HK1089769B - Pyrrolo[3,4-c]pyrazole derivatives active as kinase inhibitors - Google Patents

Description

Pyrrolo [3, 4-c ] pyrazole derivatives as kinase inhibitors

Background

Technical Field

The present invention relates to pyrrolopyrazole derivatives, processes for their preparation, pharmaceutical compositions containing them and their use as therapeutic agents, in particular for the treatment of cancer and cell proliferation disorders.

Background

Protein Kinase (PKs) dysfunction is a hallmark of many diseases. Most oncogenes and proto-oncogenes involved in human cancers encode PKs. Enhanced PKs activity has also been associated with a number of non-malignant diseases, such as benign prostate hyperplasia, familial adenomatosis, polyposis, neurofibromatosis, psoriasis, vascular smooth muscle cell proliferation associated with atherosclerosis, pulmonary fibrosis, arthritis, glomerulonephritis, and post-operative stenosis and restenosis.

PKs are also involved in inflammation and in the propagation of viruses and parasites. PKs may also play a major role in the pathogenesis and exacerbation of neurodegenerative disorders.

For a summary reference to PKs dysfunction or disorder see, for example, CurrentOption in Chemical Biology 1999, 3, 459-.

Among the several protein kinases known in the art, those involved in cancer cell growth are Aurora kinases, particularly Aurora-2.

Aurora-2 was found to be overexpressed in a range of different tumor types. The locus of this gene is located at 20q13, and this chromosomal region is frequently amplified in many cancers, including breast Cancer [ Cancer Res.1999, 59(9), 2041-4] and colon Cancer.

20q13 amplification was associated with poor prognosis in patients with non-nodal (node-negative) breast cancer, and increased Aurora-2 expression indicated poor prognosis in bladder cancer patients with decreased survival time [ J.Natl.cancer Inst., 2002, 94(17), 1320-9 ]. A general reference to the role of Aurora-2 in abnormal centrosome function in cancer may also be found in molecular cancer Therapeutics, 2003, 2, 589-.

Summary of The Invention

It is an object of the present invention to provide compounds, more particularly Aurora kinase activity, which are useful in therapy as agents against host diseases caused by and/or associated with dysregulated protein kinase activity.

It is another object of the present invention to provide compounds endowed with protein kinase inhibitory activity, more specifically Aurora kinase inhibitory activity.

The present inventors have now found that certain pyrrolopyrazoles and derivatives thereof are endowed with protein kinase inhibitory activity, for example Aurora kinase inhibitory activity.

More specifically, the compounds of the present invention are useful for the treatment of various cancers, including but not limited to: carcinomas (carcinoma) such as bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer (including small cell lung cancer), esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, prostate cancer, and skin cancer (including squamous cell carcinoma); hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic 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 fibrosarcoma and rhabdomyosarcoma; tumors of the central and peripheral nervous system, including astrocytomas, neuroblastomas, gliomas, and schwannomas; other tumors, including melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratoacanthoma (keratoxanthoma), thyroid follicular cancer, and kaposi's sarcoma.

Since PKs and Aurora kinases have a key role in the regulation of cell proliferation, these pyrrolopyrazoles are also useful in the treatment of various cell proliferation disorders, such as benign prostate hyperplasia, familial adenomatosis, polyposis, neurofibromatosis, psoriasis, vascular smooth muscle cell proliferation associated with atherosclerosis, pulmonary fibrosis, arthritis, glomerulonephritis and post-operative stenosis and restenosis.

Thus, in a first embodiment, the present invention provides a method of treating a cell proliferative disorder caused by and/or associated with altered protein kinase activity, which comprises administering to a mammal in need thereof a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof

Wherein

R is hydrogen or methyl;

R₁is hydroxy or straight or branched C₁-C₃Alkyl or alkoxy;

R₂is a hydrogen or halogen atom;

x is selected from methylene (-CH)₂-) or a fluoromethylene group (-CHF-), or a heteroatom or heteroatom group selected from oxygen (-O-) or nitrogen (-NR '-) wherein R' is a hydrogen atom, a linear or branched C₁-C₄Alkyl or C₃-C₆A cycloalkyl group.

The above methods may treat cell proliferative disorders caused by and/or associated with altered Aurora kinase activity.

In a preferred embodiment of the above method, the cell proliferative disorder is cancer.

Specific types of cancer that may be treated include carcinomas, squamous cell carcinomas, hematopoietic tumors of myeloid and lymphoid lineage, tumors of mesenchymal origin, tumors of the central and peripheral nervous system, melanomas, seminomas, teratocarcinomas, osteosarcomas, xeroderma pigmentosum, keratoacanthomas (keratoxanthomas), thyroid follicular cancer, and kaposi's sarcoma.

The invention also provides a compound of formula (I) or a pharmaceutically acceptable salt thereof

Wherein

R is hydrogen or methyl;

R₁is hydroxy or straight or branched C₁-C₃Alkyl or alkoxy;

R₂is a hydrogen or halogen atom;

x is selected from methylene (-CH)₂-) or a fluoromethylene group (-CHF-), or a heteroatom or heteroatom group selected from oxygen (-O-) or nitrogen (-NR '-) wherein R' is a hydrogen atom, a linear or branched C₁-C₄Alkyl or C₃-C₆A cycloalkyl group.

The invention also includes processes for the synthesis of the pyrrolopyrazoles of formula (I) and their pharmaceutically acceptable salts, and pharmaceutical compositions containing them.

A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description.

Detailed Description

Several heterocyclic compounds are known in the prior art as inhibitors of protein kinases.

Among these, 3-carboxamido-pyrazole and 3-ureido-pyrazole and derivatives thereof as protein kinase inhibitors are disclosed in international patent applications WO 01/12189, WO01/12188, WO 02/48114 and WO 02/70515 filed by the applicant himself.

Fused bicyclic compounds containing a pyrazole moiety and also having kinase inhibitory activity are also disclosed in WO 00/69846, WO 02/12242 and WO03/028720, all filed by the applicant of the same applicant, and in a yet unpublished PCT/EP03/04862 application (claiming priority to U.S. patent application No. 60/381092 filed 5/17/2002).

Further, aminophenyl-piperazine or aminophenyl-piperidine derivatives having inhibitory activity against prenyltransferase proteins are also disclosed in WO 02/30927 filed by Pierre fabric medical.

The compounds of the present invention fall within the general formula of the above-mentioned WO 02/12242, which is incorporated herein by reference, but are not specifically exemplified therein.

The compounds of the present invention have asymmetric carbon atoms and thus may exist as individual optical isomers, as racemic mixtures, or as other mixtures containing a majority of one of the two optical isomers, all of which are within the scope of the present invention.

Likewise, all possible isomers of the compounds of formula (I) of the present invention and mixtures thereof, as well as metabolites and pharmaceutically acceptable biological precursors (otherwise known as prodrugs) of both, are also within the scope of the present invention for use as antitumor agents.

Prodrugs refer to any covalently bonded compounds that release the active parent drug of formula (I) in vivo.

When compounds exist in tautomeric forms, either in equilibrium or predominantly in one of the forms, each form is also within the scope of the invention.

Thus, unless otherwise limited, when only one of the tautomeric forms of the following formula (Ia) or (Ib) is indicated, the other is also included within the scope of the invention:

in this specification, unless otherwise defined, the term straight or branched chain C₁-C₃Or C₁-C₄Alkyl means any of the following groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl and sec-butyl.

The term straight or branched C₁-C₃Alkoxy means any of the groups described below, such as methoxy, ethoxy, n-propoxy and isopropoxy.

The term halogen means fluorine, chlorine, bromine or iodine.

Term C₃-C₆Cycloalkyl means any of the groups mentioned below, for example cyclopropyl, cyclobutyl, cyclopentyl or cyclohexylAnd (4) a base.

It is clear that, depending on the nature of the group X, the same heterocycle linked to the phenylene group in the compound of formula (I) may represent a piperidino, 4-fluoropiperidino, piperazino, 4-alkyl-piperazino, 4-cycloalkyl-piperazino or morpholino ring.

Pharmaceutically acceptable salts of the compounds of formula (I) include acid addition salts with inorganic or organic acids, for example nitric, hydrochloric, hydrobromic, sulfuric, perchloric, phosphoric, acetic, trifluoroacetic, propionic, glycolic, lactic, oxalic, malonic, malic, maleic, tartaric, citric, benzoic, cinnamic, mandelic, methanesulfonic, isethionic and salicylic acid.

A preferred class of compounds of the invention are the derivatives of formula (I) wherein R is hydrogen or methyl; r₁Selected from hydroxy, methyl or methoxy; r₂Is a hydrogen or fluorine atom; x is selected from methylene, fluoromethylene, -O-or-NR ', wherein R' is as defined above.

Any particular compound of formula (I) or optionally a pharmaceutically acceptable salt form thereof, according to the invention is as described in the experimental section and claims.

As mentioned before, another object of the present invention is a process for the preparation of compounds of formula (I) and pharmaceutically acceptable salts thereof, said process comprising:

a) reacting a compound of formula (II) with a compound of formula (III)

Wherein R and X are as defined above, Q is lower alkyl, t-Bu represents tert-butyl and Z is hydroxy or a suitable leaving group, to give a compound of formula (IV)

b) Reacting a compound of formula (IV) under acidic conditions to obtain a compound of formula (V)

c) Reacting a compound of formula (V) with a compound of formula (VI)

Wherein R is₁And R₂As defined above, and Z' represents hydroxy or a suitable leaving group, to give a compound of formula (VII)

d) Reacting a compound of formula (VII) under basic conditions to give the corresponding compound of formula (I), if desired, converted into a pharmaceutically acceptable salt thereof.

The above process is similar and can be carried out under very common operating conditions.

The reaction between the compound of formula (II) and the compound of formula (III) according to step a) in the above process may be carried out in various conventional ways for acylating amino derivatives. For example, a compound of formula (II) may be reacted with an acid chloride compound of formula (III) wherein Z represents a suitable leaving group, such as a chlorine atom.

Preferably, the reaction is carried out at a temperature of from room temperature to about 60 deg.C in a suitable solvent such as tetrahydrofuran or dichloromethane in the presence of a proton scavenger such as triethylamine or diisopropylethylamineThe process was carried out as follows. In the compounds of formula (II), Q represents lower alkyl, e.g. C₁-C₄Alkyl, more preferably methyl or ethyl.

Deprotection at the pyrrolidine nitrogen atom is facilitated by treatment of the compound of formula (IV) with an acid according to step (b) in the above process.

The above reaction may be conveniently carried out in the presence of an inorganic or organic acid such as hydrochloric acid, trifluoroacetic acid or methanesulfonic acid in a suitable solvent such as dichloromethane, 1, 4-dioxane, a lower alcohol (e.g. methanol or ethanol) at a temperature of from room temperature to about 40 ℃ for from about 1 hour to about 48 hours.

The resulting compound of formula (V) is further reacted with a compound of formula (VI) according to step (c) of the above process. It is thus obvious to the person skilled in the art that the acylation reaction can also be carried out according to various methods and operating conditions for the preparation of carboxamides which are well known in the art.

The reaction between a compound of formula (V) and a carboxylic acid of formula (VI) wherein Z' is hydroxy may be carried out in a coupling reagent such as a carbodiimide, namely 1, 3-dicyclohexylcarbodiimide, 1, 3-diisopropylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, N-cyclohexylcarbodiimide-N '-propoxymethylpolystyrene or N-cyclohexylcarbodiimide-N' -methylpolystyrene, in a suitable solvent such as dichloromethane, chloroform, tetrahydrofuran, diethyl ether, 1, 4-dioxane, acetonitrile, toluene or N, N-dimethylformamide, at a temperature of about-10 c to reflux for a suitable time, i.e., about 30 minutes to 96 hours. The reaction is optionally carried out in the presence of a suitable catalyst, such as 4-dimethylaminopyridine, or in the presence of an additional coupling reagent, such as N-hydroxybenzotriazole.

The reaction between the compound of formula (V) and the compound of formula (VI) can also be carried out, for example, by the mixed anhydride method by using an alkyl chloroformate such as ethyl, isobutyl or isopropyl chloroformate in the presence of a tertiary base such as triethylamine, N-diisopropylethylamine or pyridine in a suitable solvent such as toluene, dichloromethane, chloroform, tetrahydrofuran, acetonitrile, diethyl ether, 1, 4-dioxane or N, N-dimethylformamide at a temperature of about-30 ℃ to room temperature.

The reaction between the compound of formula (V) and the carboxylic acid derivative of formula (VI) wherein Z' is a suitable leaving group may be carried out in the presence of a tertiary base such as triethylamine, N-diisopropylethylamine or pyridine in a suitable solvent such as toluene, dichloromethane, chloroform, diethyl ether, tetrahydrofuran, acetonitrile or N, N-dimethylformamide at a temperature of about-10 ℃ to reflux.

The compounds of formula (VI) are characterized by the presence of R₁The attached asymmetric carbon atoms, marked with asterisks

Thus, the compounds of formula (VI) may be in the form of either a single enantiomer or a mixture of individual enantiomers, i.e. a racemic mixture.

Obviously, depending on the nature of the compound of formula (VI) used in the process of the invention, it is possible to obtain therefrom the corresponding compound of formula (VII) having a suitably defined stereochemistry at this same carbon atom.

According to a preferred embodiment of the invention, step (c) is accomplished by reacting a suitable compound of formula (VI) in the form of the designated enantiomer.

Likewise, if a racemic mixture of the compound of formula (VI) is used, or when an optically pure final compound of formula (I) is desired, it is necessary to perform optical resolution of the intermediate of the compound of formula (VII) or the final compound of formula (I) according to conventional methods. For example, conventional racemate resolution techniques include, for example, fractional crystallization of diastereomeric salt derivatives or preparative chiral HPLC.

Finally, the compound of formula (VII) is deprotected on the pyrazole nitrogen atom according to step (d) of the above process by conventional methods, thus ensuring, for example, selective hydrolysis of the carbamate group.

For example, the above reaction can be carried out under basic conditions, for example, in the presence of sodium hydroxide, potassium hydroxide or lithium hydroxide, or a tertiary amine such as triethylamine, in a suitable solvent such as N, N-dimethylformamide, methanol, ethanol, tetrahydrofuran, water, and mixtures thereof. Typically, the reaction is carried out at a temperature of from room temperature to about 60 ℃ for a period of from about 30 minutes to about 96 hours.

Finally, the pharmaceutically acceptable salts of the compounds of formula (I), or the free compounds of their salts, may be prepared according to conventional methods.

The starting materials for the process of the present invention are known or can be conveniently prepared by known methods.

The preparation of the compound of formula (II) wherein Q represents ethyl is disclosed, for example, in the aforementioned international patent application WO 02/12242 (see in particular page 249, example 26; this compound is referred to therein as 5-tert-butyl 1-ethyl 3-amino-4, 6-dihydro-pyrrolo [3, 4-c ] pyrazole-1, 5-dicarboxylate).

In a similar manner, other compounds of formula (II) wherein Q represents lower alkyl rather than ethyl may also be prepared therefrom.

The compounds of the formulae (III) and (VI), for example, in which Z and Z' represent halogen atoms such as chlorine atoms, are known or can be conveniently prepared from the corresponding known carboxylic acids by conventional methods.

It is also obvious to the person skilled in the art that if the compound of formula (I) is obtained in the form of a mixture of isomers according to any of the variants corresponding to the above process, it is also within the scope of the present invention to separate it into the single isomers of formula (I) according to conventional techniques.

Pharmacology of

The compounds of formula (I) are useful as protein kinase inhibitors, particularly Aurora kinase inhibitors, and thus are useful in limiting uncontrolled proliferation of tumor cells.

In therapy, they are useful in the treatment of a variety of tumors, such as those of the tumor types previously indicated, and in addition they are useful in the treatment of other cell proliferative disorders, such as psoriasis, vascular smooth muscle cell proliferation associated with atherosclerosis, and post-operative stenosis and restenosis.

The inhibitory activity and potency of selected compounds were measured by an assay based on the use of SPA technology (Amersham Pharmacia Biotech).

The assay involves the migration of a radioactively labelled phosphate moiety to a biotinylated substrate by a kinase. The resulting 33P-labeled biotinylation product was bound to streptavidin (streptavidin) -coated SPA beads (biotin capacity 130pmol/mg) and the emitted light was measured with a scintillation counter.

Aurora-2 activity inhibition assay

Kinase activity: will be 30. mu.l buffer (HEPES 50mM pH 7.0, MgCl) in a final volume₂8 μ M biotinylated peptide (4 LRRWSSG repeats), 10 μ M ATP (0.5 μ CiP) in 10mM, 1mM DTT, 0.2mg/ml BSA, 3 μ M orthovanadate)³³γ -ATP), 7.5ng Aurora 2, inhibitor were added to each well on a 96U bottom well plate. After incubation at room temperature for 60 minutes, the reaction was stopped by adding 100. mu.l of strain suspension, capturing the biotinylated peptide.

Layering: mu.l of CsCl₂5M was added to each well and radioactivity was counted in a Top-Count device after 4 hours of standing.

IC₅₀Measurement: different concentrations of inhibitor at 0.0015-10. mu.M were tested. By application ofThe computer program GraphPad Prizm of the four parameter logistic equation analyses the experimental data:

y-bottom + (Top-bottom)/(1 +10^ ((logIC50-x) slope))

Wherein x is the logarithm of the inhibitor concentration and y is the response value; y starts at the bottom and reaches the top in an S-shape.

Ki calculation:

the experimental method comprises the following steps: the reaction was carried out in a buffer (10mM Tris, pH 7.5, 10mM MgCl) containing 3.7nM enzyme, histone and ATP (constant ratio of cold/labeled ATP 1/3000)₂0.2mg/ml BSA, 7.5mM DTT). The reaction was stopped with EDTA and the substrate was captured on a phosphomembrane (Multiscreen 96 well plate under the trade name Millipore). After thorough washing, multiscreen plates were read on a top counter. Controls for each ATP and histone concentration were measured (time zero).

Experiment design: the reaction rates were measured at four ATP, substrate (histone) and inhibitor concentrations. At corresponding ATP and substrate Km values, and inhibitor IC₅₀Values (0.3, 1, 3, 9 times Km or IC)₅₀Values) are set around 80 points of the density matrix. Preliminary time course experiments were performed in the absence of inhibitor and at different ATP and substrate concentrations, whereby a single endpoint time (10 min) within the linear range of the reaction was selected for the Ki determination experiments.

And (3) evaluating kinetic parameters: using all data (80 points), kinetic parameters were estimated by simultaneous nonlinear least squares regression according to [ eq.1] (competitive inhibitor to ATP, random mechanism):

where a ═ ATP, { B ═ substrate }, I ═ inhibitor }, Vm ═ maximum rate, and Ka, Kb, Ki are the ionization constants of ATP, substrate, and inhibitor, respectively. Alpha and beta are synergistic factors of the binding force between the substrate and ATP and the binding force between the substrate and the inhibitor respectively.

The compounds of the invention were further tested in vitro to evaluate their antiproliferative activity in cell culture and their inhibitory effect on the cell cycle.

In vitro cell proliferation assay

Human colon cancer cell line HCT-116 was seeded at 5000 cells/cm²Using F12 medium (Gibco) supplemented with 10% FCS (EuroClone, Italy), 2 mML-glutamine and 1% penicillin/streptomycin, and then maintaining the plates at 37 ℃ and 5% CO₂And 96% relative humidity. The following day, the well plates were treated in duplicate with 5 μ l of appropriately diluted compound from a 10mm mdsso stock. Two untreated control wells were included in each plate. After 72 hours of treatment, the medium was removed and cells were isolated from each well using 0.5ml of 0.05% (w/v) insulin, 0.02% (w/v) EDTA (Gibco). The samples were diluted with 9.5ml of Isoton (Coulter) and counted in a Multisizer 3 cell counter (Beckman Coulter). Data are expressed as a percentage of control wells: CTR% (treatment-blank)/(control-blank).

IC was calculated by LSW/data analysis using the Microsoft Excel sigmoidal curve fitting method₅₀The value is obtained.

In vitro cell cycle analysis

As described above, the human colon cancer cell line HCT-116 was seeded at 5000 cells/cm²And cultured in 24-well plates (Costar). Cells were treated with different concentrations of compounds for 24 hours in their exponential growth phase. In addition, the supernatant in the culture medium is collected to avoid loss of isolated apoptotic or mitotic cells. Thereafter, the cells were washed with PBS and separated by 0.05% (w/v) insulin, 0.02% (w/v) EDTA (Gibco). The insulin activity is terminated using culture medium. The adherent and non-adherent cell fractions were collected and centrifuged at 2000rpm for 10 minutes. Resuspending the cells in PBS and using a Multisizer 3 cytometer (Beckman Coulter) count. For fixation, ethanol (70%, v/v) was added and the cells were left overnight at-20 ℃.

One million of the fixed cells were centrifuged at 2000rpm for 5 minutes, washed with PBS, and then 200 μ l: 25 μ g/ml propidium iodide (Sigma) and 15 μ g/ml RNAse A (Sigma), 0.001% (v/v) Nonidet P40(Sigma) in sodium citrate (0.1% w/v, pH 7.5) for 1 hour. Samples were analyzed by flow cytometry (FACSCalibur, Beckton Dickinson) at 488nm using Cell Quest 3.0 software (Beckton Dickinson). Typically 10000 times were collected (double differentiation module DDM activated while only single cells were gated) and cell cycle curves were recorded with CellQuest (Verity software). Population cell cycle distribution was calculated using a modified model in Modfit 3.1 software (Verity software) expressed as% G0/G1, S, G2/M and polyploidy.

According to the above test, it was found that the compound of formula (I) of the present invention has a significant protein kinase inhibitory activity, such as Aurora-2 inhibitory activity. For example, Table I below reports experimental data (IC) measured as Aurora-2 kinase inhibitors for four representative compounds of the invention₅₀nM) and their anti-cell-proliferation activity (IC)₅₀nM), and its ability to block the cell cycle and induce polyploidy (% polyploidy at G2/M +200 nM).

Interestingly, the above-mentioned compounds were tested against prior art compounds which closely approximate their structure (hereinafter referred to as reference compounds), which are specifically disclosed in the aforementioned WO 02/12242 (see page 160, lines 5-7 thereof); the reference compound was named N- { 5-phenylacetyl-4, 6-dihydropyrrolo [3, 4-c ] pyrazol-3-yl } -4- (4-methylpiperazino) benzamide therein.

Reference compound (R ═ R)₁＝R₂＝H；X＝NMe)

N- { 5-phenylacetyl-4, 6-dihydropyrrolo [3, 4-c ] pyrazol-3-yl } -4- (4-methylpiperazino) benzamide;

compound (1) (R ═ R)₂＝H；R₁＝OMe；X＝NMe)

N- {5- [ (2R) -2-methoxy-2-phenylacetyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } -4- (4-methylpiperazin-1-yl) benzamide;

compound (11) (R ═ R₂＝H；R₁＝Me；X＝NMe)

N- {5- [ (2R) -2-methyl-2-phenylacetyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } -4- (4-methylpiperazin-1-yl) benzamide;

compound (6) (R ═ R₂＝H；R₁＝OMe；X＝CH₂)

N- {5- [ (2R) -2-methoxy-2-phenylacetyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } -4-piperidin-1-ylbenzamide;

compound (5) (R ═ Me; R₁＝OMe；R₂＝H；X＝NMe)

4- (3, 4-dimethylpiperazin-1-yl) -N- {5- [ (2R) -2-methoxy-2-phenylacetyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } benzamide.

TABLE I

Compound (I)	Aurora-2 inhibitory IC(nM)	Anti-cell proliferation IC(nM)	Cell cycle blockade (% ploidy at G2/M +200 nM)
				Reference compound	140	220	33
Is free of	---	---	19
				(1)	16	31	90
(11)	63	17	90
				(6)	37	30	90
(5)	18	50	90

It has been surprisingly found that the compounds of the present invention have Aurora-2 inhibitory activity which is consistently significantly superior to that of the reference compound.

It has also been found that the above compounds have both an anti-cell proliferation effect and the ability to block the cell cycle and induce polyploidy, and are all significantly superior to the reference compounds tested under the same conditions.

It follows that, as a whole, the novel compounds of formula (I) of the present invention appear to be endowed with an unexpected biological profile superior to that of WO 02/12242, which is closest to the prior art, and are therefore particularly useful in the treatment of proliferative disorders associated with dysregulated Aurora-2 kinase activity.

The compounds of the present invention may be administered as single agents, or in combination with known anti-cancer treatments such as radiation therapy or chemotherapy regimens, in combination with: agents that inhibit cell growth or cytotoxic agents, antibiotic-type agents, alkylating agents, antimetabolite agents, hormonal agents, immunological agents, interferon-type agents, cyclooxygenase inhibitors (e.g., COX-2 inhibitors), metallomatrix protease inhibitors, telomerase inhibitors, tyrosine kinase inhibitors, anti-growth factor receptor agents, anti-HER agents, anti-EGFR agents, anti-angiogenic agents (e.g., angiogenesis inhibitors), 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.

If formulated in fixed doses, such combination products may employ the compounds of the present invention in the dosage ranges described below, as well as other pharmaceutically active agents in acceptable dosage ranges.

If the combined preparation is not suitable, the compounds of formula (I) may be used sequentially with known anti-cancer agents.

The compounds of formula (I) of the present invention, which are suitable for administration to mammals such as humans, can be administered by conventional routes and dosage levels, depending on the age, weight, health and route of administration of the patient.

For example, a suitable dose for oral administration of a compound of formula (I) may be from about 10 to about 500mg per dose, 1-5 times per day. The compounds of the invention may be administered in various dosage forms, for example orally in the form of tablets, capsules, sugar-or film-coated tablets, liquid solutions or suspensions; rectally in the form of suppositories; parenteral administration, for example intramuscular administration, or administration by intravenous and/or intrathecal and/or intraspinal injection or infusion.

The invention also includes pharmaceutical compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, which may be a carrier or diluent.

Pharmaceutical compositions containing the compounds of the present invention are generally prepared according to conventional methods and then administered in the form of suitable medicaments.

For example, solid oral formulations may contain the active compound in combination with a diluent such as lactose, dextrose, sucrose (saccharose), sucrose (sucrose), cellulose, corn starch, or potato starch; lubricants, such as silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycol; binding agents, such as starch, gum arabic, gelatin, methyl cellulose, carboxymethyl cellulose or polyvinyl pyrrolidone; disintegrating agents, such as starch, alginic acid, alginates or sodium starch glycolate; an effervescent mixture; a dye; a sweetener; wetting agents such as lecithin, polysorbate, lauryl sulfate; and non-toxic and pharmacologically active substances commonly used in pharmaceutical preparations. These pharmaceutical preparations can be prepared in a known manner, for example by mixing, granulating, tabletting, sugar-coating or film-coating processes.

Liquid dispersions for oral administration may be, for example, syrups, emulsions and suspensions.

For example, syrups may contain as carrier sucrose or sucrose together with glycerol and/or mannitol and sorbitol.

Suspensions and emulsions may contain as carrier, for example, a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol.

Suspensions or solutions for intramuscular injections may contain the active compound together with a pharmaceutically acceptable carrier, for example sterile water, olive oil, ethyl oleate, glycols such as propylene glycol, and, if desired, a suitable amount of lidocaine hydrochloride.

Solutions for intravenous injection or infusion may contain, for example, sterile water as the carrier, or preferably, they may be in the form of sterile aqueous isotonic saline solutions, or propylene glycol as the carrier.

Suppositories may also contain the active compound, a pharmaceutically acceptable carrier such as cocoa butter, polyethylene glycol, polyoxyethylene sorbitan fatty acid ester surfactant or lecithin.

The following examples are intended to better illustrate the invention without, however, constituting any limitation thereto.

Examples

General procedure

Flash chromatography was performed on silica gel (Merck grade 9385, 60A). HPLC/MS was performed on a Waters X Terra RP 18 (4.6X 50mm, 3.5 μm) column using a Waters 2790HPLC system equipped with a 996 Waters PDA detector and Micromass mod. The ZQ single quadruple mass spectrometer was equipped with an Electrospray (ESI) ion source. Mobile phase A was ammonium acetate 5mM buffer (pH 5.5, acetic acid/acetonitrile 95: 5) and mobile phase B was H₂O/acetonitrile (5: 95). The gradient was from 10% to 90% B over 8 min, holding 90% B for 2 min. UV detection was performed at 220nm and 254 nm. Flow rate 1 ml/min. The injection volume was 10. mu.l. The mass range is 100-. Capillary voltage of2.5 KV; the source temperature is 120 ℃; the arc (Cone) was 10V. Retention times at 220nm or 254nm (hplcr.t.) are given in minutes. The mass is given in m/z ratio.¹H-NMR spectroscopy was performed on Mercury VX 400 equipped with a 5mm dual resonance probe (1H {15N-31P } ID _ PFG Varian), operating at 400.45 MHz.

Example 1

Preparation of 5-tert-butyl-1-ethyl-3-amino-4, 6-dihydropyrrolo [3, 4-c ] pyrazole-1, 5-dicarboxylate

A solution of ethyl chlorocarbonate (8.9ml, 93mmol) in tetrahydrofuran (THF, 250ml) was slowly added to 3-amino-4, 6-dihydropyrrolo [3, 4-c ] at 0-5 deg.C]Pyrazole-5 (1H) -carboxylic acid tert-butyl ester (20g, 89mmol) and diisopropylethylamine (DIEA, 92ml, 528mmol) in a mixture of THF (500 ml). The reaction was maintained at the same temperature for two hours, then allowed to stand to room temperature and stirred overnight. The resulting mixture was evaporated to dryness in vacuo and the resulting residue was 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 (elution: ethyl acetate/cyclohexane 4/6 to 7/3) to yield 19g of the title compound as a white solid. [ M + H ]]⁺ 297。

Example 2

Preparation of 5-tert-butyl 1-ethyl 3- { [4- (4-methylpiperazin-1-yl) benzoyl ] amino } -4, 6-dihydropyrrolo [3, 4-c ] pyrazole-1, 5-dicarboxylate

Oxalyl chloride (23.2ml, 265mmol) was added to a suspension of 4- (4-methyl-1-piperazinyl) -benzoic acid (11.7g, 53mmol) in dichloromethane (DCM, 320ml) and dimethylformamide (DMF, 0.52 ml). After 6.5 hours of reflux of the mixture, the volatiles were carefully removed under reduced pressure (the residue was taken up three times with toluene).

The resulting 4-methylpiperazino-benzoyl chloride dihydrochloride was added portionwise to a solution of 5-tert-butyl 1-ethyl 3-amino-4, 6-dihydropyrrolo [3, 4-c ] pyrazole-1, 5-dicarboxylate (13.1g, 44.3mmol) in anhydrous THF (620ml) and DIEA (54.4ml, 0.32mol) at room temperature with stirring. The resulting suspension was stirred at room temperature for 16 hours and at 40 ℃ for 1 hour.

After removal of the solvent under reduced pressure, the residue was taken up in AcOEt (600ml), and the organic layer was washed with aqueous sodium carbonate (200ml), brine (200ml) and dried over sodium sulfate.

The solvent was removed by evaporation and the residue was taken up in diethyl ether (Et)₂O, 135ml) and AcOEt (15ml), filtered and dried under vacuum at 40 ℃ to give 20g of the title compound as a white powder. [ M + H ]]⁺ 499。

By operating in a similar manner, the following compound was prepared by reacting 5-tert-butyl 1-ethyl 3-amino-4, 6 dihydropyrrolo [3, 4-c ] pyrazole-1, 5-dicarboxylate with the appropriate acid chloride derivative:

5-tert-butyl 1-ethyl 3- { [4- (4-ethylpiperazin-1-yl) benzoyl]Amino) -4, 6-dihydropyrrolo [3, 4-c]Pyrazole-1, 5-dicarboxylic acid esters; [ M + H ]]⁺ 513。

5-tert-butyl 1-ethyl 3- { [4- (4-isopropylpiperazin-1-yl) benzoyl]Amino } -4, 6-dihydropyrrolo [3, 4- (5) c]Pyrazole-1, 5-dicarboxylic acid esters; [ M + H ]]⁺ 527。

5-tert-butyl 1-ethyl 3- { [4- (4-cyclopropylpiperazin-1-yl) benzoyl]Amino } -4, 6-dihydropyrrolo [3, 4-c)]Pyrazole-1, 5-dicarboxylic acid esters; [ M + H ]]⁺ 525。

5-tert-butyl 1-ethyl 3- { [4- (3, 4-dimethylpiperazin-1-yl) benzoyl]Amino } -4, 6-dihydropyrrolo [3, 4-c)]Pyrazole-1, 5-dicarboxylic acid esters; [ M + H ]]⁺ 513。

5-tert-butyl-1-ethyl-3- [ (4-piperidin-1-ylbenzoyl) amino]-4, 6-dihydropyrrolo [3, 4-c]Pyrazole-1, 5-dicarboxylic acid esters; [ M + H ]]⁺ 484。

5-tert-butyl 1-ethyl 3- { [4- (4-fluoropiperidin-1-yl) benzoyl]Amino } -4, 6-dihydropyrrolo [3, 4-c)]The compound of pyrazole-1 and the compound of pyrazole-1,5-dicarboxylic acid esters; [ M + H ]]⁺ 502。

5-tert-butyl-1-ethyl-3- [ (4-morpholin-4-ylbenzoyl) amino]-4, 6-dihydropyrrolo [3, 4-c]Pyrazole-1, 5-dicarboxylic acid esters; [ 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-dicarboxylic acid esters; [ M + H ]]⁺ 541。

Example 3

Preparation of ethyl 3- { [4- (4-methylpiperazin-1-yl) benzoyl ] amino } -5, 6-dihydropyrrolo [3, 4-c ] pyrazole-1 (4H) -carboxylate trihydrochloride

A4N solution of hydrochloric acid in dioxane (122ml, 488mmol) was added portionwise to a stirred 5-tert-butyl 1-ethyl 3- { [4- (4-methylpiperazin-1-yl) benzoyl group]Amino } -4, 6-dihydropyrrolo [3, 4-c)]Pyrazole-1, 5-dicarboxylate (19.5g, 39.2mmol) (prepared as in example 2) in anhydrous DCM (240 ml); almost immediately, a white solid precipitated. The resulting mixture was stirred at room temperature for 24 hours; with Et₂After dilution with O (100ml), the solid was filtered and Et₂O is washed thoroughly and dried under vacuum at 50 ℃ to give 20.1g of the title compound, which is used without further purification in the next step. [ M + H ]]⁺ 399。

¹H-NMR(DMSO-d₆)δppm：1.4(t，3H)；2.8(d，3H)；3.2(m，4H)；3.5(m，2H)；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)。

The following compounds are analogously prepared starting from the appropriate intermediates, following the procedure described above:

3- { [4- (4-ethylpiperazin-1-yl) benzoyl]Amino } -5, 6-dihydropyrrolo [3, 4-c)]Pyrazole-1 (4H) -carboxylic acid ethyl ester trihydrochloride; [ M + H ]]⁺ 413。

3- { [4- (4-isopropylpiperazin-1-yl) benzoylBase of]Amino } -5, 6-dihydropyrrolo [3, 4-c)]Pyrazole-1 (4H) carboxylic acid ethyl ester trihydrochloride; [ M + H ]]⁺ 427。

3- { [4- (4-Cyclopropylpiperazin-1-yl) benzoyl]Amino) -5, 6-dihydropyrrolo [3, 4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester trihydrochloride; [ M + H ]]⁺ 425。

3- { [4- (3, 4-dimethylpiperazin-1-yl) benzoyl]Amino) -5, 6-dihydropyrrolo [3, 4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester trihydrochloride; [ M + H ]]⁺ 413。

3- [ (4-piperidin-1-ylbenzoyl) amino group]-5, 6-dihydropyrrolo [3, 4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester; [ M + H ]]⁺ 384。

3- { [4- (4-Fluoropiperidin-1-yl) benzoyl]Amino } -5, 6-dihydropyrrolo [3, 4-c)]Pyrazole-1 (4H) carboxylic acid ethyl ester trihydrochloride; [ M + H ]]⁺ 402。

3- { [4- (4-Morpholin-4-yl) benzoyl]Amino } -5, 6-dihydropyrrolo [3, 4-c)]Pyrazole-1 (4H) carboxylic acid ethyl ester trihydrochloride; [ M + H ]]⁺ 386。

3- { [4- (4-tert-butylpiperazin-1-yl) benzoyl]Amino } -5, 6-dihydropyrrolo [3, 4-c)]Pyrazole-1 (4H) -carboxylic acid ethyl ester trihydrochloride; [ M + H ]]⁺ 441。

Example 4

Preparation of ethyl 5- [ (2R) -2-methoxy-2-phenylacetyl ] -3- { [4- (4-methylpiperazin-1-yl) benzoyl ] amino } -5, 6-dihydropyrrolo [3, 4-c ] pyrazole-1 (4H) -carboxylate

Oxalyl chloride (101.2ml, 115mmol) was added to a solution of R- (-) - α -methoxyphenylacetic acid (1.91g, 11.5mmol) in DCM (90ml) and DMF (0.50 ml). After the mixture was stirred at room temperature for 16 hours, the volatiles were carefully removed under reduced pressure.

A solution of the resulting R- (-) - α -methoxyphenylacetyl chloride in DCM (20ml) was added dropwise with stirring to a solution of 3- { [4- (4-methylpiperazin-1-yl) benzoyl ] amino } -5, 6-dihydropyrrolo [3, 4-c ] pyrazole-1 (4H) -carboxylic acid ethyl ester trihydrochloride (4.5g, 8.9mmol) in DCM (400ml) and DIEA (11.8ml, 69mmol) at room temperature. The resulting solution was stirred at room temperature for 20 hours.

The reaction mixture was then washed with aqueous sodium carbonate (200mD, brine (200ml), dried over sodium sulfate, the solvent was removed by evaporation and the residue was Et₂A mixture of O (100ml) and AcOEt (10ml) was triturated, filtered and dried under vacuum at 40 ℃ to give 3.94g of the title compound as a white powder which was used without further purification in the next step. [ M + H ]]⁺ 547；

¹H-NMR(DMSO-d₆)δppm：1.3(t，3H)；2.3(d，3H)；2.6(m，4H)；3.3-3.4(m，7H)；4.3(q，2H)；4.6-4.9(m，4H)；5.1(d，1H)7.0-8.0(m，9H)；11.1(d，1H)。

The following compounds are analogously prepared starting from the appropriate intermediates, following the procedure described above:

5- [ (2R) -2-methoxy-2-phenylacetyl]-3- { [4- (4-ethylpiperazin-1-yl) benzoyl]Amino } -5, 6-dihydropyrrolo [3, 4-c)]Pyrazole-1 (4H) -carboxylic acid ethyl ester; [ M + H ]]⁺ 561。

5- { (2R) -2-methoxy-2-phenylacetyl]-3- { [4- (4-isopropylpiperazin-1-yl) benzoyl]Amino } -5, 6-dihydropyrrolo [3, 4-c)]Pyrazole-1 (4H) -carboxylic acid ethyl ester; [ M + H ]]⁺ 575。

5- [ (2R) -2-methoxy-2-phenylacetyl]-3- { [4- (4-cyclopropylpiperazin-1-yl) benzoyl]Amino } -5, 6-dihydropyrrolo [3, 4-c)]Pyrazole-1 (4H) -carboxylic acid ethyl ester; [ M + H ]]⁺ 573。

3- { [4- (3, 4-dimethylpiperazin-1-yl) benzoyl]Amino } -5- [ (2R) -2-methoxy-2-phenylacetyl]-5, 6-dihydropyrrolo [3, 4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester; [ M + H ]]⁺561。

5- [ (2R) -2-methoxy-2-phenylacetyl]-3- [ (4-piperidin-1-ylbenzoyl) amino group]-5, 6-dihydropyrrolo[3，4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester; [ M + H ]]⁺ 532。

3- { [4- (4-Fluoropiperidin-1-yl) benzoyl]Amino } -5- [ (2R) -2-methoxy-2-phenylacetyl]-5, 6-dihydropyrrolo [3, 4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester; [ M + H ]]⁺ 550。

5- [ (2R) -2-methoxy-2-phenylacetyl]-3- [ (4-morpholin-4-ylbenzoyl) amino]-5, 6-dihydropyrrolo [3, 4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester; [ M + H ]]⁺ 534。

3- { [4- (4-tert-butylpiperazin-1-yl) benzoyl]Amino) -5- [ (2R) -2-methoxy-2-phenylacetyl]-5, 6-dihydropyrrolo [3, 4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester; [ M + H ]]⁺ 589。

5- [ (2R) -2-methyl-2-phenylacetyl]-3- { [4- (4-ethylpiperazin-1-yl) benzoyl]Amino } -5, 6-dihydropyrrolo [3, 4-c)]Pyrazole-1 (4H) -carboxylic acid ethyl ester; [ M + H ]]⁺ 545。

5- [ (2R) -2-methyl-2-phenylacetyl]-3- { [4- (4-isopropylpiperazin-1-yl) benzoyl]Amino } -5, 6-dihydropyrrolo [3, 4-c)]Pyrazole-1 (4H) -carboxylic acid ethyl ester; [ M + H ]]⁺ 559。

5- [ (2R) -2-methyl-2-phenylacetyl]-3- { [4- (4-cyclopropylpiperazin-1-yl) benzoyl]Amino } -5, 6-dihydropyrrolo [3, 4-c)]Pyrazole-1 (4H) -carboxylic acid ethyl ester; [ M + H ]]⁺ 557。

3- { [4- (3, 4-dimethylpiperazin-1-yl) benzoyl]Amino } -5- [ (2R) -2-methyl-2-phenylacetyl]-5, 6-dihydropyrrolo [3, 4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester; [ M + H ]]⁺ 545。

5- [ (2R) -2-methyl-2-phenylacetyl]-3- [ (4-piperazin-1-ylbenzoyl) amino group]-5, 6-dihydropyrrolo [3, 4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester; [ M + H ]]⁺ 516。

3- { [4- (4-Fluoropiperazin-1-yl) benzoyl]Amino } -5- [ (2R) -2-methyl-2-phenylacetyl]-5, 6-dihydropyrrolo [3, 4-c]Pyrazole-1 (4H) -carboxylic acidsEthyl ester of acid; [ M + H ]]⁺ 534。

5- [ (2R) -2-methyl-2-phenylacetyl]-3- [ (4-morpholin-4-ylbenzoyl) amino]-5, 6-dihydropyrrolo [3, 4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester; [ M + H ]]⁺ 518。

3- { [4- (4-tert-butylpiperazin-1-yl) benzoyl]Amino } -5- [ (2R) -2-methyl-2-phenylacetyl]-5, 6-dihydropyrrolo [3, 4-c]Pyrazole-1 (4H) -carboxylic acid ethyl ester; [ M + H ]]⁺ 573。

5- [ (2R) -2-hydroxy-2-phenylacetyl]-3- { [4- (4-methylpiperazin-1-yl) benzoyl]Amino } -5, 6-dihydropyrrolo [3, 4-c)]Pyrazole-1 (4H) -carboxylic acid ethyl ester; [ M + H ]]⁺ 533。

Example 5

Preparation of N- {5- [ (2R) -2-methoxy-2-phenylacetyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } -4- (4-methylpiperazin-1-yl) benzamide (1)

Reacting 5- [ (2R) -2-methoxy-2-phenylacetyl]-3- { [4- (4-methylpiperazin-1-yl) benzoyl]Amino } -5, 6-dihydropyrrolo [3, 4-c)]Pyrazole-1 (4H) -carboxylic acid ethyl ester (3.94g, 7.2mmol) in methanol (MeOH, 130ml) and triethylamine (Et)₃N, 13ml) was stirred at room temperature for 16 hours (partial precipitation occurred). After separation of the solid, use Et₂O wash to yield 1.6g of the title compound. The solution was evaporated to a few ml and a second portion of solid product (1.62g) was isolated. The two fractions were combined and analyzed by LC-MS (purity around 90%, 254 and 220 nM). After chromatographic purification (short silica gel column, DCM/MeOH 45: 5), 2.83g (83%) of the title compound are obtained as a white solid. M.p 289 deg.C, 289 deg.C; [ M + H ]]⁺ 475；

¹H-NMR(DMSO-d₆)δppm：2.21(s，3H)；2.43(m，4H)；3.29(m，7H)；4.20-4.90(m，4H)；5.09(s，1H)6.80-8.00(m，9H)；10.6(br，1H)；12.09(br，1H)。

The following compound was prepared by basic hydrolysis of the compound of example 4, operating in a similar manner:

(2) n- {5- [ (2R) -2-methoxy-2-phenylacetyl]-1, 4, 5, 6-tetrahydropyrrolo [3, 4-c]Pyrazol-3-yl } -4- (4-ethylpiperazin-1-yl) benzamide; [ M + H ]]⁺ 489；

¹H-NMR(DMSO-d₆)δppm：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-phenylacetyl]-1, 4, 5, 6-tetrahydropyrrolo [3, 4-c]Pyrazol-3-yl } -4- (4-isopropylpiperazin-1-yl) benzamide; [ M + H ]]⁺ 503；

¹H-NMR(DMSO-d₆)δppm：1.3-1.3(dd，6H)；3.3-3.4(m，9H)；4.6-4.9(m，4H)；5.1(d，1H)；7.0-8.0(m，9H)；10.7(bs，1H)；12.3(bs，1H)。

(4) N- {5- [ (2R) -2-methoxy-2-phenylacetyl]-1, 4, 5, 6-tetrahydropyrrolo [3, 4-c]Pyrazol-3-yl } -4- (4-cyclopropylpiperazin-1-yl) benzamide; [ M + H ]]⁺ 501；

¹H-NMR(DMSO-d₆)δppm：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(m，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-phenylacetyl]-1, 4, 5, 6-tetrahydropyrrolo [3, 4-c]Pyrazol-3-yl } benzamide; [ M + H ]]⁺ 489；

¹H-NMR(DMSO-d₆)δppm：1.0-1.1(m，3H)；2.24(s，3H)；3.3-3.5(m，7H)；4.3-5.0(m，4H)；5.1(d，1H)；6.9-8.0(m，9H)；10.6(bs，1H)；11.9-12.6(br，1H)。

(6) N- {5- [ (2R) -2-methoxy-2-phenylacetyl]-1, 4, 5, 6-tetrahydropyrrolo [3, 4-c]Pyrazol-3-yl } -4-piperazinesPyridin-1-ylbenzamide; [ M + H ]]⁺ 460；

¹H-NMR(DMSO-d₆)δppm：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-yl) -N- {5- [ (2R) -2-methoxy-2-phenylacetyl]-1, 4, 5, 6-tetrahydropyrrolo [3, 4-c]Pyrazol-3-yl } benzamide; [ M + H ]]⁺ 478；

¹H-NMR(DMSO-d₆)δppm：1.65-2.1(m，4H)；3.15-3.6(m，7H)；4.35-5.0(m，5H)；5.1(d，1H)；6.9-8.0(m，9H)；10.4-10.7(br，1H)。

(8) N- {5- [ (2R) -2-methoxy-2-phenylacetyl]-1, 4, 5, 6-tetrahydropyrrolo [3, 4-c]Pyrazol-3-yl } -4-morpholin-4-ylbenzamide; [ M + H ]]⁺ 462；

¹H-NMR(DMSO-d₆)δppm：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(m，9H)；10.4-10.7(br，1H)。

(9)4- (4-tert-butylpiperazin-1-yl) -N- {5- [ (2R) -2-methoxy-2-phenylacetyl]-1, 4, 5, 6-tetrahydropyrrolo [3, 4-c]Pyrazol-3-yl } benzamide; [ M + H ]]⁺ 517。

(10) N- {5- [ (2R) -2-hydroxy-2-phenylacetyl]-1, 4, 5, 6-tetrahydropyrrolo [3, 4-c]Pyrazol-3-yl } -4- (4-methylpiperazin-1-yl) benzamide; [ M + H ]]⁺ 461；

¹H-NMR(DMSO-d₆)δppm：2.3(s，3H)；2.45-2.65(m，4H)；3.2-3.4(m，4H)；4.1-4.9(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-phenylacetyl]-1, 4, 5, 6-tetrahydropyrrolo [3, 4-c]Pyrazol-3-yl } -4- (4-methylpiperazin-1-yl) benzamide; [ M + H ]]⁺ 459；

¹H-NMR(DMSO-d₆)δppm：1.33(d，3H)；2.21(s，3H)；3.85-5.0(m，5H)；4.2-4.9(m，4H)；5.1(s，1H)6.8-8.0(m，9H)；10.3-10.7(br，1H)；11.8-12.5(br，1H)。

(12) N- {5- [ (2R) -2-methyl-2-phenylacetyl]-1, 4, 5, 6-tetrahydropyrrolo [3, 4-c]Pyrazol-3-yl } -4- (4-ethylpiperazin-1-yl) benzamide; [ M + H ]]⁺ 473；

¹H-NMR(DMSO-d₆)δppm：1.06(t，3H)；1.36(d，3H)；2.41(q，2H)；2.47-2.6(m，4H)；3.2-3.4(m，4H)；3.9-5.0(m，5H)；6.9-8.0(m，9H)；10.5(bs，1H)；11.9-12.5(br，1H)。

(13) N- {5- [ (2R) -2-methyl-2-phenylacetyl]-1, 4, 5, 6-tetrahydropyrrolo [3, 4-c]Pyrazol-3-yl } -4- (4-isopropylpiperazin-1-yl) benzamide; [ M + H ]]⁺ 487；

(14) N- {5- [ (2R) -2-methyl-2-phenylacetyl]-1, 4, 5, 6-tetrahydropyrrolo [3, 4-c]Pyrazol-3-yl } -4- (4-cyclopropylpiperazin-1-yl) benzamide; [ M + H ]]⁺ 485；

(15)4- (3, 4-dimethylpiperazin-1-yl) -N- {5- [ (2R) -2-phenylpropionyl]-1, 4, 5, 6-tetrahydropyrrolo [3, 4-c]Pyrazol-3-yl } benzamide; [ M + H ]]⁺ 473；

(16) N- {5- [ (2R) -2-phenylpropionyl group]-1, 4, 5, 6-tetrahydropyrrolo [3, 4-c]Pyrazol-3-yl } -4-piperidin-1-ylbenzamide; [ M + H ]]⁺ 444；

¹H-NMR(DMSO-d₆)δppm：1.37(d，3H)；1.6(s，6H)；3.2-3.4(m，4H)；3.90-4.05(m，1H)；4.1-4.9(m，4H)；6.9-8.0(m，9H)；10.4-10.7(br，1H)。

(17)4- (4-Fluoropiperidin-1-yl) -N- {5- [ (2R) -2-methyl-2-phenylacetyl]-1, 4, 5, 6-tetrahydropyrrolo [3, 4-c]Pyrazol-3-yl } benzamide; [ M + H ]]⁺ 462；

¹H-NMR(DMSO-d₆)δppm：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-methyl-2-phenylacetyl]-1, 4, 5, 6-tetrahydropyrrolo [3, 4-c]Pyrazol-3-yl } -4-morpholin-4-ylbenzamide; [ M + H ]]⁺ 446；

¹H-NMR(DMSO-d₆)δppm：1.37(d，3H)；3.2-3.4(m，4H)；3.7-3.85(m，4H)；3.9-4.1(m，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-phenylpropionyl]-1, 4, 5, 6-tetrahydropyrrolo [3, 4-c]Pyrazol-3-yl benzamide; [ M + H ]]⁺ 501。

Claims (21)

1. Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of a cell proliferative disorder caused by and/or associated with altered Aurora kinase activity in a mammal:

wherein

R is hydrogen or methyl;

R₁is hydroxy or straight or branched C₁-C₃Alkyl or alkoxy;

R₂is a hydrogen or halogen atom;

x is a divalent radical selected from methylene or fluoromethylene, or a heteroatom or heteroatom radical selected from oxygen or-NR '-wherein R' is a hydrogen atom, a linear or branched C₁-C₄Alkyl or C₃-C₆A cycloalkyl group.

2. Use according to claim 1, wherein the Aurora kinase is Aurora 2.

3. Use according to claim 1, wherein the cell proliferative disorder is selected from the group consisting of cancer, alzheimer's disease, viral infections, autoimmune diseases and neurodegenerative disorders.

4. Use according to claim 3, wherein said cancer is selected from the group consisting of malignancies of epithelial tissue origin, 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, keratoacanthoma, thyroid follicular cancer, and Kaposi's sarcoma.

5. Use according to claim 4, wherein the malignant tumour derived from epithelial tissue is a squamous cell carcinoma.

6. Use according to claim 1, wherein the cell proliferative disorder is selected from the group consisting of benign prostate hyperplasia, familial adenomatosis, polyposis, neurofibromatosis, psoriasis, vascular smooth muscle cell proliferation associated with atherosclerosis, pulmonary fibrosis, arthritis, glomerulonephritis and post-operative stenosis and restenosis.

7. The use according to claim 1, wherein said mammal is a human.

8. Use of a compound as defined in claim 1 for the manufacture of a medicament for inhibiting Aurora 2 kinase activity.

9. A compound of formula (I) or a pharmaceutically acceptable salt thereof

Wherein

R is hydrogen or methyl;

R₁is hydroxy or straight or branched C₁-C₃Alkyl or alkoxy;

R₂is a hydrogen or halogen atom;

x is a divalent radical selected from methylene or fluoromethylene, or a heteroatom or heteroatom radical selected from oxygen or-NR '-wherein R' is a hydrogen atom, a linear or branched C₁-C₄Alkyl or C₃-C₆A cycloalkyl group.

10. The compound of formula (I) according to claim 9, wherein R is hydrogen or methyl; r₁Selected from hydroxy, methyl or methoxy; r₂Is a hydrogen or fluorine atom; x is selected from methylene, fluoromethylene, -O-or-NR ', wherein R' is as defined in claim 9.

11. A compound of formula (I) according to claim 1, optionally in the form of a pharmaceutically acceptable salt thereof, selected from:

(1) n- {5- [ (2R) -2-methoxy-2-phenylacetyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } -4- (4-methylpiperazin-1-yl) benzamide;

(2) n- {5- [ (2R) -2-methoxy-2-phenylacetyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } -4- (4-ethylpiperazin-1-yl) benzamide;

(3) n- {5- [ (2R) -2-methoxy-2-phenylacetyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } -4- (4-isopropylpiperazin-1-yl) benzamide;

(4) n- {5- [ (2R) -2-methoxy-2-phenylacetyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } -4- (4-cyclopropylpiperazin-1-yl) benzamide;

(5)4- (3, 4-dimethylpiperazin-1-yl) -N- {5- [ (2R) -2-methoxy-2-phenylacetyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } benzamide;

(6) n- {5- [ (2R) -2-methoxy-2-phenylacetyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } -4-piperidin-1-ylbenzamide;

(7)4- (4-fluoropiperidin-1-yl) -N- {5- [ (2R) -2-methoxy-2-phenylacetyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } benzamide;

(8) n- {5- [ (2R) -2-methoxy-2-phenylacetyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } -4-morpholin-4-ylbenzamide;

(9)4- (4-tert-butylpiperazin-1-yl) -N- {5- [ (2R) -2-methoxy-2-phenylacetyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } benzamide;

(10) n- {5- [ (2R) -2-hydroxy-2-phenylacetyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } -4- (4-methylpiperazin-1-yl) benzamide;

(11) n- {5- [ (2R) -2-methyl-2-phenylacetyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } -4- (4-methylpiperazin-1-yl) benzamide;

(12) n- {5- [ (2R) -2-methyl-2-phenylacetyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } -4- (4-ethylpiperazin-1-yl) benzamide;

(13) n- {5- [ (2R) -2-methyl-2-phenylacetyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } -4- (4-isopropylpiperazin-1-yl) benzamide;

(14) n- {5- [ (2R) -2-methyl-2-phenylacetyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } -4- (4-cyclopropylpiperazin-1-yl) benzamide;

(15)4- (3, 4-dimethylpiperazin-1-yl) -N- {5- [ (2R) -2-phenylpropionyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } benzamide;

(16) n- {5- [ (2R) -2-phenylpropionyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } -4-piperidin-1-ylbenzamide;

(17)4- (4-fluoropiperidin-1-yl) -N- {5- [ (2R) -2-methyl-2-phenylacetyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } benzamide;

(18) n- {5- [ (2R) -2-methyl-2-phenylacetyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } -4-morpholin-4-ylbenzamide;

(19)4- (4-tert-butylpiperazin-1-yl) -N- {5- [ (2R) -2-phenylpropionyl ] -1, 4, 5, 6-tetrahydropyrrolo [3, 4-c ] pyrazol-3-yl } benzamide.

12. A process for the preparation of a compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 9, which process comprises:

a) reacting a compound of formula (II) with a compound of formula (III)

Wherein R and X are as defined in claim 9, Q is C₁-C₄Alkyl, t-Bu represents tert-butyl and Z is hydroxy or a suitable leaving group, in order to obtain a compound of formula (IV)

b) Reacting a compound of formula (IV) under acidic conditions to obtain a compound of formula (V)

c) Reacting a compound of formula (V) with a compound of formula (VI)

Wherein R is₁And R₂As defined in claim 9, and Z' represents a hydroxyl group or a suitable leaving group, so as to obtain a compound of formula (VII)

d) Reacting a compound of formula (VII) under basic conditions to give the corresponding compound of formula (I), or, alternatively, converting it into a pharmaceutically acceptable salt thereof.

13. The method according to claim 12, wherein Q is methyl or ethyl.

14. The process according to claim 12, wherein in the compounds of formulae (III) and (VI), Z and Z' are each independently a hydroxyl group or a halogen atom.

15. The method according to claim 14, wherein the halogen atom is chlorine.

16. The process according to claim 12, wherein step (b) is carried out under acidic conditions in the presence of hydrochloric acid, trifluoroacetic acid or methanesulfonic acid.

17. The process according to claim 12, wherein step (d) is carried out in the presence of sodium hydroxide, potassium hydroxide or lithium hydroxide, or a tertiary amine.

18. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, as defined in claim 9, and at least one pharmaceutically acceptable excipient, carrier and/or diluent.

19. The pharmaceutical composition according to claim 18, further comprising one or more chemotherapeutic agents.

20. A kit comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, as defined in claim 9, or a pharmaceutical composition as defined in claim 18, and one or more chemotherapeutic agents, as a combined preparation for simultaneous, separate or sequential use in anticancer therapy.

21. Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof as defined in claim 9 for the manufacture of a medicament having anti-tumour activity.