CN101360750A

CN101360750A - Method of modulating stress-activated protein kinase system

Info

Publication number: CN101360750A
Application number: CNA2006800514899A
Authority: CN
Inventors: S·D·塞维尔特; K·科森; V·谢列布里亚内
Original assignee: Intermune Inc
Current assignee: Intermune Inc
Priority date: 2005-11-23
Filing date: 2006-11-22
Publication date: 2009-02-04

Abstract

It has now been discovered that a high therapeutic effect in treating various disorders associated with enhanced activity of kinase p38 may be achieved by using a potent p38 gamma kinase inhibitor compound which also has inhibitory activity against p38gamma. Furthermore, reducing the activities of both kinase p38 gamma and kinase p38 gamma without reducing the activity of a kinase p38 gamma to such an extent that undesired side effects are observed upon administration to a subject having a disorder associated with enhanced activity of kinase p38 has been discovered to be achievable by modifying inhibitors of p38 gamma such that the modification engenders inhibitory activity against p38 gamma. Described are compounds with activity against p38 gamma and p38 gamma. Disclosed are methods of using described compounds and compositions to modulate a stress activated protein kinase (SAPK) system with an active compound, wherein the active compound exhibits inhibition of the p38 gamma and p38 gamma MAPKs. Also disclosed are methods for identifying compounds which inhibit p38 gamma and p38 gamma MAPKs and which can modulate a stress activated protein kinase (SAPK) system.

Description

The control method of stress activated protein kinase system

Invention field

[0001] the present invention relates to can be used for treating the Compounds and methods for of multiple fibrotic disease, comprise the disease that those are relevant with the kinase p 38 increased activity.

Background of invention

[0002] has realized that a large amount of chronic and acute illnesss is relevant with the perturbation of inflammatory reaction.It is generally acknowledged that a large amount of cytokines participate in this reaction, comprise IL-1, IL-6, IL-8 and TNF α.The activity that seems these cytokines adjusting inflammation may be relevant with the activation of certain enzyme pair cell signal transduction pathway, and this enzyme is the map kinase family member, generally is called p38, also is called SAPK, CSBP and RK.

[0003] to several p38 inhibitor, for example NPC 31169, SB239063, SB203580, FR-167653 and pirfenidone have carried out external and/or the body build-in test, find its effectively adjusting inflammatory reaction.

[0004] still need safe and effective medicine to treat various inflammation, for example struvite pulmonary fibrosis.

Summary of the invention

[0005] in one embodiment, provide the method for a kind of adjusting stress activated protein kinase (SAPK) system, this method comprises makes compound contact with p38 mitogen-activated protein kinase (MAPK),

The IC of the inhibition p38 γ that wherein said compound shows ₅₀At about 10pM to the scope of about 500 μ M;

The IC of the inhibition p38 α MAPK that wherein said compound shows ₅₀At least be the IC that suppresses p38 γ MAPK ₅₀2 times, 5 times or 10 times.

In another embodiment, provide the method for a kind of adjusting stress activated protein kinase (SAPK) system, this method comprises makes compound contact with p38 mitogen-activated protein kinase (MAPK),

The IC of the inhibition p38 γ MAPK that wherein said compound shows ₅₀Value at about 10pM to the scope of about 500 μ M;

The IC of the inhibition p38 α MAPK that wherein said compound shows ₅₀Value at about 10pM to the scope of about 1500 μ M.

[0006] in another embodiment, provide the method for treatment or prevention experimenter's morbid state, comprise

Discriminating is among the risk of fibrotic disease or suffers from the experimenter of fibrotic disease;

Give the experimenter with compound with the treatment or the significant quantity of prevention of fibrotic diseases;

[0007] in another embodiment, provide the method for identification of pharmaceutical active compounds, comprising:

Library of compounds is provided;

Measure a plurality of compounds in this library to the inhibition of p38 α MAPK;

Measure a plurality of compounds in this library to the inhibition of p38 γ MAPK;

From these a plurality of compounds, select at least one compound, the IC of the inhibition p38 γ MAPK that wherein said at least one compound shows ₅₀At about 10pM to the scope of about 500 μ M; And

The IC of the inhibition p38 α MAPK that wherein said at least one compound shows ₅₀At least be the IC that suppresses p38 γ MAPK ₅₀2 times, 5 times or 10 times.

[0008] in another embodiment, provide the method for identification of pharmaceutical active compounds, comprising:

Library of compounds is provided;

From these a plurality of compounds, select at least one compound, the IC of the inhibition p38 γ MAPK that wherein said at least one compound shows ₅₀At about 10pM to the scope of about 500 μ M;

[0009] in another embodiment, provide the method for identification of pharmaceutical active compounds, comprising:

Library of compounds is provided;

From these a plurality of compounds, select at least one compound, the IC of the inhibition p38 α MAPK that wherein said at least one compound shows ₅₀At about 10pM to the scope of about 1500 μ M;

[0010] in one embodiment, provide the method for regulating stress activated protein kinase (SAPK) system, this method comprises makes compound contact with p38 mitogen-activated protein kinase (MAPK),

The IC of the inhibition p38 γ that wherein said compound shows ₅₀At about 100 μ M to the scope of about 1000 μ M;

The IC of the inhibition p38 α MAPK that wherein said compound shows ₅₀Be the IC that suppresses p38 γ MAPK ₅₀At least 2 times.

[0011] in another embodiment, provide the method for treatment or prevention experimenter's morbid state, comprise

The IC of the inhibition p38 γ that wherein said compound shows ₅₀At about 100 μ M to the scope of about 1000 μ M; And

The IC of the inhibition p38 α MAPK that wherein said compound shows ₅₀Be the IC that suppresses p38 γ MAPK ₅₀At least 10 times.

[0012] in another embodiment, provide the method for identification of pharmaceutical active compounds, comprising:

Library of compounds is provided;

Measure a plurality of compounds in this library to the inhibition of p38 γ MAPK; With

From these a plurality of compounds, select at least one compound, the IC of the inhibition p38 γ MAPK that wherein said at least one compound shows ₅₀At about 10pM to the scope of about 5 μ M; And

The IC of the inhibition p38 α MAPK that wherein said at least one compound shows ₅₀Be the IC that suppresses p38 γ MAPK ₅₀At least 10 times.

[0013] in another embodiment, provide the method for identification of pharmaceutical active compounds, comprising:

Library of compounds is provided;

[0014] in another embodiment, provide the method for identification of pharmaceutical active compounds, comprising:

Library of compounds is provided;

Measure a plurality of compounds in this library to the inhibition of p38 α MAPK; With

[0015] in some above embodiment, the IC of the inhibition p38 γ MAPK that described compound shows ₅₀At about 10pM to the scope of about 5 μ M.In other embodiments, the IC of the inhibition p38 γ MAPK that shows of described compound ₅₀At about 100nM to the scope of about 500 μ M.

[0016] in some above embodiment, described compound is not selected from:

And pirfenidone.

[0017] in another embodiment, provide and have compound in structural formula I:

[0018] wherein

[0019] R is selected from H, halogen, cyano group, nitro, hydroxyl, the optional C that replaces _1-6Alkyl, the optional C that replaces _3-7Cycloalkyl, the optional C that replaces _4-10Alkyl-cycloalkyl, the optional C that replaces _2-6Thiazolinyl, the optional C that replaces _1-6Alkoxyl group, the optional C that replaces _{6 or 10}Aryl, the optional pyridyl that replaces, the optional pyrimidyl that replaces, the optional thienyl that replaces, the optional furyl that replaces, the optional thiazolyl that replaces, the optional De oxazolyl that replaces, the optional phenoxy group that replaces, the optional sulfo-phenoxy group that replaces, the optional amino-sulfonyl (optionallysubstituted sulphonamido) that replaces, the optional urea groups that replaces, the optional thioureido that replaces, the optional amide group (optionally substituted amido) that replaces, the optional ketone group that replaces, the optional carboxyl that replaces, the optional formamyl that replaces, the optional thioether that replaces, the optional sulfoxide that replaces, the optional sulfone that replaces, the optional amino that replaces, the optional alkoxy amino that replaces, the optional alkoxyl group heterocyclic radical that replaces, the optional alkylamino that replaces, the optional alkyl carboxyl that replaces, the optional carbonyl that replaces, the optional volution cycloalkyl that replaces, the optional pyrazinyl that replaces, the optional pyridazinyl that replaces, the optional pyrryl that replaces, the optional thienyl that replaces, the optional thiazolyl that replaces, the optional De oxazolyl that replaces, the optional imidazolyl that replaces, the optional De isoxazolyl that replaces, the optional pyrazolyl that replaces, the optional isothiazolyl that replaces, the optional naphthyl that replaces, the optional quinolyl that replaces, the optional isoquinolyl that replaces, the optional quinoxalinyl that replaces, the optional benzothiazolyl that replaces, the optional benzothienyl that replaces, the optional benzofuryl that replaces, optional indyl that replaces and the optional benzimidazolyl-that replaces;

[0020] or the pharmacy acceptable salt of described compound, ester, solvate or prodrug.

[0021] in one embodiment, the IC of the inhibition p38 γ that shows of formula I compound ₅₀At about 10pM to the scope of about 5 μ M.

[0022] another embodiment provides the compound with structural formula II:

[0023] wherein

[0024] R ⁴Be selected from H, halogen, cyano group, nitro, hydroxyl, the optional C that replaces _1-6Alkyl, the optional C that replaces _3-7Cycloalkyl, the optional C that replaces _4-10Alkyl-cycloalkyl, the optional C that replaces _2-6Thiazolinyl, the optional C that replaces _1-6Alkoxyl group, the optional C that replaces _{6 or 10}Aryl, the optional pyridyl that replaces, the optional pyrimidyl that replaces, the optional thienyl that replaces, the optional furyl that replaces, the optional thiazolyl that replaces, the optional De oxazolyl that replaces, the optional phenoxy group that replaces, the optional sulfo-phenoxy group that replaces, the optional amino-sulfonyl that replaces, the optional urea groups that replaces, the optional thioureido that replaces, the optional amide group that replaces, the optional ketone group that replaces, the optional carboxyl that replaces, the optional formamyl that replaces, the optional thioether that replaces, the optional sulfoxide that replaces, the optional sulfone that replaces, the optional amino that replaces, the optional alkoxy amino that replaces, the optional alkoxyl group heterocyclic radical that replaces, the optional alkylamino that replaces, the optional alkyl carboxyl that replaces, the optional carbonyl that replaces, the optional volution cycloalkyl that replaces, the optional pyrazinyl that replaces, the optional pyridazinyl that replaces, the optional pyrryl that replaces, the optional thienyl that replaces, the optional thiazolyl that replaces, the optional De oxazolyl that replaces, the optional imidazolyl that replaces, the optional De isoxazolyl that replaces, the optional pyrazolyl that replaces, the optional isothiazolyl that replaces, the optional naphthyl that replaces, the optional quinolyl that replaces, the optional isoquinolyl that replaces, the optional quinoxalinyl that replaces, the optional benzothiazolyl that replaces, the optional benzothienyl that replaces, the optional benzofuryl that replaces, optional indyl that replaces and the optional benzimidazolyl-that replaces;

[0025] or the pharmacy acceptable salt of described compound, ester, solvate or prodrug.

[0026] in one embodiment, the IC of the inhibition p38 γ that shows of formula II compound ₅₀At about 10pM to the scope of about 5 μ M.

[0027] in another embodiment, provide compound with structural formula II I:

[0028] wherein

[0029] R ²Be selected from H, halogen, cyano group, nitro, hydroxyl, the optional C that replaces _1-6Alkyl, the optional C that replaces _3-7Cycloalkyl, the optional C that replaces _4-10Alkyl-cycloalkyl, the optional C that replaces _2-6Thiazolinyl, the optional C that replaces _1-6Alkoxyl group, the optional C that replaces _{6 or 10}Aryl, the optional pyridyl that replaces, the optional pyrimidyl that replaces, the optional thienyl that replaces, the optional furyl that replaces, the optional thiazolyl that replaces, the optional De oxazolyl that replaces, the optional phenoxy group that replaces, the optional sulfo-phenoxy group that replaces, the optional amino-sulfonyl that replaces, the optional urea groups that replaces, the optional thioureido that replaces, the optional amide group that replaces, the optional ketone group that replaces, the optional carboxyl that replaces, the optional formamyl that replaces, the optional thioether that replaces, the optional sulfoxide that replaces, the optional sulfone that replaces, the optional amino that replaces, the optional alkoxy amino that replaces, the optional alkoxyl group heterocyclic radical that replaces, the optional alkylamino that replaces, the optional alkyl carboxyl that replaces, the optional carbonyl that replaces, the optional volution cycloalkyl that replaces, the optional pyrazinyl that replaces, the optional pyridazinyl that replaces, the optional pyrryl that replaces, the optional thienyl that replaces, the optional thiazolyl that replaces, the optional De oxazolyl that replaces, the optional imidazolyl that replaces, the optional De isoxazolyl that replaces, the optional pyrazolyl that replaces, the optional isothiazolyl that replaces, the optional naphthyl that replaces, the optional quinolyl that replaces, the optional isoquinolyl that replaces, the optional quinoxalinyl that replaces, the optional benzothiazolyl that replaces, the optional benzothienyl that replaces, the optional benzofuryl that replaces, optional indyl that replaces and the optional benzimidazolyl-that replaces; With

[0030] R ³Be selected from H, halogen, cyano group, nitro, hydroxyl, the optional C that replaces _1-6Alkyl, the optional C that replaces _3-7Cycloalkyl, the optional C that replaces _4-10Alkyl-cycloalkyl, the optional C that replaces _2-6Thiazolinyl, the optional C that replaces _1-6Alkoxyl group, the optional C that replaces _{6 or 10}Aryl, the optional pyridyl that replaces, the optional pyrimidyl that replaces, the optional thienyl that replaces, the optional furyl that replaces, the optional thiazolyl that replaces, the optional De oxazolyl that replaces, the optional phenoxy group that replaces, the optional sulfo-phenoxy group that replaces, the optional amino-sulfonyl that replaces, the optional urea groups that replaces, the optional thioureido that replaces, the optional amide group that replaces, the optional ketone group that replaces, the optional carboxyl that replaces, the optional formamyl that replaces, the optional thioether that replaces, the optional sulfoxide that replaces, the optional sulfone that replaces, the optional amino that replaces, the optional alkoxy amino that replaces, the optional alkoxyl group heterocyclic radical that replaces, the optional alkylamino that replaces, the optional alkyl carboxyl that replaces, the optional carbonyl that replaces, the optional volution cycloalkyl that replaces, the optional pyrazinyl that replaces, the optional pyridazinyl that replaces, the optional pyrryl that replaces, the optional thienyl that replaces, the optional thiazolyl that replaces, the optional De oxazolyl that replaces, the optional imidazolyl that replaces, the optional De isoxazolyl that replaces, the optional pyrazolyl that replaces, the optional isothiazolyl that replaces, the optional naphthyl that replaces, the optional quinolyl that replaces, the optional isoquinolyl that replaces, the optional quinoxalinyl that replaces, the optional benzothiazolyl that replaces, the optional benzothienyl that replaces, the optional benzofuryl that replaces, optional indyl that replaces and the optional benzimidazolyl-that replaces;

[0031] or the pharmacy acceptable salt of described compound, ester, solvate or prodrug.

[0032] in one embodiment, the IC of the inhibition p38 γ that shows of formula III compound ₅₀At about 10pM to the scope of about 5 μ M.

[0033] in one embodiment, the formula III compound is not 1-(the 5-tertiary butyl-2-p-methylphenyl-2H-pyrazole-3-yl)-3-[4-(2-morpholine-4-base-oxyethyl group) naphthalene-1-yl] urea (BIRB796).

[0034] another embodiment provides the compound with structural formula IV:

[0035] wherein

[0036] R ⁵And R ⁶Be selected from H, halogen, cyano group, nitro, hydroxyl, the optional C that replaces independently of one another _1-6Alkyl, the optional C that replaces _3-7Cycloalkyl, the optional C that replaces _4-10Alkyl-cycloalkyl, the optional C that replaces _2-6Thiazolinyl, the optional C that replaces _1-6Alkoxyl group, the optional C that replaces _{6 or 10}Aryl, the optional pyridyl that replaces, the optional pyrimidyl that replaces, the optional thienyl that replaces, the optional furyl that replaces, the optional thiazolyl that replaces, the optional De oxazolyl that replaces, the optional phenoxy group that replaces, the optional sulfo-phenoxy group that replaces, the optional amino-sulfonyl that replaces, the optional urea groups that replaces, the optional thioureido that replaces, the optional amide group that replaces, the optional ketone group that replaces, the optional carboxyl that replaces, the optional formamyl that replaces, the optional thioether that replaces, the optional sulfoxide that replaces, the optional sulfone that replaces, the optional amino that replaces, the optional alkoxy amino that replaces, the optional alkoxyl group heterocyclic radical that replaces, the optional alkylamino that replaces, the optional alkyl carboxyl that replaces, the optional carbonyl that replaces, the optional volution cycloalkyl that replaces, the optional pyrazinyl that replaces, the optional pyridazinyl that replaces, the optional pyrryl that replaces, the optional thienyl that replaces, the optional thiazolyl that replaces, the optional De oxazolyl that replaces, the optional imidazolyl that replaces, the optional De isoxazolyl that replaces, the optional pyrazolyl that replaces, the optional isothiazolyl that replaces, the optional naphthyl that replaces, the optional quinolyl that replaces, the optional isoquinolyl that replaces, the optional quinoxalinyl that replaces, the optional benzothiazolyl that replaces, the optional benzothienyl that replaces, the optional benzofuryl that replaces, optional indyl that replaces and the optional benzimidazolyl-that replaces;

[0037] or the pharmacy acceptable salt of described compound, ester, solvate or prodrug.

[0038] in one embodiment, the IC of the inhibition p38 γ that shows of formula IV compound ₅₀At about 10pM to the scope of about 5 μ M.

[0039] in one embodiment, formula IV compound is not

Wherein X is N.

[0040] these and other embodiment has more detailed description hereinafter.

The accompanying drawing summary

[0041] Fig. 1 illustrates the collagen level that changes with the different slow virus particles that give cell (the slow virus particle that comprises multiple coding shRNA) in inductive cell not.

[0042] Fig. 2 illustrates the collagen level that changes with the different slow virus particles that give cell (the slow virus particle that comprises multiple coding shRNA) in the cell beta induced with TGF-.

Description of Preferred Embodiments

[0043] has now found that also have active effective p38 γ kinase inhibitor compounds of inhibition, can realize high curative effect the treatment various diseases relevant with the kinase p 38 increased activity by using at p38 α.In addition, have found that, by modifying the p38 alpha inhibitor, make this modification produce inhibition activity at p38 γ, can realize reducing these two activity of kinase p 38 γ and kinase p 38 α, the activity of kinase p 38 α is not reduced to such degree simultaneously: when the experimenter who suffers from kinase p 38 increased activity diseases associated, observe undesirable side effects.

[0044] therefore, in one embodiment, provide by compound is contacted with p38 mitogen-activated protein kinase (MAPK) regulate stress activated protein kinase (SAPK) system method.The IC that preferred compound shows at p38 γ MAPK ₅₀To the scope of about 5 μ M, preferably suppress the IC of p38 γ MAPK at about 10pM ₅₀To the scope of about 1 μ M, more preferably suppress the IC of p38 γ MAPK at about 50pM ₅₀At about 50pM to the scope of about 200nM.The IC that preferred compound shows at p38 α ₅₀Value also is not less than its IC at p38 γ ₅₀Be worth 2 times.

[0045] " mitogen-activated protein kinase (mitogen-activated protein kinase, MAPK) " be to evolve to go up conservative serine/threonine kinase, participate in regulating many cell incidents.In mammalian cell, identify some MAPK groups, comprised extracellular signal-regulated kinase (ERK), p38 and SAPK/JNK.It is generally acknowledged that MAPK is activated by its specificity mapk kinase (MAPKK): ERK is activated by MEK1 and MEK2, and p38 is activated by MKK3 and MKK6, and SAPK/JNK is activated by SEK1 (being also referred to as MKK4) and MKK7 (SEK2).These MAPKK can also be activated by the multiple MAPKK kinases (MAPKKK) such as Raf, MLK, MEKK1, TAK1 and ASK1.

[0046] it is generally acknowledged that the MAPK network comprises at least 12 clones', high conservative, point to the serine-threonine kinase of proline(Pro), this kinases is by cellular stress (oxidative stress, dna damage, heat or osmotic shock, uv-radiation, ischemia-reperfusion), allogenic material (Anisomycin, Sodium metaarsenite, lipopolysaccharides, LPS) or pro-inflammatory cytokine TNF-α and IL-1 β when activating, can carry out phosphorylation and activating cells matter or endonuclear other kinases or nucleoprotein, transcription factor (referring to Underwood etc., the Fig. 1 among the 2001Prog Respir Res31:342-345) for example.

p38MAPK

[0047] " p38MAPK " used herein a member that is stress activated protein kinase family, comprise at least 4 kinds of isomer (α, β, γ, δ), wherein several being considered to the critical process of inflammatory reaction and tissue remodeling very important (Lee etc., 2000 Immunopharmacol.47:185-201).As if main kinase p 38 α in monocyte and the scavenger cell and p38 β and p38 γ (skeletal muscle) or p38 δ (testis, pancreas, prostate gland, small intestine, and sialisterium, hypophysis and suprarenal gland) compare and express more widely.Many p38MAP kinase substrates have been identified, comprise other kinases (MAPKAP K2/3, PRAK, MNK 1/2, MSK1/RLPK, RSK-B), transcription factor (ATF2/6, myocyte enhancer factor 2, nuclear factor-β, CHOP/GADD153, Elk1 and SAP-1A1) and cytoplasmic protein (microtubule removes steady albumen (stathmin)), wherein having many all is very important on physiology.

[0048] Jiang, Y. etc. report among the 1996 J Biol Chem.271:17920-17926, p38 β is characterized as closely-related 372 amino acid whose albumen with p38 α.These two is all activated p38 α and p38 β by pro-inflammatory cytokine and environmental stress, and p38 β is preferentially activated by map kinase kinases 6 (MKK6), and priority activation transcription factor 2 (ATF2).Kumar, S. etc., 1997Biochem Biophys Res Comm 235:533-538 and Stein, B. etc., reported second isomer p38 β 2 of p38 β among the 1997 J BiolChem 272:19509-19517, it contains 364 amino acid, has 73% identity with p38 α.It is generally acknowledged that p38 β is activated by pro-inflammatory cytokine and environmental stress, as if although compare with the more general tissue expression of p38 α, the p38 beta isomer p38 β 2 of second report preferentially expresses in central nervous system (CNS), heart and skeletal muscle.And, it is generally acknowledged transcriptional factors-2 (ATF-2) as the substrate of p38 β 2 liken to into the substrate of p38 α better.

[0049] Li, Z. etc., reported the evaluation of p38 γ among the 1996 Biochem Biophys Res Comm 228:334-340, Wang, X. etc., 1997 J Biol.Chem 272:23668-23674 and Kumar, S. etc. have reported the evaluation of p38 δ among the 1997 Biochem Biophys Res Comm 235:533-538.Based on its tissue expression pattern, substrate utilization, for the response of direct and indirect stimulation and to the susceptibility of kinase inhibitor, these two p38 isomer (γ and δ) have been represented unique subclass of MAPK family.It is generally acknowledged that p38 α and β are closely-related, but variant with γ and δ, γ and δ are more closely related to each other.

[0050] common, the p38MAP kinase pathways is directly or indirectly activated by cell surface receptor (for example receptor tyrosine kinase, chemokine or g protein coupled receptor), and these cell surface receptors are activated with the specific ligand (for example cytokine, chemokine or lipopolysaccharides (LPS)) of related receptors bind.Subsequently, the p38MAP kinases is activated by the phosphorylation on residue Threonine 180 and the tyrosine 182.After activation, the p38MAP kinases can make other intracellular protein phosphorylation that comprises protein kinase, and can be displaced to nucleus, locate its phosphorylation and transcriptional factors at this, cause the expression of pro-inflammatory cytokine and other the proteic expression that promotes inflammatory reaction, cell adhesion and proteolytic degradation.For example, in such as scavenger cell and monocytic myeloid lineage cell, IL-1 β and TNF α are all transcribed in response to p38 activates.These and other cytokine subsequently translation and secretion causes in the adjacent tissue and part or systemic inflammatory responses by the white corpuscle infiltration.Although reacting, this is that the normal integral part at the physiological response of cellular stress, acute or chronic cellular stress cause the overexpression of pro-inflammatory cytokine, not controlled expression or excessive and controlled expression not.This causes tissue injury again, often causes pain and weakness.

[0051] in pulmonary alveolar macrophage, p38 inhibitor SB203580 is to p38 kinase whose inhibition minimizing cytokine gene product.It is generally acknowledged that inflammatory cytokine (TNF-α, IFN-γ, IL-4, IL-5) and chemokine (IL-8, RANTES, eotaxin) can regulate or support the chronic respiratory inflammation.As if the generation of a lot of potential media of respiratory inflammation and effect depend on and stress activate map kinase system (SAPK) or p38 kinase cascade (Underwood etc., 2001 Prog Respir Res 31:342-345).Many environmental stimuluses are to the activation of p38 kinase pathways, have caused the retrofit of the inflammatory mediator (its generation be considered to be subjected to translate adjusting) to identification.In addition, have multiple inflammatory mediator can activate p38MAPK, p38MAPK can activate the MAPK downstream targets that comprises other kinases or transcription factor then, produces the potentiality of amplifying inflammatory process in the lung thus.

The downstream substrate of p38 class map kinase

[0052] The protein kinase substrate of p38 α or p38 β: map kinase activated protein kinase 2 (MAPKAPK2 or M2), map kinase interaction protein kinases (MNK1), p38 adjusting/activated protein kinase (PRAK), phospho-mitogen-activated protein kinase and stress activated protein kinase (MSK:RSK-B or RLPK).

[0053] P38 activated transcription factor: activating transcription factor (ATF)-1,2 and 6, SRF accessory protein 1 (Sap 1), CHOP (can lure the gene 153 or the GADD153 of cessation of growth cessation and dna damage into), p53, C/EBP β, myocyte enhancer factor 2C (MEF2C), MEF2A, MITF1, DDIT3, ELK1, NFAT and high mobility group protein (HBP1).

[0054] The p38 substrate of other type: cPLA2, Na ⁺/ H ⁺Exchanger isomer-1, tau protein, Keratin sulfate 8 and microtubule remove steady albumen.

[0055] Gene by the adjusting of p38 approach: c-jun, c-fos, junB, IL-1, TNF, IL-6, IL-8, MCP-1, VCAM-1, iNOS, PPAR γ, cyclo-oxygenase (COX)-2, collagenase-1 (MMP-1), collagenase-3 (MMP-13), HIV-LTR, Fgl-2, brain natriuretic peptide (BNP), CD23, CCK, kytoplasm phosphoenolpyruvate carboxykinase, cyclin D1, ldl receptor (Ono etc., 2000 Cellular Signalling 12:1-13).

P38 activatory biological results

P38 and inflammation

It is generally acknowledged that [0056] acute and chronic inflammation plays a leading role to the morbidity of numerous disease, described disease is rheumatoid arthritis, asthma, chronic obstructive pulmonary disease (COPD) and adult respiratory distress syndrome (ARDS) for example.The activation of p38 approach may play the role of a nucleus in following several: (1) is the generation of the pro-inflammatory cytokine of IL-1 β, TNF-α and IL-6 for example; (2) induce for example enzyme of COX-2, the reticular tissue under the COX-2 control pathological condition is reinvented; (3) for example the intracellular enzyme of iNOS is expressed, and iNOS regulates oxygenizement; (4) induce attachment proteins such as VCAM-1 and many other inflammation associated molecules.In addition, the p38 approach can play regulating effect in the propagation of immune system cell and differentiation.P38 can participate in GM-CSF, CSF, EPO and cell proliferation of CD40 inductive and/or differentiation.

[0057] in some animal models, studied the effect of p38 approach in inflammation related disease.SB203580 has reduced the mortality ratio of the mouse model of endotaxin induction shock to the inhibition of p38, and suppresses the development of mouse collagen-induced property sacroiliitis and rat assist agent arthritis.Current research shows that a kind of more effective p38 inhibitor SB220025 makes granulomatous vessel density produce significant dose-dependently decline.These results show that the component of p38 or p38 approach can be the treatment target of inflammatory disease.

P38 and fibrosis

[0058] uncontrolled and/or unusual extracellular matrix protein deposition causes cystic fibrosis, is the pathogenesis basis that comprises the disease of the special property sent out pulmonary fibrosis (IPF), sarcoidosis, chronic obstructive pulmonary disease (COPD) and sclerosis.P38 involves several short fibrosis approach incident (Kaminska etc., Acta Biochimica Polonica52 (2): 329-37) by its effect in TGF-beta receptor signal transduction.TGF-β is Fibrotic basis, relate to some incidents, comprise and induce collagen deposition, synthetic short fibrosis cytokine, fibroblast proliferation, myofibroblast differentiation and epithelium-mesenchyme conversion (Kaminska etc., Acta Biochimica Polonica52 (2): 329-37; Border and Noble 1994NEJM 331:1286-92) (R.Newton and N.S.Holden, Drug Discovery Today:Disease Mechanisms, the 3rd volume, the 1st phase, spring in 2006 number, 53-61 page or leaf).P38 is by other cytokine and growth factor activation that may be relevant with cystic fibrosis, and these factors comprise: il-1 β (IL-1 β), interleukin-17 (IL-17), Urogastron (EGF) and Thr6 PDGF BB (PDGF).

[0059] fibrosis, the fibrosis in liver for example, be that excess deposition owing to extracellular matrix component (especially collagen) takes place, excess deposition results from the amount imbalance (NIH guides PA-99-110, the state-run institute of alcohol abuse and alcoholism) between the matrix macromole that produce in the liver and degraded.Historically, hepatic fibrosis is considered to relate to the hepatic parenchymal non-reversible process of the progressive replacement of the ECM that is rich in collagen (Muddu etc., Int J BiochemCell Biol.2006 October 7) always.In the pulmonary fibrosis patient of acute and chronic form, all confirmed the biological chemistry evidence (Laurent, Ciba FoundSymp.1985114:222-33) that collagen deposition increases in the human body.In idiopathic pulmonary fibrosis, exist the over-drastic extracellular matrix molecules to produce, described molecule comprises collagen, cytotactin (tenascin) and proteoglycan (Noble etc., Clin Chest Med 200425:749-758).Control by fiber (potential reversible reaction) to the mechanism of the step of fibrosis (irreversible) takes place may related (Toxicol Pathol.1991 with being formed with of collagen sudden change, calcification or cross-linked proteins; 19 (4Pt 1): 526-39).As described herein with shown in the embodiment, by the discovery of RNA inhibition test, suppress p38 already, include but not limited to p38 gamma isomer (isoform), can influence the collagen generation level in the target cell that comprises inoblast.

[0060] RNA interference (RNAi) is wherein to be called the cell mechanism that the double-stranded short rna of short interfering rna or siRNA instructs the degraded of complementary RNA target.This process can cause RNA target level significantly to reduce.Elbashir and colleague (2001 Nature 411:494-8) have determined the constitutional features of typical siRNA, are the duplex of 19 base pairs, respectively have two Nucleotide overhangs at two ends.The researchist uses this form to design synthetic siRNA subsequently, wherein one of two RNA oligonucleotide and target gene complementation.These synthetic siRNA is imported the corresponding minimizing that can promote complementary mRNA degraded and corresponding protein product in the mammalian cell.The RNAi The Application of Technology makes can carry out such functional study: wherein the minimizing of specific gene expression allows with the effect of this gene of experimental study in cellular pathways or illness.

[0061] short hairpin RNA (or shRNA) is the research tool of being correlated with, and wherein changes the hair clip form into by the duplex of Elbashir and 21 Nucleotide of colleague's description.The hair clip design allows the expression cassette importing is instructed in the cell of shRNA expression by suitable transcripting promoter.The application of shRNA has been eliminated with the demand of the synthetic effective transfectional cell of siRNA (this demand can be difficult to some cell type), has therefore expanded the cell type number that can successfully be used for the test of RNAi type.The application of shRNA also allows to prolong ground/stably strike and subtracts (knockdown) mRNA target, and separates the cell mass with the transduction of shRNA expression cassette.

[0062] the stable a kind of form that imports the shRNA expression cassette is the attenuation slow virus.Moffat and colleague have described attenuation slow virus particulate library, and these attenuation slow virus particles are designed for to be stablized the shRNA expression cassette of U6 promoters driven in the genome that mixes cells infected (Moffat etc., 2006 Cell 124:1283-98).The transfer section of the engineering virus tetracycline resistance marker of also encoding, this mark can be used for selecting having the cell of the shRNA expression cassette of stable integration.

[0063] as described in the embodiment 10, prepare in 8 slow virus particles and transfered cell that contain coding region (SEQ IDNO:1-8), the different shRNA of described coding region coding target p38 α or p38 γ, separate and quantification of mrna according to embodiment 11, and according to embodiment 12 mensuration collagen levels, the result of this moment shows that the sequence that reduces p38 γ mRNA level descends relevant with the collagen accumulation that is produced.Referring to the table 1 among the embodiment 12.Therefore, for example suppress p38 γ and can cause the collagen expression level to descend, and therefore can suppress fibrosis.

P38 and apoptosis

[0064] activation of following that seems p38 and apoptosis is induced by multiple material, and for example NGF gives up the combination with Fas.L-Cysteine HCL Anhydrous (caspase) is the key of apoptosis pathway, is expressed as the inactivation proenzyme.The caspase inhibitor can be by the crosslinked blocking-up of Fas p38 activation then.But expressing excessively of dominance activatory MKK6b also may be induced caspase activity and necrocytosis.The effect of p38 in apoptosis is cell type and stimulates dependent.Promote the necrocytosis in some clone although shown the p38 signal transduction, shown that p38 strengthens survival, cell growth and differentiation in different clones.

P38 in cell cycle

[0065] p38 α crossing in yeast expressed and caused propagation significantly to slow down, and illustrates that p38 α participates in the cell growth.When handling cell, observe the proliferation slows of the mammalian cell of cultivation with p38 α/beta inhibitor SB203580.

P38 and myocardial cell's hypertrophy

[0066] studied activation and the function of p38 in myocardial cell's hypertrophy.In loose evolution, the level of p38 α and p38 β all improves, and myogen joint tissue and the room diuresis factor expression that raises have strengthened the MKK3 that excites constitutive activity and the hypertrophic responses of MKK6.In addition, the p38 signal transduction that weakens in heart promotes myocyte's differentiation via the mechanism that relates to calcineurin-NFAT signal transduction.

P38 and growth

[0067], exists about the evidence of p38 in developmental otherness effect although the p38 knock-out mice does not have viability.P38 is associated with the placenta vasculogenesis in several researchs, but does not grow related with cardiovascular.In addition, also p38 and erythropoietin expression are interrelated, point out its effect in promoting erythrocyte generates.PRAK has involved the cell development in the mouse graft recently.Discovery PRAK mRNA and p38 isomer are expressed in whole blastocyst growth course.

P38 and cytodifferentiation

[0068] found that p38 α and/or p38 β play an important role in the cytodifferentiation of several different cell types.The 3T3-L1 cytodifferentiation is that adipocyte and PC12 cytodifferentiation are that neurone all needs p38 α and/or β.Having found to be divided into haemoglobin cell and C2C112 for SKT6 by the p38 approach, to be divided into myotube be essential and enough.

P38 in old and feeble and tumor suppression

[0069] p38 has effect in tumour generation and aging.Reported already that the activation of MKK6 and MKK3 produces depended on the active old and feeble phenotype of p38MAPK.In addition, shown that the p38MAPK activity is to cause in response to telomere shortening, H ₂O ₂The reason of the aging of exposure and chronic RAS oncogene signal transduction.The common trait of tumour cell is old and feeble forfeiture, and p38 takes place relevant with tumour in some cell.Reported already that p38 in tumour activation weakened, and lost the component of p38 approach such as MKK3 and MKK6 and cause breeding the possibility that changes with tumorigenesis and increase, and irrelevant with clone of in these researchs, using or tumor inducing thing.

The p38MAP kinase inhibitor

[0070] " p38MAPK inhibitor " is for suppressing the active compound of p38.Can be by the well-known the whole bag of tricks detection compound of technician to the active restraining effect of p38.For example, can measure restraining effect (Lee etc., 1988 Int J Immunopharmacol 10:835-843 by measuring the level that suppresses the generation of lipopolysaccharides (LPS) the stimulated cells factor; Lee etc., 1993 Ann NY Acad Sci 696:149-170; Lee etc., 1994 Nature 372:739-746; Lee etc., 1999 Pharmacol Ther 82:389-397).

[0071] effort of exploitation p38MAPK inhibitor generally concentrates on to increase and renders a service.Found that SB203580 is effective p38 kinase inhibitor, its IC ₅₀Value is in nanomolar concentration (nM) scope.For example, find the IC of SB203580 ₅₀Be 48nM.Below Pyridinylimidazoles SKF 86002 (P1) of Xian Shiing and SB203582 (P2) also are the p38 kinase inhibitor.P38 inhibitor (P3-P6) shown in nearest publication (Lee etc., 2000Immunopharmacology 47:185-201) discloses hereinafter.In these inhibitor, it should be noted that the relative efficient and selectivity (the p38 IC that describe at Compound P 4 ₅₀=0.19nM), and SB 220025 (P6) is to the inhibition of the vasculogenesis that driven by inflammation.These compounds generally are considered to combination in the ATP pocket (pocket) of p38 α polypeptide, are the competitive inhibitors of cofactor ATP.

[0072] finds that also some pyrimidine imidazoles also is effective p38 kinase inhibitor, its IC ₅₀Value is inferior nanomolar concentration (Liverton etc., 1999 J.Med.Chem.42:2180-2190).It is generally acknowledged the combination in the ATP pocket of p38 α polypeptide of these compounds, and be the competitive inhibitor of essential cofactor ATP.

[0073] some diaryl urea, for example BIRB 796, also are effective inhibitor (Pargellis etc., Nature Structural Biology 9:268-272) of p38MAPK.BIRB 796 is at the IC of TNF α ₅₀Be 18nM.Diaryl urea structurally Pyridinylimidazoles and the pyrimidyl imidazoles inhibitor with p38 is completely different.Some diaryl urea is considered to the allosteric inhibitor of p38 α.It is generally acknowledged, the site of spatially shifting out the ATP binding pocket is during in conjunction with p38 α, allosteric inhibitor is induced the conformational change of the ATP binding pocket of polypeptide, this conformational change produces the ATP binding pocket suitable mutually with effective ATP combination, has explained the effectiveness of viewed this compound as the p38 alpha inhibitor thus.

[0074] two kinds of p38 inhibitor reporting in the clinical development are HEP689 (P7) and VX-745 (P8).It is reported that VX-745 is used for rheumatoid arthritis in the II clinical trial phase.Disclose the effective local anti-inflammatory activity of HRP689, it is reported that HRP689 has entered the clinical development stage, be used for the treatment of psoriatic and other dermopathic potentiality as local application to study it.

P1SK&F 86002 P2X＝N；SB 203580

P3X＝CH；L-167307

P4 P5 RWJ 68354

P6X＝H，Y＝CH；HEP 689(SB 235699) P8VX-745

P7X＝HN ₂，Y＝N；SB 220025

[0075] can be to the further argumentation of various p38 inhibitor referring to Boehm etc., 2000Exp Opin Ther Pat 10:25-37; With Salituro etc., 1999Curr Med Chem6:807-823 and Fitzgerald etc., 2003 Nature Structural Biology 10:764-769.

Have found that [0076] gamma isomer of p38 and αYi Gouti have significant structural similarity.Yet, although important residue is guarded between isomer, observe structure variation, comprise the variation in the proteic ATP binding pocket peripheral region.The modeling of above-mentioned p38 inhibitor shows, when p38 alpha inhibitor and the interaction of p38 gamma isomer, observes disadvantageous interaction on energy.Modeling also discloses, and when p38 alpha inhibitor and the interaction of p38 gamma isomer, other interaction favourable on energy is kept.

[0077] has found that, in the formula II compound some do not have disadvantageous interaction on the space, for example, at the Met109 of p38 γ and Fitzgerald etc., disadvantageous interaction on the space between the trifluoromethylbenzene base section of the compound 1 that 2003 Nature StructuralBiology 10:764-769 describe, in addition, some in the formula II compound has the substituting group that interacts with p38 γ and give positive bound energy.Have been found that only removing the trifluoromethylbenzene base section can lose the favourable bound energy that has with the substituent compound of protein-interacting.Therefore, in certain embodiments, Fitzgerald etc., trifluoromethyl substituting group in the compound 1 of 2003 Nature Structural Biology 10:764-769 is by the less substituting group displacement of less molecular volume, perhaps less by molecular volume, and with by p38 γ residue Leu89, Leu58, the hydrophobic pocket that Leu107 and Leu108 limit produces the interactional substituting group displacement of Van der Waals, perhaps less by molecular volume, and it is, perhaps less by molecular volume with the substituting group displacement that the Met109 of p38 γ produces hydrogen bonding interaction or electrostatic interaction, and with by p38 γ residue Leu89, Leu58, the hydrophobic pocket that Leu107 and Leu108 limit produces Van der Waals and interacts, and also produce the substituting group displacement of hydrogen bonding interaction or electrostatic interaction with the Met109 of p38 γ.

[0078] has found that, in the formula IV compound some do not have disadvantageous interaction on the space, for example, disadvantageous interaction on the space between the fluorophenyl part of the Met109 of p38 γ and P2, in addition, some in the formula IV compound has the substituting group that interacts with p38 γ and give positive bound energy.Therefore, in certain embodiments, the fluorophenyl substituting group of P2 is by the less substituting group displacement of molecular volume, perhaps less by molecular volume, and with by p38 γ residue Leu89, Leu58, the hydrophobic pocket that Leu107 and Leu108 limit produces the interactional substituting group displacement of Van der Waals, perhaps less by molecular volume, and it is, perhaps less by molecular volume with the substituting group displacement that the Met109 of p38 γ produces hydrogen bonding interaction or electrostatic interaction, and with by p38 γ residue Leu89, Leu58, the hydrophobic pocket that Leu107 and Leu108 limit produces Van der Waals and interacts, and also produce the substituting group displacement of hydrogen bonding interaction or electrostatic interaction with the Met109 of p38 γ.

[0079] has found that, in the formula III compound some do not have disadvantageous interaction on the space, for example, disadvantageous interaction on the space between the naphthylidene part of the replacement of the Met109 of p38 γ and BIRB 796, in addition, some in the formula III compound has the substituting group that interacts with p38 γ and give positive bound energy.Therefore, in certain embodiments, the naphthylidene of the replacement of BIRB796 is by the less substituting group displacement of molecular volume, perhaps less by molecular volume, and with by p38 γ residue Leu89, Leu58, the hydrophobic pocket that Leu107 and Leu108 limit produces the interactional substituting group displacement of Van der Waals, perhaps less by molecular volume, and with the Met109 generation hydrogen bonding interaction of p38 γ or the substituting group displacement of electrostatic interaction, perhaps less by molecular volume, and produce π-accumulation or the interactional substituting group displacement of π-positively charged ion with the Phe111 of p38 γ, perhaps less by molecular volume, and with by p38 γ residue Leu89, Leu58, the hydrophobic pocket that Leu107 and Leu108 limit produces Van der Waals and interacts, and also produce the substituting group displacement of hydrogen bonding interaction or electrostatic interaction with the Met109 of p38 γ, perhaps less by molecular volume, and with by p38 γ residue Leu89, Leu58, the hydrophobic pocket that Leu107 and Leu108 limit produces Van der Waals and interacts, and also the Phe111 with p38 γ produces π-accumulation or the interactional substituting group displacement of π-positively charged ion, and is perhaps less by molecular volume, and with by p38 γ residue Leu89, Leu58, the hydrophobic pocket that Leu107 and Leu108 limit produces Van der Waals and interacts, and produce hydrogen bonding interaction or electrostatic interaction with the Met109 of p38 γ, and also the Phe111 with p38 γ produces π-accumulation or the interactional substituting group displacement of π-positively charged ion.

[0080] preferred p38 inhibitor described herein be show higher relatively p38 γ suppress to render a service and have suppress the higher relatively curative effect (for example being used to regulate the SAPK system) that produces thus bis-epoxy for pyridine derivate and analogue.Preferably, the IC of the inhibition p38 γ MAPK that shows of the p38 inhibitor of this embodiment ₅₀At about 10pM to the scope of about 500 μ M, preferably about 10pM to about 5 μ M or about 100nM to the scope of about 500 μ M, preferably at about 50pM extremely in the scope of about 1 μ M, more preferably at about 50pM extremely in the scope of about 200nM.Preferably, the IC of the inhibition p38 α that shows of the p38 inhibitor of this embodiment ₅₀Value is to suppress the IC of p38 γ MAPK ₅₀The value at least about 2 times.More preferably, the IC of the inhibition p38 α that shows of the p38 inhibitor of this embodiment ₅₀Value is to suppress the IC of p38 γ MAPK ₅₀The value at least about 5 times.Even more preferably, the IC of the inhibition p38 α that the p38 inhibitor of this embodiment shows ₅₀Value is to suppress the IC of p38 γ MAPK ₅₀The value at least about 10 times.

[0081] preferred p38 inhibitor described herein is to show higher relatively p38 γ to suppress to render a service and have pyrimidyl imdazole derivatives and the analogue that suppresses the higher relatively curative effect (for example being used to regulate the SAPK system) of generation thus.Preferably, the IC of the inhibition p38 γ MAPK that shows of the p38 inhibitor of this embodiment ₅₀At about 10pM to the scope of about 500 μ M, preferably about 10pM to about 5 μ M or about 100nM to the scope of about 500 μ M, preferably at about 50pM extremely in the scope of about 1 μ M, more preferably at about 50pM extremely in the scope of about 200nM.Preferably, the IC of the inhibition p38 α that shows of the p38 inhibitor of this embodiment ₅₀Value is to suppress the IC of p38 γ MAPK ₅₀The value at least about 2 times.More preferably, the IC of the inhibition p38 α that shows of the p38 inhibitor of this embodiment ₅₀Value is to suppress the IC of p38 γ MAPK ₅₀The value at least about 5 times.Even more preferably, the IC of the inhibition p38 α that the p38 inhibitor of this embodiment shows ₅₀Value is to suppress the IC of p38 γ MAPK ₅₀The value at least about 10 times.

[0082] preferred p38 inhibitor described herein is to show higher relatively p38 γ to suppress to render a service and have Diarylurea derivatives and the analogue that suppresses the higher relatively curative effect (for example being used to regulate the SAPK system) of generation thus.Preferably, the IC of the inhibition p38 γ MAPK that shows of the p38 inhibitor of this embodiment ₅₀At about 10pM to the scope of about 500 μ M, preferably about 10pM to about 5 μ M or about 100nM to the scope of about 500 μ M, preferably at about 50pM extremely in the scope of about 1 μ M, more preferably at about 50pM extremely in the scope of about 200nM.Preferably, the IC of the inhibition p38 α that shows of the p38 inhibitor of this embodiment ₅₀Value is to suppress the IC of p38 γ MAPK ₅₀The value at least about 2 times.

[0083] term used herein " alkyl " is meant the unit price straight or branched group of 1-10 carbon atom, includes but not limited to methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, isobutyl-, the tertiary butyl, n-hexyl etc.

[0084] term used herein " thiazolinyl " is meant the unit price straight or branched group of 2-10 the carbon atom that contains the two keys of carbon, includes but not limited to 1-propenyl, 2-propenyl, 2-methyl isophthalic acid-propenyl, 1-butylene base, crotyl etc.

[0085] term used herein " halogen " is meant fluorine, chlorine, bromine or iodine.

[0086] term used herein " haloalkyl " is meant and is connected with one or more halogen groups on the alkyl.

[0087] term used herein " 4-nitro alkyl " is meant and is connected with one or more nitros on the alkyl.

[0088] term used herein " alkylthio " is meant and is connected with one or more sulfenyls on the alkyl.

[0089] term used herein " hydroxyalkyl " is meant and is connected with one or more hydroxyls on the alkyl.

[0090] term used herein " alkoxyl group " is meant by-O-and connects and the straight or branched alkyl of covalent bonding on parent molecule.The example of alkoxyl group includes but not limited to methoxyl group, oxyethyl group, propoxy-, isopropoxy, butoxy, n-butoxy, sec-butoxy, tert.-butoxy etc.

[0091] term used herein " alkoxyalkyl " is meant and is connected with one or more alkoxyl groups on the alkyl.

[0092] term used herein " carboxyl " is meant-COOH.

[0093] term " alkoxy carbonyl " be meant-(CO)-the O-alkyl.The example of alkoxy carbonyl includes but not limited to methoxycarbonyl, ethoxy carbonyl, propoxycarbonyl etc.

[0094] group used herein is meant and has material single, unpaired electronics, make contain this group material can with another material covalent bonding.Therefore, in this article, group is free radical not necessarily.On the contrary, group is meant more macromolecular specific part.Term " group " can exchange with term " base " and use.

[0095] substituting group used herein is derived from unsubstituted precursor structure, and in this precursor structure, one or more hydrogen atoms have been exchanged for another atom or group.When being substituted; substituting group be one or more separately or be independently selected from following group: alkyl, cycloalkyl, aryl, fused-aryl, heterocyclic radical, heteroaryl, hydroxyl, alkoxyl group, aryloxy, sulfydryl, alkylthio, arylthio, cyano group, halogen, carbonyl, thiocarbonyl, alkoxy carbonyl, nitro, silyl, three halo methylsulfonyls, trifluoromethyl and amino; comprise single substituted-amino and disubstituted amido, and protected derivative.The protecting group that can form above-mentioned substituent protectiveness derivative is well known by persons skilled in the art, and can be referring to reference, for example Greene and Wuts, Protective Groups in Organic Synthesis; John

And Sons:New York, 1999.Which kind of situation no matter, as long as substituting group is described to " the optional replacement ", then this substituting group can be replaced by above-mentioned substituting group.

[0096] term " purifying " is meant the compound of having separated from other compound, makes it comprise at least 95% measured matter when measuring.

[0097] can there be unsymmetrical carbon in the compound of describing herein.All these isomer comprise diastereomer and enantiomer and composition thereof, all are included in the scope of mentioned compound.In some cases, compound can exist with tautomeric form.All tautomeric forms all are included in the scope of mentioned compound.Equally, when compound contained thiazolinyl or alkenylene, compound may exist cis isomerism form and trans-isomerism form.The present invention includes the mixture of cis-isomeride and trans-isomer(ide) and cis-isomeride and trans-isomer(ide).Therefore, unless point out in addition clearly in the context, the compound of indication comprises all aforementioned isomeric form herein.

[0098] various forms all comprises in embodiments, comprises polymorphic form, solvate, hydrate, conformer, salt and prodrug derivatives.Polymorphic form is that chemical formula is identical but binding substances that structure is different.Solvate is the binding substances (solvent molecule combines with solute molecule or ionic) that forms by solvation.The compound of hydrate for forming by combination water.Conformation is a kind of structure of conformer that is.Conformational isomerism is that structural formula is identical but around the molecular phenomenon of atom conformation difference (conformer) of rotation key.The salt of compound can prepare by method known to those skilled in the art.For example, can make suitable alkali or acid with etc. stoichiometric compound reaction and prepare the salt of compound.Prodrug is to demonstrate the compound of its pharmacotoxicological effect afterwards carrying out bio-transformation (chemical conversion).Therefore, for instance, prodrug can be considered as containing the medicine of specialization protecting group, described specialization protecting group is used in of short duration mode, with the undesirable characteristic in change or the elimination parent molecule.Therefore, unless spell out in addition in the context, compound mentioned herein comprises all aforementioned forms.

[0099] following compound can be used for method described herein.In one embodiment, the IC of the inhibition p38 γ that shows of following compound ₅₀At about 10pM to the scope of about 5 μ M, preferably at about 100nM extremely in the scope of about 500 μ M.

[0100] embodiment provides a compounds of being represented by following structural (formula I):

[0101] wherein R is selected from H, halogen, cyano group, nitro, hydroxyl, the optional C that replaces _1-6Alkyl, the optional C that replaces _3-7Cycloalkyl, the optional C that replaces _4-10Alkyl-cycloalkyl, the optional C that replaces _2-6Thiazolinyl, the optional C that replaces _1-6Alkoxyl group, the optional C that replaces _{6 or 10}Aryl; the optional pyridyl that replaces; the optional pyrimidyl that replaces; the optional thienyl that replaces; the optional furyl that replaces; the optional thiazolyl that replaces; the optional De oxazolyl that replaces; the optional phenoxy group that replaces; the optional sulfo-phenoxy group that replaces; the optional amino-sulfonyl that replaces; the optional urea groups that replaces; the optional thioureido that replaces; the optional amide group that replaces; the optional ketone group that replaces; the optional carboxyl that replaces; the optional formamyl that replaces; the optional thioether that replaces; the optional sulfoxide that replaces; the optional sulfone that replaces; the optional amino that replaces; the optional alkoxy amino that replaces; the optional alkoxyl group heterocyclic radical that replaces; the optional alkylamino that replaces; the optional alkyl carboxyl that replaces; the optional carbonyl that replaces; the optional volution cycloalkyl that replaces; the optional pyrazinyl that replaces; the optional pyridazinyl that replaces; the optional pyrryl that replaces; the optional thienyl that replaces; the optional thiazolyl that replaces; the optional De oxazolyl that replaces; the optional imidazolyl that replaces; the optional De isoxazolyl that replaces; the optional pyrazolyl that replaces; the optional isothiazolyl that replaces; the optional naphthyl that replaces; the optional quinolyl that replaces; the optional isoquinolyl that replaces; the optional quinoxalinyl that replaces; the optional benzothiazolyl that replaces; the optional benzothienyl that replaces; the optional benzofuryl that replaces; optional indyl that replaces and the optional benzimidazolyl-that replaces.

[0102] another embodiment provides the compounds by following structural (formula II) expression:

[0103] R wherein ⁴Be selected from H, halogen, cyano group, nitro, hydroxyl, the optional C that replaces _1-6Alkyl, the optional C that replaces _3-7Cycloalkyl, the optional C that replaces _4-10Alkyl-cycloalkyl, the optional C that replaces _2-6Thiazolinyl, the optional C that replaces _1-6Alkoxyl group, the optional C that replaces _{6 or 10}Aryl; the optional pyridyl that replaces; the optional pyrimidyl that replaces; the optional thienyl that replaces; the optional furyl that replaces; the optional thiazolyl that replaces; the optional De oxazolyl that replaces; the optional phenoxy group that replaces; the optional sulfo-phenoxy group that replaces; the optional amino-sulfonyl that replaces; the optional urea groups that replaces; the optional thioureido that replaces; the optional amide group that replaces; the optional ketone group that replaces; the optional carboxyl that replaces; the optional formamyl that replaces; the optional thioether that replaces; the optional sulfoxide that replaces; the optional sulfone that replaces; the optional amino that replaces; the optional alkoxy amino that replaces; the optional alkoxyl group heterocyclic radical that replaces; the optional alkylamino that replaces; the optional alkyl carboxyl that replaces; the optional carbonyl that replaces; the optional volution cycloalkyl that replaces; the optional pyrazinyl that replaces; the optional pyridazinyl that replaces; the optional pyrryl that replaces; the optional thienyl that replaces; the optional thiazolyl that replaces; the optional De oxazolyl that replaces; the optional imidazolyl that replaces; the optional De isoxazolyl that replaces; the optional pyrazolyl that replaces; the optional isothiazolyl that replaces; the optional naphthyl that replaces; the optional quinolyl that replaces; the optional isoquinolyl that replaces; the optional quinoxalinyl that replaces; the optional benzothiazolyl that replaces; the optional benzothienyl that replaces; the optional benzofuryl that replaces; optional indyl that replaces and the optional benzimidazolyl-that replaces.

[0104] another embodiment provides the compounds by following structural (formula III) expression:

[0105] wherein

[0106] R ²And R ³Be selected from H, halogen, cyano group, nitro, hydroxyl, the optional C that replaces independently of one another _1-6Alkyl, the optional C that replaces _3-7Cycloalkyl, the optional C that replaces _4-10Alkyl-cycloalkyl, the optional C that replaces _2-6Thiazolinyl, the optional C that replaces _1-6Alkoxyl group, the optional C that replaces _{6 or 10}Aryl; the optional pyridyl that replaces; the optional pyrimidyl that replaces; the optional thienyl that replaces; the optional furyl that replaces; the optional thiazolyl that replaces; the optional De oxazolyl that replaces; the optional phenoxy group that replaces; the optional sulfo-phenoxy group that replaces; the optional amino-sulfonyl that replaces; the optional urea groups that replaces; the optional thioureido that replaces; the optional amide group that replaces; the optional ketone group that replaces; the optional carboxyl that replaces; the optional formamyl that replaces; the optional thioether that replaces; the optional sulfoxide that replaces; the optional sulfone that replaces; the optional amino that replaces; the optional alkoxy amino that replaces; the optional alkoxyl group heterocyclic radical that replaces; the optional alkylamino that replaces; the optional alkyl carboxyl that replaces; the optional carbonyl that replaces; the optional volution cycloalkyl that replaces; the optional pyrazinyl that replaces; the optional pyridazinyl that replaces; the optional pyrryl that replaces; the optional thienyl that replaces; the optional thiazolyl that replaces; the optional De oxazolyl that replaces; the optional imidazolyl that replaces; the optional De isoxazolyl that replaces; the optional pyrazolyl that replaces; the optional isothiazolyl that replaces; the optional naphthyl that replaces; the optional quinolyl that replaces; the optional isoquinolyl that replaces; the optional quinoxalinyl that replaces; the optional benzothiazolyl that replaces; the optional benzothienyl that replaces; the optional benzofuryl that replaces; optional indyl that replaces and the optional benzimidazolyl-that replaces.

[0107] in one embodiment, the formula III compound is not 1-(the 5-tertiary butyl-2-p-methylphenyl-2H-pyrazole-3-yl)-3-[4-(2-morpholine-4-base-oxyethyl group) naphthalene-1-yl] urea (BIRB796).

[0108] another embodiment provides the compounds by following structural (formula IV) expression:

[0109] wherein

[0110] R ⁵And R ⁶Be selected from H, halogen, cyano group, nitro, hydroxyl, the optional C that replaces independently of one another _1-6Alkyl, the optional C that replaces _3-7Cycloalkyl, the optional C that replaces _4-10Alkyl-cycloalkyl, the optional C that replaces _2-6Thiazolinyl, the optional C that replaces _1-6Alkoxyl group, the optional C that replaces _{6 or 10}Aryl; the optional pyridyl that replaces; the optional pyrimidyl that replaces; the optional thienyl that replaces; the optional furyl that replaces; the optional thiazolyl that replaces; the optional De oxazolyl that replaces; the optional phenoxy group that replaces; the optional sulfo-phenoxy group that replaces; the optional amino-sulfonyl that replaces; the optional urea groups that replaces; the optional thioureido that replaces; the optional amide group that replaces; the optional ketone group that replaces; the optional carboxyl that replaces; the optional formamyl that replaces; the optional thioether that replaces; the optional sulfoxide that replaces; the optional sulfone that replaces; the optional amino that replaces; the optional alkoxy amino that replaces; the optional alkoxyl group heterocyclic radical that replaces; the optional alkylamino that replaces; the optional alkyl carboxyl that replaces; the optional carbonyl that replaces; the optional volution cycloalkyl that replaces; the optional pyrazinyl that replaces; the optional pyridazinyl that replaces; the optional pyrryl that replaces; the optional thienyl that replaces; the optional thiazolyl that replaces; the optional De oxazolyl that replaces; the optional imidazolyl that replaces; the optional De isoxazolyl that replaces; the optional pyrazolyl that replaces; the optional isothiazolyl that replaces; the optional naphthyl that replaces; the optional quinolyl that replaces; the optional isoquinolyl that replaces; the optional quinoxalinyl that replaces; the optional benzothiazolyl that replaces; the optional benzothienyl that replaces; the optional benzofuryl that replaces; optional indyl that replaces and the optional benzimidazolyl-that replaces.

[0111] in one embodiment, formula IV compound is not

Wherein X is N.

[0112] it being understood that particular compound described herein can be the above member of a class in the above-mentioned classes of compounds.Compound described herein can be used for regulating stress activated protein kinase (SAPK) system.

[0113] another embodiment relates to the compound of being represented by formula I-IV of purifying.Purity can be represented with aforesaid percentage.In a preferred embodiment, account for composition total weight in purifying compounds, the purity of the compound of being represented by formula I-IV of purifying is about more than 96%, and more preferably its purity is about (weight ratio) more than 98%.

[0114] formula I-IV compound can synthesize by using various popular response known in the art.The synthesis flow that provides in

embodiment

6,7,8 and 9 is provided the synthetic example.

[0115] can also by known in the art, based on any popular response of the known synthetic schemes that is used for the dicyclo oxo pyridine, for example reaction that provides in embodiment 6 synthesizes the formula I compound of bis-epoxy for pyridine derivate.

[0116] can also pass through any popular response known in the art, comprise those reactions based on the known synthetic schemes that is used for the pyrimidyl imidazoles, for example reaction of setting forth in embodiment 7 and 9 synthesizes the formula II and the formula IV compound of pyrimidyl imdazole derivatives.

[0117] can also pass through any popular response known in the art, comprise those reactions based on the known synthetic schemes that is used for diaryl urea, for example reaction of setting forth in embodiment 8 synthesizes the formula III compound of Diarylurea derivatives.

[0118] raw material described herein can be commercially available, for known materials or can prepare by methods known in the art.In addition, the raw material do not described of this paper also can be commercially available, for known materials or can prepare by methods known in the art.

[0119] raw material can have suitable substituting group, finally to obtain having corresponding substituent target product.Perhaps, substituting group can be in synthetic any time adding, finally to obtain having corresponding substituent target product.

[0120] synthetic schemes that is shown in embodiment 6 has shown the method that can be used for preparation I compound.The synthetic schemes that is shown in embodiment 7 and 9 has shown the method that can be used for preparation formula II and formula IV compound.The synthetic schemes that is shown in embodiment 8 has shown the method that can be used for preparing the formula III compound.It should be appreciated by those skilled in the art that and to use many different building-up reactions schemes to come synthesis type I-IV compound.In addition, skilled person in the art will appreciate that and in building-up reactions, can use many different solvents, coupling agent and reaction conditions, to obtain suitable result.

[0121] those skilled in the art by shown in or known similar reaction can be appreciated that the order variation, also will appreciate that the variation of suitable reaction condition, these change applicable to aforesaid method, with preparation formula I-IV compound.

[0122] at this paper the method that is used for preparation formula I-IV compound is described; normally the organic chemistry filed technician is known in the use of protecting group, therefore, and in some cases; the flow and method of this paper may be inferred and use suitable protecting group, although these groups may not explained clearly.The introducing of these suitable protecting groups and to remove be that organic chemistry filed is well-known; Referring to for example T.W.Greene, " Protective Groups in Organic Synthesis ", Wiley (New York), 1999.Reaction product described herein can be separated by conventional means, for example extracts, distillation, chromatogram etc.

[0123] can by make suitable alkali or acid and etc. the salt of stoichiometric compound prepared in reaction formula I-IV compound, pharmacy acceptable salt for example.Similarly, can be by pharmaceutically acceptable derivates (for example ester), metabolite, hydrate, solvate and the prodrug that well known to a person skilled in the art method preparation formula I-IV compound.Therefore, another embodiment is provided as the compound of the prodrug of active compound.Usually, prodrug be metabolism in vivo (for example by metabolic conversion, for example deaminizating, take off alkyl, take off ester etc.) and provide the compound of active compound." pharmaceutically acceptable prodrug " is meant and reasonably is being suitable for compound medicinal in the patient in the medical judgment scope, there are not toxicity, stimulation, transformation reactions etc. excessively improperly, and effectively realize desired use, if possible, the pharmaceutically acceptable ester and the zwitterionic form that comprise the embodiment compound.The example of pharmaceutically acceptable prodrug type is described in T.Higuchi and V.Stella, Pro-drugs as Novel Delivery Systems, the 14th volume of A.C.S. special topic series, and Edward B.Roche edits, Bioreversible Carriers in Drug Design, American PharmaceuticalAssociation and Pergamon Press, 1987, these two pieces of documents all are attached to herein by reference.

[0124] compound described herein and binding substances can also comprise metabolite.Term used herein " metabolite " is meant the compound of embodiment or the meta-bolites of its pharmacologically acceptable salts, analogue or derivative, and it shows in external or body and embodiment compound similar activity.Compound described herein and binding substances can also comprise hydrate and solvate.Term used herein " solvate " is meant the complex compound that is formed by solute (being formula I-IV compound herein) and solvent.Be used for the biological activity that these solvents of embodiment preferably should not hinder solute.For example, solvent can be water, ethanol or acetate.By aforementioned content, the particular compound of indication or a compounds are understood to include above-mentioned various forms herein, comprise its pharmacologically acceptable salts, ester, prodrug, metabolite and solvate.

The preferential inhibitor of p38 γ in the SCREENED COMPOUND library

[0125] on the other hand, be provided for the method for identification of pharmaceutical active compounds, for example be used for determining whether compound can be used as therapeutical agent potentially, for example be used for prevention or treatment fibrotic disease (for example relevant disease) with p38 or cytokine.This method comprises measures the inhibition of a plurality of compounds to p38 γ, option table reveals the compound that higher relatively p38 γ suppresses effectiveness, the selected compound of the pacing of going forward side by side examination selects also to show the compound that higher relatively p38 α suppresses effectiveness to the inhibition of p38 α.This method also comprises measures the inhibition of a plurality of compounds to p38 γ, and option table reveals higher relatively p38 γ and suppresses to render a service and also show the compound that higher relatively p38 α suppresses effectiveness.This method also comprises measures the inhibition of a plurality of compounds to p38 α, and option table reveals higher relatively p38 α and suppresses to render a service and also show the compound that higher relatively p38 γ suppresses effectiveness.Preferably, this efficient p38 γ and p38 alpha inhibitor compound suppress the IC of p38 γ ₅₀At about 10pM to the scope of about 500 μ M, preferably about 10pM to about 5 μ M or about 100nM to the scope of about 500 μ M, more preferably at about 500pM extremely in the scope of about 1 μ M, even more preferably at about 500pM extremely in the scope of about 1 μ M.Preferably, this efficient p38 γ and p38 alpha inhibitor compound are at the IC of p38 α ₅₀Value is the IC at p38 γ ₅₀The value 2 times.More preferably, this efficient p38 γ and p38 alpha inhibitor compound are at the IC of p38 α ₅₀Value is the IC at p38 γ ₅₀The value 5 times.Even more preferably, this efficient p38 γ and p38 alpha inhibitor compound are at the IC of p38 α ₅₀Value is the IC at p38 γ ₅₀The value 10 times.A plurality of compounds to be determined are preferably selected from potential library of compounds.Can measure a plurality of compounds that are selected from this library in every way.For example, in certain embodiments, this method also comprises makes p38MAPK contact with a plurality of compounds, and determines whether this compound suppresses cytokine activity.P38MAPK is preferably selected from p38 α and p38 γ.In preferred embodiments, contact procedure is carried out external; In some preferred embodiment, contact procedure comprises makes the cell that contains p38MAPK contact with this compound.

[0126] in another embodiment, is provided for external or suppress the active method of p38MAPK in the cell in vivo.Usually, these methods are included under the condition that the p38 activity that makes in the cell is suppressed, and the cell that contains p38MAPK is contacted with the compound (for example formula I-IV compound) that effectively suppresses the amount of p38.The example of these methods embodiment below partly provides.Preferably, the IC of the inhibition p38 γ that shows of described compound ₅₀At about 10pM to the scope of about 500 μ M, preferably about 10pM to about 5 μ M or about 100nM to the scope of about 500 μ M, more preferably at about 500pM extremely in the scope of about 1 μ M, even more preferably at about 500pM extremely in the scope of about 1 μ M.Preferably, described compound is at the IC of p38 α ₅₀Value is the IC at p38 γ ₅₀The value 2 times.

[0127] method for example comprises that target compound (for example formula I-IV compound) oral administration or injection with different concns give a treated animal in the body.Give after this compound, intravenously gives lipopolysaccharides.Measure the serum TNF alpha levels and with this level contrast of control animal.Preferred compound suppresses TNF α and discharges, and therefore reduces the TNF alpha levels in the experimental animal blood sample.Preferably, the EC that discharges of the inhibition TNF α that shows of this compound ₅₀At about 10pM to the scope of about 5 μ M, more preferably at about 500pM to the scope of about 1 μ M, even more preferably at about 500pM extremely in the scope of about 1 μ M.

[0128] method of identification of pharmaceutical active compounds can also comprise the mammalian toxicity of determining selected compounds.These methods are normally well known by persons skilled in the art.The method of identification of pharmaceutical active compounds can also comprise and gives the experimenter with selected compounds, determines that simultaneously Mammals is active or is used for other purpose.In one embodiment, the experimenter suffers from inflammatory diseases or is in the middle of the risk of inflammatory diseases.Preferably, the experimenter is a Mammals, and can be the people.

Suppress the kinase whose method of p38MAP

[0129] in one embodiment, be provided at the method for regulating the SAPK system in external or the body.These methods comprise that the compound that makes SAPK regulate concentration contacts (for example by this compound is contacted with the cell or tissue that contains p38MAPK) with p38MAPK, wherein, this compound has higher relatively p38 γ to be suppressed to render a service, and suppresses the higher relatively inhibition concentration of p38MAPK corresponding to this compound.

[0130] inhibition concentration (IC) is for making the active concentration that reduces specific percentage (for example 50%, 40%, 30%, 20%, 10%) of p38MAPK on dose-response curve.For example, determine IC according to causing the p38MAPK activity to reduce by 50%, 40%, 30%, 20% and 10% concentration respectively on the dose-response curve ₅₀, IC ₄₀, IC ₃₀, IC ₂₀And IC ₁₀The compound of regulating the SAPK system suppresses the IC of p38 γ ₅₀Preferably at about 10pM to the scope of about 500 μ M, preferably about 10pM to about 5 μ M or about 100nM to the scope of about 500 μ M, more preferably at about 500pM extremely in the scope of about 1 μ M, even more preferably at about 500pM extremely in the scope of about 1 μ M.

[0131] " with cells contacting " is meant a kind of state, and under this state, compound or other composition of matter directly contact with cell or tissue, the perhaps tight biological action that must be enough to induce expectation in cell or tissue.For example, can with the interactional any-mode of compound the cell or tissue that contains p38MAPK be contacted with compound to allow p38MAPK, thereby in cell, produce the biological action of expectation.Can be for example by mixing or giving compound (formula I-IV compound for example; And/or its salt, ester, prodrug and/or intermediate, and/or comprise the pharmaceutical composition of one or more aforementioned substances) realize exposing cell or tissue.

[0132] or, can introduce compound by making the mode that the direct or indirect target of compound contains the cell or tissue of p38MAPK, realize and the contacting of cell or tissue.Can realize under compound and the p38MAPK bonded condition and the contacting of cell or tissue making.Such condition can comprise that compound and the degree of approach, pH, the temperature that contain the cell or tissue of p38 maybe can influence compound and any condition of p38MAPK bonded.

[0133] in certain embodiments, cell is contacted external with compound; In other embodiments, cell is contacted in vivo with compound.

[0134] when exposing cell in vivo, effective concentration (EC) is for according to depending on the detection of the active specific physiological response that reduces of p38MAPK, makes the active concentration that reduces specific percentage (for example 50%, 40%, 30%, 20%, 10%) of p38MAPK.This physiological response can reduce for the TNF α concentration in for example blood or other body fluid.For example, EC ₅₀, EC ₄₀, EC ₃₀, EC ₂₀And EC ₁₀Be confirmed as causing the p38MAPK activity on dose-response curve, to reduce by 50%, 40%, 30%, 20% and 10% concentration respectively according to the detection that TNF α concentration reduces.The compound of regulating the SAPK system suppresses the EC of p38 γ ₅₀Preferably at about 10pM to the scope of about 500 μ M, preferably about 10pM to about 5 μ M or about 100nM to the scope of about 500 μ M, more preferably at about 500pM extremely in the scope of about 1 μ M, even more preferably at about 500pM extremely in the scope of about 1 μ M.

[0135] in certain embodiments, the compound that provides is the form together with the pharmaceutical composition of pharmaceutically acceptable carrier.

Treat and/or prevent method

[0136] another embodiment provides the method for treatment or preventing disease such as fibrotic disease.This method comprises differentiates the experimenter fibrotic disease risk is arranged or suffered from fibrotic disease, and will treat or the compound of prevention of fibrotic diseases significant quantity gives the experimenter.In preferred embodiments, the IC of the inhibition p38 γ that shows of described compound ₅₀At about 10pM to the scope of about 500 μ M, preferably about 10pM to about 5 μ M or about 100nM to the scope of about 500 μ M, more preferably at about 500pM extremely in the scope of about 1 μ M, even more preferably at about 500pM extremely in the scope of about 1 μ M.In preferred embodiments, described compound is at the IC of p38 α ₅₀Value is the IC at p38 γ ₅₀The value 2 times.In preferred embodiments, the concentration of the blood of significant quantity generation or serum or another kind of body fluid is equal to or less than the IC that this compound suppresses p38 γ ₅₀, or IC ₄₀, or IC ₃₀, or IC ₂₀, or IC ₁₀, in addition, the blood that significant quantity produces or the concentration of serum or another kind of body fluid are equal to or less than the IC that this compound suppresses p38 α ₅₀, or IC ₄₀, or IC ₃₀, or IC ₂₀, or IC ₁₀In preferred embodiments, the inhibition TNF α excretory EC that shows of described compound ₅₀At about 10pM to the scope of about 5 μ M, more preferably at about 500pM to the scope of about 1 μ M, even more preferably at about 500pM extremely in the scope of about 1 μ M.In other preferred embodiment, the blood that significant quantity produces or the concentration of serum or another kind of body fluid are equal to or less than this compound and suppress the EC that LPS pungency TNF α discharges in body fluid ₅₀, or EC ₄₀, or EC ₃₀, or EC ₂₀, or EC ₁₀This significant quantity is preferably the about below 70% of the amount that causes undesirable side effect in the experimenter, and more preferably from about below 50%, described side effect is such as but not limited to drowsiness, gastrointestinal upset and photosensitivity fash.The compound that is used for the treatment of or prevents is preferably formula I-IV compound.

[0137] method that is used to differentiate the fibrotic disease risk or suffered from the experimenter of fibrotic disease is well known by persons skilled in the art.The example of fibrotic disease that can be by methods described herein treatments or prevention comprise the diseases associated with p38, for example with cytokine activity change diseases associated, with the SAPK system regulate diseases associated, with autoimmune disorder and fibrosis illness diseases associated.Cytokine (one or more cytokines) is preferably selected from but is not limited to IL-1 β, IL-6, IL-8 and TNF α.In one embodiment, being used for the treatment of or preventing the compound of inflammatory diseases is the kinase whose compound that suppresses in the SAPK signal transduction pathway.The example of preferred compound comprises formula I-IV compound.

[0138] term " illness relevant with p38 " is meant disease or other the harmful situation that wherein no matter directly still relates to p38MAP signal transduction of kinases approach indirectly.The examples of disorders relevant with p38 comprises IL-1 β, TNF α, IL-6 or the IL-8 imbalance that is produced by the p38 activity level that continues, prolongs, strengthens or raise or crosses and express caused illness.These illnesss include but not limited to inflammatory diseases, autoimmune disorder, fibrotic disease, destructive osteopathy, proliferative disease, infectious diseases, nerve degenerative diseases, transformation reactions, apoplexy pour into again local asphyxia, heart attack, vasculogenesis disease, organ anoxic, blood vessel hyperplasia, cardiac hypertrophy, thrombin induction platelet aggregation and with prostaglandin(PG) or the relevant illness of cyclo-oxygenase approach, for example relate to the illness of prostaglandin endoperoxide synthase.The illness relevant with p38 can comprise that any and p38 isomer are relevant or by the illness of its mediation.

[0139] " fibrosis illness ", " fiber proliferative disease ", " fibrotic disease ", " fiber proliferative disease ", " fibrosing disorders " and " fiber proliferative disorder " all are used interchangeably, and are meant that pathology or excessive gathering the with fibroblasts proliferation or active imbalance and/or collagenous tissue is illness, disease or the obstacle of feature.Usually, any this disease, obstacle or illness can both be by giving compounds for treating described herein.Fibrotic disease includes but not limited to pulmonary fibrosis, comprises idiopathic pulmonary fibrosis (IPF) and the pulmonary fibrosis that is caused by the known cause of disease, hepatic fibrosis and renal fibrosis.Other exemplary fiber illness comprises muscle skeleton fibrosis, cardiac fibrosis, postoperative adhesion, scleroderma, glaucoma and skin injury, for example keloid.

[0140] term " is regulated the SAPK system " and is meant no matter for example improve or reduce the stress activated protein kinase system activity by suppressing the p38 activity in external still body.In certain embodiments, when comparing with the p38 activity of untreated control cells, the p38 activity inhibited in the cell about 50%, about 40%, about 30%, about 20% or about 10% o'clock, the SAPK system is regulated.

[0141] used herein and cytokine activity change relevant illness and are meant the reformed illness of comparing with non-disease conditions wherein of cytokine activity, include but not limited to the illness that IL-1 β, TNF α, IL-6 or the excessive generation of IL-8 or imbalance by the cytokine activity level that causes continuing, prolong, strengthening or raise cause, this may be relevant with the p38 activity.Such illness includes but not limited to that inflammatory diseases, autoimmune disorder, fibrotic disease, destructive osteopathia, proliferative disease, infectious diseases, nerve degenerative diseases, transformation reactions, apoplexy pour into/local asphyxia, heart attack, vasculogenesis disease, organ anoxic, blood vessel hyperplasia, cardiac hypertrophy, the platelet aggregation of thrombin induction and the illness relevant with the lipoxygenase signal transduction pathway with cyclo-oxygenase again, for example with the relevant illness of prostaglandin(PG) endoperoxides synthase.The illness relevant with cytokine can comprise and IL-1 (especially IL-1 β), TNF α, IL-6 or IL-8 or can be by any other cytokine that p38 regulates relevant or by any illness of its mediation.In preferred embodiments, relevant with cytokine illness is the illness relevant with TNF α.

[0142] method described herein can also be used for the treatment of autoimmune disease and with the acute and chronic inflammation diseases associated.These diseases include but not limited to: chronic obstructive pulmonary disease (COPD), struvite pulmonary fibrosis (IPF), rheumatoid arthritis; Rheumatoid spondylitis; Osteoarthritis; Gout, other arthritis disease; Sepsis; Septic shock; Endotoxin shock; Gram negative sepsis; Toxic shock syndrome; Myofasical pain syndrome (MPS); Shigellosis; Asthma; Adult respiratory distress syndrome; Inflammatory bowel; Crohn's disease (Crohn ' s disease); Psoriatic; Eczema; Ulcerative colitis; Glomerulonephritis; Scleroderma; Chronic thyroiditis; Graves disease (Grave ' s disease); Ormond's disease (Ormond ' s disease); Autoimmunity gastritis; Myasthenia gravis; Autoimmune hemolytic anemia; The autoimmunity neutropenia; Thrombocytopenia; Pancreatic gland fibrosis; Chronic active hepatitis comprises hepatic fibrosis; Acute and chronic nephropathy; Renal fibrosis; Irritable bowel syndrome; Pyreticosis (pyresis); Restenosis; Cerebral malaria; Apoplexy and ischemic injury; Nerve injury; Alzheimer's disease (Alzheimer ' s disease); Huntington Chorea (Huntington ' s disease); Parkinson's disease (Parkinson ' s disease); Acute and chronic pain; Transformation reactions comprises rhinallergosis and allergic conjunctivitis; Cardiac hypertrophy; Chronic heart failure; Acute coronary syndrome; Emaciation; Malaria; Leprosy; Leishmaniasis; Lyme disease (Lyme disease); Reiter syndrome (Reiter ' s syndrome); Acute synovitis; Myodegeneration; Bursitis; Tendonitis; Tenosynovitis; Hernia, disruptiveness or prolapsus property disc syndrome; Osteopetrosis; Thrombosis; Silicatosis; The lung sarcosis; Bone resorption disease, for example osteoporosis or the osteopathy relevant with multiple myeloma; Cancer includes but not limited to metastatic breast cancer, colorectal carcinoma, malignant melanoma, cancer of the stomach and nonsmall-cell lung cancer; Graft-vs-host reaction; And autoimmune disease, for example multiple sclerosis, lupus and fibromyalgia; AIDS and other virus disease, for example zoster, herpes simplex I or II, influenza virus, serious acute respiratory system synthesis are levied (SARS) and cytomegalovirus infection; And diabetes.In addition, the method of embodiment can also be used for the treatment of proliferative disease (comprising optimum and neoplasm), comprise acute myelogenous leukemia, chronic lymphocytic leukemia, Kaposi sarcoma (Kaposi ' s sarcoma), multiple myeloma, breast cancer, comprise the transitivity breast cancer; Colorectal carcinoma; Metastatic carcinoma of bone etc.; Pain disease comprises neuromuscular pain, headache, cancer pain, has a toothache and arthritis pain; The vasculogenesis disease, comprise the noumenal tumour vasculogenesis, the eye neovascularization and infantile hemangioma; The illness relevant with the lipoxygenase signal transduction pathway with cyclo-oxygenase comprises the illness (comprise oedema, heating, analgesia and pain) relevant with prostaglandin endoperoxide synthase 2; The organ anoxic; The platelet aggregation of thrombin induction.In addition, methods described herein can also be used for the treatment of the protozoal disease in the animal (comprising Mammals).

[0143] experimenter can comprise one or more cell or tissues, or organism.Preferred experimenter is a Mammals.Mammals can comprise any Mammals.As limiting examples, preferred Mammals comprises ox, pig, sheep, goat, horse, camel, buffalo, cat, dog, rat, mouse and people.Highly preferred mammalian subject is behaved.Can give the experimenter with compound by any medicine approach of passing known in the art.That concrete exemplary route of administration comprises is oral, eye with, rectum, in oral cavity, part, intranasal, ophthalmology, subcutaneous, intramuscular, intravenously (inject and instil), brain, transdermal and through lung.

[0144] as the term " significant quantity " that in " treatment significant quantity " used herein and " prevention significant quantity " scope, uses be meant be enough to treat, alleviate or prevent as described in the compound amount of disease or illness, the compound amount that perhaps is enough to show detectable treatment, prevention or suppresses effect.Can for example detect this effect by disclosed mensuration in following examples.The accurate significant quantity that is used for the experimenter depends on experimenter's body weight, size and healthy state; The illness nature and extent; And selected therapeutical agent or the therapeutical agent combination that is used for administration.Can be by determine treatment and prevention significant quantity in clinician's technical ability and the routine test in the determination range at the given patient's condition.Preferably, the blood of the significant quantity of embodiment compound generation or serum or another kind of body-fluid concentration are less than suppressing the kinase whose IC of p38MAP ₅₀, IC ₄₀, IC ₃₀, IC ₂₀Or IC ₁₀Preferably, the TNF α secretion that effectively changes whole blood of the blood that produces of the significant quantity of embodiment compound or serum or another kind of body-fluid concentration reaches 10%, 15%, 20%, 30%, 40% or 50%.

[0145] for any compound, can measure or estimation treatment or prevention significant quantity in animal model (being generally rat, mouse, rabbit, dog or pig) in the cell cultures of for example tumour cell at first.Animal model can also be used for determining suitable concentration range and route of administration.Can use this information to determine the dosage and the approach of human then.

[0146] can determine treatment/prevention effectiveness and toxicity, for example ED by the standard pharmaceutical procedures in cell culture or experimental animal ₅₀(to the effective dosage of 50% mass treatment) and LD ₅₀(making the lethal dosage of 50% colony).Dosage between result of treatment and the toxic action can be used ED than for therapeutic index ₅₀/ LD ₅₀Ratio represent.Preferably has big treatment exponential pharmaceutical composition.But the pharmaceutical composition with narrow therapeutic index is also included within the scope of embodiment.Can be used for formulating the human dosage range from the data that cell cultures is measured and zooscopy obtains.The dosage that contains in these compositions is preferably comprising ED ₅₀And in the very little or nontoxic circulation composition scope of toxicity.Dosage can change in this scope with used formulation, patient's susceptibility and route of administration.

[0147] practitioner is determined dosage accurately according to the factor relevant with the patient of needs treatment.Dosage and administration are adjusted, enough active medicine levels to be provided or to keep desirable effect.The factor that can consider comprises being in a bad way property, experimenter's healthy situation, age, body weight and experimenter's sex, diet, administration time and frequency, drug regimen, reaction sensibility and the tolerance/response to treating.The depot drug product composition can every 3-4 days 1 time, 1 time or per 2 week 1 medication weekly, and this depends on the transformation period and the clearance rate of concrete preparation.

Should be appreciated that [0148] treatment described herein comprises preventing disease, relief of symptoms, slows down disease progression, reverses infringement or cure diseases.

[0149] on the one hand, the treatment to the fibrosis illness causes subject population of subjects to compare the mean survival time prolongation with untreated population of subjects.Preferably, mean survival time prolongs about more than 30 days; More preferably from about more than 60 days; More preferably from about more than 90 days; Even more preferably from about more than 120 days.Can adopt any repeatably mode to measure the prolongation of colony's survival time.One preferred aspect, can for example after with the active compound begin treatment, calculate the prolongation that the average survival length of colony is measured colony's mean survival time.Another preferred aspect, can also be for example the prolongation of average survival linear measure colony mean survival time by after the first round treatment of finishing active compound, calculating colony.

[0150] on the other hand, the treatment to the fibrosis illness causes subject population of subjects to compare the mortality ratio reduction with the population of subjects of only accepting carrier.On the other hand, the treatment to the fibrosis illness causes subject population of subjects to compare the mortality ratio reduction with untreated population of subjects.Another aspect causes subject population of subjects to compare mortality ratio with the colony of the single therapy of the medicine of the compound of accepting non-the present embodiment or its pharmacologically acceptable salts, metabolite, analogue or derivative to the treatment of fibrosis illness and reduces.Preferably, mortality ratio reduces about more than 2%; More preferably from about more than 5%; More preferably from about more than 10%; Most preferably from about more than 25%.One preferred aspect, can reduce by the mortality ratio that any repeatably mode be measured subject population of subjects.Another preferred aspect, can for example after with the active compound begin treatment, calculate in the colony dead mean number relevant in the time per unit, thereby measure the reduction of colony's mortality ratio with disease.Another preferred aspect, can also be for example after the first round treatment of finishing active compound, calculate in the colony dead mean number relevant in the time per unit with disease, thus the reduction of measurement colony mortality ratio.

[0151] on the other hand, the treatment to the fibrosis illness causes tumor growth rate to reduce.Preferably, before treatment, the tumor growth rate after the treatment reduces at least about 5%; More preferably, tumor growth rate reduces at least about 10%; More preferably reduce at least about 20%; More preferably reduce at least about 30%; More preferably reduce at least about 40%; More preferably reduce at least about 50%; Even more preferably reduce at least about 60%; Most preferably reduce at least about 75%.Can measure tumor growth rate by any repeatably metering system.One preferred aspect, according to the diameter of tumor measure of the change tumor growth rate of time per unit.

[0152] on the other hand, the treatment to the fibrosis illness causes cell proliferation speed to reduce.Preferably, the cell proliferation speed after the treatment reduces at least about 5%; More preferably at least about 10%; More preferably at least about 20%; More preferably at least about 30%; More preferably at least about 40%; More preferably at least about 50%; Even more preferably at least about 60%; Most preferably at least about 75%.Can measure cell proliferation speed by any repeatably metering system.One preferred aspect, for example measure cell proliferation speed by the somatoblast quantity of measuring in the time per unit inner tissue sample.

[0153] on the other hand, the treatment to the fibrosis illness causes the proliferative cell ratio to reduce.Preferably, the proliferative cell ratio after the treatment reduces at least about 5%; More preferably at least about 10%; More preferably at least about 20%; More preferably at least about 30%; More preferably at least about 40%; More preferably at least about 50%; Even more preferably at least about 60%; Most preferably at least about 75%.Can measure the proliferative cell ratio by any repeatably metering system.One preferred aspect, for example by determining in the tissue sample that the somatoblast number measures the proliferative cell ratio to somatoblast number not.Another preferred aspect, the proliferative cell ratio equals mitotic index.

[0154] on the other hand, the treatment to the fibrosis illness causes the size in cell proliferation scope or zone to reduce.Preferably, with respect to the size in cell proliferation scope before the treatment or zone, its size after the treatment reduces at least about 5%; More preferably reduce at least about 10%; More preferably reduce at least about 20%; More preferably reduce at least about 30%; More preferably reduce at least about 40%; More preferably reduce at least about 50%; Even more preferably reduce at least about 60%; Most preferably reduce at least about 75%.Can measure the size in cell proliferation scope or zone by any repeatably metering system.One preferred aspect, the cell proliferation scope or the zone size can with the cell proliferation scope or the zone diameter or width measurements.

[0155] method described herein can comprise the experimenter that evaluation need be treated.In a preferred embodiment, described method comprises the Mammals that evaluation need be treated.In a highly preferred embodiment, described method comprises the people that evaluation need be treated.The experimenter's that can be benefited by treatment by any indication method realizes the evaluation to the experimenter of needs treatment.For example, can comprise any combination of authentication method, identify the experimenter who needs treatment by clinical diagnosis, laboratory inspection or any other method well known by persons skilled in the art.

[0156] as described in other place of this paper, compound described herein can be made pharmaceutical composition, if needed, and can be by allowing any administration of treatment disease or illness.Preferred route of administration is an oral administration.Can take the administration of single agent form of medication, perhaps can be with the preparation of divided dose or release continuously or the compound that medication (for example pump) gives embodiment in for some time.But when giving the experimenter with the compound of embodiment, the dosage and the selected route of administration of reply compound are selected, and make and can effectively treat disease.

[0157] method of embodiment comprises that also one or more compounds described herein are used for the treatment of the purposes of disease together with one or more other therapeutical agents.Therefore, for instance, the combination of activeconstituents can for: prepare and administration simultaneously or send in combination preparation jointly (1); (2) replace or parallel sending as independent preparation; Or (3) any other combined treatment known in the art.When sending with rotational therapy, method described herein can comprise for example with independent solution, emulsion, suspensoid, tablet, pill or capsule or by giving in proper order or delivering active ingredients with the different injections in the independent syringe.Usually, in the process of alternating treatment, (promptly continuous) gives each activeconstituents of effective dose successively, and in treating at the same time, gives two or more activeconstituentss of effective dose together.Can also use the combination treatment at intermittence of various orders.

[0158] diagnostic test is regarded as the part of methods described herein.For example, can from the experimenter who suffers from fibrosis illness (for example relevant illness), obtain the biopsy sample with p38 or cytokine.Can check biopsy samples, with the p38 activity level of determining to exist in the sample (or cytokine levels); Sample is contacted with the embodiment compound of selecting, and measure p38 activity (or cytokine levels), (for example, suppress p38 or cytokine activity, it suppresses the IC of p38 γ to determine whether have the effect of expectation by compound ₅₀At about 10pM to the scope of about 500 μ M, preferably about 10pM to about 5 μ M or about 100nM to the scope of about 500 μ M, more preferably at about 50pM extremely in the scope of about 1 μ M, even more preferably at about 50pM extremely in the scope of about 1 μ M, the IC of its inhibition p38 α ₅₀Value is to suppress the IC of p38 γ ₅₀Be worth at least 2 times).Whether this check can be used for determining might be effective to this experimenter with this compounds for treating.Perhaps, sample is contacted with tagged compound (for example fluorescently-labeled compound or radiolabeled compound), sample for reference and detect fluorescence or radiated signal then is to determine the distribution of p38 in tissue sample.The biopsy samples that repeats to obtain in therapeutic process also can be used to study curative effect.According to the instruction of this specification sheets, for those of ordinary skills, it is conspicuous using other diagnostic test of compound described herein.

[0159] therefore, for instance, an embodiment provides the method for determining existence, position and/or the quantity of p38 albumen in the cell or tissue sample.This method comprises: a) the compound that makes embodiment can with p38MAPK bonded condition under the cell or tissue sample is contacted with this compound; And b) determines existence, position and/or the quantity of this compound in the cell or tissue sample, thereby determine existence, position and/or the quantity of p38MAPK in the cell or tissue sample.Can determine existence, position and/or the quantity of this compound in the cell or tissue sample by any method that discloses existence, position and/or the quantity of this compound in cell or tissue.For example, as previously mentioned, can use radioactivity or fluorescent marker method.Other method of determining existence, position and/or the quantity of compound is apparent to a skilled reader.

[0160] method that provides of another embodiment is used to determine: whether (1) compound is the useful therapeutical agent of experimenter of treatment being suffered from inflammatory diseases, or (2) severity of disease, or (3) are with the course of disease in the process of disease cushion treatment.This method comprises: a) before with the treatment of compound described herein or another kind of disease cushion, obtain the cell or tissue sample by the experimenter after the process neutralization; B) this sample is contacted with this compound; And c) determine amount with sample bonded compound, wherein the amount of the p38MAPK in the combination of compound and p38MAPK and the sample is relevant.

Specific examples with the disease of Compounds and methods for described herein treatment

COPD

[0161] chronic obstructive pulmonary disease (COPD) is characterised in that the chronic inflammatory diseases process in the lung, comprises (1) inflammatory cell (neutrophil, scavenger cell and SD8 in respiratory tract and essence ⁺The T cell) quantity increases, and (2) inflammatory cytokine and chemokine expression increase and (3) proteolytic enzyme (elastoser, kethepsin and matrix metalloproteinase MMP) quantity increases.It is generally acknowledged the generation of many potential respiratory inflammation media and MAPK or the p38 kinase cascade that effect all depends on stress-induced.Some reports are all supported the related of pf p38 kinase activation and lung plethora incident: LPS-and TNF α on lung's capillary endothelium-inductive cell-cell adhesion molecule 1 is expressed, the stimulation to the pulmonary artery cell of MMP9 activation, hypoxia inducible, press inductive IL-8 to express thoroughly and the enhanced eosinophilic granulocyte is divided a word with a hyphen at the end of a line and survived at the bronchial epithelial cell middle and high infiltration.

[0162] people (2005 Brit J Pharmacol 144:1002-10) such as Trifilieff report, a kind of adenosine receptor antagonists of combination, p38MAPK and phosphodiesterase 4 type inhibitor C GH2466 demonstrate effectively external and interior anti-inflammatory activity to the disease of for example asthma and COPD.Underwood etc. (2000 Am J Physiol Lung Cell Mol Physiol279:L895-L902) show, effective as selective p38 MAPK inhibitor SB239063 reduces the generation of pro-inflammatory cytokine (comprise IL-1 β, TNF-α, IL-6 and IL-8, they are associated with the respiratory tract fibrosis because of the ability that it regulates fibroblast proliferation and matrix generation); Cause dividing a word with a hyphen at the end of a line and activating minimizing of neutrophil in the lung.Not long ago, found that same compound can change and the relevant reaction of bleomycin inductive chronic fibrosis.Active α and the beta isomer to p38 of this inhibition is optionally.Compounds and methods for described herein can be used for treating COPD.

Pulmonary fibrosis

[0163] pulmonary fibrosis is also referred to as idiopathic pulmonary fibrosis (IPF), a matter diffusivity pulmonary fibrosis, struvite pulmonary fibrosis or fibrosing alveolitis, be inflammatory lung disease and the one group of different substantiality disease that forms feature with fibrosis tissue abnormalities between the alveolar that causes by pulmonary alveolitis, comprise that inflammatory cell infiltration is gone in the alveolar membranes and produce fibrosis.The effect of IPF is chronic, progressive and normally fatal.In suffering from patient's lung of pulmonary fibrosis, confirmed the p38MAPK activation.Many researchs about pulmonary fibrosis point out that some cytokines continuing in lung reinvented afterwards extracellular matrix component with raising of inflammatory cell with lung tissue structure with the expression that increases and accumulate relevant.Specifically, confirmed that pro-inflammatory cytokine such as TNF α and interleukin I L-1 β play a major role in the formation of pneumonia and pulmonary fibrosis.In addition, short fibrosis cytokine such as TGF β and CTGF also play crucial effects in the morbidity of pulmonary fibrosis.Matsuoka etc. (2002 Am J Physiol Lung Cell Mol Physiol 283:L103-L112) have confirmed that p38 inhibitor FR-167653 improves the pulmonary fibrosis of bleomycin inductive mouse.And, the compound pirfenidone (5-methyl isophthalic acid-phenyl-2-(1H)-pyridone) of finding a kind of anti-inflammatory with combination, anti-oxidant and anti-fibrosis effect effectively (Raghu etc., 1999 Am J Respir Crit Care Med 159:1061-1069 in the experimental model of pulmonary fibrosis and clinical study; Nagai etc., 2002 Intern Med 41:1118-1123; Gahl etc., 2002 Mol Genet Metab76:234-242; Azuma etc., 2002 Am J Respir Crit Care Med 165:A729).Compounds and methods for described herein can be used for treating pulmonary fibrosis, for example IPF.

Renal fibrosis

[0164] regardless of the character of initial infringement, renal fibrosis all is regarded as the common final approach that ephrosis is developed to end stage renal failure.Stambe etc. (2004 J Am Soc Nephrol 15:370-379) have tested (the San Francisco of Scios company in the renal fibrosis rat model, CA) the inhibitor NPC 31169 of Yan Fa activity (phosphorylation) form p38, and reported that the renal fibrosis by interstitial volume, collagen iv deposition and the evaluation of connective tissue growth mRNA level significantly reduces.Compounds and methods for described herein can be used for the treatment of renal fibrosis.

Leiomyoma

[0165] leiomyoma of uterus or fibroma are the modal pelvic tumors of women, and do not have known permanently effective available pharmacotherapy.Leiomyomatous cell proliferation and the tissue fibrosis that is characterised in that increase.In myometrium of cultivating and leiomyoma smooth muscle cell, tested the effect of pirfenidone on cell proliferation and collagen expression, find that it is effective inhibitor (Lee etc., 1998 J Clin Endocrinol Metab 83:219-223) of myometrium and leiomyoma cell proliferation.Compounds and methods for described herein can be used for treating leiomyoma.

Endomyocardial fibrosis

[0166] endomyocardial fibrosis (EMF) is a kind of disease that develops into feature with restrictive cardiomyopathy.EMF be regarded as sometimes comprising Lv Fule (

) integral part of single course of disease spectrum of endocarditis (non-tropical eosinophilic granulocyte endomyocardial fibrosis or locular wall fibroplasia endocarditis companion eosinophilia).In EMF, the potential course of disease causes the irregular fibrosis on cardiac intima surface, and conformability is descended, and finally involves the restricted physiology of myocardium intimal surface more at large.The endocardium fibrosis is mainly involved the inflow road of right and left ventricle, and can involve Atrioventricular valve, causes tricuspidal valve and mitral incompetence.Shown that the MAPK activation helps the irregular pulse atrium structural remodeling among the EMF.Compounds and methods for described herein can be used for the treatment of and/or prevent endomyocardial fibrosis.

Other inflammatory diseases

[0167] with many autoimmune disorders and with chronic inflammatory diseases diseases associated and acute reaction and kinase whose activation of p38MAP and inflammatory cytokine cross to express or imbalance connects.These diseases include but not limited to: rheumatoid arthritis, rheumatoid spondylitis; Osteoarthritis; Gout, other arthritis disease; Sepsis; Septic shock; Endotoxin shock; Gram negative sepsis; Toxic shock syndrome; Asthma; Adult respiratory distress syndrome; Chronic obstructive pulmonary disease; Chronic pneumonia; Inflammatory bowel; Crohn's disease; Psoriatic; Eczema; Ulcerative colitis; Pancreatic gland fibrosis; Hepatic fibrosis; Acute and chronic nephropathy; Irritable bowel syndrome; Pyreticosis (pyresis); Restenosis; Cerebral malaria; Apoplexy and local ischemic lesions; Nerve injury; Alzheimer's disease; Huntington Chorea; Parkinson's disease; Acute and chronic pain; Rhinallergosis; Allergic conjunctivitis; Chronic heart failure; Acute coronary syndrome; Emaciation; Malaria; Leprosy; Leishmaniasis; Lyme disease; Reiter syndrome; Acute synovitis; Myodegeneration; Bursitis; Tendonitis; Tenosynovitis; Hernia, disruptiveness or prolapsus property disc syndrome; Osteopetrosis; Thrombosis; Cancer; Restenosis; Silicatosis; The lung sarcosis; Bone resorption disease, for example osteoporosis; Graft-vs-host reaction; And autoimmune disorder, for example multiple sclerosis, lupus and fibromyalgia; AIDS and other virus disease, for example zoster, herpes simplex I or II, influenza virus and cytomegalovirus infection; And diabetes.

[0168] many researchs show, the activity that reduces p38MAP kinases, its upstream activator or its downstream effect by heredity or chemical process weakens inflammatory reaction, and prevention or minimize tissue injury (referring to for example English etc., 2002 Trends Pharmacol Sci 23:40-45; With Dong etc., 2002Annu Rev Immunol 20:55-72).Therefore, suppress also that cytokine excessive or imbalance produces and the p38 activity inhibitor that can suppress more than one pro-inflammatory cytokine can be used as anti-inflammatory agent and therapeutical agent.In addition, a large amount of diseases relevant with the inflammatory reaction of p38MAP kinases dependency show, also need to treat the effective ways of these illnesss.

[0169] Cardiovascular disorderInflammation and leukocyte activation/infiltration play a major role in outbreak that comprises the arteriosclerosis and the cardiovascular disorder of heart failure and development.Acute p38 mitogen-activated protein kinase (MAPK) approach suppresses to reduce tissue injury and the white corpuscle accumulation in myocardial ischaemia/reperfusion injury.Compounds and methods for described herein can be used for the treatment of cardiovascular disorder.

[0170] Multiple sclerosisInflammation in the central nervous system occurs in the disease such as multiple sclerosis, and causes aixs cylinder dysfunction and destruction.The interior observation of external and body shows that all nitrogen protoxide (NO) plays an important role in mediation inflammatory aixs cylinder pathology.The p38MAP kinases exposes and is activated through NO, has shown that suppressing the p38 signal transduction produces neurone and aixs cylinder survival effect.OCM and IGF-1 reduce the p38 activation in the cortical neuron after NO exposes, and improve the aixs cylinder viability in the culture that is exposed to behind the NO, and this viability is the process that depends on mitogen-activated protein kinase/extracellular signal associated kinase signal transduction.Compounds and methods for described herein can be used for the treatment of multiple sclerosis.

[0171] The primary graft does not have functionIt is relevant that nonspecific inflammation and primary graft do not have function (PNF).The inflammatory pancreas islet damages to small part and is mediated by pro-inflammatory cytokine, il-1 β (IL-1 β) and tumor necrosis factor alpha (TNF α) that described pro-inflammatory cytokine is for example produced by the scavenger cell of settling down pancreas islet.The cytokine that known p38 approach participates in monocyte-scavenger cell pedigree cell produces.P38 chemical inhibitor SB203580 has contained that to the inhibition of p38 approach IL-1 β and the TNF α in the people's pancreas islet that contacts with lipopolysaccharides (LPS) and/or inflammatory cytokine produces.Although IL-1 β is mainly produced by resident macrophage, find that pancreatic ductal cell and pancreas islet vascular endothelial cell are the another kind of IL-1 β cell sources in isolating people's pancreas islet.SB203580 is also in the expression that suppresses inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the pancreas islet of treatment.In addition, people's pancreas islet of handling with SB203580 in preceding 1 hour in transplanting demonstrates the graft function of remarkable improvement.Compounds and methods for described herein can be used for improving the graft survival of clinical pancreatic islets transplantation

[0172] Acute injury of kidneyCis-platinum is a kind of important chemotherapeutic, but can cause injury of the kidney.This acute injury of kidney part is mediated by tumor necrosis factor alpha (TNF α).Cis-platinum activates p38MAPK and brings out cancer cell-apoptosis.The p38MAPK activation causes the TNF α in ischemia injury and the scavenger cell to produce to be increased.External, cis-platinum causes the dose-dependently activation of p38MAPK in the proximal tubule cell.Suppress p38MAPK and activate the inhibition that causes TNF α generation.In vivo, with the serious kidney dysfunction of mouse appearance of single agent plus cisplatin in treatment, it is attended by, and kidney p38MAPK is active to be improved and the increase of infiltration white corpuscle.But, to compare with animal with cis-platinum+vehicle treatment, the animal for the treatment of together with cis-platinum with p38MAPK inhibitor SKF86002 demonstrates less kidney dysfunction, not too serious tissue injury and less white corpuscle.Compounds and methods for described herein can be used for the prophylaxis of acute injury of the kidney.

[0173] PeriodontitisPro-inflammatory mediator bradykinin (BK) is in the generation of stimulated in vitro interleukin-8 (IL-8) in people's gums inoblast, and plays an important role in the pathogeny of the various inflammatory diseasess that comprise periodontitis.The IL-8 that IL-8 produces and IL-1 β stimulates that specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 reduces by BK and IL-1 β combination of stimulation produces.Compounds and methods for described herein can be used for the treatment of or pre-preventing parodontitis.

[0174] in certain embodiments, the p38 inhibitor of any such use is not selected from:

And pirfenidone.

Pharmaceutical composition

[0175] although might compound described herein is individually dosed, preferably compound described herein can be made pharmaceutical composition.Therefore, aspect another, provide the pharmaceutical composition that can be used for the inventive method.More particularly, pharmaceutical composition described herein especially can be used for treatment or prevention inflammatory diseases, for example with p38 activity or cytokine activity or the relevant illness of its arbitrary combination.Pharmaceutical composition is for giving the experimenter any composition with treatment or alleviation illness in external or body or through this dual mode.In a preferred embodiment, pharmaceutical composition can vivo medicine-feeding.The experimenter can comprise one or more cell or tissues, or organism.Preferred experimenter is a Mammals.Mammals comprises any Mammals, as non-limiting example, for example is ox, pig, sheep, goat, horse, camel, buffalo, cat, dog, rat, mouse and people.Highly preferred mammalian subject is behaved.

[0176] in one embodiment, pharmaceutical composition can be prepared with pharmaceutically acceptable vehicle, and described vehicle is carrier, solvent, stablizer, assistant agent, thinner etc. for example, depends on concrete administering mode and formulation.Usually answer the compounding pharmaceutical composition obtaining the suitable mutually pH of physiology, and according to preparation and route of administration, can be to the scope of about pH 11 at about pH 3, preferably at about pH 3 to the scope of about pH 7.In alternate embodiment, preferably with pH regulator to about pH 5.0 to the scope of about pH 8.More particularly, pharmaceutical composition can comprise the compound at least a described herein of treatment or prevention significant quantity, together with one or more pharmaceutically acceptable vehicle.Randomly, pharmaceutical composition can comprise combination of compounds described herein, perhaps can comprise the second kind of activeconstituents (for example antibacterial agent or biocide) that can be used for treating or preventing infectation of bacteria.

[0177] preparation that for example is used for parenteral or oral administration is generally solid, liquor agent, emulsion or suspensoid most, and the suction preparation that is used for pulmonary administration is generally liquid or powder, and usually preferred powder formulation.Preferred pharmaceutical composition can also be mixed with the lyophilized solid of before administration, using the compatible solvent reprovision of physiology.The alternate pharmaceutical composition can also be made syrup, creme, paste, tablet etc.

[0178] term " pharmaceutically acceptable vehicle " is meant the vehicle of the medicament that is used to give all compounds as described herein.This term is meant can administration and inexcessive improper toxic any pharmaceutical excipient.

[0179] the acceptable vehicle of pharmacy part is by the concrete composition that will give and the concrete grammar decision that is used to give said composition.Therefore, there is various appropriate drug combination dosage form (referring to for example Remington ' s Pharmaceutical Sciences).

[0180] Shi Yi vehicle can be carrier molecule, comprises big, slow metabolic macromole, for example protein, polysaccharide, poly(lactic acid), polyglycolic acid, polyamino acid, amino acid copolymer and inactivation virus particle.Other exemplary vehicle comprises antioxidant, for example xitix; Sequestrant, for example EDTA; Carbohydrate, for example dextrin, hydroxy alkyl cellulose, hydroxyalkyl methyl cellulose, stearic acid; Liquid, for example oil, water, salt solution, glycerine and ethanol; Wetting agent or emulsifying agent; The pH buffer substance; Or the like.Liposome is also included within the definition of pharmaceutically acceptable vehicle.

[0181] pharmaceutical composition as herein described can be made and be suitable for expecting any form of medication.When plan orally uses, can preparation example such as tablet, pastille, dragee, water-based or oiliness suspensoid, non-aqueous solution agent, dispersible pulvis or granule (comprising micronized particle or nano particle), emulsion, hard or soft capsule, syrup or elixir.The composition that can orally use according to any method preparation plan that is used for pharmaceutical compositions known in the art, this based composition can contain one or more materials that comprises sweeting agent, seasonings, tinting material and sanitas, so that agreeable to the taste preparation is provided.

[0182] is particularly suitable for comprising for example inert diluent, for example Mierocrystalline cellulose, lime carbonate or yellow soda ash, lactose, calcium phosphate or sodium phosphate with the pharmaceutically acceptable vehicle that tablet uses; Disintegrating agent, for example polyvinylpolypyrrolidone, W-Gum or Lalgine; Wedding agent, for example polyvidone, starch, gelatin or gum arabic; And lubricant, for example Magnesium Stearate, stearic acid or talcum powder.

What [0183] tablet can be for dressing not, perhaps can be with known technology (comprising microencapsulation) dressing, postponing its disintegration and absorption in gi tract, thereby in for some time, provide continuous action.For example, can use the time-delay material separately or with paraffin, for example Zerol or Stearic diglyceride.

[0184] preparation that orally uses can also be provided as hard gelatin capsule, wherein activeconstituents mixes with inert solid diluent (for example Mierocrystalline cellulose, lactose, calcium phosphate or kaolin), perhaps make Gelseal, wherein activeconstituents mixes with non-aqueous or oil medium (for example glycerine, propylene glycol, polyoxyethylene glycol, peanut oil, whiteruss or sweet oil).

[0185] in another embodiment, pharmaceutical composition can be mixed with suspensoid, it comprises the compound of embodiment, this compound and at least a pharmaceutically acceptable mixed with excipients that is suitable for preparing suspensoid.

[0186] in another embodiment, is suitable for preparing the dispersed powders and the particle of suspensoid by adding suitable vehicle but pharmaceutical composition can be mixed with.

[0187] is fit to comprise suspension agent, for example Xylo-Mucine, methylcellulose gum, Vltra tears, sodiun alginate, polyvinylpyrrolidone, tragakanta, Sudan Gum-arabic together with the vehicle that suspensoid uses; Dispersion agent or wetting agent, the condensation product of condensation product (for example polyoxyethylene stearic acid ester), oxyethane and the long chain aliphatic alcohol of for example naturally occurring phosphatide (for example Yelkin TTS), oxirane and lipid acid (for example 17 carbon vinyloxy group hexadecanols), oxyethane and derived from the condensation product (for example polyoxyethylene 20 sorbitan monooleate) of the partial ester of lipid acid and hexitan; And thickening material, for example carbomer, beeswax, paraffinum durum or cetyl alcohol.Suspensoid can also contain one or more sanitass, for example acetate, methyl p-hydroxybenzoate and/or P-hydroxybenzoic acid n-propyl; One or more tinting materials; One or more seasoningss; And one or more sweeting agents, for example sucrose or asccharin.

[0188] pharmaceutical composition can also be oil-in-water emulsion form.Oil phase can be vegetables oil, for example sweet oil or peanut oil, mineral oil, for example whiteruss, or these mixture.Suitable emulsifying agent comprises natural gum, for example Sudan Gum-arabic and tragakanta; Natural phospholipid, for example soybean lecithin, derived from the ester or the partial ester of lipid acid; Hexitan, for example sorbitan monooleate; And the condensation product of these partial esters and oxyethane, for example polyoxyethylene 20 sorbitan monooleate.Emulsion can also contain sweeting agent and seasonings.Syrup and elixir can be prepared with sweeting agent such as glycerine, Sorbitol Powder or sucrose.Such preparation can also contain negative catalyst, sanitas, seasonings or tinting material.

[0189] in addition, pharmaceutical composition can be aseptic injection preparation form, for example sterile injectable aqueous emulsion or oiliness suspensoid.Can use those suitable dispersion agents or wetting agent and above mentioned suspension agent to prepare this emulsion or suspensoid according to known technology.Aseptic injection preparation can also be for aseptic injectable solution agent or the suspensoid in nontoxic, parenteral acceptable diluent or solvent, for example 1, and the solution in the 2-propylene glycol.

[0190] aseptic injection preparation can also be made lyophilized powder.Water, Ringer's solution and isoosmotic sodium chloride solution are arranged in operable acceptable carrier and solvent.In addition, can also be with aseptic expressed oil as solvent or suspension medium.For this reason, the expressed oil of any gentleness be can use, synthetic monoglyceride or triglyceride comprised.In addition, lipid acid, for example oleic acid can be used in the injection equally.

[0191] in order to obtain the stable water-soluble formulation of pharmaceutical composition, the pharmacy acceptable salt of compound described herein can be dissolved in organic acid or the inorganic aqueous acid 0.3M succinic acid solution for example, perhaps more preferably citric acid solution.If can not obtain soluble salt form, then can be in suitable cosolvent or cosolvent combination with compound dissolution.The example of suitable cosolvent comprises ethanol, propylene glycol, Liquid Macrogol, polysorbate80, glycerine etc., and its concentration is about 0-60% of cumulative volume.In one embodiment, active compound is dissolved among the DMSO and dilute with water.

[0192] pharmaceutical composition can also be the solution form of salt form in suitable aqueous vehicles (for example water or isotonic saline solution or glucose solution) of activeconstituents.Also proposed to pass through replacement or addition chemistry or biochemical part and reformed compound, described chemistry or biochemistry partly for example make compound be more suitable for sending (for example improving solvability, biological activity, palatability, reduction side reaction etc.) by esterification, glycosylation, PEGization etc.

[0193] in a preferred embodiment, compound described herein can be mixed with the formulation in liquid form that is applicable to the low-solubility compound and be used for oral administration.Formulation in liquid form can strengthen the oral administration biaavailability of these compounds usually.

[0194] therefore, preferred pharmaceutical composition comprises the compound described herein of treatment or prevention significant quantity, together with at least a pharmaceutically acceptable vehicle (being selected from medium chain fatty acid or its propylene glycol ester, for example edible fatty acid such as sad and propylene glycol ester capric acid lipid acid) and pharmaceutically acceptable tensio-active agent (for example polyoxyethylene (40) hydrogenated castor oil).

[0195] in the alternate preferred embodiment, can add cyclodextrin as water-soluble toughener.Preferred cyclodextrin comprise α-, β-and hydroxypropyl, hydroxyethyl, glucosyl, malt-base and the trisaccharide maltose radical derivative of γ-Huan Hujing.Particularly preferred cyclodextrin solubility enhancer is hydroxypropyl-o-cyclodextrin (BPBC), and it can join in any above-mentioned composition, with the further water-soluble feature of improving the embodiment compound.In one embodiment, composition comprises hydroxypropyl-o-cyclodextrin of about 0.1% to about 20%, 1% to about 15% hydroxypropyl-o-cyclodextrin more preferably from about, even 2.5% to about 10% hydroxypropyl-o-cyclodextrin more preferably from about.The amount of used solubility enhancer depends on the amount of the embodiment compound in the composition.

[0196] the activeconstituents total amount that contains of pharmaceutical composition is enough to realize expected effect.More particularly, in certain embodiments, pharmaceutical composition contains the treatment significant quantity, and (for example, the amount that SAPK regulates compound is the symptom of prevention or treatment inflammatory diseases or illness effectively, wherein the IC of the inhibition p38MAPK that shows of this compound ₅₀At about 100 μ M to the scope of about 1000 μ M, preferably at about 200 μ M to the scope of about 800 μ M).Can be with carrier substance combination and the total amount of the compound of production unit formulation becomes with the host and the concrete administering mode of treatment.Preferably, compositions formulated, thus the SAPK of dosage between the 0.01-100mg/kg body weight/day regulated the patient that compound is accepted said composition.

Embodiment 1

[0197] screens in external ability according to compound by p38MAP kinase inhibition ATF2 phosphorylation.The ability of compound inhibition ATF2 phosphorylation is relevant with TNF alpha expression in kinase whose restraining effect of p38MAP and the body in this external test, therefore and become the active indicator (Raingeaud of potential interior therapeutic, J. etc., 1995 J.Biol.Chem.270:7420-7426; Brinkman, M.N. etc., 1999 J.Biol.Chem.274:30882-30886; And Fuchs, S.Y. etc., J.Biol.Chem.275:12560-12564,2000).

[0198] all kinases and substrate A TF2 all derive from Upstate (Charlottesville, VA).The p38MAP kinases is to have the recombinant human full-length proteins that N-terminal GST merges, and it is at expression in escherichia coli and be purified.ATF2 is the gst fusion protein that contains at the amino acid/11 9-96 of the people ATF2 of e. coli expression.All albumen are divided into sample aliquot and are stored in-80 ℃.

[0199] use contains 25mM HEPES pH 7.5,10mM MgCl ₂, 2mMDTT, 20mM β-glycerophosphate, 0.1mM Na ₃VO ₄, 40 μ M ATP and 1.25 μ MATF2 and 6ng p38 α albumen, 12ng p38 β albumen, 1.5ng p38 γ or 0.4ngJNK2 α 2 the mensuration damping fluid carry out the p38MAP kinase assays.The compound serial dilution in DMSO, and is used the test compound of 2 μ L different concns.DMSO is only accepted in the solvent contrast.

[0200] with test compound and 20 μ l enzymes in kinase buffer liquid (25mM HEPESpH 7.5,10mM MgCl ₂, 2mM DTT, 20mM β-glycerophosphate and 0.1mMNa ₃VO ₄) in solution in room temperature preincubation 15 minutes.Obtain the final concentration of 40 μ M ATP and 1.25 μ M ATF2 by in kinase buffer liquid, adding 30 μ l substrate solutions, thus initiation reaction.With reactant in 37 ℃ of incubations 30 minutes, and by adding the EDTA termination reaction of 18 μ l 200mM.Use the ELISA method to measure the phosphorylation of ATF2 to Thr 69.Reached 1 hour in 37 ℃ of bags by high bonded 96 orifice plates with 50 μ l kinase reaction things.With 200 μ l lavation buffer solutions (25mM Tris HCl pH 8.3,192mM glycine, 0.1%SDS and 0.05% tween (Tween)-20) washing bag by plate three times.Use TBS solution (Pierce, the 37535) wash plate three times of SuperBlock then.After the sealing, with plate and 50 μ l rabbits anti-phosphoric acid ATF2 antibody (CellSignaling, 9221L, 1: 500) in 37 ℃ of incubations 30 minutes.

[0201], puts together goat anti-rabbit antibodies (Cell Signaling, 7074,1: of HRP afterwards with 50 μ l 500) in 37 ℃ of incubations 30 minutes with lavation buffer solution wash plate three times.Use the lavation buffer solution wash plate then three times, afterwards in room temperature and 50 μ l Ultra TMB-ELISA (Pierce, 34028) incubations 8 minutes.At last, add 50 μ l phosphoric acid (1M) termination reactions, and read the plate instrument with SpectraMax 250 and read the plate absorbancy in 450nm.

[0202] phosphorylation of compound inhibition ATF2 in this external test.The IC that preferred compound shows ₅₀Value is less than about 100 μ M, preferably less than about 1 μ M.

Embodiment 2

[0203] screens by the ability of the THP-1 cell release of lipopolysaccharides (LPS) stimulation at vitro inhibition TNF α according to compound.The ability that compound inhibition TNF α discharges in this external test is relevant with the inhibition of active to p38 in TNF α, and therefore becomes the active indicator of potential interior therapeutic (Lee J.C. etc., 1993Ann.N.Y.Acad.Sci.696:149-170; With 1994 Nature 372:739-746).

[0204] will derive from the THP-1 cell (TIB202) of ATCC in 37 ℃, 5%CO ₂Maintain down RPMI 1640 substratum that contain 4.5g/L glucose and add 10% foetal calf serum, 1% penicillin/streptomycin and 50 μ M beta-mercaptoethanols (MediaTech, Herndon, VA) in.

When [0205] beginning, test compound is dissolved in the RPMI substratum that contains 1%DMSO (volume/volume).Then, the compound serial dilution in the RPMI substratum, is used for all follow-up extent of dilution.Under aseptic condition, measure.The collection culture density is 6-8 * 10 ⁵The THP-1 cell of individual cell/ml, and with 10 ⁶Individual cell/ml is resuspended in the RPMI substratum.The resuspension cell of 100 μ l is added in each hole, and 100 μ l test compounds are equipped with in each hole.Prepare test compound with two times of final concentrations.Final DMSO concentration is not more than 0.5% (volume/volume).Before stimulating with lipopolysaccharides (LPS) (Sigma L-2880, the PBS mother liquor of 4mg/ml), cell and compound are at 37 ℃, 5%CO ₂Following preincubation 60 minutes.The final LPS concentration that is respectively applied for TNF α and IL-1 β release in each hole is respectively 10 μ g/ml or 30 μ g/ml.The stimulated control cell suspending liquid is not only accepted the PBS solvent.Discharge at TNF α and IL-1 β, cell mixture was cultivated respectively 18 or 48 hours.Get 150 μ l supernatant liquors and be transferred in the new plate, be stored in-20 ℃, until further analysis.Use the ELISA test kit to measure TNF α and IL-1 β level.Light-emitting device is as reading the plate device.Analyze with non-linear regression, obtain dose-response curve.IC ₅₀Calculated value is to make TNF α or IL-1 β level reduce by 50% test compound concentration.

[0206] in this external test, compound suppresses these two release of TNF α or IL-1 β or TNF α and IL-1 β.The IC that preferred compound demonstrates at TNF α and/or IL-1 β ₅₀Value is less than 100 μ M, preferably less than about 1 μ M.

Embodiment 3

[0207] screens from the ability of former generation human peripheral blood mononuclear cell (PBMC) release of lipopolysaccharides (LPS) stimulation at vitro inhibition TNF α according to compound.In this external test, the ability that compound suppresses TNF α release suppresses relevant with p38 is active, and therefore becomes the active indicator of potential interior therapeutic (2002 Osteoarthritis; Cartilage 10:961-967; And Laufer, S.A. and Wagner, G.K.2002 J Med.Chem.45:2733-2740).

[0208] differential centrifugation by carrying out through the Ficoll-HyPaque density gradient of the individual blood donor's of 3-8 name combining anteserum is isolated human peripheral blood mononuclear cell (PBMC).Isolating PBMC contain the positive monocyte of 10% the CD-14 of having an appointment, 90% lymphocyte and＜1% granulocyte and thrombocyte.Under the situation of the test compound that has and do not exist serial dilution, with duplicate GIBCO at serum-free ^TM(Invitrogen, Carlsbad cultivate on polystyrene board and with lipopolysaccharides (LPS in 37 ℃ in CA) the RPMI substratum; 50ng/ml; Sigma, St.Louis MO) stimulates PBMC (10 ⁶Individual/as ml) to reach 24 hours.Use is purchased test kit (MDSPanlabs#309700) by the TNF alpha levels in the ELISA mensuration cell conditioned medium liquid.

[0209] in this is measured, preferred compound suppresses the IC that TNF α discharges ₅₀Value is less than about 100 μ M, preferably less than about 1 μ M.

Embodiment 4

[0210] according to compound suppress in the animal model in vivo the ability that TNF α discharges screen (referring to for example Griswold D.E. etc., 1993 Drugs Exp.Clin.Res.19:243-248; Badger, A.M. etc., 1996 J.Pharmacol.Exp.Ther.279:1453-1461; Dong, C. etc., 2002 Annu.Rev.Immunol.20:55-72 (with the reference of wherein quoting); Ono, K. and Han, J.2000 Cellular Signalling 12:1-13 (with the reference of wherein quoting); And Griffiths, J.B. etc., 1999 Curr.Rheumatol Rep.1:139-148).

[0211] though do not wish to be subjected to the constraint of any concrete theory, it is generally acknowledged that this model comes from compound to the kinase whose inhibition of p38MAP to the inhibition of TNF α.

[0212] male Sprague-Dawley rat (0.2-0.35kg) is divided into 6 groups or more groups at random, and by intravenous drip or inject administration, perhaps in all cases with test compound with suitable preparation oral administration.Instil or inject finished back 30 minutes and oral administration after 1-2 hour, IV gives lipopolysaccharides intestinal bacteria/0127:B8 (0.8mg/kg).Handle back 1.5 hours collection blood samples with LPS.Use the TNF alpha levels of ELISA test kit (KRC3011C) the mensuration serum of Biosource, and compare with this level of vehicle treated contrast.

[0213] suppressing TNF α during preferred compound is measured in this body discharges.The ED that preferred compound demonstrates ₅₀Value preferably less than 400mg/kg, preferably less than 200mg/kg, preferably less than 100mg/kg, is more preferably less than 50mg/kg less than 500mg/kg, is more preferably less than 40mg/kg, is more preferably less than 30mg/kg, is more preferably less than 20mg/kg, is more preferably less than 10mg/kg.

[0214] measures the IC that compound suppresses p38 ₅₀Method comprise any method of any downstream substrate of the above-mentioned p38MAPK of permission detection by quantitative known in the art.Therefore, these methods include but not limited to detect the genetic expression that is subjected to the independent adjusting of p38 or is regulated by the gene array in addition.

Embodiment 5

Kinase assays

[0215] exists ³²P-γ-ATP exists down, determines the kinases isomer P38 γ of P38 and the activity of P38 α by the phosphorylation of ATF-2.Under the situation that has or do not exist inhibitor, determine ³²P is to the situation of mixing of ATF-2.Test compound described herein is to P38 γ and the active inhibition of P38 alpha kinase in this biochemical measurement.Compound dissolution in water or DMSO, and is used and to be suitable for diluting and as the different concns test with 0-10mM of the solvent of solvent contrast.Obtain as the enzyme P38 γ of the activation and the recombinant protein of purifying and P38 α (Upstate, Charlottesville, VA).In end reaction, use the activating enzymes of 24.8nM.Dilute enzyme before being to react in the following damping fluid (1M HEPES pH 7.4,500mM DTT, 1%Triton X-100 and 10mg/mlBSA) earlier.Be reflected in the following solution that is prepared into 2 times of stostes (1M HEPESpH 7.4,500mM DTT and 1%Triton X-100) and carry out, and the on-radiation ATP of existence 6.25 μ M ATP in the reaction (Cell Signaling, Beverly, MA).In order to determine the phosphorylation of ATF-2, with 7.5 μ M concentration ³²P-γ-ATP 3000Ci/mmol joins in each reaction.(Cell Signaling, Beverly is MA) as kinase substrate to use 3 μ M ATF-2.As the first step of combination enzyme reaction, activated protein kinase and inhibitor or suitable solvent contrast are joined in the reaction buffer, and incubation 30 minutes at room temperature.Add ATF-2 and ATP mixture and cause kinase reaction.The final volume of each reaction is 20 μ l, and at room temperature carries out 30 minutes.After 30 minutes incubations, add 80 μ l Laemmlie damping fluids.Under reductive condition, use SDS-Page (BioRad, Hercules, CA) reactant of separation 20% subsequently.Behind electrophoresis, that gel is right in room temperature ³²The exposure of spending the night of P-intensifying screen, and (Amersham Biosciences, Piscataway NJ) analyze to use phosphoroscope Storm System.The quantitative signal that obtains after background correction uses the kinase activity and the solvent contrast that do not suppress to suppress to be calculated as the inhibition percentage as 0%.Exist under the situation of different inhibitor concentration, (Synergy Software, Reading is PA) to the kinase activity mapping, to determine the EC of every kind of compound to use Kaleidagraph ₅₀, and test P38 kinases.

TNF α inductive suppresses

[0216] under the conventional organization culture condition that ATCC recommends, cultivate THP-1 (ATCC, Rockville, MD).In test preceding 18 hours, with cell with the density bed board of 500,000 cells/well in the conventional substratum that contains 1% serum and 0.25ml volume of culture of 96 well format.Compound is added (in triplicate) in each hole, all comprise the The suitable solvent contrast during each is measured.Each mensuration comprises the p38 inhibitor SB203850 of 1 μ M/ml, and (Upstate, Waltham is MA) as positive control.For inducing the TNF alpha expression, after adding compound, 1 μ g/ml LPS added to each hole in 30 minutes.Incubation by centrifugal (10 minutes, 1000rpm, Beckman desk centrifuge) sedimentation cell, and was collected a part of acellular supernatant liquor after 4 hours under conditions of tissue culture, was used for the quantitative (R﹠amp at TNF alpha specific ELISA with 10 times of extent of dilution; DSystems, Minneapolis, MN).Carry out TNF α ELISA according to the guide that manufacturers provides.Detect TNF α (pg/ml), and with it as mapping to the standardized mark of the TNF alpha expression in the solvent control is active.

The toxicity of compound of in mensuration, testing based on cell

[0217] LDH that will produce owing to the cytolemma of fragmentation discharges as Cytotoxic measurement.Use commercially available diagnostic kit (Roche Diagnostics, catalog number (Cat.No.) 1 644 793) to detect LDH by its enzymic activity.For consistent, use the THP-1 cell to determine cytotoxicity with inductive TNF alpha expression in the previous test.As before TNF α being induced the description that suppresses test, be under 1% serum and the conventional organization culture condition with 96 well format culturing cells.The compound that adds different concns, triplicate.Each is measured and uses the The suitable solvent contrast.Added behind the compound under the conventional organization culture condition culturing cell 18 hours.Behind this cultivation stage, Triton-X-100 (2% volume/volume) is added to untreated cell starts positive control, and incubation 10 minutes again, with complete lysing cell.Pass through centrifugation cell subsequently, and take out a part of supernatant liquor, analyze the LDH enzymic activity according to the explanation of manufacturers.Data are typically expressed as the % cytotoxicity, and being normalized to the Triton-X-100 lysing cell is 100% cytotoxicity.

Embodiment 6

[0218] formula (1) compound as directly over shown in flow process synthetic.The 3-acyl pyridine (compound 2) that R-replaces derives from commercial source or through the currently known methods preparation, itself and ethyl thioglycolate anionic reactive, formation thieno-[2,3-b] pyridine derivate (compound 3).Compound 3 is oxidized to N-oxide compound (compound 4), and it changes pyridone (compound 5) succeeded by with posthydrolysis the time into handling with diacetyl oxide.In the presence of venus crystals, use currently known methods, for example the method for flow process 1 uses the 4-chlorophenylboronic acid that compound 5 is carried out the N-arylation.Compound 1 is a formula I examples for compounds.Other example of formula I compound can synthesize in a similar manner.

Embodiment 7

[0219] formula (9) compound as directly over shown in flow process synthetic.Make 2-methylthio group-4-methylpyrimidine deprotonation with lithium diisopropylamine, then with suitable N, O-dimethyl hydroxyl acid amides (compound 1) is handled, and obtains corresponding ketone (compound 2).The latter reacts by the acetic acid solution with Sodium Nitrite changes α-oximinoketone (compound 3) into.Piperidines-4-the formaldehyde condensation of compound 3 and N-carbobenzoxy-(Cbz) (Cbz)-protection; obtain N-hydroxyl imidazoles (compound 4), its by reduction under typical conditions with form imidazoles intermediate (compound 5) and after connect to methylate and change N-methyl-imidazoles (compound 6) into.With after forming sulfone (compound 7), methylsulfonyl is replaced by amine, obtains aminopyridine derivative (compound 8) in oxidation.Last protecting group of sloughing piperidines nitrogen in the acetic acid solution of standard conditions such as HBr obtains the imidazoles (compound 9) that target replaces.Compound 9 is a formula II examples for compounds.Other example of formula II compound can synthesize in a similar manner.

Embodiment 8

[0220] formula (4) compound according to currently known methods (referring to J.Org.Chem., 62,17,5908-5919) as directly over shown in flow process synthetic.Pinacolone negatively charged ion and ethyl oxalate reaction obtain 5,5-dimethyl-2, the ethyl acetate of 4-dioxo caproic acid (compound 1).Compound 1 obtains pyrazoles (compound 2) with the suitable hydrazine reaction that replaces, and it changes target compound 4 into by common method.Compound 4 is formula III examples for compounds.Other example of formula III compound can synthesize in a similar manner.

Embodiment 9

[0221] formula (4) compound as directly over shown in flow process synthetic.Benzenyl amidine (compound 1) and α-chlorine ketone (compound 2) reaction by with ethanol/ammonia treatment 4-methylthiomethyl ethyl benzoate preparation obtain imidazoles (compound 3).Obtain target compound 4 in oxidation intermediates 3 backs down in standard conditions (Potassium Persulphate for example).Compound 4 is a formula IV examples for compounds.Other example of formula IV compound can synthesize in a similar manner.

Embodiment 10

[0222] short hairpin RNA (shRNA) with target p38a or p38 γ imports in the culturing cell.MISSION ^TMTRC shRNA slow virus particle derive from Sigma Aldrich (St.Louis, MO).These are designed for by U6 promoter expression shRNA from inactivation replication-defective virus particle, and allow tetracycline to select cells infected.The shRNA that is used for this research is designed for target people p38 γ (the slow virus particle that contains the coding region of SEQ ID No:1-4), people p38 α (the slow virus particle that contains the coding region of SEQ ID No:5-8).ShRNA of first contrast slow virus particle coding carries at least 5 mispairing at all known person and little musculus cdna, and not any known people of target or little musculus cdna.Second contrast slow virus particle do not expressed shRNA.The 3rd the contrast slow virus particle that is designed for expressing green fluorescent protein (GFP) is used to optimize infectious condition.

[0223] (ATCC, Rockville is MD) in 37 ℃ and 5%CO for the HFL1 cell ₂Remain in down the F12K substratum of adding L-glutaminate, 10% foetal calf serum, sodium bicarbonate (1.5g/L) and penicillin/streptomycin (MediaTech, Herndon, VA) in.Cell preceding 1 day of infection by bed board in 24 orifice plates, about 2/3 converges when infecting.For infecting, discard former substratum, be replaced by and contain 0-12 μ g/mL Polybrene (fresh culture MO) is that 1-5 adds the slow virus particle with infection multiplicity (MOI) then for Sigma Aldrich, St.Louis.After 24 hours, discard the infection substratum, be replaced by fresh culture.Infect the back step for all, cell goes down to posterity as required, with stoste is remained on 5 and converge fully between.Between metainfective 1-3 days, cell is placed under the tetracycline selection of 0.25-1 μ g/mL final concentration, described final concentration is determined based on the tetracycline susceptibility of non-infected cells.Select to keep 5-10 days, chosen process is estimated by observing with the cell that is designed for the slow-virus transfection of expressing GFP and the loss of viability of non-infected cells.The HFL1 cell is infected (virus-free) by vacation, or gives the tetracycline resistance and express one of following slow virus infection with being designed for: the shRNA (SEQ IDNO:1-4) of the shRNA of target people p38 α (SEQ ID NO:5-8), target people p38 γ, not target known person or little musculus cdna shRNA, do not have and express (contrast is viral) or green fluorescent protein.

[0224] the NIH3T3 cell (ATCC.Rockville, MD) remain on the DMEM substratum of adding L-glutaminate, 10% foetal calf serum, sodium bicarbonate (1.5g/L) and penicillin/streptomycin (MediaTech, Herndon, VA) in.Cells infected, and carry out aforesaid tetracycline and select.The NIH3T3 cell is infected (virus-free) by vacation, or gives the tetracycline resistance and express one of following slow virus infection with being designed for: the shRNA (SEQ ID NO:1-4) of the shRNA (SEQID NO:5-8) of target mouse p38 α, target mouse p38 γ, not target known person or little musculus cdna shRNA, do not have and express (contrast is viral) or green fluorescent protein.

Embodiment 11

[0225] separating mRNA and use quantitative PCR to measure the mRNA level.(Ambion, Austin TX), according to manufacturer's specification sheets, obtain RNA by about 15,000～150,000 cell to use RNaqueous 96 RNA separating kits.Use TaqMan

(NJ) with the random hexamer primer, the specification sheets according to the manufacturer changes RNA into cDNA to reverse transcription reagent for Applied Biosystems, Branchburg.According to manufacturer's specification sheets, use above cDNA, the commercially available probe primer group (TaqMan that obtains

Gene expression arrays, Applied Biosystems) and TaqMan

Universal PCR Master Mix (AppliedBiosystems, Branchburg, NJ), at Applied Biosystems 7900HT sequence detection system (Applied Biosystems, Branchburg carries out the quantitative PCR reaction on NJ), and is triplicate.Use is designed for amplification people glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Endogenous contrast), probe/primer sets of people p38 α and people p38 γ, to 3 blocks of plates of HFL1 cell manipulation.Use is designed for amplification mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Endogenous contrast), probe/primer sets of mouse p38 α and mouse p38 γ, to 3 blocks of plates of NIH3T3 cell manipulation.Data use Δ Δ Ct method to analyze, and wherein import the level of RNA and use glyceraldehyde-3-phosphate dehydrogenase as endogenous reference standardization.Cycle threshold (Ct) derives from the geometry phase of amplification figure.The level relatively of genetic expression uses following formula to obtain:

Δ Δ Ct=(mean value Ct _{Analyte GAPDH}-mean value Ct _{Contrast GAPDH})-(mean value Ct _{Analyte p38}-mean value _{Ct contrasts P38})

Wherein

Mean value Ct _{Analyte GAPDH}Average Ct value for analyte (slow virus is handled) clone that adopts GAPDH probe primer group

Mean value Ct _{Contrast GAPDH}Average Ct value for the control cells (untreated) that adopts GAPDH probe primer group

Mean value Ct _{Analyte p38}Average Ct value for analyte (slow virus is handled) clone that adopts p38 (α or γ) probe primer group

Mean value Ct _{Contrast P38}Average Ct value for the control cells (untreated) that adopts p38 probe primer group

[0226] data report is the relative p38mRNA level of the cell of slow virus processing when using the endogenous contrast of GAPDH to proofread and correct the input rna level than untreated cell.The p38mRNA level that selected shRNA construct is observed provides in embodiment 12 tables 1 below.

Embodiment 12

[0227] mensuration of collagen level.Normal HFL1 cell or the cell selected with slow virus infection and with tetracycline are in 37 ℃ and 5%CO ₂Down in the F12K substratum of adding L-glutaminate, 10% foetal calf serum, sodium bicarbonate (1.5g/L) and penicillin/streptomycin (MediaTech, Herndon, VA) middle growth.Measure for collagen, with cell with about 6 * 10 ⁵In the density bed board of the individual cells/well hungry substratum in the 2mL/ hole in 6 orifice plates (adding the F12K substratum of L-glutaminate, 0.1% foetal calf serum, sodium bicarbonate (1.5g/L) and penicillin/streptomycin).After 16-20 hour, discard former substratum, be replaced by add 20 μ g/mL xitix (EM Science, Gibbstown, NJ) and 10 μ M L-proline(Pro) (Calbiochem, L Jolla, fresh hungry substratum CA).After changing this substratum 1 hour, with 5ng/mL (10ng/ hole) transforming growth factor-beta 1 (TGF-β; Chemicon/Millipore, Billerica, MA or Sigma Aldrich, St.Louis MO) handles cell.40-48 hour removal cell culture medium after adding TGF-β, and with cell 250 μ L add 1.25%Triton X-100 (Fisher, Fair Lawn, NJ) and whole protein enzyme inhibitors mixture (Roche, Mannheim carries out cracking in phosphate-buffered saline Germany) (PBS).Use on cell curette or the transfer pipet point slave plate and scrape cell, lysate is in-20 ℃ of refrigerations.

[0228] handle in a similar manner with slow virus infection and NIH3T3 cell and the NIH3T3 cell selected with tetracycline, just (Mediatech, Herndon VA) substitute the F12K substratum by DMEM.The DMEM substratum is added other component as above F12K.

[0229] Shou Huo cell thawing Cheng Bing.The mixing in the 1.7mL micro-centrifuge tube with 200 μ L samples and isopyknic 0.5M acetate.Manufacturer's scheme that collagen concentration uses the Sircol soluble collagen to measure (Biocolor Ltd, Newtonabbey, Northern Ireland) use change is determined.In brief, add 1ml Sircol dye reagent, jolting sample 30-60 minute (time span all is constant to all samples in the test).Use desk centrifuge with the centrifugal sample of about 10,000 * g 30 minutes then.Use the transfer pipet point to remove unconjugated dyestuff from precipitation, careful operation is to avoid the disturbance precipitation.Centrifugal again sample 4 minutes is removed remaining not combination dye by in precipitating carefully with transfer pipet.Add 200 μ L manufacturers' alkaline reagents then, the jolting sample is until resolution of precipitate.Typical curve is set up by the collagen standard substance (Biocolor Ltd, Newtonabbey, Northern Ireland) that use known quantity in such scheme.100 μ L standard substance or sample are added to 96 transparent orifice plates, use μ Quant spectrophotometer (Bio-Tek, Winooski, VT) the sample absorbancy of mensuration 540nm.

[0230] as seen, p38 α or p38 γ express to reduce and cause the collagen level to reduce, and p38 α and the increase of p38 γ level cause the collagen level to increase by following table 1 and Fig. 1 and 2.Therefore, these data acknowledgements for example suppress p38 γ and can cause the collagen level to reduce, and therefore reduced fibrosis.

Table 1. adopts the p38mRNA of different shRNA constructs and the expression of collagen

Sequence	Infection multiplicity (MOI)	Inductive collagen (μ g) not	Standard deviation	The collagen (μ g) that TGF-is beta induced	Standard deviation	Relative p38 γ	Relative p38 α
Sequence	Infection multiplicity (MOI)	Inductive collagen (μ g) not	Standard deviation	The collagen (μ g) that TGF-is beta induced	Standard deviation	Relative p38 γ	Relative p38 α	SEQ ID NO：1	5	9.91	2.61	19.76	2.42	0.5	1.15
SEQ ID NO：1	2	10.60	2.36	22.91	1.18	0.52	1.36	SEQ ID NO：1	5	9.91	2.61	19.76	2.42	0.5	1.15
SEQ ID NO：1	2	10.60	2.36	22.91	1.18	0.52	1.36	SEQ ID NO：6	2	12.87	0.72	13.18	1.09	1.37	0.59
The empty carrier contrast	5	19.75	3.15	29.33	2.45	1.35	1.39	SEQ ID NO：6	2	12.87	0.72	13.18	1.09	1.37	0.59
The empty carrier contrast	5	19.75	3.15	29.33	2.45	1.35	1.39	Non-target contrast	2	21.11	1.68	31.18	2.78	1.04	1.2
Non-target contrast	5	20.56	1.85	34.67	4.95	1.19	1.19	Non-target contrast	2	21.11	1.68	31.18	2.78	1.04	1.2
Non-target contrast	5	20.56	1.85	34.67	4.95	1.19	1.19	SEQ ID NO：4	5	22.59	1.35	26.73	2.31	1.15	1.28
SEQ ID NO：2	2	25.61	2.54	46.74	2.73	1.38	1.58	SEQ ID NO：4	5	22.59	1.35	26.73	2.31	1.15	1.28
SEQ ID NO：2	2	25.61	2.54	46.74	2.73	1.38	1.58	SEQ ID NO：7	5	26.47	1.57	53.20	7.00	0.96	2.27
SEQ ID NO：7	2	34.49	3.92	60.04	5.45	1.38	1.14	SEQ ID NO：7	5	26.47	1.57	53.20	7.00	0.96	2.27

Embodiment 13

[0231] estimated of the inhibition of several compounds to p38 α and p38 γ MAPK isomer.The mensuration that use is described in the embodiment 5 of WO 2006/122154 is determined IC ₅₀Value, this patent are attached to herein by reference in full.The results are shown in Table 2.

The p38 restraining effect of the selected compound of table 2.

¹The aryl that is connected on the 2-pyridone nitrogen is a N-picoline part

²5 and 6 of bromine aryl and 2-pyridone ring condense.

NC (not calculating) is meant the value that exceeds upper limit of quantification, owing to too greatly consequently can not determine reliably

[0232] although the present invention has been made detailed description, it should be appreciated by those skilled in the art that under the situation that does not depart from true scope of the present invention, can implement various variations form of the present invention and details in order to be aware and understand purpose.Institute above-mentioned drawings attached, form, appendix, patent, patent application and publication all are attached to herein by reference.

Sequence table

＜110〉Intermune Inc. (InterMune, Inc.)

＜120〉control method of stress activated protein kinase system

<130>INTMU.028VPC

<150>US 60/793,526

<151>2006-04-20

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Claims

1. the compound of a following formula:

Wherein R is selected from H, halogen, cyano group, nitro, hydroxyl, the optional C that replaces _1-6Alkyl, the optional C that replaces _3-7Cycloalkyl, the optional C that replaces _4-10Alkyl-cycloalkyl, the optional C that replaces _2-6Thiazolinyl, the optional C that replaces _1-6Alkoxyl group, the optional C that replaces _{6 or 10}Aryl; the optional pyridyl that replaces; the optional pyrimidyl that replaces; the optional thienyl that replaces; the optional furyl that replaces; the optional thiazolyl that replaces; the optional De oxazolyl that replaces; the optional phenoxy group that replaces; the optional sulfo-phenoxy group that replaces; the optional amino-sulfonyl that replaces; the optional urea groups that replaces; the optional thioureido that replaces; the optional amide group that replaces; the optional ketone group that replaces; the optional carboxyl that replaces; the optional formamyl that replaces; the optional thioether that replaces; the optional sulfoxide that replaces; the optional sulfone that replaces; the optional amino that replaces; the optional alkoxy amino that replaces; the optional alkoxyl group heterocyclic radical that replaces; the optional alkylamino that replaces; the optional alkyl carboxyl that replaces; the optional carbonyl that replaces; the optional volution cycloalkyl that replaces; the optional pyrazinyl that replaces; the optional pyridazinyl that replaces; the optional pyrryl that replaces; the optional thienyl that replaces; the optional thiazolyl that replaces; the optional De oxazolyl that replaces; the optional imidazolyl that replaces; the optional De isoxazolyl that replaces; the optional pyrazolyl that replaces; the optional isothiazolyl that replaces; the optional naphthyl that replaces; the optional quinolyl that replaces; the optional isoquinolyl that replaces; the optional quinoxalinyl that replaces; the optional benzothiazolyl that replaces; the optional benzothienyl that replaces; the optional benzofuryl that replaces; optional indyl that replaces and the optional benzimidazolyl-that replaces.

2. the compound of a following formula:

R wherein ⁴Be selected from H, halogen, cyano group, nitro, hydroxyl, the optional C that replaces _1-6Alkyl, the optional C that replaces _3-7Cycloalkyl, the optional C that replaces _4-10Alkyl-cycloalkyl, the optional C that replaces _2-6Thiazolinyl, the optional C that replaces _1-6Alkoxyl group, the optional C that replaces _{6 or 10}Aryl; the optional pyridyl that replaces; the optional pyrimidyl that replaces; the optional thienyl that replaces; the optional furyl that replaces; the optional thiazolyl that replaces; the optional De oxazolyl that replaces; the optional phenoxy group that replaces; the optional sulfo-phenoxy group that replaces; the optional amino-sulfonyl that replaces; the optional urea groups that replaces; the optional thioureido that replaces; the optional amide group that replaces; the optional ketone group that replaces; the optional carboxyl that replaces; the optional formamyl that replaces; the optional thioether that replaces; the optional sulfoxide that replaces; the optional sulfone that replaces; the optional amino that replaces; the optional alkoxy amino that replaces; the optional alkoxyl group heterocyclic radical that replaces; the optional alkylamino that replaces; the optional alkyl carboxyl that replaces; the optional carbonyl that replaces; the optional volution cycloalkyl that replaces; the optional pyrazinyl that replaces; the optional pyridazinyl that replaces; the optional pyrryl that replaces; the optional thienyl that replaces; the optional thiazolyl that replaces; the optional De oxazolyl that replaces; the optional imidazolyl that replaces; the optional De isoxazolyl that replaces; the optional pyrazolyl that replaces; the optional isothiazolyl that replaces; the optional naphthyl that replaces; the optional quinolyl that replaces; the optional isoquinolyl that replaces; the optional quinoxalinyl that replaces; the optional benzothiazolyl that replaces; the optional benzothienyl that replaces; the optional benzofuryl that replaces; optional indyl that replaces and the optional benzimidazolyl-that replaces.

3. the compound of a following formula:

R wherein ²And R ³Be selected from H, halogen, cyano group, nitro, hydroxyl, the optional C that replaces independently of one another _1-6Alkyl, the optional C that replaces _3-7Cycloalkyl, the optional C that replaces _4-10Alkyl-cycloalkyl, the optional C that replaces _2-6Thiazolinyl, the optional C that replaces _1-6Alkoxyl group, the optional C that replaces ₆ _{Or 10}Aryl, the optional pyridyl that replaces, the optional pyrimidyl that replaces, the optional thienyl that replaces, the optional furyl that replaces, the optional thiazolyl that replaces, the optional De oxazolyl that replaces, the optional phenoxy group that replaces, the optional sulfo-phenoxy group that replaces, the optional amino-sulfonyl that replaces, the optional urea groups that replaces, the optional thioureido that replaces, the optional amide group that replaces, the optional ketone group that replaces, the optional carboxyl that replaces, the optional formamyl that replaces, the optional thioether that replaces, the optional sulfoxide that replaces, the optional sulfone that replaces, the optional amino that replaces, the optional alkoxy amino that replaces, the optional alkoxyl group heterocyclic radical that replaces, the optional alkylamino that replaces, the optional alkyl carboxyl that replaces, the optional carbonyl that replaces, the optional volution cycloalkyl that replaces, the optional pyrazinyl that replaces, the optional pyridazinyl that replaces, the optional pyrryl that replaces, the optional thienyl that replaces, the optional thiazolyl that replaces, the optional De oxazolyl that replaces, the optional imidazolyl that replaces, the optional De isoxazolyl that replaces, the optional pyrazolyl that replaces, the optional isothiazolyl that replaces, the optional naphthyl that replaces, the optional quinolyl that replaces, the optional isoquinolyl that replaces, the optional quinoxalinyl that replaces, the optional benzothiazolyl that replaces, the optional benzothienyl that replaces, the optional benzofuryl that replaces, optional indyl that replaces and the optional benzimidazolyl-that replaces;

Wherein said compound is not 1-(the 5-tertiary butyl-2-p-methylphenyl-2H-pyrazole-3-yl)-3-[4-(2-morpholine-4-base-oxyethyl group) naphthalene-1-yl] urea (BIRB 796).

4. the compound of a following formula:

R wherein ⁵And R ⁶Be selected from H, halogen, cyano group, nitro, hydroxyl, the optional C that replaces independently of one another _1-6Alkyl, the optional C that replaces _3-7Cycloalkyl, the optional C that replaces _4-10Alkyl-cycloalkyl, the optional C that replaces _2-6Thiazolinyl, the optional C that replaces _1-6Alkoxyl group, the optional C that replaces ₆ _{Or 10}Aryl, the optional pyridyl that replaces, the optional pyrimidyl that replaces, the optional thienyl that replaces, the optional furyl that replaces, the optional thiazolyl that replaces, the optional De oxazolyl that replaces, the optional phenoxy group that replaces, the optional sulfo-phenoxy group that replaces, the optional amino-sulfonyl that replaces, the optional urea groups that replaces, the optional thioureido that replaces, the optional amide group that replaces, the optional ketone group that replaces, the optional carboxyl that replaces, the optional formamyl that replaces, the optional thioether that replaces, the optional sulfoxide that replaces, the optional sulfone that replaces, the optional amino that replaces, the optional alkoxy amino that replaces, the optional alkoxyl group heterocyclic radical that replaces, the optional alkylamino that replaces, the optional alkyl carboxyl that replaces, the optional carbonyl that replaces, the optional volution cycloalkyl that replaces, the optional pyrazinyl that replaces, the optional pyridazinyl that replaces, the optional pyrryl that replaces, the optional thienyl that replaces, the optional thiazolyl that replaces, the optional De oxazolyl that replaces, the optional imidazolyl that replaces, the optional De isoxazolyl that replaces, the optional pyrazolyl that replaces, the optional isothiazolyl that replaces, the optional naphthyl that replaces, the optional quinolyl that replaces, the optional isoquinolyl that replaces, the optional quinoxalinyl that replaces, the optional benzothiazolyl that replaces, the optional benzothienyl that replaces, the optional benzofuryl that replaces, optional indyl that replaces and the optional benzimidazolyl-that replaces;

Wherein said compound is not

Wherein X is N.

5. the compound of a following formula:

Wherein, at described compound during in conjunction with ρ 38 γ, R ⁴For distance p38 γ Met109 sulfydryl partly is no less than And be no more than

Localized group.

6. the compound of claim 5, wherein R ⁴Be selected from H, halogen, cyano group, nitro, hydroxyl, the optional C that replaces _1-6Alkyl, the optional C that replaces _3-7Cycloalkyl, the optional C that replaces _4-10Alkyl-cycloalkyl, the optional C that replaces _2-6Thiazolinyl, the optional C that replaces _1-6Alkoxyl group, the optional C that replaces _{6 or 10}Aryl; the optional pyridyl that replaces; the optional pyrimidyl that replaces; the optional thienyl that replaces; the optional furyl that replaces; the optional thiazolyl that replaces; the optional De oxazolyl that replaces; the optional phenoxy group that replaces; the optional sulfo-phenoxy group that replaces; the optional amino-sulfonyl that replaces; the optional urea groups that replaces; the optional thioureido that replaces; the optional amide group that replaces; the optional ketone group that replaces; the optional carboxyl that replaces; the optional formamyl that replaces; the optional thioether that replaces; the optional sulfoxide that replaces; the optional sulfone that replaces; the optional amino that replaces; the optional alkoxy amino that replaces; the optional alkoxyl group heterocyclic radical that replaces; the optional alkylamino that replaces; the optional alkyl carboxyl that replaces; the optional carbonyl that replaces; the optional volution cycloalkyl that replaces; the optional pyrazinyl that replaces; the optional pyridazinyl that replaces; the optional pyrryl that replaces; the optional thienyl that replaces; the optional thiazolyl that replaces; the optional De oxazolyl that replaces; the optional imidazolyl that replaces; the optional De isoxazolyl that replaces; the optional pyrazolyl that replaces; the optional isothiazolyl that replaces; the optional naphthyl that replaces; the optional quinolyl that replaces; the optional isoquinolyl that replaces; the optional quinoxalinyl that replaces; the optional benzothiazolyl that replaces; the optional benzothienyl that replaces; the optional benzofuryl that replaces; optional indyl that replaces and the optional benzimidazolyl-that replaces.

7. treat or the inflammatory diseases of prevention individuality or the method for fibrotic disease for one kind, described method comprises the compound of the claim 1 that gives described individual effective dose.

8. treat or the inflammatory diseases of prevention individuality or the method for fibrotic disease for one kind, described method comprises the compound of the claim 2 that gives described individual effective dose.

9. treat or the inflammatory diseases of prevention individuality or the method for fibrotic disease for one kind, described method comprises the compound of the claim 3 that gives described individual effective dose.

10. treat or the inflammatory diseases of prevention individuality or the method for fibrotic disease for one kind, described method comprises the compound of the claim 4 that gives described individual effective dose.

11. treat or the inflammatory diseases of prevention individuality or the method for fibrotic disease for one kind, described method comprises the compound of the claim 5 that gives described individual effective dose.

12. treat or the inflammatory diseases of prevention individuality or the method for fibrotic disease for one kind, described method comprises the compound of the claim 6 that gives described individual effective dose.

13. each method among the claim 7-12, wherein said inflammatory diseases or fibrotic disease are selected from fibrosis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, struvite pulmonary fibrosis, rheumatoid arthritis; Rheumatoid spondylitis; Osteoarthritis; Gout; Sepsis; Septic shock; Endotoxin shock; Gram negative sepsis; Toxic shock syndrome; Myofasical pain syndrome (MPS); Shigellosis; Asthma; Adult respiratory distress syndrome; Inflammatory bowel; Crohn's disease; Psoriasis; Eczema; Ulcerative colitis; Glomerulonephritis; Scleroderma; Chronic thyroiditis; Graves disease; Ormond's disease; Autoimmunity gastritis; Myasthenia gravis; Autoimmune hemolytic anemia; The autoimmunity neutropenia; Thrombocytopenia; Pancreatic gland fibrosis; Chronic active hepatitis; Hepatic fibrosis; Ephrosis; Renal fibrosis, irritable bowel syndrome; Pyreticosis; Restenosis; Cerebral malaria; Apoplexy and ischemic injury; Nerve injury; Alzheimer's disease; Huntington Chorea; Parkinson's disease; Acute and chronic pain; Transformation reactions; Cardiac hypertrophy, chronic heart failure; Acute coronary syndrome; Emaciation; Malaria; Leprosy; Leishmaniasis; Lyme disease; Reiter syndrome; Acute synovitis; Myodegeneration; Bursitis; Tendonitis; Tenosynovitis; Hernia, disruptiveness or prolapsus property disc syndrome; Osteopetrosis; Thrombosis; Silicatosis; The lung sarcosis; Bone resorption disease; Cancer; Multiple sclerosis, lupus; Fibromyalgia; AIDS; Varicella zoster virus infects, herpes simplex infections; Influenza infection; Severe acute respiratory syndrome (SARS); Cytomegalovirus infection; And diabetes.

14. a method of regulating stress activated protein kinase (SAPK) system, described method comprises makes compound contact with p38 mitogen-activated protein kinase (MAPK),

The IC of the inhibition p38 γ MAPK that wherein said compound shows ₅₀Value at about 10pM to the scope of about 500 μ M; With

The IC of the inhibition p38 α MAPK that wherein said compound shows ₅₀Value is to suppress the IC of p38 γ MAPK ₅₀The value at least 2 times.

15. the method for claim 14, the IC of the inhibition p38 γ MAPK that wherein said compound shows ₅₀Value at about 10pM to the scope of about 5 μ M.

16. the method for claim 14, the IC of the inhibition p38 γ MAPK that wherein said compound shows ₅₀Value at about 100nM to the scope of about 500 μ M.

17. a method of regulating stress activated protein kinase (SAPK) system, described method comprises makes compound contact with p38 mitogen-activated protein kinase (MAPK),

The IC of the inhibition p38 α MAPK that wherein said compound shows ₅₀Value is to suppress the IC of p38 γ MAPK ₅₀The value at least 5 times.

18. the method for claim 17, the IC of the inhibition p38 γ MAPK that wherein said compound shows ₅₀Value at about 10pM to the scope of about 5 μ M.

19. the method for claim 17, the IC of the inhibition p38 γ MAPK that wherein said compound shows ₅₀Value at about 100nM to the scope of about 500 μ M.

20. a method of regulating stress activated protein kinase (SAPK) system, described method comprises makes compound contact with p38 mitogen-activated protein kinase (MAPK),

The IC of the inhibition p38 α MAPK that wherein said compound shows ₅₀Value is to suppress the IC of p38 γ MAPK ₅₀The value at least 10 times.

21. the method for claim 20, the IC of the inhibition p38 γ MAPK that wherein said compound shows ₅₀Value at about 10pM to the scope of about 5 μ M.

22. the method for claim 20, the IC of the inhibition p38 γ MAPK that wherein said compound shows ₅₀Value at about 100nM to the scope of about 500 μ M.

23. a method of regulating stress activated protein kinase (SAPK) system, described method comprises makes compound contact with p38 mitogen-activated protein kinase (MAPK),

24. the method for claim 23, wherein

The IC of the inhibition p38 γ MAPK that described compound shows ₅₀Value at about 10pM to the scope of about 5 μ M; With

The IC of the inhibition p38 α MAPK that wherein said compound shows ₅₀Value at about 10pM to the scope of about 5 μ M.

25. the method for claim 23, wherein

The IC of the inhibition p38 γ MAPK that described compound shows ₅₀Value at about 100nM to the scope of about 500 μ M; With

The IC of the inhibition p38 α MAPK that wherein said compound shows ₅₀Value at about 100nM to the scope of about 1500 μ M.

26. each method among the claim 14-25, the wherein said concentration of regulating SAPK that is contacted with is carried out the IC of the inhibition p38 γ MAPK that wherein said compound shows ₅₀Value at about 50pM to the scope of about 1 μ M.

27. each method among the claim 14-25, the wherein said concentration of regulating SAPK that is contacted with is carried out the IC of the inhibition p38 γ MAPK that wherein said compound shows ₅₀Value at about 50pM to the scope of about 200nM.

28. also comprising, each method among the claim 14-27, described method give the experimenter with described compound.

29. a treatment or the method for preventing experimenter's morbid state, described method comprises

Discriminating is among the risk of inflammatory diseases or fibrotic disease or suffers from the experimenter of inflammatory diseases or fibrotic disease; With

Give the experimenter with compound with the significant quantity of treatment or prevention inflammatory diseases or fibrotic disease;

The IC of the inhibition p38 γ MAPK that wherein said compound shows ₅₀At about 10pM to the scope of about 500 μ M; With

30. the method for claim 29, the IC of the inhibition p38 γ MAPK that wherein said compound shows ₅₀At about 10pM to the scope of about 5 μ M.

31. the method for claim 29, the IC of the inhibition p38 γ MAPK that wherein said compound shows ₅₀At about 100nM to the scope of about 500 μ M.

32. a treatment or the method for preventing experimenter's morbid state, described method comprises

The IC of the inhibition p38 α MAPK that wherein said compound shows ₅₀Be the IC that suppresses p38 γ MAPK ₅₀At least 5 times.

33. the method for claim 32, the IC of the inhibition p38 γ MAPK that wherein said compound shows ₅₀At about 10pM to the scope of about 5 μ M.

34. the method for claim 32, the IC of the inhibition p38 γ MAPK that wherein said compound shows ₅₀At about 100nM to the scope of about 500 μ M.

35. a treatment or the method for preventing experimenter's morbid state, described method comprises

36. the method for claim 35, the IC of the inhibition p38 γ MAPK that wherein said compound shows ₅₀At about 10pM to the scope of about 5 μ M.

37. the method for claim 35, the IC of the inhibition p38 γ MAPK that wherein said compound shows ₅₀At about 100nM to the scope of about 500 μ M.

38. each method among the claim 29-37, the wherein IC of the inhibition p38 γ MAPK that shows of institute's administered compound ₅₀At about 50pM to the scope of about 1 μ M.

39. each method among the claim 29-37, the wherein IC of the inhibition p38 γ MAPK that shows of institute's administered compound ₅₀At about 50pM to the scope of about 200nM.

40. the method for an identification of pharmaceutical active compounds, described method comprises:

Library of compounds is provided;

41. the method for claim 40, the IC of the inhibition p38 γ MAPK that wherein said at least one compound shows ₅₀At about 10pM to the scope of about 5 μ M.

42. the method for claim 40, the IC of the inhibition p38 γ MAPK that wherein said at least one compound shows ₅₀At about 100nM to the scope of about 500 μ M.

43. the method for an identification of pharmaceutical active compounds, described method comprises:

Library of compounds is provided;

The IC of the inhibition p38 α MAPK that wherein said at least one compound shows ₅₀Be the IC that suppresses p38 γ MAPK ₅₀At least 5 times.

44. the method for claim 43, the IC of the inhibition p38 γ MAPK that wherein said at least one compound shows ₅₀At about 10pM to the scope of about 5 μ M.

45. the method for claim 43, the IC of the inhibition p38 γ MAPK that wherein said at least one compound shows ₅₀At about 100nM to the scope of about 500 μ M.

46. the method for an identification of pharmaceutical active compounds, described method comprises:

Library of compounds is provided;

The IC of the inhibition p38 α MAPK that wherein said at least one compound shows ₅₀Be the IC that suppresses p38 γ MAPK ₅₀At least 2 times.

47. the method for claim 46, the IC of the inhibition p38 γ MAPK that wherein said at least one compound shows ₅₀At about 10pM to the scope of about 5 μ M.

48. the method for claim 46, the IC of the inhibition p38 γ MAPK that wherein said at least one compound shows ₅₀At about 100nM to the scope of about 500 μ M.

49. the method for an identification of pharmaceutical active compounds, described method comprises:

Library of compounds is provided;

50. the method for claim 49, the IC of the inhibition p38 γ MAPK that wherein said at least one compound shows ₅₀At about 10pM to the scope of about 5 μ M.

51. the method for claim 49, the IC of the inhibition p38 γ MAPK that wherein said at least one compound shows ₅₀At about 100nM to the scope of about 500 μ M.

52. the method for an identification of pharmaceutical active compounds, described method comprises:

Library of compounds is provided;

53. the method for claim 52, the IC of the inhibition p38 γ MAPK that wherein said at least one compound shows ₅₀At about 10pM to the scope of about 5 μ M.

54. the method for claim 52, the IC of the inhibition p38 γ MAPK that wherein said at least one compound shows ₅₀At about 100nM to the scope of about 500 μ M.

55. the method for an identification of pharmaceutical active compounds, described method comprises:

Library of compounds is provided;

56. the method for claim 55, the IC of the inhibition p38 γ MAPK that wherein said at least one compound shows ₅₀At about 10pM to the scope of about 5 μ M.

57. the method for claim 55, the IC of the inhibition p38 γ MAPK that wherein said at least one compound shows ₅₀At about 100nM to the scope of about 500 μ M.

58. the method for an identification of pharmaceutical active compounds, described method comprises:

Library of compounds is provided;

From these a plurality of compounds, select at least one compound, the IC of the inhibition p38 α MAPK that wherein said at least one compound shows ₅₀At about 10pM to the scope of about 1500 μ M; With

59. the method for claim 58, the IC of the inhibition p38 γ MAPK that wherein said at least one compound shows ₅₀At about 10pM to the scope of about 5 μ M.

60. the method for claim 58, the IC of the inhibition p38 γ MAPK that wherein said at least one compound shows ₅₀At about 100nM to the scope of about 1500 μ M.

61. the method for an identification of pharmaceutical active compounds, described method comprises:

Library of compounds is provided;

62. the method for claim 61, the IC of the inhibition p38 γ MAPK that wherein said at least one compound shows ₅₀At about 10pM to the scope of about 5 μ M.

63. the method for claim 61, the IC of the inhibition p38 γ MAPK that wherein said at least one compound shows ₅₀At about 100nM to the scope of about 1500 μ M.

64. the method for an identification of pharmaceutical active compounds, described method comprises:

Library of compounds is provided;

65. the method for claim 64, the IC of the inhibition p38 γ MAPK that wherein said at least one compound shows ₅₀At about 10pM to the scope of about 5 μ M.

66. the method for claim 64, the IC of the inhibition p38 γ MAPK that wherein said at least one compound shows ₅₀At about 100nM to the scope of about 1500 μ M.

67. each method among the claim 40-66, the wherein said concentration of regulating SAPK that is contacted with is carried out the IC of the inhibition p38 γ MAPK that wherein said contact compound shows ₅₀At about 50pM to the scope of about 1 μ M.

68. each method among the claim 40-66, the wherein said concentration of regulating SAPK that is contacted with is carried out the IC of the inhibition p38 γ MAPK that wherein said contact compound shows ₅₀At about 50pM to the scope of about 200nM.

69. each method among the claim 40-68, described method also comprise the mammalian toxicity of measuring selected compound.

70. also comprising, each method among the claim 40-69, described method give the compound that the experimenter selectes.

71. the method for claim 70, wherein said experimenter suffer from inflammatory diseases or fibrotic disease be in inflammatory diseases or the fibrotic disease risk among.

72. each method among the claim 14-71, wherein said compound is not selected from:

And pirfenidone.

73. a pharmaceutical composition, described pharmaceutical composition contains:

A) compound of claim 1 or claim 2 or claim 3 or claim 4 or claim 5 or claim 6; With

B) pharmaceutically acceptable carrier.

74. a treatment or prevent the method for disease as herein described or illness, described method comprise among the claim 1-6 that gives individual effective dose each compound.