A quinoline amine compound crystal form and a preparation method thereof

Abstract

The present disclosure relates to a quinoline amine compound crystal form and a preparation method thereof. Specifically, the present disclosure provides a crystal form of 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)quinolin-2-amine and a preparation method thereof. The corresponding crystal form has good stability and can be better used for clinical treatment.

Description

[0001] This application claims priority to Chinese patent application 2022114917970, filed on 2022 / 11 / 25. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This disclosure pertains to the pharmaceutical field and relates to a crystalline form of a quinolinamine compound and its preparation method. Background Technology

[0003] miR-124 is widely expressed in various tissues throughout the body, especially in brain tissue. Studies have shown that overexpression of miR-124 can promote the transformation of activated macrophages-microglia to a quiescent state, thereby inhibiting the autoimmune disease encephalomyelitis. In addition, miR-124 can promote the transformation of macrophages to the M2 type, thus exerting an anti-inflammatory effect. miR-124 also affects T cell differentiation; the levels of IFN-γ and TNFα in miR-124-treated T cells are decreased. Overexpression of miR-124 exerts its anti-inflammatory effect by downregulating STAT3 protein, thereby reducing the expression of the inflammatory cytokine IL-17 and inhibiting Th17 cell differentiation. These studies suggest that developing a novel small molecule drug that upregulates miR-124 could be used to effectively treat related inflammatory diseases.

[0004] The published related patent applications include WO2010143169A2, WO2015001518A1, WO2016009065A2, WO2017158201A1 and WO2020127843A1, etc.

[0005] WO2022247920 discloses a class of quinolinamine compounds that can upregulate miR-124, the structures of which are shown below.

[0006]

[0007] The crystal structure of a pharmaceutical active ingredient often affects its chemical and physical stability. Different crystallization and storage conditions can lead to changes in the crystal structure of the compound, sometimes even resulting in other crystal forms. Generally, amorphous drug products lack regular crystal structures and often have other defects, such as poor product stability, difficulty in filtration, easy agglomeration, and poor flowability. Therefore, studying their crystal forms is of great significance for developing drugs suitable for industrial production and with good biological activity. Summary of the Invention

[0008] This disclosure provides, in one aspect, the A crystal form of the compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine, and its X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, showing characteristic peaks at 7.237, 9.232, 13.702, 14.459, and 18.917.

[0009] In some embodiments, the A crystal form, in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ, has characteristic peaks at 7.237, 9.232, 13.702, 14.459, 18.917, 24.428, and 29.321.

[0010] In some embodiments, the A crystal form, in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ, has characteristic peaks at 7.237, 9.232, 13.702, 14.459, 18.033, 18.917, 24.428, 25.521, and 29.321.

[0011] In other embodiments, the X-ray powder diffraction pattern of the A crystal form, expressed in terms of a diffraction angle 2θ, is as follows: Figure 1 As shown.

[0012] This disclosure also provides a method for preparing the crystal form of the aforementioned compound A, said method being selected from any of the following methods:

[0013] Method 1:

[0014] (a) The compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine was mixed with solvent (1) and dissolved by stirring or heating.

[0015] (b) Add solvent (2) to induce crystallization.

[0016] The solvent (1) is selected from acetonitrile, methanol, ethanol, isopropanol, acetone, ethyl acetate, isopropyl acetate, tetrahydrofuran, methyl isopropyl ketone, dichloromethane, 10% water / methanol, 7% water / ethanol, 10% water / isopropanol or 10% water / acetone, and the solvent (2) is selected from water, cyclohexane or n-heptane.

[0017] Alternatively, method two:

[0018] (a) The compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine is mixed with solvent (3) and dissolved by stirring or heating.

[0019] (b) Crystallization,

[0020] The solvent (3) is selected from tetrahydrofuran, ethyl acetate or dichloromethane;

[0021] Alternatively, method three:

[0022] (a) The compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine was mixed with solvent (4).

[0023] (b) Stirring and mixing, wherein the solvent (4) is selected from water, cyclohexane, n-heptane, methanol, ethanol, isopropanol, dichloromethane, 1,4-dioxane, 10% water / methanol, 7% water / ethanol or 10% water / isopropanol.

[0024] This disclosure provides, in one aspect, the B crystal form of the compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine, and its X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, showing characteristic peaks at 8.205, 9.781, 12.87, 15.907, and 19.796.

[0025] In some embodiments, the B crystal form, in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ, has characteristic peaks at 8.205, 9.781, 12.870, 15.907, 19.448, 19.796, 20.264, and 23.185.

[0026] In some embodiments, the B crystal form, in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ, has characteristic peaks at 8.205, 9.781, 10.672, 12.87, 15.356, 15.907, 16.997, 19.448, 19.796, 20.264, and 23.185.

[0027] In other embodiments, the X-ray powder diffraction pattern of the B crystal form, expressed in terms of a diffraction angle 2θ, is as follows: Figure 2 As shown.

[0028] This disclosure also provides a method for preparing the crystal form of the aforementioned compound B, the method comprising:

[0029] (a) The compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine is mixed with solvent (5) and dissolved by stirring or heating.

[0030] (b) Add solvent (6) to induce crystallization.

[0031] Solvent (5) is selected from dimethyl sulfoxide, and solvent (6) is selected from water.

[0032] This disclosure provides, in one aspect, the C-crystal form of the compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine, and its X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, showing characteristic peaks at 9.296, 15.522, 18.784, 23.216, and 25.889.

[0033] In some embodiments, the C-type crystal form, in X-ray powder diffraction patterns expressed as diffraction angle 2θ, has characteristic peaks at 7.753, 9.296, 15.522, 18.784, 21.398, 23.216, and 25.889.

[0034] In some embodiments, the C-type crystal form, as expressed in X-ray powder diffraction patterns at diffraction angles of 2θ, has characteristic peaks at 7.353, 7.753, 9.296, 14.475, 15.522, 16.248, 17.320, 18.784, 21.398, 23.216, and 25.889.

[0035] In other embodiments, the X-ray powder diffraction pattern of the C-type, expressed in terms of a diffraction angle 2θ, is as follows: Figure 3 As shown.

[0036] This disclosure also provides a method for preparing the C crystal form of the aforementioned compound, the method comprising:

[0037] (a) The compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine is mixed with solvent (7) and dissolved by stirring or heating.

[0038] (b) Add solvent (8) to induce crystallization.

[0039] Solvent (7) is selected from 1,4-dioxane, and solvent (8) is selected from n-heptane.

[0040] This disclosure provides, in one aspect, the D crystal form of compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine, and its X-ray powder diffraction pattern expressed as a diffraction angle 2θ, showing characteristic peaks at 7.350, 12.084, 15.384, 18.643, and 29.312.

[0041] In some embodiments, the D crystal form, in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ, has characteristic peaks at 7.350, 12.084, 15.384, 16.260, 17.855, 18.643, 21.610, and 29.312.

[0042] In some embodiments, the D crystal form, in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ, has characteristic peaks at 7.350, 12.084, 15.384, 16.260, 17.855, 18.643, 21.610, 22.768, 24.347, 25.201, 26.038, and 29.312.

[0043] In other embodiments, the D crystal form, as expressed in X-ray powder diffraction patterns with a diffraction angle of 2θ, is as follows: Figure 4 As shown.

[0044] This disclosure also provides a method for preparing the D crystal form of the aforementioned compound, the method comprising:

[0045] (a) The compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxane-5-yl)quinoline-2-amine was mixed with solvent (9) and dissolved by stirring or heating.

[0046] (b) Add solvent (10) to induce crystallization.

[0047] Solvent (9) is selected from tetrahydrofuran, and solvent (10) is selected from water.

[0048] This disclosure provides, in one aspect, the E crystal form of the compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine, and its X-ray powder diffraction pattern expressed as a diffraction angle 2θ, which has characteristic peaks at 13.489, 18.000, 23.559, 24.276, and 26.328.

[0049] In some embodiments, the E crystal form, in X-ray powder diffraction patterns expressed as diffraction angles 2θ, has characteristic peaks at 13.489, 16.863, 18.000, 23.559, 24.276, 26.108, 26.328, and 27.094.

[0050] In some embodiments, the E crystal form, in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ, has characteristic peaks at 13.489, 14.587, 16.863, 18.000, 18.943, 23.279, 23.559, 24.276, 25.768, 26.108, 26.328, and 27.094.

[0051] In other embodiments, the X-ray powder diffraction pattern of the E crystal form, expressed in terms of a diffraction angle 2θ, is as follows: Figure 5 As shown.

[0052] This disclosure provides, in one aspect, the F-crystal form of the compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine, and its X-ray powder diffraction pattern expressed as a diffraction angle 2θ, showing characteristic peaks at 9.299, 14.439, 15.621, 16.200, and 17.314.

[0053] In some embodiments, the F crystal form, in X-ray powder diffraction patterns expressed as diffraction angle 2θ, has characteristic peaks at 7.734, 9.299, 14.439, 15.621, 16.200, 17.314, 21.395, and 25.814.

[0054] In some embodiments, the F crystal form, in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ, has characteristic peaks at 7.734, 9.299, 14.439, 15.621, 16.200, 17.314, 21.055, 21.395, 23.194, 25.814, 28.906, 34.485, and 43.544.

[0055] In other embodiments, the F crystal form, as expressed in X-ray powder diffraction patterns with a diffraction angle of 2θ, is as follows: Figure 6 As shown.

[0056] Furthermore, the X-ray powder diffraction patterns of the compounds of crystal form A, B, C, D, E, or F described in this disclosure are expressed in terms of the diffraction angle 2θ, wherein the error range of the 2θ angle is ±0.2.

[0057] In some embodiments, the method for preparing the crystal form described in this disclosure further includes any one of the steps of filtration, washing, or drying.

[0058] In some implementations, the crystallization includes, but is not limited to, agitation crystallization (dissolution crystallization, pulping crystallization) and volatilization crystallization.

[0059] In some embodiments, the drying method includes, but is not limited to, forced-air drying and vacuum drying. The drying temperature is generally 25°C to 100°C, preferably 30°C to 70°C, such as 40°C, 50°C, or 60°C.

[0060] On the other hand, this disclosure also provides a pharmaceutical composition comprising the aforementioned crystal form and a pharmaceutically acceptable excipient.

[0061] This disclosure also provides a pharmaceutical composition prepared from the aforementioned crystal form and a pharmaceutically acceptable excipient.

[0062] This disclosure also provides a method for preparing a pharmaceutical composition, including the step of mixing the aforementioned crystal form with a pharmaceutically acceptable excipient.

[0063] This disclosure also provides the use of the aforementioned crystal form or pharmaceutical composition in the preparation of a medicament for regulating miRNA levels; preferably, the miRNA is miR-124.

[0064] This disclosure also provides for use in medicaments for treating and / or preventing diseases or conditions selected from inflammation and cancer, using the aforementioned crystal form or pharmaceutical composition.

[0065] In some embodiments, the inflammation is inflammatory bowel disease. In some embodiments, the cancer is melanoma or breast cancer.

[0066] The "2θ or 2θ angle" mentioned in this disclosure refers to the diffraction angle, where θ is the Bragg angle, and the unit is ° or degree; the error range of 2θ for each characteristic peak is ±0.20 (including the case where the number has more than one decimal place after rounding), specifically -0.20, -0.19, -0.18, -0.17, -0.16, -0.15, -0.14, -0.13, -0.12, -0.11, -0.10, -0.09, -0.08, -0.07, -0.06, -0.05, -0.04, -0.03, -0.02, -0.01, 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20.

[0067] The “differential scanning calorimetry or DSC” described in this disclosure refers to measuring the temperature difference and heat flow difference between the sample and the reference material during the sample heating or isothermal process, in order to characterize all physical and chemical changes related to thermal effects and obtain phase transition information of the sample.

[0068] The drying temperature described in this disclosure is generally 25℃-100℃, preferably 30℃-70℃, and can be dried under normal pressure or reduced pressure.

[0069] The “pharmaceuticalally acceptable excipients” described in this disclosure include, but are not limited to, any adjuvant, carrier, flow aid, sweetener, diluent, preservative, dye / coloring agent, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, or emulsifier that has been approved by the U.S. Food and Drug Administration for use in humans or livestock.

[0070] The "pulping" described in this disclosure refers to a purification method that utilizes the characteristic that substances have poor solubility in solvents, but impurities have good solubility in solvents. Pulping purification can remove color, change crystal form, or remove a small amount of impurities.

[0071] The crystal forms described in this disclosure include, but are not limited to, solvates of compounds of formula (I), wherein the solvents include, but are not limited to, water. Attached Figure Description

[0072] Figure 1 The image shows the XRPD spectrum of compound 1 in crystal form A.

[0073] Figure 2 The image shows the XRPD spectrum of compound 1 in crystal form B.

[0074] Figure 3 The image shows the XRPD spectrum of the C crystal form of compound 1.

[0075] Figure 4 The image shows the XRPD spectrum of compound 1 in its D crystal form.

[0076] Figure 5 The image shows the XRPD spectrum of the E crystal form of compound 1.

[0077] Figure 6 The image shows the XRPD spectrum of the F-crystal form of compound 1. Detailed Implementation

[0078] This disclosure is further described in detail through the following embodiments and experimental examples. These embodiments and experimental examples are for illustrative purposes only and are not intended to limit the scope of this disclosure.

[0079] Test conditions of the instruments used in the experiment:

[0080] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) were expressed in 10⁻¹⁰. -6 The unit (ppm) is given. NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), with tetramethylsilane (TMS) as the internal standard.

[0081] MS measurements were performed using an Agilent 1200 / 1290 DAD-6110 / 6120 Quadrupole MS LC-MS system (manufacturer: Agilent, MS model: 6110 / 6120 Quadrupole MS). Other instruments used included a waters ACQuity UPLC-QD / SQD system (manufacturer: waters, MS model: waters ACQuity Qda Detector / waters SQ Detector) and a THERMO Ultimate 3000-Q Exactive system (manufacturer: THERMO, MS model: THERMO Q 15 Exactive).

[0082] HPLC determinations were performed using an Agilent 1260DAD high-performance liquid chromatograph (Sunfire C18 150×4.6mm column) and a Thermo U3000 high-performance liquid chromatograph (Gimini C18 150×4.6mm column).

[0083] XRPD (X-ray Powder Diffraction) was used for analysis. Measurements were performed using a BRUKER D8 X-ray diffractometer. Specific data acquisition information included: Cu anode (40 kV, 40 mA), and the radiation source was monochromatic Cu-Ka rays. Scanning mode: θ / 2θ, scanning range: 3-48°.

[0084] DSC stands for Differential Scanning Calorimetry: Measurements were performed using a METTLER TOLEDO DSC 3+ differential scanning calorimeter, with a heating rate of 10℃ / min, a temperature range of 25-300℃ or 25-350℃, and a nitrogen purging rate of 50mL / min.

[0085] TGA was performed using a METTLER TOLEDO TGA 2 thermogravimetric analyzer. The heating rate was 10℃ / min, and the specific temperature range was referenced from the corresponding chromatogram. The nitrogen purging rate was 50mL / min.

[0086] DVS (Dynamic Moisture Adsorption): Surface Measurement Systems instrinsic was used. The humidity range was 0%-95%, starting from 50%, with increments of 10%. The criterion was that the mass change dM / dT for each gradient ≤ 0.002%. The time was 360 min, and the cycle was repeated twice.

[0087] The known starting materials disclosed herein can be synthesized using or according to methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, AccelaChemBio Inc, and Darui Chemicals.

[0088] The reaction process in the examples was monitored using thin-layer chromatography (TLC). The developing solvent used in the reaction, the eluent system for column chromatography used to purify the compounds, and the developing solvent system for TLC included: A: dichloromethane / methanol system, B: n-hexane / ethyl acetate system. The volume ratio of the solvent was adjusted according to the polarity of the compounds, and small amounts of basic or acidic reagents such as triethylamine and acetic acid could also be added for adjustment.

[0089] Example 1. Synthesis of 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine (refer to the preparation method in Example 1 of application No. WO2022247920)

[0090]

[0091] 2,8-Dichloroquinoline 1a (100 mg, 0.51 mmol, Bio-Tech Pharmaceuticals) and 5-amino-2,2-difluoro-1,3-benzo[1,3]dioxane 1b (105 mg, 0.61 mmol, Shanghai Haohong) were dissolved in isopropanol (1 mL) and the mixture was heated to 90 °C for 12 hours. The reaction solution was filtered and then subjected to high performance liquid chromatography (Waters 2767-SQ Detecor2, elution system: 0.1% formic acid aqueous solution and acetonitrile, acetonitrile gradient: 65%-85%, flow rate: 30 mL / min) to obtain title compound 1 (150 mg, yield 89.0%).

[0092] MS m / z(ESI): 335.0 [M+1].

[0093] 1 H NMR (500MHz, DMSO-d6) δ9.99(s,1H),8.88(d,1H),8.17(d,1H),7.81(dd,1H),7.77(dd,1H),7.50(dd,1H),7.39(d,1H),7.32(t,1H),7.14(d,1H).

[0094] Test Example 1. Upregulation effect on miR-124

[0095] I. Experimental Materials and Instruments

[0096] 1. Dynabead Human T-Activator CD3 / CD28 for T Cell Expansion and Activation (Gibco, 11131D)

[0097] 2. Pan T Cell Isolation Kit (human) (Miltenyi, 130-096-535)

[0098] 3. Human interleukin-2 (Peprotech, 200-02-100)

[0099] 4. MicroRNA Extraction Kit (Qiagen, 217004)

[0100] 5. Small RNA Reverse Transcription Kit (miScript II RT Kit) (Qiagen, 218161)

[0101] 6. Small RNA SYBR Green PCR Kit (miScript SYBR Green PCR Kit) (Qiagen, 218073)

[0102] 7. Phosphate-buffered saline (PBS), pH 7.4 (Shanghai Yuanpei Biotechnology Co., Ltd., B320)

[0103] 8. Bovine serum albumin (BSA) (Beyotime, ST023)

[0104] 9.EDTA (0.5M), pH 8.0 (Invitrogen, AM9260G)

[0105] 10. LS Separation Columns (Miltenyi, 130-042-401)

[0106] 11. 24-well cell culture plate (Corning, 3524)

[0107] 12. 96-well plate (Corning, 3788)

[0108] 13. Cell incubator (Thermo, Steri cycle i160)

[0109] 14. Real-time quantitative PCR instrument (Applied biosystem, QuantStudio 6Flex)

[0110] 15.PCR instrument (Applied biosystem, ProFlex)

[0111] 16. 96-well clear PCR plate, 0.2 mL (Applied biosystems, N8010560)

[0112] 17. RPMI 1640 medium (Gibco, 11875119)

[0113] 18. Fetal bovine serum, FBS (Gibco, 10099-141)

[0114] 19. Magnetic rack (Invitrogen, DynaMag) TM -2)

[0115] 20. Six-well cell culture plate (Thermo, 150239)

[0116] 21. Spectrophotometer (IMPLEN, NP80)

[0117] 22. QuadroMACS Separator (Meitianni, 130-090-976)

[0118] 23. miR124-3P-F primers (custom-made by Genewiz)

[0119] 24. HSA-U6 detection primers (Tiangen, CD201-0145)

[0120] II. Experimental Procedure

[0121] The effect of the compound on miR-124 expression levels was detected in T cells activated by CD3 / CD28 antibody. After treatment with the compound, total RNA was extracted from the activated T cells, and the cDNA obtained from reverse transcription was used as a template. Quantification was performed using SYBR Green real-time PCR with specific miR-124 primers.

[0122] T cell isolation: Purchased human peripheral blood mononuclear cells (PBMCs), counted and filtered, washed once with separation buffer (PBS pH 7.4, containing 0.5% BSA and 2mM EDTA), discarded the supernatant, and divided into T cells at a ratio of 1×10⁻⁶ cells / mL. 7 Add 40 μL of buffer and 10 μL of pan T cell biotinylated antibody (pan T Cell Biotin-Antibody Cocktail) to each cell, resuspend the precipitate, mix well, and incubate at 4°C for 5 minutes. After incubation, repeat the process at 1 × 10⁻⁶ cells / cell. 7Add 30 μL of buffer and 20 μL of Pan T Cell MicroBeads Cocktail to each cell, mix well, and incubate at 4°C for 10 minutes. Pre-wash the separation column (LS column) with 3 mL of cell separation buffer, pass the cell suspension through the column, and wash the column three times with 1 mL of cell separation buffer after passing the cell suspension. Collect the effluent in a 15 mL filter tube; this is the enriched T cells. Count the cells at a ratio of 1 × 10⁻⁶. 6 Add cells at a density of 10% FBS and 40 U / mL IL-2 to RPMI 1640 medium (complete medium) and store on ice for later use.

[0123] T cell activation: per 1×10 6 Add 25 μL of activating magnetic beads to each cell. Take the corresponding amount of T cell-activating CD3 / CD28 magnetic beads and place them in a 1.5 mL filter tube. Shake the tube for approximately 30 seconds before aspirating. Wash the activated magnetic beads three times with culture medium at a volume ratio greater than 1:1. Remove all washings on the last wash and resuspend the activated magnetic beads in an equal volume of complete culture medium. Add the washed activated magnetic beads to the cell resuspension and mix thoroughly. Remove a six-well plate and add 3 mL of cells to each well. Incubate at 37°C in a 5% CO2 cell culture incubator for 2 days.

[0124] Compound preparation: The stock solution of the compound was 20 mM, diluted to 200 μM with DMSO, and then diluted 4-fold with complete culture medium to 50 μM (50×). Mix well and set aside. A 4-fold dilution with 25% DMSO served as a negative control. T cells activated for two days were pipetted to homogenize them. Using a magnetic rack with a 1.5 mL filter tube attached, the activation beads were removed, and the cell suspension was collected. After cell counting, the cells were incubated at 300 x g for 10 min, filtered, and the supernatant was discarded. The cells were resuspended to 1.02 × 10⁻⁶. 6 Add 980 μL of cell suspension and 20 μL of 50× compound to each well of a 24-well plate, bringing the final compound concentration to 1 μM. Incubate the cells at 37°C in a 5% CO2 cell culture incubator for 3 days.

[0125] RNA extraction: Collect T cells by filtration, filter at 1500 rpm for 3 minutes, wash once with PBS, and discard the supernatant after filtration. Extract total RNA from cells using a small RNA extraction kit according to the manufacturer's instructions. Add 700 μL of Trizol cell lysis buffer to the cell pellet, mix well by pipetting, and incubate at room temperature for 5 minutes. Add 140 μL of chloroform, vortex to mix, and incubate at room temperature for 3 minutes. Filter the chloroform-cell lysis buffer mixture at 12000 xg for 15 minutes at 4°C. Transfer the supernatant to a new RNase-free filter tube, add 1.5 volumes of anhydrous ethanol, and mix several times by pipetting. Transfer the solution to an RNA adsorption column and filter at 8000 xg for 15 seconds. Wash the filter column once with 700 μL of RWT solution, filter at 8000 xg for 15 seconds, wash twice with 500 μL of RPE solution, and filter at 8000 xg for 2 minutes. Place the adsorption column into a new 2 mL filter tube and filter at 12000 x g for 1 min to remove residual wash buffer. Place the adsorption column into a new 1.5 mL filter tube, add 30-50 μL of RNase-free water, and filter at 12000 x g for 2 min. The collected solution is the RNA solution; measure the RNA concentration using a spectrophotometer. Store the RNA solution at -80°C.

[0126] Reverse transcription: Place the extracted RNA template on ice. Remove the small RNA reverse transcription kit and thaw a portion of its components (containing 5×miScript HiSpec Buffer, 10×miScript nucleics Mix, and RNase-free water) at room temperature. Thaw the miScript Reverse Transcriptase mix component on ice. Each reaction (10 μL) consists of: 5×miScript HiSpec Buffer (2 μL), 10×miScript nucleics Mix (1 μL), miScript Reverse Transcriptase mix (1 μL), RNase-free water (2 μL), and RNA template (4 μL). Prepare the reaction on ice. Place the samples in a PCR instrument and set the program as follows: 37℃, 60 minutes; 95℃, 5 minutes; store at 4℃. The completed reaction sample is the cDNA sample.

[0127] Quantitative real-time PCR: The transcriptional level of miR-124 was detected using SYBR Green staining, while the transcriptional level of the housekeeping gene U6 was detected as an internal control. Thaw all reagents required for the small RNA SYBR Green PCR kit to room temperature. Dilute each cDNA sample template 10-fold with RNase-free water, then dilute 5-fold. Prepare the reaction mixture according to Table 1 below, add the reaction mixture to a 96-well PCR plate, seal with sealing film, and filter. Perform the PCR reaction on a quantitative real-time PCR instrument according to the steps in Table 2.

[0128] Table 1. Components of Real-Time PCR Reaction

[0129]

[0130] Table 2. Steps for Real-Time PCR

[0131]

[0132] Table 3 Primer list for quantitative real-time PCR detection

[0133]

[0134] Data Analysis: Based on the CT values ​​calculated by the software, the ratio of miR-124 expression level to the internal reference U6 expression level for each sample was calculated, i.e., ΔCT (test compound) = CT. miRNA-124 (Test Compound)-CT U6 (Test compound). The relative expression level is calculated using the following formula: Relative expression level (test compound) = 2 (-[ΔCT(测试化合物)-ΔCT(DMSO)]) .

[0135] Compound 1 upregulated miR-124 by 3.9-fold, demonstrating good activity in promoting miR124 upregulation.

[0136] Example 2: Preparation of Crystal Form A

[0137] Take 250 mg of compound 1, add it to 2.5 mL of acetonitrile and stir to dissolve. Add 17.5 mL of water and stir to crystallize. Slurry at room temperature for 4 days, filter, and vacuum dry to obtain solid.

[0138] X-ray powder diffraction analysis determined the product to be crystal form A. The X-ray powder diffraction data are shown in Table 4, and the X-ray powder diffraction pattern is shown below. Figure 1 As shown.

[0139] The DSC spectrum shows an endothermic peak at 177.14℃.

[0140] The TGA spectrum showed a weight loss of 0.22% between 30℃ and 100℃.

[0141] DVS testing showed that under normal storage conditions (i.e., room temperature, 60% RH), the sample's moisture absorption weight gain was approximately 0.02%; under accelerated testing conditions (i.e., 70% RH), the moisture absorption weight gain was approximately 0.02%; and under extreme conditions (i.e., 90% RH), the moisture absorption weight gain was approximately 0.18%. Furthermore, retesting of the crystal form after DVS testing showed no change in crystal form.

[0142] Table 4

[0143]

[0144]

[0145] Example 3: Preparation of Crystal Form A

[0146] Compound 1 (6 mg, 17.63 μmol) was dissolved in 0.3 mL of tetrahydrofuran. After dissolution, the solid was slowly volatilized and dried under vacuum at 45 °C for 3 hours to obtain the product.

[0147] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0148] Example 4: Preparation of Crystal Form A

[0149] Compound 1 (11 mg, 32.87 μmol) was dissolved in 0.4 mL of ethyl acetate. After dissolution, the solid was slowly evaporated and dried under vacuum at 45 °C for 3 hours to obtain the product.

[0150] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0151] Example 5: Preparation of Crystal Form A

[0152] Compound 1 (14 mg, 41.83 μmol) was dissolved in 0.5 mL of dichloromethane. After dissolution, the solid was slowly volatilized and dried under vacuum at 45 °C for 3 hours to obtain the product.

[0153] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0154] Example 6: Preparation of Crystal Form A

[0155] Compound 1 (1.70 g, 5.08 mmol) was dispersed in 35 mL of cyclohexane, stirred and slurried for 48 hours, filtered and the filter cake was collected, and dried under vacuum at 45 °C for 16 hours to obtain the product.

[0156] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0157] Example 7: Preparation of Crystal Form A

[0158] Weigh 10 mg of compound 1, add 1 ml of purified water and stir at room temperature for 3 days. After filtration, dry the solid under vacuum to obtain the product.

[0159] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0160] Example 8: Preparation of Crystal Form A

[0161] Following the method in Example 7, 10 mg of compound 1 was weighed, added to a solvent, slurried, filtered, and then dried under vacuum to obtain the product.

[0162] X-ray powder diffraction analysis showed that the product was crystal form A, and the data are shown in Table 5.

[0163] Table 5

[0164]

[0165]

[0166] Example 9: Preparation of Crystal Form A

[0167] Weigh 10 mg of compound 1, add 0.2 ml of methanol and stir at room temperature to dissolve it. Add 0.2 ml of purified water and stir to crystallize. Slurry at room temperature for 1 day. After filtration, dry the solid under vacuum to obtain the product.

[0168] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0169] Example 10: Preparation of Crystal Form A

[0170] Weigh 10 mg of compound 1, add 0.2 ml of ethanol and stir at room temperature until dissolved, then add 0.4 ml of n-heptane and stir for 1 day. The product is obtained by volatilization and crystallization.

[0171] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0172] Example 11: Preparation of Crystal Form A

[0173] Weigh 10 mg of compound 1, add 0.6 ml of isopropanol and stir at room temperature until dissolved, then add 0.4 ml of n-heptane and stir for 1 day. The product is obtained by volatilization and crystallization.

[0174] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0175] Example 12: Preparation of Crystal Form A

[0176] Weigh 10 mg of compound 1, add 0.1 ml of acetone and stir at room temperature to dissolve it. Add 0.4 ml of n-heptane and stir for 1 day. Volatilize and crystallize to obtain the product.

[0177] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0178] Example 13: Preparation of Crystal Form A

[0179] Weigh 10 mg of compound 1, add 0.1 ml of ethyl acetate and stir at room temperature until dissolved. Add 0.4 ml of n-heptane and stir for 1 day. Volatilize and crystallize to obtain the product.

[0180] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0181] Example 14: Preparation of Crystal Form A

[0182] Weigh 10 mg of compound 1, add 0.1 ml of isopropyl acetate and stir at room temperature to dissolve. Add 0.4 ml of n-heptane and stir to crystallize. Pulp at room temperature for 1 day, add 0.2 ml of n-heptane and continue pulverizing for 3 hours. After filtration, dry the solid under vacuum to obtain the product.

[0183] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0184] Example 15: Preparation of Crystal Form A

[0185] Weigh 10 mg of compound 1, add 0.1 ml of tetrahydrofuran and stir at room temperature until dissolved, then add 0.4 ml of n-heptane and stir for 1 day. The product is obtained by volatilization and crystallization.

[0186] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0187] Example 16: Preparation of Crystal Form A

[0188] Weigh 10 mg of compound 1, add 0.1 ml of methyl isopropyl ketone and stir at room temperature until dissolved. Add 0.4 ml of n-heptane and stir for 1 day. Volatilize and crystallize to obtain the product.

[0189] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0190] Example 17: Preparation of Crystal Form A

[0191] Weigh 10 mg of compound 1, add 0.4 ml of dichloromethane and stir at room temperature to dissolve it. Add 0.4 ml of n-heptane to crystallize it. Slurry at room temperature for 1 day. Add 0.2 ml of n-heptane and slurry for 3 hours. Filter and dry the solid under vacuum to obtain the product.

[0192] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0193] Example 18: Preparation of Crystal Form A

[0194] Weigh 10 mg of compound 1, add 1 ml of 10% water / methanol and stir at room temperature to dissolve. Add 0.1 ml of water and stir to crystallize. Add another 0.1 ml of water and continue stirring for 1 day. After filtration, dry the solid under vacuum to obtain the product.

[0195] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0196] Example 19: Preparation of Crystal Form A

[0197] Weigh 10 mg of compound 1, add 0.6 ml of 7% water / ethanol and stir at room temperature until dissolved, add 0.4 ml of n-heptane, and allow to evaporate and crystallize to obtain the product.

[0198] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0199] Example 20: Preparation of Crystal Form A

[0200] Weigh 10 mg of compound 1, add 0.6 ml of 10% water / isopropanol and stir at room temperature until dissolved. Add 0.4 ml of n-heptane and allow to evaporate and crystallize to obtain the product.

[0201] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0202] Example 21: Preparation of Crystal Form A

[0203] Weigh 10 mg of compound 1, add 0.1 ml of 10% water / acetone and stir at room temperature until dissolved, then add 0.4 ml of n-heptane and allow to evaporate and crystallize to obtain the product.

[0204] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0205] Example 22: Preparation of Crystal Form A

[0206] Weigh 10 mg of compound 1, add 0.2 ml of ethanol and stir to dissolve at room temperature, add 0.1 ml of purified water and stir to precipitate, continue to add 0.2 ml of purified water and stir for 1 day, filter and dry the solid under vacuum to obtain the product.

[0207] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0208] Example 23: Preparation of Crystal Form A

[0209] Weigh 10 mg of compound 1, add 0.2 ml of ethanol and stir to dissolve at room temperature. Add 0.4 ml of cyclohexane and stir for 1 day. The product is obtained by volatilization and crystallization.

[0210] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0211] Example 24: Preparation of Crystal Form A

[0212] Weigh 10 mg of compound 1, add 0.1 ml of acetone and stir to dissolve at room temperature, add 0.1 ml of purified water and stir to precipitate, add another 0.2 ml of purified water and stir for 1 day, filter and dry the solid under vacuum to obtain the product.

[0213] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0214] Example 25: Preparation of Crystal Form A

[0215] Weigh 10 mg of compound 1, add 0.1 ml of acetone and stir to dissolve at room temperature. Add 0.4 ml of cyclohexane and stir for 1 day. The product is obtained by volatilization and crystallization.

[0216] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0217] Example 26: Preparation of Crystal Form A

[0218] Weigh 10 mg of compound 1, add 0.1 ml of ethyl acetate and stir to dissolve at room temperature. Add 0.4 ml of cyclohexane and stir for 1 day. The product is then obtained by volatilization and crystallization.

[0219] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0220] Example 27: Preparation of Crystal Form A

[0221] Weigh 10 mg of compound 1, add 0.1 ml of ethyl acetate and stir to dissolve at room temperature. Add 0.4 ml of n-heptane and stir for 1 day. The product is then obtained by volatilization and crystallization.

[0222] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0223] Example 28: Preparation of Crystal Form A

[0224] Weigh 10 mg of compound 1, add 0.1 ml of methyl tert-butyl ether and stir to dissolve at room temperature. Add 0.1 ml of cyclohexane to precipitate the product. Continue to add 0.2 ml of cyclohexane and stir for 1 day. After filtration, dry the solid under vacuum to obtain the product.

[0225] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0226] Example 29: Preparation of Crystal Form A

[0227] Weigh 10 mg of compound 1, add 0.1 ml of tetrahydrofuran and stir to dissolve at room temperature. Add 0.4 ml of cyclohexane and stir for 1 day. The product is obtained by volatilization and crystallization.

[0228] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0229] Example 30: Preparation of Crystal Form A

[0230] Weigh 10 mg of compound 1, add 0.1 ml of methyl isobutyl ketone and stir to dissolve at room temperature. Add 0.4 ml of cyclohexane and stir for 1 day. The product is then obtained by volatilization and crystallization.

[0231] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0232] Example 31: Preparation of Crystal Form A

[0233] Weigh 10 mg of compound 1, add 0.1 ml of 10% water / acetone and stir to dissolve at room temperature. Add 0.1 ml of purified water to precipitate the product. Continue to add 0.2 ml of purified water and stir for 1 day. After filtration, dry the solid under vacuum to obtain the product.

[0234] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0235] Example 32: Preparation of Crystal Form A

[0236] Weigh 10 mg of compound 1, add 0.1 ml of isopropyl acetate and stir to dissolve at room temperature, then add 0.4 ml of cyclohexane and allow to evaporate and crystallize to obtain the product.

[0237] X-ray powder diffraction analysis revealed that the product is crystal form A.

[0238] Example 33: Preparation of B crystal form

[0239] Weigh 10 mg of compound 1, add 0.1 ml of dimethyl sulfoxide to dissolve it, add 0.2 ml of water to crystallize it, continue to slurry for 1 day, filter and vacuum dry to obtain a solid.

[0240] X-ray powder diffraction analysis determined the product to be crystal form B. The XRPD spectrum is shown below. Figure 2 The positions of its characteristic peaks are shown in Table 6.

[0241] The DSC spectrum shows endothermic peaks of 81.99 and 173.56 °C.

[0242] The TGA spectrum showed a weight loss of 18.35% between 30℃ and 100℃.

[0243] Table 6

[0244]

[0245]

[0246] Example 34: Preparation of C-type crystal

[0247] Weigh 30 mg of compound 1, add it to 1.2 ml of 1,4-dioxane and stir to dissolve. Add 1.8 ml of n-heptane, stir to crystallize, filter and dry under vacuum to obtain a solid.

[0248] X-ray powder diffraction analysis determined the product to be of C-type crystal form. The XRPD spectrum is shown below. Figure 3 The positions of its characteristic peaks are shown in Table 7.

[0249] The DSC spectrum shows endothermic peaks at 141.90 and 176.81 °C.

[0250] The TGA spectrum showed a weight loss of 1.89% at 30℃-85℃ and a weight loss of 5.94% at 85℃-160℃.

[0251] Table 7

[0252]

[0253]

[0254] Example 35: Preparation of D-type crystal

[0255] Weigh 30 mg of compound 1, add 0.3 ml of tetrahydrofuran and stir to dissolve at room temperature, add 1.2 ml of water and stir to crystallize, filter and vacuum dry to obtain the product solid.

[0256] X-ray powder diffraction analysis determined the product to be of crystal form D. The XRPD spectrum is shown below. Figure 4 The positions of its characteristic peaks are shown in Table 8.

[0257] The DSC spectrum shows an endothermic peak at 176.85℃.

[0258] The TGA spectrum showed a weight loss of 0.34% between 30℃ and 100℃.

[0259] Table 8

[0260]

[0261]

[0262] Example 36: Preparation of E-crystal form

[0263] Weigh 100 mg of compound 1 and disperse it in 1 mL of acetonitrile. Stir and beat for 48 hours. After filtration, collect the solid and dry it under vacuum at 45 °C to obtain the product solid.

[0264] X-ray powder diffraction analysis determined the product to be of crystal form E. The X-ray powder diffraction data are shown in Table 9, and the X-ray powder diffraction pattern is shown below. Figure 5 As shown.

[0265] The DSC spectrum shows that the endothermic peak values ​​are 88.25℃ and 177.28℃.

[0266] TGA spectra showed that the compound lost 1.80% of its weight from 30℃ to 115℃ and 5.29% of its weight from 115℃ to 230℃.

[0267] Table 9

[0268]

[0269]

[0270] Example 37: Preparation of F-type crystal

[0271] Weigh 10 mg of compound 1, add 0.4 ml of 1,4-dioxane solvent, add 0.4 ml of n-heptane and stir for 1 day to crystallize. Add 0.2 ml of n-heptane and stir for 3 hours. After filtration, evaporate the supernatant to crystallize and obtain the product solid.

[0272] X-ray powder diffraction analysis determined the product to be of crystal form F. The XRPD spectrum is shown below. Figure 6 The positions of its characteristic peaks are shown in Table 10.

[0273] Table 10

[0274]

[0275] Test Example 2: Influencing Factors

[0276] The A-type crystal was laid flat in the open, and the stability of the sample was investigated under light (4500 Lux), high temperature (40℃, 60℃), and high humidity (RH75%, RH92.5%) conditions. The sampling period was 30 days.

[0277] Table 11 Factors affecting crystal form stability

[0278]

[0279] Conclusion: The influencing factor experiment shows that the free A crystal form has good physicochemical stability after 30 days under the conditions of light, high temperature of 40℃ and 60℃, and high humidity of 75% and 92.5%.

[0280] Test Example 3: Long-Term Accelerated Test

[0281] The stability of crystal form A was investigated under conditions of 25℃ / 60%RH and 40℃ / 75%RH.

[0282] Table 12 Long-term accelerated stability of crystal forms

[0283]

[0284] Conclusion: Long-term accelerated experiments show that the A crystal form exhibits good physicochemical stability under conditions of 25℃ / 60%RH and 40℃ / 75%RH for 6 months.

Claims

1. Compound 8-chloro- N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 7.237, 9.232, 13.702, 14.459, and 18.

917.

2. The 8-chloro- according to claim 1 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 7.237, 9.232, 13.702, 14.459, 18.917, 24.428, and 29.

321.

3. The 8-chloro- according to claim 1 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 7.237, 9.232, 13.702, 14.459, 18.033, 18.917, 24.428, 25.521, and 29.

321.

4. The 8-chloro- according to claim 1 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern expressed in terms of angle is shown in Figure 1.

5. Compound 8-chloro- N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 8.205, 9.781, 12.870, 15.907, and 19.

796.

6. The 8-chloro- according to claim 5 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 8.205, 9.781, 12.870, 15.907, 19.448, 19.796, 20.264, and 23.

185.

7. The 8-chloro- according to claim 5 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 8.205, 9.781, 10.672, 12.870, 15.356, 15.907, 16.997, 19.448, 19.796, 20.264, and 23.

185.

8. The 8-chloro- according to claim 5 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern expressed in terms of angle is shown in Figure 2.

9. Compound 8-chloro- N -(2,2-difluorobenzo[ d The C-form of [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 9.296, 15.522, 18.784, 23.216, and 25.

889.

10. The 8-chloro- according to claim 9 N -(2,2-difluorobenzo[ d The C-form of [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 7.753, 9.296, 15.522, 18.784, 21.398, 23.216, and 25.

889.

11. The 8-chloro- according to claim 9 N -(2,2-difluorobenzo[ d The C-form of [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 7.353, 7.753, 9.296, 14.475, 15.522, 16.248, 17.320, 18.784, 21.398, 23.216, and 25.

889.

12. The 8-chloro- according to claim 9 N -(2,2-difluorobenzo[ d The C-form of [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern expressed in terms of angle is shown in Figure 3.

13. Compound 8-chloro- N -(2,2-difluorobenzo[ d The D-phase of [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 7.350, 12.084, 15.384, 18.643, and 29.

312.

14. The 8-chloro- according to claim 13 N -(2,2-difluorobenzo[ d The D-phase of [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 7.350, 12.084, 15.384, 16.260, 17.855, 18.643, 21.610, and 29.

312.

15. The 8-chloro- according to claim 13 N -(2,2-difluorobenzo[ d The D-phase of [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 7.350, 12.084, 15.384, 16.260, 17.855, 18.643, 21.610, 22.768, 24.347, 25.201, 26.038, and 29.

312.

16. The 8-chloro- according to claim 13 N -(2,2-difluorobenzo[ d The D-phase of [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern expressed in terms of angle is shown in Figure 4.

17. Compound 8-chloro- N -(2,2-difluorobenzo[ d The E-crystal form of [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 13.489, 18.000, 23.559, 24.276, and 26.

328.

18. The 8-chloro- according to claim 17 N -(2,2-difluorobenzo[ d The E-crystal form of [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 13.489, 16.863, 18.000, 23.559, 24.276, 26.108, 26.328, and 27.

094.

19. The 8-chloro- according to claim 17 N -(2,2-difluorobenzo[ d The E-crystal form of [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 13.489, 14.587, 16.863, 18.000, 18.943, 23.279, 23.559, 24.276, 25.768, 26.108, 26.328, and 27.

094.

20. The 8-chloro- according to claim 17 N -(2,2-difluorobenzo[ d The E-crystal form of [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern expressed in terms of angle is shown in Figure 5.

21. Compound 8-chloro- N -(2,2-difluorobenzo[ d The F-form of [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 9.299, 14.439, 15.621, 16.200, and 17.

314.

22. The 8-chloro- according to claim 21 N -(2,2-difluorobenzo[ d The F-form of [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 7.734, 9.299, 14.439, 15.621, 16.200, 17.314, 21.395, and 25.

814.

23. The 8-chloro- according to claim 21 N -(2,2-difluorobenzo[ d The F-form of [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 7.734, 9.299, 14.439, 15.621, 16.200, 17.314, 21.055, 21.395, 23.194, 25.814, 28.906, 34.485, and 43.

544.

24. The 8-chloro- according to claim 21 N -(2,2-difluorobenzo[ d The F-form of [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern expressed in terms of angle is shown in Figure 6.

25. The crystal form according to any one of claims 1-24, characterized in that... The 2 θ The error range is ±0.

2.

26. A method for preparing the A crystal form according to any one of claims 1-4, selected from any of the following methods, Method 1: (a) The compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine was mixed with solvent (1) and dissolved by stirring or heating. (b) Add solvent (2) to induce crystallization. The solvent (1) is selected from acetonitrile, methanol, ethanol, isopropanol, acetone, ethyl acetate, isopropyl acetate, tetrahydrofuran, methyl isopropyl ketone, dichloromethane, 10% water / methanol, 7% water / ethanol, 10% water / isopropanol or 10% water / acetone, and the solvent (2) is selected from water, cyclohexane or n-heptane. Alternatively, method two: (a) The compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine is mixed with solvent (3) and dissolved by stirring or heating. (b) Crystallization, The solvent (3) is selected from tetrahydrofuran, ethyl acetate or dichloromethane; Alternatively, method three: (a) The compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine was mixed with solvent (4). (b) Stirring and mixing, wherein the solvent (4) is selected from water, cyclohexane, n-heptane, methanol, ethanol, isopropanol, dichloromethane, 1,4-dioxane, 10% water / methanol, 7% water / ethanol or 10% water / isopropanol.

27. A method for preparing the B crystal form according to any one of claims 5-8, the method comprising: (a) The compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine is mixed with solvent (5) and dissolved by stirring or heating. (b) Add solvent (6) to induce crystallization. Solvent (5) is selected from dimethyl sulfoxide, and solvent (6) is selected from water.

28. A method for preparing the C-type crystal form according to any one of claims 9-12, the method comprising: (a) The compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine is mixed with solvent (7) and dissolved by stirring or heating. (b) Add solvent (8) to induce crystallization. Solvent (7) is selected from 1,4-dioxane, and solvent (8) is selected from n-heptane.

29. A method for preparing the D-type crystal form according to any one of claims 13-16, the method comprising: (a) The compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxane-5-yl)quinoline-2-amine was mixed with solvent (9) and dissolved by stirring or heating. (b) Add solvent (10) to induce crystallization. Solvent (9) is selected from tetrahydrofuran, and solvent (10) is selected from water.

30. A pharmaceutical composition comprising the crystal form as described in any one of claims 1-25, and a pharmaceutically acceptable excipient.

31. A pharmaceutical composition prepared from the crystal form and pharmaceutically acceptable excipients as described in any one of claims 1-25.

32. Use of the crystal form according to any one of claims 1-25 or the pharmaceutical composition according to claim 30 or 31 in the preparation of a medicament for treating and / or preventing miR-124-mediated diseases or conditions selected from inflammation and cancer.

33. The use according to claim 32, wherein the inflammation is selected from inflammatory bowel disease.

34. The use according to claim 32, wherein the cancer is selected from melanoma or breast cancer.

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