Design,Synthesis And Anti-Tyrosinase Activity Of Novel Kojic Acid Derivatives

Objective:With kojic acid as the lead compound,a series of kojic acid derivatives were designed and synthesized by retaining the key active site of the interaction between kojic acid and tyrosinase and using the principle of compound combination.And through screening for tyrosinase inhibitory activity,studying its mechanism of action,and evaluating its safety,we hope to develop more new,low toxicity,and efficient tyrosinase inhibitors.Methods:（1）Based on the principle of active skeleton hybridization of compound design,this study took kojic acid as the lead compound and introduced various heterocycles at the 7-position hydroxyl of its structure.Four series of compounds:series I of kojic acid-thioheterocycles（I-4a～I-4l）,series II of kojic acid-1,3,4-oxadiazoles（II-5a～II-5n）,series III of kojic acid-1,2,4-triazines（III-7a～III-7o）and series IV of kojic-coumarins（IV-6a～IV-6m）were synthesized,and their structures were identified by¹H NMR,¹³C NMR and HRMS.（2）_L-DOPA was used as the substrate and kojic acid as the positive control to study the anti-tyrosinase activity of all synthetic compounds.Moreover,this study combines various methods,such as enzyme kinetics,determination of copper ion chelating ability,fluorescence spectrometry,ANS binding with fluorescence quenching analysis,molecular docking analysis,ultraviolet spectroscopy and circular dichroism assays,to further clarify the mechanism of action between compounds and enzyme.At the same time,human normal liver cell line（LO₂）or human normal embryonic kidney cell line（HEK-293）were selected to evaluate the safety of the compound.Results:（1）Four series of derivatives were synthesized in this study:kojic acid-thioheterocyclic derivatives（I-4a～I-4l）,kojic acid-1,3,4-oxadiazole derivatives（II-5a～II-5n）,kojic acid-1,2,4-triazine derivatives（III-7a～III-7o）and kojic acid-coumarin derivatives（IV-6a～IV-6m）.A total of 54 compounds were synthesized,all of which were new compounds through literature search,and their structures were identified by ¹H NMR,¹³C NMR and HRMS.（2）The enzyme inhibition assay showed that all synthetic derivatives showed better anti-tyrosinase activity than kojic acid.Kinetic studies showed that compound I-4d was a competitive inhibitor of tyrosinase,and the inhibition constant（K_i）of compound I-4d and tyrosinase was calculated to be 1.96μM.Compounds II-5f,III-7m and IV-6f are mixed inhibitors of tyrosinase.Based on the quadratic construction of the Lineweaver-Burk double reciprocal diagram,the inhibition constants K_iand K_isof compounds with free enzymes and substrate-tyrosinase complexes were obtained respectively:II-5f(K_i=7.93μM,K_is=21.14μM),III-7m(K_i=0.73μM,K_is=1.27μM),IV-6f(K_i=1.02μM,K_is=1.15μM).（3）The mechanisms of the compounds were discussed in detail by using a variety of spectral techniques and molecular docking experiments.The research showed that the compound obtained by structural modification of kojic acid can not only chelate with the copper ion in the active center of the enzyme,but also interact with the amino acid residues in the introduced heterocyclic part,which changes the spatial structure of tyrosinase,thus exerting an efficient anti-tyrosinase activity.（4）The cytotoxicity of compounds I-4d,II-5f,III-7m and IV-6f were tested,and the results suggested that these compounds showed no obvious toxicity to the test cells.Conclusions:In this study,the hydroxypyranone part of kojic acid structure was retained,and four series of derivatives of kojic acid-thioheterocycles,kojic-1,3,4-oxadiazoles,kojic-1,2,4-triazines and kojic-coumarins were finally synthesized by introducing various heterocycles.In the anti-tyrosinase activity assay,it was found that all derivatives showed better tyrosinase inhibition than positive control kojic acid.At the same time,the mechanism and safety evaluation of the most active compounds in each series were conducted.The results showed that these compounds reduced the catalytic activity of tyrosinase by changing its spatial structure.Importantly,these compounds exhibit low cytotoxicity,so it is believed that the compounds synthesized in this study have the potential to become lead compounds for the development of new,safe,and efficient tyrosinase inhibitors in the future.