阅读说明：本技术 一种3＇，4＇，7-三羟基异黄酮的合成方法 (Synthesis method of 3 ', 4', 7-trihydroxyisoflavone ) 是由 赵勇 景临林 邵瑾 马慧萍 赵彤 于 2020-06-08 设计创作，主要内容包括：本发明公开一种3′,4′,7-三羟基异黄酮的合成方法。该方法包括：4′,7-二甲氧基异黄酮与溴素在二氯甲烷介质中混合反应,得到3′-溴-4′,7-二甲氧基异黄酮,其中,所述4′,7-二甲氧基异黄酮与溴素的摩尔比为1∶1.1～1.5,反应温度为20～30℃；3′-溴-4′,7-二甲氧基异黄酮在亚铜盐作用下与甲醇钠反应,得到3′,4′,7-三甲氧基异黄酮；3′,4′,7-三甲氧基异黄酮脱甲基,得到所述的3′,4′,7-三羟基异黄酮。与现有技术相比,本发明起始原料来源丰富,反应条件温和,选择性好,产率高,适合工业化生产。(The invention discloses a method for synthesizing 3 ', 4', 7-trihydroxyisoflavone. The method comprises the following steps: mixing 4 ', 7-dimethoxy isoflavone and bromine in a dichloromethane medium for reaction to obtain 3' -bromo-4 ', 7-dimethoxy isoflavone, wherein the molar ratio of the 4', 7-dimethoxy isoflavone to the bromine is 1: 1.1-1.5, and the reaction temperature is 20-30 ℃; reacting 3 '-bromo-4', 7-dimethoxy isoflavone with sodium methoxide under the action of cuprous salt to obtain 3 ', 4', 7-trimethoxy isoflavone; demethylating the 3 ', 4', 7-trimethoxy isoflavone to obtain the 3 ', 4', 7-trihydroxy isoflavone. Compared with the prior art, the method has the advantages of rich source of the initial raw materials, mild reaction conditions, good selectivity and high yield, and is suitable for industrial production.)

1. A method for synthesizing 3 ', 4', 7-trihydroxyisoflavone comprises the following steps:

(1) mixing 4 ', 7-dimethoxy isoflavone and bromine in a dichloromethane medium for reaction to obtain 3' -bromo-4 ', 7-dimethoxy isoflavone, wherein the molar ratio of the 4', 7-dimethoxy isoflavone to the bromine is 1: 1.1-1.5, and the reaction temperature is 20-30 ℃;

(2) reacting 3 '-bromo-4', 7-dimethoxy isoflavone with sodium methoxide under the action of cuprous salt to obtain 3 ', 4', 7-trimethoxy isoflavone;

(3) demethylating the 3 ', 4', 7-trimethoxy isoflavone to obtain the 3 ', 4', 7-trihydroxy isoflavone.

2. The method of synthesis according to claim 1, characterized in that: the cuprous salt is cuprous bromide, cuprous iodide and/or cuprous chloride.

3. The method of synthesis according to claim 1, characterized in that: 4', 7-dimethoxy isoflavone is obtained by the following method: mixing daidzein or formononetin, potassium carbonate and a methylation reagent in an organic solvent for reaction to obtain the 4', 7-dimethoxy isoflavone, wherein the molar ratio of daidzein to potassium carbonate to the methylation reagent is 1: 2-3, or the molar ratio of formononetin to potassium carbonate to the methylation reagent is 1: 1-2, and the reaction temperature is 60-70 ℃.

4. The method of synthesis according to claim 3, characterized in that: the methylating agent is dimethyl sulfate or dimethyl carbonate.

5. The method of synthesis according to claim 1, characterized in that: the reaction process of the step (2) comprises the following steps: mixing 3 '-bromo-4', 7-dimethoxy isoflavone with an organic solvent, heating to 110-130 ℃, adding cuprous salt and excessive sodium methoxide, and reacting to obtain 3 ', 4', 7-trimethoxy isoflavone, wherein the molar ratio of the 3 '-bromo-4', 7-dimethoxy isoflavone to the cuprous salt is 1: 1-1.5.

6. The method of synthesis according to claim 1, characterized in that: the reaction process of the step (3) comprises the following steps: suspending aluminum chloride in an organic solvent, cooling to 0-8 ℃, slowly dropwise adding dimethyl sulfide, uniformly mixing, adding 3 ', 4', 7-trimethoxy isoflavone, heating, reacting at 20-30 ℃ to obtain 3 ', 4', 7-trihydroxy isoflavone, wherein the molar ratio of the aluminum chloride to the dimethyl sulfide to the 3 ', 4', 7-trimethoxy isoflavone is 7-9: 1.

7. The synthesis method according to any one of claims 3 to 7, wherein: the organic solvent is acetone, acetonitrile, dichloromethane or N, N-dimethylformamide.

Technical Field

The invention belongs to the field of drug synthesis, and particularly relates to a synthesis method of 3 ', 4', 7-trihydroxyisoflavone.

Background

Isoflavones are secondary metabolites of plants and have a wide range of physiological activities such as anti-inflammatory, antiviral, anti-tumor, anti-diabetic, anti-radiation, anti-ischemia-reperfusion injury, neuroprotection, etc., and these beneficial pharmacological effects are mostly attributed to their excellent antioxidant activities, which can prevent oxidative damage by inhibiting and scavenging free radicals and reactive oxygen species (nat. prod. rep, 2019, 36 (8): 1156-1195). The study indicated that: the hydroxyl substituent in the isoflavone structure is an active group for scavenging free radicals, and the number, substitution position and substitution form of the hydroxyl group have important effects on the activity (Int J Mol Sci, 2015, 16 (6): 12891-12906).

3 ', 4', 7-trihydroxyisoflavone, the structural formula is as follows:

the isoflavone has excellent antioxidant activity, but is less distributed in natural products and has low content, so that the compounds cannot be obtained from the natural products in large quantity.

3 ', 4', 7-Trihydroxyisoflavone is one of the major secondary metabolites of daidzein in vivo (J.Agric. food Chem, 2001, 49: 3024-3033), has anti-inflammatory, anti-apoptotic (Evid. Based comparative Altern. Med, 2013, 636597), antiprotozoal (Nat. Prod, 2006, 69 (1): 43-49), antibacterial (Lett Appl Microbiol, 2015, 60, (3): 242-247), antiproliferative (J.biol. Chem.2010, M109: 094797), anti-tumor (Toxicolog, 2012, 302 (2-3): 221-232), anti-skin cancer (J.biol. Chem, 2011, 142286 (16): 14246 Eu56), anti-obesity (Res. Fonto. cheol, 2014, 240 (4): 869), etc., and can also be used as anti-dermatitis (Megchint) 1163, 92, 11620, anti-dermatitis (Biomot. RTM 1163, 1164, 190, Biotin, Psub. Chem, Psub. RT, 1164, etc., 2018, 13: 3279 and 3293). Recent studies have shown that they also have neuroprotective (Biomol Ther (Seoul), 2019, 27 (4): 363-.

The means for obtaining 3 ', 4', 7-trihydroxyisoflavone are few, and the following are mainly available: goto (Chem pharmBull (Tokyo) 2009, 57 (4): 346-360) and the like, which use resorcinol and 3, 4-dihydroxy phenylacetic acid as raw materials, at BF₃·Et₂Under the catalysis of O, dehydration reaction is carried out, and then MeSO is carried out₂In a Cl-DMF system, deoxybenzoin reaction is carried out to generate 3 ', 4', 7-trihydroxyisoflavone, although the total yield can reach 65%, BF which is inflammable, toxic, strong in irritation and strong in corrosivity is required to be used in the reaction₃·Et₂And O. Roh et al, by regioselective hydroxylation of daidzein using Streptomyces avermitilis MA-4680, 7, 3 ', 4' -trihydroxyisoflavone was produced (Journal of Bioscience and Biobioinformatics, 2009, 108 (1): 41-46). Changhyun et al used purified CYP105D7 enzyme to catalyze daidzein by the redox NADH of P.putida to produce 7, 3 ', 4' -trihydroxyisoflavone (Molecules, 2013, 18 (3): 3028-3040). However, these methods are difficult to separate and purify due to their harsh reaction conditions and low conversion rate.

Disclosure of Invention

Based on the defects of the prior art, the invention aims to provide a method for synthesizing 3 ', 4', 7-trihydroxyisoflavone suitable for industrial production.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for synthesizing 3 ', 4', 7-trihydroxyisoflavone comprises the following steps:

(1) mixing 4 ', 7-dimethoxy isoflavone and bromine in a dichloromethane medium for reaction to obtain 3' -bromo-4 ', 7-dimethoxy isoflavone, wherein the molar ratio of the 4', 7-dimethoxy isoflavone to the bromine is 1: 1.1-1.5, and the reaction temperature is 20-30 ℃;

(2) reacting 3 '-bromo-4', 7-dimethoxy isoflavone with sodium methoxide under the action of cuprous salt to obtain 3 ', 4', 7-trimethoxy isoflavone;

(3) demethylating the 3 ', 4', 7-trimethoxy isoflavone to obtain the 3 ', 4', 7-trihydroxy isoflavone.

Preferably, the cuprous salt is cuprous bromide, cuprous iodide and/or cuprous chloride.

Preferably, 4', 7-dimethoxyisoflavone can be obtained by the following method: mixing daidzein or formononetin, potassium carbonate and a methylation reagent in an organic solvent for reaction to obtain the 4', 7-dimethoxy isoflavone, wherein the molar ratio of daidzein to potassium carbonate to the methylation reagent is 1: 2-3, the molar ratio of formononetin to potassium carbonate to the methylation reagent is 1: 1-2, and the reaction temperature is 60-70 ℃.

Specifically, 20mmol of daidzein and 40mmol of potassium carbonate are suspended in 160-240 mL of acetone, 40mmol of dimethyl sulfate is added, and the reaction is carried out at 60 ℃ for 9-11 h. Cooling to 300-500 mu L of 25% (w/w) ammonia water for quenching reaction, adding 80-120 mL of water, adjusting the pH value to acidity by concentrated HCl, decompressing and removing acetone, filtering, washing to neutrality by purified water, and drying to obtain 4', 7-dimethoxy isoflavone.

Or suspending 20mmol of formononetin and 20mmol of potassium carbonate in 160-240 mL of acetone, adding 20mmol of dimethyl sulfate, and reacting at 60 ℃ for 9-11 h. Cooling to 300-500 mu L of 25% (w/w) ammonia water for quenching reaction, adding 80-120 mL of water, adjusting the pH value to acidity by concentrated HCl, decompressing and removing acetone, filtering, washing to neutrality by purified water, and drying to obtain 4', 7-dimethoxy isoflavone.

Preferably, the methylating agent is dimethyl sulfate or dimethyl carbonate.

Preferably, the reaction process of step (2) comprises: mixing 3 '-bromo-4', 7-dimethoxy isoflavone with an organic solvent, heating to 110-130 ℃, adding cuprous salt and excessive sodium methoxide, and reacting to obtain 3 ', 4', 7-trimethoxy isoflavone, wherein the molar ratio of the 3 '-bromo-4', 7-dimethoxy isoflavone to the cuprous salt is 1: 1-1.5.

Preferably, the reaction process of step (3) comprises: suspending aluminum chloride in an organic solvent, cooling to 0-8 ℃, slowly dropwise adding dimethyl sulfide, uniformly mixing, adding 3 ', 4', 7-trimethoxy isoflavone, heating, reacting at 20-30 ℃ to obtain 3 ', 4', 7-trihydroxy isoflavone, wherein the molar ratio of the aluminum chloride to the dimethyl sulfide to the 3 ', 4', 7-trimethoxy isoflavone is 7-9: 1.

Preferably, the organic solvent is acetone, acetonitrile, dichloromethane or N, N-dimethylformamide.

More specifically, the synthesis method of 3 ', 4', 7-trihydroxyisoflavone comprises the following steps,

(1) suspending 10mmol of 4 ', 7-dimethoxy isoflavone in 40-60 mL of anhydrous dichloromethane, dropwise adding 11-15 mmol of liquid bromine under stirring at room temperature, reacting for 25-35 min, stopping the reaction, adding 130-170 mL of water, reducing unreacted bromine by using sodium bisulfite, removing dichloromethane under reduced pressure, filtering, washing a filter cake with purified water, drying in the dark, and recrystallizing by using an ethyl acetate/petroleum ether mixed solution to obtain 3 ' -bromo-4 ', 7-dimethoxy isoflavone.

(2) Suspending 4-6 mmol of cuprous salt in 3-5 mL of anhydrous DMF, stirring at 20-30 ℃ in the dark for 25-35 min, adding 20-40 mL of 25% sodium methoxide/methanol solution, and continuously stirring in the dark for 0.8-1.2 h for later use. Heating 4mmol of 3 '-bromo-4', 7-dimethoxy isoflavone obtained in the step 1 and 3-5 mL of DMF to 110-130 ℃, stirring, adding the standby solution, continuing to react for 2-5 h, cooling to room temperature, pouring the reaction solution into 30-50 mL of 2M hydrochloric acid solution, removing the solvent under reduced pressure, extracting the water phase with ethyl acetate, combining the organic phases, washing once with saturated sodium chloride, drying over night with anhydrous sodium sulfate, filtering, removing the ethyl acetate under reduced pressure, and separating and purifying by silica gel column chromatography to obtain 3 ', 4', 7-trimethoxy isoflavone.

(3) Suspending 1.5-1.8 mmol of aluminum chloride in 8-12 mL of anhydrous dichloromethane, cooling to 0-8 ℃, slowly dropwise adding 1.5-1.8 mmol of dimethyl sulfide, stirring for 20-30 min, adding 0.2mmol of 3 ', 4', 7-trimethoxy isoflavone obtained in the step 2, heating to 20-30 ℃, and continuing to react for 5-8 h. Stopping the reaction, cooling to 0-4 ℃, adding 10% (w/w) hydrochloric acid solution, stirring for 2h, decompressing to remove dichloromethane, filtering, and recrystallizing a filter cake by using a methanol/water system to obtain 3 ', 4', 7-trihydroxyisoflavone.

The invention takes daidzein or formononetin with rich sources as a starting material, and obtains polyhydroxy isoflavone 3 ', 4', 7-trihydroxy isoflavone and 3 ', 4', 7, 8-tetrahydroxy isoflavone by four steps of methylation, bromination, methoxylation and demethylation, wherein the total yield reaches 60 percent (calculated by daidzein or formononetin). 4 ', 7-dimethoxy isoflavone reacts with bromine in dichloromethane to generate 3 ' -bromo-4 ', 7-dimethoxy isoflavone with good selectivity and high yield, if the reaction solvent of the reaction is replaced by ethanol or a mixed solution of ethanol and dichloromethane, even if the dosage of bromine and the reaction temperature are strictly controlled, the reaction is incomplete, or a mixture of 3 ' -bromo-4 ', 7-dimethoxy isoflavone and 3 ', 8-dibromo-4 ', 7-dimethoxy isoflavone is obtained, and the mixture can hardly be separated to obtain a single product.

The synthetic method has the advantages of mild reaction conditions, high yield and easy industrial production. The purity of the product is more than 99.0 percent monitored by UPLC, and the product can be used for pharmacological activity research.

Drawings

FIG. 1 is a UPLC chromatogram of 3 ', 4', 7-trihydroxyisoflavone.

Detailed Description

The present invention will be described in further detail with reference to examples.