Medicinal chemists try to avoid certain organic functional groups, summarized in 
an ever-growing list, in order to avoid the potential bioactivation to reactive 
metabolites. To add to that alert list, we report herein that boronic 
acid-containing compound structures, such as those found in proteasome 
inhibitors bortezomib and ixazomib, can become bioactivated to chemically 
reactive imine amide metabolites. Test compounds, ixazomib and bortezomib, were 
incubated in vitro using human liver fractions containing cytosol and microsomes 
(S9) under conventional conditions in the presence of GSH. Metabolites were then 
analyzed using LC-MS(n) with or without online hydrogen-deuterium exchange (HDX) 
liquid chromatography coupled with an LTQ-Orbitrap. The exact mass measurements 
of both the precursor and product ions were acquired through data dependent 
acquisition and compared with theoretical values of proposed fragment ions. Upon 
deboronation catalyzed by cytochrome P450 enzymes, both test compounds formed 
imine amide metabolites that were identified by high resolution exact mass 
measurements in both normal aqueous and HDX HPLC-MS analysis. GSH conjugates 
were also identified and were postulated as nucleophilic addition of GSH to the 
imine amide metabolites. All mass spectrometric and HDX measurements of these 
GSH conjugates proved that the GSH unit was added to the carbon atom of the 
imine amide partial structure, hence demonstrating the electrophilic property of 
these imine amide metabolites. The awareness of the formation of electrophilic 
imine amide metabolites from boronic acid-containing compounds, where the boron 
atom is bonded to a carbon atom adjacent to an amide nitrogen, should help in 
drug candidate design and optimization with regard to avoiding potential 
bioactivation.