Synthesis, DNA binding, topoisomerase I inhibition and antiproliferation activities of three new binuclear terpyridine platinum(II) complexes

Article abstract of DOI:10.1016/j.poly.2018.09.053

Three binuclear platinum(II) complexes, 1 ([Pt₂(L)Cl₂]Cl₂), 2 ([Pt₂(L)Br₂]Br₂) and 3 ([Pt₂(L)I₂]I₂) (where L = 1,4-bi-[4′-(4-Hydroxyphenyl)-2,2′,6′,2″-terpyridine] butane) were synthesized and characterized by nuclear magnetic resonance, elemental analysis and mass spectrometry. The crystal structure of the new ligand L was determined by single crystal X-ray crystallography, and π–π stacking interactions were revealed in the geometry. Interactions of complexes with calf thymus DNA have been investigated by UV–Vis spectroscopy and fluorescence spectroscopy. Due to the structural differences, binuclear complex 3 exhibited higher binding affinity for calf thymus DNA with a DNA-binding constant K_b of 8.72 × 10⁶ M^?1. The competitive study indicated that the ethidium bromide (EB) can displaced by complexes 1–3 from the DNA-EB conjugation. The concentration-dependence unwinding of supercoiled circular plasmid pBR322 DNA by complexes 1–3 was observed via agarose gel electrophoresis. Meanwhile, complexes displayed obvious activities for inhibition of topoisomerase I. Herein the halogen leaving groups Cl^?/Br^?/I^? presented in 1–3 showed significant effect on DNA binding and topoisomerase I inhibition activities. An evaluation of in vitro cytotoxicity for three complexes was performed by MTT assay in two cancer cell lines and normal cells as the control, respectively.

Full text of DOI:10.1016/j.poly.2018.09.053

Accepted Manuscript
Synthesis, DNA binding, topoisomerase I inhibition and antiproliferation ac-
tivities of three new binuclear terpyridine platinum(II) complexes
Keke Chai, Yihui Jiang, Tianzhi Han, Junlong Niu, Liushuang Yao, Haiyu
Zhang, Min Zeng, Li Zhang, Xuemin Duan, Jintao Wang
PII:
S0277-5387(18)30606-5
https://doi.org/10.1016/j.poly.2018.09.053
POLY 13452
DOI:
Reference:
Polyhedron
To appear in:
Received Date:
Accepted Date:
29 July 2018
19 September 2018
Please cite this article as: K. Chai, Y. Jiang, T. Han, J. Niu, L. Yao, H. Zhang, M. Zeng, L. Zhang, X. Duan, J. Wang,
Synthesis, DNA binding, topoisomerase I inhibition and antiproliferation activities of three new binuclear
terpyridine platinum(II) complexes, Polyhedron (2018), doi: https://doi.org/10.1016/j.poly.2018.09.053
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Synthesis, DNA binding, topoisomerase I inhibition and
antiproliferation activities of three new binuclear terpyridine
platinum(II) complexes
Keke Chaia, Yihui Jianga, Tianzhi Hana, Junlong Niua, Liushuang Yaoa, Haiyu
Zhanga, Min Zenga, Li Zhanga, Xuemin Duana,*, Jintao Wanga,*
Jiangxi Provincial Key Laboratory of Drug Design and Evaluation, School of
a
Pharmacy, Jiangxi Science & Technology Normal University, Nanchang 330013, P.R.
China
*
Corresponding author: Fax: +86-791-83823320, Tel.: +86-791-88537967, E-mail:
duanxuemin@126.com; jintaochem@163.com
ABSTRACT
Three binuclear platinum(II) complexes, 1 ([Pt(L)Cl2]Cl2), 2 ([Pt(L)Br2]Br2) and 3
([Pt(L)I2]I2) (where L= 1,4-bi-[4’-(4-Hydroxyphenyl)-2,2’,6’,2’’-terpyridine] butane)
were synthesized and characterized by nuclear magnetic resonance, elemental analysis
and mass spectrometry. The crystal structure of the new ligand L was determined by
single crystal X-ray crystallography, and π-π stacking interactions were revealed in
the geometry. Interactions of complexes with calf thymus DNA have been
investigated by UV-vis spectroscopy and fluorescence spectroscopy. Due to the
structural differences, binuclear complex 3 exhibited higher binding affinity for calf
thymus DNA with a DNA-binding constant Kb of 8.72 × 106 M-1. The competitive
study indicated that the ethidium bromide (EB) can displaced by complexes 1-3 from
the DNA-EB conjugation. The concentration-dependence unwinding of supercoiled

circular plasmid pBR322 DNA by complexes 1-3 was observed via agarose gel
electrophoresis. Meanwhile, complexes displayed obvious activities for inhibition of
topoisomerase I. Herein the halogen leaving groups Cl-/Br-/I- presented in 1-3 showed
significant effect on DNA binding and topoisomerase I inhibition activities. An
evaluation of in vitro cytotoxicity for three complexes was performed by MTT assay
in two cancer cell lines and normal cells as the control, respectively.
Keywords: Platinum(II) complex; DNA binding; inhibitory activity; topoisomerase I
1. Introduction
Cancer is one of the significant factors which cause of death in recent years,
accounting for nearly one-quarter deaths annually [1]. Medical treatment has a
significant effect on reducing cancer mortality and morbidity, and rates should
continue to improve with recent advances in new therapies. Following the discovered
of antitumor activity of cisplatin by Barnett Rosenberg [2], the success of the classical
structure-activity relationship platinum complexes in the treatment of cancer
stimulated scientists research interests and the non-classical platinum complexes
included binuclear or multinuclear platinum complexes were researched [3].
Terpyridine and its derivatives with plentiful potential application, such as DNA
binding agents [4], topoisomerase I inhibitors [5] and anti-tumor agents [6], are of
important ligands for constructing metal complexes. Specifically, Pt(II) terpyridine [7]
complexes as planar intercalators, have been researched for interest in recent years
due to their promising biological activity [8]. Besides the feature of the chelating
ligands, the leaving groups in the metal complexes also have significant effects on the

rates of hydrolysis, binding to biomolecule and the cytotoxic activity [9, 10]. The
cytotoxicity of some complexes in vitro can be improved when the chloride ligand
was replaced by different halogenated ligand. For instance, the [Ru(η6-p-
cymene)(L1)Cl]PF6 and [Ru(η6-p-cymene)(L1)I]PF6 (where L1 = N,N-dimethyl-N’-(2-
pyridinylmethylene)) displayed the IC50 values of 16.2 ± 0.9 and 3.0 ± 0.2 µM,
against the A2780 human ovarian carcinoma cells [11]. The bromide complex
([Ru(η6-p-cymene)(5,7-diiodo-8-quinoline)Br], IC50 = 2.92 ± 0.53 µM) was more
potent than its chloride analog ([Ru(η6-p-cymene)(5,7-diiodo-8-quinoline)Cl], IC50 =
4.60 ± 1.29 µM) against the NCI-H460 non-small cell lung carcinoma [12].
Topoisomerase I (Topo I) is a crucial nicking-closing enzyme, whose main
function is deal with DNA super-helices in chromosomes during replication and
transcription in biological cells [13, 14]. Topoisomerase I inhibition is one of the
dominating means used in cancer therapy because it could lead to DNA damage that
induce apoptosis and necrosis and stop DNA replication. It drew many researchers,
who explored a drug targeting both Topo I and DNA using endogenous metals, as
well as made efforts to reduce undesired side effects and improve the activity.
Functionalized terpyridine derivatives [15-17] have a wide range of potential
applications ranging from colorimetric metal determination to DNA binding agents,
anticancer agents and topoisomerase inhibitors. Therefore, topoisomerases are the
cellular targets of clinically important anticancer and antibacterial drugs, and
inhibition of topoisomerases has been considered as an effective strategy for the
design of many anticancer agents [18]. Inspired by the above considerations, we

designed and synthesized three binuclear Pt(II) complexes, with different leaving
groups (Scheme 1). Their DNA binding abilities, in vitro topoisomerase I inhibition
and cytotoxicity against cancer cell lines was also investigated.
2. Experimental
2.1. Reagents and instrumentation
All reagents and materials were purchased from commercial suppliers which for
scientific laboratory. 2-Acetylpyridine, p-hydroxybenzaldehyde, K2PtCl4, KBr, KI and
AgNO3, were purchased from Energy-chemical. 4’-(4-hydroxyphenyl)-2,2’,6’,2’’-
terpyridine [19] and Pt(DMSO)2Cl2 [20] were prepared according to the published
procedure. Calf thymus DNA (CT DNA) was purchased from Sigma-Aldrich.
pBR322 DNA was obtained from BBI LIFE SCIENCES and GelRed was purchased
from Biotium. Topoisomerase I (Topo I) was obtained from Takara Biomedical
Technology (Beijing). Agarose gel, ethylenediaminetetraacetic acid (EDTA), and
PBS (1×) buffer reagents was purchased from Sangon. A549 (human lung carcinoma
cell lines), HepG2 (human liver hepatocellular carcinoma), and LO2 (human
normal liver cell) were obtained from the Cell Resource Center, Peking Union
Medical College (Beijing, China).
Nuclear magnetic spectra were recorded on a Bruker AVANCE 400
spectrometer at ambient temperature. Elemental analyses of C, H and N were
measured on a Perkin-Elmer 240C elemental analyzer. High resolution mass
spectrometric analysis was carried out on a Waters Xevo G2-XS Q-TOF instrument.
A Shimadzu UV-2550 UV-vis spectrophotometer was used for UV scanning. Bio-Rad

Sub-Cell GT electrophoresis system was used for the electrophoresis experiment and
JUNYI scanner (JY04s-3c) was used for gel imaging. Stock solutions (10 mM) of
complexes 1-3 were prepared in DMSO, which were further diluted using buffer
or cell culture medium until working concentrations were achieved.
2.2. Synthesis
Ligand L (1.4-bi-[4’-(4-hydroxyphenyl)-2,2’,6’,2’’-terpyridine] butane)
The ligand was synthesized by a similar method as reported [21]. 2.16 g (36
mmol) of KOH was added into the round bottom flask with 150 mL DMF containing
3.90 g (12 mmol) dissolved 4’-(4-hydroxyphenyl)-2,2’,6’,2’’-terpyridine. Then, 1,4-
dibrombutane (0.597 mL, 5 mmol) was added dropwise to the mixture and it was
heated at 80 °C for 24 hours. The resulting mixture was percolated and the residue
was purified via flash chromatography (dichloromethane/methanol). Then the product
was obtained by reduced pressure distillation. It was dried under vacuum to give 5.22
g the white powder. Yield: 74.0%. 1H NMR (400 MHz, CDCl3, δ, ppm): 8.71 (m, 8H,
4, 7, 9, 12-tpyH), 8.68 (d, J=8 Hz, 4H, 1, 15-tpyH), 7.88 (m, 8H, 2, 3, 13,14-tpyH),
7.35 (m, 4H, 17, 21-tpyH), 7.06 (d, J=8 Hz, 4H, 18, 20-ArH), 4.15 (t, 4H, OCH2),
2.07 (m, 4H, OCH2CH2). 13C NMR (400 MHz, CDCl3, δ, ppm): 156.28, 155.67,
149.88, 148.95, 137.03, 130.64, 128.58, 123.78, 121.47, 118.43, 114.93, 67.62, 26.04.
HR–MS (Acetonitrile) m/z: calcd 705.2978 for C46H37N6O2, found 705.2981 for
[L+H]+. Elemental analysis calcd (%) for C46H36N6O2: C 78.39, H 5.15, N 11.92;
found: C 78.20, H 5.60, N 11.80.
Complex 1 ([Pt(L)Cl2]Cl2)

The complex 1 was synthesized as previously reported [22] with minor
modifications. A 40 mL CHCl3 solution containing Pt(DMSO)2Cl2 (1.06 g, 2.4 mmol)
and 1 equiv of L (0.89 g, 1.2 mmol) was refluxed under argon for 24 h. The reaction
mixture was cooled to ambient temperature, and the resulting yellow precipitate was
collected by filtration, washed with 15 mL CHCl3 and 15 mL diethyl ether. It was
dried under vacuum to give 0.76 g the faint yellow powder. Yield: 65.5%.1H NMR
(400 MHz, DMSO-d6, δ, ppm): 8.90 (m, 4H, 7 and 9-tpyH), 8.83 (d, 8H, 1, 4, 12 and
15-tpyH), 8.51 (d, 4H, 17, 21-ArH), 8.19 (d, 4H, 3, 13-tpyH), 7. 91 (d, 4H, 2, 15-
tpyH), 7.20 (t, 4H, 18, 20-ArH), 4.18 (m, 4H, CH2OCH2), 1.93 (m, 4H, OCH2CH2).
HR–MS (Acetonitrile) m/z: calcd 582.0786 for C46H36N6O2Pt2Cl2 (with two positive
charges), found 582.0781 for [1-2Cl]2+. Elemental analysis calcd (%) for
C46H36N6O2Pt2Cl4: C 44.67, H 2.93, N 6.80; found: C 44.42, H 3.26, N 6.66.
Complex 2 ([Pt(L)Br2]Br2)
Complex 2 was synthesized as previously reported [23] with modifications.
Complex 1 (0.124 g, 0.1 mmol) and AgNO3 (0.068 g, 0.4 mmol) were dissolved in a
mixed solution containing H2O (2 mL), CH3CN (10 mL) and THF (20 ml). Then the
mixture solution was stirred for 6 hours at 25 °C. After the solution was filtered to
remove the generated AgCl precipitation, excess of KBr (0.120 g, 1.0 mmol) was
added into the filtrate, which was stirred for another 6 hours. After filtration, the
filtrate was reduced under vacuum, and the collected precipitate was washed with 10
mL KBr (1 M) aqueous solution, 10 mL H2O and 10 mL CH3OH respectively. It was
dried under vacuum to give 0.07 g the yellow powder. Yield: 49.5%.1H NMR (400

MHz, DMSO-d6, δ, ppm): 9.05 (m, 4H, 7, 9-tpyH), 8.88 (d, 4H, 4, 12-tpyH), 8.81 (d,
4H, 1, 15-tpyH), 8.49 (d, 4H, 17, 21-ArH), 8.19 (d, 4H, 3, 13-tpyH), 7.88 (d, 4H, 2,
14-tpyH), 7.23 (t, 4H, 18, 20-ArH), 4.18 (m, 4H, OCH2), 1.92 (m, 4H, OCH2CH2).
HR–MS (Acetonitrile) m/z: calcd 626.0281 for C46H36N6O2Pt2Br2 (with two positive
charges), found 626.0273 for [2-2Br]2+. Elemental analysis calcd (%) for
C46H36N6O2Pt2Br4: C 39.06, H 2.57, N 5.94; found: C 38.87, H 2.89, N 5.69.
Complex 3 ([Pt(L)I2]I2)
The complex 3 was synthesized as reported [23], with minor modifications. The
methanol of complex 1 (0.248 g, 0.2 mmol) was added KI (0.166 g, 1.0 mmol) and
it’s stirred for 12 hours at room temperature. Then the color of mixture became more
and more deep. After filtration, the precipitate ware collected, and washed with 10 mL
KI (1 M) aqueous solution, 10 mL H2O, and 10 mL CH3OH. It was dried under
vacuum to give 0.166 g the dark yellow powder. Yield: 51.9%.1H NMR (400 MHz,
DMSO-d6, δ, ppm): 8.82 (m, 8H, 4, 7, 9, 12 -tpyH), 8.65 (d, 4H, 1, 15-tpyH), 8.32 (d,
4H, 17, 21-ArH), 7.96 (d, 8H, 2,3, 13,14-tpyH), 7.01 (d, 4H, 18, 20-tpyH), 7.01 (d,
4H, 18, 20-ArH), 4.30 (t, 4H, OCH2), 2.00 (m, 4H, OCH2CH2). HR–MS
(Acetonitrile) m/z: calcd 674.0142 for C46H36N6O2Pt2I2 (with two positive charges),
found 674.0135 for [3-2I]2+. Elemental analysis calcd (%) for C46H36N6O2Pt2I4: C
34.48, H 2.26, N 5.24; found: C 34.20, H 2.49, N 5.03.
2.3. Single crystal X-ray data collection and structure refinement
The monocrystal data of the ligand L (0.3 × 0.08 × 0.07 mm) were collected
using an Agilent Gemini EOS diffractometer with graphite-monochromated with Mo-

Kα radiation (λ = 1.54184) at 293 (2) K. An empiric absorption correction was
applied. All the non-hydrogen atoms were refined anisotropically and the hydrogen
atoms of organic molecule were refined in calculated positions, assigned isotropic
thermal parameters, and allowed to ride their parent atoms. All calculations were
performed using the SHELX2014 program package [24]. Crystallographic data
(excluding structure factors) for the structure of L reported in this paper have been
deposited with the Cambridge Crystallographic Data Centre with the reference
numbers 1853639. The crystal and refinement data are collected, and selective bone
distances and angles are given in Table 1.
2.4. DNA binding experiment
2.4.1 UV/Vis Absorption Titration
A pH 7.4 PBS (1×) buffer was used and UV/Vis spectra were recorded after each
addition of concentrated DNA stock to 50 μM Pt(II) complex solutions in a quartz
cuvette (path length = 1 cm) at 25 °C. The binding ability of complexes with CT DNA
can be estimated through the binding constant Kb, which was obtained by monitoring
the changes in the absorbance at the corresponding λmax with increasing
concentrations of CT DNA and is given by the ratio of slope to the y intercept in plots
[퐷푁퐴]
versus [DNA], according to the equation [25]:
휀퐴 ‒ 휀푓
[DNA]/(εA - εf) = [DNA]/(εb - εf) + 1/(Kb(εb - εf))
Where [DNA] is the concentration of DNA in base pairs, εA = Aobsd/[compound], εf =
the extinction coefficient for the free compound and εb = the extinction coefficient for
the compound in the fully bound form.

2.4.2 Fluorescence DNA titration
The competitive studies of complexes with EB have been investigated by means
of fluorescence spectroscopy in order to prove EB can be displaced via complex from
CT DNA-EB system. The CT DNA-EB mixture (5 μM EB and 10 μM CT DNA) was
prepared which is incubated at least 8 hours at 37 °C. The competitive ability of
complexes 1-3 have been obtained by recording the change of fluorescence emission
spectra when adding solution of complex step by step into the DNA-EB conjugation
solution.
The Stern–Volmer constant Ksv is used to evaluate the quenching efficiency for each
complex according to the equation[26].
I0/I = 1 + Ksv[Q]
Where I0 and I are the emission intensities in the absence and the presence of the
quencher. Ksv = the dynamic quenching constant, [Q] = the concentration of the
quencher (complexes or ligand), the dynamic quenching constant (Ksv, M−1) can be
obtained by the slope of the diagram I0/I versus [Q].
2.5. DNA unwinding experiment
A plasmid pBR322 DNA unwinding assay was used to investigate the effect of
complexes 1-3 on the structural of plasmid by agarose gel electrophoresis. Typical
reactions DNA-cleavage were carried out. DNA unwinding under various
concentrations of 1-3 was determined in buffer (10 mM KH2PO4, 10 mM NaCl, 10
mM EDTA, pH 7.2). Briefly, after incubation of pBR322 DNA (19 µM base pair
concentrations) with complex of different concentrations for 24 hours, loading buffer

(0.05% bromophenol blue, 50% glycerol, and 2 mM EDTA) was added to the
mixture. Then the samples were loaded onto a 0.8% agarose gel and electrophoresed
in TBE (1×) at a constant voltage of 120 mV for 80 mins. The gels were visualized in
the electrophoresis gel documentation and analysis system.
2.6. Topoisomerase I inhibition assay
Inhibition assay was used to investigate the effect of complex on the activity of
Topoisomerase I (Topo I) by agarose gel electrophoresis. In the experimental process,
a reaction mixture containing 19 µM (base pair concentrations) pBR322 DNA in
buffer (10 mM Tris–HCl, 50 mM KCl, 5 mM MgCl2, 0.1 mM DTT, 0.1 mM EDTA,
150 μg/ml BSA, pH 7.5) was incubated with 1 unit Topo I in the absence or in the
presence of complexes 1 (0-25 µM), 2 (0-70 µM) and 3 (0-150 µM), for 30 mins at
37 °C. Each sample was preincubated for 15 mins prior to the addition of pBR322
DNA. Loading buffer (0.2% bromophenol blue, 30% glycerol, 4.5% SDS, 0.2%
xylene cyanol) was added to the reaction mixture. The image was visualized using
analysis system.
2.7. Antitumor activity assays
A standard MTT assay was employed to evaluate the potential cytotoxicities
of L and three complexes against A549, HepG2 and LO2 cell lines. The cells
were maintained in DMEM supplemented with 10% FBS 100 μg mL−1
streptomycin, and 100 U mL−1 penicillin. The cells were cultured in a humidified
incubator, which provided an atmosphere of 5% CO2 and 95% air at a constant
temperature of 37 °C. In a typical experiment, the cells were seeded in a 96-well

flat-bottomed microplate at 10,000 cell per well in growth medium solution. The
microplate was incubated at 37 °C with 5% CO2 in a humidified incubator for 24
hours. The medium was then changed to growth medium with or without complexes
with serial concentrations. The microplate was then incubated at 37 °C with 5% CO2
in a humidified incubator for 48 hours. After 48 hours, MTT (20 μL, 5 mg/mL) was
added to each well. The microplate was reincubated at 37 °C in 5% CO2 for another 4
hours. Then the medium was carried out and 150 μL DMSO was added into each
well, the microplates were shaken for 10 mins. The absorbance at the wavelength of
570 nm was measured by a microplate reader (reference wavelength: 630 nm). The
IC50 value of each complex (the concentration that is required to reduce the
absorbance by 50% relatively to the controls) was analysed and calculated by SPSS.
3. Results and discussion
3.1. Synthesis and general characterization
The designed ligand L and complexes were prepared following the procedure
1
shown in Scheme 1, and characterized by elemental analysis, H NMR, 13C NMR
spectra along with HR-MS spectrometry. White crystals of L suitable for X-ray
diffraction studies were obtained by slow volatilized from a trichloromethane and
acetone mixture solution.
Information concerning X-ray data collection and crystal structure refinement is
summarized in Table 1, with the selected bond lengths and bond angles listed in Table
S1. The structure is shown in Fig. 1, that prove the synthetic ligand has the expected
molecular, and the obtained bond lengths and bond angles are similar to a reported

related terpyridine derivative [27]. For instance, the determined bond angles among
the three N atoms are (116.2(5) for Cl-N1-C5, 117.5(4) for C6-N2-C10 and 117.7(4)
for Cl1-N3-C15) respectively. As shown, within one terpyridine unit, the dihedral
angles between the middle pyridine ring (N2, C6-C10) and another two connected
pyridine rings, ring1 (N1, C1-C5) and ring2 (N2, C6-C10) are only 3.26° and 3.47°
respectively. This indicates that the terpyridine moieties are of good planar character.
Whereas, the dihedral angle between pyridine ring (N2, C6-C10) and the adjacent
benzene ring (C16-C21) is 25.82°. The most interesting feature of the crystal structure
is the mode of intermolecular interactions. Two monomers stacked in a head to head
fashion, with a distance of 3.703 Å between the two planes, suggesting that π-π
stacking interactions are involved in stabilising the structure.
3.2. DNA binding
3.2.1 UV-Vis titration
DNA is one of the targets for the studies of biologically important small
molecules, such as anticancer drugs. The complex interacted to calf thymus DNA (CT
DNA) by different modes, as intercalation, electrostatic and major/minor groove
binding [28]. Generally, intercalation was commonly detected between Pt-terpyridine
complexes and DNA [4]. The interaction of between 1-3 and CT DNA was
investigated in vitro by UV-vis spectroscopy, and the spectra of CT DNA in the
presence of increasing concentrations of 1-3 are shown in Fig. S1. The low-energy
absorption bands at 400 - 500 nm are tentatively assigned to the dπ (Pt) → π* (terpy)
transition, that is, the MLCT (MLCT = metal to ligand charge transfer) bands [29].

Herein the MLCT bands of complex 1-3 are observed at 435 nm (1), 437 nm (2) and
430 nm (3). Obviously, the titration results revealed apparent hypochromism of the
MLCT bands of complexes upon the addition of CT DNA, indicating strong π-π
stacking interaction between an aromatic chromophore and the base pairs of DNA
was involved. The DNA binding constants (Kb, Table 2) of complexes, were
determined from the Wolfe-Shimer equation [25], and were found to be 1.83 × 106,
3.21 × 106 and 8.72 ×106 M-1 for complexes 1-3 respectively. The obtained Kb
constants of complexes 1-3 were higher than that of the classical intercalator ethidium
bromide (EB, 1.4 × 106 M-1) [30], and another two mononuclear Pt-terpyridine
complexes [Pt(4-hydroxylthiophenolato)(2,2ʹ:6ʹ,2ʹʹ-terpyridine)]Cl (1.2 × 105 M-1)
[15] and [Pt (2,2ʹ:6ʹ,2ʹʹ-terpyridine)(SCH2CH2OH)]NO3 ( 2.0 × 105 M-1) [4]. Complex
3, with the coordinating group I-, exhibited the highest Kb constant among the
complexes. Besides, L was not involved here because the MLCT band was
unobserved.
3.2.2 Competitive binding with EB
Competitive DNA-binding studies with EB were also used to reveal the binding
abilities between compounds and CT DNA. The fluorescence emission spectra of the
prepared EB-DNA system in the presence of increasing amounts of complexes 1-3 of
different ratios of r (r = [compound]/[DNA]) were recorded. As shown in Fig. S2, the
addition of L and complexes 1-3 at diverse concentrations induced a dramatic
decrease of the fluorescence intensity of the emission band from the CT DNA-EB
system, where the L and complexes 1-3 induced the reductions of up to 4%, 43%,

44% and 56.5% respectively. The Stern-Volmer plot of DNA-EB (Fig. S3) illustrate
that the quenching of EB bound to DNA by these compounds, resulting in a decrease
in the fluorescence intensity. Obviously, the Ksv (Table 3) value of L is lowest (Ksv
value of 5.00 × 103 M-1), and complex 3 bearing the highest binding constant (Ksv
value of 7.60 × 104 M-1). The calculated Ksv values are more potent than some
reported partial intercalators, such as cis-[PtCl2(liriodenine)(DMSO)] (5.85 × 104 M-1)
[31]
and
[Pt(MAMP)(H2O)2](ClO4)2
(where
MAMP
=
2-[(N-
methylamino)methyl]pyridine, 0.19 × 104 M-1) [32]. Noteworthy, the results obtained
here are consistent well with UV-vis titration data. The results from above DNA
binding assays indicate strong binding affinities are occurred between complexes and
CT DNA, and intercalation is probably the predominant binding mode between them
[33].
3.3. DNA unwinding studies
The DNA unwinding property of complexes 1-3 was also explored by native
agarose gel electrophoresis using pBR322 plasmid DNA. It is reported that the
binding of an unwinding agent to the closed circular DNA (Form I) can reduce its
superhelical density and hence decrease its rate of migration in the agarose gel [34].
As shown in Fig. 2, the mobility of the supercoiled DNA was decreased upon
incubation with complexes 1-3. With an increasing concentration of the Pt(II)
complex, Form I gradually diminished accompanied by the increasing of Form II.
When the concentration of complex 1 reached to 5 μM, Form I was converted into
Form II completely. As a whole, the order of these complexes for DNA unwinding is

1 ≈ 2 > 3. It’s reported that the rate of hydrolysis of Cl- from Pt was replaced with Br-
and I- is fast, and the ability to leave increased order is shown to be Cl- <Br- <I- [35,
36]. The hydrolysis behavior of Pt(II) complexes in biological systems closely related
to their anticancer activity and binding nature with DNA. Additionally, aqua adducts
formation of Pt(II) complexes are of an essential step for the mode of action to bind
DNA and form a monofunctional adduct.
3.4. Topoisomerase I inhibition
Topoisomerases are well-established targets in antitumor research, and
compounds inhibiting these enzymes are potential antitumor agents. The inhibitory
effect of the Pt(II) complexes on Topo I was investigated by unwinding plasmid
pBR322 DNA. As shown in Fig. 3, with concentration of those complexes increased,
the Form I is diminished accompany with Form I is increased. As the concentration of
the complex 1 reached to 20 μM, the Form II was migrated to Form I completely,
demonstrating that the Pt(II) complex was in possession of remarkable concentration-
dependent inhibitory activity towards Topo I. While 40 μM of complex 2 or 100 μM
of 3 could completely suppress the activity of enzyme, indicating the inhibitory effect
of complex 1 was more potent than 2 and 3. Thus, the Pt(II) complexes described
herein possess potent concentration-dependent Topo I inhibitory activity and prevent
enzyme-mediated unwinding of supercoiled pBR322 plasmid DNA, indicating the
potential as anticancer agents.
3.5. Antitumor activity assays
The antiproliferative activity of three complexes against A549 and HepG2 cancer

cell lines and human normal liver LO2 cell lines were evaluated by MTT assays.
Cisplatin was used as a positive control in these studies. The half maximal inhibitory
concentrations (IC50) of the complexes are summarized in Table 4. Based on these
values, the in vitro antiproliferative activities of the complexes follow the order: 3 ≈
cisplatin > 2 > 1. Complex 3, with a coordination ion I-, shows higher cytotoxicity
than complexes 1 and 2 against two human cancer cell lines tested. Interestingly, a
relatively high selectivity towards cancer cells (HepG2) over normal cells (LO2) is
observed for complex 3, showing approximately 2-fold higher cytotoxicity against
cancer cell lines than that of LO2 cells.
4. Conclusions
A new ligand L and three binuclear complexes 1 ([Pt(L)Cl2]Cl2]), 2
([Pt(L)Br2]Br2]) and 3 ([Pt(L)I2]I2) were synthesized and the structure of L was
confirmed by X-ray crystallography. π-π stacking interaction was detected in the
geometric structure of L. The binding abilities between those complexes and CT DNA
were investigated and the result showed that the highest calculated DNA-binding
constants Kb of complex 3 reach to 8.72 × 106 M-1, which exhibited stronger DNA
binding affinity than that of the classical DNA intercalator EB and complexes 1-2.
Agarose gel electrophoresis study revealed that the unwinding of plasmid DNA and
Topo I inhibition activities of complex 1 was more potent compared to complexes 2-
3. Meanwhile, complex 3 achieved obvious anticancer activities against two cancer
cell lines, which was comparable to the drug cisplatin. Overall, our results suggest
that changing the leaving groups in the aromatic Pt(II) coordination center could be

promising strategies for developing Pt(II) polypyridyl complexes as important DNA
binders and potential anticancer agents.
Acknowledgements
We thank the National Natural Science Foundation of China (21401105), the
Research Fund for the Doctoral Program of Jiangxi Science & Technology Normal
University (3000990404), Jiangxi Provincial Key Laboratory of Drug Design and
Evaluation (20171BCD40015), Jiangxi Provincial Department of Education
(GJJ170695), Natural Science Foundation of Jiangxi Province (20181BAB213001)
and College Students' Science and Technology Innovation Project of Jiangxi Science
& Technology Normal University (20171103057).
Appendix A. supplementary data
CCDC 1853639 contains the supplementary crystallographic data for this article.
These data can be obtained free of charge via https://www.ccdc.cam.ac.uk/ , or from
the Crystallographic data for the structures in this paper have been deposited with the
Cambridge Crystallographic Database Center, CCDC, 12 Union Road, Cambridge
CB21EZ, UK. (Fax: +44-1223-336-033; E-mail: deposit@ccdc.cam.ac.uk).
Supplementary data associated with this article can be found online at
https://doi.org/10.1xxxxxxxxxxxx.
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Cl
Pt
2
2
Br(I)
Pt
N
N
N
N
N
N
O
O
N
N
N
N
OH
O
O
EtOH/H2O aq. NH3
RT
O
O
O
O
K2PtCl4
AgNO3/KBr
KI
OH
1,4-Dibromobutane
N
N
L
N
N
N
N
N
N
N
N
N
N
Pt
Pt
Cl
Br(I)
Complex 2(3)
Complex 1
Scheme 1. Synthesis of the new ligand and complexes (1-3).

Fig. 1. Thermal ellipsoid plot of L in ORTEP view.
Fig. 2. Concentration-dependent of pBR322 DNA unwinding by Pt(II) complex 1 (0–
8 μM), complex 2 (0–8 μM) and complex 3 (0–17.5 μM) in buffer (10 mM KH2PO4,
10 mM NaCl, 10 mM EDTA, pH 7.2) solutions at 37 °C for 24 h. Lane 1, DNA
control, lane 2 - 9, with Pt(II) complexes of increasing concentrations.

Fig. 3. Topo I inhibitory effect by Pt(II) complex 1 (0–35 μM), complex 2 (0–70 μM)
and complex 3 (0–150 μM). Lane 1, DNA control, Lane 2 – 10, with Pt(II) complexes
of various concentrations.
Table 1
Crystal and structure refinement data for L
Crystal data
Chemical formula
Formular weight(g mol-1)
Crystal system
Space group
a(Å)
L
C46H42N6O6
774.86
Monoclinic
R -3
46.504(5)
46.504(5)
4.8819(5)
b(Å)
c(Å)
α(o)
90
β(o)
90
γ(o)
120
V(Å3)
9143(2)
9
Z
Dc(g cm-3)
θ range (º)
µ(mm-1)
1.445
5.7030-51.4810
1.54184

F(000)
3672.0
Table 2
Crystal size(mm)
0.3 × 0.08 × 0.07
293(2)
The DNA-binding
constants (Kb) value of
complexes 1-3
Temperature(K)
Reflections collected
Independent reflections
Goodness-of-fit(GOF)
Largest difference in peak and hole(e Å -3)
5518
1523
Table
3
The
of
0.944
percentage
0.213, -0.156
0.1352, 0.1322
0.0602, 0.0921
fluorescence (%I/I0)
and the Stern-Volmer
constants (Ksv) value
a
b
R1 wR2 (I> 2σ (I))
a
b
R2 wR2 (all data)
aR1 = Σ‖Fo| − |Fc‖/Σ|Fo|.bwR2 =|Σw(|Fo|2 − |Fc|2)/Σ|w(Fo)2|1/2,
Where w=1/ [s2 (Fo2) + (0.0189P)2] where P=(Fo2+2Fc2)/3.
of ligand
L
and
complexes 1-3.
Compound ΔA/A0(%)
Kb(M-1)
Compound ΔI/I0 (%) Ksv (M-1)
1
2
3
26
32
29
1.83 × 106
3.21 × 106
8.72 × 106
L
1
2
3
4
5.00 × 103
5.85 × 104
6.56 × 104
7.60 × 104
43
44
49
Table 4 IC50 values of complexes 1-3
towards different cell lines. [a]
[a]
IC50 [μM]
IC50
values
are
drug
Compound A549
HepG2
LO2
concentrations necessary for 50%
inhibition of cell viability. Data are
presented as means ± standard
deviations of at least three
independent experiments and the
drug treatment period was 48 h.
1
23.3 ± 1.5 25.9 ± 1.8 29.6 ± 5.1
21.9 ± 2.3 22.8 ± 2.1 26.7 ± 2.1
13.9 ± 1.6 15.2 ± 1.4 36.6 ± 8.1
14.6 ± 1.3 13.8 ± 1.5 16.6 ± 2.4
2
3
cisplatin
Graphical abstract:
Three new binuclear platinum(II) complexes were synthesized and characterized.
Those complexes exhibited high binding affinities for calf thymus DNA and potent
topoisomerase I inhibitory activities, and significant cytotoxicity activities.

Graphical abstract - synopsis
Three new binuclear platinum (II) complexes were synthesized and characterized.
Those complexes exhibited high binding affinities for calf thymus DNA and potent
topoisomerase I inhibitory activities, and significant cytotoxicity activities.
Graphical abstract – pictogram