Received for publication August 15, 2007.
Revised January 27, 2008.
Accepted for publication January 28, 2008.

Biotransformation of TZB-30878, a novel 5-HT_1A agonist/5-HT₃ antagonist, in human hepatic cytochrome P450 enzymes

Abstract

TZB-30878 (3-amino-5,6,7,8-tetrahydro-2-{4-[4-(quinolin-2-yl)piperazin-1-yl]butyl}quinazolin-4(3H)-one), a novel 5-HT_1A agonist/5-HT₃ antagonist, is currently under development for the treatment of irritable bowel syndrome (IBS). 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-2-¹⁴C]TZB-30878 were determined using radio-HPLC on samples obtained after incubation with human hepatic microsomes. Eight metabolites were detected in the microsomal incubation mixture and their structures were proposed by MS techniques using TZB-30878 and two stable labeled TZB-30878 analogs, [quinoline-D₆]TZB-30878, and [piperazin-D₈]TZB-30878. LC/MS/MS 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, which 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 were 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 CYP-specific inhibitors revealed that CYP3A4 was the major enzyme responsible for the metabolism of TZB-30878. Other cytochrome P450 enzymes, such as a CYP2D6 played a minor role in its metabolism.

Key words: cytochrome P450, HPLC, human CYP enzymes, liver microsomes, mass spectrometry, metabolite identification, microsomes, stable isotopes