Research Article: Metabolite proving fungal cleavage of the aromatic core part of a fluoroquinolone antibiotic

Date Published: January 3, 2012

Publisher: Springer

Author(s): Heinz-Georg Wetzstein, Josef Schneider, Wolfgang Karl.


Liquid cultures of the basidiomycetous fungus Gloeophyllum striatum were employed to study the biodegradation of pradofloxacin, a new veterinary fluoroquinolone antibiotic carrying a CN group at position C-8. After 16 days of incubation, metabolites were purified by micro-preparative high-performance liquid chromatography. Four metabolites could be identified by co-chromatography with chemically synthesized standards. The chemical structures of three compounds were resolved by 1H-nuclear magnetic resonance spectroscopy plus infrared spectroscopy in one case. All metabolites were confirmed by high resolution mass spectrometry-derived molecular formulae. They comprised compounds in which the carboxyl group or the fluorine atom had been exchanged for a hydroxyl group. Furthermore, replacement of the CN group and the intact amine moiety by a hydroxyl group as well as degradation of the amine substituent were observed. The chemical structure of a catechol-type fluoroquinolone metabolite (F-5) could be fully defined for the first time. The latter initiated a hypothetical degradation sequence providing a unique metabolite, F-13, which consisted of the cyclopropyl-substituted pyridone ring still carrying C-7 and C-8 of pradofloxacin, now linked by a double bond and substituted by a hydroxyl and the CN group, respectively. Most likely, all reactions were hydroxyl radical-driven. Metabolite F-13 proves fungal cleavage of the aromatic fluoroquinolone core for the first time. Hence, two decades after the emergence of the notion of the non-biodegradability of fluoroquinolones, fungal degradation of all key structural elements has been proven.

Partial Text

Pradofloxacin (PRA), a new fluoroquinolone (FQ) drug, is used to treat bacterial infections in cats and dogs Litster et al. 2007;Mueller and Stephan 2007. It shares the core structure of common cyclopropyl-type FQs Domagala and Hagen 2003 but carries a cyano group at position C-8 and a bi-cyclic amine at C-7, S,S-pyrrolidinopiperidine ([1S,6S]-2,8-diazabicyclo[4.3.0]non-8-yl); the latter is also contained in moxifloxacin Petersen 2006. Concerning in vitro antibacterial activity, particularly low mutant prevention concentrations of PRA suggest a high potential for preventing the emergence of resistance under therapy Wetzstein 2005. Both substituents in combination are essential for its improved efficacy Wetzstein and Hallenbach 2011. Furthermore, the CN group facilitates hydrolytic elimination of the amine moiety (i.e., drug inactivation) under the slightly alkaline conditions present in decaying animal waste Wetzstein et al. 2009. Hence, PRA should be more readily biodegradable and thus ecologically favorable than conventional FQs.

The basic metabolic pathway of PRA, a new veterinary FQ antibacterial drug, in the brown rot fungus G. striatum was similar to schemes established for other FQs such as enrofloxacin, ciprofloxacin and moxifloxacin. Hydroxylated primary metabolites of PRA, each representing a different class of compounds (Figure 3), were generated by hydroxyl radical-based decarboxylation (F-1), defluorination (F-2) and elimination of CN (F-6). The definitive identification of a catechol-type FQ congener, compound F-5, carrying one hydroxyl group each at C-5 and C-6, is described here for the first time. This was facilitated by the CN substituent blocking C-8, in contrast to F-5 of enrofloxacin Wetzstein et al. 1997 or ciprofloxacin Wetzstein et al. 1999, for which hydroxylation of position C-5 was indistinguishable from hydroxylation of C-8. Degradation of the amine substituent is represented by F-9.

The authors are (HGW and JS) or have been (WK) employees of Bayer AG, as indicated.

HGW carried out the microbiological experiments and drafted the manuscript. JS determined the optimal experimental conditions and performed chemical analyses. WK guided structure elucidation and provided all interpretations of the chemical raw data. All authors read and approved the final manuscript.