Published reports have established that naphthoquinone compounds with similar structures to those of our library are commonly used in experimental models of oxidative stress induction. Moreover, both plumbagin and jugalone have been shown to induce oxidative stress as well. We therefore sought to assess whether ROS production associated with cytotoxicity. Indeed, the formation of ROS was induced by all of the cytotoxic compounds. However, compound-treated cells were only partially protected by the anti-oxidant enzymes SOD and/or catalase or the ROS scavenger ascorbic acid suggesting the potential role of additional cell-death inducing properties of the cytotoxic compounds. The additional cytotoxic mechanism induced by our compound library is particularly underscored by compound 5, which was shown to promote the generation of ROS but does not activate mitochondrial depolarization. This is in contrast to what has been seen in other mammalian cell model systems, where changes in mitochondrial membrane potential were observed. Plumbagin may instead be inducing cell cycle arrest through effects on cell cycle progression mediators and prosurvival molecules. Although not tested here, it is possible that our compounds are also interacting with glutathione or other thiol-containing proteins. Glutathione GSH, generated from the reduction of glutathione disulfide, is a critical molecule in resisting oxidative stress acting as a scavenger for hydroxyl radicals, singlet oxygen, and various electrophiles. In this capacity, it is possible that the electrophilic disubstituted 1, 4naphthoquinone compounds interact with GSH inhibiting its ability to U73122 scavenge ROS. This may explain the partial protection from ROS formation imparted by SOD, catalase, and ascorbic acid upon BAR501 treatment with the compounds. Indeed, quinones and naphthoquinones, including jugalone, have been shown to readily cause depletion of intracellular GSH. In summary, our small library of compounds has yielded a group of synthetically generated naphthoquinones that mediate cytotoxicity.