INFLUENCE OF SUBSTITUENTS IN FLUOROBENZENE DERIVATIVES ON THE CYTOCHROME P450-CATALYZED HYDROXYLATION AT THE ADJACENT ORTHO AROMATIC CARBONCENTER

In a previous study, the in vivo cytochrome P450-catalyzed regioselect ivity of aromatic ring hydroxylation for a series of (poly)fluorobenze nes could be quantitatively predicted by the calculated frontier orbit al density distribution in the aromatic ring [Rietjens et al. (1993) B iochemistry 32, 4801-4812]. However, the relative small fluorine, its size almost comparable to a hydrogen, is not expected to influence the regioselectivity of aromatic hydroxylation due to steric hindrance. T he aim of the present study was to investigate the influence of substi tuents larger than a hydrogen or fluorine on the possibilities for hyd roxylation at the adjacent carbon center. First, the in vivo regiosele ctivity of aromatic ring hydroxylation of a series of C4-substituted f luorobenzenes was investigated. The results obtained demonstrate that a chlorine and cyano C4 substituent do not hamper hydroxylation at the positions ortho to the C4 carbon atom. For 4-chloro- and 4-cyanofluor obenzene, the observed regioselectivity of aromatic hydroxylation corr elated with the regioselectivity predicted on the basis of the frontie r orbital density distribution. In contrast, a bromine and iodine subs tituent affected the hydroxylation at the adjacent ortho carbon center s, reducing it to respectively 40 and 6% of the calculated intrinsic r eactivity of the carbon centers. Additional experiments were performed to investigate whether the regioselectivity of the aromatic hydroxyla tion of the C4-substituted fluorobenzene model compounds was influence d by changes in the cytochrome P450 enzyme pattern. Results obtained d emonstrate that for these relatively small substrates the regioselecti vity of their hydroxylation was not significantly influenced by severa l cytochrome P450 inducers. This suggests that the active sites of the cytochromes P450 catalyzing the aromatic hydroxylation do not impose a stereoselective orientation of the aromatic rings with respect to th e iron-ore porphyrin reaction center. Thus, the working hypothesis for additional experiments was that the deviations for the regioselectivi ty of aromatic hydroxylation observed for 4-bromo- and 4-iodofluoroben zene may be ascribed to steric hindrance by the bromine and iodine sub stituents hampering the attack of the cytochrome P450 iron-ore species on the adjacent carbon centers in the benzene derivative. This workin g hypothesis was further tested by investigating whether useful steric correction factors could be derived from the results obtained with th e series C4-substituted fluorobenzenes. These correction factors shoul d make it possible to correct calculated relative reactivities of carb on sites for steric hindrance by substituents positioned ortho with re spect to the carbon to be hydroxylated. This will make it possible to better explain and predict the regioselectivities for other chlorine-, bromine-, iodine-, and cyano-containing fluorobenzenes. The in vivo r egioselectivity of aromatic ring hydroxylation of a series of five chl orine-, bromine-, iodine-, or cyano-containing fluorobenzenes did not correlate with the noncorrected calculated reactivities (r = 0.49). Ho wever, upon correction of the calculated reactivity values by using th e steric correction factors, a correlation between the observed and ca lculated regioselectivity for the substrates of the present study was obtained (r = 0.91). Together these results strongly indicate that for the fluorobenzenes studied the main factors directing the regioselect ivity of their aromatic hydroxylation are (i) the nucleophilic chemica l reactivity of the site to be hydroxylated and (ii) the steric influe nce of the substituent ortho with respect to the site of hydroxylation . This latter effect appears to be negligible for a fluorine, chlorine , and cyano substituent but significant for a bromine and iodine subst ituent.