Synthesis and anti-platelet evaluation of 2-benzoylaminobenzoate analogs

Article abstract of DOI:10.1016/j.bmc.2008.03.071

Fifty-two 2-benzoylaminobenzoate analogs were synthesized and subjected to anti-platelet aggregation assay using arachidonic acid (AA), collagen (Col), thrombin (Thr), and U46619 as inducers. The results revealed that most of 2-benzoylaminobenzoic acid derivatives showed a selectively inhibitory effect on AA-induced platelet aggregation. As a result of the 2-benzoylaminobenzoic acid derivatives (18, 44, and 46), there were no inhibitory effects on platelet aggregation induced by U46619, but these elicited an inhibitory effect on thromboxane B₂ formation at 1.0 μM. These 2-benzoylaminobenzoate analogs were therefore proposed as cyclooxygenase inhibitors.

Full text of DOI:10.1016/j.bmc.2008.03.071

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5803–5814
Synthesis and anti-platelet evaluation
of 2-benzoylaminobenzoate analogs
Pei-Wen Hsieh,a,* Shin-Zan Chiang,a Chin-Chung Wu,a Yi-Ching Lo,a,b
Yu-Tzu Shiha,b and Yang-Chang Wua,*
aGraduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, 100, Shih-Chuan 1st Road,
Kaohsiung 807, Taiwan
bDepartment of Pharmacology, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
Received 28 January 2008; revised 21 March 2008; accepted 22 March 2008
Available online 31 March 2008
Abstract—Fifty-two 2-benzoylaminobenzoate analogs were synthesized and subjected to anti-platelet aggregation assay using
arachidonic acid (AA), collagen (Col), thrombin (Thr), and U46619 as inducers. The results revealed that most of 2-benzoylamino-
benzoic acid derivatives showed a selectively inhibitory effect on AA-induced platelet aggregation. As a result of the 2-benzoylami-
nobenzoic acid derivatives (18, 44, and 46), there were no inhibitory effects on platelet aggregation induced by U46619, but these
elicited an inhibitory effect on thromboxane B2 formation at 1.0 lM. These 2-benzoylaminobenzoate analogs were therefore
proposed as cyclooxygenase inhibitors.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
inhibitory effects on arachidonic acid (AA)-induced
platelet aggregation.2,3 We have designed and synthe-
sized new substituted 2-benzoylaminobenzoate analogs
in an attempt to obtain new anti-platelet aggregation
agents. In particular, the synthetic structures that pro-
cess in the (1) substitutions at the C-4 position; (2)
length of carbon chain of the ester/amide at C-1; (3)
straight-chain and branched-chain of the ester/amide
at C-1; and (4) 20- or 20,60-disubstitution in the B ring4
were tested. Newly synthesized compounds were assayed
for in vitro inhibitory effects on platelet aggregation in-
duced by arachidonic acid (AA), collagen (Col), throm-
bin (Thr), and U46619 (Tables 1–4). To investigate the
pharmacologic mechanism of 2-benzoylaminobenzoate
analogs, the most potent compounds, including deriva-
tives 18, 44, and 46, were further assayed to test the
inhibitory effect on thromboxane (Tx) B2 formation.
We describe the synthesis, bioactivity data, and struc-
ture–activity relationship (SAR) of anti-platelet aggre-
gation related to 2-benzoylaminobenzoate analogs.
Arterial thromboembotic diseases such as acute coro-
nary syndrome and ischemic stroke are caused by plate-
let aggregation, and are the major causes of death in the
developed countries. Anti-platelet drugs (e.g., aspirin,
ticlopidine) are used to protect against myocardial
infarction, stroke, cardiovascular death, and other seri-
ous vascular events in patients with a history of previous
vascular events or known risk factors for cardiovascular
disease.1 Current anti-platelet drugs have some restric-
tions in their mode of action and efficacy, and research
and development of new generation anti-platelet agents
continue.
We previously synthesized a series of derivatives of 2-
benzoylaminobenzoic acid; we investigated their anti-
platelet aggregation, as well as inhibition of superoxide
anion generation and neutrophil elastase release of neu-
trophils.2,3 The results showed that most 2-benzoylami-
no-4-chlorobenzoic acid derivatives showed selective
2. Results and discussion
2.1. Chemistry
Keywords: 2-Benzoylaminobenzoate analogs; Anti-platelet aggrega-
tion; Cyclooxygenase; Structure–activity relationships (SAR).
*
Corresponding authors. Tel.: +886 7 3121101x2653; fax: +886 7
3114773 (P.-W.H.); tel.: +886 7 3121101x2197; fax: +886 7 3114773
(Y.-C.W); e-mail addresses: pewehs@kmu.edu.tw; yachwu@kmu.
edu.tw
Scheme 1 describes the synthesis of 52 new 2-ben-
zoylaminobenzoate analogs (12–63) via an accomplished
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.03.071

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P.-W. Hsieh et al. / Bioorg. Med. Chem. 16 (2008) 5803–5814
O
fects on AA-induced platelet aggregation (ca. 200-fold)
than aspirin, with IC50 values of 0.7, 0.9, 0.7, 0.9, and
0.5 lM, respectively. Compounds 18, 44, 45, 46, 60,
61, and 62 showed more potent inhibitory effects on
Col-induced platelet aggregation, with IC50 values of
2.3, 1.6, 1.2, 1.0, 2.2, 1.1, and 1.0 lM, respectively.
OH
R1
NH2
Anti-platelet aggregation assay data and SAR analysis
confirmed that activity depended on the substituents
on 20- or 20,60-disubstitution in the B ring, the length
of carbon chain of ester/amide at C-1, as well as the
straight-chain and branched-chain of the ester/amide
at C-1. Conversely, in our previous research,2,3 we pro-
posed that a chloro-substituent at C-4 is required for the
anti-platelet activity of 2-benzoylaminobenzoic acids.
However, changing the chloro-substituent into its isos-
teres4 (e.g., fluoro- and methyl groups) or replacement
by an electron-donating (nitro) group were slightly en-
hanced the anti-platelet aggregation activity (Table 1).
Most ethyl and propyl ester derivatives (12–21) were
more active toward anti-platelet aggregation with AA
as inducers than the corresponding isopropyl (52–54) es-
ters (Table 1). Anti-platelet aggregation activities van-
ished when the isopropyl ester was substituted at C-1,
indicating that the size of R2 and R3 enhanced bioactiv-
ity. Additionally, 20,60-difluoro derivatives (44–47) were
more potent than the 20-fluoro-substituted (12,17, and
22) and 20,60-dichloro derivatives (48–51; Table 1).5
However, the monosubstituted (20-Cl) derivatives were
more potent than the 20,60-dichloro derivatives (48–51;
Table 1), suggesting that the bulky group substituted
at 20 and 60 blocks the activity. We therefore propose
that the amide between A and B rings is required for
anti-platelet aggregation. Furthermore, amide analogs
enhanced the selectivity (AA vs Col) of platelet aggrega-
tion inhibitory effects (Tables 1–3), indicating that the
amide at the C-1 position was necessary for inhibiting
platelet aggregation induced by collagen.
a, b
a, c
O
O
OR2
NR3
R1
NH2
R1
O
NH2
+
10-11
1-9
Cl
R4
d
OR2
NH
NR3
O
O
R1
R1
NH
O
O
R4
R4
55-63
12-54
Scheme 1. The straightforward synthesis of 52 new 2-ben-
zoylaminobenzoate analogs (12–63). Reagents: (a) SO2Cl, DCM; (b)
R2OH; (c) R3NH2; (d) DCM.
2-Benzoylaminobenzoic acids inhibited AA-, but not
U46619-induced platelet aggregation, this suggests that
2-benzoylaminobenzoic acids inhibit AA conversion to
TxA2 rather than blocking the TxA2 receptor. There-
fore, compounds 18, 44, and 46 were further examined
for their inhibitory effect on AA-induced TxB2 forma-
tion (Table 4).6 The results showed compounds 18, 44,
and 46 inhibited TxB2 formation at 1.0 lM, suggesting
that 2-benzoylaminobenzoic acid analogs caused anti-
platelet aggregation effect via arachidonic pathway.
previously described pathway.2,3 Initially, 4-substituted
benzoic ester/amide derivatives 1–11 were obtained by
a simple method. These derivatives were then added
with the corresponding substituted benzoyl chlorides.2,3
The mixtures were stirred at room temperature for 16 h
to give compounds 12–63. The products (22–63) were
new compounds and fully characterized using spectro-
scopic data as shown in Section 4.
2.2. Anti-platelet activity
3. Conclusion
All synthesized 2-benzoylaminobenzoic acid analogs
(12–63) were subjected to anti-platelet aggregation assay
with AA, Col, Thr, and U46619 (a thromboxane recep-
tor inhibitor) as inducers. The results showed that all
synthesized agents did not produce an inhibitory effect
on platelet aggregation assay when Thr and U46619
were used as inducers. Tables 1–3 list the IC50 values ob-
tained with these compounds, as well as the positive
controls, aspirin, and indomethacin. Compounds 18,
34, 44, 45, and 46 exhibited more potent inhibitory ef-
The bioassay results of these compounds show dramatic
relationships between structure and anti-platelet aggre-
gation effects. Compounds 18, 44, and 46 exhibited
inhibitory effects 200-times more potent than aspirin
on platelet aggregation and TxB2 formation induced
by AA. These data suggest that the anti-platelet aggre-
gation of 2-benzoyl-4-chloroaminobenzoic acid analogs
via inhibiting arachidonic pathway. Additionally, these
could be developed as new lead compounds for anti-
platelet aggregation agents.

P.-W. Hsieh et al. / Bioorg. Med. Chem. 16 (2008) 5803–5814
5805
Table 1. Anti-platelet aggregation potencies of ethyl, propyl, butyl, and iso-propyl ester derivatives of 2-benzoylamino-4-chlorobenzoate
OR2
6
5
4
O
1
A
3
2
Cl
NH
6'
B
3'
1'
5'
O
R4
2'
4'
a,b
Anti-platelet aggregation, IC50 (lM)
Compound
Substituents
R4
R2
AA (150 lM)
Thr (0.1 U/ml)
Col (10 lg/ml)
U46619
Aspirinc
149.8 0.7a,b
0.12 0.0
3.8 1.1
1.0 0.2
3.6 0.0
4.9 1.1
4.9 1.1
1.6 0.0
0.7 0.2
3.5 0.0
2.2 0.5
23.4 6.2
3.0 0.6
2.1 0.5
7.1 0.1
4.7 1.0
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
48.8 14.3
>100
>100
>100
>100
>100
>100
>100
>100
153.2 12.0a,b
0.4 0.0
20.1 6.8
7.6 0.5
13.9 1.3
15.9 0.7
46.4 0.5
11.6 1.6
2.3 1.2
9.5 1.8
8.3 0.2
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Indomethacinc
12d
13d
14d
15d
16d
17d
18d
19d
20d
21d
22
Ethyl
Ethyl
20-F
20-Cl
20-Br
20-CH3
Ethyl
Ethyl
Ethyl
20-OCH3
20-F
20-Cl
20-Br
20-CH3
20-OCH3
20-F
20-Cl
20-Br
20-CH3
20-OCH3
20,60-di-F
20,60-di-F
20,60-di-F
20,60-di-F
20,60-di-Cl
20,60-di-Cl
20,60-di-Cl
20,60-di-Cl
20-F
Propyl
Propyl
Propyl
Propyl
Propyl
Butyl
32.1 2.1
19.5 3.8
50.4 16.7
76.2 11.7
>100
23
24
Butyl
Butyl
25
26
Butyl
Butyl
44
45
Methyl
Ethyl
0.7 0.2
0.9 0.2
0.5 0.1
1.5 0.0
12.6 2.1
68.3 14.3
38.4 12.7
>100
35.8 4.7
1.6 0.1
1.2 0.2
21.8 6.4
3.4 0.8
32.3 0.5
29.7 2.1
>100
46
47
Propyl
Butyl
48
49
Methyl
Ethyl
50
51
Propyl
Butyl
52
53
iso-Propyl
iso-Propyl
iso-Propyl
>100
82.2 8.9
>100
36.7 0.6
8.51 0.5
28.8 1.4
20-Cl
20,60-di-F
54
a Platelets were pre-incubated with DMSO (0.5%, control) or test compounds at 37 ꢁC for 3 min before the addition of the inducers.
b The IC50 values are presented as means SEM (n = 3).
c Aspirin and indomethacin, two cyclooxygenase inhibitors, were used as positive controls in platelet aggregation assay.
d The biological data of compounds 12–21 were reported in the literature.1.
4. Experimental
reaction mixture was stirred and at reflux for
8.0 h. The solvent was evaporated at reduced pres-
sure. The residue was purified by flash column chro-
matography (Si-Gel) using n-hexane to afford the
products.
4.1. General
The NMR spectra using C5D5N and CDCl3 as solvents
were obtained on a Varian NMR spectrometer (Unity
Plus 400 and Unity INOVA-500). Chemical shifts were
internally referenced to the solvent signals in CDCl3
(1H, d 7.26; 13C: d 77.0). Low-resolution EI-MS were re-
corded on a Quattro GC/MS spectrometer having a di-
rect inlet system, high-resolution ESI-MS spectra on a
Bruker Daltonics APEX II 30e spectrometer.
4.3. General procedure for the synthesis of compounds 22–
63
To compounds 1–11 (1.0 mmol) dissolved in DCM
were added the suitable benzoyl chlorides. The reac-
tion mixture was stirred at room temperature for
16 h. The solvent was evaporated at reduced pressure.
The residue was purified by column chromatography
(Si-Gel) using CHCl3/hexane (2:7) mixture to afford
the products.
4.2. General procedure for the synthesis of compounds 1–11
To a DCM solution of compounds 1–11 (1.0 mmol)
were added the suitable alcohols or amines. The

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P.-W. Hsieh et al. / Bioorg. Med. Chem. 16 (2008) 5803–5814
Table 2. Anti-platelet aggregation effects of amide derivatives of 2-benzoylamino-4-chlorobenzoate
NR3
6
5
4
O
1
A
3
2
Cl
NH
6'
B
3'
1'
5'
O
R4
2'
4'
a,b
Anti-platelet aggregation, IC50 (lM)
Compound
Substituents
R4
R3
AA (150 lM)
Thr (0.1 U/ml)
Col (10 lg/ml)
U46619
Aspirinc
149.8 0.7a,b
0.12 0.0
1.9 0.4
3.4 1.2
1.5 0.6
8.4 2.5
6.0 1.0
2.9 0.4
3.5 0.1
3.4 0.0
3.5 0.0
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
153.2 12.0a,b
0.4 0.0
8.7 1.0
4.1 0.1
3.4 2.1
10.3 0.8
11.1 2.5
1.0 0.2
2.2 0.2
1.1 0.1
1.0 0.2
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Indomethacinc
55
56
57
58
59
60
61
62
63
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Butyl
20-F
20-Cl
20-Br
20-CH3
20-OCH3
20,60-di-F
20-F
Butyl
Butyl
20-Cl
20,60-di-F
a Platelets were pre-incubated with DMSO (0.5%, control) or test compounds at 37 ꢁC for 3 min before the addition of the inducers.
b The IC50 values are presented as means SEM (n = 3).
c Aspirin and indomethacin, two cyclooxygenase inhibitors, were used as positive controls in platelet aggregation assay.
4.4. Butyl 4-chloro-2-(2-fluorobenzamido)benzoate (22)
60), 7.44 (1H, dd, J = 8.0, 1.6 Hz, H-30), 7.39 (2H, m, H-
40 50), 7.09 (1H, dd, J = 8.8, 2.0 Hz, H-5), 4.27 (2H, t,
J = 6.8 Hz, OCH2CH2CH2CH3), 1.71 (2H, m, OCH2
CH2CH2CH3), 1.42 (2H, m, OCH2CH2CH2CH3), 0.94
(3H, t, J = 7.6 Hz, OCH2CH2CH2CH3); 13C NMR
(CDCl3) d 167.43 (s, COOR), 165.35 (s, CONH), 141.87
(s, C-2), 140.75 (s, C-4), 135.61 (s, C-10), 131.81 (d, C-6),
131.51 (d, C-40), 131.17 (s, C-20), 130.54 (d, C-30),
129.08 (d, C-60), 127.05 (d, C-50), 123.14 (d, C-5), 120.29
(d, C-3), 113.79 (s, C-1), 65.47 (t, OCH2CH2CH2CH3),
30.34 (t, OCH2CH2CH2CH3), 19.07 (t, OCH2CH2CH2
CH3), 13.57 (q, OCH2CH2CH2CH3); HRESI-MS m/z
388.0485[M+Na]+ (calcdforC18H17Cl2NO3Na388.0483).
Yield (62%) from 3 and 2-fluorobenzoyl chlorides; EI-
MS (m/z, %): 351(8), 349(25) [M]+, 250(23), 249(63),
1
124(74), 123(100); H NMR (CDCl3) d 11.96 (1H, br,
NH), 9.01 (1H, d, J = 2.0 Hz, H-3), 8.05 (1H, td,
J = 8.0, 2.0 Hz, H-60), 7.99 (1H, d, J = 8.8 Hz, H-6),
7.51 (1H, m, H-40), 7.28 (1H, td, J = 8.0, 1.2 Hz, H-
50), 7.19 (1H, ddd, J = 10.4, 8.0, 1.2 Hz, H-30), 7.09
(1H, dd, J = 8.8, 2.0 Hz, H-5), 4.33 (2H, t, J = 6.4 Hz,
OCH2CH2CH2CH3), 1.75 (2H, m, OCH2CH2CH2CH3),
1.47 (2H, m, OCH2CH2CH2CH3), 0.97 (3H, t,
J = 7.2 Hz, OCH2CH2CH2CH3); 13C NMR (CDCl3) d
167.27 (s, COOR), 162.37 (s, CONH, JC–F = 2.3 Hz),
160.17 (s, C-20, JC–F = 249.3 Hz), 141.96 (s, C-2),
140.56 (s, C-4), 133.72 (d, C-40, JC–F = 9.1 Hz), 131.84
(d, C-6), 131.66 (d, C-60, JC–F = 1.9 Hz), 124.71 (d,
C-50, JC–F = 3.5 Hz), 123.12 (d, C-5), 122.22 (s, C-10,
JC–F = 12.2 Hz), 121.09 (d, C-3), 116.43 (d, C-30,
JC–F = 23.5 Hz), 114.37 (s, C-1), 65.46 (t, OCH2CH2
CH2CH3), 30.46 (t, OCH2CH2CH2CH3), 19.16 (t,
OCH2CH2CH2CH3), 13.65 (q, OCH2CH2CH2CH3);
HRESI-MS m/z 372.0777 [M+Na]+ (calcd for
C18H17ClFNO3Na 372.0779).
4.6. Butyl 4-chloro-2-(2-bromobenzamido)benzoate (24)
Yield (44%) from 3 and 2-bromobenzoyl chloride; EI-
MS (m/z, %): 411 [M]+(11), 230(38), 185(100), 184(35),
1
183(46); H NMR (CDCl3) d 11.56 (1H, s, NH), 8.98
(1H, d, J = 2.4 Hz, H-3), 7.97 (1H, d, J = 8.8 Hz, H-6),
7.62 (1H, dd, J = 8.0, 1.2 Hz, H-30), 7.57 (1H, dd,
J = 8.0, 1.6 Hz, H-60), 7.39 (1H, td, J = 8.0, 1.2 Hz, H-
50), 7.30 (1H, td, J = 8.0, 1.6 Hz, H-40), 7.08 (1H, dd,
J = 8.8, 2.4 Hz, H-5), 4.26 (2H, t, J = 6.8 Hz,
OCH2CH2CH2CH3), 1.70 (2H, m, OCH2CH2CH2CH3),
1.42 (2H, m, OCH2CH2CH2CH3), 0.93 (3H, t,
J = 7.6 Hz, OCH2CH2CH2CH3); 13C NMR (CDCl3) d
167.41(s, COOR), 166.15 (s, CONH), 141.83 (s, C-2),
140.74 (s, C-4), 137.75 (s, C-10), 133.70 (d, C-30),
131.80 (d, C-6), 131.53 (d, C-40), 128.74 (d, C-60),
127.57 (d, C-50), 123.15 (d, C-5), 120.22 (d, C-3),
119.58 (s, C-20), 113.74 (s, C-1), 65.46 (t, OCH2CH2
4.5. Butyl 4-chloro-2-(2-chlorobenzamido)benzoate (23)
Yield (52%) from 3 and 2-chlorobenzoyl chloride; EI-
MS (m/z, %): 365(4) [M]+, 351(7), 348(11), 266(11),
1
264(14), 141(54), 139(100); H NMR (CDCl3) d 11.63
(1H, br, NH), 8.99 (1H, d, J = 2.0 Hz, H-3), 7.98 (1H,
d, J = 8.8 Hz, H-6), 7.63 (1H, dd, J = 7.6, 2.0 Hz, H-

P.-W. Hsieh et al. / Bioorg. Med. Chem. 16 (2008) 5803–5814
5807
Table 3. Replacement of the 4-substitution in 2-benzoylamino-4-chlorobenzoate analogs and their anti-platelet aggregation effects
OR2
6
5
4
O
1
A
3
2
R1
NH
6'
B
3'
1'
5'
O
R4
2'
4'
a,b
Anti-platelet aggregation, IC50 (lM)
Compound
Substituents
R1
R2
R4
AA (150 lM)
Thr (0.1 U/ml)
Col (10 lg/ml)
Aspirinc
149.8 0.7a,b
0.12 0.0
1.6 0.5
1.6 0.6
3.3 0.1
2.0 0.5
31.5 5.3
4.1 1.4
21.1 5.3
0.9 0.2
1.6 0.4
3.1 0.2
5.8 1.3
10.4 2.6
4.7 1.1
2.0 0.5
2.5 0.7
4.7 1.0
>100
>100
>100
>100
>100
>100
>100
>100
87.8 3.5
77.0 1.0
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
153.2 12.0a,b
0.4 0.0
14.5 2.3
9.3 2.2
16.7 2.9
25.1 4.1
33.2 3.9
11.0 2.7
36.1 8.8
11.3 3.0
7.8 0.4
27.3 1.7
20.1 4.1
62.5 15.1
3.5 0.5
2.9 0.6
4.9 1.3
8.0 2.2
>100
Indomethacinc
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
F
Propyl
Propyl
Propyl
Propyl
Propyl
Ethyl
20-F
F
20-Cl
20-Br
20-CH3
F
F
F
20-OCH3
20-Cl
20-CH3
20-F
20-Cl
20-Br
20-CH3
20-OCH3
20-F
20-Cl
20-Br
20-CH3
20-OCH3
F
F
Ethyl
CH3
CH3
CH3
CH3
CH3
NO2
NO2
NO2
NO2
NO2
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
a Platelets were pre-incubated with DMSO (0.5%, control) or test compounds at 37 ꢁC for 3 min before addition of the inducers.
b The IC50 values are presented as means SEM (n = 3).
c Aspirin and indomethacin, two cyclooxygenase inhibitors, were used as positive controls in platelet aggregation assay.
HRESI-MS m/z 431.9976 [M+Na]+ (calcd for
C18H17BrClNO3Na 431.9978).
Table 4. Effects of Compounds 18, 44, and 46 on the TxB2 formations
in washed human platelets caused by arachidonic acid
Treatment
TxB2 (ng/3 · 108 platelets)
AA (150 lM)
4.7. Butyl 4-chloro-2-(2-methylbenzamido)benzoate (25)
Resting
0.09 0.01
58.68 1.39
DMSO (control)
Aspirin 200 lM
Indomethacin 0.5 lM
18 1 lM
18 0.5 lM
44 1 lM
Yield (67%) from 3 and 2-methylbenzoyl chloride; EI-
MS (m/z, %): 345 [M]+ (7), 228(11), 118(100), 119(96);
1H NMR (CDCl3) d 11.61 (1H, br, NH), 9.03 (1H, d,
J = 2.0 Hz, H-3), 7.97 (1H, d, J = 8.4 Hz, H-6), 7.61
(1H, dd, J = 0.8, 7.6 Hz, H-60), 7.36 (1H, td, J = 0.8,
7.2 Hz, H-40), 7.27 (2H, m, H-50 30), 7.06 (1H, dd,
J = 8.4, 2.0 Hz, H-5), 4.28 (2H, t, J = 6.8 Hz,
OCH2CH2CH2CH3), 2.56 (3H, s, CH3), 1.72 (2H, m,
OCH2CH2CH2CH3), 1.44 (2H, m, OCH2CH2CH2CH3),
0.96 (3H, t, J = 7.6 Hz, OCH2CH2CH2CH3); 13C NMR
(CDCl3) d 168.11 (s, COOR), 167.50 (s, CONH), 142.43
(s, C-2), 140.66 (s, C-4), 137.13 (s, C-20), 135.61 (s, C-10),
131.74 (d, C-6), 131.42 (d, C-40), 130.49 (d, C-30), 126.85
(d, C-60), 125.95 (d, C-50), 122.63 (d, C-5), 119.92 (d, C-
3), 113.45 (s, C-1), 65.34 (t, OCH2CH2CH2CH3), 30.32
(t, OCH2CH2CH2CH3), 20.16 (q, CH3), 19.05 (t,
OCH2CH2CH2CH3), 13.54 (q, OCH2CH2CH2CH3);
HRESI-MS m/z 368.1031 [M+Na]+ (calcd for
C19H20ClNO3Na 368.1029).
26.68 1.56***
3.09 0.90***
32.52 2.09***
62.56 1.05**
29.48 4.46***
61.18 3.76
44 0.5 lM
46 1 lM
46 0.5 lM
7.74 1.97***
45.47 16.21
Washed human platelets were pre-incubated with DMSO or test
compounds at 37 ꢁC for 3 min and then arachidonic acid (200 lM) was
added. The reactions were terminated by EDTA (2 mM) and indo-
methacin (50 lM) 5 min after the addition of AA. The platelet sus-
pensions were centrifuged for 3 min at 13,000 rpm, the thromboxane
B2 in the supernatants was assayed using enzyme immunoassay kits.
Values are presented as means SEM (n = 3). **P < 0.01, ***P < 0.001
as compared with the respective controls.
CH2CH3), 30.31 (t, OCH2CH2CH2CH3), 19.05 (t,
OCH2CH2CH2CH3), 13.56 (q, OCH2CH2CH2CH3);

5808
P.-W. Hsieh et al. / Bioorg. Med. Chem. 16 (2008) 5803–5814
4.8. Butyl 4-chloro-2-(2-methoxybenzamido)benzoate (26)
21.84 (t, OCH2CH2CH3), 10.36 (q, OCH2CH2CH3);
HRESI-MS m/z 336.0800 [M+H]+ (calcd for
C17H16ClFNO3 336.0803).
Yield (54%) from 3 and 2-methoxybenzoyl chloride; EI-
MS (m/z, %): 347 [M]+ (6), 229(21), 227(62), 137(54),
1
135(100); H NMR (CDCl3) d 12.31 (1H, br, NH), 9.09
4.11. Propyl 4-fluoro-2-(2-bromobenzamido)benzoate (29)
(1H, d, J = 2.0 Hz, H-3), 8.17 (1H, dd, J = 7.6, 1.6 Hz,
H-60), 7.92 (1H, d, J = 8.4 Hz, H-6), 7.44 (1H, m, H-40),
7.03 (2H, m, H-5, 50), 6.97 (1H, d, J = 8.0 Hz, H-30),
4.29 (2H, t, J = 6.8 Hz, OCH2CH2 CH2CH3), 4.02 (3H,
s, OCH3), 1.73 (2H, m, OCH2CH2CH2CH3), 1.46 (2H,
m, OCH2CH2CH2CH3), 0.97 (3H, t, J = 7.2 Hz,
OCH2CH2CH2CH3); 13C NMR (CDCl3) d 166.48 (s,
COOR), 164.45 (s, CONH), 157.43 (s, C-20), 142.14 (s,
C-2), 139.89 (s, C-4), 133.26 (d, C-60, JC–F = 9.1 Hz),
132.20 (d, C-40), 131.58 (d, C-6), 122.37 (d, C-5), 121.98
(s, C-10), 121.44 (d, C-3), 120.71 (d, C-50), 114.92 (s, C-
1), 111.14 (d, C-30), 64.84 (t, OCH2CH2CH2CH3), 55.27
(q, OCH3), 30.47 (t, OCH2CH2CH2CH3), 19.11 (t,
OCH2CH2CH2CH3), 13.59 (q, OCH2CH2CH2CH3);
HRESI-MS m/z 384.0979 [M+Na]+ (calcd for
C19H20ClNO4Na 384.0978).
Yield (32%) from 4 and 2-bromobenzoyl chloride; EI-MS
(m/z, %): 380(8), 379 [M]+(8), 216(17), 214(54), 185(100),
184(51), 183(38); 1H NMR (CDCl3) d 11.67 (1H, br, NH),
8.72 (1H, dd, J = 2.4, 11.6 Hz, H-3), 8.10 (1H, dd, J = 6.4,
8.8 Hz, H-6), 7.65 (1H, dd, J = 7.6, 0.8 Hz, H-30), 7.59
(1H, dd, J = 7.6, 1.6 Hz, H-60), 7.41 (1H, td, J = 7.6,
0.8 Hz, H-50), 7.32 (1H, td, J = 7.6, 1.6 Hz, H-40), 6.84
(1H, m, H-5), 4.24 (2H, t, J = 6.4 Hz, OCH2CH2CH3),
1.77 (2H, m, OCH2CH2CH3), 1.00 (3H, t, J = 7.2 Hz,
OCH2CH2CH3); 13C NMR (CDCl3) d 167.49 (s, COOR),
166.42 (s, CONH), 166.17 (s, C-4, JC–F = 252.4 Hz),
143.21 (s, C-2, JC–F = 13.0 Hz), 137.93 (s, C-10), 133.80
(d, C-30), 131.11 (d, C-6, JC–F = 10.6 Hz), 131.60 (d, C-
40), 128.86 (d, C-60), 127.64 (d, C-50), 119.65 (s, C-20),
111.81 (s, C-1), 110.30 (d, C-5, JC–F = 22.0 Hz), 107.73
(d, C-3, JC–F = 28.0 Hz), 67.08 (t, OCH2CH2CH3),
21.83 (t, OCH2CH2CH3), 10.36 (q, OCH2CH2CH3);
HRESI-MS m/z 380.0297 [M+H]+ (calcd for
C17H16BrFNO3 380.0298).
4.9. Propyl 4-fluoro-2-(2-fluorobenzamido)benzoate (27)
Yield (69%) from 4 and 2-fluorobenzoyl chloride; EI-MS
(m/z, %): 320(19), 319 [M]+ (61), 233(23), 232(82), 125(30),
1
123(100); H NMR (CDCl3) d 12.06 (1H, br, NH), 8.74
4.12. Propyl 4-fluoro-2-(2-methylbenzamido)benzoate
(30)
(1H, dd, J = 12.0, 2.4 Hz, H-3), 8.09 (1H, dd, J = 8.8,
6.4 Hz, H-6), 8.04 (1H, td, J = 7.6, 1.6 Hz,, H-60), 7.51
(1H, m, H-40), 7.28 (1H, t, J = 8.0 Hz, H-50), 7.19 (1H,
dd, J = 11.2, 8.4 Hz, H-30), 6.82 (1H, m, H-5), 4.29 (2H,
t, J = 6.8 Hz, OCH2CH2CH3), 1.79 (2H, m, OCH2CH2
CH3), 1.02 (3H, t, J = 7.2 Hz, OCH2CH2CH3); 13C
NMR (CDCl3) d 167.22 (s, COOR), 166.01 (s, C-4,
JC–F = 250.9 Hz), 162.54 (s, CONH), 160.17 (s, C-20,
JC–F = 249.4 Hz), 143.26 (s, C-2, JC–F = 12.9 Hz),
133.70 (d, C-40, JC–F = 9.1 Hz), 132.54 (d, C-6,
JC–F = 10.6 Hz), 131.64 (d, C-60, JC–F = 3.2 Hz), 124.69
Yield (46%) from 4 and 2-methylbenzoyl chloride; EI-
MS (m/z, %): 315 [M]+ (7), 119(100), 118(84), 91(40);
1H NMR (CDCl3) d 11.68 (1H, br, NH), 8.75 (1H, dd,
J = 12.0, 2.0 Hz, H-3), 8.10 (1H, dd, J = 6.4, 8.8 Hz,
H-6), 7.60 (1 H, dd, J = 1.2, 7.6 Hz, H-60), 7.38 (1H,
td, J = 1.2, 7.2 Hz, H-40), 7.29 (2H, m, H-50 30), 6.82
(1H, m, H-5), 4.25 (2H, t, J = 6.8 Hz, OCH2CH2CH3),
2.56 (3H, s, CH3), 1.78 (2H, m, OCH2CH2CH3), 1.02
(3H, t, J = 7.2 Hz, OCH2CH2CH3); 13C NMR (CDCl3)
d 168.50 (s, COOR), 167.65 (s, CONH), 166.25 (s, C-4,
JC–F = 251.5 Hz), 143.86 (s, C-2,JC–F = 13.6 Hz), 137.18
(d, C-50, JC–F = 3.8 Hz), 122.29 (s, C-10, JC–F
=
12.1 Hz), 116.43 (d, C-30, JC–F = 23.5 Hz), 112.35 (s, C-
1), 110.14 (d, C-5, JC–F = 22.0 Hz), 108.36 (d, C-3,
JC–F = 28.1 Hz), 66.99 (t, OCH2CH2CH3), 21.87 (t,
OCH2CH2CH3), 10.37 (q, OCH2CH2CH3); HRESI-MS
m/z 320.1097 [M+H]+ (calcd for C17H16F2NO3 320.1098).
(s, C-20), 135.89 (s, C-10), 131.11 (d, C-6, JC–F
=
10.6 Hz), 131.53 (d, C-40), 130.61 (d, C-30), 127.00 (d,
C-60), 126.10 (d, C-50), 111.59 (s, C-1, JC–F = 3.0 Hz),
109.89 (d, C-5, JC–F = 22.0 Hz), 107.44 (d, C-3, JC–F
=
28.0 Hz), 67.02 (t, OCH2CH2CH3), 21.89 (t, OCH2CH2
CH3), 20.24 (q, CH3), 10.42 (q, OCH2CH2CH3); HRES-
I-MS m/z 338.1168 [M+Na]+ (calcd for C18H18FNO3Na
338.1168).
4.10. Propyl 4-fluoro-2-(2-chlorobenzamido)benzoate (28)
Yield (40%) from 4 and 2-chlorobenzoyl chloride; EI-
MS (m/z, %): 337(22), 335 [M]+ (61), 248(45), 214(29),
140(100); 1H NMR (CDCl3) d 11.73 (1H, br, NH),
8.73 (1H, dd, J = 2.4, 11.6 Hz, H-3), 8.11 (1H, dd,
J = 6.4, 8.8 Hz, H-6), 7.65 (1H, dd, J = 7.2, 2.0 Hz, H-
30), 7.47 (1H, dd, J = 8.0, 1.6 Hz, H-60), 7.40 (2H, m,
H- 50 40), 6.84 (1H, m, H-5), 4.25 (2H, t, J = 6.8 Hz,
OCH2 CH2CH3), 1.78 (2H, m, OCH2 CH2CH3), 1.01
(3H, t, J = 7.2 Hz, OCH2 CH2CH3); 13C NMR (CDCl3)
d167.49 (s, COOR), 166.60 (s, CONH), 165.19 (s, C-4,
JC–F = 252.4 Hz), 143.25 (s, C-2, JC–F = 12.9 Hz),
135.78 (s, C-10), 131.09 (d, C-6, JC–F = 11.4 Hz),
131.56 (d, C-40), 131.24 (s, C-20), 130.62 (d, C-30),
129.19 (d, C-60), 127.11 (d, C-50), 111.85 (s, C-1,
JC–F = 3.0 Hz), 110.27 (d, C-5, JC–F = 22.0 Hz), 107.80
(d, C-3, JC–F = 28.0 Hz), 67.06 (t, OCH2CH2CH3),
4.13. Propyl 4-fluoro-2-(2-methoxybenzamido)benzoate
(31)
Yield (43%) from 4 and 2-methoxybenzoyl chloride; EI-
MS (m/z, %): 331 [M]+ (5), 198(13), 197(86), 137(44),
135(100); 1H NMR (CDCl3) d 12.40 (1H, br, NH),
8.83 (1H, dd, J = 12.8, 2.8 Hz, H-3), 8.17 (1H, dd,
J = 7.6, 1.6 Hz, H-60), 8.07 (1H, dd, J = 8.8, 6.8 Hz,
H-6), 7.49 (1H,,m, H-40), 7.08 (1H, td, J = 7.6, 0.8 Hz,
H-30), 7.02 (1H, d, J = 8.4 Hz, H-50), 6.79 (1H, m, H-
5), 4.28 (2H, t, J = 6.4 Hz, OCH2CH2CH3), 4.06 (3H,
s, OCH3), 1.80 (2H, m, OCH2CH2CH3), 1.04 (3H, t,
J = 7.6 Hz, OCH2CH2CH3); 13C NMR (CDCl3) d
166.70 (s, COOR), 165.89 (s, C-4, JC–F = 250.1 Hz),

P.-W. Hsieh et al. / Bioorg. Med. Chem. 16 (2008) 5803–5814
5809
164.78 (s, CONH), 157.54 (s, C-20), 143.56 (s, C-2,
JC–F = 13.0 Hz), 133.34 (d, C-40), 132.83 (d, C-6,
JC–F = 10.6 Hz), 132.35 (d, C-60), 122.32 (s, C-10),
120.92 (d, C-50), 112.92 (s, C-1, JC–F = 3.1 Hz), 111.29
(d, C-30), 109.60 (d, C-5, JC–F = 21.9 Hz), 108.82 (d, C-
3, JC–F = 28.0 Hz), 66.57 (t, OCH2CH2CH3), 55.43 (q,
OCH3), 21.98 (t, OCH2CH2CH3), 10.53 (q, OCH2CH2
CH3); HRESI-MS m/z 354.1120 [M+Na]+ (calcd for
C18H18FNO4Na 354.1118).
OCH2CH2CH3), 1.02 (3H, t, J = 7.2 Hz, OCH2CH2
CH3); 13C NMR (CDCl3) d 167.96 (s, COOR), 162.37
(s, CONH, JC–F = 2.2 Hz), 160.25 (s, C-20, JC–F
=
249.3 Hz), 145.46 (s, C-4), 141.01 (s, C-2), 133.36 (d,
C-40, JC–F = 9.1 Hz), 131.51 (d, C-60, JC–F = 2.3 Hz),
130.76 (d, C-6), 124.62 (d, C-50, JC–F = 3.8 Hz), 123.94
(d, C-5), 122.83 (s, C-10, JC–F = 12.2 Hz), 121.57 (d, C-
3), 116.42 (d, C-30, JC–F = 23.5 Hz), 113.65 (s, C-1),
66.70 (t, OCH2CH2CH3), 22.08 (q, CH3), 21.91 (t,
OCH2CH2CH3), 10.44 (q, OCH2CH2CH3); HRESI-
MS m/z 338.1169 [M+Na]+ (calcd for C18H18FNO3Na
338.1168).
4.14. Ethyl 4-fluoro-2-(2-chlorobenzamido)benzoate (32)
Yield (41%) from 5 and 2-chlorobenzoyl chloride; EI-
MS (m/z, %): 321 [M]+ (6), 141(37), 139(100), 111(54);
1H NMR (CDCl3) d 11.73 (1H, br, NH), 8.72 (1H, dd,
J = 2.4, 12.0 Hz, H-3), 8.10 (1H, dd, J = 6.4, 8.8 Hz,
H-6), 7.64 (1H, dd, J = 7.2, 1.6 Hz, H-30), 7.46 (1H,
dd, J = 7.6, 1.2 Hz, H-60), 7.39 (2H, m, H- 50 40), 6.83
(1H, m, H-5), 4.34 (2H, q, J = 6.8 Hz, OCH2CH3),
1.37 (3H, t, J = 6.8 Hz, OCH2CH3); 13C NMR (CDCl3)
d 167.73 (s, COOR), 165.89 (s, CONH), 166.46 (s, C-4,
JC–F = 251.9 Hz), 143.52 (s, C-2, JC–F = 12.9 Hz), 136.04
(s, C-10), 133.47 (d, C-6, JC–F = 10.0 Hz), 131.88 (d,
C-40), 131.53 (s, C-20), 130.92 (d, C-30), 129.48 (d,
C-60), 127.42 (d, C-50), 112.10 (s, C-1, JC–F = 2.6 Hz),
4.17. Propyl 2-(2-chlorobenzamido)-4-methylbenzoate
(35)
Yield (43%) from 6 and 2-chlorobenzoyl chloride; EI-MS
(m/z, %): 333(15), 331(45) [M]+, 244(24), 141(74),
1
139(100), 111(41); H NMR (CDCl3) d 11.57 (1H, br,
NH), 8.75 (1H, s, H-3), 7.97 (1H, d, J = 8.0 Hz, H-6),
7.64 (1H, dd, J = 7.2, 2.0 Hz, H-60), 7.47 (1H, dd,
J = 7.6, 1.6 Hz, H-30), 7.38 (2H, m, H- 40 50), 6.96 (1H,
d, J = 8.0 Hz, H-5), 4.23 (2H, t, J = 6.4 Hz, OCH2
CH2CH3), 2.45 (3H, s, CH3), 1.77 (2H, m, OCH2CH2
CH3), 1.01 (3H, t, J = 7.6 Hz, OCH2CH2CH3); 13C
NMR (CDCl3) d 168.26 (s, COOR), 166.52 (s, CONH),
145.89 (s, C-4), 141.11 (s, C-2), 136.29 (s, C-10), 131.31
(d, C-6, s, C-20), 130.79 (d, C-40), 130.59 (d, C-30),
129.08 (d, C-60), 127.09 (d, C-50), 124.02 (d, C-5), 120.88
(d, C-3), 113.09 (s, C-1), 66.79 (t, OCH2CH2CH3), 22.14
(q, CH3), 21.91 (t, OCH2CH2CH3), 10.45 (q, OCH2
CH2CH3); HRESI-MS m/z 354.0871 [M+Na]+ (calcd
for C18H18ClNO3Na 354.0873).
110.55 (d, C-5, JC–F = 22.0 Hz), 108.02 (d, C-3, JC–F
=
28.4 Hz), 61.86 (t, OCH2CH3), 14.38 (q, OCH2CH3);
HRESI-MS m/z 344.0464 [M+Na]+ (calcd for
C16H13ClFNO3Na 344.0466).
4.15. Ethyl 4-fluoro-2-(2-methylbenzamido)benzoate (33)
Yield (44%) from 5 and 2-methylbenzoyl chloride; EI-
MS (m/z, %): 301 [M]+ (10), 119(100), 118(23), 91(61);
1H NMR (CDCl3) d 11.67 (1H, br, NH), 8.75 (1H, dd,
J = 12.0, 2.8 Hz, H-3), 8.11 (1H, dd, J = 9.2, 6.4 Hz,
H-6), 7.61 (1H, dd, J = 1.2, 7.6 Hz, H-60), 7.38 (1H,
td, J = 1.2, 7.2 Hz, H-40), 7.30 (2H, m, H-50 30), 6.82
(1H, m, H-5), 4.35 (2H, q, J = 7.2 Hz, OCH2CH2CH3),
2.56 (3H, s, CH3), 1.39 (3H, t, J = 7.2 Hz,
OCH2CH2CH3); 13C NMR (CDCl3) d 168.54 (s,
4.18. Propyl 2-(2-bromobenzamido)-4-methylbenzoate
(36)
Yield (33%) from 6 and 2-bromobenzoyl chloride; EI-MS
(m/z, %): 377(11), 375(11) [M]+, 185(64), 183(64), 157(16),
1
155(16); H NMR (CDCl3) d 11.50 (1H, br, NH), 8.75
(1H, s, H-3), 7.97 (1H, d, J = 8.0 Hz, H-6), 7.65 (1H, dd,
J = 8.0, 1.2 Hz, H-30), 7.59 (1H, dd, J = 8.0, 1.6 Hz, H-
60), 7.41 (1H, td, J = 8.0, 1.2 Hz, H-50), 7.32 (1H, td,
J = 8.0, 1.6 Hz, H-40), 7.11 (1H, dd, J = 8.0, 1.2 Hz, H-
5), 4.23 (2H, t, J = 6.8 Hz, OCH2CH2CH3), 2.45 (3H, s,
CH3), 1.77 (2H, m, OCH2CH2CH3), 1.01 (3H, t,
J = 7.2 Hz, OCH2CH2CH3); 13C NMR (CDCl3) d
168.27 (s, COOR), 166.35 (s, CONH), 145.91 (s, C-4),
141.11 (s, C-2), 138.49 (s, C-10), 133.77 (d, C-30), 131.37
(d, C-6), 130.80 (d, C-40), 128.83 (d, C-60), 127.64 (d, C-
50), 124.05 (d, C-5), 120.87 (d, C-3), 119.78 (s, C-20),
113.11 (s, C-1), 66.81 (t, OCH2CH2CH3), 21.12 (q,
CH3), 21.91 (t, OCH2CH2CH3), 10.44 (q, OCH2CH2
CH3); HRESI-MS m/z 398.0370 [M+Na]+ (calcd for
C18H18BrNO3Na 398.0368).
COOR), 167.62 (s, CONH), 166.27 (s, C-4, JC–F
=
251.6 Hz), 143.87 (s, C-2,JC–F = 12.9 Hz), 137.18 (s, C-
20), 135.92 (s, C-10), 133.19 (d, C-6, JC–F = 10.6 Hz),
131.55 (d, C-40), 130.63 (d, C-30), 127.02 (d, C-60),
126.11 (d, C-50), 111.59 (s, C-1, JC–F = 3.0 Hz), 109.91
(d, C-5, JC–F = 22.0 Hz), 107.47 (d, C-3, JC–F = 28.0
Hz), 61.51 (t, OCH2CH3), 20.26 (q, CH3), 14.15 (q,
OCH2CH3); HRESI-MS m/z 324.1009 [M+Na]+ (calcd
for C17H16FNO3Na 324.1012).
4.16. Propyl 2-(2-fluorobenzamido)-4-methylbenzoate
(34)
Yield (46%) from 6 and 2-fluorobenzoyl chloride; EI-
MS (m/z, %): 315 [M]+ (33), 229(19), 228(92), 124(62),
123(100); 1H NMR (CDCl3) d 11.90 (1H, br, NH),
8.75 (1H, d, J = 1.2 Hz, H-3), 8.05 (1H, td, J = 8.0,
1.2 Hz,, H-60), 7.97 (1H, d, J = 8.0 Hz, H-6), 7.50 (1H,
m, H-40), 7.28 (1H, td, J = 8.0, 1.2 Hz, H-50), 7.19
(1H, ddd, J = 11.6, 8.4,1.2 Hz, H-30), 6.95 (1H, dd,
J = 8.0, 1.2 Hz, H-5), 4.28 (2H, t, J = 6.4 Hz,
OCH2CH2CH3), 2.43 (3H, s, CH3), 1.79 (2H, m,
4.19. Propyl 2-(2-methylbenzamido)-4-methylbenzoate
(37)
Yield (40%) from 6 and 2-methylbenzoyl chloride; EI-MS
(m/z, %): 311 [M]+ (9), 119(100), 118(46), 91(74); 1H NMR
(CDCl3) d 11.52 (1H, br, NH), 8.76 (1H, s, H-3), 7.97 (1H,
d, J = 8.0 Hz, H-6), 7.61 (1H, dd, J = 0.8, 7.6 Hz, H-60),

5810
P.-W. Hsieh et al. / Bioorg. Med. Chem. 16 (2008) 5803–5814
7.37 (1H, td, J = 0.8, 7.6 Hz, H-40), 7.28 (2H, m, H-50 30),
6.94 (1H, dd, J = 8.0, 0.8 Hz, H-5), 4.23 (2H, t,
J = 6.8 Hz, OCH2CH2CH3), 2.56 (3H, s, CH3), 2.45
(3H, s, CH3), 1.78 (2H, m, OCH2CH2CH3), 1.01 (3H, t,
J = 7.6 Hz, OCH2CH2CH3); 13C NMR (CDCl3) d
168.46 (s, COOR), 168.33 (s, CONH), 145.79 (s, C-4),
141.67 (s, C-2), 136.96 (s, C-20), 136.48 (s, C-10), 131.41
(d, C-6), 130.79 (d, C-40), 130.30 (d, C-30), 127.02 (d, C-
60), 126.04 (d, C-50), 123.63 (d, C-5), 120.61 (d, C-3),
112.90 (s, C-1), 66.71 (t, OCH2CH2CH3), 22.13 (q,
CH3), 21.92 (t, OCH2CH2CH3), 20.22 (q, CH3), 10.45
(q, OCH2CH2CH3); HRESI-MS m/z 334.1419 [M+Na]+
(calcd for C19H21NO3Na 334.1419).
J = 2.4 Hz, H-3), 8.25 (1H, d, J = 8.8 Hz, H-6), 7.94
(1H, dd, J = 8.8, 2.4 Hz, H-60), 7.68 (1H, dd, J = 7.2,
1.6 Hz, H-30), 7.48 (1H, td, J = 8.0, 1.6 Hz, H-50), 7.42
(2H, m, H-40 5), 4.32 (2H, t, J = 6.4 Hz, OCH2CH2CH3),
1.82 (2H, m, OCH2CH2CH3), 1.03 (3H, t, J = 7.6 Hz,
OCH2CH2CH3); 13C NMR (CDCl3) d 166.74 (s, COOR),
165.59 (s, CONH), 151.17 (s, C-4), 141.98 (s, C-2), 135.23
(s, C-10), 131.98 (d, C-30), 131.94 (d, C-40), 131.23 (s, C-20),
130.73 (d, C-6), 129.46 (d, C-60), 127.25 (d, C-50), 120.06
(s, C-1), 117.09 (d, C-5), 115.55 (d, C-3), 67.99 (t,
OCH2CH2CH3), 21.80 (t, OCH2CH2CH3), 10.38 (q,
OCH2CH2CH3); HRESI-MS m/z 363.0746 [M+H]+
(calcd for C17H16ClN2O5363.0748).
4.20. Propyl 2-(2-methoxybenzamido)-4-methyl-benzoate
(38)
4.23. Propyl 2-(2-bromobenzamido)-4-nitrolbenzoate (41)
Yield (45%) from 7 and 2-bromobenzoyl chloride; EI-MS
(m/z, %): 408(7), 406 [M]+ (7), 321(15), 319(15), 185(100),
184(92); H NMR (CDCl3) d 11.56 (1H, br, NH), 9.78
Yield (38%) from 6 and 2-methoxybenzoyl chloride; EI-
MS (m/z, %): 327 [M]+ (7), 193(10), 135(100), 92(33); 1H
NMR (CDCl3) d 12.20 (1H, br, NH), 8.80 (1H, d,
J = 0.8 Hz, H-3), 8.17 (1H, dd, J = 8.0, 1.2 Hz, H-60),
7.94 (1H, d, J = 8.0 Hz, H-6), 7.48 (1H, m, H-40), 7.08
(2H, td, J = 8.0, 1.2 Hz, H-50), 7.02 (2H, d, J = 8.0 Hz,
H-5), 6.92 (1H, dd, J = 1.2, 8.4 Hz, H-30), 4.26 (2H, t,
J = 6.8 Hz, OCH2CH2CH3), 4.06 (3H, s, OCH3), 2.43
(3H, s, CH3), 1.79 (2H, m, OCH2CH2CH3), 1.04 (3H,
t, J = 7.2 Hz, OCH2CH2CH3); 13C NMR (CDCl3) d
167.46 (s, COOR), 164.59 (s, CONH), 157.52 (s, C-20),
144.96 (s, C-4), 141.18 (s, C-2), 133.00 (d, C-6), 132.19
(d, C-40), 130.61 (d, C-60), 123.48 (d, C-5), 122.93 (s,
C-10), 122.21 (d, C-3), 120.89 (d, C-50), 114.37 (s, C-1),
111.31 (d, C-30), 66.32 (t, OCH2CH2CH3), 55.49 (q,
OCH3), 22.11 (q, CH3), 22.03 (t, OCH2CH2CH3),
10.56 (q, OCH2CH2CH3); HRESI-MS m/z 350.1369
[M+Na]+ (calcd for C19H21NO4Na 350.1368).
1
(1H, d, J = 2.4 Hz, H-3), 8.25 (1H, d, J = 8.8 Hz, H-6),
7.95 (1H, dd, J = 8.8, 2.4 Hz, H-5), 7.68 (1H, dd,
J = 7.6, 1.2 Hz, H-30), 7.62 (1H, dd, J = 7.6, 2.0 Hz, H-
60), 7.44 (1H, td, J = 7.6, 1.2 Hz, H-50), 7.36 (1H, td,
J = 7.6, 2.0 Hz, H-40), 4.32 (2H, t, J = 6.8 Hz,
OCH2CH2CH3), 1.82 (2H, m, OCH2CH2CH3), 1.03
(3H, t, J = 7.6 Hz, OCH2CH2CH3); 13C NMR (CDCl3)
d 166.77 (s, COOR), 166.45 (s, CONH), 151.25 (s, C-4),
142.00 (s, C-2), 137.53 (s, C-10), 133.95 (d, C-30), 131.99
(d, C-40), 131.95 (d, C-6), 129.11 (d, C-60), 127.78 (d, C-
50), 120.06 (s, C-1), 119.65 (s, C-20), 117.13 (d, C-5),
115.55 (d, C-3), 68.02 (t, OCH2CH2CH3), 21.81 (t,
OCH2CH2CH3), 10.39 (q, OCH2CH2CH3); HRESI-MS
m/z 407.0244 [M+H]+ (calcd for C17H16BrN2
O5407.0243).
4.24. Propyl 2-(2-methylbenzamido)-4-nitrolbenzoate (42)
4.21. Propyl 2-(2-fluorobenzamido)-4-nitrolbenzoate (39)
Yield (74%) from 7 and 2-methylbenzoyl chloride; EI-MS
(m/z, %): 342 [M]+ (5), 119(100), 118(88), 92(65); 1H NMR
(CDCl3) d 11.59 (1H, br, NH), 9.80 (1H, d, J = 2.4 Hz, H-
3), 8.25 (1H, dd, J = 0.4, 8.8 Hz, H-6), 7.91 (1H, dd,
J = 8.8, 2.4 Hz, H-5), 7.63 (1H, dd, J = 1.2, 8.8 Hz, H-
60), 7.41 (1H, td, J = 1.2, 7.2 Hz, H-40), 7.31 (2H, m, H-
50 30), 4.32 (2H, t, J = 6.8 Hz, OCH2CH2CH3), 2.57
(3H, s, CH3), 1.82 (2H, m, OCH2CH2CH3), 1.04 (3H, t,
J = 7.2 Hz, OCH2CH2CH3); 13C NMR (CDCl3) d
168.34(s, COOR), 166.91 (s, CONH), 151.20 (s, C-4),
142.61 (s, C-2), 137.56 (s, C-20), 135.26 (s, C-10), 131.94
(d, C-40), 131.70 (d, C-6), 130.94 (d, C-30), 127.00 (d, C-
60), 126.16 (d, C-50), 119.73 (s, C-1), 116.59 (d, C-5),
115.24 (d, C-3), 67.91 (t, OCH2CH2CH3), 21.80 (t,
OCH2CH2CH3), 20.32 (q, CH3), 10.38 (q, OCH2CH2
CH3); HRESI-MS m/z 365.1111 [M+Na]+ (calcd for
C18H18N2O5Na 365.1113).
Yield (42%) from 7 and 2-fluorobenzoyl chloride; EI-MS
(m/z, %): 346(12) [M]+, 259 (8), 135(16), 123(100), 95(68);
1H NMR (CDCl3) d 11.99 (1H, br, NH), 9.80 (1H, d,
J = 2.4 Hz, H-3), 8.24 (1H, d, J = 8.8 Hz, H-6), 8.10
(1H, td, J = 8.0, 1.6 Hz,, H-60), 7.92 (1H, dd, J = 8.8,
2.4 Hz, H-5), 7.55 (1H, m, H-40), 7.31 (1H, td, J = 8.0,
0.8 Hz, H-50), 7.20 (1H, dd, J = 10.8, 8.0 Hz, H-30), 4.36
(2H, t, J = 6.4 Hz, OCH2CH2CH3), 1.84 (2H, m,
OCH2CH2CH3), 1.04 (3H, t, J = 7.2 Hz, OCH2
CH2CH3); 13C NMR (CDCl3) d 166.43 (s, COOR),
162.48 (s, CONH, JC–F = 2.3 Hz), 160.28 (s, C-20,
JC–F = 248.6 Hz), 150.95 (s, C-4), 141.96 (s, C-2), 134.20
(d, C-40, JC–F = 9.1 Hz), 131.94 (d, C-60, JC–F = 2.3 Hz),
131.90 (d, C-6), 124.90 (d, C-50, JC–F = 3.8 Hz), 121.55
(s, C-10, JC–F = 11.4 Hz), 120.66 (s, C-1), 116.99 (d, C-
5), 116.47 (d, C-30, JC–F = 23.5 Hz), 116.24 (d, C-3),
67.89 (t, OCH2CH2CH3), 21.81 (t, OCH2CH2CH3),
10.38 (q, OCH2CH2CH3); HRESI-MS m/z 369.0861
[M+Na]+ (calcd for C17H15FN2O5Na 369.0863).
4.25. Propyl 2-(2-methoxybenzamido)-4-nitrolbenzoate
(43)
4.22. Propyl 2-(2-chlorobenzamido)-4-nitrolbenzoate (40)
Yield (38%) from 7 and 2-methoxybenzoyl chloride; EI-
MS (m/z, %): 358 [M]+ (2), 224(72), 136(11), 135(100),
92(6); H NMR (CDCl3) d 12.39 (1H, br, NH), 9.87
(1H, d, J = 2.4 Hz, H-3), 8.23 (1H, dd, J = 2.0, 8.0 Hz,
H-60), 8.17 (1H, d, J = 8.8 Hz, H-6), 7.86 (1H, dd,
1
Yield (86%) from 7 and 2-chlorobenzoyl chloride; EI-MS
(m/z, %): 362(1) [M]+, 275(14), 141(35), 139(100); 1H
NMR (CDCl3) d 11.65 (1H, br, NH), 9.78 (1H, d,

P.-W. Hsieh et al. / Bioorg. Med. Chem. 16 (2008) 5803–5814
5811
J = 2.4, 8.8 Hz, H-5), 7.51 (1H,m, H-40), 7.05 (1H, td,
J = 0.8, 8.0 Hz, H-50), 7.03 (1H, d, J = 8.4 Hz, H-30),
4.34 (2H, t, J = 6.4 Hz, OCH2CH2CH3), 4.09 (3H, s,
OCH3), 1.84 (2H, m, OCH2CH2CH3), 1.06 (3H, t,
J = 7.2 Hz, OCH2CH2CH3); 13C NMR (CDCl3) d
165.95 (s, COOR), 164.72 (s, CONH), 157.65 (s, C-20),
50.81 (s, C-4), 142.21 (s, C-2), 133.79 (d, C-40), 132.64
(d, C-6), 131.61 (d, C-60), 121.56 (s, C-1), 121.40 (s, C-
10), 121.10 (d, C-50), 116.88 (d, C-5), 116.42 (d, C-3),
111.33 (d, C-30), 67.42 (t, OCH2CH2CH3), 55.50 (q,
OCH3), 21.91 (t, OCH2CH2CH3), 10.50 (q,
OCH2CH2CH3); HRESI-MS m/z 381.1061 [M+Na]+
(calcd for C18H18N2O6Na 381.1062).
OCH2CH2CH3), 1.01 (3H, t, J = 7.6 Hz, OCH2CH2
CH3); 13C NMR (CDCl3) d 167.58 (s, COOR), 159.98
(s, C-20, JC–F = 251.6 Hz), 159.92 (s, C-60, JC–F
=
251.6 Hz), 159.01 (s, CONH), 141.71 (s, C-2), 140.96 (s,
C-4), 132.25 (d, C-40, JC–F = 10.3 Hz), 131.86 (d, C-6),
123.49 (d, C-5), 120.57 (d, C-3), 113.84 (s, C-1), 112.26
(d, C-50, JC–F = 20.5 Hz), 112.21 (d, C-30, JC–F
=
19.7 Hz), 112.11 (s, C-10), 67.27 (t, OCH2CH2CH3),
21.83 (t, OCH2CH2CH3), 10.40 (q, OCH2CH2CH3);
HRESI-MS m/z 354.0709 [M+H]+ (calcd for C17H15
ClF2NO3 354.0708).
4.29. Butyl 4-chloro-2-(2,6-difluorobenzamido)benzoate
(47)
4.26. Methyl 4-chloro-2-(2,6-difluorobenzamido)benzoate
(44)
Yield (43%) from 3 and 2,6-difluorobenzoyl chloride;
EI-MS (m/z, %): 369(10), 367 [M]+ (29), 266(24),
141(100), 113(31); H NMR (CDCl3) d 11.71 (1H, br,
1
Yield (38%) from 8 and 2,6-difluorobenzoyl chloride; EI-
MS (m/z, %): 327(10), 325 [M]+ (30), 266(18), 141(100),
NH), 9.01 (1H, d, J = 2.4 Hz, H-3), 8.00 (1H, d,
J = 8.8 Hz, H-6), 7.43 (1H, m, H-40), 7.13 (1H, dd,
J = 8.8, 2.4 Hz, H-5), 7.01 (2H, m, H-50 30), 4.30 (2H,
t, J = 6.8 Hz, OCH2CH2CH2CH3), 1.74 (2H, m,
OCH2CH2CH2CH3), 1.45 (2H, m, OCH2CH2CH2CH3),
0.97 (3H, t, J = 7.6 Hz, OCH2CH2CH2CH3); 13C NMR
1
113(52); H NMR (CDCl3) d 11.66 (1H, br, NH), 9.01
(1H, d, J = 2.0 Hz, H-3), 8.01 (1H, d, J = 8.4 Hz, H-6),
7.43 (1H, m, H-40), 7.14 (1H, dd, J = 8.4, 2.0 Hz, H-5),
7.02 (2H, m, H-50 30), 6.43 (1H, br, NHC3H8), 3.91 (3H,
s, OCH3); 13C NMR (CDCl3) d 168.01 (s, COOR),
160.03 (s, C-20, JC–F = 251.6 Hz), 159.96 (s, C-60,
JC–F = 252.3 Hz), 159.04 (s, CONH), 141.74 (s, C-2),
141.18 (s, C-4), 132.30 (d, C-40, JC–F = 10.3 Hz), 131.95
(d, C-6), 123.57 (d, C-5), 120.64 (d, C-3), 113.53 (s, C-1),
112.30 (d, C-50, JC–F = 20.4 Hz), 112.25 (d, C-30,
JC–F = 20.4 Hz), 112.15 (s, C-10), 52.62 (q, OCH3);
HRESI-MS m/z 348.0217 [M+Na]+ (calcd for
C15H10ClF2NO3Na 348.0215).
(CDCl3) d 167.59 (s, COOR), 160.00 (s, C-20, JC–F
=
251.6 Hz), 159.93 (s, C-60, JC–F = 251.6 Hz), 159.03 (s,
CONH), 141.72 (s, C-2), 140.97 (s, C-4), 132.25 (d, C-
40, JC–F = 10.3 Hz), 131.88 (d, C-6), 123.51 (d, C-5),
120.60 (d, C-3), 113.85 (s, C-1), 112.27 (d, C-50,
JC–F = 19.7 Hz), 112.22 (d, C-30, JC–F = 20.5 Hz),
112.12 (s, C-10), 65.61 (t, OCH2CH2CH2CH3), 30.45
(t, OCH2CH2CH2CH3), 19.17 (t, OCH2CH2CH2CH3),
13.67 (q, OCH2CH2CH2CH3); HRESI-MS m/z
368.0866 [M+H]+ (calcd for C18H17ClF2NO3 368.0865).
4.27. Ethyl 4-chloro-2-(2,6-difluorobenzamido)benzoate
(45)
4.30. Methyl 4-chloro-2-(2,6-dichlorobenzamido)benzoate
(48)
Yield (46%) from 1 and 2,6-difluorobenzoyl chloride;
EI-MS (m/z, %): 339 [M]+ (12), 266(33), 141(100),
114(24); H NMR (CDCl3) d 11.71 (1H, br, NH), 9.00
1
Yield (29%) from 8 and 2,6-dichlorobenzoyl chloride;
EI-MS (m/z, %): 359(12), 357 [M]+ (12), 175(94),
(1H, d, J = 2.4 Hz, H-3), 8.01 (1H, d, J = 8.4 Hz, H-6),
7.43 (1H, m, H-40), 7.13 (1H, dd, J = 8.4, 2.4 Hz, H-5),
7.01 (2H, m, H-50 30), 4.36 (2H, q, J = 7.2 Hz,
OCH2CH3), 1.39 (3H, t, J = 7.2 Hz, OCH2CH3); 13C
NMR (CDCl3) d 167.52 (s, COOR), 159.98 (s, C-20,
JC–F = 251.6 Hz), 159.92 (s, C-60, JC–F = 251.6 Hz),
159.01 (s, CONH), 141.70 (s, C-2), 140.96 (s, C-4),
132.24 (d, C-40, JC–F = 10.2 Hz), 131.92 (d, C-6),
123.47 (d, C-5), 120.56 (d, C-3), 113.82 (s, C-1), 112.26
1
173(100), 147(19), 145(28); H NMR (CDCl3) d 11.41
(1H, br, NH), 9.00 (1H, d, J = 2.0 Hz, H-3), 8.01 (1H,
d, J = 8.8 Hz, H-6), 7.34 (3H, m, H-30 40 50), 7.16 (1H,
dd, J = 8.8, 2.0 Hz, H-5), 3.90 (3H, s, OCH3); 13C
NMR (CDCl3) d 168.01 (s, COOR), 163.11 (s, CONH),
141.63 (s, C-2), 140.26 (s, C-4), 135.92 (s, C-10), 132.17
(s, C-20, d, C-60), 131.97 (d, C-40), 131.04 (d, C-6),
128.28 (d, C-30, d, C-50), 123.72 (d, C-5), 120.75 (d, C-
3), 113.62 (s, C-1), 52.61 (q, OCH3); HRESI-MS m/z
357.9807 [M+H]+ (calcd for C15H11Cl3NO3 357.9805).
(d, C-50, JC–F = 20.4 Hz), 112.21 (d, C-30, JC–F
=
19.7 Hz), 112.11 (s, C-10), 61.76 (t, OCH2CH3), 14.08
(q, OCH2CH3); HRESI-MS m/z 340.0554 [M+H]+
(calcd for C16H13ClF2NO3 340.0552).
4.31. Ethyl 4-chloro-2-(2,6-dichlorobenzamido)benzoate
(49)
4.28. Propyl 4-chloro-2-(2,6-difluorobenzamido)benzoate
(46)
Yield (32%) from 1 and 2,6-dichlorobenzoyl chloride;
EI-MS (m/z, %): 373(15), 371 [M]+(15), 175(93),
173(100), 147(18), 145(26); H NMR (CDCl3) d 11.46
(1H, br, NH), 8.99 (1H, d, J = 2.0 Hz, H-3), 8.02 (1H,
d, J = 8.8 Hz, H-6), 7.32 (3H, m, H-30 40 50), 7.14 (1H,
dd, J = 8.4, 2.0 Hz, H-5), 4.33 (2H, q, J = 7.0 Hz,
OCH2CH3), 1.37 (3H, t, J = 7.2 Hz, OCH2CH3); 13C
NMR (CDCl3) d 167.50 (s, COOR), 163.06 (s, CONH),
141.56 (s, C-2), 141.00 (s, C-4), 135.89 (s, C-10), 132.11
1
Yield (58%) from 2 and 2,6-difluorobenzoyl chloride;
EI-MS (m/z, %): 355(9), 353 [M]+ (22), 268(24),
266(54), 142(100), 141(90); 1H NMR (CDCl3)
d
11.71(1H, br, NH), 9.00 (1H, d, J = 2.0 Hz, H-3), 8.01
(1H, d, J = 8.8 Hz, H-6), 7.42 (1H, m, H-40), 7.13 (1H,
dd, J = 8.8, 2.0 Hz, H-5), 7.02 (2H, m, H-50 30), 4.26
(2H, t, J = 6.4 Hz, OCH2CH2CH3), 1.78 (2H, m,

5812
P.-W. Hsieh et al. / Bioorg. Med. Chem. 16 (2008) 5803–5814
(s, C-20, d, C-60), 131.95 (d, C-40), 131.01 (d, C-6), 128.24
(d, C-30, d, C-50), 123.63 (d, C-5), 120.66 (d, C-3), 113.90
(s, C-1), 61.78 (t, OCH2CH3), 14.04 (q, OCH2CH3);
HRESI-MS m/z 371.9962 [M+H]+ (calcd for
C16H13Cl3NO3 371.9961).
(d, C-3), 116.49 (d, C-30, JC–F = 23.1 Hz), 114.87 (s, C-1),
69.34 (d, OCH(CH3)2), 21.83 (q, OCH(CH3)2); HRESI-
MS m/z 358.0623[M+Na]+ (calcd for C17H15ClFNO3Na
358.0622).
4.35. Isopropyl 4-chloro-2-(2-chlorobenzamido)benzoate
(53)
4.32. Propyl 4-chloro-2-(2,6-dichlorobenzamido)benzoate
(50)
Yield (36%) from 9 and 2-chlorobenzoyl chloride; EI-MS
(m/z, %): 353(11), 351(17) [M]+, 266(15), 264(21), 141(38),
139(100); H NMR (CDCl3) d 11.66 (1H, br, NH), 9.00
Yield (32%) from 2 and 2,6-dichlorobenzoyl chloride;
EI-MS (m/z, %): 387(10), 385 [M]+ (10), 175(78),
173(100), 147(11), 145(15); H NMR (CDCl3) d 11.46
1
1
(1H, d, J = 2.0 Hz, H-3), 8.00 (1H, d, J = 8.8 Hz, H-6),
7.64 (1H, dd, J = 7.2, 2.0 Hz, H-60), 7.42 (3H, m, H-30 40
50), 7.11 (1H, dd, J = 8.8, 2.0 Hz, H-5), 5.21 (1H, m,
OCH(CH3)2), 1.35 (6H, d, J = 6.0 Hz,OCH(CH3)2); 13C
NMR (CDCl3) d 167.01 (s, COOR), 165.50 (s, CONH),
141.95 (s, C-2), 140.77 (s, C-4), 135.81 (s, C-10), 131.93
(d, C-40), 131.56 (d, C-6), 131.27 (s, C-20), 130.64 (d, C-
30), 129.19 (d, C-60), 127.14 (d, C-50), 123.19 (d, C-5),
120.43 (d, C-3), 114.28 (s, C-1), 69.51 (d, OCH(CH3)2),
21.75 (q, OCH(CH3)2); HRESI-MS m/z 374.0329
[M+Na]+ (calcd for C17H15Cl2NO3Na 374.0326).
(1H, br, NH), 9.00 (1H, d, J = 2.0 Hz, H-3), 8.02 (1H,
d, J = 8.8 Hz, H-6), 7.33 (3H, m, H-30 40 50), 7.15 (1H,
dd, J = 8.8, 2.0 Hz, H-5), 4.24 (2H, t, J = 6.8 Hz,
OCH2CH2CH3), 1.77 (2H, m, OCH2CH2CH3), 1.00
(3H, t, J = 7.6 Hz, OCH2CH2CH3); 13C NMR (CDCl3)
d 167.57 (s, COOR), 163.10 (s, CONH), 141.58 (s, C-2),
141.05 (s, C-4), 135.91 (s, C-10), 132.15 (s, C-20, d, C-60),
131.90 (d, C-40), 131.00 (d, C-6), 128.25 (d, C-30, d, C-
50), 123.67 (d, C-5), 120.72 (d, C-3), 113.96 (s, C-1),
67.29 (t, OCH2CH2CH3), 21.82 (t, OCH2CH2CH3),
10.40 (q, OCH2CH2CH3); HRESI-MS m/z 386.0119
[M+H]+ (calcd for C17H15Cl3NO3 386.0117).
4.36. Isopropyl 4-chloro-2-(2,6-difluorobenzamido)benzo-
ate (54)
4.33. Butyl 4-chloro-2-(2,6-dichlorobenzamido)benzoate
(51)
Yield (33%) from 9 and 2,6-difluorobenzoyl chloride;
EI-MS (m/z, %): 355 (9), 353 [M]+ (27), 268 (14), 266
(37), 153 (22), 143 (52), 141 (100); H NMR (CDCl3) d
1
Yield (26%) from 3 and 2,6-dichlorobenzoyl chloride; EI-
MS (m/z, %): 401(16), 399 [M]+ (16), 175(37), 173(100),
11.74 (1H, br, NH), 9.00 (1H, d, J = 2.0 Hz, H-3), 8.00
(1H, d, J = 8.8 Hz, H-6), 7.43 (1H, m, H-40), 7.13 (1H,
dd, J = 8.8, 2.0 Hz, H-5), 7.02 (2H, m, H-50 30), 5.22
(1H, m, OCH(CH3)2), 1.36 (6H, d, J = 6.0 Hz,OCH
(CH3)2); 13C NMR (CDCl3) d167.05 (s, COOR),
160.02 (s, C-20, JC–F = 251.5 Hz), 159.96 (s, C-60,
JC–F = 252.4 Hz), 159.03 (s, CONH), 141.71 (s, C-2),
140.84 (s, C-4), 132.21 (d, C-40, JC–F = 10.2 Hz),
131.93 (d, C-6), 123.45 (d, C-5), 120.59 (d, C-3),
114.27 (s, C-10), 112.38 (s, C-1), 112.23 (d, C-50,
JC–F = 23.5 Hz), 112.28 (d, C-30, JC–F = 19.8 Hz), 69.60
(d, OCH(CH3)2), 21.78 (q, OCH(CH3)2); HRESI-MS
m/z 376.0531 [M+Na]+ (calcd for C17H14ClF2NO3Na
376.0528).
1
147(12), 145(17); H NMR (CDCl3) d 11.45 (1H, br,
NH), 9.00 (1H, d, J = 2.0 Hz, H-3), 8.01 (1H, d,
J = 8.8 Hz, H-6), 7.33 (3H, m, H-30 40 50), 7.15 (1H, dd,
J = 8.8, 2.0 Hz, H-5), 4.28 (2H, t, J = 6.4 Hz,
OCH2CH2CH2CH3), 1.73 (2H, m, OCH2CH2CH2CH3),
1.44 (2H, m, OCH2CH2CH2CH3), 0.96 (3H, t,
J = 7.2 Hz, OCH2CH2CH2CH3); 13C NMR (CDCl3) d
167.55 (s, COOR), 163.07 (s, CONH), 141.58 (s, C-2),
141.04 (s, C-4), 135.92 (s, C-10), 132.15 (s, C-20, d, C-60),
131.92 (d, C-40), 131.00 (d, C-6), 128.28 (d, C-30, d, C-
50), 123.66 (d, C-5), 120.71 (d, C-3), 113.97 (s, C-1),
65.62 (t, OCH2CH2CH2CH3), 30.43 (t, OCH2CH2
CH2CH3), 19.16 (t, OCH2CH2CH2CH3), 13.66 (q,
OCH2CH2CH2CH3); HRESI-MS m/z 400.0276 [M+H]+
(calcd for C18H17Cl3NO3 400.0274).
4.37. 4-Chloro-2-(2-fluorobenzamido)-N-propylbenzamide
(55)
4.34. Isopropyl 4-chloro-2-(2-fluorobenzamido)benzoate
(52)
Yield (37%) from 10 and 2-fluorobenzoyl chloride; EI-MS
(m/z, %): 334 [M]+ (3), 276(16), 248(6), 123(100), 95(47);
1H NMR (CDCl3) d 11.77 (1H, br, NH), 8.78 (1H, d,
J = 2.4 Hz, H-3), 8.01 (1H, td, J = 7.6, 2.0 Hz,, H-60),
7.51 (1H, m, H-40), 7.38 (1H, d, J = 8.4 Hz, H-6), 7.28
(1H, td, J = 7.6, 1.2 Hz, H-50), 7.19 (1H, ddd, J = 10.4,
8.4,1.2 Hz, H-30), 7.01 (1H, dd, J = 8.4, 2.4 Hz, H-5),
6.48 (1H, br, NHC3H8), 3.40 (2H, q, J = 6.8 Hz,
NHCH2CH2CH3), 1.65 (2H, m, NHCH2CH2CH3), 0.99
(3H, t, J = 7.2 Hz, NHCH2CH2CH3); 13C NMR (CDCl3)
Yield (40%) from 9 and 2-fluorobenzoyl chloride; EI-MS
(m/z, %): 335(26) [M]+, 250(16), 249(46), 124(49),
1
123(100); H NMR (CDCl3) d 11.99 (1H, br, NH), 9.02
(1H, d, J = 2.0 Hz, H-3), 8.05 (1H, td, J = 7.6, 1.6 Hz,,
H-60), 8.00 (1H, d, J = 8.4 Hz, H-6), 7.52 (1H, m, H-40),
7.30 (1H, td, J = 7.6, 0.8 Hz, H-50), 7.21 (1H, ddd,
J = 10.4, 8.4, 0.8 Hz, H-30), 7.11 (1H, dd, J = 8.4,
2.0 Hz, H-5), 5.29 (1H, m, OCH(CH3)2), 1.38 (6H, d,
J = 6.0 Hz,OCH(CH3)2); 13C NMR (CDCl3) d 166.80
(s, COOR), 162.48 (s, CONH, JC–F = 2.7 Hz), 160.21 (s,
C-20, JC–F = 249.3 Hz), 141.97 (s, C-2), 140.48 (s, C-4),
133.73 (d, C-40, JC–F = 9.1 Hz), 131.93 (d, C-6), 131.69
(d, C-60, JC–F = 1.9 Hz), 124.76 (d, C-50, JC–F = 3.8 Hz),
123.13 (d, C-5), 122.38 (s, C-10, JC–F = 12.1 Hz), 121.13
d 167.98 (s, CONHC3H8), 162.43 (s, CONH, JC–F
=
2.5 Hz), 160.25 (s, C-20, JC–F = 250.0 Hz), 139.77 (s, C-
2), 138.11 (s, C-4), 133.67 (d, C-40, JC–F = 8.4 Hz),
131.42 (d, C-60, JC–F = 2.3 Hz), 127.61 (d, C-6), 124.68
(d, C-50, JC–F = 3.8 Hz), 123.25 (d, C-5), 122.29 (s,
C-10), 122.13 (d, C-3), 120.31 (s, C-1), 116.57 (d, C-30,

P.-W. Hsieh et al. / Bioorg. Med. Chem. 16 (2008) 5803–5814
5813
JC–F = 23.5 Hz), 41.79 (t, NHCH2CH2CH3), 22.65 (t,
NHCH2CH2CH3), 11.39 (q, NHCH2CH2CH3); HRES-
I-MS m/z 357.0781 [M+Na]+ (calcd for C17H16ClFN2O2
Na 357.0782).
131.50 (d, C-40), 130.57 (d, C-6), 127.68 (d, C-30), 127.09
(d, C-60), 126.15 (d, C-50), 122.85 (d, C-5), 121.13 (d, C-
3), 118.99 (s, C-1), 41.74 (t, NHCH2CH2CH3), 22.59 (t,
NHCH2CH2CH3), 20.25 (q, CH3), 11.38 (q, NHCH2
CH2CH3); HRESI-MS m/z 353.1032 [M+Na]+ (calcd
for C18H19ClN2O2Na 353.1033).
4.38. 4-Chloro-2-(2-chlorobenzamido)-N-propylbenza-
mide (56)
4.41. 4-Chloro-2-(2-methoxybenzamido)-N-propylbenza-
mide (59)
Yield (58%) from 10 and 2-chlorobenzoyl chloride; EI-
MS (m/z, %): 350 [M]+ (10), 294 (18), 292 (23), 141 (33),
139 (100); 1H NMR (CDCl3) d 11.65 (1H, br, NH), 8.85
(1H, d, J = 2.0 Hz, H-3), 7.62 (1H, dd, J = 7.2, 2.4 Hz,
H-6), 7.46 (1H, dd, J = 8.0, 1.6 Hz, H-60), 7.38 (3H, m,
H-30 40 50), 7.12 (1H, dd, J = 8.0, 2.0 Hz, H-5), 6.37 (1H,
br, NHC3H8), 3.36 (2H, q, J = 6.4 Hz, NHCH2CH2CH3),
1.63 (2H, m, NHCH2CH2CH3), 0.97 (3H, t, J = 7.6 Hz,
NHCH2CH2CH3); 13C NMR (CDCl3) d 168.07 (s,
CONHC3H8), 165.49 (s, CONH), 140.17 (s, C-2),
138.61 (s, C-4), 135.67 (s, C-10), 131.54 (d, C-40), 131.36
(s, C-20), 130.67 (d, C-6), 129.22 (d, C-30), 127.47 (d, C-
60), 127.17 (d, C-50), 123.32 (d, C-5), 121.57 (d, C-3),
119.15 (s, C-1), 41.78 (t, NHCH2CH2CH3), 22.63 (t,
NHCH2CH2CH3), 11.38 (q, NHCH2CH2CH3); HRES-
I-MS m/z 373.0487 [M+Na]+ (calcd for C17H16Cl2N2O2
Na 373.0486).
Yield (57%) from 10 and 2-methoxybenzoyl chloride; EI-
1
MS (m/z, %): 346 [M]+ (2), 288(2), 135(100), 92(22); H
NMR (CDCl3) d 11.69 (1H, br, NH), 8.72 (1H, d,
J = 1.6 Hz, H-3), 8.18 (1H, dd, J = 7.6, 1.6 Hz, H-6),
7.50 (1H, td, J = 8.8, 2.0 Hz, H-60), 7.24 (1H, d,
J = 8.4 Hz, H-40)M, 7.09 (1H, t, J = 7.2 Hz, H-30), 7.02
(1H, d, J = 8.8 Hz, H-5), 6.82 (1H, dd, J = 8.8, 1.2 Hz,
H-50), 6.64 (1H, br, NHC3H8), 4.09 (3H, s, OCH3), 3.42
(2H, q, J = 6.4 Hz, NHCH2CH2CH3), 1.69 (2H, m,
NHCH2CH2CH3), 1.02 (3H, t, J = 7.6 Hz, NHCH2
CH2CH3); 13C NMR (CDCl3) d 168.02 (s, CONHC3H8),
164.46 (s, CONH), 157.73 (s, C-20), 139.04 (s, C-2), 137.11
(s, C-4), 133.46 (d, C-40), 132.23 (d, C-6), 132.27 (d, C-60),
127.86 (d, C-5), 121.46 (d, C-3), 121.74 (s, C-10), 120.88 (d,
C-50), 111.34 (s, C-1, d, C-30), 55.66 (q, OCH3), 41.67 (t,
NHCH2CH2CH3), 22.78 (t, NHCH2CH2CH3), 11.51 (q,
NHCH2CH2CH3); HRESI-MS m/z 369.0982[M+Na]+
(calcd for C18H19ClN2O3Na 369.0982).
4.39. 4-Chloro-2-(2-bromobenzamido)-N-propylbenza-
mide (57)
Yield (33%) from 10 and 2-bromobenzoyl chloride; EI-
MS (m/z, %): 396 (14), 394 [M]+ (11), 338 (25), 336
(20), 185 (96), 183 (100), 155 (24); H NMR (CDCl3) d
4.42. 4-Chloro-2-(2,6-difluorobenzamido)-N-propylbenza-
mide (60)
1
11.64 (1H, s, NH), 8.79 (1H, d, J = 2.0 Hz, H-3), 7.63
(1H, dd, J = 7.6, 1.2 Hz, H-30), 7.55 (1H, dd, J = 7.6,
1.6 Hz, H-60), 7.40 (2H, m, H-6 50), 7.31 (1H, td,
J = 7.6, 1.6 Hz, H-40), 7.01 (1H, dd, J = 8.4, 2.0 Hz,
H-5), 6.67 (1H, br, NHC3H8), 3.33 (2H, q, J = 6.4 Hz,
NHCH2CH2CH3), 1.61 (2H, m, NHCH2CH2CH3),
0.95 (3H, t, J = 7.2 Hz, OCH2 CH2CH3); 13C NMR
(CDCl3) d 168.05 (s, CONHC3H8), 166.36 (s, CONH),
140.02 (s, C-2), 138.40 (s, C-4), 137.75 (s, C-10), 133.82
(d, C-30), 131.60 (d, C-40), 128.84 (d, C-6), 127.70 (d,
C-60), 127.69 (d, C-50), 123.31 (d, C-5), 121.34 (d, C-
3), 119.73 (s, C-20), 119.09 (s, C-1), 41.76 (t, NHCH2
CH2CH3), 22.56 (t, NHCH2CH2CH3), 11.38 (q,
NHCH2CH2CH3); HRESI-MS m/z 416.9979 [M+Na]+
(calcd for C17H16BrClN2O2Na 416.9981).
Yield (21%) from 10 and 2,6-difluorobenzoyl chloride;
EI-MS (m/z, %): 352 [M]+ (19), 294(60), 180(93),
1
142(52), 141(100); H NMR (CDCl3) d 11.75 (1H, br,
NH), 8.83 (1H, d, J = 2.0 Hz, H-3), 7.40 (2H, m, H-40
6), 7.08 (1H, dd, J = 8.4, 2.0 Hz, H-5), 6.99 (2H, m, H-50
30), 6.43 (1H, br, NHC3H8), 3.36 (2H, q, J = 6.8 Hz,
NHCH2CH2CH3), 1.63 (2H, m, NHCH2CH2CH3), 0.97
(3H, t, J = 7.2 Hz, NHCH2CH2CH3); 13C NMR (CDCl3)
d 168.02 (s, CONHC3H8), 160.01 (s, C-20, JC–F
=
251.6 Hz), 159.94 (s, C-60, JC–F = 251.6 Hz), 159.04 (s,
CONH), 139.90 (s, C-2), 138.64 (s, C-4), 132.09 (d, C-40,
JC–F = 9.9 Hz), 127.43 (d, C-6), 123.55 (d, C-5), 121.69
(d, C-3), 119.06 (s, C-1), 112.24 (d, C-50, JC–F
=
23.5 Hz), 112.20 (d, C-30, JC–F = 19.8 Hz), 112.10 (s, C-
10), 41.81 (t, NHCH2CH2CH3), 22.60 (t, NHCH2CH2
CH3), 11.36 (q, NHCH2CH2CH3); HRESI-MS m/z
375.0686 [M+Na]+ (calcd for C17H15ClF2N2O2Na
375.0688).
4.40. 4-Chloro-2-(2-methylbenzamido)-N-propylbenza-
mide (58)
Yield (40%) from 10 and 2-methylbenzoyl chloride; EI-
MS (m/z, %): 330 [M]+ (6), 271 (10), 119 (100), 91 (100);
1H NMR (CDCl3) d 11.59 (1H, br, NH), 8.81 (1H, d,
J = 2.0 Hz, H-3), 7.57 (1H, dd, J = 1.2, 7.6 Hz, H-60),
7.38 (1H, d, J = 8.4 Hz, H-6), 7.36 (1H, td, J = 1.2,
7.6 Hz, H-40), 7.27 (2H, m, H-50 30), 6.96 (1H, dd,
J = 8.4, 2.0 Hz, H-5), 6.66 (1H, br, NHC3H8), 3.34
(2H, q, J = 6.8 Hz, NHCH2CH2CH3), 2.54 (3H, s,
CH3), 1.69 (2H, m, NHCH2CH2CH3), 0.96 (3H, t,
J = 7.6 Hz, NHCH2CH2CH3); 13C NMR (CDCl3) d
168.43 (s, CONHC3H8), 168.19 (s, CONH), 140.49 (s,
C-2), 138.32 (s, C-4), 137.00 (s, C-20), 135.73 (s, C-10),
4.43. N-Butyl-4-chloro-2-(2-fluorobenzamido)benzamide
(61)
Yield (35%) from 11 and 2-fluorobenzoyl chloride; EI-MS
(m/z, %): 348 [M]+ (12), 278 (27), 276 (80), 180 (37), 123
(37), 124 (100); 1H NMR (CDCl3) d 11.76 (1H, br, NH),
8.75 (1H, d, J = 2.4 Hz, H-3), 8.00 (1H, td, J = 7.6,
1.6 Hz,, H-60), 7.51 (1H, m, H-40), 7.37 (1H, d,
J = 8.4 Hz, H-6), 7.27 (1H, td, J = 7.6, 1.2 Hz, H-50),
7.18 (1H, ddd, J = 10.2, 8.4,1.2 Hz, H-30), 6.97 (1H, dd,
J = 8.4, 2.4 Hz, H-5), 6.57 (1H, br, NHC4H9), 3.43 (2H,
q, J = 6.8 Hz, NHCH2CH2CH2CH3), 1.61 (2H, m,

5814
P.-W. Hsieh et al. / Bioorg. Med. Chem. 16 (2008) 5803–5814
NHCH2CH2CH2CH3), 1.40 (2H, m, NHCH2CH2CH2
CH3), 0.94 (3H, t, J = 7.2 Hz, NHCH2CH2CH2CH3);
13C NMR (CDCl3) d 167.96 (s, CONHC4H9), 162.44 (s,
CONH), 160.29 (s, C-20, JC–F = 250.1 Hz), 139.70 (s, C-
2), 138.02 (s, C-4), 133.66 (d, C-40, JC–F = 9.1 Hz),
131.37 (d, C-60, JC–F = 1.5 Hz), 127.69 (d, C-6), 124.66
(d, C-50, JC–F = 3.1 Hz), 123.23 (d, C-5), 122.25 (s, C-10,
JC–F = 12.1 Hz), 122.06 (d, C-3), 120.35 (s, C-1), 116.57
(d, C-30, JC–F = 22.7 Hz), 39.87 (t, NHCH2CH2CH2
CH3), 31.36 (t, NHCH2CH2CH2CH3), 20.10 (t, NHCH2
CH2CH2CH3), 13.71 (q, NHCH2CH2CH2CH3); HRES-
I-MS m/z 371.0935[M+Na]+ (calcd for C18H18ClFN2O2
Na 371.0938).
4.46. Preparation of washed human platelets
Human blood anti-coagulated with acid citrate dextrose
(ACD) was obtained from healthy human volunteers
who had not taken any drugs within the last two weeks.
The platelet suspension was then prepared according to
the washing procedure previously described.7 Platelets
were finally suspended in Tyrode0s solution containing
Ca2+ (2 mM), glucose (11.1 mM) and bovine serum albu-
min (3.5 mg/ml) at a concentration of 3 · 108 platelets/ml.
4.47. Measurement of platelet aggregation6
Platelet aggregation was measured turbidimetrically
with a light-transmission aggregometer (Chrono-Log
Co., U.S.A.). The platelet suspension was incubated
with dimethyl sulfoxide (DMSO, vehicle) or test com-
pounds at 37 ꢁC for 3 min under a stirring condition
(1200 rpm) prior to the addition of the platelet aggrega-
tion inducers. The extent of platelet aggregation was
measured as the maximal increase of light transmission
within 5 min after the addition of inducers.
4.44. N-Butyl-4-chloro-2-(2-chlorobenzamido)benzamide
(62)
Yield (45%) from 11 and 2-chlorobenzoyl chloride; EI-
MS (m/z, %): 364 [M]+ (10), 292 (48), 181 (36), 154 (25),
1
141 (48), 139 (100); H NMR (CDCl3) d 11.67 (1H, br,
NH), 8.78 (1H, d, J = 2.0 Hz, H-3), 7.60 (1H, dd,
J = 7.6, 2.0 Hz, H-60), 7.39 (4H, m, H-30 40 50 6), 7.00
(1H, dd, J = 8.4, 2.0 Hz, H-5), 6.65 (1H, br, NHC4H9),
3.37 (2H, q, J = 6.8 Hz, NHCH2CH2CH2CH3), 1.57
(2H, m, NHCH2CH2CH2CH3), 1.37 (2H, m, NHCH2
CH2CH2CH3), 0.92 (3H, t, J = 7.2 Hz, NHCH2CH2
CH2CH3); 13C NMR (CDCl3) d 168.01 (s, CONHC4H9),
165.52 (s, CONH), 139.97 (C-2), 138.35 (C-4), 135.57 (C-
10), 131.55 (C-40), 131.31 (C-20), 130.65 (C-6), 129.11 (C-
30), 127.49 (C-60), 127.15 (C-50), 123.29 (C-5), 121.38
(C-3), 119.21 (C-1), 39.85 (t, NHCH2CH2CH2CH3),
31.30 (t, NHCH2CH2CH2CH3), 20.09 (t, NHCH2CH2
CH2CH3), 13.69 (q, NHCH2CH2CH2CH3); HRESI-MS
m/z 387.0642 [M+Na]+ (calcd for C18H18Cl2N2O2Na
387.0643).
6,7
4.48. Measurement of the formation of TxB2
Because TxA2 is very unstable and rapidly converted to
more stable metabolite TxB2, we measured the latter in-
stead of TxA2. After the challenge of platelets with AA
for 5 min, EDTA (2 mM) and indomethacin (50 lM) were
added. The platelet suspensions were centrifuged for 3 min
at 13,000 rpm, the contents of TxB2 in the supernatants
were assayed using an enzyme immunoassay kit according
to the procedure described by the manufacturer.
Acknowledgment
4.45. N-Butyl-4-chloro-2-(2,6-difluorobenzamido)benz-
amide (63)
The investigation was supported by Research Grants to
P.W. Hsieh from the National Science Council of the
Republic of China (NSC94-2314-B-037-105) in Taiwan.
Yield (28%) from 11 and 2,6-difluorobenzoyl chloride;
EI-MS (m/z, %): 366 [M]+ (12), 296(30), 294(89),
182(38), 180(77), 142 (37), 141(100); H NMR (CDCl3)
1
d 11.76 (1H, br, NH), 8.78 (1H, d, J = 2.0 Hz, H-3), 7.40
(2H, m, H-40 6), 7.04 (1H, dd, J = 8.4, 2.0 Hz, H-5), 6.98
(2H, m, H-50 30), 6.57 (1H, br, NHC4H9), 3.39 (2H, q,
J = 6.8 Hz, NHCH2CH2CH2CH3), 1.58 (2H, m, NHCH2
CH2CH2CH3), 1.38 (2H, m, NHCH2CH2CH2CH3), 0.93
(3H, t, J = 7.2 Hz, NHCH2CH2CH2CH3); 13C NMR
(CDCl3) d 167.96 (s, CONHC4H9), 159.94 (s, C-20,
JC–F = 251.6 Hz), 159.88 (s, C-60, JC–F = 251.6 Hz),
159.09 (s, CONH), 139.73 (s, C-2), 138.45 (s, C-4),
132.13 (d, C-40, JC–F = 10.2 Hz), 127.59 (d, C-6), 123.54
(d, C-5), 121.55 (d, C-3), 119.09 (s, C-1), 112.24 (d, C-50,
JC–F = 20.1 Hz), 112.18 (d, C-30, JC–F = 20.1 Hz),
112.08 (s, C-10), 39.88 (t, NHCH2CH2CH2CH3), 31.28
(t, NHCH2CH2CH2CH3), 20.08 (t, NHCH2CH2CH2
CH3), 13.68 (q, NHCH2CH2CH2CH3); HRESI-MS m/z
389.0843 [M+Na]+ (calcd for C18H17ClF2N2O2Na
389.0844).
References and notes
1. Hankey, G. J.; Eikelboom, J. W. Med. J. Aust. 2003, 178,
568.
2. Hsieh, P. W.; Hwang, T. L.; Wu, C. C.; Chiang, S. Z.; Wu,
C. I.; Wu, Y. C. Bioorg. Med. Chem. Lett. 2007, 17, 1812.
3. Hsieh, P. W.; Hwang, T. L.; Wu, C. C.; Chang, F. R.;
Wang, T. W.; Wu, Y. C. Bioorg. Med. Chem. Lett. 2005, 15,
2786.
4. Patirick, G. L. An introduction to medicinal chemistry;
Oxford University Press: New York, 2005, pp. 197-198.
5. Burdick, D. J.; Marsters, J. C.; Aliagas-Martin, I.; Stanley,
M.; Beresini, M.; Clark, K.; McDowell, R. S.; Gadek, T. R.
Bioorg. Med. Chem. Lett. 2004, 14, 2055.
6. Wu, C. C.; Wang, T. W.; Wang, W. Y.; Hsieh, P. W.; Wu,
Y. C. Eur. J. Pharmacol. 2005, 527, 37.
7. Wang, W. Y.; Wu, Y. C.; Wu, C. C. Mol. Pharmacol. 2006,
70, 1380.