CN111285857A

CN111285857A - Synthesis method and anti-oxidation and anti-aging application of silibinin derivatives

Info

Publication number: CN111285857A
Application number: CN202010071261.8A
Authority: CN
Inventors: 马大友; 刘苏友; 董祎婧; 庞晨绪; 李灼阳; 彭光颖; 马正英
Original assignee: Changsha Jialimei Biotechnology Co Ltd; Central South University
Current assignee: Changsha Jialimei Biotechnology Co Ltd; Central South University
Priority date: 2020-01-21
Filing date: 2020-01-21
Publication date: 2020-06-16
Anticipated expiration: 2040-01-21
Also published as: CN111285857B

Abstract

A synthesis method and antioxidant and antiaging application of silibinin derivatives are disclosed, wherein the silibinin derivatives have structure shown as formula (I), wherein n is natural number of 1-6. The 7- (morpholine-N-alkyl) oxy-2, 3-dehydrosilybin derivative can be obtained by a selective nucleophilic substitution reaction of silybin and morpholine-N-alkyl bromide under an alkaline condition and only one-step reaction. Compared with silybin, the compound has the advantages of lower toxicity, stronger oxidation resistance, more obvious effect of prolonging the life of fruit flies, application value in the aspect of anti-aging, and good water solubility due to salification with acids such as hydrogen chloride and the like. From the aspects of activity and pharmacy, the invention has good development and application values of antioxidation and anti-aging.

Description

Synthesis method and anti-oxidation and anti-aging application of silibinin derivatives

Technical Field

The invention relates to a synthesis method and anti-oxidation and anti-aging application of silybin derivatives.

Background

Silymarin is a kind of flavonolignan compounds separated from fruit or seed of Silybum marianum of Compositae herbaceous plant, and mainly comprises 7 compounds such as silibinin (compound 1), isosilibinin, silidianin, etc. In silymarin, the content of silibinin is the highest, accounting for 50-70%, and is the main active ingredient. Silibinin and its oxidation product 2, 3-dehydrosilybin (compound 2) have wide biological activity, such as active oxygen scavenging, lipid peroxidation resisting, cardiovascular disease preventing and treating, and tumor resisting, and at present, Silibinin is mainly used in liver protecting medicine clinically.

However, silibinin and its oxidation products are poorly water soluble and have a certain cytotoxicity, so that its use in the fields of food and medicine is greatly limited. We have studied and designed to synthesize a 2, 3-dehydrosilybin derivative with a 7-position amino side chain, and a plurality of compounds in the derivatives have better antitumor activity than silybin (patent No. 201210179444.7-a 2, 3-dehydrosilybin derivative and a preparation method and application thereof).

Silybin (1), 2, 3-dehydrosilybin (2) and 7- (morpholine-N-alkyl) oxy-2, 3-dehydrosilybin derivatives (3-5)

In further research, the compounds (such as the compounds 3-5) containing the 7-morpholine-N-alkoxy side chain in the 2, 3-dehydrosilybin derivatives have lower cytotoxicity and stronger antioxidant activity than silybin, and show better effect of prolonging the life of fruit flies than the silybin. We also found that the 7- (morpholine-N-alkyl) oxy-2, 3-dehydrosilybin derivative can be obtained by a selective nucleophilic substitution reaction of silybin and morpholine-N-alkyl bromide under alkaline conditions in one step, and the method is simpler in operation and higher in yield than the method reported in the literature.

As shown in table 1, cell proliferation experimental studies showed that the above 7- (morpholin-N-alkyl) oxy-2, 3-dehydrosilybin derivatives (compounds 3-5) have lower toxicity to human umbilical vein endothelial cells and stronger scavenging ability to the active radical DPPH, compared to silybin (compound 1) and its oxidation product 2, 3-dehydrosilybin (compound 2).

Compounds 3-5 above the percent survival of human umbilical vein endothelial cells in the presence of 100. mu.M of compound and their half maximal scavenging concentration (EC) for DPPH free radicals₅₀) Higher ratio of (C) such as Compound 3 percent survival of human umbilical vein endothelial cells at 100 μ M concentration to half the concentration of scavenging DPPH free radicals (EC)₅₀) The ratio of (A) to (B) is 7.18 times and 31.6 times of silybin (compound 1) and its oxidation product 2, 3-dehydrosilybin (compound 2), respectively, and the application in resisting oxidation of 7- (morpholine-N-alkyl) oxygen-2, 3-dehydrosilybin derivatives is demonstratedThe organism is safer and more effective than silybin and its oxidation product 2, 3-dehydrosilybin.

TABLE 1 percent survival of human umbilical vein endothelial cells at 100. mu.M concentration of compound and compound's ability to scavenge DPPH free radicals (EC)₅₀，μM，EC₅₀Concentration of compound that achieves half the radical DPPH scavenging) and the ratio of the two

The influence of the compound on the life of the fruit flies is researched by taking the compound 3 as a representative. In the experiment, the medicines are uniformly distributed in the fruit fly culture medium and are set to be low, medium and high, namely the medicines account for 0.05 percent, 0.1 percent and 0.2 percent of the mass of the fruit fly culture medium. The results are shown in table 2, compound 3 at low concentration significantly extended the average life of fruit flies (elongation up to 30.6%), whereas the reference compound silibinin had no significant effect on the life of fruit flies at the same low concentration; in medium concentration, silybin shows certain activity, and the life of the fruit flies is prolonged to 10.3%; while both showed some toxicity at high concentrations, compound 3 reduced the average life span of the flies by only 9.9% compared to silybin, which reduced the average life span of the flies by 16.1%. Therefore, the 7- (morpholine-N-alkyl) oxygen-2, 3-dehydrogenation silybin derivative can effectively prolong the life of fruit flies at a proper dosage, has stronger effect than natural compound silybin, and has higher safety than silybin.

TABLE 2 influence of Compound 3 and Silibinin on the longevity of Drosophila

Moreover, the 7- (morpholine-N-alkyl) oxygen-2, 3-dehydrosilybin can form salt with acid such as hydrogen chloride and the like, so that the water-solubility is good. Therefore, the 7- (morpholine-N-alkyl) oxygen-2, 3-dehydrosilybin derivative has good development and application values of antioxidation, liver protection and anti-aging no matter from the aspects of activity or drug forming property.

Disclosure of Invention

One object of the present invention relates to a process for the preparation of compounds of formula I:

the compound shown in the formula I is a silybin derivative containing a morpholine side chain, wherein n is a natural number of 1-6.

Preferably, the 7- (morpholin-N-alkyl) oxy-2, 3-dehydrosilybin derivatives comprise, but are not limited to, the compounds represented by 3 to 5:

the technical route of the preparation method of the 7- (morpholine-N-alkyl) oxygen-2, 3-dehydrosilybin compound is as follows:

preparation of Compound (I):

dissolving silybin (compound 1) in a solvent, sequentially adding alkali and morpholine-N-alkyl halide, adding water after reaction, extracting for several times by using the solvent, combining extract liquor, adding a drying agent for drying, spin-drying the solvent, separating and purifying to obtain a compound (I), wherein the alkali is potassium carbonate, sodium carbonate, cesium carbonate and triethylamine, and the morpholine-N-alkyl halide is morpholine-N-alkyl bromide, morpholine-N-alkyl iodide and morpholine-N-alkyl chloride; the molar weight ratio of the silybin (compound 1) to the alkali is 1: 0.1-10, wherein the molar weight ratio of the silybin (compound 1) to the morpholine-N-alkyl halide is 1: 0.1-10, wherein the dosage of the solvent is 1-80 mL per 1g of silybin; the solvent is isopropanol, water, ethyl acetate, ethanol, dichloromethane, tetrahydrofuran, dimethyl sulfoxide and N, N-dimethylformamide; the reaction temperature is 0-150 ℃; the drying agent is anhydrous sodium sulfate or anhydrous magnesium sulfate; the separation and purification is separation and purification through silica gel column chromatography.

The invention also relates to the application of the compound of formula I or the pharmaceutically acceptable salt thereof as an effective active ingredient for resisting oxidation, protecting liver and resisting aging.

The pharmaceutically acceptable salt of the invention refers to a salt formed by inorganic acid or organic acid, such as hydrochloric acid, sulfuric acid, trifluoroacetic acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, oxalic acid, tartaric acid, maleic acid, citric acid or ascorbic acid, and the like; or salts formed from anions of elements such as chlorine, bromine or iodine, and the like.

Experiments such as human umbilical vein endothelial cell proliferation inhibition experiments, DPPH free radical scavenging capacity tests, and life influence tests of the compounds on fruit flies prove that the compounds have lower toxicity and stronger oxidation resistance compared with silybin, and have more remarkable effect of prolonging the life of the fruit flies and anti-aging application value. Meanwhile, the 7- (morpholine-N-alkyl) oxygen-2, 3-dehydrosilybin can form salt with acids such as hydrogen chloride and the like, so that the water-solubility is good. The compounds have good development and application values of antioxidation and anti-aging from the aspects of activity and druggability.

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

Detailed Description

Example 1

Synthesis of Compound 3

964mg (2mmol) of silibinin were dissolved in 20ml of acetone, and 776mg (4mmol) of N-bromoethylmorpholine and 276mg (2mmol) of potassium carbonate were added and stirred overnight at 60 ℃ to stop the reaction. Distilling under reduced pressure to remove acetone, adding 30ml water, extracting with ethyl acetate (3X30ml), washing the ethyl acetate extract with water and saturated sodium chloride respectively, drying with anhydrous sodium sulfate, filtering, distilling under reduced pressure to remove ethyl acetate, separating the product with silica gel column to obtain 795mg white solid with yield of 67%, mp231-232 deg.C,¹H NMR(400MHz,DMSO-d₆):δ2.41-2.62(m,4H),2.70(t,J＝ 5.6Hz,2H),3.36-3.42(m,1H),3.57-3.59(m,1H),3.57(t,J＝4.4Hz,4H),3.79(s,3H),4.19(t, J＝5.6Hz,2H),4.25-4.29(m,1H),4.96(s,1H),4.98(d,1H),6.33(s,1H),6.80-6.83(m,2H), 6.88-6.92(m,1H),7.04(s,1H),7.11-7.13(m,1H),7.80-7.84(m,2H)；HRMS(ESI)m/z [M+H]⁺calcd.for C₃₁H₃₂O₁₁N,594.1975；found 594.1982.

example 2:

human Umbilical Vein Endothelial cell (Human Umbilical Vein Endothelial Cells, HUVEC) proliferation inhibition experiment (MTT method):

(1) inoculating cells: taking HUVEC cells in logarithmic phase, sucking and uniformly mixing by using a pipette, centrifuging and collecting, preparing cell suspension, and counting the cells. HUVEC cells were performed at 3X 10 per well in a volume of 200. mu.l per well³The standard for each cell calculates the required cell volume per well. 96-well plates were seeded, setting 3 to 5 duplicate wells.

(2) Adding medicine: zero-adjustment wells (no cells and drug added), control wells (cell plus solvent, e.g. DMSO solvent, < 0.1% final DMSO concentration) and dosing groups (concentration gradient 100 μ M, 50 μ M, 25 μ M, 12.5 μ M) were set.

(3) Culturing the cells: at 37 deg.C, 5% CO₂And culturing for 48h under saturated humidity.

(4) After drug treatment, 20. mu.l of MTT reagent (200. mu.l system) was added to each well of a 96-well plate and incubated for 4h at 37 ℃ in a cell incubator. The medium in the wells was carefully discarded, 200. mu.l DMSO was added, and shaking was performed for 10 min. OD measurement with microplate reader_570nmThe value is obtained.

(5) Cell relative viability was calculated after three independent replicates of each experiment (OD values of all wells minus OD values of blank wells): the relative survival rate of the cells was defined as the mean OD value of the drug-added group/the mean OD value of the control group x 100% (see table 2).

TABLE 2 cell viability of human umbilical vein endothelial cells at different concentrations of compound (%)

Example 3

Test of the ability of Compounds to scavenge DPPH free radicals

(1) The DPPH stock solution is prepared by accurately weighing 3.5mg of DPPH reagent, dissolving with absolute ethyl alcohol, quantitatively transferring into a 10ml volumetric flask, and fixing the volume. Taking 2ml to 1000ml volumetric flask, and shaking up to obtain DPPH stock solution with the concentration of 0.0178 mmol/L.

(2) Preparing a test solution: dissolving a small amount of compounds to be detected 1-5 in absolute methanol respectively to prepare a high-concentration solution of 200 mu g/mL, and diluting with methanol for 7 times in an equal time manner to obtain a gradient-concentration methanol solution of the compounds to be detected.

(3) Color comparison: the absorbance of each compound at 517nm was measured under a UV spectrophotometer at 8 different concentrations.

(4) Data processing: and (4) drawing a compound concentration and absorbance curve, and taking a point with the clearance rate of 50% to obtain the corresponding concentration, namely EC 50. The results are shown in the following table.

TABLE 3 DPPH radical scavenging ability (EC) of the compounds₅₀，μM)

Compound (I)	Compound 1	Compound 2	Compound 3	Compound 4	Compound 5
						EC₅₀	166.39	116.72	57.07	62.87	40.92

EC₅₀Refers to the concentration of the compound at which half-maximal clearance of free radical DPPH is achieved

Example 4

Test of the effect of compound 3 and silibinin on the longevity of fruit flies:

(1) normal feeding and reproduction of fruit fly

The pure drosophila melanogaster was cultured and propagated using a subculture medium at 25 ℃ and 66% relative humidity.

(2) Preparation of culture Medium

① subculture medium preparation method comprises adding 20.87g fruit fly culture medium coarse powder (Shanghai Ruchu Biotech Co., Ltd.) into 110ml distilled water, boiling, cooling to 80 deg.C, adding 600ul propionic acid, stirring, and placing into sterilized culture tubes (about 5g each tube).

② preparation of blank control group culture medium comprises weighing 10.44g fruit fly culture medium coarse powder in 55ml distilled water, heating and boiling, slightly cooling to 80 deg.C, adding 300ul propionic acid, stirring, and packaging into sterilized culture tubes, each tube is 1.5-2.0 g.

③ preparation method of experimental group of Silybin culture medium comprises weighing 10.44g fruit fly culture medium coarse powder and Silybin (132mg, 66mg or 33mg), stirring, adding 55ml distilled water, heating to boil under stirring, slightly cooling to 80 deg.C, adding propionic acid 300ul, stirring, and packaging 1.5-2.0g per tube into sterilized culture tubes to obtain three kinds of culture medium with high, medium and low doses.

④ Compound 3 Experimental group media configuration:

A. preparation of Compound 3 propionic acid aqueous solution

a) Weighing 800mg of the compound 3, dissolving the compound in 1800ul of propionic acid solution, and adding 1800ul of water to prepare high-concentration propionic acid liquid medicine after dissolving; b) 3/7 high-concentration propionic acid liquid medicine is added with equal amount of 50% propionic acid water solution to prepare medium-concentration propionic acid liquid medicine; c) 1/3 the low-concentration propionic acid liquid medicine is prepared by adding equal amount of 50% propionic acid water solution.

B. Preparation of drug culture medium

Weighing 10.44g fruit fly culture medium coarse powder in 55ml distilled water, heating to boil, slightly cooling to 80 ℃, adding 600ul of the compound 3 propionic acid aqueous solution, stirring uniformly, and subpackaging 1.5-2g per tube into sterilized culture tubes to obtain three types of culture media with high, medium and low doses respectively.

(3) Experimental protocol

In the experimental process, female and male fruit flies are separated in order to eliminate the influence of sex difference of the fruit flies and self-generated offspring on the experimental result and facilitate counting of the quantity of the fruit flies. The specific operation is as follows: newly emerged flies within 12h were collected and placed in an empty culture tube with 60 μ l ethanol: the mixture of ethyl ether and 1:1 is used for anaesthetizing for 5min, the male and female drosophila are distinguished, and the drosophila are separately fed into different culture tubes and recovered within 15 min. The medium was changed every 5 days.

(4) Record of experiment

Culturing the fruit flies in a constant-temperature constant-humidity incubator, observing the survival state of the fruit flies every 12 hours, recording the death number of the fruit flies, drawing a table until all experimental fruit flies die, and counting the average life of each group of fruit flies.

(5) The data processing results are arranged in the following table and the experimental results are evaluated.

TABLE 4 Effect of Compounds on Drosophila longevity

Note: the algebraic sum of survival time per fruit fly divided by the number of adults was the average life span of the group of fruit flies.

Claims

1. A method for synthesizing silybin derivatives is characterized in that: the compound contains a 7-morpholine-N-alkoxy side chain, and the structure of the silybin derivative is shown as the formula (I):

in formula (I): n is a natural number of 1-6;

the synthesis method comprises the following steps:

dissolving silybin in a solvent, sequentially adding alkali and morpholine-N-alkyl halide, adding water after reaction, extracting for several times by using the solvent, combining extract liquor, adding a drying agent for drying, spin-drying the solvent, and separating and purifying to obtain a compound (I), wherein the alkali is potassium carbonate, sodium carbonate, cesium carbonate and triethylamine, and the morpholine-N-alkyl halide is morpholine-N-alkyl bromide, morpholine-N-alkyl iodide and morpholine-N-alkyl chloride; the molar weight ratio of the silybin to the alkali is 1: 0.1-10, wherein the molar weight ratio of the silybin to the morpholine-N-alkyl halide is 1: 0.1-10, wherein the dosage of the solvent is 1-80 mL per 1g of silybin; the solvent is isopropanol, water, ethyl acetate, ethanol, dichloromethane, tetrahydrofuran, dimethyl sulfoxide or N, N-dimethylformamide; the reaction temperature is 0-150 ℃; the drying agent is anhydrous sodium sulfate or anhydrous magnesium sulfate; the separation and purification is separation and purification through silica gel column chromatography.

2. The use of compounds obtained by the process for the synthesis of silibinin derivatives according to claim 1, characterized in that: relates to the application of the compound of formula (I) or the pharmaceutically acceptable salt thereof as an effective active ingredient in the aspects of antioxidation, liver protection and anti-aging; the antioxidant application comprises skin care, cancer prevention, arteriosclerosis prevention, diabetes, cataract, cardiovascular diseases, senile dementia and arthritis prevention.

3. The use of compounds obtained by a method for the synthesis of a class of silibinin derivatives according to claim 2, characterized in that: the pharmaceutically acceptable salt refers to a salt formed by inorganic acid or organic acid, or a salt formed by element anions, such as chlorine, bromine or iodine.

4. The use of compounds obtained by the process for the synthesis of silibinin derivatives according to claim 3, characterized in that: the salt formed by the inorganic acid or the organic acid is hydrochloric acid, sulfuric acid, trifluoroacetic acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, oxalic acid, tartaric acid, maleic acid, citric acid or ascorbic acid.