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Imidazopyridine holds significance as a crucial fused bicyclic heterocycle within the realm of medicinal chemistry, being acknowledged as a “privileged scaffold” owing to its extensive utility. Numerous methodologies for the synthesis of imidazo[1,2-a]pyridines have been documented, with a considerable focus on strategies aimed at functionalizing these compounds. This study aimed to explore the potential of novel imidazo[1,2-a] pyridine derivatives as agents against drug-resistant Mycobacterium tuberculosis, which poses significant challenges in tuberculosis (TB) treatment. The research involved the synthesis of substituted imidazo[1,2-a]pyridine derivatives using site identification and molecular docking techniques. Characterization of the synthesized compounds was carried out using FT-IR, LC-MS, 1H NMR, and 13C NMR analysis. Compounds 6a and 6k showed good anti-TB activity against the H37Rv strain of Mycobacterium tuberculosis, with MIC values of 0.6 μM and 0.9 μM, respectively, which were comparable with the standard drug isoniazid. These findings suggest that imidazo[1,2-a]pyridine derivatives, especially compounds 6a and 6k, have potential as agents against drug-resistant TB, providing valuable insights for ongoing efforts in developing effective TB treatments.