4-aminophenyl)-1-tert-butyl-3-1H-pyrazole-4-carboxylic
acid amide (27.1 mg, 0.1 mmol) in chloroform (5 mL) was added
2-fluoro-5-trifluoromethyl-phenyl isocyanate (22 mg, 0.11 mmol, 1.1
eq). The reaction mixture was stirred for 1 hour at room
temperature while monitored by TLC. The mixture was then evaporated
and the crude product was purified by either normal phase silica
column chromatography (100% ethyl acetate) or preparative HPLC to
give pure
5-amino-1-tert-butyl-3-{4-[3-(2-fluoro-5-trifluoromethyl-phenyl)-ureido]--
phenyl}-1H-pyrazole-4-carboxylic acid amide. .sup.1H-NMR
(DMSO-d.sub.6): ppm 1.66 (s, 9H), 5.30 (br s, 2H), 5.72 (br s, 2H),
7.11 (m, 2H), 7.44 (d, 2H, J=6.2 Hz), 7.51 (d, 2H, J=6.2 Hz), 8.52
(br s, 1H), 8.82 (m, 1H), 9.21 (br s, 1H). LC MS (m/e)=478.8
(MH+).
Example 2
5-Amino-3-{4-[3-(2-fluoro-5-trifluoromethyl-phenyl)-ureido]-phenyl}-1-meth-
yl-1H-pyrazole-4-carboxylic acid amide
[0244] Prepared as described above in Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-methyl-1H-pyrazole-4-carboxylic acid
amide to give the title compound
5-amino-3-{4-[3-(2-fluoro-5-trifluoromethyl-phenyl)-ureido]-phenyl}-1-met-
hyl-1H-pyrazole-4-carboxylic acid amide. .sup.1H-NMR
(MeOH-d.sub.4): ppm 3.63 (s, 3H), 7.34 (d, 2H, J=8.6 Hz), 7.49 (d,
2H, J=8.6 Hz), 7.62 (m, 2H), 8.64 (m, 1H). LC-MS (m/e)=437.4
(MH+).
Example 3
5-Amino-3-{4-[3-(2-chloro-5-trifluoromethyl-phenyl)-ureido]-phenyl}-1-meth-
yl-1H-pyrazole-4-carboxylic acid amide
[0245] Prepared as described above in Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-methyl-1H-pyrazole-4-carboxylic acid
amide to give the title compound
5-amino-3-{4-[3-(2-chloro-5-trifluoromethyl-phenyl)-ureido]-phenyl}-1-met-
hyl-1H-pyrazole-4-carboxylic acid amide. .sup.1H-NMR
(MeOH-d.sub.4): ppm 3.63 (s, 3H), 7.31 (d, 1H, J=8.3 Hz), 7.49 (d,
2H, J=8.6 Hz), 7.63 (m, 3H), 8.66 (s, 1H). LC-MS (m/e)=453.2
(MH+).
Example 4
5-Amino-3-{4-[3-(bis-3,5-trifluoromethyl-phenyl)-ureido]-phenyl}-1-methyl--
1H-pyrazole-4-carboxylic acid amide
[0246] Prepared as described above in Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-methyl-1H-pyrazole-4-carboxylic acid
amide to give the title compound
5-amino-3-{4-[3-(bis-3,5-trifluoromethyl-phenyl)-ureido]-phenyl}-1-methyl-
-1H-pyrazole-4-carboxylic acid amide. .sup.1H-NMR (MeOH-d.sub.4):
ppm 3.63 (s, 3H), 7.48 (m, 2H), 7.57 (s, 1H), 7.63 (m, 2H), 7.62
(m, 2H), 8.11 (s, 2H). LC-MS (m/e)=487.6 (MH+).
Example 5
5-Amino-1-methyl-3-[4-(3-naphthalen-2-yl-ureido)-phenyl]-1H-pyrazole-4-car-
boxylic acid amide
[0247] Prepared as described above in Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-methyl-1H-pyrazole-4-carboxylic acid
amide to give the title compound
5-amino-1-methyl-3-[4-(3-naphthalen-2-yl-ureido)-phenyl]-1H-pyrazole-4-ca-
rboxylic acid amide. .sup.1H-NMR (MeOH-d.sub.4): ppm 3.63 (s, 3H),
6.80 (d, 1H, J=8.4 Hz), 7.47 (d, 1H, J=8.4 Hz), 7.50 (m, 4H), 7.64
(d, 2H, J=8.6 Hz), 7.81 (m, 3H). LC-MS (m/e)=401.2 (MH+).
Example 6
5-Amino-3-{4-[3-(3-chloro-phenyl)-ureido]-phenyl}-1-methyl-1H-pyrazole-4-c-
arboxylic acid amide
[0248] Prepared as described above in Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-methyl-1H-pyrazole-4-carboxylic acid
amide to give the title compound
5-amino-3-{4-[3-(3-chloro-phenyl)-ureido]-phenyl}-1-methyl-1H-pyrazole-4--
carboxylic acid amide. .sup.1H-NMR (MeOH-d.sub.4): ppm 3.63 (s,
3H), 7.04 (m, 1H), 7.28 (m, 2H), 7.46 (d, 2H, J=8.6 Hz), 7.59 (d,
2H, J=8.6 Hz), 7.66 (s, 1H). LC-MS (m/e)=385.2 (MH+).
Example 7
5-Amino-3-[4-[3-(2,5-dichloro-phenyl)-ureido]-phenyl]-1-methyl-1H-pyrazole-
-4-carboxylic acid amide
[0249] Prepared as described above in Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-methyl-1H-pyrazole-4-carboxylic acid
amide to give the title compound
5-amino-3-4-[3-(2,5-dichloro-phenyl)-ureido]-phenyl}-1-methyl-1H-pyrazole-
-4-carboxylic acid amide. .sup.1H-NMR (MeOH-d.sub.4): ppm 3.63 (s,
3H), 7.06 (m, 1H), 7.42 (m, 1H), 7.48 (d, 2H, J=8.6 Hz), 7.63 (d,
2H, J=8.6 Hz), 8.35 (s, 1H). LC-MS (m/e)=419.0 (MH+).
Example 8
5-Amino-1-methyl-3-[4-(3-naphthalen-1-yl-ureido)-phenyl]-1H-pyrazole-4-car-
boxylic acid amide
[0250] Prepared as described above in Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-methyl-1H-pyrazole-4-carboxylic acid
amide to give the title compound
5-Amino-1-methyl-3-[4-(3-naphthalen-1-yl-ureido)-phenyl]-1H-pyrazole-4-ca-
rboxylic acid amide. .sup.1H-NMR (MeOH-d.sub.4): ppm 3.63 (s, 3H),
7.47-7.91 (m, 11H). LC-MS (m/e)=400.8 (MH+).
Example 9
5-Amino-1-methyl-3-{4-[3-(3-trifluoromethyl-phenyl)-ureido]-phenyl}-1H-pyr-
azole-4-carboxylic acid amide
[0251] Prepared as described above in Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-methyl-1H-pyrazole-4-carboxylic acid
amide to give the title compound
5-amino-1-methyl-3-{4-[3-(3-trifluoromethyl-phenyl)-ureido]-phenyl}-1H-py-
razole-4-carboxylic acid amide. .sup.1H-NMR (MeOH-d.sub.4): ppm
3.63 (s, 3H), 6.80 (d, 1H, J=8.4 Hz), 7.26 (m, 2H), 7.49 (d, 2H,
J=8.6 Hz), 7.62 (d, 2H, J=8.6 Hz), 7.93 (s, 1H). LC-MS (m/e)=419.2
(MH+).
Example 10
5-Amino-3-[4-(3-biphenyl-4-yl-ureido)-phenyl]-1-methyl-1H-pyrazole-4-carbo-
xylic acid amide
[0252] Prepared as described above in Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-methyl-1H-pyrazole-4-carboxylic acid
amide to give the title compound
5-amino-3-[4-(3-biphenyl-4-yl-ureido)-phenyl]-1-methyl-1H-pyrazole-4-carb-
oxylicacid amide as its TFA salt. .sup.1H-NMR (MeOH-d.sub.4): ppm
3.66 (s, 3H), 7.56 (m, 13H). LC-MS (m/e)=427.0 (MH+).
Example 11
5-Amino-3-{4-[3-(2,6-difluoro-phenyl)-ureido]-phenyl}-1-methyl-1H-pyrazole-
-4-carboxylic acid amide
[0253] Prepared as described above in Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-methyl-1H-pyrazole-4-carboxylic acid
amide to give the title compound
5-amino-3-{4-[3-(2,6-difluoro-phenyl)-ureido]-phenyl}-1-methyl-1H-pyrazol-
e-4-carboxylic acid amide as its TFA salt. .sup.1H-NMR
(MeOH-d.sub.4): ppm 3.66 (s, 3H), 7.08 (m, 2H), 7.31 (m, 1H), 7.49
(d, 2H, J=8.5 Hz), 7.61 (d, 2H, J=8.5 Hz). LC-MS (m/e)=386.8
(MH+).
Example 12
5-Amino-3-[4-(3-benzo[1,3]dioxol-5-yl-ureido)-phenyl]-1-methyl-1H-pyrazole-
-4-carboxylic acid amide
[0254] Prepared as described above in Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-methyl-1H-pyrazole-4-carboxylic acid
amide to give the title compound
5-amino-3-[4-(3-benzo[1,3]dioxol-5-yl-ureido)-phenyl]-1-methyl-1H-pyrazol-
e-4-carboxylic acid amide as its TFA salt. .sup.1H-NMR
(MeOH-d.sub.4): ppm 3.65 (s, 3H), 5.94 (s, 2H), 6.77 (s, 2H), 7.13
(s, 1H), 7.08 (m, 2H), 7.48 (d, 2H, J=8.6 Hz), 7.60 (d, 2H, J=8.6
Hz). LC-MS (m/e)=394.6 (MH+).
Example 13
5-Amino-1-methyl-3-[4-(3-o-tolyl-ureido)-phenyl]-1H-pyrazole-4-carboxylic
acid amide
[0255] Prepared as described above in Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-methyl-1H-pyrazole-4-carboxylic acid
amide to give the title compound
5-amino-1-methyl-3-[4-(3-o-tolyl-ureido)-phenyl]-1H-pyrazole-4-carboxylic
acid amide as its TFA salt. .sup.1H-NMR (MeOH-d.sub.4): ppm 2.32
(s, 3H), 3.66 (s, 3H), 7.06 (m, 1H), 7.23 (m, 2H), 7.47 (d, 2H,
J=8.6 Hz), 7.65 (m, 3H). LC-MS (m/e)=365.2 (MH+).
Example 14
5-Amino-1-methyl-3-[4-(3-1-tolyl-ureido)-phenyl]-1H-pyrazole-4-carboxylic
acid amide
[0256] Prepared as described above in Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-methyl-1H-pyrazole-4-carboxylic acid
amide to give the title compound
5-amino-1-methyl-3-[4-(3-m-tolyl-ureido)-phenyl]-1H-pyrazole-4-carboxylic
acid amide as its TFA salt. .sup.1H-NMR (MeOH-d.sub.4): ppm 2.34
(s, 3H), 3.65 (s, 3H), 6.87 (m, 1H), 7.18 (m, 3H), 7.46 (d, 2H,
J=8.6 Hz), 7.60 (d, 2H, J=8.6 Hz). LC-MS (m/e)=365.2 (MH+).
Example 15
5-Amino-3-{4-[3-(2,5-difluoro-phenyl)-ureido]-phenyl}-1-methyl-1H-pyrazole-
-4-carboxylic acid amide
[0257] Prepared as described above in Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-methyl-1H-pyrazole-4-carboxylic acid
amide to give the title compound
5-Amino-3-{4-[3-(2,5-difluoro-phenyl)-ureido]-phenyl}-1-methyl-1H-pyrazol-
e-4-carboxylic acid amide as its TFA salt. .sup.1H-NMR
(MeOH-d.sub.4): ppm 3.67 (s, 3H), 7.52 (d, 2H, J=8.6 Hz), 7.65 (d,
2H, J=8.6 Hz), 8.02 (m, 1H), 8.11 (m, 1H), 9.01 (m, 1H). LC-MS
(m/e)=387.2 (MH+).
Example 16
Amino-3-{4-[3-(2,4-dimethoxy-phenyl)-ureido]-phenyl}-1-methyl-1H-pyrazole--
4-carboxylic acid amide
[0258] Prepared as described above in Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-methyl-1H-pyrazole-4-carboxylic acid
amide to give the title compound
5-amino-3-{4-[3-(2,4-dimethoxy-phenyl)-ureido]-phenyl}-1-methyl-1H-pyrazo-
le-4-carboxylic acid amide as its TFA salt. .sup.1H-NMR
(MeOH-d.sub.4): ppm 3.64 (s, 3H), 3.80 (s, 3H), 3.91 (s, 3H), 6.61
(s, 1H), 7.47 (d, 2H, J=8.6 Hz), 7.58 (d, 2H, J=8.6 Hz), 7.88 (m,
2H). LC-MS (m/e)=411.4 (MH+).
Example 17
5-Amino-1-methyl-3-{4-[3-(4-phenoxy-phenyl)-ureido]-phenyl}-1H-pyrazole-4--
carboxylic acid amide
[0259] Prepared as described above in Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-methyl-1H-pyrazole-4-carboxylic acid
amide to give the title compound
5-amino-1-methyl-3-{4-[3-(4-phenoxy-phenyl)-ureido]-phenyl}-1H-pyrazole-4-
-carboxylic acid amide as its TFA salt. .sup.1H-NMR (MeOH-d.sub.4):
ppm 3.65 (s, 3H), 6.96 (m, 3H), 7.08 (m, 1H), 7.32 (m, 2H), 7.34
(m, 4H), 7.60 (d, 2H, J=8.6 Hz), 7.91 (s, 1H). LC-MS (m/e)=443.2
(MH+).
Example 18
5-Amino-1-tert-butyl-3-{4-[3-(2-chloro-5-trifluoromethyl-phenyl)-ureido]-p-
henyl}-1H-pyrazole-4-carboxylic acid amide
[0260] Prepared as described above in Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-tert-butyl-1H-pyrazole-4-carboxylic
acid amide to give the title compound
5-amino-1-tert-butyl-3-{4-[3-(2-chloro-5-trifluoromethyl-phenyl)-ureido]--
phenyl}-1H-pyrazole-4-carboxylic acid amide as its TFA salt. LC-MS
(m/e)=494.3 (MH+). R.sub.t=2.24 min.
Example 19
5-Amino-3-{4-[3-(bis-3,5-trifluoromethyl-phenyl)-ureido]-phenyl}-1-tert-bu-
tyl-1H-pyrazole-4-carboxylic acid amide
[0261] Prepared as described above in Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-tert-butyl-1H-pyrazole-4-carboxylic
acid amide to give the title compound
5-amino-3-{4-[3-(bis-3,5-trifluoromethyl-phenyl)-ureido]-phenyl}-1-tert-b-
utyl-1H-pyrazole-4-carboxylic acid amide as its TFA salt. LC-MS
(m/e)=529.2 (MH+). Rt=2.32 min.
Example 20
5-Amino-1-tert-butyl-3-[4-(3-naphthalen-1-yl-ureido)-phenyl]-1H-pyrazole-4-
-carboxylic acid amide
[0262] Prepared as described above in Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-tert-butyl-1H-pyrazole-4-carboxylic
acid amide to give the title compound
5-amino-1-tert-butyl-3-[4-(3-naphthalen-1-yl-ureido)-phenyl]-1H-pyrazole--
4-carboxylic acid amide as its TFA salt. LC-MS (m/e)=443.2 (MH+).
R.sub.t=1.94 min.
Example 21
5-Amino-1-tert-butyl-3-[4-[3-(3-chloro-phenyl)-ureido]-phenyl]-1H-pyrazole-
-4-carboxylic acid amide
[0263] Prepared as described above in Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-tert-butyl-1H-pyrazole-4-carboxylic
acid amide to give the title compound
5-amino-1-tert-butyl-3-{4-[3-(3-chloro-phenyl)-ureido]-phenyl}-1H-pyrazol-
e-4-carboxylic acid amide as its TFA salt. LC-MS (m/e)=427.0 (MH+).
R.sub.t=1.97 min.
Example 22
[0264]
5-Amino-1-tert-butyl-3-{4-[3-(2,5-dichloro-phenyl)-ureido]-phenyl}--
1H-pyrazole-4-carboxylic acid amide Prepared as described above in
Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-tert-butyl-1H-pyrazole-4-carboxylic
acid amide to give the title compound
5-amino-1-tert-butyl-3-{4-[3-(2,5-dichloro-phenyl)-ureido]-phenyl}-1H-pyr-
azole-4-carboxylic acid amide as its TFA salt. LC-MS (m/e)=461.2
(MH+). R.sub.t=2.20 min.
Example 23
[0265]
5-Amino-1-tert-butyl-3-[4-(3-naphthalen-2-yl-ureido)-phenyl]-1H-pyr-
azole-4-carboxylic acid amide Prepared as described above in
Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-tert-butyl-1H-pyrazole-4-carboxylic
acid amide to give the title compound
5-amino-1-tert-butyl-3-[4-(3-naphthalen-2-yl-ureido)-phenyl]-1H-pyrazole--
4-carboxylic acid amide as its TFA salt. LC-MS (m/e)=443.2 (MH+).
R.sub.t=2.02 min.
Example 24
5-Amino-1-tert-butyl-3-{4-[3-(3-trifluoromethyl-phenyl)-ureido]-phenyl}-1H-
-pyrazole-4-carboxylic acid amide
[0266] Prepared as described above in Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-tert-butyl-1H-pyrazole-4-carboxylic
acid amide to give the title compound
5-amino-1-tert-butyl-3-{4-[3-(3-trifluoromethyl-phenyl)-ureido]-phenyl}-1-
H-pyrazole-4-carboxylic acid amide as its TFA salt. LC-MS
(m/e)=461.4 (MH+). R.sub.t=2.04 min.
Example 25
[0267]
5-Amino-3-[4-(3-biphenyl-4-yl-ureido)-phenyl]-1-tert-butyl-1H-pyraz-
ole-4-carboxylic acid amide Prepared as described above in Example
1 starting from
5-amino-3-(4-amino-phenyl)-1-tert-butyl-1H-pyrazole-4-carboxylic
acid amide to give the title compound
5-amino-3-[4-(3-biphenyl-4-yl-ureido)-phenyl]-1-tert-butyl-1H-pyrazole-4--
carboxylic acid amide as its TFA salt. LC-MS (m/e)=469.2 (MH+).
R.sub.t=2.17 min.
Example 26
[0268]
5-Amino-1-tert-butyl-3-{4-[3-(2,6-difluoro-phenyl)-ureido]-phenyl}--
1H-pyrazole-4-carboxylic acid amide Prepared as described above in
Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-tert-butyl-1H-pyrazole-4-carboxylic
acid amide to give the title compound
5-amino-1-tert-butyl-3-{4-[3-(2,6-difluoro-phenyl)-ureido]-phenyl}-1H-pyr-
azole-4-carboxylic acid amide as its TFA salt. LC-MS (m/e)=429.4
(MH+). R.sub.t=1.67 min.
Example 27
[0269]
5-Amino-3-[4-(3-benzo[1,3]dioxol-5-yl-ureido)-phenyl]-1-tert-butyl--
1H-pyrazole-4-carboxylic acid amide Prepared as described above in
Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-tert-butyl-1H-pyrazole-4-carboxylic
acid amide to give the title compound
5-amino-3-[4-(3-benzo[1,3]dioxol-5-yl-ureido)-phenyl]-1-tert-butyl-1H-pyr-
azole-4-carboxylic acid amide as its TFA salt. LC-MS (m/e)=437.7
(MH+). R.sub.t=1.69 min.
Example 28
[0270]
5-Amino-1-tert-butyl-3-[4-(3-o-tolyl-ureido)-phenyl]-1H-pyrazole-4--
carboxylic acid amide Prepared as described above in Example 1
starting from
5-amino-3-(4-amino-phenyl)-1-tert-butyl-1H-pyrazole-4-carboxylic
acid amide to give the title compound
5-amino-1-tert-butyl-3-[4-(3-o-tolyl-ureido)-phenyl]-1H-pyrazole-4-carbox-
ylic acid amide as its TFA salt. LC-MS (m/e)=407.2 (MH+).
R.sub.t=1.77 min.
Example 29
[0271]
5-Amino-1-tert-butyl-3-[4-(3-1-tolyl-ureido)-phenyl]-1H-pyrazole-4--
carboxylic acid amide Prepared as described above in Example 1
starting from
5-amino-3-(4-amino-phenyl)-1-tert-butyl-1H-pyrazole-4-carboxylic
acid amide to give the title compound
5-amino-1-tert-butyl-3-[4-(3-r-tolyl-ureido)-phenyl]-1H-pyrazole-4-carbox-
ylic acid amide as its TFA salt. LC-MS (m/e)=407.4 (MH+).
R.sub.t=1.84 min.
Example 30
[0272]
5-Amino-1-tert-butyl-3-{4-[3-(2,5-difluoro-phenyl)-ureido]-phenyl}--
1H-pyrazole-4-carboxylic acid amide Prepared as described above in
Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-tert-butyl-1H-pyrazole-4-carboxylic
acid amide to give the title compound
5-amino-1-tert-butyl-3-{4-[3-(2,5-difluoro-phenyl)-ureido]-phenyl}-1H-pyr-
azole-4-carboxylic acid amide as its TFA salt. LC-MS (m/e)=429.0
(MH+). R.sub.t=1.95 min
Example 31
[0273]
5-Amino-1-tert-butyl-3-{4-[3-(2,4-dimethoxyo-phenyl)-ureido]-phenyl-
}-1H-pyrazole-4-carboxylic acid amide Prepared as described above
in Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-tert-butyl-1H-pyrazole-4-carboxylic
acid amide to give the title compound
5-amino-1-tert-butyl-3-{4-[3-(2,4-dimethoxyo-phenyl)-ureido]-phenyl}-1H-p-
yrazole-4-carboxylic acid amide as its TFA salt. LC-MS (m/e)=453.2
(MH+). R.sub.t=1.79 min.
Example 32
[0274]
5-Amino-1-tert-butyl-3-{4-[3-(4-phenoxy-phenyl)-ureido]-phenyl}-1H--
pyrazole-4-carboxylic acid amide Prepared as described above in
Example 1 starting from
5-amino-3-(4-amino-phenyl)-1-tert-butyl-1H-pyrazole-4-carboxylic
acid amide to give the title compound
5-amino-1-tert-butyl-3-{4-[3-(4-phenoxy-phenyl)-ureido]-phenyl}-1H-pyrazo-
le-4-carboxylic acid amide as its TFA salt. LC-MS (m/e)=485.4
(MH+). R.sub.t=2.19 min.
Example 33
[0275]
5-Amino-3-{4-[3-2-(fluoro-5-trifluoromethyl-phenyl)-ureido]-phenyl}-
-1H-pyrazole-4-carboxylic acid amide
5-Amino-1-tert-butyl-3-{4-[3-(fluoro-trifluoromethyl-phenyl)-ureido]-phen-
yl}-1H-pyrazole-4-carboxylic acid amide (48 mg, 0.1 mmol) was
dissolved in 2 mL of conc. sulfuric acid and the reaction mixture
was stirred for 30 minutes at the room temperature. The reaction
mixture was then poured in the 10 mL of ice water and product was
isolated by filtration. Crude product was then purified by prep.
HPLC to give the title compound
5-amino-3-{4-[3-2-(fluoro-5-trifluoromethyl-phenyl)-ureido]-phenyl}-1H-py-
razole-4-carboxylic acid amide as its TFA salt. .sup.1H-NMR (MeOH):
ppm 7.36 (m, 2H), 7.54 (m, 2H), 7.69 (m, 2H), 8.62 (m, 1H) LC-MS
(m/e)=423.2 (MH+). Rt=1.67 min.
Example 34
[0276]
N,N-Dimethylamino-{4-[3-(fluoro-trifluoromethyl-phenyl)-ureido]-phe-
nyl}-methyl-1H-pyrazole-4-carboxylic acid amide Prepared as
described above in Example 1 starting from
(4-amino-phenyl)-N,N-dimethylamino-methyl-1H-pyrazole-4-carboxylic
acid amide (prepared as described above in Preparation 5 starting
from
N,N-dimethylamino-methyl-(4-nitro-phenyl)-1H-pyrazole-4-carboxylic
acid amide) to give title compound
N,N-dimethylamino-{4-[3-(fluoro-trifluoromethyl-phenyl)-ureido]-phenyl}-m-
ethyl-1H-pyrazole-4-carboxylic acid amide as its TFA salt. LC-MS
(m/e)=465.2 (MH+). R.sub.t=1.88 min.
Example 35
[0277]
5-Amino-1-tert-butyl-3-{4-[3-(5-tert-butyl-isoxazol-3-yl)-ureido]-p-
henyl}-1H-pyrazole-4-carboxylic acid amide To a solution of
5-amino-3-(4-amino-phenyl)-1-tert-butyl-3-1H-pyrazole-4-carboxylic
acid amide (85 mg, 0.31 mmol) in 5 mL of anhydrous THF was added
(5-tert-butyl-isoxazol-3-yl)-carbamic acid phenyl ester (96 mg,
0.37 mmol, 1.2 eq) followed by triethylamine (37 mg, 0.37 mmol, 1.2
eq). The reaction mixture was heated under reflux for 16 hours,
then evaporated. Crude product was purified by preparative HPLC to
give 55 mg of pure
5-amino-1-tert-butyl-3-{4-[3-(5-tert-butyl-isoxazol-3-yl)-ureido]-phenyl}-
-1H-pyrazole-4-carboxylic acid amide as its trifluoroacetate salt.
.sup.1H-NMR (MeOH): ppm 1.37 (s, 3H), 1.67 (s, 3H), 6.43 (s, 1H),
7.50 (d, 2H, J=8.8 Hz), 7.62 (d, 2H, J=8.8 Hz). LC MS (m/e)=440.4
(MH+). R.sub.t=2.00 min
Biological Data
[0278] Compounds are tested for TIE-2 kinase and VEGFR kinase
inhibition activity according to one or more of the following
methods.
TIE-2 Enzyme Assay (TIE2-E)
[0279] The TIE-2 enzyme assay uses the LANCE method (Wallac) and
GST-TIE2, baculovirus expressed recombinant constructs of the
intracellular domains of human TIE2 (amino acids 762-1104, GenBank
Accession # L06139) tagged by GST). The method measures the ability
of the purified enzymes to catalyse the transfer of the
.gamma.-phosphate from ATP onto tyrosine residues in a biotinylated
synthetic peptide, D1-15 (biotin-C6-LEARLVAYEGWVAGKKKamide). This
peptide phosphorylation is detected using the following procedure:
for enzyme preactivation, GST-TIE2 is incubated for 30 mins at room
temperature with 2 mM ATP, 5 mM MgCl.sub.2 and 12.5 mM DTT in 22.5
mM HEPES buffer (pH7.4). Preactivated GST-TIE2 is incubated for 30
mins at room temperature in 96 well plates with 1 uM D1-15 peptide,
80 uM ATP, 10 mM MgC12, 0.1 mg/ml BSA and the test compound
(diluted from a 10 mM stock in DMSO, final DMSO concentration is
2.4%) in 1 mM HEPES (pH7.4). The reaction is stopped by the
addition of EDTA (final concentration 45 mM). Streptavidin
linked-APC (allophycocyanin, Molecular Probe) and Europium-labeled
anti-phosphorylated tyrosine antibody (Wallac) are then added at
the final concentration of 17 ug/well and 2.1 ug/well,
respectively. The APC signal is measured using an ARVO multilabel
counter. (Wallac Berthold Japan). The percent inhibition of
activity is calculated relative to blank control wells. The
concentration of test compound that inhibits 50% of activity
(IC.sub.50) is interpolated using nonlinear regression
(Levernberg-Marquardt) and the equation, y=Vmax(1-x/(K+x))+Y2,
where "K" is equal to the IC.sub.50. The IC.sub.50 values are
converted to pIC.sub.50 values, i.e., -log IC.sub.50 in Molar
concentration.
[0280] TIE-2 Autophosphorylation assay (TIE2-C) The TIE-2
autophosphorylation assay uses an ELISA method and a TIE2
intracellular domain/c-fins extracellular domain (TIE2/c-fms)
chimeric protein expressing mouse 3T3 cell line. This assay
measures the autophosphorylation level of TIE2 protein expressed in
cells. The cells are cultured in 96 well plates and grown in high
glucose DMEM containing 10% serum at 37.degree. C. in a humidified
10% CO2, 90% air incubator. On the day of the assay, the serum
containing medium is removed from the cells and replaced with serum
free medium for one hour. The test compound (diluted from a 10 mM
stock in DMSO, final DMSO concentration is 0.1%) is incubated with
TIE2/c-fms expressing cells for 30 minutes in serum free DMEM.
Intrinsic cellular dephosphorylation of the receptor is blocked by
the addition of the tyrosine phosphatase inhibitor, sodium
orthovanadate, from a 100 mM aqueous stock to a final concentration
of 1 mM. The culture media is removed by aspiration and the cells
incubated for 30 to 60 mins on ice with lysis buffer containing 137
mM NaCl, 2 mM EDTA, 10% glycerol, 1 mM sodium ortho vanadate,
1.times. tyrosine phosphatase inhibitor cocktail (Sigma) and
complete protease inhibitor cocktail (Roche) in 20 mM Tris-HCl
(pH8.0). The cell extracts are transferred into Rat anti-c-fms
antibody (Zymed-clone 12-2d6)(2.5 mg/ml) coated 96 well plates and
incubated for 12 hrs at 4 degrees. The extracts are removed by
aspiration and the plate, washed in a buffer comprising PBS, 0.05%
Tween-20, 0.05% NP-40 and 5% SuperBlock (Pierce) followed by
incubation with an HRP (horseradish peroxidase) conjugated
anti-phosphotyrosine antibody, (Upstate Biotech) The plates are
again washed with the aforementioned wash buffer and the
calorimetric HRP substrate, TMB is added. The reaction progresses
for 90 seconds and is stopped with the addition of 2M
H.sub.2SO.sub.4. The optical density at 450 nm derived from HRP
catalyzed TMB is measured with a plate reader capable of reading at
the appropriate wavelength (e.g. SpectroMax from Molecular
Dynamics). The percent inhibition of activity is calculated
relative to non-vanadate treated control wells. The concentration
of test compound that inhibits 50% of activity (IC.sub.50) is
interpolated using nonlinear regression (Levenberg-Marquardt) and
the equation, y=Vmax(1-x/(K-x))+Y2, where "K" is equal to the
IC.sub.50.
Tie2 Fluorescence Polarization Kinase Activity Assay (TIE2-FP)
Activation of Recombinant Tie2 Activation:
[0281] Recombinant GST-Tie2 is activated by incubating the enzyme
in 20 mM Tris-HCl, pH 7.5, 12 mM MgCl.sub.2, 100 mM NaCl, 20 .mu.M
sodium vanidate, 1 mM DTT and 300 .mu.M ATP at room temperature for
2 hours. The activation mixture is then passed through a NAP-25
desalting column (Pharmacia Biotech cat. no. 17-0852-02) to remove
the free ATP. The activated enzyme is stored as aliquots at
-80.degree. C. in 20 mM Tris-HCl, pH 7.5 and 100 mM NaCl.
Assay Conditions:
[0282] The final assay conditions are 50 mM HEPES, pH 7.5, 5% DMSO
(when screening compounds), 200 .mu.M ATP, 5 mM MgCl.sub.2, 1 mM
DTT, 50 .mu.M sodium vanidate, 1 nM activated enzyme, and 200 .mu.M
peptide. IC.sub.50's of compounds are measured under sub saturating
ATP (200 .mu.M) and varing concentrations of activated Tie2 and
peptide substrate (RFWKYEFWR-OH; MW 1873 Da, TFA salt). Panvera
Anti-phosphotyrosine antibody (Cat#P2840) and PTK Green Tracer
(Cat#P2842) are used to detect the phosphorylated peptide.
Polarization is measured on a TECAN Polarion in 138-second cycles
for 30 minutes at room temperature. IC.sub.50's are then determined
from the % polarization using normal calculation methods. The
IC.sub.50 values are converted to pIC.sub.50 values, i.e., -log
IC.sub.50 in Molar concentration.
VEGF-R2 Enzyme Assay (VEGF-E)
[0283] The VEGF enzyme assay uses the LANCE method (Wallac) and
GST-VEGFR2, baculovirus expressed recombinant constructs of the
intracellular domains of human TIE2 tagged by GST. The method
measures the ability of the purified enzymes to catalyse the
transfer of the .gamma.-phosphate from ATP onto tyrosine residues
in a biotinylated synthetic peptide,
(biotin-aminohexyl-EEEEYFELVAKKKK-NH2). This peptide
phosphorylation is detected using the following procedure:
GST-VEGFR2 is incubated for 40-60 mins at room temperature with 75
uM ATP, 5 mM MgCl.sub.2, 0.1 mM DTT, 0.1 mg/mL BSA and the test
compound (diluted from a 10 mM stock in DMSO for desired
concentration) in 100 mM HEPES buffer. The reaction is stopped by
the addition of EDTA (final concentration 50 mM). Streptavidin
linked-APC (allophycocyanin, Molecular Probe) and Europium-labeled
anti-phosphorylated tyrosine antibody (Wallac) are then added at
the final concentration of 15 nM and 1 nM, respectively. The APC
signal is measured using an ARVO multilabel counter (Wallac
Berthold, Japan). The percent inhibition of activity is calculated
relative to blank control wells. The concentration of test compound
that inhibits 50% of activity (IC.sub.50) is interpolated using
nonlinear regression (Levernberg-Marquardt) and the equation,
y=Vmax(1-x/(K+x))+Y2, where "K" is equal to the IC.sub.50. The
IC.sub.50 values are converted to pIC.sub.50 values, i.e., -log
IC.sub.50 in Molar concentration.
VEGF-R2 Enzyme Assay (VEGFR-2-E2)
[0284] The VEGF enzyme assay uses the LANCE method (Wallac) and
GST-VEGFR2, baculovirus expressed recombinant constructs of the
intracellular domains of human TIE2 tagged by GST. The method
measures the ability of the purified enzymes to catalyse the
transfer of the .gamma.-phosphate from ATP onto tyrosine residues
in a biotinylated synthetic peptide,
(biotin-aminohexyl-EEEEYFELVAKKKK-NH2). This peptide
phosphorylation is detected using the following procedure:
GST-VEGFR2 is incubated for 40-60 mins at room temperature with 75
uM ATP, 5 mM MgCl.sub.2, 0.1 mM DTT, 0.1 mg/mL BSA and the test
compound (diluted from a 10 mM stock in DMSO for desired
concentration) in 100 mM HEPES buffer. The reaction is stopped by
the addition of EDTA (final concentration 50 mM). Streptavidin
linked-APC (allophycocyanin, Molecular Probe) and Europium-labeled
anti-phosphorylated tyrosine antibody (Wallac) are then added at
the final concentration of 15 nM and 1 nM, respectively. The APC
signal is measured using an ARVO multilabel counter (Wallac
Berthold, Japan). The percent inhibition of activity is calculated
relative to blank control wells. The concentration of test compound
that inhibits 50% of activity (IC.sub.50) is interpolated using
nonlinear regression (Levernberg-Marquardt) and the equation,
y=Vmax (1-x/(K+x))+Y2, where "K" is equal to the IC.sub.50. The
IC.sub.50 values are converted to pIC.sub.50 values, i.e., -log
IC.sub.50 in Molar concentration.
VEGF-Driven Cellular Proliferation Assay: BrdU Incorporation Assay
(VEGF-C)
[0285] Human umbilical cord endothelial cells (HUVEC, Clonetics,
CC2519) are passaged in Type I collagen-coated 100-mm petridishes
in EGM-MV medium (Clonetics, CC3125) at 37 C in a humidified 5%
CO.sub.2, 95% air incubator. (HUVEC passaged more than 6 times in
vitro are discarded and not subjected to assaying.) The cells are
harvested using trypsin/EDTA, counted using a haemocytometer and
plated at 5000 cells/well in a Type I-collagen coated 96-well plate
(Becton Dickinson, 354407) in M199 medium (Gibco BRL, 12340-030)
containing 5% FBS (Hyclone, A 1115-L) and gentamicin (at 50 ug/ml,
Gibco BRL). After incubation overnight at 37.degree. C., the media
are replaced with 100 ul of M199 serum-free medium containing
compounds at various concentrations with 0.6% DMSO and gentamicin.
The compounds are diluted in serum-free M199 medium from 10 mM
stock solutions prepared in 100% DMSO. After a 30 min incubation at
37.degree. C., the cells are fed with 100 ul of serum-free M199
medium containing gentamicin, 0.2% culture-grade bovine serum
albumin (BSA, Sigma A1993) and 20 ng/ml of VEGF (R&D systems,
293-VE) or 0.6 ng/ml of basic FGF (R&D systems, 233-FB), and
cultured at 37.degree. C. for another 24 h. The cells are pulsed
with bromodeoxyuridine (BrdU at 10 uM in serum-free M199) at
37.degree. C. for an additional 24 h. The incorporation of BrdU
into the proliferating HUVEC are analyzed using BrdU Cell
Proliferation ELISA (Roche Molecular Biochemicals, 1647229)
according to the manufacturer's protocols. The optical density at
450 nm is measured with a multilabel counter (ARVO SX, Wallac). The
percent inhibition of cell growth is calculated relative to blank
control wells. The concentration of test compound that inhibits 50%
of cell growth (IC.sub.50) is interpolated using nonlinear
regression (Levernberg-Marquardt) and the equation, y=Vmax
(1-x/(K.sup.+ x))+Y2, where "K" is equal to the IC.sub.50. The
IC.sub.50 values are converted to pIC.sub.50 values, i.e., -log
IC.sub.50 in Molar concentration.
VEGFR-3--Homogenous Time-Resolved Fluorescence Assay
(VEGFR-3-HTRF)
[0286] This assay assesses Vascular Endothelial Growth Factor 3
(VEGFR3) tyrosine kinase inhibitory activity in substrate
phosphorylation assays. The assay examines the ability of small
molecule organic compounds to inhibit the tyrosine phosphorylation
of a peptide substrate.
[0287] The substrate phosphorylation assays use the VEGFR3
catalytic domain, which is expressed in Sf. 9 insect cells as an
amino-terminal GST-tagged fusion protein. The catalytic domain of
human VEGFR3 (AA residues #819-1298 based upon GenBank Accession
#XM003852) is cloned by PCR from human Placenta Marathon Ready cDNA
(Clontech). The PCR product is subcloned into pFastBac1 vector
containing an N-terminal GST tag. The resulting pFB/GSTNVEGFR3icd
vector is used to generate a recombinant baculovirus for protein
expression. The VEGFR3 catalytic domain translated sequence is:
TABLE-US-00001 MSPILGYWKI KGLVQPTRLL LEYLEEKYEE HLYERDEGDK
WRNKKFELGL EFPNLPYYID GDVKLTQSMA IIRYIADKHN MLGGCPKERA EISMLEGAVL
DIRYGVSRIA YSKDFETLKV DFLSKLPEML KMFEDRLCHK TYLNGDHVTH PDFMLYDALD
VVLYMDPMCL DAFPKLVCFK KRIEAIPQID KYLKSSKYIA WPLQGWQATF GGGDHPPKSD
LLVPRGSPEF KGLPGEVPLE EQCEYLSYDA SQWEFPRERL HLGRVLGYGA FGKVVEASAF
GIHKGSSCDT VAVKMLKEGA TASEQRALMS ELKILIHIGN HLNVVNLLGA CTKPQGPLMV
IVEFCKYGNL SNFLRAKRDA FSPCAEKSPE QRGRFRAMVE LARLDRRRPG SSDRVLFARF
SKTEGGARRA SPDQEAEDLW LSPLTMEDLV CYSFQVARGM EFLASRKCIH RDLAARNILL
SESDVVKICD FGLARDIYKD PDYVRKGSAR LPLKWMAPES IFDKVYTTQS DVWSFGVLLW
EIFSLGASPY PGVQINEEFC QRLRDGTRMR APELATPAIR RIMLNCWSGD PKARPAFSEL
VEILGDLLQG RGLQEEEEVC MAPRSSQSSE EGSFSQVSTM ALHIAQADAE DSPPSLQRHS
LAARYYNWVS FPGCLARGAE TRGSSRMKTF EEFPMTPTTY KGSVDNQTDS GMVLASEEFE
QIESRHRQES GFR
[0288] Autophosphorylation allows enzymes to be fully activated
prior to addition to peptide substrates. The assays are performed
using enzyme that has been activated by autophosphorylation via
preincubation in buffer with ATP and magnesium. Activated enzyme is
then diluted and added to titrated compound and the substrate
mix.
[0289] 200 nM VEGFR3 enzyme is activated for 45 minutes at room
temperature by incubating the enzyme in buffer containing 100 mM
HEPES (pH7.2), 75 .mu.M ATP, 0.3 mM DTT, 0.1 mg/mL BSA, and 10 mM
MgCl.sub.2. After activation, VEGFR3 is diluted 100-fold into
2.times. dilution buffer: 200 mM HEPES (pH 7.5), 0.2 mg/mL BSA, 0.6
mM DTT. 20 .mu.L of the diluted enzyme mix is added to 20 .mu.L of
2.times. substrate mix (150 .mu.M ATP, 20 mM MgCl.sub.2, 0.72 .mu.M
biotinylated peptide) in the assay plates. Final assay conditions
are: 100 mM HEPES (pH 7.2), 75 .mu.M ATP, 10 mM MgCl.sub.2, 0.1
mg/mL BSA, 0.3 mM DTT, 0.36 .mu.M biotinylated peptide, and 1 nM
VEGFR3 enzyme. Assay plates are incubated for 1.5 hours at room
temperature before the addition of 30 L 100 mM EDTA to the wells to
stop the enzymatic reaction. 40 .mu.L/well of HTRF mix are then
added to the assay plates for the detection of phosphorylated
substrate. Final assay concentrations are: 100 mM HEPES (pH7.2),
0.1 mg/mL BSA, 15 nM streptavidin-labeled allophycocyanin
(PerkinElmer), and 1 nM europium-labeled anti-phosphotyrosine
antibody (PerkinElmer). Assay plates are left unsealed and are
counted in a Wallac Multilabel Counter 1420 (PerkinElmer).
[0290] The data for dose responses are plotted as % Control
calculated with the data reduction formula 100*(U1-C2)/(C1-C2)
versus concentration of compound where U is the unknown value, C1
is the average control value obtained for DMSO, and C2 is the
average control value obtained for 0.1M EDTA. Data are fitted to
the curve described by: y=((Vmax*x)/(K+x)) where Vmax is the upper
asymptote and K is the IC50.
[0291] Example compounds were tested for TIE-2 and/or VEGF kinase
inhibitory activity. Results are shown in Table 1.
TABLE-US-00002 TABLE I Assay Ex. VEGFR-3- No TIE2-FP VEGF-C
VEGFR-2-E2 HTRF TIE-2-E2 1 +++ +++ 2 +++ 3 +++ 4 ++ 5 ++ 6 + 7 +++
8 + 9 +++ 10 ++ 11 # 12 + 13 + 14 ++ 15 + 16 + 17 ++ 18 +++ 19 +++
20 ++ 21 +++ 22 ++ 23 +++ 24 +++ 25 ++ 26 + 27 ++ 28 29 +++ 30 ++
31 + 32 ++ 33 +++ 34 + + +++ 35 +++ +++ +++ # = pIC50 < 5.0; + =
pIC.sub.50 of 5.0-6.0; ++ = pIC.sub.50 of 6.0-7.0; +++ = pIC.sub.50
> 7.0; blank = not tested
Sequence CWU 1
1
11713PRTUnknownVEGFR3 catalytic domain 1Met Ser Pro Ile Leu Gly Tyr
Trp Lys Ile Lys Gly Leu Val Gln Pro1 5 10 15Thr Arg Leu Leu Leu Glu
Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30Tyr Glu Arg Asp Glu
Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45Gly Leu Glu Phe
Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60Leu Thr Gln
Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn65 70 75 80Met
Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90
95Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
100 105 110Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu
Pro Glu 115 120 125Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys
Thr Tyr Leu Asn 130 135 140Gly Asp His Val Thr His Pro Asp Phe Met
Leu Tyr Asp Ala Leu Asp145 150 155 160Val Val Leu Tyr Met Asp Pro
Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175Val Cys Phe Lys Lys
Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185 190Leu Lys Ser
Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205Thr
Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Leu Val Pro 210 215
220Arg Gly Ser Pro Glu Phe Lys Gly Leu Pro Gly Glu Val Pro Leu
Glu225 230 235 240Glu Gln Cys Glu Tyr Leu Ser Tyr Asp Ala Ser Gln
Trp Glu Phe Pro 245 250 255Arg Glu Arg Leu His Leu Gly Arg Val Leu
Gly Tyr Gly Ala Phe Gly 260 265 270Lys Val Val Glu Ala Ser Ala Phe
Gly Ile His Lys Gly Ser Ser Cys 275 280 285Asp Thr Val Ala Val Lys
Met Leu Lys Glu Gly Ala Thr Ala Ser Glu 290 295 300Gln Arg Ala Leu
Met Ser Glu Leu Lys Ile Leu Ile His Ile Gly Asn305 310 315 320His
Leu Asn Val Val Asn Leu Leu Gly Ala Cys Thr Lys Pro Gln Gly 325 330
335Pro Leu Met Val Ile Val Glu Phe Cys Lys Tyr Gly Asn Leu Ser Asn
340 345 350Phe Leu Arg Ala Lys Arg Asp Ala Phe Ser Pro Cys Ala Glu
Lys Ser 355 360 365Pro Glu Gln Arg Gly Arg Phe Arg Ala Met Val Glu
Leu Ala Arg Leu 370 375 380Asp Arg Arg Arg Pro Gly Ser Ser Asp Arg
Val Leu Phe Ala Arg Phe385 390 395 400Ser Lys Thr Glu Gly Gly Ala
Arg Arg Ala Ser Pro Asp Gln Glu Ala 405 410 415Glu Asp Leu Trp Leu
Ser Pro Leu Thr Met Glu Asp Leu Val Cys Tyr 420 425 430Ser Phe Gln
Val Ala Arg Gly Met Glu Phe Leu Ala Ser Arg Lys Cys 435 440 445Ile
His Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Ser Glu Ser Asp 450 455
460Val Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys
Asp465 470 475 480Pro Asp Tyr Val Arg Lys Gly Ser Ala Arg Leu Pro
Leu Lys Trp Met 485 490 495Ala Pro Glu Ser Ile Phe Asp Lys Val Tyr
Thr Thr Gln Ser Asp Val 500 505 510Trp Ser Phe Gly Val Leu Leu Trp
Glu Ile Phe Ser Leu Gly Ala Ser 515 520 525Pro Tyr Pro Gly Val Gln
Ile Asn Glu Glu Phe Cys Gln Arg Leu Arg 530 535 540Asp Gly Thr Arg
Met Arg Ala Pro Glu Leu Ala Thr Pro Ala Ile Arg545 550 555 560Arg
Ile Met Leu Asn Cys Trp Ser Gly Asp Pro Lys Ala Arg Pro Ala 565 570
575Phe Ser Glu Leu Val Glu Ile Leu Gly Asp Leu Leu Gln Gly Arg Gly
580 585 590Leu Gln Glu Glu Glu Glu Val Cys Met Ala Pro Arg Ser Ser
Gln Ser 595 600 605Ser Glu Glu Gly Ser Phe Ser Gln Val Ser Thr Met
Ala Leu His Ile 610 615 620Ala Gln Ala Asp Ala Glu Asp Ser Pro Pro
Ser Leu Gln Arg His Ser625 630 635 640Leu Ala Ala Arg Tyr Tyr Asn
Trp Val Ser Phe Pro Gly Cys Leu Ala 645 650 655Arg Gly Ala Glu Thr
Arg Gly Ser Ser Arg Met Lys Thr Phe Glu Glu 660 665 670Phe Pro Met
Thr Pro Thr Thr Tyr Lys Gly Ser Val Asp Asn Gln Thr 675 680 685Asp
Ser Gly Met Val Leu Ala Ser Glu Glu Phe Glu Gln Ile Glu Ser 690 695
700Arg His Arg Gln Glu Ser Gly Phe Arg705 710
* * * * *