AZD7325 [4-amino-8-(2-fluoro-6-methoxyphenyl)-N-propylcinnoline-3-carboxamide] is a selective gamma-amino-butyric acid (GABA)A α2,3 receptor modulator intended for the treatment of anxiety through oral administration. We present an interesting metabolic cyclization and aromatization pathway leading to the tricyclic core of M9, i.e. 2-ethyl-7-(2-fluoro-6-methoxyphenyl)pyrimido[5,4-c]cinnolin-4(3H)-one. Further oxidative metabolism leads to M10 via demethylation and M42 via hydroxylation. The authentic standard of M9 has been recently synthesized to prove the novel structure of M9, as well as to verify the identification of M10 and M42 by liver microsomal incubation of the M9 standard. The aryl-aryl single bond of AZD7325 has restricted rotation thus atropisomers have been previously observed by chiral liquid chromatography. This had a direct consequence for the hydroxylation of the M9 ethyl group to M42: two diastereomers were isolated by typical reversed-phase liquid chromatography and preliminarily characterized by NMR. Either pure single diastereomer of M42 was fully converted to a 1:1 mixture after heating at 60 °C for 14 h. Metabolites M9, M10 and M42 were either minor or absent in plasma samples after a single dose, but all became major circulating metabolites after repeated doses in human and preclinical animals. The existence of these long-circulating metabolites - M9, M10, and M42 – was used to our advantage when anomalous exposure finding with a number of plasma samples occurred during a Phase-2b clinical study. The concentration of AZD7325 in these samples was unexpectedly below the lower limit of quantification which complicated data interpretation, but after examining the presence/absence of long-circulating metabolites, it was revealed that patient non-compliance was the cause of the anomalous exposure. The observation of late-occurring and long-circulating metabolites for AZD7325 demonstrates the need to collect plasma samples at steady state after repeated doses when conducting a MIST analysis. In this study, representative human plasma samples were obtained by pooling clinical multiple ascending dose (MAD) study samples collected over a 24 hours dosing interval after the final dose. Representative animal plasma samples were acquired by pooling toxicokinetic (TK) samples collected over a 24 hours dosing interval at the end of 3 month TK studies in dog, rat, and mouse. All 12 major oxidative metabolites of AZD7325 observed in the human MAD plasma were also found in dog, rat, and mouse TK plasma with greater exposure in the animals as compared to the human. This eliminated concern about human specific/disproportional metabolites, without performing an ADME study in healthy volunteers with 14C-labeled drug. The MIST analysis data successfully supported the regulatory approval for a special protocol assessment of carcinogenicity study in rodents and allow us to start the carcinogenicity study on time.