(-)-gossypol derivative, preparation method thereof and antioxidant

Abstract

The invention discloses a (-)-gossypol derivative, a preparation method thereof and an antioxidant, and relates to the technical field of chemical synthesis, the (-)-gossypol derivative comprises a (-)-gossypol-polylysine derivative and / or a pharmaceutically acceptable salt thereof, the repetitive structural unit of the (-)-gossypol-polylysine derivative comprises any one of a structural formula (I) and a structural formula (II); wherein n is equal to 25 to 40. In the novel compound synthesized by the invention, an aldehyde group in the (-)-gossypol and an amino group in the polylysine form a Schiff base compound through an imine bond, an active aldehyde group in the (-)-gossypol is closed, and meanwhile, the (-)-gossypol-polylysine derivative has relatively strong antioxidant activity and is a relatively good antioxidant and drug source.

Description

Technical Field

[0001] The invention relates to the technical field of chemical synthesis, and in particular to a (-)-gossypol derivative and a preparation method and an antioxidant thereof. Background Art

[0002] Gossypol is a yellow polyphenolic hydroxybisnaphthaldehyde compound that is widely found in the roots, stems, leaves and seeds of the mallow plant cotton, with the highest content in cottonseed kernels.

[0003] There are two enantiomers of natural gossypol, namely left-handed gossypol [(-)-gossypol] and right-handed gossypol [(+)-gossypol], which exist in different proportions in the glands of the roots, stems, leaves and seeds of cotton plants. A large number of research results show that the biological activity of (-)-gossypol is higher than that of (+)-gossypol and racemic (±)-gossypol. At present, gossypol is widely used in agriculture and medicine.

[0004] However, the toxic side effects of cottonpol should not be underestimated. For monogastric animals, a single intake of a diet with too high a cottonpol content can induce various pathological changes and even lead to death. Summary of the invention

[0005] The purpose of the present invention is to provide a (-)-gossypol derivative and a preparation method and an antioxidant thereof, in order to solve the problem in the prior art that gossypol is too toxic and has limited application in the medical field.

[0006] To achieve the above object, the present invention provides a (-)-gossypol derivative, including a (-)-gossypol-polylysine derivative and / or a pharmaceutically acceptable salt thereof, wherein the repeating structural unit of the (-)-gossypol-polylysine derivative includes any one of the structural formulas (I) and (II); in formula (I) and (II), n=25-40,

[0007]

[0008] The present invention also provides a method for preparing a (-)-gossypol derivative, comprising the following steps:

[0009] S10, mixing (-)-gossypol with an organic solvent to obtain a first solution;

[0010] S20, obtaining a polylysine source solution with a pH of 3.0 to 6.0, which is the second solution;

[0011] S30, mixing the first solution and the second solution, performing an aldehyde-amine condensation reaction, and obtaining the (-)-gossypol derivative.

[0012] In one embodiment, in step S10, the organic solvent includes at least one of methanol and ethanol; and / or,

[0013] In step S20, the polylysine source includes at least one of α-polylysine, ε-polylysine, α-polylysine hydrochloride, α-polylysine sulfate, ε-polylysine hydrochloride and ε-polylysine sulfate; and / or,

[0014] In step S30, the molar ratio of (-)-gossypol in the first solution to the free amino groups in the polylysine source in the second solution is 1:(2-2.5); and / or,

[0015] In step S30, the temperature of the aldehyde-amine condensation reaction is 50-75° C.; and / or,

[0016] In step S30, the aldehyde-amine condensation reaction time is ≥ 3 hours; and / or,

[0017] Step S30 includes: mixing the first solution and the second solution, and performing an aldehyde-amine condensation reaction in a closed environment to obtain the (-)-gossypol derivative, wherein the closed environment includes an oxygen-free environment.

[0018] In one embodiment, step S30 includes: the polylysine source is polylysine, and step S20 includes: mixing polylysine with a solvent to obtain a polylysine source solution with a pH of 3 to 6, that is, the second solution, wherein the solvent includes water and an inorganic acid solution, or an organic acid solution and an alkaline substance.

[0019] In one embodiment, the solvent comprises water and an inorganic acid solution, the inorganic acid solution comprises an aqueous hydrochloric acid solution and / or an aqueous sulfuric acid solution, the mass concentration of hydrochloric acid in the aqueous hydrochloric acid solution is 1 to 37%, the mass concentration of sulfuric acid in the aqueous sulfuric acid solution is 1 to 60%, and the mass ratio of water to polylysine is 1:(0.05 to 0.5); or,

[0020] The solvent includes an organic acid solution and an alkaline substance, wherein the organic acid solution includes an aqueous citric acid solution and / or an acetic acid solution, and the mass concentration of the acid in the organic acid solution is 0.3-3.0%; the alkaline substance includes an alkaline solid and / or an alkaline solution, the alkaline solid includes a sodium hydroxide solid and / or a potassium hydroxide solid, the alkaline solution includes a sodium hydroxide solution and / or a potassium hydroxide solution, and the mass concentration of the alkali in the alkaline solution is 1-40%.

[0021] In one embodiment, step S30 includes:

[0022] The first solution and the second solution are mixed to carry out an aldehyde-amine condensation reaction, and after the reaction is completed, the solution is filtered, and the filter residue is washed and dried to obtain the (-)-gossypol derivative;

[0023] The washing comprises washing with ethanol and deionized water in sequence;

[0024] The drying temperature is 40-60°C.

[0025] In one embodiment, in step S10, obtaining (-)-gossypol comprises the following steps:

[0026] S01, mixing acetic gossypol, an amino compound and an organic solvent, adjusting the pH to 8.0-10.0, and performing an aldehyde-amine condensation reaction to obtain an intermediate;

[0027] S02. Under anaerobic conditions, the intermediate, an organic solvent and an acid are mixed to carry out an acidolysis reaction to obtain (-)-gossypol.

[0028] In one embodiment, the organic solvent comprises at least one of ether, methanol and ethanol; and / or,

[0029] In step S01, the amino compound includes L-tyrosine methyl ester, L-tyrosine methyl ester salt, L-tryptophan methyl ester, L-tryptophan methyl ester salt, L-phenylalanine methyl ester, L-phenylalanine methyl ester salt, (+)-p-nitrophenyl-2-amino-1,3-propanediol, (+)-p-nitrophenyl-2-amino-1,3-propanediol salt, (-)-p-nitrophenyl-2-amino-1,3-propanediol, (-)-p-nitrophenyl-2-amino-1,3-propanediol salt, (+)-2-amino-3-phenyl-1-propanol, (+)-2-amino-3-phenyl-1-propanol salt, (-)-2-amino- at least one of 3-phenyl-1-propanol salt, (+)-phenethylamine, (+)-phenethylamine salt, (-)-phenethylamine, (-)-phenethylamine salt, (+)-1-hydroxy-3-propylamine, (+)-1-hydroxy-3-propylamine salt, (-)-1-hydroxy-3-propylamine, (-)-1-hydroxy-3-propylamine salt, (+)-cyclopropylamine, (+)-cyclopropylamine salt, (-)-cyclopropylamine, (-)-cyclopropylamine salt, (+)-p-bromoaniline, (+)-p-bromoaniline salt, (-)-p-bromoaniline, (-)-p-bromoaniline salt, (+)-2-aminopropane, (+)-2-aminopropane salt, (-)-2-aminopropane, (-)-2-aminopropane salt; and / or,

[0030] In step S01, the temperature of the aldehyde-amine condensation reaction is 50-75° C.; and / or,

[0031] In step S01, the aldehyde-amine condensation reaction time is 3 to 14 hours; and / or,

[0032] In step S01, the molar ratio of the acetic acid gossypol to the amino group in the amino compound is 1:(2-2.5); and / or,

[0033] In step S02, the acid comprises a mixture of glacial acetic acid and a strong acid solution, the strong acid solution comprises a hydrochloric acid aqueous solution and / or a sulfuric acid aqueous solution, the mass concentration of the hydrochloric acid aqueous solution is ≥36%, the mass concentration of the sulfuric acid aqueous solution is 50% to 60%, the volume ratio of the glacial acetic acid to the strong acid solution is (1.5 to 2.5):1, and the volume ratio of the glacial acetic acid to the organic solvent is 1:(20 to 100); and / or,

[0034] In step S02, the acid hydrolysis reaction time is ≥ 2h.

[0035] In one embodiment, step S02 includes:

[0036] Under anaerobic conditions, the intermediate, an organic solvent and an acid are mixed to perform an acid hydrolysis reaction, after the acid hydrolysis reaction is completed, an organic phase and an aqueous phase are separated, the organic phase is washed to neutrality and water in the organic phase is removed, and crystallization is performed to obtain (-)-gossypol;

[0037] The method for removing moisture from the organic phase includes using anhydrous magnesium sulfate or anhydrous sodium sulfate for adsorption.

[0038] The present invention also provides an antioxidant, comprising the aforementioned (-)-gossypol derivative.

[0039] In the technical scheme of the present invention, a new compound is synthesized, in which (-)-gossypol forms a Schiff base compound with polylysine or a polylysine salt through an imine bond, so that the active aldehyde group in the (-)-gossypol is blocked by the imine bond, thereby reducing the toxicity of the (-)-gossypol. At the same time, the (-)-gossypol derivative also has strong antioxidant properties and is a good antioxidant and drug source. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0041] Figure 1 The infrared spectra of (-)-gossypol and gossypol acetate in Example 1 provided by the present invention are compared;

[0042] Figure 2 The infrared spectra of (-)-gossypol and (-)-gossypol-α-polylysine in Example 1 provided by the present invention are compared;

[0043] Figure 3 The infrared spectra of ε-polylysine, (-)-gossypol and (-)-gossypol-ε-polylysine derivatives provided in Example 4 of the present invention are compared;

[0044] Figure 4 The ABTS removal by acetic gossypol, (-)-gossypol, (-)-gossypol-α-polylysine derivative, (-)-gossypol-ε-polylysine derivative and ascorbic acid in Examples 1 and 4 provided by the present invention + ·Results of free radical activity test.

[0045] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0046] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0047] Gossypol is a yellow polyphenolic hydroxybisnaphthaldehyde compound that is widely found in the roots, stems, leaves and seeds of the mallow plant cotton, with the highest content in cottonseed kernels.

[0048] There are two enantiomers of natural gossypol, namely left-handed gossypol [(-)-gossypol] and right-handed gossypol [(+)-gossypol], which exist in different proportions in the glands of the roots, stems, leaves and seeds of cotton plants. A large number of research results show that the biological activity of (-)-gossypol is higher than that of (+)-gossypol and racemic (±)-gossypol. At present, gossypol is widely used in agriculture and medicine.

[0049] However, the toxic side effects of cottonpol should not be underestimated. For monogastric animals, a single intake of a diet with too high a cottonpol content can induce various pathological changes and even lead to death.

[0050] In view of this, the present invention provides a (-)-gossypol derivative, including a (-)-gossypol-polylysine derivative and / or a pharmaceutically acceptable salt thereof, wherein the repeating structural unit of the (-)-gossypol-polylysine derivative includes any one of the structural formulas (I) and (II); in formula (I) and (II), n=25-40,

[0051]

[0052] In the technical scheme of the present invention, a new compound is synthesized, in which (-)-gossypol forms a Schiff base compound with polylysine or polylysine salt through an imine bond, and the active aldehyde group in (-)-gossypol is blocked, thereby weakening the toxicity of (-)-gossypol, and the (-)-gossypol-polylysine derivative also has strong antioxidant properties, and is a good antioxidant and drug source.

[0053] The polylysine or polylysine salt is a polymer formed by connecting multiple lysine units through peptide bonds formed between the amino and carboxyl groups thereof, and the number of lysine units (or degree of polymerization) n in the polylysine or polylysine salt may range from 25 to 40. That is, its molecular weight is 3218 to 5138 Da, which means that it may contain 25 to 40 lysine units.

[0054] The sources of the polylysine include at least α-polylysine, ε-polylysine, α-polylysine hydrochloride, α-polylysine sulfate, ε-polylysine hydrochloride and ε-polylysine sulfate, the formula (I) represents the structure of a (-)-gossypol-α-polylysine derivative, and the formula (II) represents the structure of a (-)-gossypol-ε-polylysine derivative.

[0055] The present invention also provides a method for preparing a (-)-gossypol derivative, comprising the following steps:

[0056] S10, mixing (-)-gossypol with an organic solvent to obtain a first solution;

[0057] S20, obtaining a polylysine source solution with a pH of 3.0 to 6.0, which is the second solution;

[0058] S30, mixing the first solution and the second solution, performing an aldehyde-amine condensation reaction, and obtaining the (-)-gossypol derivative.

[0059] In the technical scheme of the present invention, (-)-gossypol has low solubility in water but high solubility in certain organic solvents, so (-)-gossypol is dissolved in an organic solvent to obtain a first solution; a polylysine source is easily soluble in water but difficult to dissolve in an organic solvent, so a water system is used to dissolve it, and the pH of the polylysine source solution is controlled to be within the range of 3.0 to 6.0 to increase the yield of the subsequent generation of (-)-gossypol-polylysine derivatives, and the pH may be 3.0, 3.5, 4, 4.5, 5, 5.5 or 6 to obtain a second solution; the first solution and the second solution are mixed and subjected to an aldehyde amine condensation reaction, so that the active aldehyde group of the (-)-gossypol in the first solution and the amino group of the polylysine source in the second solution undergo an aldehyde amine condensation reaction to obtain the (-)-gossypol derivative. It should be noted that since a lysine molecule has two amino groups, one amino group in the lysine molecule condenses with the carboxyl group in another lysine molecule to form a peptide bond, and the other amino group becomes a side chain free amino group. Therefore, each lysine unit can provide a free amino group that reacts with the aldehyde group of the (-)-gossypol molecule; and each molecule of (-)-gossypol has two active aldehyde groups. It can be seen that one (-)-gossypol molecule can undergo an aldehyde-amine condensation reaction with the side chain amino groups of two lysine units in polylysine to form a bis-Schiff base, thereby blocking the two active aldehyde groups in the (-)-gossypol molecule, thereby achieving the effect of weakening the toxicity of (-)-gossypol.

[0060] It should be noted that (-)-gossypol can be purchased or synthesized.

[0061] In some embodiments of the present invention, in step S10, the organic solvent includes at least one of methanol and ethanol. (-)-Gossypol has a high solubility in the above organic solvents.

[0062] In some embodiments of the present invention, in step S20, the polylysine source includes at least one of α-polylysine, ε-polylysine, α-polylysine hydrochloride, α-polylysine sulfate, ε-polylysine hydrochloride and ε-polylysine sulfate. α-Polylysine refers to polylysine connected by a peptide bond formed between the α-amino group and the α-carboxyl group of lysine, wherein the main chain part of lysine (including the α-amino group and the α-carboxyl group) participates in the formation of the peptide bond. ε-Polylysine refers to polylysine that forms a peptide bond between the ε-amino group of lysine and the α-carboxyl group of another lysine molecule. The above two polylysines have good antibacterial properties and are widely used in the fields of food industry, biomedicine, etc., thereby making the prepared (-)-cottonypol derivatives have prospects for application in the field of medicine.

[0063] In some embodiments of the present invention, in step S30, the molar ratio of the (-)-gossypol in the first solution to the free amino groups in the polylysine source in the second solution is 1:(2-2.5). The molar ratio of the (-)-gossypol to the amino groups in the polylysine in the above range can ensure that the active aldehyde groups in the (-)-gossypol are completely blocked, thereby achieving the effect of weakening the toxicity of the (-)-gossypol as much as possible.

[0064] In some embodiments of the present invention, in step S30, the temperature of the aldehyde-amine condensation reaction is 50-75° C. The temperature within the above range can ensure that the aldehyde-amine condensation reaction proceeds smoothly and improve the conversion rate of the reactants.

[0065] In some embodiments of the present invention, in step S30, the time of the aldehyde-amine condensation reaction is ≥ 3 hours. The time of the aldehyde-amine condensation reaction can be 3 hours, 6 hours, 9 hours, 10 hours or even longer. The time of the aldehyde-amine condensation reaction within the above range can ensure that the aldehyde-amine condensation reaction is more complete and improve the yield of the (-)-gossypol derivative.

[0066] In some embodiments of the present invention, step S30 includes: mixing the first solution with the second solution, and performing an aldehyde-amine condensation reaction in a closed container to obtain the (-)-gossypol derivative, wherein the closed environment includes an oxygen-free environment. Carrying out the reaction in a closed environment can prevent the solvent in the system from volatilizing during the reaction, especially the organic solvent in the first solution, so as to ensure that the aldehyde-amine condensation reaction is more complete; further, when the closed environment is an oxygen-free environment, that is, by introducing nitrogen, argon or other inert gas into the closed environment to completely drive out the oxygen in the container to form an oxygen-free environment, (-)-gossypol can be prevented from being oxidized during the reaction, thereby further improving the yield.

[0067] In some embodiments of the present invention, the polylysine source is polylysine, and step S20 includes: mixing polylysine with a solvent to obtain a polylysine source solution with a pH of 3 to 6, which is the second solution, wherein the solvent includes water and an inorganic acid solution, or an organic acid solution and an alkaline substance. That is, when the polylysine source is polylysine, the polylysine can be first dissolved with water, at which time the polylysine solution is alkaline, and then an inorganic acid solution is added to directly adjust its pH to 3.0 to 6.0, so as to facilitate the aldehyde-amine condensation reaction; or the polylysine can be first dissolved with an organic acid aqueous solution, and then an alkaline substance is added to adjust the system pH to 3.0 to 6.0. Among them, the organic acid solution can provide a relatively stable pH buffer environment for the aldehyde-amine condensation reaction.

[0068] In some embodiments of the present invention, the solvent includes water and an inorganic acid solution, the inorganic acid solution includes a hydrochloric acid aqueous solution and / or a sulfuric acid aqueous solution, the mass concentration of hydrochloric acid in the hydrochloric acid aqueous solution is 1 to 37%, the mass concentration of sulfuric acid in the sulfuric acid aqueous solution is 1 to 60%, and the mass ratio of water to polylysine is 1:(0.05 to 0.5). Within the above range, the pH of the polylysine source solution can be well adjusted to 3.0 to 6.0, and the aldehyde amine condensation reaction is conducive to proceed; in addition, the mass ratio of water to polylysine can be 1:0.05, 1:0.1, 1:0.2, 1:0.3, 1:0.4 or 1:0.5, and the mass ratio within the above range can ensure that the polylysine is more completely dissolved.

[0069] In some embodiments of the present invention, the solvent includes an organic acid solution and an alkaline substance, wherein the organic acid solution includes an aqueous citric acid solution and / or an acetic acid solution, and the mass concentration of the acid in the organic acid solution is 0.3-3.0%; the alkaline substance includes an alkaline solid and / or an alkaline solution, the alkaline solid includes a sodium hydroxide solid and / or a potassium hydroxide solid, the alkaline solution includes a sodium hydroxide solution and / or a potassium hydroxide solution, and the mass concentration of the alkali in the alkaline solution is 1-40%.

[0070] In the technical solution of the present invention, the aqueous solution is a good solvent for dissolving polylysine or polylysine salt. The aqueous solution is preferably an organic acid aqueous solution. When the acidic aqueous solution is a buffered aqueous solution of citric acid or a buffered aqueous solution of acetic acid, it can provide a suitable and stable pH buffer environment for the system, and the appropriate acidic environment can also improve the solubility of polylysine in water. The mass concentration of the acid in the organic acid solution can be 0.3%, 0.7%, 1.0%, 1.5%, 2.0% or 3.0%. The mass concentration within the above range has a good buffering effect, which is conducive to improving the yield of the aldehyde-amine condensation reaction.

[0071] The pH of the reaction system can be adjusted to 3.0-6.0 by adding alkaline solid (such as sodium hydroxide solid, potassium hydroxide solid) or alkaline solution (such as high concentration sodium hydroxide solution or saturated sodium hydroxide solution, high concentration potassium hydroxide solution or saturated potassium hydroxide solution). It should be noted that after dissolving polylysine with citric acid aqueous solution (or acetic acid aqueous solution), sodium hydroxide is then added, and sodium hydroxide is ionized into hydroxide ions and sodium ions after being dissolved in the second solution. The hydroxide ions combine with hydrogen ions in the solution to increase the pH of the solution, and the sodium ions form sodium citrate with part of the citrate, so that the reaction system forms a citric acid-sodium citrate buffer environment (or the sodium ions form sodium acetate with part of the acetate, so that the reaction system forms an acetic acid-sodium acetate buffer environment), and the buffer environment can better maintain the pH of the reaction system at 3.0-6.0, so that the aldehyde amine condensation reaction is carried out in a stable weakly acidic environment. This is because if the acidity of the reaction system is too high, the amino groups in the polylysine or polylysine salt molecules will preferentially combine with the protons (hydrogen ions) in the solvent to form polylysine salts, which will be difficult to react with the aromatic aldehyde groups of (-)-gossypol, and even the target product (-)-gossypol-polylysine derivatives cannot be obtained.

[0072] In addition, since the alkaline solution is easy to drop, it can better and faster adjust the system pH to 3.0-6.0, so the alkaline substance is preferably an alkaline solution, and the mass concentration of the alkali in the alkaline solution can be 1%, 10%, 30% or 40%. If the mass concentration of the alkali is too low, more water will be introduced, reducing the yield. The alkaline solution is preferably a saturated sodium hydroxide aqueous solution or a saturated potassium hydroxide aqueous solution. This concentration can ensure that less water is introduced into the reaction system, which is conducive to the positive progress of the aldehyde-amine condensation reaction and promotes the formation of the target product.

[0073] In some embodiments of the present invention, the polylysine source is a polylysine salt, and the polylysine salt includes polylysine hydrochloride and / or polylysine sulfate. Step S20 includes: mixing the polylysine salt with a solvent to obtain a polylysine salt solution with a pH of 3.0 to 6.0, which is the second solution. Since the pH of the aqueous solution of the polylysine salt is about 5, the pH can be adjusted to be within the range of 3 to 5 or 5 to 6 by adding acid or alkali.

[0074] In some embodiments of the present invention, step S30 includes: mixing the first solution with the second solution, performing an aldehyde-amine condensation reaction, filtering after the reaction is completed, washing and drying the filter residue to obtain the (-)-gossypol derivative; the washing includes washing with ethanol and deionized water in sequence; the drying temperature is 40-60°C. The main component of the product deposited on the filter after filtering is the (-)-gossypol-polylysine derivative, and there may be unreacted (-)-gossypol, polylysine, sodium salt and other impurities. Washing with cold anhydrous ethanol (4°C) can better wash away the unreacted (-)-gossypol, and washing with a small amount of neutral deionized water can better wash away the unreacted polylysine and sodium salt. The drying temperature within the above range can prevent the product from being dehydrated or converted into other by-products due to excessive temperature.

[0075] In some embodiments of the present invention, in step S10, obtaining (-)-gossypol comprises the following steps:

[0076] S01, mixing acetic gossypol, an amino compound and an organic solvent, adjusting the pH to 8.0-10.0, and performing an aldehyde-amine condensation reaction to obtain an intermediate;

[0077] S02. Under anaerobic conditions, the intermediate, an organic solvent and an acid are mixed to carry out an acidolysis reaction to obtain (-)-gossypol.

[0078] In the technical scheme of the present invention, in step S01, the acetic gossypol includes left-handed gossypol [(-)-gossypol] and right-handed gossypol [(+)-gossypol] in different molar ratios, and in an alkaline environment of pH 8.0 to 10.0, the amino group in the amino compound and the active aldehyde group in the acetic gossypol undergo an aldehyde-amine condensation reaction to obtain an intermediate, that is, a bis-Schiff base compound; in step S02, under anaerobic conditions, the bis-Schiff base compound is hydrolyzed to (-)-gossypol, wherein the anaerobic environment can prevent (-)-gossypol from being oxidized, ensuring that the product is (-)-gossypol. It should be noted that in step S02, nitrogen or argon may be introduced first to create anaerobic conditions, then the bis-Schiff base compound and the organic solvent are added, and after sufficient mixing, an acid is finally added to carry out an acidolysis reaction; or the bis-Schiff base compound and the organic solvent may be added first to mix, then nitrogen or argon may be introduced to create anaerobic conditions, and finally an acid is added to carry out an acidolysis reaction.

[0079] In some embodiments of the present invention, the organic solvent includes at least one of ether, methanol and ethanol. The organic solvent can dissolve acetic acid gossypol and amino compounds well, ensure that the reaction raw materials react under homogeneous conditions, and improve the reaction efficiency.

[0080] In some embodiments of the present invention, in step S01, the amino compound includes L-tyrosine methyl ester, L-tyrosine methyl ester salt, L-tryptophan methyl ester, L-tryptophan methyl ester salt, L-phenylalanine methyl ester, L-phenylalanine methyl ester salt, (+)-p-nitrophenyl-2-amino-1,3-propanediol, (+)-p-nitrophenyl-2-amino-1,3-propanediol salt, (-)-p-nitrophenyl-2-amino-1,3-propanediol, (-)-p-nitrophenyl-2-amino-1,3-propanediol salt, (+)-2-amino-3-phenyl-1-propanol, (+)-2-amino-3-phenyl-1-propanol salt, (-)-2-amino-3-phenyl-1-propanol, ( -)-2-amino-3-phenyl-1-propanol salt, (+)-phenethylamine, (+)-phenethylamine salt, (-)-phenethylamine, (-)-phenethylamine salt, (+)-1-hydroxy-3-propylamine, (+)-1-hydroxy-3-propylamine salt, (-)-1-hydroxy-3-propylamine, (-)-1-hydroxy-3-propylamine salt, (+)-cyclopropylamine, (+)-cyclopropylamine salt, (-)-cyclopropylamine, (-)-cyclopropylamine salt, (+)-p-bromoaniline, (+)-p-bromoaniline salt, (-)-p-bromoaniline, (-)-p-bromoaniline salt, (+)-2-aminopropane, (+)-2-aminopropane salt, (-)-2-aminopropane, (-)-2-aminopropane salt, at least one of the following. The above-mentioned amino compounds can undergo aldehyde-amine condensation reaction with acetic gossypol well, especially the amino compounds with aromatic rings, and the formation of products can be monitored by inexpensive instruments such as infrared and ultraviolet.

[0081] It should be noted that L-tryptophan methyl ester salts include L-tryptophan methyl ester hydrochloride and L-tryptophan methyl ester sulfate, L-phenylalanine methyl ester salts include L-phenylalanine methyl ester hydrochloride and L-phenylalanine methyl ester sulfate, (+)-p-nitrophenyl-2-amino-1,3-propanediol salts include (+)-p-nitrophenyl-2-amino-1,3-propanediol hydrochloride and (+)-p-nitrophenyl-2-amino-1,3-propanediol sulfate, (-)-p-nitrophenyl-2-amino-1,3-propanediol salts include (-)-p-nitrophenyl phenyl-2-amino-1,3-propanediol hydrochloride, (-)-p-nitrophenyl-2-amino-1,3-propanediol sulfate, (+)-2-amino-3-phenyl-1-propanol salts including (+)-2-amino-3-phenyl-1-propanol hydrochloride, (+)-2-amino-3-phenyl-1-propanol sulfate, (-)-2-amino-3-phenyl-1-propanol salts including (-)-2-amino-3-phenyl-1-propanol hydrochloride, (-)-2-amino-3-phenyl-1-propanol sulfate, (+)-phenylethylamine salts including ( (+)-phenethylamine hydrochloride, (+)-phenethylamine sulfate, (-)-phenethylamine salts include (-)-phenethylamine hydrochloride, (-)-phenethylamine sulfate, (+)-1-hydroxy-3-propylamine salts include (+)-1-hydroxy-3-propylamine hydrochloride, (+)-1-hydroxy-3-propylamine sulfate, (-)-1-hydroxy-3-propylamine salts include (-)-1-hydroxy-3-propylamine hydrochloride, (-)-1-hydroxy-3-propylamine sulfate, (+)-cyclopropylamine salts include (+)-cyclopropylamine hydrochloride, (+)-cyclopropylamine sulfate , (-)-cyclopropylamine salts include (-)-cyclopropylamine hydrochloride and (-)-cyclopropylamine sulfate, (+)-p-bromoaniline salts include (+)-p-bromoaniline hydrochloride and (+)-p-bromoaniline sulfate, (-)-p-bromoaniline salts include (-)-p-bromoaniline hydrochloride and (-)-p-bromoaniline sulfate, (+)-2-aminopropane salts include (+)-2-aminopropane hydrochloride and (+)-2-aminopropane sulfate, (-)-2-aminopropane salts include (-)-2-aminopropane hydrochloride and (-)-2-aminopropane sulfate.

[0082] In some embodiments of the present invention, in step S01, the temperature of the aldehyde-amine condensation reaction is 50-75° C. The temperature of the aldehyde-amine condensation reaction can be 50° C., 55° C., 60° C., 65° C., 70° C. or 75° C. The temperature within the above range can increase the chemical reaction rate and yield.

[0083] In some embodiments of the present invention, in step S01, the aldehyde-amine condensation reaction time is 3 to 14 hours. The aldehyde-amine condensation reaction time can be 3 hours, 6 hours, 9 hours, 12 hours or 14 hours. The time within the above range can ensure that the acetic acid gossypol and the amino compound react fully, thereby increasing the yield of the acetic acid gossypol converted into the bis-Schiff base compound.

[0084] In some embodiments of the present invention, in step S01, the molar ratio of the gossypol acetate to the amino group in the amino compound is 1:(2.0-2.5). The molar ratio of the gossypol acetate to the amino group in the amino compound in the above range can ensure a higher yield of converting gossypol acetate to the bis-Schiff base compound.

[0085] In some embodiments of the present invention, in step S02, the acid is a mixed acid, including a mixture of glacial acetic acid and a strong acid solution, the strong acid solution includes a hydrochloric acid aqueous solution and / or a sulfuric acid aqueous solution, the mass concentration of the hydrochloric acid aqueous solution is ≥36%, the mass concentration of the sulfuric acid aqueous solution is 50-60%, the volume ratio of the glacial acetic acid to the strong acid solution is (1.5-2.5):1, and the volume ratio of the glacial acetic acid to the organic solvent is 1:(20-100). Glacial acetic acid can combine with (-)-gossypol to form acetic gossypol, which plays a role in protecting the (-)-gossypol produced by acid hydrolysis. The strong acid aqueous solution is used to create an acid hydrolysis environment and promote the quantitative conversion of the bis-Schiff base into (-)-gossypol. The volume ratio of the glacial acetic acid to the strong acid aqueous solution within the above range can ensure a good acid hydrolysis effect and obtain a high yield and high purity (-)-gossypol product.

[0086] In some embodiments of the present invention, in step S02, the acid hydrolysis reaction time is ≥ 2 hours. The acid hydrolysis reaction time can be 2 hours, 4 hours, 6 hours or even longer, so that the bis-Schiff base compound is fully acid-hydrolyzed into (-)-gossypol.

[0087] In some embodiments of the present invention, step S02 comprises: mixing the intermediate, the organic solvent and an acid under anaerobic conditions to perform an acid hydrolysis reaction, separating the organic phase and the aqueous phase after the acid hydrolysis reaction is completed, washing the organic phase to neutrality and removing water from the organic phase, and crystallizing to obtain (-)-gossypol;

[0088] The method for removing moisture from the organic phase includes using anhydrous magnesium sulfate or anhydrous sodium sulfate for adsorption.

[0089] In the technical solution of the present invention, since (-)-cottonpol is easily soluble in organic solvents, it is present in the organic phase. The organic phase is washed to neutrality and the moisture of the organic phase is removed to remove impurities, and preliminary purification is performed. Subsequently, (-)-cottonpol is precipitated from the organic phase by crystallization to obtain relatively pure (-)-cottonpol. The purified (-)-cottonpol can also be further recrystallized to obtain (-)-cottonpol with higher purity.

[0090] The present invention also provides an antioxidant, comprising the aforementioned (-)-gossypol derivative or the (-)-gossypol derivative prepared by the aforementioned method for preparing the (-)-gossypol derivative. Therefore, the antioxidant has all the beneficial effects of the aforementioned (-)-gossypol derivative and the method for preparing the (-)-gossypol derivative, which will not be described one by one here.

[0091] In agriculture, cotton pol is used to protect cotton plants from cotton borers. In medicine, cotton pol has the effect of inhibiting spermatogenesis and sperm activity, so it is used as an effective male contraceptive. In addition, cotton pol also has antioxidant, anti-tumor (including breast cancer, ovarian cancer, adrenocortical carcinoma and colon cancer, etc.), anti-HIV, anti-malarial, anti-parasitic and anti-fungal effects. Since the application of acetic cotton pol in clinical medicine and bioengineering fields has multiple toxic and side effects, the present invention also provides an alternative drug of acetic cotton pol, the drug includes the aforementioned (-)-cotton pol derivative or the (-)-cotton pol derivative prepared by the aforementioned preparation method of the (-)-cotton pol derivative, because the raw materials (-)-cotton pol and polylysine or polylysine salt of the (-)-cotton pol derivative can be used in the field of medicine, and the (-)-cotton pol derivative can be prepared with excipients such as starch, pregelatinized starch, dextrin, powdered sugar, magnesium stearate, lactose, calcium sulfate, calcium hydrogen phosphate, etc. to obtain antioxidant or anticancer drugs.

[0092] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0093] Example 1

[0094] A method for preparing a (-)-gossypol-α-polylysine derivative comprises the following steps:

[0095] S01. Weigh two compounds in a molar ratio of 1:2 between gossypol acetate and L-tyrosine methyl ester hydrochloride, dissolve them in methanol respectively, and mix them. Adjust the pH of the methanol solution to 8.0-10.0 with a 40% NaOH aqueous solution, place it in a constant temperature container at 55°C, and stir slowly for 12 hours. Evaporate the methanol, dissolve the product in cold ethanol at 4°C, and filter out the insoluble matter. Dry the filtrate at 50°C under ventilation to obtain a solid (-)-gossypol-bis(L-tyrosine methyl ester) Schiff base;

[0096] S02. Dissolve (-)-gossypol-bis(L-tyrosine methyl ester) Schiff base in anhydrous ether, transfer to a sealed container, introduce nitrogen to drive out the oxygen in the container, and then add glacial acetic acid and 37% concentrated HCl in a volume ratio of 2:1. The amount of glacial acetic acid added is one twentieth of the volume of anhydrous ether. Maintain the solution in a strong acidic state and hydrolyze for 5 hours under anaerobic conditions. Transfer the hydrolysis product to a separatory funnel and wash the hydrolysis product with deionized water until it is neutral to obtain an orange-yellow solution. Then treat with anhydrous sodium sulfate for 5 hours to absorb the water in the solution, filter out the insoluble matter, and place the solution in a ventilated place to air dry until crystals precipitate. Add low-boiling point (30-60°C) petroleum ether to recrystallize the crystals. After the recrystallized product is ventilated and air-dried, it is ground to obtain an orange-red powder, which is (-)-gossypol;

[0097] S10, accurately weigh (-)-gossypol, and dissolve it in anhydrous ethanol to obtain a first solution;

[0098] S20, weighing α-polylysine in a ratio of 2:1 between the amino group in the α-polylysine and (-)-gossypol, dissolving it in water, adding water twice the mass of the weighed α-polylysine, and then dropping a 37% hydrochloric acid aqueous solution to adjust the pH of the system to 4.0 to obtain a second solution; the degree of polymerization of the α-polylysine is 36;

[0099] S30, mix the above two solutions, stir slowly at a constant temperature of 50°C for 8h, and filter with filter paper. The solid on the filter paper is washed with cold ethanol at 4°C and a small amount of neutral deionized water for 3 times, and then placed at 40°C for ventilation and drying to obtain orange-red crystals, which are (-)-gossypol-α-polylysine derivatives.

[0100] Example 2

[0101] A method for preparing a (-)-gossypol-α-polylysine derivative is different from that of Example 1 in that:

[0102] In step S20, a sulfuric acid aqueous solution with a mass concentration of 50% is added dropwise to adjust the pH of the system to 4.0 to obtain a second solution.

[0103] Example 3

[0104] A method for preparing a (-)-gossypol-α-polylysine derivative is different from that of Example 1 in that:

[0105] In step S20, α-polylysine is replaced with α-polylysine hydrochloride, and α-polylysine hydrochloride is weighed in a ratio of 2:1 between the amino group in α-polylysine hydrochloride and (-)-gossypol, and dissolved in water, the mass of water added is twice the mass of the weighed α-polylysine hydrochloride, and then a hydrochloric acid aqueous solution with a mass concentration of 37% is added dropwise to adjust the pH of the system to 4.0 to obtain a second solution; the degree of polymerization of the α-polylysine hydrochloride is 36.

[0106] Example 4

[0107] A method for preparing a (-)-gossypol-ε-polylysine derivative is different from that of Example 1 in that α-polylysine is replaced by ε-polylysine, and the polymerization degree of the ε-polylysine is 36.

[0108] Example 5

[0109] A method for preparing a (-)-gossypol-ε-polylysine derivative is different from that of Example 2 in that α-polylysine is replaced by ε-polylysine, and the polymerization degree of the ε-polylysine is 36.

[0110] Example 6

[0111] A method for preparing a (-)-gossypol-ε-polylysine derivative is different from that of Example 3 in that α-polylysine hydrochloride is replaced by ε-polylysine hydrochloride, and the polymerization degree of the ε-polylysine hydrochloride is 36.

[0112] Example 7

[0113] A method for preparing a (-)-gossypol-α-polylysine derivative, wherein Example 7 differs from Example 1 in that:

[0114] In step S20, water is replaced with an equal amount of 0.8 (m / v)% citric acid aqueous solution, α-polylysine is dissolved with the citric acid aqueous solution, and then the pH of the system is adjusted to 3.0 with a 40% mass concentration of NaOH aqueous solution to obtain a second solution. The subsequent steps are the same as in Example 1.

[0115] Example 8

[0116] A method for preparing a (-)-gossypol-α-polylysine derivative, wherein Example 8 differs from Example 7 in that:

[0117] In step S20, the pH of the system is adjusted to 3.5 with a 40% mass concentration of NaOH aqueous solution to obtain a second solution. The subsequent steps are the same as those in Example 7.

[0118] Example 9

[0119] A method for preparing a (-)-gossypol-α-polylysine derivative, wherein Example 9 differs from Example 7 in that:

[0120] In step S20, the pH of the system is adjusted to 4.0 with a 40% mass concentration of NaOH aqueous solution to obtain a second solution. The subsequent steps are the same as those in Example 7.

[0121] Example 10

[0122] A method for preparing a (-)-gossypol-α-polylysine derivative, wherein Example 10 differs from Example 7 in that:

[0123] In step S20, the pH of the system is adjusted to 5.0 with a 40% mass concentration of NaOH aqueous solution to obtain a second solution. The subsequent steps are the same as those in Example 7.

[0124] Embodiment 11

[0125] A method for preparing a (-)-gossypol-α-polylysine derivative, wherein Example 11 differs from Example 7 in that:

[0126] In step S20, the pH of the system is adjusted to 6.0 with a 40% mass concentration of NaOH aqueous solution to obtain a second solution. The subsequent steps are the same as those in Example 7.

[0127] Example 12

[0128] A method for preparing a (-)-gossypol-α-polylysine derivative, wherein Example 12 differs from Example 9 in that:

[0129] In step S30, the second solution is mixed with the first solution, transferred into a sealed container, stirred slowly at a constant temperature of 50° C. for 8 hours, and filtered with filter paper. The subsequent steps are the same as those in Example 9.

[0130] Embodiment 13

[0131] A method for preparing a (-)-gossypol-α-polylysine derivative, wherein Example 13 differs from Example 12 in that:

[0132] In step S30, the second solution and the first solution are mixed, transferred into a sealed container, nitrogen is introduced to drive out the oxygen in the container, the mixture is stirred slowly at a constant temperature of 50° C. for 8 hours, and filtered with filter paper. The subsequent steps are the same as those in Example 12.

[0133] Embodiment 14

[0134] A method for preparing a (-)-gossypol-α-polylysine derivative, wherein Example 14 differs from Example 13 in that:

[0135] In step S20, the pH of the system is adjusted to 4 with a 10% mass concentration of NaOH aqueous solution to obtain a second solution. The subsequent steps are the same as those in Example 13.

[0136] Embodiment 15

[0137] The difference between Example 15 and Example 13 is that:

[0138] In step S20, the pH of the second solution is adjusted to 4 using a 1% NaOH aqueous solution to obtain a second solution. The subsequent steps are the same as those in Example 13.

[0139] Comparative Example 1

[0140] A method for preparing a (-)-gossypol-α-polylysine derivative, wherein the difference between Comparative Example 1 and Example 1 is that:

[0141] In step S20, water is replaced with an equal amount of 0.8 (m / v)% citric acid aqueous solution to obtain a second solution. The subsequent steps are the same as those in Example 1.

[0142] Comparative Example 2

[0143] A method for preparing a (-)-gossypol-α-polylysine derivative, wherein the difference between Comparative Example 2 and Example 7 is that:

[0144] In step S20, the pH of the system is adjusted to 7.0 using a 40% mass concentration NaOH aqueous solution to obtain a second solution. The subsequent steps are the same as those in Example 7.

[0145] Performance Testing

[0146] The infrared spectra of (-)-cottonpol prepared in Example 1 were compared with those of the raw material acetic cottonpol. Figure 1 And as shown in Table 1.

[0147] Table 1 Infrared analysis results of (-)-cottonpol and raw material acetic acid cottonypol

[0148]

[0149] As shown in Table 1 and Figure 1 As shown in the infrared spectrum of gossypol acetate, 3516 cm -1 It is the characteristic absorption of the hydrogen bond stretching vibration within the -OH molecule of HAC, but there is no such absorption in the infrared spectrum of (-)-gossypol, indicating that (-)-gossypol does not contain HAC.

[0150] The characteristic absorption of the stretching vibration of the aromatic aldehyde group (-C=O) of acetic gossypol and (-)-gossypol has changed. In the infrared spectrum of acetic gossypol, the characteristic absorption of the stretching vibration of the aromatic aldehyde group (-C=O) appears at 1711cm-1 The characteristic absorption of the stretching vibration of the aromatic aldehyde group (-C=O) of (-)-cottonpol appears at 1726 cm -1 .

[0151] The characteristic absorption of the stretching vibration of the naphthalene ring skeleton of acetic gossypol and (-)-gossypol is different. The characteristic absorption of the stretching vibration of the naphthalene ring skeleton of acetic gossypol appears at 1612cm -1 、1578cm -1 、1440cm -1 , and the three characteristic absorptions of the stretching vibration of the naphthalene ring skeleton of (-)-cottonpol appear at 1618 cm -1 、1516cm -1 、1450cm -1 .

[0152] In view of the obvious differences in the characteristic absorption of -C=O stretching vibration on the aromatic aldehyde group of the two compounds and the three characteristic absorptions representing the stretching vibration of the naphthalene ring skeleton, it is indicated that the naphthalene ring skeleton structure of racemic (±)-gossypol (gossypol acetate) and (-)-gossypol has changed (flipped).

[0153] The infrared spectra of (-)-gossypol prepared in Example 1 and (-)-gossypol-α-polylysine were compared. Figure 2 And as shown in Table 2.

[0154] Table 2 Infrared analysis results of (-)-gossypol and (-)-gossypol-α-polylysine

[0155]

[0156] From Table 2 and Figure 2 It can be seen that the wave number is 1563cm -1 、3240cm -1 They represent the NH in-plane bending vibration absorption and the secondary amide-NH stretching vibration absorption of the α-polylysine segment in the (-)-gossypol-α-polylysine derivative. In the infrared spectrum of (-)-gossypol, there are no absorptions at the above two locations.

[0157] The stretching vibration absorption of the aldehyde group (C=O) on the naphthalene ring of (-)-gossypol appears at 1726 cm -1 When (-)-gossypol reacts with α-polylysine to form a double Schiff base, the stretching vibration absorption representing the aldehyde group (C=O) disappears, and a new wave number of 1731 cm -1 It is the stretching vibration absorption of (-)-gossypol-α-polylysine derivative (-C=N).

[0158] In the infrared spectrum of (-)-cottonpol, three characteristic absorptions representing the stretching vibration of the naphthalene ring skeleton appear at 1618 cm -1 、1516cm-1 、1450cm -1 In the infrared spectrum of (-)-gossypol-α-polylysine derivatives, three characteristic absorptions representing the stretching vibration of the naphthalene ring skeleton appeared at 1622 cm -1 、1542cm -1 、1450cm -1 . It can be seen that after (-)-gossypol reacts with α-polylysine to form derivatives, the characteristic absorption of the stretching vibration of the naphthalene ring skeleton at two locations changes. This is attributed to the increase in the steric hindrance of the newly formed double Schiff base after the introduction of (-)-gossypol into α-polylysine. At the same time, the newly formed derivative also causes the characteristic absorption of the hydroxyl group (-OH) linked to the naphthalene ring of (-)-gossypol and (-)-gossypol-α-polylysine derivatives to change. This is manifested in: the characteristic absorption of the hydroxyl group (-OH) linked to the naphthalene ring of (-)-gossypol is 3379cm -1 The characteristic absorption of the hydroxyl group (-OH) linked to the naphthalene ring of (-)-gossypol-α-polylysine derivative is 3416 cm -1 .

[0159] The infrared spectra of the ε-polylysine, the prepared (-)-cottonpol and the (-)-cottonpol-ε-polylysine derivative in Example 4 were compared. Figure 3 And as shown in Table 3.

[0160] Table 3 Infrared analysis results of ε-polylysine, (-)-gossypol and (-)-gossypol-ε-polylysine derivatives

[0161]

[0162] As shown in Table 3 and Figure 3 It can be seen that the infrared characteristic absorption peaks of ε-polylysine are: primary amine (-NH) stretching vibration: 3431cm -1 ; (-NH) stretching vibration absorption of primary amide (3249cm -1 ) and in-plane bending vibration absorption (1531cm -1 、1564cm -1 ); (-C=O) stretching vibration absorption of primary amide: 1644cm -1 .

[0163] (-)-Gossypol-ε-polylysine infrared absorption characteristics: It retains the infrared absorption characteristics of certain groups of ε-polylysine and (-)-gossypol, such as the characteristic absorption of the primary amide bond in ε-polylysine, the naphthalene ring skeleton structure in the (-)-gossypol molecule, and the characteristic absorption of the aromatic hydroxyl groups on the naphthalene ring. Differences: The stretching vibration of the primary amine (-NH) in the ε-polylysine molecule (3431cm -1) disappeared, and the characteristic absorption of the aromatic aldehyde group in the (-)-cottonpol molecule (1726cm -1 ) disappears. This is the result of the aromatic aldehyde group (-CHO) in the (-)-gossypol molecule and the α-NH2 in the ε-polylysine forming a (-)-gossypol-ε-polylysine double Schiff base (-C=N).

[0164] ABTS removal by acetic gossypol, (-)-gossypol, (-)-gossypol-α-polylysine bis-Schiff base in Example 1 and (-)-gossypol-ε-polylysine bis-Schiff base and ascorbic acid in Example 4 + The activity of free radicals is tested, and the test results are as follows Figure 4 The specific detection method is as follows:

[0165] (1)ABTS + . Preparation of working solution: 7.0×10 -3 mol / L ABTS aqueous solution and 2.45×10 -3 mol / L K2S2O8 aqueous solution was mixed in equal volume ratio, placed in the dark, and reacted at room temperature (25°C) for 16 hours to obtain ABTS +· When using, use phosphate buffer (0.1M, pH7.4) as blank and use phosphate buffer (0.1M, pH7.4) to mix ABTS +· The stock solution was diluted to 734 nm and the absorbance was 0.70 ± 0.02, and ABTS was obtained. +· Working fluid.

[0166] (2) Take 1 mL of each solution of the samples to be tested at different concentration gradients, such as (-)-gossypol, acetic gossypol, ascorbic acid, (-)-gossypol-ε-polylysine, (-)-gossypol-α-polylysine, α-polylysine, and ε-polylysine, and add them to 3.8 mL of ABTS. +· Working solution, mix well. React at room temperature for 6 minutes, use phosphate buffer (0.1M, pH7.4) as blank, and record the absorbance value of the sample at 734nm. ABTS +· The free radical scavenging activity was calculated as follows:

[0167] ABTS +· Free radical scavenging rate (%) x = (A0-A1) / A0×100%

[0168] Where A0: 1 mL of phosphate buffer (0.1 M, pH 7.4) added to 3.8 mL of ABTS +· Absorbance value of the working solution after 6 minutes of reaction at room temperature; A1: 1 mL of the sample to be tested with different concentration gradients is added to 3.8 mL ABTS +·The absorbance value of the reaction in the working solution at room temperature for 6 minutes.

[0169] Depend on Figure 4 It can be seen that within the experimental concentration range (5-100 μmol / L), α-polylysine / ε-polylysine can clear ABTS +· The activity of free radicals is weak and can be ignored. (-)-Gossypol, acetic gossypol, ascorbic acid, (-)-gossypol-ε-polylysine derivatives, (-)-gossypol-α-polylysine derivatives can scavenge ABTS +· The half doses of free radicals (Scavenging Median Dose, SD-50) are: (-)-gossypol 6.0μmol / L, gossypol acetate 19.0μmol / L, ascorbic acid 43.8μmol / L, (-)-gossypol-ε-polylysine derivative 51.5μmol / L, (-)-gossypol-α-polylysine derivative 58.0μmol / L. [Note: The molar mass of (-)-gossypol-ε-polylysine derivative and (-)-gossypol-α-polylysine derivative are both calculated as 774.5g / mol, that is, calculated based on the mass of Schiff base formed by 1 mole of (-)-gossypol and 2 moles of lysine monomers]. That is, (-)-gossypol scavenges ABTS + The activity of free radicals is the highest, more than 3 times that of gossypol acetate; gossypol acetate removes ABTS + The free radical activity is 2.3 times that of ascorbic acid; (-)-gossypol-ε-polylysine derivatives and (-)-gossypol-α-polylysine derivatives scavenge ABTS + The free radical activity is slightly lower than that of ascorbic acid.

[0170] Although (-)-gossypol and gossypol acetate scavenged ABTS + · The activity of free radicals is high, but they are also highly toxic to organisms. Although (-)-gossypol-ε-polylysine derivatives and (-)-gossypol-α-polylysine derivatives can scavenge ABTS + The free radical activity is much lower than (-)-gossypol and acetic gossypol, but it can remove ABTS + The free radical activity is still very high; and after the dialdehyde group of (-)-gossypol is blocked by the amino group of ε-polylysine / α-polylysine, the toxic and side effects of the formed (-)-gossypol derivatives are greatly reduced, and the safety of medication can be ensured.

[0171] The yield of the (-)-gossypol derivatives prepared in Examples 1 to 16 and Comparative Example 1 was calculated using the formula: Yield of (-)-gossypol derivatives (%) = (actual yield / theoretical yield) × 100%. The results are shown in Table 5.

[0172] Table 5 Yields of (-)-gossypol derivatives prepared in Examples 1 to 16 and Comparative Example 1

[0173] Yield of (-)-gossypol derivatives (%) Example 1 70 Example 2 72 Example 3 74 Example 4 70 Example 5 73 Example 6 75 Example 7 55 Example 8 65 Example 9 72 Example 10 65 Embodiment 11 54 Example 12 83 Embodiment 13 93 Embodiment 14 82 Embodiment 15 45 Comparative Example 1 37 Comparative Example 2 38

[0174] As shown in Table 5, the yield of the (-)-cottonpol polylysine derivative obtained according to the scheme of Example 13 is the highest, which is the most optimized production process condition. That is: accurately weigh (-)-cottonpol and dissolve it with anhydrous ethanol to obtain a first solution; weigh polylysine according to the molar ratio of amino groups in polylysine to (-)-cottonpol of 2:1, dissolve it with 0.8 (m / v)% citric acid aqueous solution, and the volume of the added citric acid aqueous solution is twice the mass of the weighed polylysine. The pH of the second solution is adjusted to 4.0 with a 40% mass concentration of NaOH aqueous solution to obtain a second solution; mix the above two solutions, transfer them into a closed container, pass nitrogen gas, drive out the oxygen in the container, stir slowly at a constant temperature of 50-55°C for 8h, and filter with filter paper. The solid on the filter paper is washed with cold ethanol at 4°C and a small amount of neutral deionized water for 3 times, and then placed at 40°C for ventilation and drying to obtain orange-red (-)-cottonpol-polylysine derivative crystals.

[0175] The difference between Example 13 and Examples 14 and 15 is that the mass concentration of the NaOH aqueous solution used to adjust the pH of the citric acid aqueous solution is different. The mass concentrations of the NaOH aqueous solution used in Examples 13, 14 and 15 are 40%, 10% and 1%, respectively. The lower the mass concentration of the NaOH aqueous solution used to adjust the acidity of the system, the larger the volume of water introduced. In view of the fact that the reaction of (-)-gossypol and polylysine to form a (-)-gossypol polylysine derivative is accompanied by the generation of water, the introduction of too much water into the system inhibits the generation of the target product, resulting in a low yield of the target product.

[0176] It is not difficult to conclude from Examples 7 to 11 and Comparative Examples 1 and 2 that the acidity in the reaction system is directly related to the yield of (-)-gossypol polylysine derivatives, and too high or too low acidity is not conducive to improving the yield of (-)-gossypol polylysine derivatives. When the acidity is too high, hydrogen ions preferentially bind to the amino groups in the polylysine molecules to form polylysine salts, which are difficult to form Schiff bases with (-)-gossypol. When the acidity is too low, the oxygen atom on the aldehyde group (C=O) of the binaphthyl ring has a weak ability to bind hydrogen ions, and the ability to form carbon cations on the carbon atoms in the aldehyde group is weak, and the carbon atoms in the aldehyde group have a weak ability to bind to the lone pair of electrons of the nitrogen atom on the amino group of the polylysine molecule. It can be seen that maintaining the slightly acidic pH of the reaction system at 4.0 is most beneficial to improving the yield of (-)-gossypol polylysine derivatives.

[0177] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A (-)-gossypol derivative, characterized in that The invention comprises a (-)-gossypol-polylysine derivative and / or a pharmaceutically acceptable salt thereof, wherein the repeating structural unit of the (-)-gossypol-polylysine derivative comprises any one of the structural formulas (I) and (II); in the formulas (I) and (II), n=25-40, 2. A method for preparing a (-)-gossypol derivative as claimed in claim 1, characterized in that: The following steps are involved: S10, mixing (-)-gossypol with an organic solvent to obtain a first solution; S20, obtaining a polylysine source solution with a pH of 3.0 to 6.0, which is the second solution; S30, mixing the first solution and the second solution, performing an aldehyde-amine condensation reaction, and obtaining the (-)-gossypol derivative.

3. The method for preparing the (-)-gossypol derivative according to claim 2, characterized in that: In step S10, the organic solvent includes at least one of methanol and ethanol; and / or, In step S20, the polylysine source includes at least one of α-polylysine, ε-polylysine, α-polylysine hydrochloride, α-polylysine sulfate, ε-polylysine hydrochloride and ε-polylysine sulfate; and / or, In step S30, the molar ratio of (-)-gossypol in the first solution to the free amino groups in the polylysine source in the second solution is 1:(2-2.5); and / or, In step S30, the temperature of the aldehyde-amine condensation reaction is 50-75° C.; and / or, In step S30, the aldehyde-amine condensation reaction time is ≥ 3 h; and / or, Step S30 includes: mixing the first solution and the second solution, and performing an aldehyde-amine condensation reaction in a closed environment to obtain the (-)-gossypol derivative, wherein the closed environment includes an oxygen-free environment.

4. The method for preparing the (-)-gossypol derivative according to claim 2, characterized in that: The polylysine source is polylysine, and step S20 includes: mixing polylysine with a solvent to obtain a polylysine source solution with a pH of 3 to 6, that is, the second solution, wherein the solvent includes water and an inorganic acid solution, or an organic acid solution and an alkaline substance.

5. The method for preparing the (-)-gossypol derivative according to claim 4, characterized in that: The solvent comprises water and an inorganic acid solution, the inorganic acid solution comprises a hydrochloric acid aqueous solution and / or a sulfuric acid aqueous solution, the mass concentration of hydrochloric acid in the hydrochloric acid aqueous solution is 1 to 37%, the mass concentration of sulfuric acid in the sulfuric acid aqueous solution is 1 to 60%, and the mass ratio of water to polylysine is 1:(0.05 to 0.5); or, The solvent includes an organic acid solution and an alkaline substance, wherein the organic acid solution includes an aqueous citric acid solution and / or an acetic acid solution, and the mass concentration of the acid in the organic acid solution is 0.3-3.0%; the alkaline substance includes an alkaline solid and / or an alkaline solution, the alkaline solid includes a sodium hydroxide solid and / or a potassium hydroxide solid, the alkaline solution includes a sodium hydroxide solution and / or a potassium hydroxide solution, and the mass concentration of the alkali in the alkaline solution is 1-40%.

6. The method for preparing the (-)-gossypol derivative according to claim 2, characterized in that: Step S30 includes: The first solution and the second solution are mixed to carry out an aldehyde-amine condensation reaction, and after the reaction is completed, the solution is filtered, and the filter residue is washed and dried to obtain the (-)-gossypol derivative; The washing comprises washing with ethanol and deionized water in sequence; The drying temperature is 40-60°C.

7. The method for preparing the (-)-gossypol derivative according to claim 2, characterized in that: In step S10, obtaining the (-)-cottonpol comprises the following steps: S01, mixing acetic gossypol, an amino compound and an organic solvent, adjusting the pH to 8.0-10.0, and performing an aldehyde-amine condensation reaction to obtain an intermediate; S02. Under anaerobic conditions, the intermediate, an organic solvent and an acid are mixed to carry out an acidolysis reaction to obtain (-)-gossypol.

8. The method for preparing the (-)-gossypol derivative according to claim 7, characterized in that: The organic solvent comprises at least one of ether, methanol and ethanol; and / or, In step S01, the amino compound includes L-tyrosine methyl ester, L-tyrosine methyl ester salt, L-tryptophan methyl ester, L-tryptophan methyl ester salt, L-phenylalanine methyl ester, L-phenylalanine methyl ester salt, (+)-p-nitrophenyl-2-amino-1,3-propanediol, (+)-p-nitrophenyl-2-amino-1,3-propanediol salt, (-)-p-nitrophenyl-2-amino-1,3-propanediol, (-)-p-nitrophenyl-2-amino-1,3-propanediol salt, (+)-2-amino-3-phenyl-1-propanol, (+)-2-amino-3-phenyl-1-propanol salt, (-)-2-amino- at least one of 3-phenyl-1-propanol salt, (+)-phenethylamine, (+)-phenethylamine salt, (-)-phenethylamine, (-)-phenethylamine salt, (+)-1-hydroxy-3-propylamine, (+)-1-hydroxy-3-propylamine salt, (-)-1-hydroxy-3-propylamine, (-)-1-hydroxy-3-propylamine salt, (+)-cyclopropylamine, (+)-cyclopropylamine salt, (-)-cyclopropylamine, (-)-cyclopropylamine salt, (+)-p-bromoaniline, (+)-p-bromoaniline salt, (-)-p-bromoaniline, (-)-p-bromoaniline salt, (+)-2-aminopropane, (+)-2-aminopropane salt, (-)-2-aminopropane, (-)-2-aminopropane salt; and / or, In step S01, the temperature of the aldehyde-amine condensation reaction is 50-75° C.; and / or, In step S01, the aldehyde-amine condensation reaction time is 3 to 14 hours; and / or, In step S01, the molar ratio of the acetic acid gossypol to the amino group in the amino compound is 1:(2-2.5); and / or, In step S02, the acid comprises a mixture of glacial acetic acid and a strong acid solution, the strong acid solution comprises a hydrochloric acid aqueous solution and / or a sulfuric acid aqueous solution, the mass concentration of the hydrochloric acid aqueous solution is ≥36%, the mass concentration of the sulfuric acid aqueous solution is 50% to 60%, the volume ratio of the glacial acetic acid to the strong acid solution is (1.5 to 2.5):1, and the volume ratio of the glacial acetic acid to the organic solvent is 1:(20 to 100); and / or, In step S02, the acid hydrolysis reaction time is ≥ 2h.

9. The method for preparing a (-)-gossypol derivative according to claim 7, characterized in that: Step S02 includes: Under anaerobic conditions, the intermediate, an organic solvent and an acid are mixed to perform an acid hydrolysis reaction, after the acid hydrolysis reaction is completed, an organic phase and an aqueous phase are separated, the organic phase is washed to neutrality and water in the organic phase is removed, and crystallization is performed to obtain (-)-gossypol; The method for removing moisture from the organic phase includes using anhydrous magnesium sulfate or anhydrous sodium sulfate for adsorption.

10. An antioxidant, characterized in that The invention comprises the (-)-gossypol derivative according to claim 1 or the (-)-gossypol derivative prepared by the method for preparing the (-)-gossypol derivative according to any one of claims 2 to 9.

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