The role of intramolecular reactions and chemical degradation in the apparent biotransformation pathways of a series of SYK inhibitors

Abstract

In vitro metabolism studies of the SYK inhibitors AZ-A and AZ-B identified four unusual metabolites. M1 (m/z 411) was formed by both molecules and was common to several analogues (AZ-C to AZ-H) sharing the same core structure, appearing to derive from the complete loss of a pendent 3,4-diaminotetrahydropyran ring and pyrazole ring cleavage resulting in a non-obvious metabolite. M2-M4 were formed by AZ-A and a subset of the other compounds only and apparently resulted from a sequential loss of H₂ from parent. Initial attempts to isolate M3 for identification were unsuccessful due to sample degradation and it was subsequently found that M2 and M3 underwent sequential chemical degradation steps to M4. M4 was successfully isolated and shown by mass spectrometry and NMR spectroscopy to be a tricyclic species incorporating the pyrazole and the 3,4-diaminotetrahydropyran groups. We propose that this arises from an intramolecular reaction between the primary amine on the tetrahydropyran and a putative epoxide intermediate on the adjacent pyrazole ring, evidence for which was generated in a b-mercaptoethanol trapping experiment. The loss of the THP-moiety observed in M1 was found to be enhanced in an analogue which was unable to undergo the intra-molecular reaction step leading us to propose two possible reaction pathways originating from the reactive intermediate. Ultimately, we conclude that the apparently complex and unusual metabolism of this series of compounds likely resulted from a single metabolic activation step forming an epoxide intermediate which subsequently underwent intramolecular rearrangement and/or chemical degradation to form the final observed products. Significance Statement The current work provides an unusual biotransformation example showing the potential for intramolecular reactions and chemical degradation to give the appearance of complex metabolism arising from a single primary route of metabolism.

Keywords: kinases; metabolite identification.