Crystal form of pyrimidine heterocyclic compound and preparation method therefor

Abstract

To provide a crystal form of a pyrimidine heterocyclic compound and a pharmaceutical composition comprising the crystal form.SOLUTION: The present invention provides a crystal form A of the compound of formula (II), wherein n is 2, characterized by an X-ray powder diffraction pattern with characteristic diffraction peaks at 2θ angles of 8.514±0.200°, 14.689±0.200°, and 18.122±0.200°, and a pharmaceutical composition for the treatment of solid tumors comprising the crystal form A.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application is Chinese Patent Application Publication No. 202111062619.1, filed on September 10, 2021; Priority is claimed from the specification of Chinese Patent Application Publication No. 202211034826.0, filed on August 26, 2022.

[0002] <Technical field> The present disclosure relates to crystalline forms of certain pyrimidine heterocyclic compounds and methods for their preparation, and in particular to methods for preparing and using the compound represented by formula (II) and crystalline forms thereof. [Background technology]

[0003] RAS oncogene mutations are the most common activating mutations in human cancers, occurring in 30% of human tumors. The RAS gene family includes three subtypes (KRAS, HRAS, and NRAS), of which KRAS subtype mutations cause 85% of RAS-driven cancers. KRAS mutations are commonly found in solid tumors, such as lung adenocarcinoma, pancreatic ductal carcinoma, and colorectal cancer. In KRAS-mutated tumors, 80% of oncogene mutations occur at codon 12, with the most common mutations including p.G12D (41%), p.G12V (28%), and p.G12C (14%).

[0004] The official name of the KRAS gene is Kirsten rat sarcoma viral oncogene homolog. KRAS plays a crucial role in regulating cell proliferation signaling. Upstream cell surface receptors, such as EGFR (ErbB1), HER2 (ErbB2), ErbB3, and ErbB4, receive external signals and then transmit them downstream via RAS proteins. When inactivated, KRAS binds strongly to GDP (guanosine diphosphate). After activation by guanosine exchange factors, such as SOS1, KRAS binds to GTP (guanosine triphosphate) and becomes kinase-active. After gene mutation, KRAS transmits growth and proliferation signals independently to downstream pathways unrelated to upstream growth factor receptor signals, potentially leading to uncontrolled cell proliferation and tumor progression. Meanwhile, the presence or absence of KRAS gene mutations is also an important indicator of tumor prognosis.

[0005] KRAS was the first oncogene discovered, but it has long been considered an undruggable target. Until 2019, Amgen and Mirati Therapeutics published clinical study results for their small molecule KRAS inhibitors, AMG510 and MRTX849, respectively, confirming the clinical efficacy of KRAS inhibitors in the clinical treatment of tumors for the first time. Both AMG510 and MRTX849 are irreversible small molecule inhibitors that inhibit KRAS activity by forming irreversible covalent bonds with the cysteine ​​residues of KRAS G12C mutant proteins.

[0006] KRAS plays a crucial role in regulating cell proliferation signaling. After receiving external signals, upstream cell surface receptors, such as EGFR (ErbB1), HER2 (ErbB2), ErbB3, and ErbB4, transmit signals downstream via RAS proteins. When inactive, KRAS binds strongly to GDP (guanosine diphosphate). After activation by guanosine exchange factors, such as SOS1, KRAS binds to GTP (guanosine triphosphate) and becomes kinase-active. KRAS is an important member of the RAS family of proteins. After gene mutation, KRAS transmits growth and proliferation signals independently to downstream pathways unrelated to upstream growth factor receptor signals, potentially leading to uncontrolled cell proliferation and tumor progression. Meanwhile, the presence or absence of mutations in the KRAS gene is also an important indicator of tumor prognosis.

[0007] Statistical results show that 12-36% of lung adenocarcinomas are caused by KRAS mutations, 27-56% of colon cancers, 90% of pancreatic cancers, 21% of endometrial cancers, and 12-36% of lung adenocarcinomas are caused by KRAS, indicating a large patient population. Among KRAS gene mutations, 97% occur at amino acid residues 12 or 13. G12D, G12V, and G13D mutations are difficult to develop new drugs for, while the KRAS (G12C) mutation, in which glycine at position 12 is replaced by cysteine, offers a good opportunity for the development of covalent inhibitors. Summary of the Invention [Means for solving the problem]

[0008] The present disclosure provides a compound of formula (II): [ka]

[0009] In the formula, n is selected from 0 to 3.

[0010] In some embodiments of the present disclosure, n is selected from 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0.

[0011] In some embodiments of the present disclosure, n is selected from 0.5, 1, 1.5, 2, 2.5, and 3.

[0012] In some embodiments of the present disclosure, n is 2.

[0013] The present disclosure also provides a compound of formula (II): [ka]

[0014] In the formula, n is selected from 0 to 2.

[0015] In some embodiments of the present disclosure, n is selected from 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 2.

[0016] In some embodiments of the present disclosure, n is selected from 0.5, 1, 1.5, and 2.

[0017] In some embodiments of the present disclosure, n is 2.

[0018] The present disclosure also provides crystalline form A of the compound of formula (II), characterized by an X-ray powder diffraction pattern having characteristic diffraction peaks at 2θ angles of 8.514±0.200°, 14.689±0.200°, and 18.122±0.200°.

[0019] In some embodiments of the present disclosure, the crystalline form A has an X-ray powder diffraction pattern with characteristic diffraction peaks at 2θ angles of 6.218±0.200°, 8.514±0.200°, 12.299±0.200°, 14.689±0.200°, 16.903±0.200°, 18.122±0.200°, 18.927±0.200°, and 25.580±0.200°.

[0020] In some embodiments of the present disclosure, the crystalline form A has an X-ray powder diffraction pattern with characteristic diffraction peaks at 2θ angles of 6.218±0.200°, 8.514±0.200°, 11.663±0.200°, 12.299±0.200°, 14.689±0.200°, 16.903±0.200°, 18.122±0.200°, 18.927±0.200°, 19.364±0.200°, 20.386±0.200°, 21.914±0.200°, and 25.580±0.200°.

[0021] In some embodiments of the present disclosure, the crystalline form A has the following molecular weights: 6.218±0.200°, 8.514±0.200°, 11.663±0.200°, 12.299±0.200°, 14.689±0.200°, 16.903±0.200°, 18.122±0.200°, 18.927±0.200°, 19.364±0.200°, 20.122±0.200°, 21.122±0.200°, 22.122±0.200°, 23.122±0.200°, 24.122±0.200°, 25.122±0.200°, 26.122±0.200°, 27.122±0.200°, 28.122±0.200°, 29.122±0.200°, 30.122±0.200°, 31.122±0.200°, 32.122±0.200°, 33.122±0.200°, 34.122±0.200°, 35.122±0.200°, 36.122±0.200°, 37.122±0.200°, 38.122±0.200°, 39.122±0.200°, 40.122±0.200°, 41.122±0.200°, 42.122±0.200°, 43.122±0.200 It has a powder X-ray diffraction pattern having characteristic diffraction peaks at 2θ angles of 20.0±0.200°, 20.386±0.200°, 21.914±0.200°, 22.640±0.200°, 25.580±0.200°, 25.988±0.200°, 27.147±0.200°, and 27.715±0.200°.

[0022] In some embodiments of the present disclosure, the crystalline form A has an X-ray powder diffraction pattern with characteristic diffraction peaks at 2θ angles of 6.218°, 8.514°, 11.663°, 12.299°, 14.689°, 16.903°, 18.122°, 18.554°, 18.927°, 19.364°, 20.386°, 21.914°, 22.640°, 23.867°, 24.553°, 24.806°, 25.580°, 25.988°, 27.147°, 27.715°, 29.135°, and 31.799°.

[0023] In some embodiments of the present disclosure, the crystalline form A has characteristic diffraction peaks at 2θ angles of 8.514±0.200° and 14.689±0.200°, and optionally at 6.218±0.200°, and / or 11.663±0.200°, and / or 12.299±0.200°, and / or 16.903±0.200°, and / or 18.122±0.200°, and / or 18.554±0.200°, and / or 18.927±0.200°, and / or 19.364±0.200°, and / or 20.386±0.200°, and / or 21.914±0.200°, and / or 22.64±0.200°, and / or 23.867±0.200°, and / or 24.553±0.200°, and / or 24.806±0.200°, and / or 25.58±0.200°, and / or 25.988±0.200°, and / or 27.147±0.200°, and / or 27.715±0.200°, and / or 29.135±0.200°, and / or 31.799±0.200°.

[0024] In some embodiments of the present disclosure, the above crystalline form A is disclosed, having the XRPD pattern shown in FIG.

[0025] In some embodiments of the present disclosure, the XRPD pattern resolution data for crystalline form A is shown in Table 1. [Table 1]

[0026] In some embodiments of the present disclosure, the differential scanning calorimetry curve of crystalline form A has an endothermic peak at 115.37°C ± 3°C.

[0027] In some embodiments of the present disclosure, the DSC curve of the crystalline form A is shown in FIG.

[0028] In some embodiments of the present disclosure, the thermogravimetric analysis curve of crystalline form A has a maximum weight loss of 5.379% at 150.0±3°C.

[0029] In some embodiments of the present disclosure, the TGA curve of the crystalline form A is shown in FIG.

[0030] The present disclosure also provides a method for preparing crystalline form A of compound of formula (II), comprising: [ka]

[0031] (a) adding a compound of formula (I) to ethanol and stirring the mixture until it becomes clear;

[0032] (b) slowly add water to the system while stirring, and add seed crystals at 20-30°C;

[0033] (c) stirring at 20 to 30°C for 15 hours;

[0034] (d) slowly adding water dropwise to the reaction system at 20 to 30°C and stirring for an additional 1 to 3 hours;

[0035] (e) filtering and recovering the solids; The present invention provides a method comprising:

[0036] The present disclosure also provides the use of the above compound and its crystalline form A in the manufacture of a medicament for treating solid tumors.

[0037] In some embodiments of the present disclosure, the solid tumor is lung cancer and rectal cancer. [Effects of the Invention]

[0038] The compounds of the present disclosure have good cell growth inhibitory activity against KRASG12C mutant MIA-PA-CA-2 cell line and NCI-H358 cells. The compounds of the present disclosure have good stability in liver microsomes, hepatocytes, plasma, and whole blood, as well as good PK properties and significant antitumor effects. Crystalline Form A is stable, resistant to light and heat, and has good PK properties.

[0039] Definitions and Explanations Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings: A particular phrase or term should not be considered vague or unclear in the absence of a specific definition, but should be understood in its conventional sense. When trade names appear herein, they are intended to refer to the corresponding commercial product or its active ingredient.

[0040] Intermediate compounds of the present disclosure can be prepared by a variety of synthetic methods known to those skilled in the art, such as the specific embodiments listed below, embodiments formed by combining the specific embodiments listed below with other chemical synthetic methods, and equivalent alternative methods known to those skilled in the art. Alternative embodiments include, but are not limited to, the examples of the present disclosure.

[0041] The chemical reactions in certain embodiments of the present disclosure are accomplished in suitable solvents, which must be appropriate for the chemical transformations of the present disclosure and the reagents and materials required. To obtain compounds of the present disclosure, one skilled in the art may need to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0042] The present disclosure will be described in detail below with reference to examples, but these examples are not intended to limit the present disclosure in any way.

[0043] All solvents used in this disclosure are commercially available and can be used without further purification.

[0044] The structures of the compounds disclosed herein can be confirmed by conventional methods known to those skilled in the art. Where the present disclosure relates to the absolute configuration of a compound, the absolute configuration can be confirmed by conventional techniques in the art, such as single crystal X-ray diffraction (SXRD). In single crystal X-ray diffraction (SXRD), diffraction intensity data of a grown single crystal is collected using a Bruker D8 venture diffractometer equipped with a CuKα radiation source in a φ / ω scan mode. After the data collection, the crystal structure is further analyzed by a direct method (Shelxs97) to confirm the absolute configuration.

[0045] Compounds are named according to common naming principles in the art or by ChemDraw® software; commercially available compounds are named by their vendor directory name.

[0046] X-ray powder diffraction (XRPD) methods used in this disclosure Instrument model: Bruker D2 Phaser X-ray diffractometer

[0047] Test method: About 10-20 mg of sample was used for XRPD detection.

[0048] The detailed XRPD parameters were as follows:

[0049] Light tube: Cu, kα (λ=1.54184Å).

[0050] Light tube voltage: 30kV, light tube current: 10mA

[0051] Divergence slit: 0.60 mm

[0052] Detector slit: 5.827 mm

[0053] Anti-scatter slit: 0 mm

[0054] Scanning range: 3~40deg

[0055] Step size: 0.02 deg

[0056] Step length: 0.2 seconds

[0057] Differential Scanning Calorimetry (DSC) Method Used in This Disclosure Device model: NETZSCH DSC214 DSC21400A-0958-L

[0058] Test method: A sample (approximately 4.02 mg) was placed in a DSC aluminum pot for testing and heated from 30°C (room temperature) to 400°C at a heating rate of 10°C / min under 50 mL / min N2.

[0059] Thermogravimetric analysis (TGA) methods used in this disclosure Instrument model: TA Discovery TGA5500 Thermogravimetric Analyzer

[0060] Test method: Samples (2-5 mg) were placed in a TGA platinum pot for testing. Under 25 mL / min of N2, the samples were heated from room temperature to 350°C at a heating rate of 10°C / min, or until a 20% weight loss was achieved.

[0061] Single Crystal X-ray Diffraction Methods Used in This Disclosure Instrument model: Bruker D8 VENTURE CMOS Photon II diffractometer with helios mx multilayer monochromator

[0062] Test method: 0.0133 g of crystalline form A of compound of formula (II) was dissolved in 2 mL of acetonitrile at room temperature. The sample solution was added to a 4 mL semi-sealed sample vial and allowed to evaporate slowly at room temperature. After 10 days, colorless bulky crystals were obtained. The temperature for the diffraction experiment was T=173(2)K.

[0063] Equipment parameters:

[0064] Bruker D8 VENTURE CMOS Photon II Diffractometer with Helios MX Multilayer Monochromator

[0065] Cryogenic system: Oxford Cryostream 800 Cu: λ=1.54184Å, 2.5kW

[0066] Distance from crystal to detector: d = 45 mm

[0067] Tube voltage: 50kV

[0068] Tube current: 50mA [Brief explanation of the drawings]

[0069] [Figure 1] FIG. 1 is an XRPD pattern of crystalline form A of the compound of formula (II) using Cu-Kα radiation.

[0070] [Figure 2] FIG. 2 is a DSC curve of crystalline form A of the compound of formula (II).

[0071] [Figure 3] FIG. 3 is a TGA curve of crystalline form A of the compound of formula (II).

[0072] [Figure 4] FIG. 4 shows the change in tumor volume over time at various doses of the compound of formula (I).

[0073] [Figure 5] FIG. 5 shows the change in body weight of animals over time at various doses of the compound of formula (I).

[0074] [Figure 6] FIG. 6 is an ellipsoidal representation of the single crystal X-ray diffraction three-dimensional structure of the compound of formula (III). DETAILED DESCRIPTION OF THE INVENTION

[0075] To better understand the contents of the present disclosure, the present disclosure will be further described below in conjunction with specific examples, which are not intended to limit the contents of the present disclosure.

[0076] Example 1: Preparation of compounds of formula (I) [ka] [ka]

[0077] Step 1: Synthesis of Compound 1-2 In a 5-L three-neck flask, compound 1-1 (250 g, 2.00 mol, 1 equivalent), anhydrous potassium carbonate (690.26 g, 4.99 mol, 2.5 equivalents), and potassium iodide (331.62 g, 2.00 mol, 1 equivalent) were added to N-methylpyrrolidone (2.5 L). p-Methoxybenzyl chloride (641.36 g, 4.10 mol, 557.71 mL, 2.05 equivalents) was then added dropwise, and the reaction solution became yellow and cloudy. The resulting mixture was stirred under nitrogen in an oil bath at 120 °C for 5 hours. Six batches of the reaction solution (250 × 6) were combined and added to 20 L of water. Then, 10 L of methyl tert-butyl ether was added, and the mixture was stirred. After separation, the organic phase was recovered, and the aqueous phase was extracted with methyl tert-butyl ether (5 L × 1). The organic phases were combined, washed with saturated brine (10 L x 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 1-2. 3 L of petroleum ether was added to the crude product, and the mixture was slurried overnight. The slurry system was milky white and cloudy, and was filtered through a Buchner funnel. The filter cake was washed with petroleum ether (500 mL x 3), and compound 1-2 was obtained from the filter cake. 1H NMR (400 MHz, CDCl3) δ = 7.23-7.18 (m, 4H), 6.91-6.87(m, 1H), 6.82-6.76 (m, 4H), 6.65 -6.59(m, 2H), 4.20 (s, 4H), 3.79(s, 6H), 2.19 (s, 3H). LCMS: MS m / z = 366.1 [M+H] + .

[0078] Step 2: Synthesis of Compounds 1-3

[0079] 2,2,6,6-Tetramethylpiperidine (2.44 kg, 17.29 mol, 2.94 L, 4 equiv.) was added to anhydrous tetrahydrofuran (15 L), and the mixture was cooled to -5 to 0 °C. The system was purged with nitrogen three times, and n-butyllithium (2.5 M, 6.92 L, 4 equiv.) was added dropwise under nitrogen. The mixture was reacted at -5 to 0 °C for 15 min and then cooled to -60 °C. A solution of compound 1-2 (1.58 kg, 4.32 mol, 1 equiv.) in tetrahydrofuran (1.5 L) was added dropwise. After the addition was complete, the mixture was reacted at -65 to -60 °C for 0.5 h. N,N-dimethylformamide (3.16 kg, 43.24 mol, 3.33 L, 10 equiv.) was then quickly added, and the mixture was reacted at -60 °C for 10 min. To the reaction solution, 20 L of saturated ammonium chloride was added, and the mixture was extracted with 5 L of methyl tert-butyl ether. The layers were separated. The organic phase was washed with 20 L of saturated ammonium chloride. The aqueous phase was then extracted with 10 L of methyl tert-butyl ether. The layers were separated. The organic phases were combined, washed with saturated brine (12 L x 2), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was then slurried with petroleum ether and methyl tert-butyl ether (3 / 1, 3 L) for 5 hours and then filtered. The filter cake was collected to obtain compound 1-3. 1H NMR (400MHz, CDCl3) δ = 10.43 - 10.35 (m, 1H), 7.21-7.18 (m, 5H), 6.92 - 6.81 (m, 5H), 4.25 (s, 4H), 3.80 (s, 6H), 2.23 (s, 3H). LCMS:MS m / z = 394.2[M+H] + .

[0080] Step 3: Synthesis of Compounds 1-4

[0081] Compound 1-3 (1.17 kg, 2.83 mol, 95% purity, 1 equiv.) was added to N,N-dimethylformamide (5.7 L), and bromosuccinimide (603.35 g, 3.39 mol, 1.2 equiv.) was added in portions at 5 °C. The mixture was reacted at 5–15 °C for 1 h. 5.7 L of water was slowly added to the reaction solution, resulting in the precipitation of a solid. After stirring for 20 min, 11.4 L of water was slowly added. The mixture was stirred for an additional 40 min and then filtered. The filter cake was washed with water (2 L × 2). The crude product was slurried in 7.7 L of a 10:1 mixture of petroleum ether and methyl tert-butyl ether for 12 h and then filtered. The filter cake was washed with 500 mL of a 10:1 mixture of petroleum ether and methyl tert-butyl ether. After concentration in vacuo, the product was sparged with nitrogen for 12 hours to give compound 1-4. 1 H NMR (400MHz, CDCl3) δ = 10.39 (s, 1H), 7.17 (d, J = 8.8 Hz, 4H), 6.89 (d, J = 8.8 Hz, 1H), 6.85-6.82 (m, 4H), 4.22 (s, 4H), 3.79 (s, 6H), 2.28 (s, 3H). LCMS:MS m / z = 472.1[M+H] + , 474.1[M+H] + .

[0082] Step 4: Synthesis of Compounds 1-6

[0083] Compound 1-4 (130 g, 275.22 mmol, 1 equiv.), iodinated ketone (104.83 g, 550.44 mmol, 2 equiv.), and compound 1-5 (264.37 g, 1.38 mol, 175.08 mL, 5 equiv.) were dissolved in DMF (1.3 L). The mixture was stirred under nitrogen at 100 °C for 4 h. The reaction was cooled, filtered, and quenched by pouring into water (1.3 L). The mixture was extracted with methyl tert-butyl ether (400 mL × 2), washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, and filtered. The mother liquor was collected and concentrated to give the crude product. The crude product was slurried with petroleum ether and methyl tert-butyl ether (8 / 1, 300 mL) to give compound 1-6. The mother liquor was collected, concentrated, and purified by column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 5 / 1) to obtain the crude product. The crude product was slurried with petroleum ether and methyl tert-butyl ether (8 / 1, 100 mL) to obtain compound 1-6. The two batches of solid were mixed, slurried with petroleum ether, and filtered. The solid was collected to obtain compound 1-6. 1 H NMR (400MHz, CDCl3) δ = 10.37 (q, J = 4.0 Hz, 1H), 7.18 - 7.11 (m, 4H), 6.89 - 6.82 (m, 4H), 6.73 (d, J = 8.8 Hz, 1H), 4.36 (s, 4H), 3.81 (s, 6H), 2.37 - 2.29 (m, 3H). LCMS: MS m / z =484.0[M+Na] + .

[0084] Step 5: Synthesis of Compounds 1-8

[0085] Sodium bicarbonate (70.21 g, 1.76 mol, 60% purity, 1.8 equiv.) was added to anhydrous tetrahydrofuran (4.5 L) and cooled to -5 °C. The mixture was purged with nitrogen three times, and compound 1-7 (203.82 g, 1.76 mol, 188.72 mL, 1.8 equiv.) was added dropwise under nitrogen. After reacting at -5 to 0 °C for 10 min, n-butyllithium (2.5 M, 702.14 mL, 1.8 equiv.) was added dropwise. The mixture was reacted under nitrogen at -5 to 0 °C for an additional 10 min and then cooled to -10 °C. A solution of compound 1-6 (450 g, 975.19 mmol, 1 equiv.) in tetrahydrofuran (450 mL) was added dropwise, and the mixture was reacted at -10 °C for 10 min. The reaction solution was extracted by slowly adding 5 L of saturated ammonium chloride. The layers were separated. The organic phase was washed with 4 L of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. 4 x 900 mL of petroleum ether was added to the concentrated crude product and washed with shaking. The petroleum ether supernatant was poured off, and the crude product was concentrated in vacuo to give compound 1-8. 1 H NMR (400MHz, CDCl3) δ = 7.18-7.15 (m, 4H), 6.90 - 6.78 (m, 4H), 6.61 (d, J = 8.8 Hz, 1H), 5.72 - 5.57 (m, 1H), 4.31 (m, 4H), 3.81(s, 6H), 3.76(s, 3H), 3.56 (s, 2H), 3.50 - 3.38 (m, 1H), 2.98 - 2.93 (m, 1H), 2.38 - 2.26 (m, 3H). LCMS: MS m / z =578.1[M+H] + .

[0086] Step 6: Synthesis of Compounds 1-9

[0087] Compound 1-8 (1.15 kg, 1.77 mol, 89% purity, 1 equiv.) was added to dichloromethane (5.7 L) and N,N-dimethylformamide dimethyl acetal (337.86 g, 2.84 mol, 376.66 mL, 1.6 equiv.) was added. The mixture was reacted at 25 °C for 1 h and then cooled to 0 °C. Boron trifluoride diethyl etherate (377.27 g, 2.66 mol, 328.06 mL, 1.5 equiv.) was added dropwise at 0-5 °C, and the mixture was reacted for 10 min. LCMS showed the disappearance of the starting material and the appearance of the product MS signal. The reaction solution was extracted by slowly adding 10 L of half-saturated sodium bicarbonate solution. The layers were separated. The organic phase was washed with 5 L of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was divided equally into six portions, and 0.83 L of methyl tert-butyl ether was added to each portion. The mixture was stirred for 20 minutes to precipitate a solid. 0.5 L of a 1:1 mixture of petroleum ether and methyl tert-butyl ether was added, and the mixture was slurried overnight for 16 hours. The six portions were filtered together. The filter cake was rinsed with a 1:1 mixture of petroleum ether and methyl tert-butyl ether (800 mL x 2) and rotary evaporated to dryness. The mother liquor was purified by column chromatography (petroleum ether:ethyl acetate = 100:0 - 0:1) to give compound 1-9. 1 H NMR (400MHz, CDCl3) δ =8.43 (d, J = 0.8 Hz, 1H), 7.21 - 7.10 (m, 4H), 6.91 - 6.81 (m, 4H), 6.70 (d, J = 8.8 Hz, 1H), 5.93 (dd, J = 3.2, 14.8 Hz, 1H), 4.35 (s, 4H), 3.8(s, 3H), 3.81 (s, 6H), 3.38-3.29 (m, 1H), 2.68 (dd, J = 3.6, 16.8 Hz, 1H), 2.39 - 2.24 (m, 3H). LCMS: MS m / z =588.2[M+H] + .

[0088] Step 7: Synthesis of Compounds 1-10

[0089] Compound 1-9 (775 g, 1.32 mol, 1 equiv.) was added to tetrahydrofuran (4 L). The mixture was cooled to -60 °C and purged with nitrogen three times. Then, tri-sec-butylborohydride (1 M, 1.45 L, 1.1 equiv.) was added dropwise under nitrogen. The mixture was allowed to react at -60 °C for 10 min. The reaction solution was slowly added to 3.5 L of 1 M hydrochloric acid solution, and extracted with 2 L of water. The layers were separated. The aqueous phase was further extracted with 2 L of ethyl acetate. The organic phases were combined, and 1 L of ethyl acetate was added. The mixture was washed with 5 L of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was divided into four portions, and 200 mL of methyl tert-butyl ether was added to each portion. The mixture was stirred for 10 min, and 200 mL of petroleum ether was slowly added. After stirring for 0.5 to 1 h, a solid precipitated. An additional 1.6 L of petroleum ether was added in several portions, and the mixture was slurried for 12 hours. The slurry was filtered, and the filter cake was washed three times with 300 mL of a 10:1 mixture of petroleum ether and methyl tert-butyl ether. The solid was collected to give compound 1-10 (1.15 kg). 1 H NMR (400MHz, CDCl3) δ = 7.167-7.14(m, 4H), 6.87-6.83 (m, 4H), 6.63 (d, J = 8.8 Hz, 1H), 5.05-5.00 (m, 1H), 4.61-4.58 (m, 1H), 4.42 - 4.24 (m, 5H), 3.85-3.73 (m, 10H), 3.13-3.05 (m, 1H), 2.47 - 2.38 (m, 1H), 2.35-2.31 (m, 3H). LCMS: MS m / z = 590.3.[M+H] + .

[0090] Step 8: Synthesis of Compounds 1-11

[0091] Compound 1-10 (310 g, 525.80 mmol, 1 equiv.) was added to ethanol (1.55 L). Then, S-methylisothiourea sulfate (439.11 g, 1.58 mol, 3 equiv.) and sodium carbonate (111.46 g, 1.05 mol, 2 equiv.) were added. The mixture was reacted under nitrogen at 45-50°C (internal temperature) for 16 hours. Most of the ethanol was removed by concentration. 500 ml of water and 400 ml of ethyl acetate were added to the crude product. The mixture was stirred and adjusted to pH 3-4 with 500 ml of 1 M hydrochloric acid. An off-white solid precipitated. 600 ml of petroleum ether was further added. A large amount of off-white solid precipitated in the system while stirring. The system was filtered through a Buchner funnel, and the filter cake was washed with ethyl acetate (200 mL × 2) to obtain the product. The filter cake was dissolved in 2 L of dichloromethane. After separation, the organic phase was dried over anhydrous sodium sulfate and concentrated to give compound 1-11. 1 H NMR (400MHz, CDCl3) δ = 7.22 - 7.14 (m, 4H), 6.91 - 6.82 (m, 4H), 6.65 (dd, J = 8.4 Hz 1H), 5.12-5.08 (m, 1H), 4.97-4.91 (m, 1H), 4.67 - 4.57 (m, 1H), 4.45 - 4.22 (m, 4H), 3.88 - 3.74 (m, 6H), 3.43-3.35 (m, 1H), 2.77-2.72 (m, 1H), 2.59 (m, 3H), 2.40-2.31 (m, 3H). LCMS:MS m / z =630.2[M+H] + .

[0092] Step 9: Synthesis of Compound 1-12B

[0093] Compound 1-11 was separated by SFC (column: DAICEL CHIRALPAK AD (250 mm * 50 mm, 10 μm); mobile phase: [0.1% NH₃·H₂O EtOH]; EtOH%: 45%-45%, 6.3 min) to give compound 1-12B (peak time: 1.665 min) and compound 1-12A (peak time: 2.446 min).

[0094] Step 10: Synthesis of Compounds 1-13

[0095] Compound 1-12B (2 g, 3.18 mmol, 1 equiv.) was dissolved in dichloromethane (20 mL) and N,N-diisopropylethylamine (1.23 g, 9.53 mmol, 1.66 mL, 3 equiv.) was added. The mixture was cooled to 0-10 °C, and trifluoromethanesulfonic anhydride (1.34 g, 4.76 mmol, 786.11 μL, 1.5 equiv.) was slowly added to the system. The mixture was allowed to react at this temperature for 15 min. Saturated aqueous ammonium chloride solution (15 mL) was added. After separation, the aqueous phase was extracted with dichloromethane (15 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product. The crude product was separated by column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 0 / 1) to give compound 1-13. LCMS m / z = 762.2 [M+H] + . 1 H NMR (400MHz, CDCl3) δ = 7.21 - 7.11 (m, 4H), 6.90 - 6.80 (m, 4H), 6.66 (d, J = 8.4 Hz, 1H), 5.19-5.15 (m, 1H), 5.04 - 4.93 (m, 1H), 4.77-4.72 (m, 1H), 4.41 - 4.19 (m, 4H), 3.80 (s, 6H), 3.62-3.54 (m, 1H), 3.11 - 2.97 (m, 1H), 2.56 (s, 3H), 2.42 - 2.31 (m, 3H). LCMS:MS m / z =762.2[M+H] + .

[0096] Step 11: Synthesis of Compounds 1-15

[0097] Compound 1-13 (147 g, 186.74 mmol, 96.767% purity, 1 equivalent) was dissolved in N,N-dimethylformamide (1.5 L). N,N-Diisopropylethylamine (72.40 g, 560.23 mmol, 97.58 mL, 3 equivalents) was added, followed by compound 1-14 (42.54 g, 214.76 mmol, 1.15 equivalents, 2HCl). The mixture was heated to 50 °C and stirred for 0.5 h to give a solution of compound 1-15 in N,N-dimethylformamide. The reaction solution was used directly in the next step.

[0098] Step 12: Synthesis of Compounds 1-16

[0099] A solution of compound 1-15 (137.8 g, 187.02 mmol, 1 equiv.) in N,N-dimethylformamide (1.5 L) was added to a stirrer, and triethylamine (18.92 g, 187.02 mmol, 26.03 mL, 1 equiv.) was added. Di-tert-butyl dicarbonate (48.98 g, 224.42 mmol, 51.56 mL, 1.2 equiv.) was then added to the reaction solution, and the mixture was stirred at 18 °C for 10 h. The mixture was poured into water (1.5 L), followed by the addition of ethyl acetate (400 mL × 3) and saturated aqueous ammonium chloride solution (400 mL × 4). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give compound 1-16. 1H NMR (400 MHz, CDCl3) δ = 7.16 (d, J = 8.4 Hz, 4H), 6.85 (d, J = 8.6 Hz, 4H), 6.64 (d, J = 8.0 Hz, 1H), 5.22 (d, J = 7.2 Hz, 1H), 4.90 - 4.68 (m, 2H), 4.61 (s, 1H), 4.41 - 4.21 (m, 4H), 4.04 (s, 1H), 3.80 (s, 6H), 3.71 (s, 1H), 3.50 (d, J = 11.0 Hz, 2H), 3.30 (s, 1H), 3.24 - 3.02 (m, 2H), 2.90 (d, LCMS m / z =837.2[M+H] + .

[0100] Step 13: Synthesis of Compounds 1-17

[0101] Compound 1-16 (245 g, 278.10 mmol, 95% purity, 1 equiv.) was dissolved in anhydrous dichloromethane (2500 mL) and cooled to 0-10 °C. m-Chloroperbenzoic acid (56.46 g, 278.10 mmol, 85% purity, 1 equiv.) was then added portionwise, and the mixture was stirred at 10 °C for 0.5 h. Additional m-chloroperbenzoic acid (8.47 g, 41.71 mmol, 85% purity, 0.15 equiv.) was added, and the mixture was stirred at 10 °C for an additional 0.5 h. This reaction was treated with a 10 g batch of compound 1-16. The reaction solution was washed with saturated sodium bicarbonate (1500 mL), 5% sodium thiosulfate (1500 mL) (after passing the test with wet potassium iodide starch paper), and half-saturated brine (1500 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (ethyl acetate:petroleum ether=10%-15%-20%-30%) to give compound 1-17. 1HNMR (400 MHz,CDCl3) δ ppm 7.15 (d, J=8.00 Hz, 4 H), 6.85 (d, J=8.80 Hz, 4 H), 6.65 (d, J=8.80 Hz, 1 H), 5.27 (m, 1 H), 4.78 - 4.91 (m, 2 H), 4.61 (s, 1 H), 4.24 - 4.38 (m, 4 H), 3.90 - 4.18 (m, 2 H), 3.78 - 3.82 (m, 6 H), 3.42 - 3.70 (m, 3 H), 3.33 (br s, 1 H), 3.06 - 3.28 (m, 2 H), 2.90 (s, 3 H), 2.66 (m, 2 H), 2.29 - 2.41 (s, 3 H), 1.51 (s, 9 H). LCMS m / z =853.2[M+H] + .

[0102] Step 14: Synthesis of Compounds 1-19

[0103] Compound 1-18 (18.24 g, 114.59 mmol, 1.2 equiv.) was dissolved in anhydrous tetrahydrofuran (900 mL), and the mixture was cooled to -20 °C. Sodium tert-butoxide (11.01 g, 114.59 mmol, 1.2 equiv.) was then added, and the mixture was stirred for 15 minutes. A solution of compound 1-17 (90.5 g, 95.49 mmol, 90% purity, 1 equiv.) in anhydrous tetrahydrofuran (180 mL) was then added, and the mixture was stirred for an additional 0.5 hours. This reaction was treated with a 50 g batch of compound 1-18. The reaction was quenched by adding 1000 mL of saturated ammonium chloride to the reaction solution. After separation, the aqueous phase was extracted with ethyl acetate (1000 mL). The layers were separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (ethyl acetate:petroleum ether=20%-50%-100%) to give compound 1-19 as a white solid. LCMS m / z = 948.4 [M+H] + .

[0104] Step 15: Synthesis of Compounds 1-20

[0105] Compound 1-19 (97.00 g, 97.20 mmol, 95% purity, 1 equiv.) was dissolved in 2-methyltetrahydrofuran (500 mL) and the mixture was cooled to 0 °C. Hydrochloric acid (8 M, 614.80 mL, 50.6 equiv.) was then added dropwise to the reaction solution, and the mixture was heated to 25 °C and stirred for 2 h. Then, n-heptane (200 mL) was added, and the layers were separated. The aqueous phase was collected, and 2-methyltetrahydrofuran (300 mL) was added. The pH of the mixture was adjusted to 8-9 with sodium carbonate, and the mixture was stirred until clear. After separation, the organic phase was collected, and the aqueous phase was extracted with 2-methyltetrahydrofuran (300 mL × 2). The organic phase was collected, washed with saturated brine (200 mL), dried over anhydrous magnesium sulfate, and filtered. The organic phase was collected and concentrated to give the crude product. The crude product was dissolved in dichloromethane (1000 mL), and trifluoroacetic acid (283.73 g, 2.49 mol, 184.24 mL, 25.6 equiv.) was slowly added to the dichloromethane while maintaining the temperature below 10°C. The mixture was stirred at 25°C for 3 hours. The reaction solution was poured into 500 mL of ice water, and the mixture was stirred until clear. After separation, the organic phase was extracted with water (300 mL x 3). Two batches of the aqueous phase were combined and then extracted with dichloromethane (500 mL x 4). The organic phase was discarded. The aqueous phase was cooled to 10°C, and 500 mL of 2-methyltetrahydrofuran was added (resulting in an exotherm). The pH of the mixture was then adjusted to 9 with sodium carbonate, and 2-methyltetrahydrofuran (400 mL x 2) was added for extraction. The organic phases were combined, washed with water (300 mL×4) and saturated brine (300 mL) respectively, dried over anhydrous sodium sulfate, filtered and concentrated to give compound 1-20 as a gray solid. LCMS m / z = 608.27 [M+H] + .

[0106] Step 16: Synthesis of Compounds of Formula (I)

[0107] Compound 1-20 (58 g, 84.00 mmol, 88% purity, 1 equiv.), N,N-diisopropylethylamine (21.71 g, 168.00 mmol, 29.26 mL, 2 equiv.), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38.33 g, 100.80 mmol, 1.2 equiv.) were dissolved in DMF (600 mL), and the mixture was cooled to 0 °C. 2-Fluoroacrylic acid (6.81 g, 75.60 mmol, 0.9 equiv.) was added in several portions, and the mixture was stirred at 0 °C for 10 min. The reaction solution was poured into 900 mL of water and extracted with ethyl acetate (200 mL × 3). The organic phases were combined and washed with saturated ammonium chloride (200 mL x 3), saturated sodium carbonate (200 mL x 3), and saturated brine (200 mL), respectively, then dried over anhydrous sodium sulfate, filtered, and concentrated. The mixture was then slurried with acetonitrile and water (100 mL:150 mL) for 16 hours. The slurry was filtered to obtain the compound of formula (I). SFC analysis (column: Chiralcel OD-3, 50 x 4.6 mm I.D., 3 μm; mobile phase: A (CO2), B (methanol containing 0.05% diisopropylamine); gradient: B% = 5 to 50%, 3 min; flow rate: 3.4 mL / min; wavelength: 220 nm; pressure: 1800 psi, optical purity: 99.21%, peak time: 1.840 min) was performed. 1H NMR (400 MHz, CD3OD) δ = 6.80 - 6.68 (m, 1H), 5.73 - 5.51 (m, 1H), 5.46 - 5.19 (m, 3H), 5.05 - 4.90 (m, 3H), 4.74 - 4.58 (m, 2H), 4.37 - 4.26 (m, 1H), 4.20 - 4.06 (m, 2H), 4.05 - 3.84 (m, 3H), 3.79 - 3.59 (m, 2H), 3.54 - 3.43 (m, 1H), 3.42 - 3.35 (m, 1H), 3.31 - 3.24 (m, 1H), 3.13 - 2.89 (m, 3H), 2.82 - 2.52 (m, 2H), 2.50 - 2.42 (m, 1H), 2.41 - 2.30 (m, 5H), 2.29 - 2.18 (m, 1H).

[0108] Example 2: Single crystal X-ray diffraction analysis of the compound of formula (III) [ka] 0.0133 g of crystalline form A of compound of formula (II) was dissolved in 2 mL of acetonitrile at room temperature. The sample solution was added to a 4 mL semi-sealed sample vial and allowed to evaporate slowly at room temperature. After 10 days, colorless, bulky crystals were obtained. The crystals were collected and diffraction intensity data were collected using a single crystal X-ray diffractometer (D8-VENTURE). The crystal structure data of compound of formula (III) is shown in Table 2, and an ellipsoid diagram of the three-dimensional structure of compound of formula (III) is shown in Figure 6. [Table 2]

[0109] Example 3: Preparation of crystalline form A of the compound of formula (II) Method 1: Compound of formula (I) (0.2 g) was added to ethanol (2.4 mL) and the mixture was heated to 50° C. After dissolution, water (0.48 mL) was slowly added to the reaction solution and the mixture was stirred at 50° C. for 72 hours. After filtration, the solid was collected to obtain crystalline form A of compound of formula (II).

[0110] Method 2: Compound of formula (I) (5.0 g) was added to ethanol (4 V) and the mixture was stirred until clear. Water (1 V) was slowly added to the system, and seed crystals (0.5 g) were added at 20-30°C. The mixture was stirred at this temperature for 15 hours. Then, water (3 V) was slowly added dropwise, and the mixture was stirred for another 1-3 hours. After filtration, the filter cake was collected to obtain crystalline form A of compound of formula (II).

[0111] Example 4: Solid state stability assay of crystalline form A of compound of formula (II) In accordance with the "Guidelines for Stability Testing of Raw Materials and Preparations" (Chinese Pharmacopoeia 2015, Part 4, General Principles, Chapter 9001), the crystalline stability of crystalline form A of compound of formula (II) was investigated under conditions of high temperature (60°C, unsealed), high humidity (room temperature / relative humidity 92.5%, unsealed) and strong light (5000lx, unsealed), as well as under long-term experimental conditions (25±2°C / 60±5%RH) and accelerated experimental conditions (40±2°C / 75±5%RH).

[0112] Approximately 20 mg of crystalline form A of compound of formula (II) was weighed and placed at the bottom of a glass sample vial. The sample was spread to form a thin layer. For samples placed under high temperature and humidity conditions, the vial opening was sealed with aluminum foil, and several small holes were made in the foil to allow complete contact of the sample with the ambient air. For samples placed under strong light conditions, the vial opening was sealed with aluminum foil, and several small holes were made in the foil. Samples placed under various conditions were sampled and tested (XRPD) on days 5 and 10, respectively. The assay results were compared with the initial assay results on day 0. The assay results are shown in Table 3 below.

[0113] Crystalline Form A of the compound of formula (II) was placed in a pharmaceutical double-layered low-density polyethylene bag. Each pharmaceutical low-density polyethylene bag was tightly tied with a string and then placed in a single-layered aluminum foil bag. The single-layered aluminum foil bag was then heat-sealed. Finally, the samples were stored in plastic buckets and stored in a constant temperature and humidity chamber. The packaging used in the stability assay simulated the material storage packaging. The material was divided into 1.5 g / package as stability samples under red light. Each sample was packaged in the same way and labeled with a stability sample label. Samples placed under various conditions were sampled and tested at 3 and 6 months (XRPD). The assay results were compared with the initial assay results on day 0. The assay results are shown in Table 3 below. [Table 3]

[0114] Light control*1: The light control sample had to be placed at the same time. The light control sample was sealed with a screw cap and then completely wrapped in tin foil.

[0115] Conclusion: Crystalline form A of compound of formula (II) has good stability under conditions of high temperature, high humidity, strong light, as well as long-term and accelerated experiments.

[0116] Biological Assay Data: Assay example 1: KRAS G12C Assay of the inhibitory effect of compounds on the proliferation of mutant MIA-PA-CA-2 cells 1.1 Purpose of the assay

[0117] The compound is then added to KRAS G12C IC for growth inhibition of mutant MIA-PA-CA-2 cells 50 were assayed for.

[0118] 1.2 Reagents

[0119] The main reagents used in this assay included CellTiter-Glo (Promega, catalog number G7573).

[0120] 1.3 Equipment

[0121] The primary instrument used in this assay was a PerkinElmer EnVision multifunction microplate reader.

[0122] 1.4 Assay Method 1) Adherent cells were digested with trypsin to form a cell suspension, and the cell suspension was counted for subsequent use. 2) An appropriate amount of cells was added to a centrifuge tube, and cell culture medium was added to make up the required volume, and then the cells were seeded into a 96-well plate at a final density of 2000 cells / well (100 μL of culture medium). 3) After 24 hours of incubation, compounds were formulated to 10 mM in DMSO and serially diluted 3-fold in nine steps with DPBS (Dulbecco's Phosphate Buffered Saline), and 10 μL was added to each well in duplicate. 10 μL of DPBS per well was added to assay control wells (Con). 4) On the same day, 50 μL of CellTiter Glo was added to another cell culture plate without compound, and the fluorescence value was read using EnVision. This value was recorded as the value on day 0. 5) After 72 hours of incubation of compound-treated cells, the plate was removed and 50 μL of CellTiter Glo was added to the cell plate. Fluorescence values ​​were read using EnVision. 6) Data analysis: The inhibition rate of the cells in each well was calculated according to the following formula:

number

number

[0123] 1.5 Assay Results [Table 4]

[0124] The assay results show that the compounds of formula (I) inhibit KRAS G12C It was shown to have good inhibitory activity against cell proliferation of the mutant MIA-PA-CA-2 cell line.

[0125] Assay example 2: KRAS G12C Assay of the inhibitory effect of compounds on the proliferation of mutant H358 cells 2.1 Purpose of the assay

[0126] The compound is then added to KRAS G12C IC for growth inhibition of mutant H358 cells 50 were assayed for.

[0127] 2.2 Reagents

[0128] The main reagents used in this assay included RPMI-1640 medium, penicillin / streptomycin antibiotics purchased from Vicente, fetal bovine serum purchased from Biosera, CellTiter-Glo (cell viability chemiluminescence detection reagent) reagent purchased from Promega, and NCI-H358 cell line purchased from the Cell Bank of the Chinese Academy of Sciences.

[0129] 2.3 Equipment

[0130] The primary instrument used in this assay was a Nivo multilabel analyzer (PerkinElmer).

[0131] 2.4 Assay Method: 1) NCI-H358 cells were seeded into a white 96-well plate, with each well containing 80 μL of cell suspension and 4,000 NCI-H358 cells. The cell plate was incubated overnight in a carbon dioxide incubator. 2) The compounds to be assayed were serially diluted 5-fold using a multichannel pipette to obtain nine concentrations, namely, 2 mM to 5.12 nM. Assays were performed in duplicate. 78 μL of medium was added to the intermediate plate, and 2 μL of the serially diluted compounds was transferred to each well of the intermediate plate according to the corresponding position. After mixing the wells, 20 μL per well was transferred to the cell plate. The compound concentrations transferred to the cell plate ranged from 10 μM to 0.0256 nM. The cell plate was incubated in a carbon dioxide incubator for 5 days. A separate cell plate was prepared, and the signal value of the cell plate on the day of compound addition was read as the maximum value (Max value in the formula below) and used for data analysis. 25 μL of cell viability chemiluminescence detection reagent was added to each well of the cell plate, and the plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal. A multilabel analyzer was used to read the plate. 3) 25 μL of cell viability chemiluminescence detection reagent was added to each well of the cell plate, and the plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal. A multi-label analyzer was used to read the plate.

[0132] Data Analysis:

[0133] Raw data were converted to percent inhibition using the formula (Sample-Min) / (Max-Min)*100% and IC was calculated by curve fitting with four parameters ("log(inhibitor) vs. response--variable slope" mode in GraphPad Prism). 50The inhibitory activity of the compounds of the present disclosure against NCI-H358 cell proliferation is shown in Table 5. [Table 5]

[0134] Conclusion: The compound of formula (I) exhibits good inhibitory activity against the proliferation of NCI-H358 cells.

[0135] Assay Example 3: Metabolic Stability of Hepatocytes Purpose of the assay: The metabolic stability of the test compounds was assayed in CD-1 mice, SD rats, beagle dogs, cynomolgus monkeys, and human hepatocytes, respectively.

[0136] Assay Procedure: Several 96-well sample deposition plates were prepared and designated T0 substrate, T15 substrate, T30 substrate, T60 substrate, T90 substrate, T120 substrate, T0-MC substrate, T120-MC substrate, and blank substrate, respectively. The recovery medium and incubation medium were removed in advance and placed in a 37°C water bath for preheating. Cryopreserved hepatocytes were removed from the liquid nitrogen tank and immediately immersed in a 37°C water bath (approximately 90 seconds). After thawing and loosening, the cryopreserved hepatocytes were poured into a centrifuge tube containing 40 mL of recovery medium. The tube was gently inverted to resuspend the cells in the recovery medium. The cells were centrifuged at 100 × g for 5 minutes at room temperature, and the supernatant was removed. The hepatocytes were resuspended in an appropriate volume of incubation medium, and cell viability was calculated using trypan blue staining. 198 μL of hepatocyte suspension (0.51 × 10 6198 μL of hepatocyte-free incubation medium was added to the T0-MC and T120-MC incubation plates. All incubation plates were pre-incubated for 10 minutes in a 37°C incubator. 2 μL of working solution of the assay sample and control compound were then added, and the mixture was thoroughly mixed. The incubation plate was immediately placed on a shaker in the incubator, and the timer was started to initiate the reaction. Two replicate samples were prepared for each compound at each time point. The incubation conditions were 37°C, saturated humidity, and 5% CO2. In the assay system, the final concentration of the assay sample was 1 μM, the final concentration of the control sample was 3 μM, and the final concentration of hepatocytes was 0.5 × 10 6 The final concentration of all organic solvents was 0.96% (cells / mL), including 0.1% DMSO. At the end of the corresponding incubation time, the incubation plates were removed, and 25 μL of the compound and control compound / cell mixtures were added to sample plates containing 125 μL of stop solution (200 ng / mL tolbutamide and labetalol in acetonitrile). For blank sample plates, 25 μL of hepatocyte-free incubation medium was added directly. After sealing, all sample plates were shaken at 600 rpm for 10 minutes and then centrifuged at 3220 × g for 20 minutes. The supernatants of the assay and control samples were diluted 1:3 with ultrapure water. All samples were thoroughly mixed and analyzed by LC / MS / MS.

[0137] Assay Results: The assay results are shown in Table 6. [Table 6]

[0138] Conclusion: Metabolic assays in hepatocytes of various species showed that the compound of formula (I) has good metabolic stability.

[0139] Assay Example 4: In vitro stability assay in liver microsomes Purpose of the assay: The metabolic stability of the assay compounds was assayed in liver microsomes from CD-1 mice, SD rats, beagle dogs, cynomolgus monkeys, and humans, respectively.

[0140] Assay procedure: Two 96-well incubation plates were prepared, designated T60 and NCF60 incubation plates, respectively. 445 μL of microsome working solution (liver microsomal protein concentration of 0.56 mg / mL) was added to the T60 and NCF60 incubation plates, respectively, and then the incubation plates were pre-incubated in a 37°C water bath for approximately 10 minutes.

[0141] After preincubation, 5 μL of working solution of the assay sample or control compound was added to the T60 incubation plate and the NCF60 incubation plate, respectively, and the mixture was mixed thoroughly. The reaction was initiated by adding 50 μL of potassium phosphate buffer to each well of the NCF60 incubation plate. 180 μL of stop solution (200 ng / mL tolbutamide and 200 ng / mL labetalol in acetonitrile) and 6 μL of NADPH-regenerating system working solution were added to the TO stop plate, and 54 μL of sample was transferred from the T60 incubation plate to the TO stop plate (generation of TO sample). The reaction was initiated by adding 44 μL of NADPH-regenerating system working solution to each well of the T60 incubation plate. 54 μL of microsome working solution, 6 μL of NADPH-regenerating system working solution, and 180 μL of stop solution alone were added to a blank plate. Therefore, for the samples containing the assay or control compounds, the final reaction concentrations of the compounds, testosterone, diclofenac, and propafenone were 1 μM, the liver microsome concentration was 0.5 mg / mL, and the final reaction concentrations of DMSO and acetonitrile in the reaction system were 0.01% (v / v) and 0.99% (v / v), respectively. After incubation for an appropriate time (e.g., 5, 15, 30, 45, and 60 min), 180 μL of stop solution (200 ng / mL tolbutamide and 200 ng / mL labetalol in acetonitrile) was added to each sample well of the stop plate. 60 μL of sample was removed from the T60 incubation plate to stop the reaction. All sample plates were thoroughly shaken and then centrifuged at 3220 × g for 20 min. 80 μL of supernatant was then removed from each well and diluted with 240 μL of purified water for liquid chromatography-tandem mass spectrometry analysis. All samples were injected and analyzed by liquid chromatography-tandem mass spectrometry.

[0142] Assay Results: The assay results are shown in Table 7. [Table 7]

[0143] Conclusion: The metabolic stability assay in liver microsomes showed that the compound of formula (I) has good metabolic stability.

[0144] Assay Example 5: Stability assay in plasma Purpose of the assay: The stability of the assay compound in CD-1 mouse and human plasma, respectively.

[0145] Assay Procedure: Frozen plasma was thawed for 10-20 minutes. After complete thawing, the plasma was placed in a centrifuge and centrifuged at 3220 x g for 5 minutes to remove any suspended solids or precipitates. 96-well incubation plates were prepared and designated T0, T10, T30, T60, and T120. 98 μL of blank plasma from mouse, rat, dog, monkey, and human samples was added to the corresponding incubation plates, followed by 2 μL of working solution of the compound or control compound in duplicate. All samples were incubated in a 37°C water bath. The final incubation concentrations of the compounds and control compounds bisacodyl maleate, enalapril maleate, procaine, and provantine were 2 μM, and the final organic phase content was 2.0%. At the end of each incubation time point, the corresponding incubation plate was removed, and 400 μL of a 200 ng / mL solution of tolbutamide and labetalol in acetonitrile was added to each corresponding sample well to precipitate proteins. All sample plates were sealed, shaken thoroughly, and then centrifuged at 3220 × g for 20 minutes. 50 μL of the supernatant was removed and diluted with 100 μL of ultrapure water. All samples were mixed well and then analyzed by LC / MS / MS.

[0146] Assay Results: The assay results are shown in Table 8. [Table 8]

[0147] Conclusion: The compound of formula (I) has good stability in human and mouse plasma.

[0148] Assay Example 6: Stability assay in whole blood Purpose of the assay: The stability of the assay compounds in whole blood of CD-1 mice, SD rats, beagle dogs, and cynomolgus monkeys was assayed, respectively.

[0149] Assay Procedure: Fresh whole blood was collected from CD-1 mice, SD rats, beagle dogs, and cynomolgus monkeys using the anticoagulant EDTA-K2 on the day of the assay or the day before. Before the start of the assay, the whole blood was mixed 1:1 (v:v) with PBS, and the mixture was prewarmed in a 37°C water bath for 10–20 min. 96-well incubation plates were prepared and designated T0, T30, T60, and T240, respectively. In the corresponding incubation plates, including the T0, T30, T60, and T240 incubation plates, 2 μL of working solution of the compound or control compound was mixed in duplicate with 98 μL of blank whole blood from mice, rats, dogs, monkeys, and humans. All samples were incubated in a 37°C water bath. The final incubation concentration of the compound was 5 μM, and the final incubation concentration of the control compound was 2 μM. At the end of each incubation time point, the corresponding incubation plate was removed, and 100 μL of ultrapure water was immediately added to the corresponding sample well and mixed thoroughly. Proteins were precipitated by adding 800 μL of a 200 ng / mL solution of tolbutamide and labetalol in acetonitrile. The sample plate was sealed, shaken thoroughly, and then centrifuged at 3220 × g for 20 minutes. 150 μL of the supernatant was removed and analyzed by LC / MS / MS.

[0150] Assay Results: The assay results are shown in Table 9. [Table 9]

[0151] Conclusion: Stability assays in whole blood of various species showed that the compound of formula (I) has good stability in whole blood.

[0152] Assay Example 7: Protein Binding Rate Assay Purpose of the assay: The protein binding rate of the assay compound in plasma of CD-1 mice, SD rats, beagle dogs, cynomolgus monkeys, and humans was determined by equilibrium dialysis.

[0153] Assay procedure: Plasma samples containing 2 μM compound were prepared using the five types of plasma described above, placed in a 96-well equilibrium dialysis device, and dialyzed against phosphate buffer at 37 ± 1°C for 4 hours. Warfarin was used as a control compound in this assay. The concentrations of the assay compounds in the plasma and dialysis buffer were determined by LC-MS / MS.

[0154] Assay Results: The assay results are shown in Table 10. [Table 10]

[0155] Conclusion: Plasma binding assays in various species showed that the compound of formula (I) has a high protein unbound rate in plasma.

[0156] Assay Example 8: In vivo pharmacokinetic assay 1) Pharmacokinetics of the test compound after oral administration and intravenous injection in SD rats

[0157] The assay compounds were mixed with a 5% dimethyl sulfoxide / 95% (10% hydroxypropyl-β-cyclodextrin) solution. The mixture was vortexed and sonicated to prepare a clear solution of 1 mg / mL, which was then filtered through a microporous membrane for later use. Male SD rats aged 7 to 10 weeks were selected and administered the candidate compound solution intravenously or orally. Whole blood was collected at regular intervals and prepared to obtain plasma. Drug concentrations were analyzed by LC-MS / MS, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software (Pharsight, USA). The assay results are shown in Table 11. [Table 11]

[0158] Note: Vd ss , u is the apparent volume of distribution under unbound plasma proteins (Vd ss ,u=Vd ss / PPB(Unbound %));C max,u and AUC 0-last,u is the corresponding value under unbound plasma protein (C max,u =C max ×PPB(unbound %);AUC 0-last,u =AUC 0-last ×PPB(unbound %)).

[0159] Conclusion: PK assays showed that the compound of formula (I) has high unbound plasma exposure and good oral bioavailability in rats.

[0160] 2) Pharmacokinetic assay of crystalline form A of the compound of formula (II) by oral administration in SD rats

[0161] 109.72 mg of the assay compound was weighed and added to a glass vial. 774 μL of 0.5% methylcellulose (400 viscosity) aqueous solution was added, and the mixture was stirred for 5 minutes. 10 mL of 0.5% methylcellulose (400 viscosity) aqueous solution was added, and the mixture was stirred for 5 minutes to obtain a uniform, opaque suspension. Male SD rats aged 7 to 10 weeks were selected and orally administered the candidate compound solution. Whole blood was collected at regular intervals and prepared to obtain plasma. Drug concentrations were analyzed by LC-MS / MS, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software (Pharsight, USA). The assay results are shown in Table 12. [Table 12]

[0162] Conclusion: The PK assay showed that crystalline form A of the compound of formula (II) had high exposure in rats.

[0163] 3) Pharmacokinetics of the test compounds in CD mice by oral administration and intravenous injection

[0164] The assay compounds were mixed with a 5% dimethyl sulfoxide / 95% (10% hydroxypropyl-β-cyclodextrin) solution. The mixture was vortexed and sonicated to prepare a clear solution of 1 mg / mL, which was then filtered through a microporous membrane for later use. Male CD mice aged 7 to 10 weeks were selected and administered the candidate compound solution intravenously or orally. Whole blood was collected at regular intervals and prepared to obtain plasma. Drug concentrations were analyzed by LC-MS / MS, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software (Pharsight, USA). The assay results are shown in Table 13. [Table 13]

[0165] Note: Vd ss , u is the apparent volume of distribution under unbound plasma proteins (Vd ss ,u=Vd ss / PPB(Unbound %));C max,u and AUC 0-last,u is the corresponding value under unbound plasma protein (C max,u =C max ×PPB(unbound %);AUC 0-last,u =AUC 0-last ×PPB(unbound %)).

[0166] Conclusion: PK assays showed that the compound of formula (I) has high unbound plasma exposure and good oral bioavailability in mice.

[0167] 4) Pharmacokinetics of the compound after oral administration in beagle dogs

[0168] 680.397 mg of assay compound powder was weighed and added to 50 mL of 0.5% methylcellulose (400 viscosity) aqueous solution. The mixture was stirred for 10 minutes and sonicated for 10 minutes. 50 mL of 0.5% methylcellulose (400 viscosity) aqueous solution was added and homogenized using a homogenizer for 10 minutes. 11 mL of 0.5% methylcellulose (400 viscosity) aqueous solution was added. The mixture was sonicated for 5 minutes and stirred for 10 minutes. 358 μL of 0.5% methylcellulose (400 viscosity) aqueous solution was added and stirred for 2 minutes. Male beagle dogs over 6 months of age were selected and orally administered the candidate compound solution. Whole blood was collected at regular intervals and prepared to obtain plasma. Drug concentrations were analyzed by LC-MS / MS, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software (Pharsight, USA). The assay results are shown in Table 14. Whole blood was collected at regular intervals and prepared to obtain plasma. Drug concentrations were analyzed by LC-MS / MS method, and pharmacokinetic parameters were calculated by Phoenix WinNonlin software (Pharsight, USA). The assay results are shown in Table 14. [Table 14]

[0169] Conclusion: The PK assay showed that crystalline form A of the compound of formula (II) had high exposure in dogs.

[0170] Assay Example 9: In vivo Pharmacodynamics Assay In vivo pharmacodynamic assay of human pancreatic cancer Mia PaCa-2 cells in a subcutaneous tumor model in Balb / c nude mice

[0171] 1. Cell Culture and Tumor Tissue Preparation

[0172] Cell Culture: Human pancreatic cancer Mia PaCa-2 cells (ATCC-CRL-1420) were cultured in vitro as monolayers in DMEM medium containing 10% fetal bovine serum and 2.5% horse serum in a 37°C, 5% CO incubator. Cells were passaged twice a week by routine trypsin-EDTA digestion. When cell saturation reached 80%-90% and the required cell number was met, the cells were harvested, counted, and resuspended in an appropriate volume of PBS. Matrigel was added at a 1:1 ratio to 25×10 6 A cell suspension with a cell density of 1000 cells / mL was obtained.

[0173] Cell inoculation: 0.2mL (5×10 6 Mia PaCa-2 cells (+Matrigel, 1:1 in volume) were inoculated subcutaneously into the right dorsal region of each mouse. The mean tumor volume was 190 mm. 3 When tumor volume reached 100 μg / kg, mice were randomized into groups based on tumor volume and dosing was initiated according to the protocol in Table 15. [Table 15]

[0174] Note: PO indicates oral administration and QD indicates once daily.

[0175] 2. Tumor Measurement and Assay Indicators

[0176] Tumor diameters were measured twice a week with a vernier caliper. Tumor volume was calculated using the formula V = 0.5a × b 2 (where a and b represent the long and short diameters of the tumor, respectively).

[0177] The antitumor effect of the compound was evaluated by TGI (%) or relative tumor growth rate T / C (%). Relative tumor growth rate T / C (%) = TRTV / CRTV × 100% (TRTV: RTV in the treatment group; CRTV: RTV in the negative control group). Relative tumor volume (RTV) was calculated from the tumor measurement results using the formula RTV = Vt / V0 (where V0 is the average tumor volume measured for each group at the time of administration (i.e., D0), and Vt is the average tumor volume at a given measurement time). For TRTV and CRTV, data from the same day were used.

[0178] TGI (%) reflected the tumor growth inhibition rate: TGI (%) = [(1 - (mean tumor volume at the end of treatment in the treatment group - mean tumor volume at the start of treatment in the treatment group)) / (mean tumor volume at the end of treatment in the vehicle control group - mean tumor volume at the start of treatment in the vehicle control group)] × 100%.

[0179] 3. Assay Results

[0180] The assay results are shown in FIGS.

[0181] The results on day 22 of administration are shown in Table 16. [Table 16]

[0182] Conclusion: The compound of formula (I) has a significant tumor-inhibiting effect, and the body weight of the mice in each dose group is stable, with no obvious intolerance.

Claims

1. Compound of formula (II) 【Chemical 1】 wherein n is 2; Crystalline form A is characterized by a powder X-ray diffraction pattern having characteristic diffraction peaks at 2θ angles of 8.514±0.200°, 14.689±0.200°, and 18.122±0.200°.

2. 2. The crystalline form A of claim 1, characterized by an X-ray powder diffraction pattern having characteristic diffraction peaks at 2θ angles of 6.218±0.200°, 8.514±0.200°, 12.299±0.200°, 14.689±0.200°, 16.903±0.200°, 18.122±0.200°, 18.927±0.200°, and 25.580±0.200°.

3. 2. The crystalline form A of claim 1, characterized by an X-ray powder diffraction pattern having characteristic diffraction peaks at 2θ angles of 6.218±0.200°, 8.514±0.200°, 11.663±0.200°, 12.299±0.200°, 14.689±0.200°, 16.903±0.200°, 18.122±0.200°, 18.927±0.200°, 19.364±0.200°, 20.386±0.200°, 21.914±0.200°, and 25.580±0.200°.

4. 2. The crystalline form A of claim 1, characterized by a powder X-ray diffraction pattern having characteristic diffraction peaks at 2θ angles of 6.218°, 8.514°, 11.663°, 12.299°, 14.689°, 16.903°, 18.122°, 18.554°, 18.927°, 19.364°, 20.386°, 21.914°, 22.640°, 23.867°, 24.553°, 24.806°, 25.580°, 25.988°, 27.147°, 27.715°, 29.135°, and 31.799°.

5. 2. The crystalline form A of claim 1, having a differential scanning calorimetry curve with an endothermic peak at 115.37°C ± 3°C.

6. 2. The crystalline form A of claim 1, having a thermogravimetric analysis curve with a maximum weight loss of 5.379% at 150.0°C±3°C.

7. 6. The crystalline form A of claim 5, having a thermogravimetric analysis curve with a maximum weight loss of 5.379% at 150.0°C ± 3°C.

8. A pharmaceutical comprising crystalline form A according to any one of claims 1 to 7.

9. A pharmaceutical composition for treating a disease, comprising crystalline form A according to any one of claims 1 to 7, wherein the disease is a solid tumor.

10. The pharmaceutical composition for treating a disease according to claim 9, wherein the disease is lung cancer and rectal cancer.

11. The pharmaceutical composition for treating a disease according to claim 9, wherein the disease is rectal cancer.

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