Synthesis, in vitro biological evaluation and in silico studies of novel pyrrolidine derived thiosemicarbazones as dihydrofolate reductase inhibitors†

Dihydrofolate reductase (DHFR) is a crucial enzyme involved in folate metabolism and serves as a prime target for anticancer and antimicrobial therapies. In this study, a series of 4-pyrrolidine-based thiosemicarbazones were synthesized and evaluated for their DHFR inhibitory activity. The synthesis involved a multistep procedure starting from readily available starting materials, leading to the formation of diverse thiosemicarbazone 5(a–r) derivatives. These compounds were then subjected to in vitro assays to evaluate their inhibitory potential against DHFR enzyme. The synthesized compounds 5(a–r) exhibited potent inhibition with IC₅₀ values in the range of 12.37 ± 0.48 μM to 54.10 ± 0.72 μM. Among all the derivatives 5d displayed highest inhibitory activity. Furthermore, molecular docking and ADME studies were performed to understand the binding interactions between the synthesized compounds and the active site of DHFR. The in vitro and in silico data were correlated to identify compounds with promising inhibitory activity and favorable binding modes. This comprehensive study provides insights into the structure–activity relationships of 4-pyrrolidine-based thiosemicarbazones as DHFR inhibitors, offering potential candidates for further optimization towards the development of novel therapeutic agents.