CN111825738A - 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 PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
- C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/06—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings
- A01N43/12—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings condensed with a carbocyclic ring
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N45/00—Biocides, pest repellants or attractants, or plant growth regulators, containing compounds having three or more carbocyclic rings condensed among themselves, at least one ring not being a six-membered ring
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
- C07J71/0005—Oxygen-containing hetero ring
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
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 1-3RaSubstituted by groups; and each RaThe 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 RaFurther optionally substituted by 1-3RbIs substituted by radicals in which each R isbThe 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 is
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,
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";
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
3a structural formula is
The synthesis steps are as follows:
dichloromethane (CH) was added to a 50ml flask2Cl210ml) and betulinic acid (90mg, 0.2mmol) were added after the betulinic acid had dissolved 2-bromoethyl methyl ether (0.2ml,2.0mmol) and 1, 8-diazabicycloundecen-7-ene (0.4 mmol).
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，ppm)：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, formula 4a
R is a group
4a structural formula is:
dichloromethane (CH) was added to a 50ml flask2Cl210ml) and betulinic acid (90mg, 0.2mmol), 2-methoxyethylamine (0.04ml,0.4mmol) and 4-dimethylaminopyridine (48mg, 0.4mmol) were added after the betulinic acid was dissolved; stirring and reacting for 12h at 40 ℃, and adding tolueneThe product was isolated by silica gel column chromatography as a developing solvent in 90% yield.
Nuclear magnetic hydrogen spectrum:
1H NMR(CDCl3，ppm)：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 betulin 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:
nuclear magnetic hydrogen spectrum detection: (apparatus Bruker 400 MHz; solvent CDCl3；Me4Si)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 flask2Cl210ml) followed by betulinic acid (45.6mg, 0.10mmol) and bismuth triflate (3.28mg, 0.005 mmol).
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 ℃): the product is obtained by silica gel column chromatography with dichloromethane of 1: 4 (volume ratio) as a developing agent. M/z 457.6(M + H) +. 1H NMR (CDCl)3，ppm)：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
Pyridine (10mL) and betulin (90mg, 0.2mmol) were added to a 50mL flask, and benzoic anhydride (0.43mL,2.26mmol) and DMAP (0.138g,1.13mmol) were added after 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，ppm)：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 (1.4g) and phthalic anhydride (3.35g) were dissolved in 150mL of pyridine, and the mixture was refluxed at 120 ℃ for 10 hours,
the reaction formula is as follows:
and (3) recovery and purification: after the reaction is finished, cooling, adding a large amount of water, and extracting with dichloromethane for three times; washing with 10% hydrochloric acid three times, washing with saturated brine twice, drying over anhydrous sodium sulfate, and 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 400 MHz; solvent CDCl)3；Me4Si)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 charged with 2g of the crude product of formula 5 (4.52mmol) dissolved in 200mL of dichloromethane, and 0.99g of pyromellitic dianhydride (4.52mmol) and 1.6mL of N, N-diisopropylethylamine were added and reacted at 50 ℃ under reflux for 8 hours.
The reaction formula is as follows:
and (3) recovery and purification: the reflux recovered substance is added into 200mL of 1mol/L diluted hydrochloric acid, stirred until turbid precipitate appears, filtered by suction and washed by 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 400 MHz; solvent CDCl 3; Me4Si)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 a hydrophilic modification product, namely 20mg of a compound shown as a formula compound 8 and 60mg of KOH, adding the hydrophilic modification product into a round-bottom flask, then accurately adding 10ml of pure water, heating and stirring at 55 ℃, cooling to room temperature, transferring into a sealed bottle, adding 2ml of span, performing ultrasonic treatment for 15min to obtain a transparent and clear solution, testing by using a laser nanometer particle size analyzer, and obtaining a nanometer solution of the betulin derivative (shown as a formula 8) with very good dispersibility, wherein the particle size range is about 360nm as shown in figure 1;
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 a hydrophilic modification product, namely 20mg of a compound shown as a formula compound 12 and 20mg of KOH, adding the hydrophilic modification product into a round-bottom flask, then accurately adding 10ml of pure water, heating and stirring at 55 ℃, cooling to room temperature, transferring into a sealed bottle, adding 2ml of span, and carrying out ultrasonic treatment for 15min to obtain a transparent and clear solution, wherein the particle size range is about 230nm by using a laser nanometer particle sizer, and as shown in figure 2, the nanometer solution of the betulin derivative 9 is obtained.
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 in the formula 3, 4 and 7 is subjected to the hydrophilic modification product compound shown in the formula 9, 10 and 11 obtained by the method, and the clear and transparent nano solution is obtained through the nano solution preparation step, and the particle size test shows that the particle size is between 150-400 nm;
the unmodified compound of formula 3, 4, 7, as a control, after treatment with the nanosol preparation procedure, gave only a white precipitate.
Example 6 preparation of Metal salts of Betulol derivatives
Accurately weighing hydrophilic modification product, such as compound 6.5g shown in formula 12, and KOH 1.3g, adding into a round-bottom flask, accurately adding pure water 100ml, stirring at 95 deg.C for 1.5h, and cooling to room temperature.
Adding 500ml alcohol into the system, stirring, standing to precipitate white precipitate, filtering, and drying to obtain potassium salt of compound 12, and labeling as 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 products, such as 1.5g of the compound of formula 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 nano preparation is slowly added into water with the volume of 100-.
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
The disease index [ Σ (number of diseased leaves at each stage × number of relative stages)/(total number of examined leaves × 9) ] × 100%.
c. Controlling effect
The result of the statistics of the preventing and treating effect (%) of [ (control disease index-treating disease index)/control disease index ] × 100% on the CMV-preventing and treating effect of the hydrophilization modified products 9, 10 and 11 in the table 1
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
TABLE 3 statistics of CMV controlling effect of the compound of formula 12
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
The disease index [ Σ (number of diseased leaves at each stage × number of relative stages)/(total number of examined leaves × 9) ] × 100%.
c. Controlling effect
The control effect (%) is [ (control disease index-treatment disease index)/control disease index ] × 100%
TABLE 4 statistical results of TBSV control effects of compounds represented by formula 8
TABLE 5 statistical results of TBSV control effects of the compounds represented by formula 12
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 (12)
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 1-3RaSubstituted by groups; and each RaThe 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 RaFurther optionally substituted by 1-3RbIs substituted by radicals in which each R isbThe 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 is
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,
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.
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