17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) and steroid sulfatase (STS) are involved in the synthesis of the most potent estrogen in the human body, estradiol (E2). These enzymes are known to play a pivotal role in the progression of estrogen-dependent diseases, such as breast cancer and endometriosis. Therefore, the inhibition of 17β-HSD1 and/or STS represents a promising avenue to modulate the growth of estrogen-dependent tumors or lesions. We recently established the key role of a bromoethyl side chain added at the C3-position of a 16β-carbamoyl-benzyl-E2 nucleus to covalently inhibit 17β-HSD1. To extend the structure-activity relationship study to the C16β-position of this new selective irreversible inhibitor (PBRM), we synthesized a series of analog compounds by changing the nature of the C16β-side chain but keeping the 2-bromoethyl group at position C3. We determined their 17β-HSD1 inhibitions in T-47D cells (transformation of E1 into E2), but we did not obtain a stronger 17β-HSD1 inhibitor than PBRM. Compounds 16 and 17 were found to be more likely to bind to the catalytic site and showed a promising but moderate inhibitory activity with estimated IC50 values of 0.5 and 0.7 µM, respectively (about 10 times higher than PBRM). Interestingly, adding one or two sulfamate groups in the D-ring's surroundings did not significantly decrease compounds' potential to inhibit 17β-HSD1, but clearly improved their potential to inhibit STS. These results open the door to the development of a new family of steroid derivatives with dual (17β-HSD1 and STS) inhibiting actions. Copyright © 2021 Elsevier Inc. All rights reserved.