A crystalline form of a pyrrolidinyl urea derivative and uses thereof

Abstract

The application discloses a crystal form of a pyrrolidinyl urea derivative and application thereof. Specifically disclosed are an A crystal form and an F crystal form of a compound of formula (I) and application of the crystal forms in preparation of drugs for treating diseases related to pain, cancer, inflammation, neurodegenerative diseases and infectious diseases.

Description

[0001] This application is a divisional application of patent application no. 2021800088985, the filing date of which is 8 January 2021, and its title is “Crystal form of pyrrolidinyl urea derivative and its application”. TECHNICAL FIELD

[0002] The present application relates to a crystal form of a TrkA inhibitor and its use in the preparation of a medicament for treating diseases associated with pain, cancer, inflammation, neurodegenerative diseases and infectious diseases. BACKGROUND

[0003] Trk-associated receptor kinases (Trk) are high affinity receptor tyrosine kinases activated by a group of soluble growth factors known as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins (NT). The family consists of three members (TrkA, TrkB, TrkC). NGF, BDNF, NT-4 / 5 play an important role in many physiological regulatory processes such as signal maintenance of neuronal cells, signal transmission of neuronal cells, cell proliferation, cell differentiation, cell survival, etc. through the receptor Trk. There is much evidence that inhibitors of NGF / Trk signaling pathway are effective in many preclinical models of pain; also, inhibitors of NGF / Trk signaling pathway are effective in many preclinical models of inflammatory diseases. In addition, overexpression, activation, amplification and / or mutation of Trk kinases are associated with many tumors or cancers. Therefore, Trk has become an important therapeutic target, attracting extensive research interest. The TrkA inhibitor described in the present application can solve the treatment needs of pain, cancer, inflammation, neurodegenerative diseases and infectious diseases.

[0004] WO2015175788 patent reports a single compound having inhibitory activity on TrkA and its pharmaceutically acceptable salt. WO2012158413, WO2016116900, WO2016021629, WO2017006953 patents report a series of compounds having inhibitory activity on TrkA, including the pyrrolidinyl urea structure used in the present application. SUMMARY

[0005] The present application provides a crystal form A of the compound of formula (I), characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 9.34±0.20°, 13.40±0.20°, 14.57±0.20°, 15.59±0.20°, 16.95±0.20°, 18.71±0.20°, 19.51±0.20°, 24.07±0.20°.

[0006]

[0007] In some embodiments of the application, the X-ray powder diffraction pattern of the Form A is characterized by peaks at the following 2-theta angles: 6.06°, 8.30°, 9.05°, 9.34°, 10.52°, 11.86°, 12.34°, 13.40°, 14.25°, 14.57°, 15.30°, 15.59°, 16.95°, 17.74°, 18.45°, 18.71°, 19.51°, 19.88°, 20.33°, 21.03°, 21.60°, 22.61°, 23.64°, 24.07°, 24.53°, 25.37°, 26.41°, 27.05°, 27.74°, 28.10°, 30.48°, 34.72°, 36.84°, 37.60°.

[0008] In some embodiments of the application, the Form A has an XRPD pattern as shown in Figure 1 .

[0009] In some embodiments of the application, the Form A has an XRPD pattern as shown in

[0010] Table 1. XRPD pattern analysis data of Form A

[0011]

[0012]

[0013] In some embodiments of the application, the Form A has a DSC curve with an endothermic peak at 75.3±3.0°C, 99.6±3.0°C and 167.9±3.0°C, respectively, and an exothermic peak at 132.3±3.0°C.

[0014] In some embodiments of the application, the Form A has a DSC curve as shown in Figure 2 .

[0015] In some embodiments of the application, the Form A has a TGA curve with a weight loss of 1.75% at 55.0±3.0°C, and a weight loss of 3.08% at 100.0±3.0°C.

[0016] In some embodiments of the application, the Form A has a TGA curve as shown in Figure 3 .

[0017] The present application also provides a crystal form F of the compound of formula (I), which is characterized by an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 6.30±0.20°, 9.25±0.20°, 13.62±0.20°, 15.80±0.20°, 17.16±0.20°, 17.96±0.20°, 18.92±0.20°, 20.09±0.20°;

[0018]

[0019] In some embodiments of the present application, the X-ray powder diffraction pattern of the above-mentioned crystal form F has characteristic diffractions at the following 2θ angles: 6.30°, 8.48°, 9.25°, 9.71°, 12.57°, 13.62°, 14.46°, 15.80°, 17.16°, 17.96°, 18.67°, 18.92°, 19.69°, 20.09°, 21.26°, 22.15°, 23.68°, 24.29°, 25.58°, 26.64°, 27.32°, 27.95°, 28.28°, 30.71°, 35.16°.

[0020] In some embodiments of the present application, the above-mentioned crystal form F has an XRPD pattern as shown in Figure 4 .

[0021] In some embodiments of the present application, the XRPD pattern of the above-mentioned crystal form F has the data analysis as shown in Table 2:

[0022] Table 2. Data analysis of XRPD pattern of crystal form F

[0023]

[0024] In some embodiments of the present application, the above-mentioned crystal form F has a differential scanning calorimetry curve having an endothermic peak with a peak value at 100.0±3.0℃ and 172.7±3.0℃, and an exothermic peak with a peak value at 126.0±3.0℃, respectively.

[0025] In some embodiments of the present application, the DSC pattern of the above-mentioned crystal form F is as shown in Figure 5 .

[0026] In some embodiments of the present application, the above-mentioned crystal form F has a thermogravimetric analysis curve with a weight loss of 3.92% at 130.0℃±3.0℃.

[0027] In some embodiments of the present application, the TGA pattern of the above-mentioned crystal form F is as shown in Figure 6 .

[0028] The present application also provides the use of the above-mentioned A crystal form or F crystal form in the preparation of a medicament for treating diseases related to pain, cancer, inflammation, neurodegenerative diseases and infectious diseases.

[0029] Technical effects

[0030] The compound of the present application has a broad prospect of each crystal form of the compound, the compound of formula (I) has a significant TrkA enzyme inhibition effect, a higher plasma protein unbinding rate, a lower drug-drug interaction risk, and a better liver microsomal metabolic stability.

[0031] Definitions and explanations

[0032] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be construed to be indefinite or unclear unless specifically defined, but should be interpreted according to its ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding product or active ingredient thereof.

[0033] The intermediate compounds of the present application can be prepared by various synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by the combination of the specific embodiments with other chemical synthetic methods well known to those skilled in the art, and equivalent replacement methods well known to those skilled in the art, and preferred embodiments include but are not limited to the examples of the present application.

[0034] The chemical reactions of the specific embodiments of the present application are carried out in suitable solvents, which are chosen based on the nature of the reactants and reagents and the transformation desired. In order to obtain the compounds of the present application, it is sometimes necessary for those skilled in the art to modify or select the synthetic steps or reaction sequences based on the existing embodiments.

[0035] The compounds of the present application can be confirmed by conventional methods well known to those skilled in the art, and if the present application relates to 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 single crystal grown is collected by a Bruker D8 venture diffractometer to collect diffraction intensity data, the light source is Cu Kα radiation, the scanning mode is: After scanning and collecting relevant data, the crystal structure is further analyzed by the direct method (Shelxs97), and the absolute configuration can be confirmed.

[0036] The present application will be described in detail below through examples, which do not mean any limitation on the present application.

[0037] All solvents used in the present application are commercially available and can be used without further purification.

[0038] The solvents used in the present application are commercially available. The following abbreviations are used in the present application: EtOH stands for ethanol; MeOH stands for methanol; TFA stands for trifluoroacetic acid; TsOH stands for p-toluenesulfonic acid; mp stands for melting point; EtSO3H stands for ethanesulfonic acid; MeSO3H stands for methanesulfonic acid; THF stands for tetrahydrofuran; EtOAc stands for ethyl acetate.

[0039] The compounds are named according to the principles of nomenclature in the art or using software nomenclature, and commercially available compounds are named using the supplier's catalog name.

[0040] The X-ray powder diffractometer (XRPD) method of the present application

[0041] Instrument model: X'Pert 3 X-ray diffractometer from PANalytical

[0042] Test method: About 10 mg of sample was used for XRPD measurement.

[0043] Detailed XRPD parameters are as follows:

[0044] X-ray source: Cu, kα Kα2 / Kα1 intensity ratio: 0.5

[0045] Tube voltage: 45 kV, tube current: 40 mA

[0046] Divergence slit: fixed 1 / 8 deg

[0047] First soller slit: 0.04 rad, second soller slit: 0.04 rad

[0048] Receiving slit: none, anti-scatter slit: 7.5 mm

[0049] Measurement time: 5 min

[0050] Scan angle range: 3-40 deg

[0051] Step width angle: 0.0263 deg

[0052] Step size: 46.665 s

[0053] Sample holder rotation: 15 rpm

[0054] The X-ray powder diffractometer (XRPD) method of the present application

[0055] Diffractometer system: PNalytical XPERT-PRO ​

[0056] Detailed XRPD parameters are as follows:

[0057] Source: Cu, kα (1.5418 A) Kα2 / Kα1 intensity ratio: 0.5)

[0058] Tube voltage: 40 kV, tube current: 40 mA

[0059] Divergence slit: fixed 0.2177 deg

[0060] Scan type: continuous

[0061] Scan angle range: 3.0131-59.9791 deg

[0062] Step width angle: 0.0263 deg

[0063] Step size: 0.0260

[0064] Scan step time: 14.0927 seconds

[0065] Differential Scanning Calorimeter (DSC) method of the present invention

[0066] Instrument model: TA Q2000 / Discovery 2500 Differential Scanning Calorimeter

[0067] Test method: The sample (about 1-5 mg) was placed in a DSC aluminum pan for testing, under the condition of 50 mL / min nitrogen, the sample was heated at a rate of 10°C / min from 25°C (room temperature) to before sample decomposition.

[0068] Thermal Gravimetric Analyzer (TGA) method of the present invention

[0069] Instrument model: TA Q5000 / Discovery 5500 Thermal Gravimetric Analyzer

[0070] Test method: The sample (about 1-5 mg) was placed in a TGA aluminum pan for testing, under the condition of 10 mL / min nitrogen, the sample was heated at a rate of 10°C / min from room temperature to 350°C.

[0071] Dynamic Vapor Sorption (DVS) method of the present invention

[0072] Instrument model: Intrinsic Dynamic Vapor Sorption Analyzer

[0073] Test conditions: Sample (10-30 mg) was placed in a DVS sample pan and tested.

[0074] Detailed DVS parameters are as follows:

[0075] Temperature: 25 °C

[0076] Equilibration: dm / dt = 0.002% / min (minimum: 10 min, maximum: 180 min)

[0077] RH (%) test steps: 10 (0-90%), 5 (90-95%)

[0078] RH (%) test step range: 70-95 - 0 - 95

[0079] Hygroscopicity evaluation classification is as follows:

[0080] Hygroscopicity classification ΔW% Deliquescent Absorbs sufficient moisture to form a liquid Very hygroscopic ΔW% > 15% Hygroscopic 15% > ΔW% > 2% Slightly hygroscopic 2% > ΔW% > 0.2% Non- or almost non-hygroscopic ΔW% < 0.2%

[0081] Note: ΔW% represents the weight gain of the test product under 25 ± 1 °C and 80 ± 2% RH.

[0082] High Performance Liquid Chromatograph (HPLC) method of the present application

[0083] Detailed parameters are as follows:

[0084] BRIEF DESCRIPTION OF DRAWINGS

[0085] Figure 1 XRPD pattern of Compound A Form of Formula (I) in Cu-Ka radiation.

[0086] Figure 2 DSC pattern of Compound A Form of Formula (I).

[0087] Figure 3 TGA pattern of Compound A Form of Formula (I).

[0088] Figure 4 XRPD pattern of Compound F Form of Formula (I) in Cu-Ka radiation.

[0089] Figure 5 DSC pattern of Compound F Form of Formula (I).

[0090] Figure 6 TGA pattern of Compound F Form of Formula (I). DETAILED DESCRIPTION

[0091] For a better understanding of the present application, further description will be made with specific examples below, but the specific examples are not intended to limit the content of the present application.

[0092] Example 1: Preparation of compound of formula (I)

[0093]

[0094] Step 1: Synthesis of compound 2

[0095] Compound 1 (15 g, 60.00 mmol) and trimethylsilylethylene (12.03 g, 120.01 mmol) were dissolved in acetonitrile (150 mL), activated copper powder (190.65 mg, 3.00 mmol) was added, the reaction solution was heated to 65 °C and stirred for 15 hours. After cooling, the organic solvent was removed under reduced pressure, and the obtained crude product was separated and purified by silica gel column chromatography (eluent: 0-5% ethyl acetate / petroleum ether) to obtain compound 2. 1 H NMR (400 MHz, CDCl3): 4.18 (q, J = 7.2 Hz, 2H), 2.94-2.90 (m, 1H), 2.52-2.37 (m, 2H), 1.20 (t, J = 7.2 Hz, 3H), 0.00 (s, 9H).

[0096] Step 2: Synthesis of compound 3

[0097] Compound 2 (5 g, 14.28 mmol) was dissolved in anhydrous tetrahydrofuran (100 mL) at -30 °C, diisobutylaluminum hydride (1 M, 28.55 mL) was slowly added dropwise, and the reaction solution was slowly warmed to 20 °C and stirred for 2 hours. 60 mL of 0.5 N aqueous hydrochloric acid was added to the reaction solution, extracted with ethyl acetate (100 mL*2), the combined organic phase was washed with 200 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed under reduced pressure to obtain crude compound 3, which was used directly in the next step without further purification.

[0098] Step 3: Synthesis of compound 5

[0099] Compound 4 (8.7 g, 45.02 mmol) was dissolved in dichloromethane (60 mL), triethylamine (13.67 g, 135.06 mmol) was added, and methane sulfonyl chloride (11.35 g, 99.05 mmol) was slowly added dropwise. The reaction solution was slowly warmed to 25 °C and stirred for 3 hours. 80 mL of water was added to the reaction solution, and dichloromethane (80 mL*2) was extracted. The combined organic phase was washed with 150 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed under reduced pressure. The obtained crude product was separated and purified by silica gel column chromatography (eluent: 20-50% ethyl acetate / petroleum ether) to obtain compound 5. 1 H NMR (400 MHz, CDCl3): 7.43-7.28 (m, 5H), 3.90 (t, J = 4.4 Hz, 2H), 3.75-3.61 (m, 2H), 3.02 (dd, J = 6.4, 10.0 Hz, 2H), 2.70-2.58 (m, 2H).

[0100] Step 4: Synthesis of compound 6

[0101] Compound 5 (15 g, 42.93 mmol) was dissolved in N,N-dimethylformamide (100 mL), and sodium azide (8.37 g, 128.78 mmol) was added. The reaction solution was warmed to 100 °C and stirred for 16 hours. After cooling, 200 mL of water was added to the reaction solution, and ethyl acetate (200 mL*3) was extracted. The combined organic phase was washed with water (300 mL*2) and saturated brine (300 mL) in sequence, dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed under reduced pressure. The obtained crude product was separated and purified by silica gel column chromatography (eluent: 0-2% ethyl acetate / petroleum ether) to obtain compound 6. 1 H NMR (400 MHz, CDCl3): 7.43-7.28 (m, 5H), 3.90 (t, J = 4.4 Hz, 2H), 3.75-3.61 (m, 2H), 3.02 (dd, J = 6.4, 10.0 Hz, 2H), 2.70-2.58 (m, 2H).

[0102] Step 5: Synthesis of compound 7

[0103] Compound 6 (7 g, 28.77 mmol) was dissolved in tetrahydrofuran (60 mL), water (1.04 g, 57.55 mmol) was added, triphenylphosphine (6.79 g, 25.90 mmol) was added slowly in batches, the reaction was stirred at 25 °C until no gas was released, and then the temperature was raised to 80 °C and the stirring was continued for 1 h. After cooling, the organic solvent was removed under reduced pressure, 80 mL of 4N hydrochloric acid aqueous solution was added to the obtained crude product, extracted with 80 mL of dichloromethane, the aqueous phase was adjusted to pH about 10 with ammonia water, extracted with dichloromethane (80 mL*2), the combined organic phase was washed with 100 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed under reduced pressure to obtain the crude compound 7, which was used directly in the next step without further purification. 1 H NMR (400 MHz, CDC13): 7.35-7.24 (m, 5H), 3.64 (q, J = 13.2 Hz, 2H), 3.56 (td, J = 3.6, 6.8 Hz, 1H), 3.48-3.40 (m, 1H), 3.07-2.90 (m, 2H), 2.64 (dd, J = 4.4, 10.4 Hz, 1H), 2.31 (dd, J = 5.2, 9.6 Hz, 1H).

[0104] Step 6: Synthesis of compound 8

[0105] Compound 7 (6.4 g, 29.46 mmol) was dissolved in dichloromethane (60 mL), triethylamine (5.96 g, 58.91 mmol) and di-tert-butyl dicarbonate (7.71 g, 35.35 mmol) were added, and the reaction was continued to stir at 25 °C for 15 h. The organic solvent was removed under reduced pressure, and the obtained crude product was separated and purified by silica gel column chromatography (eluent: 0-10% ethyl acetate / petroleum ether) to obtain compound 8. 1 H NMR (400 MHz, CDC13): 7.40-7.25 (m, 5H), 4.87 (s, 1H), 4.07 (s, 1H), 3.81 (s, 1H), 3.70-3.56 (m, 2H), 3.07 (dd, J = 6.8, 10.4 Hz, 1H), 2.93-2.77 (m, 1H), 2.56-2.32 (m, 2H), 1.47 (s, 9H).

[0106] Step 7: Synthesis of compound 9

[0107] Compound 8 (8.8 g, 27.73 mmol) was dissolved in methanol (100 mL), palladium on carbon (0.5 g, 27.73 mmol, 10% purity) was added, and the reaction was stirred at 20 °C under hydrogen pressure of 15 psi for 3 h. Filtration through celite and removal of organic solvent under reduced pressure gave crude compound 9, which was used directly for the next step without further purification. 1 H NMR (400 MHz, CDC13): 7.33-7.25 (m, 5H), 5.04 (d, J = 6.4 Hz, 1H), 3.70 (s, 1H), 3.63-3.54 (m, 2H), 3.34-3.23 (m, 1H), 3.07 (t, J = 8.4 Hz, 1H), 2.82 (dd, J = 7.2, 9.6 Hz, 1H), 2.51-2.43 (m, 1H), 2.21-2.09 (m, 1H), 1.44 (s, 9H).

[0108] Step 8: Synthesis of compound 10

[0109] Compound 9 (2.35 g, 8.06 mmol) was dissolved in acetonitrile (50 mL), compound 3 (1.98 g, 6.45 mmol) was added, and the reaction was stirred at 50 °C for 15 h. After cooling, the organic solvent was removed under reduced pressure, and the resulting crude product was purified by column chromatography on silica gel (eluent: 0-30% ethyl acetate / petroleum ether) to give compound 10. 1 H NMR (400 MHz, CDC13): 7.33-7.25 (m, 5H), 5.04 (d, J = 6.4 Hz, 1H), 3.70 (s, 1H), 3.63-3.54 (m, 2H), 3.34-3.23 (m, 1H), 3.07 (t, J = 8.4 Hz, 1H), 2.82 (dd, J = 7.2, 9.6 Hz, 1H), 2.51-2.43 (m, 1H), 2.21-2.09 (m, 1H), 1.44 (s, 9H).

[0110] Step 9: Synthesis of compound 11

[0111] Compound 10 (840 mg, 2.34 mmol) was dissolved in toluene (30 mL), diisopropylethylamine (422.86 mg, 3.27 mmol) was added, a-chloroformic acid-1- chloroethyl ester (434.35 mg, 3.04 mmol) was added dropwise slowly under ice water bath condition, the reaction was warmed to 90 °C and stirred for 1 h. It was cooled, the organic solvent was removed under reduced pressure, methanol (30 mL) was added and stirred at 20 °C for 17 h. The organic solvent was removed under reduced pressure to give crude compound 11 which was used directly in the next step without further purification. MS m / z = 270.1 [M+1] + .

[0112] Step 10: Synthesis of compound 12

[0113] Compound 11 (630 mg, 2.34 mmol) was dissolved in N,N-dimethylformamide (10 mL), diisopropylethylamine (906.98 mg, 7.02 mmol) and 2-bromoethyl methyl ether (536.92 mg, 3.51 mmol) were added, the reaction was stirred at 20 °C for 64 h. The reaction was diluted with 100 mL ethyl acetate, washed with 60 mL water and 60 mL saturated brine successively, dried over anhydrous sodium sulfate, filtered and the organic solvent was removed under reduced pressure to give crude product which was separated and purified by silica gel column chromatography (eluent: 25%-60% ethyl acetate / petroleum ether) to give compound 12. 1 H NMR (400 MHz, CDC13): 6.60 (s, 1H), 6.54 (s, 1H), 5.88 (dd, J = 2.0, 2.8 Hz, 1H), 4.91 (s, 1H), 4.24 (s, 1H), 4.12-4.05 (m, 1H), 3.51 (t, J = 5.6 Hz, 2H), 3.37 (s, 3H), 3.19 (s, 1H), 3.08 (d, J = 8.0 Hz, 1H), 2.84-2.64 (m, 3H), 1.43 (s, 9H).

[0114] Step 11: Synthesis of compound 13

[0115] Compound 12 (100 mg, 305.44 μmol) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (2 mL) was added, the reaction was stirred at 20 °C for 0.5 h. The organic solvent was removed under reduced pressure to give crude compound 13 which was used directly in the next step without further purification. MS m / z = 228.1 [M+1] + .

[0116] Step 12: Synthesis of compound 15

[0117] Compound 14 (4.0 g, 14.04 mmol) was dissolved in anhydrous tetrahydrofuran (40 mL), cooled to -78 °C, oxetane (1.2 g, 16.85 mmol) was added, and a solution of n-butyllithium (2.5 M, 8.4 mL) was added dropwise slowly. The reaction was stirred at this temperature for 20 minutes. Saturated aqueous ammonium chloride solution (20 mL) was added slowly to the reaction, and ethyl acetate (50 mL*3) was used for extraction. The combined organic phase was washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed under reduced pressure. The obtained crude product was separated and purified by silica gel column chromatography (eluent: 0-20% ethyl acetate / petroleum ether) to obtain compound 15. 1 HNMR: (400 MHz, CDC13): 8.89 (s, 2H), 5.06-4.95 (m, 4H).

[0118] Step 13: Synthesis of compound 16

[0119] Compound 15 (1.8 g, 7.75 mmol) was dissolved in dichloromethane (13 mL) under an ice water bath, and a solution of diethylamine trifluoride (2.5 g, 15.50 mmol) in dichloromethane (4 mL) was added. The reaction was stirred at this temperature for 20 minutes. Water (20 mL) was added to the reaction, and ethyl acetate (50 mL*3) was used for extraction. The combined organic phase was washed with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed under reduced pressure. The obtained crude product was separated and purified by silica gel column chromatography (eluent: 0-10% ethyl acetate / petroleum ether) to obtain compound 16. 1 H NMR (400 MHz, CDC13): 8.91 (s, 2H), 5.20-5.05 (m, 4H).

[0120] Step 14: Synthesis of compound 17

[0121] Compound 16 (300 mg, 1.29 mmol) was dissolved in 1,4-dioxane (8.0 mL), and pinacol diboronic acid (392 mg, 1.54 mmol) and potassium acetate (379 mg, 3.86 mmol) were added sequentially. The reaction was replaced with nitrogen three times, and then 1,1'-bis(diphenylphosphino)ferrocene palladium dichloride (94 mg, 128.73 μmol) was added. The reaction was heated to 100 °C and stirred for 11 hours. After cooling, the organic solvent was removed under reduced pressure, and the obtained crude product was separated and purified by silica gel column chromatography (eluent: 0-50% ethyl acetate / petroleum ether) to obtain compound 17. 1 H NMR (400 MHz, CDC13): 9.11 (s, 2H), 5.24-5.05 (m, 4H), 1.37 (s, 12H).

[0122] Step 15: Synthesis of compound 19

[0123] Compound 18 (20.00 g, 184.95 mmol) and ethyl 2-cyanopropionate (23.51 g, 184.95 mmol) were dissolved in 1,4-dioxane (40 mL), the reaction was heated to 110 °C and stirred for 72 h. After cooling, the reaction was concentrated to about 20 mL, solid was precipitated, filtered, the filter cake was washed with ethyl acetate (30 mL), and the filter cake was collected to give compound 19. 1 H NMR (400 MHz, CD3OD): 7.53-7.46 (m, 2H), 7.42-7.35 (m, 3H), 1.77 (s, 3H).

[0124] Step 16: Synthesis of compound 20

[0125] Compound 19 (10.00 g, 52.85 mmol) was dissolved in N,N-dimethylformamide (150 mL), followed by the addition of N,N-diisopropylethylamine (20.49 g, 158.55 mmol) and N-phenyl bis(trifluoromethanesulfonyl)imide (19.82 g, 55.49 mmol) in sequence, and the reaction was stirred at 25 °C for 16 h. The reaction was poured into 500 mL of water, followed by extraction with ethyl acetate (150 mL*3), the organic phases were combined, washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness to give crude compound 20. 1 H NMR (400 MHz, CDCl3): 7.54-7.44 (m, 4H), 7.40-7.34 (m, 1H), 3.76 (s, 2H), 1.95 (s, 3H).

[0126] Step 17: Synthesis of compound 21

[0127] Compound 20 (320 mg, 1.14 mmol) was dissolved in a mixture of dioxane (2.5 mL) and water (0.5 mL), compound 17 (293 mg, 912.00 umol) and 1,1’- bis(diphenylphosphino)ferrocene palladium dichloride (83 mg, 114.00 umol) were added, followed by sodium carbonate (242 mg, 2.28 mmol), and the reaction was replaced with nitrogen three times, and then heated to 100 °C and stirred for 14 h. The reaction was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated and purified by column chromatography (eluent: 0-25% ethyl acetate / petroleum ether) to give compound 21. 1H NMR (400 MHz, CDC13): 9.11 (s, 2H), 7.64-7.27 (m, 5H), 5.32-4.88 (m, 4H), 3.67 (s, 2H), 2.10 (s, 3H).

[0128] Step 18: synthesis of compound of formula (I)

[0129] Compound 21 (60 mg, 184.42 umol) was dissolved in dichloromethane (5 mL), and added with triphosgene (43.78 mg, 147.54 umol) and N,N-diisopropylethylamine (71.50 mg, 553.27 umol, 96.37 uL). The reaction was stirred at 20 °C for 20 min, and added with compound 13 (139.72 mg, 184.42 umol) and N,N-diisopropylethylamine (71.50 mg, 553.27 umol, 96.37 uL). The reaction was continued to stir at 20 °C for 15 h. The organic solvent was removed under reduced pressure, and the obtained crude product was separated and purified by high performance liquid chromatography (column: Xtimate C18 150*25mm*5um; mobile phase: [water (10 mM ammonium bicarbonate) - acetonitrile]; acetonitrile%: 37% - 58%, 10.5 min) to obtain the compound of formula (I). 1 H NMR (400 MHz, CDC13): 9.11 (s, 2H), 7.64-7.27 (m, 5H), 5.32-4.88 (m, 4H), 3.67 (s, 2H), 2.10 (s, 3H). + .

[0130] Example 2: preparation of crystal form A of compound of formula (I)

[0131] Take 50 mg of compound of formula (I) into a 4.0 mL glass vial, and add 1 mL of water to make a suspension. After adding a magnetic stirrer, the suspension sample was placed on a magnetic heating stirrer (50 °C) for testing. After stirring at 50 °C for 48 h, centrifugation was performed, and the residue was placed in a vacuum drying oven (60 °C) for drying overnight to obtain crystal form A of compound of formula (I).

[0132] Take 50 mg of the compound of formula (I) into a 4.0 mL glass vial, add 1 mL of a mixed solvent of acetone and water (1:4) to make a suspension. After adding a magnetic stirrer, the sample is placed on a magnetic heating stirrer (20°C) for testing. After stirring for 96 hours at 20°C, centrifugation is performed, and the residue is placed in a vacuum drying oven (60°C) for drying overnight to obtain the crystal form A of the compound of formula (I).

[0133] Example 3: Preparation of the crystal form F of the compound of formula (I)

[0134] Take 17 g of the compound of formula (I) into a 1 L glass bottle, add 200 mL of a mixed solvent of ethanol and water (1:4) to make a suspension. The sample is placed on a magnetic heating stirrer (50°C) for testing. After stirring for 24 hours at 50°C, filtration is performed. The filter cake is collected, and the sample is subjected to the above-mentioned operation for 5 times and then placed in a vacuum drying oven (60°C) for drying overnight to obtain the crystal form F of the compound of formula (I).

[0135] Experimental Example 1: Test of TrkA enzyme activity

[0136] Experimental materials

[0137]

[0138] Kinase reaction buffer

[0139] 50 mM Hepes (pH 7.5), 5 mM MgCl2(magnesium chloride), 0.01 mM Orthovanadate (sodium vanadate), 1% BSA (bovine serum albumin), 1 mM (dithiothreitol)

[0140] Experimental method

[0141] The test uses the homogeneous time-resolved fluorescence conjugated energy transfer (HTRF) technology of Cisbio Company to detect the activity. In the detection plate, the enzyme, biotin-labeled polypeptide substrate, ATP and detection compound are mixed, and the reaction is incubated. After the reaction, ethylenediamine tetraacetic acid is added to terminate the reaction, and at the same time, Eu-labeled antibody and streptavidin-labeled XL665 are added to react and detect. The data are represented by the readings of fluorescence signals at 665 nm and 620 nm, respectively, wherein a high ratio of 665 nm / 620 nm indicates high activity, and a low ratio of 665 nm / 620 nm indicates inhibited activity. Experimental procedure

[0142] 1. Compound dilution: 3-fold dilution of the test compound, a total of 11 concentrations, the final system concentration from 10 μM to 0.17 nM;

[0143]

[0144] 1. Compound dilution: 3-fold dilution of the test compound, a total of 11 concentrations, the final system concentration from 10 μM to 0.17 nM;2. In a 10 μL reaction system with buffer of 50 mM Hepes (pH 7.5), 5 mM MgCl2, 0.01 mM sodium vanadate, 1% BSA, 1 mM DTT, 0.5 nM TrkA kinase, 0.3 μM biotin-TK peptide (biotin-labeled tyrosine kinase substrate polypeptide), 90 μM ATP, incubate at 23 °C for 90 minutes. Add 10 μL stop solution containing 20 mM EDTA, 1.34 nM phosphorylated substrate antibody, 100 nM streptavidin-labeled fluorescent molecule XL-665, incubate at 23 °C for 60 minutes, and read with multifunctional microplate reader Envision.

[0145] 3. Calculate the inhibition rate of the compound from the data read by the instrument, and then calculate the IC 50 value by using mode 205 in XLFIT5 of IDBS.

[0146] Experimental results

[0147] The results are shown in Table 3.

[0148] Table 3 IC 50 values of the compound of formula (I) for inhibiting TrkA enzyme

[0149] Compound No. TrkA IC 50 (nM) Compound of formula (I) 0.56

[0150] The results show that the compound of formula (I) has significant inhibitory effect on TrkA enzyme.

[0151] Experimental Example 2: Plasma protein binding rate (PPB) test

[0152] Purpose of the experiment

[0153] Determine the protein binding rate of the test compound in human and SD rat plasma.

[0154] Experimental operation

[0155] Human and SD rat blank plasma 796 μL (plasma purchased from Bioreclamation IVT) were added with 4 μL of test compound working solution (400 μM) or warfarin working solution (400 μM) to make the final concentration of test compound and warfarin in plasma samples both 2 μM. The samples were mixed well. The final concentration of organic phase DMSO was 0.5%; 50 μL of test compound and warfarin plasma samples were pipetted into sample receiving plates, and immediately added with corresponding volume of corresponding blank plasma or buffer to make the final volume of each sample well 100 μL, and the volume ratio of plasma:dialysis buffer was 1:1, then 400 μL of stop solution was added into these samples, which will be used as T0 samples for recovery and stability determination. The T0 samples were stored at 2-8 °C, and waited for subsequent processing together with other dialyzed samples; 150 μL of test compound and warfarin plasma samples were added into the dosing end of each dialysis well, and 150 μL of blank dialysis buffer was added into the corresponding receiving end of dialysis well. Then the dialysis plate was sealed with a gas-permeable membrane and placed in a humidified 5% CO2incubator at 37 °C with 100 rpm shaking for 4 hours. After dialysis, 50 μL of dialyzed buffer samples and dialyzed plasma samples were pipetted into new sample receiving plates. Corresponding volume of corresponding blank plasma or buffer was added into the samples to make the final volume of each sample well 100 μL, and the volume ratio of plasma:dialysis buffer was 1:1. All samples were subjected to protein precipitation and LC / MS / MS analysis, and the plasma protein unbound rate, binding rate and recovery rate were calculated by the formula: %unbound rate = 100*membrane buffer side free compound concentration / membrane plasma side total compound concentration, %protein binding rate = 100-%unbound rate, %recovery rate = 100*(membrane buffer side free compound concentration + membrane plasma side total compound concentration) / total compound measured concentration before dialysis.

[0156] Experimental results

[0157] The results are shown in Table 4.

[0158] Table 4 Human and rat plasma protein unbound rate of compound of formula (I)

[0159]

[0160] The results show that the compound of formula (I) has a high plasma protein unbound rate.

[0161] Experimental Example 3: Test of cytochrome P450 isozyme inhibitory activity

[0162] Purpose of experiment

[0163] Determine the inhibitory activity of test compound on different subtypes of human cytochrome P450 isozyme.

[0164] Experimental operation

[0165] Prepare test compound, standard inhibitor (100x final concentration) and mixed substrate working solutions; take out microsomes from -80°C freezer and thaw. Add 2 μL of test compound and standard inhibitor solutions to the corresponding wells, while add 2 μL of corresponding solvent to the no inhibitor control (NIC) and blank control (Blank) wells; next add 20 μL of mixed substrate solution to the corresponding wells, except for the Blank wells (add 20 μL of PB to the Blank wells); prepare the human liver microsomes solution (put back to the freezer immediately after use) and then add 158 μL of human liver microsomes solution to all wells; put the sample plate into 37°C water bath for pre-incubation, and then prepare the co-factor (NADPH) solution; after 10 minutes, add 20 μL of NADPH solution to all wells, mix the sample plate and then put it into 37°C water bath for incubation for 10 minutes; at the corresponding time point, add 400 μL of cold acetonitrile solution (internal standard is 200 ng / mL tolbutamide and labetalol) to stop the reaction; mix the sample plate well and then centrifuge at 4000 rpm for 20 minutes to precipitate the protein; take 200 μL of supernatant and add to 100 μL of water, mix well and then send for LC / MS / MS detection.

[0166] Results of the experiment

[0167] The results are shown in Table 5.

[0168] Table 5 IC of compound of formula (I) for P450 isozyme inhibition 50 Values

[0169]

[0170] The results show that the compound of formula (I) has a low risk of drug-drug interaction.

[0171] Experimental Example 4: Metabolic stability (MMS) study in liver microsomes

[0172] Purpose of the experiment

[0173] Test the metabolic stability of the test product in human and rat liver microsomes.

[0174] Materials for the experiment

[0175] Test product (10 mM), testosterone (control product, 10 mM), diclofenac (control product, 10 mM), propafenone (control product, 10 mM).

[0176] Buffer system

[0177] 1. 100 mM potassium phosphate buffer (pH 7.4).

[0178] 2.10 mM MgCl2.

[0179] Compound dilution

[0180] 1. Intermediate solution: 5 μL of test or control article was diluted with 45 μL of DMSO (with 450 μL of 1:1 methanol / water).

[0181] 2. Working solution: The intermediate solution was diluted with 450 μL of 100 mM potassium phosphate buffer.

[0182] NADPH regenerating system

[0183] 1. β-Phosphoamide adenine dinucleotide, from Sigma, Cat. No. N0505.

[0184] 2. Isocitric acid, from Sigma, Cat. No. 112252.

[0185] 3. Isocitrate dehydrogenase, from Sigma, Cat. No. 12002.

[0186] Preparation of liver microsomal solution (final concentration: 0.5 mg protein / mL)

[0187]

[0188] Termination solution

[0189] Cold acetonitrile containing 100 ng / mL tolbutamide and 100 ng / mL labetalol as internal standards.

[0190] Experimental procedure

[0191] Add 10 μL of test or control article working solution to all plates (T0, T5, T 10 , T 20 , T 30 , T 60 , NCF 60 ).

[0192] Distribute 680 μL / well of liver microsomal solution to the 96-well plates and then add 80 μL / well to each plate. Place the incubation plates at 37°C for approximately 10 minutes.

[0193] Add 10 μL of 100 mM potassium phosphate buffer per well to the NCF 60 plates.

[0194] After the pre-incubation was complete, 90 μL / well of NADPH Regeneration System Working Solution was dispensed onto the 96-well plate, followed by the addition of 10 μL / well to each plate to initiate the reaction.

[0195] Incubate for the appropriate time (e.g., 5, 10, 20, 30, and 60 minutes).

[0196] Add 300 μL / well of Stop Solution (100 ng / mL Tolbutamide and 100 ng / mL Labetalol, stored at 4°C) to each sample well.

[0197] Shake the sample plate for approximately 10 minutes and centrifuge at 4000 rpm for 20 minutes at 4°C.

[0198] At the time of centrifugation, add 300 μL of HPLC water to each well and take 100 μL of supernatant for LC-MS / MS analysis.

[0199] Data Analysis

[0200] The half-life T is calculated by the following equation 1 / 2 and the liver microsomal intrinsic clearance C lint(mic)

[0201]

[0202] Each gram of liver contains 45 mg of microsomal protein, and the liver weights of mice, rats, dogs, monkeys and humans are 88 g / kg, 40 g / kg, 32 g / kg, 30 g / kg and 20 g / kg, respectively.

[0203] C t is the concentration at time t, t is the incubation time, C0is the concentration at 0, k e is the elimination rate constant, Cl int(mic) is the liver microsomal intrinsic clearance, Cl int(liver) is the liver intrinsic clearance.

[0204] Experimental Results

[0205] The results are shown in Table 6.

[0206] Table 6. Intrinsic clearance of the compound of formula (I) in human and rat liver microsomes

[0207]

[0208] The results show that the compound of formula (I) has good stability of liver microsomal metabolism in both human and rat species.

Claims

1. A crystalline form of the compound of formula (I) characterized by, XRPD pattern having characteristic diffraction peaks at the following 2Θ angles: 6.06°, 8.30°, 9.05°, 9.34°, 10.52°, 11.86°, 12.34°, 13.40°, 14.25°, 14.57°, 15.30°, 15.59°, 16.95°, 17.74°, 18.45°, 18.71°, 19.51°, 19.88°, 20.33°, 21.03°, 21.60°, 22.61°, 23.64°, 24.07°, 24.53°, 25.37°, 26.41°, 27.05°, 27.74°, 28.10°, 30.48°, 34.72°, 36.84°, 37.60°; 2. The A-type crystal according to claim 1, characterized in that, The XRPD pattern is shown in Figure 1.

3. The A crystalline form of claim 1 or 2, characterized in that, The DSC curve has an endothermic peak with a peak value at 75.3±3.0°C, 99.6±3.0°C and 167.9±3.0°C, respectively, and an exothermic peak with a peak value at 132.3±3.0°C.

4. The Form A of claim 3, characterized by an X-ray powder diffraction pattern substantially in accordance with Figure 2, The DSC curve is shown in Figure 2.

5. The A-type crystal according to claim 1 or 2, characterized in that, The TGA curve has a weight loss of 1.75% at 55.0±3.0°C, and a weight loss of 3.08% at 100.0±3.0°C.

6. The Form A of claim 5, characterized by an X-ray diffraction pattern substantially in accordance with Figure 8, The TGA curve is shown in Figure 3.

7. A crystalline form of the compound of formula (I) characterized by, XRPD pattern having characteristic diffraction peaks at the following 2Θ angles: 6.30°, 8.48°, 9.25°, 9.71°, 12.57°, 13.62°, 14.46°, 15.80°, 17.16°, 17.96°, 18.67°, 18.92°, 19.69°, 20.09°, 21.26°, 22.15°, 23.68°, 24.29°, 25.58°, 26.64°, 27.32°, 27.95°, 28.28°, 30.71°, 35.16°; 8. The Form F of claim 7, characterized by an X-ray diffraction pattern substantially in accordance with Figure 18, The XRPD pattern is shown in Figure 4.

9. The Form F of claim 7 or 8, characterized by an X-ray diffraction pattern substantially in accordance with Figure 18, The DSC curve has an endothermic peak with a peak value at 100.0±3.0°C and 172.7±3.0°C, respectively, and an exothermic peak with a peak value at 126.0±3.0°C.

10. The Form F of claim 9, characterized by an X-ray diffraction pattern substantially in accordance with Figure 18. The DSC curve is shown in Figure 5.

11. The Form F of claim 7 or 8, characterized by an X-ray diffraction pattern substantially in accordance with Figure 18, The TGA curve has a weight loss of 3.92% at 130.0±3.0°C.

12. The Form F of claim 11, characterized by an X-ray diffraction pattern substantially in accordance with Figure 18. The TGA curve is shown in Figure 6.

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