1-MHMD, preparation method and application thereof

Abstract

The invention discloses a 1-MHMD, a preparation method and an application thereof. It relates to the field of biomedicine technology. The invention combines 1-methylhydantoin and metformin to form a novel compound with medical use based on the principle of drug combination. The order in which the chemical groups at both ends of R1 in XR1COR2 react with metformin hydrochloride or 1-methylhydantoin can be determined by the preparation method of intermediate I and the preparation method of intermediate II, and compound 1-MHMD can be prepared, providing multiple options for future production; compound 1-MHMD has few by-products, is easy to purify, and has high purity; the preparation method of the invention is simple, has few steps, and has low energy consumption; the invention combines 1-methylhydantoin and metformin for the first time, which can play a role in combining drug efficacy and reducing toxic side effects, and the molecular weight is ≤500, which is a compound with great development value in the fields of medicines such as lowering blood sugar, lowering blood lipids, losing weight, and treating fatty liver.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to 1-MHMD, a preparation method and application thereof. Background Art

[0002] In recent years, the prevalence of overweight and obese people in my country has continued to rise. According to the 2024 Edition of the Obesity Diagnosis and Treatment Guidelines, the prevalence of obesity in adults in my country has reached 16.4%, which has become a major public health problem. Obesity is a recognized risk factor for multiple complications. Type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD) are typical obesity-induced metabolic diseases, and the two often occur simultaneously. In terms of pathogenesis, T2DM mainly involves insulin resistance and pancreatic β-cell dysfunction. Insulin resistance refers to the body's (especially muscle, liver and adipose tissue) decreased efficiency of glucose uptake and utilization under insulin stimulation, and the relative lack of insulin, leading to hyperglycemia. At the same time, the toxic effects of long-term hyperglycemia and hyperlipidemia cause pancreatic β-cell damage and gradual decline in function. The pathogenesis of NAFLD is complex and driven by multiple factors. The classic "two-hit" theory has gradually evolved into a "multiple-hit" theory, that is, multiple metabolic and environmental factors work together to trigger the occurrence and development of NAFLD. It is currently believed that insulin resistance, abnormal lipid metabolism, oxidative stress, mitochondrial dysfunction and chronic inflammation are the key pathogenic factors.

[0003] Metformin (MET) is the first choice drug for the treatment of T2DM worldwide. It mainly achieves the effect of lowering blood sugar by improving insulin sensitivity, inhibiting hepatic gluconeogenesis, and increasing the utilization of glucose by peripheral tissues. Since its introduction, MET has been widely used in clinical practice, and its safety and effectiveness have been fully verified. However, the long-term use of MET has also brought several problems, such as gastrointestinal discomfort, vitamin B12 deficiency and other side effects. Most importantly, the double-edged sword effect of MET in lipid metabolism and liver fat accumulation requires extra caution in the clinical application of T2DM combined with NAFLD. The 2022 edition of the Manual for Clinical Diagnosis and Treatment of Metabolic-Related Fatty Liver Disease clearly states that MET is not recommended for the treatment of fatty liver. On the other hand, due to the complex pathogenesis of NAFLD, the first drug for the treatment of non-alcoholic steatohepatitis (NASH), Resmetirom, was approved worldwide only in 2024. As a selective agonist of thyroid hormone receptor-β for the liver, resmetiro is mainly used for NASH patients with moderate to severe liver fibrosis, and has limited therapeutic effect on patients with combined T2DM. Therefore, it is of great clinical value to develop a new drug that can not only produce a hypoglycemic effect, but also has additional lipid-lowering or other metabolic improvement functions, and has few toxic and side effects.

[0004] The principle of drug splicing mainly refers to obtaining a new drug molecule by splicing the structural units or pharmacophores of two or more different drugs into one molecule. This hybrid molecule usually has the functions of the original drug, or exhibits better pharmacological properties in terms of enhancing efficacy, improving selectivity, reducing toxic side effects, and improving drug metabolism. This strategy has been successfully applied in many therapeutic fields. For example, potassium clavulanate is a β-lactamase inhibitor that can protect amoxicillin from being destroyed by β-lactamase produced by bacteria, thereby improving the efficacy of amoxicillin against drug-resistant bacteria. However, in the field of metabolic disease treatment, the successful application of drug splicing is still rare, so the introduction of this innovative strategy in this field has great research value and therapeutic potential.

[0005] 1-Methylhydantoin (1-MH) is a natural monomer component extracted from toad oil, a traditional animal medicine in my country. Toad oil is a product of the Chinese forest frog, a species of the Ranidae family. Rana temporaria chensinensisDavid's fallopian tube was collected and dried. As a tonic medicine, it has a wide range of pharmacological effects such as antitussive and expectorant, anti-oxidation, lowering blood lipids, anti-aging, improving the body's immune function and enhancing stress resistance. Previous studies have shown that 1-MH has multiple pharmacological activities, including significant antitussive, anti-inflammatory, hypoglycemic and lipid-lowering effects. At the same time, 1-MH also exhibits excellent antioxidant effects and can effectively fight the damage caused by oxidative stress, which is especially important for patients with T2DM and NAFLD. On the other hand, from the perspective of pharmaceutical chemistry, the chemical structures of both 1-MH and MET are relatively simple, and they have good metabolic stability and a low risk of causing toxic reactions. Summary of the invention

[0006] The technical solution of the present invention to solve the above technical problem is to provide a 1-MHMD, wherein the structural formula of the 1-MHMD is: ; Wherein, R1 includes alkanes, and / or olefins, and / or aromatics, and / or heteroatoms containing 1-15 carbon atoms.

[0007] In order to solve the above technical problems, the present invention also provides a method for preparing 1-MHMD, comprising the following steps:

[0008] Substitution reaction of 1-methylhydantoin with XR1COR2 to obtain intermediate I; ; Intermediate I is reacted with metformin hydrochloride by base catalysis to obtain a crude product,

[0009] High-purity 1-MHMD was obtained after recrystallization:

[0010] Wherein, R2 includes alkoxy, and / or alkenyloxy, and / or aryloxy, and / or halogen, and / or sulfonyloxy, and / or hydroxyl, and / or nitro, and / or nitroso containing 1-10 carbon atoms;

[0011] Alternatively, XR1COR2 is reacted with metformin hydrochloride by base catalysis to obtain intermediate II; ; 1-methylhydantoin is reacted with intermediate II to obtain a crude product;

[0012] High-purity 1-MHMD was obtained after recrystallization:

[0013] Wherein, X is a halogen, and / or a sulfonyl group, and / or a sulfonyloxy group, and / or a hydroxyl group, and / or an acyl group, and / or a nitro group, and / or a nitroso group, and / or a diazo group, and / or an azide group.

[0014] Furthermore, the steps of subjecting 1-methylhydantoin to a substitution reaction with XR1COR2 to obtain an intermediate I; reacting the intermediate I with metformin hydrochloride by base catalysis to obtain a crude product, and obtaining high-purity 1-MHMD after recrystallization include:

[0015] Add 1-methylhydantoin and potassium carbonate to N,N-dimethylformamide solvent, heat the mixture to 45°C-100°C, and stir for 1h-3h to prepare reaction solution a;

[0016] Add XR1COR2 to the reaction solution a, heat to 52°C-100°C, and stir for 8h-16h; remove the solvent under reduced pressure to obtain a crude product;

[0017] Pour in ethyl acetate, perform ultrasonic extraction for 5 minutes to 60 minutes, filter out the precipitate, and repeat the extraction operation on the precipitate 3 to 5 times;

[0018] The organic phases were combined and the solvent was removed under reduced pressure to obtain intermediate I;

[0019] Add metformin hydrochloride to the solvent methanol at room temperature, and also add the catalyst sodium methoxide thereto, and stir the suspension at a temperature of 25°C-40°C for 20 minutes-3 hours to prepare solution b;

[0020] The intermediate I is diluted in the solvent methanol to prepare solution c;

[0021] Solution c was slowly added dropwise into solution b, and the reaction was stirred at 20°C-50°C for 8h-16h. After the reaction was completed, the solvent was removed under reduced pressure to obtain a crude product, which was then recrystallized from ethanol to obtain 1-MHMD.

[0022] Furthermore, the steps of reacting XR1COR2 with metformin hydrochloride by base catalysis to obtain intermediate II; reacting 1-methylhydantoin with intermediate II to obtain a crude product; and obtaining high-purity 1-MHMD after recrystallization include:

[0023] Add metformin hydrochloride to solvent ethanol at room temperature, and also add catalyst sodium ethoxide thereto, and stir the suspension at a temperature of 30°C-50°C for 25min-3h to prepare solution a;

[0024] Dilute XR1COR2 in methanol solvent to make solution b;

[0025] Slowly drop solution b into solution a, and stir the reaction at 20-55°C for 8-16 hours;

[0026] The solvent was removed by rotary evaporation under reduced pressure, and the intermediate II was obtained by recrystallization;

[0027] Add 1-methylhydantoin and potassium carbonate to N,N-dimethylformamide solvent, heat the mixture to 60°C-110°C and stir for 0.5h-1h to prepare reaction solution c;

[0028] Add intermediate II to reaction solution c, heat to 75-90°C and stir for 16-48h;

[0029] The solvent was removed under reduced pressure to obtain a crude product, which was recrystallized to give 1-MHMD.

[0030] The present invention also discloses the use of the 1-MHMD described above in preparing drugs for reducing blood sugar, reducing blood lipids and treating fatty liver.

[0031] The invention also discloses the use of the 1-MHMD in preparing weight loss medicine.

[0032] Compared with the prior art solutions, the present invention has the following beneficial effects:

[0033] 1. The starting material is a natural drug, the raw material is easy to synthesize, cheap and easy to obtain, and the preparation process is simple and easy to operate and suitable for production;

[0034] 2. Intermediates I and II can be stored for a long time under normal conditions;

[0035] 3. The preparation process of 1-MHMD has few by-products, little environmental pollution, and most of the reagents used are recyclable;

[0036] 4. The order in which the chemical groups at both ends of R1 in XR1COR2 react with metformin hydrochloride or 1-methylhydantoin can be determined by the preparation method of intermediate I or the preparation method of intermediate II to prepare 1-MHMD, providing multiple options for future production;

[0037] 5. 1-MHMD has few by-products, is easy to purify, and has high purity;

[0038] 6. The reaction conditions used in the present invention are all between 25°C and 100°C, which is simple to operate, requires fewer steps, and has low energy consumption;

[0039] 7. The present invention combines 1-methylhydantoin and metformin for the first time, which has the effect of combining drug effects and reducing toxic side effects, and the molecular weight is ≤500, which is a compound with great development value in the field of medicine and health;

[0040] 8. The obtained compounds can significantly inhibit the abnormal body weight and blood sugar caused by a high-fat diet, and improve insulin sensitivity and glucose tolerance;

[0041] 9. The obtained compounds can significantly improve liver damage such as dyslipidemia and fatty liver caused by a high-fat diet. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0043] Figure 1 This is the hydrogen spectrum of intermediate I (R1=CH2, R2=OCH3), the solvent is CDCl3;

[0044] Figure 2 This is the carbon spectrum of intermediate I (R1=CH2, R2=OCH3), the solvent is CDCl3;

[0045] Figure 3 This is the hydrogen spectrum of intermediate I (R1=CH2, R2=OCH2CH3), the solvent is DMSO- d 6;

[0046] Figure 4 This is the carbon spectrum of intermediate I (R1=CH2, R2=OCH2CH3), the solvent is DMSO- d 6;

[0047] Figure 5 This is the hydrogen spectrum of 1-MHMD (R1=CH2), the solvent is DMSO- d 6;

[0048] Figure 6 This is the carbon spectrum of 1-MHMD (R1=CH2), the solvent is DMSO- d 6;

[0049] Figure 7 This is the hydrogen spectrum of intermediate II (X = Cl, R1 = CH2), the solvent is DMSO- d 6;

[0050] Figure 8 This is the carbon spectrum of intermediate II (X=Cl, R1=CH2), the solvent is DMSO- d 6;

[0051] Fig. 9 This is the hydrogen spectrum of intermediate II (X = Br, R1 = CH2), the solvent is DMSO- d 6;

[0052] Fig.10 This is the hydrogen spectrum of 1-MHMD (R1=CH2) hydrochloride, the solvent is D2O;

[0053] Fig.11This is the hydrogen spectrum of 1-MHMD (R1=CH2) maleate, the solvent is D2O;

[0054] Fig.12 This is the carbon spectrum of 1-MHMD (R1=CH2) maleate, the solvent is D2O;

[0055] Fig.13 This is the ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY of 1-MHMD (R1=CH2);

[0056] Fig.14 It is the high-resolution mass spectrum of 1-MHMD (R1=CH2);

[0057] Fig.15 It is the Fourier transform infrared chromatogram of 1-MHMD (R1=CH2);

[0058] Fig.16 Effects of 1-MHMD on body weight and fasting blood glucose in HFD-induced obese mice. (A) Body weight; (B) Fasting blood glucose. x±SE, n=6 per group. #p<0.05, ##p<0.01, ### p<0.001, Chow vs. HFD; *p<0.05, **p<0.01, ***p<0.001, HFD vs. each group.

[0059] Fig.17 After 10 weeks of 1-MHMD administration, the mice were fasted for 12 hours and given 1 g / kg glucose by gavage. Blood glucose was measured by tail vein blood at 0, 15, 30, 60, 90, and 120 minutes after glucose administration, and AUC was calculated. x±SE, n=6 per group. #p<0.05, ##p<0.01, ### p<0.001, Chow vs. HFD; *p<0.05, **p<0.01, ***p<0.001, HFD vs. each group.

[0060] Fig.18 After 12 weeks of 1-MHMD administration, the rats were fasted for 6 hours and injected intraperitoneally with 0.5 U / kg insulin. Blood glucose was measured by tail vein blood at 0, 15, 30, 60, 90, and 120 minutes after injection, and AUC was calculated. x±SE, n=6 per group. #p<0.05, ##p<0.01, ### p<0.001, Chow vs. HFD; *p<0.05, **p<0.01, ***p<0.001, HFD vs. each group.

[0061] Fig.19 These are histopathological sections of mouse liver (100×, 200×).

[0062] Fig. 20Effects of 1-MHMD on plasma lipid levels in HFD-induced obese mice. (A) TG; (B) TC; (C) LDL; (D) HDL. x ± SD, n = 6 per group. #p<0.05, ##p<0.01, ### p<0.001, Chow vs. HFD; *p<0.05, **p<0.01, ***p<0.001, HFD vs. each group.

[0063] Fig.21 Effects of 1-MHMD on liver function indices in HFD-induced obese mice. (A) ALT; (B) AST. x±SD, n=6 per group. #p<0.05, ##p<0.01, ### p<0.001, Chow vs. HFD; *p<0.05, **p<0.01, ***p<0.001, HFD vs. each group.

[0064] Fig. 22 The effect of 1-MHMD on plasma MAD level in HFD-induced obese mice. x±SD, n=6 for each group. #p<0.05, ##p<0.01, ### p<0.001, Chow vs. HFD; *p<0.05, **p<0.01, ***p<0.001, HFD vs. each group.

[0065] Fig.23 The effect of 1-MHMD on the proliferation activity of AML12 cells under the action of GP. x±SD, n=4 in each group. #p<0.05, ##p<0.01, ### p<0.001, Con vs. GP; *p<0.05, **p<0.01, ***p<0.001, GP vs. each group.

[0066] Fig.24 The effect of 1-MHMD on the transcriptional levels of inflammatory factors in AML12 cells under the action of GP. x±SD, n=3 for each group. #p<0.05, ##p<0.01, ### p<0.001, Con vs. GP; *p<0.05, **p<0.01, ***p<0.001, GP vs. each group.

[0067] Fig.25 The effect of 1-MHMD on the apoptosis level of MIN6 cells under the action of GP. x±SD, n=3 for each group. #p<0.05, ##p<0.01, ### p<0.001, Con vs. GP; *p<0.05, **p<0.01, ***p<0.001, GP vs. each group.

[0068] Fig.26This is the effect of 1-MHMD on the level of ROS in MIN6 cells under the action of GP. DETAILED DESCRIPTION

[0069] The present invention provides 1-MHMD, a preparation method and application thereof, aiming to provide a 1-MHMD which can be used for preparing drugs for lowering blood sugar, lowering blood lipids, losing weight and treating fatty liver.

[0070] The 1-MHMD proposed in the present invention will be described in the following specific embodiments: 1-MHMD, the structure is as follows: ; Wherein, R1 includes alkanes, and / or olefins, and / or aromatics, and / or heteroatoms containing 1-15 carbon atoms.

[0071] The molecular weight of compound 1-MHMD is ≤500;

[0072] Compound 1-MHMD and pharmaceutically acceptable salts thereof.

[0073] 1-MHMD can be used to prepare drugs for lowering blood sugar, lowering blood lipids and treating fatty liver.

[0074] 1-MHMD can be used to prepare weight loss drugs.

[0075] The study found that 1-methylhydantoin has a significant effect on lowering blood sugar and blood lipids. Based on the principle of drug splicing, 1-methylhydantoin and metformin are combined to synthesize a new compound (1-MHMD), which is helpful to develop a new drug with good drugability and efficacy. In summary, 1-MHMD is an innovative design based on the principle of drug splicing. It has multiple effects of lowering blood sugar, lowering blood lipids, anti-inflammatory, and anti-oxidation, and has important clinical application potential. It may effectively solve the complex problems of T2DM and NAFLD patients in metabolic regulation. At present, there is no report on the synthesis of a new drug compound using 1-methylhydantoin and metformin. It is expected to become a new type of metabolic syndrome treatment drug that is efficient, safe, and multifunctional, meeting the urgent needs of current clinical treatment.

[0076] Example 1: A method for preparing 1-MHMD, comprising the following steps:

[0077] The compound 1-methylhydantoin at a molar ratio of 1 and potassium carbonate at a molar ratio of 1 were added to the solvent N,N-dimethylformamide, the molar ratio of N,N-dimethylformamide being 30, and the mixture was heated to 80° C. and stirred for 1 hour to prepare reaction solution a.

[0078] Add XR1COR2 (X=Br, R1=CH2, R2=OCH3) to the reaction solution a, heat to 85°C and stir for 16 hours. Remove the solvent under reduced pressure to obtain a crude product. Pour 5 times the amount of ethyl acetate, perform ultrasonic extraction for 60 minutes, filter out the precipitate, and repeat the extraction operation for 3 times. Combine the organic phases and remove the solvent under reduced pressure to obtain intermediate I (R1=CH2, R2=OCH3). The hydrogen spectrum is shown in Figure 1 As shown, the carbon spectrum is Figure 2 As shown, 1 H NMR (400 MHz, Chloroform- d )δ4.14 (s, 2H), 3.87 (s, 2H), 3.65 (s, 3H), 2.91 (s, 3H); 13 C NMR (100 MHz, Chloroform- d )δ169.35, 167.62,155.79, 52.57, 51.71, 39.45, 29.57.

[0079] At room temperature, metformin hydrochloride was added to the solvent methanol at a molar ratio of 1, the molar ratio of methanol was 70, and the catalyst sodium methoxide was also added thereto at a molar ratio of 1. The suspension was stirred at 40° C. for 0.5 hour to prepare solution b.

[0080] The intermediate I (R1=CH2, R2=0CH3) was diluted in the solvent methanol, with a methanol molar ratio of 40, to make solution c. Solution c was slowly added dropwise to solution b, and the reaction was stirred at room temperature for 16 hours. After the reaction was completed, the solvent was removed under reduced pressure to obtain a crude product, which was recrystallized from ethanol to obtain 1-MHMD (R1=CH2). The hydrogen spectrum is shown in Figure 5 As shown, the carbon spectrum is Figure 6 As shown, 1 H NMR (400 MHz, DMSO- d 6) δ6.79 (s, 2H), 4.23 (s, 2H), 4.04 (s, 2H), 2.99 (s, 6H), 2.89 (s, 3H). 13 CNMR (100MHz, DMSO- d 6 ) δ171.61,170.90,166.98,165.33,156.93,51.85,43.07,35.95,29.69.

[0081] Example 2: A method for preparing 1-MHMD, comprising the following steps:

[0082] The compound 1-methylhydantoin with a molar ratio of 1 and pyridine with a molar ratio of 1 were added to the solvent acetonitrile (molar ratio of 30), and the mixture was heated to 60°C and stirred for 3 hours to prepare reaction solution a. XR1COR2 (X=Cl, R1=CH2, R2=OCH3) was added to the reaction solution a, heated to 100°C and stirred for 8 hours. After the solvent was removed under reduced pressure, 10 times the amount of cyclohexane was added, and ultrasonic extraction was performed for 30 minutes, and the precipitate was filtered out. The extraction operation was repeated for 5 times. The solvent was removed under reduced pressure to obtain intermediate I (R1=CH2, R2=OCH3). 1 HNMR (400 MHz, Chloroform- d )δ4.14 (s, 2H), 3.87 (s, 2H), 3.65 (s, 3H), 2.91 (s, 3H); 13 C NMR (100 MHz, Chloroform- d )δ169.35,167.62,155.79,52.57, 51.71,39.45,29.57.

[0083] At room temperature, the compound metformin hydrochloride with a molar ratio of 1 is added to the solvent tert-butyl alcohol (molar ratio of 80), and potassium tert-butylate is also added thereto with a molar ratio of 1. The suspension is stirred at a temperature of 25°C for 3 hours to prepare solution b. After the reaction, the intermediate I (R1=CH2, R2=OCH3) is diluted in the solvent methanol with a molar ratio of 35 to prepare solution c. Solution c is slowly added dropwise to solution b, and the reaction is stirred at a temperature of 25°C for 16 hours. The organic phases are combined and the solvent is removed under reduced pressure, and 1-MHMD (R1=CH2) is obtained after recrystallization. 1 H NMR (400 MHz, DMSO- d 6) δ6.79 (s, 2H), 4.23 (s, 2H), 4.04 (s, 2H), 2.99 (s, 6H), 2.89 (s, 3H). 13 C NMR (100 MHz, DMSO) δ171.61,170.90,166.98, 165.33,156.93,51.85,43.07,35.95,29.69.

[0084] Example 3: A method for preparing 1-MHMD, comprising the following steps:

[0085] The compound 1-methylhydantoin in a molar ratio of 1 and sodium carbonate in a molar ratio of 1 were added to the solvent acetone, with the acetone molar ratio being 15. The mixture was heated to 45° C. and stirred for 1 hour to prepare the reaction solution a.

[0086] Add XR1COR2 (X=Cl, R1=CH2, R2=OCH2CH3) to the reaction solution a, heat to 50°C and stir for 16 hours. Drain the solvent completely by vacuum rotary evaporation to obtain a crude product. Pour 8 times the amount of ethyl acetate, perform ultrasonic extraction for 5 minutes, filter out the precipitate, and repeat the extraction operation 4 times. Combine the organic phases and vacuum rotary evaporate to obtain intermediate I (R1=CH2, R2=OCH2CH3). The hydrogen spectrum is shown in Figure 3 As shown, the carbon spectrum is Figure 4 As shown, 1 H NMR (400 MHz,) δ4.16 (s, 2H), 4.12 (q, J = 8.0 Hz,2H), 4.07(s, 2H),2.88 (s,3H),1.19(t, J = 8.0 Hz, 3H). 13 C NMR (100 MHz, DMSO-d6) δ169.83,167.43,155.55,61.22,51.30,39.32,29.15,13.94.

[0087] At room temperature, add the compound metformin hydrochloride at a molar ratio of 1 to the solvent ethanol, the ethanol molar ratio is 100, and sodium ethoxide is also added at a molar ratio of 1. The suspension is stirred at a temperature of 35 degrees for 1 hour to make solution b. Intermediate I (R1=CH2, R2=OCH2CH3) is diluted in the solvent ethanol, the ethanol molar ratio is 40, and the solution c is made. Solution c is slowly added dropwise to solution b, and stirred at room temperature for 16 hours. The solvent is recovered under reduced pressure, and 1-MHMD (R1=CH2) is obtained after recrystallization. 1 H NMR (400MHz, DMSO- d 6) δ 6.79 (s,2H), 4.23 (s,2H), 4.04 (s,2H), 2.99 (s,6H), 2.89 (s,3H). 13 C NMR (100 MHz, DMSO) δ171.61,170.90,166.98,165.33,156.93, 51.85, 43.07,35.95,29.69.

[0088] Example 4: A method for preparing 1-MHMD, comprising the following steps:

[0089] Add the compound 1-methylhydantoin in a molar ratio of 1 and triethylamine in a molar ratio of 2 to the solvent toluene, with a toluene molar ratio of 10, and heat the mixture to 100°C and stir for 1 hour to prepare reaction solution a. Add XR1COR2 (X=Cl, R1=CH2Ph, R2=OCH3) to the reaction solution a, heat to 65°C and stir for 16 hours. Remove the solvent by rotary evaporation to obtain a crude product. Pour 7 times the amount of acetone, perform ultrasonic extraction for 5 minutes, filter out the precipitate, and repeat the extraction operation for 5 times. Combine the organic phases and rotary evaporate under reduced pressure to obtain intermediate I (R1=CH2Ph, R2=OCH3).

[0090] At room temperature, the compound metformin hydrochloride with a molar ratio of 1 is added to the solvent ethanol, the molar ratio of ethanol is 50, and the sodium ethoxide is also added with a molar ratio of 1. The suspension is stirred at a temperature of 35 degrees for 1.5 hours to make solution b. After the reaction, the intermediate I (R1=CH2Ph, R2=OCH3) is diluted in the solvent ethanol, the molar ratio of ethanol is 25, and the solution c is made. Solution c is slowly added dropwise to solution b, and stirred at a temperature of 50 degrees for 16 hours. The solvent is removed under reduced pressure, and 1-MHMD (R1=CH2Ph) is obtained after recrystallization.

[0091] Example 5: A method for preparing 1-MHMD, comprising the following steps:

[0092] Add 1-methylhydantoin compound at a molar ratio of 1 and 1 cesium carbonate at a molar ratio of 30 to the solvent acetone, and heat the mixture to 45°C and stir for 1 hour to prepare reaction solution a. After the reaction, add XR1COR2 (X=Br, R1=CH2CH2, R2=CH3PhSO3) to the reaction solution a, heat to 52°C and stir for 16 hours. Remove the solvent by vacuum rotary evaporation to obtain a crude product. Pour 20 times the amount of chloroform, perform ultrasonic extraction for 5 minutes, filter out the precipitate, and repeat the extraction operation for 5 times. Combine the organic phases and vacuum rotary evaporation to obtain intermediate I (R1=CH2CH2, R2=CH3PhSO3).

[0093] At room temperature, the compound metformin hydrochloride with a molar ratio of 1 was added to the solvent ethanol, with a molar ratio of 120, and the catalyst sodium tert-butoxide was also added thereto with a molar ratio of 1. The suspension was stirred and reacted at room temperature for 20 minutes to prepare solution b. The intermediate I (R1=CH2CH2, R2=CH3PhSO3) was diluted in the solvent tert-butanol with a molar ratio of 45 to prepare solution c. Solution c was slowly dripped into solution b, and stirred and reacted at a temperature of 50 degrees for 8 hours. The solvent was removed under reduced pressure, and 1-MHMD (R1=CH2CH2) was obtained after recrystallization.

[0094] Example 6: A method for preparing 1-MHMD, comprising the following steps:

[0095] At room temperature, add the compound metformin hydrochloride at a molar ratio of 1 to the solvent ethanol, with a molar ratio of 40, and add the catalyst sodium ethoxide at a molar ratio of 1. Stir the suspension at a temperature of 30 degrees for 2 hours to make solution a. Dilute XR1COR2 (X=Cl, R1=CH2, R2=OCH3) in the solvent methanol, with a molar ratio of methanol of 40, to make solution b. Slowly add solution b dropwise into solution a, stir and react at a temperature of 55°C for 16 hours. Remove the solvent by vacuum rotary evaporation, and recrystallize to obtain intermediate II (X=Cl, R1=CH2); the hydrogen spectrum is shown as follows Figure 7 As shown, the carbon spectrum is Figure 8 As shown, 1 H NMR (400 MHz, DMSO- d 6)δ6.93 (s, 2H), 4.26 (s,2H), 3.07 (s,3H), 3.04 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 172.38, 167.31, 165.76, 47.13, 36.13.

[0096] Add the compound 1-methylhydantoin at a molar ratio of 1 and potassium carbonate at a molar ratio of 1 to the solvent N,N-dimethylformamide, with a molar ratio of 30, and heat the mixture to 110°C and stir for 1 hour to prepare reaction solution c. Add intermediate II (X=Cl, R1=CH2) to reaction solution c, heat to 90°C and stir for 16 hours. Remove the solvent under reduced pressure to obtain a crude product, and obtain 1-MHMD (R1=CH2) after recrystallization. 1 H NMR (400 MHz, DMSO- d 6) δ6.79(s, 2H), 4.23(s, 2H), 4.04(s, 2H), 2.99(s, 6H), 2.89(s, 3H). 13 C NMR (100 MHz, DMSO) δ171.61, 170.90, 166.98, 165.33, 156.93, 51.85, 43.07, 35.95, 29.69.

[0097] Example 7: A method for preparing 1-MHMD, comprising the following steps:

[0098] At room temperature, add the compound metformin hydrochloride at a molar ratio of 1 to the solvent methanol, with a molar ratio of 50, and the catalyst sodium methoxide at a molar ratio of 1. Stir the suspension at a temperature of 35 degrees for 3 hours to make solution a. Dilute XR1COR2 (X=Br, R1=CH2, R2=OCH3) in the solvent dichloromethane, with a molar ratio of 40, to make solution b. Drop solution b into solution a, stir and react at a temperature of 35 degrees for 16 hours. Remove the solvent by vacuum rotary evaporation, and recrystallize to obtain intermediate II (X=Br, R1=CH2); the hydrogen spectrum is shown as follows Fig. 9 As shown, 1 H NMR (400 MHz, DMSO-d6) δ 6.92 (s, 2H), 4.10 (s, 2H), 3.06 (s, 3H), 3.04 (s, 3H).

[0099] The compound 1-methylhydantoin (molar ratio: 1) and cesium carbonate (molar ratio: 1) were added to the solvent N,N-dimethylformamide (molar ratio: 50), and the mixture was heated to 60°C and stirred for 1 hour to obtain the reaction solution c. The intermediate II (X=Br, R1=CH2) was added to the reaction solution c, and the mixture was heated to 75°C and stirred for 16 hours. The solvent was removed by vacuum rotary evaporation, and 1-MHMD (R1=CH2) was obtained after recrystallization. 1 HNMR (400 MHz, DMSO- d 6) δ6.79 (s, 2H), 4.23 (s, 2H), 4.04 (s, 2H), 2.99 (s, 6H), 2.89 (s, 3H). 13 C NMR (100 MHz, DMSO) δ171.61,170.90,166.98,165.33,156.93,51.85,43.07,35.95,29.69.

[0100] Example 8: A method for preparing 1-MHMD, comprising the following steps:

[0101] At room temperature, add the compound metformin hydrochloride in a molar ratio of 1 to the solvent methanol, with a molar ratio of 100, and add the catalyst sodium methoxide in a molar ratio of 1. Stir the suspension at a temperature of 50°C for 1 hour to make solution a. Dilute XR1COR2 (X=Br, R1=CH2Ph, R2=OCH3) in the solvent methanol, with a methanol molar ratio of 40, to make solution b. Slowly drop solution b into solution a, stir at room temperature for 16 hours. After the reaction is completed, remove the solvent by vacuum rotary evaporation, and recrystallize to obtain intermediate II (X=Br, R1=CH2Ph);

[0102] Add 1 molar ratio of 1 compound 1-methylhydantoin and 1 molar ratio of potassium hydroxide to solvent N,N-dimethylformamide (molar ratio 80), heat the mixture to 85°C and stir for 1 hour to prepare reaction solution c. Add intermediate II (X=Br, R1=CH2Ph) to reaction solution c, heat and stir for 48 hours. After the reaction is completed, remove the solvent under reduced pressure and obtain 1-MHMD (R1= CH2Ph) after recrystallization.

[0103] Example 9: A method for preparing 1-MHMD, comprising the following steps:

[0104] At room temperature, add the compound metformin hydrochloride in a molar ratio of 1 to the solvent ethanol, the molar ratio is 120, and the catalyst potassium tert-butoxide is also added in a molar ratio of 1. The suspension is stirred and reacted at a temperature of 45 degrees for 25 minutes to make solution a. XR1COR2 (X=Br, R1=CH2Ph, R2=Cl) is diluted in the solvent acetone, the molar ratio is 50, and the solution b is made. Solution b is slowly added dropwise to solution a, and stirred and reacted at a temperature of 50 degrees for 8 hours. The solvent is removed by vacuum rotary evaporation, and the intermediate II (X=Br, R1=CH2Ph) is obtained by recrystallization.

[0105] The compound 1-methylhydantoin in a molar ratio of 1 and potassium carbonate in a molar ratio of 1 were added to the solvent acetonitrile, with an acetonitrile molar ratio of 15. The mixture was heated to 78°C and stirred for 0.5 hours to prepare reaction solution c. Intermediate II (X=Br, R1=CH2Ph) was added to the reaction solution c, heated to 80°C and stirred for 24 hours. The solvent was removed by vacuum rotary evaporation, and 1-MHMD (R1=CH2Ph) was obtained after recrystallization.

[0106] Example 10: A method for preparing 1-MHMD (R1=CH2) hydrochloride, comprising the following steps:

[0107] At room temperature, 1-MHMD (R1=CH2) with a molar ratio of 1 was added to solvent water, with a molar ratio of 10, and hydrochloric acid was also added thereto with a molar ratio of 1. The reaction was stirred at room temperature for 1.5 hours, and 1-MHMD (R1=CH2) hydrochloride was obtained after removing the solvent. The hydrogen spectrum is shown in FIG. Fig.10 As shown, 1 H NMR (400 MHz, Deuterium Oxide) δ4.62 (s, 2H), 4.17 (s, 2H), 3.10 (s, 6H), 2.96 (s, 3H).

[0108] Example 11: A method for preparing 1-MHMD (R1=CH2) maleate, comprising the following steps:

[0109] At room temperature, 1-MHMD (R1=CH2) with a molar ratio of 1 was added to solvent water, with a molar ratio of 150, and maleic acid was also added thereto with a molar ratio of 1. The reaction was stirred at room temperature for 2 hours, and 1-MHMD (R1=CH2) maleate was obtained after removing the solvent. The hydrogen spectrum is shown in FIG. Fig.11 As shown, the carbon spectrum is Fig.12 As shown, 1 H NMR (400 MHz, Deuterium Oxide)δ6.33(s,2H),4.67 (s,2H),4.23 (s,2H),3.16(s,6H), 3.02(s,3H). 13 C NMR (100 MHz, Deuterium Oxide) δ172.88,170.66,163.79, 160.84,157.77,157.30,134.07,51.95,40.22,36.77,29.14.

[0110] Verification experiment: 1-MHMD (R1=CH2) in Example 6 was dissolved in chromatography grade methanol to prepare a sample with a concentration of 1 mg / mL, and then subjected to ultra-high performance liquid chromatography. 18 The chromatographic column was (2.1 mm × 100 mm, 1.7 μm), and the mobile phase consisted of acetonitrile containing 0.1% formic acid (A) and water containing 0.1% formic acid (B). The flow rate was 0.4 mL / min, the injection volume was 2 μL, the pressure range was 0-15000 psi, and the column temperature was room temperature. The results are shown in Fig.13 shown.

[0111] The 1-MHMD (R1=CH2) in Example 6 was dissolved in methanol to prepare a sample with a concentration of 1 mg / mL, and then subjected to high-resolution mass spectrometry analysis. An electrospray ionization source (ESI) was used in the positive ion mode (+H + 、+Na + 、+K + ) and negative ion mode (-H + 、+HCOO - ) detection, MSE Continuum mode. The scanning parameters are as follows: cone voltage: 40 V, scanning mass range: 50-2000 Da, scanning time: 0.5 s, collision energy: 6-40 V. The results are as follows Fig.14 shown.

[0112] The 1-MHMD (R1=CH2) in Example 6 was mixed and ground with KBr in a ratio of 1:100, and Fourier transform infrared spectroscopy analysis was performed using a tablet pressing method. The results are as follows: Fig.15 shown.

[0113] Experimental verification: 1. Construction of HFD mouse model:

[0114] C57BL / 6J male mice aged 6-7 weeks (purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were divided into a normal chow group (Chow, n=6) and a high-fat diet group (HFD, n=24) and fed for 8 weeks.

[0115] 2. Mouse grouping and intervention:

[0116] 1) The mice with successful modeling were randomly divided into model control group (HFD, n=6), MET positive control group (HFD+MET, 220 mg / kg, n=6), 1-MHMD low-dose administration group (HFD+1-MHMD, 75 mg / kg, n=6) and high-dose administration group (HFD+1-MHMD, 150 mg / kg, n=6);

[0117] 2) Monitor the body weight and fasting 9-hour blood glucose of mice every week;

[0118] 3) After 10 weeks of administration, an oral glucose tolerance test (OGTT) was performed: the mice were fasted overnight, and the blood glucose at 0 min was measured by a blood glucose meter. Then, glucose solution (1 g / kg) was gavaged and the blood glucose at 15, 30, 60, 90, and 120 min was measured respectively.

[0119] 4) After 12 weeks of administration, an insulin tolerance test (ITT) was performed: the mice were fasted for 6 hours, and the blood glucose at 0 min was measured by a blood glucose meter. Then, insulin (0.5 U / kg) was intraperitoneally injected, and the blood glucose at 15, 30, 60, 90, and 120 min was measured respectively;

[0120] 5) The experiment was terminated after 16 weeks of administration: the mice were fasted but not watered for 6 hours. After anesthesia, blood samples were collected by eyeball bleeding. The supernatant was collected after centrifugation and stored at -80°C for subsequent biochemical index detection. The liver, fat and pancreatic tissues of the mice were separated, weighed and recorded. A small part of the tissue was fixed with paraformaldehyde and used for subsequent sectioning. The remaining tissue was stored at -80°C for future use.

[0121] 3. Detection of mouse plasma biochemical indicators:

[0122] 1) Plasma triglyceride (TG) detection:

[0123] (1) Add 200 μl of working solution to each well of a 96-well ELISA plate, then add 2.5 μl of distilled water, standard or plasma sample, mix well, and incubate in a 37°C incubator for 10 min.

[0124] (2) Measure the OD value of each well at a wavelength of 500 nm using an enzyme-labeled instrument and calculate according to the formula:

[0125] TG content (mmol / L) = (sample OD-blank OD) / (standard OD-blank OD) × standard concentration (mmol / L).

[0126] 2) Plasma total cholesterol (TC) test:

[0127] (1) Add 200 μl of working solution to each well of a 96-well ELISA plate, then add 2.5 μl of distilled water, standard or plasma sample, mix well, and incubate in a 37°C incubator for 10 min.

[0128] (2) Measure the OD value of each well at a wavelength of 500 nm using an enzyme-labeled instrument and calculate according to the formula:

[0129] TC content (mmol / L) = (sample OD-blank OD) / (standard OD-blank OD) × standard concentration (mmol / L).

[0130] 3) Plasma low-density lipoprotein cholesterol (LDL) detection:

[0131] (1) Add 180 μl of working solution 1 to each well of a 96-well ELISA plate, then add 2.5 μl of distilled water, standard or plasma sample, mix well, and incubate in a 37°C incubator for 5 min. Measure the OD1 value of each well at a wavelength of 546 nm on the ELISA plate.

[0132] (2) Then add 60 μl of working solution 2 to each well and incubate in a 37°C incubator for 5 min. Measure the OD2 value of each well at a wavelength of 556 nm using an ELISA reader. ΔOD = OD2 - OD1. Calculate according to the formula:

[0133] LDL content (mmol / L) = (sample ΔOD-blank ΔOD) / (standard ΔOD-blank ΔOD) × standard concentration (mmol / L).

[0134] 4) Plasma high-density lipoprotein cholesterol (HDL) test:

[0135] (1) Add 180 μl of working solution 1 to each well of a 96-well ELISA plate, then add 2.5 μl of distilled water, standard or plasma sample, mix well, and incubate in a 37°C incubator for 5 min. Measure the OD1 value of each well at a wavelength of 546 nm on the ELISA plate.

[0136] (2) Then add 60 μl of working solution 2 to each well and incubate in a 37°C incubator for 5 min. Measure the OD2 value of each well at a wavelength of 556 nm using an ELISA reader. Calculate ΔOD=OD2-OD1 according to the formula:

[0137] HDL content (mmol / L) = (sample ΔOD-blank ΔOD) / (standard ΔOD-blank ΔOD) × standard concentration (mmol / L).

[0138] 5) Plasma malondialdehyde (MDA) test (built in Nanjing):

[0139] (1) Prepare 1.5 ml Eppendorf tubes as blank tube (add 100 μl of anhydrous ethanol), standard tube (add 100 μl of 10 nM standard), assay tube (add 100 μl of serum) and control tube (add 100 μl of serum, 1 tube). Add 100 μl of reagent 1 to each tube and mix well.

[0140] (2) Add 300 μl of Reagent 2 to each tube, and add 100 μl of Reagent 3 to each tube except the control tube. Add 100 μl of 50% glacial acetic acid to the control tube.

[0141] (3) Place in a 95℃ water bath for 40 min, remove and cool under running water. Centrifuge at 4000 rpm for 10 min, take 200 μl of the supernatant and place on an ELISA plate, and measure the absorbance at 532 nm.

[0142] IV. Liver tissue embedding, sectioning and staining:

[0143] 1) Paraffin-embedded tissue sections:

[0144] (1) Sampling: The left lobe of mouse liver was fixed with 4% paraformaldehyde for 48 h.

[0145] (2) Dehydration: After paraformaldehyde fixation, the tissue was rinsed in running water overnight. Dehydrated in 70%, 80%, 90% and anhydrous ethanol for 1 h in sequence;

[0146] (3) Transparency: Place in xylene for 30 min each time;

[0147] (4) Wax dipping: Place the tissue in an oven at a constant temperature of 60°C and dip it in wax three times, each time for 40 min;

[0148] (5) The tissue is placed in a wax embedding box. After cooling, the paraffin solidifies and is embedded into a tissue wax block.

[0149] (6) Use a microtome to cut the paraffin sections into 5 μm slices, attach them to adhesive slides, and dry them at 60°C.

[0150] 2) HE staining:

[0151] (1) Dewaxing: Immerse the paraffin sections in xylene twice, 20 min each time;

[0152] (2) Hydration: Take out the slices and place them in anhydrous ethanol twice, 5 min each time, 90%, 80%, and 70% ethanol for 5 min each, and wash them in distilled water twice, 3 min each time;

[0153] (3) Staining: Use hematoxylin staining solution for 15 min, rinse with running water for 10 min, wash with distilled water for 10 s, stain with eosin for 30 s, and rinse with distilled water;

[0154] (4) Dehydration: Soak the slices in 95% ethanol for 1 min and in anhydrous ethanol for 3 min, twice;

[0155] (5) Transparency: Place in xylene twice, 10 min each time;

[0156] (6) Sealing: Let the xylene dry, seal with neutral resin, observe under a microscope, and take photos of three fields of view for each section.

[0157] 5. Cell culture:

[0158] 1) Cell culture medium: AML12 cells were cultured in DMEM medium containing 10% FBS, 100 U / ml penicillin, and 0.1 mg / ml streptomycin; MIN6 cells were cultured in DMEM medium containing 10% FBS, 100 U / ml penicillin, 0.1 mg / ml streptomycin, and 50 μM β-mercaptoethanol;

[0159] 2) Cell recovery: Take out the cells from the liquid nitrogen tank, quickly thaw them in a 37℃ water bath, transfer the cell suspension to a centrifuge tube containing 5ml culture medium, mix well, and centrifuge at 1000 rpm for 5 min. After centrifugation, discard the supernatant, resuspend the precipitate in culture medium, transfer it to a 100 mm cell culture dish, and culture it in a cell culture incubator at 37℃ and 5% CO2;

[0160] 3) Cell passaging: When the cell confluence reaches 80%, discard the culture medium, wash with PBS buffer, add 3 ml 0.25% trypsin for digestion, and immediately add 3 ml DMEM culture medium containing 10% FBS to terminate digestion when the cell morphology becomes round and the gap increases under the microscope. Gently blow to make the cells fall off the culture dish, collect the cell suspension into a centrifuge tube, and centrifuge at 1000 rpm for 5 min. After centrifugation, discard the supernatant, resuspend the precipitate with culture medium, transfer to a new culture dish, and culture in a cell culture incubator at 37°C and 5% CO2;

[0161] 4) Cell cryopreservation: When the cell confluence reaches 80-90%, digest the cells as usual and centrifuge them. After aspirating the supernatant, resuspend the precipitate with 1 ml of freezing solution and store it in a -80°C refrigerator. After overnight, transfer it to liquid nitrogen for long-term storage.

[0162] 6. AML12 cell CCK8 test:

[0163] 1) Cell inoculation: Logarithmic phase cells were collected and inoculated into 96-well plates at 5000 cells per well. The cells were divided into normal control group, GP group (40mM Glu + 400µM PA), GP+MET group (GP+MET, 2mM), GP+1-MHMD low-dose group (GP+1-MHMD, 2mM), and GP+1-MHMD high-dose group (GP+1-MHMD, 4mM), with 4 replicate wells for each concentration;

[0164] 2) Drug intervention: cells were cultured under 5% CO2 and 37°C. After overnight attachment, cells in each group were treated with GP solution and / or drugs at corresponding concentrations for 12 h.

[0165] 3) Color development: 12 hours after cell administration, add 10 μl of CCK8 solution to each well;

[0166] 4) Colorimetry: Select 450nm wavelength to measure the absorbance of each well.

[0167] 7. Detection of transcriptional levels of inflammatory factors in AML12 cells:

[0168] 1) AML12 cells in the logarithmic growth phase were selected and cultured at 8×10 5 Each well was inoculated into a six-well plate. They were divided into a normal control group, a GP group (40mM Glu + 400µM PA), a GP+MET group (GP+MET, 2mM), a GP+1-MHMD low-dose group (GP+1-MHMD, 2mM), and a GP+1-MHMD high-dose group (GP+1-MHMD, 4mM), with 3 replicate wells for each concentration.

[0169] 2) The cells were cultured at 5% CO2 and 37°C. After overnight attachment, the cells were replaced with serum-free medium for pretreatment for 6 hours. After that, the cells in each group were treated with GP solution and / or drugs at corresponding concentrations for 12 hours. After quick freezing in liquid nitrogen, the cells were stored at -80°C for subsequent RT-PCR experiments.

[0170] 8. Annexin-V / PI double staining method to analyze the apoptosis rate of MIN6 cells:

[0171] 1) MIN6 cells in the logarithmic growth phase were selected and cultured at 6.5×10 5Each well was inoculated into a six-well plate. It was divided into a normal control group, a GP group (40mM Glu + 400µM PA), a GP+MET group (GP+MET, 2mM), a GP+1-MHMD low-dose group (GP+1-MHMD, 2mM), and a GP+1-MHMD high-dose group (GP+1-MHMD, 4mM), with 3 replicate wells for each concentration;

[0172] 2) The cells were cultured under 5% CO2 and 37°C. After overnight attachment, the cells were replaced with serum-free medium for pretreatment for 6 hours. Then, the cells in each group were treated with GP solution and / or drugs at corresponding concentrations for 12 hours. The cells were collected and centrifuged at 300×g for 5 minutes, and the supernatant was discarded. The cells were washed with PBS, gently resuspended and counted;

[0173] 3) Take 1-5×10 5 The resuspended cells were centrifuged at 300×g for 5 min and the supernatant was discarded. The cells were washed once with PBS, centrifuged and the supernatant was discarded, and 500 μl of diluted 1× Annexin V Binding Buffer was added to resuspend the cells;

[0174] 4) Add 5 μl of Annexin V-FITC Reagent and 5 μl of PI Reagent (50 μg / ml) to the cell suspension;

[0175] 5) After gently vortexing to mix, incubate at room temperature in the dark for 15-20 minutes and immediately test on the instrument.

[0176] 9. Detection of reactive oxygen levels in MIN6 cells:

[0177] 1) MIN6 cells in the logarithmic growth phase were selected and cultured at 6×10 5 Each well was inoculated into a six-well plate. It was divided into a normal control group (Con), a GP group (40mM Glu + 400µM PA), a GP+MET administration group (2mM), and a GP+1-MHMD administration group (4mM), with 2 replicate wells for each concentration;

[0178] 2) The cells were cultured under 5% CO2 and 37°C. After overnight attachment, the cells were replaced with serum-free medium for pretreatment for 6 hours. After that, the cells in each group were treated with GP solution and / or drugs at corresponding concentrations for 12 hours.

[0179] 3) Remove the cell culture medium and add an appropriate volume of diluted DCFH-DA, the volume added should be sufficient to cover the cells;

[0180] 4) Incubate at 37°C for 20 min, and wash the cells three times with serum-free cell culture medium to fully remove DCFH-DA that has not entered the cells;

[0181] 5) Observe the cells immediately using a confocal laser microscope.

[0182] 10. Real time PCR experiment:

[0183] 1) Total RNA extraction:

[0184] (1) Add 600 μl Trizol lysis buffer to each well of a 6-well plate to completely cover the plate. After standing on ice for 5 min, repeatedly pipette and transfer the cells to an RNase-free 1.5 ml Eppendorf tube.

[0185] (2) Add 1 / 5 of the volume of Trizol chloroform to each tube, shake thoroughly to mix, and place on ice for 15 min. Centrifuge at 12,000 rpm and 4°C for 15 min. After centrifugation, collect the upper aqueous phase and place it in another 1.5 ml Eppendorf tube.

[0186] (3) Add an equal volume of isopropanol to the water phase, mix by inverting, and place on ice for 15 min. Centrifuge at 12,000 rpm and 4°C for 15 min. Carefully discard the supernatant. A white precipitate may be seen on the bottom of the tube.

[0187] (4) Add 500 μl of pre-cooled 75% ethanol to each tube to wash the precipitate, centrifuge at 12,000 rpm and 4°C for 15 min, and carefully discard the supernatant;

[0188] (5) Centrifuge at idle for 1 min, leave the tube open at room temperature for 5 min, and add an appropriate amount of DEPC water to dissolve the precipitate;

[0189] (6) Use agarose gel electrophoresis to detect whether RNA is degraded. If not, use an ELISA reader to detect the RNA concentration of each sample.

[0190] 2) Reverse transcription:

[0191] One-step reverse transcription, the configuration system and reaction conditions are as follows:

[0192] Components volume 5×RT MasterMix 4 μl Total RNA 2 μg <![CDATA[Nuclease-free H2O]]> Up to 20μl

[0193] Reaction temperature time 37℃ 15 min 60℃ 10 min 95℃ 3 min

[0194] 3) Real time PCR detection of target gene:

[0195] Prepare each component on ice, mix well and centrifuge. The reaction system is as follows:

[0196] Components volume Primer F (5 μM) 0.9 μl Primer R (5 μM) 0.9 μl FastStart Universal SYBR Green Master (ROX) (2×) 7.5 μl cDNA 2 μl DEPC water Up to 15 μl

[0197] result:

[0198] 1. 1-MHMD can inhibit the increase of body weight and blood glucose induced by HFD in mice:

[0199] To verify the therapeutic effect of 1-MHMD on blood glucose and body weight in obese mice, we established a HFD-induced obese mouse model. After 8 weeks of HFD feeding, 1-MHMD was administered intragastrically once a day for 16 weeks, and body weight and fasting blood glucose were measured at the same time point every week. Fig.16 As shown in (A), after 8 weeks of HFD feeding, the body weight of mice in the HFD group increased significantly compared with the Chow group (p < 0.001). After 1-MHMD intervention, the weight gain trend of mice in each drug-treated group slowed down, especially the weight of mice in the high-dose drug-treated group was significantly lower than that in the HFD group after 16 weeks of drug administration (p < 0.05), indicating that 1-MHMD has a certain regulatory effect on the weight gain caused by a high-fat diet. However, there was no significant difference in the body weight of mice between the low-dose and high-dose 1-MHMD drug-treated groups, and between the various 1-MHMD drug-treated groups and the MET drug-treated group. Fig.16 As shown in (B), before treatment, there was no significant difference in fasting blood glucose among the groups of mice fed with high-fat diet. Compared with the positive drug MET, the fasting blood glucose of mice decreased more significantly after 1-MHMD administration. On the other hand, the blood glucose of mice in the high-dose 1-MHMD administration group was also more stable throughout the experiment. At the end of the experiment, compared with the HFD group mice, the blood glucose of mice in the MET administration group and the high-dose 1-MHMD administration group was significantly reduced (p < 0.05), among which the blood glucose of mice in the high-dose 1-MHMD administration group was lower than that of MET, but there was no significant difference. In summary, 1-MHMD can effectively regulate the increase in body weight and blood glucose induced by high-fat diet in mice, and its stabilizing effect on blood glucose is even better than metformin when administered at 150 mg / kg.

[0200] 2. 1-MHMD improves glucose tolerance in HFD-induced obese mice:

[0201] When the body's ability to utilize glucose is reduced, the time curve of blood sugar levels after sugar loading rises and falls slowly, and even remains at a high level 2 hours after sugar loading. This state is called impaired glucose tolerance or impaired glucose tolerance. Fig.17 As shown in the figure, through the statistics of the area under the curve (AUC) of the OGTT experiment, it can be seen that compared with the Chow group, the AUC value of the HFD group increased significantly (p < 0.001), and compared with the HFD group, the AUC of the drug-treated mice decreased significantly, among which the AUC of the mice in the high-dose 1-MHMD group decreased the most (p < 0.05), indicating that 1-MHMD can effectively improve the abnormal glucose tolerance of mice caused by long-term high-fat diet.

[0202] 3.1-MHMD improves insulin sensitivity in HFD-induced obese mice:

[0203] like Fig.18 As shown in the figure, compared with the Chow group, the blood glucose of the HFD group mice decreased more slowly within 0-15min after receiving 0.5U / kg of insulin, indicating that their insulin sensitivity was reduced. Compared with the HFD group mice, the blood glucose of the mice in the 1-MHMD high-dose group decreased more rapidly during 0-15min, indicating that the body was more sensitive to exogenous insulin. Statistics of AUC showed that the AUC of the HFD group mice was significantly increased compared with the Chow group mice, while the AUC of the MET group and the 1-MHMD high-dose group mice was significantly decreased compared with the HFD group mice. This indicates that the HFD diet for 20 weeks led to the insulin resistance of mice, and each administration group had a certain therapeutic effect on this. MET and 1-MHMD could significantly reverse this trend when administered at high doses.

[0204] 4. Effects of 1-MHMD on liver histopathology in HFD-induced obese mice:

[0205] High-fat diet is one of the important factors causing obesity, which can cause significant changes in the tissue morphology of the liver and thus affect normal energy metabolism. Fig.19 As shown, HE staining showed that the hepatocyte morphology, central vein and portal vein regions, hepatocyte cords and hepatic sinusoids of mice in the Chow group were normal, the tissue structure was complete and transparent, and there were no pathological signs. The liver tissue in the HFD group was overall disordered, the hepatic cords were irregularly arranged, the cytoplasm was loose and lightly stained, swollen, the nuclei were condensed and pigmented, the hepatic sinusoids were compressed by hypertrophic hepatocytes, and there were scattered inflammatory cell infiltrations in the local hepatic lobules. The hepatocytes in the area around the central vein showed varying degrees of macrovesicular and microvesicular fatty degeneration, and some cell nuclei were significantly displaced, showing the morphology of typical fatty liver. After administration, the hepatocytes of mice in each group were arranged normally, the nuclear structure was clear, and the inflammatory cells were reduced, indicating that the degree of fatty degeneration and damage of the liver was significantly improved. There was almost no obvious fatty degeneration in the liver tissue of mice in the high-dose 1-MHMD administration group, indicating that 1-MHMD has excellent therapeutic potential for NAFLD.

[0206] 5. 1-MHMD alleviates lipid accumulation in HFD-induced obese mice:

[0207] Studies have shown that hyperlipidemia plays a key role in the pathogenesis of various metabolic diseases induced by obesity, including vascular sclerosis, fatty liver, insulin resistance, etc. In addition, high blood lipids can produce lipotoxicity, inhibit the secretion of insulin by β cells, and thus lead to the occurrence of diabetes. Therefore, we detected the levels of TG, TC, LDL and HDL in the plasma of each group of mice. Fig. 20As shown in (A, B, C), the plasma TG, T-CHO, and LDL of mice in the HFD group were significantly higher than those in the control group, indicating that the high-fat diet damaged the normal blood lipid levels of mice. Continuous oral administration of 1-MHMD for 16 weeks can reverse this damage. It is worth noting that in the regulation of TC and LDL, 1-MHMD showed a pharmacological effect superior to metformin in a dose-dependent manner. In addition, HDL can transport cholesterol from extrahepatic tissues to the liver for metabolism, thereby achieving the effect of clearing tissue cholesterol. Fig. 20 As shown in (D), HFD feeding caused a significant decrease in plasma HDL-C in mice, while the increased HDL-C level in the 1-MHMD-treated group indicated that it did have a positive effect on lowering blood lipids.

[0208] 6. 1-MHMD inhibited liver damage in mice caused by long-term HFD diet:

[0209] ALT and AST have different distributions in liver cells. ALT is mainly distributed in the liver cytoplasm, and its increased plasma level can reflect liver cell membrane damage; while AST is mainly distributed in the liver cytoplasm and liver cell mitochondria, and its increase indicates that liver cell damage has reached the organelle level. Fig.21 As shown, compared with the Chow group mice, HFD feeding for 24 weeks significantly increased the serum ALT and AST levels of mice, and the plasma ALT and AST levels of each drug-treated group were significantly lower than those of the HFD group mice. The therapeutic effects of each dose-treated group of 1-MHMD were better than those of the positive control drug MET.

[0210] 7. 1-MHMD reversed lipid peroxidation in mice caused by long-term HFD diet:

[0211] MDA is a byproduct of lipid peroxidation of polyunsaturated fatty acids. Free radicals attack polyunsaturated fatty acids in cell membranes to generate lipid peroxidation, which increases the concentration of MDA. The increase in its level indicates that the oxidative stress in the body is enhanced and the balance between free radical generation and removal is broken. It is one of the most common indicators for detecting lipid peroxides in cells and tissues. Fig. 22 As shown in the figure, compared with the Chow group mice, the plasma MDA level of the HFD group mice was significantly increased, indicating that the 24-week high-fat diet caused significant plasma lipid peroxidation. MET had no therapeutic effect on this, while low-dose and high-dose 1-MHMD continuous oral administration for 16 weeks completely reversed this damage, suggesting that 1-MHMD may have a beneficial effect on liver tissue by inhibiting oxidative stress.

[0212] 8. Effects of 1-MHMD on AML12 cells:

[0213] like Fig.23As shown in Figure 2, 1-MHMD treatment for 12 h significantly promoted the proliferation of hepatocytes in response to the inhibition of AML12 cell proliferation caused by glucolipid toxicity. Fig.24 As shown in the figure, compared with the GP group, 1-MHMD significantly reduced the transcription levels of classic pro-inflammatory factors such as IL-6, IL-1β, TNFα, and MCP1 in hepatocytes. In summary, 1-MHMD can inhibit the cellular inflammatory response caused by glucolipid toxicity and promote cell proliferation.

[0214] 9. Effects of 1-MHMD on MIN6 cells:

[0215] like Fig.25 As shown in the figure, compared with the Con group, GP caused a significant increase in apoptotic cells, while MET had no significant effect on this. 1-MHMD significantly inhibited cell apoptosis after administration at doses of 2 and 4 mM. ROS (reactive oxygen species) is a chemical substance with oxidative activity produced in cells. When it is produced excessively or not cleared enough, it will cause oxidative stress to cells, leading to cell damage and disease. Fig.26 As shown, 1-MHMD significantly inhibited the accumulation of ROS in pancreatic islet cells caused by GP, indicating that 1-MHMD has the effect of inhibiting pancreatic islet cell apoptosis and reducing excessive ROS in pancreatic islet cells.

[0216] In conclusion, 1-MHMD can be used in the preparation of drugs for lowering blood sugar, lowering blood lipids and treating fatty liver.

[0217] 1-MHMD can be used in the preparation of weight loss drugs.

[0218] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A 1-MHMD, characterized in that The structural formula of the 1-MHMD is: ; wherein R1 is selected from CH2, CH2CH2.

2. The 1-MHMD according to claim 1, characterized in that The 1-MHMD is a pharmaceutically acceptable salt.

3. A method for preparing 1-MHMD, characterized in that: The following steps are involved: 1-Methylhydantoin is subjected to substitution reaction with XR1COR2 to obtain intermediate I: ; Intermediate I is reacted with metformin hydrochloride by base catalysis to obtain a crude product, High-purity 1-MHMD was obtained after recrystallization: Alternatively, XR1COR2 is reacted with metformin hydrochloride by base catalysis to obtain intermediate II: ; 1-methylhydantoin is reacted with intermediate II to obtain a crude product; High-purity 1-MHMD was obtained after recrystallization; wherein X is selected from halogen atoms; R2 is selected from an alkoxy group containing 1 to 10 carbon atoms and a halogen atom.

4. The method for preparing 1-MHMD according to claim 3, characterized in that: The step of subjecting 1-methylhydantoin to a substitution reaction with XR1COR2 to obtain an intermediate I; The steps of reacting the intermediate I with metformin hydrochloride by base catalysis to obtain a crude product, and then recrystallizing to obtain high-purity 1-MHMD include: Add 1-methylhydantoin and potassium carbonate to N,N-dimethylformamide solvent, heat the mixture to 45°C-100°C, and stir for 1h-3h to prepare reaction solution a; Add XR1COR2 to the reaction solution a, heat to 52°C-100°C, and stir for 8h-16h; remove the solvent under reduced pressure to obtain a crude product; Pour in ethyl acetate, perform ultrasonic extraction for 5 minutes to 60 minutes, filter out the precipitate, and repeat the extraction operation on the precipitate 3 to 5 times; The organic phases were combined and the solvent was removed under reduced pressure to obtain intermediate I; Add metformin hydrochloride to the solvent methanol at room temperature, and also add the catalyst sodium methoxide thereto, and stir the suspension at a temperature of 25°C-40°C for 20 minutes-3 hours to prepare solution b; The intermediate I is diluted in the solvent methanol to prepare solution c; Solution c was slowly added dropwise into solution b, and the reaction was stirred at 20°C-50°C for 8h-16h. After the reaction was completed, the solvent was removed under reduced pressure to obtain a crude product, which was then recrystallized from ethanol to obtain 1-MHMD.

5. The method for preparing 1-MHMD according to claim 3, characterized in that: The method comprises reacting XR1COR2 with metformin hydrochloride by base catalysis to obtain intermediate II; reacting 1-methylhydantoin with intermediate II to obtain a crude product; The steps of obtaining high-purity 1-MHMD after recrystallization include: Add metformin hydrochloride to solvent ethanol at room temperature, and also add catalyst sodium ethoxide thereto, and stir the suspension at a temperature of 30°C-50°C for 25min-3h to prepare solution a; Dilute XR1COR2 in methanol solvent to make solution b; Slowly drop solution b into solution a, and stir the reaction at 20-55°C for 8-16 hours; The solvent was removed by rotary evaporation under reduced pressure, and the intermediate II was obtained by recrystallization; Add 1-methylhydantoin and potassium carbonate to N,N-dimethylformamide solvent, heat the mixture to 60°C-110°C and stir for 0.5h-1h to prepare reaction solution c; Add intermediate II to reaction solution c, heat to 75-90°C and stir for 16-48h; The solvent was removed under reduced pressure to obtain a crude product, which was recrystallized to give 1-MHMD.

6. Use of 1-MHMD as claimed in claim 1 in the preparation of drugs for lowering blood sugar, lowering blood lipids and treating fatty liver.

7. Use of 1-MHMD as claimed in claim 1 in the preparation of weight loss drugs.

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