Salt forms of isoquinolinone compounds that are ROCK protease inhibitors and methods for preparing same

Abstract

The present invention discloses a salt form of isoquinolinone-type compound which is a ROCK protease inhibitor and a preparation method thereof, and further includes the application of said salt form in preparing a medicine for treating glaucoma or ocular hypertension. [C1] JPEG2024501081000029.jpg3685

Description

[Technical field]

[0001] The present invention relates to a salt form of an isoquinolinone-type compound which is a ROCK protease inhibitor and a preparation method thereof, further including the application of said salt form in the preparation of a medicament for treating glaucoma or ocular hypertension. [Background technology]

[0002] RHO associated protein kinase (ROCK) belongs to serine / threonine protein kinase, is a downstream target effect molecule of RHO, and is widely expressed in the human body. RHO associated protein kinase (ROCK) is involved in the regulation of myosin light chain (MLC), which is suitable for the treatment of vasodilation, and ROCK kinase can act on the outflow tract cells of the trabecular meshwork, dilate trabecular meshwork cells, and reduce the drainage resistance of aqueous humor. Recent research has shown that ROCK kinase inhibitors can promote the repair of corneal endothelial cell damage and prevent fibrosis, and have great application prospects.

[0003] Isoquinoline sulfonamide compounds are one of the important ROCK kinase inhibitors, and currently, Fashur and K-115 (patent WO2006057397A1) on the market are both isoquinoline sulfonamide compounds. Fashur is a new drug with a wide range of pharmacological effects, an RHO kinase inhibitor, which can dilate blood vessels by increasing the activity of myosin light chain phosphatase, reduce the tension of endothelial cells, improve the microcirculation of brain tissue, cause cerebral blood steal and not aggravate it, while at the same time antagonizing anti-inflammatory factors, protecting nerves from apoptosis, and promoting nerve regeneration. The approval and potential applications of K-115 are very broad, including glaucoma, high intraocular pressure, diabetic retinal injury complications, age-related macular degeneration, corneal injury, cataract and glaucoma post-surgery recovery, etc., and at the same time, it may be expanded to a systematic drug.

[0004] Patent WO2007026664A1 reports compounds with ROCK kinase inhibitory activity, such as control compound 2. This series of compounds has relatively good enzyme activity, but needs to be improved in terms of membrane permeability, pharmacokinetics, drug formability, etc. The present invention reports a similar compound modified in structure, which has significantly improved the properties in this respect. [ka] Summary of the Invention [Means for solving the problem]

[0005] The present invention provides a compound represented by formula (II): [ka]

[0006] The present invention further provides crystalline form A of the compound of formula (II), whose X-ray powder diffraction pattern has characteristic diffraction peaks at 2θ angles of 6.33±0.20°, 10.62±0.20° and 13.11±0.20°. [ka]

[0007] In some embodiments of the present invention, the X-ray powder diffraction pattern of crystalline form A has characteristic diffraction peaks at 2θ angles of 3.30±0.20°, 6.33±0.20°, 6.55°±0.20°, 10.62±0.20°, 12.57±0.20°, and 13.11±0.20°.

[0008] In some embodiments of the present invention, the X-ray powder diffraction pattern of crystalline Form A has characteristic diffraction peaks at 2θ angles of 3.30±0.20°, 6.33±0.20°, 10.62±0.20°, 12.57±0.20°, 13.11±0.20°, 17.85±0.20°, 18.51±0.20° and 20.99±0.20°.

[0009] In some implementations of the present invention, the X-ray powder diffraction pattern of crystalline Form A has characteristic diffraction peaks at 2θ angles of 3.30±0.20°, 6.33±0.20°, 6.55±0.20°, 10.62±0.20°, 12.57±0.20°, 13.11±0.20°, 14.20±0.20°, 16.37±0.20°, 17.85±0.20°, 18.51±0.20°, 19.56±0.20°, 20.99±0.20°, 25.53±0.20° and 26.35±0.20°.

[0010] In some implementations of the present invention, the X-ray powder diffraction pattern of crystalline Form A is essentially as shown in FIG.

[0011] In some embodiments of the present invention, the X-ray powder diffraction pattern analysis data of the crystalline form A is shown in Table 1.

[0012] [Table 1]

[0013] In some embodiments of the present invention, the X-ray powder diffraction pattern of crystalline form A is detected under conditions of a Cu-Kα radiation source.

[0014] In some implementations of the present invention, the differential scanning calorimetry curve for crystalline Form A has an endothermic peak onset at 235.9±3.0° C.

[0015] In some implementations of the present invention, the differential scanning calorimetry curve of crystalline form A is essentially as shown in FIG.

[0016] In some embodiments of the present invention, the thermogravimetric analysis curve of said crystalline form A reaches a weight loss rate of 7.70% at 160.0±3.0° C.

[0017] In some implementations of the present invention, the thermogravimetric analysis curve of the A crystalline form is essentially as shown in FIG.

[0018] The present invention further provides crystalline form B of the compound of formula (II), whose X-ray powder diffraction pattern has characteristic diffraction peaks at 2θ angles of 16.48±0.20°, 16.95±0.20° and 21.87±0.20°. [ka]

[0019] The present invention further provides crystalline form B of the compound of formula (II), whose X-ray powder diffraction pattern has characteristic diffraction peaks at 2θ angles of 9.69±0.20°, 12.12±0.20° and 21.87±0.20°. [ka]

[0020] In some implementations of the present invention, the X-ray powder diffraction pattern of crystalline form B has characteristic diffraction peaks at 2θ angles of 12.12±0.20°, 16.48±0.20°, 16.95±0.20°, 17.94±0.20° and 21.87±0.20°.

[0021] In some embodiments of the present invention, the X-ray powder diffraction pattern of crystalline form B has characteristic diffraction peaks at 2θ angles of 9.69±0.20°, 12.12±0.20°, 16.48±0.20°, 16.95±0.20° and 21.87±0.20°.

[0022] In some embodiments of the present invention, the X-ray powder diffraction pattern of crystalline form B has characteristic diffraction peaks at 2θ angles of 9.69±0.20°, 12.12±0.20°, 16.48±0.20°, 16.95±0.20°, 17.94±0.20°, 19.23±0.20°, 20.37±0.20° and 21.87±0.20°.

[0023] The present invention provides crystalline form B of compound of formula (II), having an X-ray powder diffraction pattern having characteristic diffraction peaks at 2θ angles of 16.48±0.20°, 16.95±0.20°, and / or 21.87±0.20°, and / or 12.12±0.20°, and / or 17.94±0.20°, and / or 9.69±0.20°, and / or 20.37±0.20°, and / or 21.87±0.20°, and / or 4.80±0.20°, and / or 14.61±0.20°, and / or 19.23±0.20°, and / or 27.53±0.20°, and / or 28.72±0.20°, and / or 33.61±0.20°.

[0024] In some implementations of the present invention, the X-ray powder diffraction pattern of crystalline form B has characteristic diffraction peaks at 2θ angles of 4.80±0.20°, 9.69±0.20°, 12.12±0.20°, 14.61±0.20°, 16.48±0.20°, 16.95±0.20°, 17.94±0.20°, 19.23±0.20°, 20.37±0.20°, 21.87±0.20°, 27.53±0.20°, 28.72±0.20° and 33.61±0.20°.

[0025] In some embodiments of the present invention, the X-ray powder diffraction pattern of crystalline form B has characteristic diffraction peaks at 2θ angles of 4.80±0.20°, 9.69±0.20°, 12.12±0.20°, 16.48±0.20°, 16.95±0.20°, 17.94±0.20°, 19.23±0.20°, 20.37±0.20° and 21.87±0.20°.

[0026] In some implementations of the present invention, the X-ray powder diffraction pattern of crystalline form B is essentially as shown in FIG.

[0027] In some implementations of the present invention, the X-ray powder diffraction pattern analysis data for crystalline form B is shown in Table 2.

[0028] [Table 2]

[0029] In some embodiments of the present invention, the X-ray powder diffraction pattern of the B crystalline form is detected under the condition of a Cu-Kα radiation source.

[0030] In some implementations of the present invention, the differential scanning calorimetry curve for crystalline form B has an onset of one endothermic peak at 239.5±3.0° C.

[0031] In some implementations of the present invention, the differential scanning calorimetry curve of the crystalline form B is essentially as shown in FIG.

[0032] In some embodiments of the present invention, the thermogravimetric analysis curve of said crystalline form B reaches a weight loss rate of 1.30% at 200.0±3.0° C.

[0033] In some implementations of the present invention, the thermogravimetric analysis curve of the B crystalline form is essentially as shown in FIG.

[0034] The present invention further provides a process for preparing crystalline form B of compound of formula (II), comprising: (a) adding crystalline Form A of compound of formula (II) to a solvent to form a suspension; (b) stirring the suspension at 50° C. for 3 hours, filtering and drying.

[0035] Wherein, the solvent is selected from isopropyl alcohol, tetrahydrofuran, acetonitrile, 2-butanone and ethyl acetate.

[0036] In some aspects of the present invention, there is provided the application of the compound of formula (II), crystalline form A and crystalline form B, in the preparation of ROCK protease inhibitor related drugs.

[0037] In some aspects of the present invention, there is provided an application of the compound represented by the above formula (II), crystalline form A and crystalline form B, in the preparation of a medicament for treating glaucoma or ocular hypertension. Effect of the Invention

[0038] The compound represented by formula (I) significantly improves the exposure of the active drug, and at the same time significantly increases the peak concentration of the drug in the blood and the duration of action. In the acute ocular hypertension model, the compound represented by formula (I) shows good intraocular pressure lowering effect at different measured doses, and at the same time has a certain dose-dependency, and the intraocular pressure lowering amplitude and duration of action are both superior to K-115. The compound represented by formula (I) has excellent pharmacological efficacy (maximum intraocular pressure lowering effect and duration of action), and the compound represented by formula (I) has high system safety.

[0039] Definitions and Explanations Unless otherwise specified, the following terms and phrases used herein are intended to include the following meanings. If a particular phrase or term is not specifically defined, it should not be considered unclear or unclear, but should be understood according to its ordinary meaning. When a trade name appears in this specification, it is intended to refer to the corresponding product or its active ingredients.

[0040] The intermediate compounds of the present invention can be prepared by a number of synthetic methods well known to those skilled in the art, including the specific embodiments shown below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitution forms well known to those skilled in the art, and preferred embodiments include, but are not limited to, the examples of the present invention.

[0041] The chemical reactions of the specific embodiments of the present invention are completed in suitable solvents, which must be suitable for the chemical reactions of the present invention and the reagents and materials required therefor. In order to obtain the compounds of the present invention, those skilled in the art may need to modify or select synthetic steps or reaction procedures based on the existing embodiments.

[0042] The structure of the compound of the present invention can be confirmed by the usual methods well known to those skilled in the art, and the present invention relates to the absolute configuration of the compound, and the absolute configuration can be confirmed by the usual technical means in the art.For example, single crystal X-ray diffraction (SXRD) is performed by culturing single crystal and using Bruker D8 venture diffractometer to collect diffraction intensity data, the light source is CuKα radiation, scanning mode: φ / scan, and after collecting relevant data, the crystal structure can be analyzed by direct method (Shelxs97) to confirm the absolute configuration.

[0043] The present invention will be described in detail below through examples, but these examples are not intended to limit the present invention in any way.

[0044] All solvents used in this invention are commercially available and can be used without further purification.

[0045] The present invention uses the following abbreviations: rt denotes room temperature, THF denotes tetrahydrofuran, NMP denotes N-methylpyrrolidone, MeSO 3 H indicates methanesulfonic acid, DME indicates ethylene glycol dimethyl ether, DCM indicates dichloromethane, Xphos indicates 2-dicyclohexylphosphino-2'4'6'-triisopropylbiphenyl, EtOAc indicates ethyl acetate, MeOH indicates methanol, acetone indicates acetone, 2-Me-THF indicates 2-methyltetrahydrofuran, and IPA indicates isopropyl alcohol.

[0046] Compounds are named according to conventional naming principles in the art or using ChemDraw® software; commercially available compounds use the vendor catalog name.

[0047] The X-ray powder diffractometer (XRPD) method of the present invention Model number of the device: PANalytical X'pert 3 X-ray powder diffraction equipment Test method: Approximately 10 mg of sample was used for XRPD detection.

[0048] Detailed XRPD parameters are as follows: Source: Cu, Kα1=1.540598Å;Cu, Kα2=1.544426Å Light-emitting tube voltage: 40 kV, light-emitting tube current: 40 mA Scanning range: 3-40deg Step width angle: 0.0263deg Step time: 46.665 seconds

[0049] Differential Scanning Calorimetry (DSC) Method of the Present Invention Model number of the device: TA2500 Differential Scanning Calorimeter Test method: Place a sample (~1-5mg) inside the DSC aluminum plate and test. 2 Under these conditions, the sample is heated from 25°C (room temperature) at a heating rate of 10°C / min before the sample decomposes.

[0050] The Thermogravimetric Analysis (TGA) Method of the Present Invention Model number of the instrument: TAQ5000 Thermogravimetric Analyzer Test method: Place the sample (~1-5mg) inside the TGA aluminum plate, test it by opening it, and run N at 10-25mL / min. 2 Under these conditions, the sample is heated from room temperature to 350° C. at a heating rate of 10° C. / min.

[0051] The Dynamic Vapor Sorption (DVS) Method of the Present Invention Model number: SMS DVS intrinsic dynamic gas sorption meter Dynamic moisture sorption experiments consist of adsorption and desorption. Generally, when the sample weight dm / dt is ≦ 0.01% under a set relative humidity, it is considered that the moisture adsorption or desorption by the sample under the relative humidity has reached balance. Sample test temperature: T=25℃ Balance time: dm / dt: 0.01% / min Relative humidity range: 0%~95%~0%; RH(%) Humidity change per test step: 5% The hygroscopicity is rated as follows: [Table 3] Note: ΔW% indicates the weight increase due to moisture absorption of the test sample at 25±1°C and 80±2% RH. [Brief description of the drawings]

[0052] [Figure 1] FIG. 2 is an XRPD spectrum of Cu-Kα radiation of crystalline form A of compound of formula (II). [Diagram 2] FIG. 2 is a DSC spectrum of crystalline form A of the compound represented by formula (II). [Diagram 3] FIG. 2 is a TGA spectrum of crystalline form A of the compound represented by formula (II). [Figure 4] FIG. 2 is an XRPD spectrum of Cu-Kα radiation of the B crystal form of the compound of formula (II). [Diagram 5] FIG. 2 is a DSC spectrum of crystalline form B of the compound represented by formula (II). [Figure 6] FIG. 2 is a TGA spectrum of crystalline form B of the compound represented by formula (II). [Figure 7] FIG. 2 is a DVS spectrum of crystalline form B of the compound represented by formula (II). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] In order to better understand the contents of the present invention, the present invention will be further described in conjunction with specific examples below, but the specific embodiments do not limit the contents of the present invention.

[0054] Intermediate 1-4 [ka] Step 1 The internal temperature is controlled at 25-35°C, toluene (24L) is added to the reaction vessel, and 1-4a (4000g), sodium carbonate (6120g), methyl boric acid (3465g), 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl (98.66g) and tris(dibenzylacetone)dipalladium (88.03g) are added to the reaction vessel in sequence under stirring, the reaction vessel is purged with nitrogen three times, the temperature in the reaction vessel is raised to 90°C, and stirred at 90-100°C for 13 hours. Water (4L) is added to the reaction vessel to dissolve the solid, the reaction liquid is cooled to room temperature, the reaction liquid is filtered through diatomaceous earth, the filter cake is washed with methyl tert-butyl ether (4L), the washing liquid and the filtrate are combined, and then adjusted to pH=3 with concentrated hydrochloric acid (8L), and the liquid is allowed to stand to separate. Extract the lower aqueous phase with methyl tert-butyl ether (5 L), adjust the aqueous phase to pH = 9 with aqueous sodium hydroxide, extract the aqueous phase twice with ethyl acetate (12 L), and concentrate the combined organic phases in vacuo until no weight loss occurs to give 1-4b. MS-ESI calculated value [M+H] + 144, actual value 144.

[0055] Step 2 Control the temperature inside the reactor to less than 30°C, and slowly add concentrated sulfuric acid (13.36 L) to the 50 L reactor. Control the internal temperature to less than 50°C, and slowly add 1-4b (2500 g) to the reactor using a constant voltage dropping funnel. Control the internal temperature of the reactor to -10 to 0°C, and slowly add N-bromosuccinimide (3070.25 g) to the reactor in a batchwise manner, and stir the reaction solution at -10 to 8°C for 13 hours. Control the internal temperature to less than 50°C, and slowly pour the reaction solution into ice water (8 L). Control the internal temperature to less than 50°C, and slowly drop sodium hydroxide aqueous solution into the reaction solution to adjust the pH to 9. Add ethyl acetate (10 L) to the reaction solution, and stir for 10 minutes. Filter the reaction solution, and wash the filter cake with ethyl acetate (12.5 L). The aqueous phase is extracted with ethyl acetate (10 L x 3), and the combined organic phase is concentrated under reduced pressure at 45° C. The concentrated residue is pulped with n-heptane (10 L), suction filtered to obtain a brown solid, and the solid is dried in a vacuum drying oven (40° C.) to obtain 1-4c. MS-ESI calculated value [M+H] + 222, 224 Actual values: 222, 224.

[0056] Step 3 Add the solvent N,N-dimethylacetamide (10L) to the reaction vessel (50L), add 1-4c (1003.65g) under stirring, control the internal temperature to be less than 55℃, slowly add methylthiosodium (1276.97g) to the reaction vessel (50L) in batches, and stir the reaction mixture at 50℃ for 30 minutes. Increase the temperature of the reaction vessel to 120℃, at which time the internal temperature is 109℃, and stir under this condition for 12 hours. Adjust the reaction vessel temperature to 50℃, lower the temperature of the reaction liquid to 40-50℃, filter the reaction liquid through diatomaceous earth and wash with ethyl acetate (10L), add water (10L) to the filtrate, and extract the aqueous phase with ethyl acetate (5L×2). The pH of the aqueous phase is adjusted to 7 with concentrated hydrochloric acid, the aqueous phase is extracted with ethyl acetate (5 L x 2), the organic phases are combined, the organic phase is washed with water (10 L x 3), and the organic phase is concentrated under reduced pressure until the weight does not change to obtain 1-4d. MS-ESI calculated value [M+H] + 176, actual value 176.

[0057] Step 4 Dichloromethane (8 L) is added to a clean 50 L high-temperature reaction kettle and stirring is started. 1-4d (1474.02 g) is added to the reaction kettle and the oil bath temperature is adjusted to control the internal temperature of the reaction kettle at 0-10°C. Concentrated hydrochloric acid (4.38 L) is slowly added to the reaction kettle in a batch manner and the internal temperature is controlled to be 5-15°C. After the dripping of concentrated hydrochloric acid is completed, the internal temperature of the reaction kettle is lowered to -5°C. Sodium hypochlorite aqueous solution (21.35 L) is slowly dripped into the reaction kettle in a batch manner and the temperature is controlled to be 0-10°C during the whole process. The reaction liquid is filtered, the filter cake is washed with methyl tert-butyl ether (2 L x 2), and the filter cake is collected. The collected filter cake is placed in an oven to dry to obtain 1-4e. MS-ESI calculated value [M+H] + 242, actual value 242.

[0058] Step 5 Add dichloromethane (10L) to a 50L reaction vessel, start stirring, control the internal temperature to 0-10°C, weigh out 1-4e (1462.35g) and add it to the reaction vessel. Control the internal temperature to 0-10°C, weigh out N,N-diisopropylethylamine (934.63g) and slowly drop it into the reaction liquid, control the internal temperature to 0-10°C, weigh out 1-4f (1230.24g) and slowly add it to the reaction liquid in batches. After addition, increase the temperature to 10-20°C and continue stirring for 0.5 hours. Wash the organic phase with saturated ammonium chloride aqueous solution (3L x 3), and extract the combined saturated ammonium chloride aqueous solution with dichloromethane (3L). Separate the organic phase, concentrate under reduced pressure until the weight does not change, and then dry in a vacuum drying oven to obtain 1-4g. MS-ESI calculated value [M+H] + 392, actual value 392.

[0059] Step 6 Add 3.1L of anhydrous dichloromethane to a clean 5L three-neck flask and begin stirring. Weigh out 1-4g (451.34g) and add it to the reaction flask. After the solids have dissolved, place the reaction flask in an ice-water bath and lower the internal temperature of the reaction system to 0°C. Weigh out m-chloroperbenzoic acid (429.81g) and slowly add it to the 5L reaction flask in batches, maintaining the temperature at 0-10°C throughout the process. After adding m-chloroperbenzoic acid, remove the ice-water bath from the reaction flask and replace it with an oil bath, stabilize its internal temperature at 20-25°C, and stir for 12 hours. Put the reaction flask into an ice-water bath and stir for 1 hour, then filter, rinse the filter cake with dichloromethane (400mL x 2), add 10% sodium thiosulfate aqueous solution to the filtrate at 25-30°C so that the potassium iodide test paper does not turn blue, and add saturated sodium bicarbonate aqueous solution to the system so that the pH is 7. Leave to stand and stratify, leave the organic phase, extract the aqueous phase again with 3L of dichloromethane, and combine the organic phase. Wash the organic phase with 1% sodium bicarbonate aqueous solution (4L x 10), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Dry the concentrated solid in an oven to obtain 1-4h. MS-ESI calculated value [M+H] + 408, actual value 408.

[0060] Step 7 Add 1-4h (301.42 g) to a 5 L three-neck flask, add tetrahydrofuran (1.5 L), stir evenly, and when the internal temperature is 10-20 ° C, add triethylamine (206 mL) dropwise and stir evenly. When the internal temperature is 20-30 ° C, add trifluoroacetic anhydride (206 mL) dropwise and react at 20-30 ° C for 0.5 hours. Add water (1.5 L) to the reaction liquid, stir evenly, and the reaction liquid becomes cloudy. When the internal temperature is 40-50 ° C, add solid sodium hydroxide (600 g) and stir for 0.5 hours, a large amount of solid precipitates, add 1.5 L of methyl tert-butyl ether, and stir for 0.5 hours. Separate the reaction liquid, filter the upper organic phase, and obtain a solid. Pulp the solid with methyl tert-butyl ether / ethyl acetate (4.4 L, 10:1), and filter the slurry to obtain a white solid. The white solid is dried in an oven to obtain crude product 1-4 (371.52 g). Add tetrahydrofuran (7 L) to a 50 L reactor, control the internal temperature at 20-25 ° C, add crude product 1-4 (1713.19 g, combine with other batches), and stir at an internal temperature of 20-25 ° C for 0.5 hours. Add 8.6 L of water, stir at 20-25 ° C for 0.5 hours, add solid sodium hydroxide (800 g), and stir at an internal temperature of 20-25 ° C for 0.5 hours. Add methyl tert-butyl ether (8.6 L) and stir at an internal temperature of 20-25 ° C for 0.5 hours. Separate the liquid, filter the upper organic phase, leave the solid obtained by filtration, and dry the solid in an oven until it reaches a constant weight to obtain 1-4. MS-ESI calculated value [M+H] + 408, actual value 408.

[0061] Example 1: Preparation of crystalline form A of the compound of formula (II) [ka] Step 1 Start stirring and nitrogen protection, add dichloromethane (8.6L), water (8.6L), starting material 1-1 (575.34g) to the reactor in sequence. Control the internal temperature of the reactor at 10-15℃, add sodium bicarbonate (1289.36g), tetrabutyl ammonium hydrogen sulfate (133.87g) and starting material 1-2 (760.25g) to the reactor in sequence, control the internal temperature at 10-15℃ and stir for 2 hours. Allow the reaction liquid to stand and stratify, and concentrate the organic phase under reduced pressure until no more distillate flows out. Transfer the concentrate to a three-neck flask and control the internal temperature at 60-70℃ and stir for 12 hours, then naturally cool to room temperature, and quickly filter the crude product through silica gel, then rinse with dichloromethane until no product remains. The eluent is concentrated under reduced pressure to approximately 1.2 L, washed with 1% aqueous sodium bicarbonate solution (3 L), the organic phase is dried over anhydrous sodium sulfate (500 g), filtered, and then concentrated under reduced pressure until no more fractions flow out. The resulting intermediate 1-3 is used in the next step without purification. MS-ESI calculated value [M+H] + 199, actual value 199. 1 H NMR (400 MHz, CD 3 Cl) δ 7.81 - 7.79 (d, J = 8 Hz, 1H), 7.01 - 6.95 (m, 2H), 5.84 (s, 2H), 2.52 (s, 3H), 2.27 (s, 3H).

[0062] Step 2 Add 2-methyltetrahydrofuran (9702 mL) to the reactor, start stirring to control the internal temperature at 10-20°C, then add 1-4 (1078.26 g), cesium carbonate (1017.63 g) and intermediate 1-3 (631.71 g) in sequence. Raise the internal temperature of the reactor to 57-63°C and stir for 2 hours and 40 minutes, then stop the reaction. After lowering the internal temperature of the reactor to 15-25°C, add water (10.78 L) to the reaction liquid, stir and then leave to stratify, extract the resulting aqueous phase twice with 2-methyltetrahydrofuran (5390 mL x 2), and concentrate the combined organic phase to about 2.2 L under reduced pressure. Add acetone (1078 mL) and n-heptane (2156 mL) to the concentrated liquid and concentrate until no distillate flows out, and obtain a loose solid. The internal temperature is controlled at 10-30° C., the solid is pulped with mixed solvent (11.0495 L, acetone:n-heptane=1:40) and stirred three times, filtered and rinsed with n-heptane (1078 mL). The filter cake is dried under vacuum to a constant weight to obtain intermediate 1-5. MS-ESI calculated value [M+H] + 570, actual value 570. 1 H NMR (400 MHz, CD 3 Cl) δ 8.86 - 8.78 (m, 1H), 8.13 - 8.04 (m, 1H), 7.93 - 7.86 (m, 1H), 7.58 - 7.49 (m, 1H), 7.39 - 7.32 (m, 1H), 7.09 - 6.99 (m, 2H), 6.14 (s, 2H), 4.86 - 4.76 (m, 1H), 4.42 - 4.30 (m, 1H), 3.73 - 3.61 (m, 2H), 3.57 - 3.47 (m, 1H), 3.46 - 3.37 (m, 1H), 2.76 - 2.67 (m, 3H), 2.59 (s, 3H), 2.39 - 2.29 (m, 4H), 2.09 - 1.97 (m, 1H), 1.46 (s, 9H).

[0063] Step 3 Control the internal temperature at 20-30℃, add anhydrous tetrahydrofuran (4.24L) and concentrated sulfuric acid (712.43g) in 5L three-neck flask in sequence, stir evenly, and then prepare for use. Control the internal temperature at 20-30℃, add 1-5 (1062.15g) and tetrahydrofuran (4.24L) in sequence to the reactor, stir evenly, and then add the pre-prepared tetrahydrofuran sulfate solution dropwise into the system, then control the internal temperature of the reaction at 35-45℃ and stir for 4 hours, and a large amount of white precipitate will gradually precipitate in the system. Add methyl tert-butyl ether (8.48L) slowly to the reactor, control the internal temperature at 20-30℃, stir for 0.5 hours, filter the solid, pulp with mixed solvent (methyl tert-butyl ether / tetrahydrofuran=10.6L / 10.6L) and methyl tert-butyl ether (21.2L) successively for 0.5 hours at 20-30℃, and then pulp with water 5 times (21.2L×5) until the pH of the filtrate is about 7. Control the internal temperature at 20-30℃, pulp the solid in methyl tert-butyl ether (15.9L) and mixed solvent (methyl tert-butyl ether / tetrahydrofuran=10.6L / 5.3L), filter, rinse and vacuum dry below 45℃ to obtain the A crystal form of the compound of formula (II). The content of sulfate radical was found to be 9.17% by ion chromatography, which suggests that the compound represented by formula (II) also contains 0.5 sulfate units. MS-ESI calculated value [M+H] + 470, actual value 470. 1 H NMR (400 MHz, DMSO-d 6) δ 8.64 - 8.58 (m, 1H), 8.26 (d, J = 8 Hz, 1H), 7.74 - 7.62 (m, 3H), 7.16 - 7.11 (m, 1H), 7.11 - 7.05 (m, 1H), 6.08 (s, 2H), 3.94 - 3.89 (m, 1H), 3.70 - 3.55 (m, 2H), 3.55 - 3.44 (m, 1H), 3.43 - 3.27 (m, 1H), 2.59 (s, 3H), 2.48 - 2.45 (m, 3H), 2.35 - 2.29 (m, 1H), 2.29 - 2.24 (m, 3H), 2.16 - 1.90 (m, 1H).

[0064] Characterization of crystalline form A of compound of formula (II): (1) X-ray powder diffraction analysis: The test results are shown in FIG. (2) Differential thermal analysis: The test results are shown in Figure 2. (3) Thermogravimetric analysis: The test results are shown in Figure 3.

[0065] Example 2: Preparation of compounds of formula (I) [ka] Compound 1-5 (2.2 g, 3.84 mmol) was dissolved in ethyl acetate (35 mL), and a solution of hydrochloric acid in ethyl acetate (4 M, 20 mL) was added to the reaction solution, followed by stirring for 12 hours at 15° C. Saturated aqueous sodium bicarbonate solution was added to the reaction solution to adjust the pH value to 8, followed by extraction with ethyl acetate (60 mL×2), and the organic phase was dried over anhydrous sodium sulfate (5 g), filtered, and concentrated under reduced pressure. The crude product obtained was purified by high performance liquid chromatography (neutral system) to obtain the compound represented by formula (I). MS-ESI calculated value [M+H] + 470, actual value 470. 1 H NMR (400MHz, CD 3OD) δ = 8.71 (dd, J = 1.4, 8.0 Hz, 1H), 8.24 (dd, J = 1.5, 7.8 Hz, 1H), 7.81 (d, J = 7.9 Hz, 1H), 7.64 (t, J = 7.9 Hz, 1H), 7.54 (d, J = 0.9 Hz, 1H), 7.09 (s, 1H), 7.05 (d, J = 7.9 Hz, 1H), 6.14 (s, 2H), 3.77 - 3.62 (m, 3H), 3.59 - 3.50 (m, 1H), 3.20 (dd, J = 4.9, 9.5 Hz, 1H), 2.70 (d, J = 0.9 Hz, 3H), 2.53 (s, 3H), 2.32 (s, 3H), 2.30 - 2.23 (m, 1H), 1.96 - 1.86 (m, 1H).

[0066] Example 3: Preparation of crystalline form B of the compound of formula (II) An appropriate amount of crystalline form A of compound of formula (II) is weighed into a sample bottle, and a certain volume of the solvent in Table 3 is added to prepare suspensions or solutions in different single solvents. The suspensions are stirred continuously at 50°C for 3 hours, and the samples are filtered to obtain solids, which are then placed in a vacuum drying oven and vacuum dried at 45°C to remove residual solvent.

[0067] [Table 4]

[0068] Characterization of crystalline form B of compound of formula (II): (1) X-ray powder diffraction analysis: The test results are shown in FIG. (2) Differential thermal analysis: The test results are shown in Figure 5. (3) Thermogravimetric analysis: The test results are shown in Figure 6.

[0069] Example 4: Study on the hygroscopicity of the B crystalline form of compound of formula (II) Test materials: SMS DVS intrinsic dynamic gas sorption meter Testing Method: 10-15 mg of crystalline form B of the compound of formula (II) is placed in a DVS sample tray for testing. Test Results: The DVS spectrum of crystalline form B of the compound of formula (II) is shown in FIG. 7, with ΔW=0.736%. Test conclusion: The crystalline form B of the compound represented by formula (II) has a moisture weight increase rate of 0.736% at 25° C. and 80% RH, and is slightly hygroscopic.

[0070] Example 5: Study on the solid state stability of the B crystalline form of compound of formula (II) Crystalline form B of compound of formula (II) was stored at 40°C / 75%RH (sealed in a double layer LDPE bag, then heat sealed in an aluminum foil bag) for 6 months and at 25°C / 60%RH (sealed in a double layer LDPE bag, then heat sealed in an aluminum foil bag) for 12 months. Each crystal form was tested at each sampling point to determine the stability of the crystal form of the sample.

[0071] [Table 5] Conclusion: The crystalline form B of the compound of formula (II) has good stability under both accelerated and long-term tests.

[0072] Biological Test Experiment Experimental Example 1: Pharmacokinetics test in aqueous humor Purpose of the Test: The compound is a prodrug molecule containing an ester functional group, which is hydrolyzed to an active drug molecule (parent drug) by the action of ester hydrolases abundant in ocular tissues when administered by eye drop. In this experiment, the rate at which the compound produces the active drug component in the body and the exposure level of the active drug component are detected. Test materials: Male New Zealand white rabbits, aged 3-6 months, weighing 2.0-5.0 kg, were purchased from Hebei Oriental Aquaculture Co., Ltd. Eye drop sample placement: The solvent used was 1.2% hydroxypropylmethylcellulose E5 / 20.5% poloxamer P407 / 1.6% poloxamer P188. Experimental Procedure: The dose of eye drop was 0.5mg / eye, and the drug was administered to both eyes. After administration, aqueous humor was collected at 0.25h, 0.5h, 2h, 4h, 8h, and 24h, and aqueous humor samples were prepared. All samples were quantitatively detected by liquid chromatography-mass spectrometry to determine the content of the administered compound in the aqueous humor of the experimental animals. The measured concentration values ​​were used to calculate parameters such as half-life, aqueous humor drug peak concentration, aqueous humor drug peak time, and unit exposure amount based on the aqueous humor concentration-time data using the WinNonlin non-compartmental model.

[0073] [Table 6]

[0074] Conclusion: As can be seen from the experimental results, the test compound (prodrug molecule) was not detected in the aqueous humor, and the active metabolite (parent drug molecule) after ester hydrolysis was mainly detected. The compound represented by formula (I) significantly improved the exposure of the active drug, and both the peak blood drug concentration and duration of action were significantly increased.

[0075] Experimental example 2: Intraocular pressure reduction test in New Zealand rabbits with acute ocular hypertension Purpose of the Test: Acute ocular hypertension is induced in rabbits by injecting a viscoelastic agent into the anterior chamber, and the intraocular pressure-lowering effect of different concentrations of the compound of formula (I) is explored by eye drop administration. Test materials: Male New Zealand white rabbits, aged 97-127 days, weighing 2.5-3.4 kg, were purchased from Hebei Oriental Aquaculture Co., Ltd. Experimental Procedure: Fifty male New Zealand white rabbits were randomly grouped according to body weight, totaling 5 groups, 10 rabbits per group. Medical sodium hyaluronate gel was injected into the anterior chamber of the right eye of the animals in groups 1-5 at one time, 100 μL / eye, to induce the development of high intraocular pressure in the animals. Five to 15 minutes after the model was created, the right eye was instilled with the solvent, K-115, and the test product (a compound represented by formula (I) at different concentrations), and the left eye was instilled with the solvent, all in an administration volume of 50 μL / eye. The intraocular pressure of both eyes of the animals was measured before administration, and 2, 4, 6, 8, and 10 hours after administration. The experimental results are shown in Table 6.

[0076] [Table 7]

[0077] Conclusion: In the acute ocular hypertension model, the compound of formula (I) showed good ocular hypotensive effects at different doses, with a certain dose-dependency, and the ocular hypotensive amplitude and duration of action were superior to those of K-115.

[0078] Experimental Example 3: Intraocular pressure reduction and ocular toxicity test using repeated eye drops in normotensive New Zealand rabbits for 14 days Purpose of the Test: The intraocular pressure lowering effect and potential ocular toxicity of the compounds of formula (I) are explored by repeated eye drop administration for 14 days in normotensive rabbits. Test materials: Male New Zealand white rabbits, aged 97-127 days, weighing 2.63.5 kg, were purchased from Hebei Oriental Aquaculture Co., Ltd. Experimental Procedure 1: Male New Zealand white rabbits were randomly divided into 7 groups, with 6 animals in each group. The animals were randomly grouped according to their body weight. The animals in groups 1-7 were instilled with the solvent / control / test product in both eyes, with the administration volume of each being 50 μL / eye, once a day for 14 consecutive days, with the first administration day recorded as day 1. The intraocular pressure of the animals was measured before administration on day 1, and 1, 2, 4, 6, 8, and 10 hours after administration. On days 2-14, the intraocular pressure of the K-115 administration group was measured 1 hour after daily administration, and the intraocular pressure of the animals in each of the other groups was measured 4 hours after daily administration. The experimental results are shown in Tables 7, 8, and 9.

[0079] [Table 8]

[0080] [Table 9]

[0081] [Table 10]

[0082] Conclusion: Single administration of the compound of formula (I) showed superior efficacy (best intraocular pressure lowering effect and duration of action) at all doses (0.5-8.0mg / mL) tested, and was significantly superior to K-115. When administered continuously for 14 days, the compound of formula (I) could maintain a significant intraocular pressure lowering effect at a dose of 0.5mg / mL, and was still significantly superior to K-115 in the evaluation of peak (Cmax) intraocular pressure lowering effect.

[0083] Test Procedure 2: 42 male New Zealand white rabbits were randomly divided into 7 groups, with 6 rabbits in each group. The animals were randomly grouped according to their weight. The animals in groups 1-7 were instilled with saline into the left eye and with the vehicle / control / test product into the right eye, with the administration volume of each being 50 μL / eye. The administration was performed once a day for 14 consecutive days, with the first administration day recorded as day 1. The intraocular pressure (Table 10) of the animals was measured before administration on day 1, and 1, 2, 4, 6, 8, and 10 hours after administration on day 1.

[0084] Before the start of the study (Day 2 / Day 1), before each day of the dosing period (Days 1-14), and at 1, 2, 4, 24, 48, and 72 hours after the final (Day 14) dose, animals will undergo eye irritation and sodium fluorescein tests using a handheld slit lamp in both eyes (assessed with reference to the assessment score criteria).

[0085] [Table 11]

[0086] Conclusion: A single dose of the compound represented by formula (I) showed superior efficacy (highest intraocular pressure lowering effect and duration of action) at all doses measured (0.25-4.0 mg / mL), and was significantly superior to K-115.

[0087] Eye irritation tests were performed on both eyes of the animals using a handheld slit lamp before the start of the study (Day-2 to Day-1 (Day 2 / Day 1)), before the first administration every day (Days 1-14) during the administration period, and 1, 2, 4, 24, 48, and 72 hours after the final administration. The evaluation score criteria are as follows: [Table 12]

[0088] Eye irritation response evaluation: The maximum scores of cornea, iris, conjunctiva, edema and secretion are added to obtain the total score of eye irritation symptoms for each animal's eye at each time point. For the eye irritation symptom evaluation score, calculate the average score of the animals in each group at each observation time point, and judge the degree of eye irritation of each group of animals at each time point according to the table below.

[0089] Eye irritation assessment criteria: [Table 13]

[0090] Sodium fluorescein test: After each eye irritation test, a sodium fluorescein test was performed using a handheld slit lamp. The evaluation score criteria were as follows: [Table 14]

[0091] The experimental results are as follows. The eye irritation evaluation criteria were used to evaluate the eye irritation response, and the total eye irritation response score for each group at each time point was less than 3, and all were non-irritating according to the criteria classification.

[0092] During the study, animals in each group had their eyes treated with saline, and the sodium fluorescein test scores for eyes treated with vehicle, K-115, and the compound of formula (I) were all less than 1. Animals in each group showed a corneal fluorescence staining score of 1 at each treatment and individual time point, which is considered physiological staining. None of the groups showed corneal epithelial damage at each time point.

[0093] Conclusion: Under the test conditions, K-115 at a concentration of 4 mg / mL was not irritating when instilled into the eye for 14 consecutive days at 50 microliters / eye / day. Compound (I) at a concentration of 0.25-4 mg / mL was not irritating when instilled into the eye for 14 consecutive days at 50 microliters / eye / day.

[0094] Experimental example 4: Toxicokinetics experiment Purpose of the Test: The rate at which the compound produces active drug components in plasma and the exposure amount of the active drug components after 14 consecutive days of administration are detected. Test materials: Male New Zealand white rabbits, aged 3-6 months, weighing 2.0-5.0 kg, were purchased from Hebei Oriental Aquaculture Co., Ltd. Experimental Procedure: After 14 consecutive days of administration, on the 14th and 15th days, a group administered with the compound of formula (I) (8.0 mg / mL) was selected, and blood samples were collected at 0 hour (before administration) and 0.5, 1, 2, 4, 8, and 24 hours after administration. Approximately 0.8 mL of whole blood was collected from the middle ear artery or saphenous vein of the hind leg (or other suitable site) of the toxicity test animals, and dipotassium ethylenediaminetetraacetate (K 2 The blood samples were then placed in labeled blood collection tubes containing EDTA as an anticoagulant. After collection, the blood samples were centrifuged at 3,000 rpm for 10 minutes at 2°C to 8°C for 60 minutes to obtain plasma. All samples were then subjected to liquid chromatography-mass spectrometry to quantitatively detect the content of the administered compound in the plasma of the experimental animals.

[0095] [Table 15]

[0096] Conclusion: At a high dose of 8 mg / mL, the compound of formula (I) had a metabolite concentration of 0.934 ng / mL 4 hours after administration, and the metabolite concentration was below the detection limit 8 hours after administration, indicating high system safety. [CROSS REFERENCE TO RELATED APPLICATIONS]

[0097] Priority is claimed to a Chinese patent application having application number CN202011521011.6 and filing date of December 21, 2020.

Claims

1. Crystal form A of the compound represented by formula (II), wherein the X-ray powder diffraction pattern of the crystal form A has characteristic diffraction peaks at 2θ angles of 3.30±0.20°, 6.33±0.20°, 6.55±0.20°, 10.62±0.20°, 12.57±0.20°, and 13.11±0.20°. 【Chemistry 1】

2. The X-ray powder diffraction pattern of crystalline form A described in claim 1 has characteristic diffraction peaks at 2θ angles of 3.30±0.20°, 6.33±0.20°, 10.62±0.20°, 12.57±0.20°, 13.11±0.20°, 17.85±0.20°, 18.51±0.20°, and 20.99±0.20°.

3. The X-ray powder diffraction pattern of crystalline form A described in claim 1 has characteristic diffraction peaks at 2θ angles of 3.30±0.20°, 6.33±0.20°, 6.55±0.20°, 10.62±0.20°, 12.57±0.20°, 13.11±0.20°, 14.20±0.20°, 16.37±0.20°, 17.85±0.20°, 18.51±0.20°, 19.56±0.20°, 20.99±0.20°, 25.53±0.20°, and 26.35±0.20°.

4. The X-ray powder diffraction pattern of crystalline form A described in claim 2 has characteristic diffraction peaks at 2θ angles of 3.30°, 6.33°, 6.55°, 10.62°, 12.57°, 13.11°, 14.20°, 16.37°, 17.85°, 18.51°, 19.56°, 20.99°, 25.53°, and 26.35°.

5. Crystal form A described in claim 4, the analytical data of which is shown in the table below. 【Table 1】

6. Crystalline form A described in any one of claims 1 to 5, wherein the differential scanning calorimetry curve of crystal form A has an endothermic peak starting point at 235.9°C ± 3.0°C.

7. Crystalline form A described in any one of claims 1 to 5, wherein the thermogravimetric analysis curve of crystal form A reaches a weight loss rate of 7.70% at 160.0±3.0°C.

8. Crystal form B of the compound represented by formula (II), characterized in that the X-ray powder diffraction pattern of the crystal form B has characteristic diffraction peaks at 2θ angles of 12.12±0.20°, 16.48±0.20°, 16.95±0.20°, 17.94±0.20°, and 21.87±0.20°. 【Chemistry 2】

9. The B crystalline form described in claim 8, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at 2θ angles of 9.69±0.20°, 12.12±0.20°, 16.48±0.20°, 16.95±0.20°, and 21.87±0.20°.

10. The B crystalline form described in claim 9, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at 2θ angles of 9.69±0.20°, 12.12±0.20°, 16.48±0.20°, 16.95±0.20°, 17.94±0.20°, 19.23±0.20°, 20.37±0.20°, and 21.87±0.20°.

11. The B crystalline form described in claim 10, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at 2θ angles of 4.80±0.20°, 9.69±0.20°, 12.12±0.20°, 14.61±0.20°, 16.48±0.20°, 16.95±0.20°, 17.94±0.20°, 19.23±0.20°, 20.37±0.20°, 21.87±0.20°, 27.53±0.20°, 28.72±0.20°, and 33.61±0.20°.

12. Crystalline form B described in claim 11, the analytical data of which is shown in the table below. 【Table 2】 13. The crystalline form B according to any one of claims 8 to 12, wherein the differential scanning calorimetry curve of the crystalline form B has an endothermic peak starting point at 239.5±3.0°C.

14. Crystalline form B according to any one of claims 8 to 12, wherein the thermogravimetric analysis curve of said crystal form B shows a weight loss rate of 1.30% at 200.0±3.0°C.

15. A process for preparing crystalline form B of compound of formula (II), comprising the steps of: (a) adding crystalline Form A of compound of formula (II) to a solvent to form a suspension; (b) stirring the suspension at 50° C. for 3 hours, filtering and drying; Where: The X-ray powder diffraction pattern of the A crystalline form of the compound represented by formula (II) has characteristic diffraction peaks at 2θ angles of 3.30±0.20°, 6.33±0.20°, 6.55±0.20°, 10.62±0.20°, 12.57±0.20°, and 13.11±0.20°, The X-ray powder diffraction pattern of the B crystalline form of the compound represented by formula (II) has characteristic diffraction peaks at 2θ angles of 12.12±0.20°, 16.48±0.20°, 16.95±0.20°, 17.94±0.20°, and 21.87±0.20°, The method for preparing crystalline form B of the compound of formula (II), wherein the solvent is selected from isopropyl alcohol, tetrahydrofuran, acetonitrile, 2-butanone and ethyl acetate. 【Chemistry 3】

16. Use of crystalline form A according to any one of claims 1 to 7, or crystalline form B according to any one of claims 8 to 14, or crystalline form B obtained by the method according to claim 15, in the preparation of a medicament for treating glaucoma or ocular hypertension.

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