Solid form of pyrimidine derivative, and preparation method therefor and use thereof

Abstract

The present invention relates to a solid form of a pyrimidine derivative, and a preparation method therefor and a use thereof. Specifically, disclosed in the present invention is a maleate crystal form I of a compound of formula (I), the crystal form having excellent stability, solubility, hygroscopicity, druggability, etc. Also disclosed in the present invention are a preparation method for the crystal form, and a use of the crystal form in the prevention and / or treatment of CDK7-related diseases (such as cancer).

Description

A solid form of a pyrimidine derivative, its preparation method and application Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a solid form of a pyrimidine derivative, its preparation method, and its application. Background Technology

[0002] Cyclin-dependent kinases (CDKs) belong to the serine / threonine kinase family. They exert their physiological functions by binding to their corresponding cyclins to form active dimer complexes, thereby inducing cell growth and proliferation. Currently, more than 20 CDKs have been identified, which are divided into two main categories based on their functions: CDKs that regulate the cell cycle and CDKs that regulate cellular transcription. CDKs 1-6 and 14-18 are involved in cell cycle regulation, while CDKs 7-13 and 19-20 are involved in transcriptional regulation.

[0003] CDK7 is an important member of the CDK family, with its main physiological functions being the regulation of cell cycle and transcription. In the cytosol, CDK7, along with cyclin H and Mat1, forms CAK (CDKs activating kinase), participating in cell cycle regulation through phosphorylation of CDK1 / 2 / 4 / 6. In the nucleus, CDK7, as a component of the universal transcription factor TFIIH (Transcription Factor II human), participates in gene transcription during the crucial initiation stage by phosphorylating the carboxy-terminal domain of RNA polymerase II. Because CDK7 possesses the dual functions of CAK and CTD phosphorylation, it plays a vital role in cell proliferation, cell cycle, and gene transcription.

[0004] Due to its unique dual role in transcription and cell cycle processes, CDK7 is widely expressed in various types of cancer, and downregulating CDK7 activity can lead to reduced cell proliferation. More importantly, it is now generally accepted that targeted transcription can selectively restrict the synthesis of mRNAs involved in tumor growth without disrupting the transcription of housekeeping genes. Therefore, CDK7 is considered a viable and promising target for cancer therapy, attracting widespread attention. Many small molecules, such as THZ1, THZ2, CT7001, and SY-1365, have shown excellent inhibitory effects on tumor growth in preclinical studies, particularly in unmet needs in major diseases such as small cell lung cancer, triple-negative breast cancer, and pancreatic cancer where effective treatments are currently lacking. Summary of the Invention

[0005] The purpose of this invention is to provide a maleate crystal form of compound (I) with excellent stability, solubility, hygroscopicity, and pharmaceutical properties, as well as its preparation and application.

[0006] In a first aspect, the present invention provides a maleate crystal form Form I of a compound of formula (I), wherein the maleate of the compound of formula (I) has the structure shown in formula (II).

[0007] Furthermore, the X-ray powder diffraction pattern of the maleate crystal form Form I has characteristic peaks at the following 2θ values: 9.13±0.2°, 15.57±0.2°, 16.05±0.2°, 16.78±0.2°, 18.39±0.2°, 19.57±0.2°, 21.21±0.2°, 22.56±0.2°, 24.21±0.2°, and 25.62±0.2°.

[0008] In another preferred embodiment, in the maleate crystal form Form I, the molar ratio of compound of formula (I) to maleic acid is 1:1.

[0009] In another preferred embodiment, the X-ray powder diffraction pattern of the maleate crystal form Form I has characteristic peaks at the following 2θ values: 7.98±0.2°, 8.26±0.2°, 9.13±0.2°, 13.50±0.2°, 15.57±0.2°, 16.05±0.2°, 16.78±0.2°, 17.27±0.2°, 1 8.39±0.2°, 18.77±0.2°, 19.57±0.2°, 21.03±0.2°, 21.21±0.2°, 22.56±0.2°, 24.21±0.2°, 24.42±0.2°, 24.75±0.2°, 25.39±0.2°, 25.62±0.2°, 27.92±0.2°.

[0010] In another preferred embodiment, the X-ray powder diffraction pattern of the maleate crystal form Form I has characteristic peaks at the following 2θ values: 7.98±0.2°, 8.26±0.2°, 9.13±0.2°, 13.50±0.2°, 15.57±0.2°, 16.05±0.2°, 16.78±0.2°, 17.27±0.2°, 18.39±0.2°, 18.77±0.2°, 19.57±0.2°, 21.03±0.2°, 21.21±0.2°, 2 2.56±0.2°, 23.45±0.2°, 24.21±0.2°, 24.42±0.2°, 24.75±0.2°, 25.09±0.2°, 25.39±0.2°, 25.62±0.2°, 26.97±0.2°, 27.26±0.2°, 27.92±0.2°, 28.48±0.2°, 29.19±0.2°, 29.43±0.2°, 30.32±0.2°, 32.09±0.2°, 36.14±0.2°.

[0011] In another preferred embodiment, the X-ray powder diffraction pattern of the maleate crystal form Form I has characteristic peaks at the following 2θ values: 3.54±0.2°, 7.98±0.2°, 8.26±0.2°, 9.13±0.2°, 13.50±0.2°, 15.57±0.2°, 16.05±0.2°, 16.78±0.2°, 17.27±0.2°, 17.85±0.2°, 18.39±0.2°, 18.77±0.2°, 19.57±0.2°, 20.67±0.2°, 21.03±0.2°, 21.21±0.2°, 22.56±0.2°, 23.45±0.2°, 24 .21±0.2°, 24.42±0.2°, 24.75±0.2°, 25.09±0.2°, 25.39±0.2°, 25.62±0.2°, 26.97±0.2°, 27.26±0.2°, 27.92±0.2°, 28.48±0.2°, 29.19±0.2°, 29.43±0.2°, 30.32±0.2°, 31.48±0.2°, 32.09±0.2°, 32.41±0.2°, 33.54±0.2°, 36.14±0.2°, 36.53±0.2°, 37.61±0.2°, 38.56±0.2°, 39.56±0.2°.

[0012] In another preferred embodiment, the X-ray powder diffraction pattern of the maleate crystal form Form I has characteristic peaks at the following 2θ values: 3.54±0.2°, 7.98±0.2°, 8.26±0.2°, 9.13±0.2°, 13.50±0.2°, 15.57±0.2°, 16.05±0.2°, 16.78±0.2°, 17. 27±0.2°, 17.85±0.2°, 18.39±0.2°, 18.77±0.2°, 19.57±0.2°, 20.67±0.2°, 21.03±0.2°, 21.21±0.2°, 22.56±0.2°, 23.45±0.2°, 24.21±0.2°, 24.42±0.2°, 24 0.75±0.2°, 25.09±0.2°, 25.39±0.2°, 25.62±0.2°, 26.09±0.2°, 26.97±0.2°, 27.26±0.2°, 27.92±0.2°, 28.48±0.2°, 29.19±0.2°, 29.43±0.2°, 30.32±0.2°, 3 1.48±0.2°, 32.09±0.2°, 32.41±0.2°, 33.54±0.2°, 33.98±0.2°, 36.14±0.2°, 36.53±0.2°, 37.03±0.2°, 37.61±0.2°, 37.96±0.2°, 38.56±0.2°, 39.56±0.2°.

[0013] In another preferred embodiment, the X-ray powder diffraction pattern of the maleate crystal form Form I has characteristic peaks at the following 2θ values: 3.54°, 7.98°, 8.26°, 9.13°, 13.50°, 15.57°, 16.05°, 16.78°, 17.27°, 17.85°, 18.39°, 18.77°, 19.57°, 20.67°, 21.03°, 21.21°, 22.56°, 23.45°, 24.21°, 2 4.42°, 24.75°, 25.09°, 25.39°, 25.62°, 26.09°, 26.97°, 27.26°, 27.92°, 28.48°, 29.19°, 29.43°, 30.32°, 31.48°, 32.09°, 32.41°, 33.54°, 33.98°, 36.14°, 36.53°, 37.03°, 37.61°, 37.96°, 38.56°, 39.56°.

[0014] In another preferred embodiment, the X-ray powder diffraction pattern of the maleate crystal form Form I is essentially as shown in Figure 1.

[0015] In another preferred embodiment, the maleate crystal form Form I is amorphous.

[0016] In another preferred embodiment, the DSC curve of the maleate crystal form Form I has an endothermic peak at 170-185°C (preferably 175-184°C).

[0017] In another preferred embodiment, the TGA curve of the maleate crystal form Form I shows no significant weight loss before decomposition.

[0018] In another preferred embodiment, the thermogravimetric analysis spectrum and differential scanning calorimetry spectrum of the maleate crystal form Form I are basically as shown in Figure 2.

[0019] In another preferred embodiment, the maleate crystal form Form I has a water absorption weight gain of ≤0.5% at 80% RH.

[0020] In another preferred embodiment, the maleate crystal form Form I has a water absorption weight gain of ≤0.4% at 80% RH, more preferably 0.39%.

[0021] A second aspect of the present invention provides a method for preparing the maleate crystal form Form I described in the first aspect of the present invention, comprising the following steps:

[0022] 1) Dissolve the compound shown in formula (Ⅰ) in a good solvent to obtain the first solution;

[0023] 2) Add maleic acid to the first solution and react for a first time, then optionally add maleate crystal form Form I and react for a second time;

[0024] 3) Add a poor solvent to the product obtained in step 2), crystallize, filter, and dry to obtain the maleate crystal form Form I as described in the first aspect of the present invention.

[0025] In another preferred embodiment, the good solvent is selected from the group consisting of: butanone, ethyl acetate, acetonitrile, acetone, isopropanol, methanol, ethanol, and tetrahydrofuran.

[0026] In another preferred embodiment, the good solvent is selected from the group consisting of: butanone, acetonitrile, acetone, and isopropanol.

[0027] In another preferred embodiment, the undesirable solvent is selected from the group consisting of: methyl tert-butyl ether, 1,4-dioxane, isopropyl acetate, cyclohexane, water, and n-heptane.

[0028] In another preferred embodiment, the undesirable solvent is methyl tert-butyl ether.

[0029] In another preferred embodiment, the first time is 2-48 hours, more preferably 10-30 hours.

[0030] In another preferred embodiment, the second time is 2-48 hours, more preferably 10-30 hours.

[0031] In another preferred embodiment, the drying temperature is 40-60°C, more preferably 45-55°C.

[0032] In another preferred embodiment, the drying time is 2-16 hours, more preferably 4-16 hours.

[0033] A third aspect of the present invention provides a pharmaceutical composition comprising a safe and effective amount of the maleate crystal form Form I described in the first aspect of the present invention and a pharmaceutically acceptable carrier.

[0034] A fourth aspect of the present invention provides the use of the maleate crystal form Form I described in the first aspect of the present invention for the preparation of a medicament for regulating CDK7 kinase activity or for the prevention and / or treatment of CDK7-related diseases.

[0035] In another preferred embodiment, the CDK7-related diseases are selected from the group consisting of: inflammation, cancer, cardiovascular disease, infection, immune disease, and metabolic disease.

[0036] In another preferred embodiment, the cancer is selected from the group consisting of: lung cancer, breast cancer, prostate cancer, colorectal cancer, liver cancer, pancreatic cancer, ovarian cancer, leukemia, neuroblastoma, gastric cancer, kidney cancer, esophageal cancer, and uterine cancer.

[0037] In another preferred embodiment, the lung cancer is small cell lung cancer or non-small cell lung cancer.

[0038] In another preferred embodiment, the breast cancer is triple-negative breast cancer.

[0039] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0040] Figure 1 is the XRPD spectrum of maleate Form I (Lot#ET57141-3-P4) of the present invention;

[0041] Figure 2 is a DSC & TGA overlay spectrum of maleate Form I (Lot#ET57141-3-P4) of the present invention;

[0042] Figure 3 is a single crystal structure diagram of maleate Form I of the present invention;

[0043] Figure 4 is the XRPD spectrum of the compound represented by formula (Ⅰ) of this invention;

[0044] Figure 5 is the DSC and TGA superimposed spectrum of the compound represented by formula (Ⅰ) of this invention;

[0045] Figure 6 shows the compound represented by formula (I) of this invention. 1 H NMR spectrum;

[0046] Figure 7 is the M DSC spectrum of the compound represented by formula (Ⅰ) of this invention;

[0047] Figure 8 is a bar chart showing the visual solubility of the compound represented by formula (Ⅰ) of this invention;

[0048] Figure 9 shows the XRPD overlay patterns of some samples in the 96-well plate of this invention;

[0049] Figure 10 is an XRPD superimposed spectrum of salt formation in Acetone according to the present invention;

[0050] Figure 11 is an XRPD superimposed spectrum of salt formation in IPA according to the present invention;

[0051] Figure 12 is an XRPD superimposed spectrum of salt formation in EtOAc according to the present invention;

[0052] Figure 13 is an XRPD superimposed spectrum of salt formation in MEK according to the present invention;

[0053] Figure 14 is the XRPD spectrum of salt formation in ACN according to the present invention;

[0054] Figure 15 is the DSC overlay spectrum of the methanesulfonate (Lot#ET57141-16-P14) of the present invention;

[0055] Figure 16 is the XRPD pattern of the suspension-to-crystal transformation in IBA / Heptane according to the present invention;

[0056] Figure 17 is the XRPD pattern of the suspension-to-crystal transformation in ACN / MTBE according to the present invention;

[0057] Figure 18 is the XRPD pattern of the present invention, which first forms salt and then suspends and transforms crystals in Heptane;

[0058] Figure 19 is the 1H NMR spectrum of maleate Form I (Lot#ET57141-3-P4) of the present invention;

[0059] Figure 20 is the DVS spectrum of maleate Form I (Lot#ET57141-18-P1) of the present invention;

[0060] Figure 21 is a superimposed XRPD spectrum of maleate Form I (Lot#ET57141-18-P1) before and after DVS of the present invention;

[0061] Figure 22 is the XRPD spectrum of the methanesulfonate pattern 1 (Lot#ET57141-3-P8) of the present invention;

[0062] Figure 23 is the DSC & TGA spectrum of the methanesulfonate Pattern 1 (Lot#ET57141-3-P8) of the present invention;

[0063] Figure 24 is the 1H NMR spectrum of the methanesulfonate pattern 1 (Lot#ET57141-3-P8) of the present invention;

[0064] Figure 25 is an XRPD superimposed spectrum of the methanesulfonate Pattern 1 (Lot#ET57141-3-P8) of the present invention after heating;

[0065] Figure 26 shows the methanesulfonate pattern 1 (Lot#ET57141-3-P8) of the present invention after heating. 1 H NMR spectrum;

[0066] Figure 27 is the XRPD pattern of the methanesulfonate pattern 2 (Form I, Lot#ET57141-13-P13) of the present invention;

[0067] Figure 28 is the DSC & TGA spectrum of the methanesulfonate Pattern 2 (Form I, Lot#ET57141-13-P13) of the present invention;

[0068] Figure 29 is the 1H NMR spectrum of the methanesulfonate pattern 2 (Form I, Lot#ET57141-13-P13) of the present invention;

[0069] Figure 30 is an XRPD superimposed spectrum of the methanesulfonate Pattern 2 (Form I, Lot#ET57141-13-P13) of the present invention after heating;

[0070] Figure 31 is the DSC & TGA spectrum of the methanesulfonate Pattern 2 (Form I, Lot#ET57141-13-P13) of the present invention after heating;

[0071] Figure 32 is the XRPD pattern of the methanesulfonate pattern 3 (Form II, Lot#ET57141-23-P2) of the present invention;

[0072] Figure 33 is the DSC & TGA spectrum of the methanesulfonate Pattern 3 (Form II, Lot#ET57141-23-P2) of the present invention;

[0073] Figure 34 is the 1H NMR spectrum of the methanesulfonate pattern 3 (Form II, Lot#ET57141-23-P2) of the present invention;

[0074] Figure 35 is an XRPD superimposed spectrum of the methanesulfonate Pattern 3 (Form II, Lot#ET57141-23-P2) of the present invention after heating;

[0075] Figure 36 is the DSC & TGA spectrum of the methanesulfonate Pattern 3 (Form II, Lot#ET57141-23-P2) of the present invention after heating;

[0076] Figure 37 shows the DVS curve of the methanesulfonate pattern 3 (Form II, ET57141-27-P1) of the present invention;

[0077] Figure 38 is the XRPD overlay pattern of the methanesulfonate Pattern 3 (Form II, ET57141-27-P1) after DVS test of the present invention;

[0078] Figure 39 is the XRPD spectrum of the methanesulfonate Pattern 4 (Form III, Lot#ET57141-19-P1) of the present invention;

[0079] Figure 40 is the DSC & TGA spectrum of the methanesulfonate Pattern 4 (Form III, Lot#ET57141-19-P1) of the present invention.

[0080] Figure 41 shows the methanesulfonate pattern 4 (Form III, Lot#ET57141-19-P1) of this invention. 1 H NMR spectrum;

[0081] Figure 42 is an XRPD overlay of the methanesulfonate Pattern 4 (Form III, Lot#ET57141-19-P1) of the present invention after heating;

[0082] Figure 43 is the DSC & TGA spectrum of the methanesulfonate Pattern 4 (Form III, Lot#ET57141-19-P1) of the present invention after heating;

[0083] Figure 44 shows the DVS curve of the methanesulfonate Pattern 4 (Form III, ET57141-19-P1) of the present invention;

[0084] Figure 45 shows the XRPD overlay patterns before and after DVS testing of the methanesulfonate Pattern 4 (Form III, ET57141-19-P1) of the present invention;

[0085] Figure 46 is the XRPD spectrum of the methanesulfonate pattern 5 (Lot#ET57141-22-P13) of the present invention;

[0086] Figure 47 is the DSC & TGA spectrum of the methanesulfonate Pattern 5 (Lot#ET57141-22-P13) of the present invention;

[0087] Figure 48 is the 1H NMR spectrum of the methanesulfonate pattern 5 (Lot#ET57141-22-P13) of the present invention;

[0088] Figure 49 is an XRPD superimposed spectrum of the methanesulfonate Pattern 5 (Lot#ET57141-22-P13) of the present invention after heating;

[0089] Figure 50 is the DSC & TGA spectrum of the methanesulfonate Pattern 5 (Lot#ET57141-22-P13) of the present invention after heating;

[0090] Figure 51 is an XRPD overlay pattern of the methanesulfonate Pattern 5 (Lot#ET57141-22-P13) of the present invention after 7 days of storage;

[0091] Figure 52 is the XRPD spectrum of the methanesulfonate pattern 6 (Lot#ET57141-27-P1S1) of the present invention;

[0092] Figure 53 is the DSC & TGA spectrum of the methanesulfonate Pattern 6 (Lot#ET57141-27-P1S1) of the present invention;

[0093] Figure 54 is an XRPD superimposed spectrum of the methanesulfonate Pattern 6 (Lot#ET57141-27-P1S1) of the present invention after heating;

[0094] Figure 55 is the XRPD spectrum of the fumarate pattern 1 (Lot#ET57141-16-P5) of the present invention;

[0095] Figure 56 is a DSC & TGA overlay spectrum of the fumarate pattern 1 (Lot#ET57141-16-P5) of the present invention;

[0096] Figure 57 shows the fumarate pattern 1 (Lot#ET57141-16-P5) of this invention. 1 H NMR spectrum;

[0097] Figure 58 is the XRPD spectrum of maleate Form I (Lot#ET57141-18-P1) of the present invention;

[0098] Figure 59 is a microscopic image of the maleate Form I (Lot#ET57141-18-P1) of the present invention;

[0099] Figure 60 is a DSC & TGA overlay spectrum of maleate Form I (Lot#ET57141-18-P1) of the present invention;

[0100] Figure 61 is the 1H NMR spectrum of maleate Form I (Lot#ET57141-18-P1) of the present invention;

[0101] Figure 62 is the DVS spectrum of maleate Form I (Lot#ET57141-18-P1) of the present invention;

[0102] Figure 63 is an XRPD superimposed spectrum of maleate Form I (Lot#ET57141-18-P1) before and after DVS of the present invention;

[0103] Figure 64 is an XRPD overlay pattern during the optimization of the preparation conditions of maleate Form I in this invention;

[0104] Figure 65 is the PLM spectrum of maleate Form I (Lot#ET57141-26-P1) of the present invention;

[0105] Figure 66 is the XRPD spectrum of the methanesulfonate Form II (Lot#ET57141-27-P1) of the present invention;

[0106] Figure 67 is the DSC & TGA spectrum of the methanesulfonate Form II (Lot#ET57141-27-P1) of the present invention;

[0107] Figure 68 shows the methanesulfonate Form II (Lot#ET57141-27-P1) of the present invention. 1 H NMR spectrum;

[0108] Figure 69 shows the DVS curve of the methanesulfonate Form II (Lot#ET57141-27-P1) of the present invention;

[0109] Figure 70 shows the XRPD overlay spectra of the methanesulfonate Form II (Lot#ET57141-27-P1) before and after DVS testing;

[0110] Figure 71 is an XRPD overlay pattern of the optimized preparation process of Form III methanesulfonate in this invention;

[0111] Figure 72 is an XRPD overlay pattern of the preparation process of the methanesulfonate Form III (Lot#ET57141-19-P1) of the present invention;

[0112] Figure 73 is the DSC & TGA spectrum of the methanesulfonate Form III (Lot#ET57141-19-P1) of the present invention;

[0113] Figure 74 shows the methanesulfonate Form III (Lot#ET57141-19-P1) of the present invention. 1 H NMR spectrum;

[0114] Figure 75 shows the DVS curve of the methanesulfonate Form III (Lot#ET57141-19-P1) of the present invention;

[0115] Figure 76 is the XRPD overlay spectrum of the methanesulfonate Form III (Lot#ET57141-19-P1) after DVS test of the present invention;

[0116] Figure 77 is the XRPD spectrum of the compound (Lot#EW34502-101-D-P5) of formula (II) of this invention;

[0117] Figure 78 is a microscopic image of the compound (Lot#EW34502-101-D-P5) of formula (II) of this invention;

[0118] Figure 79 is the DSC and TGA superimposed spectrum of the compound (Lot#EW34502-101-D-P5) of formula (II) of the present invention;

[0119] Figure 80 is the 1H-NMR spectrum of the compound (Lot#EW34502-101-D-P5) shown in formula (II) of this invention;

[0120] Figure 81 is a bar chart of the visual solubility of the compound (Lot#EW34502-101-D-P5) shown in formula (II) of the present invention;

[0121] Figure 82 is the XRPD superimposed spectrum of the suspension-to-crystal conversion experiment under 50℃ conditions of the present invention-1;

[0122] Figure 83 is the XRPD superimposed spectrum of the suspension-to-crystal conversion experiment under 50℃ conditions of the present invention-2;

[0123] Figure 84 is the XRPD superimposed spectrum of the suspension-to-crystal conversion experiment under 25℃ conditions of the present invention-1;

[0124] Figure 85 is the XRPD superimposed spectrum of the suspension-to-crystal conversion experiment under 25℃ conditions of the present invention-2;

[0125] Figure 86 is the XRPD superimposed spectrum of the antisolvent precipitation (positive drop) experiment of the present invention-1;

[0126] Figure 87 is the XRPD superimposed spectrum of the antisolvent precipitation (positive drop) experiment of the present invention-2;

[0127] Figure 88 is the XRPD superimposed spectrum of the antisolvent precipitation (re-dropping) experiment of the present invention;

[0128] Figure 89 is an XRPD superimposed spectrum of the cooling crystallization experiment of the present invention;

[0129] Figure 90 is a superimposed XRPD spectrum of the high and low temperature cycling experiment of the present invention;

[0130] Figure 91 is the XRPD spectrum of the amorphous sample (Lot#ET57523-38-P0) of the present invention;

[0131] Figure 92 is a superimposed XRPD spectrum of the suspension-to-crystal conversion experiment at 50℃ according to the present invention;

[0132] Figure 93 is the XRPD superimposed spectrum of the suspension-to-crystal conversion experiment under 25℃ conditions of the present invention-1;

[0133] Figure 94 is the XRPD superimposed spectrum of the suspension-to-crystal conversion experiment under 25℃ conditions of the present invention-2;

[0134] Figure 95 is an XRPD superimposed spectrum of maleate Form I before and after dry grinding according to the present invention;

[0135] Figure 96 is an XRPD superimposed spectrum of maleate Form I before and after wet milling according to the present invention;

[0136] Figure 97 is an XRPD superimposed spectrum of the Form II methanesulfonate of the present invention before and after dry milling;

[0137] Figure 98 is an XRPD superimposed spectrum of the Form II methanesulfonate of the present invention before and after wet milling;

[0138] Figure 99 is an XRPD superimposed spectrum of the Form III methanesulfonate of the present invention before and after dry milling;

[0139] Figure 100 is an XRPD superimposed pattern of the Form III methanesulfonate of the present invention before and after wet milling;

[0140] Figure 101 is an XRPD superimposed image of maleate Form I before and after tableting according to the present invention;

[0141] Figure 102 is an XRPD superimposed spectrum of the Form II mesylate tablets of the present invention before and after compression;

[0142] Figure 103 is an XRPD superimposed spectrum of the Form III methanesulfonate tablets of the present invention before and after compression;

[0143] Figure 104 is an XRPD overlay pattern of the maleate Form I stability sample of the present invention;

[0144] Figure 105 is an HPLC overlay chromatogram of the maleate Form I stability sample of the present invention;

[0145] Figure 106 is an XRPD overlay pattern of the form II stability sample of methanesulfonate of the present invention;

[0146] Figure 107 is an HPLC overlay chromatogram of the form II stability sample of methanesulfonate of the present invention;

[0147] Figure 108 is an XRPD overlay pattern of the methanesulfonate Form III stability sample of the present invention;

[0148] Figure 109 is an HPLC overlay chromatogram of the form III stability sample of the methanesulfonate of this invention. Detailed Implementation

[0149] Through long-term and in-depth research, the inventors have prepared a series of salt forms and crystal forms of the compounds shown in formula (Ⅰ) with excellent stability, especially the maleate crystal form Form I. Based on this, the inventors have completed this invention.

[0150] the term

[0151] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0152] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0153] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0154] As used in this article, the term "n or more 2θ values ​​selected from the following group" refers to any positive integer including n and greater than n (e.g., n, n+1, ...), where the upper limit Nup is the number of all 2θ peaks in the group. For example, "3 or more" includes not only the positive integers of the upper limit Nup (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, ...), but also ranges such as "4 or more", "5 or more", and "6 or more".

[0155] compound

[0156] Application CN113717157A discloses a series of compounds used as CDK7 kinase inhibitors, which exhibit very good CDK7 inhibitory activity (especially selective CDK7 inhibitory activity). This patent application discloses a compound with the structural formula shown in formula (Ⅰ).

[0157] To further investigate the druggability of this compound and enable it to be used as a drug molecule in clinical trials, its salt form and solid form were studied. We screened and developed the salt form and crystal form of the compound shown in formula (I) to find stable and reliable salt and crystal forms to ensure the quality stability of the compound shown in formula (I). This will allow for better use in pharmaceuticals, drug formulations, and future clinical applications, achieving stable and controllable drug quality, better dissolution, and higher bioavailability. This will avoid safety issues such as side effects caused by toxic impurities due to drug instability, and will be of great significance for future advancements.

[0158] As confirmed below, the compound of formula (I) obtained in CN113717157A is amorphous.

[0159] Crystal form

[0160] Crystal form refers to the solid state of a drug. Drug crystal form research is essentially the study of the fundamental state of a drug. Only with a sufficient and comprehensive understanding of the crystal forms of chemical drugs can we find more suitable solid crystal forms for treating diseases. Drug crystal form can affect the physicochemical properties of drugs, directly influencing the basis for their clinical therapeutic effects. Different crystal forms of the same drug may exhibit significant differences in appearance, solubility, melting point, dissolution rate, and bioavailability, thus affecting the drug's stability, bioavailability, and efficacy. Therefore, studying the stable crystal form of a compound is of great significance.

[0161] The active ingredient of a drug generally exists in two or more crystalline forms, known as drug polymorphs. Different polymorphs have different solubilities and dissolution rates, affecting the drug's clinical therapeutic effect by causing changes in bioavailability in the body. Differences in drug polymorphs may affect its dissolution and absorption in the body, thus impacting bioavailability, clinical efficacy, and safety. Simultaneously, the stability of drug polymorphs is also crucial. To improve drug bioavailability, reduce toxicity, and enhance therapeutic efficacy, greater emphasis must be placed on drug polymorph stability. Stable polymorphs ensure the physicochemical stability of the drug dosage form during preparation and storage, maintaining good solubility and bioavailability, and ensuring equivalence between batches of the drug. The same drug often has multiple polymorphs; currently, the polymorph with better therapeutic effects and most suitable for clinical use is called the dominant drug polymorph.

[0162] Crystal Formation

[0163] The present invention also provides the following method for preparing the maleate crystal form Form I:

[0164] Using maleate crystal form Form I as the starting material, the method is selected from the group consisting of:

[0165] Method 1: Mix the compound shown in formula (II) with a solvent to obtain a suspension, crystallize at 25°C for 3 days or at 50°C for 1 day, filter, dry and characterize; the solvent is selected from one or two of n-heptane, cyclohexane, ethyl acetate, water, acetonitrile, methyl tert-butyl ether, isopropanol, 1,4-dioxane, methanol, 2-butanone, tetrahydrofuran, and acetone.

[0166] Method 2: Mix the compound shown in formula (II) with a good solvent and stir at room temperature until completely dissolved. While stirring, gradually add a poor solvent. Then stir at 25°C for 1 day, filter the resulting suspension, dry it and characterize it. The good solvent is selected from methanol, tetrahydrofuran, acetone and ethanol, and the poor solvent is selected from water, n-heptane, methyl tert-butyl ether and isopropyl acetate.

[0167] Method 3: Dissolve the compound shown in formula (II) in a good solvent at 25°C or 50°C to prepare a solution of 150-300 mg / mL. Take 0.1 or 0.2 mL of the prepared solution and add it to a poor solvent at room temperature. Then stir at 25°C for 2 days. Filter the resulting suspension, dry it and characterize it. The good solvent is selected from methanol, tetrahydrofuran, acetone and ethanol. The poor solvent is selected from water, n-heptane, methyl tert-butyl ether and isopropyl acetate.

[0168] Method 4: Dissolve the solid by adding a certain volume of good solvent to the compound shown in formula (II) at 50°C, and then filter; or add a certain volume of antisolvent at 50°C, then add good solvent to dissolve the solid, filter, transfer the filtrate to -5°C and stir for 1 day, filter the suspension, dry and characterize; the good solvent is selected from 2-butanone, acetone and ethanol, and the poor solvent is selected from acetonitrile and isopropyl acetate.

[0169] Method 5: Place the compound shown in formula (II) in a sample vial, add the selected solvent, cycle at 50℃~5℃ for 3 days, then filter and characterize the suspension. The solvent is selected from one or two of the following: n-heptane, cyclohexane, ethyl acetate, water, acetonitrile, methyl tert-butyl ether, isopropanol, 1,4-dioxane, methanol, 2-butanone, and acetone.

[0170] The temperature cycling method is as follows: ① Maintain a constant temperature of 50℃ for 2 hours → ② Cool down to 5℃ at a rate of 0.2℃ / min → ③ Maintain a constant temperature of 5℃ for 2 hours → ④ Increase the temperature to 50℃ at a rate of 0.2℃ / min. Repeat the above conditions.

[0171] Using maleate amorphous samples as starting materials, the method is selected from the following group:

[0172] Method 1: Place the amorphous sample of the compound shown in formula (II) in a sample vial, add the selected solvent, stir at 50°C for 3 days, filter and characterize; the solvent is selected from one or two of n-heptane, cyclohexane, ethyl acetate, water, acetonitrile, methyl tert-butyl ether, isopropanol, 1,4-dioxane, methanol, 2-butanone, tetrahydrofuran, and acetone.

[0173] Method 2: Place the amorphous sample of the compound shown in formula (II) in a sample vial, add the selected solvent, stir at 25°C for 3 days, filter and characterize; the solvent is selected from one or two of n-heptane, cyclohexane, ethyl acetate, water, acetonitrile, methyl tert-butyl ether, isopropanol, 1,4-dioxane, methanol, 2-butanone, tetrahydrofuran, and acetone.

[0174] Pharmaceutical Composition

[0175] The present invention also provides a pharmaceutical composition comprising a safe and effective amount of the maleate crystal form Form I and a pharmaceutically acceptable carrier.

[0176] "Safe and effective dose" means that the amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects.

[0177] Typically, the pharmaceutical composition contains 1-2000 mg of active ingredient per dose, more preferably 10-200 mg of active ingredient per dose. Preferably, "one dose" refers to one tablet or one syringe.

[0178] "Pharmaceutical acceptable carriers" refer to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity.

[0179] "Compatibility" here refers to the ability of each component in the composition to interact with and incorporate with the active ingredient of the present invention without significantly reducing the efficacy of the active ingredient.

[0180] Pharmaceutically acceptable examples of carrier components include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), and emulsifiers (such as Tween). Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0181] There are no particular limitations on the administration of the active ingredients or pharmaceutical compositions of the present invention. Representative administration methods include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular, or subcutaneous) administration.

[0182] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

[0183] In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient or carrier, such as sodium citrate or dicalcium phosphate, or mixed with one or more of the following components:

[0184] (a) Fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol and silica;

[0185] (b) Adhesives, such as hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic;

[0186] (c) Moisturizers, such as glycerin;

[0187] (d) Disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginate, certain complex silicates, and sodium carbonate;

[0188] (e) Slow solvents, such as paraffin;

[0189] (f) Absorption accelerators, such as quaternary ammonium compounds;

[0190] (g) Wetting agents, such as cetyl alcohol and glyceryl monostearate;

[0191] (h) Adsorbent, for example, kaolin; and / or

[0192] (i) Lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof.

[0193] Buffers may also be included in capsules, tablets, and pills.

[0194] The solid dosage form can also be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active ingredient from this composition can be delayed in a portion of the digestive tract. Examples of suitable encapsulating components are polymers and waxes.

[0195] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. Besides these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0196] In addition to the active ingredient, the suspension may contain suspending agents, such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0197] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0198] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–2000 mg, preferably 20–500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skills of a skilled physician.

[0199] The compounds of this invention can be administered alone or in combination with other therapeutic agents (such as antitumor drugs).

[0200] Compound (I) can also be used in combination with other known drugs for treating or improving similar symptoms. When used in combination, the original drug's administration method and dosage remain unchanged, while compound (I) is taken simultaneously or subsequently. When compound (I) is taken concurrently with one or more other drugs, a pharmaceutical composition containing one or more known drugs and compound (I) is preferred. Drug combination also includes taking compound (I) with one or more other known drugs during overlapping time periods. When compound (I) is used in combination with one or more other drugs, the dosage of compound (I) or the known drug may be lower than the dosage when they are taken alone.

[0201] Compared with the prior art, the present invention has the following main advantages:

[0202] (1) Compared with amorphous, maleate crystal form Form I has excellent chemical stability, solid-state stability and solution stability;

[0203] (2) The maleate crystal form Form I of the present invention has excellent pharmacokinetic properties, such as excellent in vivo exposure in animals and high bioavailability.

[0204] (3) The maleate crystal form Form I of the present invention has excellent purity;

[0205] (4) The maleate crystal form Form I of the present invention has excellent solubility and hygroscopicity.

[0206] (5) The maleate crystal form Form I of the present invention has excellent activity and broad-spectrum antitumor activity.

[0207] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0208] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0209] The technical solution of the present invention will be described in detail below.

[0210] The solvents and their corresponding Chinese names are shown in Table 1.

[0211] Table 1. Comparison of Chinese and English names of solvents used in the experiment

[0212] Analytical methods

[0213] 1.1 X-ray powder diffraction (XRPD)

[0214] XRPD diffraction patterns were acquired using a Bruker D2 Phaser. The sample was placed on a smooth, background-free silicon wafer for measurement. Measurement parameters are shown in Table 2.

[0215] Table 2 XRPD Method Parameters

[0216] 1.2 Polarizing Microscope (PLM)

[0217] PLM analysis was performed using an Ottoman BK-Pol optical microscope. A small amount of sample was placed on a glass slide, a drop of silicone oil was added to disperse it, a coverslip was then placed on top, and the sample was observed under the microscope.

[0218] 1.3 Differential Scanning Calorimetry (DSC)

[0219] The DSC curves were acquired using a TA instrument with a DSC 250 sensor. The testing method for the DSC 250 instrument was as follows: an appropriate amount of sample was accurately weighed into a perforated aluminum crucible, and the temperature was increased from 25°C to the final temperature at a rate of 10°C / min. Instrument parameters are shown in Table 3.

[0220] Table 3 DSC and M DSC analysis parameters

[0221] 1.4 Thermogravimetric Analysis (TGA)

[0222] TGA data were acquired using a TGA 550 instrument from TA. An appropriate amount of sample was placed in a pre-peeled aluminum crucible and heated from room temperature to 300°C at a rate of 10°C / min. The temperature program and equipment parameters of the TGA 550 instrument are shown in Table 4.

[0223] Table 4 TGA Analysis Parameters

[0224] 1.5 Proton NMR (1H-NMR)

[0225] NMR data were acquired using a Varian or Bruker 400MHz sensor. The measured parameters are shown in Table 5.

[0226] Table 5 NMR Analysis Parameters

[0227] 1.6 Dynamic Steam Adsorption Analyzer (DVS)

[0228] Moisture adsorption / desorption data were collected by DVS Intrinsic. Approximately 20–30 mg of sample was placed in a pre-peeled sample chamber and automatically weighed. The sample was dried at 40 °C / 0% RH until dm / dt ≤ 0.002%. After cooling to 25 °C and dm / dt ≤ 0.002%, the test was started using the operating parameters in Table 6.

[0229] Table 6 DVS Analysis Parameters

[0230] 1.7 High Performance Liquid Chromatography (HPLC)

[0231] Liquid chromatography analysis was performed using a Shimadzu SPD-20. See Table 7 for the liquid chromatography methods used for solubility and stability.

[0232] Table 7 HPLC Methods

[0233] Regarding crystal form examples

[0234] Example 1: Preparation of compound sample of formula (Ⅰ)

[0235] The preparation method of compound (I) is the same as that in patent application CN113717157A, and the synthetic route is as follows:

[0236] The specific steps are as follows:

[0237] 1.1 Synthesis of compound int2

[0238] Under anhydrous and oxygen-free conditions, 300 mg of compound int1, methyl fluorosulfonyl difluoroacetate (5.0 eq), and cuprous iodide (5.0 eq) were added to a 10 ml reaction tube. The mixture was heated to 100 °C and maintained at this temperature until the reaction was complete. After cooling to room temperature, the reaction was quenched with 1 M dilute hydrochloric acid. The mixture was extracted with water and ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate. The organic phase was concentrated and purified by silica gel column chromatography to give 110 mg of compound int2, with a yield of 37.3%. LC-MS [M+1]: 603.2.

[0239] 1.2 Synthesis of Equation (Ⅰ)

[0240] 110 mg of compound int2 was added to a 50 ml three-necked flask, followed by 6 ml of dichloromethane. After dissolving the compound, 2 ml of trifluoroacetic acid was added. The mixture was stirred at room temperature for 30 min. After the reaction was completed, the mixture was concentrated to remove excess solvent. The pH was then adjusted to alkaline, extracted with ethyl acetate, and evaporated to dryness. The mixture was then purified by a medium-pressure reversed-phase column (mobile phase: water, 1,4-dioxane, and methanol) to obtain 60 mg of compound (I) with a yield of 65.2%. The HPLC purity was 95%, and the LC-MS [M+1] was 503.1.

[0241] Example 2: Screening of salt types of compound (I)

[0242] 2.1 Raw material characterization

[0243] The received material (Free base, i.e., the product obtained in Example 1) was fully characterized, and the results are summarized in Table 8.

[0244] The XRPD spectrum (Figure 4) shows that the received drug substance free base (Lot#LHB-035A-132) is amorphous. The TGA curve shows two weight loss segments at 3.2% / 65-125℃ and 4.9% / 125-180℃, and the DSC curve shows corresponding endothermic peaks at 58℃ and 115℃ (Figure 5). 1 ¹H NMR (Figure 6) detected 5.35% EtOAc and 0.44% 1,4-dioxane solvent residues. ¹H DSC results (Figure 7) indicated that the glass transition temperature (Tg) of the active pharmaceutical ingredient was 54 °C.

[0245] Table 8 Characterization results of starting materials

[0246] In this invention, the amorphous form of the compound of formula (I) obtained in Example 1 was used as the raw material for the subsequent preparation of salt form and crystal form.

[0247] 2.2 Visually assessed solubility

[0248] The solubility of the compound Free base (Lot#LHB-035A-132) of formula (I) at room temperature was roughly determined visually in 14 solvents. The results are shown in Table 9 and Figure 8. The compound Free base of formula (I) showed high solubility in most of the tested solvents, >91.8 mg / mL; and low solubility in cyclohexane, n-heptane, and water, <1 mg / mL.

[0249] Table 9 shows the visual solubility results of the Free base of the compound represented by formula (Ⅰ).

[0250] 2.3 High-throughput salt formation screening

[0251] Using a slow evaporation method, phosphoric acid, succinic acid, citric acid, maleic acid, fumaric acid, L-tartaric acid, hydrochloric acid, methanesulfonic acid, oxalic acid, L-malic acid, acetic acid, benzoic acid, sulfuric acid, and p-toluenesulfonic acid were selected and reacted with the Free base of compound (I) in 96-well plates for salt type screening. Most of the obtained products were adhesive residues; only the reaction with methanesulfonic acid (2.1 eq.) yielded crystals, but the crystallinity was poor. Combined with high-throughput salt formation and reactive salt formation 1H NMR results, it was found that the Free base of compound (I) reacted with all 14 acids mentioned above with chemical shifts, indicating that it could form salts with them. Detailed information and results for the 96-well plates are shown in Table 10. XRPD results for some products are shown in Figure 9.

[0252] 2.4 Salt Form Preparation

[0253] Based on the pKa of the free base shown in formula (I) and the results of high-throughput screening, the salt formation of the free base shown in formula (I) with different acids was attempted in different solvent systems, and the obtained salts were characterized to study their solid-state properties.

[0254] 2.4.1 Salt formation screening was carried out using the direct reaction salt formation method.

[0255] Weigh approximately 50 mg of the free base (Lot#LHB-035A-132) of formula (Ⅰ) into a sample vial, and dissolve and clarify it by adding 0.5 mL (10V) of the selected solvent. Then add 1.05 eq. or 2.1 eq. of the selected acid, and react for a period of time under cyclic conditions of room temperature (16–22 °C) or high and low temperature (25–-5 °C). Add the antisolvent to the unprecipitated sample and continue the reaction. Filter, dry and characterize the resulting suspension.

[0256] 2.4.1.1 Salt formation screening in Acetone

[0257] Approximately 50 mg of the free base (Lot#LHB-035A-132) of formula (Ⅰ) was weighed into a sample vial, dissolved in 0.5 mL of Acetone, followed by the addition of 1.05 eq. of the selected acid. After reacting at 22 °C for 1 day, no obvious solid precipitation was observed. MTBE was added, and stirring was continued for 4–5 hours. The resulting suspension was filtered and dried under vacuum at 50 °C for 4–16 hours before characterization. As shown in Table 11 and Figure 10, only the crystalline salts maleate (Form I) and methanesulfonate (Pattern 1) were obtained.

[0258] Table 11 Results of salt formation screening using Acetone as solvent

[0259] 2.4.1.2 Salt formation screening in IPA

[0260] Approximately 50 mg of the free base (Lot#LHB-035A-132) of formula (Ⅰ) was weighed into a sample vial, dissolved in 0.5 mL of IPA, followed by the addition of 1.05 eq. of the selected acid. After reacting at 22 °C for 1 day, no obvious solid precipitation was observed. MTBE was added, and stirring was continued for 4–5 hours. The resulting suspension was filtered, dried under vacuum at 50 °C for 4–16 hours, and then characterized. As shown in Table 12 and Figure 11, only the crystalline salt maleate Form I was obtained.

[0261] Table 12 Results of salt formation screening using IPA as solvent

[0262] 2.4.1.3 Salt formation screening in EtOAc

[0263] Approximately 50 mg of the free base (Lot#LHB-035A-132) of formula (I) was weighed into a sample vial, and 0.5 mL of EtOAc was added to dissolve it. Then, 1.05 eq. of the selected acid was added. After reacting at 22 °C for 30 min, heptane was added to the unprecipitated sample, and stirring was continued. After reacting at 22 °C for 1 day, the resulting suspension was filtered, dried under vacuum at 50 °C for 3–4 h, and then characterized. As shown in Table 13 and Figure 12, only fumarate crystals with poor crystallinity were obtained.

[0264] Table 13 Results of salt formation screening using EtOAc as solvent

[0265] Note: ① After adding 1.05 eq. of hydrochloric acid to 6-P7, the solution is yellow and no solid precipitates. After adding another 1.05 eq. of hydrochloric acid, a yellow solid precipitates.

[0266] ②6-P3, P5 and P6 were reacted at 50℃ for 1 day, and then transferred to room temperature (16-20℃) and the antisolvent Heptane was added respectively.

[0267] 2.4.1.4 Salt formation screening in MEK

[0268] Approximately 50 mg of the free base (Lot#LHB-035A-132) of formula (Ⅰ) was weighed into a sample vial and dissolved in 0.5 mL of MEK. Subsequently, 1.1 eq. or 2.1 eq. of the selected acid was added according to pKa data. After reacting at high and low temperatures (25 to -5 °C) for 1 day, the antisolvent MTBE was added to the unprecipitated solution sample, and the high and low temperature cycling was continued for 3 days. The resulting suspension was filtered, dried under vacuum at 50 °C for 2–4 h, and then characterized. As shown in Table 14 and Figure 13, only crystalline salts of maleate and methanesulfonate were obtained.

[0269] Table 14 Results of salt formation screening using MEK as solvent

[0270] Note: ① High and low temperature cycling conditions: 1) 25℃ constant temperature for 2 hours; 2) 0.2℃ / min cooling to -5℃; 3) -5℃ constant temperature for 4 hours; 4) 0.2℃ / min heating to 25℃; repeat the above conditions.

[0271] 2.4.1.5 Salt formation screening in ACN

[0272] Approximately 50 mg of the free base (Lot#LHB-035A-132) of formula (Ⅰ) was weighed into a sample vial and dissolved in 0.5 mL of ACN. Subsequently, 1.1 eq. or 2.1 eq. of the selected acid was added according to pKa data. After reacting at room temperature (16-18℃) for 1 day, the suspension was filtered, dried under vacuum at 50℃ for 2-4 hours, and then characterized. MTBE was added to the unprecipitated sample, and the cap was slightly loosened before stirring for another 3 days. As shown in Table 15 and Figure 14, only maleate, methanesulfonate, and poorly crystallized fumarate crystalline salts were obtained.

[0273] Based solely on XRPD, it is difficult to distinguish methanesulfonate (Lot#ET57141-16-P14) as a mixed crystal of Pattern 3 and Pattern 4. The comparison diagram of DSC is shown in Figure 15.

[0274] Table 15 Results of salt formation screening using ACN as solvent at room temperature.

[0275] Based on the results of methanesulfonate and fumarate in Table 15, the preparation of fumarate and methanesulfonate was repeated under ACN / 50℃ conditions: approximately 50 mg of the free base (Lot#LHB-035A-132) of formula (Ⅰ) was weighed into a sample vial, and 1.0 mL of ACN was added to dissolve it. Subsequently, 1.1 eq. or 2.1 eq. of the selected acid was added at 50℃ according to the pKa data. Samples were taken for analysis after reacting at 50℃ for 1 to 4 days. As shown in Table 16 and Figure 14, only methanesulfonate Pattern 3 was obtained, and fumarate crystals were not obtained.

[0276] Table 16 Results of salt formation screening using ACN as solvent at 50℃

[0277] 2.4.2 Salt screening was performed using the suspension-to-crystal conversion method.

[0278] Because only maleate (Form I), methanesulfonate (Pattern 1, Pattern 2, Pattern 3, Pattern 4), and the poorly crystallized fumarate (Pattern 1) were obtained during the direct salt formation process, a method of first forming salts and then suspending them in different solvents for crystal transformation was adopted for screening. The specific method is as follows:

[0279] 2.4.2.1 Crystal Transformation in IBA / Heptane Suspension

[0280] Lot#ET57141-13-P1, P2, P7, P8, P9, P10, P12, P15, and P16 were dried under N2 conditions, then IBA and Heptane were added, and the mixtures were suspended at room temperature (16-18℃) for 1-4 days to undergo crystallization. The results are shown in Table 17 and Figure 16; no crystalline salts were obtained.

[0281] Table 17 Crystallization of suspensions in IBA / Heptane

[0282] 2.4.2.2 Attempts to induce crystallization in amorphous salts through suspension

[0283] Ten amorphous salt forms were attempted to be suspended in ACN / MTBE (1 / 4) at room temperature (~17°C) for crystallization. Samples were taken after 3 days for XRPD analysis. The results are shown in Table 18 and Figure 17. No satisfactory crystalline salts were obtained.

[0284] Table 18 Results of suspension-to-crystal transformation in ACN / MTBE

[0285] 2.4.2.3 Attempt to form salt first and then transform suspension into crystals

[0286] Approximately 50 mg of the free base (Lot#LHB-035A-132) of formula (Ⅰ) was weighed into a sample vial and dissolved in 0.5 mL of MeOH. Then, 1.1 eq. or 2.1 eq. of the selected acid was added. After reacting at room temperature (~17℃) for 2 h, the solvent was removed by rotary evaporation (water bath temperature of 30℃). The resulting residues were all adhesives. 1 mL of Heptane was added to each adhesive, and after suspension and crystallization at room temperature (~17℃) for 1 day, no solids appeared. After transfer to 50℃ for suspension and crystallization for 4 days, the results are shown in Table 19 and Figure 18. Except for the peak (~18°) of the magnetic stir bar in the XRPD of some products, only the crystal salt of methanesulfonic acid, Pattern 5, was obtained.

[0287] Table 19 Results of salt formation followed by crystal transformation in Heptane suspension.

[0288] 2.5 Characterization of Salt

[0289] 2.5.1 Characterization of Maleate Form I

[0290] The obtained maleate Form I (Lot#ET57141-3-P4) was characterized, and the results are shown in Figures 1-2 and 19-21. XRPD (Figure 1) shows that the obtained maleate is a well-crystallized crystal. The DSC curve of maleate Form I (Figure 2) shows an endothermic peak at 182℃, which should be a melting peak. TGA showed no significant weight loss before decomposition. The 1H NMR spectrum (Figure 19) shows that the free base reacts with maleic acid in an approximately 1:1 ratio to form a salt, with 0.48% MTBE and 0.43% Acetone solvent residues. Based on the above results, maleate Form I should be an amorphous form.

[0291] DVS tests were performed on a small-scale batch of maleate Form I (Lot#ET57141-18-P1), and the DVS results are shown in Figure 20. The weight gain after water absorption at 80% RH was 0.39%, indicating slight hygroscopicity. The XRPD pattern after the DVS test did not change (Figure 21).

[0292] Based on Figure 1, the data in Table A-1 is obtained.

[0293] Table A-1

[0294] Calculations show that the maleate crystal form Form I belongs to the triclinic crystal system, space group P1, and its unit cell parameters are: { α=111.1780(10)°, β=99.4490(10)°, γ=92.7800(10)°, Its single-crystal structure is shown in Figure 3.

[0295] 2.5.2 Characterization of Methanesulfonate

[0296] Methanesulfonate patterns 1-5 were obtained during the experiment and were characterized. As shown in Table 20, each pattern showed partial weight loss before decomposition, so each pattern underwent heat treatment experiments.

[0297] Table 20 Characterization results of various crystal forms of methanesulfonate

[0298] Note: DMSO is suspected to have been adsorbed during the drying process.

[0299] 2.5.2.1 Characterization of Methanesulfonate Pattern 1

[0300] As shown in the XRPD spectrum (Figure 22), methanesulfonate Pattern 1 is a poorly crystallized crystal. The DSC curve (Figure 23) shows two endothermic peaks at 75℃ and 170℃, while the corresponding TGA curves show two weight loss segments at 25-90℃ and 105-195℃, with losses of 0.3% and 7.5%, respectively. The 1H NMR results (Figure 24) indicate that the free base forms a salt with methanesulfonic acid at a ratio of approximately 1:1.5, with 6.99% MTBE remaining. Heating methanesulfonate Pattern 1 to 170℃ using TGA and immediately characterizing it reveals that the MTBE content decreases from 6.99% to 0.41% (Figure 26), and the crystals transform into an amorphous form (Figure 25). Based on these findings, methanesulfonate Pattern 1 is likely a poorly crystallized MTBE (~0.5 mol) solvate.

[0301] 2.5.2.2 Characterization of mesylate pattern 2

[0302] The XRPD spectrum (Figure 27) shows that methanesulfonate Pattern 2 is a moderately crystalline crystal. The DSC curve (Figure 28) shows two endothermic peaks at 100℃ and 183℃, and the TGA curve shows a 2.2% weight loss at 25-65℃. The 1H NMR results (Figure 29) indicate that the free base forms a salt with methanesulfonic acid in a ratio of approximately 1:2, and a suspected 0.26% DMSO solvent residue was found, possibly due to adsorption during the drying process. Based on these results, it is inferred that methanesulfonate Pattern 2 is likely a hydrate (~0.87 mol of water). It is named methanesulfonate Form I.

[0303] Methanesulfonate Pattern 2 (Form I) was heated to 120°C using DSC and characterized immediately. After dehydration upon heating, Pattern 2 quickly reabsorbed water, transforming into methanesulfonate Pattern 4 (Figures 30-31). Furthermore, repeated attempts to prepare methanesulfonate Pattern 2 failed, and methanesulfonate Pattern 4 was actually obtained. Therefore, it is inferred that methanesulfonate Pattern 2 (Form I) is a thermodynamically unstable crystalline form.

[0304] 2.5.2.3 Characterization of Methanesulfonate Pattern 3

[0305] The XRPD spectrum (Figure 32) shows that methanesulfonate Pattern 3 is a moderately crystalline crystal. The DSC curve (Figure 33) shows two endothermic peaks at 82℃ and 171℃, and the TGA curve shows a 2.2% weight loss between 21-120℃. The 1H NMR results (Figure 34) indicate that the free base forms a salt with methanesulfonic acid in a ratio of approximately 1:2, containing 0.02% ACN solvent residue. Based on these results, it is inferred that methanesulfonate Pattern 3 is likely a hydrate (~0.86 mol of water), and it is named methanesulfonate Form II.

[0306] Methanesulfonate Pattern 3 (Form II) was heated to 128°C by DSC and immediately characterized: Pattern 3 (Form II) quickly reabsorbed water after heating and dehydration, and the crystal form did not change (Figures 35-36).

[0307] DVS testing was performed on the scaled-up batch of methanesulfonate Pattern 3 (Form II, ET57141-27-P1), and the results are shown in Figure 37: Methanesulfonate Pattern 3 (Form II) exhibits hygroscopicity, showing a 6.72% weight gain upon water absorption at 80% RH. The XRPD pattern changed after DVS testing, revealing a new crystal form, which was named methanesulfonate Pattern 6 (Figure 38).

[0308] 2.5.2.4 Characterization of Methanesulfonate Pattern 4

[0309] Methanesulfonate Pattern 4 was obtained during a small-scale amplification process. The XRPD spectrum (Figure 39) shows that Methanesulfonate Pattern 4 is a moderately crystalline crystal. The DSC curve (Figure 40) shows two endothermic peaks at 96℃ and 181℃, and the TGA curve shows a weight loss of 2.9% between 21-75℃. The 1H NMR results (Figure 41) indicate that the free base reacts with methanesulfonic acid in a ratio of approximately 1:2 to form a salt, containing 0.11% MTBE solvent residue. Based on these results, it is inferred that Methanesulfonate Pattern 4 is likely a hydrate (~1.15 mol of water), and it is named Methanesulfonate Form III.

[0310] Methanesulfonate Pattern 4 (Form III) was heated to 135°C by DSC and immediately characterized: Pattern 4 quickly reabsorbed water after heating and dehydration, and the crystal form did not change (Figures 42-43).

[0311] DVS tests were performed on methanesulfonate Pattern 4 (Form III). The test results are shown in Figure 44: methanesulfonate Pattern 4 (Form III) exhibits hygroscopicity, showing a 6.78% weight gain upon water absorption at 80% RH. After the DVS test, the XRPD pattern changed to a mixed crystal of Pattern 4 and Pattern 2 (Figure 45).

[0312] 2.5.2.5 Characterization of Methanesulfonate Pattern 5

[0313] As shown in the XRPD spectrum (Figure 46), methanesulfonate Pattern 5 is a poorly crystallized crystal. The DSC curve (Figure 47) shows three endothermic peaks at 90℃, 132℃, and 176℃, and TGA shows a slow weight loss of 3.9% between 23-150℃. 1H NMR results (Figure 48) indicate that the free base forms a salt with methanesulfonic acid at a ratio of approximately 1:2.36, containing 0.22% heptane solvent residue. Characterization of methanesulfonate Pattern 5 by heating to 125℃ and 150℃ immediately using DSC reveals that Pattern 5 transforms into Pattern 4, but with poor crystallinity (Figures 49-50). Furthermore, methanesulfonate Pattern 5 is found to be unstable during storage, transforming into a mixed crystal of Pattern 4 and Pattern 5 after 7 days of storage at 2-8℃ (Figure 51). Based on these results, it is inferred that methanesulfonate Pattern 5 may be an unstable hydrate.

[0314] 2.5.2.6 Characterization of Methanesulfonate Pattern 6

[0315] Methanesulfonate Pattern 6 was obtained from methanesulfonate Pattern 3 (Form II) after DVS testing. The XRPD spectrum (Figure 52) shows that methanesulfonate Pattern 6 is a moderately crystalline crystal. The DSC curve (Figure 53) shows three endothermic peaks at 71℃, 129℃, and 186℃. TGA shows two weight loss segments at 23-55℃ and 55-110℃, with losses of 1.6% and 3.9%, respectively. Combined with the characterization results of Pattern 3, methanesulfonate Pattern 6 is likely a hydrate (~2.25 mol H2O). Immediately after heating methanesulfonate Pattern 6 to 55℃ with TGA or to 85℃ with DSC, XRPD testing showed no change in crystal form. However, after dehydration by heating to 115℃ with TGA and immediately followed by XRPD testing, the heated sample quickly absorbed water and transformed into methanesulfonate Pattern 4 (Figure 54).

[0316] 2.5.3 Characterization of fumarate

[0317] The obtained fumarate pattern 1 (Lot#ET57141-16-P5) was characterized. XRPD spectra (Figure 55) showed that the obtained fumarate pattern 1 was a poorly crystallized crystal. As shown in Figure 56, fumarate pattern 1 exhibited an endothermic peak at 197 °C, which should be a melting peak. TGA showed a weight loss of 0.75% between 25 and 120 °C. ¹H NMR spectra (Figure 57) showed that the free base formed a salt with fumaric acid at a ratio of approximately 1:0.6, with 1.23% ACN solvent residue. The poor crystallinity of fumarate pattern 1 and the inability to reproduce it suggest that incomplete salt formation was not possible.

[0318] 2.5.4 Summary of the obtained crystalline salts

[0319] A total of eight XRPD spectra of the three salts were found. A summary table is shown in Table 21.

[0320] Table 21 summarizes the characterization results of the obtained crystalline salts.

[0321] 2.6 Preparation of Salt Form

[0322] Based on the solid-state characterization results, 500-1000 mg of maleate Form I, methanesulfonate Form II, and Form III were prepared for comprehensive characterization, as well as solubility and stability testing of the dominant salt forms. The preparation conditions and characterization results are as follows:

[0323] 2.6.1 Maleate Form I

[0324] 501.65 mg of the active pharmaceutical ingredient (Lot#LHB-035A-132) was weighed and dissolved in 5 mL (10V) MEK solution. Then, 127.87 mg (1.1 eq.) of maleic acid was added, and the mixture was reacted at room temperature (16-18℃) for approximately 3 hours. No solid precipitate was observed. After adding 5 mg of maleate Form I (Lot#ET57141-3-P4) as seed crystals, the mixture was reacted under high and low temperature cycling for 1 day, filtered, and then vacuum dried at 50℃ for 4 hours for characterization.

[0325] High and low temperature cycling method: 1) Maintain temperature at -5℃ for 4 hours; 2) Increase temperature to 25℃ at 0.2℃ / min; 3) Maintain temperature at 25℃ for 4 hours; 4) Decrease temperature to -5℃ at 0.2℃ / min; Repeat the above method.

[0326] 377.70 mg of maleate Form I (ET57141-18-P1) was successfully prepared as an off-white solid with a yield of 61%. Specific information and characterization results are shown in Table 22 and Figures 58-63.

[0327] Table 22 Preparation and characterization results of maleate Form I (ET57141-18-P1)

[0328] The yield of maleate Form I prepared under the above conditions was low, only 61%. Therefore, optimization of the preparation conditions for maleate was attempted. Specific information is shown in Table 23.

[0329] Table 23 Optimization Experiment and Results of Preparation Conditions for Form I Maleate

[0330] 2.6.2 Methanesulfonate Form II

[0331] 799.88 mg of the active pharmaceutical ingredient (Lot#LHB-035A-132) was weighed and dissolved in 16 mL (20V) ACN. Then, 2.1 eq. (2.26 mL, ACN diluted 10 times) of methanesulfonic acid was added. The mixture was reacted at 50 °C for 1 day and then filtered. The resulting sample was then vacuum dried at 50 °C for 6 hours and characterized.

[0332] 817.2 mg of methanesulfonate Form II (Pattern 3, ET57141-27-P1) was successfully prepared as a yellow solid with a yield of 73.9%.

[0333] Table 24 Preparation and characterization results of methanesulfonate Form II (ET57141-27-P1)

[0334] 2.6.3 Methanesulfonate Form III

[0335] Initially, the aim was to prepare methanesulfonate Form I (Pattern 2) on a small scale using the method described in Lot#ET57141-13-P13. However, this was unsuccessful, and a new crystal form of methanesulfonate, Form III (Pattern 4), was ultimately obtained. The specific experimental methods are as follows:

[0336] 503.08 mg of the active pharmaceutical ingredient (Lot#LHB-035A-132) was weighed and added to a 25 mL reactor. It was dissolved in 5 mL (10V) MEK at -5°C, followed by the addition of 2.1 eq. (1.04 mL, MEK diluted to 2 mol / L) of methanesulfonic acid. The reaction was carried out under temperature cycling conditions for 1 and 2 days. Samples were taken for testing, revealing that the resulting product was a new crystalline form, Pattern 4, with low crystallinity and a relatively viscous consistency. The product quickly absorbed moisture and hardened after filtration. Therefore, a small amount of the suspension (Lot#ET57141-19-P1) was tested to optimize the product by adding a poor solvent. The optimized batches were Lot#ET57141-19-P2 and 19-P3. Details are shown in Table 25.

[0337] Temperature cycling method: 1) Maintain temperature at -5℃ for 4 hours; 2) Increase temperature to 25℃ at 0.2℃ / min; 3) Maintain temperature at 25℃ for 4 hours; 4) Decrease temperature to -5℃ at 0.2℃ / min; Repeat the above method.

[0338] Table 25 Optimization Experiment and Results of Methanesulfonate Preparation Conditions

[0339] Form III methanesulfonate was obtained in good condition by adding an antisolvent. Considering factors such as the product state, solvent residue, and crystallinity, the MTBE antisolvent method was adopted for the preparation of batch ET57141-19-P1. Samples were taken for testing during the MTBE addition process. The results are shown in Figure 72. The product obtained immediately after adding the antisolvent was Form I methanesulfonate (Pattern 2), but it eventually transformed into Form III methanesulfonate (Pattern 4) after 7.5 hours. Therefore, it is inferred that Form I methanesulfonate may be a thermodynamically unstable crystal form.

[0340] The sample was filtered after adding the antisolvent MTBE and continuing temperature cycling for 1 day, and then dried under vacuum at 50 °C for approximately 6.5 hours for characterization. 530.25 mg of methanesulfonate Form III (ET57141-19-P1), a yellow solid, was prepared with a yield of 85%. Specific information and characterization results are shown in Table 26 and Figure 72.

[0341] Table 26 Preparation and characterization results of methanesulfonate Form III (ET57141-19-P1)

[0342] Example 3: Preparation of maleate crystal form Form I

[0343] 3.1 Characterization of the active pharmaceutical ingredient shown in formula (II)

[0344] The materials manufactured by Wuhan WuXi AppTec New Drug Development Co., Ltd. were comprehensively characterized, and the results are summarized in Table 27. XRPD (Figure 77) and PLM (Figure 78) spectra showed that the received drug substance compound (Lot#EW34502-101-D-P5) of formula (II) had the same crystal form as the maleate Form I found in the salt type screening, and was an irregularly shaped crystal with good crystallinity. The DSC curve showed an endothermic peak at 175℃, and the TGA curve showed a weight loss of 2.0% at 100-170℃ (Figure 79). 1H-NMR (Figure 80) detected 0.41% EtOAc solvent residue.

[0345] Table 27 Characterization results of starting materials

[0346] 3.2 Visually assessed solubility

[0347] The solubility of compound (Lot#EW34502-101-D-P5) of formula (II) at room temperature was roughly determined visually in 14 solvents. The results are shown in Table 28 and Figure 81. Compound (II) showed good solubility in MeOH and THF (>170 mg / mL), but poor solubility in MTBE, IPAC, Cyclohexane, and Heptane (<1 mg / mL).

[0348] Table 28 shows the visual solubility results of the compounds represented by formula (II).

[0349] 3.3 Screening Results

[0350] 3.3.1 Using Form I as the starting material

[0351] 3.3.1.1 Suspension-to-crystal transformation

[0352] Based on the visual solubility results of the active pharmaceutical ingredient shown in Formula (II), suspension and crystallization were carried out in the selected solvents at 25°C and 50°C, respectively.

[0353] Approximately 30 mg of the active pharmaceutical ingredient (Lot#EW34502-101-D-P5) as shown in formula (II) was weighed into a sample vial, and 0.5 mL of the selected solvent was added. The resulting suspension was subjected to crystallization at 25°C for 3 days or at 50°C for 1 day, then filtered, dried, and characterized. The results are shown in Tables 29-30 and Figures 82-85. No new crystal form was obtained through this experiment.

[0354] Table 29 Results of suspension crystallization experiment at 50℃

[0355] Table 30 Results of suspension crystallization experiment at 25℃

[0356] 3.3.1.2 Antisolvent precipitation

[0357] 3.3.1.2.1 Positive Drop Experiment

[0358] Approximately 30 mg of the active pharmaceutical ingredient (Lot#EW34502-101-D-P5) of formula (II) was weighed into a sample vial and dissolved in 0.5 mL (~17V) of a good solvent at room temperature, followed by the addition of a poor solvent. The mixture was then stirred at 25°C for one day, filtered, dried, and characterized. The results are shown in Table 31 and Figures 86-87. No new crystal form was obtained through this experiment.

[0359] Table 31 Results of Antisolvent Precipitation (Positive Drop) Experiment

[0360] 3.3.1.2.2 Back-dropping experiment

[0361] Weigh an appropriate amount of the active pharmaceutical ingredient (Lot#EW34502-101-D-P5) as shown in formula (II) and dissolve it in a selected good solvent at room temperature (~25℃) or 50℃ to prepare a solution of 150~300mg / mL. Take 0.1 or 0.2 mL of the prepared solution and add it to a poor solvent at room temperature (~25℃). Then, stir at 25℃ for 2 days, filter the resulting suspension, dry it, and characterize it. The results are shown in Table 32 and Figure 88. No new crystal form was obtained through this experiment.

[0362] Table 32 Results of Antisolvent Precipitation (Reverse Dropping) Experiment

[0363] 3.3.1.3 Cooling crystallization

[0364] Single solvent cooling crystallization (P9-P11): Weigh approximately 30 mg of the active pharmaceutical ingredient (Lot#EW34502-101-D-P5) as shown in formula (II) into a sample vial, and add a certain volume of good solvent at 50°C to dissolve the solid. Filter and transfer the filtrate to -5°C.

[0365] Mixed solvent cooling crystallization (P12-P15): Weigh approximately 30 mg of the active pharmaceutical ingredient (Lot#EW34502-101-D-P5) as shown in formula (II) into a sample vial. Add a certain volume of antisolvent at 50°C, and then add a good solvent to dissolve the solid. Filter and transfer the filtrate to -5°C.

[0366] After stirring at -5℃ for 1 day, the suspension was filtered, dried, and characterized. The results are shown in Table 33 and Figure 89. No new crystal form was obtained through this experiment.

[0367] Table 33 Results of Cooling Crystallization Experiment

[0368] Note: The ET57523-36-P9 / P11 / P14 solution samples without precipitated solids were converted to evaporative crystallization. After evaporation for 3 / 4 days, all samples obtained were oily. The ET57523-36-P14 / P15 samples without precipitated solids were recovered and converted to antisolvent precipitation (reverse dropping) experiments.

[0369] 3.3.1.4 Evaporation and Crystallization

[0370] Weigh approximately 30 mg of the active pharmaceutical ingredient (Lot#EW34502-101-D-P5) as shown in Formula (II) into a sample vial. Dissolve and filter the vial in the selected solvent at room temperature (~25°C). Cover the filtrate with a sealing film, punch holes, and place it in a fume hood for slow evaporation. As shown in Table 34, the resulting product is an oily substance.

[0371] Table 34 Results of Evaporation Crystallization Experiment

[0372] 3.3.1.5 High and Low Temperature Cycling Experiment

[0373] Weigh approximately 30 mg of the active pharmaceutical ingredient (Lot#EW34502-101-D-P5) as shown in formula (II) into a sample vial, add 0.5 mL of the selected solvent, cycle at 50℃ to 5℃ for 3 days, filter the suspension and characterize it.

[0374] The temperature cycling method is as follows: ① Maintain a constant temperature of 50℃ for 2 hours → ② Decrease the temperature to 5℃ at a rate of 0.2℃ / min → ③ Maintain a constant temperature of 5℃ for 2 hours → ④ Increase the temperature to 50℃ at a rate of 0.2℃ / min. Repeat the above conditions.

[0375] The results are shown in Table 35 and Figure 90. No new crystal form was obtained through this experiment.

[0376] Table 35 Results of High and Low Temperature Cycling Experiments

[0377] 3.3.2 Using amorphous samples as starting materials

[0378] 3.3.2.1 Preparation of amorphous samples

[0379] Amorphous maleate product was prepared by grinding: 408.31 mg of active pharmaceutical ingredient (Form I, Lot#EW34502-101-D-P5) was manually dry-ground in a mortar, and XRPD analysis was performed as needed, as shown in Figure 91. After grinding for ~3 hours, 378.71 mg of amorphous sample (Lot#ET57523-38-P0) was successfully prepared. Yield: 92.8%

[0380] 3.3.2.2 Suspension-to-crystal transformation experiment at 50℃

[0381] Approximately 10 mg of the amorphous sample (Lot#ET57523-38-P0) was weighed into a sample vial, and 0.3 mL of the selected solvent was added. After stirring at 50 °C for 3 days, the sample was filtered and XRPD was performed. As shown in Table 36 and Figure 92, no new crystal form was obtained except for Form I.

[0382] Table 36 Results of suspension crystallization experiment at 50℃ - using amorphous material as starting material

[0383] Note: When the clear solution ET57523-39-P4 / P7 / P9 / P10 / P13 was transferred to 5℃ and stirred for 2 days, no solid precipitated.

[0384] 3.3.2.3 25℃ Suspension-to-Crystal Transformation Experiment

[0385] Approximately 10 mg of the amorphous sample (Lot#ET57523-38-P0) was weighed into a sample vial, and 0.3 mL of the selected solvent was added. After stirring at 25 °C for 3 days, the sample was filtered and XRPD was performed. As shown in Table 37 and Figures 93-94, no new crystal form was obtained except for Form I.

[0386] Table 37 Results of suspension crystallization experiment at 25℃ - using amorphous material as starting material

[0387] Note: After transferring the clear solution ET57523-39-P23 / P24 to 5℃ and stirring at 5℃ for 2 days, no solid precipitated.

[0388] Test Example 1: Grinding Experiment

[0389] Weigh 15-20 mg of maleate (Form I), mesylate (Form II), and form III) into a mortar and grind using both dry and wet methods. Grind manually for 5 minutes, and take samples at 1, 3, and 5 minutes for XRPD analysis. Specific information and results are shown in Tables 38-40 and Figures 95-100.

[0390] Table 38 Results of grinding experiments with maleate Form I

[0391] Table 39 Results of grinding experiments on Form II methanesulfonate

[0392] Table 40 Results of grinding experiments on Form III methanesulfonate

[0393] Test Example 2 Tablet Compression Experiment

[0394] ~30 mg of maleate Form I, mesylate Form II, and Form III were respectively subjected to tablet compression tests at a pressure of 30 MPa. The compressed samples were then subjected to XRPD analysis. The crystallinity of maleate Form I and mesylate Form II & Form III decreased after tablet compression. Furthermore, some samples of mesylate Form II & Form III showed a darker color (bright yellow) and a translucent surface after tablet compression. The experimental results are shown in Table 41 and Figures 101-103.

[0395] Table 41 Results of tablet compression test

[0396] Note: After tableting tests, some samples of mesylate Form II & Form III showed a darker color (bright yellow) and a semi-transparent surface.

[0397] Stability Study of Test Example 3

[0398] Take appropriate amounts of maleate Form I, methanesulfonate Form II and Form III respectively and spread them evenly in the sample bottle. Place them openly under five conditions for 7 days: 80℃ (3d), 60℃, 25℃ / 60%RH, 40℃ / 75%RH and 25℃ / 90±5%RH. Take samples on 3d and 7d respectively for HPLC and XRPD detection.

[0399] Stability studies have shown that:

[0400] 1) The crystal form of maleate remained unchanged under all five conditions mentioned above, but impurities (RRT = 1.29, MS) were observed at 60℃ and 80℃. +=601.3) Content increased.

[0401] 2) Methanesulfonate Form II turns into a yellow oily substance at 25℃ / 90±5%RH (3d, 7d); under two conditions of 25℃ / 60%RH and 40℃ / 75%RH, it crystallizes into methanesulfonate Form III at 3d and into methanesulfonate Pattern 6 at 7d.

[0402] 3) Form III methanesulfonate turned into a yellow oily substance at 25℃ / 90±5%RH (3d, 7d); its crystal form did not change under the other four conditions.

[0403] The results are summarized in Table 42 and Figures 104-109.

[0404] Table 42 Stability Test Results

[0405] (a) The purity values ​​above are the average of the three parallel experiments.

[0406] (b) Tested at 80℃ for 3 days; tested under the other four conditions for 7 days.

[0407] Test Example 4: Determination of the solubility of maleate crystal form Form I

[0408] Based on solid-state characterization, grinding, tableting, and stability results, the solubility of maleate Form I in water and biological solvents was determined only at 37°C, with an initial concentration of 5 mg / mL.

[0409] The specific procedure is as follows: Weigh approximately 15 mg of maleate Form I and add 3 mL each of SGF, FaSSIF, FeSSIF, and H2O. The resulting sample is stirred at 37°C in the dark for 24 hours. Samples are taken for analysis at 0.5 h, 2 h, and 24 h: the sample is drawn up with a syringe, filtered through a filter membrane, the first two drops are discarded, and a portion of the filtrate is diluted and subjected to HPLC analysis. Residual solids within the filter membrane are detected by XRPD. Three replicates are prepared for each sample.

[0410] Maleate Form I exhibits high solubility in SGF and H2O, both >5 mg / mL (24 h). It transforms into an adhesive in FaSSIF and FeSSIF. The results are summarized in Table 43.

[0411] Table 43 Solubility test results of maleate Form I in biological solvents and water

[0412] (a) The above values ​​are the mean of the three parallel experiments.

[0413] Test Example 5: Hygroscopicity Assessment of Maleate Crystal Form I

[0414] The hygroscopicity of the maleate crystal form Form I sample was evaluated using DVS. The hygroscopicity evaluation results showed that the maleate crystal form Form I sample had a weight gain of 0.39% upon absorbing water at 80% RH, indicating slight hygroscopicity.

[0415] Test Example 6: Pharmacokinetic Determination of Maleate Crystal Form I and Maleate Amorphous Samples

[0416] The maleate crystal form (Form I) and the amorphous maleate sample of compound (I) were subjected to in vivo drug metabolism experiments in rats.

[0417] 1. Drug preparation

[0418] Accurately weigh approximately 10 mg of the sample to be tested, add 10% of the converted total volume of DMSO to dissolve it, and then slowly add 90% of the total volume of 0.5% MC solvent while stirring. Sonicate and vortex to mix thoroughly to obtain a solution of the preparation that is considered to be homogeneous, with a concentration of 1 mg / mL. Prepare fresh immediately before use.

[0419] Pipe 0.2 mL of the sample into a 1.5 mL centrifuge tube and store at -80 °C for analysis of the concentration of the drug solution.

[0420] 2. Animal preparation

[0421] Animals were housed in rat cages and fasted for at least 10 hours starting the day before the experiment, but water was allowed. On the day of the experiment, each animal was weighed and marked on its tail. Blank blood samples were collected before drug administration. Blood was collected via tail vein.

[0422] 3. Administration

[0423] Route of administration: Oral administration (po)

[0424] Dosage concentration: 1 mg / ml

[0425] Dosage: 10 mg / kg

[0426] Dosage volume: 10 mL / kg

[0427] Procedure: Hold the rat upright with your left hand wearing a bite-proof glove, insert the gavage needle into the throat through the mouth, and insert the needle when you feel no obvious resistance. Then inject the drug into the stomach.

[0428] 4. Sample Collection

[0429] Whole blood (0.1-0.2 ml) was collected from test animals at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after drug administration. The blood was collected in EDTA-Na2 anticoagulant tubes, inverted 3-4 times to mix, and centrifuged at 2000g for 5 min at 4℃ to separate the supernatant plasma. The plasma was then promptly transferred to -80℃ for storage until analysis. Blood was collected via tail vein.

[0430] 5. Sample Analysis and Data Processing

[0431] 5.1 Sample Analysis

[0432] Using Shimadzu liquid chromatography and Triple Quad™ 6500 + An AB mass spectrometry method was established for the quantitative detection of analytes. The concentration of the parent drug in plasma was analyzed. The analytical results were subjected to variation control using quality control samples, with the accuracy of the quality control samples expected to be between 80% and 120%.

[0433] 5.2 Data Processing

[0434] The main pharmacokinetic parameters were calculated using a non-compartmental model in Winnonlin Phoenix software. These parameters included the area under the curve (AUC(0-t) and AUC(0-∞)) and the elimination half-life (T0). 1 / 2 ), maximum plasma concentration (C max ), time to reach maximum plasma concentration (T) max ))wait.

[0435] The pharmacokinetic data of the test samples obtained through the above tests are shown in Table 44.

[0436] Table 44 Pharmacokinetic data of maleate Form I

[0437] As shown in Table 44, compared with the amorphous maleate sample, the crystalline maleate Form I has superior pharmacokinetic properties.

[0438] Test Example 7: Maleate Crystal Form I Cell Anti-proliferation Experiment

[0439] I. Experimental Materials and Equipment:

[0440] Ovarian cancer cells A2780 and colorectal cancer cells WiDr were purchased from Beijing Beina Chuanglian Biotechnology Co., Ltd. DMEM medium (Bio-Channel), DMSO (dimethyl sulfoxide), MTT (thiazolyl blue), 0.25% EDTA-Tripsin (trypsin digestion solution), 1xPBS (phosphate buffer, pH 7.2), 96-well plates (Corning), fetal bovine serum (FBS), 10,000 U / mL penicillin-G / streptomycin, high-speed refrigerated centrifuge (EPPENDORF 5810R), and ELISA reader (Tecan Spark) were used.

[0441] II. Experimental Preparation:

[0442] 1. Cell plating

[0443] A) Tumor cells were cultured at 37°C, 5% CO2 and saturated humidity in DMEM (high glucose, containing 10% FBS and 100 U / mL penicillin-G / streptomycin) to a density of 80-90%.

[0444] B) Remove the culture medium from the 10cm petri dish;

[0445] C) Rinse the cells once with 10 ml of 1xPBS;

[0446] D) Add 4 ml of 0.25% EDTA-Tripsin and incubate at 37°C with 5% CO2 for 5 minutes to digest with trypsin. Transfer to a 15 ml centrifuge tube, centrifuge at 200g for 5 minutes, and discard the supernatant to obtain cell pellet.

[0447] E) Resuspend in 4 ml of DMEM medium, count and adjust to 50,000 cells / ml.

[0448] F) Add 100 μL of cell suspension to each well of a 96-well plate and incubate overnight at 37°C in a 5% CO2 incubator.

[0449] 2. Compound treatment

[0450] compound dilution

[0451] A) Preparation of serially diluted solutions for the test compounds: The maleate crystal form, Form I, was prepared in 1 mM stock solution. Then, 1.5 μl of the stock solution was dissolved in 1.5 ml of DMSO-free culture medium, followed by a 3-fold serial dilution with 0.1% DMSO culture medium, resulting in 9 concentrations. The concentrations of the diluted compounds are as follows:

[0452] 333.33nM, 111.11nM, 37.03nM, 12.35nM, 4.15nM, 1.37nM, 0.46nM, 0.15Nm

[0453] B) After thorough mixing, take 100 μL of the culture compound solution to replace the culture medium in the cell culture plate, with 4 replicates for each concentration;

[0454] C) Transfer the cells to an incubator and incubate for 3 days.

[0455] 3. MTT test

[0456] A) Remove the cell culture plate and add 10 μL of 5 mg / ml MTT in a biosafety cabinet;

[0457] B) Place the cell culture plate back into the incubator and continue incubation for 3 hours;

[0458] C) Remove the cell culture plate and the culture medium, add 100 μL of isopropanol (containing 0.4 mM HCl, 0.1% NP-40), and shake on a shaker at room temperature for 30 minutes;

[0459] D) Measure the absorbance value at a wavelength of 570 nm on the TECAN enzyme-linked immunosorbent assay (ELISA) instrument.

[0460] 4. Data Analysis

[0461] Calculate the %Cell Viability using the following formula:

[0462] %Cell Viability=100%×(Lum_Sample-Lum_LC) / (Lum_HC-Lum_LC)

[0463] Lum_HC:0.1% DMSO control group cell readings

[0464] Lum_Sample: Cell readings with added compounds

[0465] Lum_LC: Blank culture medium reading

[0466] The IC50 value was obtained by curve fitting using GraphPad Prism 8 software.

[0467] The cell viability data of maleate crystal form Form I obtained through the above tests are shown in Table 45.

[0468] Table 45

[0469] Based on the above cell anti-proliferation experiments, the cell activity data of maleate crystal form Form I in various cell lines are shown in Table 46.

[0470] Table 46

[0471] Table 46 shows that the maleate crystal form Form I of the compound has an effect on triple-negative breast cancer (TNBC) cell lines HCC70 and ER. + HER2 - The proliferation of 23 cell lines was inhibited, including breast cancer (BC) cell line T47D, BC palbociclib-resistant cell line xMCF-7 / palbo-R, non-small cell lung cancer (NSCLC) cell line A549, small cell lung cancer (SCLC) cell line NCI-H446, ovarian cancer (OVA) cell line OVCAR-3, colon cancer (CRC) cell line WiDr, pancreatic cancer (PDAC) cell line PANC-1, myeloid monocytic leukemia (HAMLC) cell line MV-4-11, and mantle lymphoma (MCL) cell line Mino.

[0472] In summary, this invention employed various pharmaceutically acceptable acid pairs with amorphous free bases of compound (I) for salt form screening. Multiple methods, including slow evaporation, dissolution crystallization, and suspension-to-crystallization, were used to obtain eight XRPD spectra for three salts: maleate (1), methanesulfonate (6), and fumarate (1). Anhydrous maleate (Form I) and hydrated methanesulfonate (Form II and Form III) were the dominant crystal forms. Based on the physicochemical property evaluation results of the salt form screening study, anhydrous maleate (Form I) exhibited better solid-state properties, solubility, and crystal form reproducibility; therefore, maleate was selected for further crystal form screening.

[0473] The inventors investigated the solid morphology of the maleate salt of compound (I) and screened for polymorphs of maleate salt of compound (I) in 14 solvent systems. Experiments were conducted using methods such as suspension crystallization (50℃ / 25℃), antisolvent precipitation, cooling crystallization, evaporation crystallization, and high and low temperature cycling. No new crystal forms other than maleate salt form Form I were found.

[0474] To further investigate the potential crystal forms of maleate of formula (I), an amorphous form of maleate of formula (I) was prepared by dry grinding and experiments were conducted, but no new crystal forms were found.

[0475] In summary, the stable crystal form of compound (Ⅰ) is maleate crystal form Form I.

[0476] Therefore, maleate Form I has good crystallinity, good solubility and slight hygroscopicity (0.39% / 80% RH), and is available in most solvents, thus maleate Form I has relevant properties that are acceptable for further development.

[0477] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A maleate salt crystalline form of a compound of Formula (I) Form I, characterized by, The maleate salt of the compound of formula (I) has the structure of formula (II), Further, the X-ray powder diffraction pattern of the maleate salt Form I has characteristic peaks at 2-theta values of 9.13±0.2°, 15.57±0.2°, 16.05±0.2°, 16.78±0.2°, 18.39±0.2°, 19.57±0.2°, 21.21±0.2°, 22.56±0.2°, 24.21±0.2°, 25.62±0.2°.

2. The maleate salt crystalline Form I of claim 1, characterized by, Further, the X-ray powder diffraction pattern of the maleate salt Form I has characteristic peaks at 2-theta values of 7.98±0.2°, 8.26±0.2°, 9.13±0.2°, 13.50±0.2°, 15.57±0.2°, 16.05±0.2°, 16.78±0.2°, 17.27±0.2°, 18.39±0.2°, 18.77±0.2°, 19.57±0.2°, 21.03±0.2°, 21.21±0.2°, 22.56±0.2°, 24.21±0.2°, 24.42±0.2°, 24.75±0.2°, 25.39±0.2°, 25.62±0.2°, 27.92±0.2°.

3. The maleate salt crystalline Form I of claim 1, characterized by, The X-ray powder diffraction pattern of the maleate salt Form I is substantially as shown in Figure 1.

4. The maleate salt crystalline Form I of claim 1, characterized by, The maleate salt Form I is an anhydrous crystal form.

5. The maleate salt crystalline Form I of claim 1, characterized by, The DSC curve of the maleate salt Form I has an endothermic peak at 170-185°C.

6. The maleate salt crystalline Form I of claim 1, characterized by, The maleate salt Form I has a water uptake weight gain of ≤0.5% at 80% RH.

7. A process for preparing the maleate salt crystalline form of claim 1, Form I, characterized in that, The method comprises the following steps: 1) dissolving the compound of formula (I) in a good solvent to obtain a first solution; 2) adding maleic acid to the first solution for a first time period, and then optionally adding the maleate salt Form I for a second time period; 3) adding a poor solvent dropwise to the product of step 2) to crystallize, filter and dry to obtain the maleate salt Form I of claim 1.

8. A pharmaceutical composition, characterized by, A pharmaceutical composition comprising a safe and effective amount of the maleate salt Form I of claim 1 and a pharmaceutically acceptable carrier.

9. Use of the crystalline form of maleate salt of claim 1 characterized in that, A medicament for modulating CDK7 kinase activity or preventing and / or treating a CDK7 related disease.

10. Use according to claim 9, characterized in that, The CDK7 related disease is selected from the group consisting of inflammation, cancer, cardiovascular disease, infection, immunological disease, metabolic disease. The CDK7 related disease is selected from the group consisting of inflammation, cancer, cardiovascular disease, infection, immunological disease, metabolic disease.

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