Metabolic Oxidative Cleavage of Thioesters: Evidence for the

Such an oxidative cleavage of a thioester bond has been reported to occur during metabolism of drugs or agrochemicals such as disulfiram (2), fluticas...
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Chem. Res. Toxicol. 2009, 22, 369–373

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Communication Metabolic Oxidative Cleavage of Thioesters: Evidence for the Formation of Sulfenic Acid Intermediates in the Bioactivation of the Antithrombotic Prodrugs Ticlopidine and Clopidogrel Patrick M. Dansette,* Julie Libraire, 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 December 19, 2008

Metabolic cleavage of the CO-S bond of some thioesters RCOSR′ with the formation of RCOOH requires a monooxygenase-dependent oxidative activation of this bond. The nature of the S-containing product(s) resulting from this cleavage remains unclear in most cases. This communication provides the first evidence for the formation of sulfenic acid intermediates 4a and 4b during the oxidative cleavage of the CO-S bond of thiolactone metabolites 2a and 2b of antithrombotic prodrugs, ticlopidine and clopidogrel, by rat and human liver microsomes. These intermediates have been trapped by dimedone, and the corresponding adducts 5a and 5b have been characterized by mass spectrometry (MS) and 1H and 13C NMR spectroscopy. Their formation is monooxygenase-dependent and almost completely inhibited by microsomal cytochrome P450 inhibitors. Moreover, they were also formed upon incubation with microsomes containing recombinant human P450 3A4, 3A5, 2C8, 2C9, 2C19, 2D6, or 1A2. In the presence of thiols such as mercaptoethanol, N-acetylcysteine, or glutathione, microsomal incubations of 2a led to mixed disulfides that have been characterized by MS and should result from reaction of 4a with these thiols. At high thiol concentrations, one observed in HPLC-MS the formation of a product exhibiting the MS expected for the previously described thiol metabolite 3a, a reduction product of 4a that has been reported as the pharmacologically active metabolite of ticlopidine. These data provide the first evidence for the formation of sulfenic acid reactive metabolites upon P450-catalyzed oxidative cleavage of thioesters. They also provide a first detailed mechanism for the previously described formation of pharmacologically active thiols such as 3a upon oxidative metabolism of ticlopidine and clopidogrel. Introduction Metabolism in mammals of several compounds involving a thioester function, RCO-SR′, often leads to the corresponding carboxylic acid, RCOOH (1). For some of them, this cleavage of the thioester bond does not derive from a simple hydrolysis but involves a monooxygenase-dependent oxidative activation of the thioester function (eq 1). Such an oxidative cleavage of a thioester bond has been reported to occur during metabolism of drugs or agrochemicals such as disulfiram (2), fluticasone (3), dithiopyr (4), ticlopidine (5), clopidogrel (6), and prasugrel (7). The nature of the S-containing product(s) resulting from this oxidative cleavage of the thioester bond remains unclear, even though the eventual formation of the corresponding thiol, R′SH, has been shown in the case of the three latter compounds.

Tetrahydrothienopyridines, ticlopidine 1a and clopidogrel 1b, are antithrombotic prodrugs that must be metabolized in vivo into a pharmacologically active 4-mercapto-3-piperidinylidene* To whom correspondence should be addressed. Tel: +33 142862191. Fax: +33 142868387. E-mail: [email protected].

Figure 1. Metabolic activation of ticlopidine, 1a, and clopidogrel, 1b, into their pharmacologically active thiol derivatives 3a (5) and 3b (6). Metabolite 3b was identified after trapping with acrylonitrile leading to 3′b (6).

acetic acid derivative, 3, in order to exert their activity as antagonists of the platelet ADP receptor P2Y12 (5, 6, 8, 9). Their metabolic activation occurs in two steps that are catalyzed by cytochromes P450 (Figure 1) (5, 6). The first step is a classical P450-dependent monooxygenation of the thiophene ring by NADPH and O2 (10-12) that leads to the thiolactone metabolites 2a and 2b. The formation of thiols 3a and 3b, which are proposed to be responsible for the irreversible inhibition of ADP-induced platelet aggregation, is also a cytochrome P450catalyzed reaction that requires NADPH and O2. It is presently unclear why the transformation of 2a and 2b into 3a and 3b,

10.1021/tx8004828 CCC: $40.75  2009 American Chemical Society Published on Web 01/26/2009

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respectively, that could result from a simple hydrolysis of the thioester bond of 2a and 2b requires the presence of cytochrome P450 and its cofactors, NADPH and O2. A possible explanation would be that this cleavage of the CO-S bond of thiolactones 2a and 2b could be the result of three successive reactions, (i) an S-oxidation leading to an electrophilic thiolactone S-oxide that would be much more reactive toward nucleophiles including H2O, (ii) a hydrolysis of its CO-SO bond with formation of the sulfenic acids of thiols 3a and 3b, and (iii) reduction of these sulfenic acid intermediates leading eventually to 3a and 3b. This communication reports the first evidence showing that sulfenic acid reactive intermediates are formed during P450dependent oxidation of thioesters such as 2a and 2b. These intermediates have been trapped by dimedone, a C-nucleophile usually used as a trapping agent for sulfenic acids (13, 14). They also react with thiol nucleophiles such as mercaptoethanol or glutathione to give the final reduced metabolites 3a and 3b via mixed disulfide intermediates.

Experimental Procedures Chemicals and Biochemicals. Ticlopidine was a gift from Sanofi (France); 2-oxo-ticlopidine 2a was synthesized by the method of Boigegrain et al. (15) by Lucie Durand-Gasselin. [R,S]-2-Oxoclopidogrel 2b was purchased from Toronto Research Chemicals (Toronto, Canada). All other products including enzymes were from Sigma-Aldrich (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 (16). Human liver microsomes and insect cell microsomes expressing recombinant human cytochromes P450 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 (0.1-2 µM cytochrome P450), 1 mM NADP, 15 mM glucose-6-phosphate, 2 u/mL of glucose-6-phosphate dehydrogenase, substrate (5-500 µM), and trapping agent (1-2 mM dimedone or 1-10 mM thiols) at 37 °C. 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. 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 Biobasic C18 column (100 mm × 2 mm, 3 µm) and a 20 min linear gradient of A ) ammonium acetate (10 mM, pH 4.6) to B ) CH3CN: CH3OH:H2O (7:2:1) mixture at 200 µL/min. Mass spectra were obtained by electrospray ionization (ESI) in positive 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; and m/z range for MS recorded generally between 130 and 620. ESI mass spectra in negative ionization mode detection (ESI-) were also obtained for 5a and 5b. Preparation of the Adducts 5a and 5b. Semipreparative incubations of 300 µM 2a or 2b in phosphate buffer (100 mM, pH 7.4) in a total volume of 20-30 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 2 u/mL of glucose-6-phosphate dehydrogenase) and 1 mM dimedone for 1 h at 37 °C. Then, 20 µL 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). After they were washed with 2 mL of water, the metabolites were eluted with 2 mL of CH3OH. After concentration under vacuum, the mixture was purified by three repetitive HPLC separations on a Hypersil Mos column (250 mm

Communications × 4.6 mm, 5 µm) using a 20 min gradient of A ) ammonium acetate (0.1 M, pH 4.6) to B ) CH3CN:CH3OH:H2O (7:2:1) at 1 mL/min. Collected fractions were lyophilyzed and taken twice in D2O and transferred to Shigemi tubes for NMR analysis. NMR Experiments. 1H NMR spectra of 5a and 5b (in D2O) were obtained on a Bruker Avance 500 spectrometer (500 MHz) at 27 and 7 °C, respectively. 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 and Discussion Incubation of an aerobic suspension of rat or human liver microsomes in phosphate buffer, pH 7.4, containing 300 µM 2a, 1 mM NADPH, and 1 mM dimedone for 1 h at 37 °C led to the formation of a new major product that was detected by HPLC-MS and purified by preparative HPLC. The mass spectrum (ESI+) of this product (Figure 2A) exhibited a molecular ion (isotopic cluster showing the presence of one Cl atom; m/z ) 436 [M + H+] for 35Cl) corresponding to the dimedone-adduct 5a, the expected product from an attack of dimedone on the electrophilic sulfur atom of sulfenic acid 4a. Fragmentation of this ion by MS-MS led to an ion at m/z ) 264 (for 35Cl), which results from the loss of 172 Da corresponding to the S-dimedone fragment (Figure 2A′). A detailed analysis of the 1H NMR spectrum of this metabolite, using two-dimensional (2D) NMR methods [total correlation spectroscopy, nuclear Overhauser effect spectroscopy, heteronuclear single quantum coherence (HSQC), and heteronuclear multiple-bond correlation], allowed us to assign all of the signals to the protons of the molecule and also to find the 13C chemical shifts of most carbons of the molecule (Table 1). These NMR data were in complete agreement with the structure shown for adduct 5a in Figure 3 (the 1H NMR and 2D HSQC 1H-13C NMR spectra of 5a are shown in the Supporting Information). They include characteristic 1H and 13C NMR signals at 5.71 and 128.8 ppm for the vinylic CH moiety and at 2.3 and 49.6 and 0.98 and 27.6 ppm for the CH2 and CH3 groups of the dimedone part of the molecule. Identical experiments were performed on compound 2b and gave a major new product, whose mass spectrum (ESI+, m/z ) 494 [M + H+] for 35Cl, fragments in MS-MS at 322 and 354 resulting from the loss of 2-mercapto-dimedone and dimedone) (see the Supporting Information) and 1H and 13C NMR characteristics (Table 1) were in complete agreement with the structure indicated for adduct 5b in Figure 3. In a general manner, these NMR characteristics of 5a and 5b were very similar to those previously reported for an adduct 3′b, derived from a Michael addition of the SH group of 3b to acrylonitrile (6) (Figure 1) (see Table 1). They also were in good agreement with the NMR characteristics reported for prasugrel derivatives, exhibiting similar structures (17). It is noteworthy that in the case of 2b (racemate form), the dimedone adduct 5b existed as a mixture of two couples of diastereoisomers (55:45) for which NMR signals could be assigned via the previously indicated 2D NMR methods. Identical incubations of 2a but in the absence of NADPH neither led to any formation of adduct 5a nor to any significant consumption of 2a. Moreover, identical incubations of 2a in the presence of 100 µM SKF 525A or N-benzylimidazole, which are usual microsomal cytochromes P450 inhibitors (18), led to a 90-95% inhibition of adduct 5a formation. Similar results were obtained in the case of 2b. This key role of cytochromes P450 in oxidation of 2a and 2b with formation of 5a and 5b was further confirmed by experiments using recombinant human

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Figure 2. Mass spectra of metabolites 5a and 6a. The MS of 5a and 6a were obtained after incubation of 2a with phenobarbital-pretreated rat liver microsomes in the presence of NADPH and a trapping agent (dimedone, mercaptoethanol, N-acetylcysteine, or glutathione) and analysis of the reaction mixture by HPLC-MS (conditions were described in the Experimental Procedures). (A) MS (ESI+) of 5a [as expected, the MS (ESI-) of 5a exhibited a molecular ion at m/z ) 434 [M - H]- for 35Cl); (A′) collision-induced dissociation (CID) spectrum of the ion at m/z ) 436; and (B-D) MS (ESI+) of adducts 6a with mercaptoethanol, N-acetylcysteine, or glutathione, respectively.

P450s (19) expressed under a catalytically active form in bacculovirus-infected insect cells. Actually, P450 3A4, 3A5, 2C8, 2C9, 2C19, 2D6, and 1A2 were found to efficiently catalyze the oxidation of 2a and 2b, with the formation of 5a and 5b, respectively, in the presence of NADPH and dimedone, whereas P450 2E1 was unable to catalyze this reaction. In agreement with this ability of several human microsomal cytochromes P450 to catalyze the oxidation of 2a and 2b with the formation of 5a and 5b, it was found that besides liver microsomes from rats pretreated with phenobarbital, liver

microsomes from untreated rats or from rats pretreated with dexamethasone or β-naphthoflavone were also able to efficiently catalyze these oxidations. Sulfenic acids are known to react not only with dimedone but also with other nucleophiles such as thiols with the formation of mixed disulfides (14). Accordingly, the addition of increasing concentrations (0.1-5 mM) of mercaptoethanol, N-acetylcysteine, or glutathione to incubations of 2a with aerobic rat liver microsomes in the presence of NADPH and dimedone under the aforementioned conditions led to a decrease of 5a formation

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Table 1. Comparison of the 1H and 13C NMR Characteristics of Compounds 5a and 5b (Two Diastereoisomers 5b1 and 5b2) with Those of the Previously Described Adduct 3′b (7S-Diastereoisomer) (6)a

a

Compounds 5a and 5b (acetate salts) in D2O; 3′b in CDCl3 (6).

Figure 3. Intermediate formation of sulfenic acids 4 upon metabolic oxidation of thiolactones 2 and their trapping by dimedone and thiols.

accompanied by a progressive appearance of a new product. Its mass spectrum (ESI+) exhibited a molecular ion at m/z ) 374, 459, and 603 (for 35Cl) (Figure 2), as expected for mixed disulfides 6a that result from reaction of 4a with mercaptoethanol, N-acetylcysteine, and glutathione, respectively. At the largest thiol concentrations, one observed in HPLC the formation of a product exhibiting the mass spectrum previously described for the thiol metabolite 3a (5). The aforementioned data provide the first evidence for intermediate formation of reactive sulfenic acid metabolites that have been trapped by dimedone, upon P450-catalyzed oxidative

cleavage of the thioester bond of 2a and 2b. They also provide a first detailed mechanism for the previously described formation of pharmacologically active thiol metabolites 3a and 3b after oxidative metabolism of 1a and 1b. We have found that these thiol metabolites 3 can be formed from the reaction of reactive species 4 with thiol nucleophiles such as glutathione, and a further reaction of excess thiol nucleophiles with intermediate mixed dithioethers 6 (Figure 3). Interestingly, in the two reports describing the formation of thiol metabolites 3a and 3b from 2a and 2b, the authors have used either liver S9 or microsomal fractions containing an excess of glutathione (5, 6). Such

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conditions should favor the eventual formation of 3a and 3b and disfavor the formation of glutathione adducts such as 6. Metabolites 4 and 6 both involve an electrophilic sulfur atom and should react with protein nucleophiles with possible formation of protein adducts. The possible consequences of such reactions on the pharmacological and/or toxicological effects of 1a and 1b remain to be determined. The above data provide the first evidence for the formation of a sulfenic acid reactive metabolite upon P450-catalyzed oxidative cleavage of a thioester (eq 2).

(7) (8) (9) (10)

(11)

The generality of such metabolic oxidative cleavage of thioesters is currently under study. Actually, evidence has been reported for intermediate formation of a sulfenic acid upon metabolic oxidation of similar compounds, thiazolidinediones, that involve a -S-CO-NHCO- moiety (20). Oxidation of the sulfur atom of these compounds as well as that of S-methyl N,N′-diethylthiolcarbamate, a metabolite of disulfiram (2, 20, 21), and of fatty acid thioesters (22) has been proposed as an initial step in the bioactivation of these molecules and in the oxidative cleavage of their CO-S bond. Such an S-oxidation also presumably occurs in the case of the oxidation of compounds 2a and 2b (Figure 3). Supporting Information Available: 1H NMR and 2D HSQC 1 H-13C NMR spectra for compound 5 and MS and MS/MS of compound 5b. This material is available free of charge via the Internet at http://pubs.acs.org.

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