Cytochrome P-450 mediated N-dealkylation

• Main Author: Jones, Barry Christopher
• Format: Dissertation
• Language: English
• Published: University of Surrey 1989
• Subjects: Biochemistry

An investigation into the physico-chemical and molecular mechanical properties of tertiary amines which predispose them to rapid metabolism by cytochrome P-450 has been undertaken. A series of tertiary amines based on the common 2-(2,4-dichlorophenoxy)-N-methylethanamine moiety was synthesised with different substituent groups which changed the lipophilicity, size, polarity and electronic configuration. The substituent groups employed included n-alkyl, branched alkyl, aromatic, unsaturated, and oxygenated moieties. The design of the molecules was centred around an N~methyl group which was shown to be the major site of metabolism in a selected series of the molecules. The rates of N-demethylation were used as the index by which the effect of changing the substituent group was assessed. Studies using chemical inducers and inhibitors of cytochrome P-450 indicated that the hepatic microsomal fraction obtained from rats that had been pretreated with phenobarbital was the most effective biological system for the study of the rate of metabolism of these compounds. Further investigations using purified cytochrome P450 IIB4 in a reconstituted system showed that this particular isozyme of cytochrome P-450 was capable of supporting the metabolism of these compounds indicating that in the rat hepatic microsomes it was cytochrome P450 IIB1 which was mediating the compounds' metabolism. Various physico-chemical properties including lipophilicity, pKa and the octanol/water partition coefficient at pH 7.4 (log D) were determined for each of the compounds. The X-ray crystal structures of two of the molecules were solved to provide co-ordinates that were used in the determination of various molecular mechanical parameters such as the partial charges on the atoms in the molecules. The physico-chemical parameters were correlated with biological parameters obtained from both hepatic microsomal and purified cytochrome P450 IIB4 in a reconstituted system. The biological parameters determined were the binding affinity (K3, K3 and K4), the substrate-induced cytochrome P-450 spin state change (% HS and K2), Vmax and Km. No correlation was found between these parameters derived from the different biological systems. This may have been a result of changes in the tertiary structure of the enzyme brought about- as a result of the purification process or it may have been due to differences in the primary structures of cytochrome P450 IIB1 and IIB4. Quantitative structure-activity relationships derived between these sets of parameters for the microsomal system indicated that increasing size and lipophilicity produced stronger binding of the substrate to the enzyme, however, this gave no information about the orientation of the molecule in the enzyme's active site. A correlation was found for Km which indicated that the electronic structure of the molecules may influence the orientation that the molecule adopts in the active site of the enzyme. No correlations were found for Vmax, however the ratioVmax/Km produced a number of correlations which indicated that this parameter was dependent on the electron density on the nitrogen atom and the electronic influence of the substituent group on the nitrogen atom. Very few correlations were found for the biological parameters determined using purified cytochrome P450 IIB4 in a reconstituted system. Those that were obtained went a little way in supporting the suggestion that compounds bind to cytochrome P-450 via a hydrophobic interaction.