Reactive Metabolite Trapping Screens and Potential Pitfalls: Bioactivation of a Homomorpholine and Formation of an Unstable Thiazolidine Adduct

Abstract

Successful early attrition of potential problematic compounds is of great importance in the pharmaceutical industry. The lead compound in a recent project targeting neuropathic pain was susceptible to metabolic bioactivation, which produced reactive metabolites and showed covalent binding to protein. Therefore, as a part of the backup series for this compound several structural modifications were explored to mediate the reactive metabolite and covalent binding risk. A homomorpholine containing series of compounds was identified without compromising potency. However, when these compounds were incubated with human liver microsomes in the presence of GSH, Cys-Gly adducts were identified, instead of intact GSH conjugates. This article examines the formation of the Cys-Gly adduct with AZX ([M+H]+ 486) as a representative compound for this series. The AZX-Cys-Gly-adduct ([M+H]+ 662) showed evidence of ring contraction by formation of a thiazolidine-glycine and was additionally shown to be unstable. During its isolation for structural characterization by 1H NMR spectroscopy, it was found to have decomposed to a product with [M+H]+ 446. The characterization and identification of this labile GSH-derived adduct using LC-MS/MS and 1H NMR are described, along with observations around stability. In addition, various structurally related trapping reagents were employed in an attempt to further investigate the reaction mechanism along with a methoxylamine trapping experiment to confirm the structure of the postulated reactive intermediate.

A high‐throughput glutathione trapping assay with combined high sensitivity and specificity in high‐resolution mass spectrometry by applying product ion extraction and data‐dependent neutral loss

“…Following LC‐UV analysis (Figure a) of samples from incubations conducted in the presence or absence of GSH, a major GSH adduct was identified with a mass increase of 190.0023 Da (Figure b), and the resulting MS 2 analysis was triggered by the isotope‐pattern default setting. Further structural analysis suggested that the GlyCys adduct was rearranged to form a thiazolidine adduct with a proposed structure shown in Figure c, comparable with a rearrangement that was previously reported in previous literatures . Overall, this assay successfully identified unusual GSH adducts and provided structure and semiquantitative information of glutathione adducts.…”

“…Although the above XoPI and DDA-NL will effectively identify most GSH adducts, in a few cases, structural rearranged GSH-adducts could escape identification as reported in the previous literatures 24,25 and would result in false negatives. Therefore, in our method, by employing LC-UV coupled with HRMS, even if both MS settings fail to identify GSH adducts, extra peaks in UV spectra in the presence of glutathione will be observed, and their m/z of GSH adducts appearing as unique isotopic doublet peaks will be obtained.…”

Methanol adducts leading to the identification of a reactive aldehyde metabolite of CPAQOP in human liver microsomes by ultra‐high‐performance liquid chromatography/mass spectrometry

“…The first involved GSH, the routine industry standard for trapping soft electrophilic reactive metabolites. GSH does not routinely adduct with aldehydes, though some literature data suggests this can occur . Saturated aldehydes generally require a nitrogen‐based trapping agent, such as methoxyamine, which forms a stable Schiff base adduct .…”

“…GSH does not routinely adduct with aldehydes, though some literature data suggests this can occur. [5] Saturated aldehydes generally require a nitrogen-based trapping agent, such as methoxyamine, which forms a stable Schiff base adduct. [6][7][8] The incubation of CPAQOP with GSH did not produce an adduct (data not shown); instead, only the methanol adducts were detected, confirming that the reactive intermediate was not a soft electrophilic species.…”

Detecting reactive drug metabolites for reducing the potential for drug toxicity

“…In some instances, in vitro reactive metabolite trapping assays with GSH can lead to false-negatives due to GSH adduct degradation and/or intramolecular rearrangement reactions occurring during incubations leading to modified cyclized adducts not recognized by LC--MS/MS assays since diagnostic fragmentation patterns are no longer useful [65,66]. This has been shown, for example, in studies on the bioactivation of a 1,3-disubstituted piperazine selective melanocortin receptor subtype-4 agonist, MB243 ( Figure 3) [65].…”