Novel anthranilamide-based fxa inhibitors: Drug design, synthesis and biological evaluation

Article abstract of DOI:10.3390/molecules21040491

Factor Xa (FXa) plays a significant role in the blood coagulation cascade and it has become a promising target for anticoagulation drugs. Three oral direct FXa inhibitors have been approved by the FDA for treating thrombotic diseases. By structure-activity relationship (SAR) analysis upon these FXa inhibitors, a series of novel anthranilamide-based FXa inhibitors were designed and synthesized. According to our study, compounds 1a, 1g and 1s displayed evident FXa inhibitory activity and excellent selectivity over thrombin in in vitro inhibition activities studies. Compounds 1g and 1s also exhibited pronounced anticoagulant activities in in vitro anticoagulant activity studies.

Full text of DOI:10.3390/molecules21040491

molecules
Article
Novel Anthranilamide-Based FXa Inhibitors:
Drug Design, Synthesis and Biological Evaluation
Wenzhi Wang 1,2, Jing Yuan 2, Xiaoli Fu 2, Fancui Meng 2, Shijun Zhang 2, Weiren Xu 2,
Yongnan Xu 1,* and Changjiang Huang 2,
*
1
School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China;
wangwenzhi870507@163.com
Tianjin Key Laboratory of Molecular Design and Drug Discovery, Tianjin Institute of Pharmaceutical
Research, Tianjin 300193, China; yuanj@tjipr.com (J.Y.); fuxl@tjipr.com (X.F.); mengfc@tjipr.com (F.M.);
zhangsj@tjipr.com (S.Z.); xuwr@tjipr.com (W.X.)
2
*
Correspondence: wzzyyx2016@163.com (Y.X.); huangcj@tjipr.com (C.H.);
Tel.: +86-24-2398-6445 (Y.X.); +86-22-2300-6833 (C.H.)
Academic Editor: Jean Jacques Vanden Eynde
Received: 2 February 2016; Accepted: 7 April 2016; Published: 14 April 2016
Abstract: Factor Xa (FXa) plays a significant role in the blood coagulation cascade and it has become
a promising target for anticoagulation drugs. Three oral direct FXa inhibitors have been approved by
the FDA for treating thrombotic diseases. By structure-activity relationship (SAR) analysis upon these
FXa inhibitors, a series of novel anthranilamide-based FXa inhibitors were designed and synthesized.
According to our study, compounds 1a, 1g and 1s displayed evident FXa inhibitory activity and
excellent selectivity over thrombin in in vitro inhibition activities studies. Compounds 1g and 1s also
exhibited pronounced anticoagulant activities in in vitro anticoagulant activity studies.
Keywords: thrombosis; FXa inhibitor; anthranilamide-based FXa inhibitors; docking simulation
1. Introduction
Thromboembolic diseases such as myocardial infarction (MI), pulmonary embolism (PE),
deep vein thrombosis (DVT) and ischemic strokes are the major causes of mortality all over the
world in the 21st century [
anticoagulant drugs [ ]. For example, vitamin K antagonists (VKAs), low-molecular-weight heparins
(LMWHs) and warfarin are validated in the prevention and treatment of these thrombotic disorders [ ],
but they still have many shortcomings in clinical applications including the inconvenience of frequent
monitoring, interactions with many drugs and food, and slow onset action [ ]. These shortcomings
1–3]. The typical method for treating and preventing these diseases is to use
4
5
6,7
further accelerate the need for development of new oral anticoagulants with efficacy and safety.
Factor Xa (FXa), which is located at the junction of the intrinsic and extrinsic pathways in the
coagulation cascade and catalyzes the conversion of prothrombin to thrombin, plays a pivotal role
in the blood coagulation cascade and has become a promising target for anticoagulation effects [4,8].
It is well demonstrated that selective FXa inhibitors can decrease the generation of thrombin without
affecting the existing thrombin level which means that selective FXa inhibitors can play an ideal
antithrombotic effects without influencing normal hemostasis and decrease the risk of bleeding [
Three oral direct FXa inhibitors have been approved by the FDA for treating thrombotic diseases and
several FXa inhibitors have entered the stage of clinical research or biological testing (Figure 1) [ 12 14].
9–11].
2,
–
However, they still have many drawbacks, such as drug interactions, narrow indications and long
treatment duration [15]. Therefore, new antithrombotic drugs still need to be developed for addressing
these issues.
Molecules 2016, 21, 491; doi:10.3390/molecules21040491
www.mdpi.com/journal/molecules

Molecules 2016, 21, 491
2 of 16
Figure 1. Oral direct FXa inhibitors.
From the structures above, FXa inhibitors have three components which make up the
pharmacophore: core scaffold, P1 and P4. This typical structure can help these molecules combine with
FXa, P1 locates at the S1 pocket and P4 locates at the S4 pocket (Figure 2) [16]. Presented if Figure 3
here are several examples can help us understand this more clearly [17–23].
Figure 2. X-ray crystal structure of rivaroxaban in complex with human FXa.
Interestingly, we found that for both rivaroxaban and betrixaban employed a carboxamide group
to connect the scaffold, P1 and P4. Based on analysis of the X-ray crystal structure of rivaroxaban
complexed with human FXa, it was found that there are two hydrogen bonds formed between
rivaroxaban and the residue Gly219 in FXa [12]. Similarly, betrixaban possesses two hydrogen bonds
with residues Gly218 and Gly216 in FXa [24], so we hypothesized that the key factor for FXa inhibitors
docked to human FXa might be the carboxamide group. Based on the structures of rivaroxaban,
betrixaban and darexaban, we have designed a series of novel FXa inhibitors employing anthranilamide

Molecules 2016, 21, 491
3 of 16
as the scaffold (Figure 4). Through metabolism of rivaroxaban in vivo
[
25], we can find that the
main metabolic pathway is the hydrolysis of the amide bond. We reversed the carbonyl and amino
connection order of betrixaban with the expectation that the designed compounds may display different
toxicity data or participate in different metabolic pathways in humans.
Figure 3. Examples of the molecules with typical structure.
Figure 4. Design of anthranilamide-based FXa inhibitors.

Molecules 2016, 21, 491
4 of 16
2. Results and Discussion
2.1. Synthesis
The synthetic routes used in this study are illustrated in Scheme 1. All the starting material are
commercial available. As shown in Scheme 1, the acylation [26] of 3a–3b respectively with a series of
o-nitrobenzoyl chloride 2a
provide the corresponding anilines 5a
6a–6c yielded the target compounds 1a–1x.
–
2d provided 4a
–
4h, which were subsequently reduced with Zn, NH4Cl to
–
5h in satisfied yields. Acylation [26] of 5a
–
5h respectively with
Scheme 1. Synthetic route of compounds 1a–1x. Reagents and conditions: (i) THF, K2CO3, DMAP, reflux,
2 h; (ii) Zn, NH4Cl, H2O, THF, MeOH, 40 ˝C, 2 h; (iii) THF, K2CO3, DMAP, reflux, 4 h.
2.2. Biological Activities and Discussion
2.2.1. In Vitro Inhibition Activity Studies on FXa
All the targeted compounds were evaluated in vitro for investigating their FXa inhibitory activity,
using rivaroxaban as the positive control in this assay. The assay results (Table 1) showed that several
designed compounds exhibited inhibitory activity against FXa with IC50 values at the nanomole level
from 951.3 to 23.0 nM. In particular compound 1g was the most promising FXa inhibitor in this series
with an IC50 value of 23.0 nM. The results indicated that the compounds with a 3-methyl-substituted
scaffold (1j
micromole level no matter what kind of Ar1 and Ar2 in Table 1 they linked with. When the Ar1 group
was pyridin-2(1H)-one, the compounds (1a 1l) demonstrated that the affinity with FXa ranking was
–1l, 1v–1x) possessed relative poor inhibitory activity against FXa, with IC50 values at a
–
electron withdrawing group substituted scaffold < electron donating group scaffold < non-substituted
scaffold. Through Table 1 can also be found that using 5-chlorothiophene as P1 (Ar2) the anticoagulant
activity was much better than in other cases (1a = 30.9 nM, Ki = 22.0 nM; 1g = 23.0 nM, Ki = 16.4 nM;
1s = 76.2 nM, Ki = 54.4 nM; Km = 2.4). We also show an inhibition profile figure of the most potent
compounds 1a, 1g and 1s in Figure 5.

Molecules 2016, 21, 491
5 of 16
110.00%
90.00%
70.00%
50.00%
30.00%
10.00%
Blank
0.25
0.2
3.1 nM
10.3 nM
31 nM
103.5 nM
310.4 nM
1 nM
0.15
1034.5 nM
3103.4 nM
0.1
0
0
1
2
3
4
10
20
30
40
No. 1a
Time (min)
No. 1a
lg Concentration
0.25
0.2
110.00%
90.00%
70.00%
50.00%
30.00%
10.00%
Blank
3.1 nM
10.3 nM
31 nM
103.5 nM
310.4 nM
1 Nm
0.15
1034.5 nM
3103.4 nM
0.1
0
10
No. 1g
20
30
40
0
1
2
3
4
Time (min)
No. 1g
lg Concentration
0.25
0.2
Blank
110.00%
90.00%
70.00%
50.00%
30.00%
10.00%
3.1 nM
10.3 nM
31 nM
103.5 nM
310.4 nM
1 nM
0.15
1034.5 nM
3103.4 nM
0.1
0
10
No. 1s
20
30
40
0
1
2
3
4
No. 1s
lg Concentration
Time (min)
Figure 5. The inhibition profile figures of 1a, 1g and 1s.

Molecules 2016, 21, 491
6 of 16
Table 1. The structures of the target compounds 1a–1x and their biological activity evaluation.
Compound
R
Ar1
Ar2
IC50 (nM)
1a
1b
1c
1d
1e
1f
H
30.2
H
>1000
197.3
430.1
>1000
570.5
25.0
H
5-chloro
5-chloro
5-chloro
5-methyl
5-methyl
5-methyl
3-methyl
3-methyl
1g
1h
1i
>1000
510.8
>1000
>1000
1j
1k
1l
1m
1n
1o
3-methyl
>1000
317.4
>1000
439.6
H
H
H

Molecules 2016, 21, 491
7 of 16
Table 1. Cont.
Compound
R
Ar1
Ar2
IC50 (nM)
1p
1q
1r
5-chloro
110.7
5-chloro
5-chloro
5-methyl
5-methyl
5-methyl
3-methyl
3-methyl
3-methyl
>1000
951.3
71.0
1s
1t
>1000
>1000
>1000
>1000
1u
1v
1w
1x
>1000
0.9
Rivaroxaban
2.2.2. Selectivity vs. Thrombin and Prothrombin Time (PT) Assay
To evaluate the inhibitory activity against FXa of compounds 1a
,
1g and 1s more accurately,
these compounds were chosen to assess degree of selectivity versus thrombin and the extension of the
prothrombin time (PT). Compounds 1a 1g and 1s showed no inhibition effect on thrombin, with IC50
values far higher than 10 M. They showed similar selectivity against thrombin as rivaroxaban which
is far more than 6.9 M [27]. The prothrombin time (PT) assay results are shown in Table 2, where
,
µ
µ
compounds 1g and 1s also show good anticoagulant activity, judging by their 2
24.2 µM in rat plasma and 12.8 µM and 10.4 µM in human plasma.
ˆ
PT value of 19.7 and
Table 2. The anticoagulant activity of 1g and 1s.
Compound
2 ˆ PT (µM) (Rat)
2 ˆ PT (µM) (Human)
1g
1s
19.7
24.2
0.3
12.8
10.4
0.2
Rivaroxaban
2.2.3. Docking Simulation Studies on the Interaction of Compounds 1g and 1s with FXa
Computer-based docking simulation studies were used to analyze the binding mode of
compounds 1g and 1s at the active site of FXa. As shown in Figure 6, the pyridine moiety of 1g
and morpholino unit of 1s are located at the sides of the phenyl groups of Tyr99 and Phe174 of the

Molecules 2016, 21, 491
8 of 16
S4 aryl binding pocket, while its aryl ring is oriented perpendicularly, extending across the face of
Trp215. Two hydrogen bonds are formed between 1g and the residue Gly216 and Gly218 in FXa and
one hydrogen bond is formed between 1s and the amino acid Gly218 of FXa. In the S1 pocket of FXa,
there is an interaction between the chlorine substituent of the thiophene moiety and the aromatic ring
of Tyr228 at the bottom of the S1 pocket. They thus show similar interactions with FXa as rivaroxaban
reported before [12].
Figure 6. The interactions of compounds 1g and 1s with the active site of FXa.
3. Materials and Methods
3.1. General Information
Reagents and solvents were obtained from commercial suppliers and used as received without
1
further purification. All reactions were monitored by thin layer chromatography. H-NMR spectra
(400 MHz) were recorded for DMSO-d6 solutions on an AV400 NMR (Bruker, Billerica, MA, USA),
MS were measured on a Finnigan LCQ Mass (Thermo Fisher Scientific, Cambridge, MA, USA),
HRMS were measured on a miorOTOF-QII instrument (Bruker Daltonics, Billerica, MA, USA) and
melting points (uncorrected) were determined on a YRT-3 Melting Point Tester (Precision Instrument
of Tianjin University, Tianjin, China).
3.2. Chemistry
2-Nitro-N-(4-(2-oxopyridin-1(2H)-yl)phenyl)benzamide (4a). To a stirred solution of 1-(4-aminophenyl)
pyridin-2(1H)-one (3a) (0.90 g, 4.8 mmol), K2CO3 (0.80 g, 5.8 mmol) and DMAP (0.05 g, 0.4 mmol) in
THF (20 mL), solution of 2-nitrobenzoyl chloride (2a) (1.15 g, 6.24 mmol) in THF (5 mL) was added
at room temperature and the mixture was refluxed for 2 h. The reaction mixture was cooled down
to room temperature and concentrated under reduced pressure. Then water (100 mL) was added
to the mixture and stirred for 10 min at room temperature. The resulting precipitate was collected
by filtration. The reaction was monitored by TLC with EA. White solid product (1.46 g, 90%). MS:
1
[M + H]+ 336.22. H-NMR:
δ
ppm 6.30 (t, J = 6.4 Hz, 1H), 6.47 (d, J = 9.2 Hz, 1H), 7.39 (d, J = 8.8 Hz,
2H), 7.49 (t, J = 4.4 Hz, 1H), 7.63 (d, J = 6.8 Hz, 1H), 7.76–7.80 (m, 4H), 7.89 (t, J = 7.6 Hz, 1H), 8.17 (d,
J = 8.0 Hz, 1H), 10.84 (s, NH).
5-Chloro-2-nitro-N-(4-(2-oxopyridin-1(2H)-yl)phenyl)benzamide (4b). Compound 4b was prepared from
3a and 5-chloro-2-nitrobenzoyl chloride (2b) according to the procedure described for the preparation
1
of 4a. White solid product (1.64 g, 92%). MS: [M + H]+ 370.05. H-NMR:
δ ppm 6.31 (t, J = 6.8 Hz, 1H),

Molecules 2016, 21, 491
9 of 16
6.47 (d, J = 9.2 Hz, 1H), 7.40 (d, J = 8.4 Hz, 2H), 7.50 (t, J = 2.4 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.76 (d,
J = 6.8 Hz, 2H), 7.86 (d, J = 6.4 Hz, 1H), 8.00 (s, 1H), 8.20 (d, J = 8.8 Hz, 1H), 10.88 (s, NH).
5-Methyl-2-nitro-N-(4-(2-oxopyridin-1(2H)-yl)phenyl)benzamide (4c). Compound 4c was prepared from 3a
and 5-methyl-2-nitrobenzoyl chloride (2c) according to the procedure described for the preparation
1
of 4a. White solid product (1.50 g, 89%). MS: [M + H]+ 350.42. H-NMR:
δ ppm 2.43 (s, 3H), 6.31 (t,
J = 6.4 Hz, 1H), 6.47 (d, J = 9.2 Hz, 1H), 7.38 (d, J = 8.8 Hz, 2H), 7.50 (t, J = 4.8 Hz, 1H), 7.55–7.64 (m,
3H), 7.76 (d, J = 9.2 Hz, 2H), 8.08 (d, J = 8.4 Hz, 1H), 10.78 (s, NH).
3-Methyl-2-nitro-N-(4-(2-oxopyridin-1(2H)-yl)phenyl)benzamide (4d). Compound 4d was prepared from
3a and 3-methyl-2-nitrobenzoyl chloride (2d) according to the procedure described for the preparation
1
of 4a. White solid product (1.54 g, 91%). MS: [M + H]+ 350.10. H-NMR:
δ
ppm 2.36 (s, CH3), 6.30 (t,
J = 6.4 Hz, 1H), 6.46 (d, J = 9.2 Hz, 1H), 7.38–7.40 (m, 2H), 7.49 (t, J = 8.8 Hz, 1H), 7.62 (d, J = 8.8 Hz,
1H), 7.64–7.67 (m, 2H), 7.69–7.73 (m, 1H),7.77 (d, J = 8.8 Hz, 2H), 10.87 (s, NH).
2-Nitro-N-(4-(3-oxomorpholino)phenyl)benzamide (4e). Compound 4e was prepared from 4-(4-aminophenyl)
morpholin-3-one (3b) and 2a according to the procedure described for the preparation of 4a. White
1
solid product (1.54 g, 93 %). MS: [M + H] + 342.09. H-NMR:
δ
ppm 3.71 (t, J = 4.8 Hz, CH2), 3.97 (t,
J = 4.8 Hz, CH2), 4.19 (s, 2H), 7.37 (d, J = 8.8 Hz, 2H), 7.67 (d, J = 8.8 Hz, 2H), 7.74–7.78 (m, 2H), 7.87 (t,
J = 7.6 Hz, 1H), 8.15 (d, J = 8.0 Hz, 1H), 10.72 (s, NH).
5-Chloro-2-nitro-N-(4-(3-oxomorpholino)phenyl)benzamide (4f). Compound 4f was prepared from 3b and
2b according to the procedure described for the preparation of 4a. White solid product (1.67 g, 94%).
1
MS: [M + H]+ 376.05. H-NMR:
δ
ppm 3.72 (t, J = 10.0 Hz, 2H), 3.97 (t, J = 10.0 Hz, 2H), 4.19 (s, 2H),
7.38 (d, J = 8.4 Hz, 2H), 7.66 (d, J = 8.8 Hz, 2H), 7.84 (d, J = 8.8 Hz, 2H), 7.94 (s, 1H), 8.18 (d, J = 8.8 Hz,
2H), 10.77 (s, NH).
5-Methyl-2-nitro-N-(4-(3-oxomorpholino)phenyl)benzamide (4g). Compound 4g was prepared from 3b
and 2c according to the procedure described for the preparation of 4a. White solid product (1.55 g,
1
90%). MS: [M + H]+ 355.99. H-NMR:
δ ppm 2.47 (s, CH3), 3.71 (t, J = 4.8 Hz, CH2), 3.97 (t, J = 4.8 Hz,
CH2), 4.19 (s, CH2), 7.37 (d, J = 8.8 Hz, 2H), 7.55 (d, J = 8.8 Hz, 2H), 7.57 (s, 1H), 7.67 (d, J = 8.8 Hz, 2H),
8.07 (d, J = 8.4 Hz, 1H), 10.66 (s, NH).
3-Methyl-2-nitro-N-(4-(3-oxomorpholino)phenyl)benzamide (4h). Compound 4h was prepared from 3b
and 2d according to the procedure described for the preparation of 4a. White solid product (1.53 g,
1
89%). MS: [M + H]+ 356.19. H-NMR:
δ ppm 2.36 (s, CH3), 3.72 (t, J = 4.8 Hz, CH2), 3.98 (t, J = 4.8 Hz,
CH2), 4.20 (s, CH2), 7.38 (d, J = 8.8 Hz, 2H), 7.65–7.24 (m, 5H), 10.76 (s, NH).
2-Amino-N-(4-(2-oxopyridin-1(2H)-yl)phenyl)benzamide (5a). To a 250 mL round bottom flask, 4a (1.40 g,
3.9 mmol), zinc powder (2.05 g, 31.2 mmol), NH4Cl (2.11 g, 39 mmol), methanol (30 mL), THF (30 mL)
˝
and water (15 mL) were added. The mixture stirred at 40 C for 2 h. The reaction mixture was filtered,
washed with DMF and the filtrate was concentrated under reduced pressure. Then water (200 mL)
was added to the mixture and stirred for 0.5 h. The residue was filtered and washed with water to
yield the title compound as a white solid product (1.13 g, 89%). The reaction was monitored by TLC
1
with EA. MS: [M + H]+ 306.10. H-NMR:
δ
ppm 6.30 (t, J = 6.4 Hz, 1H), 6.34 (s, NH2), 6.46 (d, J = 8.8 Hz,
1H), 6.60 (t, J = 7.2 Hz, 1H), 6.76 (d, J = 8.4 Hz, 1H), 7.21 (t, J = 7.2 Hz, 1H), 7.33–7.36 (m, 2H), 7.49 (t,
J = 8.8 Hz, 1H), 7.61–7.65 (m, 2H), 7.83 (d, J = 8.8 Hz, 2H), 10.14 (s, NH).
2-Amino-5-chloro-N-(4-(2-oxopyridin-1(2H)-yl)phenyl)benzamide (5b). Compound 5b was prepared from
4b according to the procedure described for the preparation of 5a. White solid product (1.29 g, 91%).
1
MS: [M + H]+ 340.10. H-NMR:
δ
ppm 6.30 (t, J = 5.2 Hz, 1H), 6.47 (d, J = 10.4 Hz, 1H), 6.48 (s, NH2),
6.78 (d, J = 9.2 Hz, 1H), 7.24 (d, J = 6.8 Hz, 1H), 7.35 (d, J = 8.8 Hz, 2H), 7.49 (t, J = 7.2 Hz, 1H), 7.63 (d,
J = 8.4 Hz, 1H), 7.70 (s, 1H), 7.80 (d, J = 8.8 Hz, 2H), 10.24 (s, NH).

Molecules 2016, 21, 491
10 of 16
2-Amino-5-methyl-N-(4-(2-oxopyridin-1(2H)-yl)phenyl)benzamide (5c). Compound 5c was prepared from
4c according to the procedure described for the preparation of 5a. White solid product (1.23 g, 92%).
1
MS: [M + H]+ 320.04. H-NMR:
δ ppm 2.22 (s, CH3), 6.11 (s, NH2), 6.30 (t, J = 6.4 Hz, 1H), 6.46 (d,
J = 8.8 Hz, 1H), 6.68 (d, J = 8.4 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 7.34 (d, J = 8.8 Hz, 2H), 7.44 (s, 1H),
7.49 (t, J = 8.8 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.81 (d, J = 8.8 Hz, 2H), 10.11 (s, NH).
2-Amino-3-methyl-N-(4-(2-oxopyridin-1(2H)-yl)phenyl)benzamide (5d). Compound 5d was prepared from
4d according to the procedure described for the preparation of 5a. White solid product (1.20 g, 90%).
1
MS: [M + H]+ 320.04. H-NMR:
δ ppm 2.12 (s, CH3), 6.13 (s, NH2), 6.30 (t, J = 6.8 Hz, 1H), 6.46 (d,
J = 9.2 Hz, 1H), 6.58 (t, J = 7.2 Hz, 1H), 7.15 (d, J = 7.2 Hz, 1H), 7.34 (d, J = 8.8 Hz, 2H), 7.47–7.54 (m,
2H), 7.62 (d, J = 8.4 Hz, 1H), 7.82 (d, J = 8.8 Hz, 2H), 10.17 (s, NH).
2-Amino-N-(4-(3-oxomorpholino)phenyl)benzamide (5e). Compound 5e was prepared from 4e according
to the procedure described for the preparation of 5a. White solid product (1.21 g, 93%). MS: [M + H]+
1
312.04. H-NMR:
δ
ppm 3.71 (t, J = 4.8 Hz, CH2), 3.96 (t, J = 4.8 Hz, CH2), 4.18 (s, CH2), 6.31 (s, NH2),
6.58 (t, J = 7.2 Hz, 1H), 6.74 (d, J = 8.0 Hz, 1H), 7.13 (t, J = 8.4 Hz, 1H), 7.33 (d, J = 7.2 Hz, 2H), 7.61 (d,
J = 8.8 Hz, 1H), 7.72 (d, J = 8.8 Hz, 2H), 10.03 (s, NH).
2-Amino-5-chloro-N-(4-(3-oxomorpholino)phenyl)benzamide (5f). Compound 5f was prepared from 4f
according to the procedure described for the preparation of 5a. White solid product (1.28 g, 89%). MS:
1
[M + H]+ 345.97. H-NMR:
δ ppm 3.71 (t, J = 4.8 Hz, CH2), 3.96 (t, J = 4.8 Hz, CH2), 4.19 (s, CH2),
6.46 (s, NH2), 6.78 (d, J = 8.8 Hz, 1H), 7.23 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 8.8 Hz, 2H), 7.68 (d, J = 7.6 Hz,
2H), 7.72 (s, 1H), 10.14 (s, NH).
2-Amino-5-methyl-N-(4-(3-oxomorpholino)phenyl)benzamide (5g). Compound 5g was prepared from 4g
according to the procedure described for the preparation of 5a. White solid product (1.22 g, 90%). MS:
1
[M + H]+ 326.07. H-NMR:
δ ppm 2.21 (s, CH3), 3.71 (t, J = 4.8 Hz, CH2), 3.96 (t, J = 4.8 Hz, CH2),
4.19 (s, CH2), 6.09 (s, NH2), 6.67 (d, J = 8.4 Hz, 1H), 7.03 (d, J = 8.0 Hz, 1H), 7.33 (d, J = 8.8 Hz, 2H),
7.42 (s, 1H), 7.72 (d, J = 8.8 Hz, 2H), 10.01 (s, NH).
2-Amino-3-methyl-N-(4-(3-oxomorpholino)phenyl)benzamide (5h). Compound 5h was prepared from 4h
according to the procedure described for the preparation of 5a. White solid product (1.23 g, 91%). MS:
1
[M + H]+ 326.03. H-NMR:
δ ppm 2.11 (s, CH3), 3.71 (t, J = 4.8 Hz, CH2), 3.96 (t, J = 4.8 Hz, CH2),
4.19 (s, CH2), 6.11 (s, NH2), 6.56 (t, J = 7.6 Hz, 1H), 7.13 (d, J = 6.8 Hz, 1H), 7.33 (d, J = 8.8 Hz, 2H),
7.51 (d, J = 7.6 Hz, 1H), 7.72 (d, J = 8.8 Hz, 2H), 10.06 (s, NH).
5-Chloro-N-(2-((4-(2-oxopyridin-1(2H)-yl)phenyl)carbamoyl)phenyl)thiophene-2-carboxamide (1a). To a stirred
solution of 5a (0.30 g, 0.98 mmol), K2CO3 (0.16 g, 1.18 mmol) and DMAP (0.01 g, 0.08 mmol) in THF
(10 mL), solution of 5-chlorothiophene-2-carbonyl chloride (6a) (0.23 g, 1.27 mmol) in THF (5 mL)
was added at room temperature and the mixture was refluxed for 4 h. The reaction mixture was cool
down to room temperature and water (30 mL) was added. The resulting precipitate was collected by
filtration. The authentic sample was prepared by recrystallization from DMF/MeOH. The reaction
was monitored by TLC with EA. White solid product (0.27 g, 60%), m.p. > 250 ˝C. 1H-NMR:
δ ppm
6.31 (t, J = 6.4 Hz, 1H), 6.47 (d, J = 9.2 Hz, 1H), 7.27 (d, J = 4.0 Hz, 1H), 7.32 (t, J = 7.2 Hz, 1H), 7.38 (d,
J = 8.4 Hz, 2H), 7.50 (t, J = 8.8 Hz, 1H), 7.59–7.64 (m, 3H), 7.81 (d, J = 8.4 Hz, 2H), 7.92 (d, J = 7.6 Hz, 1H),
8.22 (d, J = 8.4 Hz, 1H), 10.67 (s, NH), 11.52 (s, NH). HRMS (ESI) calcd. for C23H16ClN3O3S: [M + Na]+
m/z: 472.0499, found: 472.0482.
2-(4-Fluorobenzamido)-N-(4-(2-oxopyridin-1(2H)-yl)phenyl)benzamide (1b). Compound 1b was prepared
from 5a and 4-fluorobenzoyl chloride (6b) according to the procedure described for the preparation of
1a. White solid product (0.24 g, 56%), m.p. > 250 ˝C. 1H-NMR:
δ ppm 6.31 (t, J = 6.8 Hz, 1H), 6.47 (d,
J = 8.8 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H), 7.37–7.42 (m, 4H), 7.50 (t, J = 8.8 Hz, 1H), 7.60–7.64 (m, 2H),
7.81 (d, J = 8.8 Hz, 2H), 7.93 (d, J = 7.6 Hz, 1H), 7.96–8.00 (m, 2H), 8.39 (d, J = 8.0 Hz, 1H), 10.68 (s, NH),
11.56 (s, NH). HRMS (ESI) calcd. for C25H18FN3O3: [M + Na]+ m/z: 450.1230, found: 450.1222.

Molecules 2016, 21, 491
11 of 16
2-(4-Chlorobenzamido)-N-(4-(2-oxopyridin-1(2H)-yl)phenyl)benzamide (1c). Compound 1c was prepared
from 5a and 4-chlorobenzoyl chloride (6c) according to the procedure described for the preparation of
1a. White solid product (0.28 g, 63%), m.p. > 250 ˝C. 1H-NMR:
δ ppm 6.31 (t, J = 6.4 Hz, 1H), 6.47 (d,
J = 9.2 Hz, 1H), 7.32 (t, J = 7.2 Hz, 1H), 7.38 (d, J = 8.8 Hz, 2H), 7.50 (t, J = 8.8 Hz, 1H), 7.60–7.65 (m, 4H),
7.81 (d, J = 8.8 Hz, 2H), 7.92 (d, J = 8.4 Hz, 3H), 8.38 (d, J = 8.0 Hz, 1H), 10.68 (s, NH), 11.57 (s, NH).
HRMS (ESI) calcd. for C25H18ClN3O3: [M + Na]+ m/z: 466.0934, found: 466.0933.
5-Chloro-N-(4-chloro-2-((4-(2-oxopyridin-1(2H)-yl)phenyl)carbamoyl)phenyl)thiophene-2-carboxamide (1d).
Compound 1d was prepared from 5b and 6a according to the procedure described for the preparation
˝
of 1a. White solid product (0.31 g, 65%), m.p. > 250 C. 1H-NMR:
δ ppm 6.31 (t, J = 6.8 Hz, 1H), 6.47 (d,
J = 9.2 Hz, 1H), 7.27 (d, J = 4.0 Hz, 1H), 7.39 (d, J = 8.8 Hz, 2H), 7.50 (t, J = 8.8 Hz, 1H), 7.62–7.69 (m,
3H), 7.80 (d, J = 8.8 Hz, 2H), 7.96 (d, J = 2.4 Hz, 1H), 8.19 (d, J = 8.8 Hz, 1H), 10.73 (s, NH), 11.41 (s,
NH). HRMS (ESI) calcd. for C23H15Cl2N3O3S: [M + Na]+ m/z: 506.0109, found: 506.0104.
5-Chloro-2-(4-fluorobenzamido)-N-(4-(2-oxopyridin-1(2H)-yl)phenyl)benzamide (1e). Compound 1e was
prepared from 5b and 6b according to the procedure described for the preparation of 1a. White solid
product (0.25 g, 55%), m.p. > 250 ˝C. 1H-NMR:
δ ppm 6.30 (t, J = 6.8 Hz, 1H), 6.47 (d, J = 9.2 Hz, 1H),
7.40 (t, J = 8.8 Hz, 4H), 7.50 (t, J = 6.8 Hz, 1H), 7.62 (d, J = 1.6 Hz, 1H), 7.63 (d, J = 1.6 Hz, 1H), 7.68 (d,
J = 8.8 Hz, 2H), 7.95–7.99 (m, 3H), 7.36 (d, J = 8.8 Hz, 1H), 10.74 (s, NH), 11.44 (s, NH). HRMS (ESI)
calcd. for C25H17ClFN3O3: [M + Na]+ m/z: 484.0840, found: 484.0845.
5-Chloro-2-(4-chlorobenzamido)-N-(4-(2-oxopyridin-1(2H)-yl)phenyl)benzamide (1f). Compound 1f was
prepared from 5b and 6c according to the procedure described for the preparation of 1a. White solid
product (0.29 g, 60%), m.p. > 250 ˝C. 1H-NMR:
δ ppm 6.30 (t, J = 6.0 Hz, 1H), 6.47 (d, J = 9.6 Hz, 1H),
7.39 (d, J = 8.4 Hz, 2H), 7.50 (t, J = 6.8 Hz, 1H), 7.62–7.70 (m, 4H), 7.78 (d, J = 8.0 Hz, 2H), 7.91 (d,
J = 8.8 Hz, 2H), 7.97 (s, 1H), 8.34 (d, J = 9.6 Hz, 1H), 10.74 (s, NH), 11.45 (s, NH). HRMS (ESI) calcd. for
C25H17Cl2N3O3: [M + Na]+ m/z: 500.0545, found: 500.0539.
5-Chloro-N-(4-methyl-2-((4-(2-oxopyridin-1(2H)-yl)phenyl)carbamoyl)phenyl)thiophene-2-carboxami-de (1g).
Compound 1g was prepared from 5c and 6a according to the procedure described for the preparation
˝
of 1a. White solid product (0.27 g, 58%), m.p. > 250 C. 1H-NMR:
δ ppm 2.39 (s, CH3), 6.31 (t, J = 6.4 Hz,
1H), 6.47 (d, J = 9.2 Hz, 1H), 7.26 (d, J = 4 Hz, 1H), 7.25–7.43 (m, 3H), 7.50 (t, J = 8.8 Hz, 1H), 7.61–7.64
(m, 2H), 7.72 (s, 1H), 7.81 (d, J = 8.8 Hz, 2H), 8.08 (d, J = 8.4 Hz, 1H), 10.62 (s, NH), 11.38 (s, NH). HRMS
(ESI) calcd. for C24H18ClN3O3S: [M + Na]+ m/z: 486.0655, found: 486.0648.
2-(4-Fluorobenzamido)-5-methyl-N-(4-(2-oxopyridin-1(2H)-yl)phenyl)benzamide (1h). Compound 1h was
prepared from 5c and 6b according to the procedure described for the preparation of 1a. White solid
product (0.27 g, 61%), m.p. > 250 ˝C. 1H-NMR:
δ ppm 2.39 (s, CH3), 6.30 (t, J = 6.4 Hz, 1H), 6.47 (d,
J = 9.2 Hz, 1H), 7.37–7.44 (m, 5H), 7.49 (t, J = 8.8 Hz, 1H), 7.62 (d, J = 5.6 Hz, 1H), 7.74 (s, 1H), 7.80 (d,
J = 8.8 Hz, 2H), 7.94–7.98 (m, 2H), 8.27 (d, J = 8.4 Hz, 1H), 10.64 (s, NH), 11.43 (s, NH). HRMS (ESI)
calcd. for C26H20FN3O3: [M + Na]+ m/z: 464.1386, found: 464.1379.
2-(4-Chlorobenzamido)-5-methyl-N-(4-(2-oxopyridin-1(2H)-yl)phenyl)benzamide (1i). Compound 1i was
prepared from 5c and 6c according to the procedure described for the preparation of 1a. White solid
product (0.29 g, 63 %), m.p. > 250 ˝C. 1H-NMR:
δ ppm 2.39 (s, CH3), 6.30 (t, J = 7.2 Hz, 1H), 6.47 (d,
J = 9.2 Hz, 1H), 7.38 (d, J = 8.8 Hz, 2H), 7.43 (d, J = 8.4 Hz, 1H), 7.50 (t, J = 8.8 Hz, 1H), 7.62–7.64 (m,
3H), 7.73 (s, 1H), 7.80 (d, J = 8.8 Hz, 2H), 7.91 (d, J = 8.8 Hz, 2H), 8.25 (d, J = 8.4 Hz, 1H), 10.64 (s, NH),
11.44 (s, NH). HRMS (ESI) calcd. for C26H20ClN3O3: [M + Na]+ m/z: 480.1091, found: 480.1083.
5-Chloro-N-(2-methyl-6-((4-(2-oxopyridin-1(2H)-yl)phenyl)carbamoyl)phenyl)thiophene-2-carboxami-de (1j).
Compound 1j was prepared from 5d and 6a according to the procedure described for the preparation
˝
of 1a. White solid product (0.23 g, 50%), m.p. > 250 C. 1H-NMR:
δ
ppm 2.27 (s, CH3), 6.28 (t, J = 6.8 Hz,
1H), 6.45 (d, J = 9.2 Hz, 1H), 7.22 (d, J = 4.0 Hz, 1H), 7.30–7.34 (m, 2H), 7.37 (d, J = 7.6 Hz, 1H), 7.46–7.52

Molecules 2016, 21, 491
12 of 16
(m, 3H), 7.59 (d, J = 6.8 Hz, 1H), 7.77 (d, J = 8.8 Hz, 2H), 7.84 (d, J = 4.4 Hz, 1H), 10.10 (s, NH), 11.42 (s,
NH). HRMS (ESI) calcd. for C24H18ClN3O3S: [M + Na]+ m/z: 486.0655, found: 486.0649.
2-(4-Fluorobenzamido)-3-methyl-N-(4-(2-oxopyridin-1(2H)-yl)phenyl)benzamide (1k). Compound 1k was
prepared from 5d and 6b according to the procedure described for the preparation of 1a. White solid
product (0.22 g, 46%), m.p. > 250 ˝C. 1H-NMR:
δ ppm 2.28 (s, CH3), 6.27 (t, J = 6.8 Hz, 1H), 6.44 (d,
J = 9.2 Hz, 1H), 7.29–7.32 (m, 5H), 7.46–7.50 (m, 3H), 7.57 (d, J = 8.8 Hz, 1H), 7.77 (d, J = 8.8 Hz, 2H),
7.98–8.01 (m, 2H), 9.99 (s, NH), 11.41 (s, NH). HRMS (ESI) calcd. for C26H20FN3O3: [M + Na]+ m/z:
464.1386, found: 464.1381.
2-(4-Chlorobenzamido)-3-methyl-N-(4-(2-oxopyridin-1(2H)-yl)phenyl)benzamide (1l). Compound 1l was
prepared from 5d and 6c according to the procedure described for the preparation of 1a. White solid
product (0.22 g, 48%), m.p. > 250 ˝C. 1H-NMR:
δ ppm 2.28 (s, CH3), 6.27 (t, J = 6.4 Hz, 1H), 6.44 (d,
J = 8.8 Hz, 1H), 7.30 (d, J = 8.8 Hz, 2H), 7.36 (t, J = 7.6 Hz, 1H), 7.46–7.50 (m, 3H), 7.56–7.59 (m, 3H),
7.77 (d, J = 8.4 Hz, 2H), 7.94 (d, J = 8.4 Hz, 2H), 10.05 (s, NH), 10.42 (s, NH). HRMS (ESI) calcd. for
C26H20ClN3O3: [M + Na]+ m/z: 480.1091, found: 480.1087.
5-Chloro-N-(2-((4-(3-oxomorpholino)phenyl)carbamoyl)phenyl)thiophene-2-carboxamide (1m). Compound
1m was prepared from 5e and 6a according to the procedure described for the preparation of 1a. White
˝
solid product (0.29 g, 63%), m.p. > 250 C. 1H-NMR:
δ ppm 3.72 (t, J = 4.8 Hz, CH2), 3.97 (t, J = 4.8 Hz,
CH2), 4.19 (s, CH2), 7.27 (d, J = 4.0 Hz, 1H), 7.30 (t, J = 7.2 Hz, 1H), 7.38 (d, J = 8.8 Hz, 2H), 7.58–7.60 (m,
2H), 7.72 (d, J = 8.8 Hz, 2H), 7.91 (d, J = 7.6 Hz, 1H), 8.24 (d, J = 8.4 Hz, 1H), 10.57 (s, NH), 11.60 (s,
NH). HRMS (ESI) calcd. for C22H18ClN3O4S: [M + Na]+ m/z: 478.0604, found: 478.0613.
2-(4-Fluorobenzamido)-N-(4-(3-oxomorpholino)phenyl)benzamide (1n). Compound 1n was prepared from
5e and 6b according to the procedure described for the preparation of 1a. White solid product (0.27 g,
62%), m.p. > 250 ˝C. 1H-NMR:
δ ppm 3.72 (t, J = 4.8 Hz, CH2), 3.97 (t, J = 4.8 Hz, CH2), 4.19 (s, CH2),
7.29 (t, J = 7.6 Hz, 1H), 7.36–7.42 (m, 4H), 7.61 (t, J = 7.8 Hz, 1H), 7.71 (d, J = 8.8 Hz, 2H), 7.92 (d,
J = 8.0 Hz, 1H), 7.95–7.99 (m, 2H), 8.42 (d, J = 8.4 Hz, 1H), 10.58 (s, NH), 11.64 (s, NH). HRMS (ESI)
calcd. for C24H20FN3O4: [M + Na]+ m/z: 456.1336, found: 456.1386.
2-(4-Chlorobenzamido)-N-(4-(3-oxomorpholino)phenyl)benzamide (1o). Compound 1o was prepared from
5e and 6c according to the procedure described for the preparation of 1a. White solid product (0.30 g,
˝
1
67%), m.p. > 250 C. H-NMR:
δ ppm 3.59 (t, J = 4.8 Hz, CH2), 3.71 (t, J = 4.8 Hz, CH2), 4.19 (s,
CH2), 7.30 (t, J = 7.2 Hz, 1H), 7.37 (d, J = 8.8 Hz, 2H), 7.60–7.65 (m, 3H), 7.71 (d, J = 9.2 Hz, 2H),
7.91 (d, J = 8.4 Hz, 3H), 8.40 (d, J = 8.0 Hz, 1H), 10.58 (s, NH), 11.64 (s, NH). HRMS (ESI) calcd. for
C24H20ClN3O4: [M + Na]+ m/z: 472.1040, found: 472.1035.
5-Chloro-N-(4-chloro-2-((4-(3-oxomorpholino)phenyl)carbamoyl)phenyl)thiophene-2-carboxamide
(1p). Compound
1p was prepared from 5f and 6a according to the procedure described for the preparation of 1a. White
˝
solid product (0.32 g, 65%), m.p. > 250 C. 1H-NMR:
δ ppm 3.72 (t, J = 4.8 Hz, CH2), 3.97 (t, J = 4.8 Hz,
CH2), 4.19 (s, CH2), 7.26 (s, 1H), 7.38 (d, J = 8.8 Hz, 2H), 7.62–7.71 (m, 4H), 7.95 (d, J = 2.0 Hz, 1H), 8.22
(d, J = 8.8 Hz, 1H), 10.63 (s, NH), 11.49 (s, NH). HRMS (ESI) calcd. for C22H17Cl2N3O4S: [M + Na]+
m/z: 512.0215, found: 512.0205.
5-Chloro-2-(4-fluorobenzamido)-N-(4-(3-oxomorpholino)phenyl)benzamide
(1q). Compound 1q was prepared
from 5f and 6b according to the procedure described for the preparation of 1a. White solid product
˝
(0.29 g, 63%), m.p. > 250 C. 1H-NMR:
δ ppm 3.71 (t, J = 4.8 Hz, CH2), 3.97 (t, J = 4.8 Hz, CH2), 4.19 (s,
CH2), 7.33–7.43 (m, 4H), 7.65–7.70 (m, 3H), 7.95–7.98 (m, 3H), 8.39 (d, J = 8.8 Hz, 1H), 10.65 (s, NH),
11.54 (s, NH). HRMS (ESI) calcd. for C24H19ClFN3O4: [M + Na]+ m/z: 490.0946, found: 490.0940.
5-Chloro-2-(4-chlorobenzamido)-N-(4-(3-oxomorpholino)phenyl)benzamide (1r). Compound 1r was prepared
from 5f and 6c according to the procedure described for the preparation of 1a. White solid product
˝
(0.32 g, 66%), m.p. > 250 C. 1H-NMR:
δ
ppm 3.71 (t, J = 4.8 Hz, CH2), 3.97 (t, J = 4.8 Hz, CH2), 4.19 (s,
CH2), 7.37 (d, J = 8.8 Hz, 2H), 7.63 (d, J = 8.4 Hz, 2H), 7.67–7.72 (m, 3H), 7.91 (d, J = 8.4 Hz, 2H),

Molecules 2016, 21, 491
13 of 16
7.96 (d, J = 2.4 Hz, 1H), 8.37 (d, J = 8.8 Hz, 1H), 10.65 (s, NH), 11.52 (s, NH). HRMS (ESI) calcd. for
C24H19Cl2N3O4: [M + Na]+ m/z: 506.0650, found: 506.0644.
5-Chloro-N-(4-methyl-2-((4-(3-oxomorpholino)phenyl)carbamoyl)phenyl)thiophene-2-carboxamide
(1s). Compound
1s was prepared from 5g and 6a according to the procedure described for the preparation of 1a. White
˝
solid product (0.30 g, 63%), m.p. > 250 C. 1H-NMR:
δ ppm 2.37 (s, CH3), 3.72 (t, J = 4.8 Hz, CH2), 3.97
(t, J = 4.8 Hz, CH2), 4.19 (s, CH2), 7.25 (d, J = 3.6 Hz, 1H), 7.36–7.42 (m, 3H), 7.60 (d, J = 3.2 Hz, 1H),
7.70 (d, J = 8.8 Hz, 3H), 8.10 (d, J = 8.0 Hz, 1H), 10.52 (s, NH), 11.45 (s, NH). HRMS (ESI) calcd. for
C23H20ClN3O4S: [M + Na]+ m/z: 492.0761, found: 492.0754.
2-(4-Fluorobenzamido)-5-methyl-N-(4-(3-oxomorpholino)phenyl)benzamide (1t). Compound 1t was prepared
from 5g and 6b accordin˝g to the procedure described for the preparation of 1a. White solid product
δ ppm 2.38 (s, CH3), 3.72 (t, J = 4.8 Hz, CH2), 3.97 (t, J = 4.8 Hz,
(0.28 g, 63%), m.p. > 250 C. 1H-NMR:
CH2), 4.19 (s, CH2), 7.36–7.43 (m, 5H), 7.69–7.73 (m, 3H), 7.94–7.97 (m, 2H), 8.29 (d, J = 8.4 Hz, 1H), 10.54
(s, NH), 11.49 (s, NH). HRMS (ESI) calcd. for C25H22FN3O4: [M + Na]+ m/z: 470.1492, found: 470.1487.
2-(4-Chlorobenzamido)-5-methyl-N-(4-(3-oxomorpholino)phenyl)benzamide
(1u). Compound 1u was prepared
from 5g and 6c according to the procedure described for the preparation of 1a. White solid product
˝
(0.28 g, 60%), m.p. > 250 C. 1H-NMR:
δ ppm 2.39 (s, CH3), 3.72 (t, J = 4.8 Hz, CH2), 3.97 (t, J = 4.8 Hz,
CH2), 4.19 (s, CH2), 7.37 (d, J = 8.8 Hz, 2H), 7.42 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 8.4 Hz, 2H), 7.69–7.73
(m, 2H), 7.90 (d, J = 8.8 Hz, 2H), 8.27 (d, J = 8.4 Hz, 1H), 10.54 (s, NH), 11.51 (s, NH). HRMS (ESI) calcd.
for C25H22ClN3O4: [M + Na]+ m/z: 486.1197, found: 486.1185.
5-Chloro-N-(2-methyl-6-((4-(3-oxomorpholino)phenyl)carbamoyl)phenyl)thiophene-2-carboxamide
(1v). Compound
1v was prepared from 5h and 6a according to the procedure described for the preparation of 1a. White
˝
solid product (0.24 g, 51%), m.p. > 250 C. 1H-NMR:
δ ppm 2.22 (s, CH3), 3.68 (t, J = 4.8 Hz, CH2), 3.95
(t, J = 4.8 Hz, CH2), 4.17 (s, CH2), 7.24 (d, J = 7.6 Hz, 1H), 7.31 (d, J = 9.2 Hz, 2H), 7.39 (t, J = 8.8 Hz, 1H),
7.55 (d, J = 8.8 Hz, 1H), 7.6 (d, J = 8.8 Hz, 2H), 7.70–7.79 (m, 2H), 10.10 (s, NH), 10.29 (s, NH). HRMS
(ESI) calcd. for C23H20ClN3O4S: [M + Na]+ m/z: 492.0761, found: 492.0755.
2-(4-Fluorobenzamido)-3-methyl-N-(4-(3-oxomorpholino)phenyl)benzamide (1w). Compound 1w was prepared
from 5h and 6b accordin˝g to the procedure described for the preparation of 1a. White solid product
(0.19 g, 43%), m.p. > 250 C. 1H-NMR:
δ ppm 2.49 (s, CH3), 3.67 (t, J = 4.8 Hz, CH2), 3.94 (t, J = 4.8 Hz,
CH2), 4.16 (s, CH2), 7.27–7.36 (m, 5H), 7.47 (t, J = 7.6 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H), 7.97–8.01 (m,
2H), 9.98 (s, NH), 10.28 (s, NH). HRMS (ESI) calcd. for C25H22FN3O4: [M + Na]+ m/z: 470.1492,
found: 470.1488.
2-(4-Chlorobenzamido)-3-methyl-N-(4-(3-oxomorpholino)phenyl)benzamide
(1x). Compound 1x was prepared
from 5h and 6c according to the procedure described for the preparation of 1a. White solid product
˝
(0.21 g, 46%), m.p. > 250 C. 1H-NMR:
δ ppm 2.27 (s, CH3), 3.67 (t, J = 4.8 Hz, CH2), 3.95 (t, J = 4.8 Hz,
CH2), 4.17 (s, CH2), 7.28 (d, J = 8.8 Hz, 2H), 7.34 (t, J = 6.8 Hz, 1H), 7.47 (t, J = 8.4 Hz, 2H), 7.56 (d,
J = 8.4 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H), 7.93 (d, J = 8.0 Hz, 2H), 10.03 (s, NH), 10.29 (s, NH). HRMS
(ESI) calcd. for C25H22ClN3O4: [M + Na]+ m/z: 486.1197, found: 486.1196.
3.3. Inhibition of FXa in Vitro
The inhibition of FXa was measured using human FXa (Hyphen BioMed, Paris, France) and
chromogenic substrate CS-11(22) (Hyphen BioMed, Paris, France) in 384-well microtiter plates at room
temperature. The synthesized compounds (1a
concentration of 10 mM and then serially diluted to spanning a range of 30 nM to 100
L of FXa (56.8 nM), 16 L of Tris buffer (adjust to pH 7.4 with HCl containing 0.3 M NaCl and
50 mM Tris) and 3 L of test compound were added to the well, respectively. The negative control
–
1x) and Rivaroxaban were dissolved in DMSO at a
µM, respectively.
2
µ
µ
µ
was composed of the same mixed solutions except replacing test compound with DMSO. The positive
control was composed of the same mixed solutions except replacing test compound with rivaroxaban.

Molecules 2016, 21, 491
14 of 16
˝
After incubated˝at 37 C for 5 min, 8
µ
L of FXa substrate solution (3.5 mM) was added and then
incubated at 37 C for 25 min. The FXa activity was measured at 405 nm using a SpectraMax M5
(Molecular Devices, Sunnyvale, CA, USA). The IC50 was calculated by the software named SPSS (IBM,
North Castle, NY, USA) and the Probit function in it.
3.4. Thrombin Inhibition in Vitro of 1a, 1g and 1s
The inhibition of thrombin was measured using human FIIa (Hyphen BioMed, Paris, France)
and chromogenic substrate CS-01(38) (Hyphen BioMed, Paris, France) in 384-well microtiter plates
at room temperature. The compounds 1a
concentration of 10 mM and then serially diluted to spanning a range of 10
L of FIIa (3 NIH/mL), 20 L of Tris buffer (adjust to pH 7.4 with HCl) containing 0.3 M NaCl and
50 mM Tris and 2 L of test compound were added to the well, respectively. The negative control
,
1g and 1s and Rivaroxaban were dissolved in DMSO to a
µ
M to 100 M, respectively.
µ
2
µ
µ
µ
was composed of the same mixed solutions except replacing test compound with DMSO. The positive
control was composed of the same mixed solutions except replacing test compound with Rivaroxaban.
˝
After incubated˝at 37 C for 5 min, 3
µL of FIIa substrate solution (4 mM) was added and then
incubated at 37 C for 25 min. The FIIa activity was measured at 405 nm using a SpectraMax M5
(Molecular Devices).
3.5. Prothrombin Time (PT) Assay
A commercially available automatic coagulometer (Steellex Science Instrument Co., Ltd., Beijing,
China) was employed to measure PT. The clotting times were also measured using the instrument
itself, in accordance with the manufacturer’s instructions. Increasing concentrations of inhibitor or
solvent were added to rat (Sprague-Dawley rats, Shanchuanhong Experimental Animals Co., Ltd.,
Tianjin, China) and human (29 Years old, male, Chinese) plasma and incubated for 3 min at 37 ˝C.
Prothrombin time (PT) was determined by automatic coagulometer.
3.6. Docking Simulation Study
FXa structure was selected from the protein data bank (PDB code: 2xbv) and prepared using
Protein Preparation Wizard in Schrödinger package, including assigning bond orders, adding hydrogen
atoms, deleting water molecules, creating disulfide bonds and capping terminals. The original ligand
of the protein structure-XBV was used as the docking center to generate the receptor grid parameters.
The box size was set as 12 Å. Compounds 1g and 1s were prepared using the LigPrep module in
Schrödinger. Epik method was used to determine possible ionization state of ligands at pH 7.0
low-energy conformers were produced using OPLS-2005 force field. Molecular docking calculations
were performed by using Glide module with default parameters at standard precision in Schrödinger.
˘
2.0 and
4. Conclusions
In this study, we designed and synthesized a series of novel potent FXa inhibitors based on the
anthranilamide scaffold. In vitro inhibition activity studies showed that compounds 1a, 1g and 1s
displayed evident FXa inhibition and excellent selectivity over thrombin. Compounds 1g and 1s also
exhibited pronounced anticoagulant activity in in vitro anticoagulant activity studies. Further docking
simulation study also disclosed that the interaction of compounds 1g and 1s with FXa was very similar
to that of rivaroxaban. Therefore, compounds 1g and 1s with an anthranilamide scaffold could be
considered as lead compounds for exploring new FXa inhibitors with better medicinal effects and
further modification of this structure for better antithrombotic activity is still in progress.
Acknowledgments: This project was supported by the Scientific and Technological Plan Projects of Tianjin
(No. 12ZCZDSY01100).

Molecules 2016, 21, 491
15 of 16
Author Contributions: Wenzhi Wang conceived, designed and performed the synthetic experiment part; Xiaoli Fu
analyzed the data; Fancui Meng conceived, designed and performed the docking simulation study part;
Shijun Zhang conceived, designed and performed the pharmacological test part; Wenzhi Wang wrote the paper.
Yongnan Xu, Changjiang Huang, Weiren Xu and Jing Yuan assisted paper revision.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
Turpie, A.G. Advances in oral anticoagulation treatment: The safety and efficacy of rivaroxaban in the
prevention and treatment of thromboembolism. Ther. Adv. Hematol. 2012, 3, 309–323. [CrossRef] [PubMed]
Lee, Y.K.; Player, M.R. Developments in factor Xa inhibitors for the treatment of thromboembolic disorders.
Med. Res. Rev. 2011, 31, 202–283. [CrossRef] [PubMed]
Lau, D.H.; Alasady, M.; Brooks, A.G.; Sanders, P. Incidence and prognostic impact of new-onset atrial
fibrillation in patients with septic shock: A prospective observational study. Rev. Cardiovasc. Ther. 2010, 8,
941–948. [CrossRef] [PubMed]
4.
5.
6.
7.
8.
Leadley, R.J. Coagulation factor Xa inhibition: Biological background and rationale. Curr. Top. Med. Chem.
2001, 1, 151–159. [CrossRef] [PubMed]
Fareed, J.; Thethi, I.; Hoppensteadt, D. Old versus new oral anticoagulants: Focus on pharmacology.
Annu. Rev. Pharmacol. Toxicol. 2012, 52, 79–99. [CrossRef] [PubMed]
Bassand, J.P. Review of atrial fibrillation outcome trials of oral anticoagulant and antiplatelet agents. Europace
2012, 14, 312–324. [CrossRef] [PubMed]
Ansell, J.; Hirsh, J.; Poller, L.; Bussey, H.; Jacobson, A.; Hylek, E. The pharmacology and management of the
vitamin K antagonists. Chest 2004, 126 (Suppl. S3), 204s–233s. [CrossRef] [PubMed]
Bukowska, A.; Zacharias, I.; Weinert, S.; Skopp, K.; Hartmann, C.; Huth, C.; Goette, A. Coagulation factor
Xa induces an inflammatory signalling by activation of proteaseactivated receptors in human atrial tissue.
Eur. J. Pharmacol. 2013, 718, 114–123. [CrossRef] [PubMed]
9.
Xue, T.; Ding, S.; Guo, B.; Zhou, Y.; Sun, P.; Wang, H.; Chu, W.; Gong, G.; Wang, Y.; Chen, X.; et al. Design,
synthesis, and structure-activity and structure pharmacokinetic relationship studies of novel [6,6,5] tricyclic
fused oxazolidinones leading to the discovery of a potent, selective, and orally bioavailable FXa inhibitor.
J. Med. Chem. 2014, 57, 7770–7791. [CrossRef] [PubMed]
10. Galanis, T.; Thomson, L.; Palladino, M.; Merli, G.J. New oral anticoagulants. J. Thromb. Thrombolysis. 2011
31, 310–320. [CrossRef] [PubMed]
,
11. Bauer, K. Recent progress in anticoagulant therapy: Oral direct inhibitors of thrombin and factor Xa.
J. Thromb. Haemost. 2011, 9, 12–19. [CrossRef] [PubMed]
12. Elisabeth, P.; Susanne, R.; Alexander, S.; Dagmar, K.; Frank, M. The discovery and development of
Rivaroxaban, an oral, direct factor Xa inhibitor. Nat. Rev. Drug Discov. 2011, 10, 61–75.
13. Pancras, C.W.; Donald, J.P.; Zhang, D.L. Preclinical discovery of apixaban, a direct and orally bioavailable
factor Xa inhibitor. J. Thromb. Thrombolysis 2011, 31, 478–492.
14. Maignan, S.; Guilloteau, J.P.; Choi-Sledeski, Y.M.; Becker, M.R.; Ewing, W.R.; Pauls, H.W.; Spada, A.P.;
Mikol, V. Molecular Structures of Human Factor Xa Complexed with Ketopiperazine Inhibitors: Preference
for a Neutral Group in the S1 Pocket. J. Med. Chem. 2003, 46, 685–690. [CrossRef] [PubMed]
15. Mega, J.L.; Braunwald, E.; Wiviott, S.D.; Bassand, J.P.; Bhatt, D.L.; Bode, C.; Burton, P.; Cohen, M.; Cook, B.N.;
Fox, K.A.; et al. Rivaroxaban in patients with a recent acute coronary syndrome—Edinburgh Research
Explorer. N. Engl. J. Med. 2012, 366, 9–19. [CrossRef] [PubMed]
16. Roehrig, S.; Straub, A.; Pohlmann, J.; Lampe, T.; Pernerstorfer, J.; Schlemmer, K.H.; Reinemer, P.;
Perzborn, E. Discovery of the Novel antithrombotic Agent 5-Chloro-N-({(5S)-2-oxo-3-[4-(3-oxomorpholin-
4-yl)phenyl]-1,3-oxazolidin-5-yl}methyl)thiophene-2-carboxamide (BAY 59–7939): An Oral, Direct Factor Xa
Inhibitor. J. Med. Chem. 2005, 48, 5900–5908. [CrossRef] [PubMed]
17. Imaeda, Y.; Kuroita, T.; Sakamoto, H.; Kawamoto, T.; Tobisu, M.; Konishi, N.; Hiroe, K.; Kawamura, M.;
Tanaka, T.; Kubo, K. Discovery of Imidazo[1,5-c]imidazol-3-ones: Weakly Basic, Orally Active Factor Xa
Inhibitors. J. Med. Chem. 2008, 51, 3422–3436. [CrossRef] [PubMed]
18. Rami, A.A.; Akul, Y.M.; Umesh, R.D. Potent direct inhibitors of factor Xa based on the tetrahydroisoquinoline
scaffold. Eur. J. Med. Chem. 2012, 54, 771–783.

Molecules 2016, 21, 491
16 of 16
19. Matter, H.; Defossa, E.; Heinelt, U.; Blohm, P.M.; Schneider, D.; Müller, A.; Herok, S.; Schreuder, H.;
Liesum, A.; Brachvogel, V.; et al. Design and Quantitative Structure-Activity Relationship of
3-Amidinobenzyl-1H-indole-2-carboxamides as Potent, Nonchiral and Selective Inhibitors of Blood
Coagulation Factor Xa. J. Med. Chem. 2002, 45, 2749–2769. [CrossRef] [PubMed]
20. Yu, K.L.; Daniel, J.P.; Tian, B.L.; Tho, V.T. 7-Fluoroindazoles as Potent and Selective Inhibitors of Factor Xa.
J. Med. Chem. 2008, 51, 282–297.
21. Nagata, T.; Yoshino, T.; Haginoya, N.; Yoshikawa, K.; Nagamochi, M.; Kobayashi, S.; Komoriya, S.;
Yokomizo, A.; Muto, R.; Yamaguchi, M.; et al. Discovery of N-[(1R,2S,5S)-2-{[(5-chloroindol-2-yl)carbonyl]
amino}-5-(dimethylcarbamoyl)cyclohexyl]-5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine-2-carboxamide
hydrochloride: A novel, potent and orally active direct inhibitor of factor Xa. Bioorg. Med. Chem. 2009, 17,
1193–1206. [CrossRef] [PubMed]
22. Pruitt, J.R.; Pinto, D.J.; Estrella, M.J.; Bostrom, L.L.; Knabb, R.M.; Wong, P.C.; Wright, M.R.; Wexler, R.R.
Isoxazolines and Isoxazoles as Factor Xa Inhibitors. Bioorg. Med. Chem. Lett. 2000, 10, 685–689. [CrossRef]
23. Pinto, D.J.; Orwat, M.J.; Wang, S.; Fevig, J.M.; Quan, M.L.; Amparo, E.; Cacciola, J.; Rossi, K.A.;
Alexander, R.S.; Smallwood, A.M.; et al. Discovery of 1-[3-(Aminomethyl)phenyl]-N-[3-fluoro-21-
(methylsulfonyl)-[1,11-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC423), a Highly
Potent, Selective, and Orally Bioavailable Inhibitor of Blood Coagulation Factor Xa. J. Med. Chem. 2001, 44,
3422–3436. [CrossRef]
24. Corte, J.R.; Fang, T.N.; Pinto, D.J.; Han, W.; Hu, Z.; Jiang, X.J.; Li, Y.L.; Gauuan, J.F.; Hadden, M.; Orton, D.; et al.
Structure-activity relationships of anthranilamide-based factor Xa inhibitors containing piperidinone and
pyridinone P4 moieties. Bioorg. Med. Chem. Lett. 2008, 18, 2845–2849. [CrossRef] [PubMed]
25. Weinz, C.; Schwarz, T.; Kubitza, D.; Mueck, W.; Lang, D. Metabolism and Excretion of Rivaroxaban, an Oral,
Direct Factor Xa Inhibitor, in Rats, Dogs, and Humans. Drug. Metab. Dispos. 2008, 37, 1056–1064. [CrossRef]
[PubMed]
26. Doležal, M.; Zitko, J.; Kesetovicová, D.; Kunes, J.; Svobodová, M. Substituted N-Phenylpyrazine-2-
carboxamides: Synthesis and Antimycobacterial Evaluation. Molecules 2009, 14, 4180–4189. [CrossRef]
[PubMed]
27. Gerotziafas, T.G.; Samama, M.M. Heterogeneity of Synthetic Factor Xa Inhibitors. Curr. Pharm. Des. 2005, 11,
3855–3876. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds 1a–1x are available from the authors.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).