Identification of Metabolites of N-(5-Benzoyl-2-(4-(2-Methoxyphenyl)piperazin-1-yl)thiazol-4-yl)pivalamid e Including CYP3A4-Mediated C-Demethylation in Human Liver Microsomes with High-Resolution/High-Accuracy Tandem Masss

Abstract

KRO-105714 [ N-(5-benzoyl-2-(4-(2-methoxyphenyl)perazin-1-yl)thiazol-4-yl)pivalamide] is a 2,4,5-trisubstituted 1,3-thiazole derivative that exerts anti-atopic dermatitis activity via robust suppression of the sphingosylphosphorylcholine receptor. This study used high-resolution/high-accuracy tandem mass spectroscopy (HRMS) and recombinant cDNA-expressed cytochrome P450 (P450) iso-forms to identify the metabolic pathway and metabolites of KRO-105714 in human liver microsomes (HLMs) as therapeutic agents for inflammation. The incubation of KRO-105714 with pooled HLMs in the presence of NADPH generated four metabolites (M1-M4). The metabolites were identified using HRMS and confirmed using synthetic standards for M2 and M4. M1 and M2 were identified as monohydroxylated metabolites, and M3 and M4 were identified as O-demethyl KRO-105714 and C-demethyl KRO-105714, respectively. In the inhibition study with selective CYP3A4 inhibitors and incubation in recombinant cDNA-expressed P450 enzymes, all the metabolites of KRO-105714 were formed by CYP3A4 in HLMs. The CYP3A4-mediated formation of M4 from M2 was confirmed via incubation of M2 in HLMs. These results showed that the unusual C-demethylated metabolite M4 was generated from mono-hydroxyl metabolite M2 via a CYP3A4-mediated enzymatic reaction in HLMs.