Tyrosinase enzyme was used as a molecular target for 4-hydroxyanisole (4-HA), an alkoxyphenol, for the treatment of malignant melanoma. However, 4-HA causes serious liver damage due to its metabolism by liver P450s to cytotoxic intermediate metabolites. In the current work, we adopted for the first time a strategy in order to identify alkoxyphenols that were minimally metabolized by liver P450 metabolizing enzymes and hence to have potentially a minimal toxicity towards liver but could still form quinone reactive intermediate metabolites in melanoma cells and hence to be potentially useful for the treatment of melanoma. Ten alkoxyphenols (4-hydroxyanisole, 4-ethoxyphenol, 4-n-propoxyphenol, 4- iso-propoxyphenol, 2-iso-propoxyphenol, 4-n-butoxyphenol, 4-iso-butoxyphenol, 4-sec-butoxyphenol, 4-t-butoxyphenol, and 4-n-hexyloxyphenol) were investigated for their metabolism by mushroom tyrosinase/O2 and toxicity towards murine B16-F0 melanoma cells. The extent of glutathione depletion by rat liver P450 microsomal /NADPH/O2 /alkoxyphenol mixture was used as a surrogate marker for the liver toxicity. All the alkoxyphenols acted as substrates for tyrosinase/O2 metabolizing system and demonstrated a dose- and time-dependent toxicity towards B16-F0 melanoma cells except 2-iso-propoxyphenol. 4-n-Hexyloxyphenol demonstrated the greatest toxicity towards B16-F0 cells while depleting only 17% glutathione by rat liver microsomal preparations at its calculated LC50 lethal concentrations for B16-F0 cells whereas 4-HA demonstrated 33% glutathione depletion. At 100 μM concentrations, 4-t-butoxyphenol showed the lowest amount of glutathione depletion of 25% by microsomal P450 system whereas 4-HA showed 58% glutathione depletion. Alkoxyphenols with at least two alkyl groups derivatized at alpha carbon of alkoxy group showed the minimal rates of metabolism by tyrosinase/O2 metabolizing system. A quantitative structural toxicity relationship equation was also derived, LogLC50(μM)= -0.265(±0.064)LogP+2.482(±0.179) indicating the toxicity was increased with an increase in the lipid solubility of the alkoxyphenols. In summary, we identified 4-n-hexyloxyphenol and 4-t-butoxyphenol as two potential lead compounds for their further in vitro and in vivo toxicity and efficacy evaluation as anti-melanoma agents which is currently under way in our laboratory.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]