Ethyl-2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H- chromene-3-carboxylate (HA 14-1), a prototype small-molecule antagonist against antiapoptotic Bcl-2 proteins, decomposes to generate reactive oxygen species that induce apoptosis.

Overexpressing antiapoptotic Bcl-2 proteins to suppress apoptosis is one major mechanism via which cancer cells acquire drug resistance against cancer therapy. Ethyl-2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4 H-chromene-3-carboxylate (HA 14-1) is one of the earliest small-molecule antagonists against antiapoptotic Bcl-2 proteins. Since its discovery, HA 14-1 has been shown to be able to synergize a variety of anticancer agents. HA 14-1 also could selectively eliminate tumor cells with elevated level of Bcl-2 protein. HA 14-1, therefore, is being intensely investigated as a potential anticancer agent. Previous reports of HA 14-1 implied that it may not be stable, raising the question of whether HA 14-1 is a suitable drug candidate. The potential stability also raised the concern about whether HA 14-1 is the bioactive species. In this report, we confirm that HA 14-1 is not stable under physiological conditions: it rapidly decomposes in RPMI cell culture medium with a half-life of 15 min. This decomposition process also generates reactive oxygen species (ROS). To identify the actual candidate(s) for the observed bioactivity of HA 14-1, we characterized the structures, quantified the amount, and evaluated the bioactivities of the decomposed products. We also used ROS scavengers to explore the function of ROS. From these studies, we established that none of the decomposition products could account for the bioactivity of HA 14-1. ROS generated during the decomposition process, however, are critical for the in vitro cytotoxicity and the apoptosis induced by HA 14-1. This study demonstrates that HA 14-1 is not stable under physiological conditions and that HA 14-1 can generate ROS through its decomposition, independent of Bcl-2 antagonism. Because of its intrinsic tendency to decompose and to generate ROS, caution should be taken in using HA 14-1 as a qualified antagonist against antiapoptotic Bcl-2 proteins.