(1) is a small-molecule which demonstrated a sub-nM inhibitory potency toward the histamine
H4 receptor (H4R). However, it was found to be mutagenic in an in vitro Ames assay.
Metabolic bioactivation of 1 could potentially arise from the piperazine moiety by forming reactive
intermediates such as glyoxal, aldehyde-imine and/or iminium ion, which could all lead to genotoxicity.
The aim of this study was to investigate bioactivation of 1 to determine the potential causes of the
genotoxicity and mitigate liabilities in this scaffold.
Methods: 1 was investigated for its genotoxicity in phenobarbital and β-naphthoflavone induced
Sprague Dawley rat liver S9 fractions. Trapping agents such as o-phenylenediamine was used postincubation.
Results: Following metabolic profiling of 1, two oxidative metabolites were observed and identified in
phenobarbital- and β -naphthoflavone induced Sprague Dawley rat liver S9 fractions. Metabolic pathway
of 1 was primarily mediated by the metabolism of the piperazine moiety. The trapped glyoxal
was identified by using high resolution LC-MS instrument. Structural characterization of the trapped
glyoxal was determined by comparison of retention time, accurate mass measurement and Collision
Induced Dissociation (CID) spectra to authentic standard.
Conclusion: In the present investigation, a novel method was developed to trap glyoxal, which may
potentially be liberated from piperazine moiety. These findings led to modifications on the piperazine
ring to mitigate the bioactivation pathways leading to mutagenicity. Subsequently, the next generation
compounds with modified piperazine moiety, retained H4R inhibitory potency in vitro and were not
genotoxic in the Ames mutagenicity assay.