Screening of novel narrow-spectrum benzofuroxan derivatives for the treatment of multidrug-resistant tuberculosis through in silico, in vitro, and in vivo approaches

Tuberculosis remains a serious global health threat, exacerbated by the rise of resistant strains. This study investigates the potential of two benzofuroxan (<i>Bfx</i>) derivatives, 5n and 5b, as targeted treatments for MDR-TB using <i>in silico</i>, <i>in vitro</i>, and <i>in vivo</i> methodologies. <i>In vitro</i> analyses showed that <i>Bfx</i> compounds have significant activity against <i>Mtb</i> H37Rv, with <i>Bfx</i> 5n standing out with a MIC₉₀ of 0.09 ± 0.04 μM. Additionally, their efficacy against MDR and pre-XDR strains was superior compared to commercial drugs. These <i>Bfx</i> compounds have a narrow spectrum for mycobacteria, which helps avoid dysbiosis of the gut microbiota, and they also exhibit high selectivity and low toxicity. Synergism studies indicate that <i>Bfx</i> derivatives could be combined with rifampicin to enhance treatment efficacy and reduce its duration. Scanning electron microscopy revealed severe damage to the morphology of <i>Mtb</i> following treatment with <i>Bfx</i> 5n, showing significant distortions in the bacillary structures. Whole-genome sequencing of the 5n-resistant isolate suggests resistance mechanisms mediated by the Rv1855c gene, supported by <i>in silico</i> studies. <i>In vivo</i> studies showed that the 5n compound reduced the pulmonary load by 3.0 log₁₀ CFU/mL, demonstrating superiority over rifampicin, which achieved a reduction of 1.23 log₁₀ CFU/mL. In conclusion, <i>Bfx</i> derivatives, especially 5n, effectively address resistant infections caused by <i>Mtb</i>, suggesting they could be a solid foundation for future therapeutic developments against MDR-TB.