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Vol. 30, Issue 11, 1288-1295, November 2002
Sanofi-Synthelabo Recherche, Toulouse cedex, France
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Clopidogrel (SR25990C, PLAVIX) is a potent antiplatelet drug, which has been recently launched and is indicated for the prevention of vascular thrombotic events in patients at risk. Clopidogrel is inactive in vitro, and a hepatic biotransformation is necessary to express the full antiaggregating activity of the drug. Moreover, 2-oxo-clopidogrel has been previously suggested to be the essential key intermediate metabolite from which the active metabolite is formed. In the present paper, we give the evidence of the occurrence of an in vitro active metabolite after incubation of 2-oxo-clopidogrel with human liver microsomes. This metabolite was purified by liquid chromatography, and its structure was studied by a combination of mass spectometry (MS) and NMR experiments. MS results suggested that the active metabolite belongs to a family of eight stereoisomers with the following primary chemical structure: 2-{1-[1-(2-chlorophenyl)-2-methoxy-2-oxoethyl]-4-sulfanyl-3-piperidinylidene}acetic acid. Chiral supercritical fluid chromatography resolved these isomers. However, only one of the eight metabolites retained the biological activity, thus underlining the critical importance of associated absolute configuration. Because of its highly labile character, probably due to a very reactive thiol function, structural elucidation of the active metabolite was performed on the stabilized acrylonitrile derivative. Conjunction of all our results suggested that the active metabolite is of S configuration at C 7 and Z configuration at C 3-C 16 double bound.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Clopidogrel (SR25990C, PLAVIX) is a potent
antiaggregant and antithrombotic drug, as demonstrated in several
experimental models of thrombosis (Herbert et al., 1993a
). The drug was
launched on the market following a successful clinical evaluation
(Feliste et al., 1987) and demonstration of superior efficacy versus
aspirin in preventing thrombotic events (myocardial infarction, stroke, and vascular death) in high risk patients (CAPRIE Steering Committee, 1996).
Clopidogrel inhibits platelet aggregation ex vivo induced by ADP,
low concentrations of thrombin, or by collagen (CAPRIE Steering Committee, 1996). The specific pharmacological target of clopidogrel is
the ADP-induced platelet activation process (Herbert et al., 1993b),
and it has been described as a specific and irreversible inhibitor of
2-methyl-S-ADP binding to its platelet receptors, the
purinergic P2Y12 receptor (Savi et al., 1994a; Herbert et al., 1999;
Savi et al., 2001). Clopidogrel is not active in vitro, and a
biotransformation by the liver is necessary to allow the expression of
its antiaggregating activity (Savi et al., 1992). Therefore,
clopidogrel can be considered as a precursor of an active metabolite.
Moreover, no antiaggregating activity was found in platelet poor plasma
of SR25990C-treated animals or humans, indicating a high reactivity and
instability of the active metabolite.
Clopidogrel has an absolute S configuration at carbon 7 (see
chemical structure in Fig. 1). The
corresponding R enantiomer is totally devoid of antiaggregating
activity (Savi et al., 1994b), thus indicating the importance of the
configuration of this asymmetric carbon for the biological activity. In
previous experiments, incubation of clopidogrel with rat hepatic
microsomes was found to generate 2-oxo-clopidogrel, through a
CYP450-dependent pathway of metabolism (Savi et al., 1992, 1994b).
Similar results were obtained using human liver microsomes (Savi et
al., 2000). Despite being not active in vitro, 2-oxo-clopidogrel can
demonstrate an antiaggregating activity ex vivo, thus indicating that
the formation of the active metabolite of clopidogrel occurred
downstream to the formation of 2-oxo-clopidogrel (Savi et al., 2000).
The structure of active metabolite of another thienopyridine, CS-747,
was reported (Sugidachi et al., 2000, 2001). In another report, these
authors indicated the precise absolute configuration to express the
biological activity, since only one among the four optical isomers
showed activity in inhibiting platelet aggregation (Kazui et al.,
2001). However, to our knowledge, no structural and
stereochemical characterization data were published in detail
concerning this active metabolite.
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The objective of this work was to identify the chemical structure of the active metabolite of clopidogrel. For this purpose, metabolites generated after incubation of human liver microsomes with 2-oxo-clopidogrel and its corresponding inactive R enantiomer were isolated and purified using a two-step liquid chromatographic procedure. The biological activity of the metabolites was evaluated through the inhibition of binding of radiolabeled 2-methyl-S-ADP to rat platelets. Subsequently, the structure and stereochemistry of the metabolites were studied by a combination of mass spectrometry (MS1), NMR, and chiral supercritical fluid chromatography (chiral SFC).
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Chemicals.
Clopidogrel (SR25990C, 2-(2-chlorophenyl)-2-(2,4,5,6,7,7a
hexahydrothieno [3,2c]pyridine-5yl-acetic acid methylester
hydrogen sulfate, 7S) and its corresponding inactive levogyre
enantiomer (SR25989C, 7R) were made available from
Sanofi-Synthélabo Recherche (Toulouse, France). 2-Oxo-clopidogrel
[(7S) SR121683] and 2-oxo-SR25989C [(7R) SR121682] were prepared by
semipreparative chiral HPLC from the racemic (7R,S) SR25552, which was
synthesized by Sanofi-Synthélabo Recherche.
33P-2-Methyl-S-ADP (600 Ci/mmol) was
from PerkinElmer Life Sciences (Le Blanc Mesnil, France).
Gluthatione, reduced form, and 
-nicotinamide adenosine diphosphate,
reduced form (NADPH), were from Sigma-Aldrich (L'Ile d'Abeau,
France). All other chemicals were of analytical or HPLC grade.
Purification of Clopidogrel Metabolites from Incubation of 2-Oxo-precursors with Human Liver Microsomes. Human liver microsomes from BD Gentest (Woburn, MA) were adjusted to 0.75 mg of protein/ml in 100 mM potassium phosphate buffer, pH 7.4, containing 100 mM KF and 10 mM gluthatione. SR121682 and SR121683 were added at a final concentration of 0.1 mM, and the reaction was initiated with 1 mM NADPH. Incubation was carried out at 37°C under continuous stirring (100 rpm) and light protection using a reciprocal incubator. After 60 min, the incubation medium was cooled at 4°C and centrifuged at 10,000g for 10 min.
A first purification step for isolation of metabolites was performed by semipreparative liquid chromatography on a system from Pharmacia AB (Uppsala, Sweden). The incubation supernatants were loaded on a HR16/5 precolumn (50 × 16 mm) from Pharmacia AB, filled with a 10-µm C18 support from Millipore Corporation (Epernon, France). The precolumn was rinsed and equilibrated with a 10 mM ammonium acetate buffer (pH 6.5). The precolumn was then coupled to a 10-µm Kromasil C18 column from Akzo Nobel (Bohus, Sweden). Elution at 2.0 ml/min was performed using an acetonitrile/10 mM ammonium acetate gradient (10 to 90%) and monitored with a dual UV detection (214 nm/254 nm). Eluted fractions were collected and concentrated to 300 µl using a SpeedVac vacuum centrifuge evaporator from Savant Instruments (Holbrook, NY). The eluted fraction "H" that contains a pool of metabolites was controlled by analytical HPLC using a Superspher 60RP8-E column (125 × 4 mm) from Merck-Clevenot (Nogent/Marne, France). Isocratic elution was performed at 0.8 ml/min with 60% methanol in aqueous 0.2% acetic acid/0.1% diethylamine (pH 5.5) and monitored with UV detection at 234 nm. A second purification step using semipreparative HPLC was performed to separate individual metabolites from the fraction "H", using an Ultrabase UB225 column (250 × 4.6 mm) from SFCC (Neuilly-Plaisance, France). Elution was performed at 1 ml/min using an acetonitrile/10 mM ammonium acetate (pH 6.5) gradient (10 to 24%) with UV detection at 234 nm. The collected fractions were concentrated on the SpeedVac evaporator. Concentrations of native "H" metabolites were estimated by analytical HPLC, using ticlopidine as an external standard for quantitative calibration. Ticlopidine was chosen because it can be eluted by the same analytical HPLC method as the "H" metabolites, whereas this is not possible with the more hydrophobic clopidogrel. In addition, ticlopidine has the same thiophene and 2-chlorophenyl chromophore groups than clopidogrel. This quantification approach was validated using incubation of radiolabeled 14C-clopidogrel (35.5 µCi/mg, Isotopic Chemistry Department, Sanofi-Synthelabo Research, Alnwick, UK) to correlate the concentrations measured by UV and specific radioactivity, using the analytical control HPLC method described above.In Vitro Activity of Clopidogrel Metabolites on the Binding of
33P-2MeS-ADP Human Platelets.
Venous blood was collected on citrated tubes from human healthy
volunteers. PRP was obtained by centrifugation (120g, 10 min), and PRP samples (2 ml) were incubated for 1 h at 20°C with
the purified metabolites. Experiments on the specific binding of
33P-2MeS-ADP to human platelets were performed
using a filtration technique to separate the free from bound
33P-2MeS-ADP as previously described (Savi et
al., 1994). The preincubated PRPs were centrifuged (600g, 10 min.), then the supernatants were discarded, and the pellets were
resuspended in binding buffer (145 mM NaCl, 5 mM KCl, 0.1 mM
MgCl2, 5.5 mM glucose, 15 mM HEPES, 5 mM EDTA).
Incubations of the 2-oxo metabolic precursors (SR25552, SR121683, and
SR121682) were carried out in 0.2 ml of binding buffer, which contained
washed human platelets (0.1 × 109
platelets/ml) and 33P-2MeS-ADP (0.5 nM).
Triplicate incubations were carried out at 37°C for 15 min and were
terminated by the addition of a 3-ml ice-cold assay buffer followed by
rapid vacuum filtration over glass-fibber Filtermats 11734 from Skatron
Instruments (Sterling, VA). Filters were then washed twice with 5-ml
ice-cold incubation buffer, dried, and the radioactivity was measured
by scintillation counting. Nonspecific binding was defined as the total
binding measured in the presence of excess unlabeled ADP (1 mM), and
specific binding was defined as the difference between total binding
and nonspecific binding. The percent inhibition was expressed as
%I = (total binding 
total binding with
antagonist)/specific binding ×100.
Derivatization of Clopidogrel Metabolites with Acrylonitrile. The fraction H containing the metabolites from microsomal incubation with 2-oxo-precursors (SR25552, SR121683, or SR121682) was diluted in an excess of acrylonitrile. After overnight agitation at room temperature, the solutions were evaporated to dryness with a SpeedVac system. The derivatized metabolites were then purified either by semipreparative HPLC as described for native metabolites or by analytical HPLC system using a 5-µm Kromasil C18 column (100 × 4.6 mm) and an acetonitrile/0.1% trifluoroacetic acid gradient (18 to 25%). Concentrations of derivatized metabolites were estimated by analytical HPLC, as described above for native metabolites.
Mass Spectrometry LC/MS and LC/MS/MS of native metabolites. Fraction H from microsomal incubation with the racemic 2-oxo-precursor SR25552 was injected on a Lichrocart 60RP8E column (125 × 4 mm) from Merck-Clevenot using a HP1100 liquid chromatograph from Agilent Technologies (Waldbronn, Germany). Isocratic elution was performed with a mixture of methanol/water/acetic acid/diethylamine (40:60:0.2:0.1 v/v/v/v) at 0.7 ml/min, and UV signal was followed at 254 nm. MS data were acquired on a Finnigan LCQ instrument from Thermoquest (San Jose, CA) in positive electrospray ionization (ESI+) mode. The spray potential was set at 5.6 kV and capillary temperature at 230°C. Mass range was scanned between 100 and 900 amu. In MS/MS mode (23% of collision energy), the two parent ions obtained at m/z 356.5 and 358.5 (with 1.4 amu peak width) correspond, respectively, to the quasi-molecular ions of the metabolites containing either 35Cl or 37Cl isotope.
EI and CI mass spectrometry of methyl-derivatized metabolites. Fraction H from SR25552 was derivatized using ethereal diazomethane reaction. MS analyses using electron impact (EI) and chemical ionization (CI) were done on a Finnigan TSQ 700 mass spectrometer from Thermoquest. EI was performed at 70 eV, and scans were taken over the mass range m/z 40 to 500, whereas CI was conducted using ammonia as reactant gas, and scans were taken over the mass range of m/z 80 to 900. Both direct introductions were done using a probe with a current gradient from 50 to 800 mA in 2 min.
LC/MS of acrylonitrile-derivatized metabolites. Fraction H from SR25552 was derivatized with acrylonitrile and analyzed on the TSQ 700 mass spectrometer using the same conditions as for the native metabolic fraction H.
Chiral Supercritical Fluid Chromatography. The acrylonitrile-derivatized H metabolites from microsomal incubation with either 2-oxo-precursor SR121683 or SR121682 were analyzed by SFC using a chromatograph from Berger Instruments Inc. (Newark, DE). The system comprises a FCM-1200 fluid control module for pumping carbon dioxide and polar modifier, a TCM-2020 column thermal control module, an ALS-3150 autosampler, a DAD-4100 diode-array UV detector, an electronic back-pressure regulator, and a ChemStation software (Agilent Technologies) for instrument control and data acquisition/processing. Separation was achieved using a Chiralpak-AD column (250 × 4.6 mm) from Daicel Chemicals (Tokyo, Japan) and a carbon dioxide/isopropanol containing 0.4% triethylamine and 0.4% trifluoroacetic acid (90:10 v/v) mixture as fluid eluent. The operating conditions were 3 ml/min flow rate, 200 bar outlet pressure, and 5°C column temperature. The sample was dissolved in an isopropanol/methylene chloride (v/v) mixture and 10 µl were injected. UV detection was carried out at 220 nm.
Nuclear Magnetic Resonance.
1H (500.13 MHz) and 13C
(125.77 MHz) NMR spectra of acrylonitrile-derivatized H metabolites
from microsomal incubation with racemic 2-oxo-precursor SR25552 were
recorded on an Avance DRX500 spectrometer from Bruker (Karlsruhe,
Germany). The probe was a
1H/13C 5 mm, 3 axis
gradients (x,y,z), optimized for inverse detection. Spectra were
recorded in CDCl3 solvent in 5-mm tubes without
spinning at a temperature of 300K. Sample concentration was less than 1 mg in 0.5 ml. The residual protonated resonance of the solvent (CDCl3) was used as an internal chemical shift
standard, which was related to tetramethylsilane with chemical
shifts of 7.25 and 77 ppm, respectively, for 1H
and 13C. The pulse programs of all 2D experiments
(gradient-selected COSY, gradient-selected DQF-COSY, ROESY and gradient
selected 1H/13C HSQC) were
taken from the Bruker standard software library. Processing of the raw
data were performed using Bruker XWinNmr software running on a Silicon
Graphics Indy workstation. The pulse conditions were 90° pulse, 6.8 µs (attenuation 0db) for 1H and 5 µs (attenuation 2db) for
13C. Gradient pulses used in this study were all
shaped to a sine envelope with 1 ms duration (COSY, DQF-COSY, and
1H/13C HSQC). Spectral
width was 5530.97 Hz for proton and 34013.6 Hz for carbon.
10 G.cm-1, decoupling
13C using globally optimized alternating-phase
rectangular pulses, delay optimized for an average
1JCH-coupling constant of 131 Hz,
zero-filling up to 2K in t1, cosinus2 filter in t1 and t2.
Molecular Modeling. Conformational analysis was performed using the SYBIL software from Tripos Inc.(St. Louis, MO) running on a Silicon Graphics R10000 workstation. The goal of this modelisation was to explore the conformational space of the acrylonitrile-derivatized H metabolites. Molecular dynamics studies were carried out using a method called "high temperature" (600K) during 1 ps. Results were used to find a minimum of energy for each structure. The force field used to perform dynamic experiments and energy minimization were those of Tripos. These calculations allowed finding a privileged conformation for each molecule.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Biotransformation of 2-Oxo-clopidogrel into an Active Metabolite.
The active metabolite of clopidogrel (SR25990C) was suspected to be
formed downstream to the formation of 2-oxo-clopidogrel following
incubation of human liver microsomes with the latter. Parallel
incubations were carried out with the 2-oxo-clopidogrel (SR121683) and
its opposite inactive R enantiomer (SR121682) as a control. Only
SR121683 could demonstrate ex vivo biological activity (Savi et al.,
2000), supporting the fact that SR121683 is the 2-oxo-SR25990C and
therefore should have an S configuration at carbon 7. The metabolites
present in the supernatant of incubates were isolated in a single
fraction using semipreparative chromatography. SR121683 and SR121682
generated similar elution patterns (data not shown), indicating an
identical metabolic pathway. An inhibitory effect of
33P-2-methyl-S-ADP binding to
platelets was found only in a fraction from the SR121683 incubate,
named fraction H.
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Structural Elucidation of the Active Metabolite. It was clearly demonstrated that the biological activity was supported by the S stereochemical configuration at carbon 7 of clopidogrel. It was also shown that the two achiral chromatographic profiles of metabolic pool "H" from either oxo-precursor SR12683 or SR121682 microsomal incubates were similar (see Fig. 4). Hence, all the subsequent structural elucidation studies using nonstereoselective MS and NMR spectroscopy were carried out on metabolic pool "H" obtained by human microsomal incubation of the racemic 2-oxo-precursor (7S, 7R) SR25552.
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Mass spectrometry. LC/MS experiments were conducted on the metabolic pool "H" from SR25552. Both UV and mass detection (m/z 356-357) profiles demonstrated the presence of the four different peaks noted H1 to H4 (Fig. 4, A and B). The detected four peaks in MS had exactly the same characteristics with a molecular ion MH+ at m/z 356.5, corresponding to 34 amu more than clopidogrel (Fig. 4C). The MS/MS data obtained on quasi-molecular ions MH+ 356.5 and 358.5 containing 35Cl or 37Cl isotope, respectively, allowed the detection of fragments bearing a chlorine atom (Fig. 4, D and E). MS/MS data were the same for the four compounds and were found in accordance to the primary structure proposal and the fragmentation scheme shown in Fig. 5.
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Nuclear Magnetic Resonance. From the four diastereomer H1 to H4 metabolites generated from incubation of SR25552 with human microsomes and purified by HPLC, only H4 was shown to retain the biological activity. This underlines the importance of a specific and critical absolute configuration for the active metabolite, the exact nature of which remained to be elucidated using NMR. However the active H fraction isolated by semipreparative chromatography or obtained after subsequent semipreparative HPLC purification, appeared to be highly labile since its activity was quickly lost after overnight incubation at room temperature. This could be explained by the presence of the reactive thiol group on the proposed structure (Fig. 5). Therefore, for all subsequent NMR studies, the "H" metabolites were always stabilized by trapping the reactive thiol group with acrylonitrile a thiol specific reagent, which was chosen because it did not introduce a supplementary asymmetric carbon in the chemical structure of the compound. The resulting acrylonitrile derivatives were controlled by LC/MS before use. They always gave the four HPLC separated H1 to H4 analog peaks, all of which exhibited a quasi-molecular ion MH+ at m/z 409 thus confirming the introduction of an acrylonitrile group on the thiol function.
Assignments of the resonance to individual proton and carbon nuclei of the molecules were performed according to the following numbering NMR nomenclature (Scheme 1).
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Conformational analysis and configuration of the ethylenic bond. Molecular modeling calculations were performed taking into account NMR constraints (1H homonuclear long range couplings and nuclear Overhauser effects). Conformations corresponding to energy minima were sought. In these conformers, the piperidine group adopted a chair conformation with the SCH2CH2CN group in axial position. Indeed, it seems that the equatorial position was unfavorable for the SCH2CH2CN group (+3 kCal) due to steric constraints induced by the bulky sulfur group.
Scheme 2 shows the calculated structure for both E and Z double bond configuration, together with estimated internuclei distances. For derivatized H3 and H4 metabolites, ROESY experiments exhibit cross peak between protons 2' and 16 but no cross peak between proton 4 and 16. For derivatized H1, ROESY experiments exhibit a cross peak between protons 4 and 16 and no cross peak between protons 2' and 16. It was then deduced that H3 and H4 have the same Z configuration, H1 being of the E configuration. This is confirmed by a close examination of chemical shifts, as highlighted in Table 3. Variations on chemical shifts of protons 2' and 4 appears reverse in H1 compared with H3 and H4. This can be explained by Scheme 3:
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SFC Separation of Stereoisomer Metabolites. MS and NMR data are in accordance with Scheme 5. This chemical structure contains three stereochemical sites: two chiral centers at C 4 and C 7 and one geometric center at C 3 (ethylenic bond). Eight stereochemical isomers can be metabolically generated from incubation of a mixture of the two oxo-precursors SR121683 and SR121682 with microsomes, four diastereomers being generated from each of them. These two groups of four metabolites could not be discriminated by achiral HPLC, thus leading to identical chromatographic profiles whatever the incubated 2-oxo-enantiomer. On the other hand, using chiral SFC, these metabolites could be differentiated. To validate this hypothesis, we decided to submit separately the two 2-oxo-precursors SR121683 and SR121682 to microsomal biotransformation and the resulting two metabolic pools H were derivatized with acrylonitrile for stabilization. Then, the individual acrylonitrile-derivatised metabolites were analyzed by chiral SFC using a column packed with tris (3,5-dimethylphenyl carbamate) amylose as chiral discriminating agent, which allowed splitting of each HPLC peak into two peaks. Figure 6 shows the chiral SFC chromatogram, thus confirming the existence of 8 distinct stereoisomer (4 enantiomeric pairs) metabolites. This result again is in good agreement with the structure proposal from the MS and NMR studies.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Clopidogrel has to be administered in vivo to selectively and
irreversibly inhibits the binding of 2MeS-ADP to its platelet receptors
(Savi et al., 1994a, 2001; Herbert et al., 1999). Clopidogrel is
inactive in vitro and has to undergo metabolic activation by hepatic
cytochrome P450-1A to exhibit its antiaggregating activity (Savi et
al., 1992, 1994b). From these studies, a possible metabolic pathway
leading to the formation of active metabolite of clopidogrel was
tentatively deduced (Savi et al., 2000). In the liver, clopidogrel is
metabolized into 2-oxo-clopidogrel through a cytochrome P450-dependent pathway. This intermediate metabolite is then hydrolyzed and generates the highly labile active metabolite, which reacts as a thiol reagent with the ADP receptors on platelets when they pass through the liver.
This in situ biological effect could account for the absence of an
antiaggregating activity in the plasma. In this study, we isolated in
sufficient amounts the metabolites of Clopidogrel by incubating the
synthetic 2-oxo-clopidogrel with human liver microsomes to determine
the chemical structure and biological activity of the active
metabolite. The 2-oxo-clopidogrel was used instead of clopidogrel
because it has been shown to be generated from clopidogrel by the liver
and to show a higher antiaggregating activity ex vivo (Savi et al.,
1992, 1994b).
Incubation of (7S) 2-oxo-clopidogrel with human microsomes led to a pool of metabolites (fraction H), which exhibited a potent in vitro activity as assessed by measuring 33P-2-methyl-S-ADP binding to human platelets. This result confirmed the key role played by bio-oxidation of clopidogrel at carbon 2 as an important first step toward the formation of an active metabolite. The active fraction H was shown to be composed of four diastereoisomers only one of which (named H4) with antiplatelet activity. Moreover, parallel microsomal incubations conducted with the inactive (7R) 2-oxo-isomer gave the same HPLC and MS data patterns, whereas no biologically active metabolite could be detected in that case. Altogether, these results underlined the critical importance of a specific absolute stereochemistry and in particular the 7S configuration associated to the active metabolite. We conducted parallel experiments with either the active (7S) or (7R) 2-oxo precursors with a systematic analytical and biological measurement at each step of the purification process. The results indicated that S configuration is preserved in the active fraction since only metabolites issued from the 7S precursor retained biological activity. This strongly suggests that no, even partial, racemization reaction occurs during the incubation with microsomes and/or purification conditions.
The MS data suggested a primary chemical structure with an opened unsaturated thiophene ring, a highly reactive thiol function, and a free carboxylic group. This primary structure was the same for the four H1 to H4 metabolites. This proposed structure bore three stereochemical sites (C 7, C 4, and C 3-C 16 ethylenic bond) and could explain the multiplicity of the observed isomers. The NMR study on the four H metabolic fractions demonstrated that the only active H4 had an ethylenic bond of a Z configuration. Hence, this second stereochemistry factor following the S configuration at carbon 7 was considered to be of crucial importance for the expression of activity. Chiral SFC was able to differentiate eight peaks (two series of four diastereoisomer metabolites), each generated from the active (7S) 2-oxo-clopidogrel or its opposite inactive (7R) 2-oxo-isomer. A diagram showing the possible set of 4 enantiomeric pairs obtained from the (7S) clopidogrel and its opposite (7R) enantiomer is shown in Fig. 7.
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The absolute configuration of the stereochemical site C 4 remains the third important structural key to be determined to fully elucidate the structure of the active H4 metabolite of clopidogrel. Its nature seems to be as important for the activity as the two elucidated configurations since the fraction H3 was shown to have 7S and Z configurations like H4 but was inactive. However, due to the highly unstable character of the active metabolite H4, we have not yet been able to isolate it from human microsomal incubations in sufficient amounts to complete its full characterization by X-ray crystallography.
In conclusion, the present study elucidates the structure and stereochemistry of the active metabolite of clopidogrel generated from human liver microsomes incubated with the 2-oxo-intermediate metabolite. Only one metabolite (bearing 7S, 3Z, and 4S or 4R configuration) of the 8 isomers exhibits in vitro the antiaggregating activity of clopidogrel observed ex vivo. This clearly demonstrates that interaction of the active metabolite with its target was highly dependent on its stereochemistry. Whether this compound is the sole active metabolite of clopidogrel remains to be elucidated.
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Footnotes |
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Received February 15, 2002; accepted July 19, 2002.
Address correspondence to: Claudine Picard, 195 route d'Espagne, 31036 Toulouse cedex, France. E-mail: claudine.picard{at}sanofi-synthelabo.com
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Abbreviations |
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Abbreviations used are: MS, mass spectrometry; SFC, supercritical fluid chromatography; PRP, platelet-rich plasma; HPLC, high-performance liquid chromatography; ESI, positive electrospray ionization; amu, atomic mass unit(s); MS/MS, tandem mass spectrometry; EI, electron impact; CI, chemical ionization; DQF, double quantum-filtered correlation spectroscopy; ROESY, rotating frame nuclear Overhauser enhancement spectroscopy; HSQC, heteronuclear single quantum correlation; LC, liquid chromatrography.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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, selected ion
m/z 356.5. 
