Discovery of 4-Aryl-4H-chromenes as a New Series of Apoptosis Inducers Using a Cell- and Caspase-based High-Throughput Screening Assay. 1. Structure−Activity Relationships of the 4-Aryl Group

By applying a novel cell- and caspase-based HTS assay, 2-amino-3-cyano-7-(dimethylamino)-4-(3-methoxy-4,5-methylenedioxyphenyl)-4H-chromene (<BO>1a</BO>) has been identified as a potent apoptosis inducer. Compound <BO>1a</BO> was found to induce nuclear fragmentation and PARP cleavage, as well as to arrest cells at the G<INF>2</INF>/M stage and to induce apoptosis as determined by the flow cytometry analysis assay in multiple human cell lines (e.g. Jurkat, T47D). Through structure−activity relationship (SAR) studies of the 4-aryl group, a 4- and 7-fold increase in potency was obtained from the screening hit <BO>1a</BO> to the lead compounds 2-amino-4-(3-bromo-4,5-dimethoxyphenyl)-3-cyano-7-(dimethylamino)-4H-chromene (<BO>1c</BO>) and 2-amino-3-cyano-7-(dimethylamino)-4-(5-methyl-3-pyridyl)-4H-chromene (<BO>4e</BO>), with an EC<INF>50</INF> of 19 and 11 nM in the caspase activation assay in T47D breast cancer cells, respectively. The 2-amino-4-aryl-3-cyano-7-(dimethylamino)-4H-chromenes also were found to be highly active in the growth inhibition MTT assay, with GI<INF>50</INF> values in the low nanomolar range for compound <BO>1c</BO>. Significantly, compound <BO>1c</BO> was found to have a GI<INF>50</INF> value of 2 nM in the paclitaxel resistant, p-glycoprotein overexpressed, MES-SA/DX5 tumor cells. Functionally, compound <BO>1c </BO>was found to be a potent inhibitor of tubulin polymerization and to effectively inhibit the binding of colchicine to tubulin. These results confirm that the cell-based caspase activation assay is a powerful tool for the discovery of potent apoptosis inducers and suggest that the 4-aryl-4H-chromenes have the potential to be developed into future anticancer agents.