Novel crystal form of compound containing bi-azetidine subunit sulfonyl structure

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a novel crystal form of a compound (S)-4-((1 '-(3-hydroxy-2-phenylpropionyl)-1', 4 '-dihydro-2H, 2' H-[3, 3 '-biazetidine subunit]-1 (4H)-yl) sulfonyl) cyanobenzene. The novel crystal form provided by the invention has an obvious property especially suitable for medicines, especially a crystal form A and a crystal form C. The novel crystal form has one or more than two medicinal property advantages of stability, solubility, impurity removal capability or hygroscopicity.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a novel diazotidine subunit sulfonyl structure-containing new crystal form having PKM2 stimulating and / or PKR stimulating effects. Background Art

[0002] The related applications previously submitted by the applicant of this invention involve a Chinese invention patent application (application number CN202211606701.0) and a PCT international patent application (application number PCT / CN2022 / 140731). The entire contents of the two patent applications are incorporated into this application by full reference.

[0003] The aforementioned related application of the present invention provides a novel compound containing a diazetidinyl sulfonyl structure, and the compound "(S)-4-((1'-(3-hydroxy-2-phenylpropionyl)-1',4'-dihydro-2H,2'H-[3,3'-diazetidinyl]-1(4H)-yl)sulfonyl)benzonitrile" is one of the specific compounds provided, which has good PKR agonist and / or PKM2 inhibitory activity and is expected to be used for the prevention and treatment of PKR and / or PKM2-mediated related diseases, such as sickle cell anemia, β-thalassemia, colorectal cancer, renal cancer, pancreatic cancer, breast cancer, lung cancer, esophageal cancer, melanoma, lymphoma, glioblastoma or multiple myeloma.

[0004] The chemical structure of the compound "(S)-4-((1'-(3-hydroxy-2-phenylpropionyl)-1',4'-dihydro-2H,2'H-[3,3'-diazetidinylidene]-1(4H)-yl)sulfonyl)benzonitrile" is shown below:

[0005]

[0006] The compounds synthesized in the aforementioned related applications of the present invention are amorphous, and amorphous states often have undesirable adverse properties when used for drug development. Polymorphism is common in compounds in nature, and the properties of crystals obtained by different methods are often unpredictable, and generally not all crystal forms have properties suitable for pharmaceutical use. The purpose of the present invention is to provide a new crystal form, and the new crystal form has properties that are obviously particularly suitable for pharmaceutical use. Summary of the invention

[0007] As mentioned above, the purpose of the present invention is to study and provide a new crystalline form of the compound "(S)-4-((1'-(3-hydroxy-2-phenylpropionyl)-1',4'-dihydro-2H,2'H-[3,3'-diazetidinylidene]-1(4H)-yl)sulfonyl)benzonitrile" with obvious and particularly suitable properties for pharmaceutical use, and the new crystalline form has one or more pharmaceutical property advantages among stability, solubility, impurity removal ability or hygroscopicity.

[0008] The specific scheme provided by the present invention is as follows:

[0009] The present invention first provides a crystalline state of the compound "(S)-4-((1'-(3-hydroxy-2-phenylpropionyl)-1',4'-dihydro-2H,2'H-[3,3'-diazetidinylidene]-1(4H)-yl)sulfonyl)benzonitrile", referred to as a crystalline compound.

[0010] The dominant crystalline form of the crystalline compound is named Form A, and the X-ray powder diffraction pattern of Form A includes diffraction peaks at 2θ values ​​of 5.5±0.2°, 11.0±0.2°, 17.0±0.2°, and 20.4±0.2°. Optionally, it also includes diffraction peaks at 2θ values ​​of 21.3±0.2° and 24.2±0.2°. Optionally, it also includes diffraction peaks selected from 14.9±0.2°, 15.1±0.2°, 16.4±0.2°, 16.6±0.2°, 22.1±0.2°, 23.2±0.2°, 24.7±0.2°,

[0011] 26.3±0.2°, 27.6±0.2°, 28.6±0.2°, 29.5±0.2° 2θ values ​​at one or more diffraction peaks. Optionally, it also includes one or more diffraction peaks selected from 13.7±0.2°, 15.9±0.2°, 18.8±0.2°, 19.4±0.2°, 22.7±0.2°, 25.3±0.2°, 25.8±0.2°, 29.0±0.2°, 30.3±0.2°, 30.5±0.2° 2θ values. According to a specific embodiment of the present invention, a more specific exemplary complete X-ray powder diffraction pattern of Form A is as follows Figure 2 shown.

[0012] As shown in some specific embodiments, Form A also has Figure 4 An exemplary complete DSC graph is shown.

[0013] As shown in the DSC graph, Form A has a melting point of 153±2°C.

[0014] Form A can be obtained by stirring and slurrying the compound (S)-4-((1'-(3-hydroxy-2-phenylpropionyl)-1',4'-dihydro-2H,2'H-[3,3'-diazotidine subunit]-1(4H)-yl)sulfonyl)benzonitrile in an organic solvent, wherein the organic solvent is methanol, ethanol, isopropanol, n-butanol, tert-butanol, methyl acetate, ethyl acetate, isopropyl acetate, dimethyl carbonate, ethyl ether, isopropyl ether, methyl tert-butyl ether, butanone, methyl isobutyl ketone, n-hexane, cyclohexane, n-heptane, toluene, xylene or methyltetrahydrofuran. In a specific embodiment, the stirring time is more than 20 hours, and can be selected from 20 to 48 hours; the system temperature during the stirring is room temperature or 20 to 50°C. In another specific embodiment, the starting compound is an amorphous state, and the amorphous state can be prepared by reverse phase high pressure liquid phase after the raw materials of the compound undergo a synthetic reaction, or can be further obtained by freeze drying. The amorphous state can also be obtained by the following method: the aqueous solution containing the compound (S)-4-((1'-(3-hydroxy-2-phenylpropionyl)-1',4'-dihydro-2H,2'H-[3,3'-diazetidine subunit]-1(4H)-yl)sulfonyl)benzonitrile is concentrated under reduced pressure, DCM is added for extraction and separation, the organic phase is collected, and the organic phase is concentrated under reduced pressure to obtain the amorphous product.

[0015] The present invention also provides a hydrate of the compound "(S)-4-((1'-(3-hydroxy-2-phenylpropionyl)-1',4'-dihydro-2H,2'H-[3,3'-diazetidinylidene]-1(4H)-yl)sulfonyl)benzonitrile", which may be in crystalline form.

[0016] In some embodiments, the predominant crystalline form of the hydrate of the crystalline form is named Form C, and the X-ray powder diffraction pattern of Form C comprises diffraction peaks at 2θ values ​​of 12.0±0.2°, 12.2±0.2°, and 20.1±0.2°. Optionally, it also comprises a diffraction peak at a 2θ value of 25.7±0.2°. Optionally, it also comprises one or more diffraction peaks at 2θ values ​​selected from 9.2±0.2°, 12.4±0.2°, 17.4±0.2°, 18.5±0.2°, 19.4±0.2°, 21.0±0.2°, 21.5±0.2°, 22.4±0.2°, 24.0±0.2°, 24.6±0.2°, 27.2±0.2°, 27.8±0.2°, 28.6±0.2°, and 28.9±0.2°. Optionally, it further comprises one or more diffraction peaks at 2θ values ​​selected from 4.6±0.2°, 13.4±0.2°, 13.8±0.2°, 16.0±0.2°, 16.7±0.2°, 18.0±0.2°, 22.6±0.2°, 23.2±0.2°, 25.4±0.2°, 26.7±0.2°, 29.3±0.2°, 30.0±0.2°, and 30.4±0.2°. According to a specific embodiment of the present invention, a more specific exemplary complete X-ray powder diffraction pattern of Form C is as follows Figure 8 shown.

[0017] As shown in some specific embodiments, Form C also has Fig.10 An exemplary complete DSC graph is shown.

[0018] As shown in the DSC graph, Form C has a melting point of 103±2°C.

[0019] Hydrate Form C can be obtained by stirring and slurrying the compound (S)-4-((1'-(3-hydroxy-2-phenylpropionyl)-1',4'-dihydro-2H,2'H-[3,3'-diazotidine subunit]-1(4H)-yl)sulfonyl)benzonitrile in water. In a specific embodiment, the stirring time is more than 20 hours, and can be 20 to 48 hours; the system temperature during the stirring is room temperature or 20 to 50°C. In another specific embodiment, the starting compound is an amorphous state, and the amorphous state can be prepared by reverse high pressure liquid phase after the synthesis reaction of the raw materials of the compound, and can also be further obtained by freeze drying. The amorphous form can also be obtained by the following method: the aqueous solution containing the compound (S)-4-((1'-(3-hydroxy-2-phenylpropionyl)-1',4'-dihydro-2H,2'H-[3,3'-diazetidinylidene]-1(4H)-yl)sulfonyl)benzonitrile is concentrated under reduced pressure, DCM is added for extraction and separation, the organic phase is collected, and the organic phase is concentrated under reduced pressure to obtain the amorphous product.

[0020] On the basis of the above, the present invention also provides a pharmaceutical composition, comprising a pharmaceutically acceptable excipient and any crystalline compound provided by the present invention, or comprising a pharmaceutically acceptable excipient and any hydrate provided by the present invention. Pharmaceutically acceptable excipients can be selected according to the specific dosage form required, and pharmaceutical excipients suitable for different dosage forms are recorded in the pharmacopoeias of various countries, and can also be selected as needed by referring to the textbook "Handbook of Pharmaceutical Excipients (RC Luo et al.)".

[0021] On the basis of the above, based on the verified activity of the compounds of the present invention, the present invention also provides the use of any of the aforementioned crystalline compounds or any of the hydrates of the present invention, especially the crystalline form A or the crystalline form C, as a PKM2 agonist or a PKR agonist in the preparation of a drug for treating PKM2 or PKR related diseases, and further, in the preparation of a drug for treating liver cancer, lung cancer, colorectal cancer, glioblastoma, breast cancer, lymphoma, pancreatic cancer, renal cancer, experimental autoimmune encephalomyelitis, inflammatory bowel disease, ulcerative colitis, alopecia, alopecia or hypotrichosis, or in the preparation of a drug for treating myeloproliferative disorders. The invention also provides a method for treating any of the above-mentioned diseases, comprising administering to a patient any of the above-mentioned crystalline compounds or any of the above-mentioned hydrates of the present invention.

[0022] The crystalline compound or hydrate provided by the present invention, especially the crystal form A or the crystal form C, has excellent properties suitable for pharmaceutical use. As verified in the specific embodiments of the present invention: Crystal form A is the most stable crystalline compound, and it is also the crystal form that other crystalline or amorphous forms are most easily transformed into under various conditions. While maintaining high stability, crystal form A also has a higher solubility similar to that of the amorphous form, and is expected to provide a better exposure effect in the body after the drug preparation. Crystal form A also has excellent impurity removal ability and stronger chemical stability. Crystal form C is a hydrate crystal form, which also has high stability, and especially in an aqueous system or an environment with a high water content, crystal form C has a unique stability advantage. This special property advantage is expected to promote its use in aqueous drug dosage forms or formulation processes (such as wet granulation) that undergo water environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The XRPD pattern of the amorphous product obtained in Example 2 is shown.

[0024] Figure 2 The XRPD pattern of Form A obtained in Example 3 is shown.

[0025] Figure 3 The TGA / DSC spectrum of Form A obtained in Example 3 is shown.

[0026] Figure 4 The DSC spectrum of Form A obtained in Example 3 is shown.

[0027] Figure 5 The XRPD pattern of Form B obtained in Example 5 is shown.

[0028] Figure 6 The TGA / DSC spectrum of Form B obtained in Example 5 is shown.

[0029] Figure 7 The DSC spectrum of Form B obtained in Example 5 is shown.

[0030] Figure 8 The XRPD spectrum of Form C obtained in Example 6 is shown.

[0031] Fig. 9 The TGA / DSC spectrum of Form C obtained in Example 6 is shown.

[0032] Fig.10 The DSC spectrum of Form C obtained in Example 6 is shown.

[0033] Fig.11 The DSC spectrum of the crystal form C obtained in Example 6 is shown.

[0034] Fig.12 The HNMR comparison spectrum of Form C and Form A in Example 6 is shown.

[0035] Fig.13 The XPRD comparison diagram of the crystal form C obtained in Example 6 before and after heating and drying at 100°C is shown.

[0036] Fig.14 The TGA / DSC spectrum of Form C in Example 6 after drying at 100°C for 3h is shown.

[0037] Fig.15 The XRPD spectrum of Form D obtained in Example 7 is shown.

[0038] Fig.16 The TGA / DSC spectrum of Form D obtained in Example 7 is shown.

[0039] Fig.17The DSC spectrum of Form D obtained in Example 7 is shown. DETAILED DESCRIPTION

[0040] Unless otherwise expressly stated, the following abbreviations or terms have the following meanings:

[0041] DMF stands for N,N-dimethylformamide;

[0042] HATU stands for 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate;

[0043] DCM means dichloromethane;

[0044] XPRD means X-ray powder diffraction;

[0045] TGA means thermogravimetric analysis;

[0046] DSC stands for differential scanning calorimetry;

[0047] TGA / DSC means thermogravimetric differential scanning calorimetry simultaneous thermal analysis;

[0048] Example 1: Preparation and Synthesis of Compound (S)-4-((1'-(3-hydroxy-2-phenylpropionyl)-1',4'-dihydro-2H,2'H-[3,3'-diazetidinylidene]-1(4H)-yl)sulfonyl)benzonitrile

[0049] The preparation and synthesis steps of (S)-4-((1'-(3-hydroxy-2-phenylpropionyl)-1',4'-dihydro-2H,2'H-[3,3'-diazetidinylidene]-1(4H)-yl)sulfonyl)benzonitrile (Compound 1) are as follows:

[0050]

[0051] Step 1: Preparation of tert-butyl 1'-((4-cyanophenyl)sulfonyl)-3-hydroxy-3'-iodo-[3,3'-diazetidine]-1-carboxylate (Compound 1A):

[0052]

[0053] 3-(1-azabicyclo[1.1.0]butan-3-yl)-3-hydroxyazetidine-1-carboxylic acid tert-butyl ester (3.65 g, 16.31 mmol) and sodium iodide (4.84 g, 32.26 mmol) were dissolved in acetonitrile (50 mL), the reaction system was cooled to 0°C, and then 4-cyanobenzenesulfonyl chloride (3.90 g, 19.36 mmol) was slowly added dropwise. After the addition was completed, the reaction was stirred at 0°C for 30 minutes. After the reaction was completed, compound 1A (5.93 g, yield was 70%) was purified.

[0054] MS (ESI) m / z 420.0 (M+H-100) +

[0055] Step 2: Preparation of tert-butyl 1'-((4-cyanophenyl)sulfonyl)-3'-iodo-3-((methylsulfonyl)oxy)-[3,3'-diazetidine]-1-carboxylate (Compound 1B):

[0056]

[0057] 1'-((4-cyanophenyl)sulfonyl)-3-hydroxy-3'-iodo-[3,3'-diazetidine]-1-carboxylic acid tert-butyl ester (5.93 g, 11.42 mmol) was dissolved in dichloromethane (60 mL). After cooling the reaction system to 0°C, triethylamine (2.31 g, 22.81 mmol) and 4-dimethylaminopyridine (2.79 g, 22.81 mmol) were added, followed by slow dropwise addition of methylsulfonyl chloride (1.57 g, 13.69 mmol). After the addition was complete, the reaction was stirred at 0°C for 30 minutes. After the reaction was complete, compound 1B (5.59 g, yield was 82%) was obtained by purification.

[0058] MS (ESI) m / z 498.0 (M+H-100) +

[0059] Step 3: Preparation of tert-butyl 1'-((4-cyanophenyl)sulfonyl)-1',4'-dihydro-2H-2'H-[3,3'-diazetidinylidene]-1(4H)-carboxylate (Compound 1C):

[0060]

[0061] 1'-((4-cyanophenyl)sulfonyl)-3'-iodo-3-((methylsulfonyl)oxy)-[3,3'-diazetidine]-1-carboxylic acid tert-butyl ester (5.59 g, 9.36 mmol) was dissolved in acetic acid (50 mL). Zinc powder (3.06 g, 46.78 mmol) was then added. After the addition was completed, the reaction system was stirred at room temperature for 3 hours. After the reaction was completed, compound 1C (3.09 g, yield was 88%) was purified.

[0062] MS (ESI) m / z 276.1 (M+H-100) +

[0063] Step 4: Preparation of 4-((1',4'-dihydro-2H,2'H-[3,3'-diazetidinylidene]-1(4H)-yl)sulfonyl)benzonitrile (Compound 1D):

[0064]

[0065] Dissolve tert-butyl 1'-((4-cyanophenyl)sulfonyl)-1',4'-dihydro-2H,2'H-[3,3'-diazetidinylidene]-1(4H)-carboxylate (2.26 g, 8.23 ​​mmol) in trifluoroacetic acid (20 mL), and heat the reaction system to 60°C and stir for 1 hour. After the reaction is complete, concentrate and use directly in the next step.

[0066] MS (ESI) m / z 276.1 (M+H) +

[0067] Step 5: Preparation of (S)-4-((1'-(3-hydroxy-2-phenylpropionyl)-1',4'-dihydro-2H,2'H-[3,3'-diazetidinylidene]-1(4H)-yl)sulfonyl)benzonitrile (Compound 1):

[0068]

[0069] 4-((1',4'-dihydro-2H,2'H-[3,3'-diazetidinylidene]-1(4H)-yl)sulfonyl)benzonitrile (2.26 g, 8.21 mmol) and N,N-diisopropylethylamine (3.18 g, 24.64 mmol) were dissolved in DMF (50 mL), and HATU (4.68 g, 12.32 mmol) and (S)-3-hydroxy-2-phenylpropionic acid (1.36 g, 8.21 mmol) were added, and the reaction system was stirred at room temperature for 3 hours. After the reaction was complete, the compound 1 (2.33 g, yield was 67%) was obtained by reverse phase high pressure liquid preparative purification.

[0070] MS (ESI) m / z 424.1 (M+H) +

[0071] 1 H NMR (400MHz, DMSO-d6) δ8.19–8.13(m,2H),8.03–7.98(m,2H),7.32–7.18(m,5H),4.79(t,J=5.3Hz,1H),4.70(d,J= 12.5Hz,1H),4.33(d,J=15.0Hz,6H),4.19(d,J=14.4Hz,1H),3.88(m,1H),3.61(dd,J=8.8,5.5Hz,1H),3.45(m,1H).

[0072] Example 2: Preparation of amorphous

[0073] The purified solution containing (S)-4-((1'-(3-hydroxy-2-phenylpropionyl)-1',4'-dihydro-2H,2'H-[3,3'-diazotidine ylidene]-1(4H)-yl)sulfonyl)benzonitrile (Compound 1) (reverse high-pressure filtrate) was concentrated under reduced pressure to obtain a high-concentration aqueous solution; DCM was added for extraction and separation, and the organic phase was collected. The organic phase was concentrated under reduced pressure under heating in a water bath at 40°C to obtain an oily to white foamy solid, about 6 g. The obtained white foamy solid was detected by XRPD (X-ray powder diffractometer instrument model DX-2700, detection method conditions: diffraction light source: copper target (40KV, 30mA); scanning rate: 0.04° / s; scanning range: 3~35°(2θ)), and the detection results are as follows Figure 1 As shown, it was identified as amorphous.

[0074] Example 3: Preparation of Form A 1

[0075] Take 150 mg of the amorphous product obtained in Example 2, disperse it in 3 ml of methanol, and stir it at 150-200 r / min for 20 hours at ambient temperature (about 20-25°C). Filter to obtain a powdered solid, and dry it in vacuum at room temperature for about 16 hours. The dried solid was subjected to XRPD detection (X-ray powder diffractometer instrument model DX-2700, detection method conditions: diffraction light source: copper target (40KV, 30mA); scanning rate: 0.04° / s; scanning range: 3-35° (2θ)), and its XRPD spectrum is as follows Figure 2 As shown, Figure 2 The diffraction peak data of is shown in Table 1, and is named Form A.

[0076] Table 1 XRPD data of Form A

[0077]

[0078] The powdered solid crystal form A was tested by TGA / DSC (simultaneous thermal analyzer model Mettler Toledo TGA / DSC3 + , detection method conditions: heating rate: 10K / min; temperature range: 30~200℃), its TGA / DSC spectrum is as follows Figure 3 The TGA / DSC test results show that Form A has no obvious weight loss at 200°C. The powdered solid Form A was tested by DSC (differential scanning calorimeter model: Mettler Toledo DSC 3, test method conditions: heating rate: 10K / min; temperature range: 30-250°C), and its DSC spectrum is as follows: Figure 4 As shown. The DSC test results show that Form A has a sharp endothermic peak at 153±2℃ (onset). Combined with the above data analysis, it can be seen that Form A is a non-solvate.

[0079] In further parallel experimental studies, the methanol in the aforementioned preparation method of this embodiment was replaced with other types of organic solvents, and the remaining steps, methods, conditions and parameters remained unchanged. The prepared product was also tested by XPRD using the same method to identify the crystalline form. The results are shown in Table 2 below.

[0080] Table 2 Crystal identification results of different types of organic solvent products in Example 3

[0081]

[0082]

[0083] Example 4: Preparation of Form A 2

[0084] Take 150 mg of the amorphous product obtained in Example 2, disperse it in 4 ml of methanol, and stir it at 150-200 r / min for 48 h at 50°C. Filter to obtain a powdered solid, and dry it in vacuum at room temperature for about 16 h. The dried solid was subjected to XRPD detection (X-ray powder diffractometer instrument model DX-2700, detection method conditions: diffraction light source: copper target (40KV, 30mA); scanning rate: 0.04° / s; scanning range: 3-35° (2θ)), and its XRPD spectrum is consistent with the crystalline AXRPD obtained in Example 3.

[0085] In further parallel experimental studies, the methanol in the aforementioned preparation method of this embodiment was replaced with other types of organic solvents, and the remaining steps, methods, conditions and parameters remained unchanged. The prepared product was also tested by XPRD using the same method to identify the crystalline form. The results are shown in Table 3 below.

[0086] Table 3 Crystal identification results of different types of organic solvent products in Example 4

[0087] Organic solvents Product crystal form Organic solvents Product crystal form Organic solvents Product crystal form Ethanol Crystalline Form A Isopropyl alcohol Crystalline Form A n-Butanol Crystalline Form A Tert-Butanol Crystalline Form A Methyl acetate Crystalline Form A Ethyl acetate Crystalline Form A Isopropyl acetate Crystalline Form A Dimethyl carbonate Crystalline Form A Ether Crystalline Form A Isopropyl ether Crystalline Form A Methyl tert-butyl ether Crystalline Form A Butanone Crystalline Form A Methyl isobutyl ketone Crystalline Form A n-Hexane Crystalline Form A Cyclohexane Crystalline Form A n-Heptane Crystalline Form A Toluene Crystalline Form A Xylene Crystalline Form A Methyltetrahydrofuran Crystalline Form A

[0088] Example 5: Preparation of Form B

[0089] Take 150 mg of the amorphous product obtained in Example 2 and dissolve it in 6 ml of acetone. Filter it, seal the filtrate in a vial with a PE film, and pierce 4 holes with a 1 ml syringe needle. Let it evaporate slowly under static conditions to obtain a white solid, and filter it until there is basically no solvent residue to obtain a powdered solid. The obtained solid was subjected to XRPD detection (X-ray powder diffractometer instrument model DX-2700, detection method conditions: diffraction light source: copper target (40KV, 30mA); scanning rate: 0.04° / s; scanning range: 3~35°(2θ)), and its XRPD spectrum is as follows Figure 5 As shown, Figure 5 The diffraction peak data of is shown in Table 4, and is named Form B.

[0090] Table 4 XRPD data of Form B

[0091]

[0092]

[0093] The powdered solid crystal form B was tested by TGA / DSC (simultaneous thermal analyzer model Mettler Toledo TGA / DSC3 + , detection method conditions: heating rate: 10K / min; temperature range: 30~250℃), its TGA / DSC spectrum is as follows Figure 6 The TGA / DSC test results show that Form B has no obvious weight loss at 200°C. The powdered solid Form B was tested by DSC (differential scanning calorimeter model: Mettler Toledo DSC 3, test method conditions: heating rate: 10K / min; temperature range: 30-200°C), and its DSC spectrum is as follows Figure 7 As shown. The DSC test results show that Form B has two endothermic peaks at 92±2℃ (onset) and 153±2℃ (onset); and one exothermic peak at 106±2℃ (onset). This indicates that there is a crystal transformation process during the DSC test. Further experiments found that Form B will be transformed into a mixed crystal of Form A and Form B after vacuum drying at 25℃ for 12h, so the endothermic peak at 153±2℃ (onset) is the endothermic peak of Form A. Combined with the above data analysis, it can be seen that Form B is a non-solvate.

[0094] Example 6: Preparation of Form C

[0095] Take 150 mg of the amorphous product obtained in Example 2, disperse it in 2 ml of water, and stir it at 150-200 r / min for 20 h. Filter to obtain a powdered solid, and dry it in vacuum at room temperature for about 16 h. The dried solid was subjected to XRPD detection (X-ray powder diffractometer instrument model DX-2700, detection method conditions: diffraction light source: copper target (40 KV, 30 mA); scanning rate: 0.04° / s; scanning range: 3-35° (2θ)). The XRPD spectrum is as follows: Figure 8 As shown, Figure 8 The diffraction peak data of is shown in Table 5, and is named Form C.

[0096] Table 5 XRPD data of Form C

[0097]

[0098]

[0099] The powdered solid crystal form C was tested by TGA / DSC (simultaneous thermal analyzer model Mettler Toledo TGA / DSC3 + , detection method conditions: heating rate: 10K / min; temperature range: 30~300℃), its TGA / DSC spectrum is as follows Fig. 9 As shown. TGA / DSC test results show that Form C starts to lose weight at about 60°C, about 1.8%. Powdered solid Form C was tested by DSC (differential scanning calorimeter model: Mettler Toledo DSC 3, test method conditions: heating rate: 10K / min; temperature range: 30-230°C), and its DSC spectrum is as shown Fig.10 The DSC test results show that Form C has two endothermic peaks at 103±2℃ (onset) and 152±2℃ (onset), and one exothermic peak at 117±2℃ (onset); the DSC spectrum of Form C with increasing and decreasing temperatures ( Fig.11 ) It was confirmed that the crystal form C began to transform into the crystal form A before the temperature reached 140°C during the detection process. HNMR comparison of the crystal form C and the crystal form A ( Fig.12 ) shows that the solvent peaks of Form C and Form A are almost the same, and do not contain organic solvent peaks, so the weight loss component is water.

[0100] Powdered solid crystal form C was heated and dried at 100°C for 3 hours, and the XRPD was re-measured using the same instrument. The XPRD comparison chart of crystal form C before and after heating and drying at 100°C is as follows ( Fig.13 ) shows that the crystal form does not change before and after heating and drying at 100°C, and is still crystal form C. The TGA / DSC detection spectrum of crystal form C after heating and drying at 100°C for 3h ( Fig.14) showed that Form C began to lose weight at around 60°C, with a total weight loss of about 1.3%.

[0101] Combining the above two weight loss data, it can be known that Form C is a hydrate, wherein the hydration amount is about 1.3-1.8%.

[0102] Example 7: Preparation of Form D

[0103] Take 150 mg of the amorphous product obtained in Example 2, dissolve it in acetonitrile-acetone (1:5-5:1), filter it, seal the filtrate in a vial with a PE film, and pierce 4 holes with a 1 ml syringe needle. Slowly evaporate under static conditions to obtain a white solid. The obtained solid is subjected to XRPD detection (X-ray powder diffractometer instrument model DX-2700, detection method conditions: diffraction light source: copper target (40KV, 30mA); scanning rate: 0.04° / s; scanning range: 3-35° (2θ)), and its XRPD spectrum is as follows Fig.15 As shown, Fig.15 The diffraction peak data of is shown in Table 6, and is named as Form D.

[0104] Table 6 XRPD data of Form D

[0105]

[0106]

[0107] The powdered solid crystal form D was tested by TGA / DSC (simultaneous thermal analyzer model Mettler Toledo TGA / DSC3 + , detection method conditions: heating rate: 10K / min; temperature range: 30~200℃), its TGA / DSC spectrum is as follows Fig.16 The TGA / DSC test results show that Form D starts to lose weight at about 60°C, about 2.6%. The powdered solid Form D was tested by DSC (differential scanning calorimeter model: Mettler Toledo DSC 3, test method conditions: heating rate: 10K / min; temperature range: 30-200°C), and its DSC spectrum is as follows Fig.17 The DSC test results show that Form D has two endothermic peaks at 82±2℃ (onset) and 152±2℃ (onset); Form D has an exothermic peak at 91±2℃ (onset), and the second exothermic peak coincides with Form A. Therefore, Form D will be converted to Form A during the DSC test.

[0108] Test Example 1: Thermodynamic Stability

[0109] 80 mg of each of the above-mentioned crystal forms A, B, and C were weighed and placed in different glass dishes, and then the glass dishes were dried at 60° C. for 20 h, and then further dried at 100° C. for 12 h. The samples after the two dryings were respectively subjected to XPRD detection for crystal form identification, and the results are shown in Table 6 below.

[0110] Table 6 Thermodynamic stability investigation results of crystal forms A, B and C

[0111] Starting crystal form Crystalline Form A Form B Crystalline Form C Crystal form after drying Crystalline Form A Crystalline Form A Crystalline Form C

[0112] The results show that Form A and Form C have good high-temperature crystal stability. Good high-temperature crystal stability and high crystal melting point can maintain the stability of the crystal under local high temperature conditions during the drug preparation process (such as micronization).

[0113] Test Example 2: Water System Stability and Grinding Stability

[0114] Weigh 1000 mg of the above-mentioned crystal form A, 500 mg of each of the crystal forms A and C and mix them evenly, and conduct competition tests on the two samples under the following conditions. The samples after the competition test are subjected to XPRD detection for crystal form identification, and the results are shown in Table 7 below.

[0115] Table 7 Competitive test results of crystal forms A and C under different conditions

[0116] Starting crystal form Competition test conditions result Crystalline Form A Pulping in water at room temperature for 46 hours Crystalline Form A Crystal form A + crystal form C Pulping in water at room temperature for 46 hours Crystalline Form C Crystalline Form A Grinding Crystalline Form A Crystalline Form C Grinding Crystalline Form C

[0117] The results show that in a water system, single crystal form A can exist stably; if it coexists with crystal form C, crystal form A will turn into crystal form C. Therefore, crystal form C has better stability in a water system. After grinding, both crystal forms A and C maintain the same crystal form, indicating that both crystal forms A and C have good grinding stability, which is conducive to the crushing and grinding operation in the subsequent drug-making stage.

[0118] Test Example 3: Solubility

[0119] The amorphous product of Example 2, the crystal form A of Example 3, and the crystal form C of Example 6 were taken and their solubility in water under different pH conditions was tested respectively (1 mol / l hydrochloric acid and sodium hydroxide aqueous solution were used to adjust the required pH value, and the dissolution process control conditions were: oscillation 133 r / min + 37°C / 24h). The test results are shown in Table 8 below.

[0120] Table 8 Solubility determination results at different pH

[0121]

[0122] Note: Solubility unit is "μg / ml".

[0123] The results showed that Form A unexpectedly had a solubility similar to that of the amorphous form, which was much higher than that of Form C.

[0124] Test Example 4: Chemical Stability

[0125] The amorphous product of Example 2, the crystalline form A of Example 3, and the crystalline form C of Example 6 were subjected to chemical stability tests for content changes and crystalline form changes under various conditions. The results are shown in Table 9 below.

[0126] Table 9 Crystal stability under different conditions

[0127]

[0128]

[0129] The results show that both crystal forms A and C are prepared from amorphous forms. Comparing the chemical contents of the two at 0d, it can be seen that crystal form A has better impurity removal ability than crystal form C. Crystal forms A and C have better crystal stability and chemical stability than amorphous forms.

[0130] Test Example 5: PKR activation and PKM2 activation activity test

[0131] Activity tests have shown that the compound (S)-4-((1'-(3-hydroxy-2-phenylpropionyl)-1',4'-dihydro-2H,2'H-[3,3'-diazetidine subunit]-1(4H)-yl)sulfonyl)benzonitrile has an activity against AC activated by PKR enzyme. 50 AC with a value of 101 to 300 nM for PKM2 enzyme activation in human brain glioblastoma (U118-MG) 50 The value is 0~100nM.

[0132] The corresponding activity test methods are as follows:

[0133] Pyruvate kinase (PKR) activity assay

[0134] Reagents and consumables:

[0135] Reagent name Suppliers Part Number PKLR Var2 active human Sigma-Aldrich 9001-59-6 DMSO West Asia Reagent 20190701 Pyruvate Kinase Assay Kit Abcam ab83432

[0136] instrument:

[0137] 1. Test steps:

[0138] 1 Prepare recombinant human PKR enzyme solution with a concentration of 60 ng / well (20 μL reaction system) and add 5 μL to each well of a black transparent bottom 384-well plate.

[0139] 2 Prepare compound solutions of different concentrations, with a final concentration of 1000 / 300 / 100 nM (containing 1% DMSO), add 5 μL of compound to the 3.1-well plate, mix well, and incubate at room temperature for 10 min.

[0140] 3 Prepare the reaction system according to the kit instructions (as shown in the table below), add 10 μL to each well of the 384-well plate, and mix with the solution in 3.2.

[0141] Item μL <![CDATA[OxiRed TM Probe]]> 0.08 HRP Enzyme Mix (Lyophilized) 0.2 PK Substrate Mix (Lyophilized) 0.4 PK Assay Buffer 9.32

[0142] 4 Place the 384-well plate on an enzyme reader to detect the fluorescence value. Detect the fluorescence at 540 / 590 nm, read the value every 2 minutes, and continue measuring for 1 hour.

[0143] 5. Calculate the Slope value within the measurement time, and use the 0.1% DMSO wells as control wells to calculate the compound activation rate.

[0144] Activation rate % = (compound well Slope / control well Slope) × 100%

[0145] 6 Fitting dose-effect curve

[0146] The log value of compound concentration was used as the X-axis and the percentage activation rate was used as the Y-axis. The log (agonist) vs. response-variable slope (Variable slope) of the analysis software GraphPadPrism 5 was used to fit the dose-effect curve to obtain the AC value of each compound on PKR activity. 50 value.

[0147] Calculation formula: Y = min + (max - min) / (1 + 10^((LogAC 50 -X)×Hillslope)).

[0148] PKM2 cell activity assay

[0149] 1. Purpose of the experiment

[0150] The compounds of the present invention were tested for their effects on activation of PKM2 in human brain glioblastoma (U118-MG).

[0151] 2. Experimental Materials and Instruments

[0152] Reagents and consumables

[0153]

[0154] instrument

[0155] 3. Experimental Procedure

[0156] 3.1 The cultured U118-MG cells were digested with trypsin, seeded in a 96-well plate at a density of 2500 cells / well (100 μL), and placed in a CO2 incubator to adhere overnight.

[0157] 3.2 Dilute the test compound and add it to the 96-well plate in 3.1, add 100 μL to each well, and the final concentration of the compound is 3000 / 1000 / 300 nM (containing 1% DMSO). Incubate the cells and the compound in a 37°C, 5% CO2 incubator for 4 hours. Discard the supernatant, add 100 μL PBS to each well to wash the residual culture medium, discard the PBS, and add 100 μL Pierce TM The IP lysate was shaken for 20 min, centrifuged at 4000 g for 10 min, and the supernatant was taken for detection.

[0158] 3.3 Prepare the reaction system according to the kit instructions (as shown in the table below), add 10 μL to each well of the 384-well plate, and take 10 μL of cell lysate and mix well (20 μL reaction system).

[0159] Item μL <![CDATA[OxiRed TM Probe]]> 0.08 HRP Enzyme Mix (Lyophilized) 0.2 PK Substrate Mix (Lyophilized) 0.4 PK Assay Buffer 9.32

[0160] 3.4 Place a black transparent bottom 384-well plate on an ELISA reader to detect the fluorescence value. Detect the fluorescence at 540 / 590 nm, read the value every 2 minutes, and measure continuously for 1 hour.

[0161] 3.5 Calculate the Slope value within the measurement time, take the 0.1% DMSO well as the control well, and calculate the compound activation rate.

[0162] Activation rate % = (compound well Slope / control well Slope) × 100%

[0163] 3.6 Fitting the dose-effect curve

[0164] The log value of compound concentration was used as the X-axis and the percentage activation rate was used as the Y-axis. The log (agonist) vs. response-variable slope (Variable slope) of the analysis software GraphPadPrism 5 was used to fit the dose-effect curve to obtain the AC value of each compound on PKM2 activity. 50 value.

[0165] Calculation formula: Y = min + (max - min) / (1 + 10^((LogAC 50 -X)×Hillslope)).

[0166] The present invention is not limited to the above optional implementations, and anyone can derive other various forms of products under the enlightenment of the present invention. The above specific implementations should not be understood as limiting the scope of protection of the present invention. The scope of protection of the present invention should be based on the definition in the claims, and the description can be used to interpret the claims.

Claims

1. A crystalline compound of (S)-4-((1'-(3-hydroxy-2-phenylpropionyl)-1',4'-dihydro-2H,2'H-[3,3'-diazetidinylidene]-1(4H)-yl)sulfonyl)benzonitrile.

2. The crystalline compound according to claim 1, which is Form A, and the X-ray powder diffraction pattern of Form A comprises diffraction peaks at 2θ values ​​of 5.5±0.2°, 11.0±0.2°, 17.0±0.2°, and 20.4±0.2°.

3. The crystalline compound according to claim 2, wherein the X-ray powder diffraction pattern of Form A further comprises diffraction peaks at 2θ values ​​of 21.3±0.2° and 24.2±0.2°.

4. The crystalline compound according to claim 2 or 3, wherein the X-ray powder diffraction pattern of Form A further comprises one or more diffraction peaks at 2θ values ​​selected from 14.9±0.2°, 15.1±0.2°, 16.4±0.2°, 16.6±0.2°, 22.1±0.2°, 23.2±0.2°, 24.7±0.2°, 26.3±0.2°, 27.6±0.2°, 28.6±0.2°, and 29.5±0.2°.

5. The crystalline compound according to claim 4, wherein the X-ray powder diffraction pattern of Form A further comprises one or more diffraction peaks at 2θ values ​​selected from 13.7±0.2°, 15.9±0.2°, 18.8±0.2°, 19.4±0.2°, 22.7±0.2°, 25.3±0.2°, 25.8±0.2°, 29.0±0.2°, 30.3±0.2°, and 30.5±0.2°.

6. The crystalline compound according to claim 2, wherein Form A has an X-ray powder diffraction pattern as shown in FIG2.

7. The crystalline compound according to claim 2, wherein Form A has a melting point of 153±2°C.

8. The crystalline compound according to claim 2, wherein Form A has a DSC graph as shown in FIG4.

9. The method for preparing the crystalline compound according to any one of claims 1 to 8, characterized in that: The method comprises the following steps: taking a compound (S)-4-((1'-(3-hydroxy-2-phenylpropionyl)-1',4'-dihydro-2H,2'H-[3,3'-diazetidinylidene]-1(4H)-yl)sulfonyl)benzonitrile, dispersing the compound in an organic solvent, stirring the mixture and filtering the mixture to obtain a powdery solid, thereby obtaining the crystalline compound; The organic solvent is selected from methanol, ethanol, isopropanol, n-butanol, tert-butanol, methyl acetate, ethyl acetate, isopropyl acetate, dimethyl carbonate, ethyl ether, isopropyl ether, methyl tert-butyl ether, butanone, methyl isobutyl ketone, n-hexane, cyclohexane, n-heptane, toluene, xylene or methyltetrahydrofuran.

10. The preparation method according to claim 9, characterized in that: The stirring time is more than 20 hours, and can be 20 to 48 hours. The system temperature during the stirring is room temperature or 20 to 50°C.

11. The preparation method according to claim 9, characterized in that: The obtained compound (S)-4-((1'-(3-hydroxy-2-phenylpropionyl)-1',4'-dihydro-2H,2'H-[3,3'-diazetidinylidene]-1(4H)-yl)sulfonyl)benzonitrile is an amorphous state, and the amorphous state can be obtained by the following preparation method: The aqueous solution containing the compound (S)-4-((1'-(3-hydroxy-2-phenylpropionyl)-1',4'-dihydro-2H,2'H-[3,3'-diazetidinylidene]-1(4H)-yl)sulfonyl)benzonitrile was concentrated under reduced pressure, DCM was added for extraction and separation, the organic phase was collected, and the organic phase was concentrated under reduced pressure to obtain the amorphous product.

12. (S)-4-((1'-(3-hydroxy-2-phenylpropionyl)-1',4'-dihydro-2H,2'H-[3,3'-diazetidinylidene]-1(4H)-yl)sulfonyl)benzonitrile hydrate.

13. The hydrate according to claim 12, which is in crystalline form.

14. The hydrate according to claim 13, wherein the crystalline form is Form C, and the X-ray powder diffraction pattern of Form C comprises diffraction peaks at 2θ values ​​of 12.0±0.2°, 12.2±0.2°, and 20.1±0.2°.

15. The hydrate according to claim 14, wherein the X-ray powder diffraction pattern of the crystalline form C further comprises a diffraction peak at a 2θ value of 25.7±0.2°.

16. The hydrate according to claim 15, wherein the X-ray powder diffraction pattern of the crystalline form C further comprises one or more diffraction peaks at 2θ values ​​selected from 9.2±0.2°, 12.4±0.2°, 17.4±0.2°, 18.5±0.2°, 19.4±0.2°, 21.0±0.2°, 21.5±0.2°, 22.4±0.2°, 24.0±0.2°, 24.6±0.2°, 27.2±0.2°, 27.8±0.2°, 28.6±0.2°, and 28.9±0.2°.

17. The hydrate according to claim 16, wherein the X-ray powder diffraction pattern of the crystalline form C further comprises one or more diffraction peaks at 2θ values ​​selected from 4.6±0.2°, 13.4±0.2°, 13.8±0.2°, 16.0±0.2°, 16.7±0.2°, 18.0±0.2°, 22.6±0.2°, 23.2±0.2°, 25.4±0.2°, 26.7±0.2°, 29.3±0.2°, 30.0±0.2°, and 30.4±0.2°.

18. The hydrate according to claim 14, wherein the crystalline form C has an X-ray powder diffraction pattern as shown in FIG8.

19. The hydrate according to claim 14, wherein the crystalline form C has a melting point of 103±2°C.

20. The hydrate according to claim 14, wherein the crystalline form C has a DSC chart as shown in FIG10.

21. The method for preparing the hydrate according to any one of claims 12 to 20, characterized in that: The method comprises the following steps: taking the compound (S)-4-((1'-(3-hydroxy-2-phenylpropionyl)-1',4'-dihydro-2H,2'H-[3,3'-diazetidine substituent]-1(4H)-yl)sulfonyl)benzonitrile, dispersing it in water, stirring it and filtering it to obtain a powdery solid, thereby obtaining the hydrate.

22. The preparation method according to claim 21, characterized in that: The stirring time is more than 20 hours, and can be 20 to 48 hours. The system temperature during the stirring is room temperature or 20 to 50°C.

23. The preparation method according to claim 21, characterized in that: The obtained compound (S)-4-((1'-(3-hydroxy-2-phenylpropionyl)-1',4'-dihydro-2H,2'H-[3,3'-diazetidinylidene]-1(4H)-yl)sulfonyl)benzonitrile is an amorphous state, and the amorphous state can be obtained by the following preparation method: The aqueous solution containing the compound (S)-4-((1'-(3-hydroxy-2-phenylpropionyl)-1',4'-dihydro-2H,2'H-[3,3'-diazetidinylidene]-1(4H)-yl)sulfonyl)benzonitrile was concentrated under reduced pressure, DCM was added for extraction and separation, the organic phase was collected, and the organic phase was concentrated under reduced pressure to obtain the amorphous product.

24. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the crystalline compound according to any one of claims 1 to 8, or comprising a pharmaceutically acceptable excipient and the hydrate according to any one of claims 12 to 20.

25. Use of the crystalline compound according to any one of claims 1 to 8 or the hydrate according to any one of claims 12 to 20 as a PKM2 agonist or a PKR agonist in the preparation of a medicament for treating PKM2 or PKR related diseases.

26. Use of the crystalline compound according to any one of claims 1 to 8 or the hydrate according to any one of claims 12 to 20 in the preparation of a medicament for treating liver cancer, lung cancer, colorectal cancer, glioblastoma, breast cancer, lymphoma, pancreatic cancer, renal cancer, experimental autoimmune encephalomyelitis, inflammatory bowel disease, ulcerative colitis, alopecia, alopecia or hypotrichosis.

27. Use of the crystalline compound according to any one of claims 1 to 8 or the hydrate according to any one of claims 12 to 20 in the preparation of a medicament for treating myelodysplastic syndrome (MDS), hemoglobinopathy, sickle cell anemia (SCD), β-thalassemia, hereditary non-spherocytic hemolytic anemia, hemolytic anemia, hereditary spherocytosis, hereditary elliptocytosis, abetalipoproteinemia, paroxysmal nocturnal hemoglobinuria, acquired hemolytic anemia, congenital anemia or anemia of chronic disease.

28. Use of the crystalline compound according to any one of claims 1 to 8 or the hydrate according to any one of claims 12 to 20 in the preparation of a medicament for treating pyruvate kinase deficiency (PKD), sickle cell anemia (SCD), β-thalassemia or low-risk myelodysplastic syndrome (LR-MDS).

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