The present work reports the synthesis of new bis-oxadiazole-bearing benzo[b]thiophene derivatives as well as assessments of their antimicrobial and antitubercular activity through both in vitro and in silico approaches. The novel benzo[b]thiophene-based oxadiazole derivatives (7a−7k) were synthesized via a three-step path involving the preparation of thioacetonitrile, followed by the conversion to acetimidamide, and finally cyclization with various functionalized benzoic acids. The structural elucidation of the prepared benzo[b]thiophene compounds was established by ¹H-NMR, ¹³C-NMR, and HRMS spectroscopic analysis. In vitro antimicrobial activity against Gram-(–ve), Gram(+ve) and fungal microorganisms and antitubercular effect on the H37Rv strain were assessed for each synthesized compound. It was revealed that compound 7f showed well-established antibacterial affinities on S. aureus and P. aeruginosa with zones of inhibition of 39.95±0.82 and 37.29±0.33 mm in comparison to the reference drug gemifloxacin (37.02±0.92 and 36.10±2.17 mm). Notably, compounds 7c and 7h displayed the highest antifungal activity among the tested compounds, with selectivity towards the C. albicans ZI values of 30.15±0.28 and 31.37±1.32 mm, with respects to CLZ (ZI = 29.36±1.15 mm). Among them, oxadiazole conjugates 7a (MIC = 5.80 μM), 7f (2.89 μM), and 7h (3.10 μM) showed potent anti-TB activity on the tested H37Rv compared to the conventional drug STM. Moreover, in silico molecular docking studies indicate that these conjugates strongly interrelate and bind with the C. albicans and M. tuberculosis active sites (PDB codes: 5FSA & 4FDN). Compound 7c showed the best docking score (−11.77 kcal/mol), followed by ligand 7f (−10.68 kcal/mol), with key H-bond interacting residues such as Gly⁷⁷(A), Ser⁷⁹(A), Arg⁷⁸(A), Thr¹⁴²(A), and Tyr⁴³⁵(A). Additionally, the synthesized compounds were screened for drug-likeness based on Lipinski’s rule of five and ADME-Tox parameters. Overall, the study identifies promising oxadiazole-based inhibitors and provides a valuable insight for further optimization and development of potential antimicrobial targets.

Keywords: Benzo[b]thiophene; 1,2,4-Oxadiazole; 1,3,4-Oxadiazole; Anti-TB; Antimicrobial; Computational Studies;