Synthesis and biological evaluation of platinum(II) complexes containing (1R,2R)-N1-alkyl-1,2-diaminocyclohexane and D-(+)-camphorate ligands

Article abstract of DOI:10.1016/j.ica.2012.10.011

Five platinum(II) complexes with N-monoalkyl derivatives of 1R,2R-diaminocyclohexane as ligands and D-(+)-camphorate anion as leaving group have been synthesized and characterized by elemental analysis, IR, ¹H NMR, ESI-MS, and HRMS spectra. All

Full text of DOI:10.1016/j.ica.2012.10.011

Inorganica Chimica Acta 395 (2013) 154–159
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Synthesis and biological evaluation of platinum(II) complexes containing
(1R,2R)-N1-alkyl-1,2-diaminocyclohexane and D-(+)-camphorate ligands
Yanyan Sun a,b, Gang Xu a,b, Zhe Cao a, Shaohua Gou a,b,
⇑
a Pharmaceutical Research Center, 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
Article history:
Five platinum(II) complexes with N-monoalkyl derivatives of 1R,2R-diaminocyclohexane as ligands and
D-(+)-camphorate anion as leaving group have been synthesized and characterized by elemental analysis,
IR, 1H NMR, ESI-MS, and HRMS spectra. All complexes were evaluated for their in vitro cytotoxicity
against four human tumor cell lines and most of them showed promising cytotoxic activity, especially
compounds 3 and 4 with branched alkyl substituent at one of nitrogen atoms. Preliminary mechanism
study by flow cytometry and agarose gel electrophoresis was also carried out in comparison with cis-
platin and oxaliplatin.
Received 11 June 2012
Received in revised form 13 September
2012
Accepted 13 October 2012
Available online 2 November 2012
Keywords:
Ó 2012 Elsevier B.V. All rights reserved.
Platinum(II) complexes
Camphorate anion
Antitumor activity
Flow cytometry
DNA cleavage
1. Introduction
Norcantharidin, a demethylated form of cantharidin, has recently
been introduced as leaving group to prepare TCM-based platinum
Most of platinum-based antitumor drugs available in clinic are
adhered to the classical structure–activity relationship (SAR) sum-
marized by Cleare and Hoeschele in 1973 [1]. These cisplatin ana-
logues possess similar properties including narrow antitumor
spectrum, severe toxicity, intrinsic and acquired cross-resistance
[2–4]. Much effort has been made to overcome these drawbacks
by designing novel platinum complexes based on the non-classical
SAR. And many studies during the last two decades demonstrated
that cross-resistance between cisplatin and its analogues could be
overcome by introducing sterically hindered platinum(II) com-
plexes [5–8]. cis-Amminedichloro(2-methylpyridine)platinum(II)
(ZD0473) is a sterically hindered platinum(II) complex with a
bulky methylpyridine ligand which is responsible for its ability to
overcome platinum resistance [9]. Besides, other strategies used
in designing novel platinum-based anticancer agents have focused
on integrating biologically active ligands with platinum moieties,
such as doxorubicin [10], amino acids [11], and sugars [12].
It has been proved that traditional Chinese medicines (TCMs)
play an important role in cancer chemotherapy. And continuous at-
tempts have been made to exploit the benefits of TCM in designing
a new chemical entity with special biological activity [13–15].
complexes which showed remarkable antitumor activity [16].
Camphor, a traditional Chinese medicine, has long been used to
relieve pain, stop tickle, eliminate inflammation, and kill worm and
acariasis as early as in the Ming Dynasty. We have recently re-
ported a novel series of platinum complexes with various diamines
as carrier ligands and camphorate anion as leaving group, which
showed good antitumor activity. Among them, complex 1 contain-
ing 1R,2R-diaminocyclohexane (1R,2R-DACH) ligand showed
in vitro and in vivo antitumor activities that were superior to those
of carboplatin [17]. In this study, our strategy is to integrate the
camphorate anion with Pt-moieties containing N-monoalkyl deriv-
atives of 1R,2R-diaminocyclohexane as carrier ligands. The result-
ing compounds were shown in Scheme 1. And their biological
activity on four human tumor cell lines and preliminary mecha-
nism of action were also discussed.
2. Experimental
2.1. Materials and instruments
Potassium tetrachloroplatinate(II), 1R,2R-diaminocyclohexane
and disodium D-(+)-camphorate were obtained from a local chem-
ical company. All reagents were of high purity and used without
any further purification. Elemental analysis for C, H, and N was per-
formed with a Perkin–Elmer 1400C instrument. Elemental analysis
⇑
Corresponding author at: Pharmaceutical Research Center, School of Chemistry
and Chemical Engineering, Southeast University, Nanjing 211189, China. Tel./fax:
+86 25 83272381.
E-mail address: sgou@seu.edu.cn (S. Gou).
0020-1693/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ica.2012.10.011

Y. Sun et al. / Inorganica Chimica Acta 395 (2013) 154–159
155
H
N
H
N
2.27 (t, 1H, COCH), 2.45–2.70 (m, 3H, 2NHCH and NH2CH), 4.65–
6.44 (m, 3H, CHNH and CHNH2); IR (KBr): 3420, 3194, 3121, 2939,
1596, 1457, 1384, 1350, 1173, 1126, 801 cmꢀ1; ESI-MS: m/z (%):
549 (90), 550 (100), 551 (83) [M+H]+; HRMS: m/z [M+H]+ calcd.
R
Cl
Cl
R
2AgNO3
H2O
K2PtCl4
+
Pt
N
NH2
H2
for C19H35N2O4Pt: 550.22446, found: 550.22303. Anal. Calc. for C19
-
H34N2O4Pt: C, 41.52; H, 6.24; N, 5.10; Pt, 35.50. Found: C, 41.60; H,
NaOOC
6.18; N, 5.15; Pt, 35.29.cis-[(1R,2R)-N1-(2-methylpropyl)-1,2-
diaminocyclohexane-N,N’][(+)-camphorato-O,O’]platinum(II) (4).
White solid. Yield: 0.20 g (36%). 1H NMR (d6-DMSO/TMS): d 0.89
(d, 6H, CH(CH3)2), 0.99 (s, 6H, C(CH3)2), 1.22 (s, 3H, CCH3), 1.05–
1.89 (m, 13H, NHCH2CH(CH3)2 and CH2 of DACH and camphorato),
2.25 (t, 1H, COCH), 2.40–2.85 (m, 4H, NHCH2 and NHCH and NH2-
CH), 4.69–6.70 (m, 3H, CHNH and CHNH2); IR (KBr): 3434, 3186,
3117, 2938, 1608, 1383, 1347, 1169, 1125, 801 cmꢀ1; ESI-MS: m/z
(%): 563 (79), 564 (100), 565 (87) [M+H]+; HRMS: m/z [M+H]+ calcd.
for C20H37N2O4Pt: 564.24011, found: 564.24143. Anal. Calc. for C20-
H36N2O4Pt: C, 42.62; H, 6.44; N, 4.97; Pt, 34.61. Found: C, 42.74; H,
6.36; N, 5.01; Pt, 34.41.cis-[(1R,2R)-N1-cyclopentyl-1,2-diaminocy-
clohexane-N,N’][(+)-camphorato-O,O’]platinum(II) (5). White solid.
Yield: 0.24 g (42%). 1H NMR (d6-DMSO/TMS): d 0.95 (s, 6H, C(CH3)2),
1.25 (s, 3H, CCH3), 1.12–1.98 (m, 20H, CH2 of DACH and cyclopentyl
and camphorate), 2.22 (t, 1H, COCH), 2.55–2.78 (m, 3H, 2NHCH and
NH2CH), 4.76–6.55 (m, 3H, CHNH and CHNH2); IR (KBr): 3434 (br),
3178, 3113, 2944, 1602, 1457, 1382, 1348, 1126, 801 cmꢀ1; ESI-MS:
m/z (%): 575 (85), 576 (100), 577 (80) [M+H]+; HRMS: m/z [M+H]+
calcd. for C21H37N2O4Pt: 576.24011, found: 576.24175. Anal. Calc.
for C21H36N2O4Pt: C, 43.82; H, 6.30; N, 4.87; Pt, 33.89. Found: C,
43.90; H, 6.24; N, 4.92; Pt, 34.10.cis-[(1R,2R)-N1-cyclohexyl-1,2-
diaminocyclohexane-N,N’][(+)-camphorato-O,O’]platinum(II) (6).
White solid. Yield: 0.23 g (39%). 1H NMR (d6-DMSO/TMS): d 0.97
(s, 6H, C(CH3)2), 1.24 (s, 3H, CCH3), 1.15–1.95 (m, 22H, CH2 of DACH
and cyclohexyl and camphorate), 2.24 (t, 1H, COCH), 2.57–2.83 (m,
3H, 2NHCH and NH2CH), 4.80–6.67 (m, 3H, CHNH and CHNH2); IR
(KBr): 3431 (br), 3187, 3109, 2934, 2859, 1602, 1456, 1383, 1349,
1126, 800 cmꢀ1; ESI-MS: m/z (%): 589 (76), 590 (100), 591 (90)
[M+H]+; HRMS: m/z [M+H]+ calcd. for C22H39N2O4Pt: 590.25576,
found: 590.25425. Anal. Calc. for C22H38N2O4Pt: C, 44.81; H, 6.50;
N, 4.755; Pt, 33.09. Found: C, 44.89; H, 6.47; N, 4.805; Pt, 33.28.
2+
H
N
R
OH2
OH2
2
-
2NO3
Pt
NaOOC
N
O
O
H2
H
R
N
O
O
Pt
N
H2
R =
H
1
2
3
4
5
6
Scheme 1. The synthetic route to obtain platinum(II) complexes 1–6.
for Pt was carried out on a J-A1100 inductively coupled plasma
(ICP) spectrometer. ESI-MS and HRMS spectra were carried out in
an Agilent 6224 TOF LC/MS instrument. IR spectra were scanned
by
a
Nicolet IR200 spectrophotometer in the range of
4000–400 cmꢀ1 in KBr pellet. 1H NMR spectra were recorded on
a Bruker DRX-500 spectrometer at 500 MHz in d6-DMSO using
TMS as an internal reference. UV spectra were tested in a Shimadzu
UV-1700 instrument.
2.2. Preparation of target compounds
The general synthetic route to obtain the complexes containing
D-(+)-camphorate anion is shown in Scheme 1. The preparation of
(1R,2R)-N1-alkyl-1,2-diaminocyclohexane has been reported previ-
ously [18–20]. To a solution of K2PtCl4 (0.42 g, 1 mmol) in distilled
water (10 mL) was added (1R,2R)-N1-alkyl-1,2-diaminocyclohex-
ane (1 mmol) in distilled water (10 mL). The reaction mixture was
then stirred at 25 °C for 12 h in the dark to give yellow precipitate
as the intermediate cis-[Pt(Am)2Cl2] ((Am)2 = (1R,2R)-N1-alkyl-1,2-
diaminocyclohexane). cis-[Pt(Am)2Cl2] was then treated with two
equiv AgNO3 in distilled water (80 mL) at 40 °C for 18 h to form
an pellucid solution containing the aqua-diamine-dinitratoplati-
num intermediate by filtering off AgCl deposits. The solution was
then mixed with equimolar disodium D-(+)-camphorate at 50 °C
for 12 h stirring and the solution was concentrated to 20 mL by ro-
tary evaporation, and kept at 4 °C for several hours. The resulting
pale yellow solid was filtered off and white solid products were ob-
tained by recrystallization from water with active charcoal. Then
they were dried in vacuo to obtain the final product.cis-[(1R,2R)-
N1-butyl-1,2-diaminocyclohexane-N,N0][(+)-camphorato-O,O’]plat-
inum(II) (2). White solid. Yield: 0.22 g (39%). 1H NMR (d6-DMSO/
TMS): d 0.85 (t, 3H, CH2CH3), 0.98 (s, 6H, C(CH3)2), 1.20 (s, 3H,
CCH3), 1.10–1.89 (m, 16H, NHCH2CH2CH2CH3 and CH2 of DACH
and camphorato), 2.25 (t, 1H, COCH), 2.43–2.88 (m, 4H, NHCH2
and NHCH and NH2CH), 4.80–6.80 ppm (m, 3H, CHNH and CHNH2);
IR (KBr): 3429 (br), 3187, 3117, 2957, 1603, 1457, 1382, 1348, 1169,
1125, 909, 801 cmꢀ1; ESI-MS: m/z (%): 563 (87), 564 (100), 565 (90)
[M+H]+; HRMS: m/z [M+H]+ calcd. for C20H37N2O4Pt: 564.24011,
found: 564.24202. Anal. Calc. for C20H36N2O4Pt: C, 42.62; H, 6.44;
N, 4.97; Pt, 34.61. Found: C, 42.70; H, 6.40; N, 4.99; Pt, 34.35.cis-
[(1R,2R)-N1-isopropyl-1,2-diaminocyclohexane-N,N’][(+)-camph-
orato-O,O’]platinum(II) (3). White solid. Yield: 0.24 g (43%). 1H NMR
(d6-DMSO/TMS): d 1.02 (s, 6H, C(CH3)2), 1.22 (s, 3H, CCH3), 1.09–
1.85 (m, 18H, NHCH(CH3)2 and CH2 of DACH and camphorato),
2.3. Cell culture
The in vitro antitumor activity of resulting complexes were
tested on A549 (human non-small cell lung cancer), MCF-7 (human
breast carcinoma), HepG2 (hepatocellular carcinoma), and
HCT-116 (human colorectal cancer) cell lines, respectively. They
were all cultured in RPMI-1640 medium supplemented with 10%
fetal bovine serum (FBS), 100
l
g mLꢀ1 of streptomycin, and
100
l
g mLꢀ1 of penicillin in an atmosphere of 5% CO2 at 37 °C.
2.4. Cytotoxicity assay (IC50 values)
The in vitro cytotoxicity of all complexes (IC50 values) were
determined by MTT assay (MTT = 3-(4, 5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide). The suspension of 1000 cells
wellꢀ1 was plated in 96-well culture plates with culture medium
and was incubated for 24 h at 37 °C in a 5% CO2 incubator. The
complexes were dissolved in ultrapure water firstly and diluted
to the required concentration with culture medium. Then the di-
luted solution of complexes was added to the wells, and the cells
were incubated at 37 °C in a 5% CO2 incubator for 48 h. After that,
the cells were treated with 10
4 h cultivation. The media with MTT solution were removed with
100 L of DMSO solution. The absorbance of formazane solution
l
L MTT dye solution (5 mg mLꢀ1) for
l
was measured at 540 nm with an automatic microplate ELISA
reader. The IC50 value was determined from the chart of cell viabil-
ity (%) against dose of complex added (lM).

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Y. Sun et al. / Inorganica Chimica Acta 395 (2013) 154–159
2.5. Cell cycle analysis
The alkyl/cycloalkyl substituent at N1 position of target com-
plexes with an optically active ligand D-(+)-camphorate may pro-
duce diastereoisomeric mixtures for complexes 2–6 as shown in
Scheme 1. In order to clarify the structures of target complexes,
we have made theoretic calculations for complex 2–6 to investi-
gate which diastereoisomer is energically preferred by using
HyperChem 7.5 [25]. After building the molecule and constraining
bond angle for PtN2O2 plane, we chose AMBER as the molecular
mechanics force field which assigns atom types and parameters
to the molecule. Then amber2 was selected as the parameter set
for the AMBER force field. At last, Polak–Ribiere was applied as
the minimization algorithm to optimize the structure of the mole-
cule and calculate the minimal energy of the molecule, as for com-
plex 3, which are illustrated in Fig. 1.
It can be observed from Fig. 1 that the configuration B
(E = 87.907 kcal/mol) is more thermodynamically stable than con-
figuration A (E = 149.228 kcal/mol). Analogous calculation results
were also obtained for other complexes (data not shown). So it is
proposed that complexes 2–6 prefer to adopt configuration B
shown in Fig. 1 and Scheme 1 (red one).
A Cell Cycle and Apoptosis Analysis Kit (Beyotime Institute of
Biotechnology, China) was used in the cell cycle progression study.
Firstly, 1 ꢁ 106 HCT-116 cells were cultured in a 6-well plate and
incubated at 37 °C in 5% CO2 overnight. Then cells were treated
with the selected complexes for 12 h at the concentration of
30 lM. Successively, cells were harvested and washed twice with
PBS solution. After that, cells were fixed in cold 70% ethanol and
stored at 4 °C. On the day of analysis, ethanol was removed by cen-
trifugation and cells were washed twice with PBS, and then treated
with mixed solution of RNase and propidium iodide (PI) for 30 min
at 37 °C. The samples were processed by a flow cytometer (FAC
Scan, Becton Dickenson, USA). The results were analyzed by using
FCSEXPRESS software.
2.6. Gel electrophoresis experiment
DNA cleavage induced by complexes 1, 3, 6, and cisplatin was
investigated by agarose gel electrophoresis. pET22b plasmid DNA
Besides, considering that there is an 8-membered ring in all
compounds due to Pt coordinating with D-(+)-camphorate, we
tested the stability of all compounds in aqueous solution by UV–
Vis spectral analysis at the concentration of 0.5 mg/mL. The UV
absorption spectra in the range of 200–600 nm for compound 3
was shown in Fig. 2 and no obvious changes of absorption peak
and UV absorbance were observed within 12 h, suggesting that
compound 3 is relatively stable in aqueous solution. The similar re-
sults were obtained for other compounds in the same experimental
condition (data not shown).
(50 ng l
Lꢀ1) was used as the target in this experiment. Based on
the previous reports by Lippard and co-workers [23,24], we made
some modifications of experiment conditions in consideration of
the specificity of our compounds. Appropriate dilutions of com-
pounds were made before dissolving them in ultrapure water,
and different volumes of solutions were added to the tubes to
achieve a set of concentrations in the range of 1–100
lM. pET22b
DNA (5 L) was added to each tube, and then the mixtures of
l
tested complexes and pET22b plasmid DNA were incubated at
37 °C for 24 h. After that, the agarose gel (1% w/v) containing ethi-
dium bromide (0.01%) was prepared using TA buffer (50 mM Tris–
acetate, pH 7.5). The mixtures with loading buffer (1 lL) under-
3.2. In vitro cytotoxicity
went electrophoresis in agarose gel in TA buffer (50 mM Tris–ace-
tate, pH 7.5) at 90 V for 60 min. The bands were photographed
using a Gel Image System (Tanon 2500, China).
The in vitro cytotoxicities of complexes 2–6 were tested by MTT
assay against four human tumor cell lines including HepG2 (hu-
man hepatocellular carcinoma), MCF-7 (human breast carcinoma),
A549 (human non-small-cell lung cancer), and HCT-116 (human
colorectal cancer) after 48 h incubation, which were compared
with those of cisplatin and oxaliplatin (Table 1). The cytotoxicity
of parent compound 1 on HL-60 (leukemia), 3AO (ovarian), BEL-
7402 (hepatocarcinoma), and A549 cell lines has been tested in
our previous report [17], suggesting that the cytotoxicity of com-
plex 1 was very close to that of cisplatin against HL-60 and slightly
better than cisplatin against 3AO, however, it was far less than cis-
platin on A549 and BEL-7402 cell lines. Table 1 showed that the
complexes with D-(+)-camphorate anion, which were modified in
the substituent of nitrogen atom, generally displayed good cyto-
toxicity against selected cell lines. Based on the IC50 values, it
was noted that the compounds with a chain alkyl substituent at
one of nitrogen atoms of carrier ligands (2–4) were more potent
in vitro than those with a cycloalkyl substituent (5–6). Generally,
the alkyl substituent at the nitrogen atom increased the cytotoxic-
ity of complexes on non-small-cell lung cancer cell line (A549) and
hepatocarcinoma cell line relative to that of parent compound 1.
As for MCF-7 cell line, compounds 3 (R = isopropyl, IC50 = 6.0 -
3. Results and discussion
3.1. Synthesis and characterization
The synthesis of complexes 2–6 is shown in Scheme 1. The li-
gands (1R,2R)-N1-alkyl-1,2-diaminocyclohexane were prepared in
the previous studies [18–20]. The resulting complexes were char-
acterized by 1H NMR, MS, and IR spectra together with elemental
analysis. For IR spectra in the range of 3109–3194 cmꢀ1, the N–H
stretching vibrations of complexes 2–6 are red shifted compared
to the single amino group of the corresponding ligands due to ami-
no group coordination with Pt(II) ion. Also it is notable that the
asymmetric stretching vibrations of (C–O) signals appear between
1596 and 1608 cmꢀ1, characteristic of coordinated dicarboxylate
ligands, whereas the symmetric stretching vibrations of (C–O)
show in the range of 1382–1384 cmꢀ1 [21]. The
D
m
[m
as(C–O) ꢀ ms
(C–O)] values of these complexes are in the range of 212–
225 cmꢀ1, greater than 200 cmꢀ1, in line with the structures of
the resulting complexes with bidentate-coordinated carboxylate li-
gands [22]. 1H NMR spectroscopy of the platinum(II) complexes
was performed with d6-DMSO as the solvent, and the expected sig-
nals were accordingly observed. The broad signals of hydrogen
atoms belonging to amino groups appear in the range of d 4.65–
6.80 ppm due to amine coordination with platinum(II), shifting
high-field relative to the metal-free ligands in the range of d
8.85–9.42 ppm. Besides, the structures of all the complexes were
further determined by HRMS spectra and elemental analysis for
C, H, N, and Pt.
lM) and 4 (R = 2-methylpropyl, IC50 = 8.0 lM) showed comparable
antitumor effect to those of cisplatin and oxaliplatin, especially
compound 3, 2.3-fold less potent than oxaliplatin. With respect
to A549 cell line, the most active agent was compound 4, which re-
vealed similar cytotoxicity with positive control, about 2.0-fold
less potent than cisplatin and oxaliplatin. Besides, compound 3
had desirable antitumor effect in both HCT-116 and HepG2 cell
lines, and the IC50 values were 5.1-fold less than that of oxaliplatin
for HCT-116 and 7.7-fold less than that of cisplatin in HepG2. Gen-
erally, compounds 3 and 4 with branched alkyl substituent at one

Y. Sun et al. / Inorganica Chimica Acta 395 (2013) 154–159
157
A
2
Energy = 149.228 kcal/mol
B
2
Energy = 87.907 kcal/mol
Fig. 1. Theoretically optimized structure of complex 3 (A isomer: E = 149.228 kcal/mol; B isomer: E = 87.907 kcal/mol).
Table 1
4
3.5
3
In vitro cytotoxicity of complexes 1–6 against selected human tumor cell lines after
48 h incubation.
0 h
2 h
4 h
6 h
8 h
12 h
Compd.
R
IC50
A549b
19.7 1.8f n.d.g
(l
M)a
HCT-116c HepG2d
n.d.
MCF-7e
n.d.
1
2
3
4
5
6
H
Butyl
Isopropyl
2-Methylpropyl
Cyclopentyl
Cyclohexyl
32.0 2.2 41.8 3.6 36.7 1.9 31.3 2.0
2.5
2
12.5 1.4 11.1 0.7
7.8 0.9 15.7 1.8 13.5 1.5
>100 >100 >100
9.2 1.0
6.0 0.5
8.0 1.1
41.0 3.5
40.0 3.0 33.9 4.8 47.7 3.8 29.3 3.8
Cisplatin
Oxaliplatin
3.6 0.2
3.9 0.4
1.7 0.1
2.2 0.2
1.2 0.1
1.4 0.1
1.2 0.1
2.6 0.3
1.5
1
a
IC50 value represents the drug concentration suppressing 50% tumor cell pro-
liferation; values are mean SD from three independent experiments.
b
Human non-small-cell lung cancer cell line.
Human colorectal cancer cell line.
Human hepatocellular carcinoma cell line.
Human breast cancer cell line.
Data from L. Wang, S. Gou et al. [17].
n.d. = Not determined.
c
0.5
d
e
0
205
f
255
305
355
405
455
505
555
g
wavelength (nm)
Fig. 2. UV absorption spectra of complex 3 in aqueous solution within 12 h.
It was observed that HCT-116 tumor cells exposed to cisplatin
and oxaliplatin for 12 h were blocked in the S (44.23%) and G2/M
(37.57%) phase, respectively. It was also noted that compounds 3
and 4 produced cell accumulation in the S phase, with the popula-
tion of 38.31% and 38.39%, respectively, after 12 h exposure at a
concentration equal to that of cisplatin. Based on the investigation
we can conclude that compounds 3 and 4 inhibit tumor cell prolif-
eration mainly by inhibiting DNA synthesis similar to cisplatin, dif-
ferent from oxaliplatin.
of nitrogen atoms of carrier ligands were the most effective agents
on the selected tumor cell lines.
3.3. Cell cycle block analysis
We analyzed the DNA content of cells stained with propidium
iodide by flow cytometry to examine the effect of complexes on
HCT-116 cell cycle progression. The DNA histograms were shown
in Fig. 3, which were obtained after cells were treated with the se-
lected compounds 3 and 4 for 12 h incubation at the concentration
3.4. DNA cleavage pattern
In the study on the interaction with DNA, we introduced
agarose gel electrophoresis and used pET22b plasmid DNA as the
of 30 lM.

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Y. Sun et al. / Inorganica Chimica Acta 395 (2013) 154–159
Oxaliplatin
G0/G1
Cisplatin
G0/G1: 44.45 %
S: 17.99 %
G2/M: 37.57 %
G0/G1: 38.29 %
S:
44.23 %
G2/M: 17.48 %
G2/M
Complex 3
Complex 4
G0/G1: 33.70 %
G0/G1: 29.83 %
S:
38.31 %
S:
38.39 %
G2/M: 27.99 %
G2/M: 31.78 %
Control
G0/G1: 43.45 %
S:
26.49 %
G2/M: 30.06 %
Fig. 3. Cell cycle distribution of HCT-116 cells by FACS analysis exposed for 12 h to complexes 3 and 4, cisplatin, and oxaliplatin.
target. In order to clarify the different DNA cleavage pattern of
compounds with different substituents at one of nitrogen atoms,
complexes 1, 3, and 6 were chosen to test their ability to distort
plasmid DNA.
In Fig. 4a, as for cisplatin (lanes 2–5), the electrophoretic mobil-
ity of supercoiled DNA (form I) and open circular DNA (form II) in-
creased with the concentration of cisplatin and a little increased
density of form II was observed at high concentration (100 lM),

Y. Sun et al. / Inorganica Chimica Acta 395 (2013) 154–159
159
derivatives as carrier ligands and D-(+)-camphorate anion as the
leaving group. The in vitro cytotoxicity results showed that most
of complexes had good antitumor activity against selected cell
lines. Compounds 3 and 4, with a branched alkyl substituent at
one of nitrogen atoms, displayed not only better antitumor activity
but also broader spectrum. Besides, the branched alkyl substituent
at the nitrogen atom increased the cytotoxicity of complexes on
non-small-cell lung cancer cell line (A549) and hepatocarcinoma
cell line in comparison to that of parent compound 1. Flow cytom-
etry demonstrated that DNA synthesis phase in the cell cycle was
one of the targets for typical complexes 3, 4, and cisplatin. Besides,
representative complexes (1, 3, and 6) and positive drug in this
study possessed DNA cleaving ability in the agarose gel experi-
ment, especially complex 6, with a cycloalkyl substituent, demon-
strating a different effect (mainly degrading effect) on DNA relative
to its analogous complexes (1 and 3), but similar to cisplatin. As
such, we can conclude that alkyl and cycloalkyl substituents at
N1 position have different influences in DNA-drug interaction pat-
tern. However, the specific mechanism needs to be further
clarified.
Fig. 4. Gel electrophoretic mobility pattern of pET22b plasmid DNA incubated at
37 °C with various concentrations of tested compounds for 24 h. (a): untreated
plasmid DNA (lanes 1 and 10), concentrations of 1, 10, 50, and 100
(lanes 2–5), concentrations of 1, 10, 50, and 100 M of complex 1 (lanes 6–9); (b):
untreated plasmid DNA (lanes 1 and 14), concentrations of 1, 10, 50, and 100 M of
cisplatin (lanes 2–5), concentrations of 1, 10, 50, and 100 M of complex 3 (lanes 6–
9), concentrations of 1, 10, 50, and 100 M of complex 6 (lanes 10–13). Form I and
lM of cisplatin
l
Acknowledgements
l
l
l
The work is supported by the National Natural Science Founda-
tion of China (Project No. 21271041) and the New Drug Creation
Project of the National Science and Technology Major Foundation
of China (Project 2013ZX09402102-001-006) for financial aids to
this work. Sun would like to thank the Scientific Research Innova-
tion Project for College Graduates of Jiangsu Province (Project
CX10B_084Z) and Scientific Research Foundation of Graduate
School of Southeast University (Project YBJJ1106).
Form II indicate supercoil and unwinding (open circle) forms of pET22b,
respectively.
suggesting that cisplatin produced open circle DNA partly, and
supercoiled DNA was cleaved to produce short DNA strand. More-
over, it is noted that the total amount of forms I and II had a signif-
icant decrease in the range of 50–100
lM, indicating that plasmid
DNA was partly degraded by cisplatin.
With respect to complex 1 shown in Fig. 4a (lanes 6–9), the
electrophoretic mobility of form I and form II increased slightly
with the drug concentration and the density of open circle DNA
(form II) had a significant increase at high concentrations (50–
100 lM). Then it can be found that complex 1 mainly produced
open circle DNA and there was no obvious degradation of DNA,
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4. Conclusion
In conclusion, we designed and synthesized five novel plati-
num(II) complexes with N-monoalkyl 1R,2R-diaminocyclohexane