The evaluation and structure-activity relationships of 2-benzoylaminobenzoic esters and their analogues as anti-inflammatory and anti-platelet aggregation agents

Article abstract of DOI:10.1016/j.bmcl.2006.12.038

Forty-seven 2-benzoylaminobenzoic esters were synthesized and evaluated in anti-platelet aggregation, inhibition of superoxide anion generation, and inhibition of neutrophil elastase release assays. Most 2-benzoylamino-4-chlorobenzoic acid derivatives showed selective inhibitory effects on arachidonic acid (AA)-induced platelet aggregation. Among them, compounds 6b and 7b exhibited more potent inhibitory effects (ca. 200-fold) than aspirin. Additionally, compounds 1a and 5a showed strong inhibitory effects on neutrophil superoxide generation with IC₅₀ values of 0.65 and 0.17 μM, respectively. However, compounds 6d and 6e exhibited dual inhibitory effects on platelet aggregation and neutrophil elastase (NE) release; therefore, these two compounds may be new leads for development as anti-inflammatory and anti-platelet aggregatory agents.

Full text of DOI:10.1016/j.bmcl.2006.12.038

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1812–1817
The evaluation and structure–activity relationships of
2-benzoylaminobenzoic esters and their analogues as
anti-inflammatory and anti-platelet aggregation agents
Pei-Wen Hsieh,a Tsong-Long Hwang,b Chin-Chung Wu,a Shin-Zan Chiang,a
Chung-I Wua and Yang-Chang Wua,*
aGraduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan
bGraduate Institute of Natural Products, Chang Gung University, Taoyuan 333, Taiwan
Received 8 September 2006; revised 24 November 2006; accepted 12 December 2006
Available online 15 December 2006
Abstract—Forty-seven 2-benzoylaminobenzoic esters were synthesized and evaluated in anti-platelet aggregation, inhibition of
superoxide anion generation, and inhibition of neutrophil elastase release assays. Most 2-benzoylamino-4-chlorobenzoic acid deriv-
atives showed selective inhibitory effects on arachidonic acid (AA)-induced platelet aggregation. Among them, compounds 6b and
7b exhibited more potent inhibitory effects (ca. 200-fold) than aspirin. Additionally, compounds 1a and 5a showed strong inhibitory
effects on neutrophil superoxide generation with IC50 values of 0.65 and 0.17 lM, respectively. However, compounds 6d and 6e
exhibited dual inhibitory effects on platelet aggregation and neutrophil elastase (NE) release; therefore, these two compounds
may be new leads for development as anti-inflammatory and anti-platelet aggregatory agents.
Ó 2006 Elsevier Ltd. All rights reserved.
Arterial thromboembotic diseases, for example, acute
coronary syndrome and ischemic stroke, which are
caused by platelet aggregation, are the major causes of
death in developed countries. Antiplatelet drugs, such
as aspirin and ticlopidine, are used to protect against
myocardial infarction, stroke, cardiovascular death,
and other serious vascular events in patients with a his-
tory of previous vascular events or known risk factors
for cardiovascular disease.1 However, the current anti-
platelet drugs still have some restrictions in their mode
of action and efficacy. Therefore, research and develop-
ment of new generation antiplatelet drugs continue as
important targets.
asthma.2 In response to diverse stimuli, activated
neutrophils secrete a series of cytotoxins, such as
superoxide ðO2ÅꢀÞ, a precursor of other reactive oxy-
gen species (ROS), granule proteases, and bioactive
lipids.3–6 Thus, suppression of extensive or inappro-
priate activation of neutrophils using drugs has been
proposed as
diseases.
a way to ameliorate inflammatory
In prior studies, we synthesized 2-phenyl substituted
quinazolines and 2,8-disubstituted benzoxazinones
that exhibited significant inhibitory effects on platelet
aggregation and superoxide ðO2ÅꢀÞ generation of
neutrophils.7,8 Among them, 2-(2-bromophenyl)-8-
methoxybenzoxazinone showed concentration-depen-
dent inhibitory effects on platelet aggregation, ATP
release, P-selectin expression, and intracellular calcium
mobilization caused by thrombin. In contrast, it had
no significant effect on either SFLLRN- or GYPGKF-
induced platelet aggregation.9 In our continuing
research on anti-platelet aggregation and anti-inflamma-
tory agents, we also found that certain 2-benzoylamino-
benzoic acid derivatives exhibited higher anti-platelet
aggregation and anti-inflammatory potency than related
2-phenylbenzoxazinones.
In addition, neutrophils are known to play important
roles in a host’s defenses against invasion by micro-
organisms and in the pathogenesis of various diseases
such as rheumatoid arthritis, ischemia-reperfusion
injury, chronic obstructive pulmonary disease, and
Keywords: 2-Benzoylaminobenzoic esters; Anti-inflammatory; Anti-
platelet aggregation agents.
*
Corresponding author. Tel.: +886 7 3121101; fax: +886 7 3114773;
e-mail: yachwu@kmu.edu.tw
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.038

P.-W. Hsieh et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1812–1817
1813
Previously, 2-benzoylaminobenzoic acid derivatives,
such as phytoalexin, were isolated from Dianthus spe-
cies of the family Caryophyllaceae, while these plants
were infected by Phytophthora parasitica.10 Therefore,
we synthesized 2-benzoylaminobenzoic acid derivatives
and investigated their anti-fungal effects.11–13 Addition-
ally, such compounds were also reported recently
as anti-proliferative14 and anti-hypercholesterolemic15
agents. In the present study, we report the preparation,
preliminary pharmacological data, and structure-activity
relationships of new 2-benzoylaminobenzoic acid
derivatives.
Most 2-benzoylamino-4-chlorobenzoic acid derivatives
showed selective inhibitory effects on AA-induced
platelet aggregation. Among them, compounds 6b
and 7b exhibited the highest potency, about 200
times stronger than aspirin. Furthermore, all 2-ben-
zoylamino-3-chloro- and 2-benzoylamino-6-chloroben-
zoic acid derivatives were not active or only weakly
active.
On the basis of these results, several structure–activity
relationship conclusions for anti-platelet aggregation
activity were summarized as follows:
Forty-seven 2-benzoylaminobenzoic acid derivatives
(1a–1o, 2a–2e, 3a–3e, 3k–3o, 4a–4e, 5a, 6a–6e, 7a–
7e, and 8a) were synthesized (Schemes 1 and 2,
Tables 1 and 2). All new products were fully char-
acterized using spectroscopic data as shown in
Table 3.16,17
1. 2-Benzoylamino-4-chlorobenzoic acid derivatives
showed selective inhibitory effects on AA-induced
platelet aggregation.
2. Bioactivity was essentially abolished in the presence
of 3- or 6-chloro substitution.
3. 2-(20-Chlorobenzoylamino)-4-chlorobenzoic
acid
analogues exhibited more potent effects than those
with fluoro, bromo, methoxy, or methyl groups.
4. Among compounds 1a–1e, 6a–6e, and 7a–7e, the bio-
activity generally improved by increasing the ester
chain length.
As previously reported,7,8 benzoxazinones exhibited
selective inhibitory effects on platelet aggregation in-
duced by arachidonic acid (AA) and thrombin
(Thr). Thus, these two inducers were used in the
present work. Aspirin and indomethacin were also
assayed as positive controls.18 Bioassay results are
shown in Table 4.
Neutrophil elastase release and neutrophil superoxide
generation induced by N-formyl-L-methionyl-L-lucyl-L-
R3
O
R3
O
O
OCH3
A
OCH3
Cl
R2
NH
CH2Cl2, r.t.
16 hrs
R1
+
R2
NH2
R6
R8
O
1'
B
4'
R1
R7
a-o
R6 2'
R8
3'
R7
1-4
1a- 1o
2a- 2e
3a- 3e, 3k- 3o
4a- 4e
Scheme 1.
O
R4
O
O
A
R5
NH
R6
Cl
R4
CH2Cl2, r.t.
16 hrs
+
R6
O
R5
NH2
B
5-8
a-o
5a
6a- 6e
7a- 7e
8a
Scheme 2.

1814
P.-W. Hsieh et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1812–1817
Table 1. The 2-benzoylamino-benzoic acid derivates
that substitution of a chlorine atom on A ring resulted
in decreased bioactivity. Among this series, compounds
1aÅaꢀnd 5a showed strong inhibitory effects on neutrophil
O2 generation. The results indicated that when the
methyl ester was replaced by an ethyl ester, the inhibito-
Compound
R1
R2
R3
R6
R7
R8
1a
1b
1c
1d
1e
1f
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
F
Cl
H
H
H
H
Br
H
H
Åꢀ
ry effect on neutrophil O2 generation was increased.
CH3
OCH3
H
H
H
Moreover, compounds 1h, 1i, 5a, 6d, and 6e also
inhibited neutrophil elastase (NE) release induced by
fMLP. According to these results, the following
structure–activity relationships for anti-inflammatory
effects were proposed.
H
H
F
H
1g
1h
1i
H
Cl
Br
CH3
OCH3
H
H
H
H
H
H
1j
H
H
1k
1l
H
F
1. Chloro-substitution at position C-3, -4, or -6 in A
ring resÅuꢀlted in decreased inhibitory effects on neutro-
phil O2 generation.
2. Bioactivity was increased by replacing the methyl
ester with an ethyl ester. These results suggested that
the hydrophobicity of 2-benzoylamino-benzoic acid
derivatives plays an important role in the anti-inflam-
matory effect. On the other hand, when the ester
group was replaced by a ketone group (8a), the
anti-inflammatory activity decreased.
H
H
Cl
Br
CH3
OCH3
H
1m
1n
1o
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
3k
3l
H
H
H
H
H
H
F
Cl
H
H
H
Br
H
H
CH3
OCH3
F
H
H
H
H
H
H
Cl
Br
H
H
3. Anti-inflammatory activity depended dramatically
upon the identity and position of B ring substituents.
H
H
CH3
OCH3
H
H
H
Åꢀ
For inhibition of neutrophil O2 generation, a meta-
H
H
fluoro substituent appeared to be the most favorable.
However, the best choice for inhibition of NE release
was a meta-methyl group. Furthermore, for inhibi-
H
F
H
H
Cl
Br
CH3
OCH3
H
3m
3n
3o
4a
4b
4c
4d
4e
H
H
Åꢀ
tion of neutrophil O2 generation, C-20 substitution
H
H
resulted in higher potency than C-30 or C-40 substitu-
tion, but C-30 substitution was preferable in the NE
release inhibition assay.
H
H
F
Cl
H
H
H
Br
CH3
OCH3
H
H
H
H
In conclusion, the bioassay results of these compounds
show dramatic relationships between structure and
anti-platelet aggregation and anti-inflammation effects.
This is the first reported evaluation of the anti-platelet
aggregatory and anti-inflammatory effects of 2-ben-
zoylaminobenzoic acid derivatives. In contrast to the
anti-platelet aggregation of bezoxazinones, the 2-ben-
zoylamino-4-chlorobenzoic acid derivatives showed
selective inhibitory effects on AA-induced platelet
aggregation. These observations confirmed that the
oxazinone ring is necessary for inhibiting platelet
aggregation induced by thrombin.8 Among the tested
compounds, 6b and 7b exhibited 200-fold more potent
inhibitory effects than aspirin on AA-induced platelet
aggregation, which suggested that 2-benzoylaminoben-
zoic acid derivatives also can be designed as aspirin-
like anti-platelet agents. Additionally, compounds 1a
and 5a showed strong inhibitory effects on neutrophil
superoxide generation with IC50 values of 0.65 and
0.17 lM, respectively. The present results led to the
identification of 1a and 5a as a new generic class of
lead compounds for anti-inflammatory agents. Addi-
tionally, compounds 6d and 6e exhibited dual inhibito-
ry effects on platelet aggregation and NE release;
therefore, these two compounds may represent new
leads for development as anti-inflammatory and anti-
platelet aggregatory agents.
H
H
Table 2. The 2-benzoylamino-benzoic acid derivates
Compound
R4
R5
R6
5a
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
8a
OCH2CH3
OCH2CH3
H
F
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
F
Cl
OCH2CH3
OCH2CH3
Br
OCH2CH3
OCH2CH3
CH3
OCH3
F
OCH2CH2CH3
OCH2CH2CH3
OCH2CH2CH3
OCH2CH2CH3
OCH2CH2CH3
CH3
Cl
Br
CH3
OCH3
F
phenylaline (fMLP) were also tested as a measure of
anti-inflammatory activity.19 Results are presented in
Table 3. Comparison of series 1a–1e, 2a–2e, 3a–3e,
and 4a–4e showed that only serÅiꢀes 1a–1e exhibited inhib-
itory effects on neutrophil O2 generation, indicating

Table 3. The 1H NMR data (in CDCl3) of new compounds (d)
Compound
dH (mult., J)
2a
9.43 (1H, br d, J = 13.6 Hz, NH), 8.17 (1H, t, J = 8.0 Hz, Ar-H), 7.83 (1H, d, J = 8.0 Hz, Ar-H), 7.64 (1H, d, J = 8.0 Hz, Ar-H), 7.54 (1H, br q, J = 8.0 Hz, Ar-H), 7.25
(3H, m, Ar-H), 3.87 (3H, s, COOMe)
2b
2c
8.88 (1H, br s, NH), 7.81 (1H, br t, J = 8.0 Hz, Ar-H), 7.63 (1H, d, J = 8.0 Hz, Ar-H), 7.42 (3H, m, Ar-H), 7.25 (1H, br m, Ar-H), 3.89 (3H, s, COOMe)
8.72 (1H, br s, NH), 7.83 (1H, dd, J = 8.0, 1.6 Hz, Ar-H), 7.72 (1H, dd, J = 8.0, 1.6 Hz, Ar-H), 7.65 (1H, dd, J = 8.0, 1.6 Hz, Ar-H), 7.64 (1H, dd, J = 8.0, 1.6 Hz, Ar-H), 7.41
(1H, td, J = 8.0, 1.6 Hz, Ar-H), 7.32 (1H, td, J = 8.0, 1.6 Hz, Ar-H), 7.26 (1H, t, J = 8.0 Hz, Ar-H), 3.89 (3H, s, COOMe)
8.65 (1H, br s, NH), 7.84 (1H, dd, J = 7.6, 1.6 Hz, Ar-H), 7.68 (1H, d, J = 7.6 Hz, Ar-H), 7.63 (1H, dd, J = 8.0, 1.6 Hz, Ar-H), 7.37 (1H, dd, J = 8.0, 1.6 Hz, Ar-H), 7.27 (2H,
br t, J = 8.0 Hz, Ar-H), 7.25 (1H, t, J = 7.6 Hz, Ar-H), 3.89 (3H, s, COOMe), 2.55 (3H, s, Ar-CH3)
2d
2e
3a
3c
3l
10.52 (1H, br s, NH), 8.26 (1H, dd, J = 7.6, 1.6 Hz, Ar-H), 7.74 (1H, dd, J = 7.6, 1.6 Hz, Ar-H), 7.59 (1H, dd, J = 8.0, 1.6 Hz, Ar-H), 7.51 (1H, m, Ar-H), 7.20
(1H, t, J = 8.0 Hz, Ar-H), 7.11 (1H, td, J = 8.0, 1.6 Hz, Ar-H), 7.05 (1H, t, J = 7.6 Hz, Ar-H), 4.08 (3H, s, OMe), 3.84 (3H, s, COOMe)
11.90 (1H, br d, J = 7.6 Hz, NH), 9.01 (1H, d, J = 2.0 Hz, Ar-H), 8.06 (1H, td, J = 7.6, 2.0 Hz, Ar-H), 7.99 (1H, d, J = 8.0 Hz, Ar-H), 7.52 (1H, m, Ar-H), 7.30
(1H, t, J = 8.0 Hz, Ar-H), 7.20 (1H, dd, J = 8.0, 8.0 Hz, Ar-H), 7.10 (1H, dd, J = 8.0, 2.0 Hz, Ar-H), 3.94 (3H, s, COOMe)
11.52 (1H, br s, NH), 9.00 (1H, d, J = 2.0 Hz, Ar-H), 7.99 (1H, d, J = 8.0 Hz, Ar-H), 7.66 (1H, dd, J = 8.0, 2.0 Hz, Ar-H), 7.59 (1H, dd, J = 8.0, 2.0 Hz, Ar-H), 7.42
(1H, td, J = 8.0, 2.0 Hz, Ar-H), 7.33 (1H, td, J = 8.0, 2.0 Hz, Ar-H), 7.12 (1H, dd, J = 8.0, 2.0 Hz, Ar-H), 3.89 (3H, s, COOMe)
12.09 (1H, br s, NH), 8.99 (1H, d, J = 2.0 Hz, Ar-H), 7.99 (1H, d, J = 8.4 Hz, Ar-H), 7.96 (2H, d, J = 8.8 Hz, Ar-H), 7.49 (2H, d, J = 8.8 Hz, Ar-H), 7.09
(1H, dd, J = 8.4, 2.0 Hz, Ar-H), 3.96 (3H, s, COOMe)
3m
3n
3o
4a
12.08 (1H, br s, NH), 8.97 (1H, d, J = 2.0 Hz, Ar-H), 7.97 (1H, d, J = 8.4 Hz, Ar-H), 7.87 (2H, d, J = 8.8 Hz, Ar-H), 7.64 (2H, d, J = 8.8 Hz, Ar-H), 7.08
(1H, dd, J = 8.4, 2.0 Hz, Ar-H), 3.95 (3H, s, COOMe)
11.98 (1H, br s, NH), 8.99 (1H, d, J = 2.4 Hz, Ar-H), 7.92 (1H, d, J = 8.8 Hz, Ar-H), 7.88 (2H, d, J = 8.4 Hz, Ar-H), 7.27 (2H, d, J = 8.4 Hz, Ar-H), 7.01
(1H, dd, J = 8.8, 2.4 Hz, Ar-H), 3.95 (3H, s, COOMe), 2.39 (3H, s, Ar-CH3)
12.00 (1H, br s, NH), 9.03 (1H, d, J = 2.0 Hz, Ar-H), 8.00 (2H, d, J = 8.4 Hz, Ar-H), 7.99 (1H, d, J = 8.8 Hz, Ar-H), 7.06 (1H, dd, J = 8.8, 2.0 Hz, Ar-H),
7.01 (2H, d, J = 8.4 Hz, Ar-H), 3.96 (3H, s, COOMe), 3.88 (3H, s, Ar-CH3)
9.76 (1H, br d, J = 13.2 Hz, NH), 8.36 (1H, d, J = 8.0 Hz, Ar-H), 8.13 (1H, td, J = 8.0, 1.6 Hz, Ar-H), 7.53 (1H, m, Ar-H), 7.39 (1H, d, J = 8.0 Hz, Ar-H), 7.30
(1H, td, J = 8.0, 1.6 Hz, Ar-H), 7.22 (1H, dd, J = 8.0, 1.6 Hz, Ar-H), 7.19 (1H, dd, J = 8.0, 8.0 Hz, Ar-H), 4.00 (3H, s, COOMe)
9.41 (1H, br s, NH), 8.41 (1H, d, J = 8.0 Hz, Ar-H), 7.70 (1H, dd, J = 8.0, 1.6 Hz, Ar-H), 7.41 (4H, m, Ar-H), 7.24 (1H, dd, J = 8.0, 1.6 Hz, Ar-H), 3.96 (3H, s, COOMe)
9.30 (1H, br s, NH), 8.43 (1H, d, J = 8.0 Hz, Ar-H), 7.65 (1H, dd, J = 8.0, 1.2 Hz, Ar-H), 7.59 (1H, dd, J = 8.0, 1.2 Hz, Ar-H), 7.43 (2H, m, Ar-H), 7.34
(1H, td, J = 8.0, 1.2 Hz, Ar-H), 7.24 (1H, dd, J = 8.0, 1.2 Hz, Ar-H), 3.96 (3H, s, COOMe)
4b
4c
4d
4e
6a
6b
6c
6d
6e
7a
9.37 (1H, br s, NH), 8.45 (1H, d, J = 8.0 Hz, Ar-H), 7.52 (1H, dd, J = 8.0, 1.6 Hz, Ar-H), 7.39 (2H, m, Ar-H), 7.29 (2H, br t, J = 8.0 Hz, Ar-H), 7.22
(1H, dd, J = 8.0, 1.6 Hz, Ar-H), 3.96 (3H, s, COOMe), 2.54 (3H, s, Ar-CH3)
10.65 (1H, br s, NH), 8.39 (1H, d, J = 8.0 Hz, Ar-H), 8.27 (1H, dd, J = 8.0, 2.0 Hz, Ar-H), 7.51 (1H, ddd, J = 8.4, 8.0, 2.0 Hz, Ar-H), 7.38 (1H, t, J = 8.0 Hz, Ar-H),
7.17 (1H, br t, J = 8.0 Hz, Ar-H), 7.11 (1H, br t, J = 8.0 Hz, Ar-H), 7.03 (1H, d, J = 8.0 Hz, Ar-H), 4.10 (3H, s, OMe), 3.96 (3H, s, COOMe)
11.95 (1H, br d, J = 7.6, NH), 9.02 (1H, d, J = 2.2 Hz, Ar-H), 8.06 (1H, td, J = 8.0, 1.8 Hz, Ar-H), 8.02 (1H, d, J = 8.6 Ar-H), 7.53 (1H, m, Ar-H), 7.29 (1H, td, J = 8.0,
7.0, 1.2 Hz, Ar-H), 7.20 (1H, ddd, J = 11.6, 7.0,1.2 Hz, Ar-H), 7.11 (1H, dd, J = 8.6, 2.2 Hz, Ar-H), 4.40 (2H, q, J = 7.2 Hz, OCH2CH3), 1.41 (3H, t, J = 7.2 Hz, OCH2CH3)
11.47 (1H, s, NH), 8.82 (1H, d, J = 1.8 Hz, Ar-H), 7.82 (1H, d, J = 8.4 Hz, Ar-H), 7.47 (1H, dd, J = 7.6, 2.6, Ar-H), 7.23 (3H, m, Ar-H), 6.91 (1H, dd, J = 8.4, 1.8 Hz, Ar-H),
4.16 (2H, q, J = 7.2 Hz, OCH2CH3), 1.92 (3H, t, J = 7.2 Hz, OCH2CH3)
11.55 (1H, s, NH), 9.00 (1H, d, J = 2.2 Hz, Ar-H), 8.02 (1H, d, J = 8.8 Hz, Ar-H), 7.66 (1H, dd, J = 7.6, 1.0 Hz Ar-H), 7.59 (1H, dd, J = 7.2, 1.8 Hz, Ar-H), 7.43 (1H, m, Ar-H),
7.35 (1H, m, Ar-H), 7.13 (1H, dd, J = 8.8, 2.2 Hz, Ar-H), 4.35 (2H, q, J = 7.0 Hz, OCH2CH3), 1.38 (3H, t, J = 7.0 Hz, OCH2CH3)
11.56 (1H, s, NH), 9.03 (1H, d, J = 2.2 Hz, Ar-H), 8.01 (1H, d, J = 8.8 Hz, Ar-H), 7.60 (1H, d, J = 7.6 Hz, Ar-H), 7.36 (3H, m, Ar-H), 7.10 (1H, dd, J = 8.8, 2.2 Hz, Ar-H),
4.36 (2H, q, J = 7.0 Hz, OCH2CH3), 2.56 (3H, s, Ar-CH3), 1.39 (3H, t, J = 7.0 Hz, OCH2CH3)
12.30 (1H, s, NH), 9.09 (1H, d, J = 1.8 Hz, Ar-H), 8.18 (1H, dd, J = 7.6, 1.8 Hz, Ar-H), 7.98 (1H, d, J = 8.8 Hz, Ar-H), 7.49 (1H,, ddd, J = 8.4, 7.6,1.8 Hz, Ar-H),
7.06 (3H, m, Ar-H), 7.10 (1H, dd, J = 8.8, 1.8 Hz, Ar-H), 4.38 (2H, q, J = 7.0 Hz, OCH2CH3), 4.06 (3H, s, OMe), 1.41 (3H, t, J = 7.0 Hz, OCH2CH3)
11.95 (1H, br d, J = 7.6 Hz, NH), 9.03 (1H, d, J = 2.2 Hz, Ar-H), 8.06 (1H, td, J = 7.8, 2.0 Hz, Ar-H), 8.02 (1H, d, J = 8.6 Hz, Ar-H), 7.53 (1H, m, Ar-H), 7.31 (1H, td, J = 7.8,
1.0 Hz, Ar-H), 7.20 (1H, ddd, J = 8.6, 7.8, 1.0 Hz, Ar-H), 7.11 (1H, dd, J = 8.6, 2.2 Hz, Ar-H), 4.30 (2H, t, J = 6.8 Hz, OCH2CH2CH3), 1.80 (2H, m, OCH2CH2CH3), 1.03
(3H, t, J = 7.2 Hz, OCH2CH2CH3)
7b
7c
11.63 (1H, s, NH), 9.01 (1H, d, J = 2.2 Hz, Ar-H), 8.01 (1H, d, J = 8.6 Hz, Ar-H), 7.64 (1H, dd, J = 6.6, 2.8 Hz, Ar-H), 7.41 (3H, m, Ar-H), 7.12 (1H, d, J = 8.6, 2.2 Hz, Ar-H),
4.25 (2H, t, J = 6.0 Hz, OCH2CH2CH3), 1.78 (2H, m, OCH2 CH2CH3), 1.00 (3H, t, J = 7.4 Hz, OCH2CH2CH3)
11.56 (1H, s, NH), 9.00 (1H, d, J = 2.2 Hz, Ar-H), 8.01 (1H, d, J = 8.6 Hz, Ar-H), 7.61 (1H, dd, J = 8.0, 1.6 Hz, Ar-H), 7.47 (1H, dd, J = 7.4, 2.2 Hz, Ar-H), 7.42
(1H, m, Ar-H), 7.34 (1H, m, Ar-H), 7.12 (1H, dd, J = 8.6, 2.2 Hz, Ar-H), 4.25 (2H, t, J = 6.0 Hz, OCH2CH2CH3), 1.78 (2H, m, OCH2CH2CH3), 1.00
(3H, t, J = 7.4 Hz, OCH2CH2CH3)

1816
P.-W. Hsieh et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1812–1817
Table 4. The bioactivity data of compounds (lM)
Compound
Antiplatelet
aggregation
Anti-inflammation
Åꢀ
O2 NE
AA
Thr
generation release
Aspirinc
151.6 1.2a,b >100 >40
>40
>40
Indomethacinc 0.14 0.0
>100 >40
0.86 0.35
DPId
PMSFd
130.93 29.10
1a
1b
1c
1d
1e
1f
10.8 3.7
11.0 3.4
22.9 6.2
16.6 7.3
8.0 2.0
>100
>100 0.65 0.36 >40
>100 11.01 2.82 >40
>100 12.02 2.10 >40
>100 9.26 2.22 >40
>100 7.41 0.59 >40
>100 10.56 5.75 >40
1g
1h
1i
>100
>100
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>40
15.40 2.14
18.30 0.76
>40
>100
>100
1j
1k
1l
>100
>100
>40
>40
1m
1n
1o
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
3k
3l
>100
>100
>40
>40
>100
>100
>40
>40
>100
>100
>100
>40
>40
>40
>40
66.6 23.6
2.2 0.5
1.1 0.3
2.6 0.2
4.3 2.5
2.2 0.6
>100
>40
>40
>40
>40
>40
>40
>100
>100
>40
>40
3m
3n
3o
4a
4b
4c
4d
4e
5a
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
8a
>100
>100
>40
>40
78.2 15.4
80.8 13.6
76.4 16.7
>100
>40
>40
>40
>40
62.3 15.4
NT
>40
>100 0.17 0.03 14.35 1.92
3.8 1.1
0.8 0.0
3.6 0.0
4.9 1.1
4.9 1.1
1.6 0.0
0.7 0.0
3.5 0.0
2.2 0.5
23.4 6.2
NT
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 >40
>100 24.1 2.8
>40
>40
>40
2.83 1.05
4.31 1.55
>40
>40
>40
>40
>40
>40
a Platelets were pre-incubated with DMSO (0.5%, control), aspirin or
test compound at 37 °C for 3 min before addition of the inducer.
b The IC50 values are presented as means SEM (n = 3).
c Aspirin and indomethacin, two cyclooxygenase inhibitors, were used
as positive control in platelet aggregation assay.
d Diphenyleneiodonium (DPI) and phenylmethylsulfonyl fluoride
(PMSF) were used as positive control in anti-inflammatory assay.

P.-W. Hsieh et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1812–1817
1817
chromatography (Si-Gel) using CHCl3/hexane (2:7) mix-
ture to afford the products. Compound 2a: EI-MS m/z:
157 (100), 274 (42), 306 [M]+ (38). Compound 2b: EI-MS
m/z: 139 (100), 256 (40), 324 [M]+ (1). Compound 2c: EI-
MS m/z: 183 (100), 332 (50), 368 [M]+ (0.5). Compound
2d: EI-MS m/z: 91 (100), 119 (88), 268 (52), 304 [M]+ (0.2).
Compound 2e: EI-MS m/z: 135 (100), 284 (54), 320 [M]+
(1). Compound 3a: EI-MS m/z: 307 (18), 309 [M]+ (6).
Compound 3c: EI-MS m/z: 367 [M]+ (20), 369 (27).
Compound 3e: EI-MS m/z: 319 [M]+ (3). Compound 3l:
EI-MS m/z: 323 [M]+ (15), 325 (10). Compound 3m: EI-
MS m/z: 367 [M]+ (21), 369 (28). Compound 3n: EI-MS m/z:
303 [M]+ (18). Compound 3o: EI-MS m/z: 319 [M]+ (7).
Compound 4a: EI-MS m/z: 123 (100), 248 (40), 307 [M]+
(7). Compound 4b: EI-MS m/z: 139 (100), 264 (28), 323
[M]+ (5). Compound 4c: EI-MS m/z: 183 (100), 310 (31),
367 [M]+ (7), 369 (8). Compound 4d: EI-MS m/z: 91 (100),
119 (99), 244 (17), 303 [M]+ (2). Compound 4e: EI-MS m/z:
135 (100), 260 (17), 319 [M]+ (4).
Acknowledgment
This work was supported by a grant from the National
Science Council of the Republic of China (NSC94-2314-
B-037-105).
References and notes
1. Hankey, G. J.; Eikelboom, J. W. Med. J. Aust. 2003, 178,
568.
2. Nathan, C. Nat. Rev. Immunol. 2006, 6, 173.
3. Borregaard, N. Eur. J. Haematol. 1998, 41, 401.
4. Ross, D.; Van Bruggen, R.; Meischl, C. Microb. Infect.
2003, 5, 1307.
5. Witko-Sarsat, V.; Rieu, P.; Descamps-Latscha, B.; Lesa-
vre, P.; Halbwachs-Mecarelli, L. Lab. Invest. 2000, 80,
617.
6. Hwang, T. L.; Hung, H. W.; Kao, S. H.; Teng, C. M.; Wu,
C. C.; Cheng, S. J. Mol. Pharmacol. 2003, 64, 1419.
7. Chang, F. R.; Wu, C. C.; Hwang, T. L.; Patnam, R.; Kuo,
R. Y.; Wang, W. Y.; Lan, Y. H.; Wu, Y. C. Arch.
Pharmacol. Res. 2003, 26, 511.
8. 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.
9. Wu, C. C.; Wang, T. W.; Wang, W. Y.; Hsieh, P. W.; Wu,
Y. C. Eur. J. Pharmacol. 2005, 527, 37.
10. Kurosaki, F.; Naishi, A. Phytochemistry 1983, 22, 669.
11. Ponchet, M.; Favre-Bonvin, J.; Hauteville, M.; Ricci, P.
Phytochemistry 1988, 27, 725.
12. Hauteville, M.; Ponchet, M.; Ricci, P.; Favre-Bonvin, J.
J. Heterocycl. Chem. 1988, 25, 715.
13. Kirino, O.; Yamamoto, S.; Kato, T. Agric. Biol. Chem.
1980, 44, 2149.
14. Fenton, G.; Newton, C. G.; Wyman, B. M.; Bagge, P.;
Dron, D. I.; David, R.; Graham, D. J. J. Med. Chem.
1989, 32, 265.
15. Ogita, H.; Isobe, Y.; Takaku, H.; Sekine, R.; Goto, Y.;
Misawa, S.; Hayashi, H. Bioorg. Med. Chem. Lett. 2001,
11, 549.
16. General experimental procedure for the synthesis of
compounds 1a–1o, 2a–2e, 3a–3e, 3k–3o, and 4a–4e. To a
DCM solution of methyl anthranilate (1.0 mmol) were
added corresponding substituted benzoyl chlorides. The
reaction mixture was stirred overnight at room tempera-
ture for 16 h, respectively. The solvent was evaporated at
reduced pressure. The residue was purified by column
17. General experimental procedure for the synthesis of
compounds 5a, 6a–6e, 7a–7e, and 8a. To a DCM solution
of ethyl anthranilate, ethyl 2-amino-4-chloro-benzoate,
propyl 2-amino-4-chloro-benzoate, or 2-amino-acetophe-
none (each 1.0 mmol) was added, respectively, and then
mixed with corresponding substituted benzoyl chlorides.
The reaction mixture was stirred overnight at room
temperature for 16 h, respectively. The solvent was evap-
orated at reduced pressure. The residue was purified by
column chromatography (Si-Gel) using CHCl3/hexane
(1:4) mixture to afford the products. Compound 6a: EI-
MS m/z: 123 (100), 248 (75), 250 (22), 321 [M]+ (35),
323(12). Compound 6b: EI-MS m/z: 139 (100), 141 (33),
264 (34), 266 (23), 320 (99), 322 (65), 337[M]+ (60).
Compound 6c: EI-MS m/z: 183 (100), 185 (99), 230 (55),
381 [M]+ (15), 383 (20). Compound 6d: EI-MS m/z: 119
(100), 300 (31), 317[M]+ (12). Compound 6e: EI-MS m/z:
135 (100), 201 (16), 333 [M]+ (22), 334 (15). Compound 7a:
EI-MS m/z: 123 (100), 248 (65), 318 (49), 335 [M]+ (32).
Compound 7b: EI-MS m/z: 139 (100), 264 (22), 351 [M]+
(13), 353 (9). Compound 7c: EI-MS m/z: 185(100), 230
(44), 395 [M]+ (7), 397 (8). Compound 7d: EI-MS m/z: 119
(100), 331 [M]+ (5). Compound 7e: EI-MS m/z: 135 (100),
213 (79), 347 [M]+ (2).
18. Antiplatelet aggregation assays: see Wu, C. C.; Wang, W.
Y.; Kuo, R. Y.; Chang, F. R.; Wu, Y. C. Eur.
J. Pharmacol. 2004, 483, 187.
19. Superoxide anion formation and elastase release: see
Hwang, T. L.; Hung, H. S.; Kao, S. H.; Teng, C. M.;
Wu, C. C.; Cheng, S. J. S. Mol. Pharmacol. 2003, 64, 1.