Application of the crystalline sponge method for metabolite structure elucidation

The identification of metabolites during drug discovery and development can play a crucial role to understand their contribution to efficacy and safety. To investigate the metabolism of new drug candidates in human as early as possible, different in vitro systems from human origin are used. Standard structural elucidation tools like liquid chromatography-mass spectrometry, single-crystal X-ray diffraction or nuclear magnetic resonance spectroscopy are either limited in providing the complete structure of metabolites or require a larger amount of material not available from in vitro incubations. A new technology to overcome these limitations was introduced by Makoto Fujita in 2013 and is commonly known as the crystalline sponge method. The usage of a pre-existing crystal allows the complete crystallographic identification of small molecules at the atomic level by encapsulation of only nanogram amount of analyte material without the need for crystallization of the analyte itself. In this thesis, the structural elucidation of metabolites generated from in vitro incubations using the crystalline sponge method is described. First, the analysis of an active pharmaceutical ingredient and its known metabolites demonstrated, that the crystalline sponge technology is a valuable method to identify the complete structure of trace amounts of analytes isolated from incubation matrices. The crystallographic data provided the exact position of metabolism for phase I and phase II metabolites. The technology was further used to identify various known and unknown metabolites of the anthelmintic drug praziquantel, which has been used for treatment of the parasitic flatworm of the genus Schistosoma for several decades. Moreover, to optimize the soaking process of the crystalline sponge method, which is the key element of the technology, an affinity screening to prioritize the optimal soaking conditions for individual analytes without time-consuming X-ray diffraction measurements, was developed. ; Die Identifizierung von Metaboliten ...