阅读说明：本技术 肉桂酸酯衍生物及其作为酪氨酸酶抑制剂和凝胶剂的应用 (Cinnamate derivatives and their use as tyrosinase inhibitors and gels ) 是由 邓翀 姜祎 唐凯 张化为 黄文丽 宋小妹 王薇 颜永刚 于 2020-04-29 设计创作，主要内容包括：本发明公开了一类肉桂酸酯衍生物及其作为酪氨酸酶抑制剂和凝胶剂的应用,该衍生物的结构通式是：<Image he="169" wi="700" file="DDA0002473485050000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>式中,X、Y各自独立的代表O、S、NH中任意一种；R<Sup>1</Sup>、R<Sup>2</Sup>各自独立的代表H、OH、甲氧基、烯丙基、乙酰基中任意一种；R<Sup>3</Sup>、R<Sup>4</Sup>各自独立的代表H、OH、甲氧基、叔丁基二甲基硅氧基、乙酯基中任意一种,且R<Sup>3</Sup>、R<Sup>4</Sup>不同时为叔丁基二甲基甲硅烷基；n是2～5的整数。本发明肉桂酸酯衍生物对蘑菇酪氨酸酶二酚酶活性和B16F10小鼠黑色素瘤细胞内酪氨酸酶与黑素含量具有明显的抑制活性,可用于酪氨酸酶抑制剂的制备。同时本发明肉桂酸酯衍生物在橄榄油中能够形成稳定的凝胶,可作为小分子凝胶剂用于化妆品等。(The invention discloses cinnamate derivatives and application thereof as tyrosinase inhibitors and gels, wherein the structural general formula of the cinnamate derivatives is as follows: wherein X, Y independently represents any one of O, S, NH; r 1 、R 2 Each independently represents H, OH, methoxyl, allyl, acetyl; r 3 、R 4 Each independently represents H, OH, methoxyl, tert-butyl dimethyl siloxy, carbethoxy, and R 3 、R 4 Is not tert-butyldimethylsilyl at the same time; n is an integer of 2 to 5. The cinnamate derivative has obvious inhibition activity on mushroom tyrosinase diphenol enzyme activity and tyrosinase and melanin content in B16F10 mouse melanoma cells, and can be used for preparing tyrosinase inhibitors. Meanwhile, the cinnamate derivative can form stable gel in olive oil, and can be used as a micromolecule gel for cosmetics and the like.)

1. A cinnamate derivative is characterized in that the structural general formula of the derivative is as follows:

wherein X, Y independently represents any one of O, S, NH; r¹、R²Each independently represents H, OH, methoxyl, allyl, acetyl; r³、R⁴Each independently represents H, OH, methoxyl, tert-butyl dimethyl siloxy, carbethoxy, and R³、R⁴Is not tert-butyldimethylsilyloxy at the same time; n is an integer of 2 to 5.

2. The cinnamate derivative according to claim 1, characterized in that the derivative is any one of the following compounds 1 to 14:

compound 1: (E) -3- (2-acetyl-5-methoxyphenoxy) propyl-3- (3-methoxy-4- (prop-1-en-2-yloxy) phenyl) acrylate

Compound 2: (E) -3- (3-acetyl-4-hydroxyphenoxy) propyl-3- (4-acetoxy-3-methoxyphenyl) acrylate

Compound 3: (E) -3- (4-allyl-2-methoxyphenoxy) propyl-3- (4-acetoxy-3-methoxyphenyl) acrylate

Compound 4: (E) -4- (3- (3- (3- (2-acetyl-5-methoxyphenoxy) propoxy) -3-oxo-1-propenyl) -1, 2-benzenediacetate

Compound 5: (E) -4- (3- (3- (3- (3-acetyl-4-hydroxyphenoxy) propoxy) -3-oxo-1-propenyl) -1, 2-benzenediacetate

Compound 6: (E) -4- (3- (3- (3- (4-allyl-2-methoxyphenoxy) propoxy) -3-oxo-1-propen-1-yl) -1, 2-benzenediacetate

Compound 7: 3- (2-acetyl-5-methoxyphenoxy) propyl cinnamate

Compound 8: 3- (3-acetyl-4-hydroxyphenoxy) propyl cinnamate

Compound 9: 3- (4-allyl-2-methoxyphenoxy) propyl cinnamate

Compound 10: (E) -3- (2-acetyl-5-methoxyphenoxy) propyl 3- (4- ((tert-butyldimethylsilyl) oxy) -3-methoxyphenyl) acrylate

Compound 11: (E)3- (3-acetyl-4-hydroxyphenoxy) propyl (E) -3- (4- ((tert-butyldimethylsilyl) oxy) -3-methoxyphenyl) acrylate

Compound 12: (E) -3- (4-allyl-2-methoxyphenoxy) propyl 3- (4- ((tert-butyldimethylsilyl) oxy) -3-methylphenyl) acrylate

Compound 13: (E)3- (4-hydroxy-3-methoxyphenyl) -3- (2-acetyl-5-methoxyphenoxy) acrylic acid propyl ester

Compound 14: (E)3- (3-acetyl-4-hydroxyphenoxy) -3- (4-hydroxy-3-methoxyphenyl) acrylic acid propyl ester

3. Use of the cinnamate derivative of claim 1 for preparing a tyrosinase inhibitor.

4. Use of the cinnamate derivative of claim 1 as a small molecule gel.

Technical Field

The invention relates to a cinnamate derivative with tyrosinase activity inhibition.

Background

Tyrosinase is involved in the production of melanin and is a key enzyme that limits melanin production. Due to the abnormal activity of tyrosinase, a series of highly active quinones can be generated to cause hyperpigmentation, thereby affecting the skin beauty, such as chloasma, freckle, senile plaque, melanosis after inflammation and other melanosis, and a plurality of researches report that the melanosis hyperpigmentation even causes melanoma and early senile dementia diseases. Particularly, melanoma is characterized by high malignancy degree and high mortality, according to statistics of 2018 northern large tumor hospitals, the detection rate of melanoma in China is rapidly increased at present, and 2 ten thousand new cases occur every year. Tyrosinase plays a key role in the process of melanin generation, and has become a main target for inhibiting excessive melanin generation.

Tyrosinase inhibitors are increasingly used in pharmaceuticals and cosmetics, primarily for the prevention and treatment of pigmentation disorders. Most of the currently used tyrosinase inhibitors such as kojic acid (Contact Dermatitis,1995,32 (1): 9-13), arbutin and hydroquinone (paeonol structural analogue and its chemical modification for the research on tyrosinase inhibitory action [ D ]. university of Nanchang, 2014) still have corresponding disadvantages such as cytotoxicity, carcinogenicity, Dermatitis, neurodegenerative symptoms, allergy, leukoderma, brown yellow (Bioorganic & Medicinal Chemistry,2017, 25(5):1687-1695), and potential toxicity and instability of bone marrow, which limits their application in the medical field and food industry; in addition, lead and mercury ions have the effect of inhibiting melanin production, but can cause skin leukoplakia, nervous system disorders, vision loss, kidney damage, skin mucosa sensitivity and influence on embryonic development in some users. In view of the disadvantages of current market use of tyrosinase inhibitors, the search for safer, effective and stable tyrosinase inhibitors has been the focus of attention of researchers.

In recent years, with the increasing demand of tyrosinase inhibitors as food additives and whitening agents, a large number of high-activity tyrosinase inhibitors are successively reported, and particularly, the tyrosinase inhibitors having strong activity are obtained by performing appropriate structural modification and activity screening by using the skeleton of a natural active compound. Among them, natural active phenolic compounds such as quercetin, ferulic acid and cinnamic acid exhibit an inhibitory activity against mushroom tyrosinase (Food Chem,2019,297: 124910). IC of vanillyl ester cinnamate analogs₅₀213.2. mu.M (Bioorg Chem,2019,93: 103316). Cinnamic acid amide derivatives were slightly more active than kojic acid (78.8. mu.M) (Food Chem,2012,134(2): 1081-1087). Phenethyl cinnamates have tyrosinase inhibitory activity (J Nat Prod,2013,76(8): 1399-1405). In addition, oneSeries of studies show that the small molecular phenolic acid component and the cinnamic acid component have better tyrosinase inhibitory activity (interaction study of the composite inhibitor on tyrosinase inhibition [ D ]]Guangdong institute of medicine, 2015; inhibitory Effect of inhibitors on Mushroom tyrosinase and antibacterial Activity [ D]Building university, 2007). IC of carvacrol derivatives₅₀The value was 16.69. mu.M (Ploss One,2017,12(5): 351-.

In summary, according to the synthesis and activity research of novel tyrosinase inhibitors reported at home and abroad at present, on one hand, a part of compounds with higher activity lack cell experiments such as cytotoxicity and the like, and the reliable safety of the compounds cannot be proved; on the other hand, the inhibitory activity of tyrosinase is insufficient.

Disclosure of Invention

The invention aims to provide a safe and efficient cinnamate derivative with tyrosinase inhibitory activity and application of the cinnamate derivative.

The structural general formula of the cinnamate derivative used for solving the technical problems is as follows:

wherein X, Y independently represents any one of O, S, NH; r¹、R²Each independently represents H, OH, methoxyl, allyl, acetyl; r³、R⁴Each independently represents H, OH, methoxyl, tert-butyl dimethyl siloxy, carbethoxy, and R³、R⁴Is not tert-butyldimethylsilyloxy at the same time; n is an integer of 2 to 5.

The cinnamate derivative is preferably any one of the following compounds 1 to 14:

compound 1: (E) -3- (2-acetyl-5-methoxyphenoxy) propyl-3- (3-methoxy-4- (prop-1-en-2-yloxy) phenyl) acrylate

Compound 2: (E) -3- (3-acetyl-4-hydroxyphenoxy) propyl-3- (4-acetoxy-3-methoxyphenyl) acrylate

Compound 3: (E) -3- (4-allyl-2-methoxyphenoxy) propyl-3- (4-acetoxy-3-methoxyphenyl) acrylate

Compound 4: (E) -4- (3- (3- (3- (2-acetyl-5-methoxyphenoxy) propoxy) -3-oxo-1-propenyl) -1, 2-benzenediacetate

Compound 5: (E) -4- (3- (3- (3- (3-acetyl-4-hydroxyphenoxy) propoxy) -3-oxo-1-propenyl) -1, 2-benzenediacetate

Compound 6: (E) -4- (3- (3- (3- (4-allyl-2-methoxyphenoxy) propoxy) -3-oxo-1-propen-1-yl) -1, 2-benzenediacetate

Compound 7: 3- (2-acetyl-5-methoxyphenoxy) propyl cinnamate

Compound 8: 3- (3-acetyl-4-hydroxyphenoxy) propyl cinnamate

Compound 9: 3- (4-allyl-2-methoxyphenoxy) propyl cinnamate

Compound 10: (E) -3- (2-acetyl-5-methoxyphenoxy) propyl 3- (4- ((tert-butyldimethylsilyl) oxy) -3-methoxyphenyl) acrylate

Compound 11: (E)3- (3-acetyl-4-hydroxyphenoxy) propyl (E) -3- (4- ((tert-butyldimethylsilyl) oxy) -3-methoxyphenyl) acrylate

Compound 12: (E) -3- (4-allyl-2-methoxyphenoxy) propyl 3- (4- ((tert-butyldimethylsilyl) oxy) -3-methylphenyl) acrylate

Compound 13: (E)3- (4-hydroxy-3-methoxyphenyl) -3- (2-acetyl-5-methoxyphenoxy) acrylic acid propyl ester

Compound 14: (E)3- (3-acetyl-4-hydroxyphenoxy) -3- (4-hydroxy-3-methoxyphenyl) acrylic acid propyl ester

In the structural formula of the cinnamate derivative, R³、R⁴When each independently represents any one of H, methoxyl and carbethoxy, the synthesis method comprises the following steps:

1. mixing compound I, compound II and anhydrous K₂CO₃Adding organic solvent A into a round-bottom flask, and adding compound I, compound II and anhydrous K₂CO₃The molar ratio of (1: 1.2) - (1.5: 0.5-0.8), heating to 60-100 ℃, reacting for 2-8 hours, separating and purifying the product to obtain a compound III, wherein the reaction equation is as follows:

2. adding a compound IV and thionyl chloride into a round-bottom flask according to the mass-volume ratio of 1g: 4-8 m L, connecting a tail gas absorption device, heating to 40-80 ℃, heating to 90 ℃ after no gas is generated in the reaction, and recovering the thionyl chloride to obtain a compound V, wherein the reaction equation is as follows:

3. adding a compound III, an organic solvent B and an acid-binding agent into a round-bottom flask under the protection of nitrogen, cooling to 0 ℃, dropwise adding a compound V, wherein the molar ratio of the compound III to the acid-binding agent to the compound V is 1: 1.5-2.0: 2-2.5, heating to room temperature after dropwise adding, and reacting at room temperature for 4-5 hours. After the reaction is finished, the solvent is removed under reduced pressure, and NaHCO with the mass fraction of 5 percent is respectively used₃Extracting with water solution and saturated NaCl water solution, drying with anhydrous magnesium sulfate, and separating with 100-200 mesh silica gel column chromatography (petroleum ether: ethyl acetate ═ 1:2, V/V) to obtain compound VI, i.e. cinnamate derivative, with the following reaction equation:

in the structural formula of the cinnamate derivative, R³、R⁴When one of the groups is tert-butyldimethylsilyloxy, the synthesis method comprises the following steps:

1. compound III was synthesized according to step 1 above.

2. Adding the compound IV' and tert-butyldimethylsilyl chloride (TBDMSCl) into a round-bottomed flask containing an organic solvent A according to the molar ratio of 1: 2.5-3.5, cooling to-10 ℃, stirring until the mixture is completely dissolved, slowly dropping a dichloromethane solution of triethylamine, stirring for 1-2 hours, gradually increasing the temperature to room temperature, and continuing stirring for reaction for 3-5 hours. After the reaction was completed, the reaction mixture was washed with water, and the organic layer was then washed with MgSO₄Drying, recovering excessive solvent under reduced pressure, and separating by 100-200 mesh silica gel column chromatography (petroleum ether: ethyl acetate: 2:1, V/V) to obtain an intermediate compound. Adding intermediate compound and organic solvent A into round-bottom flask, connecting tail gas absorption device, and adding SOCl₂Heating to 40-80 ℃, reacting until no gas is generated, heating to 90 ℃ to recover thionyl chloride, cooling to room temperature, and concentrating under reduced pressure to obtain a compound V'; wherein the molar ratio of the compound IV 'to triethylamine is 1: 0.8-1, and the compound IV' and SOCl₂The mass-volume ratio of the (1 g) to the (2-5 m) L. the reaction equation is as follows:

wherein R' represents any one of H, OH, methoxyl and carbethoxy.

3. Adding a compound III, an organic solvent B and an acid-binding agent into a round-bottom flask under the protection of nitrogen, cooling to 0 ℃, dropwise adding an intermediate V ', wherein the molar ratio of the compound III to the acid-binding agent to the compound V' is 1: 1.5-2.0: 2-2.5, heating to room temperature after dropwise adding, and reacting at room temperature for 4-5 hours. After the reaction is finished, the solvent is removed under reduced pressure, and NaHCO with the mass fraction of 5 percent is respectively used₃Extracting with water solution and saturated NaCl water solution, drying with anhydrous magnesium sulfate, and separating with 100-200 mesh silica gel column chromatography (petroleum ether: ethyl acetate: 1:2, V/V) to obtain compound VI', namely cinnamate derivative, with the following reaction formula:

in the structural formula of the cinnamate derivative, R³、R⁴One of them is OH, and the other is any one of H, methoxy group and ethyl ester group, its synthesis method is that according to the above-mentioned method compound VI 'is synthesized, then under the protection of nitrogen gas compound VI' is dissolved in tetrahydrofuran, then 1 mol/L NH is slowly dropped₄Tetrahydrofuran solution of F, Compound VI' and NH₄And F is in a molar ratio of 1: 1.1-1.3, and the reaction is stirred at room temperature for 1 hour. After the reaction is finished, the solvent is recovered under reduced pressure to obtain crude products, and the crude products are respectively treated with saturated NaHCO₃The aqueous solution, deionized water and brine were washed, and the organic layer was MgSO₄Drying and purifying by 100 mesh silica gel column chromatography to obtain the compound VII, namely the cinnamate derivative, the reaction equation of which is as follows:

the organic solvent A is any one of N, N-dimethylformamide, acetone, dimethyl sulfoxide and tetrahydrofuran; the organic solvent B is any one of N, N-dimethylformamide, acetone, dichloromethane, ethyl acetate and tetrahydrofuran. The acid-binding agent is triethylamine or anhydrous pyridine.

The invention relates to application of cinnamate derivatives in preparing tyrosinase inhibitors. Experimental results prove that the cinnamate derivative has good inhibition effect on the bisphenol enzyme activity of mushroom tyrosinase, the tyrosinase activity in melanocytes and melanogenesis, so that the cinnamate derivative can be used for preparing an anti-tyrosinase inhibitor.

In addition, the cinnamate derivative can form stable gel in olive oil, and can be used as small molecule gel for cosmetics and the like.

The invention has the following beneficial effects:

1. the invention successfully synthesizes a series of cinnamate derivatives with stronger inhibitory activity to tyrosinase by utilizing the natural skeleton of active ingredients in the traditional Chinese medicine, has short synthetic route, simple operation and cheap and easily obtained reaction raw materials.

2. The cinnamate derivative synthesized by the invention has stronger inhibiting effect on the bisphenol enzyme activity of mushroom tyrosinase, the tyrosinase activity in melanocytes and melanogenesis, and can be used for preparing anti-tyrosinase activity medicaments. The tyrosinase inhibitor can be used independently or combined with other tyrosinase inhibitors, and the activity of the tyrosinase inhibitor is obviously stronger than that of the tyrosinase inhibitor kojic acid and arbutin which are widely used at present.

3. The cinnamate derivative can form stable gel in olive oil, and can be used as a small molecule gel for cosmetics and the like.

Drawings

FIG. 1 is a drawing of Compound 8¹H NMR spectrum.

FIG. 2 is a drawing of Compound 8¹³C NMR spectrum.

FIG. 3 is a drawing of Compound 11¹H NMR spectrum.

FIG. 4 is a photograph of Compound 11¹³C NMR spectrum.

FIG. 5 is a drawing of Compound 13¹H NMR spectrum.

FIG. 6 is a drawing of Compound 13¹³C NMR spectrum.

FIG. 7 is a drawing of Compound 14¹H NMR spectrum.

FIG. 8 is a drawing of Compound 14¹³C NMR spectrum.

FIG. 9 is a tyrosinase inhibition curve for compounds 8, 13, 14 and kojic acid at 10 different concentrations.

FIG. 10 shows the effect of different doses of compounds 8,11, 13 and 14 on B₁₆F₁₀Cell viability of the cells.

FIG. 11 shows the effect of different doses of compounds 8,11, 13 and 14 on B₁₆F₁₀Anti-melanogenesis inhibitory effect of cells.

FIG. 12 is a graph of Compounds 8,11, 13 and 14 vs. B₁₆F₁₀Inhibitory activity of tyrosinase in melanocytes.

Figure 13 is a photograph of compounds 8,11, 13 and 14 before and after gel formation in olive oil.

Description of the drawings: in FIGS. 10 to 12, p<0.05，**:p<0.01，***:p<0.001 (compare with model group)，^###：p<0.01 (in comparison to model group).

Detailed Description

The invention will be further described in detail with reference to the following figures and examples, but the scope of the invention is not limited to these examples.