S.H. Chan et al. / European Journal of Medicinal Chemistry 44 (2009) 2736–2740
2739
(500 MHz, CDCl3):
d
5.13 (s, 2H), 7.51 (t, 2H, J ¼ 8.0 Hz), 7.63 (t, 1H,
washing with phosphate buffered saline and centrifugation, cancer
cells were re-suspended in complete cell culture medium at
a concentration of approximately 1 ꢂ105/mL and counted manually
using a haematocytometer under an inverted microscope. Cancer
cells seeded in the 96 wells microtitre plates for 24 h were prepared
J ¼ 7.5 Hz), 7.73–7.75 (m, 2H), 7.88–7.90 (m, 2H), 8.00 (d, 2H,
J ¼ 7.5 Hz); 13C NMR (125 MHz, CDCl3):
d 44.85, 124.20, 128.79,
129.55, 132.87, 134.59, 134.70, 134.79, 135.04, 168.55, 191.62; HRMS
(ESI): Calcd. for C16H11NO3Na [M þ Na]þ, 288.0637; found 288.0641;
Melting Point ¼ 168–169 ꢁC; Yield ¼ 68% (4.1 mmol., 1.1 g).
for the
a-phthalimide ketones screening. The a-phthalimide
ketones were dissolved in molecular biology grade dimethylsulf-
oxide (DMSO). Cisplatinum was used as the positive reference and
5.1.2. 2-Phthalimide-1-p-tolylethanone 4
Prepared according to the general procedure described above
from 2-bromo-40-methylacetophenone (6 mmol, 1.28 g) and
potassium phthalimide (6 mmol, 1.11 g) and isolated as a white
added at a starting concentration of 167
m
M. Compounds were
added at a starting concentration of 176 M–179
m
mM and followed
by a serial of two-fold dilutions and incubated for a further 48 h.
The effective concentration of DMSO in each case was approxi-
mately 0.1% by volume. Untreated control received either total
complete cell culture medium or 0.1% DMSO. Afterwards, the
evaluation of possible antiproliferative potential of our synthesized
compounds was evaluated by the One Step ATP lite assay cell
proliferation kit purchased from PerkinElmer (Netherlands). The
one step ATP lite assay is a homogeneous experiment of deter-
mining the number of viable cells in culture based on quantitation
of the ATP present, which is an indicator of metabolically active
cells. The homogeneous assay procedure involves adding the single
reagent directly to cells cultured in serum-supplemented medium.
This results in cell lysis and generation of a luminescent signal
proportional to the amount of ATP present. The amount of ATP is
directly proportional to the number of cells present in culture.
solid, 1H NMR (500 MHz, CDCl3):
d 2.44 (s, 3H), 5.11 (s, 2H), 7.31 (d,
2H, J ¼ 8.5 Hz), 7.74–7.76 (m, 2H), 7.89–7.92 (m, 4H); 13C NMR
(125 MHz, CDCl3):
d 22.48, 44.79, 124.22, 128.93, 130.25, 132.64,
132.96, 134.78, 145.71, 168.64, 191.20; HRMS (ESI): Calcd. for
C17H13NO3Na [M þ Na]þ, 302.0793; found 302.0779; Melting
Point ¼ 177–177.5 ꢁC; Yield ¼ 66% (4.0 mmol., 1.1 g).
5.1.3. 2-Phthalimide-1-(4-fluoro-phenyl)ethanone 5
Prepared according to the general procedure described above
from 2-bromo-40-fluoroacetophenone (6 mmol, 1.30 g) and potas-
sium phthalimide (6 mmol, 1.11 g) and isolated as a white solid, 1H
NMR (500 MHz, CDCl3):
d 5.09 (s, 2H), 7.15–7.19 (m, 2H), 7.73–7.75 (m,
2H), 7.87–7.88(m, 2H), 8.01–8.04(m, 2H);13CNMR(125 MHz,CDCl3):
d
44.67, 116.69, 116.87, 124.20, 131.50, 132.79, 134.83, 165.84, 167.88,
168.48, 190.14; HRMS (ESI): Calcd. for C16H10NO3FNa [M þ Na]þ,
306.0542; found 306.0540; Melting Point ¼ 158–158.9 ꢁC;
Yield ¼ 45% (2.7 mmol., 0.77 g).
5.4. Morphological investigation of cancer cells
MDAMB-231 and SKHep-1 human carcinoma cells were seeded
at a concentration of 1 ꢂ105/mL. After 24 h,
a
-phthalimide ketones
5.1.4. 2-Phthalimide-1-biphenyl-4-yl-ethanone 6
Prepared according to the general procedure described above
from 2-bromo-40-phenylacetophenone (6 mmol, 1.65 g) and
potassium phthalimide (6 mmol, 1.11 g) and isolated as a white
were added (compound 4 at 179 mM and compound 5 at 177 mM)
and incubated for 48 h. Cells were then fixed with trichloroacetic
acids, washed and Sulforhodamine B stained. Any morphological
changes were compared with the vehicle control under the inver-
ted microscope.
solid, 1H NMR (500 MHz, CDCl3):
d
5.17 (s, 2H), 7.43–7.44 (m, 1H),
7.49–7.51 (m, 2H), 7.64–7.66 (m, 2H), 7.74–7.78 (m, 4H), 7.91–7.93
(m, 2H), 8.08–8.10 (m, 2H); 13C NMR (125 MHz, CDCl3):
44.90,
d
124.25, 127.99, 128.19, 129.14, 129.42, 129.70, 132.95, 133.78, 134.81,
140.30, 147.40, 168.60, 191.24; HRMS (ESI): Calcd. for C22H15NO3Na
[M þ Na]þ, 364.0950; found 364.0964; Melting Point ¼ 198–199 ꢁC;
Yield ¼ 73% (4.4 mmol., 1.5 g).
Acknowledgments
We acknowledge a donation fund from Mr. KC Lo to Dr. JCO Tang
for the establishment of Lo Ka Chung Centre for Natural Anti-cancer
Drug Development (Laboratory B), the matching fund provided by
the Hong Kong Polytechnic University (BB65) and a Niche area
grant offered by the Hong Kong Polytechnic University to Dr. KH
Lam and Dr. JCO Tang (BB8T). Drs. CH Chui and FY Lau are the
honorary tutors kindly offered by Professor J Sung and Professor
GYM Cheng from Department of Medicine and Therapeutics, Prince
of Wales Hospital, The Chinese University of Hong Kong. Dr. CH
Chui is supported by the post of ‘‘Senior Research Fellow’’ kindly
offered by Dr. JCO Tang (Niche area grant: BB8P to Dr. JCO Tang).
Special thank is due to Professor R Gambari, Drs. CH Chui and SHL
Kok who gave us valuable advice on the anticancer drug screening
experiments and their constructive criticism on this paper.
Professor R Gambari is sponsored by AIRC (Italian Association for
Cancer Research).
5.1.5. 2-Phthalimide-1-phenylpropanone 7
Prepared according to the general procedure described above
from 2-bromopropiophenone (6 mmol, 896
phthalimide (6 mmol, 1.11 g) and isolated as a white solid, 1H NMR
(500 MHz, CDCl3):
1.73 (d, 3H, J ¼ 7.5 Hz), 5.66 (q, 1H, J ¼ 7.0 Hz),
7.39 (t, 2H, J ¼ 7.5 Hz), 7.48 (t,1H, J ¼ 7.5 Hz), 7.68–7.71 (m, 2H), 7.79–
7.83 (m, 4H); 13C NMR (125 MHz, CDCl3):
15.59, 51.62, 124.18,
ml) and potassium
d
d
128.70, 129.38, 132.47, 133.71, 134.84, 135.94, 168.13, 196.79; HRMS
(ESI): Calcd. for C17H13NO3Na [M þ Na]þ, 302.0793; found 302.0801;
Melting Point ¼ 85.7–86.4 ꢁC; Yield ¼ 65% (3.9 mmol, 1.1 g).
5.2. Cell lines and cell culture
Human breast cancer MDAMB-231 and hepatoma SKHep-1 cell
lines were used for preliminary anti-cancer screening of these
compounds. Both cancer cell lines were routinely maintained in
RPMI cell culture medium supplemented with 5% heat inactivated
fetal bovine serum and penicillin/streptomycin antibiotics
(complete cell culture medium). The cells were incubated in
a humidified cell culture incubator at 37 ꢁC with 5% CO2.
Appendix A. Supplemental material
Supplementary information for this manuscript can be down-
loaded at doi: 10.1016/j.ejmech.2008.10.024.
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5.3. Antiproliferative assay of a-phthalimide ketones
Cancer cells were removed from 75 cm3 sterile cell culture flasks
with 1 fold trypsin and neutralized with fetal bovine serum. After