Enzymatic Defluorination of a Terminally Monofluorinated Pentyl Moiety: Oxidative or Hydrolytic Mechanism?

Fluorination of organic compounds plays an important role in the chemical and pharmaceutical industry and is often applied to improve physicochemical parameters or modify pharmacological properties. Although oxidative and reductive defluorination have been shown to be responsible for the metabolic degradation of organofluorine compounds, the involvement of hydrolytic mechanisms catalyzed by human enzymes has not been reported so far. Here, we investigated the enzymatic defluorination of terminally monofluorinated aliphates with [1-(5-fluoropentyl)-1H-indol-3-yl]-1-naphthalenyl-methanone (AM-2201) as a model substance. We performed in vitro biotransformation using pooled human liver microsomes (pHLM) and human recombinant cytochrome P450 (P450) assays. To elucidate the underlying mechanisms, modified incubation conditions were applied, including the use of deuterium-labeled AM-2201 (d₂-AM-2201). Identification of the main metabolites and analysis of their isotopic composition was performed by liquid chromatography coupled to time-of-flight mass spectrometry. Quantification of the metabolites was achieved with a validated method based on liquid chromatography–tandem mass spectrometry. CYP 1A2–mediated defluorination of d₂-AM-2201 revealed an isotopic pattern of the defluorinated 5-hydroxypentyl metabolite (5-HPM), indicating a redox mechanism with an aldehyde as a plausible intermediate. In contrast, formation of 5-HPM by pHLM was observed independently of the presence of atmospheric oxygen or cofactors regenerating the redox system. pHLM incubation of d₂-AM-2201 confirmed the hypothesis of a nonoxidative mechanism involved in the defluorination of the 5-fluoropentyl moiety. So far, enzymatically catalyzed, hydrolytic defluorination was only described in bacteria and other prokaryotes. The presented data prove the involvement of a hydrolytic mechanism catalyzed by human microsomal enzymes other than P450.

SIGNIFICANCE STATEMENT

Elucidating the mechanisms involved in the enzymatic detoxification of organofluorine compounds is crucial for enhancing our understanding and facilitating the design and development of drugs with improved pharmacokinetic profiles. The carbon-fluorine bond possesses a high binding energy, which suggests that nonactivated fluoroalkanes would not undergo hydrolytic cleavage. However, our study provides evidence for the involvement of a nonoxidative mechanism catalyzed by human liver enzymes. It is important to consider cytochrome P450–independent, hydrolytic defluorination when investigating the pharmacokinetic properties of fluorinated xenobiotics.