Glycyrrhetinic acid-berberine derivatives and use thereof in preparing drugs for treating metabolic diseases

Abstract

The application belongs to the technical field of biological medicine, and relates to a chemical medicine for treating metabolic diseases, in particular to glycyrrhetinic acid-berberine derivative and application thereof in preparation of a medicine for treating metabolic diseases. The chemical structure is shown as formula I, wherein n is 1-6, is a single bond or a double bond, R is O, a hydroxyl group or R' is a C1-C5 alkyl group. The glycyrrhetinic acid with wide biological activity is combined with berberine, the bioavailability and activity of berberine and glycyrrhetinic acid when used alone are significantly improved, the treatment effect on metabolic diseases, especially diabetes and hyperlipidemia is improved from mmol level to nmol level, and the safety of clinical medication of berberine is greatly improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to chemical drugs for treating metabolic diseases, specifically to glycyrrhetinic acid-berberine derivatives and their application in the preparation of drugs for treating metabolic diseases. Background Technology

[0002] Current treatments for metabolic diseases include lifestyle interventions, a balanced diet, appropriate exercise, and medication. In recent years, significant progress has been made in the use of biguanides, sulfonylureas, statins, and glucocorticoids to treat metabolic diseases. However, these medications still have limitations due to their adverse reactions, such as gastrointestinal discomfort, hypoglycemia, liver dysfunction, myalgia, weight gain, and cardiovascular damage.

[0003] Berberine is an important tetrabenzylisoquinoline alkaloid, but its clinical application in the treatment of metabolic diseases is limited by its side effects (such as gastrointestinal discomfort, nausea and vomiting) and poor bioavailability. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a glycyrrhetinic acid-berberine derivative and its application in the preparation of drugs for treating metabolic diseases. This invention combines glycyrrhetinic acid, which has broad biological activity, with berberine, significantly improving the bioavailability and activity of berberine and glycyrrhetinic acid when used alone. Furthermore, it enhances the therapeutic effect on metabolic diseases, especially diabetes and hyperlipidemia, from the mmol level to the nmol level, greatly improving the safety of clinical use of berberine.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: Firstly, a glycyrrhetinic acid-berberine derivative, the chemical structure of which is shown in Formula I.

[0006] Where n is 1 to 6, It can be a single or double bond, and R can be O, hydroxyl, or... R' is a C1-C5 alkyl group.

[0007] In some embodiments, It is a single bond, and R is a hydroxyl group or R' is a C1-C5 alkyl group.

[0008] In some embodiments, It is a double bond, and R is O.

[0009] When it is a single key, it can be It can also be used as In some embodiments, the structural formula is as follows: .

[0010] In some embodiments, R' is a C1-C5 straight-chain alkyl group.

[0011] In some embodiments, R' is a C1-C3 straight-chain alkyl group.

[0012] In some embodiments, R' is a methyl group.

[0013] In some embodiments, n is 1, 3, 4 or 6.

[0014] Secondly, a method for preparing a glycyrrhetinic acid-berberine derivative includes the step of obtaining the compound shown in Formula I according to the following reaction route;

[0015] Wherein, R and n are as described above.

[0016] In some embodiments, berberine (BBR) is reacted with acetone in a strongly alkaline environment (such as a solution of a strong base like sodium hydroxide or potassium hydroxide) to give compound 1. Compound 1 then reacts with diiodoalkane (I-CH2-(CH2)). n -CH2-I, or I-CH2-CH2-(CH2) n -I) Compound 2 is obtained by nucleophilic substitution. Compound 2 is then reacted with glycyrrhetinic acid or its derivatives by nucleophilic substitution to obtain the compound shown in Formula I.

[0017] In one or more embodiments, the nucleophilic substitution reaction of compound 1 to prepare compound 2 is carried out under reflux conditions. Specifically, acetonitrile is used as the organic solvent.

[0018] In one or more embodiments, the nucleophilic substitution reaction of compound 2 with glycyrrhetinic acid or its derivatives to prepare the compound of formula I is carried out under the following conditions: stirring at room temperature under alkaline conditions. Specifically, the alkaline conditions can be adjusted by alkaline compounds such as potassium carbonate or cesium carbonate. Specifically, the solvent used is N,N-dimethylformamide.

[0019] In some embodiments, the specific reaction route is as follows:

[0020] In step a, berberine (BBR) undergoes addition reaction with acetone in a strongly alkaline environment to yield compound 1; the strongly alkaline environment is an aqueous solution of sodium hydroxide (5N).

[0021] In step b, compound 1 undergoes nucleophilic substitution with the corresponding diiodoalkane to obtain compounds 2a-d; the nucleophilic substitution reaction is carried out in an organic solvent; the organic solvent is acetonitrile; the diiodoalkane is 1,3-diiodopropane, 1,5-diiodopentane, 1,6-diiodohexane, 1,8-diiodooctane, etc.; the reaction condition is heating under reflux.

[0022] In step c, glycyrrhetinic acid is acetylated to obtain compound 3; the acetylation reaction is carried out in an acetylation reagent, under alkaline conditions, and in an organic solvent; the acetylation reagent is acetic anhydride; the alkaline conditions and the organic solvent are pyridine.

[0023] In step d, glycyrrhetinic acid is oxidized to obtain compound 4; the oxidation reaction is carried out in an organic solvent in the presence of an oxidizing agent; the organic solvent is N,N-dimethylformamide; the oxidizing agent is pyridine dichromate or pyridine chlorochromate.

[0024] In step e, compound 2 reacts with glycyrrhetinic acid or its derivatives via a nucleophilic substitution reaction to obtain compounds 5a-d, 6a-d, and 7a-d; the nucleophilic substitution reaction is carried out under alkaline conditions in an organic solvent; the alkaline conditions are excess potassium carbonate, cesium carbonate, etc.; the organic solvent is N,N-dimethylformamide; compound 2 is 2a-d as described in step b; and glycyrrhetinic acid or its derivatives are compounds 3 and 4 as described in steps (c) and (d).

[0025] Thirdly, a pharmaceutical composition comprising the above-mentioned glycyrrhetinic acid-berberine derivative or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0026] The pharmaceutically acceptable salts described in this invention can be salts formed by the compound and inorganic acids (such as hydrochloric acid, sulfuric acid, nitric acid, etc.), or salts formed by the compound and organic acids (such as acetic acid, oxalic acid, malonic acid, lactic acid, citric acid, malic acid, tartaric acid, p-benzenesulfonic acid, fumaric acid, maleic acid, etc.).

[0027] The solvates mentioned in this invention refer to compounds formed in organic solvents, such as ethanol derivatives, ether derivatives, and acetone compounds of glycyrrhetinic acid-berberine derivatives.

[0028] The hydrates mentioned in this invention refer to compounds containing water of crystallization, and can be hemihydrates, monohydrates, dihydrates, etc.

[0029] In some embodiments, a pharmaceutical carrier is also included. Examples of the pharmaceutical carrier include aluminum stearate, lecithin, serum albumin, polyethylene glycol, colloidal silica, sodium carboxymethyl cellulose, beeswax, microcrystalline wax, isopropyl palmitate, etc.

[0030] In some embodiments, excipients are also included. These excipients include binders, fillers, lubricants, disintegrants, stabilizers, antioxidants, etc.

[0031] Fourthly, a pharmaceutical preparation comprising the above-mentioned glycyrrhetinic acid-berberine derivative, wherein the dosage form is a liquid dosage form or a solid dosage form.

[0032] Specifically, the liquid dosage form can be an oral liquid, injection, tincture, decoction, medicated wine, syrup, elixir, etc. The solid dosage form can be a tablet, granule, capsule, pellet, powder, film, etc.

[0033] Fifthly, the use of the above-mentioned glycyrrhetinic acid-berberine derivative, pharmaceutical composition, or pharmaceutical preparation in the preparation of SCD1 protein inhibitors, SREBP-1 protein inhibitors, SREBP-1c phosphorylation promoters, AMPK activators, or cholesterol inhibitors.

[0034] Sixthly, the use of the above-mentioned glycyrrhetinic acid-berberine derivative, pharmaceutical composition, or pharmaceutical preparation in the preparation of a drug for treating metabolic diseases.

[0035] Specifically, the metabolic diseases include diabetes and / or hyperlipidemia, etc.

[0036] The beneficial effects of this invention are: Pharmacological experiments have revealed that the glycyrrhetinic acid-berberine derivatives provided in this invention can regulate the protein expression levels in pathways related to metabolic diseases, thus exerting a therapeutic effect. Their main function is manifested in insulin- and high-glucose-induced cell models, where these compounds can activate the AMPK-SREBP-1c-SCD1 pathway in metabolic diseases, promote the phosphorylation of AMPK and SREBP-1c, and thereby reduce SCD1 expression. This regulates lipid metabolism, reduces cholesterol levels, inhibits phospholipid production, and suppresses inflammatory responses in the body and vascular walls, demonstrating significant therapeutic effects in chronic diseases with major metabolic abnormalities such as diabetes and its complications, and hyperlipidemia. Attached Figure Description

[0037] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0038] Figure 1 This is a graph showing the expression levels of proteins related to metabolic pathways in HepG2 (A) and HL7702 (B) cells after compound treatment in the embodiments of the present invention. Figure 2The graph shows the expression levels of proteins related to metabolic pathways in HepG2 cells after treatment with different concentrations of compound 7c in this embodiment of the invention. A is a Western blot, B is a bar chart of m-SREBP1, and C is a bar chart of SCD1. Figure 3 This is a diagram showing the effect of compound 7c inhibiting the SREBP1 pathway by activating AMPK in an embodiment of the present invention; Figure 4 The image shows the immunofluorescence results of compound 7c downregulating the expression of the mature form SREBP1 in the embodiments of the present invention. A is blank, B is insulin + glucose, C is compound 7c, and D is berberine + glycyrrhetinic acid. Figure 5 These are fluorescence microscopy images of compound 7c reducing cellular cholesterol levels in embodiments of the present invention. A is the model, B is the positive control drug (25-HC), C is 0.4 μM compound 7c, and D is 0.8 μM compound 7c. Figure 6 The following graphs show the results of in vivo experiments in this embodiment of the invention: A is the body weight curve, B is the bar chart of fat to body weight ratio, C is the bar chart of liver weight, D is the bar chart of fat (WAT) weight, E is the insulin resistance curve, and F is the glucose tolerance curve. The in vivo activity evaluation of compound 7c showed statistically significant differences compared to the model group (#P<0.05, ##P<0.01, ###P<0.001, *P<0.05, **P<0.01, ***P<0.001, *P<0.001, ***P<0.001, *P<0.05, **P<0.01, ***P<0.001, * ... Figure 7 The image shows a representative photograph (A) of the mouse liver in this embodiment of the invention and the result of the effect on fat and liver histology (B). Detailed Implementation

[0039] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0040] The reaction routes of the compounds in the following examples are shown below.

[0041]

[0042] Example 1: Preparation of berberine derivatives 2a-d Accurately weigh berberine hydrochloride (BBR) (1.0 g, 2.7 mmol) and dissolve it in 5N NaOH (10 mL) solution. While stirring, slowly add 0.8 mL of acetone dropwise. After stirring at room temperature for 1 h, filter the reaction mixture and wash with 80% MeOH until neutral to obtain intermediate 1, which can be directly proceeded to the next reaction without purification. Then, add diiodoalkane (10 mmol) to a dry MeCN solution of compound 1 (1 mmol) and heat the mixture to 82 °C for 6–12 h. Under TLC monitoring, after the reaction is complete, evaporate the solvent under reduced pressure to obtain the crude product, which is then subjected to column chromatography on silica gel (DCM / MeOH, 100 / 1). Purification at 50 / 1, v / v yielded intermediate 2a-d as a yellow powder.

[0043] Compound 2a: Yellow powder, yield 54%. 1 H NMR (400 MHz, CDCl3) δ 10.40 (s, 1H),8.01 (d, J = 9.3 Hz, 1H), 7.91 (d, J = 9.3 Hz, 1H), 7.13 (s, 1H), 6.93 (s, 1H), 6.14 (s, 2H), 5.15 (s, 2H), 4.42 (s, 3H), 4.12 (s, 3H), 3.62 – 3.52 (m, 2H), 3.34 (t, J = 6.1 Hz, 2H), 3.25 (t, J = 5.7 Hz, 2H), 2.37 – 2.25 (m, 2H). 13 C NMR(101 MHz, CDCl3) δ 150.3, 149.9, 147.4, 145.8, 145.1, 136.6, 133.6, 132.9,132.5, 125.6, 121.8, 120.1, 119.9, 109.1, 108.6, 102.1, 62.9, 58.0, 56.9,33.5, 30.7, 28.3, 5.1. ESI-MS m / z 504.4 [M - I] + . Compound 2b: Yellow powder, yield 43%. 1 H NMR (400 MHz, CDCl3) δ 10.24 (s, 1H),7.91 (q, J= 9.4 Hz, 2H), 7.08 (s, 1H), 6.89 (s, 1H), 6.11 (s, 2H), 5.09 (s,2H), 4.35 (s, 3H), 4.07 (s, 3H), 3.31 (t, J = 8.1 Hz, 2H), 3.20 (q, J = 6.8, 5.9Hz, 4H), 1.92 – 1.82 (m, 4H), 1.65 – 1.56 (m, 2H). 13 C NMR (101 MHz, CDCl3) δ150.4, 149.8, 147.3, 146.0, 145.1, 136.1, 133.9, 133.5, 133.0, 125.6, 121.8,120.2, 120.1, 109.0, 108.6, 102.2, 63.1, 58.1, 57.0, 32.4, 30.2, 29.9, 29.8,28.5, 6.6. ESI-MS m / z 532.4 [M - I] + . Compound 2c: Yellow powder, yield 47%. 1 H NMR (400 MHz, CDCl3) δ 10.29 (s, 1H),7.89 (q, J = 9.6 Hz, 2H), 7.09 (s, 1H), 6.89 (s, 1H), 6.11 (s, 2H), 5.11 (s,2H), 4.37 (s, 3H), 4.08 (s, 3H), 3.29 (t, J = 8.8 Hz, 2H), 3.21 (td, J = 6.5, 3.8Hz, 4H), 1.94 – 1.79 (m, 4H), 1.60 – 1.43 (m, 4H). 13 C NMR (101 MHz, CDCl3) δ150.2, 149.7, 147.2, 145.5, 144.4, 136.0, 134.3, 133.3, 132.9, 125.6, 121.6,120.3, 120.0, 108.9, 108.4, 102.1, 62.7, 58.0, 56.9, 33.0, 30.7, 29.7, 28.3,28.2, 6.8. ESI-MS m / z546.4 [M - I] + . Compound 2d: Yellow powder, yield 55%. 1 H NMR (400 MHz, CDCl3) δ 10.25 (s, 1H),7.89 (d, J = 10.0 Hz, 2H), 7.10 (s, 1H), 6.89 (s, 1H), 6.12 (s, 2H), 5.09 (s,2H), 4.39 (s, 3H), 4.08 (s, 3H), 3.31 – 3.25 (m, 2H), 3.25 – 3.16 (m, 4H), 1.92 – 1.81 (m, 4H), 1.56 – 1.49 (m, 2H), 1.43 – 1.35 (m, 6H). 13 C NMR (101MHz, CDCl3) δ 150.3, 149.7, 147.2, 146.1, 145.2, 136.1, 134.3, 133.6, 133.1,125.4, 122.0, 120.2, 120.1, 109.1, 108.6, 102.2, 63.2, 58.2, 57.0, 33.3,31.1, 30.3, 30.0, 29.4, 28.8, 28.5, 28.3, 7.3. ESI-MS m / z 574.5 [M - I] + . Example 2: Preparation of glycyrrhetinic acid derivative 3 Under 0°C ice bath conditions, at 18 β 4 mL of pyridine was added to a solution of glycyrrhetinic acid (GA) (400 mg, 0.86 mmol). Acetic anhydride (8 mL) was added. The mixture was heated to room temperature and stirred for 16 h until the reaction was complete. The mixture was then quenched with saturated NH4Cl and extracted with DCM (20 mL × 3). The organic phase was washed with brine, dried over Na2SO4, and the solvent was removed by rotary evaporation. The crude product was then subjected to column chromatography (hexane / ethyl acetate, 10 / 10). The compound was purified (2 / 1, v / v) to give compound 3 as a white powder (400 mg, 92%). 1 H NMR (400 MHz, CDCl3) δ 5.70 (s, 1H), 4.52 (dd, J = 11.6, 4.8 Hz, 1H), 2.80 (dt, J= 13.6, 3.3 Hz, 1H), 2.37 (s, 1H), 2.19 (dd, J = 14.1, 3.5 Hz, 1H),2.05 (s, 3H), 2.04 – 1.91 (m, 3H), 1.89 – 1.79 (m, 1H), 1.73 – 1.65 (m, 3H),1.63 – 1.56 (m, 3H), 1.45 – 1.39 (m, 4H), 1.37 (s, 3H), 1.23 (s, 3H), 1.21 –1.19 (m, 1H), 1.17 (s, 3H), 1.13 (s, 3H), 1.10 – 1.01 (m, 2H), 0.88 (s, 6H),0.83 (s, 3H), 0.82 – 0.78 (m, 1H). 13 C NMR (101 MHz, CDCl3) δ 200.4, 181.6,171.1, 169.5, 128.4, 80.6, 61.7, 55.0, 48.2, 45.5, 43.8, 43.2, 40.8, 38.8,38.0, 37.7, 36.9, 32.7, 31.9, 30.9, 28.5, 28.4, 28.0, 26.4, 26.4, 23.6, 23.3,21.3, 18.7, 17.4, 16.7, 16.4. ESI-MS m / z 513.8 [M + H] + . Example 3: Preparation of glycyrrhetinic acid derivative 4 GA (1 g, 2.1 mmol) and PDC (3.2 g, 3.15 mmol) were added to a dry DMF solution (20 mL). The mixture was stirred overnight at room temperature. After the reaction was completed by TLC monitoring, the mixture was quenched with water, extracted with ethyl acetate, washed with brine, dried over Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by column chromatography (DCM / MeOH, 20 / 1, v / v) to give compound 4 as a white powder (820 mg, 83%). 1 H NMR (400 MHz, CDCl3) δ 5.74 (s, 1H), 2.97 (ddd, J = 13.3, 7.1, 4.0 Hz, 1H), 2.64 (ddd, J= 16.1, 11.3, 7.1 Hz, 1H), 2.45 (s, 1H), 2.36 (ddd, J = 15.8, 6.2, 4.0 Hz, 1H), 2.22 (dd, J = 13.4, 4.4 Hz, 1H), 2.10 –1.99 (m, 2H), 1.98 – 1.91 (m, 1H), 1.89 – 1.81 (m, 1H), 1.73 – 1.60 (m, 3H), 1.57 – 1.52 (m, 2H), 1.48 – 1.41 (m, 3H), 1.38 (s, 3H), 1.33 – 1.26 (m, 2H), 1.28 (s, 3H), 1.23 (s, 3H), 1.18 (s, 3H), 1.11 (s, 3H), 1.07 (s, 3H), 1.03(s, 1H), 0.91 – 0.86 (m, 1H), 0.86 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 217.4,199.7, 182.0, 170.0, 128.3, 61.0, 55.3, 48.2, 47.7, 45.2, 43.8, 43.3, 40.8,39.6, 37.6, 36.6, 34.2, 32.0, 31.8, 30.8, 28.5, 28.4, 26.5, 26.3, 26.3, 23.3,21.4, 18.7, 18.5, 15.6. ESI-MS m / z 469.7 [M + H] + . Example 4: Preparation of the target product, glycyrrhetinic acid-berberine derivative GA (0.5 mmol) and K2CO3 (2.0 mmol) were added to a dry DMF solution, followed by the corresponding berberine intermediate 2a. d (0.75 mmol). After stirring overnight at room temperature, TLC monitoring was performed, followed by quenching with water, extraction with dichloromethane (DCM), washing with brine, drying on Na2SO4, and removing the solvent by rotary evaporation. The crude product was purified by column chromatography (DCM / MeOH, 50 / 1-10 / 1, v / v) to give compound 5a. d, 6a-d, and 7a-d are yellow powders.

[0044] Compound 5a: Yellow powder, yield 47%. 1H NMR (400 MHz, CDCl3) δ 10.27 (s, 1H),8.07 – 7.89 (m, 2H), 7.09 (s, 1H), 6.84 (s, 1H), 6.08 (d, J = 3.6 Hz, 2H), 5.49(s, 1H), 5.24 – 5.10 (m, 1H), 5.10 – 4.90 (m, 1H), 4.34 (s, 3H), 4.26 (t, J =5.9 Hz, 2H), 4.08 (s, 3H), 3.41 (t, J = 7.3 Hz, 2H), 3.30 – 3.09 (m, 3H), 2.72(d, J = 13.4 Hz, 1H), 2.32 – 2.16 (m, 3H), 2.06 – 1.95 (m, 3H), 1.89 (d, J = 13.3Hz, 1H), 1.82 – 1.75 (m, 1H), 1.68 – 1.55 (m, 5H), 1.45 – 1.37 (m, 3H), 1.34(s, 3H), 1.32 – 1.28 (m, 1H), 1.24 (d, J = 7.0 Hz, 2H), 1.20 – 1.14 (m, 1H),1.16 (s, 3H), 1.07 (s, 3H), 1.06 (s, 3H), 1.03 – 0.93 (m, 2H), 0.98 (s, 3H),0.77 (s, 3H), 0.76 (s, 3H), 0.67 (d, J = 11.6 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ199.9, 176.2, 169.3, 150.4, 149.7, 147.2, 146.1, 145.4, 136.4, 133.8, 132.8,132.6, 128.2, 126.0, 121.8, 120.0, 119.8, 109.0, 108.6, 102.1, 78.5, 63.6,63.1, 61.8, 58.1, 57.0, 54.8, 48.6, 45.4, 44.1, 43.2, 41.1, 39.1, 39.0, 37.6,37.0, 32.6, 31.8, 31.0, 29.9, 28.5, 28.4, 28.3, 28.0, 27.2, 26.9, 26.4, 26.3,23.4, 18.6, 17.4, 16.2, 15.5. HR-ESI-MS m / z Calculated for C 53 H 68 NO8 + [M - I] + 846.4939, found 846.4935. Compound 5b: Yellow powder, yield 46%. 1 H NMR (400 MHz, CDCl3) δ 10.26 (s, 1H),7.97 (q, J = 7.8 Hz, 2H), 7.08 (s, 1H), 6.88 (s, 1H), 6.09 (s, 2H), 5.62 (s,1H), 5.11 (s, 2H), 4.36 (s, 3H), 4.19 (dt, J = 12.6, 6.4 Hz, 1H), 4.11 – 4.05(m, 1H), 4.07 (s, 3H), 3.31 (t, J = 7.8 Hz, 2H), 3.26 – 3.15 (m, 3H), 2.74 (d, J= 13.4 Hz, 1H), 2.33 (s, 1H), 2.05 – 1.98 (m, 2H), 1.96 – 1.90 (m, 2H), 1.78 – 1.70 (m, 4H), 1.65 – 1.59 (m, 6H), 1.43 – 1.38 (m, 3H), 1.36 (s, 3H), 1.34– 1.30 (m, 3H), 1.27 – 1.23 (m, 3H), 1.20 (s, 1H), 1.15 (s, 3H), 1.11 (s, 3H), 1.10 (s, 3H), 1.06 – 1.02 (m, 1H), 0.99 (s, 3H), 0.97 – 0.91 (m, 1H), 0.79 (s, 6H), 0.69 (d, J = 11.5 Hz, 1H). 13 C NMR (101 MHz, CDCl3) δ 200.2, 176.4,169.5, 150.4, 149.7, 147.2, 146.0, 145.2, 136.0, 133.9, 133.6, 133.0, 128.4,125.7, 121.9, 120.3, 120.2, 109.0, 108.6, 102.1, 78.6, 64.0, 63.1, 61.8,58.1, 56.9, 54.9, 48.6, 45.4, 44.0, 43.2, 41.1, 39.1, 39.1, 37.7, 37.1, 32.7,31.8, 31.1, 30.8, 29.9, 29.6, 28.6, 28.5, 28.4, 28.1, 27.2, 26.4, 26.3, 26.2,23.4, 18.7, 17.4, 16.3, 15.5.HR-ESI-MS m / z Calculated for C 55 H 72 NO8 + [M - I] + 874.5252, found 874.5250. Compound 5c: Yellow powder, yield 39%. 1 H NMR (400 MHz, CDCl3) δ 10.18 (s, 1H),7.93 (q, J= 9.3 Hz, 2H), 7.08 (s, 1H), 6.87 (s, 1H), 6.08 (s, 2H), 5.56 (s,1H), 5.07 (s, 2H), 4.32 (s, 3H), 4.13 (dt, J = 12.9, 6.6 Hz, 1H), 4.06 (s, 3H),4.08 – 4.01 (m, 1H), 3.27 (t, J = 7.4 Hz, 2H), 3.22 – 3.15 (m, 3H), 2.66 (d, J =13.5 Hz, 1H), 2.29 (s, 1H), 2.02 – 1.96 (m, 2H), 1.91 – 1.87 (m, 2H), 1.71 –1.62 (m, 4H), 1.59 – 1.55 (m, 5H), 1.48 – 1.44 (m, 2H), 1.40 – 1.35 (m, 3H),1.33 (s, 3H), 1.28 (d, J = 9.6 Hz, 3H), 1.22 (s, 3H), 1.17 (s, 1H), 1.11 (s,3H), 1.06 (s, 3H), 1.06 (s, 3H), 1.03 – 0.99 (m, 1H), 0.96 (s, 3H), 0.90 (s,1H), 0.76 (s, 3H), 0.75 (s, 3H), 0.65 (d, J = 11.6 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 200.2, 176.5, 169.5, 150.4, 149.8, 147.3, 145.9, 145.0, 136.2,134.3, 133.5, 133.2, 128.4, 125.7, 121.9, 120.4, 120.3, 109.1, 108.6, 102.2,78.6, 64.2, 63.1, 61.8, 58.2, 57.1, 54.9, 48.6, 45.4, 44.0, 43.3, 41.1, 39.1,37.7, 37.1, 32.7, 31.8, 31.1, 31.0, 30.0, 29.7, 29.2, 28.7, 28.6, 28.5, 28.4,28.1, 27.3, 26.5, 26.4, 25.6, 23.4, 18.7, 17.5, 16.4, 15.6.HR-ESI-MS m / z Calculated for C 56 H 74 NO8 + [M - I] + 888.5409, found 888.5404. Compound 5d: Yellow powder, yield 32%. 1 H NMR (400 MHz, CDCl3) δ 10.61 (s, 1H),7.89 (q, J = 9.5 Hz, 2H), 7.09 (s, 1H), 6.87 (s, 1H), 6.08 (s, 2H), 5.59 (s,1H), 5.25 (s, 2H), 4.32 (s, 3H), 4.13 – 4.02 (m, 2H), 4.06 (s, 3H), 3.27 (dq, J = 16.8, 8.1 Hz, 2H), 3.20 – 3.10 (m, 3H), 2.60 (d, J= 13.2 Hz, 1H), 2.27 (s,1H), 2.05 – 1.96 (m, 2H), 1.92 – 1.83 (m, 3H), 1.82 – 1.71 (m, 2H), 1.66 –1.58 (m, 4H), 1.57 – 1.48 (m, 4H), 1.44 – 1.36 (m, 8H), 1.33 (s, 3H), 1.31 –1.26 (m, 2H), 1.23 (s, 2H), 1.17 (s, 1H), 1.11 (d, J = 11.4 Hz, 4H), 1.07 (s,3H), 1.02 (s, 3H), 0.96 (s, 3H), 0.87 – 0.83 (m, 1H), 0.77 (s, 3H), 0.74 (s,3H), 0.63 (d, J = 11.8 Hz, 1H). 13 C NMR (101 MHz, CDCl3) δ 200.1, 176.4, 169.3,150.4, 149.6, 147.2, 146.5, 146.1, 135.9, 134.1, 133.9, 133.0, 128.4, 125.4,122.2, 120.3, 120.2, 109.0, 108.5, 102.1, 78.4, 64.4, 63.1, 61.8, 57.4, 57.0,54.9, 48.5, 45.4, 44.0, 43.2, 41.1, 39.1, 39.0, 37.7, 37.0, 32.7, 31.8, 31.1,29.8, 29.6, 29.5, 29.2, 28.9, 28.7, 28.5, 28.4, 28.1, 27.2, 26.4, 26.3, 26.0,23.3, 18.6, 17.4, 16.2, 15.6.HR-ESI-MS m / z Calculated for C 58 H 78 NO8 + [M - I] + 916.5722, found 916.5720. Compound 6a: Yellow powder, yield 55%. 1 H NMR (400 MHz, CDCl3) δ 10.30 (s, 1H),7.95 (q, J= 8.8 Hz, 2H), 7.09 (s, 1H), 6.85 (s, 1H), 6.08 (s, 2H), 5.50 (s,1H), 5.23 – 4.96 (m, 2H), 4.50 (d, J = 11.0 Hz, 1H), 4.35 (s, 3H), 4.30 – 4.20(m, 2H), 4.07 (s, 3H), 3.41 (d, J = 10.9 Hz, 2H), 3.27 – 3.08 (m, 2H), 2.74 (d, J = 12.4 Hz, 1H), 2.30 (s, 1H), 2.26 – 2.15 (m, 2H), 2.03 (s, 3H), 2.02 – 1.96(m, 3H), 1.90 (d, J = 13.7 Hz, 1H), 1.85 – 1.71 (m, 2H), 1.66 – 1.56 (m, 4H),1.46 – 1.36 (m, 3H), 1.34 (s, 3H), 1.31 – 1.28 (m, 1H), 1.26 – 1.22 (m, 2H),1.16 (s, 3H), 1.11 (s, 3H), 1.07 (s, 3H), 1.01 (d, J = 13.4 Hz, 2H), 0.86 (s,6H), 0.79 (d, J = 11.5 Hz, 1H), 0.76 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 199.8,176.3, 170.9, 169.3, 150.5, 149.7, 147.2, 146.1, 145.5, 136.3, 133.8, 132.8,132.5, 128.2, 125.9, 121.8, 120.0, 119.8, 108.9, 108.6, 102.1, 80.4, 63.6,63.1, 61.7, 58.1, 57.0, 54.9, 48.5, 45.4, 44.1, 43.2, 41.0, 38.8, 37.9, 37.6,36.9, 32.6, 31.8, 31.0, 29.9, 28.5, 28.4, 28.3, 27.9, 26.9, 26.3, 26.3, 23.5,23.3, 21.2, 18.6, 17.3, 16.6, 16.3.HR-ESI-MS m / z Calculated for C 55 H 70 NO9 + [M -I] + 888.5045, found 888.5041. Compound 6b: Yellow powder, yield 53%. 1 H NMR (400 MHz, CDCl3) δ 10.27 (s, 1H),8.07 – 7.87 (m, 2H), 7.09 (s, 1H), 6.88 (s, 1H), 6.09 (s, 2H), 5.63 (s, 1H),5.11 (s, 2H), 4.50 (dd, J = 11.5, 4.7 Hz, 1H), 4.38 (s, 3H), 4.19 (dt, J = 12.8,6.5 Hz, 1H), 4.13 – 4.06 (m, 1H), 4.08 (s, 3H), 3.37 – 3.27 (m, 2H), 3.21 (t, J = 5.5 Hz, 2H), 2.76 (d, J= 13.6 Hz, 1H), 2.36 (s, 1H), 2.10 – 2.05 (m, 1H), 2.04 (s, 3H), 2.02 – 1.97 (m, 2H), 1.96 – 1.91 (m, 2H), 1.83 – 1.72 (m, 3H),1.68 – 1.60 (m, 6H), 1.45 – 1.39 (m, 2H), 1.37 (s, 3H), 1.35 – 1.30 (m, 3H),1.27 – 1.24 (m, 2H), 1.21 (s, 1H), 1.15 (s, 3H), 1.14 (s, 3H), 1.11 (s, 3H), 1.06 – 1.00 (m, 2H), 0.87 (s, 6H), 0.79 (s, 3H), 0.82 – 0.75 (m, 1H). 13 C NMR (101 MHz, CDCl3) δ 200.0, 176.4, 170.9, 169.5, 150.4, 149.8, 147.3, 146.1,145.3, 136.1, 134.0, 133.6, 133.1, 128.4, 125.7, 122.0, 120.3, 120.3, 109.0,108.6, 102.1, 80.5, 64.0, 63.2, 61.8, 58.1, 56.9, 55.0, 48.6, 45.4, 44.0,43.3, 41.1, 38.8, 38.0, 37.7, 37.0, 32.7, 31.9, 31.1, 30.8, 30.0, 29.3, 28.6,28.5, 28.4, 28.0, 26.5, 26.4, 26.3, 23.5, 23.3, 21.3, 18.7, 17.3, 16.6, 16.4.HR-ESI-MS m / z Calculated for C 57 H 74 NO8 + [M - I] + 916.5358, found 916.5355. Compound 6c: Yellow powder, yield 60%. 1 H NMR (400 MHz, CDCl3) δ 10.58 (s, 1H),7.89 (q, J= 8.9 Hz, 2H), 7.07 (s, 1H), 6.87 (s, 1H), 6.09 (s, 2H), 5.59 (s,1H), 5.23 (s, 2H), 4.48 (dd, J = 11.0, 3.6 Hz, 1H), 4.32 (s, 3H), 4.19 – 4.10(m, 1H), 4.06 (s, 3H), 4.08 – 4.01 (m, 1H), 3.27 (d, J = 6.4 Hz, 2H), 3.20 –3.12 (m, 2H), 2.72 (d, J = 13.1 Hz, 1H), 2.33 (s, 1H), 2.03 (s, 3H), 2.08 –1.94 (m, 2H), 1.93 – 1.84 (m, 3H), 1.81 – 1.74 (m, 1H), 1.71 – 1.64 (m, 3H),1.62 – 1.53 (m, 5H), 1.52 – 1.42 (m, 3H), 1.38 (d, J = 13.1 Hz, 2H), 1.34 (s,3H), 1.29 (d, J = 9.7 Hz, 2H), 1.23 (s, 2H), 1.18 (s, 1H), 1.12 (s, 3H), 1.11(s, 3H), 1.07 (s, 3H), 1.00 (d, J = 12.9 Hz, 2H), 0.86 (s, 6H), 0.80 – 0.73 (m,1H), 0.75 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 200.0, 176.5, 171.0, 169.4,150.4, 149.7, 147.2, 146.5, 146.1, 136.0, 134.0, 133.9, 133.0, 128.3, 125.5,122.2, 120.3, 120.1, 109.0, 108.6, 102.1, 80.5, 64.2, 63.1, 61.7, 57.4, 57.0,55.0, 48.6, 45.4, 44.0, 43.2, 41.0, 38.8, 38.0, 37.7, 36.9, 32.6, 31.8, 31.1,31.0, 29.8, 29.6, 29.2, 28.7, 28.5, 28.4, 28.0, 26.4, 26.3, 25.6, 23.5, 23.3,21.3, 18.6, 17.3, 16.6, 16.4. HR-ESI-MS m / z Calculated for C 58 H 76 NO9 + [M - I] + 930.5515, found 930.5515. Compound 6d: Yellow powder, yield 80%. 1 H NMR (400 MHz, CDCl3) δ 10.43 (s, 1H),7.88 (q, J = 9.6 Hz, 2H), 7.06 (s, 1H), 6.84 (s, 1H), 6.06 (s, 2H), 5.57 (s,1H), 5.16 (s, 2H), 4.43 (dd, J = 11.5, 4.4 Hz, 1H), 4.28 (s, 3H), 4.04 (d, J =8.1 Hz, 5H), 3.28 – 3.18 (m, 2H), 3.12 (s, 2H), 2.69 (d, J= 13.7 Hz, 1H), 2.29(s, 1H), 2.08 – 2.01 (m, 1H), 1.99 (s, 3H), 1.96 – 1.92 (m, 1H), 1.89 – 1.78(m, 3H), 1.77 – 1.70 (m, 1H), 1.65 – 1.56 (m, 4H), 1.54 – 1.45 (m, 4H), 1.42– 1.33 (m, 8H), 1.31 (s, 3H), 1.29 – 1.23 (m, 3H), 1.21 – 1.19 (m, 2H), 1.15(s, 1H), 1.09 (s, 3H), 1.07 (s, 3H), 1.05 (s, 3H), 1.02 – 0.93 (m, 2H), 0.82 (s, 6H), 0.74 (s, 3H), 0.78 – 0.68 (m, 1H). 13 C NMR (101 MHz, CDCl3) δ 199.9,176.4, 170.8, 169.3, 150.3, 149.6, 147.1, 146.1, 145.4, 135.8, 134.2, 133.6,132.9, 128.2, 125.5, 121.9, 120.1, 108.9, 108.4, 102.0, 80.4, 64.2, 62.9,61.6, 57.6, 57.0, 54.9, 48.4, 45.3, 43.9, 43.1, 40.9, 38.6, 37.9, 37.6, 36.8,32.5, 32.3, 31.7, 31.0, 31.0, 29.8, 29.5, 29.4, 28.9, 28.8, 28.5, 28.4, 28.3,27.9, 26.3, 26.2, 25.8, 23.4, 23.2, 21.1, 18.5, 17.2, 16.5, 16.2. HR-ESI-MS m / z Calculated for C 60 H 80 NO9 + [M - I] + 958.5828, found 958.5828. Compound 7a: Yellow powder, 90% yield. 1H NMR (400 MHz, CDCl3) δ 10.30 (s, 1H),8.03 – 7.86 (m, 2H), 7.10 (s, 1H), 6.86 (s, 1H), 6.09 (s, 2H), 5.59 (s, 1H),5.29 (s, 1H), 5.20 – 4.98 (m, 2H), 4.37 (s, 3H), 4.27 (t, J = 5.2 Hz, 2H), 4.09(s, 3H), 3.41 (t, J = 7.8 Hz, 2H), 3.29 – 3.14 (m, 2H), 2.96 – 2.86 (m, 1H),2.67 – 2.55 (m, 1H), 2.44 – 2.32 (m, 2H), 2.28 – 2.18 (m, 2H), 2.10 – 1.98(m, 3H), 1.92 (d, J = 13.5 Hz, 1H), 1.83 (dd, J = 12.1, 3.9 Hz, 1H), 1.72 – 1.59(m, 3H), 1.57 – 1.50 (m, 2H), 1.47 – 1.41 (m, 2H), 1.37 (s, 3H), 1.33 – 1.30(m, 2H), 1.23 (s, 3H), 1.17 (s, 3H), 1.13 (s, 3H), 1.09 (s, 3H), 1.05 (s,3H), 1.04 – 1.00 (m, 1H), 0.90 – 0.82 (m, 1H), 0.79 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 216.9, 199.2, 176.3, 169.8, 150.5, 149.8, 147.3, 146.2, 145.6,136.4, 133.8, 132.8, 132.6, 128.3, 125.9, 121.9, 120.1, 119.7, 109.0, 108.6,102.1, 63.6, 63.1, 61.1, 58.1, 57.0, 55.3, 48.6, 47.7, 45.2, 44.1, 43.3,41.2, 39.8, 37.7, 36.7,34.1,32.0,31.9,31.0,30.0,28.6,28.5,28.3,26.9,26.4,26.4,26.3,23.3,21.3,18.7,18.5,15.7. m / z Calculated for C 53 H 66 NO8 + [M - I] + 844.4783, found 844.4781. Compound 7b: Yellow powder, 94% yield. 1 H NMR (400 MHz, CDCl3) δ 10.23 (s, 1H),7.99 – 7.91 (m, 2H), 7.07 (s, 1H), 6.87 (s, 1H), 6.08 (s, 2H), 5.64 (s, 1H),5.08 (s, 2H), 4.33 (s, 3H), 4.20 – 4.08 (m, 2H), 4.06 (s, 3H), 3.30 (t, J = 8.1Hz, 2H), 3.22 – 3.14 (m, 2H), 2.88 (dt, J = 12.2, 5.9 Hz, 1H), 2.62 – 2.52 (m,1H), 2.42 (s, 1H), 2.33 (dt, J = 13.0, 3.9 Hz, 1H), 2.22 – 2.14 (m, 1H), 2.09(d, J= 13.1 Hz, 1H), 2.03 – 1.96 (m, 2H), 1.95 – 1.87 (m, 3H), 1.85 – 1.78 (m,1H), 1.75 – 1.71 (m, 1H), 1.65 – 1.57 (m, 3H), 1.55 – 1.49 (m, 2H), 1.46 –1.38 (m, 3H), 1.36 (s, 3H), 1.31 – 1.27 (m, 3H), 1.22 (s, 3H), 1.18 (s, 1H),1.13 (s, 6H), 1.07 (s, 3H), 1.03 (s, 3H), 1.01 (s, 1H), 0.88 – 0.81 (m, 1H), 0.79 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 216.9, 199.3, 176.3, 169.9, 150.3,149.7, 147.2, 145.9, 145.0, 136.0, 134.0, 133.5, 133.0, 128.2, 125.7, 121.8,120.3, 120.1, 108.9, 108.5, 102.1, 63.9, 63.0, 61.0, 58.1, 57.0, 55.2, 48.5,47.6, 45.1, 43.9, 43.3, 41.1, 39.7, 37.6, 36.6, 34.1, 32.0, 31.8, 31.0, 30.7,29.9, 28.6, 28.4, 28.3, 26.4, 26.4, 26.3, 26.1, 23.3, 21.3, 18.7, 18.4, 15.6.HR-ESI-MS m / z Calculated for C 55 H 70 NO8 + [M - I] + 872.5096, found 872.5093. Compound 7c: Yellow powder, yield 92%. 1 H NMR (400 MHz, CDCl3) δ 10.24 (s, 1H),7.91 (q, J= 9.3 Hz, 2H), 7.08 (s, 1H), 6.87 (s, 1H), 6.09 (s, 2H), 5.62 (s,1H), 5.09 (s, 2H), 4.34 (s, 3H), 4.14 – 4.02 (m, 2H), 4.06 (s, 3H), 3.31 –3.23 (m, 2H), 3.19 (s, 2H), 2.92 – 2.82 (m, 1H), 2.63 – 2.53 (m, 1H), 2.41(s, 1H), 2.37 – 2.29 (m, 1H), 2.11 – 2.05 (m, 1H), 2.01 – 1.95 (m, 2H), 1.93– 1.86 (m, 3H), 1.82 – 1.76 (m, 1H), 1.72 – 1.63 (m, 3H), 1.60 – 1.53 (m,3H), 1.48 – 1.38 (m, 4H), 1.35 (s, 3H), 1.30 – 1.26 (m, 3H), 1.23 (s, 2H),1.21 (s, 3H), 1.17 (s, 1H), 1.12 (s, 3H), 1.11 (s, 3H), 1.07 (s, 3H), 1.04(s, 3H), 0.99 (s, 1H), 0.85 (t, J = 6.4 Hz, 1H), 0.77 (s, 3H). 13 C NMR (101 MHz,CDCl3) δ 217.0, 199.4, 176.4, 169.9, 150.4, 149.7, 147.2, 146.1, 145.8,136.1, 134.2, 133.6, 133.1, 128.3, 125.7, 122.0, 120.3, 109.1, 108.6, 102.2,64.2, 63.4, 61.0, 58.2, 57.1, 55.3, 48.5, 47.7, 45.2, 43.9, 43.3, 41.1, 39.7,37.7, 36.6, 34.1, 32.0, 31.8, 31.1, 31.0, 30.0, 29.6, 29.2, 28.8, 28.6, 28.6,28.4, 26.5, 26.4, 26.3, 25.5, 23.3, 21.3, 18.7, 18.5, 15.7. HR-ESI-MS m / z Calculated for C 56 H 72 NO8 + [M - I] + 886.5252, found 886.5251. Compound 7d: Yellow powder, yield 68%. 1 H NMR (400 MHz, CDCl3) δ 10.57 (s, 1H),7.88 (t, J = 9.7 Hz, 2H), 7.08 (s, 1H), 6.87 (s, 1H), 6.09 (s, 2H), 5.65 (s,1H), 5.23 (s, 2H), 4.33 (s, 3H), 4.12 – 4.01 (m, 2H), 4.06 (s, 3H), 3.31 –3.21 (m, 2H), 3.15 (s, 2H), 2.93 – 2.85 (m, 1H), 2.62 – 2.52 (m, 1H), 2.41(s, 1H), 2.36 – 2.29 (m, 1H), 2.13 – 2.07 (m, 1H), 2.03 – 1.98 (m, 3H), 1.92– 1.81 (m, 4H), 1.68 – 1.58 (m, 5H), 1.56 – 1.49 (m, 5H), 1.45 – 1.41 (m,3H), 1.35 (s, 3H), 1.31 – 1.26 (m, 6H), 1.21 (s, 3H), 1.18 (s, 1H), 1.12 (s,6H), 1.07 (s, 3H), 1.03 (s, 3H), 1.00 (s, 1H), 0.87 – 0.84 (m, 1H), 0.79 (s,3H). 13C NMR (101 MHz, CDCl3) δ 217.0, 199.4, 176.4, 169.8, 150.4, 149.7,147.2, 146.4, 146.0, 135.9, 134.0, 133.9, 133.0, 128.3, 125.4, 122.1, 120.3,120.0, 109.0, 108.5, 102.1, 64.3, 63.1, 61.0, 57.5, 57.0, 55.3, 48.4, 47.7,45.2, 43.9, 43.3, 41.1, 39.7, 37.7, 36.6, 34.1, 32.0, 31.8, 31.1, 29.9, 29.6,29.5, 29.0, 28.9, 28.6, 28.6, 28.6, 28.4, 26.5, 26.4, 26.3, 25.8, 23.3, 21.3,18.7, 18.4, 15.6. HR-ESI-MS m / z Calculated for C 58 H 76 NO8 + [M - I] + 914.5565, found 914.5568. Example 6: Western blot experiment of the compound (a) Human hepatocellular carcinoma HepG2 and human normal hepatocytes HL7702 were incubated overnight in serum-free DMEM medium containing 5.5 mM glucose, and then treated with 0.8 μM glycyrrhetinic acid-berberine derivative in the presence of glucose (30 mM) and insulin (100 nM) for 24 hours.

[0045] (b) After drug treatment, the cells were washed twice with PBS. Pre-chilled 1X cell lysis buffer (PMSF protease inhibitor added fresh for use) was added, and the cells were lysed on ice for 20 min. The cells were then scraped off with a cell scraper and transferred to EP tubes.

[0046] (c) Centrifuge at 4 °C, 12000 rpm for 20 min. Collect the supernatant and heat at 95 °C for 5 min to denature it.

[0047] (d) Protein concentration was determined by the BCA method.

[0048] (e) SDS-PAGE electrophoresis. Select the separation gel concentration according to the target protein. After the separation gel solidifies, prepare a 5% stacking gel. After the gel solidifies, remove the sample comb, place it in the electrophoresis tank, and add electrophoresis buffer.

[0049] (f) Sample loading: Calculate the protein concentration according to the standard protein curve, take 30-80 μg of protein, add 4x SDS loading buffer, and bring the volume to the same level with double-distilled water. Heat at 95°C for 5 min. After loading the processed sample protein and standard protein marker, connect to the electrophoresis apparatus, maintain a constant voltage of 90V for 30 min, and then use 120V to make the bromophenol blue run to the bottom of the electrophoresis tank, then stop electrophoresis. Transfer: Remove the gel and cut out suitable separating gel and NC membrane. Assemble them in the following order: negative electrode - sponge - filter paper - separating gel - NC membrane - filter paper - sponge, gently remove air bubbles, and install them into the electrophoresis tank. Maintain a constant current of 280 mA for 3 h. Blocking and immunoreaction: After removing the NC membrane, place it in 5% skim milk powder prepared by TBS and shake slowly on a horizontal shaker for 1 h. Wash once with TBST. Prepare the primary antibody working solution according to the antibody instructions, add the primary antibody working solution to the NC membrane, and incubate on a shaker overnight at 4°C. Remove the membrane, recover the primary antibody, wash three times with TBST for 5 min each, and wash with TBS for 5 min each. Incubate the secondary antibody at room temperature for 1 h, wash three times with TBST for 5 min each, and wash with TBS for 5 min each. For color development: Prepare ECL chemiluminescence solution in advance, mix A:B = 1:1 to form the working solution, and evenly drop it onto the NC membrane. Quickly place the membrane in a gel imaging system for imaging. The results are processed and analyzed using ImageLab software.

[0050] The results are as follows Figure 1 As shown, the glycyrrhetinic acid-berberine derivative of the present invention reduces the protein expression of SCD1 and SREBP-1, increases the phosphorylation of SREBP-1c (Ser372), and inhibits de novo cholesterol synthesis. This indicates that the glycyrrhetinic acid-berberine derivative of the present invention can significantly regulate key enzymes in metabolic disease-related pathways at a concentration of nM, achieving two orders of magnitude higher efficacy than the original drug. It achieves the same therapeutic effect with a very small dosage, avoiding the gastrointestinal discomfort and other side effects caused by high doses of berberine in the treatment of metabolic diseases, thus demonstrating a high safety profile.

[0051] Compound 7c, which exhibits relatively good activity, was selected for immunoblotting experiments at different concentrations, such as... Figure 2As shown. Berberine (1 μM), glycyrrhetinic acid (1 μM), and berberine + glycyrrhetinic acid (1 μM + 1 μM) were added for comparison. The results showed that 1 μM of the corresponding doses of berberine and glycyrrhetinic acid had no activity, while compound 7c activated AMPK phosphorylation at 0.3 μM, with the activation effect increasing with concentration, reaching a significant level at 0.8 μM. Furthermore, AMPK is known to be upstream of SREBP-1c and can directly phosphorylate SREBP-1c (Ser372). Phosphorylation of SREBP-1c (Ser372) inhibits its cleavage and nuclear translocation, thereby reducing the expression of its target genes SREBP-1c and SCD1. Therefore, the compounds of this invention may reduce SCD1 expression in hepatocytes through the AMPK-SREBP-1c pathway. Western blot experiments showed that the compound of the present invention increased the phosphorylation level of AMPK(Thr172), resulting in the inhibition of SREBP-1 and SCD1 expression. The inhibitory effect increased with increasing concentration, and at 0.8 μM, it almost restored the expression level of normal cells.

[0052] Treatment with compound 7c did not significantly change the total amount of AMPK, but phosphorylated AMPK was upregulated in a dose-dependent manner. The AMPK inhibitor Cpd.C could prevent the downregulation of m-SREBP1 induced by compound 7c, indicating that compound 7c inhibits the SREBP1 pathway by activating AMPK. Figure 3 As shown.

[0053] Example 7: Immunofluorescence assay of the compound HepG2 cells were induced to form a cell model using high glucose and insulin. After drug treatment, the culture medium was discarded, and the cells were washed twice with PBS and fixed in fixative (methanol:acetone = 1:1) for 5 min. After washing with PBS for 5 min, the cells were soaked in 3% BSA (Triton X-100) for 20 min and washed with PBS-Tx for 5 min. Primary antibody blocking solution was added, and the cells were blocked overnight at 4 °C. The cells were washed with PBS-Tx for 5 min three times. Secondary antibody blocking solution was added, and the cells were blocked at 37 °C for 1 h. The cells were washed with PBS-Tx for 5 min three times. The PBS-Tx solution was discarded, and the cells were stained with DAPI for 20 min and washed with PBS-Tx for 5 min three times. The cells were observed under a laser confocal microscope.

[0054] Immunofluorescence such as Figure 4 As shown, the results indicate that under normal conditions, SREBP1 is mainly distributed in the cytoplasm; in the high glucose and insulin-induced cell models, SREBP1 can be observed to enter the nucleus, and the combined effect of berberine and glycyrrhetinic acid is negligible. However, after treatment with compound 7c of the present invention, the intranuclear fluorescence intensity of SREBP1 is weakened, indicating that it can significantly downregulate the expression of the mature form of SREBP1.

[0055] Example 8: Fillipin III staining – for detecting cholesterol content First, HepG2 cells induced by high glucose and insulin were fixed using 4% paraformaldehyde. Next, the fixed cells were stained with Fillipin III fluorescent staining agent. Fillipin III is a fluorescent staining agent that binds to cholesterol and produces a fluorescent signal. After staining, the cells were washed three times with PBS. Finally, the stained cells were observed using a fluorescence microscope. The results showed that under a fluorescence microscope, the fluorescence intensity of cells treated with 0.8 μM 7c was significantly reduced, indicating that it can reduce cholesterol content. Figure 5 As shown.

[0056] Example 9: In vivo experiment Twenty-four 8-week-old male C57 mice were randomly divided into four groups: a control group (chow), a high-fat diet model group (HFD), a low-dose group (7c, 1 mg / kg), and a high-dose group (7c, 2 mg / kg), with six mice in each group. The control group was fed a normal mouse diet (73.4% carbohydrates, 4.0% fat, and 22.6% protein), while the HFD and drug-treated groups were fed a high-fat diet (HFD; 40.0% carbohydrates, 39.9% fat, and 20.0% protein) for 12 weeks to establish an obese mouse model. After successful model establishment, the drug-treated groups were intraperitoneally injected with compound 7c every two days for six weeks. After six weeks, insulin resistance and glucose resistance were tested in each group. Subsequently, venous blood was collected from the ocular fossa, and liver tissue was harvested from the mice.

[0057] Experimental results showed that mice treated with compound 7c of this invention experienced a significant reduction in body weight, especially at a dose of 2 mg / kg, where the effect was most pronounced. Figure 6 As shown in A. Furthermore, the fat-to-body-weight ratio improved, and liver weight also decreased significantly, as... Figure 6 As shown in B, C, and D. Insulin resistance and glucose resistance experiments indicate that HFD promotes insulin resistance and reduces glucose tolerance in mice; however, treatment with 7c improved HFD-induced insulin resistance (…). Figure 6 E) and increased glucose tolerance ( Figure 6 F). From Figure 7Liver morphology and H&E staining showed that administration of 7c at a dose of 2 mg / kg reduced steatosis in the liver lobules of HFD mice and significantly inhibited high-fat diet-induced fat deposition and adipocyte enlargement. The liver structure of HFD group mice was severely damaged, with numerous vacuoles in hepatocytes. However, compared with the HFD group, 7c intake significantly reduced the degree of hepatic steatosis and the number of fat vacuoles in the liver lobules. These results indicate that compound 7c alleviates the damage to the liver and subcutaneous adipose tissue caused by a high-fat diet, suggesting its ameliorative effect on metabolic dysfunction induced by a high-fat diet in obese mice.

[0058] Example 10: Evaluation of Pharmacokinetic Parameters Construction of the standard curve: A certain amount of compound 7c was accurately weighed and dissolved in methanol (solvent volume less than 100 mL). The solution was then transferred to a 100 mL volumetric flask and diluted to the appropriate mark. The solution was shaken to ensure thorough mixing of the compound and solvent, thus preparing a 1 μmol / mL stock solution. A portion of this stock solution was taken, diluted with methanol, and diluted to the appropriate volume to prepare a 10% standard curve. -1 10 -2 10 -3 10 -4 10 -5 10 -6 A series of standard solutions at μmol / ml were prepared and stored at 4 ℃. 90 μl of blank rat plasma (i.e., untreated rat plasma) was accurately measured, and 10 μl of the standard solutions and 300 μl of an internal standard solution containing 2 nmol / ml were added. The mixture was vortexed for 10 min, centrifuged (13000 rpm) for 10 min, and 200 μl of the supernatant was collected for HPLC-HR-MS analysis. The obtained linear regression equation can be considered as a standard curve for the concentration of compound 7c in serum.

[0059] Plasma sample collection: Take appropriate amounts of compounds BBR and 7c, add DMSO, PEG, and physiological saline to dissolve them, and prepare a clear solution.

[0060] Twelve healthy female SD rats were randomly divided into four groups (BBR gavage group, BBR intravenous group, 7c gavage group, and 7c intravenous group), with three rats in each group. To ensure experimental accuracy, the rats were not fed for 12 hours before the experiment, but were given an appropriate amount of drinking water. The gavage group received the test drug directly via gavage at a concentration of 15 μmol / kg. Blood was collected from the orbital vein of the rats at regular intervals: approximately 0 min before administration, and 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h after administration. Approximately 1 mL of venous blood was collected and placed in centrifuge tubes filled with heparin sodium. The tubes were centrifuged rapidly for approximately 10 min, and the supernatant serum was collected and stored at -80 °C for later analysis.

[0061] Rats in the intravenous group were administered the test drug via tail vein injection at a concentration of 15 μmol / kg. Blood samples were collected from the orbital vein of the rats at regular intervals, marked as 0 min. Blood samples were collected at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h after drug administration. Approximately 1 mL of venous blood was collected and placed into centrifuge tubes filled with heparin sodium. The centrifuge tubes were centrifuged rapidly for approximately 10 min, and the supernatant serum was collected and stored at -80 ℃ for later analysis.

[0062] Take 100 μl of rat plasma sample, add 300 μl of internal standard solution, vortex to mix for 10 min, centrifuge (13000 rpm) for 10 min, and take 200 μl of supernatant for HPLC-HR-MS analysis. The calculation of the corresponding blood drug concentration data in the above bioavailability experiment is based on a non-compartmental model. Pharmacokinetic parameters can be calculated using DAD2.0 software.

[0063] The bioavailability of compounds BBR and 7c can be calculated using the bioavailability formula F = (AUC_ext*Dose_iv) / (AUC_iv*Dose_ext)*100%.

[0064] As can be seen from Tables 1 and 2, after oral administration of compounds BBR and 7c, the whole blood exposure AUC was greater than that of BBR, the oral relative bioavailability was 21.2%, while the oral relative bioavailability of BBR was only 9.08%, and the half-life of 7c was significantly increased.

[0065] Table 1. Pharmacokinetic parameters of compound BBR

[0066] Table 2. Pharmacokinetic parameters of compound 7c

[0067] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A glycyrrhetinic acid-berberine derivative, characterized in that, Its chemical structure is shown in Formula I. Where n is 1 to 6, It can be a single or double bond, and R can be O, hydroxyl, or... R' is a C1-C5 alkyl group; When it is a single bond, R is a hydroxyl group or ; When it is a double bond, R is 0.

2. The glycyrrhetinic acid-berberine derivative as described in claim 1, characterized in that it has the following structural formula: 。 3. The glycyrrhetinic acid-berberine derivative as described in claim 1, characterized in that, R' is a C1-C5 straight-chain alkyl group.

4. The glycyrrhetinic acid-berberine derivative as described in claim 1, characterized in that, R' is a C1-C3 straight-chain alkyl group.

5. The glycyrrhetinic acid-berberine derivative as described in claim 1, characterized in that, R' stands for methyl.

6. The glycyrrhetinic acid-berberine derivative as described in claim 1, characterized in that, n can be 1, 3, 4 or 6.

7. A method for preparing a glycyrrhetinic acid-berberine derivative, characterized in that, This includes the steps of obtaining the compound shown in Formula I via the following reaction route; Wherein, R and n are as described in claim 1.

8. The method for preparing the glycyrrhetinic acid-berberine derivative as described in claim 7, characterized in that, Berberine reacts with acetone in a strongly alkaline environment to give compound 1. Compound 1 is then nucleophilically substituted with diiodoalkane to give compound 2. Compound 2 is then nucleophilically substituted with glycyrrhetinic acid or its derivative to give the compound shown in formula I.

9. The method for preparing the glycyrrhetinic acid-berberine derivative as described in claim 7, characterized in that, The nucleophilic substitution reaction of compound 1 to prepare compound 2 was carried out under reflux conditions.

10. The method for preparing the glycyrrhetinic acid-berberine derivative as described in claim 7, characterized in that, In the nucleophilic substitution reaction of compound 1 to prepare compound 2, acetonitrile was used as the organic solvent.

11. The method for preparing the glycyrrhetinic acid-berberine derivative as described in claim 7, characterized in that, The conditions for the nucleophilic substitution reaction of compound 2 with glycyrrhetinic acid or its derivatives to prepare the compound shown in Formula I are: under alkaline conditions, with stirring at room temperature.

12. The method for preparing the glycyrrhetinic acid-berberine derivative as described in claim 11, characterized in that, The alkaline conditions are adjusted by potassium carbonate or cesium carbonate.

13. The method for preparing the glycyrrhetinic acid-berberine derivative as described in claim 11, characterized in that, In the nucleophilic substitution reaction of compound 2 with glycyrrhetinic acid or its derivatives to prepare the compound shown in Formula I, the solvent used is N,N-dimethylformamide.

14. A pharmaceutical composition, characterized in that, Including the glycyrrhetinic acid-berberine derivatives as described in any one of claims 1 to 6.

15. The pharmaceutical composition of claim 14, characterized in that, It also includes pharmaceutical carriers; Or, it may also include excipients.

16. A pharmaceutical preparation, characterized in that, The product includes the glycyrrhetinic acid-berberine derivative as described in any one of claims 1 to 6, wherein the dosage form is a liquid or a solid dosage form.

17. The use of any glycyrrhetinic acid-berberine derivative according to any one of claims 1 to 6, the pharmaceutical composition according to claim 14 or 15, or the pharmaceutical preparation according to claim 16 in the preparation of an SCD1 protein inhibitor, an SREBP-1 protein inhibitor, an SREBP-1c phosphorylation promoter, an AMPK activator, or a cholesterol inhibitor.

18. The use of a glycyrrhetinic acid-berberine derivative according to any one of claims 1 to 6, a pharmaceutical composition according to claim 14 or 15, or a pharmaceutical preparation according to claim 16 in the preparation of a medicament for treating metabolic diseases, said metabolic diseases including diabetes and / or hyperlipidemia.

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