C.-H. Tseng et al. / Bioorg. Med. Chem. 17 (2009) 6773–6779
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5.1.2. Furo[30,20:3,4]naphtho[1,2-d]imidazole (12)
5.1.7. 2-(5-Nitrofuran-2-yl)furo[30,20:3,4]naphtho[1,2-
d]imidazole (17)
To a suspension of 2 (0.20 g, 1.0 mmol) in acetic acid (30 mL)
was added ammonia acetate (0.23 g, 3.0 mmol) and paraformalde-
hyde (0.3 g). The reaction mixture was heated with stirring under
microwave irradiation (100 W) for 30 min (TLC monitoring). The
solvent was removed in vacuo and the residue suspended in H2O
(20 mL). The resulting precipitate was purified by column chroma-
tography (MeOH:CH2Cl2 1/50) and recrystallized from EtOH to give
4 as a white solid (0.13 g, 61%). Mp: 282–283 °C. 1H NMR
(400 MHz, DMSO-d6 + TFA-d): 7.32 (d, 1H, J = 2.0 Hz, 4-H), 7.75
(m, 2H, 8- and 9-H), 8.25 (d, 1H, J = 2.0 Hz, 5-H), 8.34 (m, 1H, 7-
H), 8.52 (m, 1H, 10-H), 9.68 (s, 1H, 2-H). 13C NMR (100 MHz,
DMSO-d6 + TFA-d): 105.62, 111.05, 118.79, 119.62, 121.10,
122.42, 122.54, 123.62, 127.36, 127.67, 137.45, 146.90, 149.35.
EIMS (m/z): 208 (M+), 197, 153, 126. Anal. calcd for C13H8N2O: C,
74.99; H, 3.87; N, 13.45. Found: C, 75.04; H, 3.89; N, 13.50.
From 10 and 5-nitro-2-furaldehyde diacetate as described for
12, to give a red solid (0.26 g, 82% yield). Mp: 314–315 °C. 1H
NMR (400 MHz, DMSO-d6 + TFA-d): 7.17 (d, 1H, J = 2.0 Hz, 4-H),
7.61 (m, 2H, 8- and 9-H), 7.63 (d, J = 4.0 Hz, 1H, furanyl-30-H),
7.82 (d, J = 4.0 Hz, 1H, furanyl-40-H), 8.06 (d, J = 2.0 Hz, 1H, 5-H),
8.18 (m, 1H, 7-H), 8.40 (m, 1H, 10-H). 13C NMR (100 MHz,
DMSO-d6 + TFA-d): 106.14, 112.12, 115.30, 116.33, 119.84,
120.37, 121.52, 123.18, 126.12, 127.36, 127.58, 127.85, 136.06,
143.08, 146.75, 150.20, 153.25. Anal. calcd for C17H9N3O4ꢁ0.4EtOH:
C, 63.31; H, 3.40; N, 12.44. Found: C, 63.47; H, 3.21; N, 12.21.
5.1.8. (E)-2-(2-(5-Nitrofuran-2-
yl)vinyl)furo[30,20:3,4]naphtho[1,2-d]imidazole (18)
From 10 and 3-(5-nitro-2-furyl)acrolein as described for 12, to
give a brown solid (0.25 g, 71% yield). Mp: 284–285 °C. 1H NMR
(400 MHz, DMSO-d6): 7.17 (d, J = 4.0 Hz, 1H, furanyl-30-H), 7.29
(d, 1H, J = 2.0 Hz, 4-H), 7.42 (d, 1H, J = 16.0 Hz, =CH), 7.58 (d, 1H,
J = 16.0 Hz, =CH), 7.65 (m, 2H, 8- and 9-H), 7.80 (d, J = 4.0 Hz, 1H,
furanyl-40-H), 8.22 (d, J = 2.0 Hz, 1H, 5-H), 8.30 (m, 1H, 7-H), 8.45
(m, 1H, 10-H). 13C NMR (100 MHz, DMSO-d6 + TFA-d): 105.77,
111.25, 113.37, 115.10, 117.42, 118.63, 120.20, 121.58, 122.72,
124.22, 125.16, 125.29, 127.73, 128.20, 143.80, 147.19, 150.24,
152.80, 152.90. Anal. calcd for C19H11N3O4ꢁ1.3H2O: C, 61.88; H,
3.72; N, 11.40. Found: C, 61.66; H, 3.83; N, 11.36.
5.1.3. 2-Phenylfuro[30,20:3,4]naphtho[1,2-d]imidazole (13)
From 10 and benzaldehyde as described for 12, to give a gray
solid (0.18 g, 65% yield). Mp: 313–314 °C. 1H NMR (400 MHz,
DMSO-d6 + TFA-d): 7.27 (d, 1H, J = 2.4 Hz, 4-H), 7.66–7.77 (m, 5-
H, Ar-H), 8.20–8.26 (m, 4H, Ar-H), 8.55 (m, 1H, Ar-H). 13C NMR
(100 MHz, DMSO-d6 + TFA-d): 105.56, 111.07, 118.53, 119.48,
121.04, 122.60, 123.46, 123.48, 124.49, 127.17, 127.56, 128.04
(2C), 129.92 (2C), 133.14, 146.38, 146.88, 149.35. EIMS (m/z):
302 (M+), 273, 153, 126. Anal. calcd for C19H12N2O: C, 80.27; H,
4.25; N, 9.85. Found: C, 79.97; H, 4.35; N, 9.73.
5.2. Cell culture
5.1.4. 2-(4-Nitrophenyl)furo[30,20:3,4]naphtho[1,2-d]imidazole
(14)
Raw 264.7 cells, purchased from American Type Culture Collec-
tion, USA, were cultured in DMEM, supplemented with 5% fetal bo-
vine serum, 100 units/mL of penicillin, 100 lg/mL of streptomycin,
From 10 and 4-nitrobenzaldehyde as described for 12, to give a
brown solid (0.18 g, 56% yield). Mp: 304–305 °C. 1H NMR
(400 MHz, DMSO-d6 + TFA-d): 7.30 (d, 1H, J = 2.0 Hz, 4-H), 7.72
(m, 2-H, 8- and 9-H), 8.23 (d, 1H, J = 2.0 Hz, 5-H), 8.30 (m, 1H, 7-
H), 8.43 (m, 2H, Ar-H), 8.52 (m, 2H, Ar-H), 8.58 (m, 1H, 10-H).
13C NMR (100 MHz, DMSO-d6 + TFA-d): 105.73, 111.71, 119.26,
119.79, 121.20, 122.82, 124.95 (2C), 125.44, 126.59, 127.22,
127.64, 128.80 (2C), 130.54, 144.54, 146.88, 149.31, 149.54. EIMS
(m/z): 329 (M+), 283, 255, 228. Anal. calcd for C19H11N3O3ꢁ1.2H2O:
C, 65.01; H, 3.86; N, 11.98. Found: C, 64.97; H, 3.84; N, 12.05.
2 mM L-glutamine, and 1 mM non-essential amino acids in a 10-cm
plate at a density of 1 ꢀ 106 cells/mL, at 37 °C in a humidified
atmosphere containing 5% CO2.
5.2.1. Neutrophil superoxide generation30
Compound stock solution (30 mM in DMSO) was prepared and
stored at ꢂ25 °C, and was diluted with DMSO to 1–20 mM range at
room temperature before experiment. The final percentage of
DMSO in the reaction mixture was less than 0.5% (v/v). Rat (Spra-
gue-Dawley) peripheral blood neutrophils31 were isolated and
incubated with test compounds for 5 min at 37 °C before stimula-
5.1.5. 2-(4-Formylphenyl)furo[30,20:3,4]naphtho[1,2-
d]imidazole (15)
From 10 and terephthalaldehyde as described for 12, to give a
brown solid (0.22 g, 72% yield). Mp: 253–254 °C. 1H NMR
(400 MHz, DMSO-d6 + TFA-d): 7.32 (d, 1H, J = 2.0 Hz, 4-H), 7.74
(m, 2-H, 8- and 9-H), 8.23 (m, 2H, Ar-H), 8.25 (d, 1H, J = 2.0 Hz,
5-H), 8.31 (m, 1H, 7-H), 8.42 (m, 2H, Ar-H), 8.61 (m, 1H, 10-H),
10.12 (s, 1H, CHO). 13C NMR (100 MHz, DMSO-d6 + TFA-d):
105.68, 111.37, 118.87, 119.72, 121.16, 122.79, 124.54, 125.61,
127.30, 127.74, 128.44 (2C), 128.81, 130.61 (2C), 138.53, 145.20,
146.97, 149.54, 192.62. Anal. calcd for C20H12N2O2ꢁ1.0H2O: C,
72.71; H, 4.28; N, 8.48. Found: C, 72.56; H, 4.36; N, 8.09.
tion with 1 lM formyl-Met-Leu-Phe (fMLP) for another 45 min,
respectively. Neutrophils were assessed by the determination of
b-glucuronidase and lysozyme, in the supernatant.32 In the super-
oxide anion generation experiments, neutrophils were stimulated
with fMLP (0.3 lM)/CB (5 lg/mL) or phorbol myristate (PMA;
3 nM) for 30 min in the presence of cytochrome c, and the superox-
ide anion generation was measured in terms of superoxide dismu-
tase-inhibitable cytochrome c reduction.33,34
5.2.2. Nitrite measurement
Nitrite was measured by adding 100
(1% sulfanilamide and 0.1% N-(1-naphthyl)ethylenediamine dihy-
drochloride in 5% phosphoric acid) to 100 L of medium for
5 min. The optical density at 550 nm (OD550) was measured with
a microplate reader. Concentrations were calculated by compari-
son with the OD550 of a standard solution of sodium nitrite pre-
pared in culture medium.
lL of the Griess reagent
5.1.6. 2-Furanylfuro[30,20:3,4]naphtho[1,2-d]imidazole (16)
From 10 and 2-furaldehyde as described for 12, to give a white
solid (0.21 g, 75% yield). Mp: 270–271 °C. 1H NMR (400 MHz,
DMSO-d6 + TFA-d): 6.97 (dd, J = 3.6, 1.6 Hz, 1H, furanyl-40-H),
7.23 (d, J = 2.0 Hz, 1H, 4-H), 7.65–7.70 (m, 3H, furanyl-30-, 8- and
9-H), 8.17 (d, J = 2.0 Hz, 1H, 5-H), 8.24 (m, 1H, 7-H), 8.26 (dd,
J = 1.6, 0.8 Hz, 1H, furanyl-50-H), 8.46 (m, 1H, 10-H). 13C NMR
(100 MHz, DMSO-d6 + TFA-d): 105.57, 111.16, 113.93, 116.61,
118.55, 119.56, 121.12, 122.56, 123.19, 124.15, 127.20, 127.52,
137.58, 139.21, 146.79, 148.22, 149.52. Anal. calcd for
C17H10N2O2: C, 74.44; H, 3.67; N, 10.21. Found: C, 74.12; H, 3.75;
N, 10.06.
l
5.2.3. Cell viability
To evaluate whether the suppressive effect of compounds 16–
18 on NO production is related to cell viability, Raw 264.7 cells
were treated with LPS for 24 h, and washed with 500
lL of phos-
phate buffer solution (PBS) before resuspended in 1 mL of culture