The cytotoxic and mechanistic effects of novel 10-substituted 3-methyl-1,6-diazaphenothiazines were investigated in human melanoma models. Antiproliferative activity was evaluated in vitro using the WST-1 assay in four melanoma cell lines (A375, C32, G361, and SK-MEL-28) and normal human dermal fibroblasts (HDF). Among the tested derivatives, compound 6 exhibited the most pronounced biological activity, showing the strongest growth inhibition in melanoma cells, with the lowest IC50 value against C32 cells (54 µM), while displaying lower toxicity toward normal fibroblasts. Mechanistic studies using image cytometry and immunofluorescence revealed that compound 6 profoundly disrupts melanoma cell homeostasis by suppressing cell proliferation, inducing DNA damage, and activating apoptotic cell death. These effects were accompanied by mitochondrial membrane depolarization, depletion of intracellular reduced thiols, and DNA fragmentation, indicating the involvement of oxidative stress and mitochondrial dysfunction in the observed cytotoxic response. Taken together, these results demonstrate that 10-substituted 1,6-diazaphenothiazines exert anti-melanoma activity through multiple biological mechanisms. We believe our study provides a basis for developing derivatives with optimized pharmacological properties.
Morak-Młodawska et al. (Sat,) studied this question.