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'-dinitroso)] and which were designed to be subject 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 degrees gave rise to 2-chloroethylisocyanate, bis(2-chloroethyl)
urea and bis(2-hydroxyethyl)
disulfide, whereas in the presence of DTT it afforded 2-chloroethylisocyanate, bis(2-chloroethyl)
urea, bis(2-hydroxyethyl)
disulfide,
thiirane and
2-mercaptoethanol. 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-methylthioethyl)-1-nitrosourea.
CNCC-(C) caused 20% interstrand cross-linking of lambda-
DNA in 2 hr, 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 subsequent 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) approximately equal to (M) greater than (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) greater than (C) greater than (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.