RPR-106541

Cytochromes P450 3A4, 2D6, and 2C9 metabolize a large fraction of drugs. Knowing where these enzymes will preferentially oxidize a molecule, the regioselectivity, allows medicinal chemists to plan how best to block its metabolism. We present QSAR-based regioselectivity models for these enzymes calibrated against compiled literature data of drugs and drug-like compounds. These models are purely empirical and use only the structures of the substrates, in contrast to those models that simulate a specific mechanism like hydrogen radical abstraction, and/or use explicit models of active sites. Our most predictive models use three substructure descriptors and two physical property descriptors. Descriptor importances from the random forest QSAR method show that other factors than the immediate chemical environment and the accessibility of the hydrogen affect regioselectivity in all three isoforms. The cross-validated predictions of the models are compared to predictions from our earlier mechanistic model (Singh et al. J. Med. Chem. 2003, 46, 1330-1336) and predictions from MetaSite (Cruciani et al. J. Med. Chem. 2005, 48, 6970-6979).

RPR 106541 [20R-16α,17α-[butylidenebis(oxy)]-6α,9α-difluoro-11β-hydroxy-17β-(methylthio)androsta-4-en-3-one] is an airway-selective steroid developed for the treatment of asthma.Two metabolites produced by human liver microsomes were identified as the R- and S-sulfoxide diastereomers based on liquid chromatog./mass spectrometry anal., proton NMR, and cochromatog. with standardsSulfoxide formation was shown to be cytochrome P 450 (CYP) 3A4-dependent by correlation with CYP3A4-marker nifedipine oxidase activity, inhibition by cyclosporin A and troleandomycin, and inhibition of R- (70%) and S- (64%) sulfoxide formation by anti-3A antibody.Expressed CYP2C forms catalyzed RPR 106541 sulfoxidation; however, other phenotyping approaches failed to confirm the involvement of CYP2C forms in these reactions in human liver microsomes.Expressed CYP3A4 catalyzed the formation of the sulfoxide diastereomers in a 1:1 ratio, whereas CYP3A5 displayed stereoselectivity for formation of the S-diastereomer.The high rate of sulfoxidation by CYP3A4 and the blockade of oxidative metabolism at the electronically favored 6β-position provided advantages for RPR 106541 over other substrates as an active-site probe of CYP3A4.Therefore, oxidation of RPR 106541 by various CYP3A4 substrate recognition site (SRS) mutants was assessed.In SRS-4, mutants A305V and F304A gave dramatically reduced rates of R-diastereomer formation (83 and 64% decreases, resp.), but S-diastereomer formation was affected to a lesser extent.A370V (SRS-5) gave decreased formation of the R-sulfoxide (52%) but increased formation of the S-diastereomer.In the SRS-2 region, the most dramatic change in sulfoxide ratios was observed for L210A.In conclusion, the structure of RPR 106541 imposes specific constraints on enzyme binding and activity and thus represents an improved CYP3A4 probe substrate.