Synthesis and in Vitro and in Vivo anticoagulant and antiplatelet activities of amidino- and non-amidinobenzamides

Article abstract of DOI:10.3390/molecules21050676

Three amidino- and ten non-amidinobenzamides were synthesized as 3-aminobenzoic acid scaffold-based anticoagulant and antiplatelet compounds. The anticoagulant activities of thirteen synthesized compounds 1-13, and 2b and 3b as prodrugs were preliminary evaluated by screening the prolongation of activated partial thromboplastin time (aPTT) and prothrombin time (PT) in vitro. From the aPTT results obtained, two amidinobenzamides, N-(3′-amidinophenyl)-3-(thiophen-2″-ylcarbonylamino) benzamide (1, 33.2 ± 0.7 s) and N-(4′-amidinophenyl)-3-(thiophen-2″-ylcarbonylamino) benzamide (2, 43.5 ± 0.6 s) were selected to investigate the further anticoagulant and antiplatelet activities. The aPTT results of 1 (33.2± 0.7 s) and 2 (43.5 ± 0.6 s) were compared with heparin (62.5 ± 0.8 s) in vitro at 30 μM. We investigated the effect of 1 and 2 on blood anticoagulant activity (ex vivo) and on tail bleeding time (in vivo) on mice. A tail cutting/bleeding time assay revealed that both 1 and 2 prolonged bleeding time in mice at a dose of 24.1 g/mouse and above. Compounds 1 and 2 dose-dependently inhibited thrombin-catalyzed fibrin polymerization and platelet aggregation. In addition, 1 and 2 were evaluated on the inhibitory activities of thrombin and FXa as well as the generation of thrombin and FXa in human umbilical vein endothelial cells (HUVECs). Collectively, 1 and 2 possess some antiplatelet and anticoagulant activities and offer a basis for development of a novel antithrombotic product.

Full text of DOI:10.3390/molecules21050676

molecules
Article
Synthesis and in Vitro and in Vivo Anticoagulant and
Antiplatelet Activities of Amidino- and
Non-Amidinobenzamides
Soo Hyun Lee 1,†, Wonhwa Lee 2,3,†, Jong-Sup Bae 2,* and Eunsook Ma 1,
*
1
College of Pharmacy, Catholic University of Daegu, Hayang-ro 13-13, Hayang-eup, Gyeongsan-si,
Gyeongbuk 712-702, Korea; soohyun0320@cu.ac.kr
College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, Kyungpook National University,
80 Daehak-ro, Buk-gu, Daegu 702-701, Korea; ByWonhwalee@gmail.com
Department of Biochemistry and Cell Biology, BK21 Plus KNU Biomedical Convergence Program,
School of Medicine, Kyungpook National University, Daegu 700-422, Korea
Correspondence: baejs@knu.ac.kr (J.-S.B.); masook@cu.ac.kr (E.M.); Tel.: +82-53-950-8570 (J.-S.B.);
+82-53-850-3621 (E.M.); Fax: +82-53-950-8557 (J.-S.B.); +82-53-850-3602 (E.M.)
These authors contributed equally to this work.
2
3
*
†
Academic Editor: Derek J. McPhee
Received: 1 March 2016; Accepted: 9 May 2016; Published: 21 May 2016
Abstract: Three amidino- and ten non-amidinobenzamides were synthesized as 3-aminobenzoic
acid scaffold-based anticoagulant and antiplatelet compounds. The anticoagulant activities
of thirteen synthesized compounds
1–13, and 2b and 3b as prodrugs were preliminary
evaluated by screening the prolongation of activated partial thromboplastin time (aPTT) and
prothrombin time (PT) in vitro
.
From the aPTT results obtained, two amidinobenzamides,
N-(311-amidinophenyl)-3-(thiophen-211-ylcarbonylamino) benzamide (
1
,
33.2
0.6 s) were selected to
(33.2 0.7 s)
0.8 s) in vitro at 30 µM. We investigated the
˘
0.7 s) and
11
N-(4 -amidinophenyl)-3-(thiophen-2 -ylcarbonylamino) benzamide (
investigate the further anticoagulant and antiplatelet activities. The aPTT results of
2
, 43.5
˘
1
˘
and
effect of
2
(43.5
˘
0.6 s) were compared with heparin (62.5
˘
1
and
2
on blood anticoagulant activity (ex vivo) and on tail bleeding time (in vivo) on mice. A
tail cutting/bleeding time assay revealed that both
of 24.1 g/mouse and above. Compounds and
fibrin polymerization and platelet aggregation. In addition,
activities of thrombin and FXa as well as the generation of thrombin and FXa in human umbilical
vein endothelial cells (HUVECs). Collectively, and possess some antiplatelet and anticoagulant
1
and
2 prolonged bleeding time in mice at a dose
1
2
dose-dependently inhibited thrombin-catalyzed
and were evaluated on the inhibitory
1
2
1
2
activities and offer a basis for development of a novel antithrombotic product.
Keywords: antiplatelet; anticoagulant; aPTT; amidinobenzamide; nonamidinobenzamide
1. Introduction
Thromboembolic diseases are leading causes of cardiovascular-associated morbidity and
death [
(e.g., atherosclerotic vascular disease, acute coronary syndrome) are being achieved using antiplatelet
drugs [ ], whereas venous thrombosis illness, such as deep vein thrombosis (DVT) and pulmonary
1]. Prevention and treatment of arterial thrombosis in patients with cardiovascular diseases
2
embolism (PE), are treated with anticoagulant drugs [3,4].
Several anticoagulants, such as heparins (unfractionated and low-molecular-weight heparins)
and vitamin K antagonists (e.g., warfarin), have proved to be effective in the prevention and
treatment of these thrombotic diseases, but considerable shortcomings, such as inconvenient drug
administration, unneglectable side effects for heparins, and extensive drug and food interactions
Molecules 2016, 21, 676; doi:10.3390/molecules21050676
www.mdpi.com/journal/molecules

Molecules 2016, 21, 676
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for Vitamin K antagonists, restrict their clinical use [
5
]. However, fondaparinux sodium (Arixtra®),
heparin pentasaccharide, is the first of a new class of synthetic indirect antithrombotic agents, distinct
from low molecular weight heparin and heparin. Its pharmacokinetic properties allow for a simple,
fixed-dose, once-daily regimen of subcutaneous injection, without the need for monitoring [6,7]. Blood
clotting in response to vascular injury requires the activation of zymogens in the coagulation cascade,
in which thrombin and FXa are key players. Direct FXa inhibitors such as rivaroxaban, apixaban, and
edoxaban function at the convergence of the extrinsic and intrinsic coagulation pathways without
the need for routine coagulation monitoring [8,9]. Thrombin inhibitors, such as dabigatran etexilate
(dabigatran) and argatroban, play an important role in the anticoagulation process [10,11].
In addition, aspirin, which irreversibly inhibits cyclooxygenase 1-mediated transformation of
arachidonic acid to thromboxane A2 (TXA2), and the P2Y12 antagonists, clopidogrel and prasugrel,
which selectively and irreversibly bind to the P2Y12 ADP receptor, are routinely used as antiplatelet
agents [12,13]. However, there are still some serious limitations to these agents, which include weak
inhibition of platelet function and bleeding events of aspirin [14
bleeding events of clopidogrel [16,17].
,15] and slow onset of action and
Furthermore, a combination of drugs causing antithrombosis with different antiplatelet
mechanisms was reported to improve clinical efficiency and safety. For instance, aspirin and
clopidogrel [18], aspirin plus dipyridamole [19], or aspirin plus glycoprotein IIb/IIIa-receptor
inhibitors [20], are successfully used in stroke prevention. Moreover, enhanced antiplatelet activity has
been described for the combined administration of TXA2 synthase inhibitor (Dazoxiben) plus TXA2
receptor antagonist (Sulotroban) in experimental assays [21].
In addition, the combined use of anticoagulant and antiplatelet agents showed additional benefits
over anticoagulants alone in patients with prosthetic heart valves [22] and in patients with atrial
fibrillation [23]. A number of clinical data have shown that the combination of antiplatelet agents
(e.g., aspirin or clopidogrel) and anticoagulant agents (e.g., warfarin or heparin) has an additive effect
by suppressing both blood coagulation and platelet aggregation, as well as combinations of aspirin plus
warfarin [24], aspirin plus heparin [25], warfarin plus clopidogrel [26], direct thrombin inhibitor plus
GpIIb/IIIa antagonists [27], ADP inhibitor plus FXa inhibitors [28], and warfarin plus amiodarone [29].
However, because such combinations may also increase the risk of bleeding, these will require careful
monitoring in large clinical trials of patients, with particular attention to the elderly [20].
Even though, compounds combining anticoagulant and platelet antiaggregating activities in
the same molecule are believed to be promising drugs [28]. Herein, we described the synthesis of
amidino- and non-amidinobenzamides as 3-aminobenzoic acid scaffold-based anticoagulant and
antiplatelet compounds. The antiplatelet effects on thrombin, U46619, and thrombin-catalyzed fibrin
polymerization, as well as the regulation of clotting time (aPTT and PT), were determined. Additionally,
the anticoagulant activities of synthesized compounds on direct thrombin and FXa inhibition, the
generation of thrombin, and FXa were evaluated.
2. Results and Discussion
2.1. Chemistry
Using diversely substituted 3-aminobenzoic acid as the central scaffold, a series of FXa inhibitors
was patented [30]. The FXa inhibitors containing a carboxyl group at the central phenyl ring
were reported. Considering the potential of 3-aminobenzoic acid as an anticoagulant agent,
we tried to synthesize 1,3-diamide derivatives containing the amidino- and nonamidino group
from 3-aminobenzoic acid. Scheme 1 illustrates the synthesis of amidinobenzamides 1–3.

Molecules 2016, 21, 676
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O
O
O
R
Et3N, Benzene
RT, 3 h
(COCl)2
OH
Cl
R
N
+
H2N
H
Benzene
reflux, 1h
NO2
NO2
NO2
1i, 2i, 3i (81%, 80%, 80%)
R = 3-CN, 4-CN, 4-CH2CN
O
S
1(i,ii, a)
: R = 3-CN
2(i,ii, a) : R = 4-CN
3(i,ii, a)
OH
: R = 4-CH2CN
(COCl)2
Dichloromethane
Reflux, 1h
O
S
O
O
R
Fe/NH4Cl
Methanol
R
Cl
N
NH2OH.HCl
N
H
H
Et3N,Benzene
EtOH,Et3N
Reflux, 2 h
Reflux, 7h~
S
HN
Reflux, 1 h
NH2
O
1a, 2a, 3a (64%, 71%, 61%)
1ii, 2ii, 3ii (60%, 71%, 68%)
O
O
O
R'
R''
R'''
10% Pd-C
c-HCl
N
N
H
N
H
CH3COCl, Et3N
H
S
S
60psi H2
Dichloromethane
RT, 1-2 h
S
HN
HN
HN
45 C, 2 h
O
O
O
NH
NOH
NH2
NOAc
NH2
HCl
-3
-4
(46%)
1b
1c
-3
-4
1
: R'' =
: R' = 3-
: R' = 4-
(87%)
(87%)
: R'' =
NH2
NH
NOAc
NH2
NOH
NH2
(37%)
HCl
NH
HCl
NH2
2b
2c
: R'' =
2
: R'' =
NH2
NOAc
NOH
(90%)
NH2
(51%)
3b: R' = 4- CH2
-4-CH2
3: R'' =
-4-CH2
3c: R'' =
NH2
Scheme 1. Synthetic methods of amidinobenzamides 1–3.
The first side chain was introduced through acylation of 3-nitrobenzoyl chloride with 3-cyano-,
4-cyano-, and 4-cyanomethylaniline to give 1i 3i. The nitro group of these 3-nitrobenzamides
was reduced by NH4Cl and Fe to give amines 1ii 3ii. An acylation with thiophene-2-carbonyl
chloride provided the N-(thiophene-2-carbonyl) 3- or 4-cyanophenylbenzamides 1a and 2a, and the
N-(thiophene-2-carbonyl) 4-cyanobenzylbenzamid 3a, which were converted to amidoximes 1b 3b
3b was
3c and catalytic hydrogenated
–
–
–
by treatment with hydroxylamine
converted to amidinium chloride
¨
HCl and triet-hylamine. The N–O bond of amidoximes 1b
via the O-acetyl amidoximes 1c
–
1–
3
–
over 10% Pd/C in the presence of c-HCl at 60 psi, 45 C for 2 h. In the 1H-NMR spectra, the four proton
˝
peaks of amidinium chloride in
the four proton peaks of amidinium chloride in
singlets at 9.17 and 9.36 ppm and at 8.83 and 9.28 ppm in the 1H-NMR spectra. Two NH proton peaks
of compound were confirmed by observing the correlation of NH (10.78 ppm) and CO (164.9 ppm)
1
were identified as one broad singlet at 9.45 ppm, and otherwise
2
and were identified, respectively, as two broad
3
2
of the –NHCO group and of NH (10.63 ppm) and CO (166.1 ppm) of the –CONH group in the HMBC
spectrum, respectively.
Instead of a basic amidine group, neutral substituents which improve oral bioavailability
and are still capable of maintaining hydrogen bonding with the enzyme are required for
FXa and thrombin inhibitors. Anilines containing 4-chloro-, 4-bromo-, 4-fluoro-, 4-methoxy-,

Molecules 2016, 21, 676
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and 4-morpholine as a neutral substituent were coupled with 3-nitrobenzoyl chloride to synthesize
the corresponding 3-nitrobenzamide derivatives 4i 8i, respectively, in Scheme 2. The reduction of
the nitro group of 4i 8i with NH4Cl and Fe produced the 3-aminobenzamides 4ii 8ii. The second
acylation of 4ii 8ii was performed by treatment with thiophene-2-carbonyl chloride to give
also 5-chloro-thiophene-2-carbonyl chloride to give 9–13, respectively.
–
–
–
–
4
–8
and
O
O
Fe/NH4Cl
Et3N
NH
R1-R5
Cl
H2N
R1-R5
+
Methanol,
Reflux, 7 h-
Benzene
R1 = 4-Cl, R4 = 4-OMe
R2 = 4-Br, R5 = 4-morpholine
R3 = 4-F
NO2
NO2
4i:
7i:
R4 = 4-OMe (77%)
R1 = 4-Cl (82%),
5i:
8i:
R2 = 4-Br (79%),
R5 = 4-morpholine (75%)
6i: R3 = 4-F (78%)
O
NH
R1-R5
S
COCl
S
NH
O
Et3N, Benzene
2 h, RT
O
4 9
: R1 = 4-Cl (77%,77%)
4
8
,
-
NH
R1-R5
5 10
,
: R2 = 4-Br (65%, 65%)
6, 11: R3 = 4-F (66%, 75%)
7 12
8, 13: R5 = 4-morpholine
,
: R4 = 4-OMe (67%, 88%)
Et3N, Benzene
2 h, RT
NH2
O
O
(15%, 12%)
4ii:
5ii:
R1 = 4-Cl (97%)
R2 = 4-Br (97%)
Cl
NH
R1-R5
S
6ii: R3 = 4-F (76%)
7ii:
8ii: R5 = 4-morpholine (16%)
COCl
R4 = 4-OMe (86%)
S
NH
(COCl)2
Dichloromethane
Reflux, 1 h
Cl
9
13
-
Cl
S
COOH
Scheme 2. Synthetic methods of non-amidinobenzamides 4–13.
2.2. Biology
2.2.1. Effects of Synthesized Compounds on Aptt, PT, and Tail Bleeding Time
The anticoagulant effects of fifteen synthesized compounds (2b 3b, and
aPTT and PT assays using human plasma at a concentration of 30
in Table 1. From these aPTT results, N-(31-amidinophenyl)-3-(thiophen-211-ylcarbonylamino)
benzamide 1 and N-(41-amidinophenyl)-3-(thiophen-211-ylcarbonylmino) benzamide
were selected
as target for the further anticoagulant and antiplatelet experiments. Both aPTT and PT were not
prolonged by amidoximes 2b and 3b as prodrugs of and and non-amidinobenzamides 13 at
concentrations 30 g/mL. Most of the amidine-type compounds were found to be insufficiently
,
1–13) were screened in
µg/mL and are summarized
2
1
2
4–
µ
absorbed when administered orally, because of strongly basic amidine groups [11]. Therefore, the
trend in synthetic studies seems to be shifted to non-amidine-type compounds containing weak basic
groups. However, our non-amidine compounds did not show prolongation in aPTT and PT. The
amidine group seems to be needed for the anticoagulant activity.

Molecules 2016, 21, 676
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Table 1. Anticoagulant activities of 1b–3b, and 1–13 at 30 µg/mL.
Sample No.
PT (s)
aPTT (s)
DMSO (1)
12.2
-
12.0
38.7
-
36.5
1b
2b
3b
1
11.9
14.9
32.9
46.9
2
3
15.4
13.5
51.9
41.4
4
5
13.8
12.8
40.2
41.7
6
7
12.0
11.8
46.0
41.0
8
9
12.5
12.8
43.0
44.0
10
11
12
13.0
11.4
12.7
45.1
48.2
48.9
13
12.2
34 (10 mg/mL)
61.3
85 (0.5 mg/mL)
Heparin (2)
(1)
(2)
(-): Did not experiment.
DMSO was used as the negative control.
Heparin was used as the positive control.
PT: prothrombin time, aPTT: activated partial thromboplastin time.
We investigated the effect of
bleeding time (in vivo) on mice. As shown in Table 2, aPTT in the vehicle-treated group was 23.6 ˘ 0.6 s
(mean SEM, n = 5) and in amidines and heparin, aPTT increased 33.2 0.7 s, 43.5 0.6 s and
62.5 0.8 s at dose 30 M, respectively. Although the in vitro anticoagulant activities of and were
weaker than those of heparin, aPTT was significantly prolonged by and at concentrations 20
1 and 2 on anticoagulant activities (in vitro and ex vivo) and on tail
˘
1
,
2
˘
˘
˘
µ
1
2
1
2
µM
and above, as compared to the vesicle group, while no obvious increase in PT was found. Noting that
a prolongation of aPTT suggests the inhibition of the intrinsic and/or common coagulation pathway,
and a PT prolongation suggests inhibition of the extrinsic and/or common pathway, obtained results
in this study showing prolongation of aPTT of compounds 1 and 2 suggest inhibition of the intrinsic
pathway and/or common pathway by compounds 1 and 2.
Table 2. Anticoagulant activity 1 and 2.
In Vitro Anticoagulant Assay
Sample
Dose
aPTT (s)
PT (s)
PT (INR)
Control
saline
23.6 ˘ 0.6
12.4 ˘ 0.4
1.00
5 µM
10 µM
20 µM
30 µM
40 µM
50 µM
22.2 ˘ 0.4
12.2 ˘ 0.2
12.1 ˘ 0.5
12.2 ˘ 0.4
13.1 ˘ 0.5
12.8 ˘ 0.6
12.4 ˘ 0.4
0.96
0.94
0.96
1.14
1.08
1.00
23.4 ˘ 0.5
28.5 ˘ 0.4 *
33.2 ˘ 0.7 *
45.7 ˘ 0.5 *
46.2 ˘ 0.6 *
1
5 µM
10 µM
20 µM
30 µM
40 µM
50 µM
23.8 ˘ 0.2
12.7 ˘ 0.5
12.4 ˘ 0.4
12.8 ˘ 0.3
13.0 ˘ 0.6
13.2 ˘ 0.5
12.9 ˘ 0.6
1.06
1.00
1.08
1.12
1.16
1.10
22.9 ˘ 0.5
31.4 ˘ 0.4 *
43.5 ˘ 0.6 *
50.6 ˘ 0.4 *
52.1 ˘ 0.3 *
2
20 µM
30 µM
53.3 ˘ 0.5 *
21.6 ˘ 0.4 *
3.79 *
6.59 *
Heparin
62.5 ˘ 0.8 *
27.2 ˘ 0.6 *
Each value represents the means ˘ SEM (n = 5). * p < 0.05 as compared to control.

Molecules 2016, 21, 676
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To confirm in vitro anticoagulant activity, in vivo tail bleeding times were evaluated. The average
circulating blood volume for mice is 72 mL/kg [31]. Because the average weight of the mouse used
is 27 g, the molecular weight of
of synthesized compounds (24.1, 32.1, or 40.1
of 30, 40, or 50 M in the peripheral blood. As shown in Table 3, tail bleeding times were significantly
1
or
2
is 400.88, and the average blood volume is 2 mL, the amount
µg/mouse) injected yielded a maximum concentration
µ
prolonged by compounds
the controls.
1 and 2 at concentrations 24.1 µg/mouse and above, as compared to
Table 3. In vivo bleeding time of 1 and 2.
In Vivo Bleeding Time
Sample
Dose
Tail Bleeding Time (s)
Control
saline
30.8 ˘ 0.8
24.1 µg/mouse
32.1 µg/mouse
40.1 µg/mouse
34.7 ˘ 1.1 *
44.1 ˘ 1.2 *
45.8 ˘ 1.0 *
1
24.1 µg/mouse
32.1 µg/mouse
40.1 µg/mouse
42.4 ˘ 1.0 *
51.5 ˘ 1.1 *
52.4 ˘ 1.2
2
140.0 µg/mouse
210.0 µg/mouse
51.2 ˘ 0.8 *
Heparin
68.4 ˘ 1.2 *
Each value represents the means ˘ SEM (n = 5). * p < 0.05 as compared to control.
aPTT values were also significantly prolonged by
1 and 2 at a concentration of 24.1 µg/mouse and
above ex vivo clotting times, while no obvious increase in PT values was found (Table 4). Collectively,
aPTT (in vitro and ex vivo) and tail bleeding time (in vivo) on the mice of
of
2
were longer than those
1
, suggesting that the para-amidine group is more efficient than the meta-amidine group on
anticoagulant activity.
Table 4. Ex vivo clotting time of 1 and 2.
Ex Vivo Clotting Time
Sample
Dose
aPTT (s)
PT (s)
PT (INR)
Control
saline
32.2 ˘ 0.8
12.8 ˘ 0.4
1.00
24.1 µg/mouse
32.1 µg/mouse
40.1 µg/mouse
35.4 ˘ 1.0 *
45.7 ˘ 0.8 *
46.2 ˘ 0.4 *
12.9 ˘ 0.5
13.1 ˘ 0.7
13.4 ˘ 0.4
1.02
1.06
1.12
1
24.1 µg/mouse
32.1 µg/mouse
40.1 µg/mouse
41.7 ˘ 1.2 *
49.5 ˘ 1.0 *
51.4 ˘ 0.8 *
13.2 ˘ 0.3
13.4 ˘ 0.5
13.6 ˘ 0.8
1.08
1.12
1.16
2
140.0 µg/mouse
210.0 µg/mouse
63.4 ˘ 0.9 *
25.4 ˘ 0.4 *
5.18 *
8.62 *
Heparin
75.4 ˘ 1.2 *
31.4 ˘ 0.6 *
Each value represents the means ˘ SEM (n = 5). * p < 0.05 as compared to control.
2.2.2. Effects of 1 or 2 on Thrombin-Catalyzed Fibrin Polymerization and Platelet Aggregation
The effects of
as changes in absorbance at 360 nm, as described in the Experimental Section. The results, shown in
Figure 1A, demonstrate that incubation of human plasma with or resulted in a significant decrease
1 or 2 on thrombin-catalyzed fibrin polymerization in human plasma were monitored
1
2
in the maximum rate of fibrin polymerization (Figure 1A). To eliminate the effect of sample pH, all
dilutions were performed using 50 mM TBS (pH 7.4). We also evaluated the effect of the same volume of
DMSO on human plasma; however, coagulation properties were unaffected. To confirm the antiplatelet

Molecules 2016, 21, 676
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activities of compounds
1
and
2, a thrombin-catalyzed platelet aggregation assay was performed.
As shown in Figure 1B, treatment with compounds
1 or 2 resulted in significantly inhibited mouse
platelet aggregation induced by thrombin (final concentration: 3 U/mL) in a concentration-dependent
manner. In order to exclude the possibility that the decrease of polymerization could be due to a
direct effect on thrombin leading to a decrease in fibrin generation, rather than polymerization of
fibrin formed, a reptilase-catalyzed polymerization assay was performed. Results showed that
induced a significant decrease in reptilase-catalyzed polymerization (data not shown). To confirm the
antiplatelet activities of compounds or , a U46619-(a stable thromboxane A2 analog/aggregation
1 and 2
1
2
agonist) catalyzed platelet aggregation assay was performed. The thromboxane A2 pathway is a
major contributor to the amplification of the initial platelet activation process. As shown in Figure 1C,
treatment with compounds
(final concentration: 2 M) in a concentration-dependent manner. These in vitro results were confirmed
in an ex vivo platelet aggregation assay (i.v. injection, Figure 1D). As shown in Figure 1D, treatment
with or resulted in significantly inhibited mouse platelet aggregation induced by U46619 (final
concentration: 2 M) in a concentration-dependent manner [32 33]. So far, most of the amidine-type
1 or 2 significantly inhibited human platelet aggregation induced by U46619
µ
1
2
µ
,
compounds have been reported as FXa inhibitors, and these amidine derivatives
potential as thromboxane A2 receptor antagonists.
1
and
2
also exhibited
120
90
120
90
A
B
Comp. 1
Comp. 2
Comp. 1
Comp. 2
*
*
*
*
*
*
*
*
*
*
*
*
60
60
30
30
0
Comp [μM]
Th
0
Comp [μM]
0
-
50
-
0
+
5
+
10
+
20
+
30
+
50
+
0
-
50
-
0
+
5
+
10
+
20
+
30
+
50
+
Th
120
90
100
80
60
40
20
C
D
Comp. 1
Comp. 2
Comp. 1
Comp. 2
*
*
*
*
*
*
*
*
*
*
*
60
*
30
0
Comp [μM]
U46619
0
0
-
50
-
0
+
5
+
10
+
20
+
30
+
50
+
Comp [μM]
D
50
Comp
[μM/mouse]
U46619
30
40
50
U46619 + Comp
[μM/mouse]
Figure 1. Effects of
polymerization at the indicated concentrations of
1
or
2
on fibrin polymerization in human plasma. (
or was monitored using a catalytic assay, as
described in the “Experimental Section”. The results are Vmax values expressed as percentages versus
controls; ( ) Effect of or on mouse platelet aggregation induced by 3 U/mL thrombin; ( ) The effect
of each compound on human platelet aggregation induced by 2 mM U46619; ( ) The indicated amount
of each compound concentration in DMSO was injected intravenously. The effects of each compound
A) Thrombin-catalyzed fibrin
1
2
B
1
2
C
D
on mouse platelet aggregation induced by 2
the vehicle control. Data represent the mean
triplicate. * p < 0.05 vs. Th or U46619 alone.
µ
M U46619 were monitored ex vivo. D = 0.2% DMSO is
SEM of three independent experiments performed in
˘

Molecules 2016, 21, 676
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2.2.3. Thrombin and Factor Xa (FXa) Activity
In order to determine the underlying mechanism whereby
1
and
2
mediated inhibition of
coagulation, the effect of
Figure 2A, we also investigated the effects of
dose-dependently inhibited the activity of thrombin. The direct thrombin inhibitor, argatroban was
used as a positive control. In addition, treatment with and resulted in a dose-dependent inhibition
1
and
2
on the activities of thrombin and FXa were measured. As shown in
1
and on the activity of thrombin. Compounds and
2
1
2
1
2
of amidolytic activity of FXa, indicating direct inhibition of FXa activity. Ribaroxaban, a direct FXa
inhibitor, was used as a positive control (Figure 2B).
120
90
120
90
A
B
Comp. 1
Comp. 2
Comp. 1
Comp. 2
*
*
*
*
*
*
*
*
*
*
*
*
60
60
30
30
*
*
*
*
0
0
Comp [μM]
Comp [μM]
0
Argatro
30
40
50
0
Rivaro
30
40
50
600
400
200
120
100
80
C
Comp. 1
Comp. 2
D
Comp. 1
Comp. 2
*
*
*
*
60
*
*
40
20
0
0
Comp [μM]
Comp [μM]
0
30
40
50
0
30
40
50
Figure 2. Effects of
thrombin (Th) by
Section”; (
described in the “Experimental Section”. Argatroban (A) or rivaroxaban (B) was used as a positive
control; ( ) HUVEC monolayers were pre-incubated with FVa (100 pM) and FXa (1 nM) for 10
min with the indicated concentrations of 1 or . Prothrombin was added to a final concentration
of 1 M and prothrombin activation was determined 30 min later, as described in the “Experimental
Section”; (D) HUVECs were pre-incubated with the indicated concentrations of or for 10 min. After
1
or
or
2
2
on inactivation and production of thrombin and factor Xa. (
was measured using a chromogenic assay, as described in the “Experimental
or was monitored using a chromogenic assay, as
A) Inhibition of
1
B
) Inhibition of factor Xa (FXa) by
1
2
C
2
1
2
TNF-(10 ng/mL for 6 h) stimulated HUVECs were incubated with FVIIa (10 nM) and FX (175 nM), FXa
production was determined, as described in the “Experimental Section”. D = 0.2% DMSO is the vehicle
control. * p < 0.05 vs. 0.
2.2.4. Thrombin and Factor Xa (FXa) Generation
In a previous study, Sugo et al. [34] reported that endothelial cells are able to support prothrombin
activation by FXa. In the current study, pre-incubation of HUVECs with FVa and FXa in the presence
of CaCl2 prior to addition of prothrombin resulted in production of thrombin (Figure 2C). According
to Rao et al. [35], the endothelium provides the functional equivalent of pro-coagulant phospholipids
and supports activation of FXa, and, in TNF-
in a tissue factor (TF) expression-dependent manner. Thus, we investigated the effects of
activation of FX by FVIIa. Pre-incubation with and resulted in dose-dependent inhibition of FX
α
stimulated HUVECs, activation of FX by FVIIa occurred
1
and on
2
1
2

Molecules 2016, 21, 676
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activation by FVIIa (Figure 2D). Therefore, these results suggested that
of thrombin and FXa.
1
and
2
can inhibit production
2.2.5. Cellular Viability
To determine the cellular viability of compounds
1
and
2, the cellular viability assay (MTT assay)
was performed in HUVECs treated with
1
and
2
for 24 h. Both
1
and did not affect cell viability at
2
concentration up to 100 µM (Figure 3).
120
E
Comp. 1
Comp. 2
100
80
60
40
20
0
Comp [μM]
D
5
25
50
100
Figure 3. Effects of
1 or 2 on cellular viability was measured by MTT assay. D = 0.2% DMSO is the
vehicle control.
3. Experimental Section
3.1. Chemistry
3.1.1. Reagents and Instruments
All non-aqueous reactions were performed under a dry atmosphere of nitrogen. The commercial
reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) or TCI (Tokyo, Japan). Solvents
were purified and dried prior to use. Melting points were measured on Thomas-Hoover melting point
1
apparatus (Thomas Scientific, Swedesboro, NJ, USA) and not corrected. H, 13C-NMR and HMBC
spectra were taken on a Varian 400 MHz spectrometer (Thomas Scientific, Swedesboro, NJ, USA)
in DMSO-d6. Chemical shifts (δ) are in parts per million (ppm) relative to tetramethylsilane, and
coupling constants (J) are in Hertz. DIP-MS (EI) was obtained on an Agilent 7890A-5975C GC/MSD
(Agilent Technologies, Santa Clara, CA, USA). GC/MS (EI) was obtained on a SHIMADZU QP 2010
model (Shimadzu, Kyoto, Japan) and FAB-MS was obtained on a JEOL JMS-700 Mstation (JEOL,
Tokyo, Japan). Fraction collection was performed on an EYELA fraction collector DC-1500 (Tokyo
Rikakikai, Tokyo, Japan). An analytical TLC was performed on pre-coated silica gel 60 F254 plates
(Merck, Kenilworth, NJ, USA). Solvent systems for TLC were ethyl acetate/n-hexane mixtures and 10%
methanol in dichloromethane. Column chromatography was carried out on Merck silica gel 9385
(Merck, Kenilworth, NJ, USA) (230–400 mesh).
3.1.2. General Experimental Procedures of 1i–3i
To a stirred solution of 3-nitrobenzoic acid (35.90 mmol) in anhydrous benzene (50 mL) was
dropwise added: oxalyl chloride (46.67 mmol) and then triethylamine (39.49 mmol) at room
temperature. The reaction mixture was refluxed for 1 h and the solvent and the unreacted oxalyl
chloride were evaporated off under reduced pressure and the acid chloride was used without
purification. To a solution of 3-aminobenzonitrile, 4-aminobenzonitrile, and 4-aminobenzyl cyanide
(8.62 mmol) in anhydrous benzene (50 mL) was added: acyl chloride (10.78 mmol) and triethylamine
(8.62 mmol) at room temperature and stirred for 3 h. To the reaction mixture, water was added and
extracted with ethyl acetate (50 mL ˆ 3), dried with anhydrous magnesium sulfate and filtrated. The

Molecules 2016, 21, 676
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filtrate was evaporated under reduced pressure to give crude compound, which was recrystallized
with ethyl acetate and n-hexane to give a pure white or pale yellow compound, respectively.
1
˝
3-Nitro-N-(3 -cyanophenyl) benzamide (1i). Yield: 81%; m.p.: 195–196 C; 1H-NMR (400 MHz, DMSO-d6)
: 7.62 (2H, d, J = 8.0 Hz, H-61,41), 7.86 (1H, t, J = 8.0 Hz, H-51), 8.06 (1H, t, J = 8.0 Hz, H-5), 8.26
(1H, s, H-21), 8.41 (1H, ddd, J = 7.8, 1.5, 1.1 Hz, H-6), 8.47 (1H, ddd, J = 8.2, 2.3, 1.0 Hz, H-4), 8.81
(1H, t, J = 2.0 Hz, H-2), 10.88 (1H, s, CONH); 13C-NMR (100 MHz, DMSO-d6) : 111.5 (C-31), 118.6
(C
N), 122.5 (C-61), 123.3 (C-2), 125.1(C-51), 126.5 (C-4), 127.6 (C-21), 130.3 (C-41), 134.3 (C-6), 135.7
(C-1), 139.5 (C-1 ), 147.8 (C-3), 163.8 (C=O); GC-MS (EI) m/z: 267 [M]+.
δ
δ
”
1
1
˝
3-Nitro-N-(4 -cyanophenyl) benzamide (2i). Yield: 80%; m.p.: 225–226 C; 1H-NMR (400 MHz, DMSO-d6)
δ
1 1
1 1
: 7.85 (2H, d, J = 8.0 Hz, H-3 ,5 ), 7.86 (1H, t, J = 8.0 Hz, H-5), 7.99 (2H, d, J = 8.0 Hz, H-2 ,6 ), 8.41 (1H,
d, J = 8.0 Hz, H-6), 8.46 (1H, ddd, J = 8.2, 2.3, 1.0 Hz, H-4), 8.67 (1H, t, J = 2.0 Hz1, H-2), 10.96 (1H, s,
1
1
CONH); 13C-NMR (100 MHz, DMSO-d6)
δ
: 106.6 (C-4 ), 119.6 (C N), 121.1 (C-2 ,6 ), 123.3 (C-2), 127.2
”
1
1
1
(C-4), 131.0 (C-5), 133.9 (C-3 ,5 ), 135.0 (C-6), 136.4 (C-1), 143.7 (C-1 ), 148.4 (C-3), 164.7 (C=O); GC-MS
(EI) m/z: 267 [M]+.
3-Nitro-N-(41-cyanomethylphenyl) benzamide (3i). Yield: 80%; m.p.: 197–198 ˝C; 1H-NMR (400 MHz,
DMSO-d6)
(1H, t, J = 8.0 Hz, H-5), 8.36 (1H, d, J = 8.0 Hz, H-6), 8.40 (1H, ddd, J = 8.2, 2.3, 1.0 Hz, H-4) 8.75 (1H,
δ
: 3.97 (2H, s, CH2), 7.31 (2H, d, J = 8.8 Hz, H-31,51), 7.76 (2H, d, J = 8.8 Hz, H-21,61), 7.80
t, J = 2.0 Hz, H-2), 10.60 (1H, s, CONH); 13C-NMR (100 MHz, DMSO-d6)
δ
: 21.9 (CH2), 119.3 (C N),
”
121.0 (C-211,61), 122.4 (C-2), 126.2 (C-41), 126.7 (C-4), 128.4 (C-31,51), 130.2 (C-5), 134.2 (C-6), 136.1 (C-1),
138.1 (C-1 ), 147.7 (C-3), 163.3 (C=O); GC-MS (EI) m/z: 281 [M]+.
3.1.3. General Experimental Procedures of 1ii–3ii
To a solution of 1i
amount of 1i 3i) and iron powder (3.58 mmol), and the reaction mixture was refluxed for 7 h. To the
reaction mixture, ice water was added and rotary evaporated to remove methanol under reduced
pressure. The aqueous residue was extracted with dichloromethane (30 mL 3) and the organic
–3i (1.79 mmol) in methanol (20 mL) was added: ammonium chloride (double
–
ˆ
phase was dried with anhydrous MgSO4, filtrated, and evaporated to give the crude solid, which was
recrystallized with ethyl acetate and n-hexane to give a pure white or pale-yellow solid.
1
3-Amino-N-(31-cyanophenyl) benzamide (1ii). Yield: 60%; m.p.: 166–168 ˝C; H-NMR (400 MHz,
DMSO-d6) δ: 5.35 (2H, s, NH2), 6.77 (1H, ddd, J = 8.2, 2.3, 1.0 Hz, H-4), 7.07 (1H, d, J = 7.6 Hz,
H-6), 7.10 (1H, t, J 1= 1.9 Hz, H-2), 7.17 (1H, t, J = 7.6 Hz, H-5), 7.54-7.58 (2H, m, H-41,51), 8.04 (1H, dt,
1
J = 7.2, 2.2 Hz, H-6 ), 8.24 (1H, s, H-2 ), 10.39 (1H, s, CONH); 13C-NMR (100 MHz, DMSO-d6) δ: 111.4
(C-31), 112.9 (C-2), 114.7 (C-6), 117.1 (C-4), 118.8 (C N), 122.8 (C-61), 124.7 (C-51), 126.9 (C-21), 128.9
”
1
1
(C-5), 130.1 (C-4 ), 135.2 (C-1), 140.2 (C-1 ), 148.9 (C-3), 166.8 (C=O); GC-MS (EI) m/z: 237 [M]+.
1
3-Amino-N-(41-cyanophenyl) benzamide (2ii). Yield: 71%; m.p.: 182–183 ˝C; H-NMR (400 MHz,
DMSO-d6) δ: 5.36 (2H, s, NH2), 6.77 (1H, ddd, J = 8.0, 2.0, 1.2 Hz, H-4), 7.05 (1H, d, J = 7.6 Hz,
1
1
H-6), 7.07 (1H, t, J = 1.9 Hz, H-2), 7.17 (1H, t, J = 8.0 Hz, H-5), 7.80 (2H, d, J = 8.0 Hz, H-3 ,5 ), 7.98 (2H,
1
1
1
d, J = 8.0 Hz, H-2 ,6 ), 10.48 (1H, s, CONH); 13C-NMR (100 MHz, DMSO-d6)
δ
: 105.0 (C-4 ), 112.9 (C-2),
N), 120.0 (C-2 ,6 ), 128.9 (C-5), 133.1 (C-3 ,5 ), 135.3 (C-1), 143.7 (C-1 ),
1
1
1
1
1
114.8 (C-6), 117.2 (C-4), 119.1 (C
”
148.9 (C-3), 167.0 (C=O); GC-MS (EI) m/z: 237 [M]+.
3-Amino-N-(41-cyanomethylphenyl) benzamide (3ii). Yield: 68%; m.p.: 173–174 ˝C; 1H-NMR (400 MHz,
DMSO-d6)
J = 7.6 Hz, H-6), 7.09 (1H, t, J = 1.9 Hz, H-2), 7.14 (1H, t, J = 8.0 Hz, H-5), 7.30 (2H, d, J = 8.0 Hz, H-3 ,5 ),
7.78 (2H, d, J = 8.0 Hz, H-21,61), 10.13 (1H, s, CONH); 13C-NMR (100 MHz, DMSO-d6)
: 21.9 (CH2),
δ: 3.98 (2H, s, CH2), 5.31 (2H, s, NH2), 6.74 (1H, ddd, J = 7.8, 2.2, 1.2 Hz, H-4), 7.05 (1H, d,
1
1
δ

Molecules 2016, 21, 676
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1 1 1
1 1
112.9 (C-2), 114.7 (C-6), 116.8 (C-4), 119.4 (C N), 120.6 (C-2 ,6 ), 125.9 (C-4 ), 128.3 (C-3 ,5 ), 128.7 (C-5),
”
1
135.8 (C-1), 138.8 (C-1 ), 148.8 (C-3), 166.4 (C=O); GC-MS (EI) m/z: 251 [M]+.
3.1.4. General Experimental Procedures of 1a–3a
To a solution of thiophene-2-carboxylic acid (7.8 mmol) in anhydrous dichloromethane (40 mL)
was added: triethylamine (8.6 mmol) and dropwise added oxalyl chloride (10.1 mmol) at room
temperature. The reaction mixture was refluxed for 1 h and rotary evaporated to remove
dichloromethane and oxalyl chloride, which was used next reaction without purification. To a solution
of 1ii
thiophene-2-carbonyl chloride (1.85 mmol) at room temperature and the reaction mixture was refluxed
for 1 h. To the reaction mixture, water was added and extracted with ethyl acetate (50 mL 3), dried
–3ii (1.48 mmol) in anhydrous benzene (20 mL) was added: triethylamine (1.48 mmol) and
ˆ
with anhydrous MgSO4, and filtrated. The filtrate was evaporated under reduced pressure to give a
crude compound, which was recrystallized with ethyl acetate and n-hexane to give a pure white or
pale yellow compound, respectively.
˝
N-(31-Cyanophenyl)-3-(thiophen-211-ylcarbonylamino) benzamide (1a). Yield: 64%; m.p.: 201–202 C;
1H-NMR (400 MHz, DMSO-d6) : 7.25 (1H, dd, J = 5.0, 4.0 Hz, H-411), 7.54 (1H, t, J = 8.0 Hz, H-5),
δ
7.58-7.60 (2H, m, H-41,51), 7.71 (1H, d, J = 6.8 Hz, H-6), 7.89 (1H, dd, J = 5.0, 11.0 Hz, H-511), 8.03 (1H,
11
1
dd, J = 8.0, 0.8 Hz, H-3 ), 8.07-8.09 (2H, m, H-4,6 ), 8.26 (1H, t, J = 1.2 Hz, H-2 ), 8.28 (1H, t, J = 1.8 Hz,
1
H-2), 10.47 (1H, s, NHCO), 10.61 (1H, s, CONH); 13C-NMR (100 MHz, DMSO-d6)
δ: 111.5 (C-3 ), 118.7
(C
N), 119.8 (C-2), 122.7 (C-6), 123.0 (C-4), 123.6 (C-61), 124.9 (C-51), 127.2 (C-21), 128.2 (C-5), 128.9
”
(C-411), 129.4 (C-311), 130.2 (C-41), 132.2 (C-511), 135.0 (C-1), 139.0 (C-3), 139.7 (C-211), 140.0 (C-11), 160.1
(CONH), 165.9 (NHCO); DIP-MS (EI) m/z: 347 [M]+.
˝
N-(41-Cyanophenyl)-3-(thiophen-211-ylcarbonylamino) benzamide (2a). Yield: 71%; m.p.: 214–216 C;
11
1H-NMR (400 MHz, DMSO-d6)
(1H, d, J = 6.8 Hz, H-6), 7.83 (2H, d, J = 8.0 Hz, H-3 ,5 ), 7.88 (1H, dd, J = 4.0, 0.8 Hz, H-5 ), 8.00 (2H, d,
δ: 7.25 (1H, dd, J = 5.0, 4.0 Hz, H-4 ), 7.54 (1H, t, J = 8.0 Hz, H-5), 7.71
1
1
11
1
1
11
J = 8.0 Hz, H-2 ,6 ), 8.03 (1H, ddd, J = 8.0, 2.3, 1.0 Hz, H-4), 8.09 (1H, dd, J = 4.0, 0.8 Hz, H-3 ), 8.28 (1H,
t, J = 1.9 Hz, H-2), 10.47 (1H, s, NHCO), 10.70 (1H, s, CONH); 13C-NMR (100 MHz, DMSO-d6)
: 105.4
δ
(C-41), 119.2 (C N), 119.9 (C-2), 120.2 (C-21,61), 122.8 (C-6), 123.7 (C-4), 128.2 (C-5), 128.9 (C-411), 129.4
”
(C-311), 132.2 (C-511), 133.1 (C-31,51), 135.0 (C-1), 139.1 (C-3), 139.7 (C-211), 143.5 (C-11), 160.1 (CONH),
166.1 (NHCO); DIP-MS (EI) m/z: 347 [M]+.
1
11
˝
;
N-(4 -Cyanomethylphenyl)-2-(thiophen-2 -ylcarbonylamino) benzamide (3a). Yield: 61%; m.p.: 202–203 C
1H-NMR (400 MHz, DMSO-d6) : 4.01 (2H, s, CH2), 7.25 (1H, dd, J = 5.0, 4.0 Hz, H-411), 7.34 (2H, d,
δ
J = 8.0 Hz, H-31,51), 7.52 (1H, t, J = 8.0 Hz, H-5), 7.70 (1H, d, J = 8.0 Hz, H-6), 7.81 (2H, d, J = 8.0 Hz,
H-21,61), 7.88 (1H, 1d1 d, J = 4.0, 0.8 Hz, H-511), 8.01 (1H, ddd, J = 8.0, 2.3, 1.0 Hz, H-4), 8.08 (1H, dd,
J = 4.0, 0.8 Hz, H-3 ), 8.26 (1H, t, J = 2.0 Hz, H-2), 10.36 (1H, s, NHCO), 10.43 (1H, s, CONH); 13C-NMR
(
100 MHz, DMSO-d6)
δ
: 21.9 (CH2), 1119.3 (C
”
N), 119.8 (C-2),111 20.7 (C-21,6111), 122.7 (C-6), 123.3 (C-4),
1
1
11
126.2 (C-4 ), 128.1 (C-5), 128.4 (C-3 ,5 ), 128.7 (C-4 ), 129.3 (C-3 ), 132.1 (C-5 ), 135.5 (C-1), 138.6 (C-3),
138.9 (C-1 ), 139.7 (C-2 ), 160.0 (CONH), 165.5 (NHCO); DIP-MS (EI) m/z: 361 [M]+.
1
11
3.1.5. General Experimental Procedures of 1b–31ii–3ii
To a suspension of intermediate 1a
(5 mL) was added: triethylamine (2.59 mmol) and refluxed to dissolve. The reaction mixture was
added with hydroxylamine HCl (3.45 mmol) and refluxed for 4–6 h. The mixture was evaporated to
–3a (0.86 mmol) in absolute ethanol (25 mL) and 1,4-dioxane
¨
remove the ethanol and ice water was poured into the residue. The resulting precipitate was filtrated,
washed with water, and dried under reduced pressure to give a pure white or pale yellow solid.

Molecules 2016, 21, 676
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˝
N-(31-Amidoximephenyl)-3-(thiophen-211-ylcarbonylamino) benzamide (1b). Yield: 87%; m.p.: 234–235 C
;
1H-NMR (400MHz, DMSO-d6) : 5.75 (2H, s, NH2), 7.24 (1H, dd, J = 4.9, 3.8 Hz, H-411), 7.34–7.38
δ
1
1
(2H, m, H-5, 4 ), 7.51 (1H, t, J = 7.9 Hz, H-5 ), 7.71 (1H, d, J = 7.8 Hz, H-4), 7.80 (1H, d, J = 7.6 Hz, H-6),
1
11
7.88 (1H, dd, J = 5.0, 0.8 Hz, H-511), 8.01 (1H, dd, J = 8.0, 1.2 Hz, H-61), 8.05–8.10 (2H, m, H-2 ,3 )
,
8.27 (1H, t, J = 2.0 Hz, H-2), 9.62 (1H, s, NOH), 10.35 (1H, 1s, NHCO), 10.47 (1H, s, CONH)
1 1
100 MHz, DMSO-d6) δ: 118.5 (C-2), 120.6 (C-2 ), 121.6 (C-6 ), 121.7 (C-6), 123.4 (C-4 ), 124.0 (C-4), 128.8
;
13C-NMR
(
(C-5), 128.9 (C-411), 129.4 (C-311), 130.0 (C-51), 132.8 (C-511), 134.6 (C-1), 136.2 (C-11), 139.6 (C-211), 140.5
(C-3), 151.6 (C=NOH), 160.7 (CONH), 166.1 (NHCO); FAB-MS m/z: 363 [M + 1
´
H2O]+, 380 [M]+,
381 [M + 1]+, 403 [M + Na]+.
˝
N-(41-Amidoximephenyl)-3-(thiophen-211-ylcarbonylamino) benzamide (2b). Yield: 87%; m.p.: 241–242 C
;
1H-NMR (400 MHz, DMSO-d6) : 5.76 (2H, s, NH2), 7.25 (1H, dd, J = 5.0, 4.0 Hz, H-411), 7.52 (1H, t,
δ
J = 8.0 Hz, H-5), 7.66 (2H, d, J = 8.0 Hz, H-31,51), 7.70 (1H, d, J = 7.6 Hz, H-6), 7.78 (2H, d, J = 8.0 Hz
,
H-21,61), 7.88 (1H, dd, J = 4.0, 0.8 Hz, H-511), 8.01 (1H, d, J = 7.6, Hz, H-4), 8.08 (1H, dd, J = 8.0, 0.8 Hz,
H-311), 8.25 (1H, t, J = 2.0 Hz, H-2), 9.55 (1H, s, NOH), 10.37 (1H, s, NHCO), 10.44 (1H, s, CONH);
13C-NMR (100 MHz, DMSO-d6) : 119.6 (C-21,61), 119.8 (C-2), 122.7 (C-6), 123.3 (C-4), 125.7 (C-31,51),
δ
128.2 (C-5), 128.7 (C-41), 128.9 (C-411), 129.3 (C-311), 132.1 (C-511), 135.5 (C-1), 139.9 (C-3), 139.6 (C-11),
139.7 (C-211), 150.5 (C=NOH), 160.0 (CONH), 165.5 (NHCO); FAB-MS m/z: 363 [M + 1
´
H2O]+, 380
[M]+, 381 [M + 1]+, 403 [M + Na]+.
N-(41-Amidoximemethylphenyl)-3-(thiophen-211-ylcarbonylamino) benzamide (3b). Yield: 90%; m.p.:
˝
164–165 C; 1H-NMR (400 MHz, DMSO-d6)
δ: 3.23 (2H, s, CH2), 5.37 (2H, s, NH2), 7.19–7.26 (3H, m,
1
1
11
1
1
11
H-3 ,5 ,4 ), 7.50 (1H, t, J = 7.9 Hz, H-5), 7.67–7.69 (3H, m, H-2 ,6 ,4), 7.88 (1H, d, J = 4.9 Hz, H-5 ), 8.01
11
(1H, d, J = 8.0 Hz, H-6), 8.10 (1H, d, J = 3.5 Hz, H-3 ), 8.26 (1H, s, H-2), 8.88 (1H, s, NOH), 10.29 (1H, s,
1 1
NHCO), 10.50 (1H, s, CONH); 13C-NMR (100 MHz, DMSO-d6)
δ: 37.4 (CH2), 120.5 (C2 ,6 ), 122.9 (C-6),
11
11
1
1
11
11
1
123.3 (C-4), 128.9 (C-4 ), 129.4 (C-3 ,5 ), 129.5 (C-3 ), 130.0 (C-5), 132.8 (C-5 ), 133.9 (C-4 ), 136.3 (C-1),
1
138.0 (C-3), 139.5 (C-2 ), 140.5 (C-1 ), 152.6 (C=NOH), 160.7 (CONH), 165.9 (NHCO); FAB-MS m/z: 377
[M + 1 ´ H2O]+, 394 [M]+, 395 [M + 1]+.
3.1.6. General Experimental Procedures of 1–3
To a solution of amidoximes 1b–3b (0.53 mmol) in anhydrous dichloromethane (10 mL) was
added: triethylamine (1.58 mmol) and acetyl chloride (0.6 mmol) at 0 ˝C. The reaction mixture was
stirred for 1–2 h at room temperature and ice water was added. The aqueous layer was extracted with
dichloromethane (30 mL
water. The organic layer was dried with MgSO4, filtrated, and evaporated in reduced pressure to give
the acetylated amidoximes (1c 3c), which were used next reaction without purification. To a solution
ˆ
3). The organic phase was washed with water, saturated NaHCO3, and
–
of 10% Pd-C in ethanol (10 mL) was added: acetylated amidoximes (1.0 eq.) and c-HCl (1.0 eq.) and
˝
hydrogenated for 2 h at 60 psi, 45 C. The reaction mixture was filtrated and concentrated under
reduced pressure to give a crude oily compound, which was purified to yield a white or pale yellow
solid by column chromatography.
1
11
˝
;
N-(3 -Amidinophenyl)-3-(thiophen-2 -ylcarbonylamino) benzamide
¨
HCl (
1
). Yield: 46%; m.p.: 219–220 C
1H-NMR (400 MHz, DMSO-d6) : 7.23 (1H, dd, J = 5.0, 3.8 Hz, H-411), 7.55–7.50 (2H, m, H-5,41), 7.60
δ
1
11
(1H, t, J = 7.9 Hz, H-5 ), 7.76 (1H, d, J = 8.1 Hz, H-6), 7.87 (1H, dd, J = 5.0, 1.0 Hz, H-5 ), 8.03 (1H, ddd,
J = 8.2, 2.3, 1.0 Hz, H-4), 8.08 (1H, dd, J = 8.0, 1.2 Hz, H-6 ), 8.24 (1H, dd, J = 3.7, 0.7 Hz, H-3 ), 8.32 (1H,
1
11
1
t, J = 1.8 Hz, H-2 ), 8.38 (1H, t, J = 1.8 Hz, H-2), 9.45 (4H, br s, hydrogens of amidine
¨
HCl), 10.68 (1H, s,
δ: 119.5 (C-2 ), 120.0 (C-2), 122.7 (C-6),
NHCO), 10.72 (1H, s, CONH); 13C-NMR (100 MHz, DMSO-d6)
1
123.1 (C-61), 123.7 (C-4), 125.1 (C-41), 128.1 (C-5), 128.7 (C-411), 129.0 (C-311), 129.3, 129.5 (C-51), 132.1
(C-511), 135.0 (C-1), 139.1 (C-3), 139.7 (C-211), 139.8 (C-11), 160.1 (carbon of amidine), 165.9 (CONH),

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166.3 (NHCO); FAB-MS m/z: 365 [M
´
Cl]+, 388 [M
´
Cl + Na]+; HRMS (EI): Calcd for C19H17N4O2S
[M + H]+ 365.1072, found 365.1067.
1
11
˝
N-(4 -Amidinophenyl)-3-(thiophen-2 -ylcarbonylamino) benzamide
¨
HCl (
2
). Yield: 37%; m.p.: 237–238 C
;
1H-NMR (400 MHz, DMSO-d6)
δ
: 7.24 (1H, dd, J = 5.0, 3.8 Hz, H-411), 7.53 (1H, t, J = 7.9 Hz, H-5),
7.77 (1H, d, J = 7.9 Hz, H-6), 7.88 (1H, dd, J = 5.1, 1.1 Hz, H-511), 7.90 (2H, d, J = 9.0 Hz, H-31,51),
8.05 (3H, m, H-4, H-21,61), 8.21 (1H, d, J = 3.8 Hz, H-311), 8.36 (1H, t, J = 1.8 Hz, H-2), 9.17 (2H, br s,
hydrogens of amidine
¨
HCl), 9.36 (2H, br s, hydrogens of amidine HCl), 10.63 (1H, s, NHCO), 10.78
¨
1 1 1
: 119.7 (C-2 ,6 ), 120.0 (C-2), 122.1 (C-4 ), 122.9 (C-6),
(1H, s, CONH); 13C-NMR (100 MHz, DMSO-d6)
δ
123.7 (C-41)1, 128.1 (C-5), 128.7 (C-411), 129.0 (C-311), 129.6 (C-31,51), 132.1 (C-511), 134.9 (C-1), 139.1(C-3),
1
139.7 (C-2 ), 144.2 (C-1 ), 160.1 (carbon of amidine), 164.9 (CONH), 166.1 (NHCO); FAB-MS m/z: 365
[M
´
Cl]+, 388 [M ´ Cl + Na]+, 423 [M + Na]+; HRMS (EI): Calcd for C19H17N4O2S [M + H]+ 365.1072,
found 365.1069.
N-(41-Amidinomethylphenyl)-3-(thiophen-211-ylcarbonylamino) benzamide
HCl (3). Yield: 51%; m.p.:
¨
219–220 ˝C; 1H-NMR (400 MHz, DMSO-d6) : 3.71 (2H, s, CH2), 7.23 (1H, dd, J = 5.0, 3.8 Hz, H-411),
δ
7.44 (2H, d, J = 8.6 Hz, H-31,51), 7.50 (1H, t, J = 7.9 Hz, H-5), 7.71 (1H, d, J = 7.9 Hz, H-6), 7.79 (2H,
d, J = 8.6 Hz, H-21,61), 7.87 (1H, dd, J = 5.0, 0.9 Hz, H-511), 8.02 (1H, dd, J = 8.1, 1.3 Hz, H-4), 8.30
(1H, t, J = 1.8 Hz, H-2), 8.83 (2H, br s, hydrogens of amidine HCl), 9.28 (2H, br s, hydrogens of
¨
amidine
¨
HCl), 10.37 (1H, s, NHCO), 10.57 (1H, s, CONH); 13C-NMR (100 MHz1, DMSO-d6)
δ
11 : 119.9
1
1
1
11
1
(C-2), 120.5 (C-2 ,6 ), 122.7 (C-6), 123.5 (C-2), 128.1 (C-4 ), 128.6 (C-4 ), 129.2 (C-3 ,5 ), 129.5 (C-3 ), 132.1
(C-511), 135.5 (C-1), 138.6 (C-3), 139.0 (C-11), 139.8 (C-211), 160.1 (carbon of amidine), 165.5 (CONH),
169.3 (NHCO); FAB-MS m/z: 379 [M
´
Cl]+, 402 [M
´
Cl + Na]+; HRMS (EI) Calcd. For C20H19N4O2S
[M + H]+ 379.1229, found 379.1216.
3.1.7. General Experimental Procedures 4i–8i
To a stirred solution of 3-nitrobenzoic acid (35.90 mmol) in anhydrous benzene (50 mL) was
dropwise added: oxalyl chloride (46.67 mmol) and triethylamine (39.49 mmol) at room temperature.
The reaction mixture was refluxed for 1 h, and benzene and unreacted oxalyl chloride were rotary
evaporated off under reduced pressure and used without purification. To a solution of 4-chloroaniline,
4-bromoaniline, 4-fluoroaniline, 4-methoxyaniline, and 4-mor pholinoaniline (2.16 mmol) in anhydrous
benzene (30 mL) was added: 3-nitrobenzoyl chloride (2.69 mmol) and triethylamine (2.16 mmol) at
room temperature and stirred at the same temperature for 3 h. To the reaction mixture, water was
added and extracted with ethylacetate (30 mL
filtrate was evaporated under reduced pressure to afford a crude product, which was recrystallized
ˆ
3), dried with anhydrous MgSO4, and filtrated. The
with ethyl acetate and n-hexane to give a pure white or pale yellow compound, respectively.
1
˝
N-(4 -Chlorophenyl)-3-nitrobenzamide (4i). Yield: 82%; m.p.: 175–177 C; 1H-NMR (400 MHz, DMSO-d6)
δ
1 1
1 1
: 7.45 (2H, d, J = 8.9 Hz, H-3 ,5 ), 7.82 (2H, d, J = 8.9 Hz, H-2 ,6 ), 7.85 (1H, t, J = 8.0 Hz, H-5), 8.40 (1H,
d, J = 8.0 Hz, H-6), 8.45 (1H, ddd, J = 8.2, 2.3, 1.0 Hz, H-4), 8.78 (1H, t, J = 1.9 Hz, H-2), 10.71 (1H, s,
CONH); 13C-NMR (100 MHz, DMSO-d6)
δ
: 122.1 (C-211,61), 122.4 (C-2), 126.3 (C-4), 127.8 (C-41), 128.7
1
1
(C-3 ,5 ), 130.3 (C-5), 134.2 (C-6), 136.0 (C-1), 137.7 (C-1 ), 147.7 (C-3), 163.4 (CONH); GC-MS (EI) m/z:
276 [M]+.
1
˝
N-(4 -Bromophenyl)-3-nitrobenzamide (5i). Yield: 79%; m.p.: 190–192 C; 1H-NMR (400 MHz, DMSO-d6)
: 7.75 (2H, d, J = 8.8 Hz, H-31,51), 7.77 (2H, d, J = 8.8 Hz, H-21,61), 7.85 (1H, t, J = 8.0 Hz, H-5), 8.40
(1H, d, J = 7.8 Hz, H-6), 8.45 (1H, ddd, J = 8.2, 2.3, 1.0 Hz, H-4), 8.78 (1H, t, J = 1.9 Hz, H-2), 10.69 (1H,
δ
s, CONH); 13C-NMR (100 MHz, DMSO-d6) : 115.9 (C-41), 122.4 (C-2,21,61), 126.3 (C-4), 130.2 (C-5),
δ
131.5 (C-31,51), 134.2 (C-6), 136.0 (C-1), 138.1 (C-11), 147.7 (C-3), 163.4 (CONH); GC-MS (EI) m/z: 320
[M ´ 1]+, 322 [M + 1]+.

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1
˝
N-(4 -Fluorophenyl)-3-nitrobenzamide (6i). Yield: 78%; m.p.: 190–191 C; 1H-NMR (400 MHz, DMSO-d6)
: 7.23 (2H, t, J = 8.8 Hz, H-31,61), 7.80 (2H, dd, J = 9.0, 5.1 Hz, H-21,61), 7.85 (1H, t, J = 8.0 Hz, H-5),
8.40 (1H, d, J = 7.8 Hz, H-6), 8.44 (1H, dd, J = 8.2, 2.3 Hz, H-4), 8.79 (1H, t, J = 2.0 Hz, H-2), 10.63 (1H,
δ
s, CONH); 13C-NMR (100 MHz, DMSO-d6) : 115.3 (J = 22.0 Hz, C-31,51), 122.4(J = 7.2 Hz, H-21,61),
δ
122.5 (C-2), 126.2 1(C-4), 130.2 (C-5), 134.1 (C-6), 135.0 (J = 2.6 Hz, C-11), 136.1 (C-1), 147.7 (C-3), 158.5
(J = 240.0 Hz, H-4 ), 163.2 (CONH); GC-MS (EI) m/z: 260 [M]+.
1
N-(41-Methoxyphenyl)-3-nitrobenzamide (7i). Yield: 77%; m.p.: 163–164 ˝C; H-NMR (400 MHz,
DMSO-d6)
7.83 (1H, t, J = 8.0 Hz, H-5), 8.39 (1H, d, J = 8.0 Hz, H-6), 8.43 (1H, ddd, J = 8.2, 2.3, 1.0 Hz, H-4), 8.78
(1H, t, J = 1.9 Hz, H-2), 10.47 (1H, s, CONH); 13C-NMR (100 MHz, DMSO-d6)
: 55.9 (CH3), 114.5
(C-21,61), 122.9 (C-31,51), 123.0 (C-2), 126.7 (C-4), 130.9 (C-5), 132.4 (C-11), 134.7 (C-6), 137.0 (C-1), 148.4
δ
: 3.75 (3H, s, CH3), 6.96 (2H, d, J = 9.1 Hz, H-31,51), 7.68 (2H, d, J = 9.1 Hz, H-21,61),
δ
1
(C-3), 156.5 (C-4 ), 163.5(CONH); GC-MS (EI) m/z: 272 [M]+.
˝
1
N-(411-Morpholinophenyl)-3-nitrobenzamide (8i). Yield: 75%; m.p.: 204–205 C; H-NMR (400 MHz,
DMSO-d6)
: 3.20 (4H, t, J = 0.8 Hz, H-311,511), 3.82 (4H, t, J = 0.8 Hz, H-211,611), 7.16 (2H, d, J = 8.4 Hz,
H-31,51), 7.71 (2H, J = 9.0 Hz, H-21,61), 7.83 (1H, t, J = 8.0 Hz, H-5), 8.39–8.44 (2H, m, H-4, H-6), 8.78
δ
(1H, t, J = 1.2 Hz, H-2), 10.53 (1H, s, CONH); 13C-NMR (100 MHz, DMSO-d6) : 49.9 (C-311,511), 65.6
δ
(C-211,611), 116.7 (C-31,51), 121.7 (C-21,61), 122.3 (C-2), 126.1 (C-4), 130.2 (C-5), 134.1 (C-6), 136.3 (C-1),
147.8 (C-3), 162.9 (CONH); GC-MS (EI) m/z: 327 [M]+.
3.1.8. General Experimental Procedures of 4ii–8ii
To a solution of 4i
amounts of 4i 8i) and iron powder (6.03 mmol) and refluxed for 7 h. The reaction mixture was
evaporated to give an oily residue, which was added with water and extracted with dichloromethane
(30 mL 3). The combined organic phase was dried with anhydrous MgSO4, filtrated, and the filtrate
–8i (3.37 mmol) in methanol (20 mL) was added: ammonium chloride (double
–
ˆ
was evaporated to yield a crude solid, which recrystallized with ethyl acetate and n-hexane to give a
pure white or pale yellow solid.
1
N-(41-Chlorophenyl)-3-aminobenzamide (4ii). Yield: 97%; m.p.: 160–161 ˝C; H-NMR (400 MHz,
DMSO-d6)
δ
: 5.32 (2H, s, NH2), 6.75 (1H, ddd, J = 7.9, 2.3, 1.0 Hz, H-4), 7.04 (1H, 1d, J = 8.0Hz,
1
H-6), 7.08 (1H, t, J = 2.0 Hz, H-2), 7.14 (1H, t, J = 7.7 Hz, H-5), 7.38 (2H, d, J = 8.9 Hz, 3 ,5 ), 7.80 (1H, d,
J = 8.9 Hz, H-21,61), 10.19 (1H, s, CONH); 13C-N1MR (100 MHz, DMSO-d6)
δ: 112.9 (C-2), 114.7 (C-6),
1
1
1
1
1
116.9 (C-4), 121.5 (C-2 ,6 ), 126.9 (C-5), 128.4 (C-3 ,5 ), 129.8 (C-4 ), 135.6 (C-1), 138.3 (C-1 ), 148.8 (C-3),
166.9 (CONH); GC-MS (EI) m/z: 246 [M]+.
1
˝
N-(4 -Bromophenyl)-3-aminobenzamide (5ii). Yield: 97%; m.p.: 176–177 C; 1H-NMR (400 MHz, DMSO-d6)
δ
: 5.32 (2H, s, NH2), 6.75 (1H, ddd, J = 7.9, 2.2, 0.9 Hz, H-4), 7.04 (1H, d, J = 8.0, Hz, H-6), 7.07 (1H,
t, J = 2.0 Hz, H-2), 7.14 (1H, t, J = 7.7 Hz, H-5), 7.51 (2H, d, J = 8.9 Hz, H-31,51), 7.75 (2H, d, J = 8.9 Hz
,
H-21,61), 10.18 (1H, s,1CONH); 13C-NMR (100 MHz, DMSO-d6)
δ
: 113.0 (C-2), 114.7 (C-6), 115.0 (C-41),
1
1
1
1
116.9 (C-4), 122.0 (C-2 ,6 ), 128.8 (C-5), 131.3 (C-3 ,5 ), 135.6 (C-1), 138.8(C-1 ), 148.8 (C-3), 166.5 (CONH);
GC-MS (EI) m/z: 290 [M ´ 1]+, 292 [M + 1]+.
1
˝
N-(4 -Fluorophenyl)-3-aminobenzamide (6ii). Yield: 76%; m.p.: 143–144 C; 1H-NMR (400 MHz, DMSO-d6)
: 5.29 (2H, s, NH2), 6.74 (1H, ddd, J = 7.9, 2.3, 0.9 Hz, H-4), 7.05 (1H, d, J = 7.6 Hz, H-6), 7.08 (1H,
t, J = 2.0 Hz, H-2), 7.12–7.23 (3H, m, H-5,31,51), 7.77 (2H, dd, J = 9.2, 5.1 Hz, H-21, H-61), 10.10 (1H, s,
CONH); 13C-NMR (100 MHz, DMSO-d6) : 115.8 (C-2), 117.5 (C-6), 117.9 (J = 22.0 Hz, C-31,51), 119.6
(C-4), 124.8 (J = 7.8 Hz, C-21,61), 131.6 (C-5), 138.5 (J = 2.6 Hz, H-11), 138.6 (C-1), 151.6 (C-3), 161.0
δ
δ
(J = 239.0 Hz, C-4 ), 167.1 (CONH); GC-MS (EI) m/z: 230 [M]+.
1

Molecules 2016, 21, 676
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˝
1
N-(41-Methoxyphenyl)-3-aminobenzamide (7ii). Yield: 86%; m.p.: 141–142 C; H-NMR (400 MHz,
DMSO-d6) : 3.73 (3H, s, CH3), 5.28 (2H, s, NH2), 6.73 (1H, dd, J = 7.5, 1.8 Hz, H-4), 6.90 (2H, d,
δ
J = 9.1 Hz, H-31, H-51), 7.04 (1H, d, J = 7.9 Hz, H-6), 7.08 (1H, t, J = 2.0 Hz, H-2), 7.13 (1H, t, J = 7.7 Hz
,
H-5), 7.65 (2H, d, J = 9.0 Hz, H-21,61), 9.92 (1H, s, CONH); 13C-N1MR (100 MHz, DMSO-d6)
δ: 55.1
1
1
1
1
(CH3), 112.9 (C-2), 113.7 (C-3 ,5 ), 114.6 (C-6), 116.6 (C-4), 121.8 (C-2 ,6 ), 128.7 (C-5), 132.5 (C-1 ), 136.0
(C-1), 148.7 (C-3), 155.3 (C-4 ), 165.9 (CONH); GC-MS (EI) m/z: 242 [M]+.
1
N-(411-Morpholinophenyl)-3-aminobenzamide (8ii). Yield: 16%; m.p.: 173–174 ˝C; 1H-NMR (400 MHz,
DMSO-d6) δ
: 3.06 (4H, t, J = 0.8 Hz, H-311, 511), 3.74 (4H, t, J = 0.8 Hz, H-211,611), 5.35 (2H, s, NH2),
6.73 (1H, d, J = 7.0 Hz, H-4), 6.91 (2H, d, J = 8.9 Hz, H-31,51), 7.12–7.21 (3H, m, H-2, H-5, 1H1 -6), 7.61
1
1
11
(2H, d, J = 8.9 Hz, H-2 ,6 ), 9.86 (1H, s, CONH); 13C-NMR (100 MHz, DMSO-d6)
δ: 51.8 (C-3 ,5 ), 69.0
(C-211,611), 115.9 (C-2), 117.7 (C-6), 118.1 (C-21,61), 119.5 (C-4), 124.2 (C-31, 51), 131.5 (C-5), 134.5 (C-11),
1
139.0 (C-1), 150.2 (C-4 ), 151.3 (C-3), 168.6 (CONH); GC-MS (EI) m/z: 297 [M]+.
3.1.9. General Experimental Procedures of 4–13
To a solution of thiophene-2-carboxylic acid or 5-chlorothiophene-2-carboxylic acid (7.8 mmol) in
anhydrous dichloromethane (40 mL) was added: triethylamine (8.6 mmol) and dropwise added oxalyl
chloride (10.1 mmol) at room temperature. The reaction mixture was refluxed for 1 h and evaporated
to remove the solvent and unreacted oxalyl chloride under reduced pressure. These acid chlorides
were used for the next reaction without purification. To a solution of 4ii–8ii (0.41 mmol) in anhydrous
benzene (20 mL) was added: triethylamine (0.41 mmol) and prepared thiophene-2-carbonyl chloride
or 5-chlorothiophene-2-carbonyl chloride (0.51 mmol) at room temperature. The reaction mixture was
stirred at room temperature for 2 h and evaporated to remove the benzene. To the residue, water was
added and extracted with ethyl acetate (30 mL
MgSO4, filtrated, and evaporated to prepare a crude compound, which was recrystallized with ethyl
ˆ
3). The organic phase was dried with anhydrous
acetate and n-hexane to give a pure white or pale yellow solid.
N-(41-Chlorophenyl)-3-(thiophen-211-ylcarbonylamino) benzamide ( ). Yield: 77%; m.p.: 252–253 ˝C;
4
1H-NMR (400 MHz, DMSO-d6)
δ
: 7.24 (1H, dd, J = 4.9, 3.8 Hz, H-411), 7.42 (2H, d, J =18.8 Hz, H-31,
1
1
H-5 ), 7.52 (1H, t, J = 7.9 Hz, H-5), 7.69 (1H, d, J = 7.8 Hz, H-6), 7.83 (2H, d, J = 8.9 Hz, H-2 ,6 ), 7.88 (1H,
dd, J = 4.9, 0.9Hz, H-511), 8.01 (1H, dd, J = 8.1, 1.2 Hz, H-4), 8.08 (1H, dd, J = 3.7, 1.0 Hz, H-311), 8.25
(1H, s, H-2), 10.41 (1H, s, NHCO), 10.44 (1H, s, CONH); 13C-NMR (100 MHz, DMSO-d6)
δ: 119.8 (C-2),
1
11
1
1
1
1
11
11
121.8 (C-2 ,6 ), 122.7 (C-6), 123.3 (C-4), 127.3 (C-4 ), 128.2 (C-5), 128.5 (C-3 ,5 ), 128.8 (C-4 ), 129.3 (C-3 ),
1
11
132.2 (C-5 ), 135.4 (C-1), 138.1 (C-1 ), 139.0 (C-3), 139.7 (C-2 ), 160.3 (CONH), 165.5 (NHCO); DIP-MS
(EI) m/z: 356 [M]+; HRMS (EI) Calcd. For C18H14ClN2O2S 357.0465 [M + H]+, found 357.0457.
N-(41-Bromophenyl)-3-(thiophen-211-ylcarbonylamino) benzamide (
5
). Yield: 65%; m.p.: 2631–2164 ˝C;
11
1H-NMR (400 MHz, DMSO-d6)
δ: 7.24 (1H, dd, J = 7.9, 3.8 Hz, H-4 ), 7.49–7.57 (3H, m, H-5,3 ,5 ), 7.69
(1H, d, J = 6.9 Hz, H-6), 7.77 (2H, d, J = 8.9 Hz, H-21,61), 7.88 (1H, dd, J = 5.0, 1.0 Hz, H-511), 8.01 (1H,
dd, J = 8.1, 1.2 Hz, H-4), 8.07 (1H, dd, J = 3.8, 1.0 Hz, H-311), 8.25 (1H, t, J = 1.8 Hz, H-2), 10.41 (1H,
s, NHCO), 10.44 (1H, s, CONH); 13C-NMR (100 MHz, DMSO-d6) : 115.3 (C-41), 119.8 (C-2), 122.2
δ
(C-21,61), 122.6 (C-6), 1123.3 (C-4), 128.1 (C-5),111 28.8 (C-411), 129.3 (C-311), 131.4 (C-31,51), 132.1 (C-511),
135.3 (C-1), 138.5 (C-1 ), 139.0 (C-3), 139.7 (C-2 ), 160.0 (CONH), 165.5 (NHCO); DIP-MS (EI) m/z: 400
[M ´ 1]+, 402 [M + 1]+ ; HRMS (EI) Calcd. For C18H14BrN2O2S [M + H+] 400.9959, found 400.9947.
N-(41-Fluorophenyl)-3-(thiophen-211-ylcarbonylamino) benzamide (
6
). Yield: 66%; m.p.: 236–237 ˝C;
1H-NMR (400 MHz, DMSO-d6) : 7.20 (2H, t, J = 8.9 Hz, H-31,51), 7.24 (1H, dd, J = 5.0, 3.8 Hz,
δ
H-411), 7.52 (1H, t, J = 7.9 Hz, H-5), 7.69 (1H, d, J = 7.6 Hz, H-6), 7.80 (2H, dd, J = 9.2, 5.1 Hz, H-21,61),
11
7.88 (1H, dd, J = 5.0, 1.1Hz, H-5 ), 8.00 (1H, ddd, J = 8.1, 2.1, 0.8 Hz, H-4), 8.08 (1H, dd, J = 3.8, 1.1 Hz,
H-311), 8.25 (1H, t, J = 1.9 Hz, H-2), 10.34 (1H, s, NHCO), 10.43 (1H, s, CONH); 13C-NMR (100 MHz,
DMSO-d6) δ
: 115.2 (J = 22.3 Hz, C-31,51), 119.8 (C-2), 122.1 (J = 7.4 Hz, C-21,61), 122.6 (C-6), 123.3 (C-4),

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128.2 (C-5), 128.7 (C-4111), 129.3 (C-311), 132.1 (C-511), 135.5 (J = 3.0 Hz, C-11), 138.9 (C-3), 139.8 (C-211),
158.3 (J = 238.7 Hz, C-4 ), 160.0 (CONH), 165.4 (NHCO); DIP-MS (EI) m/z: 340 [M]+ ; HRMS (EI) Calcd.
For C18H14FN2O2S [M + H]+ 341.0760, found 341.0751.
˝
N-(41-Methoxyphenyl)-3-(thiophen-211-ylcarbonylamino) benzamide (
7
). Yield: 671%; m.p.: 236–237 C;
1
1H-NMR (400 MHz, DMSO-d6)
δ
: 3.75 (3H, s, CH3), 6.93 (2H, d, J = 9.0 Hz, H-3 ,5 ), 7.24 (1H, dd, J = 4.6
,
4.0 Hz, H-411), 7.50 (1H, t, J = 7.9 Hz, H-5), 7.64–7.72 (3H, m, H-6,21,61), 7.88 (1H, d, J = 5.0 Hz, H-511),
8.00 (1H, dd, J = 8.0, 1.6 Hz, H-4), 8.11 (1H, d, J = 3.7 Hz, H-311), 8.25 (2H, t, J = 1.9 Hz, H-2), 10.17
(1H, s, NHCO), 10.46 (1H, s, CONH); 13C-NMR (100 MHz, DMSO-d6) : 55.2 (CH3), 113.7 (C-21,61),
δ
119.8 (C-2), 122.0 (C-31,51), 122.8 (C-6), 123.1 (C-4), 128.4 (C-5), 128.7 (C-411), 129.4 (C-311), 132.2 (C-511),
134.1 (C-11), 135.7 (C-1), 138.6 (C-3), 139.8 (C-211), 155.6 (C-41), 159.0 (CONH), 164.9 (NHCO); DIP-MS
(EI) m/z: 352 [M]+ ; HRMS (EI) Calcd. For C19H17N2O3S [M + H]+ 353.0960, found 353.0955.
N-(411-Morpholinophenyl)-3-(thiophen-2111-ylcarbonylamino) benzamide (
8
). Yield: 15%; m.p.: 254–255 ˝C;
2), 3.74 (4H, t, J = 0.8 Hz,
1H-NMR (400 MHz, DMSO-d6) : 3.07 (4H, t, J = 0.8 Hz, H-311 2, H-511
δ
ˆ
ˆ
H-211,611), 6.94 (2H, d, J = 9.1 Hz, H-31,51), 7.24 (1H, dd, J = 4.9, 3.9 Hz, H-4111), 7.49 (1H, t, J = 7.9 Hz,
H-5), 7.63 (2H, d, J = 9.0 Hz, H-21,61), 7.68 (1H, d, J = 7.8 Hz, H-6), 7.88 (1H, dd, J = 5.0, 0.9 Hz, H-5111),
7.98 (1H, dd, J = 8.1, 1.4 Hz, H-4), 8.07 (1H, dd, J = 3.8, 0.9 Hz, H-3111), 8.22 (1H, t, J =111.7 Hz, H-2),
11 11
10.09 (1H, s, NHCO), 10.41 (CONH); 13C-NMR (100 MHz, D)MSO-d6)
δ: 48.8 (C-3 , C-5 ), 66.1 (C-2 ,
11
1
1
1
1
11
C-6 ), 115.2 (C-2 ,6 ), 119.8 (C-2), 121.5 (C-3 ,5 ), 122.5 (C-6), 123.0 (C-4), 128.1 (C-5), 128.6 (C-4 ), 129.3
(C-311), 131.3 (C-11), 132.1 (C-511), 135.8 (C-1), 138.8 (C-3), 139.8 (C-211), 147.5 (C-41), 160.0 (CONH),
164.8 (NHCO); DIP-MS (EI) m/z: 407 [M]+; HRMS (EI) Calcd. For C22H22N2O3S [M + H]+ 408.1382,
found 408.1372.
1
11
˝
9). Yield: 77%; m.p.: 190–191 C;
N-(4 -Chlorophenyl)-3-(5-chlorothiophen-2 -ylcarbonylamino) benzamide (
1H-NMR (400 MHz, DMSO-d6) : 7.29 (1H, d, J = 4.1 Hz, H-411), 7.42 (2H, J = 8.9 Hz, H-31,51), 7.53
(1H, t, J = 7.9 Hz, H-5), 7.71 (1H, d, J = 7.8 Hz, H-6), 7.82 (2H, d, J = 8.9 Hz, H-21,61), 7.96–7.99 (2H, m,
δ
H-4,311), 8.22 (1H, t, J = 1.7 Hz, H-2), 10.41 (1H, s, NHCO), 10.51 (1H, s, CONH); 13C-NMR (100 MHz
DMSO-d6)
128.8 (C-4 ), 129.4 (C-3 ), 134.2 (C-1), 135.4 (C-3), 138.1 (C-1 ), 138.6 (C-2 ), 139.8 (C-5 ), 159.0 (CONH),
165.4 (NHCO); DIP-MS (EI) m/z: 390 [M
,
δ
: 119.8 (C-2), 121.8 (C-21,61), 122.9 (C-6), 123.4 (C-4), 127.3 (1C1 -), 128.4 (C-5), 128.5 (C-31,51),
11
11
1
11
´
1]+ ; HRMS (EI) Calcd. For C18H13Cl2N2O2S [M + H]+
391.0075, found 391.0066.
1
11
˝
N-(4 -Bromophenyl)-3-(5-chlorothiophen-2 -ylcarbonylamino) benzamide (10). Yield: 65%; m.p.: 271–272 C
1H-NMR (400 MHz, DMSO-d6)
;
,
δ
: 7.29 (1H, d, J = 4.1 Hz, H-411), 7.49–7.57 (3H, m, H-5, H-31,51), 7.71
1
1
11
(1H, d, J = 8.9 Hz, H-6), 7.77 (2H, d, J = 7.9 Hz, H-2 ,6 ), 7.95–8.01 (2H, m, H-4,3 ), 8.21 (1H, t, J = 1.7 Hz
H-2), 10.41 (1H, s, N1HCO), 10.51 (1H, s, CONH); 13C-NMR (100 MHz, DMSO-d6)
δ
: 116.1 (C-411),
1
11
11
11
1
120.5 (C-2), 122.9 (C-2 ,6 ), 123.6(C-6), 124.1 (C-4), 129.1 (C-4 ), 129.5 (C-3 ), 130.0 (C-5), 132.1 (C-3 ,5 ),
1
11
134.9 (C-1), 136.1 (C-3), 139.2 (C-1 ), 139.3 (C-2 ), 139.5 (C-5 ), 159.7 (CONH), 166.1 (NHCO); DIP-MS
(EI) m/z: 434 [M
found 434.9559.
´
1]+, 436 [M + 1]+; HRMS (EI) Calcd. For C18H14BrClN2O2S [M + H]+ 434.9570,
1
11
˝
N-(4 -Fluorophenyl)-3-(5-chlorothiophen-2 -ylcarbonylamino) benzamide (11). Yield: 75%; m.p.: 257–258 C;
1H-NMR (400 MHz, DMSO-d6) : 7.20 (2H, t, J = 8.9 Hz, H-31,51), 7.29 (1H, d, J = 4.1 Hz, H-411), 7.52
δ
(1H, t, J = 7.9 Hz, H-5), 7.71 (1H, d, J = 8.0 Hz, H-6), 7.79 (2H, dd, J = 9.2, 5.1 Hz, H-21,61), 7.96–7.99
(2H, m, H-4,311), 8.22 (1H, t, J = 1.8 Hz, H-2), 10.34 (1H, s, NHCO), 10.51 (1H, s, CONH); 13C-NMR
(100 MHz, DMSO-d6) δ
: 115.9 (J = 21.6 Hz, C-21,61), 119.9 (C-2), 122.2 (J = 7.5 Hz, C-31,51), 122.9 (C-6),
123.3 (C-4), 128.4 (C-5), 128.8 (C-4 ), 129.4 (C-3 ), 134.2 (C-1), 135.4 (C-3), 135.5 (J = 2.2 Hz, C-1 ), 138.6
(C-211), 138.9 (C-511), 158.3 (J = 248.8 Hz, C-41), 159.0 (CONH), 165.3 (NHCO); DIP-MS (EI) m/z: 374
[M]+; HRMS (EI) Calcd. For C18H14ClFN2O2S [M + H]+ 375.0370, found 375.0363.
11
11
1

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N-(41-Methoxyphenyl)-3-(5-chlorothiophen-211-ylcarbonylamino) benzamide (12). Yield: 88%; m.p.:
˝
1
234–235 C; H-NMR (400 MHz, DMSO-d6) δ
: 3.75 (3H, s, CH3), 6.93 (2H, d, J = 9.0 Hz, H-31,51),
11
1 1
7.29 (1H, d, J = 4.1 Hz, H-4 ), 7.50 (1H, t, J = 7.9 Hz, H-5), 7.66–7.71 (3H, m, H-6, H-2 ,6 ), 7.95–7.99 (2H,
11
m, H-4,3 ), 8.21 (1H, t, J = 1.7 Hz, H-2), 10.16 (1H, s, NHCO), 10.52 (1H, s, CONH); 13C-NMR (100 MHz,
1 1
1 1
DMSO-d6) δ: 55.2 (CH3), 113.7 (C-2 ,6 ), 119.8 (C-2), 121.9 (C-3 ,5 ), 122.8 (C-6), 123.1 (C-4), 128.4 (C-5),
128.7 (C-411), 129.3 (C-311), 132.2 (C-11), 134.1 (C-1), 135.7 (C-3), 138.5 (C-211), 138.9(C-511), 155.5 (C-41),
158.9 (CONH), 164.9 (NHCO); DIP-MS (EI) m/z: 386 [M]+ ; HRMS (EI) Calcd. For C19H16ClN2O3S
[M + H]+ 387.0570, found 387.0565.
N-(411-Morpholinophenyl)-3-(5-chlorothiophen-2111-ylcarbonylamino) benzamide (13). Yield: 12%; m.p.:
˝
11 11
2), 3.74 (4H, t, J = 0.8 Hz
269–270 C; 1H-NMR (400 MHz, DMSO-d6)
δ
: 3.07 (4H, t, J = 0.8 Hz, H-3 ,5
ˆ
,
H-211,611), 6.94 (2H, d, J = 9.1 Hz, H-31,51), 7.29 (1H, d, J = 4.1 Hz, H-4111), 7.50 (1H, t, J = 7.9 Hz, H-5),
7.63 (2H, d, J = 9.1 Hz, H-21,61), 7.70 (1H, d, J = 7.8 Hz, H-6), 7.94–7.96 (2H, m, H-4,3111), 8.19 (1H, t,
J = 1.8 Hz, H-2), 101.01 91(11H, s, NHCO), 10.49 (1H, s, CONH); 13C-NMR (100 MHz, DMSO-d6)
δ: 49.8
11 11
1
1
1 1
(C-3 ,5 ), 55.1 (C-2 ,6 ), 115.2 (C-2 ,6 ), 119.8 (C-2), 121.5 (C-3 ,5 ), 122.7 (C-6), 123.0 (C-4), 128.3 (C-5),
128.7 (C-5), 129.3 (C-311), 131.2 (C-11), 134.1 (C-1), 135.8 (C-3), 138.5 (C-211), 138.9 (C-511), 147.5 (C-411),
159.9 (CONH), 164.7 (NHCO); DIP-MS (EI) m/z: 441 [M]+ ; HRMS (EI) Calcd. For C22H21ClN3O3S
[M + H]+ 442.0992, found 442.0983.
3.2. Biology
3.2.1. Reagents and Instruments
Heparin was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) and tumor necrosis
factor-α (TNF-α) was purchased from Abnova Co. (Taipei, Taiwan). Anti-tissue factor (TF) anti-body
was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA) and U46619 was purchased from
Calbiochem-Novabiochem Co. (San Diego, CA, USA). Factor VIIa, X, Xa, pro-thrombin, and thrombin
were purchased from Haematologic Technologies (Essex Junction, VT, USA) and aPTT (APTT-XL)
and PT (Thromboplastin-D) assay reagent was purchased from Fisher Diagnostics (Middletown,
VA, USA). S-2222 (for Factor Xa) and S-2238 (for thrombin) were purchased from Chromogenix AB
(Mölndal, Sweden). Aargatroban (direct thrombin (factor IIa) inhibitor) and rivaroxaban (direct factor
Xa inhibitor) were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Microplate reader
(Tecan Austria GmbH, Grödig, Austria), thrombotimer (Behnk Elektronik, Norderstedt, Germany),
aggregometer (Chronlog, Havertown, PA, USA), spectrophotometer (TECAN, Männedorf, Switzerland)
were used.
3.2.2. Preparation of Plasma
Blood samples were taken in the morning from 10 healthy volunteers in fasting status (aged
between 24 and 28 years, four males and six females) without cardiovascular disorders, allergy, and
lipid or carbohydrate metabolism disorders, and untreated with drugs. All subjects gave written
informed consent before participation. Healthy subjects did not use additive substances or antioxidant
supplementation, and their diet was balanced (meat and vegetables). Human blood was collected into
sodium citrate (0.32% final concentration) and immediately centrifuged (2000
obtain plasma. The study protocol (KNUH 2012-01-010) was approved by the Institutional Review
Board of Kyungpook National University Hospitals (Daegu, Republic of Korea).
ˆ
15 min) in order to
3.2.3. In Vitro Anticoagulant Assay
aPTT and PT were determined using a Thrombotimer (Behnk Elektronik, Norderstedt, Germany),
according to the manufacturer1s instructions, as described previously [36]. In brief, citrated normal
˝
human plasma (90
assay reagent (100
µ
L) was mixed with 10
µ
L of target compounds incubated for 1 min at 37 C, aPTT
µ
L) was added and incubated for 1 min at 37 ˝C, followed by addition of 20 mM

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CaCl2 (100
was mixed with 10
reagent (200
L), which had been pre-incubated for 10 min at 37 ˝C, was then added and clotting
µ
L). Clotting times were recorded. For PT assays, citrated normal human plasma (90
µL)
µ
L of synthesized compounds stock and incubated for 1 min at 37 ˝C. PT assay
µ
time was recorded. PT results are expressed in seconds and as International Normalized Ratios (INR),
and aPTT results are expressed in seconds. INR = (PT sample/PT control)ISI, ISI = International
sensitivity index.
3.2.4. In Vivo Bleeding Time
Tail bleeding times were measured using the method described by Dejana et al. [37]. Briefly,
ICR mice were fasted overnight before experiments. One hour after intravenous administration
of synthesized compounds, tails of mice were transected at 2 mm from their tips. Bleeding time
was defined as the time elapsed until bleeding stopped. When the bleeding time exceeded 15 min,
bleeding time was recorded as 15 min for the analysis. All animals were treated in accordance with the
guidelines for the Care and Use of Laboratory Animals issued by Kyungpook National University.
3.2.5. Thrombin-Catalyzed Fibrin Polymerization
Thrombin-catalyzed polymerization was determined every 6 s for 20 min by monitoring turbidity
at 360 nm using a spectrophotometer (TECAN, Männedorf, Switzerland) at ambient temperature.
Control plasma and plasma incubated with synthesized compounds were diluted three times in
TBS (50 mM Tris-buffered physiological saline solution pH 7.4) and clotted with thrombin (final
concentration-0.5 U/mL). The maximum polymerization rate (Vmax,
curve was recorded. All experiments were performed three times.
∆mOD/min) of each absorbance
3.2.6. Platelet Aggregation Assay
Mouse platelets from platelet-rich plasma (PRP) were washed once with HEPES buffer (5 mM
HEPES, 136 mM NaCl, 2.7 mM KCl, 0.42 mM NaH2PO4, 2 mM MgCl2, 5.6 mM glucose, 1% BSA (w/v)
,
pH to 7.45). The platelet aggregation study was performed according to a previously reported
method [38]. Washed plasma was incubated with the indicated concentration of compounds
for 3 min, and followed by stimulation with thrombin (0.1 U/mL, Sigma) and TxA2 analog, U46619
(2
M) in 0.9% saline at 37 ˝C for 5 min. Platelet aggregations were recorded using an aggregometer
1 and 2
µ
(Chronolog, Havertown, PA, USA).
3.2.7. Thrombin Activity Assay
Compounds
with 150 mM NaCl. Following incubation at 37 C for 2 min, factor Xa solution (150
added, followed by incubation at 37 ˝C for 1 min. S-2238 as substrate (150
L; 1.5 mM) solution was
1 and 2 in 50 nM Tris-HCl buffer (pH 7.4) containing 7.5 mM EDTA were mixed
˝
µL; 10 U/ mL) was
µ
then added and absorbance at 405 nm was monitored for 120 s using a spectrophotometer (TECAN,
Männedorf, Switzerland).
3.2.8. Factor Xa Activity Assay
These assays were performed in the same manner as the thrombin activity assay, but using
thrombin FXa (150 µL; 1 U/mL) and S-2222 (a factor Xa substrate) as substrate.
3.2.9. Cell Culture
Primary HUVECs were obtained from Cambrex Bio Science (Charles City, IA, USA) and
were maintaine˝d using a previously described method [39
,40]. Briefly, cells were cultured until
confluent at 37 C at 5% CO2 in EBM-2 basal media supplemented with growth supplements
(Cambrex Bio Science, Charles City, IA, USA).

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3.2.10. Thrombin Generation on the Surfaces of HUVECs
Measurement of thrombin generation of HUVECs was quantitated as previously described [36].
Briefly, HUVECs were pre-incubated in 300 L containing synthesized compounds in 50 mM Tris-HCl
buffer, 100 pM FVa, and 1 nM FXa for 10 min, followed by the addition of prothrombin to a final
concentration of 1 M. After 10 min, duplicate samples (10 L each) were transferred to a 96-well plate
µ
µ
µ
containing 40 µL of 0.5 M EDTA in Tris-buffered saline per well in order to determinate prothrombin
activation. Activated prothrombin was determined by measuring the rate of hydrolysis of S2238
at 405 nm. Standard curves were prepared using amounts of purified thrombin.
3.2.11. Factor Xa Generation on the Surfaces of HUVECs
TNF-
α
(10 ng/mL for 6 h in serum-free medium) stimulated confluent monolayers of HUVECs
and for 10 min) in 96-well culture plates were
(preincubated with the indicated concentrations of
1
2
incubated with FVIIa (10 nM) in buffer B (bu˝ffer A supplemented with 5 mg/mL bovine serum
albumin (BSA) and 5 mM CaCl2) for 5 min at 37 C. FXa (175 nM) was added to the cells (final reaction
mixture volume, 100 µL) and incubated for 15 min. The reaction was stopped by the addition of buffer
A (10 mM HEPES, pH 7.45, 150 mM NaCl, 4 mM KCl, and 11 mM glucose) containing 10 mM EDTA,
and the amounts of FXa generated that were measured at 405 nm over 2 min were monitored using a
microplate reader. Initial rates of color development were converted into FXa concentrations using a
standard curve prepared with known dilutions of purified human FXa.
3.2.12. Cell Viability Assay
MTT was used as an indicator of cell viability. Cells were grown in 96-sell plates at a density
of 5 ˆ 103/well. After 24 h, cells were washed with fresh medium, followed by treatment with
compounds
1 and 2. After a 48 h incubation period, cells were washed and 100 µL of 1 mg/mL
MTT was added, followed by incubation for 4 h. Finally, 150 µL DMSO was added in order to
solubilize the formazan salt formed, the amount of which was determined by measuring the absorbance
at 540 nm using a microplate reader (Tecan Austria GmbH, Grödig, Austria). Data were expressed as
mean ˘ SEM of least three independent experiments.
3.2.13. Statistical Analysis
Data are expressed as mean SEM (standard error of the mean) of at least three independent
experiments. Statistical significance between the two groups was determined using the Student’s t-test.
Statistical significance was accepted for p values < 0.05.
4. Conclusions
Among thirteen amidino- and non-amdinobenzamides synthesized, two amidine compounds,
1 and 2, showed prolongation in aPTT in vitro and ex vivo, and in vivo bleeding time, respectively. These
compounds inhibited thrombin-catalyzed fibrin polymerization and platelet aggregation induced by
U46619. The activities of FXa and thrombin as well as the generation of thrombin and FXa in HUVECs
were dose-dependently inhibited. This study demonstrated that the amidine group in the meta or para
position of the B-ring of
1 and 2 are essential for U46619 antagonist and FXa and thrombin inhibitor.
Compounds and seem to be promising for the development of drugs combining anticoagulant
1
2
and antiplatelet activities in the same molecule. Currently, further structural optimization and the
modification of this scaffold are well under way in our laboratory.
Acknowledgments: This research was supported by Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0013516).
Author Contributions: S.H.L. and W.L. performed the research and analyzed the data; J.-S.B. and E.M. wrote the
paper. All authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2016, 21, 676
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Sample Availability: Samples of the compounds 1–13 are available from the authors.
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