A phloretin derivative and its use in drugs for treating non-alcoholic fatty liver disease

Abstract

A phloretin derivative and its use in the medicine for treating diabetes-associated non-alcoholic fatty liver, which relates to the technical field of medicine, in particular to the compound shown in formula (1) and its activity of alleviating diabetes metabolic syndrome-related non-alcoholic fatty liver; the pharmacological experimental results show that the compound shown in formula (1) can significantly alleviate hepatomegaly, reduce the presence of lipid droplets in liver tissues, restore the morphology of liver cells, and reduce the contents of total cholesterol, triglyceride and low-density lipoprotein in the blood, reduce related inflammatory indexes, and enhance antioxidant activity.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to a phloretin derivative and its medical use. Background Art

[0002] Type 2 diabetes mellitus (T2DM) is a severe chronic metabolic disease with long-term hyperglycemia, characterized by insulin resistance and insufficient insulin secretion by pancreatic β cells leading to hyperglycemia. Patients with T2DM metabolic syndrome have a high probability of suffering from diabetes metabolic syndrome-related non-alcoholic fatty liver disease. Non-alcoholic fatty liver disease can not only directly lead to decompensated cirrhosis, hepatocellular carcinoma, and recurrence in transplanted livers, but also further participate in the pathogenesis of type 2 diabetes and atherosclerosis, forming a vicious cycle. Currently, the treatment methods usually use hypoglycemic drugs for treatment in order to achieve the effect of reducing fat and weight, and control the further deterioration of fatty liver and diabetes. At present, some commonly used T2DM drugs (such as acarbose, metformin, and thiazolidinediones) are used to treat type 2 diabetes, but these drugs have a wide range of side effects including hypoglycemia, stomachache, diarrhea, flatulence, allergic reactions, etc.

[0003] Due to their unique chemical structures, natural products usually have a wide range of biological activities. Among them, chalcone compounds are widely present in many plants, and studies have shown that chalcone compounds (including phloretin) have preventive and therapeutic effects on non-alcoholic fatty liver disease related to type 2 diabetes metabolic syndrome. Its main pharmacological action mechanism is related to its inhibition of lipid metabolism-related pathways and antioxidant activity. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention aims to provide a new and more efficient phloretin derivative for alleviating non-alcoholic fatty liver disease related to diabetes metabolic syndrome, and further improve the effect by modifying known compounds.

[0005] The first aspect of the present invention is to provide a compound, and the compound is a phloretin derivative shown in formula (1):

[0006]

[0007] Further, in the compound, R is a saturated hydrocarbon group or halogen with C1 - C10;

[0008] Further, R is halogen, and more specifically, R is F.

[0009] The second aspect of the present invention is to provide a preparation method of the compound described in the first aspect, and the method is synthesized according to the following route:

[0010]

[0011] Further, the specific steps of the method are as follows:

[0012] 1) Using phloretin and 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazol-3-ium trifluoromethanesulfonate as raw materials, reacting at room temperature for 6 - 10 h under the catalysis of triethylamine and acetonitrile to obtain a monosubstituted phloretin derivative;

[0013] 2) Subsequently, through silica gel column chromatography, separating and purifying with ethyl acetate / petroleum ether as the developing agent.

[0014] Further, the molar ratio of phloretin to 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazol-3-ium trifluoromethanesulfonate is 1:1 - 1.5; preferably 1:1.3;

[0015] Further, after the reaction in step 1), the product is obtained by rotary evaporation and drying;

[0016] Further, in step 2), the ratio of ethyl acetate / petroleum ether is 1:3 - 7 (v / v); preferably 1:5 (v / v).

[0017] The third aspect of the present invention is to provide a pharmaceutical composition for treating diabetes metabolic syndrome - related non - alcoholic fatty liver, and the pharmaceutical composition contains the compound described in the first aspect and a pharmaceutically acceptable carrier.

[0018] Fourthly, the present invention also provides the application of the compound described in the first aspect in the preparation of a drug for treating non - alcoholic fatty liver. Preferably, it is for alcoholic fatty liver disease associated with diabetes.

[0019] On the other hand, the present invention provides the application of the above - mentioned compound in the preparation of a drug for fatty liver disease associated with diabetes, preferably including the application in drugs for reducing blood glucose AST, ALT, TG, CHO, LDL, SOD, MDA and / or CAT, or increasing the HDL level. Preferably, it is for the application in the preparation of a drug for increasing the HDL level in blood.

[0020] Preferably, the above - mentioned application includes reducing inflammatory damage in blood, enhancing the antioxidant level, and / or simultaneously reducing blood lipid content.

[0021] Preferably, the above - mentioned application also includes the application for reducing inflammatory damage in the liver and muscles.

[0022] Preferably, the above - mentioned application acts on diabetic patients, mammals, preferably model mice, and the model mice are db / db mice.

[0023] Compared with the prior art, the present invention has the following advantages

[0024] The present invention can significantly enhance the effect of alleviating non-alcoholic fatty liver associated with diabetic metabolic syndrome by modifying phloretin-like compounds to obtain compound (1), and has extremely high potential for anti-nonalcoholic fatty liver. Description of the Drawings

[0025] Figure 1 Effect of the compound on the liver organ index. Note: * represents: P < 0.05, *** represents P < 0.001.

[0026] Figure 2 Effect of the compound on liver HE staining.

[0027] Figure 3 Effect of the compound on blood biochemical indexes. Note: * represents: P < 0.05, ** represents P < 0.01, *** represents P < 0.001, **** represents P < 0.0001.

[0028] Figure 4 Effect of the compound on muscle and liver antioxidant indexes. Note: * represents: P < 0.05, ** represents P < 0.01, *** represents P < 0.001, **** represents P < 0.0001. Detailed Embodiments

[0029] The following further elaborates on the concept and technical effects of the present invention in combination with specific embodiments to fully understand the purpose, features, and effects of the present invention. The methods are all conventional methods unless otherwise specified. The materials can all be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0030] Example 1: Preparation of Phloretin Derivatives

[0031] Synthesize the compound shown in Formula I according to the following process:

[0032]

[0033] The specific preparation process is as follows: Add 27.4 mg (0.1 mmol) of phloretin and 42 mg (0.13 mmol) of 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazol-3-ium trifluoromethanesulfonate to a 10 ml round-bottom flask. Pipette 1 ml of acetonitrile into the flask, and then add 22 μl (0.16 mmol) of triethylamine. Stir the reaction at room temperature for 8 h. After the reaction is completed, spin-dry the reaction solution and separate and purify it by silica gel column chromatography using ethyl acetate / petroleum ether (1:5, v / v) as the eluent.

[0034] The physicochemical data of the obtained compound (1) and 1 1H-NMR, 13 13C-NMR, 19 19F NMR data are as follows:

[0035] Compound (1) 1 1H NMR (500 MHz, CDCl3) δ 10.25 (s, 1H), 7.27 (d, J = 8.9 Hz, 1H), 7.22–7.16 (m, 1H), 6.34 (s, 1H), 3.39 (t, J = 7.5 Hz, 1H), 2.99 (t, J = 7.5 Hz, 1H), 1.99 (d, J = 33.1 Hz, 1H); 13 13C NMR (126 MHz, CDCl3) δ 205.05, 163.03, 153.85, 148.46, 142.06, 130.44, 120.86, 109.64, 100.80, 45.93, 29.26; 19 19F NMR (471 MHz, CDCl3) δ 39.64, 37.35; Molecular formula: C 15 11H 12 2F2O9S2; Molecular weight: 438.3688.

[0036] Example 2: Effect of compound (1) on the liver organ index of non-alcoholic fatty liver related to diabetic metabolic syndrome

[0037] We conducted an in vivo evaluation of the compound on non-alcoholic fatty liver related to diabetic metabolic syndrome using a mouse gavage experiment. 5- to 6-week-old db / db mice and C57 / BL mice were used as the control groups. Before the formal experiment, they were acclimated to the experimental environment for one week. The animal experiment was conducted in a specific sterile environment (SPF). This study was approved by the Experimental Animal Management and Welfare Ethics Committee of Beijing Huayuan Times Technology Co., Ltd. (Ethical review batch number: HYSD2023-04). The db / db mice were randomly divided into a diabetic model control group, an acarbose group, and a compound (1) group, with 8 mice in each group. Compound (1) and acarbose were dissolved in physiological saline containing 10% DMSO. The compound (1) group was gavaged at a dose of 20 mg / kg and labeled as the compound (1) group. The acarbose group was gavaged at a dose of 20 mg / kg and labeled as the acarbose group. The normal control group and the diabetic model group were respectively gavaged with an equal amount of physiological saline; each was labeled as the control group and the model group. During the four-week continuous gavage administration period, all mice had free access to food and water, and their body weights and water intakes were recorded.

[0038] After administration, the mice were anesthetized for orbital blood collection. The collected blood was centrifuged at a low temperature of 3500 r / min. After 10 minutes, the serum was separated and aliquoted into cryopreservation tubes. After blood collection, the mice were euthanized by cervical dislocation, and liver and muscle tissue samples were obtained by dissection. Any bloodstains on the tissues were washed with physiological saline, and then the excess surface water was removed with filter paper. The organ index was calculated accordingly. A part of the liver tissue sample was fixed in a sample tube containing 4% paraformaldehyde, while the other part was stored in a freezer tube.

[0039] The results are as Figure 1 shown. The liver volume of the mice in the model group increased significantly, and there was a tendency for the liver volume of the mice in the compound (1) group to decrease (P < 0.05). There was no significant difference in the positive drug group.

[0040] Example 3: Effect of the compound on liver HE staining

[0041] HE staining: The tissues were trimmed, cut, placed in a dehydration box, and dehydrated with alcohol from 75% to 100% in sequence. Subsequently, the tissues were immersed in wax and processed using an embedding machine. After dressing, sections were cut with a paraffin slicer. The obtained sections were attached to glass slides and dried for later use. These sections were subjected to HE staining according to the following main steps: (1) treated with methanol and xylene, and then rinsed with distilled water; (2) continuously stained with Harris hematoxylin, differentiated with 1% hydrochloric acid ethanol, restored to blue with 0.6% ammonia water, and washed regularly with distilled water; (3) eosin staining continued for about 3 - 5 minutes; (4) dehydration and clearing were achieved through alcohol and xylene solutions of different concentrations before sealing with glue; (5) observed under a microscope with the assistance of photography.

[0042] The results are as Figure 2 shown. HE staining histological examination showed obvious differences between the model group and other treatment groups. In the model group, hepatocytes were severely swollen, with obvious lipid droplets in the cytoplasm. The cell nucleus was compressed and deformed to one side, the cell arrangement was disordered, and the hepatic sinusoids were enlarged. Compared with the model group, the hepatocytes treated with acarbose showed improved deformability, reduced lipid droplet vacuoles, regular cell arrangement, and alleviated fatty degeneration. Similarly, compared with the acarbose-treated group, the cell type arrangement in the compound (1) group was more orderly and there were fewer vacuoles.

[0043] Example 4 Effect of the compound on blood biochemical indexes

[0044] Blood biochemical indices: Using kits from Nanjing Jiancheng, the levels of triglyceride (TG), cholesterol (TC), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), glycated serum protein (GSP), lipopolysaccharide (LPS), and malondialdehyde (MDA) in the serum of rats in each group were quantitatively determined. Meanwhile, the activities of catalase (CAT), the level of reduced glutathione (GSH), and the activity of superoxide dismutase (T-SOD) were measured. For details, please refer to the kit instruction manual.

[0045] The results are as Figure 3 shown. To further investigate the effect of compound (1) administration on the liver of diabetic mice, we analyzed the levels of inflammation-related enzymes and fat in the blood. The results showed that compared with the model group, the levels of ALT, AST, TG, CHO, LDL, and MDA in the mice of the compound (1) and acarbose groups were significantly decreased. Compared with the model group, the levels of ALT, AST, TG, CHO, LDL, and MDA in compound (1) decreased by 16.9%, 31.5%, 17.4%, 12.7%, 21.0%, and 23.1% respectively (as shown in Table 1 below). There was no significant difference between compound (1) and acarbose in these indices. Meanwhile, the levels of HDL, T-SOD, GSH, and CAT in the mice of the compound (1) group increased by 22.1%, 20.1%, 29.8%, and 32.3% respectively. Similarly, there was no significant difference between compound (1) and acarbose in these indices. The above results indicate that both the compound (1) group and the acarbose group can reduce the inflammatory damage in the blood of diabetic mice, enhance the antioxidant level, and simultaneously decrease the blood lipid content.

[0046] Table 1

[0047]

[0048] Example 5 Effects of the compound on antioxidant indices in muscle and liver

[0049] Similarly, according to the instructions of the Nanjing Jiancheng kit, the levels of TG, TC, MDA, CAT, GSH, and SOD in the liver and muscle of mice were quantitatively determined.

[0050] The results are as Figure 4As shown, compared with the model group, compound (1) decreased the levels of MDA in the liver and muscle by 23.05% and 40.45% respectively. At the same time, the levels of T-SOD in the liver, GSH in the liver, CAT in the liver, T-SOD in the muscle, GSH in the muscle and CAT in the muscle increased by 57.2%, 34.75%, 30.58%, 47.52%, 25.84% and 28.85% respectively (Table 2). There was no significant difference between compound (1) and acarbose in these indicators. The above results indicate that both the compound (1) group and the acarbose group can reduce the inflammatory damage in the liver and muscle of diabetic mice.

[0051] Table 2

[0052]

[0053]

[0054] The embodiments described above are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

Claims

1. A compound, which is a phloretin derivative represented by formula (1): It is characterized in that R is F.

2. A method for preparing the compound according to claim 1, characterized in that, The method is synthesized according to the following route:

3. The method according to claim 2, wherein The specific steps of the method are as follows: 1) Using phloretin and 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazol-3-ium trifluoromethanesulfonate as raw materials, reacting at room temperature for 6 - 10 h under the catalysis of triethylamine and acetonitrile to obtain a mono-substituted phloretin derivative; 2) Subsequently, silica gel column chromatography is carried out, and ethyl acetate / petroleum ether is used as the eluent for separation and purification.

4. The method according to claim 3, characterized in that, The molar ratio of phloretin to 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazol-3-ium trifluoromethanesulfonate is 1:1 - 1.

5.

5. A pharmaceutical composition for treating diabetes mellitus metabolic syndrome-related non-alcoholic fatty liver, characterized in that, The pharmaceutical composition contains the compound according to claim 1 and a pharmaceutically acceptable carrier.

6. The use of the compound according to claim 1 or the compound prepared by the method according to any one of claims 2 - 4 for non-therapeutic purposes in vitro.

7. The application of the compound according to claim 1 or the compound prepared by the method according to any one of claims 2 - 4 in the preparation of a drug for treating non-alcoholic fatty liver associated with diabetic metabolic syndrome.

8. The application of the compound according to claim 1 or the compound prepared by the method according to any one of claims 2 - 4 in the preparation of a drug for reducing blood glucose AST, ALT, TG, CHO, LDL, SOD, MDA and / or CAT, or increasing the level of HDL.

9. The application of the compound according to claim 1 or the compound prepared by the method according to any one of claims 2 - 4 in the preparation of a drug for increasing the level of HDL.

10. The application according to any one of claims 7-9, characterized in that, The application includes the application of reducing inflammatory damage in the liver and muscles of diabetic patients.

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