Inhibitors of mammalian melanocyte tyrosinase: in vitro comparisons of alkyl esters of gentisic acid with other putative inhibitors.

To discover safe and effective topical skin-lightening agents, we have evaluated alkyl esters of the natural product gentisic acid (GA), which is related to our lead compound methyl gentisate (MG), and four putative tyrosinase inhibitors, utilizing mammalian melanocyte cell cultures and cell-free extracts. Desirable characteristics include the ability to inhibit melanogenesis in cells (IC50 < 100 microg/mL) without cytotoxicity, preferably due to tyrosinase inhibition. Of the six esters synthesized, the smaller esters (e.g. methyl and ethyl) were more effective enzyme inhibitors (IC50 approximately 11 and 20 microg/mL, respectively). For comparison, hydroquinone (HQ), a commercial skin "bleaching" agent, was a less effective enzyme inhibitor (IC50 approximately 72 microg/mL), and was highly cytotoxic to melanocytes in vitro at concentrations substantially lower than the IC50 for enzymatic inhibition. Kojic acid was a potent inhibitor of the mammalian enzyme (IC50 approximately 6 microg/mL), but did not reduce pigmentation in cells. Both arbutin and magnesium ascorbyl phosphate were ineffective in the cell-free and cell-based assays. MG at 100 microg/mL exhibited a minimal inhibitory effect on DHICA oxidase (TRP 1) and no effect on DOPAchrome tautomerase (TRP-2), suggesting that MG inhibits melanogenesis primarily via tyrosinase inhibition. MG and GA were non-mutagenic at the hprt locus in V79 Chinese hamster cells, whereas HQ was highly mutagenic and cytotoxic. The properties of MG in vitro, including (1) pigmentation inhibition in melanocytes, (2) tyrosinase inhibition and selectivity, (3) reduced cytotoxicity relative to HQ, and (4) lack of mutagenic potential in mammalian cells, establish MG as a superior candidate skin-lightening agent.