A pharmaceutically acceptable salt of a quinolinamine compound, a crystal form thereof and a preparation method thereof

Abstract

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

Description

[0001] This application claims priority to Chinese patent application 2022114917754, 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 pharmaceutically acceptable salt, crystal form, and preparation method of a quinolinamine compound. 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] Salt formation can improve certain undesirable physicochemical or biological properties of drugs. Developing salts with superior physicochemical or pharmaceutical properties compared to 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine is of great significance. Given the importance of the crystal form and stability of solid drugs in clinical treatment, in-depth research into the polymorphisms of pharmaceutically viable salts of 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine is also crucial for developing drugs suitable for industrial production and possessing good biological activity. Summary of the Invention

[0008] This disclosure provides, in one aspect, a pharmaceutically acceptable salt of the compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine.

[0009] In some embodiments, the pharmaceutically acceptable salt is selected from p-toluenesulfonate, phosphate, sulfate, maleate, hydrobromide and hydrochloride.

[0010] In other embodiments, the chemical ratio of the compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine to the acid molecule is 1:0.5 to 1:3, including 1:0.5, 1:1, 1:2 or 1:3.

[0011] In some embodiments, the compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine is stoichiometric in a ratio of 1:0.5 or 1:1 to the acid molecule.

[0012] This disclosure also provides a method for preparing a pharmaceutically acceptable salt of the compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine, comprising the step of forming a salt of the compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine with an acid.

[0013] In some implementations, the acid used in the salt formation reaction is selected from p-toluenesulfonic acid, phosphoric acid, sulfuric acid, maleic acid, hydrobromide, and hydrochloric acid.

[0014] In some embodiments, the solvent used for the salt formation reaction is selected from at least one of ethanol, acetonitrile, acetone, methanol, and water.

[0015] On the other hand, this disclosure also provides the p-toluenesulfonate I crystal form of compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxane-5-yl)quinoline-2-amine, and the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ, with characteristic peaks at 6.616, 8.996, 14.120, 19.138 and 22.592.

[0016] In some embodiments, the X-ray powder diffraction pattern of the p-toluenesulfonate I crystal form, expressed as a diffraction angle 2θ, has characteristic peaks at 6.616, 8.996, 14.120, 17.120, 19.138, 20.615, 22.592, and 25.033.

[0017] In some embodiments, the X-ray powder diffraction pattern of the p-toluenesulfonate I crystal form, expressed as a diffraction angle 2θ, has characteristic peaks at 6.616, 8.996, 13.304, 14.120, 17.120, 19.138, 20.615, 21.289, 22.592, 25.033, 26.101, and 26.503.

[0018] In other embodiments, the X-ray powder diffraction pattern of the p-toluenesulfonate I crystal form, expressed in terms of a diffraction angle 2θ, is as follows: Figure 1 As shown.

[0019] This disclosure also provides a method for preparing the p-toluenesulfonate I crystal form of the aforementioned compound, comprising mixing the compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxane-5-yl)quinoline-2-amine with a solvent (1), (b) adding p-toluenesulfonic acid, and crystallizing, wherein the solvent (1) is selected from at least one of ethanol, acetone, acetonitrile, methanol, and water.

[0020] On the other hand, this disclosure also provides the phosphate I crystal form of compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine, and the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ, with characteristic peaks at 6.511, 9.685, 12.660, 14.012 and 15.704.

[0021] In some embodiments, the X-ray powder diffraction pattern of the phosphate I crystal form, expressed as a diffraction angle 2θ, has characteristic peaks at 6.511, 9.685, 12.660, 14.012, 15.704, 16.517, 21.487, and 22.209.

[0022] In some embodiments, the X-ray powder diffraction pattern of the phosphate I crystal form, expressed as a diffraction angle 2θ, has characteristic peaks at 6.511, 9.685, 12.660, 14.012, 15.704, 16.517, 18.507, 21.487, 22.209, 24.493, and 25.596.

[0023] In other embodiments, the X-ray powder diffraction pattern of the phosphate I crystal form, expressed in terms of a diffraction angle 2θ, is as follows: Figure 2 As shown.

[0024] This disclosure also provides a method for preparing the phosphate I crystal form of the aforementioned compound, comprising mixing the compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine with solvent (2), (b) adding phosphoric acid, and crystallizing, wherein the solvent (2) is selected from acetonitrile.

[0025] On the other hand, this disclosure also provides the sulfate I crystal form of compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxane-5-yl)quinoline-2-amine, and the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ, which has characteristic peaks at 5.687, 11.448, 14.670, 17.315, 23.323 and 24.907.

[0026] In some embodiments, the sulfate I crystal form, as expressed in X-ray powder diffraction patterns at diffraction angles of 2θ, has characteristic peaks at 5.687, 9.540, 11.448, 14.670, 17.315, 18.906, 23.323, and 24.907.

[0027] In some embodiments, the sulfate I crystal form, as expressed in X-ray powder diffraction patterns at diffraction angles of 2θ, has characteristic peaks at 5.687, 9.540, 11.448, 12.137, 14.670, 17.315, 17.465, 18.906, 21.682, 23.323, and 24.907.

[0028] In other embodiments, the X-ray powder diffraction pattern of the sulfate I crystal form, expressed in terms of a diffraction angle 2θ, is as follows: Figure 3 As shown.

[0029] This disclosure also provides a method for preparing the sulfate I crystal form of the aforementioned compound, comprising mixing the compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine with a solvent (3), (b) adding sulfuric acid, and crystallizing, wherein the solvent (3) is selected from ethanol or acetone.

[0030] On the other hand, this disclosure also provides the sulfate II 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 in terms of diffraction angle 2θ, with characteristic peaks at 11.895, 13.102, 15.607, 16.658 and 17.967.

[0031] In some embodiments, the sulfate II crystal form, in X-ray powder diffraction patterns expressed as diffraction angle 2θ, has characteristic peaks at 6.518, 11.895, 13.102, 15.607, 16.181, 16.658, 17.967, and 24.329.

[0032] In some embodiments, the sulfate II crystal form, as expressed in X-ray powder diffraction patterns at diffraction angles of 2θ, has characteristic peaks at 6.518, 10.808, 11.291, 11.895, 13.102, 15.196, 15.607, 16.181, 16.658, 17.967, and 24.329.

[0033] In other embodiments, the X-ray powder diffraction pattern of the sulfate II crystal form, expressed in terms of a diffraction angle 2θ, is as follows: Figure 4 As shown.

[0034] This disclosure also provides a method for preparing the sulfate II crystal form of the aforementioned compound, comprising mixing the compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine with a solvent (4), (b) adding sulfuric acid, and crystallizing, wherein the solvent (4) is selected from acetonitrile.

[0035] On the other hand, this disclosure also provides the sulfate III crystal form of compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine, and the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ, with characteristic peaks at 13.502, 16.610, 17.858, 22.247 and 26.671.

[0036] In some embodiments, the sulfate III crystal form, in X-ray powder diffraction patterns expressed as diffraction angle 2θ, has characteristic peaks at 8.675, 13.502, 16.610, 17.858, 22.247, 25.379, 26.671, and 27.029.

[0037] In some embodiments, the sulfate III crystal form, as expressed in X-ray powder diffraction patterns at diffraction angles of 2θ, has characteristic peaks at 8.675, 13.502, 16.610, 17.449, 17.858, 22.247, 24.292, 25.379, 26.671, and 27.029.

[0038] In other embodiments, the X-ray powder diffraction pattern of the sulfate III crystal form, expressed in terms of a diffraction angle of 2θ, is as follows: Figure 5 As shown.

[0039] This disclosure also provides a method for preparing the sulfate III crystal form of the aforementioned compound, comprising mixing the compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine with a solvent (5), (b) adding sulfuric acid, and crystallizing, wherein the solvent (5) is selected from a mixture of water and methanol.

[0040] On the other hand, this disclosure also provides the maleate I crystal form of compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxane-5-yl)quinoline-2-amine, and the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ, with characteristic peaks at 8.101, 12.810, 16.435, 26.186 and 27.616.

[0041] In some embodiments, the maleate I crystal form, as expressed in X-ray powder diffraction patterns at diffraction angles of 2θ, has characteristic peaks at 8.101, 12.810, 16.435, 20.667, 25.309, 26.186, and 27.616.

[0042] In some embodiments, the maleate I crystal form, as expressed in X-ray powder diffraction patterns at diffraction angles of 2θ, has characteristic peaks at 8.101, 12.810, 14.598, 16.435, 20.667, 21.474, 22.460, 25.309, 26.186, and 27.616.

[0043] In other embodiments, the maleate I crystal form has an X-ray powder diffraction pattern expressed in terms of a diffraction angle of 2θ, as shown below. Figure 6 As shown.

[0044] This disclosure also provides a method for preparing the maleate I crystal form of the aforementioned compound, comprising mixing the compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine with a solvent (6), (b) adding maleic acid, and crystallizing, wherein the solvent (6) is selected from ethanol.

[0045] On the other hand, this disclosure also provides the maleate II 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 in terms of diffraction angle 2θ, with characteristic peaks at 7.817, 8.777, 11.663, 16.222 and 26.051.

[0046] In some embodiments, the maleate II crystal form, in X-ray powder diffraction patterns expressed as diffraction angle 2θ, has characteristic peaks at 7.817, 8.777, 11.663, 12.691, 16.222, 21.791, and 26.051.

[0047] In some embodiments, the maleate II crystal form, as expressed in X-ray powder diffraction patterns at diffraction angles of 2θ, has characteristic peaks at 7.817, 8.451, 8.777, 11.663, 12.691, 15.028, 16.222, 17.399, 21.791, and 26.051.

[0048] In other embodiments, the maleate II crystal form has an X-ray powder diffraction pattern expressed in terms of a diffraction angle of 2θ, as shown below. Figure 7 As shown.

[0049] This disclosure also provides a method for preparing the maleate II crystal form of the aforementioned compound, comprising mixing the compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine with a solvent (7), (b) adding maleic acid, and crystallizing, wherein the solvent (7) is selected from at least one of acetonitrile, ethanol, methanol or water.

[0050] On the other hand, this disclosure also provides the hydrobromide I crystal form of compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine, and the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ, which has characteristic peaks at 6.077, 12.258, 22.769, 24.662 and 26.579.

[0051] In some embodiments, the hydrobromide I crystal form, as expressed in X-ray powder diffraction patterns at diffraction angles of 2θ, has characteristic peaks at 6.077, 12.258, 22.769, 24.662, 25.696, 26.579, and 34.105.

[0052] In some embodiments, the X-ray powder diffraction pattern of the hydrobromide I crystal form, expressed as a diffraction angle 2θ, has characteristic peaks at 6.077, 12.258, 16.589, 20.396, 22.769, 24.662, 25.218, 25.696, 26.579, and 34.105.

[0053] In other embodiments, the X-ray powder diffraction pattern of the hydrobromide I crystal form, expressed in terms of a diffraction angle 2θ, is as follows: Figure 8 As shown.

[0054] This disclosure also provides a method for preparing the hydrobromide I crystal form of the aforementioned compound, comprising mixing the compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine with a solvent (8), (b) adding hydrobromic acid, and crystallizing, wherein the solvent (8) is selected from at least one of ethanol, acetonitrile or acetone.

[0055] On the other hand, this disclosure also provides the X-ray powder diffraction pattern of the hydrochloride I crystal form of compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine, expressed as a diffraction angle 2θ, with characteristic peaks at 13.229, 14.520, 18.561, 19.925 and 23.830.

[0056] In some embodiments, the X-ray powder diffraction pattern of the hydrochloride I crystal form, expressed as a diffraction angle 2θ, has characteristic peaks at 13.229, 14.520, 18.561, 19.925, 23.830, 24.603, and 25.325.

[0057] In some embodiments, the X-ray powder diffraction pattern of the hydrochloride I crystal form, expressed as a diffraction angle 2θ, has characteristic peaks at 13.229, 14.520, 18.561, 19.925, 21.085, 22.588, 23.830, 24.603, 25.325, and 26.708.

[0058] In other embodiments, the X-ray powder diffraction pattern of the hydrochloride I crystal form, expressed in terms of a diffraction angle 2θ, is as follows: Figure 9 As shown.

[0059] This disclosure also provides a method for preparing the hydrochloride I crystal form of the aforementioned compound, comprising mixing the compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine with a solvent (9), (b) adding hydrochloric acid, and crystallizing, wherein the solvent (9) is selected from at least one of ethanol, acetonitrile or acetone.

[0060] On the other hand, this disclosure also provides the X-ray powder diffraction pattern of the hydrochloride II crystal form of compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine, expressed as a diffraction angle 2θ, with characteristic peaks at 11.888, 13.593, 17.851, 23.977 and 26.309.

[0061] In some embodiments, the X-ray powder diffraction pattern of the hydrochloride II crystal form, expressed as a diffraction angle 2θ, has characteristic peaks at 11.888, 13.593, 17.851, 19.114, 23.977, 25.878, and 26.309.

[0062] In some embodiments, the X-ray powder diffraction pattern of the hydrochloride II crystal form, expressed as a diffraction angle 2θ, has characteristic peaks at 11.888, 13.593, 14.492, 17.851, 19.114, 20.367, 23.977, 25.878, 26.309, and 29.779.

[0063] In other embodiments, the X-ray powder diffraction pattern of the hydrochloride II crystal form, expressed in terms of a diffraction angle 2θ, is as follows: Figure 10 As shown.

[0064] This disclosure also provides a method for preparing the hydrochloride II crystal form of the aforementioned compound, comprising mixing the compound 8-chloro-N-(2,2-difluorobenzo[d][1,3]dioxacyclopentan-5-yl)quinoline-2-amine with a solvent (10), (b) adding hydrochloric acid, and crystallizing, wherein the solvent (10) is selected from at least one of ethanol, methanol or water.

[0065] Furthermore, the present disclosure provides an X-ray powder diffraction pattern of the aforementioned compound crystal form, expressed in terms of a diffraction angle of 2θ, wherein the error range of the 2θ angle is ±0.2.

[0066] In some embodiments, the method for preparing the crystal form described in this disclosure further includes any one of the steps of stirring to dissolve or heating to dissolve, filtering, washing, or drying.

[0067] In some implementations, the crystallization includes, but is not limited to, agitation crystallization (dissolution crystallization, pulping crystallization) and volatilization crystallization.

[0068] 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.

[0069] On the other hand, this disclosure also provides a pharmaceutical composition comprising the aforementioned crystal form and a pharmaceutically acceptable excipient.

[0070] This disclosure also provides a pharmaceutical composition prepared from the aforementioned crystal form and a pharmaceutically acceptable excipient.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] In some embodiments, the inflammation is inflammatory bowel disease. In some embodiments, the cancer is melanoma or breast cancer.

[0075] 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.

[0076] 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.

[0077] The drying temperature described in this disclosure is generally 25℃-100℃, preferably 30℃-70℃, and can be dried under normal pressure or reduced pressure.

[0078] 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.

[0079] 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.

[0080] The crystal forms described in this disclosure include, but are not limited to, solvates of compound 1, wherein the solvents include, but are not limited to, water. Attached Figure Description

[0081] Figure 1 This is an XRPD diagram of the p-toluenesulfonate I crystal form.

[0082] Figure 2 This is an XRPD diagram of phosphate I crystal form.

[0083] Figure 3 This is an XRPD diagram of sulfate I crystal form.

[0084] Figure 4 XRPD diagram of sulfate II crystal form.

[0085] Figure 5 This is an XRPD diagram of sulfate III crystal form.

[0086] Figure 6 XRPD diagram of maleate I crystal form.

[0087] Figure 7 XRPD diagram of maleate II crystal form.

[0088] Figure 8 XRPD diagram of hydrobromide I crystal form.

[0089] Figure 9 XRPD diagram of hydrochloride I crystal form.

[0090] Figure 10 XRPD diagram of the II crystal form of hydrochloride. Detailed Implementation

[0091] 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.

[0092] Test conditions of the instruments used in the experiment:

[0093] 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.

[0094] MS measurements were performed using an Agilent 1200 / 1290DAD-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 Ultimate3000-Q Exactive system (manufacturer: THERMO, MS model: THERMO Q 15 Exactive).

[0095] 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).

[0096] 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°.

[0097] 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℃, and a nitrogen purging rate of 50mL / min.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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)

[0103]

[0104] 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%).

[0105] MS m / z(ESI): 335.0 [M+1].

[0106] 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).

[0107] Test Example 1. Upregulation effect on miR-124

[0108] I. Experimental Materials and Instruments

[0109] 1. Dynabead Human T-Activator CD3 / CD28 for TCell Expansion and Activation (Gibco, 11131D)

[0110] 2. Pan T Cell Isolation Kit (human) (Miltenyi, 130-096-535)

[0111] 3. Human interleukin-2 (Peprotech, 200-02-100)

[0112] 4. MicroRNA Extraction Kit (Qiagen, 217004)

[0113] 5. Small RNA Reverse Transcription Kit (miScript II RT Kit) (Qiagen, 218161)

[0114] 6. Small RNA SYBR Green PCR Kit (miScript SYBR Green PCR Kit) (Qiagen, 218073)

[0115] 7. Phosphate-buffered saline (PBS), pH 7.4 (Shanghai Yuanpei Biotechnology Co., Ltd., B320)

[0116] 8. Bovine serum albumin (BSA) (Beyotime, ST023)

[0117] 9.EDTA (0.5M), pH 8.0 (Invitrogen, AM9260G)

[0118] 10. LS Separation Columns (Miltenyi, 130-042-401)

[0119] 11. 24-well cell culture plate (Corning, 3524)

[0120] 12. 96-well plate (Corning, 3788)

[0121] 13. Cell incubator (Thermo, Steri cycle i160)

[0122] 14. Real-time quantitative PCR instrument (Applied biosystem, QuantStudio 6Flex)

[0123] 15.PCR instrument (Applied biosystem, ProFlex)

[0124] 16. 96-well clear PCR plate, 0.2 mL (Applied biosystems, N8010560)

[0125] 17. RPMI 1640 medium (Gibco, 11875119)

[0126] 18. Fetal bovine serum, FBS (Gibco, 10099-141)

[0127] 19. Magnetic rack (Invitrogen, DynaMag) TM -2)

[0128] 20. Six-well cell culture plate (Thermo, 150239)

[0129] 21. Spectrophotometer (IMPLEN, NP80)

[0130] 22. QuadroMACS Separator (Meitianni, 130-090-976)

[0131] 23. miR124-3P-F primers (custom-made by Genewiz)

[0132] 24. HSA-U6 detection primers (Tiangen, CD201-0145)

[0133] II. Experimental Procedure

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] Table 1. Components of Real-Time PCR Reaction

[0142]

[0143] Table 2. Steps for Real-Time PCR

[0144]

[0145] Table 3 Primer list for quantitative real-time PCR detection

[0146]

[0147] 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)]) .

[0148] Compound 1 upregulated miR-124 by 3.9-fold, demonstrating good activity in promoting miR124 upregulation.

[0149] Example 2: Preparation of p-Toluenesulfonate I crystal form

[0150] Weigh approximately 10 mg of compound 1, dissolve it in 0.15 mL of ethanol, add p-toluenesulfonic acid solution (2 mol / L, 16.4 μL), stir to induce crystallization, centrifuge, and then dry the solid under vacuum to obtain the product.

[0151] X-ray powder diffraction analysis identified the product as p-toluenesulfonate I crystal form, and the XRPD spectrum is shown below. Figure 1 The positions of its characteristic peaks are shown in Table 4.

[0152] The DSC spectrum shows that the endothermic peak has a peak value of 207.15℃.

[0153] The TGA spectrum showed a weight loss of 0.22% between 30℃ and 150℃.

[0154] DVS testing showed that under normal storage conditions (i.e., 25°C, 60% RH), the sample's moisture absorption weight gain was approximately 0.06%; under accelerated testing conditions (i.e., 70% RH), the moisture absorption weight gain was approximately 0.09%; and under extreme conditions (i.e., 90% RH), the moisture absorption weight gain was approximately 0.23%. Furthermore, retesting of the crystal form after DVS testing showed no change in crystal form.

[0155] Table 4

[0156]

[0157] Example 3: Preparation of p-Toluenesulfonate I crystal form

[0158] Weigh approximately 10 mg of compound 1, add 0.15 mL of 10% water / methanol, add p-toluenesulfonic acid solution (2 mol / L, 16.4 μL), stir to induce crystallization, centrifuge, and dry the solid under vacuum to obtain the title product.

[0159] Example 4: Preparation of p-Toluenesulfonate I crystal form

[0160] Weigh approximately 10 mg of compound 1, dissolve it in 0.15 mL of acetone, add p-toluenesulfonic acid solution (2 mol / L, 16.4 μL), stir to induce crystallization, centrifuge, and dry the solid under vacuum to obtain the title product.

[0161] Example 5: Preparation of p-Toluenesulfonate I crystal form

[0162] Weigh approximately 10 mg of compound 1, dissolve it in 0.15 mL of acetonitrile, add p-toluenesulfonic acid solution (2 mol / L, 16.4 μL), stir to induce crystallization, centrifuge, and dry the solid under vacuum to obtain the title product.

[0163] Example 6: Preparation of Phosphate I Crystal Form

[0164] Weigh approximately 10 mg of compound 1, dissolve it in 0.15 mL of acetonitrile, add phosphoric acid (2 mol / L, 16.4 μL), stir to induce crystallization, centrifuge, and dry the solid under vacuum to obtain the product.

[0165] X-ray powder diffraction analysis identified the product as phosphate I crystal form, and the XRPD spectrum is shown below. Figure 2 The positions of its characteristic peaks are shown in Table 5.

[0166] The DSC spectrum shows that the endothermic peak has a peak value of 164.15℃.

[0167] The TGA spectrum showed a weight loss of 1.60% between 30℃ and 140℃.

[0168] Table 5

[0169]

[0170]

[0171] Example 7: Preparation of Sulfate I Crystal Form

[0172] Weigh approximately 10 mg of compound 1, dissolve it in 0.15 mL of ethanol, add sulfuric acid (2 mol / L, 16.4 μL), stir to induce crystallization, centrifuge, and then dry the solid under vacuum to obtain the product.

[0173] X-ray powder diffraction analysis identified the product as sulfate I crystal form, and the XRPD spectrum is shown below. Figure 3 The positions of its characteristic peaks are shown in Table 6.

[0174] The DSC spectrum shows that the endothermic peaks are 196.39℃ and 205.37℃.

[0175] The TGA spectrum showed a weight loss of 0.38% between 30℃ and 150℃.

[0176] Table 6

[0177]

[0178] Example 8: Preparation of Sulfate I Crystal Form

[0179] Weigh approximately 10 mg of compound 1, dissolve it in 0.15 mL of acetone, add sulfuric acid (2 mol / L, 16.4 μL), stir to induce crystallization, centrifuge, and dry the solid under vacuum to obtain the title product.

[0180] Example 9: Preparation of Sulfate II Crystal Form

[0181] Weigh approximately 10 mg of compound 1, dissolve it in 0.3 mL of acetonitrile, add sulfuric acid (2 mol / L, 16.4 μL), stir to induce crystallization, centrifuge, and then dry the solid under vacuum to obtain the product.

[0182] X-ray powder diffraction analysis identified the product as sulfate II crystal form, and the XRPD spectrum is shown below. Figure 4 The positions of its characteristic peaks are shown in Table 7.

[0183] The DSC spectrum shows that the endothermic peak has a peak value of 212.61℃.

[0184] The TGA spectrum showed a weight loss of 0.60% between 30℃ and 150℃.

[0185] DVS testing showed that under normal storage conditions (i.e., 25°C, 60% RH), the sample's moisture absorption weight gain was approximately 1.56%; under accelerated testing conditions (i.e., 70% RH), the moisture absorption weight gain was approximately 1.94%; and under extreme conditions (i.e., 90% RH), the moisture absorption weight gain was approximately 4.11%. Furthermore, retesting of the crystal form after DVS testing showed no change in crystal form.

[0186] Table 7

[0187]

[0188]

[0189] Example 10: Preparation of Sulfate III Crystal Form

[0190] Weigh approximately 10 mg of compound 1, add 0.15 mL of 10% water / methanol, add sulfuric acid (2 mol / L, 16.4 μL), stir to induce crystallization, centrifuge, and then dry the solid under vacuum to obtain the product.

[0191] X-ray powder diffraction analysis identified the product as sulfate III crystal form, and the XRPD spectrum is shown below. Figure 5 The positions of its characteristic peaks are shown in Table 8.

[0192] The DSC spectrum shows that the endothermic peak has a peak value of 123.93℃.

[0193] The TGA spectrum showed a weight loss of 2.39% between 30℃ and 100℃.

[0194] DVS testing showed that under normal storage conditions (i.e., 25°C, 60% RH), the sample's moisture absorption weight gain was approximately 0.66%; under accelerated testing conditions (i.e., 70% RH), the moisture absorption weight gain was approximately 0.76%; and under extreme conditions (i.e., 90% RH), the moisture absorption weight gain was approximately 1.45%. Furthermore, retesting of the crystal form after DVS testing showed no change in crystal form.

[0195] Table 8

[0196]

[0197] Example 11: Preparation of maleate I crystal form

[0198] Weigh approximately 10 mg of compound 1, dissolve it in 0.15 mL of ethanol, add maleic acid solution (2 mol / L, 16.4 μL), stir to induce crystallization, centrifuge, and dry the solid under vacuum to obtain the product.

[0199] X-ray powder diffraction analysis identified the product as maleate I crystal form, and the XRPD spectrum is shown below. Figure 6 The positions of its characteristic peaks are shown in Table 9.

[0200] The DSC spectrum shows that the endothermic peak has a peak value of 162.42℃.

[0201] The TGA spectrum showed a weight loss of 0.62% between 30℃ and 130℃.

[0202] Table 9

[0203]

[0204] Example 12: Preparation of maleate II crystal form

[0205] Weigh approximately 10 mg of compound 1, dissolve it in 0.15 mL of acetonitrile, add maleic acid solution (2 mol / L, 16.4 μL), stir to induce crystallization, centrifuge, and dry the solid under vacuum to obtain the product.

[0206] X-ray powder diffraction analysis identified the product as maleate II crystal form, and the XRPD spectrum is shown below. Figure 7 The positions of its characteristic peaks are shown in Table 10.

[0207] The DSC spectrum shows that the endothermic peak has a peak value of 164.59℃.

[0208] The TGA spectrum showed a weight loss of 1.61% between 30℃ and 145℃.

[0209] DVS testing showed that under normal storage conditions (i.e., 25°C, 60% RH), the sample's moisture absorption weight gain was approximately 0.09%; under accelerated testing conditions (i.e., 70% RH), the moisture absorption weight gain was approximately 0.11%; and under extreme conditions (i.e., 90% RH), the moisture absorption weight gain was approximately 1.11%. Furthermore, retesting of the crystal form after DVS testing showed no change in crystal form.

[0210] Table 10

[0211]

[0212] Example 13: Preparation of maleate II crystal form

[0213] Weigh approximately 10 mg of compound 1, add 0.15 mL of 10% water / methanol, add maleic acid solution (2 mol / L, 16.4 μL), stir to induce crystallization, centrifuge, and dry the solid under vacuum to obtain the title product.

[0214] Example 14: Preparation of maleate II crystal form

[0215] Weigh approximately 300 mg of compound 1, dissolve it in 4 mL of ethanol, add maleic acid solution (2 mol / L, 471 μL), stir to induce crystallization, centrifuge, and dry the solid under vacuum to obtain the title product.

[0216] Example 15: Preparation of maleate II crystal form

[0217] Weigh approximately 10 mg of compound 1, dissolve it in 0.15 mL of ethanol, add maleic acid solution (2 mol / L, 16.4 μL), stir to induce crystallization, centrifuge, and dry the solid under vacuum to obtain the title product.

[0218] Example 16: Preparation of Hydrobromide I Crystal Form

[0219] Weigh approximately 10 mg of compound 1, dissolve it in 0.15 mL of ethanol, add hydrobromic acid (2 mol / L, 16.4 μL), stir to induce crystallization, centrifuge, and then dry the solid under vacuum to obtain the product.

[0220] X-ray powder diffraction analysis identified the product as hydrobromide crystal form I, and the XRPD spectrum is shown below. Figure 8 The positions of its characteristic peaks are shown in Table 11.

[0221] The DSC spectrum shows that the endothermic peak has a peak value of 228.04℃.

[0222] The TGA spectrum showed a weight loss of 0.01% between 30℃ and 150℃.

[0223] DVS testing showed that under normal storage conditions (i.e., 25°C, 60% RH), the sample's moisture absorption weight gain was approximately 0.04%; under accelerated testing conditions (i.e., 70% RH), the moisture absorption weight gain was approximately 0.06%; and under extreme conditions (i.e., 90% RH), the moisture absorption weight gain was approximately 0.15%. Furthermore, retesting of the crystal form after DVS testing showed no change in crystal form.

[0224] Table 11

[0225]

[0226]

[0227] Example 17: Preparation of Hydrobromide I Crystal Form

[0228] Weigh approximately 10 mg of compound 1, dissolve it in 0.15 mL of acetonitrile, add hydrobromic acid (2 mol / L, 16.4 μL), stir to induce crystallization, centrifuge, and dry the solid under vacuum to obtain the title product.

[0229] Example 18: Preparation of Hydrobromide I Crystal Form

[0230] Weigh approximately 10 mg of compound 1, dissolve it in 0.15 mL of acetone, add hydrobromic acid (2 mol / L, 16.4 μL), stir to induce crystallization, centrifuge, and dry the solid under vacuum to obtain the title product.

[0231] Example 19: Preparation of Hydrochloride I Crystal Form

[0232] Weigh approximately 10 mg of compound 1, dissolve it in 0.15 mL of ethanol, add hydrochloric acid (2 mol / L, 16.4 μL), stir to induce crystallization, centrifuge, and then dry the solid under vacuum to obtain the product.

[0233] X-ray powder diffraction analysis determined the product to be hydrochloride crystal form I. The XRPD spectrum is shown below. Figure 9 The positions of its characteristic peaks are shown in Table 12.

[0234] The DSC spectrum shows that the endothermic peak has a peak value of 173.40℃.

[0235] The TGA spectrum showed a weight loss of 4.16% at 30℃-125℃.

[0236] DVS testing showed that under normal storage conditions (i.e., 25°C, 60% RH), the sample's moisture absorption weight gain was approximately 0.33%; under accelerated testing conditions (i.e., 70% RH), the moisture absorption weight gain was approximately 0.37%; and under extreme conditions (i.e., 90% RH), the moisture absorption weight gain was approximately 0.53%. Furthermore, retesting of the crystal form after DVS testing showed no change in crystal form.

[0237] Table 12

[0238]

[0239]

[0240] Example 20: Preparation of Hydrochloride I Crystal Form

[0241] Weigh approximately 200 mg of compound 1, dissolve it in 0.8 mL of acetone, add concentrated hydrochloric acid (12 mol / L, 53 μL), stir to induce crystallization, centrifuge, and dry the solid under vacuum to obtain the title product.

[0242] Example 21: Preparation of Hydrochloride I Crystal Form

[0243] Weigh approximately 40 mg of compound 1, dissolve it in 0.2 mL of acetone, add hydrochloric acid acetone solution (2 mol / L, 62.8 μL), stir to induce crystallization, centrifuge, and dry the solid under vacuum to obtain the title product.

[0244] Example 22: Preparation of Hydrochloride I Crystal Form

[0245] Weigh approximately 10 mg of compound 1, dissolve it in 0.15 mL of acetonitrile, add hydrochloric acid (2 mol / L, 16.4 μL), stir to induce crystallization, centrifuge, and then dry the solid under vacuum to obtain the product.

[0246] Example 23: Preparation of the II crystal form of hydrochloride

[0247] Weigh approximately 10 mg of compound 1, add 0.15 mL of 10% water / methanol, add hydrochloric acid (2 mol / L, 16.4 μL), stir to induce crystallization, centrifuge, and then dry the solid under vacuum to obtain the product.

[0248] X-ray powder diffraction analysis determined the product to be in hydrochloride form II. The XRPD spectrum is shown below. Figure 10 The positions of its characteristic peaks are shown in Table 13.

[0249] The DSC spectrum shows that the endothermic peak has a peak value of 172.83℃.

[0250] The TGA spectrum showed a weight loss of 0.40% between 30℃ and 120℃.

[0251] DVS testing showed that under normal storage conditions (i.e., 25°C, 60% RH), the sample's moisture absorption weight gain was approximately 0.08%; under accelerated testing conditions (i.e., 70% RH), the moisture absorption weight gain was approximately 0.12%; and under extreme conditions (i.e., 90% RH), the moisture absorption weight gain was approximately 0.23%. Furthermore, retesting of the crystal form after DVS testing showed no change in crystal form.

[0252] Table 13

[0253]

[0254] Test Example 2: Influencing Factors Experiment

[0255] Hydrobromide I, p-toluenesulfonate I, hydrochloride I, hydrochloride II, maleate II, phosphate I, and sulfate II crystals were laid out in open locations to investigate their stability under light (4500 Lux), high temperature (40℃, 60℃), and high humidity (RH 75%, RH 92.5%) conditions. The sampling period was 30 days.

[0256] Table 14: Factors affecting the stability of hydrobromide I crystal form

[0257]

[0258]

[0259] Table 15: Factors affecting the stability of p-toluenesulfonate I crystal form

[0260]

[0261] Table 16: Factors Affecting the Stability of Hydrochloride I Crystal Form

[0262]

[0263]

[0264] Table 17: Factors Affecting the Stability of Crystal Form of Hydrochloride II

[0265]

[0266] Table 18: Factors Affecting the Stability of Maleate II Crystal Form

[0267]

[0268]

[0269] Table 19: Factors Affecting the Stability of Phosphate I Crystal Form

[0270]

[0271] Table 20: Factors Affecting the Crystal Form Stability of Sulfate II

[0272]

[0273] Conclusion: The influencing factor experiment showed that phosphate I crystal form, sulfate II crystal form, maleate II crystal form, hydrochloride II crystal form, p-toluenesulfonate I crystal form, and hydrobromide I crystal form have good physical and chemical stability.

[0274] Test Example 3: Long-term Accelerated Stability Assessment

[0275] The stability of hydrobromide I, p-toluenesulfonate I, hydrochloride I, hydrochloride II, maleate II, phosphate I, and sulfate II was investigated under conditions of 25°C / 60%RH and 40°C / 75%RH, respectively.

[0276] Table 21: Long-term accelerated stability of hydrobromide I crystal form

[0277]

[0278] Table 22: Long-term accelerated stability of p-toluenesulfonate I crystal form

[0279]

[0280] Table 23: Long-term accelerated stability of hydrochloride I crystal form

[0281]

[0282] Table 24: Long-term accelerated stability of II hydrochloride crystal form

[0283]

[0284] Table 25: Long-term accelerated stability of maleate II crystal form

[0285]

[0286] Table 26: Long-term accelerated stability of phosphate I crystal form

[0287]

[0288] Table 27: Long-term accelerated stability of sulfate II crystal form

[0289]

[0290]

Claims

1. Compound 8-chloro- N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine, characterized in that, The pharmaceutically acceptable salts are selected from p-toluenesulfonate, phosphate, sulfate, maleate, hydrobromide and hydrochloride.

2. The medicinal salt according to claim 1, characterized in that, Compound 8-chloro- N -(2,2-difluorobenzo[ d The chemical ratio of [1,3]dioxane-5-yl)quinoline-2-amine to the acid molecule is 1:0.5 to 1:

3.

3. The medicinal salt according to claim 2, characterized in that, Compound 8-chloro- N -(2,2-difluorobenzo[ d The chemical ratio of [1,3]dioxacyclopentan-5-yl)quinoline-2-amine to the acid molecule is 1:0.5, 1:1, 1:2 or 1:

3.

4. The medicinal salt according to claim 2, characterized in that, Compound 8-chloro- N -(2,2-difluorobenzo[ d The chemical ratio of [1,3]dioxacyclopentan-5-yl)quinoline-2-amine to the acid molecule is 1:0.5 or 1:

1.

5. A method for preparing the pharmaceutically acceptable salt according to any one of claims 1-4, characterized in that, Including compound 8-chloro- N -(2,2-difluorobenzo[ d The steps of forming salts of [1,3]dioxacyclopentan-5-yl)quinoline-2-amine with acids.

6. The method according to claim 5, characterized in that, The solvent used in the salt formation reaction is selected from at least one of ethanol, acetonitrile, acetone, methanol, and water.

7. Compound 8-chloro- N -(2,2-difluorobenzo[ d [1,3]dioxane-5-yl)quinoline-2-amine p-toluenesulfonate I crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 6.616, 8.996, 14.120, 19.138, and 22.

592.

8. The compound 8-chloro- according to claim 7 N -(2,2-difluorobenzo[ d [1,3]dioxane-5-yl)quinoline-2-amine p-toluenesulfonate I crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 6.616, 8.996, 14.120, 17.120, 19.138, 20.615, 22.592, and 25.

033.

9. The compound 8-chloro- according to claim 7 N -(2,2-difluorobenzo[ d [1,3]dioxane-5-yl)quinoline-2-amine p-toluenesulfonate I crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 6.616, 8.996, 13.304, 14.120, 17.120, 19.138, 20.615, 21.289, 22.592, 25.033, 26.101, and 26.

503.

10. The compound 8-chloro- according to claim 7 N -(2,2-difluorobenzo[ d [1,3]dioxane-5-yl)quinoline-2-amine p-toluenesulfonate I crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern expressed in terms of angle is shown in Figure 1.

11. Compound 8-chloro- N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine phosphate I crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 6.511, 9.685, 12.660, 14.012, and 15.

704.

12. The compound 8-chloro- according to claim 11 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine phosphate I crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 6.511, 9.685, 12.660, 14.012, 15.704, 16.517, 21.487, and 22.

209.

13. The compound 8-chloro- according to claim 11 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine phosphate I crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 6.511, 9.685, 12.660, 14.012, 15.704, 16.517, 18.507, 21.487, 22.209, 24.493, and 25.

596.

14. The compound 8-chloro- according to claim 11 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine phosphate I crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern expressed in terms of angle is shown in Figure 2.

15. Compound 8-chloro- N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine sulfate, crystal form I, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 5.687, 11.448, 14.670, 17.315, 23.323, and 24.

907.

16. The compound 8-chloro- according to claim 15 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine sulfate, crystal form I, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 5.687, 9.540, 11.448, 14.670, 17.315, 18.906, 23.323, and 24.

907.

17. The compound 8-chloro- according to claim 15 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine sulfate, crystal form I, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 5.687, 9.540, 11.448, 12.137, 14.670, 17.315, 17.465, 18.906, 21.682, 23.323, and 24.

907.

18. The compound 8-chloro- according to claim 15 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine sulfate, crystal form I, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern expressed in terms of angle is shown in Figure 3.

19. Compound 8-chloro- N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine sulfate II crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 11.895, 13.102, 15.607, 16.658, and 17.

967.

20. The compound 8-chloro- according to claim 19 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine sulfate II crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, has characteristic peaks at 6.518, 11.895, 13.102, 15.607, 16.181, 16.658, 17.967, and 24.

329.

21. The compound 8-chloro- according to claim 19 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine sulfate II crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 6.518, 10.808, 11.291, 11.895, 13.102, 15.196, 15.607, 16.181, 16.658, 17.967, and 24.

329.

22. The compound 8-chloro- according to claim 19 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine sulfate II crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern expressed in terms of angle is shown in Figure 4.

23. Compound 8-chloro- N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine sulfate III crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 13.502, 16.610, 17.858, 22.247, and 26.

671.

24. The compound 8-chloro- according to claim 23 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine sulfate III crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 8.675, 13.502, 16.610, 17.858, 22.247, 25.379, 26.671, and 27.

029.

25. The compound 8-chloro- according to claim 23 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine sulfate III crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 8.675, 13.502, 16.610, 17.449, 17.858, 22.247, 24.292, 25.379, 26.671, and 27.

029.

26. The compound 8-chloro- according to claim 23 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine sulfate III crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern expressed in terms of angle is shown in Figure 5.

27. Compound 8-chloro- N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine maleate I crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 8.101, 12.810, 16.435, 26.186, and 27.

616.

28. The compound 8-chloro- according to claim 27 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine maleate I crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 8.101, 12.810, 16.435, 20.667, 25.309, 26.186, and 27.

616.

29. The compound 8-chloro- according to claim 27 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine maleate I crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 8.101, 12.810, 14.598, 16.435, 20.667, 21.474, 22.460, 25.309, 26.186, and 27.

616.

30. The compound 8-chloro- according to claim 27 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine maleate I crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern expressed in terms of angle is shown in Figure 6.

31. Compound 8-chloro- N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine maleate II crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 7.817, 8.777, 11.663, 16.222, and 26.

051.

32. The compound 8-chloro- according to claim 31 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine maleate II crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 7.817, 8.777, 11.663, 12.691, 16.222, 21.791, and 26.

051.

33. The compound 8-chloro- according to claim 31 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine maleate II crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 7.817, 8.451, 8.777, 11.663, 12.691, 15.028, 16.222, 17.399, 21.791, and 26.

051.

34. The compound 8-chloro- according to claim 31 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine maleate II crystal form, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern expressed in terms of angle is shown in Figure 7.

35. Compound 8-chloro- N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine hydrobromide, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 6.077, 12.258, 22.769, 24.662, and 26.

579.

36. The compound 8-chloro- according to claim 35 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine hydrobromide, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 6.077, 12.258, 22.769, 24.662, 25.696, 26.579, and 34.

105.

37. The compound 8-chloro- according to claim 35 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine hydrobromide, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 6.077, 12.258, 16.589, 20.396, 22.769, 24.662, 25.218, 25.696, 26.579, and 34.

105.

38. The compound 8-chloro- according to claim 35 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine hydrobromide, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern expressed in terms of angle is shown in Figure 8.

39. Compound 8-chloro- N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine hydrochloride, crystal form I, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 13.229, 14.520, 18.561, 19.925, and 23.

830.

40. The compound 8-chloro- according to claim 39 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine hydrochloride, crystal form I, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 13.229, 14.520, 18.561, 19.925, 23.830, 24.603, and 25.

325.

41. The compound 8-chloro- according to claim 39 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine hydrochloride, crystal form I, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 13.229, 14.520, 18.561, 19.925, 21.085, 22.588, 23.830, 24.603, 25.325, and 26.

708.

42. The compound 8-chloro- according to claim 39 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine hydrochloride, crystal form I, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern expressed in terms of angle is shown in Figure 9.

43. Compound 8-chloro- N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine hydrochloride, crystal form II, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 11.888, 13.593, 17.851, 23.977, and 26.

309.

44. The compound 8-chloro- according to claim 43 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine hydrochloride, crystal form II, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 11.888, 13.593, 17.851, 19.114, 23.977, 25.878, and 26.

309.

45. The compound 8-chloro- according to claim 43 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine hydrochloride, crystal form II, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 11.888, 13.593, 14.492, 17.851, 19.114, 20.367, 23.977, 25.878, 26.309, and 29.

779.

46. ​​The compound 8-chloro- according to claim 43 N -(2,2-difluorobenzo[ d [1,3]dioxacyclopentan-5-yl)quinoline-2-amine hydrochloride, crystal form II, characterized in that, With diffraction angle 2 θ The X-ray powder diffraction pattern expressed in terms of angle is shown in Figure 10.

47. The crystal form according to any one of claims 7-46, characterized in that... The 2 θ The error range is ±0.

2.

48. A pharmaceutical composition comprising a pharmaceutically acceptable salt as described in any one of claims 1-4, or a crystal form as described in any one of claims 7-47, and a pharmaceutically acceptable excipient.

49. A pharmaceutical composition prepared from a pharmaceutically acceptable salt as described in any one of claims 1-4, or a crystal form and pharmaceutically acceptable excipient as described in any one of claims 7-47.

50. Use of the pharmaceutically acceptable salt of any one of claims 1-4, or the crystal form of any one of claims 7-47, or the pharmaceutical composition of claims 48 or 49 as a miRNA regulator in the preparation of a medicament for treating and / or preventing a disease or condition selected from inflammation and cancer.

51. The use according to claim 50, wherein the inflammation is inflammatory bowel disease.

52. The use according to claim 50, wherein the cancer is melanoma or breast cancer.

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