Procarbazine is a 1,2-disubstituted
hydrazine derivative that is used to treat human
leukemias. The anticancer activity of
procarbazine results from bioactivation to reactive intermediates. It is first oxidized to
azoprocarbazine and further N-oxidized to a mixture of
methylazoxyprocarbazine and
benzylazoxyprocarbazine isomers. In this study the azoxyprocarbazine isomers were synthesized and purified. The cytotoxic effect of the metabolites on the L1210 murine
leukemia cell line were then evaluated in vitro by use of a colorimetric assay using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide. The results of this study showed that the
methylazoxyprocarbazine isomer was the most cytotoxic metabolite (IC50, 0.2 mM). The benzylazoxy isomer had an insignificant cytotoxic effect, and a mixture of the two isomers was intermediate in effectiveness. This assay, however, could not be used to determine the cytotoxicity of
procarbazine since the
drug itself (not the live cells) reduced the
dye. A soft-
agar clonogenic assay demonstrated that
procarbazine was cytotoxic only at higher concentrations (IC50, 1.5 mM) than
methylazoxyprocarbazine (IC50, 0.15 mM). The effect of
procarbazine and its metabolites on the survival of L1210
tumor-bearing mice was determined, and
methylazoxyprocarbazine was again the most effective compound. These studies demonstrate that the
methylazoxyprocarbazine metabolite is probably the major cytotoxic intermediate involved in the mechanism of anticancer action of
procarbazine.