A brominated myricetin derivative and its preparation method and application

Abstract

The present invention relates to a polybrominated myricetin derivative and a preparation method and application thereof, belonging to the field of chemical drugs. The polybrominated myricetin derivative is prepared by using myricetin as a raw material and performing bromination modification. The compound is tested for its hypoglycemic activity, and the results show that the compound has strong inhibitory activity against protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase, but weak inhibitory effect on human normal liver cell L-02. The compound can be used in drugs for preventing and treating diabetes, and has the potential to be developed into a hypoglycemic drug.

Description

Technical Field

[0001] The present invention belongs to the field of chemical medicines, and in particular relates to a brominated myricetin derivative and a preparation method and application thereof. Background Art

[0002] Diabetes mellitus (DM) is an endocrine and metabolic disease characterized by hyperglycemia. It is one of the most common chronic non-communicable diseases worldwide. Chronic hyperglycemia can lead to complications in multiple organs, including the liver, kidneys, muscles, and cardiovascular system, significantly impacting patients' health and well-being. Type 2 diabetes mellitus (T2DM) is the most common form of diabetes, accounting for approximately 90% of all diabetics. The etiology of T2DM is complex, with multiple contributing factors. Therefore, drugs that simultaneously act on multiple targets within the disease network may produce synergistic effects, significantly enhancing therapeutic efficacy. PTP1B and α-glucosidase are important target enzymes associated with diabetes. PTP1B inhibition enhances insulin sensitivity, while α-glucosidase inhibitors have a strong therapeutic effect on postprandial hyperglycemia. Drugs that simultaneously act on these two enzymes may have a dual-action glucose-lowering effect.

[0003] Natural products are an important source for drug development, offering advantages such as being environmentally friendly, highly effective, and low in toxicity. According to statistics, 63.1% of small molecule drugs approved between 1981 and 2019 were related to natural products, making in-depth research on natural products of great value. Myricetin compounds are widely found in nature, exhibiting a broad spectrum of activity and a simple structure. Previous studies have shown that myricetin exhibits strong inhibitory activity against PTP1B and α-glucosidase. Further bromination of myricetin revealed that brominated myricetin derivatives exhibit enhanced or comparable inhibitory activity against both PTP1B and α-glucosidase targets, with low toxicity to normal human liver cells, suggesting potential for development as hypoglycemic drugs. Summary of the Invention

[0004] The purpose of the present invention is to provide a polybrominated myricetin derivative and its preparation method and application, as well as the application of the compound in the preparation of a drug for preventing or treating diabetes, so as to solve the disadvantages that the T2DM treatment drugs currently used in clinical practice cannot effectively prevent further necrosis of pancreatic beta cells and are accompanied by many side effects, and to propose a high-efficiency, low-toxic, multi-target hypoglycemic active drug.

[0005] To achieve the above purpose, the following technical solution is adopted: a polybrominated myricetin derivative is prepared by brominating myricetin as a raw material, wherein the polybrominated myricetin derivative has the structural formula of any one of the compounds shown in 1-3:

[0006]

[0007] A method for preparing a polybrominated derivative of myricetin. The compound is prepared through a bromination reaction. The specific preparation steps are: using myricetin as a raw material, dissolving it with 5-15 parts of acetonitrile / methanol, adding 2-8 parts of N-bromosuccinimide, adding 5-30 parts of water after the reaction is completed to quench the reaction, adding 100-300 parts of ethyl acetate / dichloromethane for extraction, and preparing the ethyl acetate / dichloromethane layer by high performance liquid chromatography to obtain the target compound 1-3.

[0008] A brominated myricetin derivative is used to prepare a diabetic drug that inhibits PTP1B and α-glucosidase activity. The compound can be used directly or in the form of a pharmaceutical composition containing 0.1–99% of the compound, with the remainder being a pharmaceutical carrier.

[0009] A medicine for preventing or treating diabetes, comprising a compound of a myricetin polybrominated derivative having the structural formula 1-3 and a pharmaceutically acceptable carrier.

[0010] The present invention has significant advantages and beneficial effects compared to the prior art. As can be seen from the above technical solution, the brominated derivatives of myricetin have enhanced or maintained comparable dual-target inhibitory activity against PTP1B and α-glucosidase compared to the raw material myricetin, and have low toxicity to normal human liver cells, showing the potential for development into hypoglycemic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is the structural formula of compound 1-3;

[0012] Figure 2 This is the high-resolution mass spectrum of compound 1 of the present invention

[0013] Figure 3 This is the high-resolution mass spectrum of compound 2 of the present invention

[0014] Figure 4 This is the high-resolution mass spectrum of compound 3 of the present invention

[0015] Figure 5 This is the nuclear magnetic resonance hydrogen spectrum of compound 1 of the present invention

[0016] Figure 6 This is the carbon NMR spectrum of compound 1 of the present invention

[0017] Figure 7 This is the nuclear magnetic resonance hydrogen spectrum of compound 2 of the present invention

[0018] Figure 8 This is the carbon NMR spectrum of compound 2 of the present invention

[0019] Figure 9 This is the nuclear magnetic resonance HMBC pattern of compound 2 of the present invention

[0020] Figure 10 This is the nuclear magnetic resonance hydrogen spectrum of compound 3 of the present invention

[0021] Figure 11 This is the carbon NMR spectrum of compound 3 of the present invention

[0022] Figure 12 This is the nuclear magnetic resonance HMBC pattern of compound 3 of the present invention DETAILED DESCRIPTION

[0023] Example 1 Preparation of Compound 1

[0024] Myricetin (80 mg, 0.25 mmol) was used as the starting material and dissolved in 8 mL of acetonitrile. NBS (133.5 mg, 0.75 mmol) was added at 25 ° C and reacted for 1.5 h. The reaction was monitored by HPLC. After the reaction was completed, water was added at 0 ° C to quench the reaction. 150 mL of ethyl acetate was added and then extracted with water three times, 50 mL each time. The ethyl acetate layer was spin-dried and purified by preparative high performance liquid chromatography (38% acetonitrile-0.5‰ trifluoroacetic acid aqueous solution, C18 column) to obtain compound 1 (76.7 mg, yield: 55%).

[0025] Compound 1: yellow-green solid powder, 1 H-NMR (600MHz, DMSO-d6) δ13.41(s,1H,OH),11.26(s,1H,OH),9.75(s,1H,OH),9.49(s,1H,OH),9.34(s,2H,OH),6.62(s,1H,ArH); 13 C NMR (150 MHz, DMSO-d6) δ 176.2, 156.9, 156.5, 151.4, 150.1, 144.8, 144.2, 137.2, 136.4, 121.0, 109.9, 104.84, 101.2, 93.7, 88.0; HRESIMS m / z 550.7611 [MH]-, molecular formula is C 15 H7Br3O8.

[0026] Example 2 Preparation of Compound 2 and Compound 3

[0027] Myricetin (45 mg, 0.14 mmol) was used as the starting material and dissolved in 13 mL of methanol. NBS (76 mg, 0.42 mmol) was added at 25 ° C and reacted for 5 h. The reaction was monitored by HPLC. After the reaction was completed, water was added at 0 ° C to quench the reaction, 25 mL of dichloromethane was added, and then water was added for extraction 3 times, each time 10 mL. The dichloromethane layer was spin-dried and the dichloromethane layer was purified by preparative high performance liquid chromatography (38% acetonitrile-0.5‰ trifluoroacetic acid aqueous solution, C18 column) to prepare compound 2 (6.3 mg, yield: 9%) and compound 3 (5.7 mg, yield: 7%).

[0028] Compound 2: yellow-brown solid powder, 1 H-NMR (600MHz, DMSO-d6) δ12.53(s,1H,OH),11.65(s,1H,OH),9.73(s,1H,OH),9.33(s,1H,OH),9.27(s,1H,OH),6.60(s,1H,ArH),6.42(s,1H,ArH); 13 C NMR (150MHz, DMSO-d6) δ176.5,160.8,159.7,152.9,149.5,144.8,144.1,137.1,136.2,121.2,109.8,104.5,101.3,98.4,86.1; HRESIMS m / z 472.8506[MH] - , molecular formula is C 15 H8Br2O8.

[0029] Compound 3: yellow-brown solid powder, 1 H-NMR (600MHz, DMSO-d6) δ12.47(s,1H,OH),11.72(s,1H,OH),9.71(s,1H,OH),9.59(s,2H,OH),9.49(s,1H,OH),6.45(s,1H,ArH); 13 C NMR (150MHz, DMSO-d6) δ176.6,160.9,159.8,153.1,149.0,143.4(C×2),137.8,137.7,122.3,104.6,103.6(C×2),98.5,86.2; HRESIMS m / z 550.7612[MH] - , molecular formula is C 15 H7Br3O8.

[0030] The H NMR and C NMR data of compounds 1-3 are shown in Table 1:

[0031] Table 1. H NMR and C NMR data of compounds 1-3

[0032]

[0033] In order to further verify the beneficial effects of the compounds of the present invention, the compounds synthesized in Examples 1 and 2 were tested for their hypoglycemic activity. The specific experiments are as follows:

[0034] PTP1B enzyme inhibition activity test: The buffer solution contains 10mM Tris, 25mM NaCl, and 1mM EDTA, and the pH is adjusted to 7.1 with hydrochloric acid. The substrate is 2mM pNPP, the sample is dissolved in DMSO, oleanolic acid (50μM) is the positive control drug, and the reaction stop solution is 2M NaOH aqueous solution. The specific steps are as follows: The experiment is performed in a 96-well plate, and 6 groups are set up: negative experimental group (A), negative blank background group (A0), test sample experimental group (B), test sample background group (B0), positive drug experimental group (C), and positive drug background group (C0), with 3 parallel groups in each group. First, add 48 μL of buffered saline solution to each group of wells, then take 2 μL of blank solvent DMSO and add it to wells A and A0, 2 μL of the sample to be tested is added to wells B and B0, and 2 μL of the positive drug is added to wells C and C0. Then take 60 μL of PTP1B dilution and add it to the wells of groups A, B and C, and 60 μL of buffered saline solution to the wells of groups A0, B0 and C0. Beat to mix, and incubate in a 37°C incubator for 5 minutes. Then, add 40 μL of substrate pNPP solution to each well and place it in a 37°C incubator for 20 minutes. After the reaction is completed, add 50 μL of NaOH aqueous solution to each well to terminate the reaction. Read the absorbance value at a wavelength of 405 nm with a multifunctional microplate reader to calculate the inhibition rate of the test compound. Then use the inhibition rate calculator IC at different concentrations to calculate the inhibition rate of the test compound. 50 The results are shown in Table 2.

[0035] The α-glucosidase inhibitory activity test used 0.1M PBS as the experimental phosphate buffer solution with a pH of 6.8. The sample was dissolved in an appropriate amount of DMSO and diluted with PBS. The substrate was 2.5mM PNPG, 387.2μM acarbose was used as the positive control drug, the α-glucosidase concentration was 0.5U / mL, and the reaction stop solution was a 0.2M Na2CO3 aqueous solution. The specific steps were as follows: the experiment was performed in a 96-well plate, and six groups were set up: negative experimental group (A), negative blank background group (A0), test sample experimental group (B), test sample background group (B0), positive drug experimental group (C), and positive drug background group (C0), with three replicates per group. First, add 60 μL of PBS to each well, then take 20 μL of blank solvent and add it to wells A and A0, 20 μL of the sample to be tested is added to wells B and B0, 20 μL of the positive drug is added to the positive drug C and C0 wells, and then take 20 μL of α-glucosidase and add it to the wells of groups A, B and C, 20 μL of PBS is added to the wells of groups A0, B0 and C0, pat and mix, place in a 37°C incubator for 15 minutes, take out, add 20 μL of PNPG solution to each well, place in a 37°C incubator for 30 minutes, and after the reaction is completed, add 80 μL of Na2CO3 aqueous solution to each well to terminate the reaction. Read the absorbance value at a wavelength of 405 nm with a multifunctional microplate reader to calculate the inhibition rate of the test compound. Then use the inhibition rate calculator IC at different concentrations. 50 The results are shown in Table 2.

[0036] Table 2 Enzyme inhibition activity IC of myricetin and compounds 1-3 50 / μM

[0037]

[0038] Myricetin showed good activity in the PTP1B and α-glucosidase inhibitory activity tests. The introduction of bromine atoms may enhance the binding affinity of the compound to the target. Therefore, myricetin was used as a raw material to synthesize brominated derivatives, resulting in three bromine-containing myricetin derivatives, Compound 1, Compound 2, and Compound 3. Their inhibitory activity against PTP1B and α-glucosidase was tested. The results showed that Compounds 1-3 all showed stronger inhibitory activity than the positive drug, as shown in Table 2. Among them, Compound 3 had stronger inhibitory activity against both enzymes than myricetin.

[0039] To further explore the cytotoxicity of myricetin and its brominated derivatives, normal human hepatocytes L-02 in the logarithmic growth phase were seeded in 96-well cell culture plates at a cell density of 1×10 5The compound was diluted with culture medium to prepare a series of concentrations, applied to cells, and placed in a CO2 incubator at 37°C, 5% CO2 for 24 hours. Then 10 μL of CCK8 was added to the culture medium and incubated for 2 hours. The absorbance at a wavelength of 450 nm was measured using a multifunctional microplate reader. The data was processed and the relative viability of the cells treated with each concentration of the compound was calculated. The results showed that the cytotoxicity of the compound was low, and the IC 50 Greater than 100 μM, the results are shown in Table 3.

[0040] Table 3 Toxicity test IC of myricetin and compounds 1-3 on normal human liver cells L-02 50 / μM

[0041]

[0042] As shown in Tables 2 and 3, compounds 1-3 have strong inhibitory activity against PTP1B and α-glucosidase, two classic therapeutic targets in diabetes, and their IC values ​​for normal human liver cells L-02 are 50 All of them are greater than 100 μM, indicating that all the compounds in the present invention have multi-target hypoglycemic activity and low toxicity, and are characterized by high efficiency and low toxicity.

[0043] The above embodiments are merely examples for clarity of explanation and are not intended to limit the embodiments. Those skilled in the art will appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all embodiments here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A polybrominated derivative of myricetin, prepared by brominating myricetin as a raw material, characterized in that: The structural formula of the polybrominated myricetin derivative is any one of the compounds shown in 1-3:

2. A method for preparing a polybrominated derivative of myricetin according to claim 1, wherein the compound is prepared by a bromination reaction, and the specific preparation steps are: using myricetin as a raw material, dissolving it with 5-15 parts of acetonitrile / methanol, adding 2-8 parts of N-bromosuccinimide, adding 5-30 parts of water to quench the reaction after the reaction is completed, adding 100-300 parts of ethyl acetate / dichloromethane for extraction, and preparing the ethyl acetate layer / dichloromethane layer by high performance liquid chromatography to obtain the target compound 1-3.

3. A use of the brominated myricetin derivative according to claim 1, characterized in that: The myricetin bromide derivative is used for preparing a diabetic drug capable of resisting PTP1B and α-glucosidase activity.

4. The use of the brominated derivative of myricetin according to claim 3, characterized in that: When the compound is used as medicine, it can be used directly or in the form of a pharmaceutical composition. The pharmaceutical composition contains 0.1-99% of the compound and the rest is a pharmaceutical carrier.

5. A drug for preventing or treating diabetes, characterized in that: The medicine contains the compound according to any one of claims 1 and a pharmaceutically acceptable carrier.

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