Early rapid identification of in vivo rat metabolites of AN6414, a novel boron-containing PDE4 inhibitor by QTRAP LC/MS/MS to support drug discovery.

There is an increasing interest in in vivo metabolite identification in early drug discovery in order to (i) give a more complete picture of metabolic profile in investigational animal models, (ii) propose phase I and phase II metabolites using the same pharmacokinetic/toxicokinetic study samples, (iii) expose metabolically labile groups where chemical modifications could improve stability, and (iv) enable early safety assessment of metabolites. In the early discovery stage of our anti-inflammatory program, one novel benzoxaborole, AN6414, exhibiting both PDE4 enzyme and TNFα inhibition activities, became our primary candidate for further investigation. The traditional metabolite identifications usually require high dosed samples with long data scans and analysis. In this study, we conducted quick and more selective core-structure related precursor scans followed by daughter ion scans and identified a total of 10 major phase I and phase II metabolites using rat plasma samples from a toxicokinetic study at an oral dosing of 30 mg/kg. Plasma samples were treated with solid phase extraction (SPE) prior to LC/MS/MS. An AB SCIEX API 4000 QTRAP mass spectrometer coupled with a Shimadzu LC system was used for LC/MS/MS analysis. We found the major metabolites of AN6414 to be oxidative deboronation, protodeboronation, oxidation products and their sulfate-conjugated species. This analysis drove analoging efforts which improved the pharmacokinetic profile, namely, lowering clearance and increasing exposure relative to AN6414. Toxicity predictions by the software program DEREK suggest the identified potential metabolites to be safe.