Quadrupole linear ion-trap mass spectrometry studies on glutathione conjugates of nordihydroguaiaretic acid (NDGA) analogues reveals phenol-type analogues are without reactive metabolite-mediated toxic liability

Abstract Reactive metabolites (RMs) have been implicated in many drug-induced toxicities including hepatotoxicity. Nordihydroguaiaretic acid (NDGA) has known pharmacological properties but its use is also associated with toxicities possibly mediated by RMs. In our effort to design and prepare NDGA analogues with better safety profile through rational structural modification, we first evaluated metabolic activation potential of model compounds mimicking catechol- and phenol-type NDGA analogues. We incubated test compounds in rat liver microsomes (RLM) in the presence of glutathione as nucleophilic trapping agent. We also investigated their potential to form para-quinone methides using silver oxide. Glutathione conjugates were detected by electrospray ionization-mass spectrometry (ESI-MS) scanning for neutral loss (NL) 129 or 307 in positive ion mode or precursor ion (PI) scanning for 272 in negative ion mode and further characterized by liquid chromatography–tandem mass spectrometry (LC–MS/MS) or in a single LC-MS run using multiple reactions monitoring (MRM) as a survey scan to trigger acquisition of enhanced product ion (EPI) data. Our findings led us to subsequently design and synthesize a series of NDGA analogues for evaluating their metabolic activation potential with a goal of eliminating RMs liability. Among others, we found that catechol-type analogues were converted to ortho-quinones by cytochrome P450s. We saw no evidence of RMs by cytochrome P450s for phenol-type analogues. This suggest that phenol-type NDGA analogues might not be associated with reactive metabolites-mediated toxicities. Although a more extensive pharmacological evaluation is underway, our preliminary results revealed that pharmacological properties were not compromised.