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G0/G1
S
G2/M
100
80
60
40
20
0
13. Ouyang, N.; Williams, J. L.; Tsioulias, G. J.; Gao, J.; Iatropoulos, M. J.; Kopelovich,
L.; Kashfi, K.; Rigas, B. Cancer Res. 2006, 66, 4503.
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0
1
3
5
10
20
0
100 200 300
16. Hulsman, N.; Medema, J. P.; Bos, C.; Jongejan, A.; Leurs, R.; Smit, M. J.; de Esch, I.
J.; Richel, D.; Wijtmans, M. J. Med. Chem. 2007, 50, 2424.
Agent [1], µM
Agent [3], µM
17. Kashfi, K.; Rigas, B. Biochem. Biophys. Res. Commun. 2007, 358, 1096.
18. General: All moisture sensitive reactions were performed under inert
atmosphere (argon) using syringe–septum cap techniques. All glassware was
oven-dried. Freshly distilled anhydrous solvents were used for all reactions.
THF was distilled from sodium/benzophenone, while DCM was distilled from
calcium hydride. The 1H NMR high resolution spectra were recorded using
Varian Inova 500 MHz (1H NMR) spectrometers. All spectra were recorded in
CDCl3. Splitting patterns are described as singlet (s), doublet (d), doublet of
doublet (dd), triplet (t), quartet (q), and broad (br). The value of chemical shifts
(d) are given in ppm relative to TMS as internal standard and coupling
constants (J) are given in hertz (Hz). Thin-layer chromatography was
Figure 3. Effect of 1 and 3 on cell cycle in HT-29 colon cancer cells. Cells were
treated for 24 h with various concentrations of 1 and 3, and their cell cycle phase
distribution was determined by flow cytometry. Results are representative of two
different experiments. This study was repeated twice generating results within 10%
of those presented here.
apoptosis and inhibition of proliferation contribute more or less
equally to the inhibitory effects of 3 (Fig. 2C).
We examined whether cell growth inhibition by 1 and 3 were
attributable to changes in the phases of the cell cycle. HT-29 cells
were treated with different concentrations of 1 and 3 for 24 h after
which the population of cells were examined. Since these com-
pounds have different potency ratios, it was of interest to examine
them at a time point where they have differential effects on
proliferation and apoptosis. Therefore, 24 h was chosen as a repre-
sentative time point in this study. For both 1 and 3, there was a
dose-dependent reduction in the population of cells in G0/G1, the
S phase increased at low concentrations but was essentially
unchanged at higher concentrations, and the G2/M population
increased (Fig. 3). For example, treatment with 3 lM of 1, caused
a reduction in the population of cells in G0/G1 phase from 74.8%
to 53.2% and cells in the S phase increased from 19.4% to 38.2%,
and G2/M increased from 5.8% to 9.6%. At 10 lM of 1, G0/G1 was
reduced to 42.9%, S phase was 40.8%, and G2/M population had
increased to 16.3%. These data are consistent with a G2/M block
as has been reported for other compounds in different cell
lines.26,28,29 Agent 3 behaved similarly to 1 but at much higher
performed on 250
l silica plates (Analtech) and column chromatographic
purifications were performed on 100–200 mesh silica gel (Natland). Unless
otherwise noted, all the aldehydes and reagents herein used were acquired
from Sigma–Aldrich and Across Chemicals and used without further
purification. Extracts were dried over anhydrous Na2SO4 and filtered prior to
removal of all volatiles under reduced pressure. The mass spectra were
recorded on a JEOL JMS-SX 10217 instrument (EI).
19. 4-Acetyloxybenzyl diethyl phosphate (1): To
a solution of p-acetoxybenzyl
alcohol (1 g, 6.0 mmol) in dry THF (50 ml) was added triethyl amine (Et3N,
1.25 ml, 9.0 mmol), DMAP (75 mg, 0.6 mmol) and diethyl chlorophosphate
(0.95 ml, 6.6 mmol) over a 30 min period at room temperature under an argon
atmosphere. The resultant white heterogeneous mixture was stirred for 12 h.
After completion of the reaction as monitored by TLC, water was added and the
mixture was extracted using EtOAc. The organic layer was then separated and
washed with aq NaHCO3 and brine. The combined organic layers were dried
and concentrated under vacuo. The residue was purified by silica gel column
chromatography, eluting with 40% EtOAc in hexane to afford compound 1
(1.4 g, 82% yield). 1H NMR (CDCl3, 500 MHz): dH 7.42 (d, J = 8.0 Hz, 2H), 7.10 (d,
J = 8.0 Hz, 2H), 5.05 (d, J = 8.0 Hz, 2H), 4.05–4.12 (m, 4H), 2.31 (s, 3H), 1.31 (t,
J = 7.0 Hz, 6H). 13C NMR (CDCl3, 125 MHz): dC 16.11 (d, JCP = 6.8 Hz), 21.0, 63.91
(d, JCP = 6.1 Hz), 68.39 (d, JCP = 5.3 Hz), 121.88, 129.18, 133.74 (d, JCP = 6.8 Hz),
150.86, 169.30. 31P NMR (CDCl3, 202 MHz): dP À0.35. ESIMS: m/z 303 (M++1),
325 (M++Na).
2-Acetyloxybenzyl diethyl phosphate (2): The title compound was obtained from
2-acetoxybenzyl alcohol using the above procedure giving a 65% yield. 1H NMR
(CDCl3, 500 MHz): dH 7.41 (d, J = 8.0 Hz, 1H), 7.40 (d, J = 7.6 Hz, 1H), 7.32 (t,
J = 7.0 Hz, 1H), 7.18 (t, J = 7.0 Hz, 1H) 5.19 (s, 2H), 4.20 (m, 4H), 2.10 (s, 3H),
1.36 (t, J = 7.0 Hz, 6H). 13C NMR (CDCl3, 125 MHz): dC 15.76 (d, JCP = 6.1 Hz),
20.63, 60.98, 64.48 (d, JCP = 6.1 Hz), 119.52, 124.72, 126.66 (d, JCP = 7.6 Hz),
129.40, 129.86, 148.60, 170.43. 31P NMR (CDCl3, 202 MHz): dP À5.81. ESIMS:
m/z 303 (M++1), 349 (M++2Na).
concentrations, 100–300 lM (Fig. 3).
In summary, the antiproliferative activity of EHBPs against four
human cancer cell lines was evaluated. Compounds 1, 1a, and 1b
showed strong cell growth inhibition in all human cancer cell lines
at low micromolar concentrations. Compounds 2 and 3 were sig-
nificantly less potent than 1 in these cell lines. Compounds 1 and
3 increased apoptosis and inhibited cell proliferation in a time-
dependent manner. This was accompanied by changes in the cell
cycle, showing a G2/M arrest. Further studies are needed to evalu-
ate the anticarcinogenic properties of these compounds and their
molecular targets.
3-Acetyloxybenzyl diethyl phosphate (3): The title compound was obtained from
3-acetoxybenzyl alcohol using the procedure described for compound 1. 85%
yield, 1H NMR (CDCl3, 500 MHz): dH 7.38 (t, J = 7.6 Hz, 1H), 7.25 (d, J = 7.6 Hz,
1H), 7.14 (bs, 1H), 7.06 (dd, J = 7.6, 1.95 Hz, 1H), 5.06 (d, J = 8.2 Hz, 2H), 4.05–
4.12 (m, 4H), 2.30 (s, 3H), 1.31 (t, J = 6.5 Hz, 6H). 13C NMR (CDCl3, 125 MHz): dC
15.67 (d, JCP = 6.1 Hz), 20.67, 63.51 (d, JCP = 5.3 Hz), 67.82, 120.53, 121.27,
124.61, 129.23, 137.39 (d, JCP = 6.8 Hz), 150.44, 168.91. 31P NMR (CDCl3,
202 MHz): dP À0.46. ESIMS: m/z 303 (M++1).
20. Nagele, E.; Schelhaas, M.; Kuder, N.; Waldmann, H. J. Am. Chem. Soc. 1998, 120,
6889.
Acknowledgment
21. McLaughlin, M. Org. Lett. 2005, 7, 4875.
22. 4-(Acetyloxy)-3-methylbenzyl diethyl phosphate (1a): The title compound was
obtained from 4-(acetoxy)-3-methylbenzyl alcohol using the procedure
described for compound 1. The yield was 68%. 1H NMR (CDCl3, 500 MHz): dH
7.27 (s, 1H), 7.24 (d, J = 8.5 Hz, 1H), 7.01 (d, J = 8.5 Hz, 1H), 5.02 (d, J = 5.0, 2H),
4.07–4.10 (m, 4H), 2.32 (s, 3H), 2.19 (s, 3H), 1.31 (t, J = 7.0 Hz, 6H). 13C NMR
(CDCl3, 125 MHz): dC 15.74 (d, JCP = 6.8 Hz), 15.83, 20.47, 63.58 (d, JCP = 5.3 Hz),
68.15, 121.79, 126.26, 130.14, 130.45, 133.43 (d, JCP = 6.8 Hz), 149.07, 168.85.
31P NMR (CDCl3, 202 MHz): dP À0.47. ESIMS: m/z 317 (M++1), 339 (M++Na).
4-(Acetyloxy)-3-chlorobenzyl diethylphosphate (1b): The title compound was
obtained from 4-(acetoxy)-3-chlorobenzyl alcohol using the procedure
Supported by the National Cancer Institute through a subcon-
tract from ThermoFisher, Contract No. FBS-43312-26.
References and notes
1. Ma, X.; Yu, H. Yale J. Biol. Med. 2006, 79, 85.
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