Note: Descriptions are shown in the official language in which they were submitted.
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1,5-DIARYL SUBSTITUTED PYRAZOLES
AS p38 KINASE INHIBITORS
Descript inn
Technical Field
This invention is directed to kinases
inhibitors, and more particularly to 1,5-diaryl
substituted pyrazole compounds that, inter alia,
inhibit the activity of mitogen-activated protein
kinases, compositions of those inhibitors,
intermediates for the syntheses of those compounds,
and processes for treating pathological mitogen-
activated protein kinase activity.
Background of the Inven ion
Mitogen-activated protein (MAP) kinases are a
family of proline-directed serine/threonine kinases
that activate their substrates by dual
phosphorylation. The kinases are activated by a
variety of signals including nutritional and
osmotic stress, UV light, growth factors, endotoxin
and inflammatory cytokines.
The p38 MAP kinase group is a MAP family of
various isoforms, including p38 , p38 and p38 ,
and is responsible for phosphorylating and
activating transcription factors (e. g. ATF2, CHOP
and MEF2C) as well as other kinases (e.g. MAPKAP-2
and MApKAp-3). The p38 isoforms are activated by
bacterial lipopolysaccharide, physical and chemical
stress and by pro-inflammatory cytokines, including
tumor necrosis factor alpha (TNF- ) and
interleukin-1 (IL-1). The products of the p38
phosphorylation mediate the production of
inflammatory cytokines, including TNF and IL-1, and
cyclooxygenase-2.
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TNF-a is a cytokine produced primarily by
activated monocytes and macrophages. Excessive or
unregulated TNF production has been implicated in
mediating a number of diseases. Recent studies
indicate that TNF has a causative role in the
pathogenesis of rheumatoid arthritis. Additional
studies demonstrate that inhibition of TNF has
broad application in the treatment of inflammation,
inflammatory bowel disease, multiple sclerosis and
asthma.
TNF has also been implicated in viral
infections, such as HIV, influenza virus, and
herpes virus including herpes simplex virus type-1
(HSV-1), herpes simplex virus type-2 (HSV-2),
cytomegalovirus (CMV), varicella-zoster virus
(VZV), Epstein-Barr virus, human herpesvirus-6
(HHV-6), human herpesvirus-7 (HHV-7), human
herpesvirus-8 (HHV-8), pseudorabies and
rhinotracheitis, among others.
IL-8 is another pro-inflammatory cytokine,
which is produced by mononuclear cells,
fibroblasts, endothelial cells, and keratinocytes,
and is associated with conditions including
inflammation.
IL-1 is produced by activated monocytes and
macrophages and is also involved in the
inflammatory response. IL-1 plays a role in many
pathophysiological responses including rheumatoid
arthritis, fever and reduction of bone resorption.
TNF, IL-1 and IL-8 affect a wide variety of
cells and tissues and are important inflammatory
mediators of a wide variety of disease states and
conditions. The inhibition of these cytokines by
inhibition of the p38 kinase is of benefit in
controlling, reducing and alleviating many of these
disease states.
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Various pyrazoles have previously been
described. For example, WO 95/33727, published
December 14, 1995, describes substituted pyrazoles
as corticotropin-releasing factor (CFR) antagonists
used in the treatment of illnesses such as stress
and anxiety related disorders. WO 96/21660,
published July 18, 1996, describes substituted
pyrazoles and their use as ligands for dopamine
receptors within the body. EP 0 699 438 A2,
published March 6, 1996, describes pyrazoles and
their use as neurotensin antagonists. U.S. Patent
No. 2,833,779, to Fields et al., describes the
preparation of 1,3,5-tri-substituted pyrazoles.
U.S. Patent No. 4,957,971, to Picard et al.,
describes traps-6-[2-(N-heteroaryl-3,5-
disubstituted)pyrazol-4-yl)ethyl- or
ethenylJtetrahydro-4-hydroxy-2H-pyran-2-ones as
potent inhibitors of the enzyme 3-hydroxy-3-
methylglutaryl-coenzyme A reductase, used for
inhibiting cholesterol biosynthesis. U.S. Patent
No. 5,441,975, to Lee et al., describes pyrazoles
that are useful for the treatment of
hypercholesterolemia or atherosclerosis in mammals.
WO 93/04052, published March 4, 1993, describes
pyrazole having ACAT inhibitory activity. U.S.
5,102,893, to Picard et al., describes traps-6-[2-
(N-heteroaryl-3,5-disubstituted)pyrazol-4-yl)ethyl-
or ethenyl]tetrahydro-4-hydroxy-2H-pyran-2-ones
which axe potent inhibitors of the enzyme 3-
hydroxy-3-methylglutaryl-coenzyme A reductase and
useful as hydrolipidemic and hypocholesterolemic
agents. WO 94/22838, published October 13, 1994,
describes pyrazole compounds having angiotensin II
antagonism which are useful in preventing or
treating hypertension, congestive heart failure,
chronic renal failure, aldosteronism, and increased
intralocular pressure.
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WO 92/19615, published November 12, 1992,
describes pyrazoles, pyrazolines and
tetrahydropyridazine having fungicidal activity.
U.S. Patent No. 5,232,940, to Hatton et al.,
describes a N-Phenylpyrazole and their use against
arthropod, plant nematode, helminth and protozoan
pests. WO 95/01340, published January 12, 1995,
describes novel pyrazole compounds having
agrohorticultural bactericidal effect. U.S. Patent
No. 5,201,938, to Costales, describes novel
substituted N-pyrazolyl-1,2,4-triazolo[1,5-c]-
pyrimidine-2-sulfonamide compounds and their use as
herbicides. WO 93/09100, published May 13, 1993,
describes trizolocarboxamides with herbicidal
activity used to control blackgrass, wild oats,
crabgrass, giant (oxtail, and barnyardgrass. WO
94/29300, published December 22, 1994, describes
pyrazoles 3-substituted by a heterocyclic ring and
their use as agricultural fungicides. WO 96/37477,
published November 28, 1996, describes substituted
pyrazoles and their use against animal parasites
and pests and as insecticides, and fungicides.
Pyrazoles have also been described for use in
the treatment of inflammation. U.S. Patent No.
5,242,940, to Wachter and Murray, describes 1,5
heterocyclic pyrazoles and their use in alleviating
inflammatory and cardiovascular disorders in
mammals. U.S. Patent No. 5,134,142, to Matsuo, et
al., describes 1,5 diaryl substituted pyrazoles and
1,3 diaryl substituted pyrazoles useful in the
treatment of inflammation, pain, thrombosis and
rheumatism. U.S. Patent No. 5,466,823, to Talley
et al., describes a class of pyrazole
benzenesulfonamide compounds and their use in
treating inflammation and inflammation-related
disorders.
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The invention's pyrazolyl compounds are found
to show usefulness, inter alia, as p38 kinase
inhibitors.
Brief Summary of the Invention
In accordance with the present invention, it has
been found that certain 1,5-diaryl pyrazoles are
effective for inhibition of mitogen-activated protein
(MAP) kinases. Mitogen-activated protein kinases are
believed to be associated with, inter alia, the
mediation of a number of inflammatory diseases. In
particular, it has been found that these certain 1,5-
diaryl pyrazoles are effective for the inhibition of the
p38 MAP kinase group, a sub-family of MAP kinases. The
compounds of interest here have structures that
correspond to Formula I, below, whose substituent groups
are defined hereinafter, or a pharmaceutically
acceptable salt thereof.
Ark
N- ~
w
N ~ A RZ
Z
R'
A process for treating a host mammal having a
condition associated with pathological p38 MAP kinase
activity is also contemplated. That process comprises
administering a compound described herein in a p38 MAP
kinase enzyme-inhibiting effective amount to a mammalian
host having such a condition. The use of administration
repeated a plurality of times is particularly
contemplated.
The p38 MAP kinase sub-family have various
isoforms, including p38 , p38 and p38 and is
responsible for phosphorylating and activating
transcription factors (e. g. ATF2, CHOP and MEF2C)
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as well as other kinases (e. g. MAPKAP-2 and MAPKAP-
3). The p38 isoforms are activated by bacterial
lipopolysaccharide, physical and chemical stress
and by pro-inflammatory cytokines, including tumor
necrosis factor (TNF- ) and interleukin-1 (IL-1).
The products of the p38 phosphorylation mediate the
production of inflammatory cytokines, including
TNF- and IL-1, and cyclooxygenase-2.
Excessive or unregulated TNF production has
been implicated in mediating a number of diseases,
including rheumatoid arthritis, inflammation,
inflammatory bowel disease, multiple sclerosis,
asthma, and viral infections. IL-8 is another pro-
inflammatory cytokine, and is associated with
conditions including inflammation. Additionally,
IL-1 is involved in the inflammatory response. IL-
1 plays a role in many pathophysiological responses
including rheumatoid arthritis, fever and reduction
of bone resorption. TNF- , IL-1 and IL-8 affect a
wide variety of cells and tissues and are important
inflammatory mediators of a wide variety of disease
states and conditions. The inhibition of the
production of these cytokines by inhibition of the
p38 kinase is of benefit in controlling, reducing
and alleviating many of these disease states.
Detailed Descript~nn of Preferred EmbodimPnr~
As already noted, the present invention is
directed to compounds that inhibit the activity of
p38 MAP kinase, among other activities, as well as to
processes for using such a compound in treating a
condition mediated by that enzyme or TNF. One
embodiment of the present invention is directed to a
1,5-diaryl pyrazole compound that, inter alia,
inhibits the activity of the p38 mitogen-activated
protein kinase enzyme. That compound corresponds in
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structure to Formula I below, or a pharmaceutically-
acceptable salt thereof:
R~
I
Z
wherein A is =N- or =CH-;
Arl is an aryl group that is optionally
substituted by one or more substituents selected from
the group consisting of a halogen, hydrocarbyl,
hydrocarbyloxy, nitro, cyano, perfluorohydrocarbyl,
trifluoromethylhydrocarbyl, perfluorohydrocarbyloxy,
hydroxy, mercapto, hydroxycarbonyl, aryloxy,
arylthio, sulfonyl or sulfoxido, wherein the
subsituent on the sulfur atom is hydrocarbyl,
sulfonylamide,
wherein the substituents on the sulfonamido
nitrogen atom are hydrido or hydrocarbyl, arylamino,
arylhydrocarbyl, aryl, heteroaryloxy, heteroarylthio,
heteroarylamino, heteroarylhydrocarbyl,
hydrocarbyloxycarbonyl-hydrocarbyl, heterocyclooxy,
hydroxycarbonyl-hydrocarbyl, heterocyclothio,
heterocycloamino, cyclohydrocarbyloxy,
cyclohydrocarbylthio, heteroarylhydrocarbyloxy,
heteroarylhydrocarbylthio, heteroaryl-
hydrocarbylamino, arylhydrocarbyloxy,
arylhydrocarbylthio, arylhydrocarbylamino,
heterocyclic, heteroaryl, hydroxycarbonyl-
hydrocarbyloxy, hydrocarbyloxycarbonylhydrocarbyloxy,
hydrocarbyloyl, arylcarbonyl, arylhydrocarbyloyl,
hydrocarboyloxy, arylhydrocarboyloxy,
hydroxyhydrocarbyl, hydroxyhydrocarbyloxy,
hydrocarbylthio, hydrocarbyloxyhydrocarbylthio,
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hydrocarbyloxycarbonyl, hydroxycarbonyl-
hydrocarbyloxy, hydrocarbyloxycarbonylhydrocarbyl,
hydrocarbylhydroxycarbonylhydrocarbylthio,
hydrocarbyloxycarbonylhydrocarbyloxy,
hydrocarbyloxycarbonylhydrocarbylthio,
hydrocarbylcarbonylamino, arylcarbonylamino,
cyclohydrocarbylcarbonylamino, heterocyclo-
hydrocarbylcarbonylamino, arylhydrocarbyl-
carbonylamino, heteroarylcarbonylamino,
heteroarylhydrocarbylcarbonylamino,
heterocyclohydrocarbyloxy, hydrocarbylsulfonylamino,
arylsulfonylamino, arylhydrocarbylsulfonylamino,
heteroarylsulfonylamino, heteroarylhydrocarbyl-
sulfonylamino, cyclohydrocarbylsulfonylamino,
heterocyclohydrocarbylsulfonylamino, N-
monosubstituted or N,N-disubstituted aminohydrocarbyl
group,
wherein the substituent(s) on the amino-
hydrocarbyl nitrogen atom are selected from the
group consisting of hydrocarbyl, aryl,
arylhydrocarbyl, cyclohydrocarbyl,
arylhydrocarbyloxycarbonyl,
hydrocarbyloxycarbonyl, and hydrocarboyl, or
wherein the aminohydrocarbyl nitrogen and two
substituents attached thereto form a 5- to 8-
membered heterocyclic or heteroaryl ring group,
amino, and a N-monosubstituted or N,N-disubstituted
amino group,
wherein the substituent(s) on the amino
nitrogen are selected from the group
consisting of hydrido, hydrocarbyl, aryl,
arylhydrocarbyl, cyclohydrocarbyl,
arylhydrocarbyloxycarbonyl,
hydrocarbyloxycarbonyl, hydrocarboyl,
arylsulfonyl, and hydrocarbylsulfonyl or
wherein the amino nitrogen and two substituents
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attached thereto form a 5- to 8-membered
heterocyclic or heteroaryl ring group;
Z is selected from the group consisting of
hydrido, hydrocarbyl, halogen, carboxy, cyano, azido,
hydrocarbylsulfonyl, carbonyloxyhydrocarbyl,
carbonylamido, and -X-Y wherein
-X is -O, -S or -NQ,
-Y is hydrido, hydrocarbyl or hydrocarbylaryl,
Q is hydrido, hydrocarbyl, hydroxylhydrocarbyl,
2-, 3-, or 4-pyridylhydrocarbyl, or
arylhydrocarbyl;
R1 is selected from the group consisting of an
azido, hydrido, hydrocarbyl, amido, hydrocarbylamino,
halohydrocarbyl, perhalohydrocarbyl and an aryl
substituent that is optionally substituted by one or
more substituents selected from the group consisting
of a halogen, hydrocarbyl, hydrocarbyloxy, nitro,
cyano, perfluorohydrocarbyl, trifluoromethyl-
hydrocarbyl, hydroxy, mercapto, hydroxycarbonyl,
aryloxy, arylthio, arylamino, arylhydrocarbyl, aryl,
heteroaryloxy, heteroarylthio, heteroarylamino,
heteroarylhydrocarbyl, hydrocarbyloxycarbonyl-
hydrocarbyl, heterocyclooxy, hydroxycarbonyl-
hydrocarbyl, heterocyclothio, heterocycloamino,
cyclohydrocarbyloxy, cyclohydrocarbylthio,
cyclohydrocarbylamino, heteroarylhydrocarbyloxy,
heteroarylhydrocarbylthio, heteroaryl-
hydrocarbylamino, arylhydrocarbyloxy,
arylhydrocarbylthio, arylhydrocarbylamino,
heterocyclic, heteroaryl, hydroxycarbonyl-
hydrocarbyloxy, alkoxycarbonylalkoxy, hydrocarbyloyl,
arylcarbonyl, arylhydrocarbyloyl, hydrocarboyloxy,
arylhydrocarboyloxy, hydroxyhydrocarbyl,
hydroxyhydrocarbyloxy, hydrocarbylthio,
hydrocarbyloxyhydrocarbylthio,
hydrocarbyloxycarbonyl, hydroxycarbonyl-
hydrocarbyloxy, hydrocarbyloxy-carbonylhydrocarbyl,
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hydrocarbylhydroxycarbonyl-hydrocarbylthio,
hydrocarbyloxycarbonylhydrocarbyloxy,
hydrocarbyloxycarbonylhydrocarbylthio, amino,
hydrocarbylcarbonylamino, arylcarbonylamino,
cyclohydrocarbylcarbonylamino,
heterocyclohydrocarbylcarbonylamino,
arylhydrocarbylcarbonylamino, heteroaryl-
carbonylamino, heteroarylhydrocarbylcarbonylamino,
heterocyclohydrocarbyloxy, hydrocarbylsulfonylamino,
arylsulfonylamino, arylhydrocarbylsulfonylamino,
heteroarylsulfonylamino, heteroarylhydrocarbyl-
sulfonylamino, cyclohydrocarbylsulfonylamino,
heterocyclohydrocarbylsulfonylamino and N-
monosubstituted or N,N-disubstituted aminohydrocarbyl
group,
wherein the substituent(s) on the amino-
hydrocarbyl nitrogen atom are selected from the
group consisting of hydrocarbyl, aryl,
arylhydrocarbyl, cyclohydrocarbyl,
arylhydrocarbyloxycarbonyl,
hydrocarbyloxycarbonyl, and hydrocarboyl, or
wherein the aminohydrocarbyl nitrogen and two
substituents attached thereto form a 5- to 8-
membered heterocyclic or heteroaryl ring group;
and
R2 is selected from the group consisting of an
azido, hydrido, hydrocarbyl, amido, halohydrocarbyl,
perhalohydrocarbyl, hydrocarbyloxycarbonyl, N-
piperazinylcarbonyl, aminocarbonyl, piperazinyl and
an aryl group that is substituted by one or more
substituents, said one or more substituents being
selected from the group consisting of a halogen,
hydrocarbyl, hydrocarbyloxy, nitro, cyano,
perfluorohydrocarbyl, trifluoromethylhydrocarbyl,
hydroxy, mercapto, hydroxycarbonyl, aryloxy,
arylthio, arylamino, arylhydrocarbyl, aryl,
heteroaryloxy, heteroarylthio, heteroarylamino,
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heteroarylhydrocarbyl, hydrocarbyloxycarbonyl-
hydrocarbyl, heterocyclooxy, hydroxycarbonyl-
hydrocarbyl, heterocyclothio, heterocycloamino,
cyclohydrocarbyloxy, cyclohydrocarbylthio,
cyclohydrocarbylamino, heteroarylhydrocarbyloxy,
heteroarylhydrocarbylthio, heteroarylhydrocarby-
amino, arylhydrocarbyloxy, arylhydrocarbylthio,
arylhydrocarbylamino, heterocyclic, heteroaryl,
hydroxycarbonyl-hydrocarbyloxy, alkoxycarbonylalkoxy,
hydrocarbyloyl, arylcarbonyl, arylhydrocarbyloyl,
hydrocarboyloxy, arylhydrocarboyloxy,
hydroxyhydrocarbyl, hydroxyhydrocarbyloxy,
hydrocarbylthio, hydrocarbyloxyhydrocarbylthio,
hydrocarbyloxycarbonyl, hydroxycarbonyl-
hydrocarbyloxy, hydrocarbyloxycarbonylhydrocarbyl,
hydrocarbylhydroxycarbonyl-hydrocarbylthio,
hydrocarbyloxycarbonylhydrocarbyloxy,
hydrocarbyloxycarbonylhydrocarbylthio, amino,
hydrocarbylcarbonylamino, arylcarbonylamino,
cyclohydrocarbylcarbonylamino, heterocyclo-
hydrocarbylcarbonylamino, arylhydrocarbylcarbonyl-
amino, heteroarylcarbonylamino,
heteroarylhydrocarbylcarbonylamino,
heterocyclohydrocarbyloxy, hydrocarbylsulfonylamino,
arylsulfonylamino, arylhydrocarbylsulfonylamino,
heteroarylsulfonylamino, heteroarylhydrocarbyl-
sulfonylamino, cyclohydrocarbylsulfonylamino,
heterocyclohydrocarbylsulfonylamino and N-
monosubstituted or N,N-disubstituted aminohydrocarbyl
group,
wherein the substituent(s) on the
aminohydrocarbyl nitrogen are selected from the
group consisting of hydrocarbyl, aryl,
arylhydrocarbyl, cyclohydrocarbyl,
arylhydrocarbyloxycarbonyl,
hydrocarbyloxycarbonyl, and hydrocarboyl, or
wherein the aminohydrocarbyl nitrogen and two
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substituents attached thereto form a 5- to 8-
membered heterocyclic or heteroaryl ring group;
and
provided that when A is =CH- and Z is hydrido,
hydrocarbyl, halogen, or hydrocarboyl:
1) Arl is other than an aryl group that is
substituted by one or more substituents selected from
the group consisting of a hydrido, halogen,
hydrocarbyl, perfluorohydrocarbyloxy, nitro,
perfluorohydrocarbyl, amino, aminosulfonyl,
halohydrocarbyloxyhydrocarbyl, hydroxy,
hydrocarbylsulfonylamino , hydrocarbylsulfonly,
acetylamino, carbonylhydrocarbylamino,
perfluorohydrocarbylsulfonyl, hydrocarbylamino,
carbonyl monosubstituted amino, carbonyl,
hydrocarbylthio, hydroxyhydrocarbyl, arylhydrocarbyl,
hydrocarbyloxyhydrocarbyl, hydrocarbyloxycarbonyl,
hydrocarbyloxyarylhydrocarbyl, halohydrocarbyloxy,
hydrocarbyloxyhydrocarbyl; or
2) R1 is other than hydrido, hydrocarbyl, aryl,
haloaryl, cyanoaryl, hydroxyaryl, hydrocarbylaryl,
cyano, perfluorohydrocarbyl, hydroxyhydrocarbyl,
arylhydrocarbyl, carboxy, hydrocarbyloxycarbonyl,
hydrocarboylhydrocarbyl, aminocarbonyl,
arylhydrocarbyl-hydrocarboyl-hydrocarbyl
monosubstituted amino carbonyl, hydrocarbyl-
hydrocarboyl-hydrocarbyl monosubstituted amino
carbonyl, hydrocarbyl-hydrocarbylhydrocarboyl-
hydrocarbyl monosubstituted amino carbonyl,
hydrocarbyl-hydoxy-disubstituted amino
carbonylhydrocarbyl, or a six membered heteroaryl
group substituted by a nitrogen atom; or
3) R2 is other than hydrido, carboxy,
hydrocarbyloxycarbonyl, halogen, or aryl.
In particularly preferred practice, A is =CH- so
that a contemplated 5-substituent is a 4-pyridyl
residue, as compared to being a 4-pyrimidyl residue.
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When examined primarily on the basis of
activity, Formula I includes three particularly
preferred subclasses of compounds of high interest.
One subclass of particular interest is a preferred
class of compounds exhibit ICso in vitro activities
in the assay discussed hereinafter and shown in
Table I of about 1.5 to less than (<) 10 ~M. This
embodiment comprises compounds of Formula I or a
pharmaceutically acceptable salt thereof wherein:
Arl is an aryl group that is substituted by a
group selected from fluorine, or lower hydrocarbyl;
R1 is hydrido, or lower hydrocarbyl;
R2 is selected from the group consisting of
hydrido, lowerhydrocarbyl and aminocarbonyl;
Z is hydrido, or -X-Y;
-X is -O or -NQ;
Q is aryl lower hydrocarbyl; and
-Y is hydrido or lower hydrocarbyl.
A more preferred subclass of compounds exhibit
ICso in vitro activities in the assay discussed
hereinafter of about 1.0 to less than (<) 1.5 ~,M.
This more preferred embodiment comprises those
compounds of Formula I or a pharmaceutically
acceptable salt thereof wherein:
Arl is an aryl group that is substituted by one
or more substituents that are lower hydrocarbyl, or
halo such as flourine;
R1 is hydrido, or lower hydrocarbyl;
R2 is hydrido or lower hydrocarbyl;
Z is hydrido or -X-Y;
-X is -NQ;
Q is lower hydrocarbyl or hydroxyl lower
hydrocarbyl; and
-Y is hydrido or lower hydrocarbyl.
The most preferred subclass of compounds of
Formula I exhibit ICso in vitro activities in the
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assay discussed hereinafter and shown in Table I of
less than (<) 1.0 ~,M. This subclass of compounds
comprise those compounds of Formula I or a
pharmaceutically acceptable salt thereof wherein:
Arl is an aryl group that is substituted by one
or more substituents that are lower hydrocarbyl or
halo such as flourine or chlorine;
R1 is hydrido, or lower hydrocarbyl;
R2 is hydrido;
Z is selected cyano or -X-Y;
wherein -X is -O, or -NQ;
Q is selected from a group consisting of
hydrido, lowerhydrocarbyl, aryl lower
hydrocarbyl, hydroxyl lower hydrocarbyl,
and 3-pyridyl lower hydrocarbyl; and
-Y is hydrido, lower hydrocarbyl, or aryl lower
hydrocarbyl.
It has been found that certain 1,5-diaryl pyrazoles
are effective for inhibition of mitogen-activated
protein (MAP) kinases. Mitogen-activated protein
kinases are believed to be associated with, inter alia,
the mediation of a number of inflammatory diseases. In
particular, it has been found that these certain 1,5-
diaryl pyrazoles are effective for the inhibition of the
p38 MAP kinase group of enzymes, a sub-family of MAP.
Because of the interrelation between p38
kinase and TNF, compounds of Formula I are useful
for, but not limited to, the treatment of a
disorder or disease state in a human, or other
mammal, that is exacerbated or caused by excessive
or unregulated TNF or p38 kinase production; i.e.,
pathological p38 MAP kinase activity, by such
mammal. Accordingly, the present invention
provides not only compounds but also a method of
treating a TNF-mediated disease that comprises
administering an effective TNF-inhibiting amount of
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a compound of Formula I, or a pharmaceutically
acceptable salt or tautomer thereof.
Compounds of Formula I are also useful for,
but not limited to, the treatment of inflammation
in a subject, and for use as an antipyretic for the
treatment of fever. Compounds of the invention is
useful to treat arthritis, including but not
limited to, rheumatoid arthritis,
spondyloarthropathies, gouty arthritis,
osteoarthritis, systemic lupus erythematosus and
juvenile arthritis, osteoarthritis, gouty arthritis
and other arthritic conditions. Such compounds are
further useful for the treatment of pulmonary
disorders or lung inflammation, including adult
respiratory distress syndrome, pulmonary
sarcoidosis, asthma, silicosis, and chronic
pulmonary inflammatory disease. The compounds are
also useful for the treatment of viral and
bacterial infections, including sepsis, septic
shock, gram negative sepsis, malaria, meningitis,
cachexia secondary to infection or malignancy,
cachexia secondary to acquired immune deficiency
syndrome (AIDS), AIDS, ARC (AIDS related complex),
pneumonia, and herpesvirus.
The compounds disclosed herein are also useful
for the treatment of bone resorption diseases, such
as osteoporosis, endotoxic shock, toxic shock
syndrome, reperfusion injury, autoimmune disease
including graft vs. host reaction and allograft
rejections, cardiovascular diseases including
atherosclerosis, thrombosis, congestive heart
failure, and cardiac reperfusion injury, renal
reperfusion injury, liver disease and nephritis,
and myalgias due to infection. The compounds are
also useful for the treatment of influenza,
multiple sclerosis, cancer, diabetes, systemic
lupus erthrematosis (SLE), skin-related conditions
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such as psoriasis, eczema, burns, dermatitis,
keloid formation, and scar tissue formation.
Compounds of the invention are also useful to treat
gastrointestinal conditions such as inflammatory
S bowel disease, Crohn's disease, gastritis,
irritable bowel syndrome and ulcerative colitis.
The compounds would also be useful in the treatment
of ophthalmic diseases, such as retinitis,
retinopathies, uveitis, ocular photophobia, and of
acute injury to the eye tissue. The compounds of
the invention can also be useful for preventing the
production of cyclooxygenase-2.
Besides being useful for human treatment,
these compounds are also useful for veterinary
treatment of companion animals, exotic animals and
farm animals, including mammals, rodents, and the
like. More preferred animals include horses, dogs,
and cats.
The present compounds can also be used in co-
therapies, partially or completely, in place of
other conventional anti-inflammatory agents, such
as together with steroids, cyclooxygenase-2
inhibitors, NSAIDs, DMARDS, immunosuppressive
agents, 5-lipoxygenase inhibitors, LTB4 antagonists
2S and LTA4 hydrolase inhibitors.
As used herein, the term "TNF-mediated
disorder" refers to any and all disorders and
disease states in which TNF plays a role, either by
control of TNF itself, or by TNF causing another
monokine to be released, such as but not limited to
IL-1, IL-6 or IL-8. A disease state in which, for
instance, IL-1 is a major component, and whose
production or action, is exacerbated or secreted in
response to TNF, is therefore considered a disorder
mediated by TNF.
As used herein, the term "p38-mediated
disorder" refers to any and all disorders and
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disease states in which p38 plays a role, either by
control of p38 itself, or by p38 causing another
factor to be released, such as but not limited to
IL-1, IL-6 or IL-8. A disease state in which, for
instance, IL-1 is a major component, and whose
production or action, is exacerbated or secreted in
response to p38, is therefore considered a disorder
mediated by p38.
As TNF- has close structural homology with
TNF (also known as cachectin), and because each
induces similar biologic responses and binds to the
same cellular receptor, both TNF- and TNF- are
inhibited by the compounds of the present invention
and thus are herein referred to collectively as
"TNF~~ unless specifically delineated otherwise.
When examined first on the basis of structure
and then functional activity among the 5-(4-pyridyl)
pyrazole substituents, one observes three further
subclasses of particularly preferred compounds within
the compounds of Formula I. One subclass of
particularly preferred compounds has structures that
are represented by Formula II, below, or a
pharmaceutically acceptable salt thereof:
R~
II
wherein R3 is hydrido, or C1-C6 (lower)
hydrocarbyl;
R4 is selected from a group consisting of
hydrido, lower hydrocarbyl, aryl lower hydrocarbyl,
hydroxyl lower hydrocarbyl, and 2-pyridyl lower
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hydrocarbyl, 3-pyridyl lower hydrocarbyl or 4-
pyridyl lower hydrocarbyl;
Arl is an aryl group that is substituted by a
halogen or halo group (e.g., chlorine, fluorine and
bromine), lower hydrocarbyl, or hydrocarbyloxy
group;
R1 hydrido, or C1-C6 hydrocarbyl; and
R2 is hydrido, or lower hydrocarbyl.
One preferred group of compounds of Formula II
noted in Table I exhibit ICSO in vitro activities in
the assay discussed hereinafter of less than (<) 10
and greater than (>) 1.5 ~M. This group of compounds
comprises those compounds of Formula II or a
pharmaceutically acceptable salt thereof wherein:
Arl is an aryl group that is substituted by
lower hydrocarbyl;
R1 is lower hydrocarbyl;
R2 is hydrido;
R3 is hydrido or C1-C6 hydrocarbyl; and
R4 is C1-C6 hydrocarbyl or aryl lower
hydrocarbyl.
A more preferred class of compounds of Formula
II noted in Table I exhibits ICso in vitro activities
in the assay discussed hereinafter of about 1.0 to
less than (<) 1.5 ~,M. This group of compounds is
comprised of those compounds of Formula II or a
pharmaceutically acceptable salt thereof wherein:
Arl is an aryl group that is substituted by
lower hydrocarbyl;
R1 is lower hydrocarbyl;
R2 is hydrido;
R3 is hydrido or lower hydrocarbyl; and
R4 is lower hydrocarbyl, or hydroxyl lower
hydrocarbyl.
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The most preferred class of compounds of Formula
II noted in Table I exhibits ICso in vitro activities
in the assay discussed hereinafter of less than (<)
1.0 ~.M. This group of compounds is comprised of
those compounds of Formula II or a pharmaceutically
acceptable salt thereof wherein:
Arl is an aryl group that is substituted with a
lower hydrocarbyl or halogen (e.g., flourine or
chlorine) group;
R1 is hydrido, or lower hydrocarbyl;
R2 is hydrido;
R3 is hydrido or lower hydrocarbyl; and
R4 is aryl lower hydrocarbyl, hydroxyl lower
hydrocarbyl, or 3-pyridyl lower hydrocarbyl.
A second preferred subclass of compounds within
Formula I has structures that correspond to Formula
III, or a pharmaceutically acceptable salt thereof
wherein:
Ark
N-N
\ \
N / R
OR5
R~
III
R5 is hydrido, C1-C6 hydrocarbyl, or aryl
lower hydrocarbyl;
Arl is an aryl group that is substituted with
a halogen (e. g., chlorine, fluorine or bromine),
lower hydrocarbyl, or hydrocarbyloxy group;
R1 is C1-C6 hydrocarbyl ; and
R2 is hydrido.
Preferred compounds of Formula III exhibit
ICso in vitro activities in the assay discussed
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hereinafter and shown in Table I of less than (<)
and greater than (>) 1.0 ~,M. This group of
compounds is comprised of those compounds of
Formula III or a pharmaceutically acceptable salt
5 thereof wherein:
Arl is an aryl group that is substituted by a
lower hydrocarbyl group;
R1 is lower hydrocarbyl;
R2 is hydrido; and
10 R5 is lower hydrocarbyl or aryl lower
hydrocarbyl.
A third subclass of preferred compounds within
Formula I have structures represented by Formula IV
or a pharmaceutically acceptable salt thereof
wherein:
Ark
N-N
\ \
N ~ R
CN
R~
IV
Arl is an aryl group that is substituted by a
halogen (e. g., chlorine, fluorine or bromine),
lower hydrocarbyl or a hydrocarbyloxy group;
R1 is lower hydrocarbyl; and
R2 is hydrido or lower hydrocarbyl.
Preferred compounds of Formula IV exhibit ICso
in vitro activities in the assay discussed
hereinafter and shown in Table I of less than (<)
10 and greater than (>) 1.0 ~M. This group of
compounds comprises of those compounds of Formula
IV or a pharmaceutically acceptable salt thereof
wherein:
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Arl is an aryl group that is substituted by a
lower hydrocarbyl group;
R1 is lower hydrocarbyl; and
R2 is hydrido.
The term "hydrido" denotes a single hydrogen atom
(H). This hydrido radical can be attached, for example,
to an oxygen atom to form a hydroxyl radical or two
hydrido radicals can be attached to a carbon atom to
form a methylene (-CH2-) radical.
The word "hydrocarbyl" is used herein as a
short hand term to include straight and branched
chain aliphatic as well as alicyclic groups or
radicals that contain only carbon and hydrogen.
Thus, alkyl, alkenyl and alkynyl groups are
contemplated, whereas aromatic hydrocarbons such as
phenyl and naphthyl groups, which strictly speaking
are also hydrocarbyl groups, are referred to herein
as aryl groups or radicals, as discussed hereinafter.
Where a specific aliphatic hydrocarbyl substituent
group is intended, that group is recited; i.e.,
alkyl, methyl or dodecenyl. Exemplary hydrocarbyl
groups contain a chain of 1 to about 12 carbon atoms,
and preferably one to about 10 carbon atoms. Most
preferred are lower hydrocarbyl radicals that contain
one to about six carbon atoms.
Usual chemical suffix nomenclature is
followed when using the word "hydrocarbyl" except
that the usual practice of removing the terminal "yl"
and adding an appropriate suffix is not always
followed because of the possible similarity of a
resulting name to one or more substituents. Thus, a
hydrocarbyl ether is referred to as a
"hydrocarbyloxy" group rather than a "hydrocarboxy"
group as may possibly be more proper when following
the usual rules of chemical nomenclature. On the
other hand, a hydrocarbyl group containing a -C(O)O-
functionality is referred to as a hydrocarboyl group
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inasmuch as there is no ambiguity in using that
suffix. As a skilled worker will understand, a
substituent that cannot exist such as a C1 alkenyl
group is not intended to be encompassed by the word
"hydrocarbyl".
Where used, either alone or within other terms
such as "haloalkyl", "alkylsulfonyl", "alkoxyalkyl" and
"hydroxyalkyl", "thioalkyl", the term "alkyl" embraces
linear or branched saturated radicals having one to
about twenty carbon atoms or, preferably, one to about
twelve carbon atoms. More preferred alkyl radicals are
radicals having one to about twelve carbon atoms. Most
preferred are lower alkyl radicals having one to about
six carbon atoms. Examples of such radicals include
methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,
sec-butyl, tart-butyl, pentyl, iso-amyl, hexyl and the
like.
The term "alkenyl" embraces linear or branched
radicals having at least one carbon-carbon double bond
of two to about twenty carbon atoms or, preferably, two
to about twelve carbon atoms. More preferred alkenyl
radicals are "lower alkenyl" radicals having two to
about six carbon atoms. Examples of alkenyl radicals
include ethenyl, propenyl, allyl, propenyl, butenyl and
4-methylbutenyl. The terms "alkenyl" and "lower
alkenyl", embrace radicals having "cis" and "trans"
orientations, or alternatively, "E" and "Z"
orientations.
The term "alkynyl" embraces linear or
branched radicals having at least one carbon-carbon
triple bond of two to about twenty carbon atoms or,
preferably, two to about twelve carbon atoms. More
preferred alkynyl radicals are "lower alkynyl"
radicals having two to about six carbon atoms.
Examples of alkynyl radicals include ethynyl
(acetylenyl), propynyl, butynyl and 4-methylbutynyl.
An alkyl group is a particularly preferred
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hydrocarbyl group. For ease in understanding, alkyl
groups are utilized hereinbelow in the explanations
of the nomenclature used herein for various
substituent groups. It is to be understood, however,
that the word "alkyl" is used to stand in for the
less familiar word "hydrocarbyl", which encompasses
not only alkyl groups, but also alkenyl and alkynyl
groups.
The term "cycloalkyl" embraces saturated
carbocyclic radicals having three to about twelve
carbon atoms. More preferred cycloalkyl radicals are
"lower cycloalkyl" radicals having three to about
eight carbon atoms. Examples of such radicals
include cyclopropyl, cyclobutyl, cyclopentyl and
cyclohexyl.
The term "cycloalkylalkylene" embraces alkyl
radicals substituted with a cycloalkyl radical. More
preferred cycloalkylalkylene radicals are "lower
cycloalkylalkylene", which embrace lower alkyl radicals
substituted with a lower cycloalkyl radical as defined
above. Examples of such radicals include
cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl
and cyclohexylmethyl.
The term "cycloalkenyl" embraces partially
unsaturated carbocyclic radicals having three to twelve
carbon atoms and one or two double bonds, but not
necessarily conjugated ("cycloalkyldienyl"). More
preferred cycloalkenyl radicals are "lower cycloalkenyl"
radicals having four to about eight carbon atoms.
Examples of such radicals include cyclobutenyl,
cyclopentenyl and cyclohexenyl.
The term "cycloalkenylalkylene" embraces alkyl
radicals substituted with a cycloalkenyl radical. More
preferred cycloalkenylalkylene radicals are "lower
cycloalkenylalkylene", which embrace lower alkyl
radicals substituted with a lower cycloalkenyl radical,
as defined above. Examples of such radicals include
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cyclobutenylmethyl, cyclopentenylmethyl and
cyclohexenylmethyl.
The term "halo" or "halogen" means halogens
such as fluorine, chlorine, bromine or iodine. The term
"haloalkyl" embraces radicals wherein any one or more of
the alkyl carbon atoms is substituted with halo as
defined above. Specifically embraced are monohaloalkyl,
dihaloalkyl and polyhaloalkyl radicals. A monohaloalkyl
radical, for one example, can have either an iodo,
bromo, chloro or fluoro atom within the radical. Dihalo
and polyhaloalkyl radicals can have two or more of the
same halo atoms or a combination of different halo
radicals. "Lower haloalkyl" embraces radicals having
one to six carbon atoms. Examples of haloalkyl radicals
include fluoromethyl, difluoromethyl, trifluoromethyl,
chloromethyl, dichloromethyl, trichloromethyl,
pentafluoroethyl, heptafluoropropyl,
difluorochloromethyl, dichlorofluoromethyl,
difluoroethyl, difluoropropyl, dichloroethyl and
dichloropropyl.
The term "hydroxyalkyl" embraces linear or
branched alkyl radicals having one to about twelve
carbon atoms, any one of which can be substituted with
one or more hydroxyl radicals. More preferred
hydroxyalkyl radicals are "lower hydroxyalkyl" radicals
having one to six carbon atoms and one or more hydroxyl
radicals. Examples of such radicals include
hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl
and hydroxyhexyl.
The terms "alkoxy" and "alkyloxy" embrace
linear or branched oxy-containing radicals each having
alkyl portions of one to about twelve carbon atoms.
More preferred alkoxy radicals are "lower alkoxy"
radicals having one to six carbon atoms. Examples of
such radicals include methoxy, ethoxy, propoxy, butoxy
and tert-butoxy.
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The term "alkoxyalkyl" embraces alkyl radicals
having one or more alkoxy radicals attached to the alkyl
radical to form, for example, monoalkoxyalkyl and
dialkoxyalkyl radicals. The "alkoxy" radicals can be
further substituted with one or more halo atoms, such as
fluoro, chloro or bromo, to provide "haloalkoxy"
radicals.
The term "aryl", alone or in combination,
means a carbocyclic aromatic system containing one, two
or three rings wherein such rings can be attached
together in a pendent manner or can be fused. More
preferred aryl are 6-12 membered aryl radicals.
Examples of such radicals include phenyl, naphthyl,
tetrahydronaphthyl, indane and biphenyl. Phenyl
radicals are preferred aryl radicals.
Aryl moieties can also be substituted at a
substitutable position with one or more substituents
selected independently from alkyl, alkoxyalkyl,
alkylaminoalkyl, carboxyalkyl, alkoxycarbonylalkyl,
aminocarbonylalkyl, alkoxy, aralkoxy, hydroxyl, amino,
halo, nitro, alkylamino, acyl, cyano, carboxy,
aminocarbonyl, alkoxycarbonyl and aralkoxycarbonyl.
The term "heterocyclyl" embraces saturated,
partially unsaturated and aromatically-unsaturated
heteroatom-containing ring-shaped radicals, which can
also be called "heterocyclyl", "heterocycloalkenyl" and
"heteroaryl" respectively, where the heteroatoms are
nitrogen, sulfur and oxygen. Examples of saturated
heterocyclyl radicals include saturated 3 to 6-membered
heteromonocyclic group containing 1 to 4 nitrogen atoms
(e. g. pyrrolidinyl, imidazolidinyl, piperidino,
piperazinyl, etc.); saturated 3 to 6-membered
heteromonocyclic group containing 1 to 2 oxygen atoms
and 1 to 3 nitrogen atoms (e. g. morpholinyl, etc.);
saturated 3 to 6-membered heteromonocyclic group
containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms
(e. g., thiazolidinyl, etc.). Examples of partially
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unsaturated heterocyclyl radicals include
dihydrothiophene, dihydropyran, dihydrofuran and
dihydrothiazole. Heterocyclyl radicals can include a
tetravalent nitrogen, such as in tetrazolium and
pyridinium radicals.
The term "heteroaryl" embraces aromatically-
unsaturated heterocyclyl radicals. Examples of
heteroaryl radicals include unsaturated 5- to 10-
membered heteromonocyclic group containing 1 to 4
nitrogen atoms, for example, pyrrolyl, pyrrolinyl,
imidazolyl, pyrazolyl, pyridyl, pyrimidyl, pyrazinyl,
pyridazinyl, triazolyl (e.g., 4H-1,2,4-triazolyl, 1H-
1,2,3-triazolyl, 2H-1,2,3-triazolyl) tetrazolyl (e. g.
1H-tetrazolyl, 2H-tetrazolyl, etc.), and the like;
unsaturated condensed heterocyclyl group containing 1 to
5 nitrogen atoms, for example, indolyl, isoindolyl,
indolizinyl, benzimidazolyl, quinolyl, isoquinolyl,
indazolyl, benzotriazolyl, tetrazolopyridazinyl (e. g.,
tetrazolo[1,5-b]pyridazinyl), and the like; an
unsaturated 5- or 6-membered heteromonocyclic group
containing an oxygen atom, for example, pyranyl or
furyl; unsaturated 5- or 6-membered heteromonocyclic
group containing a sulfur atom, for example, thienyl;
unsaturated 5- to 6-membered heteromonocyclic group
containing 1 to 2 oxygen atoms and 1 to 3 nitrogen
atoms, for example, oxazolyl, isoxazolyl, oxadiazolyl
(e. g., 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, I,2,5-
oxadiazolyl) and the like; unsaturated condensed
heterocyclyl group containing 1 to 2 oxygen atoms and 1
to 3 nitrogen atoms (e. g. benzoxazolyl, benzoxadiazolyl,
etc.); unsaturated 5- or 6-membered heteromonocyclic
group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen
atoms, for example, thiazolyl, thiadiazolyl (e. g.,
1,2,4- thiadiazolyl, 1,3,4-thiadiazolyl,and 1,2,5-
thiadiazolyl) and the like; unsaturated condensed
heterocyclyl group containing 1 to 2 sulfur atoms and 1
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to 3 nitrogen atoms (e.g., benzothiazolyl or
benzothiadiazolyl) and the like.
The term "heteroaryl" also embraces radicals
where heterocyclyl radicals are fused with aryl
radicals. Examples of such fused bicyclic radicals
include benzofuran, benzothiophene, and the like.
A heterocyclyl group can have 1 to 3
substituents such as alkyl, hydroxyl, halo, alkoxy, oxo,
amino and alkylamino. The term "heterocyclylalkylene"
embraces heterocyclyl-substituted alkyl radicals. More
preferred heterocyclylalkylene radicals are "lower
heterocyclylalkylene" radicals having one to six carbon
atoms and a heterocyclyl radical.
The term "alkylthio" embraces radicals
containing a linear or branched alkyl radical, of one to
about twelve carbon atoms attached to a divalent sulfur
atom. More preferred alkylthio radicals are "lower
alkylthio" radicals having alkyl radicals of one to six
carbon atoms. Examples of such lower alkylthio radicals
are methylthio, ethylthio, propylthio, butylthio and
hexylthio.
The term "alkylthioalkylene" embraces radicals
containing an alkylthio radical attached through the
divalent sulfur atom to an alkyl radical of one to about
twelve carbon atoms. More preferred alkylthioalkylene
radicals are "lower alkylthioalkylene" radicals having
alkyl radicals of one to six carbon atoms. Examples of
such lower alkylthioalkylene radicals include
methylthiomethyl.
The term "alkylsulfinyl" embraces radicals
containing a linear or branched alkyl radical, of one to
about twelve carbon atoms, attached to a divalent -
S(=O)- radical. More preferred alkylsulfinyl radicals
are "lower alkylsulfinyl" radicals having alkyl radicals
of one to six carbon atoms. Examples of such lower
alkylsulfinyl radicals include methylsulfinyl,
ethylsulfinyl, butylsulfinyl and hexylsulfinyl.
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The term "sulfonyl", whether used alone or
linked to other terms such as "alkylsulfonyl", or
"halosulfonyl" denotes a divalent radical, -SOZ-.
"Alkylsulfonyl" embraces alkyl radicals attached to a
sulfonyl radical, where alkyl is defined as above. More
preferred alkylsulfonyl radicals are "lower
alkylsulfonyl" radicals having one to six carbon atoms.
Examples of such lower alkylsulfonyl radicals include
methylsulfonyl, ethylsulfonyl and propylsulfonyl.
The "alkylsulfonyl" radicals can be further
substituted with one or more halo atoms, such as fluoro,
chloro or bromo, to provide haloalkylsulfonyl radicals.
The term " halosulfonyl" embraces halo radicals
attached to a sulfonyl radical. Examples of such
halosulfonyl radicals include chlorosulfonyl and
bromosulfonyl.
The terms "sulfamyl", "aminosulfonyl" and
"sulfonamidyl" denote NH202S-. The term "carbonyl",
whether used alone or with other terms, such as
"alkoxycarbonyl", denotes -(C=O)-. The terms "carboxy"
or "carboxyl", whether used alone or with other terms,
such as "carboxyalkyl", denotes -C02-. The term
"carboxyalkyl" embraces alkyl radicals substituted with
a carboxy radical. More preferred are "lower
carboxyalkyl" radicals that embrace carboxy-substituted
lower alkyl radicals, as defined above. Examples of
such lower carboxyalkyl radicals include carboxymethyl,
carboxyethyl and carboxypropyl.
The term "alkoxycarbonyl" means a radical
containing an alkoxy radical, as defined above, attached
via an oxygen atom to a carbonyl radical. More
preferred are "lower alkoxycarbonyl" radicals with alkyl
portions having one to six carbons. Examples of such
lower alkoxycarbonyl (ester) radicals include
methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl,
butoxycarbonyl and hexyloxycarbonyl.
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The term " alkoxycarbonylalkylene" embraces
alkyl radicals substituted with an alkoxycarbonyl
radical as defined above. More preferred are " lower
alkoxycarbonylalkylene" radicals with alkyl portions
having one to six carbons. Examples of such lower
alkoxycarbonylalkylene radicals include
methoxycarbonylmethylene, ethoxycarbonylmethylene,
methoxycarbonylethylene and ethoxycarbonylethylene.
The term "alkylcarbonyl", includes radicals
having alkyl radicals attached to a carbonyl radical.
Examples of such radicals include methylcarbonyl,
ethylcarbonyl, propylcarbonyl, butylcarbonyl, and
pentylcarbonyl.
The term "aralkyl" embraces aryl-substituted
alkyl radicals. Preferred aralkyl radicals are " lower
aralkyl" , having lower alkyl groups substituted with
one or more aryl groups. Examples of such groups
include benzyl, diphenylmethyl, triphenylmethyl,
phenylethyl, and diphenylethyl. The aryl in such an
aralkyl group can be additionally substituted with halo,
alkyl, alkoxy, haloalkyl and haloalkoxy moieties. The
terms benzyl and phenylmethyl are interchangeable.
The term "heterocyclylalkylene" embraces
saturated, partially unsaturated and unsaturated
heterocyclyl-substituted alkyl radicals such as
pyrrolidinylmethyl, pyridylmethyl, quinolylmethyl,
thienylmethyl, furylethyl, and quinolylethyl. The
heteroaryl in heteroaralkyl (unsaturated heterocyclyl-
substituted alkyl radicals) can be additionally
substituted with halo, alkyl, alkoxy, haloalkyl and
haloalkoxy groups.
The term "aryloxy" embraces aryl radicals attached
through an oxygen atom to other radicals. The term
"aralkoxy" embraces aralkyl radicals attached through an
oxygen atom to other radicals.
The term "aminoalkyl" embraces alkyl radicals
substituted with amino radicals. More preferred are
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"lower aminoalkyl" radicals. Examples of such radicals
include aminomethyl, aminoethyl, and the like. The term
"alkylamino" denotes amino groups that are substituted
with one or two alkyl radicals. Preferred are "lower
alkylamino" radicals having alkyl portions having one to
six carbon atoms. Suitable lower alkylamino radicals
can be monosubstituted N-alkylamino or disubstituted
N,N-alkylamino, such as N-methylamino, N-ethylamino,
N,N-dimethylamino, N,N-diethylamino or the like.
The term "arylamino" denotes amino groups that
are substituted with one or two aryl radicals, such as
N-phenylamino. The "arylamino" radicals can be further
substituted on the aryl ring portion of the radical as
discussed previously for other aryl-containing radicals.
The term "aminocarbonyl" denotes an amide
group of the formula -C(=O)NH2. The term
"alkylaminocarbonyl" denotes an aminocarbonyl group that
has been substituted with one or two alkyl radicals on
the amino nitrogen atom. Preferred are "N-
alkylaminocarbonyl" and "N,N-dialkylaminocarbonyl"
radicals. More preferred are "lower N-
alkylaminocarbonyl" and "lower N,N-dialkylaminocarbonyl"
radicals with lower alkyl portions as defined above.
The term "alkylcarbonylamino" embraces amino
groups that are substituted with one or more
alkylcarbonyl radicals. More preferred
alkylcarbonylamino radicals are "lower
alkylcarbonylamino" having lower alkylcarbonyl radicals
as defined above attached to amino radicals. The term
"alkylaminoalkylene" embraces radicals having one or
more alkyl radicals attached to an aminoalkyl radical.
Tables 1 through 14 hereinafter illustrate
compounds of Formulas II, III and IV that illustrate
preferred substituent groups other than hydrido for one
of substituents Arl, R1, R2, R3, R4, and R5. The
remaining groups Arl, R1, R2, R3, R4, and R5 illustrated
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for each structure shown in a compound table are as
discussed elsewhere herein.
Table 1
Ary Ar \N-N Ar,\
N- \
R, I ~ \ \ Rt ~ ~ R,
I N ~ R2 I / I2
OR5 CN
II III 1V
Arl
\ CH3 CH3 H3C \
~i (\ ~i
1.1 2.1 ~ 4.1
3.1
H3C
HsC CHs
\ \ \ CH3 H3C \
5.1 6.1 7.1 8.1
HC
H3C CH3 H3C CH H3C CH
H3C 3
_.~
9.1 10.1 11.1 12.1
H3C CH3 H3C H C
~CH3 3
a H HsC _ ~ \ ~ \
13.1 / 14.1 ~ 15.1 I ~ CH3
16.1
CH3 CH3 CH3
H3C CH3
H3C
H3C ~ I \ I \ CH3 I \ CH3
17.1 ~ 18.1 ~ 19.1 i
20.1
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Table 2
Ar\ Ar y Ar\
N-N
R~ R~ ~ ~ \ R,
12
NR'R° CN
II III N
Art
HsC CH3 CH3
H3C
H3C CHs ~CH3 H3C CH3 CH3
H3C ~ \ HsC ~ \
1.2 ~ 2.2 ~ i 3.2
4.2
CH3 CH3
CH3 H3C CH3 H3C CH3 H3C
\ CH3 H3C ( \ \ CH3
5.2 ~ 6.2 I ~ 7.2 I ~ CH3 8.2
CH3 H3C H3C H C
3
\ \ \ \
11.2 I ~ 12.2
9.2 10.2
CH3 H3C CH3
\ i \ I \ H3C I \
13.2 / 14.2 / 15.2 16.2
H3C CH H3C
3
CH
3
17.2 18.2 19.2 20.2
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Table 3
Ark Ar~~ Are
N-N ~N_N
R' \ \ \ R' \ \ R~
NR3R'° OR5 CN
11 111 1 V
Arl
HO OH OH O
\ ~ / HO I \
1.3 / 2.3 3.3 / 4.3
HO O
O N02
OZN
\ I ~ OH I \
/ / / /
5.3 6.3 7.3 8.3
NH2
\ NOp HZN \ NHZ
~/ ~/ ~/ ~/
9.3 10.3 11.3
12.3
CI F
CI \ CI \
~\ ~/ ~/ I/
13.2 14.3 15.3 16.2
F Br
Br I \ I \ Br
F \
/ ~/
17.3 18.3 19.3 20.3 21.3
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Table 4
Ark Ark Are
N-N N-N
\ R~ I ~ ~ \ R~ R~
N~ R2 N' R2
NR3R4 ' IORS
II III IV
R~
/CHs ~ CHs /~ CHs /\/~CH
3
1.4 2.4 3,4 4.4
CHs
~/~CHs CH ~ ~CH3
3
CH3
5.4 6 CHs
4
. 7.4 8.4
CHs
CHs CHs CHs
- I CH
CH3 CHs s ~ CH
~
CH
s s
9 3
4
. 10.4 11.4 12.4
CHs CHs
/CHs CH CHs CHs CHs
3
~ ~ CH3
CH ~ / v '
s CHs
3
13.4 14.4 15.4 16.4
CHs CHs
' v 'CH ~CHa ~~'CHs ~CH3
. '
'~
s CH3 TCH3 C~H3 Hs
I7.4 18.4 19.4
20.4
CHs
~CH3
CHs
21.4
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Table 5
Ar ~ Ar ~ Are
N-N N-N
\ \ W R~ R~ R~
N
NR3R4 CN
II III IV
R1
/CHs CHs /~CH3 /\/~CH
3
1.5 2.5 3,5 4.5
CHs ~ _CHs
~CH3 ~ ~C / ~
H ~CH
3
5.5 6.5 CHs
8
5
7.5 .
CHs
~CH3 CHs CHs
CH
CHs CH ~. /~.~
3 C
s CHs Hs
CH
9.5 10.5 11.5 s 12.5
CHs CHs
/CHs CH CHs CHs CHs
s ~ ~
CHs
CH ~ ' v 'C
s Hs
3
13.5 14.5 15.5 16.5
CHs CHs
CHs I CHs CHs CHs
~ '
'' ~
CHs CHs C
H3Hs
17.5 18.5 19.5
20.5
CHs
H N
CH N.
3 ~ N i
I CHa CHs ~
CH ~ CHs
3 ~
21.5 22.5 23.5 24.5
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Table 6
Ar,
-N
N
\ ~ R,
N / R2
NR3R4
R3 11
/CH3 'CHs /~CH3 /\~CH
3
1.6 2.6 3.6 4.6
CH3 ~ _CH3
~CH3 ~~/\/~C ' ~
H ~
CHg
5.6 6.6 CH3
7.6 8.6
CH3
~~/CH3 CH3 ~CH3
CH ' T ~CH3
3 CH3 CH3 HCH3
I
9.6 10.6 11.6 C
3 12.6
CH3 CH3
/CH3 CH3 CH3 CH3 CH3
~CH ~ ~ /\iCH3
~CH
3 3 3
13.6 14.6 15.6 16.6
CH3 CH3
_CH I CH3 ~CH3 ~CH3
~ '
3 CH3 CH3 C~H3 H3
17.6 18.6 19.6
20.6
CH3
~CH3
CH3
21.6
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Table 7
Ark
N-N
~ ~Ri
12
NR3R4
R4 11
/CH3 ~CHa /~CH3 /~/~CH
1.7 2.7 3.7 3 4.7
CH3
~CH3 CH ~ ~CH3
3 CHs
5.7 6.7 CH3
7.7 8.7
CH3
~~ /CH3 CH3
CH3 - T ~,~.CH3 /~ CH3
CH3 CH3 ICHCH3
9.7 10. 11.7 3 12.7
7
CHaCH3 CH3 CH CHa CH3 CH3
/~ 3 ~ ~ CH3
~CH3 ~ ' v _CH3
CH3
13.7 14.7 15.7 16.7
CH3 CH3
~CH3 I CH3 ~~/CH3 ~CH3
CH
CH3 CH3 H3
17.7 I 8. 19.7 20.7
CH3 7
CHs ~ i ~ i ~ i
CH3
21.7 22.7 23.7 25.7
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Table 8
,ar,
\N-N
12
NR3R4
R4 II
i i ~~ i
CH3
1.8 2.8 3.8 4.8
CH3 I j CH3 I j CH3 I j CH3
CH3 CH3 CH3
5.8 6.8 7.8 8.8
CH3 I ~ ~ OOH
OOH OH
CH3 CH3
10.8 11.8 12.8
9.8
OOH OOH ~~OH OH
13.8 14.8 15.8 16.8
CH3 OH CH3
CH3 ~OH ~OH
CH
~OH CH3
17.8 18.8 19.8 20.8
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Table 9
Ar,
v
R'
r
NR'R"
R4 II
OH OH OH OIH
CHs CHs CHs CHs
1.9 CH3 2 CH3 C~ H3
9
. 3.9 4.9
OH CH3
CH3 ~CH3 OH OH
~ ~ ~ ~
~ ~
OH CH3 CH3
3
6.9
5.9 7.9 8.9
OH ~ ~'CH3 OH
~CH ' T ~CH ~CH3
_
3 OH 3 OH
9.9 10.9 11.9 12.9
OH OH
%~CH3 '~CH3 ~~CH3 ~!~CH3
O
O OH
13.9 14.9 15.9 16.9
OH OH OH CH3 OH
~CH3 ~CH3 ~CH3
'~~CH ~ _ I
3
T CH3
CH3
17.9 18.9 19.9 20.9
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Table 10
Ark
N-N
R~
R4 II
CH3 CH3 CH3 CH3
~CH3 ~ OH CH3
OH ~CH3 ~CH3 ~CH3
1.10 2.10 OOH 3.10 4.10
IN I , I ~ IN
5.10
6.10 7.10
8.10
~N
I 'N I ~ I ' I
N
9.10 10.10
11.10 12.10
N I ~ I N
13.10 14.10 15.10 16.10
IN I ; I N
17.10 18.10 19.10
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Table 11
Ark
N-N
\ ~R~
NR3R4
R4 II
.N
3
HsC I N HsC I ~ N HsC / CH
1.11 2.11 3.11 N 4.11
y
CHs W CHs ~ ~ N ~ ~ ~ N
~ N / N CHs
CHs 8.1 I
5.11 6.11 7.11
~ N CHs ~ CHs I ~ CHs I ~ N
/ ~ J .N /
N
CHs 9.11 CHs CHs 11.11 CHs 12.11
10.11
CHs W
i ~N ~ O
H3C NJ HsC ~ N H C I / HsC N
3
13.11 14.11 15.11 16.11
y
CHs I ~ CHs I ~ N H C ( ~ HsC I ~ N
H C ~ N HsC / s N
3
17.11 18.11 19.11 20.11
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Table 12
Ark
~N-N
\ \ \
N ~ R
NR3R4
R4 II
R~
I I ~ N CH3
H3C ~ N H3C ~ N
1.12 2.12 3.12
CHg ~ N I I
N ~N
CH3 CH3
4.12 5.12
6.12
CH3 I ~ CHg I ~ CHg I ~ N
~N
CH3 CH3 CH3 CHg CH3 CH3
7.12 8.12 9.12
~N
I , CHg
~N
CH3
10.12 11.12
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Table 13
Ar~~
R'
III
Rs
/CH3 ~CHa /~CH3 NCH
3
1.13 2.13 3.13 4.13
CH3
~CH3 NCH ~/CH3
~
CH
CH3
5.13 6.13 7.13 8.13
CH3
CH3 CH3 /\~ CH3
CH ~ CH3
3 CH3 CH
1
CH3 3
CH
3
9.13 10.13 11.13 12.13
CH3 CH3
/CH3 CH CHs CH3 CH3
~ ~
CH3
CH ~ ' v 'CH
3 3
3
13.13 14.13 15.13 16.13
CH3 CH3
v _CH3 I CH3 ~CH3 ~CH3
H I ' '
~
3 CH3 C
C H3 H3
17.13 18.13 19.13 20.13
3
C~CH3
CH
3
21. I 3 22.13 23.13 25.13
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Table 14
Ark
N-N
R'
OR'
RS III
i ~ i ~ i
1.14 2.14 3.14
CH3 I ~ CH3 I
CH3 CH3
4.14 5.14
6.14
CH3 I ~ CH3 I ~ CH3 i
CH3 CH3 CH3 CH3
7.14 8.14
9.14
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Treatment Process
The present invention also contemplates a process
for the treatment of a TNF-mediated disorder or a p38
kinase-mediated disorder, such as arthritis. That
process comprises administering a therapeutically-
effective amount (a p38 MAP kinase enzyme-inhibiting
effective amount) of a compound of Formula I, or a
pharmaceutically-acceptable salt thereof, to a mammalian
host having such a condition. A mixture of such
compounds can also be used. The use of administration
repeated a plurality of times is particularly
contemplated.
Also included in the family of compounds of
Formula I (and also Formulas II, III and IV) are the
pharmaceutically-acceptable salts of those compounds, as
noted previously. The term "pharmaceutically-acceptable
salts" embraces salts commonly used to form alkali metal
salts and to form addition salts of free acids or free
bases. The nature of the salt is not critical, provided
that it is pharmaceutically-acceptable. Suitable
pharmaceutically-acceptable acid addition salts of
compounds of Formula I can be prepared from an inorganic
acid or from an organic acid.
Examples of such inorganic acids are hydrochloric,
hydrobromic, hydroiodic, nitric, carbonic, sulfuric and
phosphoric acid. Appropriate organic acids can be
selected from aliphatic, cycloaliphatic, aromatic,
araliphatic, heterocyclyl, carboxylic and sulfonic
classes of organic acids. Illustrative pharmaceutically
acceptable salts are prepared from formic, acetic,
propionic, succinic, glycolic, gluconic, lactic, malic,
tartaric, citric, ascorbic, glucuronic, malefic, fumaric,
pyruvic, aspartic, glutamic, benzoic, anthranilic,
mesylic, stearic, salicylic, p-hydroxybenzoic,
phenylacetic, mandelic, embonic (pamoic),
methanesulfonic, ethanesulfonic, benzenesulfonic,
pantothenic, toluenesulfonic, 2-hydroxyethanesulfonic,
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sulfanilic, cyclohexylaminosulfonic, algenic, -
hydroxybutyric, galactaric and galacturonic acids.
Suitable pharmaceutically-acceptable base addition
salts of compounds of Formula I include metallic ion
salts and organic ion salts. More preferred metallic
ion salts include, but are not limited to appropriate
alkali metal (group Ia) salts, alkaline earth metal
(group IIa) salts and other physiological acceptable
metal ions. Such salts can be made from the ions of
aluminum, calcium, lithium, magnesium, potassium, sodium
and zinc. Preferred organic salts can be made from
tertiary amines and quaternary ammonium salts, including
in part, trimethylamine, diethylamine, N,N'-
dibenzylethylenediamine, chloroprocaine, choline,
diethanolamine, ethylenediamine, meglumine (N-
methylglucamine) and procaine. All of the above salts
can be prepared by conventional means from the
corresponding compound of Formula I by reacting for
example, the appropriate acid or base with the compound
of Formula I.
A compound of Formula I is preferably administered
in a pharmaceutical composition. Such a composition
contains a therapeutically-effective amount of a
compound of Formula I in association with at least one
pharmaceutically-acceptable carrier, adjuvant or
diluent.
Thus, also embraced within this invention is a
class of pharmaceutical compositions comprising a
compound of Formula I as active ingredient (agent or
compound ) in association with one or more non-toxic,
pharmaceutically-acceptable carriers and/or diluents
and/or adjuvants (collectively referred to herein as
"carrier" materials) and, if desired, other active
ingredients.
The active compounds of the present invention
can be administered by any suitable route, preferably
in the form of a pharmaceutical composition adapted
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to such a route, and in a dose effective for the
treatment intended. The active compounds and
composition can, for example, be administered orally,
intravascularly (IV), intraperitoneally,
subcutaneously, intramuscularly (IM) or topically.
For oral administration, the pharmaceutical
composition can be in the form of, for example, a
tablet, hard or soft capsule, lozenges, dispensable
powders, suspension or liquid. The pharmaceutical
composition is preferably made in the form of a
dosage unit containing a particular amount of the
active ingredient. Examples of such dosage units are
tablets or capsules.
The active ingredient can also be administered
by injection (IV, IM, subcutaneous or jet) as a
composition wherein, for example, saline, dextrose,
or water can be used as a suitable carrier. The pH
value of the composition can be adjusted, if
necessary, with suitable acid, base, or buffer.
Suitable bulking, dispersing, wetting or suspending
agents, including mannitol and PEG 400, can also be
included in the composition. A suitable parenteral
composition can also include a compound formulated as
a sterile solid substance, including lyophilized
powder, in injection vials. Aqueous solution can be
added to dissolve the compound prior to injection.
The amount of therapeutically active compounds
that are administered and the dosage regimen for
treating a disease condition with the compounds
and/or compositions of this invention depends on a
variety of factors, including the age, weight, sex
and medical condition of the subject, the severity of
the inflammation or inflammation related disorder,
the route and frequency of administration, and the
particular compound employed, and thus can vary
widely.
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A pharmaceutical composition can contain an
active compound at about 0.1 to 1000 mg, preferably
at about 7.0 to 350 mg. A daily dose of about 0.01
to 100 mg/kg body weight, preferably between about
0.1 and about 50 mg/kg body weight, and most
preferably between about 0.5 to 30 mg/kg body weight,
can be appropriate. The daily dose can be
administered in one to four doses per day.
In the case of skin conditions, it can be
preferable to apply a topical preparation of
compounds of this invention to the affected area two
to four times a day. For disorders of the eye or
other external tissues, e.g., mouth and skin, the
formulations are preferably applied as a topical gel,
spray, ointment or cream, or as a suppository,
containing the active ingredients in a total amount
of, for example, 0.075 to 30% w/w, preferably 0.2 to
20% w/w and most preferably 0.4 to 15% w/w.
When formulated in an ointment, the active
ingredients can be employed with either paraffinic or
a water-miscible ointment base. Alternatively, the
active ingredients can be formulated in a cream with
an oil-in-water cream base. If desired, the aqueous
phase of the cream base can include, for example at
least 30% w/w of a polyhydric alcohol such as
propylene glycol, butane-1,3-diol, mannitol,
sorbitol, glycerol, polyethylene glycol and mixtures
thereof .
The topical formulation can desirably include a
compound that enhances absorption or penetration of
the active ingredient through the skin or other
affected areas. Examples of such dermal penetration
enhancers include dimethylsulfoxide and related
analogs.
The compounds of this invention can also be
administered by a transdermal device. Preferably,
topical administration is accomplished using a patch
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either of the reservoir and porous membrane type or
of a solid matrix variety. In either case, the
active agent is delivered continuously from the
reservoir or microcapsules through a membrane into
the active agent permeable adhesive, which is in
contact with the skin or mucosa of the recipient. If
-the active agent is absorbed through the skin, a
controlled and predetermined flow of the active agent
is administered to the recipient. In the case of
microcapsules, the encapsulating agent can also
function as the membrane. The transdermal patch can
include the compound in a suitable solvent system
with an adhesive system, such as an acrylic emulsion,
and a polyester patch.
The oily phase of the emulsions of this
invention can be constituted from known ingredients
in a known manner. Although the phase can comprise
merely an emulsifier, it can comprise a mixture of at
least one emulsifier with a fat or an oil or with
both a fat and an oil. Preferably, a hydrophilic
emulsifier is included together with a lipophilic
emulsifier that acts as a stabilizer. It is also
preferred to include both an oil and a fat.
Together, the emulsifiers) with or without
stabilizers) make-up the so-called emulsifying wax,
and the wax together with the oil and fat make up the
so-called emulsifying ointment base which forms the
oily dispersed phase of the cream formulations.
Emulsifiers and emulsion stabilizers suitable for use
in the formulation of the present invention include
TweenT'" 60, SpanT"' 80, cetostearyl alcohol, myristyl
alcohol, glyceryl monostearate, and sodium lauryl
sulfate, among others.
The choice of suitable oils or fats for the
formulation is based on achieving the desired
cosmetic properties, since the solubility of the
active compound in most oils likely to be used in
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pharmaceutical emulsion formulations is very low.
Thus, the cream should preferably be a non-greasy,
non-staining and washable product with suitable
consistency to avoid leakage from tubes or other
containers. Straight or branched chain, mono- or
dibasic alkyl esters such as di-isoadipate, isocetyl
stearate, propylene glycol diester of coconut fatty
acids, isopropyl myristate, decyl oleate, isopropyl
palmitate, butyl stearate, 2-ethylhexyl palmitate or
a blend of branched chain esters can be used. These
can be used alone or in combination depending on the
properties required. Alternatively, high melting
point lipids such as white soft paraffin and/or
liquid paraffin or other mineral oils can be used.
Formulations suitable for topical administration
to the eye also include eye drops wherein the active
ingredients are dissolved or suspended in suitable
carrier, especially an aqueous solvent for the active
ingredients. The anti-inflammatory active ingredients
are preferably present in such formulations in a
concentration of 0.5 to 20%, advantageously 0.5 to
10% and particularly about 1.5% w/w.
For therapeutic purposes, the active compounds
of this combination invention are ordinarily combined
with one or more adjuvants appropriate to the
indicated route of administration. If administered
per os, the compounds can be admixed with lactose,
sucrose, starch powder, cellulose esters of alkanoic
acids, cellulose alkyl esters, talc, stearic acid,
magnesium stearate, magnesium oxide, sodium and
calcium salts of phosphoric and sulfuric acids,
gelatin, acacia gum, sodium alginate,
polyvinylpyrrolidone, and/or polyvinyl alcohol, and
then tableted or encapsulated for convenient
administration. Such capsules or tablets can contain
a controlled-release formulation as can be provided
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in a dispersion of active compound in hydroxy-
propylmethyl cellulose.
Formulations for parenteral administration can
be in the form of aqueous or non-aqueous isotonic
sterile injection solutions or suspensions. These
solutions and suspensions can be prepared from
sterile powders or granules having one or more of the
carriers or diluents mentioned for use in the
formulations for oral administration. The compounds
can be dissolved in water, polyethylene glycol,
propylene glycol, ethanol, corn oil, cottonseed oil,
peanut oil, sesame oil, benzyl alcohol, sodium
chloride, and/or various buffers. Other adjuvants
and modes of administration are well and widely known
in the pharmaceutical art.
Prex~aration of Useful Comnoun~~
Schemes I through IX hereinbelow illustrate
chemical processes and transformations that can be
useful for the preparation of compounds useful in
this invention; i.e., compounds of Formulas I,
wherein R1, and R2, substituent and Arl, are as
defined for Formula I, except where noted.
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Scheme I
O
+ R~ bases
2
Rz
O O Ar~\
R~ Ar~NHNH2 (4) N
N R2 ~ ~ R~
N
Scheme I shows the synthesis of a 1,5-diaryl
pyrazole (5) wherein a pyridine ring is attached to
5 position 5 of the pyrazole ring, R1 is lower alkyl and R2
is hydrido or lower alkyl. The synthesis is carried out
by condensing an appropriate ketone (2) with methyl
isonicotinate (1) in the presence of a suitable base to
provide the diketone 3. Examples of appropriate ketones
include acetone, methyl ethyl ketone, diethyl ketone and
the like. Examples of suitable bases include sodium
methoxide, and sodium ethoxide and the like. Suitable
solvents for this reaction include tetrahydrofuran (THF)
or methanol (MeOH) at temperatures ranging from room
temperature to reflux. Treatment of diketone 3 with an
aryl hydrazine derivative 4, in a suitable solvent at
temperatures ranging up to reflux provides a 1,5-diaryl
pyrazole, compound 5. Examples of suitable solvents for
this reaction include ethanol, acetic acid, ethanol-
acetic acid mixtures and the like. Substitution on Arl
in 5 is controlled by proper selection of the starting
hydrazine 4. When RZ of ketone 2 is hydrido, then R~ of
pyrazole 5 is hydrido.
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Scheme II
O NMe2
O
\ R2 (CH3)2NCH(OCH3)2 ( \
N~ 6 N J
7
R 1 C02Et
Ar ~ NHNH2 (4)
O O Are,
N-N
N ~ R R ~'t NHNH2 (4) ( \ \ ~ R
N J R2
7a
8
Scheme II illustrates the synthesis of a pyrazole,
compound 8 wherein RZ is a lower alkyl group and R1 is
hydrido or lower alkyl. Compounds wherein R1 is hydrido
and RZ is a lower alkyl group can be synthesized by
treatment of pyridyl ketone 6 with dimethylformamide
dimethyl acetal (DMF acetal). Examples of suitable
pyridyl ketones 6 include propionyl pyridine and
butanoyl pyridine. This reaction can be carried out in
the DMF acetal itself or in a suitable solvent such as
dimethylformamide. This provides enamine 7 which is
convertible to pyridyl pyrazole 8 by reaction with a
suitably substituted phenylhydrazine. This step can be
carried out as described for Scheme I and substitution
on Arl of pyrazole 8 is controlled by selection of a
properly substituted hydrazine. Scheme II also
describes the synthesis of pyrazoles wherein both R1 and
RZ are lower alkyl groups. This is achieved by reacting
ketone 6 with a carboxylic acid ester, such as methyl
acetate or methyl propionate or the like, in the
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presence of a base, such as sodium methoxide, in a
suitable solvent, such as methanol or tetrahydrofuran.
The resulting diketone 7a is converted to pyrazole 8 (R1
and R2 are lower alkyl) using the procedure described
above.
Scheme III
O O
CI
N / OH SOC12 ~ CI ~ \ CI
N
9
CH30H
O O
R2~, ~ CI \ ~CI-t3
2 R + N ~ ~O
11
Base
O O A~~ ~ N-N
CI \ R~ CI \ \
R2 Ar,NHNHZ (4~
12
13
Scheme III shows the synthesis of a pyrazole,
compound 13, analogs of compound 5 in which the pyridine
10 ring bears a chlorine atom at position 2. 2-
Chloropyridine-4-carboxylic acid, compound 9, is treated
with thionyl chloride in a solvent such as toluene, and
heated to reflux to give 2-chloropyridine-4-carboxylic
acid chloride, compound 10, which is then converted to
methyl 2-chloroisonicotinate, compound 11. Compound 11
is treated with a ketone 2 in the presence of a base
such as sodium methoxide in a solvent such as
tetrahydrofuran, at temperatures ranging from 25 °C up
to reflux to provide a diketone, compound 12. Treatment
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of the diketone, compound 12, with an arylhydrazine
derivative 4 in ethanol or other suitable solvent at a
temperature ranging up to reflux, provides pyrazole
compound 13. When RZ of ketone 2 is hydrido, then R2 of
13 is hydrido.
Are, Scheme IV Ar~'N-N
I \ N N R1 I \ ~ ~ R~
N ~ N~ 12
R2 R
OR4 RSS03Na RS~O O 20
base Ar , 19
18 4 ~ N-N
HOR
I \ ~ ~ R~
17 N~ ~2 NHZR3
R ~ Ar~.N-N
CI 13
NaN3 NI \ W ~ R~
R2
NHR3
Are. Ar ,/~ 15
N-N ~~N_N HZ/Pd(OH)2
\ ~ ~ R1 I \ ~ ~ R~ (when R3 = benzyl}
R2 N ~
N3 21 NH2 16
Scheme IV shows the syntheses of various pyrazole
derivatives from compound 13 by manipulations on its 2-
chloropyridine ring. The chlorine atom is.a labile
group and can be displaced with various nucleophiles to
provide 2-substituted pyridine derivatives. When
compound 13 is treated with an amine 14 at a temperature
of usually about 100 to about 200 °C and at a pressure
of about 70 to about 200 (or higher) psi in xylene,
pyrazole 15 is formed. Examplary substituents for R3 of
amine 14 are hydrogen, lower alkyl, hydroxyalkyl, or
aralkyl.
When R' is benzyl, hydrogenation of the compound
removes the benzyl group and forms the amino compound
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16. When the benzyl group bears a para methoxy
substituent, an alternative method of removal of the
benzyl group is treatment with refluxing trifluoroacetic
acid.
Treatment of compound 13 with an alcohol 17 in the
presence of a base in a suitable solvent provides
pyrazole compound 18. Examples of suitable alcohols are
benzyl alcohol and methanol. Suitable bases include
triethylamine and pyridine.
Compound 13 can also be treated with a sulfinic
acid sodium salt derivative, compound 19, in a suitable
solvent such as dimethylformamide (DMF) at an elevated
temperature to provide pyrazole compound 20. An example
of a sulfinic acid sodium salt is sodium methane
sulfinate and its reaction leads to methyl sulfone.
Finally, treatment of compound 13 with sodium azide
in a suitable solvent such as DMF, provides azido
pyridine 21.
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Scheme V
O O
CI ( ~ ~~CHs H3C~~ I '\
NJ N
28 29
O O
HaC.O I \ R~ R2~ R~
N J R2 2
Ar~NHNH2 (4)
Are, Ar~~N-N
.O \ N \ ' R1
H3C I ~ R ~" N / R2
N J R2 32
OH
31
Scheme V shows the preparation of pyrazole
compounds 31 and 32 bearing hydroxy and methoxy
5 substituents at position 2 of the pyridine ring.
Treatment of methyl 2-methoxyisonicotinate compound 29,
which is derived from methyl 2-chloroisonicotinate,
compound 28, with a ketone, compound 2, provides
compound 30, a diketone. Compound 30 is treated with an
10 arylhydrazine derivative, compound 4, under the standard
condition as described in the previous synthetic
schemes, to yield pyrazole 31. Treatment of compound 31
with an acid such as hydrochloric acid provides pyrazole
32 which bears a hydroxyl at position 2 of the pyridine
15 ring. When RZ of ketone 2 is hydrogen (hydrido), then RZ
of pyrazoles 31 and 32 is hydrogen (hydrido).
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Scheme 'VI
0
O~CH3 O
\ O~CHa
N / H2~2 I \
33 acid O~ N / 34
TMSiCN
(CH3)3NCOC1
O
O O R2~ O
1 1 O~CH
I \ R 2R I \
N ~ R2 ~ N /
CN 36 CN
ArINHNH2 4
Ar1 ~ ArI~N-N
N-N ~ ~ R1
R1 H202/base I
-----~.. N, / R
H2N~ ~O 38
Scheme VI demonstrates the syntheses of the
pyrazole compounds 37 and 38 that bear cyano and
5 carboxamido substituents, respectively, at position 2
of the pyridine ring. Methyl 2-cyanoisonicotinate 35
is synthesized from methyl isonicotinate 33 in two
steps by oxidation with hydrogen peroxide in an acid
solvent, such as acetic acid. The resulting pyridine
10 N-oxide 34 is treated with dimethylcarbamoyl chloride
in the presence of trimethylsilylcyanide to provide
ester 35. Treatment of ester 35 with ketone 2
according to general conditions described for similar
reactions in the preceding schemes gives diketone 36.
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Treatment of 36 with a substituted arylhydrazine
produces pyrazole 37. Desired substitution on Arl is
achieved by selection of the properly substituted
arylhydrazine.
Cyano pyrazole 37 is converted to the
carboxamido compound 38 by oxidation with hydrogen
peroxide in the presence of a base. Suitable bases
include sodium carbonate, potassium carbonate and
sodium hydroxide. Manipulation of substituent Rz is
effected by selection of the proper ketone 2. When Rz
of compound 2 is hydrogen, R2 in pyrazoles 37 and 38
is hydrogen (hydrido).
Scheme VII
O OH S
Base, RNCS~ ' ~ ~ NHR
N J R2 Solvent N J R2
A 39 Ar~NHNH2 (4)
Base/Solvent
HR
15 Scheme VII illustrates a two-step synthesis of
3-amino-1,5-diarylpyrazoles 40. In the first step,
4-acetylpyridine (A, R2 - H) is treated with a
suitable base to generate an enolate anion. Examples
of suitable bases include lithium hexamethyl
20 disilazide, sodium hexamethyldisilazide and lithium
diisopropylamide. Suitable solvents for this
reaction include tetrahydrofuran and diethyl ether.
The resulting intermediate enolate anion is treated
with a suitable isothiocyanate to give beta keto
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thioamide 39. Examples of suitable isothiocyanates
include ethyl isothiocyanate and
trimethylsilylisothicyanate. The reaction of
thioamide 39 with an arylhydrazine 4 leads to the
formation of pyrazole 40. This reaction is carried
out using conditions discussed in preceding examples.
Control of substitution on Arl is effected by proper
selection of the substituted phenylhydrazine.
When an alkyl isothiocyanate is employed in this
sequence, R of compound 40 is an alkyl group. When
trimethylsilyl isothiocyanate is employed, R of 40 is
a trimethylsilyl group, which is easily removed to
produce the primary amino compound where R of
compound 40 is hydrogen. Examples of reaction
conditions used to remove the silyl group are acetic
acid in water and tetrahydrofuran or aqueous sodium
bisulfate. When RZ of the starting pyridyl ketone is
a substituent other than H, that substituent becomes
the substituent Rz at the 4 position of the pyrazole
40.
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Scheme VIII
O Base, OH O
MeC02Me \ \
N / OMe ~ N~ v ~OMe
Solvent
41 DMF-DMA
Solvent
O
N.CH3
I \
N
O OMe
42
Are NHNH2
Solvent
Ar~'N,N Amidation Ar~'N-N
-- v
Solvent I \
O N J O~OMe
44
43
Saponification
Are 'N,N
DPPA Are 'N"N
NH2 ( \
N ~ OT'OH
C
B
Scheme VIII illustrates the synthesis of a 1,5-
diaryl pyrazole wherein RZ is a derivatized carboxyl
group or an amino group. An ester of isonicotinic
acid is treated with a carboxylic acid ester in the
presence of a base to produce beta keto ester 41.
Suitable esters of isonicotinic acid include the
methyl and ethyl esters and the like. Suitable
esters of the carboxylic acid also include the methyl
and ethyl esters. Bases such as sodium methoxide and
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sodium ethoxide are suitable for this reaction. The
reaction can be carried out in an alcoholic solvent
such as methanol or ethanol. Ketoester 41 is
converted to the enamine intermediate 42 by reaction
with dimethylformamide dimethylacetal either neat or
in a solvent such as dimethyl formamide. Reaction
with a properly substituted arylhydrazine 4 produces
the carboxylic acid ester 43. Reaction of this ester
with various amines, such as piperazine produces
amides 44 (R = substituted amine).
Similarly, saponification of 43 produces acid B.
Saponification can be carried out using a base such
as sodium hydroxide in an aqueous solvent such as
aqueous methanol or ethanol or the like. Acid B is
converted to amine C by reaction with
diphenylphosporyl azide (DPPA) in the presence of a
base such as triethylamine in a solvent such as
tetrahydrofuran or dioxane.
Scheme IX
0 0
Bromination ~ Br R'R"NH Base
I~ _CH3 _ I ~ ,
N ~ ~ N / Solvent
O
NI~6 NR'R"
DMF-DMA
Solvent
O
Ar~NHNH2 I ~ / N~CH3
Solvent '~
R.R.. N~ TR.R~~ CH3
2 0 48
Scheme IX illustrates the synthesis of a 4-
amino-1,5-diarylpyraozle, compound 48. In step 1,
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4-acetylpyridine is brominated with bromine in the
presence of a solvent such as 48 percent hydrobromic
acid to provide the bromoketone, compound 45. In
step 2, the reaction of compound 45 with an amine,
such N-tert-butoxycarbonylpiperazine, in the presence
of a base such as triethylamine, and in the presence
of a solvent such as DMF, provides intermediate
compound 46. Other suitable amines include
piperidine and dimethylamine. In step 3, the
reaction of intermediate compound 46 with
dimethylformamide dimethylacetal (DMF-DMA) in a
solvent such as tetrahydrofuran or dimethyformamide
provides intermediate enamine 47. In step 4, the
condensation of intermediate compound 47 with a
substituted arylhydrazine such as 4-
fluorophenylhydrazine, in a solvent such as ethanol
provides desired pyrazole, compound 48.
It should be recognized that in the above
Schemes, the pyridine ring can be replaced by a
pyrimidine ring when suitably substituted pyrimidine
starting materials are employed. Suitable starting
materials are recognizable by one skilled in the art
and their syntheses are readily accessible in the
scientific literature. For example, the ethyl ester
of pyrimidine-4-carboxylic acid can be synthesized
according to procedures described by Wong et al, J.
Org. Chem., vol. 30, p. 2398 (1965). The methyl
ester of 2-methoxypyrimidine-4-carboxylic acid is
described by Warczykowski and Wojciechowski, Pol. J.
Chem., vol. 54, pp. 335-340 (1980). The synthesis of
2-diethylaminopyrimidine-4-carboxylic acid from 2-
chloropyrimidine-4-carbonitrile is also described in
that paper. The synthesis of 2-aminopyrimidine-4-
carboxylic acid from 2-chloropyrimidine-4-
carbonitrile is described by Daves et al., in J. Het.
Chem., vol. 1, p. 130 (1964). The synthesis of 2-
chloropyrimidine-4-carbonitrile is described by D. J.
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Brown et al, Aust. J. Chem., vol. 37, pp. 155-163
(1984) and by A. E. Friesen et al., Tetrahedron, vol.
45, pp. 5151-5162 (1989). The conversion of 2-
methylpyrimidine-4-carboxylic acid methyl ester to a
1,3-diketone by reaction with the enolate anion of
acetophenone is described by T. Sakamoto, Chem.
Pharm. Bull., vol. 30, pp. 1033-1035 (1982).
Best Mode for Carryi~9 Out the Invention
Without further elaboration, it is
believed that one skilled in the art can, using the
preceding description, utilize the present
invention to its fullest extent. The following
preferred specific embodiments are, therefore, to
be construed as merely illustrative, and not
limiting of the remainder of the disclosure in any
way whatsoever.
Example 1: Preparation of
NI~ C I
3
Sten 1 ~ Prex~aration of 1 (4 purr; yinyl ) 2 methyl 1 3
propane ~ one
A mixture of 4-propionylpyridine (5g, 36.99
mmol) and dimethylformamide dimethylacetal(10 mL,
75.28 mmol) was heated to reflux overnight. After
cooling to room temperature, methanol generated in
situ was removed from reaction mixture under reduced
pressure, providing a brown color solid. The crude
product was purified by column chromatography (silica
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gel, 3:7 EtOAc/haxane) to give the named compound
(5.73 g, 81%) as a brown oil.
~,p 2:
A mixture of 1-(4-pyridinyl)-2-methyl-1,3-
propanedione (0.5 g, 2.63 mmol), 4-fluorophenyl-
hydrazine~HCl (0.5148, 3.16 mmol), and 15 M ammonium
hydroxide (0.211 mL, 3.16 mmol) in ethanol (7 mL) was
heated to reflux overnight. The resulting dark
solution was cooled to room temperature and extracted
with ethyl acetate. The organic layer was washed
with saturated sodium bicarbonate and brine, dried
over magnesium sulfate. After filtration, the
solution was concentrated to give a yellow oil. The
oil was purified by column chromatography (silica
gel, 3:7 EtOAc/hexane) to yield the titled compound
(0.3688, 58~) as a light yellow solid. MP: 135.86 °C;
Anal. Calc'd for C15H1zNaFØ1H20: C, 70.63, H, 4.82, N,
16.47; Found: C, 70.45, H, 4.51, N, 16.42.
Example 2: Preparation of
F
CH3
By following the method of Example 1 and
substituting of 3-fluorophenylhydrazine.HC1 for 4-
fluorophenylhydrazine.HC1 in step 2, the titled
product was obtained: MP: 131.11 °C. Anal. Calc'd for
C1sH12N3FØ1 H20: C, 70.63, H, 4.82, N, 16.47. Found:
C, 70.64, H, 4.97, N, 16.09.
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Example 3: Preparation of
/
HsC N_N
\
N / CH3
By following the method of Example 1 and
substituting of 3-methylphenylhydrazine.HCl for 4-
fluorophenylhydrazine.HC1 in step 2, the titled
product was obtained: MP: 141.77 °C. Anal. Calc'd for
C16H1sN3 ~ 0 ~ 05 H20: C, 76. 80, H, 6 . 08, N, 16 . 79. Found:
C, 76.$4, H, 6.27, N, 16.43.
Example 4: Preparation of
\
N N
\ \ ~
N / CHs
By following the method of Example 1 and
substituting of benzylhydrazine.HCl for 4-
fluorophenylhydrazine.HC1 in step 2, the titled
product was obtained: Anal . Calc' d for C16H15N3 ~ C.
77.08, H, 6.06, N, 16.85. Found: C, 77.03, H, 6.18,
N, 16.72.
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Example 5: Preparation of
H3C
/
N N
\ ~ \ CH3
N /
HN
OH
A mixture of 2-chloro-4-[3-methyl-1-(4-
methylphenyl)-1H-pyrazol-5-yl]pyridine (Example 41;
0.5 g, 2.53 mmol) and ethanolamine (15 mL, 248.52
mmol) was heated to 105 °C overnight (about eighteen
hours). The resulting solution was extracted with
ethyl acetate. The organic layer was washed with
brine, dried over magnesium sulfate, filtered and
concentrated under reduced pressure. The resulting
yellow oil crystallized upon standing. The crystals
were washed with diethyl ether to yield the titled
compound (0.475 g, 61 %) as white yellowish crystals.
MP: 148.20 ° C. Anal. Calc'd for C18 Hao NQ O: C, 70.11,
H, 6.54, N, 18.17. Found: C, 69.75, H, 6.71, N,
17.84.
Example 6: Preparation of
H3C
N N
\ ~ \ CH3
N / /
HN \ N
A mixture of 2-chloro-4-[3-methyl-1-(4-
methylphenyl)-1H-pyrazol-5-yl]pyridine (Example 41;
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0.5 g, 2.53 mmol) and 3-(aminomethyl)pyridine (15 mL,
147.3 mmol) was heated to 180 °C overnight (about
eighteen hours). The resulting solution was
extracted with ethyl acetate. The organic layer was
washed with brine, dried over magnesium sulfate,
filtered and concentrated under reduced pressure.
The resulting yellow oil was purified by column
chromatography (silica gel, EtOAc) to yield the
titled compound (0.378 g, 42 ~) as a white yellowish
solid. MP: 108.94 ° C. Anal. Calc'd for CZZ Hzl Ns . C,
73.97, H, 5.98, N, 19.60. Found: C, 73.86, H, 6.25,
N, 19.39.
Example 7: Preparation of
HsC /
N-N
CH3
N /
HN
By following the method of Example 5 and
substituting of benzylamine for ethanolamine, and
changing the reaction temperature from 105 °C to
160°C, the titled compound was obtained: MP: 102.10 °
C. Anal . Calc' d for C23HzzN4 ~ C, 77. 94, H, 6.26, N,
15.81. Found: C, 77.82, H, 6.60, N, 15.69.
Example 8: Preparation of
H3C
N N
CH3
N /
HN~CH3
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A mixture of 2-chloro-4-[3-methyl-1-(4-
methylphenyl)-1H-pyrazol-5-yl]pyridine (Example 41;
0.45 g, 2.3 mmol) and excess aqueous ethylamine (15
mL) was heated to 160 °C at <200 psi in xylene
overnight (about eighteen hours). The resulting
solution was extracted with ethyl acetate. The
organic layer was washed with brine, dried over
magnesium sulfate, filtered and concentrated under
reduced pressure. The resulting yellow oil was
purified by column chromatography [silica gel, ethyl
acetate (EtOAc)] to yield (0.372 g, 50 %) as a white
solid. MP: 97.15 ° C. Anal. Calc'd for C18 H2o N4 . C,
73.94, H, 6.89, N, 19.16. Found: C, 73.70, H, 6.83,
N, 18.98.
Example 9: Preparation of
H3C
N-N
CH3
N
HN~CH
3
A mixture of 2-chloro-4-[3-methyl-1-(4-
methylphenyl)-1H-pyrazol-5-yl]pyridine (Example 41;
0.5 g, 2.53 mmol) and excess methylamine (15 mL) was
heated to 170 °C at <200 psi in xylene overnight
(about eighteen hours). The resulting solution was
extracted with ethyl acetate. The organic layer was
washed with brine, dried over magnesium sulfate,
filtered and concentrated under reduced pressure.
The resulting yellow oil was purified by column
chromatography (silica gel, 3:7 EtOAc/hexane) to
yield the titled compound 0.218 g, 31 %) as a white
powder. MP: 126.34 ° C. Anal. Calc'd for C17 H18 Nq . C,
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73.06, H, 6.56, N, 19.81. Found: C, 73.06, H, 6.28,
N, 19.43.
Example 10: Preparation of
H3C
N N
CH3
N
H3C~ N ~CH3
A mixture of 2-chloro-4-[3-methyl-1-(4-
methylphenyl)-1H-pyrazol-5-yI]pyridine (Example 41;
0.5 g, 2.53 mmol) and excess dimethylamine (15 mL)
was heated to 170 °C at 60-80 psi in xylene overnight
(about eighteen hours). The resulting solution was
extracted with ethyl acetate. The organic layer was
washed with brine, dried over magnesium sulfate,
filtered and concentrated under reduced pressure.
The resulting yellow oil was purified by column
chromatography (silica gel, 3:7 EtOAc/hexane) to
yield the titled compound (0.126 g, 17 0) as a
colorless oil. Anal. Calc'd for C18 Hz° N4: C, 73.94,
H, 6.89, N, 19.16. Found: C, 74.04, H, 7.10, N,
19.03.
Example 11: Preparation of
CI /
N N
CH3
N
HN
OH
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Step l: Preparation of 2-chloro-4-[1-(4-
lor a r o - 1 ridi a
A mixture of 1-(2-chloro-4-pyridinyl)-1,3-
butanedione (from step 2, Example 29)(2.5 g, 12.69
mmol), p-chlorophenylhydrazine.HC1 (2.7 g, 15.11
mmol) and triethylamine (2.1 mL, 15.11 mmol) in
ethanol (50 mL) was heated to reflux overnight (about
eighteen hours).. The resulting dark colored solution
was cooled to room temperature and extracted with
ethyl acetate. The organic layer was washed with
saturated sodium bicarbonate and brine, dried over
magnesium sulfate, filtered and concentrated to give
2-chloro-4-[1-(4-chlorophenyl)-3-methyl-1H-pyrazol-5-
yl]pyridine as an orange colored solid (1.5 g, 39 %).
MP: 110.05 °C. This product was used in the next
step without further purification.
Step 2:
A mixture of 2-chloro-4-[1-(4-chlorophenyl)-3-
methyl-1H-pyrazol-5-yl]pyridine (0.6 g, 1.64 mmol)
and ethanolamine (15 mL, 248.52 mmol) was heated to
105 °C overnight (about eighteen hours). The
solution was extracted with ethyl acetate. The
organic layer was washed with brine, dried over
magnesium sulfate, filtered and concentrated under
reduced pressure. The resulting yellow oil was
purified by column chromatography (silica gel, EtOAc)
to yield the titled compound (0.253 g, 47 %) as a
white solid. MP: 137.57 °C. Anal. Calc'd. for
C1,HI,NqOCl: C, 62.10, H, 5.21, N, 17.04. Found: C,
62.09, H, 5.24, N, 16.70.
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Example 12: Preparation of
CI /
~I
N-N
CH3
N /
HN
By following the method of Example 11 and
substituting of benzylamine for ethanolamine, the
titled product was obtained: MP: 105.53 °C. Anal.
Calc'd for CzzH19N4C1: C, 69.95, H, 5.25, N, 14.44.
Found: C, 70.24, H, 5.07, N, 14.19.
Example 13: Preparation of
C)
/ )
N N
CH3
N
HN
A mixture of the compoiund from step 1 of
Example 11 (1 g, 3.29 mmol) and excess
phenylethylamine (15 mL) was heated to 200 °C in
xylene overnight (about eighteen hours). The
solution was extracted with ethyl acetate. The
organic layer was washed with brine, dried over
magnesium sulfate, filtered and concentrated under
reduced pressure. The resulting yellow oil was
purified by column chromatography (silica gel, 1:9
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EtOAc/hexane) to yield the titled compound (0.537 g,
42 %) as a brown oil . Anal . Calc' d for Cz3Ha1NaC1 : C,
71.03, H, 5.44, N, 14.41. Found: C, 70.91, H, 5.43,
N, 14.18.
Example 14: Preparation of
H3C
N-N
\ ~ \ CH3
v
NH2
Step 1: Preparation of 1-t3-aminophenvl) 1 3
butaned~one
To a solution of methyl 3-aminobenzoate (7g,
46.36 mmol) in THF (150 mL) was added acetone (7.58,
102 mmol). The solution was warmed to 35 °C where
sodium methoxide (3g, 55.63 mmol) was added
sequentially over 20 minutes. The mixture was
stirred for 30 minutes, and then brought to reflux
for 3 hours. The solvent was removed under reduced
pressure and the residue taken up in ethyl acetate.
The resulting solution was washed with saturated
sodium bicarbonate solution and brine, dried over
magnesium sulfate, filtered and concentrated under
reduced pressure to give l-(3-aminophenyl)-1,3-
butanedione as a brown oil (3.1 g, 37 %). This oil
was used in the next step without further
purification.
Step 2
A mixture of 1-(3-aminophenyl)-1,3-butanedione
(step 1) (.5.g, 2.82mmo1), p-tolylhydrazine HCl
(0.5338, 3.36mmol) and triethylamine (0.340mL, 3.68
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mmol) in ethanol (7 mL) was heated to reflux
overnight (about eighteen hours). The resulting dark
solution was cooled to room temperature and extracted
with ethyl acetate. The organic layer was washed
with saturated sodium bicarbonate solution and brine,
dried over magnesium sulfate, filtered, and
concentrated down to give a brown oil. The oil was
purified by column chromatography (silica gel, 1:1
EtOAc/hexane) to yield the titled compound (0.527 g,
65 %) as a yellow solid. Mp: 94.38 °C. Anal.
calc' d for C1,H1~N3: C 77.57, H 6.46, N 15. 97. Found:
C 77.14, H 6.56, N 15.77.
Example 15: Preparation of
F
N-N
NHEt
N
Step 1:
Compound 1: To a solution of LiHMDS (62.5 mL,
1.0 M in THF) at zero degrees C, was added 4-
acetylpyridine (6.06 g, 0.05 mol), with 20 mL of DMF
also being added to dissolve the anion. To this
mixture, a solution of ethyl isothiocyanate (5.49 g,
0.0625 mol) in 30 mL of dry THF was added dropwise
over 0.5 hours. The reaction mixture was stirred
overnight (about eighteen hours), while warming to
room temperature. After the addition of 500 mL of
saturated NH4C1 solution, the aqueous phase was
extracted with ethyl acetate. The organic layer was
washed with water, brine, dried over magnesium
sulfate and filtered. The filtrate was concentrated
and purified by chromatography on silica gel (ethyl
acetate/hexane, 3:1) to give 2.30 g of the product as
a yellow solid (24% yield), mp: 130-132 ° C.
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Step
A mixture of compound 1 (0.4 g, 0.002 mol), 4-
fluorophenylhydrazine hydrochloride (0.32 g, 0.002
mol) and triethylamine (0.20 g, 0.002 mol) in 10 mL
of ethanol was heated at reflux for 24 hours. After
the solvent was removed, the residue was purified by
chromatography on silica gel (ethyl acetate/hexane,
8:2) to give 0.31 g of the titled product as a yellow
solid (55% yield), mp: 130-131 ° C; Anal. Calc'd. for
C16H1sFN4~ 0.25H20: C, 67.00; H, 5.95; N, 19.53. Found:
C, 66.98; H, 5.13; N, 19.07.
Example 16: Preparation of 1-(4-fluorophenyl)-5-(4-
pyridinyl)-1H-pyrazole-4-carboxamide
F
N~N
N~ il'NHZ
O
A solution of methyl 1-(4-fluorophenyl)-5-(4-
pyridinyl)-1H-pyrazole-4-carboxylate (0.9 g, 0.003
mol) in 10 mL of methanol in a sealed tube treated
with excess liquid ammonia at low temperature. Then
the reaction mixture was heated at 80 ° C for 48
hours. Solvent was removed and the residue was
triturated with ethyl acetate to give 0.13 g of
product as a yellow solid (15% yield), mp 215-216 ° C;
Anal. Calcd. For C15H11FN90: C, 63.38; H, 3.93; N,
19.85. Found: C, 63.21; H, 3.98; N, 19.37.
Example 17: Preparation of
F
N-N
I~
N~ ~--N~NH
O
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Ste~Ll
Compound 3: To a solution of the carboxylic acid
of Example 43 (0.16 g, 0.00056 mol) in DMF, was added
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (0.12 g, 0.00062 mol), followed by the
addition of N-tert-butoxycarbonylpiperazine (0.10 g,
0.00056 mol). The reaction mixture was stirred at
room temperature for 16 hours. Water was added and
the aqueous phase was extracted with ethyl acetate.
The organic layer was washed with brine, dried over
magnesium sulfate and filtered. The filtrate was
concentrated and the crude product was purified by
recrystallization from ethyl acetate and hexane to
give 0.14 g of product as a light yellow solid (55%
yield) , mp: 233-234 ° C; Anal. Calc'd. for Cz4H26FNsp3;
C, 63.85; H, 5.8,0; N, 15.51. Found: C, 63.57; H,
6.12; N, 15.42.
Step 2:
To a solution of compound 3 (0.13 g, 0.00029
mol), above, in 5 mL of methylene chloride, were
added 2 mL of trifluoroacetic acid. The reaction
mixture was stirred at room temperature for 1 hour.
Solvent was removed and the residue was basified with
ammonium hydroxide. The aqueous phase was extracted
with ethyl acetate. The organic layer was washed
with brine, dried over magnesium sulfate and
filtered. The filtrate was concentrated and the
crude was purified by recrystallization from ethyl
acetate and ether to give 0.07 g of product as a pale
yellow solid (64% yield), mp: 184-186 ° C; Anal.
Calc'd. for C19H18FN50: C, 64.95; H, 5.16; N, 19.93.
Found: C, 64.50; H, 5.02; N, 19.86.
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Example 18: Preparation of
F
N~N
I \
N ~ N
~N
H
Step 1:
Compound 4: To a solution of 4-acetylpyridine
(18.2 g, 0.15 mol) in 30 mL of 48% HBr was added a
solution of Br2 (24.0 g, 0.15 mol) in 5 mL of 48% HBr
dropwise at 70°C. After stirring at the same
temperature for 3 hours, the suspension was cooled
and filtered. The solid was washed with a mixture of
methanol and petroleum ether to give 28.9 g of
product as a pale yellow solid (69% yield), mp: 210 °
C (dec) .
Ste~2
Compound 5: Triethylamine (7.5 mL, 0.54 mol) was
added to a suspension of compound 4 (7.8 g, 0.027
mol) and N-tert-butoxycarbonylpiperazine (5.0 g,
0.027 mol) in DMF at room temperature. After heating
at 80 ° C for 16 hours, the reaction mixture was
cooled and treated with water. The aqueous phase was
saturated with sodium chloride and extracted with
ethyl acetate. The organic layer was washed with
brine, dried over magnesium sulfate and filtered.
The filtrate was concentrated and the crude was
purified by chromatography on silica gel (ethyl
acetate) to give 2.2 g of product as a yellow oil
(27 % yield) .
Step
To a solution of compound 5 (2.0 g, 0.0065 mol)
in THF was added N, N-dimethylformamide dimethyl
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acetal (7.8 g 0.065 mol). After stirring at room
temperature for 24 hours, solvent was removed under
vacuum to give 2.5 g of crude as a dark brown oil.
This crude product was then dissolved in 50 mL of
ethanol, treated with 4-fluorophenylhydrazine and the
mixture was heated at reflux overnight (about
eighteen hours). After the removal of solvent, the
residue was partitioned between water and ethyl
acetate. The organic layer was washed with brine,
dried over magnesium sulfate and filtered. The
filtrate was concentrated and the crude (0.7 g) was
dissolved in 5 mL of methylene chloride and treated
with 2 mL of TFA. After stirring at room temperature
for 16 hours, the mixture was concentrated.
The concentrated residue was basified with 1N
NaOH, and extracted with ethyl acetate. The organic
layer was washed with brine, dried over magnesium
sulfate and filtered. The filtrate was concentrated
and purified by chromatography on silica gel
(methylene chloride/methanol, 9:1) to give 0.25 g of
the titled compound as a yellow solid, mp: 150-151 °
C; Anal. Calc'd. for C18H18FN5~ 2.0 HzO: C, 60.15; H,
6.17; N, 19.49. Found: C, 60.37; H, 5.74; N, 19.98.
Example 19: Preparation of 2-(Benzylamino)-4-[1-(3
methylphenyl)-3-methyl-1H-pyrazol-5-yl]pyridine
CH3
N-N
CH3
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A mixture of 2-chloro-4-[1-(3-methylphenyl)-3-
methyl-1H-pyrazol-5-yl]pyridine (Example 45; 1.24 g,
0.0044 mol) and benzylamine (40 mL) was heated at 180
° C for 20 hours The excess benzylamine was removed
under vacuum and the residue was partitioned between
ethyl acetate and water. The organic layer was
washed with brine, dried over magnesium sulfate and
filtered. The filtrate was concentrated and the
crude product was purified by chromatography on
silica gel (ethyl acetate/hexane, 3:7) to give 1.33 g
(85% yield) of the titled compound as a clear oil,
which solidified upon standing: mp: 73-75 ° C. Anal.
Calc'd. for C23H22N4~~~2 H20: C, 76.83; H, 6.39; N,
15.06. Found: C, 76.88; H, 6.37; N, 14.92.
Example 20: Preparation of 2-(Benzylamino)-4-[1-(3-
fluorophenyl)-3-methyl-1H-pyrazol-5-yl]pyridine
F
N-N
CH3
N
HN
A mixture of 2-chloro-4-[1-(3-fluorophenyl)-3-
methyl-1H-pyrazol-5-yl]pyridine (Example 47; 1.11 g,
0.0039 mol) and benzylamine (30 mL) was heated at 180
° C overnight (about eighteen hours). The excess
benzylamine was removed under vacuum and the residue
was partitioned between ethyl acetate and water. The
organic layer was washed with brine, dried over
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magnesium sulfate and filtered. The filtrate was
concentrated and the crude was purified by
chromatography on silica gel (ethyl acetate/hexane,
2:8) to give 1.24 g (90% yield) of the titled
compound as a clear oil, which solidified upon
standing: mp: 94-96 ° C; Anal. Calc'd. for C22H19FN4:
C, 73.72; H, 5.34; N, 15.63. Found: C, 73.62; H,
5.62; N, 15.34.
Example 21: Preparation of 2-Amino-4-[1-(3-
methylphenyl)-3-methyl-1H-pyrazol-5-yl]pyridine
A mixture of 2-benzylamino-4-[1-(3-
methylphenyl)-3-methyl-1H-pyrazol-5-yl]pyridine
(Example 19; 1.03 g, 0.0029 mol) and palladium
hydroxide (0.2 g)in acetic acid was hydrogenated
under 60 psi at 40 ° C for 9 hours. The mixture was
cooled and filtered through a pad of CeliteT"'. The
filtrate was concentrated and purified by
chromatography on silica gel (ethyl acetate/hexane,
l:l) to give 0.43 g of product as a white solid: mp:
124-125°C; Anal. Calc~d. for C16H16N4~ C. 72.70; H,
6.10; N, 21.20. Found: C, 72.41; H, 6.31; N, 20.63.
Example 22: Preparation of 2-Methoxy-4-[1-(3-
methylphenyl)-3-methyl-1H-pyrazol-5-yl]pyridine
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CH3
step 1: Preparation of methyl 2-mefihoxyison;c~~rinarP
A mixture of. methyl 2-chloroisonicotinate (5.23
g, 0.030 mol) and sodium methoxide (2.47 g, 0.045
mol) in 15 mL of dioxane was heated at reflux for 1.5
hours. After the reaction mixture was cooled, water
was added and the resulting mixture was extracted
with methylene chloride. The organic layer was
washed with brine, dried over magnesium sulfate and
filtered. The filtrate was concentrated in vacuo to
give 3.76 g (75~) of product as a yellow oil: Anal.
Calc~d. for C8H9N03: C, 57.48; H, 5.43; N, 8.38.
Found: C, 57.07; H, 5.54; N, 8.34.
ten 2 ~ PrP~paration of 1- (2-metho yp~rridyl) 1 3
butadione
To a solution of methyl 2-methoxyisonicotinate
(3.67 g, 0.022 mol) and acetone (4.85 mL, 0.066 mol)
in 30 mL of THF was added sodium methoxide (1.25 g,
0.023 mol) in one portion. The reaction mixture was
refluxed for 4 hours and then cooled to room
temperature. Water was added and the solution was
acidified to pH = 6 by acetic acid. The aqueous
phase was extracted with ethyl acetate and the
organic layer was washed with brine, dried over
magnesium sulfate and filtered. The filtrate was
concentrated to give 2.79 g (72%) of product as a
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brown solid that was used without further
purification.
Step 3: Preparation of 2-methoxy-4-[1-(3-
methyl henyl)-3-methxl-1H-gvrazol-5 ~yllpyridine~
To a suspension of 1-(2-methoxypyridyl)-1,3-
butadione (2.7 g, 0.014 mol) and 3-
methylphenylhydrazine hydrochloride (2.66 g, 0.017
mol) in 100 mL of ethanol was added triethylamine
(2.34 mL, 0.017 mol) dropwise. The reaction mixture
was heated at refluxed overnight (about eighteen
hours). Solvent was removed and the residue was
partitioned between ethyl acetate and water. Organic
layer was washed with brine, dried over magnesium
sulfate and filtered. The filtrate was concentrated
and the crude was purified by chromatography on
silica gel (ethyl acetate/hexane, 2:8) to give 2.8 g
(72%) of product as a yellow oil: Anal. Calc'd. for
C1~H1~N30: C, 73.10; H, 6.13; N, 15.04. Found: C,
72.81; H, 6.11; N, 14.68.
Example 23: Preparation of 4-[1-(3-Methylphenyl)-3-
methyl-1H-pyrazol-5-yl]-2-pyridone
CH3
N-N
U ~CH3
HN
O
To a solution of 2-methoxy-4-[1-(3-
methylphenyl)-3-methyl-1H-pyrazol-5-yl]pyridine
(Example 22; 0.56 g, 0.002 mol) in S mL of acetic
acid was added 5 mL of hydrobromic acid. The
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reaction mixture was heated at reflux for 3 hours and
then cooled to room temperature. Water was added and
the solution was basified with ammonium hydroxide.
The aqueous phase was extracted with ethyl acetate
and the organic layer was washed with brine, dried
over magnesium sulfate and filtered. The filtrate
was concentrated in vacuo to give 0.45 g (85%) of
product as a white solid: mp: 182-183 ° C; Anal.
Calc'd. for C16H15N3~~ C. 72.43; H, 5.70; N, 15.84.
Found: C, 72.37; H, 5.66; N, 15.97.
Example 24: Preparation of 2-(Phenylethylamino)-4-[1-
(3-methylphenyl)-3-methyl-1H-pyrazol-5-yl]pyridine
H3
A mixture of 2-chloro-4-[1-(3-methylphenyl)-3-
methyl-1H-pyrazol-5-yl]pyridine (Example 45; 0.5 g,
0.0018 mol) and phenethylamine (15 mL) was heated at
200 ° C for 24 hours. The excess amine was removed
under vacuum and the residue was partitioned between
ethyl acetate and water. The organic layer was
washed with brine, dried over magnesium sulfate and
filtered. The filtrate was concentrated and the
crude was purified by chromatography on silica gel
(ethyl acetate/hexane, 1:1) to give 0.48 g (74%
yield) of product as a yellow oil, which solidified
upon standing: mp: 86-87°C. Anal. Calc'd. for
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C24H24N4~ C~ 78.23; H, 6.56; N, 15.20. Found: C,
78.07; H, 6.82; N, 14.71.
Example 25: Preparation of 2-(N-
Methylphenylethylamino)-4-[1-(3-methylphenyl)-3-
methyl-1H-pyrazol-5-yl]pyridine
CH3
N-N
CH3
A mixture of 2-chloro-4-[1-(3-methylphenyl)-3-
methyl-1H-pyrazol-5-yl]pyridine (Example 45; 0.5 g,
0.0018 mol) and N-methylphenethylamine (15 mL) was
heated at 200 ° C for 24 hours. The excess amine was
removed under vacuum and the residue was partitioned
between ethyl acetate and water. The organic layer
was washed with brine, dried over magnesium sulfate
and filtered. The filtrate was concentrated and the
crude was purified by chromatography on silica gel
(ethyl acetate/hexane, 2:8) to give 0.45 g (675
yield) of product as a yellow oil: Anal. Calc~d. for
C25H26N4~ C~ 78.50; H, 6.85; N, 14.65. Found: C,
78.43; H, 6.87; N, 14.30.
Example 26: Preparation of 2-(2-Hydroxyethylamino)-4-
[1-(3-methylphenyl)-3-methyl-1H-pyrazol-5-yl]pyridine
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H3
A mixture of 2-chloro-4-[1-(3-methylphenyl)-3-
methyl-1H-pyrazol-5-yl]pyridine (Example 45; 0.5 g,
0.0018 mol) and ethanolamine (15 mL) was heated at
100 ° C for 18 hours. The reaction mixture was cooled
and treated with ethyl acetate and water. The
organic layer was washed with brine, dried over
magnesium sulfate and filtered. The filtrate was
concentrated and the crude was purified by
chromatography on silica gel (ethyl acetate) to give
0.44 g (80% yield) of product as a yellow oil: Anal.
Calc~d. for C18H2oN40: C, 70.11; H, 6.54; N, 18.17.
Found: C, 69.89; H, 6.65; N, 18.35.
Example 27: Preparation of 2-(N-Methylbenzylamino)-4-
[1-(3-methylphenyl)-3-methyl-1H-pyrazol-5-yl]pyridine
CH3
N-
H3
._
N
H~C~
A mixture of 2-chloro-4-[1-(3-methylphenyl)-3-
methyl-1H-pyrazol-5-yl]pyridine (Example 45; 1.0 g,
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0.0036 mol) and N-methylbenzylamine (20 mL) was
heated at 180 ° C for 24 hours. The excess amine was
removed under vacuum and the residue was partitioned
between ethyl acetate and water. The organic layer
was washed with brine, dried over magnesium sulfate
and filtered. The filtrate was concentrated and the
crude was purified by chromatography on silica gel
(ethyl acetate/hexane, 2:8) to give 0.88 g (66%
yield) of product as a yellow oil: Anal. Calc~d. for
C24H24N4~ C~ 78.23; H, 6.56; N, 15.20. Found: C,
78.55; H, 6.51; N, 15.21.
Example 28: Preparation of 2-Bezyloxy-4-[1-(3
methylphenyl)-3-methyl-1H-pyrazol-5-yl]pyridine
CH3
N-N
\ CHs
N
C
To a suspension of potassium hydroxide (0.67 g,
0.012 mol) and potassium carbonate (0.41 g, 0.003
mol) in 30 mL of toluene was added 2-chloro-4-[1-(3-
methylphenyl)-3-methyl-1H-pyrazol-5-yl]pyridine (0.85
g, 0.003 mol). Then benzyl alcohol (0.49 g, 0.0045
mol) was charged into the reaction mixture, followed
by the addition of tris[2-(2-methoxyethoxy)ethyl]-
amine (0.1 g, 0.0003 mol) and the mixture was heated
at reflux overnight (about eighteen hours). Toluene
was removed under vacuum and the residue was
partitioned between water and ethyl acetate.
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The organic layer was washed with brine, dried
over magnesium sulfate and filtered. The filtrate
was concentrated and the crude was purified by
chromatography on silica gel (ethyl acetate/hexane,
1:9) to give 0.82 g (66% yield) of product as a
yellow oil: Anal. Calc'd. for C23H21N3~~ C, 77.72; H,
5.96; N, 11.82. Found: C, 77.42; H, 5.90; N, 11.55.
Example 29: Preparation of 2-(Benzylamino)-4-[1-(4-
fluorophenyl)-1H-pyrazol-5-yl]pyridine
F
A mixture of 2-chloro-4-[1-(4-fluorophenyl)-1H-
pyrazol-5-yl]pyridine (Example 46; 0.56 g, 0.002 mol)
and benzylamine (20 mL) was heated at 180 ° C
overnight (about eighteen hours). The excess
benzylamine was removed under vacuum and the residue
was partitioned between ethyl acetate and water. The
organic layer was washed with brine, dried over
magnesium sulfate and filtered. The filtrate was
concentrated and the crude was purified by
chromatography on silica gel (ethyl acetate/hexane,
3:7) to give 0.63 g (89% yield) of product as a white
solid: mp: 125-127 ° C; Anal. Calc'd. for C22H1~FN4:
C, 73.24; H, 4.98; N, 16.27. Found: C, 72.89; H,
4.69; N, 15.82.
U ~/
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Example 30: Preparation of 2-(Phenylethylamino)-4-[1-
(4-fluorophenyl)-1H-pyrazol-5-yl]pyridine
A mixture of 2-chloro-4-[1-(4-fluorophenyl)-1H-
pyrazol-5-yl]pyridine (Example 46; 0.6 g, 0.002 mol)
and phenethylamine (20 mL) was heated at 190 ° C for
24 hours. The excess amine was removed under vacuum
and the residue was partitioned between ethyl acetate
and water. The organic layer was washed with brine,
dried over magnesium sulfate and filtered. The
filtrate was concentrated and the crude was purified
by chromatography on silica gel (ethyl
acetate/hexane, 2:8) to give 0.55 g (70% yield) of
product as a yellow oil: Anal. Calc~d. for C22H19FN4:
C, 73.72; H, 5.34; N, 15.63. Found: C, 73.33; H,
5.46; N, 15.22.
Example 31: Preparation of 2-Cyano-4-[1-(3-
methylphenyl)-3-methyl-1H-pyrazol-5-yl]pyridine
F
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Ste~~ 1- Preparation of methyl ison~rnr;nate N
Qxide:
To a solution of hydrogen peroxide (43 mL) in
250 mL of acetic acid was added methyl isonicotinate.
The reaction mixture was stirred at 80 ° C overnight
(about eighteen hours). The solution was
concentrated to about 50 mL, water was added and the
mixture was saturated with sodium carbonate. The
aqueous phase was extracted with methylene chloride.
The organic layer was washed with brine, dried over
magnesium sulfate and filtered. The filtrate was
concentrated and trituated with hexane. The
resulting precipitate was filtered and air-dried to
give 24.5 g (65% yield) of product as a white solid:
mp: 118-120 ° C.
f m n i ni
To a solution of methyl isonicotinate N-oxide
(20.0 g, 0.26 mol) in 200 mL of methylene chloride
was added trimethylsilyl cyanide (16.1 g, 0.32 mol),
followed by a solution of dimethylcarbamyl chloride
(17.82 g, 0.32 mol) in 50 mL of methylene chloride at
room temperature. The reaction mixture was stirred
overnight (about eighteen hours) and then treated
with 500 mL of 10% potassium carbonate solution. The
organic layer was washed with brine, dried over
magnesium sulfate and filtered. The filtrate was
concentrated to 15.2 g of crude product as a brown
solid, which was used without further purification.
Step 3- Preparation of 1-(2- cyanoisonicotinyl) 1 3
butanedione-
To a solution of methyl 2-cyanoisonicotinate
(20.0 g, 0.126 mol) in 250 mL THF was added acetone
(30.6 mL, 0.42 mol). The solution was warmed to 35 °
C and sodium methoxide (7.15 g, 0.132 mol) was added
portionwise over 20 minutes. The mixture was heated
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at reflux for 16 hours. The solvent was removed in
vacuo and the residue was dissolved in water,
acidified to pH = 6 with acetic acid. The aqueous
phase was extracted with ethyl acetate and the
organic layer was washed with brine, dried over
magnesium sulfate, filtered and concentrated in vacuo
to yield 13.0 g (58% yield) of product as a brown
solid, which was used in next step without
purification.
Step 4: Preparation of 2-cyano-4-[1-(3-methylphenyl)-
3-methyl-1H-pvrazol-5-yll~yridine
To a mixture of 1-(2-cyanoisonicotinyl)-1,3-
butanedione (1.10 g, 0.0058 mol) and 3-
methylphenylhydrazine hydrochloride (0.97 g, 0.0061
mol) in 20 mL of ethanol was added triethylamine
(0.85 mL, 0.0061 mol) dropwise. The reaction mixture
was heated at reflux overnight (about eighteen
hours). The solvent was removed and the residue was
partitioned between ethyl acetate and water. The
organic layer was washed with brine, dried over
magnesium sulfate and filtered. The filtrate was
concentrated and the crude product was purified by
chromatography on silica gel (ethyl acetate/hexane,
2:8) to give 1.30 g (87% yield) of product as a
yellow solid: mp: 128-130 ° C; Anal. Calc'd. for
C1~H14N4: C, 74.43; H, 5.14; N, 20.42. Found: C,
74.53; H, 5.22; N, 20.29.
Example 32: Preparation of 2-Cyano-4-[1-(4-
fluorophenyl)-3-methyl-1H-pyrazol-5-yl]pyridine
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F
CH3
Triethylamine (4.6 mL, 0.033 mol) was added
dropwise to a mixture of 1-(2-cyanoisonicotinyl)-1,3
butanedione (6.0 g, 0.032 mol) and 4-
fluorophenylhydrazine hydrochloride (5.37 g, 0.033
mol) in 100 mL of ethanol. The reaction mixture was
heated at reflux overnight (about eighteen hours).
The solvent was removed and the residue was
partitioned between ethyl acetate and water. The
organic layer was washed with brine, dried over
magnesium sulfate and filtered. The filtrate was
concentrated and the crude was purified by
recrystallization from ether/hexane to give 4.7 g
(53~ yield) of product as a yellow solid: mp: 94-95 °
C; Anal. Calc'd. for C16H11FN4~ C, 69.06; H, 3.98; N,
20.13. Found: C, 68.52; H, 3.89; N, 19.73.
Example 33: Preparation of {4-[1-(3-methylphenyl)-3-
methyl-1H-pyrazol-5-yl]}-2-pyridine-2-carboxamide
CH3
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To a solution of 2-cyano-4-[1-(3-methylphenyl)-
3-methyl-1H-pyrazol-5-yl]pyridine (Example 31; 0.16
g, 0.0006 mol) in 5 mL of DMSO was added hydrogen
peroxide (0.072 mL, 0.0006 mol) and potassium
carbonate (0.012 g, 0.00009 mol) at zero degrees C.
The reaction mixture was warmed up to room
temperature over 30 minutes. Water was added and the
mixture was stirred for 0.5 hours. The resulting
precipitate was collected by filtration to give 0.12
g (70~ yield) of product as a pale yellow solid: mp:
193-195 ° C; Anal. Calc'd. for C17H16Nq0: C, 69.85; H,
5.52; N, 19.16. Found: C, 69.84; H, 5.40; N, 19.11.
Example 34: Preparation of {4-[1-(4-fluorophenyl)-3
methyl-1H-pyrazol-5-yl]} pyridine-2-carboxamide
F
N-N
CH3
H2N
To a solution of 2-cyano-4-[1-(4-fluorophenyl)-
3-methyl-1H-pyrazol-5-yl]pyridine (Example 32; 1.0 g,
0.0036 mol) in 25 mL of DMSO was added hydrogen
peroxide (0.43 mL, 0.0036 mol) and potassium
carbonate (0.072 g, 0.00051 mol) at zero degrees C.
The reaction mixture was warmed up to room
temperature over 1 hour. Water was added and the
mixture was stirred for 0.5 hours. The resulting
precipitate was collected by filtration to give 0.95
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g (89% yield) of product as a pale yellow solid: mp:
168-170 ° C; Anal. Calc'd. for C16H13FN40: C, 64.86;
H, 4.42; N, 18.91. Found: C, 64.48; H, 4.27; N,
18.80.
Example 35: Preparation of
2-(Dimethylamino)-4-[1-(3-methylphenyl)-3-methyl-1H-
pyrazol-5-ylJpyridine
H3
A mixture of 2-chloro-4-(1-(3-methylphenyl)-3-
methyl-1H-pyrazol-5-yl]pyridine (Example 45; 0.91 g,
0.0032 mol) and 50 mL of liquid ammonia in 20 mL of
DMF was stirred in a sealed tube under 1800 psi, at
180 ° C for 72 hours. After the solution was cooled,
the solvent was removed under vacuum and the residue
was purified by chromatography on silica gel (ethyl
acetate/hexane, 2:8) to give 0.73 g (78% yield) of
product as a yellow crystal: mp: 84-85 ° C; Anal.
Calc'd. for C1gH20N4: C, 73.94; H, 6.89; N, 19.16.
Found: C, 73.68; H, 6.74; N, 19.13.
Example 36: Preparation of 2-(Methylsulfonyl)-4-[1-
(3-methylphenyl)-3-methyl-1H-pyrazol-5-yl]pyridine
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CH3
N-N
CH3
N
O=S=O
t
CH3
A mixture of 2-chloro-4-[1-(3-methylphenyl)-3-
methyl-1H-pyrazol-5-yl)pyridine (Example 45; 1.0 g,
0.0035 mol) and methanesulfonic acid sodium salt
(3.24 g, 0.021 mol) in 15 mL of DMF was heated at 140
° C for 24 hours. After the mixture was cooled, water
was added and the aqueous phase was extracted with
ethyl acetate. The organic layer was washed with
brine, dried over magnesium sulfate and filtered.
The filtrate was concentrated and the crude was
purified by chromatography on silica gel (ethyl
acetate/hexane, 1:1) to give 0.45 g (40~ yield) of
product as a yellow solid: mp: 140-142 ° C; Anal.
Calc'd. for C17H1~N3S02: C, 62.36; H, 5.23; N, 12.83;
S, 9.79. Found: C, 62.16; H, 5.31; N, 12.74; S,
9.86.
Example 37: Preparation of {4-[1-(3-fluorophenyl)-3-
methyl-1H-pyrazol-5-yl]} pyridine-2-azide
H3
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A mixture of 2-chloro-4-[1-(3-fluorophenyl)-3-
methyl-1H-pyrazol-5-yl]pyridine (Example 47; 0.87 g,
0.003 mol) sodium azide (0.59 g, 0.009 mol} in 15 mL
of DMF was heated at 100 ° C for 120 hours. After the
mixture was cooled, water was added and the aqueous
phase was extracted with ethyl acetate. The organic
layer was washed with brine, dried over magnesium
sulfate and filtered. The filtrate was concentrated
and the crude was purified by chromatography on
silica gel (ethyl acetate/hexane, l:l) to give 0.13 g
(15% yield) of product as a yellow solid: mp: 134-135
° C; Anal. Calc'd. for C15H11FN6: C, 61.22; H, 3.77;
N, 28.56. Found: C, 61.44; H, 3.67; N, 28.00.
Example 38: Preparation of 2-(3-Pyridylmethylamino)-
4-[1-(3-fluorophenyl)-3-methyl-1H-pyrazol-5-
yl]pyridine
F
N-N
CH3
N ~.
NH
A mixture of 2-chloro-4-[1-(3-fluorophenyl)-3-
methyl-1H-pyrazol-5-yl]pyridine (Example 47; 0.48 g,
0.0017 mol) and 3-(aminomethyl)pyridine (10 mL) was
heated at 190°C overnight (about eighteen hours). The
reaction mixture was cooled, treated with copious
amount of water and extracted with ethyl acetate.
The organic layer was washed with brine, dried over
magnesium sulfate and filtered. The filtrate was
concentrated and the crude was purified by
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chromatography on silica gel (ethyl
acetate/triethylamine, 99:1) to give 0.35 g (60~
yield) of product as a yellow oil: Anal. Calc'd. for
C21H18FN5: C, 70.18; H, 5.05; N, 19.49. Found: C,
69.78; H, 5.35; N, 18.87.
Example 39: Preparation of
F
CH3
The titled compound was synthesized using 4-
pentanoylpyridine as the starting material following
the procedures described in Example 1, except that 3-
fluorophenylhydrazine was substituted for 4-
fluorophenylhydrazine. 4-Butanoylpyridine and 4-
pentanoylpyridine both can be synthesized according
to procedures described by J. L. Born and S. Early in
J. Pharm. Sci., vol. 69, pp. 850-851, (1980). m.p.
93-94 ° C. Anal. Calc'd. for C1,H16FN3: C, 72.58; H,
5.73; N, 14.94. Found: C, 72.44; H, 5.81; N, 14.67.
Example 40: Preparation of
F
N~N
~CH3
N /
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This compound was synthesized using 4-
butanoylpyridine as the starting material following
the procedures described in Example 25, except that
3-fluorophenylhydrazine was substituted for 4-
fluorophenylhydrazine. 4-Butanoylpyridine and 4-
pentanoylpyridine both can be synthesized according
to procedures described by J. L. Born and S. Early in
J. Pharrn. Sci., vol. 69, pp. 850-851, (1980). M.p.
109-110 ° C. Anal . Calc' d. for C16H14FN3: C, 71 . 89; H,
5.28; N, 15.72. Found: C, 71.58; H, 5.18; N, 15.61.
Example 41: Preparation of 2-chloro-4-(3-methyl-1-(4-
methylphenyl)-1H-pyrazol-5-yl]pyridine
HsC /
N-N
\ ~ \ CH3
N /
~e~ 1~ pr~aration of methyl-2-ch~oro~son~cot~nate
To a solution of thionyl chloride (13.85 mL,
190.38 mmol) in toluene (50 mL) was added 2-
chloropyridine-4-carboxylic acid (15 g, 95 mmol).
The solution was heated to reflux for 3 hours. The
resulting brown color solution was cooled to room
temperature, and methanol (11.56 mL, 285.6 mmol) was
added slowly dropwise. The mixture was brought to
reflux for 15 minutes and became clear. The solution
was then cooled to room temperature and poured into
water (150 mL), basified with 50 percent sodium
hydroxide and extracted with ethyl acetate (2 X 200
ml). The organic layer was separated and washed with
brine, dried with magnesium sulfate, filtered and
concentrated to yield the titled compound (11.93 g,
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73 %) as a brown color solid. This was used in the
next step without further purification.
Eten 2~ Preparation of 1 (2 chloro 4 pyridinyl) 1 3
butaned;nnP
To a solution of methyl-2-chloroisonicotinate
(11.93 g, 69.53 mmol) in THF (200 mL) was added
acetone (14.19 mL, 153 mmol). The solution was
warmed to 35 °C when sodium methoxide (94.13 g, 76.48
mmol) was added sequentially over 20 minutes. The
mixture was stirred for 30 minutes, and then brought
to reflux for 2.5 hours. The solvent was removed
under reduced pressure and then taken up in ethyl
acetate. The resulting solution was washed with
saturated sodium bicarbonate solution and brine,
dried over magnesium sulfate, filtered and
concentrated to yield 1-(2-chloro-4-pyridinyl)-1,3-
butanedione (4.97 g, 76 %) as a light brown colored
solid. This was used in the next step without further
purification.
Step 3: Preparation of 2-chloro-4-[3-methyl-1-(4-
methvlphenyl)-1H-pyraz~l-5-yllpyr;d;nP~
A mixture of 1-(2-chloro-4-pyridinyl)-1,3-
butanedione (7 g, 35.25 mmol), para-
tolylhydrazine.HCl (6.13 g, 38.78 mmol) and
triethylamine (5.41 mL, 38.78 mmol) in methanol (100
mL) was heated to reflux overnight. The resulting
dark colored solution was cooled to room temperature
and extracted with ethyl acetate. The organic layer
was washed with saturated sodium bicarbonate and
brine, and dried over magnesium sulfate. After
filtration, the solution was concentrated to give a
brown solid. The solid was purified by column
chromatography (silica gel, 2:8 EtOAc/hexane) to
yield the titled product (3.6 g, 52 %) as a
crystalline light brown solid. MP: 100.07 °C. Anal.
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Calc'd for C16H14N3C1: C, 67.72, H, 4.97, N, 14.81.
Found: C 67.40, H, 4.89, N, 14.59.
Example 42: Preparation of 1-(4-fluorophenyl)-5-(4-
pyridinyl)-1H-pyrazole-4-carboxylate
F
N,N
N~ ~OMe
Step 1~ Preparation of compound 6
O O
~OCH3
./
N(CH3)2
6
Compound 6: To a solution of methyl
isonicotinate (25.0 g, 0.182 mol)in 100 mL of methyl
acetate was added NaOMe (10.8 g, 0.2 mol). The
reaction mixture was heated at reflux for 3 hours and
cooled to room temperature. Water was added and
extracted with ethyl acetate. The organic layer was
washed with brine, dried over magnesium sulfate and
filtered. The filtrate was concentrated and the
residue was trituated with hexane to give 14.0 g of
product as a pale yellow solid. To a solution of
this compound (13.0 g, 0.072 mol) in THF, was added
N,N-dirnethylformamide dimethyl acetal (51.9 g, 0.43
mol), and the mixture was stirred at room temperature
for 36 hours. The solvent was removed and the
residue was dissolved with water and saturated with
sodium chloride. The aqueous phase was extracted
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with methylene chloride, the organic layer was washed
with brine, dried over magnesium sulfate and
filtered. The filtrate was concentrated to afford
13.2 g of crude product as a yellow oil that was used
without further purification in the next step.
Step 2: Preparation of methyl 1-(4-fluorophenyl)-5-(4-
pyridinyl) -1H ~vrazole-4-carboxylate~
To a solution of the crude compound 6 (2.5 g
,0.011 mol) in 50 mL of a mixture of water and
ethanol (1:1), was added 4-flurophenylhydrazine
hydrochloride (1.71 g, 0.011 mol), and the solution
was stirred at 50 ° C for 2 hours. After the solvent
was removed, the residue was basified by ammonia
hydroxide. The resulting precipitate was filtered,
and air-dried to give 2.05 g of product as a yellow
solid (64% yield), mp: 119-120 ° C; Anal. Calcd. for
C16H1aFN30z: C, 64.64; H, 4.07; N, 14.13. Found: C,
64.28; H, 3.87'; N, 14.14.
Example 43: Preparation of
~I
,N
N
N~ ~OH
To a solution of methyl 1-(4-fluorophenyl)-5-(4-
pyridinyl)-1H-pyrazole-4-carboxylate (Example 42; 0.6 g,
0.002 mol) in 20 mL of ethanol were added 4.0 mL of 1N NaOH
solution. The reaction mixture was stirred at room
temperature for 16 hours. After the solvent was removed,
the residue was acidified with 1N HC1. The precipitate was
filtered and air-dried to give 0.25 g of product as a pale
yellow solid (45% yield), mp: 299-300 ° C; Anal. Calcd. for
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CisHozFN302~H20: C, 59.80; H, 4.01; N, 13.95. Found: C,
59.91; H, 3.52; N, 13.63.
Example 44: Preparation of 2-Chloro-4-[1-(3-
Methylphenyl)-3-methyl-1H-pyrazol-5-yl]pyridine
CH3
-N
_ 1, ~~~H3
N
CI
To a mixture of 1-(2-chloroisonicotinyl)-1,3-
butanedione (1.35 g, 0.068 mol) and 3-
methylphenylhydrazine hydrochloride (1.30 g, 0.0082
mol) in ethanol (40 mL) was added ammonia hydroxide
(0.96 mL, 0.0082 mol) dropwise. The reaction mixture
was heated at reflux overnight. The solvent was
removed and the residue was partitioned between ethyl
acetate and water. The organic layer was washed with
brine, dried over magnesium sulfate and filtered.
The filtrate was concentrated and the crude was
purified by chromatography on silica gel (ethyl
acetate/hexane, 2:8) to give 1.68 g (87~ yield) of
product as a yellow oil: Anal. Calcd. for C16H14C1N3:
C, 67.72; H, 4.97; N, 14.81; C1, 12.49. Found: C,
67.91; H, 5.08; N, 14.79; C1, 12.40.
Example 45: Preparation of 2-Chloro-4- [1- (4
fluorolphenyl)-1H-pyrazol-5-yl]pyridine
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F
N N
N,J
C1
~p 1: Preparation of 2-chloro-4-lN-methyl-N-
me t hoxyc a~bamo~r~~yr i di ne
To a suspension of 2-chloroisonicotinic acid
(18.0 g, 0.105 mol) in 250 mL of methylenechloride
was added 1,1'-carbonyldiimidazole (17.0 g, 0.105
mol) portionwise. The mixture was stirred for 0.5 h
and N,O-dimethylhydroxylamine hydrochloride (10.2 g,
0.105 mol) was added rapidly. The reaction mixture
was stirred at room temperature overnight. Ether was
added and the organic layer was washed with water,
dried, dried over magnesium sulfate and filtered.
The filtrate was concentrated to give 14.5 g (70~) of
product as a yellow oil. This was used in next step
without purification.
Step 2: Preparation of 2-chloro-4-acetyl~yridine
To methylmagnesium bromide (30 mL of 3.0 M in
THF) was added a solution of 2-chloro-4-(N-methyl-N-
methoxycarbamoyl)pyridine (6.0 g, 0.03 mol) in 20 mL
of THF at 0 C. The reaction mixture was stirred
overnight while allowing to warm up to room
temperature. A solution of potassium hydrogensulfate
(12 g) in 300 mL of water was added and the aqueous
phase was extracted with ethyl acetate. The organic
layer was washed with brine, dried over magnesium
sulfate and filtered. The filtrate was concentrated
4.0 g (86~) of product as a yellow oil. This was
used in next step without purification.
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~e_p 3: Preparation of 1-f2-chloroisonicot Vinyl)-2-
(dimethylaminomethvlene)ethanone
A mixture of 2-chloro-4-acetylpyridine (9.1 g,
0.058 mol) in 45 mL of N,N-dimethylformamide dimethyl
acetal was heated at reflux for 3 h. The dark
solution was cooled and treated with 300 mL of
hexane. The precipitate was collected by filtration
and air-dried to give 14.6 g of product as a brownish
solid (70~), which was used without further
purification.
Step 4: Preparation of 2-chloro-4-fl-(4-
fluoro~yl)-1H-pvrazol-5-yllpvridine
To a mixture of 1-(4-pyridyl)-2-
(dimethylaminomethylene)ethanone (2.1 g, 0.01 mol)
and 4-fluorophenylhydrazine hydrochloride (1.63 g,
0.01 mol) in 50 mL of ethanol was added 1 mL of
water. The reaction mixture was heated at reflux for
1.5 h. Solvent was removed and the residue was
partitioned between water and ethyl acetate. The
organic layer was washed with brine, dried over
magnesium sulfate and filtered. The filtrate was
concentrated and the crude was purified by
chromatography on silica gel (ethyl acetate/hexane,
2:8) to give 2.5 g (93~ yield) of product as a yellow
oil: Anal. Calcd. for C14H9C1FN3: C, 61.44; H, 3.31;
N, 15.35. Found: C, 61.52; H, 3.27; N, 14.73.
Example 46: Preparation of 2-Chloro-4-[1-(3-
fluorophenyl)-3-methyl-1H-pyrazol-5-yl]pyridine
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F
i
-N
y--CH3
N
CI
Steo 1- Preparation of 2 chloroison;r-otinic acid
A mixture of isonicotinic acid N-oxide (28.0 g,
0.2 mol) and phosphorus oxychloride (120.0 g, 0.8
mol) was heated at reflux for 7 h. The reaction
mixture was cooled and carefully poured into 600 mL
of ice water. The precipitate was collected by
filtration to give 20.6 g (65% yield) of product as a
pale yellow solid, mp: 248-249 C (lit: 250-252 C).
Step 2: Preparation of methyl 2-chlor~isonico ~r~ste
To a solution of thionyl chloride (15.0 g, 0.127
mol) in 20 mL of toluene was added 2-
chloroisonicotinic acid (10.0 g, 0.063 mol) and the
reaction was heated at reflux until gas evolution
ceased. Then a solution of methanol (7.7 mL, 0.19
mol) in 10 mL of toluene was added at room
temperature over 15 min. The reaction mixture was
then refluxed for 1 h and then cooled to room
temperature. The clear solution was poured into 100
mL of water, basified with 40% NaOH and extracted
with ethyl acetate. The organic layer was washed
with brine, dried over magnesium sulfate filtered.
The filtrate was concentrated in vacuo to give 8.2 g
(83%) of product as a brown oil which solidified upon
standing, mp: 36-37 C.
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,~~p 3: Preparation of 1-(2-chloroisonicotinyl)-1,3-
butanedione:
To solution of methyl 2-chloroisonicotinate
(12.0 g, 0.07 mol) and acetone (12.2 g, 0.21 mol) in
100 mL of dry THF at 35 C was added sodium methoxide
portionwise. After heating the reaction mixture at
reflux for 4 h, the solvent was removed. The residue
was dissolved with 500 mL of water, acidified with
acetic acid to pH = 6 and extracted with ethyl
acetate. The organic layer was washed with brine,
dried over magnesium sulfate and filtered. The
filtrate was concentrated in vacuo to give 11.46 g
(83°s yield) of product was a brown solid.
Step 4: Preparation of 2-chloro-4-f1-(3-
fluorophenyl)-3-methyl-1H ~yrazol-5vllgvridine:
To a mixture of 1-(2-chloroisonicotinyl)-1,3-
butanedione (1.35 g, 0.068 mol) and 3-
fluorophenylhydrazine hydrochloride (1.33 g, 0.0082
mol) in ethanol (40 mL) was added ammonium hydroxide
(0.96 mL, 0.0082 mol) dropwise. The reaction mixture
was heated at reflux overnight. The solvent was
removed and the residue was partitioned between ethyl
acetate and water. The organic layer was washed with
brine, dried over magnesium sulfate and filtered.
The filtrate was concentrated and the crude was
purified by chromatography on silica gel (ethyl
acetate/hexane, 2:8) to give 1.54 g (79°s yield) of
product as a yellow oil: Anal. Calcd. for
C15H11C1FN3: C, 62.62; H, 3.85; N, 14.60. Found: C,
62.34; H, 3.88; N, 14.30.
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BIOLOGICAL EVALUATION
p38 Kinase Assay
Cloning of human 3~
The coding region of the human p38a cDNA was
obtained by PCR-amplification from RNA isolated from
the human monocyte cell line THP.1. First strand
cDNA was synthesized from total RNA as follows: 2
~,g of RNA was annealed to 100 ng of random hexamer
primers in a 10 ~1 reaction by heating to 70 °C for 10
minutes followed by 2 minutes on ice. cDNA was then
synthesized by adding 1 ~1 of RNAsin (Promega,
Madison WI), 2 ~.1 of 50 mM dNTP'S, 4 ~l of 5X
buffer, 2 ~,1 of 100 mM DTT and 1 ul (200 U) of
Superscript II TM AMV reverse transcriptase. Random
primer, dNTP's and Superscript TM reagents were all
purchased from Life-Technologies, Gaithersburg, MA.
The reaction was incubated at 42 °C for 1 hour.
Amplification of p38 cDNA was performed by aliquoting
5 ~1 of the reverse transcriptase reaction into a 100
~1 PCR reaction containing the following: 80 ~1 dH2
O, 2 ~tl 50 mM dNTP's, 1 ~1 each of forward and
reverse primers (50 pmol/~tl), 10 ~.1 of lOX buffer and
1 ~1 Expand TM polymerase (Boehringer Mannheim). The
PCR primers incorporated Bam HI sites onto the 5' and
3' end of the amplified fragment, and were purchased
from Genosys. The forward and reverse primers were
5'-GATCGAGGATTCATGTCTCAGGAGAGGCCCA-3' and
5'GATCGAGGATTCTCAGGACTCCATCTCTTC-3', respectively.
The PCR amplification was carried out in a DNA
Thermal Cycler (Perkin Elmer) by repeating 30 cycles
of 94 °C for 1 minute, 60 oC for 1 minute and 68 oC
for 2 minutes. After amplification, excess primers
and unincorporated dNTP's were removed from the
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amplified fragment with a Wizard TM PCR prep
(Promega) and digested with Bam HI (New England
Biolabs). The Bam HI digested fragment was ligated
into BamHI digested pGEX 2T plasmid DNA
(PharmaciaBiotech) using T-4 DNA ligase (New England
Biolabs) as described by T. Maniatis, Molecular
Cloning: A Laboratory Manual, 2nd ed. (1989). The
ligation reaction was transformed into chemically
competent E. coli DH10B cells purchased from Life-
Technologies following the manufacturer's
instructions. Plasmid DNA was isolated from the
resulting bacterial colonies using a Promega WizardTM
miniprep kit. Plasmids containing the appropriate
Bam HI fragment were sequenced in a DNA Thermal
Cycler (Perkin Elmer) with PrismTM (Applied
Biosystems Inc.). cDNA clones were identified that
coded for both human p38a isoforms (Lee et al. Nature
372, 739). One of the clones which contained the
cDNA for p38a-2 (CSBP-2) inserted in the cloning site
of pGEX 2T, 3' of the GST coding region was
designated pMON 35802. The sequence obtained for this
clone is an exact match of the cDNA clone reported
by Lee et al. This expression plasmid allows for the
production of a GST-p38a fusion protein.
Expression of human 3~ 8a-
GST/p38a fusion protein was expressed from the
plasmid pMON 35802 in E. coli, stain DH10B (Life
Technologies, Gibco-BRL). Overnight cultures were
grown in Luria Broth (LB) containing 100 mg/ml
ampicillin. The next day, 500 ml of fresh LB was
inoculated with 10 ml of overnight culture, and grown
in a 2 liter flask at 37 °C with constant shaking
until the culture reached an absorbance of 0.8 at 600
nm. Expression of the fusion protein was induced by
addition of isopropyl b-D-thiogalactosidse (IPTG) to
a final concentration of 0.05 mM. The cultures were
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shaken for three hours at room temperature, and the
cells were harvested by centrifugation. The cell
pellets were stored frozen until protein
purification.
Purification of y~38 Kinase a~
All chemicals were from Sigma Chemical Co.
unless noted. Twenty grams of E. coli cell pellet
collected from five 1 L shake flask fermentations was
resuspended in a volume of PBS (140 mM NaCl, 2.7 mM
KCl, 10 mM Na2HP04, 1.8 mM KH2P04~ pH 7.3) up to 200
ml. The cell suspension was adjusted to S mM DTT
with 2 M DTT and then split equally into five 50 ml
Falcon conical tubes. The cells were sonnicated
(Ultrasonics model W375) with a 1 cm probe for 3 X 1
minutes (pulsed) on ice. Lysed cell material was
removed by centrifugation (12,000 x g, 15 minutes)
and the clarified supernatant applied to glutathione-
sepharose resin (Pharmacia).
~lutathione-Se~harose Affinity Chromatog~p~~rs
Twelve ml of a 50% glutathione sepharose-PBS
suspension was added to 200 ml clarified supernatant
and incubated batchwise for 30 minutes at room
temperature. The resin was collected by
centrifugation (600 x g, 5 min) and washed with 2 x
150 ml PBS/1% Triton X-100, followed by 4 x 40 ml
PBS. To cleave the p38 kinase from the GST-p38
fusion protein, the glutathione-sepharose resin was
resuspended in 6 ml PBS containing 250 units thrombin
protease (Pharmacia, specific activity > 7500
units/mg) and mixed gently for 4 hours at room
temperature. The glutathione-sepharose resin was
removed by centrifugation (600 x g, 5 min) and washed
2 x 6 ml with PBS. The PBS wash fractions and digest
supernatant containing p38 kinase protein were pooled
and adjusted to 0.3 mM PMSF.
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Mono O Anion Exchange Chromatog~r~phv
The thrombin-cleaved p38 kinase was further
purified by FPLC-anion exchange chromatography.
Thrombin-cleaved sample was diluted 2-fold with
Buffer A (25 mM HEPES, pH 7.5, 25 mM beta-
glycerophosphate, 2 mM DTT, 5% glycerol) and injected
onto a Mono Q HR 10/10 (Pharmacia) anion exchange
column equilibrated with Buffer A. The column was
eluted with a 160 ml 0.1 M-0.6 M NaCl/Buffer A
gradient (2 ml/minute flowrate). The p38 kinase peak
eluting at 200 mM NaCl was collected and concentrated
to 3-4 ml with a Filtron 10 concentrator (Filtron
Corp . ) .
Se~hacryl S100 Gel Filtration rhroma oq~~hy
The concentrated Mono Q- p38 kinase purified
sample was purified by gel filtration chromatography
(Pharmacia HiPrep 26/60 Sephacryl S100 column
equilibrated with Buffer B (50 mM HEPES, pH 7.5, 50
mM NaCl, 2 mM DTT, 5% glycerol)). Protein was eluted
from the column with Buffer B at a 0.5 ml/minute
flowrate and protein was detected by absorbance at
280 nm. Fractions containing p38 kinase (detected by
SDS-polyacrylamide gel electrophoresis) were pooled
and frozen at -80 °C. Typical purified protein yields
from 5 L E. coli shake flasks fermentations were 35
mg p38 kinase.
In Vitro Assay
The ability of compounds to inhibit human p38
kinase alpha was evaluated using two in vitro assay
methods. In the first method, activated human p38
kinase alpha phosphorylates a biotinylated substrate,
PHAS-I (phosphorylated heat and acid stable protein-
insulin inducible), in the presence of gamma 3ZP-ATP
('zP-ATP). PHAS-I was biotinylated prior to the assay
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and provides a means of capturing the substrate which
is phosphorylated during the assay. p38 Kinase was
activated by MKK6. Compounds were tested in 10 fold
serial dilutions over the range of 100 ~M to 0.001 ~M
using 1% DMSO. Each concentration of inhibitor was
tested in triplicate.
All reactions were carried out in 96 well
polypropylene plates. Each reaction well contained
25 mM HEPES pH 7.5, 10 mM magnesium acetate and 50 ~M
unlabeled ATP. Activation of p38 was required to
achieve sufficient signal in the assay. Biotinylated
PHAS-I was used at 1-2 ~tg per 50 ~l reaction volume,
with a final concentration of 1.5 ~M. Activated
human p38 kinase alpha was used at 1 ~g per 50 ~.1
reaction volume representing a final concentration of
0.3 ~M. Gamma 32P-ATP was used to follow the
phosphorylation of PHAS-I. 32P-ATP has a specific
activity of 3000 Ci/mmol and was used at 1.2 ~Ci per
50 ~1 reaction volume. The reaction proceeded either
for one hour or overnight at 30 °C.
Following incubation, 20 ~1 of reaction mixture
was transferred to a high capacity streptavidin
coated filter plate (SAM-streptavidin-matrix,
Promega) prewetted with phosphate buffered saline.
The transferred reaction mix was allowed to contact
the streptavidin membrane of the Promega plate for 1-
2 minutes. Following capture of biotinylated PHAS-I
with 32P incorporated, each well was washed to remove
unincorporated 32P-ATP three times with 2M NaCl,
three washes of 2M NaCl with 1% phosphoric, three
washes of distilled water and finally a single wash
of 95% ethanol. Filter plates were air dried and 20
~1 of scintillant was added. The plates were sealed
and counted. Results are shown in Table I.
A second assay format was also employed that is
based on p38 kinase alpha induced phosphorylation of
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EGFRP (epidermal growth factor receptor peptide, a 21
mer) in the presence of 33P-ATP. Compounds were
tested in 10 fold serial dilutions over the range of
100~M to O.OO1~M in 1% DMSO. Each concentration of
inhibitor was tested in triplicate. Compounds were
evaluated in 50~t1 reaction volumes in the presence of
25 mM Hepes pH 7.5, 10 mM magnesium acetate, 4%
glycerol, 0.4% bovine serum albumin, 0.4mM DTT, 50~,M
unlabeled ATP, 25 ~,g EGFRP (200~,M), and 0.05 ~Ci
gamma 3'P-ATP. Reactions were initiated by addition
of 0.09 ~g of activated, purified human GST-p38
kinase alpha. Activation was carried out using GST-
MKK6 (5:1,p38:MKK6) for one hour at 30 °C in the
presence of 50 ~M ATP. Following incubation for 60
minutes at room temperature, the reaction was stopped
by addition of 150 ~1 of AG 1X8 resin in 900 mM
sodium formate buffer, pH 3.0 (1 volume resin to 2
volumes, buffer). The mixture was mixed three times
with pipetting and the resin was allowed to settle.
A total of 50 ~1 of clarified solution head volume
was transferred from the reaction wells to Microlite-
2 plates. 150 ~.1 of Microscint 40 was then added to
each well of the Microlite plate, and the plate was
sealed, mixed, and counted.
Results of these p38 Kinase Assays are shown in
Table I.
TABLE I.
p38 Kinase
3 0 damp 1 a ( I C~~
1 16.8
2 37.2
4.9
4 >100
5 1.4
0.4
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7 0.01
8 0.07
0.29
10 >100
11 0.07
12 0.003
13 0.05
14 >100
15 >100
16 7,72
17 65.1
18 35.7
19 0.002
20 -
21 0.35
22 0.079
23 9.553
24 <0.01
25 2.239
26 0.156
27 >100
28 0.843
29 0.129
30 0.024
31 0,5
32 2.97
33 -
34 44.0
35 1.151
36 29,g9
37 85
38 0.219
39 1.48
40 16.5
41 14
42 2.68
43 65.1
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44 -
45 1.06
46 67 7
TNF Cell Assays
Method of Isolation of Human Peripheral Blood
Mononuclear Cells~
Human whole blood was collected in Vacutainer
tubes containing EDTA as an anticoagulant. A blood
sample (7 ml) was carefully layered over 5 ml PMN
Cell Isolation Medium (Robbins Scientific) in a 15 ml
round bottom centrifuge tube. The sample was
centrifuged at 450-500 x g for 30-35 minutes in a
swing out rotor at room temperature. After
centrifugation, the top band of cells were removed
and washed 3 times with PBS w/o calcium or magnesium.
The cells were centrifuged at 400 x g for 10 minutes
at room temperature. The cells were resuspended in
Macrophage Serum Free Medium (Gibco BRL) at a
concentration of 2 million cells/ml.
LPS Stimulation of Human PBMs:
PBM cells (0.1 ml, 2 million/ ml) were co-
incubated with 0.1 ml compound (10-0.41 ~M, final
concentration) for 1 hour in flat bottom 96 well
microtiter plates. Compounds were dissolved in DMSO
initially and diluted in TCM for a final
concentration of 0.1% DMSO. LPS (Calbiochem, 20
ng/ml, final concentration) was then added at a
volume of 0.010 ml. Cultures were incubated
overnight at 37 °C. Supernatants were then removed
and tested by ELISA for TNF-a and IL1-b. Viability
was analyzed using MTS. After 0.1 ml supernatant was
collected, 0.020 ml MTS was added to remaining 0.1 ml
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cells. The cells were incubated at 37 °C for 2-4
hours, then the O.D. was measured at 490-650 nM.
Results of these TNF Cell Assays are shown in
Table II.
TABLE II.
TNF Cell
Example ( IC~,~
1 0.1
2 4.0
3 0.6
4 >10
5 0.2
6 1.5
7 1.1
8 0.2
0.1
10 >10
11 0.1
12 0.3
13 0.1
14 >10
15 >10
16 >10
17 >10
18 >10
19 0.1
20 _
21 0.5
22 1.9
23 2.4
24 0.02
25 0.8
26 0.1
27 >10
28 >10
29 1.3
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30 10
31 1.7
32 >10
33 5.0
34 -
35 3.4
36 >10
37 2.2
38 0.6
39 1.3
40 0.7
41 2.9
42 -
43 -
44 -
45 8.1
46 -
Maintenance and Differentiation of the U937 Human
Hist~ ocy ~ Lymphoma Cel~ .Line
U937 cells (ATCC) were propagated in RPMI 1640
containing 10% fetal bovine serum, 100 IU/ml
penicillin, 100 ~g/ml streptomycin, and 2 mM
glutamine (Gibco). Fifty million cells in 100 ml
media were induced to terminal monocytic
differentiation by 24 hour incubation with 20 ng/ml
phorbol 12-myristate 13-acetate (Sigma). The cells
were washed by centrifugation (200 x g for 5 min) and
resuspended in 100 ml fresh medium. After 24-48
hours, the cells were harvested, centrifuged, and
resuspended in culture medium at 2 million cells/ml.
LPS Stimulation of TNF production by U937 Cells
U937 cells (0.1 ml, 2 million/ml) were incubated
with 0.1 ml compound (0.004-50 ~M, final
concentration) for 1 hour in 96 well microtiter
plates. Compounds were prepared as 10 mM stock
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solutions in DMSO and diluted in culture medium to
yield a final DMSO concentration of 0.1% in the cell
assay. LPS (E coli, 100 ng/ml final concentration)
was then added at a volume of 0.02 ml. After 4 hour
incubation at 37°C, the amount of TNF-a released in
the culture medium was quantitated by ELISA.
Inhibitory potency is expressed as IC50 (~M).
Results of these U937 Cell Assays are shown in
Table III.
TABLE III.
U937 Cell Assay
~xac~ple ( ICS pM)
1 10.0
2 -
3 0.66
4 -
5 -
6 -
7 _
8 0.72
9 17.88
10 -
11 0.48
12 0.20
13 0.86
14 -
15 10.0
16 >10
17 10.0
18 10.0
19 -
20 -
21 -
22 1.31
23 10.0
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24 0.05
25 -
26 0.21
27 10.0
28 2.64
29 0.24
30 -
31 1.65
32 >10
33 -
34 -
35 10.0
36 -
37 -
38 -
39 0.83
40 1.15
41 -
42 10.0
43 10.0
44 -
45 10.0
46 >10
All patent documents listed herein are
incorporated by reference.
Although this invention has been
describedwith respect to specific
embodiments,the details of these embodiments are
not to beconstrued as limitations.
