Structure Elucidation of the Metabolites of 2', 3', 5'-Tri-O-Acetyl-N6-(3-Hydroxyphenyl) Adenosine in Rat Urine by HPLC-DAD, ESI-MS and Off-Line Microprobe NMR.

Abstract

2', 3', 5'-Tri-O-acetyl-N6-(3-hydroxyphenyl) adenosine (also known as WS070117) is a new adenosine analog that displays anti-hyperlipidemic activity both in vitro and in vivo experiments as shown in many preliminary studies. Due to its new structure, little is known about the metabolism of WS070117. Hence, the in vivo metabolites of WS070117 in rat urine following oral administration were investigated. Identification of the metabolites was conducted using the combination of high-performance liquid chromatography (HPLC) coupled with diode array detector (DAD), ion trap electrospray ionization-mass spectrometry (ESI-MS), and off-line microprobe nuclear magnetic resonance (NMR) measurements. Seven metabolites were obtained as pure compounds at the sub-milligram to milligram levels. Results of structure elucidation unambiguously revealed that the phase I metabolite, N6-(3-hydroxyphenyl) adenosine (M8), was a hydrolysate of WS070117 by hydrolysis on the three ester groups. N6-(3-hydr-oxyphenyl) adenine (M7), also one of the phase I metabolites, was the derivative of M8 by the loss of ribofuranose. In addition to two phase I metabolites, there were five phase II metabolites of WS070117 found in rat urine. 8-hydroxy-N6-(3-hydroxy-phenyl) adenosine (M6) was the product of M7 by hydrolysis at position 8. The other four were elucidated to be N6-(3-O-β-D-glucuronyphenyl) adenine (M2), N8-hydroxy-N6-(3-O-sulfophenyl) adenine (M3), N6-(3-O-β-D-glucuronyphenyl) adenosine (M4), and N6-(3-O- sulfophenyl) adenosine (M5). Phase II metabolic pathways were proven to consist of hydroxylation, glucuronidation and sulfation. This study provides new and valuable information on the metabolism of WS070117, and also demonstrates the HPLC/MS/off-line microprobe NMR approach as a robust means for rapid identification of metabolites.