Thiosemicarbazones are a group of compounds that have received comprehensive investigation as
anticancer agents. The antitumor activity of the
thiosemicarbazone, 3-amino-2-pyridinecarboxaldehyde
thiosemicarbazone (3-AP;
triapine), has been extensively assessed in more than 20 phase I and II clinical trials. These studies have demonstrated that 3-AP induces
methemoglobin (metHb) formation and
hypoxia in patients, limiting its usefulness. Considering this problem, we assessed the mechanism of metHb formation by 3-AP compared with that of more recently developed
thiosemicarbazones, including
di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (
Dp44mT). This was investigated using intact red blood cells (RBCs), RBC lysates, purified
oxyhemoglobin, and a mouse model. The chelation of cellular labile
iron with the formation of a redox-active
thiosemicarbazone-
iron complex was found to be crucial for
oxyhemoglobin oxidation. This observation was substantiated using a
thiosemicarbazone that cannot ligate
iron and also by using the
chelator,
desferrioxamine, that forms a redox-inactive
iron complex. Of significance, cellular
copper chelation was not important for metHb generation in contrast to its role in preventing
tumor cell proliferation. Administration of
Dp44mT to mice catalyzed metHb and cardiac
metmyoglobin formation. However,
ascorbic acid administered together with the
drug in vivo significantly decreased metHb levels, providing a potential therapeutic intervention. Moreover, we demonstrated that the structure of the
thiosemicarbazone is of importance in terms of metHb generation, because the DpT analog, di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), does not induce metHb generation in vivo. Hence, DpC represents a next-generation
thiosemicarbazone that possesses markedly superior properties. This investigation is important for developing more effective
thiosemicarbazone treatment regimens.