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Definitions

• the present invention relates to aminoindazole derivatives active as kinase inhibitors and, more in particular, it relates to 3-aminoindazoles and derivatives thereof, to a process for their preparation, to pharmaceutical compositions comprising them and to their use as therapeutic agents, particularly in the treatment of diseases linked to disregulated protein kinases.
• PKs protein kinases
• a large share of the oncogenes and proto-oncogenes involved in human cancers code for PKs.
• the enhanced activities of PKs are also implicated in many non-malignant diseases, such as benign prostate hyperplasia, familial adenomatosis, polyposis, neuro- fibromatosis, psoriasis, vascular smooth cell proliferation associated with atherosclerosis, pulmonary fibrosis, arthritis glomerulonephritis and post-surgical stenosis and restenosis.
• PKs are also implicated in inflammatory conditions and in the multiplication of viruses and parasites. PKs may also play a major role in the pathogenesis and development of neurodegenerative disorders.
• the present inventors have now discovered that the compounds of the invention, hereinafter shortly referred to as aminoindazole derivatives, are endowed with multiple protein kinase inhibiting activity and are thus useful in therapy in the treatment of diseases associated with disregulated protein kinases.
• the compounds of this invention are useful in the treatment of a variety of cancers including, but not limited to: carcinoma such as bladder, breast, colon, kidney, liver, lung, including small cell lung cancer, esophagus, gall-bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, and skin, including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocitic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell-lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett's lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias, myelodysplastic syndrome and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosar
• these compounds are also useful in the treatment of a variety of cell proliferative disorders such as, for instance, benign prostate hyperplasia, familial adenomatosis, polyposis, neuro- fibromatosis, psoriasis, vascular smooth cell proliferation associated with atherosclerosis, pulmonary fibrosis, arthritis glomerulonephritis and post-surgical stenosis and restenosis.
• the compounds of the invention can be useful in the treatment of Alzheimer's disease, as suggested by the fact that cdk5 is involved in the phosphorylation of tau protein (J.
• the compounds of this invention may also be useful in the treatment of cancer, viral infections, prevention of AIDS development in HIN-infected individuals, autoimmune diseases and neurodegenerative disorders.
• the compounds of this invention may be useful in inliibiting tumor angiogenesis and metastasis.
• the compounds of the invention are useful as cyclin dependent kinase (cdk) inhibitors and also as inhibitors of other protein kinases such as, for instance, protein kinase C in different isoforms, Met, PAK-4, PAK-5, ZC-1, STLK-2, DDR-2, Aurora 1, Aurora 2, Bub-1, PLK, Chkl, Chk2, HER2, rafl, MEK1, MAPK, EGF-R, PDGF-R, FGF-R IGF- R, VEGF-R, PI3K, weel kinase, Src, Abl, Akt, ELK, MK-2, IKK-2, Cdc7, Nek, and thus be effective in the treatment of diseases associated with other protein kinases.
• cdk cyclin dependent kinase
• alkylamino-indazoles are disclosed in US 28939 (reissue of US 3,133,081) by Smithkline Co., as endowed with muscle relaxant and analgesic activity; among them are 3-methylamino-5-trifluoromethyl-indazole and 3-diethylamino-5- trifluoromethyl-indazole .
• Cyclic N,N'-urea derivatives bearing 3-aminoindazole groups are disclosed in Bioorg. Med. Chem. Lett. (1998), 8(7), 715-720 as HIV protease inhibitors.
• Diaryl-urea derivatives are disclosed either as p38 kinase inhibitors useful in the treatment of diseases other than cancer, as well as for treating cancerous cell growth mediated by RAF kinase, in WO 99/32111 and WO 99/32106 by Bayer Co; among the compounds specifically exemplified therein is N-[4-[(pyridyl-4-yl)oxy]phenyl]-N'-[6- chloro-(indazol-3 -yl)]-urea.
• Imidazopyridine derivatives further substituted by aryl moieties e.g. by indazolyl- aminocarbonyl-phenyl, are disclosed as platelet-activating factor (PAF) antagonists in WO 91/17162 by Pfizer Ltd.
• PAF platelet-activating factor
• Indazole compounds further substituted in position 3 by groups other than amino or derivatives thereof are disclosed in WO 01/02369 by Agouron Pharmaceuticals Inc., as possessing protein kinase inhibitory activity.
• Mercapto-cyanoacryloylamino- or alkylthio-cyanoacryloyl-amino-heterocycles are discloses as being useful in the treatment of disorders associated with increased cell growth in US 5,714,514 by Hoechst.
• Quinolylamino- and quinazolylamino-indazoles are disclosed in WO 97/03069 by Glaxo Group Ltd. as possessing protein tyrosine kinase inhibitory activity.
• Arylamino-indazoles further substituted in position 5 by heterocyclic rings are disclosed in WO 95/28400 by Glaxo Group Ltd. as possessing selective 5-HT1 agonist activity; the said compounds are thus reported to be useful in the treatment of migraine.
• Some other specific indazole derivatives are known as therapeutic agents: in particular, 3-[3-(morpholin-4-yl)propionylamino]-indazole, 3-( ⁇ , ⁇ ,-diethylamino)-propylamino-5- methoxy-indazole, 3 - [(3 -methyl)morpholin-4-yl] -propylamino- 5 -methoxy-indazole 3 - (N,N,-diethylamino)-propylamino-5-methyl-indazole and 3-[(3-methyl)morpholin-4-yl]- propylamino-5-methyl-indazole are disclosed as possessing analgesic and anti- inflammatory activity [see US 4,75
• indazoles mainly disclosed as chemical intermediates or for purposes other than therapeutic, e.g. polymer stabilizers, bleaching agents, dyes and the like, are known in the art. Among them are: 3-(ethoxycarbonylamino)-indazole [see Chemical Abstracts 92(1980):215400]; 3-acetylamino-indazole and 3-benzoylamino-indazole [see J. Org.
• 3-aminoindazole derivatives are disclosed as protein kinase inhibitors ' in the co-pending US patent application No. 09/962162 (filed in September 26, 2001, in the name of Pharmacia & Upjohn S.p.A.) which is herewith incorporated by reference.
• the present invention provides a method for treating diseases caused by and/or associated with an altered protein kinase activity, by administering to a mammal in need thereof an effective amount of a compound represented by formula (I)
• R is, in position 5 or 6 of the indazole ring, a halogen atom or an optionally substituted group selected from straight or branched C 2 -C ⁇ alkenyl, C -C ⁇ alkynyl, or aryl with from 0 to 3 hetero atoms selected from S, O and N;
• Ri is an optionally substituted group selected from -NHCOR', -NHCONR'R", -NHSO 2 R' or -NHCOOR';
• R a and Rb are, each independently, hydrogen or a straight or branched C ⁇ -C 6 alkyl group
• R' and R" are, each independently, hydrogen or an optionally substituted group selected from straight or branched C ⁇ -C 6 alkyl, C 2 -C 6 alkenyl or alkynyl, C 3 -C 6 cycloalkyl or cycloalkyl Ci-C ⁇ alkyl, aryl or aryl Ci-C ⁇ alkyl wherein aryl is as above defined;, or a 5 or 6 membered heterocyclyl or heterocyclyl Ci-C ⁇ alkyl; or, when taken together with the nitrogen atom to which they are attached, R and R" may form an optionally substituted 4 to 7 membered heterocycle, optionally containing an additional heteroatom selected from S, O or N; or isomers, tautomers, carriers, prodrugs, and pharmaceutically acceptable salts thereof.
• the disease caused by and/or associated with an altered protein kinase activity is selected from the group consisting of cancer, cell proliferative disorders, Alzheimer's disease, viral infections, auto-immune diseases and neurodegenerative disorders.
• cancer that may be treated include carcinoma, squamous cell carcinoma, hematopoietic tumors of myeloid or lymphoid lineage, tumors of mesenchymal origin, tumors of the central and peripheral nervous system, melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratoxanthoma, thyroid follicular cancer and Kaposi's sarcoma.
• the cell proliferative disorder is selected from the group consisting of benign prostate hyperplasia, familial adenomatosis polyposis, neuro-fibromatosis, psoriasis, vascular smooth cell proliferation associated with atherosclerosis, pulmonary fibrosis, arthritis glomerulonephritis and post- surgical stenosis and restenosis.
• the method object of the present invention also provides tumor angiogenesis and metastasis inhibition.
• the present invention further provides a compound represented by formula (I)
• R is, in position 5 or 6 of the indazole ring, a halogen atom or an optionally substituted group selected from straight or branched C 2 -C ⁇ alkenyl, C 2 -C 6 alkynyl, or aryl with from 0 to 3 heteroatoms selected from S, O and N;
• Ri is an optionally substituted group selected from -NHCOR 1 , -NHCONR'R", -NHSO 2 R or -NHCOOR;
• R a and R b are, each independently, hydrogen or a straight or branched Ci-C ⁇ alkyl group;
• R' and R" are, each independently, hydrogen or an optionally substituted group selected from straight or branched Ci-C ⁇ alkyl, C 2 -C ⁇ alkenyl or alkynyl, C 3 -C ⁇ cycloalkyl or cycloalkyl C ⁇ -C 6 alkyl, aryl or aryl C ⁇ -C 6 alkyl wherein ary
• the present invention includes all of the hydrates, solvates, complexes and prodrugs of the compounds of this invention.
• Prodrugs are any covalently bonded compounds, which release the active parent drug according to formula (I) in vivo.
• a chiral center or another form of an isomeric center is present in a compound of the present invention, all forms of such isomer or isomers, including enantiomers and diastereomers, are intended to be covered herein.
• Compounds containing a cliiral center may be used as a racemic mixture or as an enantiomerically enriched mixture, or the racemic mixture may be separated using well-known techniques and an individual enantiomer may be used alone.
• both the cis (Z) and trans (E) isomers are within the scope of this invention.
• compounds may exist in tautomeric forms, such as keto-enol tautomers, each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or predominantly in one form.
• R is in position 5 or 6 of the indazole group, according to the following numbering system:
• halogen atom we intend a fluorine, chlorine, bromine or iodine atom.
• straight or branched C ⁇ -C 6 alkyl group we intend any group such as, for instance, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n- pentyl, n-hexyl, and the like.
• C -C ⁇ alkenyl or alkynyl group we intend any of the aforementioned straight or branched alkyl groups, with from 2 to 6 carbon atoms, further bearing a double or triple bond.
• alkenyl or alkynyl groups of the invention are, for instance, vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3- butenyl, 2-pentenyl, 1-hexenyl, ethynyl, 2-propynyl, 4-pentynyl, and the like.
• C 3 -C 6 cycloalkyl we intend, unless otherwise indicated, any 3 to 6 membered carbocyclic ring such as, for instance, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
• aryl we intend a mono-, bi- or poly- either carbocyclic as well as heterocyclic hydrocarbon with from 1 to 4 ring moieties, either fused or linked to each other by single bonds, wherein at least one of the carbocyclic or heterocyclic rings is aromatic. From the above it is clear to the skilled person that, whereas any aryl group with 0 heteroatoms is an aromatic carbocyclic ring, any aryl group with from 1 to 3 heteroatoms is an aromatic heterocyclic ring, also known as heteroaryl group.
• the said heteroaryl groups are 5 or 6 membered rings with from 1 to 3 heteroatoms selected among nitrogen, oxygen or sulphur.
• aryl groups of the invention are, for instance, phenyl, indanyl, biphenyl, - or ⁇ -naphthyl, fluorenyl, 9,10-dihydroanthracenyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, imidazolyl, imidazopyridyl, 1,2-methylenedioxyphenyl, thiazolyl, isothiazolyl, pyrrolyl, pyrrolyl-phenyl, furyl, phenyl-furyl, benzotetrahydrofuranyl, oxazolyl, isoxazolyl, pyrazolyl, chromenyl, thienyl, benzothienyl,
• 5 or 6 membered heterocyclyl hence encompassing aromatic heterocyclic groups also referred to as aryl groups
• aryl groups we further intend a saturated or partially unsaturated 5 or 6 membered heterocycle with from 1 to 3 heteroatoms such as nitrogen, oxygen and sulfur.
• these 5 or 6 membered heterocyclyl groups optionally benzocondensed or further substituted, are 1,3-dioxolane, pyran, pyrrolidine, pyrroline, imidazolidine, pyrazolidine, pyrazoline, piperidine, piperazine, morpholine, tetrahydrofuran, and the like.
• Ri is a group -NHCONR'R" and R and R" are taken together with the nitrogen atom to which they are attached, they may also form an optionally substituted 4 to 7 membered heterocycle, optionally containing a heteroatom selected from S, O or N, in addition to the N atom directly bonded to R' and R".
• heterocyclic groups see, for instance, cyclic amino derivatives per the following table VI. From all of the above, it is clear to the skilled man that any group which name has been identified as a composite name such as, for instance, cycloalkylalkyl, arylalkyl, heterocyclylalkyl and the like, has to be intended as conventionally construed from the parts to which it derives. So far, the term heterocyclyl-alkyl stands for a straight or branched alkyl group being further substituted by a heterocyclyl group, as above defined.
• pharmaceutically acceptable salts embraces salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases.
• the nature of the salt is not critical, provided that it is pharmaceutically acceptable.
• Suitable pharmaceutically acceptable acid addition salts of compounds of the present invention may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric, and phosphoric acid.
• Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, trifluoroacetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, salicyclic, salicyclic, phydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, toluenesulfonic, 2-hydroxyethanesulfonic, sulfanilic, stearic, cyclohexylamino sulfonic, algenic
• Suitable pharmaceutically acceptable base addition salts of the compounds of the present invention include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methyl-glucamine) and procaine. All of these salts may be prepared by conventional means from the corresponding compound of the present invention by reacting, for example, the appropriate acid or base.
• a first embodiment of the invention is represented by the derivatives of formula (I) wherein R is an optionally substituted aryl group and Ri is a group -NHCOR, wherein R is as above defined.
• R is an optionally substituted aryl group and Ri is a group -NHCONR'R", wherein one of R or R" is a hydrogen atom and the remaining one of R or R" is as above defined.
• R is an optionally substituted aryl group and Ri is a group -NHCONR'R", wherein R and R" are both, as above defined, other than hydrogen.
• Another embodiment of the invention is represented by the derivatives of formula (I) wherein R is in optionally substituted aryl group and Ri is a group -NHSO 2 R, wherein R is as above defined.
• Another embodiment of the invention is represented by the derivatives of formula (I) wherein R is in optionally substituted aryl group and Ri is a group -NHCOOR, wherein
• R' is as above defined.
• R a and b are both methyl groups.
• the optionally substituted aryl group, in position 5 or 6 of the indazole ring is selected from any 5 or 6 membered aryl group with from 0 to
• Typical examples of preferred aryl groups of the invention are, for instance, phenyl, biphenyl, ⁇ - or ⁇ -naphthyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, imidazolyl, thiazolyl, isothiazolyl, pyrrolyl, furyl, benzoruranyl, oxazolyl, isoxazolyl, pyrazolyl, thienyl, benzothienyl, benzoimidazolyl, quinolinyl, isoquinolinyl, and the like.
• R a and Rb are as above defined; and, optionally, converting the thus obtained compound of formula (I) into another compound of formula (I), by: c) reacting the compound of formula (I), as per step (b) of the process, with a suitable indazole nitrogen protecting agent or, alternatively, supporting it onto a suitable polymeric resin so as to obtain a compound of formula (V) wherein Q is the above nitrogen protecting group or represents the supporting resin; d) reacting the compound of formula (V) with hydrazine monohydrate so as to get a compound of formula (VI)
• R and Q are as above defined and Ri is a group of formula -NHCONR'R"; g) deprotecting the compound of formula (XIII) being obtained according to any one of steps (f.l) or (f.2) or, alternatively, cleaving the polymeric resin so as to get the desired compound of formula (I) and, whenever desired, converting it into another compound of formula (I) and/or into a pharmaceutically acceptable salt thereof.
• step (a) of the process a compound of formula (II), preferably 4-bromo-2- fluorobenzonitrile or 5-bromo-2-fluorobenzonitrile, is reacted with hydrazine hydrate so as to get the formation of the indazole ring.
• the reaction may be carried out according to conventional methods, for instance in a lower alcohol, preferably n-butanol, at a temperature ranging from room temperature to refluxing temperature, and for a time of about 4 to about 12 hours.
• the reaction is carried out according to conventional methods, by operating in a suitable solvent, for instance dimethylformamide, at room temperature and for a time varying from about 8 to about 36 hours.
• the indazole derivative of formula (I) wherein Ri is a group is either protected at the indazole nitrogen atom or, alternatively, is supported onto a suitable polymeric resin.
• the reaction of protection may be carried out according to conventional methods well known in the art, for instance by using suitable nitrogen protecting groups such as, for instance, tert-butoxy-carbonyl (BOC) group.
• this indazole derivative may be also conveniently anchored to an inert polymeric support such as, for instance, the 2-chloro-trityl chloride resin, the trityl chloride resin, the p-nitrophenyl carbonate Wang resin or the bromo-(4- methoxyphenyl)methyl polystyrene, which are all conventionally known in this field.
• an inert polymeric support such as, for instance, the 2-chloro-trityl chloride resin, the trityl chloride resin, the p-nitrophenyl carbonate Wang resin or the bromo-(4- methoxyphenyl)methyl polystyrene, which are all conventionally known in this field.
• DIPEA diisopropylethylamine
• TAA triethylamine
• DBU l,8-diazabiciclo[5.4.0]undec-7-ene
• 2-tert-butylimino-2-diethylamino-l,3- dimethylperhydro-l,3,2-diaza-phosphorine in a suitable solvent, for instance dichloromethane, chloroform, tetrahydrofuran, dimethylformamide, dimethylacetamide, l-methyl-2-pyrrolidinone and the like.
• the reaction is carried out in l-methyl-2-pyrrolidinone at a temperature of about 20°C.
• the reaction may be performed by adding to a suspension of the resin, the base and the indazole derivative, and by stirring at a temperature of about 20°C for a suitable time, for instance up to 24 hours.
• step (d) of the process the protected- or otherwise polymer supported- derivative of formula (V) is reacted with hydrazine monohydrate in a suitable solvent, for instance water, pyridine and admixtures thereof.
• a suitable solvent for instance water, pyridine and admixtures thereof.
• the reaction is carried out in the presence of pyridine/water admixtures, at a temperature ranging from about 40°C to about 100°C and for a suitable time, for instance from 24 hours to few days, e.g. 48 hours.
• step (e) of the process the 3-amino-indazole derivatives of formula (VI) are then reacted with a suitable boronic acid of formula (VII), according to well-known Suzuki coupling conditions.
• a suitable boronic acid of formula (VII) according to well-known Suzuki coupling conditions.
• the reaction is carried out in the presence of catalytic amounts of tris(dibenzylideneacetone)dipalladium, palladium acetate, 1,1'- bis(diphenylphosphino)ferrocene- dichloropalladium, tetrakis(triphenylphosphine)palladium or bis(triphenylphosphine)palladium chloride.
• the reaction occurs by adding a suitable base, for instance cesium carbonate, potassium phosphate tribasic and the like, and a palladium ligand, for instance triphenylphosphine.
• the compound of formula (VI) is suspended in a suitable degased solvent such as toluene, N-methyl-2-pyrrolidone, dimethoxyethane, dioxane, and the like; a mixture of water and dimethoxyethane being preferred.
• a suitable degased solvent such as toluene, N-methyl-2-pyrrolidone, dimethoxyethane, dioxane, and the like; a mixture of water and dimethoxyethane being preferred.
• the compound of formula (VII), the catalyst, the base and the ligand are then added therein.
• the suspension is then brought to a suitable temperature varying from about 50°C to about 100°C whereas stirring is maintained for a time of about 8 hours to few days e.g. 48 firs.
• the reaction is carried out under inert atmosphere.
• the indazole derivative of formula (VIII) thus prepared can be then conveniently reacted according to any one of the alternative steps (f.1) or (f.2).
• step (f.l) of the process the compound of formula (VIII) is reacted with a suitable reagent of formula (IX), (X), (XI) or (XII), according to well-known methods.
• the compound of formula (VIII) may be reacted with: a compound of formula (IX) so as to get the corresponding amido derivative wherein Ri is a group -NHCOR and R' is as above defined; a compound of formula (X) to get the corresponding sulfonamido derivative wherein Ri is a group -NHSO 2 R and R is as above defined; a compound of formula (XI) to get the corresponding ureido derivative wherein Ri is a - NHCONHR group and R is as above defined; with a compound of formula (XII) to get the corresponding carbamate derivative wherein Ri is a -NHCOOR group and R' is as above defined.
• any one of the above reactions is carried out according to conventional methods normally used in the preparation of functionalized amino derivatives, by starting from the corresponding amine.
• Z represents a halogen atom and, even more preferably, a chlorine atom.
• the compound of formula (VIII) is dissolved in a suitable solvent such as dichloromethane, chloroform, dimethylformamide, tetrahydrofuran, dioxane, pyridine and admixtures thereof, and a suitable base is added such as, for instance, triethylamine, diisopropylethylamine, sodium carbonate, 1-methyl-imidazole, and the like.
• a suitable base such as, for instance, triethylamine, diisopropylethylamine, sodium carbonate, 1-methyl-imidazole, and the like.
• the compound of general formula (IX), (X) or (XII) is then added and the mixture stirred for a time of about 2 hours to about 24 hours, at a temperature ranging from about 20°C to about 50°C.
• a suitable catalyst such as dimethylamino pyridine may be optionally used.
• a further treatment with ammonium hydroxide is required so as to remove any side product being
• the compound of formula (VIII) may be reacted with a compound of formula R'R'NH (XIV), in the presence of a suitable aryl chloroformate, for instance 4-nitrophenyl- or 4-chlorophenyl-chloroformate so as to get the corresponding ureido -NHCONR'R" derivative of formula (XIII).
• a suitable aryl chloroformate for instance 4-nitrophenyl- or 4-chlorophenyl-chloroformate
• a suitable solvent such as dichloromethane, chloroform, dimethylformamide, tetrahydrofuran, dioxane and admixtures thereof
• a suitable base such as triethylamine, diisopropylethylamine, sodium carbonate, 1 -methyl imidazole and the like, together with a suitable aryl chloroformate, for instance 4-nitrophenyl- or 4-chlorophenyl- chloroformate, are added therein.
• the mixture is stirred for about 1 hour to about 12 hours at room temperature.
• the compound of formula (XIV) is then added to this suspension, and the mixture is stirred from about 12 hours to about few days, at a temperature ranging from about 20°C to about 40°C.
• step (g) of the process the compound of fo ⁇ nula (Xffl) is deprotected at the indazole nitrogen atom by working according to conventional methods, in acidic conditions.
• the compound of formula (XIII) is thus suspended in a suitable solvent such as methyl alcohol, ethyl alcohol or the like, and a concentrated solution of hydrochloric acid is added.
• the mixture is stirred for a suitable time of about 5 hours to about 15 hours at a temperature ranging from about 20°C to about 40°C; preferably at about 20°C.
• this same intermediate compound of formula (XIII) is cleaved from the resin to which it is supported.
• Resin cleavage may be carried out, for instance, in the presence of trifluoro acetic acid so as to yield the desired compound of formula (I).
• the resin is suspended in a solution of 5-95% of trifluoroacetic acid in dichloromethane or chloroform and the mixture is stirred at about 20°C for a time varying from about 5 minutes to about 3 hours.
• optional functional groups within both the starting materials, the reagents or the intermediates thereof, and which could give rise to unwanted side reactions need to be properly protected according to conventional techniques.
• the conversion of these latter into the free deprotected compounds may be carried out according to known procedures.
• Pharmaceutically acceptable salts of the compounds of formula (I) or, alternatively, their free compounds from the salts thereof my be all obtained according to conventional methods.
• the compounds of formula (IV), (VII), (IX), (X), (XI), (XII) and (XIV), are all known or easily prepared according to well- known methods.
• the compounds of formula (I) of the invention were conveniently prepared according to combinatorial chemistry techniques widely known in the art, by accomplishing the aforementioned reactions between the intermediates in a serial manner and by working under SPS conditions.
• novel compounds of the invention and the pharmaceutically acceptable salts thereof which are obtainable, for instance through a combinatorial chemistry technique as per the above process, by first reacting the compound of formula (Via)
• Q is the supporting resin (Trityl-chloride resin) with each one of the derivatives of formula (VII), as set forth in table I, so as to obtain a plurality of compounds of formula (Villa) (Villa)
• novel compounds of the invention and the pharmaceutically acceptable salts thereof which are obtainable, for instance through a combinatorial chemistry technique as per the above process, by first reacting the compound of formula (Via)
• Q is the supporting resin (Trityl-chloride resin) with each one of the derivatives of formula (VII), as set forth in table I, so as to obtain a plurality of compounds of formula (Villa)
• novel compounds of the invention and the pharmaceutically acceptable salts thereof which are obtainable, for instance through a combinatorial chemistry technique as per the above process, by first reacting the compound of formula (Via) wherein Q is the supporting resin (Trityl-chloride resin) with each one of the derivatives of formula (VII), as set forth in table I, so as to obtain a plurality of compounds of formula (Villa)
• novel compounds of the invention and the pharmaceutically acceptable salts thereof which are obtainable, for instance through a combinatorial chemistry technique as per the above process, by first reacting the compound of formula (Via)
• Q is the supporting resin (Trityl-chloride resin) with each one of the derivatives of formula (VII), as set forth in table I, so as to obtain a plurality of compounds of formula (Villa)
• novel compounds of the invention and the pharmaceutically acceptable salts thereof which are obtainable, for instance through a combinatorial chemistry technique as per the above process, by first reacting the compound of formula (Via)
• Q is the supporting resin (Trityl-chloride resin) with each one of the derivatives of formula (VII), as set forth in table I, so as to obtain a plurality of compounds of formula (Villa)
• Q is the supporting resin (Trityl-chloride resin) with each one of the derivatives of formula (VII), as set forth in table I, so as to obtain a plurality of compounds of formula (VlJIb)
• novel compounds of the invention and the pharmaceutically acceptable salts thereof which are obtainable, for instance through a combinatorial chemistry technique as per the above process, by first reacting the compound of formula (VTb)
• Q is the supporting resin (Trityl-chloride resin) with each one of the derivatives of formula (VII), as set forth in table I, so as to obtain a plurality of compounds of formula (Vlllb)
• novel compounds of the invention and the pharmaceutically acceptable salts thereof which are obtainable, for instance through a combinatorial chemistry technique as per the above process, by first reacting the compound of formula (VTb)
• Q is the supporting resin (Trityl-chloride resin) with each one of the derivatives of formula (VII), as set forth in table I, so as to obtain a plurality of compounds of formula (Vlllb)
• novel compounds of the invention and the pharmaceutically acceptable salts thereof which are obtainable, for instance through a combinatorial chemistry technique as per the above process, by first reacting the compound of formula (Vlb)
• Q is the supporting resin (Trityl-chloride resin) with each one of the derivatives of formula (VII), as set forth in table I, so as to obtain a plurality of compounds of formula (VHIb) (Vlllb)
• novel compounds of the invention and the pharmaceutically acceptable salts thereof which are obtainable, for instance through a combinatorial chemistry technique as per the above process, by first reacting the compound of formula (Vlb)
• R is, in position 5 or 6 of the indazole ring, a halogen atom or an optionally substituted group selected from straight or branched C 2 -C ⁇ alkenyl, C 2 -C ⁇ alkynyl, or aryl with from 0 to 3 heteroatoms selected from S, O and N;
• R a and R b are, each independently, hydrogen or a straight or branched Ci-C ⁇ alkyl group
• R' and R" are, each independently, hydrogen or an optionally substituted group selected from straight or branched Ci-C ⁇ alkyl, C 2 -C 6 alkenyl or alkynyl, C 3 -C ⁇ cycloalkyl or cycloalkyl Ci-C ⁇ alkyl, aryl or aryl Ci-C ⁇ alkyl wherein aryl is as above defined, or a 5 or 6 membered heterocyclyl or heterocyclyl Ci-C ⁇ alkyl; or, when taken together with the nitrogen atom to which they are attached, R and R" may form an optionally substituted 4 to 7 membered heterocycle, optionally containing an additional heteroatom selected from
• the compounds of formula (I) are active as protein kinase inhibitors and are therefore useful, for instance, to restrict the unregulated proliferation of tumor cells.
• they may be used in the treatment of various tumors such as, for instance, carcinomas, e.g. mammary carcinoma, lung carcinoma, bladder carcinoma, colon carcinoma, ovary and endometrial tumors, sarcomas, e.g. soft tissue and bone sarcomas, and the hematological malignancies such as, e.g., leukemias.
• the compounds of formula (I) are also useful in the treatment of other cell proliferative disorders such as psoriasis, vascular smooth cell proliferation associated with atherosclerosis and post-surgical stenosis and restenosis and in the treatment of Alzheimer's disease.
• the inliibiting activity of putative cdk/cyclin inhibitors and the potency of selected compounds is determined through a method of assay based on the use of the SPA technology (Amersham Pharmacia Biotech).
• the assay consists of the transfer of radioactivity labelled phosphate moiety by the kinase to a biotinylated substrate.
• the resulting 33P-labelled biotinylated product is allowed to bind to streptavidin-coated SPA beads (biotin capacity 130pmol/mg), and light emitted was measured in a scintillation counter.
• Kinetic parameter estimates were estimated by simultaneous nonlinear least-square regression using [Eq. l] (competitive inhibitor respect to ATP, random mechanism) using the complete data set (80 points):
• the selected compounds have been characterized on a panel of ser/threo kinases strictly related to cell cycle (cdk2/cyclin E, cdkl/cyclin Bl, cdk5/p25, cdk4/ cyclin Dl), and also for specificity on MAPK, PKA, EGFR, IGF1-R, and Aurora-2.
• Inhibition assay of cdk2/Cyclin E activity Kinase reaction 10 ⁇ M in house biotinylated histone HI (Sigma # H-5505) substrate, 30 ⁇ M ATP (0.3 microCi P 33 ⁇ -ATP), 4 ng GST-Cyclin E/CDK2 complex, inhibitor in a final volume of 30 ⁇ l buffer (TRIS HCl 10 mM pH 7.5, MgCl 2 10 mM, DTT 7.5 mM + 0.2 mg/ml BSA) were added to each well of a 96 U bottom. After 60 min at r.t.
• reaction was stopped by 100 ⁇ l PBS + 32 mM EDTA + 0.1% Triton X-100 + 500 ⁇ M ATP, containing 1 mg SPA beads. Then a volume of 110 ⁇ l is transferred to Optiplate.
• the inhibition assay of cdk5/p25 activity was performed according to the following protocol.
• Kinase reaction 10 ⁇ M biotinylated histone HI (Sigma # H-5505) substrate, 30 ⁇ M
• ATP 0.3 microCi P 33 ⁇ -ATP
• 15 ng CDK5/p25 complex inhibitor in a final volume of 30 ⁇ l buffer (TRIS HCl 10 mM pH 7.5, MgCl 2 10 mM, DTT 7.5 mM + 0.2 mg/ml BSA) were added to each well of a 96 U bottom. After 30 min at r.t. incubation, reaction was stopped by 100 ⁇ l PBS + 32 mM EDTA + 0.1% Triton X-100 + 500 ⁇ M ATP, containing 1 mg SPA beads. Then a volume of 110 ⁇ l is transferred to Optiplate. After 20 min.
• Capture 60 ⁇ l were transferred from each well to MultiScreen plate, to allow substrate binding to phosphocellulose filter. Plates were then washed 3 times with 150 ⁇ l/well PBS Ca ++ /Mg ++ free and filtered by MultiScreen filtration system. Detection: filters were allowed to dry at 37°C, then 100 ⁇ l/well scintillant were added and 33 P labeled Rb fragment was detected by radioactivity counting in the Top-Count instrument.
• ATP containing 1 mg SPA beads. Then a volume of 110 ⁇ l is transferred to Optiplate.
• ATP (0.04 microCi P 33 ⁇ -ATP)
• 36 ng insect cell expressed GST-EGFR inhibitor in a final volume of 30 ⁇ l buffer (Hepes 50 mM pH 7.5, MgCl 2 3 mM, MnCl 2 3 mM, DTT 1 mM, NaVO 3 3 ⁇ M + 0.2 mg/ml BSA) were added to each well of a 96 U bottom.
• reaction was stopped by 100 ⁇ l PBS + 32 mM EDTA + 0.1%
• the inhibition assay of IGF1-R activity is performed according to the following protocol.
• Kinase reaction 10 ⁇ M biotinylated MBP (Sigma cat. # M-1891) substrate, 0-20 ⁇ M inhibitor, 6 ⁇ M ATP, 1 microCi 33 P-ATP, and 22.5 ng GST-IGF1-R (pre-incubated for 30 min at room temperature with cold 60 ⁇ M cold ATP) in a final volume of 30 ⁇ l buffer (50 mM HEPES pH 7.9, 3 mM MnCl 2 , 1 mM DTT, 3 ⁇ M NaVO 3 ) were added to each well of a 96 U bottom well plate.
• Inhibition assay of Aurora-2 activity Kinase reaction 8 ⁇ M biotinylated peptide (4 repeats of LRRWSLG), 10 ⁇ M ATP (0.5 uCi P 33 g-ATP), 15 ng Aurora2, inhibitor in a final volume of 30 ⁇ l buffer (HEPES 50 mM pH 7.0, MgCl 2 10 mM, 1 mM DTT, 0.2 mg/ml BSA, 3 DM orthovanadate) were added to each well of a 96 U bottom well plate. After 30 minutes at room temperature incubation, reaction was stopped and biotinylated peptide captured by adding 100 ⁇ l of bead suspension.
• Biotin-MCM2 substrate is trans-phosphorylated by the Cdc7/Dbf4 complex in the presence of ATP traced with ⁇ 33 -ATP.
• the phosphorylated Biotin-MCM2 substrate is then captured by Streptavidin-coated SPA beads and the extent of phosphorylation evaluated by ⁇ counting.
• the inhibition assay of Cdc7/dbf4 activity was performed in 96 wells plate according to the following protocol.
• Substrate, enzyme and ATP were diluted in 50 mM HEPES pH 7.9 containing 15 mM MgCl 2 , 2 mM DTT, 3 ⁇ M NaVO 3 , 2mM glycerophosphate and 0.2mg/ml BSA.
• the solvent for test compounds also contained 10% DMSO.
• the compounds of formula (I) of the present invention suitable for administration to a mammal, e.g. to humans, can be administered by the usual routes and the dosage level depends upon the age, weight, conditions of the patient and the administration route.
• a suitable dosage adopted for oral administration of a compound of formula (I) may range from about 10 to about 500 mg pro dose, from 1 to 5 times daily.
• the compounds of the invention can be administered in a variety of dosage forms, e.g. orally, in the form of tablets, capsules, sugar or film coated tablets, liquid solutions or suspensions; rectally in the form of suppositories; parenterally, e.g. intramuscularly, or by intravenous and/or intrathecal and/or intraspinal injection or infusion.
• the compounds of the invention can be administered either as single agents or, alternatively, in combination with known anticancer treatments such as radiation therapy or chemotherapy regimen in combination with cytostatic or cytotoxic agents, antibiotic-type agents, alkylating agents, antimetabolite agents, hormonal agents, immunological agents, interferon-type agents, cyclooxygenase inhibitors (e.g.
• COX-2 inhibitors COX-2 inhibitors
• metallomatrixprotease inhibitors telomerase inhibitors
• tyrosine kinase inhibitors anti-growth factor receptor agents, anti-HER agents, anti-EGFR agents, anti- angiogenesis agents, farnesyl transferase inhibitors, ras-raf signal transduction pathway inhibitors, cell cycle inhibitors, other cdks inhibitors, tubulin binding agents, topoisomerase I inhibitors, topoisomerase II inhibitors, and the like.
• combination products employ the compounds of this invention within the dosage range described above and the other pharmaceutically active agent within the approved dosage range.
• Compounds of formula (I) may be used sequentially with known anticancer agents when a combination formulation is inappropriate.
• the present invention also includes pharmaceutical compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof in association with a pharmaceutically acceptable excipient (which can be a carrier or a diluent).
• a pharmaceutically acceptable excipient which can be a carrier or a diluent.
• the pharmaceutical compositions containing the compounds of the invention are usually prepared following conventional methods and are administered in a pharmaceutically suitable form.
• the solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, sucrose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic , magnesium or calcium stearate, and/or polyethylene glycols; binding agents, e.g. starches, arabic gum, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g.
• diluents e.g. lactose, dextrose, saccharose, sucrose, cellulose, corn starch or potato starch
• lubricants e.g. silica, talc, stearic , magnesium or calcium stearate, and/or polyethylene glycols
• binding agents e.g. starches, arabic gum, gelatin, methylcellulose, carboxymethylcellulose or polyviny
• a starch alginic, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents such as lecithin, polysorbates, laurylsulfates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations.
• Said pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film-coating processes.
• the liquid dispersions for oral administration may be e.g. syrups, emulsions and suspensions.
• the syrups may contain as carrier, for example, saccharose or saccharose with glycerin and/or mannitol and/or sorbitol.
• the suspensions and the emulsions may contain as carrier, for example, a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
• the suspension or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and, if desired, a suitable amount of lidocaine hydrochloride.
• a pharmaceutically acceptable carrier e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and, if desired, a suitable amount of lidocaine hydrochloride.
• the solutions for intravenous injections or infusions may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions or they may contain as a carrier propylene glycol.
• the suppositories may contain together with the active compound a pharmaceutically acceptable carrier, e.g. cocoa butter, polyethylene glycol
• Method I the analysis was performed on Waters X Terra 18 (4,6 x 50 mm, 3.5 ⁇ m) column using a Waters 2790 HPLC system equipped with a 996 Waters PDA detector and Micromass mod. ZQ single quadrupole mass spectrometer, equipped with an electrospray (ESI) ion source.
• Mobile phase A was ammonium acetate 5 mM buffer (pH 5.5 with acetic acid/acetonitrile 95:5), and Mobile phase B was H_O/acetonitrile (5:95).
• Packard 1312A binary pump Gilson 215 autosampler fitted with a 1ml syringe; Polymer Labs PLIOOO Evaporative Light Scattering Detector; Mcromass ZMD mass spectrometer operating in Electrospray positive ionisation mode.
• the LC eluent is split and approximately 200 ⁇ l/min enters the mass spectrometer, 800 ⁇ l/min to the ELS.
• the instruments are currently controlled using Micromass MassLynx 3.5 software under Windows NT4.0
• MS Detector m/z 150-800 at 0.5 sees/scan, 0.1 second interscan delay
• Instrument 1 Waters Symmetry C18 (19 x 50 mm, 5 ⁇ m) Column, HPLC 600 instrument equipped with a 996 Waters PDA detector and a Mcromass mod. ZMD single quadrupole mass spectrometer, electron spray ionization, positive mode. Mobile phase A was water 0.1% formic acid, and Mobile phase B was acetonitrile. Gradient from 10 to 90% B in 8 min, hold 90% B 2 min. Flow rate 20 ml/min. Instrument 2: Waters Symmetry C18 (4.6 x 50 mm, 3.5 ⁇ m) Column; HPLC 600 instrument equipped with a 996 Waters PDA detector and a Mcromass mod.
• Each code which unambiguosly identifies a single specific compound of formula (I) only, consists of three units A-M-B or, alternatively, A-M-C.
• Code A represents any R substituent, as per formula (I), being attached to the rest of the indazole moiety in position 6; each A group is represented through the proper chemical formula in the following table VII, together with its point of attachment to the rest of the molecule M.
• Code M refers to the central core of the indazole moiety which bears, in position 3, an amido group (-NHCO-) and is further substituted in position 6 by the aforementioned A group.
• Codes B and C represent the groups which are linked to the above amido portion so as to give rise to -NHCO-B or -NHCO-C groups Ri, as per formula (I).
• the compound A15-M-B19 of table X represents the 3 -amido indazole moiety M being substituted in position 6 by the group A15 and at the amido portion by the group B19; likewise, the compound A39-M-C3 of table XI (see example 8, entry 26) represents a 3-amido-indazole moiety M being substituted in position 6 by the group A39 and at the amido portion by the group C3.
• the DCM-filtrates contained some product which could be retrieved upon crystallization at -10°C.
• TT-NMR 300MHz DMSO-dg): 12.3 (s, 1H); 8.19 (s, 1H); 7,5-7.6 (s, d, 2H); 7.08 (d of d, 1H) 3.02, 2.98 (two s, 6H) 1H-NMR of the TFA-salt of N'-(6-bromo-lH-indazol-3-yl)-N,N- dimethylimidoformamide
• the resin was dried in vacuo, then weighed.
• Trityl-resin bearing N -(6-bromo-lH-indazol-3-yl)-N,N-dimethylimidoformarnide (23.44 g) with a loading of 0.74 mmol/g was stirred in a 0.2 M solution of hydrazine hydrate (H 2 N-NH 2 ⁇ 2 O)in pyridine/acetic acid 4/l(V/V)(250 ml) for 48 hours at 80°C using a mechanical overhead stirrer. An aliquot of the slurry containing 10-50 mg of resin was removed from the reaction mixture, transferred into a sinter glass frit with a valve on its bottom and washed the following way:
• the resin was dried in vacuo, then weighed. From the known amount of resin the bound indazole was determined upon cleavage using TFA whereby collecting the cleavage solutions. The cleavage was performed the following way:
• 3x 700 ml DMF The effluent from the washing vessel is collected to recover unused indazole. 3x a) 700 ml DMF; b) 700 ml H 2 O
• Triphenylphosphine (7.7 mmol, 2.02 g) was dissolved in DME (HPLC-grade) 275 ml.
• the pressure of the air in the headspace of the flask containing the solution was reduced to 20 mBar for 5 min., while being sonicated. Then headspace was filled with argon or nitrogen until ambient pressure was achieved. This process was repeated two more time to afford the solution sufficiently freed of oxygen.
• the solution was prepared from K 3 PO 4 and distilled or diionized water.
• the obtained solution was degassed and saturated with nitrogen or argon like the tripenylphosphine solution.
• the resin may either be subjected to acylation reactions or the product may be cleaved directly.
• the cleavage was performed the following way:
• the combined cleavage solutions were combined and then dried.
• the solid, which may contain residual Pd was taken up in DMSO and filtered to remove particular matter such as Pd-metal.
• the cleared DMSO solution was subjected to preparative reverse phase
• Example 6 N-(6-bromo-lH-indazol-3-yl)-2.2-dimethylpropanamide The reaction was performed in a "Mniblock" reactor (Bohdan) charged with Trityl-resin bearing 6-bromo-lH-indazol-3 -amine. To resin (23.5 mg) bearing 6-bromo-lH-indazol- 3-amine (1.2 mmol/g) was added N-methylimidazole (0.5 ml) distilled over sodium hydride, and a solution of pivaloyl chloride (0.5 mmol) in DCM (2 ml). The reaction mixture was shaken for 4 hours at room temperature.
• the resin was washed as follows: 5x a) l ml DMF; b) 1 ml H 2 O
• the resulting imids of 6-bromo-lH-indazol-3 -amine could be either isolated or converted to amides using an appropriate base such as aqueous ammonia.
• the ammonia treatment could be performed prior or post cleavage from the resin:
• Aqueous ammonium hydroxide (20%) was dissolved in ice cold dioxane to afford a solution ammonia/dioxane 1:4 N/N. This solution was added to the appropriate reactor, which was then sealed and agitated at 55°C for 48 hours. The resins were then washed
• Example 7 N-isopropyl-N'-[6-(4-methoxyphenyl)-lH-indazol-3-yllurea The reaction was performed in a "Mniblock” reactor (Bohdan) charged with trityl-resin bearing 6-(4-methoxyphenyl)-lH-indazol-3 -amine, obtained according to the procedure previously described.
• the resulting imids of 6-(4-methoxyphenyl)-lH-indazol-3 -amine could be either isolated or converted to amids using an appropriate base such as aqueous ammonia.
• the ammonia treatment could be performed prior or post cleavage from the resin:
• the cleavage was performed the following way:
• the cleavage solutions were combined and then dried.
• the resin was washed sequentially with DMF (50 ml), DCM (50 ml), DMF (50 ml), DCM (50 ml), MeOH (50 ml), DCM (50 ml), MeOH (50 ml), DCM (50 ml), MeOH (50 ml), TBME (50 ml x 2) and dried in vacuo to give the resin-bound phenyl carbamate (9.90 g, >100 % recovery).
• 75 mg of the resin (75 mg, 0.0525 mmol) in anhydrous DCM (1 ml) was added «-butylamine (38.4 mg, 0.525 mmol). The reaction mixture was shaken at room temperature for 72 hours and then isolated by filtration.
• the resin was washed sequentially with DMF (1 ml), DCM (1 ml), DMF (1 ml), DCM (1 ml), MeOH (1 ml), DCM (1 ml), MeOH (1 ml), DCM (1 ml), MeOH (1 ml), TBME (1 ml x 2) and dried in vacuo to give the resin-bound urea.
• the cleavage was performed the following way: 1x 0.5 ml 20% TFA/DCM 5 min. 4 0.2 ml 20% TFA/DCM 2 min.
• the resin was washed as follows:
• the cleavage solutions were combined and then dried.

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