The effect of varying halogen substituent patterns on the cytochrome P450 catalysed dehalogenation of 4-halogenated anilines to 4-aminophenol metabolites

Abstract

The cytochrome P450 catalysed biotransformation of 4-halogenated anilines was studied in vitro with special emphasis on the dehalogenation to 4-aminophenol metabolites. The results demonstrated that a fluorine substituent at the C4 position was more easily eliminated from the aromatic ring than a chloro-, bromo- or iodo-substituent. HPLC analysis of in vitro biotransformation patterns revealed that the dehalogenation of the C4-position was accompanied by formation of non-halogenated 4-aminophenol, without formation of NIH-shifted metabolites. Changes in the apparent V maxfor the microsomal oxidative dehalogenation appeared to correlate with the electronegativity of the halogen substituent at C4, the fluorine substituent being the one most easily eliminated. A similar decrease in the rate of dehalogenation from a fluoro- to a chloro- to a bromo- to an iodo-substituent was observed in a system with purified reconstituted cytochrome P450 IIB1, in a tertiair butyl hydroperoxide supported microsomal cytochrome P450 system as well as in a system with microperoxidase 8. This microperoxidase 8 is a haem-based mini-enzyme without a substrate binding site, capable of catalysing cytochrome P450-like reaction chemistry. Together, these results excluded the possibility that the difference in the rate of dehalogenation with a varying C4-halogen substituent arose from a change in the contribution of cytochrome P450 enzymes involved in oxidative dehalogenation with a change in the halogen substituent. Rather, they strongly suggested that the difference was indeed due to an intrinsic electronic parameter of the various C4 halogenated anilines dependent on the type of halogen substituent. Additional in vitro experiments with polyfluorinated anilines demonstrated that elimination of the C4-fluorine substituent became more difficult upon the introduction of additional electron withdrawing fluorine substituents in the aniline-ring. 19F-NMR analysis of the metabolite patterns showed that the observed decrease in 4-aminophenol formation was accompanied by a metabolic switch to 2-aminophenols and N-hydroxyanilines, while products resulting from NIH-type mechanisms were not observed. For a C4chloro-, bromo-, or iodo-substituted 2-fluoroaniline the V max for the oxidative dehalogenation was reduced by the additional electron withdrawing fluorine substituent at the C2 position in a similar way. In conclusion, the results of the present study strongly indicate that the possibilities for cytochrome P450 mediated dehalogenation of 4-halogenated anilines to 4-aminophenol metabolites are dependent on: (i) the characteristics of the halogen that has to be eliminated, the most electronegative and smallest halogen being the one most easily eliminated, and (ii) the electron-withdrawing capacities of other substituents in the aromatic ring, electron-withdrawing substituents decreasing the relative rate of the reaction. Together these data lead to the conclusion that the halogen is eliminated as a halogen anion.