Metabolism of a G Protein-Coupled Receptor Modulator, Including Two Major 1,2,4-Oxadiazole Ring-Opened Metabolites and a Rearranged Cysteine-Piperazine Adduct

Abstract

ABSTRACT:Metabolites of a G protein-coupled receptor modulator containing 1,2,4-oxadiazole and piperazine substructures were identified in vitro in human, rat, and dog hepatocyte incubates and in vivo in rat plasma, bile, urine, and feces by using 14 C-radiolabeled parent compound. Exposure coverage for the major circulating metabolites in humans at steady state and in preclinical species used in drug safety assessments was determined by using pooled plasma samples collected from a human multiple ascending dose study and a 3-month rat toxicokinetic study. Metabolites M1 and M2, which were formed by opening of the 1,2,4-oxadiazole ring, were observed as major metabolites both in vitro and in vivo across species. The carboxylic acid metabolite M2 was presumably formed through reductive N-O bond cleavage of the oxadiazole ring and subsequent hydrolysis. However, the mechanism for the formation of the unusual N-cyanoamide metabolite M1 remains uncertain. Neither M1 nor M2 had any target activity, as did parent drug P. In rat bile, rearranged Cys-piperazine and Gly-Cys-piperazine adducts, involving the formation of a five-membered heteroaromatic imidazole derivative from a six-membered piperazine ring, were observed as minor metabolites. These findings support a previously reported mechanism regarding glutathione detoxification for piperazine bioactivation products.

Structural identification of imatinib cyanide adducts by mass spectrometry and elucidation of bioactivation pathway

“…Doss et al . and Gu et al . reported that the piperazine substructure in their drug candidates formed conjugated imine (α,β‐unsaturated) substructures that were trapped with GSH and subsequently underwent a ring‐contraction rearrangement.…”

“…Also noteworthy is the formation of M519C-M519F. With a carbonyl group on the ring, it would be highly likely to form conjugated imine (α,β-unsaturated) intermediates, such as imine amides [24] or similar structures to the reported reactive species, [20,21] which are also soft electrophiles. However, conjugation with the soft nucleophile GSH was not detected in the current work.…”

“…Although these biotransformation reactions are not typically associated with reactive intermediates, there are reports describing the identification of metabolites formed due to electrophilic addition with nucleophilic 'trapping' agents (GSH, KCN, methoxylamine, etc). Doss et al [20] and Gu et al [21] reported that the piperazine substructure in their drug candidates formed conjugated imine (α,β-unsaturated) substructures that were trapped with GSH and subsequently underwent a ring-contraction rearrangement. An earlier report by Kalgutkar et al [22] described metabolic activation of a drug candidate due to the formation of imine or nitrone intermediates from the piperazine ring substructure.…”

Piperazine derivatives as dangerous abused compounds

“…Similarities have been demonstrated between these metabolic pathways and the pathways described for piperazines. Gu et al (56) confirmed in their research a previously reported mechanism regarding glutathione (GSH) detoxification for piperazine bioactivation products. They have analysed unusual re-arranged Cys-piperazine (cysteine-piperazine) and Gly-Cys-piperazine (glycine-cysteine-piperazine) adducts in rat and human liver microsomes.…”

The impact of radiochemistry in drug projects: The use of C‐14 label in the AZD8529, AZD7325, and AZD6280 projects

“…Initial metabolic studies on a mGluR2 positive allosteric modulator, AZD8529, 8 using only LCMS‐based assays, completely missed a large metabolite present in human, rat, and dog liver microsomes ( AZD8529‐M1 , Scheme 1). 9 However, when the radiolabeled compound was incubated with hepatocytes in early development, the metabolite was observed. The conversion of an oxadiazole ring to a cyanamide is an unusual transformation but is precedented; similar transformations have been previously observed for L‐454560 10 and SM‐6586 11 .…”