Design, synthesis, and biological evaluation of 4-methoxy-3-arylamido-N-(substitutedphenyl)benzamide derivatives as potential antiplatelet agents

Received: 5 August 2019

Revised: 14 November 2019

Accepted: 17 November 2019

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DOI: 10.1002/ardp.201900231

F U L L P A P E R

Design, synthesis, and biological evaluation of 4‐methoxy‐3‐

arylamido‐N‐(substitutedphenyl)benzamide derivatives as

potential antiplatelet agents

Xiujie Liu^1,2

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Xin Chen^1,2

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Kai Qiu²

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Zhihao Zhang²

¹Tianjin Key Laboratory of Organic Solar Cells

and Photochemical Conversion, Tianjin

University of Technology, Tianjin, China

Abstract

A series of 4‐methoxy‐3‐arylamido‐N‐(substitutedphenyl)benzamides 6a–u were

designed according to the splicing principle of structural design in the medicinal

chemistry theory and were synthesized in five steps: nitration, acylation, ammonia-

tion, reduction, and secondary ammoniation. The structures of the target compounds

were characterized and verified by infrared, ¹H nuclear magnetic resonance (NMR),

¹³C NMR, and electron spray ionization spectroscopy. Their in vitro antiplatelet

aggregation activities induced by adenosine diphosphate (ADP) or arachidonic acid

(AA) were assessed by Born’s method. The biological evaluation revealed that all

compounds exhibited certain levels of activities in both of the antiplatelet

aggregation assays; compounds 6c (IC₅₀= 3.84 μM) and 6f (IC₅₀= 3.12 μM) displayed

the strongest antiplatelet aggregation activities in the ADP‐induced and AA‐induced

assay, separately. Moreover, compounds that had stronger activities were chosen for

cell toxicity testing via the cell counting kit‐8 assay. The results indicated that none of

the compounds had obvious cell toxicity against L929 cells at the doses of 10 and

20 μM. It is worth pointing out that compound 6c showed the highest antiplatelet

activity and the lowest cell toxicity. In general, 4‐methoxy‐3‐arylamido‐N‐

(substitutedphenyl)benzamides have the potential to become a kind of safer and

more effective antiplatelet agents.

²School of Chemistry and Chemical

Engineering, Tianjin University of Technology,

Tianjin, China

Correspondence

Prof. Xiujie Liu and Xin Chen, School of

Chemistry and Chemical Engineering, Tianjin

University of Technology, No. 391 Binshui

West Road, Xiqing District, 300384 Tianjin,

China.

Email: liuxiujiechem@163.com (X. L.) and

chemchenxin@163.com (X. C.)

Funding information

National Science Foundation of China,

Grant/Award Number: 11341014; Committee

of Science and Technology of Tianjin of China,

Grant/Award Number: 15JCZDJC33100

K E Y W O R D S

4‐methoxy‐3‐arylamido‐N‐(substitutedphenyl)benzamides, antiplatelet aggregation activity, cell

toxicity, structure–activity relationship, synthesis

arachidonic acid pathway,^[3]and clopidogrel, which selectively

inhibits the P₂Y₁₂purinergic receptor to block the adenosine

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1

INTRODUCTION

Cardiovascular diseases (CVDs) are responsible for the largest

number of death and disability all over the world.^[1]Platelet

diphosphate pathway, are widely used as antiplatelet agents.^[4]

However, many patients are resistant to aspirin and clopidogrel,

which increases the risk of cardiovascular events.^[5,6]Moreover,

there are some serious side effects and limitations associated with

these agents, including local bleeding, gastrointestinal toxicity, weak

inhibition of platelet function (aspirin), and slow onset of action

(clopidogrel).^[7–10]Hence, developing novel antiplatelet agents is a

challenging task for researchers.

activation and aggregation play

a pivotal role in CVDs, and

antiplatelet agents are the mainstay of the treatment and prevention

of cardiovascular associated diseases.^[2]Many antiplatelet agents

have been used depending upon their various mechanisms of action.

For example, aspirin, which irreversibly inhibits the transformation of

cyclooxygenase

1 (COX‐1) to thromboxane A₂(TXA₂) in the

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Arch Pharm Chem Life Sci. 2019;e1900231.

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https://doi.org/10.1002/ardp.201900231

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LIU ET AL.

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Picotamide (Figure 1a), which simultaneously inhibits TXA₂

receptors and TXA₂synthase, but does not interfere with prosta-

glandin I₂(PGI₂) production, is an antiplatelet agent with dual

inhibitory effects.^[11–13]Compared with aspirin, it is more effective in

reducing overall mortality in type‐II diabetes associated with

peripheral arterial disease.^[14]In our previous study, a series of

4‐methoxyisophthalamides (Figure 1c) were designed and synthe-

sized by replacing the two 3‐(aminomethyl)pyridine groups with two

substituted phenyl groups. Among them, nearly 20% of compounds

showed stronger inhibitory activities than picotamide on platelet

aggregation.^[15–18]According to the results of the analysis of

pharmacological activities, steric quantitative structure–activity

relationships were established. The results showed that those

compounds had antiplatelet aggregation activities when the

1,3,4‐trisubstituted positional characteristics of the parent phenyl

ring were retained, and the absence of any positions will result in a

decrease of activity.

Compounds combining antiplatelet agents and anticoagulant agents

in the same molecule are deemed to be promising agents. Herein, we

retain the 1,3,4‐trisubstituted structure characteristics of picotamide and

take advantage of the amide group in the 2′‐position of betrixaban to

replace the aminoacyl group in the 3‐position of picotamide. In this study,

a

series of 4‐methoxy‐3‐arylamido‐N‐(substitutedphenyl)benzamides

(Figure 1d) were synthesized and their in vitro antiplatelet aggregation

activities and cell toxicities were evaluated.

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2

RESULTS AND DISCUSSION

Chemistry

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2.1

The general synthetic procedure of target compounds is depicted in

Scheme 1. Target compounds 6a–u were obtained by five steps:

nitration, acylation, ammoniation, reduction, and secondary ammo-

niation. The synthesis started with the nitration of 4‐methoxybenzoic

acid 1 resulting in 4‐methoxy‐3‐nitrobenzoic acid 2, which was

reacted with thionyl chloride to give 4‐methoxy‐3‐nitrobenzoyl

chloride 3. To a mixture of 4‐methoxy‐3‐nitrobenzoyl chloride 3 in

anhydrous tetrahydrofuran was added different substituted aniline,

providing the 4‐methoxy‐3‐nitro‐N‐(substitutedphenyl)benzamides 4

in a high yield. Reduction of compound 4 by treating with Fe and

NH₄Cl generated the 4‐methoxy‐3‐amino‐N‐(substitutedphenyl)‐

benzamides 5. Compound 5 was next reacted with the corresponding

substituted benzoyl chloride to give the final target compounds

4‐methoxy‐3‐arylamido‐N‐(substitutedphenyl)benzamides 6a–u in

anhydrous tetrahydrofuran, in good yields.

Arterial thrombosis illness, such as atherosclerotic vascular disease

and acute coronary syndrome, are treated with antiplatelet agents,^[19]

whereas prevention and treatment of venous thromboses, such as deep‐

vein thrombosis and pulmonary embolism, are achieved using anti-

coagulant agents.^[20]Betrixaban (Figure 1b), which rapidly and selectively

inhibits factor Xa, is the fifth oral anticoagulant.^[21,22]It significantly

reduces the bleeding risk for patients with renal insufficiency.^[23]A great

deal of clinical data have displayed that the combination of antiplatelet

agents and anticoagulant agents has more curative effects over

antiplatelet agents alone, as well as combinations of adenosine diphos-

phate (ADP) inhibitor plus FXa inhibitors,^[24]aspirin plus heparin,^[25]and

warfarin plus clopidogrel.^[26]

FIGURE 1 Structures of picotamide (a), betrixaban (b), previous compounds (c), and the target compounds (d)

LIU ET AL.

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SCHEME 1 Synthesis route of target compounds 6a–u. Reagents and conditions: (a) (CH₃CO)₂O, HNO₃, 0–5°C; (b) SOCl₂, reflux 8 hr, 80°C;

(c) Ar‐NH₂, anhydrous THF, r.t.; (d) Fe power, NH₄Cl, EtOH, reflux 6 hr, 80°C; (e) Ar‐COCl, anhydrous THF, r.t.

A

series of 4‐methoxy‐3‐arylamido‐N‐(substitutedphenyl)‐

compounds 6e and 6l showed better antiplatelet aggregation

activities than picotamide with IC₅₀values of 4.27 and 3.71 μM,

respectively. The best activity was seen by the compound 6c, which

had an IC₅₀value of 3.12 μM.

benzamides 6a–u were designed and synthesized by the procedures

mentioned above. The chemical structures were confirmed by

infrared (IR) spectra, nuclear magnetic resonance spectra (¹H NMR

and ¹³C NMR), and electron spray ionization mass spectra (ESI‐MS).

To sum up, compounds 6c, 6e, 6l and 6r had a stronger inhibitory

effect on platelet aggregation in both of the assays and could have

potential as broad‐spectrum antiplatelet inhibitors. Compound 6c was

found to be the most potent compound with an IC₅₀value of 3.84 μM in

the ADP‐induced assay, whereas the most significant activity was seen by

compound 6f with an IC₅₀value of 3.12 μM in the AA‐induced assay.

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2.2

Biological assays

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2.2.1

In vitro antiplatelet aggregation assay

ADP and AA were employed as inducers of platelet aggregation, and

aspirin and picotamide were used as the positive controls. The

inhibitory effect of the target compounds 6a–u on platelet aggrega-

tion was evaluated by using Born’s turbidimetric method.^[27,28]All the

results are listed in Table 1 and Figures 2 and 3.

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2.2.2

Structure–activity relationship

ADP‐induced platelet aggregation assay

In the ADP‐induced platelet aggregation assay, the compounds with

o‐trifluoromethyl (6c) and m‐trifluoromethyl (6b) groups showed the

strongest inhibitory activities than others, which meant that the

trifluoromethyl group can exert a positive effect on antiplatelet

aggregation. In contrast, the inhibitory activity of the compound with

the p‐trifluoromethyl (6a) group had been dramatically decreased,

which might be related to the position effect of the substituent

group. Meanwhile, the inhibitory activities of the compounds with o‐

isopropyl (6o), m‐isopropyl (6n), and p‐isopropyl (6m) groups were

inferior to that of the corresponding compounds, which contained

electron‐withdrawing groups. Therefore, the isopropyl group was

introduced to decrease the inhibitory activity.

ADP‐induced platelet aggregation assay

All the target compounds exhibited a certain degree of inhibitory effect

on platelet aggregation, among which compounds 6c (38.04 0.07%), 6b

(36.59 0.10%), 6i (33.75 0.06%), 6r (32.70 0.12%), 6l (31.86

0.08%), and 6e (31.44 0.04%) showed significant inhibitory activities

on platelet aggregation, and they were superior to picotamide

(30.83 0.15%). Moreover, the IC₅₀values were calculated for com-

pounds that had higher inhibitory activities. This result indicated that

compounds 6e (IC₅₀: 4.59 µM) and 6r (IC₅₀: 4.66 µM) have stronger

antiplatelet aggregation activities than picotamide (IC₅₀: 4.73 µM).

Compound 6c (IC₅₀: 3.84 µM) showed the strongest activity on platelet

aggregation.

AA‐induced platelet aggregation assay

The best activity was seen by the compounds with o‐fluoro (6f) and

m‐fluoro (6e) groups. The results demonstrated that the compounds

with the fluoro group seemed to promote an inhibitory effect in the

AA‐induced platelet aggregation assay. However, the compound with

p‐fluoro (6d) group exhibited a weaker inhibitory activity. These

results suggested that the position effect of the substituent group

might change the inhibitory effect. In addition, compounds with o‐

isopropyl (6o), m‐isopropyl (6n), and p‐isopropyl (6m) groups

AA‐induced platelet aggregation assay

Compared to the ADP‐induced platelet aggregation, compounds 6f,

6e, 6h, 6c, 6l and 6r inhibited the AA‐induced platelet aggregation

by 34.18 0.08%, 32.10 0.11%, 29.87 0.11%, 29.42 0.10%,

28.11 0.10% and 27.67 0.08%, respectively, which were much

higher than that of picotamide. In addition, the IC₅₀values were

calculated for potent compounds. This result suggested that

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TABLE 1 The effect of target compounds 6a–u on platelet aggregation induced by adenosine diphosphate (ADP) and arachidonic acid (AA)

ADP

AA

Inhibition

rate

Inhibition

rate

Compound

R₁

R₂

Dose (μΜ)

1.30

IC₅₀

–

IC₅₀

–

6a

4‐CF₃

3‐CF₃

2‐CF₃

4‐F

2‐OCH₃

4‐OCH₃

24.21 0.12*

36.59 0.10**

38.04 0.07*

22.48 0.15

31.44 0.04

28.30 0.11

20.62 0.06**

27.90 0.07**

33.75 0.06

23.40 0.12**

28.98 0.11*

31.86 0.08**

17.38 0.05*

25.17 0.04*

24.30 0.07

19.26 0.06*

26.73 0.13*

32.70 0.12

16.11 0.07**

17.44 0.13

17.10 0.10

34.51 0.12

30.83 0.15

18.49 0.06

21.38 0.05**

29.42 0.10

24.59 0.11*

32.10 0.11**

34.18 0.08

16.46 0.11*

29.87 0.11**

23.44 0.07

20.16 0.06*

16.95 0.05

28.11 0.10

15.08 0.05

16.82 0.10**

22.20 0.07

17.21 0.10

16.74 0.13

27.67 0.08*

12.94 0.08

14.03 0.11

21.63 0.10

30.97 0.11

26.55 0.06

6b

5.07

3.84

–

6c

5.06

–

6d

6e

3‐F

4.59

–

4.27

3.12

–

6f

2‐F

6g

4‐Cl

–

6h

3‐Cl

–

4.78

–

6i

2‐Cl

5.40

–

6j

4‐Br

–

6k

3‐Br

–

6l

2‐Br

5.12

–

3.71

–

6m

6n

4‐CH(CH)₃

3‐CH(CH)₃

2‐CH(CH)₃

4‐CF₃

3‐CF₃

2‐CF₃

4‐CH(CH)₃

3‐CH(CH)₃

2‐CH(CH)₃

–

6o

–

6p

–

6q

–

6r

4.66

–

4.16

–

6s

6t

–

6u

–

Aspirin

Picotamide

4.44

4.73

4.11

4.34

*p < 0.05 versus picotamide.

**p < 0.01 versus picotamide.

displayed poor inhibitory activities, which might be the negative

effect of the isopropyl group on platelet aggregation.

potent activity induced by ADP and the compound with the o‐fluoro

(6f) group exhibited the most significant activity induced by AA.

Interestingly, the effects of some compounds on the platelet

aggregation exhibited a similar pattern in both of the assays. The

compounds with the isopropyl group had weaker inhibitory activities,

and the compounds with the o‐methoxy group had better inhibitory

activities than the compounds with the p‐methoxy group.

In both of the platelet aggregation assays

In conclusion, the two platelet aggregation assays had different

compounds showing the best inhibitory activity. Among them, the

compound with the o‐trifluoromethyl (6c) group showed the most

FIGURE 2 The potent compounds

induced by adenosine diphosphate

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FIGURE 3 The potent compounds

induced by arachidonic acid

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2.2.3

In vitro cell toxicity assay on L929 cells

rates of compounds 6f (48.27% and 32.41%) and 6l (48.97% and

31.72%) were inferior to that of picotamide at the dose of 40 and

80 μM. Meanwhile, the cell survival rates of compound 6c (61.72%

and 44.83%) and 6r (58.62% and 45.86%) were superior to that of

picotamide at the dose of 40 and 80 μM.

On the basis of the in vitro antiplatelet activities screening, eight

compounds that had stronger activities were chosen for in vitro cell

toxicity tests on L929 cells via Cell Counting Kit‐8 (CCK‐8) assays.^[29,30]

The absorbance and survival rate are shown in Table 2 and Figure 4.

As shown in Figure 4, the cell survival rates were negatively

correlated with the drug concentration. The survival rates of the

tested compounds were more than 75% at the dose of 10 μM, which

indicated that none of the compounds had obvious cell toxicity.

Compounds 6c, 6f, 6l, and 6r had cell survival rates of 75.86%,

71.03%, 71.38%, and 74.14% at the dose of 20 μM, respectively,

which were higher than that of picotamide. However, the cell survival

To sum up, compound 6c demonstrated the highest antiplatelet

activity and lowest cell toxicity among target compounds. Therefore,

compound 6c could be considered as novel antiplatelet agent.

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2.3

Lipinski’s rule of five

To predict if a chemical compound could be used orally, the more

active compounds against ADP and AA were analyzed according to

TABLE 2 Cell toxicity effect on L929

Compound

Blank group

Control group

6b

Dose (μM)

–

Absorbance

0.112

0.402

0.339

0.310

0.268

0.225

0.373

0.332

0.291

0.242

0.345

0.308

0.264

0.224

0.367

0.318

0.252

0.206

0.343

0.302

Survival rate (%)

–

Compound

Dose (μM)

Absorbance

Survival rate (%)

–

10

20

40

80

10

20

40

80

10

20

40

80

10

20

40

80

10

20

78.28

68.27

53.79

38.96

90.10

75.86

61.72

44.83

80.34

67.59

52.41

38.62

87.93

71.03

48.27

32.41

79.31

65.52

40

80

10

20

40

80

10

20

40

80

10

20

40

80

10

20

40

80

0.258

0.221

0.331

0.298

0.268

0.217

0.364

0.319

0.254

0.204

0.365

0.327

0.282

0.245

0.348

0.313

0.272

0.230

50.34

37.59

75.52

64.14

53.79

36.21

86.90

71.38

48.97

31.72

87.24

74.14

58.62

45.86

81.38

69.31

55.17

40.69

6i

6c

6e

6f

6p

6r

picotamide

6h

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FIGURE 4 Cell survival rates of the

tested compounds

Lipinski’s “rule of five” using MarvinSketch (version 17.3),^[31]and all

results are shown in Tables 3 and 4.

promising candidate for the prevention and treatment of thromboembolic

disease.

The results indicated that all active compounds fulfilled the

Lipinski rule of five (Tables 3 and 4), reinforcing their promising

profile for further experimental investigations to design new

antiplatelet drugs against thromboembolic diseases.

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4

EXPERIMENTAL

Chemistry

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4.1

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General

3

CONCLUSION

4.1.1

In this study, a series of 4‐methoxy‐3‐arylamido‐N‐(substitutedphenyl)‐

benzamides 6a–u, which were the analogs of picotamide, were designed,

synthesized, and evaluated for antiplatelet activities by using ADP and

AA as platelet aggregation inducers. The obtained data demonstrated

that compounds 6c, 6e, 6l, and 6r had a stronger inhibitory effect on

platelet aggregation in both of the assays and could have potential as

broad‐spectrum antiplatelet inhibitors. In addition, compound 6c was

found to be the most potent compound with an IC₅₀value of 3.84 μM in

the ADP‐induced assay, whereas the most significant activity in the AA‐

induced assay was shown by the compound 6f with an IC₅₀value of

3.12 μM. In the subsequent in vitro cell toxicity assay, compounds 6c and

6r displayed lower cell toxicity than picotamide. Altogether the results

showed that compound 6c might warrant further evaluation as a

All chemical reagents and solvents were purchased from Tianjin,

Hengshan (China), Aladdin Industrial Corporation (China), and

Energy Chemical (China), and were used without further purification.

The biological reagents were purchased from Beyotime Biotechnol-

ogy (China).

Melting points (mp) were measured on X‐4 digital display

micromelting point apparatus and were not corrected. IR spectra

were recorded on an Avatar 370 spectrophotometer as potassium

bromide (KBr) disks. ¹H NMR (400 MHz) and ¹³C NMR (101 MHz)

spectra were recorded on an AVANCE III spectrometer in deuterated

solvents (DMSO [dimethyl sulfoxide]‐d₆). Chemical shifts (δ) are

reported in parts per million (ppm) downfield from tetramethylsilane

as internal standard, and coupling constants (J) are expressed in

TABLE 3 The calculated parameters of Lipinski’s rule of five for

TABLE 4 The calculated parameters of the Lipinski rule of five for

the more active compounds against adenosine diphosphate

the more active compounds against arachidonic acid

Lipinski’s rule of five^a

IC₅₀(μM)

4.59

HBA

4

HBD

Mw

clog P

4.25

2.80

3.22

2.97

3.23

2.87

1.02

0.95

IC₅₀(μM)

5.06

HBA

4

HBD

Mw

clog P

2.80

3.22

1.86

3.89

3.23

2.87

1.02

0.95

Compound

6b

Compound

6c

2

1

2

444.41

394.40

410.85

455.31

444.41

180.16

376.42

2

1

2

444.41

394.40

410.85

455.31

444.41

180.16

376.42

6c

3.84

4

6e

4.27

4

6e

5.40

4

6f

3.12

4

6i

4.07

4

6h

4.78

4

6l

5.12

4

6l

3.71

4

6r

4.66

4

6r

4.16

4

Aspirin

Picotamide

4.44

3

Aspirin

Picotamide

4.34

3

4.73

5

4.11

5

^aNumber of hydrogen bond acceptors (HBA) ≤ 10, number of hydrogen

bond donors (HBD) ≤ 5, clog P ≤ 5, and molecular weight (Mw) ≤ 500

^aNumber of hydrogen bond acceptors (HBA) ≤ 10, number of hydrogen

bond donors (HBD) ≤ 5, clog P ≤ 5, and molecular weight (Mw) ≤ 500.

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Hertz. Signal multiplicities are represented by the following

abbreviations: s (singlet), d (doublet), t (triplet), dd (double doublet),

q (quartet), m (multiplet). ESI‐MS spectra were obtained on a Xevo

GZQ‐Tof VHRS mass spectrometer. Analytical thin‐layer chromato-

graphy (TLC) was performed on silica gel GF₂₅₄plates. The solvents

for TLC were petroleum ether/ethyl acetate (1:1) and dichloro-

methane/methanol (10:1).

reaction mixture was hot filtered and the filtrate was extracted

with dichloromethane (50 ml). The organic phase was washed

with water (3 × 20 ml), saturated sodium chloride (3 × 20 ml), and

dried with anhydrous magnesium sulfate. Ethanol and dichlor-

omethane were evaporated under reduced pressure to give a

white solid, 3‐amino‐N‐(2‐fluorophenyl)‐4‐methoxybenzamide 5

(0.98 g, 94.86%).

The original spectra, as well as the InChI codes of the investigated

compounds, are provided as Supporting Information.

|

4.1.6

Synthesis of N‐(2‐fluorophenyl)‐4‐methoxy‐

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4.1.2

Synthesis of 4‐methoxy‐3‐nitrobenzoic acid 2

3‐(2‐methoxybenzamido)benzamide (6f)

4‐Methoxybenzoic acid 1 (3.00 g, 0.02 mol) was added to a solution of

acetic anhydride (30 ml), and the reaction mixture was stirred at 0–5°C.

Next, 68% HNO₃(6 ml) was added dropwise to the reaction mixture at

0–5°C, and the reaction mixture was stirred at room temperature for

about 2 hr. After the reaction was completed as monitored by TLC, the

reaction mixture was cooled by the addition of a water and ice mixture.

The crude solid 4‐methoxy‐3‐nitrobenzoic acid 2 was filtered off and

further purified by recrystallization using ethyl acetate (2.91 g, 74.61%).

4‐Methoxybenzoyl chloride (0.62 g, 0.036 mol) was dissolved in anhy-

drous tetrahydrofuran (15 ml). The above solution was added dropwise to

the mixture of 3‐amino‐N‐(2‐fluorophenyl)‐4‐methoxybenzamide

5

(0.95 g, 0.036 mol) and anhydrous tetrahydrofuran (15 ml) at room

temperature with continuous stirring. After 10 minutes, triethylamine

(0.5 ml), as an acid‐binding agent, was added to the reaction mixture. The

progress of the reaction was monitored by TLC. After completion,

the solvent was evaporated under reduced pressure. The resulting

residue was washed with 5% sodium hydroxide solution, dilute

hydrochloric acid (V_HCl/V_water= 1:4), and water until neutral in an ice

bath. The crude solid was filtered off and recrystallized from ethanol to

give N‐(2‐fluorophenyl)‐4‐methoxy‐3‐(4‐methoxybenzamido)benzamide

6f (1.20 g, 83.68%).

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4.1.3

Synthesis of 4‐methoxy‐3‐nitrobenzoyl

chloride (3)

4‐Methoxy‐3‐nitrobenzoic acid (1.00 g, 0.005 mol) 2 was added to the

excess quantity of thionyl chloride (10 ml) and the reaction mixture was

refluxed for 8 hr at 80°C with stirring. The reaction mixture was allowed

to cool, and excess thionyl chloride was evaporated under reduced

pressure to give 4‐methoxy‐3‐nitrobenzoyl chloride 3 (1.06 g, 96.74%).

Target compounds 6a–e and 6g–u were prepared similarly as

compound 6f.

N‐(4‐Trifluoromethylphenyl)‐4‐methoxy‐3‐(2‐methoxybenzamido)‐

benzamide (6a)

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4.1.4

Synthesis of N‐(2‐fluorophenyl)‐4‐methoxy‐

This compound was obtained as white granules (acetone); yield:

49.94%; mp: 294.6–295.2°C; IR (KBr, cm⁻¹): 3,262.25, 1,660.81,

1,596.26, 1,506.53, 1,325.49, 1,248.49, 1,157.39, 1,114.55, 1,020.14,

905.01, 828.40, and 753.88. ¹H NMR (400 MHz, DMSO) δ 10.76 (1H,

s, CONH), 10.55 (1H, s, CONH), 9.08 (1H, d, J = 1.8 Hz, 2‐H), 8.13 (1H,

dd, J = 8.5, 1.8 Hz, 6‐H), 8.02 (2H, d, J = 8.4 Hz, Ar–H), 7.78 (1H, dd,

J = 8.5, 1.8 Hz, Ar–H), 7.72 (2H, d, J = 8.5 Hz, Ar–H), 7.61 (1H, t,

J = 7.1 Hz, Ar–H), 7.31 (1H, d, J = 8.3 Hz, Ar–H), 7.26 (1H, d, J = 8.6 Hz,

5‐H), 7.17 (1H, t, J = 7.4 Hz, Ar–H), 4.10 (3H, s, OCH₃), and 4.06 (3H,

s, OCH₃); ¹³C NMR (101 MHz, DMSO) δ 166.23 (C, C═O), 162.80

(C, C═O), 157.72 (C, Ar–C), 151.40 (C, 4‐C), 143.55 (C, Ar–C), 134.28

(C, Ar–C), 131.88 (C, Ar–C), 128.25 (C, Ar–C), 127.47 (C, Ar–C),

126.34 (C, Ar–C), 126.30 (C, Ar–C), 124.30 (C, CF₃), 121.77

(C, Ar–C), 121.38 (C, Ar–C), 120.56 (C, Ar–C), 119.71 (C, 2‐C),

113.18 (C, 5‐C), 110.62 (C, Ar–C), 57.14 (C, OCH₃), and 57.02

(C, OCH₃); HR‐ESI‐MS (m/z) Calcd for C₂₃H₂₀N₂O₄F₃[M+H]⁺

445.1369. Found: 445.1375.

3‐nitrobenzamide (4)

4‐Methoxy‐3‐nitrobenzoyl chloride 3 (2.00 g, 0.009 mol) was dis-

solved in anhydrous tetrahydrofuran (15 ml). The above solvent was

added dropwise to the mixture of 2‐fluoroaniline (1.03 g, 0.009 mol)

and anhydrous tetrahydrofuran (15 ml) at room temperature with

continuous stirring. After 10 minutes, triethylamine (0.5 ml), as an

acid‐binding agent, was added to the reaction mixture. The progress

of the reaction was monitored by TLC. After completion, the solvent

was evaporated under reduced pressure. The resulting residue was

washed with 5% sodium hydroxide solution and dilute hydrochloric

acid (V_HCl/V_water= 1:4) in an ice bath. The crude solid was filtered off

and recrystallized from ethanol to give N‐(2‐fluorophenyl)‐4‐

methoxy‐3‐nitrobenzamide 4 (2.34 g, 86.98%).

|

4.1.5

Synthesis of 3‐amino‐N‐(2‐fluorophenyl)‐4‐

methoxybenzamide (5)

N‐(3‐Trifluoromethylphenyl)‐4‐methoxy‐3‐(2‐methoxybenzamido)‐

benzamide (6b)

A

mixture of N‐(2‐fluorophenyl)‐4‐methoxy‐3‐nitrobenzamide 4

(1.15 g, 0.004 mol), ammonium chloride (1.15 g, 0.029 mol), Fe

powder (1.38 g, 0.024 mol) in ethanol (50 ml) was stirred for 7 hr at

80°C. After the reaction was completed, under TLC monitoring, the

This compound was obtained as light yellow needles (acetone); yield:

55.07%; mp: 215.5–216.1°C; IR (KBr, cm⁻¹): 3,313.11, 3,261.61,

1,647.25, 1,609.13, 1,557.03, 1,498.30, 1,329.04, 1,263.28, 1,170.69,

8 of 12

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1,120.90, 1,022.28, 892.51, 803.10, 760.92, 722.80, and 589.35; ¹H

NMR (400 MHz, DMSO) δ 10.76 (1H, s, CONH), 10.52 (1H, s, CONH),

9.09 (1H, d, J = 2.0 Hz, 2‐H), 8.26 (1H, s, Ar–H), 8.13 (1H, dd, J = 8.5,

2.0 Hz, 6‐H), 8.08 (1H, d, J = 8.2 Hz, Ar–H), 7.79 (1H, dd, J = 8.6,

2.1 Hz, Ar–H), 7.64–7.57 (2H, m, Ar–H), 7.44 (1H, d, J = 7.7 Hz, Ar–H),

7.31 (1H, d, J = 8.3 Hz, Ar–H), 7.26 (1H, d, J = 8.6 Hz, 5‐H), 7.17 (1H, t,

J = 7.4 Hz, Ar–H), 4.10 (3H, s, OCH₃), and 4.06 (3H, s, OCH₃). ¹³C

NMR (101 MHz, DMSO) δ 166.13 (C, C═O), 162.79 (C, C═O), 157.71

(C, Ar–C), 151.38 (C, 4‐C), 140.69 (C, Ar–C), 134.26 (C, Ar–C), 131.89

(C, Ar–C), 130.24 (C, Ar–C), 128.25 (C, Ar–C), 127.39 (C, Ar–C),

126.04 (C, Ar–C), 124.20 (C, CF₃), 121.76 (C, Ar–C), 121.37

(C, Ar–C), 119.65 (C, 2‐C), 116.85 (C, Ar–C), 116.81 (C, Ar–C),

113.15 (C, 5‐C), 110.62 (C, Ar–C), 57.12 (C, OCH₃), and 57.00 (C,

OCH₃); HR‐ESI‐MS (m/z) Calcd for C₂₃H₂₀N₂O₄F₃[M+H]⁺445.1371.

Found: 445.1375.

N‐(3‐Fluorophenyl)‐4‐methoxy‐3‐(2‐methoxybenzamido)benzamide

(6e)

This compound was obtained as light yellow flocculent (acetone);

yield: 49.68%; mp: 270.8–271.4°C; IR (KBr, cm⁻¹): 3,302.47,

1,658.74, 1,597.23, 1,522.34, 1,482.98, 1,435.51, 1,236.60,

1,147.50, 1,017.04, 846.33, 763.61, and 687.20; ¹H NMR

(400 MHz, DMSO) δ 10.75 (1H, s, CONH), 10.39 (1H, s, CONH),

9.06 (1H, d, J = 2.0 Hz, 2‐H), 8.13 (1H, dd, J = 7.8 Hz, 6‐H), 7.77 (1H, s,

Ar–H), 7.74 (1H, d, J = 1.5 Hz, Ar–H), 7.62 (1H, t, J = 8.6 Hz, Ar–H),

7.57 (1H, d, J = 8.3 Hz, Ar–H), 7.38 (1H, dd, J = 15.3, 8.0 Hz, Ar–H),

7.31 (1H, d, J = 8.4 Hz, Ar–H), 7.25 (1H, d, J = 8.6 Hz, 5‐H), 7.18 (1H, t,

J = 7.5 Hz, Ar–H), 6.92 (1H, t, J = 7.2 Hz, Ar–H), 4.11 (3H, s, OCH₃),

and 4.06 (3H, s, OCH₃); ¹³C NMR (101 MHz, DMSO) δ 166.03

(C, C═O), 163.75 (C, Ar–C), 162.78 (C, C═O), 157.71 (C, Ar–C),

151.28 (C, 4‐C), 134.29 (C, Ar–C), 131.89 (C, Ar–C), 130.67 (C, Ar–C),

130.57 (C, Ar–C), 128.21 (C, Ar–C), 127.62 (C, Ar–C), 124.15 (C,

Ar–C), 121.78 (C, Ar–C), 121.35 (C, Ar–C), 119.62 (C, 2‐C), 116.40 (C,

Ar–C), 113.17 (C, 5‐C), 110.60 (C, Ar–C), 107.52 (C, Ar–C), 107.26 (C,

Ar–C), 57.13 (C, OCH₃), and 57.03 (C, OCH₃); HR‐ESI‐MS (m/z) Calcd

for C₂₂H₂₀N₂O₄F [M+H]⁺395.1415. Found: 395.1407.

N‐(2‐Trifluoromethylphenyl)‐4‐methoxy‐3‐(2‐methoxybenzamido)‐

benzamide (6c)

This compound was obtained as white granules (acetone); yield:

70.54%; mp: 227.2–227.6°C; IR (KBr, cm⁻¹): 3,320.74, 3,261.33,

1,660.59, 1,594.84, 1,520.99, 1,480.63, 1,312.07, 1,254.13, 1,158.22,

1,112.28, 1,022.08, 904.63, 811.86, 758.59, 680.57; ¹H NMR

(400 MHz, DMSO) δ 10.75 (1H, s, CONH), 10.04 (1H, s, CONH),

9.10 (1H, d, J = 1.7 Hz, 2‐H), 8.13 (1H, dd, J = 8.7, 2.0 Hz, 6‐H),

7.84–7.70 (3H, m, Ar–H), 7.63–7.49 (3H, m, Ar–H), 7.29 (1H, d,

J = 8.3 Hz, Ar–H), 7.24 (1H, d, J = 8.6 Hz, 5‐H), 7.16 (1H, t, J = 7.4 Hz,

Ar–H), 4.09 (3H, s, OCH₃), and 4.05 (3H, s, OCH₃); ¹³C NMR

(101 MHz, DMSO) δ 166.52 (C, C═O), 162.72 (C, C═O), 157.68 (C,

Ar–C), 151.23 (C, 4‐C), 136.58 (C, Ar–C), 134.29 (C, Ar–C), 133.53 (C,

Ar–C), 131.63 (C, Ar–C), 128.24 (C, Ar–C), 127.68 (C, Ar–C), 126.97

(C, Ar–C), 125.55 (C, Ar–C), 123.93 (C, CF₃), 122.83 (C, Ar–C),

121.75 (C, Ar–C), 121.30 (C, Ar–C), 119.64 (C, 2‐C), 113.13 (C, 5‐C),

110.62 (C, Ar–C), 57.10 (C, OCH₃), and 57.00 (C, OCH₃); HR‐ESI‐MS

(m/z) Calcd for C₂₃H₂₀N₂O₄F₃[M+H]⁺445.1378. Found: 445.1375.

N‐(2‐Fluorophenyl)‐4‐methoxy‐3‐(2‐methoxybenzamido)benzamide

(6f)

This compound was obtained as white solid (acetone); yield: 69.91%;

mp: 248.7–249.5°C; IR (KBr, cm⁻¹): 3,450.42, 3,314.74, 3,249.82,

1,659.31, 1,596.90, 1,551.71, 1,506.80, 1,303.70, 1,257.11, 1,021.19,

904.42, 818.98, 755.55, and 685.40; ¹H NMR (400 MHz, DMSO) δ

10.74 (1H, s, CONH), 10.05 (1H, s, CONH), 9.08 (1H, d, J = 1.8 Hz, 2‐

H), 8.12 (1H, d, J = 7.7 Hz, 6‐H), 7.79 (1H, dd, J = 8.6, 1.9 Hz, Ar–H),

7.63 (1H, d, J = 7.3 Hz, Ar–H), 7.58 (1H, t, J = 7.7 Hz, Ar–H), 7.34–7.21

(5H, m, 5‐H, Ar–H), 7.17 (1H, t, J = 7.6 Hz, Ar–H), 4.11 (3H, s, OCH₃),

and 4.06 (3H, s, OCH₃); ¹³C NMR (101 MHz, DMSO) δ 165.63 (C,

C═O), 162.75 (C, C═O), 157.70 (C, Ar–C), 155.07 (C, Ar–C), 151.29

(C, 4‐C), 134.27 (C, Ar–C), 131.87 (C, Ar–C), 128.17 (C, Ar–C), 127.54

(C, Ar–C), 127.00 (C, Ar–C), 124.73 (C, Ar–C), 124.69 (C, Ar–C),

124.20 (C, Ar–C), 121.76 (C, Ar–C), 121.37 (C, Ar–C), 119.77 (C, 2‐C),

116.35 (C, Ar–C), 116.15 (C, Ar–C), 113.16 (C, 5‐C), 110.61 (C, Ar–C),

57.10 (C, OCH₃), and 57.01 (C, OCH₃); HR‐ESI‐MS (m/z) Calcd for

C₂₂H₂₀N₂O₄F [M+H]⁺395.1408. Found: 395.1407.

N‐(4‐Fluorophenyl)‐4‐methoxy‐3‐(2‐methoxybenzamido)benzamide

(6d)

This compound was obtained as brown needles (ethanol); yield:

45.09%; mp: 275.7–277.1°C; IR (KBr, cm⁻¹): 3,300.60, 1,655.41,

1,597.16, 1,514.33, 1,231.16, 1,148.82, 1,018.01, 900.73, 824.04,

755.49, 690.83, and 508.79; ¹H NMR (400 MHz, DMSO) δ 10.74 (1H,

s, CONH), 10.26 (1H, s, CONH), 9.05 (1H, d, J = 1.9 Hz, 2‐H), 8.13 (1H,

d, J = 7.8 Hz, 6‐H), 7.82–7.73 (3H, m, Ar–H), 7.62 (1H, t, J = 7.5 Hz,

Ar–H), 7.31 (1H, d, J = 8.4 Hz, Ar–H), 7.26–7.21 (2H, m, 5‐H, Ar–H),

7.18 (2H, d, J = 8.3 Hz, Ar–H), 4.10 (3H, s, OCH₃), and 4.06 (3H, s,

OCH₃); ¹³C NMR (101 MHz, DMSO) δ 165.42 (C, C═O), 162.46

(C, C═O), 157.41 (C, Ar–C), 150.83 (C, 4‐C), 135.91 (C, Ar–C), 134.00

(C, Ar–C), 131.59 (C, Ar–C), 127.86 (C, Ar–C), 127.52 (C, Ar–C),

123.73 (C, Ar–C), 122.36 (C, Ar–C), 122.29 (C, Ar–C), 121.05 (C,

Ar–C), 119.30 (C, 2‐C), 115.41 (C, Ar–C), 115.19 (C, Ar–C), 112.87 (C,

5‐C), 110.27 (C, Ar–C), 56.77 (C, OCH₃), and 56.73 (C, OCH₃); HR‐

ESI‐MS (m/z) Calcd for C₂₂H₂₀N₂O₄F [M+H]⁺395.1407. Found:

395.1407.

N‐(4‐Chlorophenyl)‐4‐methoxy‐3‐(2‐methoxybenzamido)benzamide

(6g)

This compound was obtained as yellow flocculent (acetone); yield:

41.81%; mp: 313.7–315.2℃; IR (KBr, cm⁻¹): 3,297.20, 1,659.37,

1,593.81, 1,548.78, 1,504.56, 1,392.01, 1,303.76, 1,243.11, 1,092.34

1,017.81, 901.72, 816.49, and 755.03; ¹H NMR (400 MHz, DMSO) δ

10.75 (1H, s, CONH), 10.33 (1H, s, CONH), 9.05 (1H, d, J = 1.6 Hz, 2‐

H), 8.12 (1H, d, J = 7.7 Hz, 6‐H), 7.82 (2H, d, J = 8.7 Hz, Ar–H), 7.75

(1H, dd, J = 8.5, 1.7 Hz, Ar–H), 7.61 (1H, t, J = 7.7 Hz, Ar–H), 7.41 (2H,

d, J = 8.7 Hz, Ar–H), 7.31 (1H, d, J = 8.4 Hz, Ar–H), 7.24 (1H, d,

J = 8.6 Hz, 5‐H), 7.17 (1H, t, J = 7.5 Hz, Ar–H), 4.10 (3H, s, OCH₃), and

4.06 (3H, s, OCH₃); ¹³C NMR (101 MHz, DMSO) δ 165.87 (C, C═O),

162.77 (C, C═O), 157.71 (C, Ar–C), 151.22 (C, 4‐C), 138.86 (C, Ar–C),

LIU ET AL.

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134.29 (C, Ar–C), 131.88 (C, Ar–C), 128.94 (C, Ar–C), 128.19 (C,

Ar–C), 127.71 (C, Ar–C), 127.49 (C, Ar–C), 124.11 (C, Ar–C), 122.31

(C, Ar–C), 121.77 (C, Ar–C), 121.36 (C, Ar–C), 119.63 (C, 2‐C), 113.17

(C, 5‐C), 110.59 (C, Ar–C), 57.12 (C, OCH₃), and 57.03 (C, OCH₃);

HR‐ESI‐MS (m/z) Calcd for C₂₂H₂₀N₂O₄Cl [M+H]⁺411.1114. Found:

411.1111.

813.41, and 755.22; ¹H NMR (400 MHz, DMSO) δ 10.74 (1H, s,

CONH), 10.32 (1H, s, CONH), 9.05 (1H, d, J = 2.0 Hz, 2‐H), 8.12 (1H,

d, J = 7.6 Hz, 6‐H), 7.77 (2H, d, J = 8.8 Hz, Ar–H), 7.74 (1H, d,

J = 2.1 Hz, Ar–H), 7.62 (1H, t, J = 6.9 Hz, Ar–H), 7.53 (2H, d,

J = 8.9 Hz, Ar–H), 7.31 (1H, d, J = 8.3 Hz, Ar–H), 7.24 (1H, d,

J = 8.7 Hz, 5‐H), 7.18 (1H, t, J = 7.5 Hz, Ar–H), 4.11 (3H, s, OCH₃),

and 4.06 (3H, s, OCH₃); ¹³C NMR (101 MHz, DMSO) δ 165.86 (C,

C═O), 162.75 (C, C═O), 157.70 (C, Ar–C), 151.19 (C, 4‐C), 139.27 (C,

Ar–C), 134.32 (C, Ar–C), 131.86 (C, Ar–C), 128.16 (C, Ar–C), 127.67

(C, Ar–C), 124.13 (C, Ar–C), 122.65 (C, Ar–C), 121.78 (C, Ar–C),

121.29 (C, Ar–C), 119.58 (C, 2‐C), 115.53 (C, Ar–C), 113.17 (C, 5‐C),

110.59 (C, Ar–C), 57.13 (C, OCH₃), and 57.04 (C, OCH₃); HR‐ESI‐MS

(m/z) Calcd for C₂₂H₂₀N₂O₄Br [M+H]⁺457.0590. Found: 457.0589.

N‐(3‐Chlorophenyl)‐4‐methoxy‐3‐(2‐methoxybenzamido)benzamide

(6h)

This compound was obtained as white granules (acetone); yield:

41.88%; mp: 203.0–204.3℃; IR (KBr, cm⁻¹): 3,458.10, 3,312.76,

1,660.78, 1,594.22, 1,550.34, 1,481.14, 1,422.01, 1,246.19, 1,143.93,

1,086.42, 1,024.67, 830.71, 750.38, and 683.49; ¹H NMR (400 MHz,

DMSO) δ 10.76 (1H, s, CONH), 10.37 (1H, s, CONH), 9.06 (1H, d,

J = 1.8 Hz, 2‐H), 8.15 (1H, d, J = 7.8 Hz, 6‐H), 7.97 (1H, s, Ar–H), 7.76

(1H, dd, J = 8.7, 1.9 Hz, Ar–H), 7.72 (1H, d, J = 8.3 Hz, Ar–H), 7.62 (1H,

t, J = 7.8 Hz, Ar–H), 7.38 (1H, t, J = 8.1 Hz, Ar–H), 7.31 (1H, d,

J = 8.4 Hz, Ar–H), 7.25 (1H, d, J = 8.6 Hz, 5‐H), 7.17 (2H, dd, J = 13.9,

6.9 Hz, Ar–H), 4.11 (3H, s, OCH₃), and 4.06 (3H, s, OCH₃); ¹³C NMR

(101 MHz, DMSO) δ 166.01 (C, C═O), 162.77 (C, C═O), 157.70

(C, Ar–C), 151.29 (C, 4‐C), 141.39 (C, Ar–C), 134.27 (C, Ar–C), 133.39

(C, Ar–C), 131.89 (C, Ar–C), 130.71 (C, Ar–C), 128.22 (C, Ar–C),

127.54 (C, Ar–C), 124.15 (C, Ar–C), 123.54 (C, Ar–C), 121.76

(C, Ar–C), 121.35 (C, Ar–C), 120.15 (C, Ar–C), 119.63 (C, 2‐C), 119.07

(C, Ar–C), 113.14 (C, 5‐C), 110.58 (C, Ar–C), 57.12 (C, OCH₃), and

57.01 (C, OCH₃); HR‐ESI‐MS (m/z) Calcd for C₂₂H₂₀N₂O₄Cl [M+H]⁺

411.1115. Found: 411.1111.

N‐(3‐Bromophenyl)‐4‐methoxy‐3‐(2‐methoxybenzamido)benzamide

(6k)

This compound was obtained as white flocculent (DMF); yield:

53.28%; mp: 219.4–220.7℃; IR (KBr, cm⁻¹): 3,459.05, 3,310.12,

2,357.59, 1,661.11, 1,592.71, 1,549.36, 1,479.25, 1,418.17, 1,247.76,

1,142.92, 1,083.87, 1,024.92, 762.75, and 679.37; ¹H NMR

(400 MHz, DMSO) δ 10.75 (1H, s, CONH), 10.35 (1H, s, CONH),

9.06 (1H, d, J = 2.0 Hz, 2‐H), 8.13 (1H, dd, J = 8.5, 1.7 Hz, 6‐H), 8.10

(1H, s, Ar–H), 7.77 (1H, d, J = 7.5 Hz, Ar–H), 7.75 (1H, d, J = 2.1 Hz,

Ar–H), 7.62 (1H, t, J = 7.0 Hz, Ar–H), 7.36–7.23 (4H, m, 5‐H, Ar–H),

7.18 (1H, t, J = 7.4 Hz, Ar–H), 4.10 (3H, s, OCH₃), and 4.06 (3H, s,

OCH₃); ¹³C NMR (101 MHz, DMSO) δ 165.97 (C, C═O), 162.77

(C, C═O), 157.70 (C, Ar–C), 151.30 (C, 4‐C), 141.52 (C, Ar–C), 134.30

(C, Ar–C), 131.89 (C, Ar–C), 131.05 (C, Ar–C), 128.20 (C, Ar–C),

127.50 (C, Ar–C), 126.45 (C, Ar–C), 124.17 (C, Ar–C), 123.00

(C, Ar–C), 121.86 (C, Ar–C), 121.78 (C, Ar–C), 121.32 (C, Ar–C),

119.62 (C, 2‐C), 119.45 (C, Ar–C), 113.17 (C, 5‐C), 110.61 (C, Ar–C),

57.14 (C, OCH₃), and 57.03 (C, OCH₃); HR‐ESI‐MS (m/z) Calcd for

C₂₂H₂₀N₂O₄Br [M+H]⁺457.0585. Found: 457.0589.

N‐(2‐Chlorophenyl)‐4‐methoxy‐3‐(2‐methoxybenzamido)benzamide

(6i)

This compound was obtained as white needles (acetone); yield:

48.04%; mp: 215.4–216.7℃; IR (KBr, cm⁻¹): 3,314.49, 3,249.70,

1,663.43, 1,594.08, 1,550.57, 1,510.38, 1,442.65, 1,302.31, 1,254.03,

1,154.11, 1,020.19, 905.10, 753.31, and 680.57; ¹H NMR (400 MHz,

DMSO) δ 10.75 (1H, s, CONH), 9.97 (1H, s, CONH), 9.09 (1H, d,

J = 1.8 Hz, 2‐H), 8.12 (1H, d, J = 7.8 Hz, 6‐H), 7.80 (1H, dd, J = 8.5,

1.9 Hz, Ar–H), 7.61 (2H, d, J = 7.7 Hz, Ar–H), 7.56 (1H, d, J = 7.9 Hz,

Ar–H), 7.39 (1H, t, J = 7.7 Hz, Ar–H), 7.34–7.27 (2H, m, Ar–H), 7.25

(1H, d, J = 8.6 Hz, 5‐H), 7.17 (1H, t, J = 7.5 Hz, Ar–H), 4.11 (3H, s,

OCH₃), and 4.06 (3H, s, OCH₃); ¹³C NMR (101 MHz, DMSO) δ 165.58

(C, C═O), 162.77 (C, C═O), 157.70 (C, Ar–C), 151.32 (C, 4‐C), 135.84

(C, Ar–C), 134.30 (C, Ar–C), 131.88 (C, Ar–C), 129.99 (C, Ar–C),

129.72 (C, Ar–C), 128.58 (C, Ar–C), 128.23 (C, Ar–C), 127.93

(C, Ar–C), 127.63 (C, Ar–C), 127.01 (C, Ar–C), 124.06 (C, Ar–C),

121.77 (C, Ar–C), 121.34 (C, Ar–C), 119.66 (C, 2‐C), 113.16 (C, 5‐C),

110.67 (C, Ar–C), 57.12 (C, OCH₃), and 57.02 (C, OCH₃); HR‐ESI‐MS

(m/z) Calcd for C₂₂H₂₀N₂O₄Cl [M+H]⁺411.1114. Found: 411.1111.

N‐(2‐Bromophenyl)‐4‐methoxy‐3‐(2‐methoxybenzamido)benzamide

(6l)

This compound was obtained as white needles (ethanol); yield:

55.02%; mp: 227.1–228.8℃; IR (KBr, cm⁻¹): 3,454.19, 1,657.23,

1,595.64, 1,549.83, 1,508.76, 1,301.77, 1,253.93, 1,152.05, 1,020.05,

903.49, 752.30, and 677.28; ¹H NMR (400 MHz, DMSO) δ 10.74 (1H,

s, CONH), 9.93 (1H, s, CONH), 9.09 (1H, J = 1.9 Hz, 2‐H), 8.12 (1H, d,

J = 7.7 Hz, 6‐H), 7.81 (1H, d, J = 8.5 Hz, Ar–H), 7.72 (1H, d, J = 8.0 Hz,

Ar–H), 7.62 (1H, d, J = 7.4 Hz, Ar–H), 7.59 (1H, d, J = 7.7 Hz, Ar–H),

7.43 (1H, t, J = 7.6 Hz, Ar–H), 7.31 (1H, d, J = 8.4 Hz, Ar–H), 7.27–7.14

(3H, m, 5‐H, Ar–H), 4.11 (3H, s, OCH₃), and 4.06 (3H, s, OCH₃); ¹³C

NMR (101 MHz, DMSO) δ 165.16 (C, C═O), 162.43 (C, C═O), 157.38

(C, Ar–C), 150.99 (C, 4‐C), 136.94 (C, Ar–C), 133.95 (C, Ar–C), 132.78

(C, Ar–C), 131.56 (C, Ar–C), 128.60 (C, Ar–C), 128.22 (C, Ar–C),

127.95 (C, Ar–C), 127.74 (C, Ar–C), 126.75 (C, Ar–C), 123.65

(C, Ar–C), 121.44 (C, Ar–C), 121.06 (C, Ar–C), 120.41 (C, Ar–C),

119.34 (C, 2‐C), 112.85 (C, 5‐C), 110.36 (C, Ar–C), 56.81 (C, OCH₃),

and 56.71 (C, OCH₃); HR‐ESI‐MS (m/z) Calcd for C₂₂H₂₀N₂O₄Br

[M+H]⁺457.0594. Found: 457.0589.

N‐(4‐Bromophenyl)‐4‐methoxy‐3‐(2‐methoxybenzamido)benzamide

(6j)

This compound was obtained as white solid (DMF); yield: 56.90%;

mp: 316.4–317.6℃; IR (KBr, cm⁻¹): 3,262.08, 1,657.90, 1,592.66,

1,549.52, 1,501.90, 1,387.81, 1,303.09, 1,243.61, 1,017.63, 901.72,

10 of 12

LIU ET AL.

|

N‐(4‐Isopropylphenyl)‐4‐methoxy‐3‐(2‐methoxybenzamido)‐

benzamide (6m)

CH₃); ¹³C NMR (101 MHz, DMSO) δ 166.23 (C, C═O), 162.71 (C,

C═O), 157.70 (C, Ar–C), 151.05 (C, 4‐C), 145.33 (C, Ar–C), 135.83 (C,

Ar–C), 134.24 (C, Ar–C), 131.88 (C, Ar–C), 128.59 (C, Ar–C), 128.19

(C, Ar–C), 127.65 (C, Ar–C), 127.20 (C, Ar–C), 126.26 (C, Ar–C),

126.04 (C, Ar–C), 123.80 (C, Ar–C), 121.76 (C, Ar–C), 121.42

(C, Ar–C), 119.78 (C, 2‐C), 113.15 (C, 5‐C), 110.56 (C, Ar–C), 57.08

(C, OCH₃), 57.01 (C, OCH₃), 28.01 (C, CH), and 23.63 (C, 2× CH₃);

HR‐ESI‐MS (m/z) Calcd for C₂₅H₂₇N₂O₄[M+H]⁺419.1990. Found:

419.1971.

This compound was obtained as white needles (acetone); yield:

47.78%; mp: 211.3–212.5℃; IR (KBr, cm⁻¹): 3,308.28, 2,957.50,

1,662.60, 1,596.62, 1,547.70, 1,418.35, 1,244.50, 1,141.39, 1,087.45,

1,024.22, 899.18, 832.60, 751.99, 544.64; and ¹H NMR (400 MHz,

DMSO) δ 10.74 (1H, s, CONH), 10.13 (1H, s, CONH), 9.04 (1H, d,

J = 1.9 Hz, 2‐H), 8.13 (1H, dd, J = 8.6, 2.0 Hz, 6‐H), 7.75 (1H, dd,

J = 8.5, 2.0 Hz, Ar–H), 7.67 (1H, d, J = 8.4 Hz, Ar–H), 7.61 (1H, t,

J = 7.0 Hz, Ar–H), 7.31 (1H, d, J = 8.3 Hz, Ar–H), 7.23 (2H, d, J = 8.3 Hz,

Ar–H), 7.20 (1H, d, J = 8.6 Hz, 5‐H), 7.16 (1H, d, J = 7.4 Hz, Ar–H), 4.10

(3H, s, OCH₃), 4.05 (3H, s, OCH₃), 2.86 (dt, J = 13.7, 6.8 Hz, CH), and

1.20 (6H, d, J = 6.9 Hz, 2× CH₃); ¹³C NMR (101 MHz, DMSO) δ 165.62

(C, C═O), 162.73 (C, C═O), 157.70 (C, Ar–C), 151.02 (C, 4‐C), 143.96

(C, Ar–C), 137.62 (C, Ar–C), 134.23 (C, Ar–C), 131.89 (C, Ar–C),

128.15 (C, Ar–C), 128.13 (C, Ar–C), 126.71 (C, Ar–C), 123.94

(C, Ar–C), 121.74 (C, Ar–C), 121.41 (C, Ar–C), 120.97 (C, Ar–C),

119.66 (C, 2‐C), 113.12 (C, 5‐C), 110.47 (C, Ar–C), 57.06 (C, OCH₃),

56.99 (C, OCH₃), 33.40 (C, CH), and 24.44 (C, 2× CH₃); HR‐ESI‐MS

(m/z) Calcd for C₂₅H₂₇N₂O₄[M+H]⁺419.1971. Found: 419.1971.

N‐(4‐Trifluoromethylphenyl)‐4‐methoxy‐3‐(4‐methoxybenzamido)‐

benzamide (6p)

This compound was obtained as light yellow granules (acetone); yield:

78.12%; mp: 229.6–230.7℃; IR (KBr, cm⁻¹): 3,432.16, 3,282.92,

1,652.96, 1,602.74, 1,528.63, 1,420.63, 1,321.99, 1,254.94, 1,174.38,

1,112.88, 1,065.91, 1,020.24, 901.90, 839.34, 759.27, and 583.02; ¹H

NMR (400 MHz, DMSO) δ 10.49 (1H, s, CONH), 9.52 (1H, s, CONH),

8.38 (1H, d, J = 1.6 Hz, 2‐H), 8.02 (2H, d, J = 8.5 Hz, Ar–H), 7.99 (2H, d,

J = 8.6 Hz, Ar–H), 7.90 (1H, d, J = 8.5 Hz, 6‐H), 7.72 (2H, d, J = 8.5 Hz,

Ar–H), 7.25 (1H, d, J = 8.6 Hz, 5‐H), 7.07 (2H, d, J = 8.4 Hz, Ar–H), 3.93

(3H, s, OCH₃), and 3.84 (3H, s, OCH₃); ¹³C NMR (101 MHz, DMSO) δ

165.75 (C, C═O), 165.11 (C, C═O), 162.52 (C, Ar–C), 155.12 (C, 4‐C),

143.50 (C, Ar–C), 129.95 (C, Ar–C), 127.36 (C, Ar–C), 126.87

(C, Ar–C), 126.67 (C, Ar–C), 126.32 (C, Ar–C), 126.29 (C, Ar–C),

124.94 (C, Ar–C), 124.05 (C, CF₃), 123.74 (C, Ar–C), 123.57

(C, Ar–C), 120.59 (C, Ar–C), 114.23 (C, Ar–C), 111.37 (C, 5‐C),

56.63 (C, OCH₃), and 55.89 (C, OCH₃); HR‐ESI‐MS (m/z) Calcd for

C₂₃H₂₀N₂O₄F₃[M+H]⁺445.1377. Found: 445.1375.

N‐(3‐Isopropylphenyl)‐4‐methoxy‐3‐(2‐methoxybenzamido)‐

benzamide (6n)

This compound was obtained as white needles (DMF); yield: 66.67%;

mp: 244.6–245.3℃; IR (KBr, cm⁻¹): 3,310.33, 2,952.69, 1,661.54,

1,595.33, 1,552.26, 1,475.43, 1,431.18, 1,342.67, 1,287.50, 1,246.56,

1,144.82, 1,085.21, 1,022.41, 893.91, 834.05, 791.40, and 748.27; ¹H

NMR (400 MHz, DMSO) δ 10.74 (1H, s, CONH), 10.13 (1H, s, CONH),

9.07 (1H, d, J = 1.7 Hz, 2‐H), 8.14 (1H, d, J = 7.7 Hz, 6‐H), 7.76 (1H, dd,

J = 8.5, 1.9 Hz, Ar–H), 7.68–7.56 (3H, m, Ar–H), 7.30 (1H, d, J = 8.3 Hz,

Ar–H), 7.26–7.21 (2H, m, 5‐H, Ar–H), 7.17 (1H, t, J = 7.5 Hz, Ar–H),

6.97 (1H, d, J = 7.5 Hz, Ar–H), 4.10 (3H, s, OCH₃), 4.05 (3H, s, OCH₃),

2.87 (1H, dt, J = 13.7, 6.9 Hz, CH), and 1.22 (6H, d, J = 6.9 Hz, 2× CH₃);

¹³C NMR (101 MHz, DMSO) δ 165.69 (C, C═O), 162.74 (C, C═O),

157.70 (C, Ar–C), 151.07 (C, 4‐C), 149.24 (C, Ar–C), 139.85 (C, Ar–C),

134.26 (C, Ar–C), 131.89 (C, Ar–C), 128.89 (C, Ar–C), 128.13

(C, Ar–C), 128.08 (C, Ar–C), 123.99 (C, Ar–C), 122.00 (C, Ar–C),

121.77 (C, Ar–C), 121.40 (C, Ar–C), 119.68 (C, 2‐C), 118.80 (C, Ar–C),

118.44 (C, Ar–C), 113.17 (C, 5‐C), 110.52 (C, Ar–C), 57.10 (C, OCH₃),

57.03 (C, OCH₃), 34.01 (C, CH), and 24.37 (C, 2× CH₃); HR‐ESI‐MS

(m/z) Calcd for C₂₅H₂₇N₂O₄[M+H]⁺419.1987. Found: 419.1971.

N‐(3‐Trifluoromethylphenyl)‐4‐methoxy‐3‐(4‐methoxybenzamido)‐

benzamide (6q)

This compound was obtained as white needles (acetone); yield:

63.85%; mp: 240.7–242.1℃; IR (KBr, cm⁻¹): 3,305.36, 1,664.49,

1,556.44, 1,438.32, 1,327.60, 1,255.19, 1,127.01, 1,021.46, 898.14,

804.47, 742.62, and 546.61; ¹H NMR (400 MHz, DMSO) δ 10.46 (1H,

s, CONH), 9.53 (1H, s, CONH), 8.38 (1H, d, J = 2.0 Hz, 2‐H), 8.25 (1H,

s, Ar–H), 8.08 (1H, d, J = 8.3 Hz, Ar–H), 7.99 (2H, d, J = 8.8 Hz, Ar–H),

7.90 (1H, dd, J = 8.6, 2.1 Hz, 6‐H), 7.59 (1H, t, J = 8.0 Hz, Ar–H), 7.44

(1H, d, J = 7.7 Hz, Ar–H), 7.25 (1H, d, J = 8.7 Hz, 5‐H), 7.07 (2H, d,

J = 8.8 Hz, Ar–H), 3.93 (3H, s, OCH₃), and 3.84 (3H, s, OCH₃); ¹³C

NMR (101 MHz, DMSO) δ 165.65 (C, C═O), 165.12 (C, C═O), 162.51

(C, Ar–C), 155.15 (C, 4‐C), 140.64 (C, Ar–C), 130.24 (C, Ar–C), 129.96

(C, Ar–C), 127.33 (C, Ar–C), 126.86 (C, Ar–C), 126.60 (C, Ar–C),

126.32 (C, Ar–C), 126.03 (C, Ar–C), 124.96 (C, Ar–C), 124.23 (C,

CF₃), 123.33 (C, Ar–C), 120.14 (C, Ar–C), 116.83 (C, Ar–C), 114.22

(C, Ar–C), 111.40 (C, 5‐C), 56.61 (C, OCH₃), and 55.89 (C, OCH₃);

HR‐ESI‐MS (m/z) Calcd for C₂₃H₂₀N₂O₄F₃[M+H]⁺445.1381. Found:

445.1375.

N‐(2‐Isopropylphenyl)‐4‐methoxy‐3‐(2‐methoxybenzamido)‐

benzamide (6o)

This compound was obtained as white needles (acetone); yield:

65.69%; mp: 223.1–223.7℃; IR (KBr, cm⁻¹): 3,469.36, 3,271.06,

2,963.71, 1,658.36, 1,594.93, 1,552.86, 1,483.92, 1,292.56, 1,248.42,

1,139.89, 1,085.19, 1,023.38, 901.79, 829.79, 752.95, and 678.58; ¹H

NMR (400 MHz, DMSO) δ 10.74 (1H, s, CONH), 9.85 (1H, s, CONH),

9.08 (1H, d, J = 1.9 Hz, 2‐H), 8.12 (1H, d, J = 7.7 Hz, 6‐H), 7.79 (1H, d,

J = 8.5 Hz, Ar–H), 7.61 (1H, t, J = 7.7 Hz, Ar–H), 7.37 (1H, d, J = 7.6 Hz,

Ar–H), 7.32–7.13 (6H, m, 5‐H, Ar–H), 4.10 (3H, s, OCH₃), 4.05 (3H, s,

OCH₃), 3.18 (dt, J = 13.6, 6.7 Hz, 1H), and 1.17 (6H, d, J = 6.8 Hz, 2×

N‐(2‐Trifluoromethylphenyl)‐4‐methoxy‐3‐(4‐methoxybenzamido)‐

benzamide (6r)

This compound was obtained as yellow granules (acetone); yield:

70.23%; mp: 202.7–203.5℃; IR (KBr, cm⁻¹): 3,437.31, 3,304.20,

LIU ET AL.

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1,664.60, 1,602.65, 1,510.15, 1,319.89, 1,255.10, 1,107.88, 1,019.42,

838.09, 760.02, and 584.64; ¹H NMR (400 MHz, DMSO) δ 10.05 (1H,

s, CONH), 9.52 (1H, s, CONH), 8.35 (1H, d, J = 1.8 Hz, 2‐H), 7.99 (2H,

d, J = 8.8 Hz, Ar–H), 7.88 (1H, dd, J = 8.6, 1.9 Hz, 6‐H), 7.80 (1H, d,

J = 7.8 Hz, Ar–H), 7.74 (1H, t, J = 7.6 Hz, Ar–H), 7.53 (2H, t, J = 8.1 Hz,

Ar–H), 7.24 (1H, d, J = 8.7 Hz, 5‐H), 7.07 (2H, d, J = 8.8 Hz, Ar–H), 3.92

(3H, s, OCH₃), and 3.84 (3H, s, OCH₃); ¹³C NMR (101 MHz, DMSO) δ

166.18 (C, C═O), 165.11 (C, C═O), 162.52 (C, Ar–C), 155.02 (C, 4‐C),

136.57 (C, Ar–C), 133.48 (C, Ar–C), 129.96 (C, Ar–C), 127.66 (C,

Ar–C), 127.41 (C, Ar–C), 126.90 (C, Ar–C), 126.65 (C, Ar–C), 126.35

(C, Ar–C), 126.02 (C, Ar–C), 125.56 (C, Ar–C), 124.96 (C, Ar–C),

122.85 (C, CF₃), 114.21 (C, Ar–C), 111.38 (C, 5‐C), 56.58 (C, OCH₃),

and 55.86 (C, OCH₃); HR‐ESI‐MS (m/z) Calcd for C₂₃H₂₀N₂O₄F₃

[M+H]⁺445.1378. Found: 445.1375.

N‐(2‐Isopropylphenyl)‐4‐methoxy‐3‐(4‐methoxybenzamido)‐

benzamide (6u)

This compound was obtained as white needles (acetone); yield:

61.74%; mp: 151.3–152.1℃; IR (KBr, cm⁻¹): 3,426.88, 3,271.58,

2,962.86, 1,651.45, 1,604.39, 1,511.51, 1,260.55, 1,180.17, 1,127.02,

1,022.25, 902.30, 839.42, 758.04, and 561.14; ¹H NMR (400 MHz,

DMSO) δ 9.84 (1H, s, CONH), 9.50 (1H, s, CONH), 8.35 (1H, d,

J = 1.5 Hz, 2‐H), 7.99 (2H, d, J = 8.7 Hz, Ar–H), 7.90 (1H, dd, J = 8.5,

1.6 Hz, 6‐H), 7.37 (1H, d, J = 7.6 Hz, Ar–H), 7.30–7.24 (1H, m, Ar–H),

7.24–7.18 (3H, m, Ar–H, 5‐H), 7.07 (2H, d, J = 8.7 Hz, Ar–H), 3.91 (3H,

s, OCH₃), 3.84 (3H, s, OCH₃), 3.17 (1H, dt, J = 13.5, 6.7 Hz, CH), 1.16

(6H, d, J = 6.8 Hz, 2× CH₃); ¹³C NMR (101 MHz, DMSO) δ 165.79 (C,

C═O), 165.07 (C, C═O), 162.49 (C, Ar–C), 154.87 (C, 4‐C), 145.37 (C,

Ar–C), 135.76 (C, Ar–C), 129.95 (C, Ar–C), 128.66 (C, Ar–C), 127.29

(C, Ar–C), 127.24 (C, Ar–C), 126.93 (C, Ar–C), 126.90 (C, Ar–C),

126.26 (C, Ar–C), 126.04 (C, Ar–C), 125.09 (C, Ar–C), 114.22 (C,

Ar–C), 111.33 (C, 5‐C), 56.59 (C, OCH₃), 55.90 (C, OCH₃), 28.01 (C,

CH), and 23.63 (C, 2× CH₃); HR‐ESI‐MS (m/z) Calcd for C₂₅H₂₇N₂O₄

[M+H]⁺419.1993. Found: 419.1971.

N‐(4‐Isopropylphenyl)‐4‐methoxy‐3‐(4‐methoxybenzamido)‐

benzamide (6s)

This compound was obtained as yellow flocculent (acetone); yield:

60.50%; mp: 225.8–226.6℃; IR (KBr, cm⁻¹): 3,431.73, 3,292.53,

2,948.50, 1,660.07, 1,598.05, 1,521.04, 1,419.09, 1,326.16, 1,251.42,

1,178.70, 1,021.63, 903.07, 831.57, 759.93, 703.30, and 582.23; ¹H

NMR (400 MHz, DMSO) δ 10.08 (1H, s, CONH), 9.50 (1H, s, CONH),

8.33 (1H, d, J = 1.7 Hz, 2‐H), 7.98 (2H, d, J = 8.6 Hz, Ar–H), 7.86 (1H,

dd, J = 8.5, 1.7 Hz, 6‐H), 7.67 (2H, d, J = 8.4 Hz, Ar–H), 7.21 (3H, dd,

J = 8.5, 3.4 Hz, 5‐H, Ar–H), 7.07 (2H, d, J = 8.6 Hz, Ar–H), 3.91 (1H, s,

OCH₃), 3.84 (3H, s, OCH₃), 2.86 (1H, dt, J = 13.6, 6.8 Hz, CH), and

1.20 (6H, d, J = 6.9 Hz, 2× CH₃); ¹³C NMR (101 MHz, DMSO) δ 165.07

(C, 2× C═O), 162.48 (C, Ar–C), 154.83 (C, 4‐C), 144.00 (C, Ar–C),

137.54 (C, Ar–C), 129.95 (C, Ar–C), 126.91 (C, Ar–C), 126.72 (C,

Ar–C), 126.10 (C, Ar–C), 124.97 (C, Ar–C), 120.99 (C, Ar–C), 114.22

(C, Ar–C), 111.27 (C, 5‐C), 56.57 (C, OCH₃), 55.90 (C, OCH₃), 33.39

(C, CH), and 24.45 (C, 2× CH₃); HR‐ESI‐MS (m/z) Calcd for

C₂₅H₂₇N₂O₄[M+H]⁺419.1975. Found: 419.1971.

|

4.2

Biological assays

|

4.2.1

In vitro antiplatelet aggregation assay

Antiplatelet aggregation activity was determined by Born’s method

using two different platelet aggregation inducing agonists, namely,

ADP and AA. Fresh vein blood was obtained from the auricle vein of

rabbits (weight: 2–3 kg, male) with 3.8% sodium citrate as an

anticoagulant (9:1 by volume). Platelet‐rich plasma (PRP) was

obtained from samples centrifuged at 800 rpm/min for 10 min at

room temperature. The remaining blood was further centrifuged at

3,000 rmp/min for 15 min at room temperature to obtain platelet‐

poor plasma (PPP), which was used as the blank. PRP was

preincubated for 5 min at 37°C with the tested compounds or

positive control at the concentration of 1.30 μM. DMSO (0.5% v/v)

was used as a negative control (according to the pre‐experiment,

DMSO seems to have no remarkable effect on the antiplatelet

aggregation activity). Aspirin and picotamide were applied as positive

control. The platelet aggregation in PPR samples was induced by

ADP and AA, respectively, followed by recording light transmission at

maximal aggregation within 5 min. The percentage inhibition of

platelet aggregation was calculated by the following formula:

N‐(3‐Isopropylphenyl)‐4‐methoxy‐3‐(4‐methoxybenzamido)‐

benzamide (6t)

This compound was obtained as brown needles (acetone); yield:

61.91%; mp: 192.1–193.2℃; IR (KBr, cm⁻¹): 3,440.06, 3,246.80,

2,957.53, 1,650.28, 1,599.29, 1,547.53, 1,429.95, 1,340.38, 1,257.74,

1,181.74, 1,139.57, 1,025.43, 895.48, 838.99, 793.16, 752.63, and

582.79; ¹H NMR (400 MHz, DMSO) δ 10.09 (1H, s, CONH), 9.51 (1H,

s, CONH), 8.35 (1H, d, J = 1.5 Hz, 2‐H), 7.99 (2H, d, J = 8.6 Hz, Ar–H),

7.88 (1H, dd, J = 8.6, 1.5 Hz, 6‐H), 7.64 (2H, s, Ar–H), 7.26 (1H, d,

J = 8.2 Hz, Ar–H), 7.22 (1H, d, J = 8.6 Hz, 5‐H), 7.07 (2H, d, J = 8.6 Hz,

Ar–H), 6.97 (1H, d, J = 7.5 Hz, Ar–H), 3.92 (3H, s, OCH₃), 3.84 (3H, s,

OCH₃), 2.87 (dt, J = 13.7, 6.9 Hz, 1H), and 1.21 (6H, d, J = 6.9 Hz, 2×

CH₃); ¹³C NMR (101 MHz, DMSO) δ 165.20 (C, C═O), 165.09 (C,

C═O), 162.49 (C, Ar–C), 154.85 (C, 4‐C), 149.25 (C, Ar–C), 139.80 (C,

Ar–C), 129.96 (C, Ar–C), 128.89 (C, Ar–C), 126.91 (C, Ar–C), 126.14

(C, Ar–C), 124.97 (C, Ar–C), 122.04 (C, Ar–C) 118.82 (C, Ar–C),

118.47 (C, Ar–C), 114.22 (C, Ar–C), 111.27 (C, 5‐C), 56.56 (C, OCH₃),

55.89 (C, OCH₃), 34.01 (C, CH), and 24.36 (C, 2× CH₃); HR‐ESI‐MS

(m/z) Calcd for C₂₅H₂₇N₂O₄[M+H]⁺419.1985. Found: 419.1971.

S − D

S

Inhibition rate (%) =

× 100%,

where S is the platelet aggregation in the presence of solvent, and D

is the platelet aggregation in the presence of test compounds.

|

4.2.2

In vitro cell toxicity assay on L929 cells

The cell toxicity of tested compounds on mouse fibroblasts (L929)

cells was determined by CCK‐8 assay in vitro. Cells were cultivated in

a humidified 5% CO₂atmosphere at 37°C on 96‐well microplates

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(1 × 10⁴cells/well). Next, the target compounds previously dissolved

in DMSO were added to the cells at specific concentrations. After

cultivation for 48 hr, the medium was removed and replaced with

fresh Rosewell Park Memorial Institute (RPMI)‐1640 medium

(100 μl). Ultimately, the CCK‐8 solution was added to the 96‐well

microplates (10 μl per well). After cultivation for 1 hr, the absorbance

was monitored and recorded on a microplate reader (Bio‐Tek

FL × 800 fluorescence microplate reader) at 450 nm. The cell viability

was calculated by the following formula:

[4] J. D. Nordeen, A. V. Patel, R. M. Darracott, G. S. Johns, P. Taussky,

R. G. Tawk, D. A. Miller, W. D. Freeman, R. A. Hanel, J. Vasc. Interv.

Neurol. 2013, 6, 26.

[5] J. Bezin, O. H. Klungel, R. Lassalle, C. Dureau‐Pournin, N. Moore, A.

Pariente, Clin. Pharmacol. Ther. 2018, 103, 1038.

[6] C. N. Floyd, A. Ferro, Prostaglandins Other Lipid Mediat. 2015, 120, 21.

[7] R. Guthrie, Adv. Ther. 2011, 28, 473.

[8] R. Hall, C. D. Mazer, Anesth. Analg. 2011, 112, 292.

[9] P. C. Sathler, M. Santana, A. L. Lourenço, C. R. Rodrigues, P.

Abreu, L. M. Cabral, H. C. Castro, J. Enzyme Inhib. Med. Chem.

2014, 29, 527.

[10] Y. Ren, K. Patel, T. Crane, J. Extra‐Corpor. Technol. 2010, 42, 103.

[11] P. Gresele, H. Deckmyn, J. Arnout, G. Nenci, J. Vermylen, Thromb.

Haemost. 1989, 61, 479.

A_sample− A_blank

Cell viability (%) =

× 100%,

A_negative− A_blank

[12] F. M. Pulcinelli, P. Pignatelli, M. Pesciotti, S. Sebastiani, S. Parisi, P. P.

Gazzaniga, Thromb. Res. 1997, 85, 207.

where A_sampleis the absorbance of the samples (containing cells,

CCK‐8 solution, and sample solution), A_blankis the absorbance of the

blank (containing RPMI medium, CCK‐8 solution, and DMSO

solution), and A_negativeis the absorbance of the negative control

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Results were expressed as the means standard error of the means.

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The level of significance was considered at p < 0.05 and p < 0.01.

ACKNOWLEDGMENTS

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The authors are grateful to the National Science Foundation of China

(Grant No. 11341014) and the Committee of Science and Technology

of Tianjin of China (Grant No. 15JCZDJC33100) for the financial

supports and the Shenyang Pharmaceutical University of China for

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The authors declare that there are no conflicts of interests.

ORCID

SUPPORTING INFORMATION

Additional supporting information may be found online in the

Supporting Information section.

Xiujie Liu

http://orcid.org/0000-0001-5482-0360

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How to cite this article: Liu X, Chen X, Qiu K, Zhang Z. Design,

synthesis, and biological evaluation of 4‐methoxy‐3‐

arylamido‐N‐(substitutedphenyl)benzamide derivatives as

potential antiplatelet agents. Arch Pharm Chem Life Sci.

2019;e1900231. https://doi.org/10.1002/ardp.201900231

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Article Doi

Design, synthesis, and biological evaluation of 4-methoxy-3-arylamido-N-(substitutedphenyl)benzamide derivatives as potential antiplatelet agents

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