Evidences for adduct formation between intracellular non-protein thiols and nitroazoles possessing an alpha,beta-unsaturated carbonyl side chain and the effects on radiosensitization of hypoxic cells

Reactivity of a number of nitroazole derivatives bearing an alpha,beta-unsa turated carbonyl group on the side chain toward nonprotein thiols (NPSH) wa s examined both in the phosphate buffer solution and in the biological syst em. These alpha,beta-unsaturated compounds reacted with NPSH, such as gluta thione (GSH) and L-cysteine (Cys), in the buffer solution to afford the 1,4 -addition products. The reaction gave a second-order rate constant. The add ucts of methyl 4-(2'-nitroimidazol-1'-yl)crotonate (1) with GSH and Cys wer e isolated and characterized as two diastereomers (7a,b and 8a,b) in ca. 1: 1 ratio, respectively. Similarly, exposure of EMT6/KU cells to 1 at 1.0 mM for 1 h resulted in depletion of the intracellular NPSH by more than 80%. O ver 50% of the depleted NPSH was attributed to the formation of the conjuga ted diastereomeric adducts. On the other hand, incubation of EMT6/KU cells with I at 1.0 mM under hypoxic conditions before X-ray irradiation caused c oncurrently a sharp reduction of the shoulder of the dose-survival curves ( reduced the extrapolation number (n) from 8.0 to ca. 1.0) and an increase i n the slope (decreased the mean lethal dose (D-o) to ca. 50% of the control level). The observed effects of 1 on the dose-survival curves were due to the NPSH depletion through the Michael addition occurred in the cellular sy stem. A fairly linear relationship was obtained between the n value and the reduced intracellular NPSH level. It indicated that the shoulder effect of the dose-survival curves of hypoxic cells should be the result of the NPSH depletion by the alpha,beta-unsaturated carbonyl group attached to the nit roazoles. (C) 1999 Elsevier Science Ltd. All rights reserved.