Tyrosinase, an enzyme abundantly found in melanocytes, was selected as a molecular target for the development of anti-melanoma drugs. 4-Hydroxyanisole (4-HA), a simple phenolic agent, was shown to be metabolized by tyrosinase to cytotoxic o-quinones and have anti-melanoma activity. However in clinical trials, 4-HA caused severe liver damage due to its metabolism by liver P450 enzymes to the cytotoxic p-quinone/arene epoxide. In current work, we investigated 24 phenolic agents (2-, 3-, and 4-hydroxy derivatives) to identify compounds that are significantly metabolized by tyrosinase but not by the P450 enzymes and hence limiting their toxicity towards the liver. Tyrosinase/O2 metabolizing system, rat liver microsomal/P450s preparations, murine B16-F0 and human SK-MEL-28 melanoma cells were used to evaluate the metabolism of these phenols and their toxicity against melanoma cells. 4-Hydroxy derivatives showed substantial metabolism by mushroom tyrosinase/O2 metabolizing system compared to their 3-hydroxy and 2-hydroxy derivatives. The order of metabolism for 4-hydroxy derivatives by tyrosinase/O2 was 4-HA, 4-hydroxytoluene, 4-hydroxybenzoic acid>4-hydroxybenzyl alcohol>4-hydroxy benzophenone>>ethyl 4-hydroxybenzoate. Amongst the 2-, 3- and 4-hydroxy derivatives no distinctive order was observed when phenolic agents were metabolized by the P4502E1 induced rat liver microsomal preparations. The order of metabolism by rat liver microsomal preparations for 4-hydroxy derivatives was 4-HA>4-hydroxytoluene>>4-hydroxybenzophenone, 4-hydroxybenzyl alcohol, ethyl 4-hydroxybenzoate, 4-hydroxybenzoic acid. The toxicity of all 24 phenolic agents was investigated against murine B16-F0 cells and the order of toxicity (LD50 (48 h)) for 4-hydroxy derivatives was in the following order: 4-HA> ethyl 4-hydroxybenzoate>>>4-hydroxyoluene, 4-hydroxybenzyl alcohol>>4-hydroxybenzophenone>4-hydroxybenzoic acid. The toxicity of 4-hydroxy derivatives was also investigated against human SK-MEL-28 melanoma cells and a similar pattern was observed. In summary, we identified ethyl 4-hydroxybenzoate and 4-hydroxybenzyl alcohol as two potential lead anti-melanoma compounds which were minimally metabolized by rat liver microsomal preparations for further in vitro and in vivo toxicology, drug metabolism and efficacy investigations.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]