WO2002062793A1

WO2002062793A1 - Thiazole compounds as tgf-beta inhibitors

Info

Publication number: WO2002062793A1
Application number: PCT/EP2002/000991
Authority: WO
Inventors: Francoise Jeanne Gellibert
Original assignee: Glaxo Group Limited
Priority date: 2001-02-02
Filing date: 2002-01-31
Publication date: 2002-08-15
Also published as: JP2004521903A; EP1366047A1; GB0102673D0

Abstract

Therapeutically active thiazole derivatives of formula (I) wherein R1 R2, X and X1 as are defined in the specification, processes for the preparation thereof, the use thereof in therapy, particularly in the treatment of prophylaxis of disorders characterised by overexpression of transforming growth factor β (TGF-β), and pharmaceutical compositions for use in such therapy.

Description

THIAZOLE COMPOUNDS AS TGF-BETA INHIBITORS

The present invention relates to novel thiazole derivatives, processes for the preparation thereof, the use thereof in therapy, particularly in the treatment or prophylaxis of disorders characterised by overexpression of transforming growth factor β (TGF-β), and pharmaceutical compositions for use in such therapy.

TGF-β is a multi-functional cytokine which belongs to the TGF-β superfamily which includes activins/inhibins, bone morphogenetic proteins (BMPs) and TGF- βs. Three isoforms of TGF-β (TGF-β1 , TGF-β2, and TGF-β3) have been identified in mammals, each of which is encoded by a distinct gene on different chromosomes (D.A. Lawrence, Eur. Cytokine. Netw., 1996, 7(3), 363). TGF-β initiates an intracellular signalling pathway which ultimately leads to the expression of genes that regulate cell cycle, control proliferative responses, or relate to extracellular matrix proteins that mediate cell adhesion, migration and intercellular communication. TGF-β has pleitropic effects including modulation of cell growth and differentiation, extracellular matrix formation, hematopoiesis, and immunomodulation (Roberts and Spoon, Handbook of Experimental Pharmacology, 1990, 95, 419-458).

A variety of cell surface proteins and receptors are known to transduce the signals initiated by the binding of the active TGF-β ligand to its receptors. Initiation of the TGF-β signalling pathway results from the binding of the TGF-β ligand to the extracelullar domain of the type II membrane receptor (Massague, Ann. Rev. Biochem., 1998, 67, 753.). The bound type II receptor then recruits type I (Alk5) receptor into a multimeric membrane complex, whereupon active type II receptor kinase phoshorylates and activates type I receptor kinase. The function of the type I receptor kinase is to phosphorylate a receptor-associated co-transcription factor, Smad-2 or Smad-3; thereby releasing it into the cytoplasm where it binds to Smad-4. The PAI-1 gene is activated by TGF-β as a consequence of the abovementioned cellular pathway.

One approach to the treatment and/or prophylaxis of disorders characterised by the overexpression of TGF-β is inhibition of the TGF-β signal transduction. For example inhibition of the TGF-β type II receptor by overexpression of a dominant negative TGF-β type II receptor has previously been shown to prevent liver fibrosis and dysfunction in rat models (Proc. Natl. Acad. Sci, 1999, 96(5), 2345), and also to prevent progression of established liver fibrosis (Hepatology, 2000, 32, 247).

Pathological overexpression of TGF-β is known to be associated with a number of undesirable effects, leading ultimately to the development of serious pathogenic conditions (G.C. Blobe er a/., N. Engl. J. /Wed., 2000, 1350). In particular, pathological overexpression of TGF-β may cause excessive accumulation of extracellular matrix (ECM), inhibition of cell proliferation and immunosupression. Excessive accumulation of ECM is known to lead to fibrotic diseases such as tumor fibrosis, radiation-induced fibrosis, fibrosis of the liver, kidney, lung, bowel, heart, pancreas, peritoneum or other organs. Fibrosis can lead to pathologic conditions such as cirrhosis, idiopathic pulmonary fibrosis, glomerulosclerosis and hypertrophic scars.

A number of other disease states are known to be associated with variations in the expression of genes which are controlled by TGF-β including cancer development, abnormal bone function and inflammatory disorders.

The development of compounds capable of inhibiting the TGF-β intracellular pathway is seen as a desirable way to effect prophylaxis and/or treatment of the above- mentioned conditions. Compounds capable of inhibiting the TGF-β intracellular pathway and/or the expression of TGF-β may be used in the treatment of disorders the symptoms of which often lead to the development of fibrotic conditions. For example, compounds of the present invention may be useful in treating the fibrosis associated with various liver-related conditions such as hepatitis B virus (HBV), hepatitis C virus (HCV), alcohol-induced hepatitis, haemochromatosis and primary biliary cirrhosis.

The compounds of the present invention are thiazole derivatives. Other thiazole compounds have previously been described for use in alternative medicinal applications. PCT Patent Application WO 96/03392 (Searle & Co) discloses a series of substituted thiazole compounds for the treatment of inflammation and inflammation-related disorders. WO 93/15071 (SmithKline Beecham Intercredit NN.) describes a series of thiazolyl-pyridine derivatives which may be used as gastric acid secretion inhibitors. This type of compound may be useful in the treatment of gastrointestinal disorders such as gastric and duodenal ulcers, aspiration pneumonitis and Zollinger-Ellison Syndrome. US Patent No. 5,232,921 (Biziere ef a/.) discloses 2-alkylaminothiazoles having an affinity for muscarinic cholinergic receptors. None of the aforementioned patent applications describe the thiazole compounds of the present invention.

PCT Patent Application WO 00/12947 (Scios Inc.) describes the use of a series of quinazoline derivatives for treating various disorders associated with enhanced activity of kinase p38-α and/or TGF-β. The compounds described therein have been shown to inhibit the activities of both proteins and are therefore particularly useful for the treatment of conditions in which an enhanced activity towards both p38-α and TGF-β is required.

It has now been discovered that certain substituted thiazole compounds, as described below, are useful in the treatment or prophylaxis of disorders characterised by the overexpression of TGF-β. In particular, compounds of the present invention are TGF-β inhibitors which act at the TGF-β type I (Alk5) receptor level.

According to one aspect of the present invention, we provide compounds of formula (I),

wherein,

R¹ is selected from H, halo (such as fluoro, chloro, bromo), -CN, -CF₃, Cι_₄ alkyl or d_^t alkoxy;

R² is selected from -(CH₂)_n-phenyl, -(CH₂)_n-heterocyclyl, -(CH₂)_n-heteroaryl, each of which may be further substituted by one or more substituents, which may be the same or different, selected from halo (such as fluoro, chloro, bromo), -CN, -CF₃, -OH, -OCF₃, 0,-₄ alkyl or C^ alkoxy, -NO_2l -NH₂, -NR³R⁴, -CONR³R⁴, -NHCOR³, -SO₂R³, - SO₂NHR³,-O(CH₂)_mNR³R⁴;

R³ is selected from H or C^ alkyl;

R⁴ is selected from heterocyclyl or heteroaryl;

n is O, 1 , 2, 3, 4 or 5;

m is 0, 1 ,2 or 3;

X and X', which may be the same or different, are each selected from CH or N, provided that X and X' are not both N;

and salts and solvates thereof (hereinafter "compounds of the invention").

As used herein the term "alkyl" as a group or part of a group refers to a straight or branched chain saturated aliphatic hydrocarbon radical containing the specified number(s) of carbon atoms. Such alkyl groups in particular include methyl, ethyl, n- propyl, /so-propyl, n-butyl, sec-butyl, terf-butyl, pentyl and hexyl. The term "alkoxy" as a group or part of a group refers to an alkyl ether radical, wherein the term "alkyl" is defined above. Such alkoxy groups in particular include methoxy, ethoxy, n-propoxy, /so-propoxy, n-butoxy, /so-butoxy, sec-butoxy and tert- butoxy.

The terms "heterocyclyl" as a group or a part of a group refers to a stable saturated or partially saturated (i.e. non-aromatic) 3 to 6 membered monocyciic ring containing one or more hetero atoms independently selected from nitrogen, oxygen and sulfur, optionally substituted with one or more substituents, which may be the same or different, selected from halo (such as fluoro, chloro, bromo), -CN, -CF₃, -OH, -OCF₃, d^ alkyl and C^ alkoxy.

The term "heteroaryl" as a group or part of a group refers to a stable heterocyclic aromatic 6 to 14 membered monocyciic ring containing one or more hetero atoms independently selected from nitrogen, oxygen and sulfur, optionally substituted with one or more substituents, which may be the same or different, selected from halo (such as fluoro, chloro, bromo), -CN, -CF₃, -OH, -OCF₃, C^ alkyl and C_1-4 alkoxy.

The present invention also covers the physiologically acceptable salts of the compounds of formula (I). Suitable physiologically acceptable salts of the compounds of formula (I) include acid salts, for example sodium, potassium, calcium, magnesium and tetraalkylammonium and the like, or mono- or di- basic salts with the appropriate acid for example organic carboxylic acids such as acetic, lactic, tartaric, malic, isethionic, lactobionic and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids and inorganic acids such as hydrochloric, sulfuric, phosphoric and sulfamic acids and the like.

The present invention also relates to solvates of the compounds of Formula (I), for example hydrates.

Preferably, R¹ is positioned at the C(3) or C(6) position of the pyridine ring and is selected from H, halo (such as fluoro, chloro, bromo), -CN, -CF₃, C^ alkyl or C- alkoxy. More preferably R¹ is H.

Preferably, R² is-(CH₂)_n-phenyl, which may be further substituted by one or more substituents, which may be the same or different, selected from halo (such as fluoro, chloro, bromo), -CN, -CF₃, -OH, -OCF₃, C^ alkyl or C,-» alkoxy, -NO₂, -NH₂, -NR³R⁴, -CONR³R⁴, -NHCOR³, -SO₂R³, -SO₂NHR³,-O(CH₂)_mNR³R⁴.

Preferably n is 0 or 1. It will be appreciated that the present invention is intended to include compounds having any combination of the preferred groups as listed hereinbefore.

Compounds of formula (I) which are of special interest as agents useful in the treatment or prophylaxis of disorders characterised by the overexpression of TGF-β are,

[4-(2-Amino-4-pyridin-2-yl-thiazol-5-yl)-pyridin-2-yl]-phenyl-amine; [4-(2-Amino-4-pyridin-2-yl-thiazol-5-yl)-pyridin-2-yl]-(4-methoxy-phenyl)-amine; [4-(2-Amino-4-pyridin-2-yl-thiazol-5-yl)-pyridin-2-yl]-(4-fluoro-phenyl)-amine; 5-[4-(2-Amino-4-pyridin-2-yl-thiazol-5-yl)-pyridin-2-ylamino]-2-methoxy-phenol; 3-[4-(2-Amino-4-pyridin-2-yl-thiazol-5-yl)-pyridin-2-ylamino]-N-methyl-benzamide; N-{4-(2-Amino-4-pyridin-2-yl-thiazol-5-yl)-pyridin-2-ylamino]-phenyl}-acetamide; and salts and solvates thereof.

Certain compounds of formula (I) may exist in stereoisomeric forms (e.g. they may contain one or more asymmetric carbon atoms or may exhibit cis-trans isomerism). The individual stereoisomers (enantiomers and diastereoisomers) and mixtures of these are included within the scope of the present invention. Likewise, it is understood that compounds of formula (I) may exist in tautomeric forms other than that shown in the formula and these are also included within the scope of the present invention.

Salts of the compounds of the present invention may comprise acid addition salts derived from a nitrogen in the compound of formula (I). The therapeutic activity resides in the moiety derived from the compound of the invention as defined herein and the identity of the other component is of less importance although for therapeutic and prophylactic purposes it is, preferably, pharmaceutically acceptable to the patient. Examples of pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids, and organic acids, such as tarta c, acetic, trifluoroacetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic and methanesulphonic and arylsulphonic, for example β-toluenesulphonic, acids.

Compounds of formula (I) and salts and solvates thereof may be prepared by the methodology described hereinafter, constituting a further aspect of this invention. In yet a further aspect of the invention there is provided a process for the preparation of intermediate compounds of formula (C).

Intermediate compounds of formula (C) may conveniently be prepared according to the general methodology in Scheme I below: Scheme 1

Reagents and conditions (preferred): (i) R¹(C₅H₃N)CO₂Et, THF; (ii) NaHMDS, THF,- 78°C to r.t ; (iii) polymer-supported pyridinium perbromide, dioxane.r.t.

Compounds of formula (I) in which R¹ is H may conveniently be prepared from Rink resin according to the general methodology in Scheme 2 below:

Scheme 2

Reagents and conditions (preferred): (i) piperidine, DMF; r.t; (ii) Fmoc-NCS, DCM, r.t; (iii) intermediate (C), dioxane, r.t; (iv) R²NH₂₎ Pd₂(dba)₃, binap, tBuOK, dioxane, 80°C; (vi) 20%TFA, DCM, r.t.

Compounds of formula (I) in which R¹ is H may also be conveniently prepared according to the general methodology in Scheme 3 below:

Scheme 3

Reagents and conditions (preferred): (i) R (C₅H₃N)CO₂Et, NaHMDS, THF, -78°C to r.t ; (ii) polymer-supported pyridinium perbromide, dioxane or THF, r.t., (iv) thiourea, EtOH, reflux; (iii) trityl chloride, K₂CO₃, acetone, reflux., (v) R NH₂, Pd₂(dba)₃, binap, toluene, 80°C, (vi) HCI N, MeOH, reflux or TFA, DCM, r.t.

List of Abbreviations

Binap 2,2'-Bis(diphenylphosphino)-1 , 1 '-binaphthyl

DCM Dichloromethane

EtOH Ethanol

Fmoc Fluorenylmethoxycarbonyl

NaHMDS Sodium b/s(trimethylsilyl)amide

THF Tetrahydrofuran

4-Quinolinyl compounds of formula (A) may be prepared by processes analogous to those known in the art (e.g. R.H.F. Manske and M. Kulka, Org. React, 1953, 7, 59; Song er a/., J. Heterocycl. Chem., 1993, 30, 17).

2-Bromo-4-methylpyrimidine can be prepared from 2-amino-4-methylpyrimidine (commercial) as described in the literature : Mukkala, Veli-Matti;Sund, Christian; Kwiatkowski, Marek; Pasanen, Paavo; Hoegberg, Maria; et al.; Helv.Chim.Acta; 75; 5; 1992; 1621-1632.

Monosubstituted pyridyl esters, R¹(C₅H₅N)CO₂Et (where R¹ is hereinbefore defined) as described in step (ii) above may be prepared by processes analogous to those known in the art. For example, where R¹ = C(6)-OMe, Finger et al., J. Org. Chem., 1962, 27, 3965; where R¹ = C(3)-OMe, Dejardin et al., Bull. Chem. Soc. Fr., 1979, 289; where R¹ = C(5)-Br, Chambers and Marfat, Synth. Commun., 1997, 27(3), 515; and where R¹ = C(4)-CN, Heinisch and Lotsch, Heterocycles, 1987, 26(3), 731.

The compounds of the present invention have been found to inhibit phosphorylation of the Smad-2 or Smad-3 proteins by inhibition of the TGF-β type I (Alk5) receptor.

Furthermore, the compounds of the invention have been tested in the assays described herein and have been found to be of potential therapeutic benefit in the treatment and prophylaxis of disorders characterised by the overexpression of TGF- β-

Thus there is provided a compound of formula (I) or a physiologically acceptable salt or solvate thereof for use as a medicament in human or veterinary medicine, particularly in the treatment or prophylaxis of disorders characterised by the overexpression of TGF-β.

It will be appreciated that references herein to treatment extend to prophylaxis as well as the treatment of established conditions. It will further be appreciated that references herein to treatment or prophylaxis of disorders characterised by the overexpression of TGF-β, shall include the treatment or prophylaxis of TGF-β associated disease such as fibrosis, especially liver and kidney fibrosis, cancer development, abnormal bone function and inflammatory disorders, and scarring.

Other pathological conditions which may be treated in accordance with the invention have been discussed in the introduction hereinbefore. The compounds of the present invention are particularly suited to the treatment of fibrosis and related conditions.

Compounds of the present invention may be administered in combination with other therapeutic agents, for example antiviral agents for liver diseases or in combination with ACE inhibitors or Angiotensin II receptor antagonists for kidney diseases.

According to another aspect of the invention, there is provided the use of a compound of formula (I) or a physiologically acceptable salt or solvate thereof for the manufacture of a medicament for the treatment and/or prophylaxis of disorders characterised by the overexpression of TGF-β, particularly fibrosis.

In a further aspect there is provided a method for the treatment of a human or animal subject with a disorder characterised by the overexpression of TGF-β, particularly fibrosis, which method comprises administering to said human or animal subject an effective amount of a compound of formula (I) or a physiologically acceptable salt or solvate thereof. Compounds of the invention may be formulated for administration in any convenient way, and the invention therefore also includes within its scope pharmaceutical compositions for use in therapy, comprising a compound of formula (I) or a physiologically acceptable salt or solvate thereof in admixture with one or more physiologically acceptable diluents or carriers.

There is also provided according to the invention a process for preparation of such a pharmaceutical composition which comprises mixing the ingredients.

Compounds of the invention may , for example, be formulated for oral, buccal, parenteral, topical or rectal administration.

Tablets and capsules for oral administration may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, mucilage of starch or polyvinyl pyrrolidone; fillers, for example, lactose, microcrystalline cellulose, sugar, maize- starch, calcium phosphate or sorbitol; lubricants, for example, magnesium stearate, stearic acid, talc, polyethylene glycol or silica; disintegrants, for example, potato starch, croscarmellose sodium or sodium starch glycollate; or wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in the art. Oral liquid preparations 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 constitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example, sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxymethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats; emulsifying agents, for example, lecithin, sorbitan mono-oleate or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters, propylene glycol or ethyl alcohol; or preservatives, for example, methyl or propyl β- hydroxybenzoates or sorbic acid. The preparations may also contain buffer salts, flavouring, colouring and/or sweetening agents (e.g. mannitol) as appropriate.

For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner.

The compounds may also be formulated as suppositories, e.g. containing conventional suppository bases such as cocoa butter or other glycerides.

Compounds of the invention may also be formulated for parenteral administration by bolus injection or continuous infusion and may be presented in unit dose form, for instance as ampoules, vials, small volume infusions or pre-filled syringes, or in multi- dose containers with an added preservative. The compositions may take such forms as solutions, suspensions, or emulsions in aqueous or non-aqueous vehicles, and may contain formulatory agents such as anti-oxidants, buffers, antimicrobial agents and/or toxicity adjusting agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use. The dry solid presentation may be prepared by filling a sterile powder aseptically into individual sterile containers or by filling a sterile solution aseptically into each container and freeze-drying.

By topical administration as used herein, we include administration by insufflation and inhalation. Examples of various types of preparation for topical administration include ointments, creams, lotions, powders, pessaries, sprays, aerosols, capsules or cartridges for use in an inhaler or insufflator or drops (e.g. eye or nose drops).

Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents and/or solvents. Such bases may thus, for example, include water and/or an oil such as liquid paraffin or a vegetable oil such as arachis oil or castor oil or a solvent such as a polyethylene glycol. Thickening agents which may be used include soft paraffin, aluminium stearate, cetostearyl alcohol, polyethylene glycols, microcrystalline wax and beeswax.

Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilising agents, dispersing agents, suspending agents or thickening agents.

Powders for external application may be formed with the aid of any suitable powder base, for example, talc, lactose or starch. Drops may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilising agents or suspending agents.

Spray compositions may be formulated, for example, as aqueous solutions or suspensions or as aerosols delivered from pressurised packs, with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, 1 ,1 ,1 ,2,3,3,3-heptafluoropropane, 1 ,1 ,1 ,2- tetrafluorethane, carbon dioxide or other suitable gas.

Capsules and cartridges for use in an inhaler or insufflator, of for example gelatin, may be formulated containing a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.

Compounds of the invention may conveniently be administered in amounts of, for example, 0.01 to 100 mg/kg body weight, suitably 0.05 to 25 mg/kg body weight orally, one or more times a day. The precise dose will of course depend on the age and condition of the patient, the particular route of administration chosen, and is entirely within the discretion of the administering physician.

The following non-limiting Examples illustrate the present invention.

Intermediates

Intermediate 1 : 1-Pyridin-2-yl-2-r2-bromo-pyridin-4-vπ-ethanone

To a solution of 2-bromo-4-methyl-pyridine (27 g ) in dry THF (270 ml) was added ethyl picolinate (28.5 g), the resulting mixture was cooled at -78°C under argon. A solution of sodium bis-(trimethylsilyl)amide 1M in THF (345 ml) was added dropwise at -78°C and after completion the reaction mixture was allowed to warm up to room temperature and stirred overnight. The solvent was evaporated under reduced pressure and the solid residue was triturated with diethyl ether, filtered and washed with diethyl ether. The solid was then diluted with saturated NH₄CI solution and the aqueous phase was extracted with ethyl acetate. The organic layer was dried over sodium sulfate and concentrated. The resulting orange powder was washed with pentane to give the title compound as a yellow solid (33.97g). m.p : 111.2°C

¹H NMR (CDCI₃): δ 8.56 (d,1H); 8.12 (d, 1 H); 7.9 (d , 1 H); 7.7 (td,1 H);7.39-7.34 (m,1 H); 7.33 (s,1H); 7.06 (d,1H); 4.37 (s, 2H).

Intermediate 2: 2-Bromo-1 -Pvhdin-2-yl-2-f2-bromo-pyridin-4-vπ-ethanone

Intermediate 1 (280mg) was added to a suspension of the polymer-supported pyridinium perbromide (Fluka, 705mg, 1.26eq) in dioxane (2.5ml) . The reaction mixture was stirred under nitrogen at r.t. for 3h. Resin was removed by filtration, washed with dioxane (1ml) to give the bromo derivative which was used in the next step without further purification.

General solid phase synthesis of intermediate D

Stepl : Rink Argopore resin (12g, 0.64mmol/g substitution) was placed into a peptide vessel and swollen through the addition of CH₂CI₂ (100ml), DMF (100ml), iPrOH(IOOml) and CH₂CI₂ (2x100ml). The resin was then treated for 5 min with a solution of piperidine 20% in DMF (3x100ml). After washing with DMF(3x100ml) and CH₂CI₂ (4x100ml),the resin was treated with a solution of Fmoc NCS (0.2M) in CH₂CI₂ (190ml ) for 1 h at r.t. The resin was washed with CH₂CI₂ (3x, 100ml), DMF(3x, 100ml) and subsequently reacted with 20% piperidine in DMF (100ml) for 5 min at r.t to give after washing with DMF (3x100ml) and dioxane (3x100ml) the resin bound thiourea. Step2: The thiourea resin was reacted with intermediate 2 (0.18M) in dioxane (175ml) for 4h at r.t . The resin was washed with dioxane (3x100ml). A second exposure with intermediate 2 (0.18M in dioxane, 175ml) was realized and the resin was washed with CH₂CI₂ (5x100ml) and dried under a stream of nitrogen overnight. 2 mg of the resin obtained were cleaved with a solution 20%TFA/ CH₂CI₂ and 5-[2- bromo-pyridin-4-yl]-4-pyridin-2-yl-1 ,3-thiazol-2-amine was characterized by LC-MS (purity >96%). MS m/z 333,335,336 (MH+)

Step3: General procedure for the coupling with anilines

The bromo aminothiazole resin (1g) was weighed out into a polyethylene reaction vessel. Pd₂(dba)₃ (100mg,0.11mmol, 0.17eq), binap (250mg,0.4mmol, 0.62eq), and sodium tert-butoxide (1.1g,9mmol, 14eq) were added and suspended in dioxane (16ml).The reaction vessel was purged with argon for 5 min then aryl amine (20.5mmol, 32eq) was added to the suspension, and the mixture was stirred at 80°C for 15h. The resin was washed with water (15ml), DMF/ CH₂CI₂/iPrOH/ CH₂CI₂/diethyl ether (2x30ml). The coupling reaction and the subsequent wash were repeated two more times. Resin was cleaved by treatment with a solution of 20% TFA in CH₂CI₂(8ml). The sample was concentrated and purified by chromatography (silica gel, with a gradient CH₂CI₂/MeOH 95:5 to 80:20) to afford the 2-aminothiazole compound (D).

Examples

Example 1 : 5-r4-(2-Amino-4-pyridin-2-yl-thiazol-5-yl)-pyridin-2-ylamino1-2-methoxy- phenol

By following the general procedure and substituting 5-amino-2-methoxyphenol for aryl amine, the title compound was obtained as an oil (53mg). MS m/z 392 (MH+)

Example 2: 3-f4-(2-Amino-4-pyridin-2-yl-thiazol-5-v0-pyridin-2-ylaminol-N-methyl- benzamide

By following the general procedure and substituting 3-amino-N-methyl-benzamide for aryl amine, the title compound was obtained as an oil (73 mg). MS m/z 403 (MH+)

Example 3: N-f4-(2-Amino-4-pyridin-2-yl-thiazol-5-yl)-pyridin-2-ylaminol-phenyl - acetamide By following the general procedure and substituting N-(4-aminophenyl)acetamide for aryl amine, the title compound was obtained as an oil (72 mg). MS m/z 403 (MH+)

Biological Data

The compounds of Examples 1-3 were tested in vitro, using the biological assays described below. All of the compounds had an IC₅₀ value of 5 μM or below in Assay 1 , and an IC₅₀ value of 1 μM or below in Assay 2.

Assays

Assay 1

The potential for compounds of the invention to inhibit TGF-β signalling may be demonstrated, for example, using the following in vitro assay.

The assay was performed in HepG2 cells stably transfected with the PAI-1 promoter (known to be a strong TGF-β responsive promoter) linked to a luciferase (firefly) reporter gene. The compounds were selected on their ability to inhibit luciferase activity in cells exposed to TGF-β. In addition cells were transfected with a second luciferase (Renilla) gene which was not driven by a TGF-β responsive promoter and was used as a toxicity control.

(96 weli-)microplates are seeded, using a multidrop apparatus, with the stably transfected cell line at a concentration of 35000 cells per well in 200 μl of serum- containing medium. These plates are placed in a cell incubator.

18 to 24 hours later (Day 2), cell-incubation procedure is launched. Cells are incubated with TGF-β and a candidate compound (TGF-β inhibitor) at concentrations in the range 50 nM to 10 μM (final concentration of DMSO 1%). The final concentration of TGF-β (rhTGFβ-1) used in the test is 1 ng/mL. Cells are incubated with a candidate compound 15-30 mins prior to the addition of TGF-β. The final volume of the test reaction is 150 μl. Each well contains only one candidate compound and its effect on the PAI-1 promoter is monitored.

Columns 11 and 12 are employed as controls. Column 11 contains 8 wells in which the cells are incubated in the presence of TGF-β, without a candidate compound. Column 11 is used to determine the 'reference TGF-β induced firefly luciferase value' against which values measured in the test wells (to quantify inhibitory activity) may be compared. In wells A 2 to D12, cells are grown in medium without TGF-β. The firefly luciferase values obtained from these positions are representive of the 'basal firefly luciferase activity'. In wells E12 to H12, cells are incubated in the presence of TGF-β and 500 μM CPO (Cyclopentenone, Sigma), a cell toxic compound. The toxicity is revealed by decreased firefly and renilla luciferase activities (around 50 % of those obtained in column 11).

12 to 18 hours later (day 3), the luciferase quantification procedure is launched. The following reactions are performed using reagents obtained from a Dual. Luciferase Assay Kit (Promega). Cells are washed and lysed with the addition of 10 Dl of passive lysis buffer (Promega). Following agitation (15 to 30 mins), luciferase activities of the plates are read in a dual-injector luminometer (BMG lumistar). For this purpose, 50 μl of luciferase assay reagent and 50 μl of 'Stop & Glo' buffer are injected sequentially to quantify the activities of both luciferases. Data obtained from the measurements are processed and analysed using suitable software. The mean Luciferase activity value obtained in wells A11 to H11 (Column 11 , TGF-βonly) is considered to represent 100% and values obtained in wells A12 to D12 (cells in medium alone) give a basal level (0%). For each of the compounds tested, a concentration response curve is constructed from which an IC₅₀ value can be determined graphically.

Assay 2

The potential for compounds of the invention to inhibit the kinase Alk5 receptor may be demonstrated, for example, using the following in vitro assay. The kinase domain of Alk5 was cloned and expressed in a baculovirus/Sf9 cells system. The protein (amino acids 162 to 503) was 6-His tagged in C-terminus. After purification by affinity chromatography using a Ni²⁺ column, the autophosphorylation was tested. The enzyme was incubated in a medium containing : Tris 50 mM pH 7.4; NaCI 100 mM ; MgCI₂ 5 mM ; MnCI₂ 5 mM ; DTT 10 mM. The enzyme was preincubated with the compounds (0.1% DMSO final in the test) 10 minutes at 37°C. The reaction was initialised by the addition of 3 μM ATP (0.5 μCi gamma-33P-ATP). After 15 minutes at 37°C the reaction was stopped by addition of SDS-PAGE sample buffer (50 mM Tris-HCI, pH 6.9, 2.5 % glycerol, 1% SDS, 5 % beta- mercaptoethanol). The samples were boiled for 5 minutes at 95°C and run on a 12% SDS-PAGE. The dried gels were exposed to a phosphor screen over-night. Alk5 autophosphorylation was quantified using a STORM (Molecular Dynamics).

Claims

1. A compound of formula (I),

wherein,

R¹ is selected from H, halo, -CN, -CF₃, C_. alkyl or C.^ alkoxy;

R² is selected from -(CH₂)_n-phenyl, -(CH₂)_n-heterocyclyl, -(CH₂)_n-heteroaryl, each of which may be further substituted by one or more substituents, which may be the same or different, selected from halo (such as fluoro, chloro, bromo), -CN, -CF₃, -OH, -OCF_3> d.₄ alkyl or C^ alkoxy, -NO₂, -NH₂, -NR³R⁴, -CONR³R⁴, -NHCOR³, -SO₂R³, -

SO₂NHR³,-O(CH₂)_mNR³R⁴;

R³ is selected from H or C₁.₄ alkyl;

R⁴ is selected from heterocyclyl or heteroaryl n is O, 1 , 2, 3, 4 or 5; m is 0, 1 , 2 or 3;

X and X', which may be the same or different, are each selected from CH or N, provided that X and X' are not both N, and salts and solvates thereof.

2. A compound of formula (I) as claimed in claim 1 , wherein R¹ is positioned at the C(3) or C(6) position of the pyridine ring and is selected from H, halo, -CN, -CF₃, C-₁-₄ alkyl or Cι_₄ alkoxy.

3. A compound of formula (I) as claimed in claim 2, wherein R¹ is H.

4. A compound of formula (I) as claimed in any one of claims 1 to 3, wherein R² is-(CH₂)_n-phenyl, which may be further substituted by one or more substituents, which may be the same or different, selected from halo (such as fluoro, chloro, bromo), -CN, -CF₃, -OH, -OCF₃, C^ alkyl or C^ alkoxy, -NO₂, -NH₂, -NR³R⁴, - CONR³R⁴, -NHCOR³, -SO₂R³, -SO₂NHR³,-O(CH₂)_mNR³R⁴.

5. A compound of formula (I) as claimed in any one of claims 1 to 4 wherein n is 0 or1.

6. A compound of formula (I) as claimed in claim 1 selected from: [4-(2-Amino-4-pyridin-2-yl-thiazol-5-yl)-pyridin-2-yl]-phenyl-amine; [4-(2-Amino-4-pyridin-2-yl-thiazol-5-yl)-pyridin-2-yl]-(4-methoxy-phenyl)-amine; [4-(2-Amino-4-pyridin-2-yl-thiazol-5-yl)-pyridin-2-yl]-(4-fluoro-phenyl)-amine; 5-[4-(2-Amino-4-pyhdin-2-yl-thiazol-5-yl)-pyridin-2-ylamino]-2-methoxy-phenol; 3-[4-(2-Amino-4-pyridin-2-yl-thiazol-5-yl)-pyridin-2-ylamino]-N-methyl-benzamide; and N-{4-(2-Amino-4-pyridin-2-yl-thiazol-5-yl)-pyridin-2-ylamino]-phenyl}-acetamide; and salts and solvates thereof.

7. A pharmaceutical composition comprising at least one compound of formula (I) as claimed in any one of claims 1 to 6, together with a pharmaceutically acceptable diluent or carrier.

8. A compound of formula (I) as claimed in any one of claims 1 to 6, for use as a medicament.

9. The use of a compound of formula (I) as claimed in any one of claims 1 to 6, in the manufacture of a medicament for the treatment of a disorder characterised by the overexpression of TGF-β.

10. A method for the treatment of a human or animal subject with a disorder characterised by the overexpression of TGF-β, which method comprises administering to said human or animal subject an effective amount of a compound of formula (I) as claimed in any one of claims 1 to 6 or a physiologically acceptable salt or solvate thereof.

11. A process for the preparation of a compound of formula (I),

wherein,

R¹ is selected from H, halo, -CN, -CF₃, C_M alkyl or C^ alkoxy; R² is selected from -(CH₂)_n-phenyl, -(CH₂)_n-heterocyclyl, -(CH₂)_n-heteroaryl, each of which may be further substituted by one or more substituents, which may be the same or different, selected from halo (such as fluoro, chloro, bromo), -CN, -CF₃, -OH, -OCF₃, d-» alkyl or C^ alkoxy, -NO₂, -NH₂, -NR³R⁴, -CONR³R⁴, -NHCOR³, -SO₂R³, -

SO₂NHR³,-O(CH₂)_mNR³R⁴;

R³ is selected from H or C^ alkyl;

X and X', which may be the same or different, are each selected from CH or N, provided that X and X' are not both N, and salts and solvates thereof,

which process comprises:

a) treatment of a compound of formula (X) with an amine R²NH₂ in the presence of Pd₂(dba)₂ and binap,

(X)

b) subsequent removal of the protecting group from the resulting product.