MXPA02009319A - Pyridine derivatives as inhibitors of p38. - Google Patents

Abstract

The present invention relates to inhibitors of p38, a mammalian protein kinase involved cell proliferation, cell death and response to extracellular stimuli, having the formula (III), or tautomers thereof or pharmaceutically acceptable salts thereof, wherein: each of Q1 and Q2 are independently selected from 5 6 membered aromatic carbocyclic or heterocyclic ring systems, or 8 10 membered bicyclic ring systems comprising aromatic carbocyclic rings, aromatic heterocyclic rings or a combination of an aromatic carbocyclic ring and an aromatic heterocyclic ring. The invention also relates to methods for producing these inhibitors. The invention also provides pharmaceutical compositions comprising the inhibitors of the invention and methods of utilizing those compositions in the treatment and prevention of various disorders.

Description

"P38 INHIBITORS" TECHNICAL FIELD DK THE INVENTION The present invention relates to inhibitors of p38, a mammalian protein kinase that is involved in cell proliferation, cell death and response to extracellular stimuli. The invention also relates to methods for producing these inhibitors. The invention also provides pharmaceutical compositions comprising the inhibitors of the invention and methods for using said compositions in the treatment and prevention of various disease states.

BACKGROUND OF THE INVENTION Protein kinases are involved in several cellular responses to extracellular signals. Recently, a family of mitogen-activated protein kinases (MAPK) was discovered. Members of this family are the Ser / Thr kinases that activate their substrates by phosphorylation [B. Stein et al., Ann. Rep. Med. Chem., 31, pgs. 289-98 (1996)]. MAPKs are activated in turn by a variety of signals that include growth factors, cytokines, UV radiation, and stress-inducing agents. A particularly interesting MAPK is p38.

P38, also known as cytokine suppressive anti-inflammatory drug (CSBP) binding protein (cytokine suppressive anti-inflammatory drug binding protein) and RK is isolated from murine pre-B cells that are transfected with the lipopolysaccharide receptor (LPS) , CD14, and induced with LPS. p38 has been isolated and sequenced, as well as the cDNA encoding it in humans and mice. Activation of p38 has been observed in stress-stimulated cells, such as treatment with lipopolysaccharides (LPS), UV, anisomycin or osmotic shock, and by treatment with cytokines, such as IL-1 and TNF. Inhibition of p38 kinase leads to a blockage in the production of IL-1 and TNF. IL-1 and TNF stimulate the production of other proinflammatory cytokines such as IL-6 and IL-8 and have been implicated in acute and chronic inflammatory diseases and in post-menopausal osteoporosis [R. B. Ki ble et al., Endocrinol. , 136, pgs. 3054-61 (1995)]. Based on this finding, it is considered that p38, together with other MAPKs, has a role as intermediary in the cellular response to inflammatory stimuli, such as accumulation of leukocytes, activation of macrophages / monocytes, resorption of tissue, fever, acute phase responses and neutrophilia. In addition, MAPKs, such as p38, have been implicated in cancer, platelet aggregation induced by thrombin, immunodeficiency disorders, autoimmune diseases, cell death, allergies, osteoporosis and neurodegenerative disorders. P38 inhibitors have been implicated in the area of pain management by inhibiting the induction of prostaglandin-endoperoxidase synthetase-2. Other diseases associated with the overproduction of IL-1, IL-6, IL-8 or TNF have been discussed in WO 96/21654. Others have already started trials to develop drugs that specifically inhibit MAPKs. For example, PCT publication WO 95/31451 discloses pyrrazole compounds that inhibit MAPKs, and, in particular, p38. However, the efficacy of these inhibitors in vivo is still being investigated. Therefore, there is still a great need to develop other potent inhibitors specific for p38 that are useful for the treatment of various conditions associated with the activation of p38.

OBJECT OF THE INVENTION The present invention is directed to this problem by providing compounds that show to have a strong and specific inhibition of p38.

P1605 These compounds have the general formula or the pharmaceutically acceptable salts thereof. For the compounds of Formulas I and II: HET is a 5- to 7-membered heterocycle with 1 to 4 N, S or 0 atoms, heterocycle which is substituted with 1 to 3 C 1 -C 4 straight or branched chain alkyl groups. HET may be optionally substituted with halo, cyano, N (R ') 2, OR', C02R ', C0N (R') 2, S02N (R2) 2. X is O or NR '. n is 1 to 3. For compounds of Formula III: Each of Qi and Q2 are selected independently of carbocyclic or 5-6 membered aromatic heterocyclic ring systems, or 8-10 membered bicyclic ring systems comprising aromatic carbocyclic rings, aromatic heterocyclic rings or a combination of an aromatic carbocyclic ring and an aromatic heterocyclic ring. The rings that make up Qi are optionally substituted with 1 to 4 substituents, each of which is independently selected from J; halo; (C? -C4) alkyl optionally substituted with N (R ') 2, OR', C02R 'or CONR'2; O-C 1 -C 4 alkyl optionally with A, T-C (0) R ', 0P03H2, NR'2, NR'2, OR', C02R 'or C0NR'2; NR'2; 0CF3; CF3; N02; C02R '; CONR '; MR'; S (02) N (R ') 2; SCF3; CN; N (R ') C (0) R4; N (R ') C (0) OR 4; N (R ') C (O) C (O) R 4; N (R ') S (02) R4; N (R ') R4; N (R) 2; OR4; OC (0) R4; 0P (0) 3H2; ? N = CN (R ') 2- The rings that make up Q2 are substituted with J and optionally substituted with halo, straight or branched chain (C? -C4) alkyl, hydroxy, methoxy, trifluoromethyl, trifluoromethoxy, cyano, or Not me. J is a straight or branched chain C1-C4 alkyl derivative, substituted with 1-3 substituents selected from A, -T-C (0) R 'or OP03H2. A is selected from the groups: T is either O or NH. G is either NH 2 or OH. Z is either CH or N. For the compounds of Formulas I, II and III: R 'is selected from hydrogen, C?-C3 alkyl / C2-C3 alkenyl or C2-C3 alkynyl, phenyl or phenyl substituted with 1 to 3 substituents independently selected from halo, methoxy, cyano, nitro, amino, hydroxy, methyl or ethyl; or of a 5- to 6-membered heterocyclic ring system optionally substituted with 1 to 3 substituents independently selected from halo, methoxy, cyano, nitro, amino, hydroxy, methyl or ethyl. Rx is selected from hydrogen, C? -C3 alkyl / OH, or O- (C1-C3 alkyl). R3 is selected from 5-6 membered carbocyclic or heterocyclic aromatic ring systems. R4 is (C? -C4) alkyl optionally substituted with N (R ') 2, OR', C02R ', CON (R') 2, or S02N (R2) 2; a 5-6 membered carbocyclic or heterocyclic ring system optionally substituted with an alkyl group of (C? -C4) of straight or branched chain, N (R ') 2, OR', C02R ', CON (R') 2, or S02N (R2) 2; or a (C! -C) alkyl optionally substituted with the 5-6 membered carbocyclic or heterocyclic ring system optionally substituted with a straight or branched chain (C 1 -C 4) alkyl group, N (R ') 2, OR ', C02R', CON (R ') 2, or S02N (R2) 2; R2 is selected from hydrogen, alkyl of. { Cx-C3), or (C1-C3) alkenyl; each being optionally substituted with -N (R ') 2, -OR', SR ', -C (0) N (R') 2, -S (02) -N (R ') 2, -C (0 ) 0R ', or R3. W is selected from H; N (R2) S02-N (R2) 2; N (R2) S02-N (R2) (R3); N (R2) C (0) -OR2; N (R2) C (O) -N (R2) 2; N (R2) C (0) -N (R2) (R3): N (R2) C (0) -R2; N (R2) 2; C (0) -R2; CH (OH) -R2; C (O) -N (R2) 2; C (0) -OR2; or straight or branched chain alkyl of (C1-C4) optionally substituted with A, T-C (0) R ', N (R') 2,, OR ', C? 2R'; CON (R ') 2; R3; or S02N (R2) 2; or a 5-6 membered carbocyclic or heterocyclic ring system optionally substituted with N (R ') 2, OR', C02R ', CON (R') 2, or S02N (R2) 2; In another embodiment, the invention provides pharmaceutical compositions comprising the p38 inhibitors of this invention. These compositions can be used in methods to treat or prevent a variety of disorders, such as cancer, inflammatory diseases, autoimmune diseases, bone destruction, proliferation disorders, infectious diseases, viral diseases and neurodegenerative diseases. These compositions are also useful in methods for preventing cell death and hyperplasia, and can therefore be used to treat or prevent reperfusion / ischemia in stroke, heart attacks, and organic hypoxia. The compositions are also useful in methods for preventing platelet aggregation induced by thrombin. Each of these previously described methods is also part of the present invention.

DETAILED DESCRIPTION OF THE INVENTION In order that the invention described herein may be fully understood, the following detailed description is set forth. The following terms are used in the description: The term "heterocyclyl" or "heterocycle" refers to a stable 5-7 membered monocyclic heterocyclic ring which is saturated or unsaturated, and which may be optionally fused to benzene if it is monocyclic. Each heterocycle has one or more carbon atoms and one to four heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. As used in this, the terms "nitrogen and sulfur heteroatoms" include any oxidized form of nitrogen and sulfur, and the form P1605 Quaternized from any basic nitrogen. A heterocyclic radical can be attached to any endocyclic carbon or heteroatom if the formation of a stable structure results. Examples of such groups include imidazolyl, imidazolinoyl, imidazolidinyl, quinolyl, isoquinolyl, indolyl, indazolyl, indazolinolyl, perhydropyridazil, pyridazil, pyridyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazinyl, quinoxolyl, piperidinyl, piperranyl, pyrazolinyl, piperazinyl, pyrimidinyl, pyridazinyl. , morpholinyl, thiamorpholinyl, furyl, thienyl, triazolyl, thiazolyl, carbolinyl, tetrazolyl, thiazolidinyl, benzofuranoyl, thiamorpholinyl sulfone, oxazolyl, benzoxazolyl, oxopiperidinyl, oxopyrrolidinyl, oxoazepinyl, azepinyl, isoxozolyl, isothiazolyl, furazanyl, tetrahydropyranyl, tetrahydrofuranyl, thiazolyl, thiadiazoyl, dioxolyl , dioxinyl, oxathiolyl, benzodioxolyl, dithiolyl, thiophenyl, tetrahydrothiophenyl, sulfolanyl, dioxanyl, dioxolanyl, tetrahydrofurodihydrofuranyl, tetrahydropyranodihydrofuranyl, dihydropranyl, tetrahydrofurofuranyl and tetrahydropyranofuranyl. The term "pharmaceutically acceptable salts" refers to compounds according to the invention used in the form of salts derived from inorganic or organic acids and bases.

P1605 The salts of acids include, for example, the following: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, iodohydrate, 2-hydroxyethane sulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate. Salts derived from appropriate bases include salts of alkali metals (eg, sodium), alkaline earth metals (eg, magnesium), ammonium and NW4 + (wherein W is C? -C alkyl). Physiologically acceptable salts of a hydrogen atom or an amino group include salts of organic carboxylic acids such as acetic, lactic, tartaric, malic, isethionic, lactobionic and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids and inorganic acids such as hydrochloric, sulfuric, phosphoric and sulfamic acids. Physiologically acceptable salts of a compound with a hydroxy group include the anion of said compound in combination P160S with a suitable cation such as Na +, NH4 + and NW + (wherein W is a C1-C alkyl group). Pharmaceutically acceptable salts include salts of organic carboxylic acids such as ascorbic, acetic, citric, lactic, tartaric, malic, maleic, isothionic, lactobionic, p-aminobenzoic and succinic acids; organic sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid and p-toluenesulfonic acid and inorganic acids such as hydrochloric, sulfuric, phosphoric, sulfamic and pyrophosphoric acids. For therapeutic use, the salts of the compounds according to the invention will be pharmaceutically acceptable. Nevertheless, salts of acids and bases which are not pharmaceutically acceptable can also be used, for example, in the preparation or purification of a pharmaceutically acceptable compound. Preferred salts include salts formed from hydrochloric, sulfuric, acetic, succinic, citric and ascorbic acids. The term "chemically feasible" refers to the formation of bonds between atoms so that the valence of each atom is satisfied. For example, an oxygen atom with two ligatures and a carbon atom with P1605 Four ligatures are chemically feasible. The term "tautomerization" refers to the phenomenon by which a proton of an atom of one molecule moves to another atom. See, Jerry March, Advanced Organic Chemistry: Reactions, Mechanisms and Structures, Fourth Edition, John Wiley & Sons, pages 69-74 (1992). The term "tautomer" refers to the compounds produced by the proton displacement. For example, if Ri is OH in a compound of formula I, the compound can exist as a tautomer shown below: The present invention provides p38 inhibitors having the general formula: P1605 or pharmaceutically acceptable salts thereof. HET is a 5-7 membered heterocycle with 1 to 4 N, S or O atoms, heterocycle which is substituted with 1 to 3 straight or branched chain C? -C alkyl groups. HET may optionally be substituted with halo, cyano, N (R ') 2, OR', C02R ', CON (R') 2, and S02N (R) 2. X is O or NR '. n is 1 to 3. For compounds of formula III: Each of Qi and Q2 are independently selected from 5-6 membered carbocyclic or heterocyclic ring systems, or 8-10 membered bicyclic ring systems comprising rings P1S05 aromatic carbocyclics, aromatic heterocyclic rings or a combination of an aromatic carbocyclic ring and an aromatic heterocyclic ring. The rings that make up Qi are optionally substituted with 1 to 4 substituents, each of which is independently selected from J; halo; (C? -C) alkyl optionally substituted with N (R '), OR', C02R 'or C0NR'2; O-C 1 -C 4 alkyl optionally with A, T-C (0) R ', OPO 3 H 2, NR'2, NR 2, OR', C02R 'or CONR'2; NR'2; OCF3; CF3; N02; C02R '; CONR '; MR'; S (02) N (R ') 2; SCF3; CN; N (R ') C (0) R4; N (R ') C (0) OR 4; N (R ') C (O) C (O) R 4; N (R ') S (02) R4; N (R ') R4; N (R4) 2; OR4; 0C (0) R4; 0P (0) 3H2; o = C-N (R ') 2. The rings that make up Q2 are substituted with J, and optionally substituted with halo, straight or branched chain C1-C alkyl, hydroxy, methoxy, trifluoromethyl, trifluoromethoxy, cyano, or amino. J is a straight or branched chain C 1 -C 4 alkyl derivative, substituted with 1-3 substituents selected from A, -T-C (0) R 'or OP03H2. A is selected from the groups: P1605 T is either O or NH. G is either NH or OH. Z is either CH or N. For compounds of Formulas I, II and III: R 'is selected from hydrogen, C1-C3 alkyl, C2-C3 alkenyl or C2-C3 alkynyl, phenyl or phenyl substituted with to 3 substituents independently selected from halo, methoxy, cyano, nitro, amino, hydroxy, methyl or ethyl; or of a 5- to 6-membered heterocyclic ring system optionally substituted with 1 to 3 substituents independently selected from halo, methoxy, cyano, nitro, amino, hydroxy, methyl or ethyl. Rx is selected from hydrogen, C-C3 alkyl, OH, or O- (C? -C3 alkyl). R3 is selected from 5-6 membered carbocyclic or heterocyclic aromatic ring systems. R4 is (C? -C4) alkyl optionally substituted with N (R ') 2, OR', C02R ', C0N (R') 2, or S02N (R ') 2; a 5-6 membered carbocyclic or heterocyclic ring system optionally substituted with an alkyl group of (C? -C) of straight or branched chain, N (R ') 2, OR', C02R ', CON (R') 2, or S02N (R ') 2; or a (C? -C4) alkyl optionally substituted with the 5-6 membered carbocyclic or heterocyclic ring system optionally substituted with a straight or branched chain (C? -C4) alkyl group, N (R ') 2 , OR ', C02R', CON (R ') 2, or S02N (R2) 2; R2 is selected from hydrogen, (C? -C3) alkyl, or (C? -C3) alkenyl; each being optionally substituted with -N (R ') 2, -OR', SR ', -C (0) N (R') 2, -S (02) -N (R ') 2, -C (0 ) OR ', or R3. W is selected from H; N (R2) S02-N (R2) 2; N (R2) S02-N (R2) (R3); N (R2) C (0) -OR2; N (R2) C (O) -N (R2) 2; N (R2) C (0) -N (R2) (R3): N (R2) C (0) -R2; N (R2) 2; C (0) -R2; CH (0H) -R2; C (O) -N (R2) 2; C (0) -OR2; or straight or branched chain alkyl of (C? -C) optionally substituted with A, T-C (0) R ', N (R') 2,, OR ', C02R'; CON (R ') 2; R3; or S02N (R2) 2; or a 5-6 membered carbocyclic or heterocyclic ring system optionally substituted with N (R ') 2, OR', C02R ', CON (R') 2, OR S02N (R2) 2; According to a preferred embodiment, Qx is selected from phenyl or pyridyl containing from 1 to 3 substituents, wherein at least one of the substituents is in the ortho position, and the substituents are independently selected from chlorine, fluorine, bromine, CH3 , -OCH3, -OH, -CF3, -0CF3, -0 (CH2) 2CH3, NH2, 3.4- P1605 methylenedioxy, -N (CH3) 2, -NH-S (0) 2-phenyl, -NH-C (0) -CH2-4-pyridine, -NH-C (O) -CH2-morpholine, -NH-C (O) -CH2-N (CH3) 2, -NH-C (0) -CH2-piperazine, -NH-C (O) -CH2-pyrrolidine, -NH-C (O) -C (O) -morpholine , -NH-C (O) -C (O) -piperazine, -NH-C (O) -C (O) -pyrrolidine, -OC (O) CH2-N (CH3) 2, or -O- (CH2 2-N (CH3) 2. Even more preferred are phenyl and pyridyl containing at least 2 of the substituents indicated above, both being in the ortho position. Some specific examples of preferred Qx are: P1G05 605 pieos More preferably, Qx is selected from 2-fluoro-6-trifluoromethylphenyl, 2,6-difluorophenyl, 2,6-dichlorophenyl, 2-chloro-4-hydroxyphenyl, 2-chloro-4-aminophenyl, 2,6-dichloro-4 -aminophenyl, 2,6-dichloro-3-aminophenyl, 2,6-dimethyl-4-hydroxyphenyl, 2-methoxy-3,5-dichloro-4-pyridyl, 2-chloro-4,5-methylenedioxyphenyl, or chloro-4- (N-2-morpholino-acetamido) phenyl. According to a preferred embodiment, Q2 is phenyl or pyridyl, wherein the phenyl or pyridyl contains the substituent J and from 0 to 3 of other substituents, wherein each of these other substituents is independently selected from chlorine, fluorine, bromine, methyl, ethyl, isopropyl, -OCH3, -OH, -NH2, -CF3, -OCF3, -SCH3, -0CH3, -C (0) 0H, -C (0) 0CH3, -CH2NH2, -N (CH3) 2 , -CH2- P160S pyrrolidine and -CH20H. It will be apparent to one skilled in the art that the compounds of the present invention can exist as tautomers. Such tautomers may be ephemeral or isolable as a stable product. These tautomers are contemplated in the scope of the invention. These compounds are also inhibitors of p38 and are within the scope of the present invention. According to a preferred embodiment, Rx is H, n is 1, and HET is an imidazole, triazole, thiazole, oxazole, pyridyl or pyrimidyl ring substituted with one to three straight or branched chain C 1 -C 4 alkyl groups. Particularly preferred embodiments according to formula I are: Compound 11, Compound 12, Compound 13, and pieos Compound 14. Particularly preferred embodiments according to formula III are: Compound 15, Compound 16, Compound 17, Compound 18. According to another embodiment, the present invention provides methods for producing the above-identified p38 inhibitors of formulas I and II. A method for producing compound 11 is provided in Example 1. The activity of p38 inhibitors of this P160S invention can be tested in vi tro, in vivo or in a cell line. In vitro assays include assays that determine the inhibition or activity of kinase or ATPase activity of activated p38. Alternative assays in vitro quantify the ability of the inhibitors to bind to p38, and measurement can be performed either by radioactive labeling of the inhibitor prior to ligation, isolating the inhibitor / p38 complex and determining the amount of radioactive label bound, or by the performance of a competition experiment in which new inhibitors are incubated together with p38 linked to known radioligands. To determine the inhibitory effect of the compounds of this invention, cell culture assays can be used to determine the amounts of TNF, IL-1, IL-6 or IL-8 produced in whole blood or in cell fractions thereof, in treated cells. with inhibitor compared to cells treated with negative controls. The level of these cytokines can be determined by the use of commercially available ELISAs. A useful in vivo test to determine the inhibitory activity of the p38 inhibitors of this invention is the suppression of edema in the hind paw in rats with adjuvant arthritis induced by Mycobacterium butyricum. This essay is described in J. C. Boehm et al.

P1605 al., J. Med. Chem., 39, p. 3929-37 (1996), whose exposition is incorporated herein by reference. The p38 inhibitors of this invention can also be tested in animal models of arthritis, bone resorption, endotoxin shock and immune function, as described in A. M. Badger et al., J. Pharmacol. Experimental Therapeutics, 279, p. 1453-61 (1996), whose disclosure is incorporated herein by reference. The p38 inhibitors or the pharmaceutical salts thereof can be formulated in pharmaceutical compositions to be administered to animals or humans. These pharmaceutical compositions, comprising an amount of the p38 inhibitor effective to treat or prevent a condition in which p38 serves as an intermediary and a pharmaceutically acceptable excipient, constitute another embodiment of the present invention. The term "condition in which p38 serves as an intermediary," as used herein, means any disease or other condition that produces damage of which p38 is known to have a role. This includes conditions caused by the overproduction of IL-1, TNF, IL-6 or IL-8. Such conditions include, without limitation, inflammatory diseases, autoimmune diseases, bone destruction disorders, proliferation disorders, infectious diseases, neurodegenerative diseases, allergies, P1605 reperfusion / ischemia in stroke, heart attacks, angiogenic disorders, organic hypoxia, vascular hyperplasia, cardiac hypertrophy, platelet aggregation induced by thrombin, and conditions associated with prostaglandin-endoperoxidase-synthetase-2. Inflammatory diseases that can be treated or prevented include, but are not limited to, acute pancreatitis, chronic pancreatitis, asthma, allergies, and respiratory failure syndrome in adults. Autoimmune diseases that can be treated or prevented include, but are not limited to, glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Graves' disease, autoimmune gastritis, diabetes, autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, dermatitis atopic, chronic active hepatitis, myasthenia gravis, multiple sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, psoriasis or host graft intolerance. Disorders of bone destruction that can be treated or prevented include, but are not limited to, osteoporosis, osteoarthritis, and bone disorders related to multiple myeloma. Proliferation diseases that can be treated or prevented include, but are not limited to, P1605 acute myelocytic leukemia, chronic myelocytic leukemia, metastatic melanoma, Kaposi's sarcoma, and multiple myeloma. Angiogenic disorders that can be treated or prevented include solid tumors, ocular neovasculization, infantile hemangiomas. Infectious diseases that can be treated or prevented include, but are not limited to, sepsis, septic shock and Shigellosis. Viral diseases that can be treated or prevented include, but are not limited to, acute hepatitis infection (including hepatitis A, hepatitis B and hepatitis C), HIV infection and CMV retinitis. Neurodegenerative diseases that can be treated or prevented by the compounds of this invention include, but are not limited to, Alzheimer's disease, Parkinson's disease, cerebral ischemia or neurodegenerative disease caused by traumatic injury. "The conditions in which p38 serves as an intermediate" also include ischemia / reperfusion in stroke, heart attacks, myocardial ischemia, organic hypoxia, vascular hyperplasia, cardiac hypertrophy, and platelet aggregation induced by thrombin. In addition, the p38 inhibitors of this invention are also capable of inhibiting the expression of proteins P1605 inducible pro-inflammatory agents such as prostaglandin-endoperoxide synthetase-2 (PGHS-2), which is also referred to as cyclooxygenase-2 (COX-2). Therefore, other "conditions in which p38 serves as a mediator" are edema, analgesia, fever and pain, such as neuromuscular pain, headache, pain caused by cancer, tooth pain and arthritis pain. Diseases that can be treated or prevented by the p38 inhibitors of this invention can also be conveniently grouped by the cytokine (IL-1, TNF, IL-6, IL-8) that is considered responsible for the disease. Thus, diseases or conditions in which IL-1 serves as an intermediary include rheumatoid arthritis, osteoarthritis, stroke, endotoxin and / or toxic shock syndrome, inflammatory reaction induced by endotoxin, inflammatory bowel disease, tuberculosis, atherosclerosis, muscle degeneration, cachexia , psoriatic arthritis, Reiter's disease, gout, traumatic arthritis, rubella arthritis, acute synovitis, diabetes, cell disease? of the pancreas and Alzheimer's disease. Diseases or conditions in which TNF serves as a mediator include rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and P1605 other arthritic conditions, sepsis, septic shock, endotoxic shock, gram negative sepsis, toxic shock syndrome, adult respiratory distress syndrome, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcoisosis, bone resorption diseases, reperfusion injury, graft host intolerance, homologous graft rejections, fever and myalgia due to infection, secondary cachexia by infection, AIDS, ARC or malignancy, keloid formation, scab tissue formation, Crohn's disease, ulcerative colitis or pyresis. Diseases in which TNF serves as an intermediary also include viral infections, such as HIV, CMV, influenza or herpes; and veterinary viral infections, such as infections with lentiviruses, including, but not limited to, equine infectious anemia virus, caprine arthritis virus, visna virus or maedi virus; or infections with retroviruses, including feline immunodeficiency virus, bovine immunodeficiency virus or canine immunodeficiency virus. Diseases or conditions in which IL-8 serves as an intermediary include diseases characterized by a massive infiltration of neutrophils, such as psoriasis, inflammatory bowel disease, asthma, cardiac and renal reperfusion injury,P1605 respiratory failure in adults, thrombosis and glomerulonephritis. In addition, the compounds of this invention can be used topically to treat or prevent conditions caused or aggravated by IL-1 or TNF. Such conditions include inflamed joints, excema, psoriasis, conditions of skin inflammation, such as sunburn, conditions of eye inflammation such as conjunctivitis, pyresis, pain and other conditions associated with inflammation. In addition to the compounds of this invention, pharmaceutically acceptable salts of the compounds of this invention can also be employed in the compositions for treating or preventing the above-identified disorders. The pharmaceutically acceptable salts of the compounds of this invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzensulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate, cyclopentanpropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, flucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate. , hexanoate, hydrochloride, hydrobromide, iodohydrate, 2-hydroxyethane sulfonate, lactate, maleate, P1S05 malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate. Other acids, such as oxalic acid, if they are not pharmaceutically acceptable by themselves, can be used in the preparation of salts useful as intermediates in obtaining compounds of the invention and their pharmaceutically acceptable acid addition salts. Salts derived from appropriate bases include alkali metal salts (eg, sodium and potassium), alkaline earth metals (eg, magnesium), ammonium and N- (C 1 -C 4) alkyl 4+. This invention also contemplates the quaternization of any alkaline group of the compounds disclosed therein which contain nitrogen. By such quaternization products soluble or dispersible in water or oil can be obtained. Pharmaceutically acceptable excipients that can be used in these pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, whey proteins, such as human serum albumin, buffer substances such as phosphates, glycine, acid sdrbico, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts pieos or electrolytes, such as protamine sulfate, disodium phosphate, monopotassium phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, block polymers of polyethylene-polyoxypropylene, polyethylene glycol and wool grease. The compositions of the present invention may be administered orally, parenterally, by inhalation of spray, topically, rectally, nasally, by mouth, vaginally or by a graft reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synobial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally or intravenously. The sterile injection forms of the compositions of this invention can be aqueous or oleaginous suspensions. These suspensions can be formulated according to techniques known in the art using dispersing or wetting agents and suitable suspending agents. The sterile injectable preparation can also be an injectable solution or suspension P1605 sterile in a non-toxic parenterally acceptable diluent or solvent, for example a solution in 1,3-butanediol. Among the vehicles and acceptable solvents that can be used are Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils, including mono- or diglycerides, are conveniently employed as solvents or suspending media. Fatty acids, such as oleic acid and its glycerides are useful in the preparation of injectables, as are also pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain as a diluent or dispersant a long chain alcohol, such as carboxymethylcellulose or similar dispersing agents commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers commonly used in the manufacture of solid, liquid or other pharmaceutically acceptable dosage forms, may also be used for the purpose of formulation. The pharmaceutical compositions of this invention P1605 they can be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, commonly used excipients include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in the form of a capsule, useful diluents include lactose and dried corn starch. If aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents can also be added. Alternatively, the pharmaceutical compositions of this invention can be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature, and which will therefore melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols. The pharmaceutical compositions of this invention can also be administered topically, especially if the subject of the treatment includes areas or organs pieos easily accessible for topical applications, including eye, dermal, or lower intestinal tract diseases. Suitable topical formulations are easily prepared for each of these areas or organs. Topical application to the lower intestinal tract can be done in a rectal suppository formulation (see above) or in an adequate enema formulation. Topical transdermal patches can also be used. For topical applications, the pharmaceutical compositions can be formulated in a suitable ointment containing the active component suspended or dissolved in one or more excipients. Excipients for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax and water. Alternatively, the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable excipients. Suitable excipients include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, ethyl ester wax, cetearyl alcohol, 2-octyldodecanol benzyl alcohol and water.

P1605 For ophthalmic use, the pharmaceutical compositions can be formulated as micronized suspensions in isotonic sterile physiological solution with adjusted pH, or, preferably, as solutions in isotonic sterile physiological solution with adjusted pH, either with or without preservative such as benzylalconium chloride. Alternatively, for ophthalmic uses, the pharmaceutical compositions can be formulated into an ointment such as petrolatum. The pharmaceutical compositions of this invention can also be administered by aerosol or nasal inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulations and can be prepared as solutions in physiological solution, using benzyl alcohol or other suitable preservatives, absorption promoters to increase bioavailability, fluorocarbons, and / or other agents of conventional solubilization or dispersion. The amount of the p38 inhibitor that can be combined with the excipient materials to produce an individual dosage form will depend on the host treated and the particular mode of administration. Preferably, the compositions should be formulated so that a dose of the inhibitor between 0.01-100 can be administered to a patient receiving these compositions.

P1605 mg / kg of body weight / day. It should also be understood that a specific dosing and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound employed, age, body weight, general health, sex, diet, the time of administration, the rate of excretion, the combination with other drugs and the criterion of the attending physician and the severity of the particular disease treated. The amount of the inhibitor will also depend on the particular compound of the composition. According to another embodiment, the invention provides methods for treating or preventing conditions in which p38 serves as an intermediary, methods comprising the step of administering to a patient one of the pharmaceutical compositions described above. The term "patient", as used hn, means an animal, preferably a human. Preferably, said method is used to treat or prevent a selected condition of inflammatory diseases, autoimmune diseases, bone destruction disorders, proliferation disorders, infectious diseases, degenerative diseases, allergies, reperfusion / ischemia in stroke, heart attacks, angiogenic disorders, hypoxia P1605 organic, vascular hyperplasia, cardiac hypertrophy, and platelet aggregation induced by thrombin. According to another embodiment, the inhibitors of this invention are used to treat or prevent a disease or condition in which IL-1, IL-6, IL-8 or TNF serve as intermediates. Such conditions have been previously described. Depending on the particular condition to be treated or prevented in which p38 serves as an intermediary, additional drugs that are normally administ to treat or prevent that condition may be administ in conjunction with the inhibitors of this invention. For example, chemotherapeutic agents or other anti-proliferation agents may be combined with the p38 inhibitors of this invention to treat proliferative diseases. These additional agents can be administ separately from the composition containing the p38 inhibitor, as part of a multiple dosage regimen. Alternatively, these agents can be part of a single dosage form, mixed with the p38 inhibitor in a single composition. In order that the invention described hn may be more fully understood, the following examples are set forth. It must be understood that these examples are given P1605 only for illustrative purposes and which should not be construed in any way as limiting this invention.

EXAMPLE 1 Preparation of Compound 11 80% 2 • P1605 Compound 1 was dissolved in a ratio of 1:20 in a solution of concentrated sulfuric acid and glacial acetic acid (1: 4). Aqueous NaN02 was added to the solution over a period of two hours (h). The reaction mixture was stirred at 60 ° C for one hour. The solution was transferred to an equivalent of CuBr and three equivalents of HBr (the concentration of the standard solution of HBr was 48%) at 100 ° C for one hour. The reaction mixture was stirred at 100 ° C for one hour. The reaction mixture was poured on ice. Compound 2 precipitated, which was filt and further purified by chromatography. The yield of compound 2 was 90%. pieos One equivalent of compound 2 and two equivalents of methyl cyanoacetate in dimethylformamide (DMF) wdissolved. To the solution in DMF two equivalents of K2C03 wadded at 50 ° C. The reaction mixture was stirred at 50 ° C overnight. The reaction mixture was poured into a bath of HCl / crushed ice. Compound 3 was precipitated, filt and used directly for the next step. The yield of compound 3 was 90%. Compound 3 was dissolved in a 5% solution of concentrated sulfuric acid, 47.5% acetic acid and 47.5% water. The reaction mixture was stirred at 125 ° C for five hours. The reaction mixture was poured over an excess of crushed ice. Compound 4 precipitated, which was filtered and used directly for the next step without further purification. The yield of compound 4 was 90%. Compound 4 was suspended in ethyl alcohol. Concentrated HCl containing 4.5 equivalents of SnCl2 at 75 ° C was added. The reaction mixture was refluxed for 30 minutes at 75 ° C. The completion of the reaction was indicated by thin layer chromatography (TLC). The reaction solution was cooled to room temperature. The precipitate was filtered, dissolved in ethyl acetate, and the organic phase was washed with saturated K2CO3 and NaCl, and then dried with MgSO4. The solvent was separated under P1605 reduced pressure. Compound 5 was obtained pure with a yield of 90%. An equivalent of compound 5 and an equivalent of 3,6-dichloropyridazine were dissolved in tetrahydrofuran (THF) at 60 ° C. Two equivalents of potassium t-butylhydroxide were added. The reaction mixture was stirred at 60 ° C for one hour. Saturated NaCl solution and ethyl acetate were added to the reaction mixture. The pH of the aqueous phase was adjusted to 7 with HCl and the aqueous phase was extracted with ethyl acetate. The organic phase was washed twice with saturated NaCl solution and dried with MgSO.sub.0. The solvent was removed under reduced pressure. Compound 6 was purified by chromatography and obtained in 60% yield. A solution of 2,4-difluorothiophenol in THF at 0-5 ° C was added to NaH. The suspension was stirred at 0-5 ° C until the formation of bubbles ceased and the reaction mixture was a clear solution. Then, the solution was heated to 60 ° C. To this solution was added compound 6 at 60 ° C. The reaction mixture was heated to reflux until TLC indicated the total consumption of compound 6. Saturated NaCl solution and ethyl acetate were added to the reaction mixture. The organic phase was washed twice with saturated NaCl solution and dried with MgSO4. The solvent was removed under reduced pressure. Compound 7 was P1605 purified by chromatography with a 90% yield. A solution of compound 7 and aldehyde 8 in toluene was heated at reflux for 24 hours. The formed imine was purified by chromatography, dissolved in anhydrous methyl alcohol, and reduced to amine 9 with NaBH 4 in the presence of a catalytic amount of acetic acid. The reaction mixture was quenched with water and extracted with ethyl acetate. The organic layer was separated under vacuum and the crude amine was stirred with concentrated sulfuric acid solution at 100 ° C for 30 min. The amide 10 was separated by precipitation in a NaCl / crushed ice bath, filtered and directly used in the ring closure step. Amide 10 was dissolved in THF. To the solution was added an excess of DMF-DMA. The reaction solution was stirred at 70 ° C for one to two hours. The product 11 was purified by crystallization from ethyl acetate EXAMPLE 2 Cloning of p38 Kinase in Insect Cells Two cutting variants of the human p38, CSBP1 and CSBP2 kinase were identified. To amplify the cDNA confining region of CSBP2, specific oligonucleotide primers were used, using a HeLa cell library (Stratagene) as a template. The product of Polymerase chain reaction was cloned into the vector pET-15b (Novagen). The baculovirus transfer vector, pVL- (His) 6-p38, was constructed by subcloning a Xbal-BamHI fragment of pET15b- (His) 6-p38 into the complementary sites in the plasmid pVL1392 (Pharmingen). Plasmid pVL- (His) 6-p38 directed the synthesis of a recombinant protein consisting of a peptide of 23 residues (MGSSHHHHHHSSGLVPRGSHMLE, where LVPRGS represents a thrombin cleavage site) fused in frame to the N-terminus of p38, as it has been confirmed by DNA sequencing and by the N-terminal sequencing of the expressed protein. A monolayer culture of insect cells Spodoptera frugiperda (Sf9) (ATCC) was maintained in a medium of TNM-FH (Gibco BRL), supplemented with 10% fetal bovine serum in a T bottle at 27 ° C. The Sf9 cells in the logarithmic phase were co-transfected with linear viral DNA of Autographa califonica nuclear polyhidrosis virus (Pharmingen) and the transfer vector pVL- (His) 6-p38 using Lipofectin (Invitrogen). Individual clones of recombinant baculovirus were purified by plaque assay using 1% low melting point agarose.

P1605 EXAMPLE 3 Expression and Purification of the Recombinant p38 Kinase Trichoplusia ni (Tn-368) High-Five Cells (Invitrogen) were grown in suspension in the Excel-405 protein free medium (JRH Bioscience) in a shaker bottle at 27 ° C. A cell mass of these cells with a density of 1, 5 x 106 cells / ml was infected with the recombinant baculovirus previously described with infection concentrations in multiples of 5. The level of expression of the recombinant p38 was controlled by immunoblotting by scoring, using a rabbit anti-p38 antibody (Santa Cruz Biotechnology). The cell mass was harvested 72 hours after infection upon reaching the expression level of p38 its maximum. A frozen cell paste of cells expressing p38 labeled with (His) 6 was thawed in 5 volumes of Buffer A (50 mM NaH2P04, pH 8.0, 200 mM NaCl, 2 mM 3-mercaptoethanol, 10% glycerol and 0, 2 mM PMSF). After mechanical disruption of the cells in a microfluidizer, the lysate was centrifuged at 30,000 x g for 30 minutes. The supernatant was incubated in batches for 3-5 hours at 4 ° C with resin having affinity for metals Heel (Clontech) in a ratio of 1 ml of resin per 2-4 mg of the expected yield of p38. The resin was pelleted by centrifugation at 500 x g for 5 minutes P1605 and washed gently in batches with Buffer A. The resin was suspended, poured into a column (approximately 2.6 x 5.0 cm) and washed with Buffer A + 5 mM imidazole. (His) 6-p38 was eluted with Buffer A + 100 mM imidazole and subsequently dialyzed overnight 4 ° C against 2 liters of Buffer B (50 mM HEPES, pH 7.5, 25 mM ? -glycerophosphate, 5% glycerol, 2 mM DTT). The Híss marker was separated by the addition of 1.5 thrombin units (Calbiochem) per mg of p38 and incubated at 20 ° C for 2-3 hours. The thrombin was quenched by the addition of 0.2 mM PMSF, and then the entire sample was loaded onto a column with 2 ml of benzamidinagarose (American International Chemical). The fraction that passed through the column was loaded directly into a column of Q-Sepharose (Pharmacia) of 2.6 x 0.5 cm, previously equilibrated in Buffer B + 0.2 mM PMSF. P38 was eluted with 20 column volumes of linear gradient to 0.6 M NaCl in Buffer B. The peak of eluted protein was collected and dialyzed overnight at 4 ° C against Buffer C (50 mM HEPES, pH 7.5 , 5% glycerol, 50 mM NaCl, 2 mM DTT, 0.2 mM PMSF). The dialyzed protein was concentrated in a Centriprep (Amicon) at 3-4 ml and applied to a 2.6 x 100 cm column of Sephacryl S-100HR (Pharmacia). The protein was eluted with a flow rate of 35 ml / hr. The fractions of P1605 Main peak were pooled, adjusted to 20 mM DTT, concentrated at 10-80 mg / ml and frozen in aliquots at -70 ° C or used immediately.

EXAMPLE 4 Activation of p38 p38 was activated by combining 0.5 mg / ml of p38 with 0.005 mg / ml of mutant double MKK6 DD in Buffer B + 10 mM MgCl2, 2 mM ATP, 0.2 mM Na2V04 for 30 minutes at 20 ° C. Then, the activation mixture was loaded on a 1.0 × 10 cm MonoQ (Pharmacia) column and eluted with 20 volumes of gradient column at 1.0 M NaCl in Buffer B. The activated p38 eluted after the ADP and the ATP. Peak fractions of activated p38 were pooled and dialyzed against 0.2 mM Na2V0 Buffer B +, to remove NaCl. The dialyzed protein was adjusted to 1.1 M potassium phosphate by the addition of a 4.0 M standard solution and loaded onto a 1.0 x 10 cm HIC (Rainin Hydropore) column previously equilibrated in Buffer D (10% glycerol , 20 mM 3 -glycerophosphate, 2.0 mM DTT) + 1.1 M K2HP0. The protein was eluted with 20 volumes of linear gradient column at Buffer D + 50 mM K2HP04. Phosphorylated double p38 eluted as the main peak and was collected for dialysis against Buffer B + 0.2 mM Na2V0. Activated p38 was stored at -70 ° C.

P1605 EXAMPLE 5 p38 Inhibition Assays A. Inhibition of Phosphorylation of EGF Receptor Peptide This assay is carried out in the presence of 10 mM MgCL2, 25 mM 3-glycerophosphate, 10% glycerol and 100 mM HEPES buffer at pH 7.6. For a typical determination of the IC50, a standard solution containing all the above-mentioned components and activated p38 (5 nM) is prepared. The standard solution is subdivided into ampoule flasks. A fixed volume of DMSO or inhibitor in DMSO is introduced into each vial (the final concentration of DMSO in the reaction is 5%), mixed and incubated for 15 minutes at room temperature. To each vial is added EGF receptor peptide, KRELVEPLTPSGEAPNQALLR, a phosphoryl acceptor in the kinase reaction catalyzed by p38, to a final concentration of 200: M. The kinase reaction is initiated with ATP (100: M) and the ampoule flasks are incubated at 30 ° C. After 30 minutes, the reactions are turned off with equal volumes of 10% trifluoroacetic acid (TFA). The phosphorylated peptide is quantified by HPLC analysis. The separation of the phosphorylated peptide from the non-phosphorylated peptide is carried out on a reverse phase column (Deltapak, 5: M, C18 100D, part no. 011795) with a P1605 binary gradient of water and acetonitrile, each with 0.1% TFA. The IC50 (the concentration of the inhibitor that provides 50% inhibition) is determined by the graphical representation of the percentage of remaining activity as a function of the concentration of the inhibitor.

B. Inhibition of ATPase Activity This assay is carried out in the presence of 10 mM MgCl2, 25 mM 3-glycerophosphate, 10% glycerol and 100 mM Buffer HEPES at pH 7.6. For a typical Ki determination, the Km for ATP is determined, determining in the activated p38 reaction the ATPase activity of p38, in the absence of inhibitor and in the presence of two concentrations of inhibitor. Ki is determined from the reaction rate data as a function of inhibitor and ATP concentrations. A standard solution containing all the above-mentioned components and activated p38 is prepared. The standard solution is sub-divided in ampoule flasks. A fixed volume of DMSO or inhibitor in DMSO is introduced into each vial (the final concentration of DMSO in the reaction product is 2.5%), mixed and incubated for 15 minutes at room temperature. The reaction is initiated by adding several concentrations of ATP and then incubated at 30 ° C. After 30 minutes, the P1605 reactions are turned off with 50: 1 EDTA (final concentration 0.1 M), pH 8.0. The product of the ATPase activity of p38, ADP, is quantified by HPLC analysis. The separation of ADP from ATP is carried out in a reverse phase column (Supelcosil, LC-18, 3: M, part no. 5-8985) using a binary solvent gradient of the following composition: Solvent A - 0.1 M phosphate buffer containing 8 mM tetrabutylammonium acid sulfate (Sigma Chemical Co., catalog number T-7158), Solvent B - Solvent A with 30% methanol.

C. Inhibition in the production of IL-1, TNF, L-6 and IL-8; in PBMCB stimulated with LPS Serial dilutions of inhibitors are made in DMSO from a 20 mM standard solution. At least six serial dilutions are prepared, which are prepared by adding 4: 1 of a dilution to 1 ml of RPMI1640 / 10% fetal bovine serum medium. The diluted standard solutions contained the inhibitor at concentrations of 80: M, 32: M, 12.8: M, 5.12: M, 2.048: M, 0.819: M, 0.328: M, 0.131: M, 0.052: M, 0.021: M, etc. Diluted standard solutions of the inhibitor are preheated to 37 ° C before use. Erythrocytes (buffy cells) of fresh human blood are separated from other cells in a CPT Vacutainer P1605 Becton & Dickinson (containing 4 ml of blood and enough DPBS without Mg2 + / Ca2 + to fill the tube) by centrifugation at 1500 x g for 15 min. Peripheral blood mononuclear cells (PBMCs), which are located in the upper part of the gradient in the Vacutainer, are separated and washed twice with RPMI1640 medium / 10% fetal bovine serum. The PBMCs are harvested by centrifugation at 500 x g for 10 minutes. The total number of cells is determined with a Neubauer cell chamber and the concentration of the cells is adjusted to 4.8 x 106 cells / ml in the cell culture medium (RPMI1640 supplemented with 10% fetal bovine serum). Alternatively, whole blood containing an anticoagulant is used directly in the assay. 100: 1 cell suspension or whole blood is placed in each well of a plate for 96 cell culture. Then, 50: 1 of the dilutions of the standard solution of the inhibitor are added to the cells. Finally, 50: 1 of a working standard solution of lipopolysaccharide (LPS) (16 ng / ml in cell culture medium) is added to give a final concentration of 4 ng / ml LPS in the assay. The total volume of the control test with the vehicle is also adjusted to 200: 1 by adding 50: 1 of cell culture medium. Then, the PBMC cells or the whole blood are incubated during the P1605 night (for 12-15 hours) at 37 ° C / 5% C02 in a humid atmosphere. The next day, the cells are mixed with an agitator for 3-5 minutes before centrifugation at 500 x g for 5 minutes. Cell culture supernatants are harvested and analyzed by ELISA, to determine IL-lb levels (R & D Systems, Quantikine kits, # DBL50), TNF-V (BioSource, # KHC3012), IL-6 (Endogen, # EH2-IL6) and IL-8 (Endogen, # EH2-IL8) according to the manufacturer's instructions. The ELISA data are used to generate response curves to the doses, from which the IC50 values are determined). The p38 inhibitors of this invention will inhibit the phosphorylation of the EGF receptor peptide, and the production of IL-1, TNF, IL-6 and IL-8 in PEMCs stimulated by LPS or whole blood.

D. Inhibition in the production of IL-6 and IL-8; in PBMCs stimulated by IL-1 This test is carried out with PBMCs in exactly the same way as the previous test, except that 50: 1 of a working standard solution of IL-lb is added to the assay (2 ng / ml in cell culture medium) instead of the working standard solution of LPS.

P1605 Cell culture supernatants are harvested as previously described and analyzed by ELISA to determine the levels of IL-6 (Endogen, # EH2-IL6) and IL-8 (Endogen, # EH2-IL8) according to the manufacturer's instructions. The ELISA data are used to generate response curves to the doses from which the IC50 values are determined.

E. Inhibition of Prostaglandin-Endoperoxide-Synthetase-2 Induction (PGHS-2, or COX-2) in PBMCs Induced by LPS Human peripheral mononuclear cells (PBMCs) are isolated from erythrocytes (buffy coats) of fresh human blood by centrifugation in a Vacutainer CPT (Becton &Dickinson). In a cuvette for the culture of six-well tissue containing RPMl 1640 supplemented with 10% fetal bovine serum, 50 U / ml penicillin, 50: g / ml streptomycin, and 2 mM L-glutamine are seeded 15 x 10e cells. An inhibitor of the present invention is added to final concentrations of 0.2, 2.0 and 20: M in DMSO. Then, LPS is added to a final concentration of 4 ng / ml to induce the expression of the enzyme. The total volume of the culture is 10 ml / cavity. After an overnight incubation a Ios 37 ° C, 5% C02, the cells are harvested by scraping and subsequent centrifugation, then the supernatant is removed and the cells are washed twice in ice-cold DPBS (physiological solution with Dulbecco's phosphate buffer, BioWhittaker). The cells are disintegrated on ice for 10 minutes in 50: 1 cold lysis buffer (20 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% Triton-X-100, 1% deoxycholic acid, 0.1% SDS, 1 mM EDTA, 2% aprotinin (Sigma), 10: g / ml pepstatin, 10: g / ml leupeptin, 2 mM PMSF, 1 mM benzamidine, 1 mM DTT) containing 1: 1 Benzonase (Merck DNAse). The protein concentration of each sample is determined with a BCA assay (Pierce), taking as a standard bovine serum albumin. Then, the protein concentration of each sample is adjusted to 1 mg / ml with cold lysis buffer. At 100: 1 of the lysate a volume equal to 2 x loading buffer of SDS PAGE is added and the sample is boiled for 5 minutes. The proteins (30: g / lane) are fractionated according to their size in SDS PAGE gels (Novex) with gradients of 4-20% and subsequently transferred onto nitrocellulose membranes by means of electrophoresis for two hours at 100 mA in buffer. To bin transfer (25 mM Tris, 192 mM glycine) containing 20% methanol. The membrane is pretreated for 1 hour at room temperature with blocking buffer (5% dehydrated non-fat milk in DPBS supplemented with 0.1% P1605 Tween-20) and washed 3 times in DPBS / 0.1% Tween-20. The membrane is incubated overnight at 4 ° C with a 1: 250 dilution of anti-COX-2 monoclonal antibody (Transduction Laboratories) in blocking buffer. After 3 washes in DPBS / 0.1% Tween-20, the membrane is incubated for one hour at room temperature, with a 1: 1000 dilution in blocking buffer from sheep antisera to mouse Ig conjugated with horseradish peroxidase (Amersham). Then, the membrane is washed again three times in DPBS / 0.1% Tween-20, and an ECL detection system (CL-HRP SuperSignal ™ Substrate System) is used to determine the levels of COX-2 expression. While we have presented a number of embodiments of this invention in the foregoing, it is apparent that our basic constructions can be changed to provide other embodiments utilizing the methods of this invention. pieos

Claims (23)

CLAIMS:

1. A compound that has the formula: or tautomers thereof or pharmaceutically acceptable salts thereof; wherein: each of Qx and Q2 are independently selected from carbocyclic or aromatic heterocyclic ring systems of 5-6 members, or bicyclic ring systems of from 8 to 10 members comprising carbocyclic aromatic rings, aromatic heterocyclic rings or a combination of a aromatic carbocyclic ring and an aromatic heterocyclic ring; wherein: the rings that make up Qi are optionally substituted with 1 to 4 substituents, each of which is independently selected from J; halo; (C? -C4) alkyl optionally substituted with N (R ') 2, OR', C02R 'or CONR'2 / - O-C? -C alkyl optionally with A, TC (0) R', 0P03H2, NR'2, NR'2, OR ', C02R' or CONR'2; NR'2; OCF3; CF3; N02; C02R '; CONR '; MR'; S (02) N (R ') 2; SCF3; CN; N (R ') C (0) R4; N (R ') C (0) OR 4; N (R ') C (O) C (O) R 4; N (R ') S (02) R4; N (R ') R4; N (R4) 2; OR4; OC (0) R4; OP (0) 3H2; or N = C-N (R ') 2; and where: The rings that make up Q2 are replaced P1605 with J and optionally substituted with halo, straight or branched chain (C? -C4) alkyl, hydroxy, methoxy, trifluoromethyl, trifluoromethoxy, cyano, or amino; J is a C 1 -C 4 straight or branched chain alkyl derivative, substituted with 1-3 substituents selected from A, -T-C (0) R 'or OP03H2. A is selected from the groups: T is either O or NH; G is any of NH2 or OH; Z is either CH or N; R 'is selected from hydrogen, C?-C3 alkyl, C2-C3 alkenyl or C2-C3 alkynyl, phenyl or phenyl substituted with 1 to 3 substituents independently selected from halo, methoxy, cyano, nitro, amino, hydroxy, methyl or ethyl; or a 5- to 6-membered heterocyclic ring system optionally substituted with 1 to 3 substituents independently selected from halo, methoxy, cyano, nitro, amino, hydroxy, methyl or ethyl; pieos R3 is selected from 5-6 membered carbocyclic or heterocyclic aromatic ring systems; R4 is (C? -C4) alkyl optionally substituted with N (R ') 2, OR', C02R ', CON (R') 2, or S02N (R2) 2; a 5-6 membered carbocyclic or heterocyclic ring system optionally substituted with a straight or branched chain (C? -C) alkyl group, N (R ') 2, OR', C02R ', CON (R') 2 , or S0N (R) 2; or a (C? -C) alkyl optionally substituted with the 5-6 membered carbocyclic or heterocyclic ring system optionally substituted with a straight or branched chain (C? -C4) alkyl group, N (R ') 2 , OR ', C02R', C0N (R ') 2, or S02N (R2) 2; R2 is selected from hydrogen, (Cx-C3) alkyl, or (C? -C3) alkenyl; each being optionally substituted with -N (R ') 2, -OR', SR ', -C (0) N (R') 2, -S (02) -N (R ') 2, -C (0 ) OR ', or R3; and W is selected from H; N (R2) S02-N (R2) 2; N (R2) S02-N (R2) (R3); N (R2) C (0) -OR2; N (R2) C (O) -N (R2) 2; N (R2) C (0) -N (R2) (R3): N (R2) C (0) -R2; N (R2) 2; C (0) -R2; CH (0H) -R2; C (0) -N (R2) 2; C (0) -0R2; or straight or branched chain alkyl of (C? -C4) optionally substituted with A, T-C (0) R ', N (R') 2,, OR ', C02R'; C0N (R ') 2; R3; or S02N (R2) 2; or a 5-6 membered carbocyclic or heterocyclic ring system optionally substituted with N (R ') 2, OR', C02R ', C0N (R') 2, or S02N (R2) 2; P1605

2. The compound according to claim 1, characterized in that Qx is selected from phenyl or pyridyl containing from 1 to 3 substituents, wherein at least one of the substituents is in the ortho position, and the substituents are independently selected from chlorine, fluorine , bromine, -CH3, -OCH3, -OH, -CF3, -OCF3, -0 (CH2) 2CH3, NH2, 3,4-methylenedioxy, -N (CH3) 2, -NH-S (0) 2-phenyl , -NH-C (0) -CH2-4-pyridine, -NH-C (0) -CH2-morpholine, -NH-C (0) -CH2-N (CH3) 2, -NH-C (0) -CH2-piperazine, -NH-C (O) -CH2-pyrrolidine, -NH-C (O) -C (0) -morpholine, -NH-C (0) -C (O) -piperazine, -NH- C (O) -C (0) -pyrrolidine, -0-C (0) CH2-N (CH3) 2, or -O- (CH2) 2-N (CH3) 2, and wherein at least one of the substituents is in the ortho position.

3. The compound according to claim 2, characterized in that Qx contains at least two substituents, and both are in the ortho position.

4. The compound according to claim 2, characterized in that Qx is selected from: P1605 605

5. The compound according to claim 4, characterized in that Qx is selected from 2-fluoro-6-trifluoromethylphenyl, 2,6-difluorophenyl, 2,6-dichlorophenyl, 2-chloro-4-hydroxyphenyl, 2-chloro-4- aminophenyl, 2,6-dichloro-4-aminophenyl, 2,6-dichloro-3-aminophenyl, 2,6-dimethyl-4-hydroxyphenyl, 2-methoxy-3,5-dichloro-4-pyridyl, 2-chloro- 4, 5-methylenedioxyphenyl, or 2-chloro-4- (N-2-morpholinoacetamido) phenyl.

6. The compound according to claim 1, characterized in that Q2 is selected from phenyl or pyridyl, phenyl or pyridyl contains substituent J and from 0 to 3 other substituents, wherein each of these other substituents is independently selected of chlorine, fluorine, bromine, methyl, ethyl, isopropyl, -OCH3, -OH, -NH2 -CF3 / -0CF3, -SCH3, -OCH3, -C (0) 0H, -C (0) 0CH3, -CH2NH2, -N (CH 3) 2, -CH 2 -pyrrolidine and -CH 2 OH.

7. The compound according to claim 1, characterized in that said compound is the Compound 15. The compound according to claim 1, characterized in that said compound is P1605 he Compound 16. The compound according to claim 1, characterized in that said compound is the Compound 17. The compound according to claim 1, characterized in that said compound is the P1605 Compound 1

8. 11. A pharmaceutical composition, characterized in that it comprises a compound according to claim 1 and a pharmaceutically acceptable carrier. 12. A method for treating or preventing in a patient inflammatory diseases, autoimmune diseases, bone destruction disorders, proliferation disorders, infectious diseases, neurodegenerative diseases, allergies, reperfusion / ischemia in str heart attacks, angiogenic disorders, organ hypoxia , vascular hyperplasia, cardiac hypertrophy, platelet aggregation induced by thrombin or conditions associated with prostaglandin-endoperoxidase-synthetase-2, said method being characterized in that it comprises administering to said patient a composition according to claim 11 in an amount effective to inhibit p38 . 13. A method according to claim 12, characterized in that said method is used to treat or prevent an inflammatory disease selected from P1605 Acute pancreatitis, chronic pancreatitis, asthma, allergies, or respiratory distress syndrome in adults. A method according to claim 12, characterized in that said method is used to treat or prevent an autoimmune disease selected from glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Graves disease, autoimmune gastritis, diabetes, anemia autoimmune hemolytic, autoimmune neutropenia, thrombocytopenia, atopic dermatitis, chronic active hepatitis, myasthenia gravis, multiple sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, psoriasis or graft-versus-host disease. The method according to claim 12, characterized in that said method is used to treat or prevent a destructive bone disorder selected from osteoarthritis, osteoporosis or a bone disorder related to multiple myeloma. 16. A method according to claim 12, characterized in that said method is used to treat or prevent a proliferative disease selected from acute myelocytic leukemia, chronic myelocytic leukemia, metastatic melanoma, Kaposi's sarcoma, or multiple myeloma. 17. The method according to the claim P1605 12, characterized in that said method is used to treat or prevent an infectious disease selected from sepsis, septic shock or shigellosis. 18. The method according to claim 12, characterized in that said method is used to treat or prevent a viral disease selected from infection by acute hepatitis., HIV infection or CMV retinitis. The method according to claim 12, characterized in that said method is used to treat or prevent a neurodegenerative disease selected from Alzheimer's disease, Parkinson's disease, cerebral ischemia or neurodegenerative disease caused by traumatic injury. The method according to claim 12, characterized in that said method is used to treat or prevent ischemia / reperfusion in stroke or myocardial ischemia, renal ischemia, heart attacks, hypoxia organs or platelet aggregation induced by thrombin. The method according to claim 12, characterized in that said method is used to treat or prevent a condition associated with prostaglandin / endoperoxide synthetase-2 selected from edema, fever, analgesia or pain. 22. The method according to claim 21, characterized in that said pain is selected from P1605 neuromuscular pain, headache, pain from cancer, dental pain or arthritis pain. 23. The method according to claim 12, characterized in that said method is used to treat or prevent an angiogenic disorder selected from solid tumors, ocular neovascularization, or infantile hemangioma. P1605 < _ 68 SUMMARY A compound that inhibits p38, which is a proteinace involved in cell proliferation, cell death and response to extracellular stimuli. The invention also relates to methods for producing these inhibitors, to pharmaceutical compositions comprising them, and to methods for using said compositions in the treatment and prevention of various disease states. P1605