In vitro and in vivo biotransformation of WMS-1410, a potent GluN2B selective NMDA receptor antagonist.

Structural modification of the GluN2B selective NMDA receptor antagonist ifenprodil led to the 3-benzazepine WMS-1410 with similar GluN2B affinity but higher receptor selectivity. Herein the in vitro and in vivo biotransformation of WMS-1410 is reported. Incubation of WMS-1410 with rat liver microsomes and different cofactors resulted in four hydroxylated phase I metabolites, two phase II metabolites and five combined phase I/II metabolites. With exception of catechol 4, these metabolites were also identified in the urine of a rat treated with WMS-1410. However the metabolites 7, 8 and 12 clearly show that the catechol metabolite 4 was also formed in vivo. As shown for ifenprodil the phenol of WMS-1410 represents the metabolically most reactive structural element. The biotransformation of WMS-1410 is considerably slower than the biotransformation of ifenprodil indicating a higher metabolic stability. From the viewpoint of metabolic stability the bioisosteric replacement of the phenol of WMS-1410 by a metabolically more stable moiety should be favourable.

Metabolism studies of ifenprodil, a potent GluN2B receptor antagonist.

The NMDA receptor antagonist ifenprodil is an important lead structure for developing new GluN2B selective NMDA receptor antagonists. Ifenprodil itself has a high affinity to the GluN2B subunit but a poor selectivity for the NMDA receptor. This aspect and the fast biotransformation are the major drawbacks of ifenprodil. In order to optimize the development of new and more selective GluN2B (NMDA) receptor antagonists, the identification of the main metabolic pathways of ifenprodil is necessary. Herein the in vitro and in vivo phase I and phase II metabolites of ifenprodil were generated and analyzed via LC-MS(n) experiments. In vitro experiments were carried out with rat liver microsomes and various co-factors to generate phase I and phase II metabolites. The application of ifenprodil to a rat and the analysis of its urine led to the identification of diverse formed in vivo metabolites. The phenol represents the metabolically most labile structural element since glucuronide 7 and 8 appeared as main metabolites.

Synthesis, characterization, and metabolism studies of fluspidine enantiomers.

The enantiomers of the potent σ1 ligand fluspidine (1) were prepared by using chiral preparative HPLC. Synthesis of racemic tosylate 2 and subsequent separation of enantiomers yielded (R)-2 and (S)-2 in excellent enantiomeric purities. The fluspidine enantiomers (R)-1 and (S)-1 were synthesized from (R)-2 and (S)-2 by nucleophilic substitution with tetra-n-butylammonium fluoride, affording (R)-1 with 99.6 % ee and (S)-1 with 96.4 % ee. Tosylates (R)-2 and (S)-2 can also serve as precursors for the radiosynthesis of enantiomerically pure radiotracers [(18) F](R)-1 and [(18) F](S)-1. The absolute configuration of the pure enantiomers was elucidated by comparison of their CD spectra with a calculated CD spectrum of a simplified model compound. In receptor binding studies, both enantiomers displayed very high σ1 receptor affinity and selectivity against the σ2 receptor. (R)-Fluspidine ((R)-1) is the eutomer, with a Ki value of 0.57 nM and a eudysmic ratio of 4. Incubation of (R)-1 and (S)-1 with rat liver microsomes led to the identification of seven and eight metabolites, respectively. Although the S-configured enantiomer formed additional metabolite (S)-1-3, it is metabolically more stable than (R)-1.

Genome-wide DNA methylation level analysis by micellar electrokinetic chromatography and laser-induced fluorescence detection after treatment of cell lines with azacytidine and antifolates.

Methylation of DNA is a well-known epigenetic mechanism to control DNA transcription. The determination of the exact methylation level of DNA samples is of great interest due to its significant deregulation in tumor cells. Here the genome-wide DNA methylation is quantified precisely using micellar electrokinetic chromatography (MEKC) combined with laser-induced fluorescence (LIF) detection after enzymatic DNA hydrolysis and coupling of the resulting mononucleotides with BODIPY FL EDA: N-[3-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl)propionyl]ethylenediamine hydrochloride). For the validation of the method, two oligonucleotides containing 10 copies of each DNA base were designed and synthesized. In one oligonucleotides one cytosine residue was replaced with 5-methylcytosine, allowing the exact adjustment of different methylation levels between 0% and 10% by mixing appropriate amounts of these well-defined oligonucleotides. High precision, in particular of the detection factors of the single mononucleotides, was achieved because the complete analytical process, including hydrolysis, BODIPY coupling, and analysis, was considered during the calibration process. Application of this method on calf thymus DNA resulted in a methylation level of 6.94%, which is in good agreement with the values obtained with other methods. Whereas treatment of HEK293 cells with azacytidine led to considerably reduced global methylation from approximately 5.0% to 1.4%, treatment of the cells with the antifolates methotrexate and pemetrexed led to a slightly increased methylation level.