Formation and Fate of a Sulfenic Acid Intermediate in the Metabolic Activation of the Antithrombotic Prodrug Prasugrel

Abstract

Metabolic activation of the tetrahydro-thienopyridine antithrombotic prodrug, prasugrel, involves two steps: an esterase-dependent hydrolysis of its acetate function leading to thiolactone 6 and a cytochrome P450 (P450)-catalyzed oxidative cleavage of this thiolactone. This article shows that this second step involves the intermediate formation of a sulfenic acid 9 that has been trapped by dimedone during the metabolism of prasugrel by rat and human liver microsomes. The dimedone adduct has been characterized by mass spectrometry (MS) and (1)H and (13)C NMR spectroscopy. This article also describes the fate of the sulfenic acid intermediate in liver microsomes in the presence of various nucleophiles. Its reaction with a water-soluble phosphine cleanly leads to the corresponding thiol 7, which has been reported as the pharmacologically active metabolite of prasugrel. Its reaction with glutathione (GSH) leads to mixed disulfide 11, which may further react with GSH in excess to provide thiol 7. Experiments using microsomal incubations in the presence of (18)O(2) and (18)OH(2) have provided the first data on the mechanism of the P450-catalyzed oxidative cleavage of thiolactones such as 6. They indicate that sulfenic acid 9 is derived from a nucleophilic attack of H(2)O either directly on the electrophilic keto group of intermediate keto-sulfoxide 12, which is formed by P450-dependent S-oxidation of 6, or on the keto group of a cyclic sulfenic ester 13, which could derive from the rearrangement of 12. These data provide a first detailed mechanism for the metabolic activation of prasugrel to its pharmacologically active metabolites such as thiol 7.

Human Liver Cytochrome P450 Enzymes and Microsomal Thiol Methyltransferase Are Involved in the Stereoselective Formation and Methylation of the Pharmacologically Active Metabolite of Clopidogrel

“…Thiolactone 2 (a 1:1 mixture of 2a and 2b) was incubated for 15 minutes at 37°C with HLMs in the presence of NADPH and GSH (5 mM) to reduce sulfenic acid 4 to the corresponding thiols (H1-H4) (Dansette et al, 2009(Dansette et al, , 2010Zhang et al, 2013). A UPLC-Q/TOF MS study of the incubate showed the formation of four thiol isomers exhibiting a molecular ion (ESI + ) corresponding to [M + H] + that was characterized by two peaks at m/z = 356.073 and 358.071 with the ratio expected for the 35 Cl and 37 Cl isotopes.…”

Cytochrome P450-Activated Prodrugs

“…Conversion of this intermediate to the ring opened carboxy thiol product requires a P450 oxidation to cleave the ring and give a carboxy sulfenic acid product that is reductively converted to the pharmacologically active thiol drug [85]. The sulfenic acid can be chemically trapped [85–87]. As reported for clopidogrel, thioesterase opening of the thiolactone yields a thiol derivative with the double bond at a different ring position.…”

Formation, Reactivity, and Antiplatelet Activity of Mixed Disulfide Conjugates of Clopidogrel

“…It is well documented that P450-mediated bioactivation involves two consecutive oxidative steps (Dansette et al, 2010(Dansette et al, , 2012a; clopidogrel is first monoxygenated to 2-oxoclopidogrel, which is, in turn, oxidized to the AM in the second step. Although it has been argued that esterase PON1 is responsible for converting 2-oxoclopidogrel to the AM (Bouman et al, 2011), increasing evidence supports the idea that 2-oxoclopidogrel is converted to the AM via a sulfenic acid intermediate (Dansette et al, 2009(Dansette et al, , 2010(Dansette et al, , 2012a, as illustrated in Scheme 1. According to Scheme 1, 2-oxoclopidogrel is first oxidized to a sulfenic acid intermediate by P450s.…”