Late-occurring and Long-circulating Metabolites of GABA_Aα2,3 Receptor Modulator AZD7325 Involving Metabolic Cyclization and Aromatization: Relevance to MIST Analysis and Application for Patient Compliance

Abstract

AZD7325 [4-amino-8-(2-fluoro-6-methoxyphenyl)-N-propylcinnoline-3-carboxamide] is a selective GABA Aa2,3 receptor modulator intended for the treatment of anxiety disorders through oral administration. An interesting metabolic cyclization and aromatization pathway led 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 generated M10 via O-demethylation and M42 via hydroxylation. An authentic standard of M9 was synthesized to confirm the novel structure of M9 and that of M10 and M42 by liver microsomal incubation of the M9 standard. Metabolites M9, M10, and M42 were either minor or absent in plasma samples after a single dose; however, all became major metabolites in human and preclinical animal plasma after repeated doses and circulated in humans longer than 48 hours after the end of seven repeated doses. The absence of these long circulating metabolites from selected patients' plasma samples was used to demonstrate patient noncompliance as the cause of unexpected lack of drug exposure in some patients during a Phase IIb outpatient clinical study. The observation of late-occurring and longcirculating metabolites demonstrates the need to collect plasma samples at steady state after repeated doses when conducting metabolite analysis for the safety testing of drug metabolites. All 12 major nonconjugate metabolites of AZD7325 observed in human plasma at steady state were also observed in dog, rat, and mouse plasma samples collected from 3-month safety studies and at higher exposures in the animals than humans. This eliminated concern about human specific or disproportional metabolites.

Cinnoline Scaffold—A Molecular Heart of Medicinal Chemistry?

“…In addition, the diverse metabolite profile of AZD7325 was investigated [61]. In vivo studies in rat and in vitro studies in human, rat, mouse, rabbit and dog liver microsomes were performed with radiolabeled AZD7325, revealing approximately 40 metabolites [61,62].…”

Synthesis of C‐14 labeled GABAA α2/α3 selective partial agonists and the investigation of late‐occurring and long‐circulating metabolites of GABAA receptor modulator AZD7325

“…The rat metabolite profiles (for instance the plasma metabolite profile shown in Figure ) and the resulting biotransformation scheme (shown in Figure ) were determined using [ 14 C] AZD7325 , highlighting the immense value of radio‐labeling for DMPK studies. The full characterization of these metabolites is discussed by Gu et al, noting that M9 , M10 and M42 (cf. Figure ) were only seen in trace or minor amounts in vitro or in vivo after a single dose; however, that all 3 became major circulating metabolites after repeated oral doses .…”

“…The full characterization of these metabolites is discussed by Gu et al, noting that M9 , M10 and M42 (cf. Figure ) were only seen in trace or minor amounts in vitro or in vivo after a single dose; however, that all 3 became major circulating metabolites after repeated oral doses . Furthermore, this work includes the detailed investigations which led to the proposed tricyclic structure for M9, and thus also for M10 and M42 , formed via cyclisation and successive aromatization (cf.…”

“…The synthetic standard was used to unambiguously confirm the structure of M9 using high resolution ultra‐performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS). In addition, the authentic standard was used to verify M10 and M42 by incubation of the standard in human liver microsomes …”

“…In addition, the authentic standard was used to verify M10 and M42 by incubation of the standard in human liver microsomes. 29…”