Synthesis and biological evaluation of novel platinum complexes of imidazolyl-containing bisphosphonates as potential anticancer agents

Article abstract of DOI:10.1007/s00775-015-1305-z

Four novel platinum complexes, [Pt(en)]₂ZL (1), [Pt(en)]₂IPrBP (2), [Pt(en)]₂MIBP (3) and [Pt(en)]₂EIBP (4) [en = ethylenediamine; ZL = 1-hydroxy-3-(1H-imidazol-1-yl)ethane-1,1-diylbisphosphonic acid, commonly known as zoledronic acid; IPrBP = 1-hydroxy-3-(1H-imidazol-1-yl)propane-1,1-diylbisphosphonic acid; MIBP = 1-hydroxy-2-(2-methyl-1H-imidazol-1-yl)ethane-1,1-diylbisphosphonic acid; EIBP = 1-hydroxy-2-(2-ethyl-1H-imidazol-1-yl)ethane-1,1-diylbisphosphonic acid], were prepared and evaluated against five human cancer cell lines, including U2OS, A549, HCT116, MDA-MB-231 and HepG2. While exhibiting lower efficacy on the inhibition of cancer cell lines than cisplatin (CDDP), four complexes showed higher cytotoxicity than the corresponding ligands and relatively stronger cytotoxic effect on the hepatoma cell lines HepG2, and the complex 1 showed higher cytotoxicity than others on the whole. These complexes have better selectivity than the corresponding ligands in inhibiting hepatocarcinoma cells rather than normal liver cells, and the selective inhibitory effect of the complex 1 at the high concentration (100 μM) is better than that at the low concentration. Morphology studies exhibited typical characteristics of cell apoptosis and the cell cycle distribution analysis indicated that the complexes can inhibit cancer cells by inducing the cell cycle arrest at the G2/M phase, exhibiting a similar mechanism of action to CDDP. The binding interaction of complex with DNA has been explored by circular dichroism (CD) and UV-Vis absorption spectra, demonstrating these new complexes have moderate binding affinity for DNA. Graphical Abstract: Four platinum complexes based on imidazolyl-containing bisphosphonates with effective antitumor activity and low hepatotoxicity were designed and evaluated.[Figure not available: see fulltext.]

Full text of DOI:10.1007/s00775-015-1305-z

J Biol Inorg Chem
DOI 10.1007/s00775-015-1305-z
ORIGINAL PAPER
Synthesis and biological evaluation of novel platinum complexes
of imidazolyl‑containing bisphosphonates as potential anticancer
agents
Ling Qiu1 · Gaochao Lv1 · Yang Cao1 · Liping Chen1 · Hui Yang1 · Shineng Luo1 ·
Meifen Zou1 · Jianguo Lin1
Received: 7 July 2015 / Accepted: 17 October 2015
© SBIC 2015
Abstract Four novel platinum complexes, [Pt(en)]2ZL
(1), [Pt(en)]2IPrBP (2), [Pt(en)]2MIBP (3) and
[Pt(en)]2EIBP (4) [en = ethylenediamine; ZL = 1-hydroxy-
characteristics of cell apoptosis and the cell cycle distribu-
tion analysis indicated that the complexes can inhibit can-
cer cells by inducing the cell cycle arrest at the G2/M
phase, exhibiting a similar mechanism of action to CDDP.
The binding interaction of complex with DNA has been
explored by circular dichroism (CD) and UV–Vis absorp-
tion spectra, demonstrating these new complexes have
moderate binding affinity for DNA.
Graphical Abstract Four platinum complexes based on
imidazolyl-containing bisphosphonates with effective anti-
tumor activity and low hepatotoxicity were designed and
evaluated.
3-(1H-imidazol-1-yl)ethane-1,1-diylbisphosphonic
acid,
commonly known as zoledronic acid; IPrBP = 1-hydroxy-
3-(1H-imidazol-1-yl)propane-1,1-diylbisphosphonic acid;
MIBP = 1-hydroxy-2-(2-methyl-1H-imidazol-1-yl)ethane-
1,1-diylbisphosphonic acid; EIBP = 1-hydroxy-2-(2-ethyl-
1H-imidazol-1-yl)ethane-1,1-diylbisphosphonic
acid],
were prepared and evaluated against five human cancer cell
lines, including U2OS, A549, HCT116, MDA-MB-231 and
HepG2. While exhibiting lower efficacy on the inhibition
of cancer cell lines than cisplatin (CDDP), four complexes
showed higher cytotoxicity than the corresponding ligands
and relatively stronger cytotoxic effect on the hepatoma
cell lines HepG2, and the complex 1 showed higher cyto-
toxicity than others on the whole. These complexes have
better selectivity than the corresponding ligands in inhib-
iting hepatocarcinoma cells rather than normal liver cells,
and the selective inhibitory effect of the complex 1 at the
high concentration (100 μM) is better than that at the
low concentration. Morphology studies exhibited typical
O
O
O
O
P
n
R
NH2
NH2
NH2
NH2
OH
N
Pt
N
Pt
P
O
O
1: n=1, R=H;
2: n=2, R=H;
3: n=1, R=CH3;
4: n=1, R=CH2CH3
100
80
60
40
20
0
Complex 1
Complex 2
Complex 3
Complex 4
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-015-1305-z) contains supplementary
material, which is available to authorized users.
0
12
24
36
48
60
72
84
Time (h)
* Shineng Luo
luoshineng@jsinm.org
* Jianguo Lin
Keywords Platinum complex · Imidazolyl-containing
bisphosphonate · Hydroxyapatite (HA) affinity ·
Cytotoxicity · DNA binding
linjianguo@jsinm.org
1
Key Laboratory of Nuclear Medicine, Ministry of Health,
Jiangsu Key Laboratory of Molecular Nuclear Medicine,
Jiangsu Institute of Nuclear Medicine, Wuxi 214063,
People’s Republic of China
1 3

J Biol Inorg Chem
the BPs and has been widely used in the clinical treatment
of several bone diseases [21, 22]. This is mainly due to the
fact that the five-membered aromatic imidazole ring has
the best biological performance among the N-heterocyclic
compounds. Encouraged by the powerful potency of ZL
and positive results of other studies on platinum complexes
of bisphosphonates [23–27], in the present work four
novel platinum complexes were designed and synthesized
based on ZL and its analogs (Scheme 1b), [Pt(en)]2ZL (1),
[Pt(en)]2IPrBP (2), [Pt(en)]2MIBP (3) and [Pt(en)]2EIBP
(4) [en = ethylenediamine; ZL = 1-hydroxy-3-(1H-
imidazol-1-yl)ethane-1,1-diylbisphosphonic acid, com-
monly known as zoledronic acid; IPrBP = 1-hydroxy-
3-(1H-imidazol-1-yl)propane-1,1-diylbisphosphonic
acid; MIBP = 1-hydroxy-2-(2-methyl-1H-imidazol-1-yl)
ethane-1,1-diylbisphosphonic acid; EIBP = 1-hydroxy-
2-(2-ethyl-1H-imidazol-1-yl)ethane-1,1-diylbisphosphonic
acid], in the hope of finding new platinum-based drugs with
improved anticancer activity and less side effects. Their
bone-targeting ability was initially evaluated via the in vitro
hydroxyapatite (HA)-binding assay. Their cytotoxicity and
specificity were investigated in several human cancer cell
lines and normal cell lines, and the mechanism of action
was also studied systematically by means of nuclear stain-
ing, flow cytometry, circular dichroism (CD) and UV–Vis
absorption spectral techniques.
Introduction
Platinum complexes, such as cisplatin, carboplatin and
oxaliplatin, have received remarkable attention over the
past 40 years as widely used antineoplastic agents [1–3].
However, their anticancer efficacy is often limited by vari-
ous side effects, such as nephrotoxicity, hepatotoxicity,
ototoxicity, neurotoxicity and gastrointestinal reaction, due
to the lack of specificity [2–4]. In addition, the intrinsic or
acquired drug resistance also restricts the clinical applica-
tions of these drugs [5]. Therefore, it is essential to look for
novel platinum complexes with better specificity and less
toxicity. Up to now, enormous effort has been dedicated to
synthesizing thousands of new platinum complexes [6–9].
Also, noteworthy is that a number of drug targeting and
drug delivery strategies have been exploited to improve the
efficacy and reduce the shortcomings of platinum-based
drugs using a range of delivery vehicles, such as polymer
scaffolds, hormones, carbohydrates, peptides, proteins,
bisphosphonates, liposomes, micelles, and nanoparticles
[10–14].
Geminal bisphosphonates (BPs, Scheme 1a) are known
to possess strong affinity for bone mineral and other calci-
fied tissues [15]. Therefore, they have been applied widely
as therapeutic agents for several bone-related diseases, such
as osteoporosis, Paget’s disease, tumor-induced hypercal-
cemia and bone metastases [16, 17]. In addition to being
prescribed as drugs, BPs have also been studied as drug
targeting and delivery vehicles for therapeutic and diagnos-
tic applications [18–20]. Compared with other molecules,
BPs have more advantages as vehicles for targeted deliv-
ery of therapeutic agents to bone because their binding
affinities and biological activities can be tuned by changing
their substituents (R1 and R2) [21]. Therefore, combining
the bifunctional bisphosphonate and the platinum moiety
could potentially promote the specific targeting of the drug
in bone-related tumors or metastases, which will signifi-
cantly improve chemotherapy efficacy and reduce systemic
toxicity.
Materials and methods
General information
All chemicals were purchased as reagent grade and used
without further purification. The bisphosphonate ligands
ZL, IPrBP, MIBP and EIBP were prepared according to the
method as described in our previous work [28]. CDDP was
purchased from Shandong BoYuan pharmaceutical Co.,
Ltd. The human cancer cell lines U2OS (osteosarcoma),
A549 (lung cancer), HCT116 (colon carcinoma), MDA-
MB-231 (breast cancer) and HepG2 (liver carcinoma) as
well as the human normal liver cell lines L02 were obtained
from the Cell Bank of Chinese Academy of Sciences,
Zoledronic acid (ZL), one of the typical third-generation
N-heterocyclic bisphosphonates, is the most potent among
Scheme 1 Chemical structures
of generic BPs (a) and ZL
derivatives (b)
O
O
OH
OH
P
HO
P
R
OH
OH
R1
R2
OH
OH
N
(CH )
2 n
n=1, R=H, ZL;
N
P
P
n=2, R=H, IPrBP;
n=1, R=CH3, MIBP;
n=1, R=CH2CH3, EIBP
OH
OH
O
(b)
O
(a)
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J Biol Inorg Chem
Scheme 2 Syntheses of
complexes 1–4
Ag2SO4
Ba(OH)2/L
en
KI
K2PtCl4
K2PtI4
[Pt(en)I2]
[Pt(en)(OSO3)(H2O)]
O
P
O
R
O
OH
O
NH2
en: H2N
NH2
NH2
NH2
1: n=1, R=H;
2: n=2, R=H;
N
N
Pt
Pt
n
NH2
3: n=1, R=CH3;
P
O
4: n=1, R=CH2CH3
O
Shanghai, China. The reagent 3-[4,5-dimethyl-2-thiazolyl]-
2,5-diphenyl-2- tetrazolium bromide (MTT) used for cell
lysis was purchased from Sigma (St. Louis, MO, USA).
Dulbecco’s modified eagle medium (DMEM) and Roswell
Park Memorial Institute (RPMI-1640) medium were pur-
chased from Gibco Company (USA). Dimethyl sulfoxide
(DMSO), propidium iodide (PI), RNase inhibitor and Hoe-
chst 33342 were purchased from Beyotime Institute of Bio-
technology. Cell culture plates were products of Corning.
Calf thymus DNA (CT-DNA) was purchased from Sigma-
Aldrich and used as received.
Electrospray ionization–mass spectrometry (ESI–MS)
was determined using a Waters Platform ZMD4000 liquid
chromatography–mass spectrometry (LC–MS) (Waters,
USA). FT-IR spectra (4000–400 cm−1) were recorded on a
Nicolet Nexus470 IR Fourier transform spectrophotometer
using KBr pellets. Elemental analysis was carried out with
a Vario EL III Elementar analyzer. Nuclear magnetic reso-
nance (NMR) spectra were obtained on a Bruker DRX-500
spectrometer (Bruker, Germany), and the chemical shift
values were referenced to the internal tetramethylsilane
(TMS). The circular dichroism (CD) spectra were recorded
on a MOS 450 spectropolarimeter (BioLogic, France),
and the UV–Vis absorption spectra were recorded on a
UV-3600 spectrophotometer (Shimadzu, Japan).
filtration, the light green filtrate was obtained. The evapora-
tion of the filtrate under reduced pressure at 45 °C afforded
the target product [Pt(en)(H2O)(OSO3)]. Since platinum
drugs are sensitive to the light, all the experiments were
performed in the darkness.
Then, the BP ligand (0.1 mmol) [ZL 27.2 mg, IPrBP
28.6 mg, MIBP 28.6 mg, EIBP 30.0 mg] was dissolved
in H2O (2 mL) and the resulting solution was treated with
Ba(OH)2·8H2O (31.5 mg, 0.1 mmol), which was kept stir-
ring at 0 °C for 10 min. A solution of [Pt(en)(H2O)(OSO3)]
(73.8 mg, 0.2 mmol in 2 mL of H2O) was then added to the
obtained white suspension. After stirring for 6 h at 0 °C, the
suspension was treated with additional Ba(OH)2·8H2O until
the pH of reaction system reached a value of 5.0. Then, the
resulting mixture was left stirring at 4 °C overnight. After-
wards, the white precipitation (BaSO4) was removed by
centrifuge (2000 r/min, 10 min) and filtered by micropo-
rous membrane filter. The resulting filtrate was taken to
dry by lyophilization and white powders were isolated.
Unfortunately, suitable crystals of the four complexes for
single-crystal X-ray diffraction were not obtained in spite
of great efforts over many attempts. The structures of com-
plexes 1–4 have been characterized by elemental analysis,
1
IR, ESI–MS and H/13C/31P NMR spectra (Figs. S1–S4 in
the electronic supplementary material).
Complex 1: yield: 84 %, 65.3 mg. Anal. Calc. for
[Pt(en)]2ZL (C9H22N6O7P2Pt2) (%): C, 13.89; H, 2.85; N,
10.80; Found (%): C, 13.92; H, 2.91; N, 10.72. H NMR
Synthesis of platinum complexes
1
Four dinuclear platinum(II) complexes 1–4 were prepared
according to the following general procedure (Scheme 2).
At first, the important intermediate [Pt(en)(H2O)(OSO3)]
was prepared according to the reported method [29].
K2PtCl4 (0.415 g, 1 mmol) was dissolved in H2O and then
KI (1.4 g, 8 mmol) was added to the solution afforded a
deep rufous liquor, which was kept stirring for 15 min.
After that, ethylenediamine (0.057 g, 0.95 mmol) diluted
with water was added. The resulting yellow suspension was
kept under stirring for 6 h in the darkness. After filtration,
the yellow solid precipitation Pt(en)I2 was obtained. Then,
[Pt(en)I2] (0.509 g, 1 mmol) was suspended in 5 ml H2O
and Ag2SO4 (0.3058 g, 0.98 mmol) was added to the sus-
pension. The suspension was kept stirring overnight. After
(400 MHz, D2O, δ ppm): 8.48 (s, 1H, CHim), 7.28 (s, 1H,
CHim), 7.18 (s, 1H, CHim), 5.78 (d, 4H, J = 30.0 Hz, NHen),
5.23 (s, 4H, NHen), 4.44–4.39 (m, 2H, CH2), 2.50 (d, 8H,
J = 22.4 Hz, CHen). 13C NMR (100 MHz, D2O, δ ppm):
135.75, 123.93, 118.02 (Cim), 77.13 (Cbp), 49.44, 49.38
(CHen), 48.79 (CH2). 31P NMR (160 MHz, D2O, δ ppm):
34.74. ESI–MS (m/z): 776.8 (100) [M]−. Mp: 264–266 °C.
IR (KBr, cm−1): 3400 (w), 3280 (m), 1629 (m), 1566 (s),
1321 (s), 1288 (m), 1128 (s), 1052 (s), 994 (w), 570 (w),
488 (w).
Complex 2: yield: 53 %, 42.3 mg. Anal. Calc. for
[Pt(en)]2IPrBP (C10H24N6O7P2Pt2) (%): C, 15.16; H, 3.05;
N, 10.61; Found (%): C, 15.28; H, 3.17; N, 10.54. 1H NMR
(400 MHz, D2O, δ ppm): 8.72 (s, 1H, CHim), 7.52 (s, 1H,
1 3

J Biol Inorg Chem
CHim), 7.39 (s, 1H, CHim), 5.83 (s, 4H, NHen), 5.32 (s, 4H,
NHen), 4.51–4.47 (m, 2H, CH2), 2.61 (d, 8H, J = 15.6 Hz,
CHen), 2.37–2.30 (m, 2H, CH2). 13C NMR (100 MHz, D2O,
δ ppm): 134.56, 121.82, 119.53 (Cim), 76.87 (Cbp), 49.43,
49.39 (CHen), 45.49, 33.82(CH2). 31P NMR (160 MHz,
D2O, δ ppm): 34.57. ESI–MS (m/z): 793.0 [M]+. Mp: 265–
266 °C. IR (KBr, cm−1): 3455 (w), 3280 (s), 3255 (s), 3206
(s), 1630 (w), 1567 (s), 1289 (m), 1129 (s), 1052 (s), 572
(w), 451 (w).
The adsorption experiments were performed at 37 °C
in an aqueous medium and incubated for different time to
study the time effect on the bone-targeting ability of plati-
num complexes. The adsorbed amount of each complex
was determined by the difference between the initial com-
pound quantity and that contained in the supernatant solu-
tion. Then, the adsorption profile was obtained as a func-
tion of the incubation time. In detail, the platinum complex
solution (2 mL, 1.00 μM) and HA (10 mg) were mixed in a
penicillin bottle (10 mL). After 15 s of treatment in a vortex
apparatus, the suspension was maintained in a bascule bath
at 37 °C. After different time of incubation, the mixture
was centrifuged at 5000 rpm for 5 min to ensure complete
separation of the supernatant from the solid phase. Then,
the supernatant was filtered through a microporus filter
(0.22 μm pore size) and an aliquot (10 μL) of the super-
natant was removed for the quantification of the platinum
complex remaining in the solution, using the HPLC analy-
sis (PBS:acetonitrile = 8:2). Based on the standard curve,
the amount of platinum complex adsorbed onto HA can be
determined by the difference between the initial amount
and that remained in the suspension, which can be deduced
from the integral area. The adsorption percentage can also
be determined from the amount of the complex adsorbed
onto HA with respect to the initial complex in the solution.
Complex 3: yield: 75 %, 59.4 mg. Anal. Calc. for
[Pt(en)]2MIBP (C10H24N6O7P2Pt2) (%): C, 15.16; H, 3.05;
1
N, 10.61; Found (%): C, 14.64; H, 3.25; N, 10.91. H
NMR (400 MHz, D2O, δ ppm): 7.34 (s, 1H, CHim), 7.17 (s,
1H, CHim), 5.93 (d, 4H, J = 34.8 Hz, NHen), 5.37 (s, 4H,
NHen), 4.45–4.35 (m, 2H, CH2), 2.67 (s, 3H, CH3), 2.59
(m, 8H, CHen).13C NMR (100 MHz, D2O, δ ppm): 145.36,
123.73, 116.21 (Cim), 77.52 (Cbp), 49.46, 49.39 (CHen),
49.33 (CH2), 10.55 (CH3). 31P NMR (160 MHz, D2O, δ
ppm): 34.78. ESI–MS (m/z): 793.4 [M]+. Mp: 264–266 °C.
IR (KBr, cm−1): 3449 (w), 3200(m), 1640 (m), 1529 (w),
1452 (w), 1122 (s), 993 (m), 569 (w).
Complex 4: yield: 86 %, 69.4 mg. Anal. Calc. for
[Pt(en)]2EIBP (C11H26N6O7P2Pt2) (%): C, 16.38; H, 3.25;
N, 10.42; Found (%): C, 16.21; H, 3.44; N, 10.65. 1H NMR
(400 MHz, D2O, δ ppm): 7.36 (s, 1H, CHim), 7.18 (s, 1H,
CHim), 5.89 (d, 4H, J = 34.8 Hz, NHen), 5.35 (s, 4H, NHen),
4.47–4.42 (m, 2H, CH2), 3.14–3.06 (m, 2H, CH2CH3), 2.59
(m, 8H, CHen), 1.34 (s, 3H, CH2CH3). 13C NMR (100 MHz,
D2O, δ ppm): 152.34, 126.50, 119.05 (Cim), 80.27 (Cbp),
52.15, 52.08 (CHen), 51.85 (CH2), 21.03, 12.51 (CH2CH3).
31P NMR (160 MHz, D2O, δ ppm): 34.75. ESI–MS (m/z):
807.0 [M]+. Mp: 265–267 °C. IR (KBr, cm−1): 3422 (w),
3197 (m), 1525 (w), 1452 (w), 1124 (s), 1050 (s), 991 (m),
568 (s).
Cell culture and drug treatment
Except that the human normal liver cell lines L02 and
human lung cancer cell lines A549 were cultured in the
RPMI-1640 medium with 10 % heat-inactivated fetal
bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL
streptomycin, other four human cancer cell lines (U2OS,
HCT116, MDA-MB-231 and HepG2) were cultured in the
DMEM medium supplemented with 10 % heat-inactivated
FBS, 100 U/mL penicillin and 100 μg/mL streptomycin.
All these cells were plated (1 × 104 cells/mL) in 100 μL
per well in 96-well plates, respectively, and incubated in a
humidified atmosphere of 5 % CO2 and 95 % air at 37 °C
for 24 h, which allowed for the cell attachment. After 24 h,
these cells were treated with the test drug. Subsequently,
they were cultured in a humidified incubator of 5 % CO2
and 95 % air at 37 °C for another 48 or 72 h, respectively.
HA‑binding assay
To study the bone-targeting efficiency of novel platinum
complexes, an in vitro hydroxyapatite (HA)-binding assay
was set up using the high-performance liquid chromatog-
raphy (HPLC) method with the most usual detection sys-
tem and the simplest chromatography elution conditions.
First, complexes 1–4 with different concentrations (0.05–
1.0 μmol/L) were determined by HPLC and a standard
curve was plotted for each compound from the area integral
as a function of concentration (Fig. S5). The disposition
pattern of the standard curve can be described by the fol-
lowing linear equation: S = kC + b, where S is the average
integral area under HPLC peak of an aliquot of the com-
plex solution, C is the standard concentration of the com-
plex, k and b are the slope and intercept of the standard
curve, respectively.
Cytotoxicity assay
The cytotoxicity of novel platinum(II) complexes 1–4 and
corresponding ligands (ZL, IPrBP, MIBP, and EIBP) was
evaluated by means of conventional MTT assay. Briefly,
cells were seeded at a density of 7–10 × 103 cells per
well in 96-well flat-bottom plates and incubated at 37 °C
overnight to allow cells attachment. Then, the aqueous
1 3

J Biol Inorg Chem
solution of each complex diluted with the medium DMEM
or RPMI-1640 was added to each well to give the indicated
final concentration, and then each plate was incubated for
additional 48 or 72 h. After drug treatment, 20 μL MTT
(5 mg/mL in PBS) was added directly to each well and
the plate was incubated for additional 4 h at 37 °C. Subse-
quently, the medium was removed from each well and 150
μL of 50 % (v/v) DMSO was added to each well to lyse the
resulting formazan crystals. The absorbance of each well
was measured at 490 nm using a microplate reader (Bio-
Rad Model 3550, CA, USA) to evaluate the inhibition of
cell growth induced by the drug. A graph of the percent-
age of cell inhibition versus concentration can be plot-
ted and the IC50 value (concentration of drug that inhibits
cell growth by 50 %) was determined accordingly. All the
experiments were performed in triplicate and the medium
without the drug served as a control, as well as the cytotox-
icity of CDDP and [Pt(en)Cl2] evaluated by means of the
same method served as a positive control.
by trypsinization, washed with PBS, suspended with ice-
cold 70 % ethanol and incubated at 4 °C overnight. Then
the cells were centrifuged to remove ethanol, washed with
PBS, and then resuspended in 1 mL of DNA staining rea-
gent containing 50 μg/mL RNase, 50 μg/mL PI, 0.1 % Tri-
ton X-100, and 0.1 mM EDTA (pH 7.4) for 30 min at 4 °C.
Finally, the cell cycle profile in each sample was analyzed
using a FACS Calibur flow cytometer equipped with a 488-
nm argon ion laser and a Cell Quest 3.1 software. DNA
histograms obtained from Cell Quest 3.1 were further ana-
lyzed under Window system using WinMDI 2.8 software
(Joseph Trotter, Salk Institute for Biological Studies, La
Jolla, CA, USA).
DNA‑binding assay
Calf thymus (CT) DNA was dissolved in buffer solution
(5 mM Tris–HCl, 50 mM NaCl, pH 7.4) as a stock solu-
tion, which was stored at 4 °C for 24 h to reach homogene-
ous phase and used within 3 days. The solution of CT-DNA
in the buffer gives a UV absorbance ratio (A260/A280) of
about 1.8, indicating that CT-DNA is sufficiently free of
protein. The concentration of CT-DNA was determined by
measuring the UV absorption at 260 nm, taking 6600 M−1
cm−1 as its molar absorption coefficient. The complex was
dissolved in the stock solution mentioned above. The CD
spectra for CT-DNA were recorded as follows. Each sam-
ple of CT-DNA (1.0 × 10−4 M) was incubated with dif-
ferent concentrations of the platinum complex at 37 °C for
24 h in the dark, where the concentration ratio of the plati-
num complex to DNA is 0, 0.02, 0.04, 0.08, 0.10 and 0.20,
respectively. Each sample was scanned in the wavelength
range of 220–320 nm at the speed of 10 nm/min and the
buffer background was subtracted.
The absorption titration experiment was also carried out
to determine the DNA-binding constant of the complex. A
solution of corresponding complex (3.6 × 10−5 M) in the
buffer (5 mM Tris–HCl, 50 mM NaCl, pH 7.4) was incu-
bated for 2 h at 25 °C before the CT-DNA solution was
added. Then an increasing amount of CT-DNA (1.8 mM, 3
μL per time) was gradually added to the above solution.
Due correction was made for the absorbance of DNA itself,
and the UV–Vis spectrum was recorded after equilibration
of each successive addition of CT-DNA solution. The bind-
ing constant (Kb) of the complex to CT-DNA was deter-
mined by monitoring the changes in the absorption inten-
sity of the spectral band with the increasing concentration
of CT-DNA using the following equation:
Morphology study
Exponentially growing cells were seeded into 6-well plates
at a density of 2 × 105 cells per well. After 24 h incuba-
tion at 37 °C for cell attachment, the culture medium was
removed and replaced with fresh medium containing the
complex at the indicated concentration. The cells were
incubated for another 72 h. Then, the cells were observed
under the inverted light microscope and photographed by a
Panasonic Lumix DMC-FH2.
The morphological changes of cell nuclear chroma-
tin were also studied by staining with the combination of
fluorescent dyes Hoechst 33342 and propidium iodide (PI).
Cells were seeded at a density of 2 × 105 cells per well
in 6-well plates and incubated at 37 °C overnight to allow
cells attachment. Then the drug was added to each well to
give the indicated final concentration respectively. After
72 h of exposure to the drug, cells were washed with PBS
and then stained with Hoechst 33342 (50 μL, 10 μg/mL)
and PI (50 μL, 10 μg/mL). After incubation for 10 min at
37 °C, the cells were observed under the inverted fluores-
cence microscope and photographed twice (magnification
400×) using appropriate filters to examine and compare
Hoechst 33342 and PI fluorescence staining in the same
cells.
Cell cycle analysis
Cells were seeded at a density of 2 × 105 cells per well
in 6-well plates and incubated at 37 °C overnight to allow
cells attachment. Then the drugs (complexes and CDDP)
were added to each well to give the indicated final concen-
tration. After 72 h exposure to the drug, cells were collected
[DNA]/(εa − εf) = [DNA]/(εb − εf) + 1/Kb(εb − εf)
(1)
where [DNA] is the DNA concentration, εa is the extinc-
tion coefficient observed for the charge-transfer absorption
1 3

J Biol Inorg Chem
100
80
60
40
20
0
band at a given DNA concentration, εf is the extinction
coefficient of the free platinum complex, εb is the extinc-
tion coefficient of the platinum complex in the fully bound
form, and Kb is the equilibrium binding constant. A plot of
[DNA]/(εa−εf) versus [DNA] gives the binding constant Kb
as the ratio of the slope to the intercept.
Complex 1
Complex 2
Complex 3
Complex 4
Results and discussion
0
12
24
36
Time (h)
48
60
72
84
Synthesis and characterization
Four dinuclear platinum(II) complexes 1–4 were prepared
based on the ligands of ZL, IPrBP, MIBP and EIBP, respec-
tively, according to the synthetic route shown in Scheme 2.
To avoid the formation of side products, such as the mon-
onuclear species having one bisphosphonate ligand per
Pt(en) moiety, the barium salt of the bisphosphonate was
prepared at first. Then it was treated with two equivalents
of [Pt(en)(OSO3)(H2O)] to form the dinuclear species.
The structures of complexes 1–4 have been characterized
Fig. 1 Adsorption curves of complexes 1–4 on HA
1
identical variations in the H-, 13C-, and 31P-NMR spectra
with those of the complex 1. The comprehensive analyses
indicate that all the molecular configurations of these com-
plexes are not fully symmetric.
HA‑binding assay
1
by elemental analysis, ESI–MS, IR, and H/13C/31P-NMR
spectra (Figs. S1–S4). The elemental analyses of the result-
ing complexes were in line with the presence of one bis-
phosphonate and two [Pt(en)]2+ residues, respectively.
The formation of the complexes can also be deduced
from the infrared spectra. As shown in the typical spec-
tra of the ligand ZL and the corresponding complex 1
(Fig. S1), the stretching vibrations of P = O and P–OH
(νP=O and νP–OH) in the complex 1 shift from 1158 to
1128 cm−1 (Δν = 30 cm−1) and from 1015 to 994 cm−1
(Δν = 21 cm−1), respectively, due to the coordination to
the platinum moiety in comparison with the free ligand.
The existence of stretching vibration of Pt-O (νPt-O) in the
complex 1 also confirmed the formation of the complex.
Active targeting of therapeutic agents to bone is a prom-
ising strategy to reduce adverse effects and enhance effi-
cacy at the desired site, which will play an important role
in the clinical application of drugs [30, 31]. Human bone
is a tissue with unique properties due to its high content of
hydroxyapatite (HA). As BPs can target the bone surface
under active formation and resorption of HA, an in vitro
HA-binding assay was hence carried out based on the
HPLC analysis to evaluate the bone-targeting ability of Pt-
BPs complexes. This will support a strategy for targeted
delivery of drugs to bone by attaching a bone-affinity tag to
the active drug and it has been used to examine the binding
affinities of BPs, which provides an economic and accurate
measure for quantitative analysis of bone affinity [32].
Figure 1 shows the adsorption profiles of complexes
1–4 on HA as a function of time. The plots display that the
adsorption kinetics of four complexes are similar and all
follow the classical Langmuir adsorption isotherm. How-
ever, the adsorption rate and adsorption amount of com-
plexes 1 and 2 are obviously higher than those of complexes
3 and 4. The amount of complexes 1 and 2 adsorbed onto
HA increases rapidly within 8 h and reaches the maximum
uptake after about 12 h with the adsorption percentage of
98.66 and 95.91 %, respectively. This corresponds to about
0.15 mg of two complexes per gram of HA. Similarly, the
amount of complexes 3 and 4 on HA also increases rapidly
within 12 h and reaches the maximum uptake after about
24 h with the adsorption percentage of 82.45 and 77.32 %,
respectively. This clearly indicates that complexes 1 and
2 have stronger bone-targeting ability than complexes 3
and 4. This may be ascribed to the larger substituent in the
1
Compared with the free ligand ZL, the H-NMR spectrum
of complex 1 in D2O displays two new broad peaks at 5.787
and 5.227 ppm and a disorder multiplet at 2.503 ppm (Fig.
S2), which are assigned to the protons of the unequal en
groups. This indicates that the ligand ZL has been coordi-
nated to the Pt atoms and there exist two Pt(en) units. Fur-
thermore, two peaks at 49.4 and 49.3 ppm in the 13C-NMR
spectrum of complex 1 also demonstrates the existence of
two Pt(en) centers (Fig. S3). The 31P-NMR spectrum of
complex 1 shows a singlet at 34.74 ppm (Fig. S4), which
is assigned to the two phosphorus atoms of the phospho-
nic groups that are magnetically equivalent. In comparison
with the free ligand ZL, the singlet shifts to a lower field
(Δδ = 18.66 ppm). The shift to low magnetic field from the
ligand to the complex is due to the fact that the phospho-
nate groups lost the acidic protons when coordinated to the
metal ion [25]. Similarly, compared with the free ligands
(IPrBP, MIBP and EIBP), complexes 2–4 exhibit almost
1 3

J Biol Inorg Chem
side chain of 3 and 4 than in 1 and 2, which results in the
hydrophobicity of the complex increasing. Further compar-
ison among four complexes clearly shows that the affinity
of complex 1 for HA is the largest, in terms of both adsorp-
tion rate and total uptake. On the whole, the difference in
the adsorption rate and adsorption percentage of these com-
plexes can be ascribed to the difference in the side chain
R2. According to previous studies, it is known that small
differences in the R2 group can lead to substantial changes
in the three-dimensional (3D) shapes and atomic orienta-
tions of the BPs although they share a common P–C–P
structure and the same side chain OH (R1) [32, 33]. There-
fore, it is postulated that 3D configurations of these plati-
num-BPs complexes are different, which might explain the
difference observed in the HA-binding affinity and may in
turn have an influence on their pharmacological potency.
Based on the established interaction model of simpler
BPs on HA where the tridentate coordination of the P–C–P
“bone hook” and R1 (OH) domains interact with the cal-
cium site [32, 33], there is potential of additional interac-
tions between the imidazolyl moiety in these more com-
plex compounds with HA. For example, the protonated
imidazole can interact with HA through the hydrogen bond
N–H···O or N···H–O, which can optimally allow a bifur-
cate bonding arrangement. This has been demonstrated by
the optimal N–H–O bond angle that can be formed with the
–NH2 of alendronate and the distal heteroaromatic nitrogen
of ZL, which can account for their higher binding affini-
ties in comparison with those observed for other BPs [33].
Therefore, it is inferred that platinum complexes of N-het-
erocyclic BPs have higher affinities for the bone mineral,
which is essential for reducing adverse effect and enhanc-
ing specificity of drugs that act on bone-related diseases.
However, although these N-heterocyclic BPs can theoreti-
cally orient properly for hydrogen bond formation, steric
hindrance of different groups on the side chain may inter-
fere with these potential interactions, which may explain
why complexes 3 and 4 have lower binding affinities than
complexes 1 and 2. Compared with other platinum-BPs
complexes [34], complexes 1 and 2 also possess higher
affinity for HA. Since weak binding on HA may translate
lower potency that necessitates higher dose, it is postu-
lated that higher affinity for HA of complexes 1 and 2 may
increase their bone-targeting anticancer efficacy.
anticancer agent and the latter might be the active species
released from four complexes of bisphosphonate Pt(II) eth-
ylenediamines. As anticipated earlier, the cytotoxicities of
four complexes are higher than those of the correspond-
ing ligands on the whole, although they are both weaker
than those of CDDP and [Pt(en)Cl2]. This indicates that
combining the platinum moiety with the bisphosphonate
ligand will improve the anticancer efficacy of BP ligands,
but still cannot exceed that of CDDP. As shown in Figs. 2
and S6, the complexes and the ligands both produced
time- and dose-dependent effect on inhibiting the cancer
cell lines. After exposure to increasing concentration of
platinum complexes for 48 or 72 h, five cancer cells dis-
play significant loss of viability. It is notable that four com-
plexes have weaker sensitivity to the breast cancer cell lines
MDA-MB-231 and relatively stronger cytotoxic effect on
the hepatoma cell lines HepG2. Compared with complexes
2–4, the complex 1 shows higher cytotoxicity against the
cancer cell lines on the whole, implying that the side chain
R2 plays an important role in determining the biological
activity and the anticancer efficacy will decrease with the
increasing substituent. However, noteworthy is that the new
complexes did not show high cytotoxicity against the bone
cancer cells U2OS that was envisioned by our original drug
design strategy, although they showed high affinity for the
bone mineral HA. Therefore, it is deduced that these com-
plexes have little bone-targeting anticancer activity.
Platinum-based drugs in current clinical use usually
have significant side effects on the normal cells or tissues
[2–4], which will affect their practical clinical efficacy and
limit their widespread application. To test the side effect
of the new complexes designed in the present work, their
cytotoxicity against the human normal liver cell lines L02
was also investigated, in which CDDP was used as the
positive control. The corresponding ligands and [Pt(en)Cl2]
were also investigated to compare with the complexes. As
shown in Figs. 3 and S7, the inhibition effects of complexes
1–4 on the normal liver cells L02 are slightly stronger than
those of the ligands at the same concentration (100 μM) on
the whole, except that the complex 1 shows weaker cyto-
toxicity than the ligand 1. However, all the complexes show
much stronger inhibition effects on the hepatocarcinoma
cell lines HepG2 than the corresponding ligands. This indi-
cates that complexes 1–4 have better selectivity than the
corresponding ligands in inhibiting human hepatocarci-
noma cells rather than normal liver cells and hence possess
lower hepatotoxicity. Furthermore, comparison among four
complexes shows that the selective inhibitory effect of the
complex 1 is the best.
Cytotoxicity assay
The IC50 values of complexes 1–4 and corresponding
bisphosphonate ligands (ZL, IPrBP, MIBP and EIBP)
against five human cancer lines (U2OS, A549, HCT116,
MDA-MB-231 and HepG2) were measured (Fig. 2; Table
S1). CDDP and [Pt(en)Cl2] were also studied as the posi-
tive control, since the former is the most widely used
Compared with CDDP and [Pt(en)Cl2], complex 1 at
the concentration of 100 μM shows a higher cytotoxicity
against the cancer cell lines HepG2 than CDDP and [Pt(en)
Cl2] at the concentration of 20 μM, while its cytotoxicity
1 3

J Biol Inorg Chem
Comp 1
Ligand 1
CDDP
Comp 1
Ligand 1
CDDP
150
120
90
60
30
0
1-72h
1-48h
2-48h
3-48h
320
240
160
80
Pt(en)Cl2
Pt(en)Cl2
0
250
2-72h
850
680
510
340
170
0
Comp 2
Ligand 2
CDDP
Comp 2
Ligand 2
CDDP
200
150
100
50
Pt(en)Cl2
Pt(en)Cl2
0
350
3-72h
Comp 3
Ligand 3
CDDP
600
Comp 3
Ligand 3
CDDP
280
210
140
70
400
200
0
Pt(en)Cl2
Pt(en)Cl2
0
120
90
60
30
0
4-72h
400
300
200
100
0
4-48h
Comp 4
Ligand 4
CDDP
Comp 4
Ligand 4
CDDP
Pt(en)Cl2
Pt(en)Cl2
Fig. 2 Cytotoxicity of complexes 1–4 and ligands 1–4 as well as CDDP and [Pt(en)Cl2] against five human cancer cell lines
against the normal liver cells L02 is comparable to that of
[Pt(en)Cl2] and far lower than that of CDDP. However, at
the same concentration (10 μM or 20 μM), the cytotoxici-
ties of the complex 1 against HepG2 and L02 are both lower
than those of CDDP and [Pt(en)Cl2]. This suggests that
the hepatotoxicity of the new complex 1 is less than those
CDDP and [Pt(en)Cl2] although its anticancer efficacy is
not better than CDDP and [Pt(en)Cl2]. On the whole, the
new complex 1 at the high concentration (100 μM) has a
better selectivity in inhibiting hepatocarcinoma cells than at
the low concentration, and it is worthy of further investiga-
tion on the animal model to study its selective anticancer
effect thoroughly.
Morphology study
To intuitively examine the sensitivity and inhibition effect
of novel platinum complexes on the cancer cell lines,
morphologic changes of the cancer cells were also exam-
ined. In general, apoptotic cells are identified by their
1 3

J Biol Inorg Chem
(a)
(b)
100
100
80
60
40
20
0
HepG2 48 h
HepG2 72 h
L02 48 h
L02 72 h
HepG2 48 h
HepG2 72 h
L02 48 h
L02 72 h
80
60
40
20
0
L 2
100 µM
L 3
100 µM
L 4
Comp 2 Comp 3 Comp 4
100 µM 100 µM 100 µM
L 1
100 µM
Comp 1
100 µM
CDDP
20 µM
CDDP
20 µM
Pt(en)Cl2
20 µM
Pt(en)Cl2
20 µM
100 µM
Fig. 3 Selectivity of complexes 1–4 (a) and corresponding ligands 1–4 (b) in inhibiting human normal liver cells (L02) and liver cancer cells
(HepG2) in comparison with CDDP and [Pt(en)Cl2]
Fig. 4 Morphological changes of cancer cells treated by complexes 1–2 for 72 h at the indicated concentration (Left) and cell apoptosis detected
by Hoechst 33342-PI nuclear staining after exposure to the complexes 1–2 for 72 h (Right)
characteristic nuclei condensation, fragmentation and
bright staining, while normal cells demonstrate uniformly
dispersed chromatin, normal organelle and intact cell mem-
brane. Therefore, the nuclear staining assay with the com-
bination of PI and Hoechst 33342 fluorescent dyes was
used to evaluate the morphological changes of these can-
cer cell lines induced by complexes 1 and 2. As shown in
Fig. 4, the density of viable cells decreased significantly
after treating with complexes 1 and 2 for 72 h. Compared
with the control group without treatment of complexes,
1 3

J Biol Inorg Chem
these cells display opaque and different morphologies
besides the smaller density. After staining with Hoechst
33342 and PI, these cells also exhibit typical characteristics
of apoptosis, including the condensation of chromatin, the
shrinkage of nucleus, and the appearance of a few apop-
totic bodies. This indicates that complexes 1 and 2 have
cytotoxic effects on these human cancer cell lines, which
is consistent with the results of MTT assay. Notably, as
for the hepatoma cell lines HepG2, not only brilliant blue
apoptotic cells but also dead cells can be observed at the
complex concentration of 10 μM. Although the breast can-
cer cell lines MDA-MB-231 showed low sensitivity to both
complexes, it also exhibited typical apoptotic character at
the complex concentration of 40 μM. These further demon-
strated that complexes 1 and 2 cannot only inhibit the pro-
liferation of cancer cell lines, but also induce the apoptosis
of cancer cell lines.
DNA‑binding assay
DNA is known to be the primary target of many metal-
based antitumor compounds and circular dichroism (CD)
spectroscopy is a sensitive technique to trace the confor-
mational changes of DNA secondary structure. A com-
plex bound to DNA through intercalation is characterized
by the change in absorbance or intensity (hypochromism)
and red shift in wavelength, due to the intercalative mode
involving a strong stacking interaction between the aro-
matic chromophore and the DNA base pairs. The extent of
hypochromism is commonly correlated with the strength
of the intercalative interaction [36]. Hence, the interactions
between complexes 1–2 and DNA were investigated by
monitoring the CD spectra of CT-DNA upon complex for-
mation with complexes [Pt(en)]2ZL (1) and [Pt(en)]2IPrBP
(2) bound, which may be of paramount importance for
understanding their mechanism of action.
Cell cycle analysis
The CD spectra of CT-DNA complexed with the com-
plexes 1–2 are shown in Fig. 6. In general, the charac-
teristic CD spectrum of DNA exhibits a positive band
around 275 nm due to the base stacking, a negative band
around 245 nm due to the helicity of B-type DNA, and
crossover point near 258 nm, respectively [37]. As shown
in Fig. 6, the spectra exhibit characteristic features of the
canonical B-type DNA conformation within experimen-
tal error, although the intensities of both 245 and 275 nm
were altered in the presence of complex 1 or 2. It is nota-
ble that up to the concentration ratio r = 0.2, almost cor-
responding to a saturation, the CD spectra of the DNA-
platinum complexes are not very different from that of
DNA. Therefore, it is possible to conclude that the fixa-
tion of complex 1–2 on DNA does not greatly affect its
secondary structure.
With the concentration ratio (r) of platinum to CT-
DNA increasing, there is a slight intensity increase in
the positive band located at 280 nm and a small intensity
decrease in the negative band located at 250 nm, with a
very small bathochromic shift. These changes are simi-
lar to those in the CD spectra of DNA induced by CDDP
or [Pt(en)Cl2] [38, 39], suggesting that the DNA-binding
modes of these novel platinum complexes are identical
with that of CDDP or [Pt(en)Cl2]. The significant features
of these spectra are the amplitude decrease of the band
located at 225 nm and the existence of an isodichroic
point around 233 nm. This isodichroic point is also found
with other platinum complexes and seems to be useful
as a fingerprint of platinum fixation on DNA [38, 39]. In
principle, an enhancement in the positive band indicates
that the stacking of DNA base pairs increases, while the
reduction in the negative band suggests that the stability
of DNA double helix structure decreases. As shown in
Fig. 6, the increase in the ellipticity of positive band may
The cell cycle distribution of five different human cancer
cell lines induced by complexes 1 and 2 was analyzed using
a FACS Calibur flow cytometer. The percentage of cell
population at different phases was obtained from the Cell
Quest software package (Fig. 5). It is clear that after expo-
sure to increasing concentration of complexes 1 and 2, the
cell populations of five human cancer cell lines at the G0/
G1 phase all decrease remarkably, while those at the G2/M
phase all increase. This indicates that both complexes can
cause tumor cell cycle to arrest at the G2/M phase, so that
no more cells can go from G2/M to G0/G1. Combined with
the results of above study, it can be deduced that the DNA
damage caused by complexes 1 and 2 will not be recovered
at the M phase, which may be the reason for the cells apop-
tosis and death. Investigating the cell cycle distribution of
these human cancer cell lines treated by CDDP, the same
conclusion can be drawn that tumor cell cycle was arrested
at the G2/M phase (Tables S2–S6). Therefore, it is inferred
that the action mechanism of these novel dinuclear plati-
num complexes is similar to that of CDDP, which inhibits
human cancer cell lines by inducing the cell cycle to arrest
at the G2/M phase [35].
In addition, cell apoptosis induced by complexes 1 and
2 can be observed since the cell populations at the subG1
phase increase (Fig. 5), especially for the hepatoma cell
line HepG2. The percentage of HepG2 apoptotic cells
increases from 4.41 % (control) to 15.72 % (1, 2.5 μM),
14.96 % (1, 5 μM), 12.49 % (2, 2.5 μM) and 17.20 % (2,
5 μM), respectively, which are all higher than the posi-
tive control (11.28 %, CDDP 2.5 μM). Combining with
the above studies, complexes 1 and 2 are considered to be
particularly efficient for treating the hepatocarcinoma and
worthy of further investigation.
1 3

J Biol Inorg Chem
Fig. 5 Cell cycle perturbation of five cancer cell lines treated by complexes 1–2 and CDDP after 72 h, along with cell apoptosis of HepG2
induced by complexes 1–2 and CDDP
1 3

J Biol Inorg Chem
8
4
8
4
r=0.00
r=0.02
r=0.04
r=0.08
r=0.10
r=0.20
r=0.00
r=0.02
r=0.04
r=0.08
r=0.10
r=0.20
Complex 2
Complex 1
0
0
-4
-4
-8
-8
220
240
260
280
300
320
220
240
260
280
300
320
Wavelength (nm)
Wavelength (nm)
Fig. 6 CD spectra of CT-DNA in the absence and presence of increasing concentration ratios of complexes 1 and 2 (r = 0, 0.02, 0.04, 0.08, 0.10
and 0.20) in the buffer (5 mM Tris–HCl, 50 mM NaCl, pH 7.4) at 37 °C
1.0
be attributed to the additional stabilization of base stack-
0.8
ing derived from the covalent binding between Pt and
guanine or cytosine groups and other noncovalent inter-
actions upon DNA–platinum complex formation, while
the slight decrease in the ellipticity of the negative band
1.0
0.6
0.4
0.8
0.2
0.0
suggested that complexes 1 and 2 could only slightly
0.6
0
2
4
6
8
10
12
14
[DNA] 10-6
M
unwind the DNA helix. Moreover, a close examination
on the CD spectra of CT-DNA with the complexes 1 and
2 bound, respectively, shows that the changes in the CD
spectra are almost identical, implying the effects of both
complexes on DNA secondary structure are similar. Sub-
tle differences are only found in the degree of reduction
in the negative band. This may be due to a longer chain
in the BP ligand of complex 2, which results in a large
unwinding degree of the DNA helix.
0.4
0.2
0.0
210
215
220
225
230
235
240
245
wavelength/nm
Fig. 7 Absorption spectra of complex 1 with increasing concentra-
tion of CT-DNA in the buffer (5 mM Tris–HCl, 50 mM NaCl, pH 7.4)
at 25 °C. The inset shows the least-squares fit of Δεaf/Δεbf vs [DNA]
Furthermore, to determine the binding efficiency of
these novel dinuclear platinum complexes to DNA, the
intrinsic binding constant (Kb) of the complex 1 to CT-DNA
was also determined by UV–vis absorption spectral tech-
nique through keeping the concentration of the complex as
a constant and increasing the concentration of CT-DNA. If
there are strong stacking interactions between the aromatic
chromophore of the complex and the base pairs of DNA,
there must be a red shift in the absorption band. In the pres-
ence of increasing concentration of CT-DNA, a slight even
negligible hypochromism was observed in the absorption
spectra of complex 1 (Fig. 7), indicating the weak interca-
lation nature of the complex 1 and little effect on chang-
ing the DNA secondary structure. This is consistent with
the above CD spectra study. The binding constant (Kb) of
complex 1 was determined to be 9.7 × 105 M−1 from the
regression analysis, suggesting that it has a moderate bind-
ing affinity for DNA. This may also result in the weak cyto-
toxicity of these novel platinum complexes against human
cancer cell lines.
Conclusion
Four novel dinuclear platinum(II) complexes based on
imidazolyl-containing bisphosphonates have been designed
and synthesized, [Pt(en)]2ZL (1), [Pt(en)]2IPrBP (2),
[Pt(en)]2MIBP (3) and [Pt(en)]2EIBP (4). They showed
high affinity for the bone mineral hydroxyapatite, but low
targeting inhibition effect on the bone cancer cell lines.
Although their anticancer activities are lower than CDDP,
they exhibit higher cytotoxicity than the correspond-
ing ligands and relatively stronger cytotoxic effect on the
hepatoma cell lines HepG2. Four complexes have better
selectivity than the corresponding ligands in inhibiting hep-
atocarcinoma cells rather than normal liver cells, and the
selective inhibitory effect of the complex 1 at the high con-
centration (100 μM) is the best. The complexes all inhibit
1 3

J Biol Inorg Chem
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Acknowledgments The authors are grateful to the financial sup-
port from National Natural Science Foundation of China (21371082
and 21001055), Natural Science Foundation of Jiangsu Province
(BK20141102 and BK20151118), Key Medical Talent Project of
Jiangsu Province (RC2011097), Science Foundation of Health
Department of Jiangsu Province (Q201204) and Public Service Plat-
form for Science and Technology Infrastructure Construction Project
of Jiangsu Province (BM2012066).
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