Synthesis and biological activity of 4-substituted benzoxazolone derivatives as a new class of sEH inhibitors with high anti-inflammatory activity in vivo

Article abstract of DOI:10.1016/j.bmcl.2013.02.048

A series of novel 4-substituted benzoxazolone derivatives was synthesized, characterized and evaluated as human soluble epoxide hydrolase (sEH) inhibitors and anti-inflammatory agents. Some compounds showed moderate sEH inhibitory activities in vitro, and two novel compounds, 3g and 4j, exhibited the highest activities with IC₅₀ values of 1.72 and 1.07 μM, respectively. Structure-activity relationships (SARs) revealed that introduction of a lipophilic amino acid resulted in an obvious increase in the sEH inhibitory activity, especially for derivatives containing a phenyl (3d, IC₅₀ = 2.67 μM), pyrrolidine (3g, IC₅₀ = 1.72 μM), or sulfhydryl group (3e, IC₅₀ = 3.02 μM). Several compounds (3a-3g) were tested in vivo using a xylene-induced ear edema mouse model. Three compounds (3d, 3f, and 3g) showed strong anti-inflammatory activities in vivo which were higher than that of Chlorzoxazone, a reference drug widely used in the clinic. Our investigation provided a novel type of sEH inhibitor and anti-inflammatory agent that may lead to the discovery of a potential candidate for clinical use.

Full text of DOI:10.1016/j.bmcl.2013.02.048

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2380–2383
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological activity of 4-substituted benzoxazolone
derivatives as a new class of sEH inhibitors with high
anti-inflammatory activity in vivo
⇑
Li Tang , Wen-Hua Ma , Yun-Long Ma, Shu-Rong Ban, Xiu-E Feng, Qing-Shan Li
School of Pharmaceutical Science, Shanxi Medical University, Taiyuan 030001, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 4-substituted benzoxazolone derivatives was synthesized, characterized and evaluated
as human soluble epoxide hydrolase (sEH) inhibitors and anti-inflammatory agents. Some compounds
showed moderate sEH inhibitory activities in vitro, and two novel compounds, 3g and 4j, exhibited the
Received 14 November 2012
Revised 28 January 2013
Accepted 9 February 2013
Available online 19 February 2013
highest activities with IC50 values of 1.72 and 1.07
(SARs) revealed that introduction of a lipophilic amino acid resulted in an obvious increase in the sEH
inhibitory activity, especially for derivatives containing a phenyl (3d, IC50 = 2.67 M), pyrrolidine (3g,
IC50 = 1.72 M), or sulfhydryl group (3e, IC50 = 3.02 M). Several compounds (3a–3g) were tested
lM, respectively. Structure–activity relationships
l
Keywords:
4-Substituted benzoxazolone
Synthesis
sEH inhibitory activity
Anti-inflammatory activity
l
l
in vivo using a xylene-induced ear edema mouse model. Three compounds (3d, 3f, and 3g) showed strong
anti-inflammatory activities in vivo which were higher than that of Chlorzoxazone, a reference drug
widely used in the clinic. Our investigation provided a novel type of sEH inhibitor and anti-inflammatory
agent that may lead to the discovery of a potential candidate for clinical use.
Ó 2013 Elsevier Ltd. All rights reserved.
Inflammatory diseases presently cause major health problems
and are found in many common diseases.1,2 However, various
drugs available on the market cannot be used for prolonged times
due to diverse side effects such as dyspepsia, gastroduodenal ul-
cers, and gastrointestinal bleeding.3 Therefore, the development
of new and safe anti-inflammatory drugs is in high demand.
Inflammation is a multi-staged process that follows the release of
arachidonic acid and conversion to its metabolites, thus, cyclooxy-
genase (COX), lipoxygenase (LOX) are crucial action targets for
anti-inflammatory drugs, and the research on COX and LOX inhib-
itors is common occurrence. Recently, some investigation showed
that soluble epoxide hydrolase (sEH) inhibitor participated in the
inflammatory reaction.4–6 However, reports on sEH inhibitors are
rare.
sEH is an enzyme that catalyzes the hydrolysis of epoxyeicosatri-
enoic acids (EETs), which are endothelium-derived hyperpolarizing
factor candidates that mediate vascular relaxation responses and
possess anti-inflammatory properties.7 A sEH inhibitor is expected
to increaselocalized endogenous EET levels, then reduce the produc-
tion of NO, cytokines, and proinflammatory lipid mediators, and
minimize systemic hypotension to potentiate their anti-inflamma-
tory effects.8,9 Hence, inhibitors on sEH are worth exploring as phar-
maceuticals for the treatment of inflammatory.8,10–12 The major
investigation13–15 on sEH inhibitors focused on urea, amide, amino-
heterocycles and carbamate derivatives, but research on new com-
pound structures is limited. In this paper, benzoxazolone
analogues were synthesized and evaluated as sEH inhibitors
in vitro, and their anti-inflammatory activities were determined
in vivo.
Benzoxazolones are widely distributed in plants and are of
increasing interest for a variety of pharmacological properties,
such as detoxification, antibacterial, anti-HIV, anti-inflammatory,
and tranquilizers.16,17 They represent an important scaffold in
medicinal chemistry. Since benzoxazolones are active and inex-
pensive, many structural modification and preliminary bioactivity
evaluation studies have been performed based on benzoxazol-
ones.18–20 Several useful compounds have been developed and
evaluated for their cytotoxic activity, nitric oxide synthase (NOS)
inhibitory activity, analgesic properties, antibacterial properties,
and antifungal properties in recent years.19,21–23 Nevertheless,
most efforts have focused on N-, 5-, or 6-substituted benzoxazol-
ones.24 Presently, 4-substituted benzoxazolone derivatives have
been scarcely prepared, and no reports have described the sEH
inhibitory activities or anti-inflammatory activities. The pro-
nounced biological activity of benzoxazolone derivatives and the
lack of structure-bioactivity relationships prompted our investiga-
tion on 4-substituted-2(3H)-benzoxazolones.
In order to explore some biological activity compounds, four
series of 4-substituted-2(3H)-benzoxazolones derivatives were
synthesized by a simple route. It is known that estrification is an
⇑
Corresponding author. Tel.: +86 351 4690322; fax: +86 351 4690114.
E-mail address: qingshanl@yahoo.com (Q.-S. Li).
These authors contributed equally to the present work.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.048

L. Tang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2380–2383
2381
important method for structural modification.25 Hence, benzoate
and besilate, the most common modified groups, were introduced
to optimize the skeleton structure 2(3H)-benzoxazolones at 4-po-
sition. In addition, conjugation of two bioactive fragments is ac-
cepted as an effective approach for designing inhibitors, ligands,
and other drugs.26 In order to improve the water solubility and
the bioavailability, several groups with good hydrophilicity and
bioaffinity, such as glycosyl and amino acids were introduced to
obtain some bioactive compounds. All of the new compounds were
evaluated for their sEH inhibitory activities in vitro. The com-
pounds with high sEH inhibitory activities were further screened
for their anti-inflammatory activities in vivo using a xylene-in-
duced ear edema model in mice.
The synthetic routes are outlined in Scheme 1. 2-Aminoresor-
cinol (1) was reacted with bis(trichloromethyl) carbonate under a
nitrogen atmosphere at reflux to provide 4-hydroxy-benzoxazo-
lone (2) as a key intermediate. Compounds 3a–3g and 4a–4j were
prepared via the reaction of compound 2 with various amino acids
or benzoic acids, respectively, using a catalytic amount of DCC and
DMAP at room temperature. The compounds were characterized
by 1H NMR, 13C NMR, and ESI–MS. The 13C NMR results showed
that all of the compounds presented the two expected carbonyl
signals at d 150–170 ppm. Similarly, the reaction of compound 2
with phenylsulfonyl chlorides in anhydrous acetone and triethyl-
amine under refluxing conditions furnished derivatives 5a–5j.
For the preparation of compounds 6a–6c, 4-hydroxy-ben-
zoxazolone (2) was treated with acetylated glycosyl bromide in
the presence of cetrimonium bromide and NaOH under refluxing
conditions to form the intermediates, and then they were hydro-
lyzed to obtain target compounds 6a–6c. The 1H NMR data re-
vealed the existence of a glycosyl group at 3.0–4.0 ppm for H and
4.0–5.0 ppm for OH. The structures of target compounds are pre-
sented in Table 1.
The target compounds were evaluated for their sEH inhibitory
activity in vitro using fluorescent (3-phenyl-oxiranyl)-acetic acid
cyano-(6-methoxy-naphthalen-2-yl)-methyl ester (PHOME) as the
specific substrate.27 The IC50 value, which corresponded to the con-
centration required for 50% inhibition of sEH activity, was calcu-
lated. 12-[3-adamantane-1-yl-ureido]-dodecanoic acid (AUDA),
one of the most effective inhibitors of sEH, was used as the refer-
ence drug in this study. As shown in Table 2, compounds 3a, 3b,
3d, 3e, 3g, 4f, and 4j exhibited significant activity against sEH. It
was found that introduction of amino acid-substituted groups
obviously increased the sEH inhibitory activity, especially for deriv-
atives containing a phenyl (3d, IC50 = 2.67
IC50 = 1.72 M), or sulfhydryl group (3e, IC50 = 3.02
compounds 4a–4j, two compounds, 4f (IC50 = 3.0
meta-electron withdrawing group and 4j (IC50 = 1.07
l
M), pyrrolidine (3g,
M). For the
M) with a
M) with a
l
l
l
l
para-electron donor group, showed high activities. However,
among the other two series compounds, 5a–5j and 6a–6c, no one
exhibited significant activity, indicating that the introduction of
sulfonyl and glycosyl gropus failed to raise the activity. These re-
sults also illustrated that amide was important for sEH inhibitory
activity.28,29
Considering that most of the compounds 3a–3g showed signif-
icant sEH inhibitory activities in vitro, they were subjected to anti-
inflammatory activities in vivo using a xylene-induced ear edema
model in mice.30 The compounds were given to mice by oral
administration at a dose of 150 mg/kg. Chlorzoxazone, a reference
drug widely used in the clinic, was used as the positive control. As
shown in Table 3, compounds 3a, 3d, 3f, and 3g showed higher
anti-inflammatory activities than that of Chlorzoxazone.
Compounds 3d and 3g demonstrated a good correlation be-
tween the in vitro and in vivo activity. However, compound 3f,
which showed weak in vitro activity, exhibited higher activity than
OH
O
O
i
O
Cl3C
CCl3
+
N
H
NH2
O
O
OH
OH
1
2
R1
O
O
O
O
Boc
R1
N
H
N
H
COOH
N
H
3a-3f
Boc
O
O
3g
O
N
N
H
ii
ii
Boc
O
O
O
R2
O
OH
O
N
H
R2
4a-4j
O
O
O
S
N
H
R3
O
OH
Cl
S
O O
O
2
N
H
R3
5a-5j
O
iii
O O
O
O
R4
N
H
OH
HO
Glycosyl bromide
iv
O
H
O
H
R5
HO
v
H
H
6a-6c
Scheme 1. Reagents and conditions. (i) THF, N2, reflux; (ii) THF, DCC, DMAP, rt; (iii) acetone, Et3N, reflux; (iv) cetrimonium bromide, DCE, NaOH, reflux; (v) Et3N, MeOH, rt.

2382
L. Tang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2380–2383
Table 1
Structures of derivatives 3a–3g, 4a–4j, 5a–5j, 6a–6c
Compound
R1-
Compound
R2-
Compound
R3-
3a
3b
3c
3d
3e
–H
–CH3
–CH(CH3)2
–CH2Ph
–CH2 CH2SCH3
4a
4b
4c
4d
4e
–H
5a
5b
5c
5d
5e
–H
o-Cl
m-Cl
p-Cl
o-NO2
o-Cl
m-Cl
p-Cl
o-NO2
NH
3f
4f
m-NO2
5f
m-NO2
O
O
N
H
3g
4g
p-NO2
5g
p-NO2
O
N
Boc
O
6a
6b
R4-H; R5-OH
4h
4i
o-CH3
m-CH3
5h
5i
o-CH3
m-CH3
R4-OH; R5-H
OH
O
OH
R4-H;
6c
4j
p-CH3
5j
p-CH3
HO
5
O
R
OH
Table 2
The inhibitory activity of compounds 3a–3g, 4a–4j, 5a–5j and 6a–6b against recombinant human sEH in vitroa,b
Compound
Inhibition ratioc (%) (10
lg/mL)
IC50
(lM)
Compound
Inhibition ratioc (%) (10
lg/mL)
IC50 (lM)
3a
3b
3c
3d
3e
3f
3g
4a
4b
4c
4d
4e
4f
95.46
95.73
48.02
90.82
99.60
79.58
100.68
28.00
30.81
25.31
27.80
29.18
92.86
28.08
26.22
17.2
16.4
—
2.67
3.02
—
1.72
—
—
—
—
—
4i
4j
24.44
98.31
35.97
15.21
24.49
13.62
36.13
31.61
28.82
28.02
30.95
39.92
45.50
60.98
—
1.07
—
—
—
—
—
—
—
—
—
—
—
d
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
6a
6b
3.0
—
—
4g
4h
—
a
b
c
A fluorescence-based assay was performed.
AUDA was used as a positive control and was found to have an IC50 value of 68 nM.
Inhibition ratio (%) = (1ÀVsample/Vcontrol) Â 100%, where V means fluorescence intensity. Values are means of a minimum of three experiments.
D
— = no IC50 value.
Table 3
between the in vitro and in vivo activity for these compounds
maybe due to the hydrophilic and lipophilic disparity which could
influence their pharmacokinetics characters in vivo.31–33 The phar-
macokinetics study on above compounds is on the way which will
be helpful to understand their differences between the in vitro and
in vivo activity for the compounds.34
To summarize, thirty novel 4-substituted benzoxazolone deriv-
atives were prepared and tested as sEH inhibitors and anti-inflam-
matory agents. Several new compounds showed significant sEH
inhibitory activities in vitro, among them, 3g and 4j exhibited
In vivo anti-inflammatory activity of compounds 3a–3g (150 mg/kg) on a xylene-
induced ear edema model in mice
Compound Edema inhibitiona,b (%) Compound
Edema inhibition (%)
c
Control
3a
3c
3e
3g
—
Chlorzoxazone
33.2 3.9
40.7 4.4
64.0 3.3
61.0 2.5
54.5 4.2
46.5 1.7
24.8 4.0
51.5 4.0
3b
3d
3f
a
Each value represented the mean SD, n = 10 per group.
Edema inhibition (%) = (edema formation of control group—edema formation of
b
the highest activities with IC50 values of 1.72 and 1.07 lM, respec-
treated group)/edema formation of control group Â100%.
tively. The SAR analysis illustrated that introduction of an amino
acid-substituted group resulted in an obvious increase in the sEH
inhibitory activity, especially for derivatives containing phenyl
c
The does of the reference drug Chlorzoxazone was 150 mg/kg.
(3d, IC50 = 2.67
groups (3e, IC50 = 3.02
results for compounds 3a–3g showed that compounds 3d, 3f, and
3g exhibited higher anti-inflammatory activities than that of
Chlorzoxazone, a reference drug. Compounds 3d and 3g not only
displayed potent sEH inhibitory activities in vitro, they also
l
M), pyrrolidine (3g, IC50 = 1.72
lM), or sulfhydryl
those of the control and most of the synthetic analogs in vivo. To
the contrast, compound 3e with strong in vitro activity showed
low in vivo activity. Meanwhile, a similar unexpected correlation
between the in vitro and in vivo activities also existed for com-
pounds 3a and 3g. Compound 3a showed 10-fold less active than
3g in vitro, but they were equally effective in vivo. The difference
l
M). The in vivo anti-inflammatory activity

L. Tang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2380–2383
2383
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We would like to thank Professor Guanhua Du, Dr. Lianhua
Fang, and Guorong He for their help with the in vitro assays. This
work was financially supported by the National Natural Science
Foundation of China (NO. 81172938), the National ‘‘863’’ Program
of China (NO.2013AA092903), the Program for the Top Science and
Technology Innovation Teams of Higher Learning Institutions of
Shanxi province (2011), the Program for the Top Young and Mid-
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Shanxi Province, and the Science and Technology Ministry of
Shanxi province (NO.20110321076-01).
Supplementary data
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