Crystals of trifluoromethyl and chloro disubstituted sulfonamides as selective BCL-2 inhibitors

Abstract

A trifluoromethyl- and chloro-disubstituted sulfonamide-based selective BCL-2 inhibitor crystal, specifically a crystal of a compound of formula I, a preparation method, and its use in preventing and treating diseases associated with the anti-apoptotic protein BCL-2, such as cancer. #imgabs0#

Description

[0001] Citation of Related Applications

[0002] This application claims priority and benefits of Chinese Patent Application No. 202010355842.4 filed with the State Intellectual Property Office of the People's Republic of China on April 29, 2020, the entire contents of which are hereby incorporated by reference into the text in their entirety. Technical Field

[0003] The present application relates to crystals of trifluoromethyl and chlorine-disubstituted sulfonamide-type selective BCL-2 inhibitors, preparation methods, and uses thereof in preventing and treating diseases associated with the anti-apoptotic protein BCL-2, such as cancer. Background Art

[0004] BCL-2 proteins are divided into three families: BCL-2 family (whose family members include BCL-2, BCL-XL, etc.), BAX family and BH3-only family. Among them, the BCL-2 family plays an anti-apoptotic role, while members of the latter two families play a pro-apoptotic role.

[0005] Anti-apoptotic BCL-2 family proteins are associated with many diseases and are being studied as potential therapeutic drug targets. These targets for interventional therapy include, for example, BCL-2 family proteins BCL-2 and BCL-XL. Recently, inhibitors of BCL-2 family proteins have been reported in WO2012071374, WO2010138588, and WO2010065865. Although inhibitors with high binding to the target protein are taught therein, compound binding affinity is only one of many parameters to be considered. One goal is to produce compounds that preferentially bind to one protein relative to another, i.e., are selective for it. To demonstrate this selectivity, it is known that a compound exhibits high binding affinity to a specific protein and lower binding affinity to another member.

[0006] This application discloses a compound of Formula I, which exhibits high selectivity for the anti-apoptotic BCL-2 protein and the anti-apoptotic BCL-XL protein, and exhibits excellent performance in inhibiting the activity of the anti-apoptotic BCL-2 protein. Furthermore, it exhibits good liver microsomal stability and optimized pharmacokinetic parameters, suggesting promising drug development prospects.

[0007] Drugs are generally expected to possess excellent properties in terms of pharmaceutical activity, pharmacokinetics, bioavailability, melting point, stability, hygroscopicity, solubility, and the like. The crystals of the compound of Formula I developed herein exhibit low hygroscopicity and excellent stability, including stable product purity and content during storage, no crystal form changes, and ease of preparation. These crystals can meet the requirements for drug production, storage, and formulation.

[0008] Summary of the Invention

[0009] In one aspect, the present application provides a crystal of a compound of formula I,

[0010]

[0011] In some embodiments, the crystals of the compound of formula I of the present application are type A crystals, characterized in that the 2θ in the X-ray powder diffraction pattern has a peak at 5.01, 6.61, 8.12 or 20.13±0.2°; in some embodiments, the crystals are characterized in that the 2θ in the X-ray powder diffraction pattern has a peak at 5.01, 6.61, 8.12 or 20.13±0.2°. In some embodiments, the invention relates to a novel nanostructured ...

[0012] In other embodiments, the crystals of the compound of formula I of the present application are type A crystals, characterized in that the X-ray powder diffraction pattern has at least 7 or at least 8 diffraction peaks at 2θ selected from 5.01, 6.61, 8.12, 10.21, 12.88, 14.89, 16.63, 20.13 or 21.01±0.2°.

[0013] In some embodiments of the present application, the peak positions and relative intensities of the diffraction peaks in the XRPD pattern of the above-mentioned Type A crystal are shown in Table 1 below:

[0014] Table 1 XRPD pattern analysis data of type A crystal

[0015]

[0016] In some embodiments, the crystal of the compound of formula I of the present application is type A crystal, and its X-ray powder diffraction pattern is as follows: Figure 1 shown.

[0017] In some embodiments, the crystal of the compound of formula I of the present application is type A crystal, and its DSC spectrum is as follows: Figure 2 shown.

[0018] In some embodiments, the crystal of the compound of formula I of the present application is type A crystal, and its TG spectrum is as follows: Figure 3 shown.

[0019] The type A crystals of the compound of formula I may exist in the form of non-solvate crystals or solvate crystals, where the solvate refers to a solvate formed between an organic solvent and / or water and the corresponding compound.

[0020] In some embodiments, the present application provides a method for preparing Type A crystals of the compound of Formula I, comprising mixing the compound of Formula I with dichloromethane and methanol to dissolve the clear solution, and concentrating and isolating the solid. In some embodiments, the volume ratio of dichloromethane to methanol is selected from 20:1 to 100:1.

[0021] In some embodiments, in the above preparation method, the volume mass ratio of dichloromethane to the compound of formula I is 20-100 mL / g. In some embodiments, the volume mass ratio of dichloromethane to the compound of formula I is 40-60 mL / g, preferably 50-60 mL / g; the volume mass ratio of methanol to the compound of formula I is 0.1-10 mL / g, preferably 0.5-5 mL / g.

[0022] In some embodiments, in the above preparation method, reduced pressure concentration is used. For example, the preparation method of the type A crystal of the compound of formula I of the present invention comprises: dissolving the compound of formula I in a mixed solution of dichloromethane and methanol, stirring to dissolve, and concentrating under reduced pressure to obtain the type A crystal.

[0023] In some embodiments, the crystals of the compound of formula I of the present application are type B crystals, characterized in that the 2θ in the X-ray powder diffraction pattern has a peak at 5.31, 12.64, 19.08 or 24.21±0.2°; in some embodiments, it is characterized in that the 2θ in the X-ray powder diffraction pattern has a peak at 5.31, 10.65, 12.64, 14.23, 19.08, 19.91, 22.71 or 24.21±0.2°; in one In some embodiments, it is characterized in that the 2θ in the X-ray powder diffraction pattern has a peak at 5.31, 9.80, 10.65, 12.12, 12.64, 14.23, 16.04, 18.13, 19.08, 19.91, 22.71, 24.21 or 25.93±0.2°; in some embodiments, it is characterized in that the 2θ in the X-ray powder diffraction pattern has a peak at 5.31, 9.80, 10.65, 12.12, 12.64, 14.23, 16.04, 18.13, 19.08, 19.91, 22.71, 24.21 or 25.93±0.2°. .64, 13.57, 13.82, 14.23, 15.17, 16.04, 17.64, 18.13, 19.08, 19.91, 20.34, 22.71, 22.99, 23.45, 24.21, 25.65 or 25.93 ± 0.2 °; In some embodiments, it is characterized in that the 2θ in the X-ray powder diffraction pattern is 5.31, 9.50, 9.80, 10.65, 11.4 There are peaks at 1, 12.12, 12.64, 13.57, 13.82, 14.23, 15.17, 16.04, 16.64, 17.10, 17.64, 18.13, 18.33, 18.73, 19.08, 19.60, 19.91, 20.34, 21.22, 21.93, 22.71, 22.99, 23.45, 24.21, 25.65 or 25.93 ± 0.2°.

[0024] In other embodiments, the crystals of the compound of formula I of the present application are type B crystals, characterized in that the X-ray powder diffraction pattern has at least 6 or at least 7 diffraction peaks at 2θ selected from 5.31, 10.65, 12.64, 14.23, 19.08, 19.91, 22.71 or 24.21±0.2°.

[0025] In some embodiments of the present application, the peak positions and relative intensities of the diffraction peaks in the XRPD pattern of the above-mentioned Type B crystal are shown in Table 2 below:

[0026] Table 2 XRPD pattern analysis data of type B crystal

[0027]

[0028] In some embodiments, the crystal of the compound of formula I of the present application is type B crystal, and its X-ray powder diffraction pattern is as follows: Figure 4 shown.

[0029] In some embodiments, the crystal of the compound of formula I of the present application is type B crystal, and its DSC spectrum has an onset point of an endothermic peak at 179.42±5°C.

[0030] In some embodiments, the crystal of the compound of formula I of the present application is type B crystal, and its DSC spectrum is as follows: Figure 5 shown.

[0031] In some embodiments, the crystal of the compound of formula I of the present application is type B crystal, and its TG spectrum is as follows: Figure 6 shown.

[0032] The type B crystals of the compound of formula I may exist in the form of non-solvate crystals or solvate crystals, where the solvate refers to a solvate formed between an organic solvent and / or water and the corresponding compound.

[0033] On the other hand, the present application provides a method for preparing type B crystals of the compound of formula I, comprising mixing the compound of formula I with acetone and separating the solid.

[0034] In some embodiments, the volume mass ratio of acetone to the compound of formula I is 1 to 50 mL / g. In some embodiments, the volume mass ratio is 5 to 20 mL / g, preferably 10 mL / g.

[0035] In some embodiments, the method for preparing Type B crystals of the compound of Formula I described herein comprises: mixing the compound of Formula I with acetone, stirring at room temperature, collecting the precipitate by filtration, and vacuum drying to obtain the Type B crystals. In some embodiments, in the above preparation method, the vacuum drying is performed at 40-60°C.

[0036] In some embodiments, the crystals of the compound of formula I of the present application are C-type crystals, characterized in that 2θ in the X-ray powder diffraction pattern has a peak at 5.52, 7.56, 9.22, 11.04 or 17.43 ± 0.2°; in some embodiments, it is characterized in that 2θ in the X-ray powder diffraction pattern has a peak at 5.52, 7.56, 8.29, 9.22, 11.04, 15.81, 17.43, 18.51 or 22.59 ± 0.2°; in some embodiments, it is characterized in that 2θ in the X-ray powder diffraction pattern has a peak at 5.52, 7.56, 8.29, 9.22, 11.04, 15.17, 15.81, 17.43, 18.51 or 20.40 ± 0.2°; in some embodiments, it is characterized in that The characteristic is that the 2θ in the X-ray powder diffraction pattern has a peak at 5.52, 7.56, 8.29, 9.22, 11.04, 15.17, 15.81, 17.00, 17.43, 18.51, 19.70, 20.01, 20.40, 20.75 or 22.59 ± 0.2°; in some embodiments, it is characterized by an X-ray powder diffraction pattern There are peaks in 2θ at 5.52, 7.56, 8.29, 9.22, 10.66, 11.04, 12.94, 14.69, 15.17, 15.81, 16.63, 17.00, 17.43, 18.51, 19.70, 20.01, 20.40, 20.75, 22.59, 25.88, and 26.12 ± 0.2°.

[0037] In other embodiments, the crystals of the compound of formula I of the present application are type C crystals, characterized in that the X-ray powder diffraction pattern has at least 7 or at least 8 diffraction peaks at 2θ selected from 5.52, 7.56, 8.29, 9.22, 11.04, 15.81, 17.43, 18.51 or 22.59±0.2°.

[0038] In some embodiments of the present application, the peak positions and relative intensities of the diffraction peaks in the XRPD pattern of the above-mentioned Type C crystal are shown in Table 3 below:

[0039] Table 3 XRPD pattern analysis data of type C crystal

[0040]

[0041] In some embodiments, the crystal of the compound of formula I of the present application is type C crystal, and its X-ray powder diffraction pattern is as follows: Figure 7 shown.

[0042] In some embodiments, the crystal of the compound of formula I of the present application is type C crystal, and its DSC spectrum has an endothermic peak starting point at 205.65±5°C.

[0043] In some embodiments, the crystal of the compound of formula I of the present application is type C crystal, and its DSC spectrum is as follows: Figure 8 shown.

[0044] In some embodiments, the crystal of the compound of formula I of the present application is type C crystal, and its TG spectrum is as follows: Figure 9 shown.

[0045] The type C crystals of the compound of formula I may exist in the form of non-solvate crystals or in the form of solvate crystals. The solvate herein refers to a solvate formed between an organic solvent and / or water and the corresponding compound.

[0046] In some embodiments, the present application provides a method for preparing type C crystals of the compound of formula I, comprising mixing the compound of formula I with methanol and methyl tert-butyl ether, and isolating the solid.

[0047] In some embodiments, the above preparation method, wherein the volume mass ratio of methanol to the compound of formula I is 5-100 mL / g, and the volume mass ratio of methyl tert-butyl ether to the compound of formula I is 5-100 mL / g; in some embodiments, wherein the volume mass ratio of methanol to the compound of formula I is 10-50 mL / g, preferably 25 mL / g; the volume mass ratio of methyl tert-butyl ether to the compound of formula I is 10-50 mL / g, preferably 25 mL / g.

[0048] In some embodiments, the method for preparing Type C crystals of the compound of Formula I described herein comprises: mixing the compound of Formula I with methanol and methyl tert-butyl ether, stirring at room temperature, collecting the precipitate by filtration, and vacuum drying to obtain the Type C crystals. In some embodiments, in the above preparation method, the vacuum drying is performed at 40-60°C.

[0049] In some embodiments, the crystals of the compound of formula I of the present application are D-type crystals, characterized in that 2θ in the X-ray powder diffraction pattern has a peak at 4.78, 12.83, 16.24 or 22.33 ± 0.2°; in some embodiments, it is characterized in that 2θ in the X-ray powder diffraction pattern has a peak at 4.78, 10.52, 12.83, 16.24, 18.44, 19.41, 22.33 or 23.20 ± 0.2°; in some embodiments, it is characterized in that 2θ in the X-ray powder diffraction pattern has a peak at 4.78, 9.63, 10.52, 12.83, In some embodiments, the present invention comprises a peak at 2θ of 4.78, 7.46, 9.63, 10.52, 11.17, 12.83, 13.48, 14.42, 15.79, 16.24, 17.89, 18.44, 19.41, 19.61, 20.44, 22.33, 23.20, 26.48 or 27.05 ± 0.2°.

[0050] In other embodiments, the crystals of the compound of formula I of the present application are type D crystals, characterized in that the X-ray powder diffraction pattern has at least 6 or at least 7 diffraction peaks at 2θ selected from 4.78, 10.52, 12.83, 16.24, 18.44, 19.41, 22.33 or 23.20±0.2°.

[0051] In some embodiments of the present application, the peak positions and relative intensities of the diffraction peaks in the XRPD pattern of the above-mentioned D-type crystals are shown in Table 4 below:

[0052] Table 4 XRPD analysis data of type D crystals

[0053]

[0054] In some embodiments, the crystal of the compound of formula I of the present application is a D-type crystal, and its X-ray powder diffraction pattern is as follows: Figure 10 shown.

[0055] In some embodiments, the crystal of the compound of formula I of the present application is a type D crystal, and its DSC spectrum has an onset point of an endothermic peak at 176.47±5°C.

[0056] In some embodiments, the crystal of the compound of formula I of the present application is a D-type crystal, and its DSC spectrum is as follows: Figure 11 shown.

[0057] In some embodiments, the crystal of the compound of formula I of the present application is a D-type crystal, and its TG spectrum is as follows: Figure 12 shown.

[0058] The type D crystals of the compound of formula I may exist in the form of non-solvate crystals or in the form of solvate crystals, where the solvate refers to a solvate formed between an organic solvent and / or water and the corresponding compound.

[0059] On the other hand, the present application provides a method for preparing type D crystals of the compound of formula I, comprising mixing the compound of formula I with a solvent selected from tetrahydrofuran, a mixture of tetrahydrofuran and water, isopropanol or 1,4-dioxane, and then isolating the solid.

[0060] In the above preparation method, the volume mass ratio of the compound of formula I to the solvent is 5 to 200 mL / g; in some embodiments, the above volume mass ratio is 20 to 100 mL / g.

[0061] In some embodiments, in the mixture of tetrahydrofuran and water, the volume ratio of tetrahydrofuran to water is 20:1 to 0.1:1; preferably 10:1 to 0.5:1.

[0062] In some embodiments, the method for preparing the type D crystals of the compound of formula I described in the present invention comprises: mixing the compound of formula I with a solvent, stirring at room temperature, collecting the precipitate by filtration, and air drying to obtain the type D crystals.

[0063] In some embodiments, in the above preparation method, the forced air drying is performed at 40-60°C.

[0064] In some embodiments, the method for preparing type D crystals of the compound of formula I described in the present invention comprises: mixing the compound of formula I with a solvent, stirring and dissolving at room temperature, filtering to obtain a supernatant, transferring the supernatant to a clean container, and slowly evaporating the solvent at room temperature to obtain the type D crystals.

[0065] In some embodiments, the crystals of the compound of formula I of the present application are E-type crystals, characterized in that 2θ in the X-ray powder diffraction pattern has a peak at 4.22, 10.72, 15.17 or 15.65 ± 0.2°; in some embodiments, it is characterized in that 2θ in the X-ray powder diffraction pattern has a peak at 4.22, 10.72, 14.62, 15.17, 15.65, 17.54, 19.55, 19.80 or 21.50 ± 0.2°; in some embodiments, it is characterized in that 2θ in the X-ray powder diffraction pattern has a peak at 4.22, 10.72, 13.82, 14.62, 15.17, 15.65, 16.92 , 17.54, 19.55, 19.80, 21.50, 22.76, 23.35, or 26.06±0.2°; in some embodiments, it is characterized in that the 2θ in the X-ray powder diffraction pattern has a peak at 4.22, 7.99, 8.75, 9.91, 10.72, 11.66, 12.75, 13.82, 14.62, 15.17, 15.65, 16.24, 16.92, 17.54, 19.04, 19.55, 19.80, 20.18, 21.50, 22.76, 23.35, 26.06, 26.91, 29.82 or 30.56±0.2°.

[0066] In other embodiments, the crystals of the compound of formula I of the present application are E-type crystals, characterized in that the X-ray powder diffraction pattern has at least 7 or at least 8 diffraction peaks at 2θ selected from 4.22, 10.72, 14.62, 15.17, 15.65, 17.54, 19.55, 19.80 or 21.50±0.2°.

[0067] In some embodiments of the present application, the peak positions and relative intensities of the diffraction peaks in the XRPD pattern of the above-mentioned E-type crystals are shown in Table 5 below:

[0068] Table 5 XRPD pattern analysis data of E-type crystals

[0069]

[0070] In some embodiments, the crystal of the compound of formula I of the present application is E-type crystal, and its X-ray powder diffraction pattern is as follows: Figure 13 shown.

[0071] In some embodiments, the crystal of the compound of formula I of the present application is type E crystal, and its DSC spectrum has an endothermic peak starting point at 145.48±5°C.

[0072] In some embodiments, the crystals of the compound of formula I of the present application are E-type crystals, and their DSC spectrum is as follows: Figure 14 shown.

[0073] In some embodiments, the crystal of the compound of formula I of the present application is E-type crystal, and its TG spectrum is as follows: Figure 15 shown.

[0074] The E-type crystals of the compound of formula I may exist in the form of non-solvate crystals or in the form of solvate crystals. The solvate herein refers to a solvate formed between an organic solvent and / or water and the corresponding compound.

[0075] In some embodiments, the present application provides a method for preparing type E crystals of the compound of formula I, comprising mixing the compound of formula I with p-xylene, and separating and drying the solid.

[0076] In some embodiments, the drying is vacuum drying at 50-120° C., preferably 80-100° C., for 5-10 hours.

[0077] In the above preparation method, the volume mass ratio of p-xylene to the compound of formula I is 5 to 55 mL / g; in some embodiments, the above volume mass ratio is 10 to 40 mL / g, preferably 20 mL / g.

[0078] In some embodiments, the method for preparing the type E crystals of the compound of formula I described in the present invention comprises: mixing the compound of formula I with a solvent, stirring at room temperature, filtering to collect the solid precipitate, and vacuum drying the filtered solid to obtain the type E crystals.

[0079] In some embodiments, the filtered solid is vacuum dried at 50-120° C. or 80-100° C. In some embodiments, the filtered solid is vacuum dried at 50-120° C., preferably 80-100° C., for 5-10 hours.

[0080] In some embodiments, the crystal of the compound of formula I of the present application is a type F crystal, characterized in that the 2θ in the X-ray powder diffraction pattern has a peak at 4.54, 9.08, or 19.24 ± 0.2°; in some embodiments, it is characterized in that the 2θ in the X-ray powder diffraction pattern has a peak at 4.54, 9.08, 14.90, 18.25, 19.24, 22.86, or 23.50 ± 0.2°; in some embodiments, it is characterized in that the 2θ in the X-ray powder diffraction pattern has a peak at 4.54, 9.08, 13.66, 14.90, 1 In some embodiments, the present invention comprises a peak at 7.46, 18.25, 19.24, 22.86, 23.50, 24.75 or 27.51 ± 0.2°; in some embodiments, the present invention is characterized in that the 2θ in the X-ray powder diffraction pattern has a peak at 4.54, 8.20, 9.08, 13.66, 13.97, 14.51, 14.90, 15.51, 16.63, 17.46, 18.25, 19.24, 19.82, 21.11, 22.37, 22.86, 23.50, 24.75 or 27.51 ± 0.2°.

[0081] In other embodiments, the crystals of the compound of formula I of the present application are type F crystals, characterized in that the X-ray powder diffraction pattern has at least 8 or at least 9 or at least 10 diffraction peaks at 2θ selected from 4.54, 9.08, 13.66, 14.90, 17.46, 18.25, 19.24, 22.86, 23.50, 24.75 or 27.51±0.2°.

[0082] In some embodiments of the present application, the peak positions and relative intensities of the diffraction peaks in the XRPD pattern of the above-mentioned Type F crystal are shown in Table 6 below:

[0083] Table 6 XRPD pattern analysis data of type F crystal

[0084]

[0085]

[0086] In some embodiments, the crystal of the compound of formula I of the present application is type F crystal, and its X-ray powder diffraction pattern is as follows: Figure 16 shown.

[0087] In some embodiments, the crystal of the compound of formula I of the present application is type F crystal, and its DSC spectrum has an onset point of an endothermic peak at 171.37±5°C.

[0088] In some embodiments, the crystal of the compound of formula I of the present application is type F crystal, and its DSC spectrum is as follows: Figure 17 shown.

[0089] In some embodiments, the crystal of the compound of formula I of the present application is type F crystal, and its TG spectrum is as follows: Figure 18 shown.

[0090] The type F crystals of the compound of formula I may exist in the form of non-solvate crystals or in the form of solvate crystals. The solvate herein refers to a solvate formed between an organic solvent and / or water and the corresponding compound.

[0091] On the other hand, the present application provides a method for preparing type F crystals of the compound of formula I, comprising mixing the compound of formula I with acetonitrile or nitromethane and separating the solid.

[0092] In the above preparation method, the volume mass ratio of acetonitrile or nitromethane to the compound of formula I is 5 to 200 mL / g; in some embodiments, the above volume mass ratio is 20 to 100 mL / g, preferably 50 mL / g.

[0093] In some embodiments, the method for preparing Type F crystals of the compound of Formula I described herein comprises: mixing the compound of Formula I with acetonitrile or nitromethane, stirring at room temperature, collecting the precipitate by filtration, and vacuum drying to obtain the Type F crystals. In some embodiments, in the above preparation method, the vacuum drying is performed at 40-60°C.

[0094] In some embodiments, the crystals of the compound of formula I of the present application are type G crystals, characterized in that 2θ in the X-ray powder diffraction pattern has a peak at 3.84, 10.39, 13.39 or 20.63±0.2°; in some embodiments, it is characterized in that 2θ in the X-ray powder diffraction pattern has a peak at 3.84, 10.39, 11.22, 13.39, 15.55, 16.78, 20.01 or 20.63±0.2°; in some embodiments, it is characterized in that 2θ in the X-ray powder diffraction pattern has a peak at 3.84, 7.72, 9.56, 10.39, 11.22, 12.47, .39, 14.01, 15.00, 15.55, 16.78, 18.59, 19.02, 19.47, 20.01, 20.63, 22.50, 23.44, 23.69, 24.11, 25.48 or 26.47 ± 0.2 °.

[0095] In other embodiments, the crystals of the compound of formula I of the present application are type G crystals, characterized in that the X-ray powder diffraction pattern has at least 6 or at least 7 diffraction peaks at 2θ selected from 3.84, 10.39, 11.22, 13.39, 15.55, 16.78, 20.01 or 20.63±0.2°.

[0096] In some embodiments of the present application, the peak positions and relative intensities of the diffraction peaks in the XRPD pattern of the above-mentioned Type G crystals are shown in Table 7 below:

[0097] Table 7 XRPD pattern analysis data of type G crystal

[0098]

[0099]

[0100] In some embodiments, the crystal of the compound of formula I of the present application is a G-type crystal, and its X-ray powder diffraction pattern is as follows: Figure 19 shown.

[0101] The type G crystals of the compound of formula I may exist in the form of non-solvate crystals or in the form of solvate crystals. The solvate herein refers to a solvate formed between an organic solvent and / or water and the corresponding compound.

[0102] In some embodiments, the present application provides a method for preparing type G crystals of the compound of formula I, comprising mixing the compound of formula I with p-xylene, separating the solid, and not drying the obtained solid.

[0103] In the above preparation method, the volume mass ratio of p-xylene to the compound of formula I is 60 to 200 mL / g; in some embodiments, the above volume mass ratio is 80 to 100 mL / g, preferably 94 mL / g.

[0104] In one embodiment, the method for preparing the type G crystal of the compound of formula I described in the present invention comprises: mixing the compound of formula I with p-xylene, stirring at room temperature, and filtering and collecting the precipitate to obtain the type G crystal.

[0105] In some embodiments, the crystals of the compound of formula I of the present application are H-type crystals, characterized in that 2θ in the X-ray powder diffraction pattern has a peak at 4.65, 12.23, 14.09 or 22.04±0.2°; in some embodiments, it is characterized in that 2θ in the X-ray powder diffraction pattern has a peak at 4.65, 9.36, 12.23, 13.33, 14.09, 17.27, 19.37, 22.04 or 22.95±0.2°; in some embodiments, it is characterized in that 2θ in the X-ray powder diffraction pattern has a peak at 4.65, 9.36, 10.41, 12.23, 13.33, 1 18.88, 19.37, 20.58, 22.04, 22.49, 22.95, or 23.69 ± 0.2°; in some embodiments, it is characterized in that the 2θ in the X-ray powder diffraction pattern has a peak at 4.65, 7.20, 9.36, 10.41, 11.12, 12.23, 13.33, 14.09, 15.44, 16.41, 16.68, 17.27, 18.31, 18.88, 19.37, 20.58, 22.04, 22.49, 22.95, 23.69 or 25.12 ± 0.2°.

[0106] In other embodiments, the crystals of the compound of formula I of the present application are H-type crystals, characterized in that the X-ray powder diffraction pattern has at least 7 or at least 8 diffraction peaks at 2θ selected from 4.65, 9.36, 12.23, 13.33, 14.09, 17.27, 19.37, 22.04 or 22.95±0.2°.

[0107] In some embodiments of the present application, the peak positions and relative intensities of the diffraction peaks in the XRPD pattern of the above-mentioned H-type crystals are shown in Table 8 below:

[0108] Table 8 XRPD pattern analysis data of H-type crystal

[0109]

[0110]

[0111] In some embodiments, the crystal of the compound of formula I of the present application is H-type crystal, and its X-ray powder diffraction pattern is as follows: Figure 20 shown.

[0112] The H-type crystals of the compound of formula I may exist in the form of non-solvate crystals or in the form of solvate crystals. The solvate herein refers to a solvate formed between an organic solvent and / or water and the corresponding compound.

[0113] In some embodiments, the present application provides a method for preparing type H crystals of the compound of formula I, comprising mixing the compound of formula I with 4-methyl-2-pentanone and isolating the solid.

[0114] In the above preparation method, the volume mass ratio of 4-methyl-2-pentanone to the compound of formula I is 5 to 200 mL / g; in some embodiments, the above volume mass ratio is 20 to 100 mL / g, preferably 50 mL / g.

[0115] In some embodiments, the method for preparing Form H crystals of the compound of Formula I described herein comprises: mixing the compound of Formula I with 4-methyl-2-pentanone, stirring at room temperature, collecting the precipitate by filtration, and vacuum drying to obtain Form H crystals. In some embodiments, in the above preparation method, the vacuum drying is performed at 50°C.

[0116] In another aspect, the present application provides a crystalline composition, wherein the crystals of the compound of Formula I account for at least 50% by weight of the crystalline composition, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%. In a preferred embodiment, in the crystalline composition, the crystals of the compound of Formula I are selected from the group consisting of Type A crystals, Type B crystals, Type C crystals, Type D crystals, Type E crystals, Type F crystals, Type G crystals, or Type H crystals of the compound of Formula I.

[0117] On the other hand, the present application provides a crystalline composition, wherein type A crystals or type B crystals or type C crystals or type D crystals or type E crystals or type F crystals or type G crystals or type H crystals of the compound of formula I account for more than 50% by weight of the crystalline composition, preferably more than 80%, more preferably more than 90%, and most preferably more than 95%.

[0118] In another aspect, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a crystal of a compound of formula I, or a crystalline composition thereof. In some embodiments, the pharmaceutical composition of the present application further comprises a pharmaceutically acceptable excipient.

[0119] On the other hand, the present application describes a method for treating a disease associated with the anti-apoptotic protein BCL-2 in a mammal, comprising administering a therapeutically effective amount of a crystal of the compound of formula I, a crystalline composition thereof, or a pharmaceutical composition thereof to a mammal (preferably a human) in need of such treatment.

[0120] On the other hand, the present application describes the use of the crystal of the compound of formula I, its crystalline composition or its pharmaceutical composition in the preparation of a drug for preventing or treating diseases associated with the anti-apoptotic protein BCL-2.

[0121] On the other hand, the present application describes the use of the crystal of the compound of formula I, its crystalline composition or its pharmaceutical composition in preventing or treating diseases associated with the anti-apoptotic protein BCL-2.

[0122] On the other hand, the present application describes a crystal of the compound of formula I, a crystal composition thereof, or a pharmaceutical composition thereof for preventing or treating diseases associated with the anti-apoptotic protein BCL-2.

[0123] Wherein, the disease associated with the anti-apoptotic protein BCL-2 is selected from cancer, and the cancer is selected from acute lymphoblastic leukemia.

[0124] The crystals of the compound of formula I include type A crystals, type B crystals, type C crystals, type D crystals, type E crystals, type F crystals, type G crystals, or type H crystals. For example, the crystals of the compound of formula I are selected from the group consisting of type A crystals, type B crystals, type C crystals, type D crystals, type E crystals, type F crystals, type G crystals, and type H crystals of the compound of formula I.

[0125] X-ray powder diffraction (XRPD): instrument model: Bruker D2 Phaser; target tube: Cu.

[0126] Thermogravimetric analysis (TGA): instrument model: NETZSCH TG 209F3 TGA209F3A-0449-L; temperature range: 30-300°C; scanning rate: 10°C / min.

[0127] Differential scanning calorimetry (DSC): instrument model, TADSC25; temperature range: 40-220°C; scanning rate: 10°C / min.

[0128] Definition and Description

[0129] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be construed as ambiguous or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding commercial product or its active ingredient.

[0130] It should be noted that the positions or relative intensities of peaks in X-ray powder diffraction spectra may vary due to factors such as the measuring instrument, measurement method, and conditions. For any particular crystal form, the peak positions may have errors, and the measurement error of the 2θ value can be approximately ±0.2°. Therefore, this error should be taken into account when determining each crystal form, and determinations within this error are within the scope of this application.

[0131] It should be noted that for the same crystal form, the position of the DSC endothermic peak may vary due to factors such as the measuring instrument, measurement method / conditions, etc. For any specific crystal form, the position of the endothermic peak may have an error of approximately ±5°C or approximately ±3°C. Therefore, this error should be taken into account when determining each crystal form, and any error within this error is within the scope of this application.

[0132] The word "comprise" or "comprises" and its English variations such as comprises or comprising should be understood as having an open and non-exclusive meaning, ie, "including but not limited to".

[0133] "Pharmaceutically acceptable excipients" refer to inert substances that are administered together with the active ingredient and facilitate the administration of the active ingredient, including but not limited to any glidants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, disintegrants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that are acceptable for use in humans or animals (e.g., livestock). Non-limiting examples of such excipients include calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycol.

[0134] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present application or their salts and pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate administration of the compounds of the present application to an organism.

[0135] The pharmaceutical compositions of the present application can be prepared by combining the compounds of the present application with suitable pharmaceutically acceptable excipients, and can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols.

[0136] Typical routes of administration of the crystals, crystalline compositions, or pharmaceutical compositions thereof described herein include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, vaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.

[0137] The pharmaceutical composition of the present application can be manufactured by methods well known in the art, such as conventional mixing methods, dissolution methods, granulation methods, sugar-coated pill making methods, grinding methods, emulsification methods, freeze-drying methods, etc.

[0138] In some embodiments, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical composition can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These excipients enable the compounds of the present application to be formulated into tablets, pills, lozenges, dragees, capsules, liquids, gels, slurries, suspensions, and the like for oral administration to a patient.

[0139] The therapeutic dosage of the compounds of the present invention may be determined, for example, based on the specific therapeutic application, the method of administration, the patient's health and condition, and the judgment of the prescribing physician. The ratio or concentration of the compounds of the present invention in a pharmaceutical composition may not be fixed and depends on a variety of factors, including dosage, chemical properties (e.g., hydrophobicity), and route of administration.

[0140] The term "treatment" means administering the compound or formulation described herein to improve or eliminate a disease or one or more symptoms associated with the disease, and includes:

[0141] (i) inhibiting a disease or disease state, i.e., arresting its development;

[0142] (ii) ameliorating the disease or condition, i.e., causing regression of the disease or condition.

[0143] The term "prevention" means administering a compound, composition or formulation described herein to prevent a disease or one or more symptoms associated with the disease, and includes preventing the occurrence of a disease or disease state in a mammal, particularly when such mammal is susceptible to the disease state but has not yet been diagnosed as having the disease state.

[0144] With respect to a drug or pharmacologically active agent, the term "therapeutically effective amount" refers to a non-toxic amount of the drug or agent sufficient to achieve the desired effect. The determination of an effective amount varies from person to person, depending on the age and general condition of the recipient, as well as the specific active agent. The appropriate effective amount in each individual case can be determined by those skilled in the art through routine experimentation.

[0145] The therapeutically effective amount of the crystals described herein is from about 0.0001 to 20 mg / Kg body weight / day, for example, from 0.001 to 10 mg / Kg body weight / day.

[0146] The dosage frequency of the crystals described herein is determined by the needs of the individual patient, for example, once or twice a day, or more times a day. Administration can be intermittent, for example, wherein the patient receives a daily dose of the crystals over a period of several days, followed by a period of several or more days in which the patient does not receive a daily dose of the crystals.

[0147] For the purposes of description and disclosure, all patents, patent applications, and other identified publications are expressly incorporated herein by reference. These publications are provided solely for their disclosure prior to the filing date of the present application. All statements regarding the dates of these documents or the representations of their contents are based on the information available to the applicant and do not constitute any admission as to the correctness of the dates of these documents or the contents of these documents. Furthermore, any citation of these publications herein does not constitute an admission that such publications become part of the common general knowledge in the art in any country.

[0148] All solvents used in this application were commercially available and used without further purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0149] Figure 1 XRPD pattern of Form A crystals of the compound of Formula I;

[0150] Figure 2 DSC spectrum of Form A crystal of the compound of formula I;

[0151] Figure 3 TG spectrum of type A crystal of compound of formula I;

[0152] Figure 4 XRPD pattern of Type B crystals of the compound of Formula I;

[0153] Figure 5 DSC spectrum of Type B crystal of the compound of formula I;

[0154] Figure 6 TG spectrum of type B crystal of compound of formula I;

[0155] Figure 7 XRPD pattern of Type C crystals of the compound of Formula I;

[0156] Figure 8 DSC spectrum of Type C crystal of the compound of formula I;

[0157] Figure 9 TG spectrum of type C crystal of compound of formula I;

[0158] Figure 10 XRPD pattern of Form D crystals of the compound of Formula I;

[0159] Figure 11 DSC spectrum of Form D crystal of the compound of formula I;

[0160] Figure 12 TG spectrum of type D crystal of compound of formula I;

[0161] Figure 13 XRPD pattern of Form E crystals of the compound of Formula I;

[0162] Figure 14 DSC spectrum of Form E crystal of the compound of formula I;

[0163] Figure 15 TG spectrum of Form E crystal of the compound of formula I;

[0164] Figure 16 XRPD pattern of Form F crystal of the compound of Formula I;

[0165] Figure 17 DSC spectrum of Form F crystal of the compound of formula I;

[0166] Figure 18 TG spectrum of type F crystal of compound of formula I;

[0167] Figure 19 XRPD pattern of Form G crystals of the compound of Formula I;

[0168] Figure 20 XRPD pattern of Form H crystal of the compound of formula I. DETAILED DESCRIPTION

[0169] The present application will be described in more detail below by (but not limited to) the following examples and tests.

[0170] The following abbreviations are used herein: Boc stands for tert-butyloxycarbonyl; THF stands for tetrahydrofuran; and TBSCl stands for tert-butyldimethylchlorosilane.

[0171] Example 1: Preparation of compound of formula I

[0172]

[0173]

[0174] 1) Preparation of compound 7-d

[0175] Compound 1-c (198.6 g) and 1-Boc-piperazine (175.5 g) were dissolved in acetonitrile (800 mL) with stirring. The mixture was cooled to 0°C and sodium triacetoxyborohydride (532.6 g) was slowly added. The mixture was stirred at room temperature for 5 h. After the reaction was complete, water (1 L) and ethyl acetate (300 mL) were added for extraction. The organic phase was collected, dried over anhydrous Na2SO4, filtered, and concentrated to obtain compound 7-d (269.8 g).

[0176] 2) Preparation of compound 7-e

[0177] Compound 7-d (269.8 g), isopropyl alcohol (800 mL), and hydrochloric acid (36-38 wt%, 169 mL) were mixed, heated to 65°C, and reacted for 3 h. The solid precipitated upon cooling, was filtered, and dried to obtain compound 7-e (151.2 g).

[0178] Compound 7-e: 1 H NMR(500MHz,DMSO-d6),δ:7.82(s,1H),7.68(d,1H),7.36(d,1H),7.10(dd,1H) ,2.98(s,4H),2.63(d,2H),2.23(m,6H),1.89(m,2H),1.43(s,2H),0.94(s,6H).

[0179] 13 C NMR(125MHz,DMSO-d6),δ:133.1,132.6,131.9,130.9,129.3,128.6,126.5,125.7,124.9,1 24.6,122.7,60.2,49.4,44.7,35.2,29.4,28.4,27.1,25.2,21.4.ESI-MS: m / z=387.1[M+H] + .

[0180] 3) Preparation of compound 7-g

[0181] NaH (21.1 g) was dissolved in THF (100 mL), cooled to -20°C, and stirred for 10 minutes. 2-[(1H-pyrrolo[2,3-b]pyridin-5-yl)oxy]-4-bromobenzoic acid tert-butyl ester (Compound 1-d, 128.3 g) was dissolved in 200 mL of THF and slowly added dropwise to the reaction solution, maintaining the internal temperature below 0°C during the addition. After the addition was complete, the mixture was stirred for 30 minutes. A solution of TBSCl (64.7 g) in THF (200 mL) was added dropwise, maintaining the internal temperature around -10°C. The mixture was allowed to react for 30 minutes. After the reaction was complete, 500 mL of saturated sodium bicarbonate and ethyl acetate were added for extraction. The organic phase was collected, dried over anhydrous Na2SO4, filtered, concentrated, and purified by column chromatography to obtain Compound 7-f (150 g). ESI-MS: m / z = 503.1 [M+H]. + .

[0182] Compound 7-e (151.2 g), tert-butyl 2-[(1-tert-butyldimethylsilylpyrrolo[2,3-b]pyridin-5-yl)oxy]-4-bromobenzoate (compound 7-f, 197.1 g), tris(dibenzylideneacetone)dipalladium (2.7 g), [(4-(N,N-dimethylamino)phenyl]di-tert-butylphosphine (1.6 g), sodium tert-butoxide (187.4 g), and toluene (800 mL) were mixed, stirred, and protected with nitrogen. The mixture was heated to 100° C. and reacted for 24 h. After the reaction was completed, water (1 L) and ethyl acetate (300 mL) were added for extraction, and the organic phase was collected, dried over anhydrous Na2SO4, filtered, and concentrated to obtain compound 7-g (181.9 g).

[0183] Compound 7-g: 1 H NMR(500MHz,DMSO-d6),δ:7.95(s,1H),7.82(s,1H),7.65(t,2H),7.37(m,4H),6.76(d,1H),6.47(s,1H),3.14(s,2H),2.64(d ,1H),2.55(d,1H),2.19(m,5H),1.92(m,2H),1.42(t,2H),1.31(t,2H),1.22(m,9H),0.95(d,6H),0.84(s,10H),0.60(s,6H).

[0184] 13 C NMR (125MHz, DMSO-d6), δ: 164.4 156.8, 155.1, 150.3, 149.8, 146.2, 133.6, 133.4, 133.2, 132.0, 131.9,131.5,129.5,129.0,128.8,126.4,126.1,124.4,122.8,114.4,113.8,110.2,106.8,103. 3,80.1,60.6,52.6,47.1,44.7,35.2,29.4,27.9,27.2,26.7,25.4,19.0.ESI-MS:m / z=809.4[M+H] + .

[0185] 4) Preparation of compound 7-h

[0186] A mixture of compound 7-g (181.9 g), toluene (1.8 L), and trifluoroacetic acid (107 mL) was heated to 45°C and reacted for 5 h. The reaction solution was concentrated, 1.5 L of ethyl acetate was added, washed with saturated aqueous NaHCO3 and saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated. 1 L of toluene and 200 mL of ethyl acetate were added, and the mixture was heated to dissolve. The solid was cooled to precipitate, filtered, and dried to obtain compound 7-h (83.4 g).

[0187] Compound 7-h: 1 H NMR(500MHz,DMSO-d6),δ:7.98(s,1H),7.82(s,1H),7.73(d,1H),7.64(d,1H),7.46(s,1H),7.40(s,1H),7.32(d,1H),6.73(d,1H), 6.36(d,1H),6.34(s,1H),3.09(s,4H),2.64(d,1H),2.55(d,1H),2.19(m,6H),1.88(m,2H),1.42(m,2H),1.25(m,2H),0.95(m,6H).

[0188] 13 C NMR (125MHz, DMSO-d6), δ: 166.3,158.9,155.1,148.9,146.2,145.3,135.0,133.8,133.2,132.0,131.9,131.4,129.5,129.2,127.8,126.4,1 24.9,124.4,122.7,120.2,116.6,112.0,109.5,105.3,100.2,60.5,5 5.3,52.7,47.0,44.7,35.2,29.4,27.2,25.4.ESI-MS: m / z=639.2[M+H] + .

[0189] 5) Preparation of compound 8-k

[0190] 3-Nitro-4-chlorobenzenesulfonamide (35.0 g), (S)-2-(aminomethyl)-1,4-dioxane hydrochloride (30.0 g) and N,N-diisopropylethylamine (94.0 g) were dissolved in acetonitrile (400 mL), heated to 85°C, reacted for 5 h, cooled to room temperature, allowed to stand overnight, and filtered to obtain compound 8-k (46.5 g) ESI-MS: m / z = 316.1 [MH]-.

[0191] 6) Preparation of compound of formula I

[0192] Compound 7-h (10 g) and dichloromethane (100 mL) were mixed and stirred at room temperature. 4-Dimethylaminopyridine (2.8 g) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (4.4 g) were added and dissolved with stirring. Compound 8-k (5.0 g) and triethylamine (4.5 g) were added and reacted at room temperature for 3 h. The mixture was washed sequentially with 5 wt% hydrochloric acid, saturated aqueous sodium bicarbonate solution, and saturated aqueous sodium chloride solution, dried over anhydrous Na2SO4, filtered, concentrated, and purified by column chromatography to obtain an amorphous form of the compound of Formula I (8.9 g).

[0193] Compounds of formula I: 1 H NMR(500MHz,DMSO-d6),δ:11.66(s,1H),11.37(s,1H),8.59(t,1H),8.57(d,1H),8.04(d,1H) ,7.89(d,1H),7.84(dd,1H),7.70(d,1H),7.54(d,1H),7.52(m,2H),7.40(m,1H),7.11(d,1H), 6.75(dd,1H),6.40(dd,1H),6.29(d,1H),3.79(m,3H),3.65(m,2H),3.51(m,2H),3.42(m,2H) ,3.03(m,4H),2.67(d,1H),2.54(d,1H),2.17(m,6H),1.88(dd,2H),1.42(t,2H),0.96(s,6H).

[0194] 13 C NMR(125MHz,DMSO-d6),δ:164.0,158.2,154.0,147.9,146.9,145.9,144.3,135 .6,134.3,132.9,132.6,131.8,130.2,130.0,128.3,128.2,127.0,125.2,125.0 ,122.8,120.3,118.3,115.7,114.1,109.7,103.6,100.4,73.4,68.5,66.4,66. 2,58.6,45.1,44.4,43.9,34.6,29.3,29.1,27.2,24.8.ESI-MS: m / z=939.4[M+H] + .

[0195] Example 2: Preparation of Type A Crystals of Compound I

[0196] 5 g of the compound of formula I prepared in Example 1 was dissolved in 300 mL of a mixed solution of dichloromethane and methanol (dichloromethane:methanol=20:1 or 100:1, V:V), stirred until clear, and concentrated under reduced pressure to obtain type A crystals. The XRPD pattern of type A crystals is shown in FIG. Figure 1 As shown; DSC spectrum as Figure 2 As shown, the TG spectrum is as Figure 3 shown.

[0197] Example 3: Preparation of Type B Crystals of Compound I

[0198] Weigh about 200 mg of Type A crystals of Compound I into a 4 mL glass bottle, add 2 mL of acetone, and place the resulting suspension under magnetic stirring at room temperature for 5 hours. Filter and collect the precipitate, and finally vacuum dry at 40-60°C for 24 hours. This gives Type B crystals of Compound I. The XRPD pattern of Type B crystals is as follows: Figure 4 As shown; DSC spectrum as Figure 5 As shown, the TG spectrum is as Figure 6 shown.

[0199] Example 4: Preparation of Type C Crystals of Compound I

[0200] Weigh approximately 4 g of Type A crystals of Compound I into a 500 mL reaction flask, add 100 mL of methanol and 100 mL of methyl tert-butyl ether, respectively, and place the resulting suspension under magnetic stirring at room temperature for 12 hours. Filter and collect the precipitate, and vacuum dry at 40-60°C for 24 hours. This gives Type C crystals of Compound I. The XRPD pattern of Type C crystals is shown below. Figure 7 As shown; DSC spectrum as Figure 8 As shown, the TG spectrum is as Figure 9 shown.

[0201] Example 5: Preparation of Type D Crystals of Compound I

[0202] 1) Approximately 3 g of Form A crystals of Compound I were weighed into a 500 mL reaction flask. 200 mL of a tetrahydrofuran-water mixture (tetrahydrofuran:water = 10:1 to 0.5:1) was added. The resulting suspension was magnetically stirred at room temperature for 12 hours. The precipitate was collected by filtration and air-dried at 40-60°C to obtain Form D crystals of Compound I.

[0203] 2) Weigh approximately 3 g of Form A crystals of Compound I into a 250 mL reaction flask, add 100 mL of isopropanol or 1,4-dioxane, and magnetically stir the resulting suspension at room temperature for 12 hours. Collect the precipitate by filtration and air dry at 40-60°C to obtain Form D crystals of Compound I.

[0204] 3) Approximately 0.7 g of Form A crystals of Compound I was weighed into a 100 mL reaction flask, 35 mL of tetrahydrofuran was added, and the mixture was completely dissolved by magnetic stirring at room temperature. The supernatant was filtered and transferred to a clean 40 mL glass bottle. The glass bottle containing the supernatant was sealed with sealing film and punctured with several small holes. The solution was allowed to slowly evaporate at room temperature. After complete evaporation of the solvent, the solid was collected to obtain Form D crystals of Compound I.

[0205] The XRPD pattern of type D crystal is as follows Figure 10 As shown; DSC spectrum as Figure 11 As shown, the TG spectrum is as Figure 12 shown.

[0206] Example 6: Preparation of Type E Crystals of Compound I

[0207] About 20 g of Type A crystals of Compound I were weighed into a 1 L reaction flask, 400 mL of p-xylene was added, and the resulting suspension was placed under magnetic stirring at room temperature for 5 hours. The precipitate was collected by filtration and vacuum dried at 80-100° C. for 5-10 hours to obtain Type E crystals of Compound I. The XRPD pattern of Type E crystals is shown below. Figure 13 As shown; DSC spectrum as Figure 14 As shown, the TG spectrum is as Figure 15 shown.

[0208] Example 7: Preparation of Type F Crystals of Compound I

[0209] Weigh about 4 g of Type A crystals of Compound I into a 500 mL reaction flask, add 200 mL of acetonitrile or nitromethane, and place the resulting suspension under magnetic stirring at room temperature for 12 hours. Filter and collect the precipitate, and vacuum dry at 40-60°C to obtain Type F crystals of Compound I. The XRPD pattern of Type F crystals is as follows: Figure 16 As shown; DSC spectrum as Figure 17 As shown, the TG spectrum is as Figure 18 shown.

[0210] Example 8: Preparation of Type G Crystals of Compound I

[0211] About 4 g of Type A crystals of Compound I were weighed into a 500 mL reaction flask, 375 mL of p-xylene was added, the resulting suspension was placed under magnetic stirring at room temperature for 5 hours, and the precipitate was collected by filtration. The Type G crystals of Compound I were obtained. The XRPD pattern of the Type G crystals is shown below. Figure 19 shown.

[0212] Example 9: Preparation of Type H Crystals of Compound I

[0213] About 4 g of Type A crystals of Compound I were weighed into a 500 mL reaction flask, 200 mL of 4-methyl-2-pentanone was added, and the resulting suspension was magnetically stirred at room temperature for 12 hours. The precipitate was collected by filtration and vacuum dried at 50°C for 2 hours. This gave Type H crystals of Compound I. The XRPD pattern of Type H crystals is shown below. Figure 20 shown.

[0214] Example 10: Crystallization stability test

[0215] Place the test sample in an open, suitable clean container at 60°C for 10 days, and take samples on the 5th and 10th days;

[0216] The test sample is placed in a sealed container with constant humidity at 25°C and a relative humidity of 92.5% ± 5% for 10 days, and samples are taken on the 5th and 10th days;

[0217] Place the test sample in a light box equipped with fluorescent lamps or other suitable lighting devices (open) at an illumination of 4500 lx ± 500 lx for 10 days, and take samples on the 5th and 10th days;

[0218] In the above three cases, the purity of the product and the content of related substances are tested after the test sample is placed under experimental conditions.

[0219] Instrument: Thermo U3000 high performance liquid chromatograph;

[0220] Detector: Thermo VWD-3100 UV absorption detector / DAD-3000 diode array detector;

[0221] Chromatographic column: XSelect CSH C18 (4.6×150 mm, 3.5 μm);

[0222] Injection volume: 10 μL; detection wavelength: 280 nm; flow rate: 1.0 mL / min; column temperature: 30°C;

[0223] Mobile phase: Phase A: 0.1% formic acid aqueous solution, Phase B: acetonitrile, gradient elution;

[0224] Preparation of sample solution: Take an appropriate amount of this product, accurately weigh it, dissolve it in methanol and quantitatively dilute it to make a solution containing about 0.5 mg per 1 mL as the test solution.

[0225] The results are shown in Table 9 below.

[0226] Table 9

[0227]

[0228] Example 11: Moisture-attracting test

[0229] The hygroscopicity test method and conditions refer to the Guiding Principles for Hygroscopicity Testing of Pharmaceuticals in Part IV, General Chapter 9103, of the 2015 edition of the Chinese Pharmacopoeia: Hygroscopicity of a drug refers to the ability or degree to which a substance absorbs water under certain temperature and humidity conditions. The test sample is a solid API that meets pharmaceutical quality standards. The test results can be used as a reference for selecting appropriate drug packaging and storage conditions.

[0230] 1) Take a dry, stoppered glass weighing bottle (outer diameter 50 mm, height 15 mm) and place it in a suitable 25°C ± 1°C constant temperature desiccator (with ammonium chloride or saturated ammonium sulfate solution placed at the bottom) or an artificial climate box (set temperature 25°C ± 1°C, relative humidity 80% ± 2%) the day before the test, and accurately weigh the weight (m1).

[0231] 2). Take an appropriate amount of the test sample and spread it evenly in the above-mentioned weighing bottle. The thickness of the test sample is generally about 1mm. Accurately weigh the weight (m2).

[0232] 3) Open the weighing bottle and place it with the bottle cap under the above constant temperature and humidity conditions for 24 hours.

[0233] 4). Cover the weighing bottle and accurately weigh the weight (m3).

[0234] The experimental results are shown in Table 10.

[0235]

[0236] Table 10

[0237] Crystal form Moisture-induced weight gain Crystal form Moisture-induced weight gain Type A crystal 1.38wt% E-type crystal 0.11wt% B 0.30wt% Type F crystal 0.76wt% C 0.11wt% H-type crystal 0.25wt% D 0.75wt%

[0238] Test Example 1 In vitro protein binding inhibitory activity

[0239] 1.1 Screening of BCL-2 / BAK binding inhibitory activity

[0240] The 500nM Tag1-BCL-2 protein stock solution was diluted to 5nM using the dilution buffer in the kit (model: BCL-2 / BAK (BH3) BINDING ASSAY KITS, from cisbio). At the same time, the 20μM Tag2-BAK protein stock solution was diluted to 120nM. 5μL of Tag1-BCL-2 protein dilution was added to each well. Then, the DMSO-dissolved compound of formula I was added to the well using a nanoliter pipette to make the final concentration of the compound 200nM-0.0488nM, 4-fold gradient, a total of 7 concentrations. At the same time, blank control wells (without enzyme) and negative control wells (with enzyme, with solvent DMSO) were set up, and 2 replicates were set up. Finally, 5μL of Tag2-BAK protein dilution was added to each well, centrifuged and mixed, and incubated at 25°C for 15min. 100× anti-Tag1-Eu was added to the well using the detection buffer in the kit. 3+ Dilute to 1× concentration, and dilute 100× anti-Tag2-XL665 to 1× concentration. 3+ Mix the solution with anti-Tag2-XL665 at a ratio of 1:1, add 5 μL of the mixture to each well, and react at 25°C for 2 hours or more. Read the plate using a PE Envision multifunctional microplate reader (excitation 620 nm, emission 665 nm), and calculate the IC using a four-parameter fitting method. 50 (As shown in Table 11).

[0241] 1.2 Screening of BCL-XL / BAK binding inhibitory activity

[0242] The 300nM Tag1-BCL-XL protein stock solution was diluted to 2nM using the dilution buffer in the kit (model: BCL-XL / BAK (BH3) BINDING ASSAY KITS, from cisbio). At the same time, the 10μM Tag2-BAK protein stock solution was diluted to 80nM. 5μL of Tag1-BCL-XL protein dilution was added to each well. Then, the DMSO-dissolved compound of formula I was added to the well using a nanoliter pipette to make the final concentration of the compound 2000nM-0.488nM, 4-fold gradient, a total of 7 concentrations. At the same time, blank control wells (without enzyme) and negative control wells (with enzyme, with solvent DMSO) were set up, and 2 replicates were set up. Finally, 5μL of Tag2-BAK protein dilution was added to each well, centrifuged and mixed, and incubated at 25°C for 15min. 100× anti-Tag1-Eu was added to the well using the detection buffer in the kit. 3+ Dilute to 1× concentration, and dilute 100× anti-Tag2-XL665 to 1× concentration. 3+Mix the solution with anti-Tag2-XL665 at a ratio of 1:1, add 5 μL of the mixture to each well, and react at 25°C for 2 hours or more. Read the plate using a PE Envision multifunctional microplate reader (excitation 620 nm, emission 665 nm), and calculate the IC using a four-parameter fitting method. 50 (As shown in Table 11).

[0243] Table 11: Compounds of Formula I inhibit BCL-2 / BAK and BCL-XL / BAK binding activity

[0244]

[0245] Experimental Example 2 Inhibitory Effect of Compounds on RS4;11 Cell Proliferation

[0246] RS4;11 cells (from Nanjing Kebai) in good exponential growth phase were collected into a centrifuge tube and centrifuged at 1500 rpm for 3 min. The supernatant was discarded and 5 mL of complete medium (RPMI basal medium + 10 wt% fetal bovine serum (FBS)) was added with a pipette to resuspend the cells. The cells were counted using a cell counter and diluted with complete medium to adjust the cell density to 2 × 10 5 After adding an equal amount of RPMI basal medium to adjust the serum concentration to 5%, the cell density was 1×10 5 / mL seed plate. Use a spray gun to inoculate on a 96-well plate, 100μL / well, and culture in a cell culture incubator at 37℃ and 5% CO2 saturated humidity. After 24h of culture, use a nanoliter sampler to add the compound, set up 2 replicates for each concentration, and use cells without compound as negative control. After 72 hours, add CCK-8 reagent, 10μL / well, and 4 hours later, use Envision enzyme reader to detect its absorbance at 450nm, and calculate the inhibition rate. Inhibition rate (%) = (average value of negative control group - average value of experimental group) / (average value of negative control group - average value of blank group) × 100%, with the logarithm of compound concentration as the horizontal axis and inhibition rate as the vertical axis. Four-parameter analysis, fitting of dose-effect curve, and calculation of IC 50 (See Table 12).

[0247] Table 12 Inhibitory effect of compounds on RS4;11 cell proliferation

[0248]

[0249] Test Example 3 In vitro liver microstructure stability evaluation

[0250] The final 300 μL incubation system contains 30 μL of liver microsomes (protein concentration: 5 mg / mL), 30 μL of NADPH + MgCl2, 3 μL of the test compound (prepared in acetonitrile), and 237 μL of PBS buffer (pH 7.4). The organic solvent (acetonitrile) is 1% (volume ratio). Two 0.3 mL aliquots are prepared for each species (mouse, rat, and human). Prepare a 270 μL substrate and enzyme mixture in each tube. Pre-incubate with NADPH at 37°C for 5 minutes, then add 30 μL of NADPH + MgCl2. Remove 50 μL of the mixture at 0, 15, 30, and 60 minutes and terminate the reaction with 300 μL of glacial acetonitrile containing an internal standard.

[0251] To 50 μL of the incubated sample, add 300 μL of glacial acetonitrile containing the internal standard (diazepam 20 ng / mL) for precipitation. Vortex for 5 minutes and centrifuge (13,000 rpm, 20°C) for 10 minutes. Aspirate 70 μL of the supernatant, dilute and mix with 70 μL of ultrapure water, and inject 1 μL for analysis. Elimination parameters for the compound in human, rat, and mouse liver microsomes are shown in Table 13.

[0252] Table 13 In vitro liver microsomal metabolic stability of compounds (1 μM)

[0253]

[0254] Experimental Example 4 In vivo pharmacokinetic evaluation

[0255] 4.1 Pharmacokinetic evaluation in rats

[0256] SD rats weighing 180-220 g were randomly divided into groups after acclimation for 3-5 days, with 3 rats in each group, and were orally administered with a solution of the compound of formula I at a dose of 5 mg / kg.

[0257] The test animals (SD rats) were fasted for 12 h before administration and given food 4 h after administration. They had free access to water before, during and after the experiment.

[0258] After oral administration, approximately 0.2 mL of blood was collected from the eye socket at 0 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 24 h. After anticoagulation with EDTA-K2, the blood was transferred to 4°C within 30 min and centrifuged at 4000 rpm for 10 min to separate plasma. All collected plasma was immediately stored at -20°C until testing.

[0259] 50 μL of plasma sample to be tested was taken, 300 μL of acetonitrile solution containing internal standard (diazepam 20 mg / mL) was added, and the mixture was shaken and mixed for 5 minutes. The mixture was centrifuged at 13000 rpm for 10 minutes. 75 μL of supernatant was taken and diluted with 75 μL of ultrapure water. The mixture was mixed and 2 μL was taken for LC / MS / MS determination. The chromatogram was recorded.

[0260] The oral exposure of the present compound was evaluated in rats using a pharmacokinetic study. The pharmacokinetic parameters of the compound were fitted using DAS 3.2.5 software. The data are shown in Table 14 below.

[0261] Table 14 Pharmacokinetic parameters of compounds

[0262] PK parameters Formula I compound IG 5mg / kg <![CDATA[T max (h)]]> 4.00±0.00 <![CDATA[C max (ng / mL)]]> 739±226 AUC (0-24h) (ng*h / mL) 5973±2021 AUC(0-∞)(ng*h / mL) 6558±1805 <![CDATA[t 1 / 2 (h)]]> 7.62±2.78 MRT(0-t)(h) 7.35±0.45

[0263] 4.2 Pharmacokinetic evaluation in beagle dogs

[0264] Three male beagle dogs weighing 9-12 kg were orally administered with a solution of the compound of formula I at a dose of 2.5 mg / kg after a period of adaptation.

[0265] The test animals (male beagle dogs) were fasted for 12 h before administration and given food 4 h after administration. They had free access to water before, during and after the experiment.

[0266] After oral administration, approximately 0.5 mL of blood was collected from the forelimb vein at 0.25 h (15 min), 0.5 h (30 min), 1 h, 1.5 h, 2 h, 4 h, 6 h, 8 h, 10 h, 24 h, 30 h, 48 h, and 72 h. The blood was placed in an EDTA-K2 anticoagulant vacuum tube. Within 30 min, the plasma was centrifuged at 4000 rpm for 10 min at 4°C. All collected plasma was immediately stored at -20°C until testing.

[0267] 50 μL of plasma sample to be tested was taken, 300 μL of acetonitrile solution containing internal standard (diazepam 20 ng / mL) was added, and the mixture was shaken and mixed for 5 minutes. The mixture was centrifuged at 13000 rpm for 10 minutes. 75 μL of supernatant was taken and diluted with 75 μL of ultrapure water. The mixture was mixed and 1 μL was taken for LC / MS / MS determination. The chromatogram was recorded.

[0268] The oral exposure of the compound of the present application was evaluated by in vivo pharmacokinetic experiments in beagle dogs. The pharmacokinetic parameters of the compound were fitted using DAS3.2.5 software and are shown in Table 15 below.

[0269] Table 15 Pharmacokinetic parameters of compounds in beagle dogs

[0270]

[0271] Experimental Example 5 Pharmacodynamic Evaluation of the Test Substance in RS4;11 Human B Cell Leukemia Subcutaneous Transplantation Model

[0272] NOD / SCID female mice, 9-10 weeks old (age at time of tumor cell inoculation), weighing 16.3-22.0 g. Purchased from Ankai Yibo Biotechnology Co., Ltd., production license number: SCXK (Beijing) 2017-0006, animal qualification certificate number: 11402400013155. Housing environment: SPF grade. Mice were subcutaneously inoculated with 1×10 7 RS4;11 cells. The day of inoculation was defined as day 0. When the average tumor volume reached 240 mm 3 The patients were randomly divided into groups according to tumor size and the medication was performed according to Table 16 below.

[0273] Table 16 Administration route, dosage and regimen in human B cell leukemia RS4;11 subcutaneous animal model

[0274] Group n Drug administration group Dosage (mg / kg) Dosage Time of administration 1 6 Vehicle po Single 2 6 Compound of formula I (solution) 25 po Single

[0275] Note: n: number of animals; administration volume is 10 μL / g.

[0276] All clinical symptoms observed during the experiment were recorded in the original data. Tumor volume calculation formula: Tumor volume (mm 3 )=1 / 2×(a×b 2 ) (where a represents the major diameter and b represents the minor diameter). TM Data were collected using Studylog System, Inc. (version 3.1.399.19, supplier: Studylog System, Inc., San Francisco, CA, USA), including tumor diameter measurements and animal weights. Raw data, measured using a balance and vernier calipers, were imported directly into the software, and any changes in the data were recorded. The relative tumor growth rate (T / C%), representing the percentage of tumor volume or weight between the treatment and control groups at a given time point, was calculated using the following formula:

[0277] T / C%=T RTV / C RTV ×100%(T RTV : Average RTV of treatment group; C RTV : Average RTV of vehicle control group; RTV = V t / V0, V0 is the tumor volume of the animal when grouped, V t is the tumor volume of the animal after treatment).

[0278] The relative tumor inhibition rate (TGI) was calculated as follows: TGI% = (1-T / C) × 100%. (T and C are the relative tumor volume (RTV) or tumor weight (TW) of the treatment and control groups at a specific time point, respectively.)

[0279] All experimental results are expressed as mean tumor volume ± SEM (standard error of the mean). Independent sample t-tests were used to compare the relative tumor volumes of the treatment groups with those of the control group. All data were analyzed using SPSS 18.0. A p < 0.05 was considered significant. The results are shown in Table 17.

[0280] Table 17 Analysis of drug efficacy of each group in human B cell leukemia RS4;11 subcutaneous model

[0281]

[0282] Note: 1. Data are expressed as "mean ± standard error";

[0283] 2. T / C% = T RTV / C RTV ×100%; TGI%=(1-T / C)×100%.

[0284] Test Example 6 Human Platelet Toxicity Experiment (Caspase 3 Activity Assay)

[0285] Draw 10 mL of human whole blood using a sodium heparin anticoagulant tube, mix thoroughly by inverting, centrifuge at 90 g for 10 min, collect the supernatant, and continue centrifuging at 1950 g for 10 min. Discard the supernatant, resuspend and mix thoroughly with 4 mL of PBS, centrifuge at 1190 g for 5 min, discard the supernatant, resuspend and mix thoroughly with 4 mL of PBS, centrifuge at 1190 g for 5 min, discard the supernatant, resuspend the platelets in PBS and adjust the density to 2-3 × 10 8 / mL. According to 2~3×10 7Plate the platelets at a density of 100 μL / well in a 96-well plate. Add 50 μL of control buffer to the negative control wells and 50 μL of the corresponding compound to each well to a final concentration of 2.5 μM or 1 μM. Incubate in a 37°C incubator for 90 minutes. Transfer the contents of the 96-well plate to 1.5 mL centrifuge tubes. Centrifuge at 6000 g for 5 minutes at 4°C, discard the supernatant, and place on ice until ready to use. Dilute the 5× lysis buffer to a 1× lysis buffer using the water provided in the kit. Add a protease inhibitor cocktail at a ratio of 1:200 to prepare the lysis mix. Add 40 μL of the lysis mix to each centrifuge tube, resuspend the platelets at the bottom with a pipette, lyse on ice for 15-20 minutes, centrifuge at 14000 g for 10 minutes at 4°C, and aliquot the samples until ready to use. Dilute the 10× assay buffer to a 1× assay buffer using the water provided in the kit. Add the substrate Ac-DEVD-AMC at a 1:600 ​​ratio to prepare a reaction mixture. Add 5 μL of assay buffer and 40 μL of the reaction mixture to the blank control wells. Add 5 μL of control platelet lysate and 40 μL of the reaction mixture to the sample negative control wells. For the compound group, add 5 μL of platelet lysate and 40 μL of the reaction mixture. Add the 40 μL reaction mixture last, mix gently, and read the plate using a PE Envision multi-function microplate reader (excitation 360 nm, emission 460 nm). Read every 10 minutes for six times. Caspase-3 activity can be determined based on the fluorescence intensity of released AMC. The slope of the fitted line corresponding to each well represents caspase activity (all data were normalized to ABT-199). The results are shown in Table 18.

[0286] Table 18 Effects of compounds on human platelet Caspase 3 activity

[0287]

[0288] Note: The data have been normalized.

Claims

1. A crystal of a compound of formula I, characterized in that The X-ray powder diffraction pattern shows peaks at 5.01±0.2°, 6.61±0.2°, 8.12±0.2°, 10.21±0.2°, 14.89±0.2°, 16.63±0.2° and 20.13±0.2° for 2θ.

2. The crystal of the compound of formula I according to claim 1, wherein The X-ray powder diffraction pattern further comprises one or both of diffraction peaks at 2θ of 12.88±0.2° or 21.01±0.2°.

3. The crystal of the compound of formula I according to claim 1, characterized in that In the X-ray powder diffraction pattern, 2θ has peaks at 5.01±0.2°, 6.61±0.2°, 8.12±0.2°, 10.21±0.2°, 12.88±0.2°, 14.89±0.2°, 16.63±0.2°, 20.13±0.2° and 21.01±0.2°.

4. The crystal of the compound of formula I according to claim 1, characterized in that In the X-ray powder diffraction pattern, 2θ has peaks at 5.01±0.2°, 6.61±0.2°, 8.12±0.2°, 10.21±0.2°, 12.88±0.2°, 13.74±0.2°, 14.89±0.2°, 16.63±0.2°, 18.58±0.2°, 20.13±0.2°, 21.01±0.2° and 26.20±0.2°.

5. The crystal of the compound of formula I according to claim 1, characterized in that The X-ray powder diffraction pattern is shown in Figure 1.

6. A crystal of a compound of formula I, characterized in that The X-ray powder diffraction pattern has diffraction peaks at 2θ of 5.31±0.2°, 10.65±0.2°, 12.64±0.2°, 14.23±0.2°, 19.08±0.2°, 19.91±0.2° and 24.21±0.2°.

7. The crystal of the compound of formula (I) according to claim 6, characterized in that In the X-ray powder diffraction pattern, 2θ has peaks at 5.31±0.2°, 10.65±0.2°, 12.64±0.2°, 14.23±0.2°, 19.08±0.2°, 19.91±0.2°, 22.71±0.2° and 24.21±0.2°.

8. The crystal of the compound of formula I according to claim 6, wherein In the X-ray powder diffraction pattern, 2θ has peaks at 5.31±0.2°, 9.80±0.2°, 10.65±0.2°, 12.12±0.2°, 12.64±0.2°, 14.23±0.2°, 16.04±0.2°, 18.13±0.2°, 19.08±0.2°, 19.91±0.2°, 22.71±0.2°, 24.21±0.2° and 25.93±0.2°.

9. The crystal of the compound of formula I according to claim 6, characterized in that In the X-ray powder diffraction pattern, 2θ has peaks at 5.31±0.2°, 9.80±0.2°, 10.65±0.2°, 12.12±0.2°, 12.64±0.2°, 13.57±0.2°, 13.82±0.2°, 14.23±0.2°, 15.17±0.2°, 16.04±0.2°, 17.64±0.2°, 18.13±0.2°, 19.08±0.2°, 19.91±0.2°, 20.34±0.2°, 22.71±0.2°, 22.99±0.2°, 23.45±0.2°, 24.21±0.2°, 25.65±0.2° and 25.93±0.2°.

10. The crystal of the compound of formula I according to claim 6, characterized in that The 2θ values ​​in the X-ray powder diffraction pattern were 5.31±0.2°, 9.50±0.2°, 9.80±0.2°, 10.65±0.2°, 11.41±0.2°, 12.12±0.2°, 12.64±0.2°, 13.57±0.2°, 13.82±0.2°, 14.23±0.2°, 15.17±0.2°, 16.04±0.2°, 16.64±0.2°, 17.10±0.2°, 17.64±0.2°. There are peaks at .2°, 18.13±0.2°, 18.33±0.2°, 18.73±0.2°, 19.08±0.2°, 19.60±0.2°, 19.91±0.2°, 20.34±0.2°, 21.22±0.2°, 21.93±0.2°, 22.71±0.2°, 22.99±0.2°, 23.45±0.2°, 24.21±0.2°, 25.65±0.2° and 25.93±0.2°.

11. The crystal of the compound of formula I according to claim 6, characterized in that The X-ray powder diffraction pattern is shown in FIG4 .

12. The crystal of the compound of formula I according to claim 6, characterized in that The DSC spectrum has an endothermic peak starting point at 179.42±5℃.

13. The crystal of the compound of formula I according to claim 6, characterized in that The DSC spectrum is shown in Figure 5.

14. A crystal of a compound of formula I, characterized in that The X-ray powder diffraction pattern shows peaks at 5.52±0.2°, 7.56±0.2°, 8.29±0.2°, 9.22±0.2°, 11.04±0.2°, 17.43±0.2° and 18.51±0.2° for 2θ.

15. The crystal of the compound of formula I according to claim 14, characterized in that The X-ray powder diffraction pattern further comprises one or both of diffraction peaks at 2θ of 15.81±0.2°, or 22.59±0.2°.

16. The crystal of the compound of formula I according to claim 14, characterized in that In the X-ray powder diffraction pattern, 2θ has peaks at 5.52±0.2°, 7.56±0.2°, 8.29±0.2°, 9.22±0.2°, 11.04±0.2°, 15.81±0.2°, 17.43±0.2°, 18.51±0.2° and 22.59±0.2°.

17. The crystal of the compound of formula I according to claim 14, characterized in that In the X-ray powder diffraction pattern, 2θ has peaks at 5.52±0.2°, 7.56±0.2°, 8.29±0.2°, 9.22±0.2°, 11.04±0.2°, 15.17±0.2°, 15.81±0.2°, 17.43±0.2°, 18.51±0.2° and 20.40±0.2°.

18. The crystal of the compound of formula I according to claim 14, wherein In the X-ray powder diffraction pattern, 2θ has peaks at 5.52±0.2°, 7.56±0.2°, 8.29±0.2°, 9.22±0.2°, 11.04±0.2°, 15.17±0.2°, 15.81±0.2°, 17.00±0.2°, 17.43±0.2°, 18.51±0.2°, 19.70±0.2°, 20.01±0.2°, 20.40±0.2°, 20.75±0.2° and 22.59±0.2°.

19. The crystal of the compound of formula I according to claim 14, characterized in that In the X-ray powder diffraction pattern, 2θ has peaks at 5.52±0.2°, 7.56±0.2°, 8.29±0.2°, 9.22±0.2°, 10.66±0.2°, 11.04±0.2°, 12.94±0.2°, 14.69±0.2°, 15.17±0.2°, 15.81±0.2°, 16.63±0.2°, 17.00±0.2°, 17.43±0.2°, 18.51±0.2°, 19.70±0.2°, 20.01±0.2°, 20.40±0.2°, 20.75±0.2°, 22.59±0.2°, 25.88±0.2°, and 26.12±0.2°.

20. The crystal of the compound of formula I according to claim 14, wherein The X-ray powder diffraction pattern is shown in FIG7 .

21. The crystal of the compound of formula I according to claim 14, wherein The DSC spectrum has an endothermic peak starting point at 205.65±5℃.

22. The crystal of the compound of formula I according to claim 14, wherein The DSC spectrum is shown in Figure 8.

23. A crystal of a compound of formula I, characterized in that The X-ray powder diffraction pattern has diffraction peaks at 2θ of 4.78±0.2°, 10.52±0.2°, 12.83±0.2°, 16.24±0.2°, 18.44±0.2°, 22.33±0.2° and 23.20±0.2°, 24. The crystal of the compound of formula I according to claim 23, wherein In the X-ray powder diffraction pattern, 2θ has peaks at 4.78±0.2°, 10.52±0.2°, 12.83±0.2°, 16.24±0.2°, 18.44±0.2°, 19.41±0.2°, 22.33±0.2° and 23.20±0.2°.

25. The crystal of the compound of formula I according to claim 23, wherein In the X-ray powder diffraction pattern, 2θ has peaks at 4.78±0.2°, 9.63±0.2°, 10.52±0.2°, 12.83±0.2°, 13.48±0.2°, 15.79±0.2°, 16.24±0.2°, 17.89±0.2°, 18.44±0.2°, 19.41±0.2°, 19.61±0.2°, 22.33±0.2° and 23.20±0.2°.

26. The crystal of the compound of formula I according to claim 23, characterized in that In the X-ray powder diffraction pattern, 2θ has peaks at 4.78±0.2°, 7.46±0.2°, 9.63±0.2°, 10.52±0.2°, 11.17±0.2°, 12.83±0.2°, 13.48±0.2°, 14.42±0.2°, 15.79±0.2°, 16.24±0.2°, 17.89±0.2°, 18.44±0.2°, 19.41±0.2°, 19.61±0.2°, 20.44±0.2°, 22.33±0.2°, 23.20±0.2°, 26.48±0.2° and 27.05±0.2°.

27. The crystal of the compound of formula I according to claim 23, characterized in that The X-ray powder diffraction pattern is shown in FIG10 .

28. The crystal of the compound of formula I according to claim 23, wherein The DSC spectrum has an endothermic peak starting point at 176.47±5℃.

29. The crystal of the compound of formula I according to claim 23, wherein The DSC spectrum is shown in Figure 11.

30. A crystal of a compound of formula I, characterized in that The X-ray powder diffraction pattern shows peaks at 4.22±0.2°, 10.72±0.2°, 14.62±0.2°, 15.17±0.2°, 15.65±0.2°, 17.54±0.2° and 21.50±0.2° for 2θ.

31. The crystal of the compound of formula I according to claim 30, wherein The X-ray powder diffraction pattern further comprises one or both of diffraction peaks at 2θ of 19.55±0.2° or 19.80±0.2°.

32. The crystal of the compound of formula I according to claim 30, wherein In the X-ray powder diffraction pattern, 2θ has peaks at 4.22±0.2°, 10.72±0.2°, 14.62±0.2°, 15.17±0.2°, 15.65±0.2°, 17.54±0.2°, 19.55±0.2°, 19.80±0.2° and 21.50±0.2°.

33. The crystal of the compound of formula I according to claim 30, wherein In the X-ray powder diffraction pattern, 2θ has peaks at 4.22±0.2°, 10.72±0.2°, 13.82±0.2°, 14.62±0.2°, 15.17±0.2°, 15.65±0.2°, 16.92±0.2°, 17.54±0.2°, 19.55±0.2°, 19.80±0.2°, 21.50±0.2°, 22.76±0.2°, 23.35±0.2° and 26.06±0.2°.

34. The crystal of the compound of formula I according to claim 30, wherein The 2θ values ​​in the X-ray powder diffraction pattern were 4.22±0.2°, 7.99±0.2°, 8.75±0.2°, 9.91±0.2°, 10.72±0.2°, 11.66±0.2°, 12.75±0.2°, 13.82±0.2°, 14.62±0.2°, 15.17±0.2°, 15.65±0.2°, 16.24±0.2°, 16 There are peaks at .92±0.2°, 17.54±0.2°, 19.04±0.2°, 19.55±0.2°, 19.80±0.2°, 20.18±0.2°, 21.50±0.2°, 22.76±0.2°, 23.35±0.2°, 26.06±0.2°, 26.91±0.2°, 29.82±0.2° and 30.56±0.2°.

35. The crystal of the compound of formula I according to claim 30, wherein The X-ray powder diffraction pattern is shown in FIG13 .

36. The crystal of the compound of formula I according to claim 30, wherein The DSC spectrum has an endothermic peak starting point at 145.48±5℃.

37. The crystal of the compound of formula I according to claim 30, wherein The DSC spectrum is shown in Figure 14.

38. A crystal of a compound of formula I, characterized in that The X-ray powder diffraction pattern shows peaks at 4.54±0.2°, 9.08±0.2°, 14.90±0.2°, 18.25±0.2°, 19.24±0.2°, 22.86±0.2° and 23.50±0.2° for 2θ.

39. The crystal of the compound of formula I according to claim 38, wherein The X-ray powder diffraction pattern further comprises one, two or three diffraction peaks at 2θ of 13.66±0.2°, 17.46±0.2°, 24.75±0.2° or 27.51±0.2°.

40. The crystal of the compound of formula I according to claim 38, wherein In the X-ray powder diffraction pattern, 2θ has peaks at 4.54±0.2°, 9.08±0.2°, 13.66±0.2°, 14.90±0.2°, 17.46±0.2°, 18.25±0.2°, 19.24±0.2°, 22.86±0.2°, 23.50±0.2°, 24.75±0.2° and 27.51±0.2°.

41. The crystal of the compound of formula I according to claim 38, wherein In the X-ray powder diffraction pattern, 2θ has peaks at 4.54±0.2°, 8.20±0.2°, 9.08±0.2°, 13.66±0.2°, 13.97±0.2°, 14.51±0.2°, 14.90±0.2°, 15.51±0.2°, 16.63±0.2°, 17.46±0.2°, 18.25±0.2°, 19.24±0.2°, 19.82±0.2°, 21.11±0.2°, 22.37±0.2°, 22.86±0.2°, 23.50±0.2°, 24.75±0.2° and 27.51±0.2°.

42. The crystal of the compound of formula I according to claim 38, wherein The X-ray powder diffraction pattern is shown in FIG16 .

43. The crystal of the compound of formula I according to claim 38, wherein The DSC spectrum has an endothermic peak starting point at 171.37±5℃.

44. The crystal of the compound of formula I according to claim 38, wherein The DSC spectrum is shown in Figure 17.

45. A crystal of a compound of formula I, characterized in that The X-ray powder diffraction pattern has diffraction peaks at 2θ of 3.84±0.2°, 10.39±0.2°, 11.22±0.2°, 13.39±0.2°, 15.55±0.2°, 16.78±0.2° and 20.63±0.2°.

46. ​​The crystal of the compound of formula I according to claim 45, wherein In the X-ray powder diffraction pattern, 2θ has peaks at 3.84±0.2°, 10.39±0.2°, 11.22±0.2°, 13.39±0.2°, 15.55±0.2°, 16.78±0.2°, 20.01±0.2° and 20.63±0.2°.

47. The crystal of the compound of formula I according to claim 45, wherein In the X-ray powder diffraction pattern, 2θ has peaks at 3.84±0.2°, 7.72±0.2°, 9.56±0.2°, 10.39±0.2°, 11.22±0.2°, 12.47±0.2°, 13.39±0.2°, 14.01±0.2°, 15.55±0.2°, 16.78±0.2°, 19.02±0.2°, 20.01±0.2° and 20.63±0.2°.

48. The crystal of the compound of formula I according to claim 45, wherein The 2θ values ​​in the X-ray powder diffraction pattern were 3.84±0.2°, 6.69±0.2°, 7.72±0.2°, 9.56±0.2°, 10.39±0.2°, 11.22±0.2°, 12.47±0.2°, 13.39±0.2°, 14.01±0.2°, 15.00±0.2°, 15.55±0.2°, 16 There are peaks at .78±0.2°, 18.59±0.2°, 19.02±0.2°, 19.47±0.2°, 20.01±0.2°, 20.63±0.2°, 22.50±0.2°, 23.44±0.2°, 23.69±0.2°, 24.11±0.2°, 25.48±0.2° and 26.47±0.2°.

49. The crystal of the compound of formula I according to claim 45, wherein The X-ray powder diffraction pattern is shown in FIG19 .

50. A crystal of a compound of formula I, characterized in that The X-ray powder diffraction pattern shows peaks at 4.65±0.2°, 9.36±0.2°, 12.23±0.2°, 13.33±0.2°, 14.09±0.2°, 17.27±0.2° and 22.04±0.2° for 2θ.

51. The crystal of the compound of formula I according to claim 50, wherein The X-ray powder diffraction pattern further comprises one or both of diffraction peaks at 2θ of 19.37±0.2° or 22.95±0.2°.

52. The crystal of the compound of formula I according to claim 50, wherein In the X-ray powder diffraction pattern, 2θ has peaks at 4.65±0.2°, 9.36±0.2°, 12.23±0.2°, 13.33±0.2°, 14.09±0.2°, 17.27±0.2°, 19.37±0.2°, 22.04±0.2° and 22.95±0.2°.

53. The crystal of the compound of formula I according to claim 50, wherein In the X-ray powder diffraction pattern, 2θ has peaks at 4.65±0.2°, 9.36±0.2°, 10.41±0.2°, 12.23±0.2°, 13.33±0.2°, 14.09±0.2°, 17.27±0.2°, 18.88±0.2°, 19.37±0.2°, 20.58±0.2°, 22.04±0.2°, 22.49±0.2°, 22.95±0.2° and 23.69±0.2°.

54. The crystal of the compound of formula I according to claim 50, wherein In the X-ray powder diffraction pattern, 2θ has peaks at 4.65±0.2°, 7.20±0.2°, 9.36±0.2°, 10.41±0.2°, 11.12±0.2°, 12.23±0.2°, 13.33±0.2°, 14.09±0.2°, 15.44±0.2°, 16.41±0.2°, 16.68±0.2°, 17.27±0.2°, 18.31±0.2°, 18.88±0.2°, 19.37±0.2°, 20.58±0.2°, 22.04±0.2°, 22.49±0.2°, 22.95±0.2°, 23.69±0.2° and 25.12±0.2°.

55. The crystal of the compound of formula I according to claim 50, wherein The X-ray powder diffraction pattern is shown in FIG20 .

56. A crystalline composition comprising crystals of the compound of formula I according to any one of claims 1 to 55, wherein the crystals of the compound of formula I according to any one of claims 1 to 55 account for more than 50% by weight of the crystalline composition.

57. The crystalline composition of claim 56, comprising crystals of the compound of formula I according to any one of claims 1 to 55, wherein the crystals of the compound of formula I according to any one of claims 1 to 55 account for more than 80% by weight of the crystalline composition.

58. The crystalline composition of claim 56, comprising crystals of the compound of formula I according to any one of claims 1 to 55, wherein the crystals of the compound of formula I according to any one of claims 1 to 55 account for more than 90% by weight of the crystalline composition.

59. The crystalline composition of claim 56, comprising crystals of the compound of formula I according to any one of claims 1 to 55, wherein the crystals of the compound of formula I according to any one of claims 1 to 55 account for more than 95% by weight of the crystalline composition.

60. A pharmaceutical composition comprising a crystal of a compound of formula I according to any one of claims 1 to 55, or a crystalline composition according to any one of claims 56 to 59.

61. Use of a crystal of the compound of formula I according to any one of claims 1 to 55, or a crystalline composition according to claims 56 to 59, or a pharmaceutical composition according to claim 60 in the preparation of a medicament for preventing or treating a disease associated with the anti-apoptotic protein BCL-2. The use according to claim 61 , wherein the disease associated with the anti-apoptotic protein BCL-2 is cancer.

63. The use according to claim 62, wherein the cancer is acute lymphoblastic leukemia.

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