| Disruption of apoptosis - a common hallmark of cancer cells, contributes to the development of drug resistance to common cancer therapies. As the anti-apoptotic Bcl-2 proteins are commonly over-expressed in many cancers, nullifying the protective effects of these proteins can help to sensitize cancer cells to chemo/radio therapies.;One of the earliest reported small molecule antagonists against anti-apoptotic Bcl-2 proteins was ethyl-2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4 H-chromene-3-carboxylate (HA 14-1). This compound was identified via virtual screening, so no structure-activity relationship (SAR) data were available. The initial focus of the current work was to generate SAR data for HA 14-1 to improve its potency. The most potent analog designed exhibited a more than 13 fold enhancement in potency over HA 14-1. The over-expression of Bcl-2 or Bcl-XL, herein demonstrated to render Jurkat cells resistant to common anti-cancer agents, failed to induce resistance to HA 14-1 and its analogs. Moreover, positive correlations were observed between in vitro cytotoxicity of HA 14-1 and its analogs and their binding interactions with three anti-apoptotic proteins suggesting that these anti-apoptotic proteins are likely to be the cellular targets for these compounds.;During the course of this research, it was demonstrated that HA 14-1 is not stable and decomposes in cell culture medium with a half-life of 14 minutes. The decomposition process was systematically investigated and found to be base assisted and to generate reactive oxygen species (ROS). The decomposition products were isolated, characterized, and a decomposition pathway was proposed.;The decomposition pathway was subsequently utilized toward the rational design of stable analogs. The first analog designed and synthesized based on this pathway was CXL 001. This analog was demonstrated to be stable in culture medium and retain the similar characteristics of HA 14-1. However, CXL 001 was less potent than HA 14-1. A systematic SAR investigation via synthesis of 15 analogs proved fruitful and led to identification of sHA 14-1, a stable analog of HA 14-1. sHA 14-1, while free from ROS generation, retains the beneficial properties of HA 14-1, including its ability to synergize the activity of various intrinsic and extrinsic apoptotic stimuli. |
