Synthesis, characterization and biological evaluation of paeonol thiosemicarbazone analogues as mushroom tyrosinase inhibitors

A series of hydroxy- and methoxy-substituted paeonol thiosemicarbazone analogues were synthesized as potential tyrosinase inhibitors and their inhibitory effects on mushroom tyrosinase and inhibitory mechanism were evaluated. Paeonol thiosemicarbazone analogues have been found exhibiting more remarkable inhibition than their indexcompounds on mushroom tyrosinase. Among them, compound 2,4-dihydroxy acetophenone-4-phenyl-3-thiosemicarbazone (d1) had the most potent inhibition activity with the IC50 value of 0.006 ± 0.001 mM, displayed as a reversible competitive inhibitor. The inhibitory ability of o- or p-substituted acetophenone thiosemicarbazones was: di-substituted acetophenone thiosemicarbazones > mono-substituted acetophenone thiosemicarbazones > non-substituted acetophenone thiosemicarbazones. Copper ions chelation assay explained that compound d1 exhibited competitive inhibition by forming a chelate with the copper ions at the catalytic domain of tyrosinase as well as indicate a 1.5:1 binding ratio of compound d1 with copper ions. In the fluorescence spectrum study, compound d1 behaved stronger fluorescence quenching on tyrosinase towards d1–Cu2+ complex, inhibiting tyrosinase mainly by means of chelating the two copper ions in the active site. The newly synthesized compounds may serve as structural templates for designing and developing novel tyrosinase inhibitors.