Pharmacological characterization of 7-(4-(Piperazin-1-yl)) ciprofloxacin derivatives: antibacterial activity, cellular accumulation, susceptibility to efflux transporters, and intracellular activity.

Purpose: To evaluate pharmacological properties (antibacterial activity; accumulation in phagocytic cells; activity against intracellular bacteria; susceptibility to fluoroquinolone efflux transporters) of ciprofloxacin derivatives modified at C-7 of the piperazine ring.

Methods: N-acetyl- (1), N-benzoyl- (2), N-ethyl- (3), and N-benzyl- (4) ciprofloxacin were synthesized. MICs against Escherichia coli and Staphylococcus aureus were determined following CLSI guidelines. Cellular accumulation, subcellular distribution, and intracellular activity (towards S. aureus and Listeria monocytogenes) were determined in J774 mouse macrophages. Efflux in bacteria (NorA [S. aureus], Lde [L. monocytogenes]) and in macrophages (Mrp4) was assessed using the corresponding inhibitors reserpine and gemfibrozil, respectively.

Results: All derivatives were active, though less than ciprofloxacin. 2 and 3 accumulated 2-3 fold more than ciprofloxacin in mouse macrophages but remained substrates for efflux by Mrp4. 4 was insensitive to NorA and Lde, accumulated approx 50-fold more than ciprofloxacin in macrophages, was barely affected by Mrp4, localized in the soluble fraction of cells, and was equipotent to ciprofloxacin against intracellular bacteria.

Conclusions: Benzyl substitution at C7 markedly affects the pharmacological profile of ciprofloxacin with respect to recognition by efflux transporters and cellular accumulation. N-benzyl-ciprofloxacin may serve as basis for designing molecules with higher intrinsic activity while remaining poorly susceptible to efflux.