Inorganica Chimica Acta 467 (2017) 264–275

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Inorganica Chimica Acta

journal homepage: www.elsevier.com/locate/ica

Research paper

Synthesis, physicochemical properties, thermal analysis and biological

application of phosphorescent cationic iridium(III) complexes
V. Thamilarasan a, V. Sethuraman a, P. Karunakaran b, M. Sethupathi a, P. Manisankar a, C. Selvaraju b,
N. Sengottuvelan a,⇑
a
Department of Industrial Chemistry, Alagappa University, Karaikudi 630 003, India
b
National Centre for Ultrafast Processes, University of Madras, Taramani Campus, Chennai 600 113, India

a r t i c l e i n f o a b s t r a c t

Article history: Cyclometalating iridium(III) complexes with formula, [Ir(cpy)2(biim)Cl] 1, [Ir(cpy)2(bpy)Cl] 2 and
Received 22 June 2017 [Ir(cpy)2(dppe)Cl] 3 (where, cpy = 9-(pyridine-2-yl)-9H-carbazole, biim = biimidazole, bpy = 2,20 -bipyri-
Received in revised form 26 July 2017 dine, dppe = 1,2-bis(diphenylphosphino)ethane) were synthesized and characterized by elemental anal-
Accepted 30 July 2017
ysis, 1H, 13C NMR, and ESI-Mass spectral studies. The complex 1–3 show their emissions in green emitting
Available online 1 August 2017
region (kPL,max = 536–580 nm), and the emission maxima can be tuned by changing the ancillary ligand.
The phosphorescent line shape indicates that the emissions originate predominantly from 3MLCT states
Keywords:
with little admixture of ligand-based 3(p-p⁄) excited states. These complexes exhibited biexponential
Iridium(III) complexes
Photophysical
phosphorescence decays with relatively short lifetimes of 0.40–12.35 ms in solution state at room temper-
Electrochemical properties ature and the relaxation dynamics of 1–3 are O2 dependent. The electrochemical and thermal properties
DNA were systematically evaluated. Binding of these complexes (1–3) with calf thymus DNA was investigated
Protein interaction by UV–Vis, fluorescence, circular dichroic spectroscopy, viscosity measurements and computer aided
Cytotoxic activities molecular docking studies. The binding constant of complexes (1–3) with ct-DNA obtained by UV–Vis
absorption studies were 2.28  104, 1.98  104 and 2.98  104 M1, respectively. The result indicated that
the complexes (1–3) were able to bind to DNA with different binding affinity in the order: 3 > 1 > 2.
Complexes (1–3) also exhibit a good binding propensity to bovine serum albumin (BSA). Gel elec-
trophoresis assay demonstrated that complexes (1–3) cannot promote the cleavage ability of the
pBR322 plasmid DNA or plasmid pBluescript II KS+ DNA or pUC19 DNA in the presence of the reducing
agent 3-mercaptopropionic acid. The cytotoxicity studies of complexes 1–3 were tested in vitro on in
human cervical cancer cell line (HeLa) and they found to be active. We propose that the phosphorescent
iridium(III) complexes (1–3) are possible candidates for the green phosphors in organic light emitting
diodes (OLEDs) applications as well as better anticancer agents.
Ó 2017 Elsevier B.V. All rights reserved.

1. Introduction
The photoluminescence of organometallic iridium(III) com-
plexes has attracted much interest since it can be utilized for a
variety of applications such as oxygen sensors, biological probes
and phosphorescent dopants in optoelectronic devices [1–12]. In
particular, cyclometalating complexes of iridium(III) have received
great attention because of the high tunability of their emission in
terms of color and efficiency. Luminescent iridium(III) complexes
can be used to label biomaterials due their ability to either
covalently [13] or noncovalently [14] bind biological substrates.
Bound complexes typically express different luminescent proper-
⇑ Corresponding author at: DDE, Chemistry, Alagappa University, Karaikudi 630
003, Tamil Nadu, India.
E-mail address: nsvelan1975@yahoomail.com (N. Sengottuvelan).
ties than their free analogs due to changes in the rigidity and
hydrophobicity of the surrounding environment [14]. Thus, lumi-
nescent labels provide a facile method for monitoring bioconjuga-
tion reactions and quantifying the binding affinity between
different substrates [15,16]. This has been achieved by screening
a large variety of cyclometalating and ancillary ligands, often bear-
ing functional groups with electron withdrawing or releasing prop-
erties. The strong spin-orbital coupling effect of the heavy metal,
these complexes are capable of harvesting both singlet and triplet
excitons in devices to achieve internal quantum efficiency of 100 %,
when acting as the emissive material [17]. Cyclometalating car-
bazole based iridium complexes exhibiting green emission, having
unique hole transport ability, charge carrier mobility and good
thermal stability which play a very important role in the electroac-
tive and photoactive materials [18–23].

http://dx.doi.org/10.1016/j.ica.2017.07.061
0020-1693/Ó 2017 Elsevier B.V. All rights reserved.
 V. Thamilarasan et al. / Inorganica Chimica Acta 467 (2017) 264–275 265

Herein we report the synthesis of three carbazole based cationic

heteroleptic cyclometalated iridium(III) complexes: [Ir(cpy)2(biim)
Cl] 1, [Ir(cpy)2(bpy)Cl] 2 and [Ir(cpy)2(dppe)Cl] 3. The complexes
were characterized by various physicochemical techniques. Further
we describe the color tuning of iridium complexes by varying the
ancillary ligand units in complexes which may alter its photophys-
ical properties which could offer new biological sensing possibili-
ties, hence the complexes has been found to significantly perturb
its biomolecular binding, DNA cleavage and cytotoxic activities. In
the present biological study, the DNA-binding behaviors of three
Ir(III) complexes were explored by absorption, emission, circular
dichroism spectroscopy and viscosity measurements and their
affinity for bovine serum albumin (BSA) proteins involved in the
transport of metal ions and metal complexes with drugs through
the blood stream, performed by fluorescence spectroscopy. Their
abilities to induce cleavage of pBR322 DNA or plasmid pBluescript
II KS+ DNA or pUC19 DNA and in vitro cytotoxicity on human cervi-
cal cancer cell line (HeLa) were explored.

Fig. 1a. UV–Vis spectra of complexes (1–3).

2. Results and discussion

The Ir(III) l-chloro bridged dimer was synthesized by the reac-
tion of iridium trichloride hydrate with ligand cpy. Then diiridium
complexes was converted to mononuclear iridium(III) complexes
such as [(cpy)2Ir(biim)Cl] 1, [(cpy)2Ir(bpy)Cl] 2, and [(cpy)2Ir
(dppe)Cl] 3 with bidentate ligands biimidazole, 2,20 -bipyridine
and 1,2-bis(diphenylphosphino)ethane, respectively in 41–52%
yield (Scheme 1). 1H NMR, 13C NMR, FT-IR, mass and elemental
analyses of complexes (1–3) are consistent with the proposed
structure of complexes (1–3).
2.1. UV–Vis spectroscopy
The UV–Visible absorption spectra (Fig. 1a) of iridium(III) com-
plexes 1–3, show absorption band around 220–260 nm is assigned
to a typical ligand-centered states with mostly spin-allowed 1p-p⁄
transition of the cpy ligand because the corresponding transition
was also observed in the free ligand cpy based UV spectral profile
(Table 1). On the other hand, the band in the region 300–360 nm,
which is not observed in the spectrum of the free ligand, is

Scheme 1.
266 V. Thamilarasan et al. / Inorganica Chimica Acta 467 (2017) 264–275
Table 1
Photophysical parameters of iridium(III) complexes (1–3).
Complexes Absorption kabs/nm (e, 103 M1 cm1)
1 253(1077), 290(754), 345(300), 466(87), 485(11)
2 227(708), 310(602), 349(299), 386(330), 451(195)
3 231(665), 309(252), 407(98), 487(28)
*
Degassed.
assignable to the singlet metal-to-ligand charge-transfer (1MLCT)
transition. The moderately weaker absorptions above 380 nm can
be assigned to a spin-forbidden triplet metal-to-ligand charge-
transfer (3MLCT) transition [24].
2.2. Photoluminescence spectra and lifetime measurement
The photoluminescence spectra of the complexes (1–3) in
dichloromethane at room temperature are shown in Fig. 1b and
the corresponding photophysical data of the iridium complexes
are summarized in Table 1. Complexes 1–3 exhibits duel intense
PL emission at [425,580], [463,565] and [456,536] nm, respectively
[25,26]. The pronounced dual emission band for complexes shows
that there is an excitation of two separated LC transitions involving
two ligand subunits. As depicted in Fig. 1b, complexes 1–3 revealed
two distinct bands, which varied according to the anionic ancillary
ligands. The shorter-wavelength band, showing characteristics of a
short lifetime, is classified as fluorescence, while the longer wave-
length band can be assigned to the phosphorescence on the basis of
its longer lifetime and drastic quenching effect under oxygen.
It is believed that both of these emissions are dominated by p-
p⁄ transition in combination with some amount of ILCT character,
the latter, in part, incorporates the transfer of electron densities
from the ancillary to main ligand system. In the present work, sig-
nificant emission color tuning was realized (Fig. 1b); the emission
wavelength could be tuned from 536 to 580 nm. The higher wave-
length emission maxima of 3 is 44 nm blue-shifted compared to 1,
reflecting that the p-accepting character of the phosphine group
increased the associated MLCT energy levels, resulting in a blue-
shift emission. The spectroscopic results reveal an increased possi-
bility of color-tuning through changing the ancillary ligand with
Fig. 1b. PL spectra of complexes (1–3): ( ) aerated solution, ( ) argon
degassed solutions.
kmaxphos (nm) s1 s2
425,580 631 ns 972 ns
899 ns* 546 ns*
463,565 403 ns 1.08 ls
716 ns* 3.35 ls*
456, 536 1.51 ls 3.02 ls
5.08 ls* 12.35 ls*
the expected blue-shift of the spectral characteristics. To obtain a
better picture of the excited state quenching processes in these
materials, we have studied the photoluminescence decay tran-
sients using time-correlated single photon counting (TCSPC) has
been studied. It is worth noting that the nitrogen-degassed CH2Cl2
of complexes exhibit fluorescence decays were multiexponential
and the average lifetimes are reported in the Table 1. As listed in
Table 1, the relaxation dynamics of 1–3 are O2 dependent. For
example, the decay time of complex 1 was measured to be
546 ns (s2) in the degassed CH2Cl2, while it was decreased to
972 ns upon aeration. The result is consistent with the O2-quench-
ing triplet state according to the theory of electron-exchange type
(Dexter type) of energy transfer expressed as [27],
T þ 3 O2 !ko2 S0 þ 1 O2
2.3. Correlation of absorption, emission and lifetime measurements
The MLCT absorption bands above 380 nm, exhibit good spec-
tral overlap with the PL emission band [28]. This good spectral
overlap indicated that an efficient Forster or Dexter energy transfer
from the host (poly(vinylcarbazole) (PVK), to the iridium com-
plexes guests would be expected in the electroluminescent device.
The absorption bands above 420 nm are correlated to the photolu-
minescence (PL) of the complexes. It is notable that the absorption
edges are blue-shifted gradually in the order 3 < 2 < 1. The PL peaks
of the complexes are also blue-shifted gradually in the order
3 < 2 < 1 that are in good consistent with the absorption spectra.
Further the luminescent lifetimes fall between 1 and 12 ls, consis-
tent with emission from a triplet excited state. The positions of the
maximums in the excitation spectra for these complexes are very
similar to those in their absorption spectra. The results, in combi-
nation with the rather small radiative decay rate of <106 s1, leads
to unambiguously assign the dominant emission in complexes (1–
3) to be of phosphorescence character. The structured emission
spectra and long phosphorescence lifetime (Fig. 2) (described
below) suggest that the lowest triplet state of complexes (1–3) also
has ligand-centered 3p-p⁄ character (3p?p⁄ (N^C and N^N)) with
mixing of 3MLCT (dp (Ir) ? p.
2.4. Electrochemical properties
The electrochemical behavior of complexes were investigated
by cyclic voltammetry (Fig. 3) in CH2Cl2 solutions with ferroce-
nium/ferrocene (Fc+/Fc) redox couple as an internal reference and
the results are given in Table 2. The complexes 1–3 exhibit
quasi-reversible reduction processes, with reduction potentials of
the complexes stay in a range of 0.96 to 1.02. The complexes
(1–3) display a quasi-reversible one-electron oxidation couple in
the range 1.10–1.14 V assigned to a IrIII/IrIV oxidation. These values
are similar to the reported bis-cyclometalated iridium complexes
[29–31]. The redox peaks potential are almost in the same region.
The reduction peak can be assigned to the reduction of the pyridine
heterocyclic portion of the ligands. As revealed previously by
 V. Thamilarasan et al. / Inorganica Chimica Acta 467 (2017) 264–275
Fig. 2. Lifetime measurements of complexes (1–3): ( ) aerated solution,
( ) argon degassed solutions.
Fig. 3. Cyclic voltammograms of complexes (1–3).
electrochemistry and theoretical calculations [32–35] of cyclomet-
alated iridium(III) complexes, the reduction occurs primarily on
the more electron accepting heterocyclic portion of the cyclomet-
alated cpy ligands (LUMO contribution) whereas the oxidation pro-
cess is to largely involve in the Ir-cpy center (HOMO contribution).
Consistent with this conclusion, these complexes have similar
LUMO energy levels due to the same pyridyl based frame, which
makes the reduction potential of these complexes stay in a narrow
range. The HOMO and LUMO level of complexes were calculated to
be 5.63 to 5.71 eV and 4.04 to 4.06 eV respectively.
The oxidation potential of the Ir(III) complexes was translated
into the HOMO using the equation, EHOMO = (Eox + 4.8) eV, where
Table 2
Electrochemical properties of the iridium(III) complexes (1–3).
Complexes Eox (V) Ered (V)
1 1.11 0.96
2 1.14 1.02
3 1.10 1.02
267
Eox is the onset oxidation potential and the energy band gap
(DEgap) of the iridium(III) complexes deduced from the absorption
edge of the absorption spectra from DEgap + 1240/kedge, where kedge
is the onset value of the absorption spectrum. The LUMO energy
levels were estimated from the HOMO values and optical band
gaps (DEgap) by ELUMO = EHOMO + DEgap. Table 2 summarizes the
electrochemical data and the energy levels of the Ir(III) complexes.
2.5. Thermal properties
The thermal properties of the iridium complexes 1–3 were
examined by thermogravimetric analysis (TGA) under a nitrogen
atmosphere at a heating rate of 10 min (Fig. S1). The iridium(III)
complexes exhibited 5 % weight loss at 298 °C for 1, 293 °C for 2
and 276 °C for 3, which is beneficial to the long-term stability
and suitability for PhOLEDs device applications.
2.6. DNA binding studies
2.6.1. Absorption spectral studies
Transition metal complexes can bind with DNA via both cova-
lent (via replacement of a labile ligand of the complex by a nitro-
gen base of DNA such as guanine N7) and/or noncovalent
(intercalation, electrostatic or groove binding) interactions
[36,37]. Absorption titration can be used to observe the interaction
of molecules with DNA. The absorption spectra of complexes (1–3)
in the absence and presence of CT-DNA (at a constant concentra-
tion of complex 100 mM) in 2% DMSO at 25 °C by varying the con-
centration of DNA (buffer = 7.2 pH) are shown in Fig. 4. When the
amount of DNA is increased the intensity of the charge transfer
band is also changed. In the UV spectrum of 1, the band at
346 nm exhibited hyperchromism of 9.33 % and blue shifts of
1 nm suggesting the tight binding to CT-DNA. Additionally, the
hyperchromism of band are accompanied by a blue-shift of 1 nm
which is an indicative of stabilization. The behavior of complexes
2 and 3 is quite similar to 1 (hyperchromism and blue-shift). Com-
plexes 2 and 3 exhibited the hyperchromism of 8.98 and 10.21% at
344 and 335 nm, respectively with the blue shifts. The hyper-
chromism of complexes 1–3 indicate the DNA-stabilization and
the strong binding to CT DNA, possibly due to electrostatic interac-
tion or to the partial uncoiling of DNA helix structure, exposing
more bases of DNA [38]. Moreover, hyperchromic effect reflects
the corresponding changes in the secondary structure of DNA in
its conformation after the complex-DNA interaction. The results
derived from the UV titration experiments suggest that all the
complexes can bind to CT-DNA, although the exact mode of bind-
ing cannot be merely proposed by UV spectroscopic titration stud-
ies. The existence of hypochromism could be considered as
evidence for the binding of the complexes involving intercalation
between the base pairs of CT-DNA cannot be ruled out [39]. These
results indicate that these complexes interact with DNA through
groove binding.
The kmax, hyperchromism, blue shift and binding constant val-
ues of all the complexes with CT-DNA (Table 3) indicate that there
was a finite interaction between these complexes and CT-DNA. In
order to compare the affinity of the complexes toward DNA
Eox
onset (V) HOMO (eV) LUMO (eV) Egap (eV)
0.83 5.63 4.04 1.59
0.91 5.71 4.06 1.65
0.89 5.69 -4.05 1.64
268 V. Thamilarasan et al. / Inorganica Chimica Acta 467 (2017) 264–275
Fig. 4. Absorption spectra of complexes (1–3) in 5 mM Tris-HCl/50 mM NaCl buffer upon addition of DNA. Arrow shows the absorbance changing upon increase of DNA
concentration. The inner plot of [DNA/(ea  ef) vs [DNA] for the titration of DNA with complexes.
Table 3
The binding constant values of complexes (1–3).
Complexes kmax Hyperchromism H (%) Binding constant Kb (M1)
(nm)
1 346 9.33 2.28  104
2 344 8.98 1.98  104
3 335 10.21 2.98  104
quantitatively, the binding constant, Kb has been determined from
the spectroscopic titration date using the equation [40].
½DNA=ðea  ef Þ ¼ f½DNA=ðeb  ef Þg þ 1=kb ðeb  ef Þ
where ea is the extinction coefficient observed for the charge trans-
fer absorption at a given DNA concentration, ef the extinction coef-
ficient of the free complex in solution, eb the extinction coefficient
of the complex when fully bound to DNA, Kb the equilibrium bind-
ing constant, and [DNA] gives Kb as the ratio of the slope to the
intercept. The complex 3 serves as better DNA binding agent with
efficient activity compared with that of the other complexes, which
suggests that the interaction of the complexes with DNA is strong
and through groove binding. In the complex 3, the nature of the
metal phosphorus bond has a strong influence on the electron den-
sity on the metal center. It is possible to distinguish between pure
r-donor ligands and r-donor/p-acceptor ligands within a family
of complexes by a correlation of two appropriate properties of a
family of complexes when these properties are sensitive to the elec-
tron density of the metal [41]. As a result, the binding interaction of
the iridium(III) complexes with DNA follows the trend from high to
low: 3 > 1 > 2.
2.6.2. Competitive binding experiments
To further confirm the interaction between the test complex
and CT-DNA, emission experiments were carried out. Fluorescence
quenching measurements can be used to monitor metal binding
[42]. EB emits intense fluorescent light in the presence of CT
DNA due to its strong intercalation between the adjacent DNA base
pairs. It was previously reported that the enhanced fluorescence
could be quenched by the addition of another molecule [43,44].
The study involves the addition of the complexes to DNA pre-
treated with EB and the measurement of the intensity of emission.
The results from fluorescence titration spectra have been con-
firmed to be effective for characterizing the binding mode of metal
complexes to DNA [45]. The binding of complexes to DNA were
given by competitive binding experiments using complexes 1–3
as quenchers. The fluorescence measurement for complexes 1–3
showed no emission band with or without CT-DNA at ambient
temperature. The fluorescence quenching spectra of DNA-bound
EB at 598 nm by various complex concentrations are shown in
Fig. S2. Upon the addition of complexes, a significant decrease in
the fluorescence intensity of the EB-DNA system occurred, indicat-
ing the binding of complexes 1–3 to DNA. These results indicate
that complexes (1–3) could partially displace EB from the DNA-
EB system, as often observed in intercalative complex-DNA mode.
Addition of varying concentrations of complexes (1–3) to a
solution of EB-bound to CT-DNA causes lowering of the initial
emission intensity (51%, 1; 33%, 2; and 73%, 3) at diverse ‘r’ values
(Fig. S3), showing that Ir(III) complexes with EB to bind with DNA.
The spectral data indicate that complexes (1–3) efficiently displace
EB and bind to CT-DNA through intercalation [46–48]. Binding
strength of complexes (1–3) with CT-DNA has been estimated
using the Stern-Volmer equation:
I0
¼ 1 þ Ksv ½Q 
I
where, I0 and I are emission intensity in absence and presence of the
complexes. Ksv is a linear Stern-Volmer quenching constant, [Q] is
the ratio of the total concentration of complex to that of DNA. The
quenching plot illustrates that the quenching of EB bound to DNA
by the iridium(III) complexes is in good agreement with the linear
Stern-Volmer equation, which also indicated that the complexes
binds to DNA.
In the plot of I0/I vs [Complex]/[DNA], Ksv is given by the ratio of
the slope to intercept (Fig. S4) and resulting Ksv values for be
complexes 1–3 are 2.17  103, 4.83  103, 3.63  103 and
5.71  103 M1, respectively which varies in the order: 3 > 1 > 2.
The results clearly suggested that 3 have greater tendency to
replace EB relatively to other complexes. The quenching constant
value of Ir(III) complexes (1–3) may suggest that the complexes
(1–3) have intercalative mode of binding that involves a stacking
between the complex and the base pairs of DNA. The data suggest
that the interaction of 3 with DNA is the strongest, followed by 1,
and then 2, which is consistent with the above absorption spectral
results.
2.6.3. Circular dichroic spectral studies
Circular dichroic spectral studies (Fig. 5) are useful in diagnos-
ing changes in the structure of DNA during interaction of metal
complexes with DNA [49]. The CD spectrum of CT-DNA consists
of a positive band at 275 nm due to base stacking and a negative
band at 244 nm due to helicity, and is characteristic of DNA in
right-handed B-form. The groove binding interaction of small
molecules with DNA showed a little or no perturbations on the
base stacking and helicity bands, while intercalation enhances
the intensities of both bands, stabilizing the right-handed B form
conformation of CT-DNA. With the addition of complexes 1–3 to
the solution of CT-DNA there is increase in the intensity of the
 V. Thamilarasan et al. / Inorganica Chimica Acta 467 (2017) 264–275
Fig. 5. CD spectra of CT-DNA, and the interaction with 1, 2 and 3. All the spectra
were recorded in 5 mM Tris–HCl/ 50 mM NaCl buffer, pH 7.2 and 25 °C.
positive band (Fig. 5) with a blue shift of 2, 2 and 2 nm, respec-
tively, and decrease in the intensity of the negative band with a
no significant shift observed, compared to the DNA. The increase
in positive and a decrease in negative ellipticity indicate strong
conformational changes [50]. These changes are revealed the non-
intercalative mode of binding of these complexes and offer support
to their groove binding nature [51,52].
2.6.4. Viscosity measurements
To investigate the binding nature of the complexes to DNA, vis-
cosity measurements on the solutions of DNA incubated with the
complexes (1–3) have been carried out. In the viscosity measure-
ments, the rate of flow of the buffer (5 mM HCl/50 mM NaCl buffer,
pH 7.2), DNA (50 mM) and DNA with the complexes at various con-
centrations (2–12 mM) were measured. Data are presented as (g/
g0)1/3 vs. R {R = [complex]/[DNA]} where g is the viscosity of
DNA in the presence of complex and g0 is the viscosity of DNA
alone (Fig. S5). The viscosity of DNA increases greatly with increas-
ing concentration of complex, which is similar to that of the proven
intercalator EB [53]. This observation suggests that the principal
mode of DNA binding by complexes involve base-pair intercala-
Fig. 6. Molecular model of complexes (1–3) with DNA dodecamer duplex of sequence d(CGCGAATTCGCG)2(PDB ID: 1BNA) and modeling studies were carried out using Hex
6.0 software.
269
tion, with one ligand intercalating into the base pairs and the other
ligand being left outside the helix [54]. The intercalative interac-
tion with DNA is related to the molecular structure, as in this com-
plex there is a little distorted plane that may lead to the weak
intercalative mode. This result also parallels the pronounced emis-
sion enhancement of the complexes, and is comparable with the
proven classical intercalator EB. On the basis of the viscosity
results, the complexes bind with DNA through the intercalation
mode. Almost all the Ir(III) complexes enhance the viscosity and
the ability of the complexes to increase the viscosity of DNA fol-
lows the order EB > 3 > 1 > 2, iridium(III) complexes proposes to
be bound to DNA by intercalation.
2.6.5. Molecular docking studies
To elucidate the mode of interaction and binding affinity dock-
ing studies have been performed on B-DNA (PDB ID: 1BNA) in pres-
ence of complexes (1–3). The study revealed that complexes
interact with DNA via an electrostatic mode involving outside edge
stacking interaction with oxygen atom of the phosphate backbone.
The planarity of the complexes is compatible for strong p-p stack-
ing interactions and a better match of the complexes inside the
DNA strands. From the ensuing docked structures (Fig. 6) it is clear
that complex 1–3 fits well into the minor groove of the targeted
DNA and GC rich region stabilized by van der Waals interaction
and hydrophobic contacts [55]. Relative binding energy of the
docked structures of complex (1–3) was found to be 292.56,
284.32 and 295.52 eV, respectively. These are consistent with
the spectral studies and indicated that 3 have greater binding affin-
ity relative to other complexes. Overall results suggested that there
is good correlation between spectroscopic results and molecular
modeling.
2.7. BSA binding studies
The study on the interaction of drugs and their compounds with
blood plasma proteins especially with serum albumin, which is the
most abundant protein in plasma and is involved in the transport
of metal ions and metal complexes with drugs through the blood
stream, is of increasing interest [56]. Binding of these proteins
may lead to loss or enhancement of the biological properties of
the original drug, or provide paths for drug transportation [56].
Bovine serum albumin (BSA) is the most extensively studied serum
albumins. BSA (containing two tryptophans, Trp-134 and Trp-212)
which, when excited at 295 nm exhibit a strong fluorescence
270 V. Thamilarasan et al. / Inorganica Chimica Acta 467 (2017) 264–275

emission at 351 nm and 343 nm, respectively, due to the trypto-
phan residues [57].
The interaction of complexes (1–3) with serum albumins has
been studied from tryptophan emission-quenching experiments
(Fig. S6). The changes in the emission spectra of tryptophan in
BSA are primarily due to change in protein conformation, subunit
association, substrate binding or denaturation. An examination of
the spectrum showed a noteworthy decrease in fluorescence inten-
sity of complexes (1–3) at 354 nm (24 % for 1, 20% for 2 and 25%
for 3) (Fig. S7) and some interaction between the complexes and
BSA protein, due to possible changes in proteins secondary struc-
ture of BSA indicating the binding of the complexes to BSA [58].
The Stern-Volmer and Scatchard graphs may be used in order to
study the interaction of a quencher with serum albumins. Accord-
ing to Stern-Volmer quenching equation [59,60].
I0
¼ 1 þ kq s0 ½Q  ¼ 1 þ K sv ½Q 
I Scatchard graph (Fig. S8), the association binding constant to BSA
where, I0 = the initial tryptophan fluorescence intensity of BSA,
I = the tryptophan fluorescence intensity of BSA after the addition
of the quencher, kq = the quenching rate constants of BSA, Ksv = the
Stern-Volmer quenching constant, s0 = the average lifetime of BSA
without the quencher, [Q] = the concentration of the quencher
respectively,
K sv ¼ K q s 0
and, taking as fluorescence lifetime (s0) of tryptophan in BSA at
around 108 s, the Stern-Volmer quenching constant (Ksv, M1)
can be obtained by the slope of the diagram I0/I vs [Q] (Fig. S8),
and subsequently the approximate quenching constant (kq, M1 -
s1) may be calculated. The calculated values of Ksv and kq for the
interaction of the complexes with BSA are given in Table 4 and indi-
cate a good BSA binding propensity of the complexes in the order:
3 > 1 > 2. The kq values are higher than diverse kinds of quenchers
the existence of a static quenching mechanism [61,62]. Using the
Scatchard equation [59]:
 
DI=I0 DI
¼ nK  K
½Q I0
where n is the number of binding sites per albumin and K is the
association binding constant, K (M1), may be calculated from the
slope in plots (DI0/I)/[Q] vs (DI/I0) (Fig. S9) and n is given by the
ratio of the y intercept to the slope. It is obvious (Table 4) that
the coordination of iridium(III) complexes results in K value for
BSA with complex 3 exhibiting the highest K value among the other
complexes. The Stern-Volmer plot applied for the interaction with
BSA in Fig. S8 shows that the curves have fine linear relationships
(r = 0.9920–0.9968). The calculated values of Ksv and kq are given
in Table 4 and indicate their good BSA binding propensity with
complex 3 exhibiting the highest BSA quenching ability. From the
of each complex has been calculated (Table 4). The ‘n’ values of
complexes (1–3) are given Table 4. Thus the, complex 3 exhibit
higher binding affinity for BSA than other complexes which is sim-
ilar to that of DNA binding studies.
2.8. DNA cleavage studies
ð1Þ
The ability of complexes to cleave supercoiled DNA was deter-
mined by agarose gel electrophoresis. When circular plasmid
DNA in the presence of an inorganic molecule is subjected to elec-
trophoresis, relatively fast migration will be observed for the intact
supercoiled form (Form I). If scission occurs on one strand, the
supercoil will relax to generate a slower moving open circular form
(Form II). If both the strands are cleaved, a linear form (Form III)
that migrates in between Form I and Form II will be generated.
The DNA cleavage ability of complexes (1–3) were investigated

Table 4
The BSA binding constant and parameters (Ksv, kq, K, n) derived for complexes (1–3).

Complexes Ksv (M1) kq (M1 s-1) K (M1) n r

4 12
1 4.5  10 4.5  10 0.0546 1.0134 0.9922
2 3.8  104 3.8  1012 0.0352 0.4074 0.9921
3 6.8  104 6.8  1012 0.0885 1.0171 0.9968

Fig. 7. (a–e) (a) pBR322 DNA. lane 1, DNA control; lane 2, DNA + 1 (100 mM); lane 3, DNA + 2 (100 mM); lane 4, DNA + 3 (100 mM). (b). pBR322 DNA. lane 1, DNA control; lane
2, DNA + MPA (10 mM) + 1 (100 mM); lane 3, DNA + MPA (10 mM) + 2 (100 mM); lane 4, DNA + MPA (10 mM) + 3 (100 mM). (c). plasmid pBluescript II KS+ DNA. lane 1, DNA
control; lane 2, DNA + 1 (100 mM); lane 3, DNA + 2 (100 mM); lane 4, DNA + 3 (100 mM). (d). plasmid pBluescript II KS+ DNA. lane 1, DNA control; lane 2, DNA + MPA (10 mM) + 1
(100 mM); lane 3, DNA + MPA (10 mM) + 2 (100 mM); lane 4, DNA + MPA (10 mM) + 3 (100 mM). (e). pUC19 DNA. lane 1, DNA control; lane 2, DNA + 1 (100 mM); lane 3, DNA + 2
(100 mM); lane 4, DNA + 3 (100 mM); lane 5, DNA + MPA (10 mM) + 1 (100 mM); lane 6, DNA + MPA (10 mM) + 2 (100 mM); lane 7, DNA + MPA (10 mM) + 3 (100 mM).
 V. Thamilarasan et al. / Inorganica Chimica Acta 467 (2017) 264–275
Fig. 8. Cytotoxic effect of complexes (1–3) against human cervical cancer cell line (HeLa) at different concentration (A = 0.25 mM, B = 2.5 mM, C = 25 mM, D = 50 mM and
E = 100 mM).
using different DNA such as, pBR322 DNA (Fig. 7a and b), plasmid
pBluescript II KS+ (Fig. 7c and d) and pUC19 DNA (Fig. 7e). No
DNA cleavage was observed for the control DNA and DNA with
the metal complexes (1–3) or with activators 3-mercaptopropionic
acid (MPA).
2.9. Cytotoxic activities
Cytotoxic potential of newly synthesized Ir(III) complexes 1–3
was investigated on human cervical cancer cell line (HeLa). The
positive results obtained from DNA binding, protein binding and
DNA cleavage studies of the iridium(III) complexes (1–3) encour-
aged us to test its cytotoxicity against a panel of human cervical
cancer cell line (HeLa) by the MTT assay. Cytotoxic activities of
the complexes 1–3 were analysed in range (0.25–100 mM) of con-
centration by means MTT assays towards HeLa cells at 48 h. On
the basic of optical density, the cytotoxicity of complexes increases
with increasing concentration of complexes (1–3) from moderate
to high. The activities of the complexes were determined by MTT
test in vitro and the results were expressed in terms of IC50 values.
The relations of inhibition rates and complexes concentrations
against human cervical cancer cells (HeLa cell) were shown in
Fig. 8. As shown in Fig. 8, complex 3 has stronger inhibition ratios
than complexes 1 and 2 against tested human cervical cancer cells
at low concentrations. The inhibition effects were further
enhanced by increasing the concentration of complexes. At the
concentration of 100 lM, inhibition rates of the complexes (1–3)
against human cervical cancer cells reached 62.14, 27.06 and
93.22 %, respectively. The IC50 values of complexes (1–3) were
>100 for 2, 9.3 for 1 and 6.8 for 3. Also, the cytotoxicity of com-
plexes follows order 3 > 1 > 2. The cytotoxicity of complex 3 is
higher than other complexes due to their ability to effective
271
changes in the secondary structure of DNA, and their stronger
DNA binding affinity and organometallic biphosphine-based
cyclometalated complexes that are more stable and better p
accepting ability [63]. Further the surfaces that are more relevant
for biological processes and thus obtain new knowledge about
the processes with which cells ingest particles [64], and particles
can control the release behavior of bioactive molecules as well as
the surface activity, therefore providing a promising strategy to
enhance the efficiency of particulate drug-delivery systems [65].
3. Conclusions
In conclusion, we have synthesized three novel cyclometalated
iridium(III) complexes bearing cpy [(9-(pyridine-2-yl)-9H-car-
bazole] ligands and characterized. Significant mixing of the singlet
and triplet excited states is clearly observed in both the absorption
and emission spectra of the complexes can be fine tuned. The alter-
ation of emission wavelength correlates with the variation of
energy gap evaluated from the results of cyclic voltammetry. The
photoluminescence spectra of the complexes (1–3) in dichloro-
methane at room temperature shows the emission peaks in the
region 536–580 nm and lifetimes (complexes 1–3) are in the range
of microseconds (sp = 0.40–12.35 ls). The iridium complexes
shows radioactive decay rate in the order of 106 s1, leads to
unambiguously phosphorescence character. The complexes show
proper HOMO and LUMO energy gap, longer lifetimes and good
thermal stability. So, it can be used for the fabrication of good
phosphorescent OLEDs materials.
The binding properties of the complexes with CT-DNA, investi-
gated by UV–visible, fluorescence emission and CD spectroscopy,
suggests an efficient binding ability by the groove binding mode
in the order 3 > 1 > 2. Gel electrophoresis assay demonstrated the
272 V. Thamilarasan et al. / Inorganica Chimica Acta 467 (2017) 264–275
ability of the complexes 1–3 cannot promote the cleavage ability in
the presence of the reducing agent 3-mercaptopropionic acid. The
in vitro cytotoxicity of these complexes (1–3) on human cervical
cancer cell line (HeLa) indicates that complexes have the potential
to act as effective anticancer drugs. The complex 3 serves as better
DNA/BSA binding agents and with efficient cytotoxic activity com-
pared with that of the other complexes, which suggests that the
interaction of the complexes with DNA is strong. In complex 3,
the nature of the metal phosphorus bond has a strong influence
on the electron density on the metal center. It is believed that
the possibility to distinguish between pure r-donor ligands and
r-donor/p-acceptor ligands within a family of complexes by a cor-
relation of two appropriate properties of a family of complexes
when these properties are sensitive to the electron density on
the metal. Hence, from these findings, complex 3 was suggested
to demonstrate better activity and further evaluation of in vivo
anticancer activities of complex 3 is in progress.
4. Material and methods
4.1. Materials
Iridium(III) chloride, 2-ethoxyethanol were purchased from Alfa
Aesar, India. 2-bromopyridine, Agarose, carbazole, 1,2-bis
(diphenylphosphino)ethane, ethidium bromide (EB) and bovine
serum albumin (BSA) from Sigma-Aldrich (India). Potassium car-
bonate and 2,20 -bipyridine were purchased from Merk (India).
CT-DNA, pBR322 DNA, plasmid pBluescript II KS+DNA and pUC19
DNA were purchased from Genei, India. The human cervical cancer
cell line (HeLa cells) (3-[4,5-dimethylthiazol-2-yl]2,5-diphenylte-
trazolium bromide) was obtained from National Centre for Cell
Science (NCCS), Pune.
The CT-DNA stock solution was prepared by diluting DNA to
Tris-HCl/NaCl buffer (5 mM Tris-HCl, 50 mM NaCl, pH = 7.2), and
kept at 4 °C for no longer than a week. The UV absorbance at 260
and 280 nm of CT-DNA solution in Tris buffer give a ratio of 1.8–
1.9, indicating that the DNA was sufficiently free of protein. The
concentration of CT-DNA was determined from its absorption
intensity at 260 nm with a molar extinction coefficient of
6600 M1 cm1. Buffer solution was prepared using 2% DMSO.
4.2. Physical measurements
The mass spectra of the complexes were determined at Shi-
madzu QP 2000 mass spectrometer, SAIF, Lucknow, India. Elemen-
tal analysis of carbon, hydrogen and nitrogen were performed on a
Carlorerba-1106 microanalyzer. NMR spectra were measured on a
MERCURY-300BB spectrometer and chemical shift were referenced
to CDCl3 as an internal standard. The electronic spectra were
recorded on a Shimadzu UV-3101PC spectrophotometer. The fluo-
rescence spectral measurements were carried out using Fluoro-
max-4 spectrophotometer (Horiba Jobin Yvon), and time-resolved
photoluminescence decays were measured using nanosecond laser
flash photolysis (Applied photo physics) setup. The third harmon-
ics (355 nm) of a Q-switched Nd:YAG laser (Quanta-Ray, Spectra
Physics) pulse with 8 ns was used to excite the iridium complexes.
The emitted light was dispersed through a Czerny-Turner
monochromator and detected using a Hamamatsu R928 photomul-
tiplier tube. The luminescence decay were captured with an Agi-
lent infiniium digital storage oscilloscope and the data were
transferred to the computer for further analysis.
Cyclic voltammetry (CV) was performed with a CHI604D elec-
trochemical analyzer for studying the electrochemical behavior
of complexes using a three-electrode cell in which a glassy carbon
electrode was the working electrode, a platinum wire counter elec-
trode and an Ag/AgCl reference electrode in nitrogen atmosphere.
The concentration of complexes was 103 M in dichloromethane
and Tetramethylammonium hexafluorophosphate (101 M) was
used as the supporting electrode. The CD spectra were recorded
on Jasco J-810 spectropolarimeter. Molar conductivity of the com-
plexes was measured on a Elico model SX 80 conductivity bridge
using freshly prepared solution of the complexes in DCM. Thermo-
gravimetric (TG) analyses, measured from room temperature to
1000 °C at a heating rate for 10 °C min-1, were obtained by using
a Perkin-Elmer Diamond TG/derivative thermogravimetric (DTG)
instrument.
4.3. Molecular docking studies
The rigid molecular docking studies were performed by using
HEX 8.0 software [66]. The coordinates of complexes (1–3) were
drawn using Chemsketch (http://www.acdlabs.com) and saved in
mol files. The complex structure of the B-DNA dodecamer d
(CGCGAATTCGCG)2 (PDB ID: 1BDNA) was downloaded from the
protein data bank (http://www.resb.org./pdb). All calculations
were carried out on an Intel pentium 4.2.4 GHz based machine
running MS Windows XP SP2 as operating system. Visualization
of the docked pose has done by CHIMERA (www.cgl.ucsf.edu/
chimera).
4.4. Synthesis of complexes
4.4.1. [(cpy)2Ir(biim)Cl] (1)
The ligand [67] (cpy) (0.40 g, 1.6 mmol), IrCl3H2O (0.2 g,
0.67 mmol) in a mixed solvent of 2-ethoxyethanol (10 mL) and
water (3 mL) was stirred under nitrogen at 120 °C for 24 h. Cooled
to room temperature, the precipitate was collected by filtration
and washed with water, ethanol and hexane successively and then
dried in vacuum to give a cyclometalated Ir(III) m-chloro-bridged
dimer. The dimer (0.2 g, 0.17 mmol), biim (0.0771 g, 0.58 mmol)
and Na2CO3 (0.18 g, 1.75 mmol) were dissolved in 2-ethoxyethanol
(10 mL) and the mixture was then stirred under argon at 100 °C for
16 h. After cooling to room temperature, the precipitate was fil-
tered off and washed with water, ethanol and hexane. The crude
product was chromatographed on silica gel using CH2Cl2 as eluent
to afford the desired Ir(III) complex [(cpy)2Ir(biim)Cl] as green
solid. Yield: 35%. (0.18 g). M.p. 280 °C (dec). Anal. Calc. (%) for C40-
H28IrN8Cl: C, 56.62, H, 3.33, N, 13.21. Found (%): C, 56.36, H, 3.47, N,
12.98. FT-IR (KBr, m, cm1 selected peaks: 3100 s, 3037 s, 1608 s,
1175 s, 762 s (br, broad; s, sharp). UV–Vis in DCM [kmax/nm(emax/-
mol1 cm1)]: 253(1077), 290(754), 345(300), 466(87), 485(11).
1
H NMR (300 MHz, CDCl3) d (ppm): 6.15(d, J = 7.2 Hz, 1H), 6.35
(t, J = 6.5 Hz, 1H), 6.42 (m. 1H), 6.92 (m, 2H), 7.22 (m, 4H), 7.42
(m, 5H), 7.61 (m, 5H), 7.85 (m, 5H), 7.92 (m, 2H), 8.15 (m, 2H).
13
C NMR (75.46 MHz; CDCl3) d (ppm): 76.60, 77.22, 113.31,
115.38, 118.78, 119.25, 121.18, 123.34, 124.01, 126.07, 125.49,
126.40, 129.88, 136.08, 138.49, 144.24, 151.62, 156.18 and
173.56. ESI-MS (CH3OH) m/z (%): 848 [M+], 813(75) [M+Cl], 680
(10) [M+Cl, biim]. Conductivity (KM/S cm2 mol1) in DMF: 55.
The complexes [(cpy)2Ir(bpy)Cl] (2) and [(cpy)2Ir(dppe)Cl] (3)
were synthesized by following the above procedure using 2,2-
bipyridine (0.09 g, 0.58 mmol) and 1,2-bis (diphenylphosphino)
ethane (0.23 g, 0.58 mmol), respectively, instead of using
biimidazole.
4.4.2. [(cpy)2Ir(bpy)Cl] (2)
Red colour solid. Yield: 49%. (0.14 g). m.p. 188 °C (dec). Anal.
Calc. (%) for C44H30IrN6Cl: C, 60.70, H, 3.48, N, 9.65. Found (%): C,
60.44, H, 3.62, N, 9.42. FT-IR (KBr, m, cm1 selected peaks: 3035 s,
1601 s, 1173 s, 753 s (br, broad; s, sharp). UV–Vis in DCM [kmax/
nm (emax/mol1 cm1)]: 227(708), 310(602), 349(299), 386(330),
 V. Thamilarasan et al. / Inorganica Chimica Acta 467 (2017) 264–275
451(195). 1H NMR (300 MHz, CDCl3) d (ppm): 7.223–7.324 (m,
9Hs), 7.748–7.841 (m, 8H), 8.308–8.406 (m, 7H), 8.687–8.606 (m,
6H). 13C NMR (75.46 MHz; CDCl3) d (ppm): 76.62, 77.25, 114.17,
115.59, 119.54, 121.38, 122.79, 123.99, 124.35, 126.77, 136.17,
138.59, 139.20, 140.62, 142.89, 149.10, 151.51, 153.81, 157.22.
ESI-MS (CH3OH) m/z (%): 870 [M+], 835 (100) [M+Cl]. Conductiv-
ity (KM/S cm2 mol1) in DMF: 45.
4.4.3. [(cpy)2Ir(dppe)Cl] (3)
Green colour solid. Yield: 48%. (0.19 g). M.p. 178 °C (dec). Anal.
Calc. (%) for C60H46IrN4P2Cl: C, 64.76, H, 4.17, N, 5.03. Found (%): C,
64.50, H, 4.31, N, 4.80. FT-IR (KBr, m, cm1 selected peaks: 3046s,
1623s, 1120s, 1476s, 1178s, 753s, 502s (br, broad; s, sharp). UV–
Vis in DCM [kmax/nm (emax/mol1 cm1)]: 231(665), 309(252),
407(98), 487(28). 1H NMR (300 MHz, CDCl3) d (ppm): 1.60 (d,
4H), 5.91 (m, 1H), 6.23 (m, 1H), 6.45 (m, 2H), 6.85 (m, 4H), 7.15
(m, 1H), 7.25–7.21 (m, 6H), 7.49–7.40 (m, 15H), 7.58–7.51 (m,
8H), 7.69–7.62 (m, 4H). 13C NMR (75.46 MHz; CDCl3) d (ppm):
31.80, 76.57, 77.20, 110.98, 114.33, 116.34, 119.51, 120.41,
123.94, 124.95, 125.23, 126.08, 128.76, 129.55, 130.68, 131.44,
132.27, 134.46, 138.28, 139.88, 152.40. ESI-MS (CH3OH) m/z (%);
1112 (4) [M+], 1077 (100) [M+Cl]. Conductivity (KM/S cm2 mol1)
in DMF: 30.
4.5. DNA binding studies
4.5.1. Absorption spectral studies
Absorption spectra titrations were performed at room tempera-
ture in Tris HCl/NaCl buffer (5 mM/50 mM buffer, pH 7.2) to inves-
tigate the binding affinity between CT-DNA and complex. A fixed
concentration of the complex (10 mM) was titrated with increasing
amounts of DNA concentration. The intrinsic binding constant for
the interaction of complex with DNA was obtained from absorp-
tion spectral data.
4.5.2. Competitive binding experiments
The relative binding of complex to CT-DNA were determined
with an EB-bound CT-DNA solution in Tris-HCl/NaCl buffer
(5 mM/50 mM, pH = 7.2). The fluorescence on quenching effect
on addition of complex to the EB-DNA complex have been analyzed
by recording the fluorescence emission spectra with excitation at
510 nm and emission at 596–600 nm.
4.5.3. Circular dichroic spectral studies
The CD spectra of CT-DNA in the presence or absence of com-
plexes were collected after 12 h incubation of 100 mM CT-DNA with
50 mM individual complex in Tris-HCl/NaCl buffer (5 mM/50 mM,
pH 7.2) at 37 °C. For each spectrum, three scans were collected
and the background was subtracted from all of the reagents by
using a corresponding solution without CT-DNA as a reference
solution. All CD experiments were performed on a Jasco J-810 spec-
tropolarimeter at room temperature from 320 to 220 nm.
4.5.4. Viscosity measurements
The viscosity of solution relied on the time flowed through the
capillary viscometer. The viscosity measurements were conducted
by keeping the viscometer immersed in a water-bath maintained
at 25 °C ± 0.1 °C. Flow times were measured and the relative vis-
cosity was obtained by the equation: g = (t  t0)/t0, where t and
t0 represent the observed flow time of CT-DNA containing solu-
tions and buffer solution alone, respectively. The data was pre-
sented as (g/g0)1/3 versus the ratio of the concentration of
complex to that of CT-DNA ([Complex]/[CT-DNA]) [68,69].
273
4.6. BSA binding studies
Protein binding studies have been performed by tryptophan flu-
orescence quenching experiments using BSA (3 mM) in buffer (con-
taining 15 mM trisodium citrate and 150 mM NaCl at pH 7.0).
Fluorescence emission spectra were recorded from 300 to
500 nm at an excitation wavelength of 295 nm.
4.7. Cytotoxic assay
The cell line was cultured in the Dulbecco’s Modified Eagles
Medium (DMEM) supplemented with 10% fetal bovine serum
(FBS), 200 mM L-glutamine, 100 m/mL penicillin, and 10 mg/mL
streptomycin in a humidified atmosphere consisting of 5 % CO2
at 37 °C.
4.8. Evaluation of cytotoxicity
The cytotoxic effect of complex against human cervical cancer
(HeLa) cell line was evaluated by MTT [3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide] assay. Briefly, HeLa cells
were seeded (5  104 cells/well) in a 96-well plate and kept in
CO2 for attachment and growth for 24 h [70–72]. Then, the cells
were treated with various concentrations of complex dissolved in
DMSO (0.25–100 mM) and incubated for 24 h. After incubation,
the culture medium was removed and 15 lL of the MTT solution
(5 mg/mL in PBS) was added to each well. Following 4 h incubation
in dark, MTT was discarded and DMSO (100 lL/well) was added to
solubilize the purple formazan product. The experiment was car-
ried out in triplicates and the medium without complex served
as control. The absorbance was measured colorimetrically at
570 nm using an ELISA microplate reader. The percentage of cell
viability was calculated using the following formula and expressed
as IC50 = (OD value of treated cells)/(OD value of untreated cells
(control)  100
The IC50 value is calculated using linear regression from excel.
4.9. DNA cleavage of complexes (1–3)
Cleavage of supercoiled pBR322 DNA, plasmid pBluescript II KS+
DNA and pUC19 DNA by the complexes was studied by agarose gel
electrophoresis. In a typical experiment, the reaction mixtures
(25 mL total volume) containing the DNA, dissolved in 50 mM
Tris–HCl buffer, 50 mM NaCl, were treated with the metal com-
plexes in the 100 mM concentration. The reaction mixtures were
incubated at 37 °C for 4 h, and then the addition of 5 mL loading
buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glyc-
erol), samples were loaded on 0.8 % agarose gel containing EB
(1 mg/mL) in TAE (Tris-acetate-EDTA) buffer. The gel was run at
50 V for 1.30 h and photograph has been taken under UV light.
The proportion of DNA in each fraction was quantitatively esti-
mated from the intensity of each band with the BioRad Gel Doc
XR system using the Labwork software. Anaerobic run was per-
formed in a stopcock-equipped cuvette after extensive purging of
the reaction mixture with N2 prior to the addition of N2-purged
complexes. To enhance the DNA cleaving ability by the complexes,
activators MPA was used. All the experiments were carried out in
triplicate under the same conditions.
Acknowledgements
We are thankful to the University Grants Commission, New
Delhi (project No. 40-46/2011 (SR) and DST (DST-SR/FT/CS-
049/2009 for the financial support. The authors thank the SAIF
Central Drug Research Institute (CDRI), Lucknow for analyzing
the mass spectra.
274 V. Thamilarasan et al. / Inorganica Chimica Acta 467 (2017) 264–275

Appendix A. Supplementary data hydrogen production from an ionic iridium(III) complex, Chem. Mater. 17
(2005) 5712–5719.
[25] J. Jiang, J. Liang, S. Zhao, Y. Wang, J. Qi, Y. Huang, T. Xiong, D. Zhu, Syntheses,
Supplementary data associated with this article can be found, in crystal structures and luminescent properties of drum and ladder-like
the online version, at http://dx.doi.org/10.1016/j.ica.2017.07.061. organooxotin clusters with carbazole ligand, J. Cluster Sci. 28 (2017) 971–982.
[26] B.L. Li, L. Wu, Y.M. He, Q.H. Fan, The synthesis and properties of Iridium(III)-
cored dendrimers with carbazole peripherally functionalized b-diketonato
References dendrons, Dalton Trans. (2007) 2048–2057.
[27] S. Reineke, K. Walzer, K. Leo, Triplet-exciton quenching in organic
phosphorescent light-emitting diodes with Ir-based emitters, Phys. Rev. B 75
[1] E. Holder, B.M.W. Langeveld, U.S. Schubert, New trends in the use of transition
(2007) 125328.
metal-ligand complexes for applications in electroluminescent devices, Adv.
[28] P. Leriche, P. Frere, A. Cravino, O. Aleveque, J. Roncali, Triplet-exciton
Mater. 17 (2005) 1109–1121.
quenching in organic phosphorescent light-emitting diodes with Ir-based
[2] H. Yersin, Triplet emitters for OLED applications. Mechanisms of exciton
emitters, J. Org. Chem. 72 (2007) 8332–8336.
trapping and control of emission properties, Top. Curr. Chem. 241 (2004) 1–26.
[29] C.H. Yang, C. Tai, I.W. Sun, Synthesis of a high-efficiency red phosphorescent
[3] H. Sun, S. Liu, W. Lin, K.Y. Zhang, W. Lv, X. Huang, F. Huo, H. Yang, G. Jenkins, Q.
emitter for organic light-emitting diodes, J. Mater. Chem. 14 (2004) 947–
Zhao, W. Huang, Smart responsive phosphorescent materials for data
950.
recording and security protection, Nat Commun. 5 (2014) 3601.
[30] F.I. Wu, H.J. Su, C.F. Shu, L. Luo, W.G. Diau, C.H. Cheng, J.P. Duan, G.H. Lee,
[4] W. Xu, X. Zhao, W. Lv, H. Yang, S. Liu, H. Liang, Z. Tu, H. Xu, W. Qiao, Q. Zhao, W.
Tuning the emission and morphology of cyclometalated iridium complexes
Huang, Rational design of phosphorescent chemodosimeter for reaction-based
and their applications to organic light-emitting diodes, J. Mater. Chem. 15
one- and two-photon and time-resolved luminescent imaging of biothiols in
(2005) 1035–1042.
living cells, Adv. Healthc. Mater. 3 (2014) 658–669.
[31] K.A. King, P.J. Spellane, R.J. Watts, Excited-state properties of a triply ortho-
[5] W. Zhang, C. Liang, Z. He, H. Pang, Y. Wang, S. Zhao, Stable orange and white
metalated iridium(III) complex, J. Am. Chem. Soc. 107 (1985) 1431–1432.
electro phosphorescence based on spirobifluorenyltrifluoromethylpyridine
[32] J. Kalinowski, G. Giro, M. Cocchi, V. Fattori, P.D. Macro, Unusual disparity in
iridium complexes, Synth. Metals 210 (2015) 214–222.
electroluminescence and photoluminescence spectra of vacuum-evaporated
[6] S. Tobita, T. Yoshihara, Intracellular and in vivo oxygen sensing using
films of 1, 1-bis ((di-4-tolylamino) phenyl) cyclohexane, Appl. Phys. Lett. 76
phosphorescent iridium(III) complexes, Cur. Opin. Chem. Biol. 33 (2016) 39–45.
(2000) 2352–2354.
[7] W. Su, X. Wang, X. Lei, Q. Xiao, S. Huang, P. Li, Synthesis, characterization,
[33] G. Calogero, G. Giuffrida, S. Serroni, V. Ricevuto, S. Campagna, Absorption
cytotoxic activity of half-sandwich rhodium(III), and iridium(III) complexes
spectra, luminescence properties, and electrochemical behavior of
with curcuminoids, J. Organomet. Chem. 833 (2017) 54–60.
cyclometalated iridium(III) and rhodium(III) complexes with a bis
[8] S. Liu, P. Wang, Q. Zhao, H. Yang, J. Wong, H. Sun, X. Dong, W. Lin, W. Huang,
(pyridyl)triazole ligand, Inorg. Chem. 34 (1995) 541–545.
Single polymer-based ternary electronic memory material and device, Adv.
[34] G. Ge, G. Zhang, H. Guo, Y.C. Huai, D. Zou, Yellow organic light-emitting diodes
Mater. 24 (2012) 2901–2905.
based on phosphorescent iridium(III) pyrazine complexes: Fine tuning of
[9] S. Liu, Z. Lin, Q. Zhao, Y. Ma, H. Shi, M. Yi, Q. Ling, Q. Fan, C. Zhu, E. Kang, W.
emission color, Inorg. Chim. Acta 362 (2009) 2231–2236.
Huang, Flash-memory effect for polyfluorenes with on-chain iridium(III)
[35] C. Dragonetti, L. Falciola, P. Mussini, S. Righetto, D. Roberto, R. Ugo, A. Valore, F.
complexes, Adv. Funct. Mater. 21 (2011) 979–985.
De Angelis, S. Fantacci, A. Sgamellotti, M. Ramon, M. Muccini, The role of
[10] W. Xu, S. Liu, H. Sun, X. Zhao, Q. Zhao, S. Sun, S. Cheng, T. Ma, L. Zhou, W. Huang,
substituents on functionalized 1,10-phenanthroline in controlling the
FRET-based probe for fluoride based on a phosphorescent iridium(III) complex
emission properties of cationic iridium(III) complexes of interest for
containing triarylboron groups, J. Mater. Chem. 21 (2011) 7572–7581.
electroluminescent devices, Inorg. Chem. 46 (2007) 8533–8547.
[11] H. Shi, S. Liu, H. Sun, W. Xu, Z. An, J. Chen, S. Sun, X. Lu, Q. Zhao, W. Huang,
[36] F. Dimiza, F. Perdih, V. Tangoulis, I. Turel, D.P. Kessissoglou, G. Psomas,
Simple conjugated polymers with on-chain phosphorescent iridium(III)
Interaction of copper(II) with the non-steroidal anti-inflammatory drugs
complexes: toward ratiometric chemodosimeters for detecting trace
naproxen and diclofenac: synthesis, structure, DNA- and albumin-binding, J.
amounts of mercury(II), Chem. Eur. J. 16 (2010) 12158–12167.
Inorg. Biochem. 105 (2011) 476–489.
[12] W. Xu, S. Liu, X. Zhao, S. Sun, S. Cheng, T. Ma, H. Sun, Q. Zhao, W. Huang,
[37] F. Dimiza, A.N. Papadopoulos, V. Tangoulis, V. Psycharis, C.P. Raptopoulou, D.P.
Cationic iridium(III) complex containing both triarylboron and carbazole
Kessissoglou, G. Psomas, Biological evaluation of non-steroidal anti-
moieties as a ratiometric fluoride probe that utilizes a switchable triplet-
inflammatory drugs-cobalt(II) complexes, Dalton Trans. 39 (2010) 4517–4528.
singlet emission, Chem. Eur. J. 16 (2010) 7125–7133.
[38] N. Shahabadi, S. Kashanian, M. Khosravi, M. Mahdavi, Multispectroscopic DNA
[13] K.K.-W. Lo, C.-K. Chung, N. Zhu, Synthesis, photophysical and electrochemical
interaction studies of a water-soluble nickel(II) complex containing different
properties, and biological labeling studies of cyclometalated iridium(III) bis
dinitrogen aromatic ligands, Trans. Metal Chem. 35 (2010) 699–705.
(pyridylbenzaldehyde) complexes: novel luminescent cross-linkers for
[39] F. Dimiza, S. Fountoulaki, A.N. Papadopoulos, C.A. Kontogiorgis, V. Tangoulis, C.
biomolecules, Chem. Eur. J. 9 (2003) 475–483.
P. Raptopoulou, V. Psycharis, A. Terzis, D.P. Kessissoglou, G. Psomas, Non-
[14] K.K.-W. Lo, C.-K. Chung, N. Zhu, Nucleic acid intercalators and avidin probes
steroidal antiinflammatory drug–copper(II) complexes: structure and
derived from luminescent cyclometalated iridium (III)–dipyridoquinoxaline
biological perspectives, Dalton Trans. 40 (2011) 8555–8568.
and–dipyridophenazine complexes, Chem. Eur. J. 12 (2006) 1500–1512.
[40] P. Sathyadevi, P. Krishnamoorthy, R.R. Butorac, A.H. Cowley, N.S.P. Bhuvaneshc,
[15] K.K.-W. Lo, W.-K. Hui, C.-K. Chung, K.H.-K. Tsang, D.C.-M. Ng, N. Zhu, K.-K.
N.A. Dharmaraj, Effect of substitution and planarity of the ligand on DNA/BSA
Cheung, Biological labelling reagents and probes derived from luminescent
interaction, free radical scavenging and cytotoxicity of diamagnetic Ni(II)
transition metal polypyridine complexes, Coord. Chem. Rev. 249 (2005) 1434–
complexes: a systematic investigation, Dalton Trans. 40 (2011) 9690–9702.
1450.
[41] M.D. Matiur Rahman, H.Y. Liu, K. Eriks, A. Prock, W.P. Giering, Quantitative
[16] K.K.-W. Lo, C.-K. Chung, T.K.-M. Lee, L.-H. Lui, K.H.-K. Tsang, N. Zhu, New
analysis of ligand effects. Part 3. Separation of phosphorus(III) ligands into
luminescent cyclometalated iridium(III) diimine complexes as biological
pure sigma-donors and sigma-donor/pi-acceptors. Comparison of basicity and
labeling reagents, Inorg. Chem. 42 (2003) 6886–6897.
sigma-donicity, Organometallics 8 (1989) 1–7.
[17] M.A. Baldo, D.F. O’Brien, M.E. Thompson, S.R. Forrest, Excitonic singlet-triplet
[42] A. Tarushi, G. Psomas, C.P. Raptopoulou, D.P. Kessissoglou, Zinc complexes of
ratio in a semiconducting organic thin film, Phys. Rev. B 60 (1999) 14422–14428.
the antibacterial drug oxolinic acid: structure and DNA-binding properties, J.
[18] X. Zhang, Z. Chen, C. Yang, Z. Li, K. Zhang, H. Yao, J. Qin, J. Chen, Y. Cao, Highly
Inorg. Biochem. 103 (2009) 898–905.
efficient polymer light-emitting diodes using color-tunable carbazole-based
[43] B.S. Yang, J.Y. Feng, Y.Q. Li, F. Gao, Y.Q. Zhao, J.L. Wang, Spectral studies on
iridium complexes, Chem. Phys. Lett. 422 (2006) 386–390.
aluminum ion binding to the ligands with phenolic group(s): implications for
[19] T.-H. Kwon, H.S. Cho, M.K. Kim, J.-W. Kim, J.-J. Kim, K.H. Lee, S.J. Park, I.-S. Shin,
the differences between N- and C-terminal binding sites of human serum
H. Kim, D.M. Shin, Y.K. Chung, J.-I. Hong, Color tuning of cyclometalated iridium
apotransferrin, J. Inorg. Biochem. 96 (2003) 416–424.
complexes through modification of phenylpyrazole derivatives and ancillary
[44] B.C. Baguley, M. Le Bret, Quenching of DNA-ethidium fluorescence by
ligand based on ab initio calculations, Organometallics 24 (2005) 1578–1585.
amsacrine and other antitumor agents: a possible electron-transfer effect,
[20] J. Sun, W. Wu, H. Guo, J. Zhao, Visible-light harvesting with cyclometalated
Biochemistry 23 (1984) 937–943.
iridium(III) complexes having long-lived 3IL excited states and their
[45] D.S. Raja, N.S.P. Bhuvanesh, K. Natarajan, Biological evaluation of a novel water
application in triplet–triplet-annihilation based upconversion, Eur. J. Inorg.
soluble sulphur bridged binuclear copper(II) thiosemicarbazone complex, Eur.
Chem. 2011 (2011) 3165–3173.
J. Med. Chem. 46 (2011) 4584–4594.
[21] Y. You, S.Y. Park, Inter-ligand energy transfer and related emission change in
[46] H.M. Torshizi, M. Saeidifar, F. Khosravi, A. Divsalar, A.A. Saboury, F. Hassani,
the cyclometalated heteroleptic iridium complex: facile and efficient colour
DNA binding and antitumor activity of a-diimineplatinum(II) and Palladium
tuning over the whole visible range by the ancillary ligand structure, J. Am.
(II) dithiocarbamate complexes, Bioinorg. Chem. Appl. (2011) 1–11.
Chem. Soc. 127 (2005) 12438–12439.
[47] Z.C. Liu, B.D. Wang, Z.Y. Yang, Y. Li, D.D. Qin, T.R. Li, Synthesis, crystal structure,
[22] L. Ma, H. Guo, Q. Li, S. Guo, J. Zhao, Visible light-harvesting cyclometalated Ir
DNA interaction and antioxidant activities of two novel water-soluble Cu2+
(III) complexes as triplet photosensitizers for triplet–triplet annihilation based
complexes derivated from 2-oxo-quinoline-3-carbaldehyde Schiff-bases, Eur.
upconversion, Dalton Trans. 41 (2012) 10680–10689.
J. Med. Chem. 44 (2009) 4477–4484.
[23] Y. You, K.S. Kim, T.K. Ahn, D. Kim, S.Y. Park, Direct spectroscopic observation of
[48] G.M. Howe, K.C. Wu, W.R. Bauer, S.J. Lippard, Binding of platinum and
interligand energy transfer in cyclometalated heteroleptic iridium(III)
palladium metallointercalation reagents and antitumor drugs to closed and
complexes: a strategy for phosphorescence color tuning and white light
open DNAs, Biochemistry 19 (1976) 4339–4347.
generation, J. Phys. Chem. C 111 (2007) 4052–4060.
[49] M. Baldini, M. Belicchi-Ferrari, F. Bisceglie, P.P. Dall’Aglio, G. Pelosi, S. Pinelli,
[24] M.S. Lowry, J.I. Goldsmith, J.D. Slinker, R. Rohl, R.A. Pascal, G.G. Malliaras, S.
P. Tarasconi, Copper(II) complexes with substituted thiosemicarbazones of
Bernhard, Single-layer electroluminescent devices and photoinduced
 V. Thamilarasan et al. / Inorganica Chimica Acta 467 (2017) 264–275
a-ketoglutaric acid: synthesis, X-ray structures, DNA binding studies and
nuclease and biological activity, Inorg. Chem. 43 (2004) 7170–7179.
[50] P. Lincoln, E. Tuite, B. Norden, Short-circuiting the molecular wire: cooperative
binding of D-[Ru(phen)2dppz]2+ and D-[Rh(phi)2bipy]3+ to DNA, J. Am. Chem.
Soc. 119 (1997) 1454–1455.
[51] V. Uma, M. Kanthimathi, T. Weyhermuller, B. Unni Nair, Oxidative DNA
cleavage mediated by a new copper (II) terpyridine complex: crystal structure
and DNA binding studies, J. Inorg. Biochem. 99 (2005) 2299–2307.
[52] P. Uma Maheswari, M. Palaniandavar, DNA binding and cleavage properties of
certain tetrammine ruthenium(II) complexes of modified 1,10-
phenanthrolines – effect of hydrogen-bonding on DNA-binding affinity, J.
Inorg. Biochem. 98 (2004) 219–230.
[53] Q.L. Zhang, J.G. Liu, H. Chao, G.Q. Xue, L.N. Ji, NA-binding and photocleavage
studies of cobalt(III) polypyridyl complexes: [Co(phen)2IP]3+ and [Co
(phen)2PIP]3+, J. Inorg. Biochem. 83 (2001) 49–55.
[54] P. Xi, Z. Xu, F. Chen, Z. Zeng, X. Zhang, Study on synthesis, structure, and DNA-
binding of Ni, Zn complexes with 2-phenylquinoline-4-carboylhydrazide, J.
Inorg. Biochem. 103 (2009) 210–218.
[55] S. Tabassum, M. Zaki, M. Afzal, F. Arjmand, New modulated design and
synthesis of quercetin–CuII/ZnII–SnIV 2 scaffold as anticancer agents: in vitro
DNA binding profile, DNA cleavage pathway and Topo-I activity, Dalton Trans.
42 (2013) 10029–10041.
[56] C. Tan, J. Liu, H. Li, W. Zheng, S. Shi, L. Chen, L. Ji, Differences in structure,
physiological stability, electrochemistry, cytotoxicity, DNA and protein
binding properties between two Ru(III) complexes, J. Inorg. Biochem. 102
(2008) 347–358.
[57] Y. Wang, H. Zhang, G. Zhang, W. Tao, S. Tang, Interaction of the flavonoid
hesperidin with bovine serum albumin: a fluorescence quenching study, J.
Luminescence 126 (2007) 211–218.
[58] D.S. Raja, N.S.P. Bhuvanesh, K. Natarajan, A novel water soluble ligand bridged
cobalt(II) coordination polymer of 2-oxo-1,2-dihydroquinoline-3-
carbaldehyde (isonicotinic) hydrazone: evaluation of the DNA binding,
protein interaction, radical scavenging and anticancer activity, Dalton Trans.
41 (2012) 4365–4377.
[59] V. Thamilarasan, N. Sengottuvelan, A. Sudha, P. Srinivasan, A. Siva, Synthesis,
molecular structure, theoretical calculation, DNA/protein interaction and
cytotoxic activity of manganese(III) complex with 8-hydroxyquinoline, J.
Photochem. Photobiol. B 142 (2015) 220–231.
[60] A. Tarushi, E. Polatoglou, J. Kljun, I. Turel, G. Psomas, D.P. Kessissoglou,
Interaction of Zn(II) with quinolone drugs: Structure and biological evaluation,
Dalton Trans. 40 (2011) 9461–9473.
275
[61] M.T. Carter, A.J. Bard, Voltammetric studies of the interaction of tris(1,10-
phenanthroline)cobalt(III) with DNA, J. Am. Chem. Soc. 109 (1987) 7528–
7530.
[62] D.H. Johnston, K.C. Glasgow, H.H. Thorp, Electrochemical measurement of the
solvent accessibility of nucleobases using electron transfer between DNA and
metal complexes, J. Am. Chem. Soc. 117 (1995) 8933–8938.
[63] N. Malecevic, T. Srdic, S. Radulovic, D. Sladic, V. Radulovic, I. Brceski, K.
Andelkoivc, Synthesis and characterization of a novel Pd(II) complex with the
condensation product of 2-(diphenylphosphino)benzaldehyde and ethyl
hydrazinoacetate. Cytotoxic activity of the synthesized complex and related
Pd(II) and Pt(II) complexes, J. Inorg. Biochem. 100 (2006) 1811–1818.
[64] A.M. Javier, O. Kreft, A.P. Alberola, C. Kirchner, B. Zebli, A.S. Susha, E. Horn, S.
Kempter, A.G. Skirtach, A.L. Rogach, J. Radler, G.B. Sukhorukov, M. Benoit, W.J.
Parak, Combined atomic force microscopy and optical microscopy
measurements as a method to investigate particle uptake by cells, Small 2
(2006) 394–400.
[65] R. Luo, B. Neu, S.S. Venkatraman, Surface functionalization of nanoparticles to
control cell interactions and drug release, Small 8 (2012) 2585–2594.
[66] D. Mustard, D.W. Ritchie, Docking essential dynamics eigen structures,
Proteins Struct. Funct. Bioinf. 60 (2005) 269–274.
[67] V. Thamilarasan, A. Jayamani, P. Manisankar, Young-Inn Kim, N.
Sengottuvelan, Green-emitting phosphorescent iridium(III) complex:
structural, photophysical and electrochemical properties, Inorg. Chim. Acta
408 (2013) 240–245.
[68] Y. Li, G. Zhang, M. Tao, Binding properties of herbicide chlorpropham to DNA:
spectroscopic, chemometrics and modeling investigations, J. Photochem.
Photobiol. B 138 (2014) 109–117.
[69] X. Li, X.J. Li, Y.T. Li, Z.Y. Wu, C.W. Yan, Syntheses and structures of tetracopper
(II) complexes with an N-benzoate-N0 -[3-(2-hydroxylethylammino)propyl]
oxamide ligand: reactivity towards DNA, cytotoxic and antimicrobial
activities, New J. Chem. 36 (2012) 2472–2483.
[70] S.J. Mehdi, A. Ahmad, M. Irshad, N. Manzoor, M.M.A. Rizvi, Cytotoxic effect
of carvacrol on human cervical cancer cells, Biol. Med. 3 (2) (2011) 307–
312.
[71] Mustofa. Masrianif, Sunarti. Jumina, Pycnarrhena cauliflora ethanolic extract
induces apoptosis and cell cycle arrest in hela human cervical cancer cells, Int.
J. Res. Pharm. Biomed. Sci. 4 (2013) 1060–1068.
[72] C. Zhang, X. Jia, K. Wang, J. Bao, P. Li, M. Chen, J.B. Wan, H. Su, Z. Mei, C. He,
Polyphyllin VII induces an autophagic cell death by activation of the JNK
pathway and inhibition of PI3K/AKT/mTOR pathway in HepG2 cells, PLoS One
(2016) 1–5.