Biocatalytic Syntheses of Antiplatelet Metabolites of the Thienopyridines Clopidogrel and Prasugrel Using Fungal Peroxygenases

Scheme 1. Proposed mechanism of bioactivation and conversion of clopidogrel and prasugrel into their active thiol metabolites, CAM and PAM, by cytochrome P450 enzymes via formation of thiolactone, thiolactone sulfoxides and sulfenic acids as intermediates. An alternative inactivation pathway has been described to proceed via hydrolysis catalyzed by thioesterases (PON-1) leading to the formation of the endo-thiol metabolite.

Figure 1. Structures of clopidogrel active metabolites H1–H4.

Figure 2. Extracted ion chromatogram and MS and MS2 spectra of isomers of active clopidogrel metabolite (CAM) formed during conversion of 1 mM clopidogrel by 2 U mL−1 MroUPO in presence of 5 mM ascorbate, 20 mM KPi buffer (pH 7.0) and in sum 4 mM hydrogen peroxide. (A) Extracted ion chromatogram at m/z 356 (MS); (B) MS spectra of the two isomers; (C) MS2 spectra of the ion at m/z 356 with collision energy of CE20; (D) assignment for the fragmentation pattern in Figure 2C.

Figure 3. Extracted ion chromatogram and MS and MS2 spectra of isomers of active prasugrel metabolite (PAM) formed during conversion of 1 mM prasugrel by 2 U mL−1 PLE for 30 min followed by conversion with 2 U mL−1 MroUPO in the presence of 5 mM ascorbate, 20 mM KPi buffer (pH 7.0) and in sum 4 mM hydrogen peroxide. (A) Extracted ion chromatogram at m/z 350 (MS); (B) MS spectra of the two isomers; (C) MS2 spectra of the ion at m/z 350 with collision energy of CE25; (D) assignment for the fragmentation pattern in (C).

Figure 4. Kinetics of clopidogrel (A) and prasugrel (B) conversion (0.2 mmol each) by 2 U mL−1 MroUPO in presence of 5 mM ascorbate, 20 mM KPi buffer (pH 7.0) and continuous supply of hydrogen peroxide (2 mM h−1). Prasugrel was preincubated with 2 U mL−1 PLE for 30 min. Black circles are the thienopyridine substrates; black squares represent the 2-oxo metabolites and black triangles are the active metabolites. Isomeric double peaks were merged. The red stars reflect the residual UPO activity. Yellow background represents the PLE reaction; red background indicates the UPO reaction. Ordinates are the concentrations of the metabolites recalculated after calibration with isolated compounds.

Scheme 2. Proposed mechanism for product formation in the UPO-catalyzed oxygenation of clopidogrel.