Crystal form of LRRK2 inhibitor as well as preparation method and application of crystal form

Abstract

The invention belongs to the technical field of chemical engineering, and particularly relates to a crystal form of an LRRK2 inhibitor as well as a preparation method and application thereof. The invention discloses an A crystal form of an LRRK2 inhibitor and a preparation method of the A crystal form, and further discloses application of the A crystal form in preparation of drugs.

Description

[0001] Divisional application This application is a divisional application of the Chinese patent application with the application number CN202080072681.6, the application date of October 16, 2020, and the invention title of "Crystal Form of an LRRK2 Inhibitor and Its Preparation Method".

[0002] Priority application This application claims the priority of the Chinese patent application CN201910995375.9 filed on October 18, 2019, and the entire text of this priority patent application is incorporated herein by reference. Technical field

[0003] The present invention belongs to the technical field of chemical engineering, and particularly relates to a crystal form of an LRRK2 inhibitor, a preparation method thereof, and an application thereof. Background art

[0004] Mutations and overexpression of the LRRK2 kinase have increasingly been proven to be the fundamental factors inducing neurodegenerative diseases, which are mainly characterized by the selective degeneration and cell death of dopaminergic neurons in the substantia nigra region. It affects 1% of the population over 65 years old, and among them, hereditary patients account for 5-10% of the affected population. The most obvious early symptoms of this disease are shaking, slow movement, and difficulty walking. Cognitive and behavioral problems will also occur in the later stage, and dementia usually appears in the advanced stage.

[0005] Increasing evidence shows an inseparable link between mutations in leucine-rich repeat kinase 2 (LRRK2) and neurodegenerative diseases. LRRK2 is a 2527-amino acid protein involved in catalytic phosphorylation and GTP-GDP hydrolysis. The NCBI accession sequence of human LRRK2 mRNA is NM_198578.2. Evidence shows that LRRK2 phosphorylates α-synuclein at serine-129, and this phosphorylated form constitutes an important part of Lewy bodies. Additionally, single nucleotide polymorphisms in the functional domains of LRRK2 have been shown to cause common and sporadic neurodegenerative diseases. So far, researchers have identified more than 20 LRRK2 mutations in families with late-onset neurodegenerative diseases. For example, the G2019S mutation co-segregates with autosomal dominance, and it accounts for approximately 6% of familial cases and 3% of sporadic cases in Europe. The G2019S mutation occurs in a highly conserved kinase domain, so the G2019S mutation may affect kinase activity. In addition, amino acid substitutions at another residue, R1441, are also associated with neurodegenerative diseases and have been shown to increase the activity of the LRRK2 kinase. Overexpression of the mutant LRRK2 protein R1441G in transgenic mouse models is associated with reduced dopamine release, indicating that LRRK2 inhibitors can also positively regulate dopamine release and have potential utility in treating diseases characterized by reduced dopamine levels. Related data further show that LRRK2 kinase activity inhibitors can also be used to treat related neurodegenerative diseases.

[0006] Therefore, developing effective inhibitors of LRRK2 kinase and mutant LRRK2 kinase has become an important approach for treating neurodegenerative diseases currently.

[0007] The literature (ACS Med.Chem.Lett.2015,6,584-589) discloses the compound JH-II-127, which belongs to LRRK2 kinase inhibitors; the literature (J.Med.Chem.2012,55,9416-9433) discloses the compound GNE-7915, which also belongs to LRRK2 kinase inhibitors. The structural formulas are shown as follows: JH-II-127; GNE-7915. SUMMARY OF THE INVENTION

[0008] The object of the present invention is to provide a crystal form of an LRRK2 inhibitor, a preparation method and application of the crystal form, to partially solve or alleviate the above deficiencies in the prior art. The present invention specifically adopts the following technical solutions.

[0009] In the first aspect of the present invention, there is provided the A crystal form of the compound of formula (I): (I); Its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 7.06±0.20°, 8.04±0.20°, 21.16±0.20°.

[0010] In some embodiments of the present invention, the above A crystal form has characteristic diffraction peaks at the following 2θ angles in its X-ray powder diffraction pattern: 7.06±0.20°, 8.04±0.20°, 10.26±0.20°, 15.52±0.20°, 19.68±0.20°, 21.16±0.20°, 24.64±0.20°, 28.12±0.20°.

[0011] In some embodiments of the present invention, the above A crystal form has characteristic diffraction peaks at the following 2θ angles in its X-ray powder diffraction pattern: 7.061, 8.043, 8.958, 9.320, 10.262, 12.259, 13.001, 13.979, 14.942, 15.523, 17.124, 17.660, 18.339, 19.018, 19.681, 20.596, 21.160, 22.496, 23.020, 24.641, 25.058, 25.400, 26.276, 27.083, 27.721, 28.120, 30.162, 31.420, 32.303, 33.784, 34.700, 35.707, 36.745.

[0012] In some embodiments of the present invention, the above A crystal form has an XRPD pattern as Figure 1 shown.

[0013] In some embodiments of the present invention, the analysis data of the XRPD pattern of the above A crystal form is shown in Table 1.

[0014] Table 1 Analysis data of the XRPD pattern of the A crystal form of the compound of formula (I) In some embodiments of the present invention, the above A crystal form has an endothermic peak at 119.45±3 °C in its differential scanning calorimetry curve.

[0015] In some embodiments of the present invention, the above A crystal form has a DSC pattern as Figure 2 shown.

[0016] In some embodiments of the present invention, the above A crystal form has a weight loss of 0.740% at 300 °C ± 3 °C in its thermogravimetric analysis curve.

[0017] In some embodiments of the present invention, for the above-mentioned Form A, its TGA spectrum is as Figure 3 shown.

[0018] The present invention also provides a method for preparing Form A of the compound of formula (I), comprising: (a) adding the compound of formula (I) into a solvent to form a suspension; (b) stirring the above suspension at 35-45 °C for 8-16 hours; (c) centrifuging and then drying for 12-24 hours; wherein, the solvent is selected from a mixed solvent of ethanol and water, ethanol, acetone, and acetonitrile.

[0019] In some embodiments of the present invention, in the above-mentioned mixed solvent, the volume ratio of ethanol to water is 2:1.

[0020] Specifically, a preferred method is further involved, and the specific steps are as follows: S01: Add ethanol and the compound of formula (I) into a reaction kettle, bubble nitrogen for 5-10 minutes, under a nitrogen atmosphere and stirring, heat up to 80-90 °C for reflux, keep warm and stir until it becomes clear, perform hot filtration, continue to stir the filtrate and the precipitated solid at 80-90 °C until it becomes clear, stop heating, cool down to 20-25 °C, stir, filter, and wash the filter cake with ethanol; S02: Add the filter cake and ethanol into the reaction kettle in sequence, bubble nitrogen for 5-10 minutes, under a nitrogen atmosphere and stirring, heat up to 80-90 °C for reflux, keep warm and stir until it becomes clear, perform hot filtration, continue to stir the filtrate and the precipitated solid at 80-90 °C until it becomes clear, under stirring, cool down the reaction solution according to a programmed temperature drop of 10-20 °C every 1 hour to 20-25 °C, stop heating, and continue to stir at 20-25 °C for 12-15 hours; S03: Filter, wash the filter cake with ethanol several times until it is clean, collect the filter cake for vacuum drying to obtain Form A of the compound of formula (I); the X-ray powder diffraction pattern of the Form A has characteristic diffraction peaks at the following 2θ angles: 7.06 ±0.20°, 8.04 ±0.20°, 21.16±0.20°.

[0021] Specifically, a more preferred method is further included, and the specific steps are as follows: S01: Add 6 L of ethanol and 594.90 g of the compound of formula (I) into a reaction kettle, bubble nitrogen for 5-10 minutes, under a nitrogen atmosphere and stirring, heat up to 80-90 °C for reflux, keep warm and stir until it becomes clear, perform hot filtration, continue to stir the filtrate and the precipitated solid at 80-90 °C until it becomes clear, stop heating, cool down to 20-25 °C, stir for 10-30 minutes, filter, and wash the filter cake with ethanol several times; S02: Add the filter cake and 5 L of ethanol into the reaction kettle in sequence, bubble with nitrogen for 5 - 10 minutes. Under the nitrogen atmosphere and stirring, heat up to 80 - 90 °C for reflux, keep warm and stir until it becomes clear, then perform hot filtration. The filtrate and the precipitated solid continue to be stirred at 80 - 90 °C until it becomes clear. Under stirring, the reaction solution is cooled down to 20 - 25 °C according to the program of cooling 10 - 20 °C every 1 hour, stop heating, and continue to stir at 20 - 25 °C for 12 - 15 hours; (3) Filter, wash the filter cake with ethanol several times until it is clean, collect the filter cake and dry it under vacuum at -0.1 MPa and 50 - 60 °C to obtain the A crystal form of the compound of formula (I); the X-ray powder diffraction pattern of the A crystal form has characteristic diffraction peaks at the following 2θ angles: 7.06 ±0.20°, 8.04 ±0.20°, 21.16±0.20°.

[0022] The present invention also provides the use of the above A crystal form or the crystal form obtained according to the preparation method of the above A crystal form in the preparation of drugs related to LRRK2 kinase activity inhibitors.

[0023] The present invention also provides the use of the above A crystal form or the crystal form obtained according to the preparation method of the above A crystal form in the preparation of drugs for treating Parkinson's disease.

[0024] Those skilled in the art can understand that Parkinson's disease is a neurodegenerative disease.

[0025] Beneficial technical effects: The A crystal form provided by the present invention has stable crystal form, is less affected by light, heat and humidity, has good solubility, and has broad prospects for drug formation. The compound of the present invention has significant kinase inhibition, cell activity, membrane permeability and solubility for LRRK2, and also has excellent pharmacokinetic and pharmacodynamic properties. At the same time, it is found in animal experiments that the A crystal form also has the prospect of treating Parkinson's disease. Therefore, the compound and its crystal form of the present invention can provide more effective treatment for neurodegenerative diseases.

[0026] Definitions and explanations: Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A specific phrase or term should not be considered uncertain or unclear without a special definition, but should be understood according to its ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding product or its active ingredient.

[0027] 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 the combination of it with other chemical synthesis methods, and the equivalent replacement methods well-known to those skilled in the art. The preferred embodiments include but are not limited to the examples of the present invention.

[0028] 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. To obtain the compounds of the present invention, in some cases, those skilled in the art need to modify or select the synthesis steps or reaction processes based on the existing embodiments.

[0029] The present invention will be specifically described below through examples, and these examples do not mean any limitation to the present invention.

[0030] All solvents used in the present invention are commercially available and can be used without further purification.

[0031] The following abbreviations are used in the present invention: CDCl3 represents deuterated chloroform; CD3OD represents deuterated methanol; Xphos represents 2 - dicyclohexylphosphino - 2',4',6'-triisopropylbiphenyl; Pd2(dba)3 represents tris(dibenzylideneacetone)dipalladium(0).

[0032] Compounds are named manually or by ChemDraw® software, and commercially available compounds use the supplier catalog names.

[0033] The powder X - ray diffraction (XRPD) method of the present invention: Instrument model: DX - 2700BH X - ray powder diffractometer; Testing method: Approximately 10 - 20 mg of the sample is used for XRPD detection; The detailed XRPD parameters are as follows: X - ray source: Cu, k - Alpha1 (λ = 1.54184 Å); Tube voltage: 40 kV, tube current: 30 mA; Divergence slit: 1 mm; First Soller slit: 28 mm, second Soller slit: 28 mm; Receiving slit: 0.3 mm, anti - scatter slit: 1 mm; Measurement time: 0.5 s; Scanning angle range: 3 - 40 deg; Step width angle: 0.02 deg.

[0034] The differential scanning calorimetry (DSC) method of the present invention: Instrument model: Mettler Toledo DSC 1500 differential scanning calorimeter; Testing method: Take the sample (~2 mg) and place it in an aluminum DSC pan for testing. Under the condition of N2 at 50 mL / min, heat the sample from 30 °C (room temperature) to 350 °C at a heating rate of 10 °C / min.

[0035] The thermal gravimetric analysis (Thermal Gravimetric Analyzer, TGA) method of the present invention: Instrument model: TA Instruments TGA Q500 type thermogravimetric analyzer; Testing method: Take the sample (2 - 5 mg) and place it in a platinum TGA pan for testing. Under the condition of N2 at 60 mL / min, heat the sample from room temperature to 500 °C or until the weight loss is 20%.

[0036] The dynamic vapor sorption analysis (Dynamic Vapor Sorption, DVS) method of the present invention: Instrument model: SMS DVS Advantage dynamic vapor sorption instrument; Testing conditions: Take the sample (10 - 15 mg) and place it in the DVS sample tray for testing.

[0037] The detailed DVS parameters are as follows: Temperature: 25 °C; Equilibrium: dm / dt = 0.01% / min (shortest: 10 min, longest: 180 min); Drying: Dry at 0% RH for 120 min; RH(%) test step: 10%; RH(%) test step range: 0% - 90% - 0%; The classification of hygroscopicity evaluation is shown in Table 2.

[0038] Table 2 Note: △W% represents the weight gain of the test article under 25 ± 1 °C and 80 ± 2% RH. Description of the drawings

[0039] Figure 1 It is the XRPD spectrum of the Cu - Kα radiation of the crystalline form A of the compound A of formula (I); Figure 2 It is the DSC spectrum of the crystalline form A of the compound A of formula (I); Figure 3 It is the TGA spectrum of the crystalline form A of the compound A of formula (I); Figure 4 It is the DVS spectrum of the crystalline form A of the compound A of formula (I); Figure 5 Rotational test results of crystalline form A of compound A of formula (I) in rats; Figure 6 Gait adjustment results of crystalline form A of compound A of formula (I) in rats. Detailed implementation manners

[0040] To better understand the content of the present invention, the following further description is made in conjunction with specific embodiments, but the specific implementation manners do not limit the content of the present invention.

[0041] Example 1 Preparation of the compound of formula (I): Synthesis of compound 1: .

[0042] First step: Dissolve compound 1-1 (1944.77 g) in tetrahydrofuran (20 L). Under nitrogen protection, cool the temperature to 0 °C, and dropwise add an ethereal solution of methylmagnesium bromide (14.5 L, 3 M) to the reaction solution. Control the temperature at 0 - 20 °C. After the addition is complete, naturally raise the temperature to room temperature (25 °C), and react for 15 hours. After the reaction is completed, dropwise add an aqueous potassium carbonate solution (5920.00 g / 7.24 L) to the reaction solution, and control the temperature to stir below 30 °C. White solid precipitates. Stir overnight, filter the reaction solution, and collect the filtrate. Add the filter cake to ethanol (20 L) for pulping. Filter, combine the filtrates, and concentrate the filtrates under reduced pressure to obtain the crude product compound 1-2.

[0043] 1 1H NMR (400 MHz, CDCl3) δ ppm 6.58 - 7.78 (m, 1H), 1.60 (dd, J = 3.8, 13.6 Hz, 6H).

[0044] Second step: Dissolve compound 1-3 (1202.54 g) in dioxane (15 L) and water (1.5 L), then add compound 1-2 (1061.45 g), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (159.01 g), and potassium phosphate (1408.77 g). Under nitrogen protection, add palladium acetate (61.52 g), displace nitrogen three times, raise the temperature to 110 °C, and react for 15 hours. After the reaction is completed, filter the reaction solution, wash the filter cake with ethyl acetate (2 L), combine the filtrates and concentrate under reduced pressure. Add the concentrated crude product to ethyl acetate (800 mL), and slowly dropwise add hydrochloric acid / ethyl acetate to adjust the pH = 3 while stirring. Solids precipitate during the process. Filter the stirring solution, wash the filter cake with acetone (2 L), collect the filter cake, add the filter cake to acetone (3 L) and stir, filter, wash the filter cake with acetone (2 L), collect the filter cake, and dry it under reduced pressure to obtain compound 1-4 (hydrochloride).

[0045] 1 1H NMR (400 MHz, CD3OD) δ ppm 7.72 - 7.84 (m, 2H), 7.58 - 7.66 (m, 1H), 7.49 (dd, J J = 3.75, 8.00 Hz, 1H), 1.97 (s, 3H), 1.93 (s, 3H). MS-ESI calculated value [M + H] + 170, found 170.

[0046] Step 3: Dissolve Compound 1-4 (1213.05 g, hydrochloride) in N,N-dimethylformamide (12 L), add N,N-diisopropylethylamine (3.05 L) and Compound 1-5 (1299.92 g), then heat to 80 °C and react for 15 hours. After completion of the reaction, add water (20 L), extract with dichloromethane (10 L × 3), wash the organic phase once with saturated aqueous sodium chloride solution (10 L), dry over anhydrous sodium sulfate, filter, and concentrate. The crude product is purified by slurrying with n-heptane (2 L) to obtain Compound 1.

[0047] 1 1H NMR (400 MHz, CDCl3) δ ppm 11.54 (br s, 1H), 8.65 (dd, J J = 4.4, 8.6 Hz, 1H), 8.24 - 8.14 (m, 1H), 7.63 - 7.52 (m, 1H), 7.32 - 7.23 (m, 1H), 7.20 - 7.11 (m, 1H), 1.85 (s, 3H), 1.82 (s, 3H). MS-ESI calculated value [M + H] + 316, found 316.

[0048] Synthesize the compound of formula (I): .

[0049] Step 1: Dissolve Compound 2 (501.40 g) and sodium tert-butoxide (458.81 g) in tetrahydrofuran (5 L). Dropwise add iodomethane (408.98 g) at 0 °C, displace the mixture with nitrogen, and stir at 25 °C under nitrogen protection for 4 hours. After completion of the reaction, add the reaction solution to water (5 L), extract with ethyl acetate (5 L × 2), wash twice with saturated brine (15 L × 1), dry the organic phase over anhydrous sodium sulfate, filter, and concentrate to obtain Compound 3.

[0050] 1 1H NMR (400 MHz, CDCl3) δ ppm 3.84 (br d, J= 12.3Hz, 4H), 3.77 - 3.65(m, 1H), 3.20 - 3.13(m, 3H), 2.42(ddd, J = 3.1, 6.6, 9.6Hz, 2H), 2.06 - 1.99(m, 2H), 1.40 - 1.37(m, 9H).

[0051] Step 2: Add ethanol (12 L) to a 50 L reaction kettle. Under nitrogen protection and stirring, add Compound 3 (1201.17 g). Adjust the temperature of the reaction kettle to 0 - 5°C, and slowly add concentrated sulfuric acid (1005.48 g) in batches. Then raise the temperature to 25 - 30°C and stir for 15 - 18 hours. After the reaction is completed, adjust the temperature of the reaction kettle to 0 - 5°C. Under nitrogen protection and stirring, slowly add N,N - diisopropylethylamine (5.51 L) in batches. Raise the temperature to 20 - 25°C and keep stirring for 20 - 30 minutes (test the pH with pH test paper, pH is 8 - 9). Slowly add Compound 4 (831.29 g) in batches, displace with nitrogen for 5 - 10 minutes, then raise the temperature to 70 - 80°C and keep stirring for 17 - 20 hours. Stop heating. When the reaction solution cools naturally to 20 - 25°C, slowly add the reaction solution dropwise into water (20 L) in batches. After the addition is complete, a large amount of yellow solid precipitates. Stir the reaction solution at 20 - 25°C for 2 - 3 hours, filter, wash the filter cake with water (2 L) 2 - 3 times, and vacuum - dry the filter cake at - 0.1 MPa and 50°C for 15 - 16 hours. Disperse the filter cake in 14 L of n - heptane, stir at 20 - 25°C for 2 - 3 hours, filter, wash the filter cake with n - heptane (1 L each time) 2 - 3 times, and vacuum - dry the filter cake at - 0.1 MPa and 50°C for 15 - 16 hours to obtain Compound 5.

[0052] 1 H NMR(400 MHz, CDCl3) δ ppm 8.22(d, J = 8.9 Hz, 1H), 5.75(d, J = 9.0 Hz, 1H), 4.13(br d, J = 10.5 Hz, 4H), 4.02(s, 3H), 3.89 - 3.78(m, 1H), 3.29 - 3.21(m, 3H), 2.65 - 2.52(m, 2H), 2.26 - 2.14(m, 2H). MS - ESI calculated value [M + H] + 280, measured value 280.

[0053] Step 3: Under a nitrogen atmosphere, add intermediate compound 5 (451.76 g), 10% wet palladium on carbon (47.72 g), and tetrahydrofuran (4.5 L) into a 10 L autoclave. Replace the air with nitrogen 3 - 4 times and then with hydrogen 3 - 4 times. Adjust the pressure to 1 MPa. Stir at 20 - 25 °C and 1 MPa for 12 - 15 hours. Prepare the second batch in the same way. Combine the two batches of reaction solutions, filter through diatomaceous earth, wash the filter cake with 1 L of tetrahydrofuran 2 - 3 times, and collect the filtrate. Concentrate the filtrate under reduced pressure to obtain compound 6, and directly use the crude product for the next feeding step.

[0054] Step 4: Add tetrahydrofuran (9 L) into a 50 L reaction kettle. Add compound 1 (870.74 g), compound 6 (895.66 g), and sodium tert - butoxide (530.95 g) in batches under stirring at 20 - 25 °C. Bubble nitrogen for 5 - 10 minutes, then add X - Phos (39.36 g) and Pa2(dba)3 (76.94 g), and bubble nitrogen for another 5 - 10 minutes. Heat up to 70 - 80 °C and keep stirring for 13 - 16 hours. Stop heating. Cool the reaction solution to 20 - 25 °C. Filter the reaction solution through diatomaceous earth, wash the filter cake with ethyl acetate / tetrahydrofuran (v / v = 3 / 1) (4 L) 2 - 3 times. Combine the filtrates and add them into a 50 L reaction kettle. Add water (15 L), stir, let it stand for phase separation. Add ethyl acetate (10 L) to the aqueous phase for extraction, separate the layers. Add ethyl acetate (10 L) to the aqueous phase for extraction again, separate the layers. Combine the organic phases, wash with saturated brine (10 L), dry the organic phase with anhydrous sodium sulfate, filter, wash the filter cake with ethyl acetate (2 L) 2 - 3 times, combine the filtrates and concentrate under reduced pressure. Add the obtained solid into acetone (3 L), stir for 2 - 3 hours, filter, wash the filter cake with acetone (1 L) 2 times, and dry the filter cake under vacuum at - 0.1 MPa and 50 °C for 15 - 16 hours to obtain the crude product of compound 7. Add the crude product of compound 7 (930.74 g), anhydrous tetrahydrofuran (8 L), thiourea resin (935.58 g), and ethanol (0.8 L) into a 10 L reaction kettle. Bubble nitrogen for 5 - 10 minutes, heat up to 70 - 80 °C, and keep stirring for 3 - 4 hours. Filter, wash the filter cake with anhydrous tetrahydrofuran (0.4 L) 2 times, and repeat the above palladium removal operation on the filtrate twice. Filter, wash the filter cake with anhydrous tetrahydrofuran (0.4 L) 2 times, combine the filtrates and add them into a 10 L reaction kettle. Add activated carbon powder (93.11 g), bubble nitrogen for 5 - 10 minutes, heat up to 70 - 80 °C (reflux), and keep stirring for 1 - 2 hours. Filter through diatomaceous earth (500 g), wash the filter cake with anhydrous tetrahydrofuran (1 L) 2 - 3 times, combine the filtrates and concentrate to dryness under reduced pressure. Add the obtained solid into acetone (1.9 L), stir at 20 - 25 °C for 2 - 3 hours, filter, wash the filter cake with acetone (0.4 L) 2 - 3 times, and dry the filter cake under vacuum at - 0.1 MPa and 50 °C for 15 - 16 hours to obtain the compound of formula (I).

[0055] 1 1H NMR (400 MHz, CDCl3) δ ppm 10.82 (s, 1H), 8.57 (dd, J J = 4.3, 8.4 Hz, 1H), 8.14 (d, J J = 8.3 Hz, 1H), 8.05 (s, 1H), 7.46 (br t, J J = 7.8 Hz, 1H), 7.33 - 7.24 (m, 1H), 7.17 - 7.04 (m, 1H), 6.92 (s, 1H), 5.81 (d, J J = 8.3 Hz, 1H), 4.00 - 3.88 (m, 7H), 3.83 (quin, J J = 6.9 Hz, 1H), 3.24 (s, 3H), 2.53 (ddd, J J = 2.9, 6.8, 9.8 Hz, 2H), 2.22 - 2.09 (m, 2H), 1.84 (s, 3H), 1.81 (s, 3H). MS-ESI calculated value [M + H] + 529, found 529.

[0056] Example 2 Preparation of polymorph A of compound A of formula (I): Ethanol (6 L) and compound of formula (I) (594.90 g) were added to a 10 L reactor, and nitrogen was bubbled for 5 - 10 minutes. Under a nitrogen atmosphere and stirring, the temperature was raised to 80 - 90 °C (reflux), and the mixture was stirred for 1 - 2 hours until clear. It was hot-filtered (a large amount of solid precipitated). The filtrate and the precipitated solid were continued to be stirred at 80 - 90 °C until clear. Heating was stopped, the temperature was lowered to 20 - 25 °C, and the mixture was stirred for 10 - 30 minutes. It was filtered, and the filter cake was washed with ethanol (0.5 L) 2 - 3 times. Then the filter cake and ethanol (5 L) were successively added to a 10 L reactor, and nitrogen was bubbled for 5 - 10 minutes. Under a nitrogen atmosphere and stirring, the temperature was raised to 80 - 90 °C (reflux), and the mixture was stirred for 1 - 2 hours until clear. It was hot-filtered (a large amount of solid precipitated). The filtrate and the precipitated solid were continued to be stirred at 80 - 90 °C until clear. Under stirring, the reaction solution was cooled stepwise (the temperature was lowered by 10 - 20 °C every 1 hour) to 20 - 25 °C. Heating was stopped, and the mixture was continuously stirred at 20 - 25 °C for 12 - 15 hours. It was filtered, and the filter cake was rinsed with ethanol (0.5 L) 2 - 3 times. The filter cake was collected and dried under vacuum at -0.1 MPa and 50 - 60 °C to obtain polymorph A of the compound of formula (I).

[0057] 11H NMR (400 MHz, CDCl3) δ ppm 10.82 (s, 1H), 8.57 (dd, J J = 4.4, 8.4 Hz, 1H), 8.14 (d, J J = 8.3 Hz, 1H), 8.05 (s, 1H), 7.46 (t, J J = 7.9 Hz, 1H), 7.34 - 7.19 (m, 1H), 7.17 - 7.04 (m, 1H), 6.93 (s, 1H), 5.81 (d, J J = 8.4 Hz, 1H), 4.00 - 3.89 (m, 7H), 3.83 (quin, J J = 6.9 Hz, 1H), 3.24 (s, 3H), 2.53 (ddd, J J = 2.9, 6.8, 9.9 Hz, 2H), 2.23 - 2.08 (m, 2H), 1.84 (s, 3H), 1.80 (s, 3H). MS-ESI calculated value [M + H] + 529, found 529.

[0058] Example 3 Experimental Example 1: Hygroscopicity Study of Crystal Form A of Compound A of Formula (I) Experimental Materials: SMS DVS Advantage dynamic vapor sorption instrument; Experimental Method: Take 10 - 15 mg of crystal form A of compound A of formula (I) and place it in the DVS sample pan for testing.

[0059] Experimental Results: The DVS spectrum of crystal form A of compound A of formula (I) is as Figure 4 shown, ΔW = 0.86%.

[0060] Experimental Conclusion: The hygroscopic weight gain of crystal form A of compound A of formula (I) at 25 °C and 80% RH is 0.86%, showing slightly hygroscopic property.

[0061] Experimental Example 2: Solid Stability Test of Crystal Form A of Compound A of Formula (I) According to the "Guidelines for Stability Testing of Drug Substances and Dosage Forms" (General Chapter 9001, Volume IV, Chinese Pharmacopoeia 2015 Edition), investigate the stability of crystal form A of compound A of formula (I) under high temperature (60 °C, open), high humidity (room temperature / relative humidity 92.5%, open) and light (total illuminance = 1.2 × 10 6 Lux·hr / near ultraviolet = 200 w·hr / m2 , the stability under open conditions.

[0062] Weigh 10 mg of Compound A polymorph of formula (I) separately and place it at the bottom of a glass sample bottle, spreading it into a thin layer. For the samples placed under high temperature (60 °C) and high humidity (relative humidity 92.5% RH) conditions, seal the bottle mouth with aluminum foil paper and make some small holes in the aluminum foil paper to ensure that the sample can be in full contact with the ambient air, and place it in the corresponding thermostatic and humidity-controlled chamber; the light-exposed samples (open, not covered with aluminum foil paper) and the light-exposed reference samples (the whole sample bottle is covered with aluminum foil paper) are placed in the light box. Weigh 2 portions at each time point as the official test samples. In addition, weigh approximately 50 mg of Compound A polymorph of formula (I) for XRPD testing. Wrap the sample bottle with aluminum foil paper and make small holes, and also place it in the corresponding thermostatic and humidity-controlled chamber. Samples are taken for testing (XRPD) on the 5th and 10th days, and the test results are compared with the initial test results on day 0. The test results are shown in Table 3 below.

[0063] Table 3 Solid stability test results of Compound A polymorph of formula (I) Conclusion: Compound A polymorph of formula (I) has good stability under high temperature, high humidity, and strong light conditions.

[0064] Experimental Example 3: In vitro evaluation of LRRK2 kinase inhibitory activity Experimental purpose: Detect the energy signal transfer (fluorescence signal ratio of 520 nM / 485 nM) generated after the binding of the phosphate group of phosphorylated Fluorescein-ERM (LRRKtide) peptide to LanthaScreen® Tb-pERM (pLRRKtide) Antibody by homogeneous time-resolved fluorescence. Calculate the LRRK2 kinase inhibition IC 50 value.

[0065] Experimental materials: 1. Reaction solution: 10 mM 2-(4-(2-Hydroxyethyl)piperazin-1-yl)ethanesulfonic acid (pH 7.5); 2 mM magnesium chloride; 0.5 mM ethylene glycol bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid; 0.002% polyoxyethylene fatty alcohol ether; 1 mM dithiothreitol and 1% DMSO; 2. Detection solution: TR-FRET Dilution Buffer; 3. Human recombinant LRRK2 protein: Recombinant full-length human LRRK2 protein is expressed in insect Sf9 cells using a baculovirus with a GST tag; 4. Substrate: 0.4 μM Fluorescein-ERM (LRRKtide) peptide; 57 μM ATP.

[0066] Detection method: Homogeneous time-resolved fluorescence technique (HTRF); Energy resonance transfer between Fluorescein-ERM (LRRKtide) peptide and LanthaScreen® Tb-pERM (pLRRKtide) Antibody between 485 nM and 520 nM.

[0067] Experimental operation: 1. Add the DMSO solution of the compound to be tested through an Echo550 non-contact nano-liter acoustic pipetting system; 2. Prepare a mixed solution of enzyme and peptide with freshly prepared reaction solution, add it to the reaction wells, and pre-incubate at room temperature for 20 minutes; 3. Add 57 μM ATP to initiate the reaction and react at room temperature for 90 minutes; 4. Add the detection system (Fluorescein-ERM (LRRKtide) peptide, LanthaScreen® Tb-pERM (pLRRKtide) Antibody and 10 mM ethylenediaminetetraacetic acid), react at room temperature for 60 minutes, and detect the fluorescence signal with Em / Ex 520 / 485; 5. Calculate the relative enzyme activity inhibition relative to the DMSO blank through the signal ratio, and calculate the IC 50 value using the software XLfit5 to fit the curve.

[0068] Experimental results: Table 4 Test results of LRRK2 kinase inhibitory activity Conclusion: The compound of formula (I) has significant LRRK2 enzyme inhibitory activity.

[0069] Experimental Example 4: In vitro evaluation of LRRK2 cell (pSer 935) inhibitory activity Cell preparation: 1. Cell thawing Take out the 293T cells from liquid nitrogen and place them in water at 37 °C. After the ice has completely melted, transfer the cells to 5 ml of warm culture medium, centrifuge, discard the supernatant, and culture the suspended cells as new cells in the medium.

[0070] 2. Cell culture and passage Culture the 293T cells in the culture medium for two to three days.

[0071] 3. Cell freezing Put the cultured cell line into fresh culture medium and dilute the concentration to 1*10^7, then mix it with an equal volume of culture medium. Divide it into 1 mL aliquots, place them at -80 °C for one day, and transfer them to liquid nitrogen for storage.

[0072] Experimental procedures: 1. (On the first day) Seed 293T cells.

[0073] Seed 1.4×10^6 / 293T cells on a culture plate. After two days of culture, the cell number can grow to 5×10^6, so seeding N + 1 plates is sufficient for experiments on N 96-well plates.

[0074] 2. (On the second day) Transfect 293T cells.

[0075] I. Add 5 μL of 0.5 μg / μL pcmv-flag-lrrk2 to 145 μL of DMEM culture medium and mix well with a pipette; II. Add 15 μL of transfection reagent and mix well with a pipette; III. Equilibrate at room temperature for 10 minutes; IV. Add 0.5 mL of pre-warmed cell culture medium and mix well; V. Pipette 650 μL of the mixture into a 6-well plate and stir thoroughly; VI. Incubate the culture plate in a humidified incubator at 37 °C with 5% carbon dioxide for 20 - 24 hours.

[0076] 3. (On the third day) Seed 293T cells onto 96-well plates.

[0077] 4. (On the 4th day) Treat with inhibitors.

[0078] I. Centrifuge the compound; II. Add 55 μL of cell culture medium to the inhibitor plate. Preheat the plate at 37 °C; III. Transfer 50 μL of the cell culture containing the inhibitor to the cell culture plate; IV. Incubate the culture plate in a humidified incubator at 37 °C with 5% carbon dioxide for 20 - 24 hours; V. Use a pipette to draw 300 μL of the culture medium containing the inhibitor. Take 200 μL of it and add 100 μL of the decomposing agent. Seal the plate and shake it at 4 °C for 30 minutes; VI. Store the plate at -20 °C until use.

[0079] 5. (On the 5th day) MSD procedure.

[0080] I. Add 2 μL / 25 μL of labeled antibody to the MSD plate and incubate for 2 hours, (50 μL of 3.9 μg / μL Flag antibody + 2.5 mL of fetal bovine serum per plate). Centrifuge for 10 seconds (1000 rpm); II. Discard the Flag antibody and wash twice at low speed with 300 μL of wash buffer in multiple spots; III. Add 50 μL of buffer per well and incubate for 2 hours; IV. Discard the buffer and wash manually twice with 300 μL of wash buffer; V. Transfer 12.5 μL of lysis buffer and 12.5 μL of cell lysate to the MSD plate and incubate at room temperature for 1 hour; VI. Discard the lysate and wash three times at low speed with 300 μL of wash buffer in multiple spots; VII. Dilute ps935 (1:200) antibody, add 25 μL of antibody, and incubate at room temperature for 1 hour; VIII. Discard the primary antibody and wash three times at low speed with 300 μL of wash buffer in multiple spots; IX. Dilute goat anti-rabbit antibody 1:500, add 25 μL of antibody to the incubation plate, and incubate at room temperature for 1 hour; X. Discard the secondary antibody and wash three times at low speed with 300 μL of wash buffer in multiple spots. Transfer the last wash buffer to the MSD reader; XI. Collect data twice; XII. Discard the last wash solution and add 150 μL of 2-fold buffer to the plate to be read; XIII. Read the data 15 minutes after incubation for about 3 minutes.

[0081] Experimental results: Table 5 Test results of pSer935 cell inhibitory activity Conclusion: The compound of formula (I) has significant inhibitory activity against cells (pSer935).

[0082] Experimental Example 5: Pharmacokinetic evaluation of the compound Experimental purpose: To study the pharmacokinetics of the compound in C57BL / 6 mice - the ratio of drug concentrations in brain tissue and plasma.

[0083] Experimental materials: C57BL / 6 mice (male, 8 weeks old, body weight 25 g - 30 g) Experimental operations: The rodent pharmacokinetic characteristics after oral administration of the compound were tested with a standard protocol. In the experiment, the candidate compound was formulated into a 1 mg / mL suspension and administered to mice by single oral administration. The oral vehicle was an aqueous solution of 10% dimethyl sulfoxide / 10% Tween 80 / 20% polyethylene glycol 400. Male C57BL / 6 mice were used in this project and administered by oral gavage at a dose of 5 mg / kg. Whole brains were collected at 0.5, 1, 2, and 4 hours after dosing. The tissue samples were homogenized with 15 mM fetal bovine serum [fetal bovine serum (pH = 7.4) buffer: methanol (volume ratio, 2:1)] at a homogenization ratio of 1:5 (w:v), and the homogenate was divided into two aliquots, one for analysis and the other for backup. In addition, plasma was collected at 0.5, 1, 2, and 4 h after dosing. The plasma samples were separated by centrifugation at approximately 4 °C, 3000 g for 15 minutes within half an hour of collection to obtain plasma samples. The plasma samples were stored in polypropylene tubes, quickly frozen on dry ice and kept at -80 °C until LC / MS / MS analysis. An acetonitrile solution containing an internal standard was added to precipitate proteins, and the mixture was thoroughly mixed and centrifuged to take the supernatant for injection. The blood drug concentration was quantitatively analyzed by LC-MS / MS analysis method, and pharmacokinetic parameters such as peak concentration (C max ), half-life (T 1 / 2 ), time to peak concentration (T max ), area under the curve of drug concentration in different tissues over time (AUC 0-last ), and the ratio of drug concentration in brain tissue to plasma (B / P) were calculated.

[0084] The pharmacokinetic parameters in mice are shown in Table 6 below.

[0085] Table 6 Pharmacokinetic test results in vivo Conclusion: The compound of formula (I) has good in vivo pharmacokinetic properties, including good drug concentration in brain tissue and the ratio of drug concentration in brain tissue to plasma (B / P), etc.

[0086] Experimental Example 6: In vivo pharmacodynamic study of crystalline form A of the compound of formula (I) on a rat model of Parkinson's disease induced by 6-hydroxydopamine.

[0087] Experimental method: This model was established by using a stereotaxic apparatus to surgically inject 6-hydroxydopamine (6-OHDA) unilaterally into the medial forebrain bundle (MFB) to cause a model of Parkinson's disease (PD) with complete damage of dopaminergic neurons.

[0088] Experimental materials: Animals: SD rats (male, 6 - 8 weeks old, 220 - 250 g).

[0089] Supplier: Shanghai Slack Experimental Animal Co., Ltd.

[0090] Experimental design: Table 7 Table 7 Solvent: 4% dimethyl sulfoxide + 6% polyethylene glycol-15 hydroxystearate (Solutol) + 90% water.

[0091] Experimental procedure: Adaptation: Male Sprague-Dawley rats were allowed to adapt to the facility for 3 days after arrival.

[0092] 6-OHDA lesion surgery modeling: The rats were placed in a stereotaxic frame. After craniotomy, 18 μg of 6-OHDA was dissolved in 4 μL of physiological saline and perfused unilaterally at a rate of 1 μL / min.

[0093] Model screening: Apomorphine (0.5 mg / kg, subcutaneous injection)-induced rotation test was performed 2 weeks after surgery. Five minutes after injecting apomorphine, the rotation behavior of the rats was recorded for 30 minutes, and the number of 360° contralateral rotations completed within 30 minutes after injection was manually quantified. Rats with rotations > 60 r / 30 min were included in the following experiments.

[0094] Drug administration: According to the experimental design, the solvent and the levodopa group were administered once a day for 28 consecutive days, and the crystalline form A of compound (I) group was administered twice a day for 28 consecutive days.

[0095] Detection indexes: Behavioral tests (rotation test and gait adjustment) were performed at the end of the experiment.

[0096] A. Rotation test: Apomorphine (0.5 mg / kg, subcutaneous injection)-induced rotation test was performed. Five minutes after injecting apomorphine, the rotation behavior of each group of rats was recorded for 30 minutes, and the number of 360° contralateral rotations completed within 30 minutes after injection was manually quantified. The test results are shown in Figure 5 (Represented by Dunnett’s test for one-way ANOVA.) Results: The crystalline form A of compound (I) did not improve the rotation behavior of the animals.

[0097] B. Gait adjustment: The test was performed 60 minutes after drug administration. The experimenter grasped the hind limbs and one forepaw of the mouse so that the animal had to bear its weight with the strength of its contralateral side, freely adjusted the forepaw to maintain its balance, and then slowly moved laterally (12 s / 0.9 m). During forward and backward movements, the number of steps of the forepaw was manually counted. The test results are shown in Figure 6. (One-way ANOVA was represented by Dunnett’s test, ***p < 0.01, ****p < 0.001. Left Paw: ipsilateral to the lesioned brain; Right Paw: contralateral to the lesioned brain.) Results: The crystalline form A of the compound of formula (I) had a significant improvement effect on the gait adjustment behavior of rats, and its effect was similar to that of levodopa.

[0098] Experimental conclusion: The crystalline form A of the compound of formula (I) had a certain improvement effect on the behavior of rats in a Parkinson's disease (PD) model induced by 6-hydroxydopamine (6-OHDA) in rats.

[0099] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative rather than restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit and scope protected by the present invention and the claims. These all fall within the protection scope of the present invention.

Claims

1. The A crystal form of the compound of formula (I), ; characterized in that, The X-ray powder diffraction pattern of the A crystal form has characteristic diffraction peaks at the following 2θ angles: 7.06 ±0.20°, 8.04 ±0.20°, 21.16±0.20°.

2. The A crystal form according to claim 1, characterized in that, The X-ray powder diffraction pattern of the A crystal form has characteristic diffraction peaks at the following 2θ angles: 7.06 ±0.20°, 8.04 ±0.20°, 10.26 ±0.20°, 15.52 ±0.20°, 19.68 ±0.20°, 21.16 ±0.20°, 24.64 ±0.20°, 28.12±0.20°.

3. The A crystal form according to claim 2, characterized in that, The X-ray powder diffraction pattern of the A crystal form has characteristic diffraction peaks at the following 2θ angles: 7.061, 8.043, 8.958, 9.320, 10.262, 12.259, 13.001, 13.979, 14.942, 15.523, 17.124, 17.660, 18.339, 19.018, 19.681, 20.596, 21.160, 22.496, 23.020, 24.641, 25.058, 25.400, 26.276, 27.083, 27.721, 28.120, 30.162, 31.420, 32.303, 33.784, 34.700, 35.707, 36.

745.

4. The A crystal form according to claim 3, characterized in that, The XRPD pattern of the A crystal form is shown in Figure 1.

5. The A crystal form according to any one of claims 1-4, characterized in that, The differential scanning calorimetry curve of the A crystal form has an endothermic peak at 119.45±3 °C.

6. The A crystal form according to claim 5, characterized in that, The DSC pattern of the A crystal form is shown in Figure 2.

7. The A crystal form according to any one of claims 1-4, characterized in that, The thermogravimetric analysis curve of the A crystal form has a weight loss of 0.740% at 300 °C±3 °C.

8. The A crystal form according to claim 7, characterized in that, The TGA pattern of the A crystal form is shown in Figure 3.

9. A method for preparing the A crystal form of the compound of formula (I), ; characterized in that, Comprising the following steps: S01: Add ethanol and the compound of formula (I) into a reaction kettle, bubble nitrogen for 5 - 10 minutes, under a nitrogen atmosphere and with stirring, heat up to 80 - 90 °C for reflux, keep stirring until it becomes clear, perform hot filtration, continue to stir the filtrate and the precipitated solid at 80 - 90 °C until it becomes clear, stop heating, cool down to 20 - 25 °C, stir, filter, and wash the filter cake with ethanol; S02: Add the filter cake and ethanol into the reaction kettle in sequence, bubble nitrogen for 5 - 10 minutes, under a nitrogen atmosphere and with stirring, heat up to 80 - 90 °C for reflux, keep stirring until it becomes clear, perform hot filtration, continue to stir the filtrate and the precipitated solid at 80 - 90 °C until it becomes clear, under stirring, cool down the reaction solution according to a programmed temperature decrease of 10 - 20 °C every 1 hour to 20 - 25 °C, stop heating, and continue to stir at 20 - 25 °C for 12 - 15 hours; S03: Filter, wash the filter cake thoroughly with ethanol, collect the filter cake and perform vacuum drying to obtain the A crystal form of the compound of formula (I); the X-ray powder diffraction pattern of the A crystal form has characteristic diffraction peaks at the following 2θ angles: 7.06 ±0.20°, 8.04 ±0.20°, 21.16±0.20°.

10. Use of the A crystal form according to claim 1 or the A crystal form obtained by the preparation method according to claim 9 in the preparation of a medicament for treating Parkinson's disease.

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