Tyrosinase inhibitor and application thereof

Abstract

The invention relates to the technical field of chemistry, in particular to a tyrosinase inhibitor and application thereof, and the tyrosinase inhibitor comprises a compound with the structure shown in the specification or physiologically acceptable salt of the compound. The tyrosinase inhibitor provided by the invention has remarkable tyrosinase inhibition activity, can effectively block melanin synthesis, and is small in toxic and side effects and high in molecular stability.

Description

Technical Field

[0001] The present invention relates to the field of chemical technology, and in particular to a tyrosinase inhibitor and application thereof. Background Art

[0002] Hyperpigmentation is often caused by excessive melanin deposition in the skin. Tyrosinase, a key enzyme in melanin formation, is closely linked to melanin synthesis. Tyrosinase inhibitors, due to their ability to regulate melanin synthesis, have shown significant application value in a variety of fields: in medicine, they can be used to treat and prevent pigmentation disorders such as melasma; in cosmetics, they can whiten human skin; in the food industry, they can slow the loss of freshness and spoilage of fruits and vegetables due to browning; and in agriculture, because tyrosinase is a key enzyme for the survival of some insects, its inhibitors have become one of the most promising biopesticides. Therefore, the development of tyrosinase inhibitors has broad application prospects.

[0003] Tyrosinase inhibitors currently on the market can be categorized by their source as either natural extracts or chemically synthesized products. Natural extracts, such as arbutin and niacinamide, have limited inhibitory activity against tyrosinase, making them ineffective for treating stubborn pigmentation. Furthermore, arbutin exhibits irritation and potential cytotoxicity at high concentrations, limiting its inclusion in whitening products and impacting efficacy. Niacinamide's direct tyrosinase inhibition is limited, leading to skin intolerance in some individuals, such as redness and itching. Furthermore, its whitening effects are slow to manifest, requiring long-term use, making it difficult to meet the demand for rapid whitening. Chemically synthesized products, such as vitamin C derivatives like ascorbic acid 2-glucoside and magnesium ascorbyl phosphate, exhibit poor transdermal penetration, resulting in low effective concentrations in the skin and limited whitening effects. Furthermore, these products are susceptible to decomposition at high temperatures or under inappropriate pH conditions, resulting in poor stability and requiring high formulation and processing requirements, increasing the complexity and cost of R&D and production. Therefore, developing new, safe, effective, and stable tyrosinase inhibitors is crucial for addressing the shortcomings of existing products and meeting the needs of various applications. Summary of the Invention

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

[0005] The technical solution of the present invention is achieved as follows:

[0006] In a first aspect, the present invention provides a tyrosinase inhibitor, which includes a compound having a structure shown in the following formula I:

[0007] or a physiologically acceptable salt thereof;

[0008] In Formula 1:

[0009] X is NH or O;

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

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

[0012] Wherein, n is selected from an integer between 1 and 5.

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

[0014]

[0015]

[0016]

[0017] In a third aspect, the present invention provides a method for preparing the tyrosinase inhibitor, which comprises the following synthetic route:

[0018]

[0019] Wherein: 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 one of hydrogen, amino, C1-C6 alkyl, halogenated C1-C6 alkyl, cycloalkyl, halogenated cycloalkyl, aryl, substituted aryl and structural units Q1-Q12; the structural units Q1-Q12 are as follows:

[0022]

[0023] Wherein, n is selected from an integer between 1 and 5.

[0024] Furthermore, in step (1), triethoxymethane and compound M1 at 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 connected to the carbon group and the cyano group loses a hydrogen atom under the action of the acid anhydride to form a carbon anion. Due to the electron-withdrawing effect of the oxygen atom in triethylmethane, the methane carbon atom carries a partial positive charge. The formed carbon cation attacks the M1 carbon anion to generate M2.

[0025] Furthermore, in step (2), thiourea, compound M2, and sodium tert-butoxide are mixed in a molar ratio of (1.2-1.4):1:(1.2-1.4), 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 electrophilic reagent to attack the unsaturated cyano group and olefin structure to produce the final product.

[0026] In a fourth aspect, the present invention provides a use of the tyrosinase inhibitor for preparing a product containing the tyrosinase inhibitor as an active ingredient.

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

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

[0029] The IC values ​​of various compounds provided by the present invention on tyrosinase 50 The values ​​were all less than 5 μM, significantly superior to the positive control drugs kojic acid (32.0 μM) and phenylethylresorcinol (16.7 μM). They specifically inhibited tyrosinase catalytic activity and effectively blocked melanin synthesis. Compared to the control compound α-arbutin at the same concentration, their transdermal performance was significantly enhanced. Furthermore, several compounds showed no significant cytotoxicity within the concentration range of 0-256 μM and demonstrated good safety against melanocytes. Therefore, they have significant application prospects in the preparation of whitening, spot-lightening, and sunscreen cosmetics and skincare products, in the prevention and treatment of pigmentation disorders, in the killing of pests that rely on tyrosinase metabolism, and in food preservatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1 The toxicity test results of the test compound on melanoma cells (B16F10);

[0032] Figure 2 The results are the inhibitory activity assay results of the test compound on melanin production in melanoma cells (B16F10). DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the embodiments described are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0034] In some specific embodiments, the present invention provides a compound having formula I The compound of the structure shown in FIG. 1 comprises the following synthetic steps:

[0035]

[0036] In the above 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;

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

[0038] X is NH or O;

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

[0040]

[0041] Wherein, n is selected from an integer between 1 and 5.

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

[0043] Dissolve triethoxymethane (1.2 mol) and compound M1 (1 mol) in acetic anhydride and react at 110°C in the dark for 15 hours, then cool to room temperature. Monitor the reaction progress by thin-layer chromatography. Once the reaction is complete, cool the reaction system to room temperature. Remove the solvent by distillation under reduced pressure, and purify the resulting crude product by silica gel column chromatography (eluent: ethyl acetate: petroleum ether, volume ratio: 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 for 30 minutes. The reaction progress was monitored by thin-layer chromatography. After completion, the reaction system was cooled to room temperature. The solvent was removed by distillation under reduced pressure, and 10% (v / v) aqueous acetic acid was added to precipitate a solid, which was the title compound.

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

[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, the preparation method of compound M1 is as follows:

[0049] At room temperature, cyanoacetic acid (1.3 mol), YXH (i.e., Y-OH or Y-NH2, 1 mol), EDCI (1.1 mol), and DMAP (1.1 mol) were dissolved in dichloromethane and allowed to react for 5 hours. The reaction progress was monitored by thin-layer chromatography. After completion, the reaction system was filtered under reduced pressure. After dissolving in ethyl acetate, the mixture was washed sequentially with 1M HCl solution and saturated NaHCO3 solution. The ethyl acetate layer was collected, dried over anhydrous Na2SO4, and spin-dried. Compound M1 was isolated and purified by column chromatography. EDCI is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; DMAP is 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 in the dark at 110°C 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 distillation under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (eluent: ethyl acetate: petroleum ether, volume ratio 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 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 distillation under reduced pressure, and 10% (v / v) aqueous acetic acid was added to precipitate 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] The substrate cyanoacetic acid in step (1) of Example 1 was replaced with CH3-CH2-OC(O)-CH2-CN (ethyl cyanoacetate) to obtain 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 with a yield of 67.75% and a purity of 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] With reference 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 to synthesize the following intermediate compound and target compound, respectively:

[0060]

[0061]

[0062]

[0063] Example 8

[0064] Compound 8 Preparation , the steps are as follows:

[0065] (1) Take cyanoacetic acid (1.3 mol), n-pentanol (1 mol), EDCI (1.1 mol) and DMAP (1.1 mol) at room temperature, dissolve them in dichloromethane, react for 5 hours, monitor the reaction progress by thin layer chromatography, and after the reaction is complete, filter the reaction system under reduced pressure. Dissolve it in ethyl acetate, and wash it with 1M HCl solution and saturated NaHCO3 solution in sequence. Collect the ethyl acetate layer, dry it with anhydrous Na2SO4, and spin dry it. Separate and purify it by column chromatography to obtain compound M1-8 The yield is 65.24% and the purity is 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) Dissolve triethoxymethane (1.2 mol) and compound M1-8 (1 mol) in acetic anhydride, react at 110°C in the dark for 15 hours, and cool to room temperature. Monitor the reaction progress by thin-layer chromatography. After the reaction is complete, cool the reaction system to room temperature. Remove the solvent by distillation under reduced pressure, and purify the resulting crude product by silica gel column chromatography (eluent: ethyl acetate: petroleum ether, volume ratio 1:6) to obtain intermediate compound M2-8. The yield is 68.52% and the purity is 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 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 distillation under reduced pressure, and 10% (v / v) aqueous acetic acid 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 Y-OH to synthesize the following intermediate compounds and target compound respectively:

[0070]

[0071]

[0072]

[0073] Example 13

[0074] Compound 13 Preparation , the steps are as follows:

[0075] (1) Dissolve triethoxymethane (1.2 mol) and compound CN-CH2-C(O)NH2 (1 mol, cyanoacetamide) in acetic anhydride, react at 110°C in the dark for 15 hours, and cool to room temperature. Monitor the reaction progress by thin-layer chromatography. After the reaction is complete, cool the reaction system to room temperature. Distill under reduced pressure to remove the solvent, and purify the resulting crude product by silica gel column chromatography (eluent: ethyl acetate and petroleum ether in a volume ratio of 1:6) to obtain compound (No. 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 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 distillation under reduced pressure, and 10% (v / v) aqueous acetic acid was added to precipitate 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) Take cyanoacetic acid (1.3 mol), (CH3)2-CH-CH2-CH2-NH2 (1 mol), EDCI (1.1 mol) and DMAP (1.1 mol) at room temperature, dissolve them in dichloromethane, react for 5 hours, monitor the reaction progress by thin layer chromatography, and after the reaction is complete, filter the reaction system under reduced pressure. Dissolve it in ethyl acetate, and wash it with 1M HCl solution and saturated NaHCO3 solution in sequence. Collect the ethyl acetate layer, dry it with anhydrous Na2SO4, and spin dry it. Separate and purify it by column chromatography to obtain compound M1-14 The yield is 48.74% and the purity is 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) Dissolve triethoxymethane (1.2 mol) and compound M1-14 (1 mol) in acetic anhydride, react at 110°C in the dark for 15 hours, and cool to room temperature. Monitor the reaction progress by thin-layer chromatography. After the reaction is complete, cool the reaction system to room temperature. Remove the solvent by distillation under reduced pressure, and purify the resulting crude product by silica gel column chromatography (eluent: ethyl acetate: petroleum ether, volume ratio 1:6) to obtain intermediate compound M2-14. The yield is 25.26%, and the purity is 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 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 distillation under reduced pressure, and 10% (v / v) aqueous acetic acid 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 to synthesize the following intermediate compounds and target compound, respectively:

[0084]

[0085]

[0086]

[0087] Example 19

[0088] The tyrosinase inhibitory activities of compounds 1-18 were determined as follows:

[0089] To a 96-well plate, 50 μL of PBS buffer (pH 6.8), 40 μL of a 200 μM compound solution (containing 10% DMSO, v / v), and 10 μL of a mushroom tyrosinase solution (1000 U / mL in PBS, pH 6.8) were sequentially added and incubated at 37°C for 10 min to allow for full binding of the compound to the tyrosinase. The reaction was initiated by adding 100 μL of a 1 mM L-DOPA solution (dissolved in PBS, pH 6.8) to each well and incubating for another 10 min. The blank control group received 40 μL of PBS buffer (pH 6.8) instead of the compound solution. The positive control group received 40 μL of 200 μM kojic acid and 377 (phenylethyl resorcinol; purity ≥98%, analytical grade) in 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 and 20 min. The tyrosinase inhibition rate was calculated as follows:

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

[0091] Where A is the OD value of the blank control group at 20 minutes; B is the OD value of the blank control group at 0 minutes; C is the OD value of the compound group at 20 minutes; and D is the OD value of the test compound group at 0 minutes. The experiment was repeated 3 times and the average value was taken to evaluate the enzymatic activity of the compound against tyrosinase. The concentration of the test sample was used as the horizontal axis and the corresponding tyrosinase inhibition rate was used as the vertical axis. A curve was drawn and fitted to obtain a regression equation. According to the regression equation, the sample concentration corresponding to the tyrosinase inhibition rate of 50% was calculated, which is the IC value of the test sample. 50 value.

[0092] result:

[0093] Tyrosinase inhibitory activity of the compounds

[0094] Compound <![CDATA[IC 50 (μM)]]> Compound <![CDATA[IC 50 (μM)]]> Kojic acid 32.0μM 9 3.32μM 377 (phenylethyl resorcinol) 16.7 μM 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 3.75 μM 15 12.0μM 6 7.47 μM 16 20.8μM 7 >200μM 17 19.1μM 8 >200μM 18 15.2μM

[0095] From the above results, it can be seen that compounds 2, 3, 5, 6, 9, 11, 12, 13, 14, 15, 16, 17 and 18 showed good inhibitory activity against tyrosinase (IC 50The inhibitory activities of 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) were better than those of the positive control drugs kojic acid (32.0 μM) and 377 (16.7 μM). It is worth noting that the IC values ​​of compounds 3, 5, 9 and 14 were 50 The values ​​were all less than 5 μM, showing extremely strong tyrosinase inhibitory activity.

[0096] Example 20

[0097] The transdermal properties of the compounds were determined as follows:

[0098] Panamanian suckling pig skin was removed of subcutaneous fat, repeatedly rinsed with saline, and cut into a size suitable for Franz diffusion cells. Compounds 3, 5, 9, and 14, as well as a control group of α-arbutin, were dissolved in 30% (v / v) ethanol in sterile saline to prepare a 10 mM solution. Sterile saline containing 30% ethanol was also used as the receptor 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 cm 2 , with a volume of 15 mL. Place the diffusion cell in a constant temperature water bath at 32°C and stir at 600 r / min using a magnetic stirrer. Pipette 400 μL of 10 mM compound into the loading cell. At 2 h, 4 h, 8 h, and 12 h, remove 500 μL of receiving solution from the receiving cell and immediately add an equal amount of sterile saline containing 30% ethanol to keep the volume of the receiving solution constant. Measure the absorbance by UV-Vis spectrophotometer and substitute it into the standard curve to calculate the compound concentration.

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

[0101]

[0102] c n is the compound concentration of the receiving solution at the time of the nth sampling; V0 is the volume of the receiving pool, c i is the concentration of the sample; V i is the sampling volume; M is the total amount of the compound to be tested.

[0103] The results are as follows:

[0104]

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

[0106] Example 21

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

[0108] Melanoma cells (B16F10 cells) in logarithmic growth phase were taken and 5×10 5 Cells were seeded at a density of 100 cells / well in a 96-well plate, and 100 μL of DMEM medium containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin was added to each well. The experiment set up a control group (medium containing the same amount of solvent as the compound group) and 8 compound treatment groups with a concentration gradient (2μM, 4μM, 8μM, 16μM, 32μM, 64μM, 128μM, 256μM), with 6 replicates per group. The 96-well plate was placed in a 37°C incubator for 24 hours, and the supernatant was discarded. Under light-proof conditions, 100 μL of CCK-8 solution was added to each well, and incubated in a 37°C incubator for another 30 minutes. The absorbance value of each well at a wavelength of 450 nm was then measured.

[0109] The experimental results are as follows Figure 1 As shown, there was no significant difference in the activity of B16F10 cells between the treatment groups with compounds 3, 5, 9, and 14 and the control group. This result indicates that the above compounds have no significant toxicity to melanocytes within the experimental concentration range and have high biosafety.

[0110] 2. Quantitative determination of melanin content

[0111] B16F10 cells in the logarithmic growth phase were taken and 5×10 5 Cells were seeded at a density of 100 cells / well in a 6-well plate and incubated for 24 hours at 37°C. Then, 2 mL of the test compound at various concentrations (5 μM, 10 μM, and 50 μM) was added to each well (an equal volume of PBS was added to the blank control group) and incubated for another 24 hours at 37°C. The supernatant was discarded, the cells were washed with PBS, and lysed with 10% DMSO in 1M NaOH. The absorbance of the resulting lysate was measured at 405 nm, and melanin content was calculated using the following 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) can significantly inhibit the production of melanin in B16F10 cells.

[0113] From the above results, it can be seen 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 inhibitors provided by the present invention improve pigmentation problems by inhibiting the activity of tyrosinase in the skin and reducing melanin synthesis, and can be used to prepare cosmetics and skin care products for whitening, lightening and sun protection. In medicine, the tyrosinase inhibitors provided by the present invention improve diseases related to melanin metabolism by regulating melanin synthesis or inhibiting abnormal tyrosinase activity, and can be used to prepare drugs, health products and foods for preventing and treating pigmentation diseases. In agriculture, the tyrosinase inhibitors provided by the present invention inhibit the enzyme activity of certain pests that rely on tyrosinase metabolism to block their physiological functions, and can be used to prepare pesticides. In the food industry, the tyrosinase inhibitors provided by the present invention delay oxidative browning by inhibiting the activity of endogenous tyrosinase in fruits, vegetables, seafood and other ingredients, and can be used to prepare 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 in the scope of protection of the present invention.

Claims

1. A tyrosinase inhibitor, characterized in that Including compounds having the structure shown in the following formula I: or a physiologically acceptable salt thereof; In Formula 1: X is NH or O; Y is selected from one of hydrogen, amino, C1-C6 alkyl, halogenated C1-C6 alkyl, cycloalkyl, halogenated cycloalkyl, aryl, substituted aryl and structural units Q1-Q12; The structural units Q1-Q12 are as follows: Wherein, n is selected from an integer between 1 and 5.

2. The tyrosinase inhibitor according to claim 1, characterized in that The compound represented by the structure of formula I is specifically one of the following compounds 1-18:

3. A method for preparing the tyrosinase inhibitor according to claim 1, characterized in that: The following synthetic routes are included: Wherein: 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; X is NH or O; Y is selected from one of hydrogen, amino, C1-C6 alkyl, halogenated C1-C6 alkyl, cycloalkyl, halogenated cycloalkyl, aryl, substituted aryl and structural units Q1-Q12; the structural units Q1-Q12 are as follows: Wherein, n is selected from an integer between 1 and 5.

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

5. The preparation method according to claim 3, characterized in that In the 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.

6. The use of the tyrosinase inhibitor according to claim 1, characterized in that: Used for preparing products with the tyrosinase inhibitor as an active ingredient.

7. The use according to claim 6, characterized in that The product is a sunscreen, spot-lightening or whitening cosmetic.

8. The use according to claim 6, characterized in that The product is a medicine, health product or food for preventing and treating pigmentation diseases.

9. The use according to claim 6, characterized in that The product described is an insecticide.

10. The use according to claim 6, characterized in that The product is a food preservative.

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