title: Structural characterization of in vitro metabolites of the new anticancer agent EAPB0503 by liquid chromatography-tandem mass spectrometry.
abstract: EAPB0503, belonging to the imidazo[1,2-a]quinoxaline series, is an anticancer drug with antitumoral activity against a variety of tumors. Previous studies have shown that this drug undergoes demethylation and oxygenation reactions. In this paper, liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was employed to assess the structures of unknown oxygenated metabolites of EAPB0503. EAPB0503 and its identified demethylated metabolites, EAPB0502 and EAPB0603, were incubated with human, rat, dog and mouse liver microsomes, as well as human, rat and dog hepatocytes. After separation on a C8 analytical column with a gradient elution of acetonitrile-formate buffer, positive ESI-MS/MS experiments were performed. To facilitate metabolite identification, the detailed fragmentation pathways of the parent compounds were first studied using high-resolution MS/MS. Additional hydrogen/deuterium exchange LC-MS/MS experiments were used to support the identification and structural characterization of metabolites. Four hydroxylated metabolites were identified: M'4 and its demethylated derivative M'1 (OH in ortho position on the phenyl substituent in position 1), and M'6 and its demethylated derivative M'3 (OH on the imidazole ring at the C2 position). Three phase II metabolites (Met A, EAPB0602 glucuronide; Met B, M'4 glucuronide; Met C, EAPB0603 glucuronide) were also evidenced. Elucidation of the metabolite structures was performed by comparing the chromatographic behaviors (changes in retention times), by measuring the molecular masses (mass increment), by studying the MS(2) spectral patterns of metabolites with those of parent drugs and for M'1 and M'4 by co-analysis with synthetic standards. The results of the present study provided important structural information relating to the metabolism of EAPB0503.
authors.affiliation: Clinical Pharmacokinetic Laboratory, EA4215, Faculty of Pharmacy, 15 Avenue Ch. Flahault, Montpellier I University, 34093 Montpellier Cedex 5, France.
authors.forename: Florian
authors.initials: F
authors.lastname: Lafaille
authors.forename: Isabelle
authors.initials: I
authors.lastname: Solassol
authors.forename: Christine
authors.initials: C
authors.lastname: Enjalbal
authors.forename: Benjamin
authors.initials: B
authors.lastname: Bertrand
authors.forename: Pierre-Emmanuel
authors.initials: PE
authors.lastname: Doulain
authors.forename: Johanna
authors.initials: J
authors.lastname: Vappiani
authors.forename: Pierre-Antoine
authors.initials: PA
authors.lastname: Bonnet
authors.forename: Carine
authors.initials: C
authors.lastname: Deleuze-Masquéfa
authors.forename: Françoise M M
authors.initials: FM
authors.lastname: Bressolle
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry*; D002478:Cells, Cultured; D002626:Chemistry, Pharmaceutical; D002853:Chromatography, Liquid; D041961:Deuterium Exchange Measurement; D004285:Dogs; D022781:Hepatocytes / Q000187:drug effects; D006801:Humans; D006900:Hydroxylation; D008862:Microsomes, Liver / Q000187:drug effects; D011810:Quinoxalines / Q000737:chemistry*; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D000970:Antineoplastic Agents; C555838:EAPB0503; D011810:Quinoxalines
keywords: Animal and human liver microsomes and hepatocytes; Anticancer agent; EAPB0503; LC-ESI-MS/MS; Metabolite identification