Aldehyde oxidase (AOX) is a metabolic enzyme catalyzing the oxidation of aldehyde and aza-aromatic compounds and the hydrolysis of amides, moieties frequently shared by the majority of drugs. Despite its key role in human metabolism, to date only fragmentary information about the chemical features responsible for AOX susceptibility are reported and only "very local" structure-metabolism relationships based on a small number of similar compounds have been developed. This study reports a more comprehensive coverage of the chemical space of structures with a high risk of AOX phase I metabolism in humans. More than 270 compounds were studied to identify the site of metabolism and the metabolite(s). Both electronic [supported by density functional theory (DFT) calculations] and exposure effects were considered when rationalizing the structure-metabolism relationship.aldehyde oxidase | amide hydrolysis | site of metabolism | variously decorated heterocycles | structure-metabolism relationship I n recent years, the cytochromes P450 (CYP450)-mediated metabolism has been intensively studied. As a consequence, the development of new and more efficient in silico and in vitro screening systems contributed to decrease the discontinuation rates of new drugs in clinical studies by 10% in recent years (1). In particular, several models for in silico prediction of human CYP450-mediated metabolism have been reported so far (2-4) and, more recently, for human FMO3 (5). Nevertheless, a deeper understanding of non-CYP drug enzyme metabolism is advisable to further improve screening and models efficacy (6). Among all non-CYP metabolic enzymes (7), emerging importance has been attributed to human aldehyde oxidase (hAOX), a cytosolic drugmetabolizing enzyme expressed in human liver that, similarly to CYPs, contributes to new chemical entities' oxidation, but acting in the absence of NADPH cofactor. Drugs that are substrates for AOX often exhibit high metabolic clearance, resulting in low exposure and hence in decreased drug efficacy in humans. In particular, AOX catalyzes the oxidation of a wide range of azaaromatic scaffolds at the unsubstituted carbon in ortho to the nitrogen (SI Appendix, Fig. S1 A and B), usually the most electron deficient (8). Several marketed drugs are well-known substrates of AOX (e.g., methotrexate, famciclovir, and zaleplon) (9), and several compounds have failed due to undetected AOX oxidation (e. g., BIBX1382, RO-1, FK3453, and carbazeran) (10-14). The role of AOX in drug development has become more relevant in the last few years as a result of organic synthesis strategies designed to reduce cytochromes P450-related metabolism, based on chemical modifications and decorations that have subsequently increased the drug reactivity toward AOX (11,15). In addition, AOX was recently found to be responsible for the rapid hydrolysis of an amide bond in GDC-0834 (SI Appendix, Fig. S1C), a potent inhibitor of Bruton's tyrosine kinase (16). Although the development of structure-metabolism relationships (SMRs) for hAOX ...
