Shigeki Inoue

Melanogenesis appears to be a unique target to develop anti-tumour agents specific for malignant melanoma. Among the anti-melanoma compounds that we have examined, 4-S-cysteaminylphenol (4-S-CAP), a phenolic amine, was found to have the most promising antimelanoma effects. To further improve the efficacy as antimelanoma agents, we have recently synthesized enantiomers; of alpha-Me-4-S-CAP and alpha-Et-4-S-CAP. The enantiomers were found to be good substrates for tyrosinase. In vitro experiments showed that the enantiomers were highly cytotoxic to B16-F1 melanoma cells, and the cytotoxic effect was proved to be tyrosinase-dependent In the present study, in vivo cytotoxicity experiments showed that i.p. administration of R-alpha-Me-4-S-CAP and S-alpha-Et-4-S-CAP (and 4-S-CAP) strongly inhibited the subcutaneous growth of 13116 melanoma in mice, while the corresponding enantionners were much less effective. Similarly, i.p. treatment with R-alpha-Me-4-S-CAP or S-alpha-Et-4-S-CAP, but not with 4-S-CAP, caused strong depigmentation of follicular melanocytes in C57BL black mice. Among 4-S-CAP and the enantiomers, only R-alpha-Et-4-S-CAP caused a moderate decrease in blood pressure in spontaneously hypertensive rats. These results confirm that the use of enantiomers; increases the efficacy of tyrosinase-dependent cytotoxic phenolic amines.

Melanogenesis provides a unique target for the development of antitumour agents specific for malignant melanoma. Among the anti-melanoma compounds we have examined, 4-S-cysteaminylphenol (4-S-CAP), a phenolic amine, was found to have the most promising anti-melanoma effects. To further improve its efficacy as an anti-melanoma agent, we synthesized the R- and S-enantiomers (99% enantiomer excess) of alpha-methyl-4-S-cysteaminylphenol (alpha-Me-4-S-CAP) and alpha-ethyl-4-S-cysteaminylphenol (alpha-Et-4-S-CAP) by coupling 4-hydroxythiophenol with the oxazolines obtained from the (R)- and (S)-enantiomers of 2-amino-1-propanol and 2-amino-1-butanol, respectively. The enantiomers of a-Me-4-S-CAP and a-Et-4-S-CAP were found to be better substrates for tyrosinase than the natural substrate, L-tyrosine. In vitro experiments showed that all four enantiomers were highly cytotoxic to pigmented B16-F1 melanoma cells, the effect being 70-fold and 160-fold greater than that on non-pigmented B16-G4F melanoma cells and 3T3 fibroblasts, respectively. The cytotoxic effect against B16-F1 cells was completely inhibited by phenylthiourea, a tyrosinase inhibitor, or by N-acetyl-L-cysteine, which increases the intracellular reduced glutathione (GSH) level. 4-S-CAP and the enantiomers were taken up into B16-F1 cells at comparable rates, but showed varying rates of GSH depletion that were inversely correlated to the cytotoxicity. These results suggest that the use of enantiomers would increase the efficacy of tyrosinase-dependent cytotoxic phenols. (C) 2003 Lippincott Williams Wilkins.

4-S-Cysteaminylphenol (4-S-CAP), a phenolic thioether, has been evaluated for melanocytotoxicity. We have recently shown that dihydro-1,4-benzothiazine-6,7-dione (benzothiazine BQ) is the ultimate toxic metabolite produced by tyrosinase oxidation of 4-S-CAP. In this study we compared the antimelanoma effects of 4-S-CAP and its two homologues, alpha-methyl-4-S-cysteaminylphenol (alpha-Me-4-S-CAP) and 4-S-homocysteaminylphenol (4-S-Homo-CAP). Biochemical experiments showed that upon tyrosinase oxidation alpha-Me-4-S-CAP and 4-S-Homo-CAP also produced homologues of BQ which reacted rapidly with reduced glutathione (GSH) and also inhibited alcohol dehydrogenase, an SH enzyme. In vitro experiments showed that 4-S-CAP and its two homologues were taken up into B16-F1 melanoma cells at comparable rates but that 4-S-Homo-CAP was least effective in GSH deprivation, which was reflected in the low cytotoxicity of this phenol, and that the cytotoxicity of the phenols was tyrosinase dependent, as proved by the negligible effects on B16-G4F cells which have a much lower tyrosinase activity. In vivo experiments showed that direct intratumoral administration of these phenols inhibited the subcutaneous growth of B16 melanoma, with 4-S-Homo-CAP being the least effective, and that indirect intraperitoneal administration of 4-S-CAP inhibited melanoma growth much more effectively than the two homologues. These results indicate that 4-S-CAP is the most promising antimelanoma agent among the three phenols examined. (C) 1998 Lippincott-Raven Publishers.

4-S-Cysteaminylphenol (4-S-CAP) and the corresponding catechol 4-S-cysteaminylcatechol (4-S-CAC) have been evaluated for melanocytotoxicity. It was shown recently that tyrosinase oxidation of these substrates produces a violet pigment, dihydro-1,4-benzothiazine-6,1-dione (BQ). In this study we examined whether BQ is the ultimate toxic metabolite produced in melanoma cells from 4-S-CAP/4-S-CAC. Biochemical experiments showed that (1) BQ was formed by autoxidation of 4-S-CAC as well as by tyrosinase oxidation of 4-S-CAP/4-S-CAC, (2) BQ reacted rapidly with thiols such as reduced glutathione (GSH), and (3) BQ inhibited the activity of alcohol dehydrogenase, an SH enzyme. In vitro experiments showed that (1) the cytotoxicity of 4-S-CAC was mostly prevented by catalase and superoxide dismutase, (2) Ba was highly cytotoxic to B16 melanoma cells (IC50 being 3.9 mu M as compared with 507 mu M for 4-S-CAP), (3) BQ was metabolized rapidly to a GSH adduct in melanoma cells, and (4) the same GSH adduct was also formed upon incubation of melanoma cells with 4-S-CAP, the reaction being tyrosinase dependent. In vivo experiments showed that intratumoral administration of BQ (0.5 mu mol) inhibited the subcutaneous growth of B16 melanoma nearly as effectively as 4-S-CAP/4-S-CAC (20 mu mol). These results indicate that BQ is the ultimate toxic metabolite produced by tyrosinase oxidation of 4-S-CAP/4-S-CAC. BQ deprives melanoma cells of GSH and may inactivate SH enzymes essential for DNA synthesis and cell proliferation by covalent binding through their cysteine residues, thereby exerting melanocytotoxicity. Cytotoxicity of 4-S-CAC depends mostly on autoxidation producing BQ and active oxygens. (C) 1997 Elsevier Science Inc.