FT/IR spectrometer. Melting points were not corrected. Column
chromatography was performed on 200–300 mesh silica gel. Thin-
layer chromatography was conducted on Kieselgel 60 F254.
Diethyl flavone-◦6-yl phosphate (2e). A pale yellow solid. Yield:
1
74%. Mp: 67–68 C. H NMR (300 MHz, CDCl3): d 7.88–7.97
(m, 3H), 7.49–7.65 (m, 5H), 6.81 (s, 1H), 4.21–4.31 (m, 4H), 1.39
(t, J = 7.2 Hz, 6H); 13C NMR (75.4 MHz, CDCl3): d 177.4, 163.4,
153.0, 147.7 (d, J = 5.1 Hz), 131.6, 131.4, 128.9 (d, J = 3.0 Hz),
126.3, 126.1, 124.7, 119.6, 115.7 (d, J = 3.3 Hz), 106.9, 64.9 (d,
J = 6.3 Hz), 16.2 (d, J = 6.0 Hz); 31P NMR (121 MHz, CDCl3):
d -5.24; MS (ESI): m/z (%): 375 [(M+1)+, 100]. Anal. Calcd for
C19H19O6P: C, 60.96; H, 5.12. Found C, 60.96; H, 5.40. IR (KBr,
cm-1): 2988, 1643, 1498, 1393, 1261, 1235, 1181, 1026.
General procedure for the synthesis of 2a–2e. To a stirring
solution of 1 (2.5 mmol), DMAP (2.0 mmol per –OH group),
Et3N (2.0 mmol per –OH group) in anhydrous THF (30.0 mL),
a solution of ClP(O)(OEt)2 (DEPC, 30 mmol) in anhydrous THF
(20 mL) was added dropwise in an ice-water bath over 30 min.
After stirring at room temperature for 24 h under nitrogen, the
reaction mixture was diluted with EtOAc and washed with 0.5 M
HCl, 5% (w/v) K2CO3, brine and water, and then dried over
anhydrous Na2SO4. After removal of the solvent in vacuo, the
residue was purified by column chromatography on silica gel
with petroleum ether/EtOAc (4 : 1–2 : 1) as eluent to give the
corresponding product 2.
Assay procedure and determination of inhibitor IC50
CEase (porcine) was from Worthington, p-nitrophenyl butyrate
(pNPB) was from Sigma. The CEase inhibition was assayed
according to Hosie et al.21 with some modifications. CEase activity
was measured by following the hydrolysis of the colorimetric
substrate pNPB. The temperature was maintained at 25.0 0.2
◦C. All compounds and pNPB were dissolved in acetonitrile. The
final concentration of acetonitrile in the test solution was 1.5%.
First, 3.25 U of CEase (2.35 mg mL-1) were preincubated with
different concentrations of inhibitor in 0.1 M sodium phosphate
buffer (pH 7.04, containing 0.1 M NaCl) for 15 min. Then,
the pNPB (0.07 mM) was added to the reaction mixture and
the enzyme reaction was monitored for 1 min by measuring the
change in absorbance at 405 nm. The measurement was performed
in triplicate for each concentration and averaged before further
calculation. The computer program used for the analysis of data
was Origin 7.5.
Diethyl 4-oxo-2-phenyl-4H-chromen-3¢,4¢,5,7-yl phosphate (2a).
Oil. Yield: 91%. 1H NMR (300 MHz, CDCl3) d 7.85 (s, 1H), 7.53–
7.65 (m, 2H), 7.33 (s, 1H), 7.24 (s, 1H), 6.59 (s, 1H), 4.26–4.40 (m,
16H), 1.34–1.40 (m, 24H); 13C NMR (75.4 MHz, CDCl3) d 175.7,
159.9, 157.8, 153.8 (d, J = 3.4 Hz), 150.3 (d, J = 6.6 Hz), 144.2,
141.8, 128.2, 123.4, 121.7, 119.4, 113.7 (d, J = 5.5 Hz), 110.2 (d,
J = 6.3 Hz), 109.0, 105.7 (d, J = 5.4 Hz), 65.3, 65.2, 65.1, 65.0,
16.1 (m, 4C); 31P NMR(121 MHz, CDCl3) d: -6.65, -6.47, -5.88,
-5.43. MS: m/z 853(M+, 100); Anal. Calcd. for C31H46O18P4: C,
44.83; H, 5.58. Found C, 44.93; H, 5.74; IR (film, cm-1): 2919,
1654, 1419, 1330, 1273, 1105, 1029.
Hexaethyl tri-phosphoric ester of apigenin (2b). Oil. Yield:
83%. H NMR (300 MHz, CDCl3): d 7.75–7.79 (m, 2H), 7.15–
1
Mechanism of CEase inhibition
7.30 (m, 4H), 6.54 (s, 1H), 4.12–4.36 (m, 12H), 1.20–1.35 (m,
2a and 2d were assayed in 0.1 M sodium phosphate buffer
(pH 7.04, containing 0.1 M NaCl) using p-nitrophenyl butyrate
(pNPB) as the substrate to determine whether they are reversible
or irreversible inhibitors. The temperature was maintained by a
temperature controllable water bath kept at 25 0.2 ◦C. The
inhibitors and the enzyme were preincubated in the absence of
substrate for different time periods. Then, the pNPB (0.10 mM)
was added to the reaction mixture and the enzyme reaction was
monitored for 1 min by measuring the change in absorbance at
405 nm. The measurement was performed in triplicate for each
time interval and averaged before further calculation.
18H); 13C NMR (75.4 MHz, CDCl3 : d 175.7, 160.7, 157.7, 153.6
)
(d, J = 5.1 Hz), 153.1 (d, J = 6.2 Hz), 150.1 (d, J = 8.7 Hz), 127.6,
127.3, 120.3, 113.6 (d, J = 6.3 Hz), 109.9, 108.3, 105.5, 65.1, 65.0,
64.8, 64.7, 16.1 (m, 4C); MS (ESI): m/z (%): 678 [(M+1)+, 100].
Anal. Calcd for C27H37O14P3: C, 47.80; H, 5.50. Found C, 47.54;
H, 5.70. IR (film, cm-1): 2936, 1656, 1479, 1383, 1286, 1030, 988.
Tetraethyl bis-phosphoric es◦ter of chrysin (2c). A pale yellow
solid. Yield: 86%; Mp: 79–81 C (lit.19 83–84 ◦C). 1H NMR (300
MHz, CDCl3): d 7.77–7.86 (m, 2H), 7.41–7.53 (m, 3H), 7.31–7.40
(m, 1H), 7.17–7.28 (m, 1H), 6.64 (s, 1H), 4.24–4.40 (m, 8H), 1.31–
1.40 (m, 12H); 13C NMR (75.4 MHz, CDCl3): d 175.9, 161.7,
157.8, 153.7 (d, J = 8.4 Hz), 150.2 (d, J = 7.1 Hz), 131.5, 130.7,
128.8, 125.9, 113.7 (d, J = 8.6 Hz), 109.9, 108.6, 105.6 (d, J = 3.7
Hz), 65.2, 65.1, 16.1, 16.0; 31P NMR (121 MHz, CDCl3): d -6.28,
-6.52. MS (ESI): m/z (%): 526 [M+, 100]. IR (film, cm-1): 2986,
1651, 1438, 1380, 1278, 1156, 1031, 979.
Kinetic characterization of CEase inhibition
The general procedure of the kinetic characterization of CEase is
also similar to the above inhibition assays. First, 3.25 U of CEase
(2.35 mg mL-1) were preincubated with the inhibitor in sodium
phosphate buffer for 15 min. Then, different concentrations
of pNPB were added to the reaction mixture and the enzyme
reaction was monitored for 1 min by measuring the change in
absorbance at 405 nm. Triplicate sets of data were collected for
each inhibitor concentration. Kinetics of CEase inhibition was
generally analyzed by Lineweaver–Burk plots.4
Diethyl flavone-7-yl phosphate (2d). A pale yellow solid. Yield:
80%; Mp: 61–63 ◦C (lit.20 60–61 ◦C). 1H NMR (300 MHz, CDCl3):
d 8.20 (d, J = 8.7 Hz, 1H), 7.91–7.94 (m, 2H), 7.53–7.57 (m, 4H),
7.25–7.29 (m, 1H), 6.84 (s, 1H), 4.24–4.34 (m, 4H), 1.41 (dt, J1 =
7.2 Hz, J1 = 1.5 Hz, 6H); 13C NMR (75.4 MHz, CDCl3): d 177.3,
163.5, 156.8, 154.6 (d, J = 6.9 Hz), 131.6, 131.4, 128.9, 127.4, 126.1,
121.0, 117.8 (d, J = 7.5 Hz), 108.9 (d, J = 4.3 Hz), 107.5, 65.1 (d,
J = 7.6 Hz), 16.1 (d, J = 7.5 Hz); 31P NMR (121 MHz, CDCl3):
d -5.87. MS (ESI): m/z (%): 374 [M+, 100]. IR (film, cm-1): 2985,
1642, 1444, 1374, 1280, 1158, 1040, 976.
Acknowledgements
This work was supported by research grants from the National
Natural Science Foundation of China (Grant No. 20602043) and
the Fundamental Research Funds for the Central Universities.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 2530–2534 | 2533
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