Tyrosinase inhibitors and their use

Abstract

This invention provides tyrosinase inhibitors, methods for preparing them, and their uses. [Solution] The present invention provides a compound having the structure represented by formula I or a physiologically acceptable salt thereof. The tyrosinase inhibitor provided in this invention has remarkable tyrosinase inhibitory activity, can effectively block melanin synthesis, has low toxicity and side effects, and has high molecular stability. JPEG0007898705000036.jpg23170 (In formula I, X is NH or O, and Y is selected from hydrogen, amino, C1-C6 alkyl, halogenated C1-C6 alkyl, cycloalkyl, halogenated cycloalkyl, aryl, substituted aryl, and C1 to C5 alkyl groups having pyridine, pyrrole, piperidine, morpholine, thiophene, and benzothiophene.)

Description

[Technical Field]

[0001] This invention relates to the chemical technology, and more particularly to tyrosinase inhibitors and their use. [Background technology]

[0002] Pigmentation is usually caused by excessive melanin deposition in the skin. Tyrosinase, a key enzyme in melanin formation in the body, has activity closely related to melanin synthesis. Tyrosinase inhibitors, which can regulate the melanin synthesis process, have significant application value in various fields. In the pharmaceutical field, they can be used to treat and prevent pigmentation diseases such as melasma, and in the cosmetics field, they can achieve skin whitening. In the food industry, they can slow down the browning and deterioration of fruits and vegetables. Furthermore, in agriculture, since tyrosinase is an essential enzyme for the survival of some insects, its inhibitors have the potential to become one of the most promising biological insecticides. Thus, the development of tyrosinase inhibitors has an extremely wide range of application prospects.

[0003] Tyrosinase inhibitors currently on the market can be broadly classified into natural extracts and chemically synthesized compounds based on their origin. Natural extracts include arbutin and niacinamide, but arbutin has limited inhibitory activity against tyrosinase and is not sufficiently effective in improving stubborn discoloration. Furthermore, it has irritating and potentially cytotoxic properties at high concentrations, limiting its concentration in whitening products and hindering its full effectiveness. Niacinamide does not have a clear direct inhibitory effect against tyrosinase, and some users experience intolerance reactions accompanied by skin irritation such as erythema and itching. In addition, its whitening effect is slow to appear and requires long-term use, making it difficult to meet the demand for rapid whitening effects. Representative chemically synthesized compounds include vitamin C derivatives such as ascorbic acid-2-glucoside and magnesium ascorbyl phosphate. However, these substances have low transdermal absorption, resulting in insufficient effective concentrations in the skin and limited whitening effects. Furthermore, existing tyrosinase inhibitors are prone to degradation under high temperatures and inappropriate pH conditions, resulting in insufficient stability. This necessitates high standards in formulation design, increasing the difficulty and cost of research and development and manufacturing. Therefore, the development of novel tyrosinase inhibitors that are highly safe, efficient, and stable is crucial for improving upon the shortcomings of existing products and meeting the application needs in various fields. [Overview of the project]

[0004] In view of the above circumstances, the present invention provides a tyrosinase inhibitor and its use.

[0005] The technical means of the present invention are achieved as follows.

[0006] In a first aspect, the present invention provides a tyrosinase inhibitor. This tyrosinase inhibitor comprises a compound having the structure represented by the following formula I or a physiologically acceptable salt thereof. [ka] In equation I, X is either 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 respectively

Chemical formula

[0007] Furthermore, in some specific embodiments, the compound having the structure represented by the formula I is specifically one of the following compounds 1-18.

Table 2

[0008] In a third aspect, the present invention provides a method for preparing the above-mentioned tyrosinase inhibitor. The method includes the following synthetic route.

Chemical formula

Chemical formula

[0009] Furthermore, in the step (1), triethoxymethane and compound M1 are dissolved in acetic anhydride at a molar ratio of (1.2 - 1.4):1, and reacted under the conditions of 105 - 115 °C in the dark. The reaction in step (1) is an electrophilic substitution reaction. Under the action of the acid anhydride, the carbon atom bonded to the cyano group loses a hydrogen atom to form a carbanion. On the other hand, due to the electron-withdrawing property of the oxygen atom in triethylmethane, the carbon atom of methane is partially positively charged. The generated carbocation attacks the carbanion of M1 to generate M2.

[0010] Furthermore, in the step (2), thiourea, compound M2 and sodium tert-butoxide are mixed at a molar ratio of (1.2 - 1.4):1:(1.2 - 1.4), dissolved in propanol, and reacted under the conditions of 75 - 85 °C in 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, attacks the unsaturated cyano group and alkene structure, and forms the final product.

[0011] In a fourth aspect, the present invention provides the use of the tyrosinase inhibitor in the preparation of a product having the tyrosinase inhibitor as an active ingredient.

[0012] The beneficial effects of the present invention are as follows. The tyrosinase inhibitor provided by the present invention has significant tyrosinase inhibitory activity, and shows obvious advantages in terms of potential toxic side effects, easy availability of raw materials, molecular stability, etc. compared with conventional tyrosinase inhibitors.

[0013] A plurality of compounds provided by the present invention all have an IC 50The values ​​are less than 5 μM, which is significantly better than the positive control drugs kojic acid (32.0 μM) and phenylethyl resorcinol (16.7 μM). These compounds specifically inhibit the catalytic activity of tyrosinase and effectively block melanin synthesis. Furthermore, they exhibit remarkably superior skin permeability compared to the control compound α-arbutin at the same concentration. In addition, several compounds do not show clear cytotoxicity in the concentration range of 0 to 256 μM, demonstrating high safety for melanocytes. Therefore, they have extremely large potential applications in cosmetics such as skin whitening, spot treatment, and sunscreens, skincare products, preventive and therapeutic agents for pigmentation diseases, health foods, pest control agents for tyrosinase metabolism-dependent pests, and even food freshness preservatives. [Brief explanation of the drawing]

[0014] To more clearly illustrate the technical means in the embodiments of the present invention, the drawings that may be used in the description of the embodiments are briefly described below. Clearly, the drawings in the following description represent only a few embodiments of the present invention, and those skilled in the art can obtain other drawings based on these without expending any creative effort.

[0015] [Figure 1] This shows the toxicity measurement results of the test compound against melanoma cells (B16F10). [Figure 2] Measurement results of the inhibitory activity of the test compound on melanin production in melanoma cells (B16F10). [Modes for carrying out the invention]

[0016] To further clarify the object, technical means, and advantages of the present invention, the technical means in the present invention will be described clearly and completely below, and it should be noted that the described examples are only some examples of the present invention, not all examples. Based on the examples of the present invention, those skilled in the art will know that all other examples obtained without creative work also fall within the scope of the present invention. Unless specific conditions are specified in the examples, the methods were used under normal conditions or conditions suggested by the manufacturer. The reagents or equipment used are not specified by the manufacturer and are all common products obtained by commercial purchase.

[0017] In some specific embodiments, Formula I provided by the present invention [ka] Compounds having the structure represented by have a synthesis route that includes the following synthesis steps. [ka] In step (1), compound M1 is reacted with triethoxymethane to produce intermediate compound M2, and in step (2), intermediate compound M2 is reacted with thiourea to produce the target compound. In compounds M1, M2, and the compound having the structure represented by formula I, X and Y are as follows: X is either 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, respectively. [ka] Here, n is selected from integers between 1 and 5.

[0018] In some specific examples, the synthesis method for intermediate compound M2 and the target compound (a compound represented by the structural formula I) is as follows. Triethoxymethane (1.2 mol) and compound M1 (1 mol) are dissolved in acetic anhydride and reacted in the dark at 110°C for 15 hours, then cooled to room temperature. The reaction is monitored by thin-layer chromatography, and after complete reaction, the reaction system is cooled to room temperature. The solvent is removed by vacuum distillation, and the resulting crude product is purified by silica gel column chromatography (using a mixture of ethyl acetate and petroleum ether in a volume ratio of 1:6) to obtain intermediate compound M2.

[0019] Thiourea (1.2 mol), tert-butoxide sodium (1.2 mol), and intermediate compound M2 (1 mol) are mixed and dissolved with propanol. The mixture is then reacted at 80°C under nitrogen protection for 30 minutes, and the reaction is monitored by thin-layer chromatography. After complete reaction, the reaction system is cooled to room temperature. The solvent is removed by vacuum distillation, and a 10% (v / v) aqueous acetic acid solution is added to precipitate the compound. The resulting solid is the target compound.

[0020] In some specific embodiments, the present invention further provides a method for preparing compound M1 in step (1) above. This preparation method involves reacting an alcohol or amine YXH (i.e., Y-OH or Y-NH2) with cyanoacetic acid to produce compound M1. The synthesis route of compound M1 is as follows. [ka]

[0021] In some specific examples, the method for preparing compound M1 is as follows. 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 reacted for 5 hours. The reaction progress was monitored by thin-layer chromatography, and after complete reaction, the reaction system was filtered by suction under reduced pressure. After dissolution with ethyl acetate, the mixture was sequentially washed with 1 M HCl solution and saturated NaHCO3 solution. The ethyl acetate layer was collected and dried over anhydrous Na2SO4 to remove the solvent by distillation. Separation and purification by column chromatography yielded compound M1. EDCI was 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and DMAP was 4-dimethylaminopyridine.

[0022] Example 1 Compound 1: [ka] Preparation The procedure is as follows: (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, and after the reaction was complete, the reaction system was cooled to room temperature. The solvent was removed by vacuum distillation, and the resulting crude product was purified by silica gel column chromatography (using a mixture of ethyl acetate and petroleum ether in a volume ratio of 1:6) to obtain compound HOOC-C(CN)=CH-CH-O-CH2-CH3 (number M2-1). (2) Thiourea (1.2 mol), tert-butoxide sodium (1.2 mol), and intermediate compound M2-1 (1 mol) were mixed and dissolved with propanol. The mixture was then reacted at 80°C under nitrogen protection for 30 minutes, and the reaction 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 acetic acid solution was added to precipitate the compound, yielding compound 1 (yield 84.78%, purity 98.0%). 1H NMR (400 MHz, DMSO-d6) δ 8.47 (s, 1H), 8.05 (s, 1H), 8.03 (s, 2H). [M+H] + :172.2.

[0023] Example 2 Compound 2: [ka] Preparation The procedure is as follows: In step (1) of Example 1, the substrate cyanoacetic acid was changed to CH3-CH2-OC(O)-CH2-CN (ethyl cyanoacetate), and compound M2-2: [ka] We obtained (yield 82%, purity 7%). 1 H NMR (500 MHz, Chloroform-d) δ 7.87 (s, 1H), 4.27 (q, J = 7.1 Hz, 2H), 4.08 (q, J = 6.9 Hz, 2H), 1.31 (td, J = 7.0, 1.2 Hz, 6H). [M+H] + :170.2. Compound 2 was obtained by referring to step (2) of Example 1 (yield 67.75%, purity 97.5%). 1 H NMR (400 MHz, Chloroform-d) δ 7.97 - 7.92 (m, 1H), 5.00 (ttd, J = 11.5, 6.2, 2.9 Hz, 1H), 4.34-4.24 (m, 2H), 1.35 (tdd, J = 7.1, 5.0, 2.5 Hz, 3H), 1.20 (ddd, J = 6.3, 4.9, 2.6 Hz, 6H). [M+H] + :200.1.

[0024] Examples 3-7 Referring to the preparation procedures of Example 1 and Example 2, the substrate cyanoacetic acid in step (1) of Example 1 was changed to Y - O - C(O) - CH2 - CN, and the following intermediate compounds and target compounds were synthesized respectively. [Table 3] JPEG0007898705000016.jpg149170

[0025] Example 8 Compound 8: [Chemical formula] Preparation The procedure is as follows. (1) At room temperature, cyanoacetic acid (1.3 mol), n - pentanol (1 mol), EDCI (1.1 mol) and DMAP (1.1 mol) were dissolved in dichloromethane and reacted for 5 hours. The progress of the reaction was monitored by thin - layer chromatography. After the reaction was complete, the reaction system was suction - filtered under reduced pressure. After dissolving in ethyl acetate, it was washed successively with 1M HCl solution and saturated NaHCO3 solution. The ethyl acetate layer was collected, dried over anhydrous Na2SO4, and the solvent was distilled off. It was separated and purified by column chromatography to obtain Compound M1 - 8: [Chemical formula] yielding (65.24% yield, 97.3% purity). 1 H NMR (400 MHz, Chloroform - d) δ 4.20 (t, J = 6.8 Hz, 2H), 3.47 (s, 2H), 1.68 (dq, J = 9.5, 7.1 Hz, 2H), 1.42 - 1.29 (m, 4H), 0.95 - 0.86 (m, 3H). [M + H] + : 172.2. (2) Triethoxymethane (1.2 mol) and compound M1-8 (1 mol) 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, and after the reaction was complete, the reaction system was cooled to room temperature. The solvent was removed by vacuum distillation, and the resulting crude product was purified by silica gel column chromatography (the eluent was a mixture of ethyl acetate and petroleum ether in a volume ratio of 1:6), and intermediate compound M2-8: [ka] The result was obtained (yield 68.52%, purity 98.9%). 1 H NMR (400 MHz, Chloroform-d) δ 8.00 (s, 1H), 4.34 (q, J = 7.1 Hz, 2H), 4.17 (t, J = 6.7 Hz, 2H), 1.72-1.62 (m, 2H), 1.42 (t, J = 7.1 Hz, 3H), 1.33 (dq, J = 6.9, 3.7, 3.2 Hz, 4H), 0.93 - 0.84 (m, 3H). [M+H] + :212.1. (3) Thiourea (1.2 mol), tert-butoxide sodium (1.2 mol), and intermediate compound M2-8 (1 mol) were mixed and dissolved with propanol. The mixture was then reacted at 80°C under nitrogen protection for 30 minutes, and the reaction 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 acetic acid solution was added to precipitate the compound, yielding compound 8 (yield 56.73%, purity 98.5%). 1 H NMR (400 MHz, DMSO-d6) δ 12.65 (s, 1H), 8.50 (s, 1H), 8.05 (s, 1H), 7.89 (s, 1H), 4.19 (t, J = 6.6 Hz, 2H), 1.67 (p, J = 6.9 Hz, 2H), 1.31 (dh, J = 7.5, 4.0 Hz, 4H), 0.94 - 0.83 (m, 3H). [M+H] + :242.1.

[0026] Examples 9-12 Referring to the preparation procedure of Example 8, the substrate n-pentanol in step (1) was replaced with Y-OH to synthesize the following intermediate compound and target compound, respectively. [Table 4] JPEG0007898705000021.jpg86170

[0027] Example 13 Compound 13: [ka] Preparation The procedure is as follows: (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, and after the reaction was complete, the reaction system was cooled to room temperature. The solvent was removed by vacuum distillation, and the resulting crude product was purified by silica gel column chromatography (the eluent was a mixture of ethyl acetate and petroleum ether in a volume ratio of 1:6), and the compound [ka] (Number M2-13) was obtained (yield 49.1%, purity 98.8%). 1 H NMR (400 MHz, Chloroform-d) δ 7.97 (s, 1H), 4.23 (q, J = 7.1 Hz, 2H), 3.31 (qd, J = 7.3, 5.6 Hz, 2H), 1.35 (t, J = 7.1 Hz, 3H), 1.12 (t, J = 7.3Hz, 3H). [M+H] + :169.1. (2) Thiourea (1.2 mol), tert-butoxide sodium (1.2 mol), and intermediate compound M2-13 (1 mol) were mixed and dissolved with propanol. The mixture was then reacted at 80°C under nitrogen protection for 30 minutes, and 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 acetic acid solution was added to precipitate the compound, yielding compound 13 (yield 56.89%, purity 97.7%). 1 H NMR (400 MHz, DMSO-d6) δ 12.54 (s, 1H), 8.55 (s, 1H), 8.43 (t, J = 5.4 Hz, 1H), 8.25 (s, 1H), 8.09 (s, 1H), 3.20 (qd, J = 7.2, 5.3 Hz, 2H), 1.09 (t, J = 7.2 Hz, 3H). [M+H] + :199.1.

[0028] Example 14 Compound 14: [ka] Preparation The procedure is as follows: (1) At room temperature, cyanoacetic acid (1.3 mol), (CH3)2-CH-CH2-CH2-NH2 (1 mol), EDCI (1.1 mol), and DMAP (1.1 mol) were dissolved in dichloromethane and reacted for 5 hours. The reaction progress was monitored by thin-layer chromatography, and after complete reaction, the reaction system was filtered by suction under reduced pressure. After dissolution with ethyl acetate, the mixture was sequentially washed with 1 M HCl solution and saturated NaHCO3 solution. The ethyl acetate layer was collected and dried over anhydrous Na2SO4 to remove the solvent by distillation. Separation and purification were performed by column chromatography to obtain compound M1-14: [ka] The result was obtained (yield 48.74%, purity 98.9%). 1H NMR (400 MHz, Chloroform-d) δ 3.43 (s, 2H), 3.28 (dt, J = 7.9, 5.8 Hz, 2H), 1.62 (dp, J = 13.3, 6.7 Hz, 1H), 1.45-1.39 (m, 2H), 0.93-0.89 (m, 6H). [M+H] + :155.2.

[0029] (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, and after the reaction was complete, the reaction system was cooled to room temperature. The solvent was removed by vacuum distillation, and the resulting crude product was purified by silica gel column chromatography (the eluent was a mixture of ethyl acetate and petroleum ether in a volume ratio of 1:6), and intermediate compound M2-14: [ka] The result was obtained (yield 25.26%, purity 99.1%). 1 H NMR (400 MHz, Chloroform-d) δ 7.98 (s, 1H), 4.23 (q, J = 7.1 Hz, 2H), 3.33-3.24 (m, 2H), 1.56 (dp, J = 13.3, 6.7 Hz, 1H), 1.36 (dt, J = 12.1, 7.1 Hz, 5H), 0.86 (d, J = 6.7 Hz, 6H). [M+H] + :211.1.

[0030] (3) Thiourea (1.2 mol), tert-butoxide sodium (1.2 mol), and intermediate compound M1-14 (1 mol) were mixed and dissolved with propanol. The mixture was then reacted at 80°C under nitrogen protection for 30 minutes, and the reaction 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 acetic acid solution was added to precipitate the compound, yielding compound 14 (yield 26.05%, purity 97.1%). 1H NMR (400 MHz, DMSO-d) δ 12.54 (s, 1H), 8.53 (s, 1H), 8.38 (t, J = 5.4 Hz, 1H), 8.22 (s, 1H), 8.08 (s, 1H), 3.19 (ddd, J = 8.4, 6.9, 1.59 (dp, J = 13.3, 6.7 Hz, 1H), 1.42-1.32 (m, 2H), 0.88 (d, J = 6.6 Hz, 6H). [M+H] + :241.0.

[0031] Examples 15-18 Referring to the preparation procedure of Example 14, the substrate (CH3)2-CH-CH2-CH2-NH2 in step (1) was replaced with Y-NH2 to synthesize the following intermediate compound and target compound. [Table 5] JPEG0007898705000028.jpg89170

[0032] Example 19 The tyrosinase inhibitory activity of compounds 1-18 was measured. The method is as follows: 50 μL of pH 6.8 PBS buffer, 40 μL of 200 μM compound solution (containing 10% DMSO, v / v), and 10 μL of mushroom tyrosinase solution (1000 U / mL, dissolved in pH 6.8 PBS) were sequentially added to a 96-well plate, and incubated at 37°C for 10 minutes to allow sufficient binding of the compound to the tyrosinase. Subsequently, 100 μL of L-DOPA solution (1 mM, dissolved in pH 6.8 PBS) was added to each well, and incubated for another 10 minutes to initiate the reaction. For the blank control group, 40 μL of pH 6.8 PBS buffer was used instead of the compound solution. For the positive control group, 40 μL of pH 6.8 PBS buffer with 200 μM kojic acid and 377 (phenylethyl resorcinol, purity ≥98%, analytical pure) added was used instead of the compound solution. The absorbance (OD) value of each well was measured at a wavelength of 475 nm using a microplate reader. The OD values ​​at 0 min and 20 min were recorded for each group. The formula for calculating the tyrosinase inhibition rate is as follows: Tyrosinase inhibition rate (%) = [(AB) - (CD)] / (AB) × 100 In the equation, 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 compound group at 0 min. Each experiment was repeated three times, and the effect of each compound on the enzymatic activity of tyrosinase was evaluated using the average value. The concentration of the test sample was plotted on the x-axis and the corresponding tyrosinase inhibition rate on the y-axis, a curve was created, and a regression equation was obtained by fitting. Based on the regression equation, the sample concentration at which the tyrosinase inhibition rate reached 50% was calculated, and this was used as the IC for the test sample. 50 It was set as the value.

[0033] result: Results of the inhibitory activity of the compound against tyrosinase [Table 6]

[0034] As can be seen from the results above, compounds 2, 3, 5, 6, 9, 11, 12, 13, 14, 15, 16, 17, and 18 exhibit good inhibitory activity against tyrosinase (IC). 50 The values ​​are all ≤20.8 μM. Of these, the inhibitory activity 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) was superior to that of the positive control drugs kojic acid (32.0 μM) and 377 (16.7 μM). Compounds 3, 5, 9, and 14 showed IC50. 50 All values ​​were less than 5 μM, indicating extremely high tyrosinase inhibitory activity.

[0035] Example 20 The skin permeability of the compound was tested. The method is as follows: Sugar-suckling piglet hides were collected from Panama, subcutaneous fat was removed, and the hides were repeatedly washed with physiological saline and cut to a size suitable for Franz diffusion cells. Compounds 3, 5, 9, 14, and the control group α-arbutin were each 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.

[0036] A Franz diffusion cell was used, with an exposure surface diameter of 1 cm, an effective diffusion area of ​​approximately 0.785 cm², and a volume of 15 mL. The diffusion cell was placed in a constant temperature water bath at 32°C and stirred at a speed of 600 r / min using a magnetic stirrer. 400 μL of a 10 mM compound solution was added to the sample injection cell. At 2 h, 4 h, 8 h, and 12 h, 500 μL of the receptor solution was taken from the receptor cell and immediately replenished with the same volume of sterile physiological saline containing 30% ethanol to maintain a constant volume of the receptor solution. Absorbance was measured using a UV-Vis spectrophotometer, and the compound concentration was calculated by substituting it into a standard curve.

[0037] The formula for calculating cumulative transmittance (Tn) is as follows:

number

[0038] The measurement results are as follows: [Table 7]

[0039] In comparison, the 12-hour cumulative skin permeability of α-arbutin was 10.9%, compound 3 was 19.5%, compound 5 was 12.4%, compound 9 was 20.9%, and compound 14 was 35.1%. As can be seen from the results, compound 14 has significantly higher skin permeability compared to α-arbutin at the same concentration (p<0.05).

[0040] Example 21 1. Evaluation of the toxicity of compounds to melanocytes Melanoma cells (B16F10 cells) in the logarithmic growth phase were collected and 5 × 10 5 The compounds were inoculated at a density of 1 / well into 96-well plates, and 100 μL of DMEM medium containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin mixed antibiotic was added to each well. The experiment included a control group (medium containing an equal amount of solvent as the compound group) and compound treatment groups with eight concentration gradients (2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 64 μM, 128 μM, 256 μM), with six replicated wells prepared for each group. The 96-well plates were incubated in a 37°C incubator for 24 hours, after which the supernatant was removed. Under light-shielding conditions (darkness), 100 μL of CCK-8 solution was added to each well, and the plates were incubated in a 37°C incubator for a further 30 minutes. Subsequently, the absorbance of each well was measured at a wavelength of 450 nm.

[0041] As can be seen from the experimental results shown in Figure 1, there was no significant difference in the activity of B16F10 cells between the groups treated with compounds 3, 5, 9, and 14 and the control group. This result indicates that the above compounds do not show significant toxicity to melanocytes within the experimental concentration range and possess high biosafety.

[0042] 2. Quantitative measurement of melanin content B16F10 cells in the logarithmic growth phase were collected and 5 × 10⁶ cells were taken. 5 After inoculation into 6-well plates at a density of cells / well, adaptive culture was performed in a 37°C incubator for 24 hours. Subsequently, 2 mL of different concentrations (5 μM, 10 μM, 50 μM) of the test compound solution was added to each well (equal volumes of PBS solution were added to the blank control group), and incubation was performed for another 24 hours in a 37°C incubator. After removing the supernatant, the cells were washed with PBS solution, and then the cells were lysed with a 1 M NaOH solution containing 10% DMSO. The absorbance of the resulting lysate was measured at a wavelength of 405 nm, and the melanin content was calculated based on the following formula. Melanin content (%) = (Absorbance of compound-treated group ÷ Absorbance of PBS-treated group) × 100 As can be seen from the results shown in Figure 2, compared to the blank control group, compounds 3, 5, 9, and 14 (5-50 μM) all significantly inhibited melanin production in B16F10 cells.

[0043] As can be seen from the results above, compounds 3, 5, 9, and 14 specifically inhibit the catalytic activity of tyrosinase and block melanin synthesis, and since they do not reduce melanin by killing cells, they have no significant effect on B16F10 cell activity.

[0044] The tyrosinase inhibitor provided in this invention contributes to the improvement of pigmentation by suppressing tyrosinase activity in the skin and reducing melanin synthesis, and can therefore be applied to the manufacture of cosmetics and skincare products for whitening, spot improvement, and sunscreen purposes. In the pharmaceutical field, the tyrosinase inhibitor of this invention can improve diseases related to melanin metabolism by regulating melanin synthesis or suppressing abnormal tyrosinase activity, and can be applied to the manufacture of preventive and therapeutic agents for pigmentation diseases, health foods, and food products. In the agricultural field, the tyrosinase inhibitor of this invention can target specific pests that depend on tyrosinase metabolism, inhibiting their enzyme activity and blocking their physiological functions, and is expected to be applied as an insecticide. In the food industry, the tyrosinase inhibitor of this invention can delay oxidative browning by suppressing endogenous tyrosinase activity contained in ingredients such as fruits, vegetables, and seafood, and can be used as a food freshness preservative.

[0045] The above description represents only preferred embodiments of the present invention and does not limit it. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are within the scope of protection of the present invention.

Claims

1. A tyrosinase inhibitor comprising a compound having the structure represented by the following formula I or a physiologically acceptable salt thereof, 【Chemistry 1】 The compounds having the structure represented by formula I are the following compounds: Table 1 【change】 A tyrosinase inhibitor characterized by being one of the following.

2. The following synthesis pathway: 【Transformation 3】 A method for preparing a tyrosinase inhibitor according to claim 1, comprising: A preparation method characterized by reacting compound M1 with triethoxymethane in step (1) to produce an intermediate compound M2, and reacting intermediate compound M2 with thiourea in step (2) to produce the target compound.

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

4. The preparation method according to claim 2, characterized in that in step (2) above, thiourea, compound M2, and tert-butoxide sodium 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.

5. The use of the tyrosinase inhibitor according to claim 1 in the preparation of a product containing the tyrosinase inhibitor as an active ingredient.

6. The use according to claim 5, characterized in that the product is a sunscreen, a blemish remover, or a whitening cosmetic.

7. The use according to claim 5, characterized in that the product is a pharmaceutical product for preventing or treating pigmentation disorders.

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