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 (IC 50 = 17.69 ± 0.6 μM and 18.79 ± 0.64 μM, respectively) compared with topotecan (IC 50 = 27.27 ± 0.93 μM) and showed higher selectivity toward cancer cells over PCS-800-017 normal cells. Both compounds induced G 0 /G 1 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 5c via 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. • Benzo[4,5]thieno[3,2- b ]pyran derivatives exhibit strong topoisomerase I inhibitory activity. • Benzo[4,5]thieno[3,2- b ]pyran derivatives induce G 0 –G 1 cell cycle arrest and apoptosis. • Benzo[4,5]thieno[3,2- b ]pyran derivatives activate of the cGAS–STING signaling pathway. • Benzo[4,5]thieno[3,2- b ]pyran derivatives enhance the expression of pro-apoptotic and immune-related markers.