Nitrobenzyl mustard quaternary salts: a new class of hypoxia-selective cytotoxins capable of releasing diffusible cytotoxins on bioreduction.

To explore the utility of a new class of compounds, nitrobenzyl mustard quaternary salts, as hypoxia-selective prodrugs of diffusible cytotoxins. The parent compound N,N-bis(2-chloroethyl)-N-methyl-N-(2-nitrobenzyl)ammonium chloride (SN 25246) was prepared by reaction of 2-nitrobenzyl chloride with N-methyldiethanolamine, and reaction of the resulting quaternary diol with thionyl chloride at room temperature. The rate of release of mechlorethamine from this compound in the presence of cells under aerobic and hypoxic conditions was determined by trapping with diethyldithiocarbamate. Cytotoxicity was assessed by clonogenic assay of stirred suspension cultures of EMT6 cells, and also in intact and dissociated EMT6 spheroids. In vivo activity was evaluated in mice bearing SC KHT tumors. The parent compound is a stable, water-soluble compound with an E(1) of -369 mV. It releases mechlorethamine, and shows selective toxicity towards hypoxic EMT6 cells in culture, increasing with time to several 1000-fold after 4 h. It is much more active against intact than dissociated EMT6 spheroids, and shows low but statistically significant activity against KHT tumors in vivo. Nitrobenzyl mustard quaternary salts are a new class of hypoxia-selective cytotoxin. The parent compound shows very high selectivity for hypoxic cells in vitro, and undergoes reduction in hypoxic cells, releasing mechlorethamine which can back-diffuse to kill surrounding oxygenated cells.