A crystalline form of a benzothiophene-substituted fused ring compound and a method of making the same

Abstract

The present disclosure relates to a crystalline form of a benzothiophene-substituted fused ring compound and a preparation method thereof. Specifically, the present disclosure provides a crystal form E, a crystal form G and a crystal form H of 2-amino-7-fluoro-4-((5S,5aS,6S,9R)-1-fluoro-12-((1-((4-(fluoromethylidene)piperidin-1-yl)methyl)cyclopropyl)methoxy)-5-methyl-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methanonaphtho[1,8-ab]heptalen-2-yl)benzo[b]thiophene-3-carbonitrile, which has good stability and can be better used for clinical treatment.

Description

Technical Field

[0001] This disclosure belongs to the field of pharmaceutical technology and relates to a crystalline form of a benzothiophene-substituted fused-ring compound and its preparation method. Background Technology

[0002] RAS (Rapid Acid Spectroscopy) genes are among the most frequently mutated oncogenes in tumors, with approximately 30% of human malignancies associated with RAS gene mutations. The RAS family includes KRAS, NRAS, and HRAS, with KRAS mutations being the most common, accounting for about 85%. KRAS mutations are frequently found in solid tumors, exhibiting high frequency in the three leading causes of cancer death in humans—lung cancer (17%), colorectal cancer (33%), and pancreatic cancer (61%). In KRAS gene mutations, 97% involve mutations at amino acid residues 12 or 13, with G12D being a significant mutation. Data analysis of populations in Europe and America shows that G12D mutations account for 36%, 12%, and 4% of patients with pancreatic cancer, colorectal cancer, and non-small cell lung cancer, respectively.

[0003] Once activated, KRAS regulates various functions of cell proliferation, survival, migration, and metabolism through numerous downstream signaling pathways, including RAF-MEK-ERK, PI3K-AKT-mTOR, and TIAM1-RAc. Mutations in the KRAS gene result in a persistently activated protein, leading to continued activation of downstream signaling pathways and promoting tumorigenesis.

[0004] PCT / CN2023 / 109598 provides a KRAS G12D inhibitor with the chemical name 2-amino-7-fluoro-4-((5S,5aS,6S,9R)-1-fluoro-12-((1-((4-(fluoromethylidene)piperidin-1-yl)methyl)cyclopropyl)methoxy)-5-methyl-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaza-6,9-methylbridged naphtho[1,8-ab]heptan-2-yl)benzo[b]thiophene-3-carboxynitrile, having the structure shown in Formula 1.

[0005]

[0006] The crystal form of a pharmaceutical active ingredient often affects its chemical stability. Different crystallization and storage conditions can lead to changes in the crystal structure of the compound, sometimes even resulting in other crystal forms. Generally, amorphous drug products lack regular crystal structures and often have other defects, such as poor product stability, fine crystals, difficulty in filtration, easy agglomeration, and poor flowability. Polymorphism of drugs places different requirements on product storage, production, and scale-up. Therefore, in-depth research on the crystal forms of the aforementioned compounds and the improvement of their various properties is essential. Summary of the Invention

[0007] This disclosure provides a novel crystal form of the compound shown in Formula 1, which has good stability and can be better applied in clinical practice.

[0008]

[0009] The crystal form A of the compound of Formula 1 provided in this disclosure has characteristic peaks at 8.984, 12.800, 14.133, 14.675, 18.977, and 21.018 in its X-ray powder diffraction pattern expressed as a diffraction angle of 2θ.

[0010] In some embodiments, the X-ray powder diffraction pattern of crystal form A of the compound shown in Formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 8.984, 9.837, 12.800, 14.133, 14.675, 15.944, 16.635, 17.595, 18.977, 21.018, and 26.006.

[0011] In some embodiments, the X-ray powder diffraction pattern of crystal form A of the compound shown in Formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 8.984, 9.837, 12.800, 14.133, 14.675, 15.944, 16.635, 17.595, 18.977, 21.018, 22.089, 23.026, 26.006, and 29.233.

[0012] In some embodiments, the X-ray powder diffraction pattern of crystal form A of the compound shown in Formula 1, expressed in terms of diffraction angle 2θ, is as follows: Figure 1 As shown.

[0013] This disclosure also provides a method for preparing crystal form A of the compound shown in Formula 1, wherein the method is selected from any of the following methods:

[0014] Method 1: Dissolve the compound of Formula 1 in solvent I and evaporate the solvent, wherein solvent I is selected from methanol, 10% water / methanol, or 50% acetonitrile / methanol;

[0015] Method 2: Add the compound of Formula 1 to solvent II and stir. Solvent II is selected from water, acetonitrile, n-heptane, cyclohexane, isopropyl ether, 7% water / ethanol, and 50% water / methanol.

[0016] The crystal form B of the compound shown in Formula 1 provided in this disclosure has characteristic peaks at 4.651, 8.536, 11.909, 14.077, 15.584, and 20.969 in its X-ray powder diffraction pattern expressed as a diffraction angle of 2θ.

[0017] In some embodiments, the X-ray powder diffraction pattern of crystal form B of the compound shown in Formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 4.651, 8.536, 9.290, 11.909, 14.077, 15.584, 17.365, 18.878, 20.969, and 22.281.

[0018] In some embodiments, the X-ray powder diffraction pattern of crystal form B of the compound shown in Formula 1, expressed in terms of diffraction angle 2θ, is as follows: Figure 2 As shown.

[0019] This disclosure also provides a method for preparing crystal form B of the compound shown in Formula 1, the method comprising dissolving the compound of Formula 1 in tetrahydrofuran / ethanol (2:1) and subjecting the mixture to a temperature change of 40-5°C.

[0020] This disclosure also provides a method for preparing crystal form B of the compound shown in Formula 1, the method comprising the steps of dissolving the compound of Formula 1 in ethyl acetate / ethanol (1:1) and evaporating the solvent.

[0021] This disclosure also provides a method for preparing crystal form B of the compound shown in Formula 1, the method comprising the steps of adding the amorphous form of the compound of Formula 1 to ethanol and stirring.

[0022] The X-ray powder diffraction pattern of the compound of Formula 1 shown in this disclosure, expressed in terms of diffraction angle 2θ, has characteristic peaks at 8.666, 14.121, 18.947, 20.764, and 29.128.

[0023] In some embodiments, the X-ray powder diffraction pattern of crystal form C of the compound shown in Formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 8.666, 9.348, 12.575, 14.121, 16.942, 18.947, 20.764, 29.128, and 33.304.

[0024] In some embodiments, the X-ray powder diffraction pattern of crystal form C of the compound shown in Formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 8.666, 9.348, 12.575, 14.121, 16.942, 18.947, 20.764, 22.780, 23.932, 25.469, 29.128, and 33.304.

[0025] In some embodiments, the X-ray powder diffraction pattern of crystal form C of the compound shown in Formula 1, expressed in terms of diffraction angle 2θ, is as follows: Figure 3 As shown.

[0026] This disclosure also provides a method for preparing the crystal form C of the compound shown in Formula 1, the method comprising adding the compound of Formula 1 to an alcohol solvent I and stirring, wherein the alcohol solvent I is selected from ethanol, isopropanol, and n-propanol.

[0027] The crystal form D of the compound shown in Formula 1 provided in this disclosure has characteristic peaks at 7.229, 9.137, 11.664, 16.506, and 20.682 in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ.

[0028] In some embodiments, the crystal form D of the compound shown in Formula 1, as expressed in X-ray powder diffraction patterns at diffraction angles 2θ, has characteristic peaks at 5.265, 7.229, 9.137, 11.664, 16.506, 17.396, 20.682, 21.849, and 23.001.

[0029] In some embodiments, the X-ray powder diffraction pattern of the crystal form D of the compound shown in Formula 1, expressed in terms of the diffraction angle 2θ, is as follows: Figure 4 As shown.

[0030] This disclosure also provides a method for preparing crystal form D of the compound shown in Formula 1, said method being selected from any of the following methods:

[0031] Method 1: Dissolve the compound of formula 1 in 2-methyltetrahydrofuran, add methyl tert-butyl ether, and stir;

[0032] Method 2: Add the compound of Formula 1 to methyl tert-butyl ether or 50% ethyl acetate / n-heptane and stir.

[0033] The crystal form E of the compound shown in Formula 1 provided in this disclosure has characteristic peaks at 5.144, 8.390, 10.312, 13.575, and 20.130 in its X-ray powder diffraction pattern expressed as a diffraction angle of 2θ.

[0034] In some embodiments, the crystal form E of the compound shown in Formula 1, as expressed in X-ray powder diffraction patterns at diffraction angles 2θ, has characteristic peaks at 5.144, 8.390, 10.312, 13.575, 14.845, 15.568, 16.293, 17.776, 18.488, and 20.130.

[0035] In some embodiments, the X-ray powder diffraction pattern of the crystal form E of the compound shown in Formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 5.144, 8.390, 10.312, 13.575, 14.845, 15.568, 16.293, 17.776, 18.488, 20.130, 21.350, 22.029, 23.036, and 31.408.

[0036] In some embodiments, the X-ray powder diffraction pattern of the crystal form E of the compound shown in Formula 1, expressed in terms of the diffraction angle 2θ, is as follows: Figure 5 As shown.

[0037] This disclosure also provides a method for preparing crystal form E of the compound shown in Formula 1, said method being selected from any of the following methods:

[0038] Method 1: Dissolve the compound of formula 1 in solvent II, add solvent III, and stir. Solvent II is selected from acetone, ethyl acetate, tetrahydrofuran / ethanol (2:1), and solvent III is selected from MTBE and n-heptane.

[0039] Method 2: Add the compound of formula 1 to 50% ethyl acetate / n-heptane or acetone and stir;

[0040] Method 3: Dissolve the compound of Formula 1 in solvent IV and stir. Solvent IV is selected from ethyl acetate / methyl tert-butyl ether (v / v = 1:5), 2-methyltetrahydrofuran, and dichloromethane.

[0041] The crystal form F of the compound of Formula 1 provided in this disclosure has characteristic peaks at 4.782, 5.122, 9.989, 15.495, and 20.134 in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ.

[0042] In some embodiments, the X-ray powder diffraction pattern of the crystal form F of the compound shown in Formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 4.782, 5.122, 9.989, 13.135, 14.171, 15.495, 16.552, 17.396, and 20.134.

[0043] In some embodiments, the X-ray powder diffraction pattern of the crystal form F of the compound shown in Formula 1, expressed in terms of the diffraction angle 2θ, is as follows: Figure 6 As shown.

[0044] This disclosure also provides a method for preparing the crystal form F of the compound shown in Formula 1, the method comprising the steps of dissolving the compound of Formula 1 in dichloromethane, adding n-heptane, and stirring.

[0045] The crystal form G of the compound shown in Formula 1 provided in this disclosure has characteristic peaks at 10.692, 16.093, 16.447, 17.922, 24.567, and 33.426 in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ.

[0046] In some embodiments, the X-ray powder diffraction pattern of the crystal form G of the compound shown in Formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 7.724, 8.701, 10.692, 12.719, 13.325, 16.093, 16.447, 17.922, 19.340, 20.746, 21.219, 24.567, and 33.426.

[0047] In some embodiments, the X-ray powder diffraction pattern of the crystal form G of the compound shown in Formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 7.724, 8.701, 10.692, 12.719, 13.325, 16.093, 16.447, 17.922, 19.340, 19.891, 20.746, 21.219, 22.886, 23.577, 24.567, 25.386, 26.330, and 33.426.

[0048] In some embodiments, the X-ray powder diffraction pattern of the crystal form G of the compound shown in Formula 1, expressed as a diffraction angle 2θ, is as follows: Figure 7 As shown.

[0049] This disclosure also provides a method for preparing the crystal form G of the compound shown in Formula 1, the method comprising the steps of adding the amorphous form of the compound of Formula 1 to methanol and stirring.

[0050] The X-ray powder diffraction pattern of the compound of Formula 1 H provided in this disclosure, expressed as a diffraction angle 2θ, has characteristic peaks at 5.099, 7.096, 9.872, 15.784, and 19.440.

[0051] In some embodiments, the X-ray powder diffraction pattern of the crystal form H of the compound shown in Formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 5.099, 7.096, 8.528, 9.872, 11.100, 13.212, 15.784, 19.440, 20.621, 21.120, and 22.425.

[0052] Formula 1

[0053] In some embodiments, the X-ray powder diffraction pattern of the crystal form H of the compound shown in Formula 1, expressed in terms of the diffraction angle 2θ, is as follows: Figure 8 As shown.

[0054] This disclosure also provides a method for preparing the crystal form H of the compound shown in Formula 1, said method being selected from any of the following methods:

[0055] Method 1: Add the amorphous compound of Formula 1 to solvent V and stir. Solvent V is selected from p-xylene, methyl tert-butyl ether, and ethyl acetate.

[0056] Method 2: Dissolve the amorphous compound of Formula 1 in dichloromethane, add it to n-heptane, and stir.

[0057] Method 3: The amorphous form of compound 1 is added to ethyl acetate, followed by the addition of a ligand and stirring. The ligand is selected from citric acid ethanol solution, tartaric acid ethanol solution, and fumaric acid.

[0058] The X-ray powder diffraction pattern of the compound of Formula 1 provided in this disclosure, expressed in terms of diffraction angle 2θ, has characteristic peaks at 4.992, 7.006, 7.552, 15.706, 17.272, 17.696, and 21.247.

[0059] In some embodiments, the X-ray powder diffraction pattern of crystal form I of the compound shown in Formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 4.992, 7.006, 7.552, 8.416, 10.072, 13.212, 14.363, 15.706, 17.272, 17.696, and 21.247.

[0060] In some embodiments, the X-ray powder diffraction pattern of crystal form I of the compound shown in Formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 4.992, 7.006, 7.552, 8.416, 10.072, 11.177, 13.212, 14.363, 15.706, 16.515, 17.272, 17.696, 19.361, 19.968, 21.247, 22.809, and 23.500.

[0061] In some embodiments, the X-ray powder diffraction pattern of crystal form I of the compound shown in Formula 1, expressed in terms of diffraction angle 2θ, is as follows: Figure 9 As shown.

[0062] This disclosure also provides a method for preparing crystal form I of the compound shown in Formula 1, the method comprising adding the compound of Formula 1 to ethyl acetate, then adding an ethanolic acetic acid solution, and stirring.

[0063] The crystal form J of the compound shown in Formula 1 provided in this disclosure has characteristic peaks at 7.645, 14.939, 18.701, 20.045, and 23.539 in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ.

[0064] In some embodiments, the crystal form J of the compound shown in Formula 1 has characteristic peaks at 7.645, 8.682, 14.939, 17.243, 18.701, 20.045, 23.539, 24.498, and 27.032 in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ.

[0065] In some embodiments, the X-ray powder diffraction pattern of the crystal form J of the compound shown in Formula 1, expressed in terms of the diffraction angle 2θ, is as follows: Figure 10 As shown.

[0066] This disclosure also provides a method for preparing the crystal form J of the compound shown in Formula 1, the method comprising heating the crystal form I of the compound of Formula 1 to 215°C.

[0067] In some embodiments, the preparation method described in this disclosure further includes any one of the steps of crystallization, centrifugation (filtration), washing, or drying.

[0068] The crystallization methods disclosed herein include, but are not limited to, stirred crystallization, static crystallization, or evaporative crystallization. In some embodiments, the crystallization is stirred crystallization. In some embodiments, the crystallization is static crystallization.

[0069] This disclosure also provides a pharmaceutical composition comprising the aforementioned crystal form A, B, C, D, E, F, G, H, I, or J, and a pharmaceutical excipient optionally selected from pharmaceutically acceptable excipients.

[0070] This disclosure also provides a pharmaceutical composition prepared from the aforementioned crystal forms A, B, C, D, E, F, G, H, I, or J, and optionally a pharmaceutically acceptable excipient.

[0071] This disclosure also provides a method for preparing a pharmaceutical composition, comprising the step of mixing the aforementioned crystal form A, B, C, D, E, F, G, H, I, or J with a pharmaceutically acceptable excipient.

[0072] This disclosure also provides the use of the aforementioned crystal forms A, B, C, D, E, F, G, H, I, or J, or the aforementioned compositions, in the preparation for the prevention and / or treatment of cancer.

[0073] The uses described in this disclosure, wherein the cancer is selected from breast cancer, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, colorectal cancer, lung cancer, kidney cancer, liver cancer, cervical cancer, endometrial cancer, epithelial cancer, esophageal cancer, neuroblastoma, glioma, bone cancer, nasopharyngeal carcinoma, oral cancer, thyroid cancer, skin cancer, and squamous cell carcinoma; preferably, the cancer is selected from breast cancer, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, colorectal cancer, and lung cancer.

[0074] The "2θ or 2θ angle" mentioned in this disclosure refers to the diffraction angle, where θ is the Bragg angle, and the unit is ° or degree; the error range of 2θ for each characteristic peak is ±0.20 (including the case where the number has more than one decimal place after rounding), specifically -0.20, -0.19, -0.18, -0.17, -0.16, -0.15, -0.14, -0.13, -0.12, -0.11, -0.10, -0.09, -0.08, -0.07, -0.06, -0.05, -0.04, -0.03, -0.02, -0.01, 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20.

[0075] The numerical values ​​in this disclosure, such as those relating to the content of certain substances, are calculated data and inevitably contain a certain degree of error. Generally, ±10% is within the reasonable error range. The error may vary to some extent depending on the context in which it is used, but this variation shall not exceed ±10%, and may be ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%, preferably ±5%.

[0076] The starting material used in the crystal form preparation method disclosed herein can be any form of compound, including but not limited to: amorphous, arbitrary crystal form, hydrate, solvate, etc.

[0077] The drying temperature described in this disclosure is generally 25℃-100℃, preferably 40℃-70℃, and can be dried under normal pressure or reduced pressure.

[0078] The crystallization methods described in this disclosure include room temperature crystallization, cooling crystallization, solvent evaporation crystallization, and seed crystallization induction. The cooling temperature is selected from below 65°C, preferably from -10°C to 60°C. Stirring can also be performed during the crystallization process.

[0079] The “differential scanning calorimetry or DSC” described in this disclosure refers to measuring the temperature difference and heat flow difference between the sample and the reference material during the sample heating or isothermal process, in order to characterize all physical and chemical changes related to thermal effects and obtain phase transition information of the sample.

[0080] According to the description of hygroscopic characteristics and the definition of hygroscopic weight gain in the "Guiding Principles on Hygroscopicity of Drugs" in Part IV of the 2015 edition of the Chinese Pharmacopoeia,

[0081] Deliquescence: Absorbs sufficient moisture to form a liquid;

[0082] Extremely hygroscopic: the weight gain due to hygroscopic absorption is not less than 15%;

[0083] It has hygroscopic properties: the weight gain due to hygroscopic absorption is less than 15% but not less than 2%;

[0084] Slightly hygroscopic: the weight gain due to moisture absorption is less than 2% but not less than 0.2%;

[0085] It has little or no hygroscopicity: the weight gain due to moisture absorption is less than 0.2%.

[0086] The “excipients” described in this disclosure include, but are not limited to, any adjuvants, carriers, flow aids, sweeteners, diluents, preservatives, dyes / colorants, flavoring agents, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, or emulsifiers that have been approved by the U.S. Food and Drug Administration for use in humans or livestock. Attached Figure Description

[0087] Figure 1 The image shows the XRPD spectrum of crystal form A of compound 1.

[0088] Figure 2 The image shows the XRPD spectrum of crystal form B of compound 1.

[0089] Figure 3 The image shows the XRPD spectrum of crystal form C of compound 1.

[0090] Figure 4 The image shows the XRPD spectrum of crystal form D of compound 1.

[0091] Figure 5 The image shows the XRPD spectrum of crystal form E of compound 1.

[0092] Figure 6 The image shows the XRPD spectrum of crystal form F of compound 1.

[0093] Figure 7 The image shows the XRPD spectrum of crystal form G of compound 1.

[0094] Figure 8 The image shows the XRPD spectrum of crystal form H of compound 1.

[0095] Figure 9 The image shows the XRPD spectrum of crystal form I of compound 1.

[0096] Figure 10 The image shows the XRPD spectrum of crystal form J of compound 1.

[0097] Figure 11 The image shows the XRPD spectrum of compound 1 in its amorphous form. Detailed Implementation

[0098] The present disclosure will be explained in more detail below with reference to embodiments or experimental examples. The embodiments or experimental examples in the present disclosure are only used to illustrate the technical solutions in the present disclosure and are not intended to limit the substance and scope of the present disclosure.

[0099] Test conditions of the instruments used in the experiment:

[0100] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) were expressed in 10⁻¹⁰. -6 The unit (ppm) is given. NMR measurements were performed using a Bruker AVANCE NEO 500M NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), with tetramethylsilane (TMS) as the internal standard.

[0101] MS measurements were performed using an Agilent 1200 / 1290DAD-6110 / 6120 Quadrupole MS LC-MS system (manufacturer: Agilent, MS model: 6110 / 6120 Quadrupole MS), a Waters ACQuity UPLC-QD / SQD system (manufacturer: Waters, MS model: Waters ACQuity Qda Detector / Waters SQ Detector), or a THERMO Ultimate3000-Q Exactive system (manufacturer: THERMO, MS model: THERMO Q Exactive).

[0102] High-performance liquid chromatography (HPLC) analysis was performed using an Agilent HPLC 1200DAD, an Agilent HPLC 1200VWD, and a Waters HPLC e2695-2489 HPLC system.

[0103] High performance liquid chromatography (HPLC) was performed using Waters 2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP, and Gilson-281 preparative chromatographs.

[0104] Silica gel column chromatography generally uses Yantai Huanghai silica gel with a mesh size of 200-300 as the carrier.

[0105] XRPD (X-ray Powder Diffraction) was used for analysis: measurements were performed using a BRUKER D8 X-ray diffractometer. Specific data collected included: Cu anode (40 kV, 40 mA), Cu-Kα1 rays. Kα2 rays Kβ rays Scanning mode: θ / 2θ, scanning range (2θ range): 5°~45°.

[0106] DSC stands for Differential Scanning Calorimetry: Measurements were performed using a METTLER TOLEDO DSC 3+ differential scanning calorimeter with a heating rate of 10℃ / min. The specific temperature range was determined by referring to the corresponding spectra (mostly 25-250℃, 300℃, or 350℃). The nitrogen purging rate was 50mL / min.

[0107] TGA is thermogravimetric analysis: the test was performed using a METTLER TOLEDO TGA 2 thermogravimetric analyzer, with a heating rate of 10℃ / min, and the specific temperature range was referenced from the corresponding spectrum (mostly 30-350℃). The nitrogen purging rate was 50mL / min.

[0108] DVS stands for Dynamic Moisture Adsorption: The detection method is SMSDVS Advantage. At 25℃, the humidity changes from 50% to 95% to 0% to 95% to 50%, with a step of 10% (the last step is 5%). (The specific humidity range is subject to the corresponding spectrum. The methods listed here are the most commonly used methods.) The judgment standard is that dm / dt is not greater than 0.002%.

[0109] The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, AccelaChemBio Inc, and Darui Chemicals.

[0110] Unless otherwise specified in the examples, all reactions can be carried out under an argon or nitrogen atmosphere.

[0111] Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

[0112] The reaction process in the examples was monitored using thin-layer chromatography (TLC). The developing solvent used in the reaction, the eluent system used for column chromatography to purify the compounds, and the developing solvent system for TLC included: A: n-hexane / ethyl acetate system, B: dichloromethane / methanol system. The volume ratio of the solvent was adjusted according to the polarity of the compounds, and small amounts of basic or acidic reagents such as triethylamine and acetic acid could also be added for adjustment.

[0113] Example 1: Preparation of Compound 1

[0114] 2-Amino-7-fluoro-4-((5S,5aS,6S,9R)-1-fluoro-12-((1-((4-(fluoromethylidene)piperidin-1-yl)methyl)cyclopropyl)methoxy)-5-methyl-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaza-6,9-methylbridgednaphtho[1,8-ab]heptan-2-yl)benzo

[0115] [b]Thiophene-3-carboxynitrile 1

[0116]

[0117]

[0118] first step

[0119] 4-(fluoromethyl)piperidine-1-carboxylic acid tert-butyl ester 1b

[0120] 2-((fluoromethyl)sulfonyl)pyridine (4.2 g, 23.97 mmol) was dissolved in tetrahydrofuran (50 mL). A 1 M solution of bis(trimethylsilyl)amino potassium in tetrahydrofuran (30 mL) was added at -78 °C. After reacting for 30 minutes, N-tert-butoxycarbonyl-4-piperidinone 1a (5 g, 25.09 mmol, Shanghai Shaoyuan) was added. The reaction was maintained at 3 hours, then allowed to return to room temperature for 1 hour. Saturated ammonium chloride was added to quench the reaction mixture, followed by the addition of 3N hydrochloric acid (100 mL). After stirring for 1 hour, the mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered to remove the desiccant, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using eluent system B to give the title compound 1b (2 g, yield: 37%).

[0121] Step 2

[0122] 4-(Fluoromethyl)piperidine hydrochloride 1c

[0123] Compound 1b (1 g, 4.64 mmol) was dissolved in 20 mL of 4 M hydrogen chloride solution of 1,4-dioxane and stirred for 1 hour. The reaction solution was concentrated under reduced pressure to obtain crude title compound 1c (700 mg). The product was used directly in the next step of the reaction without purification.

[0124] MS m / z(ESI): 116.1 [M+1].

[0125] Step 3

[0126] 2,5,7-Trichloro-8-fluoropyrido[4,3-d]pyrimidine-4-phenol 1e

[0127] The crude compound 1d (2 g, 8 mmol, prepared according to the method in PCT / CN2023 / 109598) was dissolved in phosphorus oxychloride (25 mL), and N,N-diisopropylethylamine (5.16 g, 40 mmol) was added. The mixture was stirred at 110 °C for 14 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure. The residue was dissolved in 1,4-dioxane, and the pH was adjusted to 2-3 by adding 20% ​​potassium carbonate solution. After stirring for 2 hours, the mixture was filtered, the filter cake was washed with water, and dried to obtain the crude title compound 1e (1.5 g). The product was used directly in the next step without purification. MS m / z (ESI): 267.8 [M+1].

[0128] Step 4

[0129] (1S,2S,5R)-2-((S)-1-((2,7-dichloro-8-fluoro-4-hydroxypyridino[4,3-d]pyrimidin-5-yl)oxy)ethyl)-3,8-diazabicyclo

[0130] [3.2.1] 1g of tert-butyl octane-8-carboxylate

[0131] (1S,2S,5R)-2-((S)-1-hydroxyethyl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester 1f (370 mg, 1.44 mmol, prepared by the method disclosed in Intermediate 29 on page 164 of patent application "WO2022173678A1") was dissolved in tetrahydrofuran (10 mL). Sodium hydride (201 mg, 5.2 mmol, 60% purity) was added under ice bath. After reacting for 30 minutes, compound 1e (353 mg, 1.31 mmol) was added. The reaction was stirred for 2 hours. The reaction solution was quenched with water and concentrated under reduced pressure to obtain crude title compound 1 g (600 mg). The product was used directly in the next step without purification.

[0132] MS m / z(ESI):488.2[M+1].

[0133] Step 5

[0134] (5S,5aS,6S,9R)-2,12-dichloro-1-fluoro-5-methyl-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentazaze

[0135] -6,9-methyl-naphtho[1,8-ab]heptan-14-carboxylic acid tert-butyl ester 1h

[0136] 1 g (78 mg, 159.7 μmol) of the compound was dissolved in dichloromethane (2 mL). N,N-diisopropylethylamine (61.9 mg, 478.9 μmol) and phosphorus oxychloride (122.4 mg, 798.2 μmol) were added under ice bath conditions. The mixture was stirred for 2 hours. The reaction solution was quenched with saturated sodium bicarbonate solution. The reaction was then combined with dichloromethane (10 mL × 2). The organic phases were dried with anhydrous sodium sulfate. After filtration to remove the desiccant, the mixture was concentrated under reduced pressure to obtain the crude title compound 1h (75 mg). The product was used directly in the next reaction without purification.

[0137] MS m / z(ESI): 470.2 [M+1].

[0138] Step 6

[0139] (5S,5aS,6S,9R)-12-((1-(((tert-butyldimethylsilyl)oxy)methyl)cyclopropyl)methoxy)-2-chloro-1-fluoro-5-methyl-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaza-6,9-methylbridgednaphtho[1,8-ab]heptan-14-carboxylic acid tert-butyl

[0140] 1j base ester

[0141] Dissolve 1.4 g (6.4 mmol) of (1-(((tert-butyldimethylsilyl)oxy)methyl)cyclopropyl)methanol 1i in tetrahydrofuran (15 mL), add 2 M sodium bis(trimethylsilyl)amino in tetrahydrofuran under ice bath, and stir for 30 minutes while maintaining the temperature. Then add 20 mL of tetrahydrofuran solution of crude compound 1h (2.3 g, 4.9 mmol) under ice bath, and stir for 1 hour while maintaining the temperature. Quench the reaction solution with saturated ammonium chloride solution, extract with ethyl acetate (30 mL × 2), combine the organic phases, dry with anhydrous sodium sulfate, filter to remove the desiccant, concentrate under reduced pressure, and purify the residue by silica gel column chromatography with eluent system B to give title compound 1j (2 g, yield: 62.8%).

[0142] MS m / z(ESI): 650.2 [M+1].

[0143] Step 7

[0144] (5S,5aS,6S,9R)-2-chloro-1-fluoro-12-((1-(hydroxymethyl)cyclopropyl)methoxy)-5-methyl-5a,6,7,8,9,10-hexahydro-5H-4-oxa

[0145] -3,10a,11,13,14-pentazaza-6,9-methyl-naphtho[1,8-ab]heptan-4-carboxylic acid tert-butyl ester 1k

[0146] Compound 1j (100 mg, 153.8 μmol) was dissolved in tetrahydrofuran (4 mL), and a 1 M tetrabutylammonium fluoride tetrahydrofuran solution (187 μL) was added. The mixture was stirred for 2 hours, and the reaction solution was quenched with saturated ammonium chloride aqueous solution. The mixture was extracted with ethyl acetate (15 mL × 3), and the organic phases were combined. The mixture was washed successively with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered to remove the desiccant, and the filtrate was concentrated under reduced pressure to obtain the crude title compound 1k (82 mg). The product was used directly in the next reaction without purification. MS m / z (ESI): 536.2 [M+1].

[0147] Step 8

[0148] (5S,5aS,6S,9R)-2-chloro-1-fluoro-5-methyl-12-((1-((methanesulfonyl)oxy)methyl)cyclopropyl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaza-6,9-methylbridged naphtho[1,8-ab]heptan-14-carboxylic acid tert-butyl ester

[0149] 1l

[0150] The crude compound 1k (83 mg, 154.9 μmol) and N,N-diisopropylethylamine (60 mg, 464.2 μmol) were dissolved in dichloromethane (3 mL). Methanesulfonyl chloride (25 mg, 218.2 μmol) was added under ice bath conditions. The reaction was allowed to return to room temperature for 30 minutes. The reaction solution was quenched with saturated ammonium chloride aqueous solution. The mixture was extracted with ethyl acetate (10 mL × 3). The organic phases were combined and washed successively with water and saturated sodium chloride solution. The mixture was dried over anhydrous sodium sulfate, filtered to remove the desiccant, and the filtrate was concentrated under reduced pressure to obtain the crude title compound 1l (95 mg). The product was used directly in the next reaction without purification.

[0151] MS m / z(ESI): 614.2 [M+1].

[0152] Step 9

[0153] (5S,5aS,6S,9R)-2-chloro-1-fluoro-12-((1-((4-(fluoromethylidene)piperidin-1-yl)methyl)cyclopropyl)methoxy)-5-methyl-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaza-6,9-methylbridgednaphtho[1,8-ab]heptan-14-carboxylic acid tert-butyl

[0154] Ester 1m

[0155] The crude compound 1l (95 mg, 154.7 μmol) and compound 1c (35.5 mg, 234.5 μmol) were dissolved in acetonitrile (4 mL), and anhydrous potassium carbonate (64 mg, 463 μmol) and sodium iodide (70 mg, 467 μmol) were added. The mixture was stirred at 80 °C for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography with eluent system A to give the title compound 1m (80 mg, yield: 81.6%).

[0156] MS m / z(ESI): 633.2 [M+1].

[0157] Step 10 (5S,5aS,6S,9R)-2-(2-((tert-butoxycarbonyl)amino)-3-cyano-7-fluorobenzo[b]thiophene-4-yl)-1-fluoro-12-((1-((4-(fluoromethylidene)piperidin-1-yl)methyl)cyclopropyl)methoxy)-5-methyl-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaza-6,9-methylbridged naphtho[1,8-ab]heptacyclo-14-carboxylic acid tert-butyl ester 1o

[0158] Compound 1m (20 mg, 31.6 μmol), (3-cyano-4-(5,5-dimethyl-1,3,2-dioxoborhexacyclohex-2-yl)-7-fluorobenzo[b]thiophene-2-yl) tert-butyl carbamate 1n (19 mg, 47 μmol, prepared by the method disclosed in preparation 23 on page 39 of patent application "WO2022261154A1"), tetra(triphenylphosphine)palladium (7 mg, 6.1 μmol), and cesium carbonate (30 mg, 92 μmol) were mixed with N,N-dimethylformamide (1 mL), purged with nitrogen, and reacted at 90 °C for 2 hours. After the reaction solution was cooled to room temperature, it was diluted with ethyl acetate, dried with anhydrous sodium sulfate, filtered to remove the desiccant, and concentrated under reduced pressure to obtain crude title compound 1o (15 mg). The product was used directly in the next reaction without purification.

[0159] MS m / z(ESI): 889.2 [M+1].

[0160] Step 11

[0161] 2-Amino-7-fluoro-4-((5S,5aS,6S,9R)-1-fluoro-12-((1-((4-(fluoromethylidene)piperidin-1-yl)methyl)cyclopropyl)methoxy)-5-methyl-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaza-6,9-methylbridgednaphtho[1,8-ab]heptan-2-yl)benzo

[0162] [b]Thiophene-3-carboxynitrile 1

[0163] The crude compound 1O (15 mg, 16.9 μmol) was dissolved in acetonitrile (1 mL), and 0.5 mL of 4 M 1,4-dioxane hydrochloride solution was added under ice bath conditions. The reaction was maintained at this temperature for 0.5 hours, followed by 2 hours at room temperature. The mixture was concentrated under reduced pressure, and the residue was purified by preparative high-performance liquid chromatography (Waters-2545, column: Da Cao ODS-BIO C18, 30*150 mm, 5 μm; mobile phase: aqueous phase (10 mmol / L ammonium bicarbonate) and acetonitrile, gradient ratio: acetonitrile 54%-69% for 15 min, flow rate: 30 mL / min, column temperature: room temperature) to obtain title compound 1 (4 mg, yield: 34.4%). X-ray powder diffraction analysis showed that the product was in a free amorphous state. The XRPD spectrum is shown below. Figure 11 .

[0164] MS m / z(ESI): 687.3 [M-1].

[0165] 1 H NMR (500MHz, CD3OD): δ7.39(dd,1H),7.04(t,1H),6.51(d,1H),5.41(dd,1H),4.5–4.46(m,2H),4.40(d,1H),4.10(d,1H),3.7 0(d,1H),3.60(d,1H),3.19(s,1H),2.62–2.31(m,8H),2.09(d,3H),1.92–1.75(m,3H),1.61(d,3H),0.75(s,2H),0.52(s,2H).

[0166] Test Example 1: Biological Evaluation of GP2d and AGS Cell 3D Proliferation Inhibition Experiment

[0167] Experimental methods

[0168] GP2d cells were cultured in complete medium, namely DMEM / high glucose medium (Hyclone, SH30243.01) containing 10% fetal bovine serum (Corning, 35-076-CV). On the first day of the experiment, GP2d cells were seeded at a density of 1000 cells / well in 96-well low-absorption plates (Corning, CLS7007-24EA) using complete medium, with 90 μL of cell suspension per well. After centrifugation at 2000 rpm for 5 minutes at room temperature, the cells were incubated overnight at 37°C in a 5% CO2 cell culture incubator.

[0169] AGS cells were cultured in complete medium, namely RPMI 1640 medium (Hyclone, SH30809.01) containing 10% fetal bovine serum (Corning, 35-076-CV). On the first day of the experiment, AGS cells were seeded at a density of 1000 cells / well in 96-well low-absorption plates (Corning, CLS7007-24EA) using complete medium, with 90 μL of cell suspension per well. After centrifugation at 2000 rpm for 5 minutes at room temperature, the cells were incubated overnight at 37°C in a 5% CO2 cell culture incubator.

[0170] On the second day, 10 μL of serially diluted test compounds prepared with complete culture medium was added to each well. For GP2d cells, the final concentrations were determined by nine 5-fold serial dilutions starting from 1 μM; for AGS cells, the final concentrations were determined by nine 5-fold serial dilutions starting from 10 μM. A blank control containing 0.5% DMSO was included in both wells. The plates were incubated at 37°C in a 5% CO2 incubator for 5 days. On the seventh day, the 96-well cell culture plate was removed, and 50 μL of the test compound was added to each well. The 3D Cell Viability Assay reagent (Promega, G9682) was shaken at room temperature in the dark for 25 minutes, then mixed by pipetting and aspiration. 100 μL of the mixture was taken from each well and transferred to a white, opaque 96-well plate (PerkinElmer, 6005290). The luminescence signal value was read using a multi-functional microplate reader (PerkinElmer, EnVision2105).

[0171] The IC50 of the compound's inhibitory activity was calculated using Graphpad Prism software. 50 The values ​​are shown in Table 1 below.

[0172] Table 1. Data on the inhibitory activity of AGS and GP2d cells on 3D proliferation.

[0173]

[0174] Conclusion: The compound disclosed herein has a good inhibitory effect on the 3D proliferation of AGS and GP2d cells.

[0175] Test Example 2: Biological Evaluation of AsPC-1 Cell 3D Proliferation Inhibition Experiment

[0176] On day 1, AsPC-1 cells that had grown well and reached 70%-80% confluence were digested and resuspended in RPMI 1640 (Hyclone, SH30809.01) medium containing 10% FBS, and the cell density was adjusted to the desired level. 90 μL of cell suspension was added to each well of a U-shaped, low-adsorption 96-well plate (Corning, CLS7007-24EA), resulting in a cell density of 1500 cells / well. The cell plates were centrifuged at 2500 rpm for 5 minutes and then incubated overnight at 37°C in a 5% CO2 incubator. On day 2, the 20 mM test compound dissolved in DMSO was diluted to an initial concentration of 2 mM with DMSO, and then serially diluted 5-fold for a total of 9 concentration points. The control wells contained DMSO. The serially diluted compounds were then further diluted 20-fold with medium. 10 μL of the diluted test compound was added to each well of the cell plate, resulting in a final concentration of 10 μM, representing the 9 concentration points of the 5-fold serial dilutions. Cell culture wells containing 0.5% DMSO were set up as solvent control wells, and wells containing only culture medium and 0.5% DMSO were set as blank control wells. Each compound concentration and control well was replicated, with a final DMSO concentration of 0.5% in each well. The cell culture plates were centrifuged at 2500 rpm for 3 minutes and then incubated at 37°C in a 5% CO2 incubator for 5 days. On the seventh day, the 96-well cell culture plates were removed, and 50 μL of luminescent cell viability assay reagent was added to each well. In the 3D Cell Viability Assay (Promega, G9683), after shaking at room temperature in the dark for 25 minutes, mix thoroughly by pipetting up and down, then transfer 100 μL to each well into a white, opaque OptiPlate. TM -96-well plates (PerkinElmer, 6005290) were used to read the luminescence signal values ​​using a multi-functional microplate reader (PerkinElmer, EnVision2105).

[0177] Calculate the inhibition rate using the following formula: Inhibition rate = (Luminous value) / (Emitting value) 溶媒对照孔 -Luminescence value 受试化合物 ) / (luminous value) 溶媒对照孔 -Luminescence value 空白对照孔 ()×100%. Using GraphPadPrism software, curves were plotted based on the concentrations of the compounds and their corresponding inhibition rates, and the IC50 of the compounds was calculated. 50 value.

[0178] Table 2. Data on the 3D proliferation inhibition activity of AsPC-1 cells

[0179] Example number <![CDATA[AsPC-1 / IC 50 (nM)]]> 1 2.4

[0180] Conclusion: The compound disclosed herein has a good inhibitory effect on the 3D proliferation of AsPC-1 cells.

[0181] Test Example 3: Pharmacokinetic Evaluation

[0182] 1. Abstract

[0183] Taking balb / c nude mice as the test animals, the LC / MS / MS method was used to determine the drug concentrations in the plasma of balb / c nude mice at different times after intragastric administration (i.g.) of the compound of the example. The pharmacokinetic behavior of the compound of the present disclosure in balb / c nude mice was studied, and its pharmacokinetic characteristics were evaluated.

[0184] 2. Test Scheme

[0185] 2.1 Test Drug

[0186] Compound 1.

[0187] 2.2 Test Animals

[0188] Nine balb / c nude mice, female, were provided by Vital River Laboratory Animal Technology Co., Ltd., with the production license SCXK(Zhe)2019-0001.

[0189] 2.3 Drug Preparation

[0190] Weigh a certain amount of the test compound respectively, add 5% DMF + 45% PG + 50% (10% HS15 - pH7.4 buffer) + 400mpk SNAC, and prepare a colorless and clear solution of 4mg / mL.

[0191] 2.4 Drug Administration

[0192] The drug administration dose was 40.0mg / kg, and the drug administration volume was 10mL / kg.

[0193] 3. Operations

[0194] Before drug administration and at 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 11.0, 24.0 hours after drug administration, 0.1 mL of blood was collected from the orbital cavity, placed in an EDTA-K2 anticoagulant test tube, centrifuged at 10000 rpm for 1 minute (4°C), the plasma was separated within 1 hour, and stored at -20°C for later measurement. The blood collection to centrifugation process was carried out under ice bath conditions.

[0195] Determine the content of the test compound in the plasma of balb / c nude mice after administering drugs at different concentrations: Take 25 μL of the plasma samples of balb / c nude mice at each time after drug administration, add 200 μL of acetonitrile containing the internal standard (Compound 1: the internal standard is verapamil 100 ng / mL), vortex for 5 minutes, and centrifuge at 3700 rpm for 20 minutes. Mix the supernatant with water (1:1). Take 0.5 - 2 μL of the supernatant for LC / MS / MS analysis.

[0196] 4. Pharmacokinetic Parameter Results

[0197] Table 3. Pharmacokinetic parameters of the compounds disclosed herein in Balb / c nude mice.

[0198]

[0199] Conclusion: The compound disclosed herein exhibits favorable pharmacokinetic properties in balb / c nude mice.

[0200] Example 2 Preparation of free crystal form A

[0201] 5 mg of the compound shown in Formula 1 was dissolved in 0.175 mL of methanol, and the solvent was evaporated at room temperature to obtain a solid. X-ray powder diffraction analysis identified the product as free crystalline form A, and the XRPD spectrum is shown below. Figure 1 The positions of its characteristic peaks are shown in Table 4. The DSC spectrum shows endothermic peaks at 68.18℃, 106.18℃, and 160.02℃. The TGA spectrum shows a weight loss of 3.92% between 31℃ and 115℃.

[0202] Table 4

[0203]

[0204]

[0205] Example 3: Preparation of free crystal form A

[0206] 5 mg of the compound shown in Formula 1 was dissolved in the solvent in Table 5, and the solvent was evaporated at room temperature to obtain a solid. X-ray powder diffraction analysis showed that the product was in the free crystalline form A.

[0207] Table 5

[0208] solvent Solvent volume (mL) result 10% water / methanol 0.275 Crystal form A Acetonitrile / methanol (1:1) 0.25 Crystal form A

[0209] Example 4: Preparation of free crystal form A

[0210] Add 5 mg of the compound shown in Formula 1 to 0.5 mL of solvent, as shown in Table 6 below, and stir at room temperature for 4 days to obtain the title product.

[0211] Table 6

[0212] solvent Crystal form water A Acetonitrile A n-Heptane A 7% water / ethanol A 50% methanol / water A Cyclohexane A Isopropyl ether A

[0213] Example 5: Preparation of free crystal form B

[0214] 5 mg of the compound shown in Formula 1 was dissolved in 0.025 mL of tetrahydrofuran / ethanol (2:1), and the mixture was subjected to three cycles of heating and cooling at 40-5 °C at a rate of 1 °C / min. A solid precipitated out, was centrifuged, and dried under vacuum at 40 °C to obtain the title product. X-ray powder diffraction analysis identified this product as free crystalline form B. The XRPD spectrum is shown below. Figure 2 The positions of its characteristic peaks are shown in Table 7. The DSC spectrum shows endothermic peaks at 127.5℃ and 161.52℃. The TGA spectrum shows a weight loss of 2.72% between 30℃ and 143℃.

[0215] Table 7

[0216]

[0217] Example 6 Preparation of free crystal form B

[0218] 5 mg of the compound shown in Formula 1 was dissolved in 0.425 mL of ethyl acetate / ethanol (1:1), and the solvent was evaporated at room temperature to obtain a solid. X-ray powder diffraction analysis showed that the product was in the free crystalline form B.

[0219] Example 7 Preparation of free crystal form B

[0220] Five mg of the amorphous compound shown in Formula 1 was added to 0.075 mL of ethanol, and the mixture was stirred at room temperature to crystallize, yielding a solid. X-ray powder diffraction analysis showed that the product was in a free crystalline state, form B.

[0221] Example 8: Preparation of free crystalline form C

[0222] Add 5 mg of the compound shown in Formula 1 to 0.5 mL of ethanol, stir at room temperature for 4 days, centrifuge, and dry the solid under vacuum at 40 °C to obtain the title product. X-ray powder diffraction analysis identified this product as free crystalline form C, and the XRPD spectrum is shown below. Figure 3 The positions of its characteristic peaks are shown in Table 8. The DSC spectrum shows an endothermic peak at 155.84℃. The TGA spectrum shows a weight loss of 4.88% between 30℃ and 150℃.

[0223] Table 8

[0224]

[0225]

[0226] Example 9: Preparation of Free Crystal Form C

[0227] Add 5 mg of the compound shown in Formula 1 to 0.5 mL of solvent, as shown in Table 9 below, stir at room temperature for 4 days, centrifuge, and dry the solid under vacuum at 40 °C. X-ray powder diffraction analysis showed that the product is free crystalline form C.

[0228] Table 9

[0229] solvent Crystal form Isopropanol C n-Propanol C

[0230] Example 10 Preparation of free crystal form D

[0231] 5 mg of the compound shown in Formula 1 was dissolved in 0.1 mL of 2-methyltetrahydrofuran, and 0.5 mL of methyl tert-butyl ether was added. The mixture was stirred at room temperature to precipitate, centrifuged, and the solid was dried under vacuum at 40 °C to obtain the title product. X-ray powder diffraction analysis identified this product as free crystal form D. The XRPD spectrum is shown below. Figure 4 The positions of its characteristic peaks are shown in Table 10. The DSC spectrum shows that the endothermic peak has a peak value of 195.22℃. The TGA spectrum shows that the weight loss is 0.49% from 30℃ to 132℃.

[0232] Table 10

[0233]

[0234] Example 11 Preparation of free crystal form D

[0235] Add 5 mg of the compound shown in Formula 1 to 0.5 mL of solvent, as shown in Table 11 below, stir at room temperature for 4 days, centrifuge, and dry the solid under vacuum at 40 °C to obtain the title product.

[0236] Table 11

[0237]

[0238]

[0239] Example 12 Preparation of free crystal form D

[0240] 5 mg of the amorphous form of the compound shown in Formula 1 was dissolved in 0.4 mL of methyl tert-butyl ether, stirred to precipitate, centrifuged, and the solid was dried under vacuum at 40 °C to obtain the title product.

[0241] Example 13 Preparation of free crystal form E

[0242] 5 mg of the compound shown in Formula 1 was dissolved in 0.1 mL of acetone, and 0.5 mL of methyl tert-butyl ether was added. The mixture was stirred at room temperature to precipitate the product. After centrifugation, the solid was dried under vacuum at 40 °C to obtain the title product. X-ray powder diffraction analysis identified this product as free crystalline form E. The XRPD spectrum is shown below. Figure 5 The positions of its characteristic peaks are shown in Table 12. The DSC spectrum shows that the endothermic peak has a peak value of 188.85℃. The TGA spectrum shows that the weight loss is 2.37% from 32℃ to 160℃.

[0243] Table 12

[0244]

[0245] Example 14 Preparation of free crystal form E

[0246] 5 mg of the compound shown in Formula 1 was dissolved in solvent I in Table 13, and solvent II in Table 13 was added. The mixture was stirred at room temperature to precipitate, centrifuged, and the solid was dried under vacuum at 40°C to obtain the title product. X-ray powder diffraction analysis showed that the product was in the free crystalline form E.

[0247] Table 13

[0248]

[0249]

[0250] Example 15 Preparation of free crystal form E

[0251] Add 5 mg of the compound shown in Formula 1 to 0.5 mL of solvent, as shown in Table 14 below, stir at room temperature for 4 days, centrifuge, and dry the solid under vacuum at 40 °C to obtain the title product.

[0252] Table 14

[0253] solvent Crystal form Ethyl acetate / n-heptane (1:1) E acetone E

[0254] Example 16 Preparation of free crystal form E

[0255] 5 mg of the compound shown in Formula 1 was dissolved in 1.2 mL of ethyl acetate / methyl tert-butyl ether (v / v = 1:5), and the solvent was evaporated at room temperature to give the title product.

[0256] Example 17 Preparation of free crystal form E

[0257] 5 mg of the amorphous compound shown in Formula 1 was dissolved in 0.05 mL of solvent, as shown in Table 15 below. The mixture was stirred to precipitate, centrifuged, and the solid was dried under vacuum at 40 °C to obtain the title product.

[0258] Table 15

[0259] solvent Crystal form 2-Methyltetrahydrofuran E dichloromethane E

[0260] Example 18 Preparation of free crystal form F

[0261] 5 mg of the compound shown in Formula 1 was dissolved in 0.15 mL of dichloromethane, and 0.75 mL of n-heptane was added. The mixture was stirred at room temperature to precipitate, centrifuged, and the solid was dried under vacuum at 40 °C to obtain the title product. X-ray powder diffraction analysis identified this product as free crystalline form F. The XRPD spectrum is shown below. Figure 6The positions of its characteristic peaks are shown in Table 16. The DSC spectrum shows that the endothermic peak has a peak value of 163.53℃. The TGA spectrum shows that the weight loss is 1.70% from 32℃ to 102℃.

[0262] Table 16

[0263]

[0264]

[0265] Example 19 Preparation of free crystal form G

[0266] Add 5 mg of the amorphous form of the compound shown in Formula 1 to 0.05 mL of methanol, stir at room temperature to induce crystallization, centrifuge, and dry the solid under vacuum at 40 °C to obtain the title product. X-ray powder diffraction analysis identified this product as the free crystalline form G, and the XRPD spectrum is shown below. Figure 7 The positions of its characteristic peaks are shown in Table 17. The DSC spectrum shows that the endothermic peak has a peak value of 215.63℃. The TGA spectrum shows that the weight loss is 0.87% from 31℃ to 105℃.

[0267] Table 17

[0268]

[0269] Example 20 Preparation of free crystal form H

[0270] 5 mg of the amorphous compound shown in Formula 1 was added to 0.5 mL of p-xylene, stirred at room temperature for 3 days, centrifuged, and the solid was dried under vacuum at 40 °C to obtain the title product. X-ray powder diffraction analysis identified this product as the free crystalline form H, and the XRPD spectrum is shown below. Figure 8 The positions of its characteristic peaks are shown in Table 18. The DSC spectrum shows that the endothermic peak has a peak value of 154.82℃. The TGA spectrum shows that the weight loss is 5.82% from 31℃ to 170℃.

[0271] Table 18

[0272]

[0273] Example 21 Preparation of free crystal form H

[0274] The amorphous compound shown in Formula 1 was added to 1 mL of solvent, as shown in Table 19 below, stirred overnight at room temperature, centrifuged, and the solid was dried under vacuum at 40 °C to obtain the title product.

[0275] Table 19

[0276] The amount of the compound shown in Formula 1 (mg) solvent Solvent volume (mL) Crystal form 80 Methyl tert-butyl ether 1 H 112 Ethyl acetate 1 H

[0277] Example 22 Preparation of free crystal form H

[0278] 78 mg of the amorphous form of the compound shown in Formula 1 was dissolved in 0.8 mL of DCM and added to 4 mL of n-heptane. The solid precipitated out, and the mixture was stirred overnight at room temperature. After centrifugation, the solid was dried under vacuum at 40 °C to obtain the title product.

[0279] Example 23 Preparation of free crystal form H

[0280] Add 7 mg of the amorphous form of the compound shown in Formula 1 to 0.1 mL of ethyl acetate and 10.7 μL of 2 M citric acid ethanol solution (the ligand can be 10.7 μL of 2 M tartaric acid ethanol solution or 2.5 mg of fumaric acid), stir to induce crystallization, centrifuge, and dry the solid under vacuum at 40 °C to obtain the title product.

[0281] Example 24 Preparation of Free Crystal Form I

[0282] 7 mg of the amorphous compound shown in Formula 1 was added to 0.1 mL of ethyl acetate and 10.7 μL of 2M acetic acid-ethanol solution. The mixture was stirred to precipitate, centrifuged, and dried under vacuum to obtain the title product. X-ray powder diffraction analysis identified this product as free crystal form I. The XRPD spectrum is shown below. Figure 9 The positions of its characteristic peaks are shown in Table 20. The DSC spectrum shows endothermic peaks at 162.18℃ and 224.15℃. The TGA spectrum shows a weight loss of 4.27% between 33℃ and 140℃.

[0283] Table 20

[0284]

[0285] Example 25 Preparation of free crystal form J

[0286] Five mg of the amorphous compound shown in Formula 1 was heated to 215 °C to obtain the title product. X-ray powder diffraction analysis identified this product as free crystal form J, and the XRPD spectrum is shown below. Figure 10 The positions of its characteristic peaks are shown in Table 21.

[0287] Table 21

[0288]

[0289]

[0290] Test Example 4: Stability of Influencing Factors

[0291] The aforementioned crystal forms A, D, E, G, and H were laid out flat in the open, and the stability of the samples was investigated under high temperature (40℃, 60℃) and high humidity (RH 75%, RH 92.5%) conditions. The sampling period was 30 days.

[0292] Table 22

[0293]

[0294]

[0295]

[0296] Conclusion: Free crystalline forms A, D, E, G, and H exhibit good physical and chemical stability after 30 days under high humidity (75% RH, 92.5% RH) conditions. Under light exposure for 30 days, they also show good physical stability, but their chemical purity decreases. Free crystalline forms A, D, E, and G also exhibit good physical stability after 30 days under high temperature (40℃, 60℃) conditions, but their chemical purity decreases.

[0297] Test Example 5: Long-term accelerated test stability

[0298] The stability of the aforementioned crystal forms A, D, E, G, and H was investigated under conditions of 25℃ / 60%RH and 40℃ / 75%RH, respectively.

[0299] Table 23

[0300]

[0301]

[0302] Conclusion: Free crystalline forms A and H exhibit good physical and chemical stability after one month at 25℃ / 60%RH and 40℃ / 75%RH; free crystalline forms D, E, and G exhibit good physical and chemical stability after two months at 25℃ / 60%RH and 40℃ / 75%RH.

Claims

1. A crystal form E of the compound shown in Formula 1, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed as an angle, shows characteristic peaks at 5.144, 8.390, 10.312, 13.575, and 20.

130. 。 2. The crystal form E according to claim 1, with a diffraction angle of 2... θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 5.144, 8.390, 10.312, 13.575, 14.845, 15.568, 16.293, 17.776, 18.488, and 20.

130.

3. The crystal form E according to claim 1, with a diffraction angle of 2... θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 5.144, 8.390, 10.312, 13.575, 14.845, 15.568, 16.293, 17.776, 18.488, 20.130, 21.350, 22.029, 23.036, and 31.

408.

4. The crystal form E according to claim 1, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern expressed in terms of angle is shown in Figure 5.

5. A method for preparing crystal form E as described in any one of claims 1-4, wherein the method is selected from any one of the following methods: Method 1: Dissolve the compound of Formula 1 in solvent II, add solvent III, and stir. Solvent II is selected from acetone, ethyl acetate, and tetrahydrofuran / ethanol in a volume ratio of 2:1, and solvent III is selected from methyl tert-butyl ether and n-heptane. Method 2: Add the compound of Formula 1 to 50% ethyl acetate / n-heptane or add the compound of Formula 1 to acetone and stir; Method 3: Dissolve the compound of Formula 1 in solvent IV and stir. Solvent IV is selected from ethyl acetate / methyl tert-butyl ether, 2-methyltetrahydrofuran, and dichloromethane in a volume ratio of 1:

5.

6. A crystal form G of the compound shown in Formula 1, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed as an angle, shows characteristic peaks at 10.692, 16.093, 16.447, 17.922, 24.567, and 33.

426. 。 7. The crystal form G of the compound of formula 1 according to claim 6, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 7.724, 8.701, 10.692, 12.719, 13.325, 16.093, 16.447, 17.922, 19.340, 20.746, 21.219, 24.567, and 33.

426.

8. The crystal form G of the compound of formula 1 according to claim 6, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 7.724, 8.701, 10.692, 12.719, 13.325, 16.093, 16.447, 17.922, 19.340, 19.891, 20.746, 21.219, 22.886, 23.577, 24.567, 25.386, 26.330, and 33.

426.

9. The crystal form G according to claim 6, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern expressed in terms of angle is shown in Figure 7.

10. A method for preparing crystal form G as described in any one of claims 6-9, the method comprising adding the amorphous compound of formula 1 to methanol and stirring.

11. A crystal form H of the compound shown in Formula 1, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed as an angle, shows characteristic peaks at 5.099, 7.096, 9.872, 15.784, and 19.

440. 。 12. The crystal form H of the compound of formula 1 according to claim 11, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 5.099, 7.096, 8.528, 9.872, 11.100, 13.212, 15.784, 19.440, 20.621, 21.120, and 22.

425.

13. The crystal form H according to claim 11, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern expressed in terms of angle is shown in Figure 8.

14. A method for preparing crystal form H according to any one of claims 11-13, wherein the method is selected from any one of the following methods: Method 1: Add the amorphous compound of Formula 1 to solvent V and stir. Solvent V is selected from p-xylene, methyl tert-butyl ether, and ethyl acetate. Method 2: Dissolve the amorphous compound of Formula 1 in dichloromethane, add it to n-heptane, and stir; Method 3: The amorphous form of compound 1 is added to ethyl acetate, followed by the addition of a ligand and stirring. The ligand is selected from citric acid ethanol solution, tartaric acid ethanol solution, and fumaric acid solid.

15. The crystal form according to any one of claims 1-4, 6-9, and 11-13, wherein the 2 θ The angular error range is ±0.

20.

16. A pharmaceutical composition comprising the crystal form as described in any one of claims 1-4, 6-9, 11-13 and optionally a pharmaceutically acceptable excipient.

17. A method for preparing a pharmaceutical composition, comprising the step of mixing the crystal form according to any one of claims 1-4, 6-9, 11-13 with a pharmaceutically acceptable excipient.

18. Use of the crystal form according to any one of claims 1-4, 6-9, 11-13 or the pharmaceutical composition according to claim 16 in the preparation of a KRAS inhibitor.

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