Investigation of the in vitro metabolism of the emerging drug candidate S107 for doping-preventive purposes.
The metabolic fate of the emerging drug candidate S107, possessing the potential for misuse as performance-enhancing agent in sports, was investigated by in vitro phase I and II experiments with human microsomal and S9 liver enzymes. The metabolites were identified by liquid chromatography-mass spectrometry with electrospray ionisation in positive mode (LC-ESI-MS/MS). Their collision-induced dissociation behaviour was studied by high-resolution/high accuracy Orbitrap MS(n) analysis, supported by stable isotope labelling, H/D-exchange experiments and density functional theory calculations. Monooxygenation accounted for the main phase I metabolic transformation due to N- and S-oxidation of the 1,4-benzothiazepine core, as substantiated by chemical synthesis, selective reduction methods and characteristic APCI in source fragmentation behaviour of the metabolites. Another dominant metabolic pathway was demethylation, yielding the N- and O-demethylated metabolite, respectively. The latter was further conjugated by glucuronidation as well as sulfonation in subsequent phase II metabolic reactions, whereas the N-demethylated metabolite was not amenable to conjugation. The active drug molecule itself was converted to two glucuronic acid conjugates, which are proposed to consist of two quaternary S107-N(+)-glucuronide isomers. All glucuronides were susceptible to enzymatic hydrolysis with β-glucuronidase (Escherichia coli). A comprehensive LC-ESI-MS(/MS)-based detection method for urine was developed and its fitness for purpose was assessed. The assay can serve as a potential screening and/or confirmation method for S107 in clinical drug testing and doping control analysis in the future.
Institute of Biochemistry, Center for Preventive Doping Research, German Sport University Cologne, Cologne, Germany.
S
S
Beuck
W
W
Schänzer
M
M
Thevis
D002853:Chromatography, Liquid / Q000379:methods*; D004300:Doping in Sports / Q000517:prevention & control*; D005260:Female; D006801:Humans; D008297:Male; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013841:Thiazepines / Q000737:chemistry / Q000378:metabolism* / Q000652:urine
C540746:S-107 compound; D013841:Thiazepines