Here, we report on
7-nitro-4-(phenylthio)benzofurazan (
NBF-SPh), the most potent derivative among a set of patented anticancer 7-nitrobenzofurazans (NBFs), which have been suggested to function by perturbing
protein-
protein interactions. We demonstrate that
NBF-SPh participates in toxic redox-cycling, rapidly generating
reactive oxygen species (ROS) in the presence of molecular
oxygen, and this is the first report to detail ROS production for any of the anticancer NBFs. Oxygraph studies showed that
NBF-SPh consumes molecular
oxygen at a substantial rate, rivaling even
plumbagin,
menadione, and
juglone. Biochemical and enzymatic assays identified
superoxide and
hydrogen peroxide as products of its redox-cycling activity, and the rapid rate of ROS production appears to be sufficient to account for some of the toxicity of
NBF-SPh (LC(50) = 12.1 μM), possibly explaining why
tumor cells exhibit a sharp threshold for tolerating the compound. In cell cultures, lipid peroxidation was enhanced
after treatment with
NBF-SPh, as measured by
2-thiobarbituric acid-reactive substances, indicating a significant accumulation of ROS.
Thioglycerol rescued cell death and increased survival by 15-fold to 20-fold, but
pyruvate and
uric acid were ineffective protectants. We also observed that the redox-cycling activity of
NBF-SPh became exhausted after an average of approximately 19 cycles per
NBF-SPh molecule. Electrochemical and computational analyses suggest that partial reduction of
NBF-SPh enhances electrophilicity, which appears to encourage scavenging activity and contribute to electrophilic toxicity.