Characterization of a New Anticancer Agent, EAPB0203, and Its Main Metabolites: Nuclear Magnetic Resonance and Liquid Chromatography–Mass Spectrometry Studies

Abstract

The present study was conducted to assess the structures of the main unknown oxygenated metabolites of EAPB0203. The first step was to assign all the (1)H and (13)C NMR of both EAPB0203 and its demethylated metabolite (EAPB0202) to the corresponding atoms in their molecular structures and to elucidate the fragmentation pathways for the [M + H](+) ions of these compounds using high-resolution mass spectrometry (MS). MS/MS spectra showed that both protonated molecules possessing an even number of electrons were unexpectedly losing radicals such as H(•), CH(3)(•), or even C(7)H(7)(•) giving stable radical cations. In vitro metabolism studies were investigated in rat and dog liver microsomes and in the filamentous fungus Cunninghamella elegans. Structural elucidation of six oxygenated metabolites was performed based on the following: (i) their fragmentation pathways in liquid chromatography-MS/MS (LC-MS/MS) analyses; (ii) comparison of their changes in their molecular masses and fragment ions with those of the parent drugs; and (iii) the results of online H/D exchange experiments that provided additional evidence in differentiating hydoxylated metabolites from N-oxides. Structures of the metabolites were elucidated by LC-MS/MS and comparison with synthetic standards; structures of these standards were confirmed using one- and two-dimensional (1)H NMR spectroscopies.

<p>Metabolite Profiling in Anticancer Drug Development: A Systematic Review</p>

“…Most of the works in metabolite analysis were carried out using triple-quadrupole mass spectrometers. 28,39,55,57,63,82 The main advantages include its superior quantitative capabilities in the multiple reaction monitoring (MRM) mode and the fact that a family of metabolites can easily be identified using neutral-loss and precursor ion scans.…”

The synthesis ofN‐containing heterocyclic compounds catalyzed by copper/L‐proline

“…Quinoxalinone skeleton is a privileged core widely found in many biological and medicinal compounds. [ 57 ] In 2011, Balalaie and co‐workers reported an efficient protocol for the synthesis of indolo[1,2‐ a ]quinoxalinones 129 via CuI‐catalyzed cascade Ugi/Ullmann reaction of 2‐iodoaniline 125 , 2‐indole carboxylic acid 126 , aldehydes 127 , and isocyanides 128 (Scheme 22). [ 58 ] Although this method provided the products in good to excellent yields, and used commercially available materials as the reagents, the substrate scope of this reaction was not wide.…”

Online Simultaneous Hydrogen/Deuterium Exchange of Multitarget Gas-Phase Molecules by Electrospray Ionization Mass Spectrometry Coupled with Gas Chromatography

“…H/D exchange MS can be implemented in various ways with the most popular being to infuse a pure compound prepared in a deuterated solvent. 2,6 However, this method requires a process to separate and purify the target analyte from the sample. Meanwhile, an online H/D exchange MS method, which can directly analyze a sample, has also been reported.…”

“…Hydrogen/deuterium (H/D) exchange mass spectrometry (MS) is a widely used technique for identifying metabolites − or pharmaceutical impurities , in drug discovery and development processes. H/D exchange MS can be implemented in various ways with the most popular being to infuse a pure compound prepared in a deuterated solvent. , However, this method requires a process to separate and purify the target analyte from the sample. Meanwhile, an online H/D exchange MS method, which can directly analyze a sample, has also been reported.…”

“…This method can be implemented using both on-column and postcolumn techniques. On-column H/D exchange MS has been performed with a deuterated solvent as a mobile phase in liquid chromatography–mass spectrometry (LC–MS) − ,− or capillary electrophoresis mass spectrometry (CE-MS) . However, this method suffers from high consumption of an expensive deuterated solvent, such as D 2 O, and shifts in chromatographic retention times due to the different mobile phase–analyte–stationary phase interactions caused when two different mobile phases, such as H 2 O and D 2 O, are used.…”