WO2006018842A2

WO2006018842A2 - Inhibitors of p38-alpha kinase

Info

Publication number: WO2006018842A2
Application number: PCT/IL2005/000888
Authority: WO
Inventors: Yael Friedmann; Anat Shriki; David Engelberg
Original assignee: Jexys Pharmaceuticals Ltd.
Priority date: 2004-08-17
Filing date: 2005-08-16
Publication date: 2006-02-23
Also published as: WO2006018842A3

Abstract

The present invention relates to compounds capable of inhibiting p38a-Kinase, to pharmaceutical compositions comprising these compounds, and to methods of using the compounds for the treatment or prevention of diseases or disorders that are mediated by p38-a Kinase, particularly for the treatment or prevention of inflammatory diseases and disorders and heart conditions.

Description

INHIBITORS OF p38-α KINASE

FIELD OF INVENTION

The present invention relates to compounds capable of inhibiting p38α-kinase, to pharmaceutical compositions comprising these compounds, and to methods of using the compounds for the treatment or prevention of diseases or disorders that are mediated by p38-α kinase, particularly for the treatment or prevention of inflammatory diseases and disorders and heart conditions, as described herein.

BACKGROUND OF THE INVENTION

The Mitogen- Activated Protein (MAP) kinases are a family of proline-directed serine/threonine kinases that are activated by dual phosphorylation, and in turn phosphorylate their substrates on either Threonine-Proline or Serine-Proline sites. MAP kinases are activated in response to a variety of signals including nutritional and osmotic stress, UV light, growth factors, endotoxin and inflammatory cytokines. The p38 sub-group of MAP kinases (p38, also known as CSBP and RK) is a MAP kinase family of various isoforms, which is responsible for phosphorylating a large number of substrates, including transcription factors (e.g. ATF2, CHOP and MEF2C), other kinases (e.g. MAPKAP-2 and MAPKAP-3), tumor suppressors (e.g. p53) and translational regulators (e.g. 3EBP, PRAK).

A large number of chronic and acute conditions have been recognized to be associated with perturbation of the inflammatory response. A large number of cytokines participate in this response, including IL-I, IL-6, IL-8 and TNF. It appears that the expression, secretion and activity of these cytokines in the regulation of inflammation rely at least in part on the activation of p38. This kinase is activated by dual phosphorylation after stimulation by physiochemical stress, treatment with lipopolysaccharides or with proinflammatory cytokines such as IL-I, and TNF.

TNF and interleukins such as IL-I and IL-8 affect a wide variety of cells and tissues and are important inflammatory mediators of a wide variety of disease states and conditions. TNF-α is a cytokine produced primarily by activated monocytes and macrophages. Excessive or unregulated TNF production has been implicated in mediating a number of diseases. Recent studies indicate that TNF has a causative role in the pathogenesis of rheumatoid arthritis. Additional studies demonstrate that inhibition of TNF has broad application in the treatment of inflammation, inflammatory bowel disease, multiple sclerosis and asthma. TNF has also been implicated in viral infections, such as HIV, influenza virus, and herpes virus including herpes simplex virus type-1 (HSV-I), herpes simplex virus type-2 (HS V-2), cytomegalovirus (CMV), varicella-zoster virus (VZV), Epstein-Barr virus, human herpesvirus-6 (HHV-6), human herρesvirus-7 (HHV-7), human herpesvirus-8 (HHV- 8), pseudorabies and rhinotracheitia, among others.

IL-8 is another pro-inflammatory cytokine, which is produced by mononuclear cells, fibroblasts, endothelial cells, and keratinocytes, and is associated with pathological conditions including inflammation.

IL-I is produced by activated monocytes and macrophages and is involved in the inflammatory response. IL-I plays a role in many pathophysiological responses including rheumatoid arthritis, fever and reduction of bone resorption.

TNF, IL-I and IL-8 affect a wide variety of cells and tissues and are important inflammatory mediators of a wide variety of disease states and conditions. The inhibition of these cytokines by inhibition of the p38 kinase is of benefit in controlling, reducing and alleviating many of these disease states.

Within the past several years, p38 has been shown to comprise a group of MAP kinases designated p38-α, p38-β, p38-γ, and p38-δ. Jiang, Y., et al., (J Biol Chem (1996) 271:17920-17926) reported characterization of p38-β as a 372-amino acid protein closely related to p38-α. In comparing the activity of p38-α with that of p38-β, the authors state that while both are activated by proinflammatory cytokines and environmental stress, p38-β was preferentially activated by MAP kinase kinase-6 (MKK6) and preferentially activated transcription factor 2, thus suggesting that separate mechanisms for action may be associated with these forms. Kumar, S., et al., (Biochem Biophys Res Comm (1997) 235:533-538) and Stein, B., et al., (J Biol Chem (1997) 272:19509-19517) reported a second isoform of p38-β - p38-β2, containing 364 amino acids with 73% identity to p38-α. AU of these reports show evidence that p38-β is activated by proinflammatory cytokines and environmental stress, although the second reported p38-β isoform - p38-β2, appears to be preferentially expressed in the CNS, heart and skeletal muscle compared to the more ubiquitous tissue expression of p38-α. Furthermore, activated transcription factor-2 (ATF -2) was observed to be a better substrate for p38-β2 than for p38-α thus suggesting that separate mechanisms of action may be associated with these forms. The physiological role of p38-βl has been called into question by the latter two reports since it cannot be found in human tissue and does not exhibit appreciable kinase activity with the substrates of p38-α.

The identification of p38-γ was reported by Li, Z., et al, (Biochem Biophys Res Comm (1996) 228:334-340) and of p38-δ by Wang, X., et al., (J Biol Chem (1997) 272:23668-23674) and by Kumar, S., et al., (Biochem Biophys Res Comm (1997) 235:533-538). The data suggest that these two p38 isoforms (γ and δ) represent a unique subset of the MAPK family based on their tissue expression patterns, substrate utilization, response to direct and indirect stimuli, and susceptibility to kinase inhibitors.

Various results with regard to differential response to drugs targeting the p38 family as between p38-α and either the putative p38-βl or p38-β-2, or both were reported by Jiang, Kumar, and Stein cited above as well as by Eyers, P. A., et al., (Chem and Biol (1995) 5:321-328). An additional paper by Wang, Y., et al., (J Biol Chem (1998) 273:2161-2168) suggests the significance of such differential effects. As pointed out by Wang et al., a number of stimuli, such as myocardial infarction, hypertension, valvular diseases, viral myocarditis, and dilated cardiomyopathy lead to an increase in cardiac workload and elevated mechanical stress on cardiomyocytes. These are said to lead to an adaptive hypertrophic response, which, if not controlled, has decidedly negative consequences. Wang et al. cite previous studies which have shown that in ischemia reperfusion treated hearts, p38 MAPK activities are elevated in association with hypertrophy and programmed cell death. Wang et al. show in the cited paper that activation of p38-β activity results in hypertrophy, whereas activation of p38-α activity leads to myocyte apoptosis. Thus, selective inhibition of p38-α activity as compared to p38-β activity will be of benefit in treating conditions associated with cardiac failure. These conditions include congestive heart failure, cardiomyopathy, myocarditis, vasculitis, vascular restenosis, valvular disease, conditions associated with cardiopulmonary bypass, coronary artery bypass, grafts and vascular grafts. Further, to the extent that the α- isoform is toxic in other muscle cell types, α-selective inhibitors would be useful for conditions associated with cachexia attributed to TNF or other conditions such as cancer, infection, or autoimmune disease.

PCT applications WO98/06715, WO98/07425, WO98/28292 and WO 96/40143, describe the relationship of p38 kinase inhibitors with various disease states. As mentioned in these applications, inhibitors of p38 kinase are useful in treating a variety of diseases associated with chronic inflammation. These applications list rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions, sepsis, septic shock, endotoxic shock, Gram-negative sepsis, toxic shock syndrome, asthma, adult respiratory distress syndrome, stroke, reperfusion injury, CNS injuries such as neural trauma and ischemia, psoriasis, restenosis, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcosis, bone resorption diseases such as osteoporosis, graft- versus-host reaction, Crohn's Disease, ulcerative colitis including inflammatory bowel disease (IBD) and pyresis.

The above-referenced PCT applications disclose compounds which are p38 kinase inhibitors said to be useful in treating these disease states. These compounds are either imidazoles or are indoles substituted at the 3- or 4-position with a piperazine or piperidine ring linked through a carboxamide linkage. Additional compounds which are conjugates of piperazines with indoles are described as insecticides in WO97/26252.

U.S. patents 6,410,540, 6,541,477, and 6,696,443, and PCT applications WO 00/12074 and WO 01/64676, all assigned to Scios Inc., disclose a number of piperazine and piperidine derivatives, and their use in treating various disorders associated with enhanced activity of p38-α. The disclosed compounds include piperidine derivatives that are bound through linkers to aryl moieties. However, there is no specific disclosure or examples in the Scios patents and patent applications of compounds of Formula (I) below which contain a thioalkyl or thioaryl substituent on the phenyl ring.

SUMMARY OF THE INVENTION

It is an object of some aspects of the present invention to provide methods and compounds useful in treating diseases or conditions that are characterized by enhanced p38-α activity. These diseases or conditions include inflammation, proliferative diseases, and certain cardiovascular disorders as further described below.

Is has been surprisingly discovered that compounds of formula (I) shown below, inhibit p38 kinases, the α-isoform in particular, and are thus useful in treating diseases mediated by these activities. The compounds of formula (I) contain a piperidine moiety linked through a carbonyl or its isostere to a phenyl ring which is substituted by at least one thioalkyl or thioaryl moiety. The piperidine moiety is also linked through a methylene or its isostere to a second phenyl ring.

The compounds of the invention are particularly useful in the inhibition of the α- isoform of p38.

The compounds of formula (I) are represented by the following chemical structure:

(I) wherein R¹ is independently selected from H, C₁-

C₆ alkyl, C₁-C₆ alkylaryl and aryl; R² is independently selected from H, C₁- C₆ alkyl, Ci-C₆ alkyloxy, C₁-C₆ alkylaryl, aryl and aryloxy; n is selected from 0, 1, 2, 3 and 4; m is selected from 1, 2, 3, 4 and 5; wherein the sum of n and m cannot be greater than 5;

A¹ is CO or an isostere thereof; A² is CH₂ or an isostere thereof; or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof .

In one embodiment of formula (I), R¹ is methyl. In another embodiment of formula (I), R¹ is methyl and m is 1. In another embodiment of formula (I), R² is methoxy. In another embodiment of formula (I), R is methoxy and n is 1. In another embodiment or formula (I), R is methyl, R is methoxy and n and m are both 1. In another embodiment of formula (I), m is 1 and SR¹ is in the para position. In another embodiment of formula (I), n is 1 and R² is in the ortho position.

Isosteres of CO include but are not limited to CH₂, SO, SO₂, CH-OH , CH-NH₂, C=NH and CH-CH₃. Isosteres of CH₂ include but are not limited to CO, SO, SO₂, CH-OH , CH-NH₂, C=NH and CH-CH₃.

In one embodiment of the present invention, the compound of formula (I) is represented by the structure of formula (II), or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof

(II) wherein

R² _S m and n are as defined above for formula (I).

In one embodiment of the present invention, the compound of formula (I) is represented by the structure of formula (III), or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof.

(III) wherein R¹ and R² are as defined above for formula (I).

In a currently preferred embodiment, the compound of formula (I) is represented by the structure of formula (IV), or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof.

In another embodiment, the present invention provides pharmaceutical composition comprising as an active ingredient a compound represented by the structure of any of formulas (I)-(IV) or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof; and a pharmaceutically acceptable carrier or diluent.

In another embodiment, the present invention provides a pharmaceutical composition for treating a condition characterized by enhanced p38-α kinase activity, comprising as an active ingredient a therapeutically effective amount of a compound represented by the structure of any of formulas (I)-(IV) or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof; and a pharmaceutically acceptable diluent or carrier.

In another embodiment, the present invention provides an in-vitro method of inhibiting the activity of a p38-α kinase comprising the step of contacting a p38-α kinase with a compound represented by the structure of any of formulas (I)-(IV) or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof, in an amount effective to inhibit the activity of the p38-α kinase.

In another embodiment, the present invention provides a method of inhibiting the activity of ap38-α kinase comprising the step of contacting a cell comprising the p38- α kinase with a compound represented by the structure of any of formulas (I)-(IV) or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof, in an amount effective to inhibit the activity of the p38-α kinase.

In another embodiment, the present invention provides a method for inhibiting the activity of a p38-α kinase in a subject, comprising the step of administering to the subject a compound represented by the structure of any of formulas (I)-(IV)or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof, in an amount effective to inhibit the activity of the p38-α kinase.

In another embodiment, the present invention provides a method of treating or preventing a disease or condition mediated by p38-α kinase or associated with p38-α kinase activity, comprising the step of administering to a subject in need thereof a compound represented by the structure of any of formulas (I)-(IV) or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof, in an amount effective to treat or prevent the disease or condition.

In one embodiment, the p38-α kinase mediated disease or condition is characterized by a proinflammation response. The disease or condition characterized by inflammation can be, for example, acute respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, uveitis, acute renal failure, head trauma, ischemic/reperfusion injury, multiple sclerosis, IBD, rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis, other arthritic conditions, sepsis, septic shock, endotoxic shock, Gram-negative sepsis, toxic shock syndrome, adult respiratory distress syndrome, stroke, reperfusion injury, CNS injury, psoriasis, restenosis, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcosis, a bone resorption disease, graft-versus-host reaction, Crohn's Disease, ulcerative colitis, or pyresis.

In one embodiment, the p38-α kinase mediated disease or condition is a heart condition associated with cardiac failure. The heart condition, for example, can be congestive heart failure, cardiomyopathy or myocarditis. In another embodiment, the methods and compositions of the present invention further comprise the use of an additional therapeutic agent, for example a corticosteroid, a monoclonal antibody, or an inhibitor of cell division.

Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be understood from the following detailed description in conjunction with the drawings, of which:

FIG. 1 shows a growth curve of yeast cells harboring active p38 in the absence

(p38/cells) or presence of the indicated concentrations of Compound (IV). The known p38 inhibitors PD 169316 and SB 203580 were used as a control. A growth curve of normal yeast cells (not expressing any foreign protein) is also shown (control cells).

FIG. 2 shows a growth curve of yeast cells harboring active p38 in the absence

(p38/cells) or presence of the indicated concentrations of reference Compound (V). The known p38 inhibitor PD 169316 was used as a control. A growth curve of normal yeast cells (not expressing any foreign protein) is also shown (control cells). FIG. 3 shows p38 kinase assay in the presence of Compounds (IV) and (V). Kinase assays were performed in the presence of increasing concentrations of either compound (IV), compound (V) or PD 169316. Fig 3 A: Top panel: commassie blue staining to monitor protein levels. Bottom panel, measure of radioactivity, incorporated into ATF2 protein, a p38 substrate. Fig 3 B is a graph showing incorporation of radioactivity as a function of compound concentration.

FIG 4 is a graph showing a kinase assay with purified p38α and p38γ in the presence of compound (IV). p38γ was activated by MKK6 and a kinase assay was performed in the presence of compound (IV). Controls include non activated p38γ, activated p38γ with no inhibitor, and PD 169316.

FIG 5 is a western blot showing expression of Myogenin in L8 myoblasts, in the absence (DM) or presence of Compound (IV). Fig 5 A: Cells were cultured in differentiation medium with or without inhibitors for 60 hours. Fig 5B: Cells were cultured in the presence of Compound (IV) for 36 hours, followed by removal of the compound and additional culture for 36 hours. Bottom panels: total protein levels as measured by expression of tubulin. The known p38 inhibitors PD 169316 (PD) and SB203580 (SB) were used as controls.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of formula (I) are useful in treating conditions which are characterized by overactivity of p38 kinase, in particular the α-isoform. Conditions

"characterized by enhanced p38-α activity" include those where this enzyme is present in increased amount or wherein the enzyme has been modified to increase its inherent activity, or both. Thus, "enhanced activity" refers to any condition wherein the effectiveness of these proteins is undesirably high, regardless of the cause. Is has been surprisingly discovered that compounds of formula (I) shown below, inhibit p38 kinases, the α-isoform in particular, and are thus useful in treating diseases mediated by these activities. Thus, it is an object of some aspects of the present invention to provide methods and compounds useful in treating diseases or conditions that are characterized by enhanced p38-α activity. These diseases or conditions are characterized by a proinflammation response and can be, for example, acute respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, uveitis, acute renal failure, head trauma, ischemic/reperfusion injury, multiple sclerosis, IBD, rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis, other arthritic conditions, sepsis, septic shock, endotoxic shock, Gram-negative sepsis, toxic shock syndrome, adult respiratory distress syndrome, stroke, reperfusion injury, CNS injury, psoriasis, restenosis, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcosis, a bone resorption disease, graft- versus-host reaction, Crohn's Disease, ulcerative colitis, or pyresis. The diseases or conditions can also be a heart condition associated with cardiac failure. The heart condition, for example, can be congestive heart failure, cardiomyopathy or myocarditis.

Methods of treatment with the compounds of invention are further discussed below.

Compounds

The compounds useful in the invention contain a piperidine moiety linked through a carbonyl or its isostere to a phenyl ring which is substituted by at least one thioalkyl or thioaryl moiety. The piperidine moiety is also linked through a methylene or its isostere to a second phenyl ring.

(I) wherein

R¹ is independently selected from H, C₁- C₆ alkyl, C₁-C₆ alkylaryl and aryl; R² is independently selected from H, C₁- C₆ alkyl, C₁-C₆ alkyloxy, C₁-C₆ alkylaryl, aryl and aryloxy; n is selected from 0, 1, 2, 3 and 4; m is selected from 1, 2, 3, 4 and 5; wherein the sum of n and m cannot be greater than 5;

In one embodiment of formula (I), R¹ is methyl. In another embodiment of formula (I), R¹ is methyl and m is 1. In another embodiment of formula (I), R² is methoxy. In another embodiment of formula (I), R² is methoxy and n is 1. In another embodiment or formula (I), R¹ is methyl, R² is methoxy and n and m are both 1. In another embodiment of formula (I), m is 1 and SR¹ is in the para position. In another embodiment of formula (I), n is 1 and R² is in the ortho position.

Isosteres of CO include but are not limited to CH₂, SO, SO₂, CH-OH , CH-NH₂, C=NH and CH-CH₃.

Isosteres of CH₂ include but are not limited to CO, SO, SO₂, CH-OH , CH-NH₂, C=NH and CH-CH₃.

(H) wherein R¹, R², m and n are as defined above for formula (I). In one embodiment of the present invention, the compound of formula (I) is represented by the structure of formula (III), or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof.

(III)

1 0 wherein R and R are as defined above for formula (I). In a currently preferred embodiment, the compound of formula (I) is represented by the structure of formula (IV), or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof.

It is understood that when m is greater than 1, each R¹ can independently represent any of the definitions set forth for R¹ above. Furthermore, it is understood that when n is greater than 1 , each R can independently represent any of the definitions set forth for R above.

In accordance with the present invention and as used herein, the following terms are defined with the following meanings, unless explicitly stated otherwise.

The term "alkyl" refers to a straight or branched chain or cyclic hydrocarbon having 1-12 carbon atoms. In one embodiment, the alkyl has 1-10 carbons (designated C₁-C₁₀ alkyl). In another embodiment, the alkyl has 1-8 carbons (designated C₁-C₈ alkyl). In another embodiment, the alkyl has 1-6 carbons (designated C₁-C₆ alkyl). In another embodiment, the alkyl has 1-4 carbons (designated C₁-C₄ alkyl). Examples of alkyl groups include methyl, ethyl, 1-methylethyl, propyl, butyl, isobutyl, sec-butyl, t- butyl, pentyl, isopentyl, hexyl and the like. The alkyl cam be unsubstituted or can be substituted by one or more inert substituents as defined hereinbelow.

The term "aryl" refers to an aromatic group having at least one carbocyclic aromatic group, which may be unsubstituted or substituted by one or more inert substituents as defined hereinabove. An example of an aryl group is a phenyl ring. The term "aryl", as used herein, also encompasses heteroaryl ring systems. The term "heteroaryl" refers to an aromatic group containing one or more heteroatoms, for example oxygen, nitrogen, sulfur and the like, which ring is optionally substituted with one or more inert substituents as defined hereinbelow. Non-limiting examples of heterocyclic substituents are imidazole, thiazole, furan, quinoline, isoquinoline and the like.

The term "alkylaryl" refers to an alkyl group as described hereinabove which is bonded to an aryl group as described hereinabove. An example of an arylalkyl group is a benzyl group.

The term "alkyloxy" (also referred to as "alkoxy") refers to an alkyl group as described hereinabove, which is attached to an oxygen atom. For example a C₁-C₆ alkyloxy group refers to a C₁-C₆ straight chain, branched chain or cyclic alkyl group, which is attached to an oxygen atom. An example of an alkyloxy group is methoxy.

The term "aryloxy" refers to an aryl group as described hereinabove, which is attached to an oxygen atom. An example of an alkyloxy group is phenoxy. Any one of the groups referred to hereinabove can be unsubstituted or substituted by one or more substituents, i.e. substituents which do not interfere with the biological activity of the compounds. The term "substituted", as used herein, means that any one or more hydrogen on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency is not exceeded, and that the substitution results in a stable compound. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds. By stable compound or stable structure it is meant herein a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. Non-limiting examples of suitable substituents include but are not limited to halo, hydroxy, Ci-C₁₀ alkyl, C₂-Q₀ alkenyl, C₂-Qo alkynyl, Q-Qo alkoxy, C₇-C₁₂ aralkyl, C₇-C₁₂ alkaryl, Ci-Ci₀ alkylthio, arylthio, aryloxy, arylamino, C₃-Ci₀ cycloalkyl, C₃-Ci₀ cycloalkenyl, di(C₁-C₁₀)-alkylamino, C₂-C₁₂ alkoxyalkyl, Ci-C₆ alkylsulfmyl, Ci-Ci₀ alkylsulfonyl, arylsulfonyl, aryl, hydroxy, hydroxy(Q-Cio)alkyl, 3TyIoXy(Ci-C₁₀)alkyl, C₁-Ci₀ alkoxycarbonyl, aryloxycarbonyl, aryloyloxy, substituted alkoxy, fluoroalkyl, nitro, cyano, cyano(Ci-C₁₀)alkyl, Q-Qo alkanamido, aryloylamido, arylaminosulfonyl, sulfonamido, amidino, amido, alkylamido, dialkylamido, amino, alkylamino, dialkylamino, carobyl, carbamido, carboxy, heterocyclic radical, nitroalkyl, and -(CH₂)_Jn-Z-(C₁-C₁O alkyl), where m is 1 to 8 and z is oxygen or sulfur. Unless otherwise indicated, all chiral, enantiomeric, diastereomeric and racemic forms of the compounds described in the present invention are also included in the present invention; the compounds may also have asymmetric centers. Many geometric isomers of olefins, C- and N- double bonds and the like can also be present in the compounds described herein, and all such stable isomers (e.g, cis, trans, E, Z) are contemplated in the present invention. It will be appreciated that compounds of the present invention that contain asymmetrically substituted carbon atoms may be isolated in optically active or racemic forms. It is well known in the art how to prepare optically active forms, such as by resolution of racemic forms or by synthesis, from optically active starting materials. All chiral, enantiomeric) diastereomeric, racemic forms and all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomer form is specifically indicated.

When a bond to a substituent is shown to cross the bond connecting two atoms in a ring, then such substituent may be bonded to any atom on the ring. When a bond joining a substituent to another group is not specifically shown or the atom in such other group to which the bond joins is not specifically shown, then such substituent may form a bond with any atom on such other group.

As contemplated herein, the present invention further encompasses analogs, derivatives, isomers, pharmaceutically acceptable salts and solvates (e.g. hydrates) as well as polymorphs of the compounds defined by the present invention.

The term "isomer" includes, but is not limited to, optical isomers and analogs, structural isomers and analogs, conformational isomers and analogs, and the like. In one embodiment, this invention encompasses of various optical isomers of the compounds of the present invention. It will be appreciated by those skilled in the art that the compounds of the present invention contain at least one chiral center. Accordingly, these compounds exist in, and can be isolated in, optically-active or racemic forms. Some compounds may also exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically active, polymorphic, or stereroisomeric form, or mixtures thereof. In one embodiment, the compounds are the pure (R)-isomers. In another embodiment, the compounds are the pure (S)-isomers. In another embodiment, the compounds are a mixture of the (R) and the (S) isomers. In another embodiment, the compounds are a racemic mixture comprising an equal amount of the (R) and the (S) isomers. It is well known in the art how to prepare optically-active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase). This invention further includes solvates of the compounds described herein.

"Solvate" means a physical association of a compound of the invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances the solvate will be capable of isolation. "Solvate" encompasses both solution-phase and isolatable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates and the like. "Hydrate" is a solvate wherein the solvent molecule is water. The term "hydrate" includes but is not limited to hemihydrate, monohydrate, dihydrate, trihydrate and the like.

The term "polymorph" refers to a particular crystalline state of a substance, which can be characterized by particular physical properties such as X-ray diffraction, IR spectra, melting point, and the like.

The invention includes pharmaceutically acceptable salts of the compounds of the present invention. The term "pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N- ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

When the compound of the present invention is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like. Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.

It is to be understood that, as used herein, references to the compounds according to formula (I) of the present invention are meant to also include the pharmaceutically acceptable salts thereof. It is also to be understood that any reference to the compounds according to formula (I) also include reference to the compounds according to any of formulas (II), (III), and (IV), whose structure is generically covered by the structure of formula (I).

As used through this specification and the appended claims, the singular forms "a", "an" and "the" include the plural unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes mixtures of such compounds, reference to "the composition" or "the method" includes one or more compositions, methods and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure.

Therapeutic Use

The compounds of the invention are useful among other indications in treating conditions associated with inflammation. Thus, the compounds of formula (I) or their pharmaceutically acceptable salts are used in the manufacture of a medicament for prophylactic or therapeutic treatment of mammals, including humans, in respect of conditions characterized by excessive production of cytokines and/or inappropriate or unregulated cytokine activity on such cells as cardiomyocytes, cardiofibroblasts and macrophages.

The compounds of the invention inhibit the production of cytokines such as TNF, IL-I, IL-6 and IL-8, cytokines that are important proinflammatory constituents in many different disease states and syndromes. Thus, inhibition of these cytokines has benefit in controlling and mitigating many diseases. The compounds of the invention are shown herein to inhibit a member of the MAP kinase family variously called p38 MAPK (or p38), CSBP, or SAPK-2. The activation of this protein has been shown to accompany exacerbation of the diseases in response to stress caused, for example, by treatment with lipopolysaccharides or cytokines such as TNF and IL-I. Inhibition of p38 activity, therefore, is predictive of the ability of a medicament to provide a beneficial effect in treating diseases such as Alzheimer's, coronary artery disease, congestive heart failure, cardiomyopathy, myocarditis, vasculitis, restenosis, such as occurs following coronary angioplasty, atherosclerosis, IBD, rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions, multiple sclerosis, acute respiratory distress syndrome (ARDS), asthma, chronic obstructive pulmonary disease (COPD), silicosis, pulmonary sarcosis, sepsis, septic shock, endotoxic shock, Gram-negative sepsis, toxic shock syndrome, heart and brain failure (stroke) that are characterized by ischemia and reperfusion injury, surgical procedures, such as transplantation procedures and graft rejections, cardiopulmonary bypass, coronary artery bypass graft, CNS injuries, including open and closed head trauma, inflammatory eye conditions such as conjunctivitis and uveitis, acute renal failure, glomerulonephritis, inflammatory bowel diseases, such as Crohn's disease or ulcerative colitis, graft vs. host disease, bone resorption diseases like osteoporosis, type II diabetes, pyresis, psoriasis, cachexia, viral diseases such as those caused by HIV, CMV, and Herpes, and cerebral malaria. In one embodiment, the present invention provides a method for inhibiting the activity of a p38-α kinase in a subject, comprising the step of administering to the subject a compound represented by the structure of any of formulas (I)-(IV) or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof, in an amount effective to inhibit the activity of the p38-α kinase. In another embodiment, the present invention provides a method of treating or preventing a disease or condition mediated by p38-α kinase or associated with p38-α kinase activity, comprising the step of administering to a subject in need thereof a compound represented by the structure of any of formulas (I)-(IV) or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof, in an amount effective to treat or prevent the disease or condition.

In one embodiment, the p38-α kinase mediated disease or condition is characterized by a proinflammation response. The disease or condition characterized by inflammation can be, for example, acute respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, uveitis, acute renal failure, head trauma, ischemic/reperfusion injury, multiple sclerosis, IBD, rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis, other arthritic conditions, sepsis, septic shock, endotoxic shock, Gram-negative sepsis, toxic shock syndrome, adult respiratory distress syndrome, stroke, reperfusion injury, CNS injury, psoriasis, restenosis, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcosis, a bone resorption disease, graft- versus-host reaction, Crohn's Disease, ulcerative colitis, or pyresis.

In one embodiment, the p38-α kinase mediated disease or condition is a heart condition associated with cardiac failure. The heart condition, for example, can be congestive heart failure, cardiomyopathy or myocarditis.

In another embodiment, the present invention provides a method of inhibiting the activity of a p38-α kinase comprising the step of contacting a cell comprising the p38- α kinase with a compound represented by the structure of any of formulas (I)-(IV) or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof, in an amount effective to inhibit the activity of the p38-α kinase.

The term "treatment" or "treating" is intended to include the administration of the compound of the invention to a subject for purposes which may include prophylaxis, amelioration, prevention or cure of disorders mediated by p38. Such treatment need not necessarily completely ameliorate the inflammatory response or other responses related to the specific disorder. Further, such treatment may be used in conjunction with other traditional treatments for reducing the disease or disorder condition known to those of skill in the art.

The term "contacting" means that the compound of the present invention is introduced into a sample containing the enzyme in a test tube, flask, tissue culture, chip, array, plate, microplate, capillary, or the like, and incubated at a temperature and time sufficient to permit binding of the compound to the enzyme. Methods for contacting the samples with the compounds of the invention or other specific binding components are known to those skilled in the art and may be selected depending on the type of assay protocol to be run. Incubation methods are also standard and are known to those skilled in the art.

The methods of the invention may be provided as a "preventive" treatment before detection of, for example, an inflammatory state, so as to prevent the disorder from developing in patients at high risk for the same, such as, for example, transplant patients.

Pharmaceutical Formulations In another aspect, the present invention provides pharmaceutical composition comprising as an active ingredient a compound represented by the structure of any of formulas (I)-(IV) or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof; and a pharmaceutically acceptable carrier or diluent.

In another embodiment, the present invention provides a pharmaceutical composition for treating a condition characterized by enhanced p38-α kinase activity, comprising as an active ingredient a therapeutically effective amount of a compound represented by the structure of any of formulas (I)-(IV) or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof; and a pharmaceutically acceptable diluent or carrier. The manner of administration and formulation of the compounds useful in the invention and their related compounds will depend on the nature of the condition, the severity of the condition, the particular subject to be treated, and the judgment of the practitioner. The nature of the formulation will also depend on mode of administration. The pharmaceutical compositions of the present invention can be formulated for administration by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, topical and intranasal. Such compositions are prepared in a manner well known in the pharmaceutical art and comprise as an active ingredient at least one compound of the present invention and pharmaceutically acceptable salts and hydrates thereof as described hereinabove, further comprising an excipient or a carrier. During the preparation of the pharmaceutical compositions according to the present invention the active ingredient is usually mixed with an excipient, diluted by an excipient or enclosed within such a carrier which can be in the form of a capsule, sachet, paper or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

In preparing a formulation, it may be necessary to mill the active ingredient to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the active ingredient is substantially water soluble, the particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. The formulations can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxybenzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.

The compositions are preferably formulated in a unit dosage form. The term "unit dosage forms" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

The active ingredient is effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. It will be understood, however, that the amount of the compound actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the active ingredient of the present invention.

The tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer, which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate. The liquid forms in which the compositions of the present invention may be incorporated, for administration orally or by injection, include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Compositions for inhalation or insulation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. Preferably the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device may be attached to a face masks tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices that deliver the formulation in an appropriate manner.

Another preferred formulation employed in the methods of the present invention employs transdermal delivery devices ("patches"). Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, e.g., U.S. Pat. No. 5,023,252 incorporated herein by reference. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents. The compounds may also be administered topically, for topical conditions such as psoriasis, or in formulation intended to penetrate the skin. These include lotions, creams, ointments and the like which can be formulated by known methods.

Alternative formulations include nasal sprays, liposomal formulations, slow- release formulations, controlled-release formulations and the like, as are known in the art.

The dosages of the compounds of the invention will depend on a number of factors which will vary from patient to patient. However, it is believed that generally, the daily oral dosage will utilize 0.001-100 mg/kg total body weight, preferably from 0.01-50 mg/kg and more preferably about 0.01 mg/kg-10 mg/kg. The dose regimen will vary, however, depending on the conditions being treated and the judgment of the practitioner.

It should be noted that the compounds of formula (1) can be administered as individual active ingredients, or as mixtures of several embodiments of this formula. In addition, the inhibitors of p38 kinase can be used as single therapeutic agents or in combination with other therapeutic agents. Drugs that could be usefully combined with these compounds include natural or synthetic corticosteroids, particularly prednisone and its derivatives, monoclonal antibodies targeting cells of the immune system, antibodies or soluble receptors or receptor fusion proteins targeting immune or non¬ immune cytokines, and small molecule inhibitors of cell division, protein synthesis, or niRNA transcription or translation, or inhibitors of immune cell differentiation or activation.

Although the compounds of the invention may be used in humans, they are also available for veterinary use in treating animal subjects. The following examples are presented in order to more folly illustrate certain embodiments of the invention. They should in no way, however, be construed as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES

Example 1; Effect of Compound (IV) on Yeast Growth Yeast cells harboring active p38 show growth inhibition. These cells can be prepared in accordance with any method known to a person of skill in the art, for example the method set forth in WO 02/081746, the contents of which are incorporated in their entirety herein. Thus, a compound that inhibits the activity of the p38 pathway, can rescue the growth of yeast cells harboring active p38. Compound (IV) of the present invention, and Compound (V) (a reference compound), were evaluated for their ability to rescue the growth of yeast cells harboring active p38. Compound TIV):

Compound (V);

(V)

Materials and Methods:

Compounds (IV) and (V) were purchased from Chemical Diversity. Overnight cultures of 3 different transformants of yeast were grown in a yeast liquid growth medium at 30⁰C in a shaker. The 3 transformants are: 1. Saccharomyces cervisiae yeast cells which express an active variant of human p38 from a yeast expression vector as described in WO 02/081746. p38 activity interferes with the normal growth of the yeast. Cells expressing p38 grow in a slower rate and do not reach the final density as normal cells. 2. Saccharomyces cervisiae yeast cells that contain the same vector from which the p38 is expressed, but without the p38. This strain grows normally.

3. Saccharomyces cervisiae yeast cells that express active p38 and an active phosphatase (called PTCl) that dephosphorylates p38, thus rendering it inactive. Here too the cells grow in a normal rate. Optical density at 600nm (O.D.₆oo) of the overnight cultures was measured and cultures were all diluted to the same O.D.₆oo.

Compounds (IV) and (V) dissolved in DMSO were added to individual wells in a 96-well plate. To 6 control wells DMSO was added at the same final concentration as in the wells that contained the compounds. In addition, PD169316 and SB 203580, two known p38 inhibitors, were also added to a well and used as controls. PD 169316 and SB 203580 are commonly used in laboratory researches of p38. PD169316 and SB 203580 were also dissolved in DMSO.

A fixed volume of yeast, that express p38, at the starting O.D. was added to the wells with compounds (IV), (V), PD 169316 and SB 203580. To the control wells, containing DMSO, the 3 different stains were added at the same starting O.D.₆₀o as in the wells with compounds (IV) and (V). Each strain was added to 2 different control wells (duplicates of each control).

The content of the wells in the 96-well plate was mixed prior to the O.D.₆₀₀ measurement. The baseline absorbance at time 0 was recorded by the spectrophotometer . Cells were incubated at 30⁰C and O.D.₆oo was measured after about 24 and 48 hours.

Results

Figure 1 depicts the growth curve of yeast cells harboring active p38 in the presence of various concentrations (lμm, lOμm and 50 μm) of Compound (IV) of the present invention. Figure 2 depicts the growth curve of yeast cells harboring active p38 in the presence of various concentrations (lμm, lOμm and 100 μm) of the reference Compound (V). The Figures show the optical density of yeast as a function of time.

As can be clearly seen, Compound (IV) is more potent at rescuing the growth of yeast cells, as compared with the reference Compound (V), and seems to be less toxic to the cells. For example, Compound (V) is toxic at the high concentration

(lOOμM) inhibiting completely growth of the culture, while Compound (IV) shows an almost normal growth at 50 μm concentration. In other experiments Compound IV was used at concentrations up to 200 micromolar and manifested no toxicity.

PD169316 and SB 203580 were used in 50μM concentration, however only very minor growth rescue was observed. In other experiments concentrations were increased up to ImM but a strong growth rescue was also not observed.

Example 2: Effect of Compound (IV) on p38 activity

The effects of Compound (IV) and the reference compound (V) on p38 activity were measured directly in a p38 kinase assay.

Materials and Methods:

ATF2 is a known substrate of p38 and was used in this assay to evaluate the level of p38 activity in the presence of 0.25, 0.5, 1 and 10 μm of Compounds (IV) and (V).

Two controls were used. A) the solvent in which the compounds were dissolved (DMSO); and B) PD169316 at a 0.25μM concentration. All reactions were conducted in 1.5ml test tubes. 5 μl of p38 enzyme (0.2μg of purified recombinant hexahistidine tag-p38) and p38 inhibitors were added to the test tubes prior to the reaction. H₂O was added to the test tube that did not contain any inhibitor (lane 0). In order to initialize the reaction, 45 μl of reaction mixture were added to of the test tube.. Final reaction conditions were: 10OnM kinase, 25mM Hepes pH 7.5, 2OmM MgCl₂, 2OmM 2- glycerolphosphate, O.lmM Na₃VO₄, ImM DTT, 40μg substrate, 50μM ATP, lOμCi γ- ³²P-ATP. The kinase reactions were stopped after 10 minutes by placing the tubes on ice, applying Laemlli sample buffer and boiling at 100⁰C for 5 min. In order to activate p38 recombinant activ MKK6 (Upstate biotechnology) was used. Reaction conditions were similar to those recommended by the manufacturer except for using non radioactive ATP. Protein levels were monitored by staining the kinase assay gel with commassie blue.

Results Figure 3 shows the results of the p38 kinase assay in the presence of 0.25, 0.5,

1 and 10 μm of Compounds (IV) and (V). The top panel is a commassie blue stain to monitor protein levels, and the bottom panel is measure of radioactivity incorporated into ATF2 protein, a p38 substrate.

As can be clearly seen, Compound (IV) shows inhibition of activity of p38 at lower concentrations than Compound (V), and accordingly is a more potent inhibitor of p38 activity as compared with the reference compound (V). In the commassie blue stained gel, the upper most band (pointed to with an arrow and labeled with ATFII*), is the phosphorylated ATF2 band.

PD 169316, which is a widely used p38 inhibitor, did not rescue yeast growth at concentrations up to 50μM, but showed a strong inhibition of p38 in the in vitro kinase assay. PD169316 did not pass preclinical tests.

Example 3: Compounds (IV) and (V) inhibit p38α in vitro with high specificity and high efficiency. Compounds (IV) and (V) were next tested for their ability to inhibit p38α activity in vitro. Recombinant, purified p38-α was activated by MKK6 and was tested in a kinase assay using GST- ATF2 as a substrate. When Compounds (IV) or (V) were added to the kinase assay, either compound inhibited p38-α most efficiently (Fig. 3B). Compound (IV) was significantly a more potent p38-α inhibitor (IC₅₀ is 32.2nM) than Compound (V) (IC₅₀ is 48OnM). Notably, the IC₅₀ of PD169316 is 14.8nM (Fig.3B). Thus Compound (IV) is more potent and less toxic (in yeast) compared to Compound (V).

Example 4: Compounds (IV) is a specific inhibitor of p38-α in vitro In order to test the specificity of the inhibition of Compound (IV) towards p38- α for which it was targeted in the yeast screen, p38-γ was activated by MKK6 and a kinase assay was performed in the presence of Compound (IV). PD 169316, which is considered a very specific inhibitor of p38-α, was used as a control. As shown in Fig. 4, PD 169316 manifested some inhibitory activity over p38-γ, while Compound (IV) did not inhibit p38-γ activity even at lOμM concentration.

Example 5 -Compound (IV) inhibits p38α activity in mammalian cells in vivo and blocks differentiation of myoblasts

To assess if Compound (IV) might be active in mammalian cells their effect on differentiation of myoblasts to myotubes in culture was tested. Differentiation of myoblasts in culture was shown to be dependent on p38-α activity (Cuenda and Cohen, 1999; J Biol. Chem. 274, 4341-4346; Zetser et al., 1999 J Biol. Chem. 274, 5193- 5200. In myoblast cells p38α promotes the transcriptional activities of MyoD and Mef2c (Zetser et al., 1999; Wu et al., 2000 . MoI Cell. Biol 20, 3951-3964). It was already shown that SB203580 inhibits myogenic differentiation in several tissue culture models (Cuenda and Cohen, 1999; Zetser et al., 1999; Wu et al., 2000; Puri et al., 2000 Genes & Dev. 14, 574-584). The compounds of the present invention were used to treat L8 myoblasts. Differentiation medium added to these cells was supplemented with 10 μM of the different inhibitors and myoblasts were allowed to differentiate for 60 hours. Differentiation of L8 cells was assessed by inspecting appearance of myotubes under the microscope (not shown) and by monitoring expression levels of Myogenin (Fig. 5A). Compound (IV) inhibited the differentiation with similar efficiency to that of SB203580 and PD109316 (Fig. 5A), while not showing any toxicity. Compound (IV) also prevented the phosphorylation of EM, a known substrate of p38 (not shown).

In order to further evaluate the toxicity Compound (IV), cells were grown in the presence of the compounds for 36 hours, followed by removal of compound and additional 36 hours growth in their absence (Fig. 5B). The effect of Compound (IV) was reversible, as the cells completely recovered and differentiated following the removal of this compound. The data shows that Compound (IV) inhibited p38 in myoblasts at least as efficiently as known compounds such as SB203580 and PD 109316.

It will be appreciated by a person skilled in the art that the present invention is not limited by what has been particularly shown and described hereinabove. Rather, the scope of the invention is defined by the claims which follow.

Claims

A pharmaceutical composition for treating a condition characterized by enhanced p38-α kinase activity, comprising as an active ingredient a therapeutically effective amount of a compound represented by the structure of formula (I):

(I) wherein

R¹ is independently selected from H, C₁-C₆ alkyl, C₁-C₆ alkylaryl and aryl;

R² is independently selected from H, C₁-C₆ alkyl, C₁-C₆ alkyloxy, C₁-C₆ alkylaryl, aryl and aryloxy; n is selected from 0, 1, 2, 3 and 4; m is selected from 1, 2, 3, 4 and 5; wherein the sum of n and m cannot be greater than 5;

A¹ is CO or an isostere thereof;

A² is CH₂ or an isostere thereof; or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof; and a pharmaceutically acceptable diluent or carrier. The pharmaceutical composition according to claim 1, wherein R¹ is methyl.

3. The pharmaceutical composition according to claim 2, wherein m is 1. 4. The pharmaceutical composition according to claim 1, wherein R is methoxy.

5. The pharmaceutical composition according to claim 4, wherein n is 1. 6. The pharmaceutical composition according to claim 1, wherein R¹ is methyl, m is 1, R is methoxy and n is 1.

The pharmaceutical composition according to claim 1, wherein m is 1 and SR¹ is in the para position. 8. The pharmaceutical composition according to claim 1, wherein n is 1 and R² is in the ortho position.

9. The pharmaceutical composition according to claim 1, wherein the compound is represented by the structure of formula (II):

(H)

10. The pharmaceutical composition according to claim 1, wherein the compound is represented by the structure of formula (III):

(III)

11. The pharmaceutical composition according to claim 1 , wherein the compound is represented by the structure of formula (IV):

12. The pharmaceutical composition according to claim 1, further comprising an additional therapeutic agent.

13. The pharmaceutical composition according to claim 12, wherein said additional therapeutic agent is a corticosteroid, a monoclonal antibody, or an inhibitor of cell division.

14. An in-vitro or ex-vivo method of inhibiting the activity of a p38-α kinase comprising the step of contacting a ρ38-α kinase with a compound represented by the structure of formula (I), in an amount effective to inhibit the activity of said p38-α kinase

(I) wherein

R is independently selected from H, C₁-C₆ alkyl, C₁-C₆ alkyloxy, C₁-C₆ alkylaryl, aryl and aryloxy; n is selected from 0, 1, 2, 3 and 4; m is selected from 1, 2, 3, 4 and 5; wherein the sum of n and m cannot be greater than 5; A¹ is CO or an isostere thereof; and A² is CH₂ or an isostere thereof; or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof.

15. The method according to claim 14, wherein R¹ is methyl.

16. The method according to claim 15, wherein m is 1.

17. The method according to claim 14, wherein R² is methoxy.

18. The method according to claim 17, wherein n is 1.

19. The method according to claim 14, wherein R¹ is methyl, m is 1, R² is methoxy and n is 1.

20. The method according to claim 14, wherein m is 1 and SR¹ is in the para position.

21. The method according to claim 14, wherein n is 1 and R² is in the ortho position.

22. The method according to claim 14, wherein the compound is represented by the structure of formula (II):

(H) 23. The method according to claim 14, wherein the compound is represented by the structure of formula (III):

(HI)

24. The method according to claim 14, wherein the compound is represented by the structure of formula (IV):

25. A method for inhibiting the activity of a p38-α kinase in a subject, comprising the step of administering to said subject a compound represented by the structure of formula (I), in an amount effective to inhibit the activity of said p38-α kinase

(I) wherein

R¹ is independently selected from H, C₁-C₆ alkyl, Ci-C₆ alkylaryl and aryl;

R² is independently selected from H₅ C₁-C₆ alkyl, Ci-C₆ alkyloxy, C₁-C₆ alkylaryl, aryl and aryloxy; n is selected from 0, 1, 2, 3 and 4; m is selected from 1, 2, 3, 4 and 5; wherein the sum of n and m cannot be greater than 5; A¹ is CO or an isostere thereof; and A² is CH₂ or an isostere thereof; or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof.

26. The method according to claim 25, wherein R¹ is methyl.

27. The method according to claim 26, wherein m is 1.

28. The method according to claim 25, wherein R² is OCH₃.

29. The method according to claim 28, wherein n is 1.

30. The method according to claim 25, wherein R¹ is methyl, m is 1, R² is OCH₃ and n is 1.

31. The method according to claim 25, wherein m is 1 SR¹ is in the para position.

32. The method according to claim 25 wherein n is 1 and R² is in the ortho position.

33. The method according to claim 25, wherein the compound is represented by the structure of formula (II):

(H)

34. The method according to claim 25, wherein the compound is represented by the structure of formula (III):

(III)

35. The method according to claim 25, wherein the compound is represented by the structure of formula (IV):

36. The method according to claim 25, comprising administering a pharmaceutical composition comprising as an active ingredient the compound of formula (I); and a pharmaceutically acceptable diluent or carrier.

37. The method according to claim 25, further comprising administering an additional therapeutic agent.

38. The method according to claim 37, wherein said additional therapeutic agent is a corticosteroid, a monoclonal antibody, or an inhibitor of cell division.

39. A method of treating or preventing a disease or condition mediated by p38-α kinase or associated with p38-α kinase activity, comprising the step of administering to a subject in need thereof a compound represented by the structure of formula (I), in an amount effective to treat or prevent said disease or condition

(I) wherein

A¹ is CO or an isostere thereof; and A² is CH₂ or an isostere thereof; or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof.

40. The method according to claim 39, wherein R¹ is methyl.

41. The method according to claim 40, wherein m is 1.

42. The method according to claim 39, wherein R² is OCH₃.

43. The method according to claim 42, wherein n is 1.

44. The method according to claim 39, wherein R¹ is methyl, m is 1, R² is OCH₃ and n is 1.

45. The method according to claim 39, wherein m is 1 and SR¹ is in the para position.

46. The method according to claim 39, wherein n is 1 and R² is in the ortho position.

47. The method according to claim 39, wherein the compound is represented by the structure of formula (II):

(H)

48. The method according to claim 39, wherein the compound is represented by the structure of formula (III):

(HI)

49. The method according to claim 39, wherein the compound is represented by the structure of formula (IV):

50. The method according to claim 39, comprising administering a pharmaceutical composition comprising as an active ingredient the compound of formula (I); and a pharmaceutically acceptable diluent or carrier.

51. The method according to claim 39, wherein said disease or condition is characterized by a proinflammation response.

52. The method according to claim 51, wherein said disease or condition characterized by inflammation is selected from the group consisting of acute respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, uveitis, acute renal failure, head trauma, ischemic/reperfusion injury, multiple sclerosis, IBD, rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis, other arthritic conditions, sepsis, septic shock, endotoxic shock, Gram- negative sepsis, toxic shock syndrome, adult respiratory distress syndrome, stroke, reperfusion injury, CNS injury, psoriasis, restenosis, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcosis, a bone resorption disease, graft-versus-host reaction, Crohn's Disease, ulcerative colitis, and pyresis.

53. The method according to claim 39, wherein said disease or condition is a heart condition associated with cardiac failure.

54. The method according to claim 53, wherein said heart condition is congestive heart failure, cardiomyopathy or myocarditis. 55. The method according to claim 39, further comprising administering an additional therapeutic agent.

56. The method according to claim 55, wherein said additional therapeutic agent is a corticosteroid, a monoclonal antibody, or an inhibitor of cell division. 57. A pharmaceutical composition for treating a condition characterized by enhanced p38-α kinase activity comprising as an active ingredient a therapeutically effective amount of a compound represented by the structure of formula (IV)

or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof; and a pharmaceutically acceptable diluent or carrier.

58. The pharmaceutical composition according to claim 57, further comprising an additional therapeutic agent.

59. The pharmaceutical composition according to claim 58, wherein said additional therapeutic agent is a corticosteroid, a monoclonal antibody, or an inhibitor of cell division.

60. An in-vitro method of inhibiting the activity of a p38-α kinase comprising the step of comprising the step of contacting a p38-α kinase with compound represented by the structure of formula (IV), in an amount effective to inhibit the activity of said p38-α kinase

or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof.

61. A method for inhibiting the activity of a p38-α kinase in a subject, comprising the step of administering to said subject a compound represented by the structure of formula (IV), in an amount effective to inhibit the activity of said p38-α kinase

or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof.

62. The method according to claim 61, comprising administering a pharmaceutical composition comprising as an active ingredient a compound of formula (IV); and a pharmaceutically acceptable diluent or carrier.

63. The method according to claim 61, further comprising administering an additional therapeutic agent.

64. The method according to claim 63, wherein said additional therapeutic agent is a corticosteroid, a monoclonal antibody, or an inhibitor of cell division.

65. A method of treating or preventing a disease or condition mediated by p38-α kinase or associated with p38-α kinase activity, comprising the step of administering to a subject in need thereof a compound of represented by the structure of formula (IV), in an amount effective to treat or prevent said disease or condition

or a pharmaceutically acceptable salt, polymorph, isomer or solvate thereof.

66. The method according to claim 65, comprising administering a pharmaceutical composition comprising as an active ingredient a compound of formula (IV); and a pharmaceutically acceptable diluent or carrier.

67. The method according to claim 65, wherein said disease or condition is characterized by a proinflammation response. 68. The method according to claim 67, wherein said disease or condition characterized by inflammation is selected from the group consisting of acute respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, uveitis, acute renal failure, head trauma, ischemic/reperfusion injury, multiple sclerosis, IBD, rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis, other arthritic conditions, sepsis, septic shock, endotoxic shock, Gram- negative sepsis, toxic shock syndrome, adult respiratory distress syndrome, stroke, reperfusion injury, CNS injury, psoriasis, restenosis, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcosis, a bone resorption disease, graft- versus-host reaction, Crohn's Disease, ulcerative colitis, and pyresis.

69. The method according to claim 65, wherein said disease or condition is a heart condition associated with cardiac failure. 70. The method according to claim 69, wherein said heart condition is congestive heart failure, cardiomyopathy or myocarditis.

71. The method according to claim 65, further comprising administering an additional therapeutic agent.

72. The method according to claim 71, wherein said additional therapeutic agent is a corticosteroid, a monoclonal antibody, or an inhibitor of cell division.