Liabilities Associated with the Formation of “Hard” Electrophiles in Reactive Metabolite Trapping Screens

Abstract

Soft electrophiles (e.g., epoxides, quinones, quinone-imines, quinone-methides, etc.) generated via the oxidative bioactivation of phenyl, phenolic, amino-, and alkylphenolic substituents can be trapped with nucleophiles of comparable softness (e.g., glutathione or cysteine) in reactive metabolite screens. In contrast, hard nucleophiles such as cyanide and amines are frequently utilized to trap hard electrophiles (e.g., iminiums and aldehydes) that result from the oxidative bioactivation of cyclic (or acylic) amines and primary alcohols. In some instances, soft sulfydryl nucleophiles have also been utilized to trap aldehydes to yield cyclized thiazolidine adducts. Case studies where hard electrophiles are thought to be responsible for cytochrome P450 inactivation, genotoxicity, and/or target organ toxicity in animals have been presented. The association of hard electrophiles with immune-mediated idiosyncratic adverse drug reactions is less clear given the paucity of available examples and the fact that several marketed drugs containing cyclic amine motifs can generate hard electrophiles via α-carbon ring oxidation. This perspective examines available data associating toxicity with the formation of hard electrophilic intermediates from small molecule drugs/drug candidates. Pragmatic risk mitigation strategies around unwarranted idiosyncratic toxicity risks with drug candidates that generate hard electrophiles are also discussed against the backdrop of marketed agents that possess analogous cyclic amine framework.

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.…”

“…These settings enable identification of GSH adducts having no glutamyl‐dehydroalanyl‐glycine fragmentation. For example, glutathione conjugates hydrolyzed to other major products, such as cysteinyl‐glycine adducts due to the cleavage of the glutamyl residue; these adducts will not be observed in event 1 ; however, they will be captured and identified after examining spectra of neutral loss (NL) of dehydroalanyl‐glycine (144 Da) as shown in Figure .…”

“…An additional advantage of this method is the identification of uncommon GSH adducts. In a few reported cases, GSH adducts are rearranged and were not detected by using mass spectrometer settings based on CID pathways. As this GSH trapping assay incorporates a UV detector, this facilitates the identification of rearranged GSH adducts by combining information from UV peak and doublet mass peaks with 3 Da difference in HRMS.…”

Identifying the reactive metabolites of tyrosine kinase inhibitors in a comprehensive approach: Implications for drug‐drug interactions and hepatotoxicity

“…Avoiding structural alerts is common practice applied in the rational design of new chemical entities to prevent RM formation . As RM are not easily detected because of their high reactivity, RM screening using trapping agents such as soft and hard nucleophiles (glutathione [GSH], potassium cyanide [KCN], and methoxylamine) in metabolism studies and protein covalent binding in liver microsomal incubations are commonly used . RM‐positive derivatives are then subjected to additional medicinal chemistry modifications to redesign drug candidates with blocked or minimized bioactivation potential.…”

“…17,20 As RM are not easily detected because of their high reactivity, RM screening using trapping agents such as soft and hard nucleophiles (glutathione [GSH], potassium cyanide [KCN], and methoxylamine) in metabolism studies and protein covalent binding in liver microsomal incubations are commonly used. 30 RM-positive derivatives are then subjected to additional medicinal chemistry modifications to redesign drug candidates with blocked or minimized bioactivation potential. However, systematically removing "offending" moieties from drug candidates has recently been criticized, since the relationship between structural alerts and adverse effects is not clearcut.…”