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

Abstract

KRO-105714 [N-(5-benzoyl-2-(4-(2-methoxyphenyl)piperazin-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) isoforms 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 monohydroxyl metabolite M2 via a CYP3A4-mediated enzymatic reaction in HLMs.

In vitro intestinal permeability studies, pharmacokinetics and tissue distribution of 6-methylcoumarin after oral and intraperitoneal administration in Wistar rats

“…These results suggest that demethylation is probably the primary biotransformation for 6MC in order to increase the polarity of the molecule. This demethylation reaction is reported for other compounds such as lobeglitazone (Song et al, 2014), and for the Ostol, a coumarinic compound where the demethylation has been described as a phase I metabolic reaction of (Yuan et al, 2009). Another peak (Rt = 11.8, [M+H]+ (m/z) 657.3) was observed, but was not possible to propose a structure, because the molecular weight is not comparable with the common metabolites of coumarin structure.…”

Role of Sphingosylphosphorylcholine in Tumor and Tumor Microenvironment

“…KRO-105714 [N-(5-benzoyl-2-(4-(2-methoxyphenyl)piperazin-1-yl)thiazol-4-yl)pivalamide] inhibits SPC-induced cell proliferation. It was discovered via high-throughput screening and had anti-inflammatory activity in atopic dermatitis [166].…”