The rate-determining role of
tyrosinase makes it a critical component in the mechanism that is responsible for melanogenesis. Thirteen (Z)-5-(substituted benzylidene)-3-phenyl-2-thioxothiazolidin-4-one ((Z)-BPTT) analogs were designed based on the structural features of two potent
tyrosinase inhibitors, viz. (Z)-5-(3-hydroxy-4-methoxybenzylidene)-2-thioxothiazolidin-4-one (5-HMT) and (Z)-2-(2,4-dihydroxybenzylidene)benzo[4,5]imidazo[2,1-b]thiazol-3(2H)-one (compound I). The trisubstituted double bond geometry of the (Z)-BPTT analogs that were generated by Knoevenagel condensation was determined using vicinal 1H and 13C coupling constants in 13C NMR spectra. Four analogs, numbers 1-3 and 6, inhibited mushroom
tyrosinase 9 to 29 times more potently than
kojic acid did. Kinetic study results indicated that these four analogs inhibited mushroom
tyrosinase competitively and this was supported by docking simulation. Also, docking results using human
tyrosinase suggested that analogs 2 and 3 might be potent human
tyrosinase inhibitors. In vitro studies using B16F10 cells (a
melanoma cell line) showed that analogs 1, 2, 3, and 6 inhibited cellular
tyrosinase and
melanin production more than
kojic acid did, without perceptible cytotoxicity. In particular, analog 2, which possesses a
catechol group, exerted an extremely potent anti-melanogenic effect. In addition, analog 2 showed strong scavenging activity against DPPH and
ABTS radicals. Furthermore, analog 2 not only reduced ROS levels, which induce melanogenesis, but it also suppressed
tyrosinase and MITF (microphthalamia-associated
transcription factor)
protein levels and the expressions of melanogenesis-related genes. These results suggest that analog 2 is an efficient
tyrosinase inhibitor that alleviates melanogenesis by dual mechanisms of (i) the inhibition of melanogenesis-related
proteins and genes and (ii) the direct inhibition of
tyrosinase activity.