Mechanism of action of 2-haloethylnitrosoureas on deoxyribonucleic acid: Pathways of aqueous decomposition and pharmacological characteristics of new anticancer disulfide-linked nitrosoureas

Abstract

We have examined the pharmacological characteristics of three dinitrosated isomers of N, N′-bis[ N(2-chloroethyl)- N-carbamoyl]cystamine [CNCC-(D), 1C1G1325] differing in the relative positions of the nitroso substituents [CNCC-(C), (1,1′dinitroso); CNCC-(S), 3,3′ dinitroso); and CNCC-(M), (1,3′-dinitrosol)] and which were designed to be sunject to preferential bioreductive activation in hypoxic tumors. The decomposition products of the isomers formed under physiological conditions [both in the absence and in the presence of dithiothreitol (DDT)] were identified and quantified. For example, CNCC-(S) in phosphate buffer, pH 7.0, and 37° gave rise to 2-chloroethylisocyanate, bis(2-chloroethyl)urea and bis(2-hydroxyethyl)disulfide, whereas in the presence of DTT it afforded 2-cloroethylisocyanate, bis(2-chloroethyl)urea, bis(2-hydroxyethyl) disulfide, thiirane and 2-mer-captoethanol. Control aqueous decomposition profiles were performed with two known metabolites of CNCC, namely 3-(2-chloroethyl)-1-(2-thioethyl)-1-nitrosourea and 3-(2-chloroethyl)-1-(2-methyl-thioethyl)-1-nitrosourea. CNCC-(C) caused 20% interstrand cross-linking of λ-DNA in 2hr, whereas in the presence of DTT the extent of cross-linking increased to 38% in the same time period. In contrast, isomer (S) showed no detectable cross-linking in 7 hr. This thiol potentiation of cross-linking which is observed with other 2-chloroethylnitrosoureas is explained by nucleophilic attack at the carbonyl group and sunsequent stereoelectronically controlled decomposition of the tetrahedral intermediate. The relative extents of carbamoylating activity of the CNCC isomers were obtained using a [ 14C]-lysine assay which showed (S) ≈ (M) > (C). Inhibition of glutathione reductase for both Walker 256 resistant (WR) and Walker 256 sensitive (WS) strains showed that isomer (S) inactivated the enzyme more effectively than isomer (C) in accord with the carbamoylating activity results. The higher carbamoylators (S) and (M) also showed greater effects on the intracellular thiol pools in both WR and WS cells indicative of sulfhydryl conjugation and efflux and/or inhibition of the GSH metabolic enzymes. In vitro cytotoxicity studies with human DU 145 prostatic carcinoma cells showed the isomer cytotoxicity was (M) > (C) > (S) over a 24-hr incubation period. The reduced cytotoxic potential of CNCC-(S) in both the Walker 256 cells and in the human prostatic carcinoma cells may be a function of an interaction between GSH and the drug thereby protecting other more critical nucleophilic targets within the nucleus.