yl ] butyl } quinazolin-4 ( 3 H )-one ( TZB-30878 ) , a Novel 5-Hydroxytryptamine ( 5-HT ) 1 A Agonist / 5-HT 3 Antagonist , in Human Hepatic Cytochrome P 450 Enzymes

3-Amino-5,6,7,8-tetrahydro-2-{4-[4-(quinolin-2-yl)piperazin-1-yl]butyl}quinazolin-4(3H)-one (TZB-30878), a novel 5-hydroxytryptamine (5-HT)1A agonist/5-HT3 antagonist, is currently under development for the treatment of irritable bowel syndrome. The objective of this investigation was to obtain information on the biotransformation of TZB-30878. This compound has quinazoline, piperazine, and quinoline rings. Metabolites of [quinazoline-2C]TZB-30878 were determined using radio high-performance liquid chromatography on samples obtained after incubation with human hepatic microsomes. Eight metabolites were detected in the microsomal incubation mixture, and their structures were proposed by mass spectrometry techniques using TZB-30878 and two stable labeled TZB-30878 analogs, [quinoline-deuterium (D)6]TZB30878 and [piperazin-D8]TZB-30878. Liquid chromatography/ tandem mass spectrometry analyses suggested that the eight metabolites consisted of a cyclic metabolite (M6), four hydroxylated metabolites (M1, M2, M3, and M4) (three on quinoline ring and one on quinazoline ring), a deaminated metabolite (M5), and two metabolites (M7 and M8) that were presumably intermediates leading to the formation of the cyclic metabolite M6. Hydroxylation sites in the quinoline and quinazoline rings were predicted by electron density calculations and confirmed by comparison with authentic standards. To the best of our knowledge, N-deamination by microsomes leading to the formation of M5 appears to be novel. In addition, in vitro experiments in human liver microsomes with cytochrome P450 (P450)-specific inhibitors revealed that CYP3A4 was the major enzyme responsible for the metabolism of TZB30878. Other P450 enzymes, such as a CYP2D6, played a minor role in its metabolism. Irritable bowel syndrome (IBS) is a common multifactorial disorder with a largely undefined etiology. Symptoms of IBS, such as abdominal pain and diarrhea, commonly develop and persist in patients who are in remission from other stress-related diseases. To develop a medication for patients with IBS, we focused on the properties of 5-hydroxytryptamine (5-HT)1A agonism and 5-HT3 antagonism and synthesized a novel compound, TZB-30878 (Fig. 1). This compound was expected to have anxiolytic action via 5-HT1A agonism and to suppress intestinal motility and visceral hypersensitivity via 5-HT3 antagonism. TZB-30878 was previously shown to exhibit potent and highly selective serotonin 5-HT3 receptor antagonistic activities and also a high affinity for the 5-HT1A receptor subtype that contributes to an overall therapeutic effect (Tamaoki et al., 2007). The pharmacokinetics and excretion of TZB-30878 have been studied in vivo in rats previously (unpublished data). The results indicated that TZB-30878 is rapidly absorbed following p.o. administration, distributed to almost all the organs including brain, and eliminated into feces via bile. However, there is very limited information regarding metabolism of TZB-30878. The objective of this study was to clarify the biotransformation of TZB-30878 in humans. We characterized the chemical structures of metabolites and identified the human hepatic cytochromes P450 (P450) responsible for the metabolism of TZB-30878. Materials and Methods Chemicals. Trifluoroacetic acid, Tris, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and ammonium acetate were purchased from Nacalai Tesque Inc. (Kyoto, Japan). -Naphthoflavone, sulfaphenazole, quinidine hydrochloride, and diethyldithiocarbamic acid sodium salt (diethyldithiocarbamate) were purchased from Sigma-Aldrich (St. Louis, MO). -NADP , Article, publication date, and citation information can be found at http://dmd.aspetjournals. org. doi:10.1124/dmd.107. 018168. ABBREVIATIONS: IBS, irritable bowel syndrome; 5-HT, 5-hydroxytryptamine; TZB-30878, 3-amino-5,6,7,8-tetrahydro-2-{4-[4-(quinolin-2yl)piperazin-1-yl]butyl}quinazolin-4(3H)-one; P450, cytochrome P450; G-6-P, d-glucose 6-phosphate disodium salt; D, deuterium; HPLC, highperformance liquid chromatography; LC, liquid chromatography; MS/MS, tandem mass spectrometry; M1, 3-amino-5,6,7,8-tetrahydro-2-{4-[4(6-hydroxy-quinolin-2-yl)piperazin-1-yl]butyl}quinazolin-4(3H)-one; M2, 3-amino-5,6,7,8-tetrahydro-2-{4-[4-(7-hydroxy-quinolin-2-yl)piperazin-1yl]butyl}quinazolin-4(3H)-one; M3, 3-amino-5,6,7,8-tetrahydro-2-{4-[4-(3-hydroxy-quinolin-2-yl)piperazin-1-yl]butyl}quinazolin-4(3H)-one; M4, 3-amino-6-hydroxy-5,6,7,8-tetrahydro-2-{4-[4-(quinolin-2-yl)piperazin-1-yl]butyl}quinazolin-4(3H)-one; M5, 5,6,7,8-tetrahydro-2-{4-[4-(quinolin-2yl)piperazin-1-yl]butyl}quinazolin-4(3H)-one; M6, 2-hydroxy-2,3,4,5,7,8,9,10-octahydro[1,2]diazepino[7,1-b]quinazolin-11(1H)-one. 0090-9556/08/3605-831–840$20.00 DRUG METABOLISM AND DISPOSITION Vol. 36, No. 5 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 18168/3324488 DMD 36:831–840, 2008 Printed in U.S.A. 831 at A PE T Jornals on Jne 6, 2017 dm d.aspurnals. org D ow nladed from