TW202304892A - 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 an RAF kinase inhibitor, a preparation method thereof, and their application in treating and related diseases.

This application claims the following priority: CN202110625608.3, the application date is June 4, 2021; CN202110625603.0, the application date is June 04, 2021; CN202210583709.3, the application date is May 25, 2022.

The mitogen-activated protein kinase (MAPK) pathway is an important signal transduction pathway in the cell, which transmits the signal from the extracellular to the nucleus through the specific cascade phosphorylation of RAS/RAF/MEK/ERK, and finally leads to specific Activation of genes leading to 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 signal transduction. The RAF family includes three subtypes of ARAF, BRAF, and CRAF (RAF-1), which have a high degree of homology and similar structural domains. ARAF and CRAF mutations occur less frequently, and BRAF mutations have a higher rate. RAF inhibitors can effectively inhibit ERK phosphorylation, thereby blocking the downstream signal MEK/ERK transmission, thereby effectively blocking the RAS-ERK pathway, and have a therapeutic effect on tumor diseases of 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, they relapsed 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 applications. pan-RAF inhibitors can inhibit dimer activity and block paradoxcal activation of pathways, thereby reducing drug resistance. The pan-Raf dimer inhibitors under clinical research mainly include HM95573, TAK-580, BGB-283, LXH254, and LY3009120, etc. The development of these new RAF inhibitors is expected to overcome the drug resistance of the first-generation inhibitors, Further expand clinical applications.

The inventor disclosed a small molecule pan-RAF kinase inhibitor in the patent PCT/CN/2020/133933, the structure of which is shown in formula (I), and the 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. The small molecule inhibitor has good RAF kinase inhibitory activity and various cell anti-proliferation activities, and at the same time, the molecule has good pharmacokinetic properties, and is expected to be developed into a clinical drug.

， 其X射線粉末繞射圖譜在下列2θ角處具有特徵峰：6.03±0.20°、12.00±0.20°、14.36±0.20°、19.72±0.20°。 The present invention provides the compound N- (3-(2-((1 R ,5 S )-3-oxabicyclo[3.1.0]hex-1-yl)-6-(2-hydroxyethoxy Form A of base) 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 solutions of the present invention, the X-ray powder diffraction pattern of the above 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 solutions of the present invention, the X-ray powder diffraction pattern of the above 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 solutions of the present invention, the X-ray powder diffraction pattern of the above 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 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 solutions of the present invention, the above crystal form A has an X-ray powder diffraction pattern as shown in FIG. 1 .

In some schemes 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 the compound of formula (I) Form A serial number 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 solutions of the present invention, the above 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 crystal form A is a monohydrate of the compound of formula (I).

In some solutions of the present invention, the TGA spectrum of the above crystal form A is shown in FIG. 5 .

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

， 其X射線粉末繞射圖譜在下列2θ角處具有特徵峰：6.06±0.20°、12.06±0.20°、20.10±0.20°、21.24±0.20°。 The present invention also provides the compound 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 solutions of the present invention, the X-ray powder diffraction pattern of the above 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 solutions of the present invention, the X-ray powder diffraction pattern of the above 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 solutions of the present invention, the X-ray powder diffraction pattern of the above crystal form B is shown in FIG. 2 .

In some solutions of the present invention, the XRPD spectrum diffraction peak analysis data of the crystal form B of the compound of formula (I) is shown in Table 2. Table 2 XRPD diffraction peak analysis data of the compound of formula (I) Form B serial number 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 solutions of the present invention, the TGA spectrum of the above crystal form B is shown in FIG. 6 .

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

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

.

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

The present invention also provides a pharmaceutical composition, which comprises the above crystal form A and/or crystal form B and/or crystals, and/or amorphous substances and a pharmaceutically acceptable carrier.

The present invention also provides the application of the above crystal form A and/or crystal form B and/or crystalline and/or amorphous or pharmaceutical composition thereof in the preparation of medicines for treating RAF kinase mutation-mediated diseases.

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

The present invention also provides the application of the above crystalline form A and/or crystalline form B and/or crystalline and/or amorphous form in the preparation of a drug for treating lung cancer.

technical effect

The crystal form of the present application is stable, less affected by heat, humidity and light, and is convenient for storage and preparation. The crystal form of the present application has good pharmacokinetic properties, meets the requirements of the drug in vivo, and is suitable for use as a pharmaceutical preparation, wherein the pharmacokinetic properties can be tested 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 specific phrase or term should not be considered indeterminate or unclear if it is not specifically defined, but should be understood according to 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, within the scope of sound medical judgment, suitable for contact with human and animal tissues without undue toxicity, irritation, allergic reaction or other problematic complications, and reasonable benefit/risk than commensurate with those compounds, materials, compositions and/or dosage forms.

The term "pharmaceutically acceptable carrier" refers to any preparation or carrier medium capable of delivering 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 bases include suspending agents, viscosity builders, skin penetration enhancers and the like. Their formulations are well known to those skilled in the field of cosmetics or topical medicine.

The structures 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 the present invention pertains. Exactly the same analytical properties. Accordingly, it will be apparent to those skilled in the art to which the present invention pertains that various modifications and variations can be made in the present invention without departing from the scope and spirit of the present invention. For example, since the diffraction angle (2θ) in powder X-ray diffraction usually has an error within the range of ±0.20°, the value of the above-mentioned diffraction angle needs to be understood as including values within the range of approximately ±0.20°. Therefore, the present invention includes not only crystals with completely uniform diffraction angles in powder X-ray diffraction, but also crystals with uniform diffraction angles within an error range of ±0.20°.

As used herein, "polymorph" or "polymorph" refers to crystal 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 speed and similar properties. Depending on their temperature-stability relationship, the two polymorphs can be monotropic or tautotropic. For a single denaturation system, the relative stability between the two solid phases remains unchanged when the temperature is changed. In contrast, in tautotropic systems, there is a transition temperature where the stability of the two phases is reversed. This phenomenon in which compounds exist in different crystal structures is called pharmaceutical polymorphism.

The crystalline structures of the present invention can be prepared by various methods, including crystallization or recrystallization from a suitable solvent, sublimation, growth from a melt, solid state transformation from another phase, crystallization from a supercritical fluid, and jet spraying, etc. Techniques for crystallization or recrystallization of crystalline structures from solvent mixtures, including solvent evaporation, lowering the temperature of solvent mixtures, seeding of supersaturated solvent mixtures of the molecule and/or salt, lyophilization of solvent mixtures, addition of antisolvents to solvent mixtures, etc. .

The intermediate compound of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art of the present invention, including the specific embodiments listed below, the embodiments formed by its combination with other chemical synthesis methods, and the present invention There are equivalents known to those skilled in the art, and preferred embodiments include, but are not limited to, the examples 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 skilled in the art to which the present invention pertains to modify or select synthetic steps or reaction schemes on the basis of existing implementations.

The term "protecting group" includes, but is not limited to, "amino protecting group", "hydroxyl protecting group" or "mercapto protecting group". The term "amino protecting group" refers to a protecting group suitable for preventing side reactions at the amino nitrogen position. Representative amine protecting groups include, but are not limited to: formyl; acyl, such as alkanoyl (such as acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl, such as tris butoxycarbonyl (Boc); arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn), trityl ( Tr), 1,1-bis-(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 the hydroxy group. Representative hydroxy protecting groups include, but are not limited to: alkyl, such as methyl, ethyl, and tert-butyl; acyl, such as alkanoyl (such as acetyl); arylmethyl, such as benzyl (Bn ), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) And tertiary butyldimethylsilyl (TBS) and so on.

The structures of the compounds of the present invention can be confirmed by conventional methods known to those skilled in the art to which the present invention belongs. 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), the cultivated single crystal is collected with a Bruker D8 venture diffractometer to collect diffraction intensity data, the light source is CuKα radiation, and the scanning method is φ/ɷ scanning. After collecting relevant data, further The absolute configuration can be confirmed by solving the crystal structure by the direct method (Shelxs97).

The present invention will be specifically described by examples below, and these examples do not imply 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, tmphen represents 3,4,7,8-tetramethyl-1,10 phenanthroline, Pd ₂ dba ₃ represents tris-benzophenone- Dipalladium, Brettphos stands for (2-dicyclohexylphosphine-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl, PE stands for 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 supplier catalog name.

1. Instruments and analytical methods

1.1 X-ray powder diffraction (X-ray powder diffractometer, XRPD) method of crystal form A and crystal form B of the present invention Instrument model: PANalytical (PANalytical)-X'pert ³ Test conditions: Detailed XRPD parameters are as follows: 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 degrees Step size: 0.0263 degrees Scan time: 46.665 seconds

X-ray powder diffraction (X-ray powder diffractometer, XRPD) method of the amorphous substance of the present invention Instrument model: DX-2700BH Test conditions: The 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 will be described in detail through examples below, but it does not imply any unfavorable limitation to the present invention. The compound of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art of the present invention, including the specific embodiments listed below, the embodiments formed by its combination with other chemical synthesis methods, and the technology of the present invention There are equivalents known to those of ordinary skill in the art, and preferred embodiments include, but are not limited to, the examples of the present invention. Various changes and modifications to the specific embodiments of the present invention will be apparent to those skilled in the art to which the present invention pertains without departing from the spirit and scope of the present invention.

化合物2 Embodiment 1: the preparation of formula (I) compound

Compound 2

At -78°C, n-butyl lithium (23 mL, 2.5 M) was added to a solution of compound 1 (10 g, 51.96 mmol) in dichloromethane (100 mL), and stirred for 30 minutes. At -78°C, 1a (4.92 g, 57.16 mmol) was added to the mixture, and the mixture was heated to 25°C and stirred for 0.5 hour. Saturated ammonium chloride solution (40 mL) was added to the reaction mixture, 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

Add p-toluenesulfonic acid (11.5 g, 60.46 mmol) to a solution of 2 (6 g, 30.06 mmol) in toluene (80 mL), heat up to 110°C and stir for 16 hours. Add saturated sodium bicarbonate solution (40 mL) and ethyl acetate (100 mL) to the reaction mixture, separate the layers, wash the camera with saturated sodium bicarbonate solution (50 mL×2), dry over anhydrous sodium sulfate, concentrate, and 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 tert-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 ) 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 micromol) and tmphen (50 mg, 211.59 micromol) were placed in methyl tertiary butyl ether (20 mL). The reaction mixture was added with 4 (770 mg, 3.94 mmol) under nitrogen protection, heated to 80°C and stirred for 3 hours. The reaction solution was filtered, and the filtrate was concentrated to obtain a 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 micromol) and sodium carbonate (800 mg, 7.55 moles) in dioxane (50 mL) and water (10 mL). The reaction mixture was heated to 100° C. and stirred for 3 hours under nitrogen protection. The reaction mixture was filtered, the filtrate was concentrated, ethyl acetate (20 mL) and water (10 mL) were added, the layers were separated, the aqueous phase was extracted with ethyl acetate (10 mL×3), and the combined organic phase was dried over anhydrous sodium sulfate After concentration, it was separated by silica gel column chromatography (PE/EA=10/1 to 5/1) to obtain 6. 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 micromol), Brettphos (200 mg , 372.60 micromol) 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 micromole) in tetrahydrofuran (10 mL) was added hydrochloric acid (1 mL, 4M), and the reaction solution was stirred at 25°C for 1 hour. The reaction solution was diluted with ethyl acetate (20 mL), adjusted to pH=8 with saturated sodium bicarbonate solution, extracted with ethyl acetate (10×3 mL), the organic phase was dried over anhydrous sodium sulfate, concentrated, and thin-layer silica gel Plate (PE/EA= 1/1, V/V) separation gave 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] ⁺ . Compound of formula (I)

Compound 8 was chiralally 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 the compound 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%).

Compound 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%).

步驟1：化合物I-2 Embodiment 2: Preparation of formula (I) compound crystal form B

Step 1: Compound I-2

At room temperature, N,N-dimethylformamide (12.5 liters) was added to a 50-liter reaction kettle, compound I-1 (2.75 kg, 20.91 mol) and compound I-1-1 (3.55 kg , 31.36 moles), stirred until dissolved and clarified. Potassium carbonate (7.22 kg, 52.27 moles) was added, heated to 75-80 degrees and stirred for 16 hours. After the reaction is completed, cool the reaction solution to 25 degrees; add water (27 liters) to the reaction kettle, and transfer about 27 liters of the reaction solution into a 30-liter white barrel for temporary storage; add the remaining 27 liters of reaction solution to the kettle Add hydrochloric acid solution (6 mol/L, 8.5 L) slowly, 0.3 L each time, and adjust the pH to 3~4. A yellow solid was precipitated, accompanied by a large amount of gas generation and a small amount of exotherm. 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°C 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, dimethyl sulfide (12 liters) was added to a 50 liter reactor, compound I-2 (3.6 kg, 16.03 mol) was added, and the inner temperature rose to 70°C. After the reaction solution was clarified, a solution of sodium chloride (1.87 kg, 32.05 mol) in water (6 liters) was added, the inner temperature was raised to 80-85 degrees, and stirred for 16 hours. After the reaction was completed, the temperature was lowered to 25 degrees, water (20.5 liters) was added to the kettle, stirred for 5 minutes, and the reaction solution was extracted twice with ethyl acetate, 10 liters each time. The organic phases were combined and washed twice with saturated brine, 10 L each time. After the organic phase was dried with anhydrous sodium sulfate, it was concentrated under reduced pressure at 45°C 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, tetrahydrofuran (7.5 liters) was added to a 50-liter reaction kettle, compound I-3 (1.5 kg, 9.83 mol) and compound I-3-1 (0.926 kg, 10 mol) were added, and the inner temperature dropped to -25 degree. Under a nitrogen atmosphere, pump lithium hexamethylsilylamide (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 between -8~-5 degrees, and keep it warm for 3 hours. Lower the temperature to -25 degrees, add sodium hexamethylsilylamide (10 liters, 10 moles, tetrahydrofuran solution) into the reaction kettle (2 hours) with a peristaltic pump, 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 was completed, water (0.883 L, 49.1 mol) was slowly added to the reaction solution, and the inner temperature was controlled to be lower than 30° C. and stirred for 0.5 hours. The reaction solution was distilled under reduced pressure at 40°C in a kettle, and concentrated to obtain a crude compound I-4 solution (about 10 liters), which was directly used in the next reaction. Step 4: Compound I-5

At room temperature, add an aqueous solution of potassium hydroxide (9.6 liters, 19.2 moles) and ethanol (10 liters) to a 50 liter reaction kettle of crude compound I-4 (10 liters of solution), and the internal temperature rises to 75-80 degrees , stirred for 5 hours. After the reaction is completed, cool down to 25-30°C, slowly add aqueous hydrochloric acid solution (8 liters, 48 moles) to the reaction solution, control the temperature not to exceed 40°C, heat the inner temperature to 60°C, and stir for 1 hour. After the reaction was complete, it was concentrated to obtain a crude product solution (residue about 2 liters), which was extracted twice with ethyl acetate, 7.5 liters each. Add the extract to the reaction kettle, add silica gel (100-200 mesh, 750 g), add n-heptane (15 liters), and stir for 16 hours. After filtration, the filter cake was washed three times with n-heptane/ethyl acetate (1:1), 1 liter each time, and the organic phases were combined and concentrated under reduced pressure to obtain a crude black solid. Heat the crude product with ethanol (3 liters) to an internal temperature of 70°C, stir until it dissolves, and cool down to 20°C. 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 twice with water, 1 liter each time; add the filter cake to the mixed solution (n-heptane/ethanol, 4:1, 4.5 liters), stir for 0.5 hour, filter, filter the filter cake with n-heptane/ethanol After washing with ethanol (4:1) twice, 0.3 L each time, the filter cake was vacuum-dried at 45°C 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, tetrahydrofuran (16 L) was added into a 50 L reaction kettle, compound I-5 (3.2 kg, 15.27 mol) was added, and stirred until dissolved and clarified. The internal temperature was raised to 30°C. Under a nitrogen atmosphere, a tetrahydrofuran solution of lithium borohydride (4.58 liters, 9.16 moles) was slowly added to the kettle with a peristaltic pump (1.5 hours to complete), and the temperature was controlled not to exceed 35°C. 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 for 2 hours and stirred overnight for 16 hours. Slowly add saturated ammonium chloride (2.3 L) solution to the reaction solution, control the internal temperature below 20°C, and stir for 0.5 hours. Add n-heptane (8 liters) to the reaction solution, stir for 0.5 hours, filter the solid-liquid mixture under reduced pressure, wash the filter cake twice with n-heptane/tetrahydrofuran (1:2), 1.5 liters each time, and depressurize the filtrate Concentrate to about 8 liters, and add 16 liters of ethyl acetate. The organic phase was washed 3 times with 3 liters of water, and the aqueous phase was extracted 3 times with 2 liters of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate (2 kg), and concentrated under reduced pressure. The crude product was slurried 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 I-7

At room temperature, tetrahydrofuran (22.5 L) was added into a 50 L reaction kettle, compound I-6 (1.5 kg, 7.02 mol) was added, and stirred until dissolved and clarified. Add azodicarbonyldipiperidine (2.04 kg, 8.07 mol), stir until dissolved and clear, under nitrogen atmosphere, slowly add n-butylphosphine tetrahydrofuran solution (1.63 kg, 8.07 mol) to Kettle (1.5 hours added), control the temperature does not exceed 40 degrees. After feeding, control the internal temperature between 15 and 25 degrees, react for 2 hours, and stir overnight at room temperature for 16 hours. The reaction solution was filtered, and the filter cake was washed twice with 3 liters of methyl tertiary butyl ether, and the filtrate was concentrated to obtain a crude product. The crude product was dissolved with methyl tertiary butyl ether (15 liters), washed 3 times with hydrochloric acid (4 mol/liter), 3 liters each time, and the aqueous phase was extracted 3 times with methyl tertiary butyl ether, 3 liters each time, The combined organic phases were washed 3 times with saturated brine, 5 L each time, dried over anhydrous sodium sulfate (2 kg), and concentrated under reduced pressure to obtain a crude product. Transfer the crude product to a kettle, add n-heptane (7 liters), raise the temperature to 70°C, and stir for 0.5 hours until it dissolves. Cool down to an internal temperature of 15°C and stir for 16 hours. After filtration, the filter cake was washed once with n-heptane (1.4 L), and the filter cake was vacuum-dried 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 I-8

At room temperature, dioxane (12 liters) was added to a 50 liter reaction kettle, compound I-7-1 (2.11 kg, 17.89 mol) and potassium tertiary butoxide (2.51 kg, 22.36 mol) were added. The reaction solution was heated to 95°C, and a solution of compound I-7 (1.75 kg, 8.94 mol) in dioxane (5.5 liters) was added to the reaction solution using a constant pressure dropping funnel, and the internal temperature was controlled at 90-100°C. The addition was completed in 30 minutes, and the reaction was incubated for 30 minutes. After the reaction was completed, the temperature was lowered to 50°C, filtered with diatomaceous earth, the filter cake was washed twice with n-heptane, 2 liters each time, and the filtrates were combined and spin-dried. The obtained yellow oil was dissolved in n-heptane (20 liters), washed 3 times with 15 liters of water each time, and no compound I-7-1 remained by spotting. The organic phase was dried over anhydrous sodium sulfate (2.5 kg) and then concentrated to obtain a crude 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, dichloromethane (13 L) was added to a 50 L reaction kettle, trifluoromethylisonicotinic acid (2.6 kg, 13.6 mol) and N,N-dimethylformamide (105 ml, 1.36 mol), stir until clear. Slowly add oxalyl chloride (1.67 liters, 19.05 moles) with a constant pressure dropping funnel, and absorb the tail gas with lye. Stir for 16 hours under nitrogen atmosphere at 20°C. After the reaction was completed, the solvent in the reaction solution was removed by rotary evaporation, and the obtained trifluoromethylisonicotinyl chloride was dissolved in N,N-dimethylformamide (12 liters), and the solution was slowly dissolved in a constant-pressure dropping funnel. Add to a solution of 3-bromo-4-methylaniline (3.0 kg, 13.97 mol) and diisopropylethylamine (4.11 liter, 20.44 mol) in N,N-dimethylformamide (6 liters) , control the internal temperature between 15 and 25 degrees. After the addition was complete, the reaction was continued for 0.5 hours. After the reaction is completed, add the reaction solution to water (54 liters) to precipitate solids, control the temperature at 20-30 degrees, continue stirring for 10 minutes after the addition, filter the suspension under reduced pressure with a suction filter funnel, and wash the filter cake with water (2 liters) at a time, collecting the filter cake. Transfer the filter cake to a reaction kettle filled with n-heptane (15 liters), and stir for 16 hours at an internal temperature of 15 to 25 degrees. The reaction solution was filtered, the filter cake was washed twice with n-heptane (1 L), 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 I-9

At room temperature, tertiary butyl methyl ether (18 liters) was added to a 50-liter reaction kettle, compound I-8 (1.8 kg, 6.49 mol) and inanol borate (1.73 kg, 6.81 mol) were added, and stirred To clarify, the system was evacuated and replaced with nitrogen three times. Add tmphen (30.67 grams, 0.13 moles) and [Ir(COD)(OMe)]2 (40.02 grams, 65 millimoles) into the reaction kettle in turn, and raise the temperature to 55-60 degrees under the protection of nitrogen flow in the system, and keep stirring 2 hours. After the reaction was completed, the reaction solution was naturally cooled and stirred for 16 hours. The reaction solution was concentrated under reduced pressure to obtain the crude compound I-9. Step 10: Compound I-10

At room temperature, dioxane (16 liters) was added to a 50-liter reactor, compound 4a (2.2 kg, 6.12 moles) was added, stirred until clear, and Pd(dppf)Cl ₂ ·CH ₂ Cl ₂ (263.22 g , 0.32 mol) into the reaction kettle, nitrogen replacement 3 times. Sodium carbonate (1.37 kg, 12.89 moles) was dissolved in water (5.2 liters) until clarified and added to the reaction kettle, and the temperature was raised to 70-75 degrees. Under the protection of N ₂ , the solution compound I-9 (2.6 kg, 6.45 mol) was added into the reaction kettle with a peristaltic pump (100 ml/min), and the inner temperature was controlled at 70-75 degrees, and stirred for about 2 hours. After the reaction was completed, the reaction liquid was cooled to room temperature, and the reaction liquid was vacuum-filtered with diatomaceous earth, and the filter cake was washed twice with ethyl acetate, 2.5 liters each time. After the filtrate was concentrated under reduced pressure, ethyl acetate (25 liters) and saturated saline (20 liters) were added thereto, and a pad of diatomaceous earth was used for suction filtration under reduced pressure. After the filtrate was separated, the organic phase was washed with anhydrous sodium sulfate (2.5 kg). dry. The organic phase was transferred to a reaction kettle, and modified silica gel (3.6 kg) was added thereto, heated to 60° C. and stirred 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 yellow viscous substance, dissolve it at 50 degrees, transfer it to a 50 liters reaction kettle while it is hot, add n-heptane (13.5 liters), and raise the temperature to 60 degrees. Turn off the heating, cool the beating liquid to 20 degrees naturally, and stir for 16 hours. Suction filtration under reduced pressure, the filter cake was washed with (tertiary butyl methyl ether/n-heptane = 1/3, 2.5 liters), and the filter cake was vacuum-dried for 16 hours to obtain a crude product. Add ethanol/n-heptane (1/10, 12.5 liters) into a 50-liter reactor, add the crude product therein, heat up to 65 degrees, and keep stirring for 1 hour. Turn off the heating, let the beating cool down to 20 degrees naturally, and stir for 16 hours. Suction filtration under reduced pressure, the filter cake was washed with ethanol/n-heptane (1:10, 2 liters), and the filter cake was vacuum-dried 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 moles), raise the internal temperature to 60-70 degrees, and stir for 4-5 hours. After the reaction was completed, the heating was stopped, the reaction solution was cooled to room temperature, and the reaction solution was concentrated under reduced pressure (45°C). Add ethyl acetate (20 liters) and water (15 liters) to the crude product, add solid sodium bicarbonate to adjust the pH of the solution to 4~5, separate the layers, wash the organic phase once with saturated brine (10 liters), and wash with anhydrous sulfuric acid Sodium dried and spin-dried. Transfer to an oven for vacuum drying 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

Ethylene glycol dimethyl ether (10.5 L) was added to the 50 L reaction kettle at room temperature. Compound 1-11 (2.1 kg, 3.98 mol) was added and stirred until the solution was clear. Cool down to 5-10°C, slowly add a solution of sodium hydroxide (238.85 g, 5.97 mol) in pure water (2.1 L), and stir for 0.5 hour. After the reaction was completed, ethyl acetate (20 liters) and pure water (10 liters) were added to the reaction solution, the layers were separated, the organic phase was washed once with water (10 liters), and dried over anhydrous sodium sulfate. The organic phase was rotary evaporated, and the obtained solid was transferred to an oven and dried in 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 product of the above (I) compound (1.880 kg, 3.76 moles), and raise the internal temperature to 70-75 degrees. After stirring for 2 h, the solution was clear. Turn off the heating, cool the solution down to 20 degrees naturally, and stir for 16 hours. Suction filtration under reduced pressure, the filter cake was washed once with ethanol/water (1:1, 2 liters), and the filter cake was vacuum-dried for 32 hours to obtain Form B 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 shown in FIG. 2 .

Embodiment 3: Preparation of Formula (I) compound crystal form A

At room temperature, place the crystal form B of the compound of formula (I) in aluminum foil and expose it to air with a humidity greater than 30%. After standing for 24 hours until constant weight, the crystal form A of the compound of formula (I) was obtained, and its XRPD spectrum is basically shown in FIG. 1 .

Experimental example 4: KF moisture determination

Add methanol that can submerge the platinum electrode into the reaction cup, titrate to the end point with Karl Fischer reagent under stirring, add about 10 mg of pure water, accurate to 0.1 mg, and stir for 60 seconds; titrate to the end point with Karl Fischer reagent to obtain The titer of the Karl Fischer reagent; the water content was calibrated three times, and the relative standard deviation was calculated. Quickly add about 0.1 g of the weighed sample of compound crystal form A of formula (I) into 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 For moisture results, repeat the measurement twice.

Conclusion: The measured average water content of the crystal form A is 3.45%, which means that every 1 compound of formula (I) contains 1 molecule of water.

Embodiment 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 2mL of petroleum ether to this solution, the two solvents are significantly layered up and down, seal the nozzle with a parafilm, and open a small evaporation hole. After standing at room temperature for 10 days, most of the crystals precipitated after the solvent evaporated, and the crystals were collected for single crystal X-ray diffraction (SC-XRD) detection.

Experimental results: Table 3 Crystal structure data and determination parameters of the compound 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 1.54178 Å crystal system, space group Monocline, P2(1) Packet 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/ ^m3 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. limit 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% Approach 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 compound crystals 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 crystal 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 The torsion angle [deg] of the compound crystal 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 the single crystal is shown in Figure 3, and the results show that the single crystal is a monohydrate of the compound of formula (I) with the structure of 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 Niacinamide.

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

The purpose of the experiment: to prepare a relatively large amount of crystals by using the single crystal preparation method in Example 5, 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 are significantly layered up and down, seal the tube opening with a parafilm, and open a small evaporation hole. Stand at room temperature for 7 days, after the solvent volatilizes, crystals are precipitated, and the crystals are collected and crushed to test XRPD.

The method of X-ray powder diffraction (X-ray powder diffractometer, XRPD) in this experiment: Instrument model: DX-2700BH Test conditions: The 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: See Figure 4 for the XRPD spectrum of the single crystal of the compound of formula (I), and see Table 7 for the analytical data. Table 7 XRPD analysis data of single crystal of compound of formula (I) serial number 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).

Embodiment 7: in vitro enzyme activity test

Purpose: The inhibitory effect of compounds on cRAF enzyme activity was tested.

Experimental materials: Table 8 Experimental Materials Brand No. 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 board Greiner-781280 384 experimental board PerkinElmer-6007299

Experimental steps: (1) Compound preparation: Dilute the test compound and reference compound to 100 µM with DMSO, and perform a 3-fold serial dilution of the compound with Echo to obtain target plates with 11 concentration gradients. (2) Experimental procedure: 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 the mixture of MEK1 and ATP with the solution, add 5 µL of substrate mixture to the 384 intermediate plate; 3) Dilute the cRAF enzyme with buffer, add 5 µL to the 384 intermediate plate; 4) Transfer 5 µL of the reaction mixture with Bravo The solution was transferred to the 384 experimental plate, centrifuged for 15 seconds, and then incubated in a 23-degree incubator. 5) After 1 hour, add 5 µL of ADP-Glo to the 384 experimental 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 activity of compounds compound cRAFase IC ₅₀ (nM) Antiproliferative activity IC ₅₀ (nM) of lung cancer Calu-6 cells Anti-proliferation activity IC ₅₀ (nM) of colon cancer HCT-116 cells IC ₅₀ (nM) of ERK phosphorylation inhibitory activity in lung cancer Calu-6 cells IC ₅₀ (nM) of Inhibitory Activity of ERK Phosphorylation in Colon Cancer HCT-116 Cells Compound of formula (I) 0.6 600 1100 490 180

Explanation: In the in vitro activity tests of the above compounds (Example 7 to Example 11), all compounds (I) prepared in Example 1 were used.

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

Experimental materials: Table 10 Experimental reagent consumables name Brand No. EMEM medium Vicente-320-005-CL fetal bovine serum Biosera-FB-1058/500 0.25% trypsin Yuanpei-S310KJ Double Antibody (Penicillin, Streptomycin) Procell-PB180120 Cell Titer Glo Promega-G7573 cell plate Corning-3610 Table 11 Experimental Instruments name Brand No. 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 medium in the cell culture flask, digest the cells with trypsin and count them with Medium Dilute the cell suspension to the required cell density of 3.75× ¹⁰⁴ cells per milliliter; (3) Add 100 µL of medium to each well around the cell plate, add 80 µL of cell suspension to other wells, and put Incubate overnight in a 37°C incubator with 5% CO ₂ . Dosing: Use Echo to serially dilute and add drugs to the compound, then return the cell plate to the incubator for three days; read the plate and analyze data: add CTG and read the plate: add 20 μL to each well of the cell plate CellTiterGlo was shaken for 10 minutes in the dark, and the plate was read on Victor Nivo.

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

Example 9: HCT-116 (Kras ^G13D ) anti-proliferation activity experiment

Experimental materials: Table 12 Experimental reagent consumables name Brand No. Mc'Coy 5A Medium BI-01-075-1ACS fetal bovine serum Biosera-FB-1058/500 0.25% trypsin Yuanpei-S310KJ Double Antibody (Penicillin, Streptomycin) Procell-PB180120 Cell Titer Glo Promega-G7573 cell plate Corning-3610 Table 13 Experimental Instruments name Brand No. Cell counting plate Refinement Victor Nivo PerkinElmer

Experimental steps: Cell inoculation: (1) Cell culture medium: 89% Mc'Coy 5A, 10% fetal bovine serum and 1% penicillin-streptomycin; (2) Remove the original medium in the cell culture flask and digest the cells with trypsin For counting, dilute the cell suspension with medium to the required cell density of 2.5× ¹⁰⁴ cells per milliliter; (3) Add 100 µL of medium to each well around the cell plate, and add 80 µL of cell suspension to other wells , and cultured overnight in a 37°C incubator containing 5% CO ₂ . Dosing: serially dilute and add the compound, then return the cell plate to the incubator for three days; read the plate, analyze the data: add CTG and read the plate: add 20 μL CellTiterGlo to each well of the cell plate, Shake for 10 minutes in the dark, and read the plate on Victor Nivo.

Experimental results: Table 8 provides the antiproliferative 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 Reagent Brand No. Human ERK Phosphorylated Protein High Sensitive Detection Kit 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 No. HCT116 ATCC ATCC-HTB-132

Experimental steps and methods: 1) Resuscitate the cells and culture them to the logarithmic growth phase, digest with trypsin, plant in a 96-well plate, and incubate overnight in an incubator. 2) Add a series of gradient compounds dissolved in DMSO to the 96-well plate, and return to the incubator for 1 hour incubation. 3) Take out the cell plate, remove the supernatant, add cell lysate (containing 1% blocking peptide) and incubate at room temperature for 30 minutes. 4) Transfer 16 μL of cell lysate per well to the HTRF plate, and then add 4 μL of the prepared antibody mixture. 5) After overnight incubation, read the plate with Envision, get the fitted curve according to the ratio (the ratio of the fluorescence intensity of Ex665/Ex615) and use Graphpad's four-parameter fitting formula Y=Bottom + (Top-Bottom)/(1+10 ^((LogEC50-X)*HillSlope)) calculates the 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 Reagent Brand No. Human ERK Phosphorylated Protein High Sensitive Detection Kit 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 No. Calu6 ATCC ATCC-HTB-56

Experimental steps and methods: 1) Resuscitate the cells and culture them to the logarithmic growth phase, digest with trypsin, plant in a 96-well plate, and incubate overnight in an incubator. 2) Add a series of gradient compounds dissolved in DMSO to the 96-well plate, and return to the incubator for 1 hour incubation. 3) Take out the cell plate, add cell lysate (containing 1% blocking peptide) and incubate at room temperature for 30 minutes. 4) Transfer 16 μL of cell lysate per well to the HTRF plate, and then add 4 μL of the prepared antibody mixture. 5) After overnight incubation, read the plate with Envision, get the fitted curve according to the ratio (the ratio of the fluorescence intensity of Ex665/Ex615) and use Graphpad's four-parameter fitting formula Y=Bottom + (Top-Bottom)/(1+10 ^((LogEC50-X)*HillSlope)) calculates _EC50 .

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 has good anti-proliferation 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 is to study the pharmacokinetics (PK) of the test compound (prepared in Example 1) in mice after a single intravenous and a single oral administration.

Sample collection and preparation: After intravenous or oral administration, blood samples were collected from animals, and the actual time of blood collection was recorded. Immediately after blood samples were collected, they were transferred to labeled centrifuge tubes containing K2-EDTA, followed by centrifugation to obtain plasma. Plasma was transferred to pre-chilled centrifuge tubes, snap-frozen in dry ice, and stored in a -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 compound with a non-compartmental model. The peak concentration (C _max ) and peak time (T _max ) as well as the quantifiable final time were obtained directly 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 point 0 to the terminal time point ( AUC _0-last ), initial concentration (C ₀ ).

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

Experimental conclusion: the compound of formula (I) has better oral absorption in mice, has lower clearance rate, longer half-life and better bioavailability.

Example 13: Stability test of compound A crystal form of formula (I) under accelerated conditions for 6 months

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

Experimental equipment: constant temperature and humidity chamber, model: BSC-400, condition: 40°C/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 fastened and sealed separately, then put into an aluminum foil bag and heat-sealed, and placed in an accelerated (40 °C/75%RH), after January, February, March, and June, it was taken out to detect related substances, enantiomers, moisture, and crystal forms. Table 21 Accelerated test (40°C/75%RH) research results of compound A crystal form of formula (I) Test items Acceptance Criteria Inspection time initial value 1 month 2 months 3 months 6 months relative substance 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 map Consistent with the spectrum of the reference substance / / Consistent with the 0-day spectrum Consistent with the 0-day spectrum

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 significant change in all inspection items, and all inspection items met the requirements. Therefore, the proposed packaging and storage conditions can ensure that the samples remain stable for the proposed expiration date.

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

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

Experimental equipment: constant temperature and humidity chamber, model: BSC-400, condition: 25°C/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 fastened and sealed separately, then put into an aluminum foil bag and heat-sealed, and placed in a long-term (25 °C/60%RH) conditions, after 3 months and 6 months, it was taken out to detect related substances, enantiomers, moisture and crystal forms. Table 22 Long-term test (25°C/60%RH) research results of compound A crystal form of formula (I) Test items Acceptance Criteria Inspection time initial value 3 months 6 months relative substance 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 map Consistent with the spectrum of the reference substance Consistent with the 0-day spectrum Consistent with the 0-day spectrum

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 inspection items were in compliance with the regulations. Therefore, the proposed packaging and storage conditions can ensure that the samples remain stable for the proposed expiration date.

Example 15: Preparation of Amorphous Compound of Formula (I)

At room temperature, the compound of formula (I) (46 g) was placed in a 1 L round-bottomed flask, dichloromethane (300 mL) was added, and after fully dissolved, it was dried by rotary evaporation under vacuum to obtain the amorphous compound of (I). Its XRPD spectrum is shown in FIG. 9 .

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

Claims (18)

Compound of formula (I) N- (3-(2-((1 R ,5 S )-3-oxabicyclo[3.1.0]hex-1-yl)-6-(2-hydroxyethoxy)pyridine Form A of -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°. Form A as described in Claim 1, its X-ray powder diffraction pattern 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°. Form A as described in Claim 2, its 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°, 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°. For the crystal form A described in any one of claims 1-3, its X-ray powder diffraction pattern is shown in Figure 1. The crystal form A as described in any one of claims 1-4, which is a monohydrate of the compound of formula (I). The TGA spectrum of the crystal form A described in any one of claims 1 to 5 is shown in Figure 5. The DSC spectrum of the crystal form A described in any one of claims 1 to 6 is shown in Figure 7. Compound of formula (I) N- (3-(2-((1 R ,5 S )-3-oxabicyclo[3.1.0]hex-1-yl)-6-(2-hydroxyethoxy)pyridine Form B of -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°. Form B as described in Claim 8, its X-ray powder diffraction pattern 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°. Form B as described in Claim 9, its X-ray powder diffraction pattern 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°. For the crystal form B described in any one of claims 8-10, its X-ray powder diffraction pattern is shown in Figure 2. The TGA spectrum of the crystal form B described in any one of claims 8-11 is shown in Figure 6. For the crystal form B described in any one of claim items 8-12, its DSC spectrum is shown in Figure 8. Compound of formula (I) N- (3-(2-((1 R ,5 S )-3-oxabicyclo[3.1.0]hex-1-yl)-6-(2-hydroxyethoxy)pyridine Amorphous form of -4-yl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide,

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

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