A tyrosinase inhibitor and its application

Abstract

This invention relates to the field of chemical technology, and more particularly to a tyrosinase inhibitor and its application. The tyrosinase inhibitor comprises a compound having the structure shown or a physiologically acceptable salt thereof. The tyrosinase inhibitor provided by this invention exhibits significant tyrosinase inhibitory activity, effectively blocks melanin synthesis, and has low toxicity and high molecular stability.

Description

Technical Field

[0001] This invention relates to the field of chemical technology, and in particular to a tyrosinase inhibitor and its application. Background Technology

[0002] Hyperpigmentation is usually caused by excessive melanin deposition in the skin, and tyrosinase, as a key enzyme in melanin formation in organisms, has an activity closely related to melanin synthesis. Tyrosinase inhibitors, due to their ability to regulate the melanin synthesis process, have shown significant application value in multiple fields: in the pharmaceutical field, they can be used to treat and prevent pigmentary disorders such as melasma; in the cosmetic field, they can achieve skin whitening; in the food industry, they can delay the spoilage of fruits and vegetables caused by browning; and in the agricultural field, because tyrosinase is a key enzyme for the survival of some insects, its inhibitors have become one of the most promising biological insecticides. Therefore, the development of tyrosinase inhibitors has broad application prospects.

[0003] Currently, tyrosinase inhibitors on the market can be categorized into naturally derived products and chemically synthesized products based on their origin. Naturally derived products include arbutin and nicotinamide. Arbutin, however, has limited inhibitory activity against tyrosinase, resulting in poor efficacy in improving stubborn pigmentation and other problems. Furthermore, high concentrations can cause irritation and potential cytotoxicity, limiting its addition to whitening products and affecting its efficacy. Niacinamide's direct inhibitory effect on tyrosinase is not significant, and some users experience skin intolerance, such as redness and itching. Its whitening effect is slow, requiring long-term use to show results, making it unsuitable for rapid whitening. Chemically synthesized products, represented by vitamin C derivatives such as ascorbic acid-2-glucosidase and magnesium ascorbate phosphate, have poor transdermal permeability, leading to low effective concentrations in the skin and limited whitening effects. They are also prone to decomposition under high temperatures or unsuitable pH conditions, exhibiting poor stability and requiring sophisticated formulation processes, increasing the difficulty and cost of research and development. Therefore, developing safe, efficient, and stable novel tyrosinase inhibitors is crucial for improving the shortcomings of existing products and meeting the application needs of various fields. Summary of the Invention

[0004] In view of this, the present invention proposes a tyrosinase inhibitor and its application.

[0005] The technical solution of this invention is implemented as follows:

[0006] In a first aspect, the present invention provides a tyrosinase inhibitor comprising a compound having the structure shown in Formula I:

[0007] Or a physiologically acceptable salt;

[0008] In Formula I:

[0009] X is NH or O;

[0010] Y is selected from one of hydrogen, amino, C1-C6 alkyl, halo-C1-C6 alkyl, cycloalkyl, halo-cycloalkyl, aryl, substituted aryl and structural unit Q1-Q12;

[0011] The structural units Q1-Q12 are as follows:

[0012] Where n is selected from integers between 1 and 5.

[0013] Furthermore, in some specific embodiments, the compound with the structure shown in Formula I is specifically one of the following compounds 1-18:

[0014]

[0015]

[0016]

[0017] Thirdly, the present invention provides a method for preparing the tyrosinase inhibitor, the method comprising the following synthetic route:

[0018]

[0019] In step (1), compound M1 reacts with triethoxymethane to generate intermediate compound M2; in step (2), intermediate compound M2 reacts with thiourea to generate the target compound.

[0020] X is NH or O;

[0021] Y is selected from hydrogen, amino, C1-C6 alkyl, halo-C1-C6 alkyl, cycloalkyl, halo-cycloalkyl, aryl, substituted aryl, and structural units Q1-Q12; the structural units Q1-Q12 are as follows:

[0022]

[0023] Where n is selected from integers between 1 and 5.

[0024] Further, in step (1), triethoxymethane and compound M1 in a molar ratio of (1.2-1.4):1 are dissolved in acetic anhydride and reacted in the dark at 105-115°C. The reaction in step (1) is an electrophilic substitution reaction. The carbon atom bonded to the cyano group loses a hydrogen atom under the action of the anhydride to generate a carbanion. Due to the electron-withdrawing effect of the oxygen atom in triethylmethane, the carbon atom of methane carries a partial positive charge. The formed carbocation attacks the M1 carbanion to generate M2.

[0025] Further, in step (2), thiourea, compound M2, and sodium tert-butoxide in a molar ratio of (1.2-1.4):1:(1.2-1.4) are mixed, dissolved in propanol, and reacted at 75-85°C under an inert gas atmosphere. The reaction in step (2) is an electrophilic addition reaction. The amino group in the thiourea structure acts as an electrophile, attacking the unsaturated cyano group and the olefin structure to generate the final product.

[0026] Fourthly, the present invention provides the application of the tyrosinase inhibitor for preparing products in which the tyrosinase inhibitor is an active ingredient.

[0027] The beneficial effects of the present invention include at least the following:

[0028] The tyrosinase inhibitor provided by this invention has significant tyrosinase inhibitory activity and has obvious advantages over traditional tyrosinase inhibitors in terms of potential toxic side effects, raw material availability, and molecular stability.

[0029] The IC50 of various compounds provided by this invention on tyrosinase 50 The concentrations were all less than 5 μM, significantly superior to the positive control drugs kojic acid (32.0 μM) and phenylethyl resorcinol (16.7 μM). These compounds specifically inhibited tyrosinase catalytic activity, effectively blocking melanin synthesis. Their transdermal permeability was significantly enhanced compared to the control compound α-arbutin at the same concentration. Furthermore, many compounds showed no significant cytotoxicity in the 0-256 μM concentration range and exhibited good safety for melanocytes. Therefore, they have significant application prospects in the preparation of whitening, spot-fading, and sunscreen cosmetics and skincare products; drugs, health products, and foods for the prevention and treatment of pigmentation diseases; the control of pests dependent on tyrosinase metabolism; and as food preservatives. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 The results show the toxicity assay of the test compound against melanoma cells (B16F10).

[0032] Figure 2 The results show the inhibitory activity of the test compound on melanin production in melanoma cells (B16F10). Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0034] In some specific embodiments, the present invention provides formula I The synthetic route for the compound with the structure shown includes the following synthetic steps:

[0035]

[0036] In step (1) above, compound M1 reacts with triethoxymethane to generate intermediate compound M2; in step (2), intermediate compound M2 reacts with thiourea to generate the target compound.

[0037] In compounds M1, M2, and the compound having the structure shown in Formula I, X and Y are as follows:

[0038] X is NH or O;

[0039] Y is selected from hydrogen, amino, C1-C6 alkyl, halo-C1-C6 alkyl, cycloalkyl, halo-cycloalkyl, aryl, substituted aryl, and structural units Q1-Q12; the structural units Q1-Q12 are as follows:

[0040]

[0041] Where n is selected from integers between 1 and 5.

[0042] In some specific embodiments, the synthesis methods of intermediate compound M2 and target compound (compound with structural formula as shown in Formula I) are as follows:

[0043] Triethoxymethane (1.2 mol) and compound M1 (1 mol) were dissolved in acetic anhydride and reacted at 110 °C in the dark for 15 hours, then cooled to room temperature. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the reaction system was cooled to room temperature. The solvent was removed by vacuum distillation, and the crude product was purified by silica gel column chromatography (eluting ethyl acetate and petroleum ether in a volume ratio of 1:6) to obtain intermediate compound M2.

[0044] Thiourea (1.2 mol), sodium tert-butoxide (1.2 mol), and intermediate compound M2 (1 mol) were mixed, dissolved in propanol, and reacted at 80 °C under nitrogen protection for 30 min. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the reaction system was cooled to room temperature. The solvent was removed by vacuum distillation, and a 10% (v / v) aqueous solution of acetic acid was added, precipitating out a solid, which was the target compound.

[0045] In some specific embodiments, the present invention also provides a method for preparing compound M1 in step (1) above, which includes:

[0046] The alcohol or amine YXH (i.e., Y-OH or Y-NH2) is reacted with cyanoacetic acid to generate compound M1; the synthetic route of compound M1 is as follows:

[0047]

[0048] In some specific embodiments, compound M1 is prepared as follows:

[0049] At room temperature, 1.3 mol of cyanoacetic acid, 1 mol of YXH (i.e., Y-OH or Y-NH2), 1.1 mol of EDCI, and 1.1 mol of DMAP were dissolved in dichloromethane and reacted for 5 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the reaction system was filtered under reduced pressure. The solution was dissolved in ethyl acetate and washed successively with 1M HCl solution and saturated NaHCO3 solution. The ethyl acetate layer was collected, dried over anhydrous Na2SO4, and evaporated to dryness. The mixture was purified by column chromatography to obtain compound M1. EDCI was 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; DMAP was 4-dimethylaminopyridine.

[0050] Example 1

[0051] Compound 1 Preparation The steps are as follows:

[0052] (1) Triethoxymethane (1.2 mol) and compound HOOC-CH2-CN (1 mol, cyanoacetic acid) were dissolved in acetic anhydride and reacted at 110 °C in the dark for 15 hours, then cooled to room temperature. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the reaction system was cooled to room temperature. The solvent was removed by vacuum distillation, and the crude product was purified by silica gel column chromatography (eluting ethyl acetate and petroleum ether in a volume ratio of 1:6) to obtain compound HOOC-C(CN)=CH-CH-O-CH2-CH3 (numbered M2-1).

[0053] (2) Thiourea (1.2 mol), sodium tert-butoxide (1.2 mol), and intermediate compound M2-1 (1 mol) were mixed, dissolved in propanol, and reacted at 80 °C under nitrogen protection for 30 min. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the reaction system was cooled to room temperature. The solvent was removed by vacuum distillation, and 10% (v / v) aqueous acetic acid solution was added, precipitating out compound 1. The yield was 84.78%, and the purity was 98.0%. 1 H NMR (400MHz, DMSO-d6) δ8.47(s,1H),8.05(s,1H),8.03(s,2H). [M+H] + :172.2.

[0054] Example 2

[0055] Compound 2 Preparation The steps are as follows:

[0056] Replacing the substrate cyanoacetic acid in step (1) of Example 1 with CH3-CH2-OC(O)-CH2-CN (ethyl cyanoacetate) yields compound M2-2. Yield 82%, purity 7%. 1 HNMR(500MHz,Chloroform-d)δ7.87(s,1H),4.27(q,J=7.1Hz,2H),4.08(q,J=6.9Hz,2H),1.31(td,J=7.0,1.2Hz,6H).[M+H] + : 170.2.

[0057] Referring again to step (2) of Example 1, compound 2 was obtained. The yield was 67.75%, and the purity was 97.5%. 1 H NMR(400MHz,Chloroform-d)δ7.97–7.92(m,1H),5.00(ttd,J=11.5,6.2,2.9Hz,1H),4 .34-4.24(m,2H),1.35(tdd,J=7.1,5.0,2.5Hz,3H),1.20(ddd,J=6.3,4.9,2.6Hz,6H). [M+H] + :200.1.

[0058] Examples 3-7

[0059] Referring to the preparation steps of Examples 1 and 2, the substrate cyanoacetic acid in step (1) of Example 1 was replaced with YOC(O)-CH2-CN, and the following intermediate compound and target compound were synthesized respectively:

[0060]

[0061]

[0062]

[0063] Example 8

[0064] Compound 8 Preparation The steps are as follows:

[0065] (1) At room temperature, 1.3 mol of cyanoacetic acid, 1 mol of n-pentanol, 1.1 mol of EDCI, and 1.1 mol of DMAP were dissolved in dichloromethane and reacted for 5 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the reaction system was filtered under reduced pressure. The solution was dissolved in ethyl acetate and washed successively with 1M HCl solution and saturated NaHCO3 solution. The ethyl acetate layer was collected, dried over anhydrous Na2SO4, and evaporated to dryness. The compound M1-8 was purified by column chromatography. Yield: 65.24%, purity: 97.3%. 1 H NMR(400MHz,Chloroform-d)δ4.20(t,J=6.8Hz,2H),3.47(s,2H),1.68(dq,J=9.5,7.1Hz,2H),1.42-1.29(m,4H),0.95-0.86(m,3H).[M+H] + :172.2.

[0066] (2) Triethoxymethane (1.2 mol) and compound M1-8 (1 mol) were dissolved in acetic anhydride and reacted at 110 °C in the dark for 15 hours, then cooled to room temperature. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the reaction system was cooled to room temperature. The solvent was removed by vacuum distillation, and the crude product was purified by silica gel column chromatography (eluting solvent: ethyl acetate and petroleum ether, volume ratio 1:6) to obtain the intermediate compound M2-8. Yield: 68.52%, purity: 98.9%. 1 HNMR(400MHz,Chloroform-d)δ8.00(s,1H),4.34(q,J=7.1Hz,2H),4.17(t,J=6.7Hz,2H),1.72 -1.62(m,2H),1.42(t,J=7.1Hz,3H),1.33(dq,J=6.9,3.7,3.2Hz,4H),0.93–0.84(m,3H).[M+H] + :212.1.

[0067] (3) Thiourea (1.2 mol), sodium tert-butoxide (1.2 mol), and intermediate compound M2-8 (1 mol) were mixed, dissolved in propanol, and reacted at 80 °C under nitrogen protection for 30 min. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the reaction system was cooled to room temperature. The solvent was removed by vacuum distillation, and 10% (v / v) aqueous acetic acid solution was added to precipitate compound 8. The yield was 56.73%, and the purity was 98.5%. 1 H NMR (400MHz, DMSO-d6) δ12.65(s,1H),8.50(s,1H),8.05(s,1H),7.89(s,1H),4.19(t,J= 6.6Hz,2H),1.67(p,J=6.9Hz,2H),1.31(dh,J=7.5,4.0Hz,4H),0.94–0.83(m,3H).[M+H] + :242.1.

[0068] Examples 9-12

[0069] Referring to the preparation steps of Example 8, the substrate n-pentanol in step (1) was replaced with γ-OH, and the following intermediate compound and target compound were synthesized respectively:

[0070]

[0071]

[0072]

[0073] Example 13

[0074] Compound 13 Preparation The steps are as follows:

[0075] (1) Triethoxymethane (1.2 mol) and compound CN-CH2-C(O)NH2 (1 mol, cyanoacetamide) were dissolved in acetic anhydride and reacted at 110 °C in the dark for 15 hours, then cooled to room temperature. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the reaction system was cooled to room temperature. The solvent was removed by vacuum distillation, and the crude product was purified by silica gel column chromatography (eluting agent: ethyl acetate and petroleum ether, volume ratio 1:6) to obtain compound (Number M2-13). Yield 49.1%, purity 98.8%. 1H NMR(400MHz,Chloroform-d)δ7.97(s,1H),4.23(q,J=7.1Hz,2H),3.31(qd,J=7.3,5.6Hz,2H),1.35(t,J=7.1Hz,3H),1.12(t,J=7.3Hz,3H).[M+H] + : 169.1.

[0076] (2) Thiourea (1.2 mol), sodium tert-butoxide (1.2 mol), and intermediate compound M2-13 (1 mol) were mixed, dissolved in propanol, and reacted at 80 °C under nitrogen protection for 30 min. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the reaction system was cooled to room temperature. The solvent was removed by vacuum distillation, and 10% (v / v) aqueous acetic acid solution was added, precipitating out compound 13. The yield was 56.89%, and the purity was 97.7%. 1 H NMR (400MHz, DMSO-d6) δ12.54(s,1H),8.55(s,1H),8.43(t,J=5.4Hz,1H),8.25 (s,1H),8.09(s,1H),3.20(qd,J=7.2,5.3Hz,2H),1.09(t,J=7.2Hz,3H).[M+H] + :199.1.

[0077] Example 14

[0078] Compound 14 Preparation The steps are as follows:

[0079] (1) At room temperature, 1.3 mol of cyanoacetic acid, 1 mol of (CH3)2-CH-CH2-CH2-NH2, 1.1 mol of EDCI, and 1.1 mol of DMAP were dissolved in dichloromethane and reacted for 5 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the reaction system was filtered under reduced pressure. The solution was dissolved in ethyl acetate and washed successively with 1M HCl solution and saturated NaHCO3 solution. The ethyl acetate layer was collected, dried over anhydrous Na2SO4, and evaporated to dryness. The compound M1-14 was purified by column chromatography. Yield 48.74%, purity 98.9%. 1 H NMR(400MHz,Chloroform-d)δ3.43(s,2H),3.28(dt,J=7.9,5.8Hz,2H),1.62(dp,J=13.3,6.7Hz,1H),1.45-1.39(m,2H),0.93-0.89(m,6H).[M+H] +:155.2.

[0080] (2) Triethoxymethane (1.2 mol) and compound M1-14 (1 mol) were dissolved in acetic anhydride and reacted at 110 °C in the dark for 15 hours, then cooled to room temperature. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the reaction system was cooled to room temperature. The solvent was removed by vacuum distillation, and the crude product was purified by silica gel column chromatography (eluting solvent: ethyl acetate and petroleum ether, volume ratio 1:6) to obtain the intermediate compound M2-14. Yield 25.26%, purity 99.1%. 1 H NMR(400MHz,Chloroform-d)δ7.98(s,1H),4.23(q,J=7.1Hz,2H),3.33-3.24(m,2H),1 .56(dp,J=13.3,6.7Hz,1H),1.36(dt,J=12.1,7.1Hz,5H),0.86(d,J=6.7Hz,6H).[M+H] + :211.1.

[0081] (3) Thiourea (1.2 mol), sodium tert-butoxide (1.2 mol), and intermediate compound M1-14 (1 mol) were mixed, dissolved in propanol, and reacted at 80 °C under nitrogen protection for 30 min. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the reaction system was cooled to room temperature. The solvent was removed by vacuum distillation, and 10% (v / v) aqueous acetic acid solution was added to precipitate compound 14. The yield was 26.05%, and the purity was 97.1%. 1 H NMR (400MHz, DMSO-d) δ12.54(s,1H),8.53(s,1H),8.38(t,J=5.4Hz,1H),8.22(s,1H),8.08(s,1H),3.19( ddd,J=8.4,6.9,5.6Hz,2H),1.59(dp,J=13.3,6.7Hz,1H),1.42-1.32(m,2H),0.88(d,J=6.6Hz,6H).[M+H] + :241.0.

[0082] Examples 15-18

[0083] Referring to the preparation steps of Example 14, the substrate (CH3)2-CH-CH2-CH2-NH2 in step (1) was replaced with Y-NH2, and the following intermediate compounds and target compounds were synthesized respectively:

[0084]

[0085]

[0086]

[0087] Example 19

[0088] The inhibitory activity of compounds 1-18 against tyrosinase was determined using the following methods:

[0089] In a 96-well plate, 50 μL of PBS buffer (pH 6.8), 40 μL of 200 μM compound solution (containing 10% DMSO, v / v), and 10 μL of mushroom tyrosinase solution (1000 U / mL, dissolved in PBS pH 6.8) were added sequentially. The plate was incubated at 37°C for 10 min to allow for complete binding of the compound and tyrosinase. Then, 100 μL of L-DOPA solution (1 mM, dissolved in PBS pH 6.8) was added to each well, and incubation was continued for another 10 min to initiate the reaction. The blank control group used 40 μL of PBS buffer (pH 6.8) instead of the compound solution. The positive control group used 40 μL of 200 μM kojic acid and 377 (phenylethyl resorcinol; purity ≥98%, analytical grade) PBS buffer (pH 6.8) instead of the compound solution. The absorbance (OD) of each well was measured at 475 nm using a microplate reader. The OD values ​​for each group were recorded at 0 min and 20 min. The formula for calculating the tyrosinase inhibition rate is as follows:

[0090] Tyrosinase inhibition rate (%) = [(AB) - (CD)] / (AB) × 100

[0091] Where A is the OD value of the blank control group at 20 min; B is the OD value of the blank control group at 0 min; C is the OD value of the compound group at 20 min; and D is the OD value of the test compound group at 0 min. The experiment was repeated three times, and the average value was taken to evaluate the enzyme activity of the compound against tyrosinase. A curve was plotted with the concentration of the test sample on the x-axis and the corresponding tyrosinase inhibition rate on the y-axis, and a regression equation was obtained by fitting the curve. Based on the regression equation, the sample concentration corresponding to a tyrosinase inhibition rate of 50% was calculated, which is the IC50 of the test sample. 50 value.

[0092] result:

[0093] Results of the compound's inhibitory activity on tyrosinase

[0094] compound <![CDATA[IC 50 (μM)]]> compound <![CDATA[IC 50 (μM)]]> Kojic acid 32.0μM 9 3.32μM 377 (Phenethyl resorcinol) 16.7μM 10 >200μM 1 >200μM 11 6.92μM 2 12.6μM 12 12.2μM 3 4.63μM 13 19.1μM 4 >200μM 14 4.52μM 5 3.75μM 15 12.0μM 6 7.47μM 16 20.8μM 7 >200μM 17 19.1μM 8 >200μM 18 15.2μM

[0095] The results above show that compounds 2, 3, 5, 6, 9, 11, 12, 13, 14, 15, 16, 17, and 18 exhibited good inhibitory activity against tyrosinase (IC50). 50All values ​​were ≤20.8 μM. Among them, compounds 3 (4.63 μM), 5 (3.75 μM), 6 (7.47 μM), 9 (3.32 μM), 11 (6.92 μM), 12 (12.2 μM), 14 (4.52 μM), and 15 (12.0 μM) showed superior inhibitory activity compared to the positive controls kojic acid (32.0 μM) and 377 (16.7 μM). Notably, the IC50 values ​​of compounds 3, 5, 9, and 14 were ≤20.8 μM. 50 The values ​​were all less than 5 μM, indicating extremely strong tyrosinase inhibitory activity.

[0096] Example 20

[0097] The transdermal properties of the compound were determined using the following method:

[0098] Panamanian pig skin was collected, subcutaneous fat was removed, and the skin was repeatedly rinsed with physiological saline and cut to a size suitable for the Franz diffusion cell. Compounds 3, 5, 9, 14 and the control group α-arbutin were dissolved in sterile physiological saline containing 30% (v / v) ethanol to prepare 10 mM solutions. Simultaneously, sterile physiological saline containing 30% ethanol was used as the receiving solution.

[0099] A Franz diffusion cell was selected, with an exposed area diameter of 1 cm and an effective diffusion area of ​​approximately 0.785 cm². 2 The volume of the diffusion cell was 15 mL. The diffusion cell was placed in a constant temperature water bath at 32°C and stirred at 600 rpm using a magnetic stirrer. 400 μL of the 10 mM compound was added to the loading cell. At time points of 2 h, 4 h, 8 h, and 12 h, 500 μL of the receiving solution was removed from the receiving cell, and an equal volume of sterile physiological saline containing 30% ethanol was immediately added to maintain a constant volume of the receiving solution. The absorbance was measured using a UV-Vis spectrophotometer, and the compound concentration was calculated by substituting the absorbance into the standard curve.

[0100] The formula for calculating cumulative transmittance (Tn) is:

[0101]

[0102] c n The concentration of the compound in the receiving liquid during the nth sampling is V0; the volume of the receiving cell is c. i V represents the concentration of the sample. i is the sampling volume; M is the total amount of the compound to be tested.

[0103] The measurement results are as follows:

[0104]

[0105] In comparison, the cumulative transdermal permeability of α-arbutin after 12 hours was 10.9%, compound 3 was 19.5%, compound 5 was 12.4%, compound 9 was 20.9%, and compound 14 was 35.1%. The results indicate that compound 14 exhibits significantly enhanced transdermal performance compared to the same concentration of α-arbutin (p<0.05).

[0106] Example 21

[0107] 1. Evaluation of the toxicity of the compound to melanocytes

[0108] Melanoma cells (B16F10 cells) in the logarithmic growth phase were harvested and processed at a concentration of 5 × 10⁻⁶ cells / cells. 5 Cells were seeded at a density of 100 μL / well in 96-well plates, with 100 μL of DMEM medium containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin antibiotics added to each well. The experiment included a control group (medium containing the same volume of solvent as the compound group) and eight compound treatment groups at concentration gradients (2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 64 μM, 128 μM, and 256 μM), with six replicates per group. The 96-well plates were incubated at 37°C for 24 h, and the supernatant was discarded. Under dark conditions, 100 μL of CCK-8 solution was added to each well, and the plates were incubated at 37°C for another 30 min. The absorbance of each well was then measured at 450 nm.

[0109] Experimental results are as follows Figure 1 As shown, the activity of B16F10 cells treated with compounds 3, 5, 9, and 14 did not show significant differences compared to the control group. This result indicates that the above compounds have no significant toxicity to melanocytes within the experimental concentration range and possess high biosafety.

[0110] 2. Quantitative determination of melanin content

[0111] Take B16F10 cells in the logarithmic growth phase and use 5 × 10⁻⁶ cells. 5 Cells were seeded at a density of 100 cells / well in 6-well plates and cultured at 37°C for 24 hours. Then, 2 mL of different concentrations (5 μM, 10 μM, 50 μM) of the test compound (an equal volume of PBS was added to the blank control group) was added to each well, and the plates were incubated at 37°C for another 24 hours. The supernatant was then discarded, and the cells were washed with PBS solution. Cells were then lysed with 1M NaOH solution containing 10% DMSO. The absorbance of the resulting lysate was measured at 405 nm to calculate the melanin content using the formula: Melanin content (%) = (Absorbance of compound-treated group ÷ Absorbance of PBS-treated group) × 100.

[0112] The results are as follows Figure 2 As shown, compared with the blank control group, compounds 3, 5, 9, and 14 (5-50 μM) all significantly inhibited melanin production in B16F10 cells.

[0113] The results above show that compounds 3, 5, 9, and 14 specifically inhibit the catalytic activity of tyrosinase and block melanin synthesis, rather than reducing melanin by killing cells, and have no significant effect on the activity of B16F10 cells.

[0114] The tyrosinase inhibitor provided by this invention reduces melanin synthesis by inhibiting the activity of tyrosinase in the skin, thereby improving pigmentation problems. It can be applied to the preparation of whitening, spot-fading, and sun-protective cosmetics and skincare products. In the pharmaceutical field, the tyrosinase inhibitor provided by this invention improves diseases related to melanin metabolism by regulating melanin synthesis or inhibiting abnormal tyrosinase activity, and can be applied to the preparation of drugs, health products, and foods for the prevention and treatment of pigmentation diseases. In agriculture, the tyrosinase inhibitor provided by this invention inhibits the enzyme activity of certain pests that depend on tyrosinase metabolism, thereby blocking their physiological functions, and can be applied to the preparation of insecticides. In the food industry, the tyrosinase inhibitor provided by this invention delays oxidative browning by inhibiting the activity of endogenous tyrosinase in fruits, vegetables, seafood, and other food ingredients, and can be applied to the preparation of food preservatives.

[0115] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A tyrosinase inhibitor, characterized by, The application relates to a compound having the structure of formula I: or a physiologically acceptable salt thereof; The compound having the structure of formula I is specifically one of the following compounds 3, 5, 6, 9 and 11-18: ； 。 2. A method for preparing the tyrosinase inhibitor according to claim 1, characterized in that, The application further relates to a synthetic route as shown in the following formula: ； In step (1), compound M1 is reacted with triethoxymethane to generate intermediate compound M2; in step (2), intermediate compound M2 is reacted with thiourea to generate the target compound.

3. The preparation method according to claim 2, characterized in that, In step (1), triethoxymethane and compound M1 in a molar ratio of (1.2-1.4):1 are dissolved in acetic anhydride, and the reaction is carried out in the dark at 105-115 DEG C.

4. The preparation method according to claim 2, characterized in that, In step (2), thiourea, compound M2 and sodium tert-butoxide in a molar ratio of (1.2-1.4):1: (1.2-1.4) are mixed, dissolved in propanol, and the reaction is carried out at 75-85 DEG C under an inert gas atmosphere.

5. The use of a tyrosinase inhibitor according to claim 1, characterized in that, The application further relates to a product containing the tyrosinase inhibitor as an active ingredient.

6. Use according to claim 5, characterized in that, The product is a sunscreen, skin lightening or whitening cosmetic.

7. Use according to claim 5, characterized in that, The product is a drug for preventing and treating pigmentation diseases.

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