CN109705090A - The tartaric acid addition salt and its crystal form of the disubstituted 1H- pyrazole compound of 3,4- - Google Patents

Abstract

The present invention provides one or more 4- (2; 6- dichIoro-benzoylamino) -1H- pyrazoles -3- carboxylic acid piperidin -4- base acyl tartaric acid addition salt pharmaceutical composition; the crystal form of the tartaric acid addition salt, and the tartaric acid addition salt composition comprising crystal form are preventing and treating the purposes in the disease or illness mediated by cell cycle protein dependent kinase.

Description

The tartaric acid addition salt and its crystal form of the disubstituted 1H- pyrazole compound of 3,4-

Technical field

The present invention relates to the winestones of 4- (2,6- dichIoro-benzoylamino) -1H- pyrazoles -3- carboxylic acid piperidin -4- base amide Acid-addition salts and its crystal form, the method for preparing the acid-addition salts and its crystal form.

Background technique

Cell cycle protein dependent kinase (cyclin dependent kinase, CDK) is the pass of Cycle Regulation Key kinases participates in the physiology courses such as cell Proliferation, transcription, survival.Various CDK and cyclin are in the entire cell cycle The adjusting of expression, degradation rate and activation levels causes a series of circulation of CDK/ cyclin complexes to be formed, The formation of these compounds via discontinuous cell cycle checkpoint control pass on, thus enable fissional process after It is continuous.According to the difference of CDK function, two major classes can be broadly divided into.A kind of CDK participates in cell cycle regulating, mainly includes CDK1, CDK2, CDK4, CDK6 etc..Under no outer signals stimulation, Retinoblastoma Protein (retinoblastoma Protein, RB) in conjunction with transcription factor E2F and inhibit its activity, Inhibit proliferaton makes cell be in the G0 phase.There is silk point in the external world Split under signal stimulus, cell enters the G1 phase, and cyclin D synthesis increases, in conjunction with CDK4/6, make RB phosphorylation, past release its Inhibition to E2F, the expression such as albumen cyclin E needed for carrying out the period increase.The G1 later period, cyclin E in conjunction with CDK2, into The activity of one step phosphorylation RB, E2F is completely released, and cell limits point by G1/S, into the S phase.At this point, cyclin A replaces Cyclin E participates in DNA replication dna in conjunction with CDK2.S later period, cyclin A and CDK1 form compound, driving mitosis into Row promotes cell by the M phase, is finally completed the mitosis process of cell.

Another major class CDK participates in transcriptional regulatory, mainly including CDK7, CDK8, CDK9, CDK10, CDK11 etc..CDK7/ Cyclin H, CDK8/cyclin C, CDk9/cyclin T adjust transcription by adjusting the phosphorylation of rna plymerase ii. Wherein CDK7/cyclin H also forms the activity that CDK activated protein kinase (CDK activating kinase, CAK) adjusts CDK. RB phosphorylation is adjusted in CDK3/cyclin C, participates in G0/G1 transition, also can phosphorylated-activated 1 (activating of replicator Transcription factor 1, ATF1), improve its Transactivation level.CDK10/cyclin M by phosphorylation transcribe because Sub- Ets transcribes to adjust.

In addition, CDK2 is a very crucial cell cycle protein dependent kinase for the completion G1 phase entering the S phase, CDK2 It in conjunction with cyclin E and activates, maintains the phosphorylation of G1 later period pRb, guarantee that cell passes through the G1 phase and enters the S phase.In S The initial phase, CDK2 is passivated E2F transcription factor in conjunction with cyclin A, and the passivation of E2F is the precondition completed the S phase, E2F It is active persistently to will lead to Apoptosis.Therefore, selective inhibition CD see/cyclin A may cause the raising of E2F concentration, And then lead to stagnation or the apoptosis of cell S phase.And CDK5 is different from traditional CDK, adjusting subunit is not cyclin, But p35 and p39, present studies have shown that CDK5 also assist in the relevant proliferation of tumour generation hair, survival and an epithelium mesenchyma turn Multiple processes such as change.

In mammalian cells, CDK monomer without activity, only in conjunction with corresponding adjusting subunit cyclin after ability Become active Stable conformation.Intracellular CDK expression is general constant, but cyclin is in the different phase period of cell cycle Property expression and degradation, this expression pattern of cyclin determines the different CDK of orderly activation.Cyclin adjusts CDK's Subcellular localization, and specific substrate is identified by special anchored site, play the function of phosphorylation.

In tumour cell, cyclin is overexpressed or overactivity, CDK1 activity inhibited, upstream heading signal persistently swash The activity change that CDK can all be caused such as living.The imbalance of CDK activity can directly or indirectly cause uncontrolled cellular proliferation, genome unstable Fixed (DNA mutation increases, chromosome deficiency etc.) and chromosome instability fixed (chromosome number variation) etc., participate in tumour and send out Exhibition.Since CDK activity is required for cell division, and often there is CDK increased activity in tumour.Currently carry out it is clinical or The CDK inhibitor of preclinical study is broadly divided into ATP competitiveness and noncompetitive inhibitor according to mechanism of action difference.

Wherein, compound 4- (2,6- dichIoro-benzoylamino) -1H- pyrazoles -3- carboxylic acid piperidin-of the present invention 4- base amide (original grinds code name AT7519, hereinafter referred to as AT7519), first public is in international patent application No.PCT/ In GB2004/003179 (publication number WO 2005/012256), the compound is disclosed with cyclin dependent kinase The inhibitory activity of enzyme (CDK kinases).It can inhibit CDK1, CDK2, CDK4 and CDK9.Also, the compound also has anti-sugar The activity of former synthase kinase -3 (GSK-3).

In International Application Serial No. PCT/GB 2006/000207 (publication number WO 2006/077426), the compound is disclosed Mesylate and its crystal form, and claim that the mesylate has dissolubility more preferable, the properties such as more stable.

Summary of the invention

The purpose of the present invention is to provide one or more 4- (2,6- dichIoro-benzoylamino) -1H- pyrazoles -3- first The pharmaceutical composition of sour piperidin-4-yl acyl tartaric acid addition salt, the crystal form of the tartaric acid addition salt, and include crystalline The tartaric acid addition salt composition of formula is in preventing and treating the disease or illness mediated by cell cycle protein dependent kinase Purposes.4- (2,6- dichIoro-benzoylamino) -1H- pyrazoles -3- carboxylic acid piperidin -4- base acyl tartaric acid addition salt It is that the present inventor passes through 4- (2,6- dichIoro-benzoylamino) -1H- pyrazoles -3- carboxylic acid piperidin -4- base acyl free alkali and winestone Acid reaction is prepared, hereinafter referred to as " tartaric acid addition salt ".

First aspect present invention provides 4- (2,6- dichIoro-benzoylamino) -1H- pyrazoles -3- carboxylic acid piperidin -4- The tartaric acid addition salt of base acyl.

As a preferred embodiment, the tartaric acid addition salt is crystal form.

In some embodiments, the tartaric acid addition salt of the crystal form is the crystallization of Form I type.

The described Form I type crystallization being indicated, using the alpha-emitting X-ray powder diffraction of Cu-K (XRD) with 2 θ (°) Spectrum about has characteristic peak 17.7 ± 0.2,19.1 ± 0.2 and 26.2 ± 0.2.

Form I type crystallization as a preferred method, being indicated, using the alpha-emitting X of Cu-K with 2 θ (°) Ray powder diffraction (XRD) spectrum about in 12.7 ± 0.2,13.02 ± 0.2 or 16.4 ± 0.2 one at, have at two or at three Characteristic peak.

Form I type crystallization as a preferred method, being indicated, using the alpha-emitting X of Cu-K with 2 θ (°) Ray powder diffraction (XRD) spectrum about in 20.4 ± 0.2,21.6 ± 0.2 or 24.5 ± 0.2 one at, have at two or at three Characteristic peak.

Form I type crystallization as a preferred method, being indicated, using the alpha-emitting X of Cu-K with 2 θ (°) Ray powder diffraction (XRD) spectrum about 12.7 ± 0.2,13.3 ± 0.2,16.4 ± 0.2,17.7 ± 0.2,19.1 ± 0.2, There is characteristic peak at 20.4 ± 0.2,21.6 ± 0.2,24.5 ± 0.2 and 26.2 ± 0.2.

Form I type crystallization as a preferred method, being indicated, using the alpha-emitting X of Cu-K with 2 θ (°) Ray powder diffraction (XRD) spectrum has X-ray powder diffraction figure as shown in Figure 1.

Form I type crystallization as a preferred method, DSC endothermic peak change at about 194-200 DEG C.

In further embodiments, the crystalloid tartaric acid addition salt is the crystallization of Form II type.

Form II type crystallization as a preferred method, being indicated, using the alpha-emitting X of Cu-K with 2 θ (°) Ray powder diffraction (XRD) spectrum about has characteristic peak at 7.0 ± 0.2,10.5 ± 0.2 and 21.8 ± 0.2.

Form II type crystallization as a preferred method, being indicated, using the alpha-emitting X of Cu-K with 2 θ (°) Ray powder diffraction (XRD) spectrum about in 13.0 ± 0.2,17.4 ± 0.2 or 14.0 ± 0.2 one at, have at two or at three Characteristic peak.

Form II type crystallization as a preferred method, being indicated, using the alpha-emitting X of Cu-K with 2 θ (°) Ray powder diffraction (XRD) spectrum about in 23.5 ± 0.2,18.6 ± 0.2 or 24.8 ± 0.2 one at, have at two or at three Characteristic peak.

Form II type crystallization as a preferred method, being indicated, using the alpha-emitting X of Cu-K with 2 θ (°) Ray powder diffraction (XRD) spectrum about 7.0 ± 0.2,10.5 ± 0.2,21.8 ± 0.2,13.0 ± 0.2,17.4 ± 0.2, There is characteristic peak at 14.0 ± 0.2,23.5 ± 0.2,18.6 ± 0.2 and 24.8 ± 0.2.

Form II type crystallization as a preferred method, being indicated, using the alpha-emitting X of Cu-K with 2 θ (°) Ray powder diffraction (XRD) spectrum has X-ray powder diffraction figure as shown in Figure 4.

Form II type crystallization as a preferred method, DSC endothermic peak change at about 107-127 DEG C.

In further embodiments, the crystalloid tartaric acid addition salt is the crystallization of Form type III.

Form type III crystallization as a preferred method, being indicated, alpha-emitting using Cu-K with 2 θ (°) X-ray powder diffraction (XRD) spectrum about has characteristic peak 11.0 ± 0.2,14.4 ± 0.2,22.6 ± 0.2.

Form type III crystallization as a preferred method, being indicated, alpha-emitting using Cu-K with 2 θ (°) X-ray powder diffraction (XRD) spectrum about in 15.8 ± 0.2,18.4 ± 0.2,23.2 ± 0.2 one at, have at two or at three Characteristic peak.

Form type III crystallization as a preferred method, being indicated, alpha-emitting using Cu-K with 2 θ (°) X-ray powder diffraction (XRD) spectrum about in 16.2 ± 0.2,26.5 ± 0.2,28.2 ± 0.2 one at or two at or three at have Characteristic peak.

Form type III crystallization as a preferred method, being indicated, alpha-emitting using Cu-K with 2 θ (°) X-ray powder diffraction (XRD) spectrum about 11.0 ± 0.2,14.4 ± 0.2,22.6 ± 0.2,15.8 ± 0.2,18.4 ± 0.2, There is characteristic peak at 16.2 ± 0.2,23.2 ± 0.2,26.5 ± 0.2,28.2 ± 0.2.

Form type III crystallization as a preferred method, being indicated, alpha-emitting using Cu-K with 2 θ (°) X-ray powder diffraction (XRD) spectrum has X-ray powder diffraction figure as shown in Figure 7.

Form type III crystallization as a preferred method, DSC endothermic peak change at about 116-135 DEG C.

Another aspect of the present invention also provides the preparation method of the tartaric acid addition salt crystal form.

As a preferred embodiment, the preparation method of the Form I crystal of the tartaric acid addition salt, including will be described Tartaric acid addition salt add in a certain amount of suitable solvent, agitated centrifugation obtains solid.

Preferably, the solvent is selected from alcohols, nitrile, ketone, esters, ethers, aromatics, halogenated hydrocarbon or alkane Class solvent.

In some embodiments, the alcohols solvent is methanol, ethyl alcohol and propyl alcohol；The nitrile solvents are acetonitrile；It is described Ketones solvent is acetone and methyl iso-butyl ketone (MIBK)；The esters are ethyl acetate and isopropyl acetate；The ethers is methyl- tert Butyl ether, tetrahydrofuran and Isosorbide-5-Nitrae-dioxane；The aromatics are toluene；The halogenated hydrocarbon is methylene chloride and chloroform； The alkanes are normal heptane.

Preferably, the solvent is the mixing of the one or more arbitrary proportion of above-mentioned solvent.

Preferably, the reaction temperature is -20~60 DEG C.

In some embodiments, the temperature is preferably 25 DEG C.

As a preferred embodiment, the preparation method of the Form II crystal form of the tartaric acid addition salt, including following Method:

Method one: the tartaric acid addition salt is dissolved in a certain amount of suitable solvent, and filtering is placed on certain temperature The lower volatilization of degree obtains solid.

Preferably, the suitable solvent is preferably methanol；The temperature is preferably 50 DEG C.

Method two: the tartaric acid addition salt is stirred in suitable solvent under the high temperature conditions, is consolidated after centrifugation Body.

Preferably, the temperature is 80 DEG C；The solvent is selected from the mixed of acetylacetone,2,4-pentanedione, benzonitrile or its arbitrary proportion It closes.

Another aspect of the present invention provides a kind of pharmaceutical composition, and the tartaric acid containing therapeutically effective amount adds At salt.

The pharmaceutical composition is injection as a preferred method,.

The content of the pharmaceutical composition mesotartaric acid addition salts is 0.001%-0.1% as a preferred method, It is preferred that 0.01%-0.05%.

The pharmaceutical composition also includes pharmaceutical excipient as a preferred method, and the excipient is ring paste Essence or derivatives thereof, the cyclodextrin and its derivative are selected from hydroxypropyl-β-cyclodextrin, dihydroxy group-beta-cyclodextrin, hydroxyl second Group-beta-cyclodextrin, 2-HP-BETA-CD, 3- hydroxypropyl-β-cyclodextrin, 2,3- dihydroxypropyl-beta-cyclodextrin, 2,3,6- Three hydroxypropyl-β-cyclodextrins or Sulfobutyl ether β _ cyclodextrin sodium.Preferably Sulfobutyl ether β _ cyclodextrin sodium.

When the pharmaceutical composition is configured to injection as a preferred method, the auxiliary material further include water and Additives, the additives are not limited only to pH adjusting agent, antibacterial agent, antioxidant, cosolvent, osmotic pressure regulator, emulsification Agent, stabilizer, suspending agent one or more.The pH adjusting agent be sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, One or more of sodium bicarbonate, saleratus, preferably sodium hydroxide.

Another aspect of the present invention is there is provided a kind of product, comprising by the tartaric acid addition salt and medical additive It is prepared into aqueous solution.

There is provided a kind of using the composition or the product for another aspect of the present invention, is preventing or is controlling Treat the purposes in the drug of the disease or illness that are mediated by cell cycle protein dependent kinase.

The composition or the product as a preferred method, the purposes in prevention or treating cancer.

As a preferred method, the cancer be selected from bladder cancer, breast cancer, colon cancer, kidney, epidermal carcinoma, liver cancer, Lung cancer, cancer of the esophagus, gallbladder cancer, oophoroma, cancer of pancreas, gastric cancer, cervical carcinoma, thyroid cancer, prostate cancer or cutaneum carcinoma；Lymph spectrum It is hematopoetic tumor；Myeloid lineage hematopoetic tumor；Thyroid follcular carcinoma；Mesenchymal derivation tumour；Central or peripheral nervous system tumour； Melanoma；Seminoma；Teratocarcinoma；Osteosarcoma；Xeroderma pitmentosum；Keratoacanthoma；Thyroid follcular carcinoma；Or card wave Western sarcoma.

The cancer is selected from cancer and is selected from leukaemia, acute lymphatic leukemia, B- as a preferred method, Cell lymphoma, T- cell lymphoma, He Jiejin lymphomas, non_hodgkin lymphoma, hairy cell lymphoma, Burkett Lymphomas, acute and chronic myelogenous leukemia, myelodysplastic syndrome or promyelocytic leukemia.

As a preferred method, the cancer be selected from breast cancer, oophoroma, colon cancer, prostate cancer, cancer of the esophagus, Squamous cell carcinoma and non-small cell lung cancer.

Specific embodiment

The present inventor is surprised to find that for the first time by extensive and in-depth research.The present invention is completed on this basis.

Detailed description of the invention

Fig. 1 is the X- powder diffraction spectrum of tartaric acid addition salt Form I.

Fig. 2 is the DSC scanning figure of tartaric acid addition salt Form I.

Fig. 3 is the TGA figure of tartaric acid addition salt Form I.

Fig. 4 is the X- powder diffraction spectrum of tartaric acid addition salt Form II.

Fig. 5 is the DSC scanning figure of tartaric acid addition salt Form II.

Fig. 6 is the TGA figure of tartaric acid addition salt Form II.

Fig. 7 is the X- powder diffraction spectrum of tartaric acid addition salt Form III crystal form.

Fig. 8 is the DSC scanning figure of tartaric acid addition salt Form III crystal form.

Fig. 9 is the TGA figure of tartaric acid addition salt Form III crystal form.

Figure 10 is the DVS figure of tartaric acid addition salt Form I crystal.

Figure 11 is the DVS figure of tartaric acid addition salt Form II crystal form.

Figure 12 is the DVS figure of tartaric acid addition salt Form III crystal form.

Figure 13 is the DVS figure of mesylate.

Figure 14 is the stability diagram of tartaric acid addition salt Form I crystal.

Figure 15 is the stability diagram of tartaric acid addition salt Form II crystal form.

Figure 16 is the stability diagram of tartaric acid addition salt Form III crystal form.

Figure 17 is the stability diagram of mesylate.

Figure 18 is the PSD figure of tartaric acid addition salt Form I crystal.

Figure 19 is the PSD figure of tartaric acid addition salt Form II crystal form.

Figure 20 is the PSD figure of tartaric acid addition salt Form III crystal form.

Figure 21 is the PSD figure of mesylate.

Figure 22 is MCF-7 cell proliferation experiment figure.

Main advantages of the present invention are:

The crystal form of tartaric acid addition salt of the present invention is compared with other salt or free alkali, comprising:

Better dissolubility, this enables it to preferably apply in passages through which vital energy circulates；

Better stability, convenient for storage；

Preferably draw moist；

Better particle diameter distribution, is more suitable the needs of large-scale production, facilitates the post-processing work for simplifying production process Skill proposes high quality control；

Physically better chemical property；

The bioactivity that anticancer can be improved has better therapeutic index.

It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Present invention will be further explained below with reference to specific examples.The experiment of actual conditions is not specified in the following example Method, usually according to normal condition, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and part Number is calculated by weight.

Acquire instrument and method used in data:

X-ray powder diffraction figure of the present invention is adopted on Panalytical Empyrean x-ray powder diffraction instrument Collection.The method parameter of X-ray powder diffraction of the present invention is as follows:

X ray reflection parameter: Cu, K α

1.540598；1.544426

1 intensity of K α 2/K α: 0.50

Voltage: 45 volt (kV)

Electric current: 40 milliamperes (mA)

Scanning range: from 3.0 to 40.0 degree

Differential thermal analysis (DSC) data are picked up from TA Instruments Q2000MDSC, and instrument control software is Thermal Advantage, analysis software is Universal Analysis.1~10 milligram of sample is usually taken to be placed in capping (unless special Do not mentionlet alone bright) aluminium crucible in, sample is risen to 300 DEG C from room temperature under the protection of dry N2 with the heating rate of 10 DEG C/min, Thermal change of the TA software records sample in temperature-rise period simultaneously.In this application, fusing point is reported by initial temperature.

Thermogravimetric analysis (TGA) data are picked up from TA Instruments Q500TGA, and instrument control software is Thermal AdVantage, analysis software is Universal Analysis.It usually takes the sample of 5~15mg to be placed in platinum crucible, adopts With the mode of segmentation high resolution detection, with the heating rate of 10 DEG C/min under the protection of the dry N2 of 50mL/min by sample from room Temperature rise is to 250 DEG C or 300 DEG C, while weight change of the TA software records sample in temperature-rise period.Crystal form of the present invention it is aqueous Amount is to be speculated to calculate according to TGA weightlessness, as it is known by the man skilled in the art, TGA weightlessness is the reference of crystal form water content, but not Crystal form contained humidity subnumber can absolutely be represented.

Differential scanning calorimetric analysis (DSC) figure of the present invention acquires on TA Q2000.Differential of the present invention The method parameter for scanning thermometric analysis (DSC) is as follows:

Sweep speed: 10 DEG C/min

Protective gas: nitrogen

Thermogravimetric analysis (TGA) figure of the present invention acquires on TA Q500.Thermogravimetric analysis (TGA) of the present invention Method parameter it is as follows:

Sweep speed: 10 DEG C/min

Protective gas: nitrogen

Other instrument parameters used, unless specifically indicated, following embodiment operate at room temperature.

The system of 1 4- of embodiment (2,6- dichIoro-benzoylamino) -1H- pyrazoles -3- carboxylic acid piperidin -4- base acyl tartaric acid It is standby

A. synthetic intermediate 1

Raw material 6.31kg, n,N-Dimethylformamide (45L), carbodiimides are added into 100L reaction kettle (6.59kg), I-hydroxybenzotriazole (4.65kg) are stirred to react 0.5h, and ice-water bath is cooled to 0~10 DEG C, and starting is added portionwise Raw material 1 (4.5kg), then heats to 20-25 DEG C, is stirred to react 16h, and reaction solution is added in 270L water, and solid is precipitated, and takes out Filter, washing, liquid separation extraction merge organic phase and drying.

B. synthetic intermediate 2:

Intermediate 1 (2.5kg) is added into 100L reaction kettle, tetrahydrofuran (44kg), stirring, being warming up to 45~50 DEG C makes Dissolved clarification is added 250g 10%Pd/C catalyst, is warming up to 45-50 DEG C of reaction 16h；Filtering, drains, two is added into residue Chloromethanes (60.65kg) stirs 10-15min, and filter cake is washed in filtering, and filtrate decompression concentration is dry, obtains the crude product of intermediate 2.

C. synthetic intermediate 3:

Compound intermediate 2 (2.05kg) is added into reaction kettle, methylene chloride (15.41kg) and triethylamine (0.81kg) is cooled to 0~5 DEG C, and (1.46kg is dissolved in 0.5kg dichloro to the dichloromethane solution of instillation 2,6- dichlorobenzoyl chloride Methane), drop finishes, and reaction 3h is stirred at room temperature；To reaction solution be added 54.53kg methylene chloride, successively with 5% HCl (12.3kg × 2), saturated sodium bicarbonate aqueous solution (18.16kg × 1), saturated salt solution (19.82kg × 1) washing, organic phase anhydrous slufuric acid Sodium (1kg) dries, filters, and filtrate decompression concentration is dry；Dehydrated alcohol (4.85kg) and normal heptane are added into residue 1h is stirred at room temperature in (20.91kg), and filtering, filter cake is eluted with normal heptane (1kg), and filtration cakes torrefaction obtains intermediate 3.

D. synthetic intermediate 4:

42.11kg methylene chloride, 2.8kg intermediate 3 are added into 100L reaction kettle, trifluoro is added dropwise to reaction solution in stirring Acetic acid (6.86kg), drop finish, insulated and stirred 3-5h；Reaction solution is concentrated under reduced pressure and is done, isopropanol (11kg) is added to residue, mistake Filter, filter cake are eluted with isopropanol (1kg), and by filtration cakes torrefaction, dehydrated alcohol (6.5kg) is added in crude product, reduced after rising temperature for dissolving For temperature to 40~45 DEG C, about half solvent is evaporated off in reduced pressure, is cooled to 20~25 DEG C, is filtered, filter cake dehydrated alcohol (0.5kg) elution, is dried to obtain 2.55kg intermediate 4.

E. tartrate is synthesized:

(1) 43.89kg methylene chloride, 26.14kg dehydrated alcohol and intermediate 4 (1.65kg) are added into reaction kettle, stirs 0.08kg active carbon is added after mixing dissolution, is warming up to 35-40 DEG C and continues stirring 15 minutes, be then cooled to 20-25 DEG C；Filtering, Filtrate decompression concentration, 13.04kg methanol is added into residue, is dispersed with stirring stand-by；

(2) 0.78kg L- (+) tartaric acid is dissolved in 2.61kg methanol, 0.04kg active carbon is added, is warming up to 35-40 DEG C Continue stirring 15 minutes, is then cooled to 20-25 DEG C；Filtering, filtrate are stand-by；

(3) reaction solution in step (1) is warming up to 50-55 DEG C, instills L- (+) tartaric acid methanol solution in step 2, Drop finishes, and is maintained at 50-55 DEG C and stirs 4.5 hours；Reaction solution is cooled to 20-25 DEG C, is stirred overnight, is filtered, filter cake is used The elution of 0.53kg anhydrous methanol, is dried in vacuum overnight to get tartrate, yield 81.3%.

¹H-NMR(DMSO-D₆,400MHz)δ10.15(s,1H),8.63-8.61(d,1H),8.35(s,1H),7.60- 7.50(m,3H),4.00(m,1H),3.30-3.27(d,2H),2.95-2.89(m,2H),4.88-4.99(m,2H),2.58(br S, 1H), 2.09 (m, 1H), 1.61 (d, J=8.0,3H), 1.91-1.76 (m, 4H).

The preparation of 2 tartaric acid addition salt Form I crystal of embodiment

The tartaric acid addition salt of 601.7mg is weighed, 10mL methylene chloride is added, stirs, is then centrifuged at 25 DEG C Solid is collected, it is dry, obtain solid.The crystal form is characterized with above-mentioned X-ray image method.

Through detecting, the crystal form 2 θ values be 17.7 ° ± 0.2 °, 19.1 ° ± 0.2 °, 26.2 ° ± 0.2 °, 13.3 ° ± 0.2 °, There is characteristic peak at 12.7 ° ± 0.2 °, 16.4 ° ± 0.2 °, 21.6 ° ± 0.2 °, 20.4 ° ± 0.2 °, 24.5 ° ± 0.2 °.Its X is penetrated Line powder diffraction data such as Fig. 1, shown in table 1.

Table 1

Then, the Form I of the analysis-e/or determining tartaric acid addition salt is keen to using TA Instruments Q2000MDSC Crystal form nearby starts first endothermic peak occur at 81 DEG C, nearby starts second endothermic peak occur at 194 DEG C, specific to participate in Fig. 2.

The TGA of the crystal form has about 1.9% mass loss gradient as shown in figure 3, when being heated to 150 DEG C.

The preparation of 3 tartaric acid addition salt Form I crystal of embodiment

Solid is obtained using the condition in such as the following table 2 using method same as Example 2, is measured.

Table 2

Serial number	Material quality (mg)	Solvent	Volume (mL)	T(℃)	Gained crystal form
						1	10.6	Methanol	0.5	4	Form I
2	11	Ethyl alcohol	0.5	50	Form I
						3	10.5	Isopropanol	0.5	25	Form I
4	11.1	Acetonitrile	0.5	25	Form I
						5	10.8	Acetone	0.5	25	Form I
6	11.5	Methyl iso-butyl ketone (MIBK)	0.5	25	Form I
						7	11.4	Ethyl acetate	0.5	25	Form I
8	10.0	Isopropyl acetate	0.5	25	Form I
						9	10.2	Methyl tertiary butyl ether(MTBE)	0.5	25	Form I
10	10.7	Tetrahydrofuran	0.5	25	Form I
						11	10.9	2- methyltetrahydrofuran	0.5	25	Form I
12	10.2	Isosorbide-5-Nitrae-dioxane	0.5	25	Form I
						13	10.2	Methylene chloride	0.5	25	Form I
14	11.0	Chloroform	0.5	25	Form I
						15	10.3	Methanol/toluene 2:1	0.5	25	Form I
16	10.2	Toluene/n-heptane 1:1	0.5	25	Form I

By X- powder diffraction, DSC, TGA measurement, are Form I crystal.

The preparation of 4 tartaric acid addition salt Form II crystal form of embodiment

The tartaric acid addition salt solid for weighing about 200mg, is dissolved in 80mL methanol, fast under the conditions of 50 DEG C after filtering Speed volatilization, obtains solid.

Detected through XRPD, the sample 2 θ values be 7.0 ° ± 0.2 °, 10.5 ° ± 0.2 °, 21.8 ° ± 0.2 °, 13.0 ° ± There is characteristic peak at 0.2 °, 17.4 ° ± 0.2 °, 14.0 ° ± 0.2 °, 23.5 ° ± 0.2 °, 18.6 ° ± 0.2 °, 24.8 ° ± 0.2 °. Its X-ray powder diffraction data such as Fig. 4, shown in table 3.

Table 3

Then, the crystal form of the analysis-e/or determining tartaric acid addition salt is keen to using TA Instruments Q2000MDSC II nearby starts first endothermic peak occur at 56 DEG C, nearby starts second endothermic peak occur at 107 DEG C, near 162 DEG C Start third endothermic peak occur, referring specifically to Fig. 5.

The TGA of the crystal form has about 3.1% mass loss gradient as shown in fig. 6, when being heated to 77 DEG C.Continue When being heated to 150 DEG C, with about 3.0% mass loss gradient.

The preparation of 5 tartaric acid addition salt Form II crystal form of embodiment

Solid is obtained using the condition in such as the following table 4 using method same as Example 2, is measured.

Table 4

Serial number	Material quality (mg)	Solvent	Volume (mL)	Gained crystal form
					1	10.9	Acetylacetone,2,4-pentanedione	0.3	Form II
2	10.5	Benzonitrile	0.3	Form II

By X- powder diffraction, DSC, TGA measurement, are Form II crystal form.

The preparation of 6 tartaric acid addition salt Form III crystal form of embodiment

A certain amount of tartaric acid addition salt solid is weighed, is placed in solvent described in certain scale 5 and stirs, is collected by centrifugation solid Body, it is dry.It is detected, is tartaric acid addition salt crystal form Form III.

Table 5

Serial number	Material quality (mg)	Solvent	Volume (mL)	Gained crystal form
					1	582.7	Water	20	Form III
2	10.5	Tetrahydrofuran/water 9:1	0.5	Form III

Detected through XRPD, θ value be 11.0 ° ± 0.2 °, 14.4 ° ± 0.2 °, 22.6 ° ± 0.2 °, 15.8 ° ± 0.2 °, There is characteristic peak at 18.4 ° ± 0.2 °, 16.2 ° ± 0.2 °, 23.2 ° ± 0.2 °, 26.5 ° ± 0.2 °, 28.2 ° ± 0.2 °.Its X is penetrated Line powder diffraction data such as Fig. 7, shown in table 6.

Table 6

Then, the crystal form of the analysis-e/or determining tartaric acid addition salt is keen to using TA Instruments Q2000MDSC Form III nearby starts first endothermic peak occur at 60 DEG C, second endothermic peak nearby occurs at 116 DEG C, the crystal form DSC is as shown in Figure 8.

The TGA of the crystal form has about 5.2% mass loss gradient as shown in figure 9, when being heated to 84 DEG C.Continue When being heated to 131 DEG C, with about 2.0% mass loss gradient.

7 tartaric acid addition salt Form I crystal of embodiment draws moist measurement

Tartaric acid addition salt Form I crystal of the present invention about 10mg is taken to test it using dynamic water absorption (DVS) instrument Draw moist.Experimental result is as shown in table 7.The DVS figure of the hygroscopicity test of Form I crystal is as shown in Figure 10.

Table 7

The result shows that tartaric acid addition salt crystal form Form I of the invention increases weight 0.45% after balancing under 80% humidity, According to the defining standard for drawing moist weight gain, belong to slightly draw it is moist.

The hygroscopicity test of 8 tartaric acid addition salt Form II crystal form of embodiment:

Tartaric acid addition salt Form II crystal form of the present invention about 10mg is taken to survey using dynamic water absorption (DVS) instrument Try its draw it is moist.Experimental result is as shown in table 8.The DVS figure of the hygroscopicity test of Form II crystal form is as shown in figure 11.

Table 8

The result shows that tartaric acid addition salt Form II crystal form of the invention increases weight 0.35% after balancing under 80% humidity, According to the defining standard for drawing moist weight gain, belong to slightly draw it is moist.

The hygroscopicity test of 9 tartaric acid addition salt Form III crystal form of embodiment:

Tartaric acid addition salt Form III crystal form of the present invention about 10mg is taken to survey using dynamic water absorption (DVS) instrument Try its draw it is moist.Experimental result is as shown in table 9.The DVS figure of the hygroscopicity test of Form III crystal form is as shown in figure 12.

Table 9

The result shows that tartaric acid addition salt Form III crystal form of the invention increases weight after balancing under 80% humidity 0.29%, according to the defining standard for drawing moist weight gain, belong to slightly draw it is moist.

The hygroscopicity test of 10 mesylate of embodiment:

Using the preparation of method disclosed in the Chinese patent CN101146791 mesylate, consolidate through what detection obtained Body is consistent with the data in the patent.

Take above-mentioned mesylate about 10mg using dynamic water absorption (DVS) instrument test its draw it is moist.Experimental result such as table Shown in 10.The DVS figure of the hygroscopicity test of the mesylate is as shown in figure 13.

Table 10

The mesylate increases weight 0.62% after balancing under 80% humidity, according to the defining standard for drawing moist weight gain, belong to It is moist in slightly drawing.The result shows that the mesylate draw it is moist than tartaric acid addition salt crystal form Form I of the invention, Crystal form Form II, crystal form Form III are slightly higher.

Define that (Chinese Pharmacopoeia 9103 drug of version general rule in 2015 draws wet with draw moist weight gain about the description of moist feature is drawn Property test direction principle, experiment condition: 25 DEG C ± 1 DEG C, 80% relative humidity): deliquesce: absorbing enough moisture and form liquid

It is great draw it is moist: draw wet weight gain not less than 15%

Have draw it is moist: draw wet weight gain less than 15% but not less than 2%

Slightly draw moist: drawing wet weight gain less than 2% but not less than 0.2%

Nothing is moist almost without drawing: drawing wet weight gain less than 0.2%

The stability test of 11 tartaric acid addition salt crystal form Form I of embodiment:

Three parts of tartaric acid addition salt crystal form Form I samples are taken to be respectively placed in 25 DEG C/60%RH and 40 DEG C/75%RH constant temperature It is placed 1 day under the conditions of open placement 1 week and 80 DEG C in constant humidity cabinet, purity and XRPD are surveyed in sampling.XRPD comparing result such as Figure 14 (it is followed successively by the XRPD figure of the tartaric acid addition salt Form I crystal initial sample from top to bottom, places 25 DEG C/60%RH and 40 DEG C/75%RH under the conditions of 1 week and place 80 DEG C under the conditions of 1 day XRPD figure), result is as shown in table 11.

Table 11

Originate crystal form	Placement condition	Purity %	Crystal form variation
				Crystal form Form I	25 DEG C/60%RH	99.75	It is constant
Crystal form Form I	40 DEG C/75%RH	99.71	It is constant
				Crystal form Form I	80℃	99.34	It is constant

Tartaric acid addition salt crystal form Form I is placed 1 week and 80 DEG C at 25 DEG C/60%RH and 40 DEG C/75%RH and is transferred It sets 1 day, crystal form remains unchanged, and purity has no significant changes.The result shows that the Form I crystal of tartaric acid addition salt is with good Good stability.

The stability test of 12 tartaric acid addition salt crystal form Form II of embodiment:

Three parts of tartaric acid addition salt crystal form Form II samples are taken to be respectively placed in 25 DEG C/60%RH and 40 DEG C/75%RH constant temperature It is placed 1 day under the conditions of open placement 1 week and 80 DEG C in constant humidity cabinet, purity and XRPD are surveyed in sampling.XRPD comparing result such as Figure 15 (it is followed successively by the XRPD figure of the tartaric acid addition salt crystal form FormII initial sample from top to bottom, places 25 DEG C/60%RH and 40 DEG C/75%RH under the conditions of 1 week and place 80 DEG C under the conditions of 1 day XRPD figure), result is as shown in table 12.

Table 12

Originate crystal form	Placement condition	Purity %	Crystal form variation
				Crystal form Form II	25 DEG C/60%RH	99.77	It is constant
Crystal form Form II	40 DEG C/75%RH	99.72	It is constant
				Crystal form Form II	80℃	99.30	It is constant

At tartaric acid addition salt Form II crystal form is placed 1 week and 80 DEG C at 25 DEG C/60%RH and 40 DEG C/75%RH It places 1 day, crystal form remains unchanged, and purity has no significant changes.The result shows that tartaric acid addition salt Form II crystal form has Good stability.

The stability test of 13 tartaric acid addition salt crystal form Form III of embodiment:

Three parts of tartaric acid addition salt crystal form Form III samples are taken to be respectively placed in 25 DEG C/60%RH and 40 DEG C/75%RH perseverance It is placed 1 day under the conditions of open placement 1 week and 80 DEG C in constant temperature and humidity case, purity and XRPD are surveyed in sampling.XRPD comparing result is as schemed 16 (are followed successively by the XRPD figure of tartaric acid addition salt crystal form Form III initial sample from top to bottom, place 25 DEG C/60%RH and 40 DEG C/75%RH under the conditions of 1 week and place 80 DEG C under the conditions of 1 day XRPD figure), result is as shown in table 13.

Table 13

At tartaric acid addition salt crystal form Form III is placed 1 week and 80 DEG C at 25 DEG C/60%RH and 40 DEG C/75%RH It places 1 day, crystal form remains unchanged, and purity has no significant changes.The result shows that tartaric acid addition salt Form III crystal form has Good stability.

The stability test of 14 mesylate of embodiment:

The mesylate sample for taking three parts of methods according to disclosed in Chinese patent CN101146791 to prepare is respectively placed in 25 DEG C/60%RH and 40 DEG C/75%RH climatic chamber in it is open place 1 week and 80 DEG C under the conditions of places 1 day, sample survey purity And XRPD.XRPD comparing result such as Figure 17 (XRPD for being followed successively by mesylate initial sample from top to bottom schemes, and 25 DEG C of placement/ Under the conditions of 60%RH and 40 DEG C/75%RH 1 week and place 80 DEG C under the conditions of 1 day XRPD figure), result is as shown in table 14.

Table 14

Originate crystal form	Placement condition	Purity %	Crystal form variation
				Mesylate	25 DEG C/60%RH	99.93	It is constant
Mesylate	40 DEG C/75%RH	99.92	It is constant
				Mesylate	80℃	99.97	It is constant

Mesylate is placed 1 day at placing 1 week and 80 DEG C at 25 DEG C/60%RH and 40 DEG C/75%RH, and crystal form is kept It is constant, and purity has no significant changes.The result shows that the stability of mesylate and tartaric acid addition salt Form I of the invention Crystal form, Form II crystal form, Form III crystal form are suitable.

15 particle diameter distribution of embodiment and morphological research test:

Take tartaric acid addition salt crystal form Form I, crystal form Form II, crystal form Form III and the methanesulfonic acid of the invention Salt tests particle diameter distribution, as a result as shown in Table 15.

Table 15

Crystal form	MV(μm)	SD	D10(μm)	D50(μm)	D90μm)
						Form I crystal	12.28	11.34	0.842	6389	30.92
Form II crystal form	66.64	46.02	13.64	51.89	140.30
						Form III crystal form	348.90	236.0	70.65	317.2	655.10
Mesylate	386.7	407.3	6.80	240.3	821.4

MV: the average grain diameter calculated according to volume

D10: indicate particle diameter distribution in (volume distribution) account for 10% corresponding to partial size

D50: indicate particle diameter distribution in (volume distribution) account for 50% corresponding to partial size, also known as meso-position radius

D90: indicate particle diameter distribution in (volume distribution) account for 90% corresponding to partial size

The results showed that

As shown in figure 18, crystal form Form I volume average particle size is 12.28 microns to the PSD figure of crystal form Form I.

As shown in figure 19, crystal form Form II volume average particle size is 66.64 microns to the PSD figure of crystal form Form II.And grain Diameter narrow distribution, is almost presented a normal distribution, and particle diameter distribution is uniform.

As shown in figure 20, crystal form Form III volume average particle size is 348.90 microns to the PSD figure of crystal form Form III.And Particle diameter distribution is relatively narrow, and a normal distribution is almost presented, and particle diameter distribution is uniform.

As shown in figure 21, mesylate volume average particle size is 386.7 microns to the PSD figure of mesylate.Partial size it is larger and Particle diameter distribution is wider, and particle diameter distribution is uneven.

16 solubility test of embodiment research:

Tartaric acid addition salt crystal form Form I, crystal form Form II, crystal form Form III sample are used respectively PH6.5FaSSIF (simulated intestinal fluid under fasting state) and pure water are configured to saturated solution, in 1 hour, 4 hours and 24 small When after by high performance liquid chromatography (HPLC) method measure saturated solution in sample content.Tartaric acid addition salt of the present invention Crystal form Form I, crystal form Form II, the dissolubility data of crystal form Form III are as shown in table 16.

Table 16

The result shows that tartaric acid addition salt crystal form Form I, crystal form Form II, crystal form Form III are shown preferably Stability.

17 pharmaceutical preparation of embodiment

The parenteral compositions preparation of drug administration by injection is as follows: sulphur butyl betadex sodium is dissolved in water for injection, Then the tartrate (such as crystal form I) is added, the concentration for obtaining tartrate is 0.8-1.5 weight %.It then will be molten Liquid filtration sterilization, filling in ampoule, sealing.

18 active testing of embodiment

This experiment determines tartaric acid addition salt of the present invention to MCF-7 cell strainHJ2mm with the following method Proliferation inhibition activity.

Measuring method: first by MCF-7 cell (being purchased from ATCC) according to 1*10⁴The hole cells/ carries out 96 orifice plate bed boards, training It supports overnight (37 DEG C, 7%CO2).

4- (2,6- dichIoro-benzoylamino) -1H- pyrazoles -3- carboxylic acid piperidin -4- base amide of different salt form is diluted, Initial concentration is 100 μM, 3 times of concentration gradient dilutions.

96 orifice plates are taken out, supernatant is removed, the 4- (2,6- dichIoro-benzoylamino)-of various concentration gradient dilution is added The different salt form of 1H- pyrazoles -3- carboxylic acid piperidin -4- base amide, incubator culture 72h (37 DEG C, 7%CO2).96 orifice plates are taken out, CCK-8 color developing agent, 37 DEG C of incubation 30min is added, microplate reader read plate measures the OD value at 450nm.Data are as shown in table 17:

Table 17

Salt form	Tartaric acid addition salt	Mesylate	Hydrochloride
				EC50(nM)	446.6	~1086	898.2

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Claims (21)

1. The tartaric acid addition salt of 1.4- (2,6- dichIoro-benzoylamino) -1H- pyrazoles -3- carboxylic acid piperidin -4- base amide.
2. 2. tartaric acid addition salt as described in claim 1, which is characterized in that the tartaric acid addition salt is crystalloid.
3. 3. tartaric acid addition salt as claimed in claim 2, which is characterized in that the crystalloid tartaric acid addition salt is Form The crystallization of I type.
4. 4. tartaric acid addition salt as claimed in claim 3, which is characterized in that it includes use Cu-K α radiation, indicated with 2 θ degree X-ray powder diffraction spectrum have peak 17.7 ± 0.2,19.1 ± 0.2 and 26.2 ± 0.2.
5. 5. tartaric acid addition salt as claimed in claim 4, which is characterized in that its DSC endothermic peak changes at about 194-200 DEG C.
6. 6. tartaric acid addition salt as claimed in claim 2, which is characterized in that the crystalloid tartaric acid addition salt is Form The crystallization of II type.
7. 7. tartaric acid addition salt as claimed in claim 6, which is characterized in that it includes use Cu-K α radiation, indicated with 2 θ degree X-ray powder diffraction spectrum have peak 7.0 ± 0.2,10.5 ± 0.2 and 21.8 ± 0.2.
8. 8. tartaric acid addition salt as claimed in claim 7, which is characterized in that its DSC endothermic peak changes at about 107-127 DEG C.
9. 9. tartaric acid addition salt as claimed in claim 2, which is characterized in that the crystalloid tartaric acid addition salt is Form Type III crystallization.
10. 10. tartaric acid addition salt as claimed in claim 9, which is characterized in that it includes use Cu-K α to radiate, with 2 θ degree tables The X-ray powder diffraction spectrum shown has peak 11.0 ± 0.2,14.4 ± 0.2 and 22.6 ± 0.2.
11. 11. tartaric acid addition salt as claimed in claim 10, which is characterized in that its DSC endothermic peak changes in about 116-135 ℃。
12. 12. a kind of 4- (2,6- dichIoro-benzoylamino) -1H- pyrazoles -3- carboxylic acid piperidin -4- base amide tartaric acid for preparing adds At the method for salt Form I crystal, the preparation method includes adding to the tartaric acid addition salt in a certain amount of solvent, Agitated centrifugation obtains solid；Wherein, the solvent be alcohols, nitrile, ketone, esters, ethers, aromatics, halogenated hydrocarbon, or Person's alkane solvents；Preferably, the alcohols solvent is methanol, ethyl alcohol and propyl alcohol；The nitrile solvents are acetonitrile；The ketone Solvent is acetone and methyl iso-butyl ketone (MIBK)；The esters are ethyl acetate and isopropyl acetate；The ethers is methyl tertbutyl Ether, tetrahydrofuran and Isosorbide-5-Nitrae-dioxane；The aromatics are toluene；The halogenated hydrocarbon is methylene chloride and chloroform；It is described Alkanes are normal heptane；The heating temperature is -20~60 DEG C, preferably 25 DEG C.
13. 13. a kind of 4- (2,6- dichIoro-benzoylamino) -1H- pyrazoles -3- carboxylic acid piperidin -4- base amide tartaric acid for preparing adds At the method for salt Form II crystal form, the preparation method includes following method:

    Method one: the tartaric acid addition salt is dissolved in a certain amount of solvent (preferably methanol), and filtering is placed on certain temperature (preferably 50 DEG C) volatilization obtains solid under degree；

    Method two: by the tartaric acid addition salt, (preferably 80 DEG C) stir in acetylacetone,2,4-pentanedione or benzonitrile solvent under hot conditions It mixes, solid is obtained after centrifugation.
14. 14. a kind of 4- (2,6- dichIoro-benzoylamino) -1H- pyrazoles -3- carboxylic acid piperidin -4- base amide tartaric acid for preparing adds It is placed in a certain amount of solvent at the method for salt Form III, including by the tartaric acid addition salt, it is agitated to obtain solid；Its In, the solvent is the mixed solvent system (preferably tetrahydrofuran/aqueous systems) that water or ethers and water are formed.
15. 15. a kind of composition, it is characterised in that tartaric acid addition salt described in the claim 1-11 containing therapeutically effective amount.
16. 16. composition as claimed in claim 15, it is characterised in that the composition is injection.
17. 17. composition as claimed in claim 16, it is characterised in that the content of the tartaric acid addition salt is 0.5-2.0 weight Amount/mL, preferably 0.8-1.5 weight/mL.
18. 18. composition as claimed in claim 17, it is characterised in that also include pharmaceutical excipient, the excipient is ring paste Essence or derivatives thereof；Preferably Sulfobutyl ether β _ cyclodextrin sodium.
19. 19. a kind of product is prepared into aqueous solution comprising tartaric acid addition salt described in claim 1-11 and medical additive.
20. 20. composition described in claim 1-18, product described in claim 19, in preparation for preventing or treating by thin Purposes in the drug of disease or illness that born of the same parents' cyclin-dependent kinase mediates.
21. 21. purposes described in claim 20, it is characterized in that preparation for prevent or treating cancer drug in purposes.