Estrogens play an important role in the development of breast cancer. Inhibiting 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1)--the enzyme responsible for the last step in the biosynthesis of the most potent estrogen, estradiol (E2)--would thus allow hindering the growth of estrogen-sensitive tumors. Based on a previous study identifying 16beta-benzyl-E2 (1) as a lead compound for developing inhibitors of the transformation of estrone (E1) into E2, we modified the benzyl group of 1 to improve its inhibitory activity. Three strategies were also devised to produce compounds with less residual estrogenic activity: (1) replacing the hydroxy group by a hydrogen at position 3 (C3); (2) adding a methoxy at C2; and (3) adding an alkylamide chain known to be antiestrogenic at C7. In order to test the inhibitory potency of the new compounds, we used the human breast cancer cell line T-47D, which exerts a strong endogenous 17beta-HSD1 activity. In this intact cell model, 16beta-m-carbamoylbenzyl-E2 (4m) emerged as a potent inhibitor of 17beta-HSD1 with an IC50 value of 44 nM for the transformation of [14C]-E1 (60 nM) into [14C]-E2 (24-h incubation). In another assay aimed at assessing the unwanted estrogenic activity, a 10-day treatment with 4m at a concentration of 0.5 microM induced some proliferation (38%) of T-47D estrogen-sensitive (ER+) breast cancer cells. Interestingly, when 4m (0.5 microM) was given with E1 (0.1 nM) in a 10-day treatment, it blocked 62% of the T-47D cell proliferation induced by E1 after its reduction to E2 by 17beta-HSD1. Thus, in addition to generating useful structure-activity relationships for the development of 17beta-HSD1 inhibitors, our study demonstrates that using such inhibitors is a valuable strategy for reducing the level of E2 and consequently its proliferative effect in T-47D ER+ breast cancer cells.