Design, Synthesis, and Anticancer Assessment of Benzylated Pyrrole-Based Pyrido[2,3-d]Pyrimidines as Thymidylate Synthase Inhibitors.

Globally, colorectal cancer (CRC) is the second most common cause of cancer-related deaths and the third most common cancer. Thymidylate synthase (TS), a key enzyme involved in DNA biosynthesis, has emerged as a promising molecular target for anticancer therapy. In the present study, we designed and synthesized a series of 22 benzylated pyrrole-based pyrido[2,3-d]pyrimidines using Claisen Schmidt and Michael addition reactions, and evaluated their anticancer potential against four human cancer cell lines: HCT 116 (colorectal), A549 (lung), MCF-7 (breast), and MDA-MB-231 (triple-negative breast cancer) as well as for TS inhibitory potential. Compounds 1c and 2i exhibited potent TS inhibition with IC values of 11.50 ± 1.08 nM and 17.12 ± 0.91 nM, respectively, comparable to the standard drug raltitrexed (IC = 12.51 ± 0.91 nM). Molecular docking studies revealed stronger binding affinities of these compounds compared to raltitrexed, involving key interactions with the catalytic residue Cys195 of TS. Additionally, compounds 1c and 2i exhibited good stability in 300 ns molecular dynamics simulations along with acceptable drug-like properties and oral bioavailability. These findings suggest that compounds 1c and 2i are promising lead candidates for the development of TS inhibitors.