•Lactone- and lactol-estradiol derivatives were synthesized and characterized.•Lactone E-ring was diversified by adding a hydroxymethyl, a methylcarboxylate, a carboxy or an allyl group.•A chemical approach was developed to introduce a chemical group on hindered beta-steroid face.•Lactone and lactol derivatives inhibited 17β-HSD1 (34–60%) similarly as the natural substrate estrone (53%). To control estradiol (E2) formation, we are interested in synthesizing inhibitors of 17β-hydroxyteroid dehydrogenase type 1 (17β-HSD1). Since the results of docking experiments have shown that E2-lactone derivatives substituted in position 19 or 20 (E-ring) could generate interactions with the active site of the enzyme, we carried out their chemical synthesis. After having prepared the 16β,17β-γ-lactone-E2 in four steps starting from estrone (E1), we introduced the molecular diversity by adding a hydroxymethyl, a methylcarboxylate, a carboxy or an allyl group. The allyl derivative was used as a key intermediate to generate a hydroxyethyl side chain in α or β position. Two lactols were also obtained from two hydroxyalkyl lactones. Enzymatic assays revealed that lactone and lactol derivatives weakly inhibited 17β-HSD1 in homogenized HEK-293 cells overexpressing 17β-HSD1 (34–60% at 1μM) and in intact T-47D cells expressing 17β-HSD1 (10–40% at 10μM). This article is part of a Special Issue entitled “Synthesis and biological testing of steroid derivatives as inhibitors”.