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.
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.
College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Republic of Korea (M.S., D.L., S.K., J.-S.B., T.L., S.L.); Doping Control Center, Korea Institute of Science and Technology, Chungryang, Seoul, Republic of Korea (J.L.); and Center for Innovative Drug Library Research, Department of Chemistry, College of Natural Science, Dongguk University, Seoul, Republic of Korea (Y.-D.G.).
Min
M
Song
College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Republic of Korea (M.S., D.L., S.K., J.-S.B., T.L., S.L.); Doping Control Center, Korea Institute of Science and Technology, Chungryang, Seoul, Republic of Korea (J.L.); and Center for Innovative Drug Library Research, Department of Chemistry, College of Natural Science, Dongguk University, Seoul, Republic of Korea (Y.-D.G.).
Doohyun
D
Lee
College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Republic of Korea (M.S., D.L., S.K., J.-S.B., T.L., S.L.); Doping Control Center, Korea Institute of Science and Technology, Chungryang, Seoul, Republic of Korea (J.L.); and Center for Innovative Drug Library Research, Department of Chemistry, College of Natural Science, Dongguk University, Seoul, Republic of Korea (Y.-D.G.).
Sun
S
Kim
College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Republic of Korea (M.S., D.L., S.K., J.-S.B., T.L., S.L.); Doping Control Center, Korea Institute of Science and Technology, Chungryang, Seoul, Republic of Korea (J.L.); and Center for Innovative Drug Library Research, Department of Chemistry, College of Natural Science, Dongguk University, Seoul, Republic of Korea (Y.-D.G.).
Jong-Sup
JS
Bae
College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Republic of Korea (M.S., D.L., S.K., J.-S.B., T.L., S.L.); Doping Control Center, Korea Institute of Science and Technology, Chungryang, Seoul, Republic of Korea (J.L.); and Center for Innovative Drug Library Research, Department of Chemistry, College of Natural Science, Dongguk University, Seoul, Republic of Korea (Y.-D.G.).
Jaeick
J
Lee
College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Republic of Korea (M.S., D.L., S.K., J.-S.B., T.L., S.L.); Doping Control Center, Korea Institute of Science and Technology, Chungryang, Seoul, Republic of Korea (J.L.); and Center for Innovative Drug Library Research, Department of Chemistry, College of Natural Science, Dongguk University, Seoul, Republic of Korea (Y.-D.G.).
Young-Dae
YD
Gong
College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Republic of Korea (M.S., D.L., S.K., J.-S.B., T.L., S.L.); Doping Control Center, Korea Institute of Science and Technology, Chungryang, Seoul, Republic of Korea (J.L.); and Center for Innovative Drug Library Research, Department of Chemistry, College of Natural Science, Dongguk University, Seoul, Republic of Korea (Y.-D.G.).
Taeho
T
Lee
College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Republic of Korea (M.S., D.L., S.K., J.-S.B., T.L., S.L.); Doping Control Center, Korea Institute of Science and Technology, Chungryang, Seoul, Republic of Korea (J.L.); and Center for Innovative Drug Library Research, Department of Chemistry, College of Natural Science, Dongguk University, Seoul, Republic of Korea (Y.-D.G.) sangkyu@knu.ac.kr.
Sangkyu
S
Lee
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods; D013844:Thiazoles / Q000737:chemistry* / Q000378:metabolism*
D020033:Protein Isoforms; D013844:Thiazoles; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human