Synthesis and evaluation of benzoxazole derivatives as 5-lipoxygenase inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.08.047

5-Lipoxygenase (5-LOX) is important enzyme in the biosynthesis of leukotrienes, and is a potential target in the treatment of asthma and allergy. We designed and synthesized a series of benzoxazoles and benzothiazoles as 5-LOX inhibitors. Fourteen compoun

Full text of DOI:10.1016/j.bmc.2010.08.047

Bioorganic & Medicinal Chemistry 18 (2010) 7580–7585
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of benzoxazole derivatives
as 5-lipoxygenase inhibitors
Hyunmin Song a, Sei-Ryang Oh b, Hyeong-Kyu Lee b, Gyoonhee Han c, Joo-Heon Kim d, Hyeun Wook Chang e,
Kyung-Eun Doh a, Hee-Kyung Rhee f, Hea-Young Park Choo a,
⇑
a School of pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea
b Immune Modulator Research Center, KRIBB, Daejeon 305-806, Republic of Korea
c Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea
d Chungnam National University, Department of Chemistry, Daejeon 305-764, Republic of Korea
e College of Pharmacy, Yeungnam University, Gyongsan 712-749, Republic of Korea
f Department of Biological Science, Sookmyung Women’s University, Seoul 140-742, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
5-Lipoxygenase (5-LOX) is important enzyme in the biosynthesis of leukotrienes, and is a potential target
in the treatment of asthma and allergy. We designed and synthesized a series of benzoxazoles and ben-
zothiazoles as 5-LOX inhibitors. Fourteen compounds prepared showed the inhibition of LTC4 formation
Received 23 June 2010
Revised 25 August 2010
Accepted 26 August 2010
Available online 1 October 2010
with IC50 value of 0.12–23.88
hypersensitiveness.
lM. Also two compounds 2d and 2g showed improved airway
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Benzoxazoles
5-LOX inhibitors
Airway hypersensitiveness
1. Introduction
nuclear envelope.5,6 Then, it interacts with a small membrane pro-
tein designated 5-LOX activating protein (FLAP). This protein trans-
fers arachidonic acid to 5-LOX and this interaction between the
enzyme and FLAP being crucial for cellular leukotriene biosynthe-
sis.4,12 It was proposed that iron in the enzyme is bound by three
permanent ligands, His-372, His-550, and Ile-673, and one
exchangeable ligand, His-367. It was further proposed that a reac-
tion intermediate replaces the exchangeable ligand during the cat-
alytic cycle.5,13 Therefore, compounds with affinity for the iron
were proposed to displace the exchangeable ligand and thus inhi-
bit catalysis. Inhibition of 5-LOX provided an advantage of inde-
pendent of the evolving leukotriene receptor heterogenicity and
ligand specificity.
A lot of hydroxamic acids and N-hydroxyurea derivatives are re-
ported as iron-chelator inhibitors and Zileuton is the only 5-LOX
inhibitor in market for the treatment of bronchial asthma. How-
ever it showed hepatic toxicities and drug interactions.3,4,6 Re-
cently 2-(substituted phenyl)benzimidazole derivatives are
reported to have potent activity against IgE, cytokines, and CD23
for the treatment of allergy and asthma.14,15 Some reported 5-
LOX inhibitors containing indole or benzothiazole scaffold are
shown in Figure 1.16,17 In our study to develop novel agents for
asthma, we designed and synthesized benzoxazole compounds as
bioisosterer of benzothiazole, and proposed the affinity to 5-LOX
iron as follows.
Asthma is a predominantly inflammatory disease of the respira-
tory tract that has bronchial hyperreactivity and bronchospasm as
a result. Increased number of inflammatory cells such as mast cells,
eosinophils, neutrophils, and lymphocytes can be found in the
bronchial mucosa from asthmatic patients.1–3 5-Lipoxygenase (5-
LOX) is potential target in the treatment of asthma, atherosclerosis,
prostate cancer, and allergy, because 5-LOX is required for the pro-
duction of leukotrienes which are potent bronchocontraction and
proinflammatory mediators.4–9 Leukotrienes are short-lived, being
rapidly converted to inactive metabolites, and prolonged expres-
sion of activity is dependent on continuous biosynthesis.10 Thus
their action can be blocked by two different approaches, using tis-
sue cysteinyl luekotriene receptor antagonists (such as Pranlukast,
Zafrilukast, and Montelukast) or enzyme 5-LOX inhibition (such as
Zileuton).11
5-LOX has a molecular weight of 72–78 kDa and a single non-
heme iron in the enzyme.6 In resting cells, 5-LOX is localized in
the cytosol or in the nucleus in some cell types. 5-LOX is activated
by calcium, and once the cell is activated 5-LOX translocates to the
⇑
Corresponding author. Tel.: +82 2 3277 3042; fax: +82 2 3277 2821.
E-mail address: hypark@ewha.ac.kr (H.-Y.P. Choo).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.047

H. Song et al. / Bioorg. Med. Chem. 18 (2010) 7580–7585
7581
NH2
OH
COOEt
NH
O
HO
N
S
HO
N
O
S
NH
NH2
N
S
O
Cl
Zileuton
E6080
Figure 1. Structures of 5-LOX inhibitors.
Table 1
Fe2+
+Fe2
Structure and in vitro 5-LOX inhibition of synthesized compounds
Y
HO
NH2
R2
R3
O
N
N
R1
N
O
X
NH
H
N
O
R4
S
CH3
2a-n
Proposed affinity for the iron of 5-LOX and Zileuton, and de-
signed compounds (2a–n).
Compound
R1
R2
R3
R4
H
CH2CH3
H
CH2CH3
CH2CH3
H
CH2CH3
H
SCH3
H
H
CH(CH3)2
SCH3
H
IC50 (lM)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
H
H
Cl
Cl
NO2
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
Br
8.91
1.21
4.50
0.95
23.88
4.60
0.12
5.66
7.31
6.83
7.88
2.64
2.77
2.03
2. Results and discussion
2.1. Chemistry
CH2CH3
The reported method was employed for the synthesis of
benzoxazoles.18,19 2-Aminophenol was reacted with various
phenyl isothiocyanates to give the corresponding thioureas
(1a–n). Benzoxazoles (2a–n) were prepared by acid catalyzed ring
closure with oxidation from 2-hydroxyphenylthioureas (1a–n)
(Scheme 1).
H
H
H
H
H
H
CH3
OCH3
OCH3
OCH3
OCH3
2.2. Evaluation of in vitro 5-LOX inhibition
The 5-LOX inhibition was measured the formation of LTC4 in the
bone marrow-derived mast cells (BMMC) as explained in Section 4.
The ability of prepared compounds to inhibit 5-LOX was evaluated
2.3. Inhibition of allergen-induced airway responsiveness to
methacholine
at 1 and 10
compounds was 0.1–10
pound 2g showed 0.12
l
M as a first screening. The IC50 of the most reported
M (Table 1), and the most active com-
M.
Airway hyperresponsiveness (AHR) is a hallmark of bronchial
asthma. The in vivo efficacy of two compounds was tested in a
murine model of airway hyperresponsiveness. Mice that had been
sensitized to an allergen (ovalbumin, OVA) develop significantly
greater airway resistance in response to methacholine than mice
that have not exposed to OVA.
The ovalbumin-sensitized mice were challenged with aerosol-
ized ovalbumin repeatedly for 3 days and lung resistance was mea-
sured before and after each dose of intravenous methacholine. In
animals of the model group, airway hyperresponsiveness was
determined by a significant increase in lung resistance PD50 value
in addition to a significant increase in maximum lung resistance.
The two compounds prepared, 2d and 2g were selected for this as-
say. The two compounds showed improved airway hypersensitive-
ness as shown in Figure 2. Treatment with 2d and 2g reduced the
increases in Penh values after methacholine challenge compared
with that of the OVA-treated group.
l
l
In general, the substitution on benzoxazole ring affected the 5-
LOX inhibition. The introduction of nitro group on 5-position of
benzoxazole clearly decreased the activity (2e < 2b). However, 5-
chloro benzoxazoles 2c and 2d showed almost same activity com-
pared to their 5-H congeners 2a and 2b. The electron donating
groups, such as 5-methyl and 5-methoxy, increased the activity
(2f > 2a, 2g > 2b, and 2k > 2a). This observation could be explained
by the proposed affinity of 5-LOX iron to the ligands having the en-
hanced electron density as a result of the introduction of electron
donating X and Y to the aromatic rings.
Also, the position of the substituent Y on aromatic ring might
affect the 5-LOX inhibition, since there could be steric hindrance
for o-position. The p-ethyl group on the phenyl ring increased
the activity (2b, 2d, and 2g), but o-ethyl congener showed low
activity (2h).
H
H
NH2
OH
N
N
N
H
a
b
R
R'
R
N
R
R'
S
O
OH
1a-n
2a-n
Scheme 1. Reagents and conditions: (a) phenyl isothiocyanates, CH3OH, rt, 24 h; (b) KO2, CH3CN, rt, 12 h.

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H. Song et al. / Bioorg. Med. Chem. 18 (2010) 7580–7585
2.5. Measurement of BAL cytokines and OVA specific IgE levels
Th2 cells play a central role in the pathogenesis of allergic
bronchial asthma, since each of their characteristic cytokines such
as IL-4, IL-5, and IL-13 contributes to hallmarks of this disease,
including airway eosinophilia, increased mucus production,
production of allergen-specific IgE, and development of airway
hyperresponsiveness.
Twenty-four hours after the last challenge, the mice were sacri-
ficed via overdose of pentobarbital sodium. Blood was obtained
and centrifuged. The serum levels of OVA-specific IgE were com-
pared with the non-treated group (Fig. 4). The OVA-specific IgE
levels in serum were dramatically increased in OVA-treated mice
compared with PBS-treated mice. Treatment of 2d reduced
OVA-specific IgE levels compared with OVA-challenged group
(25.44 5.62 ng/mL vs 41.82 10.57 ng/mL; P <0.05; control and
test, respectively). Compound 2g also showed a reduction of
OVA-specific IgE levels (19.21 5.21 ng/mL).
Also the levels of interleukin IL-4, IL-5, and IL-13 in serum
were quantified in the supernatants of BAL fluids via enzyme
Figure 2. Effects on airway hyperresponsiveness (AHR). AHR was measured using
plethysmography 24 h after the last ovalbumin (OVA)-challenged mice by incre-
ments of methacholine (0–30 mg/mL). Control was only vehicle in OVA-sensitized/
challenged mice; others, compound (30 mg/kg) + OVA-sensitized/challenged mice.
2.4. Histological assay
Histopathology shows a marked peribronchial, perivascular,
and parenchymal infiltration of inflammatory cells in the OVA
group compared with control mice. Mice treated with 30 mg/kg
of each compound showed decreased tissue infiltration by mono-
nuclear cells and eosinophils compared with non-treated mice
(Fig. 3). These results were confirmed by a reduction of the total
histological score. In the compounds 2d and 2g treated OVA-in-
duced model, the infiltration of eosinophil-rich leukocytes was
attenuated compared to what was seen in OVA-treated mice.
Among two compounds prepared, 2g showed the most reduced to-
tal histological score. Zileuton showed a tendency to decrease tis-
sue infiltration by inflammatory cells, but the decrease was not
much different.
Figure 4. Effect on OVA-specific IgE levels in plasma. Plasma was collected 48 h
after the last ovalbumin (OVA)-challenged mice. Each level was analyzed using
ELISA (n = 5 or 6). Values are expressed as mean SEM (n = 6/group). *Significant
difference from NC, P <0.005 and **significant difference from OVA, P <0.05.
Figure 3. Effects on the recruitment of leukocytes in lung tissue. (A) Histological examination of lung tissue was performed 48 h after the last OVA challenge. Lung tissues
were fixed, sectioned at 4
l
m thickness, and stained with H&E solution (magnification ꢀ200). (I) OVA-induced asthmatic control, (II–V) compound treated OVA-induced
model, Montelukast (II), Zileuton (III), 2d (IV), and 2g (V), respectively. (B) Scoring the extent of inflammation by quantitative analysis of inflammatory-cell infiltration in lung
sections were performed based on the methods of Myou et al.18 OVA control, OVA-sensitized/challenged mice; others, each compound (30 mg/kg) + OVA-sensitized/
challenged mice. *Significant difference from OVA control, P <0.01.

H. Song et al. / Bioorg. Med. Chem. 18 (2010) 7580–7585
7583
immunoassays, all of which were conducted in accordance with
4. Experimental
the manufacturer’s instruction (Fig. 5). In 2d-treated group, cyto-
kine elevation was suppressed (IL-4, 163.49 48.60 ng/mL; IL-5,
110.91 23.12 ng/mL; IL-13, 71.91 13.82 ng/mL). Compound 2g
All melting points were taken in Pyrex capillaries using elec-
trothermal digital melting point apparatus (Büchi). NMR spectra
were recorded on a 400 MHz Varian FT-NMR spectrometer using
tetramethylsilane as an internal standard. Samples were dis-
solved in CDCl3, Acetone-d6 or DMSO-d6. Mass spectra data were
obtained on a Jeol JMS 700 high resolution mass spectrometer at
the Korea Basic Science Institute (Daegu). Most of the reagents
were purchased from Sigma–Aldrich Chemical Company. Purity
of synthesized compounds was determined to be P95% by ana-
lytical HPLC on a C-18 column eluted with a linear gradient of
10–90% acetonitrile in water containing 0.05% TFA (flow rate of
1 mL/min).
also caused
a reduction in IL-4 (198.34 44.00 ng/mL), IL-5
(116.92 33.55 ng/mL), and IL-13 (64.30 17.83 ng/mL).
3. Conclusion
Fourteen compounds prepared showed the 5-LOX inhibition
with IC50 value of 0.12–23.88
that have methyl group on 5-position of benzoxazole and p-ethyl
group on the phenyl ring showed 0.12 M. Selected two com-
lM. The most active compound 2g
l
pounds 2d and 2g showed improved airway hypersensitiveness.
4.1. General procedure for the preparation of benzoxazoles
2-Aminophenol (0.92 mmol) and isothiocyanates (0.92 mmol)
in methanol were stirred at room temperature for 1 day. The pre-
cipitate was collected by filtration and washed with ether to give
the corresponding thioureas. A solution of N-(2-hydroxy-5-substi-
tuted phenyl)-N0-(substituted) thiourea (1 mmol) in CH3CN (3 mL)
was added to a heterogeneous solution of potassium superoxide
(5 mmol) in CH3CN (2 mL) at room temperature under dry nitrogen
atmosphere. After being stirred well for 12 h at room temperature,
the reaction mixture was poured into cold water, and extracted
with dichloromethane. The solvent was dried over anhydrous
MgSO4, filtered, and concentrated under reduced pressure to give
product.
4.1.1. N-Phenylbenzo[d]oxazol-2-amine (2a)
White powder (31%), mp 180–182 °C; 1H NMR (Acetone-d6,
400 MHz)
d 9.47 (s, NH), 7.85 (d, J = 8.0 Hz, 2H), 7.42 (d,
J = 8.0 Hz, 1H), 7.37 (t, J = 12.0 Hz, 3H), 7.21 (t, J = 12.0 Hz, 1H),
7.11 (t, J = 12.0 Hz, 1H), 7.04 (t, J = 12.0 Hz, 1H); 13C NMR (Ace-
tone-d6, 100 MHz)
d 159.11, 148.59, 144.02, 139.98, 129.89,
124.92, 123.29, 122.68, 118.70, 117.91, 109.70; FABHRMS (m/z):
211.0871 (M++1, C13H11N2O requires 211.0875).
4.1.2. N-(4-Ethylphenyl)benzo[d]oxazol-2-amine (2b)
White powder (51%), mp 123–125 °C; 1H NMR (Acetone-d6,
400 MHz)
d 9.51 (s, NH), 7.82 (d, J = 8.0 Hz, 1H), 7.42 (d,
J = 8.0 Hz, 2H), 7.22 (d, J = 8.0 Hz, 2H), 7.04 (t, J = 12.0 Hz, 1H),
6.92 (d, J = 8.0 Hz, 1H), 6.84 (t, J = 12.0 Hz, 1H), 2.60 (q, J = 7.6 Hz,
2H), 1.19 (t, J = 7.6 Hz, 3H); 13C NMR (Acetone-d6, 100 MHz) d
159.29, 148.63, 144.14, 139.23, 137.66, 129.18, 124.86, 122.51,
118.88, 117.78, 109.63, 28.88, 16.34; FABHRMS (m/z): 239.1182
(M++1, C15H15N2O requires 239.1184).
4.1.3. 5-Chloro-N-phenylbenzo[d]oxazol-2-amine (2c)
Yellow powder (41%), mp 183–186 °C; 1H NMR (Acetone-d6,
400 MHz)
d 9.60 (s, NH), 7.82 (d, J = 8.0 Hz, 1H), 7.42 (d,
J = 8.0 Hz, 2H), 7.40 (s, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.30 (s, 3H);
13C NMR (Acetone-d6, 100 MHz) d 160.30, 147.40, 145.56, 139.55,
129.95, 129.81, 123.72, 122.41, 118.96, 117.68, 110.70; FABHRMS
(m/z): 245.0482 (M++1, C13H10ClN2O requires 245.0482).
4.1.4. 5-Chloro-N-(4-ethylphenyl)benzo[d]oxazol-2-amine (2d)
Yellow powder (27%), mp 196–198 °C; 1H NMR (Acetone-d6,
400 MHz) d 9.53 (s, NH), 7.72 (d, J = 8.0 Hz, 2H), 7.40 (s, 1H), 7.36
(d, J = 8.0 Hz, 1H), 7.22 (d, J = 8.0 Hz, 2H), 7.10 (d, J = 8.0 Hz, 1H),
2.60 (q, J = 8.0 Hz, 2H), 1.20 (t, J = 7.6 Hz, 3H); 13C NMR (Acetone-
d6, 100 MHz) d 160.49, 147.44, 145.71, 139.70, 137.22, 129.76,
129.24, 122.23, 119.16, 117.55, 110.61, 28.89, 16.31; FABHRMS
(m/z): 273.0794 (M++1, C15H14ClN2O requires 273.0795).
Figure 5. Cytokine levels in BALF of OVA-induced asthmatic model. Effect of four
compounds with positive controls (Singulair and Zileuton) on cytokine levels in
BALF. BALF was collected 48 h after the last ovalbumin (OVA)-challenged mice. Each
level was analyzed using ELISA (n = 5 or 6). (A) Interleukin (IL)-4 level, (B) IL-5 level,
(C) IL-13 level in BALF. OVA, OVA-sensitized/challenged mice; others, each
compound (30 mg/kg) + OVA-sensitized/challenged mice. Values are expressed as
mean SEM (n = 6/group). *Significant difference from NC, P <0.05 and **significant
difference from OVA, P <0.005.

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H. Song et al. / Bioorg. Med. Chem. 18 (2010) 7580–7585
4.1.5. N-(4-Ethylphenyl)-5-nitrobenzo[d]oxazol-2-amine (2e)
Yellow powder (81%), mp 161–163.5 °C; 1H NMR (Acetone-d6,
4.1.12. N-(4-Isopropylphenyl)-5-methoxybenzo[d]oxazol-2-
amine (2l)
400 MHz)
d
9.55 (s, NH), 7.84 (d, J = 8.0 Hz, 1H), 7.43 (d,
Brown powder (63%), mp 129.6–132.5 °C; 1H NMR (Acetone-d6,
400 MHz) d 9.35 (s, NH), 7.64–7.60 (m, 2H), 7.14–7.11 (m, 2H), 7.11
(d, J = 8.4 Hz, 1H), 6.87 (d, J = 2.8 Hz, 1H), 6.54 (dd, J = 8.4 and
2.8 Hz, 1H), 3.69 (s, 3H), 2.92 (m, 1H), 1.12 (s, 3H), 1.10 (s, 3H);
13C NMR (Acetone-d6, 100 MHz) d 160.07, 158.28, 145.10, 143.83,
143.02, 137.75, 127.69, 118.88, 109.52, 109.02, 102.83, 56.19,
34.33, 24.52; FABHRMS (m/z): 283.1447 (M++1, C17H19N2O2 re-
quires 283.1445).
J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 6.72 (d, J = 8.0 Hz, 2H),
2.63 (q, J = 7.6 Hz, 2H), 1.19 (t, J = 7.6 Hz, 3H); 13C NMR (Acetone-
d6, 100 MHz) d 161.42, 152.80, 145.09, 140.22, 136.78, 129.33,
119.43, 118.91, 112.75, 109.83, 28.90, 16.28; EI/MS (m/z):
283.0960 (M+, C15H13N3O3 requires 283.0957).
4.1.6. 5-Methyl-N-phenylbenzo[d]oxazol-2-amine (2f)
White powder (86%), mp 205.0–209.8 °C; 1H NMR (Acetone-d6,
400 MHz) d 9.34 (s, NH), 7.86 (m, 2H), 7.38 (m, 2H), 7.24 (d,
J = 8.0 Hz, 1H), 7.05 (m, 1H), 7.03 (m, 1H), 6.94 (d, J = 8.0 Hz, 1H),
2.40 (s, 3H); 13C NMR (Acetone-d6, 100 MHz) d 159.24, 146.75,
144.18, 140.05, 134.44, 129.88, 123.33, 123.20, 118.64, 118.25,
109.09, 21.55; FABHRMS (m/z): 225.1023 (M++1, C14H13N2O re-
quires 225.1028).
4.1.13. 5-Methoxy-N-(4-(methylthio)phenyl)benzo[d]oxazol-2-
amine (2m)
Pale brown powder (74%), mp 176.4–178.8 °C; 1H NMR (Ace-
tone-d6, 400 MHz) d 9.51 (s, NH), 7.84–7.81 (m, 2H), 7.36–7.33
(m, 2H), 7.26 (d, J = 8.8 Hz, 1H), 7.01 (d, J = 2.8 Hz, 1H), 6.69 (dd,
J = 8.8 and 2.8 Hz, 1H), 3.83 (s, 3H), 2.49 (s, 3H); 13C NMR (Ace-
tone-d6, 100 MHz)
d 159.75, 158.31, 144.91, 142.99, 137.83,
4.1.7. N-(4-Ethylphenyl)-5-methylbenzo[d]oxazol-2-amine (2g)
White powder (79%), mp 184–186.2 °C; 1H NMR (Acetone-d6,
400 MHz) d 9.21 (s, NH), 7.76–7.72 (m, 2H), 7.22–7.20 (m, 2H),
6.97–6.93 (m, 2H), 6.92–6.90 (m, 1H), 4.04 (q, J = 6.8 Hz, 2H),
2.38 (s, 3H), 1.36 (t, J = 6.8 Hz, 3H); 13C NMR (Acetone-d6,
132.29, 129.30, 119.41, 109.61, 109.26, 102.89, 56.20, 16.93; FAB-
HRMS (m/z): 287.0854 (M++1, C15H15N2O2S requires 287.0848).
4.1.14. N-(3-Bromophenyl)-5-methoxybenzo[d]oxazol-2-amine
(2n)
100 MHz)
d
159.43, 146.79, 144.29, 139.12, 137.73, 134.36,
Pale brown powder (40%), mp 211.5–213.1 °C; 1H NMR (Ace-
tone-d6, 400 MHz) d 9.31 (s, NH), 7.72 (m, 2H), 7.33 (t, J = 8.0 Hz,
1H), 7.29 (d, J = 8.8 Hz, 1H), 7.23 (m, 1H), 7.07 (d, J = 2.4 Hz, 1H),
6.72 (dd, J = 8.8 and 2.4 Hz, 1H), 3.84 (s, 3H); 13C NMR (Acetone-
d6, 100 MHz) d 159.23, 158.38, 144.56, 142.87, 141.51, 131.58,
125.93, 123.24, 121.11, 117.44, 109.83, 109.80, 102.98, 56.22; FAB-
HRMS (m/z): 319.0082 (M++1, C14H12BrN2O2 requires 319.0086).
129.15, 123.15, 118.82, 118.14, 109.02, 28.87, 21.56, 16.34; FAB-
HRMS (m/z): 253.1345 (M++1, C16H17N2O requires 253.1341).
4.1.8. N-(2-Ethylphenyl)-5-methylbenzo[d]oxazol-2-amine (2h)
Pale brown powder (92%), mp 127.3–128.4 °C; 1H NMR (Ace-
tone-d6, 400 MHz) d 8.50 (s, NH), 8.08 (d, J = 8.0 Hz, 1H), 7.30–7.
24 (m, 2H), 7.01–6.98 (m, 2H), 6.91 (d, J = 8.0 Hz, 2H), 3.28 (s,
3H), 2.78 (q, J = 7.6 Hz, 2H), 1.24 (t, J = 7.6 Hz, 3H); 13C NMR (Ace-
4.2. Determination of 5-lipoxygenase product LTC4 formation
tone-d6, 100 MHz)
d 160.66, 147.22, 144.28, 137.17, 136.12,
134.35, 129.75, 127.41, 125.36, 123.26, 122.98, 118.04, 109.03,
24.83, 21.55, 14.69; FABHRMS (m/z): 253.1346 (M++1, C16H17N2O
requires 253.1341).
Preparation of mouse bone marrow-derived mast cells (BMMC)
were obtained from male Balb/c mice and cultured for up to
4 weeks in 50% enriched medium (RPMI containing 2 mM
mine, 25 mM HEPES buffer, 2 mg/mL sodium bicarbonate,
100 units/mL penicillin G, 100 g/mL streptomycin sulfate, and
0.25
L-gluta-
4.1.9. 6-Methyl-N-(4-(methylthio)phenyl)benzo[d]oxazol-2-
amine (2i)
l
l
g/mL amphotericin B) supplemented with 10% fetal bovine
Pale brown powder (69%), mp 187.0–190.8 °C; 1H NMR (Ace-
tone-d6, 400 MHz) d 9.45 (s, NH), 7.84–7.81 (m, 2H), 7.36–7.33
(m, 2H), 7.24 (d, J = 8.0 Hz, 2H), 6.94 (d, J = 8.0 Hz, 1H), 2.49 (s,
3H), 2.39 (s, 3H); 13C NMR (Acetone-d6, 100 MHz) d 159.14,
146.77, 144.12, 137.88, 134.48, 132.22, 129.31, 123.36, 120.27,
119.37, 118.24, 109.11, 21.55, 16.94; FABHRMS (m/z): 271.0905
(M++1, C15H15N2OS requires 271.0905).
serum with IL-3 (Sigma I4144, 2 ng/mL).20 Three weeks after the
culture, more than 98% of BMMC was found in the cells as assessed
by staining method with toluidine blue.
4.3. Determination of LTC4
BMMC were suspended in the enriched medium at the cell den-
sity of 1 ꢀ 106 cells/mL, and were then incubated in a humidified
5% CO2 incubator with or without sample in DMSO (final DMSO
concentration was <0.5%) for 30 min at 37 °C. After the stimulation
with stem cell factor (SCF, Sigma S9915, 100 ng/mL) for 20 min, the
LTC4 release in supernatants was measured by an enzyme immu-
noassay kit (Cayman Chemical, Ann Arbor, MI, USA) according to
the manufacturer’s instructions. All the experiments were carried
out in triplicate and the inhibition of LTC4 release was determined
by calculating % reduction of LTC4 release.21
4.1.10. N-(3-Methoxyphenyl)-5-methylbenzo[d]oxazol-2-amine
(2j)
Pale brown powder (72%), mp 164.8–166.4 °C; 1H NMR (Ace-
tone-d6, 400 MHz) d 9.41 (s, NH), 7.62 (m, 1H), 7.36–7.33 (m,
1H), 7.27–7.23 (m, 3H), 6.93–6.57 (m, 1H), 6.65–6.62 (m, 1H),
3.80 (s, 3H), 2.40 (s, 3H); 13C NMR (Acetone-d6, 100 MHz) d
161.54, 159.14, 146.71, 144.13, 141.16, 134.47, 130.64, 123.39,
118.32, 111.08, 109.11, 108.41, 104.91, 55.59, 21.54; FABHRMS
(m/z): 255.1128 (M++1, C15H15N2O2 requires 255.1134).
4.4. Sensitization and airway challenge
4.1.11. 5-Methoxy-N-phenylbenzo[d]oxazol-2-amine (2k)
White powder (75%), mp 213.7–214.5 °C; 1H NMR (Acetone-d6,
400 MHz) d 9.43 (s, NH), 7.84–7.80 (m, 2H), 7.37–7.32 (m, 2H), 7.23
(d, J = 8.4 Hz, 1H), 6.99 (d, J = 2.4 Hz, 1H), 7.05–7.01 (m, 1H), 6.66
(dd, J = 2.4 and 8.4 Hz, 1H), 3.79 (s, 3H); 13C NMR (Acetone-d6,
Groups of mice (n = 5–6) were studied; they received the fol-
lowing treatment: (1) sham-sensitization plus challenge with
phosphate-buffered saline (PBS; ipNeb); (2) sensitization plus chal-
lenge with OVA (ovalbumin: Sigma A5503; Sigma, St. Louis, MO)
(ipNeb); (3) sensitization with OVA (ip) plus challenge with OVA
(Neb) and samples (tiarellic acid or Zileuton, po). Briefly, mice were
100 MHz)
d 159.83, 158.30, 144.95, 142.98, 139.97, 129.88,
123.26, 118.68, 109.61, 109.23, 102.90, 56.20; FABHRMS (m/z):
sensitized with intraperitoneal injection of 20
lg OVA, which was
241.0980 (M++1, C14H13N2O2 requires 241.0977).
emulsified with 2 mg of aluminum hydroxide in 100
ll of PBS

H. Song et al. / Bioorg. Med. Chem. 18 (2010) 7580–7585
7585
buffer (pH 7.4) on days 0 and 11. The mice were challenged
through the airways with OVA (1% in PBS) for 20 min using an
ultrasonic nubuilizer (NE-U12; Omron Corp., Tokyo, Japan) on days
19, 20, 21, and 25 after the initial sensitization. The mice were sac-
rificed 48 h after the last challenge (day 32) to determine the sup-
pressive effect of compounds on the airways of allergic asthma.
and stored at ꢁ70 °C before use. The amount of IL-4, IL-5, and
IL-13 in BALF was measured by a specific mouse ELISA kit (R&D
Systems; Minneapolis, MN) according to the manufacturer’s
instructions. The detection limit of the assays was 250 pg/mL.
Plasma was obtained by cardiac puncture after the tracheot-
omy. Complementary capture and detection antibody pairs for
mouse IgE antibodies were purchased from BD OptEIA (San Diego,
CA), and the IgE enzyme-linked immunosorbent assay (ELISA) was
performed according to the manufacturer’s directions. Duplicate
samples in plasma were diluted to 1:100. IgE levels in each sample
were measured the optical density readings at 450 nm, and OVA-
specific IgE concentrations were calculated from a standard curve,
which was generated in case recombinant IgE (5–2000 ng/mL) was
used.
4.5. Determination of airway responsiveness
Twenty-four hours after the final aerosol challenge AHR was
measured by using a whole-body plethysmography (OCP3000; All-
medicus, Korea).22 Each mouse was placed in a brometric phethys-
mographic chamber and challenged with aerosolized PBS, followed
by increasing concentrations of aerosolized methacholine (12.5,
25, and 50 mg/mL) for 3 min. Bronchoconstriction was recorded
for additional 5 min at each concentration. The highest Penh value
of each sample was obtained during each methacholine challenge,
and expressed as a percentage of a basal Penh value in response to
control (PBS) challenge.
Acknowledgment
This work was supported by a Grant (title: Development of new
technology for next generation, NBM1200513) from Ministry of
Knowledge Economy in Korea.
The formula for enhanced pause value (Penh) is as follows:
½expiratory timeðTeÞ=relaxation timeðRTÞ ꢁ 1ꢂ
References and notes
ꢀ ½peak expiratory flowðPEFÞ=peak inspiratory flowðPIFÞꢂ
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Forty-eight hours after the last challenge, the mice were sacri-
ficed with an overdose of pentobarbital (Sigma P3761), and a tra-
cheotomy was performed. After ice-cold PBS (0.5 mL) was
instilled into the lungs, bronchoalveolar lavage fluid (BALF) was
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nulation. BALF was centrifuged and supernatants were collected