7584
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