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Definitions

• This invention is directed to kinases inhibitors, and more particularly to 1,5-diary1 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.
• 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) .
• 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.
• Various pyrazoles have previously been described.
• 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.
• 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.
• 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 foxtail, 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.
• MAP mitogen-activated protein
• Mitogen-activated protein kinases are believed to be associated with, inter alia, the mediation of a number of inflammatory diseases.
• 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.
• a process for treating a host mammal having a condition associated with pathological p38 MAP kinase activity 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) 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) .
• TNF- tumor necrosis factor
• IL-1 interleukin-1
• the products of the p38 phosphorylation mediate the production of inflammatory cytokines, including TNF- and IL-1, and cyclooxygenase-2.
• 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.
• 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.
• 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 structure to Formula I below, or a pharmaceutically- acceptable salt thereof:
• Ar 1 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
• Z is selected from the group consisting of hydrido, hydrocarbyl, halogen, carboxy, cyano, azido, hydrocarbylsulfonyl, carbonyloxyhydrocarbyl , carbonylamido, and -X-Y wherein -X is -0, -S or -NQ,
• -Y is hydrido, hydrocarbyl or hydrocarbylaryl
• Q is hydrido, hydrocarbyl, hydroxylhydrocarbyl , 2-, 3-, or 4-pyridylhydrocarbyl, or arylhydrocarbyl ;
• R 1 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 , trifluoromethylhydrocarbyl , hydroxy, mercapto, hydroxycarbonyl, aryloxy, arylthio, arylamino, arylhydrocarbyl, aryl, heteroaryloxy, heteroarylthio, heteroarylamino, heteroarylhydrocarbyl , hydrocarbyloxycarbonyl - hydrocarbyl, heterocyclooxy, hydroxycarbonyl - hydrocarbyl, heterocyclothio, heterocycloamino, cyclohydrocarbyloxy,
• R 2 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, heteroarylhydrocarbyl , hydrocarbyloxycarbonyl - hydrocarbyl, heterocyclooxy, hydroxycarbon
• Ar 1 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, hydrocarby
• R 2 is other than hydrido, carboxy, hydrocarbyloxycarbonyl, halogen, or aryl.
• A CH- so that a contemplated 5-substituent is a 4-pyridyl residue, as compared to being a 4-pyrimidyl residue.
• Formula I includes three particularly preferred subclasses of compounds of high interest.
• One subclass of particular interest is a preferred class of compounds exhibit IC 50 in vi tro 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: Ar 1 is an aryl group that is substituted by a group selected from fluorine, or lower hydrocarbyl;
• R 1 is hydrido, or lower hydrocarbyl
• R 2 is selected from the group consisting of hydrido, lowerhydrocarbyl and aminocarbonyl ;
• Z is hydrido, or -X-Y;
• -X is -0 or -NQ;
• Q is aryl lower hydrocarbyl; and -Y is hydrido or lower hydrocarbyl .
• a more preferred subclass of compounds exhibit IC 50 in vi tro 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: Ar 1 is an aryl group that is substituted by one or more substituents that are lower hydrocarbyl, or halo such as flourine;
• R-L is hydrido, or lower hydrocarbyl
• R 2 is hydrido or lower hydrocarbyl; Z is hydrido or -X-Y; -X is -NQ;
• Q is lower hydrocarbyl or hydroxyl lower hydrocarbyl ;
• -Y is hydrido or lower hydrocarbyl.
• the most preferred subclass of compounds of Formula I exhibit IC 50 in vi tro activities in the 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: Ar ⁇ is an aryl group that is substituted by one or more substituents that are lower hydrocarbyl or halo such as flourine or chlorine;
• R 1 is hydrido, or lower hydrocarbyl
• R 2 is hydrido;
• Z is selected cyano or -X-Y; wherein -X is -0, or -NQ;
• Q is selected from a group consisting of hydrido, lowerhydrocarbyl , aryl lower hydrocarbyl, hydroxyl lower hydrocarbyl, and 3-pyridyl lower hydrocarbyl;
• -Y is hydrido, lower hydrocarbyl, or aryl lower hydrocarbyl .
• MAP mitogen-activated protein
• Mitogen-activated protein kinases are believed to be associated with, inter alia, the mediation of a number of inflammatory diseases.
• these certain 1,5- diaryl pyrazoles are effective for the inhibition of the p38 MAP kinase group of enzymes, a sub-family of MAP.
• 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.
• 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 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.
• 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 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 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.
• 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.
• 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, LTB 4 antagonists and LTA 4 hydrolase inhibitors.
• 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.
• p38-mediated disorder refers to any and all disorders and 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.
• IL-1 IL-6
• IL-8 another factor to be released
• 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.
• TNF also known as cachectin
• One subclass of particularly preferred compounds has structures that are represented by Formula II, below, or a pharmaceutically acceptable salt thereof:
• R 3 is hydrido, or (lower) hydrocarbyl ;
• R is selected from a group consisting of hydrido, lower hydrocarbyl, aryl lower hydrocarbyl, hydroxyl lower hydrocarbyl, and 2-pyridyl lower hydrocarbyl, 3-pyridyl lower hydrocarbyl or 4- pyridyl lower hydrocarbyl ;
• Ar ⁇ is an aryl group that is substituted by a halogen or halo group (e.g., chlorine, fluorine and bromine), lower hydrocarbyl, or hydrocarbyloxy group ;
• a halogen or halo group e.g., chlorine, fluorine and bromine
• R 1 hydrido, or C--C 6 hydrocarbyl
• R 2 is hydrido, or lower hydrocarbyl.
• One preferred group of compounds of Formula II noted in Table I exhibit IC 50 in vi tro 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: Ar ⁇ is an aryl group that is substituted by lower hydrocarbyl;
• R 1 is lower hydrocarbyl
• R 2 is hydrido
• R- is hydrido or hydrocarbyl; and R 4 is hydrocarbyl or aryl lower hydrocarbyl .
• a more preferred class of compounds of Formula II noted in Table I exhibits IC 50 in vi tro 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:
• Ar 1 is an aryl group that is substituted by lower hydrocarbyl ;
• R! is lower hydrocarbyl;
• R 2 is hydrido
• R3 is hydrido or lower hydrocarbyl
• R 4 is lower hydrocarbyl, or hydroxyl lower hydrocarbyl .
• the most preferred class of compounds of Formula II noted in Table I exhibits IC 50 in vi tro 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:
• Ar 1 is an aryl group that is substituted with a lower hydrocarbyl or halogen (e.g., flourine or chlorine) group;
• R 1 is hydrido, or lower hydrocarbyl
• R 2 is hydrido
• R 3 is hydrido or lower hydrocarbyl
• R 4 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:
• R 5 is hydrido, hydrocarbyl, or aryl lower hydrocarbyl ;
• Ar 1 is an aryl group that is substituted with a halogen (e.g., chlorine, fluorine or bromine), lower hydrocarbyl, or hydrocarbyloxy group;
• a halogen e.g., chlorine, fluorine or bromine
• R 1 is C- L -Cg hydrocarbyl
• R 2 is hydrido.
• Preferred compounds of Formula III exhibit IC 50 in vi tro 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 is comprised of those compounds of Formula III or a pharmaceutically acceptable salt thereof wherein:
• Ar 1 is an aryl group that is substituted by a lower hydrocarbyl group
• R 1 is lower hydrocarbyl
• R 2 is hydrido
• R 5 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:
• Ar 1 is an aryl group that is substituted by a halogen (e.g., chlorine, fluorine or bromine), lower hydrocarbyl or a hydrocarbyloxy group;
• a halogen e.g., chlorine, fluorine or bromine
• R 1 is lower hydrocarbyl
• R 2 is hydrido or lower hydrocarbyl .
• Preferred compounds of Formula IV exhibit IC 50 in vi tro 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: Ar 1 is an aryl group that is substituted by a lower hydrocarbyl group;
• Rl is lower hydrocarbyl
• R 2 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.
• 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.
• aryl groups or radicals 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.
• 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.
• hydrocarbyl group containing a -C(0)0- functionality is referred to as a hydrocarboyl group inasmuch as there is no ambiguity in using that suffix.
• a substituent that cannot exist such as a C ⁇ _ alkenyl group is not intended to be encompassed by the word "hydrocarbyl”.
• 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.
• radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl and the like.
• 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 .
• alkenyl and “lower alkenyl” embrace radicals having "cis” and “trans” orientations, or alternatively, "E” and "Z” orientations.
• 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
• alkyl group is a particularly preferred hydrocarbyl group.
• 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 .
• 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.
• 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 .
• 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 .
• 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 cyclobutenylmethyl, cyclopentenylmethyl and cyclohexenylmethyl .
• halo or halogen means halogens such as fluorine, chlorine, bromine or iodine.
• 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.
• haloalkyl radicals include fluoromethyl, difluoromethyl , trifluoromethyl , chloromethyl , dichloromethyl , trichloromethyl , pentafluoroethyl , heptafluoropropyl , difluorochloromethyl , dichlorofluoromethyl , difluoroethyl , difluoropropyl , dichloroethyl and dichloropropyl .
• 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 .
• alkoxy and “alkyloxy” embrace linear or branched oxy-containing radicals each having alkyl portions of one to about twelve carbon atoms.
• alkoxy radicals are "lower alkoxy" radicals having one to six carbon atoms. Examples of such radicals include methoxy, ethoxy, propoxy, butoxy and tert-butoxy.
• 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 .
• 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 , ammocarbonylalkyl, alkoxy, aralkoxy, hydroxyl, amino, halo, nitro, alkylamino, acyl , cyano, carboxy, aminocarbonyl , alkoxycarbonyl and aralkoxycarbonyl .
• 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.
• 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.
• heterocyclyl radicals include dihydrothiophene, dihydropyran, dihydrofuran and dihydrothiazole.
• Heterocyclyl radicals can include a tetravalent nitrogen, such as in tetrazolium and pyridinium radicals.
• heteroaryl embraces aromatically- unsaturated heterocyclyl radicals.
• 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.
• 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,
• 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 to 3 nitrogen atoms (e.g., benzothiazolyl or benzothiadiazolyl) and the like.
• thiazolyl, thiadiazolyl e.g., 1,2,4- thiadiazolyl, 1, 3 , 4-thiadiazolyl , and 1,2,5- thiadiazolyl
• unsaturated condensed heterocyclyl group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms e.g., be
• heteroaryl also embraces radicals where heterocyclyl radicals are fused with aryl radicals. Examples of such fused bicyclic radicals include benzofuran, benzothiophene, and the like.
• heterocyclyl group can have 1 to 3 substituents such as alkyl, hydroxyl, halo, alkoxy, oxo, amino and alkylamino.
• heterocyclylalkylene embraces heterocyclyl -substituted alkyl radicals. More preferred heterocyclylalkylene radicals are "lower heterocyclylalkylene” radicals having one to six carbon atoms and a heterocyclyl radical .
• 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.
• 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 .
• 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 .
• the term “sulfonyl”, whether used alone or linked to other terms such as “alkylsulfonyl " , or “halosulfonyl” denotes a divalent radical, -S0 2 -.
• 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.
• halosulfonyl embraces halo radicals attached to a sulfonyl radical. Examples of such halosulfonyl radicals include chlorosulfonyl and bromosulfonyl .
• sulfamyl denote NH 2 O 2 S- .
• carboxy or “carboxyl”, whether used alone or with other terms, such as “carboxyalkyl”, denotes -CO 2 -.
• 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 .
• 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 .
• w 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 .
• alkylcarbonyl includes radicals having alkyl radicals attached to a carbonyl radical. Examples of such radicals include methylcarbonyl , ethylcarbonyl, propylcarbonyl , butylcarbonyl , and pentylcarbonyl .
• 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.
• heterocyclylalkylene embraces saturated, partially unsaturated and unsaturated heterocyclyl -substituted alkyl radicals such as pyrrolidinylmethyl , pyridylmethyl , quinolylmethyl , thienylmethyl , furylethyl, and quinolylethyl .
• the heteroaryl in heteroaralkyl can be additionally substituted with halo, alkyl, alkoxy, haloalkyl and haloalkoxy groups.
• aryloxy embraces aryl radicals attached through an oxygen atom to other radicals.
• aralkoxy embraces aralkyl radicals attached through an oxygen atom to other radicals.
• aminoalkyl embraces alkyl radicals substituted with amino radicals. More preferred are “low aminoalkyl” radicals. Examples of such radicals include aminomethyl, aminoethyl , and the like.
• 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.
• 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.
• 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.
• 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.
• 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 Ar 1 , R 1 , R 2 , R 3 , R 4 , and R 5 .
• the remaining groups Ar 1 , R 1 , R 2 , R 3 , R 4 , and R 5 illustrated for each structure shown in a compound table are as discussed elsewhere herein.
• 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.
• compositions of Formula I are 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.
• 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.
• inorganic acids examples include 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, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, stearic, salicylic, p-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic) , methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, toluenesulfonic, 2-hydroxyethanesulfonic, 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 la) salts, alkaline earth metal (group Ila) 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.
• a pharmaceutical composition contains a therapeutically-effective amount of a compound of Formula I in association with at least one pharmaceutically-acceptable carrier, adjuvant or diluent .
• 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.
• carrier non-toxic, pharmaceutically-acceptable carriers and/or diluents and/or adjuvants
• active compounds of the present invention can be administered by any suitable route, preferably in the form of a pharmaceutical composition adapted 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.
• 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.
• a pharmaceutical composition can contain an active compound at about 0.1 to 1000 mg, preferably at about 7.0 to 350 mg.
• the daily dose can be administered in one to four doses per day.
• a topical preparation of compounds of this invention to the affected area two to four times a day.
• 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.
• the active ingredients can be employed with either paraffinic or a water-miscible ointment base.
• the active ingredients can be formulated in a cream with an oil-in-water cream base.
• 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.
• topical administration is accomplished using a patch either of the reservoir and porous membrane type or of a solid matrix variety.
• 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.
• 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.
• 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 .
• Emulsifiers and emulsion stabilizers suitable for use in the formulation of the present invention include TweenTM 60, SpanTM 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 pharmaceutical emulsion formulations is very low.
• 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.
• 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.
• the active compounds of this combination invention are ordinarily combined with one or more adjuvants appropriate to the indicated route of administration.
• 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 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.
• Scheme I shows the synthesis of a 1,5-diaryl pyrazole (5) wherein a pyridine ring is attached to position 5 of the pyrazole ring, R 1 is lower alkyl and R 2 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.
• 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.
• THF tetrahydrofuran
• MeOH methanol
• 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.
• suitable solvents for this reaction include ethanol, acetic acid, ethanol - acetic acid mixtures and the like.
• Substitution on Ar. in 5 is controlled by proper selection of the starting hydrazine 4. When R 2 of ketone 2 is hydrido, then R 2 of pyrazole 5 is hydrido.
• Scheme II illustrates the synthesis of a pyrazole, compound 8 wherein R 2 is a lower alkyl group and R 1 is hydrido or lower alkyl .
• Compounds wherein R 1 is hydrido and R 2 is a lower alkyl group can be synthesized by treatment of pyridyl ketone 6 with dimethylformamide dimethyl acetal (DMF acetal) .
• 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 step can be carried out as described for Scheme I and substitution on Ar x of pyrazole 8 is controlled by selection of a properly substituted hydrazine.
• Scheme II also describes the synthesis of pyrazoles wherein both R 1 and R 2 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 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 (R 1 and R 2 are lower alkyl) using the procedure described above .
• Scheme III shows the synthesis of a pyrazole, compound 13, analogs of compound 5 in which the pyridine ring bears a chlorine atom at position 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 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.
• R 2 of ketone 2 is hydrido
• R 2 of 13 is hydrido.
• 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.
• pyrazole 15 is formed.
• Examplary substituents for R 3 of amine 14 are hydrogen, lower alkyl, hydroxyalkyl, or aralkyl.
• R 3 When R 3 is benzyl, hydrogenation of the compound removes the benzyl group and forms the amino compound 16.
• 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.
• 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.
• DMF dimethylformamide
• An example of a sulfinic acid sodium salt is sodium methane sulfinate and its reaction leads to methyl sulfone.
• Scheme V shows the preparation of pyrazole compounds 31 and 32 bearing hydroxy and methoxy substituents at position 2 of the pyridine ring.
• Compound 30 is treated with an 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 ring.
• R 2 of ketone 2 is hydrogen (hydrido)
• R 2 of pyrazoles 31 and 32 is hydrogen (hydrido) .
• Scheme VI demonstrates the syntheses of the pyrazole compounds 37 and 38 that bear cyano and 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 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.
• Treatment of 36 with a substituted arylhydrazine produces pyrazole 37. Desired substitution on Ar- ⁇ 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 R 2 is effected by selection of the proper ketone 2.
• R 2 of compound 2 is hydrogen
• R 2 in pyrazoles 37 and 38 is hydrogen (hydrido) .
• Scheme VII illustrates a two-step synthesis of 3-amino-l, 5-diarylpyrazoles 40.
• suitable bases include lithium hexamethyl 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 thioamide 39.
• isothiocyanates examples include ethyl isothiocyanate and trimethylsilylisothicyanate .
• 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 Ar- is effected by proper selection of the substituted phenylhydrazine .
• R of compound 40 is an alkyl group.
• 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.
• R 2 of the starting pyridyl ketone is a substituent other than H, that substituent becomes the substituent R 2 at the 4 position of the pyrazole 40.
• Scheme VIII illustrates the synthesis of a 1,5- diaryl pyrazole wherein R 2 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 sodium ethoxide are suitable for this reaction.
• the reaction can be carried out in an alcoholic solvent such as methanol or ethanol.
• 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 .
• DPPA diphenylphosporyl azide
• Scheme IX illustrates the synthesis of a 4- amino-1 , 5-diarylpyraozle, compound 48.
• 4-acetylpyridine is brominated with bromine in the presence of a solvent such as 48 percent hydrobromic acid to provide the bromoketone, compound 45.
• 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.
• amines include piperidine and dimethylamine .
• 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.
• 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.
• 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.
• 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 Woj ciechowski, Pol . J. Chem . , vol. 54, pp.
• Step 1 Preparation of 1- (4-pyridinyl) -2-methyl-l , 3- propanedione
• Step 2 A mixture of 1- (4-pyridinyl) -2 -methyl -1 , 3- propanedione (0.5 g, 2.63 mmol), 4-fluorophenyl- hydrazine -HCl (0.514g, 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 .
• Step 1 Preparation of 2-chloro-4- [1- (4- chlorophenyl) -3-methyl-lH-pyrazol-5-yll pyridine
• Step 2
• Step 1 Preparation of 1- (3-aminophenyl) -1 , 3- butanedione
• Step 2
• step 1 A mixture of 1- (3-aminophenyl) -1 , 3 -butanedione (step 1) (.5g, 2.82mmol), p-tolylhydrazine HCl
• Step 1
• Step 1
• 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 C 19 H 18 FN 5 0 : C, 64.95; H, 5.16; N, 19.93. Found: C, 64.50; H, 5.02; N, 19.86.
• Example 18 Preparation of
• Step 2
• Step 3
• Step 1 Preparation of methyl 2-methoxyisonicotinate 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.
• Step 2 Preparation of 1- (2-methoxypyridyl) -1 , 3 - butadione
• Step 3 Preparation of 2-methoxy-4- [1- (3- methylphenyl ) -3 -methyl - lH-pyrazol -5 -yl1 pyridine : 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.
• Step 1 Preparation of methyl isonicotinate N- oxide :
• Step 2 Preparation of methyl 2-cyanoisonicotinate : 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 l-(2- cyanoisonicotinyl) -1 , 3- butanedione :
• Step 4 Preparation of 2 -cyano-4 - [1- (3 -methylphenyl ) - 3-methyl-lH-pyrazol-5-yl1 pyridine
• 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 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 C 17 H 16 FN 3 : C, 72.58; H, 5.73; N, 14.94. Found: C, 72.44; H, 5.81; N, 14.67.
• Step 1 preparation of methyl -2 -chloroisonicotinate
• Step 2 Preparation of 1- (2 -chloro-4 -pyridinyl) -1.3- butanedione
• Step 2 Preparation of methyl 1- (4-fluorophenyl) -5- (4- pyridinyl) -lH-pyrazole-4-carboxylate)
• Step 1 Preparation of 2 -chloro-4 - (N-methyl-N- methoxycarbamoyl ) pyridine
• 2-chloroisonicotinic acid (18.0 g, 0.105 mol) in 250 mL of methylenechloride
• 1 , 1' -carbonyldiimidazole (17.0 g, 0.105 mol) portionwise.
• the mixture was stirred for 0.5 h and N,0-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 4 Preparation of 2-chloro-4- Tl- (4- fluorophenyl) -lH-pyrazol-5-yll pyridine
• 1- (4-pyridyl) -2- (dimethylaminomethylene) ethanone 2.1 g, 0.01 mol
• 4-fluorophenylhydrazine hydrochloride 1.63 g, 0.01 mol
• 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.
• Step 1 Preparation of 2 -chloroisonicotinic acid:
• Step 2 Preparation of methyl 2-chloroisonicotinate : 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% ⁇ aOH and extracted with ethyl acetate.
• Step 4 Preparation of 2-chloro-4- Tl- (3- fluorophenyl ) -3 -methyl - lH-pyrazol -5yl1 pyridine :
• the coding region of the human p38a cDNA was obtained by PCR-amplification from RNA isolated from the human monocyte cell line THP.l.
• 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 ⁇ l reaction by heating to 70 °C for 10 minutes followed by 2 minutes on ice. cDNA was then synthesized by adding 1 ⁇ l of RNAsin (Promega, Madison WI) , 2 ⁇ l of 50 mM dNTP's, 4 ⁇ l of 5X buffer, 2 ⁇ l of 100 mM DTT and 1 ⁇ l (200 U) of
• the PCR amplification was carried out in a DNA Thermal Cycler (Perkin Elmer) by repeating 30 cycles of 94 °C for 1 minute, 60 °C for 1 minute and 68 °C for 2 minutes. After amplification, excess primers and unincorporated dNTP's were removed from the 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.
• 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 Biotech).
• 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.
• 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 shaken for three hours at room temperature, and the cells were harvested by centrifugation. The cell pellets were stored frozen until protein purification.
• IPTG isopropyl b-D-thiogalactosidse
• 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 Na 2 HP0 4 , 1.8 mM KH 2 P0 4/ pH 7.3 ) up to 200 ml.
• the cell suspension was adjusted to 5 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) .
• 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.
• 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 . ) .
• 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.
• PHAS-I phosphorylated heat and acid stable protein- insulin inducible
• gamma 32 P-ATP 32 P-ATP
• 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.
• reaction mixture was transferred to a high capacity streptavidin coated filter plate (SAM-streptavidin-matrix, Promega) prewetted with phosphate buffered saline.
• SAM-streptavidin-matrix Promega
• the transferred reaction mix was allowed to contact the streptavidin membrane of the Promega plate for 1- 2 minutes.
• each well was washed to remove unincorporated 32 P-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 ⁇ l of scintillant was added.
• 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 ILl-b. Viability was analyzed using MTS .
• 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.
• 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 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
• the amount of TNF- ⁇ released in the culture medium was quantitated by ELISA. Inhibitory potency is expressed as IC50 ( ⁇ M) .

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