Synthesis and thrombin, factor Xa and U46619 inhibitory effects of non-amidino and amidino N2-thiophenecarbonyl- and N2-tosylanthranilamides

International Journal of

Molecular Sciences

Article

Synthesis and Thrombin, Factor Xa and U46619

Inhibitory Effects of Non-Amidino and Amidino

N²-Thiophenecarbonyl- and N²-Tosylanthranilamides

Soo Hyun Lee ^1,†, Wonhwa Lee ^2,†, ThiHa Nguyen ¹, Il Soo Um ¹, Jong-Sup Bae ^2,* and

Eunsook Ma ^1,

*

1

College of Pharmacy, Catholic University of Daegu, Hayang-ro 13-13, Hayang-eup, Gyeongsan-si,

Gyeongbuk 712-702, Korea; Soohyunlee@cu.ac.kr (S.H.L.); Nguyenthiha@gmail.com (T.N.);

isu404777@cu.ac.kr (I.S.U.)

College of Pharmacy, CMRI, Research Institute of Pharmaceutical Sciences, BK21 Plus KNU Multi-Omics

Based Creative Drug Research Team, Kyungpook National University, 80 Daehak-ro, Buk-gu,

Daegu 41566, Korea; ByWonhwalee@gmail.com

2

*

Correspondence: jongsupbae@gmail.com (J.-S.B.); masook@cu.ac.kr (E.M.);

Tel.: +82-053-950-8570 (J.-S.B.); +82-053-850-3621 (E.M.)

†

These authors contributed equally to this work.

Academic Editor: Zdenek Wimmer

Received: 2 February 2017; Accepted: 25 April 2017; Published: 31 May 2017

Abstract: Thrombin (factor IIa) and factor Xa (FXa) are key enzymes at the junction of the intrinsic

and extrinsic coagulation pathways and are the most attractive pharmacological targets for the

development of novel anticoagulants. Twenty non-amidino N²-thiophencarbonyl- and N²-tosyl

anthranilamides

were synthesized to evaluate their activated partial thromboplastin time (aPTT) and prothrombin

time (PT) using human plasma at a concentration of 30 g/mL in vitro. As a result, compounds

, and 21 23 were selected to study the further antithrombotic activity. The anticoagulant

properties of , and 21 23 signiﬁcantly exhibited a concentration-dependent prolongation

1– –26

20 and six amidino N²-thiophencarbonyl- and N²-tosylanthranilamides 21

µ

5,

9

–

5,

9

–

of in vitro PT and aPTT, in vivo bleeding time, and ex vivo clotting time. These compounds

concentration-dependently inhibited the activities of thrombin and FXa and inhibited the generation

of thrombin and FXa in human endothelial cells. In addition, data showed that 5, 9, and 21–23

signiﬁcantly inhibited thrombin catalyzed ﬁbrin polymerization and mouse platelet aggregation

and inhibited platelet aggregation induced by U46619 in vitro and ex vivo. Among the derivatives

evaluated, N-(3⁰-amidinophenyl)-2-((thiophen-2⁰⁰-yl)carbonylamino)benzamide (21) was the most

active compound.

Keywords: N²-Arylcarbonyl/sulfonylanthranilamides; prothrombin time; activated partial

thromboplastin time; Thrombin; Factor Xa; U46619

1. Introduction

Thromboembolic disorders, such as cerebral thrombosis, deep vein thrombosis, unstable angina,

myocardial infarction, and thromboembolic stroke; are one of the major disorders that cause morbidities

and mortalities worldwise [

1]. Several anticoagulants, such as heparins and vitamin K antagonists,

have known to be effective in the treatment and prevention of venous thromboembolic diseases [

2].

However, there are signiﬁcant shortcomings, such as monitoring, inconvenience of drug administration,

bleeding for heparins, and such as considerable drug and food interactions for vitamin K antagonists,

therefore their clinical application was restricted [3].

Int. J. Mol. Sci. 2017, 18, 1144; doi:10.3390/ijms18061144

ww w .mdpi.com/journal/ijms

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Thrombin (factor IIa, FIIa) and factor Xa (FXa) occupy central positions in the blood coagulation

cascade and play prominent role in various thromboembolic complications. Thrombin promotes

blood clot formation by the conversion of ﬁbrinogen to insoluble ﬁbrin and activating platelets [4].

Inhibition of thrombin attenuates formation of ﬁbrin, reduces thrombin generation, and may limit

platelet aggregation. FXa is a trypsin like serine protease which plays a pivotal role in the sequence of

blood coagulation events. By directly binding to FXa active site, FXa inhibitors lead to interruption of

the intrinsic and extrinsic coagulation cascade pathways and thus to inhibition of thrombin formation

and thrombus development.

Heparin inhibits both thrombin and factor Xa indirectly via complex formation with modulation

of the activity of the serine protease inhibitor antithrombin III [5

]. Fondaparinux sodium (Arixtra^®),

synthetic heparin pentasaccharide, acts as antithrombin III (AT-III) mediated selective inhibition of FXa,

distinct from heparin. Its pharmacokinetic properties is nearly complete bioavailability subcutaneously

to allow for a simple, ﬁxed-dose, a prolonged half-life, without the need for monitoring [6,7].

Unexpectedly, fondaparinux sodium is inconvenient for long-term use and requires dose adjustment.

If the therapeutic range and coagulation monitoring are necessary, it can be monitored with FXa levels

by a chromogenic assay, particularly in hemorrhage and renal insufﬁciency, and it may also require the

control of platelet contents [8].

The ﬁrst synthetic direct thrombin inhibitor was ximelagatran, but it was not approved for

medicinal use because of its hepatotoxicity. The direct thrombin inhibitor, dabigatran etexilate mesylate

(Pradaxa^®), argatroban, and synthetic 20-amino acid peptide, bivalirudin are approved for use on the

market [9

]. Apixaban (Eliquis^®) and rivaroxaban (Xarelto^®) are oral direct FXa inhibitors. But, while

these anticoagulants carry similar side effects to warfarin, such as risk for gastrointestinal bleeding and

intracranial hemorrhage, international normalized ratio (INR) and PT monitoring are not required.

The structures of thrombin and FXa show similarities in their active sites and for this reason,

attempts have been made to develop novel synthetic compounds that exert a dual inhibition of the

two main enzymes of the coagulation cascade. A combination of thrombin and FXa inhibitory activity

in a single, synthetic, orally bioavailable, small molecular weight compound as a novel approach

to antithrombotic therapy should therefore result in potent anticoagulant with potentially superior

features over currently available therapies [10].

The first compounds to demonstrate dual inhibitor properties were BIBM 1015 and tanogitran (BIBT986)

which belong to the methylbenzimidazole series [5 ]. Hexadecasaccharide (e.g., SR123781) [11 ,12 ] is a

synthetic heparin mimetic that inhibits both FXa and thrombin via antithrombin III that was advanced

to phase I clinical trials. This dual inhibitor demonstrated superior antithrombotic properties in three

rat models of thrombosis following intravenous administration compared to selective factor Xa inhibitor,

synthetic pentasaccharide fondaparinux (Arixtra^®) [13 ]. In addition, novel anti-FXa pentasaccharides

coupled to active site thrombin inhibitors to obtain dual inhibitory effects have also been reported [14 ,15 ].

However, problems with thrombin-rebound remain with these mimetics, which appear to result from the

inability to inhibit clot-bound thrombin.

EP 217609 is a synthetic parenteral anticoagulant with a dual-action that combines a α-NAPAP

analog (direct thrombin inhibitor) and a fondaparinux analog (indirect factor Xa inhibitor) in a single

molecule without the complications of thrombin rebound [16]. In recent years several groups [17–20]

have reported the development of dual FXa and thrombin inhibitors and proposed a stronger

anticoagulant activity for such compounds compared to monoselective inhibitors.

Furthermore, some patients with cardiovascular disease have indications for anticoagulant

and dual antiplatelet therapy. It is estimated that between 5 and 10 percent of patients scheduled

for coronary artery stenting, and who thus require dual antiplatelet therapy, are receiving oral

anticoagulant, most often for atrial ﬁbrillation [21]. The concomitant use of dual antiplatelet therapy

and oral anticoagulant is referred to as triple oral antithrombotic therapy (triple therapy).

We previously reported the synthesis and anticoagulant and antiplatelet activities of

N³-aroylbenzamide derivatives. Two amidine compounds, N-(3⁰-amidino-, and N-(4⁰-amidinophenyl)-

Int. J. Mol. Sci. 2017, 18, 1144

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3-(thiophen-2⁰⁰-ylcarbonylamino)benzamide exhibited weak thrombin, FXa, and U46619 inhibitory

activities [22 ]. Anthranilamide derivatives have been studied as FXa inhibitors [23 26 ], therefore

–

anthranilic acid was considered as central ring of diamide to improve the activity. We now

report the synthesis and evaluation of the thrombin, FXa, and U46619 inhibitory activities of

non-amidino N²-thiophenecarbonyl- and N²-tosylanthranilamides and amidino N²-thiophenecarbonyl-

and N²-tosylanthranilamides.

2. Results and Discussion

2.1. Chemistry

All synthetic methods used in this study are shown in Schemes 1 –4 . Schemes 1 and 2 showed the

synthesis of non-amidine derivatives and Schemes 3 and 4 showed the synthesis of amidine derivatives.

Scheme 1 illustrated the synthesis of N²-(thiophenecarbonyl)anthranilamide derivatives

1–10, which

were prepared according to synthetic procedures for N³-(thiophenecarbonyl)benzamide derivatives [22 ].

The non-amidine type inhibitor, rivaroxaban, apixaban, and edoxaban, possess a small lipophilic

substituent, such as a methoxy or chloro or morpholine group. The 2-nitrobenzoyl chloride was coupled

with 4-substituted anilines (4-chloro, 4-bromo-, 4-fluoro-, 4-methoxy-, and 4-morpholinoaniline) as small

lipophilic compounds to give 1a–5a and these nitro amides were reduced with Fe and NH₄Cl to provide the

corresponding amino amides 1b

–

5b. Intermediates 1b–5b were then coupled with thiophene-2-carbonyl

chloride and 5-chlorothiophene-2-carbonyl chloride to furnish N²-(thiophene-2-carbonyl)anthranilamides

1–5 and N²-(5-chlorothiophene-2-carbonyl)anthranilamides 6–10, respectively.

The introduction of sulfonamide instead of one carboxamide in the linker of diamide may give

rise to differences in the possible hydrogen-binding interaction between the ligand and the residues

in the thrombin/FXa active site. The N²-arylsulfonamide series 11

–

20 were synthesized to compare

10. N²-tosylanthranilamides were synthesized as

beginning compounds for development of N²-arylsulfonamide derivatives.

The preparative route to N²-tosylanthranilamide derivatives 11

20 are shown in Scheme 2 .

with the activity of N²-arylcarboxamide series

1–

–

Anthranilic acid was reacted with p-tosyl chloride to give N-tosylbenzoic acid, followed by chlorination

with oxalyl chloride to give N-tosylbenzoyl chloride. This acid chloride was coupled with

3-cyano-, 4-cyano-, 4-cyanomethyl-, 4-chloro-, 4-bromo-, 4-methoxy-, 4-carboxy-, 4-(2-(2-thienyl)ethyl)-,

4-morpholino-, and 4-(2-oxomorpholino)aniline to give N²-tosylanthranilamide derivatives 11a

–

13a and

20, respectively. To consider the pharmacokinetic problems we replaced the amidine group with less

basic residues, aminoalkyl groups. The cyano group containing compounds 11a 13a were catalytically

hydrogenated in the presence of 10% Pd/C and c-HCl at 45 ^◦C to give aminoalkyl amides 11–13.

14–

–

O

Cl

Et₃N

OH (COCl)₂

Benzene

R

N

+

H₂N

R

H

Benzene

RT, 3 h

NO₂

1a-5a

Reflux, 1h

4a :

5a :

1a : R = 4-Cl

2a : R = 4-Br

3a : R = 4-F

R = 4-OMe

R = 4-morpholine

O

H

N

R

O

Cl

S

1-5

N

H

O

Et₃N, Benzene

RT, 2 h

1, 6:

R = 4-Cl

2, 7: R = 4-Br

3, 8:

Fe/NH₄Cl

MeOH

R

N

H

R = 4-F

4, 9: R = 4-OMe

5, 10:

Reflux, 7h

NH₂

Et₃N, Benzene

RT, 2 h

R = 4-morpholine

1b 5b

-

O

H

O

Cl

1b :

2b :

3b :

4b : R = 4-OMe

5b :

R = 4-Cl

R = 4-Br

R

N

S

Cl

R = 4-morpholine

S

=

R

4-F

Cl

N

H

6 10

-

O

Scheme 1. Synthesis of N²-(thiophenecarbonyl)anthranilamides derivatives 1–10.

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Scheme 2. Synthetic procedures of N²-tosylanthranilamide derivatives 11–20.

Scheme 3. Synthetic procedures of amidino N²-(thiophenecarbonyl)anthranilamide derivatives (21

–23).

Scheme 4. Synthetic procedures of amidino N²-tosylanthranilamide derivatives (24–26).

Int. J. Mol. Sci. 2017, 18, 1144

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Scheme 3 illustrated the synthetic procedures of amidino N²-thiophenecarbonyl anthranilamide

derivatives 21 23. The amidine group forms a bidentate salt bridge interaction with carboxylic acid

–

of Asp189 in the S1 site of FXa. The high polar amidine-derived inhibitors demonstrated potent

in vivo anticoagulant and antithrombotic activity. The ﬁrst amide bond was obtained from acylation

of 2-nitrobenzoyl chloride with 3-cyano-, 4-cyano-, and 4-cyanomethyl-aniline to afford 21a–23a.

2-Aminobenzamides 21b

–

23b were synthesized from reduction of 2-nitrobenzamides 21a

–

23a

with Fe and NH₄Cl. The treatment of 21b

–

23b with thiophene-2-carbonyl chloride prepared

the N²-(thiophene-2-carbonyl)-N-(3-cyano- or -4-cyanophenyl)benzamides 21c and 22c, and the

N²-(thiophene-2-carbonyl)-N-(4-cyanobenzyl)benzamide 23c, which were treated with hydroxylamine

HCl and triethylamine to give amidoximes 21d

reduced to amidinium chloride 21 23 via the catalytic hydrogenation of the O-acetyl amidoximes

21e

23e over 10% Pd/C in the presence of c-HCl at 60 psi, 45 ^◦C for 2 h. The four proton peaks of

–23d. The N–O bond of amidoximes 21d–23d was

–

amidine HCl salt in 21 were determined as two broad singlets at 9.08 and 9.36 ppm, and the four proton

peaks of amidine HCl salt in 22 and 23 were observed as one broad singlets at 9.36 and 8.92 ppm in

the ¹H-NMR spectra, respectively. In the HMBC spectrum, two amide NH proton peaks of 22 were

distinguished by observing the correlation of CO (167.5 ppm) and NH (10.77 ppm) of the -NHCO

group and the correlation of CO (164.9 ppm) and NH (11.53 ppm) of the -CONH group, respectively.

Scheme 4 illustrated the synthetic procedures of amidino N²-tosylanthranilamide derivatives

24

–

26. Compounds 11a

–

13a were reﬂuxed with hydroxylamine hydrochloride and triethylamine in

26b to form amidinium chloride derivatives 24 26

26c in 10% Pd/C and c-HCl at 60 psi,

ethanol to provide the amidoxime derivatives 24b

–

via the catalytic hydrogenation of the O-acetyl amidoximes 24c

–

45 ^◦C for 12 h.

2.2. Biology

2.2.1. Effects of Non-Amidino N²-Aroylanthranilamides on In Vitro Activated Partial Thromboplastin

Time (aPTT), Prothrombin Time (PT), In Vivo Tail Bleeding Time, and Ex Vivo Clotting Time

Many FXa and thrombin inhibitors containing an amidine group displayed insufﬁcient absorption

when administered orally, the reason is caused by strongly basic amidine group [27]. To improve

oral bioavailability, synthetic projects seem to be changed to synthesis of non-amidines having weak

basic groups. Rivaroxaban and apixaban are non-amidine direct FXa inhibitors, and argatroban and

dabigatran eterxilate are non-amidine direct thrombin inhibitors.

The anticoagulant effects of twenty non-amidino N²-thiophenecarbonyl anthranilamides

N²-tosylanthranilamides 11

20 were screened in aPTT and PT assays using human plasma at a concentration

of 30 g/mL in vitro and demonstrated in Table 1 . As shown in Table 1 , N-(4-morpholinophenyl)-2-

(thiophen-2-ylcarbonyl)benzamide ( ), N-(4-fluorophenyl)- 2-(5-chloro thiophen-2-ylcarbonyl)benzamide( ),

and N-(4-methoxyphenyl)-2- (5-chlorothiophen-2-ylcarbonyl)benzamide ( ) displayed very high aPTT values

(60.6, 59.3, and 65.1 s), respectively. Any N²-tosylanthranilamides 11

20 did not exhibit the prolongation

of PT and aPTT. This result demostrate that both carboxamide linker and thiophene ring are necessary

for anticoagulant activity. For the further antithrombotic experiments, thiophene compound and

-chlorothiophene compound were investigated on anticoagulant activities (in vitro and ex vivo) and

1–10 and

–

µ

5

8

9

–

5

9

on tail bleeding time (in vivo) on mouse. These two compounds were selected for further evaluations as

were the ones which showed an aPPT time higher than 60 s.

According to the Table 2, aPTT in the saline-treated group was 23.6

error of mean (SEM), n = 5) and aPTT of compounds , and heparin was respectively prolonged as

33.2 0.5, 37.6 0.3 and 53.3 0.5 s at dose 20 M. Although compounds and showed weaker

activities than those of heparin, aPTT was signiﬁcantly prolonged by and at concentrations

of over 20 M as compared to the saline-treated group. Unlike the methoxy compound , the

morpholine compound showed PT prolongation at concentrations 20 M and above as compared to

the saline-treated group.

±

0.6 s (mean

±

standard

5, 9

±

µ

5

9

5

9

µ

9

5

µ

Int. J. Mol. Sci. 2017, 18, 1144

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Table 1. Anticoagulant activities of non-amidine compounds 1–20 at 30 µg/mL.

Compound No.

aPPT (s)

PT (s)

Compound No.

aPTT (s)

PT (s)

DMSO ¹

39.0

44.5

37.4

41.6

41.7

60.6

39.9

44.9

59.3

65.1

53.3

11.1

14.1

13.2

11.8

11.6

12.8

12.2

12.7

12.5

14.7

11

12

13

14

15

16

17

18

40.2

41.2

42.3

38.6

35.8

36.2

41.5

38.4

35.8

11.8

11.7

10.9

13.6

11.3

11.0

11.7

11.0

10.7

10.8

1

2

3

4

5

6

7

8

19

20

9

10

33.6

Heparin ²

85 (30 µM)

34 (30 µM)

1

2

Dimethyl sulfoxide (DMSO) was used as the negative control. The molecular weight of heparin used as positive

control is 3500. aPTT: activated partial thromboplastin time, PT: prothrombin time.

We can remember that a prolongation of aPTT indicates the inhibition of intrinsic pathway, and a

prolongation of PT indicates the inhibition of extrinsic pathway including common pathway or not.

Therefore, the prolongation of both aPTT and PT in compound

and intrinsic and/or common pathway. The prolongation of aPTT in

pathway and/or common pathway.

5

means inhibition of the extrinsic

suggests inhibition of intrinsic

9

The most active compounds

bleeding times in vivo model. The circulating blood volume for mice is averagely 72 mL/kg [28].

Because the weight of used mouse is averagely 27 g, the molecular weights of and are 407.49

and 386.85 and the blood volume is averagely 2 mL, the amount of target compounds (24.4, 32.6,

40.8 g/mouse for and 23.2, 30.9, 38.7 g/mouse for ) injected provided a maximum concentration

of 30, 40, or 50 µM in the peripheral blood.

As shown in Table 3, compounds and

5 and 9 in aPTT in vitro evaluations will be evaluated in the tail

5

9

µ

5

µ

9

5

9 signiﬁcantly prolonged the tail bleeding times in at

concentrations 24.4 and 23.2 µg/mouse and above as compared to the control, respectively.

Table 2. In vitro anticoagulant activities of 5 and 9.

Compound No.

Dose

aPTT (s)

PT (s)

PT (INR)

Control

saline

23.6 ± 0.6

12.4 ± 0.4

1.00

5 µM

10 µM

20 µM

30 µM

40 µM

50 µM

23.7 ± 0.2

12.2 ± 0.4

12.6 ± 0.6

14.0 ± 0.3 *

15.2 ± 0.5 *

18.5 ± 0.4 *

19.1 ± 0.6

0.96

1.04

1.34 *

1.63 *

2.61 *

2.78 *

24.2 ± 0.8

33.2 ± 0.5 *

40.4 ± 0.6 *

58.5 ± 0.5 *

60.5 ± 0.7 *

5

5 µM

10 µM

20 µM

30 µM

40 µM

50 µM

23.2 ± 0.4

12.0 ± 0.2

12.2 ± 0.5

12.0 ± 0.2

11.6 ± 0.6

12.7 ± 0.5

13.0 ± 0.8

0.92

0.96

0.92

0.85

1.06

1.12

23.4 ± 0.5

37.6 ± 0.3 *

44.1 ± 0.4 *

65.6 ± 0.6 *

63.2 ± 0.8 *

9

20 µM

30 µM

53.3 ± 0.5 *

21.6 ± 0.4 *

3.79 *

6.59 *

Heparin ¹

62.5 ± 0.8 *

27.2 ± 0.6 *

1

The molecular weight of heparin used as positive control in our experiments is 3500. Each value represents the

means ± standard error of mean (SEM) (n = 5). * p < 0.05.

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Table 3. In vivo Bleeding time of 5 and 9.

Compound No.

Dose

Tail Bleeding Time (s)

Control

saline

30.8 ± 0.8

24.4 µg/mouse

32.6 µg/mouse

40.8 µg/mouse

37.4 ± 1.0 *

53.7 ± 1.2 *

61.8 ± 1.2 *

5

23.2 µg/mouse

30.9 µg/mouse

38.7 µg/mouse

140.0 µg/mouse

210.0 µg/mouse

48.2 ± 1.6 *

62.2 ± 1.4 *

65.4 ± 1.6 *

51.2 ± 1.2 *

68.4 ± 1.2 *

9

Heparin ¹

1

The molecular weight of heparin used as positive control in our experiments is 3500. Each value represents the

means ± SEM (n = 5). * p < 0.05.

The aPTT values were signiﬁcantly prolonged by both

g/mouse and above ex vivo clotting times, while the prolongation in PT was found in compound

(Table 4).

5 and 9 at concentration 24.4 and 23.2

µ

5.

Table 4. Ex vivo clotting time of 5 and 9.

Compound No.

Dose

aPTT (s)

PT (s)

PT (INR)

Control

saline

32.2 ± 0.8

12.8 ± 0.4

1.00

24.4 µg /mouse

32.6 µg/mouse

40.8 µg/mouse

38.4 ± 0.8 *

51.2 ± 1.4 *

60.8 ± 1.2 *

14.8 ± 0.4 *

17.4 ± 0.8 *

17.2 ± 0.7 *

1.42 *

2.09 *

2.03 *

5

23.2 µg/mouse

30.9 µg/mouse

38.7 µg/mouse

42.2 ± 0.6 *

57.4 ± 1.2 *

61.2 ± 1.0 *

12.6 ± 0.8

13.0 ± 0.6

12.4 ± 0.8

0.96

1.04

0.93

9

140.0 µg/mouse

210.0 µg/mouse

63.4 ± 0.9 *

25.4 ± 0.4 *

5.18 *

8.62 *

Heparin ¹

75.4 ± 1.2 *

31.4 ± 0.6 *

1

The molecular weight of heparin used as positive control in our experiments is 3500. Each value represents the

means ± SEM (n = 5). * p < 0.05.

In summary, aPTT (in vitro and ex vivo) of

9

was longer than those of

5

suggesting that methoxy

group of

9 is more effective for anticoagulant activity than morpholine group of 5, while 5-chloro

group of 5-chlorothiophene was seemed not to inﬂuence on the anticoagulant activity.

To obtain more active compounds, six amidino N²-aroylantranilamide derivatives (21

–26)

derivatives were synthesized and also screened in aPTT and PT assays using human plasma at

a concentration of 30

g/mL in vitro and were demonstrated in Table 5. In case of N²-thiophene

compounds, both aPTT and PT of 3-amidino- and 4-amidinomethyl- compounds 21 and 23 were longer

than 4-amidino compound 22. In reverse, among N²-tosyl derivatives 24

26, 4-amidino derivative 25

µ

–

showed longer aPTT than 3-amidino- and 4-amidinomethyl- compounds 24 and 26.

Table 5. Anticoagulant activities of amidine compounds 21–26 at 30 µg/mL.

Compound No.

aPTT (s)

PT (s)

Compound No.

aPTT (s)

A PT (s)

DMSO ¹

39.0

74.3

50.7

65.8

11.1

22.0

19.9

20.9

2

24

25

26

41.7

63.1

40.5

11.5

12.8

11.7

21

22

23

Heparin ²

34 (30 µM)

85 (30 µM)

1

DMSO was used as the negative control. The molecular weight of heparin used as positive control in our

experiments is 3500. aPTT: activated partial thromboplastin time, PT: prothrombin time.

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From these aPTT and PT results, 21

–

23 were further investigated on anticoagulant activities

(in vitro and ex vivo) and on tail bleeding time (in vivo) on mouse. These three compounds were

selected for further evaluations as were the ones which showed both aPPT and PT time higher than

50 s and 15 s, respectively.

As shown in Table 6, aPTT in the saline-treated group was 23.6

aPTT showed 38.5 0.4 s, 30.2 0.3 s, 38.5 0.7 s, and 53.3 0.5 s at dose 20

21 23 and heparin, respectively. aPTT of compounds 21 and 23 was signiﬁcantly prolonged at

concentrations of 10 M and above, and 22 at concentrations of 20 M and above as compared

to the saline-treated group. In addition, 21 23 signiﬁcantly showed PT prolongation (16.7 0.5,

13.9 0.3, 14.2 0.5 s) and INR (2.04, 1.32, and 1.38) at concentrations of 20 M and above as

0.4 s). These results in this study showing prolongation

±

0.6 s (mean

±

SEM, n = 5) and

±

µM in compounds

–

µ

–

±

µ

compared to the saline-treated group (12.4

±

of aPTT and PT of N²-thiophenecarbonyl anthranilamides 21

–23 suggest inhibition of both extrinsic

and intrinsic, and/or common pathway.

Table 6. In vitro anticoagulant activity of amidino N²-thiophene compounds 21–23.

Compound No.

Dose

aPTT (s)

PT (s)

PT (INR)

Control

saline

23.6 ± 0.6

12.4 ± 0.4

1.00

5 µM

10 µM

20 µM

30 µM

40 µM

50 µM

24.1 ±0.6

30.8 ± 0.5 *

38.5 ± 0.4 *

52.7 ± 0.7 *

71.4 ± 0.6 *

75.2 ± 0.5 *

12.8 ± 0.7

14.5 ± 0.5 *

16.7 ± 0.5 *

19.3 ± 0.6 *

22.7 ± 0.7 *

23.5 ± 0.5 *

1.08

1.46 *

2.04 *

2.89 *

4.27 *

4.64

21

22

5 µM

10 µM

20 µM

30 µM

40 µM

50 µM

22.9 ± 0.5

12.2 ± 0.4

0.96

1.06

1.32 *

1.87 *

3.46 *

3.70 *

23.7 ± 0.6

12.7 ± 0.4

30.2 ± 0.3 *

32.8 ± 0.5 *

48.6 ± 0.4 *

51.4 ± 0.7 *

13.9 ± 0.3 *

16.1 ± 0.7 *

20.8 ± 0.5 *

21.4 ± 0.6 *

5 µM

10 µM

20 µM

30 µM

40 µM

50 µM

24.2 ± 0.2

30.2 ± 0.5 *

38.5 ± 0.7 *

43.2 ± 0.4 *

63.8 ± 0.6 *

64.1 ± 0.4 *

12.1 ± 0.6

0.94

1.02

1.38 *

2.25 *

3.19 *

3.23 *

12.5 ± 0.4

14.2 ± 0.5 *

17.4 ± 0.4 *

20.1 ± 0.6 *

20.2 ± 0.5 *

23

20 µM

30 µM

53.3 ± 0.5 *

21.6 ± 0.4 *

3.79 *

6.59 *

Heparin ¹

62.5 ± 0.8 *

27.2 ± 0.6 *

1

The molecular weight of heparin used as positive control in our experiments is 3500. Each value represents the

means ± SEM (n = 5). * p < 0.05.

The most active compounds 21–23 in aPTT in vitro evaluations will be evaluated in the tail

bleeding times in vivo model. As shown in Table 7, tail bleeding times of compounds 21 and 22 were

signiﬁcantly prolonged in at concentrations of 24.1 g/mouse and above, and by compound 23 in at

µ

concentrations of 24.9 µg/mouse and above as compared to the control, respectively.

As shown Table 8, both aPTT and PT were dose-dependently prolonged by both 21 and 22 at

concentration of 24.1

µg/mouse and above, and by 23 at concentrations of 24.9 µg/mouse and above

ex vivo clotting times.

Int. J. Mol. Sci. 2017, 18, 1144

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Table 7. In vivo bleeding time of amidino N²-thiophene compounds 21–23.

Compound No.

Dose

Tail Bleeding Time (s)

Control

saline

30.8 ± 0.8

24.1 µg/mouse

32.1 µg/mouse

40.1 µg/mouse

49.2 ± 1.2 *

68.4 ± 1.0 *

70.4 ± 1.6 *

21

24.1 µg/mouse

32.1 µg/mouse

40.1 µg/mouse

33.1 ± 1.0 *

46.2 ± 0.6 *

48.3 ± 1.2 *

22

24.9 µg/mouse

33.2 µg/mouse

41.5 µg/mouse

42.3 ± 0.6 *

56.4 ± 1.6 *

58.2 ± 1.4 *

23

140.0 µg/mouse

210.0 µg/mouse

51.2 ± 0.8 *

Heparin ¹

68.4 ± 1.2 *

1

The molecular weight of heparin used as positive control in our experiments is 3500. Each value represents the

means ± SEM (n = 5). * p < 0.05.

Table 8. Ex vivo clotting time of amidino N²-thiophene compounds 21–23.

Compound No.

Dose

aPTT (s)

PT (s)

PT (INR)

Control

Saline

32.2 ± 0.8

12.8 ± 0.4

1.00

24.1 µg/mouse

32.1 µg/mouse

40.1 µg/mouse

48.8 ± 1.0 *

65.8 ± 0.8 *

69.4 ± 1.0 *

37.2 ± 1.2 *

47.5 ± 0.6 *

49.6 ± 0.8 *

19.4 ± 0.8 *

21.2 ± 0.6 *

23.7 ± 1.2 *

15.7 ± 0.6 *

19.8 ± 0.8 *

20.1 ± 0.8 *

2.71 *

3.36 *

4.39 *

21

22

24.1 µg/mouse

32.1 µg/mouse

40.1 µg/mouse

1.63 *

2.85 *

2.95 *

24.9 µg/mouse

33.2 µg/mouse

41.5 µg/mouse

39.8 ± 0.8 *

54.2 ± 1.2 *

56.4 ± 1.8 *

16.1 ± 0.5 *

18.2 ± 0.7 *

19.4 ± 0.6

1.73 *

2.33 *

2.71 *

23

140.0 µg/mouse

210.0 µg/mouse

63.4 ± 0.9 *

25.4 ± 0.4 *

5.18 *

8.62 *

Heparin¹⁾

75.4 ± 1.2 *

31.4 ± 0.6 *

1

The molecular weight of heparin used as positive control in our experiments is 3500. Each value represents the

means ± SEM (n = 5). * p < 0.05.

2.2.2. Thrombin and Factor Xa (FXa) Activity

To determine the fundamental mechanism of

5

,

9

and 21

–

23, the inhibitory activities of

5

,

9

and 21

–

23

on the thrombin and FXa were investigated. According to the Figure 1 A, compounds

showed in a dose-dependent inhibition of the activity of thrombin. In addition, treatment with

21 23 displayed in a dose-dependent inhibition of amidolytic activity of FXa, indicating direct inhibition

5, 9, and 21

–

5, 9, and

–

of FXa activity. Agartroban and rivaroxaban were used as a positive control, respectively (Figure 1 B).

Figure 1. Cont.

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Figure 1. Effects of

Xa. ( ) Inhibitory activity of thrombin (Th) by

assay. ( ) Inhibitory activity of FXa by or or 21

as. Argatroban ( ) or rivaroxaban ( ) were used as positive control. (

endothelial cells (HUVEC) monolayers were pre-incubated with FVa (100 pM), FXa (1 nM), and the

indicated concentrations of or or 21 23 for 10 min. Prothrombin was added to a ﬁnal concentration

of 1 M and prothrombin activation was determined 10 min later. ( ) HUVECs were pre-incubated

with the indicated concentrations of or or 21 23 for 10 min. After TNF- stimulated HUVECs were

5,

9

, and 21

–

23 on inhibitory activity and generation of thrombin and factor

or or 21 23 was evaluated using a chromogenic

23 was investigated using a chromogenic assay,

) Human umbilical vein

A

5

9

–

B

5

9

–

A

,

C

B

,

D

C

5

9

–

µ

D

5

9

–

α

incubated with FVIIa (10 nM) and FX (175 nM), FXa generation was determined. Data represent the

mean ± standard error of mean (SEM) of three independent experiments. * p < 0.05.

2.2.3. Thrombin and Factor Xa (FXa) Generation

According to ﬁndings reported by Sugo et al. [29], the endothelial cells are known to support

the prothrombin activation by FXa. In this study, human umbilical vein endothelial cells (HUVECs)

were pre-incubated with FVa and FXa in the presence of CaCl₂and the addition of prothrombin

resulted in generation of thrombin (Figure 1C). In a previous study, Rao et al. [30] reported that

the endothelium provides the functional equivalent of pro-coagulant phospholipids and supports

activation of FXa, and, in TNF-

factor (TF) expression-dependent manner. Thus, we determined the effects of

FX by FVIIa. Pre-incubation with , and 21 23 resulted in dose-dependent inhibition of FX activation

α

stimulated HUVECs, activated of FX by FVIIa occurred in a tissue

5, 9, and on activation of

5,

9

–

Int. J. Mol. Sci. 2017, 18, 1144

11 of 27

by FVIIa (Figure 1D). Therefore, these results indicated that

thrombin and FXa.

5,

9, and 21

–23 can inhibit generation of

2.2.4. Effects of

The effects of

were monitored as changes in absorbance at 360 nm. These results display that incubation of

or or 21 23 with human plasma resulted in a signiﬁcant decrease in the maximal rate of ﬁbrin

5,

9, and 21–23 on Thrombin-Catalyzed Fibrin Polymerization and Platelet Aggregation

5

or

9

or 21 23 on thrombin-catalyzed ﬁbrin polymerization in human plasma

–

5

9

–

polymerization (Figure 2 A). To delete the effect of sample pH, all samples were diluted with 50 mM

tris-buffer saline (TBS) (pH 7.4). The effect of dimethyl sulfoxide (DMSO) on human plasma was

also evaluated, however, coagulation properties were unaffected. In order to exclude the possibility

that the decrease of ﬁbrin polymerization could be due to a direct effect on thrombin leading to

a decrease in ﬁbrin generation rather than polymerization of ﬁbrin formed, reptilase-catalyzed

polymerization assay was performed. Results showed that

decrease in reptilase-catalyzed polymerization [31]. To determine the antiplatelet activities of

compounds or or 21 23, thrombin-catalyzed platelet aggregation assay was performed and the

results were shown in Figure 2B. Compounds or or 21 23 signiﬁcantly and dose-dependently

5, 9, and 21–23 induced a signiﬁcant

5

9

–

5

9

–

inhibited mouse platelet aggregation induced by thrombin (ﬁnal concentration: 3 U/mL).

2.2.5. Effects of 5, 9, and 21–23 on U46619 Catalyzed Platelet Aggregation

To determine the antiplatelet activities of

assay was carried out. As shown in Figure 2C, compounds

dose-dependently inhibited human platelet aggregation induced by U46619 (ﬁnal concentration:

M). These in vitro results were conﬁrmed in an ex vivo platelet aggregation assay (intravenous

injection, Figure 2D). As a result, or or 21 23 signiﬁcantly and dose-dependently inhibited platelet

aggregation induced by U46619 (ﬁnal concentration: 2 M). Until now, most of compounds containing

5

or

9

or 21

–23, a U46619 catalyzed platelet aggregation

5

or or 21 23 signiﬁcantly and

9

–

2

µ

5

9

–

µ

amidine group have been known as FXa inhibitor, but non-amidines

exhibited the potential as platelet aggregation inhibitor.

5 and 9, and amidines 21–23

Figure 2. Cont.

Int. J. Mol. Sci. 2017, 18, 1144

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Figure 2. Effects of

ﬁbrin polymerization at the indicated concentrations of

assay. The results are Vmax values expressed as percentages versus controls; (

21 23 on mouse platelet aggregation induced by 3 U/mL thrombin; ( ) The effect of each compound

on human platelet aggregation induced by 2 mM U46619; ( ) The indicated concentration of each

compound in dimethyl sulfoxide (DMSO) was injected intravenously. The effects of each compound

on mouse platelet aggregation induced by 2 M U46619 were monitored ex vivo. D: 0.2% DMSO. Data

5,

9

, and 21

–

23 on ﬁbrin polymerization in human plasma. (

or or 21 23 was monitored using a catalytic

) The effect of or or

A) Thrombin-catalyzed

5

9

–

B

5

9

–

C

D

µ

represent the mean ± SEM of three independent experiments performed. * p < 0.05.

2.2.6. Cellular Viability

To identify the cellular viability of

diphenyltetrazolium bromide (MTT) assay was performed in HUVECs treated with

for 24 h. Compounds , and 21 23 did not influence cell viability at concentration up to 100 µM

5,

9,

and 21–23,

3-(4,5-dimethylthiazol-2-yl)-2,5-

5,

9

, and 21 23

–

5,

9

–

(Figure 3 ). It is safe to treat with a dose higher than the effect dose that does not show cytotoxicity even at

a concentration three times higher than the effect concentration.

Figure 3. Effects of

5, 9, and 21–23 on cellular viability were determined by MTT assay. The data represent

the mean SEM of three independent experiments performed in triplicate. D: 0.2% DMSO is the control.

±

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3. Materials and Methods

3.1. Chemistry

3.1.1. Reagents and Instruments

The reagents were commercially purchased from Sigma-Aldrich (St. Louis, MO, USA) or TCI

(Tokyo, Japan). If necessary, solvents were puriﬁed and/or dried prior to use. All anhydrous

reactions were carried out under a dry atmosphere of nitrogen. Melting points (m.p.) were measured

on Thomas-Hoover melting point apparatus (Thomas Scientiﬁc, Swedesboro, NJ, USA) and not

corrected. ¹H, ¹³C-NMR and HMBC spectra were measured on a Varian 400 MHz spectrometer (Agilent

Technologies, Santa Clara, CA, USA) in DMSO-d₆, CDCl₃, or (CD₃)₂CO. Chemical shifts (δ) are in ppm

relative to tetramethylsilane, and coupling constants (J) are in Hz. DIP-MS (EI) was measured on an

Agilent 7890A-5975C GC/MSD (Agilent Technologies). GC/MS (EI) was determined on a SHIMADZU

QP 2010 model (Shimadzu, Kyoto, Japan) and FAB-MS was determined on a JEOL JMS-700 Mstation

(JEOL, Tokyo, Japan). Fraction collection was performed on an EYELA fraction collector DC-1500

(Tokyo Rikakikai, Tokyo, Japan). An analytical TLC was performed on pre-coated silica gel 60 F₂₅₄

plates (Merck, Kenilworth, NJ, USA). Solvent systems for TLC and column chromatography were

ethyl acetate/n-hexane mixtures and 10% methanol in dichloromethane. Column chromatography

was carried out on Merck silica gel 9385 (Merck).

3.1.2. General Synthetic Procedures for 1a–5a

The oxalyl chloride (38.92 mmol) and triethylamine (32.93 mmol) were added to a solution

of 2-nitrobenzoic acid (29.94 mmol) in anhydrous dichloromethane (50 mL) at room temperature.

The reaction mixture was refluxed for 1 h and the unreacted oxalyl chloride and solvent were removed

under reduced pressure and 2-nitrobenzoyl chloride was used to next reaction without purification. To a

solution of 3-aminophenylcyanide or 4-aminophenylcyanide or 4-aminobenzyl cyanide (7.19 mmol) in

anhydrous dichloromethane (30 mL) was added 2-nitrobenzoyl chloride (8.99 mmol) and triethylamine

(7.19 mmol). The reaction mixture was stirred at room temperature for 3 h and added with water,

extracted with dichloromethane (3

The filtrate was concentrated under reduced pressure to afford the oily residue, which was separated by

×

30 mL), dried with anhydrous magnesium sulfate and filtrated.

column chromatography to afford pure white or pale yellow compounds 1a–5a.

N-(4₀⁰-Chlorophenyl)-2-nitrobenzamide (1a). Yield: 85%; m.p.: 184–186 ^◦C (186–187 ^◦C [3_◦2]).

◦

N-(4 -Bromophenyl)-2-nitrobenzamide (2a) [33

,

34]. Yield: 86%; m.p.: 199–201 C (202–205 C [35]).

N-(4⁰-Fluorophenyl)-2-nitrobenzamide (3a). Yield: 78%; m.p.: 165–169 ^◦C (167–171 ^◦C [36]).

N-(4⁰-Methoxyphenyl)-2-nitrobenzamide (4a) [37]. Yield: 82%; m.p.: 153–154 ^◦C.

N-(4⁰-Morpholinophenyl)-2-nitrobenzamide (5a). Yield: 76%; m.p.: 220–221 ^◦C; H NMR

1

(400 MHz, DMSO-d₆) δ: 3.03 (4H, t, J = 4.8 Hz, H₃₀₀+ H₅₀₀), 3.71 (4H, t, J = 4.8 Hz, H₂₀₀+ H₆₀₀),

6.91 (2H, d, J = 9.0 Hz, H₃₀+ H₅₀), 7.49 (2H, d, J = 9.0 Hz, H₂₀+ H₆₀), 7.68–7.71 (2H, m, H₄+ H₆), 7.82

(1H, dt, J = 8.0, 1.1 Hz, H₅), 8.09 (1H, d, J = 7.6 Hz, H₃), 10.42 (1H, s, CONH); ¹³C NMR (100 MHz,

DMSO-d₆) δ: 48.9 (C₃₀₀, C₅₀₀), 66.0 (C₂₀₀, C₆₀₀), 115.4 (C₂₀, C₆₀), 120.8 (C₃₀, C₅₀), 124.2 (C₁), 129.3 (C₃), 130.8

(C₆), 131.0 (C₁0 ), 132.8 (C₄), 134.0 (C₅), 146.6 (C₂), 147.8 (C₄0 ), 163.4 (C=O); GC-MS (EI) m/z: 327 [M]⁺.

3.1.3. General Synthetic Procedures for 1b–5b

Compounds 1a

chloride (33.7 mmol) and iron powder (6.03 mmol) and then reﬂuxed for 7 h. The reaction mixture

was added with water and extracted with dichloromethane (3 40 mL), treated with anhydrous

–5a (3.37 mmol) were dissolved in methanol (30 mL) and stirred with ammonium

×

magnesium sulfate and ﬁltrated. The ﬁltrate was evaporated to prepare the oily residue, which was

recrystallized with ethyl acetate and n-hexane mixture to afford pure white or pale yellow compounds

1b–5b.

2-Amino-N-(4⁰-chlorophenyl)benzamide (1b). Yield: 63%; m.p.: 148–150 ^◦C (147 ^◦C [38]).

Int. J. Mol. Sci. 2017, 18, 1144

14 of 27

2-Amino-N-(4⁰-bromophenyl)benzamide (2b). Yield: 70%; m.p.: 143–145 ^◦C (139–144 ^◦C [39]).

2-Amino-N-(4⁰-ﬂuorophenyl)benzamide (3b). Yield: 62%; m.p.: 118–120 ^◦C (122 ^◦C [38]).

2-Amino-N-(4⁰-methoxyphenyl)benzamide [38] (4b). Yield: 80%; m.p.: 118–119 ^◦C_◦(121 ^◦C [38]).

1

2-Amino-N-(4⁰-morpholinophenyl)benzamide (5b). Yield: 97%; m.p.: 167–168 C; H NMR

(400 MHz, DMSO-d₆) δ: 3.06 (4H, t, J = 4.8 Hz, H₃₀₀+ H₅₀₀), 3.74 (4H, t, J = 4.8 Hz, H₂₀₀+ H₆₀₀), 6.29

(2H, s, NH₂), 6.57 (1H, dt, J = 7.7, 1.1 Hz, H₅), 6.73 (1H, dd, J = 8.2, 1.0 Hz, H₃), 6.91 (2H, d, J = 9.0 Hz,

H₃₀+ H₅₀), 7.17 (1H, dt, J = 8.4, 1.5 Hz, H₄), 7.56 (2H, d, J = 9.0 Hz, H₂₀+ H₆₀), 7.59 (1H, dd, J = 7.8,

1.8 Hz, H₆), 9.80 (1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ: 39.9 (C₃₀₀, C₅₀₀), 65.1 (C₂₀₀, C₆₀₀),

114.5 (C₁), 115.2 (C₂₀, C₆₀), 115.6 (C₃), 116.3 (C₅), 121.7 (C₃₀, C₅₀), 129.5 (C₆), 131.4 (C₁₀), 131.8 (C₄), 147.4

(C₄0 ), 149.6 (C₂), 167.4 (C=O); GC-MS (EI) m/z: 297 [M]⁺.

3.1.4. General Synthetic Procedures for 1–10

Thiophene-2-carbonyl chloride and 5-chlorothiophene-2-carbonyl chloride were prepared

according to the synthetic procedures for 1a–5a. Compounds 1b–5b (0.62 mmol) were dissolved

in anhydrous benzene (30 mL) and then triethylamine (0.62 mmol) was slowly added and

thiophene-2-carbonyl chloride (0.62 mmol) or 5-chlorothiophene-2-carbonyl chloride (0.62 mmol) was

added. The reaction mixture was reacted at room temperature for 2 h and the following procedures

were same as procedures for 1a–5a. Compounds 1–10 were recrystallized with ethyl acetate and

n-hexane mixture to obtain as pure white or pale yellow compounds.

N-(4⁰-Chlorophenyl)-2-(thiophen-2⁰⁰-ylcarbonylamino)benzamide

(

1

).Yield:

79%; m.p.:

197–199 ^◦C; ¹H NMR (400 MHz, DMSO-d₆)

δ: 7.25 (1H, dd, J = 4.9, 3.8 Hz, H₄₀₀), 7.29 (1H, dt, J = 7.7,

1.1 Hz, H₅), 7.43 (2H, d, J = 8.8 Hz, H₃₀+ H₅₀), 7.61 (1H, dt, J = 8.0, 1.2 Hz, H₄), 7.74–7.78 (3H, m,

H₂₀+ H₆₀+ H₅₀₀), 7.88–8.02 (2H, m, H₃+ H₆), 8.32 (1H, d, J = 8.0 Hz, H₃₀₀), 10.63 (1H, s, NHCO), 11.54

(1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ: 121.6 (C₃), 122.5 (C₂₀, C₆₀), 123.0 (C₁), 123.4 (C₅),

127.9 (C₄₀), 128.4 (C₃₀, C₅₀), 128.6 (C₆), 128.8 (C₄₀₀), 129.0 (C₅₀₀), 132.2 (C₄), 132.3 (C₃₀₀), 137.5 (C₂₀₀), 138.1

(C₁₀), 139.5 (C₂), 159.4 (CONH), 167.3 (NHCO); DIP-MS (EI): m/z 356 [M]⁺; HRMS (FAB): m/z calcd

for C₁₈H₁₄ClN₂O₂S [M + H]⁺357.0465, found 357.0478.

N-(4⁰-Bromophenyl)-2-((thiophen-2⁰⁰-yl)carbonylamino)benzamide (

2

). Yield: 75%; m.p.:

210–212 ^◦C; ¹H NMR (400 MHz, DMSO-d₆)

δ: 7.25 (1H, dd, J = 4.9, 3.8 Hz, H₄₀₀), 7.30 (1H, dt, J = 7.8,

1.1 Hz, H₅), 7.56 (2H, d, J = 8.9 Hz, H₃₀+ H₅₀), 7.61 (1H, dt, J = 8.0, 1.2 Hz, H₄), 7.71 (2H, d, J = 8.9 Hz,

H₂₀+ H₆₀), 7.75 (1H, dd, J = 3.7, 1.0 Hz, H₅₀₀), 7.88–8.03 (2H, m, H₃+ H₆), 8.30 (1H, d, J = 8.2 Hz, H₃₀₀),

10.64 (1H, s, NHCO), 11.51 (1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ: 116.0 (C₄₀), 121.6 (C₃),

122.9 (C₂₀, C₆₀), 123.1 (C₁), 123.4 (C₅), 128.4 (C₆), 128.8 (C₄₀₀), 129.0 (C₅₀₀), 131.5 (C₃₀, C₅₀), 132.2 (C₄),

132.3 (C₃₀₀), 138.0 (C₂₀₀), 138.1 (C₁₀), 139.5 (C₂), 159.4 (CONH), 167.3 (NHCO); DIP-MS (EI): m/z 400 [M

− 1]⁺, 402 [M + 1]⁺; HRMS (FAB): m/z calcd for C₁₈H₁₄BrN₂O₂S [M + H]⁺400.9959, found 400.9973.

N-(4⁰-Fluorophenyl)-2-((thiophen-2⁰⁰-yl)carbonylamino)benzamide (

3). Yield: 73%; m.p.:

215–216 ^◦C; ¹H NMR (400 MHz, DMSO-d₆)

δ: 7.19–7.26 (3H, m, H₃₀+ H₅₀+ H₄₀₀), 7.29 (1H, dt, J = 7.7,

1.1 Hz, H₅), 7.61 (1H, dt, J = 8.0, 1.0 Hz, H₄), 7.73–7.75 (3H, m, H₂₀+ H₆₀+ H₅₀₀), 7.89 (1H, dd, J = 5.0,

1.1 Hz, H₆), 7.92 (1H, dd, J = 7.8, 1.2 Hz, H₃), 8.36 (1H, d, J = 7.6 Hz, H₃₀₀), 10.58 (1H, s, NHCO), 11.68

(1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ: 115.9 (J = 22.3 Hz, C₃₀, C₅₀), 122.0 (C₃), 123.3 (C₁),

123.7 (J = 8.2 Hz, C₂₀, C₆₀), 124.1 (C₅), 129.1 (C₆), 129.4 (C₄₀₀), 129.6 (C₅₀₀), 133.0 (C₄, C₃₀₀), 135.5 (J = 3.0

Hz, C₁₀), 139.0 (C₂₀₀), 140.2 (C₂), 159.4 (J = 239.4 Hz, C₄₀), 160.0 (CONH), 167.9 (NHCO); DIP-MS (EI):

m/z 340 [M]⁺; HRMS (FAB): m/z calcd for C₁₈H₁₄FN₂O₂S [M + H]⁺341.0760, found 341.0781.

N-(4⁰-Methoxyphenyl)-2-(thiophen-2⁰⁰-ylcarbonylamino)benzamide (

170–172 ^◦C; ¹H NMR (400 MHz, DMSO-d₆)

4

). Yield: 77%; m.p.:

δ

: 3.73 (3H, s, CH₃), 6.96 (2H, d, J = 9.0 Hz, H₃₀+ H₅₀),

7.31–7.23 (2H, m, H₅+ H₄₀₀), 7.59–7.64 (3H, m, H₄+ H₂+ H₆₀), 7.73 (1H, dd, J = 3.7, 0.6 Hz, H₅₀₀), 7.89

(1H, dd, J = 5.0, 0.6 Hz, H₆), 7.94 (1H, d, J = 6.9 Hz, H₃), 8.43 (1H, d, J = 8.2 Hz, H₃₀₀), 10.45 (1H, s,

NHCO), 11.92 (1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ

: 55.2 (CH₃), 113.8 (C₃₀, C₅₀), 120.9 (C₃),

122.1 (C₁), 122.9 (C₂₀, C₆₀), 123.2 (C₅), 128.4 (C₆), 128.6 (C₄₀₀), 128.8 (C₅₀₀), 131.3 (C₁₀), 132.2 (C₄), 132.3

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(C₃₀₀), 138.5 (C₂₀₀), 139.6 (C₂), 156.1 (C₄₀), 159.3 (CONH), 167.0 (NHCO); DIP-MS (EI): m/z 352 [M]⁺;

HRMS (FAB): m/z calcd for C₁₉H₁₇N₂O₃S [M + H]⁺353.0960, found 353.0979.

N-(4⁰-Morpholinophenyl)-2-(thiophen-2⁰⁰⁰-ylcarbonylamino)benzamide (

5

). Yield: 76%; m.p.:

◦

246–247 C; ¹H NMR (400 MHz, DMSO-d₆)

δ: 3.09 (4H, t, J = 4.8 Hz, H₃₀₀+ H₅₀₀), 3.74 (4H, t, J = 4.8 Hz,

H₂₀₀+ H₆₀₀), 6.96 (2H, d, J = 9.0 Hz, H₃₀+ H₅₀), 7.22–7.30 (2H, m, H₅+ H₄₀₀₀), 7.57–7.61 (3H, m, H₄+

H₂₀+ H₆₀), 7.72 (1H, dd, J = 3.7, 0.6 Hz, H₅₀₀₀), 7.90 (1H, dd, J = 4.9, 0.5 Hz, H₆), 7.93 (1H, d, J = 7.3 Hz,

H₃), 8.43 (1H, d, J = 8.1 Hz, H₃₀₀₀), 10.39 (1H, s, NHCO), 11.97 (1H, s, CONH); ¹³C NMR (100 MHz,

00

DMSO-d₆)

δ

: 48.7 (C₃₀₀, C₅₀₀), 66.1 (C², C₆₀₀), 115.2 (C₃₀, C₅₀), 120.8 (C₃), 122.0 (C₁), 122.4 (C₂₀,C₆₀),

123.1 (C₅), 128.4 (C₆), 128.6 (C₄₀₀), 128.8 (C₅₀₀), 130.3 (C₁₀), 132.2 (C₄), 132.3 (C₃₀₀), 138.6 (C₂₀₀), 139.6 (C₂),

148.1 (C₄₀), 159.3 (CONH), 166.9 (NHCO); DIP-MS (EI): m/z 407 [M]⁺; HRMS (FAB): m/z calcd for

C₂₂H₂₂N₃O₃S [M + H]⁺408.1382, found 408.1368.

N-(4⁰-Chlorophenyl)-2-((5-chlorothiophen-2⁰⁰-yl)carbonylamino)benzamide (

6

). Yield: 60%; m.p.:

◦

207–209 C; ¹H NMR (400 MHz, DMSO-d₆)

δ: 7.29 (1H, d, J = 4.0 Hz, H₄₀₀), 7.32 (1H, dt, J = 7.7, 1.1 Hz,

H₅), 7.43 (2H, d, J = 8.9 Hz, H₃₀+ H₅₀), 7.60 (1H, dt, J = 8.0, 1.2 Hz, H₄), 7.64 (1H, d, J = 4.1 Hz, H₆), 7.75

(2H, d, J = 8.9 Hz, H₂₀+ H₆₀), 7.88 (1H, dd, J = 7.8, 1.0 Hz, H₃), 8.17 (1H, d, J = 8.2 Hz, H₃₀₀), 10.63 (1H, s,

NHCO), 11.44 (1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ: 122.2 (C₃), 122.4 (C₂₀, C₆₀), 123.9 (C₁),

124.2 (C₅), 127.8 (C₄₀), 128.6 (C₃₀, C₅₀), 128.7 (C₆), 129.1 (C₄₀₀), 132.2 (C₄, C₃₀₀), 134.2 (C₂₀₀), 137.4 (C₅₀₀),

137.7 (C₁₀), 138.7 (C₂), 158.4 (CONH), 167.1 (NHCO); DIP-MS (EI): m/z 390 [M

m/z calcd for C₁₈H₁₃Cl₂N₂O₂S [M + H]⁺391.0075, found 391.0089.

−

1]⁺; HRMS (FAB):

N-(4⁰-Bromophenyl)-2-((5-chlorothiophen-2⁰⁰-yl)carbonylamino)benzamide (

7). Yield: 41%; m.p.:

◦

198–199 C; ¹H NMR (400 MHz, DMSO-d₆)

δ: 7.28 (1H, d, J = 4.1 Hz, H₄₀₀), 7.32 (1H, dt, J = 7.7, 1.1 Hz,

H₅), 7.55 (2H, d, J = 8.9 Hz, H₃₀+ H₅₀), 7.60 (1H, dt, J = 8.0, 1.2 Hz, H₄), 7.64 (1H, d, J = 4.1 Hz, H₆),

7.70 (2H, d, J = 8.9 Hz, H₂₀+ H₆₀), 7.88 (1H, dd, J = 7.8, 1.3 Hz, H₃), 8.18 (1H, dd, J = 8.2, 0.8 Hz, H₃₀₀),

10.60 (1H, s, NHCO), 11.42 (1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ: 115.9 (C₄₀), 122.1 (C₃),

122.8 (C₂₀, C₆₀), 123.9 (C₁), 124.2 (C₅), 128.7 (C₆), 129.0 (C₄₀₀), 131.5 (C₃₀, C₅₀), 132.1 (C₄, C₃₀₀), 134.1 (C₂₀₀),

137.4 (C₅00 ), 138.0 (C₁0 ), 138.6 (C₂), 158.4 (CONH), 167.1 (NHCO); DIP-MS (EI): m/z 434 [M − 1]⁺, 436

[M + 1]⁺; HRMS (FAB): m/z calcd for C₁₈H₁₃BrClN₂O₂S [M + H]⁺434.9570, found 434.9549.

N-(4⁰-Fluorophenyl)-2-((5-chlorothiophen-2⁰⁰-yl)carbonylamino)benzamide (

8

). Yield: 73%; m.p.:

◦

215–216 C; ¹H NMR (400 MHz, DMSO-d₆)

δ: 7.22 (2H, t, J = 8.9 Hz, H₃₀+ H₅₀), 7.27–7.34 (2H, m,

H₅+ H₄₀₀), 7.64–7.57 (2H, m, H₄+ H₆), 7.73 (2H, dd, J = 9.1, 5.1 Hz, H₂₀+ H₆₀), 7.91 (1H, dd, J = 7.9,

1.1 Hz, H₃), 8.24 (1H, d, J = 8.1 Hz, H₃₀₀), 10.57 (1H, s, NHCO), 11.60 (1H, s, CONH); ¹³C NMR

(100 MHz, DMSO-d₆) δ: 115.2 (J = 22.3 Hz, C₂₀, C₆₀), 121.8 (C₃), 123.0 (J = 8.1 Hz, C₃₀, C₅₀), 123.5 (C₁),

123.8 (C₅), 128.6 (C₆), 128.9 (C₄₀₀), 132.2 (C₄), 134.1 (C₂₀₀), 134.8 (J = 2.2 Hz, C₁₀), 137.7 (C₅₀₀), 138.6 (C₂),

0

158.3 (CONH), 158.6 (J = 239.5 Hz, C4 ), 167.0 (NHCO); DIP-MS (EI): m/z 374 [M]⁺; HRMS (FAB): m/z

calcd for C₁₈H₁₃ClFN₂O₂S [M + H]⁺375.0370, found 375.0388.

N-(4⁰-Me_◦thoxyphenyl)-2-((5-chlorothiophen-2⁰⁰-yl)carbonylamino)benzamide (

9). Yield: 77%;

δ: 3.75 (3H, s, CH₃), 6.95 (2H, d, J = 8.8 Hz, H₃₀+ H₅₀),

m.p.: 190–191 C; ¹H NMR (400 MHz, DMSO-d₆)

7.26–7.32 (2H, m, H₅+ H₄₀₀), 7.56–7.65 (4H, m, H₄+ H₆+ H₂₀+ H₆₀), 7.94 (1H, d, J = 7.7 Hz, H₃), 8.31

(1H, d, J = 8.3 Hz, H₃₀₀), 10.45 (1H, s, NHCO), 11.87 (1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ

: 55.4 (CH₃), 114.0 (C₂₀, C₆₀), 121.6 (C₃), 123.0 (C₃₀, C₅₀), 123.1 (C₁), 123.8 (C₅), 128.7 (C₆), 128.8 (C₄₀₀),

129.1 (C₁₀), 131.5 (C₃₀₀), 132.3 (C₄), 134.4 (C₂₀₀), 138.2 (C₅₀₀), 139.9 (C₂), 156.3 (C₄₀), 158.5 (CONH), 167.1

(NHCO); DIP-MS (EI): m/z 386 [M]⁺; HRMS (FAB): m/z calcd for C₁₉H₁₆ClN₂O₃S [M + H]⁺387.0457,

found 387.0470.

0

000

N-(4 -Morpholinophenyl)-2-((5-chlorothiophen-2 -yl)carbonylamino)benzamide (10). Yield: 80%;

m.p.: 230–231 ^◦C; ¹H NMR (400 MHz, DMSO-d₆)

δ

: 3.08 (4H, t, J = 4.8 Hz, H₃₀₀+ H₅₀₀), 3.74 (4H, t,

J = 4.8 Hz, H₂₀₀+ H₆₀₀), 6.96 (2H, d, J = 9.1 Hz, H₃₀+ H₅₀), 7.23–7.32 (2H, m, H₅+ H₄₀₀₀), 7.51–7.64 (4H,

m, H₄+ H₆+ H₂₀+ H₆₀), 7.92 (1H, dd, J = 7.9, 1.1 Hz, H₃), 8.32 (1H, d, J = 8.3 Hz, H₃₀₀₀), 10.39 (1H, s,

NHCO); ¹³C NMR (100 MHz, DMSO-d₆)

δ: 51.6 (C₃₀₀, C₅₀₀), 68.9 (C₂₀₀, C₆₀₀), 118.0 (C₃₀, C₅₀), 124.1 (C₃),

125.2 (C₂₀, C₆₀), 125.6 (C₁), 126.4 (C₅), 131.2 (C₆), 131.5 (C₄₀₀), 131.7 (C₃₀₀), 133.2 (C₄), 135.0 (C₂₀₀), 137.0

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(C₁₀), 140.9 (C₅₀₀), 141.6 (C₂), 150.9 (C₄₀), 161.1 (CONH), 169.6 (NHCO); DIP-MS (EI): m/z 441[M]⁺;

HRMS (FAB): m/z calcd for C₂₂H₂₁ClN₃O₃S [M + H]⁺442.0992, found 442.0977.

3.1.5. General Synthetic Procedures for 11a–13a

To a solution of 4-(methylphenylsulfonamido)benzoyl chloride (2, 2.06 mmol) in anhydrous

benzene (30 mL) was added 3- or 4-aminobenzonitrile (220 mg, 1.87 mmol), 4-aminobenzylcyanide

(265 mg, 1.87 mmol) and triethylamine (0.1 mL). The following procedures were same as procedures

for 1a

obtain as a white powder.

N-(3’-Cyanophenyl)-2-(4”-methylphenylsulfonamido)benzamide (11a). Yield: 75%; m.p.:

–5a. Compounds 11a–13a were recrystallized with dichloromethane and n-hexane mixture to

◦

1

216–218 C; H NMR (400 MHz, DMSO-d₆) δ: 2.23 (3H, s, CH₃), 7.21 (2H, d, J = 8.4 Hz, H₃₀₀+

H₅₀₀), 7.23 (1H, t, J = 7.6 Hz, H₅), 7.36 (1H, d, J = 8.0 Hz, H₃), 7.48 (1H, t, J = 7.6 Hz, H₄), 7.54–7.65

(4H, m, H₆+ H₅₀+ H₂₀₀+ H₆₀₀), 7.69 (1H, d, J = 7.2 Hz, H₄₀), 7.88 (1H, d, J = 7.6 Hz, H₆₀), 8.08 (1H, s,

H₂₀), 10.26 (1H, s, NHSO₂), 10.53 (1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ: 21.6 (CH₃), 112.1

(C₃₀), 119.3 (CN), 122.9 (C₃), 124.1 (C₁), 125.9 (C₅), 127.6 (C₆), 127.8 (C₆₀), 128.3 (C₂₀₀, C₆₀₀), 129.8 (C₅₀),

130.4 (C₃₀₀, C₅₀₀), 130.8 (C₂₀), 133.1 (C₄), 136.6 (C₄₀), 137.1 (C₁₀₀), 137.4 (C₄₀₀), 140.0 (C₁₀), 144.3 (C₂), 167.5

(C=O); DIP-MS (EI): m/z 391 [M]⁺.

N-(4⁰-Cyanophenyl)-2-(4”-methylphenylsulfonamido)benzamide (12a). Yield: 70%; m.p.:

◦

1

253–255 C; H NMR (400 MHz, DMSO-d₆) δ: 2.22 (3H, s, CH₃), 7.12–7.26 (3H, m, H₅+ H₃₀₀+

H₅₀₀), 7.33 (1H, d, J = 7.6 Hz, H₃), 7.47 (1H, t, J = 7.6 Hz, H₄), 7.56 (2H, d, J = 7.6 Hz, H₂₀₀+ H₆₀₀), 7.67

(1H, d, J = 7.2 Hz, H₆), 7.77–7.87 (4H, m, H₂₀+ H₃₀+ H₅₀+ H₆₀), 10.16 (1H, s, NHSO₂), 10.59 (1H, s,

CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ: 21.6 (CH₃), 106.4 (C₄₀), 119.7 (CN), 121.2 (C₂₀, C₆₀), 123.1

(C₃), 125.2 (C₁), 126.4 (C₅), 127.5 (C₆), 128.0 (C₂₀₀, C₆₀₀), 130.4 (C₃₀₀, C₅₀₀), 133.1 (C₃₀, C₅₀), 133.8 (C₄), 137.2

(C₁00 , C₄00 ), 143.6 (C₁0 ), 144.3 (C₂), 167.6 (C=O); DIP-MS (EI): m/z 391 [M]⁺.

N-(4⁰-Cyanomethylphenyl)-2-(4”-methylphenylsulfonamido)benzamide (13a). Yield: 68%; m.p.:

◦

142–146 C; ¹H NMR (400 MHz, DMSO-d₆)

δ: 2.27 (3H, s, CH₃), 4.02 (2H, s, CH₂), 7.20–7.29 (3H, m, H₅

+ H₃₀₀+ H₅₀₀), 7.36 (2H, d, J = 8.8 Hz, H₃₀+ H₅₀), 7.45–7.53 (2H, m, H₃+ H₄), 7.67 (2H, d, J = 8.4 Hz,

H₂₀₀+ H₆₀₀), 7.76 (1H, dd, J = 8.0, 1.2 Hz, H₆), 10.39 (1H, s, NHSO₂), 10.59 (1H, s, CONH); ¹³C NMR

(100 MHz, DMSO-d₆) δ: 21.0 (CH₃), 21.9 (CH₂), 119.3 (CN), 121.4 (C₂₀, C₆₀), 124.0 (C₃), 124.1 (C₁), 126.8

(C₅), 127.0 (C₆), 128.4 (C₂₀₀, C₆₀₀), 129.1 (C₃₀, C₅₀), 129.8 (C₃₀₀, C₅₀₀), 132.4 (C₄), 135.9 (C₁₀), 137.2 (C₁₀₀),

137.7 (C₄00 ), 143.7 (C₂), 166.7 (C=O); GC-MS (EI) m/z: 405 [M]⁺.

3.1.6. General Synthetic Procedures for 11–13

To a suspension of 10% Pd-C (200 mg) in ethanol (50 mL) was added 1N-HCl (1 mL) and nitriles

11a–13a (1.28 mmol) and the reaction mixture was catalytically hydrogenated in parr hydrogenation

apparatus (50 psi of hydrogen) at room temperature for 6 h. When the starting material could be no

longer detected by TLC, the reaction mixture was ﬁltrated on celite pad. The ﬁltrate was concentrated

to give the oily product which was diluted in ethanol and then crystallized with diethyl ether to form

the light brown precipitate which was recrystallized with ethanol and diethyl ether mixture to afford

11–13 as a white powder.

N-(3’-Aminomethyl)phenyl-2-(4”-methylphenylsulfonamido)benzamide (11). Yield: 72%; m.p:

163–166 ^◦C; ¹H NMR (400 MHz, CD₃OD)

δ: 2.29 (3H, s, CH₃), 4.03 (2H, s, CH₂), 7.23 (1H, t, J = 7.2 Hz,

H₄), 7.27 (2H, d, J = 8.4 Hz, H₃₀₀+ H₅₀₀), 7.32 (1H, d, J = 7.6 Hz, H₄₀), 7.40–7.45 (2H, m, H₅₀+ H₆₀), 7.42

(1H, t, J = 7.6 Hz, H₅), 7.55 (1H, d, J = 8.4 Hz, H₃), 7.63 (2H, d, J = 8.4 Hz, H₂₀₀+ H₆₀₀), 7.83 (1H, d,

J = 7.6 Hz, H₆), 7.90 (1H, s, H₂0 ), 8.50 (3H, br s, aminium chloride), 10.52 (1H, s, NHSO₂), 10.69 (1H, s,

CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ: 20.9 (CH₃), 42.3 (CH₂), 121.0 (C₂₀), 121.2 (C₆₀), 121.5 (C₃),

123.6 (C₁), 124.8 (C₅), 126.8 (C₂₀₀, C₆₀₀), 128.9 (C₆), 129.2 (C₄₀), 129.8 (C₃₀₀, C₅₀₀), 132.5 (C₄, C₁₀), 134.5

(C₃₀), 137.4 (C₁₀₀), 138.5 (C₄₀₀), 143.7 (C₂), 166.8 (C=O); DIP-MS (EI): m/z 395 [M

−

HCl]⁺; HRMS (FAB):

calcd for C₂₁H₂₂N₃O₃S [M + H]⁺396.1382, found 396.1396.

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N-(4⁰-Aminomethyl)phenyl-2-(4”-methylphenylsulfonamido)benzamide (12). Yield: 81%; m.p:

92–95 ^◦C; ¹H NMR (400 MHz, CD₃OD)

: 2.29 (3H, s, CH₃), 4.03 (2H, s, CH₂), 7.18–7.24 (3H, m, H₄+

H₃₀₀+ H₅₀₀), 7.42 (1H, t, J = 8.0 Hz, H₅), 7.45–7.53 (3H, m, H₃+ H₃₀+ H₅₀), 7.61 (2H, d, J = 8.4 Hz, H₂₀₀

H₆00 ), 7.69 (2H, d, J = 8.4 Hz, H₂0 + H₆0 ), 7.80 (1H, d, J = 8.0 Hz, H₆), 8.36 (3H, br s, aminium chloride),

10.44 (1H, s, NHSO₂), 10.61 (1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

: 20.9 (CH₃), 41.8 (CH₂),

120.9 (C₂₀, C₆₀), 121.4 (C₃), 124.0 (C₁), 124.1 (C₅), 126.8 (C₂₀₀, C₆₀₀), 129.1 (C₆), 129.3 (C₃₀, C₅₀), 129.8 (C₃₀₀

δ

+

δ

,

C₅₀₀), 132.4 (C₄, C₁₀), 135.9 (C₄₀), 137.2 (C₁₀₀), 138.4 (C₄₀₀), 143.7 (C₂), 166.7 (C=O); DIP-MS (EI): m/z 395

[M − HCl]⁺; HRMS (FAB): calcd for C₂₁H₂₂N₃O₃S [M]⁺396.1382, found 396.1399.

N-(4⁰-Aminoethyl)phenyl-2-(4”-methylphenylsulfonamido)benzamide (13). Yield: 77%; m.p:

253–255 ^◦C; ¹H NMR (400 MHz, CD₃OD)

δ

: 2.27 (3H, s, CH₃), 2.98 (2H, t, J = 7.8 Hz, CH₂), 3.21 (2H,

t, J = 7.8 Hz, CH₂), 7.14 (2H, m, H₃₀+ H₅₀), 7.23 (1H, t, J = 7.6 Hz, H₄), 7.30 (2H, d, J = 8.0 Hz, H₃₀₀

H₅₀₀), 7.46–7.61 (6H, m, H₃+ H₅+ H₂₀+ H₆₀+ H₂₀₀+ H₆₀₀), 7.67 (1H, d, J = 7.6 Hz, H₆), 8.33 (3H, br s,

aminium chloride), 10.41 (1H, s, NHSO₂), 10.63 (1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

: 20.2

+

δ

(CH₃), 32.9 (CH₂), 40.7 (CH₂), 121.8 (C₂₀, C₆₀), 123.5 (C₃), 124.8 (C₁), 125.3 (C₅), 127.1 (C₂₀₀, C₆₀₀), 128.2

(C₆), 129.0 (C₃₀, C₅₀), 129.5 (C₃₀₀, C₅₀₀), 132.2 (C₄), 133.2 (C₄₀), 136.1 (C₁₀), 137.3 (C₁₀₀), 137.7 (C₄₀₀), 144.1

(C₂), 167.7 (C=O); DIP-MS (EI): m/z 409 [M

410.1538, found 410.1520.

−

HCl]⁺; HRMS (FAB): calcd for C₂₂H₂₄N₃O₃S [M + H]⁺

3.1.7. General Synthetic Procedures for 14–20

To a solution of 4-(methylphenylsulfonamido)benzoyl chloride (

2

, 1.50 mmol) was dissolved in

anhydrous benzene (30 mL) was added 4-chloroaniline or 4-bromoaniline or 4-methoxyaniline or

4-carboxyaniline or 2-(thiophen-2-yl)ethylamine or 4-morpholinoaniline or 4-(2-oxomorpholino)aniline

(1.36 mmol) and triethylamine (0.1 mL). The following procedures were same as procedures for 1a

–5a.

Compounds 14 20 were recrystallized with dichloromethane and n-hexane mixture to afford a white

–

powder, respectively.

N-(4⁰-Chlorophenyl)-2-(4"-methylphenylsulfonamido)benzamide (14).

Yield: 82%; m.p:

◦

1

220–223 C; H NMR (400 MHz, DMSO-d₆)

δ: 2.29 (3H, s, CH₃), 7.20–7.30 (3H, m, H₄+ H₃₀₀

+

H₅₀₀), 7.58 (2H, d, J = 7.6 Hz, H₃₀+ H₅₀), 7.69 (2H, d, J = 8.0 Hz, H₂₀₀+ H₆₀₀), 7.71 (2H, d, J = 7.6 Hz, H₂₀

H₆₀), 7.98 (1H, t, J = 7.6 Hz, H₅), 8.03 (1H, d, J = 8.0 Hz, H₃), 8.18 (1H, d, J = 8.0 Hz, H₆) 11.95 (1H, s,

CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ: 21.6 (CH₃), 121.2 (C₂₀, C₆₀), 123.1 (C₃), 125.2 (C₁), 126.4 (C₅),

127.5 (C₆), 128.0 (C₂₀₀, C₆₀₀), 129.9 (C₄₀), 130.4 (C₃₀₀, C₅₀₀), 133.1 (C₄, C₃₀, C₅₀), 136.8 (C₁₀₀), 137.2 (C₄₀₀),

144.3 (C₂), 167.6 (C=O); DIP-MS (EI) m/z: 400 [M]⁺; HRMS (FAB): calcd for C₂₀H₁₈ClN₂O₃S [M + H]⁺

401.0727, found 401.0741.

N-(4⁰-Bromophenyl)-2-(4"-methylphenylsulfonamido)benzamide (15).

Yield: 80%; m.p:

◦

1

211–213 C; H NMR (400 MHz, CDCl₃)

δ

: 2.26 (3H, s, CH₃), 7.12–7.21 (3H, m, H₄+ H₃₀₀+ H₅₀₀),

7.38 (2H, d, J = 8.8 Hz, H₃₀+ H₅₀), 7.43–7.51 (4H, m, H₃+ H₅+ H₂₀+ H₆₀), 7.62 (2H, d, J = 8.4 Hz, H₂₀₀

+ H₆₀₀), 7.69 (1H, d, J = 7.8 Hz, H₆) 10.1 (1H, s, NHSO₂), 10.45 (1H, s, CONH); ¹³C NMR (100 MHz,

CDCl₃) δ: 21.7 (CH₃), 118.2 (C₄₀), 122.2 (C₂₀, C₆₀), 122.7 (C₃), 122.9 (C₁), 124.3 (C₅), 126.9 (C₆), 127.5

(C₂₀₀, C₆₀₀), 129.8 (C₃₀₀, C₅₀₀), 132.3 (C₃₀, C₅₀), 133.3 (C₄), 136.4 (C₁₀), 136.5 (C₁₀₀), 138.8 (C₄₀₀), 144.0 (C₂),

166.7 (C=O); DIP-MS (EI) m/z: 444 [M]⁺; HRMS (FAB): calcd for C₂₀H₁₈BrN₂O₃S [M + H]⁺445.0222,

found 445.0239.

N-(4⁰-Methoxyphenyl)-2-(4"-methylphenylsulfonamido)benzamide (16). Yield: 87%; m.p:

◦

1

157–159 C; H NMR (400 MHz, CDCl₃) δ: 2.28 (3H, s, CH₃), 3.76 (3H, s, OCH₃), 6.95 (2H, d, J

= 8.8 Hz, H₃₀+ H₅₀), 7.21 (1H, t, J = 6.8 Hz, H₄), 7.26 (2H, d, J = 8.0 Hz, H₃₀₀+ H₅₀₀), 7.44–7.50 (2H, m, H₃

+ H₅), 7.54 (2H, d, J = 8.8 Hz, H₂₀+ H₆₀), 7.61 (2H, d, J = 8.4 Hz, H₂₀₀+ H₆₀₀), 7.77 (1H, d, J = 7.6 Hz, H₆),

10.23 (1H, s, NHSO₂), 10.81 (1H, s, CONH); ¹³C NMR (100 MHz, CDCl₃)

δ: 21.6 (CH₃), 55.9 (OCH₃),

114.4 (C₃₀, C₅₀), 121.6 (C₃), 123.4 (C₂₀, C₆₀), 124.0 (C₁), 124.6 (C₅), 127.5 (C₂₀₀, C₆₀₀), 129.6 (C₆), 130.5 (C₃₀₀

,

C₅₀₀), 131.8 (C₁₀), 133.0 (C₄), 136.5 (C₁₀₀), 138.2 (C₄₀₀), 144.4 (C₂), 156.8 (C₄₀), 167.0 (C=O) DIP-MS (EI)

m/z: 396 [M]⁺; HRMS (FAB): calcd for C₂₁H₂₁N₂O₄S [M + H]⁺397.1222, found 397.1241.

Int. J. Mol. Sci. 2017, 18, 1144

18 of 27

N-(4⁰-Carboxyphenyl)-2-(4"-methylphenylsulfonamido)benzamide (17). Yield: 85%; m.p:

◦

1

210–212 C; H NMR (400 MHz, DMSO-d₆) δ: 2.23 (3H, s, CH₃), 7.18–7.25 (3H, m, H₄+ H₃₀₀+

H₅₀₀), 7.36 (1H, d, J = 8.4 Hz, H₃), 7.47 (1H, t, J = 7.6 Hz, H₅), 7.57 (2H, d, J = 8.0 Hz, H₂₀₀+ H₆₀₀), 7.61

(2H, d, J = 8.8 Hz, H₂₀+ H₆₀), 7.86 (2H, d, J = 8.8 Hz, H₃₀+ H₅₀), 7.71 (1H, d, J = 8.0 Hz, H₆), 10.31 (1H, s,

NHSO₂), 10.52 (1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ: 21.3 (CH₃), 117.5 (C₂₀, C₆₀), 122.0

(C₃), 124.3 (C₁), 124.3 (C₅), 126.8 (C₄₀), 127.1 (C₂₀₀, C₆₀₀), 129.0 (C₆), 128.9 (C₃₀₀, C₅₀₀), 130.8 (C₃₀, C₅₀),

132.8 (C₄), 136.1 (C₁₀₀), 138.5 (C₄₀₀), 144.1 (C₂), 167.3 (C=O) DIP-MS (EI) m/z: 410 [M]⁺; HRMS (FAB):

calcd for C₂₁H₁₉N₂O₅S [M + H]⁺411.1015, found 411.1036.

N-(2-(thiophen-2⁰-yl)ethyl)-2-(4"-methylphenylsulfonamido)benzamide (18). Yield: 60%; m.p:

119–121 ^◦C; ¹H NMR (400 MHz, CDCl₃)

δ: 2.31 (3H, s, CH₃), 3.02 (2H, t, J = 6.8 Hz, CH₂), 3.45 (2H, q,

J = 6.8 Hz, CH₂), 6.91 (1H, d, J = 2.4 Hz, H₃₀), 6.96 (1H, d, J = 5.2, 3.6 Hz, H₄₀), 7.13 (1H, t, J = 7.2 Hz, H₄),

7.31 (2H, d, J = 8.0 Hz, H₃₀₀+ H₅₀₀), 7.35 (1H, dd, J = 5.2, 1,2 Hz, H₅₀), 7.42–7.53 (2H, m, H₃+ H₅), 7.61

(2H, d, J = 8.4 Hz, H₂₀₀+ H₆₀₀), 7.66 (1H, d, J = 8.0 Hz, H₆), 8.91 (1H, s, NHSO₂), 11.57 (1H, s, CONH);

¹³C NMR (100 MHz, CDCl₃)

δ: 21.0 (CH₃), 28.7 (CH₂), 40.9 (CH₂), 119.7 (C₃), 120.6 (C₁), 123.4 (C₅),

124.2 (C₅₀), 125.3 (C₄₀), 126.8 (C₂₀₀, C₆₀₀), 127.0 (C₃₀), 128.3 (C₆), 129.9 (C₃₀₀, C₅₀₀), 132.4 (C₄), 135.9 (C₁₀₀),

138.4 (C₄₀₀), 141.2 (C₂₀), 143.8 (C₂), 168.0 (C=O); DIP-MS (EI) m/z: 400 [M]⁺; HRMS (FAB): calcd for

C₂₀H₂₁N₂O₃S₂[M + H]⁺401.0994, found 401.0982.

N-(4⁰-morpholinophenyl)-2-(4"-methylphenylsulfonamido)benzamide (19). Yield: 55%; m.p:

206–209 ^◦C; ¹H NMR (400 MHz, CDCl₃)

δ: 2.29 (3H, s, CH₃), 3.09 (4H, t, J = 4.8 Hz, CH₂), 3.75 (4H, t,

J = 4.4 Hz, CH₂), 6.95 (2H, d, J = 8.8 Hz, H₃₀+ H₅₀), 7.20 (1H, t, J = 6.0 Hz, H₄), 7.26 (2H, d, J = 8.4 Hz,

H₃₀₀+ H₅₀₀), 7.45–7.53 (4H, m H₃+ H₅+ H₂₀+ H₆₀), 7.61 (2H, d, J = 8.4 Hz, H₂₀₀+ H₆₀₀), 7.77 (1H, d,

J = 7.6 Hz, H₆), 10.18 (1H, s, NHSO₂), 10.89 (1H, s, CONH); ¹³C NMR (100 MHz, CDCl₃)

δ: 21.7 (CH₃),

49.4 (2 x CH₂), 66.8 (2 x CH₂), 115.8 (C₃₀, C₅₀), 122.9 (C₃), 123.9 (C₁, C₂₀, C₆₀), 124.6 (C₅), 127.6 (C₂₀₀, C₆₀₀),

129.6 (C₆), 130.5 (C₃₀₀, C₅₀₀), 130.7 (C₁₀), 133.0 (C₄), 136.5 (C₁₀₀), 138.2 (C₄₀₀), 144.4 (C₂), 148.8 (C₄₀), 166.9

(C=O); DIP-MS (EI) m/z: 451 [M]⁺; HRMS (FAB): calcd for C₂₄H₂₆N₃O₄S [M + H]⁺452.1644, found

452.1627.

N-(4⁰-(2-oxomorpholino)phenyl)-2-(4"-methylphenylsulfonamido)benzamide (20). Yield: 44%;

m.p: 270–273 ^◦C; ¹H NMR (400 MHz, CDCl₃)

δ: 2.28 (3H, s, CH₃), 3.74 (2H, t, J = 4.8 Hz, CH₂), 3.99

(2H, t, J = 4.8 Hz, CH₂), 4.21 (2H, s, CH₂), 7.20–7.30 (3H, m, H₄+ H₃₀+ H₅₀), 7.39 (2H, d, J = 8.8 Hz, H₃₀₀

+ H₅₀₀), 7.40–7.52 (2H, m, H₃+ H₅), 7.61 (2H, d, J = 8.4 Hz, H₂₀+ H₆₀), 7.67 (2H, d, J = 8.8 Hz, H₂₀₀+ H₆₀₀),

7.77 (1H, d, J = 7.6 Hz, H₆), 10.40 (1H, s, NHSO₂), 10.61 (1H, s, CONH); ¹³C NMR (100 MHz, CDCl₃)

δ

: 21.0 (CH₃), 40.1 (CH₂), 63.5 (CH₂), 67.8 (CH₂), 117.8 (C₃₀, C₅₀), 121.2 (C₃), 124.0 (C₁), 124.1 (C₅), 125.7

(C₂₀, C₆₀), 126.8 (C₂₀₀, C₆₀₀), 129.1 (C₆), 129.8 (C₃₀₀, C₅₀₀), 132.4 (C₄), 135.9 (C₁₀), 136.4 (C₄₀), 137.2 (C₁₀₀),

137.7 (C₄₀₀), 143.8 (C₂), 166.0 (C=O), 166.6 (C=O); DIP-MS (EI) m/z: 465 [M]⁺; HRMS (FAB): calcd for

C₂₄H₂₄N₃O₅S [M + H]⁺466.1437, found 466.1454.

3.1.8. General Synthetic Procedures for 21a–23a

2-Nitrobenzoyl chloride (8.99 mmol) and triethylamine (7.19 mmol) were added to a solution of

3-aminophenylcyanide or 4-aminophenylcyanide or 4-aminobenzyl cyanide (7.19 mmol) in anhydrous

dichloromethane (30 mL) and stirred at room temperature for 3 h. The reaction was terminated with

water and extracted with dichloromethane (3

×

30 mL), dried with anhydrous magnesium sulfate and

ﬁltrated. The ﬁltrate was concentrated to give the crude oily reside, which was recrystallized with

ethyl acetate and n-hexane mixture to afford pure white or pale yellow compounds 21a–23a.

N-(3⁰-Cyanophenyl)-2-nitrobenzamide (21a). Yield: 80%; m.p.: 170–171 ^◦C; ¹H NMR (400 MHz,

DMSO-d₆)

δ

: 7.59–7.92 (6H, m, H₄+ H₅+ H₆+ H₄₀+ H₅₀+ H₆₀), 8.13 (1H, s, H₂₀), 8.18 (1H, d, J = 8.0 Hz,

H₃), 11.02 (1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ

: 111.7 (C₃₀), 118.6 (C N), 122.2 (C₆₀), 124.2

≡

(C₅₀), 124.4 (C₁), 127.5 (C₂₀), 129.3 (C₃), 130.4 (C₄₀), 131.3 (C₆), 132.0 (C₄), 134.3 (C₅), 139.6 (C₁₀), 146.3

(C₂), 164.6 (C=O); GC-MS (EI) m/z: 267 [M]⁺.

N-(4⁰-Cyanophenyl)-2-nitrobenzamide (22a). Yield: 84%; m.p.: 217–218 ^◦C; ; ¹H NMR (400 MHz,

DMSO-d₆) δ: 7.77–7.84 (6H, m, H₄+ H₆+ H₂₀+ H₃₀+ H₅₀+ H₆₀), 7.90 (1H, td, J = 7.6, 1.2 Hz, H₅), 8.19

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(1H, dd, J = 8.0, 4.0 Hz, H₃), 11.08 (1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ: 105.7 (C₄₀), 118.9

(C

(C₁0 ), 146.2 (C₂), 164.7 (C=O); GC-MS (EI) m/z: 267 [M]⁺.

N-(4⁰-Cyanomethylphenyl)-2-nitrobenzamide (23a). Yield: 76%; m.p.: 174–175 C; H NMR

(400 MHz, DMSO-d₆) : 4.01 (2H, s, CH₂), 7.35 (2H, d, J = 8.0 Hz, H₃₀+ H₅₀), 7.68 (2H, d, J = 8.0 Hz, H₂₀

≡

N), 119.6 (C₂₀, C₆₀), 124.8 (C₁), 129.3 (C₃), 131.3 (C₆), 132.1 (C₄), 133.4 (C₃₀, C₅₀), 134.3 (C₅), 142.9

◦

1

δ

+ H₆₀), 7.75–7.79 (2H, m, H₄+ H₆), 7.88 (1H, t, J = 8.0 Hz, H₅), 8.16 (1H, d, J = 8.0 Hz, H₃), 10.71 (1H, s,

CONH); ¹³C NMR (100 MHz, DMSO-d₆) δ: 21.9 (CH₂), 119.3 (C≡N), 120.0 (C₂0 , C₆0 ), 124.3 (C₁), 126.6

(C₄₀), 128.6 (C₃₀, C₅₀), 129.3 (C₃), 131.0 (C₆), 132.5 (C₄), 134.1 (C₅), 138.2 (C₁₀), 146.4 (C₂), 164.1 (C=O);

GC-MS (EI) m/z: 281 [M]⁺.

3.1.9. General Synthetic Procedures for 21b–23b

To a nitro compounds 21a

(17.9 mmol) and iron powder (3.58 mmol), and then the reaction mixture was reﬂuxed for 7 h.

The following procedures were same as synthetic procedures for 1b 5b. Compounds 21b 23b

–23a (1.79 mmol) in methanol (20 mL) was added ammonium chloride

–

were recrystallized with ethyl acetate and n-hexane mixture to provide pure white or pale-yellow

compounds 21b–23b.

2-Amino-N-(3⁰-cyanophenyl)benzamide (21b). Yield: 82%; m.p.: 158–160 ^◦C, (161–163 ^◦C [40]).

2-Amino-N-(4⁰-cyanophenyl)benzamide (22b). Yield: 64%; m.p.: 233–234 ^◦C, (231 ^◦C [40]).

2-Amino-N-(4⁰-cyanomethylphenyl)benzamide (23b). Yield: 61%; m.p.: 144–145 ^◦C; ¹H NMR

(400 MHz, DMSO-d₆)

J = 7.6 Hz, H₃), 7.18–7.22 (2H, m, H₄+ H₆), 7.30 (2H, d, J = 8.0 Hz, H₃₀+ H₅₀), 7.73 (2H, d, J = 8.0 Hz,

H₂₀+ H₆₀), 10.03 (1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

: 21.8 (CH₂), 114.6 (C₁), 115.0 (C₃),

N), 120.8 (C₂₀, C₆₀), 128.2 (C₃₀, C₅₀), 128.6 (C₄₀), 129.3 (C₆), 132.1 (C₄), 138.6 (C₁₀),

δ: 3.98 (2H, s, CH₂), 6.32 (2H, s, NH₂), 6.59 (1H, t, J = 6.8 Hz, H₅), 6.75 (1H, d,

δ

116.3 (C₅), 119.3 (C

≡

149.7 (C₂), 167.8 (C=O); DIP-MS (EI) m/z: 251 [M]⁺.

3.1.10. General Synthetic Procedures of 21c–23c

Compounds 21b–23b (1.48 mmol) were dissolved in anhydrous benzene (20 mL) and added with

triethylamine (1.48 mmol). To the stirred solution, thiophene-2-carbonyl chloride (1.85 mmol) was

added at room temperature and then reﬂuxed for 1 h. The following procedures were same as synthetic

procedures for 1–10. Compounds 21c–23c were recrystallized with ethyl acetate and n-hexane mixture

to give pure white or pale yellow compounds 21c–23c.

N-(3⁰-Cyanophenyl)-2-((thiophen-2⁰⁰-yl)carbonylamino)benzamide (21c). Yield: 98%; m.p.:

◦

1

208–209 C; H NMR (400 MHz, DMSO-d₆) δ: 7.25 (1H, dd, J = 5.0, 4.0 Hz, H₄₀₀), 7.32 (1H, td,

J = 7.6, 1.2 Hz, H₅), 7.56–7.65 (3H, m, H₄+ H₄₀+ H₆₀), 7.80 (1H, dd, J = 3.8, 0.8 Hz, H₅₀₀), 7.88–7.91 (2H,

m, H₃+ H₆), 7.99–8.02 (1H, m, H₅₀), 8.19 (1H, s, H₂₀), 8.24 (1H, dd, J = 8.2, 0.8 Hz, H₃₀₀), 10.80 (1H,

s, NHCO), 11.36 (1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ

: 111.2 (C₃₀), 118.5 (C N), 121.8

≡

(C₃), 123.3 (C₁), 124.5 (C₅), 125.2 (C₆₀), 127.3 (C₂₀), 128.2 (C₆), 128.7 (C₄₀₀), 128.8 (C₅₀₀), 129.9 (C₄₀), 132.0

(C₄), 132.1 (C₃₀₀), 137.6 (C₂₀₀), 139.2 (C₁₀), 139.3 (C₂), 159.5 (CONH), 167.5 (NHCO); DIP-MS (EI) m/z:

347 [M]⁺.

N-(4⁰-Cyanophenyl)-2-((thiophen-2⁰⁰-yl)carbonylamino)benzamide (22c). Yield: 69%; m.p.:

266–267 ^◦C; ¹H NMR (400 MHz, DMSO-d₆)

δ: 7.24 (1H, t, J = 4.0 Hz, H₄₀₀), 7.32 (1H, t, J = 7.6 Hz, H₅),

7.62 (1H, t, J = 8.0 Hz, H₄), 7.79 (1H, d, J = 3.2 Hz, H₅₀₀), 7.83 (2H, d, J = 8.0 Hz, H₃₀+ H₅₀), 7.87–7.89

(2H, m, H₃+ H₆), 7.94 (2H, d, J = 8.0 Hz, H₂₀+ H₆₀), 8.20 (1H, d, J = 8.0 Hz, H₃₀₀), 10.86 (1H, s, NHCO),

11.23 (1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ

: 105.6 (C₄₀), 119.0 (C N), 120.6 (C₃₀, C₅₀), 122.2

≡

(C₃), 123.7 (C₁), 124.2 (C₅), 128.3 (C₆), 128.9 (C₄₀₀), 129.1 (C₅₀₀), 132.2 (C₄), 132.3 (C₃₀₀), 133.1 (C₂₀, C₆₀),

137.7 (C₂00 ), 139.4 (C₂), 143.1 (C₁0 ), 159.5 (CONH), 167.6 (NHCO); DIP-MS (EI) m/z: 347 [M]⁺.

0

00

N-(4 -Cyanomethylphenyl)-2-((thiophen-2 -yl)carbonylamino)benzamide (23c). Yield: 88%; m.p.:

◦

221–222 C; ¹H NMR (400 MHz, DMSO-d₆)

δ: 4.02 (2H, s, CH₂), 7.25 (1H, dd, J = 5.0, 4.0 Hz, H₄₀₀), 7.29

(1H, dt, J = 7.7, 1.1 Hz, H₅), 7.36 (2H, d, J = 8.0 Hz, H₃₀+ H₅₀), 7.61 (1H, dt, J = 8.0, 1.2 Hz, H₄), 7.74

(2H, d, J = 8.0 Hz, H₂₀+ H₆₀), 7.75 (1H, d, J = 7.6 Hz, H₆), 7.89 (1H, d, J = 5.0 Hz, H₅₀₀), 7.93 (1H, dd,

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J = 7.8, 1.2 Hz, H₃), 8.35 (1H, dd, J = 8.2, 1.2 Hz, H₃₀₀), 10.60 (1H, s, NHCO), 11.66 (1H, s, CONH); ¹³

NMR (100 MHz, DMSO-d₆) : 21.9 (CH₂), 119.3 (C N), 121.4 (C₃), 121.5 (C₂₀, C₆₀), 122.8 (C₁), 123.4

C

δ

≡

(C₅), 126.9 (C₄₀), 128.4 (C₃₀, C₅₀), 128.8 (C₆), 129.0 (C₄₀₀), 129.0 (C₅₀₀), 132.2 (C₄), 132.3 (C₃₀₀), 137.9 (C₂₀₀),

138.3 (C₁), 139.5 (C₂), 159.4 (CONH), 167.3 (NHCO); DIP-MS (EI) m/z: 361 [M]⁺.

0

3.1.11. General Synthetic Procedures for 21d–23d

The nitriles 21c

mixture and added with triethylamine (2.59 mmol) and then reﬂuxed to dissolve. The reaction

mixture was added with hydroxylamine HCl (3.45 mmol) and reﬂuxed for 4–6 h. The ethanol and

–23c (0.86 mmol) were added in absolute ethanol (25 mL) and 1,4-dioxane (5 mL)

·

trimethylamine were removed under reduced pressure and the residue was poured with water to form

the precipitate. The obtained precipitate was ﬁltrated, washed with cold water to afford pure white or

pale yellow compounds 21d–23d.

N-(3⁰-Amidoximephenyl)-2-((thiophen-2⁰⁰-yl)carbonylamino)benzamide (21d). Yield: 94%; m.p.:

230–232 ^◦C; ¹H NMR (400 MHz, DMSO-d₆)

δ: 5.78 (2H, s, NH₂), 7.25 (1H, dd, J = 5.0, 4.0 Hz, H₄₀₀),

7.29 (1H, dt, J = 7.7, 1.1 Hz, H₅), 7.38 (1H, t, J = 8.0 Hz, H₅₀), 7.43 (1H, dt, J = 7.7, 1.4 Hz, H₄₀), 7.61 (1H,

dt, J = 7.6, 2.4 Hz, H₄), 7.68–7.71 (2H, m, H₆+ H₆₀), 7.89 (1H, dd, J = 5.0, 1.1 Hz, H₅₀₀), 7.95 (1H, dd,

J = 7.6, 1.2 Hz, H₃), 8.04 (1H, t, J = 1.7 Hz, H₂₀), 8.38 (1H, dd, J = 4.0, 0.8 Hz, H₃₀₀), 9.65 (1H, s, =NOH),

10.59 (1H, s, NHCO), 11.71 (1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ

: 116.6 (C₂₀), 122.8 (C₃),

123.4 (C₁), 124.6 (C₅), 124.9 (C₃₀), 129.2 (C₄₀₀), 129.3 (C₅₀₀), 129.8 (C₆), 132.1 (C₄), 132.8 (C₃₀₀), 134.8 (C₅₀),

138.1 (C₂₀₀), 138.7 (C₁₀), 139.3 (C₂), 159.1 (CONH), 167.7 (NHCO); FAB-MS m/z: 363 [M + 1

−

H₂O]⁺,

380 [M]⁺, 381 [M + 1]⁺, 403 [M + Na]⁺.

N-(4⁰-Amidoximephenyl)-2-(thiophen-2⁰⁰-ylcarbonylamino)benzamide (22d). Yield: 52%; m.p.:

241–243 ^◦C; ¹H NMR (400 MHz, DMSO-d₆)

δ: 5.40 (2H, s, NH₂), 7.20–7.29 (4H, m, H₅+ H₂₀+ H₆₀+

H₄₀₀), 7.56–7.64 (3H, m, H₄+ H₃₀+ H₅₀), 7.80 (1H, d, J = 7.6 Hz, H₆), 7.88 (1H, d, J = 4.0 Hz, H₅₀₀), 7.96

(1H, d, J = 7.6 Hz, H₃), 8.30 (1H, d, J = 8.4 Hz, H₃₀₀), 8.80 (1H, s, =NOH), 10.20 (1H, s, NHCO), 11.12 (1H,

s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ: 120.6 (C₂₀, C₆₀), 122.5 (C₃), 123.2 (C₁), 124.3 (C₅), 128.8

(C₃₀, C₅₀), 128.7 (C₆), 129.0 (C₄₀₀, C₅₀₀), 133.1 (C₄), 134.2 (C₃₀₀), 136.6 (C₁₀), 138.0 (C₂₀₀), 138.7 (C₂), 150.2

(C=NOH), 154.3 (CONH), 165.3 (NHCO); FAB-MS m/z: 363 [M + 1 − H₂O]⁺, 380 [M]⁺, 381 [M + 1]⁺.

N-(4⁰-Amidoximemethylphenyl)-2-(thiophen-2⁰⁰-ylcarbonylamino)benzamide (23d). Yield: 89%;

◦

m.p.: 209–211 C; ¹H NMR (400 MHz, DMSO-d₆)

δ: 3.26 (2H, s, CH₂), 5.43 (2H, s, NH₂), 7.19–7.27 (4H,

m, H₅+ H₃₀+ H₅₀+ H₄₀₀), 7.56–7.62 (3H, m, H₄+ H₂+ H₆₀), 7.73 (1H, d, J = 2.8 Hz, H₆), 7.89 (1H, d,

J = 4.0 Hz, H₅₀₀), 7.93 (1H, d, J = 7.6 Hz, H₃), 8.39 (1H, d, J = 8.4 Hz, H₃₀₀), 8.92 (1H, s, =NOH), 10.50

(1H, s, NHCO), 11.79 (1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ

: 36.7 (CH₂), 121.1 (C₂₀, C₆₀),

0

122.5 (C₃), 123.2 (C₁), 128.4 (C₄₀), 128.8 (C₃₀, C₅), 128.9 (C₆), 128.9 (C₄₀₀, C₅₀₀) 132.3 (C₄), 133.9 (C₃₀₀),

136.6 (C₁₀), 138.4 (C₂₀₀), 139.7 (C₂), 152.2 (C=NOH), 159.3 (CONH), 167.3 (NHCO); FAB-MS m/z: 377

[M + 1 − H₂O]⁺, 394 [M]⁺, 395 [M + 1]⁺, 417 [M + Na]⁺.

3.1.12. General Synthetic Procedures for 21–23

Amidoximes 21d–23d (0.80 mmol) were dissolved in anhydrous dichloromethane (15 mL) and

added with triethylamine (2.37 mmol) at room temperature and acetyl chloride (0.9 mmol) was added

◦

at 0 C. The mixture was reacted for 1–2 h at room temperature and ice water was added. The aqueous

layer was extracted with dichloromethane (3

saturated sodium bicarbonate and water. The organic layer was dried with anhydrous MgSO₄, ﬁltrated,

and concentrated in reduced pressure to give the acetyl amidoximes (21e 23e), which were used in

×

40 mL) and the organic phase was washed with water,

–

next reaction without puriﬁcation. The acetyl amidoximes (1.0 eq) was added to a suspension of 10%

Pd-C in absolute et_◦hanol (10 mL) and c-HCl (1.0 eq), and carried out catalytic hydrogenatin reaction

for 2 h at 60 psi, 45 C. The reaction mixture was ﬁltrated on celite pad and the ﬁltrate was evaporated

under reduced pressure to give a crude oily residue, which was puriﬁed to yield white compounds

21–23 by medium pressure liquid chromatography.

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N-(3⁰-Amidinophenyl)-2-((thiophen-2⁰⁰-yl)carbonylamino)benzamide

·

HCl (21). Yield: 32%; m.p.:

◦

262–264 C; ¹H NMR (400 MHz, DMSO-d₆)

δ: 7.24 (1H, dd, J = 5.0, 4.0 Hz, H₄₀₀), 7.33 (1H, t, J = 8.0 Hz,

H₅), 7.53 (1H, d, J = 8.0 Hz, H₄₀), 7.60–7.65 (2H, m, H₄+ H₅₀), 7.79 (1H, dd, J = 3.7, 0.8 Hz, H₆), 7.89

(1H, dd, J = 5.0, 0.8 Hz, H₅₀₀), 7.92 (1H, d, J = 8.0 Hz, H₆₀), 8.00 (1H, d, J = 8.3 Hz, H₃), 8.21 (1H, s,

H₂₀), 8.26 (1H, dd, J = 8.0, 0.8 Hz, H₃₀₀), 9.08 (2H, br s, hydrogens of amidinium chloride), 9.36 (2H,

br s, hydrogens of amidine

DMSO-d₆) : 120.0 (C₂₀), 122.0 (C₃), 123.5 (C₁), 123.7 (C₅), 125.7 (C₆₀), 128.3 (C₆), 128.9 (C₄₀₀), 129.0 (C₅₀₀),

129.5 (C₄₀), 132.3 (C₄), 132.4 (C₃₀₀), 137.9 (C₂₀₀), 139.2 (C₁₀), 139.5 (C₂), 159.4 (carbon of amidine), 166.0

(CONH), 167.6 (NHCO); FAB-MS m/z: 388 [M

Cl + Na]⁺, 365 [M Cl]⁺; HRMS (FAB): calcd for

C₁₉H₁₇N₄O₂S [M + H]⁺365.1072, found 365.1084.

N-(4⁰-Amidinophenyl)-2-((thiophen-2⁰⁰-yl)carbonylamino)benzamide

·

HCl), 10.86 (1H, s, NHCO), 11.45 (1H, s, CONH); ¹³C NMR (100 MHz,

δ

−

·

HCl (22). Yield: 51%; m.p.:

δ: 7.24 (1H, dd, J = 5.0, 4.0 Hz, H₄₀₀), 7.30 (1H, t, J = 8.0 Hz,

◦

211–213 C; ¹H NMR (400 MHz, DMSO-d₆)

H₅), 7.61 (1H, dt, J = 8.0, 1.2 Hz, H₄), 7.82 (1H, dd, J = 4.0, 1.2 Hz, H₆), 7.88 (2H, d, J = 8.0 Hz, H₃₀

H₅₀), 7.88 (1H, d, J = 4.0, 1.2 Hz, H₅₀₀), 7.94 (1H, dd, J = 8.0, 1.2 Hz, H₃), 7.97 (2H, d, J = 8.0 Hz, H₂₀

+

H₆₀), 8.18 (1H, dd, J = 8.0, 0.8 Hz, H₃₀₀), 9.36 (4H, br s, hydrogens of amidinium chloride), 10.77 (1H,

s, NHCO), 11.53 (1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ

: 120.2 (C₂₀, C₆₀), 122.4 (C₃), 122.5

(C₄₀), 123.6 (C₁), 124.4 (C₅), 128.3 (C₆), 128.9 (C₃₀, C₅₀, C₄₀₀), 129.2 (C₅₀₀), 132.1 (C₄), 132.2 (C₃₀₀), 137.9

(C₂₀₀), 139.7 (C₂), 143.9 (C₁₀), 159.6 (carbon of amidine), 164.9 (CONH), 167.5 (NHCO); FAB-MS m/z:

365 [M − Cl]⁺; HRMS (FAB): calcd for C₁₉H₁₇N₄O₂S [M + H]⁺365.1072, found 365.1089.

0

00

N-(4 -Amidinomethylphenyl)-2-((thiophen-2 -yl)carbonylamino)benzamide HCl (23). Yield: 42%;

·

m.p.: 172–174 C; ¹H NMR (400 MHz, DMSO-d₆)

δ: 3.69 (2H, s, CH₂), 7.24 (1H, dd, J = 5.0, 4.0 Hz, H₄₀₀),

◦

7.30 (1H, dt, J = 7.7, 1.1 Hz, H₅), 7.42 (2H, d, J = 8.0 Hz, H₃₀+ H₅₀), 7.61 (1H, dt, J = 8.0, 1.2 Hz, H₄),

7.72 (2H, d, J = 8.0 Hz, H₂₀+ H₆₀), 7.75 (1H, dd, J = 4.0, 1.2 Hz, H₆), 7.89 (1H, dd, J = 4.0, 1.2 Hz, H₅₀₀),

7.92 (1H, dd, J = 8.0, 1.2 Hz, H₆), 8.33 (1H, d, J = 8.0 Hz, H₃₀₀), 8.92 (4H, br s, hydrogens of amidinium

chloride), 10.61 (1H, s, NHCO), 11.64 (1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ: 121.4 (C₂₀,

C₆₀), 121.5 (C₃), 123.0 (C₁), 123.4 (C₅), 128.4 (C₆), 128.8 (C₄₀₀), 129.0 (C₅₀₀), 129.1 (C₃₀, C₅₀), 130.7 (C₄₀),

132.2 (C₄), 132.3 (C₃₀₀), 138.0 (C₂₀₀), 138.2 (C₁₀), 139.6 (C₂), 159.4 (carbon of amidine), 167.3 (CONH),

169.1 (NHCO); FAB-MS m/z: 379 [M

found 379.1243.

−

Cl]⁺; HRMS (FAB): calcd for C₂₀H₁₉N₄O₂S [M + H]⁺379.1229,

3.1.13. General Synthetic Procedures for 24b–26b

To a solution of 11a 13a (1.54 mmol) in ethanol (40 mL) was added with hydroxylamine

–

hydrochloride (300 mg, 4.60 mmol) and triethylamine (0.5 mL) at room temperature. The reaction

mixture was reﬂuxed for 12 h and the following procedures were same as synthetic procedure for

21b–23b. Compounds 24b–26b were recrystallized with dichloromethane and n-hexane mixture to

afford as a white powder, respectively.

N-(3⁰-Amidoximino)-2-(4"-methylphenylsulfonamido)benzamide (24b). Yield: 85%; m.p:

182–184 ^◦C; ¹H NMR (400 MHz, CDCl₃)

δ: 2.23 (3H, s, CH₃), 5.05 (2H, s, NH₂), 6.61 (1H, s, =NOH),

7.02–7.12 (4H, m, H₃+ H₄+ H₅+ H₅₀), 7.37 (2H, d, J = 8.0 Hz, H₃₀₀+ H₅₀₀), 7.50–7.60 (3H, m, H₆+

H₄₀+ H₆₀), 7.61 (2H, d, J = 8.0 Hz, H₂₀₀+ H₆₀₀), 7.85 (1H, s, H₂₀), 7.91 (1H, s, NHSO₂), 8.26 (1H, s,

CONH); ¹³C NMR (100 MHz, CDCl₃)

δ: 21.4 (CH₃), 118.3 (C₂₀), 121.9 (C₆₀), 122.2 (C₃), 122.5 (C₁), 124.0

(C₅), 124.1 (C₃₀), 127.2 (C₂₀₀, C₆₀₀), 129.4 (C₃₀₀, C₅₀₀), 129.7 (C₆), 132.9 (C₄), 133.1 (C₅₀), 136.2 (C₁₀₀), 137.5

(C₄₀₀), 138.6 (C₁₀), 143.9 (C₂), 158.8 (C=NOH), 166.8 (C=O); GC-MS (EI) m/z: 409 [M + 1-NH₂]⁺, 392

[M + 1 − NH₂OH]⁺.

N-(4⁰-Amidoximino)-2-(4"-methylphenylsulfonamido)benzamide (25b). Yield: 74%; m.p:

◦

1

213–215 C; H NMR (400 MHz, (CD₃)₂CO) δ: 2.26 (3H, s, CH₃), 6.60 (1H, s, =NOH), 7.19 (3H,

m, H₄+ H₃₀₀+ H₅₀₀), 7.54 (1H, t, J = 7.2 Hz, H₅), 7.62 (2H, d, J = 7.6 Hz, H₂₀₀+ H₆₀₀), 7.69 (1H, d,

J = 8.4 Hz, H₃), 7.75–7.88 (3H, m, H₆+ H₂₀+ H₆₀), 7.98 (2H, d, J = 8.4 Hz, H₃₀+ H₅₀), 9.72 (1H, s,

NHSO₂), 10.50 (1H, s, CONH); ¹³C NMR (100 MHz, (CD₃)₂CO)

δ

: 21.4 (CH₃), 120.9 (C₃), 122.9 (C1),

124.2 (C₅), 125.0 (C₄0 ), 128.0 (C₂0 , C₆0 ), 129.2 (C₃0 , C₅0 , C₂00 , C₆00 ), 130.6 (C₃00 , C₅00 ), 131.2 (C₆), 133.6 (C₄),

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137.3 (C₁₀₀), 139.5 (C₄₀₀), 142.0 (C₁₀), 144.8 (C₂), 168.2 (C=NOH), 168.3 (C=O); GC-MS (EI) m/z: 409 [M +

1-NH₂]⁺, 392 [M + 1 − NH₂OH]⁺.

N-(4⁰-Amidoximinobenzyl)-2-(4"-methylphenylsulfonamido)benzamide (26b). Yield: 63%; m.p:

70–74 ^◦C; ¹H NMR (400 MHz, DMSO-d₆)

δ: 2.29 (3H, s, CH₃), 3.52 (2H, s, CH₂), 7.22 (1H, t, J = 7.2 Hz,

H₄), 7.25 (2H, d, J = 7.6 Hz, H₃₀+ H₅₀), 7.26 (2H, d, J = 8.4 Hz, H₃₀₀+ H₅₀₀), 7.42–7.50 (2H, m, H₃+ H₅),

7.52 (2H, d, J = 7.6 Hz, H₂₀+ H₆₀), 7.61 (2H, d, J = 8.4 Hz, H₂₀₀+ H₆₀₀), 7.78 (1H, d, J = 8.0 Hz, H₆), 8.49

(2H, br s, NH₂), 9.50 (1H, br s, =NOH), 10.35 (1H, s, NHSO₂), 10.75 (1H, s, CONH); ¹³C NMR (100

MHz, DMSO-d₆) δ: 21.4 (CH₃), 120.9 (C₃), 122.9 (C₁), 124.2 (C₅), 125.0 (C₄₀), 128.0 (C₂₀, C₆₀), 129.2 (C₃₀,

C₅₀, C₂₀₀, C₆₀₀), 130.6 (C₃₀₀, C₅₀₀), 131.2 (C₆), 133.6 (C₄), 137.3 (C₁₀₀), 139.5 (C₄₀₀), 142.0 (C₁₀), 144.8 (C₂),

168.2 (C=NOH), 168.3 (C=O); GC-MS (EI) m/z: 423 [M + 1 − NH₂]⁺, 406 [M + 1 − NH₂OH]⁺.

3.1.14. General Experimental Procedures for 24–26

To a solution of amidoximes 24b

added triethylamine (1.18 mmol) and acetyl chloride (1.20 mmol) at 0 ^◦C. The following procedures

were same as synthetic procedures for acetylated amidoximes 21c 23c. To a suspention of 10% Pd-C

in absolute ethanol (50 mL) was added 21c 23c (1.12 mmol) and c-HCl (1.12 mmol) and the reaction

–26b (1.18 mmol) in anhydrous dichloromethane (10 mL) was

–

mixture was shaken on Paar hydrogenation apparatus for 12 h at 50 psi, 45 ^◦C and ﬁltrated on celite

pad. The ﬁltrate was evaporated under reduced pressure to provide a crude compound, which was

puriﬁed to give white compounds 24–26 by column chromatography.

N-(3⁰-Amidinophenyl)-2-(4"-methylphenylsulfonamido)benzamide

·

HCl (24). Yield: 37% m.p:

216–218 ^◦C; ¹H NMR (400 MHz, DMSO-d₆)

δ: 2.29 (3H, s, CH₃), 7.20–7.29 (3H, m, H₄+ H₃₀₀+ H₅₀₀),

7.39 (1H, d, J = 6.8 Hz, H₃), 7.55 (1H, t, J = 8.0 Hz, H₅), 7.58–7.68 (4H, m, H₃₀+ H₅₀+ H₂₀₀+ H₆₀₀),

7.86 (1H, d, J = 7.6 Hz, H₆), 7.92 (1H, d, J = 6.8 Hz, H₆₀), 8.24 (1H, s, H₂₀), 9.28 (2H, br s, hydrogens

of amidinium chloride), 9.46 (2H, s, hydrogens of amidinium chloride), 10.57 (1H, s, NHSO₂), 10.80

(1H, s, CONH); ¹³C NMR (100 MHz, CD₃OD)

δ: 20.2 (CH₃), 119.4 (C₂₀), 121.3 (C₃), 121.8 (C₁), 122.4

(C5), 123.0 (C₆₀), 124.2 (C₄₀), 125.8 (C₃₀), 126.8 (C₂₀₀, C₆₀₀), 126.9 (C₃₀₀, C₅₀₀), 129.1 (C₄), 129.2 (C₆), 129.8

(C₅₀), 130.6 (C₁₀), 132.3 (C₁₀₀), 139.9 (C₄₀₀), 142.9 (C₂), 167.3 (C=NH), 167.6 (C=O); DIP-MS (EI) m/z: 392

[M – HCl − NH₂]⁺; HRMS (FAB): calcd for C₂₁H₂₁N₄O₃S [M + H]⁺409.1334, found 409.1347.

N-(4⁰-Amidinophenyl)-2-(4"-methylphenylsulfonamido)benzamide

HCl (25). Yield: 43%; m.p:

·

250–252 ^◦C; ¹H NMR (400 MHz, DMSO-d₆)

δ: 2.27 (3H, s, CH₃), 7.23–7.32 (3H, m, H₄+ H₃₀₀+ H₅₀₀),

7.35 (1H, d, J = 8.0 Hz, H₃), 7.50 (1H, t, J = 8.4 Hz, H₅), 7.60 (2H, d, J = 8.0 Hz, H₂₀₀+ H₆₀₀), 7.78 (1H,

dd, J = 8.0, 1.2 Hz, H₆), 9.10 (2H, br s, hydrogens of amidinium chloride), 9.35 (2H, br s, hydrogens

of amidinium chloride), 10.34 (1H, s, NHSO₂), 10.73 (1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ

: 20.9 (CH₃), 120.1 (C₂₀, C₆₀), 122.2 (C₃), 122.6 (C₁), 124.5 (C₅), 125.4 (C₄₀), 126.8 (C₃₀, C₅₀), 128.3 (C₂₀₀

,

C₆₀₀), 129.3 (C₆), 129.5 (C₃₀₀, C₅₀₀), 132.4 (C₄), 136.0 (C₁₀₀), 136.7 (C₄₀₀), 143.5 (C₁₀), 143.6 (C₂), 164.8

(C=NH), 166.9 (C=O); DIP-MS (EI) m/z: 392 [M – HCl

−

NH₂]⁺; HRMS (FAB): calcd for C₂₁H₂₁N₄O₃S

[M + H]⁺409.1334, found 409.1348.

N-(4⁰-Amidinomethylphenyl)-2-(4"-methylphenylsulfonamido)benzamide

·

HCl (26). Yield: 33%;

m.p.: 172–173 ^◦C; ¹H NMR (400 MHz, DMSO-d₆)

δ: 2.28 (3H, s, CH₃), 3.67 (2H, s, CH₂), 7.23 (1H, t,

J = 8.0 Hz, H₄), 7.26 (2H, d, J = 8.0 Hz, H₃₀₀+ H₅₀₀), 7.41–7.53 (4H, m, H₃+ H₅+ H₃₀_,+ H₅₀), 7.60 (2H, d,

J = 8.4 Hz, H₂₀+ H₆₀), 7.66 (2H, d, J = 8.8 Hz, H₂₀₀+ H₆₀₀), 7.81 (1H, dd, J = 8.0, 1,2 Hz, H₆), 8.79 (2H, br

s, hydrogens of amidinium chloride), 9.28 (2H, br s, hydrogens of amidinium chloride), 10.44 (1H, s,

NHCO), 10.65 (1H, s, CONH); ¹³C NMR (100 MHz, DMSO-d₆)

δ: 20.9 (CH₃), 37.1 (CH₂), 121.2 (C₂₀,

C₆₀), 123.8 (C₃), 124.1 (C₁), 126.8 (C₃₀, C₅₀), 128.3 (C₆), 129.2 (C₂₀₀, C₆₀₀), 129.8 (C₃₀₀, C₅₀₀), 129.9 (C₄), 132.4

(C₄₀), 135.9 (C₁₀), 137.3 (C₁₀₀), 137.7 (C₄₀₀), 143.7 (C₂), 166.7 (C=NH), 169.2 (NHCO); DIP-MS (EI) m/z:

406 [M – HCl − NH₂]⁺; HRMS (FAB): calcd for C₂₂H₂₃N₄O₃S [M + H]⁺423.1491, found 423.1478.

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3.2. Biology

3.2.1. Reagents and Instruments

Heparin was purchased from Sigma (St. Louis, MO, USA) and TNF-

α

was purchased from

Abnova (Taipei, Taiwan). The molecular weight of heparin used as positive control in our experiments

is 3500. Anti-tissue factor (TF) antibody, rivaroxaban, and argatroban were purchased from Santa Cruz

Biotechnology, Inc. (Dallas, TX, USA). Thromboxane A₂(TXA₂) analog and U46619 were purchased

from Calbiochem-Novabiochem Corp. (San Diego, CA, USA). Prothrombin, thrombin, Factor VIIa,

X, and Xa were purchased from Haematologic Technologies (Essex Junction, VT, USA) and APTT-XL

and thromboplastin-D were purchased from Fisher Diagnostics (Middletown, VA, USA). S-2222 and

S-2238 were purchased as substrate of FXa and thrombin from ChromogenixAB (Mölndal, Sweden).

Aggregometer (Chronlog, Havertown, PA, USA), thrombotimer (Behnk Elektronik, Norderstedt,

Germany), microplate reader (Tecan Austria GmbH, Grödig, Austria), and spectrophotometer (TECAN,

Männedorf, Switzerland) were used.

3.2.2. Preparation of Plasma

Blood samples were collected in the morning from 10 healthy volunteers after fasting overnight

(aged between 24 and 28 years, four males and six females) without cardiovascular disorders,

allergy and lipid or carbohydrate metabolism disorders, untreated with drugs. All volunteers gave

written informed consent before participation. Human blood was collected into sodium citrate

(0.32% ﬁnal concentration) and immediately centrifuged (2000

The study protocol was approved by the Institutional Review Board of Kyungpook National University

Hospitals (Daegu, Republic of Korea).

×

g, 15 min) to prepare plasma.

3.2.3. In Vitro Anticoagulant Assay

According to the manufacturer’s instructions, as described previously [41], aPTT and PT of

compounds 5, 9, and 21–23 were measured by a Thrombotimer (Behnk Elektronik, Norderstedt,

Germany). The aPTT and PT results are expressed in seconds and PT results are also expressed

as INR. INR (International Normalized Rations) = (PT sample/PT control) ^ISI, ISI = international

sensitivity index.

3.2.4. In Vivo Bleeding Time

According to procedure reported by Dejana et al. [42], tail bleeding times were measured. Brieﬂy,

ICR mice were fasted overnight before experiments. One hour after intravenous administration of

5, 9,

and 21 23, tails of mice were transected at 2 mm from their tips. Bleeding time was deﬁned as the time

–

elapsed until bleeding stopped. When the bleeding time exceeded 10 min, in vivo bleeding time was

recorded as 10 min. All animals were treated in accordance with the guidelines for the Care and Use of

Laboratory Animals issued by Kyungpook National University.

3.2.5. Thrombin Activity Assay

According to procedure previously described [22], compounds 5, 9, and 21–23 were dissolved

in 50 nM Tris-HCl buffer (pH 7.4) containing 7.5 mM ethylenediaminetetraacetic acid (EDTA) and

added with 150 mM NaCl. After incubation at 37 ^◦C for 2 min, thrombin solution (150

µ

L; 10 U/mL)

◦

was added and incubated at 37 C for 1 min. S-2238 as substrate (150

µL; 1.5 mM) solution was

added and then absorbance was measured at 405 nm for 2 min using a spectrophotometer (TECAN,

Männedorf, Switzerland).

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3.2.6. Factor Xa Activity Assay

Factor Xa activity assay was carried out in the same method as the thrombin activity assay, but

using FXa (150 µL; 1 U/mL) and S-2222 as FXa substrate.

3.2.7. Cell Culture

Primary HUVECs were purchased from Cambrex Bio Science (Charles City, IA, USA) and

were maintained by a previously described method [43

conﬂuent at 37 C in 5% CO₂in EBM-2 basal media supplemented from Cambrex Bio Science

(Charles City, IA, USA).

,

44]. Brieﬂy, cells were cultured until

◦

3.2.8. Thrombin Generation on the Surfaces of Human Umbilical Vein Endothelial Cells (HUVECs)

The thrombin generation of HUVECs was quantitated as previously described [40]. Brieﬂy,

HUVECs were pre-incubated with synthesized compounds (300

buffer, 100 pM FVa, and 1 nM FXa for 10 min, and then added with prothrombin to a ﬁnal concentration

of 1 M. After 10 min, Each 10 L of triplicate samples was transferred to a 96-well plate containing

40 L of 0.5 M EDTA in Tris-buffered saline per well. Activated prothrombin was determined by

µL) dissolved in 50 mM Tris-HCl

µ

measuring the rate of hydrolysis of S2238 at 405 nm. Standard curves were prepared using amounts of

puriﬁed thrombin.

3.2.9. Factor Xa Generation on the Surfaces of HUVECs

The FXa generation of HUVECs was measured as previously described [41]. The amounts of FXa

generated by indicated concentrations of 5, 9, and 21–23 were measured at 405 nm over 2 min by a

microplate reader. Initial rates of color development were converted into FXa concentrations using a

standard curve prepared with known dilutions of puriﬁed human FXa.

3.2.10. Thrombin-Catalyzed Fibrin Polymerization

Thrombin-catalyzed polymerization was monitored turbidity at 360 nm every 6 s for 20 min by

using a spectrophotometer (TECAN, Männedorf, Switzerland) at ambient temperature. Both control

plasma and plasma incubated with compounds

5, 9, and 21–23 were diluted three times in TBS 50 mM

Tris-buffered saline and clotted with thrombin (ﬁnal concentration-0.5 U/mL). The maximum rate of

ﬁbrin polymerization (Vmax,

were performed in triplicate.

∆mOD/min) was recorded for each absorbance curve. All experiments

3.2.11. Platelet Aggregation

The platelet aggregation assay was measured following to the previously reported method [44].

Washed plasma was pre-incubated with the indicated concentration of compounds , and 21 23 for

3 min, and then stimulated with thrombin (0.1 U/mL, Sigma) and U46619 (2 M) in 0.9% saline for

5,

9

–

µ

5 min. Platelet aggregations were performed in an aggregometer (Chronolog, Havertown, PA, USA).

3.2.12. Cell Viability Assay

The colorimetric MTT was used for counting the number of live cells to determine cell viability.

Cells (5

washed with fresh medium, and treated with compounds

cells were washed and 100 L of 1 mg/mL MTT was added, followed by incubation for 4 h. Finally,

the resultant formazan salt was dissolved in 150 L DMSO and the absorbance intensity determinded

×

10³/well) were grown in a 96-well plate at 37 ^◦C and after incubation for 24 h, cells were

5, 9, and 21–23. After a 48 h incubation period,

µ

by a microplate reader (Tecan Austria GmbH, Grödig, Austria) with a reference wavelength of 540 nm

using. Data were expressed as means ± SEM at least three independent experiments.

Int. J. Mol. Sci. 2017, 18, 1144

25 of 27

3.2.13. Statistical Analysis

Data are expressed as means

signiﬁcance (p < 0.05) was determiced using the Student’s t-test.

±

SEM of at least three independent experiments. Statistical

4. Conclusions

Non-amidino N²-thiophenecarbonyl- and N²-tosylanthranilamides 20, and amidino N²-

1–

thiophenecarbonyl- and N²-tosylanthranilamides 21

–

26 synthesized and evaluated against PT and

aPTT in vitro. Compounds 5, 9, and 21–23 showed prolongation in aPTT in vitro and ex vivo, and

in vivo bleeding time. Compounds 5 and 21–23 exhibited prolongation in PT in vitro and ex vivo.

The activities of FXa and thrombin as well as the generation of thrombin and FXa in HUVECs

were dose-dependently inhibited but weak. These compounds inhibited thrombin-catalyzed ﬁbrin

polymerization and platelet aggregation induced by U46619. Any sulfonamide group containing

compounds 11–20, 24, and 26 did not exhibit prolongation of PT and aPTT, but 4-amidino derivative

25 showed high aPTT. The anticoagulant activity was governed to a great extent by an amidine group

at the meta or para position of phenyl ring and thiophenecarbonyl linker of 21–23.

Acknowledgments: This research was supported by Basic Science Research Program through the National

Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0013516)

and by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development

Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI15C0001).

Author Contributions: Soo Hyun Lee, Wonhwa Lee, ThiHa Nyugen, and Il Soo Um performed the research

and analyzed the data; Jong-Sup Bae and Eunsook Ma wrote the paper. All authors read and approved the

ﬁnal manuscript.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Synthesis and thrombin, factor Xa and U46619 inhibitory effects of non-amidino and amidino N2-thiophenecarbonyl- and N2-tosylanthranilamides

DOI: 10.3390/ijms18061144

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