Crystalline form of a thiophene derivative and method of preparing the same

Abstract

The present invention provides a crystalline form of a compound represented by formula (I) and a method for preparing the same.

Description

Technology Field

[0001] The present invention claims the following priority.

[0002] CN202110472705.3, Filing Date: April 29, 2021.

[0003] CN202111112439.X, Filing Date: September 18, 2021.

[0004] The present invention relates to a crystalline form of a thiophene derivative and a method for preparing the same, specifically to a crystalline form of a compound represented by formula (I) and a method for preparing the same. Background Technology

[0005] Gouty arthritis is a common and complex type of arthritis. When the body's blood uric acid concentration exceeds 7 mg / dL, uric acid is deposited in the joints, cartilage, and kidneys in the form of monosodium salts. This causes painful inflammation as the body's immune system becomes overactive (sensitive). Commonly affected areas include the big toe joint, ankle joint, and knee joint. Hyperuricemia is the pathological basis of gouty arthritis. Hyperuricemia refers to a disease characterized by abnormally high blood uric acid levels resulting from a disruption in the metabolism of purines, leading to increased uric acid synthesis or decreased uric acid excretion. Internationally, the diagnosis of HUA is defined as follows: two fasting blood uric acid levels on different days under a normal purine diet: >400 μmol / L (6.8 mg / dL) for men and >360 μmol / L (6 mg / dL) for women. It can be classified into three types: poor uric acid excretion type, overproduction type, and mixed type. According to clinical research results, 90% of primary hyperuricemia cases were found to be of the poor uric acid excretion type.

[0006] Hyperuricemia is inextricably linked to gout and is an independent risk factor for metabolic diseases (diabetes, metabolic syndrome (MS), hyperlipidemia, etc.), chronic kidney disease, cardiovascular disease, and stroke. Therefore, lowering uric acid levels in the body can be used not only to treat or prevent hyperuricemia and gout but also to reduce the risk of other complications associated with hyperuricemia.

[0007] There are two sources of purines in the human body. Endogenous purines are derived from autosynthesis or nucleolysis (about 600 mg / d), and exogenous purines are derived from dietary purine intake (about 100 mg / d). Under normal conditions, the body's uric acid pool is 1200 mg, and about 700 mg of uric acid is produced daily; two-thirds of this is excreted through the kidneys, one-third through the intestines, and a very small amount through sweat glands. Therefore, commonly used clinical uric acid-lowering drugs currently include xanthine oxidase inhibitors that inhibit the production of uric acid (e.g., allopurinol and Febuxostat) and urate inhibitors that excrete uric acid (e.g., benzbromarone and lesinurad).

[0008] Xanthine oxidase is an enzyme with low specificity that can not only catalyze hypoxanthine to produce xanthine and subsequently uric acid, but also directly catalyze xanthine to produce uric acid. Xanthine oxidase inhibitors are first-line treatments for hyperuricemia, and currently available drugs mainly include allopurinol and febuxostat. However, these drugs cannot meet the clinical needs of all patients and have relatively obvious side effects. Allopurinol is the only globally available uric acid-lowering treatment, but it can cause serious skin side effects. Severe hypersensitivity reactions associated with allopurinol are closely linked to Human Leukocyte Antigen (HLA)-B*5801, and the risk of hypersensitivity is higher in Chinese people (6% to 8%) who are HLA-B*5801 positive than in Caucasians (~2%). Although febuxostat has a superior uric acid-lowering effect compared to allopurinol, at a high dose of 80 mg / day, 40% to 52% of patients do not reach their expected uric acid-lowering target and may increase the frequency of acute gout attacks.

[0009] Clinical demand for safe and effective uric acid-lowering drugs in the market remains unmet. Effects of the invention

[0083] Effects of the invention

[0010] The present invention provides a crystalline form A of a compound represented by formula (I), characterized in that the powder X-ray diffraction spectrum has characteristic diffraction peaks at 2θ angles of 12.35±0.20°, 15.05±0.20°, 18.19±0.20°, 20.10±0.20°, 23.05±0.20°, 25.05±0.20°, 25.87±0.20°, and 27.16±0.20°.

[0011]

[0012] In some aspects of the present invention, the powder X-ray diffraction spectrum of crystalline form A has characteristic diffraction peaks at 2θ angles of 10.89±0.20°, 12.35±0.20°, 13.42±0.20°, 15.05±0.20°, 18.19±0.20°, 20.10±0.20°, 21.82±0.20°, 23.05±0.20°, 25.05±0.20°, 25.87±0.20°, 27.16±0.20°, and 30.28±0.20°.

[0013] In some aspects of the present invention, the powder X-ray diffraction spectrum of crystalline form A has characteristic diffraction peaks at 2θ angles of 6.72°, 8.94°, 10.89°, 12.35°, 13.42°, 15.05°, 17.26°, 18.19°, 18.70°, 20.10°, 21.82°, 23.05°, 24.28°, 25.05°, 25.87°, 27.16°, 29.41°, 30.28°, 30.89°, 33.58°, 36.29°, 37.29°, and 38.99°.

[0014] In some aspects of the present invention, the XRPD spectrum of the crystalline form A is basically as shown in FIG. 1.

[0015] In some aspects of the present invention, the XRPD spectrum analysis data of the crystalline form A is as shown in Table 1.

[0016]

[0017] In some aspects of the present invention, the thermogravimetric analysis curve of the crystalline form A shows a weight loss of 1.96% at 200°C ± 3°C.

[0018] In some aspects of the present invention, the TGA spectrum of the crystalline form A is as shown in FIG. 2.

[0019] In some embodiments of the present invention, the differential scanning calorimetry curve of the crystalline form A has a starting value of one endothermic peak at 244.3.0℃±2℃.

[0020] In some aspects of the present invention, the DSC spectrum of the crystalline form A is as shown in FIG. 3.

[0021] The present invention provides a crystalline form B of a compound represented by formula (I), characterized in that the powder X-ray diffraction spectrum has characteristic diffraction peaks at 2θ angles of 23.93±0.20°, 24.73±0.20°, and 26.58±0.20°.

[0022]

[0023] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystalline form B has characteristic diffraction peaks at 2θ angles of 13.02±0.20°, 14.68±0.20°, 16.44±0.20°, 19.50±0.20°, 22.69±0.20°, 23.93±0.20°, 24.73±0.20°, and 26.58±0.20°.

[0024] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystalline form B has characteristic diffraction peaks at 2θ angles of 13.02±0.20°, 14.68±0.20°, 16.44±0.20°, 19.50±0.20°, 22.69±0.20°, 23.93±0.20°, 24.73±0.20°, 25.87±0.20°, 26.58±0.20°, 28.98±0.20°, 29.34±0.20°, and 31.86±0.20°.

[0025] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystalline form B is 5.37°, 11.72°, 13.02°, 14.68°, 15.44°, 16.05°, 16.44°, 16.94°, 18.68°, 19.50°, 20.69°, 21.13°, 21.32°, 21.70°, 22.41°, 22.69°, 23.46°, 23.93°, 24.73°, 25.87°, 26.58°, 27.78°, 28.98°, 29.34°, 29.66°, 30.07°, 31.26°, 31.38°, It has characteristic diffraction peaks at 2θ angles of 31.86°, 32.73°, 33.71°, 34.02°, 34.68°, 35.41°, 36.64°, 37.30°, 37.86°, and 38.30°.

[0026] In some aspects of the present invention, the XRPD spectrum of the crystalline form B is basically as shown in FIG. 4.

[0027] In some aspects of the present invention, the XRPD spectrum analysis data of the crystalline form B is as shown in Table 2.

[0028]

[0029] The present invention provides a crystal of a compound represented by formula (I) characterized by having characteristic diffraction peaks at 2θ angles of 13.28±0.30°, 15.34±0.30°, and 25.14±0.30° in the powder X-ray diffraction spectrum.

[0030]

[0031] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystal has characteristic diffraction peaks at 2θ angles of 9.11±0.30°, 13.28±0.30°, 15.34±0.30°, 18.16±0.30°, 22.06±0.30°, 25.14±0.30°, 26.75±0.30°, and 27.25±0.30°.

[0032] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystal has characteristic diffraction peaks at 2θ angles of 9.11±0.30°, 11.21±0.30°, 13.28±0.30°, 15.34±0.30°, 18.16±0.30°, 22.06±0.30°, 23.15±0.30°, 25.14±0.30°, 25.97±0.30°, 26.75±0.30°, 27.25±0.30°, and 30.82±0.30°.

[0033] The present invention provides a crystal of a compound represented by formula (I) characterized by having characteristic diffraction peaks at 2θ angles of 13.20±0.20°, 15.26±0.20°, and 25.07±0.20° in the powder X-ray diffraction spectrum.

[0034]

[0035] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystal has characteristic diffraction peaks at 2θ angles of 13.20±0.20°, 15.26±0.20°, 18.08±0.20°, 21.99±0.20°, 25.07±0.20°, 26.66±0.20°, 28.38±0.20°, and 30.70±0.20°.

[0036] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystal has characteristic diffraction peaks at 2θ angles of 9.03±0.20°, 11.13±0.20°, 13.20±0.20°, 15.26±0.20°, 18.08±0.20°, 21.99±0.20°, 25.07±0.20°, 26.66±0.20°, 28.38±0.20°, 29.41±0.20°, 30.70±0.20°, and 38.53±0.20°.

[0037] The present invention provides a crystalline form C of a compound represented by formula (I), characterized in that the powder X-ray diffraction spectrum has characteristic diffraction peaks at 2θ angles of 13.20±0.20°, 18.08±0.20°, and 25.07±0.20°.

[0038]

[0039] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystalline form C has characteristic diffraction peaks at 2θ angles of 13.20±0.20°, 15.26±0.20°, 18.08±0.20°, 21.99±0.20°, 25.07±0.20°, 26.66±0.20°, 28.38±0.20°, and 30.70±0.20°.

[0040] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystalline form C has characteristic diffraction peaks at 2θ angles of 13.20±0.20°, 15.26±0.20°, 18.08±0.20°, 21.99±0.20°, 25.07±0.20°, 25.38±0.20°, 26.66±0.20°, and 30.70±0.20°.

[0041] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystalline form C has characteristic diffraction peaks at 2θ angles of 9.03±0.20°, 11.13±0.20°, 13.20±0.20°, 15.26±0.20°, 18.08±0.20°, 21.99±0.20°, 25.07±0.20°, 26.66±0.20°, 28.38±0.20°, 29.41±0.20°, 30.70±0.20°, and 38.53±0.20°.

[0042] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystalline form C has characteristic diffraction peaks at 2θ angles of 9.03±0.20°, 13.20±0.20°, 15.26±0.20°, 18.08±0.20°, 21.99±0.20°, 25.07±0.20°, 25.38±0.20°, 26.66±0.20°, 28.38±0.20°, 29.41±0.20°, 30.70±0.20°, and 38.53±0.20°.

[0043] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystalline form C has characteristic diffraction peaks at 2θ angles of 9.03±0.20°, 11.13±0.20°, 13.20±0.20°, 15.26±0.20°, 18.08±0.20°, 21.99±0.20°, 24.09±0.20°, 25.07±0.20°, 25.38±0.20°, 26.66±0.20°, 27.17±0.20°, 28.38±0.20°, 29.41±0.20°, 30.70±0.20°, 31.02±0.20°, and 38.53±0.20°.

[0044] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystalline form C has characteristic diffraction peaks at 2θ angles of 13.20±0.20° and 25.07±0.20°, and 18.08±0.20°, and / or 9.03±0.20°, and / or 11.13±0.20°, and / or 15.26±0.20°, and / or 18.92±0.20°, and / or 21.99±0.20°, and / or 24.09±0.20°, and / or 25.38±0.20°, and / or 26.66±0.20°, and / or 27.17±0.20°, and / or 28.38±0.20°, and / or It may have additional characteristic diffraction peaks at 29.41±0.20°, and / or 30.70±0.20°, and / or 31.02±0.20°, and / or 33.67±0.20°, and / or 35.40±0.20°, and / or 36.35±0.20°, and / or 37.26±0.20°, and / or 38.53±0.20°.

[0045] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystalline form C has characteristic diffraction peaks at 2θ angles of 9.03°, 11.13°, 13.20°, 15.26°, 18.08°, 18.92°, 21.99°, 24.09°, 25.07°, 25.38°, 26.66°, 27.17°, 28.38°, 29.41°, 30.70°, 31.02°, 33.67°, 35.40°, 36.35°, 37.26°, and 38.53°.

[0046] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystalline form C is 5.66°, 9.03°, 11.13°, 13.20°, 13.70°, 15.26°, 17.25°, 18.08°, 18.92°, 20.88°, 21.99°, 23.41°, 24.09°, 25.07°, 25.38°, 25.99°, 26.66°, 27.17°, 28.38°, 29.41°, 29.98°, 30.70°, 31.02°, 31.72°, 33.67°, 35.40°, 36.35°, 36.74°, It has characteristic diffraction peaks at 2θ angles of 37.26°, 38.53°, and 39.80°.

[0047] In some aspects of the present invention, the XRPD spectrum of the crystalline form C is basically as shown in FIG. 5.

[0048] In some embodiments of the present invention, the XRPD spectrum analysis data of the crystalline form C is as shown in Table 3.

[0049]

[0050] In some embodiments of the present invention, the thermogravimetric analysis curve of the crystalline form C shows a weight loss of 1.21% at 200°C ± 3°C.

[0051] In some aspects of the present invention, the TGA spectrum of the crystalline form C is as shown in FIG. 6.

[0052] In some aspects of the present invention, the differential scanning calorimetry curve of the crystalline form C has a starting value of one endothermic peak at 250.0℃±2℃.

[0053] In some aspects of the present invention, the DSC spectrum of the crystalline form C is as shown in FIG. 7.

[0054] The present invention provides a crystalline form D of a compound represented by formula (I), characterized in that the powder X-ray diffraction spectrum has characteristic diffraction peaks at 2θ angles of 6.71±0.20°, 11.87±0.20°, and 25.21±0.20°.

[0055]

[0056] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystalline form D has characteristic diffraction peaks at 2θ angles of 6.71±0.20°, 11.87±0.20°, 13.39±0.20°, 15.44±0.20°, 20.77±0.20°, 22.16±0.20°, 25.21±0.20°, and 27.05±0.20°.

[0057] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystalline form D has characteristic diffraction peaks at 2θ angles of 6.71±0.20°, 11.87±0.20°, 13.39±0.20°, 15.44±0.20°, 16.32±0.20°, 17.90±0.20°, 20.77±0.20°, 22.16±0.20°, 24.31±0.20°, 25.21±0.20°, 27.05±0.20°, and 27.41±0.20°.

[0058] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystalline form D is 6.45°, 6.71°, 9.22°, 10.40°, 11.61°, 11.87°, 12.53°, 13.39°, 13.82°, 15.44°, 16.32°, 17.37°, 17.90°, 18.27°, 19.07°, 19.67°, 19.90°, 20.77°, 22.16°, 24.31°, 25.21°, 26.10°, 27.05°, 27.41°, 28.50°, 29.59°, 30.10°, 30.89°, It has characteristic diffraction peaks at 2θ angles of 31.17°, 32.81°, 33.77°, 34.17°, 35.52°, 36.57°, 38.20°, and 38.68°.

[0059] In some aspects of the present invention, the XRPD spectrum of the crystalline form D is basically as shown in FIG. 8.

[0060] In some embodiments of the present invention, the XRPD spectrum analysis data of the crystalline form D is as shown in Table 4.

[0061]

[0062] In some aspects of the present invention, the thermogravimetric analysis curve of the crystalline form D shows a weight loss of 1.14% at 200°C ± 3°C.

[0063] In some aspects of the present invention, the TGA spectrum of the crystalline form D is as shown in FIG. 9.

[0064] In some aspects of the present invention, the differential scanning calorimetry curve of the crystalline form D has a starting value of one endothermic peak at 251.4℃±2℃.

[0065] In some aspects of the present invention, the DSC spectrum of the crystalline form D is as shown in FIG. 10.

[0066] The present invention provides a crystalline form E of a compound represented by formula (I), characterized in that the powder X-ray diffraction spectrum has characteristic diffraction peaks at 2θ angles of 13.28±0.20°, 15.34±0.20°, and 25.14±0.20°.

[0067]

[0068] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystalline form E has characteristic diffraction peaks at 2θ angles of 9.11±0.20°, 13.28±0.20°, 15.34±0.20°, 18.16±0.20°, 22.06±0.20°, 25.14±0.20°, 26.75±0.20°, and 27.25±0.20°.

[0069] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystalline form E has characteristic diffraction peaks at 2θ angles of 9.11±0.20°, 12.43±0.20°, 13.28±0.20°, 15.34±0.20°, 18.16±0.20°, 22.06±0.20°, 23.15±0.20°, and 25.14±0.20°.

[0070] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystalline form E has characteristic diffraction peaks at 2θ angles of 9.11±0.20°, 11.21±0.20°, 13.28±0.20°, 15.34±0.20°, 18.16±0.20°, 22.06±0.20°, 23.15±0.20°, 25.14±0.20°, 25.97±0.20°, 26.75±0.20°, 27.25±0.20°, and 30.82±0.20°.

[0071] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystalline form E has characteristic diffraction peaks at 2θ angles of 9.11±0.20°, 11.21±0.20°, 12.43±0.20°, 13.28±0.20°, 15.34±0.20°, 18.16±0.20°, 22.06±0.20°, 23.15±0.20°, 25.14±0.20°, 25.97±0.20°, 26.75±0.20°, and 27.25±0.20°.

[0072] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystalline form E has characteristic diffraction peaks at 2θ angles of 13.28±0.20° and 25.14±0.20°, and 15.34±0.20°, and / or 9.11±0.20°, and / or 10.94±0.20°, and / or 11.21±0.20°, and / or 12.43±0.20°, and / or 18.16±0.20°, and / or 22.06±0.20°, and / or 23.15±0.20°, and / or 23.35±0.20°, and / or 24.19±0.20°, and / or 25.97±0.20°, and / or It may have additional characteristic diffraction peaks at 26.75±0.20°, and / or 27.25±0.20°, and / or 28.45±0.20°, and / or 29.49±0.20°, and / or 30.82±0.20°, and / or 33.74±0.20°, and / or 36.39±0.20°, and / or 37.34±0.20°, and / or 38.57±0.20°.

[0073] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystalline form E is 9.11±0.20°, 10.94±0.20°, 11.21±0.20°, 12.43±0.20°, 13.28±0.20°, 15.34±0.20°, 18.16±0.20°, 22.06±0.20°, 23.15±0.20°, 23.35±0.20°, 24.19±0.20°, 25.14±0.20°, 25.97±0.20°, 26.75±0.20°, 27.25±0.20°, 28.45±0.20°, 29.49±0.20°, It has characteristic diffraction peaks at 2θ angles of 30.82±0.20°, 33.74±0.20°, 36.39±0.20°, 37.34±0.20°, and 38.57±0.20°.

[0074] In some aspects of the present invention, the powder X-ray diffraction spectrum of the crystalline form E is 9.11°, 10.94°, 11.21°, 12.43°, 13.28°, 15.34°, 17.39°, 18.16°, 20.18°, 18.94°, 20.95°, 22.06°, 23.15°, 23.35°, 24.19°, 25.14°, 25.97°, 26.75°, 27.25°, 28.45°, 29.49°, 30.16°, 30.82°, 33.74°, 35.45°, 36.39°, 37.34°, and 38.57°, with a 2θ It has characteristic diffraction peaks at each angle.

[0075] In some aspects of the present invention, the XRPD spectrum of the crystalline form E is basically as shown in FIG. 11.

[0076] In some aspects of the present invention, the XRPD spectrum analysis data of the crystalline form E is as shown in Table 5.

[0077]

[0078] In some aspects of the present invention, the thermogravimetric analysis curve of the crystalline form E shows a weight loss of 0.79% at 200°C ± 3°C.

[0079] In some aspects of the present invention, the TGA spectrum of the crystalline form E is as shown in FIG. 12.

[0080] In some aspects of the present invention, the differential scanning calorimetry curve of the crystalline form E has a starting value of one endothermic peak at 250.4℃±2℃.

[0081] In some aspects of the present invention, the DSC spectrum of the crystalline form E is as shown in FIG. 13.

[0082] The present invention further provides the use of crystalline forms A, B, C, D, and E of the compound represented by formula (I) in the manufacture of a drug for treating gout and hyperuricemia.

[0084] The crystalline form of the compound represented by formula (I) has stable properties, is non-hygroscopic, and has excellent medicinal potential.

[0085] Definition and explanation

[0086] Unless otherwise noted, the following terms and short phrases used in this text have the following meanings. Unless specifically defined otherwise, a specific short phrase or term should not be understood as indeterminate or unclear, but should be understood in its ordinary sense. Where a product name appears in this text, it refers to the corresponding product or its active ingredient.

[0087] The intermediate compounds of the present invention can be prepared by various synthesis methods well known to those skilled in the art and include specific embodiments exemplified below, embodiments combined by other chemical synthesis methods, and equivalent alternative forms well known to those skilled in the art; preferred embodiments include, but are not limited to, the embodiments of the present invention.

[0088] The chemical reaction of a specific embodiment of the present invention is completed in a suitable solvent, and said solvent must be suitable for the chemical change of the present invention and the reagents and materials required therefor. In order to obtain the compound of the present invention, a person skilled in the art may need to modify or select a synthesis step or reaction process based on existing embodiments.

[0089] The present invention will be described in detail through the following examples, but these examples are not intended to limit the invention.

[0090] All solvents used in the present invention are commercially available and can be used without further purification.

[0091] Compounds are named according to industry naming conventions or by ChemDraw® software, and commercially available compounds use the supplier's list names.

[0092] The structure of the compound of the present invention can be confirmed by conventional methods well known to those skilled in the art, and where the present invention relates to the absolute configuration of the compound, said absolute configuration can be confirmed by conventional technical means in the art. For example, the absolute configuration can be confirmed by single-crystal X-ray diffraction (SXRD) by collecting diffraction intensity data by collecting a cultured single crystal with a Bruker D8 venture diffractometer, using CuKα radiation as the light source and scanning mode: φ / ω scan, collecting the relevant data, and then analyzing the crystal structure using the direct method (Shelxs97).

[0093] All solvents used in the present invention are commercially available. The present invention uses the following abbreviations: -OMOM represents methoxymethyl ether; HPE represents 100% inhibition activity; ZPE represents 0% inhibition activity; and DPBS represents Dulbecco phosphate buffer.

[0094] X-ray powder diffractometer (XRFD) method of the present invention

[0095] Instrument Model: Bruker D2 PHASER X-ray Diffractometer

[0096] The detailed XRPD parameters are as follows.

[0097] Light source: Cu, k-Alphal(λ=1.54184Å)

[0098] Photovoltaic tube voltage: 30kV

[0099] Phototube current: 10mA

[0100] Diverging slit: 0.6mm

[0101] Main optical path axial solar slit: 2.5°

[0102] Secondary optical path axial solar slit: 2.5°

[0103] Detector slit: 5.827°

[0104] Anti-scattering slit: 0mm

[0105] Scan axis: θs-θd

[0106] Step size: 0.02deg

[0107] Duration per step: 0.2 seconds

[0108] Scan angle range: 3 to 40 degrees

[0109] Differential Scanning Calorimeter (DSC) analysis method of the present invention

[0110] Instrument Model: TA DSC Q2000 Differential Scanning Calorimeter

[0111] Test method: A sample (~1 mg) was taken and placed in a DSC aluminum pot for testing, and the sample was heated from 30°C to 250°C at a heating rate of 10°C / min under 50 mL / min N2 conditions.

[0112] The Thermal Gravimetric Analyzer (TGA) method of the present invention

[0113] Instrument Model: Discovery TGA 5500 Thermogravimetric Analyzer

[0114] Test method: A sample (2 to 5 mg) was taken and placed in a TGA platinum pot for testing, and the sample was heated from room temperature to 300°C at a heating rate of 10°C / min under 25 mL / min N2 conditions.

[0115] Dynamic Vapor Sorption (DVS) analysis method of the present invention

[0116] Device Model: Intrinsic Dynamic Vapor Adsorption Device

[0117] Test conditions: A sample (10 to 30 mg) was taken, placed in a DVS sample tray, and tested.

[0118] The detailed DVS parameters are as follows.

[0119] Temperature: 25℃

[0120] Balance: dm / dt=0.002% / min(min: 10 min, max: 180 min)

[0121] RH(%) Test Gradient: 10(90-0-90%), 5(90-95%)

[0122] RH(%) Test Range: 0%-95%-0%

[0123] The classification of conventional evaluations is as shown in Table 6 below.

[0124]

[0125] Note: ΔW% represents the increase in moisture-absorbing weight of the test product at 25±1℃ and 80±2%RH. Brief explanation of the drawing

[0126] Figure 1 is the XRPD spectrum of Cu-Kα radiation of crystalline form A of the compound represented by formula (I). Figure 2 is the TGA spectrum of crystalline form A of the compound represented by formula (I). Figure 3 is the DSC spectrum of crystalline form A of the compound represented by formula (I). Figure 4 is the XRPD spectrum of Cu-Kα radiation of crystalline form B of the compound represented by formula (I). Figure 5 is the XRPD spectrum of Cu-Kα radiation of the crystalline form C of the compound represented by formula (I). Figure 6 is the TGA spectrum of the crystalline form C of the compound represented by formula (I). Figure 7 is the DSC spectrum of the crystalline form C of the compound represented by formula (I). Figure 8 is the XRPD spectrum of Cu-Kα radiation of the crystalline form D of the compound represented by formula (I). Figure 9 is the TGA spectrum of crystalline form D of the compound represented by formula (I). Figure 10 is the DSC spectrum of the crystalline form D of the compound represented by formula (I). Figure 11 is the XRPD spectrum of Cu-Kα radiation of the crystalline form E of the compound represented by formula (I). Figure 12 is the TGA spectrum of the crystalline form E of the compound represented by formula (I). Figure 13 is the DSC spectrum of the crystalline form E of the compound represented by formula (I). Figure 14 is the DVS spectrum of the crystalline form C of the compound represented by formula (I). Specific details for implementing the invention

[0127] The present invention is described in detail through the following examples, but this does not imply any adverse limitations to the invention. Although the present invention has been described in detail and specific embodiments disclosed herein, it is obvious to those skilled in the art that various changes and improvements can be made to specific embodiments of the invention without departing from the gist and scope of the invention.

[0128] Example 1: Preparation of crystalline form A of the compound represented by formula (I)

[0129]

[0130] Step 1: Compound I-2 Synthesis of.

[0131] Potassium tert-butoxide (234.26 g, 2.09 mol) is added to dimethyl sulfoxide (1200 mL), stirred at room temperature until clear, and the compound at 15°C to 20°C I-1 Dimethyl sulfoxide (500 mL) solution (200 g, 1.49 mol) was added dropwise, and stirring continued for 40 minutes after the addition was completed. Carbon disulfide (113.54 g, 1.49 mol, 90.11 mL) was added dropwise, and stirring continued for 20 minutes after the addition was completed, while maintaining the temperature of the reaction solution at 20°C. Potassium tert-butoxide (100.40 g, 894.70 mmol) was slowly added, and stirring was performed for 30 minutes while maintaining the temperature of the reaction solution at 15°C to 20°C. Ethyl bromoacetate (498.05 g, 2.98 mol, 329.83 mL) was further added dropwise, and stirring was performed for 1.5 hours at the above temperature while maintaining the temperature of the reaction solution at 15°C to 20°C. Potassium carbonate (206.09 g, 1.49 mol) was added, the reaction solution was heated to 60°C, and stirring was continued for 1.5 hours. 1 L of water was added to the reaction solution, the pH was adjusted to 3 to 4 with a 6 M aqueous hydrochloric acid solution, and the mixture was extracted with ethyl acetate (1.5 L × 2). The organic phase after mixing was washed with saturated saline solution (200 mL × 3), and the organic solvent was removed under reduced pressure. To the crude product obtained, isopropanol (200 mL) was added, the mixture was stirred uniformly, left for 15 hours, filtered, and vacuum dried at 45°C for 1 hour to obtain the compound. I-2 obtained. 1 H NMR (400MHz, CDCl3) δ: 4.32 (q, J = 7.2 Hz, 2H), 4.19 (q, J = 7.2 Hz, 2H), 3.56 (s, 2H), 3.25 (t, J = 6.8 Hz, 2H), 3.19 (t, J = 14.4 Hz, 2H), 2.26 - 2.17 (m, 2H), 1.37 (t,J = 7.2 Hz, 3H), 1.27(t, J = 7.2Hz, 3H). MS m / z=364.8 [M+H] + .

[0132] Step 2: Compound I-3 Synthesis of.

[0133] compound I-2 (282 g, 773.82 mmol) was dissolved in ethanol (3.5 L), Raney nickel (99.45 g, 1.16 mol) was added, the mixture was purged with nitrogen gas three times, and the reaction was carried out at 85°C for 48 hours with stirring under a hydrogen gas pressure of 2.5 MPa. After cooling, the mixture was filtered through diatomite under nitrogen protection, and the solvent was removed from the filtrate under reduced pressure to obtain the compound. I-3 The compound was obtained and used directly in the next step of the reaction without further purification. 1 H NMR (400MHz, CDCl3) δ: 7.09 (s, 1H), 4.26 (q, J = 7.2 Hz, 2H), 3.20 (t, J = 6.8 Hz, 2H), 3.12 (t, J = 14.4 Hz, 2H), 2.20 - 2.10 (m, 2H), 1.30 (t, J = 6.8 Hz, 3H). MS m / z=247.0 [M+H] + .

[0134] Step 3: Compound I-4 Synthesis of.

[0135] compound I-340.00 g (162.42 mmol) was dissolved in 200 mL of methanol, 200 mL of an aqueous solution of sodium hydroxide (12.99 g, 324.84 mmol) was added, the reaction solution was heated to 50°C, and stirring was continued for 2 hours. The organic solvent was removed under reduced pressure, 150 mL of water was added to the residue, and the pH was adjusted to 2 to 3 with a 6 M aqueous hydrochloric acid solution, resulting in the precipitation of a large amount of white solid. The mixture was filtered, the cake was washed with 100 mL of water and 50 mL of petroleum ether, and the compound was vacuum dried at 50°C for 3 hours. I-4 obtained. 1 H NMR (400 MHz, CD3OD) δ: 7.38 (s, 1H), 3.33 - 3.17 (m, 4 H), 2.28 - 2.21 (m, 2H).

[0136] Step 4: Compound I-5 Synthesis of.

[0137] compound I-4 (35.0 g, 160.39 mmol) was dissolved in tetrahydrofuran (200 mL), carbonyldiimidazole (33.81 g, 208.51 mmol) was added, and the reaction solution was stirred for 2 hours under nitrogen gas protection; then, ammonia water (31.23 g, 240.58 mmol, 34.32 mL) was added, and the reaction solution was continuously stirred for 15 hours. The organic solvent was removed under reduced pressure, 300 mL of water was added to the obtained residue and stirred for 10 minutes, filtered, the cake was washed with 100 mL of water, and the compound was vacuum dried at 55°C for 2.5 hours. I-5 obtained. 1 H NMR (400 MHz, CDCl3) δ: 7.09 (s, 1H), 5.72 (brs, 2H), 3.30 - 3.18 (m, 4H), 2.29 - 2.19 (m, 2H).

[0138] Step 5: Compound I-6 Synthesis of.

[0139] compoundI-5 (31 g, 142.70 mmol) was dissolved in N,N-dimethylformamide (200 mL), N-bromosuccinimide (27.94 g, 156.97 mmol) was slowly added in a batch, and the reaction solution was continuously stirred at 20°C for 2 hours. The reaction solution was slowly poured into 600 mL of stirred water, and a large amount of solid precipitated; the mixture was stirred for 10 minutes, filtered, the cake was washed with 200 mL of water and 100 mL of petroleum ether, and vacuum dried at 50°C for 2 hours to obtain the compound. I-6 obtained. 1 H NMR (400MHz, CDCl3) δ: 5.62 (brs, 2H), 3.25 (t, J = 7.2 Hz, 2H), 3.04 (t, J = 14.0 Hz, 2H), 2.26 - 2.18 (m, 2H).

[0140] Step 6: Compound I-7 Synthesis of.

[0141] compound I-6 (48g, 162.09mmol) and triethylamine (32.80g, 324.18mmol, 45.12mL) were added to ethyl acetate (250mL), cooled to 0°C under nitrogen protection, trifluoroacetic anhydride (44.26g, 210.72mmol, 29.31mL) was added dropwise, the reaction solution was stirred continuously at the above temperature for 1 hour, raised to 20°C, and stirred continuously for 0.5 hours. The reaction solution was diluted with 250mL of ethyl acetate, washed sequentially with water (100mL × 2), saturated sodium bicarbonate solution (150mL), and saturated saline solution (100mL), dried with anhydrous sodium sulfate, filtered, and the organic solvent was removed under reduced pressure, and the compound I-7 The compound was obtained and used directly in the next step of the reaction without further purification. 1H NMR (400 MHz, CDCl3) δ: 3.13 - 2.97 (m, 4H), 2.30 - 2.20 (m, 2H).

[0142] Step 7: Compound I-8 Synthesis of.

[0143] compound I-7 (6.0g, 21.57mmol), compound I-7-1 (7.60g, 23.73mmol) and anhydrous potassium phosphate (9.16g, 43.15mmol) were added to ethylene glycol dimethyl ether (60mL) and water (12mL), and under the protection of nitrogen gas, Pd(dppf)Cl2 (394.64mg, 539.34μmol) was added, and the reaction solution was heated to 85°C under the protection of nitrogen gas and stirred continuously for 15 hours. After cooling, 20mL of water and 100mL of ethyl acetate were added to the reaction solution and stirred for 10 minutes, filtered, the organic phase was separated from the filtrate, the aqueous phase was extracted with ethyl acetate (30mL × 3), the organic phase after mixing was washed with saturated saline (30mL), dried with anhydrous sodium sulfate, filtered, and the organic solvent was removed under reduced pressure. Ethyl acetate (80 mL) was added to the obtained crude product, followed by the sequential addition of activated carbon (4 g) and palladium-removed silica gel (4 g). The temperature was raised to 80°C and stirred continuously for 1 hour, then cooled, filtered through diatomaceous earth, and the organic solvent was removed under reduced pressure. The obtained crude product was then slurried with tert-butyl methyl ether (25 mL) at 25°C for 0.5 hours, filtered, and the obtained cake was vacuum dried at 45°C for 1 hour to obtain a compound I-8 obtained. 1 H NMR (400MHz, CDCl3) δ: 11.18 (s, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.01 (d, J = 1.6 Hz, 1H), 6.92 - 6.90 (m, 1H), 3.24 (t, J= 14.4 Hz, 2H), 3.12 (t, J = 6.8Hz, 2H), 2.36 - 2.26 (m, 2H), 1.64 (s, 9H).

[0144] Step 8: Crystalline form A of the compound represented by formula (I) Synthesis of

[0145] compound I-8 (32g, 81.75mmol) was added to trifluoroacetic acid (250mL), and the reaction solution was reacted at 20°C for 1 hour while stirring. Water (300mL) was added to the residue obtained by removing trifluoroacetic acid under reduced pressure, slurried at room temperature for 20 minutes until completely dispersed, filtered, the cake was washed with water (200mL), and vacuum dried at 45°C for 1 hour. Crystalline form A of the compound represented by formula (I) obtained. 1 H NMR (400MHz, CD3OD) δ: 8.03 - 7.96 (m, 1H), 7.12 - 7.06 (m, 2H), 3.36 - 3.29 (m, 2H), 3.16 - 3.07 (m, 2H), 2.44 - 2.30 (m, 2H). The XRPD spectrum of crystal form A is as shown in Fig. 1, the TGA spectrum is as shown in Fig. 2, and the DSC spectrum is as shown in Fig. 3.

[0146] Example 2: Preparation of a compound represented by formula (I)

[0147]

[0148] Step 1: Compound I-4 Synthesis of.

[0149] compound I-3(2.5g, 10.15mmol) was dissolved in methanol (10mL), to which water (10mL) and sodium hydroxide (1.62g, 40.61mmol) were added. The resulting reaction solution was placed in an oil bath at 40°C and reacted for 2 hours while stirring. Half of the reaction solution was removed by vacuum concentration, and water (5mL) was added to the residue. The pH was adjusted to 2 to 3 with 6M hydrochloric acid while stirring, resulting in the precipitation of a large amount of white solid. The solid was collected by filtration and vacuum dried at 50°C for 3 hours to obtain the compound. I-4 obtained. 1 H NMR (400MHz, CDCl3) δ: 7.28 (s, 1H), 3.30 (t, J=7.0 Hz, 2H), 3.22 (t, J=14.3 Hz, 2H), 2.25 (tt, J=6.8, 13.4 Hz, 2H).

[0150] Step 2: Compound I-5 Synthesis of.

[0151] compound I-4 After dissolving (500 mg, 2.29 mmol) in dichloromethane (5 mL), carbonyldiimidazole (445.83 mg, 2.75 mmol) was added, and the resulting reaction solution was reacted for 1 hour while stirring under nitrogen gas protection. Then, the reaction solution was poured into tetrahydrofuran (5 mL) in vigorously stirred ammonia water (2.87 g, 22.91 mmol, 3.15 mL, content 28%) and reacted for 30 minutes while stirring. The reaction solution was concentrated under reduced pressure at 25°C, the residue was extracted with ethyl acetate (20 mL × 3), the organic phase was combined, and the product was rotary dried to obtain a crude product. The crude product was analyzed by silica gel column chromatography (ethyl acetate / petroleum ether = 0% to 45%) to form the compound I-5 obtained. 1H NMR (400MHz, CDCl3) δ: 7.10 (s, 1H), 5.58 (br s, 2H), 3.28 (t, J=6.9 Hz, 2H), 3.21 (t, J=14.4 Hz, 2H), 2.24 (tt, J=6.9, 13.4 Hz, 2H).

[0152] Step 3: Compound 2-4 Synthesis of

[0153] compound I-5 (320 mg, 1.47 mmol) was dissolved in DMF (3 mL), and after cooling the obtained solution to 0°C, cyanur chloride (298.81 mg, 1.62 mmol) was added. The final reaction solution was reacted under nitrogen gas protection for 2 hours while stirring (during this period, a large amount of white solid precipitated). The reaction solution was diluted with ethyl acetate (50 mL), washed with water (10 mL × 3) and saturated saline (10 mL), dried with an appropriate amount of anhydrous sodium sulfate, and filtered to remove the drying agent. The solvent was removed from the filtrate under reduced pressure to obtain the compound of the crude product. 2-4 A crude product was obtained and used directly in the next step of the reaction. 1 H NMR: (400MHz, CDCl3) δ: 7.25 (s, 1H), 3.21 (t, J=14.3 Hz, 2H), 3.09 (t, J=6.9 Hz, 2H), 2.28 (tt, J=6.8, 13.2 Hz, 2H).

[0154] Step 4: Compound 2-5 Synthesis of

[0155] compound 2-4After dissolving (290 mg, 1.46 mmol) in acetic acid (2 mL), liquid bromine (348.94 mg, 2.18 mmol, 112.56 μL) was added, and the resulting reaction solution was stirred and reacted at room temperature (25 °C) for 15 hours. The reaction solution was rotary-dried, and ethyl acetate (30 mL) was added to the residue. The pH was adjusted to 7 to 8 with saturated sodium carbonate, the organic phase was separated, and the aqueous phase was extracted with ethyl acetate (30 mL). The organic phases were combined and concentrated under reduced pressure to obtain a crude product. Compounds were identified on the crude product through silica gel column chromatography (ethyl acetate / petroleum ether = 0% to 5%). I-7 obtained. 1 H NMR: (400MHz, CDCL3) δ: 3.10-2.99 m, 4H), 2.32-2.19 (m, 2H).

[0156] Step 5: Compound 2-6 Synthesis of

[0157] compound I-7 (140 mg, 503.39 μmol), boric acid ester 2-5A After adding 178.39 mg (553.73 μmol) and potassium carbonate (139.14 mg, 1.01 mmol) to dioxane (3 mL) and water (0.6 mL), 1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (Pd(dppf)Cl2) (36.83 mg, 50.34 μmol) was added, and the mixture was placed in an oil bath at 105°C under nitrogen gas protection and reacted for 15 hours while stirring. The reaction solution was rotary-dried to obtain a crude product, and the compound was analyzed by silica gel column chromatography (ethyl acetate / petroleum ether = 0% to 25%) of the crude product. 2-6 obtained. 1H NMR: (400MHz, CHCl3) δ: 7.87 (d, J=8.0 Hz, 1H), 7.28 (d, J=1.6 Hz, 1H), 7.10 (dd, J=1.6, 8.0 Hz, 1H), 5.0 (s, 2H), 3.3 (s, 3H), 3.55(s, 3H), 3.23 (t, J=14.4 Hz, 2H), 3.13 (t, J=6.8 Hz, 2H), 2.39-2.24 (m, 2H).

[0158] Step 6: Compound 2-7 Synthesis of

[0159] compound 2-6 (105 mg, 266.90 μmol) was dissolved in tetrahydrofuran (2 mL), an aqueous solution of lithium hydroxide monohydrate (2 M, 533.80 μL) was added, and the resulting reaction solution was reacted at room temperature (25 °C) for 15 hours while stirring. The reaction solution was rotated at 40 °C to remove the tetrahydrofuran, and the residue was adjusted to pH 2 to 3 with 2 M hydrochloric acid until a large amount of solid precipitated; then, ethyl acetate (50 mL) was added and stirred, the ethyl acetate was separated, and the compound was rotary dried. 2-7 A crude product was obtained and used directly in the next step of the reaction.

[0160] Step 7: Compound represented by formula (I) Synthesis of

[0161] compound 2-7 After dissolving (105 mg, 276.77 μmol) in methanol (1 mL), hydrochloric acid (60.55 mg, 1.66 mmol, 59.36 μL) was added until the reaction solution became turbid, and the reaction was carried out at 25°C for 3 hours while stirring. The reaction solution was rotary-dried at 40°C, and the obtained residue was separated into a preparative high-performance liquid phase and purified (chromatography column: Venusil ASB Phenyl 150×30 mm×5 μm; mobile phase: [water (0.05% HCl)-ACN]; ACN%: 60% to 90%, 9 min). Compound represented by formula (I) obtained. 1H NMR (400MHz, CD3OD) δ: 8.00 (d, J=8.0 Hz, 1H), 7.13 - 7.04 (m, 2H), 3.35-3.32 (m, 2H), 3.12 (t, J=7.2 Hz, 2H), 2.45-2.30 (m, 2H);MS (ESI) m / z: 334.02 [MH] - .

[0162] Example 3: Preparation of crystalline form B of the compound represented by formula (I)

[0163] Crystalline form A (20 mg, 0.06 mmol) of the compound represented by formula (I) was added to dichloromethane (1 mL) and stirred continuously at 25°C for 120 hours. The mixture was filtered, and the cake was vacuum dried at 50°C for 2 to 5 hours to obtain crystalline form B of the compound represented by formula (I). The XRPD spectrum of crystalline form B is shown in Fig. 4.

[0164] Example 4: Preparation of crystalline form C of the compound represented by formula (I)

[0165] Crystalline form A (1.0 g, 2.98 mmol) of the compound represented by formula (I) was added to a mixed solvent of ethyl acetate (5 mL) and n-heptane (5 mL), and stirred continuously at 25°C for 72 hours. After filtration, the cake was vacuum dried at 45°C for 2 hours to obtain crystalline form C of the compound represented by formula (I). The XRPD spectrum of crystalline form C is shown in Fig. 5, the TGA spectrum is shown in Fig. 6, and the DSC spectrum is shown in Fig. 7.

[0166] Example 5: Preparation of crystalline form D of the compound represented by formula (I)

[0167] About 20 mg of crystalline form A of the compound represented by formula (I) was weighed and added to a mixed solvent of tetrahydrofuran (0.4 mL) and water (0.4 mL). The mixed solution was stirred at 50°C until the solid dissolved, then the temperature was lowered to 13°C and stirred for 72 hours. The mixture was filtered, and the cake was vacuum dried at 45°C for 2 hours to obtain crystalline form D of the compound represented by formula (I). The XRPD spectrum of crystalline form D is shown in Fig. 8, the TGA spectrum is shown in Fig. 9, and the DSC spectrum is shown in Fig. 10.

[0168] Example 6: Preparation of crystalline form E of the compound represented by formula (I)

[0169] Crystalline form A (1.0 g, 2.98 mmol) of the compound represented by formula (I) was added to a mixed solvent of tetrahydrofuran (3.3 mL) and water (6.6 mL), and the resulting mixture was stirred at 25°C for 72 hours. The mixture was filtered, and the cake was vacuum dried at 45°C for 2 hours to obtain crystalline form E of the compound represented by formula (I). The XRPD spectrum of crystalline form E is shown in Fig. 11, the TGA spectrum is shown in Fig. 12, and the DSC spectrum is shown in Fig. 13.

[0170] Example 7: of the compound represented by formula (I) Study on the hygroscopicity of crystalline C

[0171] Experimental materials:

[0172] DVS Intrinsic Dynamic Vapor Adsorption Device

[0173] Experiment Method:

[0174] 10 to 30 mg of crystalline form C of the compound represented by formula (I) was taken, placed in a DVS sample tray, and tested.

[0175] Experimental Results:

[0176] The DVS spectrum of the crystalline form C of the compound represented by formula (I) is as shown in Fig. 14, and △W=0.196%.

[0177] Experimental Conclusion:

[0178] The crystalline form C of the compound represented by formula (I) has no hygroscopicity, with a hygroscopic weight increase of 0.196% at 25°C and 80% RH.

[0179] Example 8: Solid stability test of crystalline form C of the compound represented by formula (I)

[0180] In accordance with the "General notice of the Chinese Pharmacopoeia 2015 edition Volume IV 9001" regarding the stability test guidelines for raw materials and formulations, the stability of crystalline form C of the compound represented by formula (I) was examined under conditions of high temperature (60℃, open), high humidity (room temperature / relative humidity 92.5%, open), and strong light irradiation (5000 l×, closed).

[0181] Approximately 1.5 g of the crystalline form C of the compound represented by Formula (I) in Part 12 was weighed in parallel, placed in a flat weighing flask (70×35 mm) or a disposable Petri dish, and spread thinly. Each was then left under conditions of high temperature (60°C), high humidity (25°C / 92.5% humidity), high temperature and high humidity (40°C / 75% humidity), and stable light irradiation. For samples left under high temperature and high humidity conditions, the flask opening was sealed with aluminum foil and small holes were punched in the aluminum foil to allow the sample to come into full contact with the surrounding air; for samples left under strong light irradiation conditions, the flask opening was sealed with a quartz glass lid. Samples left under high temperature (60℃) and high humidity (92.5% humidity, room temperature) conditions were taken on the 5th and 10th days for detection (appearance, related substances, and content), samples left under high temperature and high humidity (40℃ / 75% humidity) conditions were taken on the 1st, 2nd, and 3rd months for detection (appearance, related substances, and content), and samples left under light irradiation conditions had a total light intensity of 1.2×10 6A sample was taken and detected when Lux·hr was reached, and the detection result was compared with the initial detection result on day 0, and the test results are as shown in Table 7 below.

[0182]

[0183] Conclusion: The crystalline form C of the compound represented by formula (I) has excellent stability under conditions of high temperature, high humidity, strong light irradiation, and accelerated conditions.

[0184] Biological test data:

[0185] Experimental Example 1: Xanthine Oxidase Inhibitory Activity Test

[0186] 1. Experimental Objective:

[0187] We intend to evaluate the level of the compound for the inhibition of xanthine oxidase activity.

[0188] 2. Reagents

[0189] The main reagents used in this study include xanthine (Sigma, product number: X4002-1G, lot number: SLBB5664V) and xanthine oxidase (Sigma, product number: X4376-5UN, lot number: SLBQ1518V).

[0190] 3. Device

[0191] The main instrument used in this study is a multi-functional microplate reader.

[0192] 4. Experimental Method

[0193] 1) 50 μL of Dulbecco phosphate buffer (DPBS) was added to the background control well of the compound and the HPE (100% inhibition rate activity) positive control well.

[0194] 2) 2 U / mL of xanthine oxidase was diluted to 0.04 U / mL with DPBS, and 50 μL of xanthine oxidase was added to the compound activity test group wells and the ZPE (0% inhibition rate activity) negative control wells.

[0195] 3) The compound was diluted with DMSO in a 3-fold gradient at 8 points, then the compound was diluted with DPBS, and 50 μL was added to each well, which was a triple well. 50 μL of DPBS was added to each well of the HPE (100% inhibition activity) positive control well and the ZPE (0% inhibition activity) negative control well.

[0196] 4) 200 mM xanthine was diluted to 300 μM with DPBS. 100 μL of xanthine was added to each well and reacted at room temperature for 30 minutes; the final concentration of xanthine oxidase in each well was 0.01 U / mL, and the final concentration of DMSO in each well was 0.5%. The HPE (100% inhibition activity) positive control wells contained xanthine but not xanthine oxidase, the ZPE (0% inhibition activity) negative control wells contained xanthine and xanthine oxidase, and the compound background control wells contained different concentrations of the compound and xanthine but not xanthine oxidase.

[0197] 5) The absorbance value at 290 nm was detected using a spectrophotometer.

[0198] 6) Data Analysis: The inhibition rate of each well for xanthine oxidase was calculated according to the following formula.

[0199]

[0200] * OD test sample is the optical density value of the activity test group wells of compounds including the compound, xanthine, and xanthine oxidase;

[0201] OD compound control is the background optical density value of test compounds at different concentrations containing compounds and xanthine and not containing xanthine oxidase;

[0202] OD ZPEis the average value of the optical density of control wells without inhibitory activity containing 0.5% DMSO, xanthine, and xanthine oxidase;

[0203] OD HPE is the average value of the optical density of 100% inhibitory activity control wells containing 0.5% DMSO and xanthine and not containing xanthine oxidase.

[0204] 7) Using GraphPad Prism software, a log(agonist) vs. response -- variable slope nonlinear fitting analysis was performed on the inhibition rate data (inhibition rate %) of the compounds to determine the IC50 of the compounds. 50 The value was obtained, and the fitting formula is as follows.

[0205] Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)×HillSlope))

[0206] 5. Experimental Results

[0207] The experimental results are as shown in Table 8.

[0208]

[0209] Experimental Conclusion: The compound of the present invention has excellent xanthine oxidase inhibitory activity.

[0210] Experimental Example 2: Test of Inhibitory Activity of Compound Against Uric Acid Intake

[0211] 1. Experimental Objective:

[0212] In this study, we intend to evaluate the inhibitory activity of a test compound on uric acid uptake using a cell line stably transfected with the human Urat1 gene.

[0213] 2. Experimental Materials

[0214] 2.1 Cell line

[0215] A cell line stably transfected with the human Urat1 gene was prepared by Shanghai WuXi App Tec New Drug Development Co., Ltd. The cell line stably transfected with the human Urat1 gene (Urat1-MDCK) was obtained by transfecting MDCK cells with the human Urat1 gene and screening them with G418. The cell line was cultured in MEM medium containing 10% fetal bovine serum (FBS), 100 U / ml penicillin, 100 g / ml streptomycin, 2 mM L-glutamine, 1% non-essential amino acids, and 250 μg / ml G418.

[0216] 2.2 Reagents

[0217] The main reagent used in this study includes 14C-uric acid (ARC, product number: ARC-0513, lot number: 200122).

[0218] 2.3 Device

[0219] The main instrument used in this study is a liquid scintillation analyzer (Perkin Elmer, Tri-Carb 4910TR).

[0220] 3. Experimental Method

[0221] 3.1 Cell Plating

[0222] 3.1.1 Urat1-MDCK cells cultured in a T150 cell culture flask were digested with 0.25% trypsin and then diluted with fresh culture medium to a suspension of 200,000 cells / ml.

[0223] 3.1.2 Cells were seeded into 48-well cell culture plates at a density of 0.5 mL per well, and the final cell density was 100,000 cells / well.

[0224] 3.1.3 Cell culture plates were placed in an incubator at 37°C and 5% CO2 and cultured overnight.

[0225] 3.2 Treatment and Detection of Compounds

[0226] 3.2.1 The compound was diluted at four points with a 5-fold gradient in DMSO, and the concentration after dilution was 200 × final detection concentration. Then, the compound was diluted 10-fold with HBSS buffer.

[0227] 3.2.2 A 10 mM 14C-uric acid concentrated stock solution was diluted to 1 mM with HBSS buffer.

[0228] 3.2.3 After incubating the cell culture plates overnight, the cell culture medium was removed from the plates, and the cells were washed three times with HBSS buffer. Then, 90 μl of HBSS buffer was added to each well.

[0229] 3.2.4 5 μl of the diluted compound was added to each well, and cells were placed in an incubator at 37°C with 5% CO2 and cultured for 20 minutes. The DMSO content in each well was 0.5%. The test compound (10 μM) was used as a 100% inhibition control, and 0.5% DMSO was used as a 0% inhibition control.

[0230] 3.2.5 5 μl of diluted 14C-uric acid was added to each well of the cell plate, and the final concentration of uric acid in each well was 50 μM. Cells were placed in a 37°C, 5% CO2 incubator and cultured for 15 minutes. Then, the cells were washed three times with pre-cooled HBSS buffer.

[0231] 3.2.6 150 μl of 0.1 M NaOH was added to each well and the cells were lysed for 10 minutes.

[0232] 3.2.7 The cell lysis solution was collected in a liquid scintillation detection flask, and 2 ml of scintillation liquid was added to each flask for detection.

[0233] 3.2.8 The 14C content of each tube sample was detected using a liquid scintillation analyzer.

[0234] 3.2.9 Data Analysis:

[0235]

[0236] CPD is the radioactive signal value of the compound well;

[0237] HC is the average value of the radioactive signal in the 0% inhibition rate control well;

[0238] LC is the average value of the radioactive signal in the 100% inhibition control well.

[0239] 3.2.10 Using GraphPad Prism software, fit the dose-effect curve according to the following formula using the non-linear regression log(inhibitor) vs. response -- variable slope method and the IC of the compound 50 Value and IC 90 I obtained the value.

[0240] Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)×HillSlope))

[0241] 4. Experimental Results

[0242] The experimental results are as shown in Table 9.

[0243]

[0244] Experimental Conclusion: The compound of the present invention has excellent uric acid uptake inhibitory activity.

[0245] Experimental Example 3: Study of Metabolic Safety (HMS) in Hepatocytes

[0246] 1. Experimental Objective

[0247] We intend to test the metabolic stability of the test substance in human and rat liver cells.

[0248] 2. Experimental Materials

[0249] 2.1 Test Compound (10mM), Control Substance: 7-Ethoxycoumarin (30mM), 7-Hydroxycoumarin (Control Substance, 30mM)

[0250] 2.2 Cells

[0251] Cell information is as shown in Table 10.

[0252]

[0253] 2.3 Buffering System:

[0254] Thawing medium: Williams' medium E. containing 5% fetal bovine serum, 30% Percoll solution, and other auxiliary supplies.

[0255] Culture medium: Williams medium E containing 2 mM L-glutamine and 25 mM hydroxyethylpiperazine ethylsulfate (not containing phenol red).

[0256] Stop solution: Acetonitrile containing 200 ng / mL of toluenesulfonamide and labetalol as internal standards.

[0257] Dilution solution: Ultrapure water.

[0258] 3. Experimental Method

[0259] 1) An accurate amount of the positive control compound was dissolved in dimethyl sulfoxide (DMSO) and prepared as a 30 mM solution.

[0260] 2) 10 mM of the test compound and 30 mM of the positive control compound were diluted to 1 mM and 3 mM with DMSO in a 96-well plate.

[0261] 3) 1 mM of the test compound and 3 mM of the positive control compound were diluted with acetonitrile into 100 μM and 300 μM quantitative solutions.

[0262] 4) Thaw and separate the frozen cells, suspend them in culture medium, and then saturate 0.5 × 10⁶ with preheated culture medium 6 It was diluted to cells / mL.

[0263] 5) 198 μL of preheated cell suspension was added to a 96-well plate.

[0264] 6) 100 μL of stop solution (acetonitrile containing 200 ng / mL toluenesulfonamide and 200 ng / mL labetalol as internal standards) was transferred to a pre-labeled 96-well plate set.

[0265] 7) 2 μL of 100 μM test compound or 300 μM positive control quantitative solution was added to each well of a 96-well plate.

[0266] 8) For the T0 sample, after mixing for about 1 minute until uniformly turbid, 20 μL of each sample was immediately transferred to a well containing 100 μL of cold stop solution and mixed.

[0267] 9) All plates were cultured at 37°C in 5% CO2 in a 95% humidified incubator, and the reaction was started with constant shaking at about 600 rpm.

[0268] 10) After mixing the samples at 15, 30, 60, and 90 minutes, 20 μL of each sample at each time point was transferred to a well containing 100 μL of cold stop solution and then mixed.

[0269] 11) Medium control (MC) sample plates (labeled T0-MC and T90-MC) were prepared at T0 and T90 by adding the same components, excluding the cell suspension, to each well. A final concentration table was generated.

[0270] 12) At each corresponding time point, the plate was removed from the incubator and mixed with 100 μL of cold stop solution to stop the reaction.

[0271] 13) Immediately, the plates were vortexed and shaken in a plate shaker at 500 rpm for 10 minutes. Then, all sample plates were centrifuged at 3220 xg at 4°C for 20 minutes.

[0272] 14) After centrifugation, the supernatant from the 35 μL / well sample plate was transferred to another set of pre-labeled 96-well plates containing 70 μL of ultrapure water.

[0273] 15) The analysis plate was sealed and stored at 4°C until LC-MS-MS analysis was performed.

[0274] The residual rates of the test compound and the control compound were calculated using the following formula.

[0275]

[0276] Removal rate constants of test compounds and control compounds in hepatocytes by plotting the logarithm of the residual rate versus time k Calculate and removal rate k Half-life (T 1 / 2 ) and in vitro removal rate (CL int ) was calculated, and the formula is as follows.

[0277] T 1 / 2 =0.693 / k

[0278] CL int(hep) = k Cell volume per milliliter (million cells / mL)

[0279] CL int(liver) =CL int(hep) × Liver weight to body weight ratio × Number of liver cells per gram of liver

[0280] The parameters for each species in the formula are as shown in Table 11 below.

[0281]

[0282] 4. Experimental Results

[0283] The results are as shown in Table 12.

[0284]

[0285] Experimental Conclusion: The compound of the present invention is removed to a moderate degree in human liver cells and to a high degree in rat liver cells.

[0286] Experimental Example 4. Membrane Permeability MDR1 Test

[0287] 1. Experimental Objective:

[0288] MDR1-MDCK II cells are Madin-Darby canine kidney cells transfected with the human MDR1 gene capable of stable and high expression of P-gp. This study aims to test the bidirectional permeability of compounds penetrating the MDR1-MDCK II cell model and to evaluate whether they are transported by efflux.

[0289] 2. Cell Culture:

[0290] 2.5 × 10⁶ MDR1-MDCK II cells (obtained from Piet Borst, Netherlands Cancer Research Institute) 5 Cells were inoculated onto a polyethylene membrane (PET) of a 96-well insertion system at a density of cells / mL, and a fused cell monolayer was formed on the 4th to 7th day.

[0291] 3. Experimental Method

[0292] The test compounds were diluted with transport buffer (HBSS, 10 mM Hepes containing DMSO, pH 7.4) to a concentration of 2 μM (DMSO < 1%) and coated onto the top or bottom outer side of the cell monolayer. The test compounds were measured in duplicate in the A-to-B or B-to-A direction. Digoxin was also tested at 10 μM in the A-to-B or B-to-A direction, while Nadolol and Metoprolol were tested at 2 μM in the A-to-B direction. The plates were incubated without shaking for 2.5 hours in a CO2 incubator at 37 ± 1°C with a saturated humidity of 5% CO2. Additionally, the efflux ratio of each compound was measured, and quantification was performed for the test compounds and reference compounds. Analysis was performed by LC / MS / MS based on the peak area ratio of the analyte to the IS. After transport was measured, the integrity of the cell monolayer was determined using Lucifer Yellow exclusion measurements. After removing the buffer from the top chamber and the basal outer chamber, 75 μL of 100 μM fluorescein was added to the transport buffer, and 250 μL of transport buffer was added to the top chamber and the basal outer chamber. The plates were incubated for 30 minutes without shaking at 37°C, 5% CO2, and saturated humidity. After 30 minutes of incubation, 20 μL of fluorescein yellow sample was taken from the top and 60 μL of transport buffer was added. Then, 80 μL of fluorescein sample was taken from the basal outer chamber. The relative fluorescence units (RFU) of fluorescein yellow were measured at 425 / 528 nm (excitation / emission) using an Envision microplate reader.

[0293] 4. Data Calculation

[0294] Using the following formula, the apparent permeability coefficient (P app The outflow rate and recovery rate were calculated (e.g., cm / s).

[0295] Apparent permeability coefficient (Papp , cm / s) was calculated using the following formula.

[0296] P app =(dC r / d t )×V r / (A×C0)

[0297] dC r / d t ε is the cumulative concentration of the compound at the receiving end per unit time (μM / s); and V r ε is the volume of the solution at the aqueous end (the solution volumes at the top and base are 0.075 mL and 0.250 mL, respectively); and A is the relative surface area of ​​the cell monolayer (0.0804 cm²). 2 ) and; C0 is the initial concentration (nM) of the test substance at the dose level or the peak area ratio of the control substance.

[0298] The outflow rate was calculated using the following formula.

[0299] Outflow rate = P app (BA) / P app (AB)

[0300] The recovery rate was calculated using the following formula.

[0301] % recovery rate = 100 × [(V r ×C r )+(V d ×C d )] / (V d ×C0)

[0302] C0 is the initial concentration (nM) of the dosing group test substance or the peak area ratio of the control substance; V d is the volume of the administration end (the top side is 0.075 mL and the base side is 0.250 mL); C d and C r is the final concentration (nM) of the test substance at the administration and reception ends or the peak area ratio of the control substance.

[0303] The percentage of fluorescein in the basal lateral well was calculated using the following formula.

[0304]

[0305] Here, RFUApical and RFUBasolateral are the relative fluorescence unit values ​​of fluorescein in the top and basolateral wells, respectively; VApical and VBasolateral wells are the volumes of the top and basolateral wells (0.075 mL and 0.25 mL), respectively. %fluorescein must be less than 2.

[0306] 5. Experimental Results

[0307] The results are as shown in Table 13.

[0308]

[0309] Experimental Conclusion: The compound of the present invention is a highly permeable compound.

[0310] Experimental Example 5. Cytochrome P450 Isoenzyme Inhibitory Activity Test

[0311] 1. Experimental Objective

[0312] We intend to measure the inhibitory activity of the test compound against different subtypes of human cytochrome P450 isoenzymes.

[0313] 2. Experimental Method

[0314] Prepare the test compound, standard inhibitor (100× final concentration), and mixed substrate working solution; remove the frozen microsomes (purchased from Corning Inc.) from a refrigerator at -80°C and thaw them. Add 20 μL of the test compound and standard inhibitor solution to the corresponding wells, while simultaneously adding 20 μL of the corresponding solvent to the inhibitor-free control wells (NIC) and blank control wells (Blank); next, add 20 μL of the mixed substrate solution to the corresponding wells, excluding the blank wells (adding 20 μL of phosphate buffer (PB) to the blank wells); prepare the human liver microsome solution (mark the date after use and return it to the refrigerator immediately), and then add 158 μL of the human liver microsome solution to all wells; place the sample plate in a water bath at 37°C and pre-incubate, then prepare the coenzyme factor (NADPH) solution; After 10 minutes, 20 μL of NADPH solution was added to all wells and the sample plate was shaken uniformly, then placed in a 37°C water bath and incubated for 10 minutes; at the corresponding time, 400 μL of cold acetonitrile solution (internal standards were 200 ng / mL tolbutamide and labetalol) was added to stop the reaction; after mixing the sample plate uniformly, the protein was precipitated by centrifuging at 4000 rpm for 20 minutes; 200 μL of the supernatant was taken, added to 100 μL of water, shaken uniformly, and then sent to LC / MS / MS for detection.

[0315] 3. Experimental Results:

[0316] The results are as shown in Table 14.

[0317]

[0318] Experimental Conclusion: The compound of the present invention has very low inhibitory activity against CYP1A2, CYP2C19, CYP2D6, and CYP3A4-M, and moderate inhibitory activity against CYP2C9.

[0319] Experimental Example 6: Pharmacokinetics in SD Rats

[0320] 1. Experimental Objective:

[0321] We intend to test the pharmacokinetics of the compound in SD rats.

[0322] 2. Experimental Materials:

[0323] Sprague Dawley rat (male, 180 to 350 g, 6 to 10 weeks old, Beijing Vital River Laboratory Animal Technology Co., Ltd.)

[0324] 3. Experimental Method:

[0325] The compound was mixed with 5% DMSO / 10% Solutol / 85% water, stirred, and vortexed to prepare a clear solution of 0.6 mg / mL, which was used for administration to the injection group and prepared by filtration through a microporous membrane. The compound was mixed with 5% DMSO / 10% Solutol / 85% water, stirred, and vortexed to prepare a clear solution of 1 mg / mL, which was used for oral administration. Six male SD rats were divided into two groups. Animals in Group 1 received a single intravenous administration; the dose was 3 mg / kg, the solvent was 5% DMSO / 10% Solutol / 85% water, and the administration volume was 5 mL / kg. Animals in Group 2 were administered a single intragastric oral dose of 10 mg / kg of the test compound. The solvent for oral administration was 5% DMSO / 10% solutol / 85% water, and the administration volume was 10 mL / kg. Whole blood was collected at 0 (intragastric oral administration group only), 0.083 (intragastric injection only), 0.25, 0.5, 1, 2, 4, 8, and 24 hours after administration. 3200 g of whole blood was centrifuged at 4°C for 10 minutes to obtain plasma. The concentrations of the compound and uric acid (intragastric oral administration group only) in the plasma were measured using the LC / MS / MS method, and pharmacokinetic parameters such as peak concentration, peak time, clearance rate, half-life, area under the drug-time curve, and bioavailability were calculated using Phoenix WinNonlin software.

[0326] The experimental results are as shown in Table 15 below.

[0327]

[0328] Experimental Conclusion: The compound of the present invention possesses excellent pharmacokinetic properties and high oral bioavailability (%). Here, C0 is the initial concentration, and T 1 / 2 is the removal half-life, and Vd ss ε is the apparent volume of distribution in steady state, Cl is the total removal rate, and AUC 0-lastε is the area under the plasma concentration-time curve from time point 0 to the last quantifiable time point, and AUC 0-inf is the area under the plasma concentration-time curve from time point 0 to time point extrapolated to infinity, and C max is the peak concentration, and T max is peak time.

Claims

Claim 1 Crystalline form C of a compound represented by formula (I), having characteristic diffraction peaks at 2θ angles of 13.20±0.20°, 15.26±0.20°, 18.08±0.20°, 21.99±0.20°, 25.07±0.20°, 25.38±0.20°, 26.66±0.20°, and 30.70±0.20° in the powder X-ray diffraction spectrum. Claim 2 In claim 1, crystalline C having characteristic diffraction peaks at 2θ angles of 9.03±0.20°, 13.20±0.20°, 15.26±0.20°, 18.08±0.20°, 21.99±0.20°, 25.07±0.20°, 25.38±0.20°, 26.66±0.20°, 28.38±0.20°, 29.41±0.20°, 30.70±0.20°, and 38.53±0.20° in the powder X-ray diffraction spectrum. Claim 3 In paragraph 2, crystalline C having characteristic diffraction peaks at 2θ angles of 13.20°, 15.26°, 18.08°, 21.99°, 25.07°, 25.38°, 26.66°, and 30.70° in the powder X-ray diffraction spectrum. Claim 4 In paragraph 3, the powder X-ray diffraction spectrum is 5.66°, 9.03°, 11.13°, 13.20°, 13.70°, 15.26°, 17.25°, 18.08°, 18.92°, 20.88°, 21.99°, 23.41°, 24.09°, 25.07°, 25.38°, 25.99°, 26.66°, 27.17°, 28.38°, 29.41°, 29.98°, 30.70°, 31.02°, 31.72°, 33.67°, 35.40°, 36.35°, 36.74°, 37.26°, Crystalline form C having characteristic diffraction peaks at 2θ angles of 38.53° and 39.80°. Claim 5 In any one of claims 1 to 4, crystalline C having a thermogravimetric analysis curve with a weight loss of 1.21% at 200℃±3℃. Claim 6 In any one of claims 1 to 4, crystalline C having a differential scanning calorimetry curve having a starting value of one endothermic peak at 250.0℃±2℃. Claim 7 Crystalline form E of a compound represented by formula (I), having characteristic diffraction peaks at 2θ angles of 9.11±0.20°, 12.43±0.20°, 13.28±0.20°, 15.34±0.20°, 18.16±0.20°, 22.06±0.20°, 23.15±0.20°, and 25.14±0.20° in the powder X-ray diffraction spectrum. Claim 8 In claim 7, crystalline form E having characteristic diffraction peaks at 2θ angles of 9.11±0.20°, 11.21±0.20°, 12.43±0.20°, 13.28±0.20°, 15.34±0.20°, 18.16±0.20°, 22.06±0.20°, 23.15±0.20°, 25.14±0.20°, 25.97±0.20°, 26.75±0.20°, and 27.25±0.20° in the powder X-ray diffraction spectrum. Claim 9 In claim 8, crystalline form E having characteristic diffraction peaks at 2θ angles of 9.11°, 12.43°, 13.28°, 15.34°, 18.16°, 22.06°, 23.15°, and 25.14° in the powder X-ray diffraction spectrum. Claim 10 In claim 9, characteristic diffraction at 2θ angles of the powder X-ray diffraction spectrum at 9.11°, 10.94°, 11.21°, 12.43°, 13.28°, 15.34°, 17.39°, 18.16°, 18.94°, 20.18°, 20.95°, 22.06°, 23.15°, 23.35°, 24.19°, 25.14°, 25.97°, 26.75°, 27.25°, 28.45°, 29.49°, 30.16°, 30.82°, 33.74°, 35.45°, 36.39°, 37.34°, and 38.57° Crystalline E with a peak. Claim 11 In any one of claims 7 to 10, the thermogravimetric analysis curve is a crystalline E with a weight loss of 0.79% at 200℃±3℃. Claim 12 In any one of claims 7 to 10, crystalline E having a differential scanning calorimetry curve having a starting value of one endothermic peak at 250.4℃±2℃. Claim 13 A pharmaceutical composition for treating gout and hyperuricemia, comprising crystalline form C according to any one of claims 1 to 4 or crystalline form E according to any one of claims 7 to 10. Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete

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