370
Y. Zheng et al. / Dyes and Pigments 98 (2013) 367e371
Fig. 6. Fluorescence images of HUVEC incubated with 10
mM 1 and H2S. Cells treated with 1 in the (a) absence and (b) presence of 50 mM of H2S incubated for 5 min, and (c) presence
of 50 mM of H2S incubated for 30 min.
spectra were recorded using Bruker 400 MHz. Chemical shifts were
given in ppm and coupling constants (J) in Hz. UV absorption
spectra were obtained on Agilent Cary 60 UV/VIS Spectrometer.
Fluorescence emission spectra were obtained using Cary Eclipse
fluorescence spectrophotometer (Agilent).
4.2.4. Synthesis of 2-(2-methyl-4H-chromen-4-ylidene)
malononitrile (8)
A single round-bottomed flask equipped with a magnetic
stirring bar, containing 2-methyl-4H-chromen-4-one 7 (2 g,
12.5 mmol), acetic anhydride (40 mL) was charged with malo-
nonitrile (1 g, 15.15 mmol). The reaction mixture was stirred
vigorously and heated at reflux temperature for 14 h. After that,
the mixture was concentrated in vacuo to remove acetic anhy-
dride. Then water (30 mL) was added to the mixture and refluxed
for another 0.5 h. The mixture was filtered and the product
was recrystallized in ethanol to give a yellow solid. Yield: 350 mg
4.2. Synthesis
4.2.1. Synthesis of (4-azidophenyl)methanol (3)
To a solution of 4-aminobenzyl alcohol (300 mg, 2.44 mmol) in
5 mL of 10% HCl aqueous solution was added NaNO2 (201 mg,
2.92 mmol) in 3 mL aqueous solution at 0 ꢀC and stirred for 30 min.
Then NaN3 (190 mg, 2.92 mmol) in 3 mL aqueous solutionwas added
at 0 ꢀC and stirred for another hour. The reaction mixture was
warmed to 25 ꢀC, diluted with ethyl acetate, washed with water and
brine, dried over Na2SO4, concentrated in vacuo and subjected to
silica gel chromatography. A yellow oil 3 (315 mg, 86% yield) was
obtained by silica gel column chromatography using Petroleum
(13.5%). 1H NMR (CDCl3, 500 MHz)
d
8.92 (d, J ¼ 9 Hz 1H),
7.72 (t, J ¼ 7.5 Hz, 1H), 7.46 (d, J ¼ 8 Hz, 2H), 6.72 (s, 1H), 2.44
(s, 3H).
4.2.5. Synthesis of (E)-2-(2-(4-azidostyryl)-4H-chromen-4-ylidene)
malononitrile (1)
A mixture of 4-azido-benzaldehyde (155.6 mg, 0.75 mmol),
ether/EtOAc (5:1, v:v) as eluent. 1H NMR (500 MHz, CDCl3)
d 7.23 (d,
2-(2-methyl-4H-chromen-4-ylidene)malononitrile
7
(110 mg,
J ¼ 8 Hz, 2H), 6.93 (d, J ¼ 8.5 Hz, 2H), 4.51 (s, 2H), 2.04 (s, br, 1H).
0.75 mmol), piperidine (5 drops), and freshly distilled acetonitrile
(6 mL) were refluxed under argon for 24 h. The yellow precipitate
was filtered and washed with 50 mL of acetonitrile. The crude
product was purified by recrystallization from methanol to afford
compoud as a yellow solid. Yield: 110 mg (43.5%). 1H NMR (CDCl3,
4.2.2. Synthesis of 4-azido-benzaldehyde (4)
Compound 3 (306 mg, 2.06 mmol) was dissolved in 15 mL dry
CH2Cl2. Dess-Martin reagent (1.3 g, 3.08 mmol) was added and the
mixture was stirred for 2 h at room temperature, at which point
oxidation was completed. The mixture was diluted with EtOAc
(60 mL), washed with saturated Na2S2O3 (10 M), saturated aqueous
NaHCO3 (10 mL), and brine. Then organic layer was dried with
Na2SO4 and concentrated under reduced pressure. The crude
product was purified by silica gel column chromatography using
Petroleum ether/EtOAc (50:1, v:v) as eluent to afford 4 as a yellow
500 MHz)
d
8.92 (d, J ¼ 8 Hz,1H), 7.75 (t, J ¼ 8 Hz,1H), 7.60e7.55 (m,
4H, AreH), 7.46 (t, J ¼ 7.5 Hz,1H), 7.10 (d, J ¼ 8.5 Hz, 2H), 6.87 (s,1H),
6.77 (d, J ¼ 16 Hz, 1H). 13C NMR (CDCl3, 500 MHz)
d 157.3, 152.7,
152.3, 142.2, 137.6, 134.7, 131.4, 129.5, 126.0,125.9, 119.8,118.6,118.3,
117.8, 116.7, 115.6, 106.9, 63.0. HRMS (API-ES) calcd for C20H11N5O
[Mþ] 337.0964, found 337.0960.
oil. Yield: 283 mg (93.5%). 1H NMR (CDCl3, 500 MHz):
d 9.85 (s, 1H),
4.2.6. Synthesis of DCMCeNH2
7.78 (d, J ¼ 8.5 Hz, 2H), 7.06 (d, J ¼ 8.5 Hz, 2H).
A solution of 1 (30 mg, 0.089 mmol) in dry CH3CN (50 mL) was
added NaHS (996.8 mg, 17.8 mmol) which was dissolved in 5 mL
water. The reaction mixture was stirred at temperature for 1 h.
Then the mixture was filtered to get a brownish solid without
further purification. Yield: 17 mg (61.4%). 1H NMR (CDCl3, 500 MHz)
4.2.3. Synthesis of 2-methyl-4H-chromen-4-one (7)
A solution of 2-hydroxyacetophenone (5 g, 36.75 mmol) in dry
ethyl acetate (60 mL) was added sodium (4 g, 0.17 mmol), and the
reaction mixture was stirred at room temperature for 18 h. Cold
0.5 N HCl (50 mL) was added and the aqueous layer was separated.
The remained organic layer was dried and evaporated in vacuo to
obtain the crude diketone 5. A solution of the crude diketone with
several drops of concentrated HCl in methanol (40 mL) was stirred
at room temperature for 4 h. The methanol was removed in vacuo
to get the residue, followed by the addition of ethyl acetate (50 mL)
and washed with brine (50 mL). The organic layer was dried,
evaporated in vacuo and purified with silica gel chromatography
using Petroleum ether/EtOAc (9:1, v:v) as eluent to obtain 7 as a
d
8.92 (d, J ¼ 8.5 Hz, 1H), 7.72 (t, J ¼ 8 Hz, 1H), 7.57e7.53 (m, 2H),
7.47e7.42 (m, 3H), 6.81 (s, 1H), 6.70 (d, J ¼ 8.5 Hz, 2H), 6.62 (d,
J ¼ 16 Hz,1H), 4.05 (s, 2H), 13C NMR (CDCl3, 500 MHz)
d 160.2, 153.0,
152.6, 152.5, 141.1, 135.5, 131.2, 126.4, 125.0, 122.8, 119.4, 118.3, 117.7,
116.9, 114.3, 112.9, 105.2, 57.8. HRMS (ESI) calcd for C20H14N3O
[MHþ] 312.1137, found 312.1135.
4.3. Imaging of HUVEC
HUVEC were cultured in DMEM/F12 medium (Hyclone, China)
under 95% humidified atmosphere with 5% CO2 at 37 ꢀC. Besides
DMEM/F12, HUVEC culture medium contained 1% penicillin-
streptomycin and 10% fetal bovine serum which were purchase
yellow solid. Yield: 3.23 g (54.9%). 1H NMR (CDCl3, 500 MHz)
d 8.93
(d, J ¼ 8.5 Hz, 1H), 7.72 (t, J ¼ 7.5 Hz, 1H), 7.46 (d, J ¼ 8 Hz, 2H), 6.73
(s, 1H), 2.44 (s, 3H).