Crystal form of azetidine-substituted compounds

Abstract

The crystalline forms of the azetidine-substituted compounds of formula (I) and methods for their preparation further include the use of said crystalline forms in the preparation of a cancer medicament. [Formula 1] JPEG2023540388000032.jpg61170

Description

Technical Field

[0001] This application claims the following priority: CN202010952692.5, filed on September 11, 2020.

[0002] The present invention relates to a crystalline form of an azetidine-substituted compound and a method for preparing the same, and further includes the use of the crystalline form in the preparation of a cancer therapeutic agent.

Background Art

[0003] The first RAS oncogene was named as such because it was discovered in rat sarcoma. The RAS protein is a product expressed by the RAS gene and is a closely related monomeric globulin consisting of 189 amino acids with a molecular weight of 21 kDa. This protein can bind to guanosine triphosphate (GTP) or guanosine diphosphate (GDP). The activity state of the RAS protein influences cell growth, differentiation, the cytoskeleton, protein transport, and secretion, and its activity is regulated by its binding to GTP or GDP. The RAS protein is dormant, or inactive, when bound to GDP; however, when a specific upstream cell growth factor is stimulated, the RAS protein is induced to exchange GDP and bind to GTP, at which point it is called activated. The RAS protein bound to GTP can activate downstream proteins and transmit signals. The RAS protein itself possesses weak hydrolytic activity that hydrolyzes GTP, allowing it to hydrolyze GTP to GDP. This enables the conversion from an activated state to an inactive state. In this hydrolysis process, the involvement of GAP (GTPase activating proteins) is also necessary. GAP acts with the RAS protein and can greatly enhance its ability to hydrolyze GTP to GDP. Mutations in the RAS protein affect its interaction with GAP, i.e., its ability to hydrolyze GTP to GDP, keeping the RAS protein in an activated state. The activated RAS protein continues to provide growth signals to downstream proteins, ultimately causing cells to constantly grow and differentiate, eventually leading to tumor formation. The RAS gene family has many members, and among them, the subfamilies closely associated with various cancers mainly include the Kirsten rat sarcoma virus oncogene homolog (KRAS), the Harvey rat sarcoma virus oncogene homolog (HRAS), and the neuroblastoma rat sarcoma virus oncogene homolog (NRAS).Approximately 30% of human tumors are associated with mutations in certain RAS genes, with KRAS mutations being the most prominent, accounting for 86% of all RAS mutations. Regarding KRAS mutations, the most common occurrences are at the 12th glycine (G12), 13th glycine (G13), and 61st glutamine (Q61) residues, with G12 mutations accounting for 83% of these.

[0004] The G12C mutation is one of the relatively common mutations in the KRAS gene, in which the 12th glycine molecule is replaced with cysteine. The KRAS G12C mutation is the most common in lung cancer, and based on data reported in the literature (Nat Rev Drug Discov 2014;13:828-851), it is estimated to account for approximately 10% of all lung cancer patients.

[0005] The KRAS G12C mutant protein is a cutting-edge target, and there is still not much research on it. One publication (Nature. 2013; 503: 548-551) reports a type of covalent inhibitor targeting the KRAS G12C mutation, but the enzymatic activity of this type of compound is not high, and it does not exhibit significant activity at the cellular level. Another publication (Science 2016; 351: 604-608, Cancer Discov 2016; 6: 316-29) reports that one type of compound exhibits μM-class cell antiproliferative activity at the cellular level, but its structural and metabolic stability is low, making it difficult to further enhance its activity. Aside from a few literature reports, Araxes Pharma LLC has filed several patents related to KRAS G12C inhibitors. For example, International Publication Nos. 2016164675 and 2016168540 report that a certain type of quinazoline derivative has high enzyme-binding activity, exhibits μM-class cell antiproliferative activity, is structurally stable, and has a certain degree of selectivity. All of these compounds have a single acrylamide fragment and act as a Michael addition acceptor and a G12C residue on the KRAS protein to form a covalent complex. In 2018, Liu Yi et al. disclosed ARS-1620, a covalent inhibitor targeting KRAS G12C mutations, in Cell (Matthew R. Janes, Yi Liu et al., Cell, 2018, 172, 578-589). This compound possesses excellent metabolic stability, exhibits nM-class cellular antiproliferative activity at the cellular level, and can effectively suppress tumor growth in a pancreatic cancer MIA-Paca2 cell subcutaneous xenograft tumor model. In the second half of 2018, Amgen began recruiting participants for a Phase I clinical trial of the KRAS G12C inhibitor AMG 510 (NCT03600883). This is the first organic small molecule KRAS G12C inhibitor to enter clinical research. At the 2019 AACR meeting, the structural formula of AMG 510 and some pre-clinical research data were released.At the 2019 ASCO conference, the results of an early Phase I clinical trial of AMG 510 were presented, showing that AMG 510 achieved a 90% disease control rate against disease progression after chemotherapy in non-small cell lung cancer patients with KRAS G12C mutations. In addition, recruitment of Phase I clinical trial participants (NCT03785249) for MRTX849, a KRAS G12C inhibitor developed by Mirati Therapeutics, began in January 2019, and representative patents include International Publication No. 2017201161 and International Publication No. 2019099524. [ka] [Overview of the project] [Means for solving the problem]

[0006] The present invention relates to the crystalline form C of the compound of formula (I), whose X-ray powder diffraction spectrum has characteristic diffraction peaks at angles 2θ: 5.89±0.20°, 8.82±0.20°, and 17.71±0.20°. [ka] To provide.

[0007] In some solutions of the present invention, the above-mentioned crystal form C has characteristic diffraction peaks in its X-ray powder diffraction spectrum at angles 2θ: 5.89±0.20°, 8.82±0.20°, 13.19±0.20°, 14.81±0.20°, 17.71±0.20°, 18.72±0.20°, 21.58±0.20°, and 24.47±0.20°.

[0008] In some solutions of the present invention, the above crystal form C has characteristic diffraction peaks in its X-ray powder diffraction spectrum at angles 2θ: 5.89°, 8.82°, 11.50°, 12.58°, 13.19°, 14.42°, 14.81°, 17.71°, 18.72°, 20.70°, 21.58°, 23.51°, 24.47°, 25.36°, 26.58°, 27.09°, and 29.08°.

[0009] The present invention relates to an X-ray powder diffraction spectrum having characteristic diffraction peaks at angles 2θ: 5.89±0.20° and 8.82±0.20°, and further, 17.71±0.20° and / or 11.50±0.20° and / or 12.58±0.20° and / or 13.19±0.20° and / or 14.42±0.20° and / or 14.81±0.20° and / or 18.72±0. The present invention provides crystalline form C of the compound of formula (I), having characteristic peaks at 20° and / or 20.70±0.20°, and / or 21.58±0.20°, and / or 23.51±0.20°, and / or 24.47±0.20°, and / or 25.36±0.20°, and / or 26.58±0.20°, and / or 27.09±0.20°, and / or 29.08±0.20°.

[0010] In some solutions of the present invention, the above crystal form C has an XRPD spectrum as shown in Figure 3.

[0011] In some solutions of the present invention, the XRPD spectral analysis data for the above crystal form C is shown in Table 3.

[0012] [Table 1]

[0013] In some solutions of the present invention, the above crystal form C has a differential scanning calorimetry curve in which the start of the endothermic peak is at 214.7 ± 5°C.

[0014] In some solutions of the present invention, the above crystal form C has a DSC spectrum as shown in Figure 11.

[0015] In some solutions of the present invention, the above-mentioned crystalline form C exhibits a weight loss of 2.52% at 150.0±3℃ in the thermogravimetric analysis curve.

[0016] In some solutions of the present invention, the crystal form C has a TGA spectrum as shown in FIG. 10.

[0017] The present invention provides a crystal form A of a compound of formula (I) whose XRPD spectrum analysis data is as shown in Table 1.

[0018] [Table 2]

[0019] The present invention provides a crystal form A of a compound of formula (I) whose XRPD spectrum is as shown in FIG. 1.

[0020] In some solutions of the present invention, the crystal form A has a DSC spectrum as shown in FIG. 7.

[0021] In some solutions of the present invention, the crystal form A has a TGA spectrum as shown in FIG. 6.

[0022] The present invention provides a crystal form B of a compound of formula (I) whose XRPD spectrum analysis data is as shown in Table 2.

[0023] [Table 3]

[0024] The present invention provides a crystal form B of a compound of formula (I) whose XRPD spectrum is as shown in FIG. 2.

[0025] In some solutions of the present invention, the crystal form B has a DSC spectrum as shown in FIG. 9.

[0026] In some solutions of the present invention, the crystal form B has a TGA spectrum as shown in FIG. 8.

[0027] The present invention provides crystalline form D of the compound of formula (I), whose XRPD spectral analysis data is as shown in Table 4.

[0028] [Table 4]

[0029] The present invention provides crystalline form D of the compound of formula (I), whose XRPD spectrum is as shown in Figure 4.

[0030] In some solutions of the present invention, the crystal form D is as shown in Figure 13 by its DSC spectrum.

[0031] In some solutions of the present invention, the crystal form D has a TGA spectrum as shown in Figure 12.

[0032] s The present invention provides a crystalline form E of the compound of formula (I), whose XRPD spectral analysis data is as shown in Table 5.

[0033] [Table 5]

[0034] The present invention provides a crystalline form E of the compound of formula (I), whose XRPD spectrum is as shown in Figure 5.

[0035] In some solutions of the present invention, the crystal form E has a DSC spectrum as shown in Figure 15.

[0036] In some solutions of the present invention, the crystal form E has a TGA spectrum as shown in Figure 14.

[0037] In some solutions of the present invention, the above-mentioned crystalline form is used in the preparation of cancer therapeutics.

[0038] In some solutions of the present invention, the cancers include lung cancer, lymphoma, esophageal cancer, ovarian cancer, pancreatic cancer, rectal cancer, glioma, cervical cancer, urothelial carcinoma, gastric cancer, endometrial cancer, liver cancer, bile duct cancer, breast cancer, colon cancer, leukemia, and melanoma. [Effects of the Invention]

[0039] The compounds of the present invention possess excellent cell activity and selectivity, have relatively stable crystalline forms, high solubility, appropriate hygroscopicity, and good drug discovery potential.

[0040] Definition and explanation This invention adopts the following abbreviations: DMSO represents dimethyl sulfoxide; MeOH represents methanol; TFA represents trifluoroacetic acid; NCS represents N-chlorosuccinimide; PyBrOP represents bromo-tris-pyrrolidino-phosphonium hexafluorophosphate; LCMS represents high-performance liquid chromatography / mass spectrometry; XRPD represents X-ray powder diffraction; HPLC represents high-performance liquid chromatography; ACN represents acetonitrile; and H2O represents water.

[0041] XRPD Test Parameters

[0042] [Table 6]

[0043] Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) TGA and DSC spectra were collected using a thermogravimetric analyzer TA Discovery TGA5500 / Q5000 and a differential scanning calorimeter TAQ200 / Q2000 / Discovery DSC 2500, respectively. Table 7 lists the test parameters.

[0044] [Table 7]

[0045] High-performance liquid chromatography (HPLC) The purity and solubility of the samples are collected and measured using high-performance liquid chromatography (HPLC) with an Agilent 1260 HPLC system.

[0046] [Table 8]

[0047] [Table 9]

[0048] Tableting (Hydraulic Press) The tableting experiment was performed using a SYP-5BS manual tablet press, and the pressure applied to the sample was approximately 350 MPa. [Brief explanation of the drawing]

[0049] [Figure 1] This is the XRPD spectrum of crystal form A. [Figure 2] This is the XRPD spectrum of crystal form B. [Figure 3] This is the XRPD spectrum of crystal form C. [Figure 4] This is the XRPD spectrum of crystal form D. [Figure 5] This is the XRPD spectrum of crystal form E. [Figure 6] This is the TGA spectrum of crystal form A. [Figure 7] This is the DSC spectrum of crystal form A. [Figure 8] This is the TGA spectrum of crystal form B. [Figure 9] This is the DSC spectrum of crystal form B. [Figure 10] This is the TGA spectrum of crystal form C. [Figure 11] This is the DSC spectrum of crystal form C. [Figure 12] This is the TGA spectrum of crystal form D. [Figure 13] This is the DSC spectrum of crystal form D. [Figure 14] This is the TGA spectrum of crystal form E. [Figure 15] This is the DSC spectrum of crystal form E. [Figure 16] This is a three-dimensional ellipsoidal diagram of crystal form C. [Modes for carrying out the invention]

[0050] The present invention will be described in detail below through examples, but this does not mean any unfavorable limitation of the present invention. The compounds of the present invention can be prepared by various synthesis methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining with other chemical synthesis methods, and equivalence substitution methods well known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of the present invention. It will be obvious to those skilled in the art that various changes and improvements can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention. [Examples]

[0051] Example 1 Preparation of the compound of formula (I) [ka] Step 1: Synthesis of Compound 2 To a solution of compound 1 (1500 g, 4.49 mol) in ethanol (10.5 L) and water (4.5 L), potassium carbonate (1.24 kg, 8.97 mol) was added. The mixture was then heated to 50°C, and the temperature was controlled between 50 and 70°C. Hydrogen peroxide solution (1.78 kg, 15.71 mol, 30% purity) was slowly added dropwise, and after the addition was complete, the mixture was stirred at 70°C for 1 hour. The mixture was then cooled to 30°C, sodium sulfite aqueous solution (3.0 L) was added, and the mixture was stirred at 30°C for 1 hour. Tap water (30 L) was added, and the mixture was stirred at 20°C for 1 hour. The solid was filtered and dried to obtain compound 2. LCMS (ESI) m / z: 331.0 (M+1).

[0052] Step 2: Synthesis of Compound 3 To a solution of compound 2 (300 g, 0.84 mol) in acetonitrile (3.0 L), potassium hydroxide (189.98 g, 3.39 mol) and carbon disulfide (193.35 g, 2.54 mol) were added, and the reaction mixture was stirred at 25°C for 2 hours. After adding tap water (7.5 L), the pH was adjusted to 1 with 2 M hydrochloric acid (1.8 L), the precipitated solid was filtered, rinsed with water, and dried to obtain compound 3. LC-MS (ESI) m / z: 373.0 (M+1).

[0053] Step 3: Synthesis of Compound 4 To a solution of compound 3 (1000 g, 2.69 mol) in dimethyl carbonate (10.0 L), potassium carbonate (816.69 g, 5.91 mol) and tetrabutylammonium bromide (86.59 g, 268.60 mmol) were added. The reaction mixture was stirred at an internal temperature of 85-90°C for 16 hours, then cooled to 30°C. After filtering the solid, the filtered cake was diluted with 14 L of water and stirred at 10°C for 3 hours. The pH was then adjusted to 2 with 6 M hydrochloric acid, filtered, and dried to obtain the crude product (1085 g). The crude product was stirred at 20°C for 10 hours using a mixed solvent of ethyl acetate (3254 mL) and petroleum ether (4881 mL), filtered, and dried to obtain compound 4. LCMS (ESI) m / z: 387.0 (M+1).

[0054] Step 4: Synthesis of Compound 5 Compound 4 (390 g, 0.96 mol) was added to a solution of N,N-diethylazetidine-3-amine (159.70 g, 1.25 mol) in n-butanol (3510 mL). The reaction mixture was heated to 120 °C and stirred for 15 hours. The reaction mixture was cooled to 30 °C, water (4.0 L) was added, and the mixture was stratified. The aqueous phase was extracted twice with ethyl acetate (1 L * 2), and the organic phase was combined and concentrated under reduced pressure. Ethyl acetate (4 L) and water (3.0 L) were added to the concentrated residue, and the pH was adjusted to 2 with 6 M hydrochloric acid. The organic phase was separated and discarded, and the pH of the aqueous phase was adjusted to 8 with potassium carbonate (250 g). After extraction three times with ethyl acetate (2.5 L * 3), the mixture was dried with anhydrous sodium sulfate, filtered, concentrated, and dried to obtain compound 5. LCMS (ESI) m / z: 467.1 (M+1).

[0055] Step 5: Synthesis of Compound 6 To a solution of compound 5 (830 g, 1.69 mmol) in acetonitrile (2.52 L), TFA (771.02 g, 6.76 mmol) and NCS (1451.47 g, 3.38 mmol) were added. The reaction mixture was heated to 60°C and stirred for 2 hours. After quenching with saturated sodium sulfite aqueous solution (1.6 L), 1 M sodium hydroxide solution (7 L) was added dropwise, and the mixture was extracted three times with ethyl acetate (4 L x 3). The mixture was washed once with saturated brine (1 L), dried, filtered, and concentrated to obtain compound 6. LCMS (ESI) m / z: 535.0 (M+1).

[0056] Step 6: Synthesis of Compound 7 To a solution of compound 6 (400 g, 0.69 mol) in N,N-dimethylacetamide (3.2 L), N,N-diisopropylethylamine (135.05 g, 1.04 mol), compound 6-1 (142.72 g, 0.76 mol), and PyBrOP (357.23 g, 0.76 mmol) were added, and the reaction mixture was stirred at 20°C for 3 hours. After adding pure water (9.6 L) to the reaction mixture, it was filtered, and the filter cake was dried to obtain compound 7. LCMS (ESI) m / z: 703.1 (M+1).

[0057] Step 7: Synthesis of Compound 8 Compound 7 (600 g, 0.85 mol) was added to a solution of ethyl acetate (1.8 L) and a solution of 4 M methanol hydrochloride (1.8 L, 7.20 mol). The reaction mixture was stirred at 15°C for 12 hours. The reaction mixture was filtered, and the filter cake was beaten with ethyl acetate (2 L) at 15°C for 2 hours. The mixture was filtered, and the filter cake was dried to obtain compound 8. LC-MS (ESI) m / z: 603.1 (M+1).

[0058] Step 8: Synthesis of the compound of formula (I) To a solution of compound 8 (360 g, 0.53 mmol) in 2-methyltetrahydrofuran (3.6 L), water (7.2 L) and potassium carbonate (146.70 g, 1.06 mol) were added. The reaction mixture was cooled to 15°C, and compound 8-1 (57.64 g, 0.64 mol) was added dropwise. The reaction mixture was stirred at 15°C for 15 minutes. The reaction mixture was extracted twice with 2-methyltetrahydrofuran (0.5 L x 2). The organic phases were combined, washed once with saturated brine (0.5 L), dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was stirred at 40°C for 12 hours using a methanol / water = 1 / 2 (2.8 L) mixed solvent, filtered, the filter cake was heated and dried, and isopropyl alcohol (1.5 L) was added to form a suspension. The suspension was heated to 80°C and stirred at 80°C for 2 hours. Next, n-heptane (1.0 L) was added, and the reaction mixture was stirred at 80°C for 12 hours. The reaction mixture was cooled to 25°C and stirred continuously at 25°C for 12 hours. Subsequently, the mixture was filtered, and the filter cake was dried to obtain the compound of formula (I). LC-MS (ESI) m / z: 656.9 (M+1); 1 HNMR (400MHz, DMSO-d6) δ7.68(d, J=7.58Hz, 1H)7.00(s, 1H)6.82(dd, J=16.69, 10.45Hz, 1H)6.10-6.22(m, 3H)5.69-5.77(m , 1H) 4.09-4.22(m, 2H) 3.85-3.94(m, 2H) 3.79(brs, 2H) 3.71(brs, 6H) 3.60-3.65(m, 1H) 2.51-2.57(m, 4H) 0.91-0.99(m, 6H).

[0059] Example 2 Preparation of the crystalline form of compound (I) Preparation of crystalline form A of compound (I): 10 g of compound (I) was dissolved in 130 ml of isopropyl alcohol, the temperature was raised to 100°C, stirred at 100°C for 1 hour, then allowed to cool naturally to 10°C, stirred at 10°C for 9 hours, filtered, and crystalline form A was obtained.

[0060] Preparation of compound crystalline form B of formula (I): 19.0 mg of the compound of formula (I) was dissolved in 1.0 ml of tetrahydrofuran, and 3.6 ml of water (the reverse solvent) was added drop by drop to obtain crystalline form B.

[0061] Preparation of the crystalline form C of compound (I): 19.7 mg of the compound of formula (I) was dissolved in 1.0 ml of acetonitrile, and 3.7 ml of water (the reverse solvent) was added drop by drop to obtain crystalline form C.

[0062] Preparation of compound crystalline form D of formula (I): 14.7 mg of the compound of formula (I) was dissolved in 1.0 ml of ethyl acetate, and 5.8 ml of the reverse solvent n-heptane was added dropwise to obtain crystalline form D.

[0063] Compound (I) (22.0 mg) was added to 0.6 ml of isopropyl acetate / m-xylene (volume ratio 1:3) to obtain a suspension, which was equilibrated at 50°C for 2 hours, filtered, cooled to 5°C at a rate of 0.1°C / min, and then volatilized at room temperature for about 1 month to obtain crystalline form D.

[0064] 32.2 mg of the compound of formula (I) was added to 0.5 ml of acetonitrile / m-xylene (volume ratio 1:5) to obtain a suspension. The suspension was equilibrated at 50°C for 2 hours, then cooled to 5°C at a rate of 0.1°C / min, then heated to 50°C at a rate of 0.1°C / min, and further cooled to 5°C at a rate of 0.1°C / min. After volatilization at room temperature for about 1 month, crystalline form D was obtained.

[0065] Preparation of the crystalline form E of compound (I): 15.7 mg of the compound of formula (I) was dissolved in 1.0 ml of 2-methyltetrahydrofuran, and 3.8 ml of the reverse solvent n-heptane was added dropwise to obtain crystalline form E.

[0066] Example 3 Pressure stability of compound crystal form C of formula (I) The compound (I) in crystalline form C was placed in a circular mold (6 mm in diameter), and pressurized until the pressure on the sample reached approximately 350 MPa. A small piece of the sheet-like sample was then taken and directly examined using XRPD. The results showed that the crystalline form of crystalline form C remained unchanged after being compressed into a tablet (at a pressure of approximately 350 MPa).

[0067] Conclusion: Compound crystalline form C of formula (I) exhibits excellent pressure stability.

[0068] Example 4: Solid Stability Experiment of Compound Crystal Form C of Formula (I) To evaluate the solid stability of compound crystalline form C of formula (I), we investigated the influencing factors (high temperature, high humidity, and light irradiation) and the stability under conditions of 60°C / 75%RH and 40°C / 75%RH for compound crystalline form C of formula (I). Crystal form C was left for 1 week and 2 weeks, respectively, under high temperature (60°C, sealed) and high humidity (92.5% RH, wrapped in a sealing film with 5 holes). Simultaneously, it was left in a sealed state under ICH conditions (visible light irradiance reaching 1.2E+06 Lux·hrs and ultraviolet irradiance reaching 200 W·hrs / m2) under visible light and ultraviolet light (samples of the light-shielding control group were left simultaneously and wrapped in aluminum foil). At the same time, it was left for 1 and 2 months under 60°C / 75% RH conditions (wrapped in a sealing film with 5 holes), and for 1, 2, and 3 months under 40°C / 75% RH conditions (wrapped in a sealing film with 5 holes). Changes in crystal form were detected by XRPD testing of all stability samples, and HPLC testing was performed on all stability samples.

[0069] Experimental results: See Table 9.

[0070] [Table 10]

[0071] Conclusion: The compound crystal type C of formula (I) has excellent solid stability.

[0072] Example 5 Confirmation of the stereochemistry of compound (I) Crystal preparation of the compound of formula (I): Crystals of the compound of formula (I) are obtained by culturing at room temperature for 10 days using the solvent evaporation method under ethanol conditions. Referring to Figure 16, the ellipsoidal structure of the compound of formula (I) is in the S configuration. For structural data and parameters of the crystals of the compound of formula (I), refer to Tables 10-1, 10-2, 10-3, 10-4, 10-5, and 10-6.

[0073] [Table 11]

[0074] [Table 12]

[0075] [Table 13] JPEG0007836803000017.jpg255170JPEG0007836803000018.jpg251170JPEG0007836803000019.jpg123170

[0076] [Table 14] JPEG0007836803000021.jpg54170

[0077] [Table 15]

[0078] [Table 16] JPEG0007836803000024.jpg197170

[0079] Experimental Example 1: Cell Experiment Objective of the experiment The effects of compounds on cell proliferation were detected in NCI-H358, MIA-PA-CA-2, A375, SW1463, and A427 cells.

[0080] Studies have already demonstrated that the compound of formula (I) is a KRAS G12C selective inhibitor that covalently binds to the GDP-binding KRAS G12C protein, blocking the GDP / GTP exchange of the KRAS protein and maintaining the KRAS protein in an inactive state in its GDP-binding form, thereby suppressing cell and tumor growth.

[0081] [Table 17]

[0082] [Table 18]

[0083] [Table 19]

[0084] Experimental method Cell culture conditions H358 cell line medium: 89% medium RPMI1640 + 10% fetal bovine serum + 1% P / S MIA-PA-CA-2 cell culture medium: 86.5% DMEM medium + 10% fetal bovine serum + 2.5% equine serum + 1% P / S A375 cell line medium: 89% medium DMEM + 10% fetal bovine serum + 1% P / S SW1463 cell line medium: 89% medium Leibovit2 sL-15 + 10% fetal bovine serum + 1% P / S A427 cell culture medium: 89% EMEM medium + 10% fetal bovine serum + 1% P / S

[0085] cell seeding 1. The culture medium and pancreatin used in the cell subculturing process were placed in a 37°C water bath and preheated.

[0086] 2. The cells were rinsed with pancreatin, digested with pancreatin until they were detached, and then the culture medium was added and mixed to complete the digestion process.

[0087] 3. Cells were aspirated into a 15 mL centrifuge tube, and 0.01 mL of cell suspension was aspirated and added to a counting plate for counting.

[0088] 4. For all cells, an appropriate amount of cells was taken according to the cell density shown in the table below, added to a 15 mL centrifuge tube, and then supplemented with the appropriate cell culture medium up to a volume of 9 mL.

[0089] [Table 20]

[0090] 5. For the cells, following the attached microplate layout diagram, 150 μL of sterile water was added to the peripheral row of wells of the 96-well microplate, and 80 μL of cell suspension was added to each of the other wells. Then, the cell culture plate was placed in an incubator and cultured.

[0091] Preparation of compounds The compound of formula (I) was diluted from 10 mM to 2 mM using DMSO, and then subsequently diluted threefold in succession, resulting in a total of nine concentration gradients.

[0092] Two μL of the prepared compound was aspirated according to the concentration gradient and added to 78 μL of the culture medium for the corresponding cells, then mixed uniformly to obtain the compound administration solution.

[0093] 3. Following the attached microplate layout diagram, 20 μL of the compound solution was aspirated and added to each cell culture plate. The final concentration was 10 μM to 0.0015 μM, and the final DMSO concentration was 0.5%. After adding 20 μL of cell medium containing 2.5% DMSO to the control group, the cell culture plates were returned to the incubator and cultured.

[0094] Detection and Reading 1. On the day of compound treatment, the test plate (day 0) was removed from the incubator, 50 μL of Cell Titer Glo was added, the plate was centrifuged at 1000 rpm for 10 seconds, shaken at room temperature for 10 minutes, then centrifuged again at 1000 rpm for 10 seconds, and read by Envision.

[0095] 2. After incubating the cells with compounds for 72 hours, the test plates of MIA-PA-CA-2, A375, and A427 cells were removed from the incubator, 50 μL of Cell Titer-Glo was added to each well of the 96-well microplate, the plates were centrifuged at 1000 rpm for 10 seconds, shaken at room temperature for 10 minutes, centrifuged again at 1000 rpm for 10 seconds, and read using Envision.

[0096] 3. After incubating the cells with compounds for 120 hours, the NCI-H358 cell test plate was removed from the incubator, 50 μL of Cell Titer-Glo was added to each well of the 96-well microplate, the plate was centrifuged at 1000 rpm for 10 seconds, shaken at room temperature for 10 minutes, centrifuged again at 1000 rpm for 10 seconds, and read by Envision.

[0097] 4. After incubating the cells with the compound for 144 hours, the SW1463 cell test plate was removed from the incubator, 50 μL of Cell Titer-Glo was added to each well of the 96-well microplate, the plate was centrifuged at 1000 rpm for 10 seconds, shaken at room temperature for 10 minutes, centrifuged again at 1000 rpm for 10 seconds, and read using Envision.

[0098] Experimental results and analysis The results of this experiment demonstrate that the compound of formula (I) exhibits excellent inhibitory activity against the proliferation of human non-small cell lung cancer NCI-H358 cells, pancreatic cancer MIA-PA-CA-2 cells, and human rectal adenocarcinoma cells SW1463. Simultaneously, the compound of formula (I) shows weak activity against melanoma A375 cells and human lung cancer cells A427, indicating low inhibitory activity against wild-type KRAS and KRAS(G12D) proteins and a low risk of off-target effects. Detailed IC 50 For data, please refer to Table 11 below.

[0099] [Table 21]

Claims

1. A crystal of the compound of formula (I), wherein the X-ray powder diffraction spectrum has characteristic diffraction peaks at angles 2θ: 5.89±0.20°, 8.82±0.20°, 13.19±0.20°, 14.81±0.20°, 17.71±0.20°, 18.72±0.20°, 21.58±0.20°, and 24.47±0.20°. 【Chemistry 1】

2. The crystal according to claim 1, wherein the X-ray powder diffraction spectrum has characteristic diffraction peaks at angles 2θ: 5.89°, 8.82°, 11.50°, 12.58°, 13.19°, 14.42°, 14.81°, 17.71°, 18.72°, 20.70°, 21.58°, 23.51°, 24.47°, 25.36°, 26.58°, 27.09°, and 29.08°.

3. The crystal according to claim 1 or 2, wherein the differential scanning calorimetry curve has an endothermic peak starting at 214.7 ± 5°C.

4. The crystal according to any one of claims 1 to 3, wherein the weight loss at 150.0 ± 3°C in the thermogravimetric analysis curve reaches 2.52%.

5. Use of the crystal according to any one of claims 1 to 4 in the preparation of a cancer drug.

6. The use according to claim 5, wherein the cancer includes lung cancer, lymphoma, esophageal cancer, ovarian cancer, pancreatic cancer, rectal cancer, glioma, cervical cancer, urothelial carcinoma, stomach cancer, endometrial cancer, liver cancer, bile duct cancer, breast cancer, colon cancer, leukemia, and melanoma.

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