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
5 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
10 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.
Accordingly, inhibitors of these tyrosine kinases are useful for the
prevention and treatment chemotherapy of proliferative diseases
i 5 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
2o 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.
25 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
30 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
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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
l0 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
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 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
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expression. Examples of such cancers include cancers of the
brain, genitourinary tract, lymphatic system, stomach, larynx and
lung. These include histiocytic lymphoma, lung adenocaxcinoma
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.
The present invention relates to compounds which
inhibit tyrosine kinase enzymes, compositions which contain
to 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.
SUMMARY OF THE INVENTION
A compound is disclosed in accordance with formula
I:
R~
R4 ~ N
R
2
R5 X N
R3
I
or a pharmaceutically acceptable salt, hydrate or prodrug thereof,
wherein
X is N or C;
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R, is H, C1_~o alkyl, C3_6 cycloalkyl, Cs_~o aryl, halo, OH, C3_lo
heterocyclyl, or CS_io heteroaryl; said alkyl, alkenyl,
alkynyl, aryl, heteroaryl and heterocyclyl being optionally
substituted with from one to three members selected
from Ra;
R2&R3 axe independently H, C,_6 alkyl, Cs_lo aryl, C3-6 cycloalkyl,
OH, N02, -NH2, or halogen;
R4 is H, C1_lo alkyl, C3-6 cycloalkyl, Ci_6 alkoxy C2_~o alkenyl,
Cz-io alkynyl, Cs_,o aryl, C3_lo heterocyclyl, C1_6
alkoxyNR7R8, 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;
Rs is H, or C1_6 alkyl, OR, halo, NHZ or N02;
Ra is H, C1_~o alkyl, halogen, N02, OR, -NR~ NR~R8~ R~R8
Cs-io ~'Yl~ Cs-io heteroaryl or C3_lo heterocyclyl;
R is H, or C y _6 alkyl, C 1 _6 alkylR9;
R9 is Cs-io ~Yh C3-io heterocyclyl, or Cs_lo heteroaryl; and
R~&Rg are independently H, C 1 _ 1 o alkyl, C3 _6 cycloalkyl, COR,
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.
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Also disclosed is a pharmaceutical composition which
is comprised of a compound represented by the formula I:
R3
R
R5 X N
N
R
2
R~
wherein R~, 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 or preventing a
1 o 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 or preventing
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 or preventing 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 or preventing
ocular disease in which neoangiogenesis occurs is included herein,
which is comprised of administering to a mammalian patient in
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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 or preventing
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 or preventing age-related macular degeneration.
These and other aspects of the invention will be
apparent from the teachings contained herein.
1s
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.
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 ~/ (CH2)y
wherein: x plus y = from 0-10; and w plus z = from 0-9.
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The alkylene and monovalent alkyl portions) of the
alkyl group can be attached at any available point of attachment to
the cycloalkylene portion.
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
l0 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,
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 S-IO 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 naphthyl substituted
with one or two groups.
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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
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
l0 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.
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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
inventon such as amides of alkanoic(C1_6)acids, amides of aryl
acids (e.g., benzoic acid) and alkane(C,_~)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
accordance with formula I:
R3
R4 ~ N
R
2
Rs X N
1S R~
I
X is N or C;
R~ is H, C,_,o alkyl, C~_6 cycloalkyl, CS_to aryl, halo, OH, C3_~o
heterocyclyl, or CS_io heteroaryl; said alkyl, alkenyl,
alkynyl, aryl, heteroaryl and heterocyclyl being optionally
substituted with from one to three members selected
from Ra;
2S
R2&R3 are independently H, C,_6 alkyl, CS_~o aryl, C3-6 cycloalkyl,
OH, N02, -NH2, or halogen;
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R4 is H, C,_to alkyl, C3_6 cycloalkyl, C,_6 alkoxy C2_~o alkenyl,
C2_lo alkynyl, Cs_,o aryl, C3_~o heterocyclyl, C,_6
alkoxyNR~R8, NOZ, 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;
RS is H, or C1_6 alkyl, OR, halo, NH2 or N02;
Ra is H, C ~ _, o alkyl, halogen, N02, OR, -NR, NR7R8, R7R8
Cs-io ~'Yl~ Cs-io heteroaryl or C3_~o heterocyclyl,
R is H, or C 1 _6 alkyl, C, _6 alkylR9;
R9 is Cs_~o aryl, C3_lo heterocyclyl, or Cs_lo heteroaryl; and
R~&Rg are independently H, C1_lo alkyl, C3-6 cycloalkyl, COR,
CS_,o aryl, C3_,o heterocyclyl, or Cs_io 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.
In one aspect, the invention is described wherein X is C and
all other variables are as described above.
In another aspect, the invention is described wherein X is N
and all other variables are as described above.
In still another aspect, the invention is described wherein R4
is C,_,o alkyl, C3_6 cycloalkyl, CS_,o aryl, Cs_IO heteroaryl, or C3_,o
heterocyclyl, said alkyl, aryl, heteroaryl and heterocyclyl being optionally
substituted with from one to three members selected from Ra and all
other variables are as described above.
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In yet another aspect, the invention is described wherein
R~ is C1_~o alkyl, CS_~o aryl, C3_~o heterocyclyl, or CS_~o heteroaryl,
said alkyl, aryl, heteroaryl and heterocyclyl being optionally
substituted with from one to three members selected from Ra and all
other variables are as described above.
A preferred subset of compounds of the present
invention is realized when:
Rl is H, CI_lo alkyl, CS_~o aryl, C3_lo heterocyclyl, or CS_,o
heteroaryl; said alkyl, aryl, heteroaryl 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, C~_io alkyl, C3_6 cycloalkyl, CS_~o aryl, CS_lo
heteroaryl, C3_~o heterocyclyl, C1_6 alkoxyNR7R8, N02,
OH, -NH2 or said alkyl, aryl, heteroaryl and heterocyclyl
being optionally substituted with from one to three
2o members selected from Ra; and all other variables are as
described above.
Another preferred subset of compounds of the present
invention is realized when:
R, is CS_,o aryl, C3_,o heterocyclyl, or CS_~o heteroaryl; said
aryl, heteroaryl and heterocyclyl being optionally
substituted with from one to three members selected
from Ra;
R2&R3 are independently H or C,_6 alkyl;
R4 is C1_lo alkyl, Cs_,o aryl, CS_lo heteroaryl, C3_~o
heterocyclyl said alkyl, aryl, heteroaryl and
heterocyclyl being optionally substituted with from one
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to three members selected from Ra; and all other
variables are as described above.
Examples of the compounds of this invention are:
I-phenyl-5-(4-methoxyphenyl)benzimidaole,
1-phenyl-5-(4-(2-( 1-piperidinyl}ethoxy)phenyl)benzimidazole,
3-phenyl-6-(4-methoxylphenyl)imidazo [4, 5-b]pyridine,
3-phenyl-6-(4-(2-( 1-piperidinyl)ethoxy)phenyl)imidazo[4,5-b]pyridine,
io 3-phenyl-6-(4-(2-(1-piperidinyl)ethoxyphenyl)imidazo[4,5-b]pyridine,
3-(2-thiazoyl)-6-(4-(3-( 1-piperidinyl)propylphenyl)imidazo [4, 5-
b]pyridine,
1-(2-thiazoyl)-S-(4-(3-{ 1-piperidinyl)propyl)phenyl)benzimidazol,
1-(3-thiophenyl)-5-(4-(3-( 1-piperidinyl)propyl)phenyl)imidazo[4,5-
15 b]pyridine,
1-(3-thiophenyl)-5-(4-(3-(I-piperidinyl)propyl}phenyl)benzimidazole,
3-(3-thiophenyl)-6-(4-{3-(1-piperidinyl)propylphenyl)imidazo[4,5-
b]pyridine,
1-Phenyl-5-[5-(2-piperidin-1-yl-ethoxy)-pyridin-2-yl]-1 H-benzimidazole,
20 1-(4-Cyanophenyl}-5-[6-(2-piperidin-1-yl-ethoxy)-pyridin-3-yl]-1 H-
benzimidazoie,
1-Phenyl-5-[f>-{2-piperidin-1-yl-ethoxy)-pyridin-3-yl]-1 H-benzimidazole,
1-(3-Cyanophenyl)-5-[6-(2-piperidin-1-yl-ethoxy)-pyridin-3-yl]-1 H-
benzimidazole,
25 1-(3-Thiophene)-5-[6-(2-piperidin-1-yl-ethoxy)-pyridin-3-yl]-1 H-
benzimidazole,
[5-( 1-Phenyl-1 H-benzoimidazol-5-yl)-pyridin-2-yl]-(2-piperidin-1-yl-
ethyl)-amine,
[5-( 1-Phenyl-1 H-benzoimidazol-5-yl)-pyridin-2-yl]-(2-morpholin- I -yl-
30 ethyl)-amine,
4-( 1-Phenyl-1 H-benzoimidazol-5-yl)-1-(3-piperidin-1-yl-propyl)-1H-
pyridin-2-one,
4-( 1-Phenyl-1 H-benzoimida.zol-5-yl)-1-(3-piperidin-1-yl-ethyl)-1 H-
pyridin-2-one,
3 5 I -(3-Pyridyl)-5-(4-(2-( 1-piperidinyl)ethoxy)phenyl)benzimidazole,
1-(4-Pyridyl)-5-(4-(2-( 1-piperidinyl)ethoxy)phenyl)benzimidazole,
1-(3-Pyridyl)-5-[6-(2-piperidin-1-yl-ethoxy)-pyridin-3-yl]-1 H-
benzimidazole, and
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1-(4-Pyridyl)-S-[6-(2-piperidin-1-yI-ethoxy)-pyridin-3-yl]-1 H-
benzimidazole or a pharmaceutically acceptable salt, hydrate or prodrug
thereof.
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
and excretion. Other factors, more practical in nature, which are
also important in the 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
2o 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, triethanolhydroammonium,
etc. An appropriate number of counterions is associated with the
molecule to maintain overall charge neutrality. Likewise when the
3o 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
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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-
naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate,
1 o 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.
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, R1, 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
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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
l0 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, 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 inj ections. Also, various buffering
agents, preservatives and the like can be included.
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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 mglkg to about 1 mg/kg
per day.
The examples which follow illustrate the compounds
that can be synthesized but they are not limited by the compounds
in the tables nor by any particular substituents employed in the
schemes for illustrative purposes.
The compounds may be administered in conventional
dosages as a single agent or in combination with other
therapeutically active compounds. The non-limiting examples that
follow are illustrations of the compounds of the instant invention
and are not meant to limit the invention in any way.
EXAMPLE 1
Br ~ N02
Br ~ NOZ
---~ ~ H
F
4
3 ,,.
1-Bromo-4-fluoro-3-nitrobenzene (3) (1.14 mL, 9.06 mmol)
was dissolved in 5 mL of anhydrous 1-methyl-2-pyrrolidinone under
argon. Aniline (0.870 mL, 9.55 mmol) was added followed by the
addition of N,N diisopropylethylamine ( 1.90 mL, 10.9 mmol) and the
resulting solution was heated to 120 °C. After 14 h additional aniline
(0.082 mL, 0.90 mmol) was added and heating was continued for 8 h.
The reaction solution was cooled to ambient temperature, diluted with
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water and extracted with ethyl acetate (3x). The combined extracts was
washed with brine, dried over Na2S04, filtered and concentrated in
vacuo to provide 4.
'H NMR (CDC13) 8 9.46 (bs, 1H), 8.35 (d, 1H, J= 2.4 Hz), 7.45-7.40
(m, 3H), 7.29-7.25 {m, 3H), 7.10 (d, 1H, J= 9.2 Hz).
Me0
Br
~ N02 w I ~ N02
H ~ ~ H
/ \ 5 / \
Bromoaromatic 4 (0.218 g, 0.744 mmol) and 4
methoxyboronic acid {0.125 g, 0.823 mmol) were dissolved in a mixture
of dioxane (4 mL) and water (3 mL). Sodium carbonate (0.60 g, 5.7
mmol) was added and the resulting mixture was degassed and put under
argon. Tetrakis{triphenylphosphine)palladium(0) (0.043 g, 0.037 mmol)
was added and the reaction was heated to 80 °C. After 14 h the reaction
was cooled to ambient temperature, diluted with water and extracted
with ethyl actetate (3x). The combined extracts was dried with
Na2S04, filtered and concentrated to dryness. Purification by flash
column chromatography (2 x 16 cm silica gel, 6:1 hexane/ethyl acetate)
provided 5.
'H NMR (CDC13) d 9.48 (bs, 1H), 8.40 (d, IH, J = 2.4 Hz), 7.58 (dd,
1 H, J = 2.4, 9.2 Hz), 7.50 {d, 2H, 9.0 Hz), ?.43 {t, 2H, J = 9.0 Hz),
7.32-7.22 (m, 4H), 6.98 (d, 2H, J = 9.0 Hz), 3.83 (s, 3H).
Me0 , I Me0
NOZ \ ~ \ N
/NH --. /N
5 / \ 6 /
1
1-phenyl-5-(4-methoxyphenyl)benzimidaole.
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Nitroaniline 5 {0.213 g, 0.665 mmol) and palladium on
carbon ( 10%, 100 mg) were stirred in 8 mL 3 :1 EtOH/AcOH. The
reaction was put under a balloon of H2. After 2 h the reaction was
filtered through a plug of celite and the filtrate was concentrated to
dryness. The resulting residue was dissolved in 1.5 mL
trimethylorthoformate and heated to 120 ° C for 30 min. The solution
was cooled concentrated to dryness and purified by flash column
chromatography (2 x 15 cm silica gel, 1:1 hexane/ethyl acetate) which
provided 6.
1H NMR (CDC13) 8 8.14 (s, 1H), 8.04 (d, 1H, J= 0.9 Hz), 7.62-7.50 (m,
8H), 7.48 (t, 1 H, J = 7.1 Hz), 7.01 (d, 2H, J = 8.8 Hz), 3 . 87 (s, 3H);
FAB mass spectrometry [M+H]+ 30I.I; Anal. Calcd. for C2oH16N2O: C,
79.98; H, 5.37; N, 9.33. Found: C, 79.71; H, 5.48; N, 9.21.
HO
\ I \ N
~v
7
1
An oven dried flask under argon was charged with
benzimidazole 6 (0.039g, 0.13 mmol), aluminum chloride (0.175g, 1.31
mmol), and sodium iodide (0.200g, 1.33 mmol). Anhydrous acetonitrile
(1 mL) and dichloromethane (0.5 mL) were added and reaction was
heated to reflux. After 44 h the reaction was cooled to ambient
temperature, quenched with water and extracted 3 x with ethyl acetate.
The combined extracts was dried over Na2S04, filtered and concentrated
to dryness. The resulting residue was triturated with ether, filtered and
dried to provide phenol 7.
~ H NMR (CDCl3) b 9.48 (s, 1 H), 8.58 (s, 1 H), 7.93 (s, 1 H), 7.73-7.71
(m, 2H), 7.67-7.63 (m, 3H), 7.57-7.49 (m, 4H), 6.86 (d, 2H, J = 8.6
Hz).
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HO , NCO
G ,~ N
I , ~ ~ W ,>
N
7 ~ ~ 8
1-phenyl-5-(4-(2-( 1-piperidinyl)ethoxy)phenyl)benzimidazole
Benzimidazole 7 (0.025g, 0.087 mmol) and N-(2-
chloroethyl)piperidine hydrochloride ( 11 mg, 0.059 mmol) were
dissolved in anhydrous N,N-dimethylformamide (0.5 mL). Cesuim
carbonate (0.085g, 0.26 mmol) was added and the resulting mixture was
heated to 50 °C. After 2 h additional and N-(2-chloroethyl)piperidine
hydrochloride (11 mg, 0.059 mmol) was added. After 1 h the reaction
was allowed to cool, quenched with water and extracted with ethyl
acetate (3x). The combined extracts was washed with brine, dried over
Na2S04, filtered and concentrated to dryness. Purification by flash
column chromatography (2 x 16 cm silica gel, 9:1 CH2C12/MeOH)
provided 8 as a colorless oil.
' H NMR (CDC13) 8 8.14 (s, 1 H), 8.03 (d, 1 H, J = 0.9 Hz), 7.62-7.50 (m,
8H), 7.48 (t, 1 H, J = 7.2 Hz), 7.01 (d, 2H, J = 8.8 Hz), 4.21 (bt, 2H, J =
5.3 Hz), 2.87 (bs, 2H), 2.59 (bs, 4H), 1.66 (bs, 4H), 1.48 (bs, 2H);
Mass spectrometry [M+H]+ 398.3.
EXAMPLE 2
Br ~ N02 Br ~ N02
N OH N NH
5-Bromo-2-hydroxy-3-nitropyridine (9) (5.736 g, 0.0262
25 mol) and lSmL thionyl chloride were added under argon. N,N
dimethylformamide (1 mL) was then added and the solution was heated
to reflux for 1 hr. By the end of the reaction, the
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bromohydroxynitropyridine was completely dissolved in solution. After
cooling to ambient temperature, 5 mL of toluene was added, and the
solution was concentrated under vacuum. The product, 5-bromo-2-
chloro-3-nitropyridine, was a yellow crystalline solid.
The bromochloronitropyridine was dissolved in lSmL of
anhydrous 1-methyl-2-pyrrolidinone. Aniline (3.580 mL, 0.0393 mol)
was added followed by the addition of N,N -diisopropylethylamine ( 13.69
mL, 0.0786 mol) and the solution was heated to 120 °C. After 1.5 hr.,
the solution was cooled to ambient temperature and diluted with water.
The product was extracted using ethyl acetate and washed with brine.
The organic layer was then dried over sodium sulfate, filtered,
concentrated, and dried in vacuo. The crude mixture was purified
using flash column chromatography (7.5 x 16 cm silica gel, 10:1
hexane: ethyl acetate) to afford 10 .
' H NMR (CDC13) 8 10.04 (bs, i H), 8.65 (dd, 1 H, J = 2.2 Hz), 8.50(dd,
1 H, J =2.4 Hz), 7.60 (d, 2H, J =8.6 Hz), 7.40(t, 2H, J =7.5 Hz), 7.21 (t,
1 H, J =7 .3 Hz).
B ~ NOZ
I~ NH
20
Bromoaromatic 10 (30 mg, 0.102 mmol), 4-
methoxyphenylboronic acid {17 mg, 0.112 mmol) was dissolved in 0.75
mL dioxane followed by the addition of 204 ~.L of 2M sodium carbonate.
25 The vessel was flushed with argon followed by the addition of
tetrakis(triphenylphosphine)palladium(0) (6 mg, 0.005 mmol) and 0.56
mL water. The vessel was flushed again with argon and heated to 80
°C for 2.5 hr. The solution was cooled to room temperature and diluted
with water. The product was extracted with ethyl acetate and washed
30 with brine, followed by drying over sodium sulfate. The organic layer
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was concentrated, and the product dried in vacuo. The crude mixture
was purified by flash column chromatography (2.5 x 8 cm silica gel, 8:2
hexane:ethyl acetate), affording 11.
'H NMR (CDCl3) b 10.09 (bs, 1 H), 8.71 (dd, 1 H, J =2.4 Hz), 8.66 (dd,
1 H, J =2.4), 7.67 (d, 2H, J =7.9}, 7.49 (d, 2H, J =8.8 Hz), 7.4 i (t, 2H, J
=7.7 Hz), 7.18 (t, 1 H, J =.7.3 Hz), 7.00 (d, 2H, J =8.6 Hz), 3.86 (s, 3H).
Me0 , Me0
N02
N' _NH
11 ~ ~
w
3-phenyl-6-{4-methoxylphenyl)imidazo[4,5-b]pyridine
Nitroaniline 11 (1.333 g, 4.15 mmol), Zn dust(6.239 g,
95.40 mmol), and 10 mL acetic acid were mixed under argon. The
solution was heated to 60 °C for 1 hr until the solution turned light
green. The zinc was removed using vacuum filtration with celite and
washed with acetic acid. The filtrate was concentrated and 20 mL of
trimethylorthoformate was added. The solution was heated to 100 °C
for 2 hr followed by cooling to ambient temperature. The solution was
concentrated and the crude mixture was purified by flash column
chromatography(5 x 16 cm silica gel, 6:4 ethylacetate:hexane) affording
12.
' H NMR (CDCl3) 8 8.61 (dd, 1 H, J =2.0 Hz), 8.32 (s, 1 H), 8.22 (dd, 1 H,
J =2.0), 7.74 (d, 2H, J =7.9 Hz), 7.55-7.50 (m, 4H), 7.39 (t, 1 H, J
=7.3), 6.99 (d, 2H, J =8.8 Hz), 3.80 (s, 3H). Mass spectrometry [M+H]+
302.3.
EXAMPLE 3
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To the imidazopyridine 12 (202 mg, 0.670 mmol) was added a mixture
of 10 mL hydrobromic acid and 10 mL acetic acid. The solution was
stirred a room temperature for 5 min., followed by heating at 100 °C
for 17 hr. The solution was cooled to ambient temperature and
concentrated. Toluene ( 15 mL) was added and the solution was
concentrated a second time. The concentrate was placed in vacuo over
heating at 40 °C for 40 min., followed by further drying in vacuo at
ambient temperature. Purification was acheived by reverse phase
column chromatography affording 13.
'H NMR (CD30D) 8 9.45 (s, 1H), 8.82 (dd, 1H, J =1.8 Hz), 8.37 (dd,
1H, J =1.8 Hz), 7.91 (d, 2H, J =7.7 Hz), 7.68 (t, 2H, J =8.1 Hz), 7.63-
7.57 (m, 3H}, 6.95 (d, 2H, J =8.6 Hz).
NCO
G
14
3-phenyl-6-(4-(2-( 1-piperidinyl)ethoxy)phenyl)imidazo [4, 5-b]pyridine
Cesium carbonate (296 mg, .908 mmol) and 1-(2-
chloroethyl}piperidine monochlorohyrdide (84 mg, .454 mmol) were
added under argon to a flame dried round bottom flask. Imidazopyridine
13 (87 mg, .303 mmol) was dissolved in 1.5 mL of anhydrous N,N
dimethyl formamide under argon. The vessel was heated at 50 °C for
16 hr. and cooled to ambient temperature. The solution was diluted to
100mL with saturated sodium bicarbonate, and the product was
extracted using ethyl acetate. The aqueous layer was extracted a
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second time with dichloromethane w/ 3% 1-butanol. The organic layers
were washed with saturated sodium bicarbonate, and dried over sodium
sulfate. The organic layers were conentrated at aspirator pressure to
remove ethyl acetate and methylene chloride; the 1-butanol and residual
DMF were removed under high pressure. The product was purified
using flash column ehromatography(silica gel 2.5 x 32.5 cm, 10:1
methylene chloride:methanol). Excess trifluoroacetic acid was added to
the product to create the resulting salt, and the mixture was triturated
using ether. The TFA salt was dried using phosphorous pentoxide in
l0 vacuo to yield I4 ( 1.10 TFA salt).
1 H NMR (CD30D) S 8.66 (s, i H), 8.5 5 (dd, 1 H, J =2.0 Hz), 8.17 (dd,
1 H, J =2.0 Hz), 7.82 (d, 2H, J =8.6 Hz), 7.59-7.52 (m, 4H), 7.46 (t, 1 H,
J =7.5 Hz), 7.01 (d, 2H, J =8. 8 Hz), 4.85 (s, 2H), 4.15 (t, 2H, J =5.5
Hz), 2.84 (t, 2H, J =5.5 Hz), 2.62 (bs, 4H), 1.65 (m, 4H), 1.50 (m, 2H).
Anal. Calcd. for C25H26NaO~ 1.10 TFA: C, 62.35; H, 5.21; N, 10.69.
Found: C, 62.32; H, 4.93; N, 10.53.
EXAMPLE 4
& ~ OZ ~' ~ N
-NH
20
Bromoaromatic 4 (7.10 g, 24.1 mmol) and powdered zinc (36.2 g, 554
mmol, 23 equiv) were stirred in 80 mL glacial acetic acid. The mixture
was heated to 60 °C. After 1 h the reaction was cooled and filtered
through a plug of celite and concentrated to dryness. The resulting
residue was dissolved in 60 mL of formic acid and heated to 100 °C
overnight. The reaction was cooled and concentrated to dryness.
Purification by flash column chromatography (6x25 cm silica, 55:45
hexanesBtOAc) afforded 5.88 g benzimidazole 15 (89% yield). 1H
NMR(CDC13) 8 8.18 (s, 1 H), 8.05 (d, 1 H, J= 1.7 Hz), 7.60 (t, 2H, J--7.1
Hz), 7.54-7.48 (m, 3H), 7.46 (dd, 1 H, J 1.8, 8.8 Hz), 7.40 (d, 1 H,
J--8.8 Hz).
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F N\ NCO
I
r Br
16
1-Piperidineethanol ( 1.13 mL, 8.51 mmol) was dissolved in 10 mL
anhydrous DMF under Ar. The solution was cooled to 0 °C and NaH
(225 mg, 9.38 mmol) was added. After IO min the mixture was allowed
to warm to room temperature and 5-bromo-2-fluoropyridine ( 1.50 g,
8.52 mmol) was added. After 1 h the reaction was quenched with water
and extracted 3x with EtOAc. The combined extracts were dried over
Na2S04, filtered and concentrated to afford 2.20 g {91% yield) of the
alkoxypyridine 16. 'H NMR(CDCl3) 8 8.17 (d, 1H, J--2.6 Hz), 7.62 (dd,
1 H, J--2.6, 8.8 Hz), 6.67 (d, 1 H, J--8.8 Hz), 4.40 (t, 2H, J--6.0 Hz), 2.74
(t, 2H, J--5.9 Hz), 2.49 (m, 4H), 1.60 (m, 4H), 1.44 (m, 2H).
Br
N NCO / I
~N
N
~ 17
1-Phenyl-5-[6-(2-piperidin-1-yl-ethoxy)-pyridin-3-yl]-1 H-benzimidazo le
15 Benzimidazole 15 (2.91 g, 10.7 mmol), diboron pinacol ester (2.97 g,
11.7 mmol) and potassium acetate {3.14 g, 32.0 mmol) were stirred in
mL anhydrous DMF under Ar. PdCl2(dppf) (0.26 g, 0.32 mmol) was
added, solution was degassed and heated to 80 °C. After 20h the
reaction was quenched with 125 mL of water and SO mL of saturated
20 aqueous NaCI and was extracted 3 x with EtOAc. The combined
extracts were dried over Na2S04, filtered and concentrated to afford
2.77 g of unpurified boronate. The unpurified boronate (650 mg, 2.03
mmol), alkoxypyridine 16 (526 mg, 1.85 mmol), 2M Na2C03 (861 mg,
8.12 mmol), and 4 mL dioxane were added to a round bottom flask.
After flushing three times with argon, Pd(PPh3)4 ( 117 mg, .10 mmol)
was added, and the vessel was again flushed three times with argon.
The vessel was heated to 80 °C under argon. After 22 hr., the
reaction
was cooled to room temperature followed by quenching with 25mL
water. The mixture was extracted with 4x20 mL ethyl acetate, and the
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combined organic layers were washed with 1 x20mL brine. The organic
layer was dried over sodium sulfate, filtered, and concentrated.
Purification was performed using reverse phase column chromatography
{Waters 2x40mm C-18 column, HZO:acetonitrile mobile phase gradient).
The resulting oil was triturated with ether, filtered and washed with
ether, affording 16, a white TFA salt(150 mg, 16% yield). Mp: 160.5 -
162 °C. ~ H NMR(CDC13) $ 8.41 (d, I H, J =2.4 Hz), 8.19 (s, 1 H),
8.01 (d, 1 H, J =1.3 Hz), 7. 90 (dd, 1 H, J =11.0 Hz), 7.61 (m, 3 H, J
=13.6), 7.52 (m, 4H, J =31.0 Hz), 6.86 (d, 1 H, J =8.4 Hz), 4.79 (t, 2H, J
10 =9.9 Hz), 3.76 (bd, 2H, J =11.9 Hz), 3.51 (t, 2H, J =9.7 Hz), 2.80 (bt,
2H, J =23.1 Hz), 2.06 (m, 2H, J =26.2 Hz), 1.89 (s, 2H), 1.65 (s, 2H).
The following compounds can be made by literature
methods and/or in combination with methods disclosed herein.
v I \
/ \ N
I\ I
G / \ N N N
~C Z ~> >,-S
N N N",
S
N v I \ NCO
/ \ N I
I ~~ \ \ N
'~ N (
N N
S ~ / I
\
v I\
/ \ N
/ N
N~S
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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 receutor 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. S 19-
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).
L~rsis 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 1mM phenylmethylsulfonyl
fluoride (all Sigma).
Wash buff~,,r
50 mM Tris pH 7.4, 0.5 M NaCI, 5 mM DTT, 1 mM
EDTA, 0.05 % triton X- I 00, 10 % glycerol, 10 mg/ml of each
leupeptin, pepstatin and aprotinin and 1mM phenylmethylsulfonyl
fluoride.
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Dialvsis 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
X reaction buffer
200 mM Tris, pH 7.4, I.0 M NaCI, 50 mM MnCl2, IO
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).
Ston 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.
METHOD
A. Protein purification
1. Sf21 cells were infected with recombinant virus
at a multiplicity of infection of 5 virus particlesl cell and grown at
27 °C for 48 hours.
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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 ~.1 of 10 X
reaction buffer, 5 x.125 mM ATP/10 ~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.
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 Assay
Expression of VEGF receptors that mediate mitogenic
3o responses to the growth factor is largely restricted to vascular
endothelial cells. Human umbilical vein endothelial cells (IiLTVECs)
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
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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
(NLJNC # 167008).
Assay Medium
Dulbecco's modification of Eagle's medium containing
1 g/m1 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 1 X concentration are
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.
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1 OX f 3HlThymidine
[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 Lysis Solution
l0 1 N NaOH, 2% {w/v) Na2C03.
Method
1. HWEC 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
2o 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 ul/well of either Assay Medium, l OX
VEGF solution or l OX bFGF solution. Cells are then incubated at
37°CIS% C02.
4. After 24 hours in the presence of growth factors, lOX
[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
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transferred to ?-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 ICso
values between 150-650 nM. These compounds also show
selectivity over related tyrosine kinases (e.g. FGFR1 and the Src
family).
