Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
NOVEL ANGIOGENESIS INHIBITORS
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,
atherosclerosis, diabetic retinopathy or inflammatory diseases, in
mammals.
Tyrosine kinases are a class of enzymes that catalyze
the transfer of the terminal phosphate of adenosine triphospate 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.
2o Accordingly, inhibitors of these tyrosine kinases are useful for the
prevention and treatment chemotherapy of proliferative diseases
dependent on these enzymes.
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-I. 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- I
appears to modulate non-mitogenic functions such as those
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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. Monoclonal anti-VEGF antibodies inhibit the growth of
human tumors in nude mice. Although these same 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, 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
3o 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
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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 are useful in the treatment
of diseases in which neoangiogenesis is part of the overall
pathology, e.g., diabetic retinal vascularization, as well as various
forms of cancer.
Cancers which are treatable in accordance with the
present invention demonstrate high levels of gene and protein
expression. Examples of such cancers include 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.
SUMMARY OF THE INVENTION
A compound is disclosed in accordance with formula
I:
R3
R4 N
\ N/
R2
Rs N
R~
I
or a pharmaceutically acceptable salt, hydrate or prodrug thereof,
wherein
R, is H, C~_,o alkyl, C3_6 cycloalkyl, CS_,o aryl, halo, OH, C3_
,o heterocyclyl, or CS_~o heteroaryl; said alkyl, alkenyl,
alkynyl, aryl, heteroaryl and heterocyclyl being
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optionally substituted with from one to three members selected
from Ra;
RZ&R3 are independently H, C,_6 alkyl, CS_,o aryl, C3-6 cycloalkyl,
OH, N02, -NHS, or halogen;
R4 is H, C ~ _ ~ o alkyl, C3_6 cycloalkyl, C, _6 alkoxy C2_ I o
alkenyl, CZ_~o alkynyl, CS_,o aryl, C3_lo heterocyclyl, C,_6
alkoxyNR7Rg, N02, OH, -NH2 or CS_lo heteroaryl, said
alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclyl
being optionally substituted with from one to three
members selected from Ra;
RS is H, or C,_6 alkyl, OR, halo, NH2 or N02;
Ra is H, C,_lo alkyl, halogen, N02, OR, -NR~ NR7Rg~ R7Rb
CS_~o aryl, CS_~o heteroaryl or C3_~o heterocyclyl,
2o R is H, or C1_6 alkyl; and
R~&Rg are independently H, C1_,o alkyl, C3-6 cycloalkyl, COR,
COOR, COO-, CS_,o aryl, C3_~o heterocyclyl, or CS_~o
heteroaryl or NR~Rg 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.
Also disclosed is a pharmaceutical composition which
is comprised of a compound represented by the formula I:
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R3
/N
N
R2
~"i5 N
Ri
wherein Rl, R2, R3, R4 and RS 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.
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.
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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 I O 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.
Alkyl also includes a straight or branched alkyl group
which contains or is interrupted by a cycloalkylene portion.
Examples include the following:
and - (CH2)W ~ (CH2)Z
-(CH2)x U (CH2)y
wherein: x plus y = from 0-10; and w plus z = from 0-9.
The alkylene and monovalent alkyl portions) of the
alkyl group can be attached at any available point of attachment to
the cycloalkylene portion.
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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 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 (non-resonating) carbon-carbon double bonds may be
present. Preferred alkenyl groups include ethenyl, propenyl,
1 o butenyl and cyclohexenyl. As described above with respect to
alkyl, the straight, branched or cyclic portion of the alkenyl group
may 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 as rings which
are fused, e.g., naphthyl and the like. 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, with alternating
{resonating) double bonds between adjacent carbon atoms. 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 naphthyi substituted
with one or two groups.
The term heterocycle, heteroaryl or heterocyclic, as
used herein except where noted, represents a stable 5- to 7-
membered mono- or bicyclic or stable 7- to 10-membered bicyclic
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heterocyclic ring system, any ring of which may be saturated or
unsaturated, and which consists of carbon atoms and from one to
three heteroatoms selected from the group consisting of N, O and
S, and wherein the nitrogen and sulfur heteroatoms may optionally
be oxidized, and the nitrogen heteroatom may optionally be
quaternized, and including any bicyclic group in which any of the
above-defined heterocyclic 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, triazinyl,
thienyl, benzothienyl, thiamorpholinyl sulfoxide, thiamorpholinyl
sulfone, and oxadiazolyl. The term "alkoxy" refers to those groups
of the designated length in either a straight or branched
configuration and if two or more carbon atoms in length, they may
include a double or a triple bond. Exemplary of such alkoxy
groups are methoxy, ethoxy, propoxy, isopropoxy, butoxy,
isobutoxy, tertiary butoxy, pentoxy, isopentoxy, hexoxy, isohexoxy
allyloxy, propargyloxy, and the like.
The term "halogen" is intended to include the halogen
atom fluorine, chlorine, bromine and iodine.
The term "prodrug" refers to compounds which are
drug precursors which, following administration and absorption,
release the drug in vivo via some metabolic process. Exemplary
prodrugs include acyl amides of the amino compounds of this
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inventon such as amides of alkanoic(C~_~)acids, amides of aryl
acids (e.g., benzoic acid) and alkane(C1_6)dioic acids.
Tyrosine kinase dependent diseases or conditions
refers to hyperproliferative disorders which are initiated/maintained
by aberrant tyrosine kinase enzyme activity. Examples include
psoriasis, cancer, immunoregulation (graft rejection),
atherosclerosis, rheumatoid arthritis, angiogenesis (e.g. tumor
growth, diabetic retinopathy), etc.
The compounds of the present invention are in
1 o accordance with formula I:
R3
R4 N
\N/
R2
R5 N
Ri
I
or a pharmaceutically acceptable salt, hydrate or prodrug thereof,
wherein
R~ is H, C,_lo alkyl, C3_6 cycloalkyl, CS_~o aryl, halo, OH, C3_
to heterocyclyl, or CS_~o heteroaryl; said alkyl, alkenyl,
alkynyl, aryl, heteroaryl and heterocyclyl being
optionally substituted with from one to three members selected
from Ra;
R2&R3 are independently H, CI_6 alkyl, CS_lo aryl, C3_g cycloalkyl,
OH, N02, -NH2, or halogen;
R4 is H, C,_,o alkyl, C3_6 cycloalkyl, C~_6 alkoxy C2_lo
alkenyl, CZ_ ~ o alkynyl, CS_ ~ o aryl, C3_, o heterocyclyl, C ~ _6
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alkoxyNR7R~, N02, OH, -NH2 or CS_,o heteroaryl, said
alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclyl
being optionally substituted with from one to three
members selected from Ra;
10
Rs is H, or C ~ _~ alkyl, OR, halo, NH2 or N02;
Ra is H, C1_lo alkyl, halogen, N02, OR, -NR~ NR7Rg~ R7Rg
Cs-to at'Yl, Cs_lo heteroaryl or C3_~o heterocyclyl,
R is H, or C, _ f alkyl; and
R~&Rg are independently H, C,_~o alkyl, C3-( cycloalkyl, COR,
COOR, COO-, Cs_~o aryl, C3_~o heterocyclyl, or Cs_~o
heteroaryl or NR~Rg 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.
A preferred subset of compounds of the present
invention is realized when:
R1 is H, C1_~o alkyl, Cs_lo aryl, C3_,o heterocyclyl, or Cs_lo
heteroaryl; said alkyl, aryl, heteroaryi and heterocyclyl
being optionally substituted with from one to three
members selected from Ra;
R2&R3 are independently H, C,_6 alkyl, C3_6 cycloalkyl, OH, or
halogen;
R4 is H, C1_~o alkyl, C3_6 cycloalkyl, Cs_,o aryl, Cs_lo
heteroaryl, C3_~o heterocyclyl, C1_6 alkoxyNR7R8, N02,
OH, -NH2 or Cs_~o heteroaryl, said alkyl, aryl,
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heteroaryl and heterocyclyl 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:
3-(4-fluorophenyl)-6-(4-pyridyl) pyrazolo(1,5-A)pyrimidine,
3-(3-chlorophenyl)-6-(4-pyridyl) pyrazolo(1,5-A)pyrimidine,
3-(3,4-methylenedioxypheny)-6-(4-pyridyl) pyrazolo(1,5-
A)pyrimidine,
l0 3-(phenyl)-6-(4-pyrimidyl) pyrazolo(1,5-A)pyrimidine,
3-(4-fluorophenyl)-6-(4-pyrimidyl) pyrazolo(1,5-A)pyrimidine,
3-(3-chlorophenyl)-fi-(4-pyrimidyl) pyrazolo(1,5-A)pyrimidine,
3-(3-thienyl)-6-(4-pyrimidyl) pyrazolo(1,5-A)pyrimidine,
3-(3-acetamidophenyl)-6-(4-methylphenyl) pyrazolo(1,5-
A)pyrimidine,
3-{3-thienyl)-f-(4-methylphenyl) pyrazolo(1,5-A)pyrimidine,
3-(phenyl)-6-(4-methoxyphenyl) pyrazolo(1,5-A)pyrimidine,
3-(3-acetamidophenyl)-6-(4-methoxyphenyl)pyrazolo( 1,5--
A)pyrimidine,
3-(3-thienyl)-6-(4-methoxyphenyl) pyrazolo(1,5-A)pyrimidine,
3-(phenyl)-6-(4-methoxyphenyl) pyrazolo(1,5-A)pyrimidine,
3-(4-pyridyl)-6-(4-methoxyphenyl) pyrazolo(1,5-A)pyrimidine,
3-(phenyl)-6-(4-chlorophenyl) pyrazolo(1,5-A)pyrimidine.
3-(4-pyridyl)-6-(4-chlorophenyl) pyrazolo(1,5-A)pyrimidine,
3-(phenyl)-6-(4-methylphenyl) pyrazolo(1,5-A)pyrimidine,
3-(4-pyridyl)-6-(4-methylphenyl) pyrazolo(1,5-A)pyrimidine,
3-(phenyl)-6-(2-pyridyl) pyrazolo(1,5-A)pyrimidine,
3-(4-pyridyl)-6-(2-pyridyl) pyrazolo(1,5-A)pyrimidine,
3-(phenyl)-6-(4-pyrimidyl) pyrazolo(1,5-A)pyrimidine,
3-{4-pyridyl)-6-(4-pyrimidyl) pyrazolo(1,5-A)pyrimidine,
3-(phenyl)-6-(2-pyrazinyl) pyrazolo( 1,5-A)pyrimidine,
3-(4-pyridyl)-6-(2-pyrazinyl) pyrazolo(1,5-A)pyrimidine,
3-(3-pyridyl)-6-(4-methoxyphenyl) pyrazolo(1,5-A)pyrimidine,
3-(phenyl)-6-(4-pyridyl) pyrazolo(1,5-A)pyrimidine,
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3-(3-pyridyl)-6-(4-pyridyl) pyrazolo(1,5-A)pyrimidine,
3-(4 pyridyl)-6-(4-methoxyphenyl) pyrazolo( 1,5-A)pyrimidine,
3-(3-thienyl)-6-(4-methoxyphenyl) pyrazolo{1,5-A)pyrimidine,
3-(3-thienyl)-6-(4-hydroxyphenyl)pyrazolo( 1,5-A)pyrimidine,
3-(3-thienyl)-6-{4-(2-(4-morpholinyl)ethoxy)phenyl) pyrazolo(1,5-
A)pyrimidine,
3-(3-thienyl)-6-{cyclohexyl)pyrazolo (1,S-A}pyrimidine,
3-(bromo)-6-(4-methoxyphenyl) pyrazolo(1,5-A)pyrimidine,
3-(bromo}-6-(4-pyrimidyl) pyrazolo( 1,5-A)pyrimidine,
3-(phenyl)-6-(2-(3-carboxy}pyridyl) pyrazolo(I,5-A)pyrimidine,
and
3-(3-thienyl)-6-(4-pyridyl) pyrazolo(1,5-A)pyrimidine.
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
Schemes 1-3, but Schemes 1-3 are not limited by the compounds
in the tables nor by any particular substituents employed in the
schemes for illustrative purposes. The examples specifically
illustrate the application of the following schemes to specific
compounds.
Scheme 1
Ari ~ N N
O HN~ N EtOH/HOAc
Ar1--~O + H2N~2 80°C Ar2
3
Generally, a method for the preparation of 3,6-diaryl
pyrazolo(1,5-A)pyrimidines comprises mixing a commercially
available malondialdehyde compound (1), with commercially
available aminopyrazole (2) in an alcohol, such as ethanol,
methanol, isopropanol, butanol and the like, said alcohol containing
catalytic quantities of an acid, such as acetic acid, to yield (3),
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wherein Arl and Ar2, respectively, are Rq. and R1 ~ as described
above.
Scheme 2
H
Ary--C O + N' N EtOH/HOAc Are ~ N . N
1
O H2N Br 80°C
Br
1 ~ g
Ar~ .,
Ar1 ~ N Pd(PPh3)4 1 l~ N
+ Ar2-B(OH~ N
Br Na2COg Ar2
Dioxane/ 90°C
8
Scheme 2 depicts a means for making 3,6-diaryl
pyrazolo(1,5-A)pyrimidines when the desired aminopyrazole is not
commercially available. In a like manner to that described in
scheme 1 compound (8) is obtained. Treatment of (8) with a
boronic acid derivative in the presence of a palladium catalyst
provides after workup the desired material (9). Arl and Ar2 are as
described above.
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Scheme 3
0
O OMe Phi Ar~~ N~
Ar ~ + Me2N--C
OMe 115°C
15 16 17
CN H
CN ~~ ~JMe PhCH~ NH2NH~:HC1 H2N '
Me2~ ~ Ar
Ar2 + OMe 115"C 2 NM2 EtOH reflux Ar2
18 16 19 20
H Ari
O EtOH/HOAc , 'N~
H2N 1~ + Ar~~N~ g~oC~ N ~ .
i
Arp Ar2
20 17 21
Scheme 3 ilustrates another method for the preparation
of 3,7 diarylpyrazolo(1,5-A)pyrimidines. The comercially available
ketone (15) and nitrile (18) are treated seperately with
dimethylformamidedimethyLacetal (16) in refluxing toluene to give
products (17) and (19) respectively. Compound (19) is then
treated with hydrazinehydrochloride in refluxing ethanol to give the
aminopyrazole (20). Compounds (17) and (20) and then treated
with catalytic amounts of acetic acid in ethanol as described
previously giving the desired of 3,7 diarylpyrazolo(1,5-
A)pyrimidines (21). ArI and Ar2 are as described above.
The invention described herein includes a
pharmaceutical composition which is comprised of a compound of
formula I or a pharmaceutically acceptable salt or hydrate 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
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and excretion. Other factors, more practical in nature, which are
also important imthe selection, are the cost 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
to 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, triethanolhydroammonium,
etc. An appropriate number of counterions is 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.
- 20 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-
hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-
3o 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
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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.
1 o Other pharmaceutically acceptable salts include the sulfate salt
ethanolate and sulfate salts.
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, heterocyle, 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.
2o 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,
3o 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
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the art, such as glyceryl monostearate or glyeeryl 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, 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 SOOmg. 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/kg
per day.
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The compounds may be administered in conventional
dosages as a single agent or in combination with other
therapeutically active compounds.
EXAMPLE 1
Me0
i N~ N
~N
~N
3-(4 pyridyl)-6-(4-methoxyphenyl) pyrazolo(1,5-A)pyrimidine
A solution of commercially available dialdehyde (4,
12.9 mg, 0.0724 mmol) and aminopyrazole (5, 10.4mg
0.0652mmol) in ethanol was heated at 80°C for 10 hours in a test
tube containing catalytic amounts of acetic acid. The reaction was
cooled to room temperature and the yellow solid was collected by
filtration and the title compound was washed with cold ethanol and
dried (11.7 mg, 60%). Mass Spec (M+l, 303).
Me0 i
_ ~O ' HN'N ~ i N~N
~ + H2N ~ 80°C ~N
.O / ~ / 1
.N .N
4 5
2o E~S:AMPLE 2
N.N
N
~S
.o ~ ~
3-(3-thienyl)-6-(4-methoxyphenyl) pyrazolo(1,5-A)pyrimidine
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Step 1.
A solution of 4 (713 mg, 4.0 mmol) and commercially
availaible 7 (648 mg, 4.0 mmol), discussed above in ethanol (20
mL) was heated at 75°C for 4 h. The resulting white suspension
was as decribed in example 1 for 4 hours, then cooled to 20°C,
filtered, and washed with methanol (3 x 5 mL) to provide 10 as a
white powder (1.07 g, 88%, mp = 168-170°C): 1H NMR (CDC13) ~
8.79 (d, 1 H, J = 2.2 Hz), 8.74 (d, 1 H, J = 2.2 Hz), 8.12 (s, 1
H), 7.51 (d, 2 H, J = 8.8 Hz), 7.05 (d, 2 H, J = 8.8 Hz), 3.88 (s,
3 H).
Me0
O + ~L ~~ EtOH/HOAc ~ N . N
~O H2N' Br gp°C ~N
4 7
Step 2.
A suspension of (10) {250 mg, 0.82 mmol),
thiophene-3-boronic acid (11) (158 mg, 1.24 mmol), and aqueous
sodium carbonate (2 _M, 1 mL) in dioxane (5 mL) was de-gassed
by evacuating and backflushing with argon (3x).
Tetrakis(triphenyl-phosphine) palladium (20 mg, 0.017 mmol) was
added and the reaction mixture was de-gassed again. The argon
filled flask was then submerged in an oil bath pre-heated to 90°C
and was heated at that temperature fox 16 h. After cooling to
20°C, the yellow precipitate which formed was collected by
filtration and- was washed with methanol (3 x 5 mL) to provide the
title compound as a yellow powder (220 mg, 87%, mp = 191-193
°C): ~ H NMR (CDC13) ~ 8.79 (d, 1 H, J = 2.4 Hz), 8.76 (d, 1 H, J
- 2.2 Hz), 8.37 (s, 1 H), 7.90 (dd, 1 H, J = 2.9, 1.3 Hz), 7.70
(dd, 1 H, J = 4.9, 1.2 Hz), 7.54 (d, 2 H, J = 8.8 Hz), 7.43 (d, 1
H, J = 4.9, 2.9 Hz), 7.06 (d, 2 H, J = 8.8 Hz), 3.88 {s, 3H).
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~o ' I
I ~ N.N HaB~OH Pd(PPh~)4 ~ i N-N
~N ~~ + ~ v Na2C0~ -N
Br S Dioxane/ 90°C vs
11
EXAMPLE 3
HO
i N.N
~N
S
5
3-(3-thienyl)-6-(4-hydroxyphenyl)pyrazolo( 1,5-A)pyrimidine
Ethanethiol (30 mg, 36 uL) was added dropwise over
1 min to a suspension of sodium hydride (23 mg, 0.98 mmol) in
10 dry DMF (2 mL) under argon. After 15 min, the compound of
example 2 (50 mg, 0.16 mmol) was added and the reaction mixture
was heated at 150°C for 1.5 h. The resulting brown solution was
cooled, poured into water (25 mL) and washed with ethyl acetate
(2 x 25 mL). The combined organics were dried (Na2S04),
concentrated, and purified by flash chromatography (40%
EtOAc/Hexanes) to give the title compound as a yellow solid [ I 1
mg, 23%, Rf = 0.12 (40% EtOAc/Hexanes)]: 1H NMR (CD30D) b
8.96 (d, 1 H, J = 2.4 Hz), 8.85 (d, I H, J = 2.2 Hz), 8.44 (s, 1 H),
7.94 (dd, 1 H, J = 2.9, i .2 Hz), 7.74 (dd, 1 H, J = 4.9, 1.2 Hz),
7.56 (d, 2 H, J = 8.8 Hz), 7.46 (dd, 1 H, J = 4.9, 2.9 Hz), 6.94 (d,
2 H, J = 8.6 Hz).
,o Ho
I
w ~ N.N NaH w ~ N.N
~N ~ EtSH ~ 'N
DMF
S
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EXAMPLE 4
~N~O /
OJ w I i N.N
~N
S
3-(3-thienyl)-6-(4-(2-(4-morpholinyl)ethoxy)phenyl) pyrazolo(1,5-
A)pyrimidine
A solution of example 3 (11 mg, 0.038 mmol), cesium
carbonate (37 mg, 0.11 mmol), N-(2-chloroethyl)morpholine
hydrochloride (7 mg, 0.11 mmol), and sodium iodide (0.013 mmol)
to in DMF (3 mL) was heated at 60°C under argon for 16 h. The
reaction mixture was then poured into water (25 mL) and washed
with ethyl acetate (2 x 25 mL). The combined organics were dried
(NaZS04), concentrated, and purified by flash chromatography
[50% Hexanes/CHC13(NH3)] to give the title compound as a yellow
solid [10 mg, 65%, mp = 149-151°C, Rf = 0.39 (100%
CHC13(NH3))]: ~H NMR (CDC13) 8 8.77 (d, 1 H, J = 2.2 Hz), 8.75
(d, 1 H, J - 2.2 Hz), 8.36 (s, 1 H), 7.90 (dd, 1 H, J = 2.9, 1.3
Hz), 7.69 (dd, 1 H, J = 4.9, 1.3 Hz), 7.52 (d, 2 H, J = 8.8 Hz),
7.43 (d, 1 H, J = 4.9, 2.9 Hz), 7.06 (d, 2 H, J = 8.8 Hz), 4.18
(t, 2 H, J = 5.7 Hz), 3.76 (t, 4 H, J = 4.6 Hz), 2.85 (t, 2 H, J = 5.7
Hz), 2.61 (t, 4 H, J= 4.6 Hz); FAB MS (M++1) Anal Calcd. for
C22Hz2N402S : C, 65.00; H, 5.46; N, 13.78. Found C, 64.98; H,
5.55; N, 14.02.
HO ~ ~'N~.O i
,N ~N~,CI p J w ~ ~ N.N
N , OJ
~N ~ Cs2C03 'N ..
1 NaI
~ S DMF ' S
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EXAMPLE 5
~ N
N 'N
\ ~
\~
S
3-(3-thiophenyi)-7-(4-pyridyl) pyrazolo(1,5-A)pyrimidine
A 13 x 100 mm reaction tube was charged with
aminopyrazole (22) ( 16.5 mg, 0. I 00 mmol) dissolved in 0.500 mL
EtOH and vinylogous amide (23) ( 17.6 mg, 0.100 mmol) dissolved
in 0.200 mL EtOH. Glacial acetic acid ( 1 drop) was added and the
reaction was heated to 80 °C for 14 h. An additional 0.100 mL of
glacial acetic acid was added and heating was continued for an
additional 6 h. The sample was concentrated to dryness to provide
the desired title compound. Analysis by mass spectrometry
showed [M+H~+ 279.2.
~ N
H O
~~~ EtOH/HOAc ~~~
H2N \- Tl + ~ ~ ~ N . 80oC N 1' Tl
Nr.~ '
/\
S I\
S
22 23
EXAMPLE 6
i N. N
~N
S
3-(3-thienyl)-6-(cyclohexyl) pyrazolo(1,5-A)pyrimidine
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Step 1
Palladium on carbon ( 10%, 2 g) was added to a solution of
24 (5.62 g, 23.4 mmol) in ethanol ( 100 mL) under an argon
atmosphere. After evacuating and backflushing the reaction vessel
with H2 (3X), the black suspension was stirred vigorously under
an H2 filled balloon for 16 h. The reaction mixture was then
filtered through celite, washed with ethyl acetate (200 mL) and
concentrated to provide 25 as a colorless oil (5.0 g, 88%): 1 H
NMR (CDC13) d 4.18 (q, 4 H, J = 7.1 Hz), 3.13 (d, 1 H, J = 9.2
Hz), 2.08 (m, 1 H), 1.73 - 1.56 (m, 5 H), 1.35 - 1.01 (rn, 5 H),
1.26 (t, 6 H, J = 7.0 Hz).
H2
Et0 OEt 10% PdJC Et0 OEt
EtOH
O O O O
24 25
Step 2
A solution of 25 (2.0 g, 8.3 mmol) in dry THF (30 mL) at
0°C was treated with lithium aluminum hydride ( 1.0 M in THF,
16.5 mL, 16.5 mmol) over a 5 min period. The reaction mixture
was warmed gradually to 15°C over 20 min and then was re-
cooled to 0°C and quenched sequentially with water (630 uL),
aqueous sodium hydroxide ( 1 N, 630 uL), and then water (3 x 630
uL). The resulting white suspension was stirred for 15 min, dried
(Na2S04), and filtered washing with THF (100 mL) and ethyl
acetate ( 100 mL). The filtrate was concentrated to provide 26 as a
white solid ( 1.35 g, 100%): 1 H NMR (CDC13) d 3.83 (ddd, 4 H),
1.77 - 1.62 (m, 5 H), 1.57 (m, 1 H), 1.42 (m, 1 H), 1.30 - 0.96 (m,
5 H).
Et0 OEt THF
O O OH OH
25 26
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Step 3
A solution of oxalyl chloride (2.39 g, 1.64 mL, 18.8 mmol)
in CH2C12 (50 mL) at -60°C was treated with DMSO (2.94 g, 2.67
mL, 37.6 mmol) in CH2CI2 ( 10 mL) over 2 min. After 5 min, a
solution of 26 ( 1.35 g, 8.5 mmol) in CH2C12 (20 mL) was added
and the resulting suspension was maintained at -60°C for 15 min.
Triethylamine (8.6 g, 1 l .8 mL, 85 mmol) was then added and the
reaction mixture was allowed to warm to 20°C. The quenched
reaction was poured into water (200 mL) and washed with CH2C12
(2 x 100 mL). The combined organics were dried (Na2S04},
concentrated, and purified by flash chromatography (40%
Hexane/EtOAc) to provide 27 as a viscous oil [ 135 mg, 10%, R f =
0.34 (40% Hexane/EtOAc)]: 1H NMR (CDCl3) d 8.26 (s, 2 H),
2.09 (tt, 1 H), 1.85 - 1.68 (m, 6 H), 1.39 - 1.13 (m, 5 H).
oxalyl chl~de
DMSO
NEt
OH OH CHZCh O O
2G 27
Step 4
A solution of 27 (50 mg, 0.30 mmol) and 22 (47 mg, 0.30
mmol) in ethanol (5 mL) was heated at 75°C for 16 h. After
cooling, the reaction mixture was concentrated, and the crude
product was purified by flash chromatography (25%
EtOAc/Hexane) to provide 6 as a yellow solid [54 mg, 63%, Rf =
0.33 (25% EtOAc/Hexanes)]: 1H NMR (CDCl3) d 8.48 (d, 1 H, J =
2.2 Hz), 8.44 (d, 1 H, J = 1.5 Hz), 8.30 (s, 1 H), 7.86 {dd, 1 H, J =
2.9, I. l Hz), 7.66 (dd, 1 H, J = 4.9, 1.2 Hz}, 7.41 (dd, 1 H, J =
4.9, 2.9 Hz), 2.64 (m, 1 H), 2.03 - 1.80 (m, 5 H), 1.52 - 1.27 (m,
5 H); FAB MS (M++1) calcd. for 284, found 284; Anal Calcd. for
C 16H 17N3S (0.05 H20): C, 67.59; H, 6.06; N, 14.78. Found C,
67.66; H, 6.12; N, 15.14.
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HN~ N
~ N. N
H z . ---~ y
EtOH N
S O O
S
22 27
Kinase inhibition is demonstrated in accordance with
the following protocol.
VEGF RECEPTOR KINASE 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 incoporation 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
(Sf21 ) insect cells (Invitrogen) using a baculovirus expression
vector (pAcG2T, Pharmingen).
Lvsis buffer
50 mM Tris pH 7.4, 0.5 M NaCI, 5 mM DTT, 1 mM
EDTA, 0.5% triton X-100, 10 % glycerol, 10 mg/ml of each
leupeptin, pepstatin and aprotinin and 1 mM phenylmethylsulfonyl
fluoride (all Sigma).
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Wash buffer
50 mM Tris pH 7.4, 0.5 M NaCI, 5 mM DTT, I 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 NaCI, 5 mM DTT, 1 mM
EDTA, 0.05% triton X-100, 50 % glycerol, 10 mg/ml of each
leupeptin, 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 pg/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 plates
Millipore #MAFC NOB, GF/C glass fiber 96 well
plate.
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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
l0 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 ~1 of 10 X
reaction buffer, 5 ~.l 25 mM ATP/10 ~Ci [33P]ATP (Amersham),
and 5 ~,l 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.
S. 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.
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.
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Human Umbilical Vein Endothelial Cell Mitogenesis Assay
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
HUVECs
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).
Assay Medium
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
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made directly into Assay Medium immediately prior to addition to
cells.
lOX Growth factors
Solutions of human VEGF,65 (500 ng/ml; R&D
Systems) and bFGF ( 10 ng/ml; R&D Systems) are prepared in
Assay Medium.
lOX [3H~Thymidine
[Methyl-3H]Thymidine (20 Ci/mmol; Dupont-NEN) is
diluted to 80 uCi/ml in Iow-glucose DMEM.
Cell Wash Medium
Hank's balanced salt solution (Mediatech) containing 1
mg/ml bovine serum albumin (Boehringer-Mannheim).
Cell Lysis Solution
1 N NaOH, 2% (w/v) Na2C03.
Method
1. HUVEC monolayers maintained in EGM are harvested
by trypsinization and plated at a density of 4000 cells per 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%
COZ for 2 hours to allow test compounds to enter cells.
3. After the 2-hour pretreatment period, cells are
stimulated by addition of 10 ul/well of either Assay Medium, l OX
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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, l OX
[3H]Thymidine ( 10 ul/well) 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-stimulated
mitogenesis of human vascular endothelial cells in culture with ICS°
values between 150-650 nM. These compounds also show
selectivity over related tyrosine kinases (e.g. FGFRl and the Src
family).
