4
Tetrahedron
ACCEPTED MANUSCRIPT
(19.7 g, 176 mmol) was added into the flask. The mixture was
culture process. Before observation, the cells were washed 3
refluxed for 2 h and poured into ice. It was extracted with EtOAc
and the organic layer was washed with water followed by brine,
dried over Na2SO4 and concentrated to dryness. The crude
product was crystallized in a mixture of EtOH and petroleum
ether to give 6.22 g 4 as a brown solid (22% yield).
times with commercial available PBS buffer solution (pH =
7.4). Finally, 100 ꢀL of PBS solution was added and the cells
were visualized with OperettaTM from PerkinElmer. The
fluorescence images were taken under 20 × objective. Red
fluorescence of probe 1 was excited with wavelengths ranged
from 460-490 nm. The emission wavelengths were ranged
from 650 – 760 nm.
4.2.3. 2-Methyl-4H-chromen-4-one (5). Compound 4 (6.22 g,
34.9 mmol) and 3.3 mL 98% H2SO4 were refluxed in 50 mL
AcOH for 30 min. Then it was poured into ice and extracted with
DCM. The organic layer was washed with 10% Na2CO3 (aq)
followed by water, dried over Na2SO4 and concentrated to
dryness. The crude product was crystallized in a mixture of
EtOAc and petroleum ether to give 5.1 g 5 as a pale yellow solid
Acknowledgements
This work was supported by National Natural Science
Foundation of China (No. 81172936 and No. 91213303).
1
Supplementary data
(91% yield). mp 71-72oC, H NMR (400 MHz, CDCl3) δ 8.28 –
8.12 (m, 1H), 7.70 – 7.59 (m, 1H), 7.46 – 7.34 (m, 2H), 6.18(s,
1H), 2.39 (s, 3H).
Supplementary data associated with this article can be found
4.2.4. 2-(2-Methyl-4H-chromen-4-ylidene)malononitrile (6).
Compound 5 (1.0 equiv) and malononitrile (1.2 equiv) were
refluxed in Ac2O for 14 h. Then water was added into the
solution and refluxed for another 30 min. The mixture was
concentrated under reduced pressure and purified by column
chromatography using (petroleum ether: DCM = 3:1 followed by
petroleum ether: DCM = 2:1) as eluent. The crude product was
recrystallized in a mixture of EtOAc and petroleum ether to
afford 1.55 g compound 6 as an orange solid (23%). 1H NMR
(400 MHz, CDCl3) δ 8.93 (d, J = 8.5 Hz, 1H), 7.72 (t, J = 7.8 Hz,
1H), 7.54 – 7.40 (m, 2H), 6.72 (s, 1H), 2.44 (s, 3H).
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1
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