WO2022063305A1 - Crystal form of glucoside compounds and use thereof - Google Patents

Abstract

A crystal form (I) of glucoside compounds and the use thereof, and the use thereof in the preparation of a drug for treating related diseases.

Description

Crystal forms of glucosides and their applications

This application claims the following priority

CN202011032215.3, application date: September 27, 2020.

technical field

The invention relates to a crystal form of a glucoside compound and its application, as well as its application in the preparation of medicines for treating related diseases.

Background technique

Obesity, diabetes and related metabolic disorders have become important risk factors threatening human health.

Sodium-glucose cotransporters (SGLTs) are a family of glucose transporters found in the small intestinal mucosa and proximal renal tubules. The members mainly include SGLT1 and SGLT2. Transmembrane transport of glucose in the kidney. Specifically, SGLT1 is mainly distributed in the intestinal mucosal cells of the small intestine, and is also expressed in a small amount in the myocardium and kidney. Its function is mainly to regulate the intestinal absorption process of glucose. SGLT2 is expressed at high levels in the kidney and is mainly responsible for the regulation of the renal glucose reuptake process, that is, the glucose in the urine can be actively attached to the renal tubular epithelial cells during glomerular filtration and transported into the cells by the SGLT2 protein for reuse. Because the glucose transport process mediated by SGLTs does not intervene in the metabolism of sugar, thus avoiding the occurrence of adverse reactions of hypoglycemia and reducing the risk of cardiovascular diseases, 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 successively. They specifically inhibit the renal reabsorption of glucose by inhibiting SGLT2 activity, thereby increasing the excretion of glucose in the urine and normalizing blood glucose in diabetic patients. Since 2012, a number of SGLT2 inhibitors have been approved for marketing and become effective drugs for the treatment of diabetes.

In addition to inhibiting SGLT2, studies in recent years have found that appropriate inhibition of SGLT1 can prevent intestinal uptake of glucose without causing significant diarrhea or other gastrointestinal reactions. At the same time, inhibiting SGLT1 can reduce the intestinal absorption of glucose into the blood, thereby increasing the glucose concentration in the distal part of the intestine, resulting in an increase in the postprandial GLP-1 and PYY levels, thus exerting a good hypoglycemic effect and reducing the occurrence of urinary tract. Risk of infection and kidney damage, etc. In addition, by controlling the intestinal absorption of glucose, it can also reduce the total energy intake in food, and superimpose the effect of GLP-1 to reduce body weight, which can achieve the purpose 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.

In conclusion, SGLT1 inhibitors have good development prospects as novel diabetes and obesity treatment drugs. But 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, which only acts on the gastrointestinal tract, is undergoing Phase I clinical research for diabetes treatment (WO2014081660).

SUMMARY OF THE INVENTION

The present invention provides an amorphous form of the compound of formula (I), the X-ray powder diffraction (XRPD) pattern of which is shown in FIG. 1 .

In some aspects of the invention, a glass transition signal is observed at 74.9±3°C for the above-described amorphous differential scanning calorimetry curve.

In some embodiments of the present invention, the DSC spectrum of the above-mentioned amorphous is shown in FIG. 2 .

In some aspects of the present invention, the above-mentioned amorphous thermogravimetric analysis curve has a weight loss of 2.56% at 150.0±3°C.

In some aspects of the present invention, the above-mentioned amorphous TGA spectrum is shown in FIG. 3 .

The present invention also provides an amorphous preparation method of the compound of formula (I), which includes an anti-solvent addition method, a slow cooling method, a suspension stirring method, a slow volatilization method, a gas-solid infiltration method and a temperature cycle method.

In some aspects of the present invention, the above-mentioned preparation method, wherein, the anti-solvent addition method comprises:

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

2) adding an anti-solvent to the solution;

in,

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

The anti-solvent is _H2O or n-heptane.

In some schemes of the present invention, the above-mentioned preparation method, wherein, the slow cooling method comprises:

1) at 50 ° C, the compound of formula (I) is prepared into a clear solution in a solvent;

2) The solution is cooled from 50°C to 5°C at 0.1°C/min;

in,

Solvents were tert-butanol, isopropyl acetate, acetonitrile:water (v/v, 1:3) or 2-methyltetrahydrofuran:cyclohexane (v/v, 1:3).

In some aspects of the present invention, the above-mentioned preparation method, wherein, the suspension stirring method comprises:

1) adding the compound of formula (I) to a solvent to form a suspension;

2) The suspension solution is crystallized with magnetic stirring;

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-butane Ketone: 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.

In some aspects of the present invention, the above-mentioned preparation method, wherein, the slow volatilization method comprises:

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

2) Slowly volatilize and crystallize the clear solution after sealing the small holes with parafilm;

in,

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

In some aspects of the present invention, the above-mentioned preparation method, wherein, the gas-solid permeation method comprises:

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

2) The above-mentioned vial was opened and placed in an atmosphere of water, sealed and left to stand at room temperature.

In some aspects of the present invention, the above-mentioned preparation method, wherein, the temperature cycling method comprises:

1) at 50°C, the compound of formula (I) is dissolved in a solvent to form a suspension;

2) The temperature of the suspension is raised and lowered at a rate of 0.1°C/min according to the procedure of 50°C-5°C-50°C-5°C to transfer crystals;

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).

Definition and Explanation

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 indeterminate or unclear without a specific definition, but should be understood in its ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding commercial product or its active ingredient.

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, the embodiments formed by their combination with other chemical synthesis methods, and those skilled in the art. Well-known equivalents, preferred embodiments include, but are not limited to, the examples of the present invention.

The chemical reactions of specific embodiments of the present invention are carried out in suitable solvents suitable for the chemical changes of the present invention and their required reagents and materials. 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 synthetic steps or reaction schemes on the basis of the existing embodiments.

The structure of the compound 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 compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, single crystal X-ray diffraction method (SXRD), the cultured single crystal is collected by Bruker D8 venture diffractometer, the light source is CuKα radiation, and the scanning mode is:

After scanning and collecting relevant data, the crystal structure was further analyzed by the direct method (Shelxs97), and the absolute configuration could be confirmed.

The present invention will be specifically described below through examples, which do not imply any limitation to the present invention.

All solvents used in the present invention are commercially available and used without further purification.

The solvent used in the present invention is commercially available. The following abbreviations are used in the present invention: DCM stands for dichloromethane; DMF stands for N,N-dimethylformamide; DMSO stands for dimethyl sulfoxide; EtOH stands for ethanol; MeOH stands for methanol; TFA stands for trifluoroacetic acid; ATP stands for Adenosine triphosphate; HEPES for 4-hydroxyethylpiperazineethanesulfonic acid; _MgCl2 for magnesium dichloride; T3P for ₁ -propylphosphoric anhydride.

technical effect

The crystal form of the compound of the present invention has good stability and is easy to be formulated into medicine; the amorphous form of the compound of formula (I) has a significant inhibitory effect on the activities of human SGLT1 and SGLT2 transporters; The concentration and oral bioavailability are very low, and it has the pharmacokinetic properties of directly acting on the intestinal SGLT1; after a single administration of the compound of formula (I) amorphous, it can reduce the blood glucose level of mice after oral glucose in a dose-dependent manner .

X-ray powder diffraction (X-ray powder diffractometer, XRPD) method of the present invention

Instrument model: X-ray diffractometer from PANalytical

Test Method: Approximately 10 mg of sample was used for XRPD detection.

The detailed XRPD parameters are shown in Table 1:

Table 1

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

Instrument model: TA Discovery DSC 2500 Differential Scanning Calorimeter

Test method: Take a sample (about 1-5mg) and place it in an mDSC aluminum pan for testing. Under the condition of 50mL/min _N2 , heat the sample from 0°C to 250°C at a heating rate of 3°C/min.

The detailed DSC parameters are shown in Table 2:

Table 2

parameter	set value
method	modulated warming
sample tray	Aluminium pan, gland
temperature range	0℃-250℃
Scan rate (℃/min)	3
Protective gas	nitrogen

.

Thermogravimetric Analysis (Thermal Gravimetric Analyzer, TGA) method of the present invention

Instrument Model: TA Q5000/Discovery TGA 5500 Thermogravimetric Analyzer

Test method: Take a sample (about 1-5mg) and place it in a TGA aluminum pan for testing. Under the condition of 10mL/min _N2 , at a heating rate of 10°C/min, heat the sample from room temperature to 350°C.

The detailed TGA parameters are shown in Table 3:

table 3

parameter	set value
method	modulated warming
sample tray	Aluminium pan, gland
temperature range	0℃-250℃
Scan rate (℃/min)	3
Protective gas	nitrogen

.

Description of drawings

Fig. 1 is the XRPD spectrum of the amorphous Cu-Kα radiation of the compound of formula (I);

Fig. 2 is the amorphous DSC spectrogram of the compound of formula (I);

Figure 3 is an amorphous TGA spectrum of the compound of formula (I).

detailed description

In order to better understand the content of the present invention, further description will be given below in conjunction with specific embodiments, but the specific embodiments do not limit the content of the present invention.

Example 1: Preparation of compounds of formula (I)

Step 1: Synthesis of Compound C

2-Methyltetrahydrofuran (12L) was added to the reaction kettle, and compound A (1200.00g, 5.45mol, 1.0eq), compound B (1650.00g, 8.18mol, 1.5eq) and triphenylphosphine (2070.00g) were slowly added in batches , 7.91 mol, 1.45 equiv), start stirring. At 0-5°C, diisopropyl azodicarboxylate (1430.00g, 7.09mol, 1.30 equiv, 1.38L) was slowly added dropwise to the reaction kettle, and the dropwise addition was completed, and the reaction kettle was heated to an internal temperature of 10～ 20°C, stirring for 16 hours. After the completion of the reaction, magnesium chloride (4.14 kg) was slowly added in portions and stirred for 2 hours. Then, 2-methyltetrahydrofuran (12 L) was slowly added to the reaction kettle, and the mixture was stirred and diluted for 0.5 hour. Filtration, collecting the filtrate, adding water (12 L) to the filtrate, stirring for 5 minutes and then standing to separate the layers, separating the organic phase and the aqueous phase, and extracting the aqueous phase with ethyl acetate (6 L). The organic phases were combined and concentrated to dryness under reduced pressure to give crude product. Ethanol (12 L) was added to the crude product, and the inner temperature of the reaction kettle was controlled to be 15-25° C. and stirred for 16 hours. After suction filtration, the filter cake was washed with ethanol (1.2 L), the filter cake was collected, and dried 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] ⁺ .

Step 2: Synthesis of Compound D

Dichloromethane (14 L) was added to the reaction kettle, compound C (1400.00 g, 3.47 mol, 1.0 equiv) was slowly added in batches, and stirring was started. Trifluoroacetic acid (2.1 L, 28.36 mol, 8.17 equiv) was slowly added dropwise to the reaction kettle, the dropwise addition was completed, and the temperature in the reaction kettle was kept at 15-25° C. and stirred for 3 hours. After the completion of the reaction, the reaction solution was transferred to a rotary evaporator and concentrated to dryness under reduced pressure to remove dichloromethane and most of trifluoroacetic acid to obtain a crude product. The crude product was dissolved in dichloromethane (14 L), transferred to a separator, and washed with saturated sodium bicarbonate and water once each. The organic phase was separated and concentrated to dryness under reduced pressure to give a white solid. The white solid was transferred to a three-necked flask, ethyl acetate (2.8 L) was added, and the mixture was stirred for 12 hours. After suction filtration, the filter cake was washed once with ethyl acetate (1.4 L), and the filter cake was collected and dried 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] ⁺ .

Step 3: Synthesis of Compound F

Dichloromethane (8 L) was added to the reaction kettle, compound D (784.37 g, 2.59 mol, 1.0 equiv) and compound E (863.00 g, 2.59 mol, 1.0 equiv) were slowly added in batches, and stirring was started. Then slowly add triethylamine (800.89 g, 7.91 mol, 3.06 equiv, 1.10 L), control the inner temperature of the reaction kettle to be 10-20 °C, and stir for 15 minutes. Subsequently, T ₃ P (2.35 L, 3.96 mol, 1.53 equiv.) was slowly added dropwise to the reaction kettle, and the dropwise addition was completed. After the reaction, the reaction solution was transferred to a separator, and washed with water (8L*1), 0.1M hydrochloric acid (8L*1), 0.5M potassium carbonate (8L*2) and water (8L*2) in sequence. The organic phase was collected and concentrated to dryness under reduced pressure to give crude product. The crude product was dissolved in a mixed solvent of dioxane and n-heptane (1:5, 11.76L) and then transferred to the reaction kettle. The temperature in the reaction kettle was controlled to be 15-25°C, and the mixture was stirred for about 42 hours until the solid was precipitated. Suction filtration, and the filter cake was rinsed with a mixed solvent of dioxane and n-heptane (1:5, 1.56L). 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) 619.5 [M+H] ⁺ .

Step 4: Synthesis of Compound H

Dioxane (6L), water (1.5L), compound G (750.00 g, 1.49 mol, 1.0 equiv), compound F (1105.75 g, 1.19 mol, 1.2 equiv), Pd(PPh ₃ ) ₄ (86.07 g) , 0.08mol, 0.05 equiv) and potassium carbonate (411.81 g, 2.98 mol, 2.0 equiv) were sequentially added to the reaction kettle, the stirring was turned on, and nitrogen flow was introduced. The temperature in the reactor was raised to 45-55°C and stirred for 16 hours. After the completion of the reaction, the reaction solution was transferred to a rotary evaporator and concentrated to dryness under reduced pressure to obtain a crude product. Ethyl acetate (7.5 L) was added to the crude product to dissolve, transferred to a separator, water (7.5 L) was added, and the mixture was stirred for 5 minutes and then left to stand for separation. The organic phase was collected and the aqueous phase was extracted with ethyl acetate (7.5 L). The organic phases were combined and washed once with water (7.5 L). The organic phase was collected and concentrated to dryness under reduced pressure to give crude product. Methanol (15 L) was added to the crude product, heated to 55-65° C. to dissolve and clarify, then cooled to 15-25° C. and stirred for 16 hours. Filter and wash the filter cake once with methanol (15 L). The filter cake was collected, and the above operation was repeated once to obtain 1476.25 g of filter cake. The filter cake was dissolved in dichloromethane (7.4L), methanol (7.4L) and thiol silica gel (585.00g) were added in sequence, heated to 35-45°C, and stirred for 16 hours. After filtration, the filter residue was washed once with a mixed solvent of dichloromethane and methanol (1:1, 7.4 L). The filtrate was collected, and SiliaMets Thiol (585.00 g) was added, heated to 35-45°C, and stirred for 16 hours. After filtration, the filter residue was washed once with a mixed solvent of dichloromethane and methanol (1:1, 7.4 L). The filtrate was collected, and SiliaMets Thiol (585.00 g) was added, heated to 35-45°C, and stirred for 16 hours. After filtration, the filter residue was washed once with a mixed solvent of dichloromethane and methanol (1:1, 7.4L). The filtrate was transferred to a rotary evaporator, concentrated to dryness under reduced pressure, the product was collected, and dried 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] ⁺ .

Step 5: Synthesis of Compound J

HCl/EtOAc solution (4M, 10.5L) and compound H (700.00 g, 0.77 mol, 1.0 equiv) were added to the reaction kettle in turn, and stirring was started. Control the inner temperature of the reaction kettle to 15-25°C and stir for 2 hours. After the reaction was completed, ethyl acetate (7 L) was added to the reaction kettle to dilute the reaction solution, and the reaction solution was transferred to a rotary evaporator and concentrated to dryness under reduced pressure. Subsequently, ethyl acetate (7L*2) was added and concentrated to dryness under reduced pressure to obtain a crude product. Ethyl acetate (10.5 L) was added to the crude product, and the mixture was stirred at 15 to 25°C for 16 hours. Filter and wash the filter cake once with ethyl acetate (1.05 L). The filter cake was collected and dried in a vacuum oven for 16 hours to obtain compound J. The product was confirmed by LCMS, LC-MS (m/z) 815 [M+H] ⁺ .

Step 6: Synthesis of Compound of Formula (I)

Methanol (5.8 L) and compound J (580.00 g, 0.71 mol, 1.0 equiv) were sequentially added to the reaction kettle, and stirring was started. 25% NaOMe in methanol (290 mL) was then added and stirred at room temperature for 3 hours. After the completion of the reaction, the reaction solution was transferred to a rotary evaporator and concentrated to dryness under reduced pressure to obtain a crude product. Water (5.6 L) was added to the crude product, and the mixture was stirred at 10 to 20°C for 2 hours. Filter and wash the filter cake twice with water (1.12 L). The filter cake was collected, water (5.6 L) was added to the filter cake, and the mixture was stirred at 10 to 20° C. for 16 hours. Filter and wash the filter cake twice with water (1.12 L). The filter cake was collected and dried in a vacuum drying oven for 144 hours to obtain the compound of formula (I). ¹ H NMR (400 MHz, CD ₃ OD) δppm 1.09 (t, J=7.50 Hz, 3H), 1.73 (br d, J=17.26 Hz, 2H), 1.92 (br d, J=12.51 Hz, 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).

Example 2: Preparation of Amorphous Compound of Formula (I)

Table 4: Comparison table of Chinese and English names of solvents used in the test

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
IPAc	isopropyl acetate	DMSO	dimethyl sulfoxide
THF	tetrahydrofuran	H 2 O	water
2-MeTHF	2-Methyltetrahydrofuran	NMP	N-Methylpyrrolidone
Cyclohexane	Cyclohexane	m-Xylene	m-xylene
n-Pentane	n-pentane	CHCl 3	Chloroform
t-BuOH	tert-Butanol	EtOAc	Ethyl acetate
n-Propyl acetate	Propyl acetate

1. Anti-solvent addition method:

Weigh about 15-20 mg of the compound of formula (I), dissolve it in a positive solvent at 25° C. to prepare a clear solution, slowly add the corresponding anti-solvent dropwise to the above solution until a solid appears, and separate the obtained solid for XRPD testing. The results are shown in Table 5:

Table 5: Antisolvent Addition Test

*: Indicates that a clear solution is obtained after adding anti-solvent, turn to -20°C and stir until solid appears.

2. Slow cooling method:

Take about 20 mg of the compound of formula (I), dissolve it in a solvent at 50°C to prepare a clear solution, cool down at a rate of 0.1°C/min according to the procedure of 50°C-5°C, collect the precipitated solid and conduct XRPD test . The results are shown in Table 6:

Table 6: Slow cooling test

Test number	Solvent, v/v	test results
1	t-BuOH	Amorphous
2	IPAc	Amorphous
3	ACN/ H2O , 1:3	Amorphous
4 #	2-MeTHF/Cyclohexane, 1:3	Amorphous

#: Indicates that a clear solution is obtained after the temperature drops to 5°C, and the solution is placed at -20°C, and the solution is still clear, and the solid is volatilized and precipitated at room temperature.

3. Suspension stirring method:

Weigh about 15-20 mg of the compound of formula (I), prepare suspensions at 25°C or 50°C in different solvents, stir magnetically for about 6-8 days, collect the solid by centrifugation and conduct XRPD test. The results are shown in Table 7:

Table 7: Suspension Stirring Test

4. Slow volatilization method:

Weigh about 15-20 mg of the compound of formula (I), dissolve it in a solvent at 25°C to prepare a clear solution, seal 4-5 small holes with a parafilm, slowly volatilize and crystallize, collect the obtained solid and conduct XRPD test . The results are shown in Table 8:

Table 8: Slow Evaporation Test

Test number	Solvent, v/v	test results
1	EtOAc	Amorphous
2	CHCl 3	Amorphous
3	MeOH	Amorphous
4	Acetone	Amorphous
5	ACN	Amorphous
6	THF	Amorphous

5. Gas-solid infiltration method:

About 20 mg of the compound of formula (I) was weighed into a 3 ml vial, another 20 ml vial was taken and the corresponding solvent was added to it, the 3 ml vial was opened and placed in a 20 ml vial, sealed and kept at room temperature. The solids were collected and tested by XRPD. The results are shown in Table 9:

Table 9: Gas-Solid Penetration Test

Sample serial number	solvent	test results
1	H 2 O	Amorphous
2^	DCM	Amorphous
3^	EtOH	Amorphous

4^	Toluene	Amorphous
5^	THF	Amorphous

^: indicates that a clear solution was obtained, which was evaporated at room temperature to obtain a solid.

6. Temperature cycle method:

Weigh about 20 mg of the compound of formula (I), add it to the solvent at 50°C to prepare a suspension, and ramp up and down the temperature at a rate of 0.1°C/min according to the program of 50°C-5°C-50°C-5°C , the resulting solid was collected and tested by XRPD. The results are shown in Table 10:

Table 10: Temperature Cycling Test

Test number	Solvent, v/v	test results
1	n-Heptane	Amorphous
2	MTBE	Amorphous
3	2-MeTHF/n-Heptane, 1:3	Amorphous
4	NMP/ H2O , 1:5	Amorphous

Example 3: Stability test of the compound of formula (I) amorphous

Purpose:

The influence factors (high temperature, high humidity and light), long-term conditions (25°C/60%RH) and accelerated conditions (40°C/75%RH) stability of the amorphous samples of the compound of formula (I) were investigated, and the samples were evaluated solid stability.

experimental method:

Influencing factor experiment: the samples under high temperature (60°C) were placed in an open disposable petri dish, and the samples under high humidity (25°C/75%RH) were placed in an open flat weighing bottle, and then placed in different The blast drying oven and desiccator were investigated. The illuminated samples are placed in a clean disposable petri dish, spread into a thin layer, and covered with a quartz glass cover. The packaging method of the shading control sample is the same as that of the illuminated sample, but the outside of the disposable petri dish is covered with aluminum film. According to the Chinese Pharmacopoeia and ICH The requirements of Q1B were placed under illumination conditions (visible light 5000±500 lux; ultraviolet light 90 μw/cm ² ) for investigation;

Long-term and accelerated condition experiments: The samples were put into double-layer low-density polyethylene bags, each layer of low-density polyethylene bags was sealed with a zipper, and then put into aluminum foil bags for heat sealing, and then put into long-term conditions (25℃/ 60%RH) and accelerated conditions (40°C/75%RH). The experimental results are shown in Table 11:

Table 11: Amorphous solid stability test results of compounds of formula (I)

NA: No crystal form detected.

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

Example 4: Amorphous solubility test of the compound of formula (I)

Purpose:

Under the condition of 37℃, the amorphous compound of formula (I) was suspended and stirred in different pH media (pH=1.0, 2.0, 3.8, 4.5, 5.5, 6.0, 6.8, 7.4), biological solvent (SGF, FaSSIF, FeSSIF) and water Solubility after 24 hours.

experimental method:

1. Prepare different pH media:

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

b) Weigh about 2.722g of potassium dihydrogen phosphate, add it into a 100mL volumetric flask, add an appropriate amount of ultrapure water and dissolve by ultrasonic, then use ultrapure water to make up the volume, and mix to obtain a 0.2mol/L potassium dihydrogen phosphate solution;

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

d) Measure 5mL of 1.0mol/L hydrochloric acid solution, add it to a 50mL volumetric flask, add ultrapure water to volume, and mix to measure pH=0.99;

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

f) Weigh 32.9 mg of sodium acetate, add it to a 50 mL volumetric flask, add 1.13 mL of 2mol/L acetic acid solution, add an appropriate amount of ultrapure water and dissolve it by ultrasonic, then use ultrapure water to dilute to volume, mix well to measure pH=3.81 ;

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

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

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

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

k) Take 12.5mL of 0.2mol/L potassium dihydrogen phosphate solution, add it to a 50mL volumetric flask, add 9.775mL of 0.2mol/L sodium hydroxide solution, add ultrapure water to volume, mix well to measure pH=7.42.

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;

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

4. After 24 hours of equilibration, the supernatant was separated for pH and solubility testing.

The experimental results are shown in Table 12:

Table 12: Summary of 37°C solubility of compound of formula (I) amorphous in different media

Experiment number	medium	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	2.5489	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

#: y=10336110.7x+5294.3, R2=0.9999, LOQ=0.12 μg/mL, LOD=0.04 μg/mL; SGF: simulated gastric fluid; FaSSIF: simulated intestinal fluid in fasting state; FeSSIF: simulated intestinal fluid in fed state.

Conclusion: The solubility of the compound of formula (I) amorphous 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; in biological solvents (SGF, FaSSIF) , FeSSIF) in the solubility of 0.06-9.4mg/mL; the solubility in water is 0.03mg/mL.

Example 5: Mechanical stability test of the amorphous compound of formula (I)

experimental method:

1) Test XRPD after grinding the amorphous sample of the compound of formula (I) manually for about 5 minutes without adding water or solvent;

2) Add the amorphous sample of the compound of formula (I) into a circular mold (diameter 6mm), pressurize until the pressure on the sample reaches about 350MPa, and directly test the XRPD of the sample after tableting.

Experimental results:

After grinding and tableting, the crystal form of the sample did not change and was still amorphous.

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

Experimental example 1. In vitro cell activity test

1. Experimental purpose

By measuring the amount of [ ¹⁴ C]-labeled Methylα-D-glucopyranosid (methyl α-D-glucopyranoside) entering the cells of transporters with high expression of human SGLT1 and SGLT2 (Human-SGLT1 and Human-SGLT2) , to evaluate the inhibitory effect of the compound of formula (I) amorphous on the activity of human SGLT1 and SGLT2 transporters.

2. Experimental method

1) The experiment was set up with double holes, and the experiment was repeated 3 times;

2) Reagent and cell preparation;

a) Preparation of experimental buffer, the final concentration of each component is shown in Table 13:

Table 13 Buffer composition

reagent	Concentration (mM)
HEPES	10
MgCl 2	1.2
KCl	4.7
CaCl 2	2.2
NaCl	120

b) Compound preparation: The amorphous stock solution of the compound of formula (I) was at a concentration of 10 mM, diluted with DMSO.

c) Compound dilution was done by Bravo automatic pipetting workstation, and 10 concentrations were decremented 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, 0.1829, 0.0610, 0.0203 nM .

d) Isotope preparation: Dilute the isotope stock solution [ ¹⁴ C]Methylα-D-glucopyranosid with assay buffer to a working concentration of 6 μM.

e) Cell preparation: Human SGLT1 or human SGLT2 cells were seeded in a 96-well cell plate at 7×104 cells/well, and cultured overnight in a 37°C, 5% CO ₂ cell incubator.

3) Experimental process

a) Remove the 96-well plate from the incubator, shake off the medium and rinse the cells with 150 μL/well of experimental buffer, then shake off the buffer. Add 49 μL/well of new buffer.

b) Add 1 μL of serially diluted compound solutions according to the 96-well plate layout. Add 1 μL DMSO to high signal wells (HC, DMSO final concentration 1%); add 1 μL LX2761 to low signal wells (LC, LX2761 final concentration 0.4 μM).

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

d) Incubate at 37°C for 2 hours.

e) Take out the 96-well plate and aspirate the reaction system. Wash three times with pre-cooled experimental buffer, add 50 μL/well of 10% NaOH, and place on a shaker for lysing for 5 minutes.

f) Transfer the cell lysate in the 96-well plate to scintillation vials, add 2 mL of scintillation fluid to each tube, and read with a Tri-Carb liquid scintillation counter.

g) Analysis of experimental data, % inhibition rate=(mean value of high signal wells-signal value of sample wells)/(mean value of high signal wells-mean value of low signal wells)*100. Curve fitting using GraphPad Prism 5: Log(inhibitor) vs. response—Variable slope yields _IC50 .

3. The experimental results are shown in Table 14:

Table 14: In Vitro Cell Viability Test Results

compound	Human-SGLT1 IC50 (nM)	Human-SGLT2IC 50 (nM)
Amorphous compound of formula (I)	1.8±0.4	1.6±1.0

Conclusion: The amorphous compound of formula (I) has a significant inhibitory effect on the activities of human SGLT1 and SGLT2 transporters.

Experimental example 2. DMPK study in rats

1. Experimental purpose:

Male SD rats were used as test animals, and the amorphous plasma concentration of the compound of formula (I) was determined after single administration and the pharmacokinetic behavior was evaluated.

2. Experimental operation:

Six healthy adult male SD rats were selected, 3 were in the intravenous group and 3 were in the oral group. The vehicle in the intravenous injection group is 5% (w/v) hydroxypropyl β-cyclodextrin (HP-β-CD) aqueous solution. After the amorphous compound of formula (I) is mixed with an appropriate amount of the vehicle in the intravenous injection group, 0.25 mg/mL is prepared. Clear solution; Oral group solvent is 0.5% (w/v) methylcellulose/0.02% Tween 80 aqueous solution, the compound of formula (I) amorphous is mixed with an appropriate amount of oral group solvent to prepare 0.5mg/mL Homogeneous suspension. After intravenous administration of 0.5 mg/kg or oral administration of 5 mg/kg in 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 Phoenix WinNonlin software (Pharsight, USA) was used. Calculate pharmacokinetic parameters.

3. The experimental results are shown in Table 15:

Table 15: Compound PK test results

Note: _Cmax is the maximum concentration; F% is oral bioavailability; AUC _po is oral exposure; Vd _ss is volume of distribution; Cl is clearance; T _1/2 is half-life.

Conclusion: After oral administration of the compound of formula (I) in amorphous form, the plasma concentration and oral bioavailability in vivo are very low, and it has the pharmacokinetic properties of directly acting on intestinal SGLT1.

Experimental Example 3. Pharmacodynamic study of amorphous compound of formula (I) in mouse OGTT model

1. Experimental animals:

2. Experimental grouping:

Table 16: Experiment Grouping Information

3. Experimental process:

1) Animal adaptation and preparation

The experimental animals were housed in the animal room to acclimate to the environment for 1 week after arriving at the facility.

2) Administration

After the animals were weighed, fasted for 6 hours, measured fasting blood sugar (0min blood sugar) and grouped into groups, the compound of formula (I) amorphous and vehicle were orally administered respectively, followed by oral administration of 2 g/kg, 5 mL/kg glucose solution. At 15, 30, 60, 90, and 120 minutes after glucose administration, the animals were collected blood from the tail tip for blood glucose detection, and the glucose tolerance curve was drawn according to the time, and the area under the curve (AUC _{1-120 minutes} ) was calculated.

3) Data analysis:

All values will be expressed as averages. Statistical analysis was assessed using Graphpad Prism 6 one-way ANOVA with Tukey's multiple comparison test. A p-value less than 0.05 was considered statistically significant.

4. Experimental results:

Table 17: In vivo efficacy test results of glucose tolerance in mice

Note: * means p<0.05 relative to the vehicle control group, ** means p<0.01 relative to the vehicle control group, *** means p<0.001 relative to the vehicle control group and **** means p<0.001 relative to the vehicle control group 0.0001.

Conclusion: Compared with the vehicle control group, the amorphous compound of formula (I) can dose-dependently reduce the blood glucose level of mice after oral administration of glucose.

Claims (12)

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

   
2. The amorphous according to claim 1, the differential scanning calorimetry curve of which can observe a glass transition signal at 74.9±3°C.
3. The amorphous according to claim 2, its DSC spectrum is shown in Figure 2.
4. The amorphous according to claim 1, its thermogravimetric analysis curve is 2.56% weight loss at 150.0±3°C.
5. The amorphous according to claim 4, its TGA spectrum is shown in Figure 3.
6. The amorphous preparation method of the compound of formula (I) includes anti-solvent addition method, slow cooling method, suspension stirring method, slow volatilization method, gas-solid permeation method and temperature circulation method.
7. The preparation method according to claim 6, wherein the anti-solvent addition method comprises:

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

   2) adding an anti-solvent to the solution;

   in,

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

   The anti-solvent is _H2O or n-heptane.
8. preparation method according to claim 6, wherein, the slow cooling method comprises:

   1) at 50 ° C, the compound of formula (I) is prepared into a clear solution in a solvent;

   2) The solution is cooled from 50°C to 5°C at 0.1°C/min;

   in,

   Solvents were tert-butanol, isopropyl acetate, acetonitrile:water (v/v, 1:3) or 2-methyltetrahydrofuran:cyclohexane (v/v, 1:3).
9. The preparation method according to claim 6, wherein the suspension stirring method comprises:

   1) adding the compound of formula (I) to a solvent to form a suspension;

   2) The suspension solution is crystallized with magnetic stirring;

   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-butane Ketone: 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.
10. The preparation method according to claim 6, wherein the slow volatilization method comprises:

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

    2) Slowly volatilize and crystallize the clear solution after sealing the small holes with parafilm;

    in,

    The solvent is ethyl acetate, chloroform, methanol, acetone, acetonitrile or tetrahydrofuran.
11. The preparation method according to claim 6, wherein the gas-solid permeation method comprises:

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

    2) The above-mentioned vial was opened and placed in an atmosphere of water, sealed and left to stand at room temperature.
12. The preparation method according to claim 6, wherein the temperature cycling method comprises:

    1) at 50°C, the compound of formula (I) is dissolved in a solvent to form a suspension;

    2) The temperature of the suspension is raised and lowered at a rate of 0.1°C/min according to the procedure of 50°C-5°C-50°C-5°C to transfer crystals;

    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).

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