Synthesis and evaluation of benzoxazinone derivatives on activity of human neutrophil elastase and on hemorrhagic shock-induced lung injury in rats

Article abstract of DOI:10.1016/j.ejmech.2010.03.046

A new series of benzoxazinone analogs were designed, synthesized, and assayed to determine their effects on superoxide anion generation and neutrophil elastase (NE) release in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils.

Full text of DOI:10.1016/j.ejmech.2010.03.046

European Journal of Medicinal Chemistry 45 (2010) 3111e3115
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of benzoxazinone derivatives on activity of human
neutrophil elastase and on hemorrhagic shock-induced lung injury in rats
,
b
,
,
Pei-Wen Hsieh a , Huang-Ping Yu c, Yi-Ju Chang a, Tsong-Long Hwang a
*
**
a Graduate Institute of Natural Products, Chang Gung University, Tao-Yuan 333, Taiwan
b Department of Anesthesiology, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan
c College of Medicine, Chang Gung University, Taoyuan 333, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of benzoxazinone analogs were designed, synthesized, and assayed to determine their
effects on superoxide anion generation and neutrophil elastase (NE) release in formyl-L-methionyl-
L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils. Of these, compounds 6e10 showed
a potent dual inhibitory effect on NE release and superoxide anion generation. In contrast, compounds
11e15 exhibited highly selective and potent inhibitory activities on NE release. These results indicate that
the inhibitory activity on NE release in FMLP-activated human neutrophils depended on the position of
chloro-substituent in the A ring. On the other hand, 13 significantly attenuated the increase in myelo-
peroxidase (MPO) activity and edema in the lung of rats after trauma-hemorrhagic shock. Therefore,
these compounds could be developed as new NE inhibitors.
Received 17 November 2009
Received in revised form
25 March 2010
Accepted 29 March 2010
Available online 3 April 2010
Keywords:
Human neutrophil elastase
Benzoxazinones
Ó 2010 Elsevier Masson SAS. All rights reserved.
Superoxide anion
Hemorrhagic shock
Anti-inflammatory agents
1. Introduction
syndrome [3,4,5]. Of these, NE is a major secreted product of
stimulated neutrophils and a major contributor to the destruction
Neutrophils play a pivotal role in the defense of the human body
against infections. In addition, neutrophil infiltration is a common
pathological feature in acute inflammatory disorders [1]. In
response to diverse stimuli, activated neutrophils secrete a series of
cytotoxins, such as superoxide anion (Oꢀ2ꢁ), a precursor of other
reactive oxygen species, granule proteases, and bioactive lipids [2].
Of these, neutrophil elastase (NE) a 30-kDa glycoprotein chymo-
trypsin-like serine proteinase is stored in the azurophil granules of
neutrophils in its active form and is released following neutrophil
exposure to inflammatory stimuli [3]. Although a vigorous response
by neutrophils to infection and injury is necessary for host defense,
overly aggressive or inappropriate neutrophil responses can result
in deleterious inflammatory conditions and tissue destruction [1].
High concentrations of reactive oxygen species (ROS) and NE
produced by activated neutrophils in the sputum of patients has
been implicated in the pathogenesis of many acute and chronic
pulmonary diseases including asthma, chronic obstructive pulmo-
nary disease, cystic fibrosis, and acute respiratory distress
of tissue in chronic inflammatory disease [6,7]. For example, NE
exists in high concentrations in the airway secretions of patients
with chronic inflammatory airway diseases and induces over-
production of MUC5AC mucin, a major component of airway mucus
[8]. In addition, NE is present within atherosclerotic plaques where
it contributes to matrix degradation and weakening of the vessel
wall associated with complications such as aneurysm formation
and plaque rupture [9]. Furthermore, high NE activity is found in
synovial fluid from patients with non-infectious knee joint syno-
vitis and rheumatoid arthritis [10].
NE has a broad substrate specificity to break down extracellular
matrices, cytokines, clotting factors, adhesion molecules, and
components of the complement cascade [11,12,13]. Potential
substrates of NE include extracellular matrices, cytokines, clotting
factors, adhesion molecules, and components of the complement
cascade [11,12]. As a result of this broad substrate specificity and the
ability of this enzyme to produce tissue injury, NE has been
involved in the pathogenesis of various inflammatory conditions
and indeed it is often used as both a predictor and an indicator of
inflammatory disease severity [12]. Since NE is important in the
pathogenesis of inflammatory diseases, NE may represent a poten-
tial target in the development of new anti-inflammatory agents.
However, there are currently only a few agents available that
* Corresponding author. Tel.: þ886 3 211 8800x3105; fax: þ886 3 211 8643.
** Corresponding author. Tel./fax: þ886 3 2118506.
E-mail addresses: pewehs@mail.cgu.edu.tw (P.-W. Hsieh), htl@mail.cgu.edu.tw
(T.-L. Hwang).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.03.046

3112
P.-W. Hsieh et al. / European Journal of Medicinal Chemistry 45 (2010) 3111e3115
Table 2
directly modulate NE responses in clinical practice. Therefore,
research and development of a new generation of anti-inflamma-
tory agents are needed and are in progress.
Inhibitory effects of compounds 10e15 on activity of human NE in a cell-free
conditioned medium.
Compounds
PMSFb
10
11
12
13
14
15
IC50 (nM)a
Substituted benzoxazin-4-ones have been well characterized as
heterocyclic acylating agents of serine proteases [14]. These
compounds inhibit serine proteases according to a mechanism
which involves an acyl enzyme intermediate [14]. Many benzox-
azin-4-ones have been synthesized and assayed for their effects on
NE activities [15,16,17,18,19,20]. Furthermore, in a previous study,
we synthesized and evaluated the effects of a series of 2,8-
substituted benzoxazinones on the inhibition of NE release and O2ꢀꢁ
95219.3 ꢂ 19225.8
612.4 ꢂ 179.8
52.7 ꢂ 4.5
30.7 ꢂ 4.0
24.8 ꢂ 1.9
57.1 ꢂ 9.2
40.0 ꢂ 4.6
a
The IC50 values are presented as mean ꢂ S.E.M. (n ¼ 3).
b
Phenylmethylsulfonyl fluoride (PMSF) was used as a positive control in this
generation in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-
assay.
activated human neutrophils [3]. Of these compounds, most of the
2-phenyl-8-chloro-benzoxazinones (1e5) showed a dual inhibitory
effect on NE release and Oꢀ2ꢁ generation. In the present study, in
order to examine the structural determinants required for the anti-
inflammatory bioactivity of benzoxazinones and to develop more
potent and selective inhibitors of NE, a new series of chloro-
substituted 2-phenylbenzoxazinones were synthesized and phar-
macologically characterized.
pure and were characterized by spectroscopic data, as shown in the
Section 5.
3. Results and discussion
In the present study, ten chloro-substituted 2-phenyl-
benzoxazinones were synthesized and their inhibitory effects on
NE release and Oꢀ2ꢁ generation induced by FMLP were compared. All
the synthetics showed a potent inhibitory effect on NE release
(Table 1). Compounds 6e10 showed a potent dual inhibitory effect
on NE release and Oꢀ2ꢁ generation. These effects were decreased or
disappeared when the chlorine was substituted at C-5 and C-8.
Conversely, compounds 11e15, with a chloro-substitution at C-5
exhibited highly selective inhibitory effects on NE release with IC50
values of 77.1, 47.1, 80.8, 47.6, and 64.4 nM, respectively. Our results
indicate that compounds 11e15 showed more activity in the inhi-
bition of NE release than the corresponding chloro-substitution at
C-7 (6e10) and C-8 (1e5) by about 100- to 2000-fold, suggesting
that the inhibitory effect on NE release in FMLP-activated human
neutrophils depended on the position of the chloro-substituent in
the A ring. Although 2-(20-substituted-phenyl) benzoxazin-4-ones
have been well known acylating agents of serine proteases. In
addition, the substitutions at C-20 were affect acylating rate [14].
However, our results indicate the substitutions at C-20 of 5-chloro
compounds did not enhance activity significantly. We propose
5-chloro compounds may be as covalent binders of serine proteases
via a Michael addition-elimination.
2. Chemistry
In a previous SAR study, the compounds with a substitution on
the 6-position were found to be unfavorable [14]. Therefore, in the
present investigation, a series of 5-/ or 7-substituted 2-phenyl-
benzoxazinone derivates were prepared by reacting 2-amino-
4-chlorobenzoic acid and 2-amino-6-chlorobenzoic acid with the
corresponding substituted benzoyl chlorides as described in
a previous pathway [3,21]. All compounds (6e15, Table 1) were
Table 1
Anti-inflammatory activities of synthetics in FMLP-activated human neutrophils.
O
5
4
10
6
3
O
R1
6'
3'
1'
7
5'
2
9
N
1
8
R2
4'
2'
In a cell-free conditioned medium (Table 2) [22], compounds
11e15 also showed potent inhibitory effects on NE activity with IC50
values of 52.7, 30.7, 24.8, 57.1, and 40.0 nM, respectively. Therefore,
we next evaluated these analogs for their enzymatic activity on
elastase from human leukocytes [23]. The results showed that these
compounds directly inhibited elastase activity with IC50 values
below 50 nM (Table 3).
Compounds
Structures
IC50 (nM)a
R1
R2
O2ꢀꢁ generation
NE release
DPIb
969.7 ꢂ 394.6
NT
PMSFb
NTc
109518.5 ꢂ 25039.7
56450.0 ꢂ 9130.0
6030.0 ꢂ 650.0d
1890.0 ꢂ 1100.0d
1690.0 ꢂ 300.0d
gt;25000d
Elastatinalb
1
2
3
4
5
6
7
NT
8eCl
8eCl
8eCl
8eCl
8eCl
7eCl
7eCl
7eCl
7eCl
7eCl
5eCl
5eCl
5eCl
5eCl
5eCl
20eF
gt;25000d
20eCl
20eBr
20eCH3
8660.0 ꢂ 1440.0d
6560.0 ꢂ 3090.0d
17490.0 ꢂ 2690.0d
10030.0 ꢂ 590.0d
1381.8 ꢂ 129.6
2680.5 ꢂ 90.8
2002.1 ꢂ 158.5
NDe
It is well known that neutrophils are activated following
hemorrhagic shock [24], and the subsequent accumulation of
20eOCH3
20eF
1910.0 ꢂ 140.0d
1141.0 ꢂ 144.4
836.8 ꢂ 68.4
536.3 ꢂ 91.8
NDe
Table 3
Inhibitory effects of compounds 10e15 on enzymatic activity of elastase from
human leukocytes.
20eCl
8
9
20eBr
Compounds
PMSFb
10
11
12
13
14
15
IC50 (nM)a
20eCH3
20eOCH3
20eF
10
11
12
13
14
15
1206.1 ꢂ 127.1
23393.8 ꢂ 1232.9
gt;25000
gt;25000
gt;25000
227.5 ꢂ 47.4
52161.7 ꢂ 1073.4
221.5 ꢂ 59.2
30.8 ꢂ 5.8
77.1 ꢂ 15.3
20eCl
47.1 ꢂ 9.6
20eBr
80.8 ꢂ 10.3
28.7 ꢂ 6.2
20eCH3
20eOCH3
47.6 ꢂ 4.3
29.5 ꢂ 6.7
6475.5 ꢂ 1408.0
64.4 ꢂ 12.9
48.8 ꢂ 14.2
15.4 ꢂ 1.9
a
The IC50 values are presented as mean ꢂ S.E.M. (n ¼ 3).
b
a
Diphenyleneiodonium (DPI) [30], phenylmethylsulfonyl fluoride (PMSF) [31],
Elastase (EC 3.4.21.37) was incubated with compounds for 2 min. Elastase
activity was measured using ELISA reader at 405 nm, as described under Section 5.
All data are presented as mean ꢂ S.E.M. (n ¼ 3e4).
and elastatinal [32] were used as positive control in anti-inflammatory assay.
c
None test.
d
b
The biological data of compounds 1e5 came from the literature directly [11].
No data due to insolubility.
Phenylmethylsulfonyl fluoride (PMSF) was used as a positive control in this
e
assay.

P.-W. Hsieh et al. / European Journal of Medicinal Chemistry 45 (2010) 3111e3115
3113
anti-inflammatory agents. Furthermore, these agents might also
serve as a novel adjunct for improving depressed lung function
following adverse circulatory conditions.
5. Experimental
5.1. Materials and methods
Unless stated otherwise, the chemicals were acquired from
commercial sources and used without further purification. 2-
Amino-4-chlorobenzoic acid, 6-amino-4-chlorobenzoic acid, 2-
fluorobenzoyl chloride, 2-chlorobenzoyl chloride, 2-bromobenzoyl
chloride, 2-methylbenzoyl chloride, and 2-methoxybenzoyl chlo-
ride were purchased from ACROS. Compound purity was checked
by thin layer chromatography (TLC) on precoated silica gel plates
(Merck, Kieselgel 60 F254, layer thickness 0.25 mm). NMR spectra
were performed in 400 and 100 MHz for 1H and 13C, respectively. 1H
NMR: CDCl3 as solvent,
solvent,
on an API 3000Ô (Applied Biosystems) in positive or negative mode
d
¼ 7.265 ppm and 13C NMR: CDCl3 as
d
¼ 77.0 ppm. Low resolution ESI-MS spectra were obtained
(solvent: CH3OH).
5.1.1. General procedure for synthesis of chloro-substituted
2-phenylbenzoxazinones (6e15)
5-/or7-Chloro-substituted2-phenylbenzoxazinones,compounds
6e15 (Table 1), were prepared by reaction of 2-amino-4-
chlorobenzoic acid or 6-amino-4-chlorobenzoic acid, with corre-
sponding substituted benzoyl chlorides in pyridine solution as
described [3,21]. The reaction mixture was stirred at room temper-
ature for 24 h. The pyridine was evaporated at reduced pressure, and
further subjected to purification by silica column chromatography.
All products were fully characterized using spectral data.
Fig. 1. Effect of compound 13 on lung injury in rats after a sham operation (Sham) or
trauma-hemorrhage and resuscitation (T-H). Sprague-Dawley rats were treated with
compound 13 (1 mg/kg of body weight, intravenously), or an equal volume of the
vehicle (w0.2 mL, 10% DMSO). After 24 h, the MPO activity (A) and wet/dry weight
ratio (B) in the lung tissue were assayed, as described in Section 5.1. Data are shown as
mean ꢂ SEM of 6 rats in each group. Statistical analysis was performed using Student’s
t-test or one-way analysis of variance (ANOVA) followed by Tukey’s multiple-
5.1.1.1. 7-Chloro-2-(2-fluorophenyl)-4H-benzo[d][1,3]oxazin-4-one
(6). Yield: 85%, 1H NMR (400 MHz, CDCl3):
d
¼ 8.16 (1H, d,
J ¼ 8.0 Hz), 8.11 (1H, dt, J ¼ 2.0, 8.0 Hz), 7.70 (1H, d, J ¼ 2.0 Hz),
7.56 (1H, m), 7.50 (1H, dd, J ¼ 2.0, 8.0 Hz), 7.29 (1H, br.t,
J ¼ 8.0 Hz), 7.22 (1H, dd, J ¼ 8.0, 8.0 Hz). 13C NMR (100 MHz,
comparison test.
A value of p < 0.05 was considered statistically significant.
CDCl3):
d
¼ 161.5 (s, JCeF ¼ 260.0 Hz), 158.3 (s), 147.7 (s), 143.0 (s),
***p < 0.001 compared to Sham; ##p < 0.01; ###p < 0.001 compared to T-H þ Veh.
134.4 (d, JCeF ¼ 8.4 Hz), 131.2 (d), 131.2 (s), 129.8 (d), 129.2 (d),
127.2 (d), 124.3 (d), 124.3 (s), 117.3 (d, JCeF ¼ 21.2 Hz), 115.3 (s).
ESI-MS m/z 298 (100) [M þ Na]þ, 300 (31).
neutrophils in the lung is associated with lung injury [25, 26].
Therefore, regulation of the activity of blood neutrophils is
important in controlling lung damage caused by hemorrhagic
shock [27]. Herein, the protective effect of 13 in the lung injury
following trauma-hemorrhagic shock were examined. The results
showed no significant differences in the MPO activity and wet/dry
weight ratio between vehicle and compound 13 treated groups in
sham-operated animals. Administration of compound 13 (1 mg/kg
of body weight) significantly attenuated the increase in the MPO
activity and wet/dry weight ratio after trauma-hemorrhagic shock;
however, these injury parameters remained higher than in sham-
operated animals (Fig. 1). These data indicated that 13 attenuated
hemorrhagic shock-induced lung injury in rats.
5.1.1.2. 7-Chloro-2-(2-chlorophenyl)-4H-benzo[d][1,3]oxazin-4-one
(7). Yield: 75%,1H NMR (400 MHz, CDCl3):
d
¼ 8.18 (1H, d, J ¼ 8.0 Hz),
7.90 (1H, dd, J ¼ 2.0, 8.0 Hz), 7.72 (1H, d, J ¼ 2.0 Hz), 7.53 (2H, m), 7.48
(1H, dd, J ¼ 2.0, 8.0 Hz), 7.40 (1H, dt, J ¼ 2.0, 8.0 Hz). 13C NMR
(100 MHz, CDCl3):
d
¼ 158.4 (s),157.7 (s), 147.5 (s), 143.1 (s), 133.6 (s),
132.6 (d), 131.5 (d), 131.2 (d), 129.8 (d), 129.8 (s), 129.5 (d), 127.2 (d),
126.9 (d), 115.3 (s). ESI-MS m/z 314 (100) [M þ Na]þ, 316 (61).
5.1.1.3. 7-Chloro-2-(2-bromophenyl)-4H-benzo[d][1,3]oxazin-4-one
(8). Yield: 67%, 1H NMR (400 MHz, CDCl3):
d
¼ 8.20 (1H, dd, J ¼ 3.0,
8.0 Hz), 7.86 (1H, br.d, J ¼ 8.0 Hz), 7.73 (1H, dd, J ¼ 2.0, 8.0 Hz), 7.73
(1H, br.s), 7.54 (1H, br.d, J ¼ 8.0 Hz), 7.46 (1H, t, J ¼ 8.0 Hz), 7.39 (1H,
4. Conclusion
t, J ¼ 8.0 Hz). 13C NMR (100 MHz, CDCl3):
d
¼ 158.4 (s), 158.2 (s),
147.3 (s), 143.1 (s), 134.4 (d), 132.6 (d), 131.6 (s), 131.5 (d), 129.9 (d),
129.5 (d), 127.5 (d), 127.2 (d), 121.8 (s), 115.3 (s). ESI-MS m/z 360
(100) [M þ Na]þ, 358 (72).
In this study, the synthesis and pharmacologic evaluation of
a new series of chloro-substituted 2-phenylbenzoxazinones were
described. The bioassay results of these compounds showed
dramatic relationships between the structure of these compounds
and their anti-inflammatory effects. The data indicated that the
inhibition of human NE activity depended on the position of the
chloro-substituent in the A ring. Therefore, these compounds
are new NE inhibitors and could be developed as novel lead
5.1.1.4. 7-Chloro-2-(2-methylphenyl)-4H-benzo[d][1,3]oxazin-4-one
(9). Yield: 62%, 1H NMR (400 MHz, CDCl3):
d
¼ 8.16 (1H, dd, J ¼ 0.4,
8.4 Hz), 8.04 (1H, br.dd, J ¼ 2.0, 8.0 Hz), 7.68 (1H, dd, J ¼ 0.4, 2.0 Hz),
7.48 (1H, dd, J ¼ 2.0, 8.4 Hz), 7.44 (1H, dt, J ¼ 2.0, 8.0 Hz), 7.33 (2H,
br.t, J ¼ 8.0 Hz), 2.72 (3H, s). 13C NMR (100 MHz, CDCl3):
¼ 159.4
d

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P.-W. Hsieh et al. / European Journal of Medicinal Chemistry 45 (2010) 3111e3115
(s), 158.9 (s), 147.9 (s), 142.8 (s), 139.5 (s), 132.0 (d), 131.9 (s), 130.2
(d), 129.7 (d), 129.2 (s), 128.9 (d), 127.0 (d), 127.2 (d), 126.1 (d), 115.1
(s), 22.3 (q). ESI-MS m/z 294 (100) [MþNa]þ, 292 (32).
isolated with a standard method of dextran sedimentation prior to
centrifugation in a Ficoll Hypaque gradient and the hypotonic lysis
of erythrocytes. Purified neutrophils that contained gt; 98% viable
cells, as determined by the trypan blue exclusion method, were
resuspended in Ca2þ-free HBSS buffer at pH 7.4 and maintained at
4 ꢃC before use.
5.1.1.5. 7-Chloro-2-(2-methoxyphenyl)-4H-benzo[d][1,3]oxazin-4-one
(10). Yield: 58%, 1H NMR (400 MHz, CDCl3):
d
¼ 8.14 (1H, d,
J ¼ 8.4 Hz), 7.85 (1H, dd, J ¼ 2.0, 8.4 Hz), 7.68 (1H, d, J ¼ 2.0 Hz), 7.50
(1H, dt, J ¼ 2.0, 8.4 Hz), 7.46 (1H, dd, J ¼ 2.0, 8.4 Hz), 7.05 (1H, t,
J ¼ 8.4 Hz), 7.03 (1H, t, J ¼ 8.4 Hz), 3.92 (3H, s). 13C NMR (100 MHz,
5.2.2. Measurement of Oꢀ2ꢁ generation
The Oꢀ2ꢁ generation assay was based on the superoxide dis-
mutase (SOD)-inhibitable reduction of ferricytochrome c. In brief,
after supplementation with 0.5 mg/ml ferricytochrome c and 1 mM
Ca2þ, neutrophils (6 ꢄ 105/ml) were equilibrated at 37 ꢃC for 2 min
and incubated with compounds for 5 min. Cells were activated with
CDCl3):
d
¼ 159.0 (s),158.8 (s),158.7 (s),148.0 (s),142.6 (s),133.5 (d),
131.4 (d), 129.6 (d), 128.8 (d), 126.9 (d), 120.5 (d), 119.9 (s), 115.2 (s),
112.1 (d), 56.0 (q). ESI-MS m/z 310 (100) [M þ Na]þ, 312 (30).
5.1.1.6. 5-Chloro-2-(2-fluorophenyl)-4H-benzo[d][1,3]oxazin-4-one
100 nM FMLP for 10 min. When FMLP was used as a stimulant,1 mg/
(11). Yield: 64%, 1H NMR (400 MHz, CDCl3):
d
¼ 8.10 (1H, dt, J ¼ 2.0,
ml cytochalasin B (CB) was incubated for 3 min before activation.
Changes in absorbance with the reduction of ferricytochrome c at
550 nm were continually monitored in a double-beam, six-cell
positioner spectrophotometer with constant stirring (Hitachi
U-3010, Tokyo, Japan) [22,27].
8.0 Hz), 7.69 (1H, t, J ¼ 8.0 Hz), 7.60 (1H, dd, J ¼ 0.4, 8.0 Hz), 7.55
(2H, m), 7.28 (1H, t, J ¼ 8.4 Hz), 7.21 (1H, dd, J ¼ 8.4, 8.0 Hz). 13C NMR
(100 MHz, CDCl3):
d
¼ 161.4 (s, JCeF ¼ 259.3 Hz), 155.6 (s), 148.9 (s),
135.9 (s), 134.3 (d, JCeF ¼ 9.1 Hz), 131.7 (s), 131.1 (d, 2C), 126.4 (d),
126.0 (s), 124.3 (d, JCeF ¼ 3.7 Hz), 121.2 (s), 117.3 (d, JCeF ¼ 21.2 Hz),
114.7 (s). ESI-MS m/z 298 (100) [M þ Na]þ, 300 (34).
5.2.3. Measurement of NE release
Degranulation of azurophilic granules was determined by NE
release. Experiments were performed using MeO-Suc-Ala-Ala-Pro-
Val-p-nitroanilide as the NE substrate. Briefly, following supple-
5.1.1.7. 5-Chloro-2-(2-chlorophenyl)-4H-benzo[d][1,3]oxazin-4-one
(12). Yield: 51%, 1H NMR (400 MHz, CDCl3):
d
¼ 7.90 (1H, dd,
J ¼ 1.6, 8.0 Hz), 7.71 (1H, t, J ¼ 8.0 Hz), 7.61 (1H, dd, J ¼ 1.6, 8.0 Hz),
7.57 (1H, dd, J ¼ 1.6, 8.0 Hz), 7.51 (1H, dd, J ¼ 1.6, 8.0 Hz), 7.47 (1H,
dt, J ¼ 1.6, 8.0 Hz), 7.40 (1H, dt, J ¼ 1.6, 8.0 Hz). 13C NMR (100 MHz,
mentation with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 mM),
neutrophils (6 ꢄ 105/ml) were equilibrated at 37 ꢃC for 2 min and
incubated with the compounds for 5 min. Cells were activated by
CDCl3):
d
¼ 157.2 (s), 155.8 (s), 148.7 (s), 136.0 (d), 133.5 (s), 132.5
FMLP (100 nM) in the presence of CB (0.5 mg/ml), and changes in
(d), 131.4 (d), 131.3 (d), 131.3 (s), 131.2 (d), 129.7 (s), 126.9 (d), 126.4
absorbance at 405 nm were continuously monitored to determine
NE release. The results are expressed as a percentage of NE release
in the FMLP/CB-activated, drug-free control system.
(d), 114.7 (s). ESI-MS m/z 314 (100) [M þ Na]þ, 316 (64).
5.1.1.8. 5-Chloro-2-(2-bromophenyl)-4H-benzo[d][1,3]oxazin-4-one
To determine whether the compounds exhibited an inhibitory
effect on NE activity, a direct NE activity assay was performed in
a cell-free conditioned medium. Neutrophils (6 ꢄ 105/ml) were
incubated for 20 min in the presence of FMLP (100 nM)/CB
(13). Yield: 63%, 1H NMR (400 MHz, CDCl3):
d
¼ 7.85 (1H, dd,
J ¼ 1.6, 8.0 Hz), 7.72 (1H, dd, J ¼ 1.6, 8.0 Hz), 7.72 (1H, t, J ¼ 8.0 Hz),
7.62 (1H, dd, J ¼ 1.6, 8.0 Hz), 7.58 (1H, dd, J ¼ 1.6, 8.0 Hz), 7.45 (1H,
dt, J ¼ 1.6, 8.0 Hz), 7.38 (1H, dt, J ¼ 1.6, 8.0 Hz). 13C NMR (100 MHz,
(2.5 m
g/ml) at 37 ꢃC. Cells were then centrifuged at 1000 g for 5 min
CDCl3):
d
¼ 157.8 (s), 155.8 (s), 148.6 (s), 136.0 (d), 134.4 (d), 132.6
at 4 ꢃC in order to collect NE from the supernatant. The NE super-
natant was equilibrated at 37 ꢃC for 2 min and incubated with the
compounds for 5 min. Following incubation, the NE substrate,
(d), 131.7 (s), 131.4 (d), 131.4 (d), 131.3 (s), 127.5 (s), 126.4 (d), 121.8
(s), 114.7 (s). ESI-MS m/z 360 (100) [M þ Na]þ, 358 (69).
MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 mM), was added to
5.1.1.9. 5-Chloro-2-(2-methylphenyl)-4H-benzo[d][1,3]oxazin-4-one
the reaction mixtures. Changes in absorbance at 405 nm were
continuously monitored for 10 min to assay NE activity [23,27].
(14). Yield: 60%, 1H NMR (400 MHz, CDCl3):
d
¼ 8.03 (1H, br.d,
J ¼ 8.0 Hz), 7.68 (1H, t, J ¼ 8.0 Hz), 7.58 (1H, dd, J ¼ 1.2, 8.0 Hz), 7.52
(1H, dd, J ¼ 1.2, 8.0 Hz), 7.43 (1H, dt, J ¼ 1.2, 8.0 Hz), 7.32 (2H, br.t,
5.2.4. Enzyme activity assay
J ¼ 8.0 Hz), 2.72 (3H, s). 13C NMR (100 MHz, CDCl3):
d
¼ 158.9 (s),
Enzyme activity assay of NE was conducts in pH 7.4 HBSS buffer
containing 0.03 U/ml human NE (EC 3.4.21.37, Sigma, St. Louis, MO,
USA). The assay buffer was incubated with compounds for 2 min at
25 ꢃC before the addition of NE substrate MeO-Suc-Ala-Ala-Pro-
156.2 (s), 149.1 (s), 139.4 (s), 135.8 (d), 132.0 (d), 131.9 (d), 131.9
(s), 130.7 (d), 130.2 (d), 129.1 (s), 126.2 (d), 126.1 (d), 114.5 (s) 22.2
(q). ESI-MS m/z 294 (100) [M þ Na]þ, 292 (31).
Val-p-nitroanilide (200 mM). Assays were performed in 96-well
5.1.1.10. 5-Chloro-2-(2-methoxyphenyl)-4H-benzo[d][1,3]oxazin-4-
microtiter plates. The reaction mix was allowed to proceed for
30 min. Activity of human NE was measured using ELISA reader at
405 nm.
one (15). Yield: 49%, 1H NMR (400 MHz, CDCl3):
d
¼ 7.85 (1H, dd,
J ¼ 1.6, 8.0 Hz), 7.67 (1H, t, J ¼ 8.0 Hz), 7.59 (1H, br.dd, J ¼ 1.6,
8.0 Hz), 7.51 (1H, dd, J ¼ 1.6, 8.0 Hz), 7.50 (1H, dt, J ¼ 1.6, 8.0 Hz),
7.06 (1H, br.t, J ¼ 8.0 Hz), 7.03 (1H, br.t, J ¼ 8.0 Hz), 3.92 (3H, s). 13C
5.2.5. Trauma-hemorrhagic shock procedure
NMR (100 MHz, CDCl3):
d
¼ 158.7 (s), 156.4 (s), 149.3 (s), 135.7 (d),
A rat model of trauma-hemorrhagic shock was used in this
study [27,28]. Thirty-two male Sprague-Dawley rats (275e325 g)
obtained from the National Science Council, Taipei, Taiwan were
divided into 4 groups of 8 animals each, according to a table of
random numbers. They were housed in an air-conditioned room
under a reversed lightedark cycle and allowed at least 1 week to
adapt to the environment. Before initiation of the experiment, they
were starved overnight but were allowed water ad libitum. The rats
were anesthetized by isoflurane (Attane, Minrad, Bethlehem, PA)
inhalation prior to performing a 5-cm midline laparotomy in the
abdomen. The abdomen was closed in layers, and catheters were
133.5 (d), 132.6 (s), 131.3 (d), 130.7 (d), 126.2 (d), 125.1 (s), 121.3
(s), 120.5 (d), 114.6 (s), 112.1 (d), 56.0 (q). ESI-MS m/z 310 (100)
[M þ Na]þ, 312 (32).
5.2. Biological assays
5.2.1. Preparation of human neutrophils
Blood was taken from healthy human donors (20e32 years old)
by venipuncture, using a protocol approved by the Institutional
Review Board at Chang Gung Memorial Hospital. Neutrophils were

P.-W. Hsieh et al. / European Journal of Medicinal Chemistry 45 (2010) 3111e3115
3115
placed in both femoral arteries and the right femoral vein (poly-
ethylene [PE-50] tubing; Becton Dickinson, Sparks, MD). The
wounds were bathed with 1% lidocaine (Elkins-Sinn, Cherry Hill,
NJ) throughout the surgical procedure to reduce postoperative
pain. Rats were then allowed to awaken and were bled, and the
mean blood pressure was maintained at 40 mmHg. This level of
hypotension persisted until the animals could not maintain a mean
blood pressure of 40 mmHg unless additional fluid in the form of
Ringer’s lactate was administered. This time was defined as the
maximum bleed-out, and the amount of withdrawn blood was
noted. Following this, the rats were maintained at a mean blood
pressure of 40 mmHg until 40% of the maximum bleed-out volume
was returned in the form of Ringer’s lactate. The animals were then
resuscitated with four times the volume of the shed blood over
60 min with Ringer’s lactate. The time required for maximum
bleed-out was w45 min, the volume of the maximum bleed-out
was w60% of the calculated circulating blood volume [29], and the
total hemorrhage time was w90 min. Thirty minutes before the end
of the resuscitation period, the rats received compound 13 (1 mg/kg
of body weight, intravenously), or an equal volume of the vehicle
(w0.2 ml,10% DMSO, Sigma). The catheters were then removed, the
vessels ligated, and the skin incisions closed with sutures. Sham-
operated animals underwent the surgical procedure, which
included a laparotomy in addition to ligation of the femoral artery
and vein, but neither hemorrhage nor resuscitation was carried out.
Vehicle or compound 13 was also administered to sham-operated
rats after the catheters were put in place. The animals were then
returned to their cages and allowed food and water ad libitum. The
animals were sacrificed 24 h after the end of resuscitation. The
current study was approved by the Institutional Animal Care and
Use Committee of Chang Gung Memorial Hospital. All animal
experiments were performed according to the guidelines of the
Animal Welfare Act and the Guide for Care and Use of Laboratory
Animals from the National Institutes of Health, Taiwan.
to determine the MPO activity in the sample. The reaction was
stopped by adding 3 l sodium azide (30%). The light absorbance at
460 nm was read. MPO activity was determined using a curve
obtained from the MPO standard.
m
5.2.9. Statistical analysis
Results are expressed as the mean ꢂ S.E.M. Data were analyzed
using the GraphPad Prism software (GraphPad Software, San Diego,
CA). Statistical analysis was performed using Student’s t-test or
one-way analysis of variance (ANOVA) followed by Tukey’s
multiple-comparison test. A value of p < 0.05 was considered
statistically significant.
Acknowledgment
The investigation was supported by research grants to P.W.
Hsieh and T.L. Hwang from the National Science Council of the
Republic of China in Taiwan.
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adding 290
(containing 20 g/l o-dianisidine hydrochloride), and 3
(20 mM). The sample (10 l) was added to each well to start the
reaction. Standard MPO (Sigma, St. Louis, MO) was used in parallel
m
l of 50 mM phosphate buffer, 3
m
l substrate solution
ml H2O2
m
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