Application of a coumarin derivative in inhibiting tyrosinase activity

Abstract

The present invention discloses an application of a coumarin derivative in inhibiting tyrosinase activity. The chemical structural formula of the coumarin derivative is shown in formula (I): #imgabs0# wherein R1 is H, halogen, C 1～10 Alkyl, C 1～10 haloalkyl, hydroxyl; R2 is H, halogen, C 1～10 Alkyl, C 1～10 R3 is a substituted or unsubstituted phenyl group, a substituted or unsubstituted styryl group. A series of coumarin derivatives synthesized by the present invention have a good inhibitory effect on tyrosinase. Studies have shown that the inhibition of tyrosinase by such derivatives is reversible and mixed, and 3D fluorescence and CD spectrum results show that the compounds of the present invention can cause changes in the conformation of tyrosinase.

Description

Technical Field

[0001] The present invention relates to the technical field of new uses of coumarin derivatives, in particular to the use of coumarin derivatives in inhibiting tyrosinase activity. Background Art

[0002] Melanin is produced by melanocytes and is widely distributed in humans, animals, and plants. It is an important biological pigment for human skin, hair, and eye color. Melanin production leads to pigmentation and protects the skin from UV-related damage. However, excessive accumulation of melanin is a characteristic of multiple pigmentary diseases, including melasma, freckles, and melanoma. Melanogenesis is a complex process involving a series of chemical and enzymatic reactions. Tyrosinase, as the only rate-limiting enzyme in melanogenesis, is essential for melanogenesis in human skin cells. Tyrosinase is responsible for the first two catalytic steps in the melanin biosynthesis process: hydroxylation of tyrosine to L-dihydroxyphenylalanine (L-DOPA) and subsequent oxidation of L-DOPA to dopaquinone. Tyrosinase-associated proteins immediately catalyze the conversion of dopaquinone into melanin. Therefore, regulation of tyrosinase activity is essential for preventing melanin synthesis. To date, a large number of natural and synthetic tyrosinase inhibitors have been developed. However, only a few inhibitors, including arbutin and kojic acid, have been used as marketable therapeutic agents. But they still have adverse side effects, including dermatitis, cytotoxicity, and skin cancer.

[0003] Therefore, these issues prompted us to discover safer and more effective tyrosinase inhibitors. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a use of a coumarin derivative in inhibiting tyrosinase activity, which can effectively inhibit tyrosinase activity.

[0005] According to the first aspect of the present invention, a coumarin derivative is used in inhibiting tyrosinase activity, and the chemical structural formula of the coumarin derivative is shown in formula (I):

[0006]

[0007] Wherein, R1 is H, halogen, C 1～10 Alkyl, C 1～10 The halogenated alkyl and hydroxyl groups;

[0008] R2 is H, halogen, C 1～10 Alkyl, C 1～10 The halogenated alkyl and hydroxyl groups;

[0009] R3 is a substituted or unsubstituted phenyl group or a substituted or unsubstituted styryl group.

[0010] The application of the embodiments of the present invention has at least the following beneficial effects:

[0011] A series of coumarin derivatives synthesized by the present invention have good inhibitory effects on tyrosinase. Studies have found that the inhibition of tyrosinase by such derivatives is reversible and mixed, and 3D fluorescence and CD spectrum results show that the compounds of the present invention can cause changes in the conformation of tyrosinase.

[0012] According to some embodiments of the present invention, R3 is selected from at least one of the following structural formulas:

[0013]

[0014] According to some embodiments of the present invention, the method for preparing the coumarin derivative comprises the following steps:

[0015] S3, subjecting compound 3 to an esterification reaction with compound 4 to obtain a coumarin derivative;

[0016] The structural formulas of the compounds 3 and 4 are as follows:

[0017]

[0018] According to some embodiments of the present invention, the compound 3 is prepared by the following method:

[0019] S2, reacting compound 2 with hydroxylamine hydrochloride to obtain compound 2;

[0020]

[0021] According to some embodiments of the present invention, the compound 2 is prepared by the following method:

[0022] S1, subjecting compound 1 to ethyl acetoacetate to a Perkins reaction to obtain compound 2;

[0023] The structural formula of compound 1 is as follows:

[0024]

[0025] According to some embodiments of the present invention, the coumarin derivative is prepared into a tyrosinase inhibitor composition.

[0026] According to some embodiments of the present invention, the coumarin derivative is prepared as an inhibitor against enzymatic browning of fruits and vegetables.

[0027] According to some embodiments of the present invention, the coumarin derivative is prepared into a whitening cosmetic.

[0028] According to some embodiments of the present invention, the cosmetic is selected from a facial cream, a facial mask, a lotion, an essence, a toner, a facial cleanser or a shower gel.

[0029] According to some embodiments of the present invention, the coumarin derivative is prepared into a drug for preventing and treating human pigmentation diseases caused by abnormal melanin, melanoma, and other diseases that require inhibition of tyrosinase activity.

[0030] According to some embodiments of the present invention, the dosage form of the drug is selected from solid powder, aqueous solution, alcohol solution, cataplasm, injection, drip, powder injection, granule, tablet, granule, powder, oral liquid, sugar-coated tablet, film-coated tablet, enteric-coated tablet, capsule, lozenge, pill, ointment, pill, spray, pellet, orodispersible tablet or pellet.

[0031] Definitions and general terms

[0032] “C 1～10 "Alkyl" means an alkyl group having 1 to 10 carbon atoms, including C 1～10 Straight chain alkyl, C 1-10 The branched alkyl and C 3-10 The cycloalkyl group may be, for example, a straight-chain alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 carbon atoms, a branched-chain alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 carbon atoms, or a cycloalkyl group having 3, 4, 5, 6, 7, 8, 9, 10 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, cyclopropyl, methylcyclopropyl, ethylcyclopropyl, cyclopentyl, methylcyclopentyl, cyclohexyl, etc.

[0033] “C 1～10 The "haloalkyl" means a haloalkyl group having a total carbon atom number of 1 to 10, wherein the alkyl group is as defined above and is substituted by one or more halogen atoms which are the same or different, for example -CH2Cl, -CF3, -CCl3, -CH2CF3, -CH2CCl3, etc.

[0034] The "halogen" includes any one or two or more of fluorine, chlorine, bromine and iodine.

[0035] "Substituted or unsubstituted phenyl" means that at least one H in the phenyl group is optionally substituted with a corresponding group as defined herein.

[0036] "Substituted or unsubstituted styryl" has a similar definition, and optionally at least one H in the styryl is substituted with a corresponding group as defined herein.

[0037] In the present invention, "substituted or unsubstituted" means that the group may or may not be further substituted by one or more groups selected from the following: 1～10 Alkyl, halogen, C 1～10 The halogenated alkyl, C 1～10 Alkoxy, nitro, and hydroxyl groups.

[0038] The aforementioned preparation method of the present invention may also involve various post-processing operations known in the art, such as extraction, washing, filtration, column chromatography, recrystallization, etc. The present invention has no particular limitation on this, and those skilled in the art should not interpret this as a limitation on the present invention.

[0039] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0041] Figure 1 A graph showing the half-maximal inhibitory concentration of a coumarin derivative as a tyrosinase inhibitor on tyrosinase in vitro is provided for Example 30 of the present invention; (A) is an enzyme kinetic graph; (B) is a substrate kinetic graph;

[0042] Figure 2 3D fluorescence images of tyrosinase in vitro, (a) 3D fluorescence spectrum without the coumarin derivatives of Example 30 as tyrosinase treatment, (b) 3D fluorescence spectrum with the coumarin derivatives of Example 30 as tyrosinase treatment;

[0043] Figure 3 This is a CD diagram of the coumarin derivatives of Example 30 of the present invention as tyrosinase inhibitors on tyrosinase in vitro;

[0044] Figure 4 This is a molecular docking diagram of the interaction rules between coumarin derivatives and tyrosinase in Example 30 of the present invention; (a) the coumarin derivative is positioned in the active pocket; (b) is an interaction diagram between coumarin derivatives and tyrosinase. DETAILED DESCRIPTION

[0045] The following are specific embodiments of the present invention, and the technical solutions of the present invention are further described in conjunction with the embodiments, but the present invention is not limited to these embodiments.

[0046] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0047] Examples 1 to 30

[0048] Examples 1 to 30 provide a series of coumarin derivatives, the reaction equations thereof are as follows, and the preparation methods are as follows:

[0049]

[0050] S1. A mixture of compound 1 (1 mmol), ethyl acetoacetate (1.1 mmol) and piperidine (0.02 mmol) in ethanol (10 mL) was stirred at 70 ° C for 2 h. After the reaction was completed, the mixture was poured into ice water and then filtered to obtain a crude product. It was then recrystallized from ethanol to obtain compound 2.

[0051] S2: A mixture of compound 2 (1 mmol), hydroxylamine hydrochloride (3.0 mmol) and pyridine (0.04 mmol) in ethanol (10 mL) was stirred at room temperature for 20 h. The generated solid was filtered and washed with ethanol to obtain compound 3.

[0052] S3, add the compound 4 in DCM (3mL) to the ice bath solution of compound 3 containing oxime (1.0mmol) and triethylamine (1.1mmol) in DCM (3mL), then react 12 hours after being warming up to room temperature.After quenching with water, the mixture is extracted three times with sodium dichloride, washed with salt water, and dried over magnesium sulfate.Remove solvent under vacuum to obtain crude product, then purify by column chromatography to obtain corresponding coumarin derivative.

[0053] Wherein, R1, R2 and R3 are shown in Table 1:

[0054] Table 1

[0055]

[0056]

[0057]

[0058] The structures of the coumarin derivatives prepared in Examples 1 to 30 were characterized by NMR, MS and melting points. The properties, yields, NMR and mass spectrometry results of each compound are as follows:

[0059] (Example 1, C 20 H 15 NO4).White sold; Yield 67%; mp161-162℃; 11H NMR (500 MHz, Chloroform-d) δ 8.16 (s, 1H), 7.88 (d, J = 16.0 Hz, 1H), 7.62 - 7.55 (m, 4H), 7.45 - 7.38 (m, 3H), 7.38 - 7.29 (m, 2H), 6.59 (d, J = 16.0 Hz, 1H), 2.49 (s, 3H); 13 13C NMR (126 MHz, CDCl3) δ 164.32, 162.14, 159.26, 154.34, 146.84, 143.39, 134.14, 132.96, 130.85, 129.06, 129.02, 128.35, 124.95, 123.58, 118.55, 116.68, 115.29, 77.32, 77.06, 76.81, 15.94; HRMS (ESI) [M + H] + calcd. for C 20 H 15 NO4: 334.1070; found: 334.1074.

[0060] (Example 2, C 21 H 17 NO4). White solid; Yield 62%; m.p. 154 - 155 °C; 1 1H NMR (500 MHz, DMSO-d6) δ 8.34 (s, 1H), 7.90 (dd, J = 7.8, 1.6 Hz, 1H), 7.81 (d, J = 16.1 Hz, 1H), 7.69 (dd, J = 7.5, 4.0 Hz, 3H), 7.48 (d, J = 8.3 Hz, 1H), 7.42 (td, J = 7.5, 1.1 Hz, 1H), 7.27 (d, J = 7.8 Hz, 2H), 6.79 (d, J = 16.0 Hz, 1H), 2.38 (s, 3H), 2.34 (s, 3H). 13 13C NMR (126 MHz, DMSO) δ 164.18, 162.56, 159.13, 154.19, 146.68, 143.72, 141.46, 133.63, 131.70, 130.07, 130.02, 129.14, 125.45, 123.64, 118.87, 116.68, 114.93, 40.48, 40.39, 40.31, 40.23, 40.14, 40.05, 39.98, 39.88, 39.81, 39.64, 39.47, 21.56, 16.15. HRMS (ESI) [M + H] + calcd. for C 21H 17 NO4: 348.1229; found: 348.1233.

[0061] (Example 3, C 20 H 14 ClNO4). White solid; Yield 65%; m.p. 248 - 249 °C; 1 1H NMR (500 MHz, Chloroform - d) δ 8.15 (s, 1H), 7.83 (d, J = 16.0 Hz, 1H), 7.63 - 7.55 (m, 2H), 7.55 - 7.49 (m, 2H), 7.41 - 7.29 (m, 4H), 6.56 (d, J = 16.0 Hz, 1H), 2.48 (s, 3H). 13 13C NMR (126 MHz, CDCl3) δ 164.09, 162.28, 159.24, 154.32, 145.34, 143.43, 136.78, 133.02, 132.60, 129.52, 129.32, 129.07, 124.98, 123.49, 118.50, 116.69, 115.86, 77.35, 77.09, 76.84, 15.97. HRMS (ESI) [M + H] + calcd. for C 20 H 14 ClNO4: 368.0682, found: 368.0687.

[0062] (Example 4, C 20 H 14 BrNO4). White solid; Yield 69%; m.p. 182 - 183 °C; 1 1H NMR (500 MHz, Chloroform - d) δ 8.15 (s, 1H), 7.81 (d, J = 16.0 Hz, 1H), 7.62 - 7.53 (m, 4H), 7.52 – 7.43 (m, 2H), 7.38 - 7.30 (m, 2H), 6.58 (d, J = 16.0 Hz, 1H), 2.48 (s, 3H); 13C NMR (126MHz, CDCl3) δ164.10,162.31,159.26,154.34,145.44,143.45,133.03,132.29,129.71,129.09 ,125.20,124.99,123.50,118.52,116.71,115.97,77.31,77.26,77.06,76.80,15.98.HRMS(ESI)[M+H] + calcd.For C 20 H 14 BrNO4:412.0187; found:412.0181.

[0063] (Example 5, C 20 H 14 FNO4).White sold; Yield 67%; mp166-170℃; 1 H NMR(500MHz,Chloroform-d)δ8.14(s,1H),7.84(d,J=16.0Hz,1H),7.58(ddd,J=9.4,4.8, 2.3Hz,4H),7.38-7.28(m,2H),7.10(t,J=8.6Hz,2H),6.51(d,J=16.0Hz,1H),2.48(s,3H); 13 C NMR (126MHz, CDCl3) δ165.21,164.20,163.20,162.18,159.24,154.33,145.47,143.38,132.98,130.43,130.40,130.33,130.26 ,129.06,124.96,123.55,118.52,116.68,116.30,116.12,115.04,115.02,77.33,77.27,77.07,76.82,15.93; HRMS(ESI)[M+H] + calcd.forC 20 H 14 FNO4:352.0975; found:352.0982.

[0064] (Example 6, C 21 H 17 NO5).White sold; Yield 68%; mp150-152℃; 11H NMR (500 MHz, Chloroform-d) δ 8.15 (s, 1H), 7.83 (d, J = 16.0 Hz, 1H), 7.59 - 7.53 (m, 4H), 7.37 - 7.30 (m, 2H), 6.94 - 6.92 (m, 2H), 6.45 (d, J = 16.0 Hz, 1H), 3.85 (s, 3H), 2.48 (s, 3H); 13 13C NMR (126 MHz, CDCl3) δ 164.70, 161.83, 159.30, 154.32, 146.52, 143.34, 132.92, 130.12, 129.05, 126.91, 124.94, 123.67, 118.57, 116.67, 114.44, 112.54, 77.32, 77.27, 77.07, 76.81, 55.46, 15.91; HRMS (ESI) [M + H] + calcd. for C 21 H 17 NO5: 364.1178; found: 364.1182.

[0065] (Example 7, C 20 H 14 N2O6). Yellow solid; Yield 70%; m.p. 221 - 222 °C; 1 1H NMR (500 MHz, Chloroform-d) δ 8.30 – 8.27 (m, 2H), 8.16 (s, 1H), 7.91 (d, J = 16.0 Hz, 1H), 7.76 - 7.74 (m, 2H), 7.60 (ddd, J = 14.2, 8.0, 1.6 Hz, 2H), 7.39 - 7.31 (m, 2H), 6.73 (d, J = 16.1 Hz, 1H), 2.50 (s, 3H); 13 13C NMR (126 MHz, CDCl3) δ 163.40, 162.80, 159.19, 154.37, 148.78, 143.74, 143.51, 140.12, 133.13, 129.09, 128.95, 125.02, 124.29, 123.35, 119.71, 118.46, 116.75, 77.30, 77.25, 77.05, 76.79, 16.02; HRMS (ESI) [M + H] + calcd. for C 20 H 14 N2O6: 379.0923; found: 379.0927.

[0066] (Example 8, C 21 H 14 F3NO4).White sold; Yield 75%; mp169-171℃; 1 H NMR(500MHz,Chloroform-d)δ8.15(s,1H),7.88(d,J=16.1Hz,1H),7.71-7.65(m ,4H),7.61-7.56(m,2H),7.37-7.30(m,2H),6.66(d,J=16.1Hz,1H),2.49(s,3H); 13 C NMR (126MHz, CDCl3) δ163.76,162.54,159.21,154.35,144.86,143.46,133.06,129.07,128.47,126 .00,125.97,124.99,123.42,118.49,117.96,116.70,77.32,77.07,76.81,15.97; HRMS(ESI)[M+H] + calcd.for C 21 H 14 F3NO4:402.0944; found:402.0951.

[0067] (Example 9, C 20 H 15 NO5).White sold; Yield 75%; mp156-158℃; 1 H NMR (500MHz, DMSO-d6) δ10.12(d,J=23.3Hz,1H),8.33(d,J=23.1Hz,1H),7.93(d,J=8.4 Hz,1H),7.78-7.48(m,5H),6.83(d,J=8.5Hz,2H),6.61(d,J=16.0Hz,1H),2.37(s,4H); 13C NMR (126MHz, DMSO) δ164.49,162.21,160.73,159.15,154.18,146.98,143.66,133.60,131.22,130.00,125.51,125.44,123.73,118.89,11 6.68,116.30,116.26,111.91,40.56,40.47,40.39,40.30,40.22,40.13,40.06,39.97,39.89,39.80,39.63,39.46,16.12; HRMS(ESI)[M+H] + calcd.for C 20 H 15 NO5:350.1018; found:350.1025.

[0068] (Example 10, C 18 H 13 NO4).White sold; Yield 72%; mp157-159℃; 1 H NMR(500MHz,Chloroform-d)δ8.20(s,1H),8.13(dd,J=8.1,1.5Hz,2H),7.66-7.57(m,3H),7.51(t,J=7.7Hz,2H),7.39-7.30(m,2H),2.55(s,3H); 13 C NMR (126MHz, CDCl3) δ163.60,162.71,159.25,154.37,143.51,133.67,133.02,129.76,129.09 ,128.71,128.66,124.98,123.48,118.53,116.70,77.33,77.07,76.82,16.03; HRMS(ESI)[M+H] + calcd.for C 18 H 13 NO4:308.0914; found:308.0926.

[0069] (Example 11, C 19 H 15 NO4).White sold; Yield 70%; mp160-161℃; 11H NMR (500 MHz, Chloroform-d) δ 8.20 (s, 1H), 8.02 (d, J = 8.0 Hz, 2H), 7.59 (t, J = 7.6 Hz, 2H), 7.37 (d, J = 8.3 Hz, 1H), 7.35 - 7.28 (m, 3H), 2.55 (s, 3H), 2.44 (s, 3H); 13 13C NMR (126 MHz, CDCl3) δ 163.76, 162.54, 159.21, 154.35, 144.86, 143.46, 133.06, 129.07, 128.47, 126.00, 125.97, 124.99, 123.42, 118.49, 117.96, 116.70, 77.32, 77.07, 76.81, 15.97; HRMS (ESI) [M + H] + calcd. for C 19 H 15 NO4: 322.1071; found: 322.1084.

[0070] (Example 12, C 18 H 12 ClNO4). White solid; Yield 70%; m.p. 185 - 186 °C; 1 1H NMR (500 MHz, Chloroform-d) δ 8.19 (s, 1H), 8.06 (dd, J = 8.5, 1.6 Hz, 2H), 7.61 - 7.57 (m, 2H), 7.49 (dd, J = 8.4, 1.6 Hz, 2H), 7.39 - 7.31 (m, 2H), 2.55 (d, J = 1.5 Hz, 3H); 13 13C NMR (126 MHz, CDCl3) δ 162.98, 162.81, 159.23, 154.38, 143.59, 140.23, 133.12, 131.13, 129.12, 127.07, 125.02, 123.33, 118.48, 116.74, 77.31, 77.26, 77.05, 76.80, 16.09; HRMS (ESI) [M + H] + calcd. for C 18 H 12 ClNO4: 342.0525; found: 342.0536.

[0071] (Example 13, C 18 H 12BrNO4. White solid; Yield 69%; m.p. 180 - 182 °C; 1 1H NMR (500 MHz, Chloroform - d) δ 8.18 (s, 1H), 8.00 - 7.97 (m, 2H), 7.66 - 7.64 (m, 2H), 7.62 - 7.57 (m, 2H), 7.38 - 7.31 (m, 2H), 2.54 (s, 3H); 13 13C NMR (126 MHz, CDCl3) δ 163.01, 162.94, 159.20, 154.38, 143.58, 133.12, 132.11, 131.22, 129.11, 128.91, 127.53, 125.02, 123.31, 118.48, 116.73, 77.32, 77.07, 76.81, 16.08; HRMS (ESI) [M + H] + calcd. for C 18 H 12 BrNO4: 387.9997; found: 388.0004.

[0072] (Example 14, C 18 H 12 FNO4. White solid; Yield 68%; m.p. 208 - 209 °C; 1 1H NMR (500 MHz, Chloroform - d) δ 8.19 (s, 1H), 8.17 - 8.14 (m, 2H), 7.61 - 7.58 (m, 2H), 7.39 - 7.31 (m, 2H), 7.19 (t, J = 8.6 Hz, 2H), 2.55 (s, 3H); 13 13C NMR (126 MHz, CDCl3) δ 167.17, 165.13, 162.82, 162.67, 159.23, 154.39, 143.54, 133.08, 132.41, 132.33, 129.10, 125.00, 124.89, 124.86, 123.38, 118.51, 116.73, 116.08, 115.90, 77.30, 77.25, 77.04, 76.79, 16.04; HRMS (ESI) [M + H] + calcd. for C 18 H 12 FNO4: 326.0820; found: 326.0832.

[0073] (Example 15, C 19 H15 NO5). White solid; Yield 65%; m.p. 165 - 167 °C; 1 1H NMR (500 MHz, Chloroform - d) δ 8.20 (s, 1H), 8.10–8.08 (m, 2H), 7.59 (d, J = 7.7 Hz, 2H), 7.39 - 7.31 (m, 2H), 7.00 - 6.97 (m, 2H), 3.90 (s, 3H), 2.54 (s, 3H); 13 13C NMR (126 MHz, CDCl3) δ 163.93, 163.38, 162.21, 159.33, 154.36, 143.45, 132.97, 131.90, 129.08, 124.97, 123.60, 120.79, 118.57, 116.70, 114.00, 77.30, 77.25, 77.05, 76.79, 55.56, 16.00; HRMS (ESI) [M + H] + calcd. for C 19 H 15 NO5: 338.1019; found: 338.1033.

[0074] (Example 16, C 18 H 12 N2O6). Yellow solid; Yield 65%; m.p. 244 - 245 °C; 1 1H NMR (500 MHz, Chloroform - d) δ 8.38–8.36 (m, 2H), 8.32–8.29 (m, 2H), 8.19 (s, 1H), 7.64 - 7.59 (m, 2H), 7.40 - 7.33 (m, 2H), 2.58 (s, 3H); 13 13C NMR (126 MHz, CDCl3) δ 163.82, 161.81, 159.13, 154.43, 150.89, 143.74, 134.15, 133.30, 130.89, 129.16, 127.73, 125.09, 123.89, 123.80, 123.04, 118.40, 116.78, 77.30, 77.25, 77.05, 76.79, 16.20; HRMS (ESI) [M + H] + calcd. for C 19 H 15 NO5: 338.1019; found: 338.1033.

[0075] (Example 17, C19 H 12 F3NO4). White solid; Yield 69%; m.p. 168 - 170 °C; 1 1H NMR (500 MHz, DMSO - d6) δ 8.37 (s, 1H), 8.03–8.01 (m, 2H), 7.91 (dd, J = 7.8, 1.7 Hz, 1H), 7.83 - 7.81 (m, 2H), 7.71 (ddd, J = 8.8, 7.4, 1.7 Hz, 1H), 7.51 - 7.47 (m, 1H), 7.43 (td, J = 7.5, 1.1 Hz, 1H), 2.45 (s, 3H); 13 13C NMR (126 MHz, DMSO) δ 163.93, 162.59, 159.07, 154.24, 143.92, 133.74, 132.69, 131.84, 130.08, 128.58, 127.85, 125.48, 123.43, 118.83, 116.71, 40.45, 40.38, 40.29, 40.21, 40.12, 40.04, 39.95, 39.87, 39.79, 39.62, 39.45, 16.35; HRMS (ESI) [M + H] + calcd. for C 19 H 15 NO4: 402.0944; found: 402.0951.

[0076] (Example 18, C 18 H 13 NO5). White solid; Yield 68%; m.p. 159 - 160 °C; 1 1H NMR (500 MHz, DMSO - d6) δ 10.53 (s, 1H), 8.36 (s, 1H), 7.98 - 7.94 (m, 2H), 7.91 (dd, J = 7.8, 1.6 Hz, 1H), 7.74 - 7.69 (m, 1H), 7.50 (d, J = 8.3 Hz, 1H), 7.43 (t, J = 7.5 Hz, 1H), 6.96 - 6.90 (m, 2H), 2.43 (s, 3H); 13C NMR(126MHz,DMSO)δ163.08,162.98,162.77,159.17,154.20,143.71,133.63,132.28,130.02,125.46,123.71,118.94,118 .88,116.69,116.18,40.45,40.37,40.28,40.21,40.12,40.04,39.95,39.87,39.78,39.62,39.45,16.22;HRMS(ESI)[M+H] + calcd.for C 18 H 13 NO5:324.0863;found:324.08.

[0077] (August 19,C 20 H 14 ClNO5).White sold:Yield 70%mp201-203°C 1 H NMR(500MHz,Chloroform-d)δ10.48(s,1H),8.12(s,1H),7.81(d,J16.0Hz,1H),7.57-7.53(m,2H),7.51(d,J8.3 Hz,1H),7.47-7.43(m,2H),7.37(d,JS1.9Hz,1H),7.30(dd,J8.4,2.0Hz,1H),6.57(d,J16.0Hz,1H),2.47(s,3H) 13 C NMR(126MHz,CDCl3)δ164.00,161.98,158.54,154.54,145.53,142.56,139.10,133.00,132.30,129.81,12 9.70,125.68,125.24,123.42,117.09,117.08,115.88,77.31,77.26,77.05,76.80,15.87:HRMS(ESI)[M+H] + calcd.for C 20 H 14 ClNO5:383.9343;found:383.9348.

[0078] (Temperature 20,C 20 H 14 BrNO5).White sold:Yield 70%mp190-191°C 11H NMR (500 MHz, Chloroform-d) δ 10.53 (s, 1H), 8.11 (s, 1H), 7.81 (d, J = 16.0 Hz, 1H), 7.58 - 7.53 (m, 3H), 7.48 - 7.42 (m, 4H), 6.57 (d, J = 16.0 Hz, 1H), 2.47 (s, 3H); 13 13C NMR (126 MHz, CDCl3) δ 164.00, 162.00, 158.48, 154.44, 145.56, 142.65, 132.99, 132.31, 129.89, 129.71, 128.52, 127.21, 125.25, 123.68, 120.05, 117.43, 115.86, 77.30, 77.25, 77.05, 76.80, 15.88; HRMS (ESI) [M + H] + calcd. for C 20 H 14 BrNO5: 428.9282; found: 428.9286.

[0079] (Example 21, C 20 H 14 FNO5). White solid; Yield 70%; m.p. 186 - 188 °C; 1 1H NMR (500 MHz, Chloroform-d) δ 10.37 (s, 1H), 8.13 (s, 1H), 7.81 (d, J = 16.0 Hz, 1H), 7.56 (dd, J = 7.0, 5.0 Hz, 3H), 7.46 (d, J = 8.2 Hz, 2H), 7.08 (dt, J = 8.3, 1.7 Hz, 2H), 6.57 (d, J = 16.0 Hz, 1H), 2.47 (s, 3H); 13 13C NMR (126 MHz, CDCl3) δ 166.22, 164.18, 164.03, 162.06, 158.79, 155.63, 155.53, 145.49, 142.81, 133.01, 132.30, 132.20, 130.81, 130.73, 129.70, 125.23, 122.31, 122.28, 115.91, 115.27, 115.24, 113.45, 113.27, 104.53, 104.32, 77.30, 77.05, 76.79, 15.89; HRMS (ESI) [M + H] + calcd. for C 20 H 14FNO5: 367.0085; found: 367.0085.

[0080] (Example 22, C 21 H 17 NO5). White solid; Yield 70%; m.p. 198 - 201 °C; 1 1H NMR (500 MHz, Chloroform - d) δ 10.53 (s, 1H), 8.14 (s, 1H), 7.83 (d, J = 16.0 Hz, 1H), 7.59 - 7.55 (m, 2H), 7.48 - 7.45 (m, 3H), 7.20 - 7.13 (m, 2H), 6.60 (d, J = 16.0 Hz, 1H), 2.50 (s, 6H); 13 13C NMR (126 MHz, CDCl3) δ 164.11, 162.51, 159.51, 154.52, 145.32, 144.68, 143.46, 133.06, 132.28, 129.69, 128.74, 126.25, 125.15, 122.21, 116.84, 116.16, 116.06, 77.30, 77.25, 77.05, 76.80, 22.03, 15.97; HRMS (ESI) [M + H] + calcd. for C 21 H 17 NO5: 363.0336; found: 363.0335.

[0081] (Example 23, C 21 H 17 NO6). White solid; Yield 70%; m.p. 205 - 210 °C; 1 1H NMR (500 MHz, Chloroform - d) δ 10.49 (s, 1H), 8.11 (s, 1H), 7.80 (d, J = 16.0 Hz, 1H), 7.57 - 7.53 (m, 2H), 7.49 - 7.43 (m, 3H), 6.88 (dd, J = 8.6, 2.4 Hz, 1H), 6.83 (d, J = 2.4 Hz, 1H), 6.57 (d, J = 16.0 Hz, 1H), 3.90 (s, 3H), 2.47 (s, 3H); 1313C NMR(126MHz,CDCl3)δ164.16,163.91,162.61,159.61,156.44,145.25,143.58,133.08,132.27,130.14,129.68,125.13,119.67,116.12,113.44,112.20,100.53,77.30,77.25,77.05,76.79,55.93,15.93;HRMS(ESI)[M+H] + calcd.for C 21 H 17 NO6:379.0251;found:379.0285.

[0082] (Example 24, C20H15NO6). White solid; Yield 70%; m.p. 201 - 203 °C; 1 1H NMR(500MHz, DMSO - d6)δ11.45(s, 1H), 10.17(s, 1H), 8.13(s, 1H), 7.90–7.79(m, 2H), 7.67(dd, J = 8.8, 2.4Hz, 3H), 7.39(d, J = 2.2Hz, 1H), 7.25(dd, J = 8.4, 2.3Hz, 1H), 6.84(d, J = 8.3Hz, 3H), 6.66(dd, J = 15.9, 3.3Hz, 1H), 2.08(s, 3H); 13 13C NMR(126MHz, DMSO)δ168.43, 162.08, 160.05, 160.03, 155.82, 152.38, 150.06, 144.65, 141.84, 130.83, 130.57, 125.72, 124.39, 121.00, 116.20, 115.77, 113.98, 111.70, 102.29, 40.45, 40.37, 40.28, 40.21, 40.12, 40.04, 39.95, 39.87, 39.78, 39.69, 39.61, 39.45, 14.02; HRMS(ESI)[M + H] + calcd.forC 20 H 15 NO6:365.3418; found:365.3425.

[0083] (Example 25, C 20 H 14 ClNO5). White solid; Yield 70%; m.p. 201 - 203 °C;1 1H NMR (500 MHz, Chloroform-d) δ 10.16 (s, 1H), 8.06 (s, 1H), 7.82 (d, J = 16.0 Hz, 1H), 7.58 - 7.53 (m, 5H), 7.46 (d, J = 8.2 Hz, 2H), 7.33 - 7.30 (m, 1H), 6.58 (d, J = 16.0 Hz, 1H), 2.47 (s, 3H); 13 13C NMR (126 MHz, CDCl3) δ 163.98, 161.87, 158.62, 152.67, 145.61, 142.03, 132.99, 132.94, 132.31, 130.28, 129.72, 128.06, 125.26, 124.73, 119.52, 118.18, 115.82, 77.30, 77.25, 77.05, 76.79, 15.92; HRMS (ESI) [M + H] + calcd. for C 20 H 14 ClNO5: 383.9771; found: 383.9769.

[0084] (Example 26, C 20 H 14 BrNO5). White solid; Yield 65%; m.p. 171 - 172 °C; 1 1H NMR (500 MHz, Chloroform-d) δ 10.48 (s, 1H), 8.08 (s, 1H), 7.61 - 7.57 (m, 3H), 7.55 - 7.51 (m, 3H), 7.49 - 7.47 (m, 2H), 6.60 (d, J = 16.0 Hz, 1H), 2.50 (s, 3H); 13 13C NMR (126 MHz, CDCl3) δ 164.00, 161.85, 158.57, 153.14, 145.62, 141.94, 135.74, 132.99, 132.31, 131.13, 129.72, 125.26, 124.69, 120.01, 118.45, 117.55, 115.82, 77.30, 77.25, 77.04, 76.86, 76.79, 15.92; HRMS (ESI) [M + H] + calcd. for C 20 H 13 Br2NO4: 428.9282; found: 428.9284.

[0085] (Example 27, C20 H 14 FNO5). White solid; Yield 65%; m.p. 192 - 193 °C; 1 1H NMR (500 MHz, Chloroform - d) δ 10.12 (s, 1H), 8.08 (s, 1H), 7.81 (d, J = 15.9 Hz, 1H), 7.55 (d, J = 8.3 Hz, 2H), 7.45 (d, J = 8.3 Hz, 2H), 7.37 - 7.29 (m, 2H), 7.24 (d, J = 2.9 Hz, 1H), 6.57 (d, J = 16.0 Hz, 1H), 2.47 (s, 3H); 13 13C NMR (126 MHz, CDCl3) δ 163.98, 161.95, 159.87, 158.83, 157.92, 150.50, 150.48, 145.57, 142.32, 142.30, 132.99, 132.30, 129.71, 125.25, 124.70, 120.64, 120.44, 119.23, 119.16, 118.38, 118.32, 115.84, 114.19, 114.00, 77.31, 77.26, 77.05, 76.80, 15.91; HRMS (ESI) [M + H] + calcd. for C 20 H 14 FNO5: 367.0089; found: 367.0085.

[0086] (Example 28, C 21 H 17 NO5). White solid; Yield 70%; m.p. 182 - 183 °C; 1 1H NMR (500 MHz, Chloroform - d) δ 10.16 (s, 1H), 8.09 (s, 1H), 7.85 - 7.78 (m, 1H), 7.58 - 7.53 (m, 2H), 7.48 - 7.43 (m, 2H), 7.39 (dd, J = 8.4, 2.1 Hz, 1H), 7.34 (d, J = 2.1 Hz, 1H), 7.25 - 7.21 (m, 1H), 6.58 (d, J = 16.0 Hz, 1H), 2.47 (s, 3H), 2.42 (s, 3H); 13C NMR(126MHz,CDCl3)δ164.12,162.47,159.48,152.52,145.39,143.43,134.76,134.14,133.05,132.29,129.7 0.128.70,125.17,123.33,118.27,116.41,116.01,77.30,77.25,77.05,76.79,20.82,15.99:HRMS(ESI)[M+H] + calcd.for C 21 H 17 NO5:363.0342;found:3

[0087] (Local 29,C 21 H 17 NO6).White sold:Yield 70%mp251-253°C 1 H NMR(500MHz,Chloroform-d)δ10.16(s,1H),8.11(s,1H),7.81(d,J)16.0Hz,1H),7.56(d,J8.2Hz,2H),7.46(d,J8.2Hz,2H) ,7.29(d,J9.1Hz,1H),7.17(dd,J9.1,2.9Hz,1H),6.98(d,J2.9Hz,1H),6.58(d,J16.0Hz,1H),3.85(s,3H),2.48(s,3H) 13 C NMR(126MHz,CDCl3)δ164.10,162.40,159.41,156.36,148.87,145.42,143.26,133.03,132.29,132.27,129.70,12 5.19,123.72,121.08,118.86,117.75,115.99,110.54,77.30,77.25,77.05,76.80,55.89,15.95:HRMS(ESI)[M+H] + calcd.for C 21 H 17 NO6:379.0251;found:3

[0088] (Temperature 30,C 21 H 16 BrNO5).White sold:Yield 70%mp251-253°C. 1H NMR (500MHz, DMSO-d6) δ10.16(s,1H),8.64(s,1H),7.84–7.78(m,2H),7.67(d,J=8.5Hz,2H),7.60(dd,J=9.0,2.8 Hz,1H),7.54(d,J=8.9Hz,1H),6.83(d,J=8.5Hz,2H),6.67(d,J=16.0Hz,1H),5.57(d,J=8.0Hz,2H),2.59(s,3H); 13 C NMR (126MHz, DMSO) δ195.59,165.77,160.92,158.81,152.58,147.86,147.12,146.77,131.38,128.93,125.49,125.35,123.25,119.16 ,117.75,116.36,113.00,40.55,40.46,40.39,40.30,40.22,40.13,40.05,39.96,39.88,39.80,39.63,39.46,30.53; HRMS(ESI)[M+H] + calcd.for C 20 H 15 NO6:365.3443; found:365.3469.

[0089] Performance Testing

[0090] Test for tyrosinase inhibitory activity of coumarin derivatives:

[0091] 1. Preparation of reagents and standard solutions

[0092] (1) 50 mM phosphate buffer (PBS, pH 6.8): Weigh a certain amount of potassium dihydrogen phosphate and disodium hydrogen phosphate, dissolve them in ultrapure water, and use them to dissolve and dilute the reagents.

[0093] (2) Preparation of tyrosinase solution: Add an appropriate amount of 50 mM PBS to an enzyme with an activity of 50 ku to a working concentration of 13333.33 u / mL, and freeze in aliquots.

[0094] (3) Substrate preparation: Accurately weigh an appropriate amount of L-DOPA and dissolve it in 50 mM PBS solution to prepare a substrate working solution with a concentration of 0.5 mM. Vortex to mix well and prepare it fresh before each experiment.

[0095] Preparation of test drugs: Accurately weigh an appropriate amount of the drug to be tested, dissolve it in DMSO to prepare a 25.6 mM stock solution, and store it at -20°C in the dark. Dilute it with DMSO to different required concentrations (0-200 μM) before the experiment, and the DMSO content is equal to 5%.

[0096] 2. Experimental steps

[0097] (1) 10 μL of tyrosinase with a working concentration of 1333.33 U / mL, 130 μL of 50 mM phosphate buffer (pH 6.8), and 10 μL of compounds of different concentrations (coumarin oxime derivatives prepared in Example 1) were added to a 96-well plate in sequence. The blank control group was replaced with 10 μL of 5% DMSO to replace 10 μL of the compound. Kojic acid was used as a positive control. Three replicate wells were set in parallel for each group. The enzyme reaction system was placed on a microplate reader and incubated at 25° C. for 10 min.

[0098] (2) Subsequently, 50 μL of the substrate DOPA was added to the enzyme reaction system to initiate the enzyme reaction. The microplate was immediately placed on a microplate reader and measured at 25°C and 475 nm absorbance. Two sets of data, OD1 and OD2, were measured within one minute.

[0099] (3) The tyrosinase inhibitory activity of the test compound was calculated according to the following formula:

[0100] Inhibition rate (%) = [(OD2-OD)-(OD1-OD)] / OD2-OD×100%;

[0101] OD represents the absorbance value of the blank control group. Data processing: MS Excel was used to analyze and process the data, and Origin 9.1 was used to calculate the half inhibitory concentration (IC 50 ), IC 50 It represents the concentration of the test compound required to inhibit tyrosinase activity by 50% under the experimental conditions.

[0102] 3. Results Analysis

[0103] The tyrosinase inhibitory activity of the synthesized compounds was evaluated by in vitro enzymatic assay, and the results are shown in Table 2:

[0104] Table 2. Evaluation of in vitro inhibitory activity of coumarin compounds of Examples 1 to 30 against tyrosinase

[0105]

[0106]

[0107] In order to find a more active tyrosinase inhibitor, the coumarin derivatives of Examples 1 to 30 were synthesized for the first time, and kojic acid was used as a positive control to screen their anti-tyrosinase activity at 64 μM. As shown in Table 2, among Examples 1 to 18, Example 9 had the strongest inhibition on tyrosinase (61.24±0.17%), while the coumarin derivatives of other Examples 1 to 8 and 10 to 18 had weaker inhibition on tyrosinase activity (<50%). The coumarin derivative of Example 9 (IC 50 :6.40±0.39μM) has a tyrosinase inhibitory effect that is about 2.2 times stronger than that of kojic acid (14.13±0.80μM). In addition, the substituents on the styryl part also affect its anti-tyrosinase activity. Compared with Example 1 in which there is no substituent in the styryl group, compounds 4b~i show stronger anti-tyrosinase activity, indicating that the substituents including -CH3, Cl, Br, F, OCH3, NO2, -CF3 and -OH are beneficial to their inhibitory activity. The anti-tyrosinase activity results of the coumarin derivatives obtained from Examples 19 to 30 show that the introduction of substituents (Cl, Br, F, -CH3 and -OCH3) at the C-6 and C-7 positions of coumarin will have a negative impact, but the introduction of hydroxyl groups will increase their inhibitory activity. Among them, Example 30 has the highest anti-tyrosinase activity (IC 50 : 3.04±0.01μM), which was 4.6 times higher than kojic acid (14.13±0.80μM).

[0108] Enzyme kinetics experiments

[0109] The tyrosinase inhibitory activity kinetics of the active compound of Example 30 synthesized was evaluated by in vitro enzyme kinetics experiment:

[0110] 1. Preparation of reagents and standard solutions

[0111] (1) 50 mM phosphate buffer (PBS, pH 6.8): Weigh a certain amount of potassium dihydrogen phosphate and disodium hydrogen phosphate, dissolve them in ultrapure water, and use them to dissolve and dilute the reagents.

[0112] (2) Preparation of tyrosinase solution: Add an appropriate amount of 50 mM PBS to an enzyme with an activity of 50 kU to a working concentration of 13333.33 U / mL and freeze in aliquots. When needed, take out the tyrosinase solutions diluted to final concentrations of 50.0025 U / mL, 66.6700 U / mL, 83.3375 U / mL, and 100.0050 U / mL, respectively.

[0113] (3) Substrate preparation: Accurately weigh an appropriate amount of L-DOPA and dissolve it in 50 mM PBS solution to prepare a substrate working solution with a concentration of 0.5 mM. Vortex to mix well and prepare it fresh before each experiment.

[0114] Preparation of test drugs: Accurately weigh the appropriate amount of the drug to be tested, dissolve it in DMSO to prepare a 25.6 mM stock solution, and store it at -20°C in the dark. Dilute it with DMSO to different required concentrations (0-200 μM) before the experiment, and the DMSO content is equal to 5%.

[0115] 2. Experimental steps

[0116] (1) 10 μL of tyrosinase at different concentrations, 130 μL (pH 6.8) 50 mM phosphate buffer, and 10 μL of compounds at different concentrations (coumarin compounds prepared in Example 30) were added to a 96-well plate in sequence. The blank control group was replaced with 10 μL of 5% DMSO to replace 10 μL of the compound. The final concentrations of the enzyme were 50.0025 U / mL, 66.6700 U / mL, 83.3375 U / mL, and 100.0050 U / mL, respectively. Kojic acid was used as a positive control. Three replicate wells were set in parallel for each group. The enzyme reaction system was placed on a microplate reader and incubated at 25° C. for 10 min.

[0117] (2) Subsequently, 50 μL of a 0.5 mM substrate (L-DOPA) was added to the enzyme reaction system to initiate the enzyme reaction, and the absorbance change was immediately measured at a wavelength of 475 nm within one minute.

[0118] (3) Data processing: MS Excel was used to analyze and process the data. The reaction rate of the enzyme reaction system was ΔOD / min.

[0119] 3. Results Analysis

[0120] The results of enzyme kinetic inhibition type evaluation experiment are as follows Figure 1 As shown. Figure 1 It can be seen that the mechanism analysis by plotting ν versus [E] gives a family of straight lines passing through the origin Figure 1 (A), indicating that the compound of Example 30 is a reversible inhibitor.

[0121] Substrate kinetics experiments

[0122] In vitro substrate kinetics experiments were used to evaluate the tyrosinase inhibitory activity of the synthesized active compounds:

[0123] 1. Preparation of reagents and standard solutions

[0124] (1) 50 mM phosphate buffer (PBS, pH 6.8): Weigh a certain amount of potassium dihydrogen phosphate and disodium hydrogen phosphate, dissolve them in ultrapure water, and use them to dissolve and dilute the reagents.

[0125] (2) Preparation of tyrosinase solution: Add an appropriate amount of 50 mM PBS to an enzyme with an activity of 50 Ku to a working concentration of 13333.33 U / mL, and freeze in aliquots. The working dilution is 1333.33 U / mL.

[0126] (3) Substrate preparation: Accurately weigh an appropriate amount of levodopa (L-DOPA), add 50 mM PBS solution to dissolve, and prepare substrate working solutions with concentrations of (2 mM, 4 mM, 6 mM, 8 mM). Vortex to mix well and prepare freshly before each experiment.

[0127] Preparation of test drugs: Accurately weigh the appropriate amount of the drug to be tested, dissolve it in DMSO to prepare a 25.6 mM stock solution, and store it at -20°C in the dark. Dilute it with DMSO to different required concentrations (0-200 μM) before the experiment, and the DMSO content is equal to 5%.

[0128] 2. Experimental steps

[0129] (1) 10 μL of 1333.33 U / mL tyrosinase, 130 μL (pH 6.8) 50 mM phosphate buffer, and 10 μL of different concentrations of compounds (coumarin derivatives prepared in Example 30) were added to a 96-well plate in sequence. The blank control group was replaced with 10 μL of 5% DMSO to replace 10 μL of the compound. Kojic acid was used as a positive control. Three replicate wells were set in parallel for each group. The enzyme reaction system was placed on a microplate reader and incubated at 25° C. for 10 min.

[0130] (2) Subsequently, 50 μL of a 0.5 mM substrate (L-DOPA) was added to the enzyme reaction system to initiate the enzyme reaction, and the absorbance change rate was immediately measured at a wavelength of 475 nm within one minute.

[0131] (3) Data processing: MS Excel was used to analyze and process the data. The reaction rate of the enzyme reaction system was ΔOD / min.

[0132] 3. Results Analysis

[0133] The results of the substrate kinetic inhibition type evaluation experiment are as follows Figure 1 As shown, (B) is the substrate kinetic diagram drawn by the double reciprocal plotting method, Figure 1 B shows that the screened inhibitor is a mixed inhibitor, indicating that it can not only bind to tyrosinase, but also to the tyrosinase-substrate complex.

[0134] 3D Fluorescence Image

[0135] 1. Preparation of reagents and standard solutions

[0136] (1) 50 mM phosphate buffer (PBS, pH 6.8): Weigh a certain amount of potassium dihydrogen phosphate and disodium hydrogen phosphate, dissolve them in ultrapure water, and use them to dissolve and dilute the reagents.

[0137] (2) Preparation of tyrosinase solution: Add an appropriate amount of 50 mM PBS to an enzyme with an activity of 50 Ku to prepare a tyrosinase solution with a concentration of 13333.33 U / mL, and freeze in aliquots. The working dilution is 173.33 U / mL.

[0138] Preparation of test drugs: Accurately weigh the appropriate amount of the drug to be tested, dissolve it in DMSO to prepare a 1 mM stock solution, and store it at -20°C in the dark. Dilute it with DMSO to different required concentrations (0-200 μM) before the experiment, and the DMSO content is equal to 5%.

[0139] 2. Experimental steps

[0140] (1) Take out the frozen 13333.33 U / mL enzyme solution, take 39 μL of the enzyme and add it to 3 mL of 50 mM phosphate buffer, mix well, and put it into a HORIBA fluorescence spectrometer. In the experiment, the excitation wavelength and emission wavelength range are: 200-600 nm, and the slit width is 2.5 nm.

[0141] (2) Add 10 μL of compound to the measured enzyme solution, the concentration of the compound working solution is 1 mM, and put it into the HORIBA fluorescence spectrometer again. The excitation wavelength and emission wavelength range of the experiment are: 200-600 nm, and the width of the slit is 2.5 nm.

[0142] (3) The experimental data were processed and analyzed by Origin 9.1.

[0143] 3. Results Analysis

[0144] 3D fluorescence spectrum of tyrosinase ( Figure 2 )Two typical spectra appear: Figure 2 (a) represents the absorption peak of tyrosinase, and (b) represents the absorption peak presented by the addition of the compound to tyrosinase. Compare the peak change rates in the two figures with and without the addition of compound 5I. (Peak 1) has a tyrosine residue with a tryptophan residue when excited at 280nm and emitted at 332nm, and (Peak2) has a polypeptide backbone structure when excited and emitted at 232nm. Treatment with the coumarin derivative (3.33μM) of Example 30 reduced the fluorescence intensity of Peak 1 by 17.87% and the fluorescence intensity of Peak 2 by 28.19%, respectively, indicating that the binding of the compound of Example 30 to tyrosinase leads to changes in the polypeptide backbone structure of the microenvironment tyrosinase.

[0145] CD fluorescence image

[0146] 1. Preparation of reagents and standard solutions

[0147] (1) 50 mM phosphate buffer (PBS, pH 6.8): Weigh a certain amount of potassium dihydrogen phosphate and disodium hydrogen phosphate, dissolve them in ultrapure water, and use them to dissolve and dilute the reagents.

[0148] (2) Preparation of tyrosinase solution: Add an appropriate amount of 50 mM PBS to an enzyme with an activity of 50 Ku to a concentration of 13333.33 U / mL, and freeze in aliquots. The final concentration of the working dilution enzyme is 33 μM.

[0149] Preparation of test drugs: Accurately weigh the appropriate amount of the drug to be tested, dissolve it in DMSO to prepare a 10mM stock solution, and store it at -20°C in the dark. Dilute it with DMSO to different required concentrations (0-200μM) before the experiment, and the DMSO content is equal to 5%.

[0150] 2. Experimental steps

[0151] (1) At room temperature and under a constant helium flow, start the instrument in a 200 μL quartz cuvette and record the CD spectrum of the background air in the range of 190-250 nm.

[0152] (2) 190 μL of tyrosinase solution was added to a 200 μL quartz cuvette with a working concentration of 33 μM. 10 μL of chromatography-grade methanol was added and incubated for 5 minutes. The CD spectrum in the range of 190-250 nm was recorded, with methanol as the solvent background.

[0153] (3) 190 μL of tyrosinase solution with a concentration of 33 μM was added, and compound 5I was added with a final concentration of (0 μM, 33 μM, 66 μM). The mixture was incubated for 5 minutes, and CD spectral data were recorded in the range of 190-250 nm.

[0154] (4) The protein secondary structure data is obtained through CDNN on the computer.

[0155] 3. Results Analysis

[0156] The CD spectrum of the tyrosinase of the coumarin derivative of Example 30 was also monitored at a wavelength of 190 to 250 nm. Figure 3As shown. The CD spectrum has two negative bands at 210 and 222 nm, which is due to the π* electron transfer of n→π* and π→amide. The treatment of compound 5l resulted in a significant decrease in the intensity of the negative bands, indicating that the binding of the coumarin derivative of Example 30 to tyrosinase produced a conformational change. The secondary structure content of tyrosinase in the presence of the coumarin derivative of Example 30 was calculated and listed in Table 3. The treatment of the coumarin derivative of Example 30 (molar ratio 1:2) resulted in an increase in α-helix (from 7.3% to 9.6%) and β-fold (from 19.8% to 20.6%), while β-turn (from 38.7% to 29.9%) and random coil (from 36.1% to 35.5%).

[0157] Table 3 Secondary structure content of the complex of the compound of tyrosinase Example 30

[0158]

[0159] Activity Analysis of Coumarin Derivatives

[0160] The interaction between the coumarin derivative of Example 30 and tyrosinase was analyzed by molecular docking method:

[0161] Molecular docking was performed by SYBYL software using the crystal structure of Agaricus bisporus tyrosinase (PDBID: 2Y9X) obtained from the RCSB protein database. The tyrosinase protein was prepared by removing ligands and water, then adding hydrogen atoms and charges, repairing terminal residues, and generating active pockets. The coumarin derivative of Example 30 was prepared by adding charges and energy minimization using Gasteiger-Hückle. Then, the docking between the coumarin derivative of Example 30 and tyrosinase was operated in the default format, and the results were visualized by Pymol software.

[0162] In order to obtain more binding modes of the compound of Example 30 in the tyrosinase active site, molecular docking was performed using SYBYL software. Figure 4 As shown in (a), the coumarin derivative of Example 30 is well stabilized in the active site of tyrosinase in a "U-shaped" conformation, with the 4-hydroxycinnamic acid moiety embedded in it. Detailed interaction results Figure 4 (b) shows that the 6-hydroxyl group in coumarin is bound to His85 (bond length: ) forms a hydrogen bond, and the 4-hydroxyl group in cinnamic acid also forms a hydrogen bond with His259 (bond length: In addition, the 4-hydroxyl group of cinnamic acid is oriented at a distance of 1.9 and The two copper ions in the molecule help the compound anchor to the active site of tyrosinase.

[0163] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. Use of a coumarin derivative in preparing cosmetics or medicines for inhibiting tyrosinase activity, characterized in that: The chemical structural formula of the coumarin derivative is shown in formula (I): Wherein, R1 is H, hydroxyl; R2 is H, hydroxyl; The R3 is selected from at least one of the following structural formulas:

2. The use according to claim 1, characterized in that: The preparation method of the coumarin derivative comprises the following steps: S3, subjecting compound 3 to an esterification reaction with compound 4 to obtain a coumarin derivative; The structural formulas of the compounds 3 and 4 are as follows:

3. The use according to claim 2, characterized in that: The compound 3 was prepared by the following method: S2, reacting compound 2 with hydroxylamine hydrochloride to obtain compound 2; 4. The use according to claim 1, characterized in that: The coumarin derivative is prepared into a tyrosinase inhibitor composition.

5. The use according to claim 1, characterized in that: The coumarin derivative is prepared into an inhibitor against enzymatic browning of fruits and vegetables.

6. The use according to claim 1, characterized in that: The coumarin derivative is prepared into whitening cosmetics.

7. The use according to claim 6, characterized in that: The cosmetics are selected from facial cream, facial mask, lotion, essence, toner, facial cleanser or shower gel.

8. The use according to claim 1, characterized in that: The coumarin derivative is prepared into a drug for preventing and treating human pigmentation diseases caused by abnormal melanin, melanoma and other diseases requiring inhibition of tyrosinase activity.

9. The use according to claim 8, characterized in that: The dosage form of the drug is selected from solid powder, aqueous solution, alcohol solution, cataplasm, powder injection, granule, tablet, granule, powder, sugar-coated tablet, enteric coated tablet, capsule, lozenge, pill, ointment, pill, spray or orodisintegrating tablet.

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