By applying a novel cell- and caspase-based HTS assay, 2-amino-3-cyano-7-(dimethylamino)-4-(3-methoxy-4,5-methylenedioxyphenyl)-4H-chromene (1a) has been identified as a potent apoptosis inducer. Compound 1a was found to induce nuclear fragmentation and PARP cleavage, as well as to arrest cells at the G(2)/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 1a to the lead compounds 2-amino-4-(3-bromo-4,5-dimethoxyphenyl)-3-cyano-7-(dimethylamino)-4H-chromene (1c) and 2-amino-3-cyano-7-(dimethylamino)-4-(5-methyl-3-pyridyl)-4H-chromene (4e), with an EC(50) 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(50) values in the low nanomolar range for compound 1c. Significantly, compound 1c was found to have a GI(50) value of 2 nM in the paclitaxel resistant, p-glycoprotein overexpressed, MES-SA/DX5 tumor cells. Functionally, compound 1c 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.

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As a continuation of our efforts to discover and develop the apoptosis-inducing 4-aryl-4H-chromenes as novel anticancer agents, we explored the SAR of fused rings at the 7,8-positions. It was found that a five-member aromatic ring, such as pyrrolo with nitrogen at either the 7- or 9-position, is preferred. A six-member aromatic ring, such as benzo or pyrido, also led to potent compounds. The SAR of the 4-aryl group was found to be similar for chromenes with a fused ring at the 7,8-positions. These compounds were found to inhibit tubulin polymerization, indicating that cyclization of the 7,8-positions into a ring does not change the mechanism of action. Compound 2h was identified to be a highly potent apoptosis inducer with an EC50 of 5 nM and a highly potent inhibitor of cell proliferation with a GI50 of 8 nM in T47D cells.

In our continuing effort to discover and develop apoptosis inducing 4-aryl-4H-chromenes as novel anticancer agents, we explored the structure-activity relationship (SAR) of alkyl substituted pyrrole fused at the 7,8-positions. A methyl group substituted at the nitrogen in the 7-position of the pyrrole ring led to a series of potent apoptosis inducers with potency in the low nanomolar range. These compounds were also found to be low nanomolar or subnanomolar inhibitors of cell growth, and they inhibited tubulin polymerization, indicating that methylation of the 7-position nitrogen does not change the mechanism of action of these chromenes. Compound 2d was identified as a highly potent apoptosis inducer with an EC50 value of 2 nM and a highly potent inhibitor of cell growth with a GI50 value of 0.3 nM in T47D cells.

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