Synthesis, structure, and estrogenic activity of 4-amino-3-(2-methylbenzyl)coumarins on human breast carcinoma cells.

A number of coumarins exhibit interesting pharmacological activities and are therefore of therapeutic use. We report here the synthesis and the structural analysis of new N-substituted 4-amino-3-(2-methylbenzyl)coumarins (compounds 8a-8e) that present structural analogies with estrothiazine and 11- or 7-substituted 17beta-estradiol. These derivatives were tested with respect to estrogenic activity on the estrogen receptor positive (ER+) human MCF-7 breast cancer cell line. Two of the reported compounds (8a and 8b) stimulated specifically the proliferation of MCF-7 cells, but not that of estrogen receptor negative (ER-) human MDA-MB-231 breast cancer cells, suggesting that their mitogenic activity is mediated by ER. Accordingly, the stimulating effect of 8a and 8b was suppressed by the pure antiestrogen fulvestrant. Besides, 8a and 8b induced ER down-regulation similar to that produced by classical ER agonists or pure antagonists. The effects of the compounds under study on ER-mediated transcription were assessed on (ER+) MVLN cells, that is, MCF-7 cells stably transfected with a pVit-tk-Luc reporter plasmid. Derivatives 8a and 8b, and surprisingly compound 8c, enhanced ER-mediated gene transactivation in that model. Finally, no coumarin was able to compete with tritiated 17beta-estradiol ([(3)H]E(2)) for ER binding, suggesting unconventional interactions with the receptor, such as interactions with the second binding pocket or with the coactivator-binding region. To conclude, observations performed in this study on compound 8c reveal that estrogenic activity can be dissociated from enhancement of cell proliferation. Furthermore, ERE-driven transactivation of transcription seems to be a condition necessary, but not sufficient, for estrogen-induced stimulation of cell growth.