Crystal forms of glucoside compounds and their applications

Abstract

A crystalline form (I) of a glucoside compound and its application, and its application in preparing medicines for treating related diseases.

Description

[0001] This application claims the following priority

[0002] CN202011032215.3, application date: September 27, 2020. Technical Field

[0003] The invention relates to a crystal form of a glucoside compound and an application thereof, and an application thereof in preparing medicines for treating related diseases. Background Art

[0004] Obesity, diabetes and the related metabolic disorders they cause have become important risk factors threatening human health.

[0005] Sodium-glucose cotransporters (SGLTs) are a family of glucose transporters found in the small intestinal mucosa and renal proximal tubules. The main members include SGLT1 and SGLT2. Their function is to mediate the transmembrane transport of glucose in the intestine and kidney. Specifically, SGLT1 is mainly distributed in the intestinal mucosal cells of the small intestine, and is also expressed in small amounts in the myocardium and kidneys. Its main function is to regulate the intestinal absorption of glucose. SGLT2 is expressed at high levels in the kidneys and is mainly responsible for regulating the renal reuptake of glucose, that is, glucose in urine can actively attach to renal tubular epithelial cells when filtered by the glomerulus and be transported into the cell through the SGLT2 protein for reuse. Since the glucose transport process mediated by SGLTs does not intervene in sugar metabolism, it avoids the occurrence of adverse reactions of hypoglycemia and reduces the risk of causing cardiovascular diseases. Therefore, SGLTs have gradually become one of the ideal targets for the treatment of diabetes. In view of this, some SGLTs inhibitors, especially highly selective SGLT2 inhibitors, have been developed one after another. They specifically inhibit the reabsorption of glucose by the kidneys by inhibiting the activity of SGLT2, thereby increasing the excretion of glucose in the urine and normalizing the blood sugar level in diabetic patients. Since 2012, several SGLT2 inhibitors have been approved for marketing and have become effective drugs for the treatment of diabetes.

[0006] In addition to inhibiting SGLT2, studies in recent years have found that appropriate inhibition of SGLT1 can prevent the intestinal uptake of glucose without causing obvious diarrhea or other gastrointestinal reactions. At the same time, by inhibiting SGLT1, the glucose absorbed into the blood through the intestine can be reduced, thereby increasing the glucose concentration in the distal intestine, leading to increased levels of GLP-1 and PYY after meals, thereby exerting a better hypoglycemic effect and reducing the risk of urinary tract infections and renal damage. In addition, by controlling the intestinal absorption of glucose, the total energy intake in food can also be reduced. The weight-reducing effect of GLP-1 can be superimposed to achieve the goal of double weight reduction. Therefore, the development of SGLT1 inhibitors has become a new direction for the treatment of diabetes and obesity in recent years.

[0007] In summary, SGLT1 inhibitors have good development prospects as new drugs for the treatment of diabetes and obesity. However, so far, research on SGLT1 inhibitors is still in the clinical stage, and no drug has been approved for marketing. Currently, LX2761, an SGLT1 inhibitor developed by Lexicon that only acts on the gastrointestinal tract, is undergoing Phase I clinical research for the treatment of diabetes (WO2014081660).

[0008] Summary of the invention

[0009] The present invention provides an amorphous form of a compound of formula (I), whose X-ray powder diffraction (XRPD) pattern is as follows: Figure 1 shown.

[0010]

[0011] In some embodiments of the present invention, a glass transition signal can be observed at 74.9±3° C. in the differential scanning calorimetry curve of the amorphous phase.

[0012] In some embodiments of the present invention, the DSC spectrum of the amorphous Figure 2 shown.

[0013] In some embodiments of the present invention, the thermogravimetric analysis curve of the amorphous material shows a weight loss of 2.56% at 150.0±3°C.

[0014] In some embodiments of the present invention, the TGA spectrum of the amorphous Figure 3 shown.

[0015] The present invention also provides a method for preparing an amorphous form of the compound of formula (I), comprising an anti-solvent addition method, a slow cooling method, a suspension stirring method, a slow volatilization method, a gas-solid permeation method and a temperature cycle method.

[0016] In some embodiments of the present invention, the above-mentioned preparation method, wherein the anti-solvent addition method comprises:

[0017] 1) adding the compound of formula (I) into a solvent to form a clear solution;

[0018] 2) adding an antisolvent to the solution;

[0019] in,

[0020] The solvent is ethanol, acetone, tetrahydrofuran, acetonitrile, n-propyl acetate, anisole or 2-methyltetrahydrofuran;

[0021] The antisolvent was H2O or n-heptane.

[0022] In some embodiments of the present invention, the above-mentioned preparation method, wherein the slow cooling method comprises:

[0023] 1) at 50° C., preparing a clear solution of the compound of formula (I) in a solvent;

[0024] 2) The solution was cooled from 50°C to 5°C at a rate of 0.1°C / min;

[0025] in,

[0026] The solvents were tert-butyl alcohol, isopropyl acetate, acetonitrile:water (v / v, 1:3) or 2-methyltetrahydrofuran:cyclohexane (v / v, 1:3).

[0027] In some embodiments of the present invention, the above-mentioned preparation method, wherein the suspension stirring method comprises:

[0028] 1) adding the compound of formula (I) into a solvent to form a suspension;

[0029] 2) The suspension solution is stirred by magnetic force to transform the crystals;

[0030] in,

[0031] The solvent is methyl tert-butyl ether, ethyl acetate:n-heptane (v / v, 1:3), 2-methyltetrahydrofuran:cyclohexane (v / v, 1:3), methyl tert-butyl ether:n-heptane (v / v, 1:7), tetrahydrofuran:n-heptane (v / v, 1:7), ethyl acetate:n-heptane (v / v, 1:7), 2-butanone:n-heptane (v / v, 1:7), acetonitrile:water (v / v, 1:9), acetone:water (v / v, 1:12), n-heptane, water, tetrahydrofuran:n-pentane (v / v, 1:7), N-methylpyrrolidone:water (v / v, 1:5), dimethyl sulfoxide:water (v / v, 1:5) or m-xylene.

[0032] In some embodiments of the present invention, the above-mentioned preparation method, wherein the slow volatilization method comprises:

[0033] 1) dissolving the compound of formula (I) in a solvent to form a clear solution;

[0034] 2) Seal the clarified solution with a sealing film, pierce a small hole, and then slowly evaporate and crystallize;

[0035] in,

[0036] The solvent is ethyl acetate, chloroform, methanol, acetone, acetonitrile or tetrahydrofuran.

[0037] In some embodiments of the present invention, the above-mentioned preparation method, wherein the gas-solid permeation method comprises:

[0038] 1) placing the compound of formula (I) in a vial;

[0039] 2) Place the vial open in a water atmosphere, seal it and let it stand at room temperature.

[0040] In some embodiments of the present invention, the above-mentioned preparation method, wherein the temperature cycling method comprises:

[0041] 1) dissolving the compound of formula (I) in a solvent to form a suspension at 50°C;

[0042] 2) The suspension is heated and cooled at a rate of 0.1°C / min according to a program of 50°C-5°C-50°C-5°C to perform crystallization;

[0043] in,

[0044] The solvent was n-heptane, methyl tert-butyl ether, 2-methyltetrahydrofuran:n-heptane (v / v, 1:3) or N-methylpyrrolidone:water (v / v, 1:5).

[0045] Definition and Description

[0046] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

[0047] 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 substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the embodiments of the present invention.

[0048] The chemical reactions of the specific embodiments of the present invention are carried out in a suitable solvent, which must be suitable for the chemical changes of the present invention and the reagents and materials required. In order to obtain the compounds of the present invention, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction processes based on the existing embodiments.

[0049] The structure of the compound of the present invention can be confirmed by conventional methods known to those skilled in the art. If the present invention relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, single crystal X-ray diffraction (SXRD) is used to collect diffraction intensity data of the cultured single crystal using a Bruker D8 venture diffractometer, the light source is CuKα radiation, and the scanning mode is: After scanning and collecting relevant data, the crystal structure is further analyzed using the direct method (Shelxs97) to confirm the absolute configuration.

[0050] The present invention will be described in detail below by way of examples, which are not intended to limit the present invention in any way.

[0051] All solvents used in the present invention were commercially available and used without further purification.

[0052] The solvent used in the present invention can be obtained commercially. The present invention uses the following abbreviations: DCM represents dichloromethane; DMF represents N, N-dimethylformamide; DMSO represents dimethyl sulfoxide; EtOH represents ethanol; MeOH represents methanol; TFA represents trifluoroacetic acid; ATP represents adenosine triphosphate; HEPES represents 4-hydroxyethylpiperazineethanesulfonic acid; MgCl2 represents magnesium dichloride; T3P represents 1-propylphosphoric anhydride.

[0053] Technical Effects

[0054] The compound of the present invention has good crystal stability and is easy to formulate into medicine; the amorphous compound of formula (I) has a significant inhibitory effect on the activity of human SGLT1 and SGLT2 transporters; after oral administration of the amorphous compound of formula (I), the blood drug concentration and oral bioavailability in the body are very low, and it has the pharmacokinetic property of directly acting on intestinal SGLT1; after a single administration of the amorphous compound of formula (I), the blood sugar level of mice after oral administration of glucose can be reduced in a dose-dependent manner.

[0055] X-ray powder diffractometer (XRPD) method of the present invention

[0056] Instrument model: X-ray diffractometer from PANalytical

[0057] Test method: Approximately 10 mg of sample was used for XRPD detection.

[0058] The detailed XRPD parameters are shown in Table 1:

[0059] Table 1

[0060]

[0061] Modulated Differential Scanning Calorimetry (mDSC) method of the present invention

[0062] Instrument model: TA Discovery DSC 2500 Differential Scanning Calorimeter Test method: Take a sample (about 1-5 mg) and place it in an mDSC aluminum pan for testing. Under 50 mL / min N2 conditions, heat the sample from 0°C to 250°C at a heating rate of 3°C / min.

[0063] The detailed DSC parameters are shown in Table 2:

[0064] Table 2

[0065] parameter Settings method Modulated heating Sample tray Aluminum tray, gland Temperature range 0℃-250℃ Scan rate (℃ / min) 3 Protective gas Nitrogen

[0066] Thermogravimetric analysis (TGA) method of the present invention

[0067] Instrument model: TA Q5000 / Discovery TGA 5500 Thermogravimetric Analyzer Test method: Take a sample (about 1-5 mg) and place it in a TGA aluminum pan for testing. Under 10 mL / min N2 conditions, heat the sample from room temperature to 350°C at a heating rate of 10°C / min.

[0068] The detailed TGA parameters are shown in Table 3:

[0069] Table 3

[0070] parameter Settings method Modulated heating Sample tray Aluminum tray, gland Temperature range 0℃-250℃ Scan rate (℃ / min) 3 Protective gas Nitrogen BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 XRPD spectrum of amorphous Cu-Kα radiation of the compound of formula (I);

[0072] Figure 2 is the DSC spectrum of the amorphous compound of formula (I);

[0073] Figure 3 This is the TGA spectrum of the amorphous compound of formula (I). DETAILED DESCRIPTION

[0074] In order to better understand the content of the present invention, further description is given below in conjunction with specific embodiments, but the specific implementation methods are not intended to limit the content of the present invention.

[0075] Example 1: Preparation of the compound of formula (I)

[0076]

[0077] Step 1: Synthesis of Compound C

[0078] Add 2-methyltetrahydrofuran (12L) to the reactor, slowly add compound A (1200.00g, 5.45mol, 1.0 equivalent), compound B (165000g, 8.18mol, 1.5 equivalent) and diphenylphosphine (2070.00g, 7.91mol, 1.45 equivalent) in batches, and start stirring. At 0-5°C, slowly add diisopropyl azodicarboxylate (1430.00g, 7.09mol, 1.30 equivalent, 1.38L) to the reactor, and after the addition is complete, heat the reactor to an internal temperature of 10-20°C and stir for 16 hours. After the reaction is completed, slowly add magnesium chloride (4.14kg) in batches and stir for 2 hours. Then slowly add 2-methyltetrahydrofuran (12L) to the reactor and stir and dilute for 0.5 hours. Filter, collect the filtrate, add water (12L) to the filtrate, stir for 5 minutes and then stand to separate the organic phase and the aqueous phase, extract the aqueous phase with ethyl acetate (6L). Combine the organic phases and concentrate to dryness under reduced pressure to obtain a crude product. Add ethanol (12L) to the crude product, control the temperature in the reactor to 15-25°C and stir for 16 hours. Filter by suction, wash the filter cake with ethanol (1.2L), collect the filter cake, and dry it in a vacuum drying oven for 16 hours to obtain compound C. The product was confirmed by LCMS, LC-MS (m / z) 304.2 [M+H-Boc] + .

[0079] Step 2: Synthesis of Compound D

[0080] Add dichloromethane (14L) to the reactor, slowly add compound C (1400.00g, 3.47mol, 1.0 equivalent) in batches, and start stirring. Slowly add trifluoroacetic acid (2.1L, 28.36mol, 8.17 equivalent) to the reactor, and keep the temperature in the reactor at 15-25°C after the addition is complete, and stir for 3 hours. After the reaction is completed, transfer the reaction solution to a rotary evaporator, concentrate to dryness under reduced pressure, remove dichloromethane and most of the trifluoroacetic acid, and obtain a crude product. After the crude product is dissolved in dichloromethane (14L), transfer it to a separator, and wash it once with saturated sodium bicarbonate and water respectively. Separate the organic phase, concentrate to dryness under reduced pressure to obtain a white solid. Transfer the white solid to a three-necked flask, add ethyl acetate (2.8L), and stir for 12 hours. Filter by suction, wash the filter cake once with ethyl acetate (1.4L), collect the filter cake, and dry it in a vacuum drying oven for 16 hours to obtain compound D. The product was confirmed by LCMS, LC-MS (m / z) 304.0 [M+H] + .

[0081] Step 3: Synthesis of Compound F

[0082] Add dichloromethane (8L) to the reactor, slowly add compound D (784.37g, 2.59mol, 1.0 equivalent) and compound E (863.00g, 2.59mol, 1.0 equivalent) in batches, and start stirring. Then slowly add triethylamine (800.89g, 7.91mol, 3.06 equivalent, 1.10L), control the temperature in the reactor to 10-20°C, and stir for 15 minutes. Then slowly add T3P (2.35L, 3.96mol, 1.53 equivalent) to the reactor, and after the addition is complete, control the temperature in the reactor to 10-20°C and stir for 16 hours. After the reaction is completed, transfer the reaction solution to a separator and wash it thoroughly with water (8L*1), 0.1M hydrochloric acid (8L*1), 0.5M potassium carbonate (8L*2) and water (8L*2) in turn. The organic phase was collected and concentrated to dryness under reduced pressure to obtain a crude product. The crude product was dissolved in a mixed solvent of dioxane and n-heptane (1:5, 11.76 L) and transferred to a reactor. The temperature in the reactor was controlled at 15-25°C and stirred for about 42 hours until solids precipitated. The filter cake was filtered and rinsed with a mixed solvent of dioxane and n-heptane (1:5, 1.56 L). The filter cake was collected and dried in a vacuum drying oven for 16 hours to obtain compound F. The product was confirmed by LCMS, LC-MS (m / z) 6195 [M+H] + .

[0083] Step 4: Synthesis of Compound H

[0084] Add dioxane (6L), water (1.5L), compound G (750.00g, 1.49mol, 1.0 equivalent), compound F (1105.75g, 1.19mol, 1.2 equivalent), Pd(PPh3)4 (86.07g, 0.08mol, 0.05 equivalent) and potassium carbonate (411.81g, 2.98mol, 2.0 equivalent) to the reactor in sequence, start stirring, and introduce nitrogen flow. Raise the temperature in the reactor to 45-55°C and stir for 16 hours. After the reaction is completed, transfer the reaction solution to a rotary evaporator and concentrate to dryness under reduced pressure to obtain a crude product. Add ethyl acetate (7.5L) to the crude product to dissolve, transfer to a separator, add water (7.5L), stir for 5 minutes and then stand for stratification. Collect the organic phase and extract the aqueous phase with ethyl acetate (75L). Combine the organic phases and wash once with water (7.5L). Collect the organic phases and concentrate to dryness under reduced pressure to obtain a crude product. Add methanol (15L) to the crude product, heat to 55-65°C to dissolve and clarify, then cool to 15-25°C and stir for 16 hours. Filter, wash the filter cake once with methanol (15L). Collect the filter cake and repeat the above operation once to obtain 1476.25g of filter cake. Dissolve the filter cake with dichloromethane (7.4L), then add methanol (7.4L) and mercaptan silica gel (585.00g) in turn, heat to 35-45°C, and stir for 16 hours. Filter, wash the filter residue once with a mixed solvent of dichloromethane and methanol (1:1, 7.4L). Collect the filtrate, add SiliaMets Thiol (585.00g), heat to 35-45°C, and stir for 16 hours. Filter, wash the residue once with a mixed solvent of dichloromethane and methanol (1:1, 7.4 L). Collect the filtrate, add SiliaMetsThiol (585.00 g), heat to 35-45°C, and stir for 16 hours. Filter, wash the residue once with a mixed solvent of dichloromethane and methanol (1:1, 7.4 L). Transfer the filtrate to a rotary evaporator, concentrate to dryness under reduced pressure, collect the product, and dry it in a vacuum drying oven for 16 hours to obtain compound H. The product was confirmed by LCMS, LC-MS (m / z) 915 [M+H] + .

[0085] Step 5: Synthesis of Compound J

[0086] HCl / EtOAc solution (4M, 10.5L) and compound H (700.00g, 0.77mol, 1.0 equivalent) were added to the reactor in sequence and stirring was started. The temperature in the reactor was controlled at 15-25°C and stirred for 2 hours. After the reaction was completed, ethyl acetate (7L) was added to the reactor to dilute the reaction solution, and the reaction solution was transferred to a rotary evaporator and concentrated to dryness under reduced pressure. Ethyl acetate (7L*2) was then added and continued to be concentrated to dryness under reduced pressure to obtain a crude product. Ethyl acetate (10.5L) was added to the crude product and stirred at 15-25°C for 16 hours. Filter and wash the filter cake once with ethyl acetate (1.05L). Collect the filter cake and dry it in a vacuum drying oven for 16 hours to obtain compound J. The product was confirmed by LCMS, LC-MS (m / z) 815 [M+H] + .

[0087] Step 6: Synthesis of compound of formula (I)

[0088] Methanol (5.8L) and compound J (580.00g, 0.71mol, 1.0 equivalent) were added to the reactor in sequence and stirred. Subsequently, 25% NaOMe methanol solution (290mL) was added and stirred at room temperature for 3 hours. After the reaction was completed, the reaction solution was transferred to a rotary evaporator and concentrated to dryness under reduced pressure to obtain a crude product. Water (5.6L) was added to the crude product and stirred at 10-20°C for 2 hours. Filtered, the filter cake was washed twice with water (1.12L). The filter cake was collected, and water (5.6L) was added to the filter cake, and stirred at 10-20°C for 16 hours. Filtered, the filter cake was washed twice with water (1.12L). The filter cake was collected and placed in a vacuum drying oven for 144 hours to obtain a compound of formula (I). 1 H NMR (400MHz, CD3OD) δppm 1.09 (t, J=7.50Hz, 3H), 1.73 (br d, J=17.26Hz, 2H), 1.92 (br d, J=12.51Hz, 2H), 2.14 (s, 3H), 2.40-2.55 (m, 2H), 2.61 (q, J=7.42Hz, 2H), 2.68-2.85 (m, 2H), 3.35-3.48 (m, 5H), 3.53 (br s, 1H), 3.65-3.88 (m, 2H), 3.92-4.01 (m, 2H), 4.13 (d, J=9.13Hz, 1H), 4.39 (d, J=9.51Hz, 1H), 4.56 (br s, 1H), 6.85 (d, J=8.38Hz, 2H), 7.05 (d, J=8.38Hz, 2H), 7.10-7.31 (m, 5H).

[0089] Example 2: Preparation of amorphous compound of formula (I)

[0090] Table 4: Comparison table of Chinese and English names of solvents used in the experiment

[0091] English Chinese English Chinese MeOH Methanol MTBE Methyl tert-butyl ether EtOH Ethanol Anisole Anisole MEK 2-Butanone n-Heptane n-Heptane Acetone acetone Toluene Toluene ACN Acetonitrile DCM Dichloromethane IPA Isopropyl acetate DMSO Dimethyl sulfoxide THF Tetrahydrofuran <![CDATA[H2O]]> water 2-MeTHF 2-Methyltetrahydrofuran NMP N-Methylpyrrolidone Cyclohexane Cyclohexane m-Xylene Meta-Xylene n-Pentane n-Pentane <![CDATA[CHCl3]]> Chloroform t-BuOH Tert-Butanol EtOAc Ethyl acetate n-Propyl acetate Propyl acetate

[0092] 1. Anti-solvent addition method:

[0093] About 15-20 mg of the compound of formula (I) was weighed and dissolved in a positive solvent at 25°C to prepare a clear solution. The corresponding anti-solvent was slowly added dropwise to the above solution until a solid appeared. The obtained solid was separated and tested by XRPD. The results are shown in Table 5:

[0094] Table 5: Antisolvent addition test

[0095]

[0096] *: Indicates that a clear solution was obtained after adding the anti-solvent, and the mixture was stirred at -20 °C until solid appeared.

[0097] 2. Slow cooling method:

[0098] About 20 mg of the compound of formula (I) was dissolved in a solvent at 50°C to prepare a clear solution, and the temperature was lowered at a rate of 0.1°C / min according to a procedure of 50°C-5°C. The precipitated solid was collected and subjected to XRPD testing. The results are shown in Table 6:

[0099] Table 6: Slow cooling test

[0100] Test No. Solvent, v / v Test results 1 t-BuOH Amorphous 2 IPA Amorphous 3 <![CDATA[ACN / H2O,1:3]]> Amorphous 4# 2-MeTHF / Cyclohexane, 1:3 Amorphous

[0101] #: Indicates that a clear solution is obtained after the temperature is lowered to 5°C. It remains clear when placed at -20°C. It evaporates and precipitates solids when placed at room temperature.

[0102] 3. Suspension stirring method:

[0103] About 15-20 mg of the compound of formula (I) was weighed and prepared into suspensions at 25°C or 50°C in different solvents. After magnetic stirring for about 6-8 days, the solid was collected by centrifugation and subjected to XRPD testing. The results are shown in Table 7:

[0104] Table 7: Suspension stirring test

[0105]

[0106]

[0107] 4. Slow evaporation method:

[0108] About 15-20 mg of the compound of formula (I) was weighed and dissolved in a solvent at 25°C to prepare a clear solution. The solution was sealed with a sealing film and pierced with 4-5 small holes, then slowly evaporated and crystallized. The obtained solid was collected and subjected to XRPD testing. The results are shown in Table 8:

[0109] Table 8: Slow evaporation test

[0110] Test No. Solvent, v / v Test results 1 EtOAc Amorphous 2 <![CDATA[CHCl3]]> Amorphous 3 MeOH Amorphous 4 Acetone Amorphous 5 ACN Amorphous 6 THF Amorphous

[0111] 5. Gas-solid permeation method:

[0112] About 20 mg of the compound of formula (I) was weighed and placed in a 3 ml vial, and a 20 ml vial was taken and the corresponding solvent was added thereto. The 3 ml vial was placed open in a 20 ml vial, and the mixture was sealed and allowed to stand at room temperature. The solid was collected and subjected to XRPD analysis. The results are shown in Table 9:

[0113] Table 9: Gas-solid permeability test

[0114]

[0115]

[0116] ^: Indicates a clear solution, which evaporates to room temperature to obtain a solid.

[0117] 6. Temperature cycle method:

[0118] About 20 mg of the compound of formula (I) was weighed and added to a solvent at 50°C to prepare a suspension. The temperature was raised and lowered at a rate of 0.1°C / min according to a procedure of 50°C-5°C-50°C-5°C. The obtained solid was collected and subjected to XRPD testing. The results are shown in Table 10:

[0119] Table 10: Temperature cycle test

[0120] Test No. Solvent, v / v Test results 1 n-Heptane Amorphous 2 MTBE Amorphous 3 2-MeTHF / n-Heptane, 1:3 Amorphous 4 <![CDATA[NMP / H2O,1:5]]> Amorphous

[0121] Example 3: Stability test of amorphous compound of formula (I)

[0122] Purpose:

[0123] The amorphous sample of the compound of formula (I) was subjected to stability studies under influencing factors (high temperature, high humidity and light), long-term conditions (25°C / 60%RH) and accelerated conditions (40°C / 75%RH) to evaluate the solid stability of the sample.

[0124] Experimental methods:

[0125] Experiment on influencing factors: The samples under high temperature conditions (60℃) were placed in an open disposable culture dish, and the samples under high humidity conditions (25℃ / 75%RH) were placed in an open flat weighing bottle, and then placed in different air drying ovens and desiccators for inspection. The light-exposed samples were placed in a clean disposable culture dish, spread into a thin layer, and covered with a quartz glass cover. The packaging method of the light-shielding control samples was the same as that of the light-exposed samples, but the outside of the disposable culture dish was covered with aluminum film. According to the requirements of the Chinese Pharmacopoeia and ICH Q1B, they were placed under light conditions (visible light 5000±500lux; ultraviolet light 90μw / cm 2 )

[0126] Long-term and accelerated condition experiments: The samples were placed in double-layer low-density polyethylene bags, each layer of low-density polyethylene bags was sealed with buckles, and then placed in an aluminum foil bag for heat sealing, and then placed in long-term conditions (25°C / 60%RH) and accelerated conditions (40°C / 75%RH) for investigation. The experimental results are shown in Table 11:

[0127] Table 11: Results of amorphous solid stability test of compound of formula (I)

[0128]

[0129]

[0130] NA: No crystal form was detected.

[0131] Conclusion: The amorphous compound of formula (I) has good stability.

[0132] Example 4: Amorphous solubility test of compound of formula (I)

[0133] Purpose:

[0134] The solubility of the amorphous form of the compound of formula (I) after suspension and stirring for 24 hours in different pH media (pH = 1.0, 2.0, 3.8, 4.5, 5.5, 6.0, 6.8, 7.4), biological solvents (SGF, FaSSIF, FeSSIF) and water was tested at 37°C.

[0135] Experimental methods:

[0136] 1. Prepare different pH media:

[0137] a) Take 11.4 mL of glacial acetic acid, add it into a 100 mL volumetric flask, add ultrapure water to make up to volume, and mix well to obtain a 2 mol / L acetic acid solution;

[0138] b) Weigh about 2.722 g of potassium dihydrogen phosphate, add it into a 100 mL volumetric flask, add an appropriate amount of ultrapure water and ultrasonically dissolve it, then make up to volume with ultrapure water, and mix well to obtain a 0.2 mol / L potassium dihydrogen phosphate solution;

[0139] c) Take 10 mL of 1.0 mol / L sodium hydroxide solution, add it into a 50 mL volumetric flask, add ultrapure water to make up to volume, and mix well to obtain a 0.2 mol / L sodium hydroxide solution;

[0140] d) Take 5 mL of 1.0 mol / L hydrochloric acid solution, add it into a 50 mL volumetric flask, add ultrapure water to make up to volume, mix well and measure pH = 0.99;

[0141] e) Measure 5 mL of 0.1 mol / L hydrochloric acid solution, add it to a 50 mL volumetric flask, add ultrapure water to make up to volume, mix well and measure pH = 1.98;

[0142] f) Weigh 32.9 mg of sodium acetate and add it to a 50 mL volumetric flask, add 1.13 mL of 2 mol / L acetic acid solution, add appropriate amount of ultrapure water and dissolve by ultrasonication, then make up to volume with ultrapure water, mix well and measure pH = 3.81;

[0143] g) Weigh 150.1 mg of sodium acetate and add it to a 50 mL volumetric flask, add 0.7 mL of 2 mol / L acetic acid solution, add appropriate amount of ultrapure water and ultrasonically dissolve, then make up to volume with ultrapure water, mix well and measure pH = 4.49;

[0144] h) Weigh 0.325 g of sodium acetate and add it to a 50 mL volumetric flask, add 0.15 mL of 2 mol / L acetic acid solution, add appropriate amount of ultrapure water and dissolve by ultrasonication, then make up to volume with ultrapure water, mix well and measure pH = 5.47;

[0145] i) Take 12.5 mL of 0.2 mol / L potassium dihydrogen phosphate solution, add it to a 50 mL volumetric flask, add 1.4 mL of 0.2 mol / L sodium hydroxide solution, add ultrapure water to make up to volume, mix well and measure pH = 6.03;

[0146] j) Take 12.5 mL of 0.2 mol / L potassium dihydrogen phosphate solution, add it to a 50 mL volumetric flask, add 5.6 mL of 0.2 mol / L sodium hydroxide solution, add ultrapure water to make up to volume, mix well and measure pH = 6.78;

[0147] k) Take 12.5 mL of 0.2 mol / L potassium dihydrogen phosphate solution and add it to a 50 mL volumetric flask, add 9.775 mL of 0.2 mol / L sodium hydroxide solution, add ultrapure water to make up to volume, mix well and measure the pH value to 7.42.

[0148] 2. Weigh about 10 mg of the amorphous sample of the compound of formula (I) into a centrifuge tube and add 1 mL of the corresponding medium;

[0149] 3. Magnetic suspension stirring at about 750 rpm at 37°C;

[0150] 4. After equilibration for 24 hours, separate the supernatant for pH and solubility testing.

[0151] The experimental results are shown in Table 12:

[0152] Table 12: Summary of the solubility of the amorphous form of the compound of formula (I) in different media at 37°C

[0153] Experiment number medium <![CDATA[Solubility (mg / mL) # > pH after 24 hours 1 1.0 0.5877 0.76 2 2.0 8.2037 5.56 3 3.8 9.1290 4.29 4 4.5 9.5798 4.74 5 5.5 25489 5.92 6 6.0 0.0905 6.32 7 6.8 0.0559 6.75 8 7.4 0.0052 7.36 9 water 0.0279 7.37 10 SGF 9.3821 2.82 11 FaSSIF 0.0631 6.73 12 FeSSIF 0.2446 5.21

[0154] #: y = 10336110.7x + 5294.3, R2 = 0.9999, LOQ = 0.12 μg / mL, LOD = 0.04 μg / mL; SGF: simulated gastric fluid;

[0155] FaSSIF: fasting state simulated intestinal fluid; FeSSIF: fed state simulated intestinal fluid.

[0156] Conclusion: The solubility of the amorphous form of the compound of formula (I) in different pH media (pH = 1.0, 2.0, 3.8, 4.5, 5.5, 6.0, 6.8, 7.4) is 0.005-9.6 mg / mL; the solubility in biological solvents (SGF, FaSSIF, FeSSIF) is 0.06-9.4 mg / mL; and the solubility in water is 0.03 mg / mL.

[0157] Example 5: Mechanical stability test of amorphous compound of formula (I)

[0158] Experimental methods:

[0159] 1) manually grinding an amorphous sample of the compound of formula (I) for about 5 minutes without adding water or solvent and then testing XRPD;

[0160] 2) The amorphous sample of the compound of formula (I) was added into a circular mold (6 mm in diameter), and pressurized until the pressure on the sample reached about 350 MPa. The pressed sample was directly tested by XRPD.

[0161] Experimental results:

[0162] After grinding and tableting, the crystal form of the sample did not change and remained amorphous.

[0163] Conclusion: The amorphous compound of formula (I) is morphologically stable under manual grinding and tableting conditions (350 MPa).

[0164] Experimental Example 1: In vitro cell activity test

[0165] 1. Purpose of the experiment

[0166] By measuring the intracellular bands that enter cells that highly express human SGLT1 and SGLT2 (Human-SGLT1 and Human-SGLT2) transporters [ 14 The inhibitory effect of the amorphous form of the compound of formula (I) on the activity of human SGLT1 and SGLT2 transporters was evaluated by measuring the amount of Methyl α-D-glucopyranosid (methyl α-D-pyranoglucoside) labeled with C].

[0167] 2. Experimental Methods

[0168] 1) The experiment was repeated three times with duplicate wells.

[0169] 2) Reagent and cell preparation;

[0170] a) Prepare the experimental buffer solution, the final concentration of each component is shown in Table 13:

[0171] Table 13 Buffer composition

[0172] Reagents Concentration (mM) HEPES 10 <![CDATA[MgCl2]]> 1.2 KCl 4.7 <![CDATA[CaCl2]]> 2.2 NaCl 120

[0173] b) Compound preparation: The amorphous stock solution of the compound of formula (I) has a concentration of 10 mM and is diluted with DMSO.

[0174] c) Compound dilution was completed by Bravo automatic pipetting workstation, with 10 concentrations diluted 3-fold. In the final 100 μL reaction system, the detection concentrations of human SGLT1 and SGLT2 target compounds were diluted from 0.4 μM to 0.0203 nM, respectively: 400.0000, 133.3333, 44.4444, 14.8148, 4.9383, 1.6461, 0.5487, 01.1829, 0.0610, 0.0203 nM.

[0175] d) Isotope preparation: Mix the isotope storage solution [ 14 C]Methyl α-D-glucopyranosid was diluted to a working concentration of 6 μM.

[0176] e) Cell preparation: Human SGLT1 or human SGLT2 cells were seeded at 7×10 4 cells / well in a 96-well cell plate and cultured overnight in a 37° C., 5% CO 2 cell culture incubator.

[0177] 3) Experimental process

[0178] a) Take the 96-well plate out of the incubator, discard the culture medium, rinse the cells with 150 μL / well of assay buffer, discard the buffer, and add 49 μL / well of fresh buffer.

[0179] b) According to the 96-well plate layout, add 1 μL of the compound solution diluted in series, add 1 μL of DMSO to the high signal well (HC, final DMSO concentration 1%), and add 1 μL of LX2761 to the low signal well (LC, final LX2761 concentration 0.4 μM).

[0180] c) Add 50 μL of 6 μM [ 14 C]Methyl α-D-glucopyranosid isotope to a 96-well plate.

[0181] d) Incubate at 37°C for 2 hours.

[0182] e) Take out the 96-well plate, remove the reaction system, wash three times with pre-cooled experimental buffer, add 50 μL / well 10% NaOH, and place on a shaker for 5 minutes.

[0183] f) Transfer the cell lysate in the 96-well plate to scintillation tubes, add 2 mL of scintillation fluid to each tube, and read the results using a Tri-Carb liquid scintillation counter.

[0184] g) Analyze the experimental data, % inhibition rate = (average value of high signal wells - signal value of sample wells) / (average value of high signal wells - average value of low signal wells)

[0185] *100. GraphPadPrism 5 was used to perform curve fitting: Log(inhibitor) vs. response-Variable slope, and IC 50 .

[0186] 3. The experimental results are shown in Table 14:

[0187] Table 14: In vitro cell activity test results

[0188] Compound <![CDATA[Human-SGLT1IC 50 (nM)]]> <![CDATA[Human-SGLT2 IC 50 (nM)]]> The compound of formula (I) is amorphous 1.8±0.4 1.6±1.0

[0189] Conclusion: The amorphous form of the compound of formula (I) has a significant inhibitory effect on the activity of human SGLT1 and SGLT2 transporters.

[0190] Experimental Example 2: DMPK study in rats

[0191] 1. Experimental purpose:

[0192] Male SD rats were used as test animals. The amorphous blood concentration of the compound of formula (I) was measured after a single administration and its pharmacokinetic behavior was evaluated.

[0193] 2. Experimental operation:

[0194] Six healthy adult male SD rats were selected, 3 for intravenous injection group and 3 for oral administration group. The solvent of the intravenous injection group was 5% (w / v) hydroxypropyl β-cyclodextrin (HP-β-CD) aqueous solution, and the amorphous compound of formula (I) was mixed with an appropriate amount of the solvent of the intravenous injection group to prepare a 0.25 mg / mL clear solution; the solvent of the oral administration group was 0.5% (w / v) methylcellulose (methylcellulose) / 0.02% Tween 80 aqueous solution, and the amorphous compound of formula (I) was mixed with an appropriate amount of the solvent of the oral administration group to prepare a 0.5 mg / mL uniform suspension. After 0.5 mg / kg intravenous administration or 5 mg / kg oral administration to rats, plasma samples were collected at different time points, and the concentration of the compound of formula (I) was analyzed by LC-MS / MS method, and the pharmacokinetic parameters were calculated using Phoenix WinNonlin software (Pharsight, USA).

[0195] 3. The experimental results are shown in Table 15:

[0196] Table 15: Compound PK test results

[0197]

[0198] Note: C max is the maximum concentration; F% is the oral bioavailability; AUC po is the oral exposure; Vd ss is the distribution volume; Cl is the clearance rate; T 1 / 2 is the half-life.

[0199] Conclusion: After oral administration of the amorphous compound of formula (I), the blood concentration and oral bioavailability in vivo are very low, and it has the pharmacokinetic property of directly acting on intestinal SGLT1.

[0200] Experimental Example 3: Study on the efficacy of the amorphous form of the compound of formula (I) in the mouse OGTT model

[0201] 1. Experimental Animals:

[0202]

[0203] 2. Experimental groups:

[0204] Table 16: Experimental group information

[0205]

[0206] 3. Experimental process:

[0207] 1) Animal acclimatization and preparation

[0208] After arriving at the facility, the experimental animals were kept in the animal room and acclimatized for 1 week.

[0209] 2) Drug administration

[0210] The animals were weighed, fasted for 6 hours, fasting blood sugar (0-min blood sugar) was measured, and grouped. The amorphous compound of formula (I) and the solvent were orally administered, followed by 2 g / kg and 5 mL / kg glucose solution. Blood sugar was tested by blood sampling from the tail tip of the animals at 15, 30, 60, 90, and 120 minutes after the administration of glucose. The glucose tolerance curve was drawn according to the time of the blood sugar data, and the area under the curve (AUC 1-120分钟 ).

[0211] 3) Data analysis:

[0212] All values ​​will be expressed as mean values. Statistical analysis was evaluated using one-way ANOVA Tukey's multiple comparison test in Graphpad Prism 6. A p value of less than 0.05 was considered statistically significant.

[0213] 4. Experimental results:

[0214] Table 17: Results of in vivo efficacy experiments on glucose tolerance in mice

[0215]

[0216] Note: * indicates p < 0.05 relative to the vehicle control group, ** indicates p < 0.01 relative to the vehicle control group, *** indicates p < 0.001 relative to the vehicle control group and **** indicates p < 0.0001 relative to the vehicle control group.

[0217] Conclusion: Compared with the vehicle control group, a single administration of the amorphous compound of formula (I) can reduce the blood glucose level of mice after oral administration of glucose in a dose-dependent manner.

Claims

1. The amorphous form of the compound of formula (I), whose X-ray powder diffraction (XRPD) pattern is shown in FIG1 .

2. The amorphous form according to claim 1, wherein a glass transition signal can be observed at 74.9±3°C in its differential scanning calorimetry curve.

3. The amorphous form according to claim 2, whose DSC spectrum is shown in Figure 2.

4. The amorphous material according to claim 1, wherein the weight loss of the amorphous material at 150.0±3°C is 2.56%.

5. The amorphous form according to claim 4, whose TGA spectrum is shown in Figure 3.

6. A method for preparing an amorphous form of a compound of formula (I), comprising an anti-solvent addition method: 1) adding the compound of formula (I) into a solvent to form a clear solution; 2) adding an antisolvent to the solution; in, The solvent is ethanol, acetone, tetrahydrofuran, acetonitrile, n-propyl acetate, anisole or 2-methyltetrahydrofuran; The antisolvent was H2O or n-heptane.

7. A method for preparing an amorphous form of a compound of formula (I), comprising a slow cooling method: 1) at 50° C., preparing a clear solution of the compound of formula (I) in a solvent; 2) The solution was cooled from 50°C to 5°C at a rate of 0.1°C / min; in, The solvents were tert-butyl alcohol, isopropyl acetate, acetonitrile:water (v / v, 1:3) or 2-methyltetrahydrofuran:cyclohexane (v / v, 1:3).

8. A method for preparing an amorphous form of a compound of formula (I), comprising a suspension stirring method: 1) adding the compound of formula (I) into a solvent to form a suspension; 2) The suspension solution is stirred by magnetic force to transform the crystals; in, The solvent is methyl tert-butyl ether, ethyl acetate:n-heptane (v / v, 1:3), 2-methyltetrahydrofuran:cyclohexane (v / v, 1:3), methyl tert-butyl ether:n-heptane (v / v, 1:7), tetrahydrofuran:n-heptane (v / v, 1:7), ethyl acetate:n-heptane (v / v, 1:7), 2-butanone:n-heptane (v / v, 1:7), acetonitrile:water (v / v, 1:9), acetone:water (v / v, 1:12), n-heptane, water, tetrahydrofuran:n-pentane (v / v, 1:7), N-methylpyrrolidone:water (v / v, 1:5), dimethyl sulfoxide:water (v / v, 1:5) or m-xylene.

9. A method for preparing an amorphous form of a compound of formula (I), comprising a slow volatilization method: 1) dissolving the compound of formula (I) in a solvent to form a clear solution; 2) Seal the clarified solution with a sealing film, pierce a small hole, and then slowly evaporate and crystallize; in, The solvent is ethyl acetate, chloroform, methanol, acetone, acetonitrile or tetrahydrofuran.

10. A method for preparing an amorphous form of a compound of formula (I), comprising a gas-solid permeation method: 1) placing the compound of formula (I) in a vial; 2) Place the vial open in a water atmosphere, seal it and let it stand at room temperature.

11. A method for preparing an amorphous form of a compound of formula (I), comprising a temperature cycle method: 1) dissolving the compound of formula (I) in a solvent to form a suspension at 50°C; 2) The suspension is heated and cooled at a rate of 0.1°C / min according to a program of 50°C-5°C-50°C-5°C to perform crystallization; in, The solvent was n-heptane, methyl tert-butyl ether, 2-methyltetrahydrofuran:n-heptane (v / v, 1:3) or N-methylpyrrolidone:water (v / v, 1:5).

Images