Exploring 2-mercapto-N-arylacetamide analogs as promising anti-melanogenic agents: in vitro and in vivo evaluation†

Abstract

Based on the hypothesis that the 2-mercaptoacetamide moiety chelates the copper ions of tyrosinase, 2-mercapto-N-arylacetamide (2-MAA) analogs were designed and synthesized as potential tyrosinase inhibitors. Four 2-MAA analogs showed low IC₅₀ values ranging from 0.95 to 2.0 μM against mushroom tyrosinase, which was 12–26 times lower than that of kojic acid (IC₅₀ value = 24.3 μM). However, according to a copper ion chelation experiment performed, the 2-MAA analogs did not participate in chelation with copper ions. To identify the mode of inhibition of the 2-MAA analogs, kinetic studies were performed, and the results were supported by docking results. In addition, docking simulation results suggested that the 2-MAA analogs strongly inhibited tyrosinase activity because of the hydrogen bonding of the amide NH group and the hydrophobic interaction of the aryl ring instead of chelation with copper ions. In experiments using B16F10 cells, 2-MAA analogs were shown to inhibit melanin production by inhibiting cellular tyrosinase activity. Western blotting showed that in addition to directly inhibiting tyrosinase activity, analog 7 also has an anti-melanogenic effect by inhibiting the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. The 2-MAA analogs showed no appreciable cytotoxicity against HaCaT and B16F10 cells, making them suitable for dermal applications. In a depigmentation experiment using zebrafish embryos, analogs 1 and 2 showed more potent depigmentation effects than kojic acid even at 1000 times lower concentration than that of kojic acid. These results suggest that the 2-MAA analogs are promising anti-melanogenic agents that can inhibit most tyrosinases in various species.