Flavonoid compound, preparation method and application thereof, pharmaceutical composition and application thereof

Abstract

The invention provides a flavonoid compound, a preparation method and application thereof, a pharmaceutical composition and application thereof, and relates to the technical field of biological medicine. The flavonoid compound provided by the invention has excellent antioxidant activity and a remarkable GSH-Px activation effect, has low toxicity and high efficiency, is suitable for preparing various antioxidant drugs and drugs for treating diseases related to oxidative stress, has wide clinical application prospects and important drug development values, and has wide application prospects. The progress of research and development of flavonoid drugs is promoted, and a new solution is provided for antioxidant treatment. In-vitro experiments and cell model verification show that in HUVECs and AML12 cell lines, compared with kaempferol, the flavonoid compound provided by the invention has the advantages that the activity of GPX4 and the protein expression level of GPX4 are remarkably improved; the DPPH free radical scavenging capacity is obviously higher than that of kaempferol, and the oxidation resistance is obviously better than that of kaempferol; the toxicity to AML12 cells is obviously lower than that of kaempferol.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a flavonoid compound and a preparation method and application thereof, a pharmaceutical composition and application thereof. Background Art

[0002] Flavonoids play an important role in the treatment of various diseases due to their wide range of pharmacological activities and low toxicity. Among them, the flavonoid compound kaempferol (structural formula below) is mainly derived from the rhizome of the ginger plant kaempferol (kaempferolgalangaL), which is widely present in various fruits, vegetables and beverages, and can be extracted from tea, cabbage, witch hazelnut, propolis, grapefruit and other green plants. It has attracted widespread attention because of its various effects such as anti-cancer, anti-cancer, anti-inflammatory, antioxidant, antibacterial and antiviral. However, kaempferol is not good enough to activate glutathione peroxidase (GSH-Px).

[0003] Summary of the invention

[0004] In view of this, the object of the present invention is to provide a flavonoid compound and a preparation method and application thereof, a pharmaceutical composition and application thereof. The flavonoid compound provided by the present invention has excellent antioxidant activity and GSH-Px activation effect, and has low toxicity.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a flavonoid compound having a structure shown in Formula I:

[0007]

[0008] The present invention provides a method for preparing the flavonoid compound described in the above technical solution, comprising the following steps:

[0009] Compound M3 is subjected to a hydroxyl protection reaction with a first (chloromethyl) methyl ether under alkaline conditions to obtain compound M4;

[0010] Acylation reaction of compound M3a with a second (chloromethyl) methyl ether under alkaline conditions to obtain compound M3b;

[0011] The compound M4 and the compound M3b are subjected to an etherification reaction under alkaline conditions to obtain a compound M5;

[0012] The compound M5 is subjected to an oxidation reaction in the presence of an oxidant to obtain a compound M6;

[0013] Acetylation reaction of the compound M6 with acetic anhydride to obtain compound M7;

[0014] The compound M7 is subjected to epoxidation reaction with dimethyldioxane to obtain a compound M8;

[0015] hydrolyzing the compound M8 under alkaline conditions to obtain the flavonoid compound;

[0016]

[0017] Preferably, the molar ratio of the compound M3 to the first (chloromethyl) methyl ether is 1:3-4;

[0018] The temperature of the hydroxyl protection reaction is 15-20° C., and the time is 8-16 hours; the organic solvent used in the hydroxyl protection reaction includes one or more of acetone, acetonitrile, tetrahydrofuran and dichloromethane;

[0019] The molar ratio of the compound M3a to the second (chloromethyl) methyl ether is 1:1.2-1.6;

[0020] The temperature of the acylation reaction is room temperature and the time is 2 to 4 hours; the organic solvent used in the acylation reaction includes one or more of acetone, ethanol and dichloromethane.

[0021] Preferably, the molar ratio of the compound M4 to the compound M3b is 1:0.7-1.5;

[0022] The etherification reaction is carried out under reflux conditions, and the etherification reaction time is 4 to 8 hours;

[0023] The organic solvent used in the etherification reaction includes one or more of ethanol, methanol and dichloromethane.

[0024] Preferably, the oxidant comprises one or more of iodine, cyanogen chloride, benzoyl peroxide and trifluoroacetic anhydride;

[0025] The molar ratio of the compound M5 to the oxidant is 1:0.05-0.15;

[0026] The temperature of the first oxidation reaction is 80-90°C and the time is 8-16h;

[0027] The organic solvent used in the first oxidation reaction includes one or more of dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide and methanol.

[0028] Preferably, the molar ratio of the compound M6 to acetic anhydride is 1:5-7;

[0029] The acetylation reaction is carried out under reflux conditions, and the acetylation reaction time is 2 to 4 hours;

[0030] The organic solvent used in the acetylation reaction includes one or more of pyridine, dichloromethane, dimethylformamide and N,N-dimethylacetamide.

[0031] Preferably, the molar ratio of the compound M7 to dimethyldioxane is 1:5 to 15;

[0032] The epoxidation reaction is carried out at room temperature for 18 to 24 hours.

[0033] The organic solvent used in the epoxidation reaction includes one or more of dichloromethane, toluene, ethyl acetate and tetrahydrofuran.

[0034] Preferably, the hydrolysis reaction is carried out at a temperature of 35 to 45° C. and for a time of 20 to 40 minutes.

[0035] The present invention provides the use of the flavonoid compounds described in the above technical solution in the preparation of antioxidant drugs or drugs for treating and preparing diseases related to oxidative stress.

[0036] The present invention also provides a pharmaceutical composition, comprising the flavonoid compound described in the above technical solution and pharmaceutically acceptable excipients.

[0037] The flavonoid compound (denoted as flavonoid 01 substance) having the structure shown in Formula I provided by the present invention has excellent antioxidant activity and significant GSH-Px activation effect, and has low toxicity and high efficiency. It is suitable for preparing a variety of antioxidant drugs and drugs for treating diseases related to oxidative stress. It has broad clinical application prospects and important drug development value, which will promote the progress of flavonoid drug research and development and provide new solutions for antioxidant therapy.

[0038] The results of GPX4 activity assay in HUVECs cell line showed that there was no significant difference in GPX4 activity between the kaempferol-treated group and the control group; the GPX4 activity in the flavonoid 01-treated group was significantly higher than that in the control group and the kaempferol-treated group (P<0.01). The results of GPX4 activity assay in AML12 cell line showed that there was no significant difference in GPX4 activity between the kaempferol-treated group and the control group, and the GPX4 activity in the flavonoid 01-treated group was significantly higher than that in the control group and the kaempferol-treated group (P<0.01). The results of GPX4 activity assay in AML12 cell line showed that there was no significant difference in GPX4 activity between the kaempferol-treated group and the control group, and the GPX4 activity in the flavonoid 01-treated group was significantly higher than that in the control group and the kaempferol-treated group (P<0.01).

[0039] The results of GPX4 expression detection in HUVECs cell line showed that there was no significant difference in GPX4 protein expression between the kaempferol-treated group and the control group, and the GPX4 protein expression level in the flavonoid 01-treated group was significantly higher than that in the control group and the kaempferol-treated group (P<0.05), and the expression of GPX4 also increased significantly with the increase of the dose. The results of GPX4 expression detection in AML12 cell line showed that there was no significant difference in GPX4 protein expression between the kaempferol-treated group and the control group, and the GPX4 protein expression level in the flavonoid 01-treated group was significantly higher than that in the control group and the kaempferol-treated group (P<0.05), and the expression of GPX4 also increased significantly with the increase of the dose.

[0040] In HUVECs and AML12 cell lines, flavonoid 01 significantly increased the activity and protein expression level of GPX4 at high doses, which was significantly superior to kaempferol. Both the low-dose and high-dose groups of flavonoid 01 showed an enhanced effect on GPX4 activity and expression level, but the effect of the high-dose group was the most significant.

[0041] At the same concentration, the DPPH free radical scavenging ability of flavonoid 01 was significantly higher than that of kaempferol, and its antioxidant activity was significantly better than that of kaempferol.

[0042] At the same concentration, the toxicity of flavonoid 01 to AML12 cells was significantly lower than that of kaempferol.

[0043] The present invention provides a method for preparing flavonoid compounds according to the above technical solution. The process of extracting and separating natural flavonoid compounds from plants is complicated, and the product yield is low, which limits its application in drug development. Compared with the method of extracting from plants, the present invention adopts a chemical synthesis method, and the yield of flavonoid compounds is higher, ensuring the stability and consistency of flavonoid compound products, and laying a solid foundation for subsequent clinical trials and large-scale production. The preparation method provided by the present invention has few by-products, simple post-processing, simple operation, low cost, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Changes in GPX4 activity in HUVECs and AML12 cells among different treatment groups;

[0045] Figure 2 The graph shows the changes in GPX4 expression in HUVECs and AML12 cell lines among different treatment groups;

[0046] Figure 3 This is a schematic diagram of the structural docking of flavonoid compounds and GPX4 protein structure molecules provided by the present invention. DETAILED DESCRIPTION

[0047] The present invention provides a flavonoid compound having a structure shown in Formula I:

[0048]

[0049] The present invention also provides a method for preparing the flavonoid compound described in the above technical solution, comprising the following steps:

[0050] Compound M3 is subjected to a hydroxyl protection reaction with a first (chloromethyl) methyl ether under alkaline conditions to obtain compound M4;

[0051] Acylation reaction of compound M3a with a second (chloromethyl) methyl ether under alkaline conditions to obtain compound M3b;

[0052] The compound M4 and the compound M3b are subjected to an etherification reaction under alkaline conditions to obtain a compound M5;

[0053] The compound M5 is subjected to an oxidation reaction in the presence of an oxidant to obtain a compound M6;

[0054] Acetylation reaction of the compound M6 with acetic anhydride to obtain compound M7;

[0055] The compound M7 is subjected to epoxidation reaction with dimethyldioxane to obtain a compound M8;

[0056] hydrolyzing the compound M8 under alkaline conditions to obtain the flavonoid compound;

[0057]

[0058] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0059] The present invention conducts a hydroxyl protection reaction of compound M3 with a first (chloromethyl) methyl ether (MOMCl) under alkaline conditions to obtain compound M4. Specifically, compound M3, the first MOMCl, an alkaline reagent (referred to as the first alkaline reagent) and an organic solvent (referred to as the first organic solvent) are mixed, and a hydroxyl protection reaction is conducted under a protective atmosphere to obtain compound M4.

[0060] In the present invention, the molar ratio of the compound M3 to the first MOMC1 is preferably 1:3-4, more preferably 1:3.5.

[0061] In the present invention, the first alkaline agent preferably includes alkali metal carbonate and / or alkali metal hydroxide; the alkali metal carbonate preferably includes potassium carbonate and / or sodium carbonate; the alkali metal hydroxide preferably includes sodium hydroxide and / or potassium hydroxide; the first alkaline agent is preferably an anhydrous alkaline agent. In the present invention, the molar ratio of the compound M3 to the first alkaline agent is preferably 1:4-5, more preferably 1:4.5.

[0062] In the present invention, the first organic solvent preferably includes one or more of acetone, acetonitrile, tetrahydrofuran and dichloromethane, more preferably acetone; the first organic solvent is preferably an anhydrous organic solvent. In the present invention, the amount ratio of the compound M3 to the first organic solvent is preferably 1 mol: 7 to 9 L, more preferably 1 mol: 7.5 to 8 L.

[0063] In the present invention, the mixing of compound M3, the first MOMCl, the first alkaline reagent and the first organic solvent preferably includes: dissolving the first MOMCl in part of the organic solvent to obtain a first MOMCl solution; dissolving compound M3 in the remaining first organic solvent to obtain a compound M3 solution; adding the first alkaline reagent to the compound M3 solution at room temperature (20-25°C), cooling to -15--5°C (more preferably -10°C) under a protective atmosphere, stirring for 15-30min (more preferably 20-25min), and adding the first MOMCl solution dropwise at -25--15°C (more preferably -20°C). In the present invention, the concentration of the first MOMCl solution is preferably 2.0-3.0M (ie, mol / L), more preferably 2.4M. In the present invention, the concentration of the compound M3 solution is preferably 0.1-0.2M, more preferably 0.1-0.15M.

[0064] In the present invention, the protective atmosphere preferably includes argon, helium or nitrogen.

[0065] In the present invention, the temperature of the hydroxyl protection reaction is preferably 15-20°C, more preferably 16-18°C; the time of the hydroxyl protection reaction is preferably 8-16h, more preferably 10-12h. In a specific embodiment of the present invention, the progress of the hydroxyl protection reaction is preferably monitored by thin layer chromatography (TLC), and the hydroxyl protection reaction is stopped when the dots of the reactants disappear; the developing solvent used in the thin layer chromatography is preferably ethyl acetate-petroleum ether (EA-PE), and the volume fraction of ethyl acetate in the ethyl acetate-petroleum ether is preferably 20-40%, more preferably 30%.

[0066] After completing the hydroxyl protection reaction, the present invention preferably further comprises: subjecting the obtained hydroxyl protection reaction liquid to solid-liquid separation to obtain a liquid component and a solid component respectively, washing the solid component with ethyl acetate to obtain a washing liquid; combining the liquid component with the washing liquid, concentrating to constant weight, dissolving the obtained concentrate in water, adjusting the pH value to weak acidity under ice bath conditions, extracting with an organic solvent, and sequentially washing the obtained organic phase with saturated brine, drying with a desiccant, solid-liquid separation to remove the desiccant, concentrating to constant weight, and purifying with silica gel column chromatography to obtain compound M4. The present invention has no special restrictions on the two solid-liquid separations, and a solid-liquid separation method well known to those skilled in the art can be used, such as filtration. In the present invention, the number of ethyl acetate washings is preferably 2 to 4 times, and more preferably 3 times. The present invention has no special restrictions on both concentrations, and a concentration method well known to those skilled in the art can be used, such as reduced pressure concentration. In the present invention, the acid used to adjust the pH value preferably includes one or more of dilute hydrochloric acid, acetic acid, citric acid, and tartaric acid; the concentration of the dilute hydrochloric acid is preferably 0.8 to 1.5M, and more preferably 1 to 1.2M. In the present invention, the weakly acidic pH value is preferably 5 to 6, more preferably 5.5 to 5.8. In the present invention, the organic solvent for extraction preferably includes ethyl acetate and / or dichloromethane, more preferably ethyl acetate; the number of extractions is preferably 2 to 4 times, more preferably 3 times. In the present invention, the desiccant preferably includes anhydrous sodium sulfate and / or anhydrous magnesium sulfate. In the present invention, the eluent used in the silica gel column chromatography purification preferably includes ethyl acetate-petroleum ether, and the volume fraction of ethyl acetate in the ethyl acetate-petroleum ether is preferably 10 to 15%, more preferably 12%; the particle size of the silica gel used in the silica gel column chromatography purification is preferably 200 to 300 meshes.

[0067] In the present invention, the compound M3 is preferably a commercially available product or prepared. In the present invention, the preparation method of the compound M3 preferably comprises the following steps: mixing the compound M2, acetic acid (AcOH), acetic anhydride and BF3·Et2O, and performing a Friedel-Crafts acylation reaction under a protective atmosphere to obtain the compound M3;

[0068]

[0069] In the present invention, the usage ratio of compound M2 to acetic acid is preferably 1 g: 7.8 to 10.8 mL, more preferably 1 g: 8 to 10 mL, and further preferably 1 g: 9 to 9.5 mL.

[0070] In the present invention, the molar ratio of compound M2 to acetic anhydride is preferably 1:1 to 1.5, more preferably 1:1.1 to 1.4, and further preferably 1:1.2 to 1.3.

[0071] In the present invention, the molar ratio of compound M2 to BF3.Et2O is preferably 1:1 to 1.5, more preferably 1:1.1 to 1.4, and further preferably 1:1.2 to 1.3.

[0072] In the present invention, the mixing of compound M2, acetic acid, acetic anhydride and BF3·Et2O preferably comprises: adding compound M2, adding acetic acid, filling with a protective atmosphere to replace the air, and then adding acetic anhydride and BF3·Et2O at 0-4°C. In the present invention, the protective atmosphere preferably comprises argon, helium or nitrogen.

[0073] In the present invention, the Friedel-Crafts acylation reaction is preferably carried out under reflux conditions, and the time of the Friedel-Crafts acylation reaction is preferably 4 to 8 hours, more preferably 5 to 6 hours.

[0074] After completing the Friedel-Crafts acylation reaction, the present invention preferably further comprises: cooling the obtained Friedel-Crafts acylation reaction solution to room temperature, pouring it into water and letting it stand, separating the solid and the liquid, washing the obtained solid component with water, extracting the obtained aqueous phase with an organic solvent, washing the obtained organic phase with saturated brine, drying with a desiccant, removing the desiccant by solid-liquid separation, concentrating and purifying by silica gel column chromatography to obtain compound M3. In the present invention, the amount ratio of the compound M2 to water (water for pouring it into water and letting it stand) is preferably 1g:30-50mL, more preferably 1g:40mL. In the present invention, the number of water washings is preferably 1-3 times, more preferably 2 times; the amount ratio of the compound M2 to the water for a single water washing is preferably 1g:1.5-2.5mL, more preferably 1g:2mL. In the present invention, the organic solvent for extraction preferably includes ethyl acetate and / or dichloromethane, more preferably ethyl acetate; the number of extractions is preferably 2-5 times, more preferably 2-4 times. In the present invention, the desiccant preferably includes anhydrous sodium sulfate and / or anhydrous magnesium sulfate. The present invention has no special limitation on the two solid-liquid separations, and the solid-liquid separation method well known to those skilled in the art can be used, such as filtration. In the present invention, the elution method of the silica gel column chromatography purification is preferably gradient washing, and the eluents used preferably include methanol-dichloromethane and ethyl acetate-petroleum ether in sequence, and the volume fraction of methanol in the methanol-dichloromethane is preferably 0.5-2%, more preferably 1-1.5%; the volume fraction of ethyl acetate in the ethyl acetate-petroleum ether is preferably 20-40%, more preferably 30-35%; the particle size of the silica gel used for the silica gel column chromatography purification is preferably 100-200 meshes.

[0075] In the present invention, the compound M2 is preferably a commercially available product or self-made. In the present invention, the preparation method of the compound M2 preferably comprises the following steps: mixing the compound M1, an indicator, sodium cyanoborohydride and an organic solvent (referred to as the seventh organic solvent), performing a first reductive amination reaction under a protective atmosphere, and then adding an acid solution to perform a second reductive amination reaction to obtain the compound M2;

[0076]

[0077] In the present invention, the indicator preferably includes methyl orange. In the present invention, the usage ratio of the compound M1 to methyl orange is preferably 1 mol: 0.6-1 g, more preferably 1 mol: 0.6-0.8 g, and further preferably 1 mol: 0.6-0.7 g.

[0078] In the present invention, the molar ratio of the compound M1 to sodium cyanoborohydride is preferably 1:0.12-0.24, and more preferably 1:0.15-0.2.

[0079] In the present invention, the seventh organic solvent preferably includes one or more of tetrahydrofuran (THF), acetonitrile, dichloromethane and diethyl ether, and more preferably THF. In the present invention, the amount ratio of the compound M1 to the seventh organic solvent is preferably 30g:750-800mL, more preferably 30g:780-840mL, and further preferably 30g:800-825mL.

[0080] In the present invention, the protective atmosphere preferably includes argon, helium or nitrogen.

[0081] In the present invention, mixing the compound M1, the indicator, sodium cyanoborohydride and the seventh organic solvent preferably includes: dissolving the compound M1 in the seventh organic solvent at 0 to 4°C under a protective atmosphere, adding the indicator, and then adding sodium cyanoborohydride in batches. In the present invention, the temperature of the first reductive amination reaction is preferably 0 to 4°C, more preferably 0 to 2°C; the time of the first reductive amination reaction is preferably 15 to 45 minutes, more preferably 20 to 40 minutes, and further preferably 30 minutes. In the present invention, the sodium cyanoborohydride is preferably added in batches of 3 to 4 times.

[0082] In the present invention, the acid solution preferably includes a hydrochloric acid solution, and the concentration of the hydrochloric acid solution is preferably 3.5 to 4.5 M, more preferably 4 M; the acid solution is preferably added dropwise. In the present invention, the dosage ratio of the compound M1 to the acid solution is preferably 30 g: 130 to 150 mL, more preferably 30 g: 135 to 145 mL, and further preferably 30 g: 140 mL.

[0083] In the present invention, the temperature of the second reductive amination reaction is preferably 0-4°C, more preferably 0-2°C, and the time is preferably 10-18h, more preferably 12-17h, and further preferably 15-16h.

[0084] After completing the second reductive amination reaction, the present invention preferably further comprises: mixing the obtained second reductive amination reaction liquid with water, adjusting the pH value to 4.8-5.2 (more preferably 5), extracting with an organic solvent, washing the obtained organic phase with saturated brine, drying with a desiccant, removing the desiccant by solid-liquid separation, concentrating and purifying by silica gel column chromatography to obtain compound M2. In the present invention, the dosage ratio of the compound M1 to water (mixing water) is preferably 30g: 0.3-0.7L, more preferably 30g: 0.4-0.5L. In the present invention, the acid used for adjusting the pH value preferably comprises a hydrochloric acid solution, and the concentration of the hydrochloric acid solution is preferably 0.5-1.5M, more preferably 1M. In the present invention, the organic solvent for extraction preferably comprises ethyl acetate and / or dichloromethane, more preferably ethyl acetate; the number of extractions is preferably 2-5 times, more preferably 3-4 times. In the present invention, the desiccant preferably comprises anhydrous sodium sulfate and / or anhydrous magnesium sulfate. The present invention has no special limitation on the solid-liquid separation, and a solid-liquid separation method well known to those skilled in the art can be used, such as filtration. The present invention has no special limitation on the concentration, and a concentration method well known to those skilled in the art can be used, such as reduced pressure concentration. In the present invention, the eluent used in the silica gel column chromatography purification preferably includes ethyl acetate-petroleum ether, and the volume fraction of ethyl acetate in the ethyl acetate-petroleum ether is preferably 20-30%, more preferably 25%; the particle size of the silica gel used in the silica gel column chromatography purification is preferably 100-200 meshes.

[0085] In the present invention, the compound M1 is preferably a commercial product or self-made. In the present invention, the preparation method of the compound M1 preferably comprises the following steps: mixing phosphorus oxychloride and N,N-dimethylformamide (DMF) amide, performing a Vilsmeier-Haack reaction, and then adding phloroglucinol to perform an electrophilic aromatic substitution reaction to obtain compound M1.

[0086] In the present invention, the molar ratio of phloroglucinol to phosphorus oxychloride is preferably 1:2-3, more preferably 1:2-2.5. In the present invention, the molar ratio of phloroglucinol to DMF is preferably 1:2-3, more preferably 1:2-2.5. In the present invention, the phloroglucinol is preferably used in the form of a phloroglucinol solution, and the concentration of the phloroglucinol solution is preferably 0.8-1 mol / L, more preferably 0.81-0.95 mol / L, and further preferably 0.85-0.9 mol / L; the solvent in the phloroglucinol solution preferably includes one or more of dioxane, tetrahydrofuran (THF) and acetonitrile.

[0087] In the present invention, the temperature for mixing the phosphorus oxychloride and N,N-dimethylformamide (DMF) is preferably 0-4° C., more preferably 0-2° C.; the atmosphere for the mixing is preferably a protective atmosphere.

[0088] In the present invention, the temperature of the Vilsmeier-Haack reaction is preferably 0-4°C, more preferably 0-2°C; the time of the Vilsmeier-Haack reaction is preferably 0.2-0.8h, more preferably 0.4-0.5h; and the Vilsmeier-Haack reaction is preferably carried out under a protective atmosphere.

[0089] In the present invention, phloroglucinol is preferably added dropwise in the form of a phloroglucinol solution; the dropping is preferably performed under a protective atmosphere.

[0090] In the present invention, the electrophilic aromatic substitution reaction preferably includes a first electrophilic aromatic substitution reaction and a second electrophilic aromatic substitution reaction performed in sequence; the temperature of the first electrophilic aromatic substitution reaction is preferably 0-4°C, more preferably 0-2°C; the time of the first electrophilic aromatic substitution reaction is preferably 0.2-0.5h, more preferably 0.4-0.5h; the temperature of the second electrophilic aromatic substitution reaction is preferably room temperature; the time of the second electrophilic aromatic substitution reaction is preferably 12-20h, more preferably 15-16h; the electrophilic aromatic substitution reaction is preferably carried out under a protective atmosphere.

[0091] In the present invention, the protective atmosphere preferably includes argon, helium or nitrogen.

[0092] After completing the electrophilic aromatic substitution reaction, the present invention preferably further comprises: placing the obtained electrophilic aromatic substitution reaction solution in crushed ice, ultrasonically treating to obtain a clear solution, concentrating the clear solution and then performing solid-liquid separation, washing the obtained solid component with water and then drying to obtain compound M1. The present invention has no special limitation on the solid-liquid separation, and a solid-liquid separation method well known to those skilled in the art can be used, such as filtration. The present invention has no special limitation on the concentration, and the concentration can be performed until the amount of solid precipitation no longer increases, such as concentration under reduced pressure. In the present invention, the water for washing preferably comprises deionized water. In the present invention, the drying preferably comprises a solid oil pump spin-drying.

[0093] The present invention conducts an acylation reaction between compound M3a and a second (chloromethyl) methyl ether under alkaline conditions to obtain compound M3b. Specifically, compound M3a, an alkaline reagent (referred to as the second alkaline reagent), a second MOMCl and an organic solvent (referred to as the second organic solvent) are mixed to conduct an acylation reaction to obtain compound M3b.

[0094] In the present invention, the molar ratio of the compound M3a to the second MOMC1 is preferably 1:1.2-1.6, more preferably 1:1.2-1.5, and more preferably 1:1.3-1.4.

[0095] In the present invention, the second alkaline agent preferably includes alkali metal carbonate and / or alkali metal hydroxide; the alkali metal carbonate preferably includes potassium carbonate and / or sodium carbonate; the alkali metal hydroxide preferably includes sodium hydroxide and / or potassium hydroxide; the second alkaline agent is preferably an anhydrous alkaline agent. In the present invention, the molar ratio of the compound M3a to the second alkaline agent is preferably 1:3-5, more preferably 1:3-4.

[0096] In the present invention, the second organic solvent preferably includes one or more of acetone, ethanol and dichloromethane; the second organic solvent is preferably an anhydrous organic solvent. In the present invention, the amount ratio of the compound M3a to the second organic solvent is preferably 15g:300-500mL, more preferably 15g:400mL.

[0097] In the present invention, the mixing of compound M3a, a second MOMCl, a second alkaline reagent and a second organic solvent preferably comprises: dissolving compound M3a in a second organic solvent, adding the second alkaline reagent at room temperature (20 to 25°C), cooling to -4 to 4°C (more preferably 0°C), and adding the second MOMCl dropwise.

[0098] In the present invention, the temperature of the acylation reaction is preferably room temperature; the time of the acylation reaction is preferably 2 to 4 hours, more preferably 2 to 3 hours.

[0099] After completing the acylation reaction, the present invention preferably further comprises: subjecting the obtained acylation reaction liquid to solid-liquid separation, mixing the obtained liquid component with water, extracting with an organic solvent, washing the obtained organic phase with saturated salt water, drying with a desiccant, separating the solid and liquid to remove the desiccant, and concentrating to constant weight to obtain compound M3b. The present invention has no special limitation on the two solid-liquid separations, and a solid-liquid separation method well known to those skilled in the art can be used, such as filtration. In the present invention, the organic solvent for extraction preferably includes ethyl acetate and / or dichloromethane, more preferably ethyl acetate; the number of extractions is preferably 2 to 5 times, more preferably 2 to 3 times. In the present invention, the desiccant preferably includes anhydrous sodium sulfate and / or anhydrous magnesium sulfate. The present invention has no special limitation on the concentration, and a concentration method well known to those skilled in the art can be used, such as reduced pressure concentration.

[0100] After obtaining compound M4 and compound M3b, the present invention conducts an etherification reaction between the compound M4 and the compound M3b under alkaline conditions to obtain compound M5. Specifically, the compound M4, the compound M3b, an alkaline reagent (referred to as the third alkaline reagent) and an organic solvent (referred to as the third organic solvent) are mixed to conduct an etherification reaction to obtain compound M5.

[0101] In the present invention, the molar ratio of the compound M4 to the compound M3b is preferably 1:0.7-1.5, and more preferably 1:1.2-1.3.

[0102] In the present invention, the third alkaline agent preferably includes a hydroxide, more preferably includes one or more of potassium hydroxide, sodium hydroxide and ammonium hydroxide; the third alkaline agent is preferably an anhydrous alkaline agent. In the present invention, the molar ratio of the compound M4 to the third alkaline agent is preferably 1:1 to 1.5, more preferably 1:1 to 1.1.

[0103] In the present invention, the third organic solvent preferably includes one or more of ethanol, methanol and dichloromethane; the third organic solvent is preferably an anhydrous organic solvent. In the present invention, the dosage ratio of the compound M4 to the second organic solvent is preferably 14g:400-500mL, more preferably 14g:450-490mL.

[0104] In the present invention, the mixing of the compound M4, the compound M3b, the third alkaline agent and the third organic solvent preferably includes: dissolving the compound M4 and the compound M3b in the third organic solvent, and then adding the third alkaline agent.

[0105] In the present invention, the etherification reaction is preferably carried out under reflux conditions, and the time of the etherification reaction is preferably 4 to 8 hours, more preferably 5 to 6 hours.

[0106] After the etherification reaction is completed, the present invention preferably further comprises: cooling the obtained etherification reaction liquid to room temperature, adding saturated ammonium chloride solution, adding water, extracting with an organic solvent, washing the obtained organic phase with saturated salt water, drying with a desiccant, solid-liquid separation to remove the desiccant, concentrating and purifying by silica gel column chromatography in sequence to obtain compound M5. In the present invention, the organic solvent for extraction preferably comprises ethyl acetate and / or dichloromethane, more preferably ethyl acetate; the number of extractions is preferably 2 to 4 times, more preferably 3 times. In the present invention, the desiccant preferably comprises anhydrous sodium sulfate and / or anhydrous magnesium sulfate. The present invention has no special limitation on the solid-liquid separation, and a solid-liquid separation method well known to those skilled in the art can be used, such as filtering. The present invention has no special limitation on the concentration, and a concentration method well known to those skilled in the art can be used, such as reduced pressure concentration. In the present invention, the eluent used for the silica gel column chromatography purification preferably comprises ethyl acetate-petroleum ether, and the volume fraction of ethyl acetate in the ethyl acetate-petroleum ether is preferably 5 to 7%, more preferably 6%; the particle size of the silica gel used for the silica gel column chromatography purification is preferably 100 to 200 meshes.

[0107] After obtaining compound M5, the present invention performs an oxidation reaction on compound M5 in the presence of an oxidant to obtain compound M6. Specifically, compound M5, an oxidant and an organic solvent (referred to as the fourth organic solvent) are mixed and subjected to an oxidation reaction (referred to as the first oxidation reaction) to obtain compound M6.

[0108] In the present invention, the oxidant preferably includes one or more of iodine, cyanogen chloride, benzoyl peroxide and trifluoroacetic anhydride, and more preferably iodine. In the present invention, the molar ratio of the compound M5 to the oxidant is preferably 1:0.05-0.15, and more preferably 1:0.08-0.1.

[0109] In the present invention, the fourth organic solvent preferably includes one or more of dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide and methanol, more preferably dimethyl sulfoxide; the fourth organic solvent is preferably an anhydrous organic solvent. In the present invention, the dosage ratio of the compound M5 to the fourth organic solvent is preferably 14g:400-500mL, more preferably 14g:450-490mL.

[0110] In the present invention, the temperature of the first oxidation reaction is preferably 80-90°C, more preferably 85°C; the time of the first oxidation reaction is preferably 8-16h, more preferably 10-12h.

[0111] After completing the first oxidation reaction, the present invention preferably further comprises: mixing the obtained first oxidation reaction liquid with water, precipitating a solid, separating the solid and the liquid, washing the obtained solid component with water and drying it to constant weight to obtain compound M6, and the compound M6 is directly used in the next step of the reaction without purification. The present invention has no special limitation on the solid-liquid separation, and a solid-liquid separation method well known to those skilled in the art can be used, such as filtration. In the present invention, the number of times of washing is preferably 1 to 4 times, more preferably 2 to 3 times. In the present invention, the drying preferably includes spin drying.

[0112] After obtaining compound M6, the present invention performs an acetylation reaction on compound M6 and acetic anhydride to obtain compound M7. Specifically, compound M6, acetic anhydride and an organic solvent (referred to as the fifth organic solvent) are mixed and subjected to an acetylation reaction to obtain compound M7.

[0113] In the present invention, the molar ratio of the compound M6 to acetic anhydride is preferably 1:5-7, more preferably 1:5.5-6.5, and further preferably 1:6.

[0114] In the present invention, the fifth organic solvent includes one or more of pyridine, dichloromethane, dimethylformamide and N,N-dimethylacetamide, more preferably pyridine. In the present invention, the amount ratio of the compound M6 to the fifth organic solvent is preferably 7g:100-140mL, more preferably 7g:110-120mL.

[0115] In the present invention, the mixing of the compound M6, acetic anhydride and the fifth organic solvent preferably includes: dissolving the compound M6 in the fifth organic solvent and then adding acetic anhydride.

[0116] In the present invention, the acetylation reaction is preferably carried out under reflux conditions, and the time of the acetylation reaction is preferably 2 to 4 hours, more preferably 3 hours.

[0117] After completing the acetylation reaction, the present invention preferably further comprises: mixing the obtained acetylation reaction solution with water, precipitating solids, solid-liquid separation, washing the obtained solid component with water and then redissolving it, drying with a desiccant, solid-liquid separation to remove the desiccant, concentrating, and purifying by silica gel column chromatography to obtain compound M7. The present invention has no special limitation on the two solid-liquid separations, and a solid-liquid separation method well known to those skilled in the art can be used, such as filtration. In the present invention, the dosage ratio of the compound M6 to water (water for mixing with water) is preferably 7g: 600-1000mL, and more preferably 7g: 700-800mL. In the present invention, the number of water washings is preferably 1-3 times, and more preferably 2 times. In the present invention, the organic solvent for redissolution preferably includes one or more of dichloromethane, ethyl acetate, ether and acetonitrile. In the present invention, the desiccant preferably includes anhydrous sodium sulfate and / or anhydrous magnesium sulfate. The present invention has no special limitation on the solid-liquid separation, and a solid-liquid separation method well known to those skilled in the art can be used, such as filtration. The present invention has no special limitation on the concentration, and a concentration method well known to those skilled in the art can be used, such as concentration under reduced pressure. In the present invention, the eluent used in the silica gel column chromatography purification preferably includes methanol-dichloromethane, and the volume fraction of methanol in the methanol-dichloromethane is preferably 0.7-1.3%, more preferably 0.8-1%; the particle size of the silica gel used in the silica gel column chromatography purification is preferably 100-200 meshes.

[0118] After obtaining compound M7, the present invention performs an epoxidation reaction on compound M7 and dimethyldioxane (DMDO) to obtain compound M8. Specifically, compound M7, dimethyldioxane and an organic solvent (referred to as the sixth organic solvent) are mixed and subjected to an epoxidation reaction to obtain compound M8.

[0119] In the present invention, the molar ratio of the compound M7 to DMDO is preferably 1:5-15, more preferably 1:812, and further preferably 1:10. In the present invention, the DMDO is preferably used in the form of a DMDO solution, and the solvent in the DMDO solution preferably includes acetone; the concentration of the DMDO solution is preferably 0.05-0.2M, more preferably 0.1-0.15M.

[0120] In the present invention, the sixth organic solvent preferably includes one or more of dichloromethane, toluene, ethyl acetate and tetrahydrofuran, more preferably dichloromethane. In the present invention, the amount ratio of the compound M6 to the sixth organic solvent is preferably 2mmol:100-120mL, more preferably 2mmol:110mL.

[0121] In the present invention, the mixing of the compound M7, dimethyldioxane and a sixth organic solvent preferably includes: dissolving the compound M7 in the sixth organic solvent, and then adding dimethyldioxane.

[0122] In the present invention, the temperature of the epoxidation reaction is preferably room temperature; the time of the epoxidation reaction is preferably 18 to 24 hours, more preferably 20 to 22 hours.

[0123] After the epoxidation reaction is completed, the present invention preferably further comprises: concentrating the obtained epoxidation reaction solution and purifying it by silica gel column chromatography to obtain compound M8. The present invention has no special limitation on the concentration, and a concentration method well known to those skilled in the art can be used, such as rotary evaporation; the concentration temperature is preferably 25 to 35°C, more preferably 30°C. In the present invention, the eluent used for the silica gel column chromatography purification preferably includes ethyl acetate-petroleum ether, and the volume fraction of ethyl acetate in the ethyl acetate-petroleum ether is preferably 20 to 40%, more preferably 30%; the particle size of the silica gel used for the silica gel column chromatography purification is preferably 100 to 200 meshes.

[0124] In the present invention, the DMDO solution is preferably obtained by dissolving commercially available DMDO in a solvent, or is prepared in-house. In the present invention, the method for preparing the DMDO solution preferably comprises the following steps: mixing acetone, water, an alkaline agent (referred to as the fourth alkaline agent) and potassium peroxymonosulfate complex salt (Oxone), performing an oxidation reaction (referred to as the second oxidation reaction), and performing reduced pressure distillation to obtain a DMDO solution.

[0125] In the present invention, the molar ratio of acetone to Oxone is preferably 1:0.1-0.2, more preferably 1:0.15-0.18.

[0126] In the present invention, the fourth alkaline agent preferably includes alkali metal carbonate and / or alkali metal hydroxide; the alkali metal carbonate preferably includes potassium carbonate and / or sodium carbonate; the alkali metal hydroxide preferably includes sodium hydroxide and / or potassium hydroxide. In the present invention, the molar ratio of the acetone to the fourth alkaline agent is preferably 1:0.5-0.8, more preferably 1:0.6-0.7.

[0127] In the present invention, the volume ratio of acetone to water is preferably 1:0.6-0.8, more preferably 1:0.7.

[0128] In the present invention, the mixing of acetone, water, the fourth alkaline agent and Oxone preferably comprises: mixing water, acetone and the fourth alkaline agent, cooling to 0-10°C (more preferably 5°C), and adding Oxone in batches under stirring conditions. In the present invention, the adding time of Oxone is preferably 15-25 minutes, more preferably 20 minutes.

[0129] In the present invention, the temperature of the second oxidation reaction is preferably 0-10°C, more preferably 5°C; the time of the second oxidation reaction is preferably 5-15min, more preferably 10min; the second oxidation reaction is preferably carried out under stirring conditions, and the stirring speed is preferably 200-500r / min, more preferably 300-400r / min.

[0130] After completing the second oxidation reaction, the present invention preferably further comprises cooling, distilling under reduced pressure under stirring conditions, and collecting the DMDO solution. In the present invention, the cooling is preferably cold trap cooling, the cooling temperature is preferably -80 to -70°C, more preferably -78°C; the cooling time is preferably 5 to 15 minutes, more preferably 10 minutes; when the volume change in the cold trap is not obvious, the cold trap is closed and the temperature is returned to 25°C under a protective atmosphere; the cooling is preferably carried out under a protective atmosphere, and the protective atmosphere preferably includes argon, nitrogen or helium.

[0131] After obtaining compound M8, the present invention hydrolyzes the compound M8 under alkaline conditions to obtain the flavonoid compound. Specifically, compound M8 is mixed with an inorganic strong base solution and hydrolyzed under a protective atmosphere to obtain the flavonoid compound.

[0132] In the present invention, the inorganic strong base in the inorganic strong base solution preferably includes one or more of alkali metal alcoholates, alkali metal hydroxides, and alkali metal hydrides, and the alkali metal alcoholates preferably include sodium methoxide (NaOMe) and / or potassium methoxide; the alkali metal hydroxides preferably include NaOH and / or KOH; the alkali metal hydrides preferably include NaH and / or KH; the solvent in the inorganic strong base solution preferably includes one or more of methanol, ethanol and tetrahydrofuran; the inorganic strong base solution specifically preferably includes NaOMe-MeOH solution, NaOMe-EtOH solution, KOH-MeOH solution or NaH-THF solution; the concentration of the inorganic strong base solution is preferably 0.4-0.6M, more preferably 0.5M. In the present invention, the molar ratio of the compound M8 to the inorganic strong base is preferably 1:4-6, more preferably 1:4.5-5.

[0133] In the present invention, the protective atmosphere preferably includes argon, helium or nitrogen.

[0134] In the present invention, the temperature of the hydrolysis reaction is preferably 35-45° C., more preferably 40° C.; the time of the hydrolysis reaction is preferably 20-40 min, more preferably 30 min.

[0135] After the hydrolysis is completed, the present invention preferably further comprises: adjusting the pH value of the obtained hydrolyzate to 1 to 3 (more preferably 2), crystallizing, solid-liquid separation, dissolving the obtained solid component in ethyl acetate, concentrating and recrystallizing, solid-liquid separation, and drying the obtained crystals to obtain the flavonoid compound. In the present invention, the acid used for adjusting the pH value preferably includes a hydrochloric acid solution, and the concentration of the hydrochloric acid solution is preferably 0.5 to 1.5M, more preferably 1M. In the present invention, the crystallization temperature is preferably 0 to 5°C, more preferably 3 to 4°C; the crystallization time is preferably 0.5 to 2h, more preferably 1 to 1.5h. The present invention has no special restrictions on the two solid-liquid separations, and a solid-liquid separation method well known to those skilled in the art can be used, such as filtration. In the present invention, the dosage ratio of the compound M8 to ethyl acetate is preferably 1g: 20 to 35mL, more preferably 1g: 25 to 30mL. In the present invention, the volume ratio of the ethyl acetate to the concentrated solution obtained by concentration is preferably 25 to 35: 8 to 12, more preferably 30: 10. In the present invention, the recrystallization solvent preferably includes one or more of ethyl acetate, ethanol, methanol and water, and more preferably ethyl acetate. In the present invention, the number of recrystallizations is preferably 3 to 5 times, and more preferably 4 times. The present invention has no particular limitation on the drying, as long as the solvent can be completely removed.

[0136] The present invention also provides the use of the flavonoid compounds described in the above technical scheme in the preparation of antioxidant drugs or drugs for treating diseases related to oxidative stress. In the present invention, the diseases related to oxidative stress preferably include diseases related to glutathione peroxidase (GSH-Px), and more preferably include one or more of cardiovascular diseases, neurodegenerative diseases and liver diseases. The flavonoid compounds provided by the present invention have significant antioxidant activity and the ability to activate glutathione peroxidase (GSH-Px), have low toxicity and high efficiency, and have good application prospects in the preparation of antioxidant drugs and drugs for diseases related to oxidative stress.

[0137] The present invention also provides a pharmaceutical composition, comprising the flavonoid compound described in the above technical solution and a pharmaceutically acceptable excipient. The present invention has no special limitation on the pharmaceutically acceptable excipient, and the pharmaceutically acceptable excipients well known to those skilled in the art can be used. The present invention has no special limitation on the dosage form and preparation method of the pharmaceutical composition, and the dosage form and preparation method of the pharmaceutical composition well known to those skilled in the art can be used.

[0138] In order to further illustrate the present invention, the flavonoid compounds and their preparation methods and applications, and pharmaceutical compositions provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0139] Example 1

[0140]

[0141] (1) Preparation of Compound M1

[0142] At 0°C, under argon protection, 656mmol of DMF and 628mmol of phosphorus oxychloride were added, the solution became a light yellow viscous liquid, and stirring was continued for 0.5h to obtain a transparent colorless solution; 285mmol of phloroglucinol was dissolved in 317mL of dioxane, and the obtained phloroglucinol solution was added dropwise to the above transparent colorless solution, and the reaction was carried out for 0.3h until the light yellow solution became an orange-yellow solution, and the temperature was raised to room temperature, and the reaction was stirred for 16h, and the solution was poured into 1L of crushed ice, and ultrasonicated to obtain a clear solution, which was concentrated until the solid precipitate no longer increased and a large amount of yellow solid came out, and filtered, and the obtained filter cake was deionized and dried with a solid oil pump to obtain compound M1 (49.00g, yellow solid, yield 94%).

[0143] Compound M1: 1 H NMR (500MHz, DMSO-d6) δ12.53(s,2H),10.01(s,1H),5.95(s,1H). 13 CNMR(126MHz,DMSO-d6)δ191.85,169.91,169.47,104.20,94.59.LC-MS:[MH] + =181.0112.

[0144] (2) Preparation of Compound M2

[0145] At 0°C, under argon protection, compound M1 (30.00 g, 164.7 mmol) and THF 823 mL were added to obtain an orange-yellow solution. Methyl orange 100 mg was added as an indicator, and sodium cyanoborohydride 26.274 mmol was added in 3 batches. At this time, bubbles were generated and the solution became a khaki turbid liquid. After stirring for 0.5 h, 4M HCl solution 140 mL was added dropwise at a uniform rate and continued to stir for 16 h. The resulting reaction solution was poured into 0.5 L of water, and the pH value was adjusted to 5 with 1M HCl solution. EA 200 mL was added and stirred for 0.5 h. The layers were separated to obtain an organic phase and an aqueous phase, respectively. The aqueous phase was extracted with EA twice, and the organic phases were combined, washed with saturated brine, dried with sodium sulfate, and spin-dried. The mixture was purified by silica gel column chromatography (100-200 mesh silica gel, 20-30 vol% EA-PE) to obtain compound M2 (12.0 g, yellow solid, yield 47%).

[0146] Compound M2: 1 H NMR(500MHz,Chloroform-d)δ8.62(s,2H),7.75(s,1H),5.93(s,1H),1.87(s,6H). 13 C NMR(126MHz,Chloroform-d)δ154.59,153.65,102.06,95.00,9.22.LC-MS:[M+H] + =155.0706.

[0147] (3) Preparation of Compound M3

[0148] At 0°C, compound M2 (10 g, 64.8 mmol) was added, AcOH 93 mL was added, and the mixture was replaced with argon three times to obtain an orange-yellow solution. Acetic anhydride 77.8 mmol was added, and then BF3·Et2O 77.8 mmol was added, and the mixture was heated to reflux, and stirring was continued for 6 h, and then solid was precipitated after cooling to room temperature. The reaction solution was poured into 400 mL of water, allowed to stand for 0.5 h, and filtered. The filter cake was washed twice with deionized water, and the aqueous phase was extracted three times with EA. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (100-200 mesh silica gel, first eluted with 1 vol% MeOH-DCM, and then eluted with 30 vol% EA-PE) to obtain compound M3 (10.0 g, yellow solid, yield 79%).

[0149] Compound M3: 1H NMR(500MHz,Chloroform-d)δ11.49(s,1H),9.25(s,0H),2.61(s,1H),1.96(s,3H).13C NMR(126MHz,Chloroform-d)δ11.49(s,1H),9.25(s,0H),2.61(s,1H),1.96(s,3H).LC-MS:[M+H] + =197.0815.

[0150] (4) Preparation of Compound M4

[0151] Compound M3 (26.88 g, 137 mmol) was dissolved in 1.1 L acetone, and 617 mmol of anhydrous potassium carbonate was added at room temperature. Under argon protection, the temperature was lowered to -10°C and stirred for 20 min. 200 mL of acetone solution of MOMCl (480 mmol) was slowly added dropwise at -20°C, and stirred for 12 h at 16°C. The reaction endpoint was confirmed by TLC (30 vol% EA-PE), and the mixture was filtered and the filtrate was collected. The filter cake was washed with ethyl acetate for 3 times, and the washing liquid and the filtrate were combined and concentrated to dryness. 500 mL of water was added for redissolution, and pH was adjusted to 5.8 with 1 M dilute hydrochloric acid in an ice bath, extracted with ethyl acetate for 3 times, washed with saturated brine, dried over anhydrous sodium sulfate, filtered to remove sodium sulfate, concentrated to remove the solvent, and purified by silica gel column chromatography (200-300 mesh silica gel, 12 vol% EA-PE) to obtain compound M4 (13.20 g, yellow oil, yield 29%).

[0152] Compound M4: 1 H NMR (500MHz, Chloroform-d): δ6.92(s,1H,Ar-H),6.70(s,1H,Ar-H),4.69(s,2H,-CH2-),3.82(s,3H,-OCH3),3.79(s,3H,-OCH3),2.38(s,3H,-COCH3). 13 C NMR (126MHz, Chloroform-d): δ196.5(C=O),153.4,148.2,143.3,123.7,114.5,114.2(aromatic carbons),65.8(-CH2-),56.2(-OCH3),55.8(-OCH3),26.8(-COCH3).LC-MS:[M+H] + =287.1234.

[0153] (5) Preparation of Compound M3b

[0154] Compound M3a (15.00 g, 123 mmol) was added to a 1000 mL three-necked flask, and 400 mL of acetone was added. 368 mmol of potassium carbonate was added at room temperature, and the temperature was lowered to 0°C. 129 mmol of MOMCl was slowly added dropwise, and the temperature was raised to room temperature for reaction for 3 h. The mixture was filtered and the filtrate was added to 300 mL of water. The mixture was extracted with EA for 3 times (200 mL for each extraction). The organic phases were combined, washed with saturated brine, dried with sodium sulfate, filtered to remove sodium sulfate, and concentrated to constant weight to obtain compound M4a (21.74 g, yellow oil, unpurified yield>100%), which was directly used in the next step.

[0155] (6) Preparation of Compound M5

[0156] At room temperature, compound M4 (14 g, 49.24 mmol) and compound M3b 64.02 mmol were dissolved in 490 mL of anhydrous ethanol, and 49.24 mmol of potassium hydroxide was added. The mixture was heated to reflux with stirring for 6 h, cooled to room temperature, poured into saturated ammonium chloride solution 1.2 L, and then 500 mL of water was added. The mixture was extracted with ethyl acetate three times. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered to remove sodium sulfate, concentrated to remove the solvent, and purified by silica gel column chromatography (100-200 mesh silica gel, 7 vol% EA-PE) to obtain compound M5 (16.0 g, yellow solid, yield 75%).

[0157] Compound M5: 1 HNMR(500MHz,Chloroform-d)δ12.66(s,1H),7.85-7.78(m,1H),7.72-7.65(m,1H),7.61-7.55(m,3H),7.0 7-7.02(m,2H),5.20(s,2H),5.00(s,2H),4.86(s,2H),3.60(s,3H),3.46(s,6H),2.21(s,3H),2.15(s,3H). 13 C NMR(126MHz,Chloroform-d)δ193.85,160.95,160.53,159.27,155.19,143.14,130.30,128.80,124.84 ,116.57,116.34,115.90,113.25,101.43,99.35,94.21,58.36,57.75,56.21,10.41,9.54.LC-MS:[M+H] + =433.1871.

[0158] (7) Preparation of Compound M6

[0159] Add compound M5 (11.40 g, 26.4 mmol) into a 100 mL single-mouth bottle and 53 mL of DMSO to obtain a brown-yellow solution. Add 2.64 mmol of iodine and the brown-yellow solution turns into a brown solution. Heat to 85 °C and stir for 12 h. Pour into 800 mL of water. A large amount of solid precipitates. Filter and wash the filter cake twice with water. Spin dry to obtain compound M6 (8.0 g, yellow solid), which is used directly in the next reaction without purification.

[0160] Compound M6: LC-MS: [M+H] + =299.0911.

[0161] (8) Preparation of Compound M7

[0162] At room temperature, compound M6 (7 g, 23.47 mmol) was dissolved in 118 mL of pyridine, and 140.80 mmol of acetic anhydride was added to obtain a yellow solution. The temperature was raised to reflux, and stirring was continued for 3 h. The solution was poured into 800 mL of water, and a large amount of solid was produced. The solution was filtered and the filter cake was washed twice with water, and then dissolved in 1.2 L of DCM, dried over anhydrous sodium sulfate, filtered to remove sodium sulfate, concentrated to remove the solvent, and purified by silica gel column chromatography (silica gel particle size 100-200 mesh, 1 vol% MeOH-DCM) to obtain compound M7 (7.30 g, yellow solid, yield 73%).

[0163] (8) Preparation of DMDO solution

[0164] Add 350mL of water and acetone (500mL, 6.80mol), 2.24mol of sodium bicarbonate, stir for 15min, and obtain a white suspension; cool to 5°C, add 0.61mol of Oxone in batches under stirring, a large number of bubbles are generated, and the addition is completed in 20min. After the addition, stir vigorously (200-500r / min) for 30min in an ice bath, and the solution is slightly pink. Connect a cold trap, cool the cold trap at -78°C and argon atmosphere for 10min, remove the ice bath on the reaction bottle, and keep stirring. Connect a vacuum pump, perform reduced pressure distillation, collect the acetone solution of DMDO, close the cold trap when the volume change in the cold trap is not obvious, and return to room temperature under Ar gas to obtain a DMDO solution (125mL, yellow).

[0165] (9) Preparation of Compound M8

[0166] Compound M7 (840.1 mg, 1.98 mmol) was dissolved in 110 mL of anhydrous DCM. DMDO solution (100 mL, 20 mmol) was added under ice bath. After the addition, the temperature was raised to room temperature and the reaction was carried out for 20 h. After the reaction was completed, the solvent was evaporated in a 30°C water bath and purified by silica gel column chromatography (dissolved in dichloromethane, wet column loading, 100-200 mesh silica gel, 30 vol% EA-PE) to obtain compound M8 (off-white solid, 461.0 mg, yield was 53%).

[0167] (10) Preparation of flavonoid compounds (flavonoid 01 substances)

[0168] Compound M8 (1.12 g, 2.54 mmol) was added, and 25 mL of 0.5 M NaOMe-MeOH solution was added. The argon gas was replaced 3 times, and the mixture was heated to 40 ° C for 30 min. 1 M HCl solution was added to adjust the pH to 2, and the mixture was cooled at 4 ° C for 1 h. The obtained solid was redissolved in 30 mL of EA, concentrated to 10 mL, and recrystallized (the recrystallization solvent was ethyl acetate) to obtain a solid (flavonoid compound, dry weight 0.3 g) and a crystallization mother liquor. The crystallization mother liquor was repeatedly recrystallized to obtain a solid (flavonoid compound, dry weight 0.1 g). A total of 0.4 g of flavonoid compound was obtained (yield 50%, purity 97%): 1 H NMR: (500MHz, DMSO-d6)δ12.63(s,1H),10.10(s,1H),9.66(s,1H),9.34(s,1H),8 .08-8.07(d,J=8.8Hz,2H),6.95-6.94(d,J=8.9Hz,2H),2.27(s,3H),2.06(s,3H). 13 C NMR: (126MHz, DMSO-d6) δ176.09,159.47,159.14,155.09,151.46,146.49,135.50, 129.37,122.13,115.55,106.31,102.91,101.39,8.25,8.05.HRMS(ESI-MS):[M+H] + =315.0875.

[0169] Comparative Example 1

[0170] Preset Aldehyde-Ketone Condensation Reaction Route

[0171]

[0172] Compound M3 was prepared according to step (1) of Example 1.

[0173] Prepared by aldehyde-ketone condensation reaction: Add compound M3 (15.00 g, 123 mmol), add 100 mL of acetic acid to obtain a colorless clear solution. Add p-hydroxybenzaldehyde (139 mmol) and 0.5 g of acid catalyst (p-toluenesulfonic acid, p-TsOH) at room temperature to form a yellow solution. Heat to reflux and react for 3 h, cool to room temperature, add 300 mL of water to dilute, and then stir in an ice bath to precipitate a yellow solid. Filter the obtained solid and wash it with water twice (single amount 100 mL). Dissolve the obtained solid in 100 mL of ethyl acetate, wash with saturated brine, dry with anhydrous sodium sulfate, and concentrate to constant weight to obtain compound DP (21.00 g, yellow solid, yield 80%).

[0174] This synthetic route prepares compound DP via aldehyde-ketone condensation reaction.

[0175] Compound DP: 1 HNMR(500MHz,DMSO-d6)δ10.02(s,1H,-CHO),9.50(s,1H,Ar-OH),7.12(d,J=8.5H z,2H,Ar-H),6.98(d,J=8.5Hz,2H,Ar-H),6.60(s,1H,Ar-H),5.90(s,1H,Ar-OH). 13 C NMR (126MHz, DMSO-d6) δ190.85 (CHO), 159.34 (Ar-C-OH), 155.27 (Ar-C), 131.52 (Ar-CH), 126.45 (Ar-CH), 115.68 (Ar-CH), 108.32 (Ar-C), 101.05 (Ar-C). LC-MS: [MH]+=278.0937. According to the above mass spectrum and NMR data, the compound DP prepared in this comparative example is not a flavonoid compound having the structure shown in Formula I, and the target product shown in Formula I cannot be synthesized through this aldehyde-ketone condensation reaction synthesis route.

[0176] Test Example 1

[0177] Study on the Activity of Flavonoids

[0178] (1) Effects of flavonoid 01 on the cell activity of human umbilical vein endothelial cells (HUVECs) and mouse hepatocytes (AML12).

[0179] HUVECs were inoculated in a cell culture plate and cultured in a cell culture incubator. When the cells grew to 80% polymerization, the cells were treated with serum-free medium containing different concentrations (0.5-30 μg / mL) of flavonoid 01. The quadratic regression general rotation design method was used for the experiment. The dosage range of flavonoid 01 was 0.5-30 μg / mL, and the action time range of flavonoid 01 was 1-24 hours. Finally, the cell viability was determined. The detailed operation process was carried out according to the instructions of the cell viability detection kit.

[0180] AML12 was inoculated in a cell culture plate and cultured in a cell culture incubator. When the cells grew to 80% polymerization degree, the cells were treated with serum-free medium containing different concentrations of flavonoid 01 substances. The quadratic regression general rotation design method was used for the experiment. The dosage range of flavonoid 01 substances was 0.5-30 μg / mL, and the action time range of flavonoid 01 substances was 1-24 hours. Finally, the cell viability was determined. The detailed operation process was carried out according to the instructions of the cell viability detection kit.

[0181] The results showed that 30μg / mL flavonoid 01 substance showed certain toxicity to HUVECs and AML12 cells after 12h, and the toxicity increased significantly with the increase of dose and action time. However, 0.5-12μg / mL flavonoid 01 substance did not produce significant toxicity to HUVECs and AML12 cells after 24h.

[0182] (2) Effects of flavonoids 01 on the activity and expression of glutathione peroxidase 4 (GPX4).

[0183] Glutathione peroxidase 4 (GPX4) plays a key role in maintaining intracellular redox balance and preventing lipid peroxidation. Flavonoids have received extensive attention for their antioxidant properties. This implementation case was designed to evaluate the effects of flavonoid 01 substances on GPX4 activity and its expression levels and compare them with kaempferol to determine their relative efficacy. Two cell lines, HUVECs (human umbilical vein endothelial cells) and AML12 (mouse hepatocytes), were used in the experiment.

[0184] Materials and Methods

[0185] Chemical reagents: flavonoid 01 substance, kaempferol (standard product), GPX4 enzyme detection kit, anti-GPX4 antibody, β-actin antibody.

[0186] Cell lines: HUVECs (human umbilical vein endothelial cells), AML12 (mouse hepatocytes).

[0187] Treatment: HUVECs and AML12 cells were divided into the following four groups: control group, kaempferol-treated group (10 μM), flavonoid 01 low-dose group (5 μM), and flavonoid 01 high-dose group (20 μM).

[0188] GPX4 activity detection: After 24 hours of cell treatment, cell lysate was collected. GPX4 activity was detected using a GPX4 enzyme detection kit according to the manufacturer's instructions. Each group of experiments was repeated 3 times, and the data are expressed as mean ± standard deviation.

[0189] GPX4 expression detection: After treating cells, cell lysates were collected. Proteins were extracted and the expression levels of GPX4 and β-actin (internal reference) were detected using Western blot technology. Data analysis was performed using ImageJ software for grayscale analysis of protein bands, and the GPX4 expression level was normalized relative to the expression level of β-actin.

[0190] Figure 1 The results show the changes of GPX4 activity in HUVECs and AML12 cells among different treatment groups. The results of GPX4 activity assay of HUVECs cell line: There was no significant difference in GPX4 activity between the kaempferol-treated group and the control group; the GPX4 activity of the flavonoid 01-treated group was significantly higher than that of the control group and the kaempferol-treated group (P<0.01). The results of GPX4 activity assay of AML12 cell line: There was no significant difference in GPX4 activity between the kaempferol-treated group and the control group, and the GPX4 activity of the flavonoid 01-treated group was significantly higher than that of the control group and the kaempferol-treated group (P<0.01). The results of GPX4 activity assay of AML12 cell line: There was no significant difference in GPX4 activity between the kaempferol-treated group and the control group, and the GPX4 activity of the flavonoid 01-treated group was significantly higher than that of the control group and the kaempferol-treated group (P<0.01).

[0191] Figure 2 The figure shows the expression changes of GPX4 in HUVECs and AML12 cells between different treatment groups, where A is the HUVECs cell line and B is the AML12 cell line. The results of GPX4 expression detection in HUVECs cell line: There was no significant difference in GPX4 protein expression between the kaempferol-treated group and the control group, and the GPX4 protein expression level in the flavonoid 01-treated group was significantly higher than that in the control group and the kaempferol-treated group (P<0.05), and the expression of GPX4 also increased significantly with the increase of the dose. The results of GPX4 expression detection in AML12 cell line: There was no significant difference in GPX4 protein expression between the kaempferol-treated group and the control group, and the GPX4 protein expression level in the flavonoid 01-treated group was significantly higher than that in the control group and the kaempferol-treated group (P<0.05), and the expression of GPX4 also increased significantly with the increase of the dose.

[0192] In HUVECs and AML12 cell lines, flavonoid 01 significantly increased the activity and protein expression level of GPX4 at high doses, which was significantly superior to kaempferol. Both the low-dose and high-dose groups of flavonoid 01 showed an enhanced effect on GPX4 activity and expression level, but the effect of the high-dose group was the most significant.

[0193] Conclusion: Flavonoid 01 has a significant promoting effect on the activity and expression level of GPX4, especially under high-dose treatment, its effect is significantly better than kaempferol. This shows that the flavonoid 01 provided by the present invention has potential antioxidant properties and may play an important role in antioxidant therapy, providing a basis for further exploring the mechanism of flavonoid 01, and has great potential in clinical application.

[0194] (3) Antioxidant effects of flavonoids 01 and kaempferol (DPPH free radical scavenging experiment).

[0195] Solutions of different concentrations of flavonoid 01 and kaempferol (control) were prepared (10 μM, 25 μM, 50 μM, the solvent was 0.1 v / v% DMSO-water). 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) methanol solution was added and reacted in the dark for 30 min. The absorbance was measured at 517 nm using an ultraviolet spectrophotometer, and the DPPH free radical scavenging rate (mean ± SD) was calculated as follows: DPPH free radical scavenging rate (%) = (blank group absorbance - experimental group absorbance) / blank group absorbance × 100%, where the blank group absorbance refers to the absorbance of the sample containing only DPPH methanol solution but no drug (flavonoid or kaempferol); the experimental group absorbance refers to the absorbance of the sample after adding different concentrations of drugs (flavonoid 01 or kaempferol). The final results are expressed as mean ± standard deviation (SD), and the results are shown in Table 1.

[0196] Table 1 DPPH scavenging rate of flavonoid 01 and kaempferol

[0197] Concentration (μM) Flavonoids 01 substances Kaempferol 10 80％±3％ 55％±4％ 25 85％±2％ 65％±3％ 50 90％±2％ 75％±3％

[0198] The results showed that at the same concentration, the DPPH free radical scavenging ability of flavonoid 01 was significantly higher than that of kaempferol.

[0199] (4) Cytotoxicity test of flavonoids 01 and kaempferol

[0200] AML12 cells were inoculated in a 96-well plate and cultured for 24 hours. Different concentrations of flavonoid 01 and kaempferol were added and cultured for another 24 hours. CCK8 reagent was added and incubated for 2 hours. The absorbance was measured at 450 nm using an ELISA reader and the cell survival rate (mean ± SD) was calculated as follows: Cell survival rate (%) = (absorbance of the experimental group - absorbance of the blank group) / (absorbance of the control group - absorbance of the blank group) × 100%. The results are shown in Table 2.

[0201] Table 2 Cell survival rate under the action of flavonoids 01 and kaempferol

[0202] Concentration (μM) Flavonoids 01 substances Kaempferol 10 95％±2％ 85％±3％ 25 90％±3％ 75％±4％ 50 80％±4％ 60％±5％

[0203] The results showed that at the same concentration, the toxicity of flavonoid 01 to AML12 cells was significantly lower than that of kaempferol.

[0204] Test Example 2

[0205] Computer-aided evaluation of the drug-forming potential of flavonoids 01

[0206] The ADMET Evaluation functional module of the ADMETlab 3.0 platform was used to comprehensively evaluate the absorption, distribution, metabolism, excretion and toxicity (ADMET), molecular physicochemical properties and medicinal chemical properties of flavonoid 01 in the human body.

[0207] First, the macroscopic physical and chemical properties were obtained. Molecular weight (M W ) is 314.08, the number of hydrogen bond acceptors (nHA) is 6, the number of hydrogen bond donors (nHD) is 4, the water-oil partition coefficient (LogP) is 2.406, and the number of rotatable bonds (nRot) is 1.

[0208] The Lipinski Rule of Five is a concept often used in drug discovery. This rule helps predict whether a bioactive molecule is likely to have the physical and chemical properties of oral bioavailability and is often used in the initial screening of drug-like properties. According to the Lipinski rule, orally active small molecules need to meet the following property conditions: no more than 5 hydrogen bond donors; no more than 10 hydrogen bond acceptors; molecular weight less than 500 Da; water-oil partition coefficient no more than 5; and no more than 10 rotatable bonds.

[0209] Flavonoid 01 substance meets the conditions for preliminary drug-like screening. In addition, flavonoid 01 substance also passed the screening of PfizerRule, GSKRule, GoldenTriangle and PAINSADMET conditions, and the results are shown in Table 3. As can be seen from Table 3, flavonoid 01 substance performed well in all drug screening rules, indicating that it has a lower toxicity risk and better pharmacokinetic properties. These results show that flavonoid 01 substance has potential in further drug development, especially under the premise of ensuring effectiveness and safety, it can become a promising drug candidate. .

[0210] Table 3 Pharmacokinetic and toxicity screening evaluation results of flavonoid 01 substances

[0211]

[0212] The predicted bioavailability is about 30-50%, the human liver microsome stability (HLM Stability)> 30min, and the half-life (T 1 / 2 ) is 1.329, and the plasma clearance rate (CL) is 5.45 mL / min / kg, indicating that flavonoid 01 substance is a compound with a short half-life and medium clearance rate, and it also has the characteristics of high bioavailability and metabolic stability.

[0213] The flavonoid 01 substance and GPX4 protein structure (PDB ID: 7U4M) were standardized using Schrödinger Maestro 12.8 software, and the standard precision (SP) mode in molecular docking technology was used for docking simulation. Figure 3 As shown in the figure, the binding mode of flavonoid 01 substance and GPX4 protein is stable, and its docking score is -3.804, indicating that there is a strong binding affinity between the two. Further analysis shows that flavonoid 01 substance is bound to GPX4 at position 170 phenylalanine (PHE 170) and position 65 glutamic acid (GLU 65) residues through hydrogen bonding.

[0214] In summary, the flavonoid compounds provided by the present invention are significantly superior to the existing flavonoid compound kaempferol in antioxidant effect, and are less toxic to the normal mouse liver cell line AML12 than kaempferol at the same concentration. Based on CADD-assisted and molecular docking technology analysis, it is preliminarily proved that the flavonoid compounds provided by the present invention have good drugability potential and have broad application prospects.

[0215] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A flavonoid compound, characterized in that: It has the structure shown in Formula I:

2. The method for preparing flavonoid compounds according to claim 1, characterized in that: The following steps are involved: Compound M3 is subjected to a hydroxyl protection reaction with a first (chloromethyl) methyl ether under alkaline conditions to obtain compound M4; Acylation reaction of compound M3a with a second (chloromethyl) methyl ether under alkaline conditions to obtain compound M3b; The compound M4 and the compound M3b are subjected to an etherification reaction under alkaline conditions to obtain a compound M5; The compound M5 is subjected to an oxidation reaction in the presence of an oxidant to obtain a compound M6; Acetylation reaction of the compound M6 with acetic anhydride to obtain compound M7; The compound M7 is subjected to epoxidation reaction with dimethyldioxane to obtain a compound M8; hydrolyzing the compound M8 under alkaline conditions to obtain the flavonoid compound; 3. The preparation method according to claim 2, characterized in that: The molar ratio of the compound M3 to the first (chloromethyl) methyl ether is 1:3-4; The temperature of the hydroxyl protection reaction is 15-20° C., and the time is 8-16 hours; the organic solvent used in the hydroxyl protection reaction includes one or more of acetone, acetonitrile, tetrahydrofuran and dichloromethane; The molar ratio of the compound M3a to the second (chloromethyl) methyl ether is 1:1.2-1.6; The temperature of the acylation reaction is room temperature and the time is 2 to 4 hours; the organic solvent used in the acylation reaction includes one or more of acetone, ethanol and dichloromethane.

4. The preparation method according to claim 2, characterized in that: The molar ratio of the compound M4 to the compound M3b is 1:0.7-1.5; The etherification reaction is carried out under reflux conditions, and the etherification reaction time is 4 to 8 hours; The organic solvent used in the etherification reaction includes one or more of ethanol, methanol and dichloromethane.

5. The preparation method according to claim 2, characterized in that: The oxidant includes one or more of iodine, cyanogen chloride, benzoyl peroxide and trifluoroacetic anhydride; The molar ratio of the compound M5 to the oxidant is 1:0.05-0.15; The temperature of the first oxidation reaction is 80-90°C and the time is 8-16h; The organic solvent used in the first oxidation reaction includes one or more of dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide and methanol.

6. The preparation method according to claim 2, characterized in that: The molar ratio of the compound M6 to acetic anhydride is 1:5-7; The acetylation reaction is carried out under reflux conditions, and the acetylation reaction time is 2 to 4 hours; The organic solvent used in the acetylation reaction includes one or more of pyridine, dichloromethane, dimethylformamide and N,N-dimethylacetamide.

7. The preparation method according to claim 2, characterized in that: The molar ratio of the compound M7 to dimethyldioxane is 1:5-15; The epoxidation reaction is carried out at room temperature for 18 to 24 hours. The organic solvent used in the epoxidation reaction includes one or more of dichloromethane, toluene, ethyl acetate and tetrahydrofuran.

8. The preparation method according to claim 2, characterized in that: The temperature of the hydrolysis reaction is 35-45° C. and the time is 20-40 minutes.

9. A pharmaceutical composition comprising the flavonoid compound according to claim 1 and a pharmaceutically acceptable excipient.

10. Use of the flavonoid compound according to claim 1 or the pharmaceutical composition according to claim 9 in the preparation of antioxidant drugs or drugs for treating diseases related to oxidative stress.

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