CN111704612A - P crystal form of pyridine derivative, and preparation method and application thereof - Google Patents

P crystal form of pyridine derivative, and preparation method and application thereof Download PDF

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CN111704612A
CN111704612A CN202010741120.2A CN202010741120A CN111704612A CN 111704612 A CN111704612 A CN 111704612A CN 202010741120 A CN202010741120 A CN 202010741120A CN 111704612 A CN111704612 A CN 111704612A
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compound
formula
crystal form
preparation
crystal
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王颖
向永哲
李建国
周宁
付海霞
乔惠
张济兵
黄龙
刘建
岑国栋
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Zhejiang Yatai Pharmaceutical Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

Abstract

The invention discloses a P crystal form of a pyridine derivative, a preparation method and application thereof, discloses the invention, relates to the field of pharmaceutical chemical synthesis, and particularly relates to the P crystal form of a compound 1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxo-tetrahydropyrrole-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide compound, a preparation method thereof, and application thereof as a therapeutic medicament, particularly as a medicament for preventing or treating thrombosis or embolism.

Description

P crystal form of pyridine derivative, and preparation method and application thereof
Technical Field
The invention relates to the field of pharmaceutical chemistry synthesis, in particular to a polymorphism of a compound 1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxo-tetrahydropyrrole-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide compound, a preparation method thereof and application thereof as a therapeutic drug, especially as a drug for preventing or treating thrombosis or embolism.
Background
Thrombotic diseases are diseases that seriously harm human health, and are mainly classified into arterial thrombosis and venous thrombosis according to the part, condition and nature of thrombosis. Arterial thrombosis is initiated by atherosclerotic lesions and platelet activation of arterial vessel wall, and the severe clinical reactions caused by the arterial thrombosis are mainly acute myocardial infarction and cerebral apoplexy; venous thrombosis is induced by a variety of causes in the venous vasculature, which can lead to Venous Thromboembolism (VTE), which is clinically manifested primarily as Deep Venous Thrombosis (DVT) and Pulmonary Embolism (PE). VTE is the third major cardiovascular disease following acute coronary syndrome and stroke. In hospital, the VTE accounts for about 10%, the total number of symptoms VTE in European Union 6 countries is 100 ten thousand per year, and the number of death cases exceeds the sum of deaths caused by AIDS, breast cancer, prostate cancer and traffic accidents. The U.S. death cases exceed 29.6 ten thousand per year, while less than 50% of lethal PE is diagnosed before death. International guidelines have listed VTE prevention as one of the most important strategies for reducing mortality in hospitalized patients.
Evidence from large-scale clinical trials suggests that anticoagulant therapy can prevent the spread and recurrence of thrombi and further reduce the incidence and mortality of stroke, PE, etc. Therefore, anticoagulant therapy has become the core and the foundation of clinical prevention and treatment of thromboembolic diseases at present, and the development of anticoagulant drugs is always a hot spot of new drug development, especially the development of drugs taking Xa factor inhibitor as a target.
The present inventors have surprisingly obtained, through experimental trials during the course of their intensive studies, a novel polymorphic form of a 1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxotetrahydropyrrole-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide compound. It is well known that for a polymorphic form of a drug, different crystalline forms may have different chemical and physical properties, including chemical stability, solubility, optical and mechanical properties, etc., which may directly affect the handling and production processes of the drug substance and the formulation, and may affect the stability, solubility and bioavailability of the formulation, and thus, research on the polymorphic form is of great significance to the quality, safety and effectiveness of the drug formulation. For the factor Xa inhibitor, the art has a polymorphic form of the 1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxotetrahydropyrrole-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide compound which is more suitable for industrial production and has excellent physicochemical properties.
Disclosure of Invention
The invention discloses a polymorphic crystal form of a 1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxo-tetrahydropyrrole-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-formamide compound shown as a structural formula I, a preparation method of each crystal form, and application of each crystal form in preparing a medicament for treating diseases related to an Xa factor inhibitor. More specifically, the application is the application in the preparation of medicines for preventing or treating thrombus or embolism.
Figure BDA0002606783090000021
The polymorphic forms of the compound with the structure I disclosed by the invention specifically comprise forms B, M1, O, P, Q, T, U, V and X.
The present invention provides crystalline form B of the compound of formula I, having a pattern with diffraction angles, interplanar spacings and relative intensities as shown in the following table, as determined by X-ray powder measurements using Cu-ka radiation:
Figure BDA0002606783090000022
the error of the 2 θ diffraction angle was ± 0.2.
Further, the spectrum of form B has diffraction angles, interplanar spacings and relative intensities shown in the following table:
Figure BDA0002606783090000023
Figure BDA0002606783090000031
the error of the 2 θ diffraction angle was ± 0.2.
Further, form B has an X-ray powder diffraction pattern substantially as shown in figure 2.
The present invention provides crystalline form M1 of the compound of formula I, having the diffraction angles, interplanar spacings and relative intensities shown in the following table, as determined by X-ray powder measurements using Cu-ka radiation:
Figure BDA0002606783090000032
the error of the 2 θ diffraction angle was ± 0.2.
Further, the spectrum of form M1 has diffraction angles, interplanar spacings and relative intensities shown in the following table:
Figure BDA0002606783090000041
the error of the 2 θ diffraction angle was ± 0.2.
Further, form M1 has an X-ray powder diffraction pattern substantially as shown in figure 3.
In a further aspect the present invention provides crystalline form O of the compound of formula I, having a pattern, as determined by X-ray powder using Cu-ka radiation, having diffraction angles, interplanar spacings and relative intensities as shown in the following table:
Figure BDA0002606783090000042
the error of the 2 θ diffraction angle was ± 0.2.
Further, the spectrum of form O has diffraction angles, interplanar spacings and relative intensities shown in the following table:
Figure BDA0002606783090000051
the error of the 2 θ diffraction angle was ± 0.2.
Further, form O has an X-ray powder diffraction pattern substantially as shown in figure 4.
In a further aspect the present invention provides crystalline form P of the compound of formula I, having a pattern, as determined by X-ray powder using Cu-ka radiation, having diffraction angles, interplanar spacings and relative intensities as shown in the following table:
Figure BDA0002606783090000052
the error of the 2 θ diffraction angle was ± 0.2.
Further, the spectrum of form P has diffraction angles, interplanar spacings and relative intensities shown in the following table:
Figure BDA0002606783090000053
Figure BDA0002606783090000061
the error of the 2 θ diffraction angle was ± 0.2.
Further, form P has an X-ray powder diffraction pattern substantially as shown in figure 5.
In a further aspect the present invention provides a crystalline form Q of the compound of formula I, having a pattern, as determined by X-ray powder using Cu-ka radiation, having diffraction angles, interplanar spacings and relative intensities as shown in the following table:
Figure BDA0002606783090000062
the error of the 2 θ diffraction angle was ± 0.2.
Further, the spectrum of form Q has diffraction angles, interplanar spacings and relative intensities shown in the following table:
Figure BDA0002606783090000063
Figure BDA0002606783090000071
the error of the 2 θ diffraction angle was ± 0.2.
Further, form Q has an X-ray powder diffraction pattern substantially as shown in figure 6.
In a further aspect the present invention provides crystalline form T of the compound of formula I, having a pattern, as determined by X-ray powder using Cu-ka radiation, having diffraction angles, interplanar spacings and relative intensities as shown in the following table:
Figure BDA0002606783090000072
the error of the 2 θ diffraction angle was ± 0.2.
Further, the crystal form T spectrum has diffraction angles, interplanar spacings and relative intensities shown in the following table:
Figure BDA0002606783090000073
Figure BDA0002606783090000081
the error of the 2 θ diffraction angle was ± 0.2.
Further, form T has an X-ray powder diffraction pattern substantially as shown in figure 7.
In a further aspect the present invention provides crystalline form U of the compound of formula I having a pattern, as determined by X-ray powder using Cu-ka radiation, having diffraction angles, interplanar spacings and relative intensities as shown in the following table:
Figure BDA0002606783090000082
the error of the 2 θ diffraction angle was ± 0.2.
Further, the spectrum of the crystal form U has diffraction angles, interplanar spacings and relative intensities shown in the following table:
Figure BDA0002606783090000083
Figure BDA0002606783090000091
the error of the 2 θ diffraction angle was ± 0.2.
Further, form U has an X-ray powder diffraction pattern substantially as shown in figure 8.
In a further aspect the present invention provides a crystalline form V of the compound of formula I having a pattern, as determined by X-ray powder using Cu-ka radiation, having diffraction angles, interplanar spacings and relative intensities as shown in the following table:
Figure BDA0002606783090000092
Figure BDA0002606783090000101
the error of the 2 θ diffraction angle was ± 0.2.
Further, the spectrum of form V has diffraction angles, interplanar spacings and relative intensities shown in the following table:
Figure BDA0002606783090000102
the error of the 2 θ diffraction angle was ± 0.2.
Further, form V has an X-ray powder diffraction pattern substantially as shown in figure 9.
In a further aspect the present invention provides a compound of formula I in crystalline form X having a pattern, as determined by X-ray powder using Cu-ka radiation, having diffraction angles, interplanar spacings and relative intensities as shown in the following table:
Figure BDA0002606783090000103
Figure BDA0002606783090000111
the error of the 2 θ diffraction angle was ± 0.2.
Further, the crystal form X spectrum has diffraction angles, interplanar spacings and relative intensities shown in the following table:
Figure BDA0002606783090000112
Figure BDA0002606783090000121
the error of the 2 θ diffraction angle was ± 0.2.
Further, form X has an X-ray powder diffraction pattern substantially as shown in figure 10.
The invention also discloses a polymorphic form of the compound with the structure I, which specifically comprises a form C, D, F, H, I, J, L, M, R and a form S.
In a further aspect of the invention there is provided crystalline form C of the compound of formula I having a pattern, as determined by X-ray powder using Cu-ka radiation, having diffraction angles, interplanar spacings and relative intensities as shown in the table below.
Figure BDA0002606783090000122
The error of the 2 θ diffraction angle was ± 0.2.
In a further aspect the present invention provides crystalline form D of the compound of formula I, having a pattern, as determined by X-ray powder using Cu-ka radiation, having diffraction angles, interplanar spacings and relative intensities as shown in the following table:
Figure BDA0002606783090000123
Figure BDA0002606783090000131
the error of the 2 θ diffraction angle was ± 0.2.
In a further aspect the present invention provides crystalline form F of the compound of formula I having a spectrum with diffraction angles, interplanar spacings and relative intensities as indicated in the following table, as determined by X-ray powder analysis using Cu-ka radiation:
Figure BDA0002606783090000132
the error of the 2 θ diffraction angle was ± 0.2.
In a further aspect the present invention provides crystalline form H of the compound of formula I having a pattern, as determined by X-ray powder using Cu-ka radiation, having diffraction angles, interplanar spacings and relative intensities as shown in the following table:
Figure BDA0002606783090000133
the error of the 2 θ diffraction angle was ± 0.2.
In a further aspect of the invention there is provided crystalline form I of the compound of formula I having a pattern, as determined by X-ray powder using Cu-ka radiation, having diffraction angles, interplanar spacings and relative intensities as shown in the following table.
Figure BDA0002606783090000134
Figure BDA0002606783090000141
The error of the 2 θ diffraction angle was ± 0.2.
In a further aspect the present invention provides crystalline form J of the compound of formula I having a pattern, as determined by X-ray powder using Cu-ka radiation, having diffraction angles, interplanar spacings and relative intensities as shown in the following table:
Figure BDA0002606783090000142
the error of the 2 θ diffraction angle was ± 0.2.
In a further aspect the present invention provides crystalline form L of the compound of formula I, having a pattern, as determined by X-ray powder using Cu-ka radiation, having diffraction angles, interplanar spacings and relative intensities as shown in the following table:
Figure BDA0002606783090000143
the error of the 2 θ diffraction angle was ± 0.2.
In a further aspect of the invention there is provided crystalline form M of the compound of formula I having a pattern, as determined by X-ray powder using Cu-ka radiation, having diffraction angles, interplanar spacings and relative intensities as shown in the table below.
Figure BDA0002606783090000144
The error of the 2 θ diffraction angle was ± 0.2.
In a further aspect the present invention provides crystalline form R of the compound of formula I, having a pattern, as determined by X-ray powder using Cu-ka radiation, having diffraction angles, interplanar spacings and relative intensities as shown in the following table:
Figure BDA0002606783090000151
the error of the 2 θ diffraction angle was ± 0.2.
In a further aspect the present invention provides crystalline form S of the compound of formula I, having a pattern, as determined by X-ray powder using Cu-ka radiation, having diffraction angles, interplanar spacings and relative intensities as shown in the following table:
Figure BDA0002606783090000152
the error of the 2 θ diffraction angle was ± 0.2.
The invention also discloses a preparation method of the compound 1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxo-tetrahydropyrrole-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-formamide shown in the formula I:
Figure BDA0002606783090000161
under the condition of room temperature (10-25 ℃), reacting 2-methyl-4-nitroaniline serving as a starting material with 5-chloropentanoyl chloride to generate a compound a, performing intramolecular cyclization on the compound a under the alkaline condition to generate a compound b, performing intramolecular cyclization on the compound b under the action of phosphorus pentachloride to generate a compound c, further performing intramolecular cyclization on the generated compound c under the action of morpholine to generate a compound d, reducing nitro in the compound d to generate a compound e, reacting the compound e with 4-chlorobutyryl chloride again to generate a compound f, performing intramolecular cyclization on the compound f again to generate a compound g, performing aminolysis on the compound g and [ (4-methoxyphenyl) hydrazino ] ethyl chloroacetate to generate a compound h, and performing aminolysis on the compound h to obtain the compound of the formula I.
Crystalline forms B, M1, O, P, Q, T, U, V and X of the compound of formula I, 1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxotetrahydropyrrole-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide may be prepared by:
(1) adding a compound shown in a structure of a formula I into a sample bottle, adding a positive solvent, completely dissolving the compound under the condition of room temperature or reflux, directly or dropwise adding a counter solvent, and cooling to room temperature for crystallization;
(2) carrying out suction filtration, and drying at room temperature or under heating condition under normal pressure or vacuum to obtain different crystal forms; and optionally, adding the crystal form into an anti-solvent to form a suspension, stirring, filtering, and drying to obtain different crystal forms.
Wherein, the positive solvent is that the compound with the structural formula I has better solubility in the solvent, and the positive solvent is specifically selected from one or more of methanol, ethanol, tetrahydrofuran, acetone, N-dimethylformamide, dioxane, dichloromethane and ethyl acetate; preferably one or more of methanol, ethanol, dichloromethane, N-dimethylformamide and ethyl acetate; the anti-solvent is the compound with the structural formula I, which is provided by the invention, has poor solubility in the solvent, and is specifically selected from one or more of water, methyl tert-butyl ether, n-heptane, n-hexane and cyclohexane; one or more of water, methyl tert-butyl ether and n-heptane are preferred.
Further, crystalline forms B, M1, O, P, Q, T, U, V and X of the compound of formula I of the present invention can be prepared by:
(1) adding a compound shown in a structure of a formula I into a sample bottle, adding a positive solvent, completely dissolving the compound under the condition of room temperature or reflux, directly or dropwise adding a counter solvent, and cooling to room temperature for crystallization;
(2) and (4) carrying out suction filtration, and drying at room temperature or under heating condition under normal pressure or vacuum to obtain different crystal forms.
Alternatively, further, crystalline forms B, M1, O, P, Q, T, U, V and X of the compound of formula I of the present invention may be prepared by:
(1) adding a compound shown in a structure of a formula I into a sample bottle, adding a positive solvent, completely dissolving the compound under the condition of room temperature or reflux, directly or dropwise adding a counter solvent, and cooling to room temperature for crystallization;
(2) and (3) carrying out suction filtration, drying at room temperature or under heating condition under normal pressure or vacuum to obtain different crystal forms, adding the crystal forms into an anti-solvent to form a suspension, and then stirring, carrying out suction filtration and drying to obtain different crystal forms.
According to the invention, solubility tests show that the crystal form B, the crystal form M1, the crystal form O, the crystal form P, the crystal form Q, the crystal form T, the crystal form U, the crystal form V or the crystal form X of the compound shown in the formula I have good solubility; the chemical stability investigation tests of 5 days and 10 days show that the crystal form B, the crystal form M1, the crystal form O, the crystal form P, the crystal form Q, the crystal form T, the crystal form U, the crystal form V and the crystal form X of the compound shown in the formula I have excellent chemical stability. Further, physical stability investigation tests for 10 days show that the crystal form has good physical stability. Furthermore, the invention considers the moisture absorption of the amorphous, crystal form B, crystal form M1, crystal form O, crystal form P, crystal form Q, crystal form T, crystal form U, crystal form V and crystal form X of the compound shown in the formula I under the normal temperature and normal humidity conditions, and the results show that the crystal form B and the crystal form X of the invention have no moisture absorption, the crystal form M1, the crystal form O, the crystal form P, the crystal form Q, the crystal form T and the crystal form V have slight moisture absorption, and the crystal form U has moisture absorption.
The test on the effect of normal mouse APTT shows that the amorphous and various crystal forms prepared by the invention have the effect of obviously prolonging the mouse APTT value and are all obviously superior to the positive drug apixaban; compared with amorphous form, the APTT value of the crystal form B of the invention is remarkably increased (P is less than 0.01), and the APTT values of the crystal forms M1 and O, Q, X are remarkably increased (P is less than 0.05); the effects of the crystal form B, the crystal form M1, the crystal form O, the crystal form Q and the crystal form X are relatively better. The crystal forms B, M1 and O, Q, X of the compound in the formula I prepared by the invention can be used for preparing anticoagulant, thrombosis prevention or embolism treatment medicines, in particular to the anticoagulant medicines or the Xa factor inhibitor medicines.
Drawings
FIG. 1 amorphous XRPD pattern of a compound of formula I
FIG. 2 XRPD pattern of form B of compound of formula I
FIG. 3 XRPD pattern of crystalline form M1 of compound of formula I
FIG. 4 XRPD pattern of form O of compound of formula I
FIG. 5 XRPD pattern of form P of compound of formula I
FIG. 6 XRPD pattern for form Q of compound of formula I
FIG. 7 XRPD pattern for form T of compound of formula I
FIG. 8 XRPD pattern of form U of compound of formula I
FIG. 9 XRPD pattern for form V of compound of formula I
FIG. 10 XRPD pattern for form X of compound of formula I
Detailed Description
The present invention will be described in further detail with reference to examples, which are provided only for illustrating the technical solutions of the present invention and are not intended to limit the spirit and scope of the present invention.
The structure of the compound is determined by nuclear magnetic resonance1H NMR). Nuclear magnetic resonance (1H NMR) shift () is given in parts per million (ppm); nuclear magnetic resonance (1H NMR) was performed using a BrukeraVANCE-400 nuclear magnetic spectrometer using hexadeutero-dimethyl sulfoxide (CDCl)3) Internal standard is Tetramethylsilane (TMS).
Mass Spectrometry (MS) measurements were carried out using a FINNIGAN LCQAD (ESI) mass spectrometer (manufacturer: Therm, model: Finnigan LCQ advantage MAX).
The HPLC spectra were determined using an Agilent model 1260DAD, Agilent, Ach.
The term "nitrogen atmosphere" in the present invention means, for example, that a reaction flask is connected to a nitrogen balloon having a volume of 1L.
The thin silica gel layer is prepared from HSGF254 or GF254 silica gel plate.
The X-ray powder diffraction (XRPD) measurements were performed using a Panalytical Empyrean X-ray powder diffraction analyzer, with the specific parameters given in the following table:
Figure BDA0002606783090000181
thermogravimetric analysis (TGA) and Differential Scanning Calorimeter (DSC) data were collected on a TA Q500 thermogravimetric analysis and a TAQ200 differential scanning calorimeter, respectively, and the instrument parameters are listed in the following table:
Figure BDA0002606783090000191
the term "room temperature" in the present invention means a temperature between 10 ℃ and 25 ℃.
EXAMPLE 1 preparation of the Compound 1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxotetrahydropyrrol-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide
Figure BDA0002606783090000192
The preparation scheme is as follows:
Figure BDA0002606783090000193
the first step is as follows: preparation of a
2-methyl-4-nitroaniline (3g, 19.7mmol) was dissolved in dichloromethane (60ml), N-diisopropylethylamine (6.4g, 49.5mmol) was added, the temperature was reduced to 5 ℃ or less in an ice bath, 5-chloroacetyl chloride (3.7g, 23.9mmol) was added dropwise to give a reaction mixture, and the reaction mixture was allowed to react at room temperature overnight. The reaction progress was followed by thin layer chromatography, and after completion of the reaction, water was poured into the reaction solution, separated, washed with water 3 times, dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain compound a (4.2g, yellow solid) in yield: 78.8 percent.
MS m/z(ES):271.1[M+1]
The second step is that: b preparation of
A (4.2g, 15.5mmol) was dissolved in tetrahydrofuran (80ml), and sodium hydride (0.75g, 31.3mmol) was added in portions under ice bath to give a reaction mixture, which was allowed to react at room temperature overnight. The reaction progress was followed by thin layer chromatography, after completion of the reaction ice water was added to the reaction mixture in ice bath, sodium hydride was quenched, tetrahydrofuran was removed by distillation under reduced pressure, ethyl acetate was extracted 2 times, dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation under reduced pressure, and the residue was purified by column chromatography to give b (3.27g, yellow solid), yield: 90.1 percent.
MS m/z(ES)：235.1[M+1]
The third step: c preparation of
B (3.27g, 14.0mmol) was dissolved in dichloromethane (100ml), phosphorus pentachloride (8.7g, 41.8mmol) was added portionwise under ice bath to give a reaction mixture, which was reacted at 40 ℃ under reflux. When the reaction solution does not generate bubbles basically, the thin layer chromatography tracks the reaction process, after the reaction is completed, ice water is added into the reaction mixture under ice bath to quench phosphorus pentachloride, liquid separation and washing are carried out for 3 times, anhydrous magnesium sulfate is dried, filtration and reduced pressure distillation are carried out to remove the solvent, and then c (4g, yellow solid) is obtained, and the yield is as follows: 94.6 percent.
MS m/z(ES)：303.0，305.0[M+1]
The fourth step: preparation of d
Dissolving c (4g, 13.2mmol) in morpholine (40ml) to obtain a reaction mixture, refluxing the reaction mixture at 120 ℃ for 2 hours, following the progress of the reaction by thin layer chromatography, after completion of the reaction, adding ethyl acetate to dissolve, washing with water 3 times, drying over anhydrous magnesium sulfate, filtering, and distilling under reduced pressure to remove the solvent to obtain d (3.98g, black solid), yield: 95.0 percent.
MS m/z(ES)：318.1[M+1]
The fifth step: preparation of e
D (3.98g, 12.5mmol) was dissolved in ethanol (50ml), sodium sulfide nonahydrate (9g, 37.5mmol) was added, and water (20ml) was added to give a reaction mixture, which was reacted at 50 ℃ under reflux overnight. The reaction progress is tracked by thin layer chromatography, after the reaction is completed, ethanol is removed by concentration under reduced pressure, ethyl acetate is extracted for three times, organic phases are combined, anhydrous magnesium sulfate is dried, filtration is carried out, the solvent is removed by distillation under reduced pressure, and then e (3.2g, yellow solid) is obtained, and the yield: 88.9 percent.
MS m/z(ES)：288.2[M+1]
And a sixth step: preparation of f
E (3.2g, 11.1mmol) was dissolved in dichloromethane (50ml), N-diisopropylethylamine (3.6g, 27.9mmol) was added, and 4-chlorobutyryl chloride (2.4g, 17.0mmol) was added dropwise under ice bath to give a reaction mixture, which was allowed to react at room temperature overnight. The reaction progress was followed by thin layer chromatography, after completion of the reaction, water was added to the reaction solution, separated, washed 3 times with water, dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation under reduced pressure to give f (3.5g, yellow solid) in yield: 80.3 percent.
MS m/z(ES)：392.2[M+1]
The seventh step: preparation of g
F (3.5g, 8.9mmol) was dissolved in tetrahydrofuran (50ml), and sodium hydride (0.6g, 25mmol) was added portionwise under ice bath to give a reaction mixture, which was allowed to react at room temperature overnight. Tracking the reaction process by thin-layer chromatography, after the reaction is completed, adding ice water to the reaction solution to quench sodium hydride, removing tetrahydrofuran by reduced pressure distillation, adding dichloromethane to extract for 2 times, combining organic phases, drying with anhydrous magnesium sulfate, filtering, removing the solvent by reduced pressure distillation, and purifying the residue by column chromatography to obtain g (2.59g, yellow solid), wherein the yield is as follows: 81.7 percent.
MS m/z(ES)：356.2[M+1]
Eighth step: preparation of h
G (280mg, 0.79mmol) was dissolved in toluene (10ml), ethyl [ (4-methoxyphenyl) hydrazino ] chloroacetate (214mg, 0.83mmol), triethylamine (252mg, 2.5mmol) were added at room temperature to give a reaction mixture, and the reaction mixture was refluxed at 120 ℃ overnight. The progress of the reaction was followed by thin layer chromatography, and after completion of the reaction, toluene was distilled off under reduced pressure, and after the residue was dissolved by adding methylene chloride (20ml), trifluoroacetic acid (2ml) was added at room temperature to give a reaction mixture, which was allowed to react at room temperature overnight. The reaction progress was followed by thin layer chromatography, after completion of the reaction, h (300mg, yellow solid) was obtained by column chromatography purification, yield: 77.9 percent.
MS m/z(ES)：489.2[M+1]
The ninth step: preparation of the Compound of structural formula I1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxotetrahydropyrrol-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide
H (300mg, 0.61mmol) was dissolved in methanol (4ml), and aqueous ammonia (2ml) was added to give a reaction mixture, which was reacted at 70 ℃ under reflux overnight. The reaction progress was followed by thin layer chromatography and after completion of the reaction, the compound of formula I (208mg, pale yellow solid) was purified by column chromatography, yield: 73.8 percent.
MS m/z(ES)：460.2[M+1]
1H NMR(400MHz,CDCl3)7.51–7.46(m,4H),7.17(d,J＝8.5Hz,1H),6.93(d,J＝8.8Hz,2H), 6.87(br s,1H),5.54(br s,1H),4.09–4.02(m,1H),3.84–3.79(m,6H),3.45–3.32(m,2H),2.60(t,J＝ 8.0Hz,2H),2.26(s,3H),2.19–2.13(m,2H).
The sample obtained by column chromatography was subjected to solid state analysis using X-ray powder diffraction (XRPD) pattern, and found to have no distinct characteristic peak in XRPD, so that the sample was judged to be amorphous, see fig. 1. The product obtained by thin layer chromatography was analyzed by XRPD and the sample was found to be still amorphous.
EXAMPLE 2 preparation of form B of the Compound of formula I
100mg of 1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxotetrahydropyrrol-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide, a compound of the formula I prepared according to example 1, are added to a sample vial, 3ml of methanol are added, after complete dissolution by heating, cooling to room temperature, suction filtration is carried out, the precipitated crystals are collected and dried under vacuum at 150 ℃ to yield 82 mg of a pale yellow solid in 82% yield.
The X-ray powder diffraction pattern of the crystal sample is shown in figure 2, and the pattern has characteristic peaks of diffraction angles, interplanar spacings and relative intensities shown in the following table:
Figure BDA0002606783090000211
Figure BDA0002606783090000221
the DSC of the crystalline sample has a characteristic absorption peak at about 202 ℃, and TGA shows that the crystalline sample loses 0.25% of its weight when heated to 150 ℃. According to the above results, the crystal form is an anhydrous crystal form and is defined as form B.
Example 3 preparation of crystalline form M1 of the compound of formula I
30mg of crystalline form B of the compound of the formula I1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxotetrahydropyrrol-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide, prepared according to example 2, are added to a sample vial, 0.5ml of acetone are added, the resulting suspension is stirred overnight at room temperature, and the sample obtained after centrifugation is dried at room temperature to yield 26mg of a pale yellow solid in 86.7% yield.
The X-ray powder diffraction pattern of the crystal sample is shown in figure 3, and the pattern has characteristic peaks of diffraction angles, interplanar spacings and relative intensities shown in the following table:
Figure BDA0002606783090000222
the DSC of the crystalline sample has a number of characteristic endothermic/exothermic peaks at about 85 ℃, 106 ℃, 144 ℃, 149 ℃, 179 ℃ and 208 ℃, and TGA shows that the crystalline sample loses 7.40% of its weight when heated to 100 ℃. Of the crystalline sample1The HNMR test result shows that the sample contains 0.2% of acetone by mass, and the acetone can be presumed to be the adsorption solvent by combining the TGA data obtained by the heating test. The water content of the crystal sample was determined by KF method to be 7.2%. From the above results, the crystalline form is dihydrate and defined as form M1.
EXAMPLE 4 preparation of crystalline form O of the Compound of formula I
30mg of 1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxopyrrolidin-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide, a compound of the formula I prepared according to example 1, are added to a sample vial, dissolved by addition of 1ml of dichloromethane, slowly evaporated at room temperature to give a solid which is filtered off with suction, the solid obtained is heated to 180 ℃ under nitrogen and then cooled to room temperature to give 25mg of a pale yellow solid in 83.3% yield.
The X-ray powder diffraction pattern of the crystal sample is shown in figure 4, and the pattern has characteristic peaks of diffraction angles, interplanar spacings and relative intensities shown in the following table:
Figure BDA0002606783090000223
Figure BDA0002606783090000231
the DSC of the crystalline sample has overlapping endothermic peaks at about 199 ℃ and 208 ℃, and TGA shows that the crystalline sample loses 0.82% of weight when heated to 180 ℃. According to the above results, the crystal form is an anhydrous crystal form and is defined as crystal form O.
EXAMPLE 5 preparation of crystalline form P of the Compound of formula I
30mg of 1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxopyrrolidin-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide, a compound of the formula I prepared according to example 1, are added to a sample vial, dissolved by addition of 1ml of dichloromethane, slowly evaporated at room temperature to give a solid which is filtered off with suction, and the solid obtained is cooled to room temperature after heating to 205 ℃ under nitrogen to give 24mg of a pale yellow solid in a yield of 80.0%.
The X-ray powder diffraction pattern of the crystal sample is shown in figure 5, and the pattern has characteristic peaks of diffraction angles, interplanar spacings and relative intensities shown in the following table:
Figure BDA0002606783090000232
the DSC of the crystalline sample has a characteristic absorption peak at about 211 ℃, and TGA shows that the crystalline sample loses 0.24% of weight when heated to 200 ℃. According to the above results, the crystal form is an anhydrous crystal form and is defined as crystal form P.
EXAMPLE 6 preparation of crystalline form Q of the Compound of formula I
30mg of 1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxopyrrolidin-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide, a compound of the formula I prepared according to example 1, are charged into a sample vial, dissolved by addition of 1ml of dichloromethane, slowly evaporated at room temperature to give a solid which is filtered off with suction, the solid obtained is heated to 140 ℃ under nitrogen and then cooled to room temperature to give 26mg of a pale yellow solid with a yield of 86.7%.
The X-ray powder diffraction pattern of the crystal sample is shown in figure 6, and the pattern has characteristic peaks of diffraction angles, interplanar spacings and relative intensities shown in the following table:
Figure BDA0002606783090000233
Figure BDA0002606783090000241
the DSC of the crystalline sample has multiple endothermic/exothermic peaks at about 156 ℃, 168 ℃, 189 ℃, 200 ℃ and 208 ℃, and TGA shows that the crystalline sample loses 1.1% of weight when heated to 150 ℃. According to the above results, the crystal form is an anhydrous crystal form and is defined as crystal form Q.
EXAMPLE 7 preparation of crystalline form T of the Compound of formula I
30mg of the compound 1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxotetrahydropyrrol-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide compound of the formula I, prepared according to example 1, are added to a sample vial, 0.5ml of tetrahydrofuran are added and the resulting suspension is stirred overnight at 50 ℃ and, after centrifugation, dried in vacuo at room temperature. The solid obtained is dissolved in 1.5ml of pure water, the resulting suspension is stirred at 50 ℃ overnight and, after centrifugation, is dried under vacuum at room temperature to give 22mg of a pale yellow solid in 73.3% yield.
The X-ray powder diffraction pattern of the crystal sample is shown in figure 7, and the pattern has characteristic peaks of diffraction angles, interplanar spacings and relative intensities shown in the following table:
Figure BDA0002606783090000242
the DSC of the crystalline sample has a number of characteristic endothermic/exothermic peaks at about 83 ℃, 107 ℃, 148 ℃ and 208 ℃, and TGA shows that the crystalline sample loses 7.7% weight when heated to 120 ℃. Of the crystalline sample1The HNMR test result shows that the sample contains tetrahydrofuran with the mass fraction of 0.3 percent. The water content of the crystal sample was determined by KF method to be 7.3%. From the above results, the crystalline form is dihydrate and defined as form T.
EXAMPLE 8 preparation of crystalline form U of the Compound of formula I
After 15mg of crystalline form T of the compound of formula I1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxopyrrolidin-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide prepared according to example 7 was purged under nitrogen for 40min, 13.5mg of a light yellow solid was obtained in 90.0% yield.
The X-ray powder diffraction pattern of the crystal sample is shown in figure 8, and the pattern has characteristic peaks of diffraction angles, interplanar spacings and relative intensities shown in the following table:
Figure BDA0002606783090000251
the crystal sample can be quickly absorbed with water and transformed into the crystal form T after being exposed in the air again. According to the above results, the crystal form is an anhydrous crystal form, and is defined as a crystal form U.
EXAMPLE 9 preparation of form V of the Compound of formula I
30mg of the compound 1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxotetrahydropyrrol-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide compound of the formula I, prepared according to example 1, are added to a sample vial, 0.9ml of methanol is added to dissolve it completely and the solid is precipitated by slow evaporation at room temperature. After centrifugal separation, the solid obtained was heated to 120 ℃ under nitrogen and then cooled to room temperature to obtain 18mg of a pale yellow solid with a yield of 60.0%.
The X-ray powder diffraction pattern of the crystal sample is shown in figure 9, and the pattern has characteristic peaks of diffraction angles, interplanar spacings and relative intensities shown in the following table:
Figure BDA0002606783090000252
the DSC of the crystalline sample has a number of characteristic endothermic/exothermic peaks at about 168 ℃, 175 ℃ and 208 ℃, and TGA shows that the crystalline sample loses 1.9% of weight when heated to 15 ℃. Of the crystalline sample1The HNMR test result shows that the mass fraction of methanol in the sample is 0.2 percent. The water content of the crystal sample was measured by KF method to obtain 2.1% water content. From the above results, the crystal form is a hydrate crystal form, and is defined as form V.
EXAMPLE 10 preparation of form X of the Compound of formula I
200mg of 1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxopyrrolidin-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide, a compound of formula I prepared according to example 1, was added to a sample bottle, 1.6ml of N, N-dimethylformamide was added thereto, and after heating to 80 ℃ to completely dissolve it, 1.2ml of water was added dropwise, followed by natural cooling to room temperature, suction filtration, collection of precipitated crystals, and drying at 70 ℃ gave 166mg of a pale yellow solid with a yield of 83.0%.
The X-ray powder diffraction pattern of the crystal sample is shown in figure 10, and the pattern has characteristic peaks of diffraction angles, interplanar spacings and relative intensities shown in the following table:
Figure BDA0002606783090000261
the DSC of the crystalline sample has a characteristic absorption peak at about 214 ℃, and TGA shows that the crystalline sample loses 0.32% of weight when heated to 150 ℃. According to the above results, the crystal form is an anhydrous crystal form and is defined as crystal form X.
EXAMPLE 11 preparation of form B of the Compound of formula I
100mg of 1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxotetrahydropyrrol-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide, a compound of the formula I prepared according to example 1, are added to a sample vial, 10ml of ethyl acetate are added, after complete dissolution by heating, cooling to room temperature, suction filtration is carried out, the precipitated crystals are collected and dried in vacuo at 150 ℃ to yield 86mg of a pale yellow solid in 86% yield.
The powder diffraction pattern remained consistent with example 2.
Example 12 preparation of crystalline form M1 of the Compound of formula I
50mg of crystalline form B of the compound of the formula I1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxotetrahydropyrrol-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide prepared according to example 2 are added to a sample vial, 0.45ml of tetrahydrofuran are added, the resulting suspension is stirred overnight at room temperature, and the sample obtained after centrifugation is dried at room temperature to yield 42mg of a pale yellow solid in 84% yield.
The powder diffraction pattern remained consistent with example 3.
EXAMPLE 13 preparation of crystalline form O of the Compound of formula I
40mg of 1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxopyrrolidin-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide, a compound of the formula I prepared according to example 1, were added to a sample vial, 0.5ml of tetrahydrofuran and 0.6ml of ethyl acetate were added to dissolve it, the solution was slowly volatilized at room temperature to precipitate a solid, suction-filtered, the resulting solid was heated to 180 ℃ under nitrogen atmosphere and then cooled to room temperature to give 34mg of a pale yellow solid in a yield of 85%.
The powder diffraction pattern remained consistent with example 4.
EXAMPLE 14 preparation of crystalline form P of the Compound of formula I
40mg of 1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxopyrrolidin-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide, a compound of the formula I prepared according to example 1, were added to a sample vial, 0.5ml of tetrahydrofuran and 0.6ml of ethyl acetate were added and dissolved, and the solution was slowly evaporated at room temperature to precipitate a solid, which was filtered with suction, the resulting solid was heated to 205 ℃ under nitrogen atmosphere and then cooled to room temperature to obtain 28mg of a pale yellow solid with a yield of 70%.
The powder diffraction pattern remained consistent with example 5.
EXAMPLE 15 preparation of crystalline form Q of the Compound of formula I
40mg of 1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxopyrrolidin-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide, a compound of the formula I prepared according to example 1, were added to a sample vial, 0.5ml of tetrahydrofuran and 0.6ml of ethyl acetate were added and dissolved, slowly evaporated at room temperature to precipitate a solid, filtered with suction, the resulting solid was heated to 140 ℃ under nitrogen and then cooled to room temperature to give 30mg of a pale yellow solid in a yield of 75%.
The powder diffraction pattern remained consistent with example 6.
EXAMPLE 16 preparation of crystalline form T of the Compound of formula I
30mg of the compound 1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxotetrahydropyrrol-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide compound of the formula I, prepared according to example 1, are added to a sample vial, 0.6ml of acetone are added and the resulting suspension is stirred overnight at 50 ℃ and, after centrifugation, dried in vacuo at room temperature. The solid obtained is dissolved in 1.5ml of water and the resulting suspension is stirred at 50 ℃ overnight and, after centrifugation, dried under vacuum at room temperature, 24mg of a pale yellow solid are obtained in 80% yield.
The powder diffraction pattern remained consistent with example 7.
EXAMPLE 17 preparation of crystalline form U of the Compound of formula I
After 20mg of crystalline form T of the compound of formula I1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxopyrrolidin-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide prepared according to example 16 was purged under nitrogen for 40min, 18.5mg of a light yellow solid was obtained in 92.5% yield.
The powder diffraction pattern remained consistent with example 8.
EXAMPLE 18 preparation of form V of the Compound of formula I
20mg of the compound 1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxotetrahydropyrrol-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide compound of the formula I, prepared according to example 1, are added to a sample vial, 0.7ml of dioxane is added and completely dissolved, and slowly evaporated at room temperature to precipitate a solid. After centrifugal separation, the solid was heated to 120 ℃ under nitrogen and then cooled to room temperature to obtain 14mg of a pale yellow solid with a yield of 70.0%.
The powder diffraction pattern remained consistent with example 9.
EXAMPLE 19 preparation of form X of the Compound of formula I
100mg of 1- (4-methoxyphenyl) -7-oxo-6- [ 2-methyl-4- (2-oxopyrrolidin-1-yl) phenyl ] -4,5,6, 7-tetrahydro-1H-pyrazolo [3,4-c ] pyridine-3-carboxamide, a compound of formula I prepared according to example 1, was added to a sample bottle, 0.9ml of N, N-dimethylformamide and 0.5ml of water were added thereto, and after heating to 80 ℃ to completely dissolve it, it was naturally cooled to room temperature, filtered with suction, the precipitated crystals were collected and dried at 70 ℃ to obtain 73mg of a pale yellow solid with a yield of 73.0%.
The powder diffraction pattern remained consistent with example 10.
Test example 1 solubility test
In order to examine the solubility of the amorphous form prepared in example 1 of the present invention and the crystalline forms B, M1, O, P, Q, T, U, V and X prepared in examples 2 to 10, the equilibrium solubility (saturated solution) of the amorphous form prepared in example 1, the crystalline forms B, M1, O, P, Q, T, U, V and X prepared in examples 2 to 10 were measured in hydrochloric acid having a pH of 1.0(0.1N) and acetic acid-sodium acetate buffer solution having a pH of 4.0 at 25 ℃ and 37 ℃ respectively, and the results are shown in table 1 below:
TABLE 1 solubility test
Figure BDA0002606783090000281
The solubility test result shows that the amorphous form, the crystal form B, the crystal form M1, the crystal form O, the crystal form P, the crystal form Q, the crystal form T, the crystal form U, the crystal form V and the crystal form X of the compound shown in the formula I have good equilibrium solubility in hydrochloric acid with the pH value of 1.0(0.1N) and acetic acid-sodium acetate buffer solution with the pH value of 4.0 at the temperature of 25 ℃.
Test example 2 chemical stability investigation test
Amorphous samples prepared in example 1 and crystalline forms B, M1, O, P, Q, T, U, V and X prepared in examples 2-10 were placed in a clean culture dish and spread open, stability of the samples under conditions of high temperature (60 ℃), high humidity (25 ℃, RH 90% +/-5%) and strong light (4500Lx + -500 Lx) was examined, sampling time was examined for 5 days and 10 days, and the samples were placed for 10 days. Samples were taken at 5 days and 10 days, respectively, and the HPLC purity measurements are shown in Table 2 below:
TABLE 2 stability test (% purity)
Figure BDA0002606783090000291
Stability investigation results show that amorphous form, crystal form B, crystal form M1, crystal form O, crystal form P, crystal form Q, crystal form T, crystal form U, crystal form V and crystal form X shown in the structure of formula I are relatively found through stability comparison under the conditions of high temperature (60 ℃), high humidity (25 ℃, RH 90% +/-5%), strong light (4500Lx +/-500 Lx) and the like under the condition of open placement, and the crystal form B, the crystal form M1, the crystal form O, the crystal form P, the crystal form Q, the crystal form T, the crystal form U, the crystal form V and the crystal form X have no obvious change under the conditions of high humidity, high temperature and illumination, namely the chemical stability of various crystal forms is relatively good; the purity of the amorphous product under high-humidity conditions is not obviously changed, but the purity of the amorphous product under high-temperature conditions and illumination conditions is obviously reduced. The stability of the crystal forms B, M1, O, P, Q, T, U, V and X prepared in the embodiments 2-10 of the invention is obviously superior to that of the amorphous form under the conditions of illumination, high temperature and high humidity.
Test example 3 physical stability test
Sample form B prepared in example 2 (as form B0 day reference data, lot No. 20140408), sample form P prepared in example 5 (as form P0 day reference data, lot No. 20140415), sample form Q prepared in example 6 (as form Q0 day reference data, lot No. 20140418), sample form T prepared in example 7 (as form T0 day reference data, lot No. 20140421), sample form X prepared in example 10 (as form X0 day reference data, lot No. 20140427), were placed in a clean petri dish, spread open and placed, and examined for physical stability of each crystal form sample under conditions of high temperature (60 ℃), high humidity (25 ℃, RH 90% ± 5%), strong light (4500Lx ± 500Lx), and the sampling time was 10 days. Samples were taken on day 10 and analyzed by solid state X-ray powder diffraction (XRPD) spectroscopy, the powder diffraction data results of which are shown in tables 3-7 below. The result shows that under the condition of open placement and under the conditions of high temperature, high humidity, strong light and the like, after 10 days, the X-ray powder diffraction angle value (expressed by a 2 theta diffraction angle) of the crystal form B, P, Q, T, X is consistent with the reference data of the sample crystal form B, P, Q, T, X0 days, namely, the crystal form has good physical stability.
Table 3 powder diffraction angle data (2 θ diffraction angle) of form B under high temperature, high humidity, light conditions
Peak No. Form B for 0 days High temperature for 10 days Illuminating for 10 days High humidity for 10 days
1 10.898560 10.864932 10.826784 10.925483
2 12.203450 12.245896 12.168134 12.195742
3 12.500340 12.548621 12.526891 12.468519
4 14.091510 14.126846 14.094512 14.125623
5 15.742240 15.734682 15.748952 15.786221
6 15.801950 15.845612 15.795682 15.846618
7 22.703550 22.711326 22.691441 22.751343
8 25.202620 25.264521 25.231568 22.215495
9 26.122310 26.154313 26.118963 26.186513
Table 4 powder diffraction angle data (2 θ diffraction angle) of form P under high temperature, high humidity, and light conditions
Peak No. Crystal form P0 day High temperature for 10 days Illuminating for 10 days High humidity for 10 days
1 6.601970 6.654891 6.625926 6.574135
2 8.155473 8.216963 8.135899 8.152156
3 10.870700 10.826325 10.921263 10.895623
4 11.885930 11.841358 11.931625 11.862592
5 12.954900 12.892692 12.931592 13.024856
6 14.975110 14.945862 14.982356 15.025889
7 15.581570 15.558268 15.623893 15.581662
8 20.268180 20.316932 20.245811 20.285583
9 23.829680 23.871669 23.794946 23.861449
TABLE 5 powder diffraction Angle data (2 θ diffraction Angle) of form Q under high temperature, high humidity, and light conditions
Figure BDA0002606783090000301
Figure BDA0002606783090000311
Table 6 powder diffraction angle data (2 θ diffraction angle) of form T under high temperature, high humidity, and light conditions
Peak No. Crystal form T0 day High temperature for 10 days Illuminating for 10 days High humidity for 10 days
1 11.288110 11.325896 11.315859 11.294583
2 11.833850 11.824893 11.792661 11.814692
3 15.940380 15.981456 15.954826 15.895492
4 16.549990 16.574892 16.498262 16.582262
5 18.911510 18.948562 18.889262 18.894825
6 19.280690 19.248613 19.315658 19.325491
7 19.744570 19.781561 19.714592 19.754816
8 21.793840 21.845912 21.831491 21.768911
9 23.679410 23.681613 23.721464 23.644513
TABLE 7 powder diffraction Angle data (2 θ diffraction Angle) of form X under high temperature, high humidity, and light conditions
Peak No. Crystal form X0 day High temperature for 10 days Illuminating for 10 days High humidity for 10 days
1 12.763300 12.814492 12.784569 12.724561
2 14.703580 14.745659 14.684912 14.658922
3 15.083280 15.048916 15.078916 15.125912
4 15.235710 15.194823 15.214933 15.271652
5 18.271200 18.241658 18.268912 18.294816
6 20.528030 20.548916 20.531813 20.498165
7 21.667030 21.648913 21.694613 21.671492
8 22.150960 22.134686 22.214962 22.198135
9 26.184360 26.164981 26.178916 21.215921
Test example 4 moisture absorption test
Amorphous samples prepared in example 1 and crystalline forms B, M1, O, P, Q, T, U, V and X prepared in examples 2 to 10 were placed in a clean culture dish in an open manner, examined under normal temperature and humidity (25 ℃ and RH 50%) and checked for percentage mass increase, and the results are shown in the following table 6:
TABLE 8 hygroscopicity test
Figure BDA0002606783090000312
Figure BDA0002606783090000321
Test results show that under the conditions of normal temperature and normal humidity (25 ℃, RH 50%), the mass increase percentage of the crystal form B and the crystal form X of the compound of the formula I is less than 0.2 percent, namely no hygroscopicity exists; the mass increase percentage of the crystal form M1, the crystal form O, the crystal form P, the crystal form Q, the crystal form T and the crystal form V is in the range of 0.2 to 2 percent, namely the crystal form has slight hygroscopicity; the mass increase percentage of the crystal form U is more than 2 percent, namely the crystal form U has hygroscopicity.
Effect examples test of Effect of Compound of formula I and its Crystal forms on Normal mouse APTT
The purpose of the test is as follows: the effect of the crystalline forms prepared in the examples at the 5mg/kg dose on mouse APTT after administration was investigated.
1. Test materials:
1.1. medicine preparation:
the test drugs are: compound of formula I (amorphous), prepared as in example 1, provided by the synthetic research laboratory of medeton pharmaceutical ltd, light yellow solid, lot No.: 20140309, respectively;
positive control drug: apixaban, available from Shanghai Haoyuan chemical science and technology, Inc., 99.9% pure, under the accession number HM-038_ 13-20140427;
the test drugs are: form B, prepared as in example 2, was provided by the synthetic research laboratory of medton incorporated, light yellow solid, lot No.: 20140408, respectively;
the test drugs are: form M1, prepared as in example 3, was provided by the synthetic research laboratory of medeton pharmaceutical ltd, a light yellow solid, lot No.: 20140409, respectively;
the test drugs are: form O, prepared as in example 4, was provided by the synthetic research laboratory of medton incorporated, light yellow solid, lot No.: 20140414, respectively;
the test drugs are: form P, prepared as in example 5, was provided by the synthetic research laboratory of medton incorporated, light yellow solid, lot No.: 20140415, respectively;
the test drugs are: form Q, prepared as in example 6, was provided by the synthetic research laboratory of medton incorporated, light yellow solid, lot No.: 20140418, respectively;
the test drugs are: form T, prepared as in example 7, was provided by the synthetic research laboratory of medton incorporated, light yellow solid, lot No.: 20140421, respectively;
the test drugs are: form U, prepared as in example 8, was provided by the synthetic research laboratory of medton incorporated, light yellow solid, lot No.: 20140424, respectively;
the test drugs are: form V, prepared as in example 9, was provided by the synthetic research laboratory of medton incorporated, light yellow solid, lot No.: 20140425, respectively;
the test drugs are: form X, prepared as in example 10, was provided by the synthetic research laboratory of medton incorporated, light yellow solid, lot No.: 20140427, respectively;
1.2 test equipment:
a Sysmex CA7000 type full-automatic hemagglutination analyzer;
roche C501 full-automatic biochemical analyzer;
blood collection tubes, surgical scissors, syringes, and the like;
1.3 test animals:
the KM mice are 22-24 g in weight, 120 mice, half in male and female, and are provided by Woodson Biotech, Inc., and the production facility license is SCXK 2013-24. The animals are bred in animal houses after being purchased, adaptability is observed for at least 3 days, and the animals are used for experiments after being qualified for quarantine.
2.1 grouping:
the mice were grouped according to the body weight in table 9, 10 mice per group, half male and half female, no statistical difference between groups;
table 9 test groups and dosing regimens
Figure BDA0002606783090000331
2.2 APTT determination: administering the corresponding test agent (physiological saline for blank group) to each group by intragastric administration according to Table 9, taking blood from orbit in 0.5ml vacuum blood collection tube containing sodium citrate after 1h administration, and measuring APTT of each animal after blood sample collection;
3. the statistical method comprises the following steps:
the Excel is adopted for statistics, and experimental data are adopted
Figure BDA0002606783090000332
Showing that the comparison between the groups was statistically compared using the two-sided T-test method.
4. And (3) test results:
TABLE 10 Effect on normal mouse APTT
Figure BDA0002606783090000333
Figure BDA0002606783090000334
Figure BDA0002606783090000341
Note: compared to blank group＊P＜0.05；＊＊P＜0.01；
Compared with the positive group△P＜0.05；△△P＜0.01；
Compared with the group of example 1◇P＜0.05；◇◇P＜0.01。
5. And (4) conclusion:
(1) as can be seen from table 10, after 1h administration, the APTT (activated partial thromboplastin time) values of the positive group and the example groups were significantly increased (P < 0.01) compared to the blank group, which indicates that the apixaban, the example 1 and the crystal forms thereof all significantly prolonged the APTT value of the mice after 1h administration;
(2) compared with the positive group, the APTT value of each example group is remarkably increased (P is less than 0.01), which shows that the compound (amorphous) of example 1 and each crystal form thereof are superior to the positive group in prolonging the APTT value of mice;
(3) the significantly increased APTT values of the example 2 group (P < 0.01) and the examples 3,4, 6, 10 groups (P < 0.05) compared to the example 1 group illustrate the relatively better effect of the compound of example 2 in form B, the compound of example 3 in form M1, the compound of example 4 in form O, the compound of example 6 in form Q, and the compound of example 10 in form X.
It will be apparent to those skilled in the art that various modifications and variations can be made in the compounds, compositions, and methods of making the same of the present invention without departing from the spirit or scope of the invention, and therefore, the scope of the invention encompasses all modifications and variations that fall within the scope of the claims and their equivalents.

Claims (6)

1. Crystalline form P of a compound of formula I, characterized by X-ray powder measurements using Cu-k rays, having diffraction angles, interplanar spacings and relative intensities as shown in the following table:
Figure 884915DEST_PATH_IMAGE001
Peak No. Pos. [°2Th.] d-spacing [Å] Rel. Int. [%] 1 6.601970 13.38870 88.99 2 8.155473 10.84149 22.97 3 10.870700 8.13890 100.00 4 11.885930 7.44590 44.09 5 12.954900 6.83381 84.63 6 14.975110 5.91614 31.13 7 15.581570 5.68721 38.35 8 20.268180 4.38151 49.61 9 23.829680 3.73412 27.66
。
2. form P of the compound according to claim I, characterized in that it has an X-ray powder diffraction pattern substantially as shown in figure 5.
3. A process for preparing the compound of formula I of claim 1 in crystalline form P, comprising the steps of: adding 30mg of the compound shown in the structural formula I into a sample bottle, adding 1ml of dichloromethane to dissolve the compound, slowly volatilizing at room temperature to precipitate a solid, carrying out suction filtration, heating the obtained solid to 205 ℃ under the protection of nitrogen, and cooling to room temperature to obtain 24mg of light yellow solid.
4. Use of a crystalline form P of a compound of formula I according to claim 1 for the preparation of a medicament for the prevention or treatment of thrombosis or embolism.
5. Use of the crystalline form P of a compound of formula I according to claim 1 for the preparation of an anticoagulant medicament.
6. Use of the compound of formula I of claim 1 in crystalline form P for the preparation of a medicament for the treatment of a factor Xa inhibitor.
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