Short Communication NMR CHARACTERIZATION OF AN S-LINKED GLUCURONIDE METABOLITE OF THE POTENT, NOVEL, NONSTEROIDAL PROGESTERONE AGONIST TANAPROGET

Tanaproget is a first-in-class nonsteroidal progesterone receptor agonist that is being investigated for use in 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 liquid chromatography/mass spectrometry (LC/MS) and LC-tandem mass spectrometry analysis. To obtain additional structural details for this metabolite, additional quantities were generated from rat liver microsomal incubations and purified by high-performance liquid chromatography (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) one-dimensional (1D) and twodimensional (2D) NMR couplings and correlations in the metabolite spectra (due primarily to low sample concentration) precluded an unambiguous structure elucidation. Subsequent synthesis of the Sand N-glucuronides of tanaproget from tanaproget facilitated the unambiguous regioand 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. Tanaproget is a potent, first-in-class nonsteroidal progesterone receptor agonist being investigated at Wyeth for use in contraception (Fensome et al., 2005; Zhang et al., 2005). Earlier studies indicated that a major conjugated metabolite ( 10%), referred to here as M1, was formed in rat and human liver microsomal incubations, and initially characterized by LC/MS as a glucuronide of tanaproget. The glucuronic acid group was proposed to be either on the nitrogen or the sulfur of the benzoxazine-2-thione moiety of tanaproget (1, Scheme 1). Oxidative metabolism represented a minor metabolic pathway of 1 in both in vitro and in vivo studies. Analysis of the initial NMR data of M1 from rat liver microsomes confirmed that the glucuronide was covalently bound to either the Sor the N-form of the benzoxazine-2-thione moiety. However, due to an insufficient amount (approximately a few micrograms) of the sample in solution, we could not record complete scalar and dipolar 1D and 2D NMR couplings and correlations for an unambiguous structure elucidation. The NMR analysis was especially challenging because of the lack of protons in the benzoxazine-2-thione moiety, particularly in the scenario of an S-glucuronide conjugate of tanaproget, since most heteronuclear correlations between the sugar and the parent molecule would be too long range to be observed. Because of the challenges in preparing sufficient quantities of M1, the strategy turned away from liver microsome preparations to synthesis. Comparison of NMR, LC/MS, and HPLC data between synthetic compounds and M1 isolated from microsomes facilitated the unambiguous regioand stereochemical assignment of the metabolite. The present study highlights the successful approach and powerful utility of extensive NMR analysis and the availability of synthetic metabolite standards in the unambiguous identification of this Slinked metabolite. The data are critical for the drug development of tanaproget, since it would allow for biological testing of its major metabolite. Materials and Methods Chemicals. Chemicals used in the model compound syntheses, and deuterated NMR solvents were obtained from Aldrich (Milwaukee, WI). Ammonium acetate, magnesium chloride, and UDPGA were obtained from Sigma (St. Louis, MO). Solvents for extractions and chromatography were HPLC grade or ACS Reagent Grade (Mallinckrodt Baker, Phillipsburg, NJ and EMD Chemicals, Gibbstown, NJ). Synthesis of Nand S-Glucuronic Acid Derivatives of Tanaproget. A 0.5-mmol solution of tanaproget in anhydrous dimethyl formamide (5 ml) was added dropwise under a nitrogen atmosphere to a solution of 1.1 mmol of sodium hydride in dimethyl formamide (25 ml) that was cooled to approximately 70°C (dry ice). After stirring for 10 min, a 0.5-mmol solution of acetobromo-D-glucuronic acid methyl ester in dimethyl formamide (5 ml) was added dropwise. The reaction solution was then warmed to room temperature and stirred for a total of 8 h. The reaction solution was partitioned between water (100 ml) and ethyl acetate (100 ml). The aqueous layer was extracted again with ethyl acetate (100 ml). The combined organic layers were washed with saturated sodium chloride solution (100 ml) and dried (magnesium sulfate), and solvent was removed in vacuo to yield 0.350 g of crude reaction material. To a portion of this material (0.210 g) was added a solution Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.105.008763. ABBREVIATIONS: LC/MS, liquid chromatography/mass spectrometry; 1D, one-dimensional; 2D, two-dimensional; gHSQC, gradient heteronuclear single quantum coherence; gHMBC, gradient heteronuclear multiple bond correlation; HPLC, high-performance liquid chromatography; UDPGA, uridine diphosphoglucuronic acid; DMSO-d6, deuterated dimethyl sulfoxide; UGT, UDP-glucuronosyltransferase. 0090-9556/06/3408-1283–1287$20.00 DRUG METABOLISM AND DISPOSITION Vol. 34, No. 8 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 8763/3126606 DMD 34:1283–1287, 2006 Printed in U.S.A. 1283 at A PE T Jornals on July 7, 2017 dm d.aspurnals.org D ow nladed from