EP1656367A1 - 4-(heterocyclyle-phenyle fusionne)-3-phenyle ou pyrid-2-yle)pyrazoles utilises comme inhibiteurs du recepteur de alk-5 - Google Patents

4-(heterocyclyle-phenyle fusionne)-3-phenyle ou pyrid-2-yle)pyrazoles utilises comme inhibiteurs du recepteur de alk-5

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Publication number
EP1656367A1
EP1656367A1 EP04739898A EP04739898A EP1656367A1 EP 1656367 A1 EP1656367 A1 EP 1656367A1 EP 04739898 A EP04739898 A EP 04739898A EP 04739898 A EP04739898 A EP 04739898A EP 1656367 A1 EP1656367 A1 EP 1656367A1
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EP
European Patent Office
Prior art keywords
compound
formula
pyrazol
methyl
pyrazole
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP04739898A
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German (de)
English (en)
Inventor
Nerina Laboratoire GlaxoSmithKline DODIC
Frederic Laboratoire GlaxoSmithKline DONCHE
Francoise Jeanne Lab. GlaxoSmithkline GELLIBERT
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SmithKline Beecham Corp
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SmithKline Beecham Corp
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Publication of EP1656367A1 publication Critical patent/EP1656367A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings

Definitions

  • This invention relates to novel pyrazole derivatives which are inhibitors of the transforming growth factor, (“TGF”)- ⁇ signalling pathway, in particular, the phosphorylation of smad2 or smad3 by the TGF- ⁇ type I or activin-like kinase (“ALK”)-5 receptor, methods for their preparation and their use in medicine, specifically in the treatment and prevention of a disease state mediated by this 10 pathway.
  • TGF transforming growth factor
  • ALK activin-like kinase
  • TGF- ⁇ 1 is the prototypic member of a family of cytokines including the TGF- ⁇ s,
  • ALK activin like kinase
  • type II receptors The ALK receptors are distinguished from the type II receptors in that the ALK receptors (a) lack the serine/threonine rich
  • TGF- ⁇ 20 intracellular tail, (b) possess serine/threonine kinase domains that are very homologous between type I receptors, and (c) share a common sequence motif called the GS domain, consisting of a region rich in glycine and serine residues.
  • the GS domain is at the amino terminal end of the intracellular kinase domain and is critical for activation by the type II receptor.
  • the type II receptor phosphorylates the GS domain of the type I receptor for TGF- ⁇ , ALK5, in the presence of TGF- ⁇ .
  • the ALK5 in turn, phosphorylates the cytoplasmic proteins smad2 and smad3 at two carboxy terminal serines.
  • the phosphorylated s ad proteins translocate into the nucleus and activate genes that contribute to the
  • preferred compounds of this invention are selective in that they inhibit the type I receptor and thus matrix production.
  • WO02/066462, WO02/062794 and WO02/062787 disclose novel substituted pyrazole derivatives which are inhibitors of the transforming growth
  • TGF' 35 factor, ('TGF')- ⁇ signaling pathway, in particular, the phosphorylation of smad2 or smad3 by the TGF- ⁇ type I or activin-like kinase ("ALK”)-5 receptor.
  • ALK activin-like kinase
  • WO04/016606 discloses phenyl pyridyl substituted pyrazole derivatives which are inhibitors of the transforming growth factor, (“TGF”)- ⁇ signaling pathway, in particular, the phosphorylation of smad2 or smad3 by the TGF- ⁇ type I or activin-like kinase (“ALK”)-5 receptor.
  • TGF transforming growth factor
  • ALK activin-like kinase
  • WO2004/026306 describes novel pyrazole compounds which are potent inhibitors of transforming growth factor (“TGF”)- ⁇ signalling pathway.
  • TGF transforming growth factor
  • the compounds are said to be useful in the treatment of various TGF-related disease states including, for example, cancer and fibrotic diseases.
  • the invention provides the use of a compound of formula (I), a pharmaceutically acceptable salt, solvate or derivative thereof;
  • ring E is a saturated, unsaturated or aromatic 5 or 6-membered heterocycle which heterocycle in addition to carbon contains one or more ring-heteroatoms independently selected from nitrogen and oxygen, wherein the heterocycle is optionally substituted on any nitrogen atom where appropriate by one or more groups R Ea independently selected from C 1-6 alkyl and C 1-6 alkoxyC 1-6 alkyl and is optionally substituted on any carbon atom where appropriate by one or more groups R Eb independently selected from oxo, C 1-6 alkyl, C 1-6 alkoxyC 1-6 alkyl, C 1-6 alkoxy and halo; X is N or CH;
  • R 2 is hydrogen, C 1-6 alkyl, halo, cyano or perfluoroC 1-6 alkyl; and R 3 is hydrogen or halo; in the preparation of a medicament for treating or preventing a disease or condition mediated by ALK-5 inhibition.
  • the benzofused ring system including E is selected from the list: benzimidazol-6-yl, benzimidazol-5-yl, benzoxazol-6-yl, benzoxazol-5-yl, 4H- benzo[1 ,4]oxazin-3-one-6-yl, benzo[1 ,3]dioxol-5-yl, benzodioxan-6-yl, quinolin-6-yl and benzotriazol-6-yl.
  • the benzofused ring system including E is selected from the list: benzimidazol-6-yl, benzimidazol-5-yl, benzoxazol-6-yl, benzoxazol-5-yl, 4H- benzo[1 ,4]oxazin-3-one-6-yl and benzodioxan-6-yl.
  • X is N or CH. More preferably, X is N.
  • R 2 is hydrogen, C 1-6 alkyl, chloro or fluoro. More preferably R 2 is hydrogen, methyl, chloro or fluoro. More preferably still, R 2 is methyl.
  • R 3 is hydrogen
  • R 2 is methyl. More preferably when X is N and R 2 is methyl, R 3 is H.
  • R 2 is chloro. More preferably, when X is CH and R 2 is chloro, R 3 is H.
  • ALK5 inhibitor is used herein to mean a compound, other than inhibitory smads, e.g. smad ⁇ and smad7, which selectively inhibits the ALK5 receptor preferentially over p38 or type II receptors.
  • smads e.g. smad ⁇ and smad7
  • Activation of the TGF- ⁇ 1 axis and expansion of extracellular matrix are early and persistent contributors to the development and progression of chronic renal disease and vascular disease. Border W.A., et al, N. Engl. J. Med., 1994; 331(19), 1286-92.
  • TGF- ⁇ 1 plays a role in the formation of fibronectin and plasminogen activator inhibitor-1, components of sclerotic deposits, through the action of smad3 phosphorylation by the TGF- ⁇ 1 receptor ALK5.
  • TGF- ⁇ 1 has been implicated in many renal fibrotic disorders. Border W.A., et al, N. Engl. J. Med., 1994; 331 (19), 1286-92. TGF- ⁇ 1 is elevated in acute and chronic glomerulonephritis Yoshioka K., et al, Lab. Invest., 1993; 68(2), 154-63, diabetic nephropathy Yamamoto, T., et al, 1993, PNAS 90, 1814-1818., allograft rejection, HIV nephropathy and angiotensin-induced nephropathy Border W.
  • TGF- ⁇ 1 transgenic mice or in vivo transfection of the TGF- ⁇ 1 gene into normal rat kidneys resulted in the rapid development of glomerulosclerosis.
  • inhibition of TGF- ⁇ 1 activity is indicated as a therapeutic intervention in chronic renal disease.
  • TGF- ⁇ 1 and its receptors are increased in injured blood vessels and are indicated in neointima formation following balloon angioplasty Saltis J., et al, Clin. Exp. Pharmacol. Physiol., 1996; 23(3), 193-200.
  • TGF- ⁇ 1 is a potent stimulator of smooth muscle cell ("SMC") migration in vitro and migration of SMC in the arterial wall is a contributing factor in the pathogenesis of atherosclerosis and restenosis.
  • SMC smooth muscle cell
  • TGF- ⁇ receptor ALK5 correlated with total cholesterol (P ⁇ 0.001) Blann A.D., et al, Atherosclerosis, 1996; 120(1-2), 221-6.
  • TGF- ⁇ 1 is over-expressed in fibroproliferative vascular lesions, receptor- variant cells would be allowed to grow in a slow, but uncontrolled fashion, while overproducing extracellular matrix components McCaffrey T.A., et al, Jr., J. Clin. Invest., 1995; 96(6), 2667-75.
  • TGF- ⁇ 1 was immunolocalized to non-foamy macrophages in atherosclerotic lesions where active matrix synthesis occurs, suggesting that non-foamy macrophages may participate in modulating matrix gene expression in atherosclerotic remodeling via a TGF- ⁇ -dependent mechanism. Therefore, inhibiting the action of TGF- ⁇ 1 on ALK5 is also indicated in atherosclerosis and restenosis.
  • TGF- ⁇ is also indicated in wound repair.
  • Neutralising antibodies to TGF- ⁇ 1 have been used in a number of models to illustrate that inhibition of TGF- ⁇ 1 signalling is beneficial in restoring function after injury by limiting excessive scar formation during the healing process.
  • neutralising antibodies to TGF- ⁇ 1 -and TGF- ⁇ 2 reduced scar formation and improved the cytoarchitecture of the neodermis by reducing the number of monocytes and macrophages as well as decreasing dermal fibronectin and collagen deposition in rats Shah M., J. Cell. Sci., 1995, 108, 985- 1002.
  • TGF- ⁇ antibodies also improve healing of corneal wounds in rabbits Moller-Pedersen T., Curr.
  • TGF- ⁇ is also implicated in photoaging of the skin (see Fisher GJ. Kang SW. Varani J. Bata-Csorgo Z. Wan YS. Data S. Voorhees JJ. , Mechanisms of photoaging and chronological skin aging, Archives of Dermatology, 138(11): 1462-1470, 2002 Nov. and Schwartz E. Sapadin AN. Kligman LH. "Ultraviolet B radiation increases steady state mRNA levels for cytokines and integrins in hairless mouse skin- modulation by topical tretinoin", Archives if Dermatological Research, 290(3): 137-144, 1998 Mar.)
  • TGF- ⁇ is also implicated in peritoneal adhesions Saed G.M., et al, Wound Repair Regeneration, 1999 Nov-Dec, 7(6), 504-510. Therefore, inhibitors of ALK5 would be beneficial in preventing peritoneal and sub-dermal fibrotic adhesions following surgical procedures.
  • a disease or condition mediated by ALK-5 inhibition is preferably selected from the list: chronic renal disease, acute renal disease, wound healing, arthritis, osteoporosis, kidney disease, congestive heart failure, ulcers (including diabetic ulcers, chronic ulcers, gastric ulcers, and duodenal ulcers), ocular disorders, corneal wounds, diabetic nephropathy, impaired neurological function, Alzheimer's disease, atherosclerosis, peritoneal , sub-dermal adhesion and photoaging, any disease wherein fibrosis is a major component, including, but not limited to lung fibrosis and liver fibrosis, for example, hepatitis B virus (HBV), hepatitis C virus (HCV), alcohol- induced hepatitis, haemochromatosis and primary biliary cirrhosis, restenosis, retroperitoneal fibrosis, mesenteric fibrosis, endometriosis and keloids.
  • HBV hepatitis B virus
  • the disease or condition mediated by ALK-5 inhibition is fibrosis.
  • fibrosis Preferably kidney fibrosis.
  • the invention provides a compound as defined in the first aspect with the provisos that a) when the benzofused ring system including E is 1 ,3- dihydro-2H-benzimidazoI-2-one-5-yl, X is not CH; b) when the benzofused ring system including E is benzimidazol-5-yl or benzimidazol-6-yl and X is CH, R 3 is not halogen, and c) when the benzofused ring system including E is benzimidazol-6-yl and X is N, R 2 is not hydrogen.
  • the invention provides a compound as defined in the first aspect with the proviso that a) when the benzofused ring system including E is 1,3-dihydro-2H-benzimidazol-2-one-5-yl, X is not CH; and b) when the " benzofused ring system including E is benzimidazol-5-yl or benzimidazol-6-yl, R 2 is not hydrogen.
  • Example 10 4-[4-ethyl-4H-benzo[1,4]oxazin-3-one-6-yl]-3-(6-methylpyridin-2-yl)-1 H-pyrazole (Example 11 );
  • C 1-6 alkyl refers to a straight or branched chain saturated aliphatic hydrocarbon radical of 1 to 6 carbon atoms, unless the chain length is limited thereto, including, but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl and hexyl.
  • alkoxy refers to an alkyl ether radical, wherein the term “alkyl” is defined above.
  • alkoxy groups in particular include methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butQxy and tert- butoxy.
  • perfluoroalkyl as used herein includes compounds such as trifluoromethyl.
  • halo or halogen are used interchangeably herein to mean radicals derived from the elements chlorine, fluorine, iodine and bromine.
  • substituted means substituted by one or more defined groups.
  • groups may be selected from a number of alternative groups, the selected groups may be the same or different.
  • the term independently means that where more than one substituent is selected from a number of possible substituents, those substituents may be the same or different.
  • pharmaceutically acceptable derivative means any pharmaceutically acceptable salt, solvate, ester or amide, or salt or solvate of such ester or amide, of the compound of formula (I), or any other compound which upon administration to the recipient is capable of providing (directly or indirectly) a compound of formula (I) or an active metabolite or residue thereof, eg, a prodrug.
  • Preferred pharmaceutically acceptable derivatives according to the invention are any pharmaceutically acceptable salts, solvates or prodrugs.
  • Suitable pharmaceutically 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.
  • organic carboxylic acids such as acetic, lactic, tartaric, malic, isethionic, lactobionic and succinic acids
  • organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluen
  • Some of the compounds of this invention may be crystallised or recrystallised from solvents such as aqueous and organic solvents. In such cases solvates may be formed.
  • This invention includes within its scope stoichiometric solvates including hydrates as well as compounds containing variable amounts of water that may be produced by processes such as lyophilisation.
  • Compounds of the invention may exist in the form of optical isomers, e.g. diastereoisomers and mixtures of isomers in all ratios, e.g. racemic mixtures.
  • the invention includes all such forms, in particular the pure isomeric forms.
  • the different isomeric forms may be separated or resolved one from the other by conventional methods, or any given isomer may be obtained by conventional synthetic methods or by stereospecific or asymmetric syntheses.
  • the compounds of the invention may exist in one or more tautomeric forms. All tautomers and mixtures therof are included in the scope of the present invention.
  • the compounds of the invention are intended for use in pharmaceutical compositions it will readily be understood that they are each preferably provided in substantially pure form, for example at least 60% pure, more suitably at least 75% pure and preferably at least 85%, especially at least 98% pure (% are on a weight for weight basis). Impure preparations of the compounds may be used for preparing the more pure forms used in the pharmaceutical compositions; these less pure preparations of the compounds should contain at least 1 %, more suitably at least 5% and preferably from 10 to 59% of a compound of the invention.
  • Compounds of formula (la), i.e. compounds of general formula (I) where the benzofused ring system including E is benzimidazol-6-yl, may be prepared from compounds of formula (II) according to reaction scheme 1 by treating (II) with N,N- dimethylformamide dimethyl acetal in THF and acetic acid at room temperature followed by addition of hydrazine at room temperature.
  • Compounds of formula (lc), i.e. compounds of general formula (I) where the benzofused ring system including E is benzoxazol-5-yl, may be prepared from compounds of formula (V) according to reaction scheme 3 by treating (V) with hydrogen in the presence of Pd/C in a solvent such as ethanol or tetrahydrofuran at room temperature followed by treatment with a compound of formula (IV) in a suitable solvent such as ethanol at elevated temperature.
  • Compounds of formula (VI) may be prepared from compounds of formula (VII) according to reaction scheme 5 by treating (VII) with ethyl bromoacetate in a solvent such as acetone in the presence of a base such as cesium carbonate at room temperature followed by treatment with iron in acetic acid at elevated temperature.
  • a solvent such as acetone
  • a base such as cesium carbonate
  • Compounds of formula (VII) may be prepared from compounds of formula (V) according to reaction scheme 6 by treating (V) with trityl chloride in a solvent such as methylene chloride in the presence of a base such as triethylamine at room temperature.
  • the compounds of the invention may be prepared singly or as compound libraries comprising at least 2, for example 5 to 1,000 compounds, and more preferably 10 to 100 compounds.
  • Libraries of compounds of the invention may be prepared by a combinatorial 'split and mix' approach or by multiple parallel synthesis using either solution phase or solid phase chemistry, by procedures known to those skilled in the art.
  • a compound library comprising at least 2 compounds of the invention.
  • 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- ⁇ , shall include the treatment or prophylaxis of TGF- ⁇ associated disease such as fibrosis, especially liver and kidney fibrosis, cancer development, abnormal bone function and inflammatory disorders, and scarring.
  • 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.
  • other therapeutic agents for example antiviral agents for liver diseases, or in combination with ACE inhibitors or angiotensin II receptor antagonists for kidney diseases.
  • the compounds of the invention may be administered in conventional dosage forms prepared by combining a compound of the invention with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation.
  • compositions of the invention may be formulated for administration by any route, and include those in a form adapted for oral, topical or parenteral administration to mammals including humans.
  • compositions may be formulated for administration by any route.
  • the compositions may be in the form of tablets, capsules, powders, granules, lozenges, creams or liquid preparations, such as oral or sterile parenteral solutions or suspensions.
  • the topical formulations of the present invention may be presented as, for instance, ointments, creams or lotions, eye ointments and eye or ear drops, impregnated dressings and aerosols, and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams.
  • the formulations may also contain compatible conventional carriers, such as cream or ointment bases and ethanol or oleyl alcohol for lotions. Such carriers may be present as from about 1% up to about 98% of the formulation. More usually they will form up to about 80% of the formulation.
  • Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate.
  • the tablets may be coated according to methods well known in normal pharmaceutical practice.
  • 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 reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavouring or colouring agents.
  • suspending agents for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or
  • Suppositories will contain conventional suppository bases, e.g. cocoa-butter or other glyceride.
  • fluid unit dosage forms are prepared utilizing the compound and a sterile vehicle, water being preferred.
  • the compound depending on the vehicle and concentration used, can be either suspended or dissolved in the vehicle.
  • the compound can be dissolved in water for injection and filter sterilised before filling into a suitable vial or ampoule and sealing.
  • agents such as a local anaesthetic, preservative and buffering agents can be dissolved in the vehicle.
  • the composition can be frozen after filling into the vial and the water removed under vacuum.
  • the dry lyophilized powder is then sealed in the vial and an accompanying vial of water for injection may be supplied to reconstitute the liquid prior to use.
  • Parenteral suspensions are prepared in substantially the same manner except that the compound is suspended in the vehicle instead of being dissolved and sterilization cannot be accomplished by filtration.
  • the compound can be sterilised by exposure to ethylene oxide before suspending in the sterile vehicle.
  • a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compound.
  • compositions may contain from 0.1% by weight, preferably from 10-60% by weight, of the active material, depending on the method of administration. Where the compositions comprise dosage units, each unit will preferably contain from 50-500 mg of the active ingredient.
  • the dosage as employed for adult human treatment will preferably range from 100 to 3000 mg per day, for instance 1500 mg per day depending on the route and frequency of administration. Such a dosage corresponds to 1.5 to 50 mg/kg per day. Suitably the dosage is from 5 to 20 mg/kg per day.
  • the optimal quantity and spacing of individual dosages of a compound of the invention will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular mammal being treated, and that such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses of a compound of the invention given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.
  • composition comprising a compound of the invention and a pharmaceutically acceptable carrier or diluent
  • a disorder selected from chronic renal disease, acute renal disease, wound healing, arthritis, osteoporosis, kidney disease, congestive heart failure, ulcers (including diabetic ulcers, chronic ulcers, gastric ulcers, and duodenal ulcers), ocular disorders, corneal wounds, diabetic nephropathy, impaired neurological function, Alzheimer's disease, atherosclerosis, peritoneal and sub-dermal adhesion, any disease wherein fibrosis is a major component, including, but not limited to lung fibrosis and liver fibrosis, for example, hepatitis B virus (HBV), hepatitis C virus (HCV), alcohol-induced hepatitis, haemochromatosis and primary biliary cirrhosis, restenosis, retroperitoneal fibrosis, mesenteric fibrosis, endometriosis, keloids, cancer, abnormal bone function, inflammatory disorders, scarring and photoaging
  • HBV hepatitis B virus
  • HCV
  • a combination of a compound of the invention with an ACE inhibitor or an angiotensin II receptor antagonist iv) a combination of a compound of the invention with an ACE inhibitor or an angiotensin II receptor antagonist.
  • 6-Methyl-2-pyridinecarboxaldehyde (10g, 83mmol) was reacted with aniline and diphenylphosphite as described for Intermediate 14, to afford the title compound as a white solid (40g, 99.53%); m.p. 110-112°C.
  • Example 13 4-F4-ethyl-4H-benzori.41oxazin-3-one-6-v ⁇ -3-(3-chlorophenyl)-1 H- pyrazole
  • Example 13 (3g, 9.23mmol) was reacted with triethylamine (1.95ml, 13.85mmol) and trityl chloride (3.86g, 13.85mmol), the mixture was heated under reflux for 24 hours. The reaction mixture was poured into water and extracted with CH 2 CI 2 . The organic phase was dried over Na 2 SO 4 and concentrated under reduced pressure to afford, after chromatography on silica gel (cyclohexane/AcOEt 9/1), the 4-[4H- benzo[1,4]oxazin-3-one-6-yl]-3-(3-chlorophenyl)-1-trityl-1 H-pyrazole as a yellow solid.
  • the biological activity of the compounds of the invention may be assessed using the following assays: Assay 1 (Cellular transcriptional assay)
  • the potential for compounds of the invention to inhibit TGF- ⁇ 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- ⁇ 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- ⁇ .
  • cells were transfected with a second luciferase (Renilla) gene which was not driven by a TGF- ⁇ responsive promoter and was used as a toxicity control.
  • 96 well microplates were seeded, using a multidrop apparatus, with the stably transfected cell line at a concentration of 35000 cells per well in 200 ⁇ l of serum- containing medium. These plates were placed in a cell incubator.
  • Columns 11 and 12 were employed as controls. Column 11 contained 8 wells in which the cells were incubated in the presence of TGF- ⁇ , without a candidate compound. Column 11 was used to determine the 'reference TGF- ⁇ induced firefly luciferase value' against which values measured in the test wells (to quantify inhibitory activity) were compared. In wells A12 to D12, cells were grown in medium without TGF- ⁇ . The firefly luciferase values obtained from these positions are representative of the 'basal firefly luciferase activity'. In wells E12 to H12, cells were incubated in the presence of TGF- ⁇ and 500 ⁇ M CPO (Cyclopentenone, Sigma), a cell toxic compound. The toxicity was revealed by decreased firefly and renilla luciferase activities (around 50 % of those obtained in column 11).
  • CPO Cyclopentenone
  • luciferase quantification procedure was launched. The following reactions were performed using reagents obtained from a Dual Luciferase Assay Kit (Promega). Cells were washed and lysed with the addition of 10 ⁇ l of passive lysis buffer (Promega). Following agitation (15 to 30 mins), luciferase activities of the plates were read in a dual-injector luminometer (BMG lumistar). For this purpose, 50 ⁇ l of luciferase assay reagent and 50 ⁇ l of 'Stop & Glo' buffer were injected sequentially to quantify the activities of both luciferases. Data obtained from the measurements were processed and analysed using suitable software.
  • the mean Luciferase activity value obtained in wells A11 to H11 (Column 11, TGF- ⁇ only) was considered to represent 100% and values obtained in wells A12 to D12 (cells in medium alone) gave a basal level (0%).
  • a concentration response curve was constructed from which an IC 5 o value was determined graphically.
  • Kinase inhibitor compounds conjugated to fluorophores can be used as fluorescent ligands to monitor ATP competitive binding of other compounds to a given kinase.
  • This protocol details the use of a rhodamine green-labelled ligand for assays using recombinant GST-ALK5 (residues 198-503).
  • Assay buffer components 62.5 mM Hepes pH 7.5 (Sigma H-4034), 1 mM DTT (Sigma D-0632), 12.5 mM MgCI 2 (Sigma M-9272), 1.25 mM CHAPS (Sigma C-3023).
  • ALK5 was added to assay buffer containing the above components and 1 nM of the rhodamine green-labelled ligand so that the final ALK5 concentration was 10 nM based on active site titration of the enzyme.
  • the enzyme/ligand reagent 39 ⁇ l was added to each well of the previously prepared assay plates.
  • a control compound (1 ⁇ l) was added to column 12, rows E-H for the low control values.
  • the plates were read immediately on a LJL Acquest fluorescence reader (Molecular Devices, serial number AQ1048) with excitation, emission, and dichroic filters of 485nm, 530 nm, and 505 nm, respectively.
  • the fluorescence polarization for each well was calculated by the Acquest reader and then imported into curve fitting software for construction of concentration response curves.
  • the normalized response was determined relative to the high controls (1 ⁇ l DMSO in column 12, rows A-D) and the low controls (1 ⁇ l of control compound in column 12, rows E-H). An IC 50 value was then calculated for each compound
  • Example 1 4-[1-Ethyl-benzimidazol-6-yl]-3-[6-methylpyridin-2-yl]-1 H-pyrazole (Example 1 ) showed an ALK5 receptor modulator activity of 25 nM and TGF- ⁇ cellular activity of 20 nM.
  • Example 10 4-[4-Methyl-4H-benzo[1,4]oxazin-3-one-6-yl]-3-(6-methylpyridin-2-yl)-1 H-pyrazole (Example 10) showed an ALK5 receptor modulator activity of 32 nM and TGF- ⁇ cellular activity of 20 nM.

Abstract

L'invention concerne de nouveaux dérivés pyrazole qui sont des inhibiteurs de la voie de signalisation du facteur de croissance transformant TGF-β, en particulier de la phosphorylation de smad2 ou smad3 par le récepteur de TGF-β de type 1 ou de la kinase analogue à l'activine ALK-5, leurs procédés de préparation et leur utilisation en médecine, spécifiquement dans le traitement et la prévention d'un état pathologique médié par cette voie.
EP04739898A 2003-06-16 2004-06-14 4-(heterocyclyle-phenyle fusionne)-3-phenyle ou pyrid-2-yle)pyrazoles utilises comme inhibiteurs du recepteur de alk-5 Withdrawn EP1656367A1 (fr)

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WO2008006583A1 (fr) 2006-07-14 2008-01-17 Novartis Ag Dérivés de la pyrimidine en tant qu'inhibiteurs d'alk-5
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