Second-Generation Anti-Tubercular Squaramides Targeting Complex V of the Respiratory Chain of Mycobacterium tuberculosis Displaying Enhanced Metabolic Stability

Squaramides (SQA) were reported as potent antituberculosis drugs through inhibition of the mycobacterial enzyme adenosine triphosphate (ATP) synthase, which is critical for ATP synthesis. However, squaramide compounds showed high metabolic clearance (CL) despite their promising potency and selectivity. Herein, we describe lead optimization efforts to improve the potency and metabolic stability of previous lead 1f. Multiple squaramide analogues exhibited improved potency. The most potent analogue 20j expressed enhanced potency of 51 nM and moderate metabolic stability. SQA 6k displayed optimum balance of potency and metabolic stability both in vitro and in vivo. Notably, against bedaquiline-resistant Rv0678 mutants, a modest 2-fold increase in 6k minimum inhibitory concentration (MIC) was observed─reversed by complementation─versus a 16-fold shift in bedaquiline (BDQ) MIC. In a chronic tuberculosis (TB) mouse model, 6k coadministered with 1-aminobenzotriazole (ABT) exhibited bactericidal activity. These findings provide key strategies for improving potency and pharmacokinetic properties toward a tractable preclinical candidate.