Design, synthesis and bioactivity evaluation of novel dabigatran derivatives as potential thrombin inhibitors

Article abstract of DOI:10.2174/1570180813666160622081105

With the help of computer-aided drug design approach, five dabigatran derivatives were designed and synthesized. The structures of these compounds were elucidated by ¹H NMR, ¹³C NMR, HRMS analyses. All the compounds were evaluated fo

Full text of DOI:10.2174/1570180813666160622081105

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809
Letters in Drug Design & Discovery, 2016, 13, 809-817
ISSN: 1570-1808
eISSN: 1875-628X
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Design, Synthesis and Bioactivity Evaluation of Novel Dabigatran
Derivatives as Potential Thrombin Inhibitors
BENTHAM
S
CIENCE
Qianqian Liu, Yujie Ren* and Xiaodong Gao
School of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai
201418, China
Abstract: With the help of computer-aided drug design approach, five dabigatran derivatives were de-
1
signed and synthesized. The structures of these compounds were elucidated by H NMR, 13C NMR,
HRMS analyses. All the compounds were evaluated for their thrombin inhibitory activitiy in vitro
(IC50) and they showed significant thrombin inhibition with IC50 values from 2.74 to 20.31 nM. Espe-
cially, compound 7a, and 7b were potent thrombin inhibitor with IC50 values of 2.74 and 2.99 nM, re-
spectively, which were comporable to dabigatran (1.20 nM) and much better than argatroban (9.88
nM). Moreover, molecular docking study was performed to determine the inhibitory mechanism of ac-
tive compounds.
Keywords: Dabigatran derivaties, synthesis, antithrombin activity, molecular docking.
Received: April 20, 2015
Revised: February 09, 2016
Accepted: May 13, 2016
1. INTRODUCTION
of traditional anticoagulants, such as drug–drug interactions,
food interactions, slow onset and offset of action, and the
need for routine anticoagulation monitoring to maintain a
therapeutic international normalized ratio [15]. However,
treatment of thrombotic diseases with this thrombin inhibitor
presents several limitations, such as increased risk of dosage-
related hepatotoxicity [16] and hemorrhage [17]. Therefore,
further optimization of dabigatran is an interesting idea to
develop safe and effective thrombin inhibitors for prevention
and treatment of thrombotic diseases.
Thrombotic diseases are a serious threat to human life
and health worldwide. This group of disease encompasses
multiple clinical conditions, such as arterial and venous
thromboembolic disorders, depending on the type of vessels
involved [1]. Coagulation cascade factor inhibitors are pri-
marily administered to treat venous thromboembolism
(VTE) whereas antiplatelet drugs are usually used to treat
arterial thromboembolism [2]. VTE approximately affects 1
to 2 of every 1000 adults annually in Europe [3,4]. Throm-
bin, a multifunctional serine protease, plays an important
role in the blood coagulation cascade that converts soluble
fibrinogen to insoluble fibrin, activates relevant factors to
generate more thrombin and activates platelet [5-7]. The
determination of structures and the mechanism of action of
thrombin and some coagulation factors was contributing to a
better understanding of hemostasis and thrombosis [8].
Hauel [18] investigated the exact binding mode of
thrombin inhibitor by determining its crystal structure with
human ꢀ-thrombin. Benzimidazole, the central template of
the inhibitor, is bound to thrombin through hydrophobic in-
teraction with the P-pocket to perform anticoagulant activity.
And the pyridine ring in the D-pocket of dabigatran in dabi-
gatran plays a role in the positioning. Bioisosteric replace-
ment is a generally used and effective strategy to optimise
common compounds [19, 20]. We envisaged that the re-
placement of hydrogen atom in pyridyl ring with a methyl
bioisostere. The methyl group can regulate the formation of
hydrogen bonds, and it can be taken as an electron-donating
or electron-withdrawing group to modulate the binding affin-
ity between the ligand and receptor [21, 22]. Therefore, in-
troduction of methyl group to dabigatran aimed to modulate
the binding force between dabigatran and thrombin for de-
veloping new thrombin inhibitor. Our group [23] previously
reported that modification on pyridyl ring of dabigatran with
bulky and hydrophobic groups was favored for antithrombin
activity. So dabigatran was also optimized by changing its
pyridyl ring with the methyl and methoxyl group introduced
into the benzene ring to determine their bioactivity and study
the structure-activity relationship.
Information on thrombin can be used to determine potent,
selective, effective and safe thrombin inhibitors. Argatroban
is the first clinical anticoagulant that directly targets throm-
bin [9]. Direct thrombin inhibitors, such as ximelagatran
[10], argatroban [11], and dabigatran etexilate [12] (Fig. 1),
have been widely investigated for their anticoagulant proper-
ties. Among them, argatroban is administered intravenously.
And ximelagatran has been withdrawn from clinical devel-
opment because of drug-induced liver injury [13, 14]. Dabi-
gatran etexilate is the only oral direct thrombin inhibitor
available in the market, which is rapidly absorbed and con-
verted to the active form dabigatran. Dabigatran can prevent
a series of coagulation cascades, without several limitations
*Address correspondence to this author at the Haiquan Road No. 100,
Fengxian district, Shanghai, People's Republic of China;
E-mail: clab@sit.edu.cn
With the help of computer-aided drug design approach,
such as molecular model and scoring technology, dabigatran
1875-628X/16 $58.00+.00
©2016 Bentham Science Publishers

810 Letters in Drug Design & Discovery, 2016, Vol. 13, No. 8
HO
Liu et al.
O
O
NH
O
O
N
H
NH2
HN
HN
HN
H
N
HN
O
O
N
O
S
H
N
OH
Argatroban
Ximelagatran
O
O
O
O
NH2
NH2
NH
HN
in vivo converted to
N
N
HN
N
N
O
N
HO
N
N
O
O
Dabigatran
Dabigatran etexilate
Fig. (1). Chemical structures of direct thrombin inhibitors.
was modified to develop new compounds with antithrombin
activity in this work. Five new compounds were synthesized
and screened for their in vitro inhibitory activities against
human thrombin. Most of the compounds exhibited signifi-
cant inhibitory activities much better than argatroban and
within the range of dabigatran. Successful demonstration of
potent antithrombin activity of some compounds could pro-
vide guidance for the modification of dabigatran to find new
candidate of anticoagulants.
chromatography [Petroleum ether (PE)/ Ethyl acetate
(EtOAc) = 6:1] to afford pure compounds as brown red oil or
white solid.
2.1.2. General Procedure for Preparation of Compounds
3a-e
To a dichloromethane (DCM) (50 mL) solution of com-
pound 2a-e (35 mmol) containing triethylamine (TEA) (35
mmol), a DCM (40 mL) solution of the yellow solid chloride
was slowly added at room temperature and then the mixture
was stirred for 5 h until completion of the reaction. Upon
completion, the reaction mixture was washed with water and
extracted with DCM in the separatory funnel, dried by anhy-
drous sodium sulfate (Na2SO4) and concentrated under re-
duced pressure. The residue was purified by silica gel chro-
matography (PE/EtOAc =2:1) to afford pure yellow solid
compounds 3a-e.
MATERIALS AND METHODS
2.1. Methods and Chemicals
Unless otherwise noted, all the chemicals and solvents
were purchased from Darui and Titan Corporation, and used
without further purification. Reaction course was routinely
monitored by thin-layer chromatography (TLC) on silica gel
under UV light (254 nm). The products were purified by
column chromatography (200–300 mesh) with designated
solvents. The melting points were measured using a WPS-
2A digital melting point apparatus and were uncorrected.
High-resolution mass spectra (HRMS) were obtained using a
SolariX-70FT-MS Bruker spectrometer equipped with elec-
trosprayionisation (ESI) equipment. 1H and 13C NMR spectra
were obtained in deuterated chloroform (CDCl3) and deuter-
ated dimethyl sulfoxide (DMSO-d6) solution using a Bruker
Avance 400 magnetic resonance spectrometer at 400 MHz,
respectively, with TMS (tetramethylsilane) as the internal
reference.
2.1.3. General Procedure for Preparation of Compounds
4a-e
To a solution of compounds 3a-e (21mmol) in 100 mL
acetic acid and 50 mL water was added Zn (6.6g, 105mmol).
This mixture was stirred for 1h at room temperature. After
completion of the reaction, the Zn was filtered, and the mix-
ture was extracted with DCM (3ꢀ40 mL). The DCM layer
was dried by anhydrous Na2SO4, and concentrated under
reduced pressure. The residue was purified by silica gel
chromatography (PE/ EtOAc =1:1) to obtain white solid
compounds 4a-e.
2.1.1. General Procedure for Preparation of Compounds
2a-e
3-[(3-Amino-4-methylamino-benzoyl)-(2-methyl-phenyl)-
amino]-propionic Acid Ethyl Ester (4a)
Yield: 83%; mp: 138-139°C. 1H NMR (400 MHz,
CDCl3) ꢁ 7.05 (d, J = 8.2 Hz, 3H, Ar-H), 7.00 (s, 1H, Ar-H),
6.75 (s, 1H, Ar-H), 6.58 (d, J = 7.5 Hz, 1H, Ar-H), 6.14 (d, J
= 7.7 Hz, 1H, Ar-H), 3.98 (q, J = 7.1 Hz, 2H, -CH2-), 2.64 (s,
3H, -NCH3), 2.11 (s, 3H, Ar-CH3), 1.13 (t, J = 7.1 Hz, 3H, -
CH3).
Compounds 1a-e (50mmol) and ethyl acrylate 2 (6.0g,
60mmol) were mixed in a 100 mL dry round bottom flask.
trifluoromethanesulfonic acid (TfOH) (5mmol) as catalyst
was added. The mixture was stirred for 12h at reflux tem-
perature in a nitrogen atmosphere. The reaction was detected
by TLC. The unreacted ethyl acrylate was concentrated un-
der reduced pressure. The residue was purified by silica gel

Design, Synthesis and Bioactivity Evaluation of Novel Dabigatran Derivatives
Letters in Drug Design & Discovery, 2016, Vol. 13, No. 8 811
3-[(3-Amino-4-methylamino-benzoyl)-(2,3-dimethyl-
phenyl)-amino]-propionic Acid Ethyl Ester (4b)
ture was extracted with DCM (3ꢀ40 mL). The DCM layer
was dried by anhydrous Na2SO4, and concentrated under
reduced pressure. The residue was purified by silica gel
chromatography [DCM/Methanol (MeOH)=100:1] to obtain
white solid compounds 5a-e.
Yield: 92%; mp: 119-120 °C. 1H NMR (400 MHz,
CDCl3) ꢁ 6.97 (q, J = 7.4 Hz, 2H, Ar-H), 6.84 (d, J = 11.2
Hz, 2H, Ar-H), 6.60 (d, J = 7.8 Hz, 1H, Ar-H), 6.20 (d, J =
8.2 Hz, 1H, Ar-H), 4.02 (q, J = 7.1 Hz, 2H, -CH2-), 3.70(m,
2H, -CH2-), 2.73 (s, 3H, -NCH3), 2.62 (ddd, J = 15.4, 9.2, 5.9
Hz, 2H, -CH2-), 2.20 (s, 3H, Ar-CH3), 2.07 (s, 3H, Ar-CH3),
1.17 (t, J = 7.1 Hz, 3H, -CH3).
3-({2-[(4-Cyano-phenylamino)-methyl]-1-methyl-1H-
benzoimidazole-5-carbonyl}-(2-methyl-phenyl)-amino)-
propionic Acid Ethyl Ester (5a)
Yield: 45%; mp: 203-205ꢀ; 1H NMR (400 MHz,
CDCl3) ꢁ 7.66 (s, 1H, Ar-H), 7.42 (d, J = 8.6 Hz, 2H, Ar-H),
7.26 (d, J = 8.4 Hz, 1H, Ar-H), 7.24 (t, 1H, Ar-H), 7.03 (d, J
= 8.4 Hz, 1H, Ar-H), 7.00(t, 1H, Ar-H), 6.88 (d, J = 6.9 Hz,
2H, Ar-H), 6.77 (d, J = 7.8 Hz, 1H, Ar-H) , 6.68 (d, J = 8.6
Hz, 2H, Ar-H), 4.43 (d, J = 4.5 Hz, 2H, -CH2-), 4.19 (t, J =
7.4 Hz, 2H, -CH2-), 4.05 (q, J = 7.1 Hz, 2H, -CH2-), 3.64 (s,
3H, -NCH3), 2.69 (t, J = 7.4 Hz, 2H, -CH2-), 2.20 (s, 3H, Ar-
CH3), 1.19 (t, J = 7.1 Hz, 3H, -CH3).
3-[(3-Amino-4-methylamino-benzoyl)-(2, 6-dimethyl-
phenyl)-amino]-propionic Acid Ethyl Ester (4c)
Yield: 65%; mp: 151-153°C. 1H NMR (400 MHz,
CDCl3) ꢁ 7.02(m, 1H, Ar-H), 6.96 (d, J = 7.4 Hz, 2H, Ar-H),
6.80 (d, J = 1.8 Hz, 1H, Ar-H), 6.55 (dd, J = 8.2, 1.6 Hz, 1H,
Ar-H), 6.17 (d, J = 8.4 Hz, 1H, Ar-H), 4.03 (q, J = 7.1 Hz,
2H, -CH2-), 3.94 (dd, J = 10.0, 5.7 Hz, 2H, -CH2-), 2.74(m,
2H, -CH2-), 2.70 (s, 3H, -NCH3), 2.15 (s, 6H, Ar-CH3), 1.17
(t, J = 7.1 Hz, 3H, -CH3).
3-({2-[(4-Cyano-phenylamino)-methyl]-1-methyl-1H-
benzoimidazole-5-carbonyl}-(2, 3-methyl-phenyl)-amino)-
propionic Acid Ethyl Ester (5b)
3-[(3-Amino-4-methylamino-benzoyl)-(2-methoxy-phenyl)-
amino]-propionic Acid Ethyl Ester (4d)
Yield: 61%; mp: 206-207 °C; 1H NMR (400 MHz,
CDCl3) ꢁ 7.66 (s, 1H, Ar-H), 7.42 (d, J = 8.6 Hz, 2H, Ar-H),
7.26 (d, J = 8.4 Hz, 1H, Ar-H), 7.04 (s, 1H, Ar-H), 7.00 (m,
1H, Ar-H), 6.88 (d, J = 6.9 Hz, 1H, Ar-H, s, J = 6.9 Hz, 1H,
Ar-H), 6.77 (d, J = 7.8 Hz, 1H, Ar-H), 6.68 (d, J = 8.6 Hz,
2H, Ar-H), 4.43 (d, J = 4.5 Hz, 2H, -CH2-), 4.19 (t, J = 7.4
Hz, 2H, -CH2-), 4.05 (q, J = 7.1 Hz, 2H, -CH2-), 3.64 (s, 3H,
-NCH3), 2.69 (t, J = 7.4 Hz, 2H, -CH2-), 2.20 (s, 3H, Ar-
CH3), 1.19 (t, J = 7.1 Hz, 3H, -CH3).
Yield: 96%; mp: 135-137°C. 1H NMR (400 MHz,
CDCl3) ꢁ7.11 (t, J = 7.7 Hz, 1H, Ar-H), 6.97 (d, J = 7.9 Hz,
1H, Ar-H), 6.78 (d, J = 2.1 Hz, 2H, Ar-H), 6.76 (d, J = 5.1
Hz, 1H, Ar-H), 6.69 (d, J = 8.3 Hz, 1H, Ar-H), 6.21 (d, J =
8.2 Hz, 1H, Ar-H), 4.00 (dd, J = 14.3, 7.1 Hz, 4H, -CH2-),
3.69 (s, 3H, -OCH3), 2.70 (s, 3H, -NCH3), 2.69 – 2.60 (m,
2H, -CH2-), 1.16 (t, J = 7.1 Hz, 3H, -CH3).
3-[(3-Amino-4-methylamino-benzoyl)-(4-methyl-pyridin-2-
yl)-amino]-propionic Acid Ethyl Ester (4e)
3-({2-[(4-Cyano-phenylamino)-methyl]-1-methyl-1H-
benzoimidazole-5-carbonyl}-(2,6-dimethyl-phenyl)-amino)-
propionic Acid Ethyl Ester (5c)
Yield: 91%; mp: 112-113 °C. 1H NMR (400 MHz,
CDCl3) ꢁ 8.24 (d, J = 5.1 Hz, 1H, -H), 6.81 (dd, J = 7.0, 3.4
Hz, 2H, -H, Ar-H), 6.71 (d, J = 8.2 Hz, 1H, Ar-H), 6.55 (s,
1H, Ar-H), 6.28 (d, J = 8.3 Hz, 1H, -H), 4.29 (t, J = 7.4 Hz,
2H, -CH2-), 4.02 (q, J = 7.2 Hz, 2H, -CH2-), 2.76 (s, 3H, -
NCH3), 2.70 (t, J = 7.4 Hz, 2H, -CH2-), 2.11 (s, 3H, -CH3),
1.16 (t, J = 7.1 Hz, 3H, -CH3).
Yield: 49%; mp: 205-206°C; 1H NMR (400 MHz,
CDCl3) ꢁ 7.54 (s, 1H, Ar-H), 7.41 (d, J = 8.5 Hz, 2H, Ar-H),
7.25 (d, J = 5.9 Hz, 1H, Ar-H), 7.02 (t, 1H, Ar-H), 6.99 (d, J
= 6.6 Hz, 1H, Ar-H), 6.93 (d, J = 7.4 Hz, 2H, Ar-H), 6.67 (d,
J = 8.5 Hz, 2H, Ar-H), 4.42 (d, J = 4.5 Hz, 2H, -CH2-), 4.06
(dd, J = 9.0, 5.3 Hz, 2H, -CH2-), 4.03 (t, 2H, -CH2-), 3.62 (s,
3H, -NCH3), 2.79 (t, 2H, -CH2-), 2.19 (s, 6H, Ar-CH3), 1.19
(t, J = 7.1 Hz, 3H, -CH3).
2.1.4. General Procedure for Preparation of Compounds
5a-e
To a solution of (4-cyano-2-substitutedphenylamino)-
acetic acid (3.3g, 19 mmol), 1-hydroxybenzotriazole (HOBt)
(2.5g, 19 mmol) and 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide hydrochloride (EDCI) (3.6g, 19mmol) in 70
mL tetrahydrofuran (THF) and 10 mL DMF was stirred un-
der the ice baths. After 30 min, the compounds 4a-e
(16mmol) were dissolved in 40 mL tetrahydrofuran (THF),
and slowly added into this system. The mixture was stirred at
room temperature for 5h. The solvent was concentrated and
extracted with DCM (3ꢀ40 mL). The combined organic lay-
ers were washed with brine, dried over anhydrous Na2SO4
and concentrated under reduced pressure. To solution of the
residue, 40 mL acetic acid was added. The mixture was
heated to reflux for 2.5h at 120 °C. After cooled to room
temperature, the acetic acid was removed under reduced
pressure, and the residue was neutralized to PH=7-8 with
concentrated aqueous ammonia (NH3·H2O). Then the mix-
3-({2-[(4-Cyano-phenylamino)-methyl]-1-methyl-1H-
benzoimidazole-5-carbonyl}-(2-methoxy-phenyl)-amino)-
propionic Acid Ethyl Ester (5d)
Yield: 89%; mp: 200-202°C; 1H NMR (400 MHz,
CDCl3) ꢁ 7.61 (s, 1H, Ar-H), 7.40 (d, J = 8.3 Hz, 2H, Ar-H),
7.25 (d, J = 6.2 Hz, 1H, Ar-H), 7.07 (t, J = 7.7 Hz, 1H, Ar-
H), 6.99 (t, J = 8.5 Hz, 2H, Ar-H, Ar-H), 6.74 (d, J = 7.5 Hz,
1H, Ar-H), 6.72 (d, J = 7.0 Hz, 1H), 6.67 (d, J = 8.6 Hz, 2H,
Ar-H), 4.41 (d, J = 4.4 Hz, 2H, -CH2-), 4.08 (dq, J = 21.1,
7.2 Hz, 4H, -CH2-, -CH2-), 3.69 (s, 3H, -OCH3), 3.60 (s, 3H,
-NCH3), 2.70(m, 2H, -CH2-), 1.18 (t, J = 7.1 Hz, 3H, -CH3).
3-({2-[(4-Cyano-phenylamino)-methyl]-1-methyl-1H-
benzoimidazole-5-carbonyl}-(4-methyl-pyridin-2-yl)-
propionic Acid Ethyl Ester (5e)
Yield: 65%; mp: 203-204 °C; 1H NMR (400 MHz,
CDCl3) ꢁ 8.21 (d, J = 5.0 Hz, 1H, -H), 7.68 (s, 1H, Ar-H),

812 Letters in Drug Design & Discovery, 2016, Vol. 13, No. 8
Liu et al.
7.41 (d, J = 8.5 Hz, 2H, Ar-H), 7.25 (d, J = 8.9 Hz, 1H, Ar-
H), 7.04 (d, J = 8.4 Hz, 1H, Ar-H), 6.79 (d, J = 5.1 Hz, 1H, -
H), 6.69 (d, J = 8.6 Hz, 2H, Ar-H), 6.58 (s, 1H, -H), 4.46 (d,
J = 4.5 Hz, 2H, -CH2-), 4.34 (t, J = 7.3 Hz, 2H, -CH2-), 4.04
(q, J = 7.1 Hz, 2H, -CH2-), 3.67 (s, 3H, -NCH3), 2.75 (t, J =
7.3 Hz, 2H, -CH2-), 2.04 (s, 3H, -CH3), 1.18 (t, J = 7.1 Hz,
3H, -CH3).
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-
1H-benzoimidazole-5-carbonyl}-(2-methoxy-phenyl)-
amino]-propionic Acid Ethyl Ester (6d)
1
Yield: 86%; mp: 223-224°C; H NMR (400 MHz, dmso)
ꢀ 7.57 (d, J = 8.6 Hz, 2H, Ar-H), 7.39 (s, 1H, Ar-H), 7.29 (d,
J = 8.2 Hz, 1H, Ar-H), 7.18 (d, J = 4.9 Hz, 1H, Ar-H), 7.15
(s, 1H, Ar-H), 7.11 (d, J = 8.1 Hz, 2H, Ar-H), 6.88 (d, J =
8.1 Hz, 1H, Ar-H), 6.79 (d, J = 8.7 Hz, 2H, Ar-H), 4.56 (d, J
= 5.0 Hz, 2H, -CH2-), 3.94 (q, J = 7.1 Hz, 2H, -CH2-), 3.69
(s, 3H, -OCH3), 3.65 (s, 3H, -NCH3), 2.55 (t, J = 13.5, 6.8
Hz, 2H, -CH2-), 1.09 (t, J = 7.1 Hz, 3H, -CH3).
2.1.5. General Procedure for Preparation of Compounds
6a-e
Compounds 5a-e (10mmol), hydroxylamine hydrochlo-
ride (1.4g, 20mmol) and trimethylamine (Et3N) (20 mmol)
were mixed in 20mL ethanol. The mixture was refluxed for
3h at 80°C. Ethanol was then evaporated under reduced pres-
sure. To a acetic acid (HOAC) (20 mL) solution of the resi-
due, ammonium formate (1.9g, 30mmol) and 10% Pd/C
(1.9g) were added with stirring. The mixture was heated to
reflux for 5h at 120°C under a nitrogen atmosphere. After
completion of the reaction, the contents were cooled. The
solution was evaporated under vacuum, then the residue was
purified by silica gel chromatography (DCM/MeOH=7:1) to
obtain white solid compounds 6a-e.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-
1H-benzoimidazole-5-carbonyl}-(4-methyl-pyridin-2-yl)-
amino]-propionic Acid Ethyl Ester (6e)
Yield: 83%; mp: 191-193 °C; 1H NMR (400 MHz,
DMSO-d6) ꢀ 8.15 (d, J = 5.0 Hz, 1H, -H), 7.61 (d, J = 8.8
Hz, 2H, Ar-H), 7.45 (s, 1H, Ar-H), 7.34 (d, J = 7.0 Hz, 1H,
Ar-H), 7.14 (d, J = 8.6 Hz, 1H, Ar-H), 6.94 (d, J = 5.0 Hz,
1H, -H), 6.86 (s, 1H, -H), 6.82 (d, J = 8.8 Hz, 2H, Ar-H),
4.60 (d, J = 5.5 Hz, 2H, -CH2-), 4.13 (t, J = 7.1 Hz, 2H, -
CH2-), 3.94 (q, J = 7.1 Hz, 2H, -CH2-), 3.74 (s, 3H, -NCH3),
2.62 (t, J = 7.1 Hz, 2H, -CH2-), 2.06 (s, 3H, -CH3), 1.08 (t, J
= 7.1 Hz, 3H, -CH3).
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-
1H-benzoimidazole-5-carbonyl}-(2-methyl-phenyl)-amino]-
propionic Acid Ethyl Ester (6a)
2.1.6. General Procedure for Preparation of Compounds
7a-e
Yield: 62%; mp: 191-192°C; 1H NMR (400 MHz,
DMSO-d6) ꢀ 7.58 (d, J = 8.7 Hz, 2H, Ar-H), 7.45 (s, 1H, Ar-
H), 7.33 (d, J = 8.3 Hz, 1H, Ar-H), 7.28 (t, J = 5.5 Hz, 1H,
Ar-H), 7.16 (d, J = 8.6 Hz, 1H, Ar-H), 7.05 (s, 1H, Ar-H),
7.03 (d, J = 7.9 Hz, 1H, Ar-H), 6.91 (d, J = 7.7 Hz, 1H, Ar-
H), 6.81 (d, J = 8.7 Hz, 2H, Ar-H), 4.58 (d, J = 5.4 Hz, 2H, -
CH2-), 4.02 (t, J = 7.2 Hz, 2H, -CH2-), 3.95 (q, J = 7.1 Hz,
2H, -CH2-), 3.71 (s, 3H, -NCH3), 2.55 (t, J = 7.1 Hz, 2H, -
CH2-), 2.16 (s, 3H, Ar-CH3), 1.10 (t, J = 7.1 Hz, 3H, -CH3).
The compounds 6a-e (1mmol) were dissolved in 5 mL
ethanol, then a solution of sodium hydroxide(160mg,
4mmol) in 2 mL H2O was added into this system. The mix-
ture was stirred at room temperature for 1h. After completion
of the reaction, the reaction solution was neutralized to
PH=6-7 with diluted acetic acid. The solids were filtrated
and washed carefully with water, and dried to afford the
white solid compounds 7a-e.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-
1H-benzoimidazole-5-carbonyl}-(2-methyl-phenyl)-amino]-
propionic Acid (7a)
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-
1H-benzoimidazole-5-carbonyl}-(2,3-methyl-phenyl)-
amino]-propionic Acid Ethyl Ester (6b)
Yield: 80%; mp: 249-250°C; 1H NMR (400 MHz,
DMSO-d6) ꢀ(ppm) 8.95 (s, 1H, -OH), 8.71 (s, 1H, =NH),
7.68 (d, J = 8.1 Hz, 2H, Ar-H), 7.44 (d, J = 11.6 Hz, 2H, Ar-
H), 7.25 (d, J = 6.6 Hz, 2H, Ar-H), 7.12 (s, 3H, Ar-H), 6.87
(d, J = 8.0 Hz, 2H, Ar-H), 4.68 (s, 2H, -CH2-), 3.78 (s, 3H, -
NCH3), 3.67(m, 2H, -CH2-), 2.59 (s, 2H, -CH2-), 2.11 (d, J =
19.5 Hz, 3H, Ar-CH3). 13C NMR (101 MHz, DMSO-d6)
ꢀ(ppm) 173.02, 170.30, 164.86, 164.79, 164.71, 164.64,
153.61, 153.39, 142.21, 136.73, 135.23, 131.67, 130.13,
130.00, 128.11, 127.33, 123.44, 113.27, 112.26, 110.00,
45.92, 32.41, 30.59, 21.50, 18.00. ESI-HRMS: calcd for
C27H28N6O3 [M+H]+ , 485.2256, found 485.2323.
Yield: 78%; mp: 188-190°C; 1H NMR (400 MHz,
DMSO-d6) ꢀ 7.58 (d, J = 8.7 Hz, 2H, Ar-H), 7.45 (s, 1H, Ar-
H), 7.33 (d, J = 8.3 Hz, 1H, Ar-H), 7.28 (t, J = 5.5 Hz, 1H,
Ar-H), 7.16 (d, J = 8.6 Hz, 1H, Ar-H), 7.05 (s, 1H, Ar-H),
7.03 (d, J = 7.9 Hz, 1H, Ar-H), 6.91 (d, J = 7.7 Hz, 1H, Ar-
H), 6.81 (d, J = 8.7 Hz, 2H, Ar-H), 4.58 (d, J = 5.4 Hz, 2H, -
CH2-), 4.02 (t, J = 7.2 Hz, 2H, -CH2-), 3.95 (q, J = 7.1 Hz,
2H, -CH2-), 3.71 (s, 3H, -NCH3), 2.55 (t, J = 7.1 Hz, 2H, -
CH2-), 2.16 (s, 3H, Ar-CH3), 1.10 (t, J = 7.1 Hz, 3H, -CH3).
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-
1H-benzoimidazole-5-carbonyl}-(2,
amino]-propionic Acid Ethyl Ester (6c)
6-dimethyl-phenyl)-
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-
Yield: 78%; mp: 289-291°C; 1H NMR (400 MHz,
DMSO-d6)) ꢀ 7.57 (d, J = 8.5 Hz, 2H, Ar-H), 7.32 (d, J = 8.5
Hz, 1H, Ar-H), 7.27 (s, 1H, Ar-H), 7.21 (t, J = 5.6 Hz, 1H,
Ar-H), 7.17 (d, J = 8.5 Hz, 1H, Ar-H), 6.99 (d, J = 4.1 Hz,
2H, Ar-H), 6.79 (d, J = 8.6 Hz, 2H, Ar-H), 4.55 (d, J = 5.2
Hz, 2H, -CH2-), 3.95 (q, J = 7.1 Hz, 2H, -CH2-), 3.88 (t, 2H,
-CH2-), 3.70 (s, 3H, -NCH3), 2.66 (t, J = 7.4 Hz, 2H, -CH2-),
2.12 (s, 6H, Ar-CH3), 1.09 (t, J = 7.1 Hz, 3H, -CH3).
1H-benzoimidazole-5-carbonyl}-(2,
amino]-propionic Acid (7b)
3-methyl-phenyl)-
Yield: 77%; mp: 246-247 °C; 1H NMR (400 MHz,
DMSO-d6) ꢀ 8.91 (s, 1H, -OH), 8.66 (s, 1H, =NH), 7.66 (d, J
= 8.5 Hz, 2H, Ar-H), 7.51 (s, 1H, Ar-H), 7.39 (d, J = 8.3 Hz,
1H, Ar-H), 7.22 (d, J = 8.4 Hz, 1H, Ar-H), 7.11 (s, 1H, Ar-
H), 7.07 (d, J = 7.7 Hz, 1H, Ar-H), 6.95 (d, J = 7.5 Hz, 1H,
Ar-H), 6.87 (d, J = 8.7 Hz, 3H, Ar-H), 4.65 (s, 2H, -CH2-),

Design, Synthesis and Bioactivity Evaluation of Novel Dabigatran Derivatives
Letters in Drug Design & Discovery, 2016, Vol. 13, No. 8 813
4.02 (t, J = 7.2 Hz, 2H, -CH2-), 3.79 (d, J = 14.6 Hz, 3H, -
NCH3), 2.55 (t, J = 7.5 Hz, 2H, -CH2-), 2.20 (s, 3H, Ar-
CH3). 13C NMR (101 MHz, DMSO-d6) ꢁ(ppm) 172.56,
169.84, 164.33, 164.25, 164.18, 164.10, 153.07, 152.94,
143.25, 138.62, 136.40, 129.98, 129.62, 128.81, 127.99,
127.28, 125.27, 123.10, 118.94, 111.71, 109.46, 46.39,
32.11, 30.02, 20.79. ESI-HRMS: calcd for C27H28N6O3
[M+H]+ , 485.2256, found 485.2300.
2.2. Anticoagulant Assay
The thrombin inhibitory effects (IC50) of the target com-
pounds 7a-e were determined with a commercially available
chromogenic assay against human thrombin. Lyophilized
human thrombin (national standard), which was purified
from human blood, substrate and the test compounds were
dissolved and diluted to needed concentrations. Argatroban
was used as the reference. Firstly, lyophilized human throm-
bin (5.4ꢂg/mL) and tested compounds with different concen-
trations were preincubated for 10 minutes at 37°C in the 96-
well plate. Nextly the specificity substrate, Ac-FVR-AMC
(5ꢂM), was added into the preincubation mixture. Then, the
dynamic changes of fluorescence intensity were measured in
a microplate detector Envision (PerkinElmer) at room tem-
perature within 10 min. The slope of the linear enzyme dy-
namics curve during the initial stage of the reaction was re-
ferred to as the initial velocity of enzyme reaction. The inhi-
bition rates were calculated from the ultraviolet absorption
(Excitation/Emission=380nm/500nm) values of inhibited
wells and positive control wells. Finally, the IC50 values
were gained using a regression analysis method between the
concentration and inhibition rate. The inhibitory rate and the
concentration that induced a 50% inhibition of thrombin
(IC50) were calculated by the following formulas:
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-
1H-benzoimidazole-5-carbonyl}-(2,
amino]-propionic Acid (7c)
6-dimethyl-phenyl)-
Yield: 67%; mp: 253-255°C; 1H NMR (400 MHz,
DMSO-d6) ꢁ(ppm) 8.97 (s, 1H, -OH), 8.71 (s, 1H, =NH),
7.68 (d, J = 8.3 Hz, 2H, Ar-H), 7.51 (d, J = 8.4 Hz, 1H, Ar-
H), 7.38 (s, 1H, Ar-H), 7.29 (d, J = 8.5 Hz, 1H, Ar-H), 7.03
(s, 3H, Ar-H), 6.87 (d, J = 8.3 Hz, 2H, Ar-H), 4.73 (s, 2H, -
CH2-), 3.87 (m, 2H, -CH2-), 3.81 (s, 3H, -NCH3), 2.65 (t, J =
7.3 Hz, 2H, -CH2-), 2.18 (s, 6H, Ar-CH3). 13C NMR (101
MHz, DMSO-d6) ꢁ(ppm) 172.97, 169.82, 164.85, 164.77,
164.70, 164.62, 153.83, 153.22, 141.20, 136.29, 135.57,
131.14, 130.16, 129.51, 128.30, 123.76, 117.09, 112.39,
110.65, 46.21, 32.33, 30.93, 18.76. ESI-HRMS: calcd for
C28H30N6O3 [M+H]+, 499.2412, found 499.2475.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-
1H-benzoimidazole-5-carbonyl}-(2-methoxy-phenyl)-
amino]-propionic Acid (7d)
Inhibition rate (%) = [VDMSO-Vsample] / VDMSOꢀ100%
V
sample: initial velocity of compound group
VDMSO: initial velocity of blank group, treated with
DMSO alone
Inhibition rate (%) =100 / [1+10(LogIC50-X)h], h is the Hill
coefficient.
Yield: 74%; mp: 246-248°C; 1H NMR (400 MHz,
DMSO-d6) ꢁ 8.95 (s, 1H, -OH), 8.70 (s, 1H, =NH), 7.68 (d, J
= 8.2 Hz, 2H, Ar-H), 7.47 (s, 2H, Ar-H), 7.25 (d, J = 8.0 Hz,
1H, Ar-H), 7.19 (d, J = 7.8 Hz, 1H, Ar-H), 7.14 (d, J = 7.5
Hz, 1H, Ar-H), 6.92 (d, J = 8.1 Hz, 1H, Ar-H), 6.87 (d, J =
8.2 Hz, 2H, Ar-H), 6.81 (t, J = 7.2 Hz, 1H, Ar-H), 4.71 (s,
2H, -CH2-), 3.99 (t, J = 20.1 Hz, 2H, -CH2-), 3.79 (s, 3H, -
OCH3), 3.70 (s, 3H, -NCH3), 2.54 (s, 2H, -CH2-). 13C NMR
(101 MHz, DMSO-d6) ꢁ(ppm) 173.14, 171.13, 164.87,
164.79, 164.71, 164.64, 154.81, 153.42, 136.54, 132.03,
131.08, 130.29, 130.14, 129.41, 123.22, 121.19, 118.06,
112.76, 112.28, 109.96, 45.55, 32.50, 30.64, 18.92. ESI-
HRMS: calcd for C27H28N6O4 [M+H]+, 501.2205, found
501.2266.
2.4. Docking Simulations
The structure of the target compounds was constructed in
the sketch process in SYBYL-X 2.0 package running on
windows workstation. The structure was optimized using
Gasteiger–Huckel method and the Tripos force field, follow-
ing by 10000 iterations and the energy gradient limit is set to
0.005 kcal/ (mol* Å). For the protein preparation, the ligand
and water were removed from the receptor protein (PDB
entry code: 1KTS). Remaining protein was optimized using
Gasteiger–Huckel charges, following by 20 iterations and the
energy gradient limit is set to 0.05 kcal/ (mol*Å).
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-
1H-benzoimidazole-5-carbonyl}-(4-methyl-pyridin-2-yl)-
amino]-propionic Acid (7e)
Yield: 88%; mp: 229-230 °C; 1H NMR (400 MHz,
DMSO-d6) ꢁ 9.05 (s, 1H, -OH), 8.81 (s, 1H, =NH), 8.15 (d, J
= 5.0 Hz, 1H, Py-H), 7.71 (dd, J = 9.3 Hz, 3H, Ar-H), 7.62
(s, 1H, Ar-H), 7.35 (d, J = 8.5 Hz, 1H, Ar-H), 7.10 (s, 1H,
Py-H), 7.02 (d, J = 5.0 Hz, 1H, Py-H), 6.92 (d, J = 8.8 Hz,
2H, Ar-H), 4.90 (s, 2H, -CH2-), 4.13 (t, J = 7.4 Hz, 2H, -
CH2-), 3.92 (s, 3H, -NCH3), 2.62 (t, J = 7.4 Hz, 2H, -CH2-),
2.17 (s, 3H, Py-CH3). 13C NMR (101 MHz, DMSO-d6)
ꢁ(ppm) 177.67, 174.79, 169.54, 169.47, 169.40, 169.32,
160.85, 158.77, 157.81, 154.66, 153.43, 134.93, 129.49,
128.00, 127.36, 121.88, 118.10, 117.27, 116.23, 61.18,
49.98, 37.81, 36.11, 25.59. ESI-HRMS: calcd for
C26H27N7O3 [M+H]+, 486.2208, found 486.2255.
3. RESULTS AND DISCUSSION
3.1. Docking Score and Predicted Bioactivity by QSAR
Modeling
To reduce costs and save time of synthesis, computer-
aided drug design approaches were applied to predict the
activities of designed compounds. The docking scoring tech-
nology and QSAR model reported [23] were set up to obtain
the bioactivity of new designed compounds. The binding
scores and the predicted bioactivities are shown in Table 1.
Compounds with scores higher than dabigatran were selected
to be synthesized for further research.

814 Letters in Drug Design & Discovery, 2016, Vol. 13, No. 8
Liu et al.
Table 1. Binding scores and the predicted IC50 values of CoMFA.
Compound
Total-score
Predicted IC50 (nM)
CoMFA
7a
12.29
11.97
11.54
10.54
11.18
10.33
2.37
2.14
2.25
1.85
2.45
2.50
8.62
8.66
8.64
8.73
8.61
8.60
7b
7c
7d
7e
Dabigatran
NO2
O
O
O
O
O
O
NH2
O2N
NH
ClOC
HN
N
O
X
R
X
N
H
X
CF3SO3H,N2
R
R
Et3N
2a-e
1a-e
3a-e
OH
HN
O
O
O
O
O
CN
H2N
N
Zn
HOAc/H2O
(i) EDCI,HOBt,
(ii) HOAc
N
O
N
O
N
NC
NH
X
O
N
H
X
R
R
4a-e
5a-e
O
N
O
O
N
N
N
O
N
OH
HN
H2N
HN
H2N
(i) NH2OH°§HCl,Et3N
(ii) HCOONH4, Pd/C, N2
NaOH
N
NH
NH
X
X
CH3CH2OH/H2O
R
R
6a-e
7a-e
a: R=2-CH3, X=CH; d: R=2-OCH3, X=CH;
b: R=2,3-CH3, X=CH; e: R=4-CH3, X=N
c: R=2,6-CH3, X=CH;
Scheme 1. Synthetic route for the target compounds 7a-e. Reagents and conditions: (a) TfOH, N2, 100 °C, reflux, 12h; (b) CH3NH2, 75 °C,
reflux, 5h; (c) DCM, SOCl2, DMF, 45 °C, reflux, 1h; (d) DCM, Et3N, rt, 4h; (e) Zn, AcOH/H2O, rt, 1h; (f) ꢀ EDCI, HOBt, 4-cyano-2-
substituted-phenylamino)-acetic acid, THF/DMF, rt, 5h; ꢁAcOH, 120 °C, reflux, 2.5h; (g) ꢀ NH2OH·HCl, Et3N, EtOH, 80 °C, reflux, 3h; ꢁ
HCOONH4, Pd/C, AcOH, 120 °C, reflux, 5h; (h) NaOH, EtOH/H2O, rt, 1h.
3.2. Chemistry
under N2 atmosphere to obtain the amidino compounds 6a-e.
The amidino compounds 6a-e were hydrolyzed in the pres-
ence of sodium hydroxide in ethanol with water at room
temperature for 1h to obtain the target compounds 7a-e. The
structures of all the target compounds were analyzed and
confirmed by 1H NMR, 13C NMR and HRMS.
The synthesis of target compounds 7a-e is shown in
Scheme 1. According to the known methods [24], we syn-
thesized these compounds with different substituents 2a-e
using ethyl acrylate and corresponding amines 1a-e with
trifluoromethanesulfonic acid (TfOH) as a catalyst at 100°C
by refiuxing. Compounds 2a-e reacted with to 4-
(methylamino)-3-nitrobenzoyl chloride generate important
intermediates 3a-e. For preparation of the compounds 4a-e,
to reduce cost and improve efficiency, acetic acid with water
was picked as solvent at room temperature for 1h, while Li
[24] used THF with water as solvent at 80°C for 5h. With
this method, the time was short, and the yields were all more
than 90%. Compounds 5a-e were prepared by treating 4a-e
with (4-cyano-2-substitutedphenylamino)-acetic acid in the
presence of EDCI and HOBt via two steps. Cyano com-
pounds 5a-e with hydroxylamine hydrochloride and Et3N
under ethanol provided the corresponding hydroxamic acids
by refluxing at 80°C for 3h, followed by 10% Pd/C reduction
3.3. Anti-Thrombin Activity In Vitro
All synthesized compounds were evaluated for inhibitory
activity against thrombin in vitro, argatroban and dabigatran
were used as reference compounds. The IC50 values of these
compounds for thrombin inhibition were determined and the
data are shown in Table 2. Most of them showed moderate or
good thrombin inhibitory activity compared to argatroban.
Compounds 7a and 7b with IC50 values of 2.74, 2.99 nM,
exhibited better antithrombin activities comparable to dabi-
gatran (1.20 nM), which are higher than that of argatroban
(9.88 nM). Among these newly synthesized compounds, as

Design, Synthesis and Bioactivity Evaluation of Novel Dabigatran Derivatives
Letters in Drug Design & Discovery, 2016, Vol. 13, No. 8 815
Table 2. Thrombin inhibitory activity of synthesized compounds.
IC50(nM)*
Structure
Compound
O
O
O
O
O
O
O
O
O
NH2
HN
HN
HN
HN
HN
N
N
HO
HO
HO
HO
HO
N
N
N
N
N
NH
7a
2.74 0.20
NH2
N
N
NH
7b
7c
7d
7e
2.99 0.83
NH2
N
N
NH
3.42 0.56
NH2
N
N
NH
20.31 0.73
O
O
N
NH2
N
N
NH
5.24 0.95
-
-
Argatroban
Dabigatran
9.88 2.26
1.20 0.09
*) The concentration that causes a 50% inhibition of thrombin.
Asp 189
1.98
2.00
Thr 172
Arg 173
Gly219
Gly216
2.83
2.19
2.20
1.94
Ser214
export
1.85
Trp60D
Fig. (2). The comparison of the cognate ligand in the crystal structure and re-docking result of dabigatran in the docked complex by super-
imposing the coordinates of protein together.
3.4. Molecular Docking Studies
observed in 7e, introduction of methyl group at 4-position of
pyridine ring showed a little weak inhibitory activity. When
two methyl groups were attached at the ortho position, the
thrombin inhibition activity was decreased as obrserved in
compound 7c. However, compound 7d had an obviously
decreased IC50 value of 20.31 nmol/L with –OCH3 at the
ortho position of the phenyl ring. This trend presented that
strong electron-donating substituent on the ring can be det-
rimental to antithrombin activity.
In order to determine the interaction between the active
compounds and the target protein, molecular docking was
performed. As shown in Fig. 2, dabigatran was redocked to
validate the reliability of docking, with the thrombin crystal
structure (PDB ID: 1KTS) as the docking protein. The bind-
ing mode of redocked and crystallized dabigatran is almost
the same in the active site of thrombin receptor (PDB ID:

816 Letters in Drug Design & Discovery, 2016, Vol. 13, No. 8
Liu et al.
Gly218
Cys220
1.95
Glu217
ILe l74
ASP189
1.94
Trp215
Glu216
Export
1.92
Trp60
Glu97
Ala190
98
Leu99
Ser 195
Tyr60
Val 213
His57
Fig. (3). X-ray crystal structure of compound 7a in complex with thrombin in a surface representation. And docking result of the compound
7a into the binding site of thrombin protease. Hydrogen bonds are shown as yellow dashed lines, with distance unit of Å. The inhibitor (red)
and the important residues (Atom Type) are shown as stick model.
ACKNOWLEDGEMENTS
1KTS). The residues Asp189, Gly219, Gly216, Trp60D,
Thr172 and Arg173 played the important role of the inhibi-
tory activity of thrombin. For example, the H atom of the
amidine interacts with the carboxyl group of Gly219 and the
O atom of Asp189 by hydrogen bonds, respectively. The O
atom of the carbonyl group interacts with the hydroxy group
of Thr172. After validating the docking reliability, the com-
pound with the highest activity was selected for studying
probable binding mode.
This work was supported by the Science and Technology
Commission of Shanghai Municipality (13DZ1930402,
13DZ 1930403).
SUPPLEMENTARY MATERIAL
Supplementary material is available on the publishers
Web site along with the published article.
The active compounds 7a with good antithrombotic ac-
tivity in vivo were successfully docked in the active site of
the thrombin receptor, as shown in Fig. 3. It shares a similar
binding mode with dabigatran reported by the literature: ꢀ-ꢀ
interaction was formed between the Trp60D and the ben-
zimidazole, the H atom of amidino interacted with the resi-
dues Asp189 and Gly219 by two hydrogen bonds. Except for
the above-mentioned hydrogen bonds, another hydrogen
bond was formed between the O atom of the carbonyl group
with Thr172 in compound 7a. The distances of hydrogen
bonds observed were 1.88Å (Asp189=O…H-N); 2.01Å
(Gly219-O…H-N); 1.84Å (Thr172-O-H…O-H), respec-
tively. These hydrogen bonds interactions may be helpful in
the binding of dabigatran derivatives with the active site of
thrombin.
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The authors confirm that this article content has no con-
flict of interest.

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