IDENTIFICATION, SYNTHESIS AND PROPERTIES OF 5-(AZIRIDIN-1-YL)-2-NITRO-4-NITROSOBENZAMIDE, A NOVEL DNA CROSS-LINKING AGENT DERIVED FROM CB1954

-(Aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide, the active form of 5-(aziridin-1-yl)-2,4-dinitorbenzamide (CB1954), can react spontaneous ly with oxygen, and in aqueous solution yields 5-(aziridin-1-yl)-2-nit ro-4-nitrosobenzamide and hydrogen peroxide. Mild biological reducing agents such as NAD(P)H, reduced thiols and ascorbic acid rapidly re-re duced the nitroso compound to the hydroxylamine. Both compounds were e qually efficient at inducing cytotoxicity and DNA interstrand crosslin king in cells when exposed in phosphate-buffered saline (PBS). Neither agent was capable of inducing cross-links in isolated DNA. When acety l coenzyme A was included in the incubation, crosslink formation was s een with the hydroxylamine, but not with the nitroso compound. Thus, t he nitroso compound is acting as a prodrug for the hydroxylamine, and needs to be reduced to this compound to exert its cytotoxic effects. I n vivo anti-tumour tests showed that neither compound was effective in its own right. This may be due to the rapid reduction of the nitroso to the hydroxylamine, and the reaction of the hydroxylamine with serum proteins. The chemical synthesis of the 5-(aziridin-1-yl)-2-nitro-4-n itrosobenzamide, and an improved synthesis of 5-(aziridin-1-yl)-4-hydr oxylamino-2-nitrobenzamide is described. These results emphasize the p otential efficacy of the in situ activation of prodrugs such as CB1954 either by endogenous enzymes such as DT diaphorase, or by antibody di rected enzyme prodrug therapy (ADEPT).