Design, synthesis, and preliminary antiproliferative evaluation of azetidine-functionalized pyrrolo[2,3-d]pyrimidines with integrated computational studies

Azetidine-functionalized pyrrolo[2,3-d]pyrimidines were designed to examine the comparative influence of methyl-, propyl-, and butylsulfonyl substitution among final synthesized analogues on antiproliferative activity and structure-based behavior. Following in silico ADME filtering and docking-based prioritization, selected analogues were synthesized and characterized. Among the evaluated compounds, BCD17 showed the most favorable predicted docking profile toward 17β-hydroxysteroid dehydrogenase type 1 (3HB5), with a docking score of −9.130 kcal/mol and Glide energy of −69.236 kcal/mol. Molecular dynamics simulation and MM-GBSA analysis further supported the stable predicted interaction profile of BCD17, with a mean binding free energy of −109.49 ± 7.52 kcal/mol. DFT analysis indicated a balanced electronic profile compatible with its predicted structure-based performance. In vitro MTT evaluation confirmed antiproliferative activity in MCF-7 cells, with IC50 values of 0.76, 0.75, and 1.83 µM for BCD1, BCD9, and BCD17, respectively. Although BCD1 and BCD9 showed slightly lower IC50 values, BCD17 was prioritized based on its integrated computational stability, apoptosis-related response, and preliminary selectivity profile. MCF-7 cell-cycle and Annexin V/PI apoptosis analyses further supported the antiproliferative and pro-apoptotic effect of BCD17, although direct 17β-HSD1 target engagement was not experimentally confirmed. In MCF-10A cells, BCD9 and BCD17 showed comparatively lower toxicity than 5-FU. Overall, BCD17 emerged as a promising integrated lead candidate for further optimization and biological validation.