A crystal form of LRRK2 inhibitor and preparation method thereof

Abstract

The present invention discloses a crystal form of an LRRK2 inhibitor and a preparation method thereof, and also includes application of the crystal form in the preparation of drugs related to the LRRK2 kinase activity inhibitor.

Description

[0001] Priority application

[0002] This application claims the priority of Chinese invention patent application CN201910995375.9 filed on October 18, 2019, which is incorporated by reference in its entirety. Technical Field

[0003] The present invention relates to a crystal form of a compound used as an LRRK2 kinase inhibitor and a preparation method thereof, and also includes application of the crystal form in the preparation of drugs related to the LRRK2 kinase activity inhibitor. Background Art

[0004] Mutations and overexpression of LRRK2 kinase are increasingly being shown to be the root cause of neurodegenerative diseases, characterized by selective degeneration and cell death of dopaminergic neurons in the substantia nigra. It affects 1% of people over the age of 65, of which hereditary patients account for 5-10% of the onset population. In the early stages of the disease, the most obvious symptoms are shaking, slow activity and difficulty walking. Cognitive and behavioral problems also occur in the later stages, and dementia usually occurs in the late stages.

[0005] There is increasing evidence that mutations in leucine-rich repeat kinase 2 (LRRK2), a 2527 amino acid protein involved in catalytic phosphorylation and GTP-GDP hydrolysis, are inextricably linked to neurodegenerative diseases. The NCBI sequence of human LRRK2 mRNA is NM_198578.2. Evidence shows that LRRK2 phosphorylates a-synuclein at serine-129, and this phosphorylated form constitutes an important part of Lewy bodies. In addition, single nucleotide polypeptides in the functional domain 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 is co-segregated with autosomal dominant, and it causes 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 have an effect on kinase activity. In addition, amino acid substitutions at another residue, R1441, are also associated with neurodegenerative diseases and have been shown to increase LRRK2 kinase activity. Overexpression of the mutant LRRK2 protein R1441G in a transgenic mouse model is associated with reduced dopamine release, indicating that LRRK2 inhibitors can also actively regulate dopamine release and are associated with reduced dopamine release, indicating that LRRK2 inhibitors can also actively regulate dopamine release and have potential utility in treating diseases characterized by reduced dopamine levels. The relevant data further show that inhibitors of LRRK2 kinase activity 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 to treat neurodegenerative diseases. The present invention aims to invent a compound that can highly inhibit LRRK2 kinase, thereby further inventing a drug that can effectively treat neurodegenerative diseases.

[0007] The literature (ACS Med. Chem. Lett. 2015, 6, 584-589) discloses compound JH-II-127, which is a LRRK2 kinase inhibitor; the literature (J. Med. Chem. 2012, 55, 9416-9433) also discloses compound GNE-7915, which is a LRRK2 kinase inhibitor. The structural formula is as follows:

[0008]

[0009] Summary of the invention

[0010] The present invention provides a crystalline form A of a compound of formula (I),

[0011]

[0012] The invention is characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 7.06±0.20°, 8.04±0.20°, and 21.16±0.20°.

[0013] In some embodiments of the present invention, the above-mentioned crystal form A has an X-ray powder diffraction pattern having 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°, and 28.12±0.20°.

[0014] In some embodiments of the present invention, the above-mentioned A crystal form has an X-ray powder diffraction pattern with 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, 19.01 8, 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.

[0015] In some embodiments of the present invention, the above-mentioned Form A has an XRPD pattern as shown in Figure 1 shown.

[0016] In some embodiments of the present invention, the XRPD spectrum analysis data of the above-mentioned Form A is shown in Table 1:

[0017] Table 1 XRPD spectrum analysis data of the crystal form of compound A of formula (I)

[0018]

[0019]

[0020] In some embodiments of the present invention, the above-mentioned crystal form A has an endothermic peak at 119.45±3°C in its differential scanning calorimetry curve.

[0021] In some embodiments of the present invention, the above-mentioned A crystal form has a DSC spectrum as follows Figure 2 shown.

[0022] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned crystal form A shows a weight loss of 0.740% at 300°C±3°C.

[0023] In some embodiments of the present invention, the above-mentioned A crystal form has a TGA spectrum as shown in Figure 3 shown.

[0024] The present invention also provides a method for preparing the crystalline form A of the compound of formula (I), comprising:

[0025] (a) adding the compound of formula (I) into a solvent to form a suspension;

[0026] (b) stirring the suspension at 35-45° C. for 8-16 hours;

[0027] (c) drying for 12-24 hours after centrifugation;

[0028] Wherein, the solvent is selected from a mixed solvent of ethanol and water, ethanol, acetone and acetonitrile.

[0029] In some embodiments of the present invention, in the above-mentioned mixed solvent, the volume ratio of ethanol to water is 2:1.

[0030] The present invention also provides use of the above-mentioned crystal form A or a crystal form obtained according to the preparation method of the above-mentioned crystal form A in the preparation of drugs related to LRRK2 kinase activity inhibitors.

[0031] Technical Effects

[0032] The crystal form of the present invention is stable, less affected by light, heat and humidity, has good solubility, and has broad prospects for drug development. The compound of the present invention has significant kinase inhibition and cell activity as well as membrane permeability and solubility on LRRK2, and has excellent pharmacokinetic and pharmacodynamic properties. The compound of the present invention and its crystal form can provide more effective treatment for neurodegenerative diseases.

[0033] Definition and Description

[0034] 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 to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

[0035] 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, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the embodiments of the present invention.

[0036] The chemical reactions of the specific embodiments of the present invention are carried out in a suitable solvent, which must be suitable for the chemical changes of the present invention and the reagents and materials required. In order to obtain the compounds of the present invention, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction processes based on the existing embodiments.

[0037] The present invention will be described in detail below by way of examples, which are not intended to limit the present invention in any way.

[0038] All solvents used in the present invention were commercially available and used without further purification.

[0039] The present invention uses the following abbreviations: CDCl3 represents deuterated chloroform; CD3OD represents deuterated methanol; Xphos represents 2-dicyclohexylphosphine-2'4'6'-triisopropylbiphenyl; Pd2(dba)3 represents tris dibenzylideneacetone dipalladium.

[0040] Compounds are manually or The software names were used, and commercially available compounds were named using the supplier's catalog names.

[0041] Powder X-ray diffractometer (XRPD) method of the present invention

[0042] Instrument model: DX-2700BH X-ray powder diffractometer

[0043] Test Method: Approximately 10-20 mg of sample was used for XRPD testing.

[0044] The detailed XRPD parameters are as follows:

[0045] Radiation source: Cu, k-Alphal

[0046] Light tube voltage: 40kV, light tube current: 30mA

[0047] Divergence slit: 1mm

[0048] First cable slit: 28mm, second cable slit: 28mm

[0049] Receiving slit: 0.3mm, anti-scattering slit: 1mm

[0050] Measuring time: 0.5s

[0051] Scanning angle range: 3-40 degrees

[0052] Step width angle: 0.02deg

[0053] Differential Scanning Calorimeter (DSC) method of the present invention

[0054] Instrument model: Mettler Toledo DSC 1500 differential scanning calorimeter

[0055] Test method: Take a sample (~2 mg) and place it in a DSC aluminum pan for testing. Under 50 mL / min N2 conditions, heat the sample from 30°C (room temperature) to 350°C at a heating rate of 10°C / min.

[0056] Thermogravimetric analysis (TGA) method of the present invention

[0057] Instrument model: TA Instruments TGA Q500 thermogravimetric analyzer

[0058] Test method: Take a sample (2-5 mg) and place it in a TGA platinum pan for testing. Under 60 mL / min N2 conditions, heat the sample from room temperature to 500°C or 20% weight loss at a heating rate of 10°C / min.

[0059] Dynamic Vapor Sorption (DVS) method of the present invention

[0060] Instrument model: SMS DVS Advantage Dynamic Vapor Sorption Instrument

[0061] Test conditions: Take a sample (10-15 mg) and place it in the DVS sample pan for testing.

[0062] The detailed DVS parameters are as follows:

[0063] Temperature: 25℃

[0064] Balance: dm / dt = 0.01% / min (minimum: 10min, maximum: 180min)

[0065] Drying: Drying at 0% RH for 120 minutes

[0066] RH(%) test level: 10%

[0067] RH(%) test step range: 0%-90%-0%

[0068] The classification of moisture absorption evaluation is shown in Table 2:

[0069] Table 2

[0070] Hygroscopicity classification ΔW% deliquescence Absorb enough water to form a liquid Highly hygroscopic ΔW%≥15% Hygroscopic 15%>ΔW%≥2% Slightly hygroscopic 2%>ΔW%≥0.2% No or almost no hygroscopicity ΔW%<0.2%

[0071] Note: ΔW% indicates the weight gain of the test sample at 25±1℃ and 80±2%RH. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 The XRPD spectrum of the crystal form A of the compound of formula (I) using Cu-Kα radiation.

[0073] Figure 2 The DSC spectrum of the crystalline form A of compound of formula (I).

[0074] Figure 3 This is the TGA spectrum of the crystal form A of compound of formula (I).

[0075] Figure 4 This is the DVS spectrum of the crystal form A of compound of formula (I).

[0076] Figure 5 The figure is the result of the rotation test of the crystal form A of the compound of formula (I) on rats.

[0077] Figure 6 The figure shows the gait adjustment results of the crystal form A of compound of formula (I) on rats. DETAILED DESCRIPTION

[0078] In order to better understand the content of the present invention, further description is given below in conjunction with specific embodiments, but the specific implementation methods are not intended to limit the content of the present invention.

[0079] Embodiment 1

[0080] Preparation of compounds of formula (I)

[0081] Synthesis of compound 1:

[0082]

[0083] Step 1: Dissolve compound 1-1 (1944.77g) in tetrahydrofuran (20L), cool to 0°C under nitrogen protection, add methylmagnesium bromide ether solution (14.5L, 3M) to the reaction solution, control the temperature at 0-20°C, and naturally rise to room temperature 25°C after the addition is completed, and react for 15 hours. After the reaction is completed, potassium carbonate aqueous solution (5920.00g / 7.24L) is added dropwise to the reaction solution, and the temperature is controlled to be below 30°C and stirred. A white solid precipitates, stir overnight, filter the reaction solution, and collect the filtrate. Add ethanol (20L) to the filter cake for pulping. Filter, combine the filtrates, and concentrate the filtrate under reduced pressure to obtain the crude product compound 1-2.

[0084] 1H NMR (400MHz, CDCl3) δppm 6.58-7.78 (m, 1H), 1.60 (dd, J=3.8, 13.6Hz, 6H).

[0085] Step 2: 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(diphenylphosphine)-9,9-dimethyloxanthene (159.01 g) and potassium phosphate (1408.77 g). Add palladium acetate (61.52 g) under nitrogen protection, replace nitrogen three times, heat 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 filtrate and concentrate under reduced pressure. The concentrated crude product was added to ethyl acetate (800 mL), and hydrochloric acid / ethyl acetate was slowly added dropwise under stirring to adjust the pH to 3. Solid precipitated during the process. The stirred solution was filtered, the filter cake was washed with acetone (2 L), the filter cake was collected, acetone (3 L) was added to the filter cake, stirred, filtered, the filter cake was washed with acetone (2 L), the filter cake was collected, and dried under reduced pressure to obtain compound 1-4 (hydrochloride).

[0086] 1 H NMR (400 MHz, CD3OD) δppm 7.72-7.84 (m, 2H), 7.58-7.66 (m, 1H), 7.49 (dd, J=3.75, 8.00 Hz, 1H), 1.97 (s, 3H), 1.93 (s, 3H). MS-ESI calculated value [M+H] + 170, measured value 170.

[0087] 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 the reaction is completed, add water (20 L), extract with dichloromethane (10 L × 3), wash the organic phase with saturated sodium chloride aqueous solution (10 L) once, dry with anhydrous sodium sulfate, filter, and concentrate. The crude product is slurried and purified with n-heptane (2 L) to obtain compound 1.

[0088] 1 H NMR (400 MHz, CDCl3) δppm 11.54 (br s, 1H), 8.65 (dd, 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, measured value 316.

[0089] Synthetic compound of formula (I):

[0090]

[0091] Step 1: Dissolve compound 2 (501.40 g) and sodium tert-butoxide (458.81 g) in tetrahydrofuran (5 L). Add iodomethane (408.98 g) dropwise at 0 degrees Celsius, replace the mixture with nitrogen, and stir at 25 degrees Celsius for 4 hours under nitrogen protection. After the reaction is completed, add water (5 L), extract with ethyl acetate (5 L × 2), wash twice with saturated brine (15 L × 1), dry the organic phase with anhydrous sodium sulfate, filter, and concentrate to obtain compound 3.

[0092] 1 H NMR(400MHz,CDCl3)δppm 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).

[0093] Step 2: Add ethanol (12L) to a 50L reactor, add compound 3 (1201.17g) under nitrogen protection and stirring, adjust the temperature of the reactor to 0-5°C, slowly add concentrated sulfuric acid (1005.48g) in batches, raise the temperature to 25-30°C, and stir for 15-18 hours. After the reaction is completed, adjust the temperature of the reactor to 0-5°C, and slowly add N,N-diisopropylethylamine (5.51L) in batches under nitrogen protection and stirring, raise the temperature to 20-25°C, and stir for 20-30 minutes (pH paper detects pH 8-9), slowly add compound 4 (831.29g) in batches, replace with nitrogen for 5-10 minutes, then raise the temperature to 70-80°C, and stir for 17-20 hours. Stop heating. When the reaction solution is naturally cooled to 20-25°C, the reaction solution is slowly added to water (20L) in batches, and a large amount of yellow solid is precipitated after the addition is completed. The reaction solution is stirred at 20-25°C for 2-3 hours, filtered, and the filter cake is washed 2-3 times with water (2L), and the filter cake is vacuum dried at -0.1MPa and 50°C for 15-16 hours. The filter cake is dispersed in 14L of n-heptane, stirred at 20-25°C for 2-3 hours, filtered, and the filter cake is washed 2-3 times with n-heptane (1L each time), and the filter cake is vacuum dried at -0.1MPa and 50°C for 15-16 hours to obtain compound 5.

[0094] 1H NMR (400 MHz, CDCl3) δppm 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.

[0095] Step 3: Under nitrogen atmosphere, add intermediate compound 5 (451.76g), 10% wet palladium carbon (47.72g), and tetrahydrofuran (4.5L) into a 10L autoclave, replace with nitrogen 3-4 times, replace with hydrogen 3-4 times, adjust the pressure to 1MPa, and stir for 12-15 hours at 20-25°C and 1MPa. Prepare the second batch in the same way. Combine the two batches of reaction liquid, filter with diatomaceous earth, wash the filter cake with 1L tetrahydrofuran 2-3 times, and collect the filtrate. The filtrate is concentrated under reduced pressure to obtain compound 6, and the crude product is directly used for the next step.

[0096] Step 4: Add tetrahydrofuran (9 L) to a 50 L reactor, add compound 1 (870.74 g), compound 6 (895.66 g) and sodium tert-butoxide (530.95 g) in batches at 20-25 ° C with stirring, and 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 to 70-80 ° C, and stir for 13-16 hours. Stop heating. Cool the reaction solution to 20-25 ° C. The reaction solution was filtered through diatomaceous earth, and the filter cake was rinsed with ethyl acetate / tetrahydrofuran (v / v=3 / 1) (4L) 2-3 times. The combined filtrate was added to a 50L reactor, water (15L) was added and stirred, and the mixture was allowed to stand for stratification. Ethyl acetate (10L) was added to the aqueous phase, extracted, and separated. Ethyl acetate (10L) was added to the aqueous phase for extraction, and the liquids were separated. The organic phases were combined and washed with saturated brine (10L). The organic phase was dried over anhydrous sodium sulfate and filtered. The filter cake was rinsed with ethyl acetate (2L) 2-3 times. The combined filtrate was concentrated under reduced pressure, and acetone (3L) was added to the obtained solid, stirred for 2-3 hours, filtered, and the filter cake was rinsed with acetone (1L) twice. The filter cake was dried in vacuo at -0.1MPa and 50°C for 15-16 hours to obtain a crude product of compound 7. The crude product of compound 7 (930.74 g), anhydrous tetrahydrofuran (8 L), thiourea resin (935.58 g) and ethanol (0.8 L) were added to a 10 L reactor, nitrogen was bubbled for 5-10 minutes, the temperature was raised to 70-80 ° C, and the mixture was stirred for 3-4 hours. Filter, the filter cake was rinsed with anhydrous tetrahydrofuran (0.4 L) twice, and the filtrate was repeated with the above palladium removal operation twice. Filter, the filter cake was rinsed with anhydrous tetrahydrofuran (0.4 L) twice, the filtrate was combined and added to a 10 L reactor, activated carbon powder (93.11 g) was added, nitrogen was bubbled for 5-10 minutes, the temperature was raised to 70-80 ° C (reflux), and the mixture was stirred for 1-2 hours. The mixture was filtered through diatomaceous earth (500 g), and the filter cake was washed 2-3 times with anhydrous tetrahydrofuran (1 L). The combined filtrates were concentrated to dryness under reduced pressure, and acetone (1.9 L) was added to the obtained solid. The mixture was stirred at 20-25° C. for 2-3 hours, and filtered. The filter cake was rinsed 2-3 times with acetone (0.4 L). The filter cake was dried under vacuum at -0.1 MPa and 50° C. for 15-16 hours to obtain the compound of formula (I).

[0097] 1H NMR (400MHz, CDCl3) δppm 10.82 (s, 1H), 8.57 (dd, J = 4.3, 8.4Hz, 1H), 8.14 (d, J = 8.3Hz, 1H), 8.05 (s, 1H), 7.46 (br t, 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 = 8.3 Hz, 1H), 4.00-3.88 (m, 7H), 3.83 (quin, J = 6.9 Hz, 1H), 3.24 (s, 3H), 2.53 (ddd, 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, measured value 529.

[0098] Embodiment 2

[0099] Preparation of the crystal form of compound A of formula (I)

[0100] Ethanol (6 L) and the compound of formula (I) (594.90 g) were added to a 10 L reactor, and nitrogen was bubbled for 5-10 minutes. Under nitrogen atmosphere and stirring, the temperature was raised to 80-90° C. (reflux), and the mixture was stirred for 1-2 hours until the mixture was dissolved. The mixture was filtered hot (a large amount of solid was precipitated). The filtrate and the precipitated solid were stirred at 80-90° C. until the mixture was dissolved. The heating was stopped, the temperature was lowered to 20-25° C., and the mixture was stirred for 10-30 minutes. The mixture was filtered, and the filter cake was washed 2-3 times with ethanol (0.5 L). Then, the filter cake and ethanol (5L) were added to a 10L reactor in sequence, and nitrogen was bubbled for 5-10 minutes. Under nitrogen atmosphere and stirring, the temperature was raised to 80-90°C (reflux), and the mixture was stirred for 1-2 hours until the mixture was clear. The mixture was hot filtered (a large amount of solid was precipitated). The filtrate and the precipitated solid were stirred at 80-90°C until the mixture was clear. Under stirring, the reaction solution was cooled (10-20°C every hour) to 20-25°C, and the heating was stopped. The mixture was stirred for 12-15 hours at 20-25°C. The filter cake was filtered, and ethanol (0.5L) was used to rinse the filter cake 2-3 times. The filter cake was collected and dried in a vacuum at -0.1MPa and 50-60°C to obtain the A crystal form of the compound of formula (I).

[0101] 1H NMR (400MHz, CDCl3) δppm 10.82(s,1H),8.57(dd,J=4.4,8.4Hz,1H),8.14(d,J=8.3Hz,1H),8.05(s,1H) ,7.46(t,J=7.9Hz,1H),7.34-7.19(m,1H),7.17-7.04(m,1H),6.93(s,1H),5.8 1(d,J=8.4Hz,1H),4.00-3.89(m,7H),3.83(quin,J=6.9Hz,1H),3.24(s,3H),2 .53(ddd,J=2.9,6.8,9.9Hz,2H),2.23-2.08(m,2H),1.84(s,3H),1.80(s,3H). MS-ESI calculated value [M+H] + 529, measured value 529.

[0102] Embodiment 3

[0103] Experimental Example 1: Study on the Hygroscopicity of Crystalline Form A of Compound (I)

[0104] Experimental Materials:

[0105] SMS DVS Advantage Dynamic Vapor Sorption Tester

[0106] Experimental methods:

[0107] 10-15 mg of the crystalline form A of the compound of formula (I) was placed in a DVS sample tray for testing.

[0108] Experimental results:

[0109] The DVS spectrum of the crystal form A of the compound of formula (I) is as follows Figure 4 As shown, ΔW = 0.86%.

[0110] Experimental conclusion:

[0111] The weight gain of the crystal form A of the compound of formula (I) at 25° C. and 80% RH is 0.86%, which is slightly hygroscopic.

[0112] Experimental Example 2: Solid Stability Test of Crystalline Form A of Compound of Formula (I)

[0113] According to the "Guidelines for Stability Testing of APIs and Preparations" (Chinese Pharmacopoeia 2015 Edition, Part IV, General Rules 9001), the stability of the crystalline form of compound A of formula (I) was investigated under high temperature (60°C, open), high humidity (room temperature / relative humidity 92.5%, open) and light (total illumination = 1.2×10 6 Lux·hr / Near UV=200w·hr / m 2 , stability under open) conditions.

[0114] Weigh 10 mg of the crystal form A of the compound formula (I) respectively, place it at the bottom of a glass sample bottle, and spread it into a thin layer. For samples placed under high temperature (60°C) and high humidity (relative humidity 92.5% RH), seal the bottle mouth with aluminum foil, and poke some small holes in the aluminum foil to ensure that the sample can fully contact the ambient air, and place it in the corresponding constant temperature and humidity chamber; the illuminated sample (open, not covered with aluminum foil) and the illuminated reference substance (the entire sample bottle is covered with aluminum foil) are placed in the illumination box. Weigh 2 portions at each time point as formal test samples. In addition, weigh about 50 mg of the crystal form A of the compound formula (I) for XRPD testing, wrap the sample bottle with aluminum foil and poke small holes, and also place it in the corresponding constant temperature and humidity chamber. Samples were sampled and tested (XRPD) on the 5th and 10th days, and the test results were compared with the initial test results on day 0. The test results are shown in Table 3 below:

[0115] Table 3 Solid stability test results of compound A of formula (I)

[0116]

[0117] Conclusion: The crystal form A of compound of formula (I) has good stability under high temperature, high humidity and strong light conditions.

[0118] Experimental Example 3: In vitro evaluation of LRRK2 kinase inhibitory activity

[0119] Experimental purpose: To detect the phosphate groups of phosphorylated Fluorescein-ERM (LRRKtide) peptide by homogeneous time-resolved fluorescence. Energy signal transfer (520nM / 485nM fluorescence signal ratio) generated after binding of Tb-pERM (pLRRKtide) Antibody antibody. Calculate the LRRK2 kinase inhibition IC of the test compound 50 value.

[0120] Experimental Materials:

[0121] 1. Reaction solution: 10 mM hydroxyethylpiperazineethanesulfonic acid (PH7.5); 2 mM magnesium chloride; 0.5 mM ethylene glycol diethyl ether diaminetetraacetic acid; 0.002% polyoxyethylene fatty alcohol ether; 1 mM dithiothreitol and 1% DMSO;

[0122] 2. Detection solution: TR-FRET Dilution Buffer;

[0123] 3. LRRK2 human recombinant protein: Use GST tag to express recombinant full-length human LRRK2 protein in insect Sf9 cells using baculovirus;

[0124] 4. Substrate: 0.4uM Fluorescein-ERM (LRRKtide) peptide; 57uM ATP.

[0125] Detection method:

[0126] Homogeneous time-resolved fluorescence (HTRF);

[0127] Fluorescein-ERM (LRRKtide) peptide Energy resonance transfer of Tb-pERM (pLRRKtide) Antibody antibody between 485nM and 520nM.

[0128] Experimental procedures:

[0129] 1. Add the DMSO solution of the test compound using the Echo550 non-contact nanoliter acoustic pipetting system;

[0130] 2. Prepare the enzyme and peptide mixed solution with the freshly prepared reaction solution, add it to the reaction well, and pre-incubate at room temperature for 20 minutes;

[0131] 3. Add 57uM ATP to initiate the reaction and react at room temperature for 90 minutes;

[0132] 4. Add the detection system (Fluorescein-ERM (LRRKtide) peptide, Tb-pERM (pLRRKtide) Antibody and 10 mM EDTA), react at room temperature for 60 minutes, and detect the fluorescence signal with Em / Ex 520 / 485;

[0133] 5. Calculate the relative enzyme activity inhibition relative to the DMSO blank by signal ratio, and calculate the IC using the XLfit5 software to fit the curve 50 value.

[0134] Experimental results:

[0135] Table 4 LRRK2 kinase inhibitory activity test results

[0136] Test product LRRK2 kinase inhibitory activity (nM) Compound of formula (I) 5.3

[0137] Conclusion: The compound of formula (I) has significant LRRK2 enzyme inhibitory activity.

[0138] Experimental Example 4: In vitro evaluation of LRRK2 cell (pSer935) inhibitory activity

[0139] Cell preparation:

[0140] 1. Thawing of Cells

[0141] Take the 293T cells out of liquid nitrogen and place them in water at 37 degrees Celsius. 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 culture medium.

[0142] 2. Cell Culture and Passaging

[0143] 293T cells were cultured in culture medium for two to three days.

[0144] 3. Cell Freezing

[0145] Place the cultured cell line in fresh culture medium and dilute to a concentration of 1*10^7, then mix with an equal amount of culture medium. Divide into 1 mL portions, place at -80 degrees for one day, and transfer to liquid nitrogen for storage.

[0146] Experimental steps:

[0147] 1. (Day 1) 293T cell seeding

[0148] 1.4×10^6 / 293T cells are seeded on a culture plate. After two days of culture, the number of cells can grow to 5×10^6, so N+1 seed plates are enough for N 96-well plate experiments.

[0149] 2. (Next day) Transfection of 293T cells

[0150] 1. Add 5 μl of 0.5 μg / μl pCMV-FLAG-LRRK2 to 145 μl DMEM medium and mix thoroughly with a pipette;

[0151] 2. Add 15 μl of transfection reagent and mix thoroughly with a pipette;

[0152] 3. Equilibrate at room temperature for 10 minutes;

[0153] 4. Add 0.5 ml of pre-warmed cell culture medium and mix thoroughly;

[0154] 5. Add 650 μl of the mixture to a 6-well plate and stir thoroughly;

[0155] 6. Incubate the plate in a humidified incubator at 37°C with 5% CO2 for 20–24 hours.

[0156] 3. (Day 3) 293T cells were seeded onto 96-well plates

[0157] 4. (Day 4) Inhibitor Treatment

[0158] 1. Process the compound in a centrifuge;

[0159] 2. Add 55 μl of cell culture medium to the inhibitor plate. Pre-warm the plate at 37 degrees Celsius.

[0160] 3. Transfer 50 μl of cell culture containing inhibitors to the cell culture plate;

[0161] 4. Incubate the plate in a humidified incubator at 37°C with 5% carbon dioxide for 20–24 hours.

[0162] 5. Use a dropper to draw out 300 μl of the culture medium containing the inhibitor, take 200 μl of it, add 100 μl of the decomposition agent, seal the plate and shake at 4 degrees Celsius for 30 minutes;

[0163] 6. Store the plates at -20 degrees Celsius until use.

[0164] 5. (Day 5) MSD Procedure

[0165] 1. Add 2 μl / 25 μl / Flag antibody to the MSD plate and incubate for 2 hours (50 μl 3.9 μg / μl Flag antibody + 2.5 ml FBS / plate). Centrifuge for 10 seconds (1000 rpm);

[0166] Second, discard the labeled antibody and wash twice at a low speed with 300 μl / wash buffer;

[0167] 3. Add 50 μl / block buffer and incubate for 2 hours;

[0168] 4. Discard the buffer and wash manually twice with 300 μl / well of wash buffer;

[0169] 5. 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;

[0170] 6. Discard the lysate and wash three times with 300 μl / well of wash buffer at low speed;

[0171] VII. Dilute ps935 (1:200) antibody, add 25 μl / antibody, and incubate at room temperature for 1 hour;

[0172] 8. Discard the primary antibody and wash three times at multiple points and low speed with 300 μl / wash buffer;

[0173] IX. Dilute goat anti-rabbit antibody 1:500, add 25 μl / antibody to the incubation plate, and incubate at room temperature for 1 hour;

[0174] 10. Discard the secondary antibody and wash three times with 300 μl / well of wash buffer at low speed. Add the last wash buffer to the MSD reader;

[0175] 11. Collect data twice;

[0176] 12. Discard the last wash solution and add 150 μl / 2x buffer to the well plate to be read;

[0177] 13. About 3 minutes after incubation, read the data at 15 minutes.

[0178] Experimental results:

[0179] Table 5 pSer935 cell inhibitory activity test results

[0180] Test product LRRK2 cell inhibitory activity (nM) Compound of formula (I) 4.5

[0181] Conclusion: The compound of formula (I) has significant cell (pSer935) inhibitory activity.

[0182] Experimental Example 5: Pharmacokinetic Evaluation of Compounds

[0183] Experimental purpose: To study the pharmacokinetics of the compound in C57BL / 6 mice --- the ratio of drug concentration in brain tissue and plasma

[0184] Experimental materials: C57BL / 6 mice (male, 8 weeks old, weight 25g-30g)

[0185] Experimental operation:

[0186] The pharmacokinetic characteristics of the compounds after oral administration in rodents were tested by standard protocols. In the experiment, the candidate compounds were formulated into a 1 mg / mL suspension and administered to mice orally once. The oral solvent was 10% dimethyl sulfoxide / 10% Tween 80 / 20% polyethylene glycol 400 aqueous solution. Male C57BL / 6 mice were used for this project, and the oral gavage was administered at a dose of 5 mg / kg. Whole brains were collected at 0.5, 1, 2 and 4 hours after administration. Tissue samples were homogenized with 15mM 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 2 aliquots, one for analysis and the other for backup. In addition, plasma was collected 0.5, 1, 2, and 4h after administration. The plasma samples were separated from the supernatant by centrifugation at about 4°C, 3000g, and 15 minutes within half an hour of collection. Plasma samples were stored in polypropylene tubes, quickly frozen on dry ice and kept at -80°C until LC / MS / MS analysis. Proteins were precipitated by adding acetonitrile solution containing internal standard, mixed thoroughly, centrifuged and the supernatant was injected. 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 ), peak time (T max ), drug concentration in different tissues at the time of administration (AUC 0-last), brain tissue and plasma drug concentration ratio (B / P), etc.

[0187] The pharmacokinetic parameters in mice are shown in Table 6 below.

[0188] Table 6 In vivo pharmacokinetic test results:

[0189]

[0190] Conclusion: The compound of formula (I) has good in vivo pharmacokinetic properties, including good brain tissue drug concentration and brain tissue to plasma drug concentration ratio (B / P).

[0191] Experimental Example 6: In vivo pharmacodynamic study of the crystal form A of compound of formula (I) on the 6-hydroxydopamine-induced Parkinson's disease model in rats

[0192] Experimental method: This model is a Parkinson's disease (PD) model in which dopaminergic neurons are completely damaged by unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB) using a stereotaxic apparatus for surgery.

[0193] Experimental Materials:

[0194] Animals: SD rats (male, 6-8 weeks old, 220-250 g).

[0195] Supplier: Shanghai Slake Experimental Animal Co., Ltd.

[0196] Experimental design: Table 7

[0197] Table 7

[0198] Group Testing drugs Number of animals Dosage (mg / kg) Route of administration Dosing frequency 1 blank(solvent) 12 - Oral gavage Once a day 2 Levodopa (L-DOPA) 12 20 Intraperitoneal injection Once a day 3 Compound A of formula (I) 12 10 Oral gavage Twice a day

[0199] Solvent: 4% dimethyl sulfoxide + 6% polyethylene glycol-15 hydroxystearate (Solutol) + 90% water

[0200] Experimental steps:

[0201] Acclimation: Male Sprague-Dawley rats were acclimated to the facility for 3 days upon arrival.

[0202] 6-OHDA injury surgical model: Rats were placed in a stereotaxic frame. After craniotomy, 18 μg 6-OHDA was dissolved in 4 μL normal saline and perfused unilaterally at a rate of 1 μL / min.

[0203] Model screening: Apomorphine (0.5 mg / kg, subcutaneous injection)-induced rotation test was performed 2 weeks after surgery. 5 minutes after apomorphine injection, the rats' rotation behavior 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 experiment.

[0204] Administration: According to the experimental design, the vehicle and levodopa groups were administered once a day for 28 consecutive days, and the crystal form A group of compound of formula (I) was administered twice a day for 28 consecutive days.

[0205] Testing indicators: Behavioral testing (rotation test and gait adjustment) was performed at the end of the experiment.

[0206] A. Rotation test: A rotation test induced by apomorphine (0.5 mg / kg, subcutaneous injection) was performed. 5 minutes after the injection of apomorphine, the rotation behavior of rats in each group was recorded for 30 minutes, and the number of 360° contralateral rotations completed within 30 minutes after the injection was manually quantified. The test results are shown in Figure 5 (One-way ANOVA was performed using Dunnett's test.)

[0207] Results: The crystal form A of compound of formula (I) did not improve the rotation behavior of animals.

[0208] B. Gait adjustment: The test was performed 60 minutes after drug administration. The experimenter held the mouse's hind limbs and one forepaw so that the animal had to bear its weight with the force of its opposite side, freely adjust the forepaw to maintain its balance, and then move slowly sideways (12s / 0.9m). The number of steps of the forepaw was manually counted during the forward and backward movements. The test results are shown in Figure 6 (One-way ANOVA with Dunnett's test, ***p<0.01, ****p<0.001. Left paw: ipsilateral to the injured brain; right paw: contralateral to the injured brain.)

[0209] Results: The crystal form A of the compound of formula (I) significantly improved the gait adjustment behavior of rats, and its effect was similar to that of levodopa.

[0210] Experimental conclusion: The crystal form A of the compound of formula (I) has a certain improvement effect on the behavior of rats in the Parkinson's disease (PD) model induced by 6-hydroxydopamine (6-OHDA).

[0211] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. Crystal form A of the compound of formula (I), It is characterized in that 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°, 10.26 ±0.20°, 15.52 ±0.20°, 19.68 ±0.20°, 24.64 ±0.20°, and 28.12 ±0.20°.

2. The crystal form A according to claim 1, wherein the X-ray powder diffraction pattern 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.

3. According to the A crystal form described in claim 2, its XRPD spectrum is shown in Figure 1.

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

5. According to the A crystal form described in claim 4, its DSC spectrum is shown in Figure 2.

6. The crystal form A according to any one of claims 1 to 3, wherein the thermogravimetric analysis curve thereof shows a weight loss of 0.740% at 300°C±3°C.

7. The A crystal form according to claim 6, has a TGA spectrum as shown in Figure 3.

8. A method for preparing a crystalline form of compound A of formula (I), comprising: (1) Add 6L of ethanol and 594.90g of the compound of formula (I) into a 10L reactor, bubble nitrogen for 5-10 minutes, heat to 80-90°C and reflux under nitrogen atmosphere and stirring, keep stirring for 1-2 hours until the solution is clear, filter hot, continue to stir the filtrate and the precipitated solid at 80-90°C until the solution is clear, stop heating, cool to 20-25°C, stir for 10-30 minutes, filter, and wash the filter cake with 0.5L of ethanol 2-3 times; (2) Then, the filter cake and 5 L of ethanol were added to a 10 L reactor in sequence, and nitrogen was bubbled for 5-10 minutes. Under nitrogen atmosphere and stirring, the temperature was raised to 80-90°C and refluxed. The mixture was kept warm and stirred for 1-2 hours until it was dissolved. The mixture was hot filtered, and the filtrate and the precipitated solid were continued to be stirred at 80-90°C until it was dissolved. Under stirring, the reaction solution was cooled to 20-25°C by decreasing the temperature by 10-20°C every hour. The heating was stopped, and stirring was continued at 20-25°C for 12-15 hours. (3) Filtering, rinsing the filter cake with 0.5 L of ethanol 2-3 times, collecting the filter cake and drying it in a vacuum at -0.1 MPa and 50-60° C. to obtain a crystal form A of the compound of formula (I); the chemical formula of the crystal form A is as follows: ； 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°, 10.26 ±0.20°, 15.52 ±0.20°, 19.68 ±0.20°, 24.64 ±0.20°, and 28.12 ±0.20°.

9. Use of the crystal form A according to any one of claims 1 to 7 or the crystal form obtained by the method for preparing the crystal form A according to claim 8 in the preparation of drugs related to LRRK2 kinase activity inhibitors.

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