Synthesis And Inhibitory Effect Of 4-(Or 5-) Substituted Thiophene-2-carbaldehyde Thiosemicarbazone Compounds On Tyrosinase

Tyrosinase, a kind of metalloenzyme with dinuclear copper ions, is widely founded in animals, insects, and microorganisms, which was regarded as the rate-limiting enzyme in the process of the formation of melanin. Thiosemicarbazone compounds have a wide range of biological and chemical activity, which have been widely reported as tyrosinase inhibitors in recent years. In our previous work, it could be found that thiophene-2-carbaldehyde thiosemicarbazone could effectively inhibit the activity of tyrosinase, and its inhibitory mechanism on tyrosinase was investgated.In this subject, thiophene-2-carbaldehyde thiosemicarbazone was selected as the lead compound, after chemical modifications, 4-?or 5-? substituted thiophene-2-carbaldehyde thiosemicarbazone compounds 1-11 were successfully synthesized, structure of the compounds were characterized by IR, 1H NMR and MS. Enzyme kinetics and UV spectroscopy were used to study their inhibitory effort on mushroom tyrosinase and their water-soluble and fat-soluble. The results show that the inhibitory activity of compounds 1-11 are much stronger than that of kojic acid; however, only compound 6 had an 20.93% decrease of IC50 compared to his precursor. Steric hindrance generated from substituent reduces the inhibitory activity of such compounds. The introducing methoxyacetyl group of compound 6 may be an important factor which inhibitory activity would be enhanced, and methoxyacetyl group may exhibit a stronger ortho effect. Inhibition kinetics experimental results show that the compound 1, 2, 5, 6, 7 and 9 were reversible and mixed inhibitors on tyrosinase; After precursor modified, the water solubility were limitedly improved, and the extension of hydrophobic chains had a positive effect on improving lipid solubility.Spectrofluorimetry, ¹H and 13 C NMR titration, IR spectrum and UV spectrum were used to investigate the interaction between inhibitors and tyrosinase and their action sites. Experimental results of fluorescence quenching showed that static quenching were dominant in the quenching process of compounds 1-9, Quenching ability and its ability to inhibit tyrosinase were roughly identical. When the compound 1 formed complexes with copper ions, compared to compound 1, showed a weaker binding force and quenching capability, which indicated inhibitors mainly interacted with enzyme through occupy inhibitor activity site after inhibitor and copper coordination. The results of 1H NMR titration showed that sulfur atom of thiourea group of compounds 1, 5, 6 could combine with copper ions in the enzyme active center. The results of 13 C NMR titration showed that after the introducing methoxyacetyl group of compound 6, carbonyl oxygen of methoxyacetyl group could combine with copper ions in the enzyme active center, which could overcome steric hindrance to further enhance the inhibitory activity of precursor. To further verify the this conclusion, the experiment, inorganic copper ions?copper sulfate? simulating copper ions in the enzyme active center, was designed. Complex 6-Cu²⁺ was synthesized, and its UV and IR spectra were studied. The results obtained was consistent with the results of 1H NMR and 13 C NMR titration of compound 6, indirectly proved that by combining with copper ions in the enzyme active center, the introducing methoxyacetyl group of compound 6 further enhanced the inhibitory activity.In this subject, a series of new highly effective tyrosinase inhibitors were screened, and the interaction between the inhibitors and enzyme were further investigated, which provides a more scientific theory for further study.