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

Abstract

Reactivity of a number of nitroazole derivatives bearing an α,β-unsaturated carbonyl group on the side chain toward non-protein thiols (NPSH) was examined both in the phosphate buffer solution and in the biological system. These α,β-unsaturated compounds reacted with NPSH, such as glutathione (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 adducts of methyl 4-(2′-nitroimidazol-1′-yl)crotonate (1 ) with GSH and Cys were 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%. Over 50% of the depleted NPSH was attributed to the formation of the conjugated diastereomeric adducts. On the other hand, incubation of EMT6/KU cells with 1 at 1.0 mM under hypoxic conditions before X-ray irradiation caused concurrently 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 in the slope (decreased the mean lethal dose (Do) 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 system. 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 α,β-unsaturated carbonyl group attached to the nitroazoles.