Macrocyclic Rearrangement Ion Fragmentation of Glutathione Conjugates of Cyclobutane-Containing Covalent BTK Inhibitors

• DOI: 10.1021/jasms.4c00275
• Publication Date: 2025-03-17T07:32:56Z
• Authors: Cathy A. Muste; Chungang Gu; H. George Vandeveer; Simone Sciabola; Martin K. Himmelb...
• Keywords: Linker; Tripeptide

Covalent BTK-inhibitor drugs often contain reactive acrylamide warheads designed to irreversibly bind their protein targets at free thiol cysteines in the kinase active site. This reactivity also makes covalent inhibitors susceptible conjugation endogenous tripeptide glutathione (GSH), leading clearance. During lead optimization efforts for drug discovery of BTK inhibitor BIIB129, some expected GSH adducts resulted an unexpected and highly abundant rearrangement fragment ion LC-MS/MS. By examining more than 30 inhibitors, rearrangements were found be dependent on presence a cycloalkane linker that connects warhead hinge binder motif molecules. The proposed mechanism includes formation 16-membered macrocyclic intermediate between γ-glutamic acid residue (Glu) methyl-cyclobutyl cation, resulting originating from two distant parts adduct molecule separated by conjugated with cysteine between. Rich sets chemical analogues available during enabled confirmation rearrangement. Proposed was verified using derivatives: N-acetylation γ-Glu blocked rearrangement, esterification side chain shift mass rearranged ion. structures supported MS3 MS4 fragmentations. Comparisons fragmentation conjugates cis trans matched pairs suggest concerted cyclobutane stepwise methylcyclobutane linker, respectively.