Lin et al. Sci China Chem May (2016) Vol.59 No.5
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2.2.1 Synthesis of compound 2e
102.2, 55.4, 55.0, 51.5 (2C), 46.8 (2C), 33.5.
The mixture of 1-(2-methoxyphenyl) piperazine (192 mg,
1 mmol), 3-bromo-1-propyne (178 µL, 2 mmol) and K2CO3
(1.38 g, 10 mmol) in acetone (30 mL) was stirred at room
temperature for 12 h. The solvent was then evaporated un-
der reduced pressure. The residue was dissolved in H2O and
extracted with ethyl acetate. The organic layer was dried
over anhydrous MgSO4, filtered and evaporated in vacuum.
After addition of HCl/EtOH, the resulting suspension was
filtered to afford the gray solid 2e (0.2 g, 87%). m.p. 213–
215 °C. 1H NMR (600 MHz, DMSO-d6) δ: 7.05–6.90 (m,
4H), 4.16 (d, J=2.4 Hz, 2H), 3.90 (t, J=2.4 Hz, 1H), 3.80
(s, 3H), 3.54 (t, 4H), 3.23 (t, 2H), 3.15 (t, 2H).
2.2.4 Synthesis of probe 1c
To
a
solution of 2c (93 mg, 0.28 mmol) and
4-methylmorpholine (153 µL, 1.42 mmol) in dichloro-
methane (80 mL), isobutyl chloroformate (57 µL, 0.42
mmol) was slowly added with an ice-salt bath. The mixture
was stirred for 40 min, and then dichloromethane (20 mL)
with 2d (115 mg, 0.42 mmol) and TEA (1 mL) was slowly
added. Then the ice bath was removed. The solution was
stirred overnight and evaporated under reduced pressure.
The residue was purified by silica gel chromatography with
ethyl acetate/ CH3OH (50:1) to yield 55 mg of pure 1c
(36%). m.p. 165–167 °C. 1H NMR (600 MHz, DMSO-d6) δ:
8.88 (s, 1H), 8.55 (s, 1H), 8.52 (s, 1H), 7.65 (d, J=9.0 Hz,
1H), 6.92–6.83 (m, 5H), 6.68 (d, J=2.1 Hz, 1H), 3.77 (s,
3H), 3.53–3.43 (m, 6H), 2.97 (s, 4H), 2.58–2.51 (m, 6H),
1.15 (t, J=6.9 Hz, 6H). ESI-HRMS: m/z 546.2801 (M+H+).
2.2.2 Synthesis of probe 1a
To a solution of 2a (0.41g, 2mmol) and 4-methylmorpholine
(896 µL, 8 mmol) in dichloromethane (70 mL), isobutyl
chloroformate (298 µL, 2.2 mmol) was slowly added with
an ice-salt bath. The solution was stirred for 40 min and
slowly added by the dichloromethane (25 mL) with 2d (0.43
g, 1.6 mmol) and triethylamine (TEA 2.29 mL). Then, the
ice bath was removed. The solution was stirred overnight and
evaporated under reduced pressure. After addition of
HCl/EtOH, the resulting suspension was filtered. The solid
was further purified by silica gel chromatography with pe-
troleum ether/ethyl acetate (1:3) to afford 170 mg of pure 1a
(23.1%). m.p. 250–250.6 °C. 1H NMR (600 MHz,
DMSO-d6) δ: 11.22 (s, 1H), 10.06 (s, 1H), 8.95–8.93 (m,
1H), 8.83 (s, 1H), 7.85 (d, 1H), 7.04–6.98 (m, 2H), 6.95–
6.89 (m, 3H), 6.85 (d, 1H), 3.79 (s, 3H), 3.76 (q, 2H), 3.66
(d, 2H), 3.50 (d, 2H), 3.37 (q, 2H), 3.24 (q, 2H), 3.24 (q,
2H), 2.99 (t, 2H). 13C NMR (75 MHz, DMSO-d6) δ: 164.0,
162.4, 160.7, 156.3, 151.8, 148.3, 139.3, 132.0, 123.5, 120.8,
118.3, 114.4, 113.4, 111.9, 110.9, 101.8, 55.3, 54.8, 51.4
(2C), 46.8 (2C), 33.9. ESI-HRMS: m/z 424.1858 (M+H+).
2.2.5 Synthesis of probe 1d
To a mixture of 2d (126 mg, 0.5 mmol) and TEA (1.4 mL,
10 mmol) in dichloromethane (15 mL), fluorescein
isothiocyanate (FITC, 97 mg, 0.25 mmol) dissolved in
N,N-dimethylformamide (DMF, 4 mL) was added slowly
with an ice bath. Then the reaction mixture was stirred
overnight in the dark at room temperature for 3 d. The solu-
tion was removed under reduced pressure, and diluted with
ethyl acetate and methanol. The precipitation was collected
by filtration. The solid was dissolved in 2 M NaOH, and the
crude was filtered. After slow addition of 2 M HCl to the
filtrate, the resulting suspension was filtered to afford a
tawny solid 1d (80 mg, 52.3%). m.p. 188–190 °C.
ESI-HRMS: m/z 625.2091 (M+H+). H NMR (600 MHz,
1
DMSO-d6) δ: 10.21 (s, 2H), 8.28 (s, 1H), 8.03 (s, 1H), 7.76
(s, 1H), 7.71 d, 1H), 6.95–6.93 (m, 2H), 6.91–6.85 (m, 2H),
6.67–6.60 (m, 2H), 6.59–6.55 (m, 4H), 3.77 (s, 3H), 3.70 (s,
3H), 3.01 (s, 4H), 2.65 (s, 5H).
2.2.3 Synthesis of probe 1b
2.2.6 Synthesis of probe 1e
To a solution of 2b (0.4 g, 1.46 mmol) and 4-methylmor-
pholine (491 µL, 4.38 mmol) in dichloromethane (60 mL)
isobutyl chloroformate (198 µL, 1.46 mmol) was added
slowly with an ice-salt bath. The mixture was stirred for 40
min, and then dichloromethane (35 mL) with 2d (0.44 g,
1.61 mmol) and TEA (2.3 mL) was slowly added. After
removing the ice bath, the solution was stirred overnight
and removed under reduced pressure. After addition of
HCl/EtOH, the mixture was filtered, and the precipitate was
recrystallized by CH3OH and H2O to afford probe 1b (110
mg, 14.3%). m.p. 240 °C (dec.) ESI-HRMS: m/z 491.2010
To a mixture of 3c (50 mg, 0.19 mmol) and 2e (51 mg, 0.19
mmol) in water/EtOH (v/v=1:1, 2 mL), sodium ascorbate
(34 mg, 0.17 mmol) and 0.5 mol/L CuSO4 (38 µL, 0.019
mmol) in water were slowly added. The mixture was stirred
overnight in the dark at room temperature. The resulting
suspension was filtered, and the residue was purified by
silica gel chromatography with petroleum ether/ethyl ace-
tate/CH3OH (3:1:1) to yield the yellow solid 1e (50 mg,
54%). m.p. 147–148 °C. 1H NMR (600 MHz, DMSO-d6) δ:
8.47 (s, 1H), 8.42 (s, 1H), 7.63 (d, J=9.0 Hz, 1H),
6.94–6.81 (m, 5H), 6.67 (d, J=2.1 Hz, 1H), 3.76 (s, 3H),
3.72 (s, 2H), 3.48 (q, J=6.9 Hz, 4H), 2.96 (s, 4H), 2.59 (s,
4H), 1.15 (t, J=6.9 Hz, 6H). ESI-HRMS: m/z 489.2677
(M+H+).
1
(M+H+). H NMR (600 MHz, DMSO-d6) δ: 11.04 (s, 1H),
9.83 (s, 1H), 9.07–9.05 (m, 1H), 8.97 (s, 1H), 7.79 (s, 1H),
7.78 (d, 1H), 7.04–6.89 (m, 6H), 3.80 (s, 3H), 3.73–3.70 (m,
4H), 3.55–3.52 (m, 2H), 3.41–3.40 (m, 2H), 3.27–3.24 (m,
2H), 3.00–2.96 (m, 2H). 13C NMR (75 MHz, DMSO-d6) δ:
162.9, 159.7, 156.3, 154.8, 151.8, 142.3, 139.4, 137.2,
131.1, 127.4, 123.4, 120.8, 118.8, 114.4, 111.9, 110.1,
2.3 Fluorescence spectroscopy test
Buffer reagents were purchased from Aldrich and Acros and