Structural identification of imatinib cyanide adducts by mass spectrometry and elucidation of bioactivation pathway.

RATIONALE: Recent publications have reported that imatinib forms cyanide and methoxylamine adducts in vitro but without detail structural identification. The current work reports the identification of seven cyanide adducts that elucidate the bioactivation pathways and may provide hints for observed clinical adverse effects of the drug. METHODS: Imatinib was incubated with human liver microsomal proteins in the presence of a NADPH-regeneration system and the trapping agents reduced GSH, potassium cyanide and methoxylamine. Samples were analyzed by high-performance liquid chromatography (HPLC) coupled with a LTQ-Orbitrap data collection system. Chemical structures were determined and/or postulated based on data-dependent high-resolution tandem mass spectrometric (MS(n)) exact mass measurements in both positive and negative scan modes, as well as in combination with hydrogen-deuterium exchange (HDX). RESULTS: GSH and methoxylamine conjugates were either not detected or were in insufficient quantities for characterization. However, seven cyanide conjugates were identified, indicating that the piperazine and p-toluidine partial structures in imatinib can become bioactivated and subsequently trapped by the nucleophile cyanide ion. The reactive intermediates were postulated as imine and imine-carbonyl conjugate (α,ß-unsaturated) structures on the piperazine ring, and imine-methide on the p-toluidine partial structure. CONCLUSIONS: Chemical structures of seven cyanide adducts of imatinib have been identified or proposed based on high-resolution MS/MS data. Mechanisms for the formation of the conjugates were also proposed. The findings may help to understand the mechanism of hepatotoxicity of imatinib in humans.

Boronic acid-containing proteasome inhibitors: alert to potential pharmaceutical bioactivation.

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.

Identification of imine or enamine drug metabolites using online hydrogen/deuterium exchange and exact mass measurements.

RATIONALE: Drug metabolites that have imine or enamine partial structures cause extra mass-to-charge (m/z) increases in online hydrogen/deuterium exchange (HDX) in addition to hydroxyl or amine protons. Online HDX and exact mass measurement were used herein to characterize this extra increase property, and to further confirm proposed metabolite structures. METHODS: Metabolites of two proprietary compounds as well as two commercially available compounds were analyzed using aqueous and HDX liquid chromatography coupled with an LTQ-Orbitrap. The exact mass measurements of both the precursor ions and product ions were acquired through data-dependent acquisition and compared with theoretical values of proposed fragment ions. RESULTS: Analysis of exact mass measurements of metabolite product ions under both normal aqueous and HDX conditions led to the identification of the isoxazole ring opening of compound C-1, and a double-bond formation on the methylpyrrolidine ring of compound C-2 during biotransformation. In both cases, imine or enamine structures formed in the metabolites caused extra m/z increases upon HDX that contributed confirmatory information to the structure identification. The compound 3,3-diphenyl-2-ethyl-1-pyrroline also demonstrated that the methylene protons adjacent to the imine were exchanged during online HDX. CONCLUSIONS: The exchangeability of methylene protons adjacent to imine or enamine moieties proved to be useful to narrow down or even pinpoint the metabolism sites of parent drugs when high-resolution exact mass measurement and online HDX were used.

Metabolic profile of [(14)C]bendamustine in rat urine and bile: preliminary structural identification of metabolites.

Bendamustine, a bifunctional alkylating agent, is currently in clinical trials for the treatment of hematological and other malignancies. Although it has been used in the former East Germany for more than 30 years, very limited information is available on its biotransformation. The objective of this investigation was to obtain information on the structures of metabolites excreted into rat urine and bile to understand the metabolic fate of bendamustine in vivo. Metabolites of [(14)C]bendamustine hydrochloride in rat urine and bile were determined using liquid chromatography-mass spectrometry (MS) in parallel with on-line radioactivity detection in samples obtained after i.v. dosing of 3 mg/kg. A total of 17 radioactive peaks were identified in rat urine and 10 in rat bile (2 were unique to bile). Four of these metabolites had been previously reported, whereas 15 are novel. Proposed structures of all metabolites detected are based on MS(n) spectra generated from a linear ion trap mass spectrometer. These results suggest that the major metabolic pathways in rat are oxidative and/or hydrolytic dehalogenation, oxidation, carboxylic acid formation, N-dealkylation, sulfation, and glutathione and cysteine (probably via glutathione) conjugation. The cysteine-conjugated compounds are observed in their N-acetylated cysteine (mercapturic acid) forms.