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plates (2–10 · 103 cells per well) and allowed to attach
overnight. The medium was replaced with serum-free
medium containing the test compounds (0–50 lM). Cells
were incubated for 24 h at 37 ꢁC. The compound-contain-
ing medium was removed, fresh serum-complete medium
was added, and the cells were incubated for an additional
24 h. Growth inhibition was determined using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
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Org. Chem. 1996, 61, 4756.
(MTT). Cell growth is correlated with mitochondria-
mediated reduction of MTT to a purple formazan
precipitate. Growth of treated cells was compared to the
growth of the untreated controls to determine percent
growth inhibition.
11. Fox, D. N. A.; Lathbury, D.; Mahon, M. F.; Molloy, K.
C.; Gallagher, T. J. Am. Chem. Soc. 1991, 113, 2652.
12. The structures of all compounds were elucidated using 1H
(200 MHz) and 13C NMR (50 MHz) in CDCl3 unless
otherwise noted. Results are reported as ppm downfield
14. Sang, S.; Lambert, J. D.; Tian, S.; Hong, J.; Hou, Z.; Ryu,
J. H.; Stark, R. E.; Rosen, R. T.; Huang, M. T.; Yang, C.
S.; Ho, C. T. Bioorg. Med. Chem. 2004, 12, 459. RAW
264.7 murine macrophages were plated in 24-well plates
(3 · 105 cells per well) and allowed to attach overnight.
The medium was replaced with fresh, serum-free DMEM
medium containing 0.1 lCi/mL [5,6,8,9,11,12,14,15-3H](N)
arachidonic acid (NEN Life Science, Boston, MA, USA).
The cells were incubated overnight to allow absorption of
the labeled arachidonic acid, washed twice with PBS
containing 0.1% BSA, and stimulated for 1 h with 2 lg/mL
lipopolysaccharide (LPS). The medium was replaced with
new, serum-free medium containing test compounds
(20 lM) and the cells were incubated for an additional
18 h at 37 ꢁC. After incubation, the medium was collected,
centrifuged at 12,000 rpm for 10 min and radioactivity in
the extracellular fluid was measured with a scintillation
counter.
15. Ryu, J. H.; Ahn, H.; Jin Lee, H. Fitoterapia 2003, 74, 350,
RAW 264.7 cells were determined by stimulating cells
(plated in 24-well plates at 3 · 105 cells per well) for 1 h
with 2 lg/mL LPS. The medium was then replaced with
serum-free medium containing the test compounds
(20 lM) and cells were cultured for 18 h. NO production
was determined spectrophotometrically using previously-
reported methods.
1
from internal TMS: (1) H NMR (CD3CN) d 6.53 (br s,
3H), 6.33 (s, 2H), 4.68 (m, 1H), 2.84 (dd, 1H, J = 14, 6),
2.74 (dd, 1H, J = 14, 6), 2.45 (m, 2H), 2.26 (m, 1H), 1.93
(m, 1H); 13C NMR d 176.7, 144.6, 130.2, 128.0, 107.9,
80.4, 39.6, 27.6, 26.1. (2) 1H NMR (CD3CN) d 6.80 (d, 1H,
J = 8), 6.77 (d, 1H, J = 2), 6.65 (dd, 1H, J = 8, 2), 4.69 (m,
1H), 2.91 (dd, 1H, J = 14, 6), 2.81 (dd, 1H, J = 14, 6),
2.58–2.17 (m, 3H), 2.03–1.84 (m, 1H); 13C NMR (CD3CN)
d 176.6, 143.7, 142.6, 128.4, 120.5, 115.7, 114.6, 80.5, 39.5,
27.6, 26.1. (10) 1H NMR d 6.82(d, 1H, J = 8), 6.81 (d, 1H,
J = 1.8), 6.72(dd, 1H, J = 8, 1.8), 4.71 (m, 1H), 3.89 (s,
3H), 3.01 (dd, 1H, J = 14, 6), 2.83 (dd, 1H, J = 14, 6),
2.60–2.18 (m, 3H), 2.05–1.86 (m, 1H); 13C NMR d 177.1,
145.8, 145.7, 129.1, 121.1, 115.6, 110.9, 80.9, 56.1, 40.8,
28.7, 27.1. (11) 1H NMR d 6.90–6.75 (m, 3H), 4.75 (m,
1H), 3.89 (s, 3H), 3.88 (s, 3H), 3.01 (dd, 1H, J = 14, 6),
2.91 (dd, 1H, J = 14, 6), 2.60–2.20 (m, 3H), 2.01–1.86 (m,
1
1H). (17) H NMR d 6.46 (s, 2H), 4.76 (m, 1H), 3.87 (s,
6H), 3.85 (s, 3H), 3.00 (dd, 1H, J = 14, 6), 2.91 (dd, 1H,
J = 14, 6), 2.60–2.21 (m, 3H), 1.97 (m, 1H); 13C NMR d
177.0, 153.4, 131.7, 106.8, 80.7, 60.9, 56.3, 41.7, 28.7, 27.2.
13. Bold, R. J.; Virudachalam, S.; McConkey, D. J. J. Surg.
Res. 2001, 100, 11. Cells were plated in 96-well microtiter