Metabolic Profiling of S-praziquantel: Structure Elucidation Using the Crystalline Sponge Method in Combination with Mass Spectrometry and Nuclear Magnetic Resonance

Abstract

Praziquantel (PZQ) is the drug of choice for treatment of the neglected tropical disease schistosomiasis. Although the drug has been extensively used over several decades and its metabolism well studied (several oxidative metabolites are known from literature), the knowledge of the complete structure of some of its metabolites remains elusive. Conventional techniques like nuclear magnetic resonance (NMR) or liquid chromatography mass spectrometry (LC-MS) were used in the past to investigate phase I and phase II metabolites of PZQ. These techniques are either limited to provide the complete molecular structure (LC-MS) or require large amount of sample material (NMR), which are not always available when in vitro systems are used for investigation of the metabolites. In this study, we describe new structures of S-PZQ metabolites generated in vitro from human liver microsomes (HLM) using the crystalline sponge method (CS-XRD). After chromatographic separation and purification of the oxidative metabolites, ultra-performance liquid chromatography (UPLC)-quadrupole timeof-flight mass spectrometry (qTOF) analysis was conducted to narrow down the position of oxidation to a certain part of the molecule. To determine the exact position of hydroxylation, singe-crystal X-ray diffraction (SC-XRD) analysis of the crystalline sponges (CS) and absorbed analyte was used to identify the structure of S-PZQ and its metabolites. CS-XRD allowed for complete structure elucidation of the known metabolites S-trans-4'-hydroxy PZQ (M1), S-cis-4'-hydroxy PZQ (M2) and S-/R-11b-hydroxy PZQ (M6) as well as the unknown metabolites S-9-hydroxy PZQ (M3) and S-7-hydroxy S-PZQ (M4). For comparison of structural elucidation techniques, one metabolite (M3) was additionally analyzed using NMR.

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“…In comparison to conventional crystallography, the crystalline sponge method allows for absolute structure determination of small molecules without crystallization of the analyte using sample material within microgram to nanogram quantities. This is of particular importance for target analytes that are only available in trace amounts (e.g., natural products and metabolites) as well as liquid, volatile, and amorphous compounds. − …”