The most potent compounds within the

The most potent compounds within the current series of compounds were therefore , , , , and , with ESI-09 possessing the best selectivity towards the lyase reaction in comparison to the hydroxylase reaction, indeed, this compound was found to possess an IC value of 1210nM against the 17α-OHase component and 30nM against the lyase component. Compound showed the most potent inhibitory activity with an IC value of 100nM against the 17α-OHase component and 10nM against the lyase component. In comparison, KTZ was found to possess good selectivity but weaker inhibitory activity in comparison to compounds to . A detailed consideration of the IC values shows that compounds to possess potent inhibitory activity against the lyase component with an optimum activity being possessed by the ethyl (compound ) and phenyl (compound ) derivatives, whereas the butyl and the pentyl derivatives appear to possess weaker inhibitory activity. Since no crystal structure exists for P450, we developed a novel technique to model this enzyme using the substrate–haem complex approach. The methodology for the derivation of the substrate–haem complex has been previously described,, , as such, no detailed discussion is given here. In general, however, the determination of the overall representation of the P450 active site initially involved the derivation of the binding site corresponding to the 17α-OHase component using pregnenolone bound to the haem moiety—the substrate was allowed to minimise to give the approximate position of the hydrogen bonding group which would be expected to bind to the C(3) moiety within the substrate. The structure was ‘locked’ after the removal of the pregnenolone and the location of the second binding site (corresponding to the lyase component) was then determined using 17α-hydroxypregnenolone as the substrate and the structure again minimised. The haem and the two hydrogen bonding groups at the active site were then locked and 17α-hydroxypregnenolone removed to give the overall substrate–haem complex for P450. That the active site consists of two lobes where the two substrates for this enzyme bind to (at different times) was previously proposed by Laughton et al. who found that the two ‘lobes’ were orientated in such a manner so as to approximate to the L-shape with the lyase binding site being larger than the 17α-OHase binding site. The substrate–haem complex approach also shows the lyase binding site to be the larger of the two and that the overall orientation of the two hydrogen bonding groups appears to be of a similar orientation as reported by Laughton et al. (with respect to the haem). Inhibitors of P450 can also utilise these two hydrogen bonding groups and we have previously reported the use of both groups by inhibitors (such as 3,5-dichlorobenzyl imidazole) which resulted in the di-substituted compounds possessing potent inhibitory activity in comparison to mono-halogenated compounds. Modelling of compound using the substrate–haem complex approach shows that this compound is able to utilise either of the two potential hydrogen bonding groups, albeit one at a time (). However, from the consideration of the conformational analysis of the alkyl moiety within shows that conformers exist which allow the alkyl chain to undergo steric interaction with the hydrogen bonding group at the active site. We therefore propose that the increase in the steric interactions results in an overall reduction in inhibitory activity when compared to compounds containing smaller alkyl chains, for example, compound . Our hypothesis is further supported by the inhibitory activity observed within compounds and , which contain CF and F as the substituents, respectively, rather than the alkyl chain. These two compounds are hypothesised to occupy reduced conformational space, as such, they do not undergo the level steric interactions observed within compounds to . Modelling of compound within the overall enzyme complex shows that the CF moiety is far removed from the second hydrogen bonding group at the active site to undergo any steric interaction, indeed, the nearest atom is 5.7Å away from the hydrogen bonding group. Modelling the more bulky derivative of (more specifically compound , ) within the substrate–haem complex shows that as a result of the greatly reduced conformational flexibility of this inhibitor, the additional phenyl moiety is unable to undergo any steric interaction with the active site and therefore possesses greater inhibitory activity in comparison to .