Deuterated clopidogrel analogues as a new generation of antiplatelet agents

Article abstract of DOI:10.1021/ml300460t

Clopidogrel (CPG) is an antithrombotic prodrug that needs hepatic cytochrome P450 (CYP) enzymes for its bioactivation. The clinical effects of CPG have been associated with high intersubject variability and a certain level of resistance. Recently, comprehensive biotransformation studies of CPG support that the observed clinical uncertainty stems from the low bioactivation efficiency, which is attributed to extensive attritional metabolism (e.g., hydrolysis of the methyl ester functionality and oxidation of the piperidine moiety). With the goal of potentiating the desired thiophene 2-oxidation through minimal structural modification, we have adopted the strategy of targeted metabolism shift and have designed and synthesized deuterated piperidine analogues of CPG. In vitro studies showed that the prodrug activation percentages have been significantly increased for the deuterated analogues as a result of stability enhancement of the piperidine moiety. In a pharmacological study with a rat model, oral administration of the deuterated analogues also demonstrated higher inhibitory activity than that of CPG against adenosine diphosphate (ADP) induced platelet aggregation. These deuterated analogues represent a new generation of antiplatelet agents with the potential to overcome the major clinical drawbacks of CPG.

Full text of DOI:10.1021/ml300460t

Letter
pubs.acs.org/acsmedchemlett
Deuterated Clopidogrel Analogues as a New Generation of
Antiplatelet Agents
Yaoqiu Zhu,*,† Jiang Zhou,‡ and Bo Jiao§
†MetabQuest Research Laboratory, 202 Chengfu Road, Beijing 100871, People’s Republic of China
‡Analytical Instrumentation Center, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, People’s
Republic of China
§Department of Pharmacology, School of Pharmacy, Shandong University, Jinan, Shandong 250012, People’s Republic of China
S
* Supporting Information
ABSTRACT: Clopidogrel (CPG) is an antithrombotic
prodrug that needs hepatic cytochrome P450 (CYP) enzymes
for its bioactivation. The clinical effects of CPG have been
associated with high intersubject variability and a certain level
of resistance. Recently, comprehensive biotransformation
studies of CPG support that the observed clinical uncertainty
stems from the low bioactivation efficiency, which is attributed
to extensive attritional metabolism (e.g., hydrolysis of the
methyl ester functionality and oxidation of the piperidine
moiety). With the goal of potentiating the desired thiophene
2-oxidation through minimal structural modification, we have
adopted the strategy of targeted metabolism shift and have designed and synthesized deuterated piperidine analogues of CPG. In
vitro studies showed that the prodrug activation percentages have been significantly increased for the deuterated analogues as a
result of stability enhancement of the piperidine moiety. In a pharmacological study with a rat model, oral administration of the
deuterated analogues also demonstrated higher inhibitory activity than that of CPG against adenosine diphosphate (ADP)
induced platelet aggregation. These deuterated analogues represent a new generation of antiplatelet agents with the potential to
overcome the major clinical drawbacks of CPG.
KEYWORDS: Clopidogrel resistance, clinical variability, prodrug attrition, piperidine deuteration, targeted metabolic shift,
bioactivation potentiation
reported that 20−40% of patients that receive the drug showed
poor or no response to it.4,5
lopidogrel (CPG) is a thienopyridine antiplatelet prodrug
Cthat has been widely used in the treatment of
cardiovascular diseases, including atherothrombosis, unstable
angina, and myocardial infarction.1 As shown in Scheme 1,
CPG (M0) is activated through a two-step cytochrome P450
(CYP)-catalyzed process to form its active metabolite (M13).2,3
Despite being one of the most prescribed cardiovascular
medications of the past decade, CPG is associated with a high
clinical uncertainty for its antithrombotic therapy. It has been
To address the observed clinical variability and resistance, the
chemical mechanism of CPG bioactivation has been under
extensive investigation. Genetic factors that can impact the
therapeutic outcomes are being sought for the design of
personalized prodrug treatments. It has been hypothesized that
the active metabolite formation from CPG is dependent on
genetic polymorphic enzymes, and this leads to the observed
intersubject variability. However, research results have shown
that both steps of the prodrug activation are catalyzed by
various CYPs, and genetic polymorphic enzymes such as
paraoxonase-1 (PON-1) or CYP2C19 do not play deciding
roles in catalyzing the active metabolite formation.6−9
Scheme 1. Metabolic Activation and Major Attrition
Pathways of CPG
Our recent research has revealed that the first step of CPG
bioactivation, 2-oxidation of the thiophene motif leading to M2
formation, is significantly attenuated by CYP3A4/5-catalyzed
oxidation of the nonactivating piperidine motif and the
Received: December 18, 2012
Accepted: February 5, 2013
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
thiophene 3-position,8 in addition to the previously reported
methyl ester hydrolysis.10,11 Subsequently, we studied the
second step and found that the formation of M13 from M2 is
accompanied by multiple attritional pathways as well, which
further attenuate the active metabolite formation.12 On the
basis of these discoveries, the overall percentage of
bioactivation, conversion from M0 to M13, is expected to be
much lower than previously presumed (i.e., 1% or lower).
Consequently, the vulnerably low plasma exposure of M13 is
likely to be significantly impacted by multicomponent factors
(e.g., levels of attenuating enzymes, individual oxidative stress,
off-target hepatic and plasma proteins), leading to the varying
levels of therapeutic response and elusive clinical correlations.8
This slim bioactivation, on the other hand, demonstrates the
high potency of the active metabolite against its antiplatelet
target P2Y12,13 as well as a high ligand efficiency of CPG (MW
= 321), both resulting from the receptor covalent modification.
This mode of pharmacological action also poses a barrier to
medicinal chemists in discovering better analogues, because the
low chemical flexibility of CPG, toward both the bioactivating
enzymes and its molecular target, is unlikely to accommodate
major structural changes. In addition, the high chemical
reactivities of the active metabolite (M13) and its thiolactone
S-oxide precursor (M11) confer CPG with a narrow
therapeutic window,14 as will overdosing not only aggravate
the adverse effects of hemorrhage but also lead to severe drug−
drug interactions.15−17 On the basis of these understandings,
our medicinal chemistry endeavor was set to achieve
bioactivation potentiation through minimal structural change,
with possibly taking advantage of the safety records that CPG
has established over its long clinical use. The strategy we
adopted is targeted metabolism shift based on selective
deuteration.
Figure 1. Structural modification strategy and the design of the
deuterated CPG analogues.
a
Scheme 2. Synthesis of CPG and Its Deuterated Analogues
It has been reported that the absorbed CPG of an oral dose
undergoes esterase catalyzed hydrolysis to form its inactive
carboxylic acid metabolite (M1), which accounts for about 85%
of prodrug loss.11 Our earlier studies have shown biotransfor-
mations of the remaining 15% partition between the piperidine
moiety and the thiophene moiety, as mediated by CYP3A4/5
and multiple CYPs, respectively; the bioactivating thiophene 2-
oxidation is attenuated by extensive piperidine metabolism.8
Our results have also confirmed that selective inhibition of
piperidine metabolism can significantly potentiate the desired
thiophene 2-activation. On the basis of these findings, together
with the structural analyses of the attritional metabolites, we
designed deuterated piperidine analogues of CPG (d0-CPG),
namely, d2-CPG, d4-CPG, and d6-CPG (Figure 1).
a
Reagents: (a) NaOMe, MeOD, 91%; (b) NaBD4, MeOD, THF, 70
°C, 95%; (c) p-TsCl, Et3N, CH2Cl2, 97%; (d) KHCO3, CH3CN, 80
°C; (e) HCl, Et2O, 76% (two steps).; (f), HCl, H2O, 80 °C, 90%.
b
bCPG-II is the commercially available L-tartaric acid salt of 9. CPG-II
is the commercially available L-tartaric acid salt of 9.
parent compounds (ΔM0) and the amounts of the formed M2
from incubations of the four CPGs were quantified; the
bioactivation percentages were calculated as “M2/ΔM0”. As
shown in Figure 2, in HLM, the formations of M2 accounted
The chemical synthesis of the CPGs is anchored by a
Mannich condensation reaction.18 As shown in Scheme 2A, d0-
(1) and d2-CPG (2) were prepared from reactions of the
commercially available CPG-I intermediate (10) with paraf-
ormaldehyde and d2-paraformaldehyde, respectively. To obtain
d4- (3) and d6-CPG (4) (Scheme 2B), the deuterated
intermediate, d4-CPG-I (11), was first synthesized through a
coupling reaction between amine intermediate 10 and
deuterated tosylate intermediate 8. The latter was prepared in
three steps with full deuteration at its two methylene positions
(Scheme 2C). The final CPGs were converted to their
hydrochloride salts for in vitro and in vivo evaluations.
In vitro bioactivation studies of CPGs were conducted in
parallel in both human and rat liver microsomes (HLM and
RLM). Incubation conditions were adopted from the previously
reported M0-to-M2 formation for CPG.8 The turnovers of the
Figure 2. Bioactivation (M2 formation from M0) percentages (out
from the CYP catalyzed conversions) of the CPGs in human and rat
liver microsomes (HLM and RLM).
B
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ACS Medicinal Chemistry Letters
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for higher percentages of all the CYP catalyzed conversions of
M0 for d2-, d4-, and d6-CPG than for CPG (d0-CPG). In RLM,
the deuterated analogues also exhibited higher bioactivation
efficiencies with a pattern that is commensurate with the
metabolic profiles of CPG. These results show that, compared
to CPG, the deuterated analogues yield significantly more
pharmacologically desired metabolites and less attritional ones,
attributable to the reduced metabolism on the nonactivating
piperidine moiety. The overall conversion rates of the CPGs are
illustrated in Figure 3. In contrast to the significant boost of the
exploration of the in vitro−in vivo and animal−human
correlations. Currently, extensive preclinical testing, including
cross-species evaluations and animal-to-human extrapolations,
are underway for further development of these deuterated
analogues.
The in vitro and in vivo studies presented herein have
indicated that the deuterated analogues have the potential to
alleviate the major clinical drawbacks of CPG, possibly without
altering its long existing antithrombotic regimens, such as the
once-a-day low dose and the conjunction use with aspirin.
More importantly, the efficacy enhancement is unlikely to
jeopardize the clinical safety records of CPG. Currently, to
overcome the disadvantages of CPG, substituting therapies are
as follows: (1) dose increase of CPG (e.g., doubling), (2)
adjunctive use of cilostazol with CPG, and (3) use of other
antiplatelet agents, such as prasugrel or ticagrelor.21 Being
closely related to CPG, the improved deuterated analogues are
expected to stay in the narrow therapeutic window and be
potentially more beneficial than the above alternative treat-
ments. For example, compared to “CPG doubling”, the
deuterated analogues might yield similar effectiveness but
avoid some adverse complications of overdosing. This can
increase patient compliance and more safely and effectively
manage the cardiovascular risk due to low responsiveness.
These deuterated analogues also differ from vicagrel, another
CPG based preclinical antiplatelet agent, in their mechanistic
approach.19 The release of M2 from vicagrel is through facile
hydrolysis of a masked thiolactone promoiety, which could lead
to similar limitations as direct dosing of M2, including fatal
bleedings, drug−drug interactions, and other adverse effects.12
Taken together, the deuterated CPGs represent a new
generation of antiplatelet agents with the potential to address
unmet medical needs in treating cardiovascular complications,
especially the risk of recurrent ischemic events due to poor
responsiveness or nonresponsiveness to CPG.
A few general principles for medicinal chemistry have been
shaped along with these studies of CPG. First, the relationship
between the prodrug design and its treatment consistency is
dependent on the bioactivation percentage. Low active
metabolite conversion, especially with the involvement of
variable attenuating enzymes, is likely to foster clinical
uncertainties. Although considered feasible to leverage the
intersubject variability, the means of personalized prodrug
therapy can be complicated, and often misleading, because of
the tangled mechanistic roles of the genotypes. Second, as
demonstrated herein, the rational use of deuteration, a
seemingly simple medicinal chemistry approach, for drug
action enhancement, can be achieved with the understandings
of the metabolic fates and the therapeutic profiles of the drug.
Third, CPG, with its high ligand efficiency, can serve as a
prototype for a promising direction of drug discovery: covalent
receptor modification via targeted ligand bioactivation.22
Although some drugs with such a mode of action, such as
omeprazole and capecitabine, have been in clinical use, major
hurdles in this field are evident for its development. Systematic
methodologies are needed to address major challenges, such as
the following: (1) how to take the often pre-excluded
chemically reactive fragments for designing drug candidates
with such a mode of action; (2) how to explore and exploit the
biochemical features in the human body to channel the desired
bioactivation.
Figure 3. Metabolic stabilities of the CPGs in human and rat liver
microsomes (HLM and RLM).
M2 formation, the metabolic stabilities of the parent
compounds showed little changes in HLM and marginal
changes in RLM with the deuterated analogues. These results
confirm the successful utilization of the strategy of targeted
metabolism shift: selective deuteration enhances the stability of
the nonactivating moiety and shunts metabolism to the desired
fragment, leading to bioactivation potentiation (Figure 1).
The deuterated analogues were compared with CPG in vivo
for their activities against ADP-induced platelet aggregation. In
a rat model similar to the one that has been reported
previously,19 CPGs were orally administered (10 mg/kg), and
the ADP-induced platelet aggregation was determined by
Born’s method.20 From the obtained kinetic curves, the
maximal aggregation and the aggregation after 5 min of ADP
induction were obtained and are summarized in Table 1. Both
Table 1. Inhibitory Effects of the CPGs on the ADP-Induced
a
Platelet Aggregation in Rats at a Dose of 10 mg/kg
aggregation (%)
group
max.
5 min
vehicle
d0
76.0 7.8
69.8 10.9
29.4 18.8*
12.9 13.8*
60.4 9.0*
50.4 13.1*
49.1 11.9*,
50.6 13.3*
d2
#
##
d4
7.3 9.0*,
#
d6
7.0 12.8*,
a
*
Aggregation measurements 2 h after oral administration. P < 0.01 vs
#
##
vehicle group. P < 0.05. P < 0.01 vs d0 group. Data are the mean
SD, n = 8.
sets of results demonstrate that the deuterated analogues
exhibit higher antiplatelet activity than CPG. The goal of this
early stage in vivo study is to confirm that the bioactivation
potentiations observed in vitro translate to potency improve-
ments in an animal model. Due to the subtle nature of the
structural modification and the targeted efficacy enhancement,
clinical projections of the deuterated analogues require full
Recent investigations are beginning to unravel the long
overdue biochemical puzzle of CPG. On the basis of the
C
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(5) Kolandaivelu, K.; Bhatt, D. L. Overcoming resistance to
antiplatelet therapy: targeting the issue of nonadherence. Nat. Rev.
Cardiol. 2010, 7, 461−467.
previous biotransformation studies and the therapeutic analyses
of CPG, medicinal chemistry approaches were carefully
designed, and a series of deuterated analogues of CPG were
prepared. The in vitro and in vivo studies have confirmed that
the subtle structural modification of selective piperidine
deuteration yields the targeted metabolic shunt and leads to
the desired bioactivation potentiation. These deuterated
analogues hold a promise for overcoming the prominent
clinical drawbacks of CPG. The research presented here is part
of a continuous effort to use CPG as a tool compound to build
a methodology of “utilizing the human body to generate its
own medicine”. The discovery of deuterated CPGs might not
only lead to superior antithrombotic medications but, more
importantly, could also serve as an example for future chemical
therapeutics.
(6) Dansette, P. M.; Rosi, J.; Bertho, G.; Mansuy, D. Cytochromes
P450 catalyze both steps of the major pathway of clopidogrel
bioactivation, whereas paraoxonase catalyzes the formation of a minor
thiol metabolite isomer. Chem. Res. Toxicol. 2012, 25, 348−356.
(7) Gong, I. Y.; Crown, N.; Suen, C. M.; Schwarz, U. I.; Dresser, G.
K.; Knauer, M. J.; Sugiyama, D.; Degorter, M. K.; Woolsey, S.; Tirona,
R. G.; Kim, R. B. Clarifying the importance of CYP2C19 and PON1 in
the mechanism of clopidogrel bioactivation and in vivo antiplatelet
response. Eur. Heart. J. 2012, 33, 2856−2864.
(8) Zhu, Y.; Zhou, J. Identification of the significant involvement and
mechanistic role of CYP3A4/5 in clopidogrel bioactivation. ACS Med.
Chem. Lett. 2012, 3, 844−849.
(9) Kazui, M.; Nishiya, Y.; Ishizuka, T.; Hagihara, K.; Farid, N. A.;
Okazaki, O.; Ikeda, T.; Kurihara, A. Identification of the human
cytochrome P450 enzymes involved in the two oxidative steps in the
bioactivation of clopidogrel to its pharmacologically active metabolite.
Drug Metab. Dispos. 2010, 38, 92−99.
ASSOCIATED CONTENT
■
S
* Supporting Information
(10) Hagihara, K.; Kazui, M.; Kurihara, A.; Yoshiike, M.; Honda, K.;
Okazaki, O.; Farid, N. A.; Ikeda, T. A possible mechanism for the
differences in efficiency and variability of active metabolite formation
from thienopyridine antiplatelet gents, prasugrel and clopidogrel. Drug
Metab. Dispos. 2009, 37, 2145−2152.
Experimental procedures of CPG synthesis, in vitro bio-
activation and in vivo pharmacological studies, and spectral and
chromatographic data. This material is available free of charge
via the Internet at http://pubs.acs.org.
(11) Caplain, H.; Donat, F.; Gaud, C.; Necciari, J. Pharmacokinetics
of clopidogrel. Semin. Thromb. Hemostasis 1999, 25 (Suppl 2), 25−28.
(12) Zhu, Y.; Zhou, J. In Vitro Biotransformation Studies of 2-Oxo-
clopidogrel: Multiple Thiolactone Ring Opening Pathways Further
Attenuate Prodrug Activation. Chem. Res. Toxicol. 2013, 26, 179−190.
(13) Savi, P.; Pereillo, J. M.; Uzabiaga, M. F.; Combalbert, J.; Picard,
C.; Maffrand, J. P.; Pascal, M.; Herbert, J. M. Identification and
biological activity of the active metabolite of clopidogrel. Thromb.
Haemostasis 2000, 84, 891−896.
AUTHOR INFORMATION
■
Corresponding Author
*Phone:+86-10-6275-7536, 1-847-530-7238. Fax: +86-10-
6275-2632. E-mail: yaoqiu.zhu@metabquest.com.
Author Contributions
Y.Z. made the initial discovery, designed and synthesized the
deuterated CPGs, conducted the in vitro studies, analyzed the
results, and wrote the manuscript. J.Z. conducted part of the
compound characterization, including HRMS and enantiomeric
analyses. B.J. conducted in vivo studies of the CPGs.
(14) Sibbing, D.; Steinhubl, S. R.; Schulz, S.; Schomig, A.; Kastrati, A.
̈
Platelet aggregation and its association with stent thrombosis and
bleeding in clopidogrel-treated patients: initial evidence of a
therapeutic window. J. Am. Coll. Cardiol. 2010, 56, 317−318.
(15) Bhatt, D. L.; Fox, K. A. A.; Hacke, W.; Berger, P. B.; Black, H.
R.; Boden, W. E.; Cacoub, P.; Cohen, E. A.; Creager, M. A.; Easton, J.
D.; Flather, M. D.; Haffner, S. M.; Hamm, C. W.; Hankey, G. J.;
Johnston, S. C.; Mak, K.; Mas, J.; Montalescot, G.; Pearson, T. A.;
Steg, P. G.; Steinhubl, S. R.; Weber, M. A.; Brennan, D. M.; Fabry-
Ribaudo, L.; Booth, J.; Topol, E. J. Clopidogrel and aspirin versus
aspirin alone for the prevention of atherothrombotic events. N. Engl. J.
Med. 2006, 354, 1706−1717.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Richard B. Silverman (Northwestern University)
and Dr. Hongyu Zhao (Abbott Laboratories) for critical
reading and careful revision of the manuscript.
(16) Zhang, H.; Amunugama, H.; Ney, S.; Cooper, N.; Hollenberg, P.
F. Mechanism-based inactivation of human cytochrome P450 2B6 by
clopidogrel: involvement of both covalent modification of cysteinyl
residue 475 and loss of heme. Mol. Pharmacol. 2011, 80, 839−847.
(17) Chen, B. L.; Chen, Y.; Tu, J. H.; Li, Y. L.; Zhang, W.; Li, Q.;
Fan, L.; Tan, Z. R.; Hu, D. L.; Wang, D.; Wang, L. S.; Ouyang, D. S.;
Zhou, H. H. Clopidogrel inhibits CYP2C19-dependent hydroxylation
of omeprazole related to CYP2C19 genetic polymorphisms. J. Clin.
Pharmacol. 2009, 49, 574−581.
ABBREVIATIONS
■
CPG, clopidogrel; CYP, cytochrome P450; PON-1, para-
oxonase-1; HLM, human liver microsomes; RLM, rat liver
microsomes; ADP, adenosine diphosphate
REFERENCES
■
(1) Savi, P.; Herbert, J. M. Clopidogrel and ticlopidine: P2Y12
adenosine diphosphate receptor antagonists for the prevention of
atherothrombosis. Semin. Thromb. Hemostasis 2005, 31, 174−183.
(2) Pereillo, J. M.; Maftouh, M.; Andrieu, A.; Uzabiaga, M. F.; Fedeli,
O.; Savi, P.; Pascal, M.; Herbert, J. M.; Maffrand, J. P.; Picard, C.
Structure and stereochemistry of the active metabolite of clopidogrel.
Drug Metab. Dispos. 2002, 30, 1288−1295.
(3) Dansette, P. M.; Libraire, J.; Bertho, G.; Mansuy, D. Metabolic
oxidative cleavage of thioesters: evidence for the formation of sulfenic
acid intermediates in the bioactivation of the antithrombotic prodrugs
ticlopidine and clopidogrel. Chem. Res. Toxicol. 2009, 22, 369−373.
(4) Gurbel, P. A.; Bliden, K .P.; Hiatt, B. L.; O’Connor, C. M.
Clopidogrel for coronary stenting: response variability, drug resistance,
and the effect of pretreatment platelet reactivity. Circulation 2003, 107,
2908−2913.
(18) Alla, V. A.; Vyakaranam, K. A.; Sirigiri, A. K.; Bodapati, S. R.;
Billa, R. R.; Gudibandi, S. R.; Alla, R. Process for Preparation of
Clopidogrel Bisulphate Form-1. US 2007/0191609 A1, 2007.
(19) Shan, J.; Zhang, B.; Zhu, Y.; Jiao, B.; Zheng, W.; Qi, X.; Gong,
Y.; Yuan, Y.; Lv, F.; Sun, H. Overcoming clopidogrel resistance:
discovery of vicagrel as a highly potent and orally bioavailable
antiplatelet agent. J. Med. Chem. 2012, 55, 3342−3352.
(20) Born, G. V. Aggregation of blood platelets by adenosine
diphosphate and its reversal. Nature 1962, 194, 927−929.
(21) Kim, J. Y. Strategy for the treatment of clopidogrel low
responsiveness in diabetes mellitus and stent implantation. Korean
Circ. J. 2009, 39, 459−461.
(22) Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. The resurgence
of covalent drugs. Nat. Rev. Drug Discovery 2011, 10, 307−317.
D
dx.doi.org/10.1021/ml300460t | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX