CN101146791B - 4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acid piperidin-4-ylamid acid addition salts as kinase inhibitors - Google Patents

4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acid piperidin-4-ylamid acid addition salts as kinase inhibitors Download PDF

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CN101146791B
CN101146791B CN200680009244.XA CN200680009244A CN101146791B CN 101146791 B CN101146791 B CN 101146791B CN 200680009244 A CN200680009244 A CN 200680009244A CN 101146791 B CN101146791 B CN 101146791B
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acid
salt
pyrazoles
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benzoyl
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CN101146791A (en
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P·G·怀亚特
D·C·里斯
M·温科维奇
G·特里瓦撒
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Astex Therapeutics Ltd
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Abstract

The invention provides an acid addition salt of 4-(2,6-dichloro-benzoylamino)-lH- pyrazole-3-carboxylic acid piperidin-4-ylamide and crystals thereof, the salt being formed with an acid selected from methanesulphonic acid and acetic acid and mixtures thereof. Also provided are the novel uses of salts of 4-(2,6-dichloro-benzoylamino)-lH- pyrazole-3-carboxylic acid piperidin-4-ylamide, processes for the preparation of 4- (2,6-dichloro-benzoylamino)-lH-pyrazole-3-carboxylic acid piperidin-4-ylamide and its salts and novel chemical intermediates.

Description

Acid salt as the 4-(2,6-, two chloro-benzoyl-amidos) of kinase inhibitor-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides
The present invention relates to compound 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-acid salt and its crystalline form of 4-base acid amides, the method for preparing this compound and its acid salt, the new chemical intermediate that is used for the method and the therepic use of this compound and its acid salt.The invention still further relates to the new therepic use of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides and analogue thereof.
Background of invention
The inhibitor as cell cycle protein dependent kinase (CDK kinases) and GSK-3 (GSK3) is open in we previous international patent application No.PCT/GB2004/003179 (publication number WO 2005/012256) for compound 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides and its salt acid salt.
The protein kinase of being responsible for the intracellular multi-signal conductive process of control has consisted of relevant large 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) corresponding to each kinases family have been identified; The people such as Knighton, Science, 253:407-414 (1991); The people such as Hiles, Cell, 70:419-429 (1992); The people such as Kunz, Cell, 73:585-596 (1993); The 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 phosphorylation, protein-protein interaction, protein-lipid interaction and protein-polynucleotide interaction of turning.A kind of protein kinase may be subject 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 various extracellular signals (hormone, neurotransmitter, GDF etc.), cell cycle events, environment or Nutrient Stress etc. and phosphorylation occurs.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 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
The process of eukaryotic cell division can broadly be divided into when being called G1, S, G2 and M a series of continuous 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 amino acid whose homologous regions as the protein families of feature, and it is used for the 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.These complex formations go 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, namely 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 E type cyclin member's combination.As if D type cyclin helps not go down to posterity by 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 subject to the adjusting of cdk1 and A and type B cell cyclin mixture.
In the G1 phase, retinocytoma albumen (Rb) and relevant bag-shaped 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/cyclin-D mixture Hyperphosphorylationof inactivation and partly promoting.The Hyperphosphorylationof 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 the gene of cyclin E.The expression of cyclin E promotes cdk2/ cyclin E complex formation, 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 subject 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 represent to stimulate integrated to a certain extent point from the biological chemistry of Rb, Myc and p53 approach.Therefore Cdk2 and/or cdk2/ cyclin E mixture have represented and have ended in the cell of abnormal division or the restorative good treatment target spot of controlling the cell cycle.
The corner cut look also unclear really in the cell cycle for cdk3.Also do not have so far the cyclin partner of homology identified, but therefore the dominant negative regulator Form postponement G1 phase cell of cdk3 hint that cdk3 has the effect of regulating the G1/S conversion.
Although most cdks participates in the adjusting of cell cycle, evidence suggests that some cdk family member has participated in other Biochemical processes.Take cdk5 as example, 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 activates by the combination with p35/p39 protein usually.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 can be brought out by local asphyxia, excitotoxicity and beta amyloid peptide to the conversion of p25 and the subsequently adjusting of going of cdk5 activity.Thereby p25 involved for example pathogenesis of alzheimer's disease of neurodegenerative disease, therefore as having caused people's interest for the treatment target spot of these diseases.
Cdk7 is that a kind of cdc2 of having CAK 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 phosphorylation or the dephosphorylation event 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 wee1, Myt1 and Mik1.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 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.
The above has summarized the cell cycle was coordinated and driven to Cdks and related protein thereof in proliferative cell key role.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 therapeutic combination treatment, the treatment of target cdk also can provide clinical benefit in the treatment of aforementioned diseases.Anticancer therapy take cdk as target spot may have advantages of and be 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
GSK-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 dynamics, 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 identified by GSK3 is (Ser/Thr)-X-X-X-(pSer/pThr), 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 automatically suppressing the machine-processed model that can compete with the peptide substrates (S/TXXXpS/pT) of phosphorylation.Also there are data proposition GSK3 α and GSK β to be regulated 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 IRS (IRS) phosphorylation and raising with backward cytolemma.The further phosphorylation of IRS protein has started the gathering of phosphatidylinositol 3-kinase (P13K) 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 whereby GSK3 α and/or GSK β by the phosphorylation of Ser9 or ser21 respectively.The inhibition of GSK3 then triggers the rise of glycogen synthetase activity.Therefore the therapeutical agent that can suppress GSK3 can bring out and be similar to those cell responses of observing in insulin stimulating.Substrate was eukaryotic cell protein synthesis Eukaryotic initiation factor 2 B (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, have some to pass through mitogen-activated protein kinase (MAPK) path, make GSK3 by kinases for example mitogen-activated protein kinase activation protein kinase 1 (MAPKAP-K1 or RSK) phosphorylation and regulate the evidence of GSK3 activity.These data promptings GSK3 activity can be subject 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 suppressed for replying of Wnt stimulation.This can cause GSK3 substrate for example Axin, the gene product of adenomatous polyposis coli (APC) and the dephosphorylation of beta-catenin.The unusual adjusting of Wnt approach is relevant with many cancers.Sudden change in APC and/or the beta-catenin is common in colorectal carcinoma and other the tumour.Beta-catenin has also demonstrated the importance in cell attachment.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, although specificity in a organized way.GSK3 may be closely related with the medical conditions that the neuronal cell apoptosis wherein can occur via the effect of short apoptosis mechanism in regulating apoptosis.The example of these illnesss is 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.Can Hyperphosphorylationof canalicular apparatus associated protein Tau at the external GSK3 that confirmed.The Hyperphosphorylationof of Tau has destroyed the normal combination of it and 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 the phosphorylation that suppresses Tau by suppressing GSK3 can provide the method for a kind of restriction and/or prevention neurodegeneration effect.
Diffuse large B cell lymphoma (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 CDK supressor (CDKi) of cell cycle negative regulation albumen.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 demonstration of the clinical correlation analysis of these discoveries, 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 large B cell lymphoma (DLBCL), have bad Clinical symptoms, prompting can cause this unusual p27KIPl protein to lose function by the interaction with other cell cycle regulatory factors albumen.(Br.J.Cancer.1999 Jul; 80 (9): 1427-34.p27KIPl is unconventionality expression and relevant with bad clinical effectiveness in the Diffuse large B cell lymphoma.Saez A，Sanchez E，Sanchez-Beato M，Cruz MA，ChaconI，Munoz E，Camacho FI，Martinez-Montero JC，Mollejo M，Garcia JF，Piris MA.Department of Pathology，Virgen de la Salud Hospital，Toledo，Spain.)
Chronic lymphocytic leukemia
B Cell Chronic Lymphocytic Leukemia (CLL) is the modal leukemia in the western hemisphere, and approximately have 10 every year, 000 new case is diagnosed (Parker SL, 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.
With comparing as the treatment on basis take alkylating agent of previous use, add fludarabine and can cause the fully response (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.Although the fully response that reaches after treatment clinically is the initial step of improving the CLL survival rate, most of patients does not reach fully alleviation 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, Hines J, 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.Blood 88:141 a, 1996 (summary 552, suppl 1).Therefore, if want to realize the further improvement of this disease treatment, must identify the cytotoxicity with additional fludarabine and the new medicament of removing the novel mechanism of the resistance of being brought out by intrinsic CLL r factor.
About CLL patient's relatively poor and bad survival rate to therapeutic response study to such an extent that the most widely normative forecast factor is unusual p53 function, it is take point mutation or karyomit(e) 17p13 disappearance as feature.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 CYC 202 at the external apoptosis that brings out from the malignant cell of B Cell Chronic Lymphocytic Leukemia (B-CLL).
Good fortune draws the benefit administration to cause the stimulation of caspase 3 activity and the dependent decomposition of caspase-of 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.1998 Nov 15；92(10)：3804-16 Flavopiridol induces apoptosis inchronic lymphocytic leukemia cells via activation of caspase-3 without evidence of bcl-2 modulation 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
WO 02/34721 from Du Pont discloses a kind of indeno as cell cycle protein dependent kinase inhibitor [1,2-c] pyrazoles-4-ketone.
From the WO 01/81348 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.
WO 00/62778 from Bristol Myers Squibb discloses a kind of protein tyrosine kinase inhibitor equally.
From the WO 01/72745A1 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 purposes in treatment proliferative disease such as cancer, leukemia, the psoriasis etc.
WO 99/21845 from Agouron has described a kind of for suppressing cell cycle protein dependent kinase (CDK), for example the 4-aminothiazole derivs of CDK1, CDK2, CDK4 and CDK6.This invention also relates to treatment or the preventive use of the pharmaceutical composition that comprises this compound, and these compounds for treating malignant diseases by giving significant quantity and the method for Other diseases.
WO 01/53274 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.
WO 01/98290 (Pharmacia ﹠amp; Upjohn) a kind of 3-aminocarboxyl as kinases inhibitor-2-amide group-thiophene derivant is disclosed.
Disclose from the WO 01/53268 of Agouron and WO 01/02369 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 aryl or the heteroaryl ring of the 3-position of indazole ring.
WO 00/39108 and WO 02/00651 (both all belong to Du Pont medicine) have described heterogeneous ring compound, and it is trypsin-like serine protease, especially the inhibitor of Xa factor and zymoplasm.It is said this compound useful as anticoagulants agents or be used for the prevention thromboembolism.
US 2002/0091116 (people such as Zhu), WO 01/19798 and WO 01/64642 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.
US 6,127,382, WO 01/70668, WO 00/68191, WO 97/48672, WO97/19052 and WO 97/19062 (all belonging to Allergan) disclose the compound with retinoids activity that is used for the treatment of the various hyperproliferation diseases that comprise cancer separately.
WO 02/070510 (Bayer) has described a kind of amino-dicarboxylic acid compound that is used for the treatment of cardiovascular disorder.Although mentioned in general manner pyrazoles, in this part file, do not had the specific embodiment of pyrazoles.
WO 97/03071 (Knoll AG) discloses a kind of heterocyclic radical-carboxamides derivatives that is used for the treatment of central nervous system disorders.Mention in general manner the example of pyrazoles as heterocyclic radical, but do not had to disclose or exemplify concrete pyrazole compound.
WO 97/40017 (Novo Nordisk) has described the compound as the conditioning agent of Protein-tyrosine-phosphatase.
WO 03/020217 (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.
WO 01/58869 (Bristol Myers Squibb) discloses the cannabinoid receptor conditioning agent that can be used for treating various diseases.The main application of predicting is for the treatment respiratory disease, although mentioned the treatment of cancer.
WO 01/02385 (Aventis Crop Science) discloses 1-(quinolyl-4) as mycocide-1H-pyrazole derivatives.The unsubstituted pyrazoles as the 1-of synthetic intermediate is wherein disclosed.
WO 2004/039795 (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 the illnesss such as hyperlipidemia.
WO 2004/000318 (Cellular Genomics) discloses the monocycle class of various amino as kinase modulator-replacement.The compound that exemplifies without pyrazoles.
Summary of the invention
The present invention especially provides compound 4-(2,6-, two chloro-the benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-acid salt of 4-base acid amides and the crystalline form of acid salt, particularly methylsulfonic acid and acetate.
The present invention also provides the novel method for preparing this compound, its acid salt and its crystalline form and the new chemical intermediate that is used for the method.
The present invention further provides the novel therapeutic use of the analogue of the therepic use of compound and acid salt thereof and 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
Therefore, first aspect the invention provides the acid salt of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, and this salt is the salt except hydrochloride.
Derive the free alkali 4-(2,6-, two chloro-benzoyl-amidos) of these salt-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides and have formula (I):
Figure S200680009244XD00111
The compound of formula (I) can be mentioned with its chemical name in this application, perhaps for convenient, is called " this compound ", " compound of formula (I) " or " compound of the present invention ".These synonyms all refer to separately with the compound with chemical name 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides shown in the following formula (I).
The related salt of the application is the acid salt of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base amido acid.Term " salt " and " acid salt " are used interchangeably in this application.Unless context is otherwise noted, otherwise term used herein " salt " refers to acid salt.
To compound 4-(2; 6-two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides and mentioning of acid salt thereof comprise its all solvates, tautomer and isotropic substance in its scope; and in the situation that context allows, comprise its N-oxide compound, other ionic species and prodrug.
Acid salt be selected from the choosing group under the salt that forms of the acid organized: acetic acid, hexanodioic acid, Lalgine, xitix (for example L-AA), aspartic acid (for example L-Aspartic acid), Phenylsulfonic acid, phenylformic acid, dextrocamphoric acid (for example (+) dextrocamphoric acid), capric acid, sad, carbonic acid, citric acid, cyclohexane sulfamic acid, dodecylic acid, dodecyl sulphate, ethane-1, the 2-disulfonic acid, ethane sulfonic 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 Pidolidone), α ketoglutaric acid, oxyacetic acid, urobenzoic acid, isethionic acid, isopropylformic acid, lactic acid (for example (+)-Pfansteihl and (±)-DL-LACTIC ACID), lactobionic acid, dodecyl sodium sulfonate, toxilic acid, oxysuccinic acid, (-)-L MALIC ACID, propanedioic acid, methylsulfonic acid, glactaric acid, naphthene sulfonic acid (for example naphthalene-2-sulfonic acid), naphthalene-1, the 5-disulfonic acid, nicotinic acid, oleic acid, vitamin B13, oxalic acid, palmitinic acid, pamoic acid, phosphoric acid, propionic acid, sebacic acid, stearic acid, succsinic acid, sulfuric acid, tartrate (for example (+)-L-TARTARIC ACID), thiocyanic acid, toluenesulphonic acids (for example p-toluenesulphonic acids), valeric acid and former times naphthoic acid (xinafoic acid).
A subgroup of acid salt comprises the salt that forms with the acid that is selected from lower group: acetic acid, hexanodioic acid, xitix (for example L-AA), aspartic acid (for example L-Aspartic acid), caproic acid, carbonic acid, citric acid, dodecylic acid, fumaric acid, tetrahydroxyadipic acid, glucoheptonic acid, glyconic acid (for example D-glyconic acid), glucuronic acid (for example D-glucuronic acid), L-glutamic acid (for example Pidolidone), oxyacetic acid, urobenzoic acid, lactic acid (for example (+)-Pfansteihl and (±)-DL-LACTIC ACID), toxilic acid, palmitinic acid, phosphoric acid, sebacic acid, stearic acid, succsinic acid, sulfuric acid, tartrate (for example (+)-L-TARTARIC ACID) and thiocyanic acid.
More specifically, salt be be selected from methylsulfonic acid and acetic acid with and composition thereof the acid salt that forms of acid.
In one embodiment, salt is the acid salt that forms with methylsulfonic acid.
In another embodiment, salt is the acid salt that forms with acetic acid.
For convenient, the salt that is formed by methylsulfonic acid and acetic acid is called mesylate or mesylate and acetate in this article.
The salt of the present invention of solid state can be crystalline or unbodied or its mixture.
In one embodiment, salt is unbodied.
In amorphous solid, be present in usually that three-dimensional structure in the crystalline form does not exist and in amorphous form the toward each other position of molecule be random basically, such as referring to people J.Pharm.Sci. (1997) such as Hancock, 86,1).
In another embodiment, salt is crystalline basically; That is, their 50%-100% are crystalline, more especially they can at least 50% be crystalline, or at least 60% be crystalline, or at least 70% be crystalline, or at least 80% be crystalline, or at least 90% is crystalline, or at least 95% be crystalline, or at least 98% be crystalline, or at least 99% be crystalline, or at least 99.5% is crystalline, or at least 99.9% be crystalline, and for example 100% is crystalline.
In another embodiment, salt be selected from the group that is formed by following salt: 50%-100% be crystalline salt, at least 50% be crystalline salt, at least 60% be crystalline salt, at least 70% be crystalline salt, at least 80% be crystalline salt, at least 90% be crystalline salt, at least 95% be crystalline salt, at least 98% be crystalline salt, at least 99% be crystalline salt, at least 99.5% be crystalline salt and at least 99.9% be crystalline, for example 100% be crystalline salt.
More preferably, salt can be following salt (perhaps can be selected from the group that is comprised of following salt): 95%-100% is crystalline salt, for example at least 98% is crystalline salt, or at least 99% be crystalline salt, or at least 99.5% be crystalline salt, or at least 99.6% is crystalline salt, or at least 99.7% be crystalline salt, or at least 99.8% be crystalline salt, or at least 99.9% be crystalline salt, and for example 100% is crystalline salt.
Basically an example that is crystalline salt is the crystalline salt that forms with methylsulfonic acid.
Basically another example that is crystalline salt is the crystalline salt that forms with acetic acid.
The salt of the present invention of solid state can be (for example anhydrous) of solvation (for example aquation) or non--solvation.
In one embodiment, salt is non-solvation (for example anhydrous).The example of the salt of non-solvent is the crystalline salt that forms with methylsulfonic acid defined herein.
Term used herein " anhydrous " is not precluded within salt (for example crystal of salt) upward or has the possibility of some water in the salt (for example crystal of salt).For example, on the surface of salt (for example salt crystal), may there be some water, perhaps in the main body of salt (for example crystal), have a small amount of water.Usually anhydrous form per molecule compound contains the water less than 0.4 molecule, and more preferably the per molecule compound contains the water less than 0.1 molecule, for example contains the water of 0 molecule.
In another embodiment, salt is solvation.In the situation that salt is aquation, they for example can contain at the most 3 molecular crystal water, more generally contain at the most 2 molecular waters, for example 1 molecular water or 2 molecular waters.Also can form wherein the water molecule number that exists less than 1 or be not the non-stoichiometric hydrate of integer.For example, in the situation that exist less than 1 molecular water, can there be for example water of 0.4 or 0.5 or 0.6 or 0.7 or 0.8 or 0.9 molecule by the per molecule compound.
Other solvate comprises alcoholate, such as ethylate or Virahol compound.
Can pass through conventional chemical method such as Pharmaceutical Salts:Properties; Selection; and Use; P.Heinrich Stahl (editor), Camille G.Wermuth (editor), ISBN:3-90639-026-8; Hardcover; 388 pages, the method for describing in 2002 8 months is by parent compound 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-synthetic salt of the present invention of 4-base acid amides.Generally speaking, can in water or in organic solvent or in the mixture of the two, react to prepare this class salt with suitable acid by making parent compound 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides; The non-aqueous solvent of normal operation such as ether, ethyl acetate, ethanol, Virahol or acetonitrile.
On the other hand; the invention provides a kind of 4-of preparation (2; 6-two chloro-benzoyl-amidos)-method of the acid salt of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides; the method comprise form 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides free alkali in solvent (normally organic solvent) or solvent mixture solution and with this solution of acid treatment to form the precipitation of acid salt.
Can add acid with the form of solution, the solvent of described solution can be miscible with the solvent phase of dissolving free alkali.The solvent of initial dissolving free alkali can be therein undissolved solvent of its acid salt.Perhaps, the solvent that dissolves at first free alkali can be the solvent of therein at least part of dissolving of acid salt, add subsequently a kind of acid salt therein the worse different solvents of solvability so that salt is precipitated out from solution.
In the another kind of method that forms acid salt; with 4-(2; 6-two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides is dissolved in and comprises volatile acid and randomly comprise in the solvent of cosolvent; thereby form the solution with the acid salt of volatile acid, then gained solution is concentrated or evaporation is to isolate salt.The example of the acid salt that can in this way prepare is acetate.
On the other hand; the invention provides a kind of formation 4-(2; 6-dichloro-benzoyl base is amino)-method of the acid salt of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, the method comprises the compound that is used in the organic or inorganic acid treatment formula (X) except hydrochloric acid defined herein in the organic solvent:
Figure S200680009244XD00151
Removing the tert-butoxycarbonyl group and to form the acid salt of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides and organic or inorganic acid, and randomly separate the thus acid salt of formation.
Salt is common to be precipitated out once forming from organic solvent, thereby and can be by solid and solution being separated, for example being separated by filtering to separate.
By well known to a person skilled in the art that method can change into a kind of salt form of the present invention free alkali and randomly change into another kind of salt form.For example, can be by making the post of salts solution by containing the amine stationary phase (Strata-NH for example 2Post) forms free alkali.Perhaps, can process the solution of salt in water with salt decomposition and be settled out free alkali with sodium bicarbonate.Then can by above or one of the described method of this paper other parts with free alkali and another kind of acid combination.
Compare with corresponding free alkali, salt such as acid salt have many advantages.For example, compare with free alkali, salt has one or more in the following advantages, because they:
Solvability is better, and therefore will use (for example by using in the intravenous injection) for intravenously better
Has better stability (for example increasing the shelf lives);
Has better thermostability;
Alkalescence a little less than, therefore and will be used for better intravenously and use;
Has advantages of the preparation of being beneficial to;
The solubleness that in aqueous solution, has raising;
Have better physicochemical property;
The antitumour activity that can have raising; With
The therapeutic index that can have raising.
The mesylate form is advantageous particularly, because it has satisfactory stability at elevated temperatures and under high relative humidity condition, have non-hygroscopic property (as defined herein), do not have the formation of polymorphic form and hydrate, and stable under aqueous conditions.In addition, it has good water-soluble, and compares with other salt, has better physicochemical property (such as high-melting-point).
Term used herein " is stablized " or " stability " comprises chemical stability and solid-state (physics) stability.Term ' chemical stability ' means compound and can store under the normal storage condition with the form of separation or with the form of preparation, there are not or almost do not have chemical degradation or decomposition, in described preparation, compound is to be provided with for example form of mixtures of pharmaceutically acceptable carrier as herein described, thinner or assistant agent.' solid-state stability ' means compound and can store under the normal storage condition with the solid form of separation or the form of solid preparation, there is not or almost do not have solid-state conversion (for example hydration, dehydration, solvation, desolvation, crystallization, recrystallization or solid-state phase changes), in described solid preparation, compound is to be provided with for example form of mixtures of pharmaceutically acceptable carrier as herein described, thinner or assistant agent.
Term used herein " non-hygroscopic " and " non-hygroscopic property " and relational language refer to that for example 90% relative humidity condition lower time absorbed less than the water of 5% weight (with respect to they self weight) and/or at crystallized form under the high humidity and do not change and/or do not absorb water material of (internal water) in crystalline body under high relative humidity condition when being exposed to high relative humidity.
For the preparation of the preferred salt of liquid (for example water-based) pharmaceutical composition be in given liquid vehicle (for example water), have greater than 15mg/ml liquid vehicle (for example water), more generally greater than 20mg/ml, be preferably greater than 25mg/ml, more preferably greater than the acid salt (as herein defined mesylate and acetate and its mixture) of the solubleness of 30mg/ml.
On the other hand; the invention provides a kind of pharmaceutical composition; its comprise contain concentration greater than 15mg/ml, usually greater than 20mg/ml, be preferably greater than 25mg/ml, more preferably greater than the 4-(2 of 30mg/ml; 6-two chloro-benzoyl-amidos)-aqueous solution of the acid salt of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides (as herein defined mesylate and acetate and its mixture, preferred mesylate).
In a preferred embodiment; pharmaceutical composition comprise contain concentration greater than 15mg/ml, usually greater than 20mg/ml, be preferably greater than 25mg/ml, more preferably greater than the 4-(2 of 30mg/ml; 6-two chloro-benzoyl-amidos)-and the aqueous solution of the acid salt of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, described acid salt is selected from acetate or mesylate or its mixture.
On the other hand; the invention provides 4-(2; 6-two chloro-benzoyl-amidos)-and the aqueous solution of the acid salt (as herein defined mesylate and acetate and its mixture) of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, wherein aqueous solution has 2-12, for example 2-9, the pH of 4-7 more especially.
In aqueous solution defined above, acid salt can be any salt as herein described, in a preferred embodiment, is mesylate or acetate, particularly mesylate defined herein.
The present invention also provides the aqueous solution of the 4-(2,6-, two chloro-benzoyl-amidos) of protonated form-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides and one or more counter ion and optional one or more other counter ion.In one embodiment, one of counter ion are selected from methanesulfonate and acetate moiety.In another embodiment, one of counter ion are from preparation buffer reagent such as acetate defined herein.In another embodiment, can there be one or more other counter ion such as chlorion (for example from salt solution).
Therefore; the invention provides counter ion and optional one or more other counter ion such as the aqueous solution of chlorion that the 4-(2,6-, two chloro-benzoyl-amidos) of protonated form-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides and one or more are selected from methanesulfonate and acetate moiety.
Exist therein in the situation of more than one counter ion; the 4-(2 of protonated form; 6-two chloro-benzoyl-amidos)-aqueous solution of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides may contain for example mixture of methanesulfonate and acetate moiety counter ion of counter ion mixture, and randomly contain one or more other counter ion such as chlorions.
Therefore; the invention provides the 4-(2,6-, two chloro-benzoyl-amidos) of protonated form-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides and one or more and be selected from the counter ion of methanesulfonate and acetate moiety and the aqueous solution of optional one or more other counter ion such as chlorion and composition thereof.
Especially can be by in the solution that mesylate is dissolved in acetate ion (for example acetate buffer) or by forming aqueous solution in the solution that acetate is dissolved in the methanesulfonate ion.Methanesulfonate and acetate ion in solution can with 10: 1 or less, for example 10: 1 to 1: 10, be more preferably less than 8: 1 or less than 7: 1 or less than 6: 1 or less than 5: 1 or less than 4: 1 or less than 3: 1 or less than 2: 1 or less than 1: 1,1: 1 to 1: 10 methanesulfonate more especially: the acetate moiety ratio exists.In one embodiment, methanesulfonate and acetate ion in solution with 1: 1 to 1: 10, for example 1: 1 to 1: 8 or 1: 1 to 1: 7 or 1: 1 to 1: 6 or 1: 1 to 1: 5, about 1: 4.8 methanesulfonate for example: the acetate moiety ratio exists.
The aqueous solution of salt can be buffering or non-cushioned, but cushions in one embodiment.
In the situation of the acid salt that forms with methylsulfonic acid, preferred buffer reagent is the buffer reagent that is formed by acetic acid and sodium acetate, for example about 4.6 the described buffer reagent of pH value of solution.Under this pH and in acetate buffer, mesylate has the approximately solubleness of 35mg/ml.
Salt of the present invention is pharmacologically acceptable salt normally, and the example of pharmacologically acceptable salt is people such as Berge, and 1977, " Pharmaceutically Acceptable Salts, " J.Pharm.Sci., the 66th volume has discussion in the 1-19 page or leaf.Yet, also can prepare with the form of intermediate pharmaceutically unacceptable salt, then can convert it into pharmacologically acceptable salt.Therefore, the pharmaceutically unacceptable salt form of this class also consists of a part of the present invention.
Compound 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides also can form the N-oxide compound.Can form the N-oxide compound by processing corresponding amine with oxygenant such as hydrogen peroxide or peracid (for example peroxycarboxylic acid), referring to for example Advanced Organic Chemistry, JerryMarch, 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 meta-chlorine peroxybenzoic acid (MCPBA) reaction, for example in inert solvent such as methylene dichloride, react.
Derive the compound 4-(2 of acid salt of the present invention; 6-two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides can exist with multiple different tautomeric form, in this application mentioning of this compound comprised all these class forms.
More specifically, in acid salt of the present invention, the pyrazole ring of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides can exist with following two tautomeric form A and B.For easy, the structural formula among the application has only shown form A, but these structural formulas are contained two tautomeric forms.
In addition; in the situation of acid salt of the present invention; to 4-(2; 6-two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides and mentioning of its salt also comprise having the variant that one or more isotropic substances replace, and to all isotropic substances that in its scope, comprise this element 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.Diagnosis contains the radioisotopic compound of this class in the situation that may be useful.
To mentioning of the acid salt (such as mesylate) of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides also contain its any polymorphic forms, solvate (such as hydrate), mixture (such as with such as the inclusion complex of the compounds such as cyclodextrin or inclusion compound or with the mixture of metal).
The crystalline structure of the acid salt of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides
As mentioned above, the acid salt of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides can be unbodied or be crystalline basically.In a specific embodiment, salt is crystalline basically, and term " being crystalline basically " has above defined implication.Particularly mesylate and the acetate of 4-(2,6-dichloro-benzoyl base-amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides are crystalline basically.
Crystal as herein described and their crystalline structure form another aspect of the present invention.
Crystal and its crystalline structure can use comprise Single Crystal X-ray crystallography, X-ray powder diffraction (XRPD), dsc (DSC) and infrared spectroscopy for example many technology of Fourier transform infrared spectroscopy (FTIR) characterize.The behavior of the crystal under the different humidity condition can be analyzed by weighting method steam absorption research, also can analyze by XRPD.
The mensuration of the crystalline structure of compound can be undertaken by the X-ray crystallography of carrying out according to conventional methods, as herein with Fundamentals of Crystallography, C.Giacovazzo, H.L.Monaco, D.Viterbo, F.Scordari, G.Gilli, G.Zanotti and M.Catti, (International Union of Crystallography/Oxford University Press, 1992ISBN 0-19-855578-4 (p/b), 0-19-85579-2 (h/b)) those ordinary methods described in.This technology comprises analysis and the deciphering of Single Crystal X-ray diffraction.
The crystalline structure of the mesylate of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides has carried out measuring an embodiment who vide infra 2 by X-ray crystallography.
Table 2 has provided 4-(2,6-dichloro-benzoyl base is amino)-coordinate data of crystal in crystallography message file (CIF) form of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mesylate be (referring to Hall, Allen and Brown, Acta Cryst. (1991) .A47,655-685; Http:// www.iucr.ac.uk/iucr-top/cif/home.html).Others skilled in the art can use or preferably supply the file layout of alternative such as PDB file layout (for example with EBI macromolecular structure database (Hinxton, UK) consistent file layout).Yet, it is evident that with different file layouts provide or process in this table coordinate within the scope of the invention.The crystalline structure of mesylate is set forth in Fig. 1 and 2.
Found by X-ray crystallography research that mesylate has and belonged to rhombic system spacer (orthorhombic space group) such as the crystalline structure of Pbca (#61), and under 93K, has lattice parameter a=8.90 (10), b=12.44 (10), c=38.49 (4)
Figure 200680009244X_2
, α=β=γ=90 °.
Therefore, in another embodiment, the invention provides the mesylate of 4-(2,6-dichloro-benzoyl base amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, its be crystalline and:
(a) has crystalline structure given among Fig. 1 and 2; And/or
(b) has the defined crystalline structure of coordinate among this paper embodiment 2; And/or
(c) under 93K, has lattice parameter a=8.90 (10), b=12.44 (10), c=38.49 (4)
Figure 200680009244X_3
, α=β=γ=90 °; And/or
(d) has the crystalline structure that belongs to rhombic system spacer such as Pbca (#61).
Perhaps, the crystalline structure of compound can be analyzed by solid-state X-ray powder diffraction (XRPD) technology.XRPD can be according to conventional methods as herein (referring to embodiment 6) and introduce Ron Jenkins and Robert L.Snyder (John Wiley ﹠amp at X-ray powder diffraction; Sons, New York, 1996) described in those methods carry out.In the XRPD diffractogram, exist defined peak (with respect to random ground unrest) to represent that compound has crystallinity to a certain degree.
The X-ray powder diffraction figure of compound characterizes with diffraction angle (2 θ) and spacing (d) parameter of X-ray diffraction spectrum.These parameters are because of Bragg equation n λ=2d Sin θ (n=1 wherein; λ=employed negative electrode wavelength; The d=spacing; With θ=diffraction angle) and relevant.At this, because data characteristics is so that spacing, diffraction angle and overall pattern are identified very important for the crystal in the X-ray powder diffraction.Because relative intensity can change according to crystal growth direction, granular size and measuring condition, so can't the strict interpretation relative intensity.In addition, diffraction angle means to meet those of 2 θ ± 0.2 ° scope usually.The peak means main peak, comprises the peak of the middle number that is not more than the diffraction angle except above-mentioned diffraction angle.
Acetic acid and the mesylate of 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides all characterize by XRPD.
4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mesylate has X-ray powder diffraction figure substantially as shown in Figure 3.
4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base amidoacetic acid salt has X-ray powder diffraction figure substantially as shown in Figure 4.
In each case, x-ray diffractogram of powder represents with diffraction angle (2 θ), spacing (d) and relative intensity.
Therefore; in another embodiment; the invention provides is crystalline 4-(2 basically; 6-dichloro-benzoyl base is amino)-mesylate of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, the X-ray powder diffraction figure that it has is characterised in that main peak and the spacing (d) that exists diffraction angle given in the Table A (2 θ) to locate.
Table A
2Θ/° d/
Figure 200680009244X_4
16.60 5.34
18.30 4.85
18.45 4.81
19.45 4.56
22.90 3.88
X-ray powder diffraction figure preferred feature also is peak and the spacing (d) that other diffraction angle given among the existence table B (2 θ) is located.
Table B
2Θ/° d/
12.80 6.91
21.40 4.15
22.00 4.04
23.50 3.78
25.00 3.56
X-ray powder diffraction figure can also be characterised in that main peak that diffraction angle given among the existence table C (2 θ) is located and spacing (d) and intensity preferably.
Table C
2Θ/° d/
Figure 200680009244X_6
 I
4.55 19.41 12
10.80 8.19 9
12.25 7.22 15
12.80 6.91 56
13.40 6.60 12
13.55 6.53 26
[0150]
14.00 6.32 7
14.75 6.00 8
15.50 5.71 25
16.60 5.34 100
17.30 5.12 15
17.75 4.99 16
18.30 4.85 90
18.45 4.81 65
19.45 4.56 65
20.80 4.27 18
21.40 4.15 40
22.00 4.04 42
22.90 3.88 71
23.50 3.78 45
23.90 3.72 27
24.40 3.65 32
25.00 3.56 61
26.00 3.43 18
26.50 3.36 20
27.00 3.30 30
28.00 3.18 14
28.40 3.14 14
28.70 3.11 17
The present invention further provides basic mesylate for crystalline 4-(2,6-dichloro-benzoyl base-amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, it shows the peak at the diffraction angle place identical with the X-ray powder diffraction figure shown in Fig. 3.Preferred these peaks have the relative intensity identical with peak among Fig. 3.
What in a preferred embodiment, the invention provides the X-ray powder diffraction figure that has basically as shown in Figure 3 is the mesylate of crystalline 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides basically.
It is the acetate of crystalline 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides basically that the present invention also provides, and it shows the peak at the diffraction angle place identical with the X-ray powder diffraction figure shown in Fig. 4.Preferred these peaks have the relative intensity identical with peak among Fig. 4.
What in a preferred embodiment, the invention provides the X-ray powder diffraction figure that has basically as shown in Figure 4 is the acetate of crystalline 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides basically.
Crystallinity acid salt of the present invention also can characterize by dsc (DSC).
By DSC mesylate is analyzed, because the decomposition of compound demonstrates the peak at 379.8 ℃.
Therefore, on the other hand, the invention provides the mesylate of 4-(2,6-dichloro-benzoyl base amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, its for anhydrous and when carrying out DSC 379-380 ℃ for example 379.8 ℃ demonstrate endotherm(ic)peak.
By DSC acetate is analyzed, because being lost in 231.50 ℃ and demonstrating the peak of acetic acid, and because the decomposition of compound demonstrates another peak at 292.88 ℃.It is anhydrous not having these peak explanation acetates under lower temperature.
Therefore; on the other hand; the invention provides 4-(2; 6-dichloro-benzoyl base is amino)-acetate of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, it is for anhydrous and demonstrate endotherm(ic)peak at 231-232 ℃ (for example 231.50 ℃) and 292-293 ℃ (for example 292.88 ℃) when carrying out DSC.
Acid salt at 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides is in the crystalline situation basically, and a kind of monocrystalline form can be preponderated, but other crystalline form can on a small quantity and preferably exist with negligible quantity.
In a preferred embodiment; the invention provides is crystalline 4-(2 basically; 6-dichloro-benzoyl base is amino)-acid salt (for example mesylate defined herein or acetate) of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, any other crystalline form that it contains the monocrystalline of acid salt and is no more than the acid salt of 5 % by weight.
Preferably, monocrystalline particularly contains and is less than or equal to approximately other crystalline form of 1 % by weight with less than 4% or less than 3% or less than other crystalline form of 2%.More preferably, the monocrystalline form is with less than 0.9 % by weight or less than 0.8 % by weight or less than 0.7 % by weight or less than 0.6 % by weight or less than 0.5 % by weight or less than 0.4 % by weight or less than 0.3 % by weight or less than 0.2 % by weight or less than 0.1 % by weight or less than 0.05 % by weight or less than other crystalline form of 0.01 % by weight, for example other crystalline form of 0 % by weight.
Basically be that crystalline acid salt does not preferably conform to residual organic solvents or other solvent such as the water that is useful on recrystallization for example or this salt of purifying basically.
Therefore; in one embodiment; the crystal of the acid salt (for example methane-sulfonate or acetate) of 4-(2,6-dichloro-benzoyl base amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides be contain residual solvent (for example water or organic solvent) less than 10 % by weight, for example less than 5% residual solvent, for example less than 4% or less than 3% or less than 2% or less than 1% or less than the crystal of 0.5% solvent.
In one embodiment, crystalline acid salt (for example methane-sulfonate or acetate) is anhydrous, and term " anhydrous " has above defined implication.
Mesylate can exist with stable anhydrous crystal forms, although described crystalline form absorbs some surface waters under high relative humidity condition, the change of crystalline structure does not occur under this class condition.
The behavior of acid salt of the present invention under high humidity can be analyzed by weight steam absorption (GVS) method of standard, for example as described in Example 7.
Acid salt of the present invention also can by infrared spectra for example FTIR characterize.
The infrared spectra of mesylate (KBr pressed disc method) is 3233,3002,2829,1679,1632,1560,1430,1198,1037,909 and 784cm -1Characteristic peak is contained at the place.
Therefore; in another embodiment; the invention provides (preferably being crystalline basically) 4-(2; 6-dichloro-benzoyl base is amino)-mesylate of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, it is presented at 3233,3002,2829,1679,1632,1560,1430,1198,1037,909 and 784cm when using the KBr pressed disc method to analyze -1The infrared spectra of characteristic peak is contained at the place.
It is evident that from aforementioned paragraphs, acid salt of the present invention can be take multiple different the physical-chemical parameters as feature.Therefore; in a preferred embodiment; the invention provides 4-(2; 6-dichloro-benzoyl base is amino)-mesylate of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides; it is crystalline and is characterised in that any one or a plurality of (arbitrary combination) or all in the following parameters, i.e. this salt:
(a) has crystalline structure given among Fig. 1 and 2; And/or
(b) has the defined crystalline structure of coordinate among this paper embodiment 2; And/or
(c) under 93K, has lattice parameter a=8.90 (10), b=12.44 (10), c=38.49 (4)
Figure 200680009244X_7
, α=β=γ=90 °; And/or
(d) has the crystalline structure that belongs to rhombic system spacer such as Pbca (#61); And/or
(e) have and be characterised in that and have given diffraction angle (2 θ) is located among Table A and the optional table B main peak and the X-ray powder diffraction figure of spacing (d); For example wherein X-ray powder diffraction figure is characterised in that main peak, spacing (d) and the intensity that exists diffraction angle given among this paper table C (2 θ) to locate; And/or
(f) the diffraction angle place identical with the X-ray powder diffraction figure shown in Fig. 3 show the peak and randomly wherein said peak have with Fig. 3 in the identical relative intensity in peak; And/or
(g) has basically as shown in Figure 3 X-ray powder diffraction figure; And/or
(h) be anhydrous and when carrying out DSC 379-380 ℃ for example 379.8 ℃ show endotherm(ic)peaks; And/or
(i) when using the KBr pressed disc method to analyze, be presented at 3233,3002,2829,1679,1632,1560,1430,1198,1037,909 and 784cm -1The infrared spectra of characteristic peak is contained at the place.
The method of preparation 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides
In the embodiment 237 of we previous application PCT/GB2004/003179 (WO 2005/012256); disclosing 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides can prepare by the order that comprises the following steps:
(i) make 4-(2,6-two chloro-benzoyl-amidos)-1H-pyrazoles-3-formic acid and 4-amino-1-tert-butoxycarbonyl-piperidines be in the situation that exist 1-ethyl-3-(3 '-dimethylaminopropyl)-carbodiimide (EDC) and I-hydroxybenzotriazole (HOBt) to react in dimethyl formamide (DMF), the 4-(2,6-, two chloro-benzoyl-amidos) of generation N-Boc protection form-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides; With
(ii) by removing the Boc protecting group with the salt acid treatment.
Have now found that to replace using EDC and HOBt to promote the formation of amido linkage, can make the acyl chlorides of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-formic acid and the wherein protected 4-amino piperidine reaction of piperidines nitrogen.
Therefore, on the other hand, the invention provides the method for a kind of 4-of preparation (2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides or its salt, the method may further comprise the steps:
Make the compound of formula (XI):
With PG wherein be the compound of the formula (XII) of amine-protecting group:
Figure S200680009244XD00272
In the situation that exist glitch-free alkali such as triethylamine to react in the organic solvent, the compound of production (XIII):
Figure S200680009244XD00273
Remove afterwards protecting group PG, produce 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides or its salt; Randomly this salt of recrystallization is to produce crystalline form, for example crystalline form defined herein.
Amine-protecting group PG can be any protecting group that becomes known for protection amine groups under the condition used in aforesaid method.The example of protecting group and functional group is protected can be at Protective Groups in Organic Synthesis (T.Green and P.Wuts with de-protected method; The 3rd edition; JohnWiley and Sons, 1999) find in.Therefore, for example, the nitrogen of piperidine ring can protected one-tenth acid amides (NRCO-R) or carbamate (NCO-OR), for example protected one-tenth: methyl nitrosourea (NCO-CH 3); Benzyloxy acid amides (NCO-OCH 2C 6H 5,-NH-Cbz); Protected one-tenth tert.-butoxy acid amides (NCO-OC (CH 3) 3, N-Boc); 2-xenyl-2-propoxy-acid amides (NCO-OC (CH 3) 2C 6H 4C 6H 5, N-Bpoc); protected one-tenth 9-fluorenyl methoxy acid amides (N-Fmoc); protected one-tenth 6-nitro black false hellebore oxygen base acid amides (N-Nvoc); protected one-tenth 2-trimethyl silane base oxethyl acid amides (N-Teoc); protected one-tenth 2,2,2-three chloroethoxy acid amides (N-Troc); protected one-tenth allyloxy acid amides (N-Alloc), or protected one-tenth 2-(phenyl sulfonyl) oxyethyl group acid amides is (N-Psec).Other protecting group that is used for amine comprises tosyl group (tosyl group) and methane sulfonyl (methylsulfonyl) and benzyl such as p-methoxy-benzyl (PMB).Preferred amine protecting group is carbamate (NCO-OR), for example, and benzyloxy acid amides (NCO-OCH 2C 6H 5,-NH-Cbz), or tert.-butoxy acid amides (NCO-OC (CH 3) 3, N-Boc); Allyloxy acid amides (N-Alloc) or p-methoxy-benzyl (PMB).A particularly preferred protecting group PG is the tert-butoxycarbonyl that can be removed under acidic conditions.The example of protecting group and functional group is protected can be at Protective Groups in Organic Synthesis (T.Green and P.Wuts with de-protected method; The 3rd edition; JohnWiley and Sons, 1999) find in.
Hydroxyl protection precedent such as ether (OR) or ester (OC (=O) R), for example, can be protected into tertbutyl ether; Benzyl, diphenyl-methyl (diphenyl methyl) or trityl (trityl group) ether; TMS or tertiary butyl dimethylsilyl ether; Or ethanoyl ester (OC (=O) CH 3,-OAc).The aldehydes or ketones group can be protected respectively precedent such as acetal (R-CH (OR) 2) or ketal (R 2C (OR) 2), wherein by with for example primary alconol react with carbonyl (＞C=O) change into diether (＞C (OR) 2).By the aldehydes or ketones group of easily to regenerate in the situation that a large amount of excessive water of existence acid usefulness are hydrolyzed.Amine groups can be protected precedent such as acid amides (NRCO-R) or urethane (NRCO-OR), for example protect into: methyl nitrosourea (NHCO-CH 3); Benzyloxy acid amides (NHCO-OCH 2C 6H 5,-NH-Cbz); Tert.-butoxy acid amides (NHCO-OC (CH 3) 3,-NH-Boc); 2-xenyl-2-propoxy-acid amides (NHCO-OC (CH 3) 2C 6H 4C 6H 5-NH-Bpoc), (NH-Fmoc), (NH-Nvoc), 2-TMS ethyl oxygen base acid amides (NH-Teoc), 2 for 6-nitro veratryl oxygen base acid amides for 9-fluorenyl methoxy acid amides; 2,2-, three chloroethyl oxygen base acid amides (NH-Troc), allyl group oxygen base acid amides (NH-Alloc) or 2 (phenyl sulfonyl) ethyl oxygen base acid amides (NH-Psec).Other protecting group that is used for amine such as cyclic amine and heterocycle N-H group comprises tosyl group (tolylsulfonyl-base) and methylsulfonyl (methane sulfonyl) and benzyl such as p-methoxy-benzyl (PMB).Hydroxy-acid group can be protected into ester, for example protect into: C 1-7Alkyl ester (for example, methyl ester; Tertiary 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, benzyl ester; The nitrobenzyl ester); Or acid amides, for example methyl nitrosourea.Thiol group can be protected precedent such as thioether (SR), for example to protect into: the benzyl thioether; Acetylamino methyl ether (S-CH 2NHC (=O) CH 3).
Be can be in the situation of the protecting group that is removed under the acidic conditions at protecting group PG, the 4-(2,6-, two chloro-benzoyl-amidos) of specific salts form-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides can be provided selecting the acid of removing protecting group PG to select.Therefore, for example, when protecting group is the Boc group, can comes cracking Boc protecting group and produce the hydrochloride of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides with hydrochloric acid.Perhaps, and more preferably, can come cracking Boc group and produce the mesylate of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides with methylsulfonic acid.
The present invention also provides a kind of compound and the compound of formula (XII) method of reacting the intermediate of preparation formula (XIII) under condition defined herein by making formula (XI).
The present invention also provides the new chemical intermediate of a kind of formula (XI) itself.
On the other hand, the invention provides the method for a kind of 4-of preparation (2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides or its salt, the method comprises:
(i) in aprotic organic solvent, process the compound of formula (XIV) with thionyl chloride:
Figure S200680009244XD00291
This reaction is randomly carried out under heating;
(ii) in the situation that exist glitch-free alkali such as triethylamine to make the compound reaction of the product of step (i) and formula (XII), this reaction is randomly carried out the compound of production (XIII) under heating; With
(iii) from the compound of formula (XIII), remove protecting group PG, produce 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides or its salt; Randomly
(iv) this salt of recrystallization is to produce crystalline form, for example crystalline form as defined herein.
In step (i), with the reaction of thionyl chloride can be in heating, for example be heated under 80-100 ℃ the temperature and carry out.The solvent that carries out therein step (i) is aprotic organic solvent, and it can be for example aromatic hydrocarbons solvent such as toluene.Reaction in step (i) finishes, for example judge that by the disappearance of starting raw material (XIV) this reaction is finished after, can remove organic solvent, for example remove organic solvent by vapourisation under reduced pressure, produce resistates, can it is further dry, for example by azeotropic drying, produce resistates.Then can make the compound reaction of the formula (XII) in this resistates and the step (ii).
In step (ii), used glitch-free alkali.Term " glitch-free alkali " means can not form with acid (XIII) or acyl chlorides (XI) alkali such as the triethylamine of acid amides in this article.
Step (ii) is carried out under mildly heating usually, for example is heated at the most approximately 55 ℃, 50 ℃ temperature at the most more generally, for example is heated to 45 ℃-50 ℃ temperature.
In step (ii), this reaction can be carried out in polar aprotic solvent such as tetrahydrofuran (THF).
In step (iii); protecting group preferably can be by protecting group such as the Boc group that is removed with acid treatment; acid is selected to produce the salt of required 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, for example mesylate.
In step (iii) afterwards, can carry out recrystallization (for example using the 2-propyl alcohol to carry out recrystallization as solvent) to increase purity and to produce crystalline form to product.
When protecting group PG is tert-butoxycarbonyl, the step of the method (i), (ii) and do not comprise that (iii), the overall yield of any re-crystallization step surpasses 85%.In addition, the method is favourable, because it utilizes relatively simple and cheap reagent and solvent, and only uses simple recrystallization and solvent wash technology and does not need to carry out chromatography and just produce purity greater than 99% product.
Recrystallization 4-(2; 6-two chloro-benzoyl-amidos)-method of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides and salt thereof can be undertaken by method known by the technical staff-referring to for example (P.Heinrich Stahl (editor); Camille G.Wermuth (editor); ISBN:3-90639-026-8; Handbook ofPharmaceutical Salts:Propertied; Selection and Use, the 8th chapter, the Wiley-VCH of publisher).The product that obtains from organic reaction is pure when directly isolating from reaction mixture very less.If compound (or its salt) is solid, then it can recrystallization be purified and/or crystallization by carrying out with appropriate solvent.Good recrystallization solvent should dissolve an amount of material to be purified at elevated temperatures, but only dissolves a small amount of this material at a lower temperature.It is easily dissolved impurity or not dissolved impurity at low temperatures.At last, this solvent should easily be removed from the product of purifying.This often means that it has relatively low boiling point and those skilled in the art and should know recrystallization solvent for predetermined substance, if perhaps there is not available information, tests several solvents.In order to obtain the good yield of purified material, use the hot solvent of minimum to dissolve all impuritys.In practice, use than required solvent and many solvent of 3-5%, so solution not saturated.If pure compound does not contain the impurity that is insoluble to this solvent, then it can be by removing by filter and make subsequently solution crystallization.In addition, if impure compound contains the natural coloring matter that has of the non-compound of trace, then its can by in hot solution, add a small amount of decolorizing charcoal, with its filtration with its crystallization is removed.Usually spontaneous generation crystallization when cooling solution.If not so, can be by solution being cooled off below room temperature or bringing out crystallization by the monocrystalline (crystal seed) that adds pure substance.Also can be by using anti--solvent (anti-solvent) to carry out recrystallization and/or optimizing productive rate.In this case, compound is dissolved in the appropriate solvent at elevated temperatures, filters, then add required compound and have therein the other solvent of low solubility with help crystallisation.Then usually use vacuum filter to isolate crystallization, washing, then dry, for example in loft drier or by dehydrating.
In some cases; the 4-(2 of the Boc-protection of trace; 6-two chloro-benzoyl-amidos)-and 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides may even remain into after the recrystallization, and when acid salt of the present invention is dissolved in when for example being dissolved in the damping fluid in the water, it can form throw out.Therefore can with the aqueous solution of acid salt by millipore filter for example the strainer of 0.5 micron strainer or 0.4 micron or 0.3 micron strainer or more preferably 0.2 micron strainer filter, to remove any this class throw out.
As the alternative of filtering (or except filtering), can be with the aqueous solution of salt in the situation that exist acid to heat, described acid normally with the sour identical acid (for example in the situation that mesylate is methylsulfonic acid) of formation salt.Further acid treatment causes the 4-(2,6-, two chloro-benzoyl-amidos) of remaining Boc-protection-1H-pyrazoles-3-carboxylic acid piperidin-4-base amide hydrolysis and changes into required salt.
Given solvent-aqueous extraction or chromatography also can be used for removing or prevent forming the precipitation of the compound of remaining Boc-protection in the embodiment with literary composition.
Biologic activity
Compound 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides and its salt are the inhibitor of cell cycle protein dependent kinase.For example, they are to be selected from CDK1, CDK2, CDK3, CDK4, CDK5, CDK6 and CDK9, particularly CDK1, CDK2, CDK3, CDK4, CDK5 and CDK9, the inhibitor of the cell cycle protein dependent kinase of CDK1, CDK2, CDK4 and CDK9 more especially.They also are the inhibitor of CDK8 and CDK11.
4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides and its salt also have the activity of anti-GSK-3 (GSK-3).
Because their activity in adjusting or inhibition CDK and glycogen synthase kinase; estimate that 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides and its salt can be used for being provided in the cell of abnormal division the retardance cell cycle or recovers the means of the control of cell cycle.Therefore, expectation will prove that they can be used for treatment or prevention proliferative disorder such as cancer.Estimate that also they will can be used for treatment such as the illnesss such as paralysis, corticobasal degeneration and Pick's disease (Pick ' s disease), for example autoimmune disorder and neurodegenerative disease on virus infection, II type or non insulin dependent diabetes, autoimmune disorder, head trauma, apoplexy, epilepsy, neurodegenerative disease such as alzheimer's disease, motor neurone disease, the carrying out property nuclear.
Estimate that a useful morbid state and illness subgroup are comprised of virus infection, autoimmune disorder and neurodegenerative disease salt of the present invention therein.
CDK at cell cycle regulation, apoptosis, transcribe, play a role in differentiation and the CNS function.Therefore, the CDK inhibitor can be used for treating the disorders such as cancers that wherein has propagation, apoptosis or dysdifferentiation.Particularly the RB+ve tumour can be responsive especially to the CDK inhibitor.The RB-ve tumour also can be responsive to the CDK inhibitor.
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, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell lymphoma (hairy cell lymphoma) or Burkitt lymphoma; The hematopoietic system cancer of marrow pedigree (hematopoietic tumour of myeloid lineage), 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, CDK6 and CDK9, 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 CDK9.
Can utilize the Growth of Cells assay method that provides among the embodiment hereinafter or title to determine the whether cancer of the inhibition sensitivity of cell cycle protein dependent kinase of a kind of specific cancer for the method that provides in the part of " diagnostic method ".
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 occurs in the HIV-infected individuals; 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 with ischemia injury, irregular pulse, atherosclerosis, toxin-bring out 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 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, comprise that the disease of abnormal cell growth or illness are cancer in one embodiment.
One group of cancer comprises human breast carcinoma (for example primary breast cancer, axillary lymph node-negative breast cancer patients, 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 (such as the Diffuse large B cell lymphoma).
A kind of concrete cancer is chronic lymphocytic leukemia.
Another kind of concrete cancer is lymphoma mantle cell.
Another kind of concrete cancer is the Diffuse large B cell lymphoma.
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 acid salt of the present invention as the activity of the inhibitor of cell cycle protein dependent kinase and GSK-3 with the assay method that provides among the embodiment hereinafter, the activity level that shows can be used IC 50Value defines.
Therefore, for example, estimate that acid salt of the present invention can be used for alleviating or reducing the incidence of cancer.
The present invention especially also provides:
Be used for prevention or treatment by the acid salt of the 4-(2,6-, two chloro-benzoyl-amidos) beyond the hydrochloride of the morbid state of cell cycle protein dependent kinase or GSK-3 mediation or illness-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
Acid salt for the 4-(2,6-, two chloro-benzoyl-amidos) beyond the hydrochloride that suppresses tumor growth Mammals-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
Acid salt for the 4-(2,6-, two chloro-benzoyl-amidos) beyond the hydrochloride of inhibition tumor cell growth (for example Mammals)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
Prevention or treatment are by cell cycle protein dependent kinase or the morbid state of GSK-3 mediation or the method for illness; the method comprises the acid salt to the 4-(2,6-, two chloro-benzoyl-amidos) beyond its individual administration of salt hydrochlorate of needs-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
The method that in Mammals (for example people), suppresses tumor growth; the method comprises the acid salt of Mammals (for example people) being used 4-(2,6-, two chloro-benzoyl-amidos) beyond the hydrochloride that suppresses the tumor growth significant quantity-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
The method of inhibition tumor cell (tumour cell that for example in Mammals such as people, exists) growth; the method comprises the acid salt contact of 4-(2,6-, two chloro-benzoyl-amidos) beyond the hydrochloride that makes tumour cell and inhibition tumor cell growth significant quantity-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
Alleviate or reduce the method by the incidence of the morbid state of cell cycle protein dependent kinase or GSK-3 mediation or illness; the method comprises the acid salt to the 4-(2,6-, two chloro-benzoyl-amidos) beyond its individual administration of salt hydrochlorate of needs-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
Treatment comprises abnormal cell growth or the disease that caused by abnormal cell growth or the method for illness in Mammals; the method comprises the acid salt to the 4-(2,6-, two chloro-benzoyl-amidos) beyond the hydrochloride of administration inhibition abnormal cell growth significant quantity-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
In Mammals, alleviate or reduce the method for the incidence of the disease that comprises abnormal cell growth or caused by abnormal cell growth or illness; the method comprises the acid salt to the 4-(2,6-, two chloro-benzoyl-amidos) beyond the hydrochloride of administration inhibition abnormal cell growth significant quantity-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
Treatment comprises abnormal cell growth or the disease that caused by abnormal cell growth or the method for illness in Mammals; the method comprises the acid salt to the 4-(2,6-, two chloro-benzoyl-amidos) beyond the hydrochloride of administration inhibition cdk kinases (such as ckd1 or ckd2) or glycogen synthase kinase-3 activity significant quantity-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
In Mammals, alleviate or reduce the method for the incidence of the disease that comprises abnormal cell growth or caused by abnormal cell growth or illness; the method comprises the acid salt to the 4-(2,6-, two chloro-benzoyl-amidos) beyond the hydrochloride of administration inhibition cdk kinases (such as ckd1 or ckd2) or glycogen synthase kinase-3 activity significant quantity-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
The method that suppresses cell cycle protein dependent kinase or GSK-3, the method comprise that the acid salt of the 4-(2,6-, two chloro-benzoyl-amidos) that makes beyond kinases and the hydrochloride-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides contacts.
Regulate the method for cell processes (for example cell fission); the method realizes by the activity that the acid salt with the 4-(2,6-, two chloro-benzoyl-amidos) beyond the hydrochloride-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides suppresses cell cycle protein dependent kinase or GSK-3.
Be used for prevention or treat the acid salt of 4-(2,6-, two chloro-benzoyl-amidos) beyond the hydrochloride of morbid state as herein described-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
The purposes of acid salt in the preparation medicine of the 4-(2,6-, two chloro-benzoyl-amidos) beyond the hydrochloride-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, wherein said medicine is used for one or more purposes defined herein.
Comprise 4-(2,6-, two chloro-benzoyl-amidos) beyond the hydrochloride-1H-pyrazoles-3-carboxylic acid piperidin-acid salt of 4-base acid amides and the pharmaceutical composition of pharmaceutically acceptable carrier.
For the pharmaceutical composition of using with the aqueous solution form; described pharmaceutical composition comprises the 4-(2 beyond the hydrochloride; 6-two chloro-benzoyl-amidos)-acid salt of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides; the solubleness in water that this salt has is greater than 15mg/ml; usually greater than 20mg/ml; be preferably greater than 25mg/ml, more preferably greater than 30mg/ml.
Be used in the acid salt of 4-(2,6-, two chloro-benzoyl-amidos) beyond the hydrochloride in the medicine-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
Acid salt for the 4-(2,6-, two chloro-benzoyl-amidos) beyond the hydrochloride of mentioned above and the described any purposes of this paper other parts and method-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
Diagnosis and treatment be by the morbid state of cell cycle protein dependent kinase mediation or the method for illness, and the method comprises that (i) screening patient is suffering from maybe disease or the illness that whether disease suffered from or illness may be had the treatment sensitivity that the compound of anti-cell cyclin-dependent kinase activity carries out to use to determine this patient; Therefore (ii) showing in patient's disease or the situation that illness is susceptibility, then giving the acid salt of 4-(2,6-, two chloro-benzoyl-amidos) beyond patient's administration of salt hydrochlorate-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
4-(2 beyond the hydrochloride; 6-two chloro-benzoyl-amidos)-and the purposes of acid salt in the preparation medicine of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, described medicine is used for screened and be determined and suffer from disease or illness or have the disease suffered from or the patient of the danger of illness
Middle treatment or preventing disease state or illness, it is responsive that described disease or illness have a treatment that the compound of anti-cell cyclin-dependent kinase activity carries out to use.
Acid salt in the above-mentioned aspect of the present invention can be for example any salt, particularly mesylate as herein described and acetate and composition thereof, most preferably mesylate.
The lymphadenomatous treatment of B-cell lymphoma, chronic lymphocytic leukemia and Diffuse large B cell
The present invention also provides the new purposes (i.e. purposes in treatment B-cell lymphoma, chronic lymphocytic leukemia and Diffuse large B cell lymphoma) of disclosed compound in we previous application PCT/GB2004/003179 (WO2005/012256), and the content of this application is incorporated herein by reference.
More specifically, the invention provides defined formula (I among the PCT/GB2004/003179 (WO2005/012256) 0) and the compound of its subgroup, embodiment and embodiment for the preparation of the purposes in treatment B-cell lymphoma, chronic lymphocytic leukemia or the lymphadenomatous medicine of Diffuse large B cell:
Figure S200680009244XD00371
R wherein 2, R 3, X and Y in the PCT/GB2004/003179 (WO 2005/012256) definition.
The method for the treatment of B-cell lymphoma, Diffuse large B cell lymphoma and chronic lymphocytic leukemia also is provided, and the method is to use defined formula (I among this paper and the PCT/GB2004/003179 (WO 2005/012256) by the patient to this class treatment of needs 0) compound realizes.
Concrete formula (I 0) compound be in WO 2005/012256 in the formula (VIb) on the formula (VIa) on the formula (Vb) on the formula (Va) on the formula (IVa) on the formula (IV) on the formula (II) on the formula (Ib) on the 17th page, the 66th page, the 72nd page, the 74th page, the 76th page, the 77th page, the 78th page and the 79th page defined those, the compound that on the 79th page of WO 2005/012256, exemplifies in listed compound and the embodiment part.
Preferred formula (I 0) compound is 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides and its salt (for example acid salt), solvate, tautomer or N-oxide compound.
In one embodiment, salt can be hydrochloride.Hydrochloride can prepare described in the embodiment 150 of our previous application PCT/GB2004/003179 or embodiment 237; the content that this application is relevant with 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides is incorporated herein by reference.The embodiment 237 of PCT/GB2004/003179 (WO 2005/012256) comprises in this application as embodiment 11.
Therefore, according to this aspect of the invention, provide:
Be used for the treatment of defined among the PCT/GB2004/003179 (WO 2005/012256) of B-cell lymphoma and R wherein 2, R 3, defined formula (I among X and Y such as the PCT/GB2004/003179 (WO2005/012256) 0) and compound and its additive salt (for example hydrochloride) of its subgroup, embodiment and embodiment.
Be used for the treatment of defined among the PCT/GB2004/003179 (WO2005/012256) of chronic lymphocytic leukemia and R wherein 2, R 3, defined formula (I among X and Y such as the PCT/GB2004/003179 0) and compound and its additive salt (for example hydrochloride) of its subgroup, embodiment and embodiment.
Be used for the treatment of defined among the lymphadenomatous PCT/GB2004/003179 of Diffuse large B cell (WO2005/012256) and R wherein 2, R 3, defined formula (I among X and Y such as the PCT/GB2004/003179 0) and compound and its additive salt (for example hydrochloride) of its subgroup, embodiment and embodiment.
On the other hand; the invention provides the 4-(2,6-, two chloro-benzoyl-amidos) that is used for the treatment of the B-cell lymphoma-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides or its salt (for example acid salt), solvate, tautomer or N-oxide compound.
The present invention also provides the 4-(2,6-, two chloro-benzoyl-amidos) that is used for the treatment of chronic lymphocytic leukemia-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides or its salt (for example acid salt), solvate, tautomer or N-oxide compound.
The present invention also provides and has been used for the treatment of the lymphadenomatous 4-of Diffuse large B cell (2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides or its salt (for example acid salt), solvate, tautomer or N-oxide compound.
In the treatment of B-cell lymphoma, Diffuse large B cell lymphoma and chronic lymphocytic leukemia; can use 4-(2; 6-two chloro-benzoyl-amidos)-and the free alkali of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, perhaps more preferably use acid salt.Acid salt can be disclosed hydrochloride in we previous application PCT/GB2004/003179 (WO 2005/012256), or it is one of salt disclosed herein, for example with the salt of methylsulfonic acid and acetic acid.
The method for the treatment of B-cell lymphoma, Diffuse large B cell lymphoma and chronic lymphocytic leukemia also is provided; the method is to use 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides or its acid salt is realized by the patient to the treatment of this class of needs.
Pharmaceutical preparation
Although compound defined herein (formula (I for example 0) compound or 4-(2; 6-two chloro-benzoyl-amidos)-and 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides or its acid salt such as mesylate) can use separately; but preferably its form with pharmaceutical composition (for example preparation) is given, and described composition comprises this compound or its salt 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.Said composition also can comprise other treatment or prophylactic substance, for example reduces 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) and granulocyte colony-stimulating factor (G-CSF).
Therefore; the present invention also provides the as defined above method of pharmaceutical composition and pharmaceutical compositions; it comprises that the salt (particularly mesylate) with 4-defined herein (2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mixes 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 excessive 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 be " acceptable ".
Pharmaceutical composition can be anyly be suitable in oral, parenteral, part, the nose, the form of eye, ear and rectum, intravaginal or transdermal administration.Use in the situation that composition is used for parenteral, they can be formulated for intravenously, intramuscular, intraperitoneal, subcutaneous administration or directly be delivered to target organ or tissue by injection, infusion or other delivery means.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 make the combinations of substances of preparation and expection recipient's blood etc.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 large, then can make ionogenic drug molecule dissolving reach desired concn by adjusting pH.For intravenously and intramuscular administration, acceptable scope is pH 2-12, but for subcutaneous administration, acceptable scope is pH 2.7-9.0.PH value of solution by medicine salt form, strong acid/alkali example hydrochloric acid or sodium hydroxide or controlled 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.The water-miscible organic solvent and the tensio-active agent that are used in the injection formulations include but not limited to propylene glycol, ethanol, Liquid Macrogol, poly(oxyethylene glycol) 400, glycerine, N,N-DIMETHYLACETAMIDE (DMA), METHYLPYRROLIDONE (NMP; Pharmasolve), methyl-sulphoxide (DMSO), Solutol HS15, Cremophor EL, Cremophor RH 60 and Polysorbate 80.This class preparation can (but not always) be diluted before injection usually.
Be used in propylene glycol, PEG 300, ethanol, CremophorEL, Cremophor RH 60 and Polysorbate 80 in the injection formulations of commercially available acquisition and be fully and can and can combination with one another use with miscible organic solvent and the tensio-active agent of water.The gained organic formulations dilutes at least 2 times usually before using by intravenously bolus injection 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 comprised 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 at once add before use for example water for injection of sterile liquid carrier.
Pharmaceutical preparation can be by lyophilize formula (I defined herein 0) or (I) compound or its salt prepare.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).In the situation that bottle, they by with the freeze-drying plug portion clog.Can be cooled to preparation freezing and carry out lyophilize under standard conditions, then sealing adds a cover to form the lyophily preparation of stable drying.Composition has low residual moisture content usually, for example based on the weight of lyophile, less than 5 % by weight for example less than the residual moisture content of 1 % by 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.The example of antioxidant 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, p-hydroxy-benzoic acid.If necessary, aforementioned buffer reagent and glucose and sodium-chlor can be used for tension adjustment.
Extender (bulking agent) generally is used for Freeze Drying Technique to help to process and/or provide 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 completely reconstruct.Extender also can be used for making solution etc.
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 such 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 approximately 1 to approximately 5, and for example approximately 1 to approximately 3, for example approximately 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 provides 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 preparations.
In a preferred embodiment of the present invention, pharmaceutical composition is to be suitable for intravenously to use for example form by using in injection or the intravenous injection.
The sterilized powder that the pharmaceutical composition of the present invention of injecting for parenteral also can comprise pharmaceutically useful sterile aqueous or non-aqueous solution, dispersion liquid, suspension or emulsion and at once be reconstructed into before use aseptic parenteral solution or dispersion liquid.The example of suitable water-based and non-aqueous carrier, thinner, solvent or medium comprises water, ethanol, polyvalent alcohol (such as glycerine, propylene glycol, polyoxyethylene glycol etc.), carboxymethyl cellulose and their suitable mixture, vegetables oil (such as sweet oil) and injection organic ester such as ethyl oleate.Can be for example by with coating substance such as Yelkin TTS, in the situation of disperseing by keeping required granular size and by keep suitable flowability with tensio-active agent.
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 guarantee by comprising various antibacteriums and anti-mycotic agent such as p-Hydroxybenzoate, butylene-chlorohydrin, phenol, Sorbic Acid etc.May need also to comprise that isotonic agent is such 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 that is comprised 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; Buffering or buffering not) dilute (Solubilizing excipients in oraland injectable 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), METHYLPYRROLIDONE (NMP, Pharmasolve), glycerine, Cremophor EL, Cremophor RH 60 and polysorbate.Concrete non-aqueous solution is comprised of 70-80% propylene glycol and 20-30% ethanol.A concrete non-aqueous solution is comprised of 70% propylene glycol and 30% ethanol.Another is 80% propylene glycol and 20% ethanol.Usually these solvents are used in combination and usually dilute at least 2 times 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 a preferred embodiment of the present invention, pharmaceutical composition is to be suitable for intravenously to use for example form by using in injection or the intravenous injection.Use for intravenously, can be with solution with the form administration of itself or can before using, inject in the infusion bag and (contain pharmaceutically useful vehicle, such 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.
Contain formula (I 0) or (I) pharmaceutical composition of compound or its acid salt can prepare according to known technology, for example referring to Remington ' s Pharmaceutical Sciences, Mack PublishingCompany, Easton, PA, USA.
Defined formula (I among the WO 2005/012256 0) and the compound of its subgroup can described in this article and described in the application, be prepared.
Therefore, tablet composition can contain active compound and inert diluent or carrier such as sugar or sugar alcohol, for example lactose, sucrose, sorbyl alcohol or the N.F,USP MANNITOL of unitary dose; And/or derivative thinner such as yellow soda ash, calcium phosphate, calcium carbonate or Mierocrystalline cellulose or derivatives thereof such as methylcellulose gum, ethyl cellulose, Vltra tears and starch such as the W-Gum of non-sugar.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 discuss in detail 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 animal gelatin or its Equivalent synthetic or plant derivation.
Solid dosage (such as tablet, capsule etc.) can be by dressing or dressing not, but usually has dressing, for example the dressing that discharges of protectiveness film coating (for example wax or paint film) or control.Dressing (Eudragit for example TMThe desired location that the type polymkeric substance) can be designed in gi tract discharges active ingredient.Therefore, can select in order 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 that control discharges, and for example is adapted at optionally discharging under different acidity or alkaline condition in the gi tract material of the delayed release of compound.Perhaps, the dressing that substrate material or retardance discharge can adopt the form of erodible polymkeric substance (for example maleic anhydride polymer), and it is dissoluted basically continuously during by gi tract when formulation.As another kind of alternative, active compound can be formulated in the delivery system of infiltration control compound release.Infiltration discharges can be according to well known to a person skilled in the art the method preparation with other delayed release or extended release preparation.
Pharmaceutical preparation can be with the form of " patient's bag " passs the patient, and it contains the whole course for the treatment of 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 common this specification sheets not.Proved that package insert can improve the patient to the compliance of 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.
The composition of using for parenteral 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 with the sterilized powder form of porphyrize temporarily.
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 plasticity-or the waxy substance of the shaping that contains active compound.
Can adopt the form of inhalation of dust composition or liquid or powder spray agent but be used for sucking the composition of using, 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 the Powdered thinner of inert solid such as lactose usually.
Formula (I 0) or (I) compound or its acid salt generally provide 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 1 nanogram to 2 gram, for example the activeconstituents of 1 nanogram to 2 milligram.In this scope, the inferior scope of concrete compound is that (more generally 10 milligrams to 1 gram for 0.1 milligram of activeconstituents to 2 grams, for example 50 milligrams to 500 milligrams), or 1 microgram to 20 milligram (for example 1 microgram to 10 milligram, for example 0.1 milligram to 2 milligrams activeconstituents).
Active compound will be applied to the amount that enough reaches required result for the treatment of the patient (for example human or animal patient) who needs it.
Methods for the treatment of
Estimate 4-defined herein (2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides or its acid salt, particularly mesylate, acetate and hydrochloride (more especially mesylate and acetate or its mixture) or formula (I 0) compound can be used for preventing or treat morbid state or illness by cyclin dependent kinase and GSK-3 mediation.The example of this class morbid state or illness as mentioned above.
Generally compound and its salt are applied to the individuality that needs this class to use, for example human or animal patient, preferably people.
Usually with treatment or effective and general nontoxic amount administered compound and its salt of prevention.Yet, in some situation situation of life-threatening disease (for example), the benefit of administered compound and its salt may be more important than the shortcoming of any toxic effect or side effect, in this case, can think the compound or its salt that need to use the amount relevant with toxicity to a certain degree.
But the long-term application compound or its salt with keep useful result for the treatment of or only short-term use.Perhaps can use them with pulse mode or continuous mode.
The common per daily dose of compound or its salt defined herein can be every kg body weight 100 piks to 100 milligram, more generally every kg body weight 5 nanograms to 25 milligram, more generally every kg body weight 10 nanograms to 15 milligram (10 nanograms to 10 milligram for example, more generally every kilogram of 1 microgram is to 20 milligrams every kilogram, every kilogram of 1 microgram to 10 milligram for example), but as needing, also can use higher or lower dosage.Described compound or its salt can be on every day basis or is used repeating the basis, for example uses once in per 2 or 3 or 4 or 5 or 6 or 7 or 10 or 14 or 21 or 28 days.
For 60 kilograms people; the example of dosage comprises the initial dose with 4.5-10.8mg/60kg/ days (equaling 75-180ug/kg/ days); use 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; for example compound 4-(2; 6-two chloro-benzoyl-amidos)-free alkali of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides; but; if necessary, can use higher or lower dosage.For any given body weight, mg/kg dosage in proportion converts.
An example of the dosage of mesylate is: initial dose is 5.6-13.5mg/60kg/ days (equaling g/kg/ days/people of 93-225 μ), effective dose subsequently is that 55-122mg/60kg/ days (equaling 0.9-2.0mg/kg/ days/people) or effective dose are 90-345mg/60kg/ days (equaling 1.5-5.8mg/kg/ days/people), but, if necessary, can use higher or lower dosage.At last, the amount of the compound of using and the type of employed composition should match with the character of disease or physiological conditions to be treated, and are decided by the doctor.
In a concrete drug dosage schedule table, the patient will be given the transfusion of one hour described compound or its salt every day, and administration reaches 10 days, particularly reach 5 days weekly, and with the required time 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 one hour described compound or its salt 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, then give 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.
Can be used for for example treating in the treatment drug dosage schedule table of chronic lymphocytic leukemia at another, the patient is given 2-6 hour (more generally 3-5 hour) infusion, the timed interval with 6-8 days in 6 weeks repeated for 3,4 or 5 weeks.In a preferred embodiment of this planning chart, the patient is given 4 hours infusions, and is weekly, 4 weeks of administration in 6 weeks.
At last, however 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 are decided by the doctor.
Described compound or its salt can be used as the single therapy agent and is 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 from 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 inhibitors (agent of tubulin target), monoclonal antibody and signal transduction inhibitor, concrete example has cis-platinum, endoxan, Zorubicin, irinotecan, fludarabine, 5FU, taxanes, ametycin and radiotherapy.
Described compound or its salt with can be given with different separately dosage schedules and via different approach from its other therapeutical agent of in combined therapy, using together.Therefore; for example; the salt form (for example mesylate and acetate and composition thereof) of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides can be used with the solution form by the parenteral approach, and another kind of therapeutical agent can be Orally administered.
At formula (I 0) or (I) compound or its acid salt and, two, three, four or more kinds of (preferred one or two kind, more preferably a kind of) other therapeutic combination situation about using 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 (interval 1,2,3,4 or more hours for example of the longer timed interval, if perhaps need the interval longer time) to use, the character of accurate dosage and therapeutical agent matches.
Formula (I 0) or (I) compound or its acid salt also can with non-chemotherapeutic treatment 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 be with formula (I 0) or (I) compound or its acid salt and, 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 separately be prepared and be provided 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
Using formula (I 0) or (I) before compound or its acid salt, the screening patient is suffering from maybe disease or the illness that whether disease suffered from or illness may be had the treatment sensitivity that the compound of anti-cell cyclin-dependent kinase activity carries out to use to determine this patient.
For example, can analyze the biological specimen of taking from the patient with determine this patient suffering from maybe may with the illness suffered from or disorders such as cancers whether take cause the CDK overactivity cause the gene unconventionality of the active path enhanced sensitivity of normal CDK or protein expression unusually as disease or the illness of feature.Cause the unusual example of this class of the activation of CDK2 signal or enhanced sensitivity to comprise rise (Harwell RM, 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 especially sensitivity.Term used herein " 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 lacked or existed the CDC4 variant to detect cyclin E rise or p21 or p27 mark characteristics to the patient.Term " diagnosis " comprises screening.We comprise genetic marker with " mark " word, comprise for example measuring the sudden change that DNA forms to identify CDC4.Term " mark " also comprises and embodies the mark that cyclin E raises characteristics, comprises the mRNA level of enzymic activity, enzyme level, enzyme state (for example whether phosphorylation) and aforementioned protein.Tumour with cyclin E rise or p21 or p27 disappearance may be responsive especially to the CDK inhibitor.Can before treatment, preferentially screen for 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.
The 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 for the existence of CDC4 variant.Screening method is usually directed to direct Sequencing, oligonucleotide microarray analysis or mutant specific antibody.
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 Reverse transcriptive 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 then to estimate with pcr amplification cDNA by the cDNA copy that produces mRNA.The method of pcr amplification, the selection of primer and amplification condition are well known by persons skilled in the art.Nucleic acid operation and PCR carry out according to standard method, such as for example Ausubel, and the people such as F.M., Current Protocols in MolecularBiology, 2004, John Wiley ﹠amp; Sons Inc., or Innis, the 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 and operate in the 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 of mrna expression is fluorescence in situ hybridization (FISH) (referring to Angerer, 1987 Meth.EnzymoL, 152:649).
Generally speaking, in situ hybridization comprises following key step: (1) fixes tissue to be analyzed; (2) sample being carried out prehybridization processes to increase the accessibility of target nucleic acid and reduces non-specific binding; (3) with the nucleic acid hybridization in nucleic acid mixture and biological structure or the tissue; (4) carry out post-hybridization washing removing unconjugated nucleic acid fragment in the hybridization, and (5) detect the nucleic acid fragment of 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 probe is answered sufficiently long, and for example, approximately 50,100 or 200 Nucleotide is to approximately 1000 or more Nucleotide, so that can carry out specific hybrid with target nucleic acid under stringent condition.Carry out the standard method of FISH at Ausubel, the people such as F.M., Current Protocols in Molecular Biology, 2004, John Wiley ﹠amp; Sons Ine and Fluorescence In Situ Hybridization:Technical Overview by John M.S.Bartlett in Molecular Diagnosis ofCancer, 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, immunohistochemistry that can be by tumor sample, use microtiter plate solid-phase immunoassay, Western blotting, two-dimentional SDS-polyacrylamide gel electrophoresis, ELISA, flow cytometry and this area for detection of the currently known methods analysis of the specific protein protein by mrna expression.Detection method comprises the use site-specific antibodie.It will be understood by a person skilled in the art that all these classes known for detection of the technology of the disappearance of the rise of cyclin E or p21 or p27 or CDC4 variant 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. for 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 clinical pathology entity of non-Hodgkin lymphoma, and what it is characterized in that having CD5 and CD20 coexpression is little of medium sized lymphopoiesis, the invasive and clinical disease course that can not cure and t (11 frequently; 14) (q13; Q32) transposition.Excessively expressing of the cyclin D1 mRNA that finds in lymphoma mantle cell (MCL) is a kind of important diagnostic flag.The people such as Yatabe (Blood.2000 April 1; 95 (7): 2253-61) propose to comprise cyclin D1-positive as one of standard of MCL, and proposal should be probed into take this new standard the innovative treatments of this incurable disease as the basis.The people such as Jones (J Mol Diagn.2004 May; 6 (2): 84-9) developed a kind of real-time quantitative reverse transcription PCR for cyclin D1 (CCND1) expression and analyzed with assisted diagnosis lymphoma mantle cell (MCL).The people such as Howe (ClinChem.2004 January; 50 (1): the quantitative RT-PCR that 80-7) 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 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 provides.
Antifungal application
On the other hand, the invention provides the acid salt except hydrochloride of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides as the purposes of anti-mycotic agent.
4-(2; 6-two chloro-benzoyl-amidos)-acid salt (except the hydrochloride) of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides can be used for animal medicine (for example being used for the treatment of Mammals such as the people) be used for the treatment of plant (for example in agricultural and the Horticulture) or as anti-mycotic agent, for example be used as sanitas and sterilizing agent.
In one embodiment, the invention provides for the acid salt except hydrochloride at the 4-(2,6-, two chloro-benzoyl-amidos) of Mammals such as people prevention or treatment fungi infestation-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
The acid salt except hydrochloride that 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides also is provided is for the preparation of the purposes in the medicine of prevention in Mammals such as people or treatment fungi infestation.
For example, acid salt of the present invention can be applied to and suffer from the human patients that local fungal infects or has the local fungal risk of infection, described local fungal infects and is caused by following biology: mycocandida (Candida), Trichophyton (Trichophyton), Microsporon (Microsporum) or Epidermophyton (Epidermophyton), perhaps in the situation that mucosal infections causes (for example white mouth and vaginal candidiasis) by Candida albicans (Candida albicans).Also can use acid salt of the present invention to treat or to prevent by for example Candida albicans, Cryptococcus neoformans (Cryptococcusneoformans), flavus (Aspergillus flavus), aspergillus fumigatus (Aspergillus fumigatus), ball spore Pseudomonas (Coccidiodies), Paracoccidioides (Paracoccidioides), the systemic fungal infection that Histoplasma (Histoplasma) or Blastomyces (Blastomyces) cause.
On the other hand; the invention provides the antifungal composition for agricultural (comprising Horticulture); it comprises the acid salt except hydrochloride and the upper acceptable diluent or carrier of agricultural of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
The present invention also provides treatment to have the method for animal (comprising Mammals such as people), plant or the seed of fungi infestation; it comprises the location of processing described animal, plant or seed or described plant or seed with the acid salt except hydrochloride of the 4-(2,6-, two chloro-benzoyl-amidos) of significant quantity-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
The present invention also provides the method for the treatment of plant or seed fungi infestation; it comprises with the fungicide composition of the acid salt except hydrochloride that contains 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides of antimycotic significant quantity processes plant or seed.
Can measure acid salt of the present invention to the specificity of non-human CDK enzyme with differential screening assay method.The salt that acts on specifically the CDK enzyme of eukaryotic pathogens can be used as antimycotic or antiparasitic.The kinase whose inhibitor C KSI of mycocandida CDK can be used for treating moniliosis.Anti-mycotic agent can be used for resisting the infection of institute's define styles above or usually betides weakness or immunosuppressed patient is suffered from the patient's of illnesss such as diabetes or AIDS opportunistic infection as suffering from leukemia and lymphadenomatous patient, the people who accepts immunosuppressant therapy and Yi, and is used for nonimmune inhibition patient.
The described assay method in this area can be used for screening for the suitability of at least a fungi related in the Antifungi disease, and described mycosis is moniliosis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, torulosis, chromoblastomycosis, coccidioidomycosis, conidium bacterium sick (conidiosporosis), histoplasmosis, mycetoma, rhinosporidiosis, nocardiosis, pseudactinomycosis, penicilliosis, monoliasis or sporotrichosis for example.By utilizing the CDK gene by yeast clone, differential screening assay method can be used for identifying the anti-mycotic agent that has therapeutic value in the aspergillosis treatment, described yeast is aspergillus fumigatus (Aspergillus fumigatus) for example, yellow aspergillus (Aspergillusflavus), black aspergillus (Aspergillus niger), structure nest aspergillus (Aspergillus nidulans) or terreus (Aspergillus terreus), perhaps in the situation that fungal infection is mucon-mycosis, the CDK assay method can be derived from yeast rhizopus arrhizus (Phizopus arrhizus) for example, rice root fungus (Rhizopusoryzae), absidia corymbifera bacterium (Absidia corymbifera), absidia rasmosa bacterium (Absidia ramosa) or mucor pusillus (Mucorpusillus).The source of other CDK enzyme comprises pathogenic agent Pneumocystis carinii (Pneumocystis carinii).
For example, the in-vitro evaluation of the anti-mycotic activity of acid salt of the present invention can be undertaken by measuring minimal inhibitory concentration (M.I.C.), and it is the test 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 separately the certain concentration test compound, then with each flat board at 37 ℃ of lower incubation suitable times.Then check the growth whether fungi is arranged on the flat board, the M.I.C. value that record is suitable.Perhaps, but carry out turbidity measurement in the liquid medium within, the scheme of summarizing the example of this assay method can be referring to the following examples.
The interior evaluating of acid salt can be under a series of dosage levels by to inoculated fungi for example in the mouse peritoneum of Candida albicans or aspergillus flavus strain or intravenous injection or Orally administered carrying out.The activity of salt can be assessed by the growth (by histology or by obtaining the fungi of self-infection) 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.
With regard to human antifungal application; the acid salt except hydrochloride of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides can use separately or with put into practice selected pharmaceutical carrier according to expection route of administration and standard pharmaceutical and mix and use.Therefore, for example, can adopt at the described salt of preparation oral, parenteral, intravenously, intramuscular or subcutaneous administration described in above-mentioned " pharmaceutical preparation " joint.
With regard to the oral and parenteral of human patients was used, the dosage level of described salt was 0.01 to 10mg/kg (a plurality of divided dose), and this especially depends on the effectiveness of salt when using by oral or parenteral approach.The tablet of salt or capsule can for example contain 5mg to 0.5g active compound, take the circumstances into consideration to use a slice (grain), two (grain) or multi-disc (grain) at every turn.In any case the doctor will determine to be best suited for the actual dose (significant quantity) of individual patient, it will be different because of age, body weight and the response of particular patient.
Perhaps, described salt can be used with the form of suppository or vaginal suppository, and perhaps they can be with the form topical application of lotion, solution, ointment, ointment or dusting.For example, they can be impregnated in the ointment that the water-based emulsion by polyoxyethylene glycol or whiteruss forms; Perhaps they can be impregnated in the ointment that is comprised of Chinese wax or paraffinum molle alba matrix and the stablizer that adds as required and sanitas, and concentration is 1 to 10%.
Except above-mentioned therepic use, the anti-mycotic agent of developing with this differential screening assay method also can be used as sanitas in the food for example, promote the fodder additives that domestic animal weight increases or for example be used for the disinfectant preparation in sterilization hospital equipment and room for the treatment of non-living matter.In a similar manner, side by side relatively the restraining effect of Mammals CDK and insect CDK such as fruit bat CDK5 gene people such as (, (1994) FEBS Lett 356:317-21) Hellmich can be selected the enzymeinhibition agent that can distinguish people/Mammals and insect from the compound of this paper.Therefore, the present invention comprises purposes and the preparation of salt of the present invention in sterilant clearly, for example is used for the processing of insect such as fruit bat.
In another embodiment, can select some tested salt with respect to Mammals enzymeinhibition specificity according to plant CDK.For example, plant CDK is arranged in the differential screening that contains one or more people's enzymes, to select to suppress those the highest compounds of selectivity of plant enzyme.Therefore, the present invention specifically comprises the preparation for the tested salt of agricultural application, such as the preparation of the forms such as defoliant.
For agricultural and gardening purpose, salt of the present invention can be mixed with the composition forms that is suitable for specific end use and expection purpose.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 provide with dispersion powder, granule or particle or with the form of the concentrated solution of front dilution.This based composition can contain those known and acceptable conventional carrier, thinner or assistant agents in agricultural and Horticulture, and they can prepare according to conventional methods.Said composition also can be mixed other activeconstituents, for example, has compound or the another kind of mycocide of weeding or insecticidal activity.Salt and composition can be used in many ways; for example they can be applied directly to plant leaf, stem, branch, seed or root; or may be used on 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 contain the activeconstituents of 0.01 to 1 % by weight.Use for the field, the possible utility ratio of activeconstituents is the 50-5000g/ hectare.
The present invention comprises that also 4-(2,6-, two chloro-the benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-acid salt except hydrochloride of 4-base acid amides is used for the purposes of soil, seedling rice field or the irrigation water of control foxy fungi and processing plant-growth.The present invention comprises that also the acid salt except hydrochloride of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides is used for preventing stock's cereal and other purposes without plant place fungus infection.
The accompanying drawing summary
Fig. 1 is the three-dimensional structure explanation by the 4-of Single Crystal X-ray diffraction investigation mensuration (2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mesylate.
Fig. 2 is the diagram of the structure that produces of the X-ray diffraction studies of 4-(2,6-dichloro-benzoyl base amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mesylate.
Fig. 3 is the X-ray powder diffraction figure of 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mesylate.
Fig. 4 is the X-ray powder diffraction figure of 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base amidoacetic acid salt.
Fig. 5 is the DSC scintigram of 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base amidoacetic acid salt.
Embodiment
Now illustrate the present invention by the specific embodiments described in reference the following example, but do not limit the present invention.
Embodiment 1
4-(2,6-, two chloro-the benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-mesylate of 4-base acid amides and synthesizing of its crystal
The mesylate of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides can prepare by the route of synthesis shown in the following schema.
Figure S200680009244XD00571
The preparation of stage 1:4-nitro-1H-pyrazoles-3-methyl-formiate
Figure S200680009244XD00572
4-nitro-1H-pyrazoles-3-formic acid (1.117Kg, 7.11mol, 1 weight) and methyl alcohol (8.950L, 8 volumes) threading are equipped with in the 20L reactor of digital thermometer and agitator.Stirred reaction mixture under nitrogen is cooled to 0-5 ℃, goes through to add thionyl chloride (0.581L, 8.0mol, 0.52 volume) in 180 minutes, makes the gained mixture be warmed to 18-22 ℃ and stir and spend the night under this temperature, after this time 1H NMR analyzes (d 6-DMSO) Indicator Reaction is complete.In 40-45 ℃ of concentrated reaction mixture under reduced pressure, with the O for toluene residue and in 40-45 ℃ of reconcentration (3 * 2.250L under reduced pressure, 3 * 2 volumes), the 4-nitro of generation pale solid form-1H-pyrazoles-3-methyl-formiate (1.210Kg, 99.5%).
The preparation of stage 2:4-amino-1H-pyrazoles-3-methyl-formiate
Figure S200680009244XD00581
Under nitrogen, palladium/charcoal (10% wet paste, 0.170Kg, 0.14 weight) threading is equipped with in the 20L reactor of digital thermometer and agitator.In an independent container with 4-nitro-1H-pyrazoles-3-methyl-formiate (1.210Kg, 7.07mol, 1 weight) and slurries in ethanol (12.10L, 10 volumes) are warmed to 30-35 ℃, realize dissolving, under nitrogen, this solution is added in the catalyzer.After nitrogen-hydrogen cleaning sequence, introduce nitrogen atmosphere and reaction mixture is maintained 28-30 ℃, until pass through 1H NMR analyzes (d 6-DMSO) explanation react completely (5-10 hour).After the purge of gas circulation, under nitrogen, filter reaction mixture and concentrating under reduced pressure liquid, produce 4-amino-1H-pyrazoles-3-methyl-formiate (0.987Kg, 98.9%).
The preparation of stage 3:4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-methyl-formiate
Figure S200680009244XD00582
Under nitrogen, use triethylamine (0.761L, 5.46mol, 1.2 volumes), then use 2,6-dichlorobenzoyl chloride (0.710L, 4.96mol, 0.72 volume) is processed 4-amino-1H-pyrazoles-3-methyl-formiate (0.634Kg, 4.49mol, 1 weight) and at Isosorbide-5-Nitrae-two Solution in the alkane (8.90L, 9 volumes) is so that internal temperature maintains 20-25 ℃.2 of remnants, 6-dichlorobenzoyl chloride Isosorbide-5-Nitrae-two
Figure 200680009244X_9
The rinsing line (line rinse) of alkane (0.990L, 1 volume) washs, at 18-25 ℃ of lower stirred reaction mixture, until analyze (developping agent: ethyl acetate: heptane 3: 1 by TLC; R F amine0.25, R The f product0.65) show react completely (16 hours).Filter reaction mixture, with Isosorbide-5-Nitrae-two
Figure 200680009244X_10
Alkane (2 * 0.990L, 2 * 1 volumes) washing leaching cake, the filtrate of merging (redness) namely is used for carrying out the stage 4 without further separating.
The preparation of stage 4:4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-formic acid
Figure S200680009244XD00591
The ester solution in stage 3 disposable (1.099Kg, 3.50mol is in 6.00L) is added in the solution of sodium hydroxide (0.484Kg, 12.1mol) in water (6.05L).In 20-25 ℃ of lower stirred reaction mixture until TLC analyzes (developping agent: ethyl acetate: heptane 3: 1; R The f ester0.65, R The f stage 4Baseline) assaying reaction is complete.In 45-50 ℃ of lower concentrating under reduced pressure, water (9.90L) dilution oily resistates is acidified to pH1 with concentrated hydrochloric acid, temperature is maintained be lower than 30 ℃ with reaction mixture.Collect the gained throw out by filtering, water (5.00L) washing is drained at filter, uses subsequently heptane (5.00L) washing.Filter cake is packed in the 20L rotatory evaporator flask, with methylbenzene azeotropic dry fully (2 * 4.50L), obtain the 4-(2,6-dichloro-benzoyl base is amino) of yellow solid form-1H-pyrazoles-3-formic acid (1.044Kg, approximately 99.5%).
Stage 5:4-{[4-(2,6-dichloro-benzoyl base amino)-1H-pyrazoles-3-carbonyl] amino } preparation of piperidines-1-t-butyl formate
Figure S200680009244XD00592
In the flange flask that mechanical stirrer, dropping funnel and thermometer are installed (flange flask) with the product (1.0 weight) in stage 4 and toluene (10.0 volume) the suitable size of packing into.Stirring content in 16-25 ℃ under nitrogen waits a moment and adds slowly thionyl chloride (0.3 volume).Then content is heated to 80-100 ℃ and under this temperature, stir until pass through 1H NMR judgement reacts completely.If content becomes too thick and can not stir, can add other toluene (at the most 10 volumes) in this stage.In case finish, mixture is cooled to 40-50 ℃, then under vacuum, be concentrated into dried at 45-50 ℃.Then with resistates and toluene (3 * 2.0 volume) azeotropic drying.
With the solid transfer of separating in the flask of suitable size and the tetrahydrofuran (THF) of packing into (5.0 volume).Under nitrogen, stir content and add triethylamine (0.512 volume) in 16-25 ℃.4-amino-piperadine-1-t-butyl formate (0.704 weight) and tetrahydrofuran (THF) (5.0 volume) are packed in the independent flask.Stir content until reach fully dissolving, the reaction flask of then solution being packed into maintains 16-30 ℃ with temperature.Then reaction mixture is heated to 45-50 ℃ and stir content until pass through 1H NMR judgement reacts completely.Then content is cooled to 16-25 ℃ and the water of packing into (5.0 volume).The heptane (0.5 volume) that add to mix stirred content 10 minutes and separate each layer at the most.Then use tetrahydrofuran (THF): the heptane of mixing [(9: 1), 3 * 5.0 volumes] aqueous phase extracted.Merge organic phase, water (2.5 volume) washing, concentrated in 40-45 ℃ under vacuum subsequently.With resistates and toluene (3 * 5.0 volume) azeotropic and be concentrated into dried, the crude product in generation stage 5.
Then with solid transfer in the flask of suitable size, add methyl alcohol: toluene [(2.5: 97.5), 5.0 volumes], under nitrogen, stirred these slurries 3-18 hour.The filtering content thing; with toluene (2 * 0.7 volume) washing leaching cake; then under vacuum in 40-50 ℃ of drying solid, produce the 4-{[4-(2,6-dichloro-benzoyl base is amino) of pale solid form-1H-pyrazoles-3-carbonyl] amino piperidines-1-t-butyl formate.
Process in this way the product (every crowd of 0.831Kg) in two batches of stages 4, produce the altogether 4-{[4-of 2.366Kg (88.6% productive rate) (2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carbonyl] amino } piperidines-1-t-butyl formate.
The preparation of stage 6:4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mesylate
Figure S200680009244XD00611
Product (1.0 weight) and Isosorbide-5-Nitrae-two with the stage 5
Figure 200680009244X_11
In the flange flask that mechanical stirrer, dropping funnel and thermometer are installed of alkane (30.0 volume) the suitable size of packing into.Under nitrogen, stir content and be heated to 80-90 ℃.Go through 30-60 minute adding methylsulfonic acid (0.54 volume), content is heated to 95-105 ℃ subsequently, and under this temperature range, stirs until pass through 1H NMR judgement reacts completely.In case finish, content be cooled to 20-30 ℃ and by filter collecting the gained precipitation.With 2-propyl alcohol (2 * 2.0 volume) washing leaching cake; drained 3-24 hour at strainer; produce the 4-(2 of free-pouring pale solid form; 6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mesylate crude product (80.0-120.0%w/w does not proofread and correct impurity or solute).
With stages 5 product of several batches of this method processing, the details of every batch starting raw material and the amount of product provides in following table 1.
Table 1-removes to protect the productive rate in step-stage 6
Batch (4-{[4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carbonyl] amino }-piperidines-1-t-butyl formate) input amount (g) The quantum of output (g) of [4-(2,6-dichloro-benzoyl base-amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mesylate] Chemical purity (HPLC % area)
1 590.0 579.699.1％th，98.2％w/w 97.88
2 521.0 532.7103.1％th，102.2％w/w 98.09
3 523.8 511.798.5％th，97.7％w/w 98.17
[0408]
4 518.4 596.3 116.0％th，115.0％w/w 98.24
5 563.2 600.1 107.4％th，106.6％w/w 98.16
6 563.1 565.2 101.2％th，100.4％w/w 98.49
7 560.4 553.9 99.7％th，98.8％w/w 98.70
8 569.7 560.6 99.2％th，98.4％w/w 98.41
The recrystallization of stage 6a:4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mesylate
The product recrystallization in stage 6 is not more than 0.25% with the residual level of the product of the Boc-protection of guaranteeing any stage 5.The product in four batches of stages 6 that used following scheme recrystallization.
With pack into the flask of suitable size that mechanical stirrer, dropping funnel and thermometer are installed of the crude product in stage 6 and 2-propyl alcohol (10.0 volume).Content stirs under nitrogen and is heated to 75-85 ℃.Then water (at the most 2.5 volumes) is added in this content until obtain clear and bright solution.Then content is cooled to 40-60 ℃ and under vacuum, reduce approximately 50% in 40-50 ℃ of concentrated until reaction volume.With pack into flask and in 40-50 ℃ of concentrated content until remove the approximately solvent of 3.0 volumes of 2-propyl alcohol (3.0 volume).Then use 2-propyl alcohol (2 * 3.0 volume) to repeat again this method twice, check water-content.Then the gained slurries are cooled to 0-5 ℃ and under this temperature, stirred 1-2 hour.The filtering content thing is used 2-propyl alcohol (2 * 1.0 volume) washing leaching cake, drains at strainer subsequently to reach 24 hours.Solid transfer is dried to constant weight to drying tray and in 45-50 ℃ under vacuum; the 4-(2,6-dichloro-benzoyl base is amino) of generation pale solid form-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mesylate (60.0-100.0%w/w).
Four batches recrystallization productive rate is 85.6%-90.4%, and the purity of recrystallized product is 99.29%-99.39%.Recrystallization further increases purity for the second time.
The 4-(2,6-dichloro-benzoyl base is amino) that produces by this approach-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mesylate has 379.8 ℃ of fusing points (measuring by DSC).
The infrared spectra of mesylate (KBr pressed disc method) is 3233,3002,2829,1679,1632,1560,1430,1198,1037,909 and 784cm -1The place comprises characteristic peak.
Be not bound by any theory, it is believed that each infrared peak is by the following constituent that belongs to salt:
The peak: Come from:
3233cm -1 N-H
3002cm -1Aromatics C-H
2829cm -1Aliphatics C-H
1679cm -1Acid amides C=O
1632,1560cm -1Acid amides
1430cm -1Aliphatics C-H
1198cm -1 SO 2-O
1037cm -1C-Cl aromatics
909,784cm -1Aromatics C-H
Removing of the product of the Boc-protection in remaining stage 5
In some cases, when mesylate is dissolved in the acetate buffer, observe the thin throw out that the free alkali by the Boc-protection of the trace of remnants forms.Can use multiple technologies to remove this throw out or prevent this sedimentary formation, as mentioned below.
(a) filter
Use aseptic syringe needle from bottle, to extract the mixture of mesylate in the 200mM acetate buffer in the disposable syringe of 20mL, then 0.2 micron filter of clinical grade (the aseptic disposable filter of SartoriusMinisart unit) is connected to syringe.Depress at leisure plunger, with filtrate collection in clean clear and bright vial.The content of bottle is the clear colorless solution without the mesylate of particulate material.
(b) in aqueous acid, heat
Then mixture in water (10 volume) is cooled to 60 ℃ in 100 ℃ of lower heating 4 hours with mesylate and methylsulfonic acid (0.4 equivalent).Analyzing the indication mesylate by TLC exists as single component.Add 2-propyl alcohol (10 volume) and mixture is cooled to 40 ℃.Reduce in a vacuum extremely approximately 10 volumes of mixture, then add another part 2-propyl alcohol (10 volume), again mixture is reduced to 10 volumes.With this again triplicate that circulates.Cooling mixture in ice bath is collected formed solid by filtering, and is dry in a vacuum with 2-propyl alcohol (5 volume) washing, produces white to the mesylate of pale solid form.
(c) organic-water extraction
Then mixture in water (10 volume) is cooled to envrionment temperature in 100 ℃ of lower heating 3 hours with mesylate and methylsulfonic acid (0.4 equivalent).THF-heptane (9: 1,10 volumes) is added this mixture, with gained mixture vigorous stirring to produce solution.Separate each layer, then use ethyl acetate (2 * 10 volume) washing water with tetrahydrofuran (THF)-heptane (9: 1,2 * 10 volumes).2-propyl alcohol (10 volume) is added to aqueous phase, reduces in a vacuum solution and arrive approximately 5 volumes, then add another part 2-propyl alcohol (10 volume), again mixture is reduced to 5 volumes.With this again triplicate that circulates.Collect formed solid by filtering, dry in a vacuum with 2-propyl alcohol (5 volume) washing, produce white to the mesylate of pale solid form.
(d) chromatography
Use chromatographic technique that a kind of approach of removing non polar impurities from mesylate can be provided.The use of estimating inversion method will be effective especially.
Embodiment 2
Measure the crystalline structure of 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mesylate by X-ray diffraction
Prepare as described in Example 1 compound 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mesylate.The crystal that is used for diffraction experiment is by precipitating 0.05 * 0.08 * 0.14mm that has that obtains from the aqueous solution with the 2-propyl alcohol 3The colourless flap of size.Use CuK alpha-ray (λ=1.5418 of Rigaku rotating anode RU3HR
Figure 200680009244X_12
), the blue confocal optical system (Osmicblue confocal optics) of osmium and Rigaku Jupiter CCD detector collect crystallographic data under 93K.Collect image under 2 θ=15 and 90 ° in twice ω scanning, the distance of detector and crystal is 67mm.Collect by the CrystalClear software control data, process also in proportion drawing image with Dtrek.Because the high (μ=4.01mm of uptake factor -1), must use the 4th grade of fourier absorption correction that data are proofreaied and correct.Find that crystal belongs to rhombic system spacer Pbca (#61), under 93K, have lattice parameter a=8.90 (10), b=12.44 (10), c=38.49 (4)
Figure 200680009244X_13
, α=β=γ=90 °.Numeral deviation in the bracket (s.u, standard uncertainty).
Above-mentioned crystal and crystalline structure form another aspect of the present invention.
Use the direct method of in SHELXS-97, carrying out to resolve crystalline structure.In the refine of 271 crystallographic parameters that undertaken by SHELXL-97, used 20-0.9
Figure 200680009244X_14
The intensity data of 2710 individual reflections of total in (2.3＜θ＜58.87) resolution scope.Last statistical parameter is: wR2=0.2115 (all data), R1=0.0869 (data of I＞2 σ (I)), degree of fitting S=1.264.
In asymmetric cell, find the protonated free alkali of a part and a methanesulfonate negatively charged ion.The elementary composition of asymmetric cell is C 17H 21Cl 2N 5O 5S, the crystalline density of calculating is 1.49Mg/m 3Produce hydrogen atom on the geometry basis, but the position of the hydrogen atom of being combined with heteroatoms is determined by checking the Fo-Fc differential chart.The position of hydrogen atom and thermal parameter are only decided according to corresponding non-hydrogen atom.Determine the thermal motion model (referring to Fig. 1) of non-hydrogen atom by anisotropy calorifics factor.
Crystalline structure comprises in the molecule that (N15H...O7 2.690
Figure 200680009244X_15
) and five intermolecular hydrogen bondings (referring to accumulation graph Fig. 2).Three among them connect protonated piperidines nitrogen and two methanesulfonate negatively charged ion.First methanesulfonate negatively charged ion is by single H-key N12H12A...O2M 2.771
Figure 200680009244X_16
Connect, and second participation has interaction N12H12B...O1M 2.864
Figure 200680009244X_17
And N12H12B...O2M3.057 The H-key of bifurcated.Remaining mesylate oxygen O3M participates in hydrogen bond N8H8...O3M2.928
Figure 200680009244X_19
Contiguous protonated free alkali molecule is by H-key N15H15...O7 2.876
Figure 200680009244X_20
And by long connection N15H15...N2 3.562 Be connected with the accumulation of pyrazole ring with phenyl.These interactions are infinitely propagated along the b axle.Crystal accumulation comprises the 2D layer (in the ab plane) that is clipped in the methanesulfonate negatively charged ion between charged H-key and the cationic extensive network of two-layer protonated free alkali.Closely the 2D interlayer by benzyl ring accumulation and relate to and have C12...C18 3.341
Figure 200680009244X_22
Chlorine ... the interaction of phenyl and linking together along c-axis.
Being shown among Fig. 2 of this structure that produces by X-ray diffraction studies provides.
The coordinate of the atom of the structure of composition 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mesylate provides in table 2.
Table 2
Spacer: Pbca
The a that under 93K, has 5%s.u., b ﹠amp; The unit structure cell of c:
a＝ 8.9
b＝12.4
c＝38.5
α＝β＝γ＝90
Coordinate in the cif form:
loop_
_atom_site_label
_atom_site_type_symbol
_atom_site_fract_x
_atom_site_fract_y
_atom_site_fract_z
_atom_site_U_iso_or_equiv
_atom_site_adp_type
_atom_site_occupancy
_atom_site_symmetry_multiplicity
_atom_site_calc_flag
_atom_site_refinement_flags
_atom_site_disorder_assembly
_atom_site_disorder_group
S1M S 0.13517(17) 0.18539(13) 0.03193(5) 0.0286(5) Uani 1 1 d ...
O1M O 0.1193(5) 0.2208(3) -0.00409(14) 0.0326(13) Uani 1 1 d ...
O2M O 0.1551(5) 0.0681(3) 0.03330(13) 0.0331(13) Uani 1 1 d ...
O3M O 0.0151(5) 0.2217(4) 0.05453(14) 0.0368(13) Uani 1 1 d ...
C4M C 0.3036(8) 0.2420(6) 0.0475(2) 0.0355(19) Uani 1 1 d ...
H4M1 H 0.3855 0.2197 0.0329 0.053 Uiso 1 1 calc R ..
H4M2 H 0.3212 0.2181 0.0708 0.053 Uiso 1 1 calc R ..
H4M3 H 0.2959 0.3189 0.0471 0.053 Uiso 1 1 calc R ..
C11 Cl 0.26158(17) 0.18137(12) 0.34133(5) 0.0325(5) Uani 1 1 d ..
C12 Cl 0.75698(19) 0.16766(13) 0.26161(5) 0.0366(6) Uani 1 1 d ..
N1 N 0.6277(6) -0.2419(4) 0.34903(16) 0.0276(14) Uani 1 1 d ...
H1 H 0.5932 -0.3064 0.3484 0.033 Uiso 1 1 calc R ..
N2 N 0.7505(5) -0.2150(4) 0.36663(16) 0.0286(15) Uani 1 1 d ...
C3 C 0.7635(7) -0.1082(5) 0.36163(19) 0.0265(17) Uani 1 1 d ...
C4 C 0.6453(7) -0.0708(5) 0.34039(18) 0.0211(16) Uani 1 1 d ...
C5 C 0.5616(7) -0.1594(5) 0.3322(2) 0.0277(18) Uani 1 1 d ...
H5 H 0.4770 -0.1623 0.3181 0.033 Uiso 1 1 calc R ..
C6 C 0.8878(7) -0.0454(5) 0.3760(2) 0.0269(17) Uani 1 1 d ...
O7 O 0.9037(5) 0.0506(3) 0.36722(14) 0.0368(13) Uani 1 1 d ...
N8 N 0.9821(6) -0.0939(4) 0.39821(15) 0.0267(14) Uani 1 1 d ...
H8 H 0.9626 -0.1584 0.4048 0.032 Uiso 1 1 calc R ..
C9 C 1.1147(7) -0.0417(5) 0.41139(19) 0.0253(17) Uani 1 1 d ...
H9 H 1.1272 0.0261 0.3987 0.030 Uiso 1 1 calc R ..
C10 C 1.1019(8) -0.0148(5) 0.4502(2) 0.0330(18) Uani 1 1 d ...
H10A H 1.0156 0.0315 0.4540 0.040 Uiso 1 1 calc R ..
H10B H 1.0866 -0.0804 0.4633 0.040 Uiso 1 1 calc R ..
C11 C 1.2429(7) 0.0412(5) 0.4630(2) 0.0349(19) Uani 1 1 d ...
H11A H 1.2533 0.1102 0.4515 0.042 Uiso 1 1 calc R ..
H11B H 1.2355 0.0538 0.4878 0.042 Uiso 1 1 calc R ..
N12 N 1.3784(6) -0.0279(4) 0.45532(16) 0.0258(14) Uani 1 1 d ...
H12A H 1.4618 0.0069 0.4623 0.031 Uiso 1 1 calc R ..
H12B H 1.3716 -0.0892 0.4676 0.031Uiso 1 1 calc R ..
C13 C 1.3929(7) -0.0546(6) 0.4181(2) 0.0314(18) Uani 1 1 d ...
H13A H 1.4790 -0.1013 0.4147 0.038 Uiso 1 1 calc R ..
H13B H 1.4098 0.0107 0.4049 0.038 Uiso 1 1 calc R ..
C14 C 1.2538(7) -0.1097(6) 0.4049(2) 0.0356(19) Uani 1 1 d ...
H14A H 1.2425 -0.1785 0.4165 0.043 Uiso 1 1 calc R ..
H14B H 1.2639 -0.1231 0.3802 0.043 Uiso 1 1 calc R ..
N15 N 0.6215(5) 0.0371(4) 0.33108(16) 0.0256(14) Uani 1 1 d ...
H15 H 0.6768 0.0852 0.3408 0.031 Uiso 1 1 calc R ..
C16 C 0.5183(7) 0.0697(5) 0.30805(18) 0.0213(15) Uani 1 1 d ...
O17 O 0.4336(5) 0.0082(3) 0.29260(13) 0.0309(12) Uani 1 1 d ...
C18 C 0.5120(6) 0.1890(5) 0.30170(17) 0.0195(15) Uani 1 1 d ...
C19 C 0.3923(7) 0.2486(5) 0.31620(19) 0.0252(16) Uani 1 1 d ...
C20 C 0.3785(7) 0.3569(5) 0.30904(19) 0.0267(17) Uani 1 1 d ...
H20 H 0.2991 0.3957 0.3185 0.032 Uiso 1 1 calc R ..
C21 C 0.4814(7) 0.4078(5) 0.28805(19) 0.0270(17) Uani 1 1 d ...
H21 H 0.4708 0.4808 0.2834 0.032 Uiso 1 1 calc R ..
C22 C 0.6005(7) 0.3518(5) 0.27375(19) 0.0294(18) Uani 1 1 d ...
H22 H 0.6702 0.3865 0.2597 0.035 Uiso 1 1 calc R ..
C23 C 0.6142(7) 0.2425(5) 0.2807(2) 0.0286(17) Uani 1 1 d ...
Embodiment 3
The mensuration of the solubleness of 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mesylate
The 4-that studies embodiment 1 as described below (2,6-dichloro-benzoyl base the is amino)-1H-pyrazoles-3-carboxylic acid piperidin-solubleness of 4-base acid amides mesylate in the aqueous solution of pH value 1-11.
By preparing solution with diluted sodium hydroxide solution titrimetric standard hydrochloric acid to required pH.Prepared solution has the pH of 1-11.Then use the solubleness of these solution assessments 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mesylate.
In each experiment, take by weighing the solution that the 150mg mesylate enters in the transparent bottle of 5ml and adds the given pH of 3-4ml.Then at (i) envrionment temperature and (ii) average 3 hours of 4 ℃ of lower stirred solution mixtures.In each case, then the PVDF filter of solution by 0.22 μ m filtered the pH of the clear and bright solution of record gained, and the mesylate content of the standardized solution analytical solution of use UV-Vis spectrophotometer and this salt.
The dissolubility data that obtains is as shown in table 3 and 4.These data indication solubleness are pH and temperature dependent.Between pH4 and 7, obtained the optimal dissolution degree.Although obtained at first higher solubleness, observed salt after leaving standstill at ambient temperature in pH＞7 and pH＜3 time precipitation.
The mesylate dissolubility data that table 3. obtains at ambient temperature
Figure S200680009244XD00691
Figure S200680009244XD00692
In the laboratory, after 6 days, in solution 1, observe precipitation.After the filtration, measure the mesylate content of this solution and record pH.
Table 4. is in the mesylate dissolubility data of 4 ℃ of acquisitions
Sample ID Initial pH Final pH Absorbancy at 260nm [mesylate] (mg/ml)
1 0.26 0.26 0.1724 9.85
2 1.00 1.00 0.7350 39.41
3 2.51 2.51 0.735 41.49
4 4.99 4.99 0.7508 42.39
5 7.34 7.34 0.8124 45.87
6 8.17 8.17 0.7343 43.17
7 7.69 7.69 0.6700 37.87
8 9.04 7.40 0.5372 30.36
9 10.97 7.90 0.5469 30.90
[0529] Embodiment 4
The mensuration of 4-(2,6-dichloro-benzoyl base the is amino)-1H-pyrazoles-3-carboxylic acid piperidin-solubleness of 4-base acid amides mesylate in buffered soln
Under envrionment temperature and 4 ℃, carry out the research of the solubleness of mesylate in various buffering systems of embodiment 1.In first group of experiment, selected damping fluid is 200mM succinate pH5.5, Citrate trianion pH5.5 and Tris/ maleate pH5.5,6.5 and 7.5.The second group of damping fluid that utilizes comprises 200mM acetate pH4.6, glycine/methylsulfonic acid pH4.6 and glycine pH5.5.
The preparation of damping fluid
Toxilic acid, Tris, sodium hydrate buffer solution
This damping fluid prepares by 23.62g toxilic acid and 24.2g three (methylol) aminomethane are dissolved in the 1L water.Be settled to 200ml with this solution of 0.2M sodium hydroxide solution titration certain volume (50ml) to target pH and water.Preparation pH5.5,6.5 and 7.5 0.2M damping fluid.
Succsinic acid, sodium hydrate buffer solution
This damping fluid prepares by the 23.62g succsinic acid is dissolved in the 1L water.This solution with 0.2M sodium hydroxide solution titration certain volume (50ml) is settled to 200ml to target pH and water.
The 0.2M damping fluid of preparation pH5.5.
Citric acid, sodium citrate buffer solution
This damping fluid prepares by the 42.02g citric acid is dissolved in the 1L water.This solution with 0.2M citric acid three sodium solution (58.82g is in 1L water) titration certain volume (50ml) is settled to 200ml to target pH and water.The 0.2M damping fluid of preparation pH5.5.
Sodium acetate, acetate buffer
This damping fluid prepares by the 4.44g sodium acetate is dissolved in the 200ml water.This solution and water with 0.20M acetic acid solution (1.3ml is in 200ml water) titration certain volume (50ml) are settled to 200ml.The 0.20M acetate buffer of preparation pH4.6.
Glycine solution
This damping fluid prepares by the 3.01g glycine is dissolved in the 200ml water.The 0.20M glycine solution of preparation pH4.9.
Mesylate solubility studies in damping fluid
Use each pH and Laemmli buffer system Laemmli, carry out two experiments, one is carried out at ambient temperature, and one is carried out under 4 ℃.In each experiment, be added in the 4.0ml damping fluid 220mg mesylate of nominal and the stirred solution mixture.After 3 hours, stop to stir, solution is filtered, is diluted in the diluents and by HPLC and measures mesylate content by PVDF 0.22 μ m membrane filter.In whole experiment, all calculate the mesylate content of each sample with single point correction at every turn.
The result is as shown in table 5 and 6.
Table 5. is at the mesylate dissolubility data in the 200mM acetate buffer under 4 ℃
Sample ID Used damping fluid Initial pH Final pH [mesylate] (mg/ml)
1 Acetate 4.6 4.6 37.16
Table 6. is the mesylate dissolubility data in the 200mM acetate buffer at ambient temperature
Sample ID Used damping fluid Initial pH Final pH [mesylate] (mg/ml)
1 Acetate 4.6 4.5 30.65
By found that the solubleness of mesylate in acetate buffer is 32-37mg/ml.With the salts solution of acetate buffering in 121 ℃ of lower autoclavings 15 minutes, then further 40 ℃, 55 ℃ or 75 ℃ lower heated solution 10-12 days.Find that pH value of solution and 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mesylate is stable under these test conditions.Freeze-thaw stability property testing to the salts solution of acetate buffering does not cause 4-(2; 6-dichloro-benzoyl base is amino)-precipitation of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides or its salt; find that pH value of solution and 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mesylate is stable under these test conditions.
Embodiment 5
The mensuration of the stability of 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mesylate
The sample of the mesylate of embodiment 1 is stored six months time in the relative humidity atmosphere of the sealing bag that is arranged in sealed vessel under 40 ℃ temperature, 75%.When 1 month, 3 months and 6 months, carry out various tests and with purity or the physical condition of determining salt whether any change has occured.These tests are as follows:
(a) estimate color and the physical appearance of this salt.
(b) Fourier transform infrared spectroscopy (FTIR)-with reference standard spectrum relatively.
(c) measure fusing point by dsc (DSC).
(d) make analysis of electric power consumption reagent A KX measure water-content by method of coulometric analysis.
(e) by the HPLC analysing impurity.
The result of test provides in table 7.As seen, color and the appearance preservation of going through six months (test (a)) salt are constant, go through this cycle infrared spectra (test (b)) and show not variation.Therefore in test (c), fusing point does not reduce, and the degraded of salt does not occur in indication, and this point is analyzed by HPLC and obtained support, and this analysis is presented at the impurity that the zero-time is present in the sample not to be increased in test period concentration.
Therefore, this stability test proof mesylate has satisfactory stability long-time the kind.
Embodiment 6
The X-ray powder diffraction research of 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mesylate
On the Bruker D500 diffractometer of universal stage and temperature-changeable platform is installed to embodiment 1 in the mesylate of preparation carry out X-ray powder diffraction (XRPD) and analyze.Irradiating source is airtight copper pipe (CuK alpha-ray: 1.5406
Figure 200680009244X_23
), voltage and current is set in 40kV and 40mA.Used detector is scintillometer.XRPD analyzes and uses Diffrac Plus XRD Commander software 2.3.1 version to carry out.
By the about sample of this salt of 10mg being compressed into gently sampling receptacle, then floatingly gently being prepared sample to obtain smooth-flat-surface.
Step duration with 0.001 ° step-length and 1 second is collected diffractogram in 3 ° of-40 ° of 2-θ scopes.
The diffractogram of mesylate as shown in Figure 3.
The analytical results of table 7 stability test of mesylate under 40 ℃/75% relative humidity is summed up
T=0 T=1 month T=3 month T=6 month
Take out the date On October 15th, 04 On November 16th, 04 On January 14th, 05 On April 15th, 05
Analysis period 15-25 day in October, 04 16-18 day in November, 04 14-19 day in January, 05 14-19 day in January, 05
Stability test Standard
(a) proterties Wait to report Pale solid Pale solid The pale solid pale solid
(b) by FTIR, differentiate With reference standard spectrum AS76/R/001 (UATR, 4000-650cm -1) consistent With reference standard spectrum AS76/R/001 (UATR, 4000-650cm -1) consistent With reference standard spectrum AS76/R/001 (UATR, 4000-650cm -1) consistent With reference standard spectrum and reference standard spectrum AS76/R/001 (UATR, AS76/R/001 (UATR, 4000-650cm -1) consistent 4000-650cm -1) consistent
(c) by DSC, measure fusing point Wait to report (40 → 450 ℃, 10 ℃/minute, pressure cooker 367.7 ℃ of fusing heat absorptions 368.3 ℃ of fusing heat absorptions 375.5 ℃ 369.1 ℃ of fusing heat absorption fusing heat absorptions
(d) water content (AKX reagent) <0.1%w/w <0.1%w/w <0.1%w/w<0.1%w/w
(e) impurity (measuring by HPLC) total impurities RRT 0.812RRT 1.196RRT 1.226RRT 1.303RRT 1.318RRT 1.484RRT 1.526RRT 1.680RRT 1.682 Wait to report 0.52% area 0.07% area 0.01% area 0.02% area 0.02% area 0.03% area 0.15% area 0.03% area 0.19% area nd 0.49% area 0.07% area 0.02% area nd0.06% area nd0.17% area nd0.17% area nd 0.50% area 0.50% area 0.06% area 0.06% area nd nd0.02% area 0.02% area nd nd0.04% area 0.04% area 0.17% area 0.17% area 0.04% area 0.03% area 0.17% area 0.17% area nd 0.01% area
Chemical purity Wait to report 99.48% area 99.51% area 99.50% area 99.50% area
The note of table: nd=does not detect
Embodiment 7
Different relative humidity are to the impact research of 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mesylate
(i) carry out weight steam absorption (GVS) research to observe the behavior of mesylate under the different humidity condition.Used mesylate is to be prepared by hydrochloride (referring to embodiment 11) by the following method in the research.
Hydrochloride with the MeOH absorption and by the Strata-NH2 post, is used the MeOH wash-out.The fraction that will contain product reduces to produce 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides free alkali in a vacuum.MsOH (70%, in water) is added in the mixture of free alkali in MeOH, this mixture was stirred 14 hours at ambient temperature, then reduce with methylbenzene azeotropic (* 3) in a vacuum.By grind the purifying resistates with acetone, dry in vacuum drying oven, produce mesylate.
To move at the HidenIGASorp water adsorption analyser of operation CFRSorp software by the mesylate sample of aforesaid method preparation.Sample capacity is generally 10mg.As finishing water adsorption-desorption thermoisopleth as described in the following table:
Absorption Desorption
40 85
50 75
60 65
70 45
80 35
90 25
15
5
0
10
20
30
[0577]Use 12.92mg mesylate sample to carry out secondary circulation.The GVS thermoisopleth that produces proves that this salt absorbs the approximately water of 4 % by weight in 0%-90% relative humidity scope.Yet if this salt is not exposed to greater than under 80% the relative humidity, the water absorbed dose is less than 2.6%w/w.Importantly, absorbed water can be at the desorption stage yield-loss during absorption phase, and it is surface effects that this explanation water absorbs.
(ii) in an independent test, the mesylate that about 10mg is as above prepared flattens to obtain large surface area and it is positioned in the moisture eliminator that contains saturated aqueous sodium chloride at slide glass.Moisture eliminator is placed in the insulation can that is set as 40 ℃ so that approximately 75% relative humidity atmosphere to be provided.Shifted out sample in the time of 8 and 18 days, the XRPD figure of the salt that XRPD figure and the test beginning of the samples of these times is front compares.Observing XRPD figure does not change.HPLC analyzes and is presented at the degraded that salt does not occur test period.
Above test (i) although and (ii) the proof mesylate really absorb some water when being exposed to high humidity, the water loss that absorbs when relative humidity descends and the crystalline structure of salt do not have change.Therefore this mesylate is stable anhydrous salt.
Embodiment 8
The preparation of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base amidoacetic acid salt
With sodium bicarbonate (4.5g; 53.5mmol) be added to the 4-(2 that is stirring at ambient temperature; 6-two chloro-benzoyl-amidos)-solution of 1H-pyrazoles-3-carboxylic acid piperidin-4-base amide hydrochloride (20.6g, 50mmol) in water (500ml) in.Stirred the mixture 1 hour, by filter to collect formed solid and under vacuum with methylbenzene azeotropic (* 3) drying, produce the corresponding free alkali of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
1H NMR(400MHz，DMSO-d 6)δ10.20(s，1H)，8.30(s，1H)，8.25(d，1H)，7.60-7.50(m，3H)，3.70(m，1H)，3.00(d，2H)，2.50(m，2H)，1.70(d，2H)，1.50(m，2H)。
At ambient temperature; with Glacial acetic acid (15ml; 262mmol) be added in the 4-(2,6-, two chloro-benzoyl-amidos) that the stirring-1H-pyrazoles-3-carboxylic acid piperidin-suspension of 4-base acid amides (10.0g, 26.2mmol) in methyl alcohol (150ml).After 1 hour, obtain clear and bright solution, it is reduced with methylbenzene azeotropic (* 2) in a vacuum.Then (2 * 100ml) grind and drying solid in a vacuum, produce the 4-(2,6-, two chloro-benzoyl-amidos) of white solid form-1H-pyrazoles-3-carboxylic acid piperidin-4-base amidoacetic acid salt (10.3g) with acetonitrile with resistates.
1H NMR(400MHz，DMSO-d 6)δ10.20(s，1H)，8.40(d，1H)，8.35(s，1H)，7.60-7.50(m，3H)，3.85(m，1H)，3.00(d，2H)，2.60(t，2H)，1.85(s，3H)，1.70(d，2H)，1.55(m，2H)。
Embodiment 9
The differential of 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base amidoacetic acid salt scans calorimetry research
The TA instrument Q1000 that 50 self-actuated samplers have been installed in use carries out DSC research to the acetate of embodiment 8.Use indium as energy and temperature correction standard.
With 10 ℃/minute speed heated sample between 10 ℃-230 ℃.Keep 30ml/ minute nitrogen purge at sample.Use the sample capacity of 1mg-3mg, all samples is clipped in the sealing pan.The DSC scanning of acetate as shown in Figure 5.The DSC spike shown in 231.5 ℃ event, and it causes owing to losing acetic acid and being subsequently converted to free alkali.Further after the heating, this solid form is in 292.9 ℃ of decomposition/fusings.
Embodiment 10
The X-ray powder diffraction research of 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base amidoacetic acid salt
Carry out X-ray powder diffraction (XRPD) analysis according to the acetate to embodiment 8 on Bruker D500 diffractometer of the method described in the embodiment 6.
The diffractogram of this acetate as shown in Figure 4.
Embodiment 11
Synthesizing of the preparation 11A.4-nitro of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base amide hydrochloride-1H-pyrazoles-3-methyl-formiate
Figure S200680009244XD00771
At ambient temperature, thionyl chloride (2.90ml, 39.8mmol) is added in the mixture of 4-nitro-3-pyrazole carboxylic acid (5.68g, 36.2mmol) in methyl alcohol (100ml) at leisure, stirred the mixture 48 hours.Reduce in a vacuum mixture, dry by methylbenzene azeotropic, obtain 4-nitro-1H-pyrazoles-3-methyl-formiate.
11B.4-amino-1H-pyrazoles-3-methyl-formiate is synthetic
Figure S200680009244XD00772
Under nitrogen atmosphere, stirred 4-nitro-1H-pyrazoles-3-methyl-formiate (34.6mmol) and the mixture of 10%Pd/C (650mg) in EtOH (150ml) 20 hours.By Celite plug filtering mixt, reduce in a vacuum, by dry with methylbenzene azeotropic, obtain 4-amino-1H-pyrazoles-3-methyl-formiate.
(11C.4-2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-formic acid
With 2,6-dichlorobenzoyl chloride (8.2g; 39.05mmol) be added to carefully 4-amino-1H-pyrazoles-3-methyl-formiate (5g; 35.5mmol) and triethylamine (5.95ml; 42.6mmol) two
Figure 200680009244X_24
In the solution in the alkane (50ml), then at room temperature stirred 5 hours.Filter reaction mixture, process filtrate with methyl alcohol (50ml) and 2M sodium hydroxide solution (100ml), in 50 ℃ of heating 4 hours, then evaporation.100ml water is added in the resistates, then use the concentrated hydrochloric acid acidifying.By solid collected by filtration, water (100ml) washing is drained, the 4-(2,6-, two chloro-benzoyl-amidos) of generation 10.05g lilac solid form-1H-pyrazoles-3-formic acid.
(11D.4-{[4-2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carbonyl]-amino }-piperidines-1-t-butyl formate
At room temperature stir 4-(2; 6-two chloro-benzoyl-amidos)-1H-pyrazoles-3-formic acid (6.5g; 21.6mmol), 4-amino-1-BOC-piperidines (4.76g; 23.8mmol), EDC (5.0g; 25.9mmol) and the mixture of HOBt (3.5g, 25.9mmol) in DMF (75ml) 20 hours.Reduce in a vacuum reaction mixture, resistates is distributed between ethyl acetate (100ml) and saturated sodium bicarbonate aqueous solution (100ml).With salt water washing organic layer, dry (MgSO 4) and reduce in a vacuum.With resistates 5%MeOH-DCM (～30ml) absorption.Collect insoluble substance by filtering, dry in a vacuum with the DCM washing, the 4-{[4-(2,6-, two chloro-benzoyl-amidos) of generation white solid form-1H-pyrazoles-3-carbonyl]-amino }-piperidines-1-t-butyl formate (5.38g).Reduce in a vacuum filtrate; by using the column chromatography purifying resistates of 1: 2 EtOAc/ hexane-EtOAc of gradient elution; produce the 4-{[4-(2,6-, two chloro-benzoyl-amidos) of other white solid form-1H-pyrazoles-3-carbonyl]-amino }-piperidines-1-t-butyl formate (2.54g).
(11E.4-2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides
Process 4-{[4-(2 with saturated HCl-EtOAc (40ml); 6-two chloro-benzoyl-amidos)-1H-pyrazoles-3-carbonyl]-amino }-solution of piperidines-1-t-butyl formate (7.9g) in MeOH (50ml) and EtOAc (50ml), then at room temperature stir and spend the night.Because the existence of methyl alcohol, product does not have crystallization, so evaporation reaction mixture, and residue is ground with EtOAc.Collect the gained pale solid by filtering, with the EtOAc washing, (sinter) drains at sintered filter, the 4-(2,6-, two chloro-benzoyl-amidos) of generation 6.3g hydrochloride form-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.(LC/MS：R t 5.89，[M+H] +328/384)。
Biologic activity
Embodiment 12
Measure activation CDK2/ cyclin A kinase inhibiting activity assay method (IC 50)
Can utilize following scheme to measure the CDK2 kinase inhibiting activity.
To activate CDK2/ cyclin A (Brown etc., Nat.Cell Biol., 1, the 438-443 page or leaf, 1999; Lowe, E.D. etc., Biochemistry, 41, the 15625-15634 pages or leaves, 2002) at 2.5X concentration determination damping fluid (50mM MOPS pH7.2,62.5mM β-phospho-glycerol, 12.5mMEDTA, 37.5mM MgCl 2, 112.5mM ATP, 2.5mM DTT, 2.5mM sodium orthovanadate, 0.25mg/ml bovine serum albumin) is diluted to 125pM in, gets 10 μ l and 10 μ l histone substrate mixture (60 μ l ox histone h1 (Upstate Biotechnology, 5mg/ml), 940 μ l H 2O, 35 μ Ci γ 33P-ATP) mix, join in 96 orifice plates with the various diluents of 5 μ l test compounds in DMSO (at the most 2.5%).Reaction was carried out 2 to 4 hours, then used excessive ortho-phosphoric acid (5 μ l, 2%) to stop.On Millipore MAPH filter plate, from the phosphorylation histone h1, separate the γ of not being combined with histone h1 33P-ATP.Each hole of MAPH plate is moistening with 0.5% ortho-phosphoric acid, then reaction product is filtered with Millipore vacuum filtration unit and pass through the hole.After filtering, with twice of 200 μ l, 0.5% ortho-phosphoric acid debris.Filter adds 20 μ l Microscint, 20 scintillators after drying, then counts 30 seconds at Packard Topcount.
% inhibition and the drawing of calculating the CDK2 activity suppress the active needed test compound concentration (IC of 50%CDK2 in order to measure 50).
Compound 4-in above CDK2 assay method (2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides has the IC less than 0.1 μ M 50Value.
Embodiment 13
Measure activation CDK1/ cell periodic protein B kinase inhibiting activity assay method (IC 50)
CDK1/ cell periodic protein B assay method is identical with above-mentioned CDK2/ cyclin A, and different is to use CDK1/ cell periodic protein B (Upstate Discovery) and enzyme is diluted to 6.25nM.
Compound 4-in above CDK1 assay method (2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides has the IC less than 0.1 μ M 50Value.
Embodiment 14
GSK3-B kinase inhibiting activity assay method
With GSK3-β (Upstate Discovery) at 25mM MOPS, pH7.00,25mg/mlBSA, 0.0025%Brij-35,1.25% glycerine, 0.5mM EDTA, 25mM MgCl 2, be diluted to 7.5nM among 0.025% beta-mercaptoethanol, the 37.5mM ATP, get 10 μ l and mix with 10 μ l substrate mixture.The substrate mixture of GSK3-β is the 1ml aqueous solution of 12.5 μ M phosphoric acid-glycogen synthetase peptides-2 (Upstate Discovery), contains 35 μ Ci γ 33P-ATP.Enzyme and substrate are joined in 96 orifice plates with the various diluents of 5 μ l test compounds in DMSO (at the most 2.5%).Make reaction carry out 3 hours (GSK3-β), then use excessive ortho-phosphoric acid (5 μ l, 2%) to stop.The filter operation method is with above activating CDK2/ cyclin A assay method.
Compound 4-in above GSK3-B assay method (2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides has the IC less than 0.1 μ M 50Value.
Embodiment 15
Antiproliferative activity
The ability that suppresses the Growth of Cells of various kinds of cell system by measuring compound is measured the antiproliferative activity of compound.Utilize Alamar Blue assay method (Nociari, M.M, Shalev, A., Benias, P., Russo, C.Journal of Immunological Methods 1998,213,157-167) restraining effect of measurement cell growth.The method is the ability of its fluorescence-causing substance resorufin based on viable cell reduction resazurin.For each proliferation assay, cell is seeded on 96 orifice plates, it was recovered 16 hours, then add inhibitor compound and reach other 72 hours.When incubation period finishes, add 10% (v/v) Alamar Blue, then other 6 hours of incubation measures fluorescence-causing substance under 535nM ex/590nM em.In the situation that non-proliferative cell is measured, cell was kept under converging 96 hours, then add inhibitor compound 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 ofcell Cultures).
Use this assay method to have been found that the mesylate of compound 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides has the IC less than 0.11 in the HCT-116 cell 50Value.
Pharmaceutical preparation
Embodiment 16
(i) tablet
Preparation contains formula (I defined herein by the following method 0) or (I) tablet composition of compound or its acid salt: mix 50mg compound or its salt 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 by the following method capsule: mix 100mg formula (I defined herein 0) or (I) compound or its acid salt and 100mg lactose, the gained mixture is packed in the opaque hard gelatin capsule of standard.
(iii) injection I
Can prepare by the following method the parenteral composition of using by injection: with formula (I 0) or (I) compound (formula (I of salt form for example 0) or (I) compound) be dissolved in the water that contains 10% propylene glycol, produce the activity compound concentration of 1.5 % by weight.Then by filtering with solution sterilization also sealing in the ampoule of packing into.
(iv) injection II
By the following method for the preparation of the parenteral composition of injecting: with formula (I 0) or (I) compound (formula (I of salt form for example 0) or (I) compound) (2mg/ml) and N.F,USP MANNITOL (50mg/ml) be dissolved in the water, with solution sterile filtration, in pack into sealable 1ml bottle or the ampoule.
(v) injection III
Can be by the following method for the preparation of the preparation by sending in injection or the intravenous injection: with formula (I 0) or (I) compound (formula (I of salt form for example 0) or (I) compound) be dissolved in the water with the concentration of 20mg/ml.Then with bottle sealing and autoclaving.
(vi) injection IV
Can be by the following method for the preparation of the preparation by sending in injection or the intravenous injection: with formula (I 0) or (I) compound (formula (I of salt form for example 0) or (I) compound) be dissolved in (for example 0.2M acetate pH4.6) in the water that contains buffer reagent with the concentration of 20mg/ml.Then with bottle sealing and autoclaving.
(vii) subcutaneous injection preparation
By the following method for the preparation of the composition of subcutaneous administration: mix formula (I defined herein 0) or (I) compound or its acid salt and pharmaceutical grade Semen Maydis oil, produce the concentration of 5mg/ml.In composition sterilization and threading appropriate containers.
(viii) freeze-dried preparation
Formula (I defined herein with preparation 0) or (I) aliquots containig of compound or its acid salt bottle and the lyophilize of putting into 50ml.During lyophilize, in (45 ℃) the lower freezing said composition of a freezing scheme of step of using.Temperature is raised to-10 ℃ to rise again, then be reduced to-45 ℃ lower freezing, then in+25 ℃ lower first dry approximately 3400 minutes, succeeded by redrying, if temperature rises to 50 ℃ then increase step.During preliminary and redrying, pressure is arranged on 80 millitorrs.
(ix) be used for the concentrated solution that intravenously is used
The aqueous solution by preparation buffering in the 0.2M sodium acetate/acetate buffer that 4-(2,6-dichloro-benzoyl base amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides mesylate is dissolved in pH4.6 with the concentration of 20mg/ml.
Be filled in the container (such as 1 class vial) after the solution of this buffering removed particulate matter after filtration, then with its sealing (for example utilizing the sealing of Florotec plug) and fixing (for example fixing with aluminium lid).If compound and preparation are enough stable, then with preparation in one section appropriate time of 121 ℃ of lower autoclavings.If preparation is unstable to autoclaving, it can be used suitable filter sterilised and under aseptic condition, put in the sterile vials.Use for intravenously, can carry out administration with this solution form, perhaps can before using, it be injected in the infusion bag (containing pharmaceutically acceptable vehicle, such as 0.9% salt solution or 5% glucose).
Embodiment 17
The mensuration of anti-mycotic activity
Can adopt following scheme to measure the anti-mycotic activity of the salt of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
Test the effect that described salt resists one group of fungi, comprise Candida parapsilosis (Candidaparpsilosis), candida tropicalis (Candida tropicalis), Candida albicans (Candidaalbicans)-ATCC 36082 and Cryptococcus neoformans (Cryptococcus neoformans).These are being stored under 4 ℃ on the Sabourahd agar glucose inclined-plane for the examination microorganism.Each biological singlet suspension prepares according to following description: under 27 ℃, on rotary shaker, containing amino acid (Difco, Detroit, Mich.) (pH7.0) and in the yeast nitrogen liquid nutrient medium (YNB) of 0.05M morpholine propanesulfonic acid (MOPS) yeast growth is spent the night.Then centrifugal suspension is used the 0.85%NaCl washed twice, then will be through the cell suspension sonic treatment 4 seconds (Branson Sonifier, 350 types, Danbury, Conn.) of washing.Counting singlet blastospore is adjusted to desired concn in 0.85%NaCl in hematimeter.
Use the activity of the modification method mensuration test compound of liquid nutrient medium Microdilution technology.Test compound is diluted to the ratio of 1.0mg/ml in DMSO, then in the YNB liquid nutrient medium that contains MOPS (using fluconazole in contrast) of pH7.0, is diluted to 64 μ g/ml, 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 with the YNB liquid nutrient medium, prepare ten times of diluents (concentration range is 64 to 0.125 μ g/ml) of compound solution in the 2nd to 11 hole.The 1st hole is as aseptic contrast and the blank of spectrophotometry.The 12nd hole is as growth control.Inoculate 10 μ l (final inoculum number is 10 to each hole in micro plate the 2nd to 11 hole 4Individual biology/ml).With through the plate of inoculation 35 ℃ of lower incubations 48 hours.With vortex mixer (Vorte-Genie 2 Mixer, Scientific Industries, Inc., Bolemia, N.Y.) oscillating plate is after 2 minutes, with absorbancy (Automatic MicroplateReader, the DuPont Instruments of metric measurement at 420nm, Wilmington, Del.) measure the IC50 value.The IC50 terminal point is defined as comparing with control wells, demonstrating growth reduce approximately 50% lowest concentration of drug of (or more than).In turbidity measurement, this is defined as the aperture turbidity and is the lowest concentration of drug (IC50) of＜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 lower incubations 1 to 2 day, then check viability.
Embodiment 18
Control the biological assessment method of fungi infestation in the whole strain plant materials
The salt of 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides is dissolved in the acetone, then uses the acetone serial dilution 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 processing.
Then adopt following method to measure the activity of the anti-tomato epidemic disease of the compounds of this invention (phytophthora infestans (Phytophthora infestans)) with composition.Tomato (Rutgers kind) seed is grown until seedling 10-20 centimetre is high in without the peat base potting mixtures of soil.Then the compound to be tried with the 100ppm ratio sprays this plant to flowing out.After 24 hours, test plants is with the sporocyst aqueous suspension spray inoculation of tomato parasitica, and remains on open-air room and spend the night.Then this plant is transferred to the greenhouse until disease occurs at untreated control plant.
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).
Embodiment 19
Measure the general scheme of 4-(2,6-dichloro-benzoyl base the is amino)-1H-pyrazoles-3-carboxylic acid piperidin-free alkali of 4-base acid amides and the solubleness of salt
The solubleness of the various salt of 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides all can be measured by following scheme.
Working method
Free alkali (50mg, 0.131mmol) and water (0.5ml) are added in the 8ml bottle.(1 equivalent 0.131mmol) and with bottle vibrated 14-16 hour at ambient temperature to add suitable acid in the bottle.Estimate bottle after this time.If observe uniform solution, then stop experiment, can make the salt that forms thus and have conclusion greater than the solubleness of 100mg/ml.
If solid residue is arranged, then add other 0.5ml water, vibration bottle 6 hours.If formed uniform solution by this stage, then can make this salt and have conclusion greater than the solubleness of 50mg/ml.
If still have solid residue this moment, add other 1ml water, bottle at ambient temperature vibrates.If this causes uniform solution, then can make solubleness greater than the conclusion of 25mg/ml.If still have solid residue, can make the solubleness of this salt less than the conclusion of 25mg/ml.
Compare with free alkali, salt of the present invention shows one or more in the following advantages, because they:
Solvability is better, and therefore will use (for example by using in the intravenous injection) for intravenously better
Has better stability (for example increasing the shelf lives);
Has better thermostability;
Alkalescence a little less than, therefore and will be used for better intravenously and use;
Has advantages of the preparation of being beneficial to;
The solubleness that in aqueous solution, has raising;
Have better physicochemical property;
The antitumour activity that can have raising; With
The therapeutic index that can have raising.
Equivalents
Provide the purpose of above-described embodiment to be to set forth the present invention, it should be interpreted as the scope of the invention is forced any restriction.Obviously, can carry out various modifications and variations to the specific embodiments of the present invention that exemplifies among mentioned above and the embodiment in the situation that do not deviate from ultimate principle of the present invention.All these class modifications and variations are all contained by the application.

Claims (63)

  1. The acid salt of (1.4-2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, this salt is to form with the acid that is selected from methylsulfonic acid and acetic acid and composition thereof.
  2. 2. acid salt according to claim 1, it is the acid salt that forms with methylsulfonic acid.
  3. 3. acid salt according to claim 1, it is the acid salt that forms with acetic acid.
  4. 4. acid salt according to claim 2, it is crystalline, and:
    (a) has crystalline structure given among Fig. 1 and 2; And/or
    (b) has the defined crystalline structure of coordinate among this paper embodiment 2; And/or
    (c) under 93K, have lattice parameter a=8.90 (10), b=12.44 (10), α=β=γ=90 °; And/or
    (d) has the crystalline structure that belongs to the rhombic system spacer.
  5. 5.50%-100% is crystalline 4-(2; 6-dichloro-benzoyl base is amino)-mesylate of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, the X-ray powder diffraction figure that it has is characterised in that and has main peak and the spacing (d) that given diffraction angle (2 θ) is located among Table A and the table B.
  6. 6.4-(2; 6-dichloro-benzoyl base is amino)-mesylate of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, wherein X-ray powder diffraction figure is characterised in that main peak, spacing (d) and the intensity that exists diffraction angle given among this paper table C (2 θ) to locate.
  7. 7. 50%-100% according to claim 6 is crystalline 4-(2; 6-dichloro-benzoyl base-amino)-and the mesylate of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, it shows the peak at the diffraction angle place identical with the X-ray powder diffraction figure shown in Fig. 3.
  8. 8. 50%-100% according to claim 7 is the mesylate of crystalline 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin 4-base acid amides, and it has X-ray powder diffraction figure as shown in Figure 3.
  9. 9.50%-100% is crystalline 4-(2; 6-dichloro-benzoyl base is amino)-acetate of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, the X-ray powder diffraction figure that it has shows the peak at the diffraction angle place identical with the X-ray powder diffraction figure shown in Fig. 4.
  10. 10. 50%-100% according to claim 9 is the acetate of crystalline 4-(2,6-dichloro-benzoyl base amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, and wherein said peak has identical relative intensity with peak among Fig. 4.
  11. 11. 50%-100% according to claim 10 is the acetate of crystalline 4-(2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, it has X-ray powder diffraction figure as shown in Figure 4.
  12. 12. the mesylate of 4-according to claim 2 (2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, it is anhydrous and shows endotherm(ic)peak at 379-380 ℃ when carrying out DSC.
  13. 13. the mesylate of 4-according to claim 12 (2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, it is anhydrous and shows endotherm(ic)peak at 379.8 ℃ when carrying out DSC.
  14. 14. the acetate of 4-according to claim 3 (2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, it is anhydrous and shows exothermic peak at 231-232 ℃ and 292-293 ℃ when carrying out DSC.
  15. 15. the acetate of 4-according to claim 14 (2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, it is anhydrous and shows exothermic peak at 231.50 ℃ and 292.88 ℃ when carrying out DSC.
  16. 16. each defined 4-(2 in the preparation claim 1,2,4 to 8,12 or 13; 6-trichlorine benzoyl-amido)-method of the acid salt of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides; wherein said acid salt is the added methanesulfonic acid salify; the method comprises formation 4-(2; 6-two chloro-benzoyl-amidos)-solution of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides free alkali in solvent or solvent mixture, and process this solution to form the throw out of added methanesulfonic acid salify with methylsulfonic acid.
  17. 17. each defined 4-(2 in the preparation claim 1 to 15; 6-dichloro-benzoyl base is amino)-method of the acid salt of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides; the method comprises 4-(2; 6-two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides free alkali is dissolved in the solvent that comprises volatile acid and optional cosolvent; thereby form the solution of acid salt and volatile acid; then concentrated or evaporate this solution with separated salt, wherein said acid salt is that the salt and the described volatile acid that form with acetic acid are acetic acid.
  18. 18. form each defined 4-(2 in the claim 1 to 15; 6-dichloro-benzoyl base is amino)-method of the acid salt of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides; wherein said acid salt is added methanesulfonic acid salify or acetic acid additive salt, and the method comprises the methylsulfonic acid that is used in the organic solvent or the compound of acetic acid treatment formula (X):
    Figure FSB00000869321400031
    To remove the tert-butoxycarbonyl group and to form 4-(2; 6-two chloro-benzoyl-amidos)-acid salt of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides and methylsulfonic acid or acetic acid; randomly separate afterwards methylsulfonic acid or the acetic acid additive salt that forms thus, and randomly recrystallization methylsulfonic acid or acetic acid additive salt to produce crystalline form.
  19. 19. according to claim 1, each described acid salt in 2,4 to 8,12 or 13, it is the added methanesulfonic acid salify, and it has the solubleness greater than 15mg/ml in water.
  20. 20. pharmaceutical composition; it comprises and contains concentration greater than each defined 4-(2 in the claim 1 to 15 of 15mg/ml; 6-dichloro-benzoyl base is amino)-aqueous solution of the acid salt of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, described acid salt is added methanesulfonic acid salify or acetic acid additive salt.
  21. 21. pharmaceutical composition; it comprises and contains concentration greater than each defined 4-(2 in the claim 1 to 15 of 30mg/ml; 6-dichloro-benzoyl base is amino)-aqueous solution of the acid salt of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, described acid salt is added methanesulfonic acid salify or acetic acid additive salt.
  22. 22. according to claim 1, each described 4-(2 in 2,4 to 8,12 or 13; 6-dichloro-benzoyl base is amino)-aqueous solution of the acid salt of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides; wherein said acid salt is the added methanesulfonic acid salify, and wherein this aqueous solution has the pH of 2-12.
  23. 23. the aqueous solution of the acid salt of 4-according to claim 22 (2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, wherein this aqueous solution has the pH of 2-9.
  24. 24. the aqueous solution of the acid salt of 4-according to claim 23 (2,6-dichloro-benzoyl base is amino)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides, wherein this aqueous solution has the pH of 4-7.
  25. 25. aqueous solution according to claim 22, it is cushioned.
  26. 26. aqueous solution according to claim 25, wherein acid salt is the salt that forms with methylsulfonic acid, and buffer reagent is the buffer reagent that is formed by acetic acid and sodium acetate.
  27. 27. aqueous solution according to claim 26, buffer reagent wherein are the buffer reagents that is formed by acetic acid and sodium acetate, pH value of solution is approximately 4.6.
  28. 28. the method for preparation 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides or defined its methylsulfonic acid of claim 1 to 15 any one or acetic acid additive salt, the method comprises the following steps:
    Make the compound of formula (XI):
    With PG wherein be the compound of the formula (XII) of amine-protecting group:
    Figure FSB00000869321400042
    In the situation that exist glitch-free alkali to react in the organic solvent, the compound of production (XIII):
    Figure FSB00000869321400043
    Remove afterwards protecting group PG, produce 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides or its methylsulfonic acid or acetic acid additive salt, and randomly the methylsulfonic acid that forms thus of recrystallization or acetic acid additive salt to produce crystalline form.
  29. 29. method according to claim 28, wherein protecting group PG is that removing of uncle-butoxy carbonyl and protecting group is to realize with acid.
  30. 30. method according to claim 28, wherein said acid is methylsulfonic acid.
  31. 31. method according to claim 28, wherein said acid is acetic acid.
  32. 32. the method for the chemical intermediate of preparation formula (XIII), the method be such as in the claim 32 definition by making formula (XI) compound and the compound of formula (XII) react to realize.
  33. 33. the method for preparation 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides or defined its methylsulfonic acid of claim 1 to 15 any one or acetic acid additive salt, the method comprises:
    (i) in aprotic organic solvent, process the compound of formula (XIV) with thionyl chloride:
    Figure FSB00000869321400051
    This reaction is randomly carried out under heating;
    (ii) in the situation that exist glitch-free alkali to make the compound reaction of the product of step (i) and formula (XII), this reaction is randomly carried out the compound of production (XIII) under heating; With
    (iii) from the compound of formula (XIII), remove protecting group PG, produce 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides or its methylsulfonic acid or acetic acid additive salt; Randomly
    (iv) this methylsulfonic acid of recrystallization or acetic acid additive salt are to produce crystalline form.
  34. 34. method according to claim 33 wherein is being heated under 80-100 ℃ the temperature with the reaction of thionyl chloride in step (i) and is carrying out.
  35. 35. method according to claim 33, the solvent that wherein carries out therein step (i) is the aromatic hydrocarbons solvent.
  36. 36. method according to claim 35, the solvent that wherein carries out therein step (i) is toluene.
  37. 37. method according to claim 33, wherein step (ii) is carried out being heated under the highest approximately 55 ℃ the temperature.
  38. 38. method according to claim 33 is wherein reacted in step (ii) and is carried out in tetrahydrofuran (THF).
  39. 39. method according to claim 33; wherein protecting group is can be by the protecting group that is removed with acid treatment in step (iii); acid is selected to produce the salt of required 4-(2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
  40. 40. described method according to claim 39, wherein protecting group is uncle-butoxy carbonyl.
  41. 41. the salt of each defined 4-in the claim 1 to 15 (2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides for the preparation of prevention or treatment by the purposes in the medicine of the morbid state of cell cycle protein dependent kinase or GSK-3 mediation or illness.
  42. 42. the salt of each defined 4-in the claim 1 to 15 (2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides is for the preparation of the purposes in the medicine of the incidence that alleviates or reduces the morbid state that mediated by cell cycle protein dependent kinase or GSK-3 or illness.
  43. 43. the purposes of salt in the medicine that comprises abnormal cell growth or the disease that is caused by abnormal cell growth or illness for the preparation for the treatment of in Mammals of each defined 4-in the claim 1 to 15 (2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
  44. 44. the salt of each defined 4-in the claim 1 to 15 (2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides is for the preparation of the purposes in the medicine that alleviates or reduce the incidence that comprises abnormal cell growth or the disease that is caused by abnormal cell growth or illness in Mammals.
  45. 45. the salt of each defined 4-in the claim 1 to 15 (2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides is for the preparation of the purposes in the medicine that suppresses cell cycle protein dependent kinase or GSK-3.
  46. 46. the salt of each defined 4-in the claim 1 to 15 (2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides is for the preparation of the purposes in the medicine of regulating cell processes by the activity that suppresses cell cycle protein dependent kinase or GSK-3.
  47. 47. each defined 4-(2 in the claim 1 to 15; 6-two chloro-benzoyl-amidos)-and the salt of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides is for the preparation of the purposes in the medicine of prevention or treatment morbid state, and wherein said morbid state is cancer.
  48. 48. the salt of each defined 4-in the claim 1 to 15 (2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides is for the preparation of the purposes in the medicine of prevention or treatment leukemia or follicular carcinoma of thyroid.
  49. 49. the salt of each defined 4-in the claim 1 to 15 (2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides is for the preparation of prevention or treatment acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell lymphoma or Burkitt lymphoma; Or the purposes in the medicine of acute and chronic myelogenous leukemia, myelodysplastic syndromes or promyelocytic leukemia.
  50. 50. comprise each defined 4-in the claim 1 to 15 (2,6-, two chloro-the benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-salt of 4-base acid amides and the pharmaceutical composition of pharmaceutically acceptable carrier.
  51. 51.4-each defined its acid salt is for the preparation of the purposes in the medicine for the treatment of chronic lymphocytic leukemia in (2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides or the claim 1 to 15.
  52. 52.4-each defined its acid salt is for the preparation of the purposes in the lymphadenomatous medicine for the treatment of Diffuse large B cell in (2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides or the claim 1 to 15.
  53. 53.4-the acid salt of (2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides is for the preparation of the purposes in treatment chronic lymphocytic leukemia or the lymphadenomatous medicine of Diffuse large B cell.
  54. 54. 3 described purposes according to claim 5, wherein said salt is hydrochloride.
  55. 55. each defined 4-(2 in the claim 1 to 15; 6-two chloro-benzoyl-amidos)-purposes of salt in the preparation medicine of 1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides; described medicine is used for screened and be determined and suffer from disease or illness or have the disease suffered from or the patient of the danger of illness treatment or preventing disease state or illness, and it is responsive that described disease or illness have a treatment that the compound of anti-cell cyclin-dependent kinase activity carries out to use.
  56. 56. the acid salt of each defined 4-in the claim 1 to 15 (2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides is for the preparation of the purposes in the medicine that suppresses tumor growth in Mammals.
  57. 57. the purposes of acid salt in the medicine of growing for the preparation of inhibition tumor cell of each defined 4-in the claim 1 to 15 (2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides.
  58. 58. the salt of each defined 4-in the claim 1 to 15 (2,6-, two chloro-benzoyl-amidos)-1H-pyrazoles-3-carboxylic acid piperidin-4-base acid amides is for the preparation of the purposes in the medicine of prevention or treatment cancer.
  59. 59. the purposes of claim 58, cancer wherein are bladder cancer, mammary cancer, colorectal carcinoma, kidney, epidermal carcinoma, liver cancer, lung cancer, esophagus cancer, carcinoma of gallbladder, ovarian cancer, carcinoma of the pancreas, cancer of the stomach, cervical cancer, thyroid carcinoma, prostate cancer or skin carcinoma; The hematopoietic system cancer of lymph pedigree; The hematopoietic system cancer of marrow pedigree; Follicular carcinoma of thyroid; The tumour of mesenchyme origin; The tumour of central or peripheral nervous system; Spermocytoma; Teratocarcinoma; Osteosarcoma; Xeroderma pitmentosum; Keratoacanthoma; Follicular carcinoma of thyroid; Or Kaposi sarcoma.
  60. 60. the purposes of claim 58, cancer wherein is melanoma.
  61. 61. the purposes of claim 58, cancer wherein is selected from mammary cancer, ovarian cancer, colorectal carcinoma, prostate cancer, esophagus cancer, squamous cell carcinoma and nonsmall-cell lung cancer.
  62. 62. the purposes of claim 59, the hematopoietic system cancer of lymph pedigree wherein is leukemia.
  63. 63. the purposes of claim 59, cancer wherein are the hematopoietic system cancers that is selected from the lymph pedigree of chronic lymphocytic leukemia, lymphoma mantle cell and B-cell lymphoma.
CN200680009244.XA 2005-01-21 2006-01-20 4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acid piperidin-4-ylamid acid addition salts as kinase inhibitors Expired - Fee Related CN101146791B (en)

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