Antitumour imidazotetrazines—XVI: Macromolecular alkylation by 3-substituted imidazotetrazinones

Abstract

The extent of macromolecular alkylation by three imidazotetrazinones, 8-carbamoyl-3-(2-chloroethyl)imidazo[5,1- d]-1,2,3,5-tetrazin-4 (mitozolomide) and the 3-methyl CCRG 81045) and 3-ethyl (CCRG 82019) analogues has been studied both in intact cells and with isolated DNA, RNA and protein. Towards isolated DNA and RNA CCRG 81045 was about twice as reactive as mitozolomide and 5–10-fold more reactive than CCRG 82019. Two cell lines were chosen to study macromolecular alkylation, GM892A and Raji, the latter being 10–20-fold less sensitive to mitozolomide and CCRG 81045 than the former, but only one-and-a-half-fold less sensitive to CCRG 82019. Drug uptake into both cell lines was shown to be by a rapid diffusion process with a cell medium distribution ratio not far from unity. For all three agents intracellular radioactivity became associated with macromolecules, and the level found at any time is a balance between the rate of alkylation and the rate of alkyl group removal by repair processes. Both CCRG 81045 and CCRG 82019 produced approximately the same level of alkyl groups bound to DNA, RNA and protein over a 24-hr period, whereas mitozolomide produced a greater extent of alkylation. All three agents left more alkyl groups bound to DNA and RNA in GM892A than in Raji cells, but there was no difference in the level of alkyl groups remaining bound to proteins. However, in GM892A cells the overall level of alkylation of DNA by CCRG 81045 exceeded that of CCRG 82019 only after 24 hr of drug incubation despite the twenty-fold difference in potency of these agents. These results suggest that specific base alkylations rather than total macromolecular alkylation may be more important in determining relative cytotoxicity.