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  • 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
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• • A—HUMAN NECESSITIES
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  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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  • A61P27/00—Drugs for disorders of the senses
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  • A61P27/06—Antiglaucoma agents or miotics
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
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  • 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
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
  • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms 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
  • C07D413/02—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
  • 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

• the present invention relates in particular to new chemical compounds, particularly new substituted indazoles, the compositions containing them, and their use as medicaments.
• the invention relates to novel specific indazoles exhibiting anticancer activity, via the modulation of the activity of proteins, in particular kinases.
• Protein kinases are an enzyme family that catalyze the phosphorylation of hydroxyl groups of protein-specific residues such as tyrosine, serine or threonine residues. Such phosphorylations can greatly alter the function of proteins; thus, protein kinases play an important role in the regulation of a wide variety of cellular processes, including metabolism, cell proliferation, cell differentiation, cell migration, and cell survival. Among the various cellular functions in which the activity of a protein kinase is involved, some processes represent attractive targets for treating cancerous diseases as well as other diseases.
• compositions having anticancer activity in particular by acting against to kinases.
• kinases for which modulation of activity is desired Aurora2, CDK4, KDR and Tie2 are preferred.
• Indazoles are relatively unrepresented among marketed pharmaceuticals.
• Patent application WO 03/051847 discloses indazoles substituted in the 3-position by amides, which are useful for treating numerous pathologies, particularly those related to the central nervous system. Use in oncology, although claimed, has not been demonstrated.
• One of the merits of the invention is to have found that the substitution of indazole at the 6-position with an appropriate group results in a significant inhibition of the KDR and Tie2 kinases.
• Another merit of the invention is to have found that the substitution of indazole at the 3-position with a substituent NH-M-R3 with M and R3 as defined below, was decisive for obtaining an satisfactory activity on both kinases.
• the change of functional group at position 3 of indazole systematically leads to a fall in inhibitory activity of KDR and Tie2.
• A is selected from the group consisting of: H, aryl, heteroaryl, substituted aryl, substituted heteroaryl;
• Ar is selected from the group consisting of: aryl, heteroaryl, substituted aryl, substituted heteroaryl;
• L is selected from the group consisting of: bond, CO, NH, CO-NH, NH-CO, NH-SO, NH-SO 2 , NH-CO-NH-SO 2 , SO 2 NH,
• M is selected from the group consisting of: CO, NH, CO-NH, CS-NH, NH-CO, NH-SO, NH-SO 2 , CO-NH-SO 2 ,
• R3 is independently selected from the group consisting of H, alkyl, alkylene, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, substituted alkyl, substituted alkylene, substituted alkynyl, substituted aryl, substituted heteroaryl, substituted cycloalkyl, substituted heterocyclyl, alkylene, substituted alkylene, substituted alkynyl;
• R5 and R7 they can be linked together to form a cycle.
• Ar-L-A is advantageously:
• each X1, X2, X3 and X4 is independently selected from N and C-R11, wherein R11 is selected from the group consisting of: H, halogen, R2, CN, O (R2), OC (O) (R2) ), OC (O) N (R2) (R1), OS (O 2) (R2),
• N C (R 2) (R 1), N (R 2) C (O) (R 1), N (R 2) C (O) O (R 1),
• R 1, R 6 is independently selected from the group consisting of H, alkyl, alkylene, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, substituted alkyl, substituted alkylene, substituted alkynyl, substituted aryl, substituted heteroaryl, substituted cycloalkyl, substituted heterocyclyl, alkylene, substituted alkylene, substituted alkynyl; wherein, when R2 and R1 are simultaneously present on R11, they may be linked together to form a ring.
• R11 substituents selected from the group consisting of H, F, Cl, methyl, NH 2 , OCF 3 , and CONH 2 are preferred.
• R4, R5 and R7 are preferably selected from H, F, Cl, Br and methyl.
• R7 is preferably selected from the group consisting of F, Cl, Br and methyl, wherein F is more particularly preferred. Indeed, it has been found that the substitution of R7 with a fluorine atom brings a significant improvement in the biochemical activity, particularly with regard to the kinase inhibitory activity, in particular Tie2, KDR.
• LA is advantageously chosen from NH 2 , NH-A, NH-CO-NH-A and NH-SO 2 -A.
• a preferred substituent A is preferably selected from the group consisting of phenyl, pyridyl, pyrimidyl, thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, pyrazolyl, imidazolyl, indolyl, indazolyl, benziridazolyl, benzoxazolyl, and benzothiazolyl, each of the substituents precedents that may be substituted.
• a more preferred substituent A is selected from phenyl, isoxazolyl, substituted phenyl, and substituted isoxazolyl.
• A is preferably substituted by a first substituent selected from the group consisting of alkyl, halogenated alkyl, alkylene, alkynyl, aryl, O-alkyl, O-aryl, O-heteroaryl, S-alkyl, S-aryl, S- heteroaryl, each optionally substituted with a substituent selected from (C1-C3) alkyl, halogen, O- (C1-C3) alkyl.
• A is preferably substituted with a second substituent selected from the group consisting of F, Cl, Br, I1 OH, SH, SO 3 M, COOM, CN, NO 2 -CON (R8) (R9), N ( R8) (R9) CO (R8), (C1-C3) alkyl-OH, (C1-C3) alkyl-N (R8) (R9), (C1-C3) alkyl- (R10), (C1-C3) alkyl-COOH, N (R8) (R9); wherein R 8 and R 9 are independently selected from H, (C 1 -C 3) alkyl, (C 1 -C 3) alkylOH, (C 1 -C 3) alkylNH 2 , (C 1 -C 3) alkylCOOM, (C 1 -C 3 ) alkylSO 3 M; wherein when R8 and R9 are simultaneously different from H, they may be linked to form a ring; wherein M is H or an alkali metal cation selected
• a particularly effective substituent A for obtaining inhibition of kinase activity is selected from phenyl and isoxazolyl, each being substituted by at least one substituent selected from halogen, (C1-C4) alkyl, (C1-C3) halogenated alkyl, O- (C 1 -C 4) alkyl, S- (C 1 -C 4) alkyl, O- (C 1 -C 4) alkyl halogen, and S- (C 1 -C 4) alkyl halogen.
• a preferred M substituent will be advantageously selected from the group consisting of bond, CO 1 CO-NH, and SO 2.
• R3 is preferably selected from the group consisting of aryl, heteroaryl, substituted aryl, and substituted heteroaryl.
• a more preferred R 3 is substituted heteroaryl.
• substituted heteroaryls thienyl, pyrrolyl, furyl, indolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, indazolyl, pyridyl, pyrimidyl, pyrazolyl, and pyridazinyl are heteroaryls of choice.
• R4 and R5 are advantageously H. In fact, it has been observed in this case a significant improvement in the activity vis-à-vis the kinases KDR and / or Tie2, and generally an improvement in solubility.
• Acceptable products, meeting the conditions of inhibitory activity required may be selected from the group consisting of:
• R 7 is preferably a halogen, more preferably fluorine, satisfying the required inhibitory activity conditions, and having in fact an activity greater than analogs in which R 7 is different from a halogen, may be selected from the group consisting of:
• a product according to the invention may be in the form:
• a product according to the invention may be used for the manufacture of a medicament useful for treating a pathological state, in particular a cancer.
• the present invention also relates to therapeutic compositions comprising a product according to the invention, in combination with a pharmaceutically acceptable excipient according to the chosen mode of administration.
• the pharmaceutical composition may be in solid, liquid or liposome form.
• solid compositions include powders, capsules, tablets.
• Oral forms may also include solid forms protected from the acidic environment of the stomach.
• the supports used for the solid forms consist in particular of mineral supports such as phosphates, carbonates or organic supports such as lactose, celluloses, starch or polymers.
• the liquid forms consist of solutions of suspensions or dispersions. They contain as dispersive carrier either water, or an organic solvent (ethanol, NMP or others) or mixtures of surfactants and solvents or complexing agents and solvents.
• the liquid forms will preferably be injectable and therefore will have an acceptable formulation for such use.
• Acceptable injection routes of administration include intravenous, intraperitoneal, intramuscular, and subcutaneous routes, with the intravenous route usually being preferred.
• the administered dose of the compounds of the invention will be adapted by the practitioner according to the route of administration to the patient and the state of the latter.
• the compounds of the present invention may be administered alone or in admixture with other anticancer agents.
• anticancer agents we can mention:
• Platinum derivatives such as cisplatin, carboplatin or oxaliplatin
• antibiotic agents such as bleomycin, mitomycin, dactinomycin
• antimicrotubule agents such as vinblastine, vincristine, vindesine, vinorelbine, taxoids (paclitaxel and docetaxel)
• Anthracyclines such as doxorubicin, daunorubicin, idarubicin, epirubicin, mitoxantrone, losoxantrone
• topoisomerase inhibitors such as etoposide, teniposide, amsacrine, irinotecan, topotecan and tomudex
• Fluoropyrimidines such as 5-fluorouracil, I 1 UFT, floxuridine
• Cytidine analogues such as 5-azacytidine, cytarabine, gemcitabine, 6-mercaptomurine, 6-thioguanine
• Adenosine analogs such as pentostatin, cytarabine or fludarabine phosphate
• Antiviral agents such as derivatives of combretastatin or colchicine and their prodrugs.
• KDR and Tie2 are kinases for which the products of the invention will be particularly useful as inhibitors.
• KDR Keratinase insert Domain Receptor
• VEGF-R2 Vascular Endothelial Growth Factor Receptor 2
• VEGF-R2 Vascular Endothelial Growth Factor Receptor 2
• VEGF-R2 Vascular Endothelial Growth Factor Receptor 2
• VEGF-R2 mutants Millauer et al., Cancer Research, 1996, vol 56, p.1615-1620.
• the VEGF-R2 receptor appears to have no other function in the adult than that related to the angiogenic activity of VEGF. Therefore, a selective inhibitor of VEGF-R2 kinase activity should demonstrate only low toxicity.
• Tie-2 is a member of a family of tyrosine kinase receptors, specific for endothelial cells.
• Tie2 is the first tyrosine kinase receptor known to have both the agonist (angiopoietin 1 or Ang1) that stimulates receptor autophosphorylation and cell signaling [S. Davis et al (1996) Cell 87, 1161-1169] and the antagonist (angiopoietin 2 or Ang2) [PC Maisonpierre et al. (1997) Science 277, 55-60].
• Angiopoietin 1 can synergize with VEGF in the late stages of neoangiogenesis [AsaharaT. Wax. Res (1998) 233-240).
• Tie2 inhibitors may be used in situations where neovascularization is inappropriate (ie diabetic retinopathy, chronic inflammation, psoriasis, Kaposi's sarcoma, chronic neovascularization due to macular degeneration, rheumatoid arthritis, infantile hemangioma and cancers).
• neovascularization is inappropriate (ie diabetic retinopathy, chronic inflammation, psoriasis, Kaposi's sarcoma, chronic neovascularization due to macular degeneration, rheumatoid arthritis, infantile hemangioma and cancers).
• CDKs cyclin-dependent kinases
• INK4 and KIP / CIP endogenous CDK inhibitors
• normal cell growth is due to a balance between activators of CDKs (cyclins) and endogenous CDK inhibitors.
• cyclins cyclin-dependent kinases
• Cyclin E activates the Cdk2 kinase which then acts to phosphorylate the pRb protein (retinoblastoma protein) resulting in a commitment to irreversible cell division and a transition to the S phase (PL Toogood, Medicinal Research Reviews (2001), 21 (6)).
• the CDK2 and CDK3 kinase are required for progression in G1 phase and S phase entry.
• Cyclin A CDK2 plays a role in the inactivation of E2F and is necessary for the realization of the S phase (TD Davies et al (2001) Structure 9, 389-3).
• the CDK1 / cyclin B complex regulates the progression of the cell cycle between the G2 phase and the M phase.
• the downregulation of the CDK / Cyclin B complex prevents normal cells from entering S phase before the G2 phase has been correctly and completely performed.
• CAKs cyclin-dependent kinases
• halogen refers to an element selected from F, Cl, Br, and I.
• alkyl refers to a linear or branched, saturated hydrocarbon substituent having from 1 to 12 carbon atoms. Substituents methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1, 2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1-methylpentyl, 2-methylpentyl, 1-ethylbutyl, 2-ethylbutyl, 3,3-dimethylbutyl, heptyl, 1-ethylpentyl, octyl, nonyl, decyl, undecyl, and do
• alkylene refers to a linear or branched hydrocarbon substituent having one or more unsaturations, having 2 to 12 carbon atoms.
• alkynyl refers to a linear or branched hydrocarbon substituent having at least two unsaturations carried by a pair of vicinal carbon atoms having 2 to 12 carbon atoms.
• Ethynyl substituents; prop-1-ynyl; prop-2-ynyl; and but-1 -ynyl are examples of alkynyl substituents.
• aryl refers to a mono- or polycyclic aromatic substituent having from 6 to 14 carbon atoms. Phenyl, naphth-1-yl substituents; naphth-2-yl; anthracen-9-yl; 1,2,3,4-tetrahydronaphth-5-yl; and 1,2,3,4-tetrahydronaphth-6-yl are examples of aryl substituents.
• heteroaryl refers to a mono- or polycyclic heteroaromatic substituent having 1 to 13 carbon atoms and 1 to 4 heteroatoms.
• Pyrrol-1-yl substituents; pyrrol-2-yl; pyrrol-3-yl; furyl; thienyl; imidazolyl; oxazolyl; thiazolyl; isoxazolyl; isothiazolyl; 1,2,4-triazolyl; oxadiazolyl; thiadiazolyl; tetrazolyl; pyridyl; pyrimidyl; pyrazinyl; 1,3,5-triazinyl; indolyl; benzo [b] furyl; benzo [b] thienyl; indazolyl; benzimidazolyl; azaindolyl; quinolyl; isoquinolyl; carbazolyl; and acridyl are examples of heteroaryl substituents.
• heteroatom refers here to at least one divalent atom, different from carbon. NOT; O; S; and are examples of heteroatoms.
• cycloalkyl refers to a saturated or partially unsaturated cyclic hydrocarbon substituent having from 3 to 12 carbon atoms. Cyclopropyl substituents; cyclobutyl; cyclopentyl; cyclopentenyl; cyclopentadienyl; cyclohexyl; cyclohexenyl; cycloheptyl; bicyclo [2.2.1] heptyl; cyclooctyl; bicyclo [2.2.2] octyl; adamantyl; and perhydronaphthyl are examples of cycloalkyl substituents.
• heterocyclyl refers to a saturated or partially unsaturated cyclic hydrocarbon substituent having 1 to 13 carbon atoms and 1 to 4 heteroatoms.
• the saturated or partially unsaturated cyclic hydrated hydrocarbon substituent will be monocyclic and will have 4 or 5 carbon atoms and 1 to 3 heteroatoms.
• substituted refers to a substituent other than H, for example halogen; alkyl; aryl; heteroaryl, cycloalkyl; heterocyclyl; alkylene; alkynyl; OH ; O-alkyl; O-alkylene; O-aryl; O-heteroaryl; NH 2 ; NH-alkyl; NH-aryl; NH-heteroaryl; SH; S-alkyl; S-aryl; S (O 2 ) H; S (O 2 ) - alkyl; S (O 2 ) -aryl; SO 3 H; SO 3 -alkyl; SO 3 -aryl; CHO; C (O) -alkyl; C (O) - aryl; C (O) OH; C (O) O-alkyl; C (O) O-aryl; OC (O) -alkyl; OC (O) -aryl; C (O) NH
• the present invention also relates to the process for preparing the products of formula (I).
• protective groups of the amino, carboxyl and alcohol functions are those that allow to be eliminated without touching the rest of the molecule.
• amino protecting groups mention may be made of tert-butyl carbamate which can be regenerated by means of iodotrimethylsilane, acetyl which can be regenerated in an acid medium (hydrochloric acid for example).
• protecting groups of the carboxyl function mention may be made of esters (methoxymethyl ester, benzyl ester for example).
• esters (benzoylester for example) which can be regenerated in an acid medium or by catalytic hydrogenation.
• Other useful protecting groups are described by TW GREENE et al. in Protective Groups in Organic Synthesis, Third Edition, 1999, Wiley-Interscience.
• the compounds of formula (I) are isolated and can be purified by the usual known methods, for example by crystallization, chromatography or extraction.
• the compounds of formula (I) comprising a basic residue may optionally be converted to addition salts with a mineral or organic acid, by the action of such an acid in a solvent, for example an organic solvent such as an alcohol or a ketone. , an ether or a chlorinated solvent.
• a solvent for example an organic solvent such as an alcohol or a ketone. , an ether or a chlorinated solvent.
• the compounds of formula (I) comprising an acidic residue may optionally be converted into metal salts or addition salts with nitrogenous bases according to the methods known per se.
• These salts can be obtained by the action of a metal base (alkaline or alkaline earth metal, for example), ammonia, an amine or an amine salt on a compound of formula (I), in a solvent .
• the salt formed is separated by the usual methods.
• salts can be prepared by reaction between said product and a mineral or organic acid.
• Pharmaceutically acceptable salts include chlorides, nitrates, sulphates, hydrogen sulphates, pyrosulphates, bisulphates, sulphites, bisulphites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, acetates, propionates, acrylates, 4-hydroxybutyrates, caprylates, caproates, decanoates, oxalates, malonates, succinates, glutarates, adipates, pimelates, maleates, fumarates, citrates, tartrates, lactates, phenylacetates, mandelates, sebacates, suberate, benzoates, phthalates, methanesulfonates, propanesulfonates, xylenesulfonates, sa ⁇ c
• compositions may be prepared by reaction between said product and a mineral or organic base.
• Pharmaceutically acceptable bases include hydroxides of alkali or alkaline earth metal cations such as Li, Na, K, Mg, Ca, basic amine compounds such as ammonia, arginine, histidine, piperidine, morpholine, piperazine, triethylamine.
• the products according to the invention which are prepared in the form of salts, in particular of hydrochloride, can be desalified by the action of an organic or inorganic base according to known techniques.
• the LC / MS analyzes were performed on a Micromass LCT model connected to an HP 1100.
• the abundance of the products was measured using an HP G1315A diode array detector over a range of 200-600 nm and a Sedex 65 light scattering detector.
• Mass spectra mass spectra were acquired over a range of 180 to 800. The data were analyzed using the Micromass MassLynx software.
• the separation was carried out on a Hypersil BDS C18 column, 3 ⁇ m (50 ⁇ 4.6 mm), eluting with a linear gradient of 5 to 90% of acetonitrile containing 0.05% (v / v) of trifluoroacetic acid ( TFA) in water containing 0.05% (v / v) TFA in 3.5 min at a flow rate of 1 mL / min.
• the total analysis time, including the rebalancing period of the column, is 7 minutes.
• MS spectra were made in electrospray (ES + ) on a Platform II (Micromass). The main ions observed are described. The melting points were measured in capillary, on a Mettler FP62 apparatus, range 30 ° C. to 300 ° C. rise of 2 ° C per minute.
• the products can be purified by LC / MS using a Waters FractionsLynx system consisting of a Waters Model 600 gradient pump, a Waters Model 515 regeneration pump, a Waters Reagent Manager dilution pump, a car -Injector Waters Model 2700, two Rheodyne Model LabPro valves, a Waters Model 996 diode array detector, a Waters model ZMD mass spectrometer and a Gilson model 204 fraction collector.
• the system was controlled by the Waters FractionLynx software.
• the separation was carried out alternately on two Waters Symmetry columns (Ci 8 , 5 ⁇ M, 19 ⁇ 50 mm, catalog number 186000210), a column being regenerated with a water / acetonitrile mixture 95/5 (v / v) containing 0.07. % (v / v) of trifluoroacetic acid, while the other column was being separated. Elution of the columns was performed using a linear gradient of 5 to 95% acetonitrile containing 0.07% (v / v) trifluoroacetic acid in water containing 0.07% (v / v). trifluoroacetic acid, at a flow rate of 10 ml / min.
• one thousandth of the effluent is separated by an LC Packing Accurate, diluted with methyl alcohol at a flow rate of 0.5 mL / min and sent to the detectors, at a rate of 75%. to the diode array detector, and the remaining 25% to the mass spectrometer.
• the remainder of the effluent (999/1000) is sent to the fraction collector where the flow is eliminated until the mass of the expected product is detected by the FractionLynx software.
• the molecular formulas of the expected products are provided to the FractionLynx software which triggers the product collection when the detected mass signal corresponds to the [M + H] + and / or [M + Na] + ion.
• reaction mixture is stirred under an argon atmosphere for
• the meringue obtained is purified by flash chromatography, eluting with a mixture of dichloromethane, methanol and acetonitrile (96/2/2 by volume) to give 0.69 g of 3-thiophene carboxylic acid ⁇ 6- [4- (2, 3-dichlorobenzenesulfonylamino) -phenyl] -1H-indazol-3-yl ⁇ -amide having the following characteristics:
• the meringue obtained is purified by flash chromatography, eluting with a mixture of dichloromethane, methanol and acetonitrile (96/2/2 by volume) to give 0.2 g of 3-thiophene carboxylic acid ⁇ 6- [4- (2, 3-dichlorobenzenesulfonylamino) -7-fluorophenyl] -1H-indazol-3-yl ⁇ -amide, the characteristics of which are as follows:
• Example 17 1- (4- ⁇ 7-Fluoro-3 - [(furan-2-yl) -carbonylamino] -1H-indazol-6-yl ⁇ -phenyl) -3- (2-fluoro-5-trifluoromethyl-phenyl) - urea
• the solid obtained is taken up in ethyl acetate, this organic phase is washed several times with distilled water, then with a saturated aqueous sodium chloride solution, dried over magnesium sulfate and then concentrated to dryness under reduced pressure. .
• the solid obtained is chromatographed on a silica column (eluent cyclohexane / ethyl acetate 90/10 by volume). After evaporation of the fractions containing the expected product under reduced pressure, 7.89 g of tert-butyl- ⁇ -cyano-4'-trifluoro-biphenyl-4-carbamate are obtained in the form of a cream-white solid. whose characteristics are as follows:
• the NMR analyzes are carried out as follows: 1 H NMR spectrum at 400 MHz on BRUKER AVANCE DRX-400 spectrometer with chemical shifts ( ⁇ in ppm) - in dimethylsulfoxide-d6 solvent (DMSO-d6) referenced at 2.50 ppm:
• the inhibitory effect of the compounds is determined in a substrate phosphorylation assay by the KDR enzyme in vitro by a scintillation technique (96-well plate, NEN).
• the cytoplasmic domain of the human KDR enzyme was cloned as a GST fusion into the baculovirus expression vector pFastBac.
• the protein was expressed in SF21 cells and purified to about 60% homogeneity.
• a total activity control is measured in four different wells containing all the reagents ( ⁇ 33 P- [ATP], KDR and PLC ⁇ substrate) but in the absence of a compound.
• the inhibition of KDR activity with the compound of the invention is expressed as a percentage inhibition of the control activity determined in the absence of compound.
• the compound SU5614 (Calbiochem) (1 ⁇ M) is included in each plate as inhibition control.
• the human T1e2 coding sequence corresponding to amino acids of intracellular domain 776-1124 was generated by PCR using cDNA isolated from human placenta as a template. This sequence was introduced into a baculovirus expression vector pFastBacGT as a GST fusion protein.
• the inhibitory effect of the molecules is determined in a Tie2 PLC phosphorylation assay in the presence of GST-Tie2 purified to about 80% homogeneity.
• the substrate is composed of SH2-SH3 fragments of PLC expressed as GST fusion protein.
• Tie2 kinase activity was measured in 2mM MOPS buffer pH 7.2, containing 10mM MgCl 2 , 10mM MnCl 2 , 1mM DTT, 10mM glycerophosphate.
• a reaction mixture composed of 70 .mu.l of kinase buffer containing 100 ng of GST-Tie2 enzyme per well is deposited. Then 10 .mu.l of the test molecule diluted in DMSO at a concentration of 10% maximum are added. For a given concentration, each measurement is made in four copies.
• the reaction is initiated by adding 20 ⁇ l of solution containing 2 ⁇ g of GST-PLC, 2 ⁇ M of cold ATP and 1 ⁇ Ci of 33 P [ATP]. After 1 hour of incubation at 37 ° C., the reaction is stopped by adding 1 volume (100 ⁇ l) of EDTA at 200 mM. After removal of the incubation buffer, the wells are washed three times with 300 ⁇ l of PBS. The radioactivity is measured on a Wallac MicroBeta1450.
• Inhibition of Tie2 activity is calculated and expressed as percent inhibition over control activity determined in the absence of compound.
• the inhibitory effect of compounds on Aurorai and Aurora2 kinases is determined by an enzyme test using radioactivity detection.
• the kinase activity of Aurora 1 and Aurora 2 is assessed by phosphorylation of the Numa-histidine substrate in the presence of radiolabelled ATP ([ 33 P] ATP) using Flashplate 96-well plates where the nickel-chelate is attached to the plate. surface of the microplate.
• the amount of 33 P phosphate incorporated into the NuMA substrate is proportional to the activity of the Aurorai or Aurora2 enzyme.
• the proteins are produced in the Sanofi-Aventis group's protein production laboratory.
• Aurora 1 Aurora-B / INCENP-C3 recombinant complex, purified to approximately 50% of which the N-terminus of Aurora-B has been labeled with histidine.
• Aurora 2 an entire recombinant protein comprising an N-terminal histidine tail, was expressed in E. coli and purified more than 82%.
• NuMA nuclear protein that associates with the mitotic apparatus: fragment of 424 amino acids, expressed in E.coli whose N-terminus has been labeled with histidine and used as a substrate for the two enzymes Aurora .
• the microplates used are Flash-Plate, 96-well, nickel-chelate plates (Perkin Elmer, model SMP107).
• the products to be evaluated are incubated in a reaction volume of 100 ⁇ l per well, in the presence of 10 nM of Aurora 1 or Aurora 2, 500 nM of NuMA substrate in a buffer composed of 50 mM Tris / HCl (pH 7.5). mM NaCl, 5 mM MgCl 2 (Aurora-B) or 10 mM MgCl 2 (Aurora-A) and 1 mM DTT, at 37 ° C.
• each well 80 ⁇ l of the enzyme / substrate incubation buffer are distributed and then 10 ⁇ l of the product to be evaluated, in variable concentrations.
• the reaction is initiated by the addition of 1 ⁇ M of final ATP containing 0.2 ⁇ Ci of [ 33 P] ATP (10 ⁇ L). After 30 minutes of incubation, the reaction is stopped by simple removal of the reaction buffer and each well is washed twice with 300 ⁇ l of Tris / HCl buffer. The radioactivity is then measured in each well using a scintillation apparatus, Packard model, Top count.
• Aurora's enzymatic control activity is expressed as the number of times per minute obtained in 30 minutes after deduction of the background (reaction mixture not containing the enzyme). The evaluation of the various products tested is expressed as percentage inhibition of Aurora activity compared to the control.
• Two recombinant baculoviruses carrying the human sequences encoding respectively CDK4-HA (C-terminal fusion with the tag Hemaglutinin) and Cyclin D1- (His) Q are used to co-infect S / 9 insect cells. Sixty hours after the start of co-infection, the cells are harvested by centrifugation and then frozen at -20 ° C. until they are used.
• buffer A 200 mM HEPES pH 7.0, 50 mM NaCl, 2 mM MgCl 2 , 25 mM imidazole, 1 mM TCEP, 10% (w / v) glycerol, 1 mM NaF, 1 mM Na 3 VO 4 ) stirring for 1 h at 4 ° C., and centrifugation, the complex present in the lysis supernatant is purified by Nickel Affinity Chromatography (IMAC) and stored at -80 ° C.
• IMAC Nickel Affinity Chromatography
• Flashplate CDK4 / CvclineD1 assay in 96-well format A streptavidin-coated 96-well "Flashplate” plate test was used to evaluate the inhibition of the CDK4 / cyclin D1 kinase complex by the products of the invention.
• the biotinylated peptide substrate, fragment of the pRb protein, (Biotinyl-RPPTLSPIPHIPRSPYKFPSSPLR-amide) is solubilized at a concentration of 2 mM in kinase buffer (50 mM HEPES / NaOH, 1 mM NaCl, MgCl 2).
• the final volume in each well is 100 ⁇ l
• the final substrate concentration is 1.8 ⁇ M
• the final inhibitor concentrations are 10 ⁇ M, 3.33 ⁇ M, 1.1 ⁇ M, 0, 37 ⁇ M, 0.123 ⁇ M, 0.041 ⁇ M and 0.014 ⁇ M (depending on the concentration of the intermediate dilution)
• the final concentration of ATP is 1 ⁇ M
• the final amount of 33 ⁇ is 1.5 ⁇ Ci / well
• the final concentration of CDK4 complex / Cyclin D1 is 25 nM.
• the incorporation of 33 P into the substrate peptide is measured by scintillation counting with a Packard Topcount.NXT apparatus.
• the inhibitory activity of the products of the invention is evaluated by determining the concentration of inhibitor causing a

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