Toward overcoming cisplatin resistance via sterically hindered platinum(II) complexes

Article abstract of DOI:10.1016/j.ejmech.2016.02.060

A number of platinum(II) complexes with steric hindrance derived from (1R,2R)-N¹-benzylcyclohexane-1,2-diamine derivatives were designed and prepared. Biological assay indicated that most complexes showed antitumor activity against the tested c

Full text of DOI:10.1016/j.ejmech.2016.02.060

European Journal of Medicinal Chemistry 114 (2016) 141e152
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Toward overcoming cisplatin resistance via sterically hindered
platinum(II) complexes
,
b
,
,
,
, b, **
Haiyan Yu a, Shaohua Gou a *, Zhimei Wang a b, Feihong Chen a b, Lei Fang a
a Pharmaceutical Research Center and School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China
b Jiangsu Province Hi-Tech Key Laboratory for Bio-medical 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
A number of platinum(II) complexes with steric hindrance derived from (1R,2R)-N1-benzylcyclohexane-
1,2-diamine derivatives were designed and prepared. Biological assay indicated that most complexes
showed antitumor activity against the tested cancer cell lines, especially those with chloride anions as
leaving groups had compatible or superior activity to cisplatin and oxaliplatin. Complex 2a, as the most
potent agent, is also sensitive to cisplatin resistant SGC7901/CDDP cancer cell line, which has been
subsequently studied by cellular uptake, flow cytometry, gel electrophoresis and western blot assays. The
steric hindrance resulting from a pending 2-fluorobenzyl moiety of the ligand might be the key factor for
its ability to overcome cisplatin resistant cancer cells.
Article history:
Received 14 December 2015
Received in revised form
22 February 2016
Accepted 24 February 2016
Available online 27 February 2016
Keywords:
Platinum(II) complexes
Overcoming cisplatin resistance
Steric hindrance
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
resistance of cisplatin and reduce its side effects, different carrier
ligands have been designed and applied to platinum complexes.
Since the discovery of cisplatin by Rosenberg in 1965, the
application and research of platinum-based anticancer drugs have
achieved rapid development [1e3]. Cisplatin as a broad-spectrum
anticancer agent has shown great advantages in the treatment of
some solid tumors, especially in the treatment of cervix, ovary,
testicle, neck and head [4,5]. However, some side effects, including
acquired or intrinsic drug resistance, serious toxicity and low sol-
ubility in water, have greatly limited its clinical application [6].
Thus, the non-classical antitumor platinum(II) complexes such as
sterically hindered platinum(II) complexes [7], trans-platinum
complexes [8], bi- and multi-nuclear platinum(II) complexes
[9,10] have been investigated. So far, several mechanisms have been
involved in cisplatin resistance, including reduced accumulation of
cellular Pt and increased repair of DNA damage [11e13]. The
interaction of cisplatin with biological molecules has been found to
be one of the key reasons for the drug resistance [14,15] due to its
high affinity to the sulfur-containing binding sites. It is well known
that sulfur-containing biomolecules are correlated with the resis-
tance of platinum-based drugs [16e18]. In order to overcome the
Oxaliplatin, a third-generation platinum drug with 1R,2R-dia-
minocyclohexane (DACH) as carrier ligand, has shown great activity
against many tumors including those resistant to cisplatin [19e21].
The high stability of the DACH-platinum fragment in oxaliplatin
compared with the ammine in cisplatin leads to a different reaction
behavior [22e24], thus, researchers have made great efforts on
designing and synthesis of oxaliplatin derivatives [21e27]. Recent
studies demonstrated that the introduction of steric hindrance to
amine carrier ligand has a strong influence on the activity of the
resulting platinum complexes [28]. Therefore, by changing the
nature of the amine ligand, it is possible to obtain platinum(II)
complexes with potent antitumor activity, which may be active
toward cisplatin resistant cell lines as well.
In our recent research, a number of different alkyl groups as
sterically hindered moieties have been introduced to DACH skel-
eton, which were used as carrier ligands to prepare several kinds of
platinum(II) complexes [29e32]. The related studies indicated that
the platinum(II) complexes containing such ligands showed potent
antitumor activity and possessed somewhat different mechanism
from cisplatin and oxaliplatin in reaction with DNA, and exhibited
distinct cell cycle blocking and cell apoptosis. However, the N-
monosubstituted alkyl group is flexible, which seems not feasible to
offer fixed spatial resistance and may also result in the formation of
stereo isomers [29]. Hence, a rigid benzyl group has been intro-
duced to the DACH framework by connecting to one of the nitrogen
* Corresponding author. Pharmaceutical Research Center and School of Chemis-
try and Chemical Engineering, Southeast University, Nanjing 211189, China.
** Corresponding author. Pharmaceutical Research Center and School of Chemis-
try and Chemical Engineering, Southeast University, Nanjing 211189, China.
E-mail address: sgou@seu.edu.cn (S. Gou).
http://dx.doi.org/10.1016/j.ejmech.2016.02.060
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

142
H. Yu et al. / European Journal of Medicinal Chemistry 114 (2016) 141e152
atoms (Scheme 1) [33e35], and several new ligands as shown in
Fig. 1 have been obtained, which have been used to prepare a series
of platinum(II) complexes bearing the steric hindrance(Scheme 2).
Considering the significant feature of fluorine atom in drugs, we
selected it as a substituent on the phenyl ring. Herein reported are
these platinum(II) complexes (Fig. 2) of the ligands and their bio-
logical evaluation against a few human cancer cell lines including
the typical compounds against cisplatin resistant cancer cell lines.
Moreover, chemical and biological properties of the typical com-
pounds have been studied to explicit their preliminary mechanism
of action as well as the function of spatial resistance caused by the
ligand.
NMR spectroscopy has been applied to determine the frame-
work of the synthesized metal complexes. In the 1H NMR spectra,
the signals of hydrogen atoms in DACH appeared in a range of
1.24e2.50 ppm, and the chemical shifts of NHCH and NH2CH on
DACH of the complexes moved to highfield area compared with the
corresponding ligand. 13C NMR spectra were in agreement with the
proposed chemical structure of the complexes, particular those of
complexes with a fluorine atom showed splitting carbon peaks in
the area of aromatic carbons as expected. Besides, a representative
195Pt NMR spectrum (complex 2a) was recorded.
All the ESI-MS spectra of the complexes gave main peaks cor-
responding to [MþH]þ, [MꢀH]ꢀ or [MꢀCl]ꢀ ions, which were
composed of a few isotopic peaks owing to the presence of plat-
inum isotopes, suggesting the bonding between Pt(II) ions and the
ligands.
2. Results and discussion
2.1. Synthesis of ligands and their complexes
2.2. In vitro cytotoxicity assay
The preparation of the ligand (L) was carried out starting from
1R, 2R-diaminocyclohexane via several synthetic steps (Scheme 1),
in which N-mono Boc protected DACH (I) was used as the starting
material. The unprotected amino group first underwent Schiff base
condensation with benzaldehyde, followed by in situ reduction
with sodium boronhydride, and then the Boc group was removed
through hydrolysis in the presence of hydrochloric acid to give L in
its hydrochloride salts. LF1ꢀLF3 were prepared by the same way
except o-fluorobenzaldehyde, m-fluorobenzaldehyde and p-fluo-
robenzaldehyde were used as starting materials, respectively.
All ligands (L, LF1, LF2 and LF3) were characterized by elemental
analysis, IR, ESI-MS, 1H and 13C NMR spectra, which were in
agreement with the chemical structure proposed. The specific op-
tical rotations of these ligands were as left-handed as that of DACH.
Complexes 1aꢀ4a were directly prepared by the reaction of the
corresponding ligand and K2PtCl4. The rest complexes were ob-
tained by using complexes 1aꢀ4a as starting materials to react with
the corresponding silver dicarboxylate, respectively [29]. As ex-
pected, a chiral center at N1 position of DACH can be formed after
coordination of the ligand with a metal atom in the synthesized
platinum(II) complexes. The diastereoisomeric mixture could be
produced with one pendant benzyl moiety (at N1 position) in the
pseudo axial position above or below the resulting PtN2O2 square
plane. Like the pending alkyl group we reported formerly [29], S
configuration at N1 atom of the complex with a pendant benzyl
group is more thermodynamically stable than the corresponding R
configuration resulting from a steric effect of the ligand by mo-
lecular calculation, which has also been confirmed by the crystal-
lographic data of analogous metal complexes [29,36,37].
The cytotoxicity of all metal complexes against three human
cancer cell lines was evaluated by MTT method with cisplatin and
oxaliplatin as positive control. As shown in Table 1, complexes
1aꢀ4a with chloride anions as leaving groups showed strong
anticancer activity comparable or even superior to cisplatin and
oxaliplatin, whereas other complexes with dicarboxylates as leav-
ing groups, except individual cases, exhibited moderate or low
cytotoxicity against HepG2, SGC7901, and A2780 cell lines. Among
all compounds, complex 2a exhibited the strongest activity against
all the tested three cancer cell lines, remarkably superior to
cisplatin and oxaliplatin. It was 3.71 0.48-fold, 1.56 0.25-fold
and 3.66 0.56-fold as potent as cisplatin and 34.34 3.49-fold,
9.63 1.54-fold, and 8.64 1.34-fold as potent as oxaliplatin to-
wards HepG2, SGC7901, and A2780, respectively. In contrast to
complexes 2aꢀ4a with a ligand containing a fluoro substituent on
the phenyl ring, complex 1a was less active, which was about one
fifth as effective as cisplatin on SGC7901 and A2780, and one tenth
as active as cisplatin on HepG2. Although weaker than those of
complex 2a and cisplatin, the cytotoxicity of complexes 3a and 4a
was stronger than that of oxaliplatin against almost all the tested
cancer cell lines, demonstrating the importance and unique func-
tion of the fluorine atom of the ligand in our complexes. However,
complexes 1b-4e which substituted the leaving group from chlo-
ride anions to dicarboxylate species would result in the conspicu-
ous decrease of the antitumor activity of the complexes compared
with their parent compounds. This can be due to the slow disso-
ciation ability of chelating dicarboxylate from the metal atom in
comparison to chloride anions.
In the infrared spectra of the complexes, the strong absorption
at 3100 cmꢀ1 was attributed to the NeH stretching vibration that
was significantly red shifted in comparison to the corresponding
ligand. The C]O stretching vibration of complexes appeared be-
tween 1568 and 1616 cmꢀ1, blue shifting compared with the single
carboxylate group, which is characteristic of coordinated carbox-
ylate ligands. The absorption near 1250 cmꢀ1 was attributed to the
ArꢀF bonding which is an obvious sign to distinguish complexes 1a,
1b, 1c, 1d and 1e from others.
In terms of their strong inhibiting ability on the above cancer
cell lines, complexes 1aꢀ4a were further investigated against
cisplatin resistant cancer cell line (SGC7901/CDDP) and human
normal liver cell line (LO2). As showed in Table 2, complex 2a has
the potential to overcome cisplatin resistance, as its resistance in-
dex (RF) can reach 1.39, which is better than cisplatin and oxali-
platin. While complexes 1a, 3a and 4a were less effective against
SGC7901/CDDP cell than complex 2a, even junior to that of oxali-
platin. These results indicated that the position of the fluoro
Ph
Ph
NH2
N
Ph
NH
NH
PhCHO
NaBH4
EtOAc/HCl
2HCl
NH
NH
NH
NH2
Boc
Boc
Boc
I
L
Scheme 1. Synthesis of ligand L.

H. Yu et al. / European Journal of Medicinal Chemistry 114 (2016) 141e152
143
Fig. 1. Chemical structures of the ligands.
substituent has played an important role in the nature of the
complex overcoming cisplatin resistance. It is noticed that com-
plexes 2aꢀ4a are more toxic toward LO2 cell line than complex 1a
and oxaliplatin, but they are less toxic than cisplatin on normal
human cell. Upon the remarkable antitumor feature of complex 2a
as our partner reported [38], it was selected as a representative
compound for the subsequent study.
Scheme 2. Synthetic route to prepare platinum(II) complexes 1aꢀ4e.
Fig. 2. Chemical structures of the synthesized platinum(II) complexes.
Table 1
In vitro cytotoxicity of complexes 1aꢀ4e against human cancer cell lines.
Table 2
Cytotoxicity of complexes 1a¡4a against SGC7901/CDDP and LO2 cell lines.
Complex
IC50
(
m
M)a
Complex
IC50
SGC7901b
1.35 0.12 11.59 1.01
(m
M)a
HepG2b
SGC7901c
A2780d
SGC7901/CDDPc Resistant factord LO2e
cisplatin
oxaliplatin
1a
2a
3a
4a
1b
2b
3b
4b
1c
2c
3c
4c
1d
2d
3d
0.70 0.04
6.49 0.32
7.15 0.64
0.19 0.01
3.42 0.29
4.30 0.32
97.35 7.56
11.89 0.99
6.59 0.52
25.38 2.08
22.70 1.98
19.11 1.03
44.17 3.85
15.27 1.28
44.07 4.06
35.18 2.79
140.34 10.21
40.71 3.89
>200
1.35 0.12
8.29 0.76
6.72 0.53
0.87 0.06
2.90 0.12
3.02 0.21
193.48 13.11
40.03 3.87
28.11 2.17
39.42 3.76
35.25 2.95
28.36 1.99
>200
42.94 3.87
116.42 10.23
34.46 3.12
194.91 13.23
39.72 2.98
156.64 1.25
106.53 9.35
76.77 4.23
30.50 2.94
4.08 0.26
9.48 0.71
23.96 2.14
1.11 0.09
11.42 0.92
6.52 0.53
>200
1.56 0.12
8.33 0.74
25.57 1.96
36.22 3.19
23.77 1.98
8.09 0.62
9.82 0.74
>200
33.67 2.79
>200
38.48 2.91
>200
>200
120.84 10.13
30.92 3.02
cisplatin
8.59
1.50
4.27
1.39
5.77
6.54
3.67 0.31
11.18 0.87
10.19 0.98
6.55 0.43
7.54 0.65
6.27 0.59
oxaliplatin 8.29 0.76 12.46 0.93
1a
2a
3a
4a
6.72 0.53 28.71 1.85
0.87 0.06 1.21 0.08
2.90 0.12 16.73 1.27
3.02 0.21 19.74 0.94
a
IC50 is the drug concentration effective in inhibiting 50% of the cell growth
measured by the MTT assay after 72 h drug exposure. Each value is the mean of three
independent experiments.
b
SGC7901: human gastric cancer cell line.
SGC7901/CDDP: human gastric cancer cisplatin resistant cell line.
Resistant factor (RF) is defined as IC50 in SGC7901/CDDP/IC50 in SGC7901.
LO2: normal human liver cell line.
c
d
e
2.3. Cellular uptake
4d
1e
2e
3e
>200
39.36 2.75
35.54 3.08
Since it has the most potent antitumor activity in the cytotox-
icity assay, complex 2a was selected for the intracellular uptake
test. The cellular uptake was measured by treating SGC7901 and
4e
a
IC50 is the drug concentration effective in inhibiting 50% of the cell growth
measured by the MTT assay after 72 h drug exposure. Each value is the mean of three
independent experiments.
SGC7901/CDDP cells with 50
mM of complex 2a, cisplatin and
oxaliplatin for 12 h, respectively. The content of platinum in the
tumor cells was determined by the technique of ICP-MS. It can be
seen from Table 3 that complex 2a had the highest intracellular
accumulation of platinum both in SGC7901 and in SGC7901/CDDP
cells, while oxaliplatin had the lowest cell uptake values among
b
HepG2: human hepatocellular carcinoma cell line.
SGC7901: human gastric cancer cell line.
A2780: human ovarian cancer cell line.
c
d

144
H. Yu et al. / European Journal of Medicinal Chemistry 114 (2016) 141e152
Table 3
2.5. Interaction with pET22b plasmid DNA
Cellular uptake of complex 2a on SGC7901 and SGC7901/CDDP cells after 12 h
incubation.a
To understand the interaction of our complexes with DNA, the
DNA binding ability of complex 2a was investigated by gel elec-
trophoresis, in which cisplatin and oxaliplatin were taken as posi-
tive controls and plasmid DNA pET22b was used as target. DNA
chain can generally be divided into closed circular DNA or supercoil
DNA, linear DNA and open circular DNA. As shown in Fig. 5, it
mainly contains closed circular DNA and open circular DNA which
are defined as Form I and Form II, respectively.
Complex
Pt content (ng/106 cells)
SGC7901
SGC7901/CDDP
complex 2a
cisplatin
oxaliplatin
578 19
360 21
222 16
620 24
344 16
110 12
a
Intracellular accumulation of complex 2a cisplatin and oxaliplatin in SGC7901
and SGC7901/CDDP cells (per 106 cells) after 12 h incubation at a concentration of
50 mM. Results are expressed as the mean SD for three independent experiments.
After the plasmid was incubated with cisplatin at a concentra-
tion of 40
compared with the blank control. When the concentration of
cisplatin increased (160, 640 M), a gradual increase in the mobility
mM, the density of Form II and Form I was decreased
m
three compounds. The content of complex 2a taken up by the
SGC7901 cells was 2.6 fold as much as that of oxaliplatin and the
uptake of complex 2a in SGC7901/CDDP cells was 5.6 fold greater
than that of oxaliplatin. The uptake values of cisplatin in the tested
two cells are in the middle of those of complex 2a and oxaliplatin.
Based on the results from the cytotoxicity and cellular uptake tests,
it is found that complex 2a has a positive correlation between these
two tests, namely, the enhanced cellular uptake can result in the
increase of the cytotoxicity, and so do cisplatin and oxaliplatin. The
platinum accumulation of complex 2a in the tested cells was highly
compatible to its lipophilicity when compared with cispaltin and
oxaliplatin, although there have been reports about a unclear cor-
relation between cellular uptake and lipophilicity of the platinum
compounds [39,40].
of covalently closed circular DNA (Form I) was observed, accom-
panied by the obvious decrease in density at the same time, indi-
cating that cisplatin has an interaction with pET22b plasmid DNA in
a high concentration. When treated with different concentrations
of oxaliplatin, the migration of pET22b plasmid DNA increased
slightly and stripe density was reduced only a little, exhibiting that
oxaliplatin could cause DNA cleavage with a weak ability compared
with cisplatin. As for complex 2a, both Form I and Form II changed
slightly at a low concentration. With the increase of the concen-
tration, there was a significantly migration and an obvious decrease
in density in Form I. As a result, the effect of complex 2a on pET22b
plasmid DNA is similar to cisplatin and oxaliplatin, and the ability of
the effect is cisplatin > complex 2a > oxaliplatin.
2.6. Western blot analysis
2.4. Flow cytometry study
In order to character complex 2a in intrinsic mitochondrial
pathway to induce apoptosis, the expression levels of Bax, Bcl-2 and
Procaspase-3 proteins were investigated in SGC7901 cells by
Western blot method, meanwhile cisplatin and oxaliplatin were
used as positive control. As shown in Fig. 6, obvious increase of Bax
expression was observed in cells incubated with cisplatin and
complex 2a, while decrease of Bcl-2 and Procaspased-3 expression
was detected in cells treated with cisplatin, oxaliplatin and complex
2a. Thus, the complex 2a can induce cell apoptosis by intrinsic
mitochondrial pathway.
2.4.1. Apoptosis study with complex 2a
To study the apoptosis induced by complex 2a, Annexin V/PI
staining assay was used. The tested complexes were incubated with
SGC7901 and HepG2 cell lines for 24 h at the concentration of
50
m
M. Q1ꢀQ4 represents four different cell states: necrotic cells,
late apoptotic or necrotic cells, living cells and early apoptotic cells,
respectively (Fig. 3). As shown in Fig. 3a and c, increased apoptotic
rates of SGC7901 cells were detected after cells being treated with
complex 2a, cisplatin and oxaliplatin as positive controls.
Compared with untreated cells as 6.49 0.3% apoptotic rate, cells
treated with complex 2a, cisplatin and oxaliplatin showed
3. Conclusion
79.97
1.9%, 28.44
0.8% and 10.72
0.7% apoptotic rates,
In this study, a number of sterically hindered platinum(II)
complexes, with (1R,2R)-N1-benzylcyclohexane-1,2-diamine de-
rivatives as carrier ligands and chloride anions or dicarboxylates as
leaving groups, were designed and prepared. The in vitro antitumor
activity of all compounds against HepG2, SGC7901, and A2780
cancer cell lines revealed that complexes 1ae4a with chloride an-
ions showed strong anticancer activity comparable or even supe-
rior to cisplatin and oxaliplatin, whereas other complexes (1be4e)
with dicarboxylates, except individual cases, exhibited moderate or
low cytotoxicity. Further evaluation on complexes 1ae4a against
cisplatin resistant SGC7901/CDDP cancer cell line indicated com-
plex 2a is the most potent agent to overcome cisplatin resistance,
and less toxic than cisplatin to human normal liver LO2 cells. It was
found that the platinum accumulation of complex 2a in the tested
cells was highly compatible to its lipophilicity, which had a positive
correlation between its cellular uptake and cytotoxicity values.
Apoptosis study on HepG2 and SGC7901 cell lines indicated that
complex 2a could induce cancer cell death based on an apoptotic
pathway, but the mechanism of cell cycle arrest induced by com-
plex 2a was different from cisplatin. However, the interaction of
complex 2a with DNA showed that its effect on pET22b plasmid
DNA was similar to cisplatin and oxaliplatin, and western blot
respectively, including both the early and the late apoptotic cells.
The results in Fig. 3b and c showed that the apoptotic rate of HepG2
cells for complex 2a (26.61%) was slightly lower than cisplatin
(28.79%), but better than oxaliplatin (21.74%). In contrast to the
apoptotic rate of the HepG2 cell line, SGC7901 cell line was rela-
tively sensitive to complex 2a. These results indicated that complex
2a could induce cancer cell death based on an apoptotic pathway.
2.4.2. Cell cycle analysis of complex 2a
The effects of complex 2a on both SGC7901 and HepG2 cell cycle
progression were investigated by 24 h incubating at a concentra-
tion of 50 mM, cisplatin and oxaliplatin were used as positive con-
trols. As shown in Fig. 4, G1 phase, S phase and G2/M phase
represent early synthesis of DNA, DNA synthesis and late synthesis
of DNA, respectively. For SGC7901 cells, cisplatin arrested cell cycle
majors in G1 and S phases, whereas complex 2a majors in S and G2
phases compared with control. For HepG2 cells, the cell cycle arrest
induced by cisplatin and oxaliplatin majors in G2 phase, while the
cell cycle of complex 2a was arrested in G1 phase. This indicated
that the mechanism of cell cycle arrest induced by complex 2a was
clearly different from cisplatin.

H. Yu et al. / European Journal of Medicinal Chemistry 114 (2016) 141e152
145
Fig. 3. Apoptosis induced by tested complexes in SGC7901 and HepG2 cells. Cells were treated with 50
mM of complex 2a, cisplatin and oxaliplatin, respectively, for 24 h. Annexin V/
PI double-staining assays of (a) SGC7901 and (b) HepG2 cells were analyzed by flow cytometry. (c) The apoptotic rates of cells induced by tested complexes.
analysis indicated that the induced apoptosis by complex 2a was to
some extent caused by intrinsic mitochondrial pathway. Overall,
suitable steric hindrance in the platinum(II) complex can play an
important role in overcoming cisplatin resistance, and complex 2a
can be a drug candidate for further research.
Platinum contents were measured on an Optima 5300DV ICP-MS
instrument.
4.1.1. Synthesis of ligands
4.1.1.1. (1R,2R)-N1-benzyl-1,2-diaminocyclohexane (L). To a solution
of Boc protected 1R, 2R-cyclohexanediamine (8.56 g, 40 mmol) in
methanol (150 mL), benzaldehyde (8.48 g, 80 mmol) was added.
The reaction mixture was kept stirring for 2 h at room temperature,
then it was cooled in an ice water bath, followed by adding NaBH4
(4.54 g, 120 mmol) in portions, and kept stirring overnight at 50 ꢁC.
Then, 10 mL of water was added to quench the reaction, white
solids were obtained by concentrating the solution. The aqueous
phase was extracted with EtOAc (50 mL ꢂ 3). Organic extracts were
combined and washed by saturated NaCl solution and dried by
anhydrous Na2SO4. By concentrating the solution in vacuum, the
intermediate N1-monobenzyl, N2-Boc-(1R, 2R)-diaminocyclohex-
ane was obtained. Then the intermediate was dissolved in absolute
ether (75 mL) and a solution of HCl/EtOAc (1 mol/L, 50 mL) was
added slowly with stirring. The mixture was kept stirring for 12 h at
room temperature, then the resulting white solid was filtered off,
washed with absolute ether and dried in vacuum. The product was
obtained as dihydrochloride salt (L$2HCl). Yield: 56%. ½aꢃ2D5 ¼ ꢀ52.4
(c 0.44, H2O). Anal. calcd for C13H20N2$2HCl: C, 56.32; H, 8.00; N,
10.10. Found: C, 56.43; H, 8.25; N, 9.81. IR (KBr cmꢀ1): 3402, 2942,
4. Experimental section
4.1. Materials and measurements
All chemicals and solvents were of analytical reagent grade and
were used without further purification. Potassium tetra-
chloroplatinate(II) was obtained from a local chemical company
(Lingfeng Chemical Ltd.). Human cancer cell lines including
SGC7901/CDDP were obtained from Nanjing KeyGEN BioTECH
company. Elemental analyses for C, H, and N were made on a Vario
MICRO CHNOS Elemental Analyzer (Elementar). The specific optical
rotations were measured on a WZZ-2A Automatic Polarimeter.
Infrared spectra were recorded on KBr pellets on a Nicolet IR200 FT-
IR spectrometer in the range of 4000ꢀ400 cmꢀ1. 1H NMR and 13C
NMR spectra were recorded in D2O or d6-DMSO with a Bruker 300
or 500 MHz spectrometer. 195Pt NMR spectrum of a typical com-
pound was recorded on a Bruker DRX500 spectrometer. Mass
spectra were measured on an Agilent 6224 TOF LC/MS instrument.

146
H. Yu et al. / European Journal of Medicinal Chemistry 114 (2016) 141e152
Fig. 4. Cell cycle arrest induction effects of tested complexes on SGC7901 and HepG2 cells. Cell cycle analysis of (a) SGC7901 and (b) HepG2 cells treated with cisplatin, oxaliplatin
and complex 2a at 50 M for 24 h was performed by flow cytometry. (c) The percentages of G1, G2 and S phase cells following tested complexes treatment for 24 h are shown.
m

H. Yu et al. / European Journal of Medicinal Chemistry 114 (2016) 141e152
147
NMR (300 MHz, D2O):
d
¼ 1.27e1.42 (m, 2H, CH2 of DACH),
1.54e1.60 (m, 2H, CH2 of DACH), 1.84e1.76 (m, 2H, CH2 of DACH),
2.13e2.17 (d, J ¼ 12 Hz, 1H, CH2 of DACH), 2.38e2.43 (d, J ¼ 15 Hz,
1H, CH2 of DACH), 3.38e3.39 (m, 2H, NHCH2), 4.17e4.44 (m, 2H,
NHCH), 7.09e7.40 (m, 4H, ArH); 13C NMR (D2O, ppm):
d
¼ 24.9, 25.0,
28.7, 31.9, 45.3, 53.7, 61.4, 118.4, 118.6, 127.7134.5e135.0135.1,
161.7e164.7 (JCF ¼ ꢀ225 Hz). ESI-MS: m/z [MþH]þ ¼ 223.3 (100%).
4.1.1.3. (1R,2R)-N1-(3-fluorobenzyl)-1,2-diaminocyclohexane (LF2).
The synthetic procedure was analogous to that of L except using m-
fluorobenzaldehyde instead, white solid powder, yield: 64%.
½aꢃ2D5 ¼ ꢀ39.2 (c 0.42, H2O). Anal. Calcd (%) for C13H19FN2$2HCl: C,
52.89; H, 7.17; N, 9.49; Found: C, 52.78; H, 7.32; N, 9.35. IR
(KBr cmꢀ1): 3434, 2947, 2867, 1617, 1591, 1491, 1455, 1257, 1152,
1028, 875, 792, 690. 1H NMR (300 MHz, D2O):
d
¼ 1.43e1.46 (m, 2H,
CH2 of DACH), 1.64 (s, 2H, CH2 of DACH), 1.86e1.92 (m, 2H, CH2 of
DACH), 2.16e2.25 (d, J ¼ 27 Hz, 1H, CH2 of DACH), 2.46e2.50 (d,
J ¼ 12 Hz, 1H, CH2 of DACH), 3.54e3.56 (m, 2H, NHCH and NH2CH),
4.25e4.57 (dd, 2H, NHCH2), 7.24e7.56 (m, 4H, ArH); 13C NMR (D2O,
ppm):
128.3,
d
¼ 24.9, 25.1, 28.7, 32.0, 50.8e50.9, 53.9, 62.2, 119.0e119.8,
133.7e133.8,
135.0e135.1,
137.6,
163.5e166.8
(JCF ¼ ꢀ247.5 Hz). ESI-MS: m/z [MþH]þ ¼ 223.1 (100%).
4.1.1.4. (1R,2R)-N1-(4-fluorobenzyl)-1,2-diaminocyclohexane (LF3).
The synthetic procedure was the same as that of L except using p-
fluorobenzaldehyde instead, white solid powder, yield: 58%.
½aꢃ2D5 ¼ ꢀ33.1 (c 0.33, H2O). Anal. Calcd (%) for C13H19FN2$2HCl: C,
52.89; H, 7.17; N, 9.49; Found: C, 52.81; H, 7.28; N, 9.23.
IR(KBr cmꢀ1): 3437, 2944, 1604, 1513, 1451, 1231, 1162, 1024, 827,
776. 1H NMR (300 MHz, D2O):
d
¼ 1.28e1.43 (m, 2H, CH2 of DACH),
Fig. 5. Gel electrophoretic mobility pattern of pET22b plasmid DNA incubated with
different concentrations of platinum(II) complexes, (a) cisplatin, (b) oxaliplatin, (c)
complex 2a. Lanes 0e4 expressed concentrations of 0, 10, 40, 160, and 640 mM of the
tested compounds incubated with pET22b plasmid DNA. Form I and Form II represent
closed circular DNA and open circular DNA, respectively.
1.56 (s, 2H, CH2 of DACH),1.77e1.86 (m, 2H, CH2 of DACH), 2.13e2.17
(d, J ¼ 12 Hz, 1H, CH2 of DACH), 2.38e2.43 (d, J ¼ 15 Hz, 1H, CH2 of
DACH), 3.34e3.36 (m, 2H, NHCH and NH2CH2), 3.97e4.36 (dd, 2H,
NHCH2), 7.04e7.40 (m, 4H, ArH). 13C NMR (D2O, ppm):
d
¼ 22.4,
22.5, 26.2, 29.4, 48.2, 51.3, 58.4, 116.1e116.2, 126.4, 132.0,
162.3e163.9 (JCF ¼ ꢀ240 Hz). ESI-MS: m/z [MþH]þ ¼ 223.3 (100%).
4.1.2. Preparation of complexes 1aꢀ4a
Under nitrogen and protection from light, an aqueous solution
(75 mL) of K2PtCl4 (2.07 g, 5 mmol) was added to the ligand
(5 mmol) in water (5 mL). The reaction mixture was then stirred at
room temperature for 10 h and yellow solids deposited. The
products were filtered off, washed with ethanol and distilled water,
and then dried in vacuum. Greater than 95% purity of complexes
1ae4a was confirmed by elemental analysis.
Fig. 6. SGC7901 cells treated with cisplain, oxaliplatin and complex 2a at 50
mM for
24 h. Equal loading was testified by the detection of
similarly from three independent experiments.
b-actin. Results were obtained
4.1.2.1. Cis-dichloro[(1R,2R)-N1-benzyl-1,2-diaminocyclohexane-
N,N0]platinum(II) (Complex 1a): yield: 86%. ½aꢃ2D5 ¼ 142.9 (c 0.13,
DMF). Anal. calcd for C13H20Cl2N2Pt: C, 33.20; H, 4.29; N, 5.96.
Found: C, 33.41; H, 4.43; N, 5.69. IR (KBr, cmꢀ1): 3466 (br), 3120,
2935, 2860, 1580, 1452, 751, 702. 1H NMR (300 MHz, DMSO-d6):
2727, 2525, 2395, 1621, 1585, 1508, 1448, 1033, 762, 701. 1H NMR
(300 MHz, D2O):
d
¼ 1.24e1.48 (m, 2H, CH2 of DACH), 1.58e1.65 (m,
d1.12e1.16 (m, 1H, CH2 of DACH), 1.19e1.21 (m, 2H, CH2 of DACH),
2H, CH2 of DACH), 1.82e1.88 (m, 2H, CH2 of DACH), 2.18e2.22 (d,
J ¼ 12 Hz, 1H, CH2 of DACH), 2.44e2.48 (d, J ¼ 12 Hz, 1H, CH2 of
DACH), 3.47e3.58 (m, 2H, NHCH and NH2CH), 4.22e4.63 (dd, 2H,
1.24e1.26 (m, 1H, CH2 of DACH), 1.49e1.56 (m, 2H, CH2 of DACH),
1.90e1.93 (m, 2H, CH2 of DACH), 2.12e2.25 (m, 2H, NHCH and
NH2CH), 3.91e4.54 (m, 2H, NHCH2Ph), 6.11e6.16 (dd, 2H, CHNH2),
7.29e7.31 (m, 1H, CH2NH), 7.38e8.11 (m, 5H, ArH). 13C NMR (DMSO-
NHCH2), 7.41e7.51 (m, 5H, ArH); 13C NMR (D2O, ppm):
d
¼ 24.9,
25.0, 28.7, 32.0, 51.5, 53.9, 61.0, 131.9, 132.4, 132.5, 132.9. ESI-MS: m/
z [MþH] þ ¼ 205.2 (100%).
d6/TMS, ppm): d24.4, 24.7, 28.0, 31.8, 48.9, 61.9, 65.7, 128.3, 128.6,
131.1, 134.9. ESI-MS: m/z [MꢀCl]þ ¼ 435 (100%).
4.1.1.2. (1R,2R)-N1-(2-fluorobenzyl)-1,2-diaminocyclohexane (LF1).
The synthetic procedure was similar to that of L except using o-
fluorobenzaldehyde instead, white solid powder, yield: 55%.
½aꢃ2D5 ¼ ꢀ36.2 (c 0.39, H2O). Anal. calcd for C13H19FN2$2HCl: C,
52.89; H, 7.17; N, 9.49; Found: C, 53.03; H, 7.33; N, 9.26. IR (KBr
cmꢀ1): 3427, 2944, 1618, 1557, 1494, 1459, 1105, 1034, 784, 763. 1H
4.1.2.2. Cis-dichloro[(1R,2R)-N1 -(2-fluorobenzyl)-1, 2-
diaminocyclohexane-N, N0]platinum(II) (Complex 2a): yield: 97%.
½aꢃ2D5 ¼ 130.0 (c 0.13, DMF). Anal. calcd for C13H19Cl2FN2Pt: C, 31.98;
H, 3.92; N, 5.74. Found: C, 32.20; H, 4.23; N, 5.49. IR (KBr, cmꢀ1):
3138(br), 2933, 2862, 1579, 1494, 1451, 1229, 1187, 1139, 761. 1H
NMR (300 MHz, DMSO-d6):
d
¼ 0.76e0.87 (m, 1H, CH2 of DACH),

148
H. Yu et al. / European Journal of Medicinal Chemistry 114 (2016) 141e152
0.95e1.06 (m, 2H, CH2 of DACH), 1.13e1.20 (m, 1H, CH2 of DACH),
1.39e1.48 (m, 2H, CH2 of DACH), 1.75e1.82 (m, 2H, CH2 of DACH),
2.11e2.23 (m, 2H, NHCH and NH2CH), 3.75e4.55 (m, 2H, NHCH2Ar),
4.96e5.47 (dd, 2H, CHNH2) 6.75e7.45 (m, 4H, ArH), 8.89e8.93 (m,
4.1.3.2. [(1R,2R)-N1-(2-fluorobenzyl)-1,2-cyclohexanediamine-
N,N0](oxalate-O,O’)platinum(II)
(Complex
2b):
yield:
48%.
½aꢃ2D5 ¼ 89.5 (c 0.10, DMF). Anal. calcd for C15H19FN2O4Pt: C, 35.65;
H, 3.79; N, 5.54. Found: C, 35.15; H, 3.92; N, 5.23. IR (KBr, cmꢀ1):
3439 (br), 3439, 3239, 2935, 2861, 1601, 1451, 1384, 1292, 1230,
1124, 1062, 1037, 875, 778, 714. 1H NMR (300 MHz, DMSO-d6):
1H, CHNH). 13C NMR (DMSO-d6/TMS, ppm):
d
¼ 24.6, 24.7, 28.2,
31.9, 42.0, 62.4, 66.6, 115.3e115.6, 121.9e122.1, 124.9e125.0,
130.6e130.7, 134.1e134.2, 160.0e163.3 (JCF ¼ ꢀ248 Hz). 195Pt NMR
d
¼ 1.04e1.13 (m, 2H, CH2 of DACH),1.23e1.25 (m,1H, CH2 of DACH),
(DMSO-d6, ppm):
d
¼ ꢀ2349.2. ESI-MS: m/z [MꢀCl]þ ¼ 453 (100%).
1.41e1.43 (m, 1H, CH2 of DACH), 1.60 (s, 2H CH2 of DACH), 1.92e1.94
(m, 1H, CH2 of DACH), 2.16e2.18 (m, 1H, CH2 of DACH), 2.65e2.89
(m, 2H, CHNH2 and CHNH), 3.97e4.32 (m, 2H, NHCH2Ar), 5.24e5.78
4.1.2.3. Cis-dichloro[(1R,2R)-N1 -(3-fluorobenzyl)-1,2-
diaminocyclohexane-N,N0]platinum(II) (Complex 3a): yield: 85%.
½aꢃ2D5 ¼ 135.2 (c 0.24, DMF) Anal. calcd for C13H19Cl2FN2Pt: C, 31.98;
H, 3.92; N, 5.74. Found: C, 32.31; H, 4.20; N, 5.36. IR (KBr, cmꢀ1):
3267(br), 3188, 3100, 2938, 1587, 1451, 1258, 1147, 789, 754. 1H NMR
(dd, 2H, CHNH2), 6.82e7.33 (m, 4H, ArH), 7.83 (m, 1H, CH2NH). 13
C
NMR (DMSO-d6/TMS, ppm):
d
¼ 23.4, 24.2, 28.8, 34.4, 51.7, 61.9,
67.4, 124.2, 127.5e127.9, 129.9, 138.6, 139.9, 155.1e156.8
(JCF ¼ ꢀ255 Hz), 164.4. ESI-MS: m/z [MꢀH]ꢀ ¼ 504 (100%).
(300 MHz, DMSO-d6):
d
¼ 0.76e0.87 (m, 1H, CH2 of DACH),
4.1.3.3. [(1R,2R)-N1-(3-fluorobenzyl)-1,2-cyclohexanediamine-
0.95e1.06 (m, 2H, CH2 of DACH), 1.13e1.39 (m, 1H, CH2 of DACH),
1.41e1.75 (m, 2H, CH2 of DACH), 1.77e1.90 (m, 2H, CH2 of DACH),
1.98e2.23 (m, 2H, NHCH and NH2CH), 3.67e4.58 (m, 2H, NHCH2Ar),
5.01e5.48 (dd, 2H, CHNH2), 6.72e7.86 (m, 4H, ArH), 8.25e8.27 (m,
N,N0](oxalate-O,O’)platinum(II)
(Complex
3b):
yield:
24%.
½aꢃ2D5 ¼ 109.9 (c 0.14, DMF). Anal. calcd for C15H19FN2O4Pt: C, 35.65;
H, 3.79; N, 5.54. Found: C, 35.28; H, 4.01; N, 5.31. IR (KBr, cmꢀ1):
3445 (br), 3234, 2133, 2935, 2861, 1616, 1450, 1384, 1295, 1229, 778.
1H, CH2NH). 13C NMR (DMSO-d6/TMS, ppm):
d
¼ 24.4, 24.6, 28.0,
1H NMR (300 MHz, DMSO-d6):
d
¼ 1.04e1.12 (m, 2H, CH2 of DACH),
31.7, 48.2, 61.8, 65.8, 115.2e115.5, 117.7e117.9, 127.3e127.3,
130.3e130.4,137.5e137.6,160.7e163.9 (JCF ¼ ꢀ240 Hz). ESI-MS: m/z
[MꢀCl]þ ¼ 453 (100%).
1.23 (s, 1H, CH2 of DACH),1.45 (s, 1H, CH2 of DACH),1.61 (s, 2H CH2 of
DACH), 1.92 (s, 1H, CH2 of DACH), 2.15 (s, 1H, CH2 of DACH),
2.65e2.92 (m, 2H, CHNH2 and CHNH), 4.09e4.28 (m, 2H, NHCH2Ar),
5.17e5.94 (dd, 2H, CHNH2), 6.77e7.49 (m, 4H, ArH), 7.68 (m, 1H,
4.1.2.4. Cis-dichloro[(1R,2R)-N1 -(4-fluorobenzyl)-1,2-
diaminocyclohexane-N,N0]platinum(II) (Complex 4a): yield: 79%.
½aꢃ2D5 ¼ 133.1 (c 0.17, DMF). Anal. calcd for C13H19Cl2FN2Pt: C, 31.98;
H, 3.92; N, 5.74. Found: C, 32.25; H, 4.19; N, 5.31. IR (KBr, cmꢀ1):
3142(br), 3142, 2939, 2866, 1598, 1509, 1452, 1220, 1152, 976, 823,
CH2NH). 13C NMR (DMSO-d6/TMS, ppm):
d
¼ 23.8, 24.2, 27.6, 31.6,
49.5, 61.1, 64.8, 115.2e115.3, 117.5e117.7, 127.1, 130.4, 136.6e136.7,
164.0e165.4 (JCF ¼ ꢀ210 Hz), 166.4. ESI-MS: m/z [MꢀH]ꢀ ¼ 504
(100%).
773. 1H NMR (300 MHz, DMSO-d6):
d
¼ 0.76e0.87 (m, 1H, CH2 of
4.1.3.4. [(1R,2R)-N1-(4-fluorobenzyl)-1,2-cyclohexanediamine-
DACH), 0.95e1.06 (m, 2H, CH2 of DACH), 1.13e1.39 (m, 1H, CH2 of
DACH), 1.41e1.75 (m, 2H, CH2 of DACH), 1.77e1.90 (m, 2H, CH2 of
DACH), 2.12e2.25 (m, 2H, NHCH and NH2CH), 3.67e4.58 (m, 2H,
NHCH2Ar), 5.01e5.48 (dd, 2H, CHNH2), 6.72e7.86 (m, 4H, ArH),
8.25e8.27 (m, 1H, CH2NH). 13C NMR (DMSO-d6/TMS, ppm):
N,N0](oxalate-O,O’)platinum(II)
(Complex
4b):
yield:
28%.
½aꢃ2D5 ¼ 108.7 (c 0.15, DMF). Anal. calcd for C15H19FN2O4Pt: C, 35.65;
H, 3.79; N, 5.54. Found: C, 35.47; H, 3.94; N, 5.22. IR (KBr, cmꢀ1):
3435(br), 3314, 3243, 2936, 1622, 1591, 1449, 1384, 1293, 770. 1H
NMR (300 MHz, DMSO-d6):
d
¼ 0.95e1.11 (m, 2H, CH2 of DACH),
d
¼ 24.4, 24.6, 27.9, 31.8, 47.9, 61.9, 65.5, 115.2e115.5, 131.1,
1.23 (s,1H, CH2 of DACH),1.43 (s,1H, CH2 of DACH),1.59 (s, 2H CH2 of
DACH), 1.92 (s, 1H, CH2 of DACH), 2.11e2.13 (m, 1H, CH2 of DACH),
2.65e2.83 (m, 2H, CHNH2 and CHNH), 3.97e4.19 (m, 2H, NHCH2Ar),
5.32e5.78 (dd, 2H, CHNH2), 6.81e7.29 (m, 4H, ArH), 7.71e7.92 (m,
133.3e133.4, 160.7e163.9 (JCF
¼
ꢀ240 Hz). ESI-MS: m/z
[MꢀCl]þ ¼ 453 (100%).
1H, CH2NH). 13C NMR (DMSO-d6/TMS, ppm):
d
¼ 23.8, 24.2, 27.6,
4.1.3. Preparation of complexes 1be4b
31.6, 49.5, 61.2, 64.5, 115.2e115.4, 130.3, 133.2e133.3, 161.3e162.9
Complex 1a, 2a, 3a or 4a (1 mmol) was suspended in distilled
water (100 mL) and silver nitrate (0.34 g, 2 mmol) was added. The
mixture was heated to 38 ꢁC and stirred for 12 h in the lighting
shielding condition. Subsequently, AgCl deposits were filtered off,
the filtrate was mixed with an aqueous solution (15 mL) of sodium
oxalate (1.00 mmol) and heated to 35 ꢁC in the dark for 12 h. After
the reaction terminated, the solution was filtered through celite
and washed with water repeatedly, dried in vacuum, pale yellow
powders were obtained. Greater than 95% purity of complexes
1bꢀ4b was confirmed by elemental analysis.
(JCF ¼ ꢀ240 Hz), 165.5, 166.4. ESI-MS: m/z [MꢀH]þ ¼ 504 (100%).
4.1.4. Preparation of complexes 1ce4c
The synthetic procedure was similar to that of complexes 1be4b
except sodium malonate was used to take the place of sodium
oxalate.
4.1.4.1. [(1R,2R)-N1-benzyl-1,2-cyclohexanediamine-N,N0](malonato-
O,O’)platinum(II) (Complex 1c): yield: 67%. ½aꢃ2D5 ¼ 127.5 (c 0.16,
DMF). Anal. calcd for C16H22N2O4Pt: C, 38.32; H, 4.42; N, 5.59.
Found: C, 38.55; H, 4.74; N, 5.34. IR (KBr, cmꢀ1): 3417(br), 3241,
3140, 3048, 2931, 2858, 1581, 1445, 1347, 745. 1H NMR (300 MHz,
4.1.3.1. [(1R,2R)-N1-benzyl-1,2-cyclohexanediamine-N,N0](oxalate-
O,O0)platinum(II) (Complex 1b): yield: 65%. ½aꢃ2D5 ¼ 121.3 (c 0.12,
DMF). Anal. calcd for C15H20N2O4Pt: C, 36.96; H, 4.14; N, 5.75.
Found: C, 36.55; H, 4.32; N, 5.53. IR (KBr, cmꢀ1): 3433(br), 3237,
3048, 2933, 2859, 1621, 1447, 1291, 780, 745. 1H NMR (300 MHz,
DMSO-d6):
d
¼ 1.14e1.21 (m, 2H, CH2 of DACH), 1.40 (s, 2H, CH2 of
DACH), 1.65 (m, 2H, CH2 of DACH), 1.91e1.94 (m, 1H, CH2 of DACH),
2.16e2.18 (m, 1H, CH2 of DACH), 2.16e2.60 (m, 2H, CHNH and
CHNH2), 2.74 (s, 2H, (OOC)2CH2), 3.98e4.17 (m, 2H, NHCH2Ar), 4.83
DMSO-d6):
d
¼ 1.04e1.07 (m, 2H, CH2 of DACH), 1.22e1.23 (m, 2H,
(m, 1H, CH2NH), 5.43 (dd, 2H, CHNH2), 6.93e7.60 (m, 5H, ArH). 13
C
CH2 of DACH), 1.61 (s, 2H, CH2 of DACH), 1.93 (s, 1H, CH2 of DACH),
2.15 (s, 1H, CH2 of DACH), 1.93e2.15 (m, 2H, CHNH2 and CHNH), 2.89
(m, 2H, NHCH2Ar), 4.01e4.13 (dd, 2H, CHNH2), 5.72 (m, 1H, CH2NH),
NMR (DMSO-d6/TMS, ppm):
d
¼ 23.4, 24.3, 28.9, 34.5, 38.9, 56.9,
61.5, 67.4, 121.7, 124.6, 125.5, 133.9, 171.6. ESI-MS: m/z
[MþH]þ ¼ 502 (100%).
6.93e7.51 (m, 5H, ArH). 13C NMR (DMSO-d6/TMS, ppm):
d
¼ 23.6,
24.4, 28.9, 34.4, 57.2, 61.1, 66.8, 121.3, 124.3, 125.4, 133.6, 165.2. ESI-
4.1.4.2. [(1R,2R)-N1-(2-fluorobenzyl)-1,2-cyclohexanediamine-
N,N0](malonato-O,O’)platinum(II) (Complex 2c): yield: 46%.
MS: m/z [MꢀH]ꢀ ¼ 486 (100%).

H. Yu et al. / European Journal of Medicinal Chemistry 114 (2016) 141e152
149
½aꢃ2D5 ¼ 93.4 (c 0.13, DMF). Anal. calcd for C16H21FN2O4Pt: C, 37.00; H,
4.08; N, 5.39. Found: C, 37.35; H, 4.22; N, 5.17. IR (KBr, cmꢀ1): 3237
(br), 2934, 2860, 1596, 1450, 1348, 1231, 1161, 1124, 1037, 875, 778,
yield: 63%. ½aꢃ2D5 ¼ 97.6 (c 0.12, DMF). Anal. calcd for C19H25FN2O4Pt:
C, 40.79; H, 4.50; N, 5.01. Found: C, 41.24; H, 4.69; N, 4.88. IR (KBr,
cmꢀ1): 3436(br), 3237, 3140, 2938, 2862, 1595, 1563, 1451, 1381,
716. 1H NMR (300 MHz, DMSO-d6):
d
¼ 1.15e1.21 (m, 2H, CH2 of
1228, 778. 1H NMR (300 MHz, DMSO-d6):
d
¼ 1.17 (s, 3H, CH2 of
DACH), 1.39e1.47 (m, 2H, CH2 of DACH), 1.64e1.71 (m, 2H, CH2 of
DACH), 1.92e1.98 (m, 1H CH2 of DACH), 2.06e2.13 (m, 1H, CH2 of
DACH), 2.22e2.64 (m, 2H, CHNH and CHNH2), 2.75 (s, 2H,
(OOC)2CH2), 3.86e4.33 (m, 2H, NHCH2Ar), 4.92e5.03 (dd, 2H,
CHNH2), 5.49 (m, 1H, CH2NH), 6.84e7.32 (m, 4H, ArH). 13C NMR
DACH), 1.40 (s, 1H, CH2 of DACH), 1.65 (s, 2H, CH2 of DACH),
1.88e1.90 (m, 2H, CH2 of DACH), 1.95e1.98 (m, 2H CH2 of cdba),
2.23e2.28 (m, 4H CH2 of cdba), 2.65e2.86 (m, 2H, NHCH and
NH2CH), 3.97e4.36 (m, 2H, NHCH2Ar), 4.97 (dd, 2H, CHNH2), 5.53
(m, 1H, CH2NH), 6.85e7.42 (m, 4H, ArH). 13C NMR (DMSO-d6/TMS,
(DMSO-d6/TMS, ppm):
d
¼ 23.4, 24.2, 28.7, 34.4, 38.9, 51.7, 61.8,
ppm):
d
¼ 16.2, 23.4, 24.2, 28.7, 29.0, 34.3, 48.8, 51.7, 61.8, 67.4, 69.4,
67.4, 111.0e111.2, 127.6, 129.9, 138.3, 139.9, 155.1e156.8
111.0e111.1, 127.5, 129.9, 138.6e138.7, 140.1, 155.1e156.8
(JCF ¼ ꢀ212.5 Hz), 171.6. ESI-MS: m/z [MþH]þ ¼ 520 (100%).
(JCF ¼ ꢀ255 Hz), 177.4. ESI-MS: m/z [MþH]þ ¼ 560 (100%).
4.1.4.3. [(1R,2R)-N1-(3-fluorobenzyl)-1,2-cyclohexanediamine-
N,N0](malonato-O,O’)platinum(II) (Complex 3c): yield: 35%.
½aꢃ2D5 ¼ 115.9 (c 0.18, DMF). Anal. calcd for C16H21FN2O4Pt: C, 37.00;
H, 4.08; N, 5.39. Found: C, 37.25; H, 4.36; N, 5.25. IR (KBr, cmꢀ1):
3445 (br), 3241, 3133, 2934, 2861, 1718, 1593, 1450, 1384, 1278,
4.1.5.3. [(1R,2R)-N1-(3-fluorobenzyl)-1,2-cyclohexanediamine-
N,N0](1,1-cyclobutanedicarboxylato-O,O0)platinum(II) (Complex 3d):
yield: 59%. ½aꢃ2D5
¼
128.6 (c 0.18, DMF). Anal. calcd for
19H25FN2O4Pt: C, 40.79; H, 4.50; N, 5.01. Found: C, 40.65; H, 4.86;
C
N, 4.86. IR (KBr, cmꢀ1): 3444(br), 3248, 3133, 2938, 2863, 1721,
1230, 1214, 778. 1H NMR (300 MHz, DMSO-d6):
d
¼ 1.11e1.13 (m,
1592, 1450, 1384, 1277, 1230, 1213, 778. 1H NMR (300 MHz, DMSO-
2H, CH2 of DACH), 1.22 (s, 1H, CH2 of DACH),1.43e1.62 (m,1H, CH2 of
DACH), 1.70 (m, 2H CH2 of DACH), 1.88e1.91 (m, 1H, CH2 of DACH),
2.07 (s, 1H CH2 of DACH), 2.17e2.61 (m, 2H, CHNH and CHNH2), 2.74
(s, 2H, (OOC)2CH2), 4.00e4.25 (m, 2H, NHCH2Ar), 4.92e5.66 (dd,
2H, CHNH2), 7.61 (m, 1H, CH2NH), 6.71e7.51 (m, 4H, ArH). 13C NMR
d6):
d
¼ 1.10 (s, 3H, CH2 of DACH), 1.46 (s, 1H, CH2 of DACH), 1.61 (s,
2H, CH2 of DACH), 1.89e1.94 (m, 2H, CH2 of DACH), 2.15e2.26 (m,
2H CH2 of cdba), 2.28e2.50 (m, 4H CH2 of cdba), 2.63e2.85 (m, 2H,
NHCH and NH2CH), 4.03e4.27 (m, 2H, NHCH2Ar), 5.13 (dd, 2H,
CHNH2), 5.74 (m, 1H, CH2NH), 6.74e7.51 (m, 4H, ArH). 13C NMR
(DMSO-d6/TMS, ppm):
d
¼ 23.4, 24.0, 28.6, 33.8, 38.9, 57.2, 61.2,
(DMSO-d6/TMS, ppm):
d
¼ 16.5, 23.4, 24.2, 28.2, 28.7, 29.0, 34.2,
67.5, 109.0e109.1, 112.0e112.1, 112.6e112.8, 126.5, 135.1,
164.9e166.5 (JCF ¼ ꢀ240 Hz), 171.5. ESI-MS: m/z [MþH]þ ¼ 520
(100%).
48.8, 51.5, 61.8, 67.3, 111.0e111.2, 127.5, 129.9, 138.6, 140.1,
155.1e156.8 (JCF ¼ ꢀ240 Hz), 177.3. ESI-MS: m/z [MþH]þ ¼ 560
(100%).
4.1.4.4. [(1R,2R)-N1-(4-fluorobenzyl)-1,2-cyclohexanediamine-
N,N0](malonato-O,O’)platinum(II) (Complex 4c): yield: 19%.
½aꢃ2D5 ¼ 95.7 (c 0.15, DMF). Anal. calcd for C16H21FN2O4Pt: C, 37.00; H,
4.08; N, 5.39. Found: C, 37.39; H, 4.19; N, 5.13. IR (KBr, cmꢀ1):
3434(br), 3240, 2935, 2860, 1590, 1449, 1384, 1350, 1243, 1174. H
4.1.5.4. [(1R,2R)-N1-(4-fluorobenzyl)-1,2-cyclohexanediamine-
N,N0](1,1-cyclobutanedicarboxylato-O,O0)platinum(II) (Complex 4d):
yield: 28%. ½aꢃ2D5
¼
110.7 (c 0.19, DMF). Anal. calcd for
19H25FN2O4Pt: C, 40.79; H, 4.50; N, 5.01. Found: C, 40.93; H, 4.69;
C
N, 4.77. IR (KBr, cmꢀ1): 3420(br), 3240, 2938, 2862, 1568, 1459,
NMR (300 MHz, DMSO-d6):
d
¼ 1.14e1.20 (m, 2H, CH2 of DACH),
1383, 1243,1204. 1H NMR (300 MHz, DMSO-d6):
d
¼ 1.23 (s, 3H, CH2
1.31e1.40 (m, 2H, CH2 of DACH), 1.60e1.63 (m, 2H, CH2 of DACH),
1.71e1.74 (m, 1H CH2 of DACH), 1.87e1.90 (m, 1H, CH2 of DACH),
1.93e2.19 (m, 2H, CHNH and CHNH2), 2.74 (s, 2H, (OOC)2CH2),
3.98e4.19 (m, 2H, NHCH2Ar), 4.88 (s, 2H, CHNH2), 5.46 (s, 1H,
CH2NH), 6.81e7.30 (m, 4H, ArH). 13C NMR (DMSO-d6/TMS, ppm):
of DACH), 1.41 (s, 1H, CH2 of DACH), 1.62 (s, 2H, CH2 of DACH),
1.88e1.92 (m, 2H, CH2 of DACH), 1.96e1.99 (m, 2H CH2 of cdba),
2.13e2.28 (m, 4H CH2 of cdba), 2.30e2.38 (m, 2H, NHCH and
NH2CH), 3.98e4.19 (m, 2H, NHCH2Ar), 6.83e7.60 (m, 4H, ArH). 13
C
NMR (DMSO-d6/TMS, ppm):
45.0, 48.9, 56.3, 61.8, 67.6, 110.9e111.0, 119.9e120.0, 123.0, 139.6,
d
¼ 16.5, 23.4, 24.4, 28.9, 31.3, 34.8,
d
¼ 23.4, 24.3, 28.8, 34.3, 38.9, 56.3, 61.6, 67.4, 110.9e111.1, 119.9,
120.0e120.3, 123.1, 149.1, 158.6e160.2 (JCF ¼ ꢀ240 Hz), 171.6. ESI-
149.0, 158.7e160.3 (JCF
[MþH]þ ¼ 560 (100%).
¼ ꢀ240 Hz), 177.3. ESI-MS: m/z
MS: m/z [MþH]þ ¼ 520 (100%).
4.1.5. Preparation of complexes 1de4d
4.1.6. Preparation of complexes 1ee4e
The synthetic procedure was similar to that of complexes 1be4b
except sodium 1, 1-cyclobutanedicarboxylate was used to take the
place of sodium oxalate.
The synthetic procedure was similar to that of complexes 1be4b
except sodium 3-hydroxy-1,1-cyclobutanedicarboxylate was used
to take the place of sodium oxalate.
4.1.5.1. [(1R,2R)-N1-benzyl-1,2-cyclohexanediamine-N,N0](1,1-
cyclobutanedicarboxylato-O,O0) platinum(II) (Complex 1d): yield:
59%. ½aꢃ2D5 ¼ 140.1 (c 0.13, DMF). Anal. calcd for C19H26N2O4Pt: C,
42.14; H, 4.84; N, 5.17. Found: C, 42.35; H, 5.02; N, 4.88. IR (KBr,
cmꢀ1): 3419(br), 3232, 2935, 2860, 1648, 1579, 1361, 1448, 1379,
4.1.6.1. [(1R,2R)-N1-benzyl-1,2-cyclohexanediamine-N,N0](3-
hydroxy-1,1-cyclobutanedicarboxylato-O,O0)platinum(II)
(Complex
1e): yield: 50%. ½aꢃ2D5 ¼ 131.2 (c 0.14, DMF). Anal. calcd for
C
19H26N2O5Pt: C, 40.93; H, 4.70; N, 5.02. Found: C, 40.75; H, 4.59; N,
4.89. IR (KBr, cmꢀ1): 3418(br), 3200, 3100, 2934, 2860, 1582, 1446,
1237, 1119, 747. 1H NMR (300 MHz, DMSO-d6):
d
¼ 1.07e1.20 (m, 3H,
1382, 1036, 747, 662. 1H NMR (300 MHz, DMSO-d6):
d
¼ 1.13 (s, 3H,
CH2 of DACH), 1.40 (s, 1H, CH2 of DACH), 1.60e1.62 (m, 2H, CH2 of
DACH), 1.89e1.91 (m, 2H, CH2 of DACH), 1.93e2.06 (m, 2H CH2 of
cdba), 2.13e2.67 (m, 4H CH2 of cdba), 2.32e2.78 (m, 2H, NHCH and
CH2 of DACH), 1.36 (s, 1H, CH2 of DACH), 1.62 (s, 2H, CH2 of DACH),
1.90 (s, 2H, CH2 of DACH), 2.06e2.18 (m, 2H CH2 of hcdba),
2.24e2.49 (m, 3H CH2 of hcdba), 2.69e2.63 (m, 2H, NHCH and
NH2CH), 3.99e4.22 (m, 2H, NHCH2Ph), 4.65e4.85 (m, 1H, CHOH),
NH2CH), 4.01e4.17 (m, 2H, NHCH2Ar), 6.75e7.07 (m, 5H, ArH). 13
C
NMR (DMSO-d6/TMS, ppm):
d
¼ 16.5, 23.4, 24.3, 28.9, 29.0, 34.4,
6.80e7.49 (m, 5H, ArH). 13C NMR (DMSO-d6/TMS, ppm):
d
¼ 23.8,
45.8, 56.9, 61.5, 67.4, 68.3, 121.8, 124.6, 125.5, 133.9, 177.2. ESI-MS:
24.1, 28.0, 28.9, 31.6, 32.2, 41.9, 61.6, 62.8, 63.1, 72.5, 128.4, 128.7,
m/z [MþH]þ ¼ 542 (100%).
130.2, 133.9, 177.0, 177.5. ESI-MS: m/z [MꢀH]ꢀ ¼ 556 (100%).
4.1.5.2. [(1R,2R)-N1-(2-fluorobenzyl)-1,2-cyclohexanediamine-
N,N0](1,1-cyclobutanedicarboxylato-O,O0)platinum(II) (Complex 2d):
4.1.6.2. [(1R,2R)-N1-(2-fluorobenzyl)-1,2-cyclohexanediamine-
N,N0](3-hydroxy-1,1-cyclobutanedicarboxylato-O,O0)platinum(II)

150
H. Yu et al. / European Journal of Medicinal Chemistry 114 (2016) 141e152
(Complex 2e): yield: 23%. ½aꢃD25 ¼ 96.8 (c 0.17, DMF). Anal. calcd for
DMF and diluting with culture medium (DMF final concentration
<0.4%). Cisplatin and oxaliplatin were used as positive controls.
After that, cells were stained with MTT (5 mg/mL) for 4 h and
dissolved with DMSO. The UV absorption intensity was measured
with an automatic microplate ELISA reader at 490 nm. The IC50
values were calculated by the SPSS software.
C
19H25FN2O5Pt: C, 39.65; H, 4.38; N, 4.87. Found: C, 39.95; H, 4.58;
N, 4.47. IR (KBr, cmꢀ1): 3241(br), 2936, 2861, 1713, 1599, 1452, 1383,
1231, 1126, 1038, 874, 779. 1H NMR (300 MHz, DMSO-d6):
d
¼ 1.14e1.21 (m, 3H, CH2 of DACH), 1.39 (s, 1H, CH2 of DACH), 1.65
(s, 2H, CH2 of DACH), 1.81 (s, 2H, CH2 of DACH), 1.91 (s, 1H CH2 of
hcdba), 1.95e2.07 (m, 1H CH2 of hcdba), 2.22e2.39 (m, 3H CH2 of
hcdba), 2.50e2.66 (m, 2H, NHCH and NH2CH), 4.30e4.37 (m, 2H,
NHCH2Ar), 4.65 (m, 1H, CHOH), 6.84e7.40 (m, 4H, ArH). 13C NMR
4.2.3. Cellular uptake test
SGC7901 and SGC7901/CDDP cells were cultured in 6-well
plates until the cells reached about 90% confluence. Cisplatin,
(DMSO-d6/TMS, ppm):
48.9, 61.3, 61.7, 66.4, 115.1e115.3, 121.0, 124.0, 130.3, 133.6,
d
¼ 23.5, 24.2, 27.9, 28.7, 31.7, 34.3, 40.9,
oxaliplatin and complex 2a were added at a concentration of 50
respectively. After incubated for 12 h, cells were collected and
washed 3 times with cold PBS. 1 L suspended cells were taken to
measure the cell density. The rest of the cells was spun down and
digested at 65 ꢁC in 200
L 65% HNO3 for 10 h. The concentrations of
mM,
160.4e162.1 (JCF
¼
ꢀ255 Hz), 177.2, 177.5. ESI-MS: m/z
[MꢀH]ꢀ ¼ 574 (100%).
m
4.1.6.3. [(1R,2R)-N1-(3-fluorobenzyl)-1,2-cyclohexanediamine-
N,N0](3-hydroxy-1,1-cyclobutanedicarboxylato-O,O0)platinum(II)
(Complex 3e): yield: 23%. ½aꢃD25 ¼ 89.8 (c 0.11, DMF). Anal. calcd for
m
platinum were detected by ICP-MS.
C
19H25FN2O5Pt: C, 39.65; H, 4.38; N, 4.87. Found: C, 39.40; H, 4.64;
4.2.4. Apoptosis assay analysis
N, 4.51. IR (KBr, cmꢀ1): 3308(br), 2936, 2861, 1709, 1590, 1449, 1382,
The cultured SGC-7901 cells were washed with PBS (phosphate-
buffered saline), digested by trypsin solution and the suspension of
cells were diluted with medium to a certain concentration of
1 ꢂ 105 cells/mL. Cells were plated into 6-well culture plates (2 mL/
well) and cultured in 5% CO2 at 37 ꢁC overnight. A series of tested
reagents were added into each well and incubated for 24 h at 37 ꢁC
in 5% CO2, cisplatin and oxaliplatin were used as positive controls.
The apoptosis induced by platinum complexes was determined by
flow cytometry using an Annexin V-FITC Apoptosis Detection Kit
(Keygen, China). The detailed operation as follows: cells were
1211, 1146, 1038, 780, 666. 1H NMR (300 MHz, DMSO-d6):
d
¼ 1.05e1.08 (m, 3H, CH2 of DACH), 1.45 (s, 1H, CH2 of DACH), 1.61
(s, 2H, CH2 of DACH), 1.89e2.07 (m, 2H, CH2 of DACH), 2.15e2.20 (m,
2H CH2 of hcdba), 2.23e2.38 (m, 3H CH2 of hcdba), 2.40e2.63 (m,
2H, NHCH and NH2CH), 4.03e4.27 (m, 2H, NHCH2Ar), 4.45 (m, 1H,
CHOH), 6.70e7.51 (m, 4H, ArH). 13C NMR (DMSO-d6/TMS, ppm):
d
¼ 23.5, 24.1, 28.7, 28.8, 33.8, 34.2, 41.4, 56.6, 57.9, 61.7, 67.7, 109.0,
112.5e112.7, 118.7, 126.3e126.4, 155.3, 164.9e166.5 (JCF ¼ ꢀ240 Hz),
177.7. ESI-MS: m/z [MꢀH]ꢀ ¼ 574 (100%).
collected and washed twice with cold PBS, then stained with 5 mL
Annexin V-FITC for 5 min in Annexin-binding buffer (10 mM HEPES,
140 mM NaCl, 2.5 mM CaCl2, pH 7.4). Propidium Iodide was added
4.1.6.4. [(1R,2R)-N1-(4-fluorobenzyl)-1,2-cyclohexanediamine-
N,N0](3-hydroxy-1,1-cyclobutanedicarboxylato-O,O0)platinum(II)
(Complex 4e): yield: 30%. ½aꢃ2D5 ¼ 119.3 (c 0.15, DMF). Anal. calcd for
to cells with 5 mL before incubated at room temperature for 15 min.
C
19H25FN2O5Pt: C, 39.65; H, 4.38; N, 4.87. Found: C, 39.86; H, 4.51;
Fluorescence of cells was measured by flow cytometer (FAC Scan,
Becton Dickenson, USA). The results were analyzed using Cell Quest
software and appeared as percentage of normal and apoptotic cells
at various stages. FL1 and FL2 channel measured FITC and PI fluo-
rescence, respectively. The treatment of HepG2 cells was in the
similar way as mentioned above.
N, 4.55. IR (KBr, cmꢀ1): 3401 (br), 2936, 2861, 1569, 1453, 1383,
1242, 1206, 1037, 881. 1H NMR (300 MHz, DMSO-d6):
d
¼ 1.15e1.22
(m, 3H, CH2 of DACH), 1.38 (s, 1H, CH2 of DACH), 1.66 (s, 2H, CH2 of
DACH), 1.83 (s, 2H, CH2 of DACH), 1.91 (s, 1H CH2 of hcdba),
1.93e2.07 (m, 1H CH2 of hcdba), 2.20e2.28 (m, 3H CH2 of hcdba),
2.41e2.64 (m, 2H, NHCH and NH2CH), 3.97e4.28 (m, 2H, NHCH2Ar),
4.27e4.30 (m, 1H, CHOH), 6.76e7.86 (m, 4H, ArH). 13C NMR (DMSO-
4.2.5. Cell cycle measurement
d6/TMS, ppm):
d
¼ 23.4, 24.3, 28.0, 28.7, 33.8, 34.3, 41.2, 56.3, 61.7,
SGC7901 cells (1 ꢂ 106) were seeded in 6-well plate and incu-
67.4, 68.3, 110.9e111.0, 119.9, 123.0e123.1, 139.9, 149.1, 158.7e160.3
bated at 37 ꢁC in 5% CO2 overnight. Then, cells were treated with the
(JCF ¼ ꢀ240 Hz), 177.1, 177.6. ESI-MS: m/z [MꢀH]ꢀ ¼ 574 (100%).
platinum drugs (50 mM) for 24 h and cells were harvested from
adherent cultures by trypsin and washed with PBS. After that, cells
4.2. Biological studies
were fixed in cold 70% ethanol and stored at 4 ꢁC overnight. Then
cells were centrifuged and treated with 100
m
g/mL RNase for
4.2.1. Cell culture
30 min at 37 ꢁC, resuspended in 50
mg/mL propidium iodide in PBS
Four human solid tumor cell lines including HepG2 (human
hepatocellular carcinoma cell line), A2780 (human ovarian cell
line), SGC7901 (human gastric cancer cell line) and SGC7901/CDDP
(human gastric cancer cisplatin resistant cell line) as well as LO2
(normal liver cell line) were selected for the cytotoxicity study of all
synthesized platinum(II) complexes. The human solid tumor cell
lines were incubated carefully in RPMI-1640 medium or Dulbecco's
Modified Eagle Medium with 10% fetal bovine serum (FBS), strep-
for nucleic acids staining for 30 min at 4 ꢁC. The cells (1 ꢂ 104)
acquired for each sample using the Cell Quest software and
recording propidium iodide (PI) in FL2 channel. Cell cycle profiles
were performed with ModFit software. The treatment of HepG2
cells was in an analogous way as mentioned above.
4.2.6. Interaction with pET22b plasmid DNA
The interaction of complex 2a with pET22b plasmid DNA was
studied by agarose gel electrophoresis, cisplatin and oxaliplatin
tomycin (100
mg/mL), and ampicillin sodium (100 mg/mL) in an
atmosphere of 5% CO2 and 95% air at 37 ꢁC.
were used as positive controls. Solution of pET22b DNA (0.20
L) was mixed with the tested complex to achieve a set of con-
centrations ranging from 0 to 640 M. The mixture was incubated
in a water bath at 37 ꢁC for 24 h. 10
L aliquots of drug-DNA mix-
mg,
5
m
4.2.2. Cytotoxicity analysis (IC50
)
m
The toxicity studies of all synthesized platinum(II) complexes
(IC50 values) were determined by MTT assay (3-(4, 5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTT,
Sigma). About 5000e10000 cellsꢀ1 were seeded in 96-well cell
culture plates and treated for 72 h with the diluted solution of the
complexes, which were obtained by dissolving the compounds in
m
tures were loaded on agarose gel (ultra pure grade, Bio-Rad, made
up to 1% w/v) which was prepared using TA buffer (45 mM Tris-
acetate, pH 7.5) containing ethidium bromide (1 mg/mL) and un-
derwent electrophoresis under TAE buffer (0.05 M Tris base, 0.05 M
glacial acetic acid, 1 mM EDTA, pH ¼ 8.0) for 60 min at 100 V. The

H. Yu et al. / European Journal of Medicinal Chemistry 114 (2016) 141e152
151
[10] C.Z. Gao, Y. Zhang, T.S. Wang, J. Chen, Y.X. Qian, B. Yang, S.H. Gou, P. Dong,
Y.J. Zhang, Novel antitumor dinuclear platinum(II) complexes with a new
chiral tetradentate ligand as the carrier group, Appl. Organometal. Chem. 29
(2015) 481e486.
DNA bands were photographed under a Molecular Imager (Tanon,
China) under UV light.
[11] A.J. Kraker, C.W. Moore, Accumulation of cis-diamminedichloroplatinum(II)
and platinum analogs by platinum-resistant murine leukemia-cells in vitro,
Cancer Res. 48 (1988) 9e13.
4.2.7. Western blot analysis
SGC7901 cells were cultured in culture flasks until the cell
density reached 80% and incubated with 50
mM platinum com-
[12] S.C.
Mann,
P.A.
Andrews,
S.B.
Howell,
Modulation
of
cis-
plexes for 24 h at 37 ꢁC. Proteins were extracted by lysis buffer and
concentration was determined with protein assay (Thermo, Wal-
tham, MA) by BCA (bicinchoninic acid) as well as adjusted to an
equal concentration. Protein samples (20 mg/lane) were separated
in the polyacrylamide gel which was prepared by 10% sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
After that, protein samples were transferred to polyvinylidene
difluoride (PVDF) Immobilon-P membrane (Bio-Rad, USA) by
transblot apparatus (Bio-Rad, USA). Then, the membrane was
socked with 5% nonfat milk for 1 h in TBST buffer and incubated at
diamminedichloroplatinum(II) accumulation and sensitivity by forskolin and
3-isobutyl-1-methylxanthine in sensitive and resistant human ovarian-
carcinoma cells, Int. J. Cancer 49 (1991) 866e872.
[13] K.C. Micetich, D. Barnes, L.C. Erickson, A comparative-study of the cyto-
toxicity
and
DNA-damagine
effects
of
cis-(diammino)(1,
and
1-
cis-
cyclobutanedicarboxylato)-platinum(II)
diamminedichloroplatinum(II) on L1210 cells, Cancer Res. 45 (1985)
4043e4047.
[14] G.M. Lai, R.F. Ozols, R.C. Young, T.C. Hailton, Effect of glutathione on DNA-
repair in cisplatin-resistant human ovarian-cancer cell-lines, J. Natl. Cancer
Inst. 81 (1989) 535e539.
[15] T.C. Hamilton, M.A. Winker, K.G. Louie, G. Batist, B.C. Behrens, T. Tsuruo,
K.R. Grotzinger, W.M. Mckoy, R.C. Young, R.F. Ozols, Augmentation of adria-
mycin, melphalan, and cisplatin cyto-toxicity in drug-resistant and drug-
sensitive human ovarian-carcinoma cell-lines by buthionine sulfoximine
mediated glutathione depletion, Biochem. Pharmacol. 34 (1985) 2583e2586.
[16] D.M. Townsend, K.D. Tew, H. Tapiero, The importance of glutathione in human
disease, Biomed. Pharmacother. 57 (2003) 145e155.
[17] P. Mistry, L.R. Kelland, G. Abel, S. Sidhar, K.R.T. Harrap, The relationships be-
tween glutathione, glutathione-S-transferase and cytotoxicity of platinum
drugs and melphalan in eight human ovarian carcinoma cell lines, Br. J. Cancer
64 (1991) 215e220.
[18] R. Ravi, S.M. Somani, L.P. Rybak, Mechanism of cisplatin ototoxicity-
antioxidant system, Pharmacol. Toxicol. 76 (1995) 386e394.
[19] R.B. Weiss, M.C. Christian, New cisplatin analogs in development e a review,
Drugs 46 (1993) 360e377.
4 ꢁC with primary antibodies diluted in PBST (1:2000
b-actin, Santa
Cruz, USA; 1:500 for Bax, BD Pharmagin, USA; 1:500 for Bcl-2, Cell
Signal, USA) overnight. Membrane was washed with PBST for 3
times and incubated with IRDye 800 conjugated secondary anti-
body which was diluted in PBST with 1:30,000 for 1 h. Labeled
proteins were measured by Odyssey Scanning System (LiCOR.,
Lincoln, Nebraska, USA).
4.2.8. Statistical analysis
All the values are expressed as mean SD of not least than three
different determinations. P < 0.05 was defined as statistically sig-
nificant by using the Student's test.
[20] L. Pendyala, P.J. Creaven, In-vitro cytotoxicity, protein-binding, red-blood-cell
partitioning, and biotransformation of oxaliplatin, Cancer Res. 53 (1993)
5970e5976.
[21] J. Helleman, K. Burger, I.H.L. Hamelers, A.W.M. Boersma, A.I.P.M. de Kroon,
G. Stoter, K. Nooter, Impaired cisplatin influx in an A2780 mutant cell line-
Acknowledgments
evidence for
a putative, cis-configuration-specific, platinum influx trans-
porter, Cancer Biol. Ther. 5 (2006) 943e949.
[22] O. Rixe, W. Ortuzar, M. Alvarez, R. Parker, E. Reed, K. Paull, T. Fojo, Oxaliplatin,
tetraplatin, cisplatin, and carboplatin: spectrum of activity in drug-resistant
cell lines and in the cell lines of the national cancer institute's anticancer
drug screen panel, Biochem. Pharmacol. 52 (1996) 1855e1865.
[23] P. Soulie, E. Raymond, S. Brienza, E.B. Cvitkovic, Oxaliplatin: the first DACH
platinum in clinical practice, Cancer 84 (1997) 665e673.
[24] J.L. Misset, H. Bleiberg, W. Sutherland, M. Bekradda, E. Cvitkovic, Oxaliplatin
clinical activity: a review, Crit. Rev. Oncol. Hemat. 35 (2000) 75e93.
[25] M. Benedetti, G. Tamasi, R. Cini, G. Natile, X-ray structure and circular di-
chroism of pure rotamers of bis[guanosine-50-monophosphate(-1)](N, N, N0,
N0-tetramethylcyclohexyl-1, 2-diamine) platinum(II) complexes that have R, R
and S, S configurations at the asymmetric diammine, Chem. Eur. J. 9 (2003)
6122e6132.
[26] E. Petruzzella, N. Margiotta, G. Natile, J.D. Hoeschele, Reactivity of kiteplatin
with S-donor biomolecules and nucleotides, Dalton Trans. 43 (2014)
12851e12859.
[27] H.L. Cui, R. Goddard, K.R. Porschke, A. Hamacher, M.U. Kassack, Bispidine
analogues of cisplatin, carboplatin, and oxaliplatin. Synthesis, structure, and
cytotoxicity, Inorg. Chem. 53 (2014) 3371e3384.
[28] J. Holford, S.Y. Sharp, B.A. Murrer, L. Kelland, In vitro circumvention of
cisplatin resistance by the novel sterically hindered platinum complex
AMD473, Br. J. Cancer 77 (1998) 366e373.
[29] Y.Y. Sun, F. Liu, S.H. Gou, L. Cheng, L. Fang, R.T. Yin, Synthesis and in vitro
antiproliferative activity of platinum(II) complexes with N-monoalkyl 1R, 2R-
diaminocyclohexane as ligands, Eur. J. Med. Chem. 55 (2012) 297e306.
[30] Y.Y. Sun, S.H. Gou, F. Liu, L. Cheng, R.T. Yin, L. Fang, Synthesis, in vitro cyto-
toxicity, and internation with DNA of platinum(II) complexes with N-mono-
cycloalkyl derivatives of 1R, 2R-diaminocyclohexane as carrier ligands,
ChemMedChem 7 (2012) 642e649.
[31] Y.Y. Sun, R.T. Yin, S.H. Gou, J. Zhao, Antitumor platinum(II) complexes of N-
monoalkyl-1R,2R-diaminocyclohexane derivatives with alkyl groups as hin-
drance, Inorg. Biochem. 112 (2012) 68e76.
We are grateful to the New Drug Creation Project of the National
Science and Technology Major Foundation of China (Project NO.
2013ZX09402102-001-006), the National Nature Science Founda-
tion of China (Project NO. 21271041), and the Fundamental
Research Funds for the Central Universities (Project NO.
2242013K30011) for financial support to this work. This project is
also supported by the Priority Academic Program Development of
Jiangsu Higher Education Institutions.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.02.060.
References
[1] B. Rosenberg, L. Vancamp, T. Krigas, Inhibition of cell division in Escherichia
coli by electrolysis products from a platinum electrode, Nature 205 (1965)
698e699.
[2] B. Rosenberg, Platinum complexes for treatment of cancer, Interdiscip. Sci.
Rev. 3 (1978) 134e147.
[3] M. Galanski, A. Yasemi, S. Slaby, M.A. Jakupec, V.B. Arion, M. Rausch,
A.A. Nazarov, B.K. Keppler, Synthesis, crystal structure and cytotoxicity of new
oxaliplatin analogues indicating that improvement of anticancer activity is
still possible, Eur. J. Med. Chem. 39 (2004) 707e714.
[4] E. Wong, C.M. Giandomenico, Current status of platinum-based antitumor
drugs, Chem. Rev. 99 (1999) 2451e2466.
[32] H.Y. Zhang, S.H. Gou, J. Zhao, F.H. Chen, G. Xu, X. Liu, Cytotoxicity profile of
novel sterically hindered platinum(II) complexes with (1R, 2R)-N1, N2-dibutyl-
1, 2-diaminocyclohexane, Eur. J. Med. Chem. 96 (2015) 187e195.
[33] F. Liu, S.H. Gou, L. Li, Asymmetric henry reactions of aldehydes with various
nitroalkanes catalyzed by copper(II) complexes of novel chiral N-monoalkyl
cyclohexane-1, 2-diamines, Appl. Organomet. Chem. 28 (2014) 186e193.
[34] S. H. Gou, Divalent platinum complexes containing sterically hindered aryl
group, preparation method and uses thereof. WO 2014131281 A1 20140904.
[35] S. H. Gou, Bivalent platinum complexes containing aryl steric hindrance group
and manufacture and application of the same. CN 103145762 A 20130612.
[36] X.Y. Zhang, L. Cheng, J. Wang, S.H. Gou, L. Fang, Four chiral Ag(I) complexes
with dual chiral components: effect of positional isomerism, luminescence
[5] E.R. Jamieson, S.J. Lippard, Structure, recognition, and processing of cisplatin-
DNA adducts, Chem. Rev. 99 (1999) 2467e2498.
[6] G. Natile, M. Coluccia, Current status of trans-platinum compounds in cancer
therapy, Coord. Chem. Rev. 216 (2001) 383e410.
[7] L.P. Martin, T.C. Hamilton, J.R. Schilder, Platinum resistance: the role of DNA
repair pathways, Clin. Cancer Res. 14 (2008) 1291e1295.
[8] M. Coluccia, G. Natile, Trans-platinum complexes in Cancer therapy, Anti
Cancer Agents Med. Chem. 7 (2007) 111e123.
[9] C.Z. Gao, T.S. Wang, Y. Zhang, J. Chen, Y.X. Qian, B. Yang, Y.J. Zhang, Design,
synthesis and in vitro cytotoxicity of novel dinuclear platinum(II) complexes
with
a tetradentate carrier ligand, Appl. Organometal. Chem. 29 (2015)
138e142.

152
H. Yu et al. / European Journal of Medicinal Chemistry 114 (2016) 141e152
and SHG response, Inorg. Chem. Comm. 40 (2014) 97e102.
[39] I. Würtenberger, B. Angermaier, B. Kircher, R. Gust, Synthesis and in vitro
pharmacological behavior of platinum(II) complexes containing 1,2-diamino-
1- (4-fluorophenyl)-2-alkanol ligands, J. Med. Chem. 56 (2013) 7951e7964.
[40] Z.F. Chen, Q.P. Qin, J.L. Qin, Y.C. Liu, K.B. Huang, Y.L. Li, T. Meng, G.H. Zhang,
Y. Peng, X.J. Luo, H. Liang, Stabilization of G-quadruplex DNA, inhibition of
telomerase activity, and tumor cell apoptosis by organoplatinum(II) com-
plexes with oxoisoaporphine, J. Med. Chem. 58 (2015) 2159e2179.
[37] Z. P. Zhou, F. H. Chen, G. Xu, S. H. Gou, Study on the cytotoxic activity of
platinum(II) complexes of (1R,2R)-N1-cyclopentyl-1,2-cyclohexanediamine
with substituted malonate derivative. Bioorg. Med. Chem. Lett. Doi: org/
10.1016/j.bmcl. 2015.12.019.
[38] Y.Y. Xing, W.W. Bao, X.B. Fan, K.M. Liu, X.K. Li, T. Xi, A novel oxaliplatin de-
rivative, Ht-2, triggers mitochondrion-dependent apoptosis in human colon
cancer cells, Apoptosis 20 (2015) 83e91.