Indian Patents. 223408:SUBSTITUTED 3-PYRIDYL-4-ARYLPYRROLES, AND RELATED THERAPEUTIC AND PROPHYLACTIC METHODS

Title of Invention	SUBSTITUTED 3-PYRIDYL-4-ARYLPYRROLES, AND RELATED THERAPEUTIC AND PROPHYLACTIC METHODS
Abstract	This invention provides novel substituted 3-pyrldyl-4-arylpyrroles, having chemical structure (I) and pharmaceutical compositions comprising same, useful for treating disorders ameliorated by reducing TNF-a production and/or p38 activity in appropriate cells. This invention also provides therapeutic and prophylactic methods using the instant pharmaceutical compositions.

Full Text	SUBSTITUTED 3-PYRIDYL-4-ARYLPYRROLES AND RELATED THERAPEUTIC AND PROPHYLACTIC METHODS Field of the Invention This invention relates to novel substituted 3-pyridyM-aryipyrroles and their therapeutic and prophylactic uses. Disorders treated and/or prevented using these compounds include inflammatory and AIDS-related disorders. Background of the Invention TNF-a and p38-Related Disorders Inflammatory cytokines such as TNF-a are produced via the activity of kinases. Such kinases include the Cytokine Suppressive Antiinflammatory Drug-Binding Protein (CSBP)/p38 kinase, a Mrtogen-Activated Protein (MAP) kinase family of serine-threonine protein kinases. inflammatory cytokines play an important role in a number of inflammatory disorders (1), neurodegenerative disorders (10), and AIDS-related disorders (11-14). Although the precise mechanism of kinases such as p38 is unknown, p38 has been implicated in both the production of TNF-a and the signaling responses associated with the TNF-a receptor (6). Arthritis is a prime example of an inflammatory disorder, and is thus the "inflammatory disorder focused on most in this section. Arthritis affects millions of people and can strike at any joint in the human body. Its symptoms range from mild pain and inflammation in affected joints, to severe and debilitating pain and inflammation. Although the disorder is associated mainly with aging adults, it is not restricted to adults. The most common rheumatoid arthritis therapy involves the use of nonsterodial anti-inflammatory drugs (NSAID's) to alleviate symptoms. However, despite the widespread use of NSAID's. many individuals cannot tolerate the doses necessary to treat the disorder over a prolonged period of time. In addition, NSAID's merely treat the symptoms of disorder without affecting the underlying cause. Other drugs such as methotrexate, gold salts, D-penicillamine and prednisone are often used when patients fail to respond to NSAIO's. These drugs also have significant toxicities and their mechanism of action remains unknown. Monoclonal antibodies to TNF-a and receptor antagonists to interieukin 1ß (IL-1ß) have been shown to reduce symptoms of rheumatoid arthritis in small-scale human clinical trials (2). In addition to protein-based therapies, there are small molecule agents that inhibit the production of these cytokines and have demonstrated activity in animal rheumatoid arthritis models (3). Of these small molecule agents. SB 203580 has proven effective in reducing the production of TNF-a and IL-1ß in lipopolysaccharide (LPS)-stimulated human monocyte cell lines with IC80 values of 50 to 100 nM (4). In addition to in vitro testing results, SB 203580 has been shown to inhibit the production of inflammatory cytokines in rats and mice at IC50 values of 15 to 25 mg/kg (5). SB 203580 reduces the production of inflammatory cytokines by inhibiting the activity of CSBP/p38 kinase at an IC50 of 200 nM (6). Due to SB 203580's oral activity and potency in animal models, researchers have suggested that a compound with such an activity profile has potential as a viable treatment for rheumatoid arthritis (5). Pyridyipyrroles and their analogs have also been prepared as cytokine inhibitors and glucagon antagonists (7), and specifically as inhibitors of IL-1ß. TNF-a and other cytokines. Arylpyrroles (8) and triaryipyrroles (9) have also been prepared as cytokine inhibitors. The role of CSBP/p38 has been implicated recently in various neurodegenerative and AIDS-related disorders. With regard to neurodegenerative disorders, p38 has been shown to have a role in determining whether a cell survives or undergoes neuronal programmed cell death or apoptosis (10,11). Also related to AIDS, the Kaposi's sarcoma-associated herpesvirus HHV8 has been shown to encode a G protein-coupled receptor that activates p38. It has been proposed that this activation contributes to tumorigenesis and angiogenesis leading to Kaposi's sarcoma (12). Associated with AIDS is the rapid activation of p38 induced by infection of a CCR5* human T cell line by SIV, suggesting that p38 may play a role in early viral infection (13). Additionally. p38 inhibitors have been shown to block HIV replication in vitro in a manner that may be TNF-a-independent (14). Absence of Clinically Effective Agents In general, arthritis — particularly rheumatoid arthritis — and the host of other inflammatory and AIDS-related disorders all take a severe toll on those afflicted. There is a tremendous need for small molecule agents to treat these disorders. To date, however, no such agents have ever been identified and shown to be clinically effective in humans. Summary of the Invention This invention provides a compound having the structure: or a pharmaceuticalry acceptable salt thereof, wherein: (a) R1 is selected from the group consisting of (i) hydrogen, (ii) C1-5alkyl, (iii) substituted or unsubstrtuted C1-5alkylamino, (iv) N-containing C1-5alkyl heterocyde selected from thiazolidine, piperidine, morpholine, piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imidazole, (v) phenyl, (vi) phenyl independently substituted with one or more of C1-5alkyl, amino, substituted amino, nitro, nitrile and sulfone. and (vii) pyridine; (b) R2 is selected from the group consisting of (i) hydrogen, (ii) (CH2)3OH, (iii) substituted or unsubstrtuted C1-5alkyl phenyl, and (iv) N-containing C1-5alkyl heterocycle selected from thiazolidine, piperidine, morpholine, piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imidazde; (c) R3 is one or more substituents independently selected from the group consisting of hydrogen, halogen, methoxy, nitro, trifluoromethyl, hydroxy, dimethylamino and methyisulfoxide; and (d) X is either C or N. This invention also provides a second compound having the structure: or a pharmaceutically acceptable salt thereof, wherein: (a) R1 is selected from the group consisting of (i) hydrogen, (ii) C1-5alkyl, (iii) substituted or unsubstituted C1-5alkylamino, (iv) N-containing C1-5alkyl heterocycle selected from thiazolidine, piperidine, morpnoline, piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and tmidazole, (v) phenyl, (vi) phenyl independently substituted with one or more of C1-5alkyl, amino, substituted amino, nitro, nitrile and sulfone, and (vii) pyridine; (b) R2 is selected from the group consisting of (i) hydrogen, (ii) (CH2)3OH, (iii) substituted or unsubstituted C1-5alkyl phenyl, and (iv) N-containing C1-5alkyl heterocycle selected from thiazolidine, piperidine, morpholine, piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imidazole; (c) R4 is a substituted or unsubstituted heterocycle selected from pyridine, pyrimidine, furan orthiophene; and, (d) X is either C or N. This invention further provides a pharmaceutical composition comprising one of the instant compounds and a pharmaceuticaliy acceptable carrier. This invention still further provides a method of treating a subject having a disorder ameliorated by reducing TNF-a production and/or p38 activity in appropriate cells, which comprises administering to the subject a therapeutically effective dose of the instant pharmaceutical composition. Finally, this invention provides a method of preventing an inflammatory response in a subject, comprising administering to the subject a prophylactically effective amount of the instant pharmaceutical composition either preceding or subsequent to an event anticipated to cause the inflammatory response in the subject Detailed Description of the Invention This invention provides novel substituted 3-pyridyl-4-arylpyrroles. These compounds have surprising usefulness in treating disorders ameliorated by a reduction in TNF-a production and/or p38 activity, and are therefore useful for treating inflammatory disorders such as rheumatoid arthritis, as well as AIDS- related disorders. Specifically, this invention provides a first compound having the structure: or a pharmaceutically acceptable salt thereof, wherein: (a) R1 is selected from the group consisting of (i) hydrogen, (ii) C1-5alkyl, (iii) substituted or unsubstituted C1-5alkylamino, (iv) N-containing C1-5alkyl heterocycle selected from thiazolidine, piperidine, morpholine, piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imkJazole, (v) phenyl, (vi) phenyl independently substituted with one or more of C1-5alkyl, amino, substituted amino, nitro, nitrite and sulfone, and (vii) pyridine; (b) R2 is selected from the group consisting of (i) hydrogen, (ii) (CH2)3OH, (iii) substituted or unsubstituted C1-5alkyl phenyl, and (iv) N-containing C1-5alkyl heterocycle selected from thiazolidine, piperidine, morpholine, piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imidazole; (c) R3 is one or more substituents independently selected from the group consisting of hydrogen, halogen, methoxy, nitro, trifluoromethyl, hydroxy, dimethylamino and methylsulfoxide; and (d) X is either C or N. In one embodiment of the first compound: (a) R1 is selected from the group consisting of (i) hydrogen, (ii) C1-5alkyl, (iii) substituted or unsubstrtuted C1-5alkylamino, (iv) N-containing C1-5alkyl heterocycle selected from piperidine, morpholine and pyrrolidine, and (v) phenyl substituted with a substituent selected from the group consisting of amino, substituted amino, nitro and nitrile; (b) R2 is selected from the group consisting of hydrogen and (CH2)3Phenyl; (c) R3 is selected from the group consisting of halogen, nitro and trifluoromethyl; and (d) X is C. In the preferred embodiment, the first compound is selected from the group of compounds shown in Table 1. This invention also provides a second compound having the structure: or a pharmaceutically acceptable salt thereof, wherein: (a) R1 is selected from the group consisting of (i) hydrogen, (ii) C1-5alkyl, (iii) substituted or unsubstituted C1-5alkylamino. (iv) N-containing C1-5alkyl heterocycle selected from thiazolidine, piperidine, morpholine, piperazine, thiomorpholine. pyrrolidine, thiazine, pyrrole and imidazole, (v) phenyl, (vi) phenyl independently substituted with one or more of C1-5alkyl, amino, substituted amino, nitro, nitrite and sulfone, and (vii) pyridine; (b) R2 is selected from the group consisting of (i) hydrogen, (ii) (CH2)3OH, (iii) substituted or unsubstituted C1-5alkyl phenyl, and (iv) N-containing C1-5alkyl heterocycle selected from thiazolidine, piperidine, morpholine, piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imidazole; (c) R4 is a substituted or unsubstituted heterocycle selected from pyridine, pyrimidine, furan or thiophene; and, (d) X is either C or N. In one embodiment of the second compound: (a) R1 is selected from the group consisting of (i)C1-5alkyl, (ii) substituted or unsubstituted C1-5alkylamino, (iii) substituted or unsubstituted C1-5alkyl heterocyclic amino, (iv) phenyi, and (v) phenyi independently substituted with one or more of amino, substituted amino, nitro or nitrile; (b) R2 is selected from the group consisting of hydrogen and (CH2)3Phenyl; and (c) X is C. The instant compounds can be isolated and used as free bases. They can also be isolated and used as pharmaceutically acceptable salts. Examples of such salts include hydrobromic, hydroiodic, hydrochloric, perchloric, sulfuric, maleic, fumaric, malic, tartaric, citric, benzoic, mandelic, methanesulfonic, hydroethanesulfonic, benzenesulfonic, oxalic, palmoic, 2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic and saccharic. This invention further provides a pharmaceutical composition comprising one of the instant compounds and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are weN known to those skilled in the art and include, but are not limited to, from about 0.01 to about 0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propytene grycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, ethanol, alcoholic/aqueous solutions, glycerol, emulsions or suspensions, including saline and buffered media. Oral carriers can be elixirs, syrups, capsules, tablets and the like. The typical solid carrier is an inert substance such as lactose, starch, glucose, methyl-cellulose, magnesium stearate, dicaicium phosphate, rnannftol and the like. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous carriers include fluid and nutrient replenishes, electrolyte replenishers such as those based on Ringer's dextrose and the like. Preservatives and other additives can also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like. All carriers can be mixed as needed with disintegrants, diluents, granulating agents, lubricants, binders and the like using conventional techniques known in the art. This invention still further provides a method of treating a subject having a disorder ameliorated by reducing TNF-a production and/or p38 activity in appropriate cells, which comprises administering to the subject a therapeutically effective dose of the instant pharmaceutical composition. In one embodiment, the disorder is an inflammatory disorder. In another embodiment, the disorder is an AIDS-related disorder. Examples of disorders treatable by the instant pharmaceutical composition include, without limitation, rheumatoid arthritis, osteoporosis, osteoarthritis, allergic inflammation, periodontal disorder, inflammatory bowel disorder, septic shock, insulin- dependent diabetes mellitus, non-insulin-dependent diabetes, cachexia, pulmonary fibrosis, myasthenia gravis, Crohn's disease, hepatitis, primary biliary cirrhosis, acute pancreatitis, allograph rejection, glioblastoma, alopecia areta, psoriasis, ischemia, congestive heart failure, restenosis. atherosclerosis, systemic lupus erythematosus, nephritis, Guillain-Barre Syndrome, viral myocarditis, HIV replication, T-cell depletion in HIV infection, cognitive deficits induced by neuronal inflammation, multiple sclerosis, stroke, neuropathic pain, HIV dementia and Alzheimer's disease. In the preferred embodiment, the disorder is rheumatoid arthritis. As used herein, the term "subject" includes, without limitation, any animal or artificially modified animal having a disorder ameliorated by reducing TNF-a production and/or p38 activity in appropriate cells. In the preferred embodiment, the subject is a human. As used herein, "appropriate cells" include, by way of example, cells which secrete or are capable of secreting TNF-a, and cells wherein p38 has been activated. Specific examples of appropriate cells include, without limitation, monocytes, macrophages, T lymphocytes, fibroblasts. dendritic cells, Langerhans cells, Kuppfer cells and astroglial cells. Administering the instant pharmaceutical composition can be effected or performed using any of the various methods known to those skilled in the art. The instant compounds can be administered, for example, intravenously, intramuscularly, orally and subcutaneously. In the preferred embodiment, the instant pharmaceutical composition is administered orally. Additionally, administration can comprise giving the subject a plurality of dosages over a suitable period of time. Such administration regimens can be determined according to routine methods. As used herein, a "therapeutically effective dose" of a pharmaceutical composition is an amount sufficient to stop, reverse or reduce the progression of a disorder. A "prophyiactically effective dose" of a pharmaceutical composition is an amount sufficient to prevent a disorder, i.e., eliminate, ameliorate and/or delay the disorder's onset. Methods are known in the art for determining therapeutically and prophyiactically effective doses for the instant pharmaceutical composition. The effective dose for administering the pharmaceutical composition to a human, for example, can be determined mathematically from the results of animal studies. In one embodiment, the therapeuticaliy and/or prophylactically effective dose is a dose sufficient to deliver from about 0.05 mg/kg of body weight to about 200 mg/kg of body weight of the instant pharmaceutical composition, in another embodiment, the therapeuticaliy and/or prophylactically effective dose is a dose sufficient to deliver from about 0.5 mg/kg of body weight to about 50 mg/kg of body weight More specifically, in one embodiment, oral doses range from about 0.05 mg/kg to about 100 mg/kg daily. In another embodiment, oral doses range from about 0.05 mg/kg to about 50 mg/kg daily, and in a further embodiment, from about 0.05 mg/kg to about 20 mg/kg daily. In yet another embodiment, infusion doses range from about 1.0 µg/kg/min to about 1.0 x 104 µg/kg/min of inhibitor, admixed with a pharmaceutical carrier over a period ranging from about several minutes to about several days. In a further embodiment, for topical administration, the instant compound can be combined with a pharmaceutical carrier at a drug/carrier ratio of from about 0.001 to about 0.1. This invention still further provides a method of preventing an inflammatory response in a subject, comprising administering to the subject a prophylactically effective amount of the instant pharmaceutical composition either preceding or subsequent to an event anticipated to cause the inflammatory response in the subject. In the preferred embodiment, the event is an insect sting or an animal bite. As used herein, the following chemical terms shall have the meanings as set forth in this paragraph: "independently", when in reference to chemical substituents, shall mean that when more than one substituent exists, the substituents may be the same or different; "alkyl" shall mean straight, cyclic and branched-chain alkyl; "alkoxy" shall mean O-alkyl; "halogen" shall mean fluorine, chlorine, bromine or iodine; "Ph" shall mean phenyl; TCA" shall mean trichloroacetic acid; "FCS" shall mean fetal calf serum; and "RPMI" shad mean the medium from the Roswell Park Memorial Institute (Sigma cat # R0833). This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that these are only illustrative of the invention as described more fully in the dawns which follow thereafter. Additionally, throughout this application, various publications are cited. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains. Experimental Details I. General Synthetic Schemes Representative compounds of the present invention can be synthesized in accordance with the general synthetic methods described below and illustrated in the following general schemes. The products of some schemes can be used as intermediates to produce more than one of the instant compounds. In those cases, the choice of intermediates to be used to produce subsequent compounds of the present invention is a matter of discretion that is well within the capabilities of those skilled in the art. Scheme 1 can be used to produce the compounds of the invention where R1 is methyl. Compound 1a, such as a 1,2-disubstituted alkyne, can be used as the starting material for Scheme 1. 1,2-disubstituted alkynes can be prepared following known procedures. The substituents X and R3 of the compounds of the invention are determined by the substituents of Compound 1a. Compound 1a is combined with trimethytamine-N-oxide and dissolved in a dry solvent, such as THF, and cooled to 0oC. A base, such as lithium diisopropyiamide, is added and the reaction is stirred at 0oC for 1 h to give Compound 1b as the product. Scheme 2 can be used to prepare compounds of the invention where R1 is hydrogen. Compound 2a, of the type 1-[(1-isocyano-2-(3-rnethoxy- phenyl)ethenyl)sulfonyl]-4-methylbenzene can be used as the starting material for Scheme 2. Compound 2a can be prepared following known procedures. The substituent R3 of the compounds of the invention are generally determined by the substituents on the phenyl of the ethynyi group in Compound 2a; the X atom is determined by the heteroaromatic substituent of the acetate group in Compound 2b. Compound 2a is dissolved in a dry solvent, such as ethylene glycol dimethyl ether, and added dropwise to a mixture of Compound 2b, such as ethyl 4-pyridylacetate, and a base, such as potassium t-butoxide, in a dry solvent, such as ethylene glycol dimethyl ether, at OX. After the addition is complete, the reaction is warmed to 25°C and stirred for 3 h to give the intermediate Compound 2c. When R3 is methoxy, the intermediate Compound 2c can be treated with a demethylating agent, such as BBr3, in an inert solvent such as CH2CL2, at -78°C to give Compound 2d. Scheme 3 can be used to produce the compounds of the invention where R1 is hydrogen and R2 is substituted or unsubstituted. Compound 3a, a diaryl substituted a,ß-unsaturated ester, can be used as the starting material for Scheme 3. Compounds of this type can be prepared following known procedures. Compound 3b can be the other starting material, a substituted tosylmethyi isocyanide (tosMIC) derivative that can be prepared following known procedures. For example, Compound 3a can be ethyl 4-fluoro-a-[(4- pyridyl)methylene)benzeneacetic acid and Compound 3b can be 1-(4- tolylsurfonyl)-1-(3-phenylpropyl)methyl isocyanide. Compound 3a and Compound 3b are dissolved in dry solvent, such as ethylene glycol dimethyl ether, and added dropwise to a -40oC mixture of a base, such as potassium t- butoxide, in a dry solvent, such as ethylene glycol dimethyl ether. Stirring at - 40°C is continued for 1 hour before allowing the temperature to rise to -20°C. Accordingly, the resulting Compound 3c is 4-(4-fluorophenyl)-2-(3- phenylpropyl)-3-(4-pyridyl)pyrrole (Compound 27). As shown in Scheme 4, Compound 2c and Compound 3c can be used as intermediates to form other compounds of the present invention. Scheme 4 can be used to produce the compounds of the invention where R1 is substituted. Intermediate Compound 2c or Compound 3c is added portionwise to a base, such as sodium hydride, in a solvent, such as dimethylformamide, at 0°C. After the addition is complete, the reaction is stirred at 0°C an additional 15 minutes followed by portionwise addition of an alkylating agent, such as 4-(2-chloroethyl)morpholine hydrochloride. The reaction is heated to 60°C for 16 hours before the temperature is allowed to cool to 25°C to produce Compound 4a. Scheme 5 can be used to produce the compounds of the invention where R1 is substituted. When R1 is 4-nitrophenyi, the intermediate Compound 4a can be reduced using a catalyst, such as palladium on carbon in a solvent, such as ethyl acetate, to give Compound 5a. The amine Compound 5a can be treated with an acytating agent, such as acetic anhydride, in a solvent, such as water, to give the product Compound 5b. Scheme 6 can be used to produce the compounds of the invention where R2 is an alkyl chain containing heteroatoms. Intermediate Compound 3c is exposed to reducing conditions using a catalyst, such as palladium on carbon in a solvent, such as ethanol, to which a catalytic amount of acid, such as concentrated hydrochloric acid, is added to give Compound 6a. The alcohol Compound 6a can be treated with an sulfonating agent, such as mesyl chloride, and a base, such as triethylamine, in a solvent, such as CH2CI2, to give the intermediate Compound 6b. Compound 6b can then be heated with a nucleophile, such as morpholine, in a solvent, such as CH2CI2, to give Compound 6c. II. Specific Compound Syntheses Specific compounds which are representative of this invention can be prepared as per the following examples. No attempt has been made to optimize the yields obtained in these reactions. Based on the following, however, one skilled in the art would know how to increase yields through routine variations in reaction times, temperatures, solvents and/or reagents. The products of certain syntheses can be used as intermediates to produce more than one of the instant compounds. In those cases, the choice of intermediates to be used to produce compounds of the present invention is a matter of discretion that is well within the capabilities of those skilled in the art Compound 13 (0.15 g. 0.006 md) was dissolved in CH2Cl2 (50 mL) and cooled to -78°C. The mixture was stirred for 16 h, allowing the temperature to warm to 25oC. The reaction was quenched with MeOH (20 mL) and evaporated to a solid. The solid was triturated with ether and filtered to give Compound 12 (0.15 g. 79% yield). 1HNMR (DMSO-d6) d 11.91 (1H, s, NH), 9.47 (1H, br s, OH), 8.66 (2H, d, J = 8.6), 7.86-7.86 (3H, m), 7.25-7.14 (1H, m) 7.06 (1H, s) 6.80-6.66 (3H, m). *****1-[(1-isocyano-2-3-methoxyphenyl)ethenyl)sulfonyl]-4-methylbenzene (9.0 g, 0.0285 mol) was dissolved in dry DME (200 mL) and added dropwise to a cooled (0°C) mixture of ethyl 4-pyridyiacetate (9.0 g, 0.545 mol) and potassium t-butoxide (7.1 g. 0.0633 mol) in dry DME (100 mL). After the addition was complete the reaction was warmed to 25oC and stirred for 3 h. The reaction was then poured into ice water (1200 mL) and extracted into CH2CI2 (3 X 500 mL). The combined organics were dried over Na2SO4, filtered and evaporated in vacuo to give a solid. This solid was then triturated with ether and filtered to give 3.0 g of pure 3-(3-methoxyphenyl)-4-(4-Pyridyl)Pyrrole. The filtrate was again evaporated invacuo and then triturated with a 50/50 mixture of CH2CI2 and ether to give an additional 1.5 g of pure product. Finally the filtrate was evaporated in vacuo and purified on SiO2 eluting with ethyl acetate to give another 0.5 g of pure product resulting in a 70% combined yield. 1HNMR (DMSO-d6) d 11.38 (1H, br s, NH), 8.37 (2H, d, J = 6.1), 7.24-7.17 (4H, m), 7.02-7.00 (1H, m), 6.80-6.77 (3H, m), 3.68 (3H, s). 1-(4- tolylsulfonyl)-1-(3-phenylpropyl)methyl isocyanide (10.9 g, 0.0346 mol) and ethyl 4-fluoro-a-[(4-pyridyl)methyiene]benzeneacetic acid (9.4 g, 0.0346 mol) were dissolved in dry DME (250 mL) and added dropwise to a cooled (-40°C) mixture of potassium t-butoxide (9.5 g, 0.0847 mol) in dry DME (50 mL). The mixture was stirred for 1 hour allowing the temperature to rise to - 20°C. The mixture was poured into H2O (1800 mL) and extracted into CH2CI2 (3 X 500 mL). The combined organtcs were dried over Na2SO4 and evaporated invacuo to give a solid. Trituration of the solid with acetonitrle gave pure Compound 27 (6.0 g, 49% yield). 1HNMR (DMSO-d6)d 11.17 (1H, s, NH). 8.38 (2H, d, J = 5.8 Hz), 7.27-6.92 (12H. m), 2.61-2.51 (4H, m), 1.93- 1.83 (2H,m). 4-[(4-fluorophenyl)ethynyl]pyridine (2.0 g, 0.0101 mol) and trimethytamine-N- oxide (1.0 g, 0.0133 mol) were dissolved in dry THF (200 mL) and cooled to 0°C. Lithium diisopropylamide (1.5M in THF, 14 mL) was added and the reaction was stirred at 0°C for 1 h. The reaction was then quenched with H2O (20 mL) and extracted into CH2CI2 (2X100 mL). The organics were dried over Na2SO4 and evaporated in vacuo to give an oil. Purification on SiO2 eluiting with EtOAc gave 0.356 g (14% yield) of Compound 28. 1HNMR (CDCl3) d 8.41 (2H, d, J=5.4 Hz). 7.19 (2H, dd, J = 5.7, 6.0 Hz), 7.11 (2H, d, J = 5.4 Hz), 6.99 (2H, dd, J=8.7, 8.5 Hz), 6.87 (1H, d, J = 2.1 Hz). 6.69 (1H, d, J = 2.4 Hz), 3.71, (3H. s). Sodium hydride (60% in mineral oil, 0.80 g, 0.0209 mol) was washed 3 times with hexane and then dissolved in DMF (15 mL). Compound 27 (6.0 g, 0.01683 mol) was then added portionwise at 0°C with stirring. After the addition was complete, the reaction was stirred at 0°C an additional 15 minutes followed by dropwise addition of 4-fluoronitrobenzene (2.4 g, 0.170 mol). The reaction was stirred at 0°C for 1 h before the temperature was allowed to warm to 25°C. The reaction was poured into 1.5 L of water and extracted into CH2Cl2 (3 X 500 mL). The combined organics were washed with water (4 X 500 mL) and dried over Na2SO4. Evaporation invacuo gave a yellow solid which was triturated with acetonitrile, filtered and air dried to give 6.6 g (82.5 % yield) of pure Compound 30. 1H-INMR (CDCl3) d 8.52 (2H, d, J = 5.8 Hz). 8.26 (2H, d, J = 8.9 Hz). 7.46 (2H, d, J = 8.9 Hz), 7.16-7.13 (3H, m), 7.09-7.03 (4H. m), 6.95-6.82 (5H, m) 2.73-2.67 (2H, m), 2.35-2.32 (2H, m), 1.53-1.43 (2H, m). Compound 32 (0.85 g, 0.0019 mol) was stirred for 16 hours in acetic anhydride (20 mL) and H2O (50 mL). The solution was extracted into ethyl acetate (100 mL), washed with H2O (3 X 50 mL), then washed with saturated sodium bicarbonate (3 X 50 mL) and then washed again with H2O (2 X 50 mL). The organics were dried over Na2SO4 and evaporated in vacuo to give Compound 31 (0.89 g, 96% yield) isolated as an oil. 1HNMR (CDCI3) d 8.41 (2H, d, J = 5.6 Hz), 8.05 (1H, s), 7.73 (2H, d, J = 8.6 Hz), 7.28 (2H, d, J = 8.7 Hz), 7.17-7.04 (7H, m), 6.91-6.85 (4H, m), 6.79 (1H, s), 2.66-2.61 (2H, m), 2.35-2.30 (2H, m), 2.22 (3H, s), 1.59-1.51 (2H, m). Compound 30 (6.6 g. 0.0138 mol) was suspended in ethanol (200 mL) and ethyl acetate (50 mL) and exposed to reducing conditions for 16 hours on a Parr hydrogenator at 50 PSI. The mixture was filtered through Cellte and evaporated in vacuo to give an oil. Trituration of this oil with acetonitrile and filtration of the resulting solid gave 3.2 g of Compound 32. The filtrate was evaporated in vacuo and purified on SiO2 eluting with 50% ethyl acetate in hexane to give an additional 1.75 g of product (combined yield 79 %). 1HNMR (CDCI3) d 8.44 (2H, d. J = 5.9 Hz), 7.22-6.71 (16H, m), 3.85 (2H, s), 2.63-2.57 (2H, m), 2.38-2.33 (2H, m), 1.61-1.49 (2H, m). A solution of 2-(3-benzyioxypropy1)-4-(4-fluorophenyl)-3-(4-pyridyl)pyrrole (0.95 g, 0.0025 mol) in ethanol (125 mL) containing concentrated HCl (0.2 mL) was added to Pd on carbon (0.2 g). This mixture was placed in a hydrogen atmosphere for 16 hours on a Parr hydrogenator at 50 PSI. The mixture was filtered through Celite and triethyiamine (0.5 mL) was added to the resulting solution, followed by evaporation in vacuo to give a solid. The solid was extracted into ethyl acetate (100 mL) and washed with water (3 X 50 mL). The organics were dried over Na2SO4 and evaporated in vacuo to give a light yellow solid (0.7 g, 96% yield). 1HNMR (DMSO-d6) d 11.10 (1H, s, NH), 8.44 (2H,.d), 7.05 (6H, m), 6.91 (1H, d), 4.53 (1H, br s, OH). 3.49 (2H, brs), 2.64 (2H,t), 1.72 (2H,m). 4-4-fluoiophenyl-2-(3-mesyloxypropyl)-3-(4-pyridyl)pyrrole (0.25 g, 0.0007 mol) was refluxed for 16 hours in CH2CI2 (50 mL) and morpholine (0.25 mL). The solution was cooled and diluted with CH2CI2 (~100 mL). then washed with H2O (3 X 50 mL). The organics were dried over Na2SO4 and evaporated in vacuo to give an oil. This oil was purified on SiO2 eluting with 10% MeOH in CH2CI2 to give 4-(4-fluoropheny)-2-(3-morpholinopropyl)-3-(4-pyridyl)pyrrole isolated as a solid (0.088 g, 36 % yield). 1HNMR (DMSO-d6) d 11.12 (1H, s, NH), 8.42 (2H, d), 7.05 (6H, m), 6.95 (1H, d), 3.55 (4H, t), 2.62 (2H, t), 2.29 (6H, m),1.71(2H,m). 4-(4-fluorophenyl)-2-(3-hydroxypropyl)-3-(4-pyridyl)pyrrole (0.55 g, 0.0019 mol) was combined with triethylamine (0.52 mL, 0.0037 mol) in CH2CI2 (50 mL) and cooled to 10oC. Methanesulfonylchloride (0.16 mL. 0.0020 mol) was added dropwise and the resulting mixture was allowed to warm to room temperature. This mixture was diluted with CH2CI2 (50 mL) and washed with water (30 mL). The organtes were dried over Na2SO4 and evaporated invacuo to give an oil. This oil was dissolved in EtOAc and purified through a bed of SiO2 (-20 mL) eluting with EtOAc. Evaporation of the solvent in vacuo gave a yellow solid (0.63 g, 91% yield). 1HNMR (DMSO-d6) d 11.25 (1H, s, NH), 8.45 (2H, d), 7.09 (6H, m), 6.94 (1H, d), 4.19 (2H, t). 3.17 (3H, s), 2.71 (2H, t), 1.98 (2H, m). III. Biological Assays and Activity A. p38 Inhibition In-Vitro Enzyme Assay A solution (38 µL) of purified recombinant p38 (6xHis-p38 expressed in E.coli). myelin basic protein substrate (determined empirically), and a buffer of pH 7.5 (Hepes:25 mM, MgCl2:10 mM, MnCl2:10 mM) were added to 92 wells of a 96-well round bottom polypropylene plate. The amount of enzyme was determined empirically based on the linear range of the assay and the acceptable signal to noise ratio. The remaining wells were used for control ("CTRL") and background ("BKG"). The CTRL was prepared with the enzyme, substrate buffer and 2% DMSO, and the BKG was prepared with substrate buffer and 2% DMSO. A solution (12 µL) of the test compound in DMSO was added to the testing wells. Compounds were diluted to 125 µM in 10% DMSO/H2O and assayed at 25 µM where the final DMSO concentration was 2%. The ATP/33P- ATP solution (10 µL containing 50 µM unlabeled ATP and 1 µCi 33P-ATP) was added to all wells and the completed plates were mixed and incubated at 30°C for 30 min. Ice-cold 50 % TCA/10 mM sodium phosphate (60 yL) was added to each well and the plates were kept on ice for 15 min. The contents of each well were transferred to the wells of a 96-well filterptate (Millipore, MultiScreen- DP) and the filterplate was placed on a vacuum manifold fitted with a waste collection tray. The wells were washed five times with 10% TCA/10 mM sodium phosphate (200 µL) under vacuum. MicroScint-20 scintillant was added, and the plates were sealed using Topseal-S sheets and counted in a Packard TopCount scintillation counter using a 33P liquid program with color quench correction, where the output is in color quench-corrected cpm. The % inhibition of each test compound as shown in Table 2 was calculated by the following formula: % inhibition = {1- (sample -BKGy(CTRL-BKG)] x 100. Although compounds were initially tested at 20 µM. when warranted, the compounds were also tested at 4-fold increments above and below that concentration. In addition, the IC50 was calculated for some compounds using the Oeltagraph 4-parameter curve-fitting program. B. In-Vitro Whole Cell Assay for TNF-a Inhibition Freshly obtained venous blood was anticoagulated with heparin, diluted with an equal volume of phosphate buffered saline ("PBS") and placed in a sterile tube or other container. Aliquots (30 mL) of this mixture were transferred to centrifuge tubes which were underlaid with Ficoll-Hypaque (15 mL). The prepared tubes were centrifuged at 400 x g, without braking, for 30 min at room temperature. Approximately 1/2 to 2/3 of the platelet layer above the mononuclear cell band was removed with a pipette. The majority of the mononuclear cell layer was carefully removed using a pipette and these PBMC's were diluted with PBS and spun at 600 x g for 15 min. The resulting PBMC's were washed with another portion of PBS and spun at 400 x g for 10 min at room temperature. The recovered pellets were diluted in low endotoxin RPMI /1% FCS culture medium, and gave a cell concentration of 0.5-2.0 X 106 PMBC/ mL. A small volume of the suspension was removed for counting on a hemocytometer and the remaining preparation was centrifuged at 200 x g for 15 min at room temperature. The recovered pelleted PMBCs were resuspended in RPMI /1% FCS to a concentration of 1.67 x 106/mL. To run the assay, the PBMC suspension (180 µL) was transferred to duplicate wells of a 96-well flat-bottom microtiter plate and incubated for 1 h at 37°C. A solution of test compound (10 µL: prepared at 20 x the desired final concentration) was added to each well and the plate was incubated for 1 h at 37°C. A solution (10 nL) of LPS in RPMI /1 % FCS (200 ng/mL) was added and the wells were incubated overnight at 37oC. The supemate (100 µL) was removed from each well and diluted with RPMI /1% FCS (400 µL). The samples were analyzed for TNF-a using a commercial ELISA kit (Genzyme). Select compounds of the invention are listed in Table 3. The compounds were tested for their ability to inhibit TNF-a production. The IC50, nM results are listed for the indicated compounds. C. In Vivo Rodent Assay for Inhibitipn of TNF-a Production The ability of the instant compounds to inhibit LPS-induced TNF-a production was demonstrated in the following in vivo rodent assays. Mice (BALB / cJ females, Jackson Laboratories) or rats (Lewis males, Charles River) were fasted for 30 min prior to oral dosing with 5-10 mL/kg of test compound at 5-50 mg/kg. Thirty minutes after dosing, the animals were injected intraperitoneally with LPS at 1 mg/kg and returned to their cages for 1 h. Animals were anesthetized by CO2, exsanguinated by cardiac puncture and whole blood collected (0.1 - 0.7 mL). The blood was allowed to dot and serum was transferred to a centrifuge tube. This sample was centrifuged, and serum was collected, aliquoted and frozen at -80X. Samples were tested by commercial ELISA's for TNF-a (Endogen for mouse TNF-a and Biosource for rat TNF-a). The in vivo test results for select compounds of the invention are listed in Table 4. The compounds were tested for their ability to inhibit TNF-a production in mice and the data are listed as % inhibition at 25 mg/kg. The in vivo test results for select compounds of the invention are listed in Table 5. The compounds were tested for their ability to inhibit TNF-a production in mice and the data are listed as % inhibition at 10 mg/kg. References 1. C. Dinarello, et al., Inflammatory Cytokines: lnterleukin-1 and Tumor Necrosis Factor as Effector Molecules in Autoimmune Diseases, Curr. Opin. Immunol. 1991, 3, 941-48. 2. M. J. Elliot, et al., Treatment of Rheumatoid Arthritis with Chimeric Monoclonal Antibodies to Tumor Necrosis Factor a, Arthritis Rheum. 1993, 36, 1681-90. 3. J. C. Boehm, et al., 1-Substituted 4-Aryl-5-pyridinylimidazoles: A New Class of Cytokine Suppressive Drugs with Low 5-Lipoxygenase and Cyclooxygenase Inhibitory Potency, J. Med. Chem., 1996, 39, 3929-37. 4. International Publication No. WO 93/14081. 5. A. M. Badger, et al., Pharmacological Profile of SB 203580, A Selective Inhibitor of Cytokine Suppressive Binding Protein p38 Kinase, in Animal Models of Arthritis, Bone Resorption, Endotoxin Shock and Immune Function, The Journal of Pharmacology and Experimental Therapeutics, 1996, 279, 1463-61. 6. D. Griswold, et al., Pharmacology of Cytokine Suppressive Anti- Inflammatory Drug Binding Protein (CSBP), A Novel Stress-Induced Kinase, Pharmacology Communications, 1996, 7, 323-29. 7. U.S. Patent No. 5,776,954. 8. International Publication No. WO 97/05877. 9. International Publication No. WO 97/05878. 10. Davis, Roger J., et al., Opposing effects of ERK and JNK-p38 MAP Kinases on Apoptosis, Science, 1995, 270(5240), 1326-31. 11. Heidenreich, Kim A., et al., inhibition of p38 Mitogen-Activated Protein Kinase by Insulin in Cultured Fetal Neurons, J. Biol. Chem., 1996, 271(17), 9891-4. 12. Arvanitakis, L, et al., G-Protein-Coupled Receptor of Kaposi's Sarcoma- Associated Herpesvirus is a Viral Oncogene and Angiogenesis Activator. Nature, 1998, 391(6662), 86-89. 13. Pitha, Paula M., et al., Early Activation of Mitogen-Activated Protein Kinase Kinase, Extracellular Signal-Regulated Kinase. p38 Mitogen- Activated Protein Kinase, and c-Jun N-terminal Kinase in Response to Binding of Simian Immunodeficiency Virus to Jurkat T Cells Expressing CCR5 Receptor. Virology, 1998, 252(1), 210-217. 14. Bukrinsky, M., The Critical Role of p38 MAP Kinase in T Cell HIV-1 Replication, Mol. Med., 1997, 3(5), 339-346. We claim: 1. A compound having the structure: or a pharmaceutically acceptable salt thereof, wherein: (a) R1 is selected from the group consisting of (i) hydrogen, (ii) C1-5alkyl, (iii) substituted or unsubstituted C1-5alkylamino, (iv) N- containing C1-5alkyl heterocycle selected from thiazolidine, piperidine, morpholine, piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imidazole, (v) phenyl, (vi) phenyl independently substituted with one or more of C1-5alky, amino, substituted amino, nitro, nitrite and sulfone, and (vii) pyridine; (b) R2 is selected from the group consisting of (i) hydrogen, (ii) (CH2)3OH, (iii) substituted or unsubstituted C1-5alkyl phenyl, and (iv) N-containing C1-5alkyl heterocycle selected from thiazolidine, piperidine, morpholine, piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imidazole; (c) R3 is one or more substituents independently selected from the group consisting of hydrogen, halogen, methoxy, nitro, trifluoromethyl, hydroxy, dimethylamino and methylsulfoxide; and (d) X is either C or N. 2. The compound of claim 1, wherein: (a) R1 is selected from the group consisting of (i) hydrogen, (ii) C1-5alkyl, (iii) substituted or unsubstituted C1-5alkylamino, (iv) N- containing C1-5alkyl heterocycle selected from piperidine, morpholine and pyrrolidine, and (v) phenyl substituted with a substituent selected from the group consisting of amino, substituted amino, nitro and nitrile; (b) R2 is selected from the group consisting of hydrogen and (CH2)3phenyl; (c) R3 is selected from the group consisting of halogen, nitro and trifluoromethyl; and (d) X is C. 3. The compound of claim 1 having the structure: 4. The compound of claim 1 having the structure: 5. The compound of claim 1 having the structure: 6. The compound of claim 1 having the structure: 7. The compound of claim 1 having the structure: 8. The compound of claim 1 having the structure: 9. The compound of claim 1 having the structure: 10. The compound of claim 1 having the structure: 11. The compound of claim 1 having the structure: 12. The compound of claim 1 having the structure: 13. The compound of claim 1 having the structure: 14. The compound of claim 1 having the structure: 15. The compound of claim 1 having the structure: 16. The compound of claim 1 having the structure: 17. A compound having the structure: or a pharmaceutically acceptable salt thereof, wherein: (a) R1 is selected from the group consisting of (i) hydrogen, (ii) C1-5alkyl, (iii) substituted or unsubstituted C1-5alkylamino, (iv) N- containing C1-5alkyl heterocycle selected from thiazolidine, piperidine, morpholine, piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imidazole, (v) phenyl, (vi) phenyl independently substituted with one or more of C1-5alkyl, amino, substituted amino, nitro, nitrite and sulfone, and (vii) pyridine; (b) R2 is selected from the group consisting of (i) hydrogen, (ii) (CH2)3OH, (iii) substituted or unsubstituted C1-5alkyl phenyl, and (iv) N-containing C1-5alkyl heterocycle selected from thiazolidine, piperidine, morpholine, piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imidazole; (c) R4 is a substituted or unsubstituted heterocycle selected from pyridine, pyrimidine, furan or thiophene; and, (d) X is either C or N. 18. The compound of claim 17, wherein: (a) R1 is selected from the group consisting of (i) C1-5alkyl, (ii) substituted or unsubstituted C1-5alkylamino, (iii) substituted or unsubstituted C1-5alkyl heterocyclic amino, (iv) phenyi, and (v) phenyl independently substituted with one or more of amino, substituted amino, nitro or nitrile; (b) R2 is selected from the group consisting of hydrogen and (CH2)3phenyl; and (c) X is C. 19. A pharmaceutical composition comprising the compound of claim 1 or claim 17 and a pharmaceutically acceptable carrier. This invention provides novel substituted 3-pyrldyl-4-arylpyrroles, having chemical structure (I) and pharmaceutical compositions comprising same, useful for treating disorders ameliorated by reducing TNF-a production and/or p38 activity in appropriate cells. This invention also provides therapeutic and prophylactic methods using the instant pharmaceutical compositions.

Full Text

SUBSTITUTED 3-PYRIDYL-4-ARYLPYRROLES AND RELATED
THERAPEUTIC AND PROPHYLACTIC METHODS
Field of the Invention
This invention relates to novel substituted 3-pyridyM-aryipyrroles and
their therapeutic and prophylactic uses. Disorders treated and/or prevented
using these compounds include inflammatory and AIDS-related disorders.
Background of the Invention
TNF-a and p38-Related Disorders
Inflammatory cytokines such as TNF-a are produced via the activity of
kinases. Such kinases include the Cytokine Suppressive Antiinflammatory
Drug-Binding Protein (CSBP)/p38 kinase, a Mrtogen-Activated Protein (MAP)
kinase family of serine-threonine protein kinases. inflammatory cytokines play
an important role in a number of inflammatory disorders (1), neurodegenerative
disorders (10), and AIDS-related disorders (11-14). Although the precise
mechanism of kinases such as p38 is unknown, p38 has been implicated in
both the production of TNF-a and the signaling responses associated with the
TNF-a receptor (6).
Arthritis is a prime example of an inflammatory disorder, and is thus the
"inflammatory disorder focused on most in this section. Arthritis affects millions
of people and can strike at any joint in the human body. Its symptoms range
from mild pain and inflammation in affected joints, to severe and debilitating
pain and inflammation. Although the disorder is associated mainly with aging
adults, it is not restricted to adults.
The most common rheumatoid arthritis therapy involves the use of
nonsterodial anti-inflammatory drugs (NSAID's) to alleviate symptoms.
However, despite the widespread use of NSAID's. many individuals cannot
tolerate the doses necessary to treat the disorder over a prolonged period of
time. In addition, NSAID's merely treat the symptoms of disorder without
affecting the underlying cause.
Other drugs such as methotrexate, gold salts, D-penicillamine and
prednisone are often used when patients fail to respond to NSAIO's. These
drugs also have significant toxicities and their mechanism of action remains
unknown. Monoclonal antibodies to TNF-a and receptor antagonists to
interieukin 1ß (IL-1ß) have been shown to reduce symptoms of rheumatoid
arthritis in small-scale human clinical trials (2).
In addition to protein-based therapies, there are small molecule agents
that inhibit the production of these cytokines and have demonstrated activity in
animal rheumatoid arthritis models (3). Of these small molecule agents. SB
203580 has proven effective in reducing the production of TNF-a and IL-1ß in
lipopolysaccharide (LPS)-stimulated human monocyte cell lines with IC80
values of 50 to 100 nM (4).
In addition to in vitro testing results, SB 203580 has been shown to
inhibit the production of inflammatory cytokines in rats and mice at IC50 values
of 15 to 25 mg/kg (5). SB 203580 reduces the production of inflammatory
cytokines by inhibiting the activity of CSBP/p38 kinase at an IC50 of 200 nM (6).
Due to SB 203580's oral activity and potency in animal models, researchers
have suggested that a compound with such an activity profile has potential as
a viable treatment for rheumatoid arthritis (5).
Pyridyipyrroles and their analogs have also been prepared as cytokine
inhibitors and glucagon antagonists (7), and specifically as inhibitors of IL-1ß.
TNF-a and other cytokines. Arylpyrroles (8) and triaryipyrroles (9) have also
been prepared as cytokine inhibitors.
The role of CSBP/p38 has been implicated recently in various
neurodegenerative and AIDS-related disorders. With regard to
neurodegenerative disorders, p38 has been shown to have a role in
determining whether a cell survives or undergoes neuronal programmed cell
death or apoptosis (10,11).
Also related to AIDS, the Kaposi's sarcoma-associated herpesvirus
HHV8 has been shown to encode a G protein-coupled receptor that activates
p38. It has been proposed that this activation contributes to tumorigenesis and
angiogenesis leading to Kaposi's sarcoma (12).
Associated with AIDS is the rapid activation of p38 induced by infection
of a CCR5* human T cell line by SIV, suggesting that p38 may play a role in
early viral infection (13). Additionally. p38 inhibitors have been shown to block
HIV replication in vitro in a manner that may be TNF-a-independent (14).
Absence of Clinically Effective Agents
In general, arthritis — particularly rheumatoid arthritis — and the host of
other inflammatory and AIDS-related disorders all take a severe toll on those
afflicted. There is a tremendous need for small molecule agents to treat these
disorders. To date, however, no such agents have ever been identified and
shown to be clinically effective in humans.
Summary of the Invention
This invention provides a compound having the structure:

or a pharmaceuticalry acceptable salt thereof, wherein:
(a) R1 is selected from the group consisting of (i) hydrogen, (ii) C1-5alkyl, (iii)
substituted or unsubstrtuted C1-5alkylamino, (iv) N-containing C1-5alkyl
heterocyde selected from thiazolidine, piperidine, morpholine,
piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imidazole,
(v) phenyl, (vi) phenyl independently substituted with one or more of
C1-5alkyl, amino, substituted amino, nitro, nitrile and sulfone. and (vii)
pyridine;
(b) R2 is selected from the group consisting of (i) hydrogen, (ii) (CH2)3OH,
(iii) substituted or unsubstrtuted C1-5alkyl phenyl, and (iv) N-containing
C1-5alkyl heterocycle selected from thiazolidine, piperidine, morpholine,
piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imidazde;
(c) R3 is one or more substituents independently selected from the group
consisting of hydrogen, halogen, methoxy, nitro, trifluoromethyl,
hydroxy, dimethylamino and methyisulfoxide; and
(d) X is either C or N.
This invention also provides a second compound having the structure:

or a pharmaceutically acceptable salt thereof, wherein:
(a) R1 is selected from the group consisting of (i) hydrogen, (ii) C1-5alkyl, (iii)
substituted or unsubstituted C1-5alkylamino, (iv) N-containing C1-5alkyl
heterocycle selected from thiazolidine, piperidine, morpnoline,
piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and tmidazole,
(v) phenyl, (vi) phenyl independently substituted with one or more of
C1-5alkyl, amino, substituted amino, nitro, nitrile and sulfone, and (vii)
pyridine;
(b) R2 is selected from the group consisting of (i) hydrogen, (ii) (CH2)3OH,
(iii) substituted or unsubstituted C1-5alkyl phenyl, and (iv) N-containing
C1-5alkyl heterocycle selected from thiazolidine, piperidine, morpholine,
piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imidazole;
(c) R4 is a substituted or unsubstituted heterocycle selected from pyridine,
pyrimidine, furan orthiophene; and,
(d) X is either C or N.
This invention further provides a pharmaceutical composition comprising
one of the instant compounds and a pharmaceuticaliy acceptable carrier.
This invention still further provides a method of treating a subject having
a disorder ameliorated by reducing TNF-a production and/or p38 activity in
appropriate cells, which comprises administering to the subject a
therapeutically effective dose of the instant pharmaceutical composition.
Finally, this invention provides a method of preventing an inflammatory
response in a subject, comprising administering to the subject a
prophylactically effective amount of the instant pharmaceutical composition
either preceding or subsequent to an event anticipated to cause the
inflammatory response in the subject
Detailed Description of the Invention
This invention provides novel substituted 3-pyridyl-4-arylpyrroles. These
compounds have surprising usefulness in treating disorders ameliorated by a
reduction in TNF-a production and/or p38 activity, and are therefore useful for
treating inflammatory disorders such as rheumatoid arthritis, as well as AIDS-
related disorders.
Specifically, this invention provides a first compound having the
structure:

or a pharmaceutically acceptable salt thereof, wherein:
(a) R1 is selected from the group consisting of (i) hydrogen, (ii) C1-5alkyl, (iii)
substituted or unsubstituted C1-5alkylamino, (iv) N-containing C1-5alkyl
heterocycle selected from thiazolidine, piperidine, morpholine,
piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imkJazole,
(v) phenyl, (vi) phenyl independently substituted with one or more of
C1-5alkyl, amino, substituted amino, nitro, nitrite and sulfone, and (vii)
pyridine;
(b) R2 is selected from the group consisting of (i) hydrogen, (ii) (CH2)3OH,
(iii) substituted or unsubstituted C1-5alkyl phenyl, and (iv) N-containing
C1-5alkyl heterocycle selected from thiazolidine, piperidine, morpholine,
piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imidazole;
(c) R3 is one or more substituents independently selected from the group
consisting of hydrogen, halogen, methoxy, nitro, trifluoromethyl,
hydroxy, dimethylamino and methylsulfoxide; and
(d) X is either C or N.
In one embodiment of the first compound:
(a) R1 is selected from the group consisting of (i) hydrogen, (ii) C1-5alkyl, (iii)
substituted or unsubstrtuted C1-5alkylamino, (iv) N-containing C1-5alkyl
heterocycle selected from piperidine, morpholine and pyrrolidine, and (v)
phenyl substituted with a substituent selected from the group consisting
of amino, substituted amino, nitro and nitrile;
(b) R2 is selected from the group consisting of hydrogen and (CH2)3Phenyl;
(c) R3 is selected from the group consisting of halogen, nitro and
trifluoromethyl; and
(d) X is C.
In the preferred embodiment, the first compound is selected from the
group of compounds shown in Table 1.
This invention also provides a second compound having the structure:

or a pharmaceutically acceptable salt thereof, wherein:
(a) R1 is selected from the group consisting of (i) hydrogen, (ii) C1-5alkyl, (iii)
substituted or unsubstituted C1-5alkylamino. (iv) N-containing C1-5alkyl
heterocycle selected from thiazolidine, piperidine, morpholine,
piperazine, thiomorpholine. pyrrolidine, thiazine, pyrrole and imidazole,
(v) phenyl, (vi) phenyl independently substituted with one or more of
C1-5alkyl, amino, substituted amino, nitro, nitrite and sulfone, and (vii)
pyridine;
(b) R2 is selected from the group consisting of (i) hydrogen, (ii) (CH2)3OH,
(iii) substituted or unsubstituted C1-5alkyl phenyl, and (iv) N-containing
C1-5alkyl heterocycle selected from thiazolidine, piperidine, morpholine,
piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imidazole;
(c) R4 is a substituted or unsubstituted heterocycle selected from pyridine,
pyrimidine, furan or thiophene; and,
(d) X is either C or N.
In one embodiment of the second compound:
(a) R1 is selected from the group consisting of (i)C1-5alkyl, (ii) substituted or
unsubstituted C1-5alkylamino, (iii) substituted or unsubstituted C1-5alkyl
heterocyclic amino, (iv) phenyi, and (v) phenyi independently substituted
with one or more of amino, substituted amino, nitro or nitrile;
(b) R2 is selected from the group consisting of hydrogen and (CH2)3Phenyl;
and
(c) X is C.
The instant compounds can be isolated and used as free bases. They
can also be isolated and used as pharmaceutically acceptable salts. Examples
of such salts include hydrobromic, hydroiodic, hydrochloric, perchloric, sulfuric,
maleic, fumaric, malic, tartaric, citric, benzoic, mandelic, methanesulfonic,
hydroethanesulfonic, benzenesulfonic, oxalic, palmoic, 2-naphthalenesulfonic,
p-toluenesulfonic, cyclohexanesulfamic and saccharic.
This invention further provides a pharmaceutical composition comprising
one of the instant compounds and a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers are weN known to those skilled in the
art and include, but are not limited to, from about 0.01 to about 0.1 M and
preferably 0.05 M phosphate buffer or 0.8% saline. Such pharmaceutically
acceptable carriers can be aqueous or non-aqueous solutions, suspensions and
emulsions. Examples of non-aqueous solvents are propytene grycol,
polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters
such as ethyl oleate. Aqueous carriers include water, ethanol, alcoholic/aqueous
solutions, glycerol, emulsions or suspensions, including saline and buffered
media. Oral carriers can be elixirs, syrups, capsules, tablets and the like. The
typical solid carrier is an inert substance such as lactose, starch, glucose,
methyl-cellulose, magnesium stearate, dicaicium phosphate, rnannftol and the
like. Parenteral carriers include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous
carriers include fluid and nutrient replenishes, electrolyte replenishers such as
those based on Ringer's dextrose and the like. Preservatives and other additives
can also be present, such as, for example, antimicrobials, antioxidants, chelating
agents, inert gases and the like. All carriers can be mixed as needed with
disintegrants, diluents, granulating agents, lubricants, binders and the like
using conventional techniques known in the art.
This invention still further provides a method of treating a subject having
a disorder ameliorated by reducing TNF-a production and/or p38 activity in
appropriate cells, which comprises administering to the subject a
therapeutically effective dose of the instant pharmaceutical composition.
In one embodiment, the disorder is an inflammatory disorder. In another
embodiment, the disorder is an AIDS-related disorder. Examples of disorders
treatable by the instant pharmaceutical composition include, without limitation,
rheumatoid arthritis, osteoporosis, osteoarthritis, allergic inflammation,
periodontal disorder, inflammatory bowel disorder, septic shock, insulin-
dependent diabetes mellitus, non-insulin-dependent diabetes, cachexia,
pulmonary fibrosis, myasthenia gravis, Crohn's disease, hepatitis, primary
biliary cirrhosis, acute pancreatitis, allograph rejection, glioblastoma, alopecia
areta, psoriasis, ischemia, congestive heart failure, restenosis. atherosclerosis,
systemic lupus erythematosus, nephritis, Guillain-Barre Syndrome, viral
myocarditis, HIV replication, T-cell depletion in HIV infection, cognitive deficits
induced by neuronal inflammation, multiple sclerosis, stroke, neuropathic pain,
HIV dementia and Alzheimer's disease. In the preferred embodiment, the
disorder is rheumatoid arthritis.
As used herein, the term "subject" includes, without limitation, any animal
or artificially modified animal having a disorder ameliorated by reducing TNF-a
production and/or p38 activity in appropriate cells. In the preferred
embodiment, the subject is a human.
As used herein, "appropriate cells" include, by way of example, cells
which secrete or are capable of secreting TNF-a, and cells wherein p38 has
been activated. Specific examples of appropriate cells include, without
limitation, monocytes, macrophages, T lymphocytes, fibroblasts. dendritic cells,
Langerhans cells, Kuppfer cells and astroglial cells.
Administering the instant pharmaceutical composition can be effected or
performed using any of the various methods known to those skilled in the art.
The instant compounds can be administered, for example, intravenously,
intramuscularly, orally and subcutaneously. In the preferred embodiment, the
instant pharmaceutical composition is administered orally. Additionally,
administration can comprise giving the subject a plurality of dosages over a
suitable period of time. Such administration regimens can be determined
according to routine methods.
As used herein, a "therapeutically effective dose" of a pharmaceutical
composition is an amount sufficient to stop, reverse or reduce the progression
of a disorder. A "prophyiactically effective dose" of a pharmaceutical
composition is an amount sufficient to prevent a disorder, i.e., eliminate,
ameliorate and/or delay the disorder's onset. Methods are known in the art for
determining therapeutically and prophyiactically effective doses for the instant
pharmaceutical composition. The effective dose for administering the
pharmaceutical composition to a human, for example, can be determined
mathematically from the results of animal studies.
In one embodiment, the therapeuticaliy and/or prophylactically effective
dose is a dose sufficient to deliver from about 0.05 mg/kg of body weight to
about 200 mg/kg of body weight of the instant pharmaceutical composition, in
another embodiment, the therapeuticaliy and/or prophylactically effective dose
is a dose sufficient to deliver from about 0.5 mg/kg of body weight to about 50
mg/kg of body weight More specifically, in one embodiment, oral doses range
from about 0.05 mg/kg to about 100 mg/kg daily. In another embodiment, oral
doses range from about 0.05 mg/kg to about 50 mg/kg daily, and in a further
embodiment, from about 0.05 mg/kg to about 20 mg/kg daily. In yet another
embodiment, infusion doses range from about 1.0 µg/kg/min to about 1.0 x 104
µg/kg/min of inhibitor, admixed with a pharmaceutical carrier over a period
ranging from about several minutes to about several days. In a further
embodiment, for topical administration, the instant compound can be combined
with a pharmaceutical carrier at a drug/carrier ratio of from about 0.001 to
about 0.1.
This invention still further provides a method of preventing an
inflammatory response in a subject, comprising administering to the subject a
prophylactically effective amount of the instant pharmaceutical composition
either preceding or subsequent to an event anticipated to cause the
inflammatory response in the subject. In the preferred embodiment, the event
is an insect sting or an animal bite.
As used herein, the following chemical terms shall have the meanings
as set forth in this paragraph: "independently", when in reference to chemical
substituents, shall mean that when more than one substituent exists, the
substituents may be the same or different; "alkyl" shall mean straight, cyclic
and branched-chain alkyl; "alkoxy" shall mean O-alkyl; "halogen" shall mean
fluorine, chlorine, bromine or iodine; "Ph" shall mean phenyl; TCA" shall mean
trichloroacetic acid; "FCS" shall mean fetal calf serum; and "RPMI" shad mean
the medium from the Roswell Park Memorial Institute (Sigma cat # R0833).
This invention will be better understood by reference to the Experimental
Details which follow, but those skilled in the art will readily appreciate that these
are only illustrative of the invention as described more fully in the dawns which
follow thereafter. Additionally, throughout this application, various publications
are cited. The disclosure of these publications is hereby incorporated by
reference into this application to describe more fully the state of the art to which
this invention pertains.
Experimental Details
I. General Synthetic Schemes
Representative compounds of the present invention can be synthesized
in accordance with the general synthetic methods described below and
illustrated in the following general schemes. The products of some schemes
can be used as intermediates to produce more than one of the instant
compounds. In those cases, the choice of intermediates to be used to produce
subsequent compounds of the present invention is a matter of discretion that is
well within the capabilities of those skilled in the art.

Scheme 1 can be used to produce the compounds of the invention
where R1 is methyl. Compound 1a, such as a 1,2-disubstituted alkyne, can be
used as the starting material for Scheme 1. 1,2-disubstituted alkynes can be
prepared following known procedures. The substituents X and R3 of the
compounds of the invention are determined by the substituents of Compound
1a. Compound 1a is combined with trimethytamine-N-oxide and dissolved in a
dry solvent, such as THF, and cooled to 0oC. A base, such as lithium
diisopropyiamide, is added and the reaction is stirred at 0oC for 1 h to give
Compound 1b as the product.

Scheme 2 can be used to prepare compounds of the invention where R1
is hydrogen. Compound 2a, of the type 1-[(1-isocyano-2-(3-rnethoxy-
phenyl)ethenyl)sulfonyl]-4-methylbenzene can be used as the starting material
for Scheme 2. Compound 2a can be prepared following known procedures.
The substituent R3 of the compounds of the invention are generally determined
by the substituents on the phenyl of the ethynyi group in Compound 2a; the X
atom is determined by the heteroaromatic substituent of the acetate group in
Compound 2b. Compound 2a is dissolved in a dry solvent, such as ethylene
glycol dimethyl ether, and added dropwise to a mixture of Compound 2b, such
as ethyl 4-pyridylacetate, and a base, such as potassium t-butoxide, in a dry
solvent, such as ethylene glycol dimethyl ether, at OX. After the addition is
complete, the reaction is warmed to 25°C and stirred for 3 h to give the
intermediate Compound 2c. When R3 is methoxy, the intermediate Compound
2c can be treated with a demethylating agent, such as BBr3, in an inert
solvent such as CH2CL2, at -78°C to give Compound 2d.

Scheme 3 can be used to produce the compounds of the invention
where R1 is hydrogen and R2 is substituted or unsubstituted. Compound 3a, a
diaryl substituted a,ß-unsaturated ester, can be used as the starting material
for Scheme 3. Compounds of this type can be prepared following known
procedures. Compound 3b can be the other starting material, a substituted
tosylmethyi isocyanide (tosMIC) derivative that can be prepared following
known procedures.
For example, Compound 3a can be ethyl 4-fluoro-a-[(4-
pyridyl)methylene)benzeneacetic acid and Compound 3b can be 1-(4-
tolylsurfonyl)-1-(3-phenylpropyl)methyl isocyanide. Compound 3a and
Compound 3b are dissolved in dry solvent, such as ethylene glycol dimethyl
ether, and added dropwise to a -40oC mixture of a base, such as potassium t-
butoxide, in a dry solvent, such as ethylene glycol dimethyl ether. Stirring at -
40°C is continued for 1 hour before allowing the temperature to rise to -20°C.
Accordingly, the resulting Compound 3c is 4-(4-fluorophenyl)-2-(3-
phenylpropyl)-3-(4-pyridyl)pyrrole (Compound 27).
As shown in Scheme 4, Compound 2c and Compound 3c can be used
as intermediates to form other compounds of the present invention.

Scheme 4 can be used to produce the compounds of the invention
where R1 is substituted. Intermediate Compound 2c or Compound 3c is added
portionwise to a base, such as sodium hydride, in a solvent, such as
dimethylformamide, at 0°C. After the addition is complete, the reaction is
stirred at 0°C an additional 15 minutes followed by portionwise addition of an
alkylating agent, such as 4-(2-chloroethyl)morpholine hydrochloride. The
reaction is heated to 60°C for 16 hours before the temperature is allowed to
cool to 25°C to produce Compound 4a.

Scheme 5 can be used to produce the compounds of the invention
where R1 is substituted. When R1 is 4-nitrophenyi, the intermediate
Compound 4a can be reduced using a catalyst, such as palladium on carbon in
a solvent, such as ethyl acetate, to give Compound 5a. The amine Compound
5a can be treated with an acytating agent, such as acetic anhydride, in a
solvent, such as water, to give the product Compound 5b.

Scheme 6 can be used to produce the compounds of the invention
where R2 is an alkyl chain containing heteroatoms. Intermediate Compound 3c
is exposed to reducing conditions using a catalyst, such as palladium on
carbon in a solvent, such as ethanol, to which a catalytic amount of acid, such
as concentrated hydrochloric acid, is added to give Compound 6a. The alcohol
Compound 6a can be treated with an sulfonating agent, such as mesyl
chloride, and a base, such as triethylamine, in a solvent, such as CH2CI2, to
give the intermediate Compound 6b. Compound 6b can then be heated with a
nucleophile, such as morpholine, in a solvent, such as CH2CI2, to give
Compound 6c.
II. Specific Compound Syntheses
Specific compounds which are representative of this invention can be
prepared as per the following examples. No attempt has been made to
optimize the yields obtained in these reactions. Based on the following,
however, one skilled in the art would know how to increase yields through
routine variations in reaction times, temperatures, solvents and/or reagents.
The products of certain syntheses can be used as intermediates to
produce more than one of the instant compounds. In those cases, the choice
of intermediates to be used to produce compounds of the present invention is a
matter of discretion that is well within the capabilities of those skilled in the art

Compound 13 (0.15 g. 0.006 md) was dissolved in CH2Cl2 (50 mL) and cooled
to -78°C. The mixture was stirred for 16 h, allowing the temperature to warm to
25oC. The reaction was quenched with MeOH (20 mL) and evaporated to a
solid. The solid was triturated with ether and filtered to give Compound 12
(0.15 g. 79% yield). 1HNMR (DMSO-d6) d 11.91 (1H, s, NH), 9.47 (1H, br s,
OH), 8.66 (2H, d, J = 8.6), 7.86-7.86 (3H, m), 7.25-7.14 (1H, m) 7.06 (1H, s)
6.80-6.66 (3H, m).

*****1-[(1-isocyano-2-3-methoxyphenyl)ethenyl)sulfonyl]-4-methylbenzene (9.0 g,
0.0285 mol) was dissolved in dry DME (200 mL) and added dropwise to a
cooled (0°C) mixture of ethyl 4-pyridyiacetate (9.0 g, 0.545 mol) and potassium
t-butoxide (7.1 g. 0.0633 mol) in dry DME (100 mL). After the addition was
complete the reaction was warmed to 25oC and stirred for 3 h. The reaction
was then poured into ice water (1200 mL) and extracted into CH2CI2 (3 X 500
mL). The combined organics were dried over Na2SO4, filtered and evaporated
in vacuo to give a solid. This solid was then triturated with ether and filtered to
give 3.0 g of pure 3-(3-methoxyphenyl)-4-(4-Pyridyl)Pyrrole. The filtrate was
again evaporated invacuo and then triturated with a 50/50 mixture of CH2CI2
and ether to give an additional 1.5 g of pure product. Finally the filtrate was
evaporated in vacuo and purified on SiO2 eluting with ethyl acetate to give
another 0.5 g of pure product resulting in a 70% combined yield. 1HNMR
(DMSO-d6) d 11.38 (1H, br s, NH), 8.37 (2H, d, J = 6.1), 7.24-7.17 (4H, m),
7.02-7.00 (1H, m), 6.80-6.77 (3H, m), 3.68 (3H, s).
1-(4- tolylsulfonyl)-1-(3-phenylpropyl)methyl isocyanide (10.9 g, 0.0346 mol)
and ethyl 4-fluoro-a-[(4-pyridyl)methyiene]benzeneacetic acid (9.4 g, 0.0346
mol) were dissolved in dry DME (250 mL) and added dropwise to a cooled
(-40°C) mixture of potassium t-butoxide (9.5 g, 0.0847 mol) in dry DME (50
mL). The mixture was stirred for 1 hour allowing the temperature to rise to -
20°C. The mixture was poured into H2O (1800 mL) and extracted into CH2CI2
(3 X 500 mL). The combined organtcs were dried over Na2SO4 and
evaporated invacuo to give a solid. Trituration of the solid with acetonitrle
gave pure Compound 27 (6.0 g, 49% yield). 1HNMR (DMSO-d6)d 11.17 (1H,
s, NH). 8.38 (2H, d, J = 5.8 Hz), 7.27-6.92 (12H. m), 2.61-2.51 (4H, m), 1.93-
1.83 (2H,m).

4-[(4-fluorophenyl)ethynyl]pyridine (2.0 g, 0.0101 mol) and trimethytamine-N-
oxide (1.0 g, 0.0133 mol) were dissolved in dry THF (200 mL) and cooled to
0°C. Lithium diisopropylamide (1.5M in THF, 14 mL) was added and the
reaction was stirred at 0°C for 1 h. The reaction was then quenched with H2O
(20 mL) and extracted into CH2CI2 (2X100 mL). The organics were dried over
Na2SO4 and evaporated in vacuo to give an oil. Purification on SiO2 eluiting
with EtOAc gave 0.356 g (14% yield) of Compound 28. 1HNMR (CDCl3) d 8.41
(2H, d, J=5.4 Hz). 7.19 (2H, dd, J = 5.7, 6.0 Hz), 7.11 (2H, d, J = 5.4 Hz), 6.99
(2H, dd, J=8.7, 8.5 Hz), 6.87 (1H, d, J = 2.1 Hz). 6.69 (1H, d, J = 2.4 Hz), 3.71,
(3H. s).

Sodium hydride (60% in mineral oil, 0.80 g, 0.0209 mol) was washed 3 times
with hexane and then dissolved in DMF (15 mL). Compound 27 (6.0 g,
0.01683 mol) was then added portionwise at 0°C with stirring. After the
addition was complete, the reaction was stirred at 0°C an additional 15 minutes
followed by dropwise addition of 4-fluoronitrobenzene (2.4 g, 0.170 mol). The
reaction was stirred at 0°C for 1 h before the temperature was allowed to warm
to 25°C. The reaction was poured into 1.5 L of water and extracted into
CH2Cl2 (3 X 500 mL). The combined organics were washed with water (4 X
500 mL) and dried over Na2SO4. Evaporation invacuo gave a yellow solid
which was triturated with acetonitrile, filtered and air dried to give 6.6 g (82.5 %
yield) of pure Compound 30. 1H-INMR (CDCl3) d 8.52 (2H, d, J = 5.8 Hz). 8.26
(2H, d, J = 8.9 Hz). 7.46 (2H, d, J = 8.9 Hz), 7.16-7.13 (3H, m), 7.09-7.03 (4H.
m), 6.95-6.82 (5H, m) 2.73-2.67 (2H, m), 2.35-2.32 (2H, m), 1.53-1.43 (2H, m).

Compound 32 (0.85 g, 0.0019 mol) was stirred for 16 hours in acetic anhydride
(20 mL) and H2O (50 mL). The solution was extracted into ethyl acetate (100
mL), washed with H2O (3 X 50 mL), then washed with saturated sodium
bicarbonate (3 X 50 mL) and then washed again with H2O (2 X 50 mL). The
organics were dried over Na2SO4 and evaporated in vacuo to give Compound
31 (0.89 g, 96% yield) isolated as an oil. 1HNMR (CDCI3) d 8.41 (2H, d, J = 5.6
Hz), 8.05 (1H, s), 7.73 (2H, d, J = 8.6 Hz), 7.28 (2H, d, J = 8.7 Hz), 7.17-7.04
(7H, m), 6.91-6.85 (4H, m), 6.79 (1H, s), 2.66-2.61 (2H, m), 2.35-2.30 (2H, m),
2.22 (3H, s), 1.59-1.51 (2H, m).

Compound 30 (6.6 g. 0.0138 mol) was suspended in ethanol (200 mL) and
ethyl acetate (50 mL) and exposed to reducing conditions for 16 hours on a
Parr hydrogenator at 50 PSI. The mixture was filtered through Cellte and
evaporated in vacuo to give an oil. Trituration of this oil with acetonitrile and
filtration of the resulting solid gave 3.2 g of Compound 32. The filtrate was
evaporated in vacuo and purified on SiO2 eluting with 50% ethyl acetate in
hexane to give an additional 1.75 g of product (combined yield 79 %). 1HNMR
(CDCI3) d 8.44 (2H, d. J = 5.9 Hz), 7.22-6.71 (16H, m), 3.85 (2H, s), 2.63-2.57
(2H, m), 2.38-2.33 (2H, m), 1.61-1.49 (2H, m).

A solution of 2-(3-benzyioxypropy1)-4-(4-fluorophenyl)-3-(4-pyridyl)pyrrole (0.95
g, 0.0025 mol) in ethanol (125 mL) containing concentrated HCl (0.2 mL) was
added to Pd on carbon (0.2 g). This mixture was placed in a hydrogen
atmosphere for 16 hours on a Parr hydrogenator at 50 PSI. The mixture was
filtered through Celite and triethyiamine (0.5 mL) was added to the resulting
solution, followed by evaporation in vacuo to give a solid. The solid was
extracted into ethyl acetate (100 mL) and washed with water (3 X 50 mL). The
organics were dried over Na2SO4 and evaporated in vacuo to give a light
yellow solid (0.7 g, 96% yield). 1HNMR (DMSO-d6) d 11.10 (1H, s, NH), 8.44
(2H,.d), 7.05 (6H, m), 6.91 (1H, d), 4.53 (1H, br s, OH). 3.49 (2H, brs), 2.64
(2H,t), 1.72 (2H,m).

4-4-fluoiophenyl-2-(3-mesyloxypropyl)-3-(4-pyridyl)pyrrole (0.25 g, 0.0007
mol) was refluxed for 16 hours in CH2CI2 (50 mL) and morpholine (0.25 mL).
The solution was cooled and diluted with CH2CI2 (~100 mL). then washed with
H2O (3 X 50 mL). The organics were dried over Na2SO4 and evaporated in
vacuo to give an oil. This oil was purified on SiO2 eluting with 10% MeOH in
CH2CI2 to give 4-(4-fluoropheny)-2-(3-morpholinopropyl)-3-(4-pyridyl)pyrrole
isolated as a solid (0.088 g, 36 % yield). 1HNMR (DMSO-d6) d 11.12 (1H, s,
NH), 8.42 (2H, d), 7.05 (6H, m), 6.95 (1H, d), 3.55 (4H, t), 2.62 (2H, t), 2.29
(6H, m),1.71(2H,m).

4-(4-fluorophenyl)-2-(3-hydroxypropyl)-3-(4-pyridyl)pyrrole (0.55 g, 0.0019 mol)
was combined with triethylamine (0.52 mL, 0.0037 mol) in CH2CI2 (50 mL) and
cooled to 10oC. Methanesulfonylchloride (0.16 mL. 0.0020 mol) was added
dropwise and the resulting mixture was allowed to warm to room temperature.
This mixture was diluted with CH2CI2 (50 mL) and washed with water (30 mL).
The organtes were dried over Na2SO4 and evaporated invacuo to give an oil.
This oil was dissolved in EtOAc and purified through a bed of SiO2 (-20 mL)
eluting with EtOAc. Evaporation of the solvent in vacuo gave a yellow solid
(0.63 g, 91% yield). 1HNMR (DMSO-d6) d 11.25 (1H, s, NH), 8.45 (2H, d), 7.09
(6H, m), 6.94 (1H, d), 4.19 (2H, t). 3.17 (3H, s), 2.71 (2H, t), 1.98 (2H, m).
III. Biological Assays and Activity
A. p38 Inhibition In-Vitro Enzyme Assay
A solution (38 µL) of purified recombinant p38 (6xHis-p38 expressed in
E.coli). myelin basic protein substrate (determined empirically), and a buffer of
pH 7.5 (Hepes:25 mM, MgCl2:10 mM, MnCl2:10 mM) were added to 92 wells of
a 96-well round bottom polypropylene plate. The amount of enzyme was
determined empirically based on the linear range of the assay and the
acceptable signal to noise ratio. The remaining wells were used for control
("CTRL") and background ("BKG"). The CTRL was prepared with the enzyme,
substrate buffer and 2% DMSO, and the BKG was prepared with substrate
buffer and 2% DMSO.
A solution (12 µL) of the test compound in DMSO was added to the
testing wells. Compounds were diluted to 125 µM in 10% DMSO/H2O and
assayed at 25 µM where the final DMSO concentration was 2%. The ATP/33P-
ATP solution (10 µL containing 50 µM unlabeled ATP and 1 µCi 33P-ATP) was
added to all wells and the completed plates were mixed and incubated at 30°C
for 30 min. Ice-cold 50 % TCA/10 mM sodium phosphate (60 yL) was added
to each well and the plates were kept on ice for 15 min. The contents of each
well were transferred to the wells of a 96-well filterptate (Millipore, MultiScreen-
DP) and the filterplate was placed on a vacuum manifold fitted with a waste
collection tray. The wells were washed five times with 10% TCA/10 mM
sodium phosphate (200 µL) under vacuum. MicroScint-20 scintillant was
added, and the plates were sealed using Topseal-S sheets and counted in a
Packard TopCount scintillation counter using a 33P liquid program with color
quench correction, where the output is in color quench-corrected cpm. The %
inhibition of each test compound as shown in Table 2 was calculated by the
following formula: % inhibition = {1- (sample -BKGy(CTRL-BKG)] x 100.

Although compounds were initially tested at 20 µM. when warranted, the
compounds were also tested at 4-fold increments above and below that
concentration. In addition, the IC50 was calculated for some compounds using
the Oeltagraph 4-parameter curve-fitting program.
B. In-Vitro Whole Cell Assay for TNF-a Inhibition
Freshly obtained venous blood was anticoagulated with heparin, diluted
with an equal volume of phosphate buffered saline ("PBS") and placed in a
sterile tube or other container. Aliquots (30 mL) of this mixture were
transferred to centrifuge tubes which were underlaid with Ficoll-Hypaque (15
mL). The prepared tubes were centrifuged at 400 x g, without braking, for 30
min at room temperature. Approximately 1/2 to 2/3 of the platelet layer above
the mononuclear cell band was removed with a pipette. The majority of the
mononuclear cell layer was carefully removed using a pipette and these
PBMC's were diluted with PBS and spun at 600 x g for 15 min. The resulting
PBMC's were washed with another portion of PBS and spun at 400 x g for 10
min at room temperature. The recovered pellets were diluted in low endotoxin
RPMI /1% FCS culture medium, and gave a cell concentration of 0.5-2.0 X 106
PMBC/ mL. A small volume of the suspension was removed for counting on a
hemocytometer and the remaining preparation was centrifuged at 200 x g for
15 min at room temperature. The recovered pelleted PMBCs were
resuspended in RPMI /1% FCS to a concentration of 1.67 x 106/mL.
To run the assay, the PBMC suspension (180 µL) was transferred to
duplicate wells of a 96-well flat-bottom microtiter plate and incubated for 1 h at
37°C. A solution of test compound (10 µL: prepared at 20 x the desired final
concentration) was added to each well and the plate was incubated for 1 h at
37°C. A solution (10 nL) of LPS in RPMI /1 % FCS (200 ng/mL) was added
and the wells were incubated overnight at 37oC. The supemate (100 µL) was
removed from each well and diluted with RPMI /1% FCS (400 µL). The
samples were analyzed for TNF-a using a commercial ELISA kit (Genzyme).
Select compounds of the invention are listed in Table 3. The
compounds were tested for their ability to inhibit TNF-a production. The IC50,
nM results are listed for the indicated compounds.

C. In Vivo Rodent Assay for Inhibitipn of TNF-a Production
The ability of the instant compounds to inhibit LPS-induced TNF-a
production was demonstrated in the following in vivo rodent assays. Mice
(BALB / cJ females, Jackson Laboratories) or rats (Lewis males, Charles River)
were fasted for 30 min prior to oral dosing with 5-10 mL/kg of test compound at
5-50 mg/kg. Thirty minutes after dosing, the animals were injected
intraperitoneally with LPS at 1 mg/kg and returned to their cages for 1 h.
Animals were anesthetized by CO2, exsanguinated by cardiac puncture and
whole blood collected (0.1 - 0.7 mL). The blood was allowed to dot and serum
was transferred to a centrifuge tube. This sample was centrifuged, and serum
was collected, aliquoted and frozen at -80X. Samples were tested by
commercial ELISA's for TNF-a (Endogen for mouse TNF-a and Biosource for
rat TNF-a). The in vivo test results for select compounds of the invention are
listed in Table 4. The compounds were tested for their ability to inhibit TNF-a
production in mice and the data are listed as % inhibition at 25 mg/kg.
The in vivo test results for select compounds of the invention are listed
in Table 5. The compounds were tested for their ability to inhibit TNF-a
production in mice and the data are listed as % inhibition at 10 mg/kg.
References
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We claim:
1. A compound having the structure:

or a pharmaceutically acceptable salt thereof, wherein:
(a) R1 is selected from the group consisting of (i) hydrogen, (ii)
C1-5alkyl, (iii) substituted or unsubstituted C1-5alkylamino, (iv) N-
containing C1-5alkyl heterocycle selected from thiazolidine,
piperidine, morpholine, piperazine, thiomorpholine, pyrrolidine,
thiazine, pyrrole and imidazole, (v) phenyl, (vi) phenyl
independently substituted with one or more of C1-5alky, amino,
substituted amino, nitro, nitrite and sulfone, and (vii) pyridine;
(b) R2 is selected from the group consisting of (i) hydrogen, (ii)
(CH2)3OH, (iii) substituted or unsubstituted C1-5alkyl phenyl, and
(iv) N-containing C1-5alkyl heterocycle selected from thiazolidine,
piperidine, morpholine, piperazine, thiomorpholine, pyrrolidine,
thiazine, pyrrole and imidazole;
(c) R3 is one or more substituents independently selected from the
group consisting of hydrogen, halogen, methoxy, nitro,
trifluoromethyl, hydroxy, dimethylamino and methylsulfoxide; and
(d) X is either C or N.
2. The compound of claim 1, wherein:
(a) R1 is selected from the group consisting of (i) hydrogen, (ii)
C1-5alkyl, (iii) substituted or unsubstituted C1-5alkylamino, (iv) N-
containing C1-5alkyl heterocycle selected from piperidine,
morpholine and pyrrolidine, and (v) phenyl substituted with a
substituent selected from the group consisting of amino,
substituted amino, nitro and nitrile;
(b) R2 is selected from the group consisting of hydrogen and
(CH2)3phenyl;
(c) R3 is selected from the group consisting of halogen, nitro and
trifluoromethyl; and
(d) X is C.
3. The compound of claim 1 having the structure:

4. The compound of claim 1 having the structure:

5. The compound of claim 1 having the structure:

6. The compound of claim 1 having the structure:

7. The compound of claim 1 having the structure:

8. The compound of claim 1 having the structure:

9. The compound of claim 1 having the structure:

10. The compound of claim 1 having the structure:

11. The compound of claim 1 having the structure:

12. The compound of claim 1 having the structure:

13. The compound of claim 1 having the structure:

14. The compound of claim 1 having the structure:

15. The compound of claim 1 having the structure:

16. The compound of claim 1 having the structure:

17. A compound having the structure:

or a pharmaceutically acceptable salt thereof, wherein:
(a) R1 is selected from the group consisting of (i) hydrogen, (ii)
C1-5alkyl, (iii) substituted or unsubstituted C1-5alkylamino, (iv) N-
containing C1-5alkyl heterocycle selected from thiazolidine,
piperidine, morpholine, piperazine, thiomorpholine, pyrrolidine,
thiazine, pyrrole and imidazole, (v) phenyl, (vi) phenyl
independently substituted with one or more of C1-5alkyl, amino,
substituted amino, nitro, nitrite and sulfone, and (vii) pyridine;
(b) R2 is selected from the group consisting of (i) hydrogen, (ii)
(CH2)3OH, (iii) substituted or unsubstituted C1-5alkyl phenyl, and
(iv) N-containing C1-5alkyl heterocycle selected from thiazolidine,
piperidine, morpholine, piperazine, thiomorpholine, pyrrolidine,
thiazine, pyrrole and imidazole;
(c) R4 is a substituted or unsubstituted heterocycle selected from
pyridine, pyrimidine, furan or thiophene; and,
(d) X is either C or N.
18. The compound of claim 17, wherein:
(a) R1 is selected from the group consisting of (i) C1-5alkyl, (ii)
substituted or unsubstituted C1-5alkylamino, (iii) substituted or
unsubstituted C1-5alkyl heterocyclic amino, (iv) phenyi, and (v)
phenyl independently substituted with one or more of amino,
substituted amino, nitro or nitrile;
(b) R2 is selected from the group consisting of hydrogen and
(CH2)3phenyl; and
(c) X is C.
19. A pharmaceutical composition comprising the compound of claim 1 or
claim 17 and a pharmaceutically acceptable carrier.
This invention provides novel substituted 3-pyrldyl-4-arylpyrroles,
having chemical structure (I) and pharmaceutical compositions
comprising same, useful for treating disorders ameliorated by
reducing TNF-a production and/or p38 activity in appropriate cells.
This invention also provides therapeutic and prophylactic methods
using the instant pharmaceutical compositions.

Documents:

in-pct-2001-1236-kol-granted-abstract.pdf

in-pct-2001-1236-kol-granted-assignment.pdf

in-pct-2001-1236-kol-granted-claims.pdf

in-pct-2001-1236-kol-granted-correspondence.pdf

in-pct-2001-1236-kol-granted-description (complete).pdf

in-pct-2001-1236-kol-granted-examination report.pdf

in-pct-2001-1236-kol-granted-form 1.pdf

in-pct-2001-1236-kol-granted-form 18.pdf

in-pct-2001-1236-kol-granted-form 2.pdf

in-pct-2001-1236-kol-granted-form 26.pdf

in-pct-2001-1236-kol-granted-form 3.pdf

in-pct-2001-1236-kol-granted-form 5.pdf

in-pct-2001-1236-kol-granted-reply to examination report.pdf

in-pct-2001-1236-kol-granted-specification.pdf

in-pct-2001-1236-kol-granted-translated copy of priority document.pdf

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Patent Number

223408

Indian Patent Application Number

IN/PCT/2001/1236/KOL

PG Journal Number

37/2008

Publication Date

12-Sep-2008

Grant Date

10-Sep-2008

Date of Filing

23-Nov-2001

Name of Patentee

ORTHO-MCNEIL PHARMACEUTICAL, INC.

Applicant Address

U.S. ROUTE 202, RARITAN, NJ 08869-0602

Inventors:

#	Inventor's Name	Inventor's Address
1	BULLINGTON, JAMES	21 WESLEYAM DRIVE, HAMILTON SQUARE, NJ 08690

PCT International Classification Number

C07D 401/04

PCT International Application Number

PCT/US2000/11531

PCT International Filing date

2000-04-28

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/134,139	1999-05-14	U.S.A.