Dinuclear platinum(II) complexes of imidazophenanthroline-based bridging ligands as potential anticancer agents: synthesis, characterization, and in vitro cytotoxicity studies

Article abstract of DOI:10.1007/s00775-019-01656-3

Abstract: The synthesis and characterization of the dinucleating ligands 1,2-bis(2-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)phenoxy)ethane (L1) and 1,2-bis(2-(1H-imidazo[4,5-f][1, 10]phenanthrolin-2-yl)phenoxy)hexane (L2) and their dinuclear complexes [

Full text of DOI:10.1007/s00775-019-01656-3

JBIC Journal of Biological Inorganic Chemistry
https://doi.org/10.1007/s00775-019-01656-3
Dinuclear platinum(II) complexes of imidazophenanthroline‑based
bridging ligands as potential anticancer agents: synthesis,
characterization, and in vitro cytotoxicity studies
Carlson Alexander1 · N. U. Prajith1 · P. V. Priyanka1 · A. Nithyakumar1 · N. Arockia Samy1
Received: 30 October 2018 / Accepted: 27 March 2019
© Society for Biological Inorganic Chemistry (SBIC) 2019
Abstract
The synthesis and characterization of the dinucleating ligands 1,2-bis(2-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)phe-
noxy)ethane (L1) and 1,2-bis(2-(1H-imidazo[4,5-f][1, 10]phenanthrolin-2-yl)phenoxy)hexane (L2) and their dinuclear com-
plexes [Pt2(L1)Cl4] (1) and [Pt2(L2)Cl4] (2) and the in vitro cytotoxicity of the complexes against HeLa, HepG2, and MCF-7
cell lines are reported. Ligand L1 crystallizes in the orthorhombic system with the space group Pbca. The complexes 1 and
2 undergo aquation following frst-order kinetics. The MTT and trypan blue assays indicate higher cytotoxicity of the com-
plexes towards the HepG2 and MCF-7 cell lines compared to cisplatin. The AO/EB assay and fow cytometry by Annexin
V alexa fuor®488/PI double staining assay demonstrate distinct morphological changes of apoptosis in a dose dependent
manner. The cell cycle analysis shows a marked decrease in the DNA content in the G0/G1 phase with an increase in the
G2/M phase on increasing the concentration of the complexes. The potential of the complexes as anticancer agents is dem-
onstrated by their antiproliferative activity on the cell lines. The complexes interact with the major groove of DNA through
H-bonding between the imidazole N–H protons and the nucleotide residues DC`21/N4 (cytosine) for complex 1 and DT`7/
O2 (thymine) and DT`19/O2 (thymine) for complex 2, with the binding energy of −1.98 and −4.45 kcal/mol, respectively.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00775-019-01656-3) contains
supplementary material, which is available to authorized users.
* N. Arockia Samy
narockiasamy@loyolacollege.edu; narockiasamy@gmail.com
Carlson Alexander
carlson.alexander@chem.ox.ac.uk
1
Department of Chemistry, Loyola College, Chennai 600034,
India
Vol.:(0123456789)
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JBIC Journal of Biological Inorganic Chemistry
Graphical Abstract
Dinuclear Pt(II) complexes of imidazophenanthroline-based dinucleating ligands exhibit antiproliferative activity against
HeLa, HepG2, and MCF-7 cell lines
Keywords Anticancer drug · Apoptosis · DNA damage · Docking · Dinuclear platinum complexes · Imidazophenanthroline
for the treatment of chronic myelogenous leukemia, small-
cell lung cancer, and metastatic breast cancer in China [10].
Satraplatin, also known as JM216, an orally active version of
carboplatin [11], shows activity in human cancer cells with
acquired cisplatin resistance [12]. Picoplatin, also known
as JM473, retains activity against a range of cisplatin- and
oxaliplatin-resistant cells in vivo [13]. The discovery and the
development of cisplatin [14], the mechanisms of cisplatin
resistance [15], the synthetic strategies [16] and mechanism
of action [17–20] of platinum anticancer complexes, the sta-
tus of platinum anticancer drugs in the clinic and in clinical
trials [8], the resurgence of platinum-based cancer chemo-
therapy [21] the state-of-play and future of platinum drugs
[22], and the ongoing efort for the development of next
generation platinum drugs [23] are reviewed.
Introduction
Since the serendipitous discovery of the inhibition of cell
division in E. coli by cisplatin [1] and the subsequent dis-
covery of its antitumor properties [2] by Rosenberg and his
co-workers it has become the most prominent drug in cancer
chemotherapy. Cisplatin is an efective drug used in nearly
50% of all cancer patients and a forerunner in the treatment
of at least 18 distinct types of tumors [3]. However, despite
its clinical impact, it induces a number of toxic side efects
[4] and certain cancers are resistant to cisplatin therapy. In
an attempt to develop a less toxic platinum anticancer drug
carboplatin was introduced into the clinic in mid-1980s [5].
Compared with cisplatin, it is essentially devoid of nephro-
toxicity, less toxic to the gastrointestinal tract, and less neu-
rotoxic and widely used for the treatment of many types
of cancer. Oxaliplatin, the third platinum anticancer drug
approved by the FDA in 2002, shows a difering pattern of
sensitivity to that of cisplatin [6] and is active against some
cancer cells with acquired resistance to cisplatin [7].
There are extensive evidence that platinum drugs exert
their cytotoxic efects via the formation of platinum-DNA
adducts [24–26]. The bifunctional platinum-DNA cross-
links induce signifcant structural distortion in the double-
helix of DNA and impede cellular processes such as repli-
cation and transcription leading to apoptosis and cell death
[27]. Though cisplatin is widely used its efciency against
majority of the malignancies is highly limited due to its poor
circulation and delivery to the tumor as well as deactivation
Nedaplatin is used to treat cancers of the head, neck,
esophagus, and small-cell- and non-small-cell lung cancers
in Japan [8, 9]. Heptaplatin is approved for use in Korea
for the treatment of gastric cancer. Lobaplatin is approved
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JBIC Journal of Biological Inorganic Chemistry
mechanisms. Another main problem associated with the
use of the commercially available platinum complexes is
the severe side efects arising from the lack of selectivity
toward tumor cells [18].
by intercalation as anticancer agents. To improve the DNA
binding capabilities of platinum drugs we have undertaken a
research project on the development of dinuclear Pt(II) com-
plexes of imidazophenanthroline-based ligands bridged by
alkane linkers of diferent chain length. Herein we report the
syntheses, characterization, and in vitro cytotoxicity studies
of two dinuclear platinum(II) complexes (Fig. 1).
In an attempt to overcome the platinum resistance in
cancer chemotherapy ‘non-classical’ platinum complexes
[28] which include trans-platinum complexes and poly-
nuclear complexes capable of forming a diferent range of
DNA adducts and display a diferent spectrum of anticancer
activity compared to cisplatin and its analogs are developed.
trans-Platinum complexes of the general formula trans-
[PtCl2(NH3)L] (where L is a bulky, planar N-donor ligand)
have been reported to be active in vitro and in vivo [29, 30].
Polynuclear complexes containing two or more metal centers
covalently connected via appropriate linkers have emerged
as a promising class of anticancer compounds with the abil-
ity to overcome resistance in chemoresistant cancers via
new modes of action with the biomolecular targets. A host
Experimental section
Materials for in vitro assays
All reagents and plastic wares used for aseptic works were
either purchased sterile or sterilized according to litera-
ture recommendations. Propidium iodide, RNase A, MTT,
DMSO (Hybri-Max™ sterile-fltered), trypan blue, and
paraformaldehyde (Sigma-Aldrich); Dulbecco′s phosphate
bufer saline (PBS) (Himedia); agarose-type 1 and EDTA
(Merck); acridine orange and ethidium bromide (Loba Che-
mie Pvt. Ltd.); minimum essential medium (MEM), trypsin
(1:250), fetal bovine serum (FBS), and antibiotic/antimy-
cotic, 100X (Gibco); DNA ladder (100 bp), proteinase K
solution (20 mg/mL), Annexin V alexa fuor® 488/propidium
iodide dead cell apoptosis kit (Invitrogen); and DNA gel
loading dye (6X) (Thermo Scientifc) were procured and
stored according to the specifcations.
of dinuclear complexes containing trans-[PtCln(NH3)3–n
]
units bridged by alkanediamine linkers of variable length to
facilitate fexible intra- and interstrand DNA cross-links have
been reported by Farrell and his co-workers [31–34] and
reviewed by Wheate and Collins [35, 36]. The unconven-
tional DNA adducts enable diplatinum complexes to over-
come cisplatin resistant ovarian cancer cells [34]. The bind-
ing of polynuclear platinum complexes with DNA is vastly
diferent from that of cisplatin and is generally characterized
by fexible, non-directional DNA adducts, formation of a
greater percentage of interstrand to intrastrand adducts, and
the ability to induce DNA conformational changes [37, 38].
Platinum complexes of polypyridine ligands capable of
intercalating with DNA have also been studied for their anti-
tumor activities [39–42]. Aldrich-Wright and her co-workers
[43, 44] have studied dinuclear platinum complexes contain-
ing planar polyaromatic ligands which interact with DNA
Physical measurements
Infrared spectra were recorded on a Bruker Tensor II
FT-IR Spectrometer operated by Opus 7.5 software pack-
age in the range of 4000–400 cm−1. Spectra of the solid
Fig. 1 Structure of the com-
plexes [Pt2(L1)Cl4] (1) and
[Pt2(L2)Cl4] (2)
2
1
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JBIC Journal of Biological Inorganic Chemistry
samples were recorded by making transparent potassium
bromide (FT-IR grade, Fluka) pellets. CHN microanaly-
ses were carried out using a Perkin Elmer 2400 Series
II CHNS/O elemental analyzer interfaced with a Perkin
Elmer AD 6 Autobalance. Helium (99.999%) was used
as the carrier gas. The electrospray ionization (ESI) mass
spectra were recorded on a Micromass Quattro II Triple
Quadrupole mass spectrometer. The sample was dissolved
in a suitable solvent such as methanol, acetonitrile, or
water and introduced into the ESI source through a syringe
pump at the rate of 5 μL per min. The ESI capillary was
set at 3.5 kV and the cone voltage at 40 V. The average
spectrum of 6–8 scans was printed. NMR spectra were
recorded on a Bruker AVANCE III 500 MHz (AV 500)
multinuclear NMR spectrometer at 25 °C. The 13C NMR
spectra were recorded at 125 MHz at 25 °C and a standard
Synthesis of organic precursors
1,10‑Phenanthroline‑5,6‑dione
This compound was synthesized by the procedure reported
by Yamada et al. [45]. A mixture of 1,10-phenanthro-
line (6.94 g, 35 mmol) and potassium bromide (41.65 g,
350 mmol) was taken in a round bottom fask, placed in
an ice bath, and concentrated sulphuric acid (140 mL) was
added in small portions followed by the addition of con-
centrated nitric acid (70 mL) to get a homogenous solu-
tion. The resulting solution was heated for 2 h at 85 °C,
cooled to room temperature, poured into water (2.5 L),
neutralized with sodium hydrogen carbonate, and extracted
with chloroform. The chloroform extract was fash evapo-
rated and the resulting solid product was recrystallized in
hot methanol to obtain yellow needles, yield 3.97 g (54%),
mp 271 °C. Anal. Calcd for C12H6N2O2 (Mr = 210.19):
C, 68.57; H, 2.88; N, 13.33. Found: C, 68.37; H, 2.70;
N, 13.32%. IR (KBr, cm−1): 3061 νs(C–H) (aromatic),
1685 νs(C=O), 1575 νs(C=N), 1460 and 1414 νs(C=C),
739 δ(C–H). 1H NMR (500 MHz, CDCl3, 298 K): δ 7.50
(d, 2H, J = 3.5 Hz, –N–CH–CH–), 8.37 (d, 2H, J = 7 Hz,
–PyH), 8.98 (s, 2H –N–CH–). 13C NMR (125 MHz CDCl3,
298 K): δ 125.5, 127.9, 137.1, 152.7, 156.2, 178.5. FAB
MS: m/z 210 [M]+.
1
5 mm probe was used for the H and 13C NMR measure-
ments. Spectra were recorded in CDCl3 (99.9 atom % D)
or DMSO-d6 (99.90 atom % D) (Aldrich). The electronic
absorption spectra were recorded on a Shimadzu UV-2450
UV–visible spectrophotometer controlled by the UV Probe
version-2.33 software. The spectra were recorded in the
region 190–800 nm in methanol or N,N-dimethyl forma-
mide at 25 °C using a matched pair of Tefon-stoppered
quartz cell of path length 1 cm. Solvent corrections were
carried out before recording the spectra. Buchi Rotavapor
(Model R-215) was used to remove the solvent and isolate
the products at low temperature and pressure. Heto Hol-
ton cryogenic circulator bath (− 45 °C) was used to carry
out the reactions at low temperature. The circulating bath
was flled with water-ethylene glycol (3:2 v/v) for a bath
temperature of − 10 °C and pure methanol for − 45 °C.
X-ray diffraction data was collected with Bruker
AXS Kappa Apex II CCD Difractometer equipped with
graphite monochromated Mo (Kα) (λ = 0.7107 Å) radia-
tion. Crystal of suitable size fxed at the tip of the glass
fber using cyanoacrylate adhesive was mounted on the
goniometer head with the aid of video microscope and
optically centered at the goniometer axes. The automatic
cell determination routine with 36 frames at three difer-
ent orientations of the detector was employed to collect
refections for unit cell determination. Further, intensity
data for structure determination were collected through an
optimized strategy which gave an average fourfold redun-
dancy. The structures were solved by direct methods using
SIR92 and refned by full-matrix least squares techniques
using SHELXL-2014 computer program. Molecular
graphics were drawn using ORTEP3. Hydrogens on all
carbon atoms were fxed at calculated positions and refned
as riding model with C–H = 0.9 Å, Uiso(H) = 1.2Ueq(C),
whereas hydrogens on nitrogen and water oxygen were
fixed from the difference electron density peaks and
allowed to refne freely.
2,2′‑(Ethane‑1,2‑diylbis(oxy))dibenzaldehyde
It was synthesized by modifying the procedure reported
by Armstrong and Lindoy [46]. To a solution of salic-
ylaldehyde (5.0 mL, 48 mmol) in ethanol (30 mL) was
added a solution of sodium hydroxide (2 g, 50 mmol) in
water (100 mL), warmed, and 1,2-dibromoethane (2.0 mL,
24 mmol) was added followed by ethanol (20 mL) to
get a homogeneous solution. The solution was refuxed
under argon atmosphere for 100 h, cooled to 0 °C, and the
colorless needles that separated out were fltered, washed
with water, dried, and recrystallized in ether–chloroform
(1:1 v/v), yield 3.70 g (57%). Anal. Calcd for C16H14O4
(Mr = 270.28): C, 71.10; H, 5.22. Found: C, 71.02; H,
5.10%. IR (KBr, cm−1): 2869 νs(C–H), 1239 νas(C–O–C),
1061 νs(C–O–C), 756 δ(C–H) (aromatic), 2922 νas(C–H),
2866 νs(C–H) (aliphatic), 1687 νs(C=O), 1485 and 1452
νs(C=C) (aromatic ring), 756 δ(C–H). 1H NMR (500 MHz,
CDCl3, 298 K): δ 10.45 (s, 2H, –CHO), 7.86 (d, 2H,
J = 7.5 Hz, H2), 7.09 (t, 2H, J = 8.5 Hz, H3), 7.59 (t, 2H,
J = 7.7 Hz, H4), 7.07 (d, 2H, J = 8.5 Hz, H5), 4.54 (s, 4H,
–O–CH2–). 13C NMR (125 MHz, CDCl3, 298 K) δ 67.0,
112.8, 121.4, 125.2, 128.6, 135.9, 160.7, 189.3. MS: m/z
271 [M]+.
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JBIC Journal of Biological Inorganic Chemistry
2,2′‑(Hexane‑1,6‑diylbis(oxy))dibenzaldehyde
121.8, 122.6, 129.2, 131.2, 143.5, 147.3, 147.5, 156.1.
ESI–MS: m/z 651 [M]+.
To a solution of salicylaldehyde (5.0 mL, 48 mmol) in eth-
anol (30 mL) was added a solution of sodium hydroxide
(2 g, 50 mmol) in water (100 mL) and refuxed for 30 min.
A solution of 1,6-dibromohexane (3.7 mL, 24 mmol) in
ethanol (10 mL) was added dropwise to the reaction mix-
ture and refuxed for 100 h under argon atmosphere. The
solvent was fash evaporated to dryness, the residue was
washed with water, and recrystallized in ether to give color-
less needles, yield 4.00 g (51%). Anal. Calcd for C20H22O4
(Mr =326.39): C, 73.60; H, 6.79. Found: C, 73.55; H, 6.77%.
IR (KBr, cm−1): 3420 νs(N–H), 2852 νs(C–H) (aliphatic),
1682 νs(C=O), 1487 and 1456 νs(C=C) (aromatic ring),
1247 νas(C–O–C), 1036 νs(C–O–C), 760 δ(C–H). 1H NMR
(500 MHz, CDCl3, 298 K): δ 1.59 (q, 4H, –CH2–), 1.92 (q,
4H, –CH2–), 4.1 (t, 4H, J=6.5 Hz, –O–CH2–), 10.50 (s, 2H,
–CHO), 7.82 (d, 2H, J=7.5 Hz, H2), 7.01 (d, 2H, J=7.5 Hz,
H3), 7.52 (t, 2H, J=7 Hz, H4), 6.96 (t, 2H, J=8.5 Hz, H5).
13C NMR (125 MHz, CDCl3, 298 K): δ 25.8, 29.0, 68.2,
112.5, 120.5, 128.2, 124.8, 135.9, 161.4, 189.7. ESI–MS:
m/z 328 [M+H]+.
L2. Purified by column chromatography using ace-
tone–acetonitrile–acetic acid (5:4:1 v/v) as the eluent. The
yellow powder obtained was washed with water and dried in
vacuo to get bright yellow powder, yield 1.49 g (57%). Anal.
Calcd for C44H34N8O2 (Mr =706.79): C, 74.77; H, 4.85; N,
15.85. Found: C, 74.69; H, 4.81; N, 15.80%. IR (KBr, cm−1):
3412 νs(N–H), 3060 νs(C–H), 2937 νs(C–H), 1607 νs(C=N),
1239 νs(C–O–C), 1091 νs(C–O–C), 740 δ(C–H). 1H NMR
(500 MHz, DMSO-d6, 298 K): δ 1.44 (q, 4H, –CH2–),
1.76 (q, 4H, –CH2–), 3.99 (t, 4H, –O–CH2–), 6.99 (d, 2H,
J = 7.5 Hz, H4), 7.38 (m, 2H, H5), 7.10 (m, 2H, H6), 8.05
(d, 2H, J = 7.0 Hz, H7), 13.12 (s, 2H, –NH), 8.89 (d, 4H,
J=7.5 Hz, H9), 7.75 (m, 4H, H10), 8.96 (m, 4H, H11). 13
C
NMR (125 MHz, DMSO-d6, 298 K): δ 156.3, 149.4, 146.7,
131.7, 131.6, 130.4, 130.3, 124.6, 121.1, 113.0, 69.0, 28.8,
26.4. ESI–MS: m/z 706 [M]+.
Synthesis of dinuclear platinum(II) complexes
[Pt2(L1)Cl4] (1) and [Pt2(L2)Cl4] (2)
Synthesis of 1,2‑bis(2‑(1H‑imidazo[4,5‑f]
[1, 10]phenanthrolin‑2‑yl)phenoxy)ethane
(L1) and 1,6‑bis(2‑(1H‑imidazo[4,5‑f][1, 10]
phenanthrolin‑2‑yl)phenoxy)hexane (L2)
General procedure
1,2-Bis(2-(1H-imidazo[4,5-f][1,10] phenanthrolin-
2-yl)phenoxy)ethane (L1) (0.65 g, 1 mmol) (for 1) and
1,6-bis(2-(1H-imidazo[4,5-f][1,10] phenanthrolin-2-yl)phe-
noxy)hexane (L2) (0.71 g, 1 mmol) (for 2) in hydrochloric
acid (50 mL, 2 M) was added to a solution of potassium
tetrachloroplatinate(II) (1.00 g, 2.4 mmol) in water (10 mL)
and stirred for 8 h to give an yellow solid. This solid product
was fltered, washed with water, and dried in vacuo.
[Pt2(L1)Cl4] (1). Recrystallized in DMF:methanol
(1:1 v/v) to obtain a pale yellow crystalline solid,
yield 0.60 g (51%). Anal. Calcd for C40H26Cl4N8O2Pt2
(Mr =1182.65): C, 40.62; H, 2.21; N, 9.47. Found C, 40.60;
H, 2.21; N, 9.38%. IR (KBr, cm−1): 3396 νs(N–H), 1235
νas(C–O–C), 1057 νs(C–O–C), 926 γ(C–H) (pyridine ring),
740 δ(C–H). 1H NMR (500 MHz, DMSO-d6, 298 K): δ 4.98
(m, 4H, –O–CH2–), 7.17 (m, 2H, H2), 7.63 (m, 2H, H3), 7.51
(m, 2H, H4), 8.16 (m, 2H, H5), 12.38 (s, 2H, –NH), 8.82 (m,
4H, H7), 7.74 (m, 4H, H8), 9.30 (m, 4H, H9). ESI MS: m/z
1180 [M]+.
General procedure
A solution of 2,2′-(ethane-1,2-diylbis(oxy))dibenzal-
dehyde) (1.00 g, 3.7 mmol) (for L1) and 2,2′-(hexane-
1,6-diylbis(oxy))dibenzaldehyde (1.21 g, 3.7 mmol) (for L2)
in acetic acid was added to a hot solution of 1,10-phenanth-
roline-5,6-dione (2.10 g, 10 mmol) and ammonium acetate
(15.42 g, 200 mmol) in acetic acid (180 mL) under stirring
and refuxed for 12 h under an argon atmosphere. The reac-
tion mixture was cooled to room temperature, transferred
into a beaker containing 200 mL water, and neutralized by
the drop wise addition of aqueous ammonia (25%) with
stirring. The yellow product that separated out was fltered,
washed with water, and dried in vacuo.
L1. Recrystallized in methanol to obtain yellow crys-
tals, yield 1.90 g (79%). Anal. Calcd for C40H26N8O2
(Mr =650.69): C, 73.84; H, 4.03; N, 17.22. Found: C, 73.80;
H, 4.02; N, 17.20%. IR (KBr, cm−1): 3386 νs(N–H), 1235
νas(C–O–C), 1057 νs(C–O–C), 1607 νs(C=N), 805 γ(C–H)
(pyridine ring). 1H NMR (500 MHz, DMSO-d6, 298 K): δ
4.90 (s, 4H, –O–CH2–), 7.83 (d, 2H, J=7.5 Hz, H2), 7.45 (t,
4H, J=8.5 Hz, H3), 7.05 (t, 4H, J=8.0 Hz, H4), 7.50 (d, 2H,
J=7.0 Hz, H5), 11.50 (s, 2H, –NH), 7.95 (d, 2H, J=8.5 Hz,
H7), 7.25 (t, 4H, J = 8.0 Hz, H8), 8.78 (d, 2H, J = 9.5 Hz,
H9). 13C NMR (125 MHz, DMSO-d6, 298 K): 113.9, 119.2,
[Pt2(L2)Cl4] (2). Recrystallized in DMSO to obtain a
pale yellow powder, yield 0.69 g (56%). Anal. Calcd for
C44H34Cl2N8O2Pt2 (Mr = 1238.76): C, 42.66; H, 2.77; N,
9.05. Found C, 42.61; H, 2.76; N, 9.01%. IR (KBr, cm−1):
3396 νs(N–H), 2926 νs(C–H) (aliphatic), 1234 νas(C–O–C),
1087 νs(C–O–C), 751 δ(C–H). 1H NMR (500 MHz, DMSO-
d6, 298 K): δ 1.23 (m, 4H, –CH2–), 1.87 (m, 4H, –CH2–),
4.10 (m, 4H, –O–CH2–), 6.97 (m, 4H, H4 and H7), 7.46 (m,
2H, H6), 7.67 (m, 2H, H5), 9.30 (m, 4H, H9), 12.90 (m, 2H,
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JBIC Journal of Biological Inorganic Chemistry
–NH), 7.87 (m, 4H, H10), 8.97 (m, 4H, H11). MALDI-TOF:
m/z 1201 [M-Cl]+.
using fve concentrations of the complexes (0.1, 0.5, 1.0, 5.0,
and 10.0 µM) for 24 h.
Trypan blue exclusion assay
Kinetics of aquation
Cell viability using trypan blue assay was carried out by incu-
bating cells (1×106 cells/well) with diferent concentrations
(0.1, 0.5, 1.0, 5.0, and 10.0 µM) of the complexes 1 and 2 in
six-well plates at 37 °C for 3 h. The cells were harvested and
centrifuged at 250×G for 5 min and resuspended in 100 μL
phosphate bufer saline (PBS). Trypan blue solution (100 μL,
0.4%) was mixed with the above suspension and incubated
at room temperature for 3 min. The live and dead cells were
scored using a hemocytometer. The data were analyzed by
plotting percentage of viable cells against concentration.
The kinetics of aquation of the complexes 1 and 2 were stud-
ied in water using an Agilent Cary 8454 UV–visible spec-
trophotometer operated by UV–Vis Chemstation software.
Stock solutions of the complexes were prepared in DMSO
(10 mM) and an aliquot (30 μL) was diluted to 3 mL in
water in a quartz cuvette and the UV–visible spectra of each
solution was immediately recorded in the wavelength region
190–1100 nm at 1-min intervals. The wavelength at which
the absorbance changes signifcantly with time was selected
to monitor the changes in absorbance as a function of time
for the determination of rate constant [47].
MTT assay and estimation of IC50 values
The time-dependent changes in the absorbance at selected
wavelength were recorded at 30-s intervals. The absorbance
versus time data for each complex was computer-ftted to the
frst-order rate equation (Eq. 1) using Origin Pro 8.5 soft-
ware. The slope of the straight line gave the rate constant for
The colorimetric MTT assay was performed to quantify the
cytotoxicity of the complexes 1 and 2 on the cell lines. Cells
(2×104 cells/well) were treated with fve diferent concen-
trations (0.1, 0.5, 1.0, 5.0, and 10.0 μM) of the complexes
in triplicate in a 96-well plate in 150 μL GM-10 per well,
and incubated at 37 °C under 5% CO2 and 95% humidity
conditions for 48 h. GM-10 containing 0.2% DMSO was
used as the negative control. After the incubation period, the
supernatant media was replaced with 100 μL MTT solution
in PBS (0.75 mg/mL fnal concentration) and incubated at
37 °C for 4 h, and DMSO (100 μL) was added to each well
for solubilizing the MTT formazan crystals. After incubating
at room temperature for 30 min, the absorbance was meas-
ured at 570 nm using an ELISA plate reader. The IC50 values
were obtained from the percentage cell viability versus log
[C] plot of the dose–response data.
aquation (kH O) and the half-life period (t1/2) was calculated
2
using the k value (Eq. 2).
ln A = ln A0−kt
(1)
t1∕2 = 0.6931∕k
(2)
where A0 and A are the absorbance at the start of the reaction
and at time t, respectively.
Cell culture
The human breast cancer adenocarcinoma cell line MCF-
7, human liver hepatocellular carcinoma cell line HepG2,
and human cervical adenocarcinoma cell line HeLa were
procured from the National Center for Cell Sciences, Pune,
India. HeLa cells were maintained in GM-5 minimum essen-
tial medium (MEM, Gibco®) supplemented with 5% FBS
(fetal bovine serum, Gibco®) and 1% Ab/Am (Antibiotic/
antimycotic, 100X, Gibco™) and MCF-7 and HepG2 in
GM-10 (MEM, 10% FBS, and 1% Ab/Am) at 37 °C, 95%
humidity, and 5% CO2. HeLa cells were passaged thrice
while MCF-7 and HepG2 twice in a week.
AO/EB dual staining assay
The HeLa cells were seeded on sterile glass coverslips kept
on six-well plates (105 cells/well) and incubated with dif-
ferent concentrations (1 and 5 μM) of the complexes 1 and
2 at 37 °C for 24 h. The cells were then washed with PBS,
fxed using paraformaldehyde (4% w/v), counter stained with
40 μL of AO/EB solution (1 part 25 μg/mL AO in PBS and
one part 25 μg/mL EB in PBS), washed thoroughly with
PBS, and mounted on microscopic slide over 15% glycerol.
Images were captured in line mode with 20× objective using
a Zeiss LSM 700 laser scanning confocal microscope.
In vitro cytotoxicity assays
Stock solutions of the platinum complexes 1 and 2 required for
diferent assays were prepared in DMSO (sterile-fltered) and
diluted with the growth media maintaining the fnal DMSO
concentration of 0.2%. The concentration of DMSO in the
treated and the control wells was kept the same. This concen-
tration of DMSO (0.2%) was found to be non-toxic to the cell
lines. The MTT and the trypan blue assays were carried out
Annexin V alexa fuor® 488/propidium iodide apoptosis
assay
The apoptotic/necrotic cells were quantifed by fow cytom-
etry using the Annexin V alexa fuor® 488/propidium iodide
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JBIC Journal of Biological Inorganic Chemistry
dead cell apoptosis kit (Invitrogen™). The HeLa cells in a
six-well plate (1×106 cells/well) were treated with difer-
ent concentrations (1 and 5 μM) of the complexes 1 and 2
and incubated at 37 °C for 24 h. The cells were trypsinized,
washed twice with ice cold PBS, and resuspended in 100 μL
of 1X annexin V binding bufer. Annexin V alexa fuor®
488 (5 μL) and propidium iodide (1 μL, 100 μg/mL) were
added to each sample and incubated at room temperature
for 15 min. To this cell suspension was added 400 μL 1X
annexin V binding bufer, gently mixed, and analyzed using
Beckmann Coulter Mofo XDP cell sorter operated using
summit 4.2 software.
respectively. The lowest docked energy conformation was
selected as the best binding mode. The docking interactions
were visualized using PyMol 1.4.1 software.
Results and discussion
Synthesis and characterization of ligands
The condensation of 2,2′-(ethane-1,2-diylbis(oxy))dibenza-
ldehyde (1 equiv) and 2,2′-(1,6-hexanediylbis(oxy))diben-
zaldehyde with 1,10-phenanthroline-5,6-dione (2.4 equiv)
in the presence of ammonium acetate (40 equiv) in acetic
acid under argon atmosphere followed by neutralization with
ammonia yields L1 and L2, respectively (Scheme 1). The
ESI mass spectrum of L1 shows a peak at m/z 651 due to
the molecular ion [M]+. The electronic absorption spectrum
of L1 in methanol contains two absorption bands at 276
(ε=60,200 33 L/mol/cm) and 228 nm (ε=63,000 56 L/
mol/cm) due to the π–π* transitions. The ESI mass spectrum
of L2 shows a peak at m/z 706 corresponding to the molecu-
lar ion [M]+. The electronic absorption spectrum of L2 in
DMF contains absorption bands at 276 (ε=30,118 46 L/
mol/cm) and 312 nm (ε=19,308 32 L/mol/cm) assignable
to the π–π* transitions.
Cell cycle analysis
HeLa cells with a cell density of 1 × 106 cells/mL were
treated with 1.0 and 5.0 μM concentration of the complexes
1 and 2 at 37 °C for 24 h. Treated and untreated cells were
harvested in 1.5 mL microcentrifuge tube, washed with PBS,
and fxed with 1 mL of 70% ethanol at 4 °C for 30 min. Cells
were then washed twice with 1 mL PBS solution, centri-
fuged at 850×G for 10 min, and discarded the supernatant
carefully. The cell pellets were suspended in 0.2 mL PBS,
treated with 50 μL RNAse A solution (100 μg/mL), and
incubated for 30 min at 37 °C. The suspension was mixed
gently, treated with 200 μL of propidium iodide (50 μg/mL),
and kept in the dark at 4 °C. The cell cycle distribution was
measured using BD FACS Calibur™ fow cytometer and the
data were analyzed using Cellquest Pro software.
Synthesis and characterization of complexes
The reaction of the preformed ligands L1 and L2 with a
solution of potassium tetrachloroplatinate(II) in hydrochlo-
ric acid (2 M) gives the complexes 1 and 2, respectively.
The ESI mass spectrum of 1 shows the molecular ion peak
at m/z 1180 [M]+. The electronic absorption spectrum in
DMF shows bands at 280 (ε =81,500 32 L/mol/cm) and
314 nm (ε=62,500 42 L/mol/cm) assignable to the ligand
centered π–π* transitions. The absorption band at 419 nm
(ε = 7300 54 L/mol/cm) is due to the MLCT transition.
The MALDI-TOF mass spectrum shows a peak at m/z
1201 corresponding to the species [M–Cl]+. The electronic
absorption spectrum of 2 in DMF shows bands at 277
(ε=49,100 64 L/mol/cm) and 314 nm (ε=39,300 50 L/
mol/cm) assignable to the π–π* transitions and a band at
407 nm (ε=4500 46 L/mol/cm) due to the MLCT transi-
tion. The ligand centered π–π* transitions are bathochromi-
cally shifted due to coordination to the metal ions.
Molecular docking study with DNA
The molecular docking study of the complexes 1 and 2 with
DNA was performed using AutoDock 4.2 software and the
AutoDock scoring function which is an interactive molecu-
lar graphics program for the interaction, docking calcula-
tions, and to identify possible binding sites of these bio-
molecules. The energy optimized structures of the platinum
complexes as a .mol fle were used as input for AutoDock
software and were converted to .pdb format for perform-
ing the docking experiment. The crystal structure of DNA
dodecamer (5′-CGCGAATTCGCG-3′)2 (PDB ID: 1BNA)
was retrieved from the protein data bank (http://www.rcsb.
org/.pdb). The DNA was treated as a rigid target molecule
and the platinum complexes as fexible ligand molecules.
While preparing the receptor, the water molecules were
removed and polar hydrogen atoms and the Gasteiger and
Kollman partial atomic charges were added to the DNA. A
grid box of 90×90×90 dimension (total grid points per map
of 753,571) and grid spacing 0.375 Å was developed for the
docking experiment. The default docking parameters were
set except the number of evaluations and the Lamarckian
genetic algorithm runs which were set at 2.5 million and 10,
Crystal structure of the ligand L1
The ligand L1 crystallizes in the orthorhombic system
with the space group Pbca with the unit cell dimensions:
a = 29.283(2) Å, b = 17.8424(13) Å, c = 14.2023(10)
Å, α= β= γ=90°, V=7420.4 Å3, Z=8. The ORTEP and the
packing diagrams of the crystal structure illustrate zig–zag
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JBIC Journal of Biological Inorganic Chemistry
L1
L2
Scheme 1 The synthetic pathway of the organic precursors and ligands L1 and L2
arrangement of the molecules along the b-axis leading to a
ladder-like structure stabilized by aromatic π-stacking inter-
actions (C37–H—C6 3.290 Å, C36–H—C7 3.358 Å, and
C24–H—C13 Å) between the phenyl group and the imidazo-
phenanthroline aromatic rings of the neighboring molecules.
The ORTEP representation of L1 is presented in Fig. S10
(ESI). There also exists two weak intramolecular hydrogen
bondings between the imidazophenanthroline NH hydrogen
and the linker oxygen (N4–H–O1 2.580 Å and N7–H—O2
2.670 Å) stabilizing the bent geometry of the molecule. The
packing diagram, ladder-like arrangement of molecules
along the b-axis, aromatic stacking interactions between the
neighboring molecules, and intramolecular hydrogen bond-
ing are presented in Fig. S11 (ESI). The crystallographic
data are shown in Table S1 (ESI).
by UV–Vis spectroscopy at 298 K, follows frst-order kinet-
ics with the rate constants nearly ten and two times faster
than that of cisplatin [49]. The time-dependent changes in
the absorbance of the complexes 1 and 2 (λmax =276 nm) in
water at 298 K are depicted in Fig. 2. The rate constants and
t1/2 values are given in Table 1.
Cytotoxicity studies
MTT assay
On treating the HeLa, HepG-2, and MCF-7 cells lines with
0.1, 0.5, 1, 5, and 10 µM solutions of the complexes 1 and 2
for 48 h at 37 °C high mortality rate is observed at 5.0 and
10 µM concentrations. The IC50 values of the complexes 1,
2, and cisplatin on these cell lines, listed in Table 2, shows
higher lethality of the complexes towards the HepG2 and
MCF-7 cell lines compared to cisplatin. The infuence of
the spacer length on in vitro anticancer activity has been
reported for dinuclear Ru(II) [50, 51] and Au(I) [51] com-
plexes where the cytotoxicity correlates to the lipophilicity,
which increases with increasing linker length. However, in
our present study, there is no signifcant change in the cyto-
toxicity with an increase in the linker length. This similar-
ity in the antiproliferative activity of the complexes 1 and
2 could be attributed to the combined efect of the higher
lipophilicity and DNA groove binding potential (vide infra)
and a sixfold decrease in the rate of aquation of 2 compared
to that of 1. The cytotoxicity profles of the complexes and
cisplatin against the cell lines are presented in Fig. 3.
Kinetics of aquation of complexes
The aquation of platinum complexes used as anticancer
agents assumes importance since the seminal investigation
by Lippard et al. [48] who reported that the biological mech-
anism of action of cisplatin involves binding of [PtCl(OH2)
(NH3)2]+, the hemi-hydrolyzed product of cisplatin formed
in vivo, with DNA to form monofunctional adduct. In this
milieu, aquation of the complexes 1 and 2 are studied by
monitoring the changes in absorbance with time. The elec-
tronic absorption spectra of the stock solutions of complexes
1 and 2 in DMSO diluted in water feature a strong band at
276 nm and the absorbance decreases gradually with time. In
the case of complex 1 there is an isobestic point at 338 nm.
The rates of aquation of the complexes 1 and 2, determined
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JBIC Journal of Biological Inorganic Chemistry
Fig. 2 Time dependent elec-
tronic absorption spectra for the
aquation of a [Pt2(L1)Cl4] (1)
and b [Pt2(L2)Cl4] (2) and time-
dependence of the absorbance
at 276 nm for the hydrolysis of c
[Pt2(L1)Cl4] (1) and d [Pt2(L2)
Cl4] (2) in aqueous solution at
298 K
Table 1 The rate constants and t1/2 values for the aquation of com-
majority of the cells are damaged indicating lethality of the
complexes towards the cell lines (Fig. 4).
plexes
Complex
kH O (10−5/s)
t1/2 (min)
2
Morphological assessment of apoptosis by AO/EB
dual staining
[Pt2(L1)Cl4] (1)
[Pt2(L2)Cl4] (2)
29.30
5.30
39.42
217.96
The HeLa cell lines on treatment with the complexes 1 and 2
at 1 and 5 µM concentrations for 24 h at 37 °C and staining
with AO/EB show distinct morphological changes of apop-
tosis as shown in Fig. 5. At 1 µM concentration of the com-
plexes less apoptotic bodies are seen and the nuclei appear
green, while at 5 µM concentration more apoptotic bodies
with cell shrinkage and chromatin condensation are seen and
the nuclei appear bright green. The control cell lines do not
show any such morphological changes.
Table 2 IC50 value of the complexes 1 and 2 and cisplatin from MTT
assay
Complex
HeLa cells
HepG-2 cells
MCF-7 cells
[Pt2(L1)Cl4] (1)
[Pt2(L2)Cl4] (2)
Cisplatin
3.48 1.9
3.29 2.5
2.62 1.6
3.67 2.2
3.79 3.0
7.59 0.9
3.53 2.4
3.67 3.4
15.18 2.8
Flow cytometry by Annexin V alexa fuor® 488/PI
double staining
Trypan blue assay
Trypan blue exclusion assay is based on the principle that
live cells which possess intact cell membranes will exclude
the dye, whereas dead cells do not. Thus, when a cell sus-
pension is treated with trypan blue viable cells will have
clear cytoplasm, while nonviable cells a blue cytoplasm. In
this study when the HeLa, HepG-2, and MCF-7 cell lines are
treated with the complexes 1 and 2 at diferent concentra-
tions (0.1, 0.5, 1, 5, and 10 µM) for 24 h at 37 °C a decrease
in the cell proliferation is observed with increasing concen-
tration of the complexes. At 5 and 10 µM concentrations
Flow cytometry by Annexin V alexa fuor® 488 conjugate
provides a quick and reliable detection method for studying
the externalization of phosphatidylserine, one of the earliest
indicators of apoptosis. The diference in the fuorescence
intensity between apoptotic and nonapoptotic cells stained
with the fuorescent Annexin V conjugates measured by fow
cytometry is typically about 100-fold. It ofers the possibil-
ity of detecting early phases of apoptosis before the loss of
cell membrane integrity and permits measurements of the
kinetics of apoptotic death in relation to the cell cycle. The
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JBIC Journal of Biological Inorganic Chemistry
Fig. 3 MTT assay profles of the HeLa, MCF-7, and HepG2 cell lines treated with diferent concentrations (0.1, 0.5, 1, 5, and 10 μM) of the
complexes 1 and 2 for 24 h at 37 °C
Fig. 4 Trypan blue assay: cytotoxicity profles of the HeLa, MCF-7, and HepG2 cell lines incubated with diferent concentrations of the com-
plexes 1 and 2 (0.1, 0.5, 1, 5, and 10 µM) for 24 h at 37 °C
Annexin V conjugated alexa fuor® 488/PI binding assay
is used to evaluate live cells, dead cells, and apoptotic and
necrotic cells. Propidium iodide stains necrotic cells with
red fuorescence, while the apoptotic cells show green fuo-
rescence, dead cells show red and green fuorescence, and
live cells show little or no fuorescence. When the HeLa
cell lines are inoculated with the complexes 1 and 2 at 1 µM
concentration for 24 h at 37 °C followed by staining with
Annexin V alexa fuor® 488 and co-staining with PI, a signif-
icant apoptosis is observed with 21.26 and 22.63% live cells
(R3) and 37.58 and 26.55% early apoptotic cells (R4), 25.63
and 35.37% late apoptotic cells (R2), and 15.55 and 15.45%
nonviable cells (R1), respectively, compared to the control
cells. At 5 µM concentration of the complexes a pronounced
increase in apoptosis is observed with 17.26 and 11.35%
viable cells (R3), 28.75 and 14.68% early apoptotic cells
(R4), 36.35 and 51.52% late apoptotic cells (R2), and 17.63
and 22.44% dead cells (R1), respectively. The percentage
of dead cells (R1), nonviable or necrotic cells (R2), viable
or live cells (R3), and apoptotic cells (R4) are indicated in
Fig. 6. These results indicate that complex 2 induces more
apoptosis than 1 at higher concentration which could be cor-
related to the higher lipophilicity of the former containing
longer alkyl spacer. However, the IC50 values of the com-
plexes 1 and 2 are comparable due to a sixfold decrease in
the rate of aquation of 2 compared to that of 1.
Cell cycle analysis by fow cytometry
The HeLa cell lines upon inoculation with 1 µM solution
of the complexes 1 and 2 a marked decrease in the DNA
content in the G0/G1 phase from 87.32 for the control to
69.38 and 66.41% and an increase in the G2/M phase from
7.90 to 26.97 and 29.82%, respectively, is observed. At
5 µM concentration of the complexes a rapid decrease in the
DNA content in the G0/G1 phase from 87.32 for the control
to 62.01 and 57.19% with an increase in the G2/M phase
from 7.90 to 35.65 and 40.03%, respectively, is observed.
On increasing the concentration of the complexes the DNA
content in the G2/M phases increases indicating cell death
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JBIC Journal of Biological Inorganic Chemistry
Fig. 5 Fluorescence microscopic images of the HeLa cell lines incubated with the complexes 1 and 2 (1 and 5 µM concentrations) for 24 h at
37 °C and stained with AO/EB
Fig. 6 Flow cytometry examination of HeLa cell lines after treatment with the complexes 1 and 2 for 24 h at 37 °C and staining with Annexin V
alexa fuor® 488/PI (R1: dead cells, R2: nonviable or necrotic cells, R3: viable or live cells, and R4: apoptotic cells, percentage)
1 3

JBIC Journal of Biological Inorganic Chemistry
in a dose dependent manner. Our study demonstrates the
potential of the complexes 1 and 2 as anticancer agents as
they control the proliferation of the HeLa cells. The fow
cytometry for the DNA quantifcation of HeLa cells after
treating with the complexes followed by staining with PI is
shown in Fig. 7.
potential of the complex 2 compared to 1. Our study reveals
that complex 2 binds efciently with DNA than complex 1.
Conclusion
Polynuclear platinum complexes represent a new class of
anticancer drugs which could expand the realm of platinum
chemotherapy treatment. Such complexes containing two
or more platinum centers that can each covalently bind to
DNA are capable of forming a completely diferent range
of DNA adducts compared to cisplatin and its analogs. The
synthetic strategy developed in this study could be exploited
for the synthesis of a variety of imidazophenanthroline-
based bridging ligands by changing the aldehyde precursors
and linker moieties. The infuence of the spacer length on
the lipophilicity and DNA binding ability of the complexes
could be exploited to develop dinuclear complexes with dif-
ferent linker moieties. The present study has the potential
for studying the pH-dependent anticancer activity as the
imidazophenanthroline-based bridging ligand contains ion-
isable N–H proton.
Molecular docking study
The molecular docking study of the complexes 1 and 2 with
DNA (PDB ID: 1BNA) is performed to evaluate the possible
binding site, interaction mode, inhibition constant, desolva-
tion energy, and binding afnity (the data are presented in
Table 3). The complexes interact with DNA by hydrogen
bonding at the major groove through the imidazole N–H
protons of the complexes 1 and 2 and the nucleotide resi-
dues DC`21/N4 (cytosine) for 1 and DT`7/O2 (thymine) and
DT`19/O2 (thymine) for 2 at the distances of 2.0 Å and 2.2 Å
and 2.0 Å, respectively (Fig. 8). The relative binding energy
of the docked complexes 1 and 2 with DNA are −1.98 and
−4.45 kcal/mol, respectively. The inhibition constant and
the desolvation energy also support the higher binding
Fig. 7 Flow cytometry analysis of HeLa cell lines for DNA quantifcation after treating with the complexes 1 and 2 for 24 h at 37 °C and stained
with PI
Table 3 Parameters of binding
Complex
Binding energy
(kcal/mol)
Inhibition constant Desolvation
Ligand
ef-
of the complexes 1 and 2 with
(mM)
energy (kcal/mol)
DNA
ciency
[Pt2(L1)Cl4] (1)
[Pt2(L2)Cl4] (2)
−1.98
−4.45
35.09
0.55
−5.26
−6.60
0.03
0.08
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JBIC Journal of Biological Inorganic Chemistry
Fig. 8 Graphical representation of the interaction of complex 1 (top row) and 2 (bottom row) with DNA (PDB ID: 1BDNA)
Acknowledgements Partial fnancial support to Carlson Alexander
from Loyola Research Park is gratefully acknowledged. NAS thanks
SERB, New Delhi, for fnancial support. The service rendered by the
Sophisticated Analytical Instrumentation Facilities (SAIF) at IIT-
Madras and Punjab and Jawaharlal Nehru Universities for recording
ESI-TOF and MALDI-TOF mass spectra and NMR spectra is grate-
fully acknowledged. We thank Dr. P. K. Sudhadevi Antharjanam, SAIF,
IIT-Madras, Chennai, for her help in solving the crystal structure. We
thank Dr. J. John Kirubaharan and Dr. S. Rajalakshmi, Department
of Veterinary Microbiology, Madras Veterinary College, Chennai, for
their invaluable help in carrying out the in vitro studies.
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Compliance with ethical standards
Conflict of interest There are no conficts to declare.
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ESI-TOF mass spectra, 500 MHz H NMR spectra, and
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