Title of Invention	"A PYRROLO(3,4-C) PYRAZOLE COMPOUND OF FORMULA(I)"
Abstract	The present invention relates to a pyrrolo [3,4-c] pyrazole compound of formula (1) Wherein R is hydrogen or methyl; Rl is hydroxy or a straight or branched C1-C3 alkyl or alkoxy oroup; R2 is a hydrogen or halogen atom; X is a divalerit group selected from methylene (-CH2-) or fluoromethylene ( CHF- ), or it is a heteroatom or heternatornic group selected from oxygen (-0-) or nitrogen (-NR'-) wherein R' is a hydrogen atom, a straight or branched C1-C4 alkyl group; or a C3-C6 cycldallyl group; a pharmaceutically acceptable salt thereof the kind such as hereindescribed.

Title of Invention

"A PYRROLO(3,4-C) PYRAZOLE COMPOUND OF FORMULA(I)"

Abstract

The present invention relates to a pyrrolo [3,4-c] pyrazole compound of formula (1) Wherein R is hydrogen or methyl; Rl is hydroxy or a straight or branched C1-C3 alkyl or alkoxy oroup; R2 is a hydrogen or halogen atom; X is a divalerit group selected from methylene (-CH2-) or fluoromethylene ( CHF- ), or it is a heteroatom or heternatornic group selected from oxygen (-0-) or nitrogen (-NR'-) wherein R' is a hydrogen atom, a straight or branched C1-C4 alkyl group; or a C3-C6 cycldallyl group; a pharmaceutically acceptable salt thereof the kind such as hereindescribed.

Full Text	The present invention relates to "A PYRROLO [3,4-C] PYRAZOLE COMPOUND OF FORMULA (1)" BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to pyrroio-pyrazole derivatives, to a pnocess for their preparation, to pharmaceutical compositions comprising them, and to their use as therapeutic agents, particularly in the treatment of cancer and cell proliferation disorders. Discussion of the Background The malfunctioning of protein kinases (PKs) is the hallmark of numerous djseases. A large share of the oncogenes and proto-oncogenes involved in human cancers cdde 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, pulmonaryfibrosis, arthritis glomerulonephritis and post-surgical stenosis and restenosis. PKs are also implicated in inflammatory conditions and in the multiplicatinof viruses and parasites. PKs may also play a major role in the pathogenesis and Idevelopment of neyrodegenerative disorders. For a general reference to PKs malfunctioning or disregulation see, for instance. Current Opinion in Chemical Biology 1999, 3, 459 -465. atl. Cancer Inst, 2002, 94(17)i 1320-9]. For a general reference to Aurora-2 role in the abnormal centrosome function in cancer see also Molecular Cancer Therapeutics, 2003, 2, 589 - 595. SUMMARY OF THE INVENTION It is an object of the invention to provide compounds, which are useful in therapy as agents against a host of diseases caused by and/or associated to a disregulatejd protein kinase activity and, more particularly, Aurora kinases activity. It is another object to provide compounds, which are endowed with protein activity and, more particularly, Aurora kinases inhibiting activity. kinase inhibiting The present inventors have now discovered that some pyrrolo-pyrazoles, and derivatives thereof, are endowed with protein kinase inhibiting activity, e.g. Aurora kinases inhibiting activity. ; More specifically, 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, parjicreas, stomach, cervix, thyroid, prostate, and skin, including squamous cell carcinoma; hem^topoietic tumors of lymphoid lineage, including leukemia, acute lymphocitic leukemia, acute lymphoblastic non-Hodgkin's leukemia, B cell lymphoma, T-cell-lymphoma, Hodgkin's lymphoma, lymphoma, hairy cell lymphoma and Burkett's lymphoma; hematopoietic timers of myeloid lineage, including acute and chronic myelogenous leukemias, myelodysplastic syndrome and promyelocytic leukemia; tumors of mesenchyma! origin, including fibrosarcoma and neuro-fibromatosis, psoriasis, vascular smooth cell proliferation ssociated with atherosclerosis, pulmonary fibrosis, arthritis glomerulonephritis and post-surgical stenosis and restenosis. ! Accordingly, in a first embodiment, the present invention provides a method for treating cell proliferative disorders caused by and/or associated with an altered protein kinase activity, which comprises administering to a mammal in need thereof an effective amount of a compound of formula (I) (Figure Removed) wherein R is hydrogen or methyl, RT is hydroxy or a straight or branched C-,-C3 alkyl or alkoxy group; R2 is a hydrogen or halogen atom; X is a divalent group selected from methylene (-CH2-) or fluoromethylene (-CHF-), or it is a heteroatom or heteroatomic group selected from oxygen (-0-) or nitrogen (-NR1-) wherein R' is a hydrogen atom, a straight or branched C^-C^ alky! group or a C3-C6 cycloalkyl group; or a pharmaceutically acceptable salt thereof. The above method enables treatment of cell proliferative disorders caused by and/or associated with altered Aurora kinases activity. In a preferred embodiment of the method described above, the cell proliferative disorder is cancer DETAILED DESCRIPTION OF THE INVENTION Several heterocyclic compounds are known in the art as protein kinase inhibitors. Among them, 3-carboxamido-pyrazoles and 3-ureido-pyrazo!es and derivatives thereof, have been disclosed as protein kinase inhibitors in the international patent applications WO 01/12189, WO 01/12188, WO 02/48114 and WO 02/70515, all in the name of the applicant itself. ;inase inhibitory WO 03/028720 US patent self. fnm Fused bicyclic compounds comprising pyrazole moieties and possessing activity have been also disclosed in WO 00/69846, WO 02/12242 as well as and still unpublished PCT/EP03/04862 application (claiming priority application 60/381092, filed in May 17, 2002), all in the name of the applicant i In addition to the above, aminophenyl-piperazine or aminophenyl-piperidine derivatives as possessing inhibitory activity towards prenyl transferase proteins are disclosed in WO 02/30927 in the name of Pierre Fabre Medicament. The compounds of the present invention fall within the scope of the general formula of the aforementioned WO 02/12242, herewith incorporated by reference, but are not specifically exemplified therein. The compounds of formula (1) of the invention have asymmetric carbon'atoms and may therefore exisl as individual optical isomers, as racemic mixtures or as any other mixture comprising a majority of one of the two optical isomers, which are all to be intended as within the scope of the present invention. Likewise, the use as an antitumor agent of all the possible isomers and their admixtures and of both the metabolites and the pharmaceutically acceptable bio-precursors (otherwise referred to as pro-drugs) of the compounds of formula (I) are also within present invention. Prodrugs are any covalently bonded compounds, which release the acr according to formula (I), in vivo. the scope of the ve parent drug, In cases when compounds may exist in tautomeric forms, each form is contemplated as being included within this invention whether existing in equilibrium or predominantly: in one form. As such, unless otherwise provided, when only one of the following tautomeric forms of formula (la) or (Ib) is indicated, the remaining one has still to be intended as comprised within the scope of the invention: (Figure Removed) In the present description, unless otherwise specified, with the term straight or branched CrC3 or CrC4 alkyl we intend any of the groups such as, for instance, methyl, ethyl, n-propyl, isopropyl, n-butyl, isohutyl, tert-butyl and sec-butyl. With the term straight or branched C,-C3 alkoxy we intend any of the groups such as, for instance, methoxy, ethoxy, n-propoxy and isopropoxy. With the term halogen atom we intend s. fluorine, chlorine, bromine or iodine at)m. With the term C3-CS cycioalkyl we intend any group such as cycloprcpyl, cyclobutyl, cyciopentyl or cyclohexyl. Clearly, depending upon the nature of the X group, this same heterocycle being linked to the phenylene moiety of the compounds of formula (1} may represent a piperidino, 4-ftuoropiperidino, piperazino, 4-alkyl-piperazino, 4-cycloalkyl-piperazino ormorpholino ring. Pharmaceutjcally acceptabie salts of the compounds of formula (I) include the acid addition salts with inorganic or organic acids such as, for instance, nitric, hydrochloric, hydrobromic, sulfuric, perchloric, phosphoric, acetic, trifluoroacetic propionic, glycolic, lactic, oxalic, malonic, malic, maleic, tartaric, ciiric, benzole, cinnamic, mandelic, methanesulphonic, isethionic and salicylic acid. A preferred class of compounds of the invention is represented by the deriv; tives of formula (I) wherein R is hydrogen or methyl; R-, is selected from hydroxy, methyl or nelhoxy; R2 is a hydrogen or fluorine atom; X is selected from melhyene, fluoromethylene, -O-R1 is as above reported. >r-NR', wherein For a reference to any specific compound of formula (I) of the invention, optionally in the form of a pharmaceutically acceptable salt, see the experimental section and claims. As formerly indicated, a further object of the present invention is represented by the process for preparing the compounds of formula (I) and the pharmaceutically acceptable salts thereof, which process comprises: a) reacting a compound of formula (II) with a compound of formula (II!) (Figure Removed) wherein R and X are as above defined, Q is a lower alkyl group, t-Bu repres Z is hydroxy or a suitable leaving group, so as to obtain a compound of form (IV) ;nts tert-butyl and Jla (IV) 5 b) reacting the compound of formula (IV) under acidic conditions so as to obtain a compound of formula (V) lt thereof. as to obtain the pharmaceutically The above process is an analogy process, which can be carried out by wor, very well-known operative conditions. ;ing according to According to step (a) of the process, the reaction between the compounds of formula (II) and (III) can be carried out in a variety of ways, according to conventional methods for acylating j amino derivatives. As an example, the compound of formula (II) may be reacted with an acyl chloride of formula (III) wherein Z represents, as a suitable leaving group, a chlorine atom. Preferably, this reaction is carried out at a temperature ranging from room temperature to about 60°C, in a suitable solvent such as, for instance, tetrahydrofuran or c'ichloromethane, and in the presence of a proton scavenger such as triethylamine or diisopropy ethylamine. Within the compounds of formula (II) Q represents a lower alky) group, for hstance a alkyl group, more preferably methyl or ethyl. According to step (b) of the process, the compound of formula (IV) is easily deprotected at the pirrolidine nitrogen atom, by acidic treatment. acid such ble solvent such at a temperature about 1 hour to This reaction can be conveniently carried out in the presence of a mineral or organic as, for instance, hydrochloric, trifluoroacetic or methansulfonic acid, in a suit; as dichloromethane, 1,4-dioxane, a lower alcohol (e.g. methanol or ethanol), ranging from room temperature to about 40°C and for a time varying from about 48 hours. The compound of formula (V) thus obtained is further reacted, according to step (c) of the process, with a compound of formula (VI). From the above it is clear to the skilled person that also this acylation reaction may be accomplished in a variety of ways and operative conditions, which are widely known in the art for the preparation of carboxamides. The reaction between a compound of formula (V) and a carboxylic acid of formula (VI) wherein Z' is hydroxy can be carried out in the presence of a coupling agent such as, for instance, carbodiimide, i.e. 1,3-dicyclohexylcarbodiimide, 1,3-diisopropylcarbcdiimide, 1-(3- polystyrene or N-cyclohexylcarbodiimide-N'-methyl polystyrene, in a suitable dimethylaminopropyl)-3-ethylcarbodiimide, N-cyclohexylcarbodiimide-N' propyloxymethyl solvent such as, for instance, dichioromethane, chloroform, telrahydrofuran, diethyl eth^r, 1,4-dioxane, acetonitrile, toluene, or N.N-dimethylformamide at a temperature ranging fron about -10°C to reflux lor a suitable time, i.e. from about 30 minutes to about 96 hours. The said reaction is optionally carried out in the presence of a suitable catalyst, for instance 4- dimethylaminopyridine, or in the presence of a further coupling reagent such as N- hydroxybenzotriazole. ! The reaction between a compound of formula (V) and a compound of formula (VI) can be also carried out, for example, through a mixed anhydride method, by using an alkyl chloroformate, such as ethyl, iso-butyl, or iso-propyl chloroformate, in the presence of a tertiary base, such as triethylamine, N,N-diisopropylethylamine or pyridine, in a suitable solve it such as, for 3, diethyl ether, instance, toluene, dichloromethane, chloroform, tetrahydrofuran, acetonitri 1,4-dioxane, or N,N-dimethylformamide, at a temperature ranging from abou -30°C to room temperature. The reaction between a compound of formula (V) and a carboxylic derivative of formula (VI) wherein 2' is a suitable leaving group can be carried out in the presence of a tertiary base such as triethylamine, N,N-diisopropylethylamine or pyridine, in a suitable solvent, such as toluene, dichloromethane, chloroform, diethyl ether, tetrahydrofuran, acetonitrile, or N,N-dirnethylformamide, at a temperature ranging from about -10°C to reflux. The compounds of formula (VI) are characterised by the presence of an asymmetric carbon atom being linked to R, and presently noted with an asterisk O R. (VI) Hence, the compounds of formula (VI) may be either in the form of single en'antiomers or as mixtures thereof, also comprehensive of racemic mixtures. usejd in the process riroperly defined Clearly, depending upon the nature of the compound of formula (VI) being of the invention, corresponding compounds of formula (VII) having a stereochemistry at this same carbon atom could be thus obtained. According to a preferred embodiment of the invention, step (c) is carried out by reacting a suitable compound of formula (Vi), which is in a given enantiomeric form. Likewise, if racemic mixtures of the compound of formula (VI) are employed and whenever final compounds of formula (I) in optical pure form are desired, optical resolution of the intermediate compound of formula (VII) or, alternatively, of the final compound of formula (I) will be required, by working according to conventional means. Just sis an example, conventional techniques for racemate resolution include, for instarce, partitioned crystallization of diastereoisomeric salt derivatives or preparative chiral HPLC. (Figure Removed) Finally, according to step (d) of the process, the compound of formula (VII) is deprotected at the pyrazole nitrogen atom by working according to conventional methods enabling, for instance, the selective hydrolysis of the carbamate group. • instance in the as triethylamine, tetrahydrofuran, ranging from to about 96 As an example, this reaction may be carried out under basic conditions, fo presence of sodium, potassium or lithium hydroxide, or a tertiary amine such and in a suitable solvent such as N,N-dimethy!formarnide, methanol, ethanol water and mixtures thereof. Typically, the reaction is carried out at temperatures room temperature to about 60°C and for a time varying from about 30 minptes hours. Finally, pharmaceutically acceptable salts of the compounds of formula (1) or, ialternatively, the free compounds from their salts thereof may be all obtained according!to conventional methods. The starting material of the process of the invention are known or easily prepared according to knwon methods. represents ethyl is /12242 (see, in as 3-amino-4,6-iter). As an example, the preparation of the compound of formula (11) wherein Q disclosed in Jhe aforementioned international patent application WO 02 particular, example 26 at page 249; this same compound is therein named dihydro-pyrrolo[3,4-c]pyra?:Dle-1,5-dicarboxylic acid 5-tert-butyl ester 'I-ethyl e By working in an analogous way, additional compounds of formula (II) whereip Q represents a lower alkyl group other than ethyl can be thus prepared as well. i The compounds of formula (111) and (VI), for instance those wherein Z and Z' represent a halogen atom, e.g. a chlorine atom, are either known or can be easily obtained from the corresponding known carboxylic acids, by working according to conventional methods. Likewise, it is also clear to the person skilled in the art that if a compound of formula (I), prepared according to the above process comprehensive of any variant thereof, is obtained as an admixture of isomers, their separation into the single isomers of formula (I), carried out according to conventional techniques, is still within the scope of the present in/ention. PHARMACOLOGY The compounds of formula (1) are active as protein kinase inhibitors, more particularly as Aurora kinases inhibitors and are therefore useful, for instance, to restrict the unregulated proliferation ot tumor cells. In therapy, They may be used in the treatment of various tumors, such as those formerly reported, as well as in the treatment of other cell proliferative disorders such as psoriasis, vascular smooth cell proliferation associated with atherosclerosis and post-surgical stenosis and restenosis. The inhibiting activity and the potency of selected compounds is determined through a method of assay based on the use of the SPA technology (Amersham Pharmacia Bio1 ich). The assay consists of the transfer of radioactivity labelled phosphate moiety ty the kinase to a biotinylated substrate. The resulting 33P-labelied biotinylated product is al owed to bind to streptavidin-coated SPA beads (biotin capacity 130 pmol/mg), and light emitted was measured in a scintillation counter. Inhibition assay of Aurora-2 activity \\insse reaction: 8 uM biotinylated peptide (4 repeats of LRRWSLG), 10 uM ATP (0.5 uCi P^y-ATP), 7 fi ng Aurora 2, inhibitor in a finsit volume of 30 ul buffer (HEPES 50 mWI pH 7.0, to each well of a in, reaction was MgCl2 10 ruM, ! mM DTT, 0.2 mg/ml BSA. 3 uM orthovanadate) were added 96 U bottom well plate. After 60 minutes at room temperature incubati stopped and biotinylated peptide captured by adding 100 ul of bead suspension. Stratification: 100 pi of CsCI2 5 M were added to each well and let starjicf 4 hour before radioactivity was counted in the Top-Count instrument. 1C50 determination: inhibitors were tested at different concentrations ranging from 0.0015 to 10 uM. Experimental data were analyzed by the computer program GraphPaiji Prizm using the four parameter logistic equation: y = bottom+(lop-bottom)/(1--10A((loglC50-x)s\|ope)) where x is tin; logarithm of the inhibitor concentration, y is the response; y starts at bottom and goes lo top with a sigrnoid shape. Ki calculation: Experimental method: Reaction was carried out in buffer (10 mM Tris, pH 7[5, 10 mM MgCI2, 0.2 rng/ml BSA, 7.5 mM DTT) containing 3.7 nM enzyme, histone and ATP {constant ratio of cold/labeled ATP 1/3000). Reaction was stopped with EDTA and the substrate captured on phosphomembrane (Multiscreen 96 well plates from Millipore). After extensive washing, the multiscreen plates were read on a top counter. Control (time zero) for each ATP and histone concentrations was measured. Experimental design: Reaction velocities are measured at four ATP, substrate (histone) and inhibitor concentrations. An 80-point concentration matrix was designed around the respective ATP and substrate Km values, and the inhibitor IC50 values (0.3, 1, 3, 9 folJ the Km or IC50 values). A preliminary time course experiment in the absence of inhibitor and at the different ATP and substrate concentrations allows the selection of a single endpoin1 time (10 min) in the linear range of the reaction for the Ki determination experiment. Kinetic parameter estimates: Kinetic parameters were estimated by simultaneous nonlinear least-square regression using [Eq.1] (competitive inhibitor respect to ATP, random mechanism) using the complete data set (80 points): Vm * o: '-" Ka o Kb + a » Ka e> B 4- a ® Kb fin R - ® 7 0 (Kb + •+-) Ki j? [Eq.1] where A--[ATP], B=[.Substrate], l=[inhibitor], Vm^ maximum velocity, dissociation constants of ATP, substrate and inhibitor respectively, a and (3 Ka, Kb, Ki the the cooperativity factor between substrate and ATP binding and substrate and inhibitor bindinc respectively. The compounds of the invention were further tested in vitro to assess thf effect onto cell cultures and the inhibititory effect on the cell cycle. anti-proliferative In vitro cell proliferation assay The human colon cancer cell line HCT-116 was seeded at 5000 cells/cm2 in 24 wells plate (Costar) using F12 medium (Gibco) supplemented with 10% PCS (EuroClone, Italy) 2 mM L-glutarnine and 1% penicillin/streptomycin and maintained at 37°C, 5% CO2 and 96% relative humidity. The following day, plates were treated in duplicates with 5ul of dilution of compounds starting from a 10 mM stock in DMSO. Two untreal an appropriate :d control wells were included in each plate. After 72 hours of treatment, medium was withdrawn and cells detached from each well using 0.5 ml of 0.05% (w/v) Trypsin, 0,02% (w/v EDTA (Gibco). Samples were diluted with 9.5 ml of Isoton (Coulter) and counted using a Multisizer 3 cell counter (Beckman Coulter). Data were evaluated as percent of the control wells: % of CTR = (Treated - Blank)/(Control - Blank). ICSo values were calculated by LSW/Data Analysis using Microsoft Excel sigmoidal curve fitting. In vitro cell cycle analysis The human colon cancer cell line HCT-116 was seeded at 5000 ce\|ls/cm2 (Costar) and cultured as mentioned above. Cells in their exponential phase treated for ?4 hours with different concentrations of compound. Also, supernatant was collected to avoid loss of detached apoptotic or-mitotic cells. n 24 wells plate of growth were culture medium Thereafter, cells were washed using PBS and detached by 0.05% (w/v) trypsin, 0.02% (w/v EDTA (Gibco). Trypsin activity was stopped using culture medium. Adherent and non-adherent cell fractions were pooled and centrifuged at 2000 rpm for 10 minutes. Cells were re-suspended in PBS and counted using a Multisizer 3 cell counter (Beckman Coulter). For fixation, etranol was added (70 %, v/v) arid cells were kept at -20°C overnight. One million fixed cells were centrifuged at 2000 rpm for 5 minutes and washed with PBS and subsequently stained for 1 hour at room temperature in the dark by adding 2[00 Dl: 25 Dg/ml propidiurn iodide (Sigma) and 15 tig/ml RNAse A (Sigma), 0.001% (v/v) Non Jet P40 (Sigma) in sodium curate (0.1% w/v, pH 7.5). Samples were analyzed by flow cytonetry at 488 nm excitation (FACSCalibur, Beckton Dickinson) using Cell Quest 3.0 so'twere (Beckton DDM and,gating Dickinson). Typically 10000 events (activating doubles discriminate module only single cells) were collected and cell cycle profiles were recorded using CellQuest (Verity Software). Cell Cycle distribution of the population was calculated using a modified model in Modfit 3.1 software (Verity Software) and expressed in % GO/G1, S, G2/M and polyploidy. i Given the above assays, the compounds of formula (I) of the invention resulted to possess a remarkable protein kinase inhibitory activity, e.g. Aurora-2 inhibitory activity. See, as an representative nM), for their cell ;king and induce example, the following table I reporting the experimental data of fojjr compounds of the invention being tested as Aurora-2 kinase inhibitors (ICSO antiproiHerative effect (ICSO nM), and for their capability to exert cell cycle blc polyploidy (% of G2/M •+• polyploidy at 200 nM). Interestingly, these same compounds were tested in comparison to a structurally very close prior art compound, herewith defined as Reference compound, which is specifically disclosed in the aforementioned WO 02/12242 - see page 160, lines 5-7 of the same; the Reference compound was therein named as N-{5-phenylacetyl-4,6-dihydropyrrolo[3,4-c]pyrazol-3-y!}-4-(4-methylpiperazino)benzamide. R (Figure Removed) Reference Compound (R = R, = R2 = H; X = NWig) N-{5-phenylacetyl-4,6-diriydropyrrolo[314-c]pyrazoi-3-yl}-4-(4-metbylpiperazinb)benzannide; Compound (1) {R = R2 = H; R., = OMe; X = NMe) N-{5-[(2R)-2-methoxy-2-phenylethanoyl]-1,4,5,6-tetrahydropyrrolo[3,4-c]pyra^ol-3-yl}-4-(4-methylpipera2in-1-yl)benzamide; Compound (11) (R= R2= H; R,= Me;X N-{5-[(2R)-2-methyl-2-phenylethanoyl]-1l415,6-tetrahydropyrrolo[3,4-c]pyrazo;f-3-yl}-4-(4-methylpipera/.in-1-yl)benzamide; Compound (6) (R = R2 = H; R, = OMe; X = CH2) N-(5-[(2R)-2-methoxy-2-phenylethanoy!]-1l4)5l6-tBtrahydropyrrolo[3l4-c3pyraz()l-3-yl}-4-piperidin-1-ylbenzamide; Compound (5) (R = Me; R, = OMe; R2= H; X = NMe) 4-(3,4-dimethylpiperazin-1-yl)-N-{5-[(2R)-2-methoxy-2-phenylethanoyl]-1,4,5,6- tetrahydropyrrolo[3,4-c]pyrazol-3-yl}benzamide. Table I (Table Removed) Compound Reference compound none _____ __ Surprisingly, the Aurora-2 inhibitory activity of the compounds of the invention resulted to be constantly and markedly superior that that of the Reference compound. In addition, those same compounds resulted to possess a cell antiproliferative effect together with the capability to block the cell cycle and induce polyploidy, significantly superior than that of the Reference compound being tested in the same conditions. times daily. jThe compounds of the invention can be administered in a variety of dosage forms, e.g., orally, in the form tablets, capsules, sugar or film coated tablets, liquid solutions or suspensions; rectally in the form suppositories; parenterally, e.g., intramuscularly, or through intravenous and/or intrathecal and/or intraspinal injection or infusion. The present invention also includes pharmaceutical compositions comprisin formula (I) or a pharmaceutically acceptable salt thereof in association with a acceptable excipient, which may be a carrier or a diluent. 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 liclocaine hydrochloride. The solutions for intravenous injections or infusions may contain, as a carriet", sterile water or preferably they may be in the form of sterile, aqueous, isotonic, saline solutions or they may contain propylene glycol as a carrier. The suppositories may contain, together with the active compound, a ipharmaceutically acceptable carrier, e.g., cocoa butter, polyethylene glycol, a polyoxyethylene sorbitan fatty acid ester surfactant or lecithin. With the aim to better illustrate the present invention, without posing any limitation to it, the following examples are now given. EXAMPLES General Methods Flash chromatography was performed on silica gel (Merck grade 9385, 60A\ HPLC/MS was performed on a Waters X Terra RP 18 (4.6 x 50 mm, 3.5 (im) column using a Waters 2790 HPLC system equipped with a 996 Waters PDA detector and a 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 9'5:5), and Mobile phase B was H2O / acetonitrile (5:95). Gradient from 10 to 90% B in 8 minutes, hold 90% B 2 min. UV detection at 220 nm and 254 nm. Flow rate 1 ml/min. Injection volume 10 ul. Full scan, mass range from 100 to 800 amu. Capillary voltage was 2.5 KV; Source temp.was 120°C; Cone was 10V. Retention Times (HPLC r.t.) are given in minutes at 220 nm or 254 nm. Mass are given as m/z ratio. 1H-NMR spectroscopy was performed on a Mercury VX 400 operating at 400.45 MHz equipped with a 5mm double resonance probej (1H {15N-31P} ID_PFG Varian). Example 1 Preparation of 5-iert-butyl 1-ethyl 3-amino-4,S-dihydropyrrolo[3,4\|-c]pyrazole-1,5-dicarboxylate A solution of ethyl chlorocartjonate (8.9 ml, 93 mmol) in tetrahydrofuran (THF, 250 ml) was slowly added to a mixture of tert-butyl 3-amino^,6-dihydropyrrolo[3,4-q]pyrazole-5(1H)-carboxylate (20 g, 89 mmol) and diisopropylethylamine (DIEA, 92 ml, 528 mrpo!) in THF (500 ml) at 0-5°C The reaction was kept at: the same temperature for two hoursi then allowed to reach room temperature and stirred overnight. The obtained mixture was evaporated to dryness under vacuum, and the resulting residue extracted with ethyl acetate (AcOEt) and water. The organic layer was separated, dried over sodium sulfate and evaporated to dryness. The mixture was purified by flash-chromatography (eluent ethyl acetate/cycflohexane 4/6 to 7/3) to give 19 g of the title compound as a white solid. [M+H]+ 297 Example 2 Preparation of 5-tert-buty! 1-ethyl 3-{[4-(4-methylpiperazin-1-yl)benzoyl]a\|mino}-4,6-dihydropyrrolo[3,4-c]pyrazole-1,5-dicarboxylate Oxglyl chloride (23.2 ml, 265 mrnol) was added to a suspension of 4-(4-meth.yi-1-piperazinyl)- i benzole acid (11.7 g, 53 mmol) in dichloromethane (DCM, 320 ml) and dirhethylformamide (DMF, 0.52 ml). After refluxing the mixture for 6.5 hours, volatiles were carefully removed under reduced pressure (taking up the residue three times with toluene). The resulting 4-methylpiperazino-benzoyl chloride di-hydrochloride was added portionwise to a solution of 5-tert-butyl 1-ethyl 3-amino-4,6-dihydropyrrolo[3,4-c]pyrazole-" ,5-dicarboxylate (13.1 g, 44.3 rnmol) in dry THF {620 ml) and DIEA (54.4 ml, 0.32 mo!) under stirring at room temperature. The resulting suspension was stirred 16 hours at room tempemture and 1 hour at40°C After solvent removal under reduced pressure, the residue was taken up with! AcOEt (600 ml) and the organic layer washed with aqueous sodium carbonate (200 ml), brine, (200 ml) and dried over sodium sulfate. Solvent was evaporated, and the residue was triturated with a mixture of diethyl ether (Et20, 135 ml) and AcOEt (15 ml), filtered, dried under vacuum at 40°C to give 20 g of the title compound as a white powder [M->-H]+ 499. By operating in an analogous way and by reacting' 5-tert-butyl 1-etryl 3-amino-4,6-dihydropyrrolo[3l4-c]pyrazole-1,5-dicarboxylate with the appropriate acyl ch oride derivative, the following compounds were prepared: ii 5-tert-butyl 1-ethyl 3~{[4-(4-ethy]piperazin-1-yl)ben2oyl]amino}-4,6-dihydropyrr6lo[3.4- c]pyrazole~1,5-dicarboxylate; [M+H]+ 513. i 5-tert-butyl 1-ethyl 3-{l4-(4-isopropylpiperazin-1-yl)benzoyl]amino}-4,6-dihydrobyrrolo[3,4-(5)c]pyrazole-1.5-dicarboxylate; [M+Hf 527. 5-tert-butyl 1-ethyl 3-{[4-(4-cyclopropylpiperazin-1-yl)benzoyl]amino}-4,6-dihydropyrrolo[3,4-c]pyrazole-1,5-dicarboxylate; [M+H]+ 525. 5-tert-butyl 1-ethyl 3-{[4-(3,4-dimethylpiperazin-1-yl)benzoyl]amino}-4,6-dihydrppyrro!o[3,4-c]pyrazole-1,5-dicarboxylate; [M+H]+ 513. 5-tert-butyl 1 -ethyl 3-[(4-piperidin-1-ylbenzoyl)amino]-4,6-dihydropyrrolo[3,4-c pyrazole-1,5-dicarboxylate, [M+H]+ 484. 5-tert-butyl 1-ethyl 3-{[4-(4-fluoropiperidin-1-yl)benzoyl]amino]-4,S-dihydropyrr3lo[3,4-c]pyrazole-1,5 dicarboxylate; [M+Hf 502. 5-tert-butyl 1 -ethyl 3-[(4-morpholin-4-ylbenzoyl)arnino]-4,6-dihydropyrrolo[3,4-c]pyrazole-1,5-dicarbox-ylate, [M+H]+ 486. 5-tert-butyl 1-ethyl 3-{[4-(4-tert-butylpiperazin-1-yl)benzoyl]amino}-4,6-dihydropyrrolo[3,4-(5)c]pyrazole-1,5-dicarboxylate; [M+H]+ 541. of ethyl Example 3 3-f[4-(4-methylpiperazin-'l-vl)ben::ovllamino)-5,6- dihydrDpyrrolof3,4-c]pyrazole-1(4H)-carboxylate trihydrochloride d dropwise to a A 4N solution of hydrochloric acid in dioxane (122 ml, 488 mmol) was add stirred solution of 5-tert-butyl 1-ethyl 3-{[4-(4-methylpiperazin-1-yl)benzoyl]amino}-4,6-dihydropyrrolo[3,4-c)pyrazo!e-1,5-dicarboxylate (19.5 g, 39.2 mmol), as prepared in Example 2, in dry DCM (240 ml); precipitation of a white solid occurred almost immediately. The resulting mixture was stirred at room temperature for 24 hours; after dilution with Et20 (100 ml), the solid was filtered, extensively washed with Et20, and dried under vatuum at 50°C to give 20.1 g of the title compound, used in the next step without further purification. [M+Hf 399. 1H-NMR (DMSO--d6) 6 ppm: 1.4 (t, 3H); 2.8 (d, 3H); 3.2 (m. 4H); 3.5 (m, 2H)r 4.1 (m, 2H), 4.4 (q, 2H); 4 6 (m, 4H); 7.1-8.0 (m, 4H); 10.3 (bs, 2H); 10.7 (bs, 1H); 11.4 (s, 1H). By operating as above reported and by starting from the suitable intermediate, the following compounds were analogously prepared: Ethy -ethylpiperazin-1-yl)benzoyl]amino}-5,6-dihydropyrrolo[3,4-;]pyrazole-1(4H)- ethyl 5-[(2R)--?-methyl-2-phenylethanoyl]-3-{[4-(4-ethylpiperazin-1-yl)benzoyr:mino}-5,6- dihydropyrrolo[3,4-c]pyrazole-1 (4H)-carboxylate; [M+Hf 545. ethyl 5-[(2R)-2-methyl-2-phenylethanoyl]-3-{[4-(4-isopropylpiperazin-1-yl)ben^oyl]amino}-5,6- dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxy]ate; [M+H]* 559. \| ethyl 5-[(2R)-2-methyl-2-phenylethanoyl]-3-{[4-(4-cyclopropylpiperazin-1-yl)benzoyl]amino}- 5,6-dihydropyrrolo[3,4-c]pyra2ole-1 (4H)-carboxylate; [M+H]* 557. ethyl 3-{f4-(3,4-dimethylpiperazin-l-yl)benzoyl]amino}-5-[(2R)-2-methyl-2-phenylethanoy\|]-5,6- dihydropyrrolo[3,4-c]pyrazole-1 (4H)-carboxylate; [M+Hf 545. ethyl 5-[(2R)-2-methyl-2-phenylethanoyl]-3-[(4-piperidin-1-ylbenzoyl)amino]-5,6- dihydropyrrolo[3,4-c]pyrazo!e-l(4H)-carboxylate; [M+H]+ 516. ethyl 3-{[4-(4 fluoropiperidin-1-yl)benzoyl]amino}-5-[(2R)-2-methyl-2-phenyle hanoyl]-5,6- dihydropyrrolo[3,4-c]pyrazole-1 (4H)-carboxylate; [M+H]+ 534. ethyl 5-[(2R)-2-methyl-2-phenylethanoyl]-3-[(4-morpholin-4-ylbenzoyl)amino]-5,6- dihydropyrrulo[3,4-c]pyrazole-1(4H)-carboxylate; [M+H]+ 518. ethyl 3-{l4-(4-tert-butylpiperazin-1-yl)benzoyl]amino}-5-[(2R)-2-methyl-2-phenylethanoyl]-5,6- I dihydropyrro!o[3,4-c]pyrazote-1(4H)-carboxylate; [M+H]+ 573. ethyl 5-[(2R)-2-hydroxy-2-phenylethanoy]]-3-{[4-(4-methylpiperazin-1-yl)ben^oyl]amino}-5,6- dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylate; [M+H]+ 533. I the following compounds were prepared: : (2) W-{5-[(2R)-2-metho3iy-2-phenyIethanoyl]-1,4.5,6-ietrahydropyrroIo[3,4-c]pyrazol-3-yl}-4-(4-c4hylpipera?:in-1-yl)ben2:arnide; [M-t-H]* 489; 1 H-NMR (DMSO-d6) 5 ppm: 1.1 (t, 3H); 2.3 -2.7 (m, 6H); 3.2 - 3.4 (m, 7H); 4.3-6.0 (m, 4H); 5.1 (d, 1H) 6.9 - 8.0 (m, 9H); 10.6 (bs, 1H); 12.1 (br, 1H). \| (3) N-{5-[(2R)-2-methoxy-2-phenylethanoyl]-1,4,5,6-tetrahydropyrroIo 3-yl}-4-(4-isopropylpiperazin-1-yl)benzamide; [M+H]* 503; 1 H-NMR (DMSO-d6) 5 ppm: 1.3 - 1.3 (dd, 6H); 3.3 - 3.4 (m, 9H); 4.6-4.9 (r 1H); 7.0 - 8.0 (m, 9H); 10.7 (bs, 1H); 12.3 (bs, 1H). 3,4-c]pyrazol- , 4H);5.1 (d, (4) N-fS-ffaRJ-a-methoxy^-phenylethanoyll-I^.S.G-tetrahydropyrrololS^-clpyrazoI-3-yI}-4-(4-cyr,lopropylpiperazin-1-yl)benzamide; [M+H]+ 501; 1 H-NMR (DMSO-d6) 5 ppm: 0.3 - 0.5 (m, 4H); 3.2 - 3.4 (m, 7H); 3.2 - 5.0 (m, 4H); 5.1 (d, 1H); 6.8-8.2 (in, 9H); 10.5-10. 7 (br, 1H); 12.0 - 12.4 (br, 1H). (5) 4-(3,4-dimethylpiperazin-1-yl)-N-{5-[(2R)-2-methoxy-2-phenylethahoyI]-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}benzamide; [M+H]1' 489; ; 4.3-5.0 (m, 1H-NMR (DMSO-d6)6 ppm: 1.0-1.1 (m, 3H); 2.24 (s, 3H); 3.3-3.5 (m, /H 4H);5.1 (d, 1H); 6.9 - 8.0 (m, 9H); 10.6 (bs, 1H); 11.9 - 12.6 (br, 1H). (6) N^ 3-y\|}-4-piperidin-1-ylbenzamide; [M+H]+ 460; 1H-NMR (DMSO-d6) 6 ppm: 1.5 - 1.7 (m, 6H); 3.2-3.4 (m, 7H); 4.3-4.9 (m, 4H); 5.1 (d, 1H); 6.9 - 8.0 (m, 9H); 10.4 - 10.7 (br, 1H). (7) 4-(4-fluoropiperidin-1-y!)-N-{5-[(2R)-2-methoxy-2-phenylethanoy 11-1,4,5,6-tetrahydropyrrolo[3,4-cJpyrazol-3-yl}benzamide; [M--H]+ 478; 1H-NMR (DMSO-d6) 6 ppm: 1.65 - 2.1 (m, 4H); 3.15 - 3.6 (m, 7H); 4.35 - 5 (d, 1H); 6.9-8.0 (m, 9H); 10.4 - 10.7 (br, 1H). 0(m,5H); 5.1 (S) W-{5-[(2R)-2-methoxy-2-phenylethanoyI]-1,4,5,6-tetrahydropyrroio[3,4-c]pyrazol-3-yl}-4-morphorin-4-y)benz:amide; [M+H]+ 462; 1H-NMR (DMSO-d6) 5 pprn: 3.15 - 3.5 (m, 7H); 3.7-3.8 (m, 4H); 4.3-4.9 (m, H); 5.1 (d, 1H); 6.9 -8.0(rn, 9H); 10.4 - 10.7 (br, 1H). (9) 4-(4-tert-butylpiperaz:in-1-yl)-W-{5-f(2R)-2-msthoKy-2-pheny]eihanoyl]-1,4,5,6-tei:rahydrDpyrro)o[3,4-c]pyraEol-3-yl)benzarnide; [M+H] 517. i-c]pyrazol-3- 4H);4.1 -4.9 (m, (10) N-{5-[(2R)-2-hydroxy-2-phenylethanoyl]-1,4,5,6-tetrahydropyrrolo[3, yl}-4-(4-methylpiperazin-1-yl)benzamide; [M+Hf 461; 1 H-NMR (DMSO-d6) 6 ppm: 2.3 (s, 3H); 2.45 - 2.65 (m, 4H); 3.2 - 3.4 (m, 4H); 5.69 (d, 1H); 6.9 - 8.0 (m, 9H); 10.4 - 10.7 (br, 1H); 11.5-12.9 (br, 1H). (11) N-{5-[{2R)-2-methyl-2-phenylethanoyl]-1,4,5,6-tetrahydropyrrolot\|3,4-c]pyrazol-3-yl}-4-(4-rncthylpiperazin-1-y!)benzamide; [M+Hf 459; 1 H-NMR (DMSO-d6) 5 ppm: 1.33 (d, 3H); 2.21 (s, 3H); 3.85 - 5.0 (m, 5H); 4,2 - 4.9 (m, 4H); 5.1 (s, lH)6.8-8.0(m, 9H); 10.3- 10.7 (br, 1H); 11.8 - 12.5 (br, 1H). ,4-c]pyrazol-3- 6(m,4H); 3.2-, 1H). (12) N-{5-[(2R)-2-methyl-2-phenylethanoyl]-1,4)5,6-tetrahydropyrrolo[:i y\|}-4-(4-Bthylpiperazin-1-yl)benzamide; [M+H]+ 473; 1H-NMR (DMSO-d6) 5 ppm: 1.06(t,3H); 1.36(d, 3H); 2.41 (q, 2H); 2.47-2 ' 3.4 (m,4H); 3 9-5.0 (m, 5H); 6.9 - 8.0 (m, 9H); 10.5(bs, 1H); 11.9- -12.5(bi (13) N-{5-[(2R)-2-methyl-2-phenylethanoyl]-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazoI-3- y\|}-4-(4~isopropylpiperazin-1-yl)benzamide; [M+H]+ 487; \| I (14) N-{5-[(2R)-2-methyl-2-phenylethanoyl]-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazoI-3- yl}~4-(4-cyclopropylpiperazin-1-yl)benz:amide; [M+H]+ 485; (15) 4-(3,4-dirnethylpiperazin-1-yl)-N-{5-[(2R)-2-phenylpropanoyl]-1,4,£I6- tetrahydropyrrolo[3,4-c]pyrazol-3-yl}benzamide; [M+H]+ 473; (16) W-\|5-[(2R)-2-phenylpropanoyI]-1,4)5)6-tetrahydropyrroIo[3,4-c]py piperidin-1-ylbenzamide; [M+H]+ 444; 1H-NMR (DMSO-d6) 5 ppm: 1.37 (d, 3H); 1.6 (s, 6H); 3.2-3.4 (m, 4H); 3.9C 4.1 -4.9(m, 4H); 6.9-8.0 (m, 9H); 10.4-10.7 (br, 1H). a2iol-3-yl}-4- -4.05 (m, 1H); 4-(4-fluQropiperidin-1-yl)-N-{5-[(2R)-2-methy!-2-pheny]ethgnoyl]-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}benzarnide; [M+H]4 462; i 1H-NMR (DMSO-d6) 5 ppm; 1.37 (d, 3H); 1.65 - 2.05 (m, 4H); 3.15 - 3.6 (m; 7H); 3.2 - 3.4 (m. 4H); 3.85 -4,07 (m, 1H); 4.1 - 5.05 (m, 5H); 5.1 (d, 1H); 6.9 - 8.0 (m, 9H); 10.4 - 10.7 (br, 1H). (18) N-{5-[(2R)-2-methyI-2-phenylethanoyl]-1,4,5,6-tetrahydropyrrolo^,4-c]pyrazol-3- yI}-4-rnorpholin-4-ylbenzamide; [M+H]+ 446; 1 H-NMR (DMSO-d6) 6 ppm: 1.37(d, 3H); 3.2 - 3.4 (m, 4H); 3.7-3.85 (m, 4fH); 3.9-4.1 (nx 1H); 4.1 -4.95 (m, 4H); 6.9 - 8.0 (m, 9H); 10.4-10.7 (br, 1H). (19) 4-(4 tert-butylpiperazin-1-yl)-N-{5-[(2R)-2-phenylpropanoy!]-1,4,5;6- tetrahydropyrrolo[3,4-c]pyrazol-3-yl}benzamide; [M+H]+ 501. i We Claim; 1. A pyrrolo [3,4-c] pyrazole compound of formula (1) (Formula Removed) Wherein R is hydrogen or methyl; Rl is hydroxy or a straight or branched C1-C3 alkyl or alkoxy oroup; R2 is a hydrogen or halogen atom; X is a divalent group selected from methylene (-CH2-) or fluoromethylene (-CHF-), or it is a heteroatom or heternatornic group selected from oxygen (-0-) or nitrogen (-NR'-) wherein R' is a hydrogen atom, a straight or branched C1-C4 alkvl group; or a C3-C6 cycklallyl group; a pharmaceutically acceptable salt thereof the kind such as herein described. 2. A compound of formula (I) as claimed in claim 1, wherein R is hydrogen or methyl; Ri is selected from hydroxy or methyl; R2 is a hydrogen; X is selected from methylene, fluoromethylene, -O- or -NR', wherein R' is as defined in claim 1. 3. A compound of formula (I) as claimed in claim 1, optionally in the form of a pharmceutically acceptable salt thereof, selected from the group consisting of: N-(5-[(2R)-2-methoxy-2-phenylethanoyl]-l, 4, 5,6-tetrahydropyrrole (3,4 -c]pyrazol-3-yl}- 4- (4-isopropylpiperazin-l-yl) benzamide; N-{5-{(2R)-2-methoxy--2-phenylethanoyl]-l, 4,5,6-tetrahydropyrrolo[3, 4-c]pyrazol-3-yl}- 4-(4-ethylpiperazin-l-yl) benzamide; N-{5-[(2R)-2-methoxy-2-pbenylethanoyl]-l,45,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yI}- 4-(4-isopropylpiperazin-l-yl)benzamide; N-{5-[(2R)-2-methoxy-2-phenylethanoyl)-l, 4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)- 4-(4-methylpiperazin-l-yl) benzamide; 4-(3,4-dimethylpiperazin-1-yl)-N-{5-[(2R)-2-methoxy-2-phenylethanoy] -1,4,5,6- tetrahydropyrrolo[3,4-c]pyrazol-3-yl} benzamide; N-(5-[(2R)-2-methoxy-2-phenylethanoyl]- 1,4, 5,6-tetrahydropyrrolb[3, 4-c]pyrazol-3-yl)- 4-piperidin-1 -yl) benzamide; 4-(4-fluoropiperidin- l-yl)-N-{5-[(2R)-2-methoxy-2-phenylethanoyl]-1,4,5,6- tetrahydropyrrolo[3,4-c]pyrazol-3-yl} benzamide; and N-{5-[(2R)-2-methoxy-2-phenylethanoyl)-l, 4,5,6-tetrahydropyrrolo[3,4- c]pyrazoI-3-yl)-4-morpholin-4-ylbenzamide; 4-(4-tert-butytpiperazing)-N-{5-[(2R)-2-methoxy-2-phenyl ethanoyl]- 1,4,5,6-tertanyokopyroolo [3,4-c] pyraz of-3-yl} bemamide. A pharmaceutical composition comprising a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof, as defined in claim 1, and at least one pharmaceutically acceptable excipient, carrier and/or diluent. A pharmaceutical composition as claimed in claim 4 further comprising one or more chemotherapeutic agents. A kit comprising a compound or a pharmaceutically acceptable salt thereof, as defined in claim 1, or pharmaceutical compositions thereof as defined in claim 4, and one or more chemotherapeutic agents.

Full Text

The present invention relates to "A PYRROLO [3,4-C] PYRAZOLE COMPOUND OF FORMULA (1)"
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to pyrroio-pyrazole derivatives, to a pnocess for their preparation, to pharmaceutical compositions comprising them, and to their use as therapeutic agents, particularly in the treatment of cancer and cell proliferation disorders.
Discussion of the Background
The malfunctioning of protein kinases (PKs) is the hallmark of numerous djseases. A large share of the oncogenes and proto-oncogenes involved in human cancers cdde 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, pulmonaryfibrosis, arthritis glomerulonephritis and post-surgical stenosis and restenosis.
PKs are also implicated in inflammatory conditions and in the multiplicatinof viruses and parasites. PKs may also play a major role in the pathogenesis and Idevelopment of neyrodegenerative disorders.
For a general reference to PKs malfunctioning or disregulation see, for instance. Current Opinion in Chemical Biology 1999, 3, 459 -465.

atl. Cancer Inst, 2002, 94(17)i 1320-9]. For a general reference to Aurora-2 role in the abnormal centrosome function in cancer see also Molecular Cancer Therapeutics, 2003, 2, 589 - 595.
SUMMARY OF THE INVENTION
It is an object of the invention to provide compounds, which are useful in therapy as agents against a host of diseases caused by and/or associated to a disregulatejd protein kinase activity and, more particularly, Aurora kinases activity.

It is another object to provide compounds, which are endowed with protein activity and, more particularly, Aurora kinases inhibiting activity.

kinase inhibiting

The present inventors have now discovered that some pyrrolo-pyrazoles, and derivatives
thereof, are endowed with protein kinase inhibiting activity, e.g. Aurora kinases inhibiting
activity. ;
More specifically, 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, parjicreas, stomach, cervix, thyroid, prostate, and skin, including squamous cell carcinoma; hem^topoietic tumors of lymphoid lineage, including leukemia, acute lymphocitic leukemia, acute lymphoblastic

non-Hodgkin's
leukemia, B cell lymphoma, T-cell-lymphoma, Hodgkin's lymphoma,
lymphoma, hairy cell lymphoma and Burkett's lymphoma; hematopoietic timers of myeloid lineage, including acute and chronic myelogenous leukemias, myelodysplastic syndrome and promyelocytic leukemia; tumors of mesenchyma! origin, including fibrosarcoma and

neuro-fibromatosis, psoriasis, vascular smooth cell proliferation

ssociated with

atherosclerosis, pulmonary fibrosis, arthritis glomerulonephritis and post-surgical stenosis and
restenosis. !
Accordingly, in a first embodiment, the present invention provides a method for treating cell proliferative disorders caused by and/or associated with an altered protein kinase activity, which comprises administering to a mammal in need thereof an effective amount of a compound of formula (I)
(Figure Removed)

wherein
R is hydrogen or methyl,
RT is hydroxy or a straight or branched C-,-C3 alkyl or alkoxy group;
R2 is a hydrogen or halogen atom;

X is a divalent group selected from methylene (-CH2-) or fluoromethylene (-CHF-), or it is a heteroatom or heteroatomic group selected from oxygen (-0-) or nitrogen (-NR1-) wherein R' is a hydrogen atom, a straight or branched C^-C^ alky! group or a C3-C6 cycloalkyl group; or a pharmaceutically acceptable salt thereof.
The above method enables treatment of cell proliferative disorders caused by and/or associated with altered Aurora kinases activity.
In a preferred embodiment of the method described above, the cell proliferative disorder is cancer
DETAILED DESCRIPTION OF THE INVENTION
Several heterocyclic compounds are known in the art as protein kinase inhibitors. Among them, 3-carboxamido-pyrazoles and 3-ureido-pyrazo!es and derivatives thereof, have been disclosed as protein kinase inhibitors in the international patent applications WO 01/12189, WO 01/12188, WO 02/48114 and WO 02/70515, all in the name of the applicant
itself.

;inase inhibitory WO 03/028720 US patent self.
fnm
Fused bicyclic compounds comprising pyrazole moieties and possessing activity have been also disclosed in WO 00/69846, WO 02/12242 as well as and still unpublished PCT/EP03/04862 application (claiming priority application 60/381092, filed in May 17, 2002), all in the name of the applicant i

In addition to the above, aminophenyl-piperazine or aminophenyl-piperidine derivatives as possessing inhibitory activity towards prenyl transferase proteins are disclosed in WO 02/30927 in the name of Pierre Fabre Medicament.
The compounds of the present invention fall within the scope of the general formula of the aforementioned WO 02/12242, herewith incorporated by reference, but are not specifically
exemplified therein.
The compounds of formula (1) of the invention have asymmetric carbon'atoms and may therefore exisl as individual optical isomers, as racemic mixtures or as any other mixture comprising a majority of one of the two optical isomers, which are all to be intended as within the scope of the present invention.
Likewise, the use as an antitumor agent of all the possible isomers and their admixtures and of both the metabolites and the pharmaceutically acceptable bio-precursors (otherwise

referred to as pro-drugs) of the compounds of formula (I) are also within present invention.
Prodrugs are any covalently bonded compounds, which release the acr according to formula (I), in vivo.

the scope of the
ve parent drug,

In cases when compounds may exist in tautomeric forms, each form is contemplated as being included within this invention whether existing in equilibrium or predominantly: in one form.
As such, unless otherwise provided, when only one of the following tautomeric forms of formula (la) or (Ib) is indicated, the remaining one has still to be intended as comprised within the scope of the invention:
(Figure Removed)

In the present description, unless otherwise specified, with the term straight or branched CrC3 or CrC4 alkyl we intend any of the groups such as, for instance, methyl, ethyl, n-propyl, isopropyl, n-butyl, isohutyl, tert-butyl and sec-butyl.
With the term straight or branched C,-C3 alkoxy we intend any of the groups such as, for instance, methoxy, ethoxy, n-propoxy and isopropoxy.
With the term halogen atom we intend s. fluorine, chlorine, bromine or iodine at)m.
With the term C3-CS cycioalkyl we intend any group such as cycloprcpyl, cyclobutyl,
cyciopentyl or cyclohexyl.
Clearly, depending upon the nature of the X group, this same heterocycle being linked to the phenylene moiety of the compounds of formula (1} may represent a piperidino, 4-ftuoropiperidino, piperazino, 4-alkyl-piperazino, 4-cycloalkyl-piperazino ormorpholino ring.
Pharmaceutjcally acceptabie salts of the compounds of formula (I) include the acid addition salts with inorganic or organic acids such as, for instance, nitric, hydrochloric, hydrobromic, sulfuric, perchloric, phosphoric, acetic, trifluoroacetic propionic, glycolic, lactic, oxalic, malonic, malic, maleic, tartaric, ciiric, benzole, cinnamic, mandelic, methanesulphonic, isethionic and salicylic acid.

A preferred class of compounds of the invention is represented by the deriv;

tives of formula

(I) wherein R is hydrogen or methyl; R-, is selected from hydroxy, methyl or nelhoxy; R2 is a

hydrogen or fluorine atom; X is selected from melhyene, fluoromethylene, -O-R1 is as above reported.

>r-NR', wherein

For a reference to any specific compound of formula (I) of the invention, optionally in the form of a pharmaceutically acceptable salt, see the experimental section and claims.

As formerly indicated, a further object of the present invention is represented by the process for preparing the compounds of formula (I) and the pharmaceutically acceptable salts thereof, which process comprises:
a) reacting a compound of formula (II) with a compound of formula (II!)
(Figure Removed)

wherein R and X are as above defined, Q is a lower alkyl group, t-Bu repres Z is hydroxy or a suitable leaving group, so as to obtain a compound of form
(IV)

;nts tert-butyl and Jla (IV)

5 b) reacting the compound of formula (IV) under acidic conditions so as to obtain a compound of formula (V) lt thereof.

as to obtain the
pharmaceutically

The above process is an analogy process, which can be carried out by wor, very well-known operative conditions.

;ing according to

According to step (a) of the process, the reaction between the compounds of formula (II) and
(III) can be carried out in a variety of ways, according to conventional methods for acylating
j amino derivatives. As an example, the compound of formula (II) may be reacted with an acyl
chloride of formula (III) wherein Z represents, as a suitable leaving group, a chlorine atom.
Preferably, this reaction is carried out at a temperature ranging from room temperature to about 60°C, in a suitable solvent such as, for instance, tetrahydrofuran or c'ichloromethane,

and in the presence of a proton scavenger such as triethylamine or diisopropy

ethylamine.

Within the compounds of formula (II) Q represents a lower alky) group, for hstance a alkyl group, more preferably methyl or ethyl.
According to step (b) of the process, the compound of formula (IV) is easily deprotected at the pirrolidine nitrogen atom, by acidic treatment.

acid such ble solvent such at a temperature about 1 hour to
This reaction can be conveniently carried out in the presence of a mineral or organic as, for instance, hydrochloric, trifluoroacetic or methansulfonic acid, in a suit; as dichloromethane, 1,4-dioxane, a lower alcohol (e.g. methanol or ethanol), ranging from room temperature to about 40°C and for a time varying from about 48 hours.

The compound of formula (V) thus obtained is further reacted, according to step (c) of the process, with a compound of formula (VI). From the above it is clear to the skilled person that also this acylation reaction may be accomplished in a variety of ways and operative conditions, which are widely known in the art for the preparation of carboxamides.
The reaction between a compound of formula (V) and a carboxylic acid of formula (VI) wherein

Z' is hydroxy can be carried out in the presence of a coupling agent such

as, for instance,

carbodiimide, i.e. 1,3-dicyclohexylcarbodiimide, 1,3-diisopropylcarbcdiimide, 1-(3-
polystyrene or N-cyclohexylcarbodiimide-N'-methyl polystyrene, in a suitable
dimethylaminopropyl)-3-ethylcarbodiimide,

N-cyclohexylcarbodiimide-N'

propyloxymethyl solvent such as,

for instance, dichioromethane, chloroform, telrahydrofuran, diethyl eth^r, 1,4-dioxane, acetonitrile, toluene, or N.N-dimethylformamide at a temperature ranging fron about -10°C to

reflux lor a suitable time, i.e. from about 30 minutes to about 96 hours. The

said reaction is

optionally carried out in the presence of a suitable catalyst, for instance 4-
dimethylaminopyridine, or in the presence of a further coupling reagent such as N-
hydroxybenzotriazole. !
The reaction between a compound of formula (V) and a compound of formula (VI) can be also carried out, for example, through a mixed anhydride method, by using an alkyl chloroformate, such as ethyl, iso-butyl, or iso-propyl chloroformate, in the presence of a tertiary base, such as triethylamine, N,N-diisopropylethylamine or pyridine, in a suitable solve it such as, for

3, diethyl ether,
instance, toluene, dichloromethane, chloroform, tetrahydrofuran, acetonitri
1,4-dioxane, or N,N-dimethylformamide, at a temperature ranging from abou -30°C to room temperature.

The reaction between a compound of formula (V) and a carboxylic derivative of formula (VI) wherein 2' is a suitable leaving group can be carried out in the presence of a tertiary base such as triethylamine, N,N-diisopropylethylamine or pyridine, in a suitable solvent, such as toluene, dichloromethane, chloroform, diethyl ether, tetrahydrofuran, acetonitrile, or N,N-dirnethylformamide, at a temperature ranging from about -10°C to reflux.
The compounds of formula (VI) are characterised by the presence of an asymmetric carbon atom being linked to R, and presently noted with an asterisk
O
R.
(VI)
Hence, the compounds of formula (VI) may be either in the form of single en'antiomers or as mixtures thereof, also comprehensive of racemic mixtures.

usejd in the process riroperly defined
Clearly, depending upon the nature of the compound of formula (VI) being of the invention, corresponding compounds of formula (VII) having a stereochemistry at this same carbon atom could be thus obtained.

According to a preferred embodiment of the invention, step (c) is carried out by reacting a suitable compound of formula (Vi), which is in a given enantiomeric form.
Likewise, if racemic mixtures of the compound of formula (VI) are employed and whenever final compounds of formula (I) in optical pure form are desired, optical resolution of the intermediate compound of formula (VII) or, alternatively, of the final compound of formula (I) will be required, by working according to conventional means. Just sis an example, conventional techniques for racemate resolution include, for instarce, partitioned crystallization of diastereoisomeric salt derivatives or preparative chiral HPLC.
(Figure Removed)

Finally, according to step (d) of the process, the compound of formula (VII) is deprotected at the pyrazole nitrogen atom by working according to conventional methods enabling, for instance, the selective hydrolysis of the carbamate group.

• instance in the as triethylamine, tetrahydrofuran, ranging from to about 96
As an example, this reaction may be carried out under basic conditions, fo presence of sodium, potassium or lithium hydroxide, or a tertiary amine such and in a suitable solvent such as N,N-dimethy!formarnide, methanol, ethanol water and mixtures thereof. Typically, the reaction is carried out at temperatures room temperature to about 60°C and for a time varying from about 30 minptes hours.

Finally, pharmaceutically acceptable salts of the compounds of formula (1) or, ialternatively, the free compounds from their salts thereof may be all obtained according!to conventional methods.
The starting material of the process of the invention are known or easily prepared according to knwon methods.

represents ethyl is /12242 (see, in as 3-amino-4,6-iter).
As an example, the preparation of the compound of formula (11) wherein Q disclosed in Jhe aforementioned international patent application WO 02 particular, example 26 at page 249; this same compound is therein named dihydro-pyrrolo[3,4-c]pyra?:Dle-1,5-dicarboxylic acid 5-tert-butyl ester 'I-ethyl e

By working in an analogous way, additional compounds of formula (II) whereip Q represents a lower alkyl group other than ethyl can be thus prepared as well.
i
The compounds of formula (111) and (VI), for instance those wherein Z and Z' represent a halogen atom, e.g. a chlorine atom, are either known or can be easily obtained from the corresponding known carboxylic acids, by working according to conventional methods.
Likewise, it is also clear to the person skilled in the art that if a compound of formula (I), prepared according to the above process comprehensive of any variant thereof, is obtained as an admixture of isomers, their separation into the single isomers of formula (I), carried out according to conventional techniques, is still within the scope of the present in/ention.

PHARMACOLOGY
The compounds of formula (1) are active as protein kinase inhibitors, more particularly as Aurora kinases inhibitors and are therefore useful, for instance, to restrict the unregulated proliferation ot tumor cells.
In therapy, They may be used in the treatment of various tumors, such as those formerly reported, as well as in the treatment of other cell proliferative disorders such as psoriasis, vascular smooth cell proliferation associated with atherosclerosis and post-surgical stenosis and restenosis.
The inhibiting activity and the potency of selected compounds is determined through a method

of assay based on the use of the SPA technology (Amersham Pharmacia Bio1

ich).

The assay consists of the transfer of radioactivity labelled phosphate moiety ty the kinase to a

biotinylated substrate. The resulting 33P-labelied biotinylated product is al

owed to bind to

streptavidin-coated SPA beads (biotin capacity 130 pmol/mg), and light emitted was measured in a scintillation counter.
Inhibition assay of Aurora-2 activity
\\insse reaction: 8 uM biotinylated peptide (4 repeats of LRRWSLG), 10 uM ATP (0.5 uCi P^y-ATP), 7 fi ng Aurora 2, inhibitor in a finsit volume of 30 ul buffer (HEPES 50 mWI pH 7.0,
to each well of a
in, reaction was
MgCl2 10 ruM, ! mM DTT, 0.2 mg/ml BSA. 3 uM orthovanadate) were added 96 U bottom well plate. After 60 minutes at room temperature incubati stopped and biotinylated peptide captured by adding 100 ul of bead suspension.
Stratification: 100 pi of CsCI2 5 M were added to each well and let starjicf 4 hour before radioactivity was counted in the Top-Count instrument.
1C50 determination: inhibitors were tested at different concentrations ranging from 0.0015 to
10 uM. Experimental data were analyzed by the computer program GraphPaiji Prizm using the
four parameter logistic equation:
y = bottom+(lop-bottom)/(1-*-10A((loglC50-x)*s|ope))
where x is tin; logarithm of the inhibitor concentration, y is the response; y starts at bottom and
goes lo top with a sigrnoid shape.

Ki calculation:
Experimental method: Reaction was carried out in buffer (10 mM Tris, pH 7[5, 10 mM MgCI2, 0.2 rng/ml BSA, 7.5 mM DTT) containing 3.7 nM enzyme, histone and ATP {constant ratio of cold/labeled ATP 1/3000). Reaction was stopped with EDTA and the substrate captured on phosphomembrane (Multiscreen 96 well plates from Millipore). After extensive washing, the multiscreen plates were read on a top counter. Control (time zero) for each ATP and histone concentrations was measured.
Experimental design: Reaction velocities are measured at four ATP, substrate (histone) and inhibitor concentrations. An 80-point concentration matrix was designed around the respective ATP and substrate Km values, and the inhibitor IC50 values (0.3, 1, 3, 9 folJ the Km or IC50 values). A preliminary time course experiment in the absence of inhibitor and at the different ATP and substrate concentrations allows the selection of a single endpoin1 time (10 min) in the linear range of the reaction for the Ki determination experiment.
Kinetic parameter estimates: Kinetic parameters were estimated by simultaneous nonlinear least-square regression using [Eq.1] (competitive inhibitor respect to ATP, random mechanism) using the complete data set (80 points):

Vm *
o: '-" Ka o Kb + a » Ka e> B 4- a ® Kb

fin R
- ® 7 0 (Kb + •+-)
Ki j?

[Eq.1]

where A--[ATP], B=[.Substrate], l=[inhibitor], Vm^ maximum velocity, dissociation constants of ATP, substrate and inhibitor respectively, a and (3

Ka, Kb, Ki the the cooperativity

factor between substrate and ATP binding and substrate and inhibitor bindinc respectively.

The compounds of the invention were further tested in vitro to assess thf effect onto cell cultures and the inhibititory effect on the cell cycle.

anti-proliferative

In vitro cell proliferation assay
The human colon cancer cell line HCT-116 was seeded at 5000 cells/cm2 in 24 wells plate (Costar) using F12 medium (Gibco) supplemented with 10% PCS (EuroClone, Italy) 2 mM L-glutarnine and 1% penicillin/streptomycin and maintained at 37°C, 5% CO2 and 96% relative

humidity. The following day, plates were treated in duplicates with 5ul of dilution of compounds starting from a 10 mM stock in DMSO. Two untreal

an appropriate :d control wells

were included in each plate. After 72 hours of treatment, medium was withdrawn and cells

detached from each well using 0.5 ml of 0.05% (w/v) Trypsin, 0,02% (w/v

EDTA (Gibco).

Samples were diluted with 9.5 ml of Isoton (Coulter) and counted using a Multisizer 3 cell counter (Beckman Coulter). Data were evaluated as percent of the control wells: % of CTR = (Treated - Blank)/(Control - Blank).
ICSo values were calculated by LSW/Data Analysis using Microsoft Excel sigmoidal curve
fitting.

In vitro cell cycle analysis
The human colon cancer cell line HCT-116 was seeded at 5000 ce|ls/cm2 (Costar) and cultured as mentioned above. Cells in their exponential phase treated for ?4 hours with different concentrations of compound. Also, supernatant was collected to avoid loss of detached apoptotic or-mitotic cells.

n 24 wells plate of growth were culture medium Thereafter, cells

were washed using PBS and detached by 0.05% (w/v) trypsin, 0.02% (w/v EDTA (Gibco). Trypsin activity was stopped using culture medium. Adherent and non-adherent cell fractions were pooled and centrifuged at 2000 rpm for 10 minutes. Cells were re-suspended in PBS and counted using a Multisizer 3 cell counter (Beckman Coulter). For fixation, etranol was added (70 %, v/v) arid cells were kept at -20°C overnight.
One million fixed cells were centrifuged at 2000 rpm for 5 minutes and washed with PBS and subsequently stained for 1 hour at room temperature in the dark by adding 2[00 Dl: 25 Dg/ml

propidiurn iodide (Sigma) and 15 tig/ml RNAse A (Sigma), 0.001% (v/v) Non

Jet P40 (Sigma)

in sodium curate (0.1% w/v, pH 7.5). Samples were analyzed by flow cytonetry at 488 nm excitation (FACSCalibur, Beckton Dickinson) using Cell Quest 3.0 so'twere (Beckton

DDM and,gating
Dickinson). Typically 10000 events (activating doubles discriminate module
only single cells) were collected and cell cycle profiles were recorded using CellQuest (Verity Software). Cell Cycle distribution of the population was calculated using a modified model in
Modfit 3.1 software (Verity Software) and expressed in % GO/G1, S, G2/M and polyploidy.
i
Given the above assays, the compounds of formula (I) of the invention resulted to possess a remarkable protein kinase inhibitory activity, e.g. Aurora-2 inhibitory activity. See, as an

representative nM), for their cell ;king and induce
example, the following table I reporting the experimental data of fojjr compounds of the invention being tested as Aurora-2 kinase inhibitors (ICSO antiproiHerative effect (ICSO nM), and for their capability to exert cell cycle blc polyploidy (% of G2/M •+• polyploidy at 200 nM).
Interestingly, these same compounds were tested in comparison to a structurally very close prior art compound, herewith defined as Reference compound, which is specifically disclosed in the aforementioned WO 02/12242 - see page 160, lines 5-7 of the same; the Reference compound was therein named as N-{5-phenylacetyl-4,6-dihydropyrrolo[3,4-c]pyrazol-3-y!}-4-(4-methylpiperazino)benzamide. R
(Figure Removed)

Reference Compound (R = R, = R2 = H; X = NWig) N-{5-phenylacetyl-4,6-diriydropyrrolo[314-c]pyrazoi-3-yl}-4-(4-metbylpiperazinb)benzannide;
Compound (1) {R = R2 = H; R., = OMe; X = NMe)
N-{5-[(2R)-2-methoxy-2-phenylethanoyl]-1,4,5,6-tetrahydropyrrolo[3,4-c]pyra^ol-3-yl}-4-(4-methylpipera2in-1-yl)benzamide;
Compound (11) (R= R2= H; R,= Me;X
N-{5-[(2R)-2-methyl-2-phenylethanoyl]-1l415,6-tetrahydropyrrolo[3,4-c]pyrazo;f-3-yl}-4-(4-methylpipera/.in-1-yl)benzamide;
Compound (6) (R = R2 = H; R, = OMe; X = CH2)
N-(5-[(2R)-2-methoxy-2-phenylethanoy!]-1l4)5l6-tBtrahydropyrrolo[3l4-c3pyraz()l-3-yl}-4-piperidin-1-ylbenzamide;
Compound (5) (R = Me; R, = OMe; R2= H; X = NMe)
4-(3,4-dimethylpiperazin-1-yl)-N-{5-[(2R)-2-methoxy-2-phenylethanoyl]-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl}benzamide.
Table I
(Table Removed)
Compound
Reference compound
none _____
__

Surprisingly, the Aurora-2 inhibitory activity of the compounds of the invention resulted to be constantly and markedly superior that that of the Reference compound.
In addition, those same compounds resulted to possess a cell antiproliferative effect together with the capability to block the cell cycle and induce polyploidy, significantly superior than that of the Reference compound being tested in the same conditions.
times daily. jThe compounds of the invention can be administered in a variety of dosage forms, e.g., orally, in the form tablets, capsules, sugar or film coated tablets, liquid solutions or suspensions; rectally in the form suppositories; parenterally, e.g., intramuscularly, or through intravenous and/or intrathecal and/or intraspinal injection or infusion.

The present invention also includes pharmaceutical compositions comprisin formula (I) or a pharmaceutically acceptable salt thereof in association with a acceptable excipient, which may be a carrier or a diluent. 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 liclocaine hydrochloride. The solutions for intravenous injections or infusions may contain, as a carriet", sterile water or preferably they may be in the form of sterile, aqueous, isotonic, saline solutions or they may contain propylene glycol as a carrier.
The suppositories may contain, together with the active compound, a ipharmaceutically acceptable carrier, e.g., cocoa butter, polyethylene glycol, a polyoxyethylene sorbitan fatty acid ester surfactant or lecithin.
With the aim to better illustrate the present invention, without posing any limitation to it, the following examples are now given.

EXAMPLES
General Methods
Flash chromatography was performed on silica gel (Merck grade 9385, 60A\ HPLC/MS was performed on a Waters X Terra RP 18 (4.6 x 50 mm, 3.5 (im) column using a Waters 2790 HPLC system equipped with a 996 Waters PDA detector and a 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 9'5:5), and Mobile phase B was H2O / acetonitrile (5:95). Gradient from 10 to 90% B in 8 minutes, hold 90% B 2 min. UV detection at 220 nm and 254 nm. Flow rate 1 ml/min. Injection volume 10 ul. Full scan, mass range from 100 to 800 amu. Capillary voltage was 2.5 KV; Source temp.was 120°C; Cone was 10V. Retention Times (HPLC r.t.) are given in minutes at 220 nm or 254 nm. Mass are given as m/z ratio. 1H-NMR spectroscopy was performed on a Mercury VX 400 operating at 400.45 MHz equipped with a 5mm double resonance probej (1H {15N-31P} ID_PFG Varian).
Example 1
Preparation of 5-iert-butyl 1-ethyl 3-amino-4,S-dihydropyrrolo[3,4|-c]pyrazole-1,5-dicarboxylate
A solution of ethyl chlorocartjonate (8.9 ml, 93 mmol) in tetrahydrofuran (THF, 250 ml) was slowly added to a mixture of tert-butyl 3-amino^,6-dihydropyrrolo[3,4-q]pyrazole-5(1H)-carboxylate (20 g, 89 mmol) and diisopropylethylamine (DIEA, 92 ml, 528 mrpo!) in THF (500 ml) at 0-5°C The reaction was kept at: the same temperature for two hoursi then allowed to reach room temperature and stirred overnight. The obtained mixture was evaporated to dryness under vacuum, and the resulting residue extracted with ethyl acetate (AcOEt) and water. The organic layer was separated, dried over sodium sulfate and evaporated to dryness. The mixture was purified by flash-chromatography (eluent ethyl acetate/cycflohexane 4/6 to 7/3) to give 19 g of the title compound as a white solid. [M+H]+ 297
Example 2
Preparation of 5-tert-buty! 1-ethyl 3-{[4-(4-methylpiperazin-1-yl)benzoyl]a|mino}-4,6-dihydropyrrolo[3,4-c]pyrazole-1,5-dicarboxylate
Oxglyl chloride (23.2 ml, 265 mrnol) was added to a suspension of 4-(4-meth.yi-1-piperazinyl)-
i benzole acid (11.7 g, 53 mmol) in dichloromethane (DCM, 320 ml) and dirhethylformamide
(DMF, 0.52 ml). After refluxing the mixture for 6.5 hours, volatiles were carefully removed under reduced pressure (taking up the residue three times with toluene).
The resulting 4-methylpiperazino-benzoyl chloride di-hydrochloride was added portionwise to a solution of 5-tert-butyl 1-ethyl 3-amino-4,6-dihydropyrrolo[3,4-c]pyrazole-" ,5-dicarboxylate (13.1 g, 44.3 rnmol) in dry THF {620 ml) and DIEA (54.4 ml, 0.32 mo!) under stirring at room temperature. The resulting suspension was stirred 16 hours at room tempemture and 1 hour at40°C
After solvent removal under reduced pressure, the residue was taken up with! AcOEt (600 ml) and the organic layer washed with aqueous sodium carbonate (200 ml), brine, (200 ml) and dried over sodium sulfate.
Solvent was evaporated, and the residue was triturated with a mixture of diethyl ether (Et20, 135 ml) and AcOEt (15 ml), filtered, dried under vacuum at 40°C to give 20 g of the title compound as a white powder [M->-H]+ 499.
By operating in an analogous way and by reacting' 5-tert-butyl 1-etryl 3-amino-4,6-dihydropyrrolo[3l4-c]pyrazole-1,5-dicarboxylate with the appropriate acyl ch oride derivative, the following compounds were prepared:
ii
5-tert-butyl 1-ethyl 3~{[4-(4-ethy]piperazin-1-yl)ben2oyl]amino}-4,6-dihydropyrr6lo[3.4-
c]pyrazole~1,5-dicarboxylate; [M+H]+ 513. i
5-tert-butyl 1-ethyl 3-{l4-(4-isopropylpiperazin-1-yl)benzoyl]amino}-4,6-dihydrobyrrolo[3,4-(5)c]pyrazole-1.5-dicarboxylate; [M+Hf 527.
5-tert-butyl 1-ethyl 3-{[4-(4-cyclopropylpiperazin-1-yl)benzoyl]amino}-4,6-dihydropyrrolo[3,4-c]pyrazole-1,5-dicarboxylate; [M+H]+ 525.
5-tert-butyl 1-ethyl 3-{[4-(3,4-dimethylpiperazin-1-yl)benzoyl]amino}-4,6-dihydrppyrro!o[3,4-c]pyrazole-1,5-dicarboxylate; [M+H]+ 513.
5-tert-butyl 1 -ethyl 3-[(4-piperidin-1-ylbenzoyl)amino]-4,6-dihydropyrrolo[3,4-c pyrazole-1,5-dicarboxylate, [M+H]+ 484.
5-tert-butyl 1-ethyl 3-{[4-(4-fluoropiperidin-1-yl)benzoyl]amino]-4,S-dihydropyrr3lo[3,4-c]pyrazole-1,5 dicarboxylate; [M+Hf 502.
5-tert-butyl 1 -ethyl 3-[(4-morpholin-4-ylbenzoyl)arnino]-4,6-dihydropyrrolo[3,4-c]pyrazole-1,5-dicarbox-ylate, [M+H]+ 486.
5-tert-butyl 1-ethyl 3-{[4-(4-tert-butylpiperazin-1-yl)benzoyl]amino}-4,6-dihydropyrrolo[3,4-(5)c]pyrazole-1,5-dicarboxylate; [M+H]+ 541.

of

ethyl

Example 3
3-f[4-(4-methylpiperazin-'l-vl)ben::ovllamino)-5,6-

dihydrDpyrrolof3,4-c]pyrazole-1(4H)-carboxylate trihydrochloride
d dropwise to a
A 4N solution of hydrochloric acid in dioxane (122 ml, 488 mmol) was add stirred solution of 5-tert-butyl 1-ethyl 3-{[4-(4-methylpiperazin-1-yl)benzoyl]amino}-4,6-dihydropyrrolo[3,4-c)pyrazo!e-1,5-dicarboxylate (19.5 g, 39.2 mmol), as prepared in Example 2, in dry DCM (240 ml); precipitation of a white solid occurred almost immediately. The resulting mixture was stirred at room temperature for 24 hours; after dilution with Et20 (100 ml), the solid was filtered, extensively washed with Et20, and dried under vatuum at 50°C to give 20.1 g of the title compound, used in the next step without further purification. [M+Hf 399.
1H-NMR (DMSO--d6) 6 ppm: 1.4 (t, 3H); 2.8 (d, 3H); 3.2 (m. 4H); 3.5 (m, 2H)r 4.1 (m, 2H), 4.4 (q, 2H); 4 6 (m, 4H); 7.1-8.0 (m, 4H); 10.3 (bs, 2H); 10.7 (bs, 1H); 11.4 (s, 1H).
By operating as above reported and by starting from the suitable intermediate, the following compounds were analogously prepared:
Ethy -ethylpiperazin-1-yl)benzoyl]amino}-5,6-dihydropyrrolo[3,4-;]pyrazole-1(4H)-
ethyl 5-[(2R)--?-methyl-2-phenylethanoyl]-3-{[4-(4-ethylpiperazin-1-yl)benzoyr:mino}-5,6-
dihydropyrrolo[3,4-c]pyrazole-1 (4H)-carboxylate; [M+Hf 545.
ethyl 5-[(2R)-2-methyl-2-phenylethanoyl]-3-{[4-(4-isopropylpiperazin-1-yl)ben^oyl]amino}-5,6-
dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxy]ate; [M+H]* 559. |
ethyl 5-[(2R)-2-methyl-2-phenylethanoyl]-3-{[4-(4-cyclopropylpiperazin-1-yl)benzoyl]amino}-
5,6-dihydropyrrolo[3,4-c]pyra2ole-1 (4H)-carboxylate; [M+H]* 557.
ethyl 3-{f4-(3,4-dimethylpiperazin-l-yl)benzoyl]amino}-5-[(2R)-2-methyl-2-phenylethanoy|]-5,6-
dihydropyrrolo[3,4-c]pyrazole-1 (4H)-carboxylate; [M+Hf 545.
ethyl 5-[(2R)-2-methyl-2-phenylethanoyl]-3-[(4-piperidin-1-ylbenzoyl)amino]-5,6-
dihydropyrrolo[3,4-c]pyrazo!e-l(4H)-carboxylate; [M+H]+ 516.
ethyl 3-{[4-(4 fluoropiperidin-1-yl)benzoyl]amino}-5-[(2R)-2-methyl-2-phenyle hanoyl]-5,6-
dihydropyrrolo[3,4-c]pyrazole-1 (4H)-carboxylate; [M+H]+ 534.
ethyl 5-[(2R)-2-methyl-2-phenylethanoyl]-3-[(4-morpholin-4-ylbenzoyl)amino]-5,6-
dihydropyrrulo[3,4-c]pyrazole-1(4H)-carboxylate; [M+H]+ 518.
ethyl 3-{l4-(4-tert-butylpiperazin-1-yl)benzoyl]amino}-5-[(2R)-2-methyl-2-phenylethanoyl]-5,6-
I
dihydropyrro!o[3,4-c]pyrazote-1(4H)-carboxylate; [M+H]+ 573.
ethyl 5-[(2R)-2-hydroxy-2-phenylethanoy]]-3-{[4-(4-methylpiperazin-1-yl)ben^oyl]amino}-5,6-
dihydropyrrolo[3,4-c]pyrazole-1(4H)-carboxylate; [M+H]+ 533.
I
the following compounds were prepared: :
(2) W-{5-[(2R)-2-metho3iy-2-phenyIethanoyl]-1,4.5,6-ietrahydropyrroIo[3,4-c]pyrazol-3-yl}-4-(4-c4hylpipera?:in-1-yl)ben2:arnide; [M-t-H]* 489;
1 H-NMR (DMSO-d6) 5 ppm: 1.1 (t, 3H); 2.3 -2.7 (m, 6H); 3.2 - 3.4 (m, 7H); 4.3-6.0 (m, 4H);
5.1 (d, 1H) 6.9 - 8.0 (m, 9H); 10.6 (bs, 1H); 12.1 (br, 1H). |

(3) N-{5-[(2R)-2-methoxy-2-phenylethanoyl]-1,4,5,6-tetrahydropyrroIo 3-yl}-4-(4-isopropylpiperazin-1-yl)benzamide; [M+H]* 503; 1 H-NMR (DMSO-d6) 5 ppm: 1.3 - 1.3 (dd, 6H); 3.3 - 3.4 (m, 9H); 4.6-4.9 (r 1H); 7.0 - 8.0 (m, 9H); 10.7 (bs, 1H); 12.3 (bs, 1H).

3,4-c]pyrazol-
, 4H);5.1 (d,

(4) N-fS-ffaRJ-a-methoxy^-phenylethanoyll-I^.S.G-tetrahydropyrrololS^-clpyrazoI-3-yI}-4-(4-cyr,lopropylpiperazin-1-yl)benzamide; [M+H]+ 501;
1 H-NMR (DMSO-d6) 5 ppm: 0.3 - 0.5 (m, 4H); 3.2 - 3.4 (m, 7H); 3.2 - 5.0 (m, 4H); 5.1 (d, 1H); 6.8-8.2 (in, 9H); 10.5-10. 7 (br, 1H); 12.0 - 12.4 (br, 1H).

(5) 4-(3,4-dimethylpiperazin-1-yl)-N-{5-[(2R)-2-methoxy-2-phenylethahoyI]-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}benzamide; [M+H]1' 489;
; 4.3-5.0 (m,
1H-NMR (DMSO-d6)6 ppm: 1.0-1.1 (m, 3H); 2.24 (s, 3H); 3.3-3.5 (m, /H 4H);5.1 (d, 1H); 6.9 - 8.0 (m, 9H); 10.6 (bs, 1H); 11.9 - 12.6 (br, 1H).
(6) N^
3-y|}-4-piperidin-1-ylbenzamide; [M+H]+ 460;
1H-NMR (DMSO-d6) 6 ppm: 1.5 - 1.7 (m, 6H); 3.2-3.4 (m, 7H); 4.3-4.9 (m, 4H); 5.1 (d,
1H); 6.9 - 8.0 (m, 9H); 10.4 - 10.7 (br, 1H).
(7) 4-(4-fluoropiperidin-1-y!)-N-{5-[(2R)-2-methoxy-2-phenylethanoy 11-1,4,5,6-tetrahydropyrrolo[3,4-cJpyrazol-3-yl}benzamide; [M-*-H]+ 478;

1H-NMR (DMSO-d6) 6 ppm: 1.65 - 2.1 (m, 4H); 3.15 - 3.6 (m, 7H); 4.35 - 5 (d, 1H); 6.9-8.0 (m, 9H); 10.4 - 10.7 (br, 1H).

0(m,5H); 5.1

(S) W-{5-[(2R)-2-methoxy-2-phenylethanoyI]-1,4,5,6-tetrahydropyrroio[3,4-c]pyrazol-3-yl}-4-morphorin-4-y)benz:amide; [M+H]+ 462;
1H-NMR (DMSO-d6) 5 pprn: 3.15 - 3.5 (m, 7H); 3.7-3.8 (m, 4H); 4.3-4.9 (m, H); 5.1 (d, 1H); 6.9 -8.0(rn, 9H); 10.4 - 10.7 (br, 1H).
(9) 4-(4-tert-butylpiperaz:in-1-yl)-W-{5-f(2R)-2-msthoKy-2-pheny]eihanoyl]-1,4,5,6-tei:rahydrDpyrro)o[3,4-c]pyraEol-3-yl)benzarnide; [M+H]* 517.

i-c]pyrazol-3-
4H);4.1 -4.9 (m,
(10) N-{5-[(2R)-2-hydroxy-2-phenylethanoyl]-1,4,5,6-tetrahydropyrrolo[3, yl}-4-(4-methylpiperazin-1-yl)benzamide; [M+Hf 461; 1 H-NMR (DMSO-d6) 6 ppm: 2.3 (s, 3H); 2.45 - 2.65 (m, 4H); 3.2 - 3.4 (m, 4H); 5.69 (d, 1H); 6.9 - 8.0 (m, 9H); 10.4 - 10.7 (br, 1H); 11.5-12.9 (br, 1H).

(11) N-{5-[{2R)-2-methyl-2-phenylethanoyl]-1,4,5,6-tetrahydropyrrolot|3,4-c]pyrazol-3-yl}-4-(4-rncthylpiperazin-1-y!)benzamide; [M+Hf 459;
1 H-NMR (DMSO-d6) 5 ppm: 1.33 (d, 3H); 2.21 (s, 3H); 3.85 - 5.0 (m, 5H); 4,2 - 4.9 (m, 4H); 5.1 (s, lH)6.8-8.0(m, 9H); 10.3- 10.7 (br, 1H); 11.8 - 12.5 (br, 1H).

,4-c]pyrazol-3-
6(m,4H); 3.2-, 1H).
(12) N-{5-[(2R)-2-methyl-2-phenylethanoyl]-1,4)5,6-tetrahydropyrrolo[:i y|}-4-(4-Bthylpiperazin-1-yl)benzamide; [M+H]+ 473;
1H-NMR (DMSO-d6) 5 ppm: 1.06(t,3H); 1.36(d, 3H); 2.41 (q, 2H); 2.47-2 ' 3.4 (m,4H); 3 9-5.0 (m, 5H); 6.9 - 8.0 (m, 9H); 10.5(bs, 1H); 11.9-
-12.5(bi

(13) N-{5-[(2R)-2-methyl-2-phenylethanoyl]-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazoI-3-
y|}-4-(4~isopropylpiperazin-1-yl)benzamide; [M+H]+ 487; |
I
(14) N-{5-[(2R)-2-methyl-2-phenylethanoyl]-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazoI-3-
yl}~4-(4-cyclopropylpiperazin-1-yl)benz:amide; [M+H]+ 485;
(15) 4-(3,4-dirnethylpiperazin-1-yl)-N-{5-[(2R)-2-phenylpropanoyl]-1,4,£I6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl}benzamide; [M+H]+ 473;

(16) W-|5-[(2R)-2-phenylpropanoyI]-1,4)5)6-tetrahydropyrroIo[3,4-c]py piperidin-1-ylbenzamide; [M+H]+ 444;
1H-NMR (DMSO-d6) 5 ppm: 1.37 (d, 3H); 1.6 (s, 6H); 3.2-3.4 (m, 4H); 3.9C 4.1 -4.9(m, 4H); 6.9-8.0 (m, 9H); 10.4-10.7 (br, 1H).

a2iol-3-yl}-4-
-4.05 (m, 1H);

4-(4-fluQropiperidin-1-yl)-N-{5-[(2R)-2-methy!-2-pheny]ethgnoyl]-1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl}benzarnide; [M+H]4 462;
i
1H-NMR (DMSO-d6) 5 ppm; 1.37 (d, 3H); 1.65 - 2.05 (m, 4H); 3.15 - 3.6 (m; 7H); 3.2 - 3.4 (m. 4H); 3.85 -4,07 (m, 1H); 4.1 - 5.05 (m, 5H); 5.1 (d, 1H); 6.9 - 8.0 (m, 9H); 10.4 - 10.7 (br,
1H).
(18) N-{5-[(2R)-2-methyI-2-phenylethanoyl]-1,4,5,6-tetrahydropyrrolo^,4-c]pyrazol-3-
yI}-4-rnorpholin-4-ylbenzamide; [M+H]+ 446;
1 H-NMR (DMSO-d6) 6 ppm: 1.37(d, 3H); 3.2 - 3.4 (m, 4H); 3.7-3.85 (m, 4fH); 3.9-4.1 (nx 1H); 4.1 -4.95 (m, 4H); 6.9 - 8.0 (m, 9H); 10.4-10.7 (br, 1H).
(19) 4-(4 tert-butylpiperazin-1-yl)-N-{5-[(2R)-2-phenylpropanoy!]-1,4,5;6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl}benzamide; [M+H]+ 501. i

We Claim;
1. A pyrrolo [3,4-c] pyrazole compound of formula (1)
(Formula Removed)
Wherein
R is hydrogen or methyl;
Rl is hydroxy or a straight or branched C1-C3 alkyl or alkoxy oroup;
R2 is a hydrogen or halogen atom;
X is a divalent group selected from methylene (-CH2-) or fluoromethylene
(-CHF-), or it is a heteroatom or heternatornic group selected from oxygen
(-0-) or nitrogen (-NR'-) wherein R' is a hydrogen atom, a straight or
branched C1-C4 alkvl group; or a C3-C6 cycklallyl group; a
pharmaceutically acceptable salt thereof the kind such as herein
described.
2. A compound of formula (I) as claimed in claim 1, wherein R is hydrogen or methyl; Ri is selected from hydroxy or methyl; R2 is a hydrogen; X is selected from methylene, fluoromethylene, -O- or -NR', wherein R' is as defined in claim 1.
3. A compound of formula (I) as claimed in claim 1, optionally in the form of a pharmceutically acceptable salt thereof, selected from the group consisting of:
N-(5-[(2R)-2-methoxy-2-phenylethanoyl]-l, 4, 5,6-tetrahydropyrrole (3,4 -c]pyrazol-3-yl}- 4- (4-isopropylpiperazin-l-yl) benzamide;
N-{5-{(2R)-2-methoxy--2-phenylethanoyl]-l, 4,5,6-tetrahydropyrrolo[3, 4-c]pyrazol-3-yl}- 4-(4-ethylpiperazin-l-yl) benzamide; N-{5-[(2R)-2-methoxy-2-pbenylethanoyl]-l,45,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yI}- 4-(4-isopropylpiperazin-l-yl)benzamide; N-{5-[(2R)-2-methoxy-2-phenylethanoyl)-l, 4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)- 4-(4-methylpiperazin-l-yl) benzamide; 4-(3,4-dimethylpiperazin-1-yl)-N-{5-[(2R)-2-methoxy-2-phenylethanoy] -1,4,5,6- tetrahydropyrrolo[3,4-c]pyrazol-3-yl} benzamide; N-(5-[(2R)-2-methoxy-2-phenylethanoyl]- 1,4, 5,6-tetrahydropyrrolb[3, 4-c]pyrazol-3-yl)- 4-piperidin-1 -yl) benzamide; 4-(4-fluoropiperidin- l-yl)-N-{5-[(2R)-2-methoxy-2-phenylethanoyl]-1,4,5,6-
tetrahydropyrrolo[3,4-c]pyrazol-3-yl} benzamide; and N-{5-[(2R)-2-methoxy-2-phenylethanoyl)-l, 4,5,6-tetrahydropyrrolo[3,4-
c]pyrazoI-3-yl)-4-morpholin-4-ylbenzamide;
4-(4-tert-butytpiperazing)-N-{5-[(2R)-2-methoxy-2-phenyl ethanoyl]-
1,4,5,6-tertanyokopyroolo [3,4-c] pyraz of-3-yl} bemamide.
A pharmaceutical composition comprising a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof, as defined in claim 1, and at least one pharmaceutically acceptable excipient, carrier and/or diluent.
A pharmaceutical composition as claimed in claim 4 further comprising one or more chemotherapeutic agents.
A kit comprising a compound or a pharmaceutically acceptable salt thereof, as defined in claim 1, or pharmaceutical compositions thereof as defined in claim 4, and one or more chemotherapeutic agents.

Documents:

53-DELNP-2006-Abstract-(12-03-2009).pdf

53-DELNP-2006-Abstract-(23-10-2008).pdf

53-delnp-2006-abstract.pdf

53-DELNP-2006-Claims-(12-03-2009).pdf

53-DELNP-2006-Claims-(23-10-2008).pdf

53-delnp-2006-claims.pdf

53-delnp-2006-correspondence-others 1.pdf

53-DELNP-2006-Correspondence-Others-(12-03-2009).pdf

53-DELNP-2006-Correspondence-Others-(23-10-2008).pdf

53-delnp-2006-correspondence-others.pdf

53-DELNP-2006-Description (Complete)-(23-10-2008).pdf

53-delnp-2006-description (complete).pdf

53-DELNP-2006-Form-1-(12-03-2009).pdf

53-DELNP-2006-Form-1-(23-10-2008).pdf

53-delnp-2006-form-1.pdf

53-delnp-2006-form-13-(12-03-2009).pdf

53-delnp-2006-form-18.pdf

53-DELNP-2006-Form-2-(12-03-2009).pdf

53-DELNP-2006-Form-2-(23-10-2008).pdf

53-delnp-2006-form-2.pdf

53-delnp-2006-form-3.pdf

53-delnp-2006-form-5.pdf

53-DELNP-2006-GPA-(23-10-2008).pdf

53-delnp-2006-gpa.pdf

53-delnp-2006-pct-101.pdf

53-delnp-2006-pct-210.pdf

53-delnp-2006-pct-220.pdf

53-delnp-2006-pct-304.pdf

53-delnp-2006-pct-402.pdf

53-delnp-2006-pct-409.pdf

53-delnp-2006-pct-416.pdf

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Patent Number

233092

Indian Patent Application Number

53/DELNP/2006

PG Journal Number

13/2009

Publication Date

27-Mar-2009

Grant Date

26-Mar-2009

Date of Filing

03-Jan-2006

Name of Patentee

PFIZER ITALIA S.R.L

Applicant Address

VIA ROBERT KOCH, 1.2, I-20152 MILAN, ITALY.

Inventors:

#	Inventor's Name	Inventor's Address
1	PAOLA VIANELLO	VIA TREBAZIO 6, I-20145 MILAN, ITALY.
2	DANIELE FANCELLI	VIA MONTECUCCOLI 8, I-20147 MILAN, ITALY.
3	BARBARA FORTE	VIA MORANDI 5, I-20090 BUCCINASCO, ITALY.
4	JURGEN MOLL	LOCALITA SAN BARTOLOMEO, I-22070 APPIANO GENTILE, ITALY.
5	MARIO VARASI	VIA MONCUCCO 24/A, I-20142 MILAN, ITALY.

PCT International Classification Number

C07D 487/04

PCT International Application Number

PCT/EP2004/007515

PCT International Filing date

2004-07-08

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/485,814	2003-07-09	U.S.A.