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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
  • C07D231/54—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings condensed with carbocyclic rings or ring systems
  • C07D231/56—Benzopyrazoles; Hydrogenated benzopyrazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
  • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/04—Ortho-condensed systems

Definitions

• the present invention relates to novel indazole derivatives, useful for treatment of various disorders.
• the invention relates to methods for producing these compounds.
• the invention also provides pharmaceutical compositions comprising the compounds of the invention and methods of utilizing these compositions in the treatment of various disorders.
• Protein kinases are important components of intracellular signalling pathways and kinases are involved in the regulation of a variety of cellular functions.
• the MAP kinase signalling pathways are activated by engagement of a number of cell surface receptors.
• the JNK pathway is activated specifically by stress or pro-inflammatory cytokines. Activators include LPS, the cytokines tumor necrosis factor (TNF- ⁇ ) and Interleukin-1 (IL-1), osmotic shock, chemical stress and UV radiation (Cohen, P. Trends in Cell Biol. 7:353-361 1997).
• TNF- ⁇ tumor necrosis factor
• IL-1 Interleukin-1
• Targets of the JNK pathway include a number of transcription factors, such as but not exclusively c-jun and ATF-2 (Whitmarsh, A. and Davis, R. J. Mol. Med. 74:589-607 1998).
• JNK1 Three different genes: JNK1 , JNK2 and JNK3; encode the JNK family of enzymes.
• JNK1 and JNK2 are ubiquitously expressed in human tissues whereas JNK3 is selectively expressed in the brain, heart and testis (Dong, C. et al. Science 270:1-4 1998).
• JNKs 1, 2 and 3 have been selectively knocked out in mice both singularly and in combination by both gene deletion and/or transgenic expression of dominant negative forms of the kinases (Dong, C. et al Science 282:2092-2095, 1998; Yang, D. et al Immunity 9:575-585 1998; Dong, C, et al Nature 405:91-94 2000; Yang, D. et al Nature 389:865-870 1997).
• Mice with targeted disruption of the JNK3 gene develop normally and are protected from excitotoxin-induced apoptosis of neurons. This finding suggests that specific inhibitors of JNK 3 could be effective in the treatment of neurological disorders characterized by cell death such as Alzheimer's disease and stroke.
• mice disrupted in either JNK 1 or 2 also develop normally.
• Peripheral T cells from either type of mice can be activated to make JL2, but in both cases, there is a defect in Thl cell development.
• JNK1 -/- mice this is due to an inability to make gamma interferon (a key cytokine essential for the differentiation of Thl cells).
• JNK2 -/- mice produce interferon gamma but are unable to respond to the cytokine.
• JNK also plays a major role in apoptosis of cells (Davis RJ. Cell. 103:239-252, 2000). JNK is essential for UV induced apoptosis through the cytochrome C mediated pathway (Tournier, C. et al Science 288:870-874 2000). Ischemia and ischemia coupled with re- perfusion as well as restricted blood flow itself have been shown to be accompanied by activation of JNK. Cell death can be prevented with dominant negative forms of JNK transfected into cells demonstrating a potential utility for JNK in conditions characterized by stress-induced apoptosis.
• JNK Activation of the JNK pathway has been observed in a number of human tumors and transformed cell lines (Davis RJ. Cell. 103:239-252, 2000). Indeed, one of the major targets of JNK, c-jun, was originally identified as an oncogene indicating the potential of this pathway to participate in unregulated cell growth. JNK also regulates phosphorylation of p53 and thus modulates cell cycle progression (Chen T. et al Mol. Carcinogenesis 15:215-226, 1996). Inhibition of JNK may therefore be beneficial in some human cancers.
• JNK signalling especially JNK3 has been implicated in areas of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, ALS, Huntington's disease, traumatic brain injury, as well as ischemic and haemorrhaging stroke.
• JNK specific inhibitors useful in treating the various conditions associated with JNK activation.
• the invention relates to compounds of the general Formula I:
• X is CR 4 or N
• R 1 is -OR 5 , -NHCOR 6 or -NR 6 R 7 ;
• R 2 is hydrogen, -OAr 1 or -NHAr 1 wherein Ar 1 is aryl optionally substituted with one or more of R 8 , -OR 8 , -NR 8 R 9 , -CONR 8 R 9 , -COOR 8 , -NR 8 COR 9 , -SR 8 , -SO 2 R 8 ,
• R 3 is hydrogen or -NHAr 2 wherein Ar 2 is benzene optionally substituted with one or more of R 8 , -OR 8 , -NR 8 R 9 , halogen or nitro; wherein R 2 and R 3 is not simultaneously hydrogen;
• R 4 is hydrogen, NO 2 , CN, C 1-3 alkyl or halogen
• R 5 is -CH ⁇ r 1 wherein Ar 1 is aryl optionally substituted with one or more of R 8 , -OR 8 , - NR 8 R 9 , -CONR 8 R 9 , -COOR 8 , -NR 8 COR 9 , -SR 8 , -SO 2 R 8 , -SO 2 NR 8 R 9 , -NR 8 SO 2 R 9 , halogen, cyano, or nitro;
• R 6 and R 7 are each independently hydrogen, C 1-6 alkyl, C 2-6 alkenyl, (C -8 cycloalkyl)C 0-
• B is R 8 , -COOR 8 , -COR 8 , -NHCOR 8 , -NR 8 R 9 , -CONR 8 R 9 , -OR 8 , -SO 2 NR 8 R 9 , CN, oxo or halogen;
• R 8 and R 9 each independently are hydrogen, Ci- ⁇ alkyl, - ⁇ fluoroalkyl, or hydroxy .
• alkyl includes both straight and branched chain alkyl groups.
• C ⁇ -6 alkyl may be methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-buty], t-butyl, n-pentyl, i-pentyl, t-pentyl, neo-pentyl, and hexyl.
• C 3-8 cycloalkyl includes a non- aromatic, completely saturated cyclic aliphatic hydrocarbon group containing 3 to 8 atoms. Examples of said cycloalkyl include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
• alkenyl includes both straight and branched chain alkenyl groups but references to individual alkenyl groups such as 2-butenyl is specific for the straight chain version only. Unless otherwise stated, the term “alkenyl” advantageously refers to chains with 2 to 5 carbon atoms, preferably 3 to 4 carbon atoms. C 2 - 6 alkenyl may be ethenyl, propenyl, 2-methylpropenyl, butenyl and 2- butenyl.
• heterocycle includes a 3- tolO- membered non-aromatic partially or completely saturated hydrocarbon group, which contains one or two rings and at least one heteroatom.
• heterocycle include, but are not limited to pyrrolidinyl, pyrrolidonyl, piperidinyl, piperazinyl, morpholinyl, oxazolyl, 2-oxazolidonyl, tetrahydrofuranyl, 4H-l,3-benzodioxinyl, tetrahydropyranyl or 4-oxo-l,4-dihydroquinolinyl.
• 5- or 6-membered heterocyclic ring containing 1 or 2 heteroatoms independently selected from N, O and S" and "R 6 and R 7 form together a 4-, 5- or 6-membered heterocyclic ring containing 1 or 2 heteroatoms independently selected from N, O and S" include, but are not limited to piperidinyl, piperazinyl and morpholinyl.
• aryl may be a C,- C 1 aromatic hydrocarbon and includes, but is not limited to, benzene, naphthalene, indene, anthracene, phenanthrene.
• heteroaryl may be a monocyclic heteroaromatic, or a bicyclic fused-ring heteroaromatic group.
• heteroaryl include, but are not limited to, pyridyl, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, pyrazolyl, benzofuryl, indolyl, isoindolyl, benzimidazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, tetrazolyl or triazolyl.
• halogeno may be fluor, chlorine, bromine or iodine.
• C ⁇ . 6 fluoroalkyl may be an alkyl substituted with one or more fluorine atoms.
• fluoroalkyl include, but are not limited to, monofluoromethyl, trifluoromethyl, difluoromethyl and trifluoroethyl.
• X is CR 4 or N
• R 1 is -OR 5 , -NHCOR 6 or -NR 6 R 7 ;
• R 2 is hydrogen, or -OAr 1 , wherein Ar 1 is aryl optionally substituted with -SO 2 R 8 or one or more halogen;
• R 3 is hydrogen or -NHAr 2 wherein Ar 2 is benzene substituted one or more halogen;
• R 4 is hydrogen or NO 2 ;
• R 5 is -CH 2 Ar 1 wherein Ar 1 is aryl substituted with one or more halogen; R and R are each independently hydrogen, C 1-6 alkyl, (C -8 cycloalkyl)C 0-6 alkyl, arylCo-
• B is -COR 8 , -NHCOR 8 , -OR 8 , or halogen
• R 8 is hydrogen or C ⁇ -6 alkyl.
• R 1 is NR 6 R 7 .
• R andR is selected from C 1-6 alkyl, arylC 0-6 alkyl, heteroaryl Co- 6 alkyl and heterocycleCo -6 alkyl and said C 1-6 alkyl, arylC 0-6 alkyl, heteroarylCo -6 alkyl, heterocycleCo -6 alkyl may be substituted with one or more B.
• said B is selected from -NHCOR 8 , -OR 8 , and halogen.
• R 1 is -NHCOR 6
• R 6 is selected from (C 3-8 cycloalkyl)C 0-6 alkyl, heteroarylC 0- 6 alkyl and heterocycleC 0 . 6 alkyl.
• R 1 is -NR 6 R 7 and R 6 and R 7 are each independently selected from hydrogen, . 6 alkyl, (C 3-8 cycloalkyl)C 0-6 alkyl, and heteroarylCo -6 alkyl.
• the present invention relates to the use of compounds of formula I as hereinbefore defined as well as to the salts thereof.
• Salts for use in pharmaceutical formulations will be pharmaceutically acceptable salts, but other salts may be useful in the production of the compounds of formula I. Such salts are possible, includes both pharmaceutically acceptable acid and base addition salts.
• a suitable pharmaceutically acceptable salt of a compound of Formula I is, for example, an acid-addition salt of a compound of Formula I which is sufficiently basic, for example an acid-addition salt with an inorganic or organic acid such as hydrochloric; or, for example a salt of a compound of Formula I which is sufficiently acidic, for example an alkali or alkaline earth metal salt, or a salt with an organic base.
• Some compounds of formula I may have chiral centres and/or geometric isomeric centres (E- and Z- isomers), and it is to be understood that the invention encompasses all such optical, diastereoisomers and geometric isomers.
• Certain compounds of the present invention may exist as tautomers. It is to be understood that the present invention encompasses all such tautomers.
• the present invention relates to novel pyridine derivatives, which are inhibitors of c-Jun N- terminal kinases (JNKs). JNKs have been implicated in mediating a number of disorders.
• the invention relates to methods for producing these inhibitors.
• the invention also provides pharmaceutical compositions comprising the inhibitors of the invention, uses of the inhibitors of the invention in the manufacture of medicaments and methods of utilizing the compositions in the treatment of various disorders.
• a pharmaceutical composition comprising a compound of formula I, as a free base or a pharmaceutically acceptable salt, solvate or solvate of salt thereof, for use in the prevention and/or treatment of conditions associated with c-Jun N-terminal kinases (JNKs).
• JNKs c-Jun N-terminal kinases
• the composition may be in a form suitable for oral administration, for example as a tablet, for parenteral injection as a sterile solution or suspension.
• the above compositions may be prepared in a conventional manner using pharmaceutically carriers or diluents.
• Suitable daily doses of the compounds of formula I in the treatment of a mammal, including man are approximately 0.01 to 250 mg kg bodyweight at peroral administration and about 0.001 to 250 mg/kg bodyweight at parenteral administration.
• the typical daily dose of the active ingredients varies within a wide range and will depend on various factors such as the relevant indication, the route of administration, the age, weight and sex of the patient and may be determined by a physician.
• a compound of formula I or a pharmaceutically acceptable salt, solvate or solvate of salt thereof, can be used on its own but will usually be administered in the form of a pharmaceutical composition in which the formula I compound/salt/solvate (active ingredient) is in association with a pharmaceutically acceptable diluent or carrier.
• the pharmaceutical composition may comprise from 0.05 to 99 %w (per cent by weight), for example from 0.10 to 50 %w, of active ingredient, all percentages by weight being based on total composition.
• a diluent or carrier includes water, aqueous polyethylene glycol, magnesium carbonate, magnesium stearate, talc, a sugar (such as lactose), pectin, dextrin, starch, tragacanth, microcrystalline cellulose, methyl cellulose, sodium carboxymethyl cellulose or cocoa butter.
• a composition of the invention can be in tablet or injectable form.
• the tablet may additionally comprise a disintegrant and/or may be coated (for example with an enteric coating or coated with a coating agentsuch as hydroxypropyl methylcellulose).
• the invention further provides a process for the preparation of a pharmaceutical composition of the invention which comprises mixing a compound of formula I, or a pharmaceutically acceptable salt, solvate or solvate of salt thereof, a hereinbefore defined, with a pharmaceutically acceptable diluent or carrier.
• An example of a pharmaceuticall composition of the invention is an injectable solution containing a compound of the invention, or a a pharmaceutically acceptable salt, solvate or solvate of salt thereof, as hereinbefore defined, and sterile water, and, if necessary, either sodium hydroxide or hydrochloric acid to bring the pH of the final composition to about pH 5, and optionally a surfactant to aid dissolution.
• Liquid solution comprising a compound of formula I, or a salt thereof, dissolved in water.
• the compounds of Formula I have activity as medicaments.
• the compounds of formula I are potent JNK inhibitors and preferred compounds are selective JNK inhibitors.
• the present invention provides a compound of Formula I for use as a medicament.
• the present invention provides a compound of Formula I for use in the prevention or treatment of conditions associated with JNK activation.
• the present invention provides a method of treating or preventing conditions associated with JNK activation comprising the administration of a therapeutically effective amount of a compound of Formula I to a mammal (particularly a human including a patient) in need thereof.
• the present invention provides the use of a compound of Formula I in the manufacture of a medicament for the treatment of conditions associated with JNK activation.
• Conditions that may be treated by the compounds of this invention, according to Formula I, or a pharmaceutical composition containing the same, include any condition associated with JNK activation.
• Conditions associated with JNK activation include but are not limited to: central or peripheral neurological degenerative disorders including Alzheimer's disease, cognitive disorders, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Frontotemporal dementia Parkinson's Type, Parkinson dementia complex of Gaum, HIV dementia, corticobasal degeneration, dementia pugilistica, Down's syndrome, postencephelatic parkinsonism, progressive supranuclear palsy, Pick's Disease, Niemann- Pick's Disease, epilepsy, a peripheral neuropathy, spinal cord injury, head trauma; autoimmune diseases including Multiple Sclerosis, inflammatory bowel disease, Crohn's disease, rheumatoid arthritis, asthma, septic shock, transplant rejection; cardiovascular diseases including stroke, arterosclerosis, myocardial infarction, myocardial reperfusion injury; cancer including breast-,
• JNK inhibitors of the instant invention may be capable of inhibiting the expression of inducible pro-inflammatory proteins. Therefore other conditions, which may be treated by the compounds of this invention, include edema, analgesia, fever and pain, such as neuromuscular pain, headache, cancer pain, dental pain and arthritis pain.
• the term “therapy” also includes “prevention” unless there are specific indications to the contrary.
• the terms “therapeutic” and “therapeutically” should be construed accordingly.
• condition means any disorder and disease associated with JNK activity.
• the compounds of formula I or salt thereof are also useful as pharmacological tools in the development and standardisation of in vitro and in vivo test systems for the evaluation of the effects of JNK inhibitor related activity in laboratory animals such as cats, dogs, rabbits, monkeys, rats and mice, as part of the search for new therapeutics agents.
• R 6 is as defined in formula (I) or is protected derivatives thereof, ' or, optionally after reaction (a) reduction of the formed amide bond to yield a compound of formula (I) or,
• n, R 6 and R 7 are as defined in formula (I) or are protected derivatives thereof and
• Lg represents a leaving group, and optionally thereafter in any of the reaction: • removing any protecting groups present • forming a pharmaceutically acceptable salt.
• a representative compound of Formula (JJ) is tert-butyl 3-amino-6-[(2- chlorophenyl)amino]-lH-indazole-l-carboxylate.
• reaction of compounds (II) and (III) can be carried out using standard chemistry, for example, acylation of amine groups.
• acylations can be performed by reacting the amine group with the corresponding acid in an inert solvent such as DMF in the presence of coupling reagents such as 1,3-dicyclohexylcarbodiimide, l-(3-dimethylaminopropyl)-3- ethylcarbodiimide, ⁇ ATU or TBTU.
• reaction of compounds (II) and (III) can also be carried out under amine acylation conditions such as reacting the amine group with the corresponding acid chloride in an inert solvent, such as toluene in the presence of an amine, or in pyridine.
• amine acylation conditions such as reacting the amine group with the corresponding acid chloride in an inert solvent, such as toluene in the presence of an amine, or in pyridine.
• the formed amide bond can optionally be reduced to the corresponding amine derivatives with a reducing agent such as lithium aluminiumhydride or diborane in an inert solvent such as tetrahydrofuran.
• a reducing agent such as lithium aluminiumhydride or diborane in an inert solvent such as tetrahydrofuran.
• reaction of compounds (II) and (IV) can be carried out under reductive amination conditions, for example using acid/sodium cyanoborohydride, sodium triacetoxyborohydride or sodium borohydride.
• the reaction may be carried out in a solvent such as methanol at ambient temperature.
• Suitable leaving groups Lg include halogen, in particular bromine and chlorine, or a p-toluenesulphonate or mesylate group.
• the reaction may be carried out in a suitable solvent, for example an alcohol solvent such as butanol, at elevated temperature such as reflux.
• Suitable leaving groups L include halogen such as fluoro or a methoxy group.
• reaction of compounds (VJJ) and (VIH) can be carried out in the presence of a base in a solvent such as dimethylformamide at elevated temperature, for example at 60°C.
• a palladium catalyst such as Pd(dba) and BINAP in an inert solvent such as toluene in the presence of a base at elevated temperature as described in JACS 1996, 118, 7215-7216.
• Suitable leaving groups L' include halogen, triflate or methoxy.
• LX Compounds of formula (LX) can be prepared by reaction of a compound of formula (X), where L' represents a leaving group such as a halogen atom or an ether and R* is as defined above:
• VLTJ a compound of formula (VLTJ) in a suitable solvent, such as dimethylformamide, at an elevated temperature, such as 40-100°C, ideally at 80°C, with a suitable base, such as caesium carbonate.
• a suitable solvent such as dimethylformamide
• a suitable base such as caesium carbonate.
• Compounds of formula (HI), (IV), (V) and (VH) are either commercially available or can be prepared using standard chemistry.
• Mass spectra (El) were recorded on a Finigan MAT SSQ 710 spectrometer.
• LC-MS were recorded on a Waters Alliance 2790 + ZMD spectrometer equipped with software Mass Lynx 3.5.
• Example 4 N (6-FIuoro-4H-l,3-benzodioxin-8-yl)methyl]-4-phenoxy-lH ndazol-3-amine a) 6-Fluoro-4H- l,3-benzodioxine-8-carbaldehyde Anhydrous sodium carbonate (400 mg) was added to 8-(chloromethyl)-6-fluoro-4H-l,3- benzodioxine (260 mg) in dimethylsulphoxide (2 mL). The mixture was heated to 130°C for 24 h after which it was cooled and added to water (10 mL). The mixture was extracted with dichloromethane (3 x 5 mL), the combined organic layers dried (magnesium sulphate) and evaporated in vacuo to yield the title compound as a colourless oil (203 mg).
• Example 7 N-(2,5-Dimethoxybenzyl)-5-nitro-4-phenoxy-lfir-indazol-3-amine hydrochIoride a) 6-Fluoro-3-nitro-2-phenoxybenzonitrile and 2-fluoro-3-nitro-6-phenoxybenzonitrile 2,6-Difluoro-3-nitro-benzonitrile (10 g) and phenol (5.1 g) were dissolved in dimethylformamide (80 mL). Sodium hydride (60% in mineral oil) (2.2 g) was added and the mixture stirred for 10 h at room temperature under nitrogen. Both regioisomers were formed in a 2: 1 ratio in favour of the 2-phenoxy substituted desired product.
• N-(2, 5-Dimethoxybenzyl)-5-nitro-4-phenoxy-lH-indazol-3-amine hydrochloride A suspension of 5-nitro-4-phenoxy-lH-indazol-3-amine (200 mg) and 2,5- dimethoxybenzaldehyde (123 mg) in acetonitrile (3 mL) and methanol (1 mL) was stirred at room temperature under nitrogen. After 1 h, sodium borohydride (56 mg) was added and the suspension was stirred for 12 h. Isopropyl alcohol (3 mL) was added then 2M aqueous hydrochloric acid (3 mL). The reaction mixture was evaporated to dryness and the residue partitioned between dichloromethane and water.
• Methyl 2,6-difluorobenzoate (2 g), phenol (1.09 g), caesium carbonate (3.79 g), and dimethylformamide (10 mL) were loaded in a 100 mL round bottom flask and the resulting suspension heated to 80°C for 6 h. After cooling the reaction to room temperature, water (30 mL) was added and the mixture extracted with dichloromethane (2 x 30 mL). The combined organic extracts were washed with brine (30 mL), dried (magnesium sulphate), filtered and evaporated in vacuo.
• Example 29 3-[( ⁇ 6-[(2-chlorophenyl)amino]-lH-indazol-3-yl ⁇ amino)carbonyl]benzoic acid LiO ⁇ (1 M, 0.2 ml) was added to a solution of methyl 3-[( ⁇ 6-[(2-chlorophenyl)amino]-lH- indazol-3-yl ⁇ amino)carbonyl]benzoate (18 mg) in T ⁇ F (3ml), after stirring at room temperature overnight the mixture was concentrated under vacuum and the residue purified by preparative reversed phase column chromatography (Symmetry C8, methanol / 0.1% aqueous trifluoroacetic acid) to yield the titled compound (9 mg) as a white solid.
• MS (APCI) m/z 407 (M + ), 409 [(M+2) + ]
• a scintillation proximity assay (SPA) based on the inhibition of JNK3 catalyzed transfer of the ⁇ -phosphate group of [ ⁇ - ⁇ P] ATP to biotinylated ATF2, has been set up to identify inhibitory compounds.
• the resulting 33 P-labeled biotinylated ATF2 is trapped on SPA beads surface coated with streptavidin.
• the assay is performed in 96-well plates. Test compounds made up at 10 mM in DMSO and 1:3 serial dilutions are made in 100% DMSO. These serial dilutions are then diluted 1:10 in assay buffer (50 mM MOPS pH 7.2, 150 mM, NaCl, 0.1 mM EGTA, 1 mM DTT, 6.25 mM ⁇ -glycerolphosphate) and 10 ⁇ l are transferred to assay plates (results in 2% DMSO final concentration in assay).
• assay buffer 50 mM MOPS pH 7.2, 150 mM, NaCl, 0.1 mM EGTA, 1 mM DTT, 6.25 mM ⁇ -glycerolphosphate
• JNK3/ATP enzyme solution (1.18 U/ml JNK3, 20 ⁇ M ATP, 2 mM Mg(Ac)2, 0.01 % Brfj-35 in assay buffer) was added. The mixtuie was pre-incubated for 10 minutes at ambient temperature.
• reaction was terminated by the addition of 200 ⁇ l per well of stop buffer/bead mix (0.4 mg/ml streptavidin coated SPA-beads in 50 mM EDTA, pH 7.6). Plates were sealed with a plastic cover and centrifuged (2000 rpm, 5 minutes) to settle the beads followed by counting in a Wallac 1450 microbetaTM.
• the IC 50 values were calculated as the concentration of test compound at which the ATF2 phosphorylation is reduced to 50% of the control value.
• Typical K, values for the compounds of the present invention are in the range of about 0.001 to about 10,000 nM. Other values for K, are in the range of about 0.001 to about 1000 nM. Further values for K, are in the range of about 0.001 nM to about 300 nM.

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