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K.-B. Huang et al. / European Journal of Medicinal Chemistry 63 (2013) 76e84
2.8, 1.4 Hz, 1H), 6.72 (d, J ¼ 8.0 Hz, 1H), 6.68e6.61 (m, 2H), 4.19 (d,
J ¼ 21.1 Hz, 1H), 4.11e4.03 (m, 2H), 4.00e3.93 (m, 1H), 3.88 (dd,
J ¼ 8.2, 7.1 Hz, 1H), 3.79 (s, 3H), 3.78 (s, 3H), 2.76e2.72 (m, 2H),
4.3.3. X-ray crystallography
The single crystals were mounted on glass fibres, and crystal data
were collected on a Agilent SuperNova CCD diffractometer equipped
2.72e2.63 (m, 2H), 1.21 (t, J ¼ 7.1 Hz, 3H), 1.12 (t, J ¼ 7.1 Hz, 3H). 13
C
with graphite monochromated Mo K
a
radiation (
l
¼ 0.71073 A) at
ꢀ
NMR (126 MHz, CDCl3)
d
156.41 (s), 148.97 (d, J ¼ 43.0 Hz), 148.80
room temperature. Absorption correction was applied by using the
multiscan program SADABS [37]. The structures were solved with
direct methods and refined using SHELX-97 programs [38]. The non-
hydrogen atoms were located in successive difference Fourier syn-
thesis. The final refinement was performed by full-matrix least-
squares methods with anisotropic thermal parameters for non-
hydrogen atoms on F2. The hydrogen atoms were added theoret-
ically and riding on the concerned atoms. The crystallographic data
and refinement details of the structures are summarized Table 2.
(s), 147.38 (s), 136.37 (s), 132.41 (s), 123.38 (d, J ¼ 4.6 Hz), 122.60 (d,
J ¼ 2.4 Hz), 120.60 (s), 111.99 (s), 111.23 (s), 63.27 (s), 63.08 (d,
J ¼ 6.9 Hz), 62.77 (d, J ¼ 7.0 Hz), 62.06 (s), 55.91 (s), 55.77 (s), 50.07
(d, J ¼ 16.2 Hz), 35.83 (s), 16.35 (d, J ¼ 5.7 Hz), 16.25 (d, J ¼ 5.7 Hz).
ESI þ MS: m/z 408.12 [Lf þ H]þ.
4.3.2. Synthesis of the platinum complexes
General procedure for the synthesis of the platinum complexes:
to a solution of cis-Pt(DMSO)2Cl2 (84 mg, 0.2 mmol) was added Laef
(0.2 mmol) dissolved in 30 mL mixtures of anhydrous dichloro-
methane and ethanol (1:1). The mixture was stirred in the dark at
room temperature for 1 day. The resulting yellow solution was fil-
tered and crystals were obtained by slow evaporation of filtrate
solution.
4.4. Cytotoxicity assay
The cell lines MG-63, SK-OV-3, HepG2 and HL-7702 were
obtained from the Shanghai Cell Bank in the Chinese Academy of
Sciences. Tumour cell lines were grown in the RPMI-1640 medium
supplemented with 10% (v/v) foetal bovine serum, 2 mM glutamine,
100 U/mL penicillin, and 100 U/mL streptomycin at 37 ꢀC, in a highly
humidified atmosphere of 95% air/5% CO2. The cytotoxicity of the
title compounds against MG-63, SK-OV-3, HepG2 cell lines was
examined by the microculture tetrazolium (MTT) assay [39]. The
experiments were carried out using reported procedure [40]. The
growth inhibitory rate of treated cells was calculated using the data
from three replicate tests as (ODcontrol ꢁ ODtest)/ODcontrol ꢂ 100%.
The compounds were incubated with various cell lines for 48 h at
five different concentrations of complex dissolved in fresh media;
the range of concentrations used is dependent on the complex. The
final IC50 values were calculated by the Bliss method (n ¼ 5). All tests
were independently repeated at least three times.
Data for 1: 1H NMR (500 MHz, d6-DMSO)
d 9.31 (s, 1H), 8.32e
8.19 (m, 1H), 7.56 (d, J ¼ 7.9 Hz, 1H), 7.31e7.26 (m, 1H), 6.52 (d,
J ¼ 2.2 Hz, 1H), 6.37e6.33 (m, 1H), 6.14 (dd, J ¼ 8.4, 2.2 Hz, 1H), 5.78
(s, 2H), 5.01 (d, J ¼ 24.0 Hz,1H), 4.34e4.12 (m, 1H), 4.06 (dq, J ¼ 14.1,
7.1 Hz, 2H), 3.95e3.87 (m, 1H), 1.19 (t, J ¼ 7.0 Hz, 3H), 1.06e1.01 (m,
3H). ES-MS: m/z 629.04 [M þ H]þ. Elemental analysis calculated: C,
32.63; H, 3.31; N, 4.30; Found: C, 32.55; H, 3.28; N, 4.35.
Data for 2: 1H NMR (500 MHz, d6-DMSO)
d
9.31 (d, J ¼ 5.1 Hz,
1H), 8.29 (td, J ¼ 7.8, 1.4 Hz, 1H), 7.47 (d, J ¼ 8.0 Hz, 1H), 7.31 (t,
J ¼ 4.5 Hz, 1H), 6.86e6.83 (m, 1H), 6.73 (d, J ¼ 7.9 Hz, 1H), 5.99 (d,
J ¼ 1.7 Hz, 1H), 5.92 (d, J ¼ 10.5 Hz, 2H), 4.89 (d, J ¼ 20.5 Hz, 1H),
4.19e4.13 (m, 2H), 4.10e4.00 (m, 4H), 3.63 (dd, J ¼ 14.3, 7.2 Hz, 2H),
1.24e1.20 (m, 3H), 1.14 (t, J ¼ 7.0 Hz, 3H). ESI-MS: m/z 643.04
[M þ H]þ. Elemental analysis calculated: C, 33.72; H, 3.36; N, 4.28;
Found: C, 33.65; H, 3.42; N, 4.35.
4.5. Apoptosis assays by flow cytometry assay
Data for 3: 1H NMR (500 MHz, d6-DMSO)
d
9.31 (d, J ¼ 4.5 Hz,
1H), 8.23e8.18 (m, 1H), 7.65 (d, J ¼ 8.0 Hz, 1H), 7.60e7.56 (m, 1H),
6.81e6.79 (m, 2H), 6.67 (dd, J ¼ 7.9, 1.4 Hz, 1H), 5.95e5.94 (m, 2H),
5.14 (d, J ¼ 19.7 Hz, 1H), 4.29 (dt, J ¼ 10.2, 7.0 Hz, 1H), 4.21e4.10 (m,
3H), 3.13 (dt, J ¼ 15.4, 6.6 Hz, 1H), 2.99 (d, J ¼ 3.6 Hz, 3H), 1.26 (t,
J ¼ 7.1 Hz, 3H), 1.18 (t, J ¼ 7.0 Hz, 3H). ESI-MS: m/z 657.06 [M þ H]þ.
Elemental analysis calculated: C, 34.40; H, 3.76; N, 4.38; Found: C,
34.45; H, 3.78; N, 4.46.
The ability of platinum complexes 2, 5 to induce apoptosis is
evaluated in MG-63 cell line using Annexin V conjugated with FITC
and propidium iodide (PI) counterstaining by flow cytometry.
MG-63 cells of exponential growth were inoculated in 6-well plates
and cultured for 12 h before the platinum compounds were added
to give the indicated final concentrations. After 48-h incubation,
cells were harvested, washed twice in phosphate-buffered saline
Data for 4: 1H NMR (500 M Hz, d6-DMSO)
d
9.30 (d, J ¼ 4.7 Hz,
(PBS), and resuspended in 100 mL binding buffer (including
1H), 8.32e8.22 (td, J ¼ 7.7, 1.6 Hz, 1H), 7.70e7.65 (m, 1H), 7.56 (d,
J ¼ 7.9 Hz, 1H), 6.62e6.58 (m, 1H), 6.38 (d, J ¼ 2.5 Hz, 1H), 6.15 (dd,
J ¼ 8.6, 2.5 Hz, 1H), 5.03 (d, J ¼ 23.9 Hz, 1H), 4.38e4.22 (m, 2H), 4.18
(m, 1H), 4.11 (m, 1H), 3.98e3.87 (s, 3H), 3.84e3.74 (s, 3H), 1.19 (t,
J ¼ 7.0 Hz, 3H), 1.05 (t, J ¼ 7.0 Hz, 3H). ESI þ MS: m/z 645.09
[M þ H]þ. Elemental analysis calculated: C, 33.42; H, 3.73; N, 4.43;
Found: C, 33.50; H, 3.75; N, 4.36.
140 mmol/L NaCl, 2.5 mmol/L CaCl2 and 10 mmol/L Hepes/NaOH,
pH 7.4) at a concentration of 1 ꢂ 106 cells/mL. Then cells were
incubated with 5
10 mmol/L NaCl, 1% bovine serum albumin, 0.02% NaN3 and
50 mmol/L Tris, pH 7.4) and 10 L PI (20 g/mL) for 15 min at room
m
L of Annexin Vꢁ FITC (in buffer including
m
m
temperature in the dark. Cells were kept shielded from light before
being analyzed by flow cytometry using a BectoneDickinson
FACSCalibur.
Data for 5: 1H NMR (500 MHz, d6-DMSO)
d 9.51 (m, 1H), 8.57
(dd, J ¼ 17.3, 5.3 Hz, 1H), 7.84 (d, J ¼ 8.0 Hz, 1H), 7.42 (d, J ¼ 7.9 Hz,
1H), 6.82 (d, J ¼ 9.4 Hz, 1H), 6.79 (d, J ¼ 4.2 Hz, 1H), 6.72 (d,
J ¼ 8.3 Hz, 1H), 4.89 (d, J ¼ 20.4 Hz, 1H), 4.55e4.30 (m, 2H), 4.08e
4.01 (m, 2H), 3.75 (d, J ¼ 3.6 Hz, 2H), 3.68 (s, 3H), 3.64 (s, 3H), 1.27
(q, J ¼ 6.8 Hz, 3H), 1.02 (t, J ¼ 6.9 Hz, 3H). ESI-MS: m/z 659.07
[M þ H]þ. Elemental analysis calculated: C, 34.80; H, 3.90; N, 4.35;
Found: C, 34.71; H, 3.95; N, 4.32.
4.6. Cell-cycle analysis
MG-63 cell lines were maintained in Dulbecco’s modified Eagle’s
medium with 10% foetal calf serum in 5% CO2 at 37 ꢀC. Cells were
harvested by trypsinization and rinsed with PBS. After cen-
trifugation, the pellet (105ꢁ106 cells) was suspended in 1 mL of PBS
and kept on ice for 5 min. The cell suspension was then fixed by the
dropwise addition of 9 mL precooled (4 ꢀC) 100% ethanol with vio-
lent shaking. Fixed samples were kept at 4 ꢀC until use. For staining,
cells were centrifuged, resuspended in PBS, digested with 150 mL of
Data for 6: 1H NMR (500 MHz, d6-DMSO)
d
9.40 (dd, J ¼ 40.4,
5.6 Hz, 1H), 8.61 (d, J ¼ 5.6 Hz, 1H), 7.76e7.62 (m, 1H), 6.84 (s, 2H),
6.81 (d, J ¼ 9.4 Hz, 1H), 6.72 (dd, J ¼ 8.0, 1.6 Hz, 1H), 5.15 (d,
J ¼ 19.6 Hz, 1H),
d 4.54 (m, 1H), 4.27 (m, 1H), 4.15 (m, 2H), 3.68 (s,
3H), 3.64 (s, 3H), 1.28 (t, J ¼ 7.0 Hz, 3H), 1.02 (t, J ¼ 6.9 Hz, 3H).
ESI-MS: m/z 673.12 [M þ H]þ. Elemental analysis calculated: C,
35.45; H, 4.10; N, 4.28; Found: C, 35.48; H, 4.15; N, 4.23.
RNase A (250 mg/mL), and treated with 150 mL of propidium iodide
(PI) (0.15 mM), then incubated for 30 min at 4 ꢀC. PI-positive cells
were counted with a FACScan Fluorescence-activated cell sorter
Huang, Ke-Bin
