Design, synthesis, biological evaluation and molecular docking studies of dabigatran analogs as potential thrombin inhibitors

Article abstract of DOI:10.1007/s13738-015-0743-4

A series of fluorinated dabigatran analogs were designed and synthesized. All the target compounds were characterized by ¹H NMR, ¹³C NMR, and FT-ICR-MS. The thrombin inhibitory activities of the new synthesized compounds were also evaluated in vitro. The results show that compound 12a has the highest IC₅₀ of thrombin inhibition (IC₅₀ = 5.41 nM). Moreover, molecular docking simulation was carried out to elucidate the conformations of the compounds and key amino acid residues at the active site of thrombin protein. The results show there is an appropriate relationship between IC₅₀ and the docking scores for compounds 12a-e. We suggest that the hydrogen bond interaction between Asp189, Gly219 of thrombin and the compounds appear to play major role in thrombin inhibition.

Full text of DOI:10.1007/s13738-015-0743-4

J IRAN CHEM SOC (2016) 13:347–357
DOI 10.1007/s13738-015-0743-4
ORIGINAL PAPER
Design, synthesis, biological evaluation and molecular docking
studies of dabigatran analogs as potential thrombin inhibitors
Hai‑Feng Chen1 · Ming‑Hui Dong1 · Yu‑Jie Ren1 · Fei Wang1
Received: 6 July 2015 / Accepted: 15 September 2015 / Published online: 28 September 2015
© Iranian Chemical Society 2015
Abstract A series of fluorinated dabigatran analogs were
designed and synthesized. All the target compounds were
characterized by 1H NMR, 13C NMR, and FT-ICR-MS. The
thrombin inhibitory activities of the new synthesized com-
pounds were also evaluated in vitro. The results show that
compound 12a has the highest IC50 of thrombin inhibition
(IC50 = 5.41 nM). Moreover, molecular docking simula-
tion was carried out to elucidate the conformations of the
compounds and key amino acid residues at the active site of
thrombin protein. The results show there is an appropriate
relationship between IC50 and the docking scores for com-
pounds 12a–e. We suggest that the hydrogen bond interac-
tion between Asp189, Gly219 of thrombin and the com-
pounds appear to play major role in thrombin inhibition.
(e.g., heparins) and vitamin K antagonists such as warfa-
rin [2]. Recently, the introduction of new oral anticoagu-
lants on the market has enabled the availability of new
options. The first oral anticoagulant drug dabigatran etex-
ilate is a novel, potent, nonpeptidic direct thrombin inhibi-
tor [3]. The orally administered double prodrug is hydro-
lyzed in vivo by esterases into the active form dabigatran
[4] (Fig. 1b). Dabigatran reversibly binds to the active site
of both free and clot-bound thrombin, thereby effectively
preventing the formation of fibrin clots [5]. Dabigatran
is approved in over 70 countries for stroke prevention in
patients with atrial fibrillation and for thrombosis preven-
tion after orthopedic hip and knee surgery [6]. However,
this new anticoagulant has several disadvantages, includ-
ing low oral bioavailability and probability of hemorrhage
when used in large dose [7, 8].
Keywords Dabigatran analogs · Thrombin inhibition ·
Molecular docking
To date, fluorinated drugs account for a considerable
proportion of drugs used in clinical treatment [9–13]. With
the introduction of fluorine atoms or fluoride groups, physi-
cal and chemical properties of drug molecules could be
changed [14], which consequently alters the pharmacoki-
netic properties of drug molecules. Changes in the struc-
tural conformation of compounds may enhance their capac-
ity to bind with ligands and target proteins, as well as their
selectivity for target proteins. Fluorination could block
metabolic pathways of drugs in a particular site, change the
rate of their metabolism, prolong their effects in the body,
and improve their metabolic stability [10]. Furthermore, the
introduction of fluorine into drug molecules may enhance
membrane permeability [15] and specific loci formed with
target proteins during hydrophobic interaction [16]. Since
the introduction of 5-fluorouracil, the addition of fluorine
atoms or fluoride groups into drug molecules has gradually
become an important research strategy in drug design and
structural transformation [17].
Introduction
Thromboembolic disease, which occurs in the arterial or
venous circulation, is a major cause of mortality and mor-
bidity in the developed world and is caused by excessive
stimulation of coagulation [1]. The most widely used drugs
for antithrombotic therapy are indirect thrombin inhibitors
* Yu-Jie Ren
clab@sit.edu.cn
1
School of Chemical and Environmental Engineering,
Shanghai Institute of Technology, No. 100, Haiquan Road,
Fengxian District, Shanghai 201400, China
1 3

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J IRAN CHEM SOC (2016) 13:347–357
O
O
O
N
NH2
N
HN
N
N
N
N
O
O
O
(A) Dabigatran etexilate
O
NH2
NH
HN
N
N
HO
N
Dabigatran
(B)
Fig. 1 Structures of dabigatran etexilate and dabigatran
According to the literature, derivatization by an elec-
tron-donating group at the N–H position of benzimidazole
resulted in good biological activity [18, 19]. Therefore, to
combine the benefits of dabigatran etexilate and fluorine
atoms during treatment, fluoride groups were introduced
into dabigatran molecule to modify its structure. This mod-
ification was expected to generate a series of fluoride-mod-
ified dabigatran analogs that could maximize the selected
characteristics of fluorine atoms and improve the shortcom-
ings of dabigatran etexilate. Consequently, more efficient
and safer novel anticlotting drug candidate compounds are
provided.
Fig. 2 Design strategy of fluorinated thrombin inhibitor derivatives
compounds and for the determination of the most important
parameters controlling these properties.
Researches on novel compounds and their develop-
ment have become more efficient with advancements in
computer-aided simulation and design. In this study, we
designed and synthesized a novel series of fluoride dabi-
gatran analogs using dabigatran as the reference molecule,
studied their antithrombin activities, and validated their
activity using molecular modeling approaches. Initially,
docking experiments with designed structures at the active
site (PDB entry code: 1KTS) [3] were performed in silico
to assess their binding (docking scores) with respect to
that of dabigatran. Dabigatran etexilate has a basic ben-
zimidazole ring and a benzamidine group that are inter-
linked using amino linker. According to the literature [20],
3-(pyridyl-2-amino) propionic acid ethyl ester and its ana-
logs account for its bioactivity. Therefore, we replaced the
pyridine ring with a benzene ring and introduced various
fluoride groups at different sites (Fig. 2). The effects of
introducing fluorine atoms on anticoagulant activity were
determined. Throughout this research, both strategies of
preferential selectivity toward thrombin and the addition
of a fluorine group release moiety were combined. Then,
novel analogs of fluorinated dabigatran were synthesized
and evaluated for their antithrombin activity. Docking stud-
ies were performed for validation of the observed phar-
macological properties of the investigated antithrombin
Experimental
General
All chemicals and solvents were purchased from Darui
and Titan Corporation and used without further purifica-
tion. Melting points were measured on WRS-1B and were
uncorrected. NMR spectra were carried out using a Brucker
Avance 500 MHz NMR spectrometer using TMS as inter-
nal reference. Chemical shifts are reported in δ scale (ppm).
Mass spectra of the synthesized compounds were recorded
on a Brucker FT-ICR-MS. Elemental analysis was recorded
on Germany Vario EL III Elementar.
General procedure for the synthesis of ethyl
3‑((fluorinated‑phenyl)‑amino) propanoate (3a–e)
To a solution of commercial fluorinated benzenamine (1a–
e) (0.05 mol) and ethyl acrylate (0.09 mol) without solvent,
trifluoromethanesulfonic acid (0.005 mol) was added. The
resulting mixture was heated to 100 °C for 24 h. The pro-
gress of the reaction was monitored by thin layer chro-
matography using petroleum ether/dichloromethane (2:1)
as the solvent system. The crude mixture was purified by
silica gel column chromatography eluting 5:1 petroleum
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349
ether: dichloromethane to give the intermediate com-
Ethyl 3‑(N‑(3, 5‑difluorophenyl)‑4‑(methylamino)‑3‑nitroben‑
pounds, 3a–e.
zamido)propanoate (8b) Orange–yellow solid; yield:
25 %; m.p.: 140.7–141.6 °C; H NMR (500 MHz, chlo-
1
Synthetic procedure for 4‑(ethylamino)‑3‑nitrobenzoic
acid (6) A 250-mL round-bottom flask was charged with
4-chloro-3-nitrobenzoic acid (20 g, 0.1 mol) and a solution
of ethylamine in water (60 %, 86 mL, 0.8 mol).The mixture
was refluxed at 80 °C for 5 h. The solution was adjusted to
pH 4–5 with acetic acid. The precipitations of yellow solids
were filtered to get products 5 (19.6 g, 100 %).
roform-d) δ 8.21 (d, J = 2.0 Hz, 1H), 7.45 (dd, J = 9.0,
1.9 Hz, 1H), 6.74–6.64 (m, 4H), 4.17 (t, J = 7.1 Hz, 2H),
4.10 (q, J = 7.1 Hz, 2H), 3.01 (d, J = 5.1 Hz, 3H), 2.70
(t, J = 7.1 Hz, 2H), 1.23 (t, J = 7.1 Hz, 3H). FT-MS m/z
Calcd. for C19H19FN3O5 ([M+H] +): 408.13655, Found:
408.13663.
Ethyl 3‑(4‑(methylamino)‑3‑nitro‑N‑(3‑(trifluoromethyl)
Synthetic procedure for 4‑(ethylamino)‑3‑nitrobenzoyl
chloride (7) A 250-mL round-bottom flask was charged
with 4-(ethylamino)-3-nitrobenzoic acid (9.0 g, 0.046 mol).
30 mL of dichloromethane and 5 drops of N,N-dimethylfor-
mamide were added in one portion. The mixture was stirred
at room temperature (30 °C). Thionyl chloride (20 mL,
0.23 mol) was added dropwise via addition funnel. The
reaction solution was stirred at room temperature for 5–6 h.
The progress of the reaction was monitored by thin layer
chromatography using ethyl acetate: petroleum ether (1:1)
as solvent system. The reaction solvent was evaporated in
vacuo to dryness to give the product 6 without further opera-
tion.
phenyl)benzamido)propanoate (8c) Orange–yellow oil;
yield: 70 %; H NMR (500 MHz, chloroform-d) δ 8.17 (d,
1
J = 1.6 Hz, 1H), 7.45–7.39 (m, 1H), 7.29–7.21 (m, 1H),
6.91 (td, J = 6.3, 1.6 Hz, 2H), 6.85 (dt, J = 7.6, 1.6 Hz,
1H), 6.62 (d, J = 7.2 Hz, 1H), 4.18 (t, J = 5.8 Hz, 2H),
4.07 (q, J = 5.7 Hz, 2H), 2.97 (d, J = 4.1 Hz, 3H), 2.69
(t, J = 5.8 Hz, 2H), 1.20 (t, J = 5.7 Hz, 3H). FT-MS m/z
Calcd. for C20H20F3N3O5 ([M+H] +): 440.14278, Found:
440.14178.
Ethyl 3‑(4‑(methylamino)‑3‑nitro‑N‑(4‑(trifluoromethyl)
phenyl)benzamido)propanoate (8d) Orange–yellow oil;
yield: 45 %; 1H NMR (500 MHz, chloroform-d) δ 8.21 (d,
J = 1.9 Hz, 1H), 7.57 (d, J = 8.3 Hz, 2H), 7.41 (dd, J = 9.0,
1.7 Hz, 1H), 7.29–7.25 (m, 2H), 6.65 (d, J = 9.0 Hz,
1H), 4.23 (t, J = 7.1 Hz, 2H), 4.09 (q, J = 7.1 Hz, 2H),
3.01 (d, J = 5.1 Hz, 3H), 2.73 (t, J = 7.1 Hz, 2H), 1.22
(t, J = 7.1 Hz, 3H). FT-MS m/z Calcd. for C20H20F3N3O5
([M+H] +): 440.14278, Found: 440.14373.
General procedure for the synthesis of ethyl
3‑(N‑(flourinated‑phenyl)‑4‑(ethylamino)‑3‑
nitrobenzamido) propanoate (8a–e)
To a vigorously stirred mixture of 3 (0.03 mol) and triethyl-
amine (0.03 mol) in anhydrous dichloromethane (50 mL) at
room temperature, 4-(ethylamine)-3-nitrobenzoyl-chloride
(0.03 mol) in anhydrous dichloromethane was added drop-
wise for 30 min. The reaction mixture was further stirred
for 5 h at room temperature. The progress of the reaction
was monitored by thin layer chromatography using ethyl
acetate: petroleum ether (1:1) as the solvent system. The
reaction mixture was washed with aqueous sodium chloride
solution three times and dried over anhydrous sodium sul-
fate. The crude product was purified by silica gel column
chromatography eluting 2:1 petroleum ether: ethyl acetate
to give the products 8a–e.
Ethyl 3‑(4‑(methylamino)‑3‑nitro‑N‑(4‑(trifluoromethoxy)
phenyl)benzamido)propanoate (8e) Orange–yellow oil;
yield: 77 %; 1H NMR (500 MHz, chloroform-d) δ 8.15 (d,
J = 2.0 Hz, 1H), 7.41 (dd, J = 9.0, 1.9 Hz, 1H), 7.16 (s,
4H), 6.63 (d, J = 9.0 Hz, 1H), 4.19 (t, J = 7.1 Hz, 2H),
4.06 (q, J = 7.1 Hz, 2H), 2.99 (d, J = 5.1 Hz, 3H), 2.71
(t, J = 7.1 Hz, 2H), 1.21 (t, J = 7.1 Hz, 3H). FT-MS m/z
Calcd. for C20H20F3N3O6 ([M+H] +): 456.13770, Found:
456.14187.
General procedure for the synthesis of ethyl 3‑(3‑amin
o‑N‑(fluorinated‑phenyl)‑4‑(methylamino) benzamido)
propanoate (9a–e)
Ethyl 3‑(N‑(3‑fluorophenyl)‑4‑(methylamino)‑3‑nitroben‑
zamido)propanoate (8a) Red–orange solid; yield: 68 %;
1
m.p.: 79.5–80.4 °C; H NMR (500 MHz, chloroform-d) δ
A round bottom flask was charged with a solution of com-
pound 8 (20 mmol) in tetrahydrofuran/water (40/80 mL).
Zn powder (0.1 mol) and NH4Cl (10 mmol) were added in
turn. The reaction mixture was refluxed at 80 °C overnight
under the atmosphere of nitrogen. The reaction process was
monitored by thin layer chromatography using ethyl ace-
tate: petroleum ether (1:1) as the solvent system. The mix-
ture was filtered through a pad to remove the Zn powder.
8.17 (d, J = 2.0 Hz, 1H), 7.45–7.38 (m, 1H), 7.30–7.21 (m,
1H), 6.91 (td, J = 7.9, 2.0 Hz, 2H), 6.85 (dt, J = 9.5, 2.0 Hz,
1H), 6.62 (d, J = 9.0 Hz, 1H), 4.18 (t, J = 7.2 Hz, 2H),
4.07 (q, J = 7.1 Hz, 2H), 2.97 (d, J = 5.1 Hz, 3H), 2.69
(t, J = 7.2 Hz, 2H), 1.20 (t, J = 7.1 Hz, 3H). FT-MS m/z
Calcd. for C19H20FN3O5 ([M+H] +): 390.14598, Found:
390.14826.
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J IRAN CHEM SOC (2016) 13:347–357
The residue solution was extracted with dichloromethane
three times. The combined organic layer was washed with
an aqueous solution of sodium chloride solution three times
and dried over anhydrous sodium sulfate. The crude prod-
uct was purified by silica gel chromatography eluting 1:1
ethyl acetate: petroleum ether to give product 9.
J = 7.1 Hz, 2H), 2.65 (s, 3H), 2.56 (t, J = 7.0 Hz, 2H), 1.11
(t, J = 7.1 Hz, 3H). FT-MS m/z Calcd. for C20H22F3N3O4
([M+H] +): 426.16352, Found: 426.16751.
General procedure for the synthesis of compounds 10a–e
A round bottom flask was charged with a solution of
2-((4-cyanophenyl) amino) acetic acid (12 mmol), EDCI
(12 mmol) and HoBt (12 mmol) in tetrahydrofuran/N,N-
dimethylformamide (70/10 mL). The reaction mixture was
stirred at ice bath for 35 min. Then a solution of compound
9 (10 mmol) in tetrahydrofuran (50 mL) was added drop-
wise and kept under stirring at room temperature. The reac-
tion solution was kept stirred for overnight. The reaction
solution was evaporated in vacuo to dryness. The residual
was dissolved in dichloromethane (100 mL), washed with
water three times, and dried over anhydrous sodium sul-
fate. The organic layer was evaporated in vacuo to dryness.
Then the residual was dissolved in acetic acid (70 mL) and
refluxed at 120 °C for 2–3 h. The reaction solution was
adjusted to pH 9–10 with ammonium hydroxide and stirred
for 30 min. The reaction solution was extracted with dichlo-
romethane three times, washed with water three times, and
dried over anhydrous sodium sulfate. The reaction process
was monitored by thin layer chromatography using dichlo-
romethane: methyl alcohol (20:1) as the solvent system. The
crude product was purified by silica gel chromatography
eluting 2:1 ethyl acetate: petroleum ether to give product 10.
Ethyl 3‑(3‑amino‑N‑(3‑fluorophenyl)‑4‑(methylamino)ben‑
zamido)propanoate (9a) Brown oil; yield: 90 %; 1H NMR
(500 MHz, chloroform-d) δ 7.19 (q, J = 7.5 Hz, 1H), 6.85 (d,
J = 10.5 Hz, 4H), 6.77–6.73 (m, 1H), 6.32 (d, J = 8.3 Hz,
1H), 4.17 (t, J = 7.3 Hz, 2H), 4.08 (q, J = 7.1 Hz, 2H),
3.35 (s, 3H), 2.80 (s, 3H), 2.71 (t, J = 7.3 Hz, 2H), 1.22
(t, J = 7.1 Hz, 3H). FT-MS m/z Calcd. for C19H22FN3O3
([M+H] +): 360.17180, Found: 360.17403.
Ethyl 3‑(3‑amino‑N‑(3,5‑difluorophenyl)‑4‑(methylamino)
benzamido)propanoate (9b) Red–brown oil; yield: 89 %;
1H NMR (500 MHz, DMSO-d6) δ 7.02 (t, J = 9.2 Hz, 1H),
6.94–6.86 (m, 2H), 6.72 (s, 1H), 6.48 (dd, J = 8.2, 1.6 Hz,
1H), 6.16 (d, J = 8.3 Hz, 1H), 4.57 (s, 2H), 4.06 (t, J = 7.0 Hz,
2H), 3.99 (q, J = 7.1 Hz, 2H), 2.68 (d, J = 4.7 Hz, 3H), 2.59
(t, J = 7.0 Hz, 2H), 1.14 (t, J = 7.1 Hz, 3H). FT-MS m/z
Calcd. for C19H21F2N3O3 ([M+H] +): 378.16237, Found:
378.16437.
Ethyl 3‑(3‑amino‑4‑(methylamino)‑N‑(3‑(trifluoromethyl)
phenyl)benzamido)propanoate (9c) Gray–green oil; yield:
93 %; 1H NMR (500 MHz, DMSO-d6) δ 7.49 (dd, J = 15.5,
7.7 Hz, 3H), 7.36 (d, J = 7.3 Hz, 1H), 6.72–6.67 (m, 1H),
6.31 (dd, J = 8.2, 1.8 Hz, 1H), 6.08 (d, J = 8.3 Hz, 1H),
5.02 (s, 1H), 4.52 (s, 2H), 4.07 (t, J = 6.9 Hz, 2H), 3.94 (q,
J = 7.1 Hz, 2H), 2.64 (s, 3H), 2.56 (t, J = 6.9 Hz, 2H), 1.11
(t, J = 7.1 Hz, 3H). FT-MS m/z Calcd. for C20H22F3N3O3
([M+H] +): 410.16860, Found: 410.16733.
Ethyl 3‑(2‑(((4‑cyanophenyl)amino)methyl)‑N‑(3‑fluorophenyl)
‑1‑methyl‑1H‑benzo[d]imidazole‑5‑carboxamido)pro‑
panoate (10a) White–gray solid; yield: 89 %; m.p.:
168.3–168.9 °C; 1H NMR (500 MHz, DMSO-d6) δ 7.51
(s, 1H), 7.47 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 8.4 Hz,
1H), 7.31–7.26 (m, 1H), 7.24–7.21 (m, 1H), 7.17 (d,
J = 10.2 Hz, 1H), 6.98 (dt, J = 11.2, 5.1 Hz, 2H), 6.83 (d,
J = 8.8 Hz, 2H), 5.76 (s, 1H), 4.59 (d, J = 5.5 Hz, 2H),
4.11 (t, J = 7.1 Hz, 2H), 3.99 (q, J = 7.1 Hz, 2H), 3.75 (s,
3H), 2.67–2.58 (m, 2H), 1.14 (t, J = 7.1 Hz, 3H). FT-MS
m/z Calcd. for C28H26FN5O3 ([M+H] +): 500.20924,
Found: 500.21132.
Ethyl 3‑(3‑amino‑4‑(methylamino)‑N‑(4‑(trifluoromethyl)
phenyl)benzamido)propanoate (9d) Gray–green solid;
1
yield: 83 %; m.p.: 91.3–92.0 °C; H NMR (500 MHz,
DMSO-d6) δ 7.63 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.3 Hz,
2H), 6.69 (d, J = 1.8 Hz, 1H), 6.36 (dd, J = 8.2, 1.7 Hz,
1H), 6.09 (d, J = 8.3 Hz, 1H), 5.05 (s, 1H), 4.53 (s, 2H),
4.07 (t, J = 7.0 Hz, 2H), 3.95 (q, J = 7.1 Hz, 2H), 2.65 (d,
J = 3.6 Hz, 3H), 2.56 (t, J = 7.0 Hz, 2H), 1.11 (t, J = 7.1 Hz,
3H). FT-MS m/z Calcd. for C20H22F3N3O3 ([M+H] +):
410.16860, Found: 410.16827.
Ethyl 3‑(2‑(((4‑cyanophenyl)amino)methyl)‑N‑(3,5‑difluorop
henyl)‑1‑methyl‑1H‑benzo[d]imidazole‑5‑carboxamido)pro‑
panoate (10b) Light yellow solid; yield: 78 %; m.p.: 171.2–
172.0 °C; 1H NMR (500 MHz, chloroform-d) δ 7.68–7.64 (m,
1H), 7.41 (d, J = 8.7 Hz, 2H), 7.28 (dd, J = 8.4, 1.4 Hz, 1H),
7.11 (d, J = 8.5 Hz, 1H), 6.70 (d, J = 8.7 Hz, 2H), 6.62 (dd,
J = 7.4, 2.0 Hz, 2H), 6.57 (tt, J = 8.6, 2.1 Hz, 1H), 5.67 (s,
1H), 4.48 (d, J = 4.6 Hz, 2H), 4.19 (t, J = 7.1 Hz, 2H), 4.09
(q, J = 7.1 Hz, 2H), 3.69 (s, 3H), 2.71 (t, J = 7.1 Hz, 2H),
1.23–1.19 (m, 3H). FT-MS m/z Calcd. for C28H25F2N5O3
([M+H] +): 518.19982, Found: 518.20475.
Ethyl 3‑(3‑amino‑4‑(methylamino)‑N‑(4‑(trifluoromethoxy)
phenyl)benzamido)propanoate (9e) Red–brown oil; yield:
84 %; 1H NMR (500 MHz, DMSO-d6) δ 7.27 (d, J = 8.8 Hz,
2H), 7.22 (d, J = 9.0 Hz, 2H), 6.69 (d, J = 1.8 Hz, 1H),
6.34 (dd, J = 8.2, 1.8 Hz, 1H), 6.08 (d, J = 8.3 Hz, 1H),
5.02 (s, 1H), 4.51 (s, 2H), 4.03 (t, J = 7.0 Hz, 2H), 3.94 (q,
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J IRAN CHEM SOC (2016) 13:347–357
351
Ethyl 3‑(2‑(((4‑cyanophenyl)amino)methyl)‑1‑methyl‑N‑(3
‑(trifluoromethyl)phenyl)‑1H‑benzo[d]imidazole‑5‑carbox‑
amido)propanoate (10c) Red oil; yield: 63 %; H NMR
Ethyl 3‑(2‑(((4‑carbamimidoylphenyl)amino)methyl)‑N‑(3‑
fluorophenyl)‑1‑methyl‑1H‑benzo[d]imidazole‑5‑car‑
boxamido)propanoate (11a) White solid; yield: 47 %;
1
1
(500 MHz, chloroform-d) δ 7.65 (s, 1H), 7.45–7.39 (m,
3H), 7.36 (d, J = 8.7 Hz, 2H), 7.30 (dd, J = 14.3, 6.6 Hz,
1H), 7.27–7.20 (m, 2H), 7.08 (d, J = 8.2 Hz, 1H), 6.71 (d,
J = 8.7 Hz, 2H), 5.63 (s, 1H), 4.48 (d, J = 4.4 Hz, 2H), 4.26
(t, J = 7.1 Hz, 2H), 4.08 (q, J = 7.1 Hz, 2H), 3.69 (s, 3H),
2.74 (t, J = 7.1 Hz, 2H), 1.22 (t, J = 7.1 Hz, 3H). FT-MS
m/z Calcd. for C29H26F3N5O3 ([M+H] +): 550.20605,
Found: 550.20209.
m.p.: 108.4–109.1 °C; H NMR (500 MHz, DMSO-d6) δ
8.91 (s, 3H), 7.70 (d, J = 8.1 Hz, 2H), 7.51 (s, 1H), 7.40
(d, J = 8.4 Hz, 1H), 7.24 (q, J = 7.9 Hz, 1H), 7.18 (t,
J = 10.1 Hz, 2H), 7.01–6.94 (m, 2H), 6.87 (d, J = 8.7 Hz,
2H), 4.63 (d, J = 5.5 Hz, 2H), 4.10 (t, J = 7.1 Hz, 2H), 3.98
(q, J = 7.1 Hz, 2H), 3.77 (s, 3H), 2.61 (t, J = 7.1 Hz, 2H),
1.31–1.22 (m, 3H). FT-MS m/z Calcd. for C28H29FN6O3
([M+H] +): 517.23579, Found: 517.23672.
Ethyl 3‑(2‑(((4‑cyanophenyl)amino)methyl)‑1‑methyl‑N‑(4‑
(trifluoromethyl)phenyl)‑1H‑benzo[d]imidazole‑5‑carboxa‑
mido)propanoate (10d) Orange oil; yield: 75 %; 1H NMR
(500 MHz, chloroform-d) δ 7.67 (s, 1H), 7.47–7.38 (m, 4H),
7.23 (dt, J = 8.3, 2.5 Hz, 1H), 7.18 (d, J = 8.3 Hz, 2H), 7.07
(dd, J = 8.4, 5.0 Hz, 1H), 6.71 (d, J = 8.7 Hz, 2H), 5.60
(s, 1H), 4.48 (d, J = 4.5 Hz, 2H), 4.26 (t, J = 7.1 Hz, 2H),
4.07 (q, J = 7.1 Hz, 2H), 3.68 (d, J = 4.1 Hz, 3H), 2.73 (t,
J = 7.1 Hz, 2H), 1.22–1.19 (m, 3H). FT-MS m/z Calcd. for
C29H26F3N5O3 ([M+H] +): 550.20605, Found: 550.20365.
Ethyl3‑(2‑(((4‑carbamimidoylphenyl)amino)methyl)‑N‑(3,5
‑difluorophenyl)‑1‑methyl‑1H‑benzo[d]imidazole‑5‑car‑
boxamido)propanoate (11b) White solid; yield: 57 %;
1
m.p.: 103.5–104.6 °C; H NMR (500 MHz, DMSO-d6) δ
8.89 (s, 1H), 8.68 (s, 1H), 7.66 (d, J = 8.7 Hz, 2H), 7.55
(s, 1H), 7.47–7.42 (m, 2H), 7.22 (d, J = 8.4 Hz, 1H), 7.05
(dd, J = 13.2, 4.3 Hz, 2H), 6.87 (d, J = 8.7 Hz, 2H), 4.65
(d, J = 5.4 Hz, 2H), 4.10 (t, J = 7.0 Hz, 2H), 4.00 (q,
J = 7.1 Hz, 2H), 3.78 (s, 3H), 2.64 (t, J = 7.0 Hz, 2H), 1.14
(t, J = 7.1 Hz, 3H). FT-MS m/z Calcd. for C28H28F2N6O3
([M+H] +): 535.22637, Found: 535.22759.
Ethyl 3‑(2‑(((4‑cyanophenyl)amino)methyl)‑1‑methyl‑N‑(
4‑(trifluoromethoxy)phenyl)‑1H‑benzo[d]imidazole‑5‑car‑
boxamido)propanoate(10e) Pinkoil;yield:81%;1HNMR
(500 MHz, chloroform-d) δ 7.65 (s, 1H), 7.41 (d, J = 8.4 Hz,
2H), 7.20 (d, J = 8.3 Hz, 1H), 7.09 (d, J = 8.8 Hz, 2H), 7.04
(t, J = 8.7 Hz, 3H), 6.70 (d, J = 8.5 Hz, 2H), 5.66 (s, 1H),
4.46 (d, J = 4.1 Hz, 2H), 4.22 (t, J = 7.1 Hz, 2H), 4.06 (q,
J = 7.1 Hz, 2H), 3.67 (s, 3H), 2.71 (t, J = 7.0 Hz, 2H), 1.21
(t, J = 7.1 Hz, 3H). FT-MS m/z Calcd. for C29H26F3N5O4
([M+H] +): 550.20605, Found: 566.20678.
Ethyl 3‑(2‑(((4‑carbamimidoylphenyl)amino)methyl)‑1
‑methyl‑N‑(3‑(trifluoromethyl)phenyl)‑1H‑benzo[d]imida‑
zole‑5‑carboxamido)propanoate (11c) White solid; yield:
46 %; m.p.: 169.8–170.4 °C; 1H NMR (500 MHz, DMSO-
d6) δ 8.88 (s, 4H), 7.67 (d, J = 8.5 Hz, 3H), 7.53–7.48 (m,
2H), 7.44 (d, J = 6.1 Hz, 2H), 7.40 (d, J = 8.5 Hz, 1H),
7.17 (d, J = 8.4 Hz, 1H), 6.86 (d, J = 8.7 Hz, 2H), 4.64 (d,
J = 5.2 Hz, 2H), 4.14 (t, J = 6.8 Hz, 2H), 3.76 (s, 3H), 2.63
(t, J = 6.8 Hz, 2H), 1.11 (t, J = 7.1 Hz, 3H). FT-MS m/z
Calcd. for C29H29F3N6O3 ([M+H] +): 567.23260, Found:
567.23802.
General procedure for the synthesis of compound 11a–e
A round-bottom flask was charged with a solution of
compound 10 (5 mmol), hydroxylamine hydrochlo-
ride (10 mmol), and triethylamine (10 mmol) in ethanol
(50 mL). The reaction mixture was refluxed at 80 °C for
4–5 h. Then the reaction mixture was evaporated in vacuo
to dryness to afford the crude intermediate which was used
without further purification in the next step. The crude
intermediate was dissolved in acetic acid (50 mL). Then
Pd/C (5 %, 1.0 g) and ammonium formate (15 mmol) were
added in turn. The reaction mixture was refluxed at 120 °C
for 5–6 h under the protection of nitrogen. The reaction
process was monitored by thin layer chromatography using
dichloromethane: methyl alcohol (10:1) as the solvent sys-
tem. The crude product was purified by silica gel chroma-
tography eluting 7:1 dichloromethane: methyl alcohol to
give product 11.
Ethyl 3‑(2‑(((4‑carbamimidoylphenyl)amino)methyl)‑1‑
methyl‑N‑(4‑(trifluoromethyl)phenyl)‑1H‑benzo[d]imi‑
dazole‑5‑carboxamido)propanoate (11d) White solid;
yield: 41 %; m.p.: 154.1–155.0 °C; 1H NMR (501 MHz,
DMSO-d6) δ 7.61 (dd, J = 8.4, 3.8 Hz, 4H), 7.53 (s, 1H),
7.40 (t, J = 7.7 Hz, 3H), 7.20 (d, J = 8.5 Hz, 1H), 6.84
(d, J = 8.7 Hz, 2H), 4.62 (d, J = 5.4 Hz, 2H), 4.15 (t,
J = 7.0 Hz, 2H), 3.97 (q, J = 7.1 Hz, 2H), 3.76 (s, 3H), 2.64
(t, J = 7.0 Hz, 2H), 1.12 (t, J = 7.1 Hz, 3H). FT-MS m/z
Calcd. for C29H29F3N6O3 ([M+H] +): 567.23260, Found:
567.23573.
Ethyl 3‑(2‑(((4‑carbamimidoylphenyl)amino)methyl)‑
1‑methyl‑N‑(4‑(trifluoromethoxy)phenyl)‑1H‑benzo[d]
imidazole‑5‑carboxamido)propanoate (11e) White solid;
1
yield: 43 %; m.p.: 152.6–153.8 °C; H NMR (500 MHz,
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J IRAN CHEM SOC (2016) 13:347–357
DMSO-d6) δ 8.87 (s, 4H), 7.66 (d, J = 6.2 Hz, 2H), 7.51
(s, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.31 (d, J = 8.8 Hz,
2H), 7.25 (d, J = 8.5 Hz, 2H), 7.18 (d, J = 8.4 Hz, 1H),
6.87 (d, J = 8.6 Hz, 2H), 4.64 (d, J = 5.4 Hz, 2H), 4.11 (t,
J = 6.9 Hz, 2H), 3.96 (q, J = 7.1 Hz, 2H), 3.77 (s, 3H), 2.62
(t, J = 6.9 Hz, 2H), 1.12 (t, J = 7.1 Hz, 3H). FT-MS m/z
Calcd. for C29H29F3N6O4 ([M+H] +): 583.22752, Found:
583.23286.
3‑(2‑(((4‑Carbamimidoylphenyl)amino)methyl)‑1‑m
ethyl‑N‑(3‑(trifluoromethyl)phenyl)‑1H‑benzo[d]imi‑
dazole‑5‑carboxamido)propanoic acid (12c) White
solid; yield: 86 %; m.p.: 235.2–237.7 °C; 1H NMR
(500 MHz, DMSO-d6) δ 9.12 (s, 1H), 8.88 (s, 1H), 7.85
(d, J = 8.5 Hz, 1H), 7.78 (s, 1H), 7.74 (d, J = 8.2 Hz, 3H),
7.52 (t, J = 9.1 Hz, 2H), 7.46 (d, J = 7.4 Hz, 2H), 6.93 (d,
J = 8.8 Hz, 2H), 5.02 (s, 2H), 4.11–4.06 (m, 2H), 3.97 (s,
3H), 2.58 (t, J = 7.0 Hz, 2H). 13C NMR (125 MHz, DMSO-
d6) δ 172.45, 168.57, 164.12 (q, J = 7.7 Hz), 153.54, 152.18,
143.12, 133.66, 133.46, 132.46, 130.35, 130.05, 129.71,
129.38, 125.47, 124.87, 124.72, 123.74, 122.16, 114.58,
114.51, 114.42, 112.24, 55.92, 46.18, 32.03, 31.50. FT-MS
m/z Calcd. for C27H25F3N6O3 ([M+H] +):539.20130,
Found: 539.19987. Anal. calcd for C27H25F3N6O3 (538.19):
C, 60.22; H, 4.68; N, 15.61. Found: C, 60.26; H, 4.63; N,
15.11.
General procedure for the synthesis of compound 12a–e
A round-bottom flask was charged with a solution of com-
pound 11 (0.2 mmol), sodium hydroxide (0.6 mmol) in
6 mL of water and 3 mL of ethanol. The reaction mixture
was stirred at ambient temperature for 2 h. The mixture was
neutralized with acetic acid. The precipitate was isolated and
washed with water and ether to afford the zwitterionic title
compound 12.
3‑(2‑(((4‑Carbamimidoylphenyl)amino)methyl)‑1‑methyl‑N
‑(4‑(trifluoromethyl)phenyl)‑1H‑benzo[d]imidazole‑5‑car‑
boxamido)propanoic acid (12d) White solid; yield: 81 %;
3‑(2‑(((4‑Carbamimidoylphenyl)amino)methyl)‑
N‑(3‑fluorophenyl)‑1‑methyl‑1H‑benzo[d]imidazole‑5‑car‑
boxamido)propanoic acid (12a) White solid; yield: 99 %;
1
m.p.: 254.6–256.6 °C; H NMR (500 MHz, DMSO-d6) δ
1
m.p.: 243.4–246.3 °C; H NMR (500 MHz, DMSO-d6) δ
9.13 (s, 1H), 8.86 (s, 1H), 7.84 (d, J = 8.7 Hz, 1H), 7.75–
7.72 (m, 3H), 7.62 (d, J = 8.5 Hz, 2H), 7.45 (dd, J = 13.8,
8.6 Hz, 3H), 6.92 (d, J = 8.9 Hz, 2H), 5.02 (s, 2H), 4.09
(t, J = 7.2 Hz, 2H), 3.97 (s, 3H), 2.56 (t, J = 7.3 Hz,
2H). 13C NMR (125 MHz, DMSO-d6) δ 172.36, 168.36,
164.13, 164.05, 163.98, 153.59, 152.10, 146.12, 133.56,
133.51, 129.69, 128.89, 128.62, 127.13, 126.81, 126.34,
125.80, 125.10, 122.40, 112.36, 112.27, 55.91, 46.21,
32.01, 31.59, 18.30. FT-MS m/z Calcd. for C27H25F3N6O3
([M+H] +):539.20130, Found: 539.19900. Anal. calcd for
C27H25F3N6O3 (538.19): C, 60.22; H, 4.68; N,15.61. Found:
C, 60.27; H, 4.65; N, 15.49.
9.12 (d, J = 5.3 Hz, 1H), 8.89 (d, J = 4.0 Hz, 1H), 7.85 (d,
J = 8.7 Hz, 1H), 7.74 (d, J = 8.8 Hz, 2H), 7.70 (s, 1H), 7.48
(d, J = 8.7 Hz, 1H), 7.29–7.24 (m, 2H), 7.06–7.00 (m, 2H),
6.93 (d, J = 8.9 Hz, 2H), 5.01 (s, 2H), 4.06 (t, J = 7.3 Hz,
2H), 3.97 (s, 3H), 2.57 (t, J = 7.4 Hz, 2H). 13C NMR
(125 MHz, DMSO-d6) δ 172.47, 169.07, 164.29–163.97
(m), 163.09, 160.66, 153.41, 152.55, 144.63, 144.53, 130.67,
130.58, 129.67, 124.40, 115.21, 114.98, 113.88, 113.67,
111.97, 110.90, 46.14, 32.07, 30.77. FT-MS m/z Calcd. for
C26H25FN6O3 ([M+H] +): 489.20449, Found: 489.20405.
Anal. calcd for C26H25FN6O3 (488.20): C, 63.92; H, 5.16;
N,17.20. Found: C, 63.75; H, 5.07; N, 16.93.
3‑(2‑(((4‑Carbamimidoylphenyl)amino)methyl)‑1‑meth
yl‑N‑(4‑(trifluoromethoxy)phenyl)‑1H‑benzo[d]imida‑
zole‑5‑carboxamido)propanoic acid (12e) White solid;
3‑(2‑(((4‑Carbamimidoylphenyl)amino)methyl)‑N‑(3,5‑di
fluorophenyl)‑1‑methyl‑1H‑benzo[d]imidazole‑5‑carbox‑
amido)propanoic acid (12b) White solid; yield: 57 %;
1
yield: 98 %; m.p.: 215.3–216.9 °C; H NMR (500 MHz,
1
m.p.: 229.1–232.6 °C; H NMR (500 MHz, DMSO-d6) δ
DMSO-d6) δ 9.12 (s, 1H), 8.85 (s, 0H), 7.83 (d,
J = 8.4 Hz, 1H), 7.74–7.70 (m, 3H), 7.43 (d, J = 7.5 Hz,
1H), 7.38 (d, J = 8.5 Hz, 2H), 7.24 (d, J = 8.1 Hz, 2H),
6.91 (d, J = 8.8 Hz, 2H), 5.02 (s, 2H), 4.03 (t, J = 6.0 Hz,
2H), 3.97 (s, 3H), 2.54 (t, J = 7.0 Hz, 2H). 13C NMR
(125 MHz, DMSO-d6) δ 172.37, 168.28, 164.13, 164.06,
163.98, 153.51, 152.12, 146.53, 141.43, 133.76, 133.37,
129.98, 129.69, 128.88, 125.71, 123.62, 121.71, 121.07,
118.52, 114.54, 112.24, 46.23, 31.97, 31.57. FT-MS m/z
Calcd. for C27H25F3N6O4 ([M+H] +):555.19621, Found:
555.19270. Anal. calcd for C27H25F3N6O4 (554.19): C,
58.48; H, 4.54; N,15.16. Found: C, 58.88; H, 4.57; N,
15.09.
9.13 (s, 1H), 8.89 (s, 1H), 7.88 (d, J = 8.7 Hz, 1H), 7.75
(d, J = 8.6 Hz, 3H), 7.51 (d, J = 8.6 Hz, 1H), 7.19–7.06
(m, 3H), 6.94 (d, J = 8.8 Hz, 2H), 5.02 (s, 2H), 4.06 (t,
J = 7.2 Hz, 2H), 3.98 (s, 3H), 2.60 (t, J = 7.3 Hz, 2H).
13C NMR (125 MHz, DMSO-d6) δ 172.44, 168.28,
164.14 (q, J = 7.7 Hz), 163.30, 163.15, 160.85, 160.70,
153.64, 152.21, 145.12, 133.62, 133.42, 129.72, 129.20,
125.57, 114.54, 114.47, 112.18, 111.91, 102.88, 46.03,
32.00, 31.52, 20.99. FT-MS m/z Calcd. for C26H24F2N6O3
([M+H] +): 507.19507, Found: 507.19301. Anal. calcd for
C26H24F2N6O3 (506.19): C, 61.65; H, 4.78; N,16.59. Found:
C, 61.60; H, 4.86; N, 16.63.
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353
Thrombin assay
activity (IC50) was calculated. All measurements were per-
formed by six experiments; the mean values of both deter-
minations are presented.
Different dilutions of the test compounds dissolved in
DMSO were preincubated for 10 min at 37 °C. The national
standard, lyophilized human thrombin (5.4 μg/mL), which
was purified from human blood was purchased. After that,
Ac-FVR-AMC (5 μM), a specific fluorogenic thrombin
substrate, was added to the above system. We detected the
dynamic changes in relative fluorescence intensity using
an Envision microplate reader (PerkinElmer) at room tem-
perature within 10 min. The slope of the linear enzyme
dynamics curve during the initial stage of the reaction was
referred to as the initial velocity of enzyme reaction. The
known thrombin inhibitor dabigatran was used as a posi-
tive control. Instrument settings included excitation wave-
length, 355 nm; and emission wavelength, 460 nm. Each
well was measured 20 times every 20 s for about 10 min.
The change in fluorescence within a predetermined time
was measured under these conditions. The reaction kinetic
curve slope (Vmax) was as an activity indicator, and the
concentration that induced a 50 % inhibition of thrombin
Results and discussion
Chemistry
Given the high pharmacological activity profile of thrombin
inhibitory drugs and fluorinated compounds, we designed
and synthesized fluorinated thrombin inhibitor derivatives
through the synthesis process depicted in Scheme 1. The
main intermediate ethyl 3-((fluorinated-phenyl)-amino)
propanoate (3) was synthesized from commercially avail-
able fluorinated anilines (1) in excess ethyl acrylate with
the catalyst trifluoromethanesulfonic acid at 100 °C.
Compound 4-(substituted-amino)-3-nitrobenzoic acid (6)
was synthesized in good yield by the reaction between
4-chloro-3-nitrobenzoic acid and substituted amino deriva-
tives in water without a solvent [21]. The intermediate
Scheme 1 Reagents, conditions, and range of yields: (a) trifluo-
romethanesulfonic acid, yields: 38–98 %. (b) Reflux. (c) Thio-
nyl chloride, N,N-dimethylformamide, dichloromethane, yields:
100 %. (d) Triethylamine, dichloromethane, yields: 24–79 %. (e)
Zn powder, ammonium chloride, tetrahydrofuran/water, yields:
59–98 %. (f) (I) 2-((4-cyanophenyl) amino) acetic acid, EDCI, HoBt,
tetrahydrofuran/N,N-dimethylformamide. (II) Acetic acid, ammonium
hydroxide, yields: 46–89 %. (g) (I) hydroxylamine hydrochloride, tri-
ethylamine, ethanol. (II) Pd/C, ammonium formate, acetic acid, nitro-
gen, yields: 54–88 %. (h) Sodium hydroxide, ethanol/water, yields:
42–99 %
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Table 1 The results obtained from docking studies and in vitro evaluations of compounds 12a–e on inhibitory activities of Thrombin
Compound no.
Rf
Total-score
10.3459
H–Ba
4
aa H–Bb
H–B lengthc
IC50(mean SD)d/(nM/L)
5.41 0.082
12a
3-F
Asp189,Gly219,Thr172,Gly216
2.05,1.92,
2.42,2.79
12b
12c
3,5-F2
3-CF3
10.3356
10.8250
3
3
Asp189,Gly219,
Tyr60A
2.04,1.99,
2.30
10.49 1.24
12.3 0.71
Asp189,Gly219,
Tyr60A
1.98,1.98,
2.70
12d
4-CF3
10.2052
10.3130
11.5436
2
2
2
Asp189,Gly219
Asp189,Gly219
Asp189,Gly219
2.01,1.91
2.02,1.94
1.99,1.96
228.3 14.07
249.7 26.29
2.61 0.84
12e
Dabigatrane
4-OCF3
a
The number of hydrogen bond
b
c
d
e
Amino acids involving in hydrogen bond
The distance between amino acids and compounds 4a–e
Values are expressed as the mean standard error of the mean of six experiments
Reference drug
4-(substituted-amino)-3-nitrobenzoyl chloride (7) was syn-
thesized from compound (6) and thionyl chloride in dichlo-
romethane using N,N-dimethylformamide as a catalyst. The
important intermediate ethyl 3-(N-(fluorinated-phenyl)-4-
(substituted-amino)-3-nitrobenz-amido) propanoate (8) was
synthesized from the intermediate compound (3) and com-
pound (7) in dichloromethane using triethylamine as base.
The main intermediate ethyl 3-(3-amino-N-(fluorinated-
synthesized compounds, 12a was the most potent inhibi-
tor against thrombin (IC50 = 5.41 nM), with activity value
similar to the reference drug dabigatran (IC50 = 2.61 nM).
Table 1 shows the half-maximal inhibitory (IC50) value of
the test compounds. When fluorine group was attached at
the meta and para positions of benzene nucleus, thrombin
inhibitory activities decreased, as observed in 12b, 12c,
12d, and 12e with IC50 values of 10.49, 12.3, 228.3, and
249.7 nM, respectively. When the electron-withdrawing
group (–CF3) was placed at the meta and para positions
of the modified benzene nucleus, inhibitory activities
decreased to 12.3 and 228.3 nM. Compound 12e had a
long-range withdrawing group (–OCF3) at the para position
and remarkably decreased IC50 values of 249.7 nM.
phenyl)-4-(substituted-amino)-benzamido)
propanoate
(9) was obtained using the reductant zinc powder from
compound (8) in tetrahydrofuran with water as solvent.
The main intermediate compound (10) was obtained from
compounds (9) by condensation reactions [3]. However, the
most important intermediate compound (11) was obtained
from intermediate (10) through Pinner reaction [22]. The
target compound (12) was obtained from compound (11)
by hydrolysis. The structures of products 12a–e were deter-
mined from their 1H NMR and 13C NMR spectra, FT-ICR-
MS data, as well as elemental analysis data.
Molecular modeling
To get a better understand of the binding mode of the
inhibitors in the active site of thrombin protein, molecular
docking was performed using the Surflex-Dock module in
SYBYL-X 2.0 (Tripos, Inc., USA). Initially, the perfor-
mance of docking software was tested by redocking experi-
ment. For this purpose, crystal structure of protein with the
cocrystallized ligand was redocked. In Fig. 3a, it can be
recognized that the redocking result and the cocrystallized
structure are basically similar, except the slight rotation of
carboxylic acid moiety at the end of pyridine ring. It can
be inferred that the rational of the program and the dock-
ing results are reliable. The crystal structure of the throm-
bin receptor complex was retrieved from the RCSB Protein
Data Bank (PDB entry code: 1KTS) [3]. Before the dock-
ing process, the original ligand was extracted, and the other
natural ligands and water molecules were removed from the
crystal structure. Subsequently, the protein was prepared by
Biological evaluation based on statistical analysis and P
values
With dabigatran as reference, thrombin inhibitory activi-
ties of synthesized fluorinated dabigatran analogs were
explored using a commercially available chromogenic
assay according to a reported assay [3]. Moreover, all the
mentioned compounds were stable enough in biological
conditions, and no decomposition was observed. The cor-
responding IC50 values for thrombin inhibition are shown
in Table 1. Screening data revealed that all test compounds
inhibit thrombin with IC50 values in the nanomole range.
They showed inhibition toward thrombin, but this inhibi-
tion was weaker than the compared dabigatran. Among the
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355
using the Biopolymer module implemented in Sybyl. The
polar hydrogen atoms were added, and Gasteiger–Huckel
charges were assigned to protein atoms. All the dabigatran
analogs were optimized using Tripos force field [23] and
Gasteiger–Huckel charges [24]; the structural energy
minimization was terminated when using Powell gradi-
ent algorithm with a convergence criterion of 0.005 kcal/
(mol*A) reached [25] and a maximum of 10,000 iterations.
Then, the ligands docked in the corresponding protein’s
binding site by an empirical scoring function in Surflex-
Dock. Surflex-Dock total scores, which were expressed
in –log10(Kd) units to represent binding affinities, were
applied to estimate the ligand–receptor interactions of
newly designed molecules.
The results of docking process including total-score,
hydrogen bond, amino acids involving in hydrogen bond,
and the distance between the amino acid residues and the
compounds 12a–e are outlined in Table 1. The in vitro data
acquired for these compounds demonstrated that thrombin
inhibition would be improved by varying the benzene cores
and their substitution. Dabigatran analogs 12a–e docked
into thrombin by means of total-score fitness function in
Sybyl-X2.0. Among the docking structures, the best dock-
ing solutions for each compound were considered (Figs. 3, 4,
and 5). It is noted that total-score of dabigatran was 11.5436,
which was greater than the other scores (Table 1). The results
also show there is an appropriate relationship between IC50
and the docking score for compounds 12a–e. Moreover, the
investigation of the ligand interaction mode of the docked
analogs clearly demonstrates that the number of hydrogen
bonds and hydrogen bond distances appear to play major
role in thrombin inhibition; especially, all the compounds
formed strong hydrogen bonds with Asp189 and Gly219. As
shown in Figs. 3, 4, and 5, dabigatran and compounds 12a–e
were docked in the binding cavity, and this series of com-
pound shared a similar binding mode with thrombin. The
benzimidazole ring formed π–π stacking interactions with
the conserved Trp60D and by a hydrophobic interaction in
the same P-pocket, which may be crucial to the binding of
dabigatran analogs with thrombin protein. Electrostatic inter-
actions and hydrogen bonds were observed between the ami-
dino and the following important amino acids: Asp189 and
Gly219. Remarkably, we found that different substituents
in benzene ring lead to a different biological activity, due
Fig. 3 a Redocking result of
dabigatran (atom type color)
into the binding site of thrombin
protease (PDB entry: 1KTS)
and the cocrystallized structure
(green) for compound complex.
b The docked structure of dabi-
gatran in the active site pocket
of thrombin (PDB entry: 1KTS)
in stick views. Carbon, oxygen,
nitrogen, and hydrogen atoms
are distinguished by white, red,
blue, and cyan colors, respec-
tively. The hydrogen bonds are
displayed by red dashed lines
Fig. 4 A mode of compound
12a (IC50 = 5.41 nM) binding
in the thrombin protease (PDB
code 1KTS). Fluorine is distin-
guished by green color
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J IRAN CHEM SOC (2016) 13:347–357
Fig. 5 The docked structure of
12b (a), 12c (b), 12d (c) and
12e (d) in the active site pocket
of thrombin (PDB entry: 1KTS)
in solvent surface views
to its steric effects. As can be seen clearly from Fig. 5a–d,
with the increasing volume of the substituents group (3,5-
F2 < 3-CF3 < 4-OCF3), the whole substituted benzene ring
gradually moved out of the activity pocket. The same sub-
stituent (3-CF3) in para positions of benzene nucleus had far
greater influence on activity than in the meta position (12c,
12.3 nM > 12d, 228.3 nM). In the para position, the biologi-
cal activity of compound 12e is lower than the activity of
12d because of the bulky –OCF3 group.
evaluated for their thrombin inhibitory activity in vitro.
Biological results revealed that the target compounds
exhibited antithrombin activity at nanomolar concentra-
tion. Notably, compounds 12a showed comparable throm-
bin inhibitory activity (IC50 = 5.41 nM) in contrast to
dabigatran (IC50 = 2.6 nM). Furthermore, the molecular
docking was carried out, and the results of docking process
including total-score, hydrogen bond, amino acids involv-
ing in hydrogen bond, and the distance between the resi-
dues and the compounds 12a–12e are calculated. Based on
the theoretical and experimental results, the bulky group
substituted in benzene ring would decrease the biological
activity of the anticoagulant, especially in the para position.
The hydrogen bond interaction of the nitrogen of the amid-
ino in compounds in the active site of the thrombin appears
to play major role in thrombin inhibition.
Conclusions
In summary, we developed an easy methodology for the
synthesis of fluorinated dabigatran analogs. As the synthe-
sis routine, compounds (12a–12e) were synthesized and
1 3

J IRAN CHEM SOC (2016) 13:347–357
357
Acknowledgments This work is supported by the Science
and Technology Commission of Shanghai Municipality (Nos.
13142201001, 13DZ1930402, 13DZ 1930403).
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