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The oxidative N -demethylation of several p -substituted phenyl triazenes has been determined in vitro under carefully controlled conditions. Three of these compounds had been reported not to be appreciably demethylated, one of them still possessing antitumour activity. The per cent demethylation figures obtained for the tested compounds ranged from about 20 to 60 per cent and was not dependent on the nature of the solvent used to keep the drug in solution. The enzymatic reaction proved to be strongly inhibited by diazonium cations either generated by spontaneous hydrolysis of the drug or added to the incubation mixture. The reaction is also inhibited at various degrees by the monomethyl derivatives produced by demethylation of the dimethyl triazenes. The inhibition by diazonium and monomethyl derivatives can not account for the observed time-course of demethylation, which occurs for all the tested compounds only in the first minutes of incubation. The possible occurrence of other reaction(s), competing with the demethylation process, the impossibility of correctly expressing kinetic parameters for demethylation, and the possible relevance of these observations for the activity of triazenes in vivo , are pointed out.

DM-CONH2, a dimethyltriazene active in prolonging the survival time of mice bearing TLX5 lymphoma, requires metabolic activation by liver homogenate supernatant and cofactors in order to exert in vitro cytotoxic effects on the same tumor cells, as determined by in vivo bioassay of their viability. From the examination of the metabolites produced during these in vitro experiments, it is found that in vitro cytotoxicity is attributable to the generation of MM-CONH2 by oxidative N-demethylation of DM-CONH2. Also the generation of DM-COO− is observed, although this compound is not cytotoxic in vitro. The in vivo effects of DM-CONH2 and CM-COOK on TLX5 lymphoma are not caused exclusively by cytotoxic effects of the drugs, since they are evident also when no reduction in the number or viability of peritoneal tumor cells is evident, whereas these parameters are significantly reduced by MM-CONH2. The increase in survival time of mice bearing TLX5 lymphoma caused by the dimethyltriazenes used appears to be caused by the drugs without being subjected to metabolic activation, with a mechanism different from cytotoxicity for tumor cells.