Design, synthesis and docking studies of new benzothiophene and benzothienopyran derivatives as topoisomerase I inhibitors with potential immunomodulatory action via cGAS-STING signaling pathway.

Cancer remains a major global health challenge, and the development of new therapeutic agents is urgently needed to overcome poor selectivity and multi-drug resistance. Targeting DNA replication enzymes such as topoisomerase I (Topo I), which regulates DNA topology during replication and transcription, represents an effective chemotherapeutic strategy. This study aimed to design and synthesize new benzothiophene and benzothienopyran derivatives as potential Topo I inhibitors with additional immunomodulatory activity. Four series of derivatives including benzothiophene (2a-c) and benzo[4,5]thieno[3,2-b]pyran (3a-c, 4a-c, and 5a-c) were synthesized, characterized, and biologically evaluated. In vitro cytotoxicity screening against the NCI-60 cancer cell panel demonstrated broad and potent activity, with several compounds showing 70 to >100% growth inhibition. The most active derivatives were further assessed in a five-dose NCI assay and a DNA relaxation assay for Topo I inhibition. Compounds 2c and 5c exhibited superior Topo I inhibition (IC50 = 17.69 ± 0.6 μM and 18.79 ± 0.64 μM, respectively) compared with topotecan (IC50 = 27.27 ± 0.93 μM) and showed higher selectivity toward cancer cells over PCS-800-017 normal cells. Both compounds induced G0/G1 cell cycle arrest and apoptosis in SR leukemia cells. Notably, compound 5c activated the cGAS-STING pathway and modulated apoptosis-related markers. Molecular docking studies rationalized the superior Topo I inhibitory potential of compound 5cvia forming strong interaction with the Topo I-DNA complex. Molecular dynamics (MD) simulation studies demonstrated remarkable positional stability of compound 5c with no significant structural perturbations in the Topo I active site. Additionally, drug-likeness and pharmacokinetic properties, predicted by SwissADME, indicated that 5c complies with Lipinski's and Veber's rules of oral bioavailability showing a bioavailability score of 0.55. These findings highlight compound 5c as a promising Topo I inhibitor with mechanistically validated multimodal anticancer activity, superior inhibitory potency compared with topotecan, and enhanced selectivity toward cancer cells over normal CD8+ cells, supporting its potential for further anticancer drug development.