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

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-MS(n) analysis, the metabolites were demonstrated to be 2-benzoylpyridine-4-ethyl-3-semicarbazone and N (3)-ethyl-N (1)-[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.

Simultaneous determination of the novel thiosemicarbazone anti‐cancer agent, Bp4eT, and its main phase I metabolites in plasma: Application to a pilot pharmacokinetic study in rats

“…N 3 ‐ethyl‐ N 1 ‐[phenyl(pyridin‐2‐yl)methylene]formamidrazone was prepared according to a previously published procedure (Stariat et al ., ), with only a slight modification in the purification process. Briefly, hydrogen peroxide (0.5 mL; 30%) was added to 10 mL of a Bp4eT solution (50 mg/mL in ACN) and the mixture was stirred at laboratory temperature (20°C) for 1 h. The solvent was evaporated and N 3 ‐ethyl‐ N 1 ‐[phenyl(pyridin‐2‐yl)methylene]formamidrazone was separated using column chromatography using silica with hexane–chloroform–triethylamine (12:5:1, v/v/v) as the mobile phase.…”

“…1b), amidrazone (N 3 -ethyl-N 1 -[phenyl(pyridin-2-yl)methylene]formamidrazone, M2; Fig. 1c) and hydroxylated amidrazone metabolites were suggested (Stariat et al, 2012).…”

Preparation and structural determination of four metabolites of senkyunolide I in rats using ultra performance liquid chromatography/quadrupole-time-of-flight tandem mass and nuclear magnetic resonance spectra

“…The identification of the drug’s metabolites is indispensable in this process [19]. Liquid chromatography (LC) coupled with tandem mass spectrometry has become a powerful tool to study drug metabolism because of its superior sensitivity and specificity [20, 21].…”

Thiosemicarbazonep-Substituted Acetophenone Derivatives Promote the Loss of MitochondrialΔψ, GSH Depletion, and Death in K562 Cells

“…TSCs 3a–3e (Scheme 1) were obtained with a good yield after purification of the resulting reaction mixture by elution through a silica gel chromatography column. Compounds 3a–3e were characterized via the combined use of HRESIMS, 1D nuclear magnetic resonance (NMR) and 2D NMR, and subsequent comparison with data available in the literature [25–27]. The 1D NMR spectra of general structure 3 exhibited signals at δ H / δ C 2.23–2.33 (s, 3H)/13.7–13.9 from the methyl group; 4.97–5.00 (d, 2H)/48.4–48.8 from the methylene group; 7.85–7.04/165.4–115.7 from the aromatic rings; and δ H 8.73–8.82 and 7.86–8.22 from the NH groups; in addition, the signals δ C 143.3–147.4 and 178.3–178.7 originated from the C=N and C=S groups, respectively.…”