Explanation for Main Features of Structure–Genotoxicity Relationships of Aromatic Amines by Theoretical Studies of Their Activation Pathways in CYP1A2

Abstract

Aromatic and heteroaromatic amines (ArNH(2)) represent a class of potential mutagens that after being metabolically activated covalently modify DNA. Activation of ArNH(2) in many cases starts with N-hydroxylation by P450 enzymes, primarily CYP1A2. Poor understanding of structure-mutagenicity relationships of ArNH(2) limits their use in drug discovery programs. Key factors that facilitate activation of ArNH(2) are revealed by exploring their reaction intermediates in CYP1A2 using DFT calculations. On the basis of these calculations and extensive analysis of structure-mutagenicity data, we suggest that mutagenic metabolites are generated by ferric peroxo intermediate, (CYP1A2)Fe(III)-OO(-), in a three-step heterolytic mechanism. First, the distal oxygen of the oxidant abstracts proton from H-bonded ArNH(2). The subsequent proximal protonation of the resulting (CYP1A2)Fe(III)-OOH weakens both the O-O and the O-H bonds of the oxidant. Heterolytic cleavage of the O-O bond leads to N-hydroxylation of ArNH(-) via S(N)2 mechanism, whereas cleavage of the O-H bond results in release of hydroperoxy radical. Thus, our proposed reaction offers a mechanistic explanation for previous observations that metabolism of aromatic amines could cause oxidative stress. The primary drivers for mutagenic potency of ArNH(2) are (i) binding affinity of ArNH(2) in the productive binding mode within the CYP1A2 substrate cavity, (ii) resonance stabilization of the anionic forms of ArNH(2), and (iii) exothermicity of proton-assisted heterolytic cleavage of N-O bonds of hydroxylamines and their bioconjugates. This leads to a strategy for designing mutagenicity free ArNH(2): Structural alterations in ArNH(2), which disrupt geometric compatibility with CYP1A2, hinder proton abstraction, or strongly destabilize the nitrenium ion, in this order of priority, prevent genotoxicity.

Property-Based Design in the Optimisation of Benzamide Glucokinase Activators

“…This highlights the complex effects of multiple, small structural changes against multiple parameters and, in our view, confirms that predicting the best overall compound within a narrow region of chemical space remains a significant challenge, and underlines the continued importance of generating screening data throughout optimisation campaigns [26]. Through extensive profiling of the compounds identified by the above exercise, most notably in rat and dog pharmacokinetic studies and highfat-fed female Zucker rat OGTTs, compounds (29) and (30) were selected as the most promising candidates for further evaluation (Table 1.…”

“…Of more importance in the selection between (29) and (30) was consideration of the solid state characteristics of the compounds. During the course of this campaign, most compounds were isolated and tested in their amorphous form.…”

“…As part of this evaluation, and in preparation to develop the goals of a backup campaign, we undertook a full assessment of potential risks associated with the profile of AZD1092 (30). Two key issues were identified.…”

“…In this respect, we set a target for P app to be greater than 100 nm s À1 . Finally, we sought to achieve the above while matching or surpassing the technical profile of AZD1092 (30).…”

“…Moreover, fragments which would maintain features required for GK potency (such as 2-aminopyridine) were known to be inactive in the Ames test [30], giving confidence that suitable alternatives to 1-methyl-3-aminopyrazole could be found.…”