Salts of heterocyclic antitumor compounds and their crystalline forms

Abstract

The present invention provides a crystalline form of the free base of Compound I, a salt of Compound I, and a crystalline form of a salt of Compound I. The crystalline form and salt form have excellent effects in terms of physical stability, chemical stability, hygroscopicity, etc., and have relatively good clinical application value, making them excellent candidate forms for further drug development. [Formula 1] JPEG2025539054000034.jpg30169

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS The present invention claims priority to an invention patent application filed in China on November 11, 2022, entitled "Salts of heterocyclic antitumor compounds and crystalline forms thereof," with application number 202211410146.4, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of crystalline drugs. Specifically, the present invention relates to a crystalline form or salt of the compound (6-((1-benzoylpiperidin-4-yl)amino)-2-isopropoxypyrimidin-4-yl)((3R,4R)-4-(3,4-dihydroisoquinolin-2(1H)-yl)-3-hydroxypiperidin-1-yl)methanone (hereinafter referred to as Compound I or the compound of Formula I) as a PRMT5 inhibitor, and a method for preparing the same. The present invention also relates to the use of a crystalline form or salt of Compound I in the prevention and / or treatment of PRMT5-related diseases and / or conditions. [Background technology]

[0003] PRMT5, a type of protein arginine methyltransferase (PRMT), is a novel antitumor target associated with epigenetic modifications. It has several aliases, including Hsl7, Jbp1, Skb1, Capsuleen, and Dart5. PRMT5 is a key enzyme for arginine monomethylation and symmetric dimethylation. A growing body of literature has demonstrated that protein arginine methyltransferases play important roles in various biological processes, such as cell growth, proliferation, apoptosis, and metastasis.

[0004] Protein arginine methyltransferases (PMAs) transfer a methyl group from S-adenosylmethionine (AdoMet or SAM) to arginine residues in histones or other proteins to form methylarginine and S-adenosylhomocysteine ​​(SAH). Currently, nine members of this family (PRMTs 1-9) have been identified. PRMTs can be classified into three types based on the manner in which they catalyze arginine methylation: type I PRMTs, including PRMT1, PRMT2, PRMT3, PRMT4, PRMT6, and PRMT8, catalyze monomethylarginine (MMA) and asymmetric dimethylarginine (aDMA); type II PRMTs, including PRMT5 and PRMT9, catalyze MMA and symmetric dimethylarginine (sDMA); and type III PRMTs, including PRMT7, catalyze only MMA. As an epigenetic enzyme, PRMT5 symmetrically methylates arginine residues on histones or non-histone substrates, affecting multiple target genes and signaling pathways. It plays an important role in protein methylation, including variable shear, post-transcriptional regulation, RNA processing, cell proliferation, cell differentiation, apoptosis, and tumorigenesis. Substances that selectively inhibit PRMT5 could potentially act as potent new anti-cancer drugs. The development of new drugs targeting PRMT5 has a positive effect on filling the gap in addressing unmet clinical needs. Summary of the Invention [Problem to be solved by the invention]

[0005] Chinese Patent Application No. 202210517649.5 and International Patent Application No. PCT / CN2022 / 092346 describe compounds that are PRMT5 inhibitors and methods for preparing them, including Compound I (the structure of which is shown below) and methods for preparing it. [ka]

[0006] Compound I is an effective PRMT5 inhibitor. In vitro enzyme activity inhibitory activity studies show that it has a relatively strong inhibitory effect on PRMT5 enzyme. In addition, human B-cell non-Hodgkin's lymphoma Z-138 cell line subcutaneous xenograft test in NOD / SCID female mice shows that it has excellent tumor inhibitory effect. Therefore, Compound I may be a promising compound for preventing and / or treating PRMT5-mediated diseases.

[0007] At present, there are no reports on the crystalline forms of compound I and its salts. Comprehensive and systematic screening of crystalline polymorphs and salt forms is an essential and important research topic. Therefore, it is necessary to further screen the crystalline forms of compound I and its salts, and develop crystalline forms or salt forms suitable for large-scale production, so as to provide more and better options for subsequent drug development. [Means for solving the problem]

[0008] One object of the present invention is to provide crystalline form A of compound I, which has an X-ray powder diffraction (XRPD) pattern expressed in 2θ angles using Cu-Kα radiation, with characteristic peaks at 4.0±0.2°, 18.4±0.2°, 20.3±0.2°, and 21.8±0.2°.

[0009] Preferably, the crystalline form A has an XRPD pattern using Cu-Kα radiation, which has characteristic peaks at 4.0±0.2°, 14.7±0.2°, 15.8±0.2°, 18.4±0.2°, 20.3±0.2°, and 21.8±0.2° in 2θ angles.

[0010] More preferably, the crystalline form A has an XRPD pattern using Cu-Kα radiation, expressed in 2θ angles, with characteristic peaks at 4.0±0.2°, 7.1±0.2°, 13.6±0.2°, 14.7±0.2°, 15.8±0.2°, 18.4±0.2°, 20.3±0.2°, 21.8±0.2°, and 27.7±0.2°.

[0011] More preferably, said crystalline form A has an XRPD pattern essentially as shown in FIG.

[0012] In some embodiments of the present invention, the differential scanning calorimetry (DSC) pattern of the crystalline form A has an endothermic peak at around 85.0°C to 150.0°C.

[0013] Preferably, the DSC pattern of the crystalline form A has an endothermic peak at 85.0°C±5°C to 150.0°C±5°C.

[0014] More preferably, said crystalline form A has a DSC pattern essentially as shown in FIG.

[0015] In some embodiments of the present invention, the thermogravimetric analysis (TGA) pattern of Form A has a weight loss of about 2.0% near 100.0°C and further has a weight loss of about 3.5% near 150.0°C.

[0016] Preferably, the TGA pattern of said crystalline form A has a weight loss of about 2.0% at 100.0°C ± 5°C and further has a weight loss of about 3.5% at 150.0°C ± 5°C.

[0017] More preferably, said crystalline form A has a TGA pattern essentially as shown in FIG.

[0018] Another object of the present invention is to provide a salt of Compound I, which is a hydrochloride, hydrobromide, phosphate, sulfate, methanesulfonate, p-toluenesulfonate, camphorsulfonate (e.g., L-camphorsulfonate), oxalate, maleate, tartrate (e.g., L-tartrate), fumarate, citrate, malate (e.g., L-malate), glycolate, or benzoate salt of Compound I.

[0019] Another object of the present invention is to provide a hydrochloride salt of Compound I (for example, Compound I-1), in which the molar ratio of Compound I to hydrochloric acid to form a salt is 1:2. [ka]

[0020] Another object of the present invention is to provide a crystalline form A of the hydrochloride salt of Compound I, in which the molar ratio of Compound I to hydrochloric acid to form a salt is 1:2.

[0021] In some embodiments of the present invention, the hydrochloride salt crystalline Form A of Compound I has an XRPD pattern using Cu-Kα radiation, with characteristic peaks at 4.4±0.2°, 15.3±0.2°, 18.0±0.2°, 18.8±0.2°, and 19.3±0.2° 2θ angles.

[0022] Preferably, the hydrochloride crystalline form A of Compound I has an XRPD pattern using Cu-Kα radiation, which has characteristic peaks at 4.4±0.2°, 11.5±0.2°, 12.7±0.2°, 15.3±0.2°, 18.0±0.2°, 18.8±0.2°, 19.3±0.2°, and 21.6±0.2° in 2θ angles.

[0023] More preferably, the hydrochloride crystalline Form A of Compound I has an XRPD pattern using Cu-Kα radiation, expressed in 2θ angles, with characteristic peaks at 4.4±0.2°, 8.5±0.2°, 11.5±0.2°, 12.7±0.2°, 14.0±0.2°, 15.3±0.2°, 16.7±0.2°, 18.0±0.2°, 18.8±0.2°, 19.3±0.2°, 20.9±0.2°, 21.6±0.2°, and 23.3±0.2°.

[0024] More preferably, the hydrochloride salt crystalline form A of Compound I has an XRPD pattern essentially as shown in FIG.

[0025] In some embodiments of the present invention, the DSC pattern of the hydrochloride salt crystalline Form A of Compound I has an endothermic peak at around 149.4°C.

[0026] Preferably, the DSC pattern of the hydrochloride crystalline form A of Compound I has an endothermic peak at 149.4°C ± 5°C.

[0027] More preferably, the hydrochloride salt crystalline form A of Compound I has a DSC pattern essentially as shown in FIG.

[0028] In some embodiments of the present invention, the TGA pattern of the hydrochloride salt crystalline Form A of Compound I has a weight loss of about 0.3% near 100.0°C and further has a weight loss of about 16.3% near 235.0°C.

[0029] Preferably, the TGA pattern of the hydrochloride salt crystalline Form A of Compound I has a weight loss of about 0.3% at 100.0°C ± 5°C and further has a weight loss of about 16.3% at 235.0°C ± 5°C.

[0030] More preferably, the hydrochloride salt crystalline Form A of Compound I has a TGA pattern essentially as shown in FIG.

[0031] Another object of the present invention is to provide a crystalline form A of the oxalate salt of Compound I, which has an XRPD pattern expressed in 2θ angles using Cu-Kα radiation, with characteristic peaks at 4.5±0.2°, 8.4±0.2°, 18.8±0.2°, 20.1±0.2°, and 21.2±0.2°.

[0032] Preferably, the oxalate crystalline Form A of Compound I has an XRPD pattern using Cu-Kα radiation, with characteristic peaks at 4.5±0.2°, 6.1±0.2°, 8.4±0.2°, 10.4±0.2°, 16.8±0.2°, 18.8±0.2°, 20.1±0.2°, and 21.2±0.2° in 2θ angles.

[0033] More preferably, Compound I oxalate crystalline Form A has an XRPD pattern using Cu-Kα radiation, expressed in 2θ angles, having characteristic peaks at 4.5±0.2°, 5.2±0.2°, 6.1±0.2°, 8.4±0.2°, 10.4±0.2°, 15.8±0.2°, 16.3±0.2°, 16.8±0.2°, 17.6±0.2°, 18.8±0.2°, 20.1±0.2°, 21.2±0.2°, 22.3±0.2°, and 23.0±0.2°.

[0034] More preferably, the oxalate crystalline Form A of Compound I has an XRPD pattern essentially as shown in FIG.

[0035] In some embodiments of the present invention, the DSC pattern of crystalline Form A of the oxalate salt of Compound I has an endothermic peak at around 157.2°C.

[0036] Preferably, the DSC pattern of the oxalate crystalline form A of Compound I has an endothermic peak at 157.2°C ± 5°C.

[0037] More preferably, the oxalate crystalline Form A of Compound I has a DSC pattern essentially as shown in FIG.

[0038] In some embodiments of the present invention, the TGA pattern of crystalline Form A of the oxalate salt of Compound I has a weight loss of about 4.0% near 150.0°C.

[0039] Preferably, the TGA pattern of crystalline Form A of the oxalate salt of Compound I has a weight loss of about 4.0% at 150.0°C ± 5°C.

[0040] More preferably, the oxalate crystalline Form A of Compound I has a TGA pattern essentially as shown in FIG.

[0041] Another object of the present invention is to provide a tartrate salt of Compound I (e.g., L-tartrate, i.e., Compound I-2), in which the molar ratio of Compound I to tartaric acid to form a salt is 1:1.5. [ka]

[0042] Another object of the present invention is to provide a crystalline form A of the tartrate salt (e.g., L-tartrate) of Compound I, wherein the molar ratio of Compound I to tartaric acid in the crystalline form A is 1:1.5.

[0043] In some embodiments of the present invention, the tartrate salt of Compound I, Form A, has an XRPD pattern using Cu-Kα radiation, with characteristic peaks at 15.5±0.2°, 16.0±0.2°, 18.4±0.2°, and 20.5±0.2° 2θ angles.

[0044] Preferably, the crystalline Form A of the tartrate salt of Compound I has an XRPD pattern using Cu-Kα radiation, which has characteristic peaks at 5.8±0.2°, 8.8±0.2°, 11.7±0.2°, 15.5±0.2°, 16.0±0.2°, 18.4±0.2°, and 20.5±0.2° in 2θ angles.

[0045] More preferably, the crystalline Form A of the tartrate salt of Compound I has an XRPD pattern using Cu-Kα radiation, expressed in 2θ angles, with characteristic peaks at 3.0±0.2°, 5.8±0.2°, 8.8±0.2°, 11.7±0.2°, 15.5±0.2°, 16.0±0.2°, 16.7±0.2°, 18.4±0.2°, 20.5±0.2°, and 21.2±0.2°.

[0046] More preferably, the crystalline Form A of the tartrate salt of Compound I has an XRPD pattern essentially as shown in FIG.

[0047] In some embodiments of the present invention, the DSC pattern of Form A of the tartrate salt of Compound I has an endothermic peak at around 174.8°C.

[0048] Preferably, the DSC pattern of the tartrate salt of Compound I, crystalline Form A, has an endothermic peak at 174.8°C ± 5°C.

[0049] More preferably, the crystalline Form A of the tartrate salt of Compound I has a DSC pattern essentially as shown in FIG.

[0050] In some embodiments of the present invention, the TGA pattern of Form A of the tartrate salt of Compound I has a weight loss of about 5.8% near 150.0°C.

[0051] Preferably, the TGA pattern of crystalline Form A of the tartrate salt of Compound I has a weight loss of about 5.8% at 150.0°C ± 5°C.

[0052] More preferably, the crystalline Form A of the tartrate salt of Compound I has a TGA pattern essentially as shown in FIG.

[0053] Another object of the present invention is to provide a p-toluenesulfonic acid salt of Compound I (e.g., Compound I-3), in which the molar ratio of Compound I to p-toluenesulfonic acid to form a salt is 1:2. [ka]

[0054] Another object of the present invention is to provide a crystalline form A of p-toluenesulfonate of Compound I, wherein the molar ratio of Compound I to p-toluenesulfonic acid in the salt formation is 1:2.

[0055] In some embodiments of the present invention, the p-toluenesulfonate salt of Compound I, crystalline Form A, has an XRPD pattern using Cu-Kα radiation, with characteristic peaks at 4.0±0.2°, 8.1±0.2°, 18.3±0.2°, and 20.0±0.2° 2θ angles.

[0056] Preferably, the p-toluenesulfonate crystalline form A of Compound I has an XRPD pattern using Cu-Kα radiation, which has characteristic peaks at 2θ angles of 4.0±0.2°, 8.1±0.2°, 11.7±0.2°, 12.7±0.2°, 14.6±0.2°, 18.3±0.2°, 20.0±0.2°, and 23.1±0.2°.

[0057] More preferably, the p-toluenesulfonate crystalline form A of Compound I has an XRPD pattern using Cu-Kα radiation, expressed in 2θ angles, with characteristic peaks at 4.0±0.2°, 6.2±0.2°, 8.1±0.2°, 8.8±0.2°, 11.7±0.2°, 12.7±0.2°, 14.6±0.2°, 16.3±0.2°, 17.5±0.2°, 18.3±0.2°, 19.0±0.2°, 20.0±0.2°, and 23.1±0.2°.

[0058] More preferably, the p-toluenesulfonate crystalline form A of Compound I has an XRPD pattern essentially as shown in FIG.

[0059] In some embodiments of the present invention, the DSC pattern of the p-toluenesulfonate salt of Compound I, crystalline Form A, has endothermic peaks at around 88.5°C and 150.1°C.

[0060] Preferably, the DSC pattern of the p-toluenesulfonate crystalline form A of Compound I has endothermic peaks at 88.5°C±5°C and 150.1°C±5°C.

[0061] More preferably, the p-toluenesulfonate crystalline form A of Compound I has a DSC pattern essentially as shown in FIG.

[0062] In some embodiments of the present invention, the TGA pattern of p-toluenesulfonate crystalline Form A of Compound I has a weight loss of about 5.7% near 130.0°C and further has a weight loss of about 4.7% near 220.0°C.

[0063] Preferably, the TGA pattern of the crystalline form A of p-toluenesulfonate salt of Compound I has a weight loss of about 5.7% at 130.0°C±5°C and further has a weight loss of about 4.7% at 220.0°C±5°C.

[0064] More preferably, the p-toluenesulfonate crystalline form A of Compound I has a TGA pattern essentially as shown in FIG.

[0065] Another object of the present invention is to provide a crystalline form B of p-toluenesulfonate of Compound I, wherein the molar ratio of Compound I to p-toluenesulfonic acid in the crystalline form B is 1:2.

[0066] In some embodiments of the present invention, the p-toluenesulfonate crystalline form B of Compound I has an XRPD pattern using Cu-Kα radiation, with characteristic peaks at 4.3±0.2°, 19.1±0.2°, and 20.2±0.2° 2θ angles.

[0067] Preferably, the p-toluenesulfonate crystalline form B of Compound I has an XRPD pattern using Cu-Kα radiation, which has characteristic peaks at 4.3±0.2°, 9.8±0.2°, 11.5±0.2°, 19.1±0.2°, and 20.2±0.2° in 2θ angles.

[0068] More preferably, the p-toluenesulfonate crystalline form B of Compound I has an XRPD pattern using Cu-Kα radiation, which has characteristic peaks at 2θ angles of 4.3±0.2°, 4.9±0.2°, 9.8±0.2°, 11.5±0.2°, 17.2±0.2°, 19.1±0.2°, 20.2±0.2°, and 21.5±0.2°.

[0069] More preferably, the p-toluenesulfonate crystalline form B of Compound I has an XRPD pattern using Cu-Kα radiation, which has characteristic peaks at 2θ angles of 4.3±0.2°, 4.9±0.2°, 9.0±0.2°, 9.8±0.2°, 11.5±0.2°, 17.2±0.2°, 19.1±0.2°, 20.2±0.2°, 21.5±0.2°, and 25.7±0.2°.

[0070] Even more preferably, the p-toluenesulfonate crystalline form B of Compound I has an XRPD pattern expressed in 2θ angles using Cu-Kα radiation, with characteristic peaks at 4.3±0.2°, 4.9±0.2°, 7.1±0.2°, 9.0±0.2°, 9.8±0.2°, 11.5±0.2°, 12.8±0.2°, 17.2±0.2°, 19.1±0.2°, 20.2±0.2°, 21.5±0.2°, and 25.7±0.2°.

[0071] Most preferably, the p-toluenesulfonate crystalline form B of Compound I has an X-ray powder diffraction pattern essentially as shown in FIG. 13 or FIG.

[0072] In some embodiments of the present invention, the DSC pattern of the p-toluenesulfonate crystalline form B of Compound I has an endothermic peak at about 192.1°C, and preferably further has an endothermic peak at about 124.8°C.

[0073] Preferably, the DSC pattern of the p-toluenesulfonate crystalline form B of Compound I has an endothermic peak at 192.1±5°C, and preferably further has an endothermic peak at 124.8±5°C.

[0074] More preferably, the p-toluenesulfonate crystalline form B of Compound I has a DSC pattern essentially as shown in FIG.

[0075] In some embodiments of the present invention, the TGA pattern of p-toluenesulfonate crystalline Form B of Compound I has a weight loss of about 2.0% near 167.3°C and further has a weight loss of about 5.2% near 233.7°C.

[0076] Preferably, the TGA pattern of p-toluenesulfonate crystalline Form B of Compound I has a weight loss of about 2.0% at 167.3°C ± 5°C and further has a weight loss of about 5.2% at 233.7°C ± 5°C.

[0077] More preferably, the p-toluenesulfonate crystalline form B of Compound I has a TGA pattern essentially as shown in FIG.

[0078] In some embodiments of the present invention, the p-toluenesulfonate salt of Compound I having crystalline form B (eg, the p-toluenesulfonate salt having a salt formation ratio of 1:2) is a hydrate, preferably a monohydrate.

[0079] Another object of the present invention is to provide a pharmaceutical composition comprising the hydrochloride, hydrobromide, phosphate, sulfate, methanesulfonate, p-toluenesulfonate, camphorsulfonate (e.g., L-camphorsulfonate), oxalate, maleate, tartrate (e.g., L-tartrate), fumarate, citrate, malate (e.g., L-malate), glycolate, or benzoate of Compound I.

[0080] Another object of the present invention is to provide a pharmaceutical composition comprising crystalline Form A of Compound I, crystalline Form A of the hydrochloride salt of Compound I, crystalline Form A of the oxalate salt of Compound I, crystalline Form A of the tartrate salt (e.g., the L-tartrate salt) of Compound I, crystalline Form A of the p-toluenesulfonate salt of Compound I, or crystalline Form B of the p-toluenesulfonate salt of Compound I.

[0081] Another object of the present invention is to provide use of the hydrochloride, hydrobromide, phosphate, sulfate, methanesulfonate, p-toluenesulfonate, camphorsulfonate (e.g., L-camphorsulfonate), oxalate, maleate, tartrate (e.g., L-tartrate), fumarate, citrate, malate (e.g., L-malate), glycolate or benzoate salt of Compound I or the pharmaceutical composition comprising a salt of Compound I in the manufacture of a medicament for preventing and / or treating a PRMT5-mediated disease and / or condition.

[0082] Another object of the present invention is to provide use of the hydrochloride, hydrobromide, phosphate, sulfate, methanesulfonate, p-toluenesulfonate, camphorsulfonate (e.g., L-camphorsulfonate), oxalate, maleate, tartrate (e.g., L-tartrate), fumarate, citrate, malate (e.g., L-malate), glycolate or benzoate salt of Compound I or the pharmaceutical composition comprising a salt of Compound I in the manufacture of a medicament for preventing and / or treating a tumor disease.

[0083] Preferably, the tumor disease is a solid tumor or a hematological tumor, preferably a malignant solid tumor or a hematological tumor.

[0084] More preferably, the tumor disease is lymphoma.

[0085] Another object of the present invention is to provide use of the above pharmaceutical composition comprising crystalline Form A of Compound I, crystalline Form A of the hydrochloride salt of Compound I, crystalline Form A of the oxalate salt of Compound I, crystalline Form A of the tartrate salt of Compound I, crystalline Form A of the p-toluenesulfonate salt of Compound I, or crystalline Form B of the p-toluenesulfonate salt of Compound I, or a crystalline form of a base of Compound I, in the manufacture of a medicament for the prevention and / or treatment of a PRMT5-mediated disease and / or condition.

[0086] Another object of the present invention is to provide use of the above pharmaceutical composition comprising crystalline Form A of Compound I, crystalline Form A of the hydrochloride salt of Compound I, crystalline Form A of the oxalate salt of Compound I, crystalline Form A of the tartrate salt of Compound I, crystalline Form A of the p-toluenesulfonate salt of Compound I, or crystalline Form B of the p-toluenesulfonate salt of Compound I, or a crystalline form of a base of Compound I, in the manufacture of a medicament for preventing and / or treating a tumor disease.

[0087] Preferably, the tumor disease is a solid tumor or a hematological tumor, preferably a malignant solid tumor or a hematological tumor.

[0088] More preferably, the tumor disease is lymphoma.

[0089] Another object of the present invention is to provide a pharmaceutical composition comprising the hydrochloride, hydrobromide, phosphate, sulfate, methanesulfonate, p-toluenesulfonate, camphorsulfonate (e.g., L-camphorsulfonate), oxalate, maleate, tartrate (e.g., L-tartrate), fumarate, citrate, malate (e.g., L-malate), glycolate or benzoate salt of Compound I, or a salt of Compound I, for the prevention and / or treatment of PRMT5-mediated diseases and / or conditions.

[0090] Another object of the present invention is to provide a pharmaceutical composition comprising the hydrochloride, hydrobromide, phosphate, sulfate, methanesulfonate, p-toluenesulfonate, camphorsulfonate (e.g., L-camphorsulfonate), oxalate, maleate, tartrate (e.g., L-tartrate), fumarate, citrate, malate (e.g., L-malate), glycolate or benzoate of Compound I, or a salt of Compound I, for the prevention and / or treatment of tumor diseases.

[0091] Preferably, the tumor disease is a solid tumor or a hematological tumor, preferably a malignant solid tumor or a hematological tumor.

[0092] More preferably, the tumor disease is lymphoma.

[0093] Another object of the present invention is to provide a pharmaceutical composition comprising crystalline form A of Compound I, crystalline form A of the hydrochloride salt of Compound I, crystalline form A of the oxalate salt of Compound I, crystalline form A of the tartrate salt of Compound I, crystalline form A of the p-toluenesulfonate salt of Compound I, or crystalline form B of the p-toluenesulfonate salt of Compound I, or a crystalline form of a base of Compound I, for preventing and / or treating PRMT5-mediated diseases and / or conditions.

[0094] Another object of the present invention is to provide a pharmaceutical composition comprising crystalline Form A of Compound I, crystalline Form A of the hydrochloride salt of Compound I, crystalline Form A of the oxalate salt of Compound I, crystalline Form A of the tartrate salt of Compound I, crystalline Form A of the p-toluenesulfonate salt of Compound I, or crystalline Form B of the p-toluenesulfonate salt of Compound I, or a crystalline form of a base of Compound I, for preventing and / or treating tumor diseases.

[0095] Preferably, the tumor disease is a solid tumor or a hematological tumor, preferably a malignant solid tumor or a hematological tumor.

[0096] More preferably, the tumor disease is lymphoma.

[0097] Another object of the present invention is to provide a method for preventing and / or treating a PRMT5-mediated disease and / or condition, comprising the step of administering a prophylactically and / or therapeutically effective amount of the hydrochloride, hydrobromide, phosphate, sulfate, methanesulfonate, p-toluenesulfonate, camphorsulfonate (e.g., L-camphorsulfonate), oxalate, maleate, tartrate (e.g., L-tartrate), fumarate, citrate, malate (e.g., L-malate), glycolate or benzoate salt of compound I, or a pharmaceutical composition comprising a salt of compound I, to an individual in need thereof.

[0098] Another object of the present invention is to provide a method for preventing and / or treating a tumor disease, comprising the step of administering a prophylactically and / or therapeutically effective amount of the hydrochloride, hydrobromide, phosphate, sulfate, methanesulfonate, p-toluenesulfonate, camphorsulfonate (e.g., L-camphorsulfonate), oxalate, maleate, tartrate (e.g., L-tartrate), fumarate, citrate, malate (e.g., L-malate), glycolate or benzoate salt of compound I, or the pharmaceutical composition containing a salt of compound I, to an individual in need thereof.

[0099] Preferably, the tumor disease is a solid tumor or a hematological tumor, preferably a malignant solid tumor or a hematological tumor.

[0100] More preferably, the tumor disease is lymphoma.

[0101] Another object of the present invention is to provide a method for preventing and / or treating PRMT5-mediated diseases and / or conditions, comprising the step of administering to an individual in need thereof a prophylactically and / or therapeutically effective amount of the above-mentioned pharmaceutical composition comprising crystalline Form A of Compound I, hydrochloride crystalline Form A of Compound I, oxalate crystalline Form A of Compound I, tartrate crystalline Form A of Compound I, p-toluenesulfonate crystalline Form A of Compound I, or p-toluenesulfonate crystalline Form B of Compound I, or a base crystalline form of Compound I.

[0102] Another object of the present invention is to provide a method for preventing and / or treating a tumor disease, comprising the step of administering to an individual in need thereof a prophylactically and / or therapeutically effective amount of the above-mentioned pharmaceutical composition containing crystalline Form A of Compound I, crystalline Form A of the hydrochloride salt of Compound I, crystalline Form A of the oxalate salt of Compound I, crystalline Form A of the tartrate salt of Compound I, crystalline Form A of the p-toluenesulfonate salt of Compound I, or crystalline Form B of the p-toluenesulfonate salt of Compound I, or a crystalline form of Compound I.

[0103] Preferably, the tumor disease is a solid tumor or a hematological tumor, preferably a malignant solid tumor or a hematological tumor.

[0104] More preferably, the tumor disease is lymphoma.

[0105] For the treatment of tumor diseases, the hydrochloride, hydrobromide, phosphate, sulfate, methanesulfonate, p-toluenesulfonate, camphorsulfonate (e.g., L-camphorsulfonate), oxalate, maleate, tartrate (e.g., L-tartrate), fumarate, citrate, malate (e.g., L-malate), glycolate, or benzoate salts of Compound I, or crystalline Form A of Compound I, hydrochloride crystalline Form A of Compound I, oxalate crystalline Form A of Compound I, tartrate crystalline Form A of Compound I, p-toluenesulfonate crystalline Form A of Compound I, or p-toluenesulfonate crystalline Form B of Compound I, can be used in combination with other therapeutic agents (e.g., chemotherapeutic agents, biological therapeutic agents, etc.) or in combination with other therapeutic modalities, including, but not limited to, radiotherapy. [Effects of the Invention]

[0106] The present invention provides, for the first time, multiple crystalline forms and salt forms of Compound I. Some crystalline forms and salt forms provided by the present invention have excellent effects in at least one aspect, such as physical stability, chemical stability, hygroscopicity, etc., and have relatively good clinical application value, making them excellent candidate forms for further drug development. [Brief explanation of the drawings]

[0107] [Figure 1] 1 is an XRPD pattern of crystalline form A of Compound I. [Figure 2] 1 shows the DSC and TGA patterns of crystalline form A of Compound I. [Figure 3] 1 is an XRPD pattern of crystalline Form A of the hydrochloride salt of Compound I. [Figure 4] 1 shows the DSC and TGA patterns of crystalline Form A of the hydrochloride salt of Compound I. [Figure 5] 1 is an XRPD pattern of crystalline Form A of the oxalate salt of Compound I. [Figure 6] 1 shows the DSC and TGA patterns of crystalline Form A of the oxalate salt of Compound I. [Figure 7] 1 is an XRPD pattern of crystalline Form A of the tartrate salt of Compound I. [Figure 8] 1 shows the DSC and TGA patterns of crystalline Form A of the tartrate salt of Compound I. [Figure 9] 1 is a 1H NMR pattern of crystalline Form A of the tartrate salt of Compound I. [Figure 10] 1 is an XRPD pattern of crystalline form A of p-toluenesulfonate salt of Compound I. [Figure 11] 1 shows the DSC and TGA patterns of p-toluenesulfonate crystalline form A of Compound I. [Figure 12] 1 is a 1H NMR pattern of crystalline form A of p-toluenesulfonate salt of Compound I. [Figure 13] 1 is an XRPD pattern of crystalline form B of the p-toluenesulfonate salt of Compound I. [Figure 14] 1 is a 1H NMR pattern of p-toluenesulfonate crystalline form B of Compound I. [Figure 15] 1 is an XRPD pattern of crystalline form B of the p-toluenesulfonate salt of Compound I. [Figure 16] 1 shows the DSC and TGA patterns of p-toluenesulfonate crystalline form B of Compound I. [Figure 17] 1 is an XRPD pattern of amorphous Compound I. DETAILED DESCRIPTION OF THE INVENTION

[0108] The technical solution of the present invention will be described in more detail below with reference to specific examples. The following examples are merely intended to illustrate and explain the present invention, and should not be construed as limiting the scope of the claims of the present invention. Any technology realized based on the above content of the present invention is included within the scope of the claims of the present invention.

[0109] Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available or may be prepared by known methods.

[0110] Test equipment used in the experiment 1. Differential scanning calorimetry analyzer TA Discovery 2500 (TA, US): After weighing, the sample was placed in a perforated DSC Tzero sample tray and heated to the final temperature at a rate of 10 °C / min. The nitrogen gas purge rate in the furnace was 50 mL / min.

[0111] 2. Thermogravimetric analyzer TA Discovery 55 (TA, US): The sample was placed in an equilibrated open aluminum sample tray and automatically weighed in the TGA furnace. The sample was heated to the final temperature at a rate of 10 °C / min. The nitrogen gas purge rate at the sample was 60 mL / min, and the nitrogen gas purge rate at the balance was 40 mL / min.

[0112] 3. Synchronous thermal analyzer NETZSCH STA449F3 (NETZSCH, GER): Measurement was performed according to the thermal analysis method 0661 of the Pharmacopoeia of the People's Republic of China, 2015 edition, Part 4 General Provisions, with a range of 26°C to 350°C and a scanning rate of 20.0 K / min.

[0113] 4. X-ray powder diffractometer Bruker D8 Advance (Bruker, GER): 2θ scan angle 3°~45°, scan step size 0.02°, exposure time 0.12 s. During sample testing, the light pipe voltage and current were 40 kV and 40 mA, respectively, and the sample tray was a zero-background sample tray.

[0114] 5. X-ray powder diffractometer Panalytical Empyrean (Panalytical, NL): The 2θ scan angle was 3° to 45°, the scan step size was 0.013°, and the exposure time was 20.4 seconds. The light pipe voltage and current during sample testing were 45 kV and 40 mA, respectively, and the sample tray was a zero-background sample tray.

[0115] 6. X-ray powder diffractometer: Bruker D2 PHASER type X-ray diffractometer: Cu target, pipe voltage (30 kV), pipe current (10 mA), 2θ scan range 2–40°, scan speed 0.10 s, step size 0.020°.

[0116] 7. Nuclear magnetic analysis ( 1 1 H NMR): Solid samples were dissolved in deuterated dimethyl sulfoxide (DMSO-d6) solvent and subjected to nuclear magnetic analysis on a Bruker AVANCE-III (Bruker, GER).

[0117] 8. The instrument parameters for ion chromatography (IC) ICS 5000 (Thermo Fisher, US) are shown below.

[0118] [Table 1] 9. Dynamic moisture sorption / desorption analysis (DVS) was measured using DVS Intrinsic (SMS, UK): the test was performed in gradient mode with a humidity change of 50%-95%-0%-50%, with a humidity change of 10% for each gradient within the 0%-90% range. The gradient endpoint was determined using the dm / dt method, with dm / dt less than 0.002% and maintained for 10 minutes. After the test was completed, the sample was subjected to XRPD analysis to determine whether the solid form had changed.

[0119] 10. Nuclear magnetic resonance ( 1 H NMR): Bruker AVANCE NEO 400 MHz.

[0120] 11. Liquid chromatography mass spectrometry (LC-MS): WATERS ACQUITY UPLC H-Class PLUS or / and SQD2.

[0121] Example 1: Preparation of Compound I Preparation of (6-((1-benzoylpiperidin-4-yl)amino)-2-isopropoxypyrimidin-4-yl)((3R,4R)-4-(3,4-dihydroisoquinolin-2(1H)-yl)-3-hydroxypiperidin-1-yl)methanone (Compound I) Step 1: Preparation of 6-((1-(tert-butoxycarbonyl)piperidin-4-yl)amino)-2-chloropyrimidine-4-formic acid [ka]

[0122] Methyl 6-((1-(tert-butoxycarbonyl)piperidin-4-yl)amino)-2-chloropyrimidine-4-formate (500 mg, 1.347 mmol, 1.0 equiv.) was dissolved in acetonitrile (5 mL), and potassium trimethylsilanol (0.1729 g, 1.347 mmol, 1.0 equiv.) was added and stirred at 25° C. for 1 hour. After completion of the reaction determined by LC-MS, the mixture was concentrated, dissolved in 10% dichloromethane-methanol solution, filtered, and the filtrate was concentrated to give the title compound (480 mg, yield 99.8%).

[0123] LC-MS (ESI) [M+H] + = 357.2.

[0124] Step 2: Preparation of 6-((1-(tert-butoxycarbonyl)piperidin-4-yl)amino)-2-isopropoxypyrimidine-4-formic acid [ka]

[0125] 6-((1-(tert-butoxycarbonyl)piperidin-4-yl)amino)-2-chloropyrimidine-4-formic acid (480 mg, 1.35 mmol, 1.0 equiv.) was dissolved in isopropanol (5 mL) and potassium tert-butoxide (628.8 mg, 5.4 mmol, 4.0 equiv.) was added. The mixture was stirred at 90°C for 3 hours. After completion of the reaction determined by LC-MS, the mixture was concentrated and purified by preparative TLC (MeOH:DCM=10%, v / v) to give the title compound (529 mg, 99.3% yield).

[0126] LC-MS (ESI) [M+H] + = 381.3.

[0127] Step 3: Preparation of tert-butyl 4-((6-((3R,4R)-4-(3,4-dihydroisoquinolin-2(1H)-yl)-3-hydroxypiperidine-1-carbonyl)-2-isopropoxypyrimidin-4-yl)amino)piperidine-1-formate [ka]

[0128] 6-((1-(tert-Butoxycarbonyl)piperidin-4-yl)amino)-2-isopropoxypyrimidine-4-formic acid (200 mg, 0.526 mmol, 1.0 equiv.) was dissolved in N,N-dimethylformamide (2 mL), and (3R,4R)-4-(3,4-dihydroisoquinolin-2(1H)-yl)piperidin-3-ol (146.6 mg, 0.631 mmol, 1.2 equiv), HATU (299.8 mg, 0.789 mmol, 1.5 equiv), and N,N-diisopropylethylamine (203.8 mg, 1.577 mmol, 3.0 equiv) were added, followed by stirring at room temperature for 2 hours. After the completion of the reaction was confirmed by LC-MS, the product was extracted, concentrated, and separated by reverse phase chromatography to obtain the compound (80 mg, yield 25.6%).

[0129] LC-MS (ESI) [M+H] + = 595.4.

[0130] Step 4: Preparation of ((3R,4R)-4-(3,4-dihydroisoquinolin-2(1H)-yl)-3-hydroxypiperidin-1-yl)(2-isopropoxy-6-(piperidin-4-ylamino)pyrimidin-4-yl)methanone [ka]

[0131] Tert-butyl 4-((6-((3R,4R)-4-(3,4-dihydroisoquinolin-2(1H)-yl)-3-hydroxypiperidine-1-carbonyl)-2-isopropoxypyrimidin-4-yl)amino)piperidine-1-formate (80 mg, 0.134 mmol, 1.0 equiv.) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (0.5 mL) was added, and the mixture was stirred at 25° C. for 1 hour. After completion of the reaction determined by LC-MS, the mixture was concentrated to give the title compound (70 mg crude product).

[0132] LC-MS (ESI) [M+H] + = 495.3.

[0133] Step 5: Preparation of (6-((1-benzoylpiperidin-4-yl)amino)-2-isopropoxypyrimidin-4-yl)((3R,4R)-4-(3,4-dihydroisoquinolin-2(1H)-yl)-3-hydroxypiperidin-1-yl)methanone [ka]

[0134] ((3R,4R)-4-(3,4-Dihydroisoquinolin-2(1H)-yl)-3-hydroxypiperidin-1-yl)(2-isopropoxy-6-(piperidin-4-ylamino)pyrimidin-4-yl)methanone (70 mg, 0.142 mmol, 1.0 equiv.) was dissolved in dichloromethane (1 mL), N,N-diisopropylethylamine (54.77 mg, 0.425 mmol, 3.0 equiv) was added, and the mixture was stirred in an ice bath for 15 min. Benzoyl chloride (21.96 mg, 0.156 mmol, 1.1 equiv) was then added, and the mixture was gradually warmed to room temperature and stirred for 2 h. After completion of the reaction by LC-MS, the mixture was concentrated to give the crude product, which was separated by reverse phase chromatography to give compound I (37 mg, 43.7% yield).

[0135] Measured by the above instruments 10 and 11. LC-MS (ESI) [M+H] + = 599.3; 1 H NMR (400 MHz, CDCl3) δ 7.49-7.36 (m, 5H), 7.14 (m, 3H), 7.07-6.95 (m, 1H), 6.21 (d, J = 22.4 Hz, 1H), 5.32-5.12 (m, 1H), 5.09-4.55 (m, 3H), 4.29 (m, 1H), 3.97 (m, 1H), 3.85-3.59 (m, 3H), 3.27-2.87 (m, 6H), 2.85-2.53 (m, 3H), 2.07-2.01 (m, 3H), 1.77-1.51 (m, 3H), 1.41-1.28 (m, 6H).

[0136] The XRPD pattern of Compound I measured by the above Instrument 4 is shown in FIG.

[0137] Example 2: Preparation of salts of Compound I Compound I prepared in Example 1 is used as sample, and 29.9 mg and 2 equivalents of 15 kinds of acidic compounds are weighed, added to a certain amount of selected solvent, suspended at room temperature for 3 days, centrifuged the suspension, and vacuum dried the solid at room temperature.The obtained solid is carried out XRPD test by above-mentioned instrument 5, and the result is shown in the following table.

[0138] [Table 2] As can be seen from the table, after screening, hydrochloride crystalline form A, p-toluenesulfonate crystalline form A, p-toluenesulfonate crystalline form B, tartrate crystalline form A and oxalate crystalline form A were found.

[0139] Example 3: Preparation of Crystalline Form A of Compound I A 19.8 mg sample of compound I prepared in Example 1 was weighed and suspended in a mixed solvent of 0.2 mL of ethyl acetate and 0.8 mL of cyclohexane at room temperature for 7 days. The suspension was centrifuged and dried to obtain a solid, which was designated as crystalline form A of compound I.

[0140] The XRPD pattern of crystalline form A of compound I measured by the above-mentioned instrument 4 is shown in Figure 1, and the diffraction peak data are as follows. The DSC and TGA patterns measured by the above-mentioned instruments 1 and 2 are shown in Figure 2.

[0141] [Table 3] Example 4: Hydrochloride Crystalline Form A of Compound I 28.5 mg (approximately 0.05 mmol) of Compound I prepared in Example 1 was weighed as a sample and added to 1 mL of a tetrahydrofuran / cyclohexane mixed solvent (v / v, 1:1) together with 2 equivalents of hydrochloric acid (calculated as hydrogen chloride). The mixture was stirred at room temperature for 2 days, the suspension was centrifuged, and the solid was dried to obtain a novel hydrochloride crystalline form of Compound I, which was named Compound I hydrochloride crystalline form A.

[0142] The result of ion chromatography measured by the above-mentioned instrument 8 showed that the chloride ion content was 10.2%, which was consistent with the theoretical content of 2 equivalents of chloride ion (10.4%), indicating that the salt formation ratio of compound I with hydrochloric acid (calculated as hydrogen chloride) was 1:2.

[0143] The XRPD pattern of the hydrochloride crystalline form A of Compound I measured by the above-mentioned Instrument 5 is shown in FIG. 3, and the diffraction peak data are as follows. The DSC and TGA patterns measured by the above-mentioned Instruments 1 and 2 are shown in FIG. 4.

[0144] [Table 4] Example 5: Crystalline Form A of the Oxalate Salt of Compound I 29.6 mg (approximately 0.05 mmol) of Compound I prepared in Example 1 was weighed as a sample and added to 1 mL of a mixed solvent of ethanol / n-heptane (v / v, 3:7) together with 2 equivalents of oxalic acid. The mixture was stirred at room temperature for 3 days, the suspension was centrifuged, and the solid was dried to obtain a novel oxalate crystalline form of Compound I, which was named Compound I oxalate crystalline form A.

[0145] The XRPD pattern of Compound I oxalate crystalline Form A measured by Instrument 5 is shown in FIG. 5, with the diffraction peak data as shown below. The DSC and TGA patterns measured by Instruments 1 and 2 are shown in FIG. 6.

[0146] [Table 5] Example 6: Crystalline Form A of the Tartrate Salt of Compound I 29.9 mg (approximately 0.05 mmol) of Compound I prepared in Example 1 was weighed as a sample, and added to 1 mL of a tetrahydrofuran / cyclohexane mixed solvent (v / v, 1:1) together with 2 equivalents of tartaric acid. The mixture was suspended at room temperature for 3 days. The suspension was centrifuged and the solid was dried to obtain a novel tartrate crystalline form of Compound I, which was named Compound I tartrate crystalline form A.

[0147] The XRPD pattern of Compound I tartrate salt crystalline Form A measured by Instrument 5 is shown in Figure 7, and the diffraction peak data are as follows: DSC and TGA patterns measured by Instruments 1 and 2 are shown in Figure 8; and the nuclear magnetic spectrum measured by Instrument 7 is shown in Figure 9, showing a signal peak of tartaric acid at 4.25 ppm. Integration results indicated that the salt formation ratio of Compound I to tartaric acid was 1:1.5.

[0148] [Table 6] Example 7: p-Toluenesulfonate Crystalline Form A of Compound I A 29.8 mg (approximately 0.05 mmol) sample of Compound I prepared in Example 1 was weighed out and added to 1 mL of methyl tert-butyl ether together with 2 equivalents of p-toluenesulfonic acid. The mixture was stirred at room temperature for 3 days, the suspension was centrifuged, and the solid was dried to obtain a novel crystalline form of p-toluenesulfonate of Compound I, which was named p-toluenesulfonate crystalline form A of Compound I.

[0149] The XRPD pattern of p-toluenesulfonate crystalline form A of compound I measured by instrument 5 is shown in Figure 10, and the diffraction peak data are as follows: DSC and TGA patterns measured by instruments 1 and 2 are shown in Figure 11; and the nuclear magnetic spectrum measured by instrument 7 is shown in Figure 12, showing signal peaks of p-toluenesulfonic acid at 2.27 ppm, 7.10 ppm, and 7.45 ppm. The integration result showed that the salt formation ratio of compound I to p-toluenesulfonic acid was 1:2.

[0150] [Table 7] Example 8: p-Toluenesulfonate Crystalline Form B of Compound I A 29.9 mg (approximately 0.05 mmol) sample of Compound I prepared in Example 1 was weighed out and added to 1 mL of a tetrahydrofuran / cyclohexane mixed solvent (v / v, 1:1) together with 2 equivalents of p-toluenesulfonic acid. The mixture was stirred at room temperature for 3 days, the suspension was centrifuged, and the solid was dried to obtain a novel crystalline form of p-toluenesulfonate of Compound I, which was named p-toluenesulfonate crystalline form B of Compound I.

[0151] The XRPD pattern of p-toluenesulfonate crystalline form B of compound I measured by instrument 5 is shown in Figure 13, with the diffraction peak data as shown below. The nuclear magnetic spectrum measured by instrument 7 is shown in Figure 14, with signal peaks of p-toluenesulfonic acid at 2.27 ppm, 7.10 ppm, and 7.45 ppm. The integration result showed that the salt formation ratio of compound I to p-toluenesulfonic acid was 1:2.

[0152] [Table 8] Example 9: p-Toluenesulfonate Crystalline Form B of Compound I Method 1: Using the compound I prepared in Example 1 as a sample, 2.9 g (approximately 5 mmol) was weighed out and added to 45 mL of acetone together with 2.5 equivalents of p-toluenesulfonic acid. The mixture was stirred at room temperature for 2 hours. The suspension was centrifuged and the solid was dried to obtain a p-toluenesulfonate crystalline form of compound I. The XRPD pattern measured by instrument 6 is shown in Figure 15, which is identified as p-toluenesulfonate crystalline form B. The DSC and TGA patterns measured by instrument 3 are shown in Figure 16. Based on the TGA weight loss results, it was determined that crystalline form B contained one molecule of bound water.

[0153] Method 2: 598.1 mg of Compound I prepared in Example 1 was weighed as a sample and added together with 343.9 mg of p-toluenesulfonic acid to 20 mL of a mixed solvent of methyl tert-butyl ether / acetone (v / v, 1:1), followed by suspension at room temperature for 2 d. The suspension was separated by suction filtration, and the solid was dried under vacuum at 40°C to similarly obtain the above p-toluenesulfonate crystalline form B.

[0154] These results indicate that for p-toluenesulfonate crystalline form B, the solvent system used in the salt formation reaction can be further simplified from the MTBE / acetone binary solvent to a single acetone solvent.

[0155] Test Example 1: Evaluation of PRMT5 enzyme activity inhibitory activity 1. Test method: A 1x enzyme reaction buffer (10 mM Tris 8.0 (Sigma, Cat. No. T2694-1L), 0.01% Tween-20 (Sigma, Cat. No. P2287-100ML), 1 mM DTT (Sigma, Cat. No. D0632-10G)) was prepared. PRMT5 (Active Motif, Cat. No. 31921) and [H]-SAM (PerkinElmer, Cat. No. NET155V001MC) were added to 1x enzyme reaction buffer to prepare a 25 / 15x mixed solution (final PRMT5 concentration: 5 nM, final [H]-SAM concentration: 0.3 μM). 15 μL of this solution was transferred to a 384-well microwell plate (Corning 384-well Polypropylene Storage Microplates, Cat. No. 3657) containing various concentrations of compounds (final DMSO concentration: 1%) and incubated at room temperature for 60 minutes. The polypeptide substrate GL-27 (Ac-SGRGKGGKGLGKGGAKRHRKVGG-K) (Biotin) (GL Biochem, Cat. No. 342095) was added to the 1x enzyme reaction buffer to prepare a 25 / 10x substrate solution. Then, 10 μL of the polypeptide substrate solution (final polypeptide substrate concentration: 100 nM) was added and the reaction was allowed to proceed at room temperature for 120 minutes. The reaction was then stopped by adding 5 μL of 6x ice-cold SAM (Sigma, Cat. No. A7007-100MG) solution (final SAM concentration: 0.125 mM). 25 μL of the reaction mixture was transferred to a FlashPlate (Streptavidin FlashPlate HTS PLUS, High Capacity, 384-well, Perkin Elmer, Cat. No. SMP410A001PK) and incubated at room temperature for 1 hour. After washing the plate three times with distilled water containing 0.1% Tween-20, the microwell plate was read for CPM data (counts per minute) using a MicroBeta instrument.After obtaining the raw CPM data for different concentrations of the compound, the data were normalized according to the following formula: Inh% = (Max - Sample) / (Max - Min) × 100% to obtain the enzyme activity inhibition rate Inh% for each concentration point (where Max is the CPM value of the positive well containing the enzyme, Min is the CPM value of the negative well without the enzyme, and Sample is the CPM value of the sample well treated with the compound). The inhibition rate Inh% (Y) corresponding to each concentration (X) was then entered into Excel, and the XLfit plug-in was used to fit the four-parameter fitting equation Y = Bottom + (Top - Bottom) / (1 + (IC). 50 The half inhibitory concentration (IC) of each compound is calculated by ( / X) × HillSlope) 50 values ​​were calculated.

[0156] 2. Test results: The compound of Example 1 of the present invention exhibited an IC 50 The IC value was 6.40 nM, indicating excellent biological activity. Compound 13 disclosed in WO2020182018A1 was used as a control compound (see Example 13 on pages 33-35 of the WO2020182018A1 specification for the structure and preparation method of the control compound). 50 The value was 115.00 nM. [ka]

[0157] Test Example 2: Evaluation of growth inhibitory activity on human B-cell non-Hodgkin's lymphoma Z-138 cells 1. Experimental materials and equipment 1) Cell lines and culture methods

[0158] [Table 9] 2) Culture media and reagents

[0159] [Table 10] 3) 384-well plate Corning® 384-well clear flat bottom white polystyrene microwell plate (with cover, sterile), Corning, part number: 3765.

[0160] 4)Equipment 2104 EnVision plate reader, PerkinElmer, Vi-Cell XR Cell Counter, beckmancoulter.

[0161] 2. Experimental Methods and Steps 1)Cell culture The cells were resuscitated and cultured in a 5% CO2 incubator at 37°C according to the culture conditions shown in the table above. They were passaged regularly, and after about two generations, cell lines showing good growth conditions were used for plating.

[0162] 2) Cell plating I. Remove the cells from the incubator, transfer the cell suspension to a 50 mL centrifuge tube, and centrifuge at 800-1000 rpm for 3-5 minutes. Discard the supernatant. Add an appropriate volume of medium to the centrifuge tube and resuspend the cells uniformly by gently pipetting. Count the cells using a Vi-Cell XR cell counter.

[0163] II. Based on the measured cell density, the cell suspension was adjusted to an appropriate concentration.

[0164] III. 40 μL / well of the cell suspension was added to a 384-well plate to give 700 cells / well, and an equal volume of culture medium without cells was added to blank control wells.

[0165] 3) Compound Preparation and Dosing I. Compounds were dissolved in 100% DMSO to prepare stock solutions at a concentration of 10 mM.

[0166] II. A stock solution with a concentration of 10 mM was taken and diluted with DMSO to a 2 mM solution, and this was used as the starting concentration for a 9-point 4x gradient dilution with DMSO.

[0167] III. 200 nL of the gradient concentration solutions of the above compounds were taken and added to each well. 200 nL of DMSO was added to the blank control wells and DMSO control wells so that the final DMSO concentration was 0.5%.

[0168] IV. The cell plates were incubated in a carbon dioxide incubator for 5 days (120 h).

[0169] 4) Preparation and testing of reagents The measurement was carried out according to the instructions for the Promega CellTiter-Glo luminescent cell activity detection reagent kit (Promega-G7573).

[0170] I. CellTiter-Glo buffer was dissolved and allowed to stand at room temperature.

[0171] II. Lyophilized CellTiter-Glo substrate was allowed to warm to room temperature.

[0172] III. The CellTiter-Glo working solution was prepared by adding CellTiter-Glo buffer to one tube of CellTiter-Glo substrate to dissolve the substrate.

[0173] IV. Gently vortex to dissolve thoroughly.

[0174] V. The cell culture plate was removed and allowed to equilibrate to room temperature.

[0175] VI. After uniform mixing, 25 μL of CellTiter Glo reagent was added to each well, and the mixture was shaken for 10 minutes in the dark and then incubated for 10 minutes.

[0176] VII. The luminescence signal was detected using a 2104 EnVision plate reader.

[0177] 3. Data analysis The inhibition rate (IR) of the test compound was calculated using the following formula:

[0178] IR(%)=(1-(RLU 化合物 -RLU ブランク対照 ) / (RLU DMSO -RLU ブランク対照 )) × 100%, The drug inhibition curve was plotted using XLFit, and IC 50 The values ​​were calculated using the following four-parameter model [fit=(A+((BA) / (1+((C / x)^D))))].

[0179] 4. Experimental Results: The compound of Example 1 of the present invention exhibited an IC 50 The IC value of the control compound was 0.0466 μM, which indicated excellent biological activity. Similarly, compound 13 disclosed in WO2020182018A1 was used as a control compound, and the IC value of the control compound was measured by the same test method. 50 The value was 2.7096 μM.

[0180] Test Example 3: Stability test The stability studies of the p-toluenesulfonate crystalline form B, tartrate crystalline form A and hydrochloride crystalline form A of the present invention were carried out under high temperature (60°C), high humidity (25°C / 92.5%RH), light illumination (25°C / 4500 Lux) and accelerated storage (40°C / 75%RH). Samples were taken on 7 days and 15 days, respectively, and the results are shown below.

[0181] [Table 11] The results showed that the p-toluenesulfonate crystalline form B and the tartrate crystalline form A were both stable under high temperature, high humidity, light illumination, and accelerated conditions for 15 days and did not undergo crystal transformation, while the hydrochloride crystalline form A underwent crystal transformation after 7 and 15 days under high humidity conditions and was stable under all other conditions.

[0182] Test Example 4: Hygroscopicity Study Using the above-mentioned instrument 9, dynamic moisture adsorption / desorption analysis was carried out on the p-toluenesulfonate crystalline form B, the tartrate crystalline form A and the hydrochloride crystalline form A of the present invention to study the hygroscopicity of each crystalline form. The results are shown below.

[0183] [Table 12] The results showed that, in order of hygroscopicity from lowest to highest, the p-toluenesulfonate crystalline form B < the hydrochloride crystalline form A < the tartrate crystalline form A, and that p-toluenesulfonate crystalline form B was almost not hygroscopic.

[0184] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the scope of the spirit and principles of the present invention should be included within the scope of the claims of the present invention.

Claims

1. Crystalline Form A of Compound I, the structure of which is shown below: 【Chemistry 1】 The crystalline form A has an XRPD pattern expressed in 2θ angles using Cu-Kα radiation, which has characteristic peaks at 4.0±0.2°, 18.4±0.2°, 20.3±0.2°, and 21.8±0.2°; Preferably, the crystalline form A has an XRPD pattern using Cu-Kα radiation, which has characteristic peaks at 2θ angles of 4.0±0.2°, 14.7±0.2°, 15.8±0.2°, 18.4±0.2°, 20.3±0.2°, and 21.8±0.2°; More preferably, the crystalline form A has an XRPD pattern using Cu-Kα radiation, expressed in 2θ angles, with characteristic peaks at 4.0±0.2°, 7.1±0.2°, 13.6±0.2°, 14.7±0.2°, 15.8±0.2°, 18.4±0.2°, 20.3±0.2°, 21.8±0.2°, and 27.7±0.2°; More preferably, the crystalline form A is characterized by having an XRPD pattern essentially as shown in Figure 1. Crystalline Form A of Compound I.

2. The DSC pattern of the crystalline form A has an endothermic peak at around 85.0°C to 150.0°C, Preferably, the DSC pattern of the crystalline form A has an endothermic peak at 85.0°C ± 5°C to 150.0°C ± 5°C; More preferably, the crystalline form A is characterized by having a DSC pattern essentially as shown in Figure 2. Crystalline form A of compound I according to claim 1.

3. the TGA pattern of the crystalline form A has a weight loss of about 2.0% around 100.0°C and further has a weight loss of about 3.5% around 150.0°C; Preferably, the TGA pattern of crystalline form A has a weight loss of about 2.0% at 100.0°C ± 5°C and further has a weight loss of about 3.5% at 150.0°C ± 5°C; More preferably, the crystalline form A is characterized by having a TGA pattern essentially as shown in Figure 2. Crystalline form A of compound I according to claim 1 or 2.

4. A salt of Compound I, the structure of which is shown below: 【Chemistry 2】 the salt is a hydrochloride, hydrobromide, phosphate, sulfate, methanesulfonate, p-toluenesulfonate, camphorsulfonate (e.g., L-camphorsulfonate), oxalate, maleate, tartrate (e.g., L-tartrate), fumarate, citrate, malate (e.g., L-malate), glycolate, or benzoate salt of Compound I; A salt of Compound I.

5. The hydrochloride salt of Compound I, the structure of which is shown below: 【Transformation 3】 In the hydrochloride, the salt formation ratio of Compound I to hydrochloric acid is 1:2; The hydrochloride salt of Compound I.

6. Hydrochloride crystalline Form A of Compound I, the structure of which is shown below: 【Chemistry 4】 In the hydrochloride crystalline form A, the salt formation ratio of Compound I to hydrochloric acid is 1:

2. Crystalline Form A of the hydrochloride salt of Compound I.

7. The hydrochloride crystalline form A of Compound I has an XRPD pattern using Cu-Kα radiation, which has characteristic peaks at 2θ angles of 4.4±0.2°, 15.3±0.2°, 18.0±0.2°, 18.8±0.2°, and 19.3±0.2°; Preferably, the hydrochloride crystalline form A of Compound I has an XRPD pattern using Cu-Kα radiation, expressed in 2θ angles, with characteristic peaks at 4.4±0.2°, 11.5±0.2°, 12.7±0.2°, 15.3±0.2°, 18.0±0.2°, 18.8±0.2°, 19.3±0.2°, and 21.6±0.2°; More preferably, the hydrochloride crystalline form A of Compound I has an XRPD pattern using Cu-Kα radiation, expressed in 2θ angles, with characteristic peaks at 4.4±0.2°, 8.5±0.2°, 11.5±0.2°, 12.7±0.2°, 14.0±0.2°, 15.3±0.2°, 16.7±0.2°, 18.0±0.2°, 18.8±0.2°, 19.3±0.2°, 20.9±0.2°, 21.6±0.2°, and 23.3±0.2°; More preferably, the hydrochloride crystalline form A of Compound I is characterized by having an XRPD pattern essentially as shown in Figure 3.

7. The hydrochloride crystalline form A of compound I according to claim 6.

8. The DSC pattern of the hydrochloride crystalline form A of Compound I has an endothermic peak at around 149.4°C, Preferably, the DSC pattern of the hydrochloride crystalline form A of Compound I has an endothermic peak at 149.4°C ± 5°C; More preferably, the hydrochloride crystalline form A of Compound I is characterized by having a DSC pattern essentially as shown in Figure 4.

8. Crystalline form A of the hydrochloride salt of compound I according to claim 6 or 7.

9. the TGA pattern of the hydrochloride crystalline form A of Compound I has a weight loss of about 0.3% around 100.0°C and further has a weight loss of about 16.3% around 235.0°C; Preferably, the TGA pattern of the hydrochloride salt crystalline Form A of Compound I has a weight loss of about 0.3% at 100.0°C ± 5°C and further has a weight loss of about 16.3% at 235.0°C ± 5°C; More preferably, the hydrochloride crystalline form A of Compound I is characterized by having a TGA pattern essentially as shown in Figure 4. The hydrochloride crystalline form A of compound I according to any one of claims 6 to 8.

10. Crystalline Form A of the oxalate salt of Compound I, the structure of Compound I is shown below: 【Transformation 5】 The oxalate crystalline form A of Compound I has an XRPD pattern using Cu-Kα radiation, which has characteristic peaks at 2θ angles of 4.5±0.2°, 8.4±0.2°, 18.8±0.2°, 20.1±0.2°, and 21.2±0.2°; Preferably, the oxalate crystalline form A of Compound I has an XRPD pattern using Cu-Kα radiation, expressed in 2θ angles, with characteristic peaks at 4.5±0.2°, 6.1±0.2°, 8.4±0.2°, 10.4±0.2°, 16.8±0.2°, 18.8±0.2°, 20.1±0.2°, and 21.2±0.2°; More preferably, the oxalate crystalline Form A of Compound I has an XRPD pattern using Cu-Kα radiation, expressed in 2θ angles, having characteristic peaks at 4.5±0.2°, 5.2±0.2°, 6.1±0.2°, 8.4±0.2°, 10.4±0.2°, 15.8±0.2°, 16.3±0.2°, 16.8±0.2°, 17.6±0.2°, 18.8±0.2°, 20.1±0.2°, 21.2±0.2°, 22.3±0.2°, and 23.0±0.2°; More preferably, the oxalate crystalline Form A of Compound I is characterized by having an XRPD pattern essentially as shown in FIG.

5. Crystalline Form A of the oxalate salt of Compound I.

11. The DSC pattern of oxalate crystalline form A of Compound I has an endothermic peak at around 157.2°C, Preferably, the DSC pattern of the oxalate crystalline form A of Compound I has an endothermic peak at 157.2°C ± 5°C; More preferably, the oxalate crystalline Form A of Compound I is characterized by having a DSC pattern essentially as shown in FIG.

6.

11. Crystalline Form A of the oxalate salt of Compound I according to claim 10.

12. the TGA pattern of the oxalate crystalline form A of Compound I has a weight loss of about 4.0% at around 150.0°C; Preferably, the TGA pattern of crystalline Form A of the oxalate salt of Compound I has a weight loss of about 4.0% at 150.0°C ± 5°C; More preferably, the oxalate crystalline Form A of Compound I is characterized by having a TGA pattern essentially as shown in FIG.

6.

12. Crystalline Form A of the oxalate salt of Compound I according to claim 10 or 11.

13. The tartrate salt of Compound I, the structure of which is shown below: 【Transformation 6】 In the tartrate salt, the salt formation ratio of Compound I to tartaric acid is 1:1.5; Tartrate salt of Compound I.

14. Crystalline Form A of the tartrate salt of Compound I, the structure of Compound I is shown below: 【Transformation 7】 In the tartrate salt crystalline form A, the salt formation ratio of Compound I to tartaric acid is 1:1.5; Crystalline Form A of the tartrate salt of Compound I.

15. The tartrate salt crystalline form A of Compound I has an XRPD pattern using Cu-Kα radiation, which has characteristic peaks at 2θ angles of 15.5±0.2°, 16.0±0.2°, 18.4±0.2°, and 20.5±0.2°; Preferably, the crystalline Form A of the tartrate salt of Compound I has an XRPD pattern using Cu-Kα radiation, expressed in 2θ angles, with characteristic peaks at 5.8±0.2°, 8.8±0.2°, 11.7±0.2°, 15.5±0.2°, 16.0±0.2°, 18.4±0.2°, and 20.5±0.2°; More preferably, the crystalline Form A of the tartrate salt of Compound I has an XRPD pattern using Cu-Kα radiation, expressed in 2θ angles, with characteristic peaks at 3.0±0.2°, 5.8±0.2°, 8.8±0.2°, 11.7±0.2°, 15.5±0.2°, 16.0±0.2°, 16.7±0.2°, 18.4±0.2°, 20.5±0.2°, and 21.2±0.2°; More preferably, the crystalline Form A of the tartrate salt of Compound I is characterized by having an XRPD pattern essentially as shown in Figure 7. Crystalline Form A of the tartrate salt of Compound I according to claim 14.

16. The DSC pattern of the tartrate salt crystalline form A of Compound I has an endothermic peak at around 174.8°C, Preferably, the DSC pattern of the tartrate salt crystalline form A of Compound I has an endothermic peak at 174.8°C ± 5°C; More preferably, the crystalline Form A of the tartrate salt of Compound I is characterized by having a DSC pattern essentially as shown in Figure 8.

16. Crystalline Form A of the tartrate salt of Compound I according to claim 14 or 15.

17. the TGA pattern of the tartrate salt crystalline form A of Compound I has a weight loss of about 5.8% at around 150.0°C; Preferably, the TGA pattern of crystalline Form A of the tartrate salt of Compound I has a weight loss of about 5.8% at 150.0°C ± 5°C; More preferably, the crystalline Form A of the tartrate salt of Compound I is characterized by having a TGA pattern essentially as shown in Figure 8. Crystalline Form A of the tartrate salt of Compound I according to any one of claims 14 to 16.

18. The p-toluenesulfonic acid salt of Compound I, the structure of which is shown below: 【Transformation 8】 In the p-toluenesulfonic acid salt, the salt formation ratio of Compound I to p-toluenesulfonic acid is 1:

2. p-Toluenesulfonic acid salt of Compound I.

19. Crystalline Form A of the p-toluenesulfonate salt of Compound I, the structure of Compound I being shown below: 【Chemistry 9】 The p-toluenesulfonate crystalline form A is characterized in that the salt formation ratio of Compound I to p-toluenesulfonic acid is 1:

2. Crystalline Form A of the p-toluenesulfonate salt of Compound I.

20. The p-toluenesulfonate crystalline form A of Compound I has an XRPD pattern measured using Cu-Kα radiation, which has characteristic peaks at 2θ angles of 4.0±0.2°, 8.1±0.2°, 18.3±0.2°, and 20.0±0.2°; Preferably, the p-toluenesulfonate crystalline form A of Compound I has an XRPD pattern using Cu-Kα radiation, which has characteristic peaks at 2θ angles of 4.0±0.2°, 8.1±0.2°, 11.7±0.2°, 12.7±0.2°, 14.6±0.2°, 18.3±0.2°, 20.0±0.2°, and 23.1±0.2°; More preferably, the p-toluenesulfonate crystalline form A of Compound I has an XRPD pattern using Cu-Kα radiation, expressed in 2θ angles, with characteristic peaks at 4.0±0.2°, 6.2±0.2°, 8.1±0.2°, 8.8±0.2°, 11.7±0.2°, 12.7±0.2°, 14.6±0.2°, 16.3±0.2°, 17.5±0.2°, 18.3±0.2°, 19.0±0.2°, 20.0±0.2°, and 23.1±0.2°; More preferably, the p-toluenesulfonate crystalline form A of Compound I is characterized by having an XRPD pattern essentially as shown in FIG.

10.

20. The crystalline form A of p-toluenesulfonate salt of Compound I according to claim 19.

21. The DSC pattern of the p-toluenesulfonate crystalline form A of Compound I has endothermic peaks at around 88.5°C and 150.1°C, Preferably, the DSC pattern of the p-toluenesulfonate crystalline form A of Compound I has endothermic peaks at 88.5°C ± 5°C and 150.1°C ± 5°C; More preferably, the p-toluenesulfonate crystalline form A of Compound I is characterized by having a DSC pattern essentially as shown in FIG.

11.

21. Crystalline Form A of p-toluenesulfonate salt of Compound I according to claim 19 or 20.

22. the TGA pattern of the p-toluenesulfonate crystalline form A of Compound I has a weight loss of about 5.7% at around 130.0°C and a weight loss of about 4.7% at around 220.0°C; Preferably, the TGA pattern of crystalline Form A of the p-toluenesulfonate salt of Compound I has a weight loss of about 5.7% at 130.0°C ± 5°C and further has a weight loss of about 4.7% at 220.0°C ± 5°C; More preferably, the p-toluenesulfonate crystalline form A of Compound I is characterized by having a TGA pattern essentially as shown in FIG.

11.

22. Crystalline form A of p-toluenesulfonate salt of Compound I according to any one of claims 19 to 21.

23. p-toluenesulfonate crystalline form B of Compound I, the structure of Compound I being shown below: 【Chemistry 10】 In the p-toluenesulfonate crystalline form B, the salt formation ratio of Compound I to p-toluenesulfonic acid is 1:

2. p-Toluenesulfonate Crystalline Form B of Compound I.

24. The p-toluenesulfonate crystalline form B of Compound I has an XRPD pattern expressed in 2θ angles using Cu-Kα radiation, which has characteristic peaks at 4.3±0.2°, 19.1±0.2°, and 20.2±0.2°; Preferably, the p-toluenesulfonate crystalline form B of Compound I has an XRPD pattern using Cu-Kα radiation, which has characteristic peaks at 4.3±0.2°, 9.8±0.2°, 11.5±0.2°, 19.1±0.2°, and 20.2±0.2° in 2θ angles; More preferably, the p-toluenesulfonate crystalline form B of Compound I has an XRPD pattern using Cu-Kα radiation, which has characteristic peaks at 2θ angles of 4.3±0.2°, 4.9±0.2°, 9.8±0.2°, 11.5±0.2°, 17.2±0.2°, 19.1±0.2°, 20.2±0.2°, and 21.5±0.2°; More preferably, the p-toluenesulfonate crystalline form B of Compound I has an XRPD pattern using Cu-Kα radiation, expressed in 2θ angles, with characteristic peaks at 4.3±0.2°, 4.9±0.2°, 9.0±0.2°, 9.8±0.2°, 11.5±0.2°, 17.2±0.2°, 19.1±0.2°, 20.2±0.2°, 21.5±0.2°, and 25.7±0.2°; Even more preferably, the p-toluenesulfonate crystalline form B of Compound I has an XRPD pattern using Cu-Kα radiation, expressed in 2θ angles, having characteristic peaks at 4.3±0.2°, 4.9±0.2°, 7.1±0.2°, 9.0±0.2°, 9.8±0.2°, 11.5±0.2°, 12.8±0.2°, 17.2±0.2°, 19.1±0.2°, 20.2±0.2°, 21.5±0.2°, and 25.7±0.2°; Most preferably, the p-toluenesulfonate crystalline form B of Compound I is characterized by having an XRPD pattern essentially as shown in FIG. 13 or FIG.

15.

24. The crystalline form B of p-toluenesulfonate salt of Compound I according to claim 23.

25. the DSC pattern of the p-toluenesulfonate crystalline form B of Compound I has an endothermic peak at about 192.1°C, and preferably further has an endothermic peak at about 124.8°C; Preferably, the DSC pattern of the p-toluenesulfonate crystalline form B of Compound I has an endothermic peak at 192.1±5°C, and preferably further has an endothermic peak at 124.8±5°C; More preferably, the p-toluenesulfonate crystalline form B of Compound I is characterized by having a DSC pattern essentially as shown in FIG.

16.

25. Crystalline Form B of p-toluenesulfonate salt of Compound I according to claim 23 or 24.

26. the TGA pattern of the p-toluenesulfonate crystalline form B of Compound I has a weight loss of about 2.0% at around 167.3°C and further has a weight loss of about 5.2% at around 233.7°C; Preferably, the TGA pattern of crystalline Form B of the p-toluenesulfonate salt of Compound I has a weight loss of about 2.0% at 167.3°C ± 5°C and further has a weight loss of about 5.2% at 233.7°C ± 5°C; More preferably, the p-toluenesulfonate crystalline form B of Compound I is characterized by having a TGA pattern essentially as shown in FIG.

16.

26. The crystalline form B of p-toluenesulfonate of Compound I according to any one of claims 23 to 25.

27. The crystalline form B of p-toluenesulfonate of Compound I is a hydrate, preferably a monohydrate.

27. The crystalline form B of p-toluenesulfonate of Compound I according to any one of claims 23 to 26.

28. A pharmaceutical composition comprising a salt of compound I or a crystalline form thereof according to any one of claims 4 to 27.

29. Use of a salt of compound I according to any one of claims 4 to 27 or a crystalline form thereof or a pharmaceutical composition according to claim 28 in the manufacture of a medicament for the prevention and / or treatment of PRMT5-mediated diseases and / or conditions.

30. Use of a salt of compound I according to any one of claims 4 to 27 or a crystalline form thereof or a pharmaceutical composition according to claim 28 in the manufacture of a medicament for the prevention and / or treatment of tumor diseases, Preferably, the tumor disease is a solid tumor or a hematological tumor, preferably a malignant solid tumor or a hematological tumor; More preferably, the tumor disease is lymphoma. use.

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