Elevated glutathione (GSH) levels have been detected in many tumors compared with the healthy, surrounding tissue. Often, this GSH up-regulation is associated with drug resistance. The prodrugs 6-(2-acetylvinylthio)guanine (AVTG) and 6-(2-acetylvinylthio)purine (AVTP) contain a novel butenone moiety that allows the prodrugs to react selectively with sulfhydryl nucleophiles to release the chemotherapeutic drug 6-thioguanine (6-TG) or 6-mercaptopurine (6-MP), respectively. The cellular uptake and metabolism of trans-AVTG in two human renal carcinoma cell lines that were used as models were rapid and associated with depletion of intracellular GSH. Formation of 6-TG from trans-AVTG correlated positively with intracellular GSH concentrations, and was significantly reduced by diethyl maleate pretreatment. Intracellular concentrations of 6-TG after incubations with trans-AVTG were significantly higher than the 6-TG concentrations obtained after incubations with equimolar concentrations of 6-TG; thus, the prodrug delivered more 6-TG to the cell than did 6-TG itself. Cytotoxicity studies demonstrated that AVTG and AVTP had similar IC 50 values that were comparable with those of 6-TG, but were significantly lower than those of 6-MP. Furthermore, after in vivo treatment of mice with the prodrugs, no reduction was observed in circulating white blood cell counts, whereas white blood cell counts of mice treated with equimolar or 60% lower doses of 6-TG were reduced by 50 to 60%. Collectively, the results show that AVTG and AVTP are novel potential chemotherapeutic agents that may target tumors with up-regulated levels of GSH, and may exhibit less systemic toxicity than the parent thiopurines.The narrow therapeutic index associated with most chemotherapeutic agents often leads to toxicity in nontarget tissues (Hoekman et al., 1999). An approach commonly used to decrease systemic toxicity of chemotherapeutics is prodrug design. Prodrugs are pharmacologically inactive compounds that are metabolized in vivo to yield active drugs. Because quantitative and/or qualitative biochemical differences exist between tumors and the healthy surrounding tissue, it may be possible to selectively deliver drugs to the tumor by designing prodrugs that are metabolized more rapidly or in greater quantities in the tumor than in the surrounding tissue (Dubowchik and Walker, 1999). Thus, prodrugs may decrease adverse drug reactions and systemic toxicity, and increase tissue selectivity and therapeutic efficacy of the parent drug.Glutathione (GSH) plays a major role in the detoxification of numerous chemotherapeutics (Schröder et al., 1996). Changes in GSH content and levels of glutathione S-transferases (GSTs), GSH peroxidase, and GSH reductase (GR) have been detected in tumors (Di Ilio et al., 1991Gajewska et al., 1995;Institoris et al., 1995;Blair et al., 1997). Moreover, increased levels of GSH have been linked with drug resistance (Mickisch et al., 1990;Yang et al., 1992), and up-regulation of the isoform of GST is often associated with dr...