Note: Descriptions are shown in the official language in which they were submitted.
CA 02336848 2001-O1-09
WO 00/02871
TITLE OF THE INVENTION
NOVEL ANGIOGENESIS INHIBITORS
PCTNS99/15200
BACKGROUND OF THE INVENTION
The present invention relates to compounds which
inhibit tyrosine kinase enzymes, compositions which contain
tyrosine kinase inhibiting compounds and methods of using
tyrosine kinase inhibitors to treat tyrosine kinase-dependent
diseases/conditions such as neoangiogenesis, cancer, tumor
growth, atherosclerosis, age related macular degeneration,
diabetic retinopathy or inflammatory diseases, in mammals.
Tyrosine kinases are a class of enzymes that catalyze
the transfer of the terminal phosphate of adenosine triphosphate to
tyrosine residues in protein substrates. Tyrosine kinases are
believed, by way of substrate phosphorylation, to play critical roles
in signal transduction for a number of cell functions. Though the
exact mechanisms of signal transduction is still unclear, tyrosine
kinases have been shown to be important contributing factors in
cell proliferation, carcinogenesis and cell differentiation.
Solid tumors which are treated by the present
invention are cancers such as cancers of the brain, genitourinary
tract, lymphatic system, stomach, larynx and lung. These include
histiocytic lymphoma, lung adenocarcinoma and small cell lung
cancers. Additional examples include cancers in which
overexpression or activation of Raf activating oncogenes (e.g., K-
ras, erb-B) is observed. More particularly, such cancers include
pancreatic and breast carcinoma.
Accordingly, inhibitors of these tyrosine kinases are
useful for the prevention and treatment of proliferative diseases
dependent on these enzymes.
- 1-
CA 02336848 2001-O1-09
wo ooio2sm rc~rius99nszoo
For example, a method of treatment described herein
relates to neoangiogenesis. Neoangiogenesis occurs in
conjunction with tumor growth and in certain diseases of the eye.
It is characterized by excessive activity of vascular endothelial
growth factor.
Vascular endothelial growth factor (VEGF) binds the
high affinity membrane-spanning tyrosine kinase receptors KDR
and Flt-1. Cell culture and gene knockout experiments indicate
that each receptor contributes to different aspects of angiogenesis.
KDR mediates the mitogenic function of VEGF whereas Flt-1
appears to modulate non-mitogenic functions such as those
associated with cellular adhesion. Inhibiting KDR thus modulates
the level of mitogenic VEGF activity.
Vascular growth in the retina leads to visual
degeneration culminating in blindness. VEGF accounts for most of
the angiogenic activity produced in or near the retina in diabetic
retinopathy. Ocular VEGF mRNA and protein are elevated by
conditions such as retinal vein occlusion in primates and
decreased p02 levels in mice that lead to neovascularization.
Intraocular injections of anti-VEGF monoclonal antibodies or
VEGF receptor immunofusions inhibit ocular neovascularization
in both primate and rodent models. Regardless of the cause of
induction of VEGF in human diabetic retinopathy, inhibition of
ocular VEGF is useful in treating the disease.
Expression of VEGF is also significantly increased in
hypoxic regions of animal and human tumors adjacent to areas of
necrosis. VEGF is also upregulated by the expression of the
oncogenes ras, raf, src and mutant p53 (all of which are relevant to
targeting cancer). Monoclonal anti-VEGF antibodies inhibit the
growth of human tumors in nude mice. Although these same
- 2-
CA 02336848 2001-O1-09
WO 00/02871 PCT/US99/15200
tumor cells continue to express VEGF in culture, the antibodies do
not diminish their mitotic rate. Thus tumor-derived VEGF does
not function as an autocrine mitogenic factor. Therefore, VEGF
contributes to tumor growth in vivo by promoting angiogenesis
through its paracrine vascular endothelial cell chemotactic and
mitogenic activities. These monoclonal antibodies also inhibit the
growth of typically less well vascularized human colon cancers in
athymic mice and decrease the number of tumors arising from
inoculated cells. Viral expression of a VEGF-binding construct of
Flk-1, Flt-1, the mouse KDR receptor homologue, truncated to
eliminate the cytoplasmic tyrosine kinase domains but retaining a
membrane anchor, virtually abolishes the growth of a
transplantable glioblastoma in mice presumably by the dominant
negative mechanism of heterodimer formation with membrane
spanning endothelial cell VEGF receptors. Embryonic stem cells,
which normally grow as solid tumors in nude mice, do not produce
detectable tumors if both VEGF alleles are knocked out. Taken
together, these data indicate the role of VEGF in the growth of solid
tumors. Inhibition of KDR or Flt-1 is implicated in pathological
neoangiogenesis, and these receptors are useful in the treatment of
diseases in which neoangiogenesis is part of the overall pathology,
e.g., inflammation, diabetic retinal vascularization, as well as
various forms of cancer. The compounds of the instant invention
represent novel structures for the inhibition of KDR kinase.
SUMMARY OF THE INVENTION
A compound is disclosed in accordance with formula
I:
- 3-
CA 02336848 2001-O1-09
WO 00/02871 PCTNS99/15200
R3
N
R2 ~ \~R4
R
I
or a pharmaceutically acceptable salt, hydrate or prodrug thereof,
wherein
X is O or S;
R1 is H, C1-10 alkyl, Cg_g cycloalkyl, C5_10 aryl, halo, CF3,
C3-10 heterocyclyl, or C5_10 heteroaryl; said alkyl,
alkenyl, alkynyl, aryl, heteroaryl and heterocyclyl
being optionally substituted with from one to three
members selected from Ra;
R2 is H, C1_g alkyl, C~_10 aryl, C5_10 heteroaryl, Cg-g
cycloalkyl; said alkyl, aryl, heteroaryl or cycloalkyl
optionally substituted with from one to three members
selected from Ra;
R3 is C1_g alkyl, C5_10 aryl, C5_10 heteroaryl, Cg_6
cycloalkyl; said alkyl, aryl, heteroaryl or cycloalkyl
optionally substituted with from one to three members
selected from Ra;
- 4-
CA 02336848 2001-O1-09
WO 00/02871 PCT/US99/15200
R4 is H, C1_10 alkyl, C3_g cycloalkyl, C1_6 alkoxy, C2_10
alkenyl, C2_10 alkynyl, C5_10 aryl, Cg_10 heterocyclyl,
C1_g alkoxyNR7Rg, N02, OH, -NH2 or C5_10
heteroaryl, said alkyl, cycloalkyl, alkenyl, alkynyl,
aryl, heteroaryl and heterocyclyl being optionally
substituted with from one to three members selected
from Ra;
Ra is H, C1_10 alkyl, halogen, CF3~ N02, NHC(O)R*, OR, -
NR~ NR7Rg~ R7Rg~ C5_10 aryl, C5_10 aralkyl, C5_10
heteroaryl or Cg_10 heterocyclyl, said aralkyl, aryl and
heteroaryl optionally substituted with 1-2 groups of
N02, halo, C5_10 aryl, C1_6 alkoxy, C1_6 alkyl or CFg,
R* is H, or C1_g alkyl, NHC(O)CHR(C5_10 aralkyl), the aryl
ring of the aralkyl being optionally substituted
with 1-3 groups of OH, C1_g alkyl, or halo,
R is H, or C1_6 alkyl; and
R7&Rg are independently H, C1_10 alkyl, C3_6 cycloalkyl, COR,
COOR, C02, C5_10 aryl, C3_10 heterocyclyl, or C5_10
heteroaryl or NR7Rg can be taken together to form a
heterocyclic 5-10 membered saturated or unsaturated
ring containing, in addition to the nitrogen atom,
one to two additional heteroatoms selected from the
group consisting of N, O and S.
- 5-
CA 02336848 2001-O1-09
PCT/US99/15200
WO 00/02871
Also disclosed is a pharmaceutical composition which
is comprised of a compound represented by the formula I:
R3
N
R2 ~ \~R4
R
I
wherein Rl, R2, R3 and R4 are described as above or a
pharmaceutically acceptable salt or hydrate or prodrug thereof in
combination with a carrier.
Also included is a method of treating a tyrosine kinase
dependent disease or condition in a mammal which comprises
administering to a mammalian patient in need of such treatment
a tyrosine kinase dependent disease or condition treating amount
of a compound of formula I or a pharmaceutically acceptable salt,
hydrate or pro-drug thereof.
Also included is a method of treating cancer in a
mammalian patient in need of such treatment which is comprised
of admininstering to said patient an anti-cancer effective amount
of a compound of formula I or a pharmaceutically acceptable salt,
hydrate or pro-drug thereof.
Also included in the present invention is a method of
treating diseases in which neoangiogenesis is implicated, which is
comprised of administering to a mammalian patient in need of
such treatment a compound of formula I or a pharmaceutically
acceptable salt, hydrate or pro-drug thereof in an amount which is
effective for reducing neoangiogenesis.
- 6-
CA 02336848 2001-O1-09
WO 00/02871 PCTNS99/15200
More particularly, a method of treating ocular disease
in which neoangiogenesis occurs is included herein, which is
comprised of administering to a mammalian patient in need of
such treatment a compound of formula I or a pharmaceutically
acceptable salt hydrate or pro-drug thereof in an amount which is
effective for treating said ocular disease.
More particularly, a method of treating retinal
vascularization is included herein, which is comprised of
administering to a mammalian patient in need of such treatment
a compound of formula I or a pharmaceutically acceptable salt,
hydrate or pro-drug thereof in an amount which is effective for
treating retinal vascularization. Diabetic retinopathy is an
example of a disease in which neoangiogenesis or retinal
vascularization is part of the overall disease etiology. Also
included is a method of treating age-related macular degeneration.
These and other aspects of the invention will be
apparent from the teachings contained herein.
DETAILED DESCRIPTION OF THE INVENTION
The invention is described herein in detail using the
terms defined below unless otherwise specified.
The term "alkyl" refers to a monovalent alkane
(hydrocarbon) derived radical containing from 1 to 10 carbon atoms
unless otherwise defined. It may be straight, branched or cyclic.
Preferred straight or branched alkyl groups include methyl, ethyl,
propyl, isopropyl, butyl and t-butyl. Preferred cycloalkyl groups
include cyclopropyl, cyclobutyl, cycloheptyl, cyclopentyl and
cyclohexyl.
CA 02336848 2001-O1-09
WO 00/02871 PCT/US99/15200
When substituted alkyl is present, this refers to a
straight, branched or cyclic alkyl group as defined above,
substituted with 1-3 groups of Ra, described herein.
The term "alkenyl" refers to a non-aromatic
hydrocarbon radical, straight, branched or cyclic, containing from
2 to 10 carbon atoms and at least one carbon to carbon double bond.
Preferably one carbon to carbon double bond is present, and up to
four non-aromatic carbon-carbon double bonds may be present.
Preferred alkenyl groups include ethenyl, propenyl, butenyl and
cyclohexenyl. As described above with respect to alkyl, the
straight, branched or cyclic portion of the alkenyl group rnay
contain double bonds and may be substituted with one to three
groups of Ra, when a substituted alkenyl group is provided.
The term "alkynyl" refers to a hydrocarbon radical
straight, branched or cyclic, containing from 2 to 10 carbon atoms
and at least one carbon to carbon triple bond. Up to three carbon-
carbon triple bonds may be present. Preferred alkynyl groups
include ethynyl, propynyl and butynyl. As described above with
respect to alkyl, the straight, branched or cyclic portion of the
alkynyl group may contain triple bonds and may be substituted
with 1-3 groups of Ra, when a substituted alkynyl group is
provided.
Aryl refers to 5-10 membered aromatic rings e.g.,
phenyl, substituted phenyl and like groups as well bicyclic rings
such as naphthyl. Aryl thus contains at least one ring having at
least 5 atoms, with up to two such rings being present, containing
up to 10 atoms therein. The preferred aryl groups are phenyl and
naphthyl. Aryl groups may likewise be substituted with 1-3 groups
of Ra as defined herein. Preferred substituted aryls include phenyl
and naphthyl substituted with one or two groups.
_ g_
CA 02336848 2001-O1-09
WO 00/02871 PCT/US99/15200
As used herein, "aralkyl" is intended to mean an aryl
or heteroaryl moiety, as defined herein, attached through a C1-6
alkyl linker, where alkyl is defined above. Examples of aralkyls
include, but are not limited to, benzyl, naphthylmethyl,
phenylpropyl, 2-pyridylmethyl, 2-imidazolylethyl, 2-
quinolinylmethy, 2-imidazolylmethyl and the like.
The term heterocycle, heteroaryl or heterocyclic, as
used herein except where noted, represents a stable 5- to 7-
membered mono- or 7- to 10-membered bicyclic heterocyclic ring
system, any ring of which may be saturated or unsaturated,
aromatic or non-aromatic, and which consists of carbon atoms and
from one to three heteroatoms selected from the group consisting of
N, O and S. The nitrogen and sulfur heteroatoms may optionally
be oxidized, and the nitrogen heteroatom may optionally be
quaternized. Heterocycles include any bicyclic group in which any
of the above-defined rings is fused to a benzene ring. The
heterocyclic ring may be attached at any heteroatom or carbon
atom which results in the creation of a stable structure. The
heterocycle, heteroaryl or heterocyclic may be substituted with 1-3
groups of Ra. Examples of such heterocyclic elements include
piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-
oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, pyrrolyl, 4-piperidonyl,
pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl,
imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl,
oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl,
thiazolyl, thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl,
indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, thiadiazoyl,
benzopyranyl, benzothiazolyl, benzoxazolyl, furyl, tetrahydrofuryl,
tetrahydropyranyl, thiophenyl, imidazopyridinyl, tetrazolyl,
- 9-
CA 02336848 2001-O1-09
WO 00/02871 PCT/US99/15200
triazinyl, thienyl, benzothienyl, thiamorpholinyl sulfoxide,
thiamorpholinyl sulfone, and oxadiazolyl.
The term "alkoxy" refers to a substituent with an alkyl
group of the designated length in either a straight or branched
configuration, and may include a double or a triple bond, which is
attached via an oxygen molecule. Examples of such alkoxy groups
are methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy,
tertiary butoxy, pentoxy, isopentoxy, hexoxy, isohexoxy allyloxy,
propargyloxy, vinyloxy and the like.
The term "halo" or "halogen" is intended to include
the halogen atoms fluorine, chlorine, bromine and iodine.
Tyrosine kinase dependent diseases or conditions
refers to hyperproliferative disorders which are
initiated/maintained by aberrant expression of the activating
ligands (e.g. VEGF) of the tyrosine kinases. Examples include
psoriasis, cancer, immunoregulation (graft rejection),
atherosclerosis, rheumatoid arthritis, angiogenesis (e.g. tumor
growth, diabetic retinopathy), age related macular degeneration,
etc.
One aspect of the invention is realized when X is O
and all other variables are as originally described.
.Another aspect of the invention is realized when X is S
and all other variables are as originally described.
Still another aspect of the invention is realized when
R3 and R4, independently, are C5_10 aryl or C5_10 heteroaryl
optionally substituted with 1-3 groups of Ra.
Yet another aspect of the invention is realized when:
Rl is H, C1_10 alkyl, C~_10 aryl, halo, CFg, or C5_10
heteroaryl; said alkyl, aryl, and heteroaryl being
-10-
CA 02336848 2001-O1-09
WO 00/02871 PC'T/US99/15200
optionally substituted with from one to three
members selected from Ra;
R2 is H, C1_g alkyl or C5-10 aryl, said alkyl or aryl
optionally substituted with one to three members
selected from Ra;
R3 & R4 are independently C1_10 alkyl, C5_10 aryl, or C5_10
heteroaryl, said alkyl, aryl and heteroaryl being
optionally substituted with from one to three
members selected from Ra;and all other variables are
as described above.
Examples of the compounds of this invention are:
2-(2-(3-hydroxy)napthyl)-4-pheny-5-trifluoroacetamidooxazole;
2-(2-(3-hydroxy)napthyl)-4-(3-thiophenyl)-5-acetamidooxazole;
2-(2-(3-hydroxy)napthyl)-4-pheny-5-acetamidooxazole;
2-(2-(3-hydroxy)napthyl)-4-(3-thiophenyl)-5-trifluoroacetamido-
oxazole;
2-(2-(2-hydroxy-4-methoxy)phenyl)-4-phenyl-5-acetamidooxazole;
2-(2-(2-hydroxy-4-methyl)phenyl)-4-phenyl-5-acetamidooxazole;
2-(2-(2-hydroxy)phenyl)-4-phenyl-5-acetamidooxazole;
2-(5-isoquinolinyl)-4-phenyl-5-acetamidooxazole;
2-(2-(3-hydroxy)napthyl)-4-(3-thiophenyl)-5-acetamidooxazole;
2-(2-(3-hydroxy)napthyl)-4-phenyl-5-acetamidooxazole;
2-(3-(5-phenyl)pyridyl)-4-phenyl-5-acetamidooxazole;
2-(3-(5-(3-vitro)phenyl)pyridyl)-4-phenyl-5-acetamidooxazole;
2-(3-( 5-( 1-naphthyl)pyridyl)-4-phenyl-5-acetamidooxazole;
-11-
CA 02336848 2001-O1-09
WO 00/02871 PCT/US99/15200
2-(3-(5-(4-methyl)phenyl)pyridyl)-4-phenyl-5-acetamidooxazole;
2-(3-(5-(4-methoxy)phenyl)pyridyl)-4-phenyl-5-acetamidooxazole;
2-(3-(5-(3-chloro)phenyl)pyridyl)-4-phenyl-5-acetamidooxazole;
2-(3-(5-(3-methoxy)phenyl)pyridyl)-4-phenyl-5-acetamidooxazole;
2-(3-(5-(3-fluoro)phenyl)pyridyl)-4-phenyl-5-acetamidooxazole;
2-(3-(5-(2-naphthyl)pyridyl)-4-phenyl-5-acetamidooxazole;
2-( 3-( 5-( 2-trifluoromethyl)phenyl )pyridyl)-4-phenyl-5-
acetamidooxazole;
2-(2-hydroxy)phenyl-4-pheny-5-acetamidooxazole;
2-(2-hydroxy)phenyl-4-pheny-5-benzamidooxazole;
2-(2-hydroxy)phenyl-4-pheny-5-valeramidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-phenyl-5-acetamidooxazole;
2-(2-hydroxy-(4-( 3-nitro)-phenyl))-phenyl-4-phenyl-5-acetamidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-phenyl-5-acetamidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-phenyl-5-acetamidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-phenyl-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-phenyl-5-acetamidooxazole;
2-( 2-hydroxy-( 4-( 3'-m ethoxy)-phenyl)-phenyl-4-phenyl-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-phenyl-5-acetamidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-phenyl-5-
acetamidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-phenyl-5-
acetamidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-thiophenyl-5-acetamidooxazole;
2-( 2-hydroxy-( 4-( 3-nitro)-phenyl ) )-phenyl-4-thiophenyl-5-
acetamidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-thiophenyl-5-acetamidooxazole;
-12-
CA 02336848 2001-O1-09
WO 00/02871 PCTNS99/15200
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-thiophenyl-5-
acetamidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-thiophenyl-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-thiophenyl-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-thiophenyl-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-thiophenyl-5-
acetamidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-thiophenyl-5-
acetamidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-thiophenyl-5-
acetamidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-(3-pyridyl)-5-acetamidooxazole;
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(3-pyridyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-(3-pyridyl)-5-acetarnidooxazole;
2-( 2-hydroxy-(4-(4'-methyl )-phenyl )-phenyl-4-( 3-pyridyl )-5-
acetamidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(3-pyridyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(3-pyridyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-(3-pyridyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(3-pyridyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(3-pyridyl)-5-
acetamidooxazole;
-13-
CA 02336848 2001-O1-09
WO 00/02871 PCTNS99/15200
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(3-pyridyl)-5-
acetamidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-(3-chlorophenyl)-5-acetamidooxazole
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(3-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-(3-chlorophenyl)-5-
acetamidooxazole;
2-( 2-hydroxy-( 4-( 4'-methyl)-phenyl)-phenyl-4-( 3-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(3-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(3-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-(3-chlorophenyl)-5-
acetamidooxazole;
2-( 2-hydroxy-( 4-( 3'-fluoro)-phenyl )-phenyl-4-( 3-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(3-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(3-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-(2-chlorophenyl)-5-acetamidooxazole;
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(2-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-(2-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(2-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(2-chlorophenyl)-5-
acetamidooxazole;
-14-
CA 02336848 2001-O1-09
WO 00/02871 PCT1US99/15200
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(2-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-(2-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(2-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(2-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(2-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-(4-chlorophenyl)-5-acetamidooxazole
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(4-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(1-naphthyl))-phenyl-4-(4-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(4-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(4-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(4-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-(4-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(4-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(4-chlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(4-chlorophenyl)-5-
acetamidooxazole;
-15-
CA 02336848 2001-O1-09
PCT/US99/15200
WO 00/02871
2-(2-hydroxy-4-phenyl)-phenyl-4-(4-trifluoromethylphenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3-vitro)-phenyl))-phenyl-4-(4-trifluoromethylphenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-(4-trifluoromethylphenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(4-trifluoromethylphenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(4-trifluoromethylphenyl)-
5-acetamidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(4-trifluoromethylphenyl)-5-
acetamidooxazole;
2-( 2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-(4-trifluoromethylphenyl)-
5-acetamidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(4-trifluoromethylphenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(4-trifluoromethylphenyl)-
5-acetamidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(4-
trifluoromethylphenyl)-5-acetamidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-(3-methoxyphenyl)-5-acetamidooxazole;
2-(2-hydroxy-(4-(3-vitro)-phenyl))-phenyl-4-(3-methoxyphenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-(3-methoxyphenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(3-methoxyphenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(3-methoxyphenyl)-5-
acetamidooxazole;
-16-
CA 02336848 2001-O1-09
PCTNS99/15200
WO 00/02871
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(3-methoxyphenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-(3-methoxyphenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(3-methoxyphenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(3-methoxyphenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(3-methoxyphenyl)-
5-acetamidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-(3-thiophenyl)-5-acetamidooxazole;
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(3-thiophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-(3-thiophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(3-thiophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(3-thiophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(3-thiophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-(3-thiophenyl)-5-
acetamidooxazole;
2-( 2-hydroxy-( 4-( 3'-fluoro)-phenyl )-phenyl-4-( 3-thiophenyl)-5-
acetamidooxazole;
2-( 2-hydroxy-( 4-( 2-naphthyl)-phenyl )-phenyl-4-( 3-thiophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(3-thiophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-(2-thiophenyl)-5-acetamidooxazole
-17-
CA 02336848 2001-O1-09
WO 00/02871 PCT/US99/15200
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(2-thiophenyl)-5-
acetamidooxazole;
2-( 2-hydroxy-(4-( 1-naphthyl ))-phenyl-4-( 2-thiophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(2-thiophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(2-thiophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(2-thiophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-(2-thiophenyl)-5-
acetamidooxazole;
2-( 2-hydroxy-(4-( 3'-fluoro)-phenyl )-phenyl-4-( 2-thiophenyl )-5-
acetamidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(2-thiophenyl)-5-
acetamidooxazole;;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(2-thiophenyl)-5-
acetamidooxazole
2-( 2-hydroxy-4-phenyl )-phenyl-4-( 2,6-dichlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(2,6-dichlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-(2,6-dichlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(2,6-dichlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(2,6-dichlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(2,6-dichlorophenyl)-5-
acetamidooxazole;
-18-
CA 02336848 2001-O1-09
WO 00/02871 PCT/US99115200
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-(2,6-dichlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(2,6-dichlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(2,6-dichlorophenyl)-5-
acetamidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(2,6-
dichlorophenyl)-5-acetamidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-phenyl-5-benzamidooxazole;
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-phenyl-5-benzamidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-phenyl-5-benzamidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-phenyl-5-benzamidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-phenyl-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-phenyl-5-benzamidooxazole;
2-{2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-phenyl-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-phenyl-5-benzamidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-phenyl-5-
benzamidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-phenyl-5-
benzamidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-thiophenyl-5-benzamidooxazole
2-(2-hydroxy-(4-( 3-nitro)-phenyl))-phenyl-4-thiophenyl-5-
benzamidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-thiophenyl-5-benzamidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-thiophenyl-5-
benzamidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-thiophenyl-5-
benzamidooxazole;
-19-
CA 02336848 2001-O1-09
WO 00/02871
PCT1US99/15200
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-thiophenyl-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-thiophenyl-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-thiophenyl-5-
benzamidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-thiophenyl-5-
benzamidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-thiophenyl- 5-
benzamidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-( 3-pyridyl)-5-benzamidooxazole;
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(3-pyridyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-(3-pyridyl)-5-benzamidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(3-pyridyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(3-pyridyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(3-pyridyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-(3-pyridyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(3-pyridyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(3-pyridyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(3-pyridyl)-5-
benzamidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-(3-chlorophenyl)-5-benzamidooxazole;
- 20 -
CA 02336848 2001-O1-09
WO 00/02871 PCT/US99/15200
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(3-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-(3-chlorophenyl)-5-
benzamidooxazoie;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(3-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(3-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-{3-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-{3'-methoxy)-phenyl)-phenyl-4-(3-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(3-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(3-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(3-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-(2-chlorophenyl)-5-benzamidooxazole;
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(2-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-(2-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-{2-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(2-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(2-chlorophenyl)-5-
benzamidooxazole;
-21-
CA 02336848 2001-O1-09
WO 00/02871
PCT/US99/15200
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-(2-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(2-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(2-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(2-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-(4-chlorophenyl)-5-benzamidooxazole
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(4-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-(4-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(4-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(4-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(4-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-(4-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(4-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(4-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(4-chlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-(4-trifluoromethylphenyl)-5-
benzamidooxazole;
-22-
CA 02336848 2001-O1-09
WO 00/02871
PCTNS99/15200
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(4-trifluoromethylphenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-(4-trifluoromethylphenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(4-trifluoromethylphenyl)-5-
benzamidooxazole;
2-( 2-hydroxy-( 4-( 4'-methoxy )-phenyl)-phenyl-4-( 4-trifluorom ethylphenyl )-
5-benzamidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(4-trifluoromethylphenyl)-5-
benzamidooxazole;
2-( 2-hydroxy-( 4-( 3'-m ethoxy)-phenyl )-phenyl-4-( 4-trifluorom ethylphenyl
)-
5-benzamidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(4-trifluoromethylphenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(4-trifluoromethylphenyl)-
5-benzamidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(4-
trifluoromethylphenyl)-5-benzamidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-(3-methoxyphenyl)-5-benzamidooxazole;
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(3-methoxyphenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-(3-methoxyphenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(3-methoxyphenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(3-methoxyphenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(3-methoxyphenyl)-5-
benzamidooxazole;
-23-
CA 02336848 2001-O1-09
WO 00/02871
PCT/US99/15200
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-(3-methoxyphenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(3-methoxyphenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(3-methoxyphenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-( 2-trifluoromethyl)-phenyl)-phenyl-4-(3-methoxyphenyl)-
5-benzamidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-(3-thiophenyl)-5-benzamidooxazole;
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(3-thiophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-(3-thiophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(3-thiophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(3-thiophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(3-thiophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-(3-thiophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(3-thiophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(3-thiophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(2-trifluorornethyl)-phenyl)-phenyl-4-(3-thiophenyl)-5-
benzamidooxazole;
2-( 2-hydroxy-4-phenyl )-phenyl-4-( 2-thiophenyl )-5-benzamidooxazole
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(2-thiophenyl)-5-
benzamidooxazole;
-24-
CA 02336848 2001-O1-09
WO 00/02871 PCT/US99/15200
2-(2-hydroxy-(4-(1-naphthyl))-phenyl-4-(2-thiophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(2-thiophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(2-thiophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(2-thiophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-(2-thiophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(2-thiophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(2-thiophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(2-thiophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-( 2,6-dichlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-( 3-nitro)-phenyl))-phenyl-4-(2,6-dichlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(1-naphthyl))-phenyl-4-(2,6-dichlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(2,6-dichlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(2,6-dichlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(2,6-dichlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-(2,6-dichlorophenyl)-5-
benzamidooxazole;
-25-
CA 02336848 2001-O1-09
WO 00/02871
PCTNS99/15200
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(2,6-dichlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(2,6-dichlorophenyl)-5-
benzamidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(2,6-
dichlorophenyl)-5-benzamidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-phenyl-5-valeramidooxazole;
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-phenyl-5-valeramidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-phenyl-5-valeramidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-phenyl-5-
valeramidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-phenyl-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-phenyl-5-valeramidooxazole;
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-phenyl-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-phenyl-5-valeramidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-phenyl-5-
valeramidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-phenyl-5-
valeramidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-thiophenyl-5-valeramidooxazole;
2-(2-hydroxy-(4-(3-vitro)-phenyl))-phenyl-4-thiophenyl-5-
valeramidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-thiophenyl-5-valeramidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-thiophenyl-5-
valeramidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-thiophenyl-5-
valeramidooxazole;
-26-
CA 02336848 2001-O1-09
PCT1US99/15200
WO 00/02871
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-thiophenyl-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-thiophenyl-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-thiophenyl-5-
valeramidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-thiophenyl-5-
valeramidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-thiophenyl-5-
valeramidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-(3-pyridyl)-5-valeramidooxazole
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(3-pyridyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-(3-pyridyl)-5-valeramidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(3-pyridyl)-5-
valeramidooxazole;
2-( 2-hydroxy-(4-( 4'-methoxy)-phenyl)-phenyl-4-(3-pyridyl )-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(3-pyridyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3'-methoxy)-phenyl}-phenyl-4-(3-pyridyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(3-pyridyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(3-pyridyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(3-pyridyl)-5-
valeramidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-(3-chlorophenyl)-5-valeramidooxazole;
-27-
CA 02336848 2001-O1-09
WO 00/02871
PCT/US99/15200
2-( 2-hydroxy-( 4-( 3-nitro)-phenyl) )-phenyl-4-( 3-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-(3-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(3-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(3-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(3-chlorophenyl)-5-
valeramidooxazole;
2-( 2-hydroxy-( 4-( 3' -methoxy)-phenyl )-phenyl-4-( 3-chlorophenyl )-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(3-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(3-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(3-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-(2-chlorophenyl)-5-valeramidooxazole;
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(2-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-(2-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(2-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(2-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(2-chlorophenyl)-5-
valeramidooxazole;
-28-
CA 02336848 2001-O1-09
WO 00/02871
PCTNS99/15200
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-(2-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(2-chlorophenyl)-5-
valeramidooxazole;
2-( 2-hydroxy-(4-( 2-naphthyl )-phenyl)-phenyl-4-( 2-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(2-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-(4-chlorophenyl)-5-valeramidooxazole;
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(4-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-(4-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(4-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(4-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(4-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-(4-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(4-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(4-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(4-chlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-(4-trifluoromethylphenyl)-5-
valeramidooxazole;
-29-
CA 02336848 2001-O1-09
WO 00!02871
PCTNS99/15200
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(4-trifluoromethylphenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-(4-trifluoromethylphenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(4-trifluoromethylphenyl)-5-
valeramidooxazole;
2-( 2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(4-trifluoromethylphenyl)-
5-valeramidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(4-trifluoromethylphenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-(4-trifluoromethylphenyl)-
5-valeramidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(4-trifluoromethylphenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(4-trifluoromethylphenyl)-
5-valeramidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(4-
trifluoromethy!phenyl)-5-valeramidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-( 3-methoxyphenyl)-5-valeramidooxazole;
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(3-methoxyphenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-( 1-naphthyl))-phenyl-4-(3-methoxyphenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(3-methoxyphenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(3-methoxyphenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(3-methoxyphenyl)-5-
valeramidooxazole;
-30-
CA 02336848 2001-O1-09
WO 00/02871
PCT/US99/15200
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-(3-methoxyphenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(3-methoxyphenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(3-methoxyphenyl)-5-
valeramidooxazole; '
2-( 2-hydroxy-(4-( 2-trifluoromethyl)-phenyl)-phenyl-4-( 3-methoxyphenyl)-
5-valeramidooxazole;
2-(2-hydroxy-4-phenyl)-phenyl-4-(3-thiophenyl)-5-valeramidooxazole;
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(3-thiophenyl)-5-
valeramidooxazole;
2-( 2-hydroxy-(4-( 1-naphthyl ))-phenyl-4-( 3-thiophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(3-thiophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(3-thiophenyl)-5-
valeramidooxazole;
2-( 2-hydroxy-(4-( 3'-chloro)-phenyl)-phenyl-4-( 3-thiophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-(3-thiophenyl)-5-
valeramidooxazole;
2-( 2-hydroxy-(4-( 3'-fluoro)-phenyl)-phenyl-4-( 3-thiophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(3-thiophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(3-thiophenyl)-5-
valeramidooxazole;
2-( 2-hydroxy-4-phenyl)-phenyl-4-( 2-thiophenyl )-5-valeramidooxazole;
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(2-thiophenyl)-5-
valeramidooxazole;
-31-
CA 02336848 2001-O1-09
WO 00/02871
PCT/US99/15200
2-(2-hydroxy-(4-(1-naphthyl))-phenyl-4-(2-thiophenyl)-5-
valeramidooxazole;
2-( 2-hydroxy-( 4-( 4'-methyl)-phenyl )-phenyl-4-( 2-thiophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(2-thiophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(2-thiophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-( 3'-methoxy)-phenyl)-phenyl-4-(2-thiophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(2-thiophenyl)-5-
valeramidooxazole;
2-( 2-hydroxy-(4-( 2-naphthyl)-phenyl)-phenyl-4-( 2-thiophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(2-thiophenyl)-5-
valeramidooxazole;
2-( 2-hydroxy-4-phenyl)-phenyl-4-( 2, 6-dichlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3-nitro)-phenyl))-phenyl-4-(2,6-dichlorophenyl)-5-
valeramidooxazole;
2-( 2-hydroxy-( 4-( 1-naphthyl))-phenyl-4-( 2,6-dichlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(4'-methyl)-phenyl)-phenyl-4-(2,6-dichlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(4'-methoxy)-phenyl)-phenyl-4-(2,6-dichlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3'-chloro)-phenyl)-phenyl-4-(2,6-dichlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(3'-methoxy)-phenyl)-phenyl-4-(2,6-dichlorophenyl)-5-
valeramidooxazole;
-32-
CA 02336848 2001-O1-09
WO 001028?1
PCT/US99115200
2-(2-hydroxy-(4-(3'-fluoro)-phenyl)-phenyl-4-(2,6-dichlorophenyl)-5-
valeramidooxazole;
2-(2-hydroxy-(4-(2-naphthyl)-phenyl)-phenyl-4-(2,6-dichlorophenyl)-5-
valeramidooxazole and
2-(2-hydroxy-(4-(2-trifluoromethyl)-phenyl)-phenyl-4-(2,6-
dichlorophenyl)-5-valeramidooxazole.
Schemes 1-3 for preparing the novel compounds of this
invention are presented below. The examples which follow the
schemes illustrate the compounds that can be synthesized by these
schemes. The schemes, however, are not limited by the
compounds listed nor by any particular substituents employed for
illustrative purposes. The examples specifically illustrate the
application of the following schemes to specific compounds.
Schemes 1 and 2 demonstrate to generalized protocols
for the preparation of the required oxazoles from nitriles and
diamides respectively. Scheme 3 exemplifies the synthesis of a
lactam substituted compound. The reaction conditions employed
are apparent from the specific examples that follow. Alternative
conditions and protocols would be apparent to those skilled in the
art.
-33-
CA 02336848 2001-O1-09
PCT/US99115200
WO 00/02871
SC
Oxazoles from amide nitriles
R~ O
R1 ~ Coupling
+ R
CN R gen NC ~ z
H2N HO 2 H
1
N
R~ O CH3COCI/MeS03H HN ' O~ R2
NC ~ ~ R2 CH2CI2 O
H
-34-
CA 02336848 2001-O1-09
PCT/US99/15200
WO 00/02$?I
Oxazoles from diamides
piperidine ~' NH2
-- NHFmoc pMF
agent O
NHFmoc
HO~
O R~ O
" NHFmoc ~ NH2
N iC ~ piperidine ~.-- H R
H R1 DMF
O
coupling agent N R TFAA/TFA
2
R C02H ~ H ~ CH2CI2
R~ O
i N
N R3COCI/DIPEA ~ ~ R
HN O
HN O~ R2 solvent
O O R
CFg 3
-indicates a polymeric support
-35-
CA 02336848 2001-O1-09
PCTNS99115200
W O 00/02871
10
R O
O H2N CN
HO / ~ --- NC /
C ~N /
C 'N
C
R C R N -N
N HN ~ O \ ~ \
H2N O \ ~ \
O'
CI
R C R H2N
I N -N I N -N
N O \ ~ \ N O \ ~ \
O O
The amino thiazoles can be prepared as described in
"Reactions of a-amino- and a-acylaminothioamides with
aluminum chloride. Synthesis of some imidazole and thiazole
derivatives." Nyitrai, Jozsef; Lempert, Karoly. Acta Chim.
-36-
CA 02336848 2001-O1-09
PCTNS99115200
WO 00/02871
(Budapest) (1972), 73(1), 43-61, or "Cyclization of w-chloro-co-
acylamido acetophenones." Drach,B. S.; Dolgushina, I. Yu.;
Sinitsa, A. D. Inst. Org. Khim., Kiev, USSR. Khim.
Geterotsikl. Soedin. (1974), (7)~ 928-31. Conversion of the
aminothiazoles to the lactam thiazoles can proceed in a similar
manner described above for the corresponding amino oxazoles.
The invention described herein includes a
harmaceutical composition which is comprised of a compound of
P
formula I or a pharmaceutically acceptable salt, hydrate or
prodrug thereof in combination with a carrier. As used herein the
terms "pharmaceutically acceptable salts" and "hydrates" refer to
those salts and hydrated forms of the compound which would be
apparent to the pharmaceutical chemist, i.e., those which
favorably affect the physical or pharmacokinetic properties of the
compound, such as solubility, palatability, absorption, distribution,
metabolism and excretion. Other factors, more practical in
nature, which are also important in the selection, are the coat of
the raw materials, ease of crystallization, yield, stability, solubility,
hygroscopicity and flowability of the resulting bulk drug.
When a compound of formula I is present as a salt or
hydrate which is non-pharmaceutically acceptable, this can be
converted to a salt or hydrate form which is pharmaceutically
acceptable in accordance with the present invention.
When the compound is negatively charged, it is
balanced by a counterion, e.g., an alkali metal cation such as
sodium or potassium. Other suitable counterions include calcium,
magnesium, zinc, ammonium, or alkylammonium cations such
as tetramethylammonium, tetrabutylammonium, choline,
triethylhydroammonium, meglumine, triethanol-
hydroammonium, etc. An appropriate number of counterions is
-37-
CA 02336848 2001-O1-09
WO 00/02871
PCT/US99/15200
associated with the molecule to maintain overall charge neutrality.
Likewise when the compound is positively charged, e.g.,
protonated, an appropriate number of negatively charged
counterions is present to maintain overall charge neutrality.
Pharmaceutically acceptable salts also include acid
addition salts. Thus, the compound can be used in the form of
salts derived from inorganic or organic acids or bases. Examples
include acetate, adipate, alginate, aspartate, benzoate,
benzenesulfonate, bisulfate, butyrate, citrate, camphorate,
camphorsulfonate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,
glycerophosphate, hemisulfate, heptanoate, hexanoate,
hydrochloride, hydrobromide, hydroiodide, 2-hydroxy-
ethanesulfonate, lactate, maleate, methanesulfonate, 2-
naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate,
persulfate, 3-phenylpropionate, picrate, pivalate, propionate,
succinate, tartrate, thiocyanate, tosylate and undecanoate. Base
salts include ammonium salts, alkali metal salts such as sodium
and potassium salts, alkaline earth metal salts such as calcium
and magnesium salts, salts with organic bases such as
dicyclohexylamine salts, N-methyl-D-glucamine, and salts with
amino acids such as arginine, lysine, and so forth. Also, the basic
nitrogen-containing groups may be quaternized with such agents
as lower alkyl halides, such as methyl, ethyl, propyl, and butyl
chloride, bromides and iodides; dialkyl sulfates like dimethyl,
diethyl, dibutyl; and diamyl sulfates, long chain halides such as
decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides,
aralkyl halides like benzyl and phenethyl bromides and others.
Other pharmaceutically acceptable salts include the sulfate salt
ethanolate and sulfate salts.
-38-
CA 02336848 2001-O1-09
W O 00/02871
PCTNS99/15200
The compounds of the present invention, may have
asymmetric centers and occur as racemates, racemic mixtures
and as individual diastereomers, or enantiomers with all isomeric
forms being included in the present invention. When any variable
(e.g., aryl, heteroaryl, Rl, etc)occurs more than one time in any
constituent or in Formula I, its definition on each occcurence is
independent of its definition at every other occurrence, unless
otherwise stated.
The compounds of the invention can be formulated in
a pharmaceutical composition by combining the compound with a
pharmaceutically acceptable carrier. Examples of such
compositions and carriers are set forth below.
The compounds may be employed in powder or
crystalline form, in solution or in suspension. They may be
administered orally, parenterally (intravenously or
intramuscularly), topically, transdermally or by inhalation.
Thus, the carrier employed may be, for example,
either a solid or liquid. Examples of solid carriers include lactose,
terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium
stearate, stearic acid and the like. Examples of liquid carriers
include syrup, peanut oil, olive oil, water and the like. Similarly,
the carrier for oral use may include time delay material well
known in the art, such as glyceryl monostearate or glyceryl
distearate alone or with a wax.
Topical applications may be formulated in carriers such as
hydrophobic or hydrophilic bases to form ointments, creams, lotions, in
aqueous, oleaginous or alcoholic liquids to form paints or in dry diluents
to form powders. Such topical formulations can be used to treat ocular
diseases as well as inflammatory diseases such as rheumatoid arthritis,
-39-
CA 02336848 2001-O1-09
PCTNS99/15200
WO 00/02871
psoriasis, contact dermatitis, delayed hypersensitivity reactions and the
like.
Examples of oral solid dosage forms include tablets,
capsules, troches, lozenges and the like. The size of the dosage form will
vary widely, but preferably will be from about 25 mg to about 500mg.
Examples of oral liquid dosage forms include solutions, suspensions,
syrups, emulsions, soft gelatin capsules and the like. Examples of
injectable dosage forms include sterile injectable liquids, e.g., solutions,
emulsions and suspensions. Examples of injectable solids would
include powders which are reconstituted, dissolved or suspended in a
liquid prior to injection.
In injectable compositions, the carrier is typically
comprised of sterile water, saline or another injectable liquid, e.g.,
peanut oil for intramuscular injections. Also, various buffering
agents, preservatives and the like can be included.
For the methods of treatment disclosed herein,
dosages can be varied depending upon the overall condition of the
patient, the nature of the illness being treated and other factors.
An example of a suitable oral dosage range is from about 0.1 to
about 80 mg/kg per day, in single or divided doses. An example of
a suitable parenteral dosage range is from about 0.1 to about 80
mg/kg per day, in single or divided dosages, administered by
intravenous or intramuscular injection. An example of a topical
dosage range is from about 0.1 mg to about 150 mg, applied
externally from about one to four times a day. An example of an
inhalation dosage range is from about 0.01 mg/kg to about 1 mg/l;g
per day.
The compounds may be administered in conventional dosages as a
single agent or in combination with other therapeutically active
compounds.
-40-
CA 02336848 2001-O1-09
WO 00/02871
EXAMPLE 1
PCT/US99/15200
2 (2 (3 hvdroxv)nanthvl) 4 nhenvl-5-trifluoracetamidooxazole
Ph
N
HN
p I \ \
CF3 HO / /
4
Step 1
O ~ O
Ph
NC~ NH2 OH I \ \ NC ~ I \ \
HCI ~ HO / / ~ HO / /
3
1 2
2-amino-2-phenylacetonitrile hydrochloride (1) (3.0 g,
l8mmol) and 2- hydroxy-3-napthoic acid (2) (5.1 g, 27mmo1) were
dissolved in 50mL of dimethylformamide. To this solution was added
benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate
(14 g, 27mmo1) and diisopropylethylamine (14 ml, 8lmmol) and the
reaction allowed to proceed with stirring overnight. The reaction was
then concentrated and the oil taken up into ethylacetate and washed
with water (3X), NaCl solution and the organics dried over sodium
sulfate. Flash chromatography provided the desired product (3).
1H NMR (CDC13) d 7.95 (s, 1H), 7.24-7.7 (m, 10H), 7.15 (brd, 1H), 6.38 (d,
1H).
-41-
CA 02336848 2001-O1-09
PCTNS99115200
WO 00/02871
SteQ2_
The amide nitrite (3) from above (1.9 g, 6.3mmo1) was
treated with trifluoraceticanhydride (8.9 ml, 63mmo1) in dichloroethane
containing 2% trifluoracetic. After 30 minutes the reaction was
concentrated. Residue was taken up into toluene and concentrated,
rocess was repeated to furnish slightly impure (4) which was used in
P
the next step without further purification. 1H NMR (CDCl3) d 8.4 (s, 1H),
7.7-7.9 (m, 4H), 7.3-7.5 (m, 6H), 3.0 (brs, 1H).
EXAMPL 2
P
N
H
CH3 HO
5
The 2-(2-(3-hydroxy)napthyl)-4-phenyl-5-
trifluoracetamidooxazole (4) from above (2.5g, 6.3mmmol) was dissovled
in toluene (65m1) and treated sequentially with diisopropylethylamine
(2.2m1, 12.6mmo1) and acetylchloxide (0.90m1, 12.6mmo1) and stirred at
room temperature for 2 hours. The reaction was diluted with
Ethylacetate and washed with H20 (2X), NaCI and dried over Na2S04.
After recrystallization from MeOHIEtOAc a white solid was obtained.
1H NMR (DMSO-d6) d 8.5 (s, 1H), 8.2 (d, J = 8.2Hz, 1H), 7.83 (m, 3H), '1.38-
7.54 (m, 6H), 2.18 (brs, 3H). MS (M++1) 345.
-42-
CA 02336848 2001-O1-09
PCTNS99/15200
WO 00/02871
ExAIVIPLE 3
2-(2-(3-hydroxy)napthyl)-4-(3-thiophenyl)-5-trifluoroacetamido-
ox z 1
8
,tepl
2-amino-2-(3-thiophenyl) acetonitrile hydrochloride
N~
CHO HC~ NH2
6
Sodium cyanide (3.77g, 'l7mmol) was dissolved in
water (40m1) followed by ammonium chloride (4.538, 84.7mmo1).
Thiophene-3-carboxaldehyde (Aldrich, 8.66g, 77mmol) was
dissolved in MeOH (60m1) and added via addition funnel to the
rapidly stirring solution in a steady stream. The reaction was
-43-
CA 02336848 2001-O1-09
PCTNS99I15200
WO 00102871
allowed to proceed at room temperature overnight. The reaction
was diluted with saturated NaHC03 and extracted with ,
diethylether (3X100m1). Organics were combined and washed with
saturated NaCl and dried over Na2S04. Organics were
concentrated and the in a minimum amount of ether and filtered.
The filtrate was satured with HCl gat at 0°C. The solids that
formed were filtered and washed with ether to give after drying a
yellow solid (6) which was used without further purification.
1H NMR (DMSO-dg) d 9.8 (brs,3H), 7.91 (m, 1H), 7.70 (m, 1H), 7.43
(m, 1H), 6.04 (s, 1H).
Std S
~O
S O
NC
NC I ~ OH w w ~ ~ / /
I / / --~,- HO
HCI NH2 HO
6
The 2-amino-2-(3-thiophenyl) acetonitrile hydrochloride (6)
(1.6 , 9.17 mmol) was dissolved in DMF (90m1) from above was treated
g
with 2- hydroxy-3-napthoic acid (2.588, 13.8mmo1), benzotriazol-1-yloxy-
tri yrrolidinophosphonium hexafluorophosphate (7.2 g, 13.8mmo1), 1-
P
h droxybenzotriazole (1.9g, 13.8mmol) and diisopropylethylamine (7 ml,
Y
40mmol) the reaction was stirred overnight at room temperature. After
l8hours the DMF was removed, the residue was taken up into EtOAc
and washed successively with 1N HCI, H20, saturated NaHC03 and
saturated NaCI. After purification by chromatography, product (7) was
isolated.
-44-
CA 02336848 2001-O1-09
WO 00/02871
PCT/US99/15200
1H NMR (CDCl3) d 10.8 ( s, 1H), 7.99 (s, 1H), 7.64-7.76 (m, 3H), 7.47-7.53
(m, 2H), 7.22-7.36 (m, 4H), 7.04 (brd, J = 7.7 Hz, 1H), 6.49 (d, J = 8 Hz,
1H).
Ste
The amide nitrile (7) from above (280mg, 0.93mmo1) was
dissolved in CH2Cl2 and treated with trifluoraceticanhydride (2m1) and
trifluoracetic (0.5m1). The reaction was allowed to proceed for 4 hours at
which point it was concentrated. The solids that remained were filtered
with the aid of CH2C12 and washed further with CH2Cl2 to give the
desired product.
1H NMR (CDC13) d 10.7 (s, 1H), 8.37 (s, 1H), 7.96 (brs, 1H), 7.82 (d, J= 8.4
Hz, 1H), 7.72 (d, J = 8.3 Hz, 1H), 7.68 (m, 1H), 7.3-7.52 (m, 6H).
EXAMPLE 4
2-(2-(3-h Iroxv)narthvl)-4-(3-thioDhen'~1)-5-a~Ptamidooxazole
S
N
~ of \ \
O ~ ,
CH3 HO
9
The oxazole (8) (271mg, 0.67mmol) from above was
suspended in toluene (lOml) and treated with acetyl chloride (0.14m1,
2.Olmmol), and diisopropylethylamine (0.35m1, 2.Olmmol). The reaction
was allowed to stir overnight at room temperature. Reaction was diluted
with ethylacetate and washed with saturated NaHC03 and saturated
-45-
CA 02336848 2001-O1-09
WO 00/02$71 PCT/US99/15200
NaCI and dried over MgS04. The crude product was suspended in
MeOH and treated with 1N NaOH (3m1) for l5minutes. Cloudy solution
was filtered and the filtrate was neutralized with 1N HCI. The solids
that formed were filtered washed with MeOH and dried under vacuum
to yield the desired product (9). HRMS (M++1) found 351.0803 calculated
351.0803 for C1gH15N203S.
EXAMPLE 5
1 f2 (2 Amino auinolin 3 vl) 4 phenvl-oxazol-5-vll-nvrrolidin-2-one
O
N
O
H2N N
Ste
2 chloro-auinoline-3-carboxvlic acid
/ N CI
OH
O
-46-
CA 02336848 2001-O1-09
WO 00/02871 PCTNS99/15200
To a cold solution LDA (60mL, 120mmol, 2M solution) in THF
(400mL) was added 2-chloroquinoline in THF(100mL) at such a rate to
maintain temperature <?OoC. The reaction was stirred for 2 hours at
which point C02 was bubbled through the solution until the internal
temperature reached -78oC (-69oC to -?8oC). The reaction was then
allowed to gradually warm to room temperature overnight. After
concentration to dryness, the residue taken up into diethylether and
water. The layers were then separated, the aqueous phase acidified
with 6N HCl and the solids collected. This material was used without
further purification.
St_ ep 2
N CI
N CN
I I
O
f9luinoline acid from above was suspended in CH2C12 (100mL) and cooled
to -lOoC. biphenyl phosphinic chloride was then added followed by
dropwise addition of EtgN. The reaction was allowed to proceed with
warming to OoC for one hour. The amino nitrile, suspended in CH2Cl2
(50mL) containing EtgN (1 equiv.), was added to the solution and reaction
stirred overnight with warming to room temperature. The reaction was
then concentrated to a semisolid and the residue partitional between
EtOAc and water. The layers were separated and the organics washed
-47-
CA 02336848 2001-O1-09
WO 00/02871 PCTNS99/15200
with 0.5N HCl, water, NaHCOg (sat) water, and dried (MgS04). After
the volatiles had been removed, NMR indicated that starting material
remained in the residue. Therefore, the solids were stirred in NaHCOg
(sat). The solids were then refiltered and taken up into EtOAc, washed
with water, and dried (MgS04). The desired material was obtained by
flash chromatography (10% EtOAc/CH2C12).
Step 3
N-f2-(2-Chloro-auinolin-3- ly )-4-,phenyl-oxazol 5 yll 2 2 2 trifluoro
acetamide
-C F3
-I
Amide nitrile from Step 2 was dissolved in a mixture of
CH2C12/trifluoroacetic anhydride/trifluoroacetic acid (58/40/2) and
stirred at room temperature overnight. The reaction was then
concentrated and the residue taken up into EtOAc. This EtOAc solution
was then washed with water, aqueous NaHC03 (sat) and brine. The
organics were dried with MgS04 and concentrated to afford the desired
product as a solid that was used without further purification.
-48-
CA 02336848 2001-O1-09
WO 00/02871 PCTNS99/15200
Step 4
4- Chloro-N-f2-(2-chloro-quinolin-3=yl)-4-phenvl-oxazol-5- l~tyramide
~N CI
O
O
N / N
H
CI
The requisite oxazole was dissolved in CH2C12 {100mL) and treated with
4-chloro-butyrl chloride and diisopropylethyl amine. After 3 hours of
reflex, TLC indicated that starting material still present. An additional
eqiuvalent of acid chloride and base were added and the reaction
refluxed for an additional two hours. The reaction was then cooled to
room temperature and concentrated to a yellow gum. The residue was
taken up into EtOAc and water. The layers were then separated and the
organics washed with aqueous NaHCOg (sat), brine, and dried (MgS04).
Flash LC (5% EtOAc/ CH2Cl2) gave the desired product.
Step 5
1-[2-(2-chloro-auinolin-3-vl, )~4-nhenvl-oxazol-5=yll ~vrrolidin 2 one
-49-
CA 02336848 2001-O1-09
WO 00/02871 PCTNS99/15200
N CI
O
O
N
The amide was dissolved in CHC13/Et3N (1:1, 30mL) and heated to reflux
overnight. The reaction was then concentrated to a yellow gum and the
residue taken up into EtOAc and water. The layers were then seperated
and the organics washed with brine and dried (MgS04}. The material
was used without further purification.
Step 6
1-12-(2-Amino-auinolin-3-vl)-4- henyl-oxazol-5-yll-pyrrolidin-2-one
The chloroquinoline derivative from above was suspended in NH3 (1) in a
glass bomb. The cap was placed on the vessel and the mixture heated to
80oC overnight. The reaction was cooled to -78oC and contents poured
into a beaker to facilitate evaporation of the NH3 (1). The solid residue
-50-
CA 02336848 2001-O1-09
WO 00/02871 PGT/US99/15200
that remained was partitioned between EtOAc and water. The organics
were washed with brine and dried (MgS04). Flash LC (20010
EtOAc/CH2C12) gave the desired product plus an amount of recovered
starting material.
EXAMPLE 6
1 f4 Phenvl-2-(5-thiophen-3-Yl_-nvridin-3- ~Ll)-oxazol-5-vli-nvrrolidin-2-one
~P 1
5-Thiophen-3~~1-nicotinic acid methyl ester
N
i
S \ ~ ~ O
v
OCH3
Methyl-3-bromo-nicotinate and 3-thiophene boronic acid were dissolved
in degassed dioxane (25mL). To the homogeneous solution was added
Pd((Ph3)4P)). The reaction was heated to 90oC for 18 hours and then
-51-
CA 02336848 2001-O1-09
WO 00/02871 PCT/US99/15200
cooled to room temperature and concentrated. The residue was taken up
into EtOAc and water. The layers were then separated and the organics
washed with brine and dried (MgS04). Flash LC (60% Hexanes/EtOAc)
gave the desired product.
Step 2
5-Thiophen-3 yl-nicotinic acid
N
S ~ ~ ~ OH
il
O
The ester from above was suspened in MeOH (20mL), treated with 1N
NaOH and stirred at room temperature for 1 hr. The reaction was then
concentrated to dryness and the residue dissolved in water and
neutralized with 1N HCl. The solids were filtered and dried over P205 at
50oC for 10 hours. This material was used without further purification.
Ste~3
N (Cyano nhen~rl methyl)- 5-thionhen-3-yl-nicotinamide
- 52-
CA 02336848 2001-O1-09
WO 00/02871 PC'TNS99/15200
N
O
_/
HN CN
The acid from above and the benzyl amino nitrile were treated with EDC-
HCl and HOAt in DMF ( lOmL). The reaction was allowed to proceed
overnight at room temperature. The reaction was then concentrated
and the residue taken up into EtOAc and water. The layers were
separated and the organics washed with aqueous NaHC03 (sat), water,
and dried (MgS04). Flash LC (50% CH2C12/EtOAc) gave the desired
product.
Step 4
4 Phenyl 2 (5 thionhen-3-vl-p,~~din-3-vl)-oxazol-5-vlamine
The amide nitrite (0.064mg, 0.2mmo1) was partially dissolved in
dichlorethane (5mL). MeS03H (0.039mL, 0.6mmo1) was then added and
the reaction allowed to proceed overnight at room temperature. The
reaction was diluted with CH2C12 and extracted with aqueous NaHC03
-53-
CA 02336848 2001-O1-09
WO 00/02871 PCT/US99/15200
(sat), water and then dried (MgS04). The desired product was isolated
by flash LC (40% EtOAc/ CH2Cl2) to give a pale yellow solid. The
material was triturated with Et20, filtered and dried over P205.
Step 5
4-Chloro-N-f4-Phen~-2-(5-thiophen-3-3rl-pyridin-3-Yl)-oxazol-5-vll-
butvramide
~~CI
Amino oxazole (319mg, l.Ommo1) from above was suspended in CHClg
(75mL) and treated with DMAP (24mg, 0.02mmo1), Et3N (0.280mL,
2.Ommol), and 4-chloro-butyrlchloride (0.22mL, 2.Ommo1). The reaction
was then heated to 80oC for 6hr wherein TLC indicated starting material
still present. Two equivalents of acid chloride and Et3N were then added
and the reaction heated for an additional two hours. The reaction was
then cooled to room temperature and concentrated to dryness. The
residue was partitioned between EtOAc and NaHCOg. The layers were
then separated and organics washed with water and dried over MgS04.
Flash LC with CH2C12 followed by 2% EtOAc/CH2C12 yield the product
as a yellow gum which was used directly in the next step.
Step 6
1-f 4-Phenyl-2-(5-thiophen-3-y-1-~~ridin-3-yl)-oxazol-5-vll-pyrrolidin-2-one
-54-
CA 02336848 2001-O1-09
WO 00/02871 PCT/US99115200
7
Chloro amide from above (0.38g, 0.896mmo1) was treated with Et3N
(5mL) and a small amount of CHCl3 until homogeneous. The reaction
was heated to 90oC for 2hr. The reaction was then cooled to room
temperature and concentrated to dryness. The residue was partitioned
between EtOAc and aqueous HCl (0.5N). The organics were washed
further with water, brine, and dried (MgS04). Flash LC 5%EtOAc/
CH2Cl2 gave an off white solid that was triturated with diethylether and
dried over P205.
EXAMPLE 7
1 (2 (HvdroxX, 5 methoxv phenyl) 4-phenyl-oxazol-5-vll-nvrrolidin-2-one
This compound was prepared in a manner analogous to that described
above.
-55-
CA 02336848 2001-O1-09
WO 00/02871 PCT/US99/15200
Kinase inhibition is demonstrated in accordance with
the following protocol.
VEGF RECEPTOR HINASE ASSAY
VEGF receptor kinase activity is measured by
incorporation of radio-labeled phosphate into polyglutamic acid,
tyrosine, 4:1 (pEY) substrate. The phosphorylated pEY product is
trapped onto a filter membrane and the incorporation of radio-
labeled phosphate quantified by scintillation counting.
MATERIALS
VEGF receptor kinase
The intracellular tyrosine kinase domains of human
KDR (Terman, B.I. et al. Oncogene (1991) vol. 6, pp. 1677-1683.) and
Flt-1 (Shibuya, M. et al. Oncogene ( 1990) vol. 5, pp. 519-524) were
cloned as glutathione S-transferase (GST) gene fusion proteins.
This was accomplished by cloning the cytoplasmic domain of the
KDR kinase as an in frame fusion at the carboxy terminus of the
GST gene. Soluble recombinant GST-kinase domain fusion
proteins were expressed in Spodoptera frugiperda (SfZl) insect
cells (Invitrogen) using a baculovirus expression vector {pAcG2T,
Pharmingen).
Lvsis buffer
50 mM Tris pH 7.4, 0.5 M NaCl, 5 mM DTT, 1 mM
EDTA, 0.5% triton X-100, 10 % glycerol, 10 mg/ml of each
leupeptin, pepstatin and aprotinin and 1mM phenylmethylsulfonyl
fluoride (all Sigma).
-56-
CA 02336848 2001-O1-09
WO 00/02871 PCT/US99/15200
Wash buffer
50 mM Tris pH 7.4, 0.5 M NaCI, 5 mM DTT, 1 mM
EDTA, 0.05% triton X-100, 10 % glycerol, 10 mg/ml of each
leupeptin, pepstatin and aprotinin and 1mM phenylmethylsulfonyl
fluoride.
Dialysis buffer
50 mM Tris pH 7.4, 0.5 M NaCl, 5 mM DTT, 1 mM
EDTA, 0.05% triton X-100, 50 % glycerol, 10 mg/ml of each
Ieupeptin, pepstatin and aprotinin and 1mM phenylmethylsuflonyl
fluoride.
10 X reaction buffer
200 mM Tris, pH 7.4, 1.0 M NaCI, 50 mM MnCl2, 10
mM DTT and 5 mg/ml bovine serum albumin (Sigma).
Enzyme dilution buffer
50 mM Tris, pH 7.4, 0.1 M NaCI, 1 mM DTT, 10 %
glycerol, 100 mg/ml BSA.
10 X Substrate
750 ~.g/ml poly (glutamic acid, tyrosine; 4:1) (Sigma).
Stop solution
30% trichloroacetic acid, 0.2 M sodium pyrophosphate
(both Fisher).
Wash solution
15% trichloroacetic acid, 0.2 M sodium pyrophosphate.
Filter~lates
Millipore #MAFC NOB, GF/C glass fiber 96 well plate.
-57-
CA 02336848 2001-O1-09
WO 00/02871 PCT/US99/15200
METHOD
A . Protein purification
1. Sf21 cells were infected with recombinant virus
at a multiplicity of infection of 5 virus particles/ cell and grown at
27 °C for 48 hours.
2. All steps were performed at 4°C. Infected cells
were harvested by centrifugation at 1000 X g and lysed at 4 °C for 30
minutes with 1/10 volume of lysis buffer followed by centrifugation
at 100,000Xg for 1 hour. The supernatant was then passed over a
glutathione Sepharose column (Pharmacia) equilibrated in lysis
buffer and washed with 5 volumes of the same buffer followed by 5
volumes of wash buffer. Recombinant GST-KDR protein was
eluted with wash buffer/10 mM reduced glutathione (Sigma) and
dialyzed against dialysis buffer.
B. VEGF receptor kinase assay
1. Add 5 ~.1 of inhibitor or control to the assay in 50%
DMSO.
2. Add 35 ~,l of reaction mix containing 5 pl of 10 X
reaction buffer, 5 ~.1 25 mM ATP/10 p.Ci [33P]ATP (Amersham),
and 5 ~.1 10 X substrate.
3. Start the reaction by the addition of 10 ~.l of KDR (25
nM) in enzyme dilution buffer.
4. Mix and incubate at room temperature for 15
minutes.
5. Stop by the addition of 50 ~,1 stop solution.
6. Incubate for 15 minutes at 4°C.
7. Transfer a 90 ~.1 aliquot to filter plate.
-58-
CA 02336848 2001-O1-09
WO 00/02871 PCT/US99/15200
8. Aspirate and wash 3 times with wash solution.
9. Add 30 ~,1 of scintillation cocktail, seal plate and
count in a Wallac Microbeta scintillation counter.
Human Umbilical Vein Endothelial Cell Mito~enesis Assav
Expression of VEGF receptors that mediate mitogenic
responses to the growth factor is largely restricted to vascular
endothelial cells. Human umbilical vein endothelial cells
(HUVECs) in culture proliferate in response to VEGF treatment
and can be used as an assay system to quantify the effects of KDR
kinase inhibitors on VEGF stimulation. In the assay described,
quiescent HUVEC monolayers are treated with vehicle or test
compound 2 hours prior to addition of VEGF or basic fibroblast
growth factor (bFGF). The mitogenic response to VEGF or bFGF is
determined by measuring the incorporation of [3H]thymidine into
cellular DNA.
Materials
HUVE s
HUVECs frozen as primary culture isolates are
obtained from Clonetics Corp. Cells are maintained in Endothelial
Growth Medium (EGM; Clonetics) and are used for mitogenic
assays at passages 3-7.
Culture Plates
NUNCLON 96-well polystyrene tissue culture plates
(NUNC #167008).
Assav Medium
-59-
CA 02336848 2001-O1-09
WO 00/02871 PCTNS99/15200
Dulbecco's modification of Eagle's medium containing
1 g/ml glucose (low-glucose DMEM; Mediatech) plus 10% (v/v) fetal
bovine serum (Clonetics).
Test Compounds
Working stocks of test compounds are diluted serially
in 100% dimethylsulfoxide (DMSO) to 400-fold greater than their
desired final concentrations. Final dilutions to 1X concentration
are made directly into Assay Medium immediately prior to
addition to cells.
_10X Growth factors
Solutions of human VEGF165 (500 ng/ml; R&D
Systems) and bFGF ( 10 ng/ml; R&D Systems) are prepared in
Assay Medium.
~0X f~ilThvmidine
[Methyl-3H]Thymidine (20 Ci/mmol; Dupont-NEN) is
diluted to 80 uCi/ml in low-glucose DMEM.
Cell Wash Medium
Hank's balanced salt solution (Mediatech) containing
1 mg/ml bovine serum albumin (Boehringer-Mannheim).
Cell L~sis Solution
1 N NaOH, 2% (w/v) Na2C03.
Method
1. HLTVEC monolayers maintained in EGM are
harvested by trypsinization and plated at a density of 4000 cells per
- 60 -
CA 02336848 2001-O1-09
PCT/US99/15200
WO 00/02871
100 ul Assay Medium per well in 96-well plates. Cells are growth-
arrested for 24 hours at 37°C in a humidified atmosphere
containing 5% C02.
2, Growth-arrest medium is replaced by 100 ul Assay
Medium containing either vehicle (0.25% [v/v] DMSO) or the
desired final concentration of test compound. All determinations
are performed in triplicate. Cells are then incubated at 37°C/5%
C02 for 2 hours to allow test compounds to enter cells.
3, After the 2-hour pretreatment period, cells are
stimulated by addition of 10 uUwell of either Assay Medium, 10X
VEGF solution or lOX bFGF solution. Cells are then incubated at
37°C/5% C02.
4, After 24 hours in the presence of growth factors, 10X
[3H]Thymidine (10 uUwell) is added.
5. Three days after addition of [3H]thymidine, medium is
removed by aspiration, and cells are washed twice with Cell Wash
Medium (400 ul/well followed by 200 ul/well). The washed,
adherent cells are then solubilized by addition of Cell Lysis Solution
( 100 ul/well) and warming to 37°C for 30 minutes. Cell lysates are
transferred to 7-ml glass scintillation vials containing
150 ul of water. Scintillation cocktail (5 ml/vial) is added, and cell-
associated radioactivity is determined by liquid scintillation
spectroscopy.
Based upon the foregoing assays the compounds of
formula I are inhibitors of VEGF and thus are useful for the
inhibition of neoangiogenesis, such as in the treatment of occular
disease, e.g., diabetic retinopathy and in the treatment of cancers,
e.g., solid tumors. The instant compounds inhibit VEGF-
-61-
CA 02336848 2001-O1-09
WO 00/02871
PCTNS99/15200
stimulated mitogenesis of human vascular endothelial cells in
culture with IC50 values between 0.01 - 5.0 ~.M. These compounds
also show selectivity over related tyrosine kinases (e.g. FGFRl and
the Src family).
-62-
