4-(2,4-dihydroxyphenyl)thiazole-2-carboxamide derivatives, methods of preparation and uses

Abstract

The application provides a 4-(2,4-dihydroxyphenyl)thiazole-2-carboxylic amide derivative and a preparation method and application thereof, and relates to the technical field of organic chemistry.The application provides a 4-(2,4-dihydroxyphenyl)thiazole-2-carboxylic amide derivative compound.The 4-(2,4-dihydroxyphenyl)thiazole-2-carboxylic amide derivative has the functions of inhibiting tyrosinase and melanin production, and has the characteristics of low toxicity and high safety.The preparation method can obtain the 4-(2,4-dihydroxyphenyl)thiazole-2-carboxylic amide derivative at a high yield.

Description

Technical Field

[0001] This invention belongs to the field of organic chemistry technology, specifically relating to 4-(2,4-dihydroxyphenyl)thiazole-2-carboxyamide derivatives, their preparation methods, and applications. Background Technology

[0002] Tyrosinase is the rate-limiting enzyme that inhibits melanin synthesis. Widely distributed in microorganisms, plants, animals, and humans, it is a copper-containing metalloenzyme that catalyzes the oxidation of tyrosine into dopa, dopaquinone, dopachrome, and dihydroxyindole / dihydroxyindole carboxylic acid, ultimately forming melanin. Tyrosinase inhibitors are also used in the preparation of some drugs for skin diseases related to melanin pigmentation. Tyrosinase inhibitors can also delay enzymatic browning in fruits and vegetables and are widely used in food preservation and biopesticides.

[0003] Kojic acid, hydroquinone, tranexamic acid, arbutin, and resveratrol are tyrosinase inhibitors widely used in cosmetics and dermatology. However, these drugs all have limitations in efficacy and / or safety issues. For example, hydroquinone, as a skin-whitening agent, often causes skin irritation, and long-term use can lead to extrinsic okra, allergic contact dermatitis, conjunctival melanosis, nail discoloration, and corneal degeneration.

[0004] US Patent 5254575 discloses a method for preparing 5-4-(4-hydroxyphenyl)-thiazol-2-carboxylic acid amine. Under nitrogen protection, boron tribromide is added in portions to a dichloromethane suspension containing 4-(4-methoxyphenyl)-thiazol-2-carboxamide. After stirring at room temperature, the mixture is heated to reflux, cooled, and water is added. The precipitate is filtered, and the collected solid is dried under vacuum and recrystallized in methanol to obtain the target compound. This synthetic method requires boron tribromide, a strong acid with high toxicity, difficult post-processing, significant health hazards, and requires highly precise experimental procedures.

[0005] International patent WO2012 / 117097 discloses a method for preparing N-(3-methoxyphenyl)-N-methyl-2-(3-methylphenyl)-1,3-thiazol-5-carboxamide. 2-Bromomethyl-3-methoxyphenyl-methyl-1,3-thiazol-5-carboxamide, m-tolueneboronic acid, cesium carbonate, and tetrakis(triphenylphosphine)palladium are dissolved in oxygen-free DME-ethanol-water, and then heated to 150°C for 15 minutes under microwave irradiation. The reaction mixture is cooled to room temperature and quenched with ethyl acetate. The aqueous layer is extracted with ethyl acetate. The combined organic layers are washed with brine, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue is purified on silica gel to obtain the target compound. This synthetic method involves expensive reagents, poor reagent stability, and the strong alkalinity of cesium carbonate, which can corrode materials and pose safety hazards.

[0006] In their article (Mann, T., Gerwat, W., Batzer, J. et al. Inhibition of Human Tyrosinase Requires Molecular Motifs Distinctively Different from Mushroom Tyrosinase[J]. Journal of Investigative Dermatology. 2018, 138(7):1601-1608. DOI:10.1016 / j.jid.2018.01.019), Mann et al. discovered through high-throughput screening that thiazolylresorcinol can effectively inhibit melanin production in vivo. Based on this, they conducted a structure-activity relationship (SAR) study on thiazolylresorcinol and ultimately synthesized the peptide Thiamidol, with an IC50 of 1.1 μmol / L. Subsequent studies have shown that applying a double layer of 0.2% Thiamidol twice daily or applying 4% hydroquinone cream before bedtime for 90 days significantly improves melasma. However, the Thiamidol group experienced mild adverse reactions, but two participants (8%) developed allergic contact dermatitis, indicating that Thiamidol still has some toxicity and safety concerns.

[0007] Existing tyrosinase inhibitors still exhibit certain toxicity and safety concerns, and their preparation costs are high. Therefore, there is an urgent need to provide a new substance that effectively inhibits tyrosinase and melanin, along with an efficient and safe preparation method for this substance. Summary of the Invention

[0008] This invention addresses the problems and shortcomings of existing technologies by providing a 4-(2,4-dihydroxyphenyl)thiazole-2-carboxyamide derivative, its preparation method, and its application in whitening cosmetics.

[0009] Using Thiamidol as a positive control, the inhibitory effects of the 4-(2,4-dihydroxyphenyl)thiazole-2-carboxyamide derivative on tyrosinase in B16 cells, its inhibitory effect on melanin production, cytotoxicity, and skin-whitening effect on zebrafish were tested. The results showed that the 4-(2,4-dihydroxyphenyl)thiazole-2-carboxyamide derivative prepared in this invention significantly inhibited tyrosinase and melanin production, and also exhibited greater safety in use.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] On one hand, the present invention provides a 4-(2,4-dihydroxyphenyl)thiazole-2-carboxyamide derivative as described in the following formula.

[0012] or ;

[0013] Wherein, R is selected from any one of alkyl, alkoxy, and aryl; n is 0 or 1;

[0014] R1, R2, R3, R4, and R5 are each independently selected from any one of H, alkyl, alkoxy, halogen, hydroxyl, amino, phenyl, nitro, and trifluoromethyl.

[0015] Preferably, R is selected from C 1-10 Alkyl, C 1-10 Any one of alkoxy and aryl groups;

[0016] R1 is selected from H and C. 1-10 Alkyl, C 1-10 Any one of alkoxy, halogen, hydroxyl, amino, phenyl, nitro, and trifluoromethyl;

[0017] R2 is selected from H and C. 1-10 Alkyl, C 1-10 Any one of alkoxy, halogen, hydroxyl, amino, phenyl, nitro, and trifluoromethyl;

[0018] R3 is selected from H and C. 1-10 Alkyl, C 1-10 Any one of alkoxy, halogen, hydroxyl, amino, phenyl, nitro, and trifluoromethyl;

[0019] R4 is selected from H and C. 1-10 Alkyl, C 1-10 Any one of alkoxy, halogen, hydroxyl, amino, phenyl, nitro, and trifluoromethyl;

[0020] R5 is selected from H and C. 1-10 Alkyl, C 1-10 Any one of alkoxy, halogen, hydroxyl, amino, phenyl, nitro, and trifluoromethyl.

[0021] More preferably, R is selected from C 1-8 Alkyl, C 1-8 Any one of alkoxy and aryl groups;

[0022] R1 is selected from H and C. 1-8 Alkyl, C 1-8 Any one of alkoxy, halogen, hydroxyl, amino, phenyl, nitro, and trifluoromethyl;

[0023] R2 is selected from H and C. 1-8 Alkyl, C 1-8 Any one of alkoxy, halogen, hydroxyl, amino, phenyl, nitro, and trifluoromethyl;

[0024] R3 is selected from H and C. 1-8 Alkyl, C 1-8 Any one of alkoxy, halogen, hydroxyl, amino, phenyl, nitro, and trifluoromethyl;

[0025] R4 is selected from H and C. 1-8 Alkyl, C 1-8 Any one of alkoxy, halogen, hydroxyl, amino, phenyl, nitro, and trifluoromethyl;

[0026] R5 is selected from H and C. 1-8 Alkyl, C 1-8 Any one of alkoxy, halogen, hydroxyl, amino, phenyl, nitro, and trifluoromethyl.

[0027] More preferably, R is selected from C 2-6 Alkyl, C 2-6 Any one of alkoxy and aryl groups;

[0028] R1 is selected from H and C. 2-6 Alkyl, C 2-6 Any one of alkoxy, halogen, hydroxyl, amino, phenyl, nitro, and trifluoromethyl;

[0029] R2 is selected from H and C. 2-6 Alkyl, C 2-6 Any one of alkoxy, halogen, hydroxyl, amino, phenyl, nitro, and trifluoromethyl;

[0030] R3 is selected from H and C. 2-6 Alkyl, C 2-6 Any one of alkoxy, halogen, hydroxyl, amino, phenyl, nitro, and trifluoromethyl;

[0031] R4 is selected from H and C. 2-6 Alkyl, C 2-6 Any one of alkoxy, halogen, hydroxyl, amino, phenyl, nitro, and trifluoromethyl;

[0032] R5 is selected from H and C. 2-6 Alkyl, C 2-6 Any one of alkoxy, halogen, hydroxyl, amino, phenyl, nitro, and trifluoromethyl.

[0033] More preferably, R is selected from C 3-5 Alkyl, C 3-5 Any one of cycloalkyl and aryl;

[0034] R1 is selected from H and C. 3-5 Alkyl, C 3-5 Any one of alkoxy, halogen, hydroxyl, and phenyl;

[0035] R2 is selected from H and C. 3-5 Alkyl, C 3-5 Any one of alkoxy, halogen, hydroxyl, and phenyl;

[0036] R3 is selected from H and C. 3-5 Alkyl, C 3-5 Any one of alkoxy, halogen, hydroxyl, and phenyl;

[0037] R4 is selected from H and C. 3-5 Alkyl, C 3-5 Any one of alkoxy, halogen, hydroxyl, and phenyl;

[0038] R5 is selected from H and C. 3-5 Alkyl, C 3-5 Any one of alkoxy, halogen, hydroxyl, and phenyl.

[0039] More preferably, the 4-(2,4-dihydroxyphenyl)thiazole-2-carboxyamide derivative is selected from...

[0040] , , , , , ,

[0041] , , ,

[0042] Any one of them.

[0043] Unless otherwise stated, the term "alkyl" as used herein includes branched and straight-chain saturated aliphatic hydrocarbon groups having a specific number of carbon atoms, including all isomers. Common abbreviations for alkyl groups include methyl (e.g., "Me" or CH3), ethyl (e.g., "Et" or CH2CH3), propyl (e.g., "Pr" or CH2CH2CH3), butyl (e.g., "Bu" or CH2CH2CH2CH3," etc.). For example, "C..." 1-4 Alkyl (or "C1-C4 alkyl") refers to a straight-chain or branched alkyl group having a specific number of carbon atoms, including all isomers. 1-4 Alkyl groups include n-, iso-, secondary and tert-butyl, n- and isopropyl, ethyl and methyl. The term "C" is used in this context. 1-10 "Alkyl" and similar terms have similar meanings.

[0044] The term "aryl" refers to aromatic monocyclic and polycyclic ring systems, in which the carbon rings are fused together or linked together by single bonds. Common aryl groups include phenyl, naphthyl, and biphenylene.

[0045] On the other hand, the present invention provides a method for preparing the 4-(2,4-dihydroxyphenyl)thiazole-2-carboxyamide derivative, comprising the following steps:

[0046] Step S1: Mix 2,4-dihydroxyacetophenone, copper bromide and organic reagents, heat under reflux to obtain the oily crude product 2,4-dihydroxybromoacetophenone;

[0047] Step S2: Mix ethyl thiooxazone, 2,4-dihydroxybromoacetophenone with organic reagent A and heat under reflux; dissolve the crude product in organic reagent B and filter, concentrate, then add organic reagent C for recrystallization, precipitate crystals, filter, and obtain ethyl 4-(2,4-dihydroxyphenyl)thiazol-2-carboxylate.

[0048] Step S3: Ethyl 4-(2,4-dihydroxyphenyl)thiazole-2-carboxylate is mixed with an aqueous sodium hydroxide solution; after reacting at room temperature, the pH is adjusted to acidic, and a solid is precipitated to obtain the carboxylic acid compound 4-(2,4-dihydroxyphenyl)thiazole-2-carboxylic acid;

[0049] Step S4: Dissolve 4-(2,4-dihydroxyphenyl)thiazol-2-carboxylic acid in N,N-dimethylformamide, and then add amine compounds, benzotriazol-1-yl-oxotripyrrolylphosphine hexafluorophosphate, and N,N-diisopropylethylamine in sequence; react at room temperature, and separate and purify to obtain the target compound.

[0050] Preferably, in step S1, the molar ratio of 2,4-dihydroxyacetophenone to copper bromide is 1:1.5-2.

[0051] More preferably, in step S1, the molar ratio of 2,4-dihydroxyacetophenone to copper bromide is 1:1.8.

[0052] Preferably, in step S1, the organic solvent is selected from at least one of dichloromethane, ethyl acetate, n-hexane, and petroleum ether.

[0053] More preferably, in step S1, the organic solvent is ethyl acetate.

[0054] Preferably, in step S1, the reaction time for heating and reflux is 3-8 hours.

[0055] More preferably, in step S1, the reaction time for heating and reflux is 5 hours.

[0056] Preferably, in step S1, the temperature of the heating reflux is 80-90°C; more preferably, it is 85°C.

[0057] In step S1 of this invention, as the heating and reflux reaction proceeds, the black copper bromide is reduced to white cuprous bromide. When a large amount of white precipitate is produced and the solution turns amber, the reaction is considered complete. After the reaction is complete, the mixture is filtered while hot to remove the cuprous bromide precipitate. The obtained filtrate is then evaporated under reduced pressure to remove the solvent, yielding an oily crude product, 2,4-dihydroxybromoacetophenone. Since this substance has poor stability, it can be used directly in subsequent reactions without further purification.

[0058] Preferably, in step S2, the molar ratio of the thiooxazone ethyl ester to the 2,4-dihydroxyacetophenone in step S1 is equivalent.

[0059] Preferably, in step S2, the organic solvent A is selected from at least one of methanol and ethanol.

[0060] More preferably, in step S2, the organic solvent A is ethanol.

[0061] Preferably, in step S2, the reaction time for heating and reflux is 4-8 hours.

[0062] More preferably, in step S2, the reaction time for heating and reflux is 6 hours.

[0063] Preferably, in step S2, the temperature of the heating reflux is 80-90°C; more preferably, it is 85°C.

[0064] Preferably, in step S2, the organic solvent B is selected from at least one of dichloromethane, ethyl acetate, n-hexane, and petroleum ether.

[0065] More preferably, in step S2, the organic solvent is ethyl acetate.

[0066] Preferably, in step S2, the organic solvent C is selected from at least one of dichloromethane, ethyl acetate, n-hexane, and petroleum ether.

[0067] More preferably, in step S2, the organic solvent C is petroleum ether.

[0068] Preferably, in step S3, the molar ratio of ethyl 4-(2,4-dihydroxyphenyl)thiazole-2-carboxylate to sodium hydroxide is 1:2-4.

[0069] More preferably, in step S3, the molar ratio of ethyl 4-(2,4-dihydroxyphenyl)thiazole-2-carboxylate to sodium hydroxide is 1:3.

[0070] Preferably, in step S3, the room temperature reaction time is 10-50 min.

[0071] More preferably, in step S3, the room temperature reaction time is 30 min.

[0072] Preferably, in step S3, adjusting the pH to acidic is done using a hydrochloric acid solution.

[0073] Preferably, in step S3, the concentration of the hydrochloric acid solution is 5-15 mol / L.

[0074] More preferably, in step S3, the concentration of the hydrochloric acid solution is 10 mol / L.

[0075] Preferably, in step S3, the pH is 3-5.

[0076] More preferably, in step S3, the pH is 4.

[0077] Preferably, in step S4, the molar ratio of 4-(2,4-dihydroxyphenyl)thiazol-2-carboxylic acid, amine compounds, benzotriazol-1-yl-oxotripyrrolylphosphine hexafluorophosphate and N,N-diisopropylethylamine is 1:1-1.3:1-1.3:1-1.2.

[0078] More preferably, in step S4, the molar ratio of 4-(2,4-dihydroxyphenyl)thiazol-2-carboxylic acid, amine compounds, benzotriazol-1-yl-oxotripyrrolylphosphine hexafluorophosphate and N,N-diisopropylethylamine is 1:1.1:1.1:1.

[0079] Preferably, in step S4, the amine compound is selected from at least one of n-propylamine, isopropylamine, tert-butylamine, cyclopropylamine, aniline, p-methoxyaniline, m-methoxyaniline, p-bromoaniline, benzylamine, p-hydroxyaniline, p-chloroaniline, m-chloroaniline, o-methylaniline, 2,4-dimethylaniline, 2,4-dichloroaniline, p-nitroaniline, and m-hydroxyaniline.

[0080] Preferably, in step S4, the room temperature reaction time is 3-8 hours.

[0081] More preferably, in step S4, the room temperature reaction time is 5 hours.

[0082] Preferably, in step S4, the separation and purification method is column chromatography.

[0083] Finally, this invention provides the application of 4-(2,4-dihydroxyphenyl)thiazole-2-carboxyamide derivatives in cosmetic preparation.

[0084] Preferably, the cosmetic used in the application is a whitening product.

[0085] More preferably, the cosmetics used in the application are not limited to serums, lotions, creams, body lotions, sunscreens, and sunscreen sprays.

[0086] Compared with the prior art, the present invention has the following beneficial effects:

[0087] (1) The 4-(2,4-dihydroxyphenyl)thiazole-2-carboxyamide derivatives with different structures provided by the present invention can effectively inhibit the production of tyrosinase and melanin, and have significant whitening effects; and each compound has low toxicity and high safety in use.

[0088] (2) The significant efficacy of the 4-(2,4-dihydroxyphenyl)thiazole-2-carboxyamide derivative of this invention in skin care and whitening was verified by its inhibitory effect on B16 cell tyrosinase, its inhibitory effect on melanin production, its cytotoxicity, and its whitening effect on zebrafish. This provides a basis for the development and application of whitening products based on the 4-(2,4-dihydroxyphenyl)thiazole-2-carboxyamide derivative.

[0089] (3) The present invention successfully prepared 4-(2,4-dihydroxyphenyl)thiazol-2-carboxyamide derivatives with different structures by reacting 4-(2,4-dihydroxyphenyl)thiazol-2-carboxylic acid, amine compounds, benzotriazol-1-yl-oxotripyrrolylphosphine hexafluorophosphate and N,N-diisopropylethylamine under specific feed ratio conditions. The products have high yields. Attached Figure Description

[0090] Figure 1 The hydrogen spectrum of ethyl 4-(2,4-dihydroxyphenyl)thiazole-2-carboxylate prepared in Example 2 of this invention.

[0091] Figure 2 The carbon spectrum of ethyl 4-(2,4-dihydroxyphenyl)thiazole-2-carboxylate prepared in Example 2 of this invention.

[0092] Figure 3 The photoluminescence spectrum of 4-(2,4-dihydroxyphenyl)thiazole-2-carboxylic acid prepared in Example 3 of this invention is shown.

[0093] Figure 4 The carbon spectrum of 4-(2,4-dihydroxyphenyl)thiazole-2-carboxylic acid prepared in Example 3 of this invention.

[0094] Figure 5 The hydrogen spectrum of compound M1 prepared in Example 4 of this invention.

[0095] Figure 6 This is the carbon spectrum of compound M1 prepared in Example 4 of the present invention.

[0096] Figure 7The hydrogen spectrum of compound M2 prepared in Example 5 of this invention.

[0097] Figure 8 The carbon spectrum of compound M2 prepared in Example 5 of this invention.

[0098] Figure 9 The hydrogen spectrum of compound M3 prepared in Example 6 of this invention.

[0099] Figure 10 The carbon spectrum of compound M3 prepared in Example 6 of this invention.

[0100] Figure 11 The hydrogen spectrum of compound M4 prepared in Example 7 of this invention.

[0101] Figure 12 The carbon spectrum of compound M4 prepared in Example 7 of this invention.

[0102] Figure 13 The hydrogen spectrum of compound M5 prepared in Example 8 of this invention.

[0103] Figure 14 The carbon spectrum of compound M5 prepared in Example 8 of this invention.

[0104] Figure 15 The hydrogen spectrum of compound M6 prepared in Example 9 of this invention.

[0105] Figure 16 The carbon spectrum of compound M6 prepared in Example 9 of this invention.

[0106] Figure 17 The hydrogen spectrum of compound M7 prepared in Example 10 of this invention.

[0107] Figure 18 The carbon spectrum of compound M7 prepared in Example 10 of this invention.

[0108] Figure 19 The hydrogen spectrum of compound M8 prepared in Example 11 of this invention.

[0109] Figure 20 The carbon spectrum of compound M8 prepared in Example 11 of this invention.

[0110] Figure 21 The hydrogen spectrum of compound M9 prepared in Example 12 of this invention.

[0111] Figure 22 The carbon spectrum of compound M9 prepared in Example 12 of this invention.

[0112] Figure 23 The hydrogen spectrum of compound M10 prepared in Example 13 of this invention.

[0113] Figure 24 The carbon spectrum of compound M10 prepared in Example 13 of this invention.

[0114] Figure 25 The IC50 inhibitory concentrations of the compounds of this invention on enzymes in B16 cell lysates are shown to be [missing information]. 50 Effect impact diagram.

[0115] Figure 26 This is a graph showing the effect of the various compounds of the present invention on the melanin content of B16 cells.

[0116] Figure 27 This is a graph showing the effect of the various compounds of this invention on the cytotoxicity of B16 cells.

[0117] Figure 28 This is a graph showing the effect of the various compounds of this invention on Hacat cell toxicity.

[0118] Figure 29 These are zebrafish photographs taken during the zebrafish melanin inhibition experiment of the compounds of this invention.

[0119] Figure 30 The figure shows the experimental results of the effects of the various compounds of this invention on the melanin content of zebrafish. Detailed Implementation

[0120] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0121] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0122] The present invention will be described below with reference to specific embodiments, but the scope of the present invention should not be limited to the following embodiments. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0123] In embodiments of the present invention, "rt" represents room temperature.

[0124] Example 1

[0125]

[0126] 10 g (65.8 mmol) of 2,4-dihydroxyacetophenone and 25 g of copper bromide (CuBr2, 112.4 mmol) were added to a round-bottom flask containing ethyl acetate (100 mL). The mixture was then heated under reflux for 5 hours. As the reaction proceeded, the black copper bromide was reduced to white cuprous bromide. The reaction was considered complete when a large amount of white precipitate was observed and the solution turned amber. After the reaction was complete, the mixture was filtered while hot to remove the cuprous bromide precipitate. The resulting filtrate was then evaporated under reduced pressure to remove the solvent, yielding 7.5 g (70%-80%) of the oily crude product 2,4-dihydroxybromoacetophenone. Due to its poor stability, this substance can be used directly in subsequent reactions without further purification.

[0127] Example 2

[0128]

[0129] 12 g (52.4 mmol) of thiooxazone ethyl ester and 7 g of 2,4-dihydroxybromoacetophenone were added to 100 mL of ethanol. The mixture was heated under reflux for 6 hours, and the reaction was monitored by TLC. After the reaction was complete, the solvent was removed by rotary evaporation to obtain the crude product. The crude product was then dissolved in ethyl acetate and filtered. After concentration, petroleum ether was added for recrystallization, and white crystals precipitated. After filtration and waiting for the solvent to evaporate, approximately 9.6 g of 4-(2,4-dihydroxyphenyl)thiazolium-2-carboxylate was obtained, with a yield of 70%. 1 H NMR (400 MHz, DMSO-d6) δ [ppm] = 10.42 (s, 1H), 9.64 (s,1H), 8.29 (s, 1H), 7.88 (d, J = 8.6 Hz, 1H), 6.45 (d, J = 2.3 Hz, 1H), 6.38(dd, J = 8.6, 2.4 Hz, 1H), 4.41 (q, J = 7.1 Hz, 2H), 1.36 (t, J = 7.1 Hz, 3H); 13 C10 NMR (101 MHz, DMSO-d6) δ [ppm] = 159.9, 159.3, 156.8, 155.8, 154.4, 130.2, 120.6, 111.7, 107.7, 103.3, 62.5, 14.5. The 1H NMR spectrum of ethyl 4-(2,4-dihydroxyphenyl)thiazole-2-carboxylate is shown below. Figure 1 The carbon spectrum can be found here. Figure 2 .

[0130] Example 3

[0131]

[0132] 9.6 g (36.4 mmol) of ethyl 4-(2,4-dihydroxyphenyl)thiazole-2-carboxylic acid was added to a reaction flask, and 4.4 g (110 mmol) of sodium hydroxide was dissolved in 100 mL of water. The prepared sodium hydroxide aqueous solution was slowly added under stirring. After stirring at room temperature for 30 minutes, 25 mL of 10 M hydrochloric acid solution was slowly added dropwise to acidify to pH 4. The precipitated solid was filtered under vacuum and dried to obtain approximately 9 g of yellow solid 4-(2,4-dihydroxyphenyl)thiazole-2-carboxylic acid (yield 95%). 1 H NMR (400MHz, DMSO- d6 ) δ [ppm] = 10.83 (s, 1H), 9.57 (s, 1H), 9.20 (s, 1H), 7.98 (d, J= 2.0 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 6.39 (t, J = 1.9 Hz, 1H), 6.37 –6.32 (m, 1H); 13 C NMR (101 MHz, DMSO- d6 δ [ppm] = 159.0, 156.8, 153.5, 153.5, 153.4, 129.5, 112.7, 111.6, 107.7, 103.4. The 1H NMR spectrum of 4-(2,4-dihydroxyphenyl)thiazol-2-carboxylic acid is shown below. Figure 3 The carbon spectrum can be found here. Figure 4 .

[0133] Example 4

[0134]

[0135] 250 mg (1.0 mmol) of 4-(2,4-dihydroxyphenyl)thiazol-2-carboxylic acid was dissolved in 4 mL of N,N-dimethylformamide (DMF). Then, 1.1 mmol of n-propylamine, 400 mg (1.1 mmol) of benzotriazol-1-yl-oxotripyrrolidinephosphine hexafluorophosphate, and 110 μL (1 mmol) of N,N-diisopropylethylamine were added sequentially. The reaction was carried out at room temperature for 5 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was purified by column chromatography to obtain a white solid M1, with a yield of 45%. 1 H NMR (400 MHz, DMSO- d6) δ[ppm] = 10.21 (s, 1H), 9.59 (s, 1H), 8.92 (t, J = 6.2 Hz, 1H), 8.13 (s, 1H), 8.01 (d, J = 8.5 Hz, 1H), 6.44 (d, J = 2.4 Hz, 1H), 6.36 (dd, J = 8.6, 2.3Hz, 1H), 3.26 (q, J = 6.8 Hz, 2H), 1.62 – 1.51 (m, 2H), 0.89 (t, J = 7.4 Hz, 3H); 13 C NMR (101 MHz, DMSO- d6 δ [ppm] = 162.4, 159.5, 159.1, 156.7, 153.4, 130.6, 118.8, 112.0, 107.5, 103.4, 41.3, 22.9, 11.9. The proton NMR spectrum of compound M1 is shown below. Figure 5 The carbon spectrum can be found here. Figure 6 .

[0136] Example 5

[0137]

[0138] 250 mg (1.0 mmol) of 4-(2,4-dihydroxyphenyl)thiazol-2-carboxylic acid was dissolved in 4 mL of N,N-dimethylformamide (DMF). Then, 1.1 mmol of isopropylamine, 400 mg (1.1 mmol) of benzotriazol-1-yl-oxotripyrrolylphosphine hexafluorophosphate, and 110 μL (1 mmol) of N,N-diisopropylethylamine were added sequentially. The reaction was carried out at room temperature for 5 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was purified by column chromatography to obtain a white solid M2, with a yield of 48%. 1 H NMR (400 MHz, DMSO- d6 ) δ[ppm] = 10.21 (s, 1H), 9.58 (s, 1H), 8.65 (d, J = 8.4 Hz, 1H), 8.14 (s, 1H), 8.04 (d, J = 8.5 Hz, 1H), 6.48 – 6.42 (m, 1H), 6.36 (dd, J = 8.5, 2.3 Hz,1H), 4.22 – 4.07 (m, 1H), 1.23 (d, J = 6.6 Hz, 6H); 13 C NMR (101 MHz, DMSO- d6δ [ppm] = 162.5, 159.0, 158.7, 156.6, 153.4, 130.7, 118.8, 112.1, 107.5, 103.3, 41.6, 22.6. The proton NMR spectrum of compound M2 is shown below. Figure 7 The carbon spectrum can be found here. Figure 8 .

[0139] Example 6

[0140]

[0141] 250 mg (1.0 mmol) of 4-(2,4-dihydroxyphenyl)thiazol-2-carboxylic acid was dissolved in 4 mL of N,N-dimethylformamide (DMF). Then, 1.1 mmol of tert-butylamine, 400 mg (1.1 mmol) of benzotriazol-1-yl-oxotripyrrolylphosphine hexafluorophosphate, and 110 μL (1 mmol) of N,N-diisopropylethylamine were added sequentially. The reaction was carried out at room temperature for 5 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was purified by column chromatography to obtain a white solid M3, with a yield of 61%. 1 H NMR (400 MHz, DMSO- d 6) δ[ppm] = 10.16 (s, 1H), 9.55 (s, 1H), 8.11 (s, 1H), 7.99 (d, J = 8.6 Hz, 1H), 7.92 (s, 1H), 6.42 (d, J = 2.3 Hz, 1H), 6.35 (dd, J = 8.6, 2.4 Hz, 1H), 1.43(s, 9H); 13 C NMR (101 MHz, DMSO- d 6) δ [ppm] = 163.1, 159.0, 159.0, 156.6, 153.1, 130.7, 119.1, 112.2, 107.5, 103.3, 51.7, 28.9, 28.9, 28.9. The proton NMR spectrum of compound M3 is shown below. Figure 9 The carbon spectrum can be found here. Figure 10 .

[0142] Example 7

[0143]

[0144] 250 mg (1.0 mmol) of 4-(2,4-dihydroxyphenyl)thiazol-2-carboxylic acid was dissolved in 4 mL of N,N-dimethylformamide (DMF). Then, 1.1 mmol of cyclopropylamine, 400 mg (1.1 mmol) of benzotriazol-1-yl-oxotripyrrolylphosphine hexafluorophosphate, and 110 μL (1 mmol) of N,N-diisopropylethylamine were added sequentially. The reaction was carried out at room temperature for 5 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was purified by column chromatography to obtain a white solid M4, with a yield of 45%. 1 H NMR (400 MHz, DMSO- d6 ) δ[ppm] = 10.21 (s, 1H), 9.60 (s, 1H), 8.92 (dd, J = 7.5, 4.4 Hz, 1H), 8.16 (d,J = 2.2 Hz, 1H), 8.04 (d, J = 8.6 Hz, 1H), 6.54 – 6.44 (m, H), 6.43 – 6.35 (m, H), 2.86 – 2.98 (m, 1H), 0.85 – 0.70 (m, 4H); 13 C NMR (101 MHz, DMSO- d6 δ[ppm] = 162.1, 162.1, 160.9, 159.1, 159.1, 156.6, 153.5, 130.7, 118.8, 112.0, 112.0, 107.5, 107.5, 103.4, 103.3, 23.4, 6.2, 6.2. The proton NMR spectrum of compound M4 is shown below. Figure 11 The carbon spectrum can be found here. Figure 12 .

[0145] Example 8

[0146]

[0147] 250 mg (1.0 mmol) of 4-(2,4-dihydroxyphenyl)thiazol-2-carboxylic acid was dissolved in 4 mL of N,N-dimethylformamide (DMF). Then, 1.1 mmol of aniline, 400 mg (1.1 mmol) of benzotriazol-1-yl-oxotripyrrolylphosphine hexafluorophosphate, and 110 μL (1 mmol) of N,N-diisopropylethylamine were added sequentially. The reaction was carried out at room temperature for 5 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was purified by column chromatography to obtain a white solid M5 in 65% yield. 1 H NMR (400 MHz, DMSO- d6) δ [ppm]= 10.61 (s, 1H), 10.22 (s, 1H), 9.60 (s, 1H), 8.24 (s, 1H), 8.16 (d, J = 8.6Hz, 1H), 7.93 – 7.82 (m, 2H), 7.45 – 7.36 (m, 2H), 7.24 – 7.13 (m, 1H), 6.45(d, J = 2.4 Hz, 1H), 6.38 (dd, J = 8.6, 2.4 Hz, 1H); 13 C NMR (101 MHz, DMSO- d 6) δ [ppm] = 161.9, 159.1, 158.3, 156.7, 153.6, 138.4, 131.0, 129.2, 124.8, 121.4, 120.0, 112.1, 107.5, 103.3. The proton NMR spectrum of compound M5 is shown below. Figure 13 The carbon spectrum can be found here. Figure 14 .

[0148] Example 9

[0149]

[0150] 250 mg (1.0 mmol) of 4-(2,4-dihydroxyphenyl)thiazol-2-carboxylic acid was dissolved in 4 mL of N,N-dimethylformamide (DMF). Then, 1.1 mmol of p-methoxyaniline, 400 mg (1.1 mmol) of benzotriazol-1-yl-oxotripyrrolylphosphine hexafluorophosphate, and 110 μL (1 mmol) of N,N-diisopropylethylamine were added sequentially. The reaction was carried out at room temperature for 5 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was purified by column chromatography to obtain a white solid, M6, in 60% yield. 1 H NMR (400 MHz, DMSO- d 6)δ [ppm] = 10.53 (s, 1H), 10.24 (s, 1H), 9.62 (s, 1H), 8.22 (s, 1H), 8.15 (d,J = 8.6 Hz, 1H), 7.80 – 7.72 (m, 2H), 7.03 – 6.93 (m, 2H), 6.46 (d, J = 2.0Hz, 1H), 6.39 (dd, J = 8.6, 2.3 Hz, 1H), 3.77 (s, 3H); 13 C NMR (101 MHz, DMSO- d6) δ [ppm] = 162.2, 159.1, 158.0, 156.7, 156.5, 153.6, 131.3, 131.0, 123.0, 123.0, 119.7, 114.3, 114.3, 112.1, 107.5, 103.3, 55.7. The proton NMR spectrum of compound M6 is shown below. Figure 15 The carbon spectrum can be found here. Figure 16 .

[0151] Example 10

[0152]

[0153] 250 mg (1.0 mmol) of 4-(2,4-dihydroxyphenyl)thiazol-2-carboxylic acid was dissolved in 4 mL of N,N-dimethylformamide (DMF). Then, 1.1 mmol of m-methoxyaniline, 400 mg (1.1 mmol) of benzotriazol-1-yl-oxotripyrrolylphosphine hexafluorophosphate, and 110 μL (1 mmol) of N,N-diisopropylethylamine were added sequentially. The reaction was carried out at room temperature for 5 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was purified by column chromatography to obtain a white solid M7, with a yield of 62%. 1 H NMR (400 MHz, DMSO- d 6)δ [ppm] = 10.57 (s, 1H), 10.25 (s, 1H), 9.63 (s, 1H), 8.25 (s, 1H), 8.18 (d,J = 8.6 Hz, 1H), 7.55 (t, J = 2.3 Hz, 1H), 7.53 – 7.47 (m, 1H), 7.30 (t, J =8.1 Hz, 1H), 6.77 – 6.73 (m, 1H), 6.48 (d, J = 2.4 Hz, 1H), 6.41 (dd, J =8.6, 2.4 Hz, 1H), 3.78 (s, 3H); 13 C NMR (101 MHz, DMSO- d 6) δ [ppm] = 161.9, 159.9, 159.2, 158.3, 156.7, 153.6, 139.5, 131.0, 130.0, 120.1, 113.5, 112.1, 110.5, 107.5, 106.9, 103.3, 55.5. The proton NMR spectrum of compound M7 is shown below. Figure 17 The carbon spectrum can be found here. Figure 18 .

[0154] Example 11

[0155]

[0156] 250 mg (1.0 mmol) of 4-(2,4-dihydroxyphenyl)thiazol-2-carboxylic acid was dissolved in 4 mL of N,N-dimethylformamide (DMF). Then, 1.1 mmol of p-bromoaniline, 400 mg (1.1 mmol) of benzotriazol-1-yl-oxotripyrrolylphosphine hexafluorophosphate, and 110 μL (1 mmol) of N,N-diisopropylethylamine were added sequentially. The reaction was carried out at room temperature for 5 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was purified by column chromatography to obtain a white solid M8 in 66% yield. 1 H NMR (400 MHz, DMSO- d 6) δ[ppm] = 10.8 (s, 1H), 10.3 (s, 1H), 9.6 (s, 1H), 8.3 (s, 1H), 8.2 (d, J = 8.5Hz, 1H), 8.0 – 7.9 (m, 2H), 7.7 – 7.6 (m, 2H), 6.5 (d, J = 2.3 Hz, 1H), 6.4 (dd, J = 8.5, 2.3 Hz, 1H); 13 C NMR (101 MHz, DMSO- d 6) δ [ppm] = 161.6, 159.2, 158.4, 156.7, 153.7, 137.8, 132.0, 132.0, 131.0, 123.3, 123.3, 120.2, 116.7, 112.1, 107.5, 103.3. The proton NMR spectrum of compound M8 is shown below. Figure 19 The carbon spectrum can be found here. Figure 20 .

[0157] Example 12

[0158]

[0159] 250 mg (1.0 mmol) of 4-(2,4-dihydroxyphenyl)thiazol-2-carboxylic acid was dissolved in 4 mL of N,N-dimethylformamide (DMF). Then, 1.1 mmol of benzylamine, 400 mg (1.1 mmol) of benzotriazol-1-yl-oxotripyrrolylphosphine hexafluorophosphate, and 110 μL (1 mmol) of N,N-diisopropylethylamine were added sequentially. The reaction was carried out at room temperature for 5 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was purified by column chromatography to obtain a white solid, M9, in 55% yield. 1H NMR (400 MHz, DMSO- d 6) δ [ppm]= 10.21 (s, 1H), 9.59 (s, 1H), 9.50 (t, J = 5.7 Hz, 1H), 8.16 (s, 1H), 8.01(d, J = 8.6 Hz, 1H), 7.34 (q, J = 8.0, 7.4 Hz, 4H), 7.25 (dd, J = 7.9, 4.8Hz, 1H), 6.48 – 6.39 (m, 1H), 6.39 – 6.27 (m, 1H), 4.51 (d, J = 6.1 Hz, 2H); 13 C NMR (101 MHz, DMSO- d 6) δ [ppm] = 162.0, 159.7, 159.1, 156.7, 153.5, 139.6, 130.6, 128.8, 127.9, 127.8, 127.4, 119.0, 112.0, 107.5, 103.4, 43.0, 40.6, 40.3, 40.1, 39.9, 39.7, 39.5, 39.3. The proton NMR spectrum of compound M9 is shown below. Figure 21 The carbon spectrum can be found here. Figure 22 .

[0160] Example 13

[0161]

[0162] 250 mg (1.0 mmol) of 4-(2,4-dihydroxyphenyl)thiazol-2-carboxylic acid was dissolved in 4 mL of N,N-dimethylformamide (DMF). Then, 1.1 mmol of p-hydroxyaniline, 400 mg (1.1 mmol) of benzotriazol-1-yl-oxotripyrrolylphosphine hexafluorophosphate, and 110 μL (1 mmol) of N,N-diisopropylethylamine were added sequentially. The reaction was carried out at room temperature for 5 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was purified by column chromatography to obtain a white solid, M10, in 29% yield. 1 H NMR (400 MHz, DMSO- d6)δ [ppm] = 10.44 (s, 1H), 10.22 (s, 1H), 9.60 (s, 1H), 9.40 (s, 1H), 8.21 (s,1H), 8.14 (d, J = 8.5 Hz, 1H), 7.67 – 7.60 (m, 2H), 6.84 – 6.77 (m, 2H), 6.49 – 6.44 (m, 1H), 6.42 – 6.37 (m, 1H); 13 C NMR (101 MHz, DMSO- d 6) δ [ppm] = 162.4, 159.1, 157.8, 156.7, 154.7, 153.6, 130.9, 129.8, 123.2, 119.5, 115.6, 112.1, 107.5, 103.4. The proton NMR spectrum of compound M10 is shown below. Figure 23 The carbon spectrum can be found here. Figure 24 .

[0163] Comparative Example 1

[0164] According to the preparation method of supplementary material W630: N-4-(2,4-dihydroxyphenyl)thiazolyl-2-yl)isobutyramide in the literature "Structure-activity relationships of thiazolyl resorcinols, potent and selective inhibitors of human tyrosinase":

[0165] 89 g (260 mmol) of 2-bromo-2',4'-dimethoxycarbonyloxyacetophenone was refluxed and boiled in 1000 mL of ethanol with 37.5 g (260 mmol) of N-isobutylthiourea and 32 g (380 mmol) of sodium bicarbonate for 0.5 h. After cooling, 41 g (0.93 mol) of sodium hydroxide was added to 250 mL of water and mixed. The mixture was stirred at room temperature for 30 min, then 300 mL of water was added, and the pH was adjusted to 3 with 2 mol of hydrochloric acid. The precipitate was filtered and recrystallized from ethanol to give 56 g of the target product, with a yield of 37.6%.

[0166] The structural formula of the target product compound in Comparative Example 1 is as follows:

[0167]

[0168] Comparative Example 2

[0169] The difference from Example 5 is that the reaction temperature is different; the reaction is carried out at 60°C. All other aspects are the same as in Example 5.

[0170] 250 mg (1.0 mmol) of 4-(2,4-dihydroxyphenyl)thiazol-2-carboxylic acid was dissolved in 4 mL of N,N-dimethylformamide (DMF). Then, 1.1 mmol of n-propylamine, 400 mg (1.1 mmol) of benzotriazol-1-yl-oxotripyrrolylphosphine hexafluorophosphate, and 110 μL (1 mmol) of N,N-diisopropylethylamine were added sequentially. The reaction was carried out at 60 °C for 5 h. The reaction was monitored by TLC. After the reaction was complete, compound M2 was obtained by column chromatography with a yield of 20%.

[0171] Comparative Example 3

[0172] The difference from Example 5 is that the molar ratio of N,N-diisopropylethylamine is different, being 2 mmol; otherwise, it is the same as in Example 5.

[0173] 250 mg (1.0 mmol) of 4-(2,4-dihydroxyphenyl)thiazol-2-carboxylic acid was dissolved in 4 mL of N,N-dimethylformamide (DMF). Then, 1.1 mmol of n-propylamine, 400 mg (1.1 mmol) of benzotriazol-1-yl-oxotripyrrolylphosphine hexafluorophosphate, and 220 μL (2 mmol) of N,N-diisopropylethylamine were added sequentially. The reaction was carried out at room temperature for 5 h. The reaction was monitored by TLC. After the reaction was complete, M2 was obtained by column chromatography with a yield of 31%.

[0174] Effect testing experiment

[0175] Experiment 1: Cytotoxicity Experiment

[0176] B16 cell culture: Taking a T25 culture flask as an example, the cell suspension, thawed at 37°C, was seeded into a culture flask containing 3 mL of culture medium and placed in a 37°C, 5% CO2 incubator. Cell status was observed periodically. When cells reached 80-90% confluence in the culture flask, they were digested with 1 mL of trypsin-EDTA (0.25%) into centrifuge tubes for subsequent experiments and passage.

[0177] Determination of B16 cell viability: First, the B16 cell density was adjusted to 1×10⁻⁶. 5Cells were seeded at 100 μL / well in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. After cell monolayer adhesion, high-concentration (25 μmol / L) and low-concentration (2.5 μmol / L) sample solutions of compound M1-M10 were prepared and added at 100 μL / well to 96-well plates as sample groups. The control group consisted of cells cultured in blank 1640 medium without sample, and the blank group consisted of cells cultured in blank 1640 medium only. 100 μL of MTT (0.5 mg / mL) solution was added to each well, and after incubation for 4 h, 100 μL of DMSO was added and the plates were shaken at 37°C for 10 min to allow crystals to dissolve. The absorbance was measured at 492 nm using a microplate reader, and cell viability was calculated using the following formula. Calculations were performed using GraphPad Prism 8. All data are expressed as mean ± SEM of three independent experiments. The results are shown in Table 1.

[0178] ;

[0179] The absorbances of the sample group, control group, and blank group were A1, A2, and A0, respectively.

[0180] Table 1. Effects of compounds on B16 cell survival.

[0181]

[0182] According to Table 1 above, except for M8, all compounds have low toxicity below 25 μM and will not affect subsequent experiments.

[0183] Experiment 2: Anti-tyrosinase activity experiment

[0184] Determination of tyrosinase activity in B16 cells: The density of B16 cells in good growth condition was adjusted to 1×10⁶. 5 Cells were cultured at 100 μL / well in 96-well plates at 37°C and 5% CO2 for 24 h. After cell monolayer attachment, sample solutions of various concentrations were prepared and added at 100 μL / well in 96-well plates. The control group consisted of cells cultured in blank DMEM medium at 37°C and 5% CO2 for 48 h. The cell supernatant was discarded, and the cells were washed once with PBS. 50 μL of 1% Triton-X100 aqueous solution was added to each well, and the plates were immediately frozen at -80°C for 30 min. After freezing, the cells were thawed in a water bath at 37°C to completely rupture them. 100 μL / well of 1 mg / mL L-DOPA solution (0.01 g L-DOPA powder dissolved in 10 mL PBS) was added. A blank control group was set up, consisting of wells without B16 cells. The reaction was carried out at 37°C for 2 h. The absorbance at 475 nm was measured using a microplate reader. The results are shown in Table 2.

[0185] Table 2. Antityrosinase activity of the compounds at 25 μM and 2.5 μM.

[0186]

[0187] Note: Nd indicates that the compound is highly toxic and was not measured.

[0188] According to Table 2 above, compounds M2 and M4 showed the best inhibitory effects, with inhibition rates of (85.76±1.54)% and (84.34±1.91)% at a concentration of 25 μmol / L, and (59.48±0.63)% and (62.12±2.01)% at a concentration of 2.5 μmol / L, demonstrating their inhibitory effect on tyrosinase.

[0189] Furthermore, the inhibitory effects of compounds M2 and M4, which showed better efficacy in this batch of compounds, on enzyme inhibition IC50 in B16 cell lysates were examined. 50 Tyrosinase was extracted from B16 cells, and the half-maximal inhibitory concentration (WMC) of the compound against the enzyme was determined in vitro. The results are as follows: Figure 25 As shown, Thiamidol's IC50... 50 The value was 1.72 ± 0.23 μM. The tyrosinase inhibition rates of M2 and M4 were not significantly different. The IC50 value of M2 was 1.72 ± 0.23 μM. 50 The value is 1.39±0.09μM, IC of M4 50 The value was 1.51 ± 0.05 μM. There was no significant difference between M2 and M4.

[0190] Experiment 3: Melanin Inhibition Experiment

[0191] Determination of melanin content in B16 cells: The density of B16 cells in good growth condition was adjusted to 1.2 × 10⁻⁶. 5Cells were cultured at a concentration of 1 / mL, with 0.5 mL added to each well of a 24-well plate, and incubated at 37°C with 5% CO2 for 24 hours. After cell adhesion, sample solutions of various concentrations were prepared using DMEM medium and added to each well of the 24-well plate at 0.5 mL. A DMEM group was set up as a control group, and cells were cultured in blank DMEM medium throughout the entire process to promote the increase of melanin content in B16 cells. In addition, the blank group (Con) was cultured in blank 1640 medium throughout the entire process and incubated at 37°C with 5% CO2 for 48 hours. Discard the cell supernatant, wash once with PBS, then separate from the plate with trypsin / EDTA solution, centrifuge at 3000 rpm for 10 min, add 100 μL of 10% DMSO in 1 mol / L NaOH solution, place in an 80℃ water bath for 30 min to completely dissolve the cell clumps, shake appropriately, and then aspirate the solution from each well into a 96-well plate. Measure the absorbance at 405 nm using a microplate reader and calculate the melanin content.

[0192] like Figure 26 M2 and M4 showed strong inhibitory effects on melanin at concentrations of 1 μmol / L and 2 μmol / L, respectively.

[0193] Experiment 4: Evaluation of the toxicity and safety of the compounds

[0194] The toxicity and safety of the compound will be evaluated by assessing its effects on the growth status of B16 and Hacat cells. The culture of Hcacat cells is similar to that of B16 cells, except that Hacat cells grow slightly slower and require DMEM medium.

[0195] B16 cell viability was determined using the method described above, with sample concentrations of 2.5, 25, 50, 75, and 100 μmol / L selected. The effect of the most active compound on B16 cell viability was investigated at these five concentrations. The control group (no compound was added) was cultured in 100 μmol / L DMSO and blank 1640 medium.

[0196] The determination of Hacat cell viability is similar to that of B16. First, the Hacat cell density is adjusted to 8 × 10⁶. 4 The concentration of the compound was 100 μL / well, and all other conditions were kept constant. The effects of the most active compound on the survival rate of Hacat cells at concentrations of 25, 50, 75, and 100 μmol / L were measured. Hacat cells in the control group were cultured in DMEM medium.

[0197] Firstly, it has cytotoxic effects on B16 cells, such as... Figure 27It is evident that no significant toxicity was observed at concentrations <50 μmol / L; however, at concentrations ≥50 μmol / L, the toxicity of compounds M2 and Thiamidol increased sharply, showing a significant difference compared to the control group (P < 0.0001), and compound M4 also showed a significant difference (P < 0.001), indicating that the toxicity of M4 in this concentration range was not as high as that of M2 and Thiamidol. Figure 27 In the figures, each column represents the B16 cell survival rate results from left to right at control, 2.5 μmol / L, 25 μmol / L, 50 μmol / L, 75 μmol / L, and 100 μmol / L concentrations, respectively.

[0198] The toxicity of compounds Thiamidol, M2, and M4 to Hacat cells, such as Figure 28 As shown, no significant toxicity was observed at a concentration of 25 μmol / L. When the concentration was ≥50 μmol / L, all three groups showed a sudden decrease in cell viability. Thiamidol showed a significant decrease in cell viability, with a slight decrease in the 50 μmol / L group compared to the control group (P < 0.05), and a decrease in the 75 and 100 μmol / L groups compared to the control group (P < 0.0001). However, the decrease in M2 and M4 was not significant, and no significant toxicity was observed at 50 μmol / L. Figure 28 In the figures, each column represents the Hacat cell viability results from left to right at control, 25 μmol / L, 50 μmol / L, 75 μmol / L, and 100 μmol / L concentrations, respectively.

[0199] Experiment 5: Zebrafish Melanin Inhibition Experiment

[0200] First, the toxicity of the compound to zebrafish embryos needed to be determined. Zebrafish embryos at 24 hours post-fertilization were selected and partitioned into 24-well plates at a ratio of 10 embryos / well. The compound was then dissolved in DMSO and diluted with embryo culture medium to the set concentrations for all treatments. The plates were then stored at a growth temperature of 28.5°C for 48 hours. A blank control group (zebrafish embryo culture medium, Control) was also included. At the experimental endpoint, the tested concentrations were ensured to have no significant toxicity (survival rate >90%).

[0201] Healthy zebrafish embryos developed to 24 hpf were placed in 24-well plates at a ratio of 10 embryos / well. 2 mL of the test compound solution was added to each well, with three replicates per group, and the plates were cultured for another 48 hours. At the end of the experiment, zebrafish with normal phenotype and behavior were selected from each experimental group, fixed onto slides with 1% methylcellulose, and their number and morphology of melanocytes in the somatic (trunk) regions were observed under a stereomicroscope, and photographs were taken. The analysis process for melanin on the zebrafish skin surface is as follows. We first uploaded the files to ImageJ, and after measurement, we saved and processed the data using GraphPad Prism 8.0.

[0202] like Figure 29 As shown, Thiamidol and compound M2 exhibited significant melanin-inhibiting effects at a concentration of 80 μmol / L. With increasing concentration, at 125 μmol / L, their melanin-inhibiting effects were further enhanced, and the inhibitory effect was far superior to that of arbutin at 150 μmol / L. No melanin was visible to the naked eye in the zebrafish.

[0203] Analyze the optical density (OD) value to obtain Figure 30 The inhibitory effects of Thiamidol and M2 were more clearly and specifically demonstrated. The relative optical density of the positive control group (150 μmol / L arbutin) was (64.57 ± 3.18)%, which was significantly different from that of the blank control group (P < 0.0001), indicating that the arbutin concentration was appropriate. At a concentration of 80 μmol / L, the optical density values ​​of Thiamidol and compound M2 were almost identical to those of the blank control group (P < 0.0001) and 150 μmol / L arbutin. At a concentration of 125 μmol / L, the relative optical density values ​​of Thiamidol and compound M2 were (29.27 ± 5.79)% and (29.23 ± 5.54)%, respectively.

[0204] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A 4-(2,4-dihydroxyphenyl)thiazole-2-carboxyamide derivative, characterized in that, 4-(2,4-dihydroxyphenyl)thiazole-2-carboxyamide derivatives are selected from 、 、 、 、 、 、 、 、 Any one of them.

2. The method for preparing the 4-(2,4-dihydroxyphenyl)thiazole-2-carboxyamide derivative according to claim 1, characterized in that, Includes the following steps: Step S1: Mix 2,4-dihydroxyacetophenone, copper bromide and organic reagents, heat under reflux to obtain the oily crude product 2,4-dihydroxybromoacetophenone; Step S2: Mix ethyl thiooxazone, 2,4-dihydroxybromoacetophenone with organic reagent A and heat under reflux; dissolve the crude product in organic reagent B and filter, concentrate, then add organic reagent C for recrystallization, precipitate crystals, filter, and obtain ethyl 4-(2,4-dihydroxyphenyl)thiazol-2-carboxylate. Step S3: Ethyl 4-(2,4-dihydroxyphenyl)thiazole-2-carboxylate is mixed with an aqueous sodium hydroxide solution; after reacting at room temperature, the pH is adjusted to acidic, and a solid is precipitated to obtain the carboxylic acid compound 4-(2,4-dihydroxyphenyl)thiazole-2-carboxylic acid; Step S4: Dissolve 4-(2,4-dihydroxyphenyl)thiazol-2-carboxylic acid in N,N-dimethylformamide, and then add amine compounds, benzotriazol-1-yl-oxotripyrrolylphosphine hexafluorophosphate, and N,N-diisopropylethylamine in sequence; react at room temperature, and separate and purify to obtain the target compound; the molar ratio of 4-(2,4-dihydroxyphenyl)thiazol-2-carboxylic acid, amine compounds, benzotriazol-1-yl-oxotripyrrolylphosphine hexafluorophosphate, and N,N-diisopropylethylamine is 1:1-1.3:1-1.3:1-1.

2.

3. The preparation method according to claim 2, characterized in that, In step S1, the molar ratio of 2,4-dihydroxyacetophenone to copper bromide is 1:1.5-2; in step S1, the organic solvent is selected from at least one of dichloromethane, ethyl acetate, n-hexane, and petroleum ether; in step S1, the reflux reaction time is 3-8 hours and the temperature is 80-90°C. In step S2, the molar ratio of thiooxazone ethyl ester to 2,4-dihydroxyacetophenone in step S1 is equivalent; in step S2, the heating and reflux reaction time is 4-8 hours and the temperature is 80-90℃. In step S2, organic solvent A is selected from at least one of methanol and ethanol; organic solvent B is selected from at least one of dichloromethane, ethyl acetate, n-hexane, and petroleum ether; and organic solvent C is selected from at least one of dichloromethane, ethyl acetate, n-hexane, and petroleum ether.

4. The preparation method according to claim 2, characterized in that, In step S3, the molar ratio of ethyl 4-(2,4-dihydroxyphenyl)thiazole-2-carboxylate to sodium hydroxide is 1:2-4; in step S3, the reaction time at room temperature is 10-50 min; in step S3, the pH is 3-5. In step S4, the amine compound is selected from at least one of n-propylamine, isopropylamine, tert-butylamine, cyclopropylamine, aniline, p-methoxyaniline, m-methoxyaniline, p-bromoaniline, benzylamine, p-hydroxyaniline, p-chloroaniline, m-chloroaniline, o-methylaniline, 2,4-dimethylaniline, 2,4-dichloroaniline, p-nitroaniline, and m-hydroxyaniline; In step S4, the reaction time at room temperature is 3-8 hours.

5. The use of the 4-(2,4-dihydroxyphenyl)thiazole-2-carboxyamide derivative of claim 1 in the preparation of cosmetics.

Images