362
Y. Zhang et al. / European Journal of Medicinal Chemistry 53 (2012) 356e363
(M þ 1)þ. Anal. calcd for C19H14BrN3O3: C 55.36, H 3.42, N 10.19,
medium freshly before drug administration. Cell lines were seeded
into 96-well plates at density of 8000 cells/well. 24 h after seeding,
each compound dilution was added in duplicate and incubation
continued at 37 ꢂC in a humidified atmosphere containing 5% CO2.
found: C 55.38, H 3.44, N 10.16.
6.2.16. 7-((1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-4H-
chromen-4-one (5p)
After 48 h, add 20 mL MTT (Sigma) at 5 mg/mL in PBS (filter ster-
Color: Yellow; m.p. 188.0e188.2 ꢂC; Yield: 86%; 1H NMR:
ilized, light protected, and stored at 4 ꢂC) per well, and after 4 h of
incubation at 37 ꢂC, MTT is converted to a blue formazan product by
mitochondrial succinate dehydrogenase. This product was eluted
from cells by addition of 150 mL of DMSO. The absorbance at
570 nm was determined by an ELISA using a ELX800 microplate
spectrophotometer. The IC50 value was defined as the concentration
at which 50% of the cells could survive.
(300 MHz, CDCl3)
d
: 5.27 (s, 2H), 5.51 (s, 2H), 6.28 (d, 1H, J ¼ 6.3 Hz),
7.01 (m, 2H), 7.16 (m, 2H), 7.51 (m, 2H), 7.60 (s, 1H), 7.78 (d, 1H,
J ¼ 6 Hz), 8.11 ppm (d, 1H, J ¼ 8.4 Hz); 13C NMR (75 MHz, CDCl3)
d:
53.01, 62.46, 101.51, 112.28, 114.97, 120.01, 121.98, 124.74, 126.15,
127.97, 128.15, 129.77, 129.84, 135.07, 136.17, 155.11, 158.10, 162.45,
176.90 ppm; ESI-MS: m/s (%): 412.24 (M þ 1)þ. Anal. calcd for
C19H14BrN3O3: C 55.36, H 3.42, N 10.19, found: C 55.30, H 3.43, N 10.18.
6.3.2. Methodology of the apoptosis assay by annexin V-FITC
binding
6.2.17. 3-((4-((4-Oxo-4H-chromen-7-yloxy)methyl)-1H-1,2,3-
triazol-1-yl)methyl)benzonitrile (5q)
After staining with annexin V-FITC and PI using the Annexin
V-FITC apoptosis detection kit (Beyotime, China), cells were detec-
ted by flow cytometry to assess the membrane and nuclear events
during apoptosis. Briefly, A-549 cells (1 ꢃ 105/2 mL per well) were
seeded in six-well plates and treated with compound 5m at
Color: White; m.p. 186.0e186.9 ꢂC; Yield: 46%; 1H NMR:
(300 MHz, CDCl3)
d
: 5.31 (s, 2H), 5.61 (s, 2H), 6.29 (d, 1H, J ¼ 6.3 Hz),
7.02 (m, 2H), 7.53 (m, 2H), 7.58 (s, 1H), 7.68 (m, 2H), 7.79 (d, 1H,
J ¼ 6 Hz), 8.12 ppm (d, 1H, J ¼ 8.7 Hz); ESI-MS: m/s (%): 359.63
(M þ 1)þ. Anal. calcd for C20H14N4O3: C 67.03, H 3.94, N 15.63,
found: C 67.02, H 3.98, N 15.67.
concentrations of 1, 2, 4 and 8
mM, respectively and incubation
continued at 37 ꢂC in a humidified atmosphere containing 5% CO2.
After 48 h, cells were collected, washed once with PBS, and resus-
6.2.18. 4-((4-((4-Oxo-4H-chromen-7-yloxy)methyl)-1H-1,2,3-
triazol-1-yl)methyl)benzonitrile (5r)
pended in 195
mL of annexin V-FITC binding buffer which was then
added to 5 L of annexin V-FITC and incubated at room temperature
m
Color: White; m.p. 218.0e218.9 ꢂC; Yield: 69%; 1H NMR:
in the dark for 10 min. Subsequently, the buffer was added to 10 mL
(300 MHz, CDCl3)
d
: 5.30 (s, 2H), 5.63 (s, 2H), 6.29 (d, 1H, J ¼ 5.7 Hz),
of PI and incubated at 4 ꢂC in the dark for 5 min. The samples were
7.01 (d, 2H, J ¼ 9.6 Hz), 7.38 (d, 2H, J ¼ 8.1 Hz), 7.68 (m, 3H), 7.79 (d,
analyzed by a FACScan flow cytometer (Becton Dickinson, USA).
1H, J ¼ 6 Hz), 8.12 ppm (d,1H, J ¼ 8.7 Hz); 13C NMR (75 MHz, CDCl3)
d:
47.21, 62.29, 101.32, 112.64, 114.00, 117.56, 119.19, 121.67, 124.89,
126.81, 127.43, 128.76, 130.31, 131.20, 132.78, 136.29, 154.65, 158.05,
162.54,176.90 ppm; ESI-MS: m/s (%): 359.63 (M þ 1)þ. Anal. calcd for
C20H14N4O3: C 67.03, H 3.94, N 15.63, found: C 67.02, H 3.91, N 15.61.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (Grant No. 21172259) and Shanghai Rising-Star
Program (Grant No. 09QA1407000). We also thanks to the Key
laboratory of drug research for special environments, PLA.
6.2.19. 7-((1-(3-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-4H-
chromen-4-one (5s)
Color: White; m.p. 184.1e184.7 ꢂC; Yield: 64%; 1H NMR:
Appendix A. Supporting information
(300 MHz, CDCl3)
d
: 5.31 (s, 2H), 5.68 (s, 2H), 6.29 (d, 1H, J ¼ 6 Hz),
7.01 (m, 2H), 7.62 (m, 2H), 7.70 (s, 1H), 7.79 (d, 1H, J ¼ 6 Hz), 8.11 (m,
CCDC-777465 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge via the URL
CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (þ44) 1223
The purities of resultant compounds determined by HPLC with
two quite different systems can be obtained from the Supplemen-
tary Data.
1H), 8.18 (m, 1H), 8.25 ppm (d, 1H, J ¼ 2.1 Hz); 13C NMR (75 MHz,
CDCl3) d: 46.85, 61.98, 101.51, 112.97, 114.96, 119.11, 121.67, 124.45,
126.80, 127.13, 128.46, 130.73, 131.18, 132.78, 136.05, 154.43, 158.05,
162.50,176.91 ppm; ESI-MS: m/s (%): 379.11 (M þ 1)þ. Anal. calcd for
C19H14N4O5: C 60.32, H 3.73, N 14.81, found: C 60.30, H 3.71, N 14.86.
6.2.20. 7-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)-4H-
chromen-4-one (5t)
Color: White; m.p. 178.5e179.6 ꢂC; Yield: 70%; 1H NMR:
Appendix B. Supplementary material
(300 MHz, CDCl3)
d
: 5.31 (s, 2H), 5.69 (s, 2H), 6.29 (d, 1H, J ¼ 6 Hz),
7.01 (m, 2H), 7.20 (m, 2H), 7.40 (d, 1H, J ¼ 8.7 Hz), 8.11 (m, 1H), 7.60
(s,1H), 7.79 (d,1H, J ¼ 6 Hz), 8.12 ppm (d, 1H, J ¼ 8.7 Hz); ESI-MS: m/
s (%): 379.11 (M þ 1)þ. Anal. calcd for C19H14N4O5: C 60.32, H 3.73, N
14.81, found: C 60.30, H 3.72, N 14.86.
Supplementary data associated with this article can be found, in
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