Design, synthesis and biological evaluation of demethylcantharidate-linked platinum(II) complexes of N-monoalkyl-1R,2R-diaminocyclohexane derivatives

Article abstract of DOI:10.1016/j.ica.2017.03.033

Nine platinum(II) complexes with N-monoalkyl 1R,2R-DACH derivatives as carrier ligands and demethylcantharidate as a leaving group were synthesized and spectrally characterized. All the complexes showed considerable cytotoxicity against tested human cancer cell lines: A549, HCT116 HepG-2 and MCF7 cell lines. Especially, complex 2 exhibited potent cytotoxicity against A549 (1.01?μM) and HCT116 (0.83?μM) cell lines, and showed no cross-resistance to cisplatin against SGC7901/CDDP cell line (RF?=?1.44). In addition, the typical compounds were further studied by flow cytometric analysis and western blot method. The results indicated that they induced apoptosis by a mitochondrial-dependent pathway, which were similar to cisplatin.

Full text of DOI:10.1016/j.ica.2017.03.033

Inorganica Chimica Acta 462 (2017) 188–194
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Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Research paper
Design, synthesis and biological evaluation of demethylcantharidate-
linked platinum(II) complexes of N-monoalkyl-1R,2R-
diaminocyclohexane derivatives
⇑
Gang Xu, Jing Lin, Wanchun Li, Jian Zhao, Shaohua Gou
Pharmaceutical Research Center, School of Chemistry and Chemical Engineering and Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, Southeast University,
Nanjing 211189, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Nine platinum(II) complexes with N-monoalkyl 1R,2R-DACH derivatives as carrier ligands and demethyl-
cantharidate as a leaving group were synthesized and spectrally characterized. All the complexes showed
considerable cytotoxicity against tested human cancer cell lines: A549, HCT116 HepG-2 and MCF7 cell
Received 13 February 2017
Accepted 21 March 2017
Available online 23 March 2017
lines. Especially, complex
2
exhibited potent cytotoxicity against A549 (1.01
M) cell lines, and showed no cross-resistance to cisplatin against SGC7901/CDDP cell line
RF = 1.44). In addition, the typical compounds were further studied by flow cytometric analysis and
lM) and HCT116
(0.83 l
Keywords:
(
Platinum(II) complexes
Demethylcantharidate
N-monoalkyl-1R,2R-diaminocyclohexane
Cytotoxicity
western blot method. The results indicated that they induced apoptosis by a mitochondrial-dependent
pathway, which were similar to cisplatin.
Ó 2017 Published by Elsevier B.V.
Apoptosis
1
. Introduction
Cisplatin has been widely used in the treatment of testicular,
may generate a novel strategy to combine the two kinds of old
drugs together synergistically [17]. The platinum(II) complexes
containing camphorato, derivative of long used TCM camphor,
showed promising in vitro and in vivo anticancer activity [18,19].
Cantharidin is another TCM used for the treatment of liver, lung
and intestinal cancers for a long time. Demethylcantharidin is a
synthetic analogue with similar biological activity and much less
toxic than cantharidin. Combination platinum(II) moieties with
demethylcantharidate as a leaving group have resulted in a series
of novel complexes. The relative platinum complexes showed more
potent antitumor activity against some cancer cell lines than cis-
platin/oxaliplatin and had no cross-resistance with cisplatin. More-
ovarian, bladder and head/neck tumors for decades since its first
approval by FDA in 1978 [1]. After the success of cisplatin, a large
number of anticancer platinum complexes have been studied.
Among them, carboplatin and oxaliplatin have been accepted
worldwide, along with five other platinum(II) complexes gained
approval regionally in different nations (nedaplatin and miriplatin
in Japan, lobaplatin and dicycloplatin in China, heptaplatin in
Korea) [2]. However, these clinical used platinum drugs possess
similar drawbacks, including severe side effects and intrinsic
and/or acquired resistance [3–5]. Several strategies have been
employed in order to overcome these drawbacks, among which
application of bioactive ligands is a promising one in the design
of novel platinum complexes. Such resulting complexes may have
dual mechanisms of action, i.e., one from the proper biologically
active ligand and the other from Pt pharmacophore [6–12].
over, they possessed
a dual antitumor mechanism, namely,
inhibition of PP2A (demethylcantharidin) and platination of DNA
(platinum moiety) [20–22].
So far, numerous platinum complexes containing 1R,2R-
diaminocyclohexane (1R,2R-DACH) and its derivatives as carrier
ligands have been reported after oxaliplatin was approved world-
wide. In our previous research [23–28], a number of platinum(II)
complexes of N-monoalkyl 1R,2R-DACH as carrier ligands have
been investigated and some of these compounds showed better
anticancer activity towards some cancer cell lines than their coun-
terparts with 1R,2R-DACH as ligand. To continue our research, a
series of platinum(II) complexes were obtained in this study with
the above mentioned ligands and demethylcantharidate as a leav-
ing ligand. The in vitro cytotoxicity of the synthesized complexes
Traditional Asian medicine, especially traditional Chinese med-
icine (TCM), is a treasure yet to be efficiently explored [13,14].
Artemisinin [15] as antimalarial agent and arsenic trioxide [16]
for the use in leukaemia is one of modern drugs successfully mined
from traditional Chinese medicine. The platinum-TCM conjugates
⇑
Corresponding author.
E-mail address: sgou@seu.edu.cn (S. Gou).
http://dx.doi.org/10.1016/j.ica.2017.03.033
020-1693/Ó 2017 Published by Elsevier B.V.
0

G. Xu et al. / Inorganica Chimica Acta 462 (2017) 188–194
189
against six human solid cancer lines was studied. Additionally, the
mechanism of cellular death of typical complexes was also investi-
gated in this paper.
(25%) isotopes. Elemental analysis data of C, H and N for each com-
plex were in good fit with the empirical formula proposed.
Notably, complexes 2 and 3 had an extra stereogenic carbon
center along with other three stereogenic centers [24]; however,
no effort was devoted to isolating the pure enantiomers in this
study. Therefore, both R and S configurations at the a-carbon atom
are present in the final compounds.
2
. Results and discussion
2.1. Synthesis and characterization
2.2. In vitro cytotoxicity studies
Complexes 1–9 were synthesized via Scheme 1. The intermedi-
ates M1–M9 were obtained by a routine method described by our
previous study [23–25]. Demethylcantharidate was prepared by a
Diels-Alder reaction between furan and maleic anhydride, followed
by hydrogenation using Pd/C as catalyst [20]. All intermediates
The cytotoxicity of complexes 0–9 was determined by MTT
assays against A549, HCT116, HepG-2 and MCF7 human cancer cell
lines. The in vitro and in vivo suppression of growth of cancer cells
(especially hepatocellular carcinoma cells) by complex 0 were
reported before by Yee-Ping Ho’s group [20], and the in vitro cyto-
toxicity of complex 0 was measured in this paper as another posi-
tive control, along with cisplatin and oxaliplatin. The result of
cytotoxicity against four cancer cell lines are expressed as IC50 val-
ues listed in Table 1.
According to the IC50 values, most of the synthesized complexes
showed comparable or better cytotoxicity to cisplatin and oxali-
platin against A549, HCT116 and MCF7 cancer cell lines. However,
only complex 2 possessed comparable cytotoxicity against HepG-2
cell line with positive controls.
It is noted that the antiproliferative properties of the synthe-
sized platinum complexes with N-monoalkylsubstituted 1R,2R-
DACH as carrier ligands against A549 and HCT116 cancer cells
were enhanced compared with complex 0. Significantly, complex
2 were 3–4 times more potent than cisplatin/oxaliplatin against
A549 cell line and 2–3 folds more potent than cisplatin/oxaliplatin
against HCT116 cell line. Furthermore, complexes 5 and 7 had bet-
ter cytotoxicity against MCF7 cell line than complex 0, and com-
plex 5 was slightly more potent than cisplatin against MCF7 cell
line. However, the HepG-2 cell line was much less responsive to
our complexes than complex 0, which indicated that the steric hin-
derance in the carrier ligands might reduce the cytotoxicity of the
platinum complex against HepG-2 cell line.
1
were characterized by H NMR, FT-IR, and ESI-MS spectra together
with microanalysis. Platinum(II) complexes 1–9 were spectrally
1
13
characterized by H NMR, C NMR (complexes 2 and 7), FT-IR
and ESI-MS spectra as well as element analyses.
In the IR spectra of all platinum complexes, the NAH stretching
ꢀ1
vibrations appeared between 3202 and 3440 cm , shifting to
lower frequencies than those of the free alkyl amine. The as(C@O)
signal of the complexes appears in the range from 1650 to
m
ꢀ
1
1
622 cm , characteristic signals of coordinated dicarboxylates,
ꢀ1
and the CAO signal appeared in the range of 1311–1325 cm
All H NMR and C NMR data are compatible to the molecular
structures proposed in Scheme 1. In the H NMR spectra of com-
.
1
13
1
plexes 1–9, the broad signals of hydrogen atoms belonging to
amino groups appear in the range of d 6.17–6.50 ppm due to amine
coordination with metal ions, shifting to high-field compared with
the metal-free ligands in the range of d 8.80–9.40 ppm (appeared
6
only when d -DMSO was used as NMR solvent, complexes 2 and
7). Moreover, the signals of CAH protons connected to the amino
groups were observed between 2.48 and 2.94 ppm, clearly shifting
high-field relative to the corresponding signals (d ꢁ 3.5 ppm) in the
+
free ligands. Besides, all the complexes showed 100% of [M+H] or
+
[
M+Na] peaks in the ESI-MS spectroscopy, which have three main
194
195
196
ion peaks due to the existence of
Pt (33%),
Pt (34%) and
Pt
Scheme 1. The synthetic method and related chemical complexes studied in this work.

1
90
G. Xu et al. / Inorganica Chimica Acta 462 (2017) 188–194
Table 1
In vitro cytotoxicity of complexes 1–9 and positive controls against human tumor cell lines.
Complex
IC50
(
l
M)a
A549b
HCT116c
HepG-2d
MCF7e
Cisplatin
Oxaliplatin
3.65 ± 0.38
3.98 ± 0.51
21.2 ± 1.7
11.41 ± 0.98
1.01 ± 0.05
42.12 ± 3.21
> 100
15.77 ± 1.34
8.17 ± 0.96
20.81 ± 1.76
9.32 ± 0.84
32.12 ± 2.22
1.72 ± 0.19
2.21 ± 0.24
8.23 ± 0.78
> 100
0.83 ± 0.04
9.22 ± 1.23
16.28 ± 2.01
3.41 ± 0.23
> 100
1.22 ± 0.10
1.42 ± 0.12
0.52 ± 0.03
84.38 ± 5.11
7.55 ± 1.08
55.60 ± 4.30
99.04 ± 7.44
> 100
67.82 ± 4.33
> 100
13.82 ± 1.09
29.60 ± 1.83
1.18 ± 0.22
2.61 ± 0.18
9.30 ± 1.01
25.7 ± 1.87
11.44 ± 2.11
31.61 ± 2.88
9.10 ± 4.40
0.85 ± 0.07
22.81 ± 1.86
2.41 ± 0.19
> 100
0
1
2
3
4
5
6
7
8
9
5.21 ± 1.03
6.62 ± 0.44
18.37 ± 1.26
13.39 ± 1.34
a
IC50 is the drug concentration effective in inhibiting 50% of the cell growth measured by the MTT assay after 48 h drug exposure; Values represent the mean ± SD from
three intendent experiments.
b
Human non-small-cell lung cancer cell line.
Human colorectal carcinoma cell line.
Human hepatocellular carcinoma cell line.
Human breast carcinoma cell line.
c
d
e
Intrinsic and/or acquired resistance is the main drawback of
platinum-based drugs. It is reported that there are three main
mechanisms of Pt resistance, namely, (i) reduced drug uptake
and/or increased drug efflux; (ii) detoxification by intracellular thi-
ols such as glutathione; (iii) increased repair and/or tolerance of
DNA–cisplatin adducts [29,30]. The drug resistance may arise from
one or more of the above-mentioned mechanisms.
control, while complex 2 showed the highest population of apop-
totic cells (39.6%) among all tested complexes, about 1.1-fold
higher than cisplatin (35.2%) and 1.5-fold higher than oxaliplatin
(25.6%). The result of apoptosis study is consistent with that of
in vitro cytotoxicity assay against HCT116 cells, which indicates
that the tested compounds may cause cancer cell death through
an apoptotic mechanism.
The cytotoxicity of complex 2 against cisplatin-sensitive and
cisplatin-resistant gastric carcinoma cell lines (SGC7901 and
SGC7901/CDDP) was further investigated with cisplatin as positive
control, in order to find out whether our platinum complexes can
overcome the cisplatin resistance. The IC50 data are listed in
Table 2. The result showed that complex 2 was devoid of cross-
resistance to cisplatin with a remarkable decrease in RF value
2
.4. Western blot analysis
In order to explore more about the apoptotic mechanism, west-
ern blot assay was employed to determine the expressions of four
apoptotic-related proteins (Bax, Bcl-2, Procapase-3 and Cleaved-
caspase-3) of HCT116 and MCF7 cell lines treated with complex
(
1.44) compared with cisplatin (10.00), and complex 2 had better
2
, complex 7 and cisplatin. As shown in Fig. 3, remarkable increase
cytotoxicity against SGC7901/CDDP cell lines than cisplatin.
of Bax expression and decrease of Bcl-2 expression were observed
in both HCT116 and MCF7 cells treated with cisplatin, complex 2
and complex 7 compared with negative control. Furthermore, the
expressions of Procaspase-3 in two cancer cell lines were signifi-
cantly decreased, while the expressions of Cleaved-caspase-3 were
up-regulated. The result of western bolt analysis suggested that
platinum(II) complex 2 and 7 could induce cell apoptosis through
an intrinsic mitochondrial-dependent pathway similar to cisplatin.
2.3. Apoptosis study
Apoptosis is a programmed cell death. The apoptotic analysis of
complex 2, complex 7, cisplatin and oxaliplatin was carried out
using an AnnexinV-FITC/PI assay. The tested complexes were incu-
bated with HCT-116 cells for 24 h at 50 lM, and the results are
shown in Figs. 1 and 2. Q1 (top left quarter), Q2 (top right quarter),
Q3 (lower left quarter) and Q4 (lower right quarter) of the diagram
stand for the unnatural death cells, late apoptotic/necrotic cells,
normal cells, and early apoptotic cells, respectively.
As shown in Figs. 1 and 2, complex 2 and complex 7 greatly
increased the population of apoptotic cells compared with negative
3
. Conclusion
In the present study, a series of platinum(II) complexes with N-
monoalkylsubstituted 1R,2R-DACH as carrier ligands and
demethylcantharidate as a leaving ligand were synthesized and
characterized. According to the result of in vitro cytotoxicity,
almost all the synthesized complexes showed considerable cyto-
toxicity against tested human cancer cell lines, especially A549,
HCT116 and MCF7 cell lines. Notably, complex 2 was the most
promising one among these complexes, which showed 2–4 times
higher cytotoxicity against A549 and HCT116 cells than cisplatin.
Moreover, complex 5 and complex 7 exhibited slightly potent or
similar cytotoxicity against MCF7 compared to positive controls,
while most of the complexes only had moderate responses to
HepG-2 cell lines. Further in vitro assay against SGC7901 and
SGC7901/CDDP indicated that complex 2 had no cross-resistance
to cisplatin with a smaller RF value than 2.00. The flow cytometric
analysis suggested that complex 2 and complex 7 cause cell death
by inducing apoptosis, which were similar to cisplatin and oxali-
Table 2
Further in vitro assay of complexes 2 and 7 with cisplatin as positive controls against
human cancer cell lines.
M)a
IC50 (l
Complex
SGC7901b
SGC7901/CDDPc
RFd
Cisplatin
2
3.83 ± 0.18
8.77 ± 0.81
38.32 ± 1.77
12.69 ± 1.21
10.00
1.44
a
IC50 is the drug concentration effective in inhibiting 50% of the cell growth
measured by the MTT assay after 48 h drug exposure.
b
Human gastric carcinoma cell lines.
Cisplatin-resistant human gastric carcinoma cell lines.
RF (resistance factor), equals the IC50 of the resistant cells divided by the sen-
c
d
sitive ones.

G. Xu et al. / Inorganica Chimica Acta 462 (2017) 188–194
191
Fig. 1. Flow cytometric analysis of the distribution of HCT116 cell lines untreated or treated with cisplatin, oxaliplatin, complexes 2 and 7 at 50 lm for 24 h.
4
. Experimental details
.1. Chemistry
All solvents and reagents were of analytical purity and used
6
4
2
0
0
0
0
N e ga tive C o n tro l
C isp la tin
O xa lip atin
C om p lex 2
4
C om p lex 7
without further purification. Potassium tetrachloroplatinate(II)
and 1R,2R-diaminecyclohexane were obtained from Shangdong
Boyuan Chem. Co. Ltd., China. IR spectra were carried out on KBr
ꢀ1
pellet in the range of 4000–400 cm with a Nicolet IR200 FT-IR
1
13
spectrometer. H NMR and C NMR spectra were measured on a
Bruker DRX500 spectrometer. C, H and N elemental analyses were
performed on a Vario MICRO CHNOS Elemental Analyze (Elemen-
tar). ESI-MS analysis was performed with an Agilent Accurate Mass
6
224 TOF LC/MS system.
4.1.1. Preparation of complexes 1–9
3
4.1.1.1. General procedure. A solution of AgNO (2 mmol, 5 mL
deionized water) was added into a suspension of the correspond-
ing intermediates M1–M9 (1 mmol) in 50 mL water, and the sus-
pension was vigorously stirred at 50 °C under a nitrogen
e a rly
la te
to ta l
atmosphere in darkness for 16 h. The AgCl deposit was filtered
off, then a solution of sodium demethylcantharidate (1 mmol) in
Fig. 2. Results of flow cytometric analysis of the distribution of HCT116 cell lines
untreated or treated with cisplatin, oxaliplatin, complexes 2 and 7 at 50 m for
4 h.
l
5
4
2
mL water was added into the filtrate and stirred for 24 h at
0 °C in darkness. The volume of water was reduced down to
mL approximately, and the product slowly precipitated.
2
platin. Furthermore, western blot analysis indicated that both
complexes had a similar intrinsic mitochondrial apoptotic pathway
to cisplatin. In summary, the results indicated that the steric hin-
drance of carrier ligand has an impact on the biological activity
of the platinum(II) complexes, and both N-(2-butyl)-1R,2R-DACH
and N-cyclobutyl-1R,2R-DACH are proper carrier ligands in this
type of anticancer platinum(II) complexes, and complex 2 is a
promising candidate worth further investigation.
Complex 1: White powder; Yield 55%; IR (KBr): 3434 s (br),
2942 s, 2871 s, 2426 s, 2356 s, 1634vs, 1455 s, 1384vs, 1324 s,
1256 s, 1261 s, 1144 m, 1055 m, 1040 m, 992 s, 932 s, 820 s,
ꢀ1
1
807 s, 668 m, 584 m cm
2
; H NMR (D O/TSP, ppm): 1.04–1.10
(m, 6H, CH(CH ) ), 1.16–2.17 (m, 12H, 4CH of DACH and 2CH of
3
2
2
2
C H O ), 2.49–3.02 (m, 3H, NHCH and 2CH of DACH), 3.58–3.71
8
8 5
1
3
(m, 2H, CHOCH), 4.72 (m, 2H, 2CHCOO); C NMR (d -DMSO,
6
ppm): 22.0 (2CH ), 23.9, 24.4 (C3 and C4), 30.6, 31.5, 32.4 (C2, C5
3

1
92
G. Xu et al. / Inorganica Chimica Acta 462 (2017) 188–194
Fig. 3. The expression of apoptosis-related proteins was determined by western-blot assay. HCT116 and MCF7 cells treated with cisplatin, complex 2 and complex 7 at 50
for 12 h, and equal loading was testified by the detection of b-actin. Results were obtained similarly from three independent experiments.
lM
0
0
0
0
and C3 /C4 ), 56.8, 58.1 (C1 /C6 and NHCH(CH
3
)
2
), 61.2, 63.7 (C1
1258 s, 1213 s, 1142 m, 994 s, 932 s, 901 s, 820 s, 807 s, 667 m,
0
0
+
ꢀ1
and C6), 83.1 (C2 /C5 ), 184.4 (C@O); ESI-MS m/z: [M+H] = 536.1
100%), [M+Na] = 558.2 (45%); Anal. calcd. for C17
535.1): C 38.13, H 5.27, N 5.23; found: C 38.01, H 5.29, N 5.08.
Complex 2: White powder; Yield 33%; IR (KBr): 3431 s (br),
202 s, 2939 s, 2873 s, 2360 s, 2340 s, 1629vs, 1453 s, 1384vs,
323vs, 1259 s, 1213 m, 1172 m, 1145 m, 1060 m, 1033 m, 993 s,
580 s cm ; 1H NMR (D
2
O/TSP, ppm): 0.81–0.95 (m, 6H, CH
of DACH, 2CH of C and
), 2.49–2.69 (m, 4H, NHCH and 2CH of DACH),
3.61–3.69 (m, 2H, CHOCH), 4.72–4.73 (m, 2H, 2CHCOO); C NMR
+
(
H
28
N
2
O
5
Pt
(CH )
3 2
), 1.03–2.12 (m, 13H, 4CH
2
2
8 8 5
H O
(
CH of CH(CH
3
)
2
2
13
3
1
9
(d
6
-DMSO, ppm): 21.8, 24.1, 24.6 (2CH
3
0
, C3 and C4), 28.7, 29.6,
0
0
0
30.4, 33.4 (CH(CH
NHCH
MS m/z: [M+H] = 550.1 (75%), [M+Na] = 572.1 (100%); Anal. calcd.
for C18 Pt (549.2): C 39.34, H 5.50, N 5.10; found: C 39.18, H
5.54, N 4.85.
)
3 2
, C2, C5 and C3 /C4 ), 55.7, 58.9 (C1 /C6 and
ꢀ1
0
0
33 s, 902 m, 867 m, 820 s, 668 m, 581 s cm ; 1H NMR (d
CHCH CH ), 1.22–2.12
and 2H of CH CHCH
), 2.49–2.86 (m, 3H, NHCH and 2CH of DACH), 3.58–3.70 (m,
6
-
2
), 62.7, 66.8 (C1 and C6), 81.9 (C2 /C5 ), 180.3 (C@O); ESI-
+
+
DMSO/TMS, ppm): 0.97–1.16 (m, 6H,CH
3
2
3
(
m, 14H, 4CH
2
of DACH and 2CH
2
of C
8
8
H O
5
3
2
-
30 2 5
H N O
CH
3
2
H, CHOCH), 4.66–4.75 (m, 2H, 2CHCOO), 6.17–6.36 (m, 3H, CHNH
2
Complex 6: Pale yellow powder; Yield 46%; IR (KBr):3434 s (br),
2945 s, 2868 s, 2359 s, 1622vs, 1464 s, 1384vs, 1311 s, 1253 m,
1213 m, 1142 m, 1056 m, 1036 m, 995 s, 932 s, 901 m, 820 s,
1
3
and CHNH);
C NMR (d
6
2 3
-DMSO, ppm): 11.1 (CH CH ), 18.2
(
3
CHCH ), 24.2, 24.5 (C3 and C4), 26.4, 28.8, 31.0, 31.9 (C2, C5,
0
0
0
0
ꢀ1
1
C3 /C4 and CH
2
CH
3
), 56.3 (C1 /C6 ), 59.1, 60.7, 66.7 (C1, C6 and
788 m, 657 m, 573 s cm
C(CH ), 1.05–2.12 (m, 12H, 4CH
2.50–2.87 (m, 4H, NHCH and 2CH of DACH), 3.44–3.89 (m, 2H,
CHOCH), 4.54–4.71 (m, 2H, 2CHCOO); C NMR (d
23.8, 24.3 (C3 and C4), 27.6, 29.1, 30.6, 31.5, 33.4 (3CH
;
H NMR (D
2
O/TSP, ppm): 0.87 (s, 9H,
0
0
NHCHCH ), 81.6 (C2 /C5 ), 183.8 (C@O); ESI-MS m/z: [M
3
)
3 3
2
of DACH and 2CH
2
8 8 5
of C H O ),
+
+
+H] = 550.2 (21%), [M+Na] = 572.0 (100%); Anal. calcd. for C18
H
30
-
2
1
3
N
4
2
O
5
Pt (549.2): C 39.34, H 5.50, N 5.10; found: C 39.13, H 5.29, N
6
-DMSO, ppm):
, C2, C5,
), 54.4 (C1 /C6 ), 60.2, 61.4, 67.6 (NHCH , C1
.97.
3
0
0
0
0
Complex 3: White powder; Yield 75%; IR (KBr): 3424 s (br),
943 s, 2871 s, 2360 s, 2340 s, 1629vs, 1454 s, 1384vs, 1323 s,
259 s, 1213 m, 1175 m, 1144 m, 1059 m, 1031 m, 993 s, 933 s,
C3 /C4 and C(CH
3
0
)
3
2
0
+
2
1
8
0
and C6), 80.1 (C2 /C5 ), 178.6 (C@O); ESI-MS m/z: [M+H] = 564.0
(100%); Anal. calcd. for C19
32 2 5
H N O Pt (563.2): C 40.49, H 5.72, N
ꢀ
1
1
20 s, 808 s, 668 m, 576 s cm
.97 (m, 6H,CH CHCH CH CH
of C and 4H of CH
H, NHCH and 2CH of DACH), 3.42–3.70 (m, 2H, CHOCH), 4.72–
.74 (m, 2H, 2CHCOO); 13C NMR (d
-DMSO, ppm): 14.1 (CH CH ),
), 23.8, 24.2 (C3 and C4), 29.3,
;
H NMR (D
), 1.06–2.15 (m, 16H, 4CH
CHCH CH CH ), 2.49–2.79 (m,
2
O/TSP, ppm): 0.81–
4.97; found: C 40.15, H 5.81, N 4.69.
3
2
2
3
2
of DACH
Complex 7: White powder; Yield 71%; IR (KBr): 3434 s (br),
2942 s, 2360 s, 1622vs, 1384vs, 1322 s, 1258 s, 1213 m, 1142 m,
1056 m, 1036 m, 995 s, 930 s, 900 m, 820 s, 791 m, 665 m,
and 2CH
2
8
8
H O
5
3
2
2
3
3
4
1
3
5
ꢀ1
1
6
2
3
582 s cm ; H NMR (d
DACH and 2CH of C
3H, NHCH and 2CH of DACH), 3.62–3.70 (m, 2H, CHOCH), 4.70–
6
-DMSO/TMS): 1.05–2.12 (m, 18H, 4CH
H O and 6H of cyclobutyl), 2.48–2.85 (m,
8 8 5
2
of
9.2, 21.1 (CH
1.0, 31.9, 32.4 (C2, C5, C3 /C4 and CH
9.4, 60.1, 63.4 (C1, C6 and NHCHCH
2
CH
3
and CHCH
3
0
2
0
0
0
2
CH
2 3
CH ), 56.1 (C1 /C6 ),
0
0
13
3
), 79.7 (C2 /C5 ), 180.9
4.73 (m, 2H, 2CHCOO), 6.25–6.50 (m, 3H, CHNH
2
and CHNH);
C
+
+
00
(C@O); ESI-MS m/z: [M+H] = 564.2 (34%), [M+Na] = 586.0
NMR (d
6
-DMSO, ppm): 14.1(C3 ), 24.2, 24.5 (C3 and C4), 27.6,
0
0
00
00
(
100%); Anal. calcd. for C19
H
32
N
2
O
5
Pt (563.2): C 40.49, H 5.72, N
29.2, 31.0, 32.3/32.5 (C2, C5, C3 /C4 and C2 /C4 ), 58.1, 60.4,
66.7, 68.9 (C1 /C6 , C1, C6 and C1 ), 80.9 (C2 /C5 ), 183.1(C@O);
ESI-MS m/z: [M+H] = 548.1 (100%), [M+Na] = 570.0 (58%); Anal.
calcd. for C18 Pt (547.2): C 39.49, H 5.15, N 5.12; found: C
39.12, H 5.07, N 4.97.
0
0
00
0
0
4
.97; found: C 39.99, H 5.57, N 4.78.
+
+
Complex 4: Pale yellow powder; Yield 46%; IR (KBr): 3435 s (br),
119 s, 2940 s, 2870 s, 2426 m, 2360 m, 1650vs, 1384vs, 1336 s,
256 s, 1212 s, 1144 m, 1059 m, 1040 m, 993 s, 932 s, 902 m,
3
1
8
3
C
28 2 5
H N O
ꢀ
1
1
21 s, 666 m, 582 s cm
;
H NMR (D
), 1.04–2.12 (m, 16H, 4CH
and CH CH CH ), 2.49–2.80 (m, 4H, NHCH
2
O/TSP, ppm): 0.88–0.95 (t,
of DACH and 2CH of
and 2CH of
Complex 8: White powder; Yield 78%; IR (KBr): 3434 s(br),
2
H,CH CH
3
2
2
3209 s, 2944 s, 2869 s, 2360 s, 2340 s, 1634vs, 1448 m, 1383vs,
ꢀ1
H
8 8
O
5
3
2
2
2
1323 s, 1258 s, 1213 m, 1175 m, 820 s, 806 s, 668 m, 581 s cm ;
1
DACH), 3.59–3.68 (m, 2H, CHOCH), 4.57–4.72 (m, 2H, 2CHCOO);
H NMR (D O/TSP, ppm): 1.01–2.17 (m, 20H, 4CH
2
2
of DACH and
1
3
C NMR (d
6
-DMSO, ppm): 13.6 (CH
2
CH
3
), 19.5 (CH
2
CH
3
), 25.1,
2CH of C H O and 8H of cyclopentyl), 2.57–2.79 (m, 3H, NHCH
2
8 8 5
0
0
2
CH
5.4 (C3 and C4), 29.1, 30.7, 31.2, 33.2 (C2, C5, C3 /C4 and NHCH
2
0
-
and 2CH of DACH), 3.58–3.66 (m, 2H, CHOCH), 4.62–4.70 (m, 2H,
0
0
13
00
2
0
), 49.5 (NHCH
2
), 57.1 (C1 /C6 ), 60.2, 63.5 (C1, C6), 78.6 (C2 /
2CHCOO); C NMR (d
6
-DMSO, ppm): 22.4, 22.6, 24.1, 24.3 (C3 ,
+
00
0
0
00
C5 ), 177.5 (C@O); ESI-MS m/z: [M+H] = 550.1 (100%), [M
C4 , C3 and C4), 27.9, 30.0, 31.9, 34.8, 35.0 (C2, C5, C3 /C4 , C2
and C5 ), 57.5, 59.4, 61.7, 66.6 (C1 /C6 , C1, C6 and C1 ), 77.9
(C2 /C5 ), 178.2(C@O);ESI-MS m/z: [M+H] = 562.2(88%), [M
+Na] = 583.9(88%); Anal. calcd. for
+
00
0
0
00
+Na] = 572.2 (83%); Anal. calcd. for C18
30
H N
2
O
5
Pt (549.2): C
0
0
+
3
9.34, H 5.50, N 5.10; found: C 39.09, H 5.59, N 4.98.
Complex 5: Pale yellow powder; Yield 42%; IR(KBr): 3435 s (br),
+
19 30 2 5
C H N O Pt (561.2): C
2
946 s, 2870 s, 2360 s, 2341 s, 1634vs, 1455 s, 1383vs, 1322 s,
40.64, H 5.39, N 4.99; found: C 40.22, H 5.31, N 4.73.

G. Xu et al. / Inorganica Chimica Acta 462 (2017) 188–194
193
Complex 9: White powder; Yield 66%; IR (KBr): 3440 s(br),
105 s, 2934 s, 2857 s, 2360 s, 2341 s, 1635vs, 1449 s, 1382vs,
322 s, 1258 s, 1212 m, 1168 m, 1059 m, 1031 m, 993 s, 933 s,
with annexin V/FITC and propidium iodide (PI) in binding buffers
(10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl , pH = 7.4). After
3
1
8
2
5 min incubation at room temperature, the fluorescence was mon-
itored with a flow cytometer (FAC Scan, Becton Dickenson, USA) at
an excitation wavelength of 488 nm. Results were analyzed with
CellQuest Pro software and are represented as the percentage of
normal and apoptotic cells at various stages. Results were analyzed
with CellQuest Pro software and are represented as the percentage
of normal and apoptotic cells at various stages.
ꢀ1
1
99 m, 866 m, 820 s, 806 s, 667 m, 582 s cm
of DACH and 2CH
0H of cyclohexyl), 2.62–2.94 (m, 3H, NHCH and 2CH of DACH),
;
H NMR (D
of C H O and
8 8 5
2
O/
TSP): 1.02–2.16 (m, 22H, 4CH
1
3
2
2
1
3
.60–3.71 (m, 2H, CHOCH), 4.72–4.73 (m, 2H, 2CHCOO); C NMR
0
0
00
00
6
(d -DMSO, ppm): 24.0, 24.3, 25.1, 26.0, 26.2 (C3 , C4 , C5 , C3
0
0
00
00
and C4), 29.9, 31.8, 32.8, 34.9, 35.2 (C2, C5, C3 /C4 , C2 and C6 ),
0
0
00
0
0
5
1
C
5
9.8, 61.3, 63.4, 66.9 (C1 /C6 , C1, C6 and C1 ), 78.6 (C2 /C5 ),
+
80.3(C@O); ESI-MS m/z: [M+H] = 576.2 (100%); Anal. calcd. for
4.2.4. Western blot
20
H
32
N
2
5
O Pt (575.2): C 41.74, H 5.60, N 4.87; found: C 41.33, H
HCT116 and MCF7 cells were cultured in culture flasks to reach
the cell density of 80%. 50 lM of platinum complex was added and
.51, N 4.66.
incubated at 37 °C for 12 h. Proteins were extracted by lysis buffer
and concentration was determined with protein assay (Thermo,
Waltham, MA) by BCA (bicinchoninic acid) and then adjusted to
4
4
.2. Biological studies
.2.1. Cell culture
same concentration. The samples (20
lg/lane) were separated in
Four human solid tumor cell lines including human non-small
1
0% sodium dodecyl sulfate polyacrylamide gel electrophoresis
cell lung cancer cell line (A549, ATTC No.: CCL-185), human col-
orectal carcinoma cell line (HCT-116, ATCC No.: CCL-247), human
hepatocellular carcinoma cell line (HepG-2, ATCC No.: HB-8065)
and human breast carcinoma cell line (MCF7, ATCC No.: HTB-22;
MDA-MB-231, ATCC No.: HTB-26) were used in the cytotoxicity
test of all the synthesized platinum(II) complexes. Furthermore,
complex 2 and complex 7 were selected to determine the cytotox-
icity against gastric carcinoma cell line (SGC-7901, KG026) and cis-
platin-resistant gastric carcinoma cell line (SGC-7901/CDDP,
KG309), and both of these two cancer cell lines were purchased
from Keygen Biotec Co., Ltd. (China). The cells were cultured in
(
(
SDS–PAGE), and then transferred onto polyvinylidene difluoride
PVDF) Immobilon-P membrane (Bio-Rad, USA) by a transblot
apparatus (Bio-Rad, USA). The membrane was blocked with 5%
nonfat milk in TBST buffer for 1 h, followed by overnight incuba-
tion at 4 °C with primary antibodies diluted in PBST (1:500 for
Bax, BD Pharmagin, USA; 1:500 for Bcl-2, Cell Signal, USA;
1
:2000 b-actin, Santa Cruz, USA). Membrane was washed with
PBST for 3 times and incubated with IRDye 800 conjugated sec-
ondary antibody which was diluted in PBST with 1:30,000 for
1
h. Labeled proteins were measured by Odyssey Scanning System
LiCOR., Lincoln, Nebraska, USA).
(
RPMI-1640 medium with 10% fetal bovine serum (FBS), 100
mL of streptomycin, and 100 g/mL of penicillin in an atmosphere
of 5% CO at 37 °C.
lg/
l
Acknowledgements
2
The authors thank the National Natural Science Foundation of
China (project No. 21271041), Natural Science Foundation of
Jiangsu Province (project No. BK20130603), the New Drug Creation
Project of the National Science and Technology Major Foundation
of China (project No. 2013ZX09402102-001-006) and the Funda-
mental Research Funds for the Central Universities (project No.
4
.2.2. In vitro cytotoxicity test
Cytotoxicity of all the compounds including cisplatin and oxali-
platin were determined by MTT assay (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide; Sigma). The standard proce-
dure of MTT assay is described briefly as follows: the suspension of
5
000 cells per well was plated in 96-well culture plates with cul-
ture medium and incubated for 24 h in a water-atmosphere (5%
CO ) at 37 °C. Then the diluted solution of platinum complexes
with desired concentration (dissolving in water and diluting with
culture medium) was added to the wells. 10 L of a freshly diluted
2
242016k30020) for financial aids to this work. Dr. Xu would like
to thank Nanjing University for the Open Project Program of State
Key Laboratory of Analytical Chemistry for Life Science (project No.
SKLACLS1416).
2
l
MTT solution (5 mg/mL in PBS) were added to each well after 48 h
of incubation and the plates were incubated at 37 °C in a humidi-
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