Synthesis method and application of 4',7-o-dimethyl quercetin
4',7-O-dimethylquercetin was synthesized by reacting rutin with iodomethane and purifying with silica gel, which solved the problems of low synthesis yield and high toxicity, enhanced its photosensitizing effect, and verified its application potential in photodynamic therapy.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHANGZHOU UNIV
- Filing Date
- 2023-12-01
- Publication Date
- 2026-05-26
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of tumor drugs, specifically relating to a method for synthesizing 4',7-O-dimethylquercetin and its application. Background Technology
[0002] The basic method of photodynamic therapy is to use visible light, near-infrared light, or ultraviolet light as an excitation source. The excited-state photosensitizer reacts with tumor cells through physical and biochemical reactions, thereby killing the tumor. When endogenous or exogenous photosensitizers located in biological tissues are irradiated with light of a certain wavelength, they absorb the energy carried by photons and transition from the ground state to the excited state. The photosensitizer in the excited state is very reactive and will quickly undergo physical or chemical de-excitation to release energy and return to the ground state. In the physical de-excitation process, visible fluorescence is produced, and we can use the fluorescence spectrum of this process to infer the related symptoms. The chemical de-excitation process can produce a large amount of reactive oxygen species, with singlet oxygen accounting for a large proportion. Reactive oxygen species interact with various biological macromolecules, destroying cell structure and affecting cell function, thereby killing related tumors and producing a therapeutic effect.
[0003] Quercetin is a natural flavonoid compound with various biological activities, including anti-inflammatory, antibacterial, antioxidant, and anticancer effects. Its strong ability to scavenge free radicals in the body is the basis for its antioxidant effect, and this ability is believed to be related to the number and position of the phenolic hydroxyl groups. Among them, 3... ， 4 ， The ortho-dihydroxyl group at position 7 is the most important, while the phenolic hydroxyl group at position 7 has strong acidity, which is also conducive to quercetin exerting its antioxidant effect [Research progress on structure-activity relationship of antioxidant activity of flavonoids (review) [J]. Journal of Anhui Agricultural University, 2002, 29(3):265-270.;The relative antioxidant activities of plant-derived polyphenolic flavonoids: free radical research: vol 22, no 4. Free Radical Research.], therefore 4 ， and 7 ，Methylation of hydroxyl groups can significantly reduce the antioxidant capacity of quercetin. On the other hand, studies have shown that quercetin can generate excessive amounts of ROS under the action of X-rays or 365nm ultraviolet light, and methylation of phenolic hydroxyl groups can greatly enhance this ability [Relationship between flavonoid structure and reactive oxygen species generation upon ultraviolet and X-ray irradiation[J]. Journal of Photochemistry and Photobiology A: Chemistry, 2019, 384: 112044.].
[0004] 4',7-O-dimethylquercetin is a natural compound found in Cyperus rotundus and Phellodendron amurense. It is primarily prepared by isolation and purification from Cyperus rotundus and possesses antibacterial and anti-inflammatory properties. Currently, several synthetic or semi-synthetic methods exist, including: 1. Direct reaction of quercetin with iodomethane: This method is simple but has low selectivity and poor yield. 2. Direct reaction of quercetin with diazomethane or dimethyl sulfate: This method yields good results, but the substrates are highly toxic to humans and the environment. 3. Direct decomposition or synthesis of phytolaccaside, 3-(acetyloxy)-4-methoxybenzoyl chloride, etc.: This method is simple and produces high-purity products, but the substrates are very expensive. Summary of the Invention
[0005] Based on the above-mentioned technical problems, the present invention aims to provide a method for synthesizing 4',7-O-dimethylquercetin and its application.
[0006] One of the objectives of this invention is to provide a method for synthesizing 4',7-O-dimethylquercetin.
[0007] The structural formula of 4',7-O-dimethylquercetin provided by this invention is as follows:
[0008] .
[0009] The synthesis method of 4',7-O-dimethylquercetin is as follows:
[0010] (1) Dissolve rutin in N,N-dimethylformamide, add potassium carbonate, and add iodomethane dropwise while stirring in an ice bath. After the addition is complete, stop stirring and allow the mixture to react naturally at a temperature of 12-72 hours. After the reaction is complete, add excess distilled water to the reaction solution, filter, wash with water, and dry to obtain the intermediate product; wherein the molar ratio of rutin:potassium carbonate:iodomethane is 1:10:6-10.
[0011] To improve the yield of the final product, the optimal reaction time for step (1) is 48-72 h; the concentration of rutin in N,N-dimethylformamide is 1 mol / L.
[0012] (2) The intermediate product was added to an acidic solution and heated under reflux. After the reaction solution was cooled, it was filtered, precipitated, washed with water, and dried to obtain a yellowish-brown mixed product.
[0013] The acidic solution can be sulfuric acid or hydrochloric acid. In a specific embodiment of the present invention, the acidic solution used is a sulfuric acid solution with a mass fraction of 0.5%.
[0014] (3) The mixed product was dissolved in methanol and mixed with silica gel. The mixture was then evaporated and purified by dry chromatography column loading to obtain 4',7-O-dimethylquercetin.
[0015] The eluent is a mixed solution of ethyl acetate, petroleum ether, and glacial acetic acid, with a ratio ranging from 1:50:5.1 to 1:10:1.1.
[0016] The final product was identified by NMR as 4',7-O-dimethylquercetin. The yield was over 25% when the intermediate product was hydrolyzed for more than 48 hours as the raw material.
[0017] A second objective of this invention is to provide the application of 4',7-O-dimethylquercetin in the preparation of photosensitizers for photodynamic therapy, wherein the wavelength of the ultraviolet light is 350-380 nm.
[0018] In some specific embodiments of the present invention, the ultraviolet light wavelength used is 365 nm. Test results show that 4',7-O-dimethylquercetin can generate excessive ROS under appropriate doses and wavelengths of ultraviolet light irradiation and react with the detection probe, leading to a change in absorbance.
[0019] In vitro cell experiments showed that 4',7-O-dimethylquercetin can generate or induce excessive ROS in HepG2 cells under appropriate doses and wavelengths of ultraviolet radiation, and induce excessive cell apoptosis, thus preliminarily verifying its feasibility as a photosensitizer for photodynamic therapy.
[0020] 4',7-O-dimethylquercetin can be used as an active ingredient and a pharmaceutically acceptable carrier to prepare drug formulations, such as solutions, liposomes, and nanodelivery systems.
[0021] This invention creatively proposes a method for the chemical synthesis of 4',7-O-dimethylquercetin using a partially substituted rutin intermediate. Its photosensitizing effect was also verified for the first time, showing a significantly enhanced effect compared to quercetin. This provides an experimental basis for the development of 4',7-O-dimethylquercetin drugs and further expands the application of natural drugs in antitumor sensitization therapy, with broad application value in fields such as radiomedicine and biology. Attached Figure Description
[0022] Figure 1 Yield curves of 4',7-O-dimethylquercetin synthesized at different reaction times;
[0023] Figure 2 The NMR spectrum of 4',7-O-dimethylquercetin synthesized in the example;
[0024] Figure 3 The absorbance values of superoxide anion, hydroxyl radical, and singlet oxygen were detected in Example 2.
[0025] Figure 4 The 4',7-O-dimethylquercetin concentration-cell viability curve is shown.
[0026] Figure 5 The relative ROS content in cell supernatant;
[0027] Figure 6 The effect of 4',7-O-dimethylquercetin on the proliferation of HepG2 cells after UV irradiation. Detailed Implementation Example 1
[0028] Synthesis of 4',7-O-dimethylquercetin:
[0029] (1) Dissolve 3.0 mmol of rutin in 30 ml of N,N-dimethylformamide, add 30.0 mmol of potassium carbonate, cool to 0°C in an ice-salt bath, add 1.9 ml of iodomethane dropwise over 30 min with stirring, stop stirring after the addition is complete and allow to rise naturally to room temperature, let stand for 48 h, add excess distilled water to the reaction solution, precipitate out, filter and wash with water, dry in an oven at 60°C to obtain the intermediate product.
[0030] (2) Add the intermediate product to 300 ml of 0.5% sulfuric acid solution, heat under reflux for 1 h, cool the reaction solution and filter, wash the precipitate with water and dry under vacuum to obtain a yellowish-brown mixed product.
[0031] (3) Take 0.5g of the mixed product and separate and purify it by chromatography to obtain 4',7-O-dimethylquercetin.
[0032] This embodiment also examines the intermediate products obtained by allowing the reaction to stand for 12h, 24h, 36h, 60h, and 72h in step (1) and collecting the products in steps (2) and (3).
[0033] The calculated yields of the products synthesized from different intermediates in this embodiment are as follows: Figure 1 As shown, the reaction time of 48 hours was chosen based on both efficiency and yield analysis. The intermediate product, after being allowed to stand for 48 hours, yielded a 25.4% yield.
[0034] Nuclear magnetic resonance spectrum as follows Figure 2 As shown: 1H-NMR (300MHz, deuterated DMSO): 3.353 (H2O, s), 3.872 (s, 3H, -OCH3), 3.855 (s, 3H, -OCH3), 12.452 (s, 1H, 5-OH), 9.590 (s, 1H, 3-OH), 0.333 (s, 1H, 3-OH). , -OH),7.720(s,1H,8-H),7.681(d,1H,6 , -H), 7.100(d,1H,5 , -H), 6.737(s,1H,2) , -H), 6.362 (s, 1H, 6-H). 1.242 is a petroleum ether impurity peak, and 2.502 is a deuterated DMSO solvent peak. Therefore, the compound was identified as 4',7-O-dimethylquercetin. Example 2
[0035] Relative determination of superoxide anion content
[0036] 10 μmol of the compound was dissolved in 10 mL of 1 mM hydroxylamine hydrochloride in ethanol, and the solution was heated under an 8 W 365 nm UV lamp (irradiance of 11.5 mW / cm²). 2 (The same applies below) Irradiate at 13 cm for 1 hour. Then add 1 ml of 1 mM p-aminobenzenesulfonic acid solution and incubate at 25°C for 20 min, followed by the addition of 1 ml of 1 mM α-naphthylamine solution. Incubate at 30°C for 30 min. Perform the same operation on the unirradiated group and measure the absorbance at 530 nm. Superoxide anion can oxidize hydroxylamine hydrochloride to generate NO2-. NO2- reacts with p-aminobenzenesulfonic acid and α-naphthylamine to form a red azo compound. The product has a characteristic absorption peak at 530 nm. O2 can be detected by the increase in absorbance. 2 - The content of.
[0037] Relative detection of hydroxyl radicals
[0038] 10 μmol of the compound was dissolved in 10 mL of 0.1 μM Rhodane Red B (RB) ethanol solution at pH 4 and exposed to a 365 nm UV lamp with a power of 8 W (irradiance of 11.5 mW / cm²). 2 Rhodane Red B can react with hydroxyl radicals to reduce absorbance. The absorbance is measured at 554 nm, and the content of ·OH can be detected based on the decrease in absorbance.
[0039] Relative detection of singlet oxygen
[0040] 10 μmol of the compound was dissolved in 10 mL of 0.1 mM 9,10-xyleneanthracene (DPA) ethanol solution, and the solution was heated under an 8 W 365 nm UV lamp (irradiance of 11.5 mW / cm²). 2 DPA can react with singlet oxygen to form stable internal oxides. The amount of decrease in absorbance at 355 nm can be used to detect O. 1 The content of. Example 3
[0041] 4',7-O-dimethylquercetin administration for 24 h affected the IC50 of HepG2 cells. 50
[0042] HepG2 cells were cultured with 0, 6.25, 12.5, 25, 50, 100, and 200 μM 4',7-O-dimethylquercetin, respectively. Cell viability after 24 h was detected by the CCK8 assay. The results are as follows: Figure 4 As shown. IC was obtained through GraphPad Prism analysis. 50 = 63.82±3.3 μM. Example 4
[0043] ROS assay of cell supernatant
[0044] Cells were divided into four groups in a 6-well plate: (1) PBS group: 20 μL of PBS was added; (2) PBS+: 20 μL of PBS was added to the cells, and the cells were irradiated with 365 nm ultraviolet light for 5 min; (3) Quercetin+: 20 μL of 4',7-O-dimethylquercetin solution was added to make the drug concentration reach 10 μM; (4) 4',7-O-dimethylquercetin+: 20 μL of 4',7-O-dimethylquercetin solution was added to make the drug concentration reach 10 μM, and the cells were irradiated with 365 nm ultraviolet light for 5 min. 24 hours later, the ROS content in the cell supernatant was detected using the Beyotime reactive oxygen species detection kit. The ROS content of the PBS group was set to 1, and the relative content of the other groups was calculated. The results are shown in the table below. Figure 5 . Example 5
[0045] CCK8 cell proliferation experiment
[0046] HepG2 cells were divided into four groups in a 6-well plate: (1) PBS group: 20 μL of PBS was added; (2) PBS+: 20 μL of PBS was added to the cells, and the cells were irradiated with 365 nm ultraviolet light for 5 min; (3) Quercetin+: 20 μL of 4',7-O-dimethylquercetin solution was added to make the drug concentration reach 10 μM; (4) 4',7-O-dimethylquercetin+: 20 μL of 4',7-O-dimethylquercetin solution was added to make the drug concentration reach 10 μM, and the cells were irradiated with 365 nm ultraviolet light for 5 min. Cell proliferation was measured at 12, 24, 36, 48, and 60 h. The results are as follows. Figure 6 .
[0047] The above experimental results show that 4',7-O-dimethylquercetin can excite excess ROS through ultraviolet light, thereby exerting the effect of PDT photosensitizer.
Claims
The application of 1,4',7-O-dimethylquercetin in the preparation of photosensitizers for photodynamic therapy, characterized in that, The excitation light of the photosensitizer in the photodynamic therapy is ultraviolet light with a wavelength of 350-380nm.
2. The application according to claim 1, characterized in that, The photodynamic therapy photosensitizer is a pharmaceutical preparation made with 4',7-O-dimethylquercetin as the active ingredient and a pharmaceutically acceptable carrier.
3. The application according to claim 1, characterized in that, The method for synthesizing the 4',7-O-dimethylquercetin includes the following specific steps: (1) Dissolve rutin in N,N-dimethylformamide, add potassium carbonate, and add iodomethane dropwise while stirring at 0°C. After the addition is complete, stop stirring, allow the temperature to rise naturally to room temperature, and let the reaction stand for 12-72 hours. After the reaction is complete, add excess distilled water to the reaction solution, filter, wash with water, and dry to obtain the intermediate product; wherein, the molar ratio of rutin:potassium carbonate:iodomethane is 1:10:6-10. (2) Add the intermediate product to an acidic solution and heat under reflux. After cooling the reaction solution, filter, wash with water, and dry to obtain a mixed product. (3) The mixed product was dissolved in methanol and mixed with silica gel. After being evaporated to dryness, it was purified by dry chromatography column loading to obtain 4',7-O-dimethylquercetin.
4. The application according to claim 3, characterized in that, In step (1), the molar concentration of rutin in N,N-dimethylformamide is 1 mol / L.
5. The application according to claim 3, characterized in that, The reaction time for step (1) is 48-72 hours.
6. The application according to claim 3, characterized in that, Step (2) The acidic solution is a sulfuric acid solution with a mass fraction of 0.5%.
7. The application according to claim 3, characterized in that, Step (2) The heating reflux time is 0.5-1h.
8. The application according to claim 3, characterized in that, The eluent for separation and purification by chromatography column is a mixed solution of ethyl acetate, petroleum ether, and glacial acetic acid in a volume ratio of 1:10-50:1.1-5.1.