Bioactivation of Clopidogrel and Prasugrel: Factors Determining the Stereochemistry of the Thiol Metabolite Double Bond

Article abstract of DOI:10.1021/acs.chemrestox.5b00133

The antithrombotics of the tetrahydrothienopyridine series, clopidogrel and prasugrel, are prodrugs that must be metabolized in two steps to become pharmacologically active. The first step is the formation of a thiolactone metabolite. The second step is a further oxidation with the formation of a thiolactone sulfoxide whose hydrolytic opening leads to a sulfenic acid that is eventually reduced into the corresponding active cis thiol. Very few data were available on the formation of the isomer of the active cis thiol having a trans configuration of the double bond, the most striking result in that regard being that both cis and trans thiols were formed upon the metabolism of clopidogrel by human liver microsomes in the presence of glutathione (GSH), whereas only the cis thiol was detected in the sera of patients treated with this drug. This article shows that trans thiols are also formed upon the microsomal metabolism of prasugrel or its thiolactone metabolite in the presence of GSH and that metabolites having the trans configuration of the double bond are only formed when microsomal incubations are done in the presence of thiols, such as GSH, N-acetyl-cysteine, and mercaptoethanol. Intermediate formation of thioesters resulting from the reaction of GSH with the thiolactone sulfoxide metabolite appears to be responsible for trans thiol formation. Addition of human liver cytosol to the microsomal incubations led to a dramatic decrease of the formation of the trans thiol metabolites. These data suggest that cytosolic esterases would accelerate the hydrolytic opening of thiolactone sulfoxide intermediates and disfavor the formation of thioesters resulting from the reaction of these intermediates with GSH that is responsible for trans isomer formation. This would explain why trans thiols have not been detected in the sera of patients treated with clopidogrel.

Full text of DOI:10.1021/acs.chemrestox.5b00133

Article
pubs.acs.org/crt
Bioactivation of Clopidogrel and Prasugrel: Factors Determining the
Stereochemistry of the Thiol Metabolite Double Bond
Patrick M. Dansette,* Dan Levent, Assia Hessani, and Daniel Mansuy
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Universite
Paris Cite, 45 Rue des Saints-Peres, 75270 Paris Cedex 06, France
́
Paris Descartes, Sorbonne
́
̀
ABSTRACT: The antithrombotics of the tetrahydrothienopyridine series, clopidogrel and prasugrel, are prodrugs that must be
metabolized in two steps to become pharmacologically active. The first step is the formation of a thiolactone metabolite. The
second step is a further oxidation with the formation of a thiolactone sulfoxide whose hydrolytic opening leads to a sulfenic acid
that is eventually reduced into the corresponding active cis thiol. Very few data were available on the formation of the isomer of
the active cis thiol having a trans configuration of the double bond, the most striking result in that regard being that both cis and
trans thiols were formed upon the metabolism of clopidogrel by human liver microsomes in the presence of glutathione (GSH),
whereas only the cis thiol was detected in the sera of patients treated with this drug. This article shows that trans thiols are also
formed upon the microsomal metabolism of prasugrel or its thiolactone metabolite in the presence of GSH and that metabolites
having the trans configuration of the double bond are only formed when microsomal incubations are done in the presence of
thiols, such as GSH, N-acetyl-cysteine, and mercaptoethanol. Intermediate formation of thioesters resulting from the reaction of
GSH with the thiolactone sulfoxide metabolite appears to be responsible for trans thiol formation. Addition of human liver
cytosol to the microsomal incubations led to a dramatic decrease of the formation of the trans thiol metabolites. These data
suggest that cytosolic esterases would accelerate the hydrolytic opening of thiolactone sulfoxide intermediates and disfavor the
formation of thioesters resulting from the reaction of these intermediates with GSH that is responsible for trans isomer
formation. This would explain why trans thiols have not been detected in the sera of patients treated with clopidogrel.
intermediates are efficiently reduced by ascorbate or
phosphines with quantitative formation of thiols 5.14−17 They
are also reduced by thiols including glutathione (GSH) in two
steps: a nucleophilic attack of the thiol on the electrophilic
sulfur atom of the sulfenic acid function leading to mixed
disulfides 6 and a reduction of these disulfides by a second thiol
molecule (Figure 1).14−19 Reduction of the disulfides can also
be done by glutaredoxin and thioredoxin.20,21 Recently, it has
been shown that thiolactone sulfoxides 3 are formed as
intermediates during the oxidative activation of thienopyridine
antithrombotic drugs 1. These intermediates were found to act
as bis-electrophiles that were trapped by two nucleophiles,
NuH (amine, thiol) and Nu′H (cyclopentane-1,3-dione
(CPDH), dimedone, thiol), the first one being able to react
with the CO carbon of the thiolactone sulfoxide and the second
one with the sulfur atom of the intermediate sulfenic acid, 8.22
This led to the expected bis-adducts 9 (Figure 1) that were
detected by HPLC-MS. One of them was completely
INTRODUCTION
■
The antithrombotic drugs of the tetrahydrothienopyridine
series, clopidogrel (Plavix, Iscover) 1a, and prasugrel (Effient)
1b (Figure 1), are prodrugs that must be metabolized in vivo
into the corresponding pharmacologically active 4-mercapto-3-
piperidinyliden acetic acid derivatives 5a and 5b, respectively,
to exert their activity as antagonists of the platelet receptor
P2Y12.1−6 The first step of their metabolic activation leads to
thiolactone metabolites 2a and 2b, respectively (Figure 1).6−9
The second step of the metabolic activation of 1a and 1b,
leading to the active metabolites 5a and 5b, respectively, is not
a simple hydrolysis of the thioester bond of intermediate
thiolactones 2a and 2b. Actually, it has been recently shown
that such a simple hydrolysis of thiolactones 2a and 2b, which
is catalyzed by thioesterases like paraoxonase-1, only leads to an
endo isomer of 5a and 5b in which the double bond has
migrated into the piperidine ring (7a and 7b in Figure 1).10−13
Formation of the active cis metabolite diastereomers 5 occurs in
two steps, a P450-catalyzed oxidative opening of the
thiolactone ring of 2 with the formation of intermediate
sulfenic acids 4 and a reduction of 4 into the corresponding
thiols 5 (Figure 1).14−17 These electrophilic sulfenic acid
Received: March 31, 2015
© XXXX American Chemical Society
A
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Figure 1. Detailed pathway of 1a and 1b bioactivation into active thiols 5a and 5b, and different reactions occurring upon the reaction of
intermediates 3 with nucleophiles.
Figure 2. Different thiol stereoisomers formed upon the metabolism of 1a in vivo in humans and in vitro in the presence of human liver microsomes,
NADPH, and GSH. HLM means human liver microsomes.
1
characterized by H and 13C NMR spectroscopy using 1D and
This article describes a study of the stereochemistry of the
double bond of the thiol metabolites formed upon oxidative
activation of 1a and 1b by human liver microsomes in the
presence of various thiols or other reducing agents, with or
without human liver cytosol. It shows that trans thiols are
formed in the in vitro metabolism of prasugrel under conditions
leading to trans thiols in the case of clopidogrel. It also shows
that the factors controlling the stereochemistry of the thiol
double bond are almost identical in the metabolism of 1a and
1b. Finally, it reports that the trans thiols are only formed when
GSH or other thiols such as ME or NAC are present in the
incubation medium and that their formation is strongly reduced
in the presence of cytosol. These data give a first explanation
for the exclusive formation of cis thiol 5a in the in vivo
metabolism of clopidogrel, and the formation of a 5a−5a′
mixture in the in vitro metabolism (human liver microsomes +
GSH) of clopidogrel.
2D methods.22
The metabolism of clopidogrel by human liver microsomes
in the presence of NADPH and GSH was reported to lead to
the two diastereomers of the active cis thiol 5a and to the two
diastereomers of its inactive isomer 5a′, in which the double
bond has a trans configuration (Figure 2).1,6,19,22 Both 5a and
5a′ were also formed when GSH was replaced with other thiols
such as cysteine, N-acetyl-cysteine(NAC), or mercaptoethanol
(ME).18,19,22 By contrast, the metabolism of 1a by human liver
microsomes in the presence of NADPH and ascorbate only led
to the cis thiols 5a,11,19 and in the sera of patients treated with
1a, one only found these cis thiols 5a (Figure 2).23−25 In the
case of prasugrel, 1b, there was no information available in the
literature about the possible formation of trans thiols 5b′ after
in vitro metabolism of 1b in the presence of human liver
microsomes. These data lead to the following questions: (1) are
trans thiol metabolites 5b′ also formed after in vitro metabolism
of prasugrel by human liver microsomes in the presence of
GSH? (2) What is the mechanism of formation of the trans
thiols 5′? (3) What are the factors determining the stereo-
chemistry of the double bond of the thiol metabolites of
clopidogrel in vitro and in vivo?
EXPERIMENTAL PROCEDURES
■
Chemicals and Biochemicals. [7S]-2-Oxo-clopidogrel
(SR121883), 2a, was a gift of Sanofi-Aventis (Chilly-Mazarin, France).
2-Oxo-prasugrel base (racemate), 2b, was obtained from Twisun
Pharma (Shangai, China). All other products including enzymes were
from Sigma-Aldrich (St. Quentin Fallavier, France).
B
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Figure 3. HPLC-MS study of thiol diastereomers 5b′, (1) and (2), and 5 b, (3) and (4), upon the metabolism of 2b by human liver microsomes in
the presence of various thiols or reductants. Small amounts of the endo isomer 7b were also detected (see, for instance, panels B and C). Panels A−E
show reconstructed chromatograms at m/z = 350.1216 obtained on the HPLC−HRMS system using 2 mM GSH, NAC, ME, ascorbate, or TCEP
(panels A, B, C, D, and E, respectively) (MA: peak area). Panel F shows the MS2 spectra obtained on the ion trap instrument at m/z = 350 at 25 eV
for the diastereomers of thiols 5b′ (1 and 2), 5b (3 and 4), and for 7b.
Microsomal Incubations. Human liver microsomes (pool, 10 mg
protein/mL) and human liver cytosol (20 mg protein/mL) were
obtained from BD-Gentest (Le Pont de Claix, France). Typical
incubations were performed in 200 μL of potassium phosphate buffer
(0.1M, pH 7.4) containing human liver microsomes (1 mg protein/
mL), 2a or 2b (100 μM), and various nucleophiles (1 to 10 mM) with
or without an NADPH generating system (1 mM NADP, 15 mM
glucose-6-phosphate, and 2 u/mL of glucose-6-phosphate dehydrogen-
ase) at 37 °C for 30 min. In the case of incubations with 2a, 10 mM
KF was added to inhibit the esterase-dependent hydrolysis of its
methyl ester function.1 Incubations performed with various KF
concentrations (from 0 to 100 mM) showed that such a KF
concentration variation only led to very minor changes of the 5a′/5a
ratio. In incubations performed in the presence of human liver cytosol,
20 μL of human liver cytosol was added. Reactions were stopped by
adding one-half volume of CH3CN containing 8% CH3COOH.
Proteins were removed by centrifugation at 13000g.
CH3CN/H2O/HCOOH (980:18:2)) at 220 μL/min). Mass spectra
were obtained by electrospray ionization in positive ionization mode
detection (ESI+) 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 300 and 700 (except for exploratory
experiments with a wider range 300−1000). MS2 energy was tested
between 20 and 40 eV and was 35 eV. For all products, the indicated
parent ions corresponded to M + H+.
HPLC−HRMS Studies. HPLC−HRMS studies were performed on
a Thermo Exactive Orbitrap spectrometer coupled to a Thermo Accela
600 HPLC (Thermo, Les Ulis France) system using the same column
and gradient as those described above. ESI spray positive; tune, sheath
gas 10, auxiliary gas 6, capillary temperature 250 °C, capillary voltage
65 V, tube lens 120 V, skimmer 22 V, vaporizer 300 °C; and mass
range 300−1000, and HCD (higher-energy collisional dissociation)
and CID (collision induced dissociation) disabled, resolution 60000.
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 Shimadzu
Shimpack C18 column (75 × 2.1 mm, 2.3 μm (Shimadzu, Marne La
RESULTS
■
Stereochemistry of the Thiol Metabolites Formed
upon the Oxidation of Prasugrel by Human Liver
Microsomes in the Presence of Various Reducing
Agents. HPLC-MS studies of incubations of 1b or 2b with
human liver microsomes in the presence of NADPH and 1 mM
́
Vallee, France)), and a gradient of A+B starting at 0% B for 1 min then
increasing linearly to 50% B in 13 min, then to 95% B in 1 min, and
back to 0% B at 17.5 min, and equilibration at 0% B for 5.5 min (A = 2
mM ammonium acetate plus 0.2% HCOOH, pH 4.6, and B =
C
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ascorbic acid led to only two products exhibiting the MS and
MS2 characteristics previously reported for the diastereomers of
the cis thiol 5b15,26−28 (Figure 3D). Replacing ascorbic acid
with GSH or another thiol such as cysteine, NAC, or ME, led to
a clearly different thiol metabolites pattern with the appearance
of two additional products (Figures 3A, B, and C) exhibiting
identical MS and MS2 characteristics (Figure 3F). An HPLC−
HRMS study definitely showed that these two additional
products were isomers of 5b (Table1). Their MS2 spectra,
presence of NADPH and ascorbate (data not shown), as UV
irradiation of conjugated acids with a cis configuration of the
double bond is known to lead to a cis/trans isomerization of
this double bond.29 This was also in complete agreement with
the almost identical results previously described for the
formation of both cis 5a and trans 5a′ upon microsomal
metabolism of 1a or 2a in the presence of GSH, NAC, or
ME,1,6,19,22 and the exclusive formation of 5a in the presence of
ascorbic acid instead of GSH.11,19
Replacement of GSH with the reductant Tris(carboxyethyl)-
phosphine (TCEP) led to the exclusive formation of the cis
diastereomers 5b (Figure 3E), as when ascorbic acid was used
instead of GSH. This result is in agreement with literature data
showing that sulfenic acids are efficiently reduced by TCEP
with the formation of the corresponding thiols.17
Table 1. HRMS Characteristics of the Main Metabolites
Formed upon Incubations of 1b or 2b by Human Liver
Microsomes in the Presence of GSH
a
m/z (MH+)
obs.
calc.
chemical formula
structure
Metabolism of 2b by Human Liver Microsomes in the
Presence of GSH. Previous studies have shown the formation
of dithioethers 6a, in addition to thiols 5a and 5a′, during the
metabolism of 1a by human liver microsomes in the presence
of GSH or other thiols such as ME.14,18,19 More recently, bis-
adducts 9a and 9b (Nu = Nu′ = SCH2CH2OH) have also been
detected during the metabolism of 1a and 1b, respectively, by
human liver microsomes in the presence of ME.22 These
dithioethers19 and bis-adducts22 were a mixture of cis and trans
diastereomers.
350.1216
350.1213
655.1893
944.2623
334.1443
350.1221
350.1221
655.1902
944.2635
334.1449
C18H21FNO3S+
C18H21FNO3S+
5b
5b′
6b
9b
+
C28H36FN4O9S2
+
C38H51FN7O14S3
b
+
C18H21FNO4
alcohol
a
Conditions are described in Experimental Procedures. Mass (in Da)
observed (obs.) or calculated (calc.) according to the indicated
formula for the molecular ion. Alcohol diastereomers resulting from
the replacement of the SH group of 5b and/or 5b′ with an OH group.
b
An HPLC−MS study of incubations of 2b with human liver
microsomes in the presence of NADPH and 2 mM GSH
showed the formation of several GSH adducts, in addition to
thiols 5b and 5b′ (Figure 4). The major ones were
characterized by peaks at m/z = 655 (Figure 4A). They
exhibited MS2 spectra (fragments at m/z = 526, 348, and 317
corresponding to a loss of pyroglutamate, GSH and SSG,
respectively) (Figure 4B) and HRMS data (Table 1) in
which were identical to those of 5b but clearly different (Figure
3F) from that of the previously described endo isomer, 7b,
which was formed in very low amounts (Figure 3B and 3C),
suggested that they were the trans diastereomers 5b′. This was
in agreement with the appearance of the HPLC peaks of the
5b′ diastereomers upon UV irradiation of cis 5b derived from
the incubation of 2b with human liver microsomes in the
Figure 4. HPLC-MS study of the products found upon the incubation of 2b with human liver microsomes in the presence of GSH. A: reconstructed
chromatograms obtained on the HPLC−HRMS system, at m/z = 350 (upper panel), 655 (middle panel), and 944 (lower panel). B and C: MS2
spectra at m/z = 655 and 944, respectively.
D
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Figure 5. Kinetics of oxoprasugrel metabolism by human liver microsomes in the presence of NADPH and 2 mM GSH, without (A) or with (B)
human liver cytosol. Black triangle, cis thiols 5b; open diamond, monoadducts 6b; open circle, trans thiols 5b′; black square, bis-adducts 9b.
Ordinates are the areas of the HPLC−HRMS peaks of the different metabolites. In the absence of authentic samples of the metabolites for
constructing calibration curves, this gave a qualitative idea of the metabolite kinetics. The indicated values are the means of two independent
experiments, each in duplicate; there was less than 20% variation between the obtained values.
Figure 6. Kinetics of oxoclopidogrel metabolism by human liver microsomes in the presence of NADPH and 2 mM GSH, without (A) or with (B)
human liver cytosol. Black triangle, cis thiols 5a; open diamond, monoadducts 6a; open circle, trans thiols 5a′; black square, bis-adducts 9a. Ordinates
are the areas of the HPLC−HRMS peaks of the different metabolites (see the legend for Figure 5 concerning this point).
agreement with structure 6b (with SR′ = SG). All components
of the two broad peaks at m/z = 655 exhibited identical HRMS
and MS2 characteristics. GSH bis-adducts (m/z = 944),
corresponding to the ME bis-adducts previously reported in
the metabolism of 2b by human liver microsomes in the
presence of ME, were also detected. Their MS2 (fragments at
m/z = 815, 686, and 605 corresponding to the loss of
pyroglutamate, 2 pyroglutamates, and GSSH, respectively,
Figure 4C) and HRMS characteristics (Table 1) were in
agreement with the structure of 9b (with Nu = Nu′ = SG).
However, the HPLC column used in this study, that led to a
clear separation of the four cis and trans diastereomers of 6b
and 9b (with Nu = Nu′ = SCH2CH2OH) observed in the
metabolism of 2b by human liver microsomes in the presence
of ME,22 did not allow us to separate the stereoisomers of 6b
and 9b (with Nu = Nu′ = SG).
Other metabolites not containing the GS moiety were also
formed in amounts comparable to those of 6b. Their MS2 and
HRMS characteristics (m/z = 334.1443, Table 1) indicated that
they were the alcohol diastereomers resulting from the
replacement of the SH group of 5b with an OH group. The
formation of these metabolites upon in vivo metabolism of 1b
was already reported.26,27
only appeared after 5 min (Figure 5A). After 30 min, the cis/
trans thiols ratio was around 4. Dithioethers 6b and bis-adducts
9b (with SR′ = SG) appeared as a mixture of unresolved
stereoisomers. The first ones reached a plateau after about 20
min, whereas the second ones were always formed in small
amounts (Figure 5A).
Quite interestingly, very different results were observed when
identical incubations were done in the presence of human liver
cytosol. Under those conditions, cis thiols 5b were almost the
only products of the reaction, as very minor amounts of trans
thiols 5b′ were formed (5b/5b′ ≥ 15), and mono- and bis-
GSH adducts were only formed in very low amounts (Figure
5B). The great decrease of dithioethers 6b formation in the
presence of human liver cytosol was in agreement with previous
data showing that cytosolic reductases efficiently catalyze the
reduction of these dithioethers into thiols 5b.20
Metabolism of 2a by Human Liver Microsomes in the
Presence of GSH. An analogous study of the reaction of oxo-
clopidogrel 2a with human liver microsomes in the presence of
NADPH and 2 mM GSH, by HPLC−MS and HPLC−HRMS,
showed the formation of the same kind of metabolites. cis and
trans thiols 5a and 5a′ and dithioethers 6a were the major
products of this reaction. They were already reported to be
formed in the metabolism of 2a by human liver microsomes in
the presence of GSH.1,6,19,22 We could also detect the minor
cis thiols 5b were already formed during the first minutes of
the reaction, whereas significant amounts of trans thiols 5b′
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formation of the GSH bis-adducts 9a (Nu = Nu′ = SG) that
were characterized by MS2 and HRMS (molecular ion MH+ at
m/z = 950.2136, calculated value 950.2132).
adducts 9 derived from 2a and 2b in the presence of ME or
NAC, are mixtures of cis and trans isomers. Moreover, it has
been reported that dithioethers 6a formed in the presence of
ME were also a mixture of cis and trans isomers.19,22
In a manner similar to what was observed in the case of 2b,
in the absence of human liver cytosol, cis and trans thiols 5a and
5a′ were formed in a cis/trans ratio around 4, in addition to
important amounts of dithioethers 6a and small amounts of bis-
adducts 9a (Figure 6A), whereas, in the presence of human
liver cytosol, cis thiols 5a were almost the only observed
products, trans thiols 5a′ and intermediates 6a and 9a (with
SR′ = SG) being formed in very low amounts (Figure 6B).
The formation of trans thiol diastereomers that only occurred
in the presence of thiol nucleophiles could be due to the
particular reactivity of intermediate thioesters 9 (Nu = Nu′ =
SR′) derived from the initial reaction of thiol nucleophiles on
the thioester function of thiolactone sulfoxides 3, when
compared to that of intermediate carboxylic acids or amides
derived from the initial reaction of 3 with water or amines.
Three mechanisms have been tentatively proposed for the cis−
trans isomerization of the thioester 9 double bond.22 They are
based on the greater tendency of thioesters to give intermediate
enolates, vinylidene−ketenes, or ketenes after deprotonation,
elimination of GSH, or reversible Michael-type addition of the
thiol on the conjugated double bond of 9, respectively.22
Intermediates 8 (Figure 7) also contain a thioester function and
could lead to a cis−trans isomerization of their double bond.
However, they also contain a very electrophilic sulfenic acid
function and should only exist as transient species in very low
concentrations.
DISCUSSION
■
The aforementioned results show for the first time that the
stereochemistry of the double bond of the thiols formed upon
the metabolism of 2b by human liver microsomes in the
presence of GSH or other thiols or reductants is highly similar
to that previously described for the thiols derived from the
metabolism of 2a under identical conditions. In both cases, only
cis thiols are formed in the presence of ascorbic acid or TCEP,
whereas cis and trans thiols 5 and 5′ are formed in the presence
of GSH or other thiols such as NAC or ME.
The detection of only the cis thiol diastereomers 5a in the
sera of patients treated with clopidogrel, under conditions
allowing a complete separation of the cis and trans isomers,23−25
while high amounts of GSH are present in the hepatocytes,
could be explained, at least in part, by the above data on the
role of human liver cytosol on the 5a/5a′ ratio observed after
the metabolism of 2a by human liver microsomes in the
presence of GSH. Addition of human liver cytosol to
incubations of 2a or 2b with human liver microsomes in the
presence of NADPH and GSH resulted in dramatic changes in
products formation (Figures 5 and 6). Formation of trans thiols
5′ drastically decreased in the presence of cytosol, and one also
noted a clear decrease in the formation of intermediates 6 (SR′
= SG) and 9 (Nu = Nu′ = SG). The almost exclusive formation
of cis thiols 5a upon the metabolism of 2a by human liver
microsomes in the presence of 2 mM GSH and human liver
cytosol would explain the detection of only cis thiols 5a in the
sera of patients treated with clopidogrel. Globally, it seems that
the presence of human liver cytosol would disfavor the
formation of thioester intermediates 9 (Nu = Nu′ = SR′)
that are responsible for the trans isomer formation. This should
occur at the level of the opening of the thiolactone sulfoxide
ring of intermediate 3. In the presence of GSH (or other thiols
such as ME), two nucleophiles, GSH and H2O, are in
competition for the reaction with the thioester CO group of
3 and opening of the thiolactone sulfoxide ring (Figure 7).
Reaction of H2O should lead to intermediates toward thiol 5
with a cis configuration of the double bond, whereas reaction
with GSH (or other thiols such as ME) would lead to thioester
intermediates such as 9 able to interconvert the stereochemistry
of their double bond and to give a mixture of cis and trans thiols
5 and 5′. A possible role of cytosol would be to increase the
rate of opening of 3 by H2O and thus to favor the formation of
4 relative to that of 9 (with Nu = SG). Cytosolic esterases,
especially thioesterases, could be at the origin of these observed
effects of cytosol. This is likely as the thioesterase PON-1 has
been shown to catalyze the hydrolytic opening of the tautomer
of thiolactone 2a, in which the double bond has migrated into
the piperidine ring, leading to 7a.10,11,13 Compound 3a is
structurally close to the tautomer of thiolactone 2a, and its CO
carbon is more electrophilic than that of the 2a tautomer. The
Table 2 summarizes the data described above or previously
published about the stereochemistry of the double bond of
Table 2. Stereochemistry of the Double Bond of Thiols 5 or
of Various Adducts upon the Metabolism of 2a and 2b by
Human Liver Microsomes in the Presence of Various
Nucleophiles or Reductants
presence of trans
a
isomers
nucleophile or
reductant
products
2a
- (14)
2b
dimedone
dimedone adducts
- (15)
ascorbate
thiols 5
- (10, 11,
and 18)
- (this
work)
R1R2NH +
CPDH
bis adducts 9, (Nu = N R1R2, - (22)
Nu′ = CPD
- (22)
GSH
thiols 5
+ (19)
+ (19)
+ (22)
+ (this
work)
ME
thiols 5
+ (this
work)
- bis adducts 9 (Nu = Nu′ =
+ (22)
SCH2CH2OH)
- 6 (R′S = SCH2CH2OH)
+ (19 and
22)
+ (22)
a
- means that only products with the cis configuration of the double
bond have been observed (trans/cis ratio <0.05); + means that cis and
trans isomers were formed. The reference number of publications
mentioning the formation of the indicated products are given in
parentheses.
thiols or of adducts formed upon the metabolism of 2a or 2b by
human liver microsomes in the presence of various nucleophiles
(or reductants) used to trap thiolactone sulfoxides 3 and/or
sulfenic acids 4. It clearly shows that significant amounts of
trans isomers are only formed when thiols such as GSH, NAC,
and ME are present in the incubation medium. Actually, in their
absence, the monoadducts formed in the presence of
dimedone, the bis-adducts 9 (Nu = NR1R2; Nu′ = CPD)
obtained in the presence of an amine (NH3 or piperidine) and
CPDH, and thiols 5 formed in the presence of ascorbic acid or
TCEP are only cis diastereomers. By contrast, thiols derived
from 2a and 2b in the presence of GSH, NAC, or ME, and bis-
F
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Figure 7. Competition between water and GSH for the opening of intermediate 3 explaining the different stereochemistries of the thiol 5 double
bond observed upon the metabolism of 1a and 1b in different in vitro conditions or in vivo (in the case of 1a).
(3) Hasegawa, M., Sugidachi, A., Ogawa, T., Isobe, T., Jakubowski, J.
A., and Asai, F. (2005) Stereoselective inhibition of human platelet
aggregation by R-138727, the active metabolite of CS-747 (prasugrel,
LY640315), a novel P2Y12 receptor inhibitor. Thromb. Haemostasis 94,
593−598.
nature of the cytosolic esterases possibly involved in the
catalysis of hydrolysis of 3a remains to be determined. Because
of the very similar behavior of 2a and 2b in the above-described
reactions, one would expect that significant amounts of trans
thiols 5b should not be formed in vivo in patients treated with
1b.
(4) Testa, B., and Kramer, S. D. (2009) The biochemistry of drug
metabolism–an introduction: part 5. Metabolism and bioactivity.
Chem. Biodiversity 6, 591−684.
AUTHOR INFORMATION
■
(5) Farid, N. A., Kurihara, A., and Wrighton, S. A. (2010)
Metabolism and disposition of the thienopyridine antiplatelet drugs
ticlopidine, clopidogrel, and prasugrel in humans. J. Clin. Pharmacol.
50, 126−142.
Corresponding Author
*Tel: +33 142862191. Fax: +33 142868387. E-mail: patrick.
dansette@parisdescartes.fr.
(6) Maffrand, J. (2012) The story of clopidogrel and its predecessor,
ticlopidine: Could these major antiplatelet and antithrombotic drugs
be discovered and developed today? C. R. Chim. 15, 737−743.
(7) Dalvie, D. K., Kalgutkar, A. S., Khojasteh-Bakht, S. C., Obach, R.
S., and O’Donnell, J. P. (2002) Biotransformation reactions of five-
membered aromatic heterocyclic rings. Chem. Res. Toxicol. 15, 269−
299.
Funding
Funding for this research was provided by University Paris
Descartes, Sorbonne Paris Cite, and the French Centre
́
National de Recherche Scientifique.
Notes
The authors declare no competing financial interest.
(8) Kazui, M., Nishiya, Y., Ishizuka, T., Hagihara, K., Farid, N. A.,
Okazaki, O., Ikeda, T., and Kurihara, A. (2009) Identification of the
human cytochrome P450 enzymes involved in the two oxidative steps
in the bioactivation of clopidogrel to its pharmacologically active
metabolite. Drug Metab. Dispos. 38, 92−99.
(9) Williams, E. T., Jones, K. O., Ponsler, G. D., Lowery, S. M.,
Perkins, E. J., Wrighton, S. A., Ruterbories, K. J., Kazui, M., and Farid,
N. A. (2008) The biotransformation of prasugrel, a new thienopyr-
idine prodrug, by the human carboxylesterases 1 and 2. Drug Metab.
Dispos. 36, 1227−1232.
ABBREVIATIONS
■
CPDH, cyclopentane-1,3-dione; ESI+, electron spray ionization
in positive ionization mode detection; HPLC-MS, high
performance liquid chromatography−mass spectrometry;
HRMS, high resolution mass spectrometry; GSH, glutathione;
ME, mercaptoethanol; NAC, N-acetyl cysteine; TCEP, tris-
(carboxyethyl)-phosphine; PON-1, paraoxonase-1; P450, cyto-
chrome P450
(10) Dansette, P. M., Rosi, J., Bertho, G., and Mansuy, D. (2011)
Paraoxonase-1 and clopidogrel efficacy. Nat. Med. 17, 1040−1041.
(11) Dansette, P. M., Rosi, J., Bertho, G., and Mansuy, D. (2012)
Cytochromes P450 catalyze both steps of the major pathway of
clopidogrel bioactivation whereas paraoxonase catalyzes the formation
of a minor thiol metabolite isomer. Chem. Res. Toxicol. 25, 348−356.
(12) Dansette, P. M., Rosi, J., Debernardi, J., Bertho, G., and Mansuy,
D. (2012) Metabolic activation of prasugrel: nature of the two
competitive pathways resulting in the opening of its thiophene ring.
Chem. Res. Toxicol. 25, 1058−1065.
REFERENCES
■
(1) Pereillo, J. M., Maftouh, M., Andrieu, A., Uzabiaga, M. F., Fedeli,
O., Savi, P., Pascal, M., Herbert, J. M., Maffrand, J. P., and Picard, C.
(2002) Structure and stereochemistry of the active metabolite of
clopidogrel. Drug Metab. Dispos. 30, 1288−1295.
(2) Sugidachi, A., Asai, F., Yoneda, K., Iwamura, R., Ogawa, T.,
Otsuguro, K., and Koike, H. (2001) Antiplatelet action of R-99224, an
active metabolite of a novel thienopyridine-type G(i)-linked P2T
antagonist, CS-747. Br. J. Pharmacol. 132, 47−54.
G
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Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology
Article
(13) Gong, I., Crown, N., Suen, C., Schwarz, U., Dresser, G., Knauer,
M., Sugiyama, D., Degorter, M., Woolsey, S., Tirona, R., and Kim, R.
(2012) Clarifying the importance of CYP2C19 and PON1 in the
mechanism of clopidogrel bioactivation and in vivo antiplatelet
response. Eur. Heart J. 33, 2856−2464.
States Patent US 6,610,708 B1, p 94, Sankyo Company Ltd., Ube
Industries Ltd., Japan.
(14) Dansette, P. M., Libraire, J., Bertho, G., and Mansuy, D. (2009)
Metabolic oxidative cleavage of thioesters: evidence for the formation
of sulfenic acid intermediates in the bioactivation of the antithrombotic
prodrugs ticlopidine and clopidogrel. Chem. Res. Toxicol. 22, 369−373.
(15) Dansette, P. M., Thebault, S., Bertho, G., and Mansuy, D.
(2010) Formation and fate of a sulfenic acid intermediate in the
metabolic activation of the antithrombotic prodrug prasugrel. Chem.
Res. Toxicol. 23, 1268−1274.
(16) Hagihara, K., Kazui, M., Kurihara, A., Iwabuchi, H., Ishikawa, M.,
Kobayashi, H., Tanaka, N., Okazaki, O., Farid, N. A., and Ikeda, T.
(2010) Biotransformation of prasugrel, a novel thienopyridine
antiplatelet agent, to the pharmacologically active metabolite. Drug
Metab. Dispos. 38, 898−904.
(17) Mansuy, D., and Dansette, P. M. (2011) Sulfenic acids as
reactive intermediates in xenobiotic metabolism. Arch. Biochem.
Biophys. 507, 174−185.
(18) Zhang, H., Lau, W., and Hollenberg, P. (2012) Formation of the
thiol conjugates and active metabolite of clopidogrel by human liver
microsomes. Mol. Pharmacol. 82, 302−309.
(19) Zhang, H., Lauver, D., Lucchesi, B., and Hollenberg, P. (2013)
Formation, reactivity, and anti-platelet activity of mixed disulfide
conjugates of clopidogrel. Mol. Pharmacol. 83, 848−856.
(20) Hagihara, K., Kazui, M., Kurihara, A., Kubota, K., and Ikeda, T.
(2011) Glutaredoxin and thioredoxin can be involved in producing the
pharmacologically active metabolite of a thienopyridine antiplatelet
agent, prasugrel. Drug Metab. Dispos. 39, 208−214.
(21) Hagihara, K., Kazui, M., Kurihara, A., Ikeda, T., and Izumi, T.
(2012) Glutaredoxin is involved in the formation of the pharmaco-
logically active metabolite of clopidogrel from its GSH conjugate. Drug
Metab. Dispos. 40, 1854−1859.
(22) Dansette, P. M., Levent, D., Hessani, A., Bertho, G., and
Mansuy, D. (2013) Thiolactone sulfoxides as new reactive metabolites
acting as bis-electrophiles: implication in clopidogrel and prasugrel
bioactivation. Chem. Res. Toxicol. 26, 794−802.
(23) Tuffal, G., Roy, S., Lavisse, M., Brasseur, D., Schofield, J.,
Delesque Touchard, N., Savi, P., Bremond, N., Rouchon, M. C.,
Hurbin, F., and Sultan, E. (2011) An improved method for specific
and quantitative determination of the clopidogrel active metabolite
isomers in human plasma. Thromb. Haemostasis 105, 696−705.
(24) Karazniewicz-Lada, M., Danielak, D., Tezyk, A., Zaba, C., Tuffal,
́
G., and Gowka, F. (2012) HPLC-MS/MS method for the
simultaneous determination of clopidogrel, its carboxylic acid
metabolite and derivatized isomers of thiol metabolite in clinical
samples. J. Chromatogr., B 911, 105−112.
(25) Furlong, M. T., Savant, I., Yuan, M., Scott, L., Mylott, W.,
Mariannino, T., Kadiyala, P., Roongta, V., and Arnold, M. E. (2013) A
validated HPLC-MS/MS assay for quantifying unstable pharmacolog-
ically active metabolites of clopidogrel in human plasma: application to
a clinical pharmacokinetic study. J. Chromatogr., B 926, 36−41.
(26) Smith, R. L., Gillespie, T. A., Rash, T. J., Kurihara, A., and Farid,
N. A. (2007) Disposition and metabolic fate of prasugrel in mice, rats,
and dogs. Xenobiotica 37, 884−901.
(27) Farid, N. A., Smith, R. L., Gillespie, T. A., Rash, T. J., Blair, P. E.,
Kurihara, A., and Goldberg, M. J. (2007) The disposition of prasugrel,
a novel thienopyridine, in humans. Drug Metab. Dispos. 35, 1096−
1104.
(28) Farid, N. A., McIntosh, M., Garofolo, F., Wong, E., Shwajch, A.,
Kennedy, M., Young, M., Sarkar, P., Kawabata, K., Takahashi, M., and
Pang, H. (2007) Determination of the active and inactive metabolites
of prasugrel in human plasma by liquid chromatography/tandem mass
spectrometry. Rapid Commun. Mass Spectrom. 21, 169−179.
(29) Asai, F., Sudigashi, A., Ikeda, T., Kuroki, Y., Inoue, T., Iwamura,
R., and Shibakawa, N. (2003) Cyclic Amino Compounds, United
H
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