TWI812259B - Crystal form of raf kinase inhibitor and preparation method thereof - Google Patents

Abstract

The invention provides a crystal form of a RAF kinase inhibitor and a preparation method thereof, and specifically discloses a crystal form of a compound N-(3-(2-((1R,5S)-3-oxabicyclo[3.1.0]) hex-1-yl)-6-(2-hydroxyethoxy)pyridin-4-yl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide shown as formula (I) and preparation method thereof.

Description

Crystal form of RAF kinase inhibitor and preparation method thereof

The invention discloses a crystal form of a RAF kinase inhibitor, a preparation method thereof, and their application in the treatment of related diseases.

This application claims the following priority rights: CN202110625608.3, application date June 4, 2021; CN202110625603.0, application date June 4, 2021; CN202210583709.3, application date is May 25, 2022.

The mitogen-activated protein kinase (MAPK) pathway is an important signal transduction pathway in cells. This pathway transmits signals from the outside of the cell into the nucleus through the specific cascade phosphorylation of RAS/RAF/MEK/ERK, ultimately leading to specific Activation of genes causes cell proliferation, apoptosis or differentiation. RAF is a very important serine/threonine protein kinase in the RAS/RAF/MEK/ERK signaling pathway. It is located downstream of RAS and can be activated by RAS. RAF activation can phosphorylate MEK protein and complete downstream signaling. The RAF family includes three subtypes: ARAF, BRAF, and CRAF (RAF-1), which share a high degree of homology and similar structural domains. ARAF and CRAF mutations occur less frequently, and BRAF mutation rate is higher. RAF inhibitors can effectively inhibit ERK phosphorylation, thereby blocking downstream signal MEK/ERK transmission, thereby effectively blocking the RAS-ERK pathway and having a therapeutic effect on tumor diseases in the MAPK pathway. Therefore, RAF kinase has become an important target for clinical treatment of tumors.

The first-generation BRAF kinase inhibitors Vemurafenib, Dabrafenib and Encorafenib have been approved by the FDA for the treatment of cancers with B-Raf ^V600E mutations. Although Vemurafenib, Dabrafenib and Encorafenib showed promising efficacy in the treatment of B-Raf ^V600E mutant melanoma, relapse occurred within one year due to acquired drug resistance. A new generation of pan-Raf inhibitors can overcome drug resistance and is being developed to expand the scope of clinical application. Pan-RAF inhibitors can inhibit dimer activity and block paradoxcal activation of the pathway, thereby reducing drug resistance. Pan-Raf dimer inhibitors under clinical research mainly include HM95573, TAK-580, BGB-283, LXH254, and LY3009120. The development of these new RAF inhibitors is expected to overcome the resistance of the first-generation inhibitors. Further expand clinical application.

The inventor disclosed a small molecule pan-RAF kinase inhibitor in patent PCT/CN/2020/133933. Its structure is shown in formula (I), and its chemical name is N -(3-(2-((1 R ,5 S )-3-oxabicyclo[3.1.0]hex-1-yl)-6-(2-hydroxyethoxy)pyridin-4-yl)-4-methylphenyl)-2 -( Trifluoromethyl)isonicotinamide. This small molecule inhibitor has good RAF kinase inhibitory activity and various cell anti-proliferative activities. At the same time, the molecule has good pharmacokinetic properties and is expected to be developed into a clinical drug.

The invention provides compound N- (3-(2-((1 R ,5 S )-3-oxabicyclo[3.1.0]hex-1-yl)-6-(2-hydroxyethoxy) of formula (I) Form A of pyridin-4-yl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide, , its X-ray powder diffraction pattern has characteristic peaks at the following 2θ angles: 6.03±0.20°, 12.00±0.20°, 14.36±0.20°, 19.72±0.20°.

In some aspects of the invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic peaks at the following 2θ angles: 6.03±0.20°, 9.63±0.20°, 12.00±0.20°, 14.36±0.20°, 19.02 ±0.20°, 19.72±0.20°, 20.73±0.20°, 22.10±0.20°, 24.08±0.20°.

In some aspects of the invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic peaks at the following 2θ angles: 6.03±0.20°, 7.65±0.20°, 9.63±0.20°, 12.00±0.20°, 14.09 ±0.20°, 14.36±0.20°, 14.82±0.20°, 15.28±0.20°, 15.95±0.20°, 16.31±0.20°, 17.50±0.20°, 19.02±0.20°, 19.72±0.20°, 20.73±0.20°, 22.10 ±0.20°, 23.29±0.20°, 24.08±0.20°, 25.53±0.20°, 26.73±0.20°, 27.34±0.20°, 28.51±0.20°, 29.04±0.20°, 31.04±0.20°, 33.17±0.20°, 33.72 ±0.20°, 34.28±0.20°, 39.63±0.20°.

In some aspects of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic peaks at the following 2θ angles: 12.00±0.20°, and/or 6.03±0.20°, and/or 7.65±0.20°, and/or 9.63±0.20°, and/or 14.09±0.20°, and/or 14.36±0.20°, and/or 14.82±0.20°, and/or 15.28±0.20°, and/or 15.95±0.20°, and/ or 16.31±0.20°, and/or 17.50±0.20°, and/or 19.02±0.20°, and/or 19.72±0.20°, and/or 20.73±0.20°, and/or 22.10±0.20°, and/or 23.29 ±0.20°, and/or 24.08±0.20°, and/or 25.53±0.20°, and/or 26.73±0.20°, and/or 27.34±0.20°, and/or 28.51±0.20°, and/or 29.04±0.20 °, and/or 31.04±0.20°, and/or 33.17±0.20°, and/or 33.72±0.20°, and/or 34.28±0.20°, and/or 39.63±0.20°.

In some aspects of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A is as shown in Figure 1.

In some aspects of the present invention, the XRPD spectrum diffraction peak analysis data of the crystal form A of the compound of formula (I) is shown in Table 1. Table 1 XRPD diffraction peak analysis data of crystal form A of the compound of formula (I) No. 2θ angle [°] Interplanar spacing [Å] intensity[count] Relative strength[%] 1 6.03 14.66 2742.60 50.56 2 7.65 11.56 502.08 9.26 3 9.63 9.18 777.14 14.33 4 12.00 7.37 3339.66 61.56 5 14.09 6.29 497.94 9.18 6 14.36 6.17 460.57 8.49 7 14.82 5.98 123.30 2.27 8 15.28 5.80 515.57 9.50 9 15.95 5.56 509.01 9.38 10 16.31 5.43 617.58 11.38 11 17.50 5.07 111.41 2.05 12 19.02 4.67 1676.43 30.90 13 19.72 4.50 5424.76 100.00 14 20.73 4.28 622.90 11.48 15 22.10 4.02 895.02 16.50 16 23.29 3.82 442.31 8.15 17 24.08 3.70 678.20 12.50 18 25.53 3.49 370.44 6.83 19 26.73 3.33 274.51 5.06 20 27.34 3.26 171.59 3.16 twenty one 28.51 3.13 452.71 8.35 twenty two 29.04 3.07 214.05 3.95 twenty three 31.04 2.88 562.60 10.37 twenty four 33.17 2.70 258.62 4.77 25 33.72 2.66 101.68 1.87 26 34.28 2.62 89.31 1.65 27 39.63 2.27 253.68 4.68

In some aspects of the present invention, the above-mentioned crystal form A contains water molecules, and the molar ratio of the compound of formula (I) to water molecules is 1:1.

In some aspects of the present invention, the above-mentioned crystal form A is a monohydrate of the compound of formula (I).

In some aspects of the present invention, the TGA spectrum of the above-mentioned crystal form A is shown in Figure 5.

In some aspects of the present invention, the DSC spectrum of the above-mentioned crystal form A is shown in Figure 7.

The present invention also provides the compound of formula (I) N- (3-(2-((1 R ,5 S )-3-oxabicyclo[3.1.0]hex-1-yl)-6-(2-hydroxy Form B of ethoxy)pyridin-4-yl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide, , its X-ray powder diffraction pattern has characteristic peaks at the following 2θ angles: 6.06±0.20°, 12.06±0.20°, 20.10±0.20°, 21.24±0.20°.

In some aspects of the invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic peaks at the following 2θ angles: 6.06±0.20°, 12.06±0.20°, 15.94±0.20°, 18.95±0.20°, 20.10 ±0.20°, 20.74±0.20°, 21.24±0.20°, 22.20±0.20°, 24.20±0.20°.

In some aspects of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic peaks at the following 2θ angles: 6.06±0.20°, 7.77±0.20°, 9.61±0.20°, 12.06±0.20°, 14.15 ±0.20°, 14.93±0.20°, 15.48±0.20°, 15.94±0.20°, 16.94±0.20°, 17.62±0.20°, 18.95±0.20°, 20.10±0.20°, 20.74±0.20°, 21.24±0.20°, 22.20 ±0.20°, 23.27±0.20°, 24.20±0.20°, 25.65±0.20°, 26.83±0.20°, 27.42±0.20°, 28.17±0.20°, 28.90±0.20°, 30.96±0.20°, 31.54±0.20°, 37.07 ±0.20°, 37.91±0.20°.

In some aspects of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B is as shown in Figure 2.

In some aspects of the present invention, the diffraction peak analysis data of the XRPD spectrum of the crystalline form B of the compound of formula (I) is shown in Table 2. Table 2 XRPD diffraction peak analysis data of compound Form B of formula (I) No. 2θ angle [°] Interplanar spacing [Å] intensity[count] Relative strength[%] 1 6.06 14.59 4109.29 100.00 2 7.77 11.38 378.80 9.22 3 9.61 9.20 260.17 6.33 4 12.06 7.34 2933.91 71.40 5 14.15 6.26 268.31 6.53 6 14.93 5.93 141.86 3.45 7 15.48 5.72 348.08 8.47 8 15.94 5.56 687.62 16.73 9 16.94 5.23 167.73 4.08 10 17.62 5.03 225.98 5.50 11 18.95 4.68 1866.25 45.42 12 20.10 4.42 3615.94 87.99 13 20.74 4.28 677.26 16.48 14 21.24 4.18 175.37 4.27 15 22.20 4.00 1082.49 26.34 16 23.27 3.82 274.91 6.69 17 24.20 3.68 663.20 16.14 18 25.65 3.47 595.44 14.49 19 26.83 3.32 265.97 6.47 20 27.42 3.25 290.03 7.06 twenty one 28.17 3.17 278.72 6.78 twenty two 28.90 3.09 220.77 5.37 twenty three 30.96 2.89 397.99 9.69 twenty four 31.54 2.84 130.72 3.18 25 37.07 2.43 54.15 1.32 26 37.91 2.37 98.22 2.39

In some aspects of the present invention, the TGA spectrum of the above-mentioned crystal form B is shown in Figure 6.

In some aspects of the present invention, the DSC spectrum of the above-mentioned crystal form B is shown in Figure 8.

The present invention also provides a crystal of the compound of formula (I) whose X-ray powder diffraction pattern has characteristic peaks at the following 2θ angles: 6.03±0.20°, 7.65±0.20°, 9.63±0.20°, 12.00±0.20°, 19.72 ±0.20°.

In some aspects of the present invention, the X-ray powder diffraction pattern of the crystal of the compound of formula (I) has characteristic peaks at the following 2θ angles: 6.03±0.20°, 7.65±0.20°, 9.63±0.20°, 12.00± 0.20°, 19.72±0.20°, 22.10±0.20°.

In some aspects of the present invention, the X-ray powder diffraction pattern of the crystal of the compound of formula (I) has characteristic peaks at the following 2θ angles: 6.03±0.20°, 7.65±0.20°, 9.63±0.20°, 12.00± 0.20°, 19.72±0.20°, 22.10±0.20°, 24.08±0.20°.

In some aspects of the present invention, the X-ray powder diffraction pattern of the crystal of the compound of formula (I) has characteristic peaks at the following 2θ angles: 6.03±0.20°, 7.65±0.20°, 9.63±0.20°, 12.00± 0.20°, 19.72±0.20°, 22.10±0.20°, 24.08±0.20°, 31.04±0.20°.

The invention also provides the compound of formula (I) N- (3-(2-((1 R ,5 S )-3-oxabicyclo[3.1.0]hex-1-yl)-6-(2-hydroxyethyl) Amorphous substance of oxy)pyridin-4-yl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide, .

In some aspects of the present invention, the X-ray powder diffraction pattern of the above-mentioned amorphous substance is as shown in Figure 9.

The present invention also provides a pharmaceutical composition, which contains the above-mentioned crystalline form A and/or crystalline form B and/or crystals, and/or amorphous substance, and a pharmaceutically acceptable carrier.

The present invention also provides the use of the above-mentioned crystalline form A and/or crystalline form B and/or crystalline and/or amorphous substance or pharmaceutical composition thereof in the preparation of drugs for the treatment of RAF kinase mutation-mediated diseases.

In some aspects of the invention, the disease is lung cancer.

The present invention also provides the use of the above-mentioned crystalline form A and/or crystalline form B and/or crystalline and/or amorphous form in the preparation of drugs for treating lung cancer.

Technical effect

The crystal form of the present application is stable, less affected by heat, humidity, and light, and is easy to store and prepare. The crystal form of the present application has good pharmacokinetic properties, meets the requirements of the drug in the body, and is suitable for use as a pharmaceutical preparation. The pharmacokinetic properties can be used in preclinical studies such as SD rats and beagle dogs. Measured in animal experiments.

Definition and Description

Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be considered uncertain or unclear in the absence of a specific definition, but should be understood in its ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding trade name or its active ingredient.

As used herein, the term "pharmaceutically acceptable" means suitable for contact with human and animal tissue within the scope of reasonable medical judgment without undue toxicity, irritation, allergic reactions or other problematic complications, and with reasonable benefit/risk Comparable to those compounds, materials, compositions and/or dosage forms.

The term "pharmaceutically acceptable carrier" refers to any preparation or carrier medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or patient. Representative carriers include water, oil, vegetables and Minerals, cream base, lotion base, ointment base, etc. These matrices include suspending agents, viscosifiers, transdermal penetration enhancers, etc. Their preparations are well known to those skilled in the field of cosmetics or topical medicine.

The structure of the crystals, crystals or crystal forms disclosed or claimed in the present invention may appear similar but not identical within a reasonable error range depending on experimental conditions, purity, equipment and other constant variables known to those skilled in the art to which this invention belongs. Exactly the same analytical properties. Accordingly, it will be apparent to one of ordinary skill in the art to which this invention belongs that various modifications and changes can be made within the present invention without departing from the scope and spirit of the invention. For example, the diffraction angle (2θ) in powder X-ray diffraction usually produces an error within the range of ±0.20°, so the value of the above diffraction angle needs to be understood to include a value within the range of approximately ±0.20°. Therefore, the present invention includes not only crystals with completely consistent diffraction angles in powder X-ray diffraction, but also crystals with consistent diffraction angles within an error range of ±0.20°.

As used herein, "polymorph" or "polymorph" refers to crystalline forms that have the same chemical composition but different spatial arrangements of the molecules, atoms, and/or ions that make up the crystal. Although polymorphs have the same chemical composition, they differ in packing and geometric arrangement and may exhibit different physical properties such as melting point, shape, color, density, hardness, deformability, stability, solubility, dissolution velocity and similar properties. Depending on their temperature-stability relationship, two polymorphs can be monotropic or tautotropic. For a unidenaturing system, the relative stability between the two solid phases remains unchanged when the temperature changes. In contrast, in tautotropic systems, there is a transition temperature at which the stability of the two phases switches. The phenomenon of a compound existing in different crystal structures is called drug polymorphism.

The crystalline structures of the present invention can be prepared by a variety of methods, including crystallization or recrystallization from a suitable solvent, sublimation, growth from a melt, solid state conversion from another phase, crystallization from a supercritical fluid, and jet spraying. Techniques for crystallizing or recrystallizing a crystalline structure from a solvent mixture, including solvent evaporation, lowering the temperature of the solvent mixture, seeding of a supersaturated solvent mixture of the molecule and/or salt, lyophilizing the solvent mixture, adding an antisolvent to the solvent mixture, etc. .

The intermediate compound of the present invention can be prepared by a variety of synthetic methods well known to those with ordinary knowledge in the technical field of the present invention, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and the present invention. There are equivalent alternatives well known to those skilled in the art, and preferred implementations include, but are not limited to, the embodiments of the present invention.

The chemical reactions of the specific embodiments of the present invention are completed in a suitable solvent, and the solvent must be suitable for the chemical changes of the present invention and the required reagents and materials. In order to obtain the compounds of the present invention, it is sometimes necessary for those with ordinary knowledge in the technical field to which the present invention belongs to modify or select the synthesis steps or reaction procedures based on the existing embodiments.

The term "protecting group" includes, but is not limited to, "amine protecting group", "hydroxy protecting group" or "thiol protecting group". The term "amine protecting group" refers to a protecting group suitable for preventing side reactions at the nitrogen position of an amine group. Representative amino protecting groups include, but are not limited to: formyl; carboxyl, such as alkyl (such as acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl, such as tris grade butoxycarbonyl (Boc); arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn), trityl ( Tr), 1,1-di-(4'-methoxyphenyl)methyl; silyl groups such as trimethylsilyl (TMS) and tertiary butyldimethylsilyl (TBS) etc. The term "hydroxyl protecting group" refers to a protecting group suitable for preventing side reactions of hydroxyl groups. Representative hydroxyl protecting groups include, but are not limited to: alkyl groups, such as methyl, ethyl, and tertiary butyl; hydroxyl groups, such as alkyl groups (such as acetyl groups); arylmethyl groups, such as benzyl (Bn ), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tertiary butyldimethylsilyl (TBS), among others.

The structure of the compound of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention involves the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, single crystal X-ray diffraction (SXRD) uses a Bruker D8 venture diffractometer to collect diffraction intensity data on the cultured single crystal. The light source is CuKα radiation, and the scanning mode is: φ/ɷ scanning. After collecting relevant data, further The absolute configuration can be confirmed by analyzing the crystal structure using the direct method (Shelxs97).

The present invention will be described in detail through examples below. These examples do not mean any limitation to the present invention.

The solvent used in the present invention is commercially available.

The following abbreviations are used in the present invention: Pd(dppf)Cl ₂ ·DCM stands for [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride-dichloromethane complex, [Ir( COD)OMe] ₂ represents cyclooctadiene methoxyiridium dimer, tmfen represents 3,4,7,8-tetramethyl-1,10phenanthroline, Pd ₂ dba ₃ represents tris-diphenylacetone- Dipalladium, Brettphos represents (2-dicyclohexylphosphine-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl, PE represents petroleum ether, EA stands for ethyl acetate and DMSO stands for dimethylsulfoxide.

The compounds of the present invention are named according to the conventional naming principles in this field or using ChemDraw® software, and the commercially available compounds adopt the names of suppliers' catalogs.

1. Instruments and analytical methods

1.1 X-ray powder ^diffraction (X-ray powder diffractometer, X-ray generator: Cu, kα, (λ=1.54060Å) Tube voltage: 45 kV, tube current: 40 mA. Scattering slit: 1 mm Detector slit: 0.3 mm Anti-scattering slit: 1 mm Scanning range : 3-40 degree step size: 0.0263 degree Scan time: 46.665 seconds

X-ray powder diffraction (XRPD) method of amorphous matter of the present invention Instrument model: DX-2700BH Test conditions: Detailed XRPD parameters are as follows: X-ray generator: Cu, kα, (λ=1.54184Å) Tube voltage: 40 kV, tube current: 30 mA. Scattering slit: 1 mm Detector slit: 0.3 mm Anti-scatter slit: 1 mm Scanning range: 3-40 degrees Step size: 0.02 degrees Scan time: 0.5 seconds

The present invention is described in detail below through examples, which do not mean any adverse limitations to the present invention. The compounds of the present invention can be prepared by a variety of synthetic methods well known to those with ordinary knowledge in the technical field to which the present invention belongs, including the specific embodiments listed below, embodiments formed by their combination with other chemical synthesis methods, and the technology to which the present invention belongs. Equivalents will be known to those skilled in the art, and preferred embodiments include, but are not limited to, the embodiments of the present invention. It will be apparent to those of ordinary skill in the art that various changes and improvements can be made to the specific embodiments of the invention without departing from the spirit and scope of the invention.

Example 1: Preparation of compounds of formula (I) Compound 2

To a solution of compound 1 (10 g, 51.96 mmol) in dichloromethane (100 mL) at -78°C, n-butyllithium (23 mL, 2.5 M) was added and stirred for 30 minutes. At -78°C, 1a (4.92 g, 57.16 mmol) was added to the mixture, and the temperature was raised to 25°C and stirred for 0.5 hours. Saturated ammonium chloride solution (40 mL) was added to the reaction mixture, and extracted with dichloromethane (40 mL×2). The combined extracts were dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography (PE/EA=10 /1 to 5/1, V/V) to isolate compound 2. ¹ HNMR (400MHz, CDCl ₃ ) δ 7.72 (t, J =7.8 Hz, 1H), 7.47 (d, J =7.2 Hz, 1H), 7.28 (d, J =7.2 Hz, 1H), 4.35 (s, 1H ), 4.24 - 4.14 (m, 2H), 4.05 - 4.00 (m, 1H), 3.96 - 3.92 (m, 1H), 2.45 (td, J =8.8, 13.0 Hz, 1H), 2.32 - 2.22 (m, 1H ). Compound 3

To a solution of 2 (6 g, 30.06 mmol) in toluene (80 mL), p-toluenesulfonic acid (11.5 g, 60.46 mmol) was added, and the temperature was raised to 110°C and stirred for 16 hours. Add saturated sodium bicarbonate solution (40 mL) and ethyl acetate (100 mL) to the reaction mixture, separate the liquids, wash with saturated sodium bicarbonate solution (50 mL × 2), dry over anhydrous sodium sulfate, and concentrate. Compound 3 was separated by silica gel column chromatography (PE/EA=50/1 to 20/1, V/V). ¹ HNMR (400MHz, CDCl ₃ ) δ 7.64 (t, J =7.8 Hz, 1H), 7.23 (dd, J =5.2, 7.8 Hz, 2H), 6.68 (quin, J =2.0 Hz, 1H), 5.08 (dt , J =2.0, 5.0 Hz, 2H), 4.91 (dt, J =2.0, 5.0 Hz, 2H). Compound 4

To a mixture of potassium tertiary butoxide (3.71 g, 33.04 mmol), 2a (7.3 g, 33.17 mmol) was added DMSO (50 mL). Under nitrogen protection, the reaction mixture was stirred at 25°C for 1 hour. A solution of 3 (1 g, 5.51 mmol) in DMSO (5 mL) was added to the reaction solution, and the reaction mixture was heated to 70°C and stirred for 6 hours. Water (180 mL) was added to the reaction mixture, extracted with ethyl acetate (30 × 3 mL), the organic phase was dried over anhydrous sodium sulfate, concentrated, and subjected to silica gel column chromatography (PE/EA=20/1, V/V ) was separated to obtain compound 4. ¹ HNMR (400 MHz, CDCl ₃ ) δ 7.46 (t, J =7.8 Hz, 1H), 7.05 (d, J =7.8 Hz, 1H), 6.88 (d, J =7.6 Hz, 1H), 4.11 - 4.04 ( m, 2H), 3.89 - 3.78 (m, 2H), 2.08 (ddd, J =2.8, 5.2, 8.0 Hz, 1H), 1.32 (dd, J =4.4, 8.0 Hz, 1H), 1.07 (t, J = 4.6 Hz, 1H). Compound 5

3a (1.08 g, 4.25 mmol), [Ir(COD)OMe] ₂ (70 mg, 105.60 μmol) and tmfen (50 mg, 211.59 μmol) were placed in methyl tert-butyl ether (20 mL). Under nitrogen protection, 4 (770 mg, 3.94 mmol) was added to the reaction mixture, heated to 80°C and stirred for 3 hours. The reaction solution was filtered, and the filtrate was concentrated to obtain crude compound 5. MS (ESI): m/z 240.3 [M-84+2H] ⁺ . Compound 6

Compound 5 (1.2 g, 3.73 mmol), 4a (1.4 g, 3.90 mmol) and Pd(dppf)Cl ₂ ·DCM (300 mg, 367.36 μmol) and sodium carbonate (800 mg, 7.55 mmol) were mixed Mol) in dihexane (50 mL) and water (10 mL). The reaction mixture was heated to 100°C under nitrogen protection and stirred for 3 hours. Filter the reaction mixture, concentrate the filtrate, add ethyl acetate (20 mL) and water (10 mL), separate the layers, extract the aqueous phase with ethyl acetate (10 mL×3), and dry the combined organic phase with anhydrous sodium sulfate After concentration, 6 was separated by silica gel column chromatography (PE/EA=10/1 to 5/1). ¹ HNMR (400 MHz, CDCl ₃ ) δ 8.92 (d, J =5.0 Hz, 1H), 8.20 (s, 1H), 8.11 (s, 1H), 7.94 (d, J =5.0 Hz, 1H), 7.60 - 7.51 (m, 2H), 7.32 (d, J =7.8 Hz, 1H), 7.10 (d, J =1.2 Hz, 1H), 6.90 (d, J =1.2 Hz, 1H), 4.19 - 4.14 (m, 2H ), 3.95 - 3.87 (m, 2H), 2.27 (s, 3H), 2.24 - 2.17 (m, 1H), 1.48 - 1.41 (m, 1H), 1.20 - 1.15 (m, 1H). Compound 7

To a solution of 6 (900 mg, 1.90 mmol) and 5a (400 mg, 2.27 mmol) in toluene (30 mL) was added Pd ₂ dba ₃ (180 mg, 196.57 μmol), Brettphos (200 mg , 372.60 micromoles) and cesium carbonate (1.26 g, 3.87 mmol). Under nitrogen protection, the reaction mixture was heated to 110°C and stirred for 4 hours. The reaction solution was filtered, and the filtrate was concentrated and separated by silica gel column chromatography (PE/EA=20/1 to 6/1) to obtain compound 7. ¹ HNMR (400 MHz, DMSO-d ₆ ) δ 10.65 (s, 1H), 8.95 (d, J =5.0 Hz, 1H), 8.31 (s, 1H), 8.14 (d, J =4.6 Hz, 1H), 7.70 (dd, J =2.0, 8.2 Hz, 1H), 7.59 (d, J =2.2 Hz, 1H), 7.29 (d, J =8.4 Hz, 1H), 6.68 (s, 1H), 6.49 (s, 1H ), 4.31 (br d, J =2.8 Hz, 1H), 4.07 - 3.97 (m, 2H), 3.90 - 3.86 (m, 2H), 3.79 - 3.69 (m, 2H), 2.17 (s, 3H), 2.15 - 2.09 (m, 1H), 1.33 (dd, J =3.8, 7.8 Hz, 1H), 1.13 (t, J =7.2 Hz, 1H), 0.96 - 0.91 (m, 1H), 0.80 (s, 9H), 0.00 (s, 6H). Compound 8

To 7 (300 mg, 488.81 μmol) in tetrahydrofuran (10 mL) was added hydrochloric acid (1 mL, 4 M), and the reaction was stirred at 25°C for 1 hour. After diluting the reaction solution with ethyl acetate (20 mL), adjust pH=8 with saturated sodium bicarbonate solution, extract with ethyl acetate (10 × 3 mL), dry the organic phase with anhydrous sodium sulfate, concentrate, and apply a thin layer of silica gel The plate (PE/EA= 1/1, V/V) was separated to obtain compound 8. ¹ HNMR (400 MHz, DMSO-d ₆ ) δ 10.70 (s, 1H), 9.06-8.95 (d, J =5.0 Hz, 1H), 8.37 (s, 1H), 8.23-8.18 (d, J =4.2 Hz , 1H), 7.77-7.71 (dd, J =2.2, 8.4 Hz, 1H), 7.68-7.64 (d, J =2.2 Hz, 1H), 7.39-7.31 (d, J =8.4 Hz, 1H), 6.73 ( s, 1H), 6.59-6.54 (d, J =1.0 Hz, 1H), 4.86-4.81 (t, J =5.2 Hz, 1H), 4.35-4.30 (t, J =5.2 Hz, 2H), 4.13 - 4.03 (m, 2H), 3.83 - 3.70 (m, 4H), 2.24 (s, 3H), 2.22-2.08 (ddd, J =2.4, 5.0, 7.8 Hz, 1H), 1.40-1.32 (dd, J =3.6, 7.8 Hz, 1H), 1.02-0.96 (t, J =4.4 Hz, 1H). MS (ESI) m/z: 500.4 [M+H] ⁺ . Compounds of formula (I)

Compound 8 was chiral separated by SFC (chiral column DAICEL CHIRALCEL OJ-H (250mm*30mm, 5μm), mobile phase A: ethanol (containing 0.05% diisopropylethylamine); mobile phase B: carbon dioxide) to obtain 8A ( retention time 1.487 min) and compounds of formula (I) (retention time 1.590 min). 8A is an enantiomer of the compound of formula (I).

Compound 8A: ¹ HNMR (400 MHz, DMSO-d ₆ ) δ 10.70 (s, 1H), 9.06-8.95 (d, J =5.0 Hz, 1H), 8.37 (s, 1H), 8.23-8.18 (d, J =4.2 Hz, 1H), 7.77-7.71 (dd, J =2.2, 8.4 Hz, 1H), 7.68-7.64 (d, J =2.2 Hz, 1H), 7.39-7.31 (d, J =8.4 Hz, 1H) , 6.73 (s, 1H), 6.59-6.54 (d, J =1.0 Hz, 1H), 4.86-4.81 (t, J =5.2 Hz, 1H), 4.35-4.30 (t, J =5.2 Hz, 2H), 4.13 - 4.03 (m, 2H), 3.83 - 3.70 (m, 4H), 2.24 (s, 3H), 2.22-2.08 (ddd, J =2.4, 5.0, 7.8 Hz, 1H), 1.40-1.32 (dd, J =3.6, 7.8 Hz, 1H), 1.02-0.96 (t, J =4.4 Hz, 1H). MS (ESI) m/z: 500.4 [M+H] ⁺ , 100% (ee%).

Compounds of formula (I): ¹ HNMR (400 MHz, DMSO-d ₆ ) δ 10.70 (s, 1H), 9.06-8.95 (d, J =5.0 Hz, 1H), 8.37 (s, 1H), 8.23-8.18 ( d, J =4.2 Hz, 1H), 7.77-7.71 (dd, J =2.2, 8.4 Hz, 1H), 7.68-7.64 (d, J =2.2 Hz, 1H), 7.39-7.31 (d, J =8.4 Hz , 1H), 6.73 (s, 1H), 6.59-6.54 (d, J =1.0 Hz, 1H), 4.86-4.81 (t, J =5.2 Hz, 1H), 4.35-4.30 (t, J =5.2 Hz, 2H), 4.13 - 4.03 (m, 2H), 3.83 - 3.70 (m, 4H), 2.24 (s, 3H), 2.22-2.08 (ddd, J =2.4, 5.0, 7.8 Hz, 1H), 1.40-1.32 ( dd, J =3.6, 7.8 Hz, 1H), 1.02-0.96 (t, J =4.4 Hz, 1H). MS (ESI) m/z: 500.4 [M+H] ⁺ , 99.7% (ee%).

Example 2: Preparation of Form B of Compound of Formula (I) Step 1: Compound I-2

At room temperature, add N,N-dimethylformamide (12.5 liters) into a 50-liter reaction kettle, add compound I-1 (2.75 kg, 20.91 mol) and compound I-1-1 (3.55 kg , 31.36 mol), stir until dissolved and clear. Then add potassium carbonate (7.22 kg, 52.27 mol), heat to 75~80 degrees and stir for 16 hours. After the reaction is completed, cool the reaction solution to 25 degrees; add water (27 liters) to the reaction kettle, transfer about 27 liters of the reaction solution to a 30-liter white bucket for temporary storage; add the remaining 27 liters of reaction solution in the kettle. Slowly add hydrochloric acid solution (6 mol/L, 8.5 liters), 0.3 liters each time, and adjust the pH to 3~4. A yellow solid precipitates, accompanied by the generation of a large amount of gas and a small amount of heat. The solid-liquid mixture was filtered under reduced pressure, and the filter cake was washed three times with 2 liters of water each time. The remaining 27 liters of reaction solution were treated in the same way. After the filter cakes were combined, they were vacuum dried at 45 degrees for 24 hours to obtain compound I-2. ¹ HNMR (400MHz, CDCl ₃ ) δ 14.32 (br s, 1H), 7.42 (dd, J =7.4, 9.0 Hz, 1H), 7.12 (d, J =9.0 Hz, 1H), 6.53 (dd, J =1.4 , 7.4 Hz, 1H), 4.26 - 4.18 (m, 2H), 1.28 (t, J =7.2 Hz, 3H). Step 2: Compound I-3

At room temperature, add dimethyl sulfoxide (12 liters) into a 50-liter reaction kettle, add compound I-2 (3.6 kg, 16.03 mol), and raise the internal temperature to 70 degrees. After the reaction solution is clarified, add a solution of sodium chloride (1.87 kg, 32.05 mol) in water (6 liters), raise the internal temperature to 80-85 degrees, and stir for 16 hours. After the reaction is completed, the temperature drops to 25 degrees, water (20.5 liters) is added to the kettle, stirred for 5 minutes, and the reaction solution is extracted with ethyl acetate twice, 10 liters each time. Combine the organic phases and wash twice with saturated brine, 10 liters each time. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure at 45 degrees to obtain the product compound I-3. ¹ HNMR (400MHz, CDCl ₃ ) δ 7.74 (t, J =7.8 Hz, 1H), 7.43 (d, J =7.6 Hz, 1H), 7.34 (d, J =8.0 Hz, 1H), 3.94 (s, 2H ). Step 3: Compound I-4

At room temperature, add tetrahydrofuran (7.5 liters) into a 50-liter reaction kettle, add compound I-3 (1.5 kg, 9.83 moles) and compound I-3-1 (0.926 kg, 10 moles), and the internal temperature drops to -25 degree. Under a nitrogen atmosphere, pump lithium hexamethylsilamide (10 liters, 10 moles, tetrahydrofuran solution) into the kettle, control the internal temperature not to exceed -15 degrees, and complete the addition in about 3 hours. Raise the internal temperature to -8~-5 degrees and keep the reaction for 3 hours. Lower the temperature to -25 degrees, use a peristaltic pump to add sodium hexamethylsilamide (10 liters, 10 moles, tetrahydrofuran solution) into the reaction kettle (complete the addition in 2 hours), and control the internal temperature not to exceed -15 degrees. . Raise the internal temperature to 20~25 degrees and stir for 16 hours. After the reaction is completed, slowly add water (0.883 liters, 49.1 moles) to the reaction solution, and stir for 0.5 hours while controlling the internal temperature below 30 degrees. The reaction solution was distilled under reduced pressure at 40 degrees in the kettle, and concentrated to obtain a crude product solution of compound I-4 (about 10 liters), which was directly used in the next reaction. Step 4: Compound 1-5

At room temperature, add an aqueous solution of potassium hydroxide (9.6 liters, 19.2 moles) and ethanol (10 liters) into a 50-liter reaction kettle of crude compound I-4 (10 liters of solution), and the internal temperature rises to 75~80 degrees. , stir for 5 hours. After the reaction is completed, cool down to 25~30 degrees, slowly add hydrochloric acid aqueous solution (8 liters, 48 moles) to the reaction solution, control the temperature not to exceed 40 degrees, heat the internal temperature to 60 degrees, and stir for 1 hour. After the reaction is completed, concentrate to obtain a crude product solution (remaining liquid is about 2 liters), and extract twice with ethyl acetate, each time 7.5 liters. Add the extract to the reaction kettle, add silica gel (100-200 mesh, 750 grams), add n-heptane (15 liters), and stir for 16 hours. Filter, wash the filter cake three times with n-heptane/ethyl acetate (1:1), 1 liter each time, combine the organic phases, and concentrate under reduced pressure to obtain a crude black solid product. Heat the crude product with ethanol (3 liters) to an internal temperature of 70 degrees, stir until dissolved and clear, then cool to 20 degrees. After a large amount of solid precipitates, add water (9 liters) to the suspension and stir for 0.5 hours. Filter, wash the filter cake with water twice, 1 liter each time; add the filter cake to the mixed solution (n-heptane/ethanol, 4:1, 4.5 liters), stir for 0.5 hours, filter, and wash the filter cake with n-heptane/ethanol. Wash twice with ethanol (4:1), 0.3 liters each time, and vacuum dry the filter cake at 45 degrees for 16 hours to obtain compound I-5. ¹ HNMR (400MHz, CDCl ₃ ) δ 8.09 (dd, J =0.8, 7.8 Hz, 1H), 7.64 (t, J =7.8 Hz, 1H), 7.18 (dd, J =0.8, 7.8 Hz, 1H), 4.43 (dd, J =4.6, 9.2 Hz, 1H), 4.31 (d, J =9.2 Hz, 1H), 3.04 - 2.89 (m, 1H), 2.14 (dd, J =4.4, 7.8 Hz, 1H), 1.56 - 1.44 (m, 1H). Step: 5: Compound 1-6

At room temperature, add tetrahydrofuran (16 liters) into a 50-liter reaction kettle, add compound I-5 (3.2 kg, 15.27 mol), and stir until dissolved and clear. The internal temperature rises to 30 degrees. Under a nitrogen atmosphere, use a peristaltic pump to slowly add the tetrahydrofuran solution of lithium borohydride (4.58 liters, 9.16 moles) into the kettle (complete the addition in 1.5 hours). Control the temperature not to exceed 35 degrees. The temperature of the jacket is controlled at 10 degrees. After the feeding is completed, the internal temperature is controlled between 25 degrees and 35 degrees. The reaction is kept warm for 2 hours and stirred overnight for 16 hours. Slowly add saturated ammonium chloride (2.3 liters) solution to the reaction solution, control the internal temperature below 20 degrees, and stir for 0.5 hours. Add n-heptane (8 liters) to the reaction solution and stir for 0.5 hours. The solid-liquid mixture is filtered under reduced pressure. The filter cake is washed twice with n-heptane/tetrahydrofuran (1:2), 1.5 liters each time. The filtrate is decompressed. Concentrate to about 8 liters and add 16 liters of ethyl acetate. The organic phase was washed three times with water, 3 liters each time, and the aqueous phase was extracted with ethyl acetate three times, 2 liters each time. The organic phases were combined, dried over anhydrous sodium sulfate (2 kg), and concentrated under reduced pressure. The crude product was beaten with n-heptane/ethyl acetate (10:1, 6.4 liters), stirred for 0.5 hours, filtered, and the filter cake was washed once with n-heptane/ethyl acetate (10:1, 3.2 liters). After vacuum drying for 16 hours, compound I-6 was obtained. ¹ HNMR (400MHz, DMSO-d ₆ ) δ 7.76 (t, J =7.8 Hz, 1H), 7.59 (d, J =7.8 Hz, 1H), 7.25 (d, J =7.8 Hz, 1H), 4.84 - 4.62 (m, 2H), 4.09 (dd, J =6.2, 12.4 Hz, 1H), 3.88 - 3.71 (m, 2H), 3.50 (ddd, J =4.8, 8.8, 11.8 Hz, 1H), 1.82 - 1.62 (m , 1H), 1.29 (dd, J =4.0, 8.8 Hz, 1H), 0.92 (dd, J =4.0, 6.2 Hz, 1H). Step 6: Compound 1-7

At room temperature, add tetrahydrofuran (22.5 liters) into a 50-liter reaction kettle, add compound I-6 (1.5 kg, 7.02 mol), and stir until dissolved and clear. Add azodimethyldipiperidine (2.04 kg, 8.07 mol) and stir until dissolved and clear. Under nitrogen atmosphere, use a peristaltic pump to slowly add n-butylphosphine solution in tetrahydrofuran (1.63 kg, 8.07 mol). In the cauldron (completed in 1.5 hours), control the temperature not to exceed 40 degrees. After the feeding is completed, control the internal temperature between 15 and 25 degrees, react for 2 hours, and stir at room temperature overnight for 16 hours. The reaction liquid was filtered, the filter cake was washed twice with methyl tertiary butyl ether, 3 liters each time, and the filtrate was concentrated to obtain a crude product. Dissolve the crude product with methyl tertiary butyl ether (15 liters), wash with hydrochloric acid (4 mol/L) three times, 3 liters each time, and extract the aqueous phase with methyl tertiary butyl ether three times, 3 liters each time. The combined organic phases were washed three times with saturated brine, 5 liters each time, dried over anhydrous sodium sulfate (2 kg), and concentrated under reduced pressure to obtain a crude product. Transfer the crude product to the kettle, add n-heptane (7 liters), raise the temperature to 70 degrees, and stir for 0.5 hours until dissolved. Cool down to internal temperature 15 degrees and stir for 16 hours. Filter, wash the filter cake once with n-heptane (1.4 liters), and dry the filter cake under vacuum for 16 hours to obtain compound I-7. ¹ HNMR (400MHz, CDCl ₃ ) δ 7.46 (t, J =7.8 Hz, 1H), 7.04 (dd, J =0.8, 7.8 Hz, 1H), 6.87 (dd, J =0.8, 7.7 Hz, 1H), 4.10 - 4.04 (m, 2H), 3.86 - 3.78 (m, 2H), 2.07 (ddd, J =2.8, 5.2, 8.0 Hz, 1H), 1.32 (dd, J =4.4, 8.2 Hz, 1H), 1.07 (t , J =4.8 Hz, 1H). Step 7: Compound 1-8

At room temperature, add dihexane (12 liters) into a 50-liter reaction kettle, and add compound I-7-1 (2.11 kg, 17.89 moles) and tertiary potassium butoxide (2.51 kg, 22.36 moles). The reaction solution was heated to 95 degrees, and a solution of compound I-7 (1.75 kg, 8.94 mol) in dihexane (5.5 liters) was added into the reaction solution using a constant pressure dropping funnel, and the internal temperature was controlled to 90-100 degrees. Complete the addition in 30 minutes and keep warm for 30 minutes. After the reaction is completed, cool down to 50 degrees, filter with diatomaceous earth, wash the filter cake twice with n-heptane, 2 liters each time, combine the filtrate and spin dry. The obtained yellow oil was dissolved in n-heptane (20 liters) and washed with water three times, 15 liters each time. The spot plate showed that no compound I-7-1 remained. The organic phase was dried over anhydrous sodium sulfate (2.5 kg) and concentrated to obtain crude product compound I-8. ¹ HNMR (400 MHz, CDCl ₃ ) δ 7.45 (t, J = 7.8 Hz, 1H), 6.66 - 6.48 (m, 2H), 4.45 - 4.29 (m, 2H), 4.21 - 4.07 (m, 2H), 3.95 - 3.81 (m, 2H), 3.75 - 3.64 (m, 2H), 2.13 - 2.02 (m, 1H), 1.42 - 1.34 (m, 1H), 1.25 - 1.17 (m, 9H), 1.10 - 1.02 (m, 1H). Step 8: Compound 4a

At room temperature, add methylene chloride (13 liters) into a 50-liter reaction kettle, add trifluoromethylisonicotinic acid (2.6 kg, 13.6 mol) and N,N-dimethylformamide (105 ml, 1.36 mol), stir until clear. Use a constant pressure dropping funnel to slowly add oxalyl chloride (1.67 liters, 19.05 moles), and absorb the exhaust gas with alkali solution. Stir under nitrogen atmosphere at 20 degrees for 16 hours. After the reaction is completed, the solvent in the reaction solution is rotary evaporated, and the obtained trifluoromethylisonicotinyl chloride is dissolved in N,N-dimethylformamide (12 liters). The solution is slowly added using a constant pressure dropping funnel. To a solution of 3-bromo-4-methylaniline (3.0 kg, 13.97 mol) and diisopropylethylamine (4.11 L, 20.44 mol) in N,N-dimethylformamide (6 L) was added Medium, control the internal temperature between 15 and 25 degrees. After the addition is completed, the reaction is continued for 0.5 hours. After the reaction is completed, add the reaction solution to water (54 liters) to precipitate the solid. Control the temperature at 20 to 30 degrees. After the addition, continue stirring for 10 minutes. Use a suction funnel to filter the suspension under reduced pressure, and wash the filter cake with water. (2 L) at a time and collect the filter cake. Transfer the filter cake to a reaction kettle filled with n-heptane (15 liters). Keep the internal temperature at 15 to 25 degrees and stir for 16 hours. The reaction solution was filtered, the filter cake was washed twice with n-heptane (1 liter), and the filter cake was vacuum dried for 16 hours to obtain compound 4a. ¹ HNMR (400 MHz, CDCl ₃ ) δ 8.93 (d, J = 4.8 Hz, 1H), 8.11 (s, 1H), 8.01 (br s, 1H), 7.96 - 7.86 (m, 2H), 7.49 (br d , J = 8.2 Hz, 1H), 7.26 (d, J = 8.2 Hz, 1H), 2.41 (s, 3H). Step 9: Compound 1-9

At room temperature, add tertiary butyl methyl ether (18 liters) into a 50-liter reaction kettle, add compound I-8 (1.8 kg, 6.49 moles) and zonacol borate (1.73 kg, 6.81 moles), and stir. To clarify, the system was evacuated and replaced with nitrogen three times. Add tmfen (30.67 g, 0.13 mole) and [Ir(COD)(OMe)]2 (40.02 g, 65 mmol) into the reaction kettle respectively. The system is heated to 55-60 degrees under nitrogen flow protection and kept stirred. 2 hours. After the reaction is completed, the reaction solution is naturally cooled and stirred for 16 hours. The reaction solution was concentrated under reduced pressure to obtain crude product compound I-9. Step 10: Compound I-10

At room temperature, add dihexane (16 liters) into a 50-liter reaction kettle, add compound 4a (2.2 kg, 6.12 mol), stir until clear, and add Pd(dppf)Cl ₂ ·CH ₂ Cl ₂ (263.22 g , 0.32 mol) was added to the reaction kettle and replaced with nitrogen gas 3 times. Dissolve sodium carbonate (1.37 kg, 12.89 mol) in water (5.2 liters) until it becomes clear, then add it to the reaction kettle and raise the temperature to 70-75 degrees. Under _N2 protection, add solution compound I-9 (2.6 kg, 6.45 mol) into the reaction kettle using a peristaltic pump (100 ml/min), control the internal temperature to 70-75 degrees, and stir for about 2 hours. After the reaction is completed, the reaction solution is cooled to room temperature, and the reaction solution is filtered under reduced pressure through a pad of diatomaceous earth. The filter cake is washed twice with 2.5 liters of ethyl acetate each time. After the filtrate was concentrated under reduced pressure, ethyl acetate (25 liters) and saturated brine (20 liters) were added to it, and filtered under reduced pressure with diatomaceous earth. After the filtrate was separated, the organic phase was treated with anhydrous sodium sulfate (2.5 kg). dry. Transfer the organic phase to the reaction kettle, add modified silica gel (3.6 kg), heat to 60 degrees and stir for 16 hours. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. Add tertiary butyl methyl ether (4.5 liters) to the obtained yellow viscous substance. After it is dissolved at 50 degrees, transfer it to a 50-liter reaction kettle while it is hot, add n-heptane (13.5 liters), and raise the temperature to 60 degrees. Turn off the heating, let the beating liquid naturally cool down to 20 degrees, and stir for 16 hours. Filter under reduced pressure, wash the filter cake with (tertiary butyl methyl ether/n-heptane = 1/3, 2.5 liters), and vacuum dry the filter cake for 16 hours to obtain a crude product. Add ethanol/n-heptane (1/10, 12.5 liters) to a 50-liter reaction kettle, add the crude product, raise the temperature to 65 degrees, and keep stirring for 1 hour. Turn off the heating, let the beat naturally cool down to 20 degrees, and stir for 16 hours. Filter under reduced pressure, wash the filter cake with ethanol/n-heptane (1:10, 2 liters), and dry the filter cake under vacuum for 16 hours to obtain compound I-10. ¹ HNMR (400 MHz, CDCl ₃ ) δ 8.90 (d, J = 4.8 Hz, 1H), 8.24 (br s, 1H), 8.15 (s, 1H), 7.95 (br d, J = 3.8 Hz, 1H), 7.63 (br d, J = 8.2 Hz, 1H), 7.44 (s, 1H), 7.32 - 7.27 (m, 1H), 6.53 (s, 1H), 6.47 (s, 1H), 4.41 (t, J = 5.2 Hz, 2H), 4.20 - 4.07 (m, 2H), 3.94 - 3.84 (m, 2H), 3.74 (t, J = 5.2 Hz, 2H), 2.25 (s, 3H), 2.12 (ddd, J = 2.6, 5.0, 7.8 Hz, 1H), 1.41 (dd, J = 4.2, 8.0 Hz, 1H), 1.24 (s, 9H), 1.08 (t, J = 4.4 Hz, 1H). Step 11: Compound I-11

At room temperature, add formic acid (12 liters) into a 50-liter reaction kettle, add compound I-10 (2.4 kg, 4.32 mol), raise the internal temperature to 60 to 70 degrees, and stir for 4 to 5 hours. After the reaction is completed, stop heating, cool the reaction solution to room temperature, and concentrate the reaction solution under reduced pressure (45 degrees). Add ethyl acetate (20 liters) and water (15 liters) to the crude product, add sodium bicarbonate solid to adjust the pH of the solution to 4~5, separate the liquids, wash the organic phase once with saturated brine (10 liters), and then use anhydrous sulfuric acid After sodium drying, spin dry. Transfer to an oven and vacuum dry for 16 hours to obtain compound I-11. ¹ HNMR (400 MHz, CDCl ₃ ) δ 8.87 (d, J = 4.8 Hz, 1H), 8.41 (s, 1H), 8.17 - 8.06 (m, 2H), 7.93 (br d, J = 4.4 Hz, 1H) , 7.62 - 7.52 (m, 1H), 7.46 (s, 1H), 7.32 - 7.21 (m, 1H), 6.56 (s, 1H), 6.50 (s, 1H), 4.64 - 4.45 (m, 4H), 4.22 - 4.03 (m, 2H), 3.94 - 3.83 (m, 2H), 2.25 (s, 3H), 2.12 (ddd, J = 2.6, 5.0, 7.8 Hz, 1H), 1.40 (dd, J = 4.2, 7.8 Hz , 1H), 1.09 (t, J = 4.4 Hz, 1H). Step 12: Compound (I) Form B

At room temperature, add ethylene glycol dimethyl ether (10.5 liters) into a 50-liter reaction kettle. Add compound I-11 (2.1 kg, 3.98 mol) and stir until the solution is clear. Cool the temperature to 5~10 degrees, slowly add a solution of sodium hydroxide (238.85 g, 5.97 mol) in pure water (2.1 liters), and stir for 0.5 hours. After the reaction is completed, add ethyl acetate (20 liters) and pure water (10 liters) to the reaction solution, separate the layers, wash the organic phase once with water (10 liters), and dry over anhydrous sodium sulfate. The organic phase was rotary evaporated, and the solid obtained was transferred to an oven and dried under vacuum for 16 hours to obtain a crude compound of formula (I). ¹ HNMR (400 MHz, CDCl ₃ ) δ 8.93 (d, J = 4.8 Hz, 1H), 8.18 (s, 1H), 8.14 (s, 1H), 7.96 (br d, J = 4.8 Hz, 1H), 7.60 (br d, J = 8.2 Hz, 1H), 7.52 (s, 1H), 7.32 (d, J = 8.2 Hz, 1H), 6.63 (s, 1H), 6.57 (s, 1H), 4.54 - 4.46 (m , 2H), 4.20 - 4.11 (m, 2H), 4.02 - 3.96 (m, 2H), 3.96 - 3.86 (m, 2H), 2.29 (s, 3H), 2.16 (ddd, J = 2.6, 5.0, 7.8 Hz , 1H), 1.41 (dd, J = 4.2, 7.9 Hz, 1H), 1.14 (t, J = 4.4 Hz, 1H).

At room temperature, add ethanol (9 liters) and pure water (9 liters) into a 50-liter reaction kettle, add the crude compound (I) above (1.880 kg, 3.76 mol), and raise the internal temperature to 70-75 degrees. After stirring for 2 h, the solution became clear. Turn off the heating, cool the solution to 20 degrees naturally, and stir for 16 hours. Filter under reduced pressure, wash the filter cake once with ethanol/water (1:1, 2 liters), and dry the filter cake under vacuum for 32 hours to obtain the B crystal form of compound (I). ¹ HNMR (400 MHz, DMSO-d ₆ ) δ 10.70 (s, 1H), 9.00 (d, J = 4.8 Hz, 1H), 8.37 (s, 1H), 8.20 (d, J = 4.8 Hz, 1H), 7.75 (dd, J = 2.2, 8.3 Hz, 1H), 7.70 - 7.59 (m, 1H), 7.35 (d, J = 8.4 Hz, 1H), 6.73 (s, 1H), 6.57 (s, 1H), 4.83 (t, J = 5.6 Hz, 1H), 4.30 (t, J = 5.2 Hz, 2H), 4.16 - 4.00 (m, 2H), 3.87 - 3.67 (m, 4H), 2.24 (s, 3H), 2.18 ( ddd, J = 2.6, 5.0, 7.8 Hz, 1H), 1.39 (dd, J = 3.8, 7.8 Hz, 1H), 0.98 (t, J = 4.2 Hz, 1H). Its XRPD spectrum is basically as shown in Figure 2.

Example 3: Preparation of crystal form A of the compound of formula (I)

At room temperature, the crystal form B of the compound of formula (I) is placed in aluminum foil and exposed to air with a humidity greater than 30%. Leave it for 24 hours until it reaches constant weight, and obtain the crystal form A of the compound of formula (I). Its XRPD spectrum is basically as shown in Figure 1.

Experimental Example 4: KF Moisture Determination

Add methanol that can immerse the platinum electrode into the reaction cup, titrate to the end point with Karl Fischer reagent while stirring, add about 10 mg of pure water, accurate to 0.1 mg, stir for 60 seconds; titrate with Karl Fischer reagent to the end point, get Titer of Karl Fischer reagent; moisture calibration was performed a total of 3 times, and the relative standard deviation was calculated. Quickly add about 0.1 g of the weighed crystal form A sample of the compound of formula (I) to the above methanol solution. The extraction time is 60 seconds and the stirring time is 60 seconds. Add Karl Fischer reagent and titrate to the end point to obtain the sample's Moisture results were measured twice.

Conclusion: The average water content of crystal form A was measured to be 3.45%, which is approximately 1 molecule of water per 1 molecule of compound of formula (I).

Example 5: Preparation of single crystal of compound of formula (I)

Experimental method: Dissolve 30 mg of the compound of formula (I) in 2 mL of acetone, and put the dissolved solution into a centrifuge tube (capacity: 10 mL). Slowly add 2 mL of petroleum ether to this solution. The two solvents will clearly separate up and down. Seal the mouth of the tube with a sealing film and open a few small evaporation holes. Let it stand at room temperature for 10 days. When the solvent evaporates and most of the crystals precipitate, collect the crystals for single crystal X-ray diffraction (SC-XRD) detection.

Experimental results: Table 3 Crystal structure data and measurement parameters of compounds of formula (I) compound Compound of formula (I) monohydrate Molecular formula C ₂₆ H ₂₆ F ₃ N ₃ O ₅ molecular weight 517.50 temperature 173(2)K Wavelength 1.54178Å crystal system, space group Monoclinal, P2(1) Chip package size a= 11.5998(2) Å alpha= 90 deg. b= 6.87130(10) Å beta= 90.6560(10) deg. c= 14.7592(3) Å gamma= 90 deg. Volume 1176.32(4)Å ³ Z, calculated density 2,1.461 Mg/m ³ absorption coefficient 1.001mm ^-1 F(000) 540 Crystal size 0.220 x 0.180 x 0.160mm Data collection angle range 2.994 to 68.176 deg. restriction index -13＜=h＜=13,-8＜=k＜=8,-17＜=l＜=16 Reflection Collection/Unique 13356/4211 [R(int)=0.0247] Angular completeness=67.68 98.9% Treatment method F2 full matrix least squares method data/limits/parameters 4211/1/349 Goodness of fit on F2 1.042 Final R index [I>2sigma(I)] R1= 0.0287, wR2= 0.0797 R index (all data) R1= 0.0290, wR2= 0.0801 absolute structural parameters 0.01(4) extinction index n/a Maximum diffraction peaks and holes 0.220 and -0.165e. Å ^-3 Table 4 Atomic coordinates (× 10 ⁴ ) and equivalent isotropic shift parameters (Å ² × 10 ³ ) of the crystal of the compound of formula (I) x y z U(eq) F(1) 1188(2) 3800(3) 863(1) 50(1) C(1) 6717(2) 9262(4) 6371(2) 33(1) N(1) 5507(1) 4005(3) 7502(1) 24(1) O(1) 7369(1) 7769(2) 5929(1) 28(1) F(3) 2022(1) 1971(4) 1840(1) 63(1) C(3) 5031(2) 7582(4) 5561(2) 37(1) C(26) 1044(2) 2218(4) 1370(1) 32(1) N(3) -980(2) 2653(3) 1540(1) 26(1) O(3) 6277(1) 3709(3) 10324(1) 35(1) F(2) 962(2) 724(3) 800(1) 50(1) C(2) 5504(2) 8477(3) 6413(2) 32(1) N(2) 190(1) 2583(3) 4890(1) 20(1) O(2) 5482(1) 1820(2) 8697(1) 32(1) O(4) -1772(1) 2398(3) 4852(1) 29(1) C(4) 5581(2) 6298(3) 6268(1) 23(1) C(9) 3764(2) 2303(3) 7902(1) 23(1) C(8) 3154(2) 3191(3) 7205(1) 20(1) C(7) 3763(2) 4485(3) 6644(1) 21(1) C(6) 4907(2) 4873(3) 6814(1) 21(1) C(5) 6849(2) 5903(3) 6119(2) 27(1) O(5) 4173(2) 2143(3) 10656(2) 63(1) C(11) 6683(2) 2213(4) 8873(1) 30(1) C(12) 6867(2) 2154(4) 9884(1) 31(1) C(13) 1902(2) 2834(3) 7012(1) 19(1) C(14) 1564(2) 2780(3) 6100(1) 20(1) C(15) 417(2) 2556(3) 5835(1) 19(1) C(16) -420(2) 2356(3) 6498(1) 22(1) C(17) -78(2) 2392(3) 7404(1) 23(1) C(18) 1064(2) 2633(3) 7690(1) 21(1) C(19) 1318(2) 2727(3) 8699(1) 26(1) C(20) -849(2) 2524(3) 4461(1) 20(1) C(21) 19(2) 2401(3) 1979(1) 24(1) C(22) 148(2) 2347(3) 2914(1) 22(1) C(23) -828(2) 2608(3) 3440(1) 20(1) C(24) -1868(2) 2933(3) 2989(1) 24(1) C(25) -1906(2) 2909(3) 2054(1) 26(1) C(10) 4933(2) 2759(3) 8008(1) 23(1) Table 5 Bond length (Å) and bond angle [deg] of compound crystals of formula (I) F(1)-C(26) 1.333(3) C(1)-O(1) 1.436(3) C(1)-C(2) 1.510(3) N(1)-C(10) 1.320(3) N(1)-C(6) 1.362(3) O(1)-C(5) 1.445(3) F(3)-C(26) 1.334(3) C(3)-C(2) 1.497(4) C(3)-C(4) 1.502(3) C(26)-F(2) 1.330(3) C(26)-C(21) 1.504(3) N(3)-C(21) 1.333(2) N(3)-C(25) 1.333(3) O(3)-C(12) 1.430(3) C(2)-C(4) 1.515(3) N(2)-C(20) 1.356(2) N(2)-C(15) 1.416(2) O(2)-C(10) 1.357(2) O(2)-C(11) 1.440(2) O(4)-C(20) 1.224(2) C(4)-C(6) 1.495(3) C(4)-C(5) 1.515(3) C(9)-C(8) 1.384(3) C(9)-C(10) 1.399(3) C(8)-C(7) 1.410(3) C(8)-C(13) 1.497(2) C(7)-C(6) 1.373(3) C(11)-C(12) 1.505(3) C(13)-C(14) 1.399(2) C(13)-C(18) 1.410(3) C(14)-C(15) 1.390(2) C(15)-C(16) 1.393(3) C(16)-C(17) 1.391(3) C(17)-C(18) 1.396(3) C(18)-C(19) 1.515(2) C(20)-C(23) 1.509(2) C(21)-C(22) 1.386(3) C(22)-C(23) 1.391(3) C(23)-C(24) 1.389(3) C(24)-C(25) 1.381(3) O(1)-C(1)-C(2) 105.03(18) C(10)-N(1)-C(6) 116.65(16) C(1)-O(1)-C(5) 108.85(15) C(2)-C(3)-C(4) 60.66(14) F(2)-C(26)-F(1) 106.40(16) F(2)-C(26)-F(3) 106.6(2) F(1)-C(26)-F(3) 106.5(2) F(2)-C(26)-C(21) 113.04(19) F(1)-C(26)-C(21) 111.9(2) F(3)-C(26)-C(21) 111.97(16) C(21)-N(3)-C(25) 116.27(16) C(3)-C(2)-C(1) 116.4(2) C(3)-C(2)-C(4) 59.83(16) C(1)-C(2)-C(4) 106.92(19) C(20)-N(2)-C(15) 127.87(16) C(10)-O(2)-C(11) 119.38(16) C(6)-C(4)-C(3) 122.60(17) C(6)-C(4)-C(2) 122.62(18) C(3)-C(4)-C(2) 59.51(17) C(6)-C(4)-C(5) 118.41(18) C(3)-C(4)-C(5) 114.17(18) C(2)-C(4)-C(5) 104.87(18) C(8)-C(9)-C(10) 118.09(18) C(9)-C(8)-C(7) 117.32(17) C(9)-C(8)-C(13) 123.76(18) C(7)-C(8)-C(13) 118.92(16) C(6)-C(7)-C(8) 120.31(17) N(1)-C(6)-C(7) 122.38(18) N(1)-C(6)-C(4) 115.04(17) C(7)-C(6)-C(4) 122.57(17) O(1)-C(5)-C(4) 106.11(17) O(2)-C(11)-C(12) 107.48(17) O(3)-C(12)-C(11) 111.49(18) C(14)-C(13)-C(18) 119.49(16) C(14)-C(13)-C(8) 116.64(16) C(18)-C(13)-C(8) 123.82(16) C(15)-C(14)-C(13) 122.09(17) C(14)-C(15)-C(16) 119.06(16) C(14)-C(15)-N(2) 116.25(16) C(16)-C(15)-N(2) 124.68(15) C(17)-C(16)-C(15) 118.67(16) C(16)-C(17)-C(18) 123.52(17) C(17)-C(18)-C(13) 117.18(16) C(17)-C(18)-C(19) 118.37(16) C(13)-C(18)-C(19) 124.43(16) O(4)-C(20)-N(2) 124.03(17) O(4)-C(20)-C(23) 119.82(15) N(2)-C(20)-C(23) 116.14(15) N(3)-C(21)-C(22) 124.80(18) N(3)-C(21)-C(26) 114.15(16) C(22)-C(21)-C(26) 121.04(17) C(21)-C(22)-C(23) 118.23(16) C(24)-C(23)-C(22) 117.45(17) C(24)-C(23)-C(20) 117.41(16) C(22)-C(23)-C(20) 125.13(16) C(25)-C(24)-C(23) 119.66(18) N(3)-C(25)-C(24) 123.54(17) N(1)-C(10)-O(2) 119.79(17) N(1)-C(10)-C(9) 125.21(17) O(2)-C(10)-C(9) 115.00(17) Table 6 Twist angle [deg] of the crystal of the compound of formula (I) C(2)-C(1)-O(1)-C(5) -28.9(2) C(4)-C(3)-C(2)-C(1) 95.0(2) O(1)-C(1)-C(2)-C(3) -46.0(3) O(1)-C(1)-C(2)-C(4) 18.1(2) C(2)-C(3)-C(4)-C(6) 111.5(2) C(2)-C(3)-C(4)-C(5) -93.6(2) C(3)-C(2)-C(4)-C(6) -111.4(2) C(1)-C(2)-C(4)-C(6) 137.4(2) C(1)-C(2)-C(4)-C(3) -111.2(2) C(3)-C(2)-C(4)-C(5) 109.59(19) C(1)-C(2)-C(4)-C(5) -1.6(2) C(10)-C(9)-C(8)-C(7) 0.5(3) C(10)-C(9)-C(8)-C(13) 179.80(18) C(9)-C(8)-C(7)-C(6) -2.0(3) C(13)-C(8)-C(7)-C(6) 178.68(18) C(10)-N(1)-C(6)-C(7) -0.1(3) C(10)-N(1)-C(6)-C(4) 179.02(18) C(8)-C(7)-C(6)-N(1) 1.9(3) C(8)-C(7)-C(6)-C(4) -177.22(18) C(3)-C(4)-C(6)-N(1) -172.0(2) C(2)-C(4)-C(6)-N(1) -99.8(2) C(5)-C(4)-C(6)-N(1) 34.0(3) C(3)-C(4)-C(6)-C(7) 7.2(3) C(2)-C(4)-C(6)-C(7) 79.4(3) C(5)-C(4)-C(6)-C(7) -146.79(19) C(1)-O(1)-C(5)-C(4) 28.2(2) C(6)-C(4)-C(5)-O(1) -156.53(16) C(3)-C(4)-C(5)-O(1) 47.4(2) C(2)-C(4)-C(5)-O(1) -15.5(2) C(10)-O(2)-C(11)-C(12) 143.6(2) O(2)-C(11)-C(12)-O(3) -66.4(2) C(9)-C(8)-C(13)-C(14) -142.6(2) C(7)-C(8)-C(13)-C(14) 36.7(3) C(9)-C(8)-C(13)-C(18) 39.9(3) C(7)-C(8)-C(13)-C(18) -140.8(2) C(18)-C(13)-C(14)-C(15) 0.8(3) C(8)-C(13)-C(14)-C(15) -176.75(18) C(13)-C(14)-C(15)-C(16) -0.8(3) C(13)-C(14)-C(15)-N(2) 178.16(18) C(20)-N(2)-C(15)-C(14) -175.32(19) C(20)-N(2)-C(15)-C(16) 3.6(3) C(14)-C(15)-C(16)-C(17) 0.2(3) N(2)-C(15)-C(16)-C(17) -178.7(2) C(15)-C(16)-C(17)-C(18) 0.5(3) C(16)-C(17)-C(18)-C(13) -0.4(3) C(16)-C(17)-C(18)-C(19) 177.6(2) C(14)-C(13)-C(18)-C(17) -0.2(3) C(8)-C(13)-C(18)-C(17) 177.18(19) C(14)-C(13)-C(18)-C(19) -178.11(19) C(8)-C(13)-C(18)-C(19) -0.7(3) C(15)-N(2)-C(20)-O(4) -1.4(4) C(15)-N(2)-C(20)-C(23) 179.01(18) C(25)-N(3)-C(21)-C(22) 1.4(3) C(25)-N(3)-C(21)-C(26) -177.1(2) F(2)-C(26)-C(21)-N(3) -59.7(3) F(1)-C(26)-C(21)-N(3) 60.4(3) F(3)-C(26)-C(21)-N(3) 179.9(2) F(2)-C(26)-C(21)-C(22) 121.6(2) F(1)-C(26)-C(21)-C(22) -118.2(2) F(3)-C(26)-C(21)-C(22) 1.2(3) N(3)-C(21)-C(22)-C(23) -1.2(3) C(26)-C(21)-C(22)-C(23) 177.3(2) C(21)-C(22)-C(23)-C(24) -0.8(3) C(21)-C(22)-C(23)-C(20) 177.78(18) O(4)-C(20)-C(23)-C(24) 12.7(3) N(2)-C(20)-C(23)-C(24) -167.74(19) O(4)-C(20)-C(23)-C(22) -165.9(2) N(2)-C(20)-C(23)-C(22) 13.7(3) C(22)-C(23)-C(24)-C(25) 2.5(3) C(20)-C(23)-C(24)-C(25) -176.22(18) C(21)-N(3)-C(25)-C(24) 0.4(3) C(23)-C(24)-C(25)-N(3) -2.4(3) C(6)-N(1)-C(10)-O(2) 179.06(18) C(6)-N(1)-C(10)-C(9) -1.5(3) C(11)-O(2)-C(10)-N(1) 0.6(3) C(11)-O(2)-C(10)-C(9) -178.89(19) C(8)-C(9)-C(10)-N(1) 1.3(3) C(8)-C(9)-C(10)-O(2) -179.24(19)

Conclusion: The unit cell diagram of this single crystal is shown in Figure 3. The results show that this single crystal is a monohydrate of the compound of formula (I), and its structure is N -(3-(2-((1 R ,5 S )-3) -oxabicyclo[3.1.0]hex-1-yl)-6-(2-hydroxyethoxy)pyridin-4-yl)-4-methylphenyl)-2-(trifluoromethyl)iso Nicotinamide.

Example 6: XRPD of single crystal of compound of formula (I)

Experimental purpose: Use the single crystal preparation method in Example 5 to prepare a larger amount of crystals and measure their XRPD.

Experimental method: Dissolve 100 mg of the compound of formula (I) in 2 mL of acetone, and put the dissolved solution into a centrifuge tube (capacity: 10 mL). Slowly add 2 mL of petroleum ether to this solution. The two solvents will be separated into distinct upper and lower layers. Seal the mouth of the tube with a sealing film and open a few small evaporation holes. Let it stand at room temperature for 7 days until the solvent evaporates and crystals precipitate. Collect the crystals and crush them for XRPD testing.

The X-ray powder diffractometer (XRPD) method in this experiment: Instrument model: DX-2700BH Test conditions: Detailed XRPD parameters are as follows: X-ray generator: Cu, kα, (λ=1.54184Å) Tube voltage: 40 kV, tube current: 30 mA. Scattering slit: 1 mm Detector slit: 0.3 mm Anti-scatter slit: 1 mm Scanning range: 3-40 degrees Step size: 0.02 degrees Scan time: 0.5 seconds

Experimental results: The XRPD spectrum of the single crystal of the compound of formula (I) is shown in Figure 4, and the analytical data is shown in Table 7. Table 7 XRPD pattern analysis data of single crystal of compound of formula (I) No. 2θ angle [°] Interplanar spacing [Å] intensity[count] Relative strength [%] 1 6.00 14.73 8351 19.3 2 7.60 11.62 1291 3.0 3 9.58 9.23 1394 3.2 4 11.98 7.38 13541 31.3 5 14.04 6.30 2865 6.6 6 14.32 6.18 3550 8.2 7 14.76 6.00 900 2.1 8 15.22 5.82 1473 3.4 9 15.88 5.58 3809 8.8 10 16.28 5.44 3565 8.2 11 17.44 5.08 693 1.6 12 18.94 4.68 12116 28.0 13 19.66 4.51 43310 100.0 14 20.64 4.30 4056 9.4 15 22.02 4.03 5721 13.2 16 23.20 3.83 3528 8.1 17 24.04 3.70 6125 14.1 18 25.50 3.49 5116 11.8 19 26.64 3.34 2528 5.8 20 27.26 3.27 2054 4.7 twenty one 28.48 3.13 4655 10.7 twenty two 28.94 3.08 2890 6.7 twenty three 31.02 2.88 8384 19.4 twenty four 33.08 2.70 2267 5.2 25 33.62 2.66 1984 4.6 26 34.26 2.62 1302 3.0 27 39.60 2.27 4135 9.5

Conclusion: The single crystal form of the compound of formula (I) is consistent with the crystal form A of the compound of formula (I).

Example 7: In vitro enzyme activity test

Experiment purpose: The inhibitory effect of the compound on cRAF enzyme activity was detected.

Experimental materials: Table 8 Experimental materials Brand number cRAF protein Creative BioMart-RAF1-416H MEK1 protein Invitrogen-PR3984A ADP-Glo Kinase Assay Kit Promega-V9102 Tris-HCl, pH 7.4 Sigma-T2663-1L MgCl ₂ Sigma-63020-1L NaCl Sigma-S5150 DTT Invitrogen-P2325 Triton X-100 Sigma-X100 H ₂ O Gibco-15230-162 384 middle plate Greiner-781280 384 Experimental Board PerkinElmer-6007299

Experimental steps: (1) Compound preparation: Use DMSO to dilute the test compound and reference compound to 100 µM, and use Echo to perform a 3-fold gradient dilution of the compound to obtain a target plate of 11 concentration gradients. (2) Experimental flow: 1) Buffer preparation: 50 mM Tris-HCl (pH 7.4), 3.5 mM MgCl ₂ , 150 mM NaCl, 1 mM DTT, 0.02% Triton X-100, H ₂ O; 2) Use buffer Prepare a mixture of MEK1 and ATP, and add 5 µL of the substrate mixture to the 384 intermediate plate; 3) Dilute the cRAF enzyme with buffer and add 5 µL to the 384 intermediate plate; 4) Use Bravo to transfer 5 µL of the reaction mixture Pour the solution into the 384 experimental plate, centrifuge for 15 seconds and then place it in a 23°C incubator for incubation. 5) After 1 hour, add 5 µL ADP-Glo to the 384 test plate, shake, centrifuge for 15 seconds, and then place it in a 23-degree incubator for incubation. 6) After 40 minutes, add 10 µL of kinase detection reagent to the 384 experimental plate, shake, centrifuge for 15 seconds, and then place it in a 23-degree incubator for incubation. 7) After 1 hour, read the plate on Envision.

Experimental results: Table 9 provides the inhibitory activity of the compounds of the present invention on cRAF enzyme. Table 9 In vitro activities of compounds compound cRAF enzyme IC ₅₀ (nM) Antiproliferative activity of lung cancer Calu-6 cells IC ₅₀ (nM) Anti-proliferative activity of colon cancer HCT-116 cells IC ₅₀ (nM) ERK phosphorylation inhibitory activity IC ₅₀ (nM) in lung cancer Calu-6 cells ERK phosphorylation inhibitory activity IC ₅₀ (nM) in colon cancer HCT-116 cells Compounds of formula (I) 0.6 600 1100 490 180

Note: In the in vitro activity test of the above compounds (Example 7 to Example 11), the compound (I) prepared in Example 1 was used.

Example 8: Calu-6 (Kras ^Q61K ) anti-proliferative activity experiment

Experimental materials: Table 10 Experimental reagents and consumables Name Brand number EMEM medium Vicente-320-005-CL fetal bovine serum Biosera-FB-1058/500 0.25% trypsin Yuanpei-S310KJ Double antibodies (penicillin, streptomycin) Procell-PB180120 CellTiterGlo Promega-G7573 cell plate Corning-3610 Table 11 Experimental instruments Name Brand number Cell counting plate Refinement Victor Nivo PerkinElmer

Experimental steps: Cell inoculation: (1) Cell culture medium: 89% EMEM, 10% fetal bovine serum and 1% penicillin-streptomycin; (2) Remove the original culture medium in the cell culture flask, digest the cells with trypsin and count them with Dilute the cell suspension with culture medium to the cell density required for plating: 3.75×10 ⁴ cells per ml; (3) Add 100 µL culture medium to each well around the cell plate, add 80 µL cell suspension to other wells, and place Cultivate overnight in a 37°C incubator containing 5% _CO2 . Add medicine: Use Echo to perform gradient dilution and add medicine on the compound, then put the cell plate back into the incubator for three days; Read the plate and analyze the data: Add CTG and read the plate: Add 20 μL to each well of the cell plate CellTiterGlo, protected from light, shaken for 10 minutes, and read the plate on a Victor Nivo.

Experimental results: Table 9 provides the anti-proliferative activity of the compounds of the present invention on Calu-6 cells.

Example 9: Anti-proliferative activity experiment of HCT-116 (Kras ^G13D )

Experimental materials: Table 12 Experimental reagents and consumables Name Brand number Mc'Coy 5A Medium BI-01-075-1ACS fetal bovine serum Biosera-FB-1058/500 0.25% trypsin Yuanpei-S310KJ Double antibodies (penicillin, streptomycin) Procell-PB180120 CellTiterGlo Promega-G7573 cell plate Corning-3610 Table 13 Experimental instruments Name Brand number Cell counting plate Refinement Victor Nivo PerkinElmer

Experimental steps: Cell inoculation: (1) Cell culture medium: 89% Mc'Coy 5A, 10% fetal calf serum and 1% penicillin-streptomycin; (2) Remove the original culture medium in the cell culture flask and digest the cells with trypsin Count and use culture medium to dilute the cell suspension to the cell density required for plating: 2.5 × 10 ⁴ cells per ml; (3) Add 100 µL culture medium to each well around the cell plate, and add 80 µL cell suspension to other wells. , placed in a 37°C incubator containing 5% _CO2 and cultured overnight. Adding drugs: Make gradient dilutions and adding drugs to the compounds, then put the cell plate back into the incubator for three days; Read the plate and analyze the data: Add CTG and read the plate: Add 20μL CellTiterGlo to each well of the cell plate, Incubate in the dark for 10 minutes and read the plate on a Victor Nivo.

Experimental results: Table 8 provides the anti-proliferative activity of the compounds of the present invention on HCT-116 cells.

Example 10: HCT116 (Kras ^G13D ) ERK phosphorylation inhibition test

Experimental materials: Table 14 Reagent consumables Reagents Brand number Highly sensitive detection kit for human ERK phosphorylated protein Cisbio-64AERPEH RPMI1640 medium Gibco-22400089 fetal bovine serum Hyclone-SV30087.03 96 HTRF Microplate Cisbio-66PL96025 96 microwell plate COSTAR-3599 DMSO Sigma-D2650-100mL 0.05% Trypsin-EDTA Gibco-25300-062 Table 15 Main instruments instrument Manufacturer Model biological safety cabinet AIRTECH BSC-1304IIA2 carbon dioxide incubator Thermo 311 cell counter BECKMAN Vi-cellXR microplate reader PerkinElmer Envision centrifuge Eppendorf Centrifuge 5810R Table 16 Cell information cell name Source Item number HCT116 ATCC ATCC-HTB-132

Experimental procedures and methods: 1) Resuscitate the cells and culture them to the logarithmic growth phase, digest them with trypsin, seed them in a 96-well plate, and incubate them in an incubator overnight. 2) Add a series of gradient compounds dissolved in DMSO into the 96-well plate, put it back into the incubator and incubate for 1 hour. 3) Take out the cell plate, remove the supernatant, add cell lysis solution (containing 1% blocking peptide) and incubate at room temperature for 30 minutes. 4) Transfer 16 μL of cell lysis solution to each well into the HTRF plate, and then add 4 μL of the prepared antibody mixture. 5) After incubating overnight, use Envision to read the plate. Obtain the fitted curve according to the ratio (ratio of Ex665/Ex615 fluorescence intensity) and fit it according to the four-parameter fitting formula of Graphpad Y=Bottom + (Top-Bottom)/(1+10 ^((LogEC50-X)*HillSlope)) calculates EC50.

Experimental results: Table 8 provides the inhibitory activity of the compounds of the present invention on HCT-116 ERK phosphorylation.

Example 11: Calu-6 (Kras ^Q61K ) ERK phosphorylation inhibition test

Experimental materials: Table 17 Reagent consumables Reagents Brand number Highly sensitive detection kit for human ERK phosphorylated protein Cisbio-64AERPEH RPMI1640 medium Gibco-22400089 fetal bovine serum Hyclone-SV30087.03 96 HTRF Microplate Cisbio-66PL96025 96 microwell plate COSTAR-3599 DMSO Sigma-D2650-100mL 0.05% Trypsin-EDTA Gibco-25300-062 Table 18 Main instruments instrument Manufacturer Model biological safety cabinet AIRTECH BSC-1304IIA2 carbon dioxide incubator Thermo 311 cell counter BECKMAN Vi-cellXR microplate reader PerkinElmer Envision centrifuge Eppendorf Centrifuge 5810R Table 19 Cell information cell name Source Item number Calu6 ATCC ATCC-HTB-56

Experimental steps and methods: 1) Resuscitate the cells and culture them to the logarithmic growth phase, digest them with trypsin, seed them in a 96-well plate, and incubate them in an incubator overnight. 2) Add a series of gradient compounds dissolved in DMSO into the 96-well plate, put it back into the incubator and incubate for 1 hour. 3) Take out the cell plate, add cell lysis solution (containing 1% blocking peptide) and incubate at room temperature for 30 minutes. 4) Transfer 16 μL of cell lysis solution to each well into the HTRF plate, and then add 4 μL of the prepared antibody mixture. 5) After incubating overnight, use Envision to read the plate. Obtain the fitted curve according to the ratio (ratio of Ex665/Ex615 fluorescence intensity) and fit it according to the four-parameter fitting formula of Graphpad Y=Bottom + (Top-Bottom)/(1+10 ^((LogEC50-X)*HillSlope)) calculates EC ₅₀ .

Experimental results: Table 9 provides the inhibitory activity of the compounds of the present invention on Calu-6 ERK phosphorylation.

Conclusion: The compound of formula (I) has strong inhibitory activity on cRAF enzyme, and also has good anti-proliferative activity and ERK phosphorylation inhibitory activity on Calu-6 cells and HCT-116 cells.

Example 12: Pharmacokinetic study of single intravenous and oral administration in mice

The purpose of this experiment was to study the pharmacokinetics (PK) of the test compound (prepared in Example 1) in mice after a single intravenous and single oral administration.

Sample collection and preparation: After intravenous injection or oral administration, animal blood samples were collected and the actual blood collection time was recorded. After blood samples are collected, they are immediately transferred to labeled centrifuge tubes containing K2-EDTA, and then centrifuged to collect plasma. Transfer plasma to pre-chilled centrifuge tubes, flash freeze in dry ice, and store in -70 ± 10°C ultra-low temperature freezer until LC-MS/MS analysis.

Pharmacokinetic data analysis: Use pharmacokinetic software to process the plasma drug concentration data of the compounds using a non-compartmental model. The peak concentration (C _max ), peak time (T _max ) and quantifiable end time are directly obtained from the plasma concentration-time diagram. The following pharmacokinetic parameters were calculated using the log-linear trapezoidal method: half-life (T _1/2 ), apparent volume of distribution (V _dss ) and clearance (Cl), area under the time-plasma concentration curve from 0 to the end time point ( AUC _0-last ), initial concentration (C ₀ ).

Experimental results: Table 20 Pharmacokinetic parameters of the compound of the present invention after single intravenous and oral administration in mice Mouse PK parameters Compounds of formula (I) PK intravenous injection Dosage (mg/kg/day) 2 Clearance rate (ml/kg/min) 25.8 Apparent volume of distribution (L/kg) 2.75 Exposure AUC (nanomol·hour) 2614 Half-life T _1/2 (hours) 1.43 oral Dosage (mg/kg/day) 100 Maximum plasma concentration (namolar) 25000 Peak time (hours) 1 Exposure AUC _0-last (namolar hours) 177513 Oral bioavailability (%) 131

Experimental conclusion: The compound of formula (I) is better absorbed orally in mice, has a lower clearance rate, a longer half-life, and good bioavailability.

Example 13: Stability test of crystalline form A of compound of formula (I) under 6-month accelerated conditions

Experiment purpose: The stability of the crystal form A of the compound of formula (I) under packaging accelerated conditions was investigated.

Experimental instrument: Constant temperature and humidity chamber, model: BSC-400, conditions: 40℃/75%RH.

Experimental method: Take an appropriate amount of compound A crystal form of formula (I) and put it into a double-layer low-density polyethylene bag. Each layer of low-density polyethylene bag is buckled and sealed separately, then put into an aluminum foil bag and heat-sealed, and placed in an accelerated (40 ℃/75%RH) conditions, take out and detect relevant substances, enantiomers, moisture and crystal forms after January, February, March and June. Table 21 Accelerated test (40℃/75%RH) research results of crystal form A of compound of formula (I) Test items acceptance criteria Inspection time initial value 1 month 2 months 3 months 6 months related substances Total impurities should be ≤ 2.0% 0.57% 0.57% 0.56% 0.55% 0.55% Enantiomers ≤ 0.25% Not detected / / Not detected Not detected Moisture 3.0% ~ 4.0% 3.55% 3.54% 3.62% 3.73% 3.65% Crystal form Should be consistent with the 0-day chart Consistent with reference standard spectrum / / Consistent with the 0-day chart Consistent with the 0-day chart

Experimental conclusion: The crystal form of compound A of formula (I) was placed under accelerated test conditions (40°C/75%RH) for 6 months, and there was no obvious change in all inspection items, and all tests were in compliance with regulations. Therefore, the proposed packaging and storage conditions ensure that the sample remains stable during the proposed shelf life.

Example 14: Long-term test (25°C/60%RH) stability of crystal form A of compound of formula (I)

Experiment purpose: The stability of the crystal form A of the compound of formula (I) under packaging accelerated conditions was investigated.

Experimental instrument: Constant temperature and humidity chamber, model: BSC-400, conditions: 25℃/60%RH.

Experimental method: Take an appropriate amount of compound A crystal form of formula (I) and put it into a double-layer low-density polyethylene bag. Each layer of low-density polyethylene bag is buckled and sealed separately, then put into an aluminum foil bag and heat-sealed, and placed for a long time (25 ℃/60%RH) conditions, take out the relevant substances, enantiomers, moisture and crystal forms after 3 months and 6 months. Table 22 Research results of long-term test (25℃/60%RH) of crystal form A of compound of formula (I) Test items acceptance criteria Inspection time initial value 3 months 6 months related substances Total impurities should be ≤ 2.0% 0.93% 0.86% 0.91% Enantiomers ≤ 0.25% Not detected Not detected Not detected Moisture 3.0% ~ 4.0% 3.45% 3.76% 3.67% Crystal form Should be consistent with the 0-day chart Consistent with reference standard spectrum Consistent with the spectrum on day 0 Consistent with the spectrum on day 0

Experimental conclusion: The crystal form of compound A of formula (I) was placed under long-term test conditions (25°C/60%RH) for 6 months, and there was no significant change in all inspection items, and all tests were in compliance with regulations. Therefore, the proposed packaging and storage conditions ensure that the sample remains stable during the proposed shelf life.

Example 15: Preparation of amorphous compound of formula (I)

At room temperature, place the compound of formula (I) (46 g) in a 1 L round-bottomed flask, add methylene chloride (300 mL), and after fully dissolving, vacuum rotary evaporate to dryness to obtain the amorphous compound of (I). Its XRPD spectrum is shown in Figure 9.

Figure 1 is the XRPD spectrum of crystal form A of the compound of formula (I). Figure 2 is the XRPD spectrum of crystal form B of the compound of formula (I). Figure 3 is a unit cell diagram of a single crystal of the compound of formula (I). Figure 4 is the XRPD spectrum of a single crystal of the compound of formula (I). Figure 5 is the TGA spectrum of crystal form A of the compound of formula (I). Figure 6 is the TGA spectrum of crystal form B of the compound of formula (I). Figure 7 is the DSC spectrum of crystal form A of the compound of formula (I). Figure 8 is the DSC spectrum of crystal form B of the compound of formula (I). Figure 9 is the XRPD spectrum of the amorphous form of the compound of formula (I).

Claims (18)

A compound of formula (I) N- (3-(2-((1 R ,5 S )-3-oxabicyclo[3.1.0]hex-1-yl)-6-(2-hydroxyethoxy) Form A of pyridin-4-yl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide,

Its X-ray powder diffraction pattern has characteristic peaks at the following 2θ angles: 6.03±0.20°, 12.00±0.20°, 14.36±0.20°, and 19.72±0.20°. The X-ray powder diffraction pattern of the crystal form A described in claim 1 has characteristic peaks at the following 2θ angles: 6.03±0.20°, 9.63±0.20°, 12.00±0.20°, 14.36±0.20°, 19.02±0.20 °, 19.72±0.20°, 20.73±0.20°, 22.10±0.20°, 24.08±0.20°. The X-ray powder diffraction pattern of the crystal form A described in claim 2 has characteristic peaks at the following 2θ angles: 6.03±0.20°, 7.65±0.20°, 9.63±0.20°, 12.00±0.20°, 14.09±0.20 °, 14.36±0.20°, 14.82±0.20°, 15.28±0.20°, 15.95±0.20°, 16.31±0.20°, 17.50±0.20°, 19.02±0.20°, 19.72±0.20°, 20.73±0.20°, 22.10±0.20 °, 23.29±0.20°, 24.08±0.20°, 25.53±0.20°, 26.73±0.20°, 27.34±0.20°, 28.51±0.20°, 29.04±0.20°, 31.04±0.20°, 33.17±0.20°, 33.72±0.20 °, 34.28±0.20°, 39.63±0.20°. The X-ray powder diffraction pattern of the crystalline form A described in any one of claims 1 to 3 is shown in Figure 1. Crystal form A as described in any one of claims 1 to 3, which is a monohydrate of the compound of formula (I). The TGA spectrum of the crystalline form A described in any one of claims 1 to 3 is shown in Figure 5. The DSC spectrum of the crystalline form A described in any one of claims 1 to 3 is shown in Figure 7. A compound of formula (I) N- (3-(2-((1 R ,5 S )-3-oxabicyclo[3.1.0]hex-1-yl)-6-(2-hydroxyethoxy) Form B of pyridin-4-yl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide,

Its X-ray powder diffraction pattern has characteristic peaks at the following 2θ angles: 6.06±0.20°, 12.06±0.20°, 20.10±0.20°, and 21.24±0.20°. The X-ray powder diffraction pattern of Form B as described in claim 8 has characteristic peaks at the following 2θ angles: 6.06±0.20°, 12.06±0.20°, 15.94±0.20°, 18.95±0.20°, 20.10±0.20 °, 20.74±0.20°, 21.24±0.20°, 22.20±0.20°, 24.20±0.20°. The X-ray powder diffraction pattern of the crystal form B described in claim 9 has characteristic peaks at the following 2θ angles: 6.06±0.20°, 7.77±0.20°, 9.61±0.20°, 12.06±0.20°, 14.15±0.20 °, 14.93±0.20°, 15.48±0.20°, 15.94±0.20°, 16.94±0.20°, 17.62±0.20°, 18.95±0.20°, 20.10±0.20°, 20.74±0.20°, 21.24±0.20°, 22.20±0.20 °, 23.27±0.20°, 24.20±0.20°, 25.65±0.20°, 26.83±0.20°, 27.42±0.20°, 28.17±0.20°, 28.90±0.20°, 30.96±0.20°, 31.54±0.20°, 37.07±0.20 °, 37.91±0.20°. The X-ray powder diffraction pattern of the crystal form B described in any one of claims 8 to 10 is shown in Figure 2. The TGA spectrum of the crystal form B described in any one of claims 8 to 10 is shown in Figure 6. The DSC spectrum of the crystalline form B described in any one of claims 8 to 10 is shown in Figure 8. A compound of formula (I) N- (3-(2-((1 R ,5 S )-3-oxabicyclo[3.1.0]hex-1-yl)-6-(2-hydroxyethoxy) Amorphous substance of pyridin-4-yl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide,

The amorphous substance as claimed in claim 14, wherein its X-ray powder diffraction pattern is as shown in Figure 9. A pharmaceutical composition comprising the crystal form/amorphous substance described in any one of claims 1 to 15 and a pharmaceutically acceptable carrier. The use of a crystalline form/amorphous substance as described in any one of claims 1 to 15 or a pharmaceutical composition as described in claim 16 in the preparation of a drug for treating RAF kinase mutation-mediated diseases. The application as described in claim 17, wherein the disease is lung cancer.