Salts, crystal forms of tricyclic compounds containing pyrimidine groups and their preparation methods

Abstract

The salt forms, crystal forms of tricyclic compounds containing pyrimidine groups and their preparation methods are disclosed, specifically the salt forms, crystal forms of the compound of formula (I) and their preparation methods.

Description

[0001] This application claims the following priority

[0002] CN202010757207.9, filing date: July 31, 2020. Technical Field

[0003] The present invention relates to salts, crystal forms of a class of tricyclic compounds containing pyrimidine groups and preparation methods thereof. Background Art

[0004] Receptor tyrosine kinase c-Met, also known as hepatocyte growth factor (HGF) receptor, is a transmembrane receptor encoded by the MET gene with autophosphorylation activity. It is a unique subclass in the receptor tyrosine kinases (RTKs) family and is mainly produced in epithelial cells. HGF is the only high-affinity ligand for c-Met and is widely present in various human tissues and organs.

[0005] Studies have found that c-Met is highly expressed in many tumor cells. For example, high expression of c-Met has been observed in cancer cells such as hepatocellular carcinoma, gastric cancer, ovarian cancer, non-small cell lung cancer, and renal cancer. Moreover, the overexpression of c-Met is closely related to the formation and prognosis of various tumors. The overactivation of the HGF / c-Met pathway will cause the activation of downstream signaling pathways, thus inducing cancer. In addition, the overexpression of HGF and c-Met will also lead to drug resistance responses of the EGFR, RAS-RAF-MEK, and Akt-mTOR signaling pathways to related inhibitors, which is an important mechanism for tumor cell escape. For example, in non-small cell lung cancer with EGFR activating mutations, the overexpression of HGF causes c-Met phosphorylation, thereby activating the downstream PI3K-Akt pathway and leading to drug resistance of cells to EGFR inhibitors. Similarly, the upregulation and secretion of HGF in the tumor microenvironment will lead to drug resistance of cells to RAS inhibitors.

[0006] After blocking the abnormally activated HGF / c-Met signaling pathway in tumor cells, a series of changes will occur in tumor cells, such as changes in cell morphology, slowed proliferation, reduced tumorigenicity, and decreased invasion ability. Therefore, developing a highly active c-Met inhibitor can provide an effective treatment method for various primary c-Met signaling pathway abnormalities and drug-resistant c-Met abnormally expressed tumors.

[0007] Currently, the main intervention therapies for the c-Met pathway are as follows: ① Therapeutic antibodies: Bind to HGF or c-Met, and inhibit the c-Met pathway by interfering with the interaction between HGF and c-Met; ② Small molecule tyrosine kinase inhibitors: Inhibit the kinase activity of c-Met or other kinases that play important roles in the cancer process; ③ Molecules similar to HSP90 inhibitors: Block the c-Met pathway by affecting the stability or expression of c-Met protein; ④ Functional molecules that interfere with the downstream effectors of the c-Met pathway.

[0008] Currently, the main c-Met small molecule inhibitors under clinical investigation are Crizotinib, Tepotinib (EMD1214063), Capmatinib, Volitinib, Cabozantinib (XL-184), and ARQ-197, etc. Although these drugs have shown good therapeutic effects clinically, some drugs have deficiencies such as high molecular clinical dosing, relatively large clinical side effects, and low drug stability. Therefore, the development of novel c-Met inhibitors with high activity, high selectivity, and good drug-likeness remains an unmet clinical need. Summary of the Invention

[0009] The present invention provides a pharmaceutically acceptable salt of the compound of formula (I),

[0010]

[0011] wherein the pharmaceutically acceptable salt is maleate, sulfate, methanesulfonate, p-toluenesulfonate, or fumarate.

[0012] The present invention provides a hydrochloride salt of the compound of formula (I), and its structure is shown as formula (I-1),

[0013]

[0014] wherein n is 0.9 - 1.1.

[0015] In some embodiments of the present invention, the structure of the above-mentioned hydrochloride salt is shown as formula (II),

[0016]

[0017] The present invention provides polymorph A of the compound of formula (II), and its X-ray powder diffraction pattern with CuKα radiation has characteristic diffraction peaks at the following 2θ angles: 4.68 ± 0.20°, 12.94 ± 0.20°, 14.12 ± 0.20°, and 21.86 ± 0.20°.

[0018]

[0019] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystalline form A with CuKα radiation has characteristic diffraction peaks at the following 2θ angles: 4.68±0.20°, 12.94±0.20°, 14.12±0.20°, 17.56±0.20°, 21.86±0.20°, 23.54±0.20°, and 28.48±0.20°.

[0020] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystalline form A with CuKα radiation has characteristic diffraction peaks at the following 2θ angles: 4.68±0.20°, 12.94±0.20°, 14.12±0.20°, 17.56±0.20°, 17.96±0.20°, 21.86±0.20°, 22.92±0.20°, 23.54±0.20°, 25.28±0.20°, 26.04±0.20°, 26.54±0.20°, and 28.48±0.20°.

[0021] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystalline form A with CuKα radiation has characteristic diffraction peaks at the following 2θ angles: 4.681°, 6.100°, 8.962°, 9.381°, 10.865°, 11.364°, 12.941°, 14.119°, 16.940°, 17.559°, 17.961°, 18.398°, 18.939°, 20.606°, 20.959°, 21.861°, 22.379°, 22.918°, 23.541°, 24.083°, 25.281°, 25.817°, 26.042°, 26.541°, 27.639°, 28.480°, 29.481°, 30.521°, 31.039°, 32.816°, 33.261°, 35.181°.

[0022] In some embodiments of the present invention, the XRPD pattern of the above-mentioned crystalline form A is as Figure 1 shown.

[0023] In some embodiments of the present invention, the diffraction peak data of the XRPD pattern of the above-mentioned crystalline form A are shown in Table 1.

[0024] Table 1 XRPD Diffraction Peak Data of Compound of Formula (II) in Crystalline Form A

[0025]

[0026] In some embodiments of the present invention, the differential scanning calorimetry curve (DSC) of the above-mentioned crystalline form A has an endothermic peak at 264.9°C ± 3°C.

[0027] In some embodiments of the present invention, the differential scanning calorimetry (DSC) curve of the above-mentioned crystalline form A has endothermic peaks at 100.9°C ± 3°C and 264.9°C ± 3°C.

[0028] In some embodiments of the present invention, the DSC pattern of the above-mentioned crystalline form A is as Figure 2 shown.

[0029] In some embodiments of the present invention, the thermogravimetric analysis (TGA) curve of the above-mentioned crystalline form A shows a weight loss of 5.39% at 160.0°C ± 3°C.

[0030] In some embodiments of the present invention, the TGA pattern of the above-mentioned crystalline form A is as Figure 3 shown.

[0031] The present invention provides crystalline form B of the compound of formula (II), and its X-ray powder diffraction pattern with CuKα radiation has characteristic diffraction peaks at the following 2θ angles: 4.80 ± 0.20°, 14.28 ± 0.20°, 20.22 ± 0.20° and 24.89 ± 0.20°,

[0032]

[0033] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystalline form B with CuKα radiation has characteristic diffraction peaks at the following 2θ angles: 4.80 ± 0.20°, 13.68 ± 0.20°, 14.28 ± 0.20°, 19.68 ± 0.20°, 20.22 ± 0.20°, 22.20 ± 0.20°, 24.89 ± 0.20° and 28.76 ± 0.20°.

[0034] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystalline form B with CuKα radiation has characteristic diffraction peaks at the following 2θ angles: 4.80 ± 0.20°, 13.68 ± 0.20°, 14.28 ± 0.20°, 18.02 ± 0.20°, 19.68 ± 0.20°, 20.22 ± 0.20°, 22.20 ± 0.20°, 23.56 ± 0.20°, 24.89 ± 0.20°, 27.50 ± 0.20°, 28.04 ± 0.20° and 28.76 ± 0.20°.

[0035] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystalline form B with CuKα radiation has characteristic diffraction peaks at the following 2θ angles: 4.797°, 8.262°, 9.520°, 11.661°, 13.680°, 14.279°, 16.520°, 17.323°, 18.017°, 18.521°, 19.679°, 20.221°, 21.539°, 22.199°, 22.822°, 23.562°, 24.157°, 24.889°, 25.444°, 26.159°, 27.501°, 28.038°, 28.760°, 29.719°, 31.360°, 31.979°, 32.459°, 33.540°, 34.422°, 34.839°, 35.501° and 36.382°.

[0036] In some embodiments of the present invention, the XRPD pattern of the above-mentioned crystalline form B is as Figure 4 shown.

[0037] In some embodiments of the present invention, the diffraction peak data of the XRPD pattern of the above-mentioned crystalline form B are shown in Table 2.

[0038] Table 2 XRPD Diffraction Peak Data of the Crystalline Form B of the Compound of Formula (II)

[0039]

[0040]

[0041] In some embodiments of the present invention, the differential scanning calorimetry curve (DSC) of the above-mentioned crystalline form B has endothermic peaks at 257.7 ± 3 °C and 268.9 ± 3 °C.

[0042] In some embodiments of the present invention, the differential scanning calorimetry curve (DSC) of the above-mentioned crystalline form B has endothermic peaks at 60.2 ± 3 °C, 257.7 ± 3 °C and 268.9 ± 3 °C.

[0043] In some embodiments of the present invention, the DSC pattern of the above-mentioned crystalline form B is as Figure 5 shown.

[0044] In some embodiments of the present invention, the thermogravimetric analysis (TGA) curve of the above-mentioned crystalline form B shows a weight loss of 5.10% at 110.0 °C ± 3 °C.

[0045] In some embodiments of the present invention, the TGA pattern of the above-mentioned crystalline form B is as Figure 6 shown.

[0046] The present invention also provides a method for preparing the above-mentioned crystalline form B, which comprises the following steps:

[0047] 1) Dissolve the crystalline form A of the compound of formula (II) in a solvent, and cool the resulting solution to a certain temperature;

[0048] 2) Filter, and vacuum-dry the filter cake;

[0049] wherein the solvent is a mixed solvent of ethanol: water = 1:1, and the certain temperature is 20°C to 30°C.

[0050] The present invention also provides the maleate salt of the compound of formula (I), the structure of which is shown in formula (III),

[0051]

[0052] The present invention also provides the crystalline form C of the compound of formula (III), the X-ray powder diffraction pattern of which with CuKα radiation has characteristic diffraction peaks at any of the following sets of 2θ angles: 3.82 ± 0.20°, 15.30 ± 0.20°, 16.38 ± 0.20°, 16.82 ± 0.20°, 20.02 ± 0.20°, 22.84 ± 0.20°, 23.72 ± 0.20° and 28.44 ± 0.20°;

[0053]

[0054] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above crystalline form C with CuKα radiation has characteristic diffraction peaks at the following 2θ angles: 3.82 ± 0.20°, 10.72 ± 0.20°, 14.24 ± 0.20°, 15.30 ± 0.20°, 16.38 ± 0.20°, 16.82 ± 0.20°, 20.02 ± 0.20°, 20.84 ± 0.20°, 22.84 ± 0.20°, 23.72 ± 0.20°, 26.90 ± 0.20° and 28.44 ± 0.20°.

[0055] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystalline form C with CuKα radiation has characteristic diffraction peaks at the following 2θ angles: 3.819°, 7.619°, 10.720°, 11.401°, 13.015°, 13.840°, 14.240°, 15.300°, 16.379°, 16.818°, 17.401°, 18.602°, 19.198°, 20.020°, 20.841°, 22.583°, 22.841°, 23.720°, 24.191°, 25.259°, 25.679°, 26.899°, 27.341°, 28.441°, 29.580°, 30.221°, 30.802°, 31.297°, 32.262°, 33.225°, 34.423°, 35.160°, 36.936°, 38.241° and 38.980°.

[0056] In some embodiments of the present invention, the XRPD pattern of the above-mentioned crystalline form C is as Figure 7 shown.

[0057] In some embodiments of the present invention, the diffraction peak data of the XRPD pattern of the above-mentioned crystalline form C are shown in Table 3.

[0058] Table 3 XRPD Diffraction Peak Data of Compound of Formula (III) in Crystalline Form C

[0059]

[0060]

[0061] In some embodiments of the present invention, the differential scanning calorimetry curve (DSC) of the above-mentioned crystalline form C has endothermic peaks at 119.3 ± 3 °C and 174.8 ± 3 °C.

[0062] In some embodiments of the present invention, the DSC pattern of the above-mentioned crystalline form C is as Figure 8 shown.

[0063] In some embodiments of the present invention, the thermogravimetric analysis (TGA) curve of the above-mentioned crystalline form C shows a weight loss of 3.84% at 120.0 °C ± 3 °C.

[0064] In some embodiments of the present invention, the TGA pattern of the above-mentioned crystalline form C is as Figure 9 shown.

[0065] Technical Effects

[0066] The crystal form of the present application is stable, less affected by heat, humidity, and light, and is convenient for formulation. The crystal form of the present application has good pharmacokinetic properties and is suitable for use as a drug, and the pharmacokinetic properties can be measured in preclinical animal experiments such as SD rats and beagle dogs.

[0067] Definitions and Explanations

[0068] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be considered indefinite or unclear without a specific definition, but should be understood in its ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding commodity or its active ingredient.

[0069] The intermediate compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by their combination with other chemical synthesis methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples of the present invention.

[0070] The chemical reactions in 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, sometimes those skilled in the art need to modify or select the synthesis steps or reaction processes on the basis of the existing embodiments.

[0071] The term "protecting group" includes, but is not limited to, "amino protecting group", "hydroxy protecting group" or "mercapto protecting group". The term "amino protecting group" refers to a protecting group suitable for preventing side reactions at the nitrogen position of the amino group. Representative amino protecting groups include, but are not limited to: formyl; acyl groups, such as alkanoyl groups (such as acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl groups, such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyl groups, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl groups, such as benzyl (Bn), trityl (Tr), 1,1-bis-(4'-methoxyphenyl)methyl; silyl groups, such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), etc. The term "hydroxy 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 groups, such as methyl, ethyl and tert-butyl; acyl groups, such as alkanoyl groups (such as acetyl); arylmethyl groups, such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups, such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), etc.

[0072] The structures of the compounds of the present invention can be confirmed by conventional methods well-known to those skilled in the art. If the present invention relates to the absolute configuration of a compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, single crystal X-ray diffraction (SXRD) method. The single crystal grown is used to collect diffraction intensity data with a Bruker D8 venture diffractometer. The light source is CuKα radiation, and the scanning mode is φ / ω scanning. After collecting relevant data, the crystal structure is further analyzed by the direct method (Shelxs97) to confirm the absolute configuration.

[0073] The present invention will be specifically described below by way of examples, which do not imply any limitation to the present invention.

[0074] All solvents used in the present invention are commercially available and can be used without further purification.

[0075] The solvents used in the present invention are commercially available.

[0076] The following abbreviations are used in the present invention:

[0077] μL: microliter; μM: micromole per liter; nM: nanomole per liter; mm: millimeter; Pd(dppf)Cl2 represents dichloropalladium [1,1'-bis(diphenylphosphino)ferrocene]; DMF represents N,N-dimethylformamide; DIPEA represents N,N-diisopropylethylamine; DMSO represents dimethyl sulfoxide; Boc2O represents di-tert-butyl dicarbonate; TMSCl represents trimethylchlorosilane.

[0078] The compounds of the present invention are named according to the conventional naming principles in the art or using software naming, and commercially available compounds use the supplier catalog names.

[0079] 1. Instruments and analysis methods

[0080] 1.1 X-ray powder diffraction (XRPD) method of the present invention

[0081] Instrument model: DX-2700BH

[0082] Test conditions: The detailed XRPD parameters are as follows:

[0083] X-ray generator: Cu, kα,

[0084] Tube voltage: 40 kV, tube current: 30 mA.

[0085] Scattering slit: 1 mm

[0086] Detector slit: 0.3 mm

[0087] Anti-scattering slit: 1 mm

[0088] Scanning range: 3 - 40 degrees

[0089] Step size: 0.02 degrees

[0090] Step length: 0.5 seconds

[0091] 1.2 Differential Scanning Calorimeter (DSC) method of the present invention

[0092] Instrument model: TA Instruments Discovery DSC 2500 and Q200 differential scanning calorimeters

[0093] Test conditions: Take 1 - 5 mg of the sample and place it in a covered (unless otherwise specified) aluminum crucible, and perform the test under the protection of dry N2 at 50 mL / min

[0094] Method: Heat from 25°C to the set test temperature at a heating rate of 10°C / min

[0095] 1.3 Thermal Gravimetric Analyzer (TGA) method of the present invention

[0096] Instrument model: TA Instruments Q5000 and Discovery TGA 5500 thermal gravimetric analyzers

[0097] Test conditions: Take the sample (2 - 5 mg) and place it in a TGA platinum pan, and perform the test under the protection of dry N2 at 50 mL / min

[0098] Method: Room temperature to 350°C, heating rate of 10°C / min

[0099] 1.5 Chloride ion detection and analysis method of the present invention

[0100] Test procedure: Take a certain amount of the standard product and prepare a standard product solution with a determined concentration, and its chloride ion concentration is D STD , then take a certain amount W SPL of the compound to be tested, and prepare a solution with a volume V SPL of the solvent. Detect the two solutions by ion chromatography, and obtain the peak area of the standard product as A STD , and the peak area of the compound to be tested is A SPL .

[0101] Calculation method:

[0102]

[0103] D STD : Standard chloride ion concentration of the reference substance (μg / mL);

[0104] W SPL : Sampling amount of the test solution (mg);

[0105] V SPL : Volume of the solvent consumed for diluting the test sample (mL);

[0106] A STD : Peak area of the reference substance sample after liquid chromatography detection;

[0107] A SPL : Peak area of the sample to be tested after liquid chromatography detection. Description of the Drawings

[0108] Figure 1 is the XRPD spectrum of polymorph A of the compound of formula (II).

[0109] Figure 2 is the DSC spectrum of polymorph A of the compound of formula (II).

[0110] Figure 3 is the TGA spectrum of polymorph A of the compound of formula (II).

[0111] Figure 4 is the XRPD spectrum of polymorph B of the compound of formula (II).

[0112] Figure 5 is the DSC spectrum of polymorph B of the compound of formula (II).

[0113] Figure 6 is the TGA spectrum of polymorph B of the compound of formula (II).

[0114] Figure 7 is the XRPD spectrum of polymorph C of the compound of formula (III).

[0115] Figure 8 is the DSC spectrum of polymorph C of the compound of formula (III).

[0116] Figure 9 is the TGA spectrum of polymorph C of the compound of formula (III). Detailed Description of the Invention

[0117] To better understand the content of the present invention, the following further describes it in combination with specific embodiments, but the specific implementation manners do not limit the content of the present invention.

[0118] Example 1: Preparation of the compound of formula (I)

[0119]

[0120] Compound 1:

[0121]

[0122] Dissolve tert-butyl 4-(hydroxymethyl)piperidine-1-carboxylate (50 g, 232.25 mmol) in 800 mL of anhydrous dichloromethane, add DIPEA (60.10 g, 465.04 mmol, 81 mL), and slowly add methanesulfonyl chloride (31.08 g, 271.32 mmol, 21 mL) dropwise at 0 °C. After the addition is complete, stir the mixture under a nitrogen protection environment at 27 °C for 1 hour. Wash the reaction solution three times with 200 mL of 0.5 mol / L hydrochloric acid aqueous solution and then with 300 mL of saturated sodium bicarbonate aqueous solution. Separate the organic phase, dry it over anhydrous sodium sulfate, filter, and concentrate by rotary evaporation to obtain Compound 1. 1 1H NMR (400 MHz, CDCl3) δ = 4.14 (br s, 2H), 4.07 (d, J = 6.4 Hz, 2H), 3.01 (s, 3H), 2.71 (brt, J = 12.4 Hz, 2H), 1.97 - 1.83 (m, 1H), 1.74 (br d, J = 12.8 Hz, 2H), 1.46 (s, 9H), 1.32 - 1.14 (m, 2H). LCMS (ESI): m / z: 238.1 [M - 55].

[0123] Compound 2:

[0124]

[0125] Dissolve Compound 1 (109 g, 371.53 mmol), 2-chloro-5-hydroxypyrimidine (40.25 g, 308.37 mmol), and potassium carbonate (85.24 g, 616.75 mmol) in 1000 mL of DMF. Stir the mixture under a nitrogen protection environment at 80 °C for 16 hours. Concentrate the reaction solution by rotary evaporation to remove the organic solvent. Add 400 mL of water to the remaining residue and extract it three times with 300 mL of ethyl acetate each time. Combine the organic phases, dry them over anhydrous sodium sulfate, filter, and concentrate by rotary evaporation. Purify the residue by column chromatography (eluting with petroleum ether:ethyl acetate = 50:1 - 5:1) to obtain the crude product. Then stir the crude product with 60 mL of a petroleum ether:ethyl acetate = 5:1 mixed solvent at 25 °C for 15 minutes and filter. Wash the filter cake with a petroleum ether:ethyl acetate = 5:1 mixed solvent (10 mL / time, washing three times) and concentrate by rotary evaporation to obtain Compound 2. 11H NMR (400 MHz, DMSO-d6) δ = 8.53 (s, 2H), 4.02 (d, J = 6.5 Hz, 2H), 3.96 (br d, J = 12.2 Hz, 2H), 2.87 - 2.62 (m, 2H), 2.01 - 1.87 (m, 1H), 1.80 - 1.66 (m, 2H), 1.39 (s, 9H), 1.10 - 1.02 (m, 2H). LCMS (ESI): m / z: 272.0 [M - 55].

[0126] Compound 3:

[0127]

[0128] Dissolve compound 2 (34 g, 103.72 mmol) and 3-hydroxymethylphenylboronic acid (16 g, 105.29 mmol) in 250 mL of dioxane and 50 mL of water, add sodium carbonate (33 g, 311.35 mmol) and Pd(dppf)Cl2 (3 g, 4.10 mmol). Stir the mixture under a nitrogen protection environment at 90 °C for 12 hours, and rotary evaporate the reaction solution to remove the organic solvent. Add 100 mL of water to the remaining residue, and then extract it three times with 100 mL of ethyl acetate each time. Combine the organic phases and rotary evaporate them. The residue is stirred with 200 mL of a mixed solvent of petroleum ether:ethyl acetate = 1:1 for half an hour and then filtered. Wash the filter cake with a mixed solvent of petroleum ether:ethyl acetate = 1:1 (50 mL / time, wash three times) to obtain compound 3. 1 1H NMR (400 MHz, CDCl3) δ = 8.46 (s, 2H), 8.34 (br s, 1H), 8.28 (br s, 1H), 7.47 (br s, 2H), 4.80 (br s, 2H), 4.20 (br s, 2H), 3.95 (br d, J = 5.9 Hz, 2H), 2.77 (br s, 2H), 2.02 (br s, 1H), 1.85 (br d, J = 13.7 Hz, 2H), 1.48 (s, 9H), 1.32 (1.45 - 1.12, m, 2H). LCMS (ESI): m / z: 400.1 [M + 1].

[0129] Compound 4:

[0130]

[0131] Compound 3 (44 g, 110.14 mmol) was dissolved in 400 mL of dichloromethane, and then DIPEA (57.13 g, 442.08 mmol, 77 mL) was added. Methanesulfonyl chloride (51.80 g, 452.20 mmol, 35 mL) was slowly added at 0 °C. After the addition, the reaction mixture was stirred at 20 °C for 4 h. Then, 300 mL of dichloromethane was added to the reaction mixture, and it was washed three times with 300 mL of saturated aqueous sodium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 100:1 - 10:1) to obtain Compound 4. 1 H NMR (400 MHz, CDCl3) δ = 8.49 - 8.44 (m, 2H), 8.39 (s, 1H), 8.38 - 8.29 (m, 1H), 7.50 - 7.44 (m, 2H), 4.71 - 4.67 (m, 2H), 4.19 (brs, 2H), 3.96 (d, J = 6.4 Hz, 2H), 2.77 (br t, J = 12.2 Hz, 2H), 2.08 - 1.98 (m, 1H), 1.85 (br d, J = 12.6 Hz, 2H), 1.48 (s, 9H), 1.39 - 1.29 (m, 2H). LCMS (ESI): m / z: 418.0 [M+1].

[0132] Compound 5:

[0133]

[0134] Uracil (20 g, 178.43 mmol) was dissolved in 200 mL of DMSO, and potassium carbonate (29.59 g, 214.12 mmol) and 4-bromo-2-fluoro-1-nitrobenzene (39.25 g, 178.43 mmol) were added. The mixture was stirred at 80 °C under a nitrogen atmosphere for 2 h. After the reaction mixture was cooled to 20 °C, the pH was adjusted to 4 with 2 M aqueous hydrochloric acid. Then, 1.5 L of water was added to the reaction mixture. The resulting mixture was filtered, and the filter cake was washed with water (100 mL × 2) and dried in vacuo to obtain Compound 5. 1 H NMR (400 MHz, DMSO-d6) δ = 11.69 (s, 1H), 8.16 - 8.10 (m, 2H), 8.02 - 7.95 (m, 1H), 7.89 (d, J = 7.9 Hz, 1H), 5.82 (dd, J = 2.1, 8.0 Hz, 1H).

[0135] Compound 6:

[0136]

[0137] At 25 °C, iron powder (4.47 g, 80.11 mmol) was added to a mixture of compound 5 (5 g, 16.02 mmol) and acetic acid (100 mL). The mixture was stirred at 90 °C for 1 h. After the reaction solution was cooled to room temperature, it was filtered. The filtrate was concentrated under reduced pressure and then water (50 mL) was added. The pH was adjusted to 8 with 2 M aqueous sodium hydroxide solution, and then extracted three times with 100 mL of a mixed solvent of dichloromethane:methanol = 10:1. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. The residue was purified by column chromatography (dichloromethane:methanol = 1:0 - 20:1) to obtain compound 6. 1 H NMR (400 MHz, DMSO-d6) δ = 11.29 (d, J = 1.6 Hz, 1H), 7.38 (d, J = 7.9 Hz, 1H), 7.31 - 7.21 (m, 2H), 6.72 (d, J = 8.6 Hz, 1H), 5.61 (dd, J = 2.3, 7.8 Hz, 1H), 5.54 (s, 2H). LCMS (ESI) m / z: 282.1 [M+1].

[0138] Compound 7:

[0139]

[0140] A mixture of compound 6 (2.9 g, 10.28 mmol) and polyphosphoric acid (15 g) was stirred at 170 °C for 2 h. After the reaction solution was cooled to room temperature, water (40 mL) was added to the reaction solution. The reaction solution was adjusted to pH 5 - 6 with saturated aqueous sodium carbonate solution. The resulting suspension was filtered, and the filter cake was concentrated to dryness. Ethyl acetate (20 mL) was added, and the mixture was stirred at room temperature and then filtered. The filter cake was dried in vacuo to obtain compound 7. 1 H NMR (400 MHz, DMSO-d6) δ = 8.77 (d, J = 7.7 Hz, 1H), 8.24 (d, J = 1.6 Hz, 1H), 7.51 - 7.40 (m, 2H), 6.14 (d, J = 7.8 Hz, 1H); LCMS (ESI) m / z: 264.2 [M+1].

[0141] Compound 8:

[0142]

[0143] Compound 7 (2.3 g, 8.71 mmol) and compound 4 (4.35 g, 10.41 mmol) were dissolved in DMF (25 mL), and cesium carbonate (5.67 g, 17.41 mmol) and potassium iodide (1.44 g, 8.66 mmol) were added. The mixture was stirred at 100 °C for 0.5 h, and the reaction solution was concentrated to remove the organic solvent. The remaining residue was added with 15 mL of water, and then extracted three times with 20 mL of a mixed solvent of dichloromethane:methanol = 10:1. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. The residue was purified by column chromatography (eluted with dichloromethane:methanol = 1:0 to 20:1) to obtain compound 8. 1 H NMR (400 MHz, DMSO-d6) δ = 8.90 (d, J = 7.8 Hz, 1H), 8.63 (s, 2H), 8.41 - 8.30 (m, 2H), 8.26 - 8.10 (m, 1H), 7.62 - 7.55 (m, 1H), 7.53 - 7.41 (m, 3H), 6.37 (d, J = 7.8 Hz, 1H), 5.44 (s, 2H), 4.06 (d, J = 6.4 Hz, 2H), 3.98 (br d, J = 11.9 Hz, 2H), 2.75 (br s, 2H), 1.97 (br dd, J = 6.8, 12.6 Hz, 1H), 1.76 (br d, J = 11.0 Hz, 2H), 1.41 (s, 9H), 1.17 (dq, J = 4.3, 12.3 Hz, 2H).

[0144] Compound 9:

[0145]

[0146] Compound 8 (3 g, 4.65 mmol) and zinc cyanide (2.20 g, 18.74 mmol) were dissolved in a reaction flask containing dimethylformamide (45 mL). At room temperature, zinc powder (775 mg, 11.85 mmol), bis(diphenylphosphino)ferrocene (775 mg, 1.40 mmol), and bis(dibenzylideneacetone)palladium (650.00 mg, 709.83 μmol) were added to the reaction flask. After purging with nitrogen, the reaction was stirred at 100 °C under a nitrogen atmosphere for 1 h. After the reaction was completed, it was cooled to room temperature, ammonia water (20 mL) was added to the reaction solution flask, and the reaction solution was extracted three times with 50 mL of dichloromethane each time. The combined extracted organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue obtained by concentration was purified by silica gel column chromatography (eluted with dichloromethane / methanol = 1 / 0 to 100 / 1) to obtain compound 9. 11H NMR (400 MHz, DMSO-d6) δ = 8.92 (d, J = 7.7 Hz, 1H), 8.63 (s, 1H), 8.57 (s, 1H), 8.36 (s, 1H), 8.20 (br d, J = 7.7 Hz, 1H), 7.83 - 7.71 (m, 2H), 7.54 - 7.48 (m, 1H), 7.47 - 7.41 (m, 1H), 6.47 (d, J = 7.7 Hz, 1H), 5.47 (s, 2H), 4.06 (d, J = 6.4 Hz, 2H), 3.98 (br d, J = 9.5 Hz, 2H), 2.74 (s, 2H), 1.98 (br s, 1H), 1.76 (br d, J = 10.5 Hz, 2H), 1.40 (s, 9H), 1.24 - 1.10 (m, 2H); LCMS (ESI) m / z: 592.5 [M+1].

[0147] Compound 10:

[0148]

[0149] At room temperature, compound 9 (0.9 g, 1.52 mmol) was added to a reaction flask containing anhydrous dichloromethane (3 mL). Trifluoroacetic acid (13.86 g, 121.55 mmol, 9.00 mL) was added to the reaction flask. The reaction was stirred at room temperature (25 °C) for 15 minutes. The reaction solution was concentrated under reduced pressure to obtain the trifluoroacetate salt of compound 10. 1 1H NMR (400 MHz, CD3OD) δ = 8.74 (d, J = 7.7 Hz, 1H), 8.53 (s, 2H), 8.41 (s, 1H), 8.30 (d, J = 0.9 Hz, 1H), 8.24 (d, J = 7.9 Hz, 1H), 7.79 - 7.75 (m, 1H), 7.74 - 7.69 (m, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.45 (t, J = 7.7 Hz, 1H), 6.38 (d, J = 7.8 Hz, 1H), 5.59 (s, 2H), 4.12 (d, J = 6.0 Hz, 2H), 3.48 (brd, J = 12.7 Hz, 2H), 3.13 - 3.04 (m, 2H), 2.31 - 2.18 (m, 1H), 2.13 (br d, J = 13.2 Hz, 2H), 1.73 - 1.58 (m, 2H); LCMS (ESI) m / z: 492.4 [M+1].

[0150] To the trifluoroacetate of Compound 10 (335 mg, 569.64 μmol) was added saturated aqueous sodium bicarbonate solution (20 mL), and the mixture was extracted with dichloromethane:methanol = 10:1 (20 mL × 3 times). The combined organic phases were dried over sodium sulfate, filtered, and concentrated to dryness. The obtained Compound 10 was directly used for the next step.

[0151] Compound of formula (I) and its hydrochloride salt:

[0152]

[0153] At room temperature, Compound 10 (0.15 g, 305.16 μmol) and isobutene oxide (812 mg, 11.26 mmol, 1 mL) were added to a reaction flask containing N,N-dimethylformamide (2 mL). Potassium carbonate (90 mg, 651.21 μmol) was added to the reaction flask. The reaction was stirred at 80 °C for 2 hours. After the reaction was completed, the reaction mixture was filtered, and the filter cake was collected and dried under reduced pressure to obtain the crude product of the compound of formula (I). To 41.3 mg of the crude product of the compound of formula (I) were successively added water (10 mL), acetonitrile (5 mL), and hydrochloric acid aqueous solution (1 mol / L, 0.1 mL), and the mixture was stirred at 25 °C for 30 minutes. The mixture was concentrated under reduced pressure to obtain the hydrochloride salt of the compound of formula (I). 1 H NMR (400 MHz, DMSO-d6) δ = 8.92 (d, J = 7.8 Hz, 1H), 8.63 (s, 2H), 8.57 (s, 1H), 8.36 (s, 1H), 8.20 (d, J = 7.8 Hz, 1H), 7.83 - 7.72 (m, 2H), 7.54 - 7.48 (m, 1HI), 7.47 - 7.38 (m, 1H), 6.47 (d, J = 7.8 Hz, 1H), 5.47 (s, 2H), 4.03 (br d, J = 5.9 Hz, 2H), 2.96 (br d, J = 11.1 Hz, 2H), 2.18 (s, 2H), 2.11 (br t, J = 11.1 Hz, 2H), 1.77 - 1.63 (m, 3H), 1.41 - 1.27 (m, 2H), 1.08 (s, 6H); LCMS (ESI) m / z: 564.3 [M + 1].

[0154] Example 2: Preparation of Crystal Form A of the Compound of Formula (II)

[0155]

[0156] The compound of formula (I) (0.5 g, 0.887 mmol, 1 eq) was added to a mixed solvent of ethanol:water = 5:1 (12.5 mL). The mixture was heated to 70 - 80 °C, and an aqueous hydrochloric acid solution of 1 mol / L (1 mL, 1.13 eq) was added to the mixture at 70 - 80 °C. The mixture was stirred at 70 - 80 °C for 30 minutes and then cooled to 20 - 30 °C, and stirred at 20 - 30 °C for 12 hours and then filtered. The filter cake was washed with ethanol (0.5 mL × 3) and dried to constant weight to obtain crystalline form A of the compound of formula (II), with a chloride ion content of 5.57%. Its XRPD pattern is shown in Figure 1 and its DSC pattern is shown in Figure 2 and its TGA pattern is shown in Figure 3 . 1 1H NMR (400 MHz, DMSO-d6) δ = 9.24 - 8.88 (m, 2H), 8.68 - 8.62 (m, 2H), 8.58 (s, 1H), 8.35 (s, 1H), 8.20 (d, J = 7.8 Hz, 1H), 7.80 - 7.74 (m, 2H), 7.55 - 7.51 (m, 1H), 7.48 - 7.42 (m, 1H), 6.47 (d, J = 7.8 Hz, 1H), 5.47 (s, 2H), 5.30 - 5.23 (m, 1H), 4.19 - 4.04 (m, 2H), 3.69 - 3.41 (m, 2H), 3.27 - 3.00 (m, 4H), 2.23 - 2.01 (m, 1H), 1.97 - 1.66 (m, 4H), 1.27 (s, 6H).

[0157] Example 3: Preparation of crystalline form B of the compound of formula (II)

[0158] Crystalline form A of the compound of formula (II) (2 g, 3.33 mmol, 1 eq) was added to a mixed solvent of ethanol:water = 1:1 (66 mL). The mixture was heated to 66 °C and stirred at 66 °C for 30 minutes and then cooled to 20 - 30 °C, and stirred at 20 - 30 °C for 12 hours and then filtered. The filter cake was washed with ethanol (2 mL × 3) and dried to constant weight to obtain crystalline form B of the compound of formula (II), with a chloride ion content of 5.69%. Its XRPD pattern is shown in Figure 4 and its DSC pattern is shown in Figure 5 and its TGA pattern is shown in Figure 6 . 11H NMR (400 MHz, DMSO-d6) δ = 9.35 - 9.04 (m, 1H), 8.95 (d, J = 7.6 Hz, 1H), 8.71 - 8.62 (m, 2H), 8.59 (s, 1H), 8.36 (s, 1H), 8.20 (brd, J = 8.1 Hz, 1H), 7.82 - 7.73 (m, 2H), 7.53 (br d, J = 7.3 Hz, 1H), 7.48 - 7.42 (m, 1H), 6.47 (d, J = 7.8 Hz, 1H), 5.47 (s, 2H), 5.27 (s, 1H), 4.20 - 4.03 (m, 2H), 3.65 (m, 2H), 3.24 - 3.00 (m, 4H), 2.22 - 2.03 (m, 1H), 1.96 - 1.69 (m, 4H), 1.27 (s, 6H).

[0159] Example 4: Preparation of Crystal Form C of the Compound of Formula (m)

[0160]

[0161] The compound of formula (I) (0.45 g, 0.798 mmol, 1 eq) was added to a mixed solvent of ethanol:water = 5:1 (11.2 mL). The mixture was heated to 70 - 80 °C, and an aqueous solution of maleic acid (0.8 mL, 1 eq, 1 mol / L) was added to the mixture at 70 - 80 °C. The mixture was stirred at 70 - 80 °C for 30 minutes and then cooled to 20 - 30 °C, and stirred at 20 - 30 °C for 12 hours and then filtered. The filter cake was washed with ethanol (0.5 mL × 3) and dried to a constant weight to obtain the compound of formula (III).

[0162] The compound of formula (III) (0.3 g, 0.441 mmol) was added to a mixed solvent of ethanol:water = 1:1 (4 mL). The mixture was heated to 60 - 70 °C, and stirred at 60 - 70 °C for 30 minutes and then cooled to 20 - 30 °C, and stirred at 20 - 30 °C for 12 hours and then filtered. The filter cake was washed with ethanol (0.5 mL × 3) and dried to a constant weight to obtain Crystal Form C of the compound of formula (III). The XRPD pattern is shown in Figure 7 and the DSC pattern is shown in Figure 8 and the TGA pattern is shown in Figure 9 . 11H NMR (400 MHz, DMSO-d6) δ = 8.93 (d, J = 7.8 Hz, 1H), 8.65 (s, 2H), 8.58 (s, 1H), 8.36 (s, 1H), 8.21 (d, J = 7.8 Hz, 1H), 7.81 - 7.74 (m, 2H), 7.55 - 7.50 (m, 1H), 7.49 - 7.42 (m, 1H), 6.48 (d, J = 7.8 Hz, 1H), 6.03 (s, 2H), 5.47 (s, 2H), 5.28 - 5.17 (m, 1H), 4.20 - 4.04 (m, 2H), 3.70 - 3.43 (m, 2H), 3.07 (m, 4H), 2.23 - 2.00 (m, 1H), 1.90 (m, 2H), 1.82 - 1.65 (m, 2H), 1.25 (s, 6H).

[0163] Example 5: Preparation of the Sulfate Salt of the Compound of Formula (I)

[0164] The compound of formula (I) (0.45 g, 0.798 mmol, 1 eq) was added to a mixed solvent of ethanol:water = 5:1 (11.2 mL). The mixture was heated to 70 - 80 °C, and an aqueous sulfuric acid solution (0.4 mL, 0.5 eq) of 1 mol / L was added to the mixture at 70 - 80 °C. The reaction solution did not clear. An aqueous sulfuric acid solution (0.4 mL, 0.5 eq) of 1 mol / L was continuously added to the mixture at 70 - 80 °C, and the reaction solution still did not clear. After the mixture was stirred at 70 - 80 °C for 30 minutes, the reaction solution still did not clear.

[0165] Example 6: Preparation of the Methanesulfonate Salt of the Compound of Formula (I)

[0166] The compound of formula (I) (0.45 g, 0.798 mmol, 1 eq) was added to a mixed solvent of ethanol:water = 5:1 (11.2 mL). The mixture was heated to 70 - 80 °C, and an aqueous methanesulfonic acid solution (0.8 mL, 1 eq) of 1 mol / L was added to the mixture at 70 - 80 °C. After the mixture was stirred at 70 - 80 °C for 30 minutes, it was cooled to 20 - 30 °C and stirred at 20 - 30 °C for 12 hours, and no solid precipitated.

[0167] Example 7: Preparation of the p-Toluenesulfonate Salt of the Compound of Formula (I)

[0168] The compound of formula (I) (0.45 g, 0.798 mmol, 1 eq) was added to a mixed solvent of ethanol:water = 5:1 (11.2 mL). The mixture was heated to 70 - 80 °C, and an aqueous solution of methanesulfonic acid (0.8 mL, 1 eq) at 1 mol / L was added to the mixture at 70 - 80 °C. After the mixture was stirred at 70 - 80 °C for 30 minutes, it was cooled to 20 - 30 °C and stirred at 20 - 30 °C for 12 hours, and no solid was precipitated.

[0169] Example 8: Preparation of the fumarate salt of the compound of formula (I)

[0170] The compound of formula (I) (0.45 g, 0.798 mmol, 1 eq) was added to a mixed solvent of ethanol:water = 5:1 (11.2 mL). The mixture was heated to 70 - 80 °C, and fumaric acid (93 mg, 1 eq) was added to the mixture at 70 - 80 °C. After the mixture was stirred at 70 - 80 °C for 30 minutes, the reaction solution did not become clear.

[0171] Example 9: Stability experiment of polymorph B of the compound of formula (II)

[0172] 1. Experimental purpose:

[0173] To investigate the stability of polymorph B of the compound of formula (II) under stress conditions (high temperature, high humidity and light) and accelerated conditions (40 °C / 75% RH and 60 °C / 75% RH), and to evaluate the solid stability of polymorph B.

[0174] 2. Experimental method:

[0175] Approximately 20 mg of polymorph B of the compound of formula (II) was accurately weighed and placed in a dry and clean glass bottle. Three portions were weighed and labeled as S1 - condition - time, S2 - condition - time, and S3 - condition - time, respectively, and spread into a thin layer as the test samples. They were placed under stress test conditions (60 °C, 25 °C / 92.5% RH, light, light control) and accelerated conditions (40 °C / 75% RH and 60 °C / 75% RH), and the samples were exposed completely. Samples were taken for analysis at 5 days and 10 days under 60 °C, 25 °C / 92.5% RH, light, light control, and at 1 month, 2 months, and 3 months under accelerated conditions. The analysis method is shown in Table 4.

[0176] Table 4

[0177]

[0178]

[0179] At the time point of investigation, take out the corresponding test samples, cover them with bottle caps. Take out the samples at 0 day from the refrigerator, and analyze them after the samples return to room temperature. The test samples labeled as S1 - condition - time are used for the determination of content and related substances; the test samples labeled as S2 - condition - time are used as backup samples; the test samples labeled as S3 - condition - time are used for XRPD detection.

[0180] 3. Experimental results:

[0181] 1) The analysis results of the content and related substances of the B - form stability samples are shown in Table 5.

[0182] Table 5

[0183]

[0184] 2) The experimental results of the solid stability of B - form are shown in Table 6.

[0185] Table 6

[0186]

[0187] * Light - exposed samples (visible light intensity 5000 lux and ultraviolet intensity 90 μw / cm 2 , open - mouthed); ** It is necessary to place a light - exposed control sample at the same time. After the light - exposed control sample is packaged in the same way as the light - exposed sample, it is then completely wrapped with tin foil.

[0188] Conclusion: The B - form of the compound of formula (II) has good stability.

[0189] Example 10: Hygroscopicity experiment of the B - form of the compound of formula (II)

[0190] 1. Experimental purpose:

[0191] To evaluate the hygroscopicity of the B - form of the compound of formula (II) by the pharmacopoeia method.

[0192] 2. Test procedure:

[0193] 1) Take two dry stoppered glass weighing bottles (outer diameter 50 mm, height 30 mm), place them in a drug stability test chamber (set temperature at 25 °C, relative humidity at 80%) to equilibrate.

[0194] 2) Weigh precisely the weight m1 of the weighing bottle after equilibration.

[0195] 3) Take an appropriate amount of the B - form sample, spread it evenly in the above two weighing bottles respectively. The thickness of the sample is generally about 1 mm, and weigh the total weight m2 precisely.

[0196] 4) Open the weighing bottles and place them together with the bottle caps under the above constant temperature and humidity conditions for 24 hours.

[0197] 5) Cover the weighing bottle with its lid, and accurately weigh the total weight \(m_3\).

[0198] 3. Calculation and judgment basis

[0199] Calculation: Percentage of weight gain = \((m_3 - m_2) / (m_2 - m_1)\times100\%\)

[0200] Judgment basis:

[0201] Description of Hygroscopic Properties Hygroscopic Weight Gain Deliquescence Absorbing Sufficient Water to Form a Liquid Highly Hygroscopic Hygroscopic Weight Gain Not Less than 15% Hygroscopic Hygroscopic Weight Gain Less than 15% but Not Less than 2% Slightly Hygroscopic Hygroscopic Weight Gain Less than 2% but Not Less than 0.2% No or Almost No Hygroscopicity Hygroscopic Weight Gain Less than 0.2%

[0202] 4. Experimental results:

[0203] The hygroscopicity test results of polymorph B are shown in Table 7.

[0204] Table 7

[0205]

[0206] Conclusion: Polymorph B of the compound of formula (II) has no or almost no hygroscopicity.

[0207] Biological activity

[0208] 1.1 In vitro activity test

[0209] Biochemical experiment:

[0210] Experimental purpose:

[0211] To detect the inhibitory effect of the compound on the activity of c-Met enzyme.

[0212] Experimental materials:

[0213] c-Met Kinase Enzyme System is purchased from Promega. Envision multi-label analyzer (PerkinElmer).

[0214] Experimental method:

[0215] Dilute the enzyme, substrate, ATP and inhibitor with the kinase buffer in the kit.

[0216] The test compound was serially diluted 5-fold with a multi-channel pipette to the 8th concentration, i.e., from 50 μM to 0.65 nM, with a final DMSO concentration of 5%, and a double replicate experiment was set up. 1 μL of each concentration gradient of the inhibitor, 2 μL of c-Met enzyme (4 ng), and 2 μL of the mixture of substrate and ATP (10 μM ATP, 0.2 μg / μL Poly E4Y1 (poly E4Y1)) were added to the microplate. At this time, the final concentration gradient of the compound was from 10 μM to 0.13 nM. The reaction system was incubated at 30 °C for 60 minutes. After the reaction, 5 μL of ADP-Glo reagent was added to each well and the reaction continued at 30 °C for 40 minutes. After the reaction ended, 10 μL of kinase detection reagent was added to each well and the reaction was carried out at 30 °C for 30 minutes, and then chemiluminescence was read using a PerkinElmer Envision multimode plate reader with an integration time of 0.5 seconds.

[0217] Data analysis:

[0218] The original data was converted into inhibition rate using the equation (Sample - Min) / (Max - Min)*100%, and the IC 50 value could be obtained by four-parameter curve fitting (obtained in the log(inhibitor) vs. response--Variable slope mode in GraphPad Prism). Table 8 provides the c-Met enzymatic inhibitory activity of the compounds of the present invention.

[0219] EBC-1 cell proliferation experiment:

[0220] Experimental materials:

[0221] MEM medium, fetal bovine serum, penicillin / streptomycin antibiotics were purchased from Vicente. The EBC-1 cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd. Envision multimode plate reader (PerkinElmer).

[0222] Experimental method:

[0223] EBC-1 cells were seeded in white 96-well plates, 80 μL of cell suspension per well, which contained 3000 EBC-1 cells. The cell plates were incubated overnight in a carbon dioxide incubator.

[0224] The compound to be tested was serially diluted 5-fold to the 8th concentration using a multi-channel pipette, i.e., diluted from 2 mM to 26 nM, and a double-replicate experiment was set up. 78 μL of culture medium was added to the middle plate, and then 2 μL of the gradient-diluted compound per well was transferred to the middle plate according to the corresponding positions. After mixing, 20 μL per well was transferred to the cell plate. The cell plate was placed in a carbon dioxide incubator and cultured for 3 days. Another cell plate was prepared, and the signal value was read on the day of drug addition as the Max value for data analysis. 25 μL of Promega CellTiter-Glo was added to each well of this cell plate and incubated at room temperature for 10 minutes to stabilize the luminescence signal. A PerkinElmer Envision multimode plate reader was used for reading.

[0225] 25 μL of Promega CellTiter-Glo reagent was added to each well of the cell plate and incubated at room temperature for 10 minutes to stabilize the luminescence signal. A PerkinElmer Envision multimode plate reader was used for reading.

[0226] Data analysis:

[0227] The original data was converted into inhibition rate using the equation (Sample - Min) / (Max - Min)*100%, and the IC 50 value could be obtained by four-parameter curve fitting (in the "log(inhibitor) vs. response - Variable slope" mode in GraphPad Prism). Table 8 provides the inhibitory activities of the compounds of the present invention against the proliferation of EBC-1 cells.

[0228] Hs746T cell proliferation experiment:

[0229] Experimental materials:

[0230] DMEM medium was purchased from Gibco, and fetal bovine serum was purchased from Hyclone. The Hs746T cell line was purchased from ATCC. An Envision multimode plate reader (PerkinElmer).

[0231] Experimental method:

[0232] Hs746T cells were seeded in a 384-well plate, 50 μL of cell suspension per well, which contained 1500 Hs746T cells. The cell plate was placed in a carbon dioxide incubator and cultured overnight.

[0233] The compound to be tested was serially diluted 9-fold by 3 using a Tecan, and a double-replicate experiment was set up and added to the 384-well cell plate, with the final concentration of the compound ranging from 1000 nM to 0.15 nM. The cell plate was placed in a carbon dioxide incubator and cultured for 4 days.

[0234] Four days later, 25 μL of Promega CellTiter-Glo reagent was added to each well of the cell plate and incubated at room temperature for 10 minutes to stabilize the luminescence signal. Readings were taken using a PerkinElmer Envision multimode plate reader.

[0235] Data analysis:

[0236] The Xlfit software was used to automatically fit the dose-response curves of the compounds and calculate the IC 50 values. The High control was the value of the DMSO-treated wells, and the Low control was the value of the cell-free medium wells. Table 8 provides the inhibitory activities of the compounds of the present invention against the proliferation of Hs746T cells.

[0237] Experimental results: See Table 8:

[0238] Table 8

[0239]

[0240] Conclusion: The hydrochloride salts of the compounds of formula (I) have strong inhibitory activity against c-Met enzyme and strong anti-proliferative activity against EBC-1 cells and Hs746T cells.

[0241] 2.2 Pharmacokinetic studies of single intravenous and oral administrations in mice and dogs

[0242] This experiment was designed to study the pharmacokinetics (PK) of the test compounds in different species after single intravenous and single oral administrations.

[0243] Sample collection and preparation:

[0244] After intravenous injection or oral administration, animal blood samples were collected and the actual blood collection times were recorded. After blood sample collection, they were immediately transferred to labeled centrifuge tubes containing K2-EDTA, and then centrifuged to obtain plasma. The plasma was transferred to pre-cooled centrifuge tubes, snap-frozen in dry ice, and stored in a -70 ± 10 °C ultra-low temperature freezer until LC-MS / MS analysis.

[0245] Pharmacokinetic data analysis:

[0246] Pharmacokinetic software was used to process the plasma drug concentration data of the compounds using a non-compartmental model. The peak concentration (C max ) and peak time (T max ) and the quantifiable end time were directly obtained from the plasma concentration-time curve. 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 rate (Cl), area under the time-plasma concentration curve (AUC 0-last ) from the 0 time point to the end time point, and initial concentration (C0).

[0247] Experimental results:

[0248] See Tables 9 and 10.

[0249] Experimental conclusion:

[0250] The hydrochloride salt of the compound of formula (I) has good oral absorption in mice, with a low clearance rate, a long half-life, and good bioavailability; the compound has good oral absorption in dogs, a long half-life, and high bioavailability.

[0251] Table 9 Pharmacokinetic parameters of the compound of the present invention after single intravenous and oral administrations to mice

[0252]

[0253] Table 10 Pharmacokinetic parameters of the compound of the present invention after single intravenous and oral administrations to dogs

[0254]

[0255] T 1 / 2 : half-life; AUC 0-last : area under the curve.

Claims

1. Polymorph A of the compound of formula (II), the X-ray powder diffraction pattern of which with Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 4.68 ± 0.20°, 12.94 ± 0.20°, 14.12 ± 0.20° and 21.86 ± 0.20°, 2. The polymorph A according to claim 1, the X-ray powder diffraction pattern of which with Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 4.68 ± 0.20°, 12.94 ± 0.20°, 14.12 ± 0.20°, 17.56 ± 0.20°, 21.86 ± 0.20°, 23.54 ± 0.20° and 28.48 ± 0.20°.

3. The polymorph A according to claim 1, the X-ray powder diffraction pattern of which with Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 4.68 ± 0.20°, 12.94 ± 0.20°, 14.12 ± 0.20°, 17.56 ± 0.20°, 17.96 ± 0.20°, 21.86 ± 0.20°, 22.92 ± 0.20°, 23.54 ± 0.20°, 25.28 ± 0.20°, 26.04 ± 0.20°, 26.54 ± 0.20° and 28.48 ± 0.20°.

4. The polymorph A according to claim 1, the X-ray powder diffraction pattern of which with Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 4.681°, 6.100°, 8.962°, 9.381°, 10.865°, 11.364°, 12.941°, 14.119°, 16.940°, 17.559°, 17.961°, 18.398°, 18.939°, 20.606°, 20.959°, 21.861°, 22.379°, 22.918°, 23.541°, 24.083°, 25.281°, 25.817°, 26.042°, 26.541°, 27.639°, 28.480°, 29.481°, 30.521°, 31.039°, 32.816°, 33.261° and 35.181°.

5. The polymorph A according to claim 1, the XRPD pattern of which is shown in Figure 1.

6. The polymorph A according to any one of claims 1 to 5, the differential scanning calorimetry curve of which has an endothermic peak at 264.9 °C ± 3 °C.

7. The polymorph A according to claim 6, the DSC pattern of which is shown in Figure 2.

8. Polymorph B of the compound of formula (II), the X-ray powder diffraction pattern of which with Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 4.80 ± 0.20°, 14.28 ± 0.20°, 20.22 ± 0.20° and 24.89 ± 0.20°, 9. The crystalline form B according to claim 8 has characteristic diffraction peaks in the X-ray powder diffraction pattern of Cu Kα radiation at the following 2θ angles: 4.80 ± 0.20°, 13.68 ± 0.20°, 14.28 ± 0.20°, 19.68 ± 0.20°, 20.22 ± 0.20°, 22.20 ± 0.20°, 24.89 ± 0.20°, and 28.76 ± 0.20°.

10. The crystalline form B according to claim 8 has characteristic diffraction peaks in the X-ray powder diffraction pattern of Cu Kα radiation at the following 2θ angles: 4.80 ± 0.20°, 13.68 ± 0.20°, 14.28 ± 0.20°, 18.02 ± 0.20°, 19.68 ± 0.20°, 20.22 ± 0.20°, 22.20 ± 0.20°, 23.56 ± 0.20°, 24.89 ± 0.20°, 27.50 ± 0.20°, 28.04 ± 0.20°, and 28.76 ± 0.20°.

11. The crystalline form B according to claim 8 has characteristic diffraction peaks in the X-ray powder diffraction pattern of Cu Kα radiation at the following 2θ angles: 4.797°, 8.262°, 9.520°, 11.661°, 13.680°, 14.279°, 16.520°, 17.323°, 18.017°, 18.521°, 19.679°, 20.221°, 21.539°, 22.199°, 22.822°, 23.562°, 24.157°, 24.889°, 25.444°, 26.159°, 27.501°, 28.038°, 28.760°, 29.719°, 31.360°, 31.979°, 32.459°, 33.540°, 34.422°, 34.839°, 35.501°, and 36.382°.

12. The XRPD pattern of the crystalline form B according to claim 8 is as shown in Figure 4.

13. The differential scanning calorimetry curve of the crystalline form B according to any one of claims 8 to 12 has endothermic peaks at 257.7°C ± 3°C and 268.9°C ± 3°C.

14. The DSC pattern of the crystalline form B according to claim 13 is as shown in Figure 5.

15. The crystalline form C of the compound of formula (III) has characteristic diffraction peaks in the X-ray powder diffraction pattern of Cu Kα radiation at the following 2θ angles: 3.82 ± 0.20°, 15.30 ± 0.20°, 16.38 ± 0.20°, 16.82 ± 0.20°, 20.02 ± 0.20°, 22.84 ± 0.20°, 23.72 ± 0.20°, and 28.44 ± 0.20°; 16. The crystalline form C according to claim 15 has characteristic diffraction peaks in the X-ray powder diffraction pattern of Cu Kα radiation at the following 2θ angles: 3.82 ± 0.20°, 10.72 ± 0.20°, 14.24 ± 0.20°, 15.30 ± 0.20°, 16.38 ± 0.20°, 16.82 ± 0.20°, 20.02 ± 0.20°, 20.84 ± 0.20°, 22.84 ± 0.20°, 23.72 ± 0.20°, 26.90 ± 0.20° and 28.44 ± 0.20°.

17. The crystalline form C according to claim 15, wherein the X-ray powder diffraction pattern with Cu Kα radiation has characteristic diffraction peaks at the following 2θ angles: 3.819°, 7.619°, 10.720°, 11.401°, 13.015°, 13.840°, 14.240°, 15.300°, 16.379°, 16.818°, 17.401°, 18.602°, 19.198°, 20.020°, 20.841°, 22.583°, 22.841°, 23.720°, 24.191°, 25.259°, 25.679°, 26.899°, 27.341°, 28.441°, 29.580°, 30.221°, 30.802°, 31.297°, 32.262°, 33.225°, 34.423°, 35.160°, 36.936°, 38.241° and 38.980°.

18. The crystalline form C according to claim 15, wherein its XRPD pattern is as shown in Figure 7.

Images