Design, synthesis and antithrombotic evaluation of novel dabigatran prodrugs containing methyl ferulate

Article abstract of DOI:10.1016/j.bmcl.2013.01.126

A novel series of prodrugs containing dabigatran and methyl (E)-3-(4-hydroxy-2-methoxyphenyl)propenoate (methyl ferulate) were synthesized. All of them reveal the effect of thrombin-induced anti-platelet aggregation in vitro. In addition, in vivo experiment shows that one of the target compounds, X-2 (ED₅₀ = 3.7 ± 1.0 μmol/kg) possesses a more potent activity for inhibiting venous thrombosis than that of dabigatran etexilate (ED₅₀ = 7.8 ± 1.5 μmol/kg).

Full text of DOI:10.1016/j.bmcl.2013.01.126

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2089–2092
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and antithrombotic evaluation of novel dabigatran
prodrugs containing methyl ferulate
Xiao-Zhi Yang a, Xiao-Juan Diao a, Wen-Hui Yang b, Feng Li c, Guang-Wei He c, Guo-Qing Gong b,
Yun-Gen Xu a,
⇑
a Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China
b Department of Pharmacology, China Pharmaceutical University, Nanjing 210009, China
c Hefei Yi Gong Pharmaceutical Co., Ltd, Hefei 230088, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of prodrugs containing dabigatran and methyl (E)-3-(4-hydroxy-2-methoxyphenyl)pro-
penoate (methyl ferulate) were synthesized. All of them reveal the effect of thrombin-induced anti-
platelet aggregation in vitro. In addition, in vivo experiment shows that one of the target compounds,
Received 29 October 2012
Revised 15 January 2013
Accepted 30 January 2013
Available online 8 February 2013
X-2 (ED50 = 3.7 1.0
l
mol/kg) possesses a more potent activity for inhibiting venous thrombosis than
mol/kg).
that of dabigatran etexilate (ED50 = 7.8 1.5
l
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Dabigatran
Ferulic acid
Methyl ferulate
Thrombin inhibitors
Anti-platelet aggregation
Prevention and treatment of thrombosis
Thrombosis, which could occur in the arterial or the venous cir-
culation, is one of the medical spotlights nowadays. Because of the
difference of pathophysiology between arterial thrombosis and ve-
nous thrombosis, the drugs used for treating those two symptoms
are different. Generally, arterial thrombosis is treated with anti-
platelet drugs, while venous thrombosis is treated with drugs
which target factors of the coagulation cascade, including anti-
thrombin drugs.
Platelets have a variety of cell-surface receptors which partici-
pate in mediating the activation of them, including thromboxane
A2 receptor (TXA2R), protease-activated receptor 1 (PAR1), and
ADP receptor P2Y12.1 As the key factor of arterial thrombosis, plate-
let is closely correlated with the thrombin, which is an important
factor of the coagulation cascade. This relation mainly comes from
two parts: firstly, PAR-1, as a new target on platelet, needs throm-
bin to activate it;2 secondly, the adhesion and aggregation of plate-
lets could activate the coagulation cascade to increase the amount
of thrombin. In the meanwhile, from the perspective of clinical
cases, there are also several evidences for this relation: although
warfarin, dabigatran etexilate and rivaroxaban are typical
anticoagulant drugs, they have also been used for the prevention
of arterial thromboembolic events;3 similarly, the anti-platelet
drug aspirin can also be used for the venous thromboembolic
events.4
Dabigatran etexilate (Fig. 1) is an oral prodrug of dabigatran.
After oral administration, it could be hydrolysed to generate dabig-
atran. In vivo, dabigatran (Ki = 4.5 nM) could bind to human throm-
bin selectively and reversibly to realize a strong and long-lasting
anticoagulant effect.5 Dabigatran etexilate, firstly approved by
European Medicines Agency on March 27, 2008, was used for the
prevention of venous thromboembolic events in patients who
had undergone total hip- or knee-replacement surgery.6 Recently,
its another indication for the prevention of stroke and systemic
embolism in patients with nonvalvular atrial fibrillation had been
approved by FDA.
Ferulic acid, (E)-3-(4-hydroxy-2-methoxyphenyl)propenoate as
shown in Figure 1, is one of the components of asafoetida, the dried
latex from the giant fennel (Ferula communis). It has long been re-
ported to have the inhibitory effect on platelet aggregation.7 Its
pharmacological activity is mainly due to the following reasons.
Firstly, it could inhibit [Ca2+] channel on the surface of cell mem-
brane, and impede phospholipase C on releasing the amount of
[Ca2+],8 so as to lessen the combination of ADP with P2Y12
.
Secondly, it could also inhibit the synthesis of TXA2,9 which could
bind with TXA2R to activate the platelets.
In order to combine both the therapeutic benefits of dabigatran
and ferulic acid, we coupled dabigatran with methyl ferulate to
⇑
Corresponding author. Tel./fax: +86 25 83271244.
E-mail addresses: xu_yungen@hotmail.com, xyg@cpu.edu.cn (Y.-G. Xu).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.126

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X.-Z. Yang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2089–2092
O
O
CH3
CH3
N
N
N
NH
NH2
N
CH3
O
N
Dabigatran etexilate
O
O
OH
H3CO
OH
H3CO
H3COOC
HOOC
Methyl ferulate
Ferulic acid
O
CH3
O
CH3
N
N
NH
N
NH2
N
O
H3CO
H3COOC
N
O
O
X-2
Figure 1. Structures of dabigatran etexilate, ferulic acid, methyl ferulate and X-2.
CH3
N
CH3
N
O
O
N
NH2
NH
O
N
O
N
N
HN
R1~7
a
N
HN
NH2
. 2HCl
N
N
O
CH3
O
CH3
O
O
a1~a7
1
b
O
CH3
N
CH3
N
N
HN
O
R1~7
O
O
NH2
N
c
O
O
HN
R1~7
N
N
NH2
N
N
N
O
OCH3
COOCH3
N
OH
O
O
b1~b7
X-1~X-7
R1: tert-butyl, R2: n-hexyl, R3: ethyl, R4: benzyl,
R5: n-pentyl, R6: isopropyl, R7:methyl
Scheme 1. Reagents, conditions and range of yields: (a) R1–R7 chloroformate, K2CO3, THF/H2O, yields: 57–74%; (b) NaOH, EtOH/H2O, yields: 70–92%; (c) methyl ferulate,
EDCI, DMAP, DMF, yields: 54–69%.
afford a series of mutual prodrugs. We expected that these ester
bonds of dabigatran—methyl ferulate could be hydrolyzed in vivo
by esterase to release dabigatran and ferulic acid, which exert a
synergistical inhibitory effect on venous or arterial thrombosis
via two routes–antiplatelet and antithrombin. The inhibitory effect
of X-2 for venous thrombosis will be discussed in this paper and its
inhibitory effect for arterial thrombosis is still under study.
The synthetic route for target compounds is depicted in
Scheme 1. 3-({2-[(4-Carbamimidoyl-phenylamino)methyl]-1-
methyl-1H-benzoimidazole-5-carbonyl}pyridin-2-yl-amino)-pro-
pionic acid ethyl ester hydrochloride (1)5 reacted, respectively
with seven different chloroformates in the presence of potassium
carbonate to afford carbonate derivatives a1–a7.10 Then these
intermediates were hydrolyzed into carboxylic acid derivatives
b1–b7 by sodium hydroxide.10 Reactions of b1–b7 with methyl
ferulate in the presence of 1-ethyl-3-(3-dimethylaminopropyl)car-
bodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine
(DMAP) yielded the target compounds: X-1–X-7.

X.-Z. Yang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2089–2092
2091
Table 1
Structures of target compounds X-1–X-7 and their activities for inhibiting thrombin-induced rabbit platelet aggregation after the
incubation with rabbit liver microsomes
a
Compd
R
IC50
(lM)
Dabigatran etexilate
/
0.337 0.021
0.607 0.039
0.290 0.060
0.453 0.014
0.687 0.045
0.206 0.039
0.784 0.012
0.841 0.024
X-1
X-2
X-3
X-4
X-5
X-6
X-7
tert-Butyl
n-Hexyl
Ethyl
Benzyl
n-Pentyl
Isopropyl
Methyl
a
Drug concentration to achieve half-maximal inhibition of rabbit platelet aggregation induced by thrombin. Data were
expressed as mean SD from six independent experiments.
Table 2
8
6
4
2
0
X-2
The inhibitory effects on ADP-induced rabbit platelet aggregation of X-2, ferulic acid
and dabigatran etexilate
Dabigatran etexilate
Compd
X-2
Ferulic acid
2.7 0.6
Dabigatran etexilate
/
a
IC50 (mM)
5.8 1.0
a
Drug concentration to achieve half-maximal inhibition of rabbit platelet
ED50=7.8 1.5 mol/kg
aggregation induced by ADP. Data were expressed as mean SD from six inde-
pendent experiments.
In addition, we analyzed the inhibitory effect of compound X-2
along with two reference compounds (dabigatran etexilate and
ferulic acid) on ADP-induced platelet aggregation.14 In this experi-
ment, compound X-2 and dabigatran etexilate were incubated in
rabbit liver microsomal suspensions before the activity assay.12
The results show that compound X-2 (IC50 = 5.8 1.0 mM) and
ferulic acid (IC50 = 2.7 0.6 mM) display their activities, respec-
tively (Table 2), while dabigatran etexilate reveals no inhibitory
effect.
Based on the results obtained above, we speculated that the
activity for inhibiting thrombosis of X-2 might originate from
two parts: the anti-thrombin effect of dabigatran and the anti-
platelet aggregation activity of ferulic acid (Fig. 3). The rabbit
thrombin-induced platelet aggregation assay confirmed the inhib-
itory effects of X-1–X-7 on thrombin. Since dabigatran was the
only one active moiety which could be tested in this assay, X-1–
X-7 showed the similar activities. And the data of ADP-induced
platelet aggregation test confirmed the anti-platelet aggregation
activity of X-2. The lower activity of X-2 than that of ferulic acid
might be due to the incomplete twice hydrolysis of X-2 in vitro.
The result of X-2 in the venous thrombosis model indicated a syn-
ergistic inhibitory effect on venous thrombosis via two routes-anti-
platelet and antithrombin, which indirectly reflected the
relationship between thrombin and platelet effecting on venous
thrombosis.
ED50=3.7 1.0 mol/kg
0.0
0.5
1.0
1.5
2.0
lg [compound dose] ( mol/kg i.g.)
Figure 2. Dose–response curve for the antithrombotic efficacy of X-2 and dabig-
atran etexilate (0.1–20 mg/kg) in clot weight in rat venous thrombosis model
(Reyers).13 Average dry clot weight in the vehicle-treated was 8.2 1.9 mg. Values
are expressed as mean SD of eight animals/group.
All synthesized compounds, along with the reference com-
pound dabigatran etexilate, were evaluated in rabbit thrombin-in-
duced platelet aggregation assay.11 Due to the property of prodrug,
they were all incubated in rabbit liver microsomal suspensions be-
fore test in vitro.12 Most of tested compounds reveal similar activ-
ities (IC50 of each compound is revealed in Table 1).
Compounds X-2 (IC50 = 0.29
inhibitory effect on the rat venous thrombosis model (Reyers).13
The result reveals that compound X-2 (ED50 = 3.7 1.0 mol/kg)
(Fig. 2) has a more potent activity for inhibiting thrombosis
in vivo than that of dabigatran etexilate (ED50 = 7.8 1.5 mol/kg).
lM) was chosen to investigate its
l
In conclusion, a series of novel mutual prodrugs of dabigatran
and methyl ferulate were designed, synthesized and evaluated.
l

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X.-Z. Yang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2089–2092
X-2
O
CH3
O
CH3
N
N
N
HN
NH2
N
H3CO
O
N
O
O
H3CO
in vivo
hydrolysis
O
Dabigatran
Ferulic acid
H3CO
CH3
N
NH
N
OH
HN
NH2
N
N
HOOC
HOOC
O
Ca2+
-
-
-
Thrombin
Platelet
ADP
-
Venous Thrombosis
Figure 3. Proposed metabolic route of compound X-2.
After preliminary pharmacological screening in vitro, further test
in vivo shows that the compound X-2 (ED50 = 3.7 1.0 mol/kg)
has a stronger activity for inhibiting thrombosis than that of dabig-
atran etexilate (ED50 = 7.8 1.5 mol/kg). In addition, the result of
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Financial support from the National Natural Science Foundation
of China (Grant No. 21272277) and the Enterprise Innovation Drug
Incubation Base of the Ministry of Science and Technology, China
(Grant No. 2012ZX09401-006) are gratefully acknowledged. Dr.
Yajuan Xiao is greatly acknowledged for proofreading this
manuscript.
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Supplementary data
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Supplementary data (Experimental details and compound char-
acterizations) associated with this article can be found, in the on-
line version, at http://dx.doi.org/10.1016/j.bmcl.2013.01.126.