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Realised by ALMS™
developer of the AIDS-HIV Reference project
Abstract No.: TuP-088
Session: Drug Discovery and Development
Presentation date: Tue, Aug 29, 2006
Presentation time: 14:30 – 16:00

Complete Characterization of ML3403 Metabolites from Liver Microsomes Using QTOF and LC-SPE-cryo NMR/MS

Gabriela Zurek1, Holger Scheible2, Stefan Laufer2, Bernd Kammerer2, Wolfgang Albrecht3, Markus Godejohann4, Manfred Spraul4

1 Bruker Daltonik GmbH, Bremen, Germany
2 University Tuebingen, Tuebingen, Germany
3 Merckle GmbH, Ulm, Germany
4 Bruker Biospin GmbH, Karlsruhe, Germany

Correspondence address: Gabriela Zurek, Bruker Daltonik GmbH, Fahrenheitstr. 4, Bremen, 28359 Germany.

Web site: http://www.bdal.de

Keywords: Mass Spectrometry, QTOF; Metabolism, Metabolites; NMR; Xenobiotics.

Novel aspect: Rapid identification of new metabolites from in vitro assays in early stage of drug development process.


A detailed characterization of a new chemical entity (NCE) and its metabolites already early in the drug discovery process is recommended to determine whether any potentially reactive or toxic metabolites are formed. ML3403, a 2-aminopyridin-4-yl imidazole derivative containing a thioether moiety, is a potent and selective inhibitor of p38 MAP kinase, a key enzyme in response to inflammatory cytokines, endotoxins and osmotic stress. The biotransformation of ML3403 in liver microsomes has been investigated with the target to completely characterize as many metabolites as possible. QTOF data with accurate mass and true isotopic pattern of product ions is an important tool for the elucidation of characteristic fragmentation patterns. LC-SPE-cryo NMR/MS was applied to confirm the structural hypotheses from the MS data.

In-vitro microsomal incubations were carried out using mouse liver microsomes at a concentration of 50 µmol/L ML3403 at incubation times of 0 - 90 min. The samples were analyzed by RP-LC coupled to a micrOTOF-Q (Bruker Daltonik GmbH, Bremen). The detection of metabolites in the incubations was achieved by comparison to a reference incubation at t=0min using the eXpose algorithm. Targeted LC/MS/MS and pseudo MS3 with in-source collision induced dissociation (IS-CID) of the metabolites were performed. Parent and product ion spectra were evaluated using generate molecular formula to determine the elemental compositions. ML3403 and major metabolites were also analysed by 1D and 2-D-NMR. LC-SPE-NMR-MS was applied to an up-scaled (100 Ámol/L) version of the microsomal incubation

In total, 10 different metabolites of ML3403 were detected in the mouse liver microsmal incubation. The major metabolite of ML3403 is its sulfoxide ML3603 that is formed almost instantaneously and quantitatively. The MS/MS fragmentation behaviour of ML3403 and ML3603 was investigated in detail by assignment of molecular formulae to all fragment ions leading to the creation of a structural hypothesis. The accurate mass and true isotopic pattern information of product ions was the key for a fast and reliable interpretation. At later incubation times (> 10min), ML3403 and ML3603 are further metabolized. The metabolites were characterized. This intelligent combination of modern MS- and NMR-technology allowed the complete assignment of metabolite structures. The overall result is the generation of the metabolic profile of ML3403.