Received for publication December 13, 2005.
Revised May 5, 2006.
Accepted for publication May 5, 2006.

NMR Characterization of an S-Linked Glucuronide Metabolite of the Potent, Novel, Nonsteroidal Progesterone Agonist Tanaproget

Abstract

Tanaproget is a first-in-class nonsteroidal progesterone receptor agonist which is being investigated for contraception. A major in vitro and in vivo metabolite of tanaproget formed in humans was initially characterized as a glucuronide of tanaproget. However, whether the glucuronide was linked to the nitrogen or sulfur of the benzoxazine-2-thione group in tanaproget could not be determined by LC/MS and LC/MS/MS analysis. In order to obtain additional structural details for this metabolite, additional quantities were generated from rat liver microsomal incubations and purified by HPLC for NMR analysis. The NMR data for the metabolite confirmed that the glucuronide was covalently bound to either the sulfur or the nitrogen of the benzoxazine-2-thione moiety. The lack of key through-bond (scalar) and through-space (dipolar) 1- and 2-D NMR couplings and correlations in the metabolite spectra (due primarily to low sample concentration) precluded an unambiguous structure elucidation. Subsequent synthesis of the S- and N-glucuronides of tanaproget from tanaproget and a protected form of glucuronic acid facilitated the unambiguous regio- and stereochemical assignment of the metabolite by comparison of 1D NMR chemical shifts and scalar coupling constants, 2D NMR correlations, and HPLC and LC/MS characteristics between the synthetic compounds and the metabolite. From extensive comparison of the spectral and chromatographic data of the microsomally-derived metabolite and the synthetic compounds, the metabolite has been determined to be the S-()-D-glucuronide of tanaproget.

Key words: glucuronidation, HPLC, liver microsomes, mass spectrometry, metabolite identification