Crystal form of thiadiazolylpyrimidine compounds and methods of making the same

Abstract

The application discloses three crystal forms of a thiophene pyrimidine compound A and a preparation method of the three crystal forms, wherein the crystal form I is a 3-hydrochloride salt, the crystal form II and the crystal form III are 2-hydrochloride salts, the three crystal forms have good stability and solubility, have a solubility of greater than or equal to 4 mg / mL in 5% glucose aqueous solution, and meet the basic requirement of solubility of a compound for preparation of a freeze-dried preparation for injection. The three crystal forms of the thiophene pyrimidine compound A prepared by the application have good stability and solubility, can be prepared into a freeze-dried preparation for clinical injection, and are used for effective treatment of patients with lymphoma, myeloma and lymphatic leukemia in the late stage, drug resistance and the like.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the crystal forms of thiophene pyrimidine compounds and their preparation methods. Background Technology

[0002] Hematologic malignancies have a high incidence rate; in the United States alone, approximately 184,720 new cases of hematologic malignancies were diagnosed in 2023. New cases of leukemia, lymphoma, and myeloma accounted for approximately 9.4% of all new cancer diagnoses in the US in 2023. Specifically, leukemia accounted for 32%, lymphoma for 48%, and myeloma for 19%. It is estimated that 1,629,474 people in the US have or are recovering from hematologic malignancies, including leukemia, lymphoma, myeloma, myelodysplastic syndromes, and myeloproliferative neoplasms (Leukemia and Lymphoma Society 2023). Statistics from the Chinese Anti-Cancer Association show that approximately 84,000 new lymphoma patients are diagnosed in my country each year, with over 47,000 deaths, and this number is increasing at a rate of 5% annually. In addition, approximately 10,000-15,000 new myeloma patients and over 20,000 new cases of acute and chronic lymphocytic leukemia are diagnosed in my country each year. Currently, there are no effective drugs for patients with advanced relapsed or drug-resistant lymphoma, myeloma, and lymphocytic leukemia.

[0003] Phosphoinositol 3-kinase (PI3K) and histone deacetylase (HDAC) are important targets for tumor cell survival. HDAC inhibitors exert inhibitory effects on multiple targets of tumor cell messengers through epigenetic regulatory mechanisms. PI3K inhibitors and HDAC inhibitors have significant anti-cancer effects, which have been clinically validated (Ho, T et al., Journal of Medicinal Chemistry 63, 12460-12484, 2020; Zhang, M et al., Chemical Science 11, 5855-5865, 2020; Vanhaesebroeck, B et al., Nature Reviews Drug Discovery 20, 741-769, 2021). Several known inhibitors of phosphoinositol 3-kinase and histone deacetylase, including Copanisib, Alpelisib, Idelalisib, Voronostat, and Belinostat, which have been approved by the US FDA, cannot simultaneously inhibit phosphoinositol 3-kinase and histone deacetylase, and are less effective in treating refractory or relapsed hematologic malignancies, thus failing to meet the increasing clinical needs.

[0004] Thiophene pyrimidine compounds (Compound A) are dual-target inhibitors of HDAC (histone deacetylase) and PI3K (phosphoinositol 3-kinase). By selectively inhibiting the synergistic tumor cell messenger core protein kinase target PI3K and the epigenetic target HDAC, they disrupt the tumor cell messenger network, thereby exerting a potent killing effect on various tumor cells. This inhibitor has shown strong and effective inhibition of tumor growth in various hematologic and solid xenograft tumor animal models, particularly in various hematologic B-cell malignancies, and its safety evaluation experiments have demonstrated good safety. It can be used for the effective treatment of patients with advanced relapsed or drug-resistant lymphoma, myeloma, and lymphocytic leukemia. Its structural formula is as follows:

[0005]

[0006] However, this compound has low solubility, and when prepared as an injectable clinical formulation, it may cause drug precipitation. Summary of the Invention

[0007] Therefore, the purpose of this invention is to provide stable crystal forms of thiophene pyrimidine compounds to improve their solubility.

[0008] To achieve the above-mentioned objectives, the present invention includes the following technical solutions.

[0009] On the one hand, the present invention provides a crystal form I of a thiophene pyrimidine compound, represented by a 2θ angle, which has characteristic peaks at 4.77°, 9.52°, 14.28°, 21.12°, 23.66°, 25.21° and 28.67° in the X-ray powder diffraction pattern, with an error of ±0.2°;

[0010] The structural formula of the thiophene pyrimidine compound is as follows:

[0011]

[0012] In some embodiments, represented by a 2θ angle, the crystalline form I of the thiophene pyrimidine compound exhibits characteristic peaks in its X-ray powder diffraction pattern at 4.77°, 7.06°, 7.32°, 9.52°, 10.58°, 14.28°, 18.45°, 18.84°, 20.86°, 21.12°, 21.70°, 22.40°, 23.66°, 25.21°, 25.92°, 26.88°, 28.17°, 28.67°, 28.99°, 29.78°, 30.49°, and 32.87°, with an error of ±0.2°.

[0013] In some embodiments, represented by a 2θ angle with an error of ±0.2°, the characteristic peaks of crystal form I of the thiophene pyrimidine compound in the X-ray powder diffraction pattern and the relative intensities of each characteristic peak are as follows:

[0014]

[0015]

[0016] In some embodiments, the X-ray powder diffraction pattern of crystal form I of the thiophene pyrimidine compound is as follows: Figure 1-1 As shown.

[0017] In some embodiments, the differential scanning calorimetry (DSC) curve of crystal form I of the thiophene pyrimidine compound includes an exothermic peak at 220.9 ± 0.5 °C.

[0018] In some embodiments, the differential scanning calorimetry (DSC) curves of crystal form I of the thiophene pyrimidine compound are as follows: Figure 2 As shown.

[0019] Secondly, the present invention provides a crystal form II of a thiophene pyrimidine compound, denoted by a 2θ angle, which has characteristic peaks at 5.57°, 19.28°, 21.85°, 22.51°, 23.02°, 24.10°, 24.88°, 26.17°, 27.98° and 29.50° in its X-ray powder diffraction pattern, with an error of ±0.2°;

[0020] The structural formula of the thiophene pyrimidine compound is as follows:

[0021]

[0022] In some embodiments, represented by a 2θ angle, the crystalline form II of the thiophene pyrimidine compound exhibits characteristic peaks in its X-ray powder diffraction pattern at 5.57°, 10.73°, 12.33°, 14.77°, 17.53°, 19.28°, 21.29°, 21.85°, 22.51°, 23.02°, 23.69°, 24.10°, 24.88°, 25.48°, 26.17°, 27.68°, 27.98°, 29.50°, and 29.93°, with an error of ±0.2°.

[0023] In some embodiments, represented by a 2θ angle with an error of ±0.2°, the characteristic peaks of crystal form II of the thiophene pyrimidine compound in the X-ray powder diffraction pattern and the relative intensities of each characteristic peak are as follows:

[0024]

[0025]

[0026]

[0027] In some embodiments, the X-ray powder diffraction pattern of crystal form II of the thiophene pyrimidine compound is as follows: Figure 3 As shown.

[0028] In some embodiments, the differential scanning calorimetry (DSC) curves of crystal form II of the thiophene pyrimidine compound include endothermic peaks at 79.5 ± 0.5 °C, 141.9 ± 0.5 °C, and 207.7 ± 0.5 °C.

[0029] In some embodiments, the differential scanning calorimetry (DSC) curves of crystal form II of the thiophene pyrimidine compound are as follows: Figure 4 As shown.

[0030] Thirdly, the present invention provides a crystalline form III of a thiophene pyrimidine compound, represented by a 2θ angle, which has characteristic peaks at 10.47°, 15.67°, 21.23°, 21.44°, 22.45°, 23.06°, 25.73°, and 29.63° in its X-ray powder diffraction pattern, with an error of ±0.2°;

[0031] The structural formula of the thiophene pyrimidine compound is as follows:

[0032]

[0033] In some embodiments, represented by a 2θ angle, the crystalline form III of the thiophene pyrimidine compound exhibits characteristic peaks in its X-ray powder diffraction pattern at 10.47°, 14.72°, 15.28°, 15.67°, 16.95°, 18.79°, 20.55°, 21.23°, 21.44°, 22.45°, 22.71°, 23.06°, 24.73°, 25.73°, 27.77°, 27.99°, 29.63°, 30.14°, and 31.25°, with an error of ±0.2°.

[0034] In some embodiments, represented by a 2θ angle with an error of ±0.2°, the characteristic peaks of crystal form III of the thiophene pyrimidine compound in the X-ray powder diffraction pattern and the relative intensities of each characteristic peak are as follows:

[0035]

[0036]

[0037] In some embodiments, the X-ray powder diffraction pattern of crystal form III of the thiophene pyrimidine compound is as follows: Figure 5 As shown.

[0038] In some embodiments, the differential scanning calorimetry (DSC) curve of crystal form III of the thiophene pyrimidine compound includes endothermic peaks at 96.1 ± 0.5 °C and 213.4 ± 0.5 °C.

[0039] In some embodiments, the differential scanning calorimetry (DSC) curves of crystal form III of the thiophene pyrimidine compound are as follows: Figure 6 As shown.

[0040] Fourthly, the present invention provides a method for preparing crystal form I of the thiophene pyrimidine compound, comprising the following steps:

[0041] The mixture of methanol, purified compound A and water was stirred evenly, and hydrogen chloride / methyl tert-butyl ether solution was slowly added at a temperature of 0℃-10℃. The mixture was then stirred at a temperature of 0℃-10℃ for 15-25 hours, filtered, washed and dried to obtain crystal form I of the thiophene pyrimidine compound.

[0042] The slow addition of the hydrogen chloride / methyl tert-butyl ether solution takes 2-3 hours.

[0043] The concentration of hydrogen chloride in the hydrogen chloride / methyl tert-butyl ether solution is 4 mol / L-6 mol / L;

[0044] The structural formula of compound A is as follows:

[0045]

[0046] In some embodiments, the method for preparing crystal form I of the thiophene pyrimidine compound includes the following steps:

[0047] A mixture of methanol, purified compound A, and water was stirred at 20°C-30°C for 1.5-2.5 hours. A solution of hydrogen chloride / methyl tert-butyl ether was slowly added at 4°C-6°C, and then stirred at 4°C-6°C for 18-22 hours. The mixture was then filtered, washed, and dried to obtain crystal form I of the thiophene pyrimidine compound.

[0048] In some embodiments, the mass ratio of methanol to water is 100-130:1, preferably 105-125:1; preferably 110-120:1; preferably 114-118:1.

[0049] In some embodiments, the mass ratio of the purified compound A to methanol is 1:11-14.

[0050] In some embodiments, the mass ratio of the purified compound A to methanol is 1:12-13.

[0051] In some embodiments, the mass ratio of the purified compound A to the hydrogen chloride / methyl tert-butyl ether solution is 1:2-5.

[0052] In some embodiments, the mass ratio of the purified compound A to the hydrogen chloride / methyl tert-butyl ether solution is 1:3-4.

[0053] In some embodiments, the purification method for the purified compound A includes the following steps:

[0054] Dimethyl sulfoxide and compound A were stirred at 45℃-55℃ for 1.5-2.5 hours. Methanol and compound A seed crystals were added for the first time at 45℃-55℃, and the mixture was stirred for 1.5-2.5 hours. Methanol was added for the second time, and the mixture was stirred for 1.5-2.5 hours. The mixture was then cooled to 20℃-30℃ and stirred for 1.5-2.5 hours. The mixture was then filtered, washed, and dried to obtain purified compound A.

[0055] In some embodiments, the purification method for the purified compound A is as follows:

[0056] Dimethyl sulfoxide and compound A were stirred at 48℃-52℃ for 1.8-2.2 hours. Methanol and compound A seed crystals were added for the first time at 48℃-52℃, and the mixture was stirred for 1.8-2.2 hours. Methanol was added for the second time, and the mixture was stirred for 1.8-2.2 hours. The mixture was then cooled to 22℃-27℃ and stirred for 1.8-2.2 hours. The mixture was then filtered, washed, and dried to obtain purified compound A.

[0057] In some embodiments, the mass ratio of dimethyl sulfoxide to compound A is 4-9:1, preferably 5-8:1; more preferably 6-7:1.

[0058] In some embodiments, the mass ratio of methanol added initially to compound A is 1-2:1.

[0059] In some embodiments, the mass of the added compound A seed crystals is 0.4%-0.5% of the mass of compound A.

[0060] In some embodiments, the mass ratio of methanol added a second time to compound A is 12-20:1, preferably 14-18:1, and more preferably 15-17:1.

[0061] Fourthly, the present invention provides a method for preparing crystal form II of the thiophene pyrimidine compound, comprising the following steps:

[0062] Crystal form I of the thiophene pyrimidine compound was added to a mixed solution of ethanol and water, stirred at 20°C-30°C for 72-120 hours, filtered, washed, and dried to obtain crystal form II of the thiophene pyrimidine compound.

[0063] In some embodiments, the stirring time is 84-108 hours, preferably 90-102 hours, preferably 94-98 hours, and preferably 95-97 hours.

[0064] In some embodiments, the volume ratio of ethanol to water is 2-4:1, preferably 2.5-3.5:1.

[0065] In some embodiments, the ratio of the ethanol and water mixture to crystal form I of the thiophene pyrimidine compound is 8-12 mL: 1 g, preferably 9-11 mL: 1 g.

[0066] Sixthly, the present invention provides a method for preparing crystal form III of the thiophene pyrimidine compound, comprising the following steps:

[0067] Methanol was added to crystal form I of the thiophene pyrimidine compound, and the mixture was stirred at 20°C-30°C for 8-12 minutes, then stirred at 45°C-55°C for 25-35 minutes. The mixture was then cooled to 0°C-10°C and stirred for 2-4 hours. After filtration, washing, and drying, crystal form III of the thiophene pyrimidine compound was obtained.

[0068] In some embodiments, the method for preparing crystal form III of the thiophene pyrimidine compound includes the following steps:

[0069] Methanol was added to crystal form I of the thiophene pyrimidine compound, and the mixture was stirred at 22°C-27°C for 9-11 minutes, then stirred at 48°C-52°C for 28-32 minutes. The mixture was then cooled to 4°C-6°C and stirred for 2.5-3.5 hours. After filtration, washing, and drying, crystal form III of the thiophene pyrimidine compound was obtained.

[0070] In some embodiments, the ratio of crystal form I of the thiophene pyrimidine compound to methanol is 1g:16-25mL, preferably 1g:18-22mL, and more preferably 1g:19-21mL.

[0071] In some embodiments, the cooling rate to 0°C-10°C is 0.08-0.12°C / minute.

[0072] This invention, through process optimization, prepared three crystal forms of thiophene pyrimidine compound A, where crystal form I is a 3-hydrochloride salt, and crystal forms II and III are 2-hydrochloride salts. All three crystal forms exhibit good stability and solubility, with a solubility of ≥4 mg / mL in a 5% glucose aqueous solution, meeting the basic solubility requirements for preparing lyophilized injectable formulations. Crystal forms I and III show significantly higher solubility in 5% glucose solution than crystal form II, which improves the dissolution rate and enhances the stability of the drug solution before lyophilization, preventing drug precipitation even after prolonged storage. These crystal forms are more suitable for preparing lyophilized injectable formulations and are considered superior crystal forms.

[0073] The three crystal forms of thiophene pyrimidine compound A prepared by this invention have good stability and solubility, and can be prepared into lyophilized formulations for clinical injection, for the effective treatment of patients with lymphoma, myeloma and late-stage relapse of lymphocytic leukemia or those who are drug-resistant.

[0074] Crystal form I of the present invention can be prepared from the free base of compound A in a methanol / water / hydrogen chloride / methyl tert-butyl ether solution system; crystal form II can be prepared from crystal form I by pulping in EtOH / water; and crystal form III can be prepared from crystal form I by slow cooling in MeOH. The preparation processes of the three crystal forms are very simple and suitable for industrial production. Attached Figure Description

[0075] Figure 1-1 The XRPD pattern of crystal form I of compound (I) prepared by the method in Example 1 is shown.

[0076] Figure 1-2 The XRPD pattern of crystal form I of compound (I) prepared by method 1 in Comparative Example 1 is shown.

[0077] Figure 1-3 The XRPD pattern of compound (I) solid prepared by method 2 in Comparative Example 1.

[0078] Figure 2 The image shows the DSC spectrum of crystal form I of compound (I) prepared by the method in Example 1.

[0079] Figure 3 The XRPD pattern of crystal form II of compound (I) prepared by the method in Example 2 is shown.

[0080] Figure 4 The DSC spectrum of crystal form II of compound (I) prepared by the method in Example 2 is shown.

[0081] Figure 5 The XRPD pattern of crystal form III of compound (I) prepared by the method in Example 3 is shown.

[0082] Figure 6The DSC spectrum of crystal form III of compound (I) prepared by the method in Example 3 is shown. Detailed Implementation

[0083] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments are merely illustrative of the present invention and should not be construed as limiting the invention.

[0084] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0085] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0086] In this invention, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0087] The following are specific examples.

[0088] In the following examples, the operating parameters for X-ray powder diffraction (XRPD) analysis are set as follows:

[0089] Tube:Cu:K-Alpha

[0090] Generator: Voltage: 40kV; Current: 40mA.

[0091] Scan Scope: 3 to 40 degrees;

[0092] Sample rotation speed: 15 rpm.

[0093] Scanning rate:10deg / min or others.

[0094] The method used in differential scanning calorimetry (DSC) is as follows:

[0095] The samples in the perforated aluminum pot were tested under nitrogen protection at a rate of 50 mL / min, heating from 25 °C to 250 °C at a rate of 10 °C / min.

[0096] The method for determining chloride ion content is as follows:

[0097] Adopting the General Rules of the 2020 Edition of the Chinese Pharmacopoeia <0701> The chlorine content of each crystal form of compound (I) was determined by potentiometric titration, and the results were calculated using the gravimetric content correction.

[0098] Example 1. Preparation of crystal form I of compound (I)

[0099]

[0100] Compound (I) can be prepared from compound A, which is prepared according to the synthesis method in Chinese invention patent CN104292242B.

[0101] Purification of Compound A: Dimethyl sulfoxide (56.8 kg) and Compound A (8.7 kg) were added to a reaction vessel. The reaction vessel temperature was adjusted to 50°C and stirred for 2 hours until the material was completely dissolved. The solution was transferred to a crystallization vessel through a pipeline filter. At 50°C, methanol (13 kg) and Compound A seed crystals (0.04 kg) were added to the crystallization vessel and stirred for 2 hours. Methanol (139 kg) was added over 5 hours at a rate not exceeding 30 kg / hour, and stirred for 2 hours. The reaction vessel temperature was then lowered to 25°C over 5 hours at a rate of 5°C / hour, and stirred for 2 hours. The solid was obtained by filtration, washed three times with methanol, and dried to obtain 8.39 kg of purified Compound A.

[0102] Preparation of compound (I) crystal form I: Methanol (104.4 kg), the dried product of compound A obtained above (8.39 kg), and water for injection (0.9 kg) were added to a reaction vessel. The temperature of the reaction vessel was adjusted to 25°C and stirred for 2 hours. The temperature of the reaction vessel was adjusted to 5°C, and a hydrogen chloride / methyl tert-butyl ether solution (5 mol / L concentration, 31 kg) was slowly added (about 12 kg / hour). The mixture was stirred at 5°C for 20 hours. The solid was obtained by filtration. The solid was washed three times with methyl tert-butyl ether and dried to obtain 9.14 kg of compound (I) crystal form I solid, with a molar yield of 89.7% and a chlorine content of 17%.

[0103] The XRPD pattern of crystal form I of compound (I) is shown in [reference needed]. Figure 1-1 The peak positions are listed in Table 1; the DSC spectrum is shown in [reference needed]. Figure 2 .

[0104] Table 1

[0105]

[0106]

[0107] Preparation of crystal forms of compound (I) in Comparative Example 1 (crystal forms are not constant)

[0108] Operation 1: After cooling the purified compound A (8.78 kg) in a suspension of methanol (126 kg) and dichloromethane (290 kg) to 0-10 °C, add 32 kg of 2.2 M hydrochloric acid / methanol solution below 10 °C. Stir until the solid is completely dissolved, then filter and transfer the filtrate to a clean room. Add 140 kg of methyl tert-butyl ether dropwise at 5-10 °C with stirring. Continue stirring at this temperature for 7 hours after the addition is complete. After filtration, wash the resulting solid with 44 kg of methyl tert-butyl ether to obtain a wet product (11.34 kg). Dry the wet product under vacuum at 40-50 °C for 20 hours, then under vacuum at 50-60 °C for 30 hours until the moisture content is ≤5%, yielding a pale yellow solid compound (I) (10.04 kg, yield 94.1%, chlorine content 16%). Its XRPD spectrum is shown below. Figure 1-2 It is crystal form I.

[0109] Step 2: Take the purified compound A (600g) and repeat the preparation method of Step 1 to obtain a pale yellow solid compound (I) (594g, yield 78.2%, chlorine content 16%), whose XRPD spectrum is shown in [Figure 1]. Figure 1-3 It is an unknown crystal form.

[0110] The preparation methods of Example 1 and Comparative Example 1 were repeated, and the impurity content and crystal form of multiple batches of crystalline solids prepared were tested. The method for detecting the impurity content is as follows:

[0111] The purity, content, and related substances of compound I were determined by reversed-phase high-performance liquid chromatography (HPLC). The content was calculated by external standard method, and the content of individual impurities was calculated by principal component external standard method with correction factor.

[0112] 1. Chromatographic conditions

[0113] Column: Agilent Zorbax SB-C18 (150 mm × 4.6 mm, 3.5 μm); Mobile phase A: trifluoroacetic acid-water (0.05:100), Mobile phase B: trifluoroacetic acid-acetonitrile (0.05:100); Detection wavelength: 250 nm; Flow rate: 1.5 ml / min; Injection volume: 5 μl; Column temperature: 30 ℃; Injection tray temperature: 15 ℃; Run time: 50 min.

[0114] Elution gradient:

[0115]

[0116] Diluent:

[0117] Diluent 1: Acetonitrile-water (50:50); Diluent 2: Acetonitrile; Diluent 3: Pure water.

[0118] Wash solution for injections: acetonitrile-water (50:50).

[0119] 2. Solution preparation

[0120] (1) Diluent 1 / Blank Solution: Acetonitrile-Water (50:50): Mix 500ml of acetonitrile and 500ml of water thoroughly. Label this solution as "BLK".

[0121] Diluent 2: Acetonitrile

[0122] Diluent 3: Pure water

[0123] (2) Preparation of the content reference solution (0.44 mg / ml of compound I solution)

[0124] Prepare two parallel solutions. Accurately weigh approximately 22 mg of compound I reference standard and place them in 50 mL volumetric flasks. First, add 25 mL of diluent 2 to dissolve the compound, sonicate for 5 minutes, allow to stand at room temperature, then dilute to the mark with diluent 3, mix well, sonicate again to dissolve, and cool to room temperature. Label these solutions as Reference Solution-1 (A-STD-1) and Reference Solution-2 (A-STD-2).

[0125] (3) Related substance reference solution (0.022 mg / ml of compound I solution)

[0126] Prepare two parallel solutions. Transfer 5 ml of the concentration reference solution (A-STD-1) to a 100 ml volumetric flask. Dissolve and dilute to the mark with diluent 1, and mix well. Label them as impurity reference solution-1 (R-STD-1) and impurity reference solution-2 (R-STD-2).

[0127] (4) Related substances test solution (2.2 mg / ml of compound I solution)

[0128] Prepare two parallel test solutions. Accurately weigh approximately 55 mg of the test sample and place it in a 25 ml volumetric flask. First, add 12.5 ml of diluent 2 to dissolve the sample, sonicate for 5 minutes, allow to stand at room temperature, then dilute to the mark with diluent 3, mix well, sonicate again to dissolve, and cool to room temperature. Label these solutions as Related Substances Test Solution-1 (R-SPL-1) and Related Substances Test Solution-2 (R-SPL-2).

[0129] (5) Content test solution (0.44 mg / ml of compound I solution)

[0130] Prepare two parallel test solutions. Accurately transfer 5 ml of R-SPL-1 test solution into a 25 ml volumetric flask. Dissolve and dilute to the mark with diluent 1, and mix well. Label them as test solution-1 (A-SPL-1) and test solution-2 (A-SPL-2).

[0131] 3. Calculation method:

[0132] content:

[0133] Calculate the content of the test sample using the following formula:

[0134] Content (%, w / w) = (V_SPL × A_SPL) / (W_SPL × RF) × 100%

[0135] In the formula:

[0136] ASPL represents the peak area of ​​compound I in the test solution.

[0137] WSPL represents the sample weight (mg) of compound I in the test solution.

[0138] VSPL represents the dilution volume (ml) of the test solution.

[0139] RF represents the response factor of compound I.

[0140] Calculate the weight content of free base of compound I in the test sample using the following formula:

[0141] Free base weight content (%, w / w) = weight content (%, w / w) × M_F / M_S

[0142] In the formula:

[0143] ASPL represents the peak area of ​​compound I in the test solution.

[0144] WSPL represents the sample weight (mg) of compound I in the test solution.

[0145] VSPL represents the dilution volume (ml) of the test solution.

[0146] RF represents the response factor of compound I.

[0147] MS represents the molecular weight of compound I, 616.95.

[0148] MF represents the molecular weight of compound A, 507.57.

[0149] Calculate the weight content (on a dried basis) of the test sample using the following formula:

[0150] Weight content (on dried basis) (%, w / w) = (V_SPL × A_SPL) / (W_SPL × RF × (100% - KF - RS)) × 100%

[0151] In the formula:

[0152] ASPL represents the peak area of ​​compound I in the test solution.

[0153] WSPL represents the sample weight (mg) of compound I in the test solution.

[0154] VSPL represents the dilution volume (ml) of the test solution.

[0155] RF represents the response factor of compound I.

[0156] KF represents the water content (%) of the test sample of compound I.

[0157] RS represents the total residual solvent in Compound I (the calculated solvent percentage not less than LOQ) (%).

[0158] Calculate the free alkali content (on a dried basis) of the test sample using the following formula:

[0159] Free alkali weight content (on dried basis) (%, w / w) = (Free alkali weight content (%, w / w)) / ((100% - KF - RS)) × 100%

[0160] In the formula:

[0161] ASPL represents the peak area of ​​compound I in the test solution.

[0162] WSPL represents the sample weight (mg) of compound I in the test solution.

[0163] VSPL represents the dilution volume (ml) of the test solution.

[0164] RF represents the response factor of compound I.

[0165] KF represents the water content (%) of the test sample of compound I.

[0166] RS represents the total residual solvent in Compound I (the calculated solvent percentage not less than LOQ) (%).

[0167] (1) Impurity content

[0168] Compare the chromatogram of the test sample with the blank chromatogram. Calculate the single impurity for peaks with an integral area of ​​not less than 0.02% (LOD) using the following formula:

[0169] Single hybrid = (V_S × A_S) / (W_S × RF_AVE × RRF) × 100%

[0170] In the formula:

[0171] AS represents the peak area of ​​impurities in the related substance test solution.

[0172] WS represents the weight (mg) of the related substance test sample.

[0173] RFAVE represents the average response factor of compound I in six consecutive injections of R-STD-1 solution.

[0174] VS represents the dilution volume (ml) of the related substance test solution.

[0175] RRF represents the relative response factor for each impurity, with K-acid at 1.23, K-ester at 1.16, and other unspecified impurities at 1.00.

[0176] (2) Total impurity content

[0177] Sum all individual impurities of 0.05% or higher.

[0178] (3) Purity

[0179] Purity of Compound I = 100% - Total Impurities

[0180] The test results are compared in Table 2 below: Comparative Example 1 involved adding a methanol / dichloromethane suspension of compound A to a hydrochloric acid / methanol solution at 0-10°C, followed by the dropwise addition of a methyl tert-butyl ether solution for crystallization. This process resulted in inconsistent crystal forms across different batches, and the impurity content was relatively high. The improved process in Example 1 involved reacting compound A with a hydrogen chloride / methyl tert-butyl ether solution in the presence of methanol and water, followed by crystallization. This method effectively controlled the impurity content and maintained consistent crystal forms across multiple batches.

[0181] Table 2

[0182]

[0183] Example 2. Preparation of crystal form II of compound (I)

[0184]

[0185] 900 mL of EtOH was added to a 2 L reactor, followed by 300 mL of purified water. 120 g of compound (I) crystal form I was added, and the mixture was stirred at 25 °C for 96 hours. After filtration, 200 mL of EtOH was added to wash the filter cake. The mixture was then vacuum dried at 50 °C for 20 hours to obtain 108 g of compound (I) crystal form II solid, with a molar yield of 90% and a chlorine content of 12%.

[0186] The XRPD pattern of crystal form II of compound (I) is shown in [reference needed]. Figure 3 The peak positions are listed in Table 3; the DSC spectrum is shown in Table 3. Figure 4 .

[0187] Table 3

[0188]

[0189]

[0190] Example 3. Preparation of crystal form III of compound (I)

[0191]

[0192] 10g of crystal form I of compound (I) and 200ml of MeOH were added to a 500ml reactor. The mixture was stirred at 25℃ for 10 minutes, then at 50℃ for 30 minutes. The temperature was slowly lowered from 50℃ to 5℃ at a rate of 0.1℃ / min and stirred for 3 hours. The mixture was filtered, and 20mL of MeOH was added to wash the filter cake. The mixture was then vacuum dried at 50℃ for 20 hours to obtain 9.2g of crystal form III of compound (I), with a molar yield of 92% and a chlorine content of 12%.

[0193] The XRPD pattern of crystal form III of compound (I) is shown in [reference needed]. Figure 5 The peak positions are listed in Table 4; the DSC spectrum is shown in Table 4. Figure 6 .

[0194] Table 4

[0195]

[0196]

[0197] Example 4. Comparison of chloride ion content and solubility of crystal forms I, II, and III

[0198] The solubility determination method is as follows: Weigh 5 mg of the sample of the crystal form to be tested into a liquid chromatography vial, add 1 mL of 5% glucose solution. If the compound is basically dissolved, add an appropriate amount of solid compound; otherwise, proceed directly to the next step. Add a stir bar and stir at 25°C / 700 rpm for 24 hours. Transfer the sample to a filtered centrifuge tube, centrifuge at 14000 rpm for 10 min, dilute 100 times, and then analyze the sample content in the liquid chromatography system and calculate the solubility. The content determination method is the same as the content determination method in Example 1.

[0199] The chloride ion content was determined according to the general rules of the 2020 edition of the Chinese Pharmacopoeia. <0701> The chlorine content of the crystalline sample was determined by potentiometric titration, and the result was calculated using the weight content correction. The weight content determination method is described in Example 1.

[0200] The results are shown in Table 5 below: Crystal form I is 3 mg / mL hydrochloride, while crystal forms II and III are 2 mg / mL hydrochloride. All three crystal forms have a solubility of ≥4 mg / mL in 5% glucose aqueous solution, meeting the basic solubility requirement for preparing lyophilized injectable formulations (the minimum requirement is 4 mg / mL). If the solubility is too low, it can easily lead to excessively long dissolution time of the compound during the formulation preparation process, drug precipitation after prolonged standing of the solution, or even incomplete dissolution, which is not conducive to the preparation of lyophilized injectable formulations. Among the three crystal forms, crystal forms I and III have much higher solubility in 5% glucose solution than crystal form II, making them easier to prepare lyophilized injectable formulations and thus superior crystal forms.

[0201] Table 5

[0202] Crystal form I 17 9 3 hydrochloride Crystal form II 12 4 2 hydrochloride Crystal form III 12 7 2 hydrochloride

[0203] Example 5. Stability of sample with crystal form I of compound (I)

[0204] The sample of compound (I) crystal form I was placed in a double-layer pharmaceutical low-density polyethylene bag, each layer was tied with a cable tie, silica gel desiccant (product / desiccant: 5 / 1) was added, and then placed in a single-layer aluminum foil bag and heat-sealed. Finally, it was placed in a plastic bucket and placed under different stability test conditions for a certain period of time. The impurity content of the sample was then tested and compared with the test results of the 0-day sample.

[0205] The experimental results are shown in Table 6 below: Compound (I) crystal form I has good stability.

[0206] Table 6

[0207] 0 days 0.18 0.17 0.35 25℃ / 60%RH, January 0.20 0.17 0.37 5℃±3, January 0.18 0.17 0.35 -20℃±5℃, January 0.17 0.17 0.34

[0208] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0209] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A crystalline form I of a thiophene pyrimidine compound, characterized in that, Represented by the 2θ angle, it has characteristic peaks at 4.77°, 9.52°, 14.28°, 21.12°, 23.66°, 25.21° and 28.67° in the X-ray powder diffraction pattern, with an error of ±0.2°; The structural formula of the thiophene pyrimidine compound is as follows: 。 2. The crystal form I of the thiophene pyrimidine compound according to claim 1, characterized in that, Represented by the 2θ angle, it exhibits characteristic peaks at 4.77°, 7.06°, 7.32°, 9.52°, 10.58°, 14.28°, 18.45°, 18.84°, 20.86°, 21.12°, 21.70°, 22.40°, 23.66°, 25.21°, 25.92°, 26.88°, 28.17°, 28.67°, 28.99°, 29.78°, 30.49°, and 32.87° in the X-ray powder diffraction pattern, with an error of ±0.2°.

3. The crystal form I of the thiophene pyrimidine compound according to claim 2, characterized in that, Expressed as 2θ angle, with an error of ±0.2°, its characteristic peaks and relative intensities in the X-ray powder diffraction pattern are as follows: 。 4. The crystal form I of the thiophene pyrimidine compound according to claim 1, characterized in that, Its X-ray powder diffraction pattern is shown in Figure 1-1.

5. Crystal form I of the thiophene pyrimidine compound according to any one of claims 1-4, characterized in that, Its differential scanning calorimetry curve includes an exothermic peak at 220.9 ± 0.5 °C.

6. The crystal form I of the thiophene pyrimidine compound according to claim 5, characterized in that, The differential scanning calorimetry curve is shown in Figure 2.

7. A thiophene pyrimidine compound, crystal form III, characterized in that, Represented by the 2θ angle, it has characteristic peaks at 10.47°, 15.67°, 21.23°, 21.44°, 22.45°, 23.06°, 25.73°, and 29.63° in the X-ray powder diffraction pattern, with an error of ±0.2°; The structural formula of the thiophene pyrimidine compound is as follows: 。 8. The crystalline form III of the thiophene pyrimidine compound according to claim 7, characterized in that, Represented by the 2θ angle, it exhibits characteristic peaks at 10.47°, 14.72°, 15.28°, 15.67°, 16.95°, 18.79°, 20.55°, 21.23°, 21.44°, 22.45°, 22.71°, 23.06°, 24.73°, 25.73°, 27.77°, 27.99°, 29.63°, 30.14°, and 31.25° in the X-ray powder diffraction pattern, with an error of ±0.2°.

9. The crystalline form III of the thiophene pyrimidine compound according to claim 8, characterized in that, Expressed as 2θ angle, with an error of ±0.2°, its characteristic peaks and relative intensities in the X-ray powder diffraction pattern are as follows: 。 10. The crystalline form III of the thiophene pyrimidine compound according to claim 7, characterized in that, Its X-ray powder diffraction pattern is shown in Figure 5.

11. The crystalline form III of the thiophene pyrimidine compound according to any one of claims 7-10, characterized in that, Its differential scanning calorimetry curve includes endothermic peaks at 96.1±0.5℃ and 213.4±0.5℃.

12. The crystalline form III of the thiophene pyrimidine compound according to claim 11, characterized in that, The differential scanning calorimetry curve is shown in Figure 6.

13. A method for preparing crystal form I of a thiophene pyrimidine compound according to any one of claims 1-6, characterized in that, Includes the following steps: The mixture of methanol, purified compound A and water was stirred evenly, and hydrogen chloride / methyl tert-butyl ether solution was slowly added at a temperature of 0℃-10℃. The mixture was then stirred at a temperature of 0℃-10℃ for 15-25 hours, filtered, washed and dried to obtain crystal form I of the thiophene pyrimidine compound. The slow addition of the hydrogen chloride / methyl tert-butyl ether solution takes 2-3 hours. The concentration of hydrogen chloride in the hydrogen chloride / methyl tert-butyl ether solution is 4 mol / L-6 mol / L; The structural formula of compound A is as follows: 。 14. The method for preparing crystal form I of the thiophene pyrimidine compound according to claim 13, characterized in that, Includes the following steps: A mixture of methanol, purified compound A, and water was stirred at 20°C-30°C for 1.5-2.5 hours. A solution of hydrogen chloride / methyl tert-butyl ether was slowly added at 4°C-6°C, and then stirred at 4°C-6°C for 18-22 hours. The mixture was then filtered, washed, and dried to obtain crystal form I of the thiophene pyrimidine compound.

15. The method for preparing crystal form I of the thiophene pyrimidine compound according to claim 13 or 14, characterized in that, The mass ratio of methanol to water is 100-130:1; and / or, The mass ratio of the purified compound A to methanol is 1:11-14; and / or, The mass ratio of the purified compound A to the hydrogen chloride / methyl tert-butyl ether solution is 1:2-5.

16. The method for preparing crystal form I of the thiophene pyrimidine compound according to claim 15, characterized in that, The mass ratio of methanol to water is 105-125:

1.

17. The method for preparing crystal form I of the thiophene pyrimidine compound according to claim 16, characterized in that, The mass ratio of methanol to water is 110-120:

1.

18. The method for preparing crystal form I of the thiophene pyrimidine compound according to claim 17, characterized in that, The mass ratio of methanol to water is 114-118:

1.

19. The method for preparing crystal form I of the thiophene pyrimidine compound according to claim 15, characterized in that, The mass ratio of the purified compound A to methanol is 1:12-13.

20. The method for preparing crystal form I of the thiophene pyrimidine compound according to claim 15, characterized in that, The mass ratio of the purified compound A to the hydrogen chloride / methyl tert-butyl ether solution is 1:3-4.

21. The method for preparing crystal form I of the thiophene pyrimidine compound according to claim 13 or 14, characterized in that, The purification method for the purified compound A includes the following steps: Dimethyl sulfoxide and compound A were stirred at 45℃-55℃ for 1.5-2.5 hours. Methanol and compound A seed crystals were added for the first time at 45℃-55℃, and the mixture was stirred for 1.5-2.5 hours. Methanol was added for the second time, and the mixture was stirred for 1.5-2.5 hours. The mixture was then cooled to 20℃-30℃ and stirred for 1.5-2.5 hours. The mixture was then filtered, washed, and dried to obtain purified compound A.

22. The method for preparing crystal form I of the thiophene pyrimidine compound according to claim 21, characterized in that, The mass ratio of the dimethyl sulfoxide to compound A is 4-9:1; and / or, The mass ratio of methanol to compound A added in the first addition is 1-2:1; and / or The mass of the added compound A seed crystals is 0.4%-0.5% of the mass of compound A; and / or, The mass ratio of methanol added the second time to compound A was 12-20:

1.

23. The method for preparing crystal form I of the thiophene pyrimidine compound according to claim 22, characterized in that, The mass ratio of dimethyl sulfoxide to compound A is 5-8:

1.

24. The method for preparing crystal form I of the thiophene pyrimidine compound according to claim 23, characterized in that, The mass ratio of dimethyl sulfoxide to compound A is 6-7:

1.

25. The method for preparing crystal form I of the thiophene pyrimidine compound according to claim 22, characterized in that, The mass ratio of methanol added the second time to compound A was 14-18:

1.

26. The method for preparing crystal form I of the thiophene pyrimidine compound according to claim 25, characterized in that, The mass ratio of methanol added the second time to compound A is 15-17:

1.

27. A method for preparing crystal form III of a thiophene pyrimidine compound according to any one of claims 7-12, characterized in that, Includes the following steps: Methanol was added to crystal form I of the thiophene pyrimidine compound, and the mixture was stirred at 20℃-30℃ for 8-12 minutes, then stirred at 45℃-55℃ for 25-35 minutes. The mixture was then cooled to 0℃-10℃, kept at this temperature and stirred for 2-4 hours, filtered, washed, and dried to obtain crystal form III of the thiophene pyrimidine compound. The crystal form I of the thiophene pyrimidine compound is the crystal form I of the thiophene pyrimidine compound according to any one of claims 1-6.

28. The method for preparing crystal form III of the thiophene pyrimidine compound according to claim 27, characterized in that, The ratio of crystal form I of the thiophene pyrimidine compound to methanol is 1 g: 16-25 mL; and / or, The cooling rate to 0℃-10℃ is 0.08-0.12℃ / minute.

29. The method for preparing crystal form III of the thiophene pyrimidine compound according to claim 28, characterized in that, The ratio of crystal form I of the thiophene pyrimidine compound to methanol is 1g:18-22mL.

30. The method for preparing crystal form III of the thiophene pyrimidine compound according to claim 29, characterized in that, The ratio of crystal form I of the thiophene pyrimidine compound to methanol is 1g:19-21mL.

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