Triprotic site-specific acid–base equilibria and related properties of fluoroquinolone antibacterials

The complete macro- and microequilibrium analyses of six fluoroquinolone drugs – ciprofloxacin, enrofloxacin, norfloxacin, pefloxacin, ofloxacin and moxifloxacin – are presented. Previous controversial literature data are straightened up, the protonation centers are unambiguously identified, and the protonation macro- and microconstant values are reported. The macroconstants were determined by 1H NMR-pH titrations while the microconstants were determined by a multi-modal spectroscopic-deductive methodology, in which methyl ester derivatives were synthesized and their NMR-pH titration data contributed to the evaluation of all the microconstants. The full 1H, 13C and 15N NMR assignments, NMR-pH profiles, macro- and microprotonation schemes and species-specific diagrams are included. Our studies show that the fluoroquinolones have three protonation centers: the carboxylate group, the N-1′ and N-4′ piperazine nitrogens and concentration of the uncharged microspecies is way below the values published earlier. The results could be well interpreted in terms of structural properties. The protonation macro- and microconstant values allow the pre-planned method development in techniques such as capillary zone electrophoresis and also, the interpretation of fluoroquinolone mechanism of biological action, including the pharmacokinetic properties, and antibacterial activities that are all heavily influenced by the states of protonation.

► The protonation centers of fluoroquinolone antibacterials are unambiguously identified.
► The protonation macro- and microconstants of fluoroquinolones were determined.
► The results could be well explained in terms of their structural properties.
► Protonation states of these drugs are crucial for binding of bacterial DNA gyrase.