CN101146794A - Pyrazole derivatives for the inhibition of CDK and GSK - Google Patents

Abstract

The invention provides compounds of the formula (I), or salts, tautomers, N-oxides or solvates thereof wherein: R1 is selected from: (a) 2,6-dichlorophenyl; (b) 2,6-difluorophenyl; (c) a 2,3,6-trisubstituted phenyl group wherein the substituents for the phenyl group are selected from fluorine, chlorine, methyl and methoxy; (d) a group R0; (e) a group R a; (f) a group Rlb; (g) a group Rlc; (h) a group Rld; and 0) 2,6-difluorophenylamino ; wherein R )0, r R> llaa, T Rj I1bD, T R) I1cC, r R> Iida, r R 2zaa, r R>22bD and RJ are as defined in the claims. The compounds have activity as inhibitors of cdk kinase (such as cdkl or cdk2) and glycogen synthase kinase-3 activity.

Description

Be used to suppress the pyrazole derivatives of CDK and GSK

The pyrazole compound, this compounds that the present invention relates to suppress or regulate the kinase activity of cell cycle protein dependent kinase (CDK) and glycogen synthase kinase (GSK) is in treatment or prevent by kinase mediated morbid state or the purposes in the illness and have kinase inhibition or regulate active new compound.The pharmaceutical composition that comprises this compounds and new chemical intermediate also are provided.

Background of invention

The protein kinase of being responsible for the intracellular multiple signal conductive process of control has constituted relevant big enzyme family on the structure.(Hardie, G. and Hanks, S. (1995) The Protein KinaseFacts Book.I and II.Academic Press, San Diego, CA).Kinases can be according to the substrate (for example, protein-tyrosine, protein-serine/threonine, lipid etc.) of its phosphorylation and is divided into different families.Common sequence die body (for example, Hanks, S.K., Hunter, T., FASEB J., 9:576-596 (1995) have been identified corresponding to each kinases family; People such as Knighton, Science, 253:407-414 (1991); People such as Hiles, Cell, 70:419-429 (1992); People such as Kunz, Cell, 73:585-596 (1993); People such as Garcia-Bustos, EMBO J., 13:2352-2361 (1994)).

Protein kinase can characterize according to its regulation mechanism.These mechanism comprise, for example, and self phosphorylation, by other kinase whose commentaries on classics phosphorylation, protein-protein interaction, protein-lipid interaction and protein-polynucleotide interaction.A kind of protein kinase may be subjected to the adjusting of mechanism more than one.

Kinases is regulated many different cell processes by phosphate group being added into target protein, includes but not limited to propagation, differentiation, apoptosis, mobility, transcribes, translates and other signalling process.These phosphorylation events play a part to regulate or regulate and control the molecule ON/OFF switch of target protein biological function.Target protein responds that various extracellulars signal (hormone, neurotransmitter, growth and differentiation factor etc.), cell cycle events, environment or nutrition are coerced etc. and phosphorylation takes place.The effect of suitable protein kinase in signal path be activate or deactivation (directly or indirectly) for example metabolic enzyme, regulate albumen, acceptor, cytoskeletal protein, ionic channel or pump or transcription factor.The not controlled signal that is caused by the control defective of protein phosphorylation effect has related to numerous disease, comprises for example disease and the illness of inflammation, cancer, transformation reactions/asthma, immune disease and illness, central nervous system, and vasculogenesis.

Cell cycle protein dependent kinase

When eukaryotic cell splitted process can broadly be divided into a series of successive that is called G1, S, G2 and M mutually.Verified, by the cell cycle not simultaneously the correct process key of phase depend on the protein families that is called as cell cycle protein dependent kinase (cdks) and be called regulation and control on not on the same group its homologous protein partner's the room and time of cyclin.Cdks is the homology serine-threonine kinase protein of cdc2 (being also referred to as cdk1), and it can utilize ATP as substrate in the phosphorylation of the not homopolypeptide of sequence dependent background.Cyclin is one, and this homologous region is called as " cyclin box " to comprise about 100 protein familieses that amino acid whose homologous region is a feature, and it is used for combination of homospecificity cdk partner protein matter and definition selectivity.

Various cdks and cyclin cause the circulation of a series of cdk/ cyclin mixtures to form in the adjusting of expression level, degradation rate and the activation levels of whole cell cycle, and wherein cdks is the enzymatic activation.The formation of these mixtures is gone down to posterity via the check point control of discontinuous cell cycle, and fissional process can be continued.Can not satisfy necessary biological chemistry standard in given cell cycle check point, promptly can't form essential cdk/ cyclin mixture, can cause the cell cycle to be ended and/or apoptosis.Unusual cell proliferation as shown in the cancer, often is attributable to the disappearance of correct cell cycle control.Therefore suppressing the cdk enzymic activity provides a kind of cell of abnormal division that makes to stop division and/or killed method.The diversity of cdks and cdk mixture and they provide the potential treatment target of the wide spectrum of selecting based on defined biological chemistry theory the decisive role of mediated cell in the cycle.

From the process of G1 phase to S phase of cell cycle mainly by cdk2, cdk3, cdk4 and cdk6 by regulating with D and combining of E type cyclin member.As if D type cyclin helps not go down to posterity by the G1 restriction point, and cdk2/ cyclin E mixture is the key of changing to the S phase from G1.Need to be considered to cdk2/ cyclin A mixture by S phase and the process that enters G2 subsequently.Mitotic division and trigger its G2 to the conversion of M phase, both all are subjected to the adjusting of cdk1 and A and type B cell cyclin mixture.

In the G1 phase, retinocytoma albumen (Rb) and relevant bag shape albumen (pocket protein) is p130 for example, is the substrate of cdk (2,4 and 6)/cyclin mixture.Process by G1 since Rb and p130 by cdk (4/6) thereby/the excessive phosphorylation inactivation of cyclin-D mixture and partly promoting.The excessive phosphorylation of Rb and p130 causes for example release of E2F of transcription factor, and therefore causes by G1 and the necessary gene of process that enters the S phase, for example expression of gene of cyclin E.The expression of cyclin E promotes the formation of cdk2/ cyclin E mixture, and its further phosphorylation by Rb is amplified or kept the E2F level.Cdk2/ cyclin E mixture is the required protein of other dna replication dna of phosphorylation also, NPAT for example, and it relates to the histone biosynthesizing.The process of G1 and the conversion of G1/S also are subjected to being included in the adjusting of the Myc approach of the mitotic division primary stimuli in the cdk2/ cyclin E approach.Cdk2 also is related via the dna damage response pathway that p53 regulates and p53 mediates of p21 level.P21 is the protein inhibitor of cdk2/ cyclin E, therefore can block or postpone the G1/S conversion.Therefore cdk2/ cyclin E mixture can be represented to stimulate the point that is integrated to a certain extent from the biological chemistry of Rb, Myc and p53 approach.Therefore Cdk2 and/or cdk2/ cyclin E mixture have been represented the good treatment target spot of ending or recover the control of cell cycle in the cell of abnormal division.

The corner cut look also unclear really in the cell cycle for cdk3.Also do not have homologous cyclin partner to be identified so far, but the negative adjusting form of the dominance of cdk3 postpones G1 phase cell, hint that therefore cdk3 has the effect of regulating the G1/S conversion.

Though most cdks participates in regulation of Cell Cycle, evidence suggests that some cdk family member has participated in other Biochemical processes.With cdk5 is example, and it is that the correct growth of neurone is necessary, and has participated in for example phosphorylation of Tau, NUDE-1, synapsin1, DARPP32 and Munc18/Syntaxin1A mixture of several neuronic protein.Neuronic cdk5 is usually by activating with the p35/p39 combination of proteins.Yet the Cdk5 activity can be by in conjunction with p25 (the brachymemma type of a kind of p35) adjusting of making a return journey.P35 is to the conversion of p25 and cdk5 is active subsequently goes to regulate and can be brought out by local asphyxia, excitotoxicity and beta amyloid peptide.Thereby p25 involved for example pathogenesis of alzheimer's disease of neurodegenerative disease, therefore as having caused people's interest at these treatment of diseases target spots.

Cdk7 is that a kind of cdc2CAK of having is active and in conjunction with the nucleoprotein of cyclin H.Cdk7 has been accredited as the composition of TFII H transcription complex, and it has C-end structure territory (CTD) activity of rna plymerase ii.This HIV-1 transcriptional regulatory with the bio-chemical pathway that is mediated by Tat is relevant.Cdk8 is in conjunction with cyclin C and participate in the phosphorylation of the CTD of rna plymerase ii.Similarly, cdk9/ cyclin-T1 mixture (P-TEFb mixture) participates in the control of rna plymerase ii to extending.The HIV-1 genome also needs PTEF-b by viral trans-acting factor Tat via the interactive transcription activating of itself and cyclin T1.Therefore cdk7, cdk8, cdk9 and P-TEFb mixture are the potential target spots of antiviral therapy.

On molecular level, the mediation of cdk/ cyclin complex activity needs the phosphorylation or the dephosphorylation incident of a series of pungencys and inhibition.The Cdk phosphorylation is finished by one group of cdk activated protein kinase (CAK) and/or such as the kinases of weel, Mytl and Mikl.Dephosphorylation is by phosphoesterase cdc25 (a﹠amp for example; C), pp2a or KAP finish.

The activity of Cdk/ cyclin mixture can further be regulated by the endogenous cell protein inhibitor of two families: Kip/Cip family or INK family.INK protein-specific ground is in conjunction with cdk4 and cdk6.P16 ^Ink4(being also referred to as MTS1) is the potential tumor suppressor gene, and it is undergone mutation in a large amount of primary carcinoma or lacks.Kip/Cip family comprises such as p21 ^{Cip1, waf1}, p27 ^Kip1And p57 ^Kip2In protein.As discussed previously, p21 is induced by p53 and can deactivation cdk2/ cyclin (E/A) and cdk4/ cyclin (D1/D2/D3) mixture.Having observed atypical low-level p27 in mammary cancer, colorectal carcinoma and prostate cancer expresses.On the contrary, the cross expression of cyclin E in solid tumor shows relevant with patient's prognosis mala.The mistake expression of cyclin D1 is relevant with esophagus cancer, mammary cancer, squamous cell carcinoma and nonsmall-cell lung cancer.

Summarize Cdks and related protein thereof above and in proliferative cell, coordinated and driven the key role of cell cycle.Some wherein biochemical route of playing a crucial role of cdks have also been described.Therefore, a kind of therapeutics that adopts target one class cdks or specific cdks of exploitation monotherapy for the treatment of hyperplasia such as cancer may be in demand.It is also conceivable that the cdk inhibitor is used for the treatment of other illness for example virus infection, autoimmune disease and neurodegenerative disease.When with existing or new therapeutical agent combined therapy, the treatment of target cdk also can provide clinical benefit in the treatment of aforementioned diseases.The anticancer therapy that with cdk is target spot may have the advantage that is better than multiple existing antineoplastic agent because they not with the dna direct effect, therefore reduced the danger of secondary tumor development.

Glycogen synthase kinase

Glycogen synthase kinase-3 (GSK3) is a kind of serine-threonine kinase, and there is (GSK3 α ﹠amp in the isotype with two kinds of wide expression in the mankind; β GSK3 β).GSK3 participates in fetal development, protein synthesis, cell proliferation, cytodifferentiation, microtubule kinetics, cell mobility and apoptosis.Thereby GSK3 relates to the process of morbid state, for example diabetes, cancer, alzheimer's disease, apoplexy, epilepsy, motor neurone disease and/or injury of head.(CDK) is closely related for phylogenetic GSK3 and cell cycle protein dependent kinase.

The peptide substrates consensus sequence of being discerned by GSK3 is (Ser/Thr)-X-X-X-(pSer/pThr), and wherein X is arbitrary amino acid (in (n+1), (n+2), (n+3) position), and pSer and pThr are respectively phosphorylation Serine and phosphorylation Threonine (n+4).GSK3 is at (n) position first Serine of phosphorylation or Threonine.Phosphoric acid-the Serine of (n+4) position or phosphoric acid-Threonine are that startup GSK3 is necessary to obtain maximum substrate conversion.GSK3 α is at Ser21, or GSK3 β causes the restraining effect of GSK3 in the phosphorylation of Ser9.The N-terminal that the GSK3 phosphorylation has been drawn in sudden change and peptide competition research is by suppressing the machine-processed model that can compete with the peptide substrates (S/TXXXpS/pT) of phosphorylation automatically.Also there are data proposition GSK3 α and GSK β to regulate subtly by the phosphorylation of tyrosine 279 and 216 respectively.These residues cause the reduction of kinase activity in the body to the sudden change of Phe.The x-ray crystal structure of GSK3 β helps to disclose all aspects that GSK3 activates and regulates.

The GSK3 structure Mammals insulin replies approach a part and can phosphorylation and therefore deactivation glycogen synthetase.Thus, active and the synthetic possible means that have been regarded as resisting II type or non insulin dependent diabetes (NIDDM) of incremental adjustments glycogen thus of incremental adjustments glycogen synthetase by suppressing GSK3, this illness are that a kind of wherein body tissue becomes and insulin stimulating had the illness of tolerance.Cell in liver, fat or the muscle tissue triggers the Regular Insulin that replying of Regular Insulin is incorporated into the extracellular insulin receptor.It causes proteinic phosphorylation of IRS (IRS) and raising to cytolemma subsequently.The proteinic further phosphorylation of IRS has started the gathering of phosphatidylinositol 3-kinase (PI3K) to cytolemma, and it can discharge second messenger's phosphatidylinositols 3,4, the 5-triphosphoric acid.It promotes 3-lipositol dependence protein matter kinases 1 (PDK1) and the common location of protein kinase B (PKB or Akt) on film, and PDK1 activates PKB there.PKB can come phosphorylation and suppress GSK3 α and/or GSK β whereby by the phosphorylation of Ser9 or ser21 respectively.The inhibition of GSK3 then triggers the active rise of glycogen synthetase.Therefore the therapeutical agent that can suppress GSK3 can bring out and be similar to observed those cell responses in insulin stimulating.Substrate was eukaryotic cell protein synthesis initiation factor 2B (eIF2B) in another of GSK3 was individual.EIF2B passes through phosphorylation and inactivation, therefore can the biosynthesizing of arrestin matter.Therefore, the inhibition of GSK3, for example by " Mammals rapamycin target protein " inactivation (mTOR), but the biosynthesizing of upregulated protein matter.At last, there are some to pass through mitogen-activated protein kinase (MAPK) path, make GSK3 by kinases mitogen-activated protein kinase activated protein kinase 1 (MAPKAP-K1 or RSK) phosphorylation and regulate the active evidence of GSK3 for example.These data promptings GSK3 activity can be subjected to the regulation and control that cell fission, Regular Insulin and amino acid stimulate.

Known GSK3 β also is a kind of important component in the vertebrates Wnt signal pathway.Verified this bio-chemical pathway is very important and can regulate cell proliferation in the healthy tissues for normal embryo development.GSK3 is to be suppressed for replying of Wnt stimulation.This can cause GSK3 the substrate for example gene product and the white dephosphorylation of beta-catenin of Axin, adenomatous polyposis coli (APC).The unusual adjusting of Wnt approach is relevant with many cancers.Sudden change during APC and/or beta-catenin are white is common in colorectal carcinoma and other the tumour.Beta-catenin has also demonstrated the importance in cell attachment in vain.Therefore GSK3 also can regulate the cell attachment process to a certain extent.Except the biochemical route of having described, also there be the phosphorylation of GSK3 by cyclin D1 to participate in fissional adjusting, participate in for example data of the phosphorylation of c-Jun, CCAAT/ enhancer binding protein α (C/EBP α), c-Myc and/or other substrate such as nuclear factor of activated T cells (NFATc), heat shock factor-1 (HSF-1) and c-AMP response element binding protein (CREB) of transcription factor.GSK3 also demonstrates in regulating apoptosis and works, though specificity in a organized way.GSK3 may be closely related with the medical conditions that the neuronal cell apoptosis wherein can take place via the effect of short apoptosis mechanism in regulating apoptosis.These examples of disorders are injury of head, apoplexy, epilepsy, alzheimer's disease and motor neurone disease, stein-leventhal syndrome, the sex change of cortex basal nuclei and Pick's disease.At the external excessively phosphorylation canalicular apparatus associated protein Tau of GSK3 that confirmed.The excessive phosphorylation of Tau has been destroyed it and has been combined with the normal of microtubule, and can cause the formation of Tau microfilament in the cell.It is believed that constantly accumulating of these microfilaments can finally cause neuron dysfunction and sex change.Therefore can provide the method for a kind of restriction and/or prevention neurodegeneration effect by suppressing phosphorylation that GSK3 suppresses Tau.

Diffuse type large B cell lymphoid tumor (DLBCL)

The process of cell cycle is to regulate and control by cyclin, cell cycle protein dependent kinase (CDk) with as the combined action of the proteic CDK supressor of cell cycle negative regulation (CDKi).P27KIP1 is CDKi crucial in the cell cycle regulating, and its degraded is that the conversion of G1/S is necessary.Although in the lymphocyte of propagation, lack the expression of p27KIP1, reported that some aggressive B cell lymphomas demonstrate unusual p27KIP1 dyeing.In the lymphoma of this type, found the high expression level that p27KIP1 is unusual.In single argument and multivariate analysis, the clinical correlation analysis of these discoveries shows that the expression of high-caliber p27KIP1 is bad prognostic indicator in this type tumour.These results show has unusual p27KIP1 to express in the diffuse type large B cell lymphoid tumor (DLBCL), have bad Clinical symptoms, prompting is by causing this unusual p27KIPI protein to lose function with the proteic interaction of other cell cycle regulatory factors.(Br.J.Cancer.1999Jul; 80 (9): 1427-34.p27KIPl is unconventionality expression and relevant with bad clinical effectiveness in the diffuse type large B cell lymphoid tumor.Saez?A，Sanchez?E，Sanchez-Beato?M，Cruz?MA，ChaconI，Munoz?E，Camacho?FI，Martinez-Montero?JC，Mollej?o?M，Garcia?JF，Piris?MA.Department?of?Pathology，Virgen?de?la?Salud?Hospital，Toledo，Spain.)

Chronic lymphatic leukemia

B cell chronic lymphatic leukemia (CLL) is the modal leukemia in the western hemisphere, and has every year 10,000 new cases to be diagnosed (Parker SL approximately, Tong T, Bolden S, Wingo PA:Cancer statistics, 1997.Ca.Cancer.J.Clin.47:5, (1997)).With respect to the leukemia of other form, total prognosis bona of CLL is even the patient in late period also has the survival median in 3 years.

Compare with the treatment based on alkylating agent of previous use, add fludarabine (fludarabine) and can cause the response fully (27% pair 3%) of higher rate and the survival time that gets nowhere (33 pairs 17 months) as the initial treatment of CLL patient with sympotoms.Though the response fully that reaches after treatment clinically is an initial step of improving the CLL survival rate, most of patients does not reach alleviation fully or fludarabine is not reacted.Moreover, the CLL patient of useful fludarabine treatment all recur at last, thereby make it can only be used as simple releiving property medicament (Rai KR, Peterson B, Elias L, Shepherd L, HinesJ, Nelson D, Cheson B, Kolitz J, Schiffer CA:A randomized comparison offludarabine and chlorambucil for patients with previously untreatedchronic lymphocytic leukemia.A CALGB SWOG, CTG/NCI-C and ECOGInter-Group Study.Blood88:141a, 1996 (summary 552, suppl1).Therefore, if want to realize the further improvement of this disease treatment, must identify cytotoxicity with additional fludarabine and the new medicament of removing the chemical sproof new role mechanism of bringing out by inherent CLL r factor.

About CLL patient's relatively poor and bad survival rate to therapeutic response study to such an extent that the normative forecast factor the most widely is unusual p53 function, it is a feature with point mutation or karyomit(e) 17p13 disappearance.Really, in fact there is not reaction in those have CLL patient's the case series of multiple single institution of unusual p53 function, to be proved to be to the treatment of alkylating agent or purine analogue.A kind of introducing of having the ability to overcome the drug-fast therapeutical agent relevant with the p53 sudden change of CLL may be for becoming the important advance of this disease treatment.

The inhibitor good fortune of cell cycle protein dependent kinase draws benefit (flavopiridol) and CYC202 at the external apoptosis that brings out from the malignant cell of B cell chronic lymphatic leukemia (B-CLL).

Good fortune draws the benefit administration to cause the dependent decomposition of caspase-to active stimulation of caspase3 and p27 (kip1), and p27 is the negative regulatory factor of a kind of cell cycle, and it is crossed in B-CLL and expresses.(Blood.1998Nov15；92(10)：3804-16Flavopiridol?induces?apoptosis?inchronic?lymphocytic?leukemia?cells?via?activation?of?caspase-3withoutevidence?of?bcl-2modulation?or?dependence?on?functional?p53.Byrd?JC，Shinn?C，Waselenko?JK，Fuchs?EJ，Lehman?TA，Nguyen?PL，Flinn?IW，Diehl?LF，Sausville?E，Grever?MR)。

Prior art

WO02/34721 from Du Pont discloses a kind of indeno as cell cycle protein dependent kinase inhibitor [1,2-c] pyrazoles-4-ketone.

From the WO01/81348 of Bristol Myers Squibb described 5-sulphur-, sulfinyl-and sulfonyl pyrazole also [3,4-b]-pyridine as the purposes of cell cycle protein dependent kinase inhibitor.

WO00/62778 from Bristol Myers Squibb discloses a kind of protein tyrosine kinase inhibitor equally.

From the WO01/72745A1 of Cyclacel described 4-heteroaryl-pyrimidine and preparation thereof that 2-replaces, the pharmaceutical composition that comprises them and its as the purposes of cell cycle protein dependent kinase (CDK) inhibitor with and in the treatment proliferative disease purposes in cancer, leukemia, the psoriatic etc. for example.

WO99/21845 from Agouron has described a kind of cell cycle protein dependent kinase (CDK) that is used to suppress, for example the 4-aminothiazole derivs of CDK1, CDK2, CDK4 and CDK6.This invention also relates to the treatment of the pharmaceutical composition that comprises this compound or preventive use and these compounds for treating malignant diseases by giving significant quantity and the method for other disease.

WO01/53274 from Agouron discloses a kind of compound as the CDK kinase inhibitor, and it can comprise and is connected to the phenyl ring that is replaced by acid amides that contains the N heterocyclic group.

WO01/98290 (Pharmacia ﹠amp; Upjohn) a kind of 3-aminocarboxyl-2-amido-thiophene derivant as kinases inhibitor is disclosed.

Disclose from the WO01/53268 of Agouron and WO01/02369 that for example the compound of cell proliferation is regulated or suppressed to cell cycle protein dependent kinase or Tyrosylprotein kinase by the arrestin kinases.The compound of Agouron has directly or is connected to via CH=CH or CH=N group the aryl or the heteroaryl ring of the 3-position of indazole ring.

WO00/39108 and WO02/00651 (both all belong to Du Pont medicine) have described heterogeneous ring compound, and it is a trypsin-like serine protease, especially the inhibitor of Xa factor and zymoplasm.It is said that this compound can be used as anti-coagulant or is used to prevent thromboembolism.

US2002/0091116 (people such as Zhu), WO01/19798 and WO01/64642 disclose not on the same group the heterogeneous ring compound as the Xa factor inhibitor separately.Disclose and exemplified the pyrazolecarboxamide compounds that some 1-replace.

US6,127,382, WO01/70668, WO00/68191, WO97/48672, WO97/19052 and WO97/19062 (all belonging to Allergan) disclose separately be used for the treatment of the various hyperproliferation diseases that comprise cancer have an active compound of retinoids.

WO02/070510 (Bayer) has described a kind of amino-dicarboxylic acid compound that is used for the treatment of cardiovascular disorder.Though mentioned pyrazoles prevailingly, in this part file, do not had the specific embodiment of pyrazoles.

WO97/03071 (Knoll AG) discloses a kind of heterocyclic radical-carboxamides derivatives that is used for the treatment of central nervous system disorders.Mentioned the example of pyrazoles prevailingly, but do not had to disclose or exemplify concrete pyrazole compound as heterocyclic radical.

WO97/40017 (Novo Nordisk) has described the compound as the conditioning agent of Protein-tyrosine-phosphatase.

WO03/020217 (Univ.Connecticut) discloses a kind of cannabin(e) (cannabinoid) receptor modulators pyrazoles 3-acid amides that is used for the treatment of nervous disorders.Wherein having described (the 15th page) this compound can be used in the cancer chemotherapy, but and does not know that as carcinostatic agent whether effectively or its whether administration for other purpose this compound.

WO01/58869 (Bristol Myers Squibb) discloses the cannabinoid receptor conditioning agent that can be used for treating multiple disease.The main application of being predicted is for the treatment respiratory disease, although mentioned the treatment of cancer.

WO01/02385 (Aventis Crop Science) discloses 1-(the quinolyl-4)-1H-pyrazole derivatives as mycocide.The unsubstituted pyrazoles as the 1-of synthetic intermediate is wherein disclosed.

WO2004/039795 (Fujisawa) discloses the acid amides as the pyrazole group that comprises the 1-replacement of inhibitors of apolipoprotein b secretion.It is said that this compound can be used for treating illnesss such as hyperlipidemia.

WO2004/000318 (Cellular Genomics) discloses the monocycle class as the various amino-replacement of kinase modulator.No pyrazoles exemplify compound.

The application WO2005/012256 that awaits the reply that we are previous, it is open after the application's priority date, discloses as 3 of CDK and GSK-3 kinase inhibitor 4-disubstituted pyrazol compound.

Summary of the invention

The invention provides and have that cell cycle protein dependent kinase suppresses or regulate activity and glycogen synthase kinase-3 (GSK3) suppresses or regulates active compound, and expect that it can be used for preventing or treating by kinase mediated morbid state or illness.

Therefore, for example, can predict the incidence that The compounds of this invention will can be used for alleviating or reducing cancer.

In first aspect, the invention provides formula (I) compound:

Or its salt, compounds tautomeric, solvate and N-oxide compound;

Wherein:

R ¹Be 2, the 6-dichlorophenyl;

R ^2aAnd R ^2bBe hydrogen;

And R ³Group for following formula:

R wherein ⁴Be C _1-4Alkyl.

Term " alkyl " comprises straight chain and branched alkyl group.

C _1-4Alkyl group can be C ₁, C ₂, C ₃Or C ₄Alkyl group.

At C _1-4Can comprise following subgroup in the alkyl group group:

.C _1-3Alkyl group;

.C _1-2Alkyl group;

.C _2-3Alkyl group; With

.C _2-4Alkyl group.

A concrete subgroup is C _1-3Alkyl.

Concrete C _1-4Alkyl group is methyl, ethyl, sec.-propyl, normal-butyl, isobutyl-and the tertiary butyl.

Another C _1-4The subgroup of alkyl group comprises methyl, ethyl, sec.-propyl and n-propyl.

A preferred group is a methyl.

The R that other is concrete ₄Group is ethyl and sec.-propyl.

Therefore, preferred compounds of the invention are 4-(2,6-two chloro-benzamidos)-1H-pyrazoles-3-formic acid (1-methylsulfonyl-piperidin-4-yl)-acid amides.

The present invention also provides:

. formula (I) compound or its any subgroup or example as herein defined, it is used for prevention or treatment by cell cycle protein dependent kinase or glycogen synthase kinase-3 disease states mediated or illness.

. a kind of prevention or treatment be by the method for cell cycle protein dependent kinase or glycogen synthase kinase-3 disease states mediated or illness, and this method comprises the patient who formula defined herein (I) compound or its any subgroup or example is needed they.

. a kind of method that alleviates or reduce by cell cycle protein dependent kinase or glycogen synthase kinase-3 disease states mediated or illness incidence, this method comprise the patient who formula defined herein (I) compound or its any subgroup or example is needed they.

. a kind of in Mammals treatment comprise or result from the disease of abnormal cell growth or the method for illness, this method comprises and gives formula defined herein (I) compound or its any subgroup or the example that Mammals suppresses the abnormal cell growth significant quantity.

. a kind of in Mammals, alleviate or reduce comprise or result from the disease of abnormal cell growth or the method for illness incidence, this method comprises and gives formula defined herein (I) compound or its any subgroup or the example that Mammals suppresses the abnormal cell growth significant quantity.

. a kind of in Mammals treatment comprise or result from the disease of abnormal cell growth or the method for illness, this method comprises and gives Mammals with suppressing formula defined herein (I) compound of cdk kinases (for example cdk1 or cdk2) or the active significant quantity of glycogen synthase kinase-3 or its any subgroup or example.

. a kind of in Mammals, alleviate or reduce comprise or result from the disease of abnormal cell growth or the method for illness incidence, this method comprises and gives Mammals with suppressing formula defined herein (I) compound of cdk kinases (for example cdk1 or cdk2) or the active significant quantity of glycogen synthase kinase-3 or its any subgroup or example.

. a kind of method that suppresses cell cycle protein dependent kinase or glycogen synthase kinase-3, this method comprise that the kinase inhibition compound that makes kinases and formula (I) defined herein or its any subgroup or example contact.

. the method for a kind of adjusting cell processes (for example cell fission), this method realizes by the activity of using formula (I) compound defined herein or its any subgroup or example to suppress cell cycle protein dependent kinase or glycogen synthase kinase-3.

. formula defined herein (I) compound or its any subgroup or example, it is used for prevention or the defined morbid state of treatment literary composition.

. the purposes that formula defined herein (I) compound or its any subgroup or example are used to produce medicine, wherein this medicine is used for any one or multiple purposes defined herein.

. a kind of pharmaceutical composition, it comprises formula defined herein (I) compound or its any subgroup or example and pharmaceutically acceptable carrier.

. a kind of pharmaceutical composition that is suitable for the oral administration form, it comprises formula defined herein (I) compound or its any subgroup or example and pharmaceutically acceptable carrier.

. be used for formula defined herein (I) compound or its any subgroup or the example of medicine.

. a kind of diagnosis and treatment be by the method for cell cycle protein dependent kinase disease states mediated or illness, and this method comprises that (i) screening patient is suffering from the whether treatment sensitivity to adopting the active compound of anti-cell cyclin-dependent kinase to be carried out of the morbid state that maybe may suffer from or illness to determine this patient; (ii) be instructed to give patient's formula defined herein (I) compound or its any subgroup or example when being responsive when disease of patient or illness.

. formula defined herein (I) compound or its any subgroup or example are used for the treatment of or prevent purposes in the medicine of disease of patient state or illness, wherein said patient to be examined and to be defined as suffering from morbid state or illness with treatment sensitivity that the active compound of anti-cell cyclin-dependent kinase carries out or the risk of suffering from these diseases or illness is arranged in production.

. be used to suppress formula defined herein (I) compound or its any subgroup or the example of mammal tumor growth.

. be used for suppressing formula defined herein (I) compound or its any subgroup or the example of growth of tumour cell (as Mammals).

. be used for suppressing the method for Mammals (as the mankind) tumor growth, this method comprises formula defined herein (I) compound or its any subgroup or the example that gives Mammals (as the mankind) inhibition tumor growth significant quantity.

. be used for suppressing the method for tumour cell (such as being present in for example mankind's tumour cell of Mammals) growth, this method comprises formula defined herein (I) compound or its any subgroup or the example that makes tumour cell contact inhibition tumor growth significant quantity.

. be used for the compound defined herein of described and described purposes in this paper other places and method above any.

General preferred and definition

In this application, unless indication is arranged in the context in addition, indication formula (I) compound comprises that all subgroups and the term ' subgroup ' of formula (I) are included in defined all preferably selections, specific embodiments, embodiment and particular compound herein as herein defined.

Any formula of indication in this article (I) also refers to any subgroup and its any preferred selection and embodiment of the compound in formula (I) scope, unless context has indication in addition.

Salt, solvate, tautomer, isomer, N-oxide compound, ester, prodrug and isotropic substance

Formula (I) compound and mentioning of its subgroup are also comprised its ionic species, salt, solvate, isomer, tautomer, N-oxide compound, ester, prodrug, isotropic substance and protected form, as discussed below those of example.Preferred its salt or tautomer or isomer or N-oxide compound or solvate; More preferably its salt or tautomer or N-oxide compound or solvate.

Many formulas (I) compound can exist with the form of salt, for example acid salt, perhaps can exist with salt such as carboxylate salt, sulfonate and the phosphatic form of organic or inorganic alkali in some cases.All these class salt include within the scope of the invention, to the salt form that comprises compound of mentioning of formula (I) compound.

Can use conventional chemical process such as Pharmaceutical Salts:Properties, Selection, and Use, P.Heinrich Stahl (editor), Camille G.Wermuth (editor), ISBN:3-90639-026-8, Hardcover, 388 pages, described method is by the synthetic salt of the present invention of the parent compound that contains alkalescence or acidic moiety in 2002 8 months.Generally speaking, free acid that this class salt can be by making these compounds or alkali form and suitable alkali or acid are reacted in water or in organic solvent or in the mixture of the two and are prepared; General non-aqueous media such as ether, ethyl acetate, ethanol, Virahol or the acetonitrile of using.

Can form acid salt with various acid (mineral acid and organic acid).The example of acid salt comprises the salt that forms with the acid that is selected from down group: acetate; 2; the 2-dichloro acetic acid; hexanodioic acid; Lalgine; xitix (for example L-xitix); the L-aspartic acid; Phenylsulfonic acid; phenylformic acid; the 4-acetylamino benzoic acid; butyric acid; (+) dextrocamphoric acid; camphor-sulfonic acid; (+)-(1S)-camphor-10-sulfonic acid; capric acid; caproic acid; sad; styracin; citric acid; cyclohexane sulfamic acid; dodecyl sulphate; ethane-1; the 2-disulfonic acid; ethyl sulfonic acid; the 2-ethylenehydrinsulfonic acid; formic acid; fumaric acid; tetrahydroxyadipic acid; 2; the 5-resorcylic acid; glucoheptonic acid; the D-glyconic acid; glucuronic acid (for example D-glucuronic acid); L-glutamic acid (for example L-L-glutamic acid); α-Yang Daiwuersuan; oxyacetic acid; urobenzoic acid; Hydrogen bromide; hydrochloric acid; hydroiodic acid HI; isethionic acid; (+)-L-lactic acid; (±)-DL-lactic acid; lactobionic acid; toxilic acid; oxysuccinic acid; (-)-L MALIC ACID; propanedioic acid; (±)-DL-amygdalic acid; methylsulfonic acid; naphthalene-2-sulfonic acid; naphthalene-1; the 5-disulfonic acid; 1-hydroxyl-2-naphthoic acid; nicotinic acid; nitric acid; oleic acid; vitamin B13; oxalic acid; palmitinic acid; pounce on nurse acid; phosphoric acid; propionic acid; the L-Pyrrolidonecarboxylic acid; Whitfield's ointment; 4-amino-Whitfield's ointment; sebacic acid; stearic acid; succsinic acid; sulfuric acid; tannic acid; (+)-L-tartrate; thiocyanic acid; right-toluenesulphonic acids; undecylenic acid and valeric acid, and the amino acid of acidylate and Zeo-karb.

A concrete group of salt is made up of the salt that forms with acetate, hydrochloric acid, hydroiodic acid HI, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succsinic acid, toxilic acid, oxysuccinic acid, isethionic acid, fumaric acid, Phenylsulfonic acid, toluenesulphonic acids, methylsulfonic acid (methanesulfonic), ethyl sulfonic acid, naphthene sulfonic acid, valeric acid, acetate, propionic acid, butyric acid, propanedioic acid, glucuronic acid and lactobionic acid.

A subgroup of salt is made up of the salt that forms with hydrochloric acid, acetate, methylsulfonic acid, hexanodioic acid, L-aspartic acid and DL-lactic acid.

Another subgroup of salt is made up of acetate, mesylate, esilate, DL-lactic acid salt, adipate, D-glucuronate, D-gluconate and hydrochloride.

The preferred salt that is used for preparing liquid (for example water-based) composition of formula as herein described (I) compound and subgroup and example is to have greater than 10 μ g/ml liquid vehicles (for example water), more generally greater than 0.5mg/ml, be preferably greater than the salt of the solubleness of 1mg/ml at given liquid vehicle (for example water).

In one embodiment of the invention, a kind of pharmaceutical composition is provided, and it comprises and contains concentration greater than 10 μ g/ml liquid vehicles (for example water), usually greater than 0.5mg/ml, formula as herein described (I) compound of salt form that is preferably greater than 1mg/ml and the aqueous solution of subgroup and example thereof.

If compound is an anionic property, perhaps have can be anionic property functional group (for example-COOH can be-COO), then can with suitable salt forming cation.The example of suitable inorganic cation includes but not limited to alkalimetal ion such as Na ⁺And K ⁺, alkaline earth metal cation such as Ca ²⁺And Mg ²⁺And other positively charged ion such as Al ³⁺Suitable organic cations example includes but not limited to ammonium ion (that is NH, ₄ ⁺) and substituted ammonium ion (for example, NH ₃R ⁺, NH ₂R ₂ ⁺, NHR ₃ ⁺, NR ₄ ⁺).The example of the substituted ammonium ion that some are suitable is derived from those of following material: ethamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, quadrol, thanomin, diethanolamine, piperazine, benzylamine, phenylbenzyl amine, choline, meglumine and tromethane, and amino acid, as Methionin and arginine.An example of common quaternary ammonium ion is N (CH ₃) ⁴⁺

Contain under the situation of amine functional group at formula (I) compound, these compounds can form quaternary ammonium salt, for example by according to well known to a person skilled in the art that method and alkylation reactions form quaternary ammonium salt.This class quaternary ammonium compound is in the scope of formula (I).

The salt form of compound of the present invention is pharmacologically acceptable salt normally, and the example of pharmacologically acceptable salt is at Berge etc., and 1977, " Pharmaceutically Acceptable Salts, " J.Pharm.Sci., the 66th volume has discussion in the 1-19 page or leaf.Yet pharmaceutically unacceptable salt also can be produced with intermediate forms, can convert it into pharmacologically acceptable salt then.The pharmaceutically unacceptable salt form of this class for example can be used for purifying or separates compound of the present invention, and they have also formed a part of the present invention.

Formula (I) compound that contains amine functional group also can form the N-oxide compound.This paper also comprises the N-oxide compound to the mentioning of formula (I) compound of containing amine functional group.

Contain at compound under the situation of several amine functional groups, one or more nitrogen-atoms can oxidized formation N-oxide compound.The object lesson of N-oxide compound has the N-oxide compound of tertiary amine or nitrogenous heterocyclic nitrogen-atoms.

Can form the N-oxide compound by handling corresponding amine with oxygenant such as hydrogen peroxide or peracid (for example peroxycarboxylic acid), referring to for example Advanced Organic Chemistry, Jerry March, the 4th edition, Wiley Interscience, page or leaf.More specifically, can pass through L.W.Deady (Syn.Comm.1977,7, working method 509-514) prepares the N-oxide compound, wherein amine compound and-chlorine peroxybenzoic acid (MCPBA) reaction, for example in inert solvent such as methylene dichloride, react.

Formula (I) compound can exist with many different rotamerism form and tautomeric forms, and mentioning of formula (I) compound comprised all these class forms.For fear of doubt, can exist and only specifically describe or provided under a kind of situation with one of several rotamerism forms or tautomeric form at compound, all other rotamerism forms or tautomeric form are also included within the formula (I).

For example, in formula (I) compound, the pyrazoles ring can exist with the form of following two kinds of tautomeric form A and B.For easy, general formula (I) has provided form A, but should think that described general formula comprises two kinds of tautomeric forms.

Other example of tautomeric form for example comprise ketone group-, enol-and enolate-form, for example following tautomer centering: ketone group/enol (providing below), imines/enamine, acid amides/imino-alcohol, amidine/amidine, nitroso-group/oxime, thioketones/alkene mercaptan and nitro/acid-nitro (aci-nitro).

Unless context has requirement in addition, otherwise contain one or more chiral centres (R therein for example at formula (I) compound ⁴Be in the situation of compound of 2-butyl) and the situation that can exist with the form of two or more optically active isomers under, mentioning of formula (I) compound comprised its all rotational isomerism forms (for example enantiomorph, epimer and diastereomer), the mixture that described rotational isomerism form is single optically active isomer or two or more optically active isomers (for example racemic mixture).

Can characterize and differentiate optically active isomer (promptly with its optically active, for+and-isomer, or d and l isomer) or can characterize them with " R and S " nomenclature that Cahn, Ingold and Prelog set up according to its absolute stereo chemistry, referring to Advanced Organic Chemistry, JerryMarch, the 4th edition, John Wiley ﹠amp; Sons, New York, 1992, the 109-114 pages or leaves, also referring to Cahn, Ingold﹠amp; Prelog, Angew.Chem.Int.Ed.Engl., 1966,5,385-415.

Can be with comprise that chiral chromatography (chromatography of carrying out) separates optically active isomer in interior many technology on chiral support, this class technology is known in those skilled in the art.

Alternative as chiral chromatography; separating optical isomers by the following method: form diastereomeric salt as (+)-tartrate, (-)-Pyrrolidonecarboxylic acid, (-)-two-toluyl-L-tartrate, (+)-amygdalic acid, (-)-oxysuccinic acid and (-)-camphorsulfonic acid with chiral acid; separate diastereomer with selective freezing (preferentialcrystallisation), then salt is dissociated into the single enantiomer of free alkali.

Under the situation that formula (I) compound exists with two or more optically active isomer forms, a kind of enantiomorph in a pair of enantiomorph can be better than another kind of enantiomorph, for example, is better than another kind of enantiomorph aspect biologic activity.Therefore, in some cases, only may need with a kind of in a pair of enantiomorph or only use a kind of in the multiple diastereomer as therapeutical agent.Therefore, the invention provides and contain formula (I) compound compositions with one or more chiral centres, wherein at least 55% formula (I) compound of (for example at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%) exists with the form of single optical isomer (for example enantiomorph or diastereomer).In a general embodiment, 99% or more (for example whole basically) of formula (I) total amount of compound can exist with the form of single optical isomer (for example enantiomorph or diastereomer).

Compound of the present invention comprises having the compound that one or more isotropic substances replace, to all isotropic substances that comprise this element in its scope of mentioning of concrete element.For example, mentioning in its scope of hydrogen comprised ¹H, ²H (D) and ³H (T).Similarly, mentioning in its scope of carbon and oxygen comprised respectively ¹²C, ¹³C and ¹⁴C with ¹⁶O and ¹⁸O.

Isotropic substance can be radioactive or inactive.In one embodiment of the invention, compound does not contain radio isotope.Preferably this compounds is used for the treatment of purposes.Yet in another embodiment, compound can contain one or more radio isotope.Containing the radioisotopic compound of this class may be useful under the situation of diagnosis.

Ester is also included within formula (I) scope as the carboxylicesters and the acyloxyate of formula (I) compound that has hydroxy-acid group or hydroxyl.The example of ester has and contains group-C (=O) compound of OR, wherein R is ester substituting group, for example C _1-7Alkyl, C _3-20Heterocyclic radical or C _5-20Aryl, preferred C _1-7Alkyl.The object lesson of ester group includes but not limited to-C (=O) OCH ₃,-C (=O) OCH ₂CH ₃,-C (=O) OC (CH ₃) ₃With-C (=O) OPh.The example of acyloxy (oppositely ester) group with-OC (=O) R represents, wherein R is acyloxy substituting group, for example C _1-7Alkyl, C _3-20Heterocyclic radical or C _5-20Aryl, preferred C _1-7Alkyl.The object lesson of acyloxy includes but not limited to-OC (=O) CH ₃(acetoxyl group) ,-OC (=O) CH ₂CH ₃,-OC (=O) C (CH ₃) ₃,-OC (=O) Ph and-OC (=O) CH ₂Ph.

Formula (I) also comprise any polymorphic form of compound, the solvate of compound (for example hydrate), mixture (for example with such as the inclusion complex of compounds such as cyclodextrin or inclusion compound or with the mixture of metal) and the prodrug of compound." prodrug " means any compound of the biologically active cpds that for example is converted to formula (I) in vivo.

For example, some prodrugs are esters (for example, physiology acceptable be easy to metabolic ester) of active compound.In metabolic process, the cleaved generation active medicine of ester group (C (=O) OR).This class ester can form by the esterification of any hydroxy-acid group in the parent compound for example (C (=O) OH), under suitable situation, any other active group in the parent compound is protected in advance, if desired, goes protection afterwards again.

The example that this class is easy to metabolic ester comprise formula-C (=O) those of OR, wherein R is:

C _1-7Alkyl

(for example ,-Me ,-Et ,-nPr ,-iPr ,-nBu ,-sBu ,-iBu ,-tBu);

C _1-7Aminoalkyl group

(for example, amino-ethyl; 2-(N, N-diethylamino) ethyl; 2-(4-morpholino) ethyl); With

Acyloxy-C _1-7Alkyl

(for example, acyloxy methyl;

The acyloxy ethyl;

Oxy acid methyl neopentyl;

Acetoxy-methyl;

1-acetoxyl group ethyl;

1-(1-methoxyl group-1-methyl) ethyl-ketonic oxygen base ethyl;

1-(benzoyloxy) ethyl; Isopropoxy-ketonic oxygen ylmethyl;

1-isopropoxy-ketonic oxygen base ethyl; Cyclohexyl-ketonic oxygen ylmethyl;

1-cyclohexyl-ketonic oxygen base ethyl;

Cyclohexyl oxygen base-ketonic oxygen ylmethyl;

1-cyclohexyl oxygen base-ketonic oxygen base ethyl;

(4-THP trtrahydropyranyl oxygen base) ketonic oxygen ylmethyl;

1-(4-THP trtrahydropyranyl oxygen base) ketonic oxygen base ethyl;

(4-THP trtrahydropyranyl) ketonic oxygen ylmethyl; With

1-(4-THP trtrahydropyranyl) ketonic oxygen base ethyl).

In addition, some prodrugs are produced the compound (for example, in ADEPT, GDEPT, LIDEPT etc.) of active compound or the further chemical reaction generation of warp active compound by the enzymatic activation.For example, prodrug can be sugar derivatives or other glucosides binding substances, perhaps can be amino acid ester derivative.

Biological activity

Formula (I) compound and its subgroup are the inhibitor of cell cycle protein dependent kinase.For example, compound of the present invention be cell cycle protein dependent kinase, particularly be selected from CDK1, CDK2, CDK3, CDK4, CDK5, CDK6 and CDK9, more especially be selected from the inhibitor of the cell cycle protein dependent kinase of CDK1, CDK2, CDK3, CDK4, CDK5 and CDK9.

Preferred compound is to suppress one or more to be selected from the compound of CDK kinases, for example CDK1 and/or the CDK2 of CDK1, CDK2, CDK4 and CDK9.

Compound of the present invention also has the activity of anti-glycogen synthase kinase-3 (GSK3).

Because their activity in adjusting or inhibition CDK and glycogen synthase kinase estimate that compound of the present invention can be used for being provided at the means of ending the control of cell cycle or recovery cell cycle in the cell of abnormal division.Therefore, estimate that described compound will can be used for treatment or prevention proliferative disease such as cancer.Estimate that also compound of the present invention will can be used for treatment such as paralysis, the sex change of cortex basal nuclei and Pick's disease illnesss such as (Pick ' s disease), for example autoimmune disorder and neurodegenerative disease on virus infection, II type or non insulin dependent diabetes, autoimmune disorder, injury of head, apoplexy, epilepsy, neurodegenerative disease such as alzheimer's disease, motor neurone disease, the carrying out property nuclear.

Estimate that morbid state and illness subgroup that compound of the present invention is useful therein be made up of virus infection, autoimmune disorder and neurodegenerative disease.

CDK at cell cycle, apoptosis, transcribe, work in the adjusting of differentiation and CNS function.Therefore, the CDK inhibitor can be used for treating the disease cancer for example that wherein has propagation, apoptosis or prosoplasia.Particularly the RB+ve tumour may be responsive especially to the CDK inhibitor.The RB-ve tumour also may be to CDK inhibitor sensitivity.

Example that can repressed cancer includes but not limited to: cancer, for example bladder cancer, mammary cancer, colorectal carcinoma (for example colorectal carcinoma such as adenocarcinoma of colon and adenoma of colon), kidney, epidermal carcinoma, liver cancer, lung cancer, for example exocrine pancreas cancer (exocrine pancreatic carcinoma), cancer of the stomach, cervical cancer, thyroid carcinoma, prostate cancer or skin carcinoma, for example squamous cell carcinoma of gland cancer, small cell lung cancer and nonsmall-cell lung cancer, esophagus cancer, carcinoma of gallbladder, ovarian cancer, carcinoma of the pancreas for example; The hematopoietic system cancer of lymph pedigree (hematopoietic tumors of lymphoid lineage), for example leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, B-cell lymphoma (as the diffuse type large B cell lymphoid tumor), T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hair cell lymphoma or Burkitt lymphoma; The hematopoietic system cancer of marrow pedigree (hematopoietic tumour of myeloidlineage), for example acute and chronic myelogenous leukemia, myelodysplastic syndromes or promyelocytic leukemia; Follicular carcinoma of thyroid; The tumour of mesenchyme origin, for example fibrosarcoma or rhabdosarcoma; The tumour of central or peripheral nervous system, for example astrocytoma, neuroblastoma, neurospongioma or schwannoma; Melanoma; Spermocytoma; Teratocarcinoma; Osteosarcoma; Xeroderma pitmentosum; Keratoacanthoma (keratoctanthoma); Follicular carcinoma of thyroid; Or Kaposi sarcoma.

Cancer can be to the cell cycle protein dependent kinase of any or multiple CDK1 of being selected from, CDK2, CDK3, CDK4, CDK5 and CDK6, for example one or more are selected from the cancer of inhibition sensitivity of CDK kinases, for example CDK1 and/or the CDK2 of CDK1, CDK2, CDK4 and CDK5.

Can utilize the cell growth measurement method or the title that provide among the embodiment hereinafter to determine for the method that provides in the part of " diagnostic method " whether a kind of specific cancer is the cancer of the inhibition sensitivity of cell cycle protein dependent kinase.

Also known CDK apoptosis, propagation, break up and transcribe in play a role, therefore the CDK inhibitor also can be used for treating cancer following disease in addition: virus infection, for example simplexvirus, poxvirus, Epstein-Barr virus, sindbis virus, adenovirus, HIV, HPV, HCV and HCMV; Prevention AIDS takes place in the individuality that HIV-infects; Chronic inflammatory disease, for example glomerulonephritis, rheumatoid arthritis, psoriatic, inflammatory bowel and the autoimmune diabetes of systemic lupus erythematous, autoimmunization mediation; Cardiovascular disorder is cardiac hypertrophy, restenosis, atherosclerosis for example; Neurodegenerative disease, for example alzheimer's disease, dementia, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, Duchenne-Arandisease and the cerebellar degeneration relevant with AIDS; Glomerulonephritis; Myelodysplastic syndrome, myocardial infarction, apoplexy and the reperfusion injury relevant, irregular pulse, atherosclerosis, toxin-bring out with ischemia injury or with alcohol relevant hepatopathy, blood disease, for example chronic anaemia and aplastic anemia; The degenerative disease of musculoskeletal system, for example osteoporosis and sacroiliitis, aspirin sensitive sinusitis paranasal sinusitis, cystic fibrosis, multiple sclerosis, kidney disease and cancer pain.

Have been found that also some cell cycle protein dependent kinase inhibitors can be used in combination with other carcinostatic agent.For example, the cell cycle protein dependent kinase inhibitor good fortune draws benefit (flavopiridol) to be used from combined therapy with other carcinostatic agent one.

Therefore, in pharmaceutical composition, purposes or the method that is used for the treatment of the disease that comprises abnormal cell growth or illness of the present invention, the disease or the illness that comprise abnormal cell growth are cancer in one embodiment.

One group of cancer comprises human breast carcinoma (for example primary breast cancer, lymphoglandula negative breast cancer, mammary gland infiltration duct adenocarcinoma, non-endometrial-like mammary cancer (non-endometrioid breast cancers)); And lymphoma mantle cell.In addition, other cancer has colorectal carcinoma and carcinoma of endometrium.

Another subgroup of cancer comprises the hematopoietic system cancer of lymph pedigree, for example leukemia, chronic lymphocytic leukemia, lymphoma mantle cell and B-cell lymphoma (as the diffuse type large B cell lymphoid tumor).

A kind of concrete cancer is a chronic lymphocytic leukemia.

Another kind of concrete cancer is a lymphoma mantle cell.

Another kind of concrete cancer is the diffuse type large B cell lymphoid tumor.

Another subgroup of cancer comprises mammary cancer, ovarian cancer, colorectal carcinoma, prostate cancer, esophagus cancer, squamous cell carcinoma and nonsmall-cell lung cancer.

Can measure the activity of compound of the present invention as the inhibitor of cell cycle protein dependent kinase and glycogen synthase kinase-3 with the assay method that provides among the embodiment hereinafter, the activity level that given compound showed can be used IC ₅₀Value defines.Preferred The compounds of this invention is IC ₅₀Value less than 1 micromole, be more preferably less than 0.1 micromolar compound.

The advantage of The compounds of this invention

The compound of formula defined herein (I) and its subgroup for example compound 4-(2,6-two chloro-benzamidos)-1H-pyrazoles-3-formic acid (1-methane sulfonyl-piperidin-4-yl)-acid amides have the advantage of the compound that is better than prior art.

The compounds of this invention may have the physico-chemical property that is suitable for oral contact.

The compounds of this invention in the HCT-116 cell to the IC of Transcription ₅₀Be higher than IC to proliferation function ₅₀: therefore, for example, to the IC of Transcription ₅₀Be higher than IC to proliferation function ₅₀About 100 times.This is favourable, thereby because compound can tolerate its administration and the long time of administration under higher level that allows better.

Particularly, with respect to the compound of prior art, formula (I) compound exhibits goes out improved oral administration biaavailability.The ratio (F) of the plasma exposure value of compound and the plasma exposure value of compound when intravenously (i.v.) administration when oral administration biaavailability may be defined as the by oral route administration of representing with per-cent.

The compound particularly advantageous of oral administration biaavailability (F value) greater than 30%, more preferably greater than 40%, because they can be used by Orally administered but not parenteral, perhaps not only can be by Orally administered but also can be used by parenteral.

For example, compound 4-(2,6-two chloro-benzamidos)-1H-pyrazoles-3-formic acid (1-methane sulfonyl-piperidin-4-yl)-acid amides has the 40-50% bioavailability during to the mouse administration with oral route.

Compound of the present invention; for example compound 4-(2; 6-two chloro-benzamidos)-and 1H-pyrazoles-3-formic acid (1-methane sulfonyl-piperidin-4-yl)-acid amides, have higher kinases (CDK2) vitro inhibition activity and cancerous cell line is had stronger anti-proliferative effect.In addition, compound has than low activity and to the selectivity of CDK2 GSK3 β and surpasses GSK3 β.Therefore the effect of compound mainly is to influence the cell cycle by suppressing CDK, and and without suppress the additional result that for example insulin sensitivity, somatomedin effect are produced owing to GSK3 β.Compound have clearer cell cycle inhibition feature and still less from side effect via the additional result of GSK3 β.Compound 4-(2,6-two chloro-benzamidos)-1H-pyrazoles-3-formic acid (1-methane sulfonyl-piperidin-4-yl)-acid amides and its 2, relatively the listing in following examples 11 of the biological characteristics of 6-difluoro benzamido analogue.

The method of preparation formula (I) compound

In all other parts of this part and the application, unless context has prompting in addition, indication formula (I) also comprises its subgroup and the embodiment that all are defined herein.When mentioning R ¹And R ³When group or any other " R " group, the definition of described group is as mentioned above with as described in the following paragraph of the application, unless context has requirement in addition.

Formula (I) compound can be according to synthetic method known to the skilled preparation with by following and prepare as the described method of our application PCT/GB2004/003179 (WO2005/012256) (its content is incorporated in herein as a reference).

For example, formula (I) compound can be prepared by the reaction sequence shown in the flow process 1.

The raw material that is used for the synthesis path shown in the flow process 1 is 4-nitro-pyrazoles-3-carboxylic acid (X), and it can obtain from commercial channels or can be by the nitrification preparation of the unsubstituted pyrazoles carboxylic compound of corresponding 4-.

Flow process 1

By in the presence of acid catalyst or thionyl chloride with suitable alcohol ethanol synthesis for example, nitro-pyrazole carboxylic acid (X) is changed into corresponding ester (XI), for example methyl esters or ethyl ester (being depicted as its ethyl ester).Reaction can at room temperature use the alcohol that is used for esterification to carry out as solvent.

Nitro-ester (XI) can be reduced into corresponding amine (XII) by being used for that nitro is changed into amino standard method.Therefore, for example, nitryl group can be reduced into amine by carrying out hydrogenation with Pd/carbon catalyst.Hydrogenation can at room temperature for example carry out in the ethanol in solvent.

The amine that is produced (XII) can by with formula R ¹The acyl chlorides of COCl non-interfering alkali for example triethylamine in the presence of reaction change into acid amides (XIII).Reaction can for example carried out in the dioxan in polar solvent under about room temperature.Acyl chlorides can be by handling carboxylic acid R with thionyl chloride ¹CO ₂H, or pass through in the presence of the dimethyl formamide of catalytic amount, to react with oxalyl chloride, or prepare by the sylvite of acid and the reaction of oxalyl chloride.

As the alternative method of the method for above-mentioned use acyl chlorides, amine (XII) can by with carboxylic acid R ¹CO ₂H is being generally used for forming the reaction in the presence of the acid amides coupling reagent of peptide key type and is changing into acid amides (XIII).The example of these reagent comprises 1,3-dicyclohexylcarbodiimide (DCC) (people such as Sheehan, J.Amer.Chem Soc.1955,77,1067), 1-ethyl-3-(3 '-dimethylamino-propyl)-carbodiimide (is referred to herein as EDC or EDAC, but be also referred to as EDCI and WSCDI in the art) (people such as Sheehan, J.Org.Chem., 1961,26,2525), based on the coupler of uronium (uronium) O-(7-azepine benzo triazol-1-yl)-N for example, N, N ', the coupler of N '-tetramethyl-urea hexafluorophosphate (HATU) and Ji Yu Phosphonium is 1-benzotriazole oxygen base three-(tetramethyleneimine-1-base) Phosphonium hexafluorophosphate (PyBOP) (people such as Castro, Tetrahedron Letters, 1990 for example, 31,205).Based on the coupler of carbodiimide advantageously with 1-hydroxyl-7-azepine benzotriazole (HOAt) (L A.Carpino, J.Amer.Chem.Soc., 1993,115,4397) or I-hydroxybenzotriazole (HOBt) (people such as Konig, Chem.Ber., 103,708,2024-2034) be used in combination.Preferred coupler comprises EDC (EDAC) and the DCC with HOAt or HOBt combination.

Coupled reaction is usually at nonaqueous aprotic solvent, for example in acetonitrile, dioxan, methyl-sulphoxide, methylene dichloride, dimethyl formamide or the N-crassitude, or randomly contains in the aqueous solvent of one or more mixable cosolvent and carries out.Reaction can at room temperature be carried out, and perhaps when the reactivity of reactant is low (for example have electron-withdrawing group for example the situation of the aniline of the electron deficiency of sulfuryl amine group under) carries out under the temperature that suitably raises.Reaction can be at non-interfering alkali, and for example tertiary amine such as triethylamine or N carry out under the existence of N-diisopropylethylamine.

Acid amides (XIII) subsequently by the use alkali metal hydroxide for example sodium-hydroxide treatment be hydrolyzed into carboxylic acid (XIV).Saponification reaction can for example alcohol (for example methyl alcohol) carries out and reaction mixture is heated to non-extreme temperature usually with organic cosolvent, for example the highest about 50-60 ℃.

Carboxylic acid (XIV) then can be by using above-mentioned acid amides formation condition and amine R ³-NH ₂React and the compound of a conversion accepted way of doing sth (I).Therefore, for example, the acid amides coupled reaction can be carried out in polar solvent such as DMF in the presence of EDC and HOBt.

Another synthetic wherein R ^2bFor the general approach of formula (I) compound of hydrogen is shown in the flow process 2.

Flow process 2

In flow process 2, use above-mentioned acid amides formation condition with nitro-pyrazoles-carboxylic acid (X) or its activatory derivative for example chloride of acid and amine R ³-NH ₂Reaction is to produce nitro-pyrazoles-acid amides (XV), and then with the standard method of reduction nitryl group, the method for hydrogenation of for example above-mentioned use Pd/C catalyzer is reduced into corresponding aminocompound (XVI) for it.

Then with amine (XVI) and formula R ¹-CO ₂The carboxylic acid of H or its activatory derivative for example chloride of acid or acid anhydrides above about flow process 1 described acid amides formation condition under coupling.Therefore, for example, as the alternative method of using chloride of acid, coupled reaction can for example carried out among the DMF with production (I ') compound in solvent in the presence of EDAC (EDC) and the HOBt, and it is equivalent to wherein R ^2bIt is formula (I) compound of hydrogen.

Formula (I) compound also can by with suitable sulfonylation agent, for example for example methane sulfonyl chloride reaction and of SULPHURYL CHLORIDE from formula (XVII) compound.

The compound of display type (XVII) transforms the illustrative reaction sequence of the sulfonyl-derivatives of an accepted way of doing sth (I) and lists in the flow process 3.

Flow process 3

Shown in flow process 3, R wherein ³For having alkylsulfonyl group-SO ₂R ⁴Formula (I) compound (being the compound of formula (XIX)) of piperidine ring can be by with the compound and the SULPHURYL CHLORIDE R of formula (XVII) ⁴SO ₂Cl (for example methane sulfonyl chloride) interference alkali for example diisopropylethylamine in the presence of react and prepare.Reaction is at room temperature for example carried out in dioxan and the methylene dichloride in non-aqueous aprotic solvent usually.

Formula R ⁴SO ₂The SULPHURYL CHLORIDE of Cl can obtain from commercial source, maybe can make by several different methods.For example, alkyl sulfonyl chloride can make by the following method: with the reaction of haloalkane and S-WAT and aqueous organic solvent for example in water/dioxan heating then in the presence of DMF, handle with the generation SULPHURYL CHLORIDE to form corresponding sulfonic acid with thionyl chloride.

In an optional preparation method, mercaptan R ⁴SH/R ^4aSH can react to produce required SULPHURYL CHLORIDE with saltpetre and sulfuryl chloride.

In many above-mentioned reactions, one or more groups that may need protection react on undesirable position of molecule preventing.The example of blocking group, and the method for protecting and go to protect functional group are found in Protective Groupsin Organic Synthesis (T.Green and P.Wuts; 3rd Edition; John Wiley and Sons, 1999).For example, amine groups can protected precedent such as acid amides (NRCO-R) or urethanum (NRCO-OR), for example, protected one-tenth: methyl nitrosourea (NHCO-CH ₃); Benzyloxy acid amides (NHCO-OCH ₂C ₆H ₅,-NH-Cbz); Protected one-tenth tert.-butoxy acid amides (NHCO-OC (CH ₃) ₃,-NH-Boc); 2-biphenyl-2-propoxy-acid amides (NHCO-OC (CH ₃) ₂C ₆H ₄C ₆H ₅,-NH-Bpoc); protected one-tenth 9-fluorenyl methoxy acid amides (NH-Fmoc); protected one-tenth 6-nitro black false hellebore oxygen base acid amides (NH-Nvoc); protected one-tenth 2-trimethyl silane base oxethyl acid amides (NH-Teoc); protected one-tenth 2,2,2-three chloroethoxy acid amides are (NH-Troc); protected one-tenth allyloxy acid amides (NH-Alloc), or protected one-tenth 2 (phenyl sulfonyl) oxyethyl group acid amides (NH-Psec).Other protecting group of amine (for example cyclammonium and heterocyclic N-H group) comprise tosyl group (tosyl group) and methane sulfonyl (methylsulfonyl) group and benzyl group for example right-mehtoxybenzyl (PMB) group.Hydroxy-acid group can protected one-tenth ester, for example, and protected one-tenth: C _1-7Alkyl ester (for example, methyl esters; The tert-butyl ester); C _1-7Haloalkyl ester (for example, C _1-7The tri haloalkyl ester); Three C _1-7Alkyl tin groups, alkyl silane groups-C _1-7Alkyl ester; Or C _5-20Aryl-C _1-7Alkyl ester (for example, benzene methyl; The oil of mirbane methyl esters); Or protected one-tenth acid amides, for example, methyl nitrosourea.Thiol group can protected precedent such as thioether (SR), for example, protect: the phenmethyl thioether; Acetylamino methyl ether (S-CH ₂NHC (=O) CH ₃).

Employed new chemical intermediate among aforesaid method and the embodiment (for example formula (XIII), (XIV), (XV) and new compound (XVI)) has been represented another aspect of the present invention.

The method of purifying

Compound can separate and purifying by many methods well known to those skilled in the art, and the example of these methods comprises chromatographic technique for example column chromatography (for example flash chromatography) and HPLC.Preparation type LC-MS is a kind of purifying the organic molecule for example standard and effective means of compound described herein of being used for.Can change the method for liquid chromatography (LC) and mass spectrum (MS), so that crude product separates better and improves the detection of MS to sample.The optimization of preparation type gradient LC method comprises change pillar, volatility eluent and properties-correcting agent and gradient.The method that optimization prepares the method for type LC-MS, use it for purifying compounds then is known in the art.These class methods are described in RosentreterU, Huber U.; Optimal fraction collecting in preparative LC/MS; J CombChem.; 2004; 6 (2), 159-64 and Leister W, Strauss K, Wisnoski D, Zhao Z, Lindsley C., Development of a custom high-throughput preparative liquidchromatography/massspectrometer platformfor the preparativepurification and analytical analysis of compound libraries; J Comb Chem.; 2003; 5 (3); Among the 322-9.

One is come this type systematic of purifying compounds to be described in following experimental section via preparation type LC-MS, but it will be understood by those skilled in the art that and can use other system and method different with described system and method.Particularly, can utilize the method replacement inversion method described herein for preparing type LC based on positive.Most of preparation type LC-MS system utilizes anti-phase LC and volatile acid modification agent because this method for micromolecular purifying very effectively and eluent and the cation electrodeposition mass spectrometry of spraying compatible.Also can adopt other chromatogram scheme, the positive LC that summarizes in for example above-mentioned analytical procedure, alternate buffering moving phase, alkaline properties-correcting agent wait these compounds of purifying.

Pharmaceutical preparation

Though active compound can be used separately, but preferably its form with pharmaceutical composition (for example preparation) is given, described composition comprises at least a active compound of the present invention and one or more pharmaceutically useful carriers, assistant agent, vehicle, thinner, weighting agent, buffer reagent, stablizer, sanitas, lubricant or other material well known to those skilled in the art, and randomly comprises other treatment or prophylactic substance; For example reduce or alleviate the material of some side effects relevant with chemotherapy.The object lesson of this class material comprises that extended period that antiemetic and prevention or the minimizing neutrophilic leukocyte relevant with chemotherapy reduce and prevention result from the material of the complication that red corpuscle or leucocyte level reduce, for example erythropoietin (EPO), rHuGM-CSF (GM-CSF), granulocyte colony-stimulating factor (G-CSF).

Therefore, the present invention also provides the method for pharmaceutical composition and pharmaceutical compositions as defined above, and it comprises at least a active compound is as defined above mixed mutually with one or more pharmaceutically useful carriers as herein described, vehicle, buffer reagent, assistant agent, stablizer or other material.

Term used herein " pharmaceutically useful " relates to and is suitable for contacting with the tissue of individual (for example people) in the rational medicine determination range and does not produce over-drastic toxicity, stimulation, transformation reactions or other problem or complication, have compound, material, composition and/or the formulation of rational benefit/risk ratio.Every kind of carrier, vehicle etc. also must with preparation in the compatible meaning of other composition on be " acceptable ".

Therefore, on the other hand, the invention provides formula defined herein (I) compound and its subgroup of pharmaceutical compositions.

Pharmaceutical composition can be anyly be suitable in oral, parenteral, part, the nose, the form of eye, ear and rectum, intravaginal or transdermal administration.Under composition was used for situation that parenteral uses, they can be used for intravenously, intramuscular, intraperitoneal, subcutaneous administration or directly be delivered to target organ or tissue by injection, infusion or other delivery means by preparation.Send and to realize by bolus injection, short-term infusion or long-term infusion, and can send or by utilizing suitable infusion pump to realize via passive.

Be suitable for the pharmaceutical preparation that parenteral uses and comprise water-based and non-aqueous aseptic parenteral solution, its can contain antioxidant, buffer reagent, fungistat, cosolvent, ORGANIC SOLVENT MIXTURES, cyclodextrin complexing agent, emulsifying agent (being used to form and stable emulsion) but, be used to form the liposome component of liposome, the gelation polymer that is used to form polymeric gel, freezing drying protective agent and in particular for the activeconstituents of stable meltable form and combinations of substances that preparation and expection recipient's blood etc. is opened.Be used for the form that pharmaceutical preparation that parenteral uses also can adopt water-based and non-aqueous sterile suspension, it can comprise suspending agent and thickening material (R.G.Strickly, Solubilizing Excipients in oral and injectableformulations.Pharmaceutical Research, 21 volumes (2) 2004, the 201-230 page or leaf).

If the pH value difference of the pKa of medicine and preparation is apart from enough big, then can make ionogenic drug molecule dissolving reach desired concn by adjusting pH.For intravenously and intramuscular administration, acceptable scope is pH2-12, but the acceptable scope of subcutaneous administration is pH2.7-9.0.PH value of solution by medicine salt form, strong acid/alkali example hydrochloric acid or sodium hydroxide or control by the buffered soln that glycine, Citrate trianion, acetate, maleate, succinate, Histidine, phosphoric acid salt, three (methylol) aminomethane (TRIS) or carbonate form by including but not limited to.

The normal combination of using aqueous solution and water-miscible organic solvent/tensio-active agent (being cosolvent) in injection formulations.Being used in water-miscible organic solvent in the injection formulations and tensio-active agent comprises but is not limited to propylene glycol, ethanol, Liquid Macrogol, poly(oxyethylene glycol) 400, glycerine, N,N-DIMETHYLACETAMIDE (DMA), N-N-methyl-2-2-pyrrolidone N-(NMP; Pharmasolve), methyl-sulphoxide (DMSO), Solutol HS15, Cremophor EL, Cremophor RH60 and Polysorbate 80.This class preparation is usually can (but not always) not diluted before injection.

Be used in propylene glycol, PEG300, ethanol, CremophorEL, Cremophor RH60 and Polysorbate 80 in the injection formulations of commercially available acquisition and be fully and can and can combination with one another use with the miscible organic solvent of water and tensio-active agent.The gained organic formulations dilutes at least 2 times usually before dense notes of intravenously or intravenous infusion.

Perhaps, can be by reaching the water-soluble of increase with the cyclodextrin complexing.

The spherical vesicle of sealing of that liposome is made up of the water-based core of outer field lipid duplicature and internal layer and have＜overall diameter of 100 microns.According to the hydrophobicity degree, if medicine is enclosed or embedded in the liposome, appropriate hydrophobic medicine can be by liposome dissolving.If drug molecule becomes the part of lipid duplicature, hydrophobic drug also can be by liposome dissolving, and in the case, hydrophobic drug is dissolved in the lipid part of lipid bilayer.Common Liposomal formulation contain water and-phosphatide of 5-20mg/ml, isotonic agent (isotonicifier), pH5-8 damping fluid, and randomly contain cholesterol.

Preparation may reside in the ampoule and bottle that unitary dose or multi-dose container for example seal, and can be stored under lyophilize (freeze-drying) condition, only needs add at once before use for example water for injection of sterile liquid carrier.

Pharmaceutical preparation can prepare by lyophilize formula (I) compound or its acid salt.Lyophilize refers to the working method of freeze-dried composition.Therefore, freeze-drying and lyophilize are in this article as synonym.Usual method is with compound dissolution and with the clarification of gained preparation, sterile filtration and change under aseptic condition and be suitable in the cryodesiccated container (for example bottle).Under the situation of bottle, they by with the freeze-drying plug portion clog.Can be cooled to preparation freezing and carry out lyophilize under standard conditions, sealing be added a cover to form stable exsiccant lyophily preparation then.Composition has low residual moisture content usually, for example based on the weight of lyophile, less than 5 weight % for example less than the residual moisture content of 1 weight %.

Freeze-dried preparation can contain other vehicle, for example thickening material, dispersion agent, buffer reagent, antioxidant, sanitas and tension regulator.Common buffer reagent comprises phosphoric acid salt, acetate, Citrate trianion and glycine.Examples of antioxidants comprises xitix, sodium bisulfite, sodium metabisulphite, thioglycerin, thiocarbamide, Yoshinox BHT, butylated hydroxyanisol and edetate.Sanitas can comprise alkyl ester, phenol, butylene-chlorohydrin, benzylalcohol, Thiomersalate, benzalkonium chloride and the cetylpyridinium chloride of phenylformic acid and its salt, Sorbic Acid and its salt, right-hydroxy-benzoic acid.If necessary, aforementioned buffer reagent and glucose and sodium-chlor can be used for tension adjustment.

Extender generally is used for Freeze Drying Technique to help to process and/or provide the volume and/or the mechanical integrity of lyophilize piece.Extender means the thinner of solid granular soluble in water, and when with the compound or its salt lyophilize, it provides physically stable lyophilize piece, more excellent freeze-drying method and rapidly and reconstruct completely.Extender also can be used for making solution etc. to open.

Water-soluble bulk can be any cryodesiccated pharmaceutically useful inert solids that are generally used for.This class extender comprises that for example sugar is as glucose, maltose, sucrose and lactose; Polyvalent alcohol such as sorbyl alcohol or N.F,USP MANNITOL; Amino acid such as glycine; Polymkeric substance such as polyvinylpyrrolidone; With polysaccharide such as dextran.

The weight of extender and the weight ratio of active compound are generally about 1 to about 5, for example about 1 to about 3, for example about 1 to 2.

Perhaps, they can be provided with the solution form that can be concentrated and be sealed in the suitable bottle.The sterilization of formulation can be by filtering or realizing at the autoclaving in the suitable stage of process for preparation by bottle and its content.The preparation that is provided may need further dilution or preparation before sending, for example be diluted to suitable aseptic infusion bag.

Interim blending type injection solution and suspension can be from aseptic powder, particle and tablet preparation.

In an embodiment preferred of the present invention, pharmaceutical composition is to be suitable for intravenously to use the form of for example using by injection or infusion intravenously.

Be used for the sterilized powder that the pharmaceutical composition of the present invention of parenteral injection also can comprise pharmaceutically useful sterile aqueous or non-aqueous solution, dispersion liquid, suspension or emulsion and be reconstructed into aseptic parenteral solution or dispersion liquid before use at once.The example of suitable water-based and non-aqueous carrier, thinner, solvent or medium comprises water, ethanol, polyvalent alcohol (as glycerine, propylene glycol, polyoxyethylene glycol etc.), carboxymethyl cellulose and their suitable mixture, vegetables oil (as sweet oil) and injection organic ester such as ethyl oleate.Can be for example by use coating substance such as Yelkin TTS, in the dispersive situation by keeping required granular size and by using tensio-active agent to keep suitable flowability.

Composition of the present invention also can contain assistant agent such as sanitas, wetting agent, emulsifying agent and dispersion agent.The prevention of microbial process can for example p-Hydroxybenzoate, butylene-chlorohydrin, phenol, Sorbic Acid wait and guarantee by comprising various antibacteriums and anti-mycotic agent.May need also to comprise that isotonic agent is as sugar, sodium-chlor etc.The prolongation of injectable medicament forms absorbs and can postpone the material that absorbs such as aluminum monostearate and gelatin and realize by comprising.

If compound is unstable or have low solubility in aqueous vehicles in aqueous vehicles, it can be mixed with the enriched material in organic solvent.Then enriched material is diluted to low concentration in aqueous systems, and can be enough stable in the short period of time in the administration process.Therefore, on the other hand, a kind of pharmaceutical composition is provided, and it comprises the non-aqueous solution of being made up of one or more organic solvents fully, its can with this form carry out administration or more generally before using with suitable intravenous vehicles (salt solution, glucose; Buffered or buffered not) dilute (Solubilizing excipients in oraland inj ectable formulations, Pharmaceutical Research, 21 (2), 2004, the 201-230 pages or leaves).The example of solvent and tensio-active agent has propylene glycol, PEG300, PEG400, ethanol, N,N-DIMETHYLACETAMIDE (DMA), N-N-methyl-2-2-pyrrolidone N-(NMP, Pharmasolve), glycerine, Cremophor EL, Cremophor RH60 and polysorbate.Concrete non-aqueous solution is made up of 70-80% propylene glycol and 20-30% ethanol.A concrete non-aqueous solution is made up of 70% propylene glycol and 30% ethanol.Another is 80% propylene glycol and 20% ethanol.Usually these solvents are used in combination and dilute at least 2 times usually before intravenously bolus injection or intravenous infusion.The common amount of intravenously bolus injection preparation be～50% glycerine, propylene glycol, PEG300, PEG400 and～20% ethanol.The common amount of intravenous infusion preparation be～15% glycerine, 3%DMA and～10% propylene glycol, PEG300, PEG400 and ethanol.

In an embodiment preferred of the present invention, pharmaceutical composition is to be suitable for intravenously to use the form of for example using by injection or infusion intravenously.Use for intravenously, can with solution with itself form administration or can before using, inject in the infusion bag and (contain pharmaceutically useful vehicle) as 0.9% salt solution or 5% glucose.

In another preferred embodiment, pharmaceutical composition is to be suitable for the form that subcutaneous (s.c.) uses.

Be suitable for Orally administered pharmaceutical dosage form and comprise tablet, capsule, Caplet, pill, lozenge, syrup, solution, powder, granule, elixir and suspensoid, Sublingual tablet, wafer or patch and buccal bioadhesive tablet.

The pharmaceutical composition that contains formula (I) compound can be prepared according to known technology, for example referring to Remington ' s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, USA.

Therefore, tablet composition can contain unit dose of active compound and inert diluent or carrier such as sugar or sugar alcohol, for example lactose, sucrose, sorbyl alcohol or N.F,USP MANNITOL; And/or non-sugared deutero-thinner such as yellow soda ash, calcium phosphate, lime carbonate or Mierocrystalline cellulose or derivatives thereof such as methylcellulose gum, ethyl cellulose, Vltra tears and starch such as W-Gum.Tablet also can contain this class standard composition such as tackiness agent and granulation agent such as polyvinylpyrrolidone, disintegrating agent (for example expandable cross-linked polymer such as cross-linked carboxymethyl cellulose), lubricant (for example stearate), sanitas (for example p-Hydroxybenzoate), antioxidant (for example BHT), buffer reagent (for example phosphoric acid salt or citrate buffer agent) and effervescent such as Citrate trianion/bicarbonate mixture.This class vehicle is known, need not to go through at this.

Capsule can be various glutoid or soft gelatin form and the active ingredient that can contain solid, semisolid or liquid form.Gelatine capsule can be formed by the Equivalent of animal gelatin or its synthetic or plant derivation.

Solid dosage (for example tablet, capsule etc.) can be by dressing or dressing not, but has dressing usually, for example the dressing of protectiveness film coating (for example wax or paint film) or sustained release.Dressing (Eudragit for example ^TMThe type polymkeric substance) desired location that can be designed in gi tract discharges active ingredient.Therefore, can select so that in gi tract, degrade under certain pH condition dressing, thereby optionally discharge compound under one's belt or in ileum or duodenum.

Replace dressing or except dressing, medicine can be present in the solid substrate, this solid substrate comprises the material of sustained release, for example is adapted at optionally discharging under different acidity or alkaline condition in the gi tract material that the delay of compound discharges.Perhaps, the dressing that substrate material or retardance discharge can adopt the form of erodible polymkeric substance (for example maleic anhydride polymkeric substance), when formulation during by gi tract its basically by continuously by corrosion.As another kind of alternative, active compound can be formulated in the delivery system of infiltration control compound release.Infiltration discharges and other postpones to discharge or extended release preparation can be according to well known to a person skilled in the art the method preparation.

Pharmaceutical composition comprises about 1% to about activeconstituents of 95%, preferred about 20% to about 90%.Pharmaceutical composition of the present invention can be a unit dosage form for example, as the form of ampoule, bottle, suppository, drageeing, tablet or capsule.

Being used for Orally administered pharmaceutical composition can obtain by activeconstituents is mixed with solid carrier, if desired with the gained granulating mixture, and if desired or essential, after adding suitable vehicle, mixture is processed into tablet, drageeing core or capsule.Also it may be incorporated in and allow in the plastic carrier of activeconstituents with metered amount diffusion or release.

Also compound of the present invention can be mixed with solid dispersion.Solid dispersion is two or more solid superfine disperse phase uniformly.Solid solution (molecularity dispersion system)-a kind of solid dispersion type-known can be used in the pharmaceutical technology (referring to (Chiou and Riegelman, J.Pharm.Sci., 60, and can be used for increasing dissolution rate and increase the bioavailability of medicament of poorly water-soluble 1281-1300 (1971)).

The solid dispersion of medicine is generally by scorification or solvent evaporated method preparation.For scorification, heating character be generally semi-solid and waxy material (vehicle) so that drug melt and dissolving then harden by being cooled to extremely low temperature.The comminuted solids dispersion is sieved then, with mixed with excipients, encloses in the hard gelatin capsule or is pressed into tablet.Perhaps, use and to have surface-active and carrier self-emulsifying makes solid dispersion directly enclose in the hard gelatin capsule with the form of melt.When the melt cool to room temperature, in capsule, form solid filler.

Solid solution also can be by being dissolved in medicine and required vehicle in aqueous solution or the pharmaceutically useful organic solvent, using pharmaceutically acceptable method such as spraying drying to remove then to desolvate and make.If desired, can adjust gained solid granular size, randomly with mixed with excipients and make tablet or be packed in the capsule.

A kind of particularly suitable polymkeric substance auxiliary material for preparing this class solid dispersion or solid solution is polyvinylpyrrolidone (PVP).

The invention provides a kind of pharmaceutical composition, it comprises is unbodied solid solution basically, and described solid solution comprises

(a) a kind of formula (I) compound, for example compound of embodiment 1; With

(b) be selected from down the polymkeric substance of organizing:

Polyvinylpyrrolidone (polyvidone), cross-linked polyvinylpyrrolidone (Crospovidone), Vltra tears, hydroxypropylcellulose, polyoxyethylene, gelatin, cross linked polyacrylate (carbomer), carboxymethyl cellulose, cross-linked carboxymethyl cellulose (croscarmellose), methylcellulose gum, Sipacril 2739OF, the sodium of alkylmethacrylate polymer and water-soluble salt such as methacrylic acid and alkylmethacrylate polymer and ammonium salt, cellacefate, hydroxypropylmethylcellulose phthalate and propylene glycol alginate;

Wherein said compound is about 1: 1 to about 1: 6 with the ratio of described polymkeric substance, for example 1: 3, and the methylene dichloride/alcoholic acid mixture spraying drying of its mixture, preferred 1: 1 ratio and getting by one of chloroform or methylene dichloride and one of methyl alcohol or ethanol.

The present invention also provides the solid dosage that comprises above-mentioned solid solution.Solid dosage comprises tablet, capsule and chewable tablet.Known vehicle can mix with solid solution so that required formulation to be provided.For example, capsule can contain and (a) disintegrating agent and lubricant or (b) disintegrating agent, lubricant and tensio-active agent blended solid solution.Tablet can contain and at least a disintegrating agent, lubricant, tensio-active agent and glidant blended solid solution.Chewable tablet can contain and extender, lubricant and other if desired sweeting agent (as artificial sweetening agent) and suitable correctives blended solid solution.

Pharmaceutical preparation can be with the form of " patient's bag " passs the patient, and it contains the whole course of treatment in unitary package (being generally Blister Package).Tell the tradition prescription of patient's medicine supply with pharmacist wherein and compare from supply in enormous quantities, patient's bag has advantage because patient's total energy is used the package insert that is included in patient's bag, and in patient's prescription this specification sheets not usually.Proved that package insert can improve the compliance of patient to doctor's indication.

Be used for the local composition that uses and comprise ointment, ointment, sprays, patch, gelifying agent, liquid drops and implant (insert) (for example intraocular implant).This based composition can be prepared according to currently known methods.

Being used for the composition that parenteral uses provides with the form of sterile aqueous or oily solution or particulate suspension usually, perhaps can provide to be reconstructed with sterile water for injection temporarily with the sterilized powder form of porphyrize.

The example that is used for the preparation that rectum or intravaginal use comprises vaginal suppository and suppository, and it can be for example formed by the plasticity-or the waxy substance of the shaping that contains active compound.

Be used to suck the composition of using and adopt the form that can suck powder composition or liquid or powder spray agent, and can use powder inhalation device or aerosol drug delivery device to use with standard form.This class device is known.In order to use by suction, powder formulation comprises active compound and Powdered thinner of inert solid such as lactose usually.

Formula (I) compound generally provides with unit dosage form, like this since, it contains enough compounds usually so that the biologic activity of desired level to be provided.For example, preparation can contain the activeconstituents of 1ng to 2g, and for example 1ng is to the activeconstituents of 2mg.In this scope, the inferior scope of concrete compound is 0.1mg to the activeconstituents of 2g (more generally be 10mg to 1g, for example 50mg is to 500mg), or 1 μ g is to 20mg (for example 1 μ g is to 10mg, and for example 0.1mg is to the activeconstituents of 2mg).

For oral compositions, unit dosage form can contain 1mg to 2g, more generally 10mg to 1g, for example 50mg to 1g, for example 100mg to the active compound of 1g.

Active compound will be applied to the patient (for example human or animal patient) who needs it with the significant quantity that enough reaches required result of treatment.

Methods of treatment

Estimate that formula (I) compound defined herein and its subgroup can be used for preventing or treating by cell cycle protein dependent kinase and glycogen synthase kinase-3 disease states mediated or illness.This class morbid state or examples of disorders are as mentioned above.

Usually give this compound to the patient who needs administration, for example human or animal's sufferer, preferably people.

Usually with treatment or the effective and general nontoxic amount administered compound of prevention.Yet, in some situation situation of life-threatening disease (for example), the benefit of using formula (I) compound may be more important than the shortcoming of any toxic effect or side effect, in this case, can think the compound that need use the amount relevant with toxicity to a certain degree.

But the long-term application compound with keep useful result of treatment or only short-term use.Perhaps can be with pulse mode or continuous mode administered compound.

The common per daily dose of formula (I) compound can be every kg body weight 100 piks to 100 milligram, more generally be every kg body weight 5 nanograms to 25 milligram, more generally be every kg body weight 10 nanograms to 15 milligram (10 nanograms to 10 milligram for example, more generally be that every kilogram 1 microgram is to 20 milligrams every kilogram, every kilogram 1 microgram to 10 milligram for example), but, also can use higher or lower dosage as needing.Formula (I) compound can or repeat on every day basis to use on the basis, uses once in for example per 2 or 3 or 4 or 5 or 6 or 7 or 10 or 14 or 21 or 28 days.

Compound of the present invention can be Orally administered in dosage range, for example uses with the oral dose of 1-1500mg, 2-800mg or 5-500mg, for example 2-200mg or 10-1000mg, and the object lesson of dosage comprises 10,20,50 and 80mg.But compound is used once every day or once.But compound continuous administration (promptly do not use less than interrupting every day during treatment plan).Perhaps, compound can be interrupted and use (promptly during whole treatment plan, given for some time as a week in continuous administration, interrupt for some time then as a week, and then continuous administration for some time is as week etc.).Relate to the example that is interrupted the treatment plan use and comprise wherein and using a week to cyclicity, interrupt a week; Or used for two weeks, interrupt a week; Or used for three weeks, interrupt a week; Or used for two weeks, interrupted for two weeks; Or around using, interrupted for two weeks; Or use a week, interrupted for three weeks, for example carry out one or more these circulations, for example 2,3,4,5,6,7,8,9 or 10 or more a plurality of this round-robin scheme.

For 60 kilograms people, the example of dosage comprises with the initial dose of 4.5-10.8mg/60kg/ days (equaling 75-180 μ g/kg/ days), uses formula defined herein (I) compound with the effective dose of 44-97mg/60kg/ days (equaling 0.7-1.6mg/kg/ days) or the effective dose of 72-274mg/60kg/ days (equaling 1.2-4.6mg/kg/ days) subsequently, but, if desired, can use higher or lower dosage.For any given body weight, mg/kg dosage in proportion converts.

In a concrete drug dosage schedule table, the patient will be given the transfusion of little up-to-date style (I) compound every day, and administration reaches 10 days, particularly reach 5 days weeks, and with required time at interval as two to around, particularly per three weeks are once carried out repetitive therapy.

More specifically, the transfusion that the patient can be given little up-to-date style (I) compound every day reaches 5 days, and per three weeks are once carried out repetitive therapy.

In another concrete drug dosage schedule table, the patient is given 30 minutes to 1 hour transfusion, give then variable period for example 1-5 hour for example 3 hours keep infusion.

In another concrete drug dosage schedule table, the patient is given 12 hours to 5 days continuous infusion, particularly 24 hours to 72 hours continuous infusion.

At last, the amount of institute's administered compound and the type of composition therefor should match with the character of disease or physiological conditions to be treated, and should be decided by the doctor.

Formula defined herein (I) compound and its subgroup can be used as the single therapy agent and are applied, perhaps they can with other other be used for the treatment of particular disease states for example one of the compound of neoplastic disease cancer as hereinbefore defined use in the combined therapy mode.Other can with compound of the present invention together (no matter be simultaneously or with the different timed intervals) use or the therapeutical agent that uses or the example of therapy include, but are not limited to topoisomerase enzyme inhibitor, alkylating agent, metabolic antagonist, DNA wedding agent, microtubule inhibitor (agent of tubulin target), monoclonal antibody and signal transduction inhibitor, concrete example has cis-platinum, endoxan, Zorubicin, irinotecan, fludarabine, 5FU, taxanes, ametycin and radiotherapy.

For with the situation of the CDK inhibitor of other therapeutic combination, can give two or more therapy with different separately dose plans and via different approach.

Formula (I) compound and one, two, three, four or the situation of more kinds of (preferred one or two kind, more preferably a kind of) other therapeutical agent combined administration under, compound can be simultaneously or sequential application.When sequential application, they can be with the in-plant timed interval (for example going through 5-10 minute time) or with the longer timed interval (for example at interval 1,2,3,4 or more hours, use perhaps if desired at interval for more time), the character of accurate dose scheme and therapeutical agent matches.

For with the situation of the CDK inhibitor of other therapeutic combination, can give two or more therapy with different separately dose plans and via different approach.

Formula (I) compound and one, two, three, four or the situation of more kinds of (preferred one or two kind, more preferably a kind of) other therapeutical agent combined administration under, compound can be simultaneously or sequential application.When sequential application, they can be with the in-plant timed interval (for example going through 5-10 minute time) or with the longer timed interval (for example at interval 1,2,3,4 or more hours, use perhaps if desired at interval for more time), the character of accurate dose scheme and therapeutical agent matches.

Compound of the present invention also can with non-chemotherapeutics such as radiotherapy, photodynamic therapy, gene therapy; Perform the operation and keep on a diet and use together.

For with the combined therapy of another kind of chemotherapeutics, can with formula (I) compound and one, two, three, four or more other therapeutical agents for example be mixed with together and contain one, two, three, four or the formulation of more kinds of therapeutical agents.In a yes-no decision, each therapeutical agent can be separated to prepare and provide together with the form of medicine box, and described medicine box randomly has their working instructions.

Those skilled in the art can understand used dosage regimen and combined therapy by his or her common sense.

Diagnostic method

Before using formula (I) compound, the screening patient is suffering from the whether treatment sensitivity to adopting the active compound of anti-cell cyclin-dependent kinase to be carried out of the morbid state that maybe may suffer from or illness to determine this patient.

Whether for example, can analyze the biological specimen of taking from the patient is to be the disease or the illness of feature unusually to cause the CDK overactivity or to cause the gene unconventionality or the protein expression of the active path enhanced sensitivity of normal CDK to determine that this patient is suffering from the illness that maybe may suffer from or disorders such as cancers.Cause the unusual example of this class of the activation of CDK2 signal or enhanced sensitivity to comprise rise (HarwellRM, Mull BB, Porter DC, the Keyomarsi K. of cyclin E; J Biol Chem.2004 March 26; 279 (13): 12695-705) or p21 or p27 disappearance, or have CDC4 variant (Rajagopalan H, Jallepalli PV, Rago C, Velculescu VE, Kinzler KW, Vogelstein B, LengauerC.; Nature.2004 March 4; 428 (6978): 77-81).Rise with CDC4 sudden change or cyclin E is particularly crossed the tumour of expression or p21 or p27 disappearance to CDK inhibitor sensitivity especially.Term " rise " comprises and express to raise or cross and express, and comprises gene amplification (being a plurality of gene copies) and transcribes effect and expression increase that high reactivity and activation (comprising the activation that sudden change causes) cause.

Therefore, can carry out diagnostic test to detect the mark characteristics that cyclin E rise or p21 or p27 lacked or existed the CDC4 variant to the patient.Term " diagnosis " comprises screening.We comprise genetic marker with " mark " speech, comprise that for example measuring DNA forms to identify the sudden change of CDC4.Term " mark " also comprises and embodies the mark that cyclin E raises characteristics, comprises enzymic activity, enzyme level, enzyme state (for example whether phosphorylation) and aforementioned proteinic mRNA level.Tumour with cyclin E rise or p21 or p27 disappearance may be responsive especially to the CDK inhibitor.Can before treatment, preferentially screen at the rise of cyclin E or the disappearance of p21 or p27 tumour.Therefore, can carry out diagnostic test to detect the mark characteristics of cyclin E rise or p21 or p27 disappearance to the patient.

Diagnostic test is usually with carrying out on the biological specimen that is selected from tumor biopsy sample, blood sample (separation of the tumour cell that comes off and enrichment), ight soil biopsy, phlegm, chromosome analysis, Pleural fluid, peritoneal fluid or urine.

People such as Rajagopalan find (Nature.2004 March 4; 428 (6978): 77-81) there be sudden change (people such as Spruck, Cancer Res.2002 August 15 in CDC4 (being also referred to as Fbw7 or Archipelago) in human colorectal cancer and carcinoma of endometrium; 62 (16): 4535-9).The evaluation of carrying the individuality of CDC4 sudden change means that this patient is particularly suitable for treating with the CDK inhibitor.Can before treatment, preferentially screen tumour at the existence of CDC4 variant.Screening method is usually directed to direct order-checking, oligonucleotide microarray analysis or mutant specific antibody.

The evaluation and the sudden change of analysing protein and the method for rise are well known to a person skilled in the art.Screening method includes, but are not limited to standard method such as reversed transcriptive enzyme polymerase chain reaction (RT-PCR) or in situ hybridization.

In the screening of using RT-PCR to carry out, the mRNA level in the tumour is to estimate with pcr amplification cDNA then by the cDNA copy that produces mRNA.The method of pcr amplification, selection of primers and amplification condition are well known by persons skilled in the art.Nucleic acid operation and PCR carry out according to standard method, as for example Ausubel, and people such as F.M., Current Protocols in MolecularBiology, 2004, John Wiley ﹠amp; Sons Inc., or Innis, people such as M.A., PCR Protocols:a guide to methods and applications, 1990, Academic Press is described in the San Diego.Relate to the reaction of nucleic acid technology and operate in people such as Sambrook, 2001, the 3 editions, MolecularCloning:A Laboratory Manual also has description among the Cold Spring Harbor Laboratory Press.Perhaps, can use the test kit (for example RocheMolecular Biochemicals) of the commercially available acquisition of RT-PCR, or U.S. Patent No. 4,666,828; 4,683,202; 4,801,531; 5,192,659,5,272,057,5,882,864 and 6,218, the method described in 529 is incorporated herein by reference them.

An example estimating the hybridization in situ technique that mRNA expresses be fluorescence in situ hybridization (FISH) (referring to Angerer, 1987Meth.EnzymoL, 152:649).

Usually, in situ hybridization comprises following key step: (1) fixes tissue to be analyzed; (2) sample is carried out accessibility and the minimizing non-specific binding of prehybridization processing to increase target nucleic acid; (3) with the nucleic acid hybridization in nucleic acid mixture and biological structure or the tissue; (4) carry out post-hybridization washing to remove the nucleic acid fragment that unconjugated nucleic acid fragment and (5) in the hybridization detect hybridization.The probe that uses in this class is used is mark normally, for example carries out mark with radio isotope or fluorescent indicator.Preferred probes is answered sufficiently long, and for example, about 50,100 or 200 Nucleotide are to about 1000 or more a plurality of Nucleotide, so that can carry out specific hybrid with target nucleic acid under stringent condition.The standard method of carrying out FISH is at Ausubel, people such as F.M., Current Protocols in Molecular Biology, 2004, John Wiley ﹠amp; Sons Ine and Fluorescence In Situ Hybridization:TechnicalOverview by John M.S.Bartlett in Molecular Diagnosis of Cancer, Methods and Protocols, the 2nd edition; ISBN:1-59259-760-2; In March, 2004, the 077-088 page or leaf; Among the Series:Methods in Molecular Medicine description is arranged.

Perhaps, can be used for the proteinic currently known methods analysis of detection specificity by mRNA expressed protein product by the immunohistochemistry of tumor sample, solid-phase immunoassay, Western blotting, two-dimentional SDS-polyacrylamide gel electrophoresis, ELISA, flow cytometry and this area of use microtiter plate.Detection method comprises the use site-specific antibodie.The technology that it will be understood by a person skilled in the art that the disappearance of the known rise that is used to detect cyclin E of all these classes or p21 or p27 or CDC4 variant is all applicable to this situation.

Therefore, all these technology also can be used for identifying the tumour that is particularly suitable for compounds for treating of the present invention.

Tumour with the rise of CDC4 sudden change or cyclin E, particularly mistake expression or p21 or p27 disappearance may be responsive especially to the CDK inhibitor.Preferentially before treatment, express (Harwell RM, Mull BB, Porter DC, Keyomarsi K. at the rise of cyclin E, particularly mistake; J Biol Chem.2004 March 26; 279 (13): 12695-705) or p21 or p27 disappearance or CDC4 variant tumour is screened (Rajagopalan H, Jallepalli PV, Rago C, Velculescu VE, Kinzler KW, Vogelstein B, Lengauer C.; Nature.2004 March 4; 428 (6978): 77-81).

The diagnostic test of available this paper general introduction selects to suffer from the patient of lymphoma mantle cell (MCL) to treat with compound of the present invention.MCL is a kind of special clinicopathologic entity of non-Hodgkin lymphoma, and what it is characterized in that having CD5 and CD20 coexpression is little of medium sized lymphopoiesis, invasive and the clinical disease course that can not cure and frequent t (11; 14) (q13; Q32) transposition.Expressing excessively of the cyclin D1 mRNA that finds in lymphoma mantle cell (MCL) is a kind of important diagnostic mark.People such as Yatabe (Blood.2000 April 1; 95 (7): 2253-61) propose to comprise cyclin D1-positive, and proposal should be the innovative treatments that this incurable disease is probed on the basis with this new standard as one of standard of MCL.People such as Jones (J Mol Diagn.2004 May; 6 (2): 84-9) developed a kind of real-time quantitative reverse transcription PCR that is used for cyclin D1 (CCND1) expression and analyzed with assisted diagnosis lymphoma mantle cell (MCL).People such as Howe (ClinChem.2004 January; 50 (1): 80-7) quantitative RT-PCR that uses real-time quantitative RT-PCR to assess the expression of cyclin D1 mRNA and the cyclin D1 mRNA that discovery is normalized to CD19 mRNA is used in blood, marrow and the tissue and diagnoses MCL.Perhaps, can use the diagnostic test of above-outlined to select to suffer from the patient of mammary cancer to treat with the CDK inhibitor.Tumour cell common overexpressing cell cyclin E and showed cell cyclin E in mammary cancer, cross to express (people such as Harwell, Cancer Res, 2000,60,481-489).Therefore, particularly mammary cancer can be treated with the CDK inhibitor that this paper provided.

Antifungal application

On the other hand, the invention provides formula (I) compound and the purposes of its subgroup as herein defined as anti-mycotic agent.

Formula as defined herein (I) compound and its subgroup can be used for animal medicine (for example being used for for example people's treatment of Mammals), or be used for the processing (for example agricultural and gardening) of plant, or as general anti-mycotic agent, for example as sanitas and sterilizing agent.

In a specific embodiments, the invention provides and be used to prevent or treat Mammals for example formula as defined herein (I) compound and its subgroup of people's fungi infestation.

Also provide formula as defined herein (I) compound and its subgroup to be used for preventing or to treat for example purposes of the medicine of people's fungi infestation of Mammals in manufacturing.

For example, compound of the present invention can infect the local fungal that is caused by Candida (Candida), Trichophyton (Trichophyton), microsporum (Microsporum) or Epidermophyton organisms such as (Epidermiophyton) of suffering from or have risk of infection, or the human patients administration of the mucosal infections (for example white mouth and vaginal candidiasis) that is caused by Candida albicans (Candida albicans).Also can give the systemic fungal infection that compounds for treating of the present invention or prevention are caused by for example Candida albicans, Cryptococcus neoformans (Cryptococcus neoformans), flavus (Aspergillus flavus), Aspergillus fumigatus (Aspergillus fumigatus), Coccidioides (Coccidiodies), Paracoccidioides (Paracoccidioides), Histoplasma (Histoplasma) or Blastomyces (Blastomyces).

On the other hand, the invention provides the antifungal composition of agricultural (comprising gardening) purposes, it comprises formula (I) compound and its subgroup and agriculture acceptable diluent or the carrier gone up as herein defined.

The present invention further provides a kind of treatment or processing and suffered from the method for animal (comprising for example people of Mammals), plant or the seed of fungi infestation, this method comprises the place of handling described animal, plant or seed or described plant or seed place with formula as herein defined (I) compound of significant quantity and its subgroup.

The present invention also provides a kind of method for the treatment of plant or seed fungi infestation, and this method comprises with the fungicide composition of comprising of antimycotic significant quantity of formula defined herein (I) compound and its subgroup handles plant or seed.

But the usage variance screening is analyzed and is selected that non-human CDK enzyme is had specific The compounds of this invention.The compound that acts on the CDK enzyme of eukaryotic pathogens specifically can be used as antimycotic or antiparasitic.Candida CDK kinase inhibitor CKSI can be used for treating moniliosis.Anti-mycotic agent can be used for resisting the defined infection type in front or often betides weak and immunosuppressant patient for example suffers from leukemia and lymphadenomatous patient, accepts the people of immunosuppressant treatment and suffers from procatarxis venereal disease disease for example diabetes or the patient of AIDS and the opportunistic infection among the non-immunosuppressant patient.

The described analytical method in this area can be used for screening may be used to suppress for example medicine of the relevant fungi of moniliosis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, torulosis, chromoblastomycosis, coccidioidomycosis, conidium bacterium disease (conidiosporosis), histoplasmosis, mycetoma, rhinosporidiosis, nocardiosis, pseudactinomycosis, penicilliosis, moniliosis (monoliasis) or sporotrichosis of at least a and mycosis.Described differential screening analysis can be used for identifying the anti-mycotic agent that may have therapeutic value in the treatment of aspergillosis, described analysis and utilization is cloned in yeast Aspergillus fumigatus (Aspergillus fumigatus) for example, flavus (Aspergillus flavus), aspergillus niger (Aspergillusniger), the CDK gene of Aspergillus nidulans (Aspergillus nidulans) or terreus (Aspergillus terreus), or when fungal infection was mucormycosis (muconnycosis), CDK analyzes can be derived from yeast unrooted rhizopus (Rhizopus arrhizus) for example, Rhizopus oryzae (Rhizopus oryzae), absidia corymbifera (Absidia corymbifera), absidia rasmosa (Absidia ramosa) or Mucor pusillus (Mucorpusillus).The source of other CDK enzyme comprises pathogenic agent Pneumocystis carinii (Pneumocystiscarinii).

For example, the in-vitro evaluation of the anti-mycotic activity of compound can be undertaken by measuring minimal inhibitory concentration (M.I.C.), and it is the testing compound concentration that specified microorganisms can't be grown in suitable medium.In practice, inoculate for example type culture of Candida albicans to a series of agar plates that mix the specific concentrations test compounds separately, then with each flat board at 37 ℃ of following incubation suitable times.Check then whether fungi growth is arranged on the flat board, the M.I.C. value that record is suitable.Perhaps, but carry out nephelometric analysis in the liquid medium within, the scheme of summarizing the example of this analytical procedure can be referring to the following examples.

The interior evaluating of compound can under a series of dosage level by in the mouse peritoneum of inoculated fungi, for example Candida albicans or aspergillus flavus strain or intravenous injection or oral administration carry out.The activity of compound can be assessed (by histology or by obtaining the fungi of self-infection) by the growth of monitor therapy and the fungi infestation of untreated mouse group.Active can provide the 50% dosage level (PD that protects from infection lethal effect according to compound ₅₀) measure.

For human antifungal application, as herein defined formula (I) compound and its subgroup can be individually or with put into practice selected pharmaceutical carrier mixing administration according to expection route of administration and standard pharmaceutical.Therefore, for example, can adopt at preparation oral administration, parenteral, intravenously, intramuscular or the subcutaneous administration described in above-mentioned " pharmaceutical preparation " joint.

With regard to and parenteral admin oral to human patients, the dosage level of antifungal compound of the present invention is 0.01 to 10mg/kg (dosage that separates), the effectiveness when this depends on compound oral administration or parenteral administration.The tablet of compound or capsule can for example contain 5mg to 0.5g active compound, take the circumstances into consideration administration a slice (grain), two (grain) or multi-disc (grain) at every turn.In any case the doctor will determine to be suitable for most the actual dose (significant quantity) of individual patient, it will be different because of age, body weight and the response of particular patient.

Perhaps, antifungal compound can be with the form administration of suppository or vaginal suppository, and perhaps they can be with the form topical application of lotion, solution, creme, ointment or dusting.For example, they can be impregnated in the creme of being made up of the water-based emulsion of polyoxyethylene glycol or whiteruss; Perhaps they can be impregnated in the ointment of being made up of Chinese wax or paraffinum molle alba matrix and the stablizer that adds as required and sanitas, and concentration is 1 to 10%.

Except that above-mentioned therepic use, the anti-mycotic agent of developing with this differential display analysis also can be used as the sanitas in the food, the fodder additives that promotion domestic animal weight increases, or be used to handle the disinfectant preparation of non-living matter, for example be used to sterilize hospital equipment and room.In a similar manner, the arranged side by side relatively restraining effect of Mammals CDK and insect CDK (for example fruit bat CDK5 gene) (people such as Hellmich, (1994) FEBS Lett 356:317-21), can from The compounds of this invention, select the inhibitor of the enzyme that can distinguish people/Mammals and insect.Therefore, the present invention comprises purposes and the preparation of The compounds of this invention in sterilant clearly, for example is used for the processing of insect such as fruit bat.

In another embodiment, the CDK inhibitor that can select some to be tried with respect to the inhibition specificity of mammalian enzyme according to plant CDK.For example, plant CDK is arranged in the differential screening analysis that contains one or more people's enzymes, with those the highest compounds of selectivity of selecting plant enzyme is suppressed.Therefore, the present invention specifically comprises the preparation of the CDK inhibitor of the present invention that is used for agricultural application, for example form such as defoliant.

For agricultural and gardening purpose, The compounds of this invention can be mixed with the composition forms that is suitable for specific end use and intended purposes.Therefore, compound can be used with the form of dusting powder or granule, seed dressing, the aqueous solution, dispersion liquid or emulsion, immersion liquid, sprays, aerosol or smoke substance.Composition also can be with dispersion powder, granule or particle, or provides with the form of the concentrated solution of preceding dilution.These compositions can comprise those known and acceptable conventional carrier, thinner or auxiliary agents in agricultural and gardening, and they can be made according to conventional methods.Said composition also can be mixed other activeconstituents, for example, has the compound or the another kind of mycocide of weeding or insecticidal activity.Compound and composition can be used in many ways; for example they can directly be applied to plant leaf, stem, branch, seed or root; maybe can be administered to soil or other growth medium, and they not only can eradicate disease, and preventability ground protective plant or seed are not under fire.For example, said composition can comprise the activeconstituents of 0.01 to 1 weight %.Use for the field, the possible rate of application of activeconstituents is 50 to 5000 gram/hectares.

The present invention also comprises the purposes that formula defined herein (I) compound and its subgroup are used to control wood-destroying fungi and handle soil, seedling rice field or the irrigation water of plant-growth.The present invention comprises that also formula defined herein (I) compound and its subgroup are used to prevent that stock's cereal and other no plant place from the purposes of fungi infestation taking place.

Embodiment

Set forth the present invention without limitation referring now to the specific embodiments described in the following example.

In an embodiment, use following abbreviation.

AcOH acetate

The BOC tert-butoxycarbonyl

CDI 1, the 1-carbonyl dimidazoles

DMAW90 solvent mixture: DCM:MeOH, AcOH, H ₂O (90: 18: 3: 2)

DMAW120 solvent mixture: DCM:MeOH, AcOH, H ₂O (120: 18: 3: 2)

DMAW240 solvent mixture: DCM:MeOH, AcOH, H ₂O (240: 20: 3: 2)

The DCM methylene dichloride

The DMF dimethyl formamide

The DMSO methyl-sulphoxide

EDC 1-ethyl-3-(3 '-dimethylamino-propyl)-carbodiimide

Et ₃The N triethylamine

The EtOAc ethyl acetate

Et ₂The O ether

HOAt1-hydroxyl azepine benzotriazole

HOBt1-hydroxybenzotriazole

The MeCN acetonitrile

MeOH methyl alcohol

P.E. sherwood oil

SiO ₂Silica gel

TBTU N, N, N ', N '-tetramethyl--O-(benzotriazole-1-yl) urea a tetrafluoro borate

The THF tetrahydrofuran (THF)

The explanation of analysis mode LC-MS system and method

In these embodiments, prepared compound be liquid chromatography by using following system and operational condition and mass spectroscopy qualitatively.When existence has different isotopic atoms and has quoted single quality, the quality that compound is quoted be single isotopic mass (just ³⁵Cl; ⁷⁹Br or the like).Use a plurality of systems as described below, these systems are equipped with and carry out under closely similar operational condition.Used operational condition is described in down.

Waters Platform LC-MS system:

HPLC system: Waters2795

Mass detector: Micromass Platform LC

PDA detector: Waters2996PDA

The acidic conditions of analyzing:

Elutriant A:H ₂O (0.1% formic acid)

Elutriant B:CH ₃CN (0.1% formic acid)

Gradient: 5-95% elutriant B, went through 3.5 minutes

Flow velocity: 0.8ml/ minute

Pillar: Phenomenex Synergi 4 μ MAX-RP80A, 2.0x50mm

The long acidic conditions of analyzing:

Elutriant A:H ₂O (0.1% formic acid)

Elutriant B:CH ₃CN (0.1% formic acid)

Gradient: 05-95% elutriant B, went through 15 minutes

Flow velocity: 0.4ml/ minute

Pillar: Phenomenex Synergi 4 μ MAX-RP80A, 2.0x150mm

Platform MS condition:

Capillary voltage: 3.6kV (when bearing ES is 3.40kV)

Awl voltage: 25V

Come source temperature: 120 ℃

Sweep limit: 100-800amu

Ionization mode: positive electron spray(ES) or

Negative electricity spraying or

Positive ﹠amp; The negative electricity spraying

Waters Fractionlynx L C-MS system:

HPLC system: 2767 automatic samplers-2525, two gradient pumps

Mass detector: Waters ZQ

PDA detector: Waters 2996 PDA

The acidic conditions of analyzing:

Elutriant A:H ₂O (0.1% formic acid)

Elutriant B:CH ₃CN (0.1% formic acid)

Gradient: 5-95% elutriant B, went through 4 minutes

Flow velocity: 2.0ml/ minute

Pillar: Phenomenex Synergi 4 μ MAX-RP80A, 4.6x50mm

Fractionlynx MS condition:

Capillary voltage: 3.5kV (when bearing ES is 3.2kV)

Awl voltage: 25V (when bearing ES is 30V)

Come source temperature: 120 ℃

Sweep limit: 100-800amu

Ionization mode: positive electron spray(ES) or

Negative electricity spraying or

Positive ﹠amp; The negative electricity spraying

The LC-MS system of quality indication purifying

Preparation type LC-MS is a kind of purifying the organic molecule for example standard and effective means of compound described herein of being used for.Can change the method for liquid chromatography (LC) and mass spectrum (MS), so that crude product separates better and improves the detection of MS to sample.The optimization of preparation type gradient LC method comprises change pillar, volatility eluent and properties-correcting agent and gradient.It is known in this field optimizing the method for preparing type LC-MS method, adopts it to come purifying compounds.These class methods are described in Rosentreter U, Huber U.; Optimal fraction collecting inpreparative LC/MS; J CombChem.; 2004; 6 (2), 159-64 and Leister W, Strauss K, Wisnoski D, Zhao Z, Lindsley C., Development of a custom high-throughput preparative liquidchromatography/massspectrometer platformfor the preparativepurification and analytical analysis of compound libraries; J Comb Chem.; 2003; 5 (3); Among the 322-9.

One is come this type systematic of purifying compounds as described below via preparation type LC-MS, but it will be understood by those skilled in the art that and can use other system and method different with described system and method.Particularly, can utilize the method replacement inversion method described herein for preparing type LC based on positive.Most of preparation type LC-MS system utilizes anti-phase LC and volatile acid modification agent because this method for micromolecular purifying very effectively and eluent and the cation electrodeposition mass spectrometry of spraying compatible.Also can adopt other chromatogram scheme, the positive LC that summarizes in for example above-mentioned analytical procedure, alternate buffering moving phase, alkaline properties-correcting agent wait these compounds of purifying.

Preparation type LC-MS system:

Waters Fractionlynx system:

. hardware:

2767 double loops automatic sampler/fraction collector

2525 preparation pumps

Be used for the CFO (chromatographic column fluid tissue device (fluidic organiser)) that post is selected

RMA (Waters reagent manager), pump (make up pump) as a supplement

Waters ZQ mass spectrograph

Waters 2996 photodiode arrangement detectors

Waters ZQ mass spectrograph

. software:

Masslynx4.0

.Waters MS operational condition:

Capillary voltage: 3.5kV (when bearing ES is 3.2kV)

Awl voltage: 25V

Come source temperature: 120 ℃

Amplifier: 500V

Sweep limit: 125-800amu

Ionization mode: positive electron spray(ES) or

The negative electricity spraying

Agilent 1100LC-MS preparation system:

. hardware:

Automatic sampler: 1100 series " prepALS "

Pump: be used for 1100 series " QuatPump " that preparative liquid flows 1100 series " PrepPump " of gradient and is used for flowing to preparative liquid pumping properties-correcting agent

UV detector: 1100 series " MWD " multiwavelength detector

MS detector: 1100 series " LC-MSD VL "

Fraction collector: 2x " Prep-FC "

Make-up pump: " Waters RMA "

The active demultiplexer of Agilent

. software:

Chemstation：Chem32

.Agilent MS operational condition:

Capillary voltage: 4000V (when bearing ES is 3500V)

Cracking voltage/gain: 150/1

Dry gas flow velocity: 13.0 liters/minute

Gas temperature: 350 ℃

Spraying gun pressure: 50psig

Sweep limit: 125-800amu

Ionization mode: positive electron spray(ES) or

The negative electricity spraying

Chromatographic condition:

. pillar:

1. low pH chromatography:

Phenomenex?Synergy?MAX-RP，10μ，100x21.2mm

(perhaps use Thermo Hypersil-Keystone HyPurityAquastar for compound than high polarity, 5 μ, 100x21.2mm)

2. high pH chromatography:

Phenomenex?Luna?C18(2)，10μ，100x21.2mm

(perhaps use Phenomenex Gemini, 5 μ, 100x21.2mm)

. elutriant:

1. low pH chromatography:

Solvent orange 2 A: H ₂O+0.1% formic acid, pH～1.5

Solvent B:CH ₃CN+0.1% formic acid

2. high pH chromatography:

Solvent orange 2 A: H ₂O+10mM NH ₄HOH+NH ₄OH, pH=9.2

Solvent B:CH ₃CN

. supplementing solvent:

MeOH+0.2% formic acid (being used for two kinds of chromatography types)

. method:

According to analytical results, select only preparative scale chromatography type.Common convention is to use the chromatography type (low or high pH) that is suitable for compound structure most to carry out analysis mode LC-MS.In case analytical results turns out to be good chromatography, select a kind of appropriate preparation method of same type.Common operational conditions low and high pH chromatographic process is:

Flow velocity: 24ml/ minute

Gradient: usually, all gradients all have the initial 0.4 minute step of using 95%A+5%B.Then according to analytical results, select 3.6 minutes gradients in case obtain good separation (for example be used for keeping in early days compound from 5% to 50%B; Keep in the middle of being used for compound from 35% to 80%B or the like).

Washing: the washing step that when gradient finishes, carries out 1.2 minutes

Reequilibrate: the system that the reequilibrate step of carrying out 2.1 minutes is used for moving with preparation next time

Replenished flow velocity: 1ml/ minute

. solvent:

All compounds are dissolved among 100%MeOH or the 100%DMSO usually.

From the information that is provided, those skilled in the art can be by preparation type LC-MS purifying compound as herein described.

The raw material of each embodiment all has commercially available, except as otherwise noted.

Embodiment 1

1A.4-nitro-1H-pyrazoles-3-methyl-formiate

At room temperature (2.90ml, (5.68g is 36.2mmol) in the mixture in MeOH (100ml) and stirred the mixture 48 hours 39.8mmol) slowly to add to 4-nitro-3-pyrazole carboxylic acid with thionyl chloride.Enriched mixture and by obtaining the 4-nitro-1H-pyrazoles-3-methyl-formiate of white solid in a vacuum with the methylbenzene azeotropic drying.

¹H?NMR(400MHz，DMSO-d ₆)δ14.4(s，1H)，8.9(s，1H)，3.9(s，3H)。

1B.4-amino-1H-pyrazoles-3-methyl-formiate

Under nitrogen atmosphere, stirred 4-nitro-1H-pyrazoles-3-methyl-formiate and the mixture of 10%Pd/C in EtOH 20 hours.By the plug of celite filtering mixt, concentrate in a vacuum and by obtaining 4-amino-1H-pyrazoles-3-methyl-formiate with the methylbenzene azeotropic drying.

¹H?NMR(400MHz，MeOD)δ7.2(s，1H)，3.9(s，3H)。

(1C.4-2,6-two chloro-benzamidos)-1H-pyrazoles-3-formic acid

With 2,6-dichlorobenzoyl chloride (8.2g; 39.05mmol) be added to 4-amino-1H-pyrazoles-3-methyl-formiate (5g carefully; 35.5mmol) and triethylamine (5.95ml; 42.6mmol) in the solution in dioxan (50ml), at room temperature stirred then 5 hours.Filter reaction mixture is handled filtrate with methyl alcohol (50ml) and 2M sodium hydroxide solution (100ml), heats 4 hours down in 50 ℃, then evaporation.The water of 100ml is added in the resistates, use the concentrated hydrochloric acid acidifying then.By solid collected by filtration, water (100ml) washs and drains 4-(2,6-two chloro-the benzamidos)-1H-pyrazoles-3-formic acid that obtains 10.05g lavender solid state.(LC/MS：R _t2.26，[M+H] ⁺300/302)。

(1D.4-{[4-2,6-two chloro-benzamidos)-1H-pyrazoles-3-carbonyl]-amino }-piperidines-1-formic acid uncle fourth Ester

At room temperature stir 4-(2,6-two chloro-benzamidos)-1H-pyrazoles-3-formic acid (6.5g, 21.6mmol), 4-amino-1-BOC-piperidines (4.76g, 23.8mmol), EDC (5.0g, 25.9mmol) and HOBt (3.5g, 25.9mmol) mixture in DMF (75ml) is 20 hours.Concentrated reaction mixture distributes resistates between ethyl acetate (100ml) and saturated sodium bicarbonate aqueous solution (100ml) in a vacuum.With salt water washing organic layer, dry (MgSO ₄) and concentrate in a vacuum.With resistates be dissolved in 5%MeOH-DCM (～30ml) in.Collect insolubles by filtering, obtain 4-{[4-(2,6-two chloro-the benzamidos)-1H-pyrazoles-3-carbonyl of white solid with DCM washing and vacuum-drying]-amino }-piperidines-1-t-butyl formate (5.38g).Concentrated filtrate and use gradient elution 1 by column chromatography in a vacuum: the 2EtOAc/ hexane obtains 4-{[4-(2,6-two chloro-the benzamidos)-1H-pyrazoles-3-carbonyl of white solid in addition to EtOAc purifying resistates]-amino }-piperidines-1-t-butyl formate (2.54g).

(1E.4-2,6-two chloro-benzamidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base amide hydrochloride

Handle 4-{[4-(2,6-two chloro-benzamidos)-1H-pyrazoles-3-carbonyl with saturated HCI-EtOAc (40ml)]-amino-solution of piperidines-1-t-butyl formate (7.9g) in MeOH (50ml) and EtOAc (50ml) then stirring at room temperature spend the night.Because the existence of methyl alcohol, product does not have crystallization, so evaporation reaction mixture, and resistates is ground with EtOAc.By filter collecting the gained white solid, with the EtOAc washing and on strainer (sinter), drain and obtain 6.3g4-(2,6-two chloro-benzamidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base amide hydrochloride.(LC/MS：R _t5.89，[M+H] ⁺：382/384)。

(1F.4-2,6-two chloro-benzamidos)-1H-pyrazoles-3-formic acid (1-methane sulfonyl-piperidin-4-yl)-acyl Amine

Diisopropylethylamine (2.2mmol) is added in 4-(2,6-two chloro-the benzamidos)-1H-pyrazoles-3-carboxylic acid piperidin-mixture of 4-base amide hydrochloride (1mmol) in acetonitrile (10ml), then add methane sulfonyl chloride (1mmol).Mixture at room temperature stirred concentrated in a vacuum then in 16 hours.Resistates is distributed between ethyl acetate and water, separate each layer and use salt water washing organic moiety, dry (MgSO ₄) and the concentrated in a vacuum title compound that obtains.IM+H] ⁺460?Rt?2.67。LC/MS.r.t.2.67 minute; M/z460.11

¹HNMR：(400MHz，DMSO-d ₆)δ13.51(s，1H)，10.20(s，1H)，8.50(d，J＝8.0Hz，1H)，8.41(s，1H)，7.66-7.56(m，3H)，3.95-3.89(m，1H)，3.61(d，J＝12.0Hz，2H)，2.92(s，3H)，2.84(t，J＝12.0Hz，2H)，1.89-1.86(m，2H)，1.79-1.70(m，2H)

Embodiment 2

4-(2,6-two chloro-benzamidos)-1H-pyrazoles-3-formic acid (1-sec.-propyl alkylsulfonyl-piperidin-4-yl)-acid amides

Title compound still replaces methane sulfonyl chloride with the sec.-propyl SULPHURYL CHLORIDE according to the method described in the embodiment 1 and makes by preparation type LC/MS purifying.R.t.2.83 minute; M/z488.22.

¹HNMR：(400MHz，DMSO-d6)δ13.42(s，1H)，10.16(s，1H)，8.46(d，J＝8.0Hz，1H)，8.35(s，1H)，7.60-7.51(m，3H)，3.92-3.87(m，1H)，3.65(d，J＝12.0Hz，2H)，3.35-3.27(m，1H)，2.95(t，J＝12.0Hz，2H)，1.80-1.76(m，2H)，1.66-1.59(m，2H)，1.22(d，J＝8.0Hz，6H)

Embodiment 3

4-(2,6-two chloro-benzamidos)-1H-pyrazoles-3-formic acid (1-ethyl-alkylsulfonyl-piperidin-4-yl)-acid amides

But title compound is according to the method described in the embodiment 1 with ethyl chloride replacement methane sulfonyl chloride and by (1: 1-0: 1) the column chromatography purifying of wash-out makes with P.E.-EtOAc.R.t.2.74 minute; M/z474.17.

¹H?NMR(400MHz，DMSO-d ₆)δ13.45(s，1H)，10.17(s，1H)，8.51(d，J＝8.0Hz，1H)，8.37(s，1H)，7.60-7.51(m，3H)，3.91-3.85(m，1H)，3.61(d，J＝12.0Hz，2H)，3.04(q，J＝8.0Hz，2H)，2.86(t，J＝12.0Hz，2H)，1.80-1.78(m，2H)，1.69-1.60(m，2H)，1.21(t，J＝8.0Hz，3H)

Embodiment 4

4-(2,6-two chloro-benzamidos)-1H-pyrazoles-3-formic acid (1-propyl group-alkylsulfonyl-piperidin-4-yl)-acid amides

Title compound still replaces methane sulfonyl chloride with the propane SULPHURYL CHLORIDE according to the method described in the embodiment 1 and makes by preparation type LC/MS purifying.R.t.3.11 minute; M/z488.18.

¹H?NMR：(400MHz，DMSO-d ₆)δ13.42(s，1H)，10.15(s，1H)，8.46(d，J＝8.0Hz，1H)，8.36(s，1H)，7.60-7.51(m，3H)，3.91-3.84(m，1H)，3.60(d，J＝12.0Hz，2H)，3.00(t，J＝8.0Hz，2H)，2.85(t，J＝12.0Hz，2H)，1.82-1.78(m，2H)，1.72-1.62(m，4H)，0.99(t，J＝8.0Hz，3H)。

Biological activity

Embodiment 5

Activatory CDK2/ cyclin A kinase inhibiting activity (IC ₅₀ ) mensuration

Adopt following scheme to measure the kinase inhibiting activity of The compounds of this invention.

With activatory CDK2/ cyclin A (people such as Brown, Nat.CellBiol., 1, PP438-443,1999; Lowe, E.D. waits people Biochemistry, and 41, pp15625-15634,2002) measure damping fluid (50mM MOPS pH7.2,62.5mM β-phospho-glycerol, 12.5mMEDTA, 37.5mM MgCl at 2.5 dual intensities ₂, 112.5mM ATP, 2.5mM DTT, 2.5mM sodium orthovanadate, 0.25mg/ml be diluted to 125pM bovine serum albumin), get 10 μ l and 10 μ l the histone substrate mixture (60 μ l ox histone h1s (and Upstate Biotechnology, 5mg/ml), 940 μ l H ₂O, 35 μ Ci γ ³³P-ATP) mix, add in 96 orifice plates with the different diluent of 5 μ l test compounds in DMSO (maximum 2.5%).Reaction was carried out 2 to 4 hours, used excessive ortho-phosphoric acid (5 μ l, 2%) termination reaction then.On Millipore MAPH screen plate, from the phosphorylation histone h1, isolate the γ that is not attached in the histone h1 ³³P-ATP.The aperture of MAPH plate is moistening with 0.5% ortho-phosphoric acid, pass through these hole filtering reaction products with the Millipore vacuum filtering system then.After the filtration, with twice of the 0.5% ortho-phosphoric acid debris of 200 μ l.After the strainer drying, add the Microscint20 scintillator of 20 μ l, on Packard Topcount, counted 30 seconds then.

Calculate active percent inhibition of CDK2 and drawing, suppress 50% the active needed concentration (IC of CDK2 to measure testing compound ₅₀).

Embodiment 6

Activatory CDK1/ cell periodic protein B kinase inhibiting activity (IC ₅₀ ) mensuration

CDK1/ cell periodic protein B assay method is identical with above-mentioned CDK2/ cyclin A, except using CDK1/ cell periodic protein B (Upstate Discovery) and this enzyme being diluted to the 6.25nM.

Compound of the present invention has the IC less than 20 μ M ₅₀Value or restraining effect to CDK2 activity at least 50% is provided when the concentration of 10 μ M.In CDK2 or CDK1 assay method, preferred compound of the present invention has the IC less than 1 μ M ₅₀Value.

Embodiment 7

The GSK3-B kinase inhibiting activity is analyzed

With GSK3-β (Upstate Discovery) at 25mM MOPS, pH7.00,25mg/mlBSA, 0.0025%Brij-35,1.25% glycerine, 0.5ml EDTA, 25mM MgCl ₂, be diluted to 7.5nM in 0.025% beta-mercaptoethanol, 37.5mM ATP, get 10 μ l and mix with 10 μ l substrate mixture.The substrate mixture of GSK3-β contains 35 μ Ci γ for being dissolved in 1ml ³³12.5 μ M phosphorylation glycogen synthetase peptides-2 (Upstate Discovery) in the water of p-ATP.Enzyme and substrate and the 5 μ l test compounds various diluents in DMSO (at the most 2.5%) are joined in 96 orifice plates together.Make reaction carry out 3 hours (GSK3-β), use excessive ortho-phosphoric acid (5 μ l, 2%) to stop then.The same above activatory CDK2/ cyclin A assay method of filter operation method.

Embodiment 8

Antiproliferative activity

The antiproliferative activity of The compounds of this invention can be measured the inhibition ability of the cell growth of various kinds of cell system by measuring compound.The cell growth-inhibiting utilize Alamar Blue assay method measure (Nociari, M.M, Shalev, A., Benias, P., Russo, C.Journal of ImmunologicalMethods 1998,213,157-167).This method is based on the ability that viable cell is reduced to resazurin its fluorescence-causing substance resorufin.For each proliferation assay, on 96 orifice plates, it was recovered 16 hours cell inoculation, add inhibitor compound then and reach other 72 hours.When incubation period finishes, add 10% (v/v) Alamar Blue, other 6 hours of incubation is measured fluorescence-causing substance then under 535nM ex/590nM em.Under the situation that non-proliferative cell is measured, cell was kept under converging 96 hours, add inhibitor compound then and reach other 72 hours.As above measure viable count by Alamar Blue assay method.Each clone all obtains from ECACC (European zooblast preservation center, European Collection of cell Cultures).

Particularly, with HCT-116 clone (the ECACC preserving number: 91091005) test compound of the present invention of derived from human colorectal carcinoma.

Find that in this assay method many compounds of the present invention have the IC less than 20 μ M ₅₀Value, preferred compound has the IC less than 1 μ M ₅₀Value.

Embodiment 9

The mensuration of oral administration biaavailability

Oral administration biaavailability that can following mensuration formula (I) compound.

With test compound according to following dosage level and dosage formulation with solution form intravenously with by oral administration to the balb/c mouse;

● 1mg/kg, intravenously is formulated in 10%DMSO/90% (2-hydroxypropyl)-beta-cyclodextrin (25%w/v); With

● 5mg/kg, oral, be formulated among 10%DMSO/20% water/70%PEG200.

Each time point after administration, blood sampling places the heparinization test tube, collects blood plasma and partly is used for analyzing.After carrying out albumen precipitation, analyze, by sample being carried out quantitatively with the standard correction curve ratio that makes up at test compound with LC-MS/MS.With standard method by area (AUC) under blood plasma level-time plot calculated curve.Oral administration biaavailability in order to following Equation for Calculating per-cent form:

According to this method, compound 4-(2,6-two chloro-benzamidos)-1H-pyrazoles-3-formic acid (1-methane sulfonyl-piperidin-4-yl)-acid amides during to the mouse administration, has the bioavailability of 40-50% with oral route.

Embodiment 10

Heteroplastic transplantation (Xenograph) research

The compound of embodiment 1 has antitumor action in the nude mice of having implanted the clone that derives from human tumor.When can cause the inhibiting oral dose administration to the knubble biological mark, the treatment of embodiment 1 compound has produced the restraining effect to tumor growth in the allogeneic of subcutaneous implantation.These biomarkers comprise for example inhibition of the proteic phosphorylation of retinoblastoma of substrate of cell cycle protein dependent kinase.The compound of embodiment 1 (comprising the long term administration in several weeks) with multiple different scheme administration the time is effective.

Embodiment 11

Comparing embodiment

With the biological activity of the compound (it comprises 2,6-dichlorophenyl group) of embodiment 1 and its 2, the biological activity of 6-difluorophenyl analogue compares.2,6-difluorophenyl analogue is described in the embodiment 131 of we application PCT/GB2004/003179 (publication number WO2005/012256) early, and it has following structure

More particularly, the anti-CDK2 kinases and the activity of GSK3 beta kinase and the ability of its inhibition HCT-116 human colon cancer cell propagation that have compared compound.These kinase inhibiting activities and HCT-116 suppress active and are to use above-mentioned analytical procedure to measure, and the result is presented in the following table.

	The compound of prior art (embodiment 131 of PCT/GB2004/003179)	The compound of embodiment 1
			CDK2IC 50	0.0022μM	43％@0.0003μM
GSK3βIC 50	0.014μM	0.22μM
			The IC of HCT-116 cell proliferation 50	0.74μM	0.11μM

The compound of the embodiment of the present application 1 is compared with its difluoro analogue has following advantage:

. compare with its difluoro analogue, the compound of embodiment 1 is eager to excel 6-7 doubly to the anti-proliferative effect of human colon carcinoma HCT-116 clone.

. compare with its difluoro analogue, the compound of embodiment 1 has higher vitro kinase (CDK2) and suppresses active.

. the compound of embodiment 1 is lower than its two fluoro-analogue (0.014 μ M) to the activity (0.22 μ M) of GSK3 β.

. compare with its difluoro analogue (～6 times), the compound of embodiment 1 has 200 times of higher CDK inhibition/GSK3 beta selectives (〉).

Pharmaceutical preparation

Embodiment 12

(i) tablet formulation

Preparation contains the tablet composition of formula (I) compound by the following method: mix 50mg compound and 197mg as the lactose (BP) of thinner and 3mg as the Magnesium Stearate of lubricant, be pressed into tablet with currently known methods.

(ii) capsule

Prepare capsule by the following method: mix 100mg formula (I) compound and 100mg lactose, the gained mixture is packed in the opaque hard gelatin capsule of standard.

(iii) injectable formulation I

Can prepare the parenteral composition of using by injection by the following method: formula (I) compound (for example formula of salt form (I) compound) is dissolved in the water that contains 10% propylene glycol, produces the activity compound concentration of 1.5 weight %.Then by filtering with solution sterilization also sealing in the ampoule of packing into.

(iv) injectable formulation II

The preparation parenteral composition that is used for injecting by the following method: with formula (I) compound (for example formula of salt form (I) compound) (2mg/ml) and N.F,USP MANNITOL (50mg/ml) be dissolved in water, with solution sterile filtration, in pack into sealable 1ml bottle or the ampoule.

V) injectable formulation III

Can prepare by the following method and be used for: formula (I) compound (for example formula of salt form (I) compound) is dissolved in water with the concentration of 20mg/ml by injection or the preparation sent of infusion intravenously.Then with bottle sealing and autoclaving.

Vi) injectable formulation IV

Can prepare by the following method and be used for: formula (I) compound (for example formula of salt form (I) compound) is dissolved in the water (for example 0.2M acetate pH4.6) that contains buffer reagent with the concentration of 20mg/ml by injection or the preparation sent of infusion intravenously.Then with bottle sealing and autoclaving.

(vii) subcutaneous injection preparation

Preparation is used for the composition of subcutaneous administration by the following method: hybrid (I) compound and pharmaceutical grade Semen Maydis oil, the concentration of generation 5mg/ml.In composition sterilization and threading appropriate containers.

Viii) freeze-dried preparation

The aliquots containig of formula (I) compound of preparation is put into bottle and the lyophilize of 50ml.During lyophilize, use the freezing said composition of a freezing scheme of step down at (45 ℃).Temperature is raised to-10 ℃ to rise again, be reduced to then-45 ℃ down freezing, then in+25 ℃ first down dry about 3400 minutes, succeeded by redrying, if temperature rises to 50 ℃ then increase step.During preliminary and redrying, pressure is set at 80 millitorrs.

(ix) solid solution preparation

The concentration of (for example 16 or 20%) is dissolved in methylene dichloride/ethanol (1: 1) with 5 to 50% with the compound of embodiment 1, uses corresponding to condition given in the following table the solution spray drying.Given data comprise the concentration (analysis) of compound in spray-dired solid of the compound concentrations of embodiment 1, the entrance and exit temperature of spray-dryer, spray-dired solid overall yield, embodiment 1 and the size-grade distribution of being made up of spray-dired solid (P.S.D.) in the table.

Lot number	Strength of solution w/v	Temperature in	Temperature out	The % productive rate	Analyze (mg/g)	PSD (scope) (μ m)
							BR1A	16％	140℃	80℃	87.00	246.41	4.46-52.76
BR1B	16％	180℃	80℃	97.00	246.65	14.83-91.70
							BR2A	20％	160℃	80℃	99.40	239.60	15.86-85.01
BR3A	20％	180℃	100℃	79.50	246.64	15.09-91.84

The compound of embodiment 1 and the solid solution of PVP directly can be put in glutoid or HPMC (Vltra tears) capsule, or with pharmaceutically acceptable vehicle such as extender, glidant or dispersant.This capsule can contain 2mg-200mg, for example 10,20 and the compound of 80mg embodiment 1.

Embodiment 13

The mensuration of anti-mycotic activity

Can adopt following method to measure the anti-mycotic activity of formula (I) compound.

Test compounds is resisted the effect of one group of fungi, comprises Candida parapsilosis (Candidaparpsilosis), Oidium tropicale (Candida tropicalis), Bai Nianse pearl bacterium (Candidaalbicans)-ATCC36082 and Cryptococcus neoformans (Cryptococcus neoformans).These test microbes are being stored under 4 ℃ on the Sabourahd agar glucose inclined-plane.Each biological singlet suspension prepares according to following description: under 27 ℃, on rotary shaker, containing amino acid (Difco, Detroit Mich.) spends the night yeast growth in the yeast nitrogen liquid nutrient medium (YNB) of (pH7.0) and 0.05M morpholine propanesulfonic acid (MOPS).Centrifugal then suspension is used the 0.85%NaCl washed twice, then will through the cell suspending liquid of washing with sonic treatment 4 seconds (Branson Sonifier, 350 types, Danbury, Conn.).Counting singlet blastospore is adjusted to desired concn in 0.85%NaCl in hematimeter.

Use the activity of the modification method mensuration testing compound of liquid nutrient medium Microdilution technology.Testing compound is diluted to the ratio of 1.0mg/ml in DMSO, in the YNB liquid nutrient medium that contains MOPS (using fluconazole in contrast) of pH7.0, is diluted to 64 μ g/ml then, so that the working solution of every kind of compound to be provided.Use 96 orifice plates, prepare the 1st and 3 to 12 holes, prepare ten times of diluents (concentration range is 64 to 0.125 μ g/ml) of compound solution in the 2nd to 11 hole with the YNB liquid nutrient medium.The 1st hole is as the aseptic contrast and the blank of spectrophotometry.The 12nd hole is as growth control.(final inoculum number is 104 biologies/ml) to inoculate 10 μ l to each hole in micro plate the 2nd to 11 hole.Will be through the plate of inoculation 35 ℃ of following incubations 48 hours.With vortex mixer (Vorte-Genie 2 Mixer, Scientific Industries, Inc., Bolemia, N.Y.) the vibration plate is after 2 minutes, with absorbancy (Automatic MicroplateReader, the DuPont Instruments of metric measurement at 420nm, Wilmington Del.) measures the IC50 value.The IC50 terminal point is defined as comparing with control wells, demonstrating the lowest concentration of drug that growth reduces about 50% (or more than).In turbidity measurement, this is defined as the lowest concentration of drug (IC50) of aperture turbidity for＜50% contrast.Minimum molten born of the same parents' concentration (MCC) is determined by the following method: all apertures of culture transferring 96 orifice plates on Sabourahd agar glucose (SDA) plate, 35 ℃ of following incubations 1 to 2 day, check viability then.

Embodiment 14

The biological assessment method of fungi infestation in the whole strain plant materials of control

In acetone, then use the acetone serial dilution formula (I) compound dissolution to obtain the desired concn scope.Decide according to pathogenic agent, by adding the 0.05%Tween-20 of 9 volumes ^TMThe aqueous solution or 0.01%Triton X-100 ^TMObtain the final volume of handling.

Use composition to adopt following method to measure the activity of the anti-tomato eqpidemic disease of The compounds of this invention (phytophthora infestans (Phytophthora infestans)) then.Make the growth in the peat base potting mixtures that does not have soil of tomato (Rutgers kind) seed high up to seedling 10-20 centimetre.Compound to be tried with the 100ppm ratio sprays this plant to flowing out then.After 24 hours, test plants is with the sporocyst waterborne suspension spray inoculation of tomato parasitica, and remains on open-air room and spend the night.Then this plant being transferred to the greenhouse takes place on untreated control plant up to disease.

Also adopt similar method to test the activity of Powdery Mildew, speckled leaf blotch (wheat septoria (Septoria tritici)) and the wheat glume blight (Leptosphaeria nodorum) of The compounds of this invention opposing brown rust of wheat (Puccinia Puccinia), wheat (Ervsiphe vraminis) and wheat (Monon kind).

Equivalents

Provide the purpose of the foregoing description to be to set forth the present invention, it should be interpreted as the scope of the invention is forced any restriction.Obviously, can be under the situation that does not deviate from ultimate principle of the present invention the specific embodiments of the present invention that is exemplified among mentioned above and the embodiment be carried out various modifications and variations.All these class modifications and variations are all contained by the application.

Claims (30)

1. formula (I) compound:

Or its salt, tautomer, solvate and N-oxide compound;

Wherein:

R ¹Be 2, the 6-dichlorophenyl;

R ^2aAnd R ^2bBe hydrogen;

And R ³Group for following formula:

R wherein ⁴Be C _1-4Alkyl.

2. according to the compound of claim 1, R wherein ⁴Be C _1-3Alkyl.

3. according to the compound of claim 2, R wherein ⁴It is methyl.

4. according to the compound of claim 2, R wherein ⁴It is ethyl.

5. according to the compound of claim 2, R wherein ⁴It is n-propyl.

6. according to the compound of claim 2, R wherein ⁴It is sec.-propyl.

7. according to each compound in the aforementioned claim, it is not the form of salt or N-oxide compound.

8. according to each compound in the claim 1 to 6, it is the form of salt, solvate or N-oxide compound.

9. be used for preventing or treat the compound any according to claim 1 to 8 by cell cycle protein dependent kinase or glycogen synthase kinase-3 disease states mediated or illness.

10. prevention or treatment are by the method for cell cycle protein dependent kinase or glycogen synthase kinase-3 disease states mediated or illness, and this method comprises gives the individuality that needs it with compound administration any in the claim 1 to 8.

11. alleviate or reduce the method by the sickness rate of cell cycle protein dependent kinase or glycogen synthase kinase-3 disease states mediated or illness, this method comprises gives the individuality that needs it with compound administration any in the claim 1 to 8.

12. treatment comprises or results from the disease of abnormal cell growth or the method for illness in Mammals, this method comprises gives Mammals with suppressing compound administration any in the claim 1 to 8 of abnormal cell growth significant quantity.

13. alleviate or reduce the method for sickness rate that comprises or result from the disease of abnormal cell growth or illness in Mammals, this method comprises gives Mammals with suppressing compound administration any in the claim 1 to 8 of abnormal cell growth significant quantity.

14. treatment comprises or results from the disease of abnormal cell growth or the method for illness in Mammals, this method comprises gives Mammals with suppressing compound administration any in the claim 1 to 8 of cdk kinases (for example cdk1 or cdk2) or glycogen synthase kinase-3 active significant quantities.

15. alleviate or reduce the method for sickness rate that comprises or result from the disease of abnormal cell growth or illness in Mammals, this method comprises gives Mammals with suppressing compound administration any in the claim 1 to 8 of cdk kinases (for example cdk1 or cdk2) or glycogen synthase kinase-3 active significant quantities.

16. suppress the method for cell cycle protein dependent kinase or glycogen synthase kinase-3, this method comprises contacts kinase inhibition compound any in kinases and the claim 1 to 8.

17. regulate the method for cell processes (for example cell fission), this method realizes by the activity of using compound any in the claim 1 to 8 to suppress cell cycle protein dependent kinase or glycogen synthase kinase-3.

18. be used for preventing or treating any one compound of claim 1 to 8 of morbid state described herein.

19. any described compound is used for the purposes of the medicine of one or more purposes defined herein in the claim 1 to 8 in production.

20. comprise compound any in the claim 1 to 8 and the pharmaceutical composition of pharmaceutically acceptable carrier.

21. be suitable for the pharmaceutical composition of oral administration form, it comprises compound any in the claim 1 to 8 and pharmaceutically useful carrier.

22. be used for any one compound in the claim 1 to 8 of medicine.

23. be used for the compound any above-mentioned and described any purposes of this paper other parts and method according to claim 1 to 8.

24. a method for preparing each compound in the claim 1 to 8, this method comprise formula (XVIT) compound:

With a kind of suitable introducing SO ₂R ⁴The sulfonylation agent of group (for example SULPHURYL CHLORIDE such as methane sulfonyl chloride) reaction.

25. diagnose and treat the method by cell cycle dependant kinase disease states mediated or illness for one kind, this method comprises that (i) screening patient is to determine that this patient is suffering from morbid state or the illness that maybe may suffer from and whether employing had the treatment sensitivity that the active compound of anti-cell cyclin-dependent kinase is carried out; (ii) be instructed to the patient be used compound any in the claim 1 to 8 when being responsive when disease of patient or illness.

26. any one compound is used for the treatment of or prevents purposes in the medicine of disease of patient or illness, wherein said patient to be examined and to be defined as suffering from morbid state or illness with treatment sensitivity that the active compound of anti-cell cyclin-dependent kinase carries out or the risk of suffering from these diseases or illness is arranged in production in the claim 1 to 8.

27. be used for suppressing any one compound in the claim 1 to 8 of Mammals tumor growth.

28. be used for suppressing any one compound in the claim 1 to 8 of growth of tumour cell (for example Mammals).

29. suppress the method for tumor growth in the Mammals (for example people), this method comprises uses any one compound in the claim 1 to 8 that suppresses the tumor growth significant quantity to Mammals (for example people).

30. suppress the method for tumour cell (for example being present in for example tumour cell of philtrum of Mammals) growth, this method comprises that compound any in the claim 1 to 8 that makes tumour cell and inhibition tumor growth significant quantity contacts.