Structural identification of imatinib cyanide adducts by mass spectrometry and elucidation of bioactivation pathway Austin C. Li, Erya Yu, Steven C. Ring and James P. Chovan, Rapid Commun. Mass Spectrom. 2014, 28, 123-134 Dortmund, 04.02.2014 Lisa Marie Krieger Lisa Marie Krieger Dortmund, 04.02.2014 Contents • Introduction Imatinib • Methods LC/MS/MS • Results Identification of different adducts • Discussion • Conclusion 2 Introduction Lisa Marie Krieger Dortmund, 04.02.2014 Imatinib • Drug against chronic myolegenous leukemia (CML) and gastrointestinal stromal tumors (GIST) • Metabolized by CYP3A enzymes • Hepatotoxicity 2 - 5 %; 4 patients died; mechanisms unknown • Hepatic metabolism → reactive metabolites of imatinib Mando et al. World J. Gastroenterol. 2007, 13, 6608; Buyukberber et al. Med. Oncol. 2010, 27, 768; Backman et al. Drug Metab. Dispos. 2013, 41, 50. Vorlesung Med. Chem. Prof. Dr. Rauh, TU Dortmund WS 2013/14 3 Methods • Lisa Marie Krieger Dortmund, 04.02.2014 Incubation in vitro Imatinib, HLM (Human Liver Microsome) proteins, NADPH-regenerating system, trapping agents (GSH, KCN, methoxylamine) • LC/MS/MS HPLC and LTQ-Orbitrap mass spectrometer with ESI interface Positive and negative mode with different mobile phases (formic acid / ammonium acetate) • Generation of product ions Collision-induced dissociation (CID) and higher energy collision-induced dissociation (HCD) (detecting low-mass ions) www.lookfordiagnosis.com (02.02.2014); http://planetorbitrap.com/data/fe/image/LTQXL_Schema%281%29.png (02.02.2014) 4 Results Lisa Marie Krieger Dortmund, 04.02.2014 Ion chromatograms of cyanide conjugates • Seven cyanide adducts • No GSH adducts 5 Results Lisa Marie Krieger Dortmund, 04.02.2014 Identification of cyanide adduct M519B of imatinib • Exact mass measurement Substitution of H by cyano group • Elimination of hydrogen cyanide 6 Results Lisa Marie Krieger Dortmund, 04.02.2014 Identification of cyanide adduct M519B of imatinib • No modifications of A, B, C or D ring systems Addition to piperazine M. Marull, B. Rochat. Fragmentation study of imatinib and characterization of new imatinib metabolites by liquid chromatography-triple-quadrupole and linear ion trap mass spectrometers. J. Mass Spectrom. 2006, 41, 390 7 Results Lisa Marie Krieger Dortmund, 04.02.2014 Identification of cyanide adduct M519B of imatinib • Exact position unknown 8 Results Lisa Marie Krieger Dortmund, 04.02.2014 Identification of cyanide adduct M519B of imatinib • Reverse Diels-Alder fragmentation Cyano group on C-3/5 • HDX experiment 9 Results Lisa Marie Krieger Dortmund, 04.02.2014 Identification of cyanide adduct M519B of imatinib 10 Results Lisa Marie Krieger Dortmund, 04.02.2014 Identification of cyanide adduct M519B of imatinib Reverse Diels-Alder 11 Results Lisa Marie Krieger Dortmund, 04.02.2014 Identification of cyanide adduct M519B of imatinib • Peak-1/2 show nearly identical product ion spectra to M519B 12 Results Lisa Marie Krieger Dortmund, 04.02.2014 Identification of cyanide adduct M505 of NN-demethylated imatinib • m/z 201 and 84: • m/z 394: modification on piperazine demethylated product ions • m/z 423: cyano group at C-3/C-5 13 Results Lisa Marie Krieger Dortmund, 04.02.2014 Identification of cyanide adduct M505 of NN-demethylated imatinib • Loss of N-methyleneethenamine (reverse Diels-Alder dissociation) • HDX experiment Fragmentation pathway 505 → 477 → 422 14 Results Lisa Marie Krieger Dortmund, 04.02.2014 Identification of cyanide adduct M519A of imatinib • No modification on ring D and E • Conjugation on ring A, B or C 15 Results Lisa Marie Krieger Dortmund, 04.02.2014 Identification of cyanide adduct M519A of imatinib 16 Results Lisa Marie Krieger Dortmund, 04.02.2014 Identification of cyanide adduct M519A of imatinib • Addition of cyano group on p-toluidine ring (C) partial structure • Bioactivated via imine-methide intermediate → electrophilic • Michael addition of cyanide ion to methyl carbon (C-31) through imine-methide intermediate • Dehydration of hydroxylated metabolite T. A. Baillie. Future of toxicology – metabolic activation and drug design: Challenges and opportunities in chemical toxicology. Chem. Res. Toxicol. 2006, 19, 889 17 Results Lisa Marie Krieger Dortmund, 04.02.2014 Identification of cyanide adducts M519C to M519F of imatinib m/z 519.2244 m/z 519.2245 m/z 519.2243 m/z 519.2246 18 Discussion Lisa Marie Krieger Dortmund, 04.02.2014 Bioactivation mechanism • P450-catalyzed oxidation and subsequent desaturation Imine and imine-methide intermediates • Soft electrophile (imine-methide) intermediate reacts with hard nucleophile (cyanide ion) Steric effect caused by large pyridine-pyrimidine partial structure 19 Discussion Lisa Marie Krieger Dortmund, 04.02.2014 Bioactivation in relation to observed clinical adverse effects • Elevation of transaminase and bilirubin (5.1 %; 3.5 % of patients) Clinical indicative events of liver toxicity • No previous investigation into hepatotoxicity mechanism Substitution with alkyl groups on piperazine moiety Retaining pharmacological potency while reducing irreversible covalent protein binding Blocking formation of imine intermediate 20 Conclusion Lisa Marie Krieger Dortmund, 04.02.2014 Structural determination of cyanide adducts Bioactivation of piperazine and p-toluidine substructures Proposal of mechanism Deeper understanding of metabolism and toxicology of imatinib in humans 21 Lisa Marie Krieger Dortmund, 04.02.2014 Thank you for your kind attention! 22 Results Lisa Marie Krieger Dortmund, 04.02.2014 Identification of cyanide adducts M519C to M519F of imatinib 23 Discussion Lisa Marie Krieger Dortmund, 04.02.2014 Bioactivation mechanism • Carbonyl group on the ring Conjugated imine intermediates (soft electrophiles) • No conjugation detected with GSH (soft nucleophile) Cyanide adducts were not formed via conjugated imine intermediate • Possibility of conjugated imine intermediate cannot be ruled out 24
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