Crystal Forms of Thiophene Derivatives and Their Preparation Methods

Abstract

Provided are a crystalline form of the compound represented by formula (I) and a method for preparing the same. #imgabs0#

Description

[0001] The present invention claims the following priority:

[0002] CN202110472705.3, application date: April 29, 2021.

[0003] CN202111112439.X, application date: September 18, 2021. Technical Field

[0004] The present invention relates to a crystal form of a thiophene derivative and a preparation method thereof, and particularly to a crystal form of a compound represented by formula (I) and a preparation method thereof. Background Art

[0005] Gouty arthritis is a common and complex type of arthritis. When uric acid levels in the blood exceed 7 mg / dL, uric acid deposits in the form of its monosodium salt in the joints, cartilage, and kidneys, leading to an overactive immune system (hyperactivity) and painful inflammation. Common sites of flare-ups include the big toe, ankle, and knee joints. Hyperuricemia is the pathological basis of gouty arthritis. Hyperuricemia refers to a disorder in the metabolism of purines, resulting in increased uric acid synthesis or decreased excretion, leading to abnormally high blood uric acid levels. The international diagnosis of HUA is defined as a fasting blood uric acid level >400 μmol / L (6.8 mg / dL) in men and >360 μmol / L (6 mg / dL) in women on two different days, taken on a normal purine diet. Uric acid can be categorized as malarial excretion, hyperuricemia, and mixed. Clinical studies have shown that 90% of cases of primary hyperuricemia fall under the malarial excretion type.

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

[0007] Purines in the human body come from two sources: endogenous purines, derived from internal synthesis or nucleic acid degradation (approximately 600 mg / day), and exogenous purines, derived from dietary purine intake (approximately 100 mg / day). Under normal circumstances, the body's uric acid pool is 1200 mg, and approximately 700 mg of uric acid is produced daily. Two-thirds of this is excreted via the kidneys, one-third via the intestines, and a very small amount is excreted via the sweat glands. Therefore, currently, commonly used uric acid-lowering drugs include xanthine oxidase inhibitors (such as allopurinol and febuxantane) that inhibit uric acid production and Urat1 inhibitors (such as benzbromarone and lecithin) that inhibit uric acid excretion.

[0008] Xanthine oxidase is a low-specificity enzyme that can catalyze the conversion of hypoxanthine to xanthine, which in turn produces uric acid, and can also directly catalyze the conversion of xanthine to uric acid. Xanthine oxidase inhibitors are first-line treatments for hyperuricemia, with allopurinol and febuxostat currently the main marketed drugs. However, these drugs do not meet the clinical needs of all patients and have significant side effects. Allopurinol is the only uric acid-lowering drug available worldwide, but it can cause serious skin adverse events. While febuxostat has a superior uric acid-lowering effect than allopurinol, even at a high dose of 80 mg / day, 40% to 52% of patients fail to achieve the desired uric acid-lowering target, and acute gout attacks are more likely to occur.

[0009] There is still an unmet clinical need for safe and effective uric acid-lowering drugs in the market. Summary of the Invention

[0010] The present invention provides a crystalline form A of a compound of formula (I), characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 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°, 27.16±0.20°,

[0011]

[0012] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 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°, 30.28±0.20°.

[0013] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 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 embodiments of the present invention, the XRPD pattern of the above-mentioned Form A is substantially as follows Figure 1 shown.

[0015] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned Form A is shown in Table 1:

[0016] Table 1. XRPD pattern analysis data of Form A

[0017]

[0018]

[0019] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned crystal form A shows a weight loss of 1.96% at 200°C±3°C.

[0020] In some embodiments of the present invention, the TGA spectrum of the above-mentioned A crystal form is as follows Figure 2 shown.

[0021] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned crystal form A has an onset value of an endothermic peak at 244.3.0°C±2°C.

[0022] In some embodiments of the present invention, the DSC spectrum of the above-mentioned crystal form A is as follows: Figure 3 shown.

[0023] The present invention provides a crystal form B of the compound of formula (I), characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 23.93±0.20°, 24.73±0.20°, 26.58±0.20°,

[0024]

[0025] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned B crystal form has characteristic diffraction peaks at the following 2θ angles: 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°, 26.58±0.20°.

[0026] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic diffraction peaks at the following 2θ angles: 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°.

[0027] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B has characteristic diffraction peaks at the following 2θ angles: 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°, 2 3.46°, 23.93°, 24.73°, 25.87°, 26.58°, 27.78°, 28.98°, 29.34°, 29.66°, 30.07°, 31.26°, 31.38°, 31.86°, 32.73°, 33.71°, 34.02°, 34.68°, 35.41°, 36.64°, 37.30°, 37.86°, 38.30°.

[0028] In some embodiments of the present invention, the XRPD pattern of the above-mentioned Form B is substantially as follows Figure 4 shown.

[0029] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned Form B are shown in Table 2:

[0030] Table 2. XRPD pattern analysis data of Form B

[0031]

[0032]

[0033] The present invention provides a crystal of a compound of formula (I), characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 13.28±0.30°, 15.34±0.30°, 25.14±0.30°,

[0034]

[0035] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal has characteristic diffraction peaks at the following 2θ angles: 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°, 27.25±0.30°.

[0036] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal has characteristic diffraction peaks at the following 2θ angles: 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°, 30.82±0.30°.

[0037] The present invention provides a crystal of a compound of formula (I), characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 13.20±0.20°, 15.26±0.20°, 25.07±0.20°,

[0038]

[0039] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal has characteristic diffraction peaks at the following 2θ angles: 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°, 30.70±0.20°.

[0040] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal has characteristic diffraction peaks at the following 2θ angles: 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°, 38.53±0.20°.

[0041] The present invention provides a crystal form C of a compound of formula (I), characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 13.20±0.20°, 18.08±0.20°, 25.07±0.20°,

[0042]

[0043] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2θ angles: 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°, 30.70±0.20°.

[0044] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2θ angles: 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°, 30.70±0.20°.

[0045] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2θ angles: 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°, 38.53±0.20°.

[0046] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2θ angles: 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°, 38.53±0.20°.

[0047] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2θ angles: 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°.

[0048] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2θ angles: 13.20±0.20°, 25.07±0.20°, and can also be at 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 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° have characteristic diffraction peaks.

[0049] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2θ angles: 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°.

[0050] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form C has characteristic diffraction peaks at the following 2θ angles: 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°, 38.53°, and 39.80°.

[0051] In some embodiments of the present invention, the XRPD pattern of the above-mentioned Form C is substantially as follows Figure 5 shown.

[0052] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned Form C are shown in Table 3:

[0053] Table 3. XRPD pattern analysis data of Form C

[0054]

[0055]

[0056] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned Form C shows a weight loss of 1.21% at 200°C±3°C.

[0057] In some embodiments of the present invention, the TGA spectrum of the above-mentioned C crystal form is as follows Figure 6 shown.

[0058] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned crystal form C has an onset value of an endothermic peak at 250.0°C±2°C.

[0059] In some embodiments of the present invention, the DSC spectrum of the above-mentioned crystal form C is as follows Figure 7 shown.

[0060] The present invention provides a crystal form D of a compound of formula (I), characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 6.71±0.20°, 11.87±0.20°, 25.21±0.20°,

[0061]

[0062] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned D crystal form has characteristic diffraction peaks at the following 2θ angles: 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°, 27.05±0.20°.

[0063] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned D crystal form has characteristic diffraction peaks at the following 2θ angles: 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°, 27.41±0.20°.

[0064] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned D crystal form has characteristic diffraction peaks at the following 2θ angles: 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°, 31.17°, 32.81°, 33.77°, 34.17°, 35.52°, 36.57°, 38.20°, 38.68°.

[0065] In some embodiments of the present invention, the XRPD pattern of the above-mentioned D crystal form is substantially as follows Figure 8 shown.

[0066] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned D crystal form are shown in Table 4:

[0067] Table 4. XRPD pattern analysis data of Form D

[0068]

[0069] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned D crystal form shows a weight loss of 1.14% at 200°C±3°C.

[0070] In some embodiments of the present invention, the TGA spectrum of the above-mentioned D crystal form is as follows Figure 9 shown.

[0071] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned D crystal form has an onset value of an endothermic peak at 251.4°C±2°C.

[0072] In some embodiments of the present invention, the DSC spectrum of the above-mentioned D crystal form is as follows: Figure 10 shown.

[0073] The present invention provides a crystalline form E of a compound of formula (I), characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 13.28±0.20°, 15.34±0.20°, 25.14±0.20°,

[0074]

[0075] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned E crystal form has characteristic diffraction peaks at the following 2θ angles: 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°, 27.25±0.20°.

[0076] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned E crystal form has characteristic diffraction peaks at the following 2θ angles: 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°, 25.14±0.20°.

[0077] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned E crystal form has characteristic diffraction peaks at the following 2θ angles: 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°, 30.82±0.20°.

[0078] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned E crystal form has characteristic diffraction peaks at the following 2θ angles: 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°, 27.25±0.20°.

[0079] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned E crystal form has characteristic diffraction peaks at the following 2θ angles: 13.28±0.20°, 25.14±0.20°, and can also be at 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 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° have characteristic diffraction peaks.

[0080] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned E crystal form has characteristic diffraction peaks at the following 2θ angles: 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°, 23.3 5±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°, 30.82±0.20°, 33.74±0.20°, 36.39±0.20°, 37.34±0.20°, 38.57±0.20°.

[0081] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned E crystal form has characteristic diffraction peaks at the following 2θ angles: 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°.

[0082] In some embodiments of the present invention, the XRPD pattern of the above-mentioned E crystal form is substantially as follows Figure 11 shown.

[0083] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned E crystal form are shown in Table 5:

[0084] Table 5. XRPD pattern analysis data of Form E

[0085]

[0086] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned E crystal form shows a weight loss of 0.79% at 200°C±3°C.

[0087] In some embodiments of the present invention, the TGA spectrum of the above-mentioned E crystal form is as follows Figure 12 shown.

[0088] In some embodiments of the present invention, the differential scanning calorimetry curve of the above-mentioned E crystal form has an onset value of an endothermic peak at 250.4°C±2°C.

[0089] In some embodiments of the present invention, the DSC spectrum of the above-mentioned E crystal form is as follows Figure 13 shown.

[0090] The present invention also provides the use of crystal form A, crystal form B, crystal form C, crystal form D and crystal form E of the compound of formula (I) in the preparation of drugs for treating gout and hyperuricemia.

[0091] Technical Effects

[0092] The compound of formula (I) has stable crystal properties, is non-hygroscopic, and has good prospects for drug development.

[0093] Definition and Description

[0094] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be construed as ambiguous or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding commercial product or its active ingredient.

[0095] The intermediate compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0096] The chemical reactions of the present invention are carried out in suitable solvents that are compatible with the chemical transformations of the present invention and the reagents and materials required. To obtain the compounds of the present invention, it may sometimes be necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0097] The present invention will be described in detail below through examples, which are not intended to limit the present invention in any way.

[0098] All solvents used in the present invention were commercially available and used without further purification.

[0099] Compounds are named according to the conventional nomenclature in this field or Software naming, commercially available compounds use supplier catalog names.

[0100] The structures of the compounds of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compounds, the absolute configuration can be confirmed by conventional techniques in the art. For example, single crystal X-ray diffraction (SXRD) is used to collect diffraction intensity data on the cultured single crystal using a Bruker D8 venture diffractometer, using CuKα radiation as the light source and scanning mode: After scanning and collecting relevant data, the crystal structure is further analyzed using the direct method (Shelxs97) to confirm the absolute configuration.

[0101] The solvents used in the present invention are commercially available. The present invention uses the following abbreviations: -OMOM stands for methyl oxymethyl ether; HPE stands for 100% inhibitory activity; ZPE stands for 0% inhibitory activity; DPBS stands for Dulbecco's phosphate buffered saline.

[0102] The powder X-ray diffractometer (XRPD) method of the present invention

[0103] Instrument model: Bruker D2 PHASER X-ray diffractometer

[0104] The detailed XRPD parameters are as follows:

[0105] Radiation source: Cu, k-Alphal

[0106] Light tube voltage: 30kV

[0107] Light tube current: 10mA

[0108] Divergence slit: 0.6mm

[0109] Axial Soller slit of main optical path: 2.5°

[0110] Secondary optical path axial Soller slit: 2.5°

[0111] Detector slit: 5.827°

[0112] Anti-scatter slit: 0mm

[0113] Scan axis: θs-θd

[0114] Step size: 0.02 degrees

[0115] Each step dwell time: 0.2 seconds

[0116] Scanning angle range: 3-40 degrees

[0117] Differential Scanning Calorimeter (DSC) method of the present invention

[0118] Instrument model: TA DSC Q2000 Differential Scanning Calorimeter Test method: Take a sample (~1 mg) and place it in a DSC aluminum pan for testing. Under 50 mL / min N2 conditions, the sample is heated from 30°C to 250°C at a heating rate of 10°C / min.

[0119] Thermogravimetric analysis (TGA) method of the present invention

[0120] Instrument model: DISCOVERY TGA 5500 Thermogravimetric Analyzer Test method: Take a sample (2-5 mg) and place it in the TGA platinum pot for testing. Under 25 mL / min N2 conditions, the sample is heated from room temperature to 300°C at a heating rate of 10°C / min.

[0121] Dynamic Vapor Sorption (DVS) method of the present invention

[0122] Instrument model: Intrinsic dynamic vapor adsorption instrument

[0123] Test conditions: Take a sample (10-30 mg) and place it in the DVS sample tray for testing.

[0124] The detailed DVS parameters are as follows:

[0125] Temperature: 25℃

[0126] Balance: dm / dt = 0.002% / min (minimum: 10 min, maximum: 180 min)

[0127] RH (%) test gradient: 10 (90-0-90%), 5 (90-95%)

[0128] RH (%) test range: 0%-95%-0%

[0129] The classification of moisture absorption evaluation is shown in Table 6 below:

[0130] Table 6. Moisture absorption evaluation classification

[0131] Hygroscopicity classification ΔW% deliquescence Absorb enough water to form a liquid Highly hygroscopic ΔW%≥15% Hygroscopic 15%>ΔW%≥2% Slightly hygroscopic 2%>ΔW%≥0.2% No or almost no hygroscopicity ΔW%<0.2%

[0132] Note: ΔW% indicates the weight gain of the test sample at 25±1℃ and 80±2%RH. BRIEF DESCRIPTION OF THE DRAWINGS

[0133] Figure 1 The XRPD spectrum of Form A of the compound of formula (I) using Cu-Kα radiation.

[0134] Figure 2 This is the TGA spectrum of Form A of the compound of formula (I).

[0135] Figure 3 This is the DSC spectrum of Form A of the compound of formula (I).

[0136] Figure 4 The XRPD spectrum of Form B of the compound of formula (I) using Cu-Kα radiation.

[0137] Figure 5 The XRPD spectrum of Form C of the compound of formula (I) using Cu-Kα radiation.

[0138] Figure 6 This is the TGA spectrum of Form C of the compound of formula (I).

[0139] Figure 7 This is the DSC spectrum of Form C of the compound of formula (I).

[0140] Figure 8The XRPD spectrum of the D-form of the compound of formula (I) using Cu-Kα radiation.

[0141] Figure 9 This is the TGA spectrum of the D crystal form of the compound of formula (I).

[0142] Figure 10 This is the DSC spectrum of the crystal form D of the compound of formula (I).

[0143] Figure 11 The XRPD spectrum of the E crystal form of the compound of formula (I) using Cu-Kα radiation.

[0144] Figure 12 This is the TGA spectrum of the E crystal form of the compound of formula (I).

[0145] Figure 13 This is the DSC spectrum of Form E of the compound of formula (I).

[0146] Figure 14 This is the DVS spectrum of Form C of the compound of formula (I). DETAILED DESCRIPTION

[0147] The present invention is described in detail below by way of examples, but is not intended to limit the present invention in any way. While the present invention has been described in detail herein, and specific embodiments thereof have been disclosed, it will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0148] Example 1: Preparation of Crystalline Form A of Compound (I)

[0149]

[0150] Step 1: Synthesis of compound I-2

[0151] Potassium tert-butoxide (234.26 g, 2.09 mol) was added to dimethyl sulfoxide (1200 mL) and stirred at room temperature until the mixture became clear. A solution of compound I-1 (200 g, 1.49 mol) in dimethyl sulfoxide (500 mL) was then added dropwise at 15-20°C. Stirring was continued for 40 minutes after the addition was complete. Carbon disulfide (113.54 g, 1.49 mol, 90.11 mL) was then added dropwise, maintaining the reaction temperature above 20°C. Stirring was continued for 20 minutes after the addition was complete. Potassium tert-butoxide (100.40 g, 894.70 mmol) was then slowly added, maintaining the reaction temperature at 15-20°C and stirring for 30 minutes. Ethyl bromoacetate (498.05 g, 2.98 mol, 329.83 mL) was then added dropwise, maintaining the reaction temperature at 15-20°C and stirring was continued for 1.5 hours. Potassium carbonate (206.09 g, 1.49 mol) was added, and the reaction mixture was heated to 60°C and stirred for 1.5 hours. 1 L of water was added to the reaction mixture, and the pH was adjusted to 3-4 with 6 M aqueous hydrochloric acid. The mixture was extracted with ethyl acetate (1.5 L x 2). The combined organic phases were washed with saturated brine (200 mL x 3). The organic solvent was removed under reduced pressure, and the crude product was added with isopropanol (200 mL). The mixture was stirred evenly, allowed to stand for 15 hours, filtered, and dried under vacuum at 45°C for 1 hour to obtain compound I-2. 1 HNMR(400MHz, CDCl3)δ:4.32(q,J=7.2Hz,2H),4.19(q,J=7.2Hz,2H),3.56(s,2H),3.25(t,J=6.8Hz,2H),3 .19(t,J=14.4Hz,2H),2.26-2.17(m,2H),1.37(t,J=7.2Hz,3H),1.27(t,J=7.2Hz,3H).MSm / z=364.8[M+H] + .

[0152] Step 2: Synthesis of compound I-3

[0153] Compound I-2 (282 g, 773.82 mmol) was dissolved in ethanol (3.5 L) and Raney nickel (99.45 g, 1.16 mol) was added. The atmosphere was replaced with nitrogen three times and stirred at 85°C under a hydrogen pressure of 2.5 MPa for 48 hours. The mixture was cooled and filtered through celite under nitrogen. The solvent was removed from the filtrate under reduced pressure to obtain compound I-3, which was used directly in the next reaction without further purification. 1H NMR (400MHz, CDCl3) δ: 7.09 (s, 1H), 4.26 (q, J = 7.2Hz, 2H), 3.20 (t, J = 6.8Hz, 2H), 3 .12(t,J=14.4Hz,2H),2.20-2.10(m,2H),1.30(t,J=6.8Hz,3H).MSm / z=247.0[M+H] + .

[0154] Step 3: Synthesis of compound I-4

[0155] Compound I-3 (40.00 g, 162.42 mmol) was dissolved in methanol (200 mL). A 200 mL aqueous solution of sodium hydroxide (12.99 g, 324.84 mmol) was added. The reaction mixture was heated to 50°C and stirred 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 2-3 with 6M aqueous hydrochloric acid. A large amount of white solid precipitated. The filter cake was filtered, washed with 100 mL of water and 50 mL of petroleum ether, and dried under vacuum at 50°C for 3 hours to obtain compound I-4. 1 H NMR (400MHz, CD3OD) δ: 7.38 (s, 1H), 3.33-3.17 (m, 4H), 2.28-2.21 (m, 2H).

[0156] Step 4: Synthesis of compound I-5

[0157] Compound I-4 (35.0 g, 160.39 mmol) was dissolved in tetrahydrofuran (200 mL), and carbonyldiimidazole (33.81 g, 208.51 mmol) was added. The reaction solution was stirred under nitrogen for 2 hours. Aqueous ammonia (31.23 g, 240.58 mmol, 34.32 mL) was added, and the reaction solution was stirred for 15 hours. The organic solvent was removed under reduced pressure, and 300 mL of water was added to the resulting residue. The mixture was stirred for 10 minutes and filtered. The filter cake was washed with 100 mL of water and dried under vacuum at 55°C for 2.5 hours to obtain compound I-5. 1 H NMR (400MHz, CDCl3) δ: 7.09 (s, 1H), 5.72 (brs, 2H), 3.30-3.18 (m, 4H), 2.29-2.19 (m, 2H).

[0158] Step 5: Synthesis of Compound I-6

[0159] Compound I-5 (31 g, 142.70 mmol) was dissolved in N,N-dimethylformamide (200 mL), and N-bromosuccinimide (27.94 g, 156.97 mmol) was slowly added in portions. The reaction solution was stirred at 20°C for 2 hours. The reaction solution was slowly poured into 600 mL of stirred water, resulting in the precipitation of a large amount of solid. The mixture was stirred for 10 minutes and filtered. The filter cake was washed with 200 mL of water and 100 mL of petroleum ether, and dried under vacuum at 50°C for 2 hours to obtain compound I-6. 1 H NMR (400MHz, CDCl3) δ: 5.62 (brs, 2H), 3.25 (t, J = 7.2Hz, 2H), 3.04 (t, J = 14.0Hz, 2H), 2.26-2.18 (m, 2H).

[0160] Step 6: Synthesis of Compound I-7

[0161] Compound I-6 (48 g, 162.09 mmol) and triethylamine (32.80 g, 324.18 mmol, 45.12 mL) were added to ethyl acetate (250 mL). The mixture was cooled to 0°C under nitrogen and trifluoroacetic anhydride (44.26 g, 210.72 mmol, 29.31 mL) was added dropwise. The reaction solution was stirred at this temperature for 1 hour, then heated to 20°C and stirred for 0.5 hours. The reaction solution was diluted with 250 mL of ethyl acetate and washed sequentially with water (100 mL × 2), saturated sodium bicarbonate solution (150 mL), and saturated brine (100 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed under reduced pressure to obtain compound I-7, which was used directly in the next reaction without further purification. 1 H NMR (400MHz, CDCl3) δ: 3.13-2.97 (m, 4H), 2.30-2.20 (m, 2H).

[0162] Step 7: Synthesis of Compound I-8

[0163] Compound I-7 (6.0 g, 21.57 mmol), compound I-7-1 (7.60 g, 23.73 mmol) and anhydrous potassium phosphate (9.16 g, 43.15 mmol) were added to ethylene glycol dimethyl ether (60 mL) and water (12 mL). Pd(dppf)Cl2 (394.64 mg, 539.34 μmol) was added under nitrogen protection. The reaction solution was heated to 85°C under nitrogen protection and stirred for 15 hours. After cooling, 20 mL of water and 100 mL of ethyl acetate were added to the reaction solution, stirred for 10 minutes, filtered, and the organic phase was separated from the filtrate. The aqueous phase was extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the organic solvent was removed under reduced pressure. The crude product was added with ethyl acetate (80 mL), followed by activated carbon (4 g) and palladium-removing silica gel (4 g). The temperature was raised to 80°C and stirred for 1 hour. The mixture was cooled, filtered through celite, and the organic solvent was removed under reduced pressure. The crude product was further slurried with tert-butyl methyl ether (25 mL) at 25°C for 0.5 hour, filtered, and the filter cake was dried under vacuum at 45°C for 1 hour to obtain compound I-8. 1 HNMR (400MHz, CDCl3) δ: 11.18 (s, 1H), 7.86 (d, J = 8.0Hz, 1H), 7.01 (d, J = 1.6Hz, 1H), 6.92-6 .90(m,1H),3.24(t,J=14.4Hz,2H),3.12(t,J=6.8Hz,2H),2.36-2.26(m,2H),1.64(s,9H).

[0164] Step 8: Synthesis of Crystalline Form A of Compound (I)

[0165] Compound I-8 (32 g, 81.75 mmol) was added to trifluoroacetic acid (250 mL), and the reaction solution was stirred at 20°C for 1 hour. The trifluoroacetic acid was removed under reduced pressure, and water (300 mL) was added to the resulting residue. The mixture was slurried at room temperature for 20 minutes until completely dispersed, filtered, and the filter cake was washed with water (200 mL) and dried in vacuo at 45°C for 1 hour to obtain Form A of the compound of formula (I). 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 pattern of Form A is shown in Figure 2. Figure 1 As shown, the TGA diagram is as follows Figure 2 As shown in the DSC diagram Figure 3 shown.

[0166] Example 2: Preparation of compound of formula (I)

[0167]

[0168] Step 1: Synthesis of compound I-4

[0169] Compound I-3 (2.5 g, 10.15 mmol) was dissolved in methanol (10 mL), and water (10 mL) and sodium hydroxide (1.62 g, 40.61 mmol) were added. The resulting reaction solution was stirred in a 40°C oil bath for 2 hours. The reaction solution was concentrated to half under reduced pressure, and water (5 mL) was added to the residue. The pH was adjusted to 2-3 with 6M hydrochloric acid while stirring, resulting in the precipitation of a large amount of white solid. The solid was collected by filtration and dried under vacuum at 50°C for 3 hours to obtain compound I-4. 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).

[0170] Step 2: Synthesis of compound I-5

[0171] Compound I-4 (500 mg, 2.29 mmol) was dissolved in dichloromethane (5 mL), followed by the addition of carbonyldiimidazole (445.83 mg, 2.75 mmol). The resulting reaction mixture was stirred under nitrogen for 1 hour. The mixture was then poured into vigorously stirred ammonia (2.87 g, 22.91 mmol, 3.15 mL, 28% content) in tetrahydrofuran (5 mL) and stirred for 30 minutes. The reaction mixture was concentrated under reduced pressure at 25°C, and the residue was extracted with ethyl acetate (20 mL x 3). The organic phases were combined and dried to give the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-45%) to afford compound I-5. 1 H NMR (400MHz, CDCl3) δ: 7.10 (s, 1H), 5.58 (br s, 2H), 3.28 (t, J = 6.9Hz, 2H), 3.21 (t, J = 14.4Hz, 2H), 2.24 (tt, J = 6.9, 13.4Hz, 2H).

[0172] Step 3: Synthesis of Compound 2-4

[0173] Compound I-5 (320 mg, 1.47 mmol) was dissolved in DMF (3 mL). The resulting solution was cooled to 0°C, and then cyanuric chloride (298.81 mg, 1.62 mmol) was added. The reaction mixture was stirred under nitrogen for 2 hours (during which a large amount of white solid precipitated). The reaction mixture was diluted with ethyl acetate (50 mL), then washed with water (10 mL × 3) and saturated brine (10 mL). The organic phase was dried over anhydrous sodium sulfate and filtered to remove the desiccant. The solvent was removed from the filtrate under reduced pressure to obtain crude compound 2-4, which was used directly in the next 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).

[0174] Step 4: Synthesis of Compound 2-5

[0175] Compound 2-4 (290 mg, 1.46 mmol) was dissolved in acetic acid (2 mL), and then liquid bromine (348.94 mg, 2.18 mmol, 112.56 μL) was added. The resulting reaction solution was stirred at room temperature at 25°C for 15 hours. The reaction solution was spin-dried, and ethyl acetate (30 mL) was added to the residue. The pH was then adjusted to 7-8 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. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-5%) to obtain compound I-7. 1 H NMR: (400MHz, CDCL3) δ: 3.10-2.99m, 4H), 2.32-2.19(m, 2H).

[0176] Step 5: Synthesis of Compounds 2-6

[0177] Compound I-7 (140 mg, 503.39 μmol), boronate ester 2-5A (178.39 mg, 553.73 μmol), and potassium carbonate (139.14 mg, 1.01 mmol) were added to dioxane (3 mL) and water (0.6 mL). 1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (Pd(dppf)Cl2) (36.83 mg, 50.34 μmol) was then added. The mixture was stirred in an oil bath at 105°C under nitrogen for 15 hours. The reaction mixture was dried to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 0-25%) to afford compound 2-6. 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=14.4Hz,2H),3.13(t,J=6.8Hz,2H),2.39-2.24(m,2H).

[0178] Step 6: Synthesis of Compounds 2-7

[0179] Compound 2-6 (105 mg, 266.90 μmol) was dissolved in tetrahydrofuran (2 mL), and then a 2M aqueous lithium hydroxide solution (533.80 μL) was added. The resulting reaction solution was stirred at 25°C for 15 hours. The tetrahydrofuran was removed by vortexing the reaction solution at 40°C. The pH of the residue was adjusted to 2-3 with 2M hydrochloric acid, resulting in the precipitation of a large amount of solid. Ethyl acetate (50 mL) was added and stirred, and the ethyl acetate was separated and dried to obtain compound 2-7. The crude product was used directly in the next reaction.

[0180] Step 7: Synthesis of compound of formula (I)

[0181] Compound 2-7 (105 mg, 276.77 μmol) was dissolved in methanol (1 mL), and hydrochloric acid (60.55 mg, 1.66 mmol, 59.36 μL) was added. The reaction mixture became turbid and stirred at 25°C for 3 hours. The reaction mixture was dried at 40°C, and the resulting residue was purified by preparative HPLC (column: Venusil ASB Phenyl 150*30 mm*5 μm; mobile phase: [water (0.05% HCl)-ACN]; ACN%: 60%-90%, 9 min) to obtain the compound of formula (I). 1 H NMR (400MHz, CD3OD) δ: 8.00 (d, J = 8.0Hz, 1H), 7.13-7.04 (m, 2H), 3.35-3.32 (m, 2H), 3.12 (t, J = 7.2Hz, 2H), 2.45-2.30 (m, 2H); MS (ESI) m / z: 334.02 [MH] - .

[0182] Example 3: Preparation of Crystalline Form B of Compound (I)

[0183] Crystalline Form A of the compound of formula (I) (20 mg, 0.06 mmol) was added to dichloromethane (1 mL), and then stirred at 25°C for 120 hours. Filtered, the filter cake was dried under vacuum at 50°C for 2-5 hours to obtain Crystalline Form B of the compound of formula (I). The XRPD pattern of Crystalline Form B is shown below. Figure 4 shown.

[0184] Example 4: Preparation of Crystalline Form C of Compound (I)

[0185] Form A of the compound of formula (I) (1.0 g, 2.98 mmol) was added to a mixed solvent of ethyl acetate (5 mL) and n-heptane (5 mL), and then stirred at 25°C for 72 hours. The mixture was filtered and the filter cake was dried under vacuum at 45°C for 2 hours to obtain Form C of the compound of formula (I). The XRPD pattern of Form C is shown in FIG. Figure 5 As shown, the TGA diagram is as follows Figure 6 As shown in the DSC diagram Figure 7 shown.

[0186] Example 5: Preparation of Crystalline Form D of Compound of Formula (I)

[0187] About 20 mg of Form A of the compound of formula (I) was weighed and added to a mixed solvent of tetrahydrofuran (0.4 mL) and water (0.4 mL). The mixture was stirred at 50°C until the solid dissolved, then cooled to 13°C and stirred for 72 hours. Filtered, the filter cake was dried under vacuum at 45°C for 2 hours to obtain Form D of the compound of formula (I). The XRPD pattern of Form D is shown below. Figure 8 As shown, the TGA diagram is as follows Figure 9 As shown in the DSC diagram Figure 10 shown.

[0188] Example 6: Preparation of Crystalline Form E of Compound of Formula (I)

[0189] Crystalline Form A of the compound of formula (I) (1.0 g, 2.98 mmol) 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 filter cake was dried under vacuum at 45°C for 2 hours to obtain Crystalline Form E of the compound of formula (I). The XRPD pattern of Crystalline Form E is shown below. Figure 11 As shown, the TGA diagram is as follows Figure 12 As shown in the DSC diagram Figure 13 shown.

[0190] Example 7: Hygroscopicity study of the crystalline form C of compound of formula (I)

[0191] Experimental Materials:

[0192] DVS Intrinsic Dynamic Vapor Sorption Instrument

[0193] Experimental methods:

[0194] 10-30 mg of the crystalline form C of the compound of formula (I) was placed in a DVS sample tray for testing.

[0195] Experimental results:

[0196] The DVS spectrum of the crystal form of compound C of formula (I) is as follows Figure 14 As shown, ΔW = 0.196%.

[0197] Experimental conclusion:

[0198] The weight gain of the crystal form C of the compound of formula (I) at 25° C. and 80% RH was 0.196%, indicating that it was non-hygroscopic.

[0199] Example 8: Solid Stability Test of Crystal Form C of Compound of Formula (I)

[0200] In accordance with the "Guidelines for Stability Testing of APIs and Preparations" (Chinese Pharmacopoeia 2015 Edition, Part IV, General Chapter 9001), the stability of the crystalline form C of compound of formula (I) under high temperature (60°C, open), high humidity (room temperature / relative humidity 92.5%, open) and strong light (5000 lx, sealed) conditions was investigated.

[0201] Weigh 12 portions of the crystalline form of compound C of formula (I) in parallel, approximately 1.5 g each, and place them in a flat weighing bottle (70*35 mm) or a disposable petri dish, spreading them into a thin layer. Place them under high temperature (60°C), high humidity (25°C / 92.5% humidity), high temperature and high humidity (40°C / 75% humidity), and light stability conditions. For samples placed under high temperature and high humidity conditions, seal the bottle mouth with aluminum foil and poke small holes in the foil to ensure that the sample is fully in contact with the ambient air; for samples placed under strong light conditions, seal them with a quartz glass lid. For samples placed under high temperature (60°C) and high humidity (92.5% humidity, room temperature), sample testing (appearance, related substances, and content) was performed on the 5th and 10th day. For samples placed under high temperature and high humidity (40°C / 75% humidity), sample testing (appearance, related substances, and content) was performed on the 1st, 2nd, and 3rd month. Samples placed under light irradiation were tested when the total illumination reached 1.2×10 6 Lux·hr sampling was performed and the test results were compared with the initial test results on day 0. The test results are shown in Table 7 below:

[0202] Table 7 Solid stability test results of compound of formula (I)

[0203]

[0204] Conclusion: The crystal form C of compound of formula (I) has good stability under the influencing factors of high temperature, high humidity, strong light conditions and accelerated conditions.

[0205] Biological test data:

[0206] Experimental Example 1: Xanthine oxidase inhibition activity test

[0207] 1. Purpose of the experiment

[0208] The compounds were evaluated for their level of inhibition of xanthine oxidase activity.

[0209] 2. Reagents

[0210] The main reagents used in this study include xanthine (Sigma, catalog number: X4002-1G, batch number: SLBB5664V) and xanthine oxidase (Sigma, catalog number: X4376-5UN, batch number: SLBQ1518V).

[0211] 3. Instruments

[0212] The main instrument used in this research is a multifunctional microplate reader.

[0213] 4. Experimental Methods

[0214] 1) Add 50 μL of Dulbecco's phosphate buffered saline (DPBS) to the compound background control wells and HPE (100% inhibitory activity) positive control wells.

[0215] 2) Dilute 2 U / mL xanthine oxidase to 0.04 U / mL with DPBS, and add 50 μL of xanthine oxidase to the compound activity test wells and ZPE (0% inhibition activity) negative control wells.

[0216] 3) Dilute the compound in DMSO in a 3-fold serial dilution series for 8 points. Then, dilute the compound in DPBS and add 50 μL to each well in triplicate. Add 50 μL of DPBS to each well for the HPE (100% inhibitory activity) positive control well and the ZPE (0% inhibitory activity) negative control well.

[0217] 4) Dilute 200 mM xanthine to 300 μM with DPBS. Add 100 μL of xanthine to each well and incubate at room temperature for 30 minutes. The final concentration of xanthine oxidase in each well is 0.01 U / mL, and the final concentration of DMSO in each well is 0.5%. HPE (100% inhibition activity) positive control wells contain xanthine but no xanthine oxidase. ZPE (0% inhibition activity) negative control wells contain xanthine and xanthine oxidase. Compound background control wells contain various concentrations of compound and xanthine but no xanthine oxidase.

[0218] 5) Use a spectrophotometer to detect the absorbance at 290 nm.

[0219] 6) Data analysis: Calculate the inhibition rate of xanthine oxidase in each well according to the following formula:

[0220]

[0221] *OD test sample is the optical density value of the compound activity test well, containing compound, xanthine and xanthine oxidase;

[0222] OD compound control is the background optical density value of the test compound at different concentrations, including compound and xanthine, without

[0223] Xanthine oxidase;

[0224] OD ZPE : is the average optical density value of the control wells with no inhibitory activity, containing 0.5% DMSO, xanthine and xanthine

[0225] oxidase;

[0226] OD HPE The optical density values are the average of the 100% inhibition control wells containing 0.5% DMSO and xanthine but without xanthine oxidase.

[0227] 7) GraphPad Prism software was used to perform log(agonist) vs. response--Variable slope nonlinear fitting analysis on the inhibition rate data (inhibition rate%) of the compound to obtain the IC 50 The fitting formula is:

[0228] Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)*HillSlope))

[0229] 5. Experimental Results

[0230] The experimental results are shown in Table 8.

[0231] Table 8. Xanthine oxidase inhibitory activity test results of compounds

[0232] Compound number <![CDATA[XO IC 50 (nM)]]> Compound of formula (I) 20.7

[0233] Experimental conclusion: The compound of the present invention has good xanthine oxidase inhibitory activity.

[0234] Experimental Example 2: Inhibitory activity test of compounds on uric acid uptake

[0235] 1. Purpose of the experiment

[0236] In this study, a human Urat1 gene stably transfected cell line was used to evaluate the inhibitory activity of the test compounds on uric acid uptake.

[0237] 2. Experimental Materials

[0238] 2.1 Cell lines

[0239] A stably transfected human Urat1 cell line was constructed by Shanghai WuXi AppTec Pharmaceutical Development Co., Ltd. The stably transfected human Urat1 cell line (Urat1-MDCK) was derived from MDCK cells transfected with the human Urat1 gene and selected using G418. The cell line was cultured in MEM supplemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin, 100 μg / ml streptomycin, 2 mM L-glutamine, 1% non-essential amino acids, and 250 μg / ml G418.

[0240] 2.2 Reagents

[0241] The main reagents used in this study include 14C-uric acid (ARC, product number: ARC-0513, batch number: 200122).

[0242] 2.3 Instruments

[0243] The main instrument used in this study was a liquid scintillation analyzer (Perkin Elmer, Tri-Carb 4910TR).

[0244] 3. Experimental Methods

[0245] 3.1 Cell plating

[0246] 3.1.1Urat1-MDCK cells cultured in a T150 cell culture flask were digested with 0.25% trypsin and diluted with fresh culture medium to adjust the suspension to 200,000 cells / ml.

[0247] 3.1.2 Seed the cells into a 48-well cell culture plate, 0.5 ml per well, and the final cell density is 100,000 cells / well.

[0248] 3.1.3 Place the cell culture plate in a 37°C, 5% CO2 incubator and culture overnight.

[0249] 3.2 Compound handling and detection

[0250] 3.2.1 Dilute the compound 5-fold in DMSO to 4 dilution points, with the diluted concentration being 200× the final assay concentration. Then dilute the compound 10-fold in HBSS buffer.

[0251] 3.2.2 Dilute the 10 mM 14C-uric acid concentrated stock solution to 1 mM with HBSS buffer.

[0252] 3.2.3 After overnight culture in the cell culture plate, remove the cell culture medium from the culture plate, wash the cells three times with HBSS buffer, and add 90 μl HBSS buffer to each well.

[0253] 3.2.4 Add 5 μl of the diluted compound to each well and incubate the cells in a 37°C, 5% CO2 incubator for 20 minutes. The DMSO content in each well is 0.5%. Use the test compound (10 μM) as a 100% inhibition control, and 0.5% DMSO as a 0% inhibition control.

[0254] 3.2.5 Add 5 μl of diluted 14C-uric acid to each well of the cell plate. The final concentration of uric acid in each well is 50 μM. Place the cells at 37°C.

[0255] Incubate in a 5% CO2 incubator for 15 minutes and then wash the cells three times with pre-chilled HBSS buffer.

[0256] 3.2.6 Add 150 μl of 0.1 M NaOH to each well and lyse the cells for 10 minutes.

[0257] 3.2.7 Collect the cell lysate into liquid scintillation test vials and add 2 ml of scintillation fluid to each vial for testing.

[0258] 3.2.8 Detect the 14C content of each sample using a liquid scintillation analyzer.

[0259] 3.2.9 Data Analysis:

[0260] Inhibition rate % = (HC-CPD) / (HC-LC) × 100%*

[0261] *CPD is the radioactive signal value of the compound well;

[0262] HC is the average radioactive signal of the 0% inhibition control well;

[0263] LC is the average radioactive signal of 100% inhibition control wells.

[0264] 3.2.10 Using GraphPad Prism software, the dose-effect curve was fitted using the nonlinear regression log(inhibitor) vs. response--Variable slope method according to the following formula, and the IC value of the compound was obtained. 50 Value and IC 90 value.

[0265] Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)*HillSlope))

[0266] 4. Experimental Results

[0267] The experimental results are shown in Table 9.

[0268] Table 9. Inhibitory activity of compounds on uric acid uptake

[0269] Compound number <![CDATA[IC 50 (μM)]]> Compound of formula (I) 2.24

[0270] Experimental conclusion: The compound of the present invention has good inhibitory activity on uric acid uptake

[0271] Experimental Example 3: Metabolic Stability (HMS) Study in Hepatocytes

[0272] 1. Purpose of the experiment

[0273] The metabolic stability of the test articles was tested in human and rat hepatocytes.

[0274] 2. Experimental Materials

[0275] 2.1 Test compound (10 mM), reference substance: 7-ethoxycoumarin (30 mM), 7-hydroxycoumarin (30 mM)

[0276] 2.2 Cells

[0277] The cell information is shown in Table 10.

[0278] Table 10. Cell information

[0279] Hepatocytes Cell viability Supplier Cat No. Rat hepatocytes 85％ BioreclamationIVTM00005 human hepatocytes 84％ Bioreclamation IVTX008001

[0280] 2.3 Buffer system:

[0281] Thawing medium: Williams' medium E containing 5% fetal bovine serum and 30% Percoll solution and other supplementary supplies.

[0282] Incubation medium: Williams medium E (without phenol red) containing 2 mM L-glutamine and 25 mM hydroxyethylpiperazineethanesulfonic acid.

[0283] Stop solution: acetonitrile containing 200 ng / mL tosylbutamide and labetalol as internal standard.

[0284] Dilution solution: ultrapure water.

[0285] 3. Experimental Methods

[0286] 1) Dissolve an accurate amount of the positive control compound in dimethyl sulfoxide (DMSO) to prepare a 30 mM solution.

[0287] 2) 10 mM test compound and 30 mM positive control compound were diluted to 1 mM and 3 mM with DMSO in a 96-well plate.

[0288] 3) 1 mM of the test compound and 3 mM of the positive control compound were diluted with acetonitrile to 100 μM and 300 μM quantitative solutions.

[0289] 4) Thaw the frozen cells, separate and suspend them in culture medium, and then dilute them to 0.5×10 6 cells / mL.

[0290] 5) Add 198 μL of pre-warmed cell suspension to a 96-well plate.

[0291] 6) Transfer 100 μL of stop solution (acetonitrile containing 200 ng / mL tosylbutamide and 200 ng / mL labetalol as internal standard) to a pre-labeled 96-well plate.

[0292] 7) Add 2 μL of 100 μM test compound or 300 μM positive control quantification solution in duplicate to each well of a 96-well plate.

[0293] 8) For T0 samples, mix to achieve a uniform suspension for approximately 1 minute, then immediately transfer 20 μL of each sample to a well containing 100 μL of ice-cold stop solution and mix.

[0294] 9) Incubate all plates at 37°C in a 95% humidified incubator with 5% CO2 and start the reaction with constant shaking at approximately 600 rpm.

[0295] 10) At 15, 30, 60, and 90 minutes, mix the samples, then transfer 20 μL of each sample to a well containing 100 μL of ice-cold stop solution at each time point, followed by mixing.

[0296] 11) Prepare medium control (MC) sample plates (labeled T0-MC and T90-MC) at T0 and T90 by adding the same components to each well except for the cell suspension. Generate a table of final concentrations.

[0297] 12) At each corresponding time point, stop the reaction by removing the plate from the incubator and mixing with 100 μL of ice-cold stop solution.

[0298] 13) Immediately vortex the plate on a plate shaker at 500 rpm for 10 minutes. Then, centrifuge all sample plates at 3220 x g for 20 minutes at 4°C.

[0299] 14) After centrifugation, transfer 35 μL / well of the supernatant from the sample plate to another set of pre-labeled 96-well plates containing 70 μL of ultrapure water.

[0300] 15) Seal the assay plate and store at 4°C until LC-MS-MS analysis.

[0301] The residual rates of the test compound and the control compound were calculated using the following formula:

[0302]

[0303] The elimination rate constant k of the test compound and the control compound in the hepatocytes was calculated by plotting the logarithm of the residual rate against the time, and the half-life (T 1 / 2 ) and in vitro intrinsic clearance (CL int ), the formula is as follows:

[0304] T 1 / 2 =0.693 / k

[0305] CL int(hep) = k / million cells / mL

[0306] CL int(liver) =CL int(hep) × liver weight to body weight ratio × number of hepatocytes per gram of liver

[0307] The parameters of various attributes in the formula are shown in Table 11:

[0308] Table 11. Various attribute parameters

[0309]

[0310] 4. Experimental Results

[0311] The results are shown in Table 12.

[0312] Table 12 Intrinsic clearance of compounds in human and rat liver

[0313]

[0314] Experimental conclusion: The compound of the present invention has a moderate clearance in human hepatocytes and a high clearance in rat hepatocytes.

[0315] Test Example 4. Membrane Permeability MDR1 Test

[0316] 1. Experimental purpose:

[0317] MDR1-MDCK II cells are Madin-Darby canine kidney cells transfected with the human MDR1 gene, which stably overexpress P-gp. The objectives of this study were to test the bidirectional permeability of compounds across the MDR1-MDCK II cell model and to assess whether they are transported via efflux.

[0318] 2. Cell Culture:

[0319] MDR1-MDCK II cells (obtained from Piet Borst, Netherlands Cancer Institute) were cultured at 2.5×10 5 Cells were seeded onto polyethylene membrane (PET) in a 96-well insert system at a density of 100 cells / mL and allowed to form a confluent cell monolayer after 4-7 days.

[0320] 3. Experimental Methods

[0321] Test compounds were diluted in transport buffer (HBSS, 10 mM Hepes with DMSO, pH 7.4) to a concentration of 2 μM (DMSO <1%) and applied to the apical or basolateral side of the cell monolayer. Test compounds were assayed in duplicate in the A to B or B to A directions. Digoxin was also tested at 10 μM in the A to B or B to A directions, while nadolol and metoprolol were tested at 2 μM in the A to B direction. Plates were incubated for 2.5 hours in a CO2 incubator at 37 ± 1°C in a humidified atmosphere of 5% CO2 without shaking. The efflux ratio of each compound was determined, and test and reference compounds were quantified. Analysis was performed by LC / MS / MS based on the analyte / IS peak area ratio. Following the transport assay, cell monolayer integrity was determined using a Lucifer Yellow exclusion assay. Remove the buffer from the apical and basolateral chambers, then add 75 μL of 100 μM Lucifer Yellow in transport buffer and 250 μL of transport buffer to the apical and basolateral chambers, respectively. Incubate the plate at 37°C, 5% CO2, and saturated humidity for 30 minutes without shaking. After the 30-minute incubation, withdraw a 20 μL Lucifer Yellow sample from the apical chamber, and then add 60 μL of transport buffer. Then, collect an 80 μL Lucifer Yellow sample from the basolateral chamber. Measure the relative fluorescence units (RFU) of Lucifer Yellow at 425 / 528 nm (excitation / emission) using an Envision microplate reader.

[0322] 4. Data calculation

[0323] The apparent permeability coefficient (P app ,cm / s), efflux rate and recovery rate.

[0324] Apparent permeability coefficient (P app ,cm / s) is calculated using the following formula:

[0325] P app =(dC r / d t )×V r / (A×C0)

[0326] dC r / d t is the cumulative concentration of the compound at the receiving end per unit time (μM / s); V r is the volume of the solution at the receiving end (the volume of the solution at the apical and basal ends is 0.075 mL and 0.250 mL, respectively); A is the relative surface area of the cell monolayer (0.0804 cm 2 ); C0 is the starting concentration of the test substance at the dosing end (nM) or the peak area ratio of the reference substance.

[0327] The efflux ratio is calculated using the following formula:

[0328] Exclusion ratio = P app (BA) / P app (AB)

[0329] The recovery rate was calculated using the following formula:

[0330] % recovery = 100 × [(V r ×C r )+(V d ×C d )] / (V d ×C0)

[0331] C0 is the starting concentration of the test substance at the dosing end (nM) or the peak area ratio of the reference substance; V d is the volume of the dosing end (0.075 mL on the apical side and 0.250 mL on the basal side); C d and C r are the final concentration (nM) of the test sample at the dosing end and the receiving end or the peak area ratio of the reference sample.

[0332] The percentage of Lucifer Yellow in the basolateral wells was calculated using the following formula:

[0333]

[0334] Where RFU APICAL and RFU Basolateral are the relative fluorescence units of Lucifer Yellow in the apical and basolateral wells, respectively; VAPICAL and VBasolateral wells are the volumes of the apical and basolateral wells, respectively (0.075 mL and 0.25 mL). % Lucifer Yellow should be less than 2.

[0335] 5. Experimental Results

[0336] The results are shown in Table 13.

[0337] Table 13 Data on the permeability of compounds to MDR1 cell membrane

[0338] Compound number <![CDATA[P app (AB)(10 -6 cm / s)]]> <![CDATA[P app (BA)(10) -6 cm / s)]]> External exclusion ratio Compound of formula (I) 26.42 6.63 0.25

[0339] Experimental conclusion: The compound of the present invention is a highly permeable compound.

[0340] Test Example 5. Cytochrome P450 isoenzyme inhibition activity test

[0341] 1. Purpose of the experiment

[0342] The inhibitory activity of the test compounds against different subtypes of human cytochrome P450 isoforms was determined.

[0343] 2. Experimental Methods

[0344] Prepare the test compound, standard inhibitor (100× final concentration) and mixed substrate working solution; take out the microsomes (purchased from Corning Inc) frozen in a -80°C freezer and thaw them. Add 20 μL of the test compound and standard inhibitor solution to the corresponding wells, and add 20 μL of the corresponding solvent to the inhibitor-free control well (NIC) and blank control well (Blank); then add 20 μL of the mixed substrate solution to the corresponding wells, except for the Blank well (add 20 μL of phosphate buffer (PB) to the Blank well); prepare the human liver microsome solution (mark the date after use and put it back in the refrigerator immediately), and then add 158 μL of the human liver microsome solution to all wells; place the above sample plate in a 37°C water bath for pre-incubation, and then prepare the coenzyme factor (NADPH) solution; after 10 minutes, add 20 μL NADPH solution was added to all wells, the sample plate was shaken, and then incubated in a 37°C water bath for 10 minutes. At the corresponding time point, 400 μL of cold acetonitrile solution (with 200 ng / mL tolbutamide and labetalol as internal standards) was added to terminate the reaction. After the sample plate was mixed evenly, it was centrifuged at 4000 rpm for 20 minutes to precipitate the protein. 200 μL of supernatant was added to 100 μL of water, shaken, and sent for LC / MS / MS analysis.

[0345] 3. Experimental Results

[0346] The results are shown in Table 14.

[0347] Table 14 IC of compounds inhibiting P450 isozymes 50 value

[0348]

[0349] Experimental conclusion: The compounds of the present invention have very low inhibitory activity against CYP1A2, CYP2C19, CYP2D6 and CYP3A4-M, and have moderate inhibitory activity against CYP2C9.

[0350] Test Example 6: Pharmacokinetics in SD rats

[0351] 1. Experimental purpose:

[0352] Pharmacokinetics of test compounds in SD rats

[0353] 2. Experimental Materials:

[0354] Sprague Dawley rats (male, 180-350 g, 6-10 weeks old, purchased from Beijing Weitonglihua)

[0355] 3. Experimental methods:

[0356] The compound was mixed with 5% DMSO / 10% Solutol / 85% water, stirred and vortexed to prepare a clear solution at 0.6 mg / mL for injection. The solution was then filtered through a microporous filter and used for later use. The compound was mixed with 5% DMSO / 10% Solutol / 85% water, stirred and vortexed to prepare a clear solution at 1 mg / mL for oral administration. Six male Sprague-Dawley rats were divided into two groups. Group 1 received a single intravenous dose of 3 mg / kg in 5% DMSO / 10% Solutol / 85% water (5 mL / kg). Group 2 received a single oral gavage of 10 mg / kg of the test compound in 5% DMSO / 10% Solutol / 85% water (10 mL / kg). Whole blood was collected at 0 (oral gavage only), 0.083 (iv injection only), 0.25, 0.5, 1, 2, 4, 8, and 24 hours after administration. Whole blood was centrifuged at 3200 g for 10 min at 4°C to obtain plasma. The concentrations of the compound and uric acid (only in the oral administration group) in the plasma were determined by LC / MS / MS. Pharmacokinetic parameters such as peak concentration, time to peak concentration, clearance, half-life, area under the concentration-time curve, and bioavailability were calculated using Phoenix WinNonlin software.

[0357] The experimental results are shown in Table 15 below:

[0358] Table 15. Pharmacokinetic data of the compound of formula (I) in rats

[0359]

[0360] Experimental conclusion: The compound of the present invention has good pharmacokinetic properties and high oral bioavailability. 1 / 2For the elimination half-life, Vd ss is the steady-state apparent distribution volume, Cl is the total clearance, AUC 0-last AUC is the area under the plasma concentration-time curve from time 0 to the last quantifiable time point. 0-inf C is the area under the plasma concentration-time curve from time 0 to infinity, max is the peak concentration, T max Peak time.

Claims

1. Form C of the compound of formula (I), having an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 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°, 30.70±0.20°, 2. The crystal form C according to claim 1, has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 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°.

3. The crystal form C according to claim 2, wherein its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 13.20°, 15.26°, 18.08°, 21.99°, 25.07°, 25.38°, 26.66°, and 30.70°.

4. The crystalline form C according to claim 3, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 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°, 38.53°, and 39.80°.

5. The crystal form C according to any one of claims 1 to 4, whose XRPD pattern is shown in FIG5 Show.

6. The crystal form C according to any one of claims 1 to 4, wherein the thermogravimetric analysis curve thereof shows a weight loss of 1.21% at 200°C ± 3°C.

7. The crystal form C according to claim 6, whose TGA spectrum is shown in Figure 6.

8. The crystal form C according to any one of claims 1 to 4, wherein the differential scanning calorimetry curve thereof has an endothermic peak starting value at 250.0°C±2°C.

9. The crystal form C according to claim 8, whose DSC spectrum is shown in Figure 7.

10. Form E of the compound of formula (I), having an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2θ angles: 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°, 25.14±0.20°, 11. The crystalline form E according to claim 10, wherein its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 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°.

12. The crystal form E according to claim 11, wherein its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 9.11°, 12.43°, 13.28°, 15.34°, 18.16°, 22.06°, 23.15°, and 25.14°.

13. The crystalline form E according to claim 12, wherein its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 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°.

14. The crystal form E according to any one of claims 10 to 13, whose XRPD pattern is shown in Figure 11.

15. The crystal form E according to any one of claims 10 to 13, wherein the thermogravimetric analysis curve thereof shows a weight loss of 0.79% at 200°C ± 3°C.

16. The crystal form E according to claim 15, whose TGA spectrum is shown in Figure 12.

17. The crystal form E according to any one of claims 10 to 13, wherein the differential scanning calorimetry curve thereof has an endothermic peak with an onset value at 250.4°C ± 2°C.

18. The crystal form E according to claim 17, whose DSC spectrum is shown in Figure 13.

19. Use of the crystal form C according to any one of claims 1 to 9 or the crystal form E according to any one of claims 10 to 18 in the preparation of drugs for treating gout and hyperuricemia.

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