阅读说明：本技术 桦木醇衍生物及其亲水性修饰产物，纳米溶液及其制备方法 (Betulin derivative and hydrophilic modification product thereof, nano solution and preparation method thereof ) 是由 朱水芳 田志清 王晨光 姜帆 于 2020-04-17 设计创作，主要内容包括：本发明“桦木醇衍生物及其亲水性修饰产物,纳米溶液及其制备方法”,特征在于,是桦木醇或其衍生物在其3位羟基与苯甲酸衍生物中的羧基发生酯化反应的产物；将所述亲水性修饰产物分散于水中,并加入氢氧化金属盐,在25-90℃条件下搅拌30-300分钟,自然冷却至室温,使化合物形成羧基金属盐；加入反应体系体积的0.1％-10％的表面活性剂,使用超声波处理0-60min,即得桦木醇及其衍生物的亲水性修饰产物的纳米胶体水溶液,其分散性好,粒径范围理想,易于被生物体吸收。(The invention relates to a betulin derivative and its hydrophilic modification product, nanometer solution and its preparation method, characterized by, it is the product that its 3-position hydroxyl and carboxyl in the benzoic acid derivative take esterification reaction in betulin or its derivative; dispersing the hydrophilic modification product in water, adding metal hydroxide salt, stirring at 25-90 ℃ for 30-300 minutes, and naturally cooling to room temperature to enable the compound to form carboxyl metal salt; adding surfactant 0.1-10% of the volume of the reaction system, and treating with ultrasonic for 0-60min to obtain nanometer colloid water solution of hydrophilic modification product of betulin and its derivatives, which has good dispersibility, ideal particle size range, and easy absorption by organism.)

1. Betulin derivatives represented by formulae 3 to 7:

wherein R is selected from C1-6 alkyl, aryl, heteroaryl, aryl-C1-2 alkyl, heteroaryl-C1-2 alkyl, C3-10 cycloalkyl, C3-10 cycloalkyl-C1-2 alkyl, ethynyl, amino or vinyl, each of which is optionally substituted with 1-3R_aSubstituted by groups; and each R_aThe groups are independently halogen, C1-6 alkyl, fluoro-substituted C1-6 alkyl, fluoro-substituted C1-6 alkoxy, aryl, heteroaryl, C1-6 alkoxy, -CN, -NO₂、-OH、＝O、NH₂-C (O) -O-C1-6 alkyl or-SiMe 3, wherein R_aFurther optionally substituted by 1-3R_bIs substituted by radicals in which each R is_bThe groups are independently halogen, C1-6 alkyl, C1-6 alkoxy, -CN, -NO₂Or-OH；

The benzoic acid derivative is selected from: ortho-phthalic acid, meta-phthalic acid, para-phthalic acid, phthalic anhydride, 1,2, 4-benzenetricarboxylic acid, 1,3, 5-benzenetricarboxylic acid, benzenetetracarboxylic acid, pyromellitic anhydride, benzenepentacarboxylic acid, benzenehexacarboxylic anhydride.

2. Betulin derivatives according to claim 1, wherein R isOr phenyl;

the structure of the betulin derivative shown in formula 3 is shown as formula 3a,

the betulin derivative represented by formula 4 is represented by formula 4a,

formula 7 shows betulin derivatives such as formula 7a,

3. a method for synthesizing betulin derivatives represented by the formula 3a, which comprises the steps of,

adding dichloromethane and betulinic acid into a flask, adding 2-bromoethyl methyl ether and 1, 8-diazabicycloundec-7-ene after the betulinic acid is dissolved, stirring and reacting for 2 days at room temperature, and separating by silica gel column chromatography with toluene as developing agent to obtain the product of the compound shown in formula 3a

A method for synthesizing betulin derivatives represented by the formula 4a, which comprises the steps of,

adding dichloromethane and betulinic acid into a flask, adding 2-methoxyethylamine and 4-dimethylaminopyridine after the betulinic acid is dissolved, stirring and reacting for 8-12h at 40 ℃, and separating by silica gel column chromatography with toluene as a developing agent to obtain a product, namely the compound shown as the formula 4a

A method for synthesizing betulin derivatives represented by the formula 5, which comprises the steps of,

in a reaction flask, white birch alcohol is dissolved in dichloromethane, bismuth trifluoromethanesulfonate (Bi (OTf))₃) Refluxing and reacting for 4 hours at 50 ℃; after the reaction is finished, cooling, washing for 3 times by saturated sodium bicarbonate, washing for one time by saturated sodium chloride, drying by anhydrous sodium sulfate, and performing rotary evaporation to obtain a product, namely a compound shown as a formula 5,

a method for synthesizing betulin derivatives represented by the following formula 6, which comprises the steps of:

adding dichloromethane into a flask, then adding betulinic acid and bismuth trifluoromethanesulfonate, heating and refluxing, and stirring for 2 hours; cooling the reaction system to room temperature, adding saturated sodium bicarbonate solution for washing, and drying an organic phase by using anhydrous sodium sulfate; then filtering and distilling under reduced pressure, wherein the volume ratio of the mixture is 1: 4, separating the mixture of petroleum ether and dichloromethane serving as a developing agent by silica gel column chromatography to obtain a compound shown as a formula 6,

a method for synthesizing betulin derivatives represented by the following formula 7, which comprises the steps of: adding pyridine and betulin into a flask, adding benzoic anhydride and 4-Dimethylaminopyridine (DMAP) after the betulinic acid is dissolved, stirring and reacting for 2-4h at the temperature of 60 ℃, and washing with water and dichloromethane respectively to obtain a product, namely the compound shown as the formula 7a

4. A hydrophilic modification product of betulin and its derivatives, characterized in that betulin, betulinic acid, or a product obtained by esterification of the 3-position hydroxyl group of betulin derivative according to claim 1 or 2 with a carboxyl group of a benzoic acid derivative molecule.

5. The hydrophilic modification product according to claim 4, wherein the benzoic acid derivative is phthalic anhydride,

the hydrophilic modification product of formula 5 is shown as formula 8,

the hydrophilic modification product of formula 3a is shown as formula 9,

the hydrophilic modification product of formula 4a is shown as formula 10,

the hydrophilic modification product of formula 7a is shown in formula 11,

6. the hydrophilic modification product according to claim 4, wherein the benzoic acid derivative is pyromellitic dianhydride, the modification product of formula 5 is represented by formula 12,

7. use of a betulin derivative according to any one of claims 1 to 2 and/or a hydrophilic modification product of a betulin derivative according to any one of claims 4 to 6 for the preparation of an antiviral agent, wherein the betulin derivative and/or the hydrophilic modification product is used as an active ingredient of the antiviral agent.

8. The use according to claim 7, wherein the hydrophilic modification product of betulin derivatives according to any one of claims 4 to 6 is used as an active ingredient of the antiviral agent in the form of a nano solution having a particle size of 150-400 nm.

9. A method for preparing an antiviral agent, comprising preparing a nano solution by using the hydrophilic modification product of betulin derivatives according to any one of claims 4 to 6 as an active ingredient of an antiviral agent, through the steps of:

dispersing the hydrophilic modification product of the betulin derivative in water, adding 1-10 times of equivalent of metal hydroxide salt, stirring at 30-90 deg.C for 30-300 min, naturally cooling to room temperature, adding 0.1-10% surfactant of reaction system volume, treating with ultrasonic wave for 0-60min to obtain nanometer colloid water solution,

the surfactant is selected from: polyethylene glycol, alkyl glucoside, fatty glyceride, sorbitan fatty acid, polysorbate, stearic acid, sodium dodecyl benzene sulfonate and lecithin;

preferably, the metal hydroxide salt refers to sodium hydroxide or potassium hydroxide.

10. A method for hydrophilic modification of betulin and betulin derivatives as claimed in any of claims 4 to 6, wherein betulin or betulin derivatives are esterified at the hydroxyl group at the 3-position with a carboxyl group in a benzoic acid derivative;

the esterification reaction conditions are as follows:

dissolving betulin or its derivative and benzoic acid derivative in solvent, refluxing at 40-130 deg.C for 8-12 hr in the presence of catalyst, and recovering and purifying to obtain hydrophilic modified product.

Preferably, the solvent is pyridine and/or dichloromethane;

the catalyst is as follows: dicyclohexylcarbodiimide, N, N' -diisopropylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 1, 8-diazabicycloundecen-7-ene, N, N-diisopropylethylamine, 4-dimethylaminopyridine.

Preferably, the molar ratio of betulin or betulin derivative and benzoic acid derivative is 1: 1; the mass ratio of the betulin or the derivative thereof to the solvent is 2.5-6.8g:120-220 ml; the molar ratio of the betulin or the derivative thereof to the catalyst is 1:1-1: 5.

11. The hydrophilicity modification method according to claim 10, wherein the betulin derivative is a compound represented by formula 5, the benzoic acid derivative is phthalic anhydride, and the esterification reaction conditions are as follows:

dissolving the compound shown in the formula 5, 4-dimethylamino pyridine and phthalic anhydride in pyridine, performing reflux reaction at the temperature of 100 ℃ and 140 ℃ for 8-12 hours, and recovering and purifying to obtain the compound shown in the formula 8, wherein the reaction formula is as follows:

preferably, the ratio of the mass sum of the reaction raw materials to the volume of the pyridine is 5-6.8g: 120-180 ml; and the molar ratio of the compound represented by formula 5 to 4-dimethylaminopyridine is 1:1-1: 5;

preferably, the ratio of the mass sum of the reaction raw materials to the volume of the solvent pyridine is 5.35 g: 150 ml;

preferably, the recovery and purification steps are as follows:

after the reaction is finished, cooling, adding a large amount of water, and extracting for 2-4 times by using dichloromethane; washing with 10% hydrochloric acid for 2-4 times, washing with saturated salt water for 1-3 times, and drying with anhydrous sodium sulfate; separating by column chromatography to obtain the compound shown in formula 8.

12. The hydrophilic modification method according to claim 10, wherein the betulin derivative is a compound represented by formula 5, the benzoic acid derivative is pyromellitic dianhydride, and the esterification reaction conditions are as follows:

dissolving the compound shown in the formula 5 in dichloromethane, adding pyromellitic dianhydride and N, N-diisopropylethylamine, carrying out reflux reaction at 40-60 ℃ for 8-12 hours, and recovering and purifying to obtain the compound shown in the formula 12, wherein the reaction formula is as follows:

the molar ratio of the compound shown in the reaction raw material formula 5 to the pyromellitic dianhydride is 1:1, and the volume ratio of the mass sum of the reaction raw materials to the solvent dichloromethane is 2.5-3.5 g: 180-220 ml; the mass-volume ratio of the compound shown in the formula 5 to the N, N-diisopropylethylamine is 1.8-2.2g:1.4-18 ml;

preferably, the ratio of the sum of the masses of the reaction raw materials to the volume of the solvent dichloromethane is 2.99 g: 200 ml; and the mass-to-volume ratio of the compound shown in the formula 5 to the N, N-diisopropylethylamine is 2g: 1.6 ml.

Preferably, the recovery and purification steps are as follows:

adding the reflux recovered substance into 1mol/L diluted hydrochloric acid with the same volume as the dichloromethane, stirring until turbid precipitate appears, performing suction filtration, and washing with water; dissolving the filter cake into an acetic acid solution with a half volume of the dichloromethane, heating to 80-120 ℃, adding water with the same volume as the dichloromethane, cooling, performing suction filtration, washing with a 50% acetic acid aqueous solution, methanol and dichloromethane respectively, and drying to obtain the compound shown in the formula 12.

Technical Field

The invention relates to a chemical pharmaceutical method, in particular to a betulin derivative and a hydrophilic modification product thereof, a nano solution and a preparation method.

Background

Betulin (formula 1), also called betulin, betulin and betulin, is a rich and naturally occurring triterpene, and widely exists in birch, pomegranate bark and leaf, spine date seed, asparagus and other plants. It has antiviral, antiinflammatory, protein dissolution inhibiting, hair growth promoting, and damaged hair luster improving effects.

However, due to the existence of pentacyclic triterpene structure in the molecule, the water solubility of betulin is generally poor, so that the betulin is difficult to absorb by organisms and influences the exertion of the biological activity, and the application of the betulin is limited.

Disclosure of Invention

Based on the needs in the field, the invention provides a betulin derivative which has good hydrophilicity and is easy to prepare into a nano solution, a hydrophilic modification and a preparation method thereof: .

The technical scheme of the invention is as follows:

betulin derivatives represented by formulae 3 to 7:

wherein R is selected from C1-6 alkyl, aryl, heteroaryl, aryl-C1-2 alkyl, heteroaryl-C1-2 alkyl, C3-10 cycloalkyl, C3-10 cycloalkyl-C1-2 alkyl, ethynyl, amino or vinyl, each of which is optionally substituted with 1-3R_aSubstituted by groups; and each R_aThe groups are independently halogen, C1-6 alkyl, fluoro-substituted C1-6 alkyl, fluoro-substituted C1-6 alkoxy, aryl, heteroaryl, C1-6 alkoxy, -CN, -NO₂、-OH、＝O、NH₂-C (O) -O-C1-6 alkyl or-SiMe 3, wherein R_aFurther optionally substituted by 1-3R_bIs substituted by radicals in which each R is_bThe groups are independently halogen, C1-6 alkyl, C1-6 alkoxy, -CN, -NO₂or-OH;

the benzoic acid derivative is selected from: ortho-phthalic acid, meta-phthalic acid, para-phthalic acid, phthalic anhydride, 1,2, 4-benzenetricarboxylic acid, 1,3, 5-benzenetricarboxylic acid, benzenetetracarboxylic acid, pyromellitic anhydride, benzenepentacarboxylic acid, benzenehexacarboxylic anhydride.

Preferably, wherein R isOr phenyl;

the structure of the betulin derivative shown in formula 3 is shown as formula 3a,

the betulin derivative represented by formula 4 is represented by formula 4a,

formula 7 shows betulin derivatives such as formula 7a,

a method for synthesizing betulin derivatives represented by the formula 3a, which comprises the steps of,

adding dichloromethane and betulinic acid into a flask, adding 2-bromoethyl methyl ether and 1, 8-diazabicycloundec-7-ene after the betulinic acid is dissolved, stirring and reacting for 2 days at room temperature, and separating by silica gel column chromatography with toluene as a developing agent to obtain the product shown in formula 3 a.

A method for synthesizing betulin derivatives represented by the formula 4a, which comprises the steps of,

adding dichloromethane and betulinic acid into a flask, adding 2-methoxyethylamine and 4-dimethylaminopyridine after the betulinic acid is dissolved, stirring and reacting for 8-12h at 40 ℃, and separating by silica gel column chromatography with toluene as a developing agent to obtain a product shown in formula 4 a.

A method for synthesizing betulin derivatives represented by the formula 5, which comprises the steps of,

in a reaction flask, white birch alcohol is dissolved in dichloromethane, bismuth trifluoromethanesulfonate (Bi (OTf))₃) Refluxing and reacting for 4 hours at 50 ℃; after the reaction is finished, cooling, washing for 3 times by using saturated sodium bicarbonate, washing for one time by using saturated sodium chloride, drying by using anhydrous sodium sulfate, and performing rotary evaporation to obtain a product shown in a formula 5;

a method for synthesizing betulin derivatives represented by the following formula 6, which comprises the steps of:

adding dichloromethane into a flask, then adding betulinic acid and bismuth trifluoromethanesulfonate, heating and refluxing, and stirring for 2 hours; cooling the reaction system to room temperature, adding saturated sodium bicarbonate solution for washing, and drying an organic phase by using anhydrous sodium sulfate; then filtering and distilling under reduced pressure, wherein the volume ratio of the mixture is 1: 4, separating by silica gel column chromatography by using petroleum ether dichloromethane mixed solution as a developing agent to obtain a product.

A method for synthesizing betulin derivatives represented by the following formula 7, which comprises the steps of: adding pyridine and betulin into a flask, adding benzoic anhydride and 4-Dimethylaminopyridine (DMAP) after the betulinic acid is dissolved, stirring at 60 ℃ for reacting for 2-4h, and washing with water and dichloromethane respectively to obtain a product such as 7 a.

A hydrophilic modification product of betulin and its derivatives is characterized in that betulin or its derivatives and a carboxyl group in a benzoic acid derivative molecule are subjected to esterification reaction on a 3-position hydroxyl group of the molecule to obtain a product;

the betulin derivative is selected from compounds represented by formula 2 to formula 7:

the hydrophilic modification product obtained by the invention has hydrophilic groups and hydrophobic groups in molecules, has good dispersibility,

is beneficial to preparing liquid preparation with good dispersibility and higher antiviral bioactivity.

Preferably, the benzoic acid derivative is phthalic anhydride,

the hydrophilic modification product of formula 5 is shown as formula 8,

the hydrophilic modification product of formula 3a is shown as formula 9,

the hydrophilic modification product of formula 4a is shown as formula 10,

the hydrophilic modification product of formula 7a is shown in formula 11,

the benzoic acid derivative is pyromellitic dianhydride, the modified product of the formula V is shown as the formula 12,

the use of the betulin derivative and/or the hydrophilic modification product thereof for the production of an antiviral agent is characterized in that the betulin derivative and/or the hydrophilic modification product thereof is used as an active ingredient of an antiviral agent.

Preferably, the hydrophilic modification product of the betulin derivative is used as the active ingredient of the antiviral medicament, and the medicament is a nano solution with the particle size of 150-400 nm.

A method for preparing antiviral agent comprises using hydrophilic modification product of betulin derivative as active ingredient of antiviral agent, and preparing into nanometer solution by the following steps:

dispersing the hydrophilic modification product of the betulin derivative in water, adding 1-10 times of equivalent of metal hydroxide salt, stirring at 30-90 deg.C for 30-300 min, naturally cooling to room temperature, adding 0.1-10% surfactant of reaction system volume, treating with ultrasonic wave for 0-60min to obtain nanometer colloid water solution,

the surfactant is selected from: polyethylene glycol, alkyl glucoside, fatty glyceride, sorbitan fatty acid, polysorbate, stearic acid, sodium dodecyl benzene sulfonate and lecithin.

Preferably, the metal hydroxide salt refers to sodium hydroxide or potassium hydroxide.

The hydrophilic modified betulin or the derivative thereof designed and synthesized based on the invention has both hydrophilic groups and hydrophobic groups in the molecule, so that the antiviral solution with ideal particle size range, good dispersibility and remarkably improved antiviral activity can be obtained by simple ultrasonic treatment or stirring treatment without adding complex high-molecular auxiliary agents and only by adding metal hydroxide salt and surfactant.

In another aspect of the present invention, there is provided a method for hydrophilic modification of betulin and derivatives thereof, comprising the steps of subjecting betulin or derivatives thereof to an esterification reaction with a carboxyl group of a benzoic acid derivative at the 3-position hydroxyl group thereof;

the betulin derivative is selected from compounds shown in formulas 2-7.

Preferably, the esterification reaction conditions are:

dissolving betulin or its derivative and benzoic acid derivative in solvent, refluxing at 40-130 deg.C for 8-12 hr in the presence of catalyst, and recovering and purifying to obtain hydrophilic modified product.

Preferably, the solvent is pyridine and/or dichloromethane;

the catalyst is as follows: dicyclohexylcarbodiimide, N, N' -diisopropylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 1, 8-diazabicycloundecen-7-ene, N, N-diisopropylethylamine, 4-dimethylaminopyridine.

Preferably, the molar ratio of betulin or betulin derivative and benzoic acid derivative is 1: 1; the mass ratio of the betulin or the derivative thereof to the solvent is 2.5-6.8g:120-220 ml; the molar ratio of the betulin or the derivative thereof to the catalyst is 1:1-1: 5.

In some embodiments, the betulin derivative is a compound represented by formula 5, the benzoic acid derivative is phthalic anhydride, and the esterification reaction conditions are as follows:

dissolving the compound shown in the formula 5, 4-dimethylamino pyridine and phthalic anhydride in pyridine, performing reflux reaction at the temperature of 100 ℃ and 140 ℃ for 8-12 hours, and recovering and purifying to obtain the compound shown in the formula 8, wherein the reaction formula is as follows:

preferably, the ratio of the mass sum of the reaction raw materials to the volume of the pyridine is 5-6.8g: 120-180 ml; and the molar ratio of the compound represented by formula 5 to 4-dimethylaminopyridine is 1:1-1: 5;

preferably, the ratio of the mass sum of the reaction raw materials to the volume of the solvent pyridine is 5.35 g: 150 ml;

preferably, the recovery and purification steps are as follows:

after the reaction is finished, cooling, adding a large amount of water, and extracting for 2-4 times by using dichloromethane; washing with 10% hydrochloric acid for 2-4 times, washing with saturated salt water for 1-3 times, and drying with anhydrous sodium sulfate; separating by column chromatography to obtain the compound shown in formula 8.

In some embodiments, the betulin derivative is a compound represented by formula 5, the benzoic acid derivative is pyromellitic dianhydride, and the esterification reaction conditions are as follows:

dissolving the compound shown in the formula 5 in dichloromethane, adding pyromellitic dianhydride and N, N-diisopropylethylamine, carrying out reflux reaction at 40-60 ℃ for 8-12 hours, and recovering and purifying to obtain the compound shown in the formula 12, wherein the reaction formula is as follows:

the molar ratio of the compound shown in the reaction raw material formula 5 to the pyromellitic dianhydride is 1:1, and the volume ratio of the mass sum of the reaction raw materials to the solvent dichloromethane is 2.5-3.5 g: 180-220 ml; the mass-volume ratio of the compound shown in the formula 5 to the N, N-diisopropylethylamine is 1.8-2.2g:1.4-18 ml;

preferably, the ratio of the sum of the masses of the reaction raw materials to the volume of the solvent dichloromethane is 2.99 g: 200 ml; and the mass-to-volume ratio of the compound shown in the formula 5 to the N, N-diisopropylethylamine is 2g: 1.6 ml.

Preferably, the recovery and purification steps are as follows:

adding the reflux recovered substance into 1mol/L diluted hydrochloric acid with the same volume as the dichloromethane, stirring until turbid precipitate appears, performing suction filtration, and washing with water; dissolving the filter cake into an acetic acid solution with a half volume of the dichloromethane, heating to 80-120 ℃, adding water with the same volume as the dichloromethane, cooling, performing suction filtration, washing with a 50% acetic acid aqueous solution, methanol and dichloromethane respectively, and drying to obtain the compound shown in the formula 12.

Experimental data show that the water solubility and dispersibility of the modified product obtained by the invention are greatly improved, and the antiviral effect is obviously improved.

Drawings

Fig. 1 shows, by way of example: the invention obtains the test result of the laser nanometer particle size analyzer of the nanometer solution of the modified product (the compound shown in the formula 8);

fig. 2 shows, by way of example: the invention obtains the test result of the laser nanometer particle size analyzer of the nanometer solution of the modified product (the compound shown in the formula 12).

Detailed Description

The following are merely exemplary embodiments and are not to be construed as limiting the invention. From the present disclosure, those skilled in the art can, in light of the following examples, derive other embodiments within the scope of the invention as claimed.

Experimental reagent:

betulin represented by formula 1, purchased from "alatin";

formula 2 is betulinic acid, available from "alatin";

other chemical agents are commercially available unless otherwise specified.