Nitric oxide (NO) prodrugs such as O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) are a growing class of promising NO-based cancer therapeutics. The anti-cancer activity of JS-K was established by a number of in vitro and in vivo studies. Nitric oxide release from JS-K is proposed to occur through a nucleophilic aromatic substitution by glutathione (GSH) to form a spontaneously NO-releasing diazeniumdiolate anion, a reaction that is catalyzed by glutathione S-transferase (GST), a phase II detoxification enzyme that is frequently over-expressed in cancer tissue. While a GSH/GST-mediated mechanism accounts for the selective accumulation of NO in the cancer tissue, the origin of differences in the anti-proliferative activity among various O2-2,4-(dinitrophenyl) diazeniumdiolates remains unclear. Small structural perturbations to JS-K appear to significantly lower the anti-proliferative activity, suggesting the involvement of discriminating cellular factors, whose participation could be important as a nitric oxide-independent pathway for anti-cancer activity. In order to probe the relationship between structure and activity, a number of structural analogues of JS-K were synthesized; their chemical and biological properties were compared with those of JS-K. First, half-lives of each of these diazeniumdiolate prodrugs in the presence of GSH were determined using UV-visible spectrophotometry. Next, a similar experiment was carried out in the presence of catalytic amounts of various isoforms of GST. The homopiperazine JS-36-25 and the 2,5-dimethylpiperazine JS-49-128 were found to react at a diminished rate in the presence of GSH and GSH/GST. Finally, these diazeniumdiolate prodrugs were tested for their in vitro anti-proliferative activity against a number of human cancer cell lines. JS-36-25 displayed potent and comparable cytotoxic activity to JS-K against a number of cancer cell lines. In conclusion, we report that JS-36-25 is a nitric oxide prodrug that shows anti-cancer activity that is comparable with that of JS-K but with a diminished reactivity towards GSH/GST that may prove advantageous in its development as an anti-cancer agent.

99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA