Overcoming clopidogrel resistance: Discovery of vicagrel as a highly potent and orally bioavailable antiplatelet agent

Article abstract of DOI:10.1021/jm300038c

A series of optically active 2-hydroxytetrahydrothienopyridine derivatives were designed and synthesized as prodrugs of clopidogrel thiolactone in order to overcome clopidogrel resistance. The final compounds were evaluated for their inhibitory effect on ADP-induced platelet aggregation in rats. Compound 9a was selected for further in vitro and in vivo metabolism studies, since its potency was comparable to that of prasugrel and was much higher than that of clopidogrel. Preliminary pharmacokinetic study results showed that the bioavailability of clopidogrel thiolactone generated from 9a was 6-fold higher than that generated from clopidogrel, implying a much lower clinically effective dose for 9a in comparison with clopidogrel. In summary, 9a (vicagrel) holds great promise as a more potent and a safer antiplatelet agent that might have the following advantages over clopidogrel: (1) no drug resistance for CYP2C19 poor metabolizers; (2) lower dose-related toxicity due to a much lower effective dose; (3) faster onset of action.

Full text of DOI:10.1021/jm300038c

Article
pubs.acs.org/jmc
Overcoming Clopidogrel Resistance: Discovery of Vicagrel as a
Highly Potent and Orally Bioavailable Antiplatelet Agent
Jiaqi Shan,†,‡ Boyu Zhang,†,‡ Yaoqiu Zhu,§ Bo Jiao,∥ Weiyi Zheng,⊥ Xiaowei Qi,# Yanchun Gong,†,‡
Fang Yuan,# Fusheng Lv,# and Hongbin Sun*,†,‡
†State Key Laboratory of Natural Medicines and ‡Center of Drug Discovery, College of Pharmacy, China Pharmaceutical University,
24 Tongjia Xiang, Nanjing 210009, China
§MetabQuest Research and Consulting, 1720 Oak Avenue, Suite 403, Evanston, Illinois 60201, United States
∥Department of Pharmacology, School of Pharmacy, Shandong University, 44 Wenhua West Road, Jinan 250012, China
⊥Concord Pharmatech Co., Ltd., 9 Xinglong Road, Nanjing 211800, China
#Jiangsu Vcare PharmaTech Co., Ltd., 15 Wanshou Road, Nanjing 211800, China
S
* Supporting Information
ABSTRACT: A series of optically active 2-hydroxytetrahy-
drothienopyridine derivatives were designed and synthesized
as prodrugs of clopidogrel thiolactone in order to overcome
clopidogrel resistance. The final compounds were evaluated for
their inhibitory effect on ADP-induced platelet aggregation in
rats. Compound 9a was selected for further in vitro and in vivo metabolism studies, since its potency was comparable to that of
prasugrel and was much higher than that of clopidogrel. Preliminary pharmacokinetic study results showed that the bioavailability
of clopidogrel thiolactone generated from 9a was 6-fold higher than that generated from clopidogrel, implying a much lower
clinically effective dose for 9a in comparison with clopidogrel. In summary, 9a (vicagrel) holds great promise as a more potent
and a safer antiplatelet agent that might have the following advantages over clopidogrel: (1) no drug resistance for CYP2C19
poor metabolizers; (2) lower dose-related toxicity due to a much lower effective dose; (3) faster onset of action.
irreversibly inhibiting P2Y12 receptor, a subtype of adenosine
diphosphate (ADP) receptors on the platelet membrane. It is a
prodrug requiring two-step metabolic conversion by the
cytochrome P450 (CYP) system to generate clopidogrel active
metabolite (AM, 5) via clopidogrel thiolactone (4a) (Scheme
1).2 However, nonresponsiveness or poor responsiveness to
clopidogrel therapy occurs in up to 30% of Caucasian patients
(∼2% are poor metabolizers (PMs), ∼26% are intermediate
metabolizers (IMs)),3 especially in PMs carrying CYP2C19 2*
loss-of-function polymorphism, leading to lower levels of the
active metabolite of clopidogrel, less inhibition of platelets, and
a 1- to 5-fold higher risk for death, myocardial infarction, and
stroke in comparison with noncarriers.4,5 This refers to the
concept of clopidogrel resistance (CR). In 2010, the FDA put a
blackbox warning on clopidogrel to make patients and
healthcare professionals aware that CYP2C19 PMs are at
high risk of treatment failure. Currently, clinical practice to
overcome CR includes (1) the adjunctive use of cilostazol, (2)
increasing the dose of clopidogrel, (3) the use of new
antiplatelet agents, such as prasugrel (2) and ticagrelor (3)
(Figure 1).6 However, the above approaches may increase the
risk of major and fatal bleedings and reduce patient
compliance.7 Taken together, there are still unmet medical
needs for the treatment of ACS and its complications, especially
INTRODUCTION
■
Clopidogrel (1) (Figure 1) is an oral antiplatelet agent used to
prevent blood clots in coronary artery disease, peripheral
vascular disease, and cerebrovascular disease. Currently, dual
treatment with aspirin and clopidogrel is the cornerstone of
antiplatelet therapy for patients with acute coronary syndrome
(ACS) and prevention of thrombotic events after percutaneous
coronary intervention (PCI) with stenting.1 The drug works by
Received: January 10, 2012
Published: March 19, 2012
Figure 1. Structures of P2Y12 receptor antagonists.
© 2012 American Chemical Society
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Scheme 1. Metabolic Activation of Clopidogrel and Prodrug
Design
Scheme 2. Synthesis of Optically Active 2-
Hydroxytetrahydrothienopyridine Derivatives 9a−v
a
the clinical management of CR. Novel antiplatelet agents with
fast onset of action, less interindividual variability, and low risk
of bleeding are urgently needed.
a
Reagents and conditions: (a) 4-nitrobenzenesulfonyl chloride,
CH2Cl2, Et3N, DMAP (cat.), 0 °C; (b) KHCO3, CH3CN, rt; (c)
(R2CO)2O, CH3CN, Et3N, 0 °C to rt, or R2COCl, THF, Et3N or
NaH. For R1 and R2, see Table 1.
Given the fact that clopidogrel is among the most widely
prescribed drugs worldwide and its long-term safety profile has
been established after 14 years of clinical use, new drug
discovery based on this old drug would be highly desirable. As
Nobel laureate James Black said, “The most fruitful basis for the
discovery of a new drug is to start with an old drug.” We
envisioned that, like the metabolic pattern of prasugrel, ester
prodrugs 9 might be readily converted to clopidogrel
thiolactone (4a) by esterase-mediated hydrolysis and sub-
sequently to clopidogrel AM (5) through only one CYP-
dependent step (Scheme 1). In this regard, 9 could be ideal
drug candidates for overcoming CR without increasing the risk
of bleeding and other side effects associated with the new
antiplatelet agents. Herein, we report the identification of a
series of 2-hydroxytetrahydrothienopyridine derivatives as
novel antiplatelet agents. We also describe the detailed
structure−activity relationships (SARs) of this series of
compounds. Preliminary efficacy, toxicity, and pharmacokinetic
studies led to the discovery of vicagrel (9a) as a highly potent
and orally bioavailable antiplatelet agent.
methyl 2-(2-chlorophenyl)-2-[(4-nitrobenzenesulfonyl)oxy]-
acetate and racemic methyl 2-(2-chlorophenyl)-2-[(4-
nitrobenzenesulfonyl)oxy]acetate, respectively. The optical
purity (% ee) of compounds 9a−v and (R)-9a was determined
by chiral HPLC.
Inhibition of ADP-Induced Platelet Aggregation in
Rats and SAR Analysis. Twenty-five 2-hydroxytetrahydro-
thienopyridine derivatives were evaluated for their inhibitory
effect on ADP-induced platelet aggregation in rats at dose of 3
mg/kg. Clopidogrel (1) and prasugrel (2) were used as positive
controls. ADP-induced platelet aggregation was determined by
Born’s method.8 The assay results are summarized in Table 1.
In our preliminary tests, although clopidogrel showed strong
potency at a dose of 10 mg/kg (data not shown), it was almost
inactive at a dose of 3 mg/kg. Prasugrel was the most potent
one among this set of test compounds. Compound 9a exhibited
very strong inhibitory effect on platelet aggregation and was
only slightly less potent than prasugrel. 2-Chloro substitution
seemed to be critical for the potency (e.g., 9a−c,l−o), since
both 2-hydrogen and 2-fluoro substitution resulted in
significant loss of potency (e.g., 9u, 9v). 2-Hydroxy ester
moiety of the tetrahydrothienopyridine ring also had a
significant impact on potency. Interestingly, when the size of
2-hydroxy alkyl esters increased, the potency decreased
accordingly (e.g., potency in the order 9a > 9b > 9c > 9d >
9e), suggesting that the hindrance at the 2-ester groups might
reduce their hydrolysis rates and thus result in lower
concentrations of clopidogrel AM. The same tendency was
also observed with carbonic acid esters (e.g., potency, 9m > 9n
> 9p > 9q). It was notable that the potency of carbonic acid
ester 9m was similar to that of alkyl ester 9a, indicating that
carbonic acid esters could also be converted to clopidogrel AM
as well as alkyl esters. Not surprisingly, clopidogrel thiolactone
(4a) was a potent antiplatelet agent, albeit less potent than its
RESULTS AND DISCUSSION
■
Chemistry. Optically pure (R)-methyl 2-(2-substitued-
phenyl)-2-[(4-nitrobenzenesulfonyl)oxy]acetates 7a−c were
prepared via reaction of mandelic acid and 2-substituted
mandelic acids with 4-nitrobenzenesulfonyl chloride in the
presence of triethylamine at low temperature (Scheme 2). N-
Alkylation of 5,6,7,7a-tetrahydrothieno[3,2-c]pyridin-2(4H)-
one hydrochloride (8) with 7a−c in the presence of potassium
bicarbonate afforded thiolactones 4a−c. Reaction of 4a−c with
acyl anhydrides, acyl chlorides, chlorocarbonic acid esters, or N-
substituted carbamic chlorides in the presence of triethylamine
or sodium hydride gave optically active 2-hydroxytetrahydro-
thienopyridine derivatives 9a−v with (S)-configuration. The
enantiomer of 9a ((R)-9a) and the racemic mixture of 9a
((R,S)-9a) were prepared starting from optically pure (S)-
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Table 1. Inhibitory Effect of 2-Hydroxytetrahydrothienopyridine Derivatives on ADP-Induced Platelet Aggregation in Rats at a
a
Dose of 3 mg/kg
d
compd
R1
R2
% ee
99.0
platelet aggregation (%)
b
1
73.7 5.2
c
2
32.1 9.3**
48.6 14.6**
34.6 13.5**
46.7 15.4**
52.8 7.7**
63.4 16.2*
70.0 23.0
60.1 11.9**
75.6 7.0
4a
98.1
99.1
96.5
96.3
99.1
99.5
93.5
100
9a
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
methyl
9b
ethyl
9c
n-propyl
9d
tert-butyl
9e
tert-amyl
9f
phenyl
9g
4-NO2-phenyl
4-MeO-phenyl
2-AcO-phenyl
benzyl
9h
96.9
96.0
93.5
98.7
97.7
97.0
97.3
97.5
95.5
93.7
89.0
95.5
95.7
72.0
86.9
98.7
82.6 9.1
9i
77.9 4.9
9j
53.8 10.4**
84.7 8.7
9k
styryl
9l
pyridine-3-yl
methoxy
45.1 9.0**
38.6 9.1**
53.0 11.6**
50.2 9.3**
65.6 23.2
72.2 10.5
75.4 4.5
9m
9n
ethoxy
9o
isopropoxy
isobutoxy
benzyloxy
phenoxymethyl
dimethylamino
pyrrolidine-1-yl
methyl
9p
9q
9r
9s
67.7 17.7
80.9 9.2
9t
9u
81.1 6.0
9v
F
methyl
62.9 12.8*
68.4 19.6
63.7 8.0*
75.7 7.7
(R)-9a
(R,S)-9a
vehicle
a
Assay details are described in Experimental Section. Aggregation data refer to ex vivo measurements 2 h after oral administration. (∗) P < 0.05. (∗∗)
b
c
P < 0.01 vs vehicle. Data are the mean SD, n = 10. Clopidogrel hydrogen sulfate was used as a positive control. Prasugrel free base was used as a
positive control. ee refers to enantiomeric excess.
d
acetic ester 9a. Aromatic acid esters (e.g., 9f, 9g, 9h, 9i) were
not as potent as alkyl esters, except for nicotinate ester 9l which
exhibited strong potency. Substituted carbamate esters were
inactive in this assay (e.g., 9s, 9t). Preliminary tests had shown
that 9a seemed to be the most promising drug candidate
among this series of compounds, and thus, its enantiomer ((R)-
9a) and racemic mixture ((R,S)-9a) were synthesized and
biologically evaluated in order to investigate the effect of
configuration on potency. In contrast to 9a, (R)-9a was almost
inactive, while (R,S)-9a had very weak activity. On the basis of
the above results, 9a was selected for further in vitro and in vivo
metabolism studies.
In Vitro Metabolic Activation Studies on Clopidogrel
and 9a in Rat Liver Microsomes (RLMs). Clopidogrel (1)
could be metabolically activated to form its active metabolite
(AM, 5) via the thiolactone intermediate (4a) under in vitro
incubation conditions with liver microsomes9 or cDNA-
expressed P450 isozymes10 (Figure 2). The active metabolite
(5) is chemically labile probably because of the reactive thiol
function. Although it was reported that 5 might be stable at 4
°C for 24 h in quenched incubation mixtures,10 for the
convenience of LC−MS/MS-based qualitative and quantitative
analyses as well as further purification for NMR studies, 5 was
often derivatized in the incubation mixture with a variety of
reagents that were reactive toward the thiol function including
glutathione (GSH), acrylonitrile, 3′-methoxyphenacyl bromide,
N-ethylmaleimide, and dimedone.10,11 In this study, glutathione
was used to derivatize 5 to form the active metabolite−
glutathione disulfide adducts (AMGS, Figure 2). To test the
metabolic activation of 9a and potential formation of the active
metabolite (5), rat liver microsomal incubation was conducted
for 9a in parallel with clopidogrel in the presence of NADPH
and glutathione. After quenching and centrifugation, the
supernatant was analyzed by LC−MS/MS. Incubation of
clopidogrel with rat liver microsomes in the presence of GSH
leads to NADPH-dependent formation of metabolites (Figure
3A) that exhibited a molecular ion at m/z of 661 and a product
ion spectrum that is in complete agreement with the formation
of the clopidogrel AMGS disulfide adducts. LC−MS/MS
analysis of the supernatant resulting from incubation of 9a with
rat liver microsomes in the presence of GSH reveals NADPH-
dependent formation of glutathione adducts at the same LC
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Figure 2. Proposed pathways of active metabolite and active metabolite−glutathione adduct formation via in vitro metabolic activation of clopidogrel
and 9a upon incubation with RLM in the presence of GSH and NADPH.
Figure 3. LC−MS-selective ion monitoring chromatogram (SIM, M + H+ = 661) of active metabolite−glutathione (AMGS−1/2/3/4) adducts in
RLM incubation of clopidogrel (A) or 9a (B) for 30 min.
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Figure 4. Averaged molecular ion (MS, top) spectrum and product ion (MS2, bottom) spectrum of the glutathione (AMGS) adducts detected in rat
liver microsomal incubation of 9a in the presence of NADPH and glutathione. The inset is proposed fragmentation pathways of the molecular ion
[M + H]+ of AMGS at m/z of 661.
retention time as the AMGS adducts formed in clopidogrel
incubation (Figure 3, B). The molecular ion (MS) spectrum
(Figure 4, top) and product ion (MS2) spectrum (Figure 4,
bottom) of the glutathione adducts detected in rat liver
microsomal incubation of 9a were almost identical to those of
the AMGS adducts in clopidogrel incubation (data not shown).
The MS spectra of the glutathione adducts show the typical
isotopic pattern that is in accordance with the monochloro-
containing nature of the AMGS adducts. Fragmentation
analysis (Figure 4, inserted) based on the exclusive product
ions resulting from collision-induced dissociation (CID) of the
molecular ion [M + H]+ of m/z 661 (MS2 spectrum) confirms
the formation of the AMGS adducts with the presence of both
moieties of 9a and glutathione as well as the disulfide bond
formation. The metabolic activation of 9a or clopidogrel
confers on the structure of the active metabolite two additional
stereochemical sites: one chiral center adjacent to the thio
function and one geometric center of the ethylenic double
bond, which provide the basis for the formation of four
diastereomers.12 Chromatographic separation resolved the four
diastereomeric AMGS adducts formed from 9a into four peaks
with retention times of 9.41 min (AMGS−1), 9.84 min
(AMGS−2), 10.02 min (AMGS−3), and 10.63 min (AMGS−
4) (Figure 3B). On the basis of the LC−MS/MS studies of in
vitro rat liver microsomal incubation of 9a (see Supporting
Information) paralleled to clopidogrel, the metabolic activation
pathways of 9a are proposed in Figure 2. As shown in the first
step, the acetate ester function of 9a undergoes hydrolysis
probably by esterases presented in rat liver microsomes and
gets deacetylated. The formed 2-hydroxytetrahydrothienopyr-
idine (10) is a tautomer of thiolactone 4a, which is the
metabolic intermediate resulting from the first oxidative
activation of clopidogrel (1). The metabolic activation
pathways of clopidogrel and 9a converge after the first steps
through this 2-hydroxytetrahydrothienopyridine−thiolactone
tautomerization and share the subsequent pathways including
the second step of NAPDH-dependent oxidative ring-opening
of 4a that eventually lead to active metabolite formation
(Figure 2).
Pharmacokinetic Parameters of Clopidogrel Thiolac-
tone after Oral Administration of Clopidogrel or 9a to
Rats. The dose of clopidogrel or 9a at 24 μmol kg−1 was orally
administered to SD male rats. Blood was collected at 0 h
(before dosing) and 0.25, 0.5, 1, 2, 4, 6, 8, 24 h postdose. The
dose of clopidogrel thiolactone at 8 μmol kg−1 was intravenous
administration to SD male rats to determine the conversion
rate or bioavailability of clopidogrel or 9a to clopidogrel
thiolactone. Blood was collected at 0 h (before dosing) and
0.083, 0.167, 0.5, 1, 2, 4, 6, 8, 24 h postdose. After sample
cleanup, the plasma samples were subjected to LC−MS/MS
analysis to determine the plasma concentrations of clopidogrel
thiolactone (Figure 5). After oral dosing of clopidogrel, the
C
max, Tmax, t1/2, and AUC0−∞ of clopidogrel thiolactone in
plasma were 6.93 3.36 μg L−1, 0.583 0.382 h, 2.48 0.466
h, and 32.2 10.9 μg·h L−1, respectively. After oral dosing of
,
9a, the Cmax, Tmax t1/2, and AUC0−∞ of clopidogrel thiolactone
in plasma were 67.2 42.3 μg L−1, 1.17 0.764 h, 2.19 1.68
h, and 211 119 μg·h L−1, respectively. The aforementioned
data proved that after oral administration, 9a could be readily
converted into clopidogrel thiolactone, and bioavailability of
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and was much higher than that of clopidogrel. In vitro
metabolism studies revealed that, like clopidogrel, 9a could be
readily converted to clopidogrel active metabolite by rat liver
microsomes. Preliminary pharmacokinetic study results showed
that the bioavailability of clopidogrel thiolactone generated
from 9a was 6-fold higher than that generated from clopidogrel,
implying a much lower clinically effective dose and thus lower
dose-related toxicity for 9a in comparison with clopidogrel.
In summary, 9a holds great promise as a more potent and a
safer antiplatelet agent that might have the following advantages
over clopidogrel: (1) no drug resistance for CYP2C19 poor
metabolizers; (2) lower dose-related toxicity due to a much
lower effective dose; (3) faster onset of action due to its
metabolic activation mechanism. From our point of view, the
bleeding risk of 9a should be manageable, since under a proper
dosing range, the efficacy of 9a would be equal to that of
clopidogrel, and thus, in theory, the bleeding risk of 9a should
not be higher than that of clopidogrel. Further preclinical trials
on 9a (vicagrel) are currently being conducted in our
laboratories to prove the above predictions.
Figure 5. Plasma concentrations of clopidogrel thiolactone after oral
administration of clopidogrel or 9a to SD rats at a dose of 24 μmol
kg−1.
clopidogrel thiolactone generated from 9a was 6-fold higher
than that generated from clopidogrel at the same dose.
Single-Dose Toxicity of 9a. Single-dose toxicity studies on
9a were performed in GLP laboratories. No mouse (n = 10
males, 10 females) died after receiving 5000 mg/kg 9a by oral
gavage in the 14-day observation period. Liver injury was
observed, and no adverse effects were observed in other organs.
After oral administration of 9a to beagle dogs, the no-observed-
adverse-effect level (NOAEL) was 300 mg/kg and the maximal
tolerance dose (MTD) was higher than 2000 mg/kg. The
above data indicate that 9a has very low acute toxicity.
EXPERIMENTAL SECTION
■
Chemistry. Materials and General Methods. All commercially
available solvents and reagents were used without further purification.
Melting points were determined with a Buchi capillary apparatus and
̈
1
were not corrected. H and 13C NMR spectra were recorded on an
ACF* 300Q Bruker or ACF* 500Q Bruker spectrometer in CDCl3,
with Me4Si as the internal reference, or in DMSO-d6. Low- and high-
resolution mass spectra (LRMS and HRMS) were recorded in electron
impact mode. Reactions were monitored by TLC on silica gel 60 F254
plates (Qingdao Ocean Chemical Company, China). Column
chromatography was carried out on silica gel (200−300 mesh,
Qingdao Ocean Chemical Company, China). The purity of all final
compounds was determined to be ≥95% by analytical HPLC
(equipment: Agilent 1100 system with a VWD G1314A UV detector;
column, Chiralpak IC, 4.6 mm × 250 mm). Detailed analytical HPLC
conditions for each final compound are described in the following
section.
CONCLUSIONS
■
Although in 2010, the FDA issued a “boxed warning” of
reduced effectiveness of clopidogrel in patients carrying
CYP2C19 loss-of-function alleles, there is still debate on
whether there is a significant association between CYP2C19
genotype and cardiovascular events in patients receiving
clopidogrel.5,13,14 Nevertheless, it seems no doubt that
CYP2C19 loss-of-function alleles lead to clopidogrel resistance.
In this regard, the cardiovascular risk associated with
clopidogrel resistance would be definitely an unnegligible
factor, especially for severe ACS patients with PCI, since
platelet-mediated thrombotic events might be amplified in the
presence of both plaque disruption and interventional
procedures.15 On the other hand, most of the study population
involved in the large-scale clinical trials on clopidogrel were
Caucasians among whom the percent of CYP2C19 poor
metabolizers is only about 2%. By contrast, the percent of
CYP2C19 poor metabolizers in East Asians is much higher at
15−23%.16 That is, the meta-analysis results13,14 based on those
clinical trials may not truly reflect the risk associated with
clopidogrel resistance, especially for East Asian patients. Large-
scale clinical trials addressing clopidogrel resistance are
warranted to define the risk of major adverse cardiovascular
outcomes among CYP2C19 poor metabolizers treated with
clopidogrel.
( R ) - M e t h y l 2 - ( 2 - C h l o r o p h e n y l ) - 2 - ( 4 -
nitrophenylsulfonyloxy)acetate (7a). To a stirred mixture of
(R)-2-hydroxy-2-(2-chlorophenyl)acetate (98.4 g, 0.49 mol, 99.0% ee)
and Et3N (91 mL, 0.65 mol) in CH2Cl2 (500 mL) at 0 °C was slowly
added a solution of 4-nitrobenzenesulfonyl chloride (120 g, 0.54 mol)
in CH2Cl2 (500 mL). After being stirred for 4 h at the same
temperature, the mixture was quenched with water. The organic layer
was separated, dried over anhydrous sodium sulfate, and concentrated
under reduced pressure. The residue was recrystallized in MeOH to
afford the title compound as a light yellow solid (154.5 g, 82.0% yield),
98.1% ee (Chiral HPLC analytical conditions: Chiralpak IC, 4.6 mm ×
250 mm, eluting with 50% n-hexane + 50% i-PrOH, flow rate 0.5 mL/
1
min, oven temperature 25 °C, detection UV 220 nm). H NMR (300
MHz, CDCl3): δ 8.30 (d, 2 H, J = 8.9 Hz), 8.07 (d, 2 H, J = 8.9 Hz),
7.39−7.21 (m, 4 H), 6.39 (s, 1 H), 3.57 (s, 3 H). ESI-MS m/z 408.0
[M + Na]+.
(S)-Methyl 2-(2-Chlorophenyl)-2-(2-oxo-7,7a-dihydrothieno-
[3,2-c]pyridin-5(2H,4H,6H)-yl)acetate (4a). To a solution of (R)-
methyl 2-(2-chlorophenyl)-2-(4-nitrophenylsulfonyloxy)acetate (58.1
g, 0.15 mol) in CH3CN (500 mL) were added 5,6,7,7a-
tetrahydrothieno[3,2-c]pyridin-2(4H)-one hydrochloride (32.3 g,
0.17 mol) and potassium bicarbonate (37.8 g, 0.38 mol). After being
stirred at room temperature for 26 h under N2 atmosphere, the
mixture was filtered and the liquid was concentrated under reduced
pressure. The residue was purified by column chromatography to give
a yellow oil, which was recrystallized in EtOH to afford the title
compound as a white solid (35.4 g, 70% yield), mp 146−148 °C,
98.1% ee (determined after conversion to 9a). [α]D19 +114.0° (c 0.5,
MeOH). 1H NMR (300 MHz, CDCl3): δ 7.54−7.51 (m, 1 H), 7.44−
In the present study, prodrug design based on clopidogrel
thiolactone metabolite was mainly aimed at overcoming
clopidogrel resistance. Therefore, 25 2-hydroxytetrahydrothie-
nopyridine derivatives were synthesized and evaluated for their
inhibitory effect on ADP-induced platelet aggregation in rats.
The animal study results showed that some compounds (e.g.,
9a, 9b, 9j, 9l, 9m, 9n, 9o) exhibited potent activity as
antiplatelet agents. Compound 9a was selected for further
studies, since its potency was comparable with that of prasugrel
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same as those for 9a). [α]D20 +38.00° (c 0.50, MeOH). H NMR (300
MHz, CDCl3): δ7.69−7.23 (m, 4 H), 6.26 (s, 1 H), 4.90 (s, 1 H), 3.72
(s, 3 H), 3.59 (ABq, 2 H, J = 14.1 Hz), 2.88−2.87 (m, 2 H), 2.79−2.77
(m, 2 H), 1.30 (s, 9 H). 13C NMR (75 MHz, CDCl3): δ 175.1, 171.2,
150.0, 134.6, 133.7, 129.9, 129.7, 129.3, 129.0, 127.1, 125.5, 111.3,
67.7, 52.0, 50.2, 48.1, 39.0, 26.9, 24.9. ESI-MS m/z 422.2 [M + H]+.
HRMS calcd for C21H25NO4SCl [M + H]+ m/z 422.1198, found
422.1193.
1
7.41 (m, 1 H), 7.32−7.26 (m, 2 H), 6.02 (s, 1 H), 4.91 (s, 1 H), 4.16
(m, 1 H), 3.92 (m, 1 H), 3.73 (s, 3 H), 3.25 (d, 1 H, J = 12.3 Hz), 3.03
(d, 1 H, J = 12.7 Hz), 2.65−2.59 (m, 1 H), 2.37−2.31 (m, 1 H), 1.93−
1.79 (m, 1 H). 13C NMR (75 MHz, CDCl3): δ 198.4, 170.7, 167.1,
134.8, 132.8, 130.0, 129.7, 127.1, 126.7, 67.2, 52.2, 51.5, 51.0, 49.6,
33.8. ESI-MS m/z 338.1 [M + H]+. HRMS calcd for C16H17NO3SCl
[M + H]+ m/z 338.0618, found 338.0626.
(S)-Methyl 2-(2-Acetoxy-6,7-dihydrothieno[3,2-c]pyridin-
5(4H)-yl)-2-(2-chlorophenyl)acetate (9a). To a stirred mixture of
4a (6.5 g, 19 mmol, 97.5% ee) and Et3N (5.4 mL, 38.5 mmol) in
CH3CN (100 mL) at 0 °C was slowly added acetic anhydride (3.6 mL,
38.5 mmol). The mixture was stirred for 2 h at room temperature and
quenched with water. Then AcOEt (100 mL) was added to the
mixture and the organic layer was washed with saturated sodium
bicarbonate solution, brine and dried over anhydrous sodium sulfate.
The organic layer was concentrated under reduced pressure and the
residue was purified by column chromatography to afford a light
yellow oil, which was recrystallized in EtOH to afford the title
compound as a white solid (6.8 g, 93.2% yield), mp 73−75 °C, 99.1%
ee (Chiral HPLC analytical conditions: Chiralpak IC, 4.6 mm × 250
mm, eluting with 92% n-hexane + 8% THF + 0.1% Et2NH, flow rate
0.5 mL/min, oven temperature 25 °C, detection UV 254 nm). [α]D23
(S)-5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-
tetrahydrothieno[3,2-c]pyridin-2-yl 2,2-Dimethylbutanoate
(9e). Following a procedure similar to that described for the
preparation of 9d except that an equivalent amount of 2,2-
dimethylbutanoyl chloride was used in place of pivaloyl chloride, the
title compound was obtained as a white solid in 74.9% yield, mp 98−
100 °C, 99.5% ee (Chiral HPLC analytical conditions: same as those
for 9a). [α]D20 +36.00° (c 0.50, MeOH). 1H NMR (300 MHz, CDCl3):
δ 7.69−7.22 (m, 4 H), 6.25 (s, 1 H), 4.90 (s, 1 H), 3.71 (s, 3 H), 3.59
(ABq, 2 H, J = 14.3 Hz), 2.88−2.87 (m, 2 H), 2.78−2.76 (m, 2 H),
1.64 (q, 2 H, J = 7.3 Hz), 1.26 (s, 6 H), 0.88 (t, 3 H, J = 7.4 Hz). 13C
NMR (75 MHz, CDCl3): δ 174.7, 171.2, 150.0, 134.7, 133.7, 129.9,
129.7, 129.4, 129.1, 127.1, 125.5, 111.4, 67.8, 52.1, 50.3, 48.1, 43.1,
33.3, 24.9, 24.4, 9.2. ESI-MS m/z 436.2 [M + H]+. HRMS calcd for
C22H27NO4SCl [M + H]+, m/z 436.1352, found 436.1349.
1
+45.00° (c 1.0, MeOH). H NMR (300 MHz, CDCl3): δ 7.70−7.24
(S)-5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-
tetrahydrothieno[3,2-c]pyridin-2-yl Benzoate (9f). Following a
procedure similar to that described for the preparation of 9a except
that an equivalent amount of benzoic anhydride was used in place of
acetic anhydride, the title compound was obtained as a white solid in
52.0% yield, mp 84−86 °C, 93.5% ee (Chiral HPLC analytical
conditions: same as those for 9b). [α]D20 +34.00° (c 0.50, MeOH). 1H
NMR (300 MHz, CDCl3): δ 8.17−7.26 (m, 9 H), 6.42 (s, 1 H), 4.95
(s, 1 H), 3.73 (s, 3 H), 3.68−3.57 (m, 2 H), 2.93−2.82 (m, 4 H). 13C
NMR (75 MHz, CDCl3) δ 163.5, 149.9, 134.7, 133.9, 130.2, 130.0,
129.8, 129.5, 128.6, 128.5, 127.2, 125.9, 112.1, 67.8, 52.2, 50.4, 48.2,
25.0. ESI-MS m/z 442.1 [M + H]+. HRMS calcd for C23H21NO4SCl
[M + H]+ m/z 442.0891, found 442.0880.
(m, 4 H), 6.26 (s, 1 H), 4.92 (s, 1 H), 3.72 (s, 3 H), 3.60 (ABq, 2 H, J
= 14.2 Hz), 2.90 (s, 2H), 2.79−2.65 (m, 2 H), 2.26 (s, 3 H). 13C NMR
(75 MHz, CDCl3): δ 170.7, 167.2, 149.1, 134.2, 133.3, 129.4, 129.3,
128.9, 128.8, 126.6, 125.3, 111.5, 67.3, 51.6, 49.8, 47.6, 24.5, 20.2. ESI-
MS m/z 380.0 [M + H]+. HRMS calcd for C18H19NO4SCl [M + H]+
m/z 380.0723, found 380.0737.
(S)-5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-
tetrahydrothieno[3,2-c]pyridin-2-yl Propionate (9b). Following
a procedure similar to that described for the preparation of 9a except
that an equivalent amount of propionic andydride was used in place of
acetic anhydride, the title compound was obtained as a colorless oil in
66.0% yield, 96.5% ee (Chiral HPLC analytical conditions: Chiralpak
IC, 4.6 mm × 250 mm, eluting with 90% n-hexane + 10% i-PrOH +
0.1% Et2NH, flow rate 0.5 mL/min, oven temperature 25 °C,
detection UV 254 nm). [α]D20 +36.00° (c 0.50, MeOH). 1H NMR (300
MHz, CDCl3): δ 7.69−7.26 (m, 4 H), 6.26 (s, 1 H), 4.91 (s, 1 H),
3.72 (s, 3 H), 3.59 (ABq, 2 H, J = 14.2 Hz), 2.88−2.87 (m, 2 H),
2.78−2.76 (m, 2 H), 2.55 (q, 2 H, J = 7.7 Hz), 1.23 (t, 3 H, J = 7.4
Hz). 13C NMR (75 MHz, CDCl3): δ 171.2, 149.8, 123.7, 130.0, 129.8,
129.5, 129.1, 127.2, 125.6, 111.7, 106.2, 67.8, 52.2, 50.3, 48.2, 27.4,
25.0, 21.1, 8.8. ESI-MS m/z 394.1 [M + H]+. HRMS calcd for
C19H21NO4SCl [M + H]+ m/z 394.0883, found 394.0880.
(S)-5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-
tetrahydrothieno[3,2-c]pyridin-2-yl Butyrate (9c). Following a
procedure similar to that described for the preparation of 9a except
that an equivalent amount of butyric anhydride was used in place of
acetic anhydride, the title compound was obtained in 41.0% yield,
96.3% ee (Chiral HPLC analytical conditions: same as those for 9b).
[α]D20 +32.00° (c 0.50, MeOH). 1H NMR (300 MHz, CDCl3): δ 7.69−
7.24 (m, 4 H), 6.25 (s, 1 H), 4.90 (s, 1 H), 3.72 (s, 3 H), 3.58 (ABq, 2
H, J = 14.3 Hz), 2.89−2.86 (m, 2 H), 2.78−2.76 (m, 2 H), 2.52−2.47
(m, 2 H), 1.74 (q, 2 H, J = 5.2 Hz), 1.00 (t, 3 H, J = 5.2 Hz). 13C
NMR (75 MHz, CDCl3): δ 171.2, 170.4, 149.7, 134.7, 133.8, 130.0,
129.8, 129.4, 129.2, 127.1, 125.7, 111.8, 67.9, 52.1, 50.3, 48.2, 35.8,
25.0, 18.2, 13.5. ESI-MS m/z 408.1 [M + H]+. HRMS calcd for
C20H23NO4SCl [M + H]+ m/z 408.1035, found 408.1036.
(S)-5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-
tetrahydrothieno[3,2-c]pyridin-2-yl 4-Nitrobenzoate (9g). Fol-
lowing a procedure similar to that described for the preparation of 9d
except that an equivalent amount of 4-nitrobenzoyl chloride was used
in place of pivaloyl chloride, the title compound was obtained as a
yellow solid in 26.0% yield, mp 100−102 °C, 100% ee (Chiral HPLC
analytical conditions: Chiralpak IC, 4.6 mm × 250 mm, eluting with
50% n-hexane + 50% i-PrOH + 0.1% Et2NH, flow rate 0.5 mL/min,
oven temperature 25 °C, detection UV 254 nm). [α]D20 +30.00° (c
1
0.50, MeOH). H NMR (300 MHz, CDCl3): δ 8.18 (s, 4 H), 7.70−
7.26 (m, 4 H), 6.47 (s, 1 H), 4.95 (s, 1 H), 3.73 (s, 3 H), 3.70−3.57
(m, 2 H), 2.95−2.91 (m, 2 H), 2.84−2.82 (m, 2 H). 13C NMR (75
MHz, CDCl3): δ 171.3, 161.7, 151.0, 149.2, 134.8, 133.9, 133.6, 131.3,
130.0, 129.8, 129.5, 127.2, 126.6, 123.8, 112.6, 67.8, 67.3, 52.2, 51.6,
51.1, 50.3, 49.7, 48.1, 29.7, 25.1. ESI-MS m/z 487.0 [M + H]+. HRMS
calcd for C23H20N2O6SCl [M + H]+ m/z 487.0736, found 487.0731.
(S)-5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-
tetrahydrothieno[3,2-c]pyridin-2-yl 4-Methoxybenzoate (9h).
Following a procedure similar to that described for the preparation of
9d except that an equivalent amount of 4-methoxybenzoyl chloride
was used in place of pivaloyl chloride, the title compound was obtained
as a yellow oil in 30.0% yield, 96.9% ee (Chiral HPLC analytical
conditions: same as those for 9g). [α]D20 +26.00° (c 0.50, MeOH). 1H
NMR (300 MHz, CDCl3): δ 8.09−8.06 (m, 2 H), 7.68−7.26 (m, 4
H), 6.95−6.92 (m, 2 H), 6.38 (s, 1 H), 4.92 (s, 1 H), 3.85 (s, 5 H),
3.71−3.59 (m, 5 H), 2.90−2.79 (m, 4 H). 13C NMR (75 MHz,
CDCl3): δ 171.2, 164.5, 163.1, 149.9, 134.6, 133.7, 132.7, 132.3, 129.9,
129.7, 129.4, 127.1, 125.7, 121.2, 120.6, 114.1, 113.6, 111.8, 67.8, 55.4,
52.0, 50.3, 48.1, 25.0. ESI-MS m/z 472.2 [M + H]+, 494.2 [M + Na]+.
HRMS calcd for C24H23NO5SCl [M + H]+ m/z 472.0993, found
472.0985.
(S)-5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-
tetrahydrothieno[3,2-c]pyridin-2-yl Pivalate (9d). To a solution
of 4a (337.5 mg, 1.0 mmol, 97.5% ee) in THF (15 mL) was added
Et3N (836 μL, 5.9 mmol). After being stirred for 10 min, the mixture
was cooled to 0 °C and pivaloyl chloride (738 μL, 6.1 mmol) was
added. After being stirred at 25 °C for 4 h, the mixture was poured
into saturated sodium bicarbonate solution (60 mL) and extracted
with EtOAc (30 mL). The organic layer was washed with brine, dried
over anhydrous sodium sulfate, and concentrated under vacuum. The
residue was purified by column chromatography to give the title
compound in 85% yield, 99.1% ee (Chiral HPLC analytical conditions:
(S)-5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-
tetrahydrothieno[3,2-c]pyridin-2-yl 2-Acetoxybenzoate (9i).
Following a procedure similar to that described for the preparation
3348
dx.doi.org/10.1021/jm300038c | J. Med. Chem. 2012, 55, 3342−3352

Journal of Medicinal Chemistry
Article
of 9d except that an equivalent amount of 2-(chlorocarbonyl)phenyl
acetate was used in place of pivaloyl chloride, the title compound was
obtained as a pale yellow oil in 56.0% yield, 96.0% ee (Chiral HPLC
analytical conditions: Chiralpak IC, 4.6 mm × 250 mm, eluting with
85% n-hexane + 15% THF + 0.1% Et2NH, flow rate 0.5 mL/min, oven
temperature 25 °C, detection UV 254 nm). [α]D20 +14.00° (c 0.50,
CHCl3). 1H NMR (300 MHz, CDCl3): δ 8.15−7.39 (m, 4 H), 7.36−
7.14 (m, 4 H), 6.37 (s, 1 H), 4.93 (s, 1 H), 3.72 (s, 3 H), 3.66−3.54
(m, 2 H), 2.93−2.90 (m, 2 H), 2.81−2.79 (m, 2 H), 2.34 (s, 3 H). 13C
NMR (75 MHz, CDCl3): δ 171.17, 169.47, 161.32, 151.12, 149.32,
135.73, 134.71, 133.54, 132.12, 130.71, 129.92, 129.79, 129.45, 129.35,
127.12, 126.14, 124.09, 121.66, 118.97, 117.34, 112.61, 67.65, 52.12,
50.12, 48.04, 24.93, 20.93. ESI-MS m/z 500 [M + H]+, 522 [M +
Na]+. HRMS calcd for C25H23NO6SCl [M + H]+ m/z 500.0931, found
500.0935.
used in place of pivaloyl chloride, the title compound was obtained in
39.0% yield, 97.0% ee (Chiral HPLC analytical conditions: same as
those for 9b). [α]D20 +24.00° (c 0.50, CHCl3). H NMR (300 MHz,
1
CDCl3): δ 7.68−7.23 (m, 4 H), 6.29 (s, 1 H), 4.90 (s, 1 H), 3.90 (s, 3
H), 3.72 (s, 3 H), 3.58 (ABq, 2 H, J = 14.2 Hz), 2.90−2.83 (m, 2 H),
2.78−2.75 (m, 2 H). 13C NMR (75 MHz, CDCl3): δ 171.2, 153.3,
150.4, 134.7, 133.7, 129.9, 129.8, 129.6, 129.4, 127.1, 126.0, 112.6,
67.8, 55.8, 52.1, 50.3, 48.1, 25.1. ESI-MS m/z 396.1 [M + H]+. HRMS
calcd for C18H19NO5SCl [M + H]+ m/z 396.0672, found 396.0675.
(S)-Methyl 2-(2-Chlorophenyl)-2-(2-(ethoxycarbonyloxy)-
6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetate (9n). Follow-
ing a procedure similar to that described for the preparation of 9d
except that an equivalent amount of ethyl carbonochloridate was used
in place of pivaloyl chloride, the title compound was obtained as a
white solid in 74.7% yield, mp 42−44 °C, 97.3% ee (Chiral HPLC
analytical conditions: same as those for 9b). [α]D20 +40.00° (c 0.50,
MeOH). 1H NMR (300 MHz, CDCl3): δ 7.68−7.24 (m, 4 H), 6.30 (s,
1 H), 4.90 (s, 1 H), 4.32 (q, 2 H, J = 7.1, 14.3 Hz), 3.72 (s, 3 H), 3.58
(ABq, 2 H, J = 14.2 Hz), 2.90−2.86 (m, 2 H), 2.78−2.76 (m, 2 H),
1.37 (t, 3 H, J = 7.2 Hz). 13C NMR (75 MHz, CDCl3): δ 171.5, 157.3,
152.9, 135.0, 134.0, 130.2, 130.1, 129.8, 129.7, 127.4, 126.2, 112.7,
68.1, 65.7, 52.4, 50.5, 48.4, 25.4, 14.4. ESI-MS m/z 410.1 [M + H]+,
432.1 [M + Na]+. HRMS calcd for C19H21NO5SCl [M + H]+ m/z
410.0836, found 410.0829.
(S)-Methyl 2-(2-Chlorophenyl)-2-(2-(2-phenylacetoxy)-6,7-
dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetate (9j). Following a
procedure similar to that described for the preparation of 9d except
that an equivalent amount of 2-phenylacetyl chloride was used in place
of pivaloyl chloride, the title compound was obtained as a yellow oil in
46.0% yield, 93.5% ee (Chiral HPLC analytical conditions: same as
those for 9b). [α]D20 +14.00° (c 0.50, MeOH). H NMR (300 MHz,
1
CDCl3): δ 7.68−7.64 (m, 1 H), 7.41−7.23 (m, 8 H), 6.26 (s, 1 H),
4.89 (s, 1 H), 3.82 (s, 2 H), 3.71 (s, 3 H), 3.57 (ABq, 2 H, J = 14.3
Hz), 2.88−2.86 (m, 2 H), 2.77−2.75 (m, 2 H). 13C NMR (75 MHz,
CDCl3): δ 171.2, 168.3, 149.6, 134.7, 133.7, 132.7, 129.9, 129.8, 129.4,
129.2, 129.1, 128.7, 127.4, 127.1, 125.8, 111.9, 67.8, 52.1, 50.2, 48.1,
40.8, 29.6, 24.9. ESI-MS m/z 456.2 [M + H]+, 478.2 [M + Na]+.
HRMS calcd for C24H23NO4SCl [M + H]+ m/z 456.1041, found
456.1036.
(S)-Methyl 2-(2-Chlorophenyl)-2-(2-(isopropoxycarbony-
loxy)-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetate (9o).
Following a procedure similar to that described for the preparation
of 9d except that an equivalent amount of isopropyl carbonochloridate
was used in place of pivaloyl chloride, the title compound was obtained
in 94.3% yield, 97.5% ee (Chiral HPLC analytical conditions: same as
those for 9b). [α]D20 +34.00° (c 0.50, MeOH). H NMR (300 MHz,
1
(S)-5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-
tetrahydrothieno[3,2-c]pyridin-2-yl Cinnamate (9k). Following a
procedure similar to that described for the preparation of 9d except
that an equivalent amount of cinnamoyl chloride was used in place of
pivaloyl chloride, the title compound was obtained as a yellow solid in
34.6% yield, mp 122−124 °C, 98.7% ee (Chiral HPLC analytical
conditions: Chiralpak IC, 4.6 mm × 250 mm, eluting with 90% n-
hexane + 10% THF + 0.1% Et2NH, flow rate 0.5 mL/min, oven
temperature 25 °C, detection UV 254 nm). [α]D20 +14.00° (c 0.50,
CDCl3): δ 7.68−7.24 (m, 4 H), 6.30 (s, 1 H), 5.01−4.93 (m, 1 H),
4.90 (s, 1 H), 3.72 (s, 3 H), 3.57 (ABq, 2 H, J = 14.2 Hz), 2.89−2.86
(m, 2 H), 2.78−2.77 (m, 2 H), 1.36 (d, 6 H, J = 6.2 Hz). 13C NMR
(75 MHz, CDCl3): δ 171.2, 152.0, 150.5, 134.7, 133.7, 130.3, 129.9,
129.8, 129.4, 127.1, 125.7, 112.2, 73.9, 67.8, 52.1, 50.3, 48.1, 25.4, 21.6.
ESI-MS m/z 424.1 [M + H]+. HRMS calcd for C20H23NO5SCl [M +
H]+ m/z 424.0989, found 424.0985.
(S)-Methyl 2-(2-Chlorophenyl)-2-(2-(isobutoxycarbonyloxy)-
6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetate (9p). Follow-
ing a procedure similar to that described for the preparation of 9d
except that an equivalent amount of isobutyl carbonochloridate was
used in place of pivaloyl chloride, the title compound was obtained in
68.6% yield, 95.5% ee (Chiral HPLC analytical conditions: same as
1
CHCl3). H NMR (300 MHz, CDCl3): δ 7.85 (d, 1 H, J = 15.9 Hz),
7.71−7.69 (m, 1 H), 7.58−7.55 (m, 2 H), 7.42−7.39 (m, 4 H), 7.32−
7.24 (m, 2 H), 6.56 (d, 1 H, J = 15.9 Hz), 6.35 (s, 1 H), 4.92 (s, 1 H),
3.72 (s, 3 H), 3.66−3.48 (m, 2 H), 2.90−2.89 (m, 2 H), 2.81−2.79
(m, 2 H). 13C NMR (75 MHz, CDCl3): δ 171.2, 163.6, 147.3, 134.7,
133.9, 130.9, 129.8, 129.4, 128.9, 128.6, 128.3, 127.1, 125.8, 116.0,
111.7, 67.8, 52.1, 50.3, 48.1, 25.0. ESI-MS m/z 468.2 [M + H]+, 490.2
[M + Na]+. HRMS calcd for C25H23NO4SCl [M + H]+ m/z 468.1032,
found 468.1036.
those for 9b). [α]D20 +16.00° (c 0.50, MeOH). H NMR (300 MHz,
1
CDCl3): δ 7.42−7.26 (m, 4 H), 6.29 (s, 1 H), 4.90 (s, 1 H), 4.25 (d, 2
H, J = 6.6 Hz), 3.72 (s, 3H), 3.58 (ABq, 2 H, J = 14.2 Hz), 2.90−2.86
(m, 2 H), 2.78−2.76 (m, 2 H), 2.05−2.01 (m, 1 H), 0.98 (d, 6 H, J =
6.7 Hz). 13C NMR (75 MHz, CDCl3): δ 171.5, 153.0, 150.8, 135.0,
134.0, 130.2, 130.1, 129.8, 129.7, 127.4, 126.1, 112.6, 75.6, 68.1, 52.4,
50.5,, 48.4, 28.0, 25.4, 19.1. ESI-MS m/z 460.3 [M + Na]+. HRMS
calcd for C21H25NO5SCl [M + H]+ m/z 438.1150, found 438.1142.
(S)-Methyl 2-(2-(Benzyloxycarbonyloxy)-6,7-dihydrothieno-
[3,2-c]pyridin-5(4H)-yl)-2-(2-chlorophenyl)acetate (9q). Follow-
ing a procedure similar to that described for the preparation of 9d
except that an equivalent amount of benzyloxy carbonochloridate was
used in place of pivaloyl chloride, the title compound was obtained in
81.9% yield, 93.7% ee (Chiral HPLC analytical conditions: same as
(S)-5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-
tetrahydrothieno[3,2-c]pyridin-2-yl Nicotinate (9l). Following a
procedure similar to that described for the preparation of 9d except
that an equivalent amount of nicotinoyl chloride was used in place of
pivaloyl chloride, the title compound was obtained as a yellow solid in
38.8% yield, mp 92−94 °C, 97.7% ee (Chiral HPLC analytical
conditions: Chiralpak IC, 4.6 mm × 250 mm, eluting with 50% n-
hexane + 50% i-PrOH + 0.1% Et2NH, flow rate 0.5 mL/min, oven
temperature 25 °C, detection UV 254 nm). [α]D20 +34.00° (c 0.50,
1
MeOH). H NMR (500 MHz, CDCl3): δ 9.34 (m, 1 H), 8.84−8.83
those for 9b). [α]D20 +12.00° (c 0.50, CHCl3). H NMR (300 MHz,
1
(m, 1 H), 8.41−8.38 (m, 1 H), 7.69 (m, 1 H), 7.45−7.25 (m, 4 H),
6.45 (s, 1 H), 4.93 (s, 1 H), 3.71 (s, 3 H), 3.69−3.58 (m, 2 H), 2.93−
2.91 (m, 2 H), 2.82−2.80 (m, 2 H). 13C NMR (75 MHz, CDCl3): δ
171.16, 162.17, 154.07, 151.19, 149.18, 137.54, 134.68, 133.59, 129.89,
129.78, 129.43, 129.32, 127.11, 126.33, 124.66, 123.47, 112.43, 67.73,
52.11, 50.24, 48.05, 24.93. ESI-MS m/z 443.1 [M + H]+, 465.1 [M +
Na]+. HRMS calcd for C22H20N2O4SCl [M + H]+ m/z 443.0839,
found 443.0832.
CDCl3): δ 7.42−7.24 (m, 4 H), 6.30 (s, 1 H), 5.26 (s, 2 H), 4.90 (s, 1
H), 3.72 (s, 3 H), 3.58 (ABq, 2 H, J = 14.3 Hz), 2.90−2.86 (m, 2 H),
2.78−2.76 (m, 2 H). 13C NMR (75 MHz, CDCl3): δ 171.2, 152.6,
150.4, 134.7, 134.3, 133.7, 129.9, 129.8, 129.5, 129.4, 128.9, 128.7,
128.6, 127.1, 127.0, 126.0, 112.5, 70.9, 67.8, 52.1, 50.3, 48.1, 25.1. ESI-
MS m/z 472.1 [M + H]+, 494.1 [M + Na]+. HRMS calcd for
C24H23NO5SCl [M + H]+ m/z 472.0996, found 472.0985.
(S)-Methyl 2-(2-Chlorophenyl)-2-(2-(methoxycarbonyloxy)-
6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetate (9m). Follow-
ing a procedure similar to that described for the preparation of 9d
except that an equivalent amount of methyl carbonochloridate was
(S)-Methyl 2-(2-Chlorophenyl)-2-(2-(2-phenoxyacetoxy)-6,7-
dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetate (9r). Following a
procedure similar to that described for the preparation of 9d except
that an equivalent amount of 2-phenoxyacetyl chloride was used in
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place of pivaloyl chloride, the title compound was obtained as a yellow
solid in 36.0% yield, mp 104−106 °C, 89.0% ee (Chiral HPLC
analytical conditions: same as those for 9b), [α]D20 +32.00° (c 0.50,
115.8, 115.5, 111.9, 64.3, 52.2, 50.0, 48.0, 24.9, 20.7. ESI-MS m/z
364.1 [M + H]+. HRMS calcd for C18H19NO4SF [M + H]+ m/z
364.1019, found 364.1029.
1
MeOH). H NMR (300 MHz,CDCl3): δ 7.68−7.65 (m, 1 H), 7.42−
(R)-Methyl 2-(2-Acetoxy-6,7-dihydrothieno[3,2-c]pyridin-
5(4H)-yl)-2-(2-chlorophenyl)acetate ((R)-9a). Following a proce-
dure similar to that described for the preparation of 9a except that an
equivalent amount of (R)-methyl-2-(2-chlorophenyl)-2-(2-oxo-7,7a-
dihydrothieno[3,2-c]pyridin-5(2H,4H,6H)-yl)acetate was used in
place of 4a, the title compound was obtained in 93.1% yield, 98.7%
ee (Chiral HPLC analytical conditions: same as those for 9a). [α]D23
7.24 (m, 5 H), 7.04−6.99 (m, 1 H), 6.95−6.92 (m, 2 H), 6.32 (s, 1 H),
4.90 (s, 1 H), 4.82 (s, 2 H), 3.71 (s, 3 H), 3.59 (ABq, 2 H, J = 14.3
Hz), 2.90−2.86 (m, 2 H), 2.78−2.77 (m, 2 H). 13C NMR (75 MHz,
CDCl3): δ 171.1, 165.8, 157.5, 148.7, 134.7, 133.5, 129.9, 129.8, 129.6,
129.4, 129.2, 127.1, 126.1, 122.1, 114.8, 112.4, 67.7, 65.1, 52.1, 50.2,
48.0, 24.9. ESI-MS m/z 472.2 [M + H]+, 494.2 [M + Na]+. HRMS
calcd for C24H23NO5SCl [M + H]+ m/z 472.0993, found 472.0985.
(S)-Methyl 2-(2-Chlorophenyl)-2-(2-(dimethylcarbamoy-
loxy)-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetate (9s). To
a solution of 4a (337.5 mg, 1.0 mmol, 97.5% ee) in DMF (15 mL) was
added NaH (44 mg, 1.1 mmol, 60% dispersion in mineral oil) at 0 °C.
After the mixture was stirred at the same temperature for 0.5 h,
dimethylcarbamic chloride (386.5 μL, 4.2 mmol) was added to the
mixture and stirred at 25 °C for 2 h. Then the mixture was poured into
60 mL of 10% KH2PO4 aqueous solution and extracted with EtOAc.
The organic layer was washed with brine, dried over anhydrous
sodium sulfate, and concentrated under vacuum. The residue was
purified by column chromatography to afford a yellow oil, which was
recrystallized in EtOH to give the title compound as a white solid
(183.6 mg, 45% yield), mp 96−98 °C, 95.5% ee (Chiral HPLC
analytical conditions: same as those for 9l). [α]D20 +34.00° (c 0.50,
MeOH). 1H NMR (500 MHz, CDCl3): δ 7.69−7.26 (m, 4 H), 6.19 (s,
1 H), 4.91 (s, 1 H), 3.72 (s, 3 H), 3.68−3.56 (m, 2 H), 3.03 (s, 3 H),
2.99 (s, 3 H), 2.89−2.85 (m, 2 H), 2.76−2.73 (m, 2 H). 13C NMR (75
MHz, CDCl3): δ 171.2, 151.0, 134.6, 134.3, 133.8, 130.5, 129.9, 129.7,
129.3, 129.0, 127.1, 125.3, 111.4, 67.8, 52.1, 50.4, 48.1, 36.8, 36.3, 25.0.
ESI-MS m/z 409.2 [M + H]+. HRMS calcd for C19H22N2O4SCl [M +
H]+ m/z 409.0992, found 409.0989.
1
−44.00° (c 1.0, MeOH). H NMR (300 MHz, CDCl3): δ 7.69−7.23
(m, 4 H), 6.25 (s, 1 H), 4.90 (s, 1 H), 3.72 (s, 3 H), 3.58 (ABq, 2 H, J
= 14.2 Hz), 2.89−2.86 (m, 2 H), 2.78−2.76 (m, 2 H), 2.26 (s, 3 H).
ESI-MS m/z 380.0 [M + H]+.
Methyl 2-(2-Acetoxy-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-
yl)-2-(2-chlorophenyl)acetate ((R,S)-9a). Following a procedure
similar to that described for the preparation of 9a except that an
equivalent amount of (R,S)-methyl-2-(2-chlorophenyl)-2-(2-oxo-7,7a-
dihydrothieno[3,2-c]pyridin-5(2H,4H,6H)-yl)acetate was used in
place of 4a, the title compound was obtained in 92.1% yield. 1H
NMR (300 MHz, CDCl3): δ 7.25−7.68 (m, 4 H), 6.26 (s, 1 H), 4.90
(s, 1 H), 3.72 (s, 3 H), 3.58 (ABq, 2 H, J = 14.2 Hz), 2.89−2.78 (m, 2
H), 2.78−2.76 (m, 2 H), 2.26 (s, 3 H). ESI-MS m/z 380.0 [M + H]+.
Inhibition of ADP-Induced Platelet Aggregation in Rats.
Male Wistar rats weighing 200−250 g were used (purchased from the
Animal Experimental Center, Shandong University, Shandong, China)
and subject to a 12 h/12 h light/dark cycle in an acclimatized room,
receiving water and regular food for rodents ad libitum. The animals
were divided into 28 groups of 10 animals each. Compounds were
orally administered at a dose of 3 mg/kg or CMC as a vehicle. Two
hours after administration, the animals were anesthetized with
pentobarbital sodium, a surgical incision through the abdominal wall
was made, and blood samples were collected from the abdominal aorta
with Plus blood collection tubes (Becton, Dickinson and Company,
U.K.) for platelet aggregation tests according to Born’s method.8 ADP
(1.2 μM, Sigma Chemical Co.) was used as agonists for platelet
aggregation. Platelet aggregation was measured using an aggregometer
(560 Ca, Chrono-Log Co., U.S.).
(S)-5-(1-(2-Chlorophenyl)-2-methoxy-2-oxoethyl)-4,5,6,7-
tetrahydrothieno[3,2-c]pyridin-2-yl Pyrrolidine-1-carboxylate
(9t). Following a procedure similar to that described for the
preparation of 9s except that an equivalent amount of pyrrolidine-1-
carbonyl chloride was used in place of dimethylcarbamic chloride, the
title compound was obtained as a white oil in 46.0% yield, 95.7% ee
(Chiral HPLC analytical conditions: same as those for 9g). [α]D22
Production of the Active Metabolite (5) from in Vitro
Metabolic Activation of 9a or Clopidogrel in Rat Liver
Microsomal Incubation. Materials. Molecular-biology-grade
potassium phosphate monobasic, potassium hydroxide, tri-
1
+19.00° (c 1.0, MeOH). H NMR (300 MHz, CDCl3): δ 7.40−7.22
(m, 4 H), 6.20 (s, 1 H), 4.89 (s, 1 H), 3.76−3.61 (m, 4 H), 3.54−3.44
(m, 5 H), 2.89−2.77 (m, 2 H), 2.75−2.62 (m, 2 H), 2.04−1.87 (m, 4
H). 13C NMR (75 MHz, CDCl3): δ 171.3, 151.8, 151.0, 134.7, 133.9,
130.0, 129.7, 129.3, 129.1, 128.6, 127.1, 125.2, 111.5, 67.9, 52.1, 50.4,
48.2, 46.6, 46.3, 29.7, 25.7, 25.1, 24.9. ESI-MS m/z 435.1 [M + H]+.
HRMS calcd for C21H24N2O4SCl [M + H]+ m/z 435.1145, found
435.1148.
chloroacetic acid, L-glutathione, and β-nicotinamide adenine
dinucleotide 2′-phosphate (reduced, NADPH) were purchased
from Sigma-Aldrich (St. Louis, MO). Pooled Sprague−Dawley
rat liver microsomes (RLM, protein concentration, 20 mg/mL)
were purchased from Research Institute for Liver Diseases
(RILD, Shanghai, China).
(S)-Methyl 2-(2-Acetoxy-6,7-dihydrothieno[3,2-c]pyridin-
5(4H)-yl)-2-phenylacetate (9u). Following a procedure similar to
that described for the preparation of 9a except that an equivalent
amount of (S)-methyl 2-(2-oxo-7,7a-dihydrothieno[3,2-c]pyridin-5-
(2H,4H,6H)-yl)-2-phenylacetate (4b) was used in place of 4a, the title
compound was obtained as a colorless oil in 93.0% yield, 72.0% ee
Protocol. Test compound 9a and clopidogrel were prepared as 10
mM DMSO stock solutions. Each in vitro incubation mixture contains
1.0 mg/mL rat liver microsomes, 1.0 mM NADPH, 20 μM 9a or
clopidogrel, and 10 mM glutathione in a final volume of 200 μL of
potassium phosphate (100 mM) buffer (pH 7.4). Control samples
were made by replacing NADPH with potassium phosphate buffer. All
the samples were incubated in a 37 °C shaking water bath for 30 min.
Each incubation mixture was quenched with 30 μL of ice-cold
trichloroacetic acid solution (10%, w/v) and kept on ice for 5 min
before being centrifuged at 12 000 rpm for 10 min on a 5810R
centrifuge (Eppendorf AG, Hamburg, Germany) to fully pelletize the
precipitated proteins. The supernatant was injected into the LC−MS/
MS instrument to determine the formation of active metabolite (5)
production in the form of glutathione adducts.
LC−MS/MS Analysis of Active Metabolite (5) Formation.
Materials. Chromatography-grade H2O, MeOH, and formic acid
were all purchased from Sigma-Aldrich (St. Louis, MO).
Instrumentation and Conditions. Chromatographic analysis of the
supernatant resulting from the above incubation was conducted on a
Surveyor HPLC system consisting of an autosampler, a MS pump, and
a photodiode array detector (Thermo Fisher Scientific, Waltham, MA)
using a Zorbax SB-phenyl column (4.6 mm × 75 mm, 3.5 μm, Agilent
1
(Chiral HPLC analytical conditions: same as those for 9a). H NMR
(300 MHz, CDCl3): δ 7.69−7.25 (m, 5 H), 6.25 (s, 1 H), 4.32 (s, 1
H), 3.72 (s, 3 H), 3.54 (s, 2 H), 2.91−2.47 (m, 4 H), 2.26 (s, 3 H).
13C NMR (75 MHz, CDCl3): δ 167.7, 128.9, 128.7, 111.9, 72.5, 52.2,
50.3, 48.1, 24.4, 20.7. ESI-MS m/z 346.1 [M + H]+. HRMS calcd for
C18H20NO4S [M + H]+ m/z 346.1113, found 346.1125.
(S)-Methyl 2-(2-Acetoxy-6,7-dihydrothieno[3,2-c]pyridin-
5(4H)-yl)-2-(2-fluorophenyl)acetate (9v). Following a procedure
similar to that described for the preparation of 9a except that an
equivalent amount of (S)-methyl 2-(2-fluorophenyl)-2-(2-oxo-7,7a-
dihydrothieno[3,2-c]pyridin-5(2H,4H,6H)-yl)acetate (4c) was used in
place of 4a, the title compound was obtained as a colorless oil in 86.0%
yield, 86.9% ee (Chiral HPLC analytical conditions: same as those for
1
9a). H NMR (300 MHz, CDCl3): δ 7.59−7.07 (m, 4 H), 6.27 (s, 1
H), 4.80 (s, 1 H), 3.74 (s, 3 H), 3.64 (s, 2 H), 2.97−2.47 (m, 4 H),
2.26 (s, 3 H). 13C NMR (75 MHz, CDCl3): δ 171.0, 167.7, 162.6,
159.3, 149.6, 130.3, 130.2, 130.1, 130.0, 129.0, 125.6, 124.5, 124.4,
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Technologies, Santa Clara, CA) at room temperature. The volume of
each injection was 20 μL. The mobile phase consisted of H2O (solvent
A, containing 0.1% formic acid) and MeOH (solvent B, containing
0.1% formic acid) and was delivered at 750 μL/min. The initial
composition of solvent B was maintained at 10% for 2 min and then
increased in a linear manner to 50% at 7 min and 90% at 17 min. It
was then maintained at 90% solvent B for 1 min and finally decreased
to 10% at 19 min. The column was allowed to equilibrate at 10%
solvent B for 1 min before the ending of the 20 min gradient elution
program for next injection. The scan range of the photodiode array
detector was set to 220−400 nm. Mass spectrometry (MS) analysis
was performed on a Thermo LCQ ion trap mass spectrometer
(Thermo Fisher Scientific, Waltham, MA), which was interfaced to the
above HPLC system. The HPLC eluate was split after the photodiode
array detector, and 10% (75 μL/min) eluate was injected onto the
mass spectrometer. MS analysis was conducted using a standard
electrospray ionization (ESI) source operating in positive ionization
mode. Source operating conditions were 4.5 kV spray voltage, 225 °C
heated capillary temperature, 20 V capillary voltage, and sheath gas
flow at 40 (arbitrary unit), respectively. The MS experiment
parameters including the nitrogen gas flow rate, capillary voltage,
and the tube lens voltages were all tuned and optimized to give
maximum detection sensitivities using a clopidogrel standard solution
(10 μM in MeOH/H2O, 1/1, v/v). The MS full scans were monitored
over a mass range of m/z 300−700. Product ion (MS2) scans were
generated via collision-induced dissociation (CID) with helium using
normalized collision energy of 60% and a precursor ion isolation width
of m/z 2.0. Data were centroid and processed in Qual Browser
(Thermo Fisher Scientific). The active metabolite (5) formed via
metabolic activation of 9a or clopidogrel was detected and analyzed in
its more stable glutathione-derivatized form of active metabolite−
glutathione adducts (AMGS−1/2/3/4). Fragmentations were pro-
posed based on plausible protonation sites, subsequent isomerization,
and even electron species, as well as bond saturation. Product ion
spectra comparison between the parent and AMGS adducts further
aided in the confirmation of active metabolite structure.
Pharmacokinetic Studies. Sprague−Dawley male rats (SPF
grade) were used to determine oral bioavailability and PK parameters
of the clopidogrel and 9a following oral and intravenous
administration. In this study, clopidogrel thiolactone (4a) was
measured by LC/MS/MS analysis to determine the PK parameters
for both clopidogrel and 9a. PK parameters were calculated using a
noncompartmental model. Solutions of clopidogrel and 9a in N,N-
dimethylacetamide (DMA)/polyethylene glycol 400 (PEG 400) (v/v
5:95) at 1.26 and 1.14 mg/mL were prepared for oral dosing. A
solution of clopidogrel thiolactone in DMA/PEG 400/saline (v:v:v
5:15:80) at 0.54 mg/mL was prepared for intravenous administration
to determine the oral bioavailability of both clopidogrel and 9a to be
converted to clopidogrel thiolactone. The solution formulations of
clopidogrel thiolactone along with clopidogrel and 9a were
administered separately by bolus injection into a tail vein and by
oral gavage. Blood samples (about 0.20 mL) were collected from the
retro-orbital plexus from three animals in each treatment group at 0
(predose), 0.083, 0.167. 0.5, 1, 2, 4, 6, 8, and 24 h for iv and 0
(predose), 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h for po. Calibration standards
with clopidogrel thiolactone concentrations from 0.5 to 1000 ng/mL
were prepared by serial dilution in pretreated naive rat plasma. A
portion (25 μL) of each calibration standard and unknown study
sample was mixed with 25 μL of acetonitrile containing the internal
standard followed by addition of 200 μL of MTBE into each sample.
All samples were vortex-mixed for 3 min. The mixture was then
centrifuged at 15700g at 4 °C for 3 min. The supernatants containing
the organic component for each sample were used for analysis. The
lower limit of quantitation was 0.5 ng/mL.
v; chiral HPLC chromatograms for 9a, (R)-9a, and (R,S)-9a.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +86-25-83271198. Fax: +86-25-83271198. E-mail:
hbsun2000@yahoo.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by the “111 Project” from the
Ministry of Education of China and the State Administration of
Foreign Expert Affairs of China (Grant No. 111-2-07).
ABBREVIATIONS USED
■
ACS, acute coronary syndrome; PCI, percutaneous coronary
intervention; ADP, adenosine diphosphate; CYP, cytochrome
P450; AM, active metabolite; PM, poor metabolizer; IM,
intermediate metabolizer; CR, clopidogrel resistance; RLM, rat
liver microsome; GSH, glutathione; AMGS, active metabolite−
glutathione disulfide
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ASSOCIATED CONTENT
■
S
* Supporting Information
LC−MS/MS study report on in vitro rat liver microsomal
incubation of 9a; 1H and 13C NMR spectra for compounds 9a−
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