CN115368352B - Glucokinase agonist and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a glucokinase agonist, a preparation method and application thereof. The invention is prepared by using two leucine derivatives and four different organic matters as initial raw materials through a corresponding method, and the obtained compound has high purity. Through a series of experiments, the species compound obtained by the invention can regulate the activity of glucokinase, thereby promoting insulin secretion and regulating glycogen metabolism, and finally achieving the purpose of reducing blood sugar.

Description

A kind of glucokinase agonist and its preparation method and application

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to a glucokinase agonist and its preparation method and application.

Background technique

In recent years, diabetes in China has shown a rapid growth trend, and the incidence rate of diabetes has increased from 0.6% in 1980 to 12.8% at present. According to the latest data released by the National Health and Medical Commission, the number of diabetics in my country has reached 114 million, of which type 2 diabetes accounts for nearly 90% of the diabetic population. Diabetic complications involve multiple organs such as blood vessels, eyes, kidneys, and feet. Severe complications cause disability and high mortality, seriously affect the health of patients, and bring heavy burdens to individuals, families, and society. Diabetes is a chronic metabolic disease. Although there are many ways to treat diabetes, in the long-term blood sugar control of patients, two to three drugs are often used in combination to fully control blood sugar. However, the rate of blood sugar control in diabetic patients is still high. Not high, drugs targeting new targets or new treatments need to be developed.

Glucokinase (glucokinase, GK) is one of the hexokinase isoenzymes and a key rate-limiting enzyme in the process of glucose metabolism. It is mainly distributed in pancreatic islets and liver cells. GK can convert glucose entering cells into 6 Glucose phosphate is a sensor of glucose in the body. When blood sugar rises after a meal and exceeds the threshold of GK, GK will be activated to start a chain reaction of lowering blood sugar in the body, including promoting insulin secretion in the pancreas and improving liver glucose metabolism in the liver. GK loss-of-function mutation is one of the pathogenic causes of monogenic diabetes, also known as Maturity Onset Diabetes of the Youth Type 2 (MODY2). The reduction of GK activity is also one of the pathogenic causes of type 2 diabetes. Some studies have found that activating GK through exogenous drugs can promote insulin secretion and enhance the ability of the liver to clear glucose in the blood, thereby exerting a hypoglycemic effect, so GK is activated. as a potential treatment strategy for type 2 diabetes. GK agonists can improve glucose-stimulated insulin secretion in pancreatic islets, and in the liver, GK agonists can promote hepatic glycogen synthesis, thereby reducing blood sugar and maintaining blood sugar homeostasis.

Based on the above, it is expected that an effective new application of GKA in the treatment of diabetes will bring new therapeutic means to alleviate the occurrence and development of diabetes.

Contents of the invention

In view of the above problems, the present invention provides a glucokinase agonist and its preparation method and application. The preparation method of the glucokinase agonist of the invention is simple, the yield is high, and the obtained product can effectively lower blood sugar.

The purpose of the present invention and the solution to its technical problems are achieved by adopting the following technical solutions.

One aspect of the present invention provides a glucokinase agonist, having a structural formula such as formula I:

I

Wherein, R1 is selected from any one of benzothiazolyl, 5-methylpyridyl, N-methylpyrazolyl, and 6-fluorobenzothiazolyl; R2 is hydrogen, wherein the structural formulas of R1 are for , /> , /> , .

The object of the present invention and the solution to its technical problems are also achieved by adopting the following technical solutions.

Another aspect of the present invention provides a kind of method for preparing glucokinase agonist, the method comprises the following steps:

S1: Dissolve leucine derivatives and organic matter in dichloromethane, then add 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride Mix and stir with N,N-diisopropylethylamine, then add dichloromethane to dilute the reaction solution, add water to extract to obtain the first organic phase and the first aqueous phase, and add the first aqueous phase to extract with ethyl acetate to obtain the second Two organic phases and a second aqueous phase, the first organic phase and the second organic phase are dried, filtered, concentrated, and purified by column chromatography to obtain the first product;

S2: Dissolve the above-mentioned first product in dichloromethane at 0°C, then add trifluoroacetic acid to stir the reaction, after the end, add ethyl acetate to the solution, first extract with water, then adjust the pH of the aqueous phase to > 8, and then use ethyl acetate Ester extraction, the ethyl acetate phase is dried, filtered and concentrated to obtain the second product;

S3: Dissolve the above-mentioned second product and phthalic anhydride in acetic acid and stir, remove the reaction solution by distillation under reduced pressure, extract with ethyl acetate and saturated sodium bicarbonate solution, filter, evaporate and concentrate the organic phase for column Purification by chromatography afforded the final product.

Preferably, the leucine derivative in step S1 is Boc-D-leucine or N-tert-butoxycarbonyl-leucine monohydrate.

Preferably, the organic matter in step S1 is selected from 2-aminobenzothiazole, 2-amino-5-picoline, N-methyl-3-aminopyrazole, 2-amino-6-fluorobenzothiazole of any kind.

Preferably, the molar ratio of the leucine derivative to the organic matter in step S1 is 1:1, and the 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethane The molar ratio of carbodiimide hydrochloride to N,N-diisopropylethylamine is 1:3:6.

Preferably, the stirring conditions in step S1 are: the temperature is room temperature, and the time is 2-6 hours.

Preferably, the stirring conditions in step S2 are: the temperature is room temperature, and the time is 2-24 hours.

Preferably, the molar ratio of the second product to phthalic anhydride in step S3 is 1:1.

Preferably, the stirring conditions in step S3 are: the temperature is 120° C., and the time is 4 hours.

The object of the present invention and the solution to its technical problems are also achieved by adopting the following technical solutions.

Another aspect of the present invention provides the application of glucokinase agonist, and described glucokinase agonist is above-mentioned glucokinase agonist or the glucokinase agonist obtained according to above-mentioned preparation method, and described application comprises in the preparation of diabetes mellitus The application in the treatment of drugs, the application in the abnormal glucose metabolism diseases related to the impairment of glucokinase, or the application in the field of improving glucose metabolism related diseases by activating glucokinase.

By virtue of the above technical solutions, the present invention has at least the following advantages:

1. The method adopted in the present invention is to use the virtual high-throughput drug screening technology to calculate the binding ability between the molecules in the compound library and the target GK allosteric regulatory site by means of molecular simulation, screen out possible effective candidate compounds, and chemically synthesize them Afterwards, the candidate compounds were tested for GK enzyme kinetics to verify their effects on GK activity. After repeatedly modifying and synthesizing candidate compounds and detecting GK enzyme kinetics, find out the compound that stimulates GK activity, and then confirm the effect of the compound on dynamic insulin secretion by islet perfusion experiment, and then in the wild type and high-fat high-glucose Diet-induced obese and diabetic mice in vivo confirmed that these compounds that can activate GK have the effect of lowering blood sugar.

2. The preparation method of the present invention is easy to operate, and the yield of the obtained compound is high.

3. The present invention has obtained five compounds of different structures by chemical synthesis, which all use B leucine derivatives as raw materials, and obtain five compounds with different structures by reacting with different organic substances. It can be seen from experiments that the compounds of the present invention The five compounds can reduce blood sugar by increasing GK enzyme activity, promoting glucose-stimulated insulin secretion, and regulating liver glycogen metabolism.

4. The compound of the present invention is characterized in improving glucose-stimulated insulin secretion. In a certain concentration range, the main feature is to increase the maximum value of insulin secretion while not changing the stimulation threshold of glucose. The promoting effect is dependent on glucose concentration, and when the glucose concentration is low, the compound of the present invention has no promoting effect on insulin secretion, thus avoiding the problem of hypoglycemia that may be caused during application.

The above description is only an overview of the technical solutions of the present invention. In order to understand the technical means of the present invention more clearly and implement them according to the contents of the description, the preferred embodiments of the present invention will be described in detail below.

Description of drawings

Fig. 1 is wild-type GK expression plasmid;

Fig. 2 is the R447W mutant GK expression plasmid;

3 is a graph showing the influence of the product AR-GK-01 obtained according to Example 1 of the present invention on the activity of human GK enzyme;

Figure 4 is a graph showing the influence of the product AR-GK-05 obtained according to Example 2 of the present invention on the activity of human GK enzyme;

Figure 5 is a graph showing the influence of the product AR-GK-08 obtained in Example 3 of the present invention on the activity of human GK enzyme;

Fig. 6 is a graph showing the influence of the product AR-GK-10 obtained according to Example 4 of the present invention on the activity of human GK enzyme;

Figure 7 is a graph showing the influence of the product AR-GK-25 obtained in Example 5 of the present invention on the activity of human GK enzyme;

Figure 8 is a graph showing the influence of glucose concentration on GK enzyme activity by adding 1 μM, 2 μM, and 10 μM AR-GK-01 respectively;

Figure 9 is a graph showing the influence of glucose concentration on GK enzyme activity by adding 0.5 μM, 1 μM, 2 μM, and 5 μM AR-GK-05 respectively;

Figure 10 is a graph showing the influence of glucose concentration on GK enzyme activity by adding 5 µM AR-GK-08;

Figure 11 is a graph showing the influence of glucose concentration on the enzyme activity of GK mutation (R447W) in monogenic diabetes compared with wild-type GK;

Figure 12 is a graph showing the effect of glucose concentration on the enzyme activity of GK mutation (R447W) in wild-type and monogenic diabetes by adding 10 µM AR-GK-01;

Figure 13 is a graph showing the effect of glucose concentration on GK mutation (R447W) enzyme activity in monogenic diabetes mellitus by adding 10 µM AR-GK-01 compared with the blank control;

Figure 14 is a graph showing the effect of 0.1 µM and 0.5 µM AR-GK-01 on glucose-stimulated insulin secretion (GSIS);

Figure 15 is a graph showing the effect of 2 µM and 10 µM AR-GK-01 on glucose-stimulated insulin secretion (GSIS);

Figure 16 is a comparison of the effects of 0.5 µM AR-GK-01 and the reference glucokinase agonist MK-0941 on glucose-stimulated insulin secretion (GSIS);

Figure 17 is a comparison of the effect of dose escalation of AR-GK-01 and the reference glucokinase agonist MK-0941 on 2.8 mM glucose-stimulated insulin secretion (GSIS);

Figure 18 is a comparison of the effect of dose escalation of AR-GK-01 and the reference glucokinase agonist MK-0941 on 3.5 mM glucose-stimulated insulin secretion (GSIS);

Fig. 19 is a graph showing the influence of AR-GK-01 on the blood glucose of wild-type mice over time;

Figure 20 is a graph showing the effect of AR-GK-01 on blood glucose changes over time in obese or diabetic mice induced by a high-fat diet;

Figure 21 is a glycogen standard curve;

Figure 22 shows the effect of AR-GK-01 on liver glycogen in wild-type mice and SUR1 knockout mice at a dose of 20 mg/kg body weight;

Fig. 23 is a graph showing the change of plasma AR-GK-01 concentration in mice after intragastric administration of AR-GK-01 (20 mg/kg body weight).

Detailed ways

In order to make the technical means, creative features, goals and effects achieved by the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments It is only some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

According to the glucokinase agonist of the present invention, it has a structural formula such as formula I:

. Wherein, R1 is selected from any one of benzothiazolyl, 5-methylpyridyl, N-methylpyrazolyl, and 6-fluorobenzothiazolyl; R2 is hydrogen; wherein, the structural formulas of R1 are for /> , /> , /> , .

The method for preparing glucokinase agonist according to the present invention, comprises the following steps:

S1: Dissolve leucine derivatives and organic matter in dichloromethane, then add 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride Mix and stir with N,N-diisopropylethylamine, then add dichloromethane to dilute the reaction solution, add water to extract to obtain the first organic phase and the first aqueous phase, and add the first aqueous phase to extract with ethyl acetate to obtain the second Two organic phases and the second aqueous phase, the first organic phase and the second organic phase are dried, filtered, concentrated and purified by column chromatography to obtain the first product; the leucine derivative is Boc-D-leucine or N-tert-butoxycarbonyl-leucine monohydrate; organic matter selected from 2-aminobenzothiazole, 2-amino-5-picoline, N-methyl-3-aminopyrazole, 2-amino-6 -any one of fluorobenzothiazoles; the molar ratio of leucine derivatives to said organic matter is 1:1, said 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)- The molar ratio of 3-ethylcarbodiimide hydrochloride to N,N-diisopropylethylamine is 1:3:6; the stirring conditions are: the temperature is room temperature, and the time is 2-6h;

S2: Dissolve the above-mentioned first product in dichloromethane at 0°C, then add trifluoroacetic acid to stir the reaction, after the end, add ethyl acetate to the solution, first extract with water, then adjust the pH of the aqueous phase to > 8, and then use ethyl acetate Ester extraction, the ethyl acetate phase is dried, filtered, and concentrated to obtain the second product; the stirring conditions are: the temperature is room temperature, and the time is 2-24h;

S3: Dissolve the above-mentioned second product and phthalic anhydride in acetic acid and stir, remove the reaction solution by distillation under reduced pressure, extract with ethyl acetate and saturated sodium bicarbonate solution, filter, evaporate and concentrate the organic phase for column Chromatographic purification was carried out to obtain the final product; the molar ratio of the second product to phthalic anhydride was 1:1; the stirring conditions were: temperature 120°C, time 4h.

The general synthetic route that obtains according to above-mentioned description is:

Example 1 (R)-N-(benzo[d]thiazol-2-yl)-2-(1,3-dioxoisoindoline-2-yl)-4-methylpentanamide (AR- GK-01) Preparation

The preparation route is as follows:

Concrete preparation steps are as follows:

Dissolve Boc-D-leucine (500 mg, 2 mmol) and 2-aminobenzothiazole (300 mg, 2 mmol) in 4 mL of dichloromethane, then add 1-hydroxybenzotriazole (HOBT) (135 mg, 1 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (575 mg, 3 mmol) and N,N-diisopropyl Ethylamine (DIPEA) (1.04 mL, 6 mmol). After the reaction mixture was stirred at room temperature for 4 h, the reaction solution was diluted with 50 ml of dichloromethane and extracted with 50 ml of water. Then 50 mL of ethyl acetate was added to the aqueous phase for extraction. The two organic phases were dried over Na ₂ SO ₄ , filtered, concentrated, and purified by silica gel column chromatography with ethyl acetate:n-hexane=1:8 to obtain 20D-26 as a white powder (504 mg, yield 69%) .

At 0°C, 2 mL of trifluoroacetic acid (TFA ) was slowly added dropwise to a solution of compound 20D-26 (486 mg, 1.34 mmol) in dichloromethane, and the reaction solution was stirred at room temperature for 12 h. After the reaction, add ethyl acetate (50 mL) to the solution, and extract with water (extract 4 times, 50 mL each time, aqueous solution pH<4). Then adjust the pH value of the aqueous phase to make pH>8, and then Extraction was carried out with ethyl acetate (3 times, 20 mL each). The ethyl acetate phase was dried over _Na2SO4 , filtered and concentrated to give 20D-26-T as _a white powder.

20D-26-T (150 mg, 0.57 mmol) and phthalic anhydride (84 mg, 0.57 mmol) were dissolved in CH ₃ COOH (4 mL), stirred at 120°C for 4 h, and the reaction was removed by distillation under reduced pressure. solution, extracted with ethyl acetate and saturated NaHCO ₃ solution. _The organic phase was dried over _Na2SO4 . After filtration, evaporation and concentration, purification by silica gel column chromatography with ethyl acetate:n-hexane=1:5 gave (R)-N-(benzo[d]thiazol-2-yl)-2-(1, 3-Dioxoisoindolin-2-yl)-4-methylpentanamide (AR-GK-01) was a white powder (74 mg, 33% yield).

ESI-MS m/z: 393.1 [M+H] ⁺ ; C ₂₁ H ₁₉ N ₃ O ₃ S; ¹ H NMR(MeOD, 400 MHz) δ 1.00 (dd,6H, J =6.6, 14.5 Hz), 1.50 -1.58 (m,1H), 1.99-2.08 (m, 1H), 2.39-2.49 (m, 1H),5.18 (dd, 1H, J =4.6, 14.7 Hz), 7.29 (t, 1H, J =7.9 Hz ), 7.41 (t, 1H, J =7.9Hz), 7.69 (d, 1H, J =8.1 Hz), 7.83-7.87 (m, 3H), 7.89-7.92 (m, 2H); ¹³ C NMR(MeOD, 100 MHz) δ 171.01 (1C), 169.42 (2C), 160.23 (1C), 149.37 (1C), 135.73(2C), 133.16 (2C), 132.00 (1C), 127.27 (1C), 125.03 (1C), 124. 45 (2C), 122.37(1C), 121.57(1C), 53.54(1C), 38.33(1C), 26.45(1C), 23.58(1C), 21.36(1C).

Example 2 (S)-N-(benzo[d]thiazol-2-yl)-2-(1,3-dioxoisoindoline-2-yl)-4-methylpentanamide (AR- GK-05) Preparation

The preparation route is as follows:

Concrete preparation steps are:

Dissolve N-tert-butoxycarbonyl-leucine monohydrate (5000 mg, 20 mmol) and 2-aminobenzothiazole (3000 mg, 20 mmol) in 4 mL of dichloromethane, then add 1-hydroxybenzene Triazole (HOBT) (1350 mg, 10 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (5750 mg, 30 mmol) and N, N-Diisopropylethylamine (DIPEA) (10.4 mL, 60 mmol). After the reaction mixture was stirred at room temperature for 4 h, the reaction solution was diluted with 50 ml of dichloromethane and extracted with 50 ml of water. Then 50 mL of ethyl acetate was added to the aqueous phase for extraction. The two organic phases were dried over _Na2SO4 , filtered, concentrated, and purified by silica gel column chromatography with n- _hexane : ethyl acetate = 1:8 to obtain 20L-26 as a white powder (6 g, yield 97% ). Subsequent reaction was the same as that of compound AR-GK-01 to obtain compound 20L-26-T as a white powder (5 g, yield 97%), and then reacted to obtain compound (S)-N-(benzo[d]thiazole-2- yl)-2-(1,3-dioxoisoindolin-2-yl)-4-methylpentanamide (AR-GK-05) was a white powder (225 mg, yield 57%).

ESI-MS m/z: 393.1 [M+H] ⁺ ; C ₂₁ H ₁₉ N ₃ O ₃ S; 1H NMR(MeOD, 400 MHz) δ 0.99(dd, 6H, J =6.6, 14.5 Hz), 1.49- 1.59 (m,1H), 2.01-2.08 (m, 1H), 2.38-2.46 (m,1H), 5.18 (dd, 1H, J =4.6, 11.5 Hz), 7.29 (t, 1H, J =7.9 Hz) , 7.41 (t, 1H, J =7.9 Hz), 7.69 (d, 1H, J =8.0 Hz), 7.83-7.87 (m, 3H), 7.89-7.92 (m, 2H); ¹³ C NMR(MeOD, 100 ( 2C), 122.37(1C), 121.58(1C), 53.54(1C), 38.33(1C), 26.45(1C), 23.58(1C), 21.36(1C).

Example 3 (R)-2-(1,3-dioxaindoline-2-yl)-4-methyl-N-(5-methylpyridin-2-yl)pentanamide (AR-GK -08) Preparation

The preparation route is as follows:

Concrete preparation steps are as follows:

Boc-D-leucine (3000 mg, 12 mmol) and 2-amino-5-picoline (1302 mg, 12 mmol) were dissolved in 4 mL of dichloromethane, and then 1-hydroxybenzotriazole ( HOBT) (810 mg, 6 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (3450 mg, 18 mmol) and N,N-diiso Propylethylamine (DIPEA) (2.08 mL, 12 mmol). After the reaction mixture was stirred at room temperature for 4 h, the reaction solution was diluted with 50 ml of dichloromethane and extracted with 50 ml of water. Then 50 mL of ethyl acetate was added to the aqueous phase for extraction. The two organic phases were dried over Na ₂ SO ₄ , filtered, concentrated, and purified by silica gel column chromatography with ethyl acetate:n-hexane=1:8 to obtain 20D-17 as a white powder (923 mg, yield 4% ).

At 0°C, 4 mL of trifluoroacetic acid (TFA ) was slowly added dropwise to a solution of compound 20D-17 (923 mg, 2.87 mmol) in dichloromethane, and the reaction solution was stirred at room temperature for 12 h. After the reaction, ethyl acetate (50 mL) was added to the solution, and extracted with water (4 times of extraction, 50 mL each time, pH of the aqueous solution was <4). Then adjust the pH value of the aqueous phase to make the pH>8, and then extract with ethyl acetate (3 times, 20 mL each). The ethyl acetate phase was dried over Na ₂ SO ₄ , filtered, and concentrated to give 20D-17-T as a white powder (630 mg, 99% yield).

20D-17-T (221 mg, 1 mmol) and phthalic anhydride (148 mg, 1 mmol) were dissolved in CH ₃ COOH (4 mL), stirred at 120°C for 4 h, and the reaction was removed by distillation under reduced pressure solution, extracted with ethyl acetate and saturated NaHCO ₃ solution. _The organic phase was dried over _Na2SO4 . After filtration, evaporation and concentration, purification by silica gel column chromatography with ethyl acetate:n-hexane=1:5 gave (R)-2-(1,3-dioxaindolin-2-yl)-4 -Methyl-N-(5-methylpyridin-2-yl)pentanamide (AR-GK-08) was a white powder (274 mg, 78% yield).

ESI-MS m/z:351.16 [M+H]+; C ₂₀ H ₂₁ N ₃ O ₃ ; 1H NMR(MeOD, 400 MHz) δ 0.96 (t,6H, J=7.0 Hz), 1.45-1.56 (m , 1H), 1.94-2.02 (m, 1H), 2.27 (s, 3H), 2.42-2.50(m, 1H), 5.10 (dd, 1H, J=4.2, 11.4 Hz), 7.59 (d, 1H, J =8.0 Hz), 7.82-7.90 (m,5H), 8.08 (s, 1H); 13C NMR(MeOD, 100 MHz) δ 170.20 (1C), 169.60 (2C), 150.41(1C), 148.73 (1C), 140.15 (2C), 135.68 (2C), 133.14 (1C), 131.15 (1C), 124.40(2C), 115.73 (1C), 54.26 (1C), 38.33 (1C), 26.59 (1C), 23.60 (1C), 21.32(1C), 17.71(1C).

Example 4 (R)-2-(1,3-dioxoindoline-2-yl)-4-methyl-N-(1-methyl-1H-pyrazol-3-yl)pentanamide (AR-GK-10) Preparation

The preparation route is as follows:

Concrete preparation steps are as follows:

Dissolve Boc-D-leucine (3000 mg, 12 mmol) and N-methyl-3-aminopyrazole (1.05 mL, 12 mmol) in 4 mL of dichloromethane, then add 1-hydroxybenzotriazole (HOBT) (810 mg, 6 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (3450 mg, 18 mmol) and N,N-di Isopropylethylamine (DIPEA) (2.08 mL, 12 mmol). After the reaction mixture was stirred at room temperature for 4 h, the reaction solution was diluted with 50 ml of dichloromethane and extracted with 50 ml of water. Then 50 mL of ethyl acetate was added to the aqueous phase for extraction. The two organic phases were dried on Na ₂ SO ₄ , filtered, concentrated, and purified by silica gel column chromatography with ethyl acetate:n-hexane=1:3 to obtain 20D-25 as a white powder (3.27 g, yield 75% ).

At 0°C, 4 mL of trifluoroacetic acid (TFA ) was slowly added dropwise to a solution of compound 20D-25 (3.27 g, 10.31 mmol) in dichloromethane, and the reaction solution was stirred at room temperature for 12 h. After the reaction, ethyl acetate (50 mL) was added to the solution, and extracted with water (4 times of extraction, 50 mL each time, pH of the aqueous solution was <4). Then adjust the pH value of the aqueous phase to make the pH>8, and then extract with ethyl acetate (3 times, 20 mL each). The ethyl acetate phase was dried over Na ₂ SO ₄ , filtered, and concentrated to give 20D-25-T as a white powder (2.16 g, 97% yield).

20D-25-T (300 mg, 1.43 mmol) and phthalic anhydride (211 mg, 1.42 mmol) were dissolved in CH ₃ COOH (4 mL), stirred at 120°C for 4 h, and the reaction was removed by distillation under reduced pressure. solution, extracted with ethyl acetate and saturated NaHCO ₃ solution. _The organic phase was dried over _Na2SO4 . After filtration, evaporation and concentration, silica gel column chromatography was performed with ethyl acetate:n-hexane=1:1 to obtain (R)-2-(1,3-dioxoindoline-2-yl)-4 -Methyl-N-(1-methyl-1H-pyrazol-3-yl)pentanamide (AR-GK-10) was a white powder (382 mg, 79% yield).

ESI-MS m/z:340.15 [M+H]+; C18H20N4O3; 1H NMR(MeOD, 400 MHz) δ 0.96(t, 6H, J=7.4 Hz), 1.45-1.53 (m, 1H), 1.91-1.99 (m, 1H), 2.42-2.49 (m, 1H),3.76 (s, 3H), 5.04 (dd, 1H, J=4.6, 11.7 Hz), 6.43 (d, 1H, J=2.2 Hz), 7.42 ( ( 1C), 132.44 (2C), 124.34 (2C), 98.99 (1C), 53.84 (1C), 38.77 (1C), 38.39 (1C), 26.54 (1C), 23.61 (1C), 21.29 (1C).

Example 5 (R)-2-(1,3-dioxoindolin-2-yl)-N-(6-fluorobenzo[d]thiazol-2-yl)-4-methylpentanamide (AR-GK-25) Preparation

The preparation route is as follows:

Concrete preparation steps are as follows:

Dissolve Boc-D-leucine (5000 mg, 20 mmol) and 2-amino-6-fluorobenzothiazole (3360 mg, 20 mmol) in 4 mL of dichloromethane, then add 1-hydroxybenzotriazole (HOBT) (1350 mg, 10 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (5750 mg, 30 mmol) and N,N-di Isopropylethylamine (DIPEA) (10.4 mL, 60 mmol). After the reaction mixture was stirred at room temperature for 4 h, the reaction solution was diluted with 50 ml of dichloromethane and extracted with 50 ml of water. Then 50 mL of ethyl acetate was added to the aqueous phase for extraction. The two organic phases were dried over _Na2SO4 , filtered, concentrated, and purified by silica gel column chromatography with ethyl acetate:n _- hexane=1:8 to obtain 20D-27 as a white powder (3.3 g, yield 43% ).

At 0°C, 4 mL of trifluoroacetic acid (TFA ) was slowly added dropwise to compound 20D-27 (3.3 g, 8.65 mmol) in dichloromethane, and the reaction solution was stirred at room temperature for 12 h. After the reaction, in the solution Ethyl acetate (50 mL) was added to it, and extracted with water (4 extractions, 50 mL each time, pH of the aqueous solution <4). Then adjust the pH value of the aqueous phase to make the pH>8, and then extract with ethyl acetate (3 times, 20 mL each). The ethyl acetate phase was dried over Na ₂ SO ₄ , filtered, and concentrated to give 20D-27-T as a white powder (2 g, 82% yield).

20D-27-T (250 mg, 0.89 mmol) and phthalic anhydride (131 mg, 0.89 mmol) were dissolved in CH ₃ COOH (4 mL), stirred at 120°C for 4 h, and the reaction was removed by distillation under reduced pressure solution, extracted with ethyl acetate and saturated NaHCO ₃ solution. _The organic phase was dried over _Na2SO4 . After filtration, evaporation and concentration, use ethyl acetate:n-hexane=1:7 for silica gel column chromatography purification to obtain (R)-2-(1,3-dioxaindolin-2-yl)-N -(6-Fluorobenzo[d]thiazol-2-yl)-4-methylpentanamide (AR-GK-25) was a white powder (157 mg, 43% yield).

ESI-MS m/z: 411.11 [M+H] ⁺ ; C ₂₁ H ₁₈ FN ₃ O ₃ S; 1H NMR(DMSO, 400 MHz) δ 0.90(dd, 6H, J =6.6, 20.5 Hz), 1.38- 1.51 (m, 1H), 2.00-2.13 (m, 2H), 5.10 (dd, 1H, J =4.9, 10.4 Hz), 7.27 (td, 1H, J =2.6, 9.0 Hz), 7.69 (dd, 1H, ( 1C), 157.49 (1C), 134.63 (2C), 131.71 (2C), 123.29 (2C), 114.40 (1C), 114.16 (1C), 108.33 (1C), 108.06 (1C), 50.96 (1C), 36.95 ( 1C), 24.44 (1C), 23.21 (1C), 21.02 (1C).

Example 6 GK Enzyme Kinetic Detection

1. Expression and purification of GK protein

1.1. Expression of GK protein

(1) Construction of prokaryotic expression system:

The target gene was synthesized according to standard procedures, using the E. coli expression vector plasmid pGEX-3X:

a. Insert the wild-type GK protein gene at the restriction site BamHI / EcoRI to construct the recombinant expression vector pGEX-3X-GK-GST, and select for ampicillin (Amp) resistance gene, with GST tagged protein. See Figure 1 for the plasmid map and the insertion position of the GK gene.

b. GK mutant type (R447W) plasmid construction: Wild-type GK prokaryotic expression plasmid pGEX-3X-GK-GST was subjected to point mutation according to the standard procedure of point mutation, and GK mutant type R447W plasmid pGEX-3X-R447W-GST was constructed, with restriction restriction site Point BamHI / EcoRI , ampicillin (Amp) resistance gene, with GST tag protein. See Figure 2 for the plasmid map and the insertion position of the mutant GK.

(2) Transformation into expression bacteria

a. Add 2 microliters of pGEX-3X-GCK plasmid to 50 microliters of BL21 (DE 3) competent medium on the ultra-clean workbench, mix in ice bath for 30 minutes.

b. Heat shock: put the mixture into a water bath, heat shock in a water bath at 42°C for 90 seconds, then take it out quickly.

c. After ice bathing for 3 minutes, add 900 microliters of antibiotic-free liquid LB medium to the mixture on a clean bench and place it on a shaker at 37°C at 220 rpm for 45 minutes.

d. Centrifuge at 2000 rpm for 2 min.

e. Discard most of the supernatant on the ultra-clean workbench, keep about 50 microliters of culture medium to resuspend the bacteria, suck out the solid LB culture plate with ampicillin (Amp) antibiotic (1 mg/mL), and roll the ball to coat Spread the bacterial solution evenly, and place it upside down in a 37°C incubator for overnight cultivation.

(3) Induced expression

a. Operate in an ultra-clean workbench, pick a single colony from the culture plate, add 5 mL of liquid LB medium containing Amp antibiotics (1 mg/mL), culture overnight at 37°C on a shaker at 220 rpm.

b. Operate in an ultra-clean workbench, transfer the overnight cultured bacterial solution to an Erlenmeyer flask containing 200mL liquid LB medium, add Amp antibiotics, and cultivate on a shaker at 37°C and 220 rpm.

c. After culturing for about 3-4 hours, draw 1 mL of the bacterial solution to detect the OD600 value, until the OD600 is between 0.6-0.8, add the inducer IPTG (100 mM) into the Erlenmeyer flask at a ratio of 1:1000 to induce expression , 37°C, 220 rpm for about 12 hours.

d. Collect the bacterial liquid: 4°C, 12000 rpm, centrifuge for 20 min, discard the supernatant. Wash once with PBS, centrifuge at 44°C, 12000rmp for 20 min, and discard the supernatant.

1.2. Purification of GK protein

The recombinant protein GK carries a GST tag, and the protein is purified using Beyond GST Tag Protein Purification Kit.

a. According to the wet weight of each gram of bacterial pellet, add 4 ml (2-5 ml is acceptable) of lysis buffer to fully resuspend the bacteria.

b. Add lysozyme to a final concentration of 1 mg/ml, mix well, and place in an ice-water bath or on ice for 30 min.

Note: Lysozyme can be prepared into 100 mg/ml mother solution with lysate, and added before temporary use. After the lysozyme is prepared into the mother solution, it can be properly divided and stored at -20ºC.

c. Sonicate the bacteria on ice. The ultrasonic power was 200-300W, each ultrasonic treatment was 10 s, and the interval was 10 s, and a total of 6 ultrasonic treatments were performed.

d. Centrifuge at 10,000g at 4ºC for 20-30 minutes, collect the supernatant of the bacterial lysate and place it in an ice-water bath or on ice. You can take 20 microliters of the supernatant for subsequent detection.

e. Take 1 ml of BeyoGold™ GST-tag Purification Resin that is well mixed, centrifuge at 4ºC (1000g×10 s), discard the storage solution, add 0.5 ml of lysis buffer to the gel to resuspend and equilibrate the gel, and centrifuge at 4ºC ( 1000g×10 s) Discard the liquid, repeat the balance 1~2 times, and discard the liquid. Add about 4 ml of bacterial lysate supernatant to it, and shake slowly at 4°C for 60 min on a side-swing shaker or a horizontal shaker.

f. Put the mixture of lysate and BeyoGold™ GST-tag Purification Resin into the empty affinity chromatography column tube (3ml) provided in this kit. Note: You can also take 1 ml of 50% BeyoGold™ GST-tag Purification Resin, which is mixed evenly, and pack it into the column, then equilibrate it with 0.5 ml lysis buffer for 2~3 times, then add about 4 ml of the bacterial lysate supernatant, and then you can put the flow-through After the solution was collected, repeat loading to the column 3-5 times to fully bind the target protein.

g. Open the cover at the bottom of the purification column, let the liquid in the column flow out under the action of gravity, and collect about 20 microliters of flow-through liquid for subsequent analysis.

h. Wash the column 5 times, add 0.5-1 ml of lysis buffer each time, and collect about 20 microliters of liquid passing through the column each time for subsequent analysis and detection. In the process of washing the column and the next step of elution, the Bradford method can be used to simply and quickly detect the protein content in each washing solution and eluent, so as to consider increasing or decreasing the number of washing and elution.

i. Elute the target protein 6-10 times, each time with 0.5 ml of elution buffer. Collect each eluate into separate centrifuge tubes. The collected eluate is the purified GST-tagged protein sample.

Note: The composition of the elution buffer is 50 mM Tris, 150 mM NaCl, 10 mM GSH, pH 8.0.

1.3. GK enzyme kinetic experiment

(1) Solution configuration and protein concentration determination:

a. Prepare the working reagents at the following concentrations, namely MgCl ₂ : 6 mM; BSA: 0.1%; KCl: 150 mM; HEPES: 100 mM; NADP ⁺ : 1 mM; G-6-PDH: 5 unit/ml; DTT : 2 mM; ATP: 5 mM;

b. Prepare 10 ml premix, namely 1 ml MgCl ₂ (60 mM) + 1 ml BSA (1%) + 1.5 ml KCl (1 mM) + 1 ml HEPES (1 M) + 1 ml NADP ⁺ (10 mM) + 10 µl G-6-P-GDH (5 unit/ml) + 0.2 ml ATP (250 mM) + 20 µl DTT (1 M) + 4.27 ml H ₂ O, pH 7.4. Heat the master mix in a 37°C water bath for 5 minutes for GK enzyme dilution.

c. Determine the protein concentration of the sample according to the instructions of the protein concentration determination kit (Beiyuntian, P0007).

d. Determining the effect of compounds on GK enzyme activity.

e. Concentration of WT-GST-GK: explore according to the concentration of the pre-experiment, and dilute with the master mix.

f. The concentration of the compound to be tested: dilute the compound to 1 mM, and then add it to the 96-well plate for doubling dilution with the premix;

g. Reaction system (100 microliters):

(2) Glucose dose curve:

10 μl of test compound (fixed concentration, adjusted according to test requirements) + 40 μl master mix + 40 μl glucose (different concentrations) + 10 μl WT-GST-GK (diluted by master mix)

(3) Compound effectiveness testing:

40 μl glucose (working concentration 5 mM) + 40 μl master mix + 10 μl test compound (different concentration) + 10 μl WT-GST-GK (diluted by master mix);

(4) Detection: After low-speed centrifugation and mixing, incubate in a 37°C incubator for 30 minutes, put it into a microplate reader for detection, and set the detection wavelength at 340 nm to read every 30 seconds at room temperature for 25 minutes.

(5) Analyzing data: After exporting the data to an Excel table, subtract the initial value from the value of the last cycle to obtain the corresponding values of different concentrations of drugs and input them into Graphpad Prism 8 software for analysis, using Allostericsigmoidal or log(agonist) vs. The regression line fitting of the response equation was used to estimate and analyze the enzyme kinetic parameters.

1.4. Experimental results

(1) The agonistic effect of five compounds on GK

A total of 5 GK agonist compounds, including AR-GK-01, AR-GK-05, AR-GK-08, AR-GK-10, and AR-GK-25, were detected for the kinetics of GK enzymes. In the case of a fixed glucose concentration (5 mM), the concentration-dose curves of different compounds were tested, and the results are shown in Figures 3-7. From the results in Figure 3-7, it can be seen that AR-GK-01, AR-GK-05, AR-GK-08, AR-GK-10, and AR-GK-25 have agonistic effects on GK enzymes.

Due to the different agonistic effects of each compound on GK, single or different concentrations of the three compounds AR-GK-01, AR-GK-05 and AR-GK-08 were selected for glucose dose curve detection, that is, the concentration of the compound was fixed, and the detection Changes in enzyme kinetics under different concentrations of glucose to further determine the effect of the compound, the results are shown in Figures 8-10. It was found that the substrate (glucose) concentration S _0.5 when the enzymatic reaction reached half of the maximum speed when the concentration of compound AR-GK-01 was 1 µM, 2 µM, and 10 µM was 3.155 mM, 2.633 mM, and 1.355 mM, respectively. Compound AR- When the concentration of GK-05 was 0.5 µM, 1 µM, 2 µM and 5 µM, the substrate (glucose) concentration S _0.5 when the enzymatic reaction reached half of the maximum speed was 7.733 mM, 6.698 mM, 5.534 mM, 3.364 mM, respectively, compound AR - The substrate (glucose) concentration S _0.5 at which the enzymatic reaction reached half the maximum speed at a GK-08 concentration of 5 µM was 3.163 mM, respectively. This shows that AR-GK-01, AR-GK-05 and AR-GK-08 all have a significant stimulating effect on GK activity, and the effect is dependent on the concentration gradient, and the stimulating effect of AR-GK-01 is stronger, as The best compound screened in this experiment.

(2) The agonistic effect of AR-GK-01 on GK with MODY2 pathogenic mutation (R447W):

In order to verify whether the GK agonist AR-GK-01 also has an agonistic effect on the mutant GK that causes monogenic diabetes, we chose the R447W mutant GK for expression and protein purification, and studied the enzyme kinetics of the mutant GK.

As shown in Figure 11, compared with the normal wild-type GK, the glucose S _0.5 of the R447W mutant GK was significantly increased, from 8.99 mM of the normal GK in the paired test detected at the same time to 17.04 mM, suggesting that the R447W mutation caused GK enzyme activity to be impaired .

As shown in Figure 12, 10 µM AR-GK-01 corrected the impaired GK activity, and the S _0.5 of R447W mutant glucose decreased from 17.04 mM glucose to 5.317 mM, while the wild-type GK decreased at 10 µM AR-GK- Under the stimulation of 01, the S _0.5 of glucose decreased from 8.99 mM glucose to 3.898 mM, indicating that AR-GK-01 can not only stimulate the activity of normal wild-type GK, but also stimulate the mutant GK with impaired enzyme function.

As shown in Figure 13, 10 µM AR-GK-01 not only decreased the S _0.5 of glucose in R447W mutant GK, but also increased the Vmax of R447W mutant GK, and improved the h index, indicating that AR-GK-01 can Repairing GK with impaired function due to mutation can be used to treat monogenic diabetes caused by GK loss-of-function mutation.

Example 7 Effect of GK agonist on glucose-stimulated insulin secretion

(1) Islet isolation

a. After the mouse was anesthetized, it was fixed on the operating board, the abdominal cavity was opened, and the entrance of the bile duct into the duodenum was blocked with a hemostat, and then a 2 mg/ml collagenase solution was injected into the bile duct with a 5 ml syringe and a 31.5-gauge needle. Stop when the pancreas is fully inflated.

b. Peel off the pancreas, clean up fat and non-pancreatic tissues, transfer the pancreas to a 50 ml centrifuge tube, pour 3 ml of 2 mg/ml collagenase solution, and shake in a 37°C water bath for 4 to 5 minutes.

c. Immediately after shaking, add Hanks buffer to 50 ml in a 50 ml centrifuge tube, the centrifuge tube rotates at 2000 rpm, remove the supernatant after centrifugation, add 5 ml Histopaque-1119 solution to the remaining tissue pellet, and shake to mix.

d. Slowly add 5 ml of Histopaque-1077 solution, then slowly add 5 ml of Hanks buffer solution, and the rotation speed of the centrifuge tube is 2000 rpm.

e. Remove the islet tissue from the layered liquid, pick and purify islets under a dissecting microscope, wash and culture.

(2) Islet culture.

a. Preparation of RPMI1640 culture medium: glucose 10 mM, glutamine 2 mM, fetal bovine serum 10%, sodium bicarbonate 2g/L, penicillin 100 units/ml, streptomycin 10 µg/ml, adjust the pH to 7.2.

b. Use RPMI1640 culture medium to culture the isolated and purified islets in an incubator at 37°C, 5% CO ₂ and 95% air humidity for 1-2 days.

(3) Islet perfusion experiment

a. Buffer preparation: 115 mM NaCl, 24 mM NaHCO ₃ , 5 mM KCl, 1 mM MgCl ₂ and 2.5 mM CaCl ₂ , adjust the pH to 7.4, and then add 0.25% bovine serum albumin.

b. Prepare stimulation solutions of 25 mM glucose, 2.8 mM glucose, and 3.5 mM glucose.

c. Manually pick up 120 islets of the same size, place them in the islet chamber, and place them in a water bath according to the experimental requirements.

d. Place the prepared reaction solution in a water bath (37°C) and insert the corresponding injection tube. Table 1 below shows the perfusion procedure:

Table 1 Perfusion program

Note: A is buffer solution, B is stimulation solution, C is 30 mM potassium chloride, curves 1 to 6 are ramp perfusion, A and B are gradually mixed, A gradually decreases from 100% to 0, and B gradually increases from 0 up to 100%.

e. Collect the secreted insulin by Collector Fraction Waters in a 96-deep-well plate, set 1 mL/min to collect the reaction solution, and store it in a -20°C refrigerator until hormone determination.

(4) Detection of insulin secretion value.

a. Take 10 μL/well of the solution collected in the 96-well plate in the islet perfusion experiment, and transfer it into a 384-well plate;

b. Add the antibody according to the instructions of the HTRF insulin assay kit, shake and mix well, and incubate at room temperature for 2 hours, read with the HTRF program of BMG’s Clariostar microplate reader, calculate the hormone secretion value according to the standard curve, and confirm the stimulation by measuring insulin secretion effects of drugs or drugs on insulin secretion.

(5) Experimental results.

The islet perfusion test stimulated by glucose concentration ramp (the speed of concentration increase is 0.5 mM/min) proved that the curve characteristic of glucose-stimulated insulin secretion (GSIS) is that the threshold value of glucose concentration is about 7 mM, and Vmax is maintained at 10 ng/120 The level of islets/min. As shown in Figure 14, after adding 0.1 µM and 0.5 µM AR-GK-01 at the same time as the GSIS test, 0.1 µM was consistent with the blank control and had no effect on promoting GSIS. When the concentration of AR-GK-01 was raised to 0.5 µM, the Vmax of GSIS increased to a level of 15 ng/120 islets/min, which was about 50% higher, while the threshold value did not change. When the concentration of AR-GK-01 was further increased to 2 µM (Fig. 15), Vmax increased to 19 ng/120 islets/min without changing the GSIS threshold, from 0.5 µM AR-GK- 01 increased by about 40%. But when the concentration of AR-GK-01 increased to 10 µM, the threshold of GSIS decreased from 7 mM to 4 mM, Vmax did not increase further, and returned to the level of 0.5 µM AR-GK-01 (Figure 15), so , in the concentration range of AR-GK-01 from 0.1 µM to 2 µM, the maximum insulin secretion increased with the concentration of AR-GK-01, but the threshold of GSIS did not change, indicating that AR-GK-01 promoted GSIS in This concentration range is glucose dependent. Figure 16 shows the significant difference between AR-GK-01 and another glucokinase agonist MK-0941, the same 0.5 µM, AR-GK-01 is mainly characterized by increasing Vmax without changing the glucose threshold of GSIS, while MK- 0941 significantly lowers the threshold of GSIS glucose and also lowers Vmax.

In order to further verify that the promotion of GSIS by AR-GK-01 is dependent on glucose concentration, two experiments on the promotion effect of AR-GK-01 on insulin secretion under low glucose concentrations were selected, and they were also compared with MK-0941. Figure 17 and Figure 18 When the glucose solution concentration is maintained at 2.8 mM or 3.5 mM during the perfusion process, this glucose concentration cannot stimulate insulin secretion, and the baseline of the insulin secretion curve remains at a low level. On this basis, the compound concentration climbing test is performed , the rate of concentration increase is 0.125 µM/min. Figure 17 shows that in the case of 2.8 mM background glucose, AR-GK-01, even at the maximum level of 5 µM, does not have the effect of stimulating insulin secretion at low concentrations of glucose (2.8 mM), while MK-0941 significantly increases Insulin secretion, the stimulation threshold is 0.5 µM, and the stimulation effect reaches the maximum at 1.75 µM. Figure 18 shows the situation when the background concentration of glucose is 3.5 mM. Similarly, even at the maximum level of 5 µM, AR-GK-01 does not have the effect of stimulating insulin secretion at a low concentration of glucose (3.5 mM), only at 0.875 There was a slight increase in insulin secretion between ~1.25 µM, but it was not statistically different from baseline insulin secretion. While MK-0941 significantly increased insulin secretion, the threshold and maximum stimulation concentration was consistent with the background glucose concentration of 2.8 mM. It shows that GK, which acts as a blood glucose sensor, does not stimulate insulin secretion when glucose is at a low level, and AR-GK-01 has no further promotion effect on insulin secretion, which again shows that AR-GK-01 is glucose-dependent. The possibility of hypoglycemia caused by the drug itself is avoided.

Embodiment 8 live animal test

1. Oral glucose tolerance test (OGTT) in normal diet mice

1.1. Solvent configuration:

(1) Configuration of AR-GK-01 solution:

Dissolve 6 mg AR-GK-01 in 30 μL DMSO, then dissolve in 60 μL castor oil, and finally dilute to 3 mL with 0.5% sodium carboxymethylcellulose (CMC), with a final concentration of 2 mg/mL.

(2) CMC-DMSO-castor oil mixed solution (solvent control):

Add 60 μL castor oil to 30 μL DMSO, and then dilute to 3 mL with 0.5% sodium carboxymethylcellulose (CMC).

1.2. Experimental process:

Male normal (WT) mice were equally divided into two groups. Fasting began at 8:30 in the morning. After 4 hours, blood glucose was measured at 12:30 in the afternoon. After that, AR-GK-01 (dose of 20 mg/kg body weight), the control group was given CMC-DMSO-castor oil mixed solution (solvent control with the same volume), blood glucose was measured 1 hour later, followed by OGTT, glucose (dose 2.0 g/Kg body weight) was given intragastrically, and then measured every 30 min A blood sugar.

2. Obesity/diabetes model induced by high fat and high sugar and OGTT test:

2.1. Preparation of obesity/diabetes mouse model induced by high glucose and high fat:

By feeding 60% high-fat feed and 7.5% sucrose water, after 4 months of feeding, the body weight of the mice increased from 26.3 g to 49.1 g, while blood sugar increased, and the obesity/diabetes mouse model was successfully established. The following tables 2 and 3 are the nutritional components of normal feed and high-fat feed respectively.

Table 2 Normal feed

Table 3 High fat feed

2.2. Solution configuration:

(1) Configuration of AR-GK-01 solution:

Dissolve 8 mg AR-GK-01 in 40 μL DMSO, then dissolve in 80 μL castor oil, and finally dilute to 4 mL with 0.5% sodium carboxymethylcellulose (CMC).

(2) CMC-DMSO-castor oil mixed solution (solvent control):

Add 80 μL castor oil to 40 μL DMSO, and then dilute to 4 mL with 0.5% sodium carboxymethylcellulose (CMC).

2.3. Experimental process:

Male obese mice were divided into two groups. Fasting began at 9:00 am, and blood glucose was measured 4 hours later at 13:00 pm. The experimental group was given AR-GK-01 (20 mg/kg body weight), and the control group was given CMC -DMSO-castor oil mixed solution (same volume as solvent control), blood glucose was measured 1 hour later, followed by OGTT, intragastric administration of glucose (dose 2.0 g/Kg body weight), and then blood glucose was measured every 30 min.

3. Test results:

Figure 19 shows the improvement effect of AR-GK-01 on the OGTT of normal mice at a dose of 20 mg/kg body weight, and the blood glucose levels of the drug group were significantly lower at 30 min, 60 min, 90 min and 150 min after intragastric administration of glucose Compared with the solvent control group, it is suggested that AR-GK-01 can improve glucose tolerance. Figure 20 shows the improvement effect of AR-GK-01 on the glucose tolerance of obese/diabetic mice induced by high-fat and high-sugar diet. It was 10.9±0.5 mM (n=15), and the 4-hour fasting blood glucose of normal mice was 8.8±0.4 mM (n=11), p<0.01. AR-GK-01 significantly improved the OGTT of obese/diabetic mice at a dose of 20 mg/kg body weight, showing OGTT 60 min, 90 min, 120 min, 150 min, 180 min and 210 min Min blood glucose was significantly lower than that of the solvent control group, indicating that AR-GK-01 significantly improved the glucose tolerance of obese/diabetic mice.

Example 9 Effect of AR-GK-01 on Hepatic Glycogen Metabolism

1. Mouse liver glycogen detection method:

1.1. Reagents and instruments

1) Amyloglucosidase (Sigma): 15 U/mL

2) Glucose oxidase (macklin): 200 U/mL

3) HRP (Solarbio): 100 U/mL

4) ABTS (macklin): 1 mM

5) 50 mM sodium phosphate (pH 7.4/pH 4.5)

6) NaOH: 0.2 N

7) TCA: 5%, 10%

8) 96-well plate

9) CLARIOstar multi-functional microplate reader

10) Configuration of master mix: 10 μL 100 U/mL HRP stock solution and 20 μL 200 U/mL Glucoseoxidase stock solution to 970 μL 50 mM sodium phosphate (pH 7.5) to prepare 1 mL.

2. Extraction and detection of liver glycogen

2.1. Extraction of glycogen

1) Weigh about 50 mg of frozen or fresh mouse liver into a 1.5mL conical EP tube, add 50 μL of 10% TCA and 200 μL of 5% TCA, use a tissue homogenizer to homogenize on ice, and homogenize thoroughly After slurrying, use a centrifuge to centrifuge at 5000 rpm for 10 min.

2) After centrifugation, take the supernatant and place it in an EP tube, and temporarily store the precipitate at 4°C. Add an equal volume of pre-cooled 95% ethanol solution to the supernatant, mix thoroughly and let it stand for 10 minutes (it can also be extended or overnight at 4°C), and centrifuge with a centrifuge at 5000 rpm for 10 minutes.

3) Discard the supernatant after centrifugation, absorb the remaining supernatant and dry the precipitate, add 200 μL ultrapure water to dissolve the precipitate, and bathe in boiling water for 2 min.

2.2. Glycogen standard curve:

1) Dilute the 4 mg/mL glycogen standard in 50 mM sodium phosphate (pH 4.5) (prepare fresh glycogen standard before use), double dilution, take 50 μL of each concentration and place in EP tube, 5 μL of 15 U/mL Amyloglucosidase (Sigma) was added to each tube, and the samples were reacted at a constant temperature of 55°C for 20 min.

2) Take 30 μL of the reactants in step 1 and place them in a 96-well plate, add the same volume of premix solution, mix well, and then incubate in a 37°C incubator for 45 min.

3) Take 20 μL of the incubation solution and add 100 μL of 1 mM ABTS, and incubate at 37°C for 30 min.

4) In the CLARIOstar multi-functional microplate reader, the absorbance value at the wavelength of 405 nm was detected to determine the glycogen content, and the analysis was carried out in the Graphpad Prism software, and the regression line fitting of the equation was used for the analysis to obtain the reaction curve as shown in Figure 21.

2.3. Detection of liver glycogen:

1) Dilute the extracted glycogen sample with 50 mM sodium phosphate (pH 4.5) solution for a certain number of times, then take 50 μL and place it in an EP tube, add 5 μL of 15 U/mL Amyloglucosidase (Sigma) to each tube, The samples were reacted at a constant temperature of 55 °C for 20 min.

2) Take 30 μL of the reactants in step 1 and place them in a 96-well plate, add the same volume of premix solution, mix well, and then incubate in a 37°C incubator for 45 min.

3) Take 20 μL of the incubation solution and add 100 μL of 1 mM ABTS, and read the results with a microplate reader (405nm) after 30 min in a 37°C incubator.

4) Compare the ΔOD of each sample to the standard curve to determine and infer the glycogen present in the sample (use only values within the range of the standard curve).

2.4 Calculation method of liver glycogen content

Liver glycogen content and liver wet weight

Calculate the results obtained by the glycogen standardization of the samples and the wet weight of the liver:

Total liver glycogen (mg/g wet weight) = glycogen (mg) / tissue weight (g)

3. Animal test:

3.1, 2 mg/mL AR-GK-01 solution preparation:

Dissolve 20 mg AR-GK-01 in 100 μL DMSO, then dissolve in 200 μL castor oil, and finally dilute to 10 mL with 0.5% sodium carboxymethylcellulose (CMC).

3.2. Experimental process:

5 WT mice and 5 SUR1-knockout mice were prepared, and AR-GK-01 (20 mg/Kg) was gavaged for 3 consecutive days after blood glucose was measured at 10:00 every morning. At 13:00 p.m. on the third day, the liver tissue was collected, liver glycogen was extracted and its content was detected. In addition, 5 normally fed WT and SUR1 knockout mice were sacrificed at 13:00 pm to take liver tissue as a control, and liver glycogen was extracted and its content was detected.

3.3. Test results:

As shown in Figure 22, when the dose of AR-GK-01 was 20 mg/kg body weight, the effect on wild-type mice (vehicle control group n=6, administration group n=4) and SUR1 knockout mice (SUR1- KO, solvent control group n=6, administration group n=4) the effect of liver glycogen. AR-GK-01 administration for 3 days had no effect on liver glycogen in WT mice. The liver glycogen of SUR1-KO mice was significantly higher than that of WT mice, and AR-GK-01 reduced the liver glycogen of SUR1-KO mice to restore it to the level of WT mice, indicating that AR-GK-01 has Hepatic glycogen metabolism is affected.

Example 10 Detection of Drug Metabolism and Tissue Distribution in AR-GK-01 Mice

1. Preparation of 2 mg/mL AR-GK-01 solution:

Dissolve 20 mg AR-GK-01 in 100 μL DMSO, then dissolve in 200 μL castor oil, and finally dilute to 10 mL with 0.5% sodium carboxymethylcellulose (CMC).

2. Experimental process:

Drug metabolism experiment: 3 C57BL/6J male 8-week-old mice were starved overnight, orally administered 2 mg/mL of AR-GK-01 solution, the volume of administration was 100 μL/10 g of mouse body weight, respectively. Before, 15, 30, 60, 120, 240 and 480 minutes after administration, 100 μL of blood was collected from the orbit, placed in an anticoagulant tube, centrifuged at 12,000 rpm for 2 minutes, and the upper layer of plasma was taken out and stored in a -80 degree refrigerator for use to detect drug concentrations.

Tissue distribution: 3 C57BL/6J male 8-week-old mice were starved overnight, orally administered 2 mg/mL of AR-GK-01 solution, the volume of administration was 100 μL/10 g of mouse body weight, 60 minutes after administration Finally, the pancreas and liver tissues were taken out, and the residual blood was cleaned in normal saline, then placed in cryopreservation tubes and stored in a -80°C refrigerator for detection of drug concentration.

3. LC/MS/MS analysis method of AR-GK-01

3.1. Sample pretreatment method

1) Plasma sample: Add 200 µL MeOH/ACN (1/1, v/v) to 20 µL plasma, vortex and mix well, then centrifuge (15000rpm, 5min), take 20 µL supernatant + 20 µL ACN/H ₂ O (1/1,v/v) Mix well and inject for analysis.

2) Tissue samples: After weighing the tissue, add 5 times the volume of MeOH/CAN (1/1, v/v) to homogenate according to the weight, vortex and mix, and then centrifuge (15000rpm, 5min) to obtain tissue homogenate, take 40 µL supernatant was injected for analysis.

3.2. Chromatographic method

Column: Waters ACQUITY UPLCr BEH Phenyl 1.7 µm (2.1*50 mm, 1.7 µm)

Flow rate: 0.5 mL/min

A: 5 mM NH ₄ OAc in H ₂ O with 0.1% FA B: 0.1% FA in ACN/MeOH (9/1, v/v)

The gradient is shown in Table 4 below

Table 4 Gradient

Autosampler temperature: 10°C

Column temperature: 45°C

3.3. Mass spectrometry method

Mass spectrometry parameters are shown in Table 5.

Table 5 Mass Spectrometry Parameters

4. Experimental results:

As shown in Table 6, the blood drug concentration (ng/mL) of AR-GK-01 in three mice at different times after administration, the blood drug concentration increased significantly at 0.25 hours, and the peak value appeared at 0.5 hour, and then the concentration gradually increased. decreased, and there were still drug residues in only 8 hours, as shown in Figure 23. Table 7 shows the pharmacokinetic parameters. The half-life of AR-GK-01 is 2.93 hours, and the peak time is 0.33 hours. When the dose is 20 mg/Kg body weight, the maximum plasma concentration is 1273 ng/mL. Table 8 shows the tissue distribution in the liver and pancreas, both in the liver and pancreas.

Table 6. Mouse plasma drug concentration (ng/mL) at different times

The above description is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this field Those skilled in the art, without departing from the scope of the technical solution of the present invention, may use the method and technical content disclosed above to make some changes or modifications to equivalent embodiments with equivalent changes, but if they do not depart from the technical solution of the present invention, Any simple modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims (1)

1. the application of a glucokinase agonist in the preparation of glucokinase impaired glucose metabolism disease or abnormal glucose metabolism disease treatment drug, characterized in that, the glucokinase agonist is: .