1268
Chem. Res. Toxicol. 2010, 23, 1268–1274
Formation and Fate of a Sulfenic Acid Intermediate in the Metabolic Activation of the Antithrombotic Prodrug Prasugrel† Patrick M. Dansette,* Ste´phanie The´bault, Gildas Bertho, and Daniel Mansuy Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, UniVersite´ Paris Descartes, 45 rue des Saints-Pe`res, 75270 Paris Cedex 06, France ReceiVed April 9, 2010
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 1H and 13C 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 18O2 and 18OH2 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 H2O either directly on the electrophilic keto group of intermediate ketosulfoxide 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. Introduction Tetrahydro-thienopyridines, ticlopidine 1a and clopidogrel 1b, are antithrombotic prodrugs that must be metabolized in ViVo into a pharmacologically active 4-mercapto-3-piperidinylideneacetic acid derivative, 4, in order to exert their activity as antagonists of the platelet ADP receptor P2Y12 (1-3). Their metabolic activation occurs in two steps that are catalyzed by cytochromes P450 (P450s) (Figure 1) (1, 2). The first step is a classical P450-dependent monooxygenation of the thiophene ring by NADPH and O2 (4-6) that leads to the thiolactone metabolites 2a and 2b. Formation of thiols 4a and 4b, which are proposed to be responsible for the irreversible inhibition of ADP-induced platelet aggregation, also requires the involvement of a P450-catalyzed oxidative step with the consumption of NADPH and O2. We have recently shown that this step is a P450-catalyzed cleavage of the thiolactone ring of metabolites 2 leading to the formation of reactive sulfenic acid intermediates 3, which have been trapped by dimedone (7). In the presence of glutathione (GSH) in excess, these sulfenic acids are reduced into the corresponding thiols 4 (7). Prasugrel, 5, is the newest member of the class of tetrathydrothienopyridine antithrombotic prodrugs. It is also an irreversible † This mechanism for the formation of 7 from 6 was also proposed by P.M.D. and D.M. in a poster at the Biological Reactive Intermediates (BRI VII) Symposium, Tucson (USA), January 6, 2006, and the results in agreement with this mechanism, described in this article, have been presented by the authors of this article in a poster at a meeting of the French Society of Medicinal Chemistry, Paris, February 5, 2010. * Corresponding author. Patrick M. Dansette, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Universite´ Paris Descartes, 45 rue des Saints-Pe`res, 75270 Paris Cedex 06, France. Tel: +33 142862191. Fax: +33 142868387. E-mail:
[email protected].
inhibitor of the platelet receptor P2Y12 after metabolic conversion in ViVo to a pharmacologically active 4-mercapto-3piperidinyliden-acetic acid derivative 7 (8, 9) that is analogous to 4a and 4b (Figure 2). Metabolic conversion of 5 to 7 occurs in two steps: (i) an esterase-catalyzed hydrolysis of 5 leading to the thiolactone metabolite 6 and (ii) a cleavage of the thioester bond of 6 leading to 7 (10). The precise nature of this second step is not known presently, even if it has been shown that it involves an oxidation catalyzed by P450 enzymes (P450 3A, P450 2B6, P450 2C9, or P450 2C19 in humans) (10-12). On the basis of the mechanism recently reported for the metabolic transformation of thiolactones 2a and 2b derived from ticlopidine and clopidogrel, respectively, into active thiols 4a and 4b (7), it was tempting to propose (13) that the formation of 7 from 6 would involve three steps: (i) a P450-catalyzed Soxidation of 6 leading to a very reactive, electrophilic thiolactone S-oxide, (ii) a hydrolysis of its CO-SO bond with the formation of the sulfenic acid of 7, and (iii) a reduction of this sulfenic acid leading eventually to 7. This article reports that a sulfenic acid intermediate that was trapped by the specific trapping agent for sulfenic acids, dimedone, is formed during metabolic activation of prasugrel by liver microsomes. It also describes the first data on the mechanism of formation of this sulfenic acid and on the fate of such xenobiotic-derived sulfenic acid intermediates in liver microsomes in the presence of various nucleophiles or reducing agents, such as dimedone, phosphines, and thiols. These data should allow us to better understand the bioactivation of antithrombotic tetrahydro-thienopyridine prodrugs and the different steps involved in their metabolic transformation into the
10.1021/tx1001332 2010 American Chemical Society Published on Web 06/17/2010
Formation and Fate of a Sulfenic Acid Intermediate
Chem. Res. Toxicol., Vol. 23, No. 7, 2010 1269
Figure 1. Mechanism of the metabolic activation of ticlopidine, 1a, and clopidogrel, 1b.
Figure 2. Metabolism of prasugrel into thiol 7.
corresponding 4-mercapto-3-piperidinyliden-acetic acid metabolites 4 and 7. During the preparation of this manuscript, data reporting the formation of mixed disulfides between thiol 7 and 2-nitro-5thiobenzoic acid or GSH upon the metabolism of thiolactone 6 by recombinant P450 3A4 in the presence of NADPH and 2-nitro-5-thiobenzoic acid or GSH have appeared in the online version of an article (14). Our data are in agreement with these results.
Experimental Procedures Chemicals and Biochemicals. Prasugrel base (racemate) was obtained from Bepharm (Shangai, China). 18OH2 (97% enriched in 18 O) and 18O2 (95% 18O) were purchased from Eurisotop (Saclay, France). All other products including enzymes were from SigmaAldrich (St Quentin Fallavier, France). Microsomal Incubations. Microsomes (2 nmol P450/mg protein) were prepared from the liver of rats pretreated by phenobarbital (1 g/L drinking water for 7 days) as described previously (15). Human liver microsomes were obtained from BD-Gentest (Le Pont de Claix, France). Typical incubations were performed in potassium phosphate buffer (0.1M, pH 7.4) containing microsomes (0.1 to 2 µM P450), 1 mM NADP, 15 mM glucose-6-phosphate, 2 u/mL of glucose-6-phosphate dehydrogenase, prasugrel (5-500 µM), and trapping agent (1-2 mM dimedone, 1 mM tris-carboxyethylphosphine (TCEP), or 1-10 mM GSH) at 37 °C. Some incubations were also performed in the presence of N-ethyl-maleimide (NEM) or N-methyl-maleimide (NMM) (0.1 to 4 mM) as thiol trapping agents. Reactions were stopped either by adding one-half volume of CH3CN/CH3COOH (9:1) and centrifugation of precipitated proteins (12000g, 10 min) or by solid phase extraction using Oasis columns (Waters, St Quentin en Yvelines, France) (1 mL loading, 1 mL water wash, and 1 mL CH3OH elution), evaporation of the solvent with N2, and redissolution in HPLC mobile phase. Experiments in 18OH2 were done under identical conditions except that concentrated liver microsomes and glucose-6-phosphate dehydrogenase in 9 µL of buffer in 16OH2 were added to a buffer containing all of the other reactants that had been lyophilized and redissolved in 200 µL of 18OH2, to start the reaction. Thus, the final incubation medium was 92% enriched in 18OH2. Experiments in 18O2 were performed in a 10 mL test tube closed with a rubber septum containing the microsomal incubations in 16OH2 described above. After degassing by several cycles of vacuum and argon, 2
mL of 18O2 was injected into the test tube with a gastight syringe to start the reaction. HPLC-MS Studies. HPLC-MS studies were performed on a Surveyor HPLC instrument coupled to a LCQ Advantage ion trap mass spectrometer (Thermo, Les Ulis, France), using a Gemini C18 column (100 × 2 mm, 3 µm; Phenomenex), and a 20 min linear gradient of 80% A-20% B mixture to 100% B in 20 min (A ) 10 mM ammonium acetate, pH 4.6, and B ) CH3CN/CH3OH/H2O (7:2:1)) at 200 µL/min. Mass spectra were obtained by electrospray ionization (ESI) in positive or negative ionization mode detection under the following conditions: source parameters, sheath gas, 20; auxiliary gas, 5; spray voltage, 4.5 kV; capillary temperature, 200 °C; capillary voltage, 15 V; m/z range for MS recorded generally between 300 and 700 (except for exploratory experiments with a wider range 300-800). Preparation of the Dimedone Adducts 8. Semipreparative incubations of 300 µM 5 in phosphate buffer (100 mM, pH 7.4) in a total volume of 50 to 60 mL were performed with rat liver microsomes from phenobarbital pretreated rats (1 mg protein/ml) in the presence of a NADPH generating system (15 mM glucose6-phosphate, 1 mM NADP, and 2u/mL of glucose-6-phosphate dehydrogenase) and 1 mM dimedone for 1 h at 37 °C. Then 20 µL of CH3COOH/mL incubation was added, and the medium was centrifuged at 3000g for 10 min. The supernatant was loaded on a SepPak C18 classic column (Waters, St Quentin en Yvelines, France). After washing with 2 mL of water, the metabolites were eluted with 2 mL of CH3OH. After concentrating under vacuum, the mixture was purified by 5 repetitive HPLC separations on a Hypersil Mos column (250 × 4.6 mm, 5 µm) using a 20 min gradient of A ) ammonium acetate (0.1M, pH 4.6) to B ) CH3CN/ CH3OH/H2O (7:2:1) at 1 mL/min. Collected fractions were lyophilized and taken twice in D2O and transferred to capillary tubes for NMR analysis. Because of proton exchange, another batch was neutralized with NH4HCO3 and dissolved in 400 µL of CD3CN containing 10% H2O. NMR Experiments. 1H NMR spectra of 8 (acetate salt in D2O or base in CD3CN/H2O (9:1)) were obtained on a Bruker Avance 500 spectrometer (500 MHz) at 27 °C. Chemical shifts (δ) are given in ppm relative to (CH3)4Si. Abbreviations used for singlet, doublet, doublet of doublets, broad singlet, and massif are s, d, dd, bs, and m, respectively.
Results Microsomal Oxidation of Prasugrel in the Presence of Dimedone. Incubation of an aerobic suspension of rat or human
1270
Chem. Res. Toxicol., Vol. 23, No. 7, 2010
liver microsomes in phosphate buffer, pH 7.4, containing 100 µM prasugrel, 5, for 0.5 h at 37 °C, led to an almost complete transformation of 5 into thiolactone 6, as shown by an HPLCMS study. Accordingly, the mass spectrum (ESI+) of metabolite 6, which exhibited a molecular ion at m/z ) 332 [M + H+], and its MS-MS spectrum were in good agreement with the MS data previously reported for 6 (16). Actually, the prasugrel base used in this study was a racemate, and its thiolactone metabolite 6 appeared as a diastereomeric mixture of two racemic pairs of enantiomers corresponding to two peaks of equal intensity in HPLC. Identical incubations of 5 in the absence of liver microsomes did not lead to the formation of 6. Hydrolysis of the ester function of 5 leading to 6 has been shown to be efficiently catalyzed by esterases (17) including microsomal esterases (18). When the incubation was performed in the presence of 1 mM NADPH and 1 mM dimedone, a Cnucleophile usually used as a trapping agent for sulfenic acids (19, 20), two new major products were formed in almost equal quantities. These products appeared as not completely separated HPLC peaks and exhibited identical mass spectra (ESI+) with a molecular ion at m/z ) 488 [M + H+] (Figure 3) corresponding to the dimedone adduct 8 (Figure 4), the expected product from a nucleophilic attack of dimedone on the electrophilic sulfur atom of sulfenic acid 9. Fragmentation of this ion by MS-MS led to a major ion at m/z ) 317, which results from the loss of the S-dimedone fragment. The ESI- mass spectrum of 8 exhibited a molecular ion at m/z ) 486 [M - H+], as expected for a carboxylic acid. After semipreparative incubations and purification by HPLC, the mixture of the two diastereomers was studied by 1H NMR, using two-dimensional NMR methods (correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), heteronuclear multiple bond correlation (HMBC), and heteronuclear single quantum coherence spectroscopy (HSQC)). This study allowed us to assign all of the signals to the protons of the molecules and to find the 13C chemical shifts of most of their carbons. These NMR data were in complete agreement with the structure shown in Figure 4 for the dimedone adduct 8 existing as a 50/50 diastereomer mixture. Moreover, the NMR data found for compound 8 were in good agreement with those previously reported for the dimedone adduct from ticlopidine sulfenic acid metabolite (7) at least for the protons and 13C that can be compared (Table 1). This is true for the characteristic 1 H and 13C NMR signals that appeared at 5.36 and 5.46 ppm for the vinylic protons of the diastereomers of 8 and at 118.2 and 118.6 for their vinylic carbons. The data for the dimedone part were similar to those of the ticlopidine analogous adduct (7). For instance, the methyl protons of 8 and of the ticlopidine adduct appeared as singlets (6H) at 0.87 and 0.98 ppm, respectively; the corresponding 13C signals appeared at 27.4 and 27.6 ppm, respectively. It is noteworthy that under the above-mentioned conditions microsomal oxidation of 5 in the presence of dimedone almost exclusively led to adduct 8 in a yield (based on consumed 5) of about 90%. Microsomal Oxidation of Prasugrel in the Presence of P- and S-Nucleophiles. The active metabolite of prasugrel, which could be derived from the reduction of sulfenic acid 9 and which has been previously observed upon the metabolism of prasugrel in ViVo, was thiol 7 (10, 11, 16). However, we did not detect any formation of 7 in incubations of prasugrel with liver microsomes in the presence of NADPH and in the presence or absence of dimedone (no peak corresponding to the expected mass spectrum with a molecular ion at m/z ) 350). Then
Dansette et al.
Figure 3. Mass spectra (ESI+) of metabolites 8 (A), 7 (B), 10 (C), and 11 (D). Their MS data were obtained after the incubation of 5 with phenobarbital-pretreated rat liver microsomes in the presence of NADPH and a trapping agent (dimedone, TCEP, TCEP and NEM, and glutathione, respectively), and analysis of the reaction mixture by HPLC-MS (conditions described in Experimental Procedures). A′, B′, C′, D′: collision induced dissociation (CID) spectra of the molecular ion of 8, 7, 10, and 11, respectively.
identical incubations were performed with 100 µM prasugrel, 1 mM NADPH, and a reducing agent, the water-soluble phosphine P(CH2CH2COOH)3 (TCEP) (1 mM), since phosphines were previously reported to reduce sulfenic acids to the
Formation and Fate of a Sulfenic Acid Intermediate
Chem. Res. Toxicol., Vol. 23, No. 7, 2010 1271
Figure 4. Formation of a sulfenic acid upon microsomal metabolism of prasugrel and the fate of this intermediate in the presence of C-, P-, and S-nucleophiles.
corresponding thiols (21-24). Under those conditions, prasugrel was metabolized with the major formation of thiol 7 that appeared as a 50/50 mixture of diastereomers leading to two HPLC peaks with very close retention times. Their mass spectra (ESI+) were identical and characterized by a molecular ion at m/z 350 [M + H+] with MS2 fragments at m/z ) 316 (loss of SH2), 206, 177, and 172 (Figure 3). These MS characteristics were highly similar to those previously reported for metabolite 7 in the literature (16). When identical microsomal incubations with prasugrel, NADPH, and TCEP were performed in the presence of 1 mM N-ethylmaleimide (NEM), a well-known trapping agent for thiols, the major product detected by HPLC was 10, the NEM adduct of thiol 7 (Figure 4). Compound 10 appeared as a 50:50 mixture of diastereomers that were characterized by a mass spectrum (ESI+) exhibiting a molecular ion at m/z ) 475 [M + H+] and MS2 fragments at m/z ) 318 and 206 (16). Besides C-nucleophiles such as dimedone and P-nucleophiles such as TCEP, another class of nucleophiles known to efficiently react with sulfenic acid intermediates are thiols. Thus, incubation of aerobic rat liver microsomes with 100 µM prasugrel, 1 mM NADPH, and 1 mM glutathione (GSH), for 30 min at 37 °C, led to new major products whose mass spectra exhibited a molecular ion at m/z ) 655, as expected for mixed disulfide diastereomers 11 resulting from the reaction of 9 with GSH, and MS2 fragments at m/z ) 637, 526, 382, 348, and 317 (Figure 3). Metabolite 11 also appeared as a mixture of diastereomers corresponding to four peaks (two major peaks of equal intensity and two minor peaks) in HPLC. At larger GSH concentrations (between 2 and 5 mM), increasing amounts of thiol 7 were formed at the expense of 11. Microsomal incubations of 100 µM prasugrel 5, in the presence of 1 mM NADPH and 1 mM dimedone and increasing concentrations of TCEP (from 1 to 5 mM), showed a progressive decrease of the formation of 8 with a concomitant appearance of only one new product, thiol 7. Similar competition experiments on microsomal oxidation of 5 in the presence of 1 mM dimedone and increasing concentrations of GSH (from 80 µM to 5 mM) led to a progressive decrease of the formation of 8
with the concomitant increase of only two new products, thiol 7 and the GSH adduct 11. The 7/11 ratio increased upon increasing the GSH concentration; 11 was the only new product formed with 80 µM GSH, whereas the highest amount of 7 was observed with 5 mM GSH. These data showed that TCEP and GSH are competing with dimedone for the reaction with the intermediate 9. They are in agreement with the formation of 7 mainly deriving from sulfenic acid 9 as shown in Figure 4. The aforementioned data provide a clear evidence for the intermediate formation of sulfenic acid 9 upon aerobic microsomal incubation of prasugrel in the presence of NADPH. The formation of 9 was shown from its reactions with the three different nucleophiles used as trapping agents. In the presence of the C-nucleophile dimedone, the stable adduct 8 was formed from direct reaction of the nucleophilic carbon of dimedone on the electrophilic sulfur atom of 9. Similarly, in the presence of GSH, the glutathione adduct 11 was formed from the reaction of the nucleophilic sulfur atom of GSH on the sulfur atom of 9. In the presence of the water-soluble phosphine TCEP, a nucleophilic attack of its phosphorus atom on the sulfur atom of 9 should lead to the pentavalent phosphorus intermediate shown in Figure 5, which should decompose with the formation of phosphine oxide OdP(CH2CH2COOH)3 and thiol 7 (Figure 5). Such a mechanism has been previously shown for the reduction of stable sulfenic acid by phosphines (24). Globally, the latter reaction is a reduction of sulfenic acid 9 into the corresponding thiol 7 by the soluble phosphine, TCEP, a wellknown reducing agent (25). When used in large excess (5 mM), GSH also acted globally as a reducing agent that transformed 9 into 7. However, this reaction occurred via the disulfide intermediate 11, a nucleophilic attack of GSH on the cysteinyl sulfur atom of 11 leading to 7 and GSSG. Mechanism of Microsomal P450-Dependent Oxidative Cleavage of the C-S Bond of Thiolactone 6: Use of 18O2 and 18OH2. The formation of sulfenic acid 9 in liver microsomes in the presence of NADPH is catalyzed by P450s, as the addition of 100 µM N-benzyl-imidazole, a well-known inhibitor of these hemoproteins (26), to the above-mentioned microsomal incuba-
1272
Chem. Res. Toxicol., Vol. 23, No. 7, 2010
Dansette et al.
Table 1. Comparison of the 1H and 13C NMR Characteristics of Dimedone Adducts Derived from Prasugrel, 8 (Two Diastereomers 8a and 8b), and from Ticlopidine (Previously Described in Ref 7)a
ticlopidine adduct 1
atom Nb
δH
2
3.56 (dd, 1H) 4.73 (dd, 1H) 4.71 (bs, 1H) 2.15, 2.22 (m, 2H) 3.52 (d, 1H) 3.69 (bd, 1H) 4.57 (bs, 2H) 7.6 (dd, 1H) 7.50 (dt, 1H) 7.44 (t, 1H) 7.58 (dd, 1H) 5.71 (bs, 1H) 2.30 (bs, 4H) 0.98 (s, 6H)
4 5 6 7 10 11 12 13 14 19 21 23 24
13
isomer 8a 1
isomer 8b 13
1
δ C
δH
δ C
54.8 2.85 3.27 38.8 4.85 26.5 1.70 1.93 48.7 2.65 2.70 58.1 4.61 130.7 7.28 132.5 7.08 127.9 7.14 133.7 7.36 128.8 5.46 49.6 2.19 27.6 0.87 2.22 0.74 0.87
(d, 1H) (d, 1H) (bs, 1H) (d, 1H) (m, 1H) (m, 1H) (m, 1H) (s, 1H) (d, 1H) (m, 1H) (m, 1H) (d, 1H) (s, 1H) (bs, 4H) (s, 6H) (m, 1H) (m, 2H) (m, 2H)
53.5 3.0 (d, 1H) 3.44 (d, 1H) 41.0 4.85 (bs, 1H) 29.5 1.66 (d, 1H) 1.87 (m, 1H) 46.9 2.51 (m, 1H) 2.54 (m, 1H) 71.7 4.57 (s, 1H) 130.2 7.28 (d, 1H) 122.1 7.08 (m, 1H) 124.1 7.14 (m, 1H) 130.9 7.36 (d, 1H) 118.2 5.36 (s, 1H) 45.9 2.19 (bs, 4H) 27.4 0.87 (s, 6H) 17.6 2.22 (m, 1H) 0.74 (m, 2H) 10.8 0.87 (m, 2H)
δH
13
δ C 55 41.0 29.3 44.5 71.9 130.2 122.1 124.1 130.9 118.6 45.9 27.4 17.6 10.8
a
Compound 8 (base form) in CD3CN/H2O (9:1); ticlopidine adduct (acetate salt) in D2O. The spectrum of 8 (acetate form) in D2O was also obtained; however, under these conditions, the signal of H7 was absent because of an exchange with D2O. The H7 signal was present in the spectrum of 8 (base form) in CD3CN/H2O (9:1). Compounds 8a and 8b appeared as a 1:1 mixture, but the combination of 2D spectra allowed us to assign the signals to each isomer (see Supporting Information).
Figure 5. Possible mechanism for the reduction of 9 by TCPE according to ref 24.
tions of prasugrel in the presence of NADPH and dimedone almost completely inhibited the formation of 8 (>95% inhibition). Involvement of P450s in this microsomal oxidation of 6 is in agreement with the literature data indicating that P450 3A, P450 2B6, P450 2C9, and P450 2C19 are involved in the bioactivation of prasugrel into its pharmacologically active metabolite (11, 12). Two pathways were a priori possible for the formation of 9 from the oxidation of 6: (1) a hydrolytic opening of the thiolactone ring of 6 leading to 7, followed by a P450-dependent S-hydroxylation of 7, or (2) a P450-dependent oxidation of 6 leading to the corresponding keto-sulfoxide 12 shown in Figure 4, followed by a hydrolysis of its very reactive CO-SO bond. A priori, three pathways could be considered for the hydrolytic opening of the five-membered ring of intermediate keto-sulfoxide 12 (Figure 6): (a) a nucleophilic attack of H2O
Figure 6. Possible pathways for the formation of sulfenic acid 9 from keto-sulfoxide 12.
on its keto group leading directly to 9 after the protonation of the SO moiety, (b) a rearrangement of keto-sulfoxide 12 coming from an intramolecular attack of the O- atom on the keto group, with the formation of a six-membered cyclic sulfenic ester 13 (Figure 6), followed by the reaction of H2O on the keto group, or (c) the same rearrangement but followed by reaction of H2O on the electrophilic sulfur atom of the six-membered sulfenic ester. In pathways a and b, one of the two oxygen atoms of the carboxylate group of 9 is coming from H2O, whereas in pathway c, one of the oxygen atoms of the carboxylate group of 9 should come from O2 during P450-dependent sulfoxidation of 6. Labeling experiments using either 18O2 or 18OH2 were performed in order to further study the mechanism of the formation 9 and to discriminate among pathways a, b, or c. Incubations of 100 µM prasugrel 5 with rat liver microsomes in the presence of 1 mM dimedone and 18O2 were performed either in the presence or in the absence of 1 mM NADPH. A study of the reaction mixtures by HPLC-MS and MS2 showed no significant incorporation of 18O into thiolactone 6 (experiments without NADPH) or into the dimedone adduct 8 (experiments with NADPH) (eq 1, RLM ) rat liver microsomes). The levels of 18O incorporation were measured on the basis of an analysis of the MS peaks of 8 at m/z ) 488 and 490. Identical incubation under 16O2 but in 18OH2 showed no significant 18O incorporation into thiolactone 6; however, they showed a major incorporation of 18O (>85%) into the dimedone adduct 8 (eq 2), which from MS2 data was localized in the carboxylic acid group. The control experiment showed that similar incubations of adduct 8 with or without rat liver microsomes in 18OH2 did not lead to any significant incorporation of 18O in 8 (
