Analytical and Bioanalytical Chemistry

, Volume 403, Issue 1, pp 309–321 | Cite as

LC-MS/MS identification of the principal in vitro and in vivo phase I metabolites of the novel thiosemicarbazone anti-cancer drug, Bp4eT

  • Ján Stariat
  • Vít Šesták
  • Kateřina Vávrová
  • Milan Nobilis
  • Zuzana Kollárová
  • Jiří Klimeš
  • Danuta S. Kalinowski
  • Des R. Richardson
  • Petra Kovaříková
Original Paper

Abstract

The iron chelator, 2-benzoylpyridine-4-ethyl-3-thiosemicarbazone (Bp4eT), was identified as a lead compound of the 2-benzoylpyridine thiosemicarbazone series, which were designed as potential anti-cancer agents. This ligand has been shown to possess potent anti-proliferative activity with a highly selective mechanism of action. However, further progress in the development of this compound requires data regarding its metabolism in mammals. The aim of this study was to identify the main in vitro and in vivo phase I metabolites of Bp4eT using liquid chromatography tandem mass spectrometry (LC-MS/MS). Two metabolites were detected after incubation of this drug with rat and human liver microsomal fractions. Based on LC-MSn analysis, the metabolites were demonstrated to be 2-benzoylpyridine-4-ethyl-3-semicarbazone and N3-ethyl-N1-[phenyl(pyridin-2-yl)methylene]formamidrazone, with both resulting from the oxidation of the thiocarbonyl group. The identity of these metabolites was further shown by LC-MS/MS analysis of these latter compounds which were prepared by oxidation of Bp4eT with hydrogen peroxide and their structures confirmed by nuclear magnetic resonance and infrared spectra. Both the semicarbazone and the amidrazone metabolites were detected in plasma, urine, and feces after i.v. administration of Bp4eT to rats. In addition, another metabolite that could correspond to hydroxylated amidrazone was found in vivo. Thus, oxidative pathways play a major role in the phase I metabolism of this promising anti-tumor agent. The outcomes of this study will be further utilized for: (1) the development and validation of the analytical method for the quantification of Bp4eT and its metabolites in biological materials; (2) to design pharmacokinetic experiments; and to (3) evaluate the potential contribution of the individual metabolites to the pharmacodynamics/toxico-dynamics of this novel anti-proliferative agent.

Figure

a LC-MS chromatogram of the analysis of the sample from in vivo experiment, b proposed metabolic pathway of Bp4eT and c MS/MS fragmentation of the parent compound and metabolites M1-M3

Keywords

2-Benzoylpyridine-4-ethyl-3-thiosemicarbazone Bp4eT Anti-cancer Anti-tumor chemotherapeutic LC-MS 

Supplementary material

216_2012_5766_MOESM1_ESM.pdf (355 kb)
ESM 1(PDF 354 kb)

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Ján Stariat
    • 1
  • Vít Šesták
    • 1
  • Kateřina Vávrová
    • 2
  • Milan Nobilis
    • 1
  • Zuzana Kollárová
    • 1
  • Jiří Klimeš
    • 1
  • Danuta S. Kalinowski
    • 3
  • Des R. Richardson
    • 3
  • Petra Kovaříková
    • 1
  1. 1.Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy in Hradec KrálovéCharles University in PragueHradec KrálovéCzech Republic
  2. 2.Department of Inorganic and Organic Chemistry, Faculty of Pharmacy in Hradec KrálovéCharles University in PragueHradec KrálovéCzech Republic
  3. 3.Iron Metabolism and Chelation Program, Department of Pathology and Bosch InstituteUniversity of SydneySydneyAustralia

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