Synthesis of the stabilized active metabolite of clopidogrel

Article abstract of DOI:10.1016/j.tet.2014.04.037

The convergent synthesis of the stabilized active metabolite of clopidogrel was achieved in eleven steps from commercially available 1,2,3,6- tetrahydropyridine and 2-bromo-3′-methoxy acetophenone (MPBr). This synthetic route used a standard Horner-Wadsworth-Emmons reaction allowing the introduction of a Z exocyclic double bond. A selective hydrolysis of an acrylic methyl ester moiety, isolated by an efficient and reliable preparative chiral chromatography at gram scale, released the title compound with a 98% LC purity and d.e. >99%.

Full text of DOI:10.1016/j.tet.2014.04.037

Tetrahedron xxx (2014) 1e8
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Tetrahedron
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Synthesis of the stabilized active metabolite of clopidogrel
,
a
b
b
ꢀ b
Guillaume Bluet a , Jorg Blankenstein , Eric Brohan , Celine Prevost , Michel Cheve ,
*
ꢀ
ꢀ
a
a
ꢀ
Joseph Schofield , Sebastien Roy
a SANOFI R&D, Isotope Chemistry and Metabolite Synthesis (ICMS)dSCP DSAR/DD Paris, 1 avenue Pierre Brossolette, Chilly-Mazarin 91385 Cedex,
France
b SANOFI R&D, Analytical Sciences e SCP LGCR AnSci Paris, 13 quai Jules Guesdes, Vitry-sur-Seine, 94403 Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The convergent synthesis of the stabilized active metabolite of clopidogrel was achieved in eleven steps
from commercially available 1,2,3,6-tetrahydropyridine and 2-bromo-30-methoxy acetophenone (MPBr).
This synthetic route used a standard HornereWadswortheEmmons reaction allowing the introduction of
a Z exocyclic double bond. A selective hydrolysis of an acrylic methyl ester moiety, isolated by an efficient
and reliable preparative chiral chromatography at gram scale, released the title compound with a 98% LC
purity and d.e. >99%.
Received 17 March 2014
Received in revised form 7 April 2014
Accepted 10 April 2014
Available online xxx
Keywords:
Clopidogrel
Ó 2014 Elsevier Ltd. All rights reserved.
Thienopyridine
Metabolism
HornereWadswortheEmmons
Preparative chiral HPLC
1. Introduction
is opened, via the sulfinate intermediate, 30 to give the active me-
tabolite species 4. Two additional elements of stereogenicity are
Clopidogrel 1 is an oral antiplatelet agent from the thienopyr-
idine family indicated for the prevention of artherothrombotic
events (Fig. 1).1 Clopidogrel is a P2Y12-ADP receptor antagonist and
a prodrug, requiring two distinct metabolic transformations to
produce the active metabolite responsible for the anti-aggregating
effect.
thus added to the stereocenter already present at position 7: a new
stereocenter at position 4 and a stereogenic exocyclic double bond,
suggesting that the active metabolite may be one component of
a mixture of diastereoisomers.
In vitro metabolism studies on 3 showed that the metabolic
transformation led to a mixture of four stereoisomers, referred to as
H1, H2, H3 and H4. All four were isolated and characterized: iso-
mers H1 and H2 4a are the E compounds while isomers H3 and H4
4b have Z configuration at the exocyclic double bond (Scheme 2).3
The thiol moiety of the active metabolite 4 binds specifically and
irreversibly to cysteine residues of the platelet P2Y12 purinergic
receptor, thus inhibiting ADP-mediated platelet activation and
aggregation.
COOCH
H
3
N
7(S)
S
Cl
1
In addition, it has been demonstrated by analysis of clinical
samples that only metabolites H4 and H3 are formed in vivo, and
that H4 was found to be the only biologically active isomer.4 These
various metabolism studies showed that the expression of the anti-
aggregant activity of clopidogrel depends on the configuration of
the three stereochemical sites: C7, C3eC16 and C4. The S configu-
ration at C7 and the Z configuration of the C3eC16 double bond are
considered to be crucial for expression of activity and only isomers
H3 and H4 had these features.
Fig. 1. Chemical structure of clopidogrel.
The main metabolic pathway converts 1 into an inactive car-
boxylic acid derivative 2 by hydrolysis (Scheme 1). In vitro studies
showed that the pharmacologically active metabolite is generated
by a two-step hepatic pathway involving multiple cytochrome
P450 isoenzymes.2 The first step involves oxidation of 1 to the in-
active 2-oxo-clopidogrel intermediate 3. Next, the thiolactone ring
Since only metabolite H4 was active in vivo, it appeared that
the activity was also closely linked to the R or S configuration at
* Corresponding author. Tel.: þ33 160497254; fax: þ33 160497640; e-mail ad-
dress: Guillaume.bluet@sanofi.com (G. Bluet).
0040-4020/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2014.04.037
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2
G. Bluet et al. / Tetrahedron xxx (2014) 1e8
CO H
2
H
N
7(S)
Cl
S
esterases
2
CO Me
2
H
carboxylic acid metabolite (inactive)
N
7(S)
Cl
S
1
oxidation
CYP450
CO Me
H
2
CO Me
2
H
7(S)-clopidogrel
oxidation
CYP450
N
N
7(S)
Cl
O
O
+
S
S
Cl
4
O
3
3'
7(S)-2-oxoclopidogrel (inactive)
H
N
CO Me
2
HO C
2
7(S)
HS
Cl
4
4
clopidogrel active metabolites
(mixture of diastereoisomers)
Scheme 1. Metabolic pathway of clopidogrel.
O
N
OMe
O
N
OMe
CO H
2
H
H
CO Me
2
H
CO Me
2
16
16
Z
E
3
7
H
N
HO C
2
N
7
3
3
7
7
+
O
HS
Cl
4
4
S
HS
Cl
S
Cl
4
Cl
4b
Z diastereoisomers H3 & H4
4a
E diastereoisomers H1 & H2
1
3
Scheme 2. Active metabolites of clopidogrel: possible set of four stereoisomers generated from clopidogrel 1.
C4. At the beginning of this project, the absolute configuration at
C4 in H4 had not been clearly elucidated. Further structural
analysis performed concomitantly with this study, allocated the
(R) configuration to the C4 centre in active metabolite H4 5a
(Scheme 3).5
Takahashi et al., showed that direct trapping of metabolites H3
and H4 in human plasma with 2-bromo-30-methoxy acetophenone
6 as alkylating agent overcomes this instability and allows quan-
tification of the derivatized stable forms of both active metabolite 7
and inactive metabolite 8 in clinical samples (Scheme 4).6
H
H
CO Me
2
HO C
2
N
7(S)
Cl
5a
active metabolite H4
O
N
OMe
O
N
OMe
7(S) / 4(R)
HS
4(R)
CYP450
CYP450
7(S)
7(S)
O
in vivo
S
in vivo
S
Cl
+
Cl
1
3
H
H
CO Me
2
HO C
2
N
7(S)
Cl
inactive metabolite H3
7(S) / 4(S)
HS
4(S)
5b
Scheme 3. In vivo metabolism: structure of both active metabolite H4 5a and inactive metabolite H3 5b of 1.
The presence of a free thiol group makes the clopidogrel me-
tabolites H3 and H4 unstable in human plasma, hampering their
quantification in clopidogrel-treated patients.
Derivatized active metabolite 7 has never been synthetized
preparatively by chemical means to the best of our knowledge.
Previous work relied on microsomal incubation with human liver
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G. Bluet et al. / Tetrahedron xxx (2014) 1e8
3
O
Br
H
H
CO Me
2
H
H
CO Me
2
HO C
2
N
HO C
2
N
H
H
CO Me
2
MeO
S
Cl
S
Cl
6
HO C
2
N
O
+
O
acetonitrile, buffer (pH9)
microsomal incubation
(human plasma)
HS
Cl
4a
H3 and H4 mixture
(human plasma)
MeO
MeO
7
8
active stabilized metabolite
MP-H4
inactive stabilized metabolite
MP-H3
Scheme 4. Derivatization of clopidogrel active metabolite with MPBr.
microsomes of 3.4 Unfortunately, this process was hampered by low
yields, needed extensive purifications and was not suitable for
a reliable and efficient preparative scale synthesis. Only small
amounts of 7 could be obtained.
In order to support clinical studies to assess concentrations of
MPBr derivative of 5a in human plasma, larger quantities of 7 were
needed, leading us to design a chemical synthesis.
As shown in Scheme 6, epoxide 12 was prepared from com-
mercially available 1,2,3,6-tetrahydropyridine 14 in two-steps as
described in the literature (route 1).8 Free thiol 13 was obtained
from commercially available 2-bromo-30-methoxy acetophenone
(MPBr) 16 in three steps: displacement of the bromide with po-
tassium thioacetate afforded the corresponding S-acetate 17, which
was protected as its 1,3-dioxolane, 18 using diethylene glycol and
triethyl orthoformate in the presence of catalytic amount of p-TSA.
Hydrolysis of 18 with 1 M NaOH in MeOH delivered free thiol 13 in
up to 75% yield over three steps (route 2).9
2. Results and discussion
Regioselective opening of epoxide 12 with 2 equiv of 13 in
acetonitrile in the presence of 1 M sodium hydroxide led to the
formation of a 90:10 mixture of stereo-regioisomers 19 (major) and
20 (minor) from which desired compound 19 was easily isolated by
chromatography on silica gel in 77% yield (route 3).10 Alcohol 19
was then subjected to a Swern oxidation to afford ketone 11 in up to
72% yield after chromatography on silica gel. This procedure
allowed the oxidation of the hydroxyl group without risk to the
sulfur moiety, a side reaction, which we observed with the
DesseMartin reagent.
Our retrosynthetic analysis of 7 is illustrated in Scheme 5. The
diastereoisomer 7 could be obtained by a SN2 reaction with ster-
eoinversion, between enantiopure 7(R)-chloromandelic acid
methyl ester 9 and piperidine derivative 10, a step based on the
current industrial synthesis of clopidogrel.7 The exocyclic Z double
bond might then be obtained through a HornereWittigeEmmons
(HWE) reaction between ketone 11 and a suitable coupling partner,
such as an acrylic acid ester derivative. Compound 11 could be
prepared by a regioselective opening of BOC-oxopiperidine 12 with
a suitable protected thiol 13.
SN2
HWE
H
H
H
CO Me
2
HO C
2
N
7(S)
HO C
2
NH
H
CO Me
2
O
S
Cl
S
Cl
S
O
7(R)
Cl
+
O
O
O
MP-H4
7
9
MeO
MeO
10
O
O
N
O
O
O
S
N
O
12
+
O
O
O
O
MeO
11
SH
MeO
13
Scheme 5. Retrosynthetic scheme.
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4
G. Bluet et al. / Tetrahedron xxx (2014) 1e8
O
O
NH
(a)
N
O
(b)
N
O
(1)
O
14
15
12
O
O
O
O
O
O
O
(c)
Br
(d)
(e)
O
O
O
(2)
S
S
SH
O
O
16
17
13
18
O
O
HO
O
O
N
O
S
O
(f)
N
O
+
+
13
12
(3)
S
O
HO
O
anti-20
anti-19
O
(g)
O
O
N
O
S
O
O
11
O
Scheme 6. (a) BOC2O, 1,4-dioxan, 10% aqueous NaHCO3, 16 h (97%); (b) m-CPBA, CH2Cl2, 12 (71%); (c) KSAc, abs EtOH. in acetone, 2 h at room temperature (100%); (d) diethylene
glycol, HC(OEt)3, pTSA(5%), 60 ꢀC 24 h (77%); (e) 1 M NaOH, MeOH, 2 h at room temperature (97%); (f) MeCN, 1 M NaOH, 95 ꢀC, 16 h, diastereoisomers not depicted; (g) oxalyl
chloride, CH2Cl2, DMSO, ꢁ78 ꢀC, NEt3 (72%).
The next step concerned the formation of the C16eC3 exocyclic
double bond with Z configuration. To this end, we first envisaged
introducing the Z acrylic acid moiety via a standard HWE reaction
on 11 with methyl diethylphosphono acetate 21 (Scheme 7).
employing NaH as base at 70 ꢀC led to lower yield and unchanged
selectivity (entry 2).
Tooursurprise, theStilleGennari procedure, commonlyemployed
as a variant of the HWE process to produce Z-a,b-unsaturated esters
O
CO Me
2
O
H
O
Z
16
16
MeO C
2
O
N
O
H
N
O
N
O
E
3
3
S
S
S
(a)
O
O
O
+
O
(EtO)2P(O)CH2CO2Me
21
O
O
O
O
O
11
22
23
Scheme 7. HWE reaction of 11 with 21 (see Table 1).
As expected, under standard conditions, using NaH as base in
THF, a mixture of E/Z isomers was obtained, in which the target Z
isomer 22 was isolated in 30% yield as the minor component. With
the objective of improving the Z selectivity and ultimately the yield,
we modified the experimental parameters. A variety of different
bases and solvents were tested, and the results obtained in the
HWE reaction of 11 and 21 are summarized in Table 1. Selectivity
and yield were comparable when using either KOH, K2CO3 or
NaHMDS as base (entry 3, 4 and 5). Replacing THF with toluene and
with a high degree of selectivity, in the presence of phosphonate 24,
gave again mainly the E isomer 23 (Table 1, entry 6).11a
The Z selectivity could probably be improved by changing other
experimental parameters, such as employing Ando’s protocol.11b
However, the standard HWE conditions applied (Table 1, entry 1)
were reliable and efficient enough for our aim to produce the tar-
geted Z isomer 22.
Next, it was necessary to introduce the third stereogenic centre of
(S) configuration at position 7. For this purpose, the 1,3-dioxolane
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G. Bluet et al. / Tetrahedron xxx (2014) 1e8
5
Table 1
HWE reaction of 11 with 21
efficient enough to prepare sizeable quantities of the stabilized
active metabolite 7 of clopidogrel allowing to support a large
clinical program.
Entry
Base/solvent/t ꢀC/time
Ratio
Yield (22)b
(22)/(23)a
1
2
3
4
5
6
NaH/THF/0 ꢀC to rt/2.5 h
NaH/toluene/70 ꢀC/2.5 h
KOH or K2CO3/THF/rt/2 h
NaH/THF/ꢁ20 ꢀC/8 h
1/2.5
1/3
1/3
1/3
1/3
30%
25%
Not isolated
20%
22%
4. Experimental section
4.1. General
NaHMDS/THF/ꢁ78 ꢀC/5 h
KHMDS, 18-crown-6,
1/10
Not isolated
Compounds 14, 16, 21 and 24 were purchased from Aldrich and
used as received. Compound 9 was obtained from SANOFI, In-
dustrial Affairs, Sisteron, France. Solvents and reagents were pur-
chased from commercial sources. Analytical TLC was performed on
Merck (silica gel 60 F254) 5ꢂ10 cm plates and visualized with UV
light (254 nm). 1H and 13C NMR spectra were recorded from CDCl3
solution with a Bruker DPX 200 (200 MHz) or a Bruker AVANCE 600
(600 MHz) spectrometer. 13C NMR spectra were obtained via DEPTQ
(CF3CH2O)2P(O)CH2CO2Me
(24)/THF/ꢁ78 ꢀC/4 h
The bold values signifies the best result obtained.
a
Determined by 1H NMR.
Isolated yield.
b
and BOC protecting groups in 22 were simultaneously removed
using 50% trifluoroacetic acid (TFA) in dichloromethane at room
temperature to furnish the secondary amine 25 (Scheme 8). The
piperidine nitrogen of 25 was then reacted in a SN2 reaction with
complete stereoinversion with enantiopure 7(R)-chloromandelic
acid methyl ester 9 in acetonitrile in the presence of potassium bi-
carbonate.12 This reaction afforded a 1/1 mixture of diastereoisomers
26 and 27 corresponding to the stabilized methyl ester form of active
metabolite 7 and inactive metabolite 8, respectively.
experiment. Chemical shifts are given in ppm as
d values with
reference to CDCl3. Flash chromatography was conducted on silica
gel cartridges with an Armen Spot Prep automated purification
system. Chiral preparative liquid chromatography was performed
with a chiral preparative ChiralpakÒ AD column as specified in the
experimental procedure. HPLC analyses were recorded on Hitachi
Elite LabChromÒ (UV 2400) as specified in the experimental pro-
H
H
H
CO Me
2
H
H
CO Me
2
MeO C
2
NH
MeO C
2
N
7(S)
MeO C
2
N
7(S)
Cl
3
(b)
(a)
S
S
Cl
22
+
S
O
O
CO Me
2
O
O
S
O
7(R)
O
Cl
Cl
9
O
O
O
25
eMP-H4
26
eMP-H3
27
Scheme 8. (a) TFA/CH2Cl2 then saturated aqueous NaHCO3 (93%); (b) 9 (LC purity 99% (by area); 99% e.e.) in CH3CN then 22, KHCO3 (2 equiv), 48e72 h.
Considerable effort was needed to separate diastereoisomers 26
and 27 in order to isolate the target compound 26. We succeeded in
isolating both diastereoisomers by preparative chiral chromatog-
raphy at gram scale (Scheme 9). By applying this method to 3.4 g of
1/1 mixture of 26/27 as depicted in Scheme 9 below, 1.3 g of the
desired compound 26 were isolated with a chemical purity of 98%
and with an optical purity of 99% d.e. (HPLC at 254 nm) .
cedure. LC-MS/HRMS were recorded with a LC-MS/IT-TOF Shi-
madzhu system using a Kromasil 150ꢂ3.0 mm, C18, 3.5
mm
analytical column with a mixture of acetonitrile and 20 mM am-
monium acetate (pH 4.6) buffer as mobile phase.
4.2. tert-Butyl 7-oxa-4-azabicyclo[4.1.0]heptane-4-
carboxylate (12)
HPLC-MS data and NMR spectra of stereoisomer 26 were in
accordance with the structure and corresponding data obtained for
1,2,3,6-Tetrahydropyridine 14 (19.7 g, 237 mmol) was dissolved
in 1,4-dioxan (20 mL) and 10% aqueous sodium carbonate (20 mL)
was added. The solution was cooled to 0 ꢀC and di-tert-butyl
dicarbonate (54.3 g, 249 mmol) was added in portions to the stirred
solution. The ice-bath was removed and the resulting clear yellow
solution was stirred overnight at room temperature. The reaction
mixture was diluted with brine and ether. The ether phase was
separated, and dried over anhydrous sodium sulfate, filtered and
concentrated in vacuo to give 15 as a colourless oil used in the next
compound
7 generated from incubation of 3 with human
microsomes.4
The remaining step, the selective hydrolysis of the acrylic
methyl ester moiety of 26 to release 7, was achieved selectively and
in acceptable yield, using a 37% HCl aqueous solution, a method
previously used in this series.13
The desired metabolite 7 was obtained in up to 52% yield after
purification on silica gel with 98.6% LC purity and with an optical
purity of 99% d.e. (Scheme 10).
step without purification (42 g, 97%); 1H NMR (200 MHz, CDCl3)
d:
1.46 (s, 9H), 2.1(br s, 2H), 3.44 (t, J¼5.8 Hz, 2H), 3.84(m, 2H), 5.63
3. Conclusion
(m, 1H), 5.75 (m, 1H).
tert-Butyl-1,2,3,6-tetrahydropyridine-1-carboxylate 15 (30 g,
164 mmol) was then dissolved in dichloromethane (200 mL) and
cooled to 0 ꢀC. A solution of m-chloroperbenzoic acid (70% w/w,
46.4 g, 188 mmol) in dichloromethane (200 mL) was added
dropwise over 30 min. The ice-bath was removed and the
resulting colourless mixture stirred overnight. The solution was
We have described a convergent synthesis of the stabilized ac-
tive metabolite 7 of clopidogrel, which was isolated, with chemical
purity of 98% and with an optical purity of 99% d.e. Although this
eleven steps synthesis was hampered by the low yield of the Hor-
nereWadswortheEmmons reaction, the synthesis was robust and
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6
G. Bluet et al. / Tetrahedron xxx (2014) 1e8
H
H
CO Me
2
H
H
CO Me
2
MeO C
2
N
7(S)
3
MeO C
2
N
7(S)
3
S
Cl
4(R)
+
S
Cl
4(S)
O
O
26
27
27 26
O
O
eMP-H4
26
eMP-H3
27
Inj 1
crude 1/1 mixture: 3.4g
preparative LC: 2 injections of 1.7g
26
eMP-H4
eMP-H3
26
27
mixture 26 / 27
1.3g isolated:41% yield
27
Scheme 9. Separation of crude 1/1 mixture of eMP-H4 26/eMP-H3 27 by preparative chiral LC on Chiralpak AD.
6.93e7.00 (m, 1H), 7.16e7.40 (m, 3H); 13C NMR (150 MHz, CDCl3)
H
H
H
CO Me
2
H
CO Me
2
197.9, 193.9, 157.8, 135.7, 128.6, 121.9, 119.7, 110.2, 52.1, 33.5, 29.7.
MeO C
2
N
HO C
2
N
7(S)
Cl
7(S)
Cl
S
S
HCl 37%
4(R)
aq-
4(R)
4.4. 2-(3-Methoxyphenyl)-1,3-dioxolanylmethanethiol (13)
O
O
Compound 17 was dissolved in diethylene glycol (105 mL, 2 mol)
and triethyl orthoformate (145 mL, 0.9 mol) was added, followed by
the addition of a catalytic amount of p-toluenesulfonic acid (1.1 g,
6.3 mmol). The reaction mixture was heated at 60 ꢀC and stirred for
24 h. The greenish solution was cooled to room temperature and
the solvents concentrated in vacuo. The oily residue was diluted
with brine and extracted with ether. The organic layer was dried
over anhydrous sodium sulfate, filtered and evaporated to yield
41 g of a greenish oil. Purification by chromatography on silica gel
(eluent pentane/diethylether 6/4) afforded 25.6 g (77% yield) of 1-
O
O
eMP-H4
26
MP-H4
7
Scheme 10. Hydrolysis of ester 26 to free acid 7.
filtered to remove salts and washed with 5% aqueous potassium
carbonate, brine and three times with saturated aqueous sodium
thiosulfite. The organic layer was separated, dried over anhydrous
sodium sulfate, filtered and evaporated in vacuo to give 12 as
a clear yellow oil. The crude product was subjected to flash
chromatography (SiO2, pentane/diethylether 70/30) to afford the
pure title compound 12 (23 g, 71%). Rf¼0.31 (SiO2, pentane/
((2-(3-methoxyphenyl)-1,3-dioxolan-2-yl)sulfanyl)
ethan-1-one
18: Rf¼0.42 (SiO2, pentane/diethylether 7/3); GCeMS (CI): MþHþ
at 268.9 (12.5%) with fragments at 87.0 (100%), 179.1 (25%) and
209.0 (10%), 1H NMR (200 MHz, CDC13)
d: 2.3 (s, 3H), 3.45 (s, 2H),
diethylether 7/3); 1H NMR (200 MHz, CDC13)
d
: 1.32 (s, 9H),
3.82 (m, 5H, eOCH3e, eCH2Oe), 4.07 (m, 2H, eCH2Oe), 6.83e6.87
1.67e2.0 (m, 2H), 2.9e3.35 (m, 4H), 3.51e3.78 (m, 2H); 13C NMR
(m,1H), 7.07e7.35 (m, 3H); 13C NMR (50 MHz, CDCl3)
d: 197.9,193.9,
(150 MHz, CDCl3)
36.4, 28.4, 24.4.
d: 154.8, 79.8, 50.6, 50.1 (br), 42.8, 41.9, 37.9,
157.8, 135.7, 128.6, 121.9, 119.7, 110.2, 52.1, 33.5, 29.7.
Compound 18 was dissolved in methanol (200 mL) and 1 M
sodium hydroxide (105 mL, 105 mmol) was added. The reaction
mixture was stirred at room temperature for 2 h. The solvent was
evaporated in vacuo and brine was added, followed by extraction
with diethylether. The organic layer was dried on anhydrous so-
dium sulfate and evaporated to yield 21 g (97%) of the unstable title
compound 13, used in the next step without purification. Rf¼0.59
(SiO2, pentane/diethylether 7/3); GCeMS (CI): MþHþ at 227 (100%)
with fragments at 193.3 (37%), 183.3 (70%), 149.2 (25%), 119.2 (20%).
4.3. 2-(Acetylsulfanyl)-1-(3-methoxyphenyl)ethan-1-one
(10)14
To a suspension of potassium thioacetate (18.7 g, 163 mmol) in
absolute ethanol (100 mL) was added dropwise a solution of 30-
methoxyphenacyl bromide (MP-Br) 16 (25 g, 109 mmol) in acetone
(80 mL). The reaction mixture was then stirred for 2 h at room
temperature. The dark yellow solution was filtered to remove KBr
and the filtrate evaporated to dryness. The residue was dissolved in
ether and the solution washed with water dried over anhydrous
sodium sulfate, filtered and concentrated in vacuo to afford 17 in
quantitative yield (25 g). Rf¼0.63 (SiO2, pentane/diethylether 7/3);
4.5. tert-Butyl 3-hydroxy-4-[2-((3-methoxyphenyl)-1,3-
dioxolan-2-yl]methylsulfanyl)piperidine-1-carboxylate (19)
To a stirred solution of epoxide 12 (7.3 g, 37 mmol) in acetoni-
trile (150 mL) was added dropwise a solution of 13 (16.6 g,
1H NMR (200 MHz, CDC13)
d: 2.21 (s, 3H), 3.65 (s, 3H), 4.20 (s, 2H),
Please cite this article in press as: Bluet, G.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.04.037

G. Bluet et al. / Tetrahedron xxx (2014) 1e8
7
73.3 mmol) in acetonitrile (100 mL) followed by 1 M aqueous so-
dium hydroxide solution (74 mL, 74 mmol). The mixture was heated
at 95 ꢀC for 24 h. The progress of the reaction was monitored by TLC
(SiO2, pentane/ether 6/4). The solvent was evaporated in vacuo and
brine was added, followed by extraction with diethylether. The
organic layers were dried over anhydrous sodium sulfate, filtered
and evaporated to afford a crude mixture of regioisomers 19 and 20.
Purification by flash column chromatography on silica gel (eluent
pentane/diethylether 6/4) gave 12 g of the target compound 19
(77% yield) as a clear yellow gum. Rf¼0.35 (SiO2, pentane/dieth-
The organic layer was separated, dried over sodium sulfate, fil-
tered and evaporated to afford 10 g of a clear yellow oil (1:3 mixture
of E 23 and Z 22 isomers). Purification by chromatography on silica
gel using pentane/diethylether 60/40 allowed the isolation of 2.3 g
of the target Z isomer 22 (25%). Rf (Z, 22)¼0.6, Rf (E, 23)¼0.5 (SiO2,
pentane/diethylether 1/1); 1H NMR (600 MHz, CDC13), 22(Z)
d: 1.44
(s, 9H), 1.90 (m, 1H, eCHeqe), 1.99 (br s, 1H, eCHaxe), 2.93 (d,
J¼18 Hz, 1H, eSeCH2e), 3.16 (d, J¼18 Hz, 1H, eSeCH2e), 3.18 (br s,
1H, eNeCHaxe), 3.69 (s, 3H, eCO2CH3), 3.81 (s, 3H, eOCH3), 3.84
(d, J¼18 Hz, 2H, eCH2Oe), 3.96 (br s, 1H, eNeCHeqe), 4.11 (m, 2H,
eOCH2e), 5.28 (s, 1H, eSeCHe), 5.73 (s, 1H, eCH]), 6.83 (m, 1H,
Ar), 7.04 (m, 1H, Ar), 7.07 (m, 1H, Ar), 7.24 (m, 1H, Ar); 13C NMR
ylether 6/4, Rf 20¼0.23); 1H NMR (600 MHz, CDC13)
d: 1.45 (s, 9H),
1.5 (m,1H, eCHaxe), 1.95 (m, 1H, eCHeqe), 2.53 (m, 1H, SeCHaxe),
2.60 (m, 1H, NeCHaxe), 2.71 (br s, 1H, NeCHaxe), 3.07 (d, J¼15 Hz,
1H, eSeCH2e), 3.19 (d, J¼14.4 Hz, 1H, eSeCH2e), 3.40 (br s, 1H,
eCHaxeOH), 3.81 (s, 3H, eOCH3e), 3.88 (m, 2H, eCH2Oe), 4.05 (br
s, 1H, eNeCHeqe), 4.15 (m, 2H, eCH2Oe), 4.28 (m, 1H,
eNeCHeqe), 6.86 (m, 1H, Ar), 7.01 (m, 1H, Ar), 7.04 (m, 1H, Ar), 7.25
(150 MHz, CDCl3), 22(Z) d: 166.5, 159.6, 154.4, 143.3, 129.2, 118.2,
113.9, 111.4, 109.2, 80.0, 65.3, 55.1, 51.1, 41.1, 40.5, 32.2, 28.6. HRMS
(ESIþ), 22(Z): m/z calcd for C24H33NO7S [MþNa]þ: 502.1870; found,
502.1820.
1H NMR (600 MHz, CDC13), 23(E)
d: 1.45 (s, 9H), 1.86 (m, 1H,
(m, 1H, Ar); 13C NMR (150 MHz, CDCl3)
d: 159.6, 154.6, 142.8, 129.4,
eCHeqe), 2.08 (br s, 1H, eCHaxe), 2.84 (q, J¼12 Hz, 2H, eSeCH2e),
3.26 (br s, 1H, eNeCHaxe), 3.61 (m, 1H, eCHeqe), 3.73 (s, 3H,
eCO2CH3), 3.82 (s, 3H, eOCH3), 3.84e3.86 (m, 3H), 3.94 (d, J¼18 Hz,
1H), 4.07e4.14 (m, 2H, eOCH2e), 5.48 (d, J¼18 Hz, 1H, eSeCHe),
5.67 (s,1H, eCH]), 6.85 (m,1H, Ar), 7.02 (s,1H, Ar), 7.05 (m,1H, Ar),
118.0, 114.0, 111.4, 109.2, 79.9, 71.1, 65.5, 65.3, 55.3, 52.6, 41.1, 31.5,
28.4; LC-MS: 97% LC purity (by area); ESMS m/z (%): 448 (100)
[MþNaþ]; 464 (34) [MþKþ]; 326(35) [MꢁBOCþHþ]; 308(38)
[326ꢁH2O]]; 264(85) [308ꢁC2H4O].
7.28 (m, 1H, Ar); 13C NMR (150 MHz, CDCl3), 23(E)
d: 166.6, 159.6,
154.3, 143.2, 129.3, 118.1, 116.3, 113.9, 111.3, 109.5, 79.8, 65.3, 65.1,
55.2, 51.3, 47.6, 41.1, 40.4, 31.8, 28.4; HRMS (ESIþ), 23(E): m/z calcd
for C24H33NO7S [MþNa]þ: 502.1870; found, 502.1829.
4.6. tert-Butyl-4-[2-((3-methoxyphenyl)-1,3-dioxolan-2-yl]
methylsulfanyl)-3-oxopiperidine-1-carboxylate (11)
Under a nitrogen atmosphere, a solution of dimethylsulphoxide
(6 mL, 84 mmol) in dichloromethane (8 mL) was added to a stirred
solution of oxalyl chloride (3.7 mL, 42.3 mmol) in dichloromethane
(150 mL) at ꢁ78 ꢀC; The reaction mixture was stirred for 45 min
before 19 (12 g, 28 mmol) in dichloromethane (100 mL) was slowly
added such that the temperature did not rise above ꢁ65 ꢀC. The
mixture was stirred for a further 45 min before triethylamine
(20 mL, 141 mmol) was added. This reaction mixture was stirred for
an additional 1 h and poured onto a mixture of ice and saturated
brine followed by extraction with diethylether. The organic layers
were combined, dried over anhydrous sodium sulfate, filtered and
concentrated to give an orange oil. Purification by flash column
chromatography on silica gel using pentane/diethylether 70/30 as
eluent afforded 8.5 g of 11 (72%) as an unstable yellow oil stored at
ꢁ20 ꢀC. Rf¼0.5 (SiO2, pentane/diethylether 1/1); 1H NMR (600 MHz,
4.8. Methyl 7(S)-7-((2-chlorophenyl)-2-(3(Z)-3-methoxy-2-
oxoethylidene)-4-(2-(3-methoxyphenyl)-2-oxoethyl)sulfanyl
piperidin-1-yl)acetate mixture of diastereoisomers as acrylic
methyl ester form eMp-H4 (26) and eMp-H3 (27)
Under a nitrogen atmosphere, a stirred solution of compound 22
(2.3 g, 5 mmol) in dichloromethane (30 mL) cooled at 0 ꢀC was
treated dropwise with a 50% solution of trifluoroacetic acid (7.4 mL,
96 mmol) in dichloromethane (15 mL). The clear yellow reaction
mixture was then stirred for 24 h at room temperature. The prog-
ress of the reaction was monitored by TLC on silica gel using CH2Cl2/
MeOH/NH4OH (9/1/0.1) as eluent. The mixture was concentrated in
vacuo and the residue diluted with water, basified to pH 8 with
a saturated aqueous solution of NaHCO3 and extracted several
times with dichloromethane. The organic layers were combined,
dried over anhydrous sodium sulfate and the solvent evaporated to
afford 1.5 g of the unstable crude amine 25 (93%) used in the next
step without purification. ES-MS m/z (%): 336 (100) [MþHþ]; 358
(10) [MþNaþ]; 374 (5) [MþKþ]; HRMS: m/z calcd for C17H21NO4S
[MþH]þ: 336.1264; found, 336.1287.
CDC13) d: 1.47 (s, 9H), 2.03 (m, 1H, eCHeqe), 2.27 (m, 1H, eCHaxe),
2.95 (d, J¼18.6 Hz, 1H, eSeCH2e), 3.07 (m, 1H, eSeCH2e), 3.39 (br
s, 1H, NeCHaxe), 3.44 (m, 1H, eSeCHaxe), 3.72 (br s, 1H,
eNeCHeqe), 3.82 (s, 3H, eOCH3), 3.85 (m, 2H, eCH2eOe),
4.08e4.14 two signals mixed up (m, 2H, eOeCH2e) and (d,
J¼22 Hz, 1H, COeCH2eNe), 4.24 (d, J¼22 Hz, 1H, eCOeCH2eNe),
6.84 (m, 1H, Ar), 7.01(m, 1H, Ar), 7.04 (m, 1H, Ar), 7.25 (m, 1H, Ar);
A 40% solution of 7-(R)-chloromandelic acid methyl ester (7.35 g,
7.8 mmol) 9 in dichloromethane was concentrated by solvent re-
moval under a stream of nitrogen. The clear residual oil was dis-
solved in acetonitrile (15 mL) and a solution of compound 25 (1.5 g,
4.5 mmol) in acetonitrile (3 mL) was added dropwise, followed by
the addition of KHCO3 (0.9 g, 9 mmol), in one portion. The reaction
mixture was stirred for 72 h, under nitrogen atmosphere at room
temperature, and the resultant yellow precipitate was filtered off,
and washed with acetonitrile and ethyl acetate. The combined
washings and filtrate were evaporated to afford 4 g of a crude or-
ange oil. Purification by chromatography on silica gel (pentane/
diethylether or heptane/ethyl acetate, 70/30) led to 2 g of a mixture
of both diastereoisomers eMP-H4 26 and eMP-H3 27.
13C NMR (150 MHz, CDCl3)
d: 159.5, 154.2, 142.8, 129.3, 118.2, 114.1,
111.4, 109.2, 80.5, 65.4, 65.3, 55.3, 48.3, 40.8, 29.7, 28.3.
4.7. tert-Butyl-3(Z)-3-((2-methoxy-2-oxoethylidene)-4-[2-(3-
methoxyphenyl)-1,3-dioxolan-2-yl]methylsulfanyl) piperi-
dine-1-carboxylate (22)
Under a nitrogen atmosphere, a suspension of NaH (60% w/w in
mineral oil, 1 g, 25 mmol) in anhydrous THF (30 mL) was treated
dropwise with methyl diethylphosphono acetate 21 (4.4 mL,
23 mmol) between 0 ꢀC and ꢁ5 ꢀC, and the mixture was stirred for
1 h at 0 ꢀC. A solution of ketone 11 (8 g, 19 mmol) in THF (40 mL)
was then added dropwise and the reaction mixture allowed to stir
at room temperature until TLC (SiO2 pentane/diethylether 6/4)
showed that all the ketone 11 was consumed (2 h). The mixture was
concentrated in vacuo and the residue was diluted with CH2Cl2 and
brine.
The diastereoisomers 26 and 27 were then separated and iso-
lated by preparative chiral chromatography using a Chiralpak AD
column (Di 100 mm, dp20 mm); eluent EtOH/MeOH/Heptane (15/
15/70); flow rate: 400 mL/min; detector: 254 nm. In this way 1.3 g
of the desired diastereoisomer 26 (39%) and 1.4 g (41%) of
Please cite this article in press as: Bluet, G.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.04.037

8
G. Bluet et al. / Tetrahedron xxx (2014) 1e8
diastereoisomer 27 were isolated as a clear yellow oil and as a white
254 nm; diastereoisomeric purity for MP-H4 7 (4R, 7S): 99%; HPLC
solid, respectively.
analysis: Shim-pack XR-ODS II, 2.2
m
m (75ꢂ2 mm) eluent: am-
Chiral HPLC analysis Chiralpak AD-H 5
mm (250ꢂ4.6 mm); elu-
monium acetate bufferþacetonitrile, gradient; 35 ꢀC; flow rate:
ent EtOH/MeOH/Heptane (15/15/70); flow rate: 1.5 mL/min;0 de-
0.5 mL/min; detector: 254 nm; HPLC purity (by area): 98.6%.
tector: 254 nm; acrylic ester eMP-H3 27 retention factor k1 ¼4;
1H NMR (600 MHz, CDC13)
d
: 1.93 (d, J¼14.4 Hz, 1H, eCHeqe),
1
0
acrylic ester eMP-H4 26 k2 ¼7.6. H NMR (600 MHz, CDCl3), eMP-H4
2.15 (br m, 1H, eCHaxe), 2.64e2.73 (m, 2H, eCHaxe, eCHeqe),
3.15 (d, J¼12 Hz, 1H, eCH2e), 3.66 (d, J¼12 Hz, 1H, eCH2e), 3.71 (s,
3H, eCO2CH3), 3.82 (s, 3H, eOCH3), 3.93 (d, J¼15.8 Hz, 1H, eCH2e),
4.06 (d, J¼15.6 Hz, 1H, eCH2e), 4.81 (s, 1H, eCHeCO2CH3e), 5.21
(m, 1H, eSeCHe), 5.74 (s, 1H, eCH]), 7.06 (m, 1H, Ar), 7.27e7.35
(m, 3H, Ar), 7.40e7.48 (m, 3H, Ar), 7.61 (m, 1H, Ar); 13C NMR
26
d: 1.91 (d, J¼14.6 Hz, 1H, eCHeqe), 2.17 (m, 1H, eCHaxe),
2.61e2.74 (m, 2H, eCHaxe, eCHeqe), 3.12 (d, J¼12.3 Hz, 1H,
eCH2e), 3.60 (s, 3H, eCO2CH3), 3.64 (d, J¼12.1 Hz, 1H, eCH2e), 3.71
(s, 3H, eCO2CH3), 3.86 (s, 3H, eOCH3), 3.96 (d, J¼15.8 Hz, 1H,
eCH2e), 4.07 (d, J¼15.8 Hz, 1H, eCH2e), 4.80 (s, 1H,
eCHeCO2CH3e), 5.29 (m, 1H, eSeCHe), 5.72 (s, 1H, eCH]), 7.11
(m, 1H, Ar), 7.25e7.30 (br m, 2H, Ar), 7.35e7.40 (m, 2H, Ar),
7.44e7.49 (m, 2H, Ar), 7.59 (m, 1H, Ar); 13C NMR (150 MHz, CDCl3),
(150 MHz, CDCl3) d: 194.5, 171.1, 159.8, 136.9, 134.8, 133.3, 130.1,
129.4, 127.3, 121.1, 119.9, 116.4, 112.7, 67.9, 55.4, 54.7, 52.2, 46.0, 39.8,
38.6, 31.0; HRMS (ESIþ): m/z calcd for C25H26NO6SCl [MþH]þ:
504.1242; found, 504.1148.
eMP-H4 26 d: 194.7, 171.0, 166.5, 159.9, 154.4, 137.3, 134.9, 1333.3,
129.8, 129.6, 127.2, 121.1, 120.0, 116.6, 112.8, 68.0, 58.4, 55.5, 54.5,
52.3, 51.3, 46.1, 40.0, 38.8, 31.0; HRMS (ESIþ), eMP-H4 26: m/z calcd
for C26H28NO6SCl [MþNa]þ: 540.1218.; found, 540.1250.
Acknowledgements
1H NMR (600 MHz, CDC13), eMP-H3 27
d
: 1.97 (d, J¼14.4 Hz, 1H,
We are grateful to Dr. Ruiz-Montes, J., Dr. Zard, L. Bonnet, A.
(SANOFI R&D, SCP-LGCR/CD Paris) and Dr. Perard, S. (SANOFI R&D,
ICMSdSCP DSAR/DD Paris) for helpful discussions and suggestions.
eCHeqe), 2.25 (m, 1H, eCHaxe), 2.75e2.89 (m, 2H, CHaxe,
eCHeqe), 2.91 (d, J¼12 Hz, 1H, eCH2e), 3.55 (d, J¼12 Hz, 1H,
eCH2e), 3.59 (s, 3H, eCO2CH3), 3.70 (s, 3H, eCO2CH3), 3.85 (s, 3H,
eOCH3), 3.94 (d, J¼15.8 Hz, 1H, eCH2e), 4.06 (d, J¼15.6 Hz, 1H,
eCH2e), 4.80 (s, 1H, eCHeCO2CH3e), 5.28 (m, 1H, eSeCHe), 5.60
(s,1H, eCH]), 7.10 (m,1H, Ar), 7.25e7.30 (br m, 2H, Ar), 7.35 (m,1H,
Ar), 7.41 (m, 1H, Ar), 7.47 (m, 1H, Ar), 7.61 (m, 1H, Ar); 13C NMR
ꢀ
References and notes
1. Mafrand, J. P. C.R. Chim. 2012, 15, 737e743 Clopidogrel is mainly marketed as
clopidogrel bisulfate (þ)-(S)-clopidogrel hydrogen sulphate, most commonly
under the trade name PlavixÒ
.
(150 MHz, CDCl3), eMP-H3 27 d: 194.4, 171.1, 166.4, 159.9, 154.7,
2. Dansette, P.; Rosi, J.; Bertho, G.; Mansuy, D. Chem. Res. Toxicol. 2012, 25,
348e356.
137.3, 134.7, 133.3, 129.9, 129.3, 127.2, 121.2, 119.9, 116.6, 112.8, 67.9,
55.5, 54.3, 52.2, 51.3, 46.6, 39.8, 38.8, 31.2; HRMS (ESIþ), eMP-H3 27:
m/z calcd for C26H28NO6SCl [MþH]þ: 518.1399; found, 518.1413.
3. (a) Savi, P.; Pereillo, J. M.; Uzabiaga, M. F.; Combalbert, J.; Picard, C.; Maffrand, J.
P.; Pascal, M.; Herbert, J. M. Thromb. Haemostasis 2000, 84, 891e896; (b) For
a comprehensive mechanism of metabolic oxidation of clopidogrel see also:
Zhang, H.; Lau, W. C.; Hollenberg, P. F. Mol. Pharmacol. 2012, 82, 302e309.
4. Tuffal, G.; Roy, S.; Lavisse, M.; Brasseur, D.; Schofield, J.; Delesque-Touchard, N.;
Savi, P.; Bremond, N.; Rouchon, M. C.; Hurbin, F.; Sultan, E. Thromb. Haemostasis
2011, 105, 696e705.
5. SANOFI R & D, unpublished results. It’s worth noting that metabolite H2 (di-
astereoisomer E) is also active in vitro but not circulating in vivo.
6. Takahashi, M.; Pang, H.; Kawabata, K.; Farid, N. A.; Kurihara, A. J. Pharm. Biomed.
Anal. 2008, 48, 1219e1224.
7. Current synthetic process of 7(S)-clopidogrel: Descamps, J.; Radisson, J. U.S.
Patent 5, 204, 469, 1993.
8. Zhao, S.; Ghosh, A.; D’Andrea, S. V.; Freeman, J. P.; VonVoigtlander, P. F.; Carter,
D. B.; Smith, M. W.; Blinn, J. R.; Szmusczkovicz, J. Heterocycles 1994, 39,
163e170.
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Beckers, T. Bioorg. Med. Chem. Lett. 2007, 17, 4746e4752.
10. CIBA-GEIGY A.G. French Patent 2,363,559, 1977.
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J. K. Tetrahedron Lett. 2003, 44, 7735e7740; (b) Ando, K. J. Org. Chem. 1997, 62,
1934e1939.
12. Bousquet, A.; Musolino, A. French Patent 9, 802,082, 1999. The 7(R)-chlor-
omandelic acid methyl ester 3, methyl 2(R)-2-(2-chlorophenyl)-2-((4-chlor-
ophenyl)sulfonyl)oxy)ethanoate was supplied by SANOFI, industrial affairs,
4.9. 2-(((3Z,4R)-1(7S-1-(2-Chlorophenyl)-2-methoxy-2-
oxoethyl)-4-(2-(3-methoxyphenyl)-2-oxoethyl)sulfanyl)piper-
idin-3-ylidene)acetic acid, stabilized active metabolite MP-H4
(7)
Acrylic methyl ester eMP-H4 26 (1 g, 2 mmol) isolated by chiral
preparative chromatography as described in Section 4.8, was dis-
solved in 37% aqueous HCl (50 mL) at room temperature. The re-
action mixture was then stirred at 40e45 ꢀC for 36 h. The greenish
solution was allowed to reach room temperature and cooled at 0 ꢀC.
A saturated solution of NaHCO3 was carefully added dropwise to
adjust the pH to 7e8 and the reaction mixture extracted with ethyl
acetate and dichloromethane.
The organic layers were combined, dried over anhydrous so-
dium sulfate, filtered and the solvent evaporated to afford a green
oil. Purification by chromatography on silica gel (CH2Cl2/MeOH, 94/
6) afforded 0.5 g of an off-white solid corresponding to the ex-
pected stabilized active metabolite MP-H4 7 of clopidogrel (52%
yield).
Sisteron, France. This compound is conditioned as
dichloromethane.
13. Ruiz-Montes, J.; Zard, L.; Ducandas, V.; Bonnet, A., Elter, M.; Benchekroun, M.
SANOFI R & D, unpublished results.
14. Adamczewska, Y. Z.; Barker, J. M.; Huddleston, P. R.; Wood, M. L. Synth. Commun.
1996, 26, 1083e1096.
a 40% solution in
Chiral HPLC analysis: Chiralpak AD-H 5
m
m (250ꢂ4.6 mm) elu-
ent EtOH/Heptane 30/70; 40 ꢀC; flow rate: 1 mL/min; detector:
Please cite this article in press as: Bluet, G.; et al., Tetrahedron (2014), http://dx.doi.org/10.1016/j.tet.2014.04.037