CN111825738B - Betulin derivative and hydrophilic modification product thereof, nano solution and preparation method thereof - Google Patents

Betulin derivative and hydrophilic modification product thereof, nano solution and preparation method thereof Download PDF

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CN111825738B
CN111825738B CN202010303555.9A CN202010303555A CN111825738B CN 111825738 B CN111825738 B CN 111825738B CN 202010303555 A CN202010303555 A CN 202010303555A CN 111825738 B CN111825738 B CN 111825738B
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betulin
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hydrophilic modification
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CN111825738A (en
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朱水芳
田志清
王晨光
姜帆
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China Inspection Science And Technology Beijing Group Co ltd
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Abstract

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.

Description

Betulin derivative and hydrophilic modification product thereof, nano solution and preparation method thereof
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.
Figure BDA0002454933280000011
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:
Figure BDA0002454933280000012
Figure BDA0002454933280000021
wherein R is selected from C1-6 alkyl, aryl, heteroaryl, aryl-C1-2 alkyl, heteroaryl-C1-2 alkyl, C3-10 cycloalkyl-C1-2 alkyl, ethynyl, amino or ethenyl, each of which is optionally substituted with 1-3R a Substituted by groups; and each R a The groups are independently halogen, C1-6 alkyl, fluoro-substituted C1-6 alkoxy, aryl, heteroaryl, C1-6 alkoxy, -CN, -NO 2 、-OH、＝O、NH 2 -C (O) -O-C1-6 alkyl or-SiMe 3, wherein R a Further optionally substituted by 1-3R b Is substituted with radicals wherein each R b The radicals are independently halogen, C1-6 alkyl, C1-6 alkoxy, -CN, -NO 2 or-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 is
Figure BDA0002454933280000022
Or 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,
Figure BDA0002454933280000023
Figure BDA0002454933280000031
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 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 shown in 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 was dissolved in dichloromethane, bismuth trifluoromethanesulfonate (Bi (OTf) was added 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 7a.
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:
Figure BDA0002454933280000041
the hydrophilic modification product obtained by the invention has both hydrophilic groups and hydrophobic groups in molecules, has good dispersibility,
is beneficial to preparing a liquid preparation with good dispersibility and higher antiviral biological activity.
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,
Figure BDA0002454933280000051
the benzoic acid derivative is pyromellitic dianhydride, the modified product of the formula V is shown as the formula 12,
Figure BDA0002454933280000052
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.
The hydrophilic modification product of betulin derivative is preferably used as active ingredient of the antiviral agent in the form of nano solution with particle diameter 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-diazabicycloundec-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; 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 100-140 ℃ for 8-12 hours, and recovering and purifying to obtain the compound shown in the formula 8, wherein the reaction formula is as follows:
Figure RE-GDA0002675542190000071
preferably, the ratio of the mass sum of the reaction raw materials to the volume of the pyridine is 5-6.8g:120-180ml; 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 5.35g:150ml;
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 saline 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:
Figure BDA0002454933280000072
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 2.5-3.5g:180 to 220ml; and the mass-to-volume ratio of the compound shown in the formula 5 to N, N-diisopropylethylamine is 1.8-2.2g;
preferably, the ratio of the sum of the masses of the reaction raw materials to the volume of the solvent dichloromethane is 2.99g:200ml; and the mass-to-volume ratio of the compound shown in the formula 5 to the N, N-diisopropylethylamine is 2g:1.6ml.
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.
Example 1 preparation of betulin derivatives represented by the formulae 3 to 7
(1) Exemplary derivatives of formula 3, such as formula 3a,
r is a group
Figure BDA0002454933280000081
3a structural formula is
Figure BDA0002454933280000082
The synthesis steps are as follows:
dichloromethane (CH) was added to a 50ml flask 2 Cl 2 10 ml) and betulinic acid (90mg, 0.2mmol), after betulinic acid had dissolved, 2-bromoethyl methyl ether (0.2ml, 2.0mmol) and 1,8-diazabicycloundec-7-ene (0.4 mmol) were added.
After stirring the reaction mixture at room temperature for 2 days, the product was isolated by silica gel column chromatography using toluene as a developing agent, and the yield was 70%.
And (3) detecting a nuclear magnetic hydrogen spectrum result: 1H NMR (CDCl) 3 ，δppm)：0.76(3H,s),0.84(3H,s),0.87(3H, s),0.91(3H,s),0.95(3H,s),0.97(3H,s),1.03(3H,s),3.20(1H,dd),3.38(3H,s), 3.60(2H,t),3.93(1H,s)，4.40(2H,ddt),4.60(1H,s)4.75(1H,d)。m/z＝515.4(M+H) + 。
(2) Exemplary derivatives of formula 4, such as formula 4a
R is a group
Figure BDA0002454933280000091
4a has the structural formula:
Figure BDA0002454933280000092
dichloromethane (CH) was added to a 50ml flask 2 Cl 2 10 ml) and betulinic acid (90mg, 0.2mmol), after the betulinic acid is dissolved, 2-methoxyethylamine (0.04ml, 0.4 mmol) and 4-dimethylaminopyridine (48mg, 0.4 mmol) are added; after stirring and reacting for 12h at the temperature of 40 ℃, the product is obtained by silica gel column chromatography with toluene as a developing agent, and the yield is 90%.
Nuclear magnetic hydrogen spectrum:
1H NMR(CDCl 3 ，δppm)：0.76(3H,s),0.84(3H,s),0.87(3H,s),0.91(3H,s), 0.95(3H,s),0.97(3H,s),1.03(3H,s),3.20(1H,dd),3.38(3H,s),3.42(2H,t),3.93 (1H,s)，4.40(2H,ddt),4.60(1H,s),4.75(1H,d)。m/z＝514.4(M+H) + 。
(3) Synthesis of derivative represented by formula 5
The synthesis steps are as follows:
a500 mL reaction flask was taken, 5g of birch alcohol was dissolved in 300mL of dichloromethane, and bismuth trifluoromethanesulfonate (Bi (OTf) 3 ) 200mg, refluxing and reacting at 50 ℃ for 4 hours;
after the reaction is finished, cooling, washing 3 times by saturated sodium bicarbonate, washing once by saturated sodium chloride, drying by anhydrous sodium sulfate, and performing rotary evaporation to obtain a crude product shown in a formula 5, wherein the reaction formula is as follows:
Figure BDA0002454933280000101
nuclear magnetic hydrogen spectrum detection: (apparatus Bruker 400MHz; solvent CDCl 3 ；Me 4 Si)0.76(3H,s),0.80(3H, s),0.85(3H,s),0.91(3H,s),0.93(3H,s),0.97(6H,s),3.20(1H),3.44(1 H,d),3.53(1H,s),3.78(1H,dd)；m/z 442(M+H) + (Agilent 6520)
(4) Synthesis of derivative represented by formula 6
The synthesis method comprises the following steps:
dichloromethane (CH) was added to a 50ml flask 2 Cl 2 10 ml), followed by the addition of betulinic acid (45.6mg, 0.10mmol) and bismuth trifluoromethanesulfonate (3.28mg, 0.005mmol).
The reaction mixture was refluxed under heating and stirred for 2 hours, and after the reaction mixture was cooled to room temperature, 20ml of a saturated sodium bicarbonate solution was added and washed, and the organic phase was dried over anhydrous sodium sulfate. Then filtered and distilled under reduced pressure, with petroleum ether (40-60 ℃): dichloromethane = 1:4 (volume ratio) as a developing solvent and the product is obtained by silica gel column chromatography separation. M/z =457.6 (M + H) +.1H NMR (CDCl) 3 ，δppm)： 0.76(3H,s),0.84(3H,s),0.87(3H,s),0.91(3H,s),0.95(3H,s),0.97(3H,s), 1.03(3H,s),3.20(1H,dd),3.93(1H,s).
(5) Synthesis of exemplary derivative 7a of formula 7
R group is phenyl, and the structural formula of 7a is
Figure BDA0002454933280000102
Pyridine (10 ml) and betulin (90mg, 0.2mmol) were charged into a 50ml flask, and benzoic anhydride (0.43mL, 2.26mmol) and DMAP (0.138g, 1.13mmol) were added after the betulinic acid was dissolved. After stirring and reacting for 3h at the temperature of 60 ℃, washing with water and dichloromethane respectively to obtain the product with the yield of 90%.
And (3) product detection: 1H NMR (CDCl) 3 ，δppm)：0.76(3H,s),0.84(3H,s),0.87(3H,s),0.91 (3H,s),0.95(3H,s),0.97(3H,s),1.03(3H,s),3.20(1H,dd),3.38(3H,s),3.60(2H, t),3.93(1H,s)，4.40(2H,ddt),4.60(1H,s),4.75(1H,d),7.49-7.43(2H,m),7.60 -7.55(1H,m),8.09-8.05(2H,m)。m/z＝547.4(M+H) + 。
Example 2 hydrophilization modification of betulin derivatives
Esterification reaction in a 500mL reaction flask, 2g of the crude product of formula 5, 4-dimethylaminopyridine (1.4 g) and phthalic anhydride (3.35 g) were dissolved in 150mL of pyridine, and the mixture was refluxed at 120 ℃ for 10 hours,
the reaction formula is as follows:
Figure BDA0002454933280000111
and (3) recovery and purification: after the reaction is finished, cooling, adding a large amount of water, and extracting with dichloromethane for three times; three times with 10% hydrochloric acid, two times with saturated brine, drying over anhydrous sodium sulfate, column chromatography (dichloromethane: methanol =40: 1) to obtain the compound represented by formula 8 in 85% yield according to the molar ratio of the product to the starting betulin.
Nuclear magnetic hydrogen Spectroscopy (apparatus Bruker 400MHz; solvent CDCl) 3 ；Me 4 Si)0.76(3H,s),0.84(3H,s),0.85 (3H,s),0.91(3H,s),0.95(3H,s),0.97(6H,s),3.20(1H),3.44(1H,d), 3.53(1H,s),3.78(1H,dd),7.49–7.61(2H,m),7.65–7.74(1H,m),7.83–7.93 (1H,m).m/z＝589.4(M+H) + 。
Example 3 hydrophilization modification product of betulin derivative
Esterification reaction: a500 mL reaction flask was taken, 2g of the crude product represented by formula 5 (4.52 mmol) was dissolved in 200mL of methylene chloride, and 0.99g of pyromellitic dianhydride (4.52 mmol) and 1.6mL of N, N-diisopropylethylamine were added and reacted at 50 ℃ under reflux for 8 hours.
The reaction formula is as follows:
Figure BDA0002454933280000121
and (3) recovery and purification: adding the reflux recovered substance into 200mL of 1mol/L diluted hydrochloric acid, stirring until turbid precipitate appears, performing suction filtration, and washing with water. The filter cake was dissolved in 100mL of acetic acid solution, heated to 100 ℃ and 200mL of water was added.
Cooling, vacuum filtering, washing with 50% acetic acid water solution, methanol and dichloromethane respectively, and drying to obtain compound 12 with yield of 85% according to molar ratio of product to initial betulin.
Nuclear magnetic Hydrogen Spectroscopy (apparatus Bruker 400MHz; solvent CDCl3; me4 Si) 0.76 (3H, s), 0.84 (3H, s), 0.85 (3H, s), 0.91 (3H, s), 0.95 (3H, s), 0.97 (6H, s), 3.32 (1H), 3.44 (1H, d), 3.65 (1H, s), 4.66 (1H, dd), 7.49-7.61 (2H, M), 7.88 (1H, s), 7.98 (1H, s), M/z =678.4 (M + H) + 。
Example 4 preparation of nano-solution of hydrophilic modification product of betulin and its derivatives
The preparation method of the nano solution comprises the following steps: accurately weighing hydrophilic modification products, such as a compound represented by a formula 8, 20mg and KOH 60mg, adding into a round-bottom flask, accurately adding pure water, 10ml, heating and stirring at 55 ℃ for 1h, cooling to room temperature, transferring into a sealed bottle, adding 2ml of span, performing ultrasound for 15min to obtain a transparent and clear solution, testing by using a laser nanometer particle size analyzer, wherein the particle size range is about 360nm as shown in figure 1, so that a nano solution of the betulin derivative (represented by the formula 8) is obtained, and the dispersibility of the nano solution is very good;
comparison: the derivative shown in the formula 5 (without hydrophilic modification) cannot form a transparent and clear nano solution after being treated by a nano solution preparation step, but generates a white precipitate.
Example 5 preparation of nano-solution of hydrophilic modification product of betulin and its derivatives
The preparation method of the nano solution comprises the following steps: accurately weighing hydrophilic modification products, such as 20mg of compound shown as formula 12 and 20mg of KOH, adding into a round-bottom flask, accurately adding 10ml of pure water, heating and stirring at 55 ℃ for 1h, cooling to room temperature, transferring into a sealed bottle, adding 2ml of span, performing ultrasound for 15min to obtain a transparent and clear solution, and measuring the particle size range to be about 230nm by using a laser nanometer particle size analyzer, as shown in figure 2, thus obtaining a nanometer solution of betulin derivative 9.
Comparison: the derivative shown in the formula 5 cannot form a transparent and clear nano solution after being treated by the nano solution preparation step, but generates a white precipitate.
The compound shown as the formula 3,4,7 is a hydrophilic modification product obtained by the method, namely the compound shown as the formula 9, 10 and 11, is subjected to the nano solution preparation step to obtain a clear and transparent nano solution, and the particle size test shows that the particle size is 150-400 nm;
an unmodified compound of formula 3,4,7 as a control, treated with the nanosolution preparation procedure, gave only a white precipitate.
Example 6 preparation of Metal salts of Betulol derivatives
Accurately weighing hydrophilic modification products, such as 6.5g of a compound shown as a compound 12, 1.3g of KOH, adding into a round-bottom flask, accurately adding 100ml of pure water, stirring at 95 ℃ for 1.5h, and cooling to room temperature.
Adding 500ml of alcohol into the system, stirring, standing to separate out a white precipitate, filtering and drying to obtain the potassium salt of the compound 12, wherein the label is 12-K.
Example 7 preparation of concentrated formulation of betulin derivative
For convenient transportation and use, the betulin derivative can be prepared into concentrated aqueous solution, and can be diluted with water and stirred to form nanometer solution. The preparation method comprises the following steps:
accurately weighing hydrophilic modification product, such as 1.5g of compound 9-K obtained in example 6, adding into a round-bottom flask, adding 100ml of pure water, heating and stirring at 55 ℃ for 1h, cooling to room temperature, transferring into a sealed bottle, and adding 2ml of Tween to obtain milky white solution, i.e. concentrated nanometer preparation.
When in use, the concentrated nanometer preparation is slowly added into water with the volume of 100-500, and the mixture is continuously stirred during the adding process, so that transparent and clear nanometer solution is obtained.
The nano solution is formed without a large amount of auxiliary agents and special equipment, and is convenient to use in various occasions and various crowds.
Experimental examples Effect of Water solubility improvement on antiviral Activity of Compounds
Selecting Benzen tobacco with similar growth vigor at 4-5 leaf stage, fully grinding 0.15g fresh diseased leaf with obvious symptom with 30mL double distilled water, inoculating 0.5% virus inoculation liquid by diatomite friction, and inoculating 2 leaves to each strain.
Treatment groups:
150ppm nano-solution and non-nano-solution treatment group, 300ppm nano-solution and non-nano-solution treatment group: spraying the mixture 4 days and 11 days after inoculation.
Prevention group:
150ppm nano solution and non-nano solution prevention group, 300ppm nano solution and non-nano solution prevention group: spraying the pesticide to plant, inoculating virus after 4 days, and spraying the pesticide for the second time after 7 days
20 strains were set for each concentration treatment group. The prevention and treatment effects are calculated at 7 th, 19 th and 15 th after inoculation, and the investigation results are shown in tables 1-4: a CMV disease grading standard:
grade 0-no symptoms.
Grade 1-mild symptoms appeared in inoculated leaves.
2-stage-one to two systematic leaves are clear and deformed.
Grade 3-most upper leaves are leafy, chlorosis or deformed.
Grade 4-leaf of the whole plant, severe deformation or necrosis, and severe dwarfing of diseased plants.
b. Index of disease condition
Disease index = [ Σ (number of diseased leaves at each stage × relative stage number)/(total investigated leaf number × 9) ] × 100%.
c. Controlling effect
Control effect (%) = [ (control disease index-treatment disease index)/control disease index ] × 100% CMV control effect statistics of hydrophilization modified products 9, 10, 11 in Table 1
Figure BDA0002454933280000141
It can be seen that the control effect of the hydrophilic modification products provided by the invention is obviously higher than that of betulin, and the hydrophilic modification products show the same similar results on controlling TBSV.
TABLE 2 statistics of CMV controlling effect of the compounds of formula 8
Figure BDA0002454933280000142
Figure BDA0002454933280000151
TABLE 3 statistics of CMV control Effect of Compounds of formula 12
Figure BDA0002454933280000152
TBSV disease grading standard:
grade 0-no symptoms.
Grade 1-mild symptoms appeared in inoculated leaves.
2-stage-one to two systematic leaves are clear and deformed.
Grade 3-most upper leaves are deformed or main side veins are necrotic, and diseased plants are dwarfed.
Grade 4-the whole plant is severely deformed or necrotic.
b. Index of disease condition
Disease index = [ ∑ (number of diseased leaves at each stage × relative stage value)/(total number of examined leaves × 9) ] × 100%.
c. Controlling effect
Control effect (%) = [ (control disease index-treatment disease index)/control disease index ] × 100%
TABLE 4 statistical results of TBSV control effects of compounds represented by formula 8
Figure BDA0002454933280000161
TABLE 5 statistical results of TBSV control effects of the compounds represented by formula 12
Figure BDA0002454933280000162
Exemplary experimental data of tables 1 to 5 show that the hydrophilic modification product of the hydrophilic modified betulin derivative according to the present invention has stronger antiviral activity against viruses;
moreover, the nano preparation obtained by the nano solution preparation process provided by the invention has obviously improved intracellular antiviral activity under the same concentration.

Claims (8)

1. A betulin derivative represented by formula 4 a:
Figure FDA0003989837490000011
2. 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
Figure FDA0003989837490000012
3. A hydrophilic modification product of betulin derivative, characterized in that it is a product obtained by esterification of the 3-hydroxy group of betulin derivative of claim 1 with a carboxyl group of phthalic anhydride, as shown in formula 10
Figure FDA0003989837490000013
4. Use of the hydrophilic modification product of a betulin derivative according to claim 3, in the preparation of an antiviral agent, wherein the hydrophilic modification product of a betulin derivative is used as an active ingredient of an antiviral agent.
5. The use according to claim 4, wherein the antiviral agent is in the form of a nanosolution having a particle size of 150-400 nm.
6. A method for preparing an antiviral agent, characterized in that the hydrophilic modification product of betulin derivative according to claim 3 is used as an active ingredient of an antiviral agent to prepare a nano solution by 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;
the metal hydroxide salt refers to sodium hydroxide or potassium hydroxide.
7. A method for producing a hydrophilic modification product of a betulin derivative according to claim 3, wherein the betulin derivative according to claim 1 is subjected to an esterification reaction at the hydroxyl group at the 3-position thereof with a carboxyl group of a phthalic anhydride molecule;
the esterification reaction conditions are as follows:
dissolving betulin or its derivatives and phthalic anhydride 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;
the solvent is pyridine and/or dichloromethane;
the catalyst is as follows: dicyclohexylcarbodiimide, N, N' -diisopropylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 1,8-diazabicycloundec-7-ene, N, N-diisopropylethylamine, 4-dimethylaminopyridine.
8. The hydrophilic modification method according to claim 7, wherein the molar ratio of betulin derivative to phthalic anhydride is 1:1; the mass ratio of the betulin derivative to the solvent is 2.5-6.8g; the molar ratio of the betulin derivative to the catalyst is 1:1-1:5.
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