HK1164283A - Triazolopyridine derivatives as p38 map kinase inhibitors - Google Patents

Description

Triazolopyridine derivatives as P38MAP kinase inhibitors

Technical Field

The present invention relates to compounds and compositions that are p38 MAPK inhibitors, which are useful as anti-inflammatory agents in the treatment of respiratory diseases, as well as other diseases.

Background

Mitogen-activated protein kinases (MAPKs) constitute a family of serine/threonine kinases directed to proline, which activate their substrates by dual phosphorylation. There are 4 known human isoforms of p38MAP kinase: p38 α, p38 β, p38 γ and p38 δ. The p38 kinases, which are also known as cytokine inhibitory anti-inflammatory drug binding proteins (CSBP), Stress Activated Protein Kinases (SAPK) and RK, are responsible for phosphorylation (Stein et al, Ann. Rep. Med. chem., 1996, 31, 289-298) and activated transcription factors (such as ATF-2, MAX, CHOP and C/ERPb) as well as other kinases (such as MAPKAP-K2/3 or MK2/3) and are themselves activated by physical and chemical stresses (e.g. UV, osmotic stress), pro-inflammatory cytokines and bacterial Lipopolysaccharides (LPS) (Herlaar E. & Brown. Z., Molecular Medicine Today, 1999, 5, 439-447). The phosphorylated product of p38 has been shown to mediate the production of inflammatory cytokines, including tumor necrosis factor alpha (TNF α), interleukin- (IL-) -1, and cyclooxygenase-2 (COX-2). IL-1 and TNF α are also known to stimulate the production of other proinflammatory cytokines such as IL-6 and IL-8.

IL-1 and TNF α are biological substances produced by a variety of cells, such as monocytes or macrophages. IL-1 has been shown to mediate a variety of biological activities thought to be important for immune regulation and other physiological states such as inflammation (e.g., Dinarello et al, Rev. infection. disease, 1984, 6, 51). Excessive or unregulated TNF production (especially TNF α) has been implicated in mediating or exacerbating a number of diseases, and it is believed that TNF can cause or contribute to the general inflammatory effects. IL-8 is a chemokine produced by several cell types, including monocytes, fibroblasts, endothelial cells and keratinocytes. Its production from endothelial cells is induced by IL-1, TNF or Lipopolysaccharide (LPS). IL-8 stimulates a number of functions in vitro. It has been shown to have chemoattractant properties for neutrophils, T-lymphocytes and basophils. Increased production of IL-8 also causes chemotaxis of neutrophils to sites of inflammation in vivo.

The synthesis and/or action of several other pro-inflammatory proteins (e.g., IL-6, GM-CSF, COX-2, collagenase, and stromelysin) also require inhibition of signal transduction by p38 (as well as IL-1, TNF, and IL-8 described above), which is expected to be a highly effective mechanism for modulating excessive and destructive activation of the immune system. This expectation is supported by the potent and diverse anti-inflammatory activities described for the p38 kinase inhibitor (Badger et al, j. pharm. exp. thera., 1996, 279, 1453-. In particular, p38 kinase inhibitors have been described as potential therapeutic agents for rheumatoid arthritis. In addition to the link between p38 activation and chronic inflammation and arthritis, there is also data suggesting a role for p38 in the pathogenesis of airway diseases, particularly COPD and asthma. Stress stimuli, including tobacco smoke, infections or oxidation products, can cause inflammation within the pulmonary environment. Inhibitors of p38 have been shown to inhibit LPS and ovalbumin-induced airway TNF-. alpha., IL-1. beta., IL-6, IL-4, IL-5 and IL-13(Haddad et al, Br. J. Pharmacol., 2001, 132(8), 1715-; 1724; Underwood et al, am. J. physiol. Lung cell. mol.2000, 279, 895-. In addition, they significantly inhibit neutrophilia and MMP-9 release in LPS, ozone or cigarette smoke animal models. There is also a large body of preclinical data demonstrating the potential benefit of lung-related inhibition of p38 kinase (Lee et al, Immunopharmacology, 2000, 47, 185-. Thus, therapeutic inhibition of p38 activation may be important in the regulation of airway inflammation.

Chopra et al have reviewed the involvement of the p38 MAPK pathway in different diseases (Expert Opinion on Investigational Drugs, 2008, 17(10), 1411-. It is believed that the compounds of the invention are useful for the treatment of p38 mediated diseases such as: asthma, chronic or acute bronchoconstriction, bronchitis, acute lung injury and bronchiectasis, pulmonary hypertension, tuberculosis, lung cancer, general inflammation (e.g., inflammatory bowel disease), arthritis, neuroinflammation, pain, fever, fibrotic diseases, lung disorders and diseases (e.g., hyperoxic alveolar injury), cardiovascular diseases, post-ischemic reperfusion injury and congestive heart failure, cardiomyopathy, stroke, ischemia, reperfusion injury, renal reperfusion injury, cerebral edema, neurotrauma and brain trauma, neurodegenerative disorders, central nervous system disorders, liver diseases and nephritis, gastrointestinal disorders, ulcer diseases, crohn's disease, eye diseases, ophthalmic disorders, glaucoma, acute injury and eye trauma to eye tissue, diabetes, diabetic nephropathy, skin-related disorders, myalgia due to infection, myalgia due to inflammation, and congestive heart failure, Influenza, endotoxic shock, toxic shock syndrome, autoimmune diseases, graft rejection, bone resorption diseases, multiple sclerosis, psoriasis, eczema, disorders of the female reproductive system, pathological (but not malignant) conditions (such as hemangiomas, angiofibromas of the nasopharynx and avascular necrosis), benign and malignant tumors/neoplasias (including cancers, leukemias, lymphomas), Systemic Lupus Erythematosus (SLE), angiogenesis (including neoplasias), hemorrhage, coagulation, radiation damage and/or metastasis. Chronic release of active TNF can cause cachexia and anorexia, and TNF can be fatal. TNF has also been implicated in infectious diseases. These diseases include, for example, malaria, mycobacterial infection, and meningitis. These also include viral infections such as HIV, influenza virus and herpes virus (including herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), Cytomegalovirus (CMV), varicella-zoster virus (VZV), Epstein-Barr virus, human herpes virus 6(HHV-6), human herpes virus 7(HHV-7), human herpes virus 8 (HHV-8)); pseudorabies, rhinotracheitis, etc.

Known P38 kinase inhibitors have been reviewed in J.Hanson (Expert Opinions on Therapeutic Patents 1997, 7, 729-. P38 kinase inhibitors containing a triazolopyridine group are known in the art, for example WO07/091152, WO04/072, WO 06/018727.

Disclosure of Invention

The compounds of the invention are inhibitors of p38 mitogen-activated protein kinases ("p 38 MAPK", "p 38 kinase" or "p 38"), including p38 α kinase, and are inhibitors of cytokine and chemokine production, including TNF α and IL-8 production. They have a number of therapeutic uses for the treatment of inflammatory diseases, especially allergic and non-allergic airway diseases, more particularly obstructive or inflammatory airway diseases such as chronic obstructive pulmonary disease ("COPD") and asthma. They are therefore particularly suitable for pulmonary delivery (by nasal or mouth inhalation).

According to the present invention there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof:

wherein:

R¹is C₁-C₆Alkyl radical, C₃-C₆Cycloalkyl, optionally substituted phenyl, optionally substituted 5-or 6-membered monocyclic heteroaryl, or a group of formula (II)

Wherein n is 1 or 2; and R is³And R⁴Independently is H or C₁-C₆Alkyl, or R³And R⁴Together with the nitrogen to which they are attached form a 6-membered heterocyclic ring, optionally containing a further heteroatom selected from N and O;

y is-O-or-S (O)_p-, wherein p is 0, 1 or 2;

a is optionally substituted divalent arylene, or monocyclic or bicyclic heteroarylene, or C having 5 or 6 ring atoms₃-C₆Divalent cycloalkylene radicals, or piperidylene radicals, wherein the ring nitrogen is attached to R²NHC(＝O)W-；

W is a bond, -NH-or-C (R)^A)(R^B) -, wherein R^AAnd R^BIndependently H, methyl, ethyl, amino, hydroxy or halogen; and is

R²Is a group of formula (IIIA), (IIIB) or (IIIC):

wherein

m is 0 or 1;

q is 0, 1, 2 or 3;

t is-N or-CH;

R⁵is H or F;

R⁷is-CH₃、-C₂H₅、-CH₂OH、-CH₂SCH₃、-SCH₃or-SC₂H₅；

R⁸is-CH₃or-C₂H₅(ii) a And is

Each occurrence of R⁶Independently is H, C₁-C₆Alkyl, hydroxy or halogen; or a single occurrence of R⁶Is a radical of the formula (IVA), (IVB) or (IVC)

While any other occurrence of R⁶Independently is H, C₁-C₆Alkyl, hydroxy or halogen;

wherein n and p are as defined above;

and wherein at R⁶In

R^61aAnd R^61bIs H, alkyl, or R^61aAnd R^61bMay be combined with the nitrogen to which they are attached to form a heterocyclic ring, optionally containing other heteroatoms selected from N and O.

In another aspect, the invention includes pharmaceutical compositions comprising a compound of the invention and one or more pharmaceutically acceptable carriers and/or excipients. Particularly preferred are compositions suitable for pulmonary administration by inhalation.

In another aspect, the invention includes the use of a compound of the invention for the treatment of a disease or condition that benefits from inhibition of p38MAP kinase activity. Preferred uses are the treatment of obstructive or inflammatory airway diseases. All forms of obstructive or inflammatory airways diseases may potentially be treated using the compounds of the present invention, in particular those selected from the group consisting of: chronic eosinophilic pneumonia, asthma, COPD including chronic bronchitis, emphysema or dyspnea associated or not with COPD, COPD characterized by irreversible progressive airway obstruction, Adult Respiratory Distress Syndrome (ARDS), exacerbation of airway hyperreactivity consequent to other drug treatments, and airway diseases associated with pulmonary hypertension (pulmony hypertension), chronic inflammatory diseases including cystic fibrosis, bronchiectasis and pulmonary fibrosis (idiopathic). Efficacy is expected when p38 kinase inhibitors are administered topically to the lung (e.g., by inhalation and intranasal delivery) or by systemic routes (e.g., oral, intravenous, and subcutaneous delivery).

Detailed Description

Scientific terms

The term "(C) as used herein_a-C_b) Alkyl "(wherein a and b are integers) represents a straight or branched chain alkyl group having a to b carbon atoms. Thus, for example, when a is 1 and b is 6, the term includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl and n-hexyl.

The term "carbocyclic" as used herein denotes monocyclic, bicyclic or tricyclic groups having up to 16 ring atoms (all of which are carbon), including aryl and cycloalkyl groups.

The term "cycloalkyl" as used herein denotes a monocyclic saturated carbocyclic group having 3 to 8 carbon atoms, including, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

The term "divalent cycloalkylene group" denotes a cycloalkyl group having 2 unsaturations, such as the following 1, 3-cyclopentylene and 1, 4-cyclohexylene groups:

the unlimited term "aryl" as used herein denotes a monocyclic or bicyclic carbocyclic aromatic group and includes groups having 2 monocyclic carbocyclic aromatic rings directly connected by covalent bonds. Examples of such groups are phenyl, biphenyl and naphthyl.

The term "divalent arylene" denotes a monocyclic or bicyclic aryl group having 2 unsaturations, such as the following 1, 3-phenylene or 1, 4-phenylene group:

or the following 1, 4-naphthyl (1, 4-naphthyl):

the unrestricted term "heteroaryl" as used herein denotes a monocyclic or bicyclic aromatic group containing one or more heteroatoms selected from S, N and O, and includes groups having 2 such monocyclic rings, or one such monocyclic ring and one monocyclic aromatic ring, which are directly linked by a covalent bond. Representative examples of such groups are thienyl, benzothienyl, furyl, benzofuryl, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isothiazolyl, benzisothiazolyl, pyrazolyl,Azolyl, benzoAzolyl radical, isoAzolyl, benzisoylAn oxazolyl group, an isothiazolyl group, a triazolyl group, a benzotriazolyl group, a thiadiazolyl group,Oxadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolyl and indazolyl.

The unlimited term "heterocyclyl" or "heterocyclic" as used herein includes "heteroaryl" as defined above, and in its non-aromatic meaning refers to a monocyclic, bicyclic or tricyclic non-aromatic group containing one or more heteroatoms selected from S, N and O, as well as groups consisting of monocyclic non-aromatic groups containing one or more such heteroatoms which are covalently linked to another such group or to a monocyclic carbocyclic group. Examples of such groups are pyrrolyl, furyl, thienyl, piperidinyl, imidazolyl,Azolyl radical, isoOxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridyl, pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl, morpholinyl, benzofuranyl, pyranyl, isofuranylOxazolyl, benzimidazolyl, methylenedioxyphenyl, ethylenedioxyphenyl, maleimido and succinimido.

The term "divalent heteroarylene" denotes a monocyclic or bicyclic heteroaryl group having 2 unsaturations, such as the following:

the following are presently preferred

Unless otherwise indicated in the context in which it appears, the term "substituted" as applied herein to any aryl or heteroaryl moiety means substituted with at least one substituent selected, for example, from: (C)₁-C₆) Alkyl, (C)₁-C₆) Fluoroalkyl, (C)₁-C₆) Alkoxy (including methylenedioxy and ethylenedioxy substitution at adjacent carbon atoms of the aromatic ring), (C)₁-C₆) Fluoroalkoxy, (C)₁-C₆) Alkoxy radical- (C₁-C₆) Alkyl, benzyloxy- (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy radical- (C₁-C₆) Alkoxy, benzyloxy- (C)₁-C₆) Alkoxy, hydroxy (C)₁-C₆) Alkyl, hydroxy (C)₁-C₆) Alkoxy, mercapto (C)₁-C₆) Alkyl, (C)₁-C₆) Alkylthio, cyclopropyl, halo (including fluoro and chloro), O-benzyl, nitro, nitrile (cyano), -COOH, tetrazolyl, -COOR^A、-COR^A、-SO₂R^A、-CONH₂、-SO₂NH₂、-CONHR^A、-SO₂NHR^A、-CONR^AR^B、-SO₂NR^AR^B、-NH₂、-NHR^A、-NR^AR^B、-OCONH₂、-OCONHR^A、-OCONR^AR^B、-NHCOR^A、-NHCOOR^A、-NR^BCOOR^A、-NHSO₂OR^A、-NR^BSO₂OR^A、-NHCONH₂、-NR^ACONH₂、-NHCONHR^B、-NR^ACONHR^B、-NHCONR^AR^Bor-NR^ACONR^AR^BWherein R is^AAnd R^BIndependently is (C)₁-C₄) Alkyl, or R^AAnd R^BWhen attached to the same nitrogen, a cyclic amino group such as morpholinyl, piperidinyl or piperazinyl may be formed together with the nitrogen. The "optional substituent" may be one of the substituents included in the above description.

The compounds of the present invention may exist in one or more geometric, optical, enantiomeric, diastereomeric and tautomeric forms, including, but not limited to, cis and trans forms, E-and Z-forms, R-, S-and meso-forms, keto-and enol-forms. Unless otherwise indicated, reference to a particular compound includes all such isomeric forms, including racemic and other mixtures thereof. Such isomers may be separated from their mixtures, where appropriate, by the use or adaptation of known methods, such as chromatographic techniques and recrystallization techniques. Such isomers may be prepared by application or by known methods (e.g., asymmetric synthesis) where appropriate.

The term "salt" as used herein includes base addition salts, acid addition salts and ammonium salts. As briefly mentioned above, the acidic compounds of the present invention may form salts, including pharmaceutically acceptable salts, with bases such as: alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; alkaline earth metal hydroxides such as calcium hydroxide, barium hydroxide and magnesium hydroxide; organic bases such as N-methyl-D-glucamine, choline tris (hydroxymethyl) amino-methane, L-arginine, L-lysine, N-ethylpiperidine, dibenzylamine and the like. Those basic compounds of the present invention may form salts, including pharmaceutically acceptable salts, with inorganic acids such as hydrohalic acids, e.g., hydrochloric or hydrobromic acid, sulfuric acid, nitric acid or phosphoric acid, and the like, and with organic acids such as acetic acid, trifluoroacetic acid, tartaric acid, succinic acid, fumaric acid, maleic acid, malic acid, salicylic acid, citric acid, methanesulfonic acid, p-toluenesulfonic acid, benzoic acid, benzenesulfonic acid, glutamic acid, lactic acid, and mandelic acid, and the like. Those compounds (I) having a basic nitrogen may also form quaternary ammonium salts with pharmaceutically acceptable counter ions, such as ammonium chloride, ammonium bromide, ammonium acetate, ammonium formate, ammonium p-toluenesulfonate, ammonium succinate, ammonium hemi-succinate, ammonium naphthalene-disulfonate, ammonium methanesulfonate, ammonium trifluoroacetate, ammonium xinafoate and the like. For a review of Salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: properties, Selection, and Use (Wiley-VCH, Weinheim, Germany, 2002).

It is envisioned that the compounds of the present invention may be prepared in the form of hydrates and solvates. Any reference herein, including in the claims herein, to "a compound of interest" or "a compound of the invention" and the like, includes salts, hydrates, and solvates of such compounds. The term 'solvate' is used herein to describe a molecular complex comprising a compound of the invention and a stoichiometric amount of one or more pharmaceutically acceptable solvent molecules, such as ethanol. When the solvent is water, the term 'hydrate' is used.

Individual compounds of the invention may exist in several polymorphic forms and may be obtained in different crystal habits.

The compounds may also be administered in the form of prodrugs thereof. Thus, certain derivatives of the compounds, which may themselves be active or which may themselves have little or no pharmacological activity, may be converted to compounds of the invention having the desired activity when administered into the body or onto the body surface, for example by hydrolytic cleavage. Such derivatives are referred to as 'prodrugs'. For further information on the use of prodrugs, see: pro-drugs as Novel Delivery Systems, Vol.14, ACSSymposium Series (T.Higuchi and V.J.Stella) and Bioreversible Carriersindeug Design, Pergamon Press, 1987(E.B.Roche, American pharmaceutical Association; C.S.Larsen and J).Design addressing of drivers, see Textbook of Drug Design and Discovery, 3 rd edition, 2002, Taylor and Francis).

Prodrugs according to the invention may be prepared, for example, by replacing appropriate functional groups present in the compounds of formula (I) with certain moieties known to those skilled in the art as 'pro-moieties' (for example as described in Design of produgs by h. Such an example may be a carboxyl group (such as-CO-O-CH used in a pivampicillin prodrug of ampicillin₂-O-CO-tBu), amides (-CO-NH-CH)₂-NAlk₂) Or amidines (-C (═ N-O-CH)₃)-NH₂) A prodrug of (a).

In the compounds of the invention, the divalent group-W- [ A ] -Y-may be, for example, any of the corresponding groups in the compounds of the following specific examples. For example, the group may be one of the following formulas (B) - (J):

a subclass of compounds of the invention has formula (IA):

wherein V, V ', X and X' are independently-CH or-N ═ N; and R is¹、R²Y and W are as defined for formula (I). Within this subclass, the compound can have the formula (IA)¹)：

Wherein Y is O or S, and R¹And R²As defined for formula (I).

Another subclass of compounds of the invention has formula (IB):

wherein U is CH or N, and R¹、R²Y and W are as defined for formula (I), with the proviso that when U is N, then W is not NH. Within this subclass, the compound can have the formula (IB)¹)：

Wherein Y is O or S, and R¹And R²As defined for formula (I).

Another subclass of compounds of the invention has formula (IC):

wherein Y is O or S, R²As defined in claim 1, and R¹Is a phenyl group as defined above for formula (I), a 5 or 6 membered monocyclic heteroaryl group, or a group of formula (II).

Among the compounds of the present invention are those of the above formulae (IA), (IA)¹)、(IB)、(IB¹) And (IC), specific presently preferred structural features include the following:

R¹may be a group of formula (II) as defined with respect to formula (I) above,wherein the group-NR³R⁴Is morpholinyl.

R¹It may be isopropyl or 2, 6-dichlorophenyl.

R²May be a group of formula (IIIC) as defined for formula (I) above wherein R⁷And R⁸Each is methyl.

R²Can have formula (IIID), (IIIE), (IIIF) or (IIIG):

R²may be a group of formula (IIIA) as defined for formula (I) above wherein m is 0.

R²May be a group of formula (IIIB) as defined for formula (I) above wherein (a) T is-CH ═ and R⁵H; or (b) T is-N ═ and R⁵H; or (c) T is-CH ═ and R⁵＝F。

Optional substituents in the divalent group a include: -CN, -F, -Cl, -Br, -NO₂、-OH、-SO₂C₁-C₂Alkyl, -SO₂C₁-C₂Fully or partially fluorinated alkyl, C₁-C₄Alkyl, fully or partially fluorinated C₁-C₄Alkyl radical, C₁-C₄Alkoxy, fully or partially fluorinated C₁-C₄Alkoxy radicals and-SCF₃。

The divalent group a may be a 6-membered ring (such as phenylene or pyridylene) which is attached to Y and W in the 1, 3 (meta) or 1, 4 (para) orientation.

The divalent group A may be cyclohexylene attached to W and Y in the trans-1, 4 orientation.

W may be-CH₂-。

Practicality of use

As noted above, the compounds of the present invention are p38 MAPK inhibitors and thus may be useful in the treatment of diseases or conditions which benefit from inhibition of the p38 enzyme. Such diseases and conditions are known from the literature and several have been mentioned above. However, the compounds are generally useful as anti-inflammatory agents, especially for the treatment of respiratory diseases. In particular, the compounds may be used for the treatment of Chronic Obstructive Pulmonary Disease (COPD), chronic bronchitis, pulmonary fibrosis, pneumonia, Acute Respiratory Distress Syndrome (ARDS), emphysema or smoking-induced emphysema, intrinsic (non-allergic asthma) and extrinsic (allergic) asthma, mild asthma, moderate asthma, severe asthma, steroid resistant asthma, neutrophilic asthma, bronchitic asthma, exercise-induced asthma, occupational asthma and asthma induced following bacterial infection, cystic fibrosis, pulmonary fibrosis and bronchiectasis.

Composition comprising a metal oxide and a metal oxide

As mentioned above, the compounds to which the present invention relates are p38 kinase inhibitors and are useful in the treatment of several diseases, such as inflammatory diseases of the respiratory tract. Examples of such diseases are mentioned above and include asthma, rhinitis, allergic airway syndrome, bronchitis and chronic obstructive pulmonary disease.

It will be understood that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy. The optimal dose level and frequency of administration is determined by clinical trials as required in the pharmaceutical arts. In general, for oral administration, the daily dosage will range from about 0.001mg to about 100mg/kg of human body weight, often from 0.01mg to about 50mg/kg, for example from 0.1 to 10mg/kg, in single or divided doses. In general, for administration by inhalation, the daily dose will range from about 0.1 μ g to about 1mg/kg of human body weight, preferably 0.1 μ g to 50 μ g/kg, in single or divided doses. On the other hand, in some cases, it may be necessary to use dosages outside these limits. For the purposes of the present invention, administration by inhalation is preferred.

The compounds of the invention may be prepared for administration by any route consistent with their pharmacokinetic properties. Orally administrable compositions may be in the form of tablets, capsules, powders, granules, lozenges, liquid or gel preparations, such as oral, topical or sterile parenteral solutions or suspensions. Tablets and capsules for oral administration may be in unit dose presentation form and may contain conventional excipients such as: binders, such as syrup, acacia, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone; fillers, for example lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silicon dioxide; a disintegrant, such as potato starch, or an acceptable wetting agent, such as sodium lauryl sulfate. The tablets may be coated according to methods well known in ordinary pharmaceutical practice. Liquid preparations for oral use may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid formulations may contain conventional additives such as: suspending agents, such as sorbitol, syrup, methylcellulose, glucose syrup, gelatin, hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters (such as glycerol), propylene glycol or ethanol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, where appropriate, conventional flavouring or colouring agents.

For topical application to the skin, the medicament may be formulated as a cream, lotion or ointment. Cream or ointment formulations which may be used for the medicament are conventional formulations well known in the art, for example as described in standard textbooks of pharmacy (e.g. the british pharmacopoeia).

The active ingredient may also be administered parenterally in a sterile medium. Depending on the vehicle and concentration used, the agent may be suspended or dissolved in the vehicle. Advantageously, adjuvants (such as local anesthetics, preservatives, and buffering agents) can also be dissolved in the vehicle.

However, for the treatment of inflammatory diseases of the respiratory tract, the compounds of the invention may also be formulated for inhalation, for example as a nasal spray, or a dry powder or aerosol inhaler. For inhalation delivery, the active compound is preferably in particulate form. These microparticles can be prepared by a variety of techniques including spray drying, freeze drying and micronization. Aerosol may be generated, preferably propellant-driven metered dose aerosols or propellant-free administration of micronized active compounds (from e.g. inhalation capsules or other "dry powder" delivery systems), using e.g. a pressure-driven jet nebulizer or an ultrasonic nebulizer.

For example, the compositions of the present invention may be prepared as a suspension for delivery from a nebulizer, or as an aerosol in a liquid propellant for use in, for example, a Pressurized Metered Dose Inhaler (PMDI). Propellants suitable for use in PMDI are known to the skilled artisan and include CFC-12, HFA-134a, HFA-227, HCFC-22 (CCl)₂F₂) And HFA-152 (CH)₄F₂And isobutane).

In a preferred embodiment of the invention, the composition of the invention is in the form of a dry powder for delivery using a Dry Powder Inhaler (DPI). Many types of DPI are known.

Microparticles for administration delivery may be formulated with excipients that aid delivery and release. For example, in a dry powder formulation, microparticles may be formulated with large carrier particles that facilitate flow from the DPI to the lung. Suitable carrier particles are known and include lactose particles; they may have a mass median diameter (mass median aerodynamic diameter) of more than 90 μm.

In the case of aerosol-based formulations, one example is:

the active compound may be administered as previously described, depending on the inhaler system used. In addition to the active compounds, the administration forms may additionally contain excipients, for example propellants (for example Freon for metered-dose aerosols), surface-active substances, emulsifiers, stabilizers, preservatives, flavorings, fillers (for example lactose for powder inhalers) or, if appropriate, further active compounds.

For inhalation purposes, a wide variety of systems are available that generate and administer aerosols having the most appropriate particle size, using inhalation techniques appropriate to the patient. Except for the use of adapter (spacer), spreader) and pear-shaped containers (e.g. for example) And automatic means for emitting a puff sprayBesides, for metered dose aerosols, in particular in the case of powder inhalers, a wide variety of solutions are available (e.g. for exampleOr an inhaler such as described in EP- ｃA-0505321). Additionally, the compounds of the present invention may be delivered in a multi-compartment device, allowing for the delivery of a co-agent.

Combination of

Other compounds may be combined with the compounds to which the present invention relates for the prevention and treatment of inflammatory diseases, especially respiratory diseases. Thus, the present invention also relates to pharmaceutical compositions comprising a therapeutically effective amount of a compound of the present invention and one or more other therapeutic agents. Therapeutic agents suitable for use in combination therapy with the compounds of the present invention include, but are not limited to: (1) corticosteroids such as fluticasone propionate, fluticasone furoate, mometasone furoate, beclomethasone dipropionate, ciclesonide, budesonide, GSK685698, GSK870086, QAE 397, QMF 149, TPI-1020; (2) beta 2-adrenergic receptor agonists such as salbutamol (salbutamol), salbutamol (albuterol), terbutaline, fenoterol, and long-acting beta 2-adrenergic receptor agonists such as salmeterol, indacaterol, formoterol (including formoterol fumarate), arformoterol, carmoterol, GSK 642444, GSK159797, GSK 159802, GSK 597501, GSK678007, AZD 3199; (3) a corticosteroid/long-acting β 2 agonist combination, such as salmeterol/fluticasone propionate (Advair/Seretide), formoterol/budesonide (Symbiport), formoterol/fluticasone propionate (Flutiform), formoterol/ciclesonide, formoterol/mometasone furoate, indacaterol/QAE 397, GSK159797/GSK 685698, GSK 159802/GSK 685698, GSK 642444/GSK 685698, GSK 791597/GSK 870086, GSK 159802/GSK 870086, GSK 6444/GSK 870086, arformoterol/ciclesonide; (4) anticholinergics, e.g., muscarinic-3 (M3) receptor antagonists such as ipratropium bromide, tiotropium bromide, Aclidinium (LAS-34273), NVA-237, GSK 233705, Darotropium, GSK 573719, GSK961081, QAT 370, QAX 028; (5) dual pharmacology M3-anticholinergic/β 2-adrenergic receptor agonists such as GSK 961081; (6) leukotriene modulators, e.g., leukotriene antagonists such as montelukast, zafirlukast or pranlukast, or leukotriene biosynthesis inhibitors such as zileuton or BAY-1005, or LTB4 antagonists such as amalriban, or FLAP inhibitors such as GSK 2190914, AM-103; (7) phosphodiesterase-IV (PDE-IV) inhibitors (oral or inhaled), such as roflumilast, cilomilast, omiclast, ONO-6126, Tetomilast, tofastemilast, UK 500,001, GSK 256066; (8) antihistamines, such as selective histamine-1 (H1) receptor antagonists, e.g., fexofenadine, citirizine, loratadine, or astemizole, or dual H1/H3 receptor antagonists such as GSK 835726, GSK 1004723; (9) antitussives such as codeine or dextromethorphan (dextromethorphan); (10) mucolytics, such as N-acetyl cysteine or fudosteine; (11) expectorant/mucokinetic (mucokinetic) modulators, such as ambroxol, hypertonic solutions (e.g. saline or mannitol) or surfactants; (12) peptide mucolytic agents, such as recombinant human deoxyriboribozyme I (alfa-streptokinase and rhdnase) or spirodiclodin; (13) antibiotics such as azithromycin, tobramycin and aztreonam; (14) non-selective COX-1/COX-2 inhibitors, such as ibuprofen or ketoprofen; (15) COX-2 inhibitors, such as celecoxib and rofecoxib; (16) VLA-4 antagonists, such as those described in WO97/03094 and WO 97/02289; (17) TACE inhibitors and TNF- α inhibitors, such as anti-TNF monoclonal antibodies, e.g., infliximab for injection and CDP-870 and TNF receptor immunoglobulin molecules, e.g., Enbrel; (18) matrix metalloproteinase inhibitors, such as MMP-12; (19) human neutrophil elastase inhibitors, such as ONO-6818 or those described in WO2005/026124, WO2003/053930, and WO 06/082412; (20) a2b antagonists such as those described in WO 2002/42298; (21) modulators of chemokine receptor function, such as antagonists of CCR3 and CCR 8; (22) compounds that modulate the action of other prostanoid receptors, such as thromboxane a2 antagonists; DP1 antagonists such as MK-0524, CRTH2 antagonists such as ODC9101 and AZD1981, and mixed DP1/CRTH2 antagonists such as AMG 009; (23) PPAR agonists, including PPAR α agonists (such as fenofibrate), PPAR δ agonists, PPAR γ agonists, e.g., pioglitazone, rosiglitazone and balaglitazone; (24) methylxanthines such as theophylline or aminophylline, and methylxanthine/corticosteroid combinations such as theophylline/budesonide, theophylline/fluticasone propionate, theophylline/ciclesonide, theophylline/mometasone furoate, and theophylline/beclometasone dipropionate; (25) a2a agonists such as those described in EP1052264 and EP 1241176; (26) CXCR2 or IL-8 antagonists such as SCH 527123 or GSK 656933; (27) IL-R signaling modulators such as kineret and ACZ 885; (28) MCP-1 antagonists such as ABN-912.

Synthesis method

The compounds of the invention can be prepared according to the routes illustrated in schemes 1-6.

Scheme 1

The compound of the general formula (I-a) can be prepared from the compound of the general formula (IX) as follows:

wherein R is 0 or 1, Y is S or O, and R^aAs in formula (I) with respect to R¹As defined.

By reaction with an amine of the general formula (X):

R^bNH₂(X)，

wherein R is^bAs in formula (I) with respect to R²As defined.

In the presence of a base such as diisopropylethylamine or triethylamine, using a suitable coupling agent such as 2- (7-aza-1H-benzotriazol-1-yl) -1, 1, 3, 3-tetramethylureaHexafluorophosphate, 1-ethyl-3- (3' -dimethylaminopropyl) carbodiimide or dicyclohexylcarbodiimide. The reaction may be carried out in a suitable solvent such as dichloromethane, N-dimethylformamide or tetrahydrofuran at a range of temperatures, preferably at room temperature.

Alternatively, the compound of formula (I-a) may be prepared from a compound of general formula (IX) as follows: with a suitable halogenating agent, such as oxalyl chloride or thionyl chloride, in the presence or absence of a solvent, such as dichloromethane or N, N-dimethylformamide, at a temperature in the range of preferably room temperature to 100 c, followed by reaction with an amine of the general formula (X) in a suitable solvent, such as tetrahydrofuran, using a suitable base, such as diisopropylethylamine, at a temperature in the range of preferably room temperature to 80 c.

The compounds of general formula (IX) can be prepared from compounds of general formula (V) as follows:

wherein X is a suitable leaving group (such as fluoride, bromide or iodide)

By reaction with a compound of general formula (VIII):

wherein R is H or alkyl, using a suitable catalyst such as copper (I) iodide, copper (I) chloride, palladium acetate, tetrakis (triphenylphosphine) palladium (0) or dichloro (1, 1' -bis (diphenylphosphino) ferrocene) palladium (II), in the presence or absence of a suitable ligand such as (2, 9-dimethyl) -1, 10-phenanthroline, proline, 1, 2-cyclohexyldiamine or phosphine, using a base such as cesium carbonate, potassium hydroxide, potassium carbonate or sodium tert-butoxide. The reaction can be carried out in a suitable solvent such as toluene, N-methylpyrrolidone or N, N-dimethylformamide, at a temperature in the range (preferably 40 to 150 ℃); if R is an alkyl group, hydrolysis is then carried out according to methods known to those skilled in the art.

The compounds of formula (V) can be prepared from compounds of formula (IV) as follows:

using a suitable oxidant, such as chloramine T, lead tetraacetate or iodobenzene (III) diacetate, in a suitable solvent, such as dichloromethane or ethanol, at a temperature range (preferably room temperature to 100 ℃).

The compounds of formula (IV) can be prepared from compounds of formula (II) as follows:

by reaction with an aldehyde of the general formula (III) in a suitable solvent, such as ethanol or tetrahydrofuran, at a temperature in the range (preferably room temperature to 80 ℃):

R^aCHO(III)。

alternatively, compounds of formula (IV) may be prepared from compounds of formula (II) as follows:

with a compound of formula (VI) in the presence of a base such as diisopropylethylamine in a suitable solvent such as dichloromethane or acetonitrile at a temperature range (preferably from room temperature to 150 ℃) using a suitable acylating/dehydrating agent such as triphenylphosphine/trichloroacetonitrile:

R^aCO₂H(VI)。

alternatively, compounds of formula (IV) may be prepared from compounds of formula (VII) as follows:

using suitable dehydrating agents (such as Burgess's reagent, triphenylphosphine and hexachloroethane, phosphorus oxychloride, acetic acid) or Mitsunobu conditions (diethyl azodicarboxylate/triphenylphosphine/trimethylazidosilane) in the presence or absence of a suitable solvent (such as tetrahydrofuran, toluene or NMP) at a temperature in the range (preferably room temperature to 120 ℃).

The compounds of formula (VII) can be prepared from compounds of formula (II) by reaction with carboxylic acids of formula (VI) as described for the preparation of compounds of formula (I-a) from compounds of formula (IX).

Scheme 2

The compounds of the general formula (I-b) can be prepared from the compounds of the general formula (XII) as follows:

(XII) in which R^aAs in formula (I) with respect to R¹The definition of the content of the compound is as follows,

by dissolving in a suitable solvent (such as dimethyl sulfoxide, 1, 4-diAlkane or acetonitrile) with a compound of formula (XIII) in the presence of a base such as diisopropylethylamine at a temperature in the range (preferably room temperature to 100 ℃):

wherein R is^bAs in formula (I) with respect to R²As defined.

The compounds of formula (XIII) can be prepared from amines of formula (X) according to known literature methods (e.g.WO 2006/009741, EP 1609789).

Alternatively, the compounds of formula (I-b) can be prepared from compounds of formula (XII) as follows: with an amine of the general formula (X) using a suitable coupling agent, such as phosgene, diphosgene or triphosgene, in a suitable solvent, such as dichloromethane, toluene, tetrahydrofuran or acetonitrile, using a suitable base, such as triethylamine, pyridine or diisopropylethylamine, at a temperature in the range of from 0 to 100 ℃.

The compounds of formula (XII) can be prepared from the compounds of formula (V) as follows:

with a compound of general formula (XI) using a suitable base such as cesium carbonate, potassium hydroxide, potassium carbonate or sodium tert-butoxide, in the presence or absence of a suitable catalyst such as copper (I) iodide or bromide and a suitable ligand such as (2, 9-dimethyl) -1, 10-phenanthroline, proline or 1, 2-cyclohexyldiamine:

the reaction can be carried out in a suitable solvent such as toluene, N-methylpyrrolidone, methanol or N, N-dimethylformamide at a temperature in the range of preferably 40 to 150 ℃.

Scheme 3

The compounds of the general formula (I-c) can be prepared from the compounds of the general formula (XVI) as follows:

as described for the preparation of compounds of formula (I-b) from compounds of formula (XII), with compounds of general formula (XIII) or (X).

The compounds of general formula (XVI) can be prepared from the compounds of general formula (XV) by deprotection according to methods known to the person skilled in the art:

wherein R is^cMay be a suitable protecting group or H.

The compounds of general formula (XV) can be prepared from the compounds of general formula (V) as follows:

as described for the preparation of compounds of formula (XII) from compounds of formula (XI), with compounds of general formula (XIV):

scheme 4

The compounds of formula (I-d) can be prepared from compounds of formula (XIX) as follows:

as described for the preparation of compounds of formula (I-b) from compounds of formula (XII), with compounds of general formula (XIII) or (X).

The compounds of the general formula (XIX) can be prepared from the compounds of the general formula (XVIII) as follows:

in the presence of a catalyst such as palladium on charcoal, platinum oxide or raney nickel, in the presence or absence of an acid such as hydrochloric acid or acetic acid, in a suitable solvent such as methanol, ethanol or ethyl acetate, at a temperature in the range (preferably room temperature to 80 ℃) with a suitable reducing agent such as tin (II) chloride, iron or hydrogen.

Alternatively, compounds of formula (XIX) can be prepared from compounds of formula (V) as follows:

the compound of formula (XVII) is reacted with a suitable metallated species, such as butyl lithium, magnesium chloride or isopropyl magnesium chloride, in a suitable solvent, such as tetrahydrofuran or diethyl ether, at a temperature range, preferably-78 ℃ to room temperature:

wherein R is^dIs H.

As described for the preparation of the compound of the general formula (XIX) from the compound of the general formula (V), by reaction with a compound of the general formula (XVII) (wherein R is^dIs O) reaction, it is possible to prepare the compounds of the general formula (XVIII) from the compounds of the general formula (V).

Scheme 5

The compounds of formula (I-e) can be prepared from compounds of formula (XXII) as follows:

with a compound of formula (XXIII) in the presence or absence of a suitable catalyst (such as potassium iodide or sodium iodide) in a suitable solvent (such as dichloromethane, tetrahydrofuran or acetonitrile) at a temperature in the range (preferably from room temperature to 80 ℃) using a suitable base (such as diisopropylethylamine, potassium carbonate, sodium carbonate or sodium hydride):

compounds of formula (XXIII) can be prepared from amines of formula (X) according to known literature procedures (e.g., Migliara et al, Farmaco (1992), 47(1), 111-19).

The compounds of formula (XXII) can be prepared from compounds of formula (XXI) by deprotection according to methods known to those skilled in the art:

wherein R is^cAre suitable protecting groups.

As described for the preparation of the compound of formula (XII) from the compound of formula (XI), by reaction with a compound of general formula (XX),

compounds of formula (XXI) can be prepared from compounds of formula (V):

scheme 6

The compounds of formula (I-f) can be prepared from compounds of formula (XXVI) by reaction with an amine of formula (X) as described for the preparation of compounds of formula (I-a) from compounds of formula (IX):

the compounds of formula (XXVI) can be prepared from compounds of formula (XXV) by deprotection according to methods known to those skilled in the art:

wherein R is an alkyl group.

As described for the preparation of the compound of formula (XII) from the compound of formula (XI), the compound of formula (XXV) can be prepared from the compound of formula (XXIV) by reaction with the compound of formula (V):

scheme 7

The compounds of general formula (V) may be prepared as described in chem.soc.jpn., 1980, 53, 2007-11.

Scheme 8

By reaction with an amine of the general formula (X):

R^bNH₂(X)，

wherein R is^bIs as in the above formula (I) with respect to R²The definition of the content of the compound is as follows,

compounds of formula (I-g) can be prepared from compounds of formula (XXIX):

the compounds of formula (XXIX) can be prepared from compounds of formula (XXVIII) by deprotection according to methods known to those skilled in the art:

wherein R is^cIs an alkyl group.

The compound of formula (XXVIII) can be prepared from the compound of formula (XXVII) as follows:

in a suitable solvent such as tetrahydrofuran, DMF or diethyl ether, at a temperature in the range preferably from room temperature to 80 ℃ using a suitable base such as lithium hexamethyldisilazide or sodium hydride, with a suitable compound R^dX (XXXI) reaction.

By reaction with an alcohol of formula (XXX),

R^cOH(XXX)

compounds of formula (XXVII) can be prepared from compounds of formula (IX):

wherein R is 1, Y is S or O, and R^aAs in formula (I) with respect to R¹As defined.

General experimental details

Abbreviations used in the experimental section:

aq. is aqueous;

DCM ═ dichloromethane;

DIPEA ═ diisopropylethylamine;

DMF ═ N, N-dimethylformamide;

DMSO ═ dimethyl sulfoxide;

EDCI HCl ═ 1-ethyl-3- (3' -dimethylaminopropyl) carbodiimide hydrochloride;

EtOAc ═ ethyl acetate;

EtOH ═ ethanol;

FCC ═ flash column chromatography;

h is h;

HATU ═ 2- (7-aza-1H-benzotriazol-1-yl) -1, 1, 3, 3-tetramethylureaA hexafluorophosphate salt;

HOBt ═ 1-hydroxy-benzotriazole;

HPLC ═ high performance liquid chromatography;

LCMS ═ liquid chromatography mass spectrometry;

MeCN ═ acetonitrile;

MeOH ═ methanol;

min is minutes;

NMR ═ nuclear magnetic resonance;

RT ═ room temperature;

rt ═ retention time;

sat. (saturated);

SCX-2 ═ strong cation exchange chromatography;

TFA ═ trifluoroacetic acid;

THF ═ tetrahydrofuran.

The nomenclature of the structures was specified using Autonom 2000 naming software from MDL Inc.

All reactions were carried out under nitrogen atmosphere unless otherwise indicated.

NMR spectra were obtained on a Varian Unity Inova 400 spectrometer (with a 5mm inverted detection triple resonance probe operating at 400 MHz), or on a Bruker Avance DRX 400 spectrometer (with a 5mm inverted detection triple resonance TXI probe operating at 400 MHz), or on a Bruker Avance DPX 300 spectrometer (with a standard 5mm dual frequency probe operating at 300 MHz). Migration is given in ppm relative to tetramethylsilane. NMR spectra were assigned using a datached Spectrum analysis version 4.0.b 21.

In the case of purification of the product by flash column chromatography, 'flash silica' denotes silica gel for chromatography, 0.035-0.070mm (220-Chaperone (Companion) purification system or Biotage SP1 purification system was used. All solvents and commercial reagents were used as received.

The compounds purified by preparative HPLC were purified using a C18-reverse phase column (100X 22.5mm inner diameter, Genesis column, 7 μm particle size), or phenyl-hexyl column (250X21.2mm inner diameter, Gemini column, 5 μm particle size), with UV detection at 230 or 254nm at a flow rate of 5-20mL/min, and gradient elution with 100-0 to 0-100% water/acetonitrile (containing 0.1% TFA or 0.1% formic acid) or water/MeOH (containing 0.1% TFA or 0.1% formic acid). Fractions containing the desired product (identified by LCMS analysis) were combined, the organic fraction was removed by evaporation, and the remaining aqueous fraction was lyophilized to give the final product. The product purified by preparative HPLC was isolated as formate or TFA salt unless otherwise indicated.

The Liquid Chromatography Mass Spectrometry (LCMS) and HPLC systems used were:

method 1

Waters ZMD quadrupole mass spectrometer with C18-reversed phase column (30X 4.6mm Phenomenex Luna 3 μm particle size) with eluent A: water + 0.1% formic acid; b: acetonitrile + 0.1% formic acid. Gradient:

detection-MS, ELS, UV (200 μ Ι _ split to MS with in-line ultraviolet detector). MS ionization method-electrospray (positive and negative ions).

Method 2

Waters Platform LC quadrupole mass spectrometer with C18-reversed phase column (30X 4.6mm Phenomenex Luna 3 μm particle size) and eluent A: water + 0.1% formic acid; b: acetonitrile + 0.1% formic acid. Gradient:

detection-MS, ELS, UV (200. mu.L split to MS with internal ultraviolet detector).

MS ionization method-electrospray (positive and negative ions).

Method 3

Phenomenex Gemini C18-reverse phase column (250X 21.20mm 5 μm particle size), eluent A: water + 0.1% formic acid; b: acetonitrile + 0.1% formic acid. Gradient-95% A/5% B to 5% A/95% B over 15min, flow rate 18 mL/min. Detection-an in-line ultraviolet detector set at a wavelength of 220 nM.

Method 4

Waters Micromass ZQ2000 with C18-reverse phase column (100X 3.0mm Higgins Clipeus, 5 μm particle size) eluting as A: water + 0.1% formic acid; b: acetonitrile + 0.1% formic acid. Gradient:

detection-MS, ELS, UV (100. mu.L split to MS with in-line ultraviolet detector). MS ionization method-electrospray (positive ion).

Method 5

Waters Platform LC quadrupole mass spectrometer with C18-reversed phase column (30X 4.6mm Phenomenex Luna 3 μm particle size) and eluent A: water + 0.1% formic acid; b: methanol + 0.1% formic acid. Gradient:

detection-MS, ELS, UV (200. mu.L split to MS with in-line HP1100 DAD detector). MS ionization method-electrospray (positive and negative ions).

Method 6

Phenomenex Gemini C18-reverse phase column (250X 21.20mm 5 μm particle size), eluent A: water + 0.1% formic acid; b: methanol + 0.1% formic acid. Gradient-95% A/5% B to 5% A/95% B over 15min, flow rate 18 mL/min. Detection-an in-line ultraviolet detector set at a wavelength of 254 nM.

Method 7

Waters Micromass ZQ2000 with C18-reverse phase column (100X 3.0mm Higgins Clipeus, 5 μm particle size) eluting as A: water + 0.1% formic acid; b: methanol + 0.1% formic acid. Gradient:

detection-MS, ELS, UV (100. mu.L split to MS with in-line ultraviolet detector). MS ionization method-electrospray (positive ion).

Method 8

Waters ZMD quadrupole mass spectrometer with C18-reversed phase column (30X 4.6mm Phenomenex Luna 3 μm particle size) with eluent A: water + 0.1% formic acid; b: methanol + 0.1% formic acid. Gradient:

detection-MS, ELS, UV (200. mu.L split to MS with in-line Waters 996 DAD detector). MS ionization method-electrospray (positive and negative ions).

Method 9

Waters Micromass ZQ2000 with Acquity BEH C18 column (50X 2.1mm, 1.7 μm particle size) eluting as A: water + 0.1% formic acid; b: methanol + 0.1% formic acid. Gradient:

detection-MS, UV PDA. MS ionization method-electrospray (positive/negative).

Method 10

A Waters Quatro Microtriple quadrupole mass spectrometer with C18-reverse phase column (100X 3.0mm Higgins Clipeus, 5 μm particle size) with eluent A: water + 0.1% formic acid; b: methanol + 0.1% formic acid. Gradient:

detection-MS, ELS, UV (100. mu.L split to MS with in-line ultraviolet detector). MS ionization method-electrospray (positive/negative).

Method 11

Waters ZMD quadrupole mass spectrometer with C18-reverse phase column (Acquity BEH C18 reverse phase column, 1.7um, 100X2.1mm) eluent A: water + 0.1% formic acid; b: acetonitrile + 0.1% formic acid. Gradient:

detection-MS, UV PDA. MS ionization method-electrospray (positive/negative).

Example 1

N-cyclopropylmethyl-3- {3- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] - [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl } -benzamide

a.N- (5-iodo-pyridin-2-yl) -N' - [1- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] -methylene- (E) -yl ] -hydrazine

To a suspension of 5-iodo-2-hydrazinopyridine (6.62g, 0.028mmol) in EtOH (90mL) was added 3- (2-morpholin-4-yl-ethoxy) -benzaldehyde (6.63g, 0.028 mmol). The reaction was heated under nitrogen at reflux for 2h and then cooled to room temperature. The resulting precipitate was collected by filtration and washed with EtOH to give the title compound (10.93g, 86%) as a white solid. LCMS (method 1): rt 2.37min, m/z 453[ MH ]⁺]。¹H NMR(300MHz，CD₃OD)：δ8.24(1H，d，J 2.2)，7.90-7.84(2H，m)，7.36-7.18(3H，m)，7.12(1H，d，J 8.8)，6.94-6.89(1H，m)，4.19(2H，t，J 5.4)，3.72(4H，t，J 4.6)，2.83(2H，t，J 5.4)，2.62(4H，t，J 4.6)。

6-iodo-3- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] - [1, 2, 4] triazolo [4, 3-a ] pyridine

Example 1, step a (10.93g, 24.16 mmol)) To a solution in DCM (100mL)/EtOH (15mL) was added PhI (OAc)₂(10.74g, 33.35 mmol). The reaction was stirred at room temperature for 72h, diluted with DCM (25mL), then washed with aqueous sodium hydroxide (1M, 25mL) and brine (25 mL). Drying (MgSO)₄) The organic layer was filtered and the filtrate was concentrated in vacuo. With Et₂The residue was triturated with O (10mL) and the resulting solid collected by filtration to give the title compound (9.98g, 92%) as an off-white solid. LCMS (method 1): rt 2.11min, m/z 451[ MH ]⁺]。¹H NMR(400MHz，DMSO-d₆)：δ8.69(1H，s)，7.71(1H，d，J 9.5)，7.60(1H，d，J 9.5)，7.53(1H，t，J 7.9)，7.47-7.41(2H，m)，7.18(1H，d，J 8.1)，4.21(2H，t，J 5.7)，3.59(4H，t，J 4.5)，2.75(2H，t，J 5.7)，2.45(4H，m)。

c.3- {3- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] - [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl } -benzoic acid

The reactor was charged with example 1, step b (400mg, 0.888mmol), 3-mercaptobenzoic acid (205mg, 1.24mmol), [1, 1' -bis (diphenylphosphino) ferrocene]1: 1 Complex of dichloropalladium (II) with DCM (138mg, 0.168mmol), cesium carbonate (578mg, 1.777mmol) and DMF (3 mL). The reaction was degassed under argon (x 3). The reaction was heated at 90 ℃ for 24h, then cooled to room temperature, filtered, and the filtrate was concentrated in vacuo to give the title compound (422mg, quant.). LCMS (method 2): rt 2.20min, m/z 477 MH⁺]。

d.N-cyclopropylmethyl-3- {3- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] - [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl } -benzamide

The reactor was charged with example 1, step c (213mg, 0.447mmol), cyclopropanemethylamine (32mg, 0.447mmol), HATU (212mg, 0.558mmol) and DMF (3 mL). DIPEA (225. mu.L, 1.34mmol) was added,the reaction was stirred at room temperature for 24h then concentrated in vacuo and the crude residue was directly purified by reverse phase preparative HPLC (method 3) to give the title compound (92mg, 39%) as an off-white solid. LCMS (method 4): rt 6.55min, m/z 530[ MH⁺]。¹H NMR(400MHz，CDCl₃)：δ8.39(1H，s)，7.89(1H，s)，7.75-7.65(2H，m)，7.51-7.30(5H，m)，7.17(1H，d，J 9.6)，7.08(1H，d，J 8.4)，6.85(1H，m)，4.25(2H，t，J 5.2)，3.79(4H，t，J 4.4)，3.26(2H，t，J 6.2)，3.00(2H，t，J 5.2)，2.77(4H，m)，1.07-0.95(1H，m)，0.52-0.44(2H，m)，0.24-0.18(2H，m)。

The following examples were prepared using a method similar to that used in example 1.

Example 10

1- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -3- (4- {3- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] - [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl } -phenyl) -urea

3- (6-bromo- [1, 2, 4] triazolo [4, 3-a ] pyridin-3-yl) -phenol

Boron tribromide (1M in DCM, 13.1mL, 13.1mmol) was added slowly to the stirred 3- (3-benzyloxy-phenyl) -6-bromo- [1, 2, 4] at-78 deg.C]Triazolo [4, 3-a]Pyridine (1g, 2.63mmol) in DCM (5 mL). The reaction was allowed to warm to room temperature, stirred for 24h, and saturated NaHCO₃The solution was treated until pH > 7. The product was extracted with DCM: MeOH 9: 1(mL) and dried (MgSO)₄) And concentrated in vacuo. The crude residue was purified by FCC (DCM: MeOH, 1: 0 to 9: 1) to give the title compound (350mg, 46%) as a yellow solid. LCMS (method 2): rt 2.65min, m/z 290 and 292[ MH)⁺]。

6-bromo-3- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] - [1, 2, 4] triazolo [4, 3-a ] pyridine

Example 10 step a (250mg, 1.21mmol), N- (2-bromoethyl) morpholine hydrochloride (306mg, 1.32mmol) and K₂CO₃A mixture of (500mg, 3.62mmol) in DMF (20mL) was heated at 50 ℃ for 24 h. The reaction was then cooled to room temperature and concentrated in vacuo. The residue was purified by FCC (DCM: MeOH, 1: 0 to 9: 1) to give the title compound (479mg, 98%) as a yellow oil.¹H NMR(300MHz，CDCl₃)：δ8.44(1H，dd，J 1.7，1)，7.74-7.69(1H，m)，7.50(1H，t，J 7.9)，7.39-7.35(2H，m)，7.35-7.30(1H，m)，7.14-7.08(1H，m)，4.21(2H，t，J 5.6)，3.74(4H，t，J 4.6)，2.85(2H，t，J 5.6)，2.63-2.56(4H，m)。

4- {3- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] - [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl } -aniline

The title compound was prepared in analogy to example 1 step c, using example 10 step b and 4-amino-benzene-thiol. LCMS (method 2): rt 2.14min, m/z 448[ MH)⁺]。

1- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -3- (4- {3- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] - [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl } -phenyl) -urea

A mixture of example 10, step c (315mg, 0.703mmol), 5- (2, 2, 2-trichloroethoxycarbonyl) amino-3-tert-butyl-1-p-tolyl-1H-pyrazole (299mg, 0.703mmol) and DIPEA (116. mu.L, 0.675mmol) in DMSO (3mL) was heated at 55 ℃ for 24H. The reaction was concentrated in vacuo and the residue was purified by reverse phase preparative HPLC (method 3) to give the title compound (202mg, 41%) as an off-white solid. LCMS (method 4): rt 8.46min, m/z 703[ MH⁺]。¹H NMR(400MHz，CDCl₃)：δ9.30(1H，br s)，8.34(1H，br s)，8.16(1H，s)，7.49-7.41(3H，m)，7.37(1H，d，J 9.6)，7.29(2H，d，J 8.4)，7.25-7.20(2H，m)，7.18(2H，d，J 8)，7.09-7.04(2H，m)，6.90(2H，d，J 8)，6.44(1H，s)，4.15(2H，t，J 5.4)，3.72(4H，t，J 4.5)，2.88(2H，t，J 5.4)，2.64(4H，t，J 4.4)，2.11(3H，s)，1.30(9H，s)。

Using a method similar to that used in example 10 (steps c-d), the following examples were prepared:

example 12

1- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -3- (6- {3- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] - [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl } -pyridin-3-yl) -urea

3- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] -6- (5-nitro-pyridin-2-ylsulfanyl) - [1, 2, 4] triazolo [4, 3-a ] pyridine

To a solution of example 1, step b (300mg, 0.67mmol) in THF (5mL) at 0 deg.C was added dropwise in Et₂2M isopropyl magnesium chloride in O (335. mu.L, 0.67 mmol). The reaction was stirred at 0 ℃ for 1h, then 2, 2' -dithiobis (5-nitro-pyridine) (227mg, 0.73mmol) was added. The reaction was allowed to warm to rt and stirred for an additional 1h, then diluted with EtOAc (10 mL). The organic layer was washed with 1M NaOH (10mL), brine (10mL) and dried (MgSO)₄) And concentrated in vacuo. Purification by reverse phase preparative HPLC (method 3) gave the title compound (50mg, 16%) as a yellow solid. LCMS (method 1): rt 2.33min, m/z 479[ MH⁺]。¹H NMR(400MHz，CD₃OD)：δ9.16(1H，d，J 2.6)，8.80(1H，t，J1.2)，8.41(1H，dd，J 8.9，2.7)，7.90(1H，dd，J 9.6，1)，7.60-7.53(2H，m)，7.50-7.45(3H，m)，7.25-7.20(1H，m)，4.27(2H，t，J 5.4)，3.73(4H，t，J 4.7)，2.94(2H，t，J 5.4)，2.70(4H，t，J 4.5)。

6- {3- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] - [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl } -pyridin-3-ylamine

Example 12 step a (50mg, 0.11mmol) was dissolved in EtOH (10mL) and treated with Pd/C (10%) (10 mg). The reaction was stirred at room temperature under a hydrogen atmosphere for 72 h. The catalyst was filtered under nitrogen and the filtrate was concentrated in vacuo to give the title compound (31mg, 66%). LCMS (method 1): rt 1.94min, m/z 449[ MH⁺]。

c.1- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -3- (6- {3- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] - [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl } -pyridin-3-yl) -urea

The title compound was prepared in a similar manner to example 10 step d using example 12 step b. LCMS (method 4): rt 8.06min, m/z 704[ MH)⁺]。¹H NMR(400MHz，CDCl₃)：δ8.70(1H，br s)，8.57(1H，s)，8.25(1H，dd，J 8.7，2.7)，7.99(1H，d，J 2.7)，7.73(1H，br s)，7.63(1H，d，J 9.5)，7.47(1H，t，J 8)，7.35-7.23(6H，m)，7.09(1H，dd，J 8.4，2.5)，7.04(2H，d，J 8)，6.45(1H，s)，4.20(2H，t，J 5.4)，3.73(4H，t，J 4.6)，2.91(2H，t，J 5.4)，2.67(4H，s)，2.22(3H，s)，1.34(9H，s)。

Example 13

1- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -3- (5- {3- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] - [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl } -pyridin-2-yl) -urea

5- {3- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] - [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl } -pyridin-2-ylamine

The title compound was prepared in analogy to example 1, step c, using 2-amino-5-mercaptopyridine dihydrochloride. LCMS (method 1): rt 1.77min, m/z 449[ MH⁺]。

1- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -3- (5- {3- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] - [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl } -pyridin-2-yl) -urea

The title compound was prepared in a similar manner to example 10 step d using example 13 step a. LCMS (method 4): rt 8.25min, m/z 704[ MH⁺]。¹H NMR(400MHz，CD₃OD)：δ8.31(1H，s)，7.84(1H，dd，J 8.7，2.4)，7.81-7.77(1H，m)，7.74(1H，d，J 2.4)，7.55(1H，t，J 7.9)，7.41-7.33(5H，m)，7.30(2H，d，J 8.1)，7.22(1H，dd，J 8.4，2.4)，7.03(1H，d，J 8.6)，6.51(1H，s)，4.26(2H，t，J 5.4)，3.75(4H，t，J 4.6)，2.97(2H，t，J 5.4)，2.74(4H，t，J 4.6)，2.26(3H，s)，1.34(9H，s)。

Example 14

1- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -3- (trans-4- {3- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] - [1, 2, 4] triazolo [4, 3-a ] pyridin-6-yloxy } -cyclohexyl) -urea

(trans-4- {3- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] - [1, 2, 4] triazolo [4, 3-a ] pyridin-6-yloxy } -cyclohexyl) -carbamic acid tert-butyl ester

A mixture of example 1, step b (234mg, 0.52mmol), copper (I) iodide (9mg, 0.052mmol), 1, 10-phenanthroline (19mg, 0.104mmol), caesium carbonate (335mg, 1.04mmol) and trans-4-boc-aminocyclohexanol (560mg, 2.6mmol) in toluene (3mL) was heated at 110 ℃ for 72h under argon. The suspension was cooled to room temperature, diluted with EtOAc (10mL), and filtered through HiFlo. The filtrate was concentrated in vacuo and the residue was purified by reverse phase preparative HPLC (method 3) to give the title compound (205mg, 39%) as an off-white solid. LCMS (method 2): rt 2.53min, m/z 538[ MH ]⁺]。

b. Trans-4- {3- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] - [1, 2, 4] triazolo [4, 3-a ] pyridin-6-yloxy } -cyclohexylamine trifluoroacetate salt

A solution of example 14, step a (196mg, 0.178mmol) in TFA (2mL) and DCM (10mL) was stirred at room temperature for 0.5h, then concentrated in vacuo to give the title compound (quantitative yield). LCMS (method 2): rt 0.38min, M/z 438[ M-CF₃CO₂ ⁺]。

c.1- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -3- (trans-4- {3- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] - [1, 2, 4] triazolo [4, 3-a ] pyridin-6-yloxy } -cyclohexyl) -urea

The title compound was prepared in a similar manner to example 10 step d using example 14 step b. LCMS (method 4): rt 7.84min, m/z 693[ MH⁺]。¹H NMR(400MHz，CDCl₃)：δ7.76(1H，d，J 2)，7.61(1H，d，J 9.9)，7.47(1H，t，J 8.1)，7.34-7.27(4H，m)，7.13(2H，d，J 8.1)，7.10-7.03(2H，m)，6.86(1H，s)，6.28(1H，s)，5.49(1H，d，J 7.6)，4.19(2H，t，J 5.4)，4.06-3.97(1H，m)，3.76-3.67(5H，m)，2.88(2H，t，J 5.4)，2.62(4H，t，J 4.4)，2.29(3H，s)，2.07(4H，d，J 11.4)，1.65-1.53(2H，m)，1.33(9H，s)，1.29-1.16(2H，m)。

The following examples were prepared using a method similar to that used in example 14.

Example 17

N- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -2- (4- {3- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] - [1, 2, 4] triazolo [4, 3-a ] pyridin-6-yloxy } -piperidin-1-yl) -acetamide

3- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] -6- (piperidin-4-yloxy) - [1, 2, 4] triazolo [4, 3-a ] pyridine

The title compound was prepared in analogy to example 14 steps a-b using N-Boc-4-hydroxypiperidine, followed by purification on an SCX-2 column. LCMS (method 5): rt 0.38, 1.87min, m/z 424[ MH⁺]。

N- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -2-chloro-acetamide

A solution of 5-tert-butyl-2-p-tolyl-2H-pyrazol-3-ylamine (886mg, 3.86mmol), pyridine (465. mu.L, 5.80mmol), and chloroacetyl chloride (462. mu.L, 5.80mmol) in DCM (5mL) was stirred at room temperature for 2H. The reaction mixture was diluted with saturated aqueous sodium bicarbonate and DCM, and the resulting aqueous layer was extracted 2 times with DCM. Drying (MgSO)₄) The combined organic layers were concentrated in vacuo to give the title compound (1.17g, 100%) as a yellow solid. LCMS (method 5): rt4.41min, m/z 306[ MH⁺]。

c.N- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -2- (4- {3- [3- (2-morpholin-4-yl-ethoxy) -phenyl ] - [1, 2, 4] triazolo [4, 3-a ] pyridin-6-yloxy } -piperidin-1-yl) -acetamide

A solution of example 17 step a (32mg, 0.076mmol), example 17 step b (23mg, 0.076mmol), potassium iodide (4mg, 0.002mmol) and potassium carbonate (13mg, 0.091mmol) in acetonitrile (5mL) was heated to reflux for 2.5 h. The reaction mixture was cooled to room temperature, concentrated in vacuo, and purified 2 times by reverse phase preparative HPLC (method 6) to give the title compound (5mg, 9%) as a white solid. LCMS (method 4): delta Rt 6.21min, m/z 693[ MH⁺]。¹H NMR(400MHz，CD₃OD)：7.92(1H，d，J 2.0)，7.76(1H，d，J 9.9)，7.58(1H，t，J 8.1)，7.43-7.32(7H，m)，7.22(1H，dd，J 8.4，2.3)，6.51(1H，s)，4.36-4.29(1H，m)，4.26(2H，t，J 5.4)，3.72(4H，t，J 4.6)，3.10(2H，s)，2.89(2H，t，J 5.4)，2.72-2.62(6H，m)，2.44-2.32(5H，m)，1.85(2H，m)，1.61-1.53(2H，m)，1.33(9H，s)。

Example 18

N- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -2- {4- [3- (3-hydroxy-phenyl) - [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl ] -phenyl } -acetamide

The title compound was prepared in a similar manner to example 10, step a, using example 8. LCMS (method 7): rt 12.01min, m/z 589[ MH⁺]。¹H NMR(400MHz，CD₃OD)：δ8.35(1H，t，J 1.2)，7.74(1H，dd，J 9.6，1.0)，7.45-7.36(1H，m)，7.37-7.32(3H，m)，7.24-7.20(4H，m)，7.25-7.07(4H，m)，6.99(1H，ddd，J8.2，2.4，1.1)，6.32(1H，s)，3.58(2H，s)，2.31(3H，s)，1.31(9H，s)。

Example 19

N- { 5-tert-butyl-2- [4- (2-morpholin-4-yl-ethoxy) -phenyl ] -2H-pyrazol-3-yl } -2- [4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl) -phenyl ] -acetamide

5-tert-butyl-2- [4- (2-morpholin-4-yl-ethoxy) -phenyl ] -2H-pyrazol-3-ylamine

A solution of 4- (5-amino-3-tert-butyl-pyrazol-1-yl) -phenol (WO2005/110994, 0.462g, 2mmol), 2-morpholin-4-yl-ethanol (0.327g, 2.5mmol) and triphenylphosphine (1.05g, 4mmol) in THF (5mL) was treated with diisopropyl azodicarboxylate (0.808mg, 4mmol) under a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 16h, then diluted with diethyl ether. The organic layer was washed with water. The resulting aqueous layer was basified with potassium carbonate and extracted with EtOAc. The resulting organic layer was washed with aqueous citric acid and the resulting aqueous layer was basified with potassium carbonate and extracted with EtOAc (3 times).The obtained organic layer was dried (Na)₂SO₄) Evaporated to dryness, then sonicated with diethyl ether (10mL) and filtered to give the title compound (0.297g, 43%) as a white solid. LCMS (method 5): rt 0.41, 2.16min, m/z 345[ MH⁺]。¹H NMR(400MHz，CDCl₃)：δ7.46-7.39(2H，m)，6.98-6.93(2H，m)，5.50(1H，s)，4.15-4.08(2H，m)，3.75(4H，t，J 4.5)，3.63(2H，bs)，2.83-2.78(2H，m)，2.59(4H，t，J 4.3)，1.34-1.26(9H，m)。

N- { 5-tert-butyl-2- [4- (2-morpholin-4-yl-ethoxy) -phenyl ] -2H-pyrazol-3-yl } -2- [4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl) -phenyl ] -acetamide

The title compound was prepared in a similar manner to example 1, steps c-d, using 3-mercaptoacetic acid and example 19, step a. LCMS (method 7): rt 9.09min, m/z 654[ MH ]⁺]。¹HNMR(400MHz，CDCl₃)：8.12(1H，s)，7.70(1H，d，J 9.6)，7.22(2H，d，J8.0)，7.20-7.09(5H，m)，7.12-7.05(1H，m)，6.93-6.86(2H，m)，6.57(1H，s)，4.17(2H，t，J 5.7)，3.73(4H，t，J 4.5)，3.64(2H，s)，3.41-3.32(1H，m)，2.84(2H，t，J 5.7)，2.59(4H，t，J 4.4)，1.56(3H，s)，1.54(3H，s)，1.32(9H，s)。

Example 20

N- [ 5-tert-butyl-2- (4-hydroxy-phenyl) -2H-pyrazol-3-yl ] -2- [4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl) -phenyl ] -acetamide

a.N- { 5-tert-butyl-2- [4- (tert-butyl-dimethyl-silanyloxy) -phenyl ] -2H-pyrazol-3-yl } -2- [4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl) -phenyl ] -acetamide

In a similar manner to example 19 step b, 5-tert-butyl-2- [4- (tert-butyl-dimethyl-silanyloxy) -phenyl]-2H-pyrazol-3-ylamine (US2006/035922), to prepare the title compound. LCMS (method 5): rt 4.96min, m/z 655 MH⁺]。¹H NMR(300MHz，CD₃OD)：δ8.47(1H，s)，7.65(1H，dd，J＝9.60，1.01Hz)，7.35-7.24(3H，m)，7.22-7.17(4H，m)，6.89-6.84(2H，m)，6.30(1H，s)，3.59(2H，s)，2.99(1H，m)，1.48(3H，s)，1.45(3H，s)，1.30(9H，s)，0.99(9H，s)，0.20(6H，s)。

N- [ 5-tert-butyl-2- (4-hydroxy-phenyl) -2H-pyrazol-3-yl ] -2- [4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl) -phenyl ] -acetamide

A solution of example 20, step a (80mg, 0.12mmol) and triethylamine trihydrofluoride (40mg, 0.24mmol) in THF (2mL) was stirred at room temperature for 24h, then diluted with EtOAc, washed with saturated aqueous sodium bicarbonate, and dried (Na₂SO₄) And evaporated. The residue was purified by FCC (DCM/MeOH 100/0 to 95/5), dissolved in EtOAc, washed with aqueous citric acid and brine, and dried (Na)₂SO₄) Evaporation and drying in vacuo at 40 ℃ gave the title compound (45mg, 68%) as a pink solid. LCMS (method 7): rt 11.15min, m/z 541 MH⁺]。¹H NMR(400MHz，CDCl₃)：δ8.23(1H，s)，7.91(1H，s)，7.74(1H，d，J 9.5)，7.25-7.14(5H，m)，7.05(2H，d，J 8.4)，6.85(2H，t，J8.6)，6.51(1H，s)，3.64(2H，s)，3.47-3.38(1H，m)，1.55(3H，s)，1.53(3H，s)，1.31(9H，s)。

Example 21

1- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -3- [4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-yloxy) -cyclohexyl ] -urea

a. Isobutyric acid N' - (5-fluoro-pyridin-2-yl) -hydrazide

A solution of 5-fluoro-2-hydrazino-pyridine (0.59g, 4.65mmol), isobutyric acid (528mg, 6mmol) and HOBt hydrate (153mg, 1mmol) in DCM (10mL) was treated with EDCI HCl (1.15g, 6 mmol). The reaction mixture was stirred at room temperature for 40min, poured onto saturated aqueous sodium bicarbonate (40mL), extracted with 4 parts DCM and dried (Na)₂SO₄) Evaporated and purified by FCC (DCM/EtOAc 9/1 to 3/7) to give the title compound (0.42g, 46%). LCMS (method 8): rt 2.46min, m/z 198[ MH⁺]。

6-fluoro-3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridines

A solution of example 21 step a (0.41g, 2.08mmol), triphenylphosphine (763mg, 2.91mmol) and triethylamine (0.87mL, 6.24mmol) in THF (5mL) at 0 deg.C was treated with 1, 2-hexachloroethane (690mg, 2.91 mmol). The reaction mixture was stirred at 0 ℃ for 40min, then at room temperature for 20min, quenched with water, extracted 2 times with EtOAc and dried (Na)₂SO₄) Evaporated and purified 2 times by FCC (cyclohexane/EtOAc 1/0 to 1/1) to give the title compound (274mg, containing 20% PPh)₃O, 58%) as a white solid. LCMS (method 5): rt 2.58min, m/z 180[ MH⁺]。

4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-yloxy) -cyclohexylamine

A solution of trans-4-amino-cyclohexanol (402mg, 3.6mmol) and potassium tert-butoxide (395mg, 3.6mmol) in toluene (1mL) and 1, 3-dimethyl-3, 4, 5, 6-tetrahydro-2 (1H) -pyrimidinone (0.5mL) was heated at 80 deg.C under argon for 20min, then step b (274mg, containing 20% PPh)₃O, 1.2 mmol). The reaction mixture was stirred for 1h, quenched with water, and extracted with 3 parts EtOAc. On an SCX column (MeOH to 1N NH in MeOH)₃) The aqueous and combined organic layers were purified above and passed through FCC (DCM/containing 0.1% NH)₃MeOH 100/0 to 85/15) to give the title compound (235mg, 71%). LCMS (method 5): rt 1.86min, m/z 275[ MH ]⁺]。

1- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -3- [4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-yloxy) -cyclohexyl ] -urea

In a similar manner to example 10, step d, starting from example 21, step c, two were usedThe title compound was prepared using an alkane instead of DMSO. LCMS (method 7): rt 11.66min, m/z 530[ MH⁺]。¹H NMR(400MHz，CDCl₃)：δ7.60(1H，d，J 9.9)，7.37(1H，s)，7.34(2H，d，J 8.2)，7.19(2H，d，J 8.1)，7.03(1H，dd，J 9.9，2.0)，6.37(1H，s)，6.23(1H，s)，5.12(1H，d，J 7.5)，4.07(1H，m)，3.73(1H，m)，3.31-3.22(1H，m)，2.34(3H，s)，2.12-2.05(4H，m)，1.68-1.56(2H，m)，1.51(3H，s)，1.49(3H，s)，1.54-1.31(2H，m)，1.34(9H，s)。

Example 22

N- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -2- [4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-yloxy) -cyclohexyl ] -acetamide

[4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-yloxy) -cyclohexyl ] -acetic acid ethyl ester

The title compound was prepared in analogy to example 21 step c, starting from trans- (4-hydroxy-cyclohexyl) -acetic acid ethyl ester (Krieg et al, Journal fur Praktische Chemie1987, 329(6), 1123-30)). LCMS (method 5): rt 4.16-4.23min, m/z 346[ MH⁺]。

[4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-yloxy) -cyclohexyl ] -acetic acid

A solution of example 22, step a (50mg, 0.145mmol) and 1N sodium hydroxide (0.5mL, 0.5mmol) in MeOH (2mL) was stirred at room temperature for 16 h. The reaction mixture was treated with saturated aqueous sodium bicarbonate, evaporated to dryness and purified on reverse phase FCC (water/MeOH 100/0 to 0/100) to give 2 fractions. The first eluted fraction was acidified to pH5 with 1N HCl solution and extracted with EtOAc. Drying (MgSO)₄) The organic layer was filtered, combined with the second eluted fraction from chromatography and concentrated in vacuo. The resulting residue was dissolved in MeOH/EtOAc (1: 1, 10mL), filtered, and evaporated to dryness to give the title compound as a pale brown solid (27mg, 59%). LCMS (method 5): rt 3.58-3.65min, m/z 318 MH⁺]。

c.N- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -2- [4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-yloxy) -cyclohexyl ] -acetamide

The title compound was prepared in analogy to example 1 step d, starting from example 22 step b. LCMS (method 10): rt 11.59min, m/z 529[ MH⁺]。¹H NMR(400MHz，CDCl₃)：δ7.67(1H，d，J 9.9)，7.38(1H，s)，7.36-7.25(4H，m)，7.20(1H，m)，7.04(1H，dd，J 9.8，2.0)，6.60(1H，s)，4.10-3.98(1H，m)，3.3.28(1H，m)，2.41(3H，s)，2.21(2H，d，J 6.5)，2.16(3H，m)，1.93(4H，m)，1.52(3H，s)，1.51(3H，s)，1.34(9H，s)，1.23-1.07(2H，m)。

Example 23

N- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -2- [4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-yloxy) -phenyl ] -acetamide

a.6-iodo-3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridines

The title compound was prepared in analogy to example 1 steps a-b, using 2-methyl-propanal. LCMS (method 5): rt 3.43min, m/z 288[ MH⁺]。¹H NMR(300MHz，CD₃OD)：8.75(1H，s)，7.58(1H，dd，J 9.6，1.5)，7.52(1H，dd，J 9.6，1.1)，3.68-3.49(1H，m)，1.50(3H，s)，1.47(3H，s)。

2- [4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-yloxy) -phenyl ] -acetamide

A solution of example 23, step a (430mg, 1.5mmol), tert-butyl 2- (4-hydroxyphenyl) acetate (WO2008/024746, 621mg, 3mmol), cesium carbonate (978mg, 3mmol), copper (I) chloride (74mg, 0.75mmol) and 2, 2, 6, 6-tetramethylheptanedione (28mg, 0.15mmol) in N-methylpyrrolidone (2mL) was heated at 115 deg.C for 1 h. The reaction mixture was cooled to room temperature, diluted with diethyl ether and washed with water. The aqueous layer was extracted with EtOAc and DCM and dried (Na)₂SO₄) The combined organic layers were filtered and concentrated in vacuo. The resulting oil was purified on a SCX-2 column, eluted with EtOAc, MeOH, then 1N ammonia in MeOH. The ammonia fraction was concentrated in vacuo and passed through FCC (DCM/NH containing)₃MeOH 10/0 to 9/1 followed by EtOAc) 2 times to give the title compound as an orange gum (44mg, 8%). LCMS (method 8): rt4.30min, m/z 368[ MH⁺]。

[4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-yloxy) -phenyl ] -acetic acid

A solution of example 23, step b (44mg, 0.12mmol), TFA (1mL), and anisole (1mL) in DCM (1mL) was stirred at room temperature for 1h, then evaporated in vacuo. The resulting residue was suspended in diethyl ether (3mL), filtered, and dried in vacuo to give the title compound (38mg, quantitative). LCMS (method 5): rt 3.65min, m/z 312[ MH⁺]。

d.N- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -2- [4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-yloxy) -phenyl ] -acetamide

In a similar manner to example 1, step dExample 23 beginning with step c, the title compound was prepared. LCMS (method 10): rt 11.60min, m/z 523[ MH⁺]。¹H NMR(400MHz，CDCl₃)：7.77(1H，d，J 9.9)，7.66(1H，s)，7.29(1H，s)，7.26-7.15(4H，m)，7.14(2H，d，J 8.1)，7.05(1H，dd，J 9.9，2.0)，6.98(2H，d，J 8.1)，6.60(1H，s)，3.67(2H，s)，3.27(1H，m)，2.39(3H，s)，1.52(3H，s)，1.50(3H，s)，1.33(9H，s)。

Example 24

N- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -2- [4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl) -phenyl ] -propionamide

[4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl) -phenyl ] -acetic acid methyl ester

To 4- (3-isopropyl- [1, 2, 4)]Triazolo [4, 3-a]Pyridin-6-ylsulfanyl) -phenyl]Acetic acid (prepared in a similar manner to example 1 step c) (209mg, 0.639mmol) in MeOH (20mL), concentrated HCl (25 μ L) was added and the reaction was stirred at reflux for 5 h. The reaction was cooled to room temperature, concentrated in vacuo, and taken up in DCM (5mL) and saturated NaHCO₃Partitioned (5mL), the organic layer separated and dried (MgSO)₄) And concentrated in vacuo to give the title compound (218 mg). LCMS (method 1): rt 3.04min, m/z342[ MH⁺]。

2- [4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl) -phenyl ] -propionic acid methyl ester

A solution of the product from example 24 step b (218mg, 0.637mmol) in THF (5mL) was cooled to 0 deg.C and LiHMDS (1M in hexanes, 702. mu.L, 0.702mmol) was added. After 10min, iodomethane (43 μ L, 0.702mmol) was added and stirring continued at 0 ℃ for 0.5h, then slowly warmed to room temperature while stirring overnight. With saturated NH₄The reaction was quenched with Cl solution (5mL) and Et₂O (2 × 10mL) extraction, drying (MgSO)₄) And concentrated in vacuo. Purification by chromatography using 0-10% DCM/MeOH gave the title compound (69.2mg, 30%). LCMS (method 1): rt 3.25min, m/z 356[ MH⁺]。

c.2- [4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl) -phenyl ] -propionic acid

A solution of the product from example 24 step c (69.2mg, 0.194mmol) in 1M NaOH (1mL) and MeOH (2mL) was stirred at room temperature for 3.5 h. The reaction was quenched with acetic acid (1mL) and concentrated in vacuo to afford the title compound. LCMS (method 1): rt 3.11min, m/z342[ MH⁺]。

d.N- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -2- [4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl) -phenyl ] -propionamide

The title compound was prepared in a similar manner to example 1 step d, starting from example 24 step c. LCMS (method 4): rt 5.25min, m/z 553[ MH ]⁺]。¹H NMR(400MHz，MeOD)：δ8.53(1H，dd，J 1.6，1.0Hz)，7.66(1H，dd，J 9.5，1.0Hz)，7.34(2H，m)，7.31(1H，dd，J 9.5，1.6Hz)，7.23(2H，m)，7.09(4H，s)，6.27(1H，s)，3.72(1H，q，J 7.1Hz)，3.55(1H，m)，2.31(3H，s)，1.47(6H，2xd，J6.9Hz)，1.40(3H，d，J 7.1Hz)，1.30(9H，s)。

Example 25

1- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -3- [4- (3-cyclopentyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-yloxy) -cyclohexyl ] -urea

The title compound was prepared in a similar manner to example 21. LCMS (method 11): rt4.59min, M/z 555[ M ]⁺]。¹H NMR(400MHz，MeOD)：δ7.88(1H，dd，J 2.1，0.8)，7.60(1H，dd，J 9.9，0.8)，7.30(4H，m)，7.25(1H，dd，9.9，2.1)，6.27(1H，s)，4.32(1H，m)，3.57(2H，m)，2.38(3H，s)，2.20(2H，m)，2.10(2H，m)，1.87(9H，m)，1.56(2H，m)，1.35(1H，m)，1.28(9H，s)。

Example 26

1- [4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-yloxy) -cyclohexyl ] -3- [5- (1-methyl-1-methylsulfanyl-ethyl) -2-p-tolyl-2H-pyrazol-3-yl ] -urea

The title compound was prepared in a similar manner to example 1 step d, starting from example 21 step c. LCMS (method 11): rt4.59min, M/z 561[ M ]⁺]。¹H NMR(400MHz，MeOD)：δ7.85(1H，dd，J 2.1，0.8)，7.57(1H，dd，J 9.9，0.8)，7.30(4H，m)，7.21(1H，dd，9.9，2.1)，6.41(1H，s)，4.32(1H，m)，3.56(1H，m)，(1H，sept.，J 6.9)，2.38(3H，s)，2.11(2H，m)，1.98(2H，m)，1.91(3H，s)，1.61(6H，s)，1.56(2H，m)，1.42(6H，d，J 6.9)，1.35(3H，m)。

Example 27

N- { 5-tert-butyl-2- [3- (2-pyridin-4-yl-ethoxy) -phenyl ] -2H-pyrazol-3-yl } -2- [4- (3-isopropyl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-ylsulfanyl) -phenyl ] -acetamide

The title compound was prepared in a similar manner as example 1, step d. LCMS (method 11): rt 3.81min, m/z 646[ MH⁺]。¹H NMR(400MHz，CDCl₃)：δ8.53-8.49(2H，m)，8.04(1H，s)，7.64(1H，d，J 9.6)，7.26(1H，s NH)，7.24-7.10(7H，m)，7.02(1H，dd，J 9.6，1.6)，6.87(1H，t，J 2.4)，6.82(1H，ddd，J 8.4，2.4，1.0)，6.63(1H，d，J 7.9)，6.57(1H，s)，4.19(2H，t，J 6.4)，3.62(2H，s)，2.36-3.28(1H，m)，3.08(2H，t，J 6.4)，1.51(6H，d，6.9)，1.29(9H，s)。

Example 28

Example 28 was prepared using a method similar to that used in example 1.

LCMS (method 11): rt 5.12min, m/z 525[ MH ]⁺]。¹H NMR(400MHz，CDCl₃)：δ1.36(9H，s)，1.54(6H，d，J 8Hz)，2.41(3H，s)，3.37(1H，q，J 8Hz)，6.71(1H，s)，7.12-7.16(1H，m)，7.28-7.33(2H，m)，7.35-7.46(5H，m)，6.68-7.73(1H，m)，7.78(1H，s)，7.91(1H，s)，8.12(1H，s)。

Example 29

1- (5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl) -3- [4- (3-pyridin-2-yl- [1, 2, 4] triazolo [4, 3-a ] pyridin-6-yloxy) -cyclohexyl ] -urea

In a similar manner to example 10, step d, use twoThe title compound was prepared by substituting DMSO with an alkane and heating at 80 ℃. LCMS (method 7): rt 4.95min, m/z 565[ MH)⁺]。¹H NMR(400MHz，DMSO)：δ.9.36(1H，dd，J 2.3，0.8)，8.74(1H，ddd，J4.9，1.8，1.1)，8.34(1H，dt，J 8.1，1.1，1.1)，8.03-7.97(1H，m)，7.91(1H，s，NH)，7.86(1H，dd，J 9.8，0.8)，7.48(1H，ddd，J 7.5，4.9，1.1)，7.37(1H，dd，J 9.8，2.3)，7.31-7.24(4H，m)，6.52(1H，d，J 7.4 NH)，6.21(1H，s)，4.34-4.24(1H，m)，3.52-3.40(1H，m)，2.32(3H，s)，2.12-2.00(2H，m)，1.92-1.84(2H，m)，1.56-1.44(2H，m)，1.34-1.22(2H，m)，1.20(9H，s)。

Biological assay

p38 kinase assay

The human recombinant p38 enzyme (expressed in E.coli and activated by incubation with MKK6 enzyme (Calbiochem # 559324)) was used as a source of enzyme activity.

The assay was performed in a high binding, clear, flat bottom 96 well assay plate that had been coated with recombinant ATF-2(Biosource # PHF 0043). The experimental compound was incubated with p38 kinase for 2h, and then the kinase assay was started as follows: ATP was added to give a test concentration of 250. mu.M. Phosphorylation of ATF-2 was detected and quantified using ELISA. This involves successive incubations in the presence of anti-phospho-ATF 2, biotinylated anti-IgG and streptavidin-HRP. Incubation with HRP chromogenic substrate (TMB) gave an absorbance proportional to the amount of phosphorylated substrate produced. Absorbance was measured using a multiwell plate reader.

Compounds were diluted in DMSO and then added to assay buffer, with a final DMSO concentration of 1% in the assay.

1C is₅₀Defined as the concentration at which a particular compound achieves 50% inhibition of the control.

The results are shown in table 1:

TABLE 1

Examples	p38 alpha inhibition
		Example 1	+
Example 2	++
		Example 3	++
Example 4	++++
		Example 5	++
Example 6	++++
		Example 7	++++
Example 8	+++
		Example 9	+++
Example 10	+++
		Example 11	+
Example 12	+++
		Example 13	++
Example 14	+++
		Example 15	++
Example 16	+++
		Example 17	+
Example 18	++++
		Example 19	+
Example 20	+++
		Example 21	+++
Example 22	+
		Example 23	++++
Example 24	++
		Example 25	++++
Example 26	+++
		Example 27	+++
Example 28	+++
		Example 29	+++

In Table 1 above, p38 α binding potency (IC) is indicated as follows₅₀Value): < 2000-500nM '+'; < 500-100nM '++'; 10- < 100nM '++'; < 10nM '+++'. All compounds tested showed an IC of < 2000nM₅₀A value; NT: not tested.

p38 functional test

Inhibition of cellular p38 reduces TNF α release, a functional response that can be quantified by measuring the amount of TNF α in the supernatant of LPS-activated THP-1 cells, an immortalized monocyte cell line, or Peripheral Blood Mononuclear Cells (PBMCs) isolated from freshly drawn human blood.

Cells seeded in 96-well plates were pretreated as follows: inhibitor p38 was added for 1h followed by Lipopolysaccharide (LPS) to activate cytokine production and release. The amount of TNF α released into the cell supernatant was quantified using the R & D Systems enzyme-linked immunosorbent assay (ELISA) kit (product DY210) according to the manufacturer's instructions.

Compounds were diluted in DMSO prior to addition, with the final DMSO concentration in the assay being 0.3%. To EC₅₀Defined as the concentration at which a given compound achieves 50% inhibition of the control.

The results are shown in table 2:

TABLE 2

Examples	EC50
		Example 1	+
Example 2	++
		Example 3	+++
Example 4	+++
		Example 5	++
Example 6	+++
		Example 7	++++
Example 8	++
		Example 9	+
Example 10	+++
		Example 11	++
Example 12	+++
		Example 13	+++
Example 14	+++
		Example 15	+
Example 16	+
		Example 17	+
Example 18	+++
		Example 19	+
Example 20	+++
		Example 21	+++
Example 22	+
		Example 23	++++
Example 24	NT
		Example 25	NT
Example 26	NT
		Example 27	NT
Example 28	NT
		Example 29	NT

In Table 2 above, EC is indicated as follows₅₀The value: 500nM '+' < 7000-; < 500-100nM '++'; 10- < 100nM '++'; < 10nM '+++'. All compounds tested showed an EC of < 2000nM₅₀A value; NT: not tested.

Biological assay

Preclinical mouse model of COPD inflammation (tobacco smoke induced pneumonia).

Previous studies have determined that 24h after final Tobacco Smoke (TS) exposure with TS exposure daily for 4 or 11 consecutive days (this time point was used in the studies reported herein), the number of inflammatory cells recovered in bronchoalveolar lavage (BAL) was significantly increased.

The protocol for exposing mice to TS, the protocol for obtaining bronchoalveolar lavage (BAL), and the protocol for preparing cytospin smear slides for differential cell counts are as follows.

Mice were exposed to TS daily for 4 or 11 consecutive days

In this exposure protocol, mice were exposed in 5 groups in a single clear polycarbonate chamber (27cmx16cmx12 cm). TS from the cigarette was allowed to enter the exposure chamber at a flow rate of 100 ml/min. To minimize any potential problems caused by repeated exposure to high levels of TS (6 cigarettes), the exposure of mice to TS was gradually increased over the exposure period to a maximum of 6 cigarettes. For the 4 day exposure, the exposure plan used was as follows:

day 1: 4 cigarettes (about 32min exposure)

Day 2: 4 cigarettes (about 32min exposure)

Day 3: 6 cigarettes (about 48min exposure)

Day 4: 6 cigarettes (about 48min exposure)

For 11 day exposure, the exposure plan used was as follows:

day 1: 2 cigarettes (about 16min exposure)

Day 2: 3 cigarettes (about 24min exposure)

Day 3: 4 cigarettes (about 32min exposure)

Day 4: 5 cigarettes (about 40min exposure)

Day 5-11: 6 cigarettes (about 48min exposure)

Another group of mice was exposed to air based on the same length of time each day as a control (no TS exposure).

Bronchoalveolar lavage (BAL) assay

Bronchoalveolar lavage was performed as follows: the catheter was inserted into the trachea using a Portex nylon intravenous cannula (pink luer) shortened to about 8 mm. Phosphate Buffered Saline (PBS) was used as lavage fluid. A volume of 0.4ml was gently dropped and aspirated 3 times using a 1ml syringe, then placed into eppendorf tubes and kept on ice before later measurement.

Cell counting:

the lavage fluid is separated from the cells by centrifugation, and the supernatant decanted and frozen for later analysis. The cell pellet was resuspended in a known volume of PBS and the total cell number was calculated by counting stained (Turks stained) aliquots under the microscope using a hemocytometer.

Differential cell counts were performed as follows:

diluting the residual cell pellet to about 10⁵Cells/ml. A volume of 500. mu.l was placed in the funnel of a cytospin smear slide and centrifuged at 800rpm for 8 min. Air-drying the slides and following the instructions of the Specifications, dryingStaining was performed with a 'Kwik-Diff' solution (Shandon). After drying and coverslipping, differential cells were counted using light microscopy. Using light microscopy, up to 400 cells were counted by an unbiased operator. Cells were identified using standard morphometric techniques.

Medical treatment

Rodents such as mice and rats are forced to install nasal breathing devices and thus oral delivery of experimental materials such as therapeutic agents for inhalation will not result in good lung exposure. As a result, delivery of the therapeutic agent to the lungs of the rodent is typically achieved by inhalation through intranasal, intratracheal, or systemic aerosol exposure within the chamber.

The laboratory method uses a large amount of experimental material and is generally intended for inhalation toxicology studies, not pharmacological efficacy studies. Since almost all experimental material is delivered to the lungs, intratracheal administration is a very effective delivery method, but it is entirely an invasive technique. In particular, for studies in mice, its technical requirements are also very high, since the diameter of the trachea is very small. The intranasal route is less invasive than the intratracheal route and is therefore particularly suitable for repeated administration studies, such as the 4-11 day mouse model described below. After intranasal administration, approximately 50% of the administered dose is delivered to the lungs (Eyles JE, Williamson ED and Alpar HO.1999, Int J Pharm, 189 (1): 75-9).

As an alternative to oral inhalation, mice were administered vehicle intranasally (0.2% Tween 80 in saline), example 10 (30. mu.g/kg), example 10 (100. mu.g/kg) or example 10 (300. mu.g/kg). Mice in the control group received vehicle 1 hour per day before exposure to air (up to 50 minutes per day). TS exposure was performed for 4 days. BAL was performed 24h after final TS exposure.

Data management and statistical analysis

All results are expressed as a single data point for each animal and the mean value for each group is calculated. Since the normality test was positive, the data were subjected to a one-way analysis of variance test (ANOVA), followed by Bonferroni correction for multiple comparisons to test for significance between treatment groups. A "p" value of < 0.05 was considered statistically significant. Percent inhibition of cell data was automatically calculated within the Excel spreadsheet program using the following formula:

using the above equation, the inhibition data for the other parameters is calculated manually.

Drawings

FIG. 1 is a bar graph illustrating the effect of intranasal administration of vehicle (0.2% Tween 80 in saline), example 10 (30. mu.g/kg), example 10 (100. mu.g/kg) or example 10 (300. mu.g/kg) to experimental mice 24 hours after final exposure on the number of tobacco smoke-induced BAL cells.

FIG. 2 is a bar graph illustrating the effect of intranasal administration of vehicle (0.2% Tween 80 in saline), example 10 (30. mu.g/kg), example 10 (100. mu.g/kg) or example 10 (300. mu.g/kg) to experimental mice 24 hours after final exposure on the number of tobacco smoke-induced BAL neutrophils.

As shown in figure 1, example 10 at 30, 100 or 300 μ g/kg significantly inhibited TS-induced BAL cell influx when administered by the intranasal route. Similar results were observed for BAL neutrophils (figure 2). The results demonstrate a clear anti-inflammatory effect in the lungs of TS exposed mice.

Claims (17)

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof:

wherein:

R¹is C₁-C₆Alkyl radical, C₃-C₆Cycloalkyl, optionally substituted phenyl, optionally substituted 5-or 6-membered monocyclic heteroaryl, or a group of formula (II)

Wherein n is 1 or 2, and R³And R⁴Independently is H or C₁-C₆Alkyl, or R³And R⁴Together with the nitrogen to which they are attached form a 6-membered heterocyclic ring, optionally containing a further heteroatom selected from N and O;

y is-O-or-S (O)_p-, wherein p is 0, 1 or 2;

a is optionally substituted divalent arylene, or monocyclic or bicyclic heteroarylene, or C having 5 or 6 ring atoms₃-C₆Divalent cycloalkylene radicals, or piperidylene radicals, wherein the ring nitrogen is attached to R²NHC(＝O)W-；

W is a bond, -NH-or-C (R)^A)(R^B) -, wherein R^AAnd R^BIndependently H, methyl, ethyl, amino, hydroxy or halogen; and is

R²Is a group of formula (IIIA), (IIIB) or (IIIC):

wherein

m is 0 or 1;

q is 0, 1, 2 or 3;

t is-N or-CH;

R⁵is H or F;

R⁷is-CH₃；-C₂H₅；-CH₂OH、-CH₂SCH₃；-SCH₃or-SC₂H₅；

R⁸is-CH₃or-C₂H₅(ii) a And is

Each occurrence of R⁶Independently is H, C₁-C₆Alkyl, hydroxy or halogen; or a single occurrence of R⁶Is a radical of the formula (IVA), (IVB) or (IVC)

While any other occurrence of R⁶Independently is H, C₁-C₆Alkyl, hydroxy or halogen;

wherein n and p are as defined above;

and wherein at R⁶In

R^61aAnd R^61bIs H, alkyl, or R^61aAnd R^61bMay be combined with the nitrogen to which they are attached to form a heterocyclic ring, optionally containing other heteroatoms selected from N and O.

2. A compound as claimed in claim 1 wherein the divalent group-W- [ a ] -Y-has one of the following formulae (B) to (J):

3. a compound as claimed in claim 1 having formula (IA):

wherein:

v, V ', X and X' are independently-CH or-N; and is

R¹、R²Y and W are as defined in claim 1.

4. A compound as claimed in claim 1 having formula (IA)¹)：

Wherein Y is O or S, and R¹And R²As defined in claim 1.

5. A compound as claimed in claim 1 having formula (IB):

wherein:

u is CH or N, and;

R¹、R²y and W are as defined in claim 1, with the proviso that when U is N, then W is not NH.

6. A compound as claimed in claim 1 having formula (IB)¹)：

Wherein Y is O or S, and R¹And R²As defined in claim 1.

7. A compound as claimed in claim 1 having formula (IC):

wherein Y is O or S, R²As defined in claim 1, and R¹Is phenyl, 5-or 6-membered monocyclic heteroaryl or a radical of the formula (II) as defined in claim 1.

8. A compound as claimed in any one of the preceding claims whereinR¹Is a radical of the formula (II) as defined in claim 1, in which the radical-NR³R⁴Is morpholinyl.

9. A compound as claimed in any one of claims 1 to 7 wherein R is¹Is isopropyl or 2, 6-dichlorophenyl.

10. A compound as claimed in any one of the preceding claims wherein R is²Is a radical of the formula (IIIC) as defined in claim 1, in which R⁷And R⁸Each is methyl.

11. A compound as claimed in any one of claims 1 to 9 wherein R is²Having formula (IIID), (IIIE), (IIIF) or (IIIG):

12. a compound as claimed in any one of claims 1 to 9 wherein R is²Is a radical of formula (IIIA) as defined in claim 1, in which m is 0.

13. A compound as claimed in any one of claims 1 to 9 wherein R is²Is a radical of formula (IIIB) as defined in claim 1, wherein (a) T is-CH ═ and R⁵H; or (b) T is-N ═ and R⁵H; or (c) T is-CH ═ and R⁵＝F。

14. A pharmaceutical composition comprising a compound as claimed in any one of the preceding claims together with one or more pharmaceutically acceptable carriers and/or excipients.

15. A composition as claimed in claim 14 which is suitable for pulmonary administration by inhalation.

16. Use of a compound as claimed in any one of claims 1 to 13 for the treatment of a disease or condition which benefits from inhibition of p38MAP kinase activity.

17. The use as claimed in claim 16, wherein the disease or condition is chronic eosinophilic pneumonia, asthma, COPD, Adult Respiratory Distress Syndrome (ARDS), exacerbation of airway hyperreactivity consequent to other drug therapy or airway disease associated with pulmonary hypertension.