Synthesis, biological evaluation, and mechanistic investigation of tetrahydrothieno[2,3-c]pyridine derivatives as anti-Gram-negative bacterial agents

The escalating threat of drug-resistant Gram-negative bacterial infections necessitates urgent development of novel therapeutics. We reported the design, synthesis, and evaluation of a series of tetrahydrothieno[2,3-c]pyridine derivatives derived from the hit compound IMB-H4. Five analogues demonstrated potent in vitro activity against Escherichia coli (E. coli ATCC 25922), Acinetobacter baumannii (A. baumannii ATCC 19606), Klebsiella pneumoniae (K. pneumoniae BAA 1706), and the clinical isolates of multidrug-resistant (MDR). The optimized compound 5 demonstrated 2- to 8-fold enhanced potency with reduced cytotoxicity, yielding a superior selectivity index (SI) than IMB-H4, and could significantly prolong survival time and improve survival rate of E. coli-infected G. mellonella larvae and ICR mice in vivo. Compound 5 induced outer membrane (OM) damage and the formation of filamentous cells in E. coli. Mechanistic studies revealed that it could bind to the unfolded BamA protein to inhibit its interaction with BamD, it could also bind to the folded BamA protein to affect its function, thereby affecting the assembly of outer membrane proteins (OMPs) on OM. In addition, compound 5 also perturbed the integrity of cell walls, correlating with filamentous morphology, though the precise target(s) remain unidentified. Collectively, this series of derivatives represents a promising new class of multi-target antibiotics with a unique polypharmacological profile, worth further development.