1. We investigated the mechanisms responsible for the in vivo instability of a benzofurazan compound BI-94 (NSC228148) with potent anti-cancer activity. 2. BI-94 was stable in MeOH, water, and in various buffers at pHs 2.5-5, regardless of the buffer composition. In contrast, BI-94 was unstable in NaOH and at pHs 7-9, regardless of the buffer composition. BI-94 disappeared immediately after spiking into mice, rat, monkey, and human plasma. BI-94 stability in plasma can be only partially restored by acidifying it, which indicated other mechanisms in addition to pH for BI-94 instability in plasma. 3. BI-94 formed adducts with the trapping agents, glutathione (GSH) and N-acetylcysteine (NAC), in vivo and in vitro via nucleophilic aromatic substitution reaction. The kinetics of adduct formation showed that neutral or physiological pHs enhanced and accelerated GSH and NAC adduct formation with BI-94, whereas acidic pHs prevented it. Therefore, physiological pHs not only altered BI-94 chemical stability but also enhanced adduct formation with endogenous nucleophiles. In addition, adduct formation with human serum albumin-peptide 3 (HSA-T3) at the Cys34 position was demonstrated. 4. In conclusion, BI-94 was unstable at physiological conditions due to chemical instability and irreversible binding to plasma proteins.
Keywords: Glutathione; LC-MS/MS; N-acetylcysteine; irreversible protein binding; isotopic filtering.