阅读说明：本技术 一种芦荟松的合成方法及其用途 (Synthesis method and application of aloe floss ) 是由 王雪松 李友宾 曾婷婷 王燕 徐俊裕 谭银丰 龚晶雯 王春锋 于 2021-06-28 设计创作，主要内容包括：本发明提供一种芦荟松的合成方法及其用途,将化合物A、氯化氢溶液和异丙醇混合,所述化合物A和氯化氢的摩尔比为1：4.0～4.5,加热至40-50℃搅拌反应1-2h；反应后减压浓缩除去溶剂,得到浓缩物,将浓缩物用柱层析纯化,得到目标产物芦荟松；所述化合物A的结构如式(a)所示：本发明芦荟松的合成方法,成本低、容易合成、产率高,较好工业化生产。(The invention provides a synthesis method and application of aloe floss, which is characterized in that a compound A, a hydrogen chloride solution and isopropanol are mixed, wherein the molar ratio of the compound A to the hydrogen chloride is 1: 4.0-4.5, heating to 40-50 ℃, stirring and reacting for 1-2 h; concentrating under reduced pressure after reaction to remove the solvent to obtain a concentrate, and purifying the concentrate by column chromatography to obtain a target product, namely the dried aloe floss; the structure of the compound A is shown as the formula (a): the synthesis method of the aloe floss has low cost and is easyHigh synthesis and yield, and better industrialized production.)

1. The method for synthesizing the aloe floss is characterized by comprising the following steps of:

mixing a compound A, a hydrogen chloride solution and isopropanol, wherein the molar ratio of the compound A to the hydrogen chloride is 1: 4.0-4.5, heating to 40-50 ℃, stirring and reacting for 1.4-1.6 h;

concentrating under reduced pressure after reaction to remove the solvent to obtain a concentrate, and purifying the concentrate by column chromatography to obtain a target product, namely the dried aloe floss;

the structure of the compound A is shown as the formula (a):

2. the method of synthesizing aloe vera pine as claimed in claim 1 wherein the molar ratio of isopropanol to compound a is 30-40: 1.

3. the method of claim 1, wherein the solvent for the column chromatography is a mixture of petroleum ether and ethyl acetate.

4. The method of claim 3, wherein the petroleum ether and ethyl acetate are present in a volume ratio of 1-5: 1.

5. The method of claim 4, wherein the petroleum ether and ethyl acetate are present at a volume ratio of 5: 1.

6. The method for synthesizing aloe vera pine as claimed in claim 1, wherein the molar ratio of compound a to hydrogen chloride is 1:4.

7. the method of synthesizing an aloe vera pine as claimed in claim 1 wherein the agitation reaction conditions: the reaction was stirred for 1.5h with heating to 45 ℃.

8. The method of synthesizing an aloe vera pine as claimed in claim 1 wherein the method of synthesizing compound a:

mixing the compound B, sodium hydroxide and dimethyl sulfoxide, reacting for 7.5-8.5h at 20-30 ℃ under stirring, extracting with ethyl acetate and water after reaction, taking an ethyl acetate layer, concentrating under reduced pressure to remove a solvent to obtain a concentrate, and purifying the concentrate by column chromatography, wherein the solvent for column chromatography is prepared by mixing petroleum ether and ethyl acetate at a volume ratio of 4.8-5.2: 1 to obtain a compound A;

the structure of the compound B is shown as the formula (B):

the molar ratio of the compound B to the sodium hydroxide is 1: 2.2-2.8;

the mol/L ratio of the compound B to the dimethyl sulfoxide is 1: 4.0-4.2.

9. The method of synthesizing an aloe vera pine as claimed in claim 8 wherein the method of synthesizing compound B:

mixing the compound C, malic acid, 4-dimethylaminopyridine and dichloromethane, stirring in an ice bath to react to 0 ℃, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, and stirring to react for 5.8-6.2h at 20-30 ℃;

extracting with dichloromethane and water after reaction, collecting dichloromethane layer, concentrating under reduced pressure to remove solvent to obtain concentrate, purifying the concentrate by column chromatography, wherein the solvent for column chromatography is prepared by mixing petroleum ether and ethyl acetate at volume ratio of 5.8-6.2: 1 to obtain compound B;

the structure of the compound C is shown as the formula (C):

the molar ratio of the compound C, malic acid and 4-dimethylaminopyridine is 1: 1.0-1.1: 1.0-1.1;

the mol/L of the molar volume ratio of the compound C to the dichloromethane is 1: 4.0-4.2.

10. Tyrosinase inhibitor or whitening agent, characterized in that it comprises the target product aloe floss obtained by the synthesis method according to any one of claims 1 to 9.

Technical Field

The invention relates to the technical field of tyrosinase inhibitors, and particularly relates to a synthetic method and application of aloe floss.

Background

Tyrosinase is a key enzyme in melanogenesis, produced only by melanocytes, and is produced and processed in the endoplasmic reticulum and golgi apparatus, transported to melanosomes, and synthesized as melanin in the melanosomes. Thus, regulation of melanin synthesis by inhibiting tyrosinase is the primary means of preventing pigmentation. At present, many tyrosinase inhibitors such as Hydroquinone (HQ), arbutin, kojic acid, azelaic acid, L-ascorbic acid, ellagic acid, tranexamic acid, etc. have been used as whitening agents, but have certain disadvantages. Hydroquinone (HQ) in addition to mutagenizing mammalian cells, has several adverse effects including contact dermatitis, allergies, temporary erythema, burning, tingling, vitiligo, petechiae nails, hypopigmentation and brown yellow. Arbutin, a prodrug of hydroquinone, is a natural ingredient that reduces or inhibits tyrosinase activity and thus inhibits the synthesis of melanin. However, the natural form of arbutin is chemically unstable, releases hydroquinone, breaks down into benzene-containing metabolites, and is potentially toxic to bone marrow. Kojic acid has limited use in cosmetics due to its carcinogenicity and instability during storage; l-ascorbic acid is sensitive to heat and is easily deteriorated; ellagic acid is insoluble in water and therefore poorly bioavailable, while inhibition of the melanogenesis pathway for tranexamic acid has not been established. Therefore, there is an urgent need to develop novel tyrosinase inhibitors having pharmaceutical properties.

Aloe is widely used in the fields of skin whitening, moisture retention, sun protection, skin aging delaying, slimming, weight reduction and other beauty and health care, and is known as a green skin care product and a natural beauty master. Aloesin is an active ingredient for whitening, antioxidation, sun protection and anti-aging, and is taken into cosmetic raw material catalogues. However, the aloesin has extremely low content in aloe, the cost of separation and purification is too high, and on the other hand, because the chemical synthesis of the C-glycoside part in the molecule is difficult, the industrialization is not easy, and the wide application of the aloesin is limited. Aloe pine (aloesosone) is an aglycone of aloesin and is also a unique chemical component in the aloe epidermis. The content of the aloe floss in the aloe is low, and the research on related pharmacological activity is also less. In view of the difficulty in obtaining natural aloe pine, the development of a cheap, simple and high-yield synthetic method of aloe pine is of great significance.

Disclosure of Invention

In view of this, the invention provides a synthetic method of aloe floss, which has the advantages of low cost, easy synthesis, high yield and better industrial production.

The technical scheme of the invention is realized as follows:

a synthesis method of aloe floss comprises the following steps:

mixing a compound A, a hydrogen chloride solution and isopropanol, wherein the molar ratio of the compound A to the hydrogen chloride is 1: 4.0-4.5, heating to 40-50 ℃, stirring and reacting for 1.4-1.6 h; preferably, the molar ratio of compound a to hydrogen chloride is 1:4, the stirring reaction conditions are as follows: heating to 45 deg.C, stirring, reacting for 1.5h, concentrating under reduced pressure to remove solvent to obtain concentrate, and purifying the concentrate by column chromatography to obtain target product aloes pine;

the structure of the compound A is shown as the formula (a):

further, the molar ratio of the isopropanol to the compound A is 30-40: 1.

further, the solvent for column chromatography is prepared by mixing petroleum ether and ethyl acetate at a volume ratio of 1-5: 1, preferably 5: 1.

Further, the synthesis method of the compound A comprises the following steps: mixing the compound B, sodium hydroxide and dimethyl sulfoxide, reacting for 7.5-8.5h at 20-30 ℃ under stirring, extracting with ethyl acetate and water after reaction, taking an ethyl acetate layer, concentrating under reduced pressure to remove a solvent to obtain a concentrate, and purifying the concentrate by column chromatography, wherein the solvent for column chromatography is prepared by mixing petroleum ether and ethyl acetate according to a volume ratio of 4.8-5.2: 1 to obtain the compound A.

The structure of the compound B is shown as the formula (B):

the molar ratio of the compound B to the sodium hydroxide is 1: 2.2-2.8;

the mol/L ratio of the compound B to the dimethyl sulfoxide is 1: 4.0-4.2.

Further, the synthesis method of the compound B comprises the following steps:

mixing the compound C, malic acid, 4-dimethylaminopyridine and dichloromethane, stirring in an ice bath to react to 0 ℃, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, and stirring to react for 5.8-6.2h at 20-30 ℃;

extracting with dichloromethane and water after reaction, collecting dichloromethane layer, concentrating under reduced pressure to remove solvent to obtain concentrate, purifying the concentrate by column chromatography, wherein the solvent for column chromatography is prepared by mixing petroleum ether and ethyl acetate at volume ratio of 5.8-6.2: 1 to obtain compound B;

the structure of the compound C is shown as the formula (C):

the molar ratio of the compound C, malic acid and 4-dimethylaminopyridine is 1: 1.0-1.1: 1.0-1.1;

the mol/L of the molar volume ratio of the compound C to the dichloromethane is 1: 4.0-4.2.

Tyrosinase inhibitor or whitening agent, comprising the target product aloe floss obtained by the synthesis method of any one of the invention.

Compared with the prior art, the invention has the beneficial effects that:

the invention takes cheap and easily available 3, 5-dihydroxytoluene as raw material, and synthesizes aglycone aloeswood of aloeswood by acylation reaction, esterification reaction, substitution reaction and the like, wherein, under the condition of isopropanol solvent, a certain proportion of compound A and hydrogen chloride are adopted for reaction, and certain reaction temperature and reaction time are combined to ensure that the reaction is complete, and the target product aloeswood is obtained after purification, the activity of the tyrosinase inhibiting activity of the aloeswood is 4 times that of the aloeswood, and the aloeswood has smaller molecules and smaller polarity than the aloeswood, and can enter cells more easily, and the whitening effect is better than the aloeswood; and on the basis of relatively high yield, the method is low in cost, environment-friendly, easy to synthesize and capable of realizing better industrial production. The aloe floss prepared by the method can be better applied to preparing tyrosinase inhibitors or whitening agents.

Drawings

FIG. 1 is a hydrogen spectrum of the synthesized aloe vera pine of example 1 of the present invention;

fig. 2 is a carbon spectrum of the synthesized aloe vera pine of example 1 of the present invention.

Detailed Description

In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.

The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.

The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.

The compound used in the present invention has the chinese name shown in table 1 below:

TABLE 1 Compound Chinese name

The compound A of the invention is a compound 1-4;

the compound B of the invention is a compound 1-3;

the compound C of the invention is a compound 1-2.

In the embodiment of the invention, the reactant HCl is prepared from commercially available concentrated hydrochloric acid which is a hydrogen chloride solution containing HCl with the mass concentration of 38%.

Synthetic route of aloe floss

Example 1 Synthesis of Aloe pine

(1) Synthesis of intermediates

Synthesis of Compound 1-1

A100 mL round bottom flask was charged with the compound 3, 5-dihydroxytoluene (10mmol, 1.24g) and acetic acid (8mmol, 0.48g) and boron trifluoride etherate (2.48mL, 20mmol) and reacted under magnetic stirring in an oil bath at 80 ℃ for 12 h. Cooling to room temperature after the reaction is finished, extracting with ethyl acetate and water for three times, taking an ethyl acetate layer, and adding saturated NaHCO₃To the solution (10ml), the mixture was stirred for 10min to complete the reaction of acetic acid in the solution. Extracting with ethyl acetate and water for three times, collecting ethyl acetate layer, concentrating under reduced pressure to remove solvent to obtain concentrate, adding ethyl acetate to dissolve the concentrate, adding appropriate amount of petroleum ether, standing, and recrystallizing. After suction filtration, a yellow powder solid, compound 1-1, is obtained.

Synthesis of Compound 1-2

A200 mL round-bottomed flask was charged with compound 1-1(10mmol, 1.66g) and 35mL of dichloromethane, DIPEA (8.4mL, 65mmol), magnetically stirred in an ice bath to 0 ℃ and then added with MEM-Cl (10mmol, 1.25mL), magnetically stirred at room temperature for 12 h. Extracting with ethyl acetate and water for three times after reaction, collecting ethyl acetate layer, concentrating under reduced pressure to remove solvent, and purifying by column chromatography (petroleum ether: ethyl acetate: 6: 1, v/v) to obtain oily liquid, i.e. compound 1-2.

Synthesis of Compounds 1-3

In a 100ml round bottom flask were added compound 1-2(10mmol, 2.54g), malic acid (10mmol, 2.19g), DMAP (1.22g, 10mmol) and dichloromethane (40ml) and the reaction was magnetically stirred in an ice bath to 0 ℃. EDC.HCl (3.82g, 20mmol) was added and the reaction stirred magnetically at room temperature for 6 h. After the reaction, dichloromethane and water are used for extraction, extraction is carried out for three times, a dichloromethane layer is taken, the solvent is removed by decompression and concentration, the obtained concentrate is purified by column chromatography (petroleum ether: ethyl acetate: 6: 1, v/v) to obtain colorless oily liquid, namely the compound 1-3(1.72g, the yield is 45%).

Synthesis of Compounds 1-4

In a 200ml round bottom flask were added compounds 1-3(10mmol,3.82g) and NaOH (0.96g, 24mmol) and DMSO (40ml) and the reaction was stirred magnetically at room temperature for 8 h. After the reaction is finished, ethyl acetate and water are used for extraction for three times, an ethyl acetate layer is taken, the solvent is removed through decompression and concentration, a concentrate is obtained and purified through column chromatography (petroleum ether: ethyl acetate: 5:1, v/v), and decompression and concentration are carried out, so that yellow oily liquid, namely the compound 1-4(1.92g, the yield is 50%) is obtained.

(2) Synthesis of aloe floss

In a 200ml round bottom flask were added compounds 1-4(10mmol, 3.82g) and concentrated hydrochloric acid (3.4ml, 40mmol as HCl) and isopropanol (327mmol, 20ml) and the reaction was heated to 45 ℃ and magnetically stirred for 1.5 h. After the reaction, most of the solvent was removed by concentration under reduced pressure to obtain a concentrate, which was purified by column chromatography (petroleum ether: ethyl acetate: 5:1, v/v), and concentrated under reduced pressure to obtain a white solid, i.e., compound 1-5(1.49g, yield 64%).

Nuclear magnetic data of aloe pine:¹H NMR(400MHz,DMSO-d₆)δ10.61(s,1H),6.62(s,1H),6.60(s,1H),6.03(s,1H),3.85(s,2H),2.65(s,3H),2.21(s,3H)；¹³C NMR(100MHz,DMSO-d₆)δ202.87,178.22,161.09,160.62,159.27,141.65,116.70,114.39,112.92,100.58,47.58,29.89,22.48.

example 2 Synthesis of Aloe pine

On the basis of the embodiment 1, the synthesis process parameters of the aloe floss in the step (2) are adjusted, and the method specifically comprises the following steps:

compound 1-4(10mmol) and concentrated HCl (40 mmol as HCl) and isopropanol (300mmol) were added to a 200mL round bottom flask and heated to 40 deg.C and reacted with magnetic stirring for 1.6 h. Concentrating under reduced pressure to remove most of solvent after reaction, purifying by column chromatography (petroleum ether: ethyl acetate: 5:1, v/v), and concentrating under reduced pressure to obtain white solid, i.e. compound 1-5, with yield of 62%.

Example 3 Synthesis of Aloe pine

The difference between this example and example 1 is the synthesis of the aloe floss in step (2), specifically:

compound 1-4(10mmol) and concentrated HCl (45 mmol as HCl) and isopropanol (400mmol) were added to a 200mL round bottom flask and heated to 50 deg.C and reacted with magnetic stirring for 1.4 h. Concentrating under reduced pressure to remove most of solvent after reaction, purifying by column chromatography (petroleum ether: ethyl acetate: 5:1, v/v), and concentrating under reduced pressure to obtain white solid, i.e. compound 1-5, with yield of 63%.

Comparative example 1

On the basis of example 1, isopropanol in step (2) was replaced by 1, 4-dioxane, acetone, methanol, dimethyl sulfoxide. The results are given in table 2 below:

when 1, 4-dioxane is used as a solvent, the yield is 50%, and the solid precipitated during the reaction is an impure product and is inconvenient to purify again. When acetone is used as the solvent, the yield is 60% lower than that of isopropanol, and acetone is an easily toxic chemical. When methanol was used as the solvent, the yield was 20%. When dimethyl sulfoxide is used as a solvent, the reaction hardly occurs. The invention adopts isopropanol as a reaction solvent, which is not only beneficial to full reaction, but also relatively safe.

Table 2 results of comparative solvent tests

Comparative example 2

On the basis of example 1, the amount of HCl added in step (2), i.e., the amount of concentrated hydrochloric acid added, was adjusted. The results are given in table 3 below: when the molar ratio of the compounds 1-4 to HCl is 1:3, the reaction is incomplete; when the molar ratio of the compounds 1 to 4 to HCl is 1:5, the yield is rather reduced compared to example 1, i.e. the amount of concentrated HCl added is increased. According to the invention, a certain amount of HCl is added, so that the reaction efficiency is improved, and the cost is saved.

TABLE 3 HCl addition comparative test results

Comparative example 3

On the basis of example 1, the stirring reaction time of the aloe floss synthesis step of step (2) was adjusted. The results are given in table 4 below: when the reaction time is 0.5h or 1h, the reaction is incomplete; the reaction time is 2h or 2.5h, and the reaction by-products are obviously increased. The invention preferably selects a certain reaction time, not only the reaction is sufficient, but also the generation of byproducts is reduced, and the reaction efficiency is improved.

TABLE 4 reaction time comparative test results

Comparative example 4

On the basis of example 1, the stirring reaction temperature in the step (2) of synthesizing aloe floss was adjusted. The results are given in table 5 below: the reaction temperature is low or high, and the reaction effect is not good enough. The invention adopts a certain reaction temperature, so that the reaction is sufficient, the reaction efficiency is effectively improved, and the energy consumption is also saved.

TABLE 5 results of comparative test of reaction temperature

Test examples

Compounds 1-6 (Aloe Vera pine) from example 1 were assayed for tyrosinase inhibiting activity

Adding phosphate buffer solution, sample solution (Aloe pine), and tyrosine solution into 96-well plate according to Table 6, heating at 37 deg.C for 10min, adding tyrosinase solution, mixing, heating at 37 deg.C for 10min, and measuring absorbance A at 475nm with enzyme labeling instrument_a、A_b、A_c、A_dThen, the inhibition rate of tyrosinase by the sample was calculated according to the following formula.

Inhibition rate ═ 1- (A)_d-A_c/(A_b-A_a)]*100％

TABLE 6 composition of reaction solution

TABLE 7 inhibition of tyrosinase Activity by Aloe Vera pine

The test results show (table 7) that the tyrosinase inhibitory activity of the aloes pine prepared by the method is 4 times that of aloesin, the aloes pine has no glycosyl, the polarity is lower than that of the aloesin, the molecules are also lower than that of the aloesin and are easy to be absorbed by cells, and the aloes pine is easy to be chemically synthesized, so that the aloes pine can be applied to preparation of tyrosinase inhibitors or whitening agents.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.