5,2''-di-O-alkylated gentamicin C1a analogs potentiate rifampicin against multidrug-resistant gram-negative bacteria

O-alkylated gentamicin C1a analogs were prepared in 3-5 steps from gentamicin C1a using phase transfer-catalyzed O-alkylation. The O-alkylated gentamicin C1a analogs lost their standalone antibacterial activity but instead synergized the outer membrane-impermeable antibiotic rifampicin. 5,2''-di-O-alkylated-gentamicin C1a analogs 4 and 6 bearing biphenyl- or octyl- but not phenyl-substituents synergized rifampicin against a panel of Gram-negative bacteria. At concentrations of 8 μg/mL of 4 or 6 (~ 6 mmol), Gram-positive-selective antibiotic rifampicin was 32- to 512-fold potentiated against Gram-negative pathogens including multidrug-resistant Acinetobacter baumannii, Escherichia coli, and Pseudomonas aeruginosa isolates. Time-kill studies elucidated that the dual combination of 4 and rifampicin was bacteriostatic in A. baumannii AB031. The proposed mechanism of outer membrane permeabilization was verified by the reduced rifampicin potentiation in the presence of Mg²⁺, as well as the absence of activity and synergy with rifampicin in Gram-positive bacteria. These findings indicate that di-O-alkylated gentamicin C1a analogs 4 and 6 are two new members of aminoglycoside-based outer membrane permeabilizers capable of potentiating rifampicin against Gram-negative pathogens.