A flavonoid compound, preparation method and application thereof, pharmaceutical composition and application thereof

Abstract

The present invention provides a flavonoid compound and a preparation method and application thereof, a pharmaceutical composition and application thereof, and relates to the field of biomedicine technology. The flavonoid compound provided by the present invention has excellent antioxidant activity and significant GSH-Px activation effect, has low toxicity and high efficiency, is suitable for preparing a variety of antioxidant drugs and drugs for treating diseases related to oxidative stress, has broad clinical application prospects and important drug development value, will promote the progress of flavonoid drug research and development, and provide new solutions for antioxidant therapy. In vitro experiments and cell model verification show that: in HUVECs and AML12 cell lines, compared with kaempferol, the flavonoid compound provided by the present invention significantly increased the activity of GPX4 and its protein expression level; the ability to scavenge DPPH free radicals is significantly higher than that of kaempferol, and the antioxidant property is significantly better than that of kaempferol; the toxicity to AML12 cells is significantly 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, 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 primarily derived from the rhizomes of the ginger plant Kaempferol (kaempferolgalangaL). It is widely present in various fruits, vegetables, and beverages and can be extracted from tea, cabbage, witch hazelnuts, propolis, grapefruit, and other green plants. It has attracted widespread attention due to its various anti-cancer, anti-inflammatory, antioxidant, antibacterial, and antiviral effects. However, kaempferol's ability to activate glutathione peroxidase (GSH-Px) is not good enough.

[0003] Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a flavonoid compound and its preparation method and application, a pharmaceutical composition and its application. 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 reacted with a first (chloromethyl) methyl ether under alkaline conditions to carry out a hydroxyl protection reaction to obtain compound M4;

[0010] Compound M3a is acylated with a second (chloromethyl) methyl ether under basic 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 oxidizing agent 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 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 compound M4 to 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 elemental 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 8-16 hours;

[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 1:5-15;

[0032] The epoxidation reaction temperature is room temperature and the time is 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 temperature is 35-45° C., and the time is 20-40 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 provided by the present invention having the structure shown in Formula I (denoted as flavonoid 01 substance) 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. It will promote the progress of flavonoid drug research and development and provide new solutions for antioxidant treatment.

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

[0039] GPX4 expression assays in HUVECs showed that there was no significant difference in GPX4 protein expression between the kaempferol-treated and control groups, while the flavonoid 01-treated group showed significantly higher GPX4 protein expression than both the control and kaempferol-treated groups (P<0.05). GPX4 expression also increased significantly with increasing doses. GPX4 expression assays in AML12 cells showed that there was no significant difference in GPX4 protein expression between the kaempferol-treated and control groups, while the flavonoid 01-treated group showed significantly higher GPX4 protein expression than both the control and kaempferol-treated groups (P<0.05). GPX4 expression also increased significantly with increasing doses.

[0040] In both HUVECs and AML12 cell lines, flavonoid 01 significantly increased GPX4 activity and protein expression at high doses, demonstrating a significantly superior effect compared to kaempferol. Both low- and high-dose groups of flavonoid 01 demonstrated enhanced GPX4 activity and expression, with the high-dose group showing the most significant effect.

[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 as described in the above technical solution. The extraction and isolation process of natural flavonoid compounds from plants is complex, and the product yield is low, limiting their application in drug development. Compared with plant extraction methods, the present invention utilizes a chemical synthesis method, which achieves a higher yield of flavonoid compounds, ensures the stability and consistency of the flavonoid compound product, and lays 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 Figure 3 Changes in GPX4 activity in HUVECs and AML12 cells among different treatment groups;

[0045] Figure 2 Figure 2 is the expression change of GPX4 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 reacted with a first (chloromethyl) methyl ether under alkaline conditions to carry out a hydroxyl protection reaction to obtain compound M4;

[0051] Compound M3a is acylated with a second (chloromethyl) methyl ether under basic 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 oxidizing agent 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 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] In the present invention, compound M3 is subjected to a hydroxyl protection reaction 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 carried out 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 an alkali metal carbonate and / or an 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; and 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 ratio of 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 15-30 minutes (more preferably 20-25 minutes), 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 (i.e., 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 to 20° C., more preferably 16 to 18° C.; the time of the hydroxyl protection reaction is preferably 8 to 16 h, more preferably 10 to 12 h. 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 spots 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 to 40%, more preferably 30%.

[0066] After completing the hydroxyl protection reaction, the present invention preferably further comprises: subjecting the resulting hydroxyl protection reaction solution to solid-liquid separation to obtain a liquid component and a solid component, washing the solid component with ethyl acetate to obtain a washing solution; combining the liquid component and the washing solution, concentrating to constant weight, dissolving the resulting concentrate in water, adjusting the pH to a weakly acidic state in an ice bath, extracting with an organic solvent, and sequentially washing the resulting organic phase with saturated brine, drying with a desiccant, performing solid-liquid separation to remove the desiccant, concentrating to constant weight, and purifying by silica gel column chromatography to obtain compound M4. The present invention does not specifically limit the two solid-liquid separations; solid-liquid separation methods well known to those skilled in the art, such as filtration, can be employed. In the present invention, the number of ethyl acetate washes is preferably 2 to 4 times, more preferably 3 times. The present invention does not specifically limit the two concentrations; concentration methods well known to those skilled in the art, such as reduced pressure concentration, can be employed. In the present invention, the acid used for pH adjustment preferably comprises 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 1.5 M, more preferably 1 to 1.2 M. In the present invention, the pH value of the weak acid 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 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 10 to 15%, more preferably 12%; the particle size of the silica gel used for the silica gel column chromatography purification is preferably 200 to 300 mesh.

[0067] In the present invention, the compound M3 is preferably a commercially available product or prepared. In the present invention, the method for preparing 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:7.8-10.8 mL, more preferably 1 g:8-10 mL, and even more preferably 1 g:9-9.5 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 even more 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 even more 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 displace 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 resulting Friedel-Crafts acylation reaction solution to room temperature, pouring it into water and allowing it to stand, performing solid-liquid separation, washing the resulting solid component with water, extracting the resulting aqueous phase with an organic solvent, and sequentially washing the resulting organic phase with saturated brine, drying with a desiccant, performing solid-liquid separation to remove the desiccant, concentrating, and purifying by silica gel column chromatography to obtain compound M3. In the present invention, the ratio of compound M2 to water (water used for pouring into water and allowing it to stand) is preferably 1 g:30-50 mL, more preferably 1 g:40 mL. In the present invention, the number of water washes is preferably 1-3, more preferably 2; the ratio of compound M2 to water used for a single water wash is preferably 1 g:1.5-2.5 mL, more preferably 1 g:2 mL. In the present invention, the organic solvent for extraction preferably includes ethyl acetate and / or dichloromethane, more preferably ethyl acetate; and the number of extractions is preferably 2-5, more preferably 2-4. 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 a 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. 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 mesh.

[0075] In the present invention, the compound M2 is preferably a commercially available product or self-produced. 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 (denoted 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 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 even more 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, 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, more preferably THF. In the present invention, the ratio of compound M1 to the seventh organic solvent is preferably 30 g:750-800 mL, more preferably 30 g:780-840 mL, and even more preferably 30 g:800-825 mL.

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

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

[0082] In the present invention, the acid solution preferably comprises a hydrochloric acid solution, the concentration of which is preferably 3.5 to 4.5 M, more preferably 4 M. The acid solution is preferably added dropwise. In the present invention, the ratio of compound M1 to the acid solution is preferably 30 g:130 to 150 mL, more preferably 30 g:135 to 145 mL, and even more 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 10-18 hours, more preferably 12-17 hours, and further preferably 15-16 hours.

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

[0085] In the present invention, the compound M1 is preferably a commercially available product or self-produced. 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.; and 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 carried out 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 drying to obtain compound M1. The present invention has no particular 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 particular 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 used for washing preferably comprises deionized water. In the present invention, the drying preferably comprises spin drying with a solid oil pump.

[0093] In the present invention, compound M3a is subjected to an acylation reaction with 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 carry out 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 even more preferably 1:1.3-1.4.

[0095] In the present invention, the second alkaline agent preferably includes an alkali metal carbonate and / or an 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; and the second alkaline agent is preferably an anhydrous alkaline agent. In the present invention, the molar ratio of 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 ratio of compound M3a to the second organic solvent is preferably 15 g:300-500 mL, more preferably 15 g:400 mL.

[0097] In the present invention, the mixing of compound M3a, a second MOMCl, a second alkaline reagent and a second organic solvent preferably includes: dissolving compound M3a in the second organic solvent, adding the second alkaline reagent at room temperature (20-25°C), cooling to -4-4°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 the acylation reaction is completed, the present invention preferably further comprises: solid-liquid separation of the resulting acylation reaction liquid, mixing the resulting liquid component with water, extracting with an organic solvent, washing the resulting organic phase with saturated brine, drying with a desiccant, performing solid-liquid separation to remove the desiccant, and concentrating to a constant weight to obtain compound M3b. The present invention does not specifically limit the two solid-liquid separations; solid-liquid separation methods well known to those skilled in the art may be employed, such as filtration. 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 5, more preferably 2 to 3. In the present invention, the desiccant preferably comprises anhydrous sodium sulfate and / or anhydrous magnesium sulfate. The present invention does not specifically limit the concentration; concentration methods well known to those skilled in the art may be employed, such as reduced pressure concentration.

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

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

[0102] In the present invention, the third alkaline agent preferably comprises a hydroxide, more preferably 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 ratio of compound M4 to the second organic solvent is preferably 14 g: 400-500 mL, more preferably 14 g: 450-490 mL.

[0104] In the present invention, the mixing of the compound M4, the compound M3b, a third alkaline agent and a third organic solvent preferably includes: dissolving the compound M4 and the compound M3b in a 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 etherification reaction time 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 resulting etherification reaction liquid to room temperature, adding saturated ammonium chloride solution, adding water, extracting with an organic solvent, and sequentially washing the resulting organic phase with saturated brine, drying with a desiccant, performing solid-liquid separation to remove the desiccant, concentrating, and purifying by silica gel column chromatography 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, more preferably 3. In the present invention, the desiccant preferably comprises anhydrous sodium sulfate and / or anhydrous magnesium sulfate. The present invention does not specifically limit the solid-liquid separation; any solid-liquid separation method known to those skilled in the art, such as filtration, can be employed. The present invention does not specifically limit the concentration; any concentration method known to those skilled in the art, such as reduced pressure concentration, can be employed. In the present invention, the eluent used for the silica gel column chromatography purification preferably comprises ethyl acetate-petroleum ether, with the volume fraction of ethyl acetate in the ethyl acetate-petroleum ether preferably being 5 to 7%, more preferably 6%. The silica gel used for the silica gel column chromatography purification preferably has a particle size of 100 to 200 mesh.

[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 comprises one or more of elemental iodine, cyanogen chloride, benzoyl peroxide, and trifluoroacetic anhydride, more preferably elemental iodine. In the present invention, the molar ratio of the compound M5 to the oxidant is preferably 1:0.05-0.15, 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 ratio of compound M5 to the fourth organic solvent is preferably 14 g:400-500 mL, more preferably 14 g:450-490 mL.

[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 8-16 h, more preferably 10-12 h.

[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 a constant weight to obtain compound M6, and the compound M6 is directly used in the next reaction without purification. The present invention is not particularly limited to 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 (denoted 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 ratio of compound M6 to the fifth organic solvent is preferably 7 g:100-140 mL, more preferably 7 g:110-120 mL.

[0115] In the present invention, the mixing of the compound M6, acetic anhydride and a 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 acetylation reaction time is preferably 2 to 4 hours, more preferably 3 hours.

[0117] After the acetylation reaction is completed, the present invention preferably further comprises: mixing the resulting acetylation reaction solution with water to precipitate a solid, performing solid-liquid separation, washing the resulting solid component with water and then redissolving it, drying it with a desiccant, performing solid-liquid separation to remove the desiccant, concentrating it, and purifying it by silica gel column chromatography to obtain compound M7. The present invention does not specifically limit the two solid-liquid separations; solid-liquid separation methods well known to those skilled in the art may be employed, such as filtration. In the present invention, the ratio of compound M6 to water (water for mixing with water) is preferably 7 g:600-1000 mL, more preferably 7 g:700-800 mL. In the present invention, the number of water washes is preferably 1-3 times, more preferably 2 times. In the present invention, the organic solvent for redissolution preferably includes one or more of dichloromethane, ethyl acetate, diethyl ether, and acetonitrile. In the present invention, the desiccant preferably includes anhydrous sodium sulfate and / or anhydrous magnesium sulfate. The present invention does not specifically limit the solid-liquid separation; solid-liquid separation methods well known to those skilled in the art may be employed, such as filtration. The present invention is not particularly limited to the concentration, and any concentration method familiar to those skilled in the art may be used, such as concentration under reduced pressure. In the present invention, the eluent used for 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 for the silica gel column chromatography purification is preferably 100-200 mesh.

[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 (denoted 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 compound M7 to DMDO is preferably 1:5-15, more preferably 1:812, and even more preferably 1:10. In the present invention, DMDO is preferably used in the form of a DMDO solution, wherein 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 2 mmol:100-120 mL, more preferably 2 mmol:110 mL.

[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 completing the epoxidation reaction, the present invention preferably further comprises: concentrating the resulting epoxidation reaction solution and then purifying it by silica gel column chromatography to obtain compound M8. The present invention has no particular 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 mesh.

[0124] In the present invention, the DMDO solution is preferably obtained by dissolving commercially available DMDO in a solvent, or is homemade. In the present invention, the method for preparing the DMDO solution preferably comprises the following steps: mixing acetone, water, an alkaline reagent (referred to as the fourth alkaline reagent), and potassium peroxymonosulfate (Oxone), conducting an oxidation reaction (referred to as the second oxidation reaction), and performing reduced pressure distillation to obtain the 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 comprises an alkali metal carbonate and / or an alkali metal hydroxide; the alkali metal carbonate preferably comprises potassium carbonate and / or sodium carbonate; and the alkali metal hydroxide preferably comprises sodium hydroxide and / or potassium hydroxide. In the present invention, the molar ratio of 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 step of mixing acetone, water, a fourth alkaline agent, and Oxone preferably comprises: mixing the water, acetone, and fourth alkaline agent, cooling the mixture to 0-10°C (more preferably 5°C), and adding Oxone in batches while stirring. In the present invention, the Oxone is preferably added for 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 5-15 min, more preferably 10 min; the second oxidation reaction is preferably carried out under stirring conditions, and the stirring speed is preferably 200-500 r / min, more preferably 300-400 r / min.

[0130] After the second oxidation reaction is completed, 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 performed by cooling in a cold trap, 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 performed 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, the 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 an alkali metal alcoholate, an alkali metal hydroxide, and an alkali metal hydride, and the alkali metal alcoholate preferably includes sodium methoxide (NaOMe) and / or potassium methoxide; the alkali metal hydroxide preferably includes NaOH and / or KOH; the alkali metal hydride preferably includes 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 to 0.6 M, more preferably 0.5 M. In the present invention, the molar ratio of the compound M8 to the inorganic strong base is preferably 1:4 to 6, more preferably 1:4.5 to 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 to 45° C., more preferably 40° C.; the time of the hydrolysis reaction is preferably 20 to 40 min, more preferably 30 min.

[0135] After the hydrolysis is completed, the present invention preferably further comprises: adjusting the pH of the resulting hydrolyzed solution to 1-3 (more preferably 2), crystallizing, performing solid-liquid separation, dissolving the resulting solid component in ethyl acetate, concentrating, recrystallizing, performing solid-liquid separation, and drying the resulting crystals to obtain the flavonoid compound. In the present invention, the acid used for pH adjustment preferably comprises a hydrochloric acid solution, and the concentration of the hydrochloric acid solution is preferably 0.5-1.5 M, more preferably 1 M. In the present invention, the crystallization temperature is preferably 0-5°C, more preferably 3-4°C; the crystallization time is preferably 0.5-2 hours, more preferably 1-1.5 hours. The present invention does not specifically limit the two solid-liquid separations; any solid-liquid separation method known to those skilled in the art can be used, such as filtration. In the present invention, the ratio of compound M8 to ethyl acetate is preferably 1 g:20-35 mL, more preferably 1 g:25-30 mL. In the present invention, the volume ratio of ethyl acetate to the concentrated solution obtained by concentration is preferably 25-35:8-12, more preferably 30:10. In the present invention, the recrystallization solvent preferably includes one or more of ethyl acetate, ethanol, methanol, and water, more preferably ethyl acetate. In the present invention, the number of recrystallizations is preferably 3 to 5 times, more preferably 4 times. The drying method is not particularly limited in the present invention, 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 solution 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 disease, neurodegenerative disease, and liver disease. 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 efficacy, and have great 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 does not specifically limit the pharmaceutically acceptable excipient; pharmaceutically acceptable excipients well known to those skilled in the art may be used. The present invention does not specifically limit the dosage form and preparation method of the pharmaceutical composition; dosage forms and preparation methods well known to those skilled in the art may be used.

[0138] To further illustrate the present invention, the flavonoid compounds, preparation methods, applications, and pharmaceutical compositions provided by the present invention are described in detail below with reference to the following examples. However, these examples 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, 656 mmol of DMF and 628 mmol of phosphorus oxychloride were added, and the solution became a light yellow viscous liquid. Stirring was continued for 0.5 h to obtain a transparent colorless solution; 285 mmol of phloroglucinol was dissolved in 317 mL of dioxane, and the obtained phloroglucinol solution was added dropwise to the above transparent colorless solution. The reaction was carried out for 0.3 h until the light yellow solution turned into an orange-yellow solution. The temperature was raised to room temperature, and the reaction was stirred for 16 h. The solution was poured into 1 L of crushed ice and sonicated to obtain a clear solution. The solution was concentrated until the solid precipitate no longer increased and a large amount of yellow solid was produced. The solution was filtered, and the filter cake was deionized with water and dried using a solid oil pump to obtain compound M1 (49.00 g, 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, compound M1 (30.00 g, 164.7 mmol) and 823 mL of THF were added to give an orange-yellow solution. 100 mg of methyl orange was added as an indicator, and 26.274 mmol of sodium cyanoborohydride was added in three batches. Bubbles were generated at this point, and the solution turned into a khaki turbid liquid. After stirring for 0.5 h, 140 mL of 4 M HCl solution was added dropwise at a constant rate, and stirring was continued for 16 h. The resulting reaction solution was poured into 0.5 L of water, and the pH was adjusted to 5 with 1 M HCl solution. 200 mL of EA was added and stirred for 0.5 h. The layers were separated to give an organic phase and an aqueous phase, respectively. The aqueous phase was extracted twice with EA. The organic phases were combined, washed with saturated brine, dried over sodium sulfate, and spin-dried. The product was purified by silica gel column chromatography (100-200 mesh silica gel, 20-30 vol% EA-PE) to give compound M2 (12.0 g, yellow solid, 47% yield).

[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) and 93 mL of AcOH were added, and the atmosphere was replaced with argon three times to obtain an orange-yellow solution. Acetic anhydride (77.8 mmol) was added, followed by BF3·Et2O (77.8 mmol), and the mixture was heated to reflux. Stirring was continued for 6 h, and then cooled to room temperature, whereupon a solid precipitated. The reaction solution was poured into 400 mL of water, allowed to stand for 0.5 h, and filtered. The resulting filter cake was washed twice with deionized water, and the aqueous phase was extracted three times with EA. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (100-200 mesh silica gel, eluted first with 1 vol% MeOH-DCM, then 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 of 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 an acetone solution of MOMCl (480 mmol) was slowly added dropwise at -20°C and stirred at 16°C for 12 h. The reaction endpoint was confirmed by TLC (30 vol% EA-PE). The mixture was filtered and the filtrate was collected. The filter cake was washed three times with ethyl acetate. The washing liquid and the filtrate were combined and concentrated to dryness. 500 mL of water was added for re-dissolution, and the pH was adjusted to 5.8 with 1 M dilute hydrochloric acid in an ice bath. The mixture was extracted with ethyl acetate three 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 3 h. The mixture was filtered and the filtrate was added to 300 mL of water. The mixture was extracted three times with EA (200 mL per time). The organic phases were combined, washed with saturated brine, dried over 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 used directly in the next reaction.

[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 added with 500 mL of water. The mixture was extracted three times with ethyl acetate. 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 give 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] Compound M5 (11.40 g, 26.4 mmol) was added to a 100 mL single-necked bottle and DMSO 53 mL to obtain a brown-yellow solution. 2.64 mmol of elemental iodine was added, and the brown-yellow solution turned into a brown solution. The solution was heated to 85 ° C. and stirred for 12 h. The solution was poured into 800 mL of water. A large amount of solid precipitated. The solution was filtered and the filter cake was washed twice with water and dried to obtain compound M6 (8.0 g, yellow solid), which was 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. It was then dissolved in 1.2 L of DCM and dried over anhydrous sodium sulfate. The sodium sulfate was removed by filtration, and the solvent was removed by concentration. The solution was 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 350 mL of water, 500 mL of acetone (6.80 mol), and 2.24 mol of sodium bicarbonate, and stir for 15 minutes to obtain a white suspension. Cool to 5°C and add 0.61 mol of Oxone in portions while stirring, generating a large amount of bubbles. Add the mixture over 20 minutes. After the addition is complete, stir vigorously (200-500 rpm) in an ice bath for 30 minutes, until the solution turns slightly pink. Connect a cold trap and cool the trap to -78°C under argon for 10 minutes. Remove the ice bath from the reaction flask and continue stirring. Connect a vacuum pump and perform vacuum distillation to collect the DMDO acetone solution. Close the trap when the volume change in the trap is no longer noticeable and return the temperature to room temperature under argon to obtain a DMDO solution (125 mL, 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 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 atmosphere was replaced with argon three 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 resulting solid was re-dissolved 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 recrystallized repeatedly 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 via aldehyde-ketone condensation reaction: Compound M3 (15.00 g, 123 mmol) and 100 mL of acetic acid were added to yield a colorless, clear solution. p-Hydroxybenzaldehyde (139 mmol) and 0.5 g of an acid catalyst (p-toluenesulfonic acid, p-TsOH) were added at room temperature to form a yellow solution. The mixture was heated to reflux and reacted for 3 h. The mixture was cooled to room temperature, diluted with 300 mL of water, and then stirred in an ice bath to precipitate a yellow solid. The resulting solid was filtered and washed twice with 100 mL of water. The resulting solid was dissolved in 100 mL of ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to constant weight to yield compound DP (21.00 g, yellow solid, 80% yield).

[0174] This synthetic route prepares compound DP via an 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 (126 MHz, DMSO-d6) δ 190.85 (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. Based on the above mass spectrometric and NMR data, it can be seen that Compound DP prepared in this comparative example is not a flavonoid compound having the structure represented by Formula I, and the target product represented by Formula I cannot be synthesized via this aldehyde-ketone condensation reaction synthesis route.

[0176] Test Example 1

[0177] Study on the Activity of Flavonoids

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

[0179] HUVECs were seeded in cell culture plates and cultured in a cell incubator. When the cells reached 80% polymerization, they were treated with serum-free medium containing varying concentrations of Flavonoid 01 (0.5-30 μg / mL). A quadratic regression universal rotation design was used for the experiments. The dose range of Flavonoid 01 was 0.5-30 μg / mL, and the duration of exposure ranged from 1 to 24 hours. Cell viability was then measured. The detailed procedures were performed according to the instructions of the cell viability assay kit.

[0180] AML12 cells were seeded into cell culture plates and cultured in a cell incubator. When the cells reached 80% polymerization, they were treated with serum-free medium containing varying concentrations of Flavonoid 01. A quadratic regression universal rotation design was used for the experiment. The dose range of Flavonoid 01 was 0.5 to 30 μg / mL, and the duration of exposure ranged from 1 to 24 hours. Cell viability was then measured. The detailed procedures were performed according to the instructions of the cell viability assay kit.

[0181] The results showed that flavonoid 01 exhibited a certain degree of toxicity to HUVECs and AML12 cells after treatment with 30 μg / mL for 12 hours, and the toxicity increased significantly with increasing dose and treatment time. However, flavonoid 01 at 0.5-12 μg / mL did not produce significant toxicity to HUVECs and AML12 cells after treatment with 0.5-12 μg / mL for 24 hours.

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

[0183] Glutathione peroxidase 4 (GPX4) plays a key role in maintaining intracellular redox homeostasis and preventing lipid peroxidation. Flavonoids have garnered significant attention for their antioxidant properties. This study aimed to evaluate the effects of flavonoids on GPX4 activity and expression levels and to compare them with kaempferol to determine their relative potency. Two cell lines, HUVECs (human umbilical vein endothelial cells) and AML12 (mouse hepatocytes), were used.

[0184] Materials and methods

[0185] Chemical reagents: flavonoid 01 substance, kaempferol (standard), 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 assay: After 24 hours of treatment, cell lysates were collected and GPX4 activity was assayed using a GPX4 enzyme assay kit according to the manufacturer's instructions. Each experiment was repeated three times, and data are presented as mean ± SD.

[0189] GPX4 expression assay: After cell treatment, cell lysates were collected and protein was extracted. Western blot analysis was performed to determine the expression levels of GPX4 and β-actin (an internal control). ImageJ software was used for grayscale analysis of protein bands. GPX4 expression levels were normalized to β-actin expression.

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

[0191] Figure 2 Figure 2 shows changes in GPX4 expression in HUVECs and AML12 cells between different treatment groups. (A) HUVECs cell line, B AML12 cell line. GPX4 expression in the HUVECs cell line showed no significant difference in GPX4 protein expression between the kaempferol-treated and control groups. However, GPX4 protein expression in the flavonoid 01-treated group was significantly higher than in both the control and kaempferol-treated groups (P < 0.05), and GPX4 expression increased significantly with increasing doses. GPX4 expression in the AML12 cell line showed no significant difference in GPX4 protein expression between the kaempferol-treated and control groups. However, GPX4 protein expression in the flavonoid 01-treated group was significantly higher than in both the control and kaempferol-treated groups (P < 0.05), and GPX4 expression increased significantly with increasing doses.

[0192] In both HUVECs and AML12 cell lines, flavonoid 01 significantly increased GPX4 activity and protein expression at high doses, demonstrating a significantly superior effect compared to kaempferol. Both low- and high-dose groups of flavonoid 01 demonstrated enhanced GPX4 activity and expression, with the high-dose group showing the most significant effect.

[0193] Conclusion: Flavonoid 01 significantly promoted GPX4 activity and expression, particularly at high doses, where its effect was significantly superior to that of kaempferol. This suggests that the flavonoid 01 provided by the present invention possesses potential antioxidant properties and may play an important role in antioxidant therapy. This provides a basis for further exploration of the mechanism of action of flavonoid 01 and demonstrates its potential for clinical application.

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

[0195] Solutions of flavonoid 01 and kaempferol (control) at varying concentrations (10 μM, 25 μM, and 50 μM, in 0.1 v / v% DMSO-water) were prepared. A methanolic solution of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) was added, and the mixture was allowed to react in the dark for 30 min. The absorbance was measured at 517 nm using a UV spectrophotometer, and the DPPH free radical scavenging rate (mean ± SD) was calculated using the following formula: DPPH free radical scavenging rate (%) = (blank absorbance - experimental absorbance) / blank absorbance × 100%. The blank absorbance refers to the absorbance of the sample containing only the DPPH methanolic solution without the drug (flavonoid or kaempferol); the experimental absorbance refers to the absorbance of the sample after adding the drug (flavonoid 01 or kaempferol) at varying concentrations. The results are presented as mean ± standard deviation (SD) and are shown in Table 1.

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

[0197] Concentration (μM) Flavonoid 01 substance 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 experiments of flavonoids 01 and kaempferol

[0200] AML12 cells were seeded in 96-well plates and cultured for 24 hours. Flavonoids 01 and kaempferol at varying concentrations were added and cultured for another 24 hours. CCK8 reagent was added, and after a 2-hour incubation, absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated (mean ± SD) using the following formula: Cell viability (%) = (absorbance of experimental group - absorbance of blank group) / (absorbance of control group - absorbance of blank group) × 100%. Results are shown in Table 2.

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

[0202] Concentration (μM) Flavonoid 01 substance 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-assisted evaluation of the druggability potential of flavonoids 01

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

[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 frequently used concept in drug discovery. This rule helps predict whether a bioactive molecule is likely to possess the physicochemical properties necessary for oral bioavailability and is often used in initial drug-likeness screening. According to the Lipinski Rule, orally active small molecules must meet the following property requirements: no more than five hydrogen bond donors; no more than ten hydrogen bond acceptors; a molecular weight less than 500 Da; a water-oil partition coefficient no greater than 5; and no more than ten rotatable bonds.

[0209] Flavonoid 01 met the initial drug-likeness screening criteria. Furthermore, it passed the Pfizer Rule, GSK Rule, Golden Triangle, and PAINSADMET screening criteria, as shown in Table 3. As can be seen in Table 3, Flavonoid 01 performed well across all drug screening criteria, demonstrating a low toxicity risk and excellent pharmacokinetic properties. These results suggest that Flavonoid 01 has potential for further drug development, particularly as a promising drug candidate, while ensuring both efficacy and safety.

[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 ) was 1.329, and the plasma clearance (CL) was 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 of molecular docking technology was used for docking simulation. Figure 3 As shown, the binding pattern of flavonoid 01 to the GPX4 protein is stable, with a docking score of -3.804, indicating strong binding affinity between the two. Further analysis shows that flavonoid 01 is bound to the phenylalanine 170 (PHE 170) and glutamic acid 65 (GLU 65) residues of GPX4 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 demonstrated 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing flavonoid compounds, characterized in that: The following steps are involved: Compound M3 is reacted with a first (chloromethyl) methyl ether under alkaline conditions to carry out a hydroxyl protection reaction to obtain compound M4; Compound M3a is acylated with a second (chloromethyl) methyl ether under basic 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 oxidizing agent 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 compound M8; The compound M8 is hydrolyzed under alkaline conditions to obtain a flavonoid compound having a structure shown in Formula I; Formula I.

2. The preparation method according to claim 1, 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.

3. The preparation method according to claim 1, 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.

4. The preparation method according to claim 1, characterized in that The oxidant comprises one or more of elemental 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 oxidation reaction temperature is 80-90°C and the time is 8-16 hours; The organic solvent used in the oxidation reaction includes one or more of dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide and methanol.

5. The preparation method according to claim 1, 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.

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

7. The preparation method according to claim 1, characterized in that The temperature of the hydrolysis reaction is 35-45° C., and the time is 20-40 minutes.

8. Use of the flavonoid compound prepared by the preparation method according to any one of claims 1 to 7 in the preparation of an agent for promoting the activity and expression level of GPX4.

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