CN111440068B

CN111440068B - Cinnamate derivative and application thereof as tyrosinase inhibitor and gel

Info

Publication number: CN111440068B
Application number: CN202010355992.5A
Authority: CN
Inventors: 邓翀; 姜祎; 唐凯; 张化为; 黄文丽; 宋小妹; 王薇; 颜永刚
Original assignee: Shaanxi University of Chinese Medicine
Current assignee: Shaanxi University of Chinese Medicine
Priority date: 2020-04-29
Filing date: 2020-04-29
Publication date: 2023-08-11
Anticipated expiration: 2040-04-29
Also published as: CN111440068A

Abstract

The invention discloses a cinnamic acid ester derivative and application thereof as a tyrosinase inhibitor and a gel, wherein the structural general formula of the derivative is as follows:wherein X, Y each independently represents any one of O, S, NH; r is R ¹ 、R ² Each independently represents any one of H, OH, methoxy, allyl and acetyl; r is R ³ 、R ⁴ Each independently represents one of H, OH, methoxy, tert-butyldimethylsilyloxy and ethyl, and R ³ 、R ⁴ Not both tert-butyldimethylsilyl; n is an integer of 2 to 5. The cinnamate derivative has obvious inhibition activity on the activity of mushroom tyrosinase diphenol enzyme and the content of tyrosinase and melanin in melanoma cells of B16F10 mice, 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 small molecular gel for cosmetics and the like.

Description

Cinnamate derivative and application thereof as tyrosinase inhibitor and gel

Technical Field

The invention relates to cinnamate derivatives with tyrosinase activity inhibition function.

Background

Tyrosinase is involved in melanin production and is a key enzyme limiting melanin production. Because of the abnormal activity of tyrosinase, a series of high-activity quinones can be generated to cause hyperpigmentation, so that the aesthetic appearance of skin is affected, such as chloasma, freckle, senile plaque, postinflammatory melasma and other melanin pigmentation diseases, and various researches report that the melanin hyperpigmentation even causes melanoma and early-onset senile dementia diseases. In particular, melanoma is characterized by high malignancy degree and high mortality rate, and according to the statistics of the hospital for large North tumors of 2018, the detection rate of melanoma in China is rapidly increased at present, and 2 ten thousand new cases are generated each year. Tyrosinase, however, plays a key role in the melanogenesis process and has become a major target for inhibiting melanin overproduction.

Tyrosinase inhibitors are increasingly used in medicine and cosmetics, mainly for the prevention and treatment of pigmentation disorders. The compounds of tyrosinase inhibitors such as kojic acid (Contact Dermatitis,1995,32 (1): 9-13), arbutin and hydroquinone (structural analogues of paeonol and chemical modifications thereof have a corresponding disadvantage for the study of tyrosinase inhibition [ D ]. University of nanchang, 2014), such as cytotoxicity, carcinogenicity, dermatitis, neurodegenerative symptoms, allergies, leukoplakia, brown yellow (Bioorganic & Medicinal Chemistry,2017, 25 (5): 1687-1695), and potential toxicity and instability to bone marrow, which limits their use in the medical field and the food industry; in addition, lead and mercury ions have the effect of inhibiting melanogenesis, but can lead to white spots on the skin, nervous system disorders, vision loss, kidney damage, skin mucosa sensitivity and the influence on embryo development in some users. In view of the many shortcomings of the current market using tyrosinase inhibitors, the search for safer, more effective and more stable tyrosinase inhibitors has been the focus of attention of researchers.

In recent years, along with the increasing demands of tyrosinase inhibitors as food additives and whitening agents, a large number of tyrosinase inhibitors with high activity are reported in succession, and particularly, attention is paid to the fact that a natural active compound skeleton is utilized for carrying out proper structural modification and activity screening to obtain the tyrosinase inhibitor with stronger activity. Wherein, natural active phenolic compounds such as quercetin, ferulic acid and cinnamic acid show inhibitory activity on mushroom tyrosinase (Food Chem,2019, 297:124910). IC of cinnamate vanillyl ester analogue ₅₀ 213.2. Mu.M (Bioorg Chem,2019, 93:103316). Cinnamic acid amide derivatives are slightly more active than kojic acid (78.8. Mu.M) (Food Chem 2012,134 (2): 1081-1087). Phenethyl cinnamate has tyrosinase inhibitory activity (J Nat Prod,2013,76 (8): 1399-1405). In addition, a series of researches show that the micromolecular phenolic acid component and the cinnamic acid component have better tyrosinase inhibition activity (interaction research of the composite inhibitor on tyrosinase inhibition [ D ]]Guangdong college of medicine, 2015; inhibitory Effect of inhibitors on Mushroom tyrosinase and antibacterial Activity [ D ]]University of Xiamen, 2007). IC of carvacrol derivative ₅₀ The value was 16.69. Mu.M (Plos One,2017,12 (5): 351-359).

In summary, according to the synthesis and activity research of novel tyrosinase inhibitors reported at home and abroad at present, on one hand, partial compounds with higher activity lack cell experiments such as cytotoxicity and the like, and the reliable safety cannot be confirmed; on the other hand, tyrosinase inhibitory activity was 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 cinnamic acid ester derivative used for solving the technical problems is shown as follows:

wherein X, Y each independently represents any one of O, S, NH; r is R ¹ 、R ² Each independently represents any one of H, OH, methoxy, allyl and acetyl; r is R ³ 、R ⁴ Each independently represents one of H, OH, methoxy, tert-butyldimethylsilyloxy and ethyl, and R ³ 、R ⁴ Not both tert-butyldimethylsilyloxy; n is an integer of 2 to 5.

The above cinnamate derivative is further 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-benzenediacetic acid ester

Compound 7:3- (2-acetyl-5-methoxyphenoxy) cinnamic acid propyl ester

Compound 8:3- (3-acetyl-4-hydroxyphenoxy) cinnamic acid propyl ester

Compound 9:3- (4-allyl-2-methoxyphenoxy) cinnamic acid propyl ester

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- (2-acetyl-5-methoxyphenoxy) acrylic acid 3- (4-hydroxy-3-methoxyphenyl) propyl ester

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

In the structural formula of the cinnamate derivative, R ³ 、R ⁴ When each independent represents any one of H, methoxy and ethyl, the synthesis method comprises the following steps:

1. compound I, compound II and anhydrous K ₂ CO ₃ Adding an organic solvent A into a round bottom flask, and adding a compound I, a compound II and anhydrous K ₂ CO ₃ The molar ratio of (2) is 1:1.2-1.5:0.5-0.8, the temperature is raised to 60-100 ℃, the reaction is carried out for 2-8 hours, the product is separated and purified, the compound III is obtained, and the reaction equation is as follows:

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

3. under the protection of nitrogen, adding the compound III, the organic solvent B and the acid binding agent into a round bottom flask, cooling to 0 ℃, dropwise adding the 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, and after dropwise adding, heating to room temperature, and reacting for 4-5 hours at room temperature. After the reaction, the solvent was removed under reduced pressure, and NaHCO was used in an amount of 5% by mass, respectively ₃ Extracting 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, wherein the reaction equation is as follows:

in the structural formula of the cinnamate derivative, R ³ 、R ⁴ When one of them 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-butyldimethyl chlorosilane (TBDMSCl) into a round-bottom flask containing the organic solvent A according to the mol ratio of 1:2.5-3.5, cooling to-10 ℃, stirring until the compound IV' and the tert-butyldimethyl chlorosilane are completely dissolved, slowly dripping a dichloromethane solution of triethylamine, stirring for 1-2 hours, gradually heating to room temperature, and continuing stirring for reacting for 3-5 hours. Water is used after the reaction is finishedThe organic layer was washed with MgSO ₄ Drying, recovering excess solvent under reduced pressure, and separating by 100-200 mesh silica gel column chromatography (petroleum ether: ethyl acetate=2:1, V/V) to obtain intermediate compound. Adding the intermediate compound and organic solvent A into a round bottom flask, connecting a tail gas absorbing device, and adding SOCl ₂ Heating to 40-80 ℃, heating to 90 ℃ after no gas is generated during the reaction, recovering thionyl chloride, cooling to room temperature, and concentrating under reduced pressure to obtain a compound V'; wherein the mol ratio of the compound IV 'to the triethylamine is 1:0.8-1, and the compound IV' to the SOCl ₂ The mass-volume ratio of (2) is 1 g:2-5 mL. The reaction equation is as follows:

wherein R' represents any one of H, OH, methoxy and ethyl.

3. Under the protection of nitrogen, adding the compound III, the organic solvent B and the acid binding agent into a round bottom flask, 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, and after dropwise adding, heating to room temperature, and reacting at room temperature for 4-5 hours. After the reaction, the solvent was removed under reduced pressure, and NaHCO was used in an amount of 5% by mass, respectively ₃ Extracting 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, wherein the reaction equation is as follows:

in the structural formula of the cinnamate derivative, R ³ 、R ⁴ When one of them is OH and the other is any one of H, methoxy and ethyl ester group, its synthetic method is: the compound VI 'is synthesized according to the method, then the compound VI' is dissolved in tetrahydrofuran under the protection of nitrogen, and 1mol/LNH is slowly added dropwise ₄ Tetrahydrofuran solution of F, compound VI' with NH ₄ The molar ratio of F is 1:1.1-1.3, and the reaction is stirred for 1 hour at room temperature. Recovering solvent under reduced pressure after the reaction is finished to obtain crude products, respectively using saturated NaHCO ₃ The organic layer was washed with MgSO, with aqueous, deionized and brine ₄ After drying, the compound VII, namely the cinnamate derivative, is obtained through purification by 100-mesh silica gel column chromatography, and the reaction equation 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, methylene dichloride, ethyl acetate and tetrahydrofuran. The acid binding agent is triethylamine or anhydrous pyridine.

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

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

The beneficial effects of the invention are as follows:

1. the invention utilizes the natural skeleton of the active ingredients in the traditional Chinese medicine to successfully synthesize a series of cinnamate derivatives with strong inhibition activity on tyrosinase, and has the advantages of short synthesis path, simple operation, low-cost and easily-obtained reaction raw materials.

2. The cinnamate derivative synthesized by the invention has strong inhibition effect on the bisphenol enzyme activity of mushroom tyrosinase and the tyrosinase activity and melanogenesis in melanocytes, and can be used for preparing anti-tyrosinase activity medicines. Can be used alone or in combination with other tyrosinase inhibitors, and has activity obviously stronger than that of the currently widely used tyrosinase inhibitors kojic acid and arbutin.

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 diagram of Compound 8 ¹ H NMR spectrum.

FIG. 2 is a diagram of Compound 8 ¹³ C NMR spectrum.

FIG. 3 is a diagram of Compound 11 ¹ H NMR spectrum.

FIG. 4 is a diagram of Compound 11 ¹³ C NMR spectrum.

FIG. 5 is a diagram of Compound 13 ¹ H NMR spectrum.

FIG. 6 is a diagram of Compound 13 ¹³ C NMR spectrum.

FIG. 7 is a diagram of Compound 14 ¹ H NMR spectrum.

FIG. 8 is a diagram of Compound 14 ¹³ C NMR spectrum.

FIG. 9 is a graph of tyrosinase inhibition by compounds 8, 13, 14 and kojic acid at 10 different concentrations.

FIG. 10 is a graph showing the results of compound 8,11, 13 and 14 versus B at various doses ₁₆ F ₁₀ Cell viability of the cells.

FIG. 11 is a graph showing the results of compounds 8,11, 13 and 14 for B at different doses ₁₆ F ₁₀ Cell melanogenesis inhibiting effect.

FIG. 12 is Compound 8,11, 13 and 14 vs B ₁₆ F ₁₀ Inhibitory Activity of tyrosinase in melanocytes.

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

Description: in fig. 10-12, p<0.05，**:p<0.01，***:p<0.001 (in contrast to the model set), ^### ：p<0.01 (in contrast to the model set).

Detailed Description

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

Example 1

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

1. 1.66g (10.0 mmol) of paeonol (compound I-1) and 0.83g (6.0 mmol) of K are weighed out ₂ CO ₃ Placed in a round bottom flask, 100mL of acetone was added as a solvent, stirred at room temperature for 0.5h, 2.08g (15.0 mmol) of 3-bromo-1-propanol (compound II-1) was added, the mixture was heated to 80℃for reflux reaction for 8h, cooled to room temperature after the reaction was completed, the solvent was removed under reduced pressure, and purified by silica gel column chromatography to give 2.691g of crystalline compound III-1 in a yield of 65% at m.p.56 ℃.

2. 1.96g (10 mmol) of Compound IV-1 and 15mL of thionyl chloride were added to a round-bottomed flask, a tail gas absorbing device was connected, the temperature was raised to 80℃until no gas was generated, and after the reaction, the temperature was raised to 90℃to recover thionyl chloride, to obtain Compound V-1.

3. 2.24g (10 mmol) of compound III-1 and 4.77g (20 mmol) of compound V-1 are weighed under the ice bath and nitrogen protection condition, placed in a round bottom flask, 50mL of dichloromethane is added as a solvent, 10mL of dichloromethane solution containing 1.5g (15 mmol) of triethylamine is slowly added dropwise under the stirring condition, and the temperature is raised to room temperature after the dropwise addition, and the reaction is carried out for 4 hours at room temperature. After the reaction, the solvent was removed under reduced pressure, and NaHCO was used in an amount of 5% by mass, respectively ₃ Extracting 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 3.92g white crystal compound VI-1, namely compound 1, m.p. 110-111 ℃, yield 56%, wherein the structural characterization data are as follows: ¹ H NMR(400MHz,CDCl ₃ )δ:7.85(d,J＝8.7Hz,1H),7.66(t,J＝10.9Hz,1H),7.26(d,J＝2.0Hz,1H),7.12(d,1H),7.05(d,J＝8.0Hz,1H),6.53(dd,J＝8.7,1H),6.46(d,J＝2.2Hz,1H),6.38(d,J＝15.9Hz,1H),4.44(t,J＝6.2Hz,2H),4.18(t,J＝6.1Hz,2H),3.86(t,J＝6.7Hz,6H),2.61(s,3H),2.32(s,3H)； ¹³ C NMR(100MHz,CDCl ₃ )δ:197.71,168.97,166.82,164.54,160.22,151.45,141.53,132.91,117.94,111.26,105.18,99.06,77.14,76.82,65.14,61.43,55.99,55.66,32.27,28.78,20.78；IR(cm ^-1 ):2976.23,2331.06,1720.48,1605.26,1580.56,1551.26,1414.64,1279.38,1246.35,1225.36,1030.18,1002.64,750.42,716.24。

example 2

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

In step 1 of this example, paeonol in step 1 of example 1 was replaced with equimolar 2, 4-dihydroxyacetophenone (compound I-2), and the other steps were the same as in step 1 of example 1, to obtain 3.52g of compound III-2 as white crystals in 85% yield. In step 3 of this example, compound III-1 of step 3 of example 1 was replaced with equimolar compound III-2, and the other steps were the same as in step 3 of example 1, to give 4.71g of compound VI-2 as a white solid, i.e., compound 2, in 65% yield, m.p.143-144 ℃ and structural characterization data as follows: ¹ H NMR(400MHz,CDCl ₃ )δ:12.74(s,1H),7.66(s,1H),7.64(d,J＝1.2Hz,1H),7.63(t,1H),7.17(s,1H),7.13(d,J＝1.9Hz,1H),7.10(dd,J＝3.8,1.8Hz,2H),7.06(s,1H),7.04(s,1H),6.45(d,J＝2.5Hz,1H),6.44-6.42(m,2H),6.40(s,1H),6.36(s,1H),4.40(t,J＝6.2Hz,2H),4.14(t,J＝6.1Hz,2H),3.88(s,6H),2.55(s,3H),2.23(s,3H)2.19-2.16(m,2H)； ¹³ C NMR(100MHz,CDCl ₃ )δ:202.77,168.98,166.86,165.34,165.28,151.44,141.51,132.45,121.40,114.01,108.15,101.31,77.14,76.82,64.80,61.22,55.99,28.49,26.39,20.79；IR(cm ^-1 ):2962.35,2889.58,2359.82,1757.29,1715.34,1632.21,1509.73,1377.54,1327.59,1253.68,1189.7,1170.32,1160.73,1063.45,998.03,844.99,819.08,598.06。

example 3

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

In step 1 of this example, paeonol in step 1 of example 1 was replaced with equimolar eugenol (compound I-3), and the other steps were the same as in step 1 of example 1, to obtain 2.58g of white crystalline compound III-3 in 88% yield. In step 3 of this example, the compound III-1 of step 3 of example 1 was replaced with equimolar compound III-3, and the other steps were the same as those of step 3 of example 1, to give 3.78g of compound VI-3 as white crystals, i.e., compound 3, in a yield of 68%, m.p.79 to 80 ℃, as represented by the structural characterization data: ¹ H NMR(400MHz,CDCl ₃ )δ:7.64(d,J＝16.0Hz,1H),7.26(s,1H),7.10(s,2H),7.05(d,J＝8.0Hz,1H),6.84(d,J＝8.7Hz,1H),6.71(d,J＝6.1Hz,1H),6.38(d,J＝16.0Hz,1H),5.95(d,J＝16.8,6.7Hz,1H),5.08(d,J＝17.0Hz,2H),4.42(t,J＝5.1Hz,2H),4.14(t,J＝6.3Hz,2H),3.86(d,J＝7.8Hz,3H),3.33(d,J＝6.7Hz,1H),2.24(dd,J＝8.1,4.5Hz,2H),1.57(s,1H)； ¹³ C NMR(100MHz,CDCl ₃ )δ:168.97,168.89,151.42,149.51,146.58,144.21,141.44,137.73,133.42,133.30,123.34,120.49,118.28,115.79,113.51,112.38,111.22,77.47,77.15,76.83,65.82,61.65,55.99,39.92,28.78；IR(cm ^-1 ):2920.34,2360.96,1725.26,1606.95,1552.89,1417.52,1319.77,1247.40,1175.28,1139.46,1064.98,995.54,786.96,710.54,610.51。

example 4

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

In step 2 of this example, the compound IV-1 in step 2 of example 1 was replaced with an equimolar compound IV-2, and the other steps were the same as those of step 2 of example 1, to obtain 2.58g of the white crystalline compound V-2 in 88% yield. In step 3 of this example, the compound V-1 in step 3 of example 1 was replaced with an equimolar compound V-2, and the other steps were the same as those of step 3 of example 1, to obtain 4.87g of a white crystalline compound VI-4, namely, compound 4,the yield is 71%, the m.p.126-127 ℃, and the structural characterization data are as follows: ¹ H NMR(400MHz,CDCl ₃ )δ:7.86(d,J＝1.0Hz,1H),7.83(d,J＝1.0Hz,1H),7.65(s,1H),7.61(s,1H),7.41(s,1H),7.39(s,1H),7.36(s,1H),7.26(s,1H),7.23(s,1H),7.21(s,1H),6.53(d,J＝10.0Hz,2H),6.45(s,2H),6.40(s,1H),6.36(s,1H),4.43(t,J＝6.2Hz,3H),4.17(t,J＝6.1Hz,3H),3.84(d,J＝1.0Hz,3H),2.61(d,J＝1.0Hz,3H),2.30(d,J＝2.4Hz,3H),2.27(m,J＝6.1Hz,2H),2.17(s,2H)； ¹³ C NMR(100MHz,CDCl ₃ )δ:197.93,177.73,166.58,164.56,160.23,143.35,142.50,132.92,124.10,122.87,118.93,105.26,99.02,77.33,76.81,65.09,61.48,55.67,32.27,28.73,20.76；IR(cm ^-1 ):2955.64,1780.24,1750.35,1630.24,1580.64,1496.51,1410.38,1382.36,1366.35,1010.94,820.64,704.52,650.32。

example 5

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

In step 1 of this example, the paeonol (compound I-1) in example 4 was replaced with equimolar 2, 4-dihydroxyacetophenone (compound I-2), and the other steps were the same as in step 1 of example 4, to obtain compound III-2 in 61% yield. In step 3 of this example, the compound III-1 of step 3 of example 4 was replaced with equimolar compound III-2, and the other steps were the same as in step 3 of example 4, to give 4.3g of compound VI-5 as a white solid, i.e., compound 5, in 61% yield, m.p.:88-89 ℃ as structural characterization data: ¹ H NMR(400MHz,CDCl ₃ )δ:12.74(s,1H),7.66-7.58(m,2H),7.41(d,J＝2.1Hz,1H),7.39(d,J＝2.1Hz,1H),7.36(d,J＝2.1Hz,1H),7.23(s,2H),7.22(d,J＝8.4Hz,1H),6.45(d,J＝2.5Hz,1H),6.43(d,J＝8.4Hz,1H),6.38(d,J＝8.4Hz,1H),4.39(t,J＝6.2Hz,2H),4.13(t,J＝6.1Hz,2H),2.56(s,3H),2.32-2.29(m,6H),2.25-2.16(m,2H),1.57(s,2H)； ¹³ C NMR(100MHz,CDCl ₃ )δ:202.81,168.30,166.65,165.34,165.27,143.26,142.49,132.48,124.07,119.01,108.12,101.35,77.15,76.83,64.78,61.33,28.45,26.35,20.80,20.76；IR(cm ^-1 ):3412.74,3072.89,2963.33,2899.11,2621.23,1770.16,1701.95,1630.20,1559.66,1420.12,1375.31,1251.94,1195.70,1054.45,998.21,899.14,827.47,710.46,601.39.

example 6

Synthesis of Compound 6: (E) -4- (3- (3- (3- (4-allyl-2-methoxyphenoxy) propoxy) -3-oxo-1-propen-1-yl) -1, 2-benzenediacetic acid ester

In step 1 of this example, paeonol (compound I-1) in example 4 was replaced with equimolar eugenol (compound I-3), and the other steps were the same as in step 1 of example 4, to obtain 2.58g of compound III-3 in 88% yield. In step 3 of this example, the compound III-1 of step 3 of example 4 was replaced with equimolar compound III-3, and the other steps were the same as in step 3 of example 4, to give 3.25g of compound VI-1 as a white solid, i.e., compound 6, in 60% yield, m.p.: 74-75 ℃ and structural characterization data as follows: ¹ H NMR(400MHz,CDCl ₃ )δ:7.63(d,J＝16.0Hz,1H),7.41(dd,J＝8.5Hz,1H),δ7.39(d,J＝8.5Hz,1H),δ7.35(d,J＝16.0Hz,1H),7.22(d,J＝8.4Hz,1H),6.83(d,J＝8.4Hz,1H),6.71(d,J＝6.4Hz,1H),6.38(d,J＝6.4Hz,1H),5.95(m,J＝6.7Hz,1H),5.07(t,J＝14.5Hz,1H),4.17(t,2H),3.82(d,J＝14.5Hz,9H),3.32(s,1H),2.79(s,1H),2.30(s,3H),2.11-1.97(m,2H),1.63(s,2H),1.25-1.22(m,2H)； ¹³ C NMR(100MHz,CDCl ₃ )δ:168.27,168.20,166.67,146.56,142.97,137.74,133.27,120.50,119.25,115.78,113.49,112.34,77.45,77.13,76.81,65.78,61.74,55.97,39.93,28.75,20.81,20.77；IR(cm ^-1 ):2941.43,2890.54,2364.27,2327.87,1968.56,1642.92,1603.43,1564.35,1415.99,1258.56,1206.17,1058.54,961.62,832.27,711.23,613.01。

example 7

Synthesis of Compound 7:3- (2-acetyl-5-methoxyphenoxy) propyl cinnamic acid ester

In step 2 of this example, compound IV-1 in step 2 of example 1 was replaced with equimolar cinnamic acid (compound IV-3), and the other steps were the same as in example 1 to obtain compound V-3. In step 3 of this example, the compound V-1 of step 3 of example 1 was replaced with an equimolar compound V-3, and the other steps were the same as those of step 3 of example 1, to give 4.55g of compound VI-7 as a white solid, i.e., compound 7, in 65% yield, m.p.: 75-76 ℃ C., structural characterization data: ¹ H NMR(400MHz,CDCl ₃ )δ:7.90(d,J＝10.4Hz,1H),7.70(d,J＝15.9Hz,1H),7.57(d,J＝15.9Hz,1H),7.41(d,J＝18.29Hz,1H),7.27(s,1H),6.87(s,1H),6.68(d,J＝18.29Hz,2H),4.42(d,J＝5.9Hz,2H),4.17(d,J＝5.9Hz,2H),2.40-2.15(m,2H),1.60(d,J＝9.3Hz,2H),1.25(s,2H)； ¹³ C NMR(100MHz,CDCl ₃ )δ:195.99,167.03,165.32,162.99,145.23,134.09,129.09,128.56,123.68,116.90,109.62,77.45,77.13,76.81,65.05,61.11,29.70,28.59；IR(cm ^-1 ):2941.43,2890.54,2364.27,2327.87,1968.56,1642.92,1603.43,1564.35,1415.99,1258.56,1206.17,1058.54,961.62,832.27,711.23,613.01。

example 8

Synthesis of Compound 8:3- (3-acetyl-4-hydroxyphenoxy) cinnamic acid propyl ester

In step 2 of this example, the paeonol (compound I-1) in example 7 was replaced with equimolar 2, 4-dihydroxyacetophenone (compound I-2), and the other steps were the same as in step 1 of example 7, to obtain compound III-2 in 61% yield. In step 3 of this example, compound III-1 of step 3 of example 7 was replaced with equimolar compound III-2, the other steps being similar to those of example 7In the same way, 4.60g of compound VI-8 are obtained as a white solid, i.e. compound 8, in 64% yield, m.p.: 117℃and with structural characterization data: ¹ H NMR(400MHz,CDCl ₃ ) δ:12.74 (s, 1H), 7.63 (s, 1H), 7.22 (d, j=8.2 hz, 2H), 7.08 (d, j=8.2 hz, 2H), 7.06 (s, 1H), 7.02 (d, j=15.9 hz, 1H), 6.84 (d, j=8.6 hz, 1H), 6.46 (d, j=8.6 hz, 1H), 6.29 (d, j=15.9 hz, 1H), 4.38 (t, j=2 hz, 2H), 4.13 (t, j=6.1 hz, 2H), 3.81 (s, 3H), 2.54 (s, 3H), 2.34-2.09 (m, 2H), as shown in fig. 1; ¹³ C NMR(100MHz,CDCl ₃ ) δ:196.00,167.03,165.33,163.00,145.24,134.09,129.09,128.56,123.68,116.90,109.62,77.45,77.13,76.81,65.04,61.10,29.70,28.58, as shown in FIG. 2; IR (cm) ^-1 ):3414.76,2910.35,2360.96,1720.21,1606.95,1552.89,1415.66,1319.77,1247.40,1175.28,1138.83,1062.98,995.54,766.96,710.38,620.15。

Example 9

Synthesis of Compound 9:3- (4-allyl-2-methoxyphenoxy) cinnamic acid propyl ester

In step 1 of this example, paeonol in step 1 of example 7 was replaced with equimolar eugenol (compound I-3), and the other steps were the same as in step 1 of example 7, to obtain 2.58g of white crystalline compound III-3 in 88% yield. In step 3 of this example, compound III-1 of step 3 of example 7 was replaced with equimolar compound III-3, and the other steps were the same as in example 7 to give 3.49g of compound VI-9, compound 9, in 63% yield, m.p.: 115 ℃ as structural characterization data: ¹ H NMR(400MHz,CDCl ₃ )δ:7.69(d,1H),7.36(s,1H),7.53(s,J＝8.5Hz,2H),7.22(d,J＝8.2Hz,2H),7.08(d,J＝8.2Hz,2H),7.06(d,J＝8.5Hz,2H),7.02(d,J＝14.5Hz,1H),6.92(s,J＝14.5Hz,1H),6.85(d,J＝8.6Hz,1H),6.72(d,J＝5.5Hz2H),6.44(d,J＝16.0Hz,1H),5.95(m,J＝5.5Hz,1H),5.08(d,J＝16.0Hz,1H),4.42(t,J＝6.2Hz,2H),4.14(t,J＝6.3Hz,2H),3.85(s,3H),3.34(d,J＝6.6Hz,2H),2.30-2.19(m,2H),1.57(s,1H)；IR(cm ^-1 ):3218,2966.26,2359.96,1771.99,1706.48,1629.35,1554.31,1417.05,1249.95,1228.36,1009.59,837.03,813.41,711.19,608.22。

example 10

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

In step 2 of this example, 13.95g (0.09 mol) of t-butyldimethylchlorosilane (TBDMSCl) and 5.9g (0.03 mol) of Compound IV-1 were charged into a round-bottomed flask containing 100mL of N, N-dimethylformamide, the temperature was lowered to-10℃and stirred until it was completely dissolved, 120mL of a methylene chloride solution containing 8.08g (0.08 mol) of triethylamine was slowly dropped, and after stirring for 1 hour, the temperature was gradually raised to room temperature and the stirring reaction was continued for 4 hours. After the reaction was completed, the organic layer was washed with water and was dried over MgSO ₄ Drying, and then concentrating under reduced pressure to recover the excess solvent, and purifying by 100-200 mesh silica gel column chromatography (petroleum ether: ethyl acetate=1:2, V/V) to obtain the intermediate compound as a white powder (yield 90%). The resulting white powder was added to a round bottom flask containing 20mL of N, N-dimethylformamide, connected to a tail gas absorber, and 15mL of SOCl was added ₂ Heating to 80 ℃, heating to 90 ℃ after no gas is generated during the reaction, recovering thionyl chloride, cooling to room temperature, concentrating under reduced pressure to obtain a yellow solid compound V' -1, and directly carrying out further reaction. In step 3 of this example, the compound I-1 of step 3 of example 1 was replaced with equimolar compound V' -1, and the other steps were the same as in step 3 of example 1, to give 3.45g of compound VI-10 as a white solid, i.e., compound 10 in 63% yield, m.p:115℃as structural characterization data: ¹ H NMR(400MHz,CDCl ₃ )δ:7.85(d,J＝8.8Hz,1H),7.63(d,J＝15.9Hz,1H),7.26(s,1H),7.03(dd,J＝5.4,2.0Hz,3H),6.84(d,J＝8.7Hz,1H),6.53(dd,J＝8.8,2.3Hz,1H),6.46(d,J＝2.3Hz,1H),6.29(d,J＝15.9Hz,1H),4.42(t,J＝6.2Hz,2H),4.18(t,J＝6.2Hz,2H),3.84(d,J＝4.1Hz,8H),2.61(d,J＝2.5Hz,3H),2.36-2.20(m,2H),1.58(d,J＝1.2Hz,1H),1.01-0.95(m,9H)； ¹³ C NMR(100MHz,CDCl ₃ )δ:202.75,167.30,165.38,165.30,151.26,147.69,145.32,132.43,115.46,113.99,110.76,108.17,101.31,77.45,77.13,76.81,64.85,60.96,55.50,28.55,26.38,25.73,18.58；IR(cm ^-1 ):3891.33,2855.73,2362.10,1700.70,1610.32,1552.48,1415.97,1272.62,904.53,842.09,710.08。

example 11

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

In step 1 of this example, compound I-1 of step 1 of example 10 was replaced with equimolar compound I-2, and the other steps were the same as step 1 of example 10 to give compound III-2. In step 3 of this example, compound III-1 of step 3 of example 10 was replaced with an equimolar amount of compound III-2, and the other steps were the same as in step 3 of example 10, to give 3.31g of compound VI' -11 as a white solid, i.e., compound 11, in 62% yield, m.p:79-80 ℃ as structural characterization data: ¹ H NMR(400MHz,CDCl ₃ ) δ:12.74 (s, 1H), 7.62 (s, 1H), 7.61 (d, 1H), 7.02 (s, 2H), 6.84 (d, j=8.6 hz, 1H), 6.45 (d, j=8.5 hz, 2H), 6.29 (d, j=15.9 hz, 1H), 4.39 (t, j=6.2 hz, 2H), 4.14 (t, j=6.2 hz, 2H), 3.83 (s, 3H), 2.55 (s, 3H), 2.27-2.10 (m, 2H), 1.57 (s, 2H), 0.99 (s, 9H), as shown in fig. 3; ¹³ C NMR(100MHz,CDCl ₃ ) δ:173.23,165.21,132.37,128.22,122.48,120.80,115.46,114.19,111.31,107.65,100.64,77.52,76.98,76.8,65.04,60.98,55.58,26.31,25.65, as shown in FIG. 4; IR (cm) ^-1 ):3238.39,2944.03,2891.54,2855.82,2622.35,2359.74,1701.44,1608.34,1553.95,1416.34,1272.61,1152.55,905.83,842.02,710.98,612.37。

Example 12

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

In step 1 of this example, compound I-1 of step 1 of example 10 was replaced with equimolar compound I-3, and the other steps were the same as step 1 of example 10 to give compound III-3. In step 3 of this example, compound III-3 was used in place of compound III-1 in step 3 of example 10 in equimolar amounts, and the other steps were the same as in step 3 of example 10 to give 3.42g of compound VI' -12 as a white solid, i.e., compound 12, in 61% yield, m.p.155-156 ℃ as structural characterization data: ¹ H NMR(400MHz,CDCl ₃ )δ:12.54(s,1H),7.61(d,J＝7.9Hz,1H),7.36(s,1H),7.22(d,J＝8.5Hz,1H),7.08(d,J＝8.2Hz,1H),7.06(d,J＝8.5Hz,1H),7.02(d,J＝8.2Hz,1H),6.92(s,J＝8.5Hz,1H),6.46(d,J＝14.5Hz,1H),4.39(t,J＝6.2Hz,2H),4.11(t,J＝6.2Hz,2H),3.86(s,3H),2.54(s,3H),2.31(d,J＝2.5Hz,2H),2.21(s,1H),2.21(s,1H),1.58(s,1H)； ¹³ C NMR(100MHz,CDCl ₃ )δ:202.75,165.39,165.29,164.89,151.27,147.71,145.31,132.41,121.19,115.48,108.45,108.15,77.43,76.15,64.86,60.95,55.76,37.31,25.63；IR(cm ^-1 ):2861.34,256.19,1745.26,1680.59,1671.64,1630.29,1601.34,1580.61,850.32,790.25,751.38,680.24。

example 13

Synthesis of Compound 13: (E) 3- (4-hydroxy-3-methoxyphenyl) propyl-3- (2-acetyl-5-methoxyphenoxy) acrylate

2.0g (3.82 mmol) of Compound VI' -11 are dissolved in 10mL of tetrahydrofuran under nitrogen and 4.5mL of 1mol/LNH are slowly added dropwise ₄ F, stirring and reacting at room temperature for 1 hour. Recovering solvent under reduced pressure after the reaction is finished to obtain crude products, respectively using saturated NaHCO ₃ The organic layer was washed with MgSO, with aqueous, deionized and brine ₄ After drying, purification by 100-mesh silica gel column chromatography gave 1.16g of compound VII-13 as a white powder, i.e., compound 13 in 75% yield, m.p is 127-128 ℃, structural characterization data: ¹ H NMR(400MHz,CDCl ₃ ) Delta 12.76 (s, 2H), 7.61 (s, 1H), 7.08 (d, j=8.2 Hz, 1H), 7.06 (d, j=8.5 Hz, 1H), 7.02 (d, j=8.2 Hz, 1H), 6.92 (s, j=8.5 Hz, 1H), 6.90 (s, 1H), 6.46 (d, 1H), 6.30 (s, 1H), 6.25 (s, 1H), 5.96 (d, j=1.4 Hz), 4.38 (t, j=6.2 Hz, 2H), 4.13 (t, j=6.2 Hz, 2H), 3.92 (s, 3H), 2.55 (s, 3H), 2.20 (m, j=6.2 Hz, 2H), as shown in fig. 5; ¹³ C NMR(100MHz,CDCl ₃ ) δ:202.74,168.54,166.47,165.28,142.04,142.25,132.36,123.67,118.72,107.66,101.83,77.43,77.11,76.79,64.79,61.10,28.10,26.50,26.12,20.68, as shown in FIG. 6; IR (cm) ^-1 ):3434.96,3238.39,2955.44,2893.19,2856.26,1699.01,1628.08,1509.92,1469.06,1280.11,1265.24,1180.25,1020.38,903.50。

Example 14

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

2.0g (3.8 mmol) of compound VI' -12 are dissolved in 10mL of tetrahydrofuran under nitrogen, and 4.5mL of 1mol/L NH are slowly added dropwise ₄ F, stirring and reacting at room temperature for 1 hour. Recovering solvent under reduced pressure after the reaction is finished to obtain crude products, respectively using saturated NaHCO ₃ The organic layer was washed with MgSO, with aqueous, deionized and brine ₄ Purification by 100 mesh silica gel column chromatography gave 1.06g of compound VII-14 as a white powder, i.e. compound 14 in 68% yield, m.p.: 152-153 ℃ structural characterization data: ¹ H NMR(400MHz,CDCl ₃ ) Delta 12.76 (s, 1H), 7.65 (d, 1H), 7.10 (d, j=1.0 hz, 1H), 7.07 (d, j=8.2 hz, 1H), 6.92 (d, j=8.2 hz, 1H), 6.84 (d, j=8.5 hz, 1H), 6.44 (d, j=8.5 hz, 1H), 6.30 (s, 1H), 6.29-6.24 (m, 1H), 4.40 (d, j=6.4 hz, 2H), 4.15 (d, j=6.3 hz, 2H), 3.93 (s, 3H), 3.33 (d, j=6.7 hz, 3H), 2.70-2.48 (m, 2H), 2.19 (s, 3H), 2.04 (s, 1H), 1.31-1.21 (m, 2H), as shown in fig. 7; ¹³ C NMR(100MHz,CDCl ₃ )δ:197.63,197.61,194.74,164.61,164.60,164.46,133.25,120.62,105.07,105.06,105.03,100.0,99.04,77.33,76.81,66.69,55.65,55.62,32.01,30.99, as shown in fig. 8; IR (cm) ^-1 ):3585.91,3414.30,1653.24,1630.48,1582.63,1430.25,1120.56,840.65,730.28,708.51,620.65。

Example 15

Evaluation of tyrosinase inhibitory Activity of Compounds 1 to 14 of the present invention

1. Evaluation of Mushroom tyrosinase inhibitory Activity

Levodopa was used to study the tyrosinase inhibitory activity of compounds 1-14 against mushroom tyrosinase, and the tyrosinase inhibitory activity of the target compounds was screened at concentrations of 1mM, 0.5mM, 0.25mM and 0.125mM, respectively, and at inhibition rates (I%) and IC at 1mM concentrations ₅₀ The inhibitory activity was evaluated. The specific test method is as follows:

the activity of bisphenol tyrosinase of compounds 1-14 was tested using levodopa as substrate and kojic acid as positive control. Mushroom tyrosinase (Sigma-aldrich, 2687U mg) ^-1 ) For activity determination. Respectively dissolving the compounds 1 to 14 in DMSO to obtain inhibitor solutions; 160. Mu.L of phosphate buffer (pH 6.86) and 20. Mu.L of mushroom tyrosinase (final concentration 33.3U mL) were sequentially added to a 96-well plate ^-1 ) And 10. Mu.L of inhibitor solution (final concentration of 1mM, final volume concentration of DMSO 5.0%) were pre-incubated at room temperature for 10min, and 10. Mu.L of L-dopa (final concentration of 0.2 mM) was added thereto, and the enzyme reaction was monitored at 475nm for 1min by means of a microplate reader to give absorbance A. The inhibition rate of tyrosinase reaction was calculated as follows:

tyrosinase activity (I%) = [ (a) _sample -A _blank )/(A _control -A _blank )]×100

A _sample Absorbance of the sample; a is that _blank Absorbance for blank; a is that _control The results of bisphenol enzyme inhibition activity of mushroom tyrosinase of compounds 1 to 14 are shown in Table 1 as absorbance of positive control.

Table 1 tyrosinase inhibition ratio (I%) of Compounds 1 to 14

The results in Table 1 show that compounds 1 to 14 have tyrosinase inhibitory activities to different degrees, and the inhibition rate of tyrosinase is greater than that of the parent compounds (paeonol, eugenol, cinnamic acid, ferulic acid and caffeic acid) at the concentration of 1mM, wherein the inhibition rates of compounds 1,3, 5, 6, 8, 10, 11, 12,13 and 14 are all more than 60%, and the compounds show moderate inhibition activities; the inhibition rates of compound 8, compound 14, and compound 13 were 80.1%,80.6%, and 82.4%, respectively.

To evaluate the inhibitory activity of compounds 1 to 14, IC was calculated by comparing the activity with that of the parent compounds (paeonol, eugenol, cinnamic acid, ferulic acid and caffeic acid) ₅₀ Values (see table 2) and inhibition curves for compounds 8, 13 and 14 at 10 different concentrations were obtained by Graphpad prism8.0 software (see figure 9).

TABLE 2 Compounds 1 to 14 and IC of the parent Compounds ₅₀ Value (mu M)

The results in Table 2 show that the IC of the parent compound ₅₀ The values range from 154.6. Mu.M to 1135.8. Mu.M. IC of Compounds 1, 4, 5, 6, 8, 10, 11, 12,13, 14 ₅₀ The values were all lower than the parent compound, indicating that the activity was better than the parent compounds (paeonol, eugenol, cinnamic acid, ferulic acid and caffeic acid). Wherein, IC of Compounds 13 and 14 ₅₀ 13.98. Mu.M and 15.16. Mu.M respectively, lower than the positive drug kojic acid (30.83. Mu.M) and 2 times and 2.3 times the inhibition activity of the positive drug kojic acid respectively.

2. Cell experiment

(1) Compound 8, 13 and 14 vs B ₁₆ F ₁₀ Cytotoxicity of cells

B ₁₆ F ₁₀ Melanoma cells (1.5 x 10 ⁵ cell/mL), pancreatin digests cells, counts to adjust cell concentration to 1 x 10 ⁵ /mL. The 96-well plate was transferred into an incubator for cultivation (37 ℃,5% CO) ₂ ) With DMEM medium (containing 100IU/mL penicillin and 100. Mu.g/mL streptomycin) 37℃and 5% CO ₂ An incubator. After the cells adhere to the wall, the drugs were added for 24 hours according to each experimental group. Compounds 8,11, 13 and 14 were dissolved in DMSO and diluted with medium to 200. Mu.M, 100. Mu.M, 50. Mu.M, 25. Mu.M, 12.5. Mu.M, respectively, and added to cell culture plates for further incubation for 24h. After the completion of the incubation, 10. Mu.L of 5mg/mL MTT solution (note that no air bubbles can be generated) was added to each well, and incubated in an incubator for 5 hours. After the incubation is completed, the culture solution in the well is sucked away. 150 μl DMSO was added to each well and the mixture was shaken on a shaker for 10min at low speed to allow sufficient dissolution of the crystallized product. Simultaneously setting three groups of blank holes, MTT and DMSO, and detecting absorbance values of all holes at 490nm by an enzyme-labeling instrument. The MTT method measures cell viability and the results are calculated as a percentage of the control.

In B way ₁₆ F ₁₀ Melanoma cells were used as subjects to determine and evaluate the cell viability of compounds 8,11, 13 and 14, and four compounds were applied to cells at concentrations of 0, 25, 50, 100 and 200 μm for 24h, respectively, and positive groups were controlled with arbutin and kojic acid, and the experimental results are shown in fig. 10. From the figure, arbutin, compounds 8 and 11 all showed some cytotoxicity at 200. Mu.M, whereas compounds 13 and 14 were safer than kojic acid and arbutin at a concentration of 100. Mu.M, and the results of two-factor analysis showed that compounds 13 and 14 showed a higher degree of cytotoxicity than the blank group at 50. Mu.M (alpha.<0.05) cell viability was not significantly different, indicating that it was highly safe.

(2) Cell melanogenesis assay and evaluation

B ₁₆ F ₁₀ Melanoma cells (1.5 x 10 ⁵ cell/mL) at 37℃and 5% CO ₂ Is inoculated in a cell culture plate for incubation for 24 hours, and is activated by 1 mu M alpha-MSH to increase melanin production. Then incubated with compounds 8,11, 13 and 14 (200. Mu.M, 100. Mu.M, 50. Mu.M and 25. Mu.M, respectively) in cell culture medium for 24h, washed with PBS for 2-3 times to remove the medium contents, and with 200. Mu.L 1The melanin is dissolved by incubation with N NaOH. The melanin content was determined spectrophotometrically at 405nm wavelength using an enzyme-labeled instrument. Each well was repeated three times. Model group 1 μΜ α -MSH (cytokinin) was added to enhance melanin content and stabilize melanogenesis content in melanocytes. Kojic acid and arbutin are used as positive control. The calculation formula is as follows:

melanogenesis inhibition rate (I%) = [ (a) _sample -A _blank )/(A _control -A _blank )]×100

A _sample OD of test compound for sample blank control blank x sample ₄₀₅ Absorbance; a is that _blank OD of blank ₄₀₅ Absorbance; a is that _control OD representing control ₄₀₅ Absorbance. The results are shown in FIG. 13.

The anti-melanogenesis inhibitory assay results of fig. 11 show that compounds 8,11, 13 and 14 have a significant difference from the model group and exhibit a degree of concentration dependence within a concentration level of 200 μm, and that compounds 8,11 and 14 exhibit a very significant statistical difference from the α -MSH model group at a level of 100 μm, with inhibition rates of 19.62%, 20.59% and 23.83%, respectively, with the highest anti-melanogenesis activity at compound 14, compounds 11, 8 times.

(3) Measurement and evaluation of cell tyrosinase Activity

To determine cellular tyrosinase activity, B was used ₁₆ F ₁₀ Melanoma cells were placed in 48-well plates, each well containing 1X 10 cells ⁵ Individual cells, containing 1 μM α -MSH and each concentration gradient sample for 48h (B ₁₆ F ₁₀ Cells were treated with 1. Mu.M. Alpha. -MSH to activate melanin production in cells, and after 24 hours incubation with different concentrations of sample, kojic acid and arbutin (positive control), cells were washed 2-3 times with PBS and then disrupted with 100. Mu.L of lysis buffer consisting of 50mM PBS (90. Mu.L, pH6.8, 0.1mM, PMSF (5. Mu.L) and 1% Triton X-100 (5. Mu.L). The resulting lysed cells were frozen at-80℃for 30min and then centrifuged at 12000rpm for 30min at 4 ℃. Then 80. Mu.L of lysate was added and the supernatant was mixed with 20. Mu.L of 2mM L-DOPA (L-DOPA) and incubated in 96-well plates for 30min at 37 ℃.The optical density at 475nm was measured with a microplate reader. Each group was repeated three times.

Tyrosinase activity inhibition rate (I%) = [ (a) _sample -A _blank )/(A _control -A _blank )]×100

A _sample OD of test compound for sample blank control blank x sample ₄₇₅ Absorbance; a is that _blank OD of blank ₄₇₅ Absorbance; a is that _control OD representing control ₄₇₅ Absorbance.

Different dose levels of Compounds 8,11, 13 and 14 vs B ₁₆ F ₁₀ The inhibitory activity of the cell tyrosinase activity is shown in fig. 12. Wherein the blank group is normal B ₁₆ F ₁₀ Cells, results indicate that 4 compounds have significant differences (α) at 200 μM and 100 μM levels compared to control group<0.05 Compound 13 showed moderate inhibitory activity at 100 μm level, but compounds 8,11 and 14 showed strong tyrosinase inhibitory activity, especially with compounds 11 and 14 having the highest inhibitory activity, higher than positive drug kojic acid and arbutin (19.21% and 20.45%) at the same concentration level, with inhibition rates of 20.59% and 23.83%, respectively, and a degree of concentration dependence.

In conclusion, the comprehensive results of the mushroom tyrosinase activity inhibition experiments and the related cell experiments show that the compounds 8 and 11 have higher inhibition activity on mushroom tyrosinase; when the level of 100 mu M is 100 mu M, the cell activities of the compounds 13 and 14 are higher than that of positive control medicine kojic acid, the positive control medicine kojic acid is safer, and in the cell tyrosinase inhibitory activity, the inhibitory activity of the compound 14 is strongest within the range of 50-100 mu M; compounds 8 and 13 were slightly better active at low concentration levels than compound 14 in terms of anti-melanogenesis activity.

Example 16

30mg of each of the compounds 8,11, 13, 14 was added to the sample bottle, and 1mL of olive oil was added, heated to complete dissolution, and then allowed to stand at room temperature for 10 minutes. As can be seen from fig. 13, all of the compounds 8,11, 13, 14 form stable gels in olive oil (compounds 8,11, 13, 14 are in sequence from left to right), and can be used as small molecule gels for cosmetics and the like.

Claims

1. The cinnamic acid ester derivative is characterized by being any one of the following compounds 1, 4,6, 8, 13 and 14:

Compound 8:3- (3-acetyl-4-hydroxyphenoxy) cinnamic acid propyl ester

。

2. Use of a cinnamate derivative of claim 1 in the preparation of a tyrosinase inhibitor.

3. Use of the cinnamate derivative of claim 1 in the preparation of a small molecule gel.