CN114746431A

CN114746431A - Crystal form of LRRK2 inhibitor and preparation method thereof

Info

Publication number: CN114746431A
Application number: CN202080072681.6A
Authority: CN
Inventors: 吴凌云; 王才林; 郭唐漾; 徐雄彬; 陈曙辉
Original assignee: Guizhou Inoqini Technology Co ltd
Current assignee: Guizhou Inoqini Technology Co ltd
Priority date: 2019-10-18
Filing date: 2020-10-16
Publication date: 2022-07-12
Also published as: WO2021073600A1

Abstract

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

Description

Crystal form of LRRK2 inhibitor and preparation method thereof

Priority application

This application claims priority from chinese patent application CN201910995375.9 filed in 2019,

month

10, 18, which is incorporated by reference in its entirety.

Technical Field

The invention relates to a crystal form of a compound serving as an LRRK2 kinase inhibitor and a preparation method thereof, and also relates to application of the crystal form in preparation of drugs related to LRRK2 kinase activity inhibitors.

Background

Mutations and overexpression of LRRK2 kinase are increasingly proven to be the underlying factor in the induction of neurodegenerative diseases, characterized primarily by selective degeneration and cell death of dopaminergic neurons in the substantia nigra region. Affects 1% of the population over 65 years of age, with hereditary patients accounting for 5-10% of the population with disease. Early in the disease, the most obvious symptoms are manifested by shaking, slow movement and difficulty walking. Cognitive and behavioral problems also occur in the later stages, and dementia usually occurs in the later stages.

There is increasing evidence that leucine repeat-rich kinase 2(LRRK2) mutations are inseparably linked to neurodegenerative diseases, and LRRK2 is a 2527 amino acid protein involved in catalytic phosphorylation and GTP-GDP hydrolysis. The NCBI participating sequence of human LRRK2mRNA is NM _ 198578.2. Evidence suggests that LRRK2 phosphorylates a-synuclein at serine-129, and this phosphorylated form constitutes an important part of the lewy body. In addition, single nucleotide polypetides in the functional domain of LRRK2 have been shown to cause common and sporadic neurodegenerative diseases. To date, researchers have identified more than 20 LRRK2 mutations in families with delayed neurodegenerative diseases. For example, the G2019S mutation co-segregates with autosomal dominance and it results in about 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. Furthermore, amino acid substitutions at another residue R1441 are also associated with neurodegenerative diseases and have been shown to increase the activity of LRRK2 kinase. Overexpression of mutant LRRK2 protein R1441G in a transgenic mouse model was associated with decreased dopamine release, indicating that LRRK2 inhibitors can also positively modulate dopamine release and are associated with treatment as well as decreased dopamine release, indicating that LRRK2 inhibitors can also positively modulate dopamine release and have potential utility in treating diseases characterized by decreased dopamine levels. The relevant data further show that inhibitors of LRRK2 kinase activity may also be useful in the treatment of related neurodegenerative diseases.

Therefore, the development of potent inhibitors of LRRK2 kinase as well as mutant LRRK2 kinase is now an important approach for the treatment of neurodegenerative diseases. The invention aims to invent a compound capable of highly inhibiting LRRK2 kinase, thereby further inventing a medicine capable of well treating neurodegenerative diseases.

The literature (ACS Med. chem. Lett.2015,6,584-589) discloses the compound JH-II-127, belonging to the LRRK2 kinase inhibitor; the literature (J.Med.chem.2012,55,9416-9433) also discloses the compound GNE-7915 as belonging to LRRK2 kinase inhibitors. The structural formula is as follows:

disclosure of Invention

The present invention provides crystalline form A of the compound of formula (I),

the X-ray powder diffraction pattern is characterized by having characteristic diffraction peaks at the following 2 theta angles: 7.06 plus or minus 0.20 degrees, 8.04 plus or minus 0.20 degrees and 21.16 plus or minus 0.20 degrees.

In some embodiments of the present invention, the form a has an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2 θ angles: 7.06 plus or minus 0.20 degrees, 8.04 plus or minus 0.20 degrees, 10.26 plus or minus 0.20 degrees, 15.52 plus or minus 0.20 degrees, 19.68 plus or minus 0.20 degrees, 21.16 plus or minus 0.20 degrees, 24.64 plus or minus 0.20 degrees and 28.12 plus or minus 0.20 degrees.

In some embodiments of the present invention, the form a has an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2 θ 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.

In some embodiments of the invention, the form a is as shown in figure 1 with an XRPD pattern.

In some embodiments of the invention, the XRPD pattern analysis data for form a is as shown in table 1:

TABLE 1 XRPD pattern analysis data for form A of Compound of formula (I)

In some embodiments of the present invention, the above form a has an endothermic peak at 119.45 ± 3 ℃ in its differential scanning calorimetry curve.

In some embodiments of the present invention, the DSC pattern of form a is as shown in figure 2.

In some embodiments of the invention, the above form a, has a thermogravimetric analysis curve with a weight loss of 0.740% at 300 ℃ ± 3 ℃.

In some embodiments of the invention, the TGA profile of the crystalline form a is shown in figure 3.

The present invention also provides a process for preparing the crystalline form a of compound of formula (I) comprising:

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

(b) stirring the suspension at 35-45 deg.C for 8-16 hr;

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

wherein the solvent is selected from a mixed solvent of ethanol and water, ethanol, acetone and acetonitrile.

In some embodiments of the present invention, the volume ratio of ethanol to water in the mixed solvent is 2: 1.

The invention also provides an application of the crystal form A or the crystal form obtained by the preparation method of the crystal form A in preparation of drugs related to LRRK2 kinase activity inhibitors.

Technical effects

The invention has stable crystal form, small influence by light, heat and humidity, good solubility and wide patent medicine prospect. The compound has obvious kinase inhibition and cell activity, membrane permeability and solubility on LRRK2, and has excellent pharmacokinetic and pharmacodynamic properties. The compounds of the present invention and their crystalline forms provide more effective treatments for neurodegenerative diseases.

Definitions and explanations

As used herein, the following terms and phrases are intended to have the following meanings unless otherwise indicated. A particular phrase or term should not be considered as ambiguous or unclear without special definition, but rather construed in a generic sense. When a trade name appears herein, it is intended to refer to its corresponding commodity or its active ingredient.

The intermediate compounds of the present invention may 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 combinations thereof with other chemical synthetic methods, and equivalents thereof known to those skilled in the art, with preferred embodiments including, but not limited to, the examples of the present invention.

The chemical reactions of the embodiments of the present invention are carried out in a suitable solvent that is compatible with the chemical changes of the present invention and the reagents and materials required therefor. In order to obtain the compounds of the present invention, it is sometimes necessary for a person skilled in the art to modify or select the synthesis steps or reaction schemes based on the existing embodiments.

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

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

The invention employs the following abbreviations: CDCl₃Represents deuterated chloroform; CD (compact disc)₃OD represents deuterated methanol; xphos represents 2-dicyclohexylphosphine-2 ', 4 ', 6 ' -triisopropylbiphenyl; pd₂(dba) ₃Represents tris-dibenzylideneacetone dipalladium.

The compounds are prepared by hand or

The software names, and the commercial compounds are under the supplier catalog name.

The powder X-ray diffraction (XRPD) method of the invention

The instrument model is as follows: DX-2700BH X-ray powder diffractometer

The test method comprises the following steps: approximately 10-20mg of sample was used for XRPD detection.

The detailed XRPD parameters are as follows:

ray source: cu, k-alpha

Voltage of light pipe: 40kV, light tube current: 30mA

Divergent slit: 1mm

First rope tension slit: 28mm, second cable slit: 28mm

Receiving a slit: 0.3mm, anti-scatter slit: 1mm

Measuring time: 0.5s

Scanning angle range: 3-40deg

Step width angle: 0.02deg

Differential thermal analysis (DSC) method of the present invention

The instrument model is as follows: mettler Toledo DSC 1500 type differential scanning calorimeter

The test method comprises the following steps: a sample (. about.2 mg) was placed in a DSC aluminum pan for testing at 50mL/min N₂The sample was heated from 30 deg.C (room temperature) to 350 deg.C at a ramp rate of 10 deg.C/min.

The present invention is a Thermal Gravimetric Analysis (TGA) method

The instrument model is as follows: TA Instruments TGA Q500 thermogravimetric analyzer

The test method comprises the following steps: a sample (2-5mg) was placed in a TGA platinum pan for testing at 60mL/min N₂Under the condition, the sample is heated from room temperature to 500 ℃ or the weight loss is 20 percent at the heating rate of 10 ℃/min.

The invention relates to a Dynamic Vapor adsorption analysis (DVS) method

The instrument model is as follows: SMS DVS Advantage dynamic vapor adsorption appearance

And (3) testing conditions are as follows: samples (10-15mg) were placed in DVS sample trays for testing.

The detailed DVS parameters are as follows:

temperature: 25 deg.C

Balancing: dm/dt ═ 0.01%/min (shortest: 10min, longest: 180min)

And (3) drying: drying at 0% RH for 120min

RH (%) test step: 10 percent of

RH (%) test step range: 0 to 90 to 0 percent

Hygroscopicity evaluation classifications are given in table 2:

TABLE 2

Moisture absorption classification	ΔW％
Deliquescence	Absorb sufficient water to form liquid
Is very hygroscopic	ΔW％≥15％
Has moisture absorption	15％>ΔW％≥2％
Slightly hygroscopic	2％>ΔW％≥0.2％
No or almost no hygroscopicity	ΔW％<0.2％

Note: Δ W% represents the moisture pick-up weight of the test article at 25. + -. 1 ℃ and 80. + -. 2% RH.

Drawings

Figure 1 is an XRPD pattern of Cu-ka radiation of compound form a of formula (I).

Figure 2 is a DSC profile of form a of compound of formula (I).

Figure 3 is a TGA profile of the crystalline form a of compound of formula (I).

Figure 4 is a DVS spectrum of form a of compound of formula (I).

Figure 5 is the results of a rotational test of form a of compound of formula (I) on rats.

Figure 6 shows the results of gait modulation in rats of form a of compound of formula (I).

Detailed Description

For better understanding of the present invention, the following description is given with reference to specific examples, but the present invention is not limited to the specific embodiments.

Example one

Preparation of Compounds of formula (I)

Synthesis of compound 1:

the first step is as follows: dissolving the compound 1-1(1944.77g) in tetrahydrofuran (20L), cooling to 0 ℃ under the protection of nitrogen, dropwise adding a methyl magnesium bromide ether solution (14.5L,3M) into the reaction solution, controlling the temperature to be 0-20 ℃, naturally heating to room temperature of 25 ℃ after dropwise adding, and reacting for 15 hours. After the reaction, an aqueous potassium carbonate solution (5920.00g/7.24L) was added dropwise to the reaction solution, and the mixture was stirred at a temperature of 30 ℃ or lower to precipitate a white solid, stirred overnight, the reaction solution was filtered, and the filtrate was collected. Adding ethanol (20L) into the filter cake, and pulping. Filtering, combining the filtrates, and concentrating the filtrate under reduced pressure to obtain crude product compound 1-2.

¹H NMR(400MHz,CDCl ₃)δppm 6.58-7.78(m,1H),1.60(dd,J＝3.8,13.6Hz,6H)。

The second step is that: compound 1-3(1202.54g) was dissolved in dioxane (15L) and water (1.5L), and then Compound 1-2(1061.45g), 4, 5-bis (diphenylphosphino) -9, 9-dimethylxanthene (159.01g) and potassium phosphate (1408.77g) were added. Palladium acetate (61.52g) was added under nitrogen gas, nitrogen gas was replaced three times, and the reaction was carried out at 110 ℃ for 15 hours. After the reaction was completed, the reaction solution was filtered, and the filter cake was washed with ethyl acetate (2L), and the filtrates were combined and concentrated under reduced pressure. Ethyl acetate (800mL) was added to the concentrated crude product, hydrochloric acid/ethyl acetate was slowly added dropwise while stirring to adjust pH 3, during which a solid precipitated, the stirred solution was filtered, the filter cake was washed with acetone (2L), the filter cake was collected, the filter cake was added to acetone (3L), stirred, filtered, the filter cake was washed with acetone (2L), the filter cake was collected, and the filter cake was dried under reduced pressure to obtain compound 1-4 (hydrochloride).

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

The third step: compounds 1 to 4(1213.05g, HCl salt) were dissolved in N, N-dimethylformamide (12L), and N, N-diisopropylethylamine (3.05L) and compounds 1 to 5(1299.92g) were added, followed by heating to 80 ℃ and reaction for 15 hours. After the reaction, water (20L) was added, extraction was performed with dichloromethane (10L. times.3), and the organic phase was washed once with a saturated aqueous sodium chloride solution (10L), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by slurrying with n-heptane (2L) to give compound 1.

¹H NMR(400MHz,CDCl ₃) δ ppm 11.54(br s,1H),8.65(dd, J ═ 4.4,8.6Hz,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.

Synthesis of a compound of formula (I):

the first step is as follows: compound 2(501.40g), sodium tert-butoxide (458.81g) was dissolved in tetrahydrofuran (5L). Methyl iodide (408.98g) was added dropwise at 0 deg.C, the mixture was replaced with nitrogen, and stirred at 25 deg.C for 4 hours under nitrogen. After completion of the reaction, the reaction mixture was extracted with water (5L), ethyl acetate (5L. times.2), washed twice with saturated brine (15L. times.1), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give compound 3.

¹H NMR(400MHz,CDCl ₃)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)。

The second step is that: adding ethanol (12L) into a 50L reaction kettle, adding compound 3(1201.17g) under the protection of nitrogen and stirring, adjusting the temperature of the reaction kettle to 0-5 ℃, slowly adding concentrated sulfuric acid (1005.48g) in batches, heating to 25-30 ℃, and stirring for 15-18 hours. After the reaction is finished, the temperature of the reaction kettle is adjusted to 0-5 ℃, N-diisopropylethylamine (5.51L) is slowly added in batches under the protection of nitrogen and stirring, the temperature is increased to 20-25 ℃, the mixture is kept warm and stirred for 20-30 minutes (the pH is detected by pH test paper to be 8-9), the compound 4(831.29g) is slowly added in batches, nitrogen is replaced for 5-10 minutes, then the temperature is increased to 70-80 ℃, and the mixture is kept warm and stirred for 17-20 hours. The heating was stopped. When the reaction solution is naturally cooled to 20-25 ℃, slowly dripping water (20L) into the reaction solution in batches, and separating out a large amount of yellow solid after dripping. Stirring the reaction solution at 20-25 deg.C for 2-3 hr, filtering, washing the filter cake with 2L water for 2-3 times, and vacuum drying the filter cake at-0.1 MPa and 50 deg.C for 15-16 hr. Dispersing the filter cake in 14L of n-heptane, stirring for 2-3 hours at 20-25 ℃, filtering, washing the filter cake with n-heptane (1L each time) for 2-3 times, and vacuum drying the filter cake at-0.1 MPa and 50 ℃ for 15-16 hours to obtain the compound 5.

¹H NMR(400MHz,CDCl ₃) δ ppm 8.22(d, J ═ 8.9Hz,1H),5.75(d, J ═ 9.0Hz,1H),4.13(br d, J ═ 10.5Hz,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, found 280.

The third step: under a nitrogen atmosphere, the intermediate compound 5(451.76g), 10% wet palladium carbon (47.72g), and tetrahydrofuran (4.5L) were charged into a 10L autoclave, and then replaced with nitrogen 3 to 4 times and hydrogen 3 to 4 times, the pressure was adjusted to 1MPa, and the mixture was stirred at 20 to 25 ℃ and 1MPa for 12 to 15 hours. The second batch was prepared in the same manner. The two batches were combined, filtered over celite, the filter cake was washed 2-3 times with 1L tetrahydrofuran, and the filtrate was collected. The filtrate was concentrated under reduced pressure to give compound 6, and the crude product was used directly in the next step.

The fourth step: tetrahydrofuran (9L) was charged into a 50L reactor, Compound 1(870.74g), Compound 6(895.66g), and sodium t-butoxide (530.95g) were added in portions with stirring at 20-25 deg.C, nitrogen sparged for 5-10 minutes, followed by addition of X-Phos (39.36g) and Pa₂(dba) ₃(76.94g), bubbling with nitrogen for 5-10 min, heating to 70-80 deg.C, stirring for 13-16 h. The heating was stopped. The temperature of the reaction liquid is reduced to 20-25 ℃. Filtering the reaction solution through diatomite, leaching a filter cake with ethyl acetate/tetrahydrofuran (v/v ═ 3/1) (4L) for 2-3 times, combining the filtrates, adding the combined filtrates into a 50L reaction kettle, adding water (15L), stirring, standing for layering, adding ethyl acetate (10L) into an aqueous phase, extracting, separating, combining organic phases, washing with saturated saline solution (10L), drying the organic phases with anhydrous sodium sulfate, filtering, leaching the filter cake with ethyl acetate (2L) for 2-3 times, concentrating the combined filtrates under reduced pressure, adding acetone (3L) into an obtained solid, stirring for 2-3 hours, filtering, leaching the filter cake with acetone (1L) for 2 times, and vacuum-drying at-0.1 MPa and 50 ℃ for 15-16 hours to obtain a crude product of the compound 7. Adding the crude product (930.74g) of the compound 7, anhydrous tetrahydrofuran (8L), thiourea resin (935.58g) and ethanol (0.8L) into a 10L reaction kettle, bubbling nitrogen for 5-10 minutes, heating to 70-80 ℃, and stirring for 3-4 hours under heat preservation. The filtrate was filtered, the filter cake was rinsed 2 times with anhydrous tetrahydrofuran (0.4L), and the above palladium removal was repeated twice on the filtrate. Filtering, leaching the filter cake with anhydrous tetrahydrofuran (0.4L) for 2 times, combining the filtrates, adding into a 10L reaction kettle, adding activated carbon powder (93.11g), bubbling with nitrogen for 5-10 min, heating to 70-80 deg.C (refluxing), and stirring for 1-2 hr under heat preservation. Filtering with diatomite (500g), washing the filter cake with anhydrous tetrahydrofuran (1L) for 2-3 times, combining the filtrates, concentrating under reduced pressure to dryness, adding acetone (1.9L) into the obtained solid, stirring at 20-25 deg.C for 2-3 hr, filtering, rinsing the filter cake with acetone (0.4L) for 2-3 times, and vacuum drying at-0.1 MPa and 50 deg.C for 15-16 hr to obtain the compound of formula (I).

¹H NMR(400MHz,CDCl ₃) δ ppm 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 ═ 7.8Hz,1H),7.33-7.24(m,1H),7.17-7.04(m,1H),6.92(s,1H),5.81(d, J ═ 8.3Hz,1H),4.00-3.88(m,7H),3.83(quin, J ═ 6.9Hz,1H), dd 3.24(s,3H),2.53(d, J ═ 2.9,6.8,9.8, 2H),2.22-2.09(m,2H),1.84(s,3H),1.81(s,3H), 2.8 (s, 3H). MS-ESI calculated value [ M + H%] ⁺529, measured value 529.

Example two

Preparation of compound A crystal form of formula (I)

Adding ethanol (6L) and a compound (594.90g) of formula (I) into a 10L reaction kettle, bubbling with nitrogen for 5-10 minutes, heating to 80-90 deg.C (refluxing) under nitrogen atmosphere and stirring, stirring for 1-2 hours while maintaining temperature until clear, heat-filtering (a large amount of solid is precipitated), continuing to stir the filtrate and precipitated solid at 80-90 deg.C until clear, stopping heating, cooling to 20-25 deg.C while stirring for 10-30 minutes, filtering, washing the filter cake with ethanol (0.5L) for 2-3 times, sequentially adding the filter cake and ethanol (5L) into the 10L reaction kettle, bubbling with nitrogen for 5-10 minutes, heating to 80-90 deg.C (refluxing) under nitrogen atmosphere and stirring, stirring for 1-2 hours while maintaining temperature until clear, heat-filtering (a large amount of solid is precipitated), continuing to stir the filtrate and precipitated solid at 80-90 deg.C until clear, and (3) under stirring, carrying out programmed temperature reduction (temperature reduction of 10-20 ℃ every 1 hour) on the reaction liquid to 20-25 ℃, stopping heating, and continuing stirring for 12-15 hours under 20-25 ℃. Filtering, leaching the filter cake with ethanol (0.5L) for 2-3 times, collecting the filter cake, and vacuum drying at-0.1 MPa and 50-60 deg.C to obtain crystal form A of compound of formula (I).

¹H NMR(400MHz,CDCl ₃) δ ppm 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),5.81(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), 3.84 (s, 3H). MS-ESI calculated value [ M + H%] ⁺529, found value 529.

EXAMPLE III

Experimental example 1: study of hygroscopicity of Crystal form A of Compound of formula (I)

Experimental materials:

SMS DVS Advantage dynamic vapor adsorption instrument

The experimental method comprises the following steps:

10-15mg of the compound A crystal form of the formula (I) is placed in a DVS sample tray for testing.

The experimental results are as follows:

the DVS spectrum of the form a of compound of formula (I) is shown in figure 4, with Δ W of 0.86%.

And (4) experimental conclusion:

the compound form a of formula (I) has a moisture pick-up weight of 0.86% at 25 ℃ and 80% RH, which is slightly hygroscopic.

Experimental example 2: solid stability test of Compound A Crystal form of formula (I)

According to the guidelines of the stability tests of pharmaceutical raw materials and preparations (9001 of the four pharmacopoeias 2015 edition), the crystal form a of the compound of the formula (I) is examined at high temperature (60 ℃, open), high humidity (92.5% of room temperature/relative humidity), and illumination (total illumination is 1.2 × 10 ═ 1⁶Lux hr/near ultraviolet of 200w hr/m²Open) condition.

10mg of compound A crystal form of the compound shown in the formula (I) is weighed respectively, placed at the bottom of a glass sample bottle and spread into a thin layer. Sealing the bottle mouth of a sample placed under the conditions of high temperature (60 ℃) and high humidity (relative humidity is 92.5 percent RH) by using aluminum foil paper, pricking small holes on the aluminum foil paper to ensure that the sample can be fully contacted with ambient air, and placing the sample in a corresponding constant temperature and humidity box; the illuminated sample (open, not covered with foil) and the illuminated control (the entire sample bottle covered with foil) were placed in an illumination box. 2 portions of the sample were weighed at each time point, and the sample was used as a full-scale test sample. The compound of formula (I) in crystalline form a, also called approximately 50mg, was used for XRPD testing, and the sample vials were wrapped with aluminum foil paper and punctured with small holes, again placed in the corresponding thermostated and humidistat cabinet. The samples were sampled and tested (XRPD) at day 5, day 10 and the results compared to the initial test results at day 0, as shown in table 3 below:

TABLE 3 solid stability test results for Compound A form of formula (I)

And (4) conclusion: the crystal form A of the compound shown in the formula (I) has good stability under the conditions of high temperature, high humidity and strong light.

Experimental example 3: in vitro evaluation of LRRK2 kinase inhibitory Activity

Purpose of the experiment: detection of phosphate groups of phosphorylated fluoroescein-ERM (LRRKtide) peptide and phosphate groups by homogeneous time-resolved fluorescence

The Tb-pERM (pLRRKtide) Antibody transfers the energy signal (520nM/485nM fluorescence signal ratio) upon binding. Calculating LRRK2 kinase inhibition IC of test compound₅₀The value is obtained.

Experimental materials:

1. reaction solution: 10mM hydroxyethylpiperazine ethanesulfonic acid (pH 7.5); 2mM magnesium chloride; 0.5mM ethylene glycol diethyl ether diamine tetraacetic acid; 0.002% polyoxyethylene fatty alcohol ether; 1mM dithiothreitol and 1% DMSO;

2. detection solution: TR-FRET Dilution Buffer;

LRRK2 human recombinant protein: expressing recombinant full-length human LRRK2 protein in insect Sf9 cells with baculovirus using GST tag;

4. substrate: 0.4uM fluoroescein-ERM (LRRKtide) peptide; 57uM ATP.

The detection method comprises the following steps:

homogeneous time-resolved fluorescence technology (HTRF);

Fluorescein-ERM (LRRKtide) peptide polypeptides and methods of use

Tb-pERM (pLRRKtide) Antibody shifts between 485nM and 520nM in energy resonance.

Experimental operation:

1. adding a DMSO solution of the compound to be tested through an Echo550 non-contact nano-liter level acoustic wave pipetting system;

2. preparing an enzyme and polypeptide mixed solution by using a freshly prepared reaction solution, adding the enzyme and polypeptide mixed solution into a reaction hole, and pre-incubating for 20 minutes at room temperature;

3. adding 57uM ATP to initiate reaction, and reacting for 90 minutes at room temperature;

4. adding a detection system (fluoroescein-ERM (LRRKtide) peptide polypeptide,

Tb-pERM (pLRRKtide) Antibody and 10mM EDTA), reacting at room temperature for 60 minutes, and detecting a fluorescence signal by Em/Ex 520/485;

5. relative enzyme activity inhibition to DMSO blank was calculated from the signal ratio and IC calculated using software XLfit5 fitting curve₅₀The value is obtained.

The experimental results are as follows:

TABLE 4 LRRK2 kinase inhibitory Activity test results

Test article	LRRK2 kinase inhibitory Activity (nM)
A compound of formula (I)	5.3

And (4) conclusion: the compound of formula (I) has significant LRRK2 enzyme inhibitory activity.

Experimental example 4: in vitro evaluation of LRRK2 cell (pSer935) inhibitory Activity

Cell preparation:

1. cell thawing

293T cells were removed from liquid nitrogen and placed in water at 37 ℃. After the ice was completely melted, the cells were transferred to 5 ml of warm culture medium, centrifuged, the supernatant was discarded, and the suspended cells were cultured in a medium as new cells.

2. Culture and passaging of cells

293T cells were cultured in medium for two to three days.

3. Cell freezing

The cultured cell lines were placed in fresh medium and diluted to 1 x 10^7 and then mixed with equal amounts of medium. The solution is divided into 1mL portions, placed at 80 ℃ below zero for one day and transferred to liquid nitrogen for storage.

The experimental steps are as follows:

(first day) seeding of 293T cells

1.4 x 10^6/293T cells were seeded on one plate, and after two days of culture, the cell number could grow to 5 x 10^6, so seed N +1 plates were sufficient for N experiments with 96-well plates.

(next day) transfection of 293T cells

Firstly, adding 5 microliter of 0.5 microgram/microliter of pcmv-flag-lrrk2 to 145 microliter of DMEM culture solution, and uniformly mixing by using a straw;

adding 15 microliters of transfection reagent, and uniformly mixing with a straw;

thirdly, balancing the mixture for 10 minutes at room temperature;

fourthly, adding 0.5 ml of preheated cell culture medium and uniformly mixing;

fifthly, dripping 650 microliter of the mixture into a 6-hole plate, and stirring thoroughly;

sixthly, the culture plate is cultured in a humidifying incubator containing 5% carbon dioxide at 37 ℃ for 20-24 hours.

(third day) 293T cells were plated onto 96-well plates

(day 4) inhibitor treatment

Firstly, treating the compound by using a centrifugal machine;

second, add 55 microliters of cell culture medium to the inhibitor plate. Preheating the plate at 37 ℃;

thirdly, transferring 50 microliter of cells containing the inhibitor to a cell culture plate;

fourthly, culturing the culture plate in a humidifying incubator containing 5% carbon dioxide at 37 ℃ for 20-24 hours;

fifthly, extracting 300 microliters of culture solution containing the inhibitor by using a dropper, taking 200 microliters of the culture solution, adding 100 microliters of the decomposition agent into the culture solution, sealing the plate, and then shaking for 30 minutes at 4 ℃;

and sixthly, storing the plate at-20 ℃ until the plate is used.

(day 5) MSD procedure

First, 2 μ/25 μ l/labeled antibody was added to the MSD plate and incubated for 2 hours (50 μ l 3.9 μ g/μ l Flag antibody +2.5 ml fetal bovine serum/disc). Centrifugation for 10 seconds (1000 rpm);

secondly, discarding the marker antibody, and washing twice with 300 microliter/washing buffer solution at low speed and multiple points;

thirdly, adding 50 microliter of buffer solution per block, and incubating for 2 hours;

fourthly, discarding the buffer solution, and manually washing for 2 times by using 300 microliter/washing buffer solution;

transferring 12.5 microliters of lysis buffer and 12.5 microliters of cell lysate to an MSD plate, and incubating for 1 hour at room temperature;

sixthly, discarding the lysate, and washing for 3 times at a low speed by using 300 microliters/washing buffer solution at multiple points;

seventhly, diluting the ps935(1:200) antibody, adding 25 microliter/antibody, and incubating for 1 hour at room temperature;

eighthly, discarding the first antibody, and washing 3 times by using 300 microliters/washing buffer solution at a plurality of points and a low speed;

ninthly, diluting the goat anti-rabbit antibody at a ratio of 1:500, adding 25 microliters/antibody to the incubation plate, and culturing for 1 hour at room temperature;

ten, discard the secondary antibody, wash 3 times at low speed with 300. mu.l/wash buffer at multiple spots. The last washing buffer is sent to the MSD reader;

eleven, collecting data twice;

twelve, abandoning the last washing liquid, and adding 150 microliter/2 times of buffer solution to the pore plate to be read;

thirteen, about 3 minutes after incubation, data were read at 15 minutes.

The experimental results are as follows:

TABLE 5 results of the pSer935 cell inhibitory Activity test

Test article	LRRK2 cell inhibitory Activity (nM)
A compound of formula (I)	4.5

And (4) conclusion: the compound of formula (I) has significant cell (pSer935) inhibitory activity.

Experimental example 5: compound pharmacokinetic evaluation

Purpose of the experiment: study of Compound pharmacokinetics- -brain tissue to plasma drug concentration ratio in C57BL/6 mice

Experimental materials: c57BL/6 mouse (Male, 8 weeks old, weight 25g-30g)

And (3) experimental operation:

rodent pharmacokinetic profiles following oral administration of the compounds were tested in a standard protocol, and the candidate compounds were formulated in a 1mg/mL suspension for single oral administration to mice. The oral vehicle is 10% dimethyl sulfoxide/10% tween 80/20% polyethylene glycol 400 water solution. Male C57BL/6 mice were used for this program and administered orally by gavage at a dose of 5 mg/kg. At 0.5, 1, 2 and 4 hours post-dose, whole brains will be collected. Tissue samples were diluted with 15mM fetal bovine serum [ fetal bovine serum (pH 7.4) buffer: methanol (volume ratio, 2: 1)]Homogenizing at a homogenization ratio of 1:5 (w: v) and dividing the homogenate into 2 aliquots, one for analysis and the other for backup. In addition, 0.5, 1, 2, 4h plasma was collected after administration, and plasma samples were collected within half an hourThe supernatant was separated by centrifugation at about 4 ℃ for 15 minutes at 3000g to obtain a plasma sample. Plasma samples were stored in polypropylene tubes, snap frozen on dry ice and kept at-80 ℃ until LC/MS analysis. Adding acetonitrile solution containing internal standard to precipitate protein, mixing, centrifuging, collecting supernatant, sampling, quantitatively analyzing blood concentration by LC-MS/MS analysis method, and calculating drug substitution parameters such as peak concentration (C)_max) Half life (T)_1/2) Time to peak (T)_max) Drug concentration (AUC) of different tissues at the time of administration_0-last) Brain tissue and plasma drug concentration ratio (B/P), etc.

The pharmacokinetic relevant parameters in mice are shown in table 6 below.

Table 6 in vivo pharmacokinetic testing results:

and (4) conclusion: the compound of the formula (I) has good in vivo pharmacokinetic properties, including good brain tissue drug concentration, brain tissue and plasma drug concentration ratio (B/P) and the like.

Experimental example 6: in vivo pharmacodynamic study of crystal form A of compound (I) on Parkinson disease model of 6-hydroxydopamine-induced rat

The experimental method comprises the following steps: the model is a Parkinson Disease (PD) model in which a dopaminergic neuron is completely damaged by performing an operation using a stereotaxic instrument and injecting 6-hydroxydopamine (6-OHDA) to the Medial Forebrain Bundle (MFB) on a single side.

Experimental materials:

animals: SD rats (male, 6-8 weeks old, 220-.

The supplier: shanghai slyke laboratory animals Co., Ltd

Experiment design: TABLE 7

TABLE 7

Group of	Testing drugs	Number of animals	Dosage (mg/kg)	Route of administration	Frequency of administration
1	Blank (solvent)	12	-	Oral gavage	Once a day
2	Levodopa (L-DOPA)	12	20	Abdominal injection	Once a day
3	Crystal form A of compound of formula (I)	12	10	Oral gavage	Twice a day

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

The experimental steps are as follows:

the method is suitable for the following steps: male SD rats were acclimated in the facility for 3 days after arrival.

6-OHDA injury surgery modeling: rats were placed in a stereotactic frame and, after craniotomy, 18 μ g of 6-OHDA was dissolved in 4 μ L of physiological saline and single-sided perfused at 1 μ L/min.

Model screening: apomorphine (0.5mg/kg, subcutaneously injected) induced spin test was performed 2 weeks after surgery. After 5min of apomorphine injection, the rat rotational behavior was recorded for 30min, and the number of 360 ° contralateral rotations completed within 30min after manual dosing injection was included in the following experiments for rats rotated >60r/30 min.

Administration: according to the experimental design, the vehicle and levodopa group are continuously administered once a day for 28 days, and the compound A crystal form group of the formula (I) is continuously administered twice a day for 28 days.

Detection indexes are as follows: behavioral testing (rotation test and gait adjustment) was performed at the end of the experiment.

A. And (3) rotation test: apomorphine (0.5mg/kg, injected subcutaneously) induced spin test was performed. After 5min of apomorphine injection, the rotation behavior of each group of rats was recorded for 30min, and the number of 360 ° contralateral rotations completed within 30min after manual dosing. The results of the tests are shown in FIG. 5 (one-way ANOVA is expressed in terms of the Dunnett's test.)

As a result: the compound A crystal form of the formula (I) has no improvement on the rotation behavior of animals.

B. Gait adjustment: the test was performed 60 minutes after the administration. The experimenter grasps the mouse's hind limbs and a fore paw so that the animal must bear its weight with its contralateral force, freely adjust the fore paw to maintain its balance, and then slowly move sideways (12s/0.9 m). In the forward and backward movement, the number of steps of the front paw is manually counted. The test results are shown in FIG. 6. (one-way analysis of variance is expressed in the Dennet test (Dunnett's test); p < 0.01; P < 0.001. Left Paw (Left Paw): ipsilateral to the injured brain; right Paw (Rignt Paw): contralateral to the injured brain.)

As a result: the crystal form A of the compound shown in the formula (I) has obvious improvement effect on gait regulation behaviors of rats, and the effect is similar to that of levodopa.

The experimental conclusion is that: the compound A crystal form of the formula (I) has certain improvement effect on the behavior of rats in a Parkinson Disease (PD) model of 6-hydroxydopamine (6-OHDA) induced rats.

While the present invention has been described with reference to the embodiments shown in the drawings, the present invention is not limited to the embodiments, which are illustrative and not restrictive, and it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

A crystal form A of a compound of formula (I),

the X-ray powder diffraction pattern is characterized by having characteristic diffraction peaks at the following 2 theta angles: 7.06 plus or minus 0.20 degrees, 8.04 plus or minus 0.20 degrees and 21.16 plus or minus 0.20 degrees.
Form a according to claim 1 having an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2 Θ angles: 7.06 plus or minus 0.20 degrees, 8.04 plus or minus 0.20 degrees, 10.26 plus or minus 0.20 degrees, 15.52 plus or minus 0.20 degrees, 19.68 plus or minus 0.20 degrees, 21.16 plus or minus 0.20 degrees, 24.64 plus or minus 0.20 degrees and 28.12 plus or minus 0.20 degrees.
Form a according to claim 2 having an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2 Θ 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.
The crystalline form a according to claim 3 having an XRPD pattern as shown in figure 1.
The crystalline form A according to any one of claims 1 to 4, which has an endothermic peak at 119.45 ± 3 ℃ in its differential scanning calorimetry curve.
The crystalline form A according to claim 5, having a DSC profile as shown in figure 2.
The crystalline form A according to any one of claims 1 to 4, having a thermogravimetric analysis curve with a weight loss of 0.740% at 300 ℃ ± 3 ℃.
The crystalline form a according to claim 7 having a TGA profile as shown in figure 3.
A process for preparing a crystalline form a of compound of formula (I) comprising:

(a) adding a compound of formula (I) to a solvent to form a suspension;

(b) stirring the suspension at 35-45 ℃ for 8-16 hours;

(c) centrifuging and drying for 12-24 hours;

wherein the solvent is selected from a mixed solvent of ethanol and water, ethanol, acetone and acetonitrile.
The preparation method of crystalline form a according to claim 9, wherein the volume ratio of ethanol to water in the mixed solvent is 2: 1.
Use of the crystalline form a according to any one of claims 1 to 8 or obtained by the process for its preparation according to claim 9 or 10 for the preparation of a medicament related to an inhibitor of LRRK2 kinase activity.