DNA(CT), protein(BSA) binding studies, anti-oxidant and cytotoxicity studies of new binuclear Ni(II) complexes containing 4(N)-substituted thiosemicarbazones

Article abstract of DOI:10.1016/j.ica.2016.09.006

Four new Ni(II) complexes [Ni₂(Msal-tsc)₂(μ-dppm)] (1), [Ni₂(Msal-mtsc)₂(μ-dppm)] (2), [Ni₂(Msal-etsc)₂(μ-dppm)] (3) and [Ni₂(Msal-ptsc)₂(μ-dppm)] (4) containing 3-met

Full text of DOI:10.1016/j.ica.2016.09.006

Accepted Manuscript
Research paper
DNA(CT), Protein(BSA) binding studies, anti-oxidant and cytotoxicity studies
of new binuclear Ni(II) complexes containing 4(N)- substituted thiosemicarba-
zones
G. Kalaiarasi, C. Umadevi, A. Shanmugapriya, P. Kalaivani, F. Dallemer, R.
Prabhakaran
PII:
S0020-1693(16)30507-2
http://dx.doi.org/10.1016/j.ica.2016.09.006
ICA 17251
DOI:
Reference:
Inorganica Chimica Acta
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 June 2016
1 September 2016
5 September 2016
Please cite this article as: G. Kalaiarasi, C. Umadevi, A. Shanmugapriya, P. Kalaivani, F. Dallemer, R. Prabhakaran,
DNA(CT), Protein(BSA) binding studies, anti-oxidant and cytotoxicity studies of new binuclear Ni(II) complexes
containing 4(N)- substituted thiosemicarbazones, Inorganica Chimica Acta (2016), doi: http://dx.doi.org/10.1016/
j.ica.2016.09.006
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DNA(CT), Protein(BSA) binding studies, anti-oxidant and cytotoxicity studies of
new binuclear Ni(II) complexes containing 4(N)- substituted thiosemicarbazones
c
G. Kalaiarasi,a C. Umadevi,a A. Shanmugapriya,a P. Kalaivani,b F. Dallemer and
R. Prabhakaran a*
a Department of Chemistry, Bharathiar University, Coimbatore 641 046, India
b Department of Chemistry, Nirmala College for Women, Bharathiar University, Coimbatore
641018
c Laboratoire Chimie Provence-CNRS UMR7246, Université of Aix-Marseille I, II and III –
CNRS, Campus Scientifique de Saint-Jérôme, Avenue Escadrille Normandie-Niemen, F-
13397 Marseille Cedex 20, France
Abstract
Four New Ni(II) complexes [Ni2(Msal-tsc)2(ꢀ-dppm)] (1), [Ni2(Msal-mtsc)2(ꢀ-dppm)] (2),
[Ni2(Msal-etsc)2(ꢀ-dppm)]
(3)
and
[Ni2(Msal-ptsc)2(ꢀ-dppm)]
(4)
containing
3-methoxy-salicylaldehyde-4(N)-substituted thiosemicarbazones having the general formula
[H2-MSal-tsc-R],( where R=H, Me, Et or Ph) and 1,1ꢁ-bis(diphenylphosphino)methane
(dppm) were synthesized and characterized by various spectral, analytical techniques and X-
ray crystallography. Crystallographic studies of the complexes 1 and 3 revealed the square
planar geometry around the nickel ion. The binding ability of the complexes with CT-DNA
was carried out by absorption / emission titration and which an intercalative mode of binding
was observed. Further, their ability to interact with BSA protein has been explored.
Investigations of antioxidation properties showed that all the compounds have strong radical
scavenging properties. Further, anticancer activity of the compounds on the lung cancer cell
line A549 has also been investigated. From the results it is observed that the complexes (2
(IC50=23 µM), 3 (IC50=12 µM) & 4 (IC50=20 µM)) exhibited a significant anticancer activity
over complex 1(IC50=26 µM), ligands, NiCl2.6H2O and the standard drug, Cisplatin(IC50=25
µM).
Keywords: Nickel(II) thiosemicarbazones; Spectroscopy; X-ray crystallography; CT-
DNA/BSA binding; Anti-Oxidant activity; Cytotoxicity;
Corresponding author. Tel.: +91-422-2428319; Fax: +91-422-2422387.
E-mail address: rpnchemist@gmail.com (R. Prabhakaran)

1. Introduction
Thiosemicarbazones are an important class of compounds due to their promising
pharmacological properties, such as trypanocidal activity [1], antitubercular activity [2], and
antitumor activities [3,4]. In recent years, a number of thiosemicarbazone derivatives have
been synthesized and their antitumor activity against a broad spectrum of chemotherapeutic
properties were also evaluated [5,6]. Moreover, the complexes consisted of transition metals
and thiosemicarbazone ligands usually possess more potent pharmacological effects than the
thiosemicarbazone ligands alone [7,8]. Some thiosemicarbazones have shown to increase
their biological activity by their ability to form chelates with specific metal ions [9]. Among
the transition metals, nickel, being a essential element because several hydrogenases and
carbon monoxide dehydrogenases [10] contain such nickel ion as their active site. Nickel(II)
thiosemicarbazone complexes has been more extensively studied and their anticancer,
antifungal and antibacterial activities were reported [11-16]. Phosphines are one of the most
important classes of ligands [17]. The design of polydentate phosphines to impose specific
coordination geometries upon one or more metal centers has been an active area of research
for the last three decades [18]. Though the synthesis of new chelating bis(phosphines) and the
development of their metal complexes is an important area in the field of catalysis [19],
however their biological properties were relatively unexplored. The development of
multinuclear complexes as anticancer agents has recently generated tremendous interest [20].
The development of transition metal complexes with DNA binding ability has been an active
area of research due to their potential use as drugs, regulators of gene expression and DNA
structural probes [21-31]. Metal complexes show their anticancer effects through binding to
DNA. Generally DNA is the primary intracellular target of anti-cancer drugs, so the
interaction between small molecules and DNA can often cause DNA damage in cancer cells,
blocking the division of cancer cells and resulting in cell death [32,33]. In addition to study
the interaction of metal complexes with DNA, it is also equally important to study the
binding behaviour of metal complexes with albumin proteins because of the fact that many
drugs, including anticancer, anti-inflammatory, and general anesthetics, are transported in the
blood by combining with serum albumin [34]. The studies on interaction of protein with
metal complexes can provide information of the structural features that determine the
therapeutic effectiveness of the complex and have become an interesting field in recent years
[35]. Therefore, it is absolutely essential and interest to study the interaction behaviors of the
metal complexes with DNA and protein (bovine serum albumin). It is also known that the

uncontrolled production of free radical reactive oxygen species such as superoxide anion can
induce DNA damage in humans. Such a damage to DNA has been suggested to contribute to
aging and various diseases including cardiovascular, cancer and chronic inflammation[36,37].
This has prompted us to test the synthesized complexes as free radical scavengers against
DPPH radical, superoxide anion radical and Fe2+ion. Further, the in vitro anticancer activities
of the compounds were also determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-2H-tetrazolium bromide) assay against human lung cancer cell line (A549). In this
line, analogues nickel complexes are found to be potent in various therapeutic applications
[38-40]. With the above objectives in mind, herein we are reporting the synthesis,
characterization, crystallography, DNA, protein binding, antioxidant and cytotoxicity studies
of new binuclear nickel(II) complexes containing thiosemicarbazones and 1,1ꢁ
bis(diphenylphosphino)methane.
2. Results and discussion
2.1 Synthesis of new nickel(II) complexes
The reactions of two equivalents [NiCl2.6H2O] with two equivalents of
3-methoxy-4(N)-substituted thiosemicarbazones (H2L)1–4 and
one equivalent
1,1ꢁ-bis(diphenylphosphino)methane [dppm] in 1:1 ethanol/dichloromethane resulted in the
formation of new complexes 1-4 (Scheme 1), where the substituted thiosemicarbazones acted
as a tridentate ONS donor ligand. The analytical data of which confirmed the stoichiometry
of the complexes [Ni2(Msal-tsc-R)2(ꢀ-dppm)] (where R= H, CH3, C2H5 and C6H5). The
structures of the complexes (1 and 3) were confirmed by the X-ray crystallography. The new
complexes (1–4) are soluble in common organic solvents such as dichloromethane,
chloroform, benzene, acetonitrile, ethanol, methanol, dimethylformamide and
dimethylsulfoxide.
N
R
N
N
Ni
O
PPh2
OCH3
S
H
[Ethanol/CH2Cl2]
Stirring, 24 hours
OH
S
OCH3
R
Ph2P
PPh2
[NiCl2.6H2O]
+
+
N
H
OCH3
N
N
H
PPh2
S
O
Ni
R
N
N
N
H
R = H
(1)
R = CH3
R = C2H5
R = C6H5
(2)
(3)
(4)
Scheme 1. Synthesis of new Ni(II) complexes

2.2 Spectroscopic Studies
The infrared spectra of the ligands (H2L)1–4 exhibited ꢂ(OH) vibration in the region
3310–3458 cm-1, which disappeared completely upon complexation with nickel showing
deprotonation prior to coordination through oxygen atom in all the four complexes (1–4). It is
further confirmed with the upfield shift of 33-50 cm-1 for ꢂ(C–O) [41,42]. An azomethine
nitrogen ꢂ(C=N) band appeared at the region 1536–1593 cm-1 in the ligands has been shifted to
higher frequency in all the complexes (1592-1611 cm-1) indicating the coordination of
azomethine nitrogen atoms [41]. A sharp band appeared at 771-795 cm−1 corresponding to a
ꢂC=S vibration in the ligands disappeared completely in all the complexes and a new band
appeared around 731–738 cm−1 corresponding to a possible ꢂC–S vibration, indicating
coordination of a thiolate sulphur atom after enolisation followed by deprotonation [41-43].
Moreover, the characteristic absorption bands corresponding to the presence of
diphenylphosphinomethane were also present in the expected region [44,45]. The electronic
spectra of nickel(II) complexes displayed two to four bands in the region around 263-418 nm
(Fig. S1). The band appeared at 263-268 nm has been assigned to intra ligand transition and
3
the bands at 313-371 nm to LMCT (3A2g ꢃ T2g) and the shoulder at 418-429 nm to
3
1
forbidden (3A2g ꢃ T1g) transition [42,45]. The H-NMR spectra of the ligands (H2L)1–4 and
the corresponding complexes (1-4) recorded in DMSO and CDCl3 at the field strength of 400
MHz showed all the expected signals (Fig. S2-S9). In the spectra of (H2L)1–4, a sharp singlet
corresponding to the phenolic –OH group has appeared at ꢄ 11.34-11.76 ppm. However, this
singlet completely disappeared in all the complexes confirmed the involvement of phenolic
oxygen in coordination [41,43,46]. The spectra of (H2L)1–4 showed a singlet at ꢄ 9.13-10.0
ppm corresponding to (N(2)H-C=S) group [41,43], but in the complexes 1-4 there was no
resonance attributable to N(2)H, indicating the coordination of thiolate sulphur atom of the
ligand in the anionic form after deprotonation at N(2) [41,46]. In the spectra of H2L1–4 and the
complexes, a complex multiplet appeared at ꢄ 6.3–7.98 ppm was assigned to aromatic
protons of the ligands and diphenylphosphinomethane [41,43] and a singlet corresponding to
the –OCH3 group occurred at ꢄ 3.59–3.81 ppm range [41,43]. Two singlets observed at ꢄ
8.37–8.50 ppm and ꢄ 8.37–9.20 ppm have been assigned to azomethine and terminal -NH
protons of the ligands (H2L)2–4 [41,43]. Two broad singlets appeared at ꢄ 7.8 and ꢄ 8.0 ppm
corresponding to NH2 protons of H2L1 [41]. Whereas, in the complexes a singlet at ꢄ 4.45 -
5.29 ppm was assigned to the terminal NH protons of the ligands. Though the spectra of 1
and 4 showed a doublet at ꢄ 7.835- 7.857 & ꢄ 8.122–8.144 ppm corresponding to the

azomethine proton [41], the same has been mixed in the aromatic region for 2 and 3. In the
spectra of H2L2 and the complex 2, a doublet was observed around ꢄ 2.91–2.99 ppm due to
the methyl group of protons. A triplet observed around ꢄ 1.13–1.14 ppm in the spectra of
H2L3 and the complex 3, was assigned to the presence of methyl group of protons [41].
Further, a multiplet at ꢄ 3.25 –3.58 ppm was also observed corresponding to the methylene
protons of H2L3 and complex 3 [41]. In addition, a triplet was observed in the spectra of all
the complexes at ꢄ 4.0-4.16 ppm due to the presence of methelene protons of the 1,1ꢁ-
bis(diphenylphosphino)methane ligand [44].
2.3 X-ray crystallography
In order to confirm the exact structure of the complexes, X-ray crystallographic
analyses has been done for the new complexes (1 and 3). Complex 1 and 3 crystallized in the
monoclinic space group P21. The crystallographic data, selected bond distances and angles are
listed in Tables 1 and 2. In the dimeric complexes 1 and 3, each nickel atom is coordinated by
a tridentate binegative ligand through phenolic oxygen, N1 hydrazinic nitrogen and thiolate
sulphur atom by forming a six member and another five member ring with Ni–S bond
distances of 2.1370(7) Å and 2.139(1) Å, respectively, Ni–O bond distances of 1.838(2) Å
and 1.852(2) Å, respectively and Ni–N bond distances of 1.884(2) Å and 1.885(3) Å,
respectively [42,43]. The remaining binding site is occupied by the phosphorous atom of 1,1ꢁ-
bis(diphenylphosphino)methane unit with (Ni(1)–P(1)) bond distances of 2.1974(5) Å and
2.2075(8) Å, respectively) [45] with the bite angle [S(1)–Ni(1)–N(1)] of 87.68(5)° for 1 and
86.88(9) ° for 3. The [S(1)–Ni(1)–O(1)] bond angles found are 174.66(5) ° for 1 and
174.48(8) ° for 3 [42,43] and [P(1)–Ni(1)–N(1)] bond angles found are 174.99(5) ° for 1 and
176.95(9) ° for 3 [45] which deviate considerably from the ideal angle of 180 ° causing
significant distortion in the square planar geometry of the complexes. Two of these
symmetrical units are bridged through dppm ligand in the complexes 1 and 3.
In complex 1, an intermolecular hydrogen bonding was found between the imine
nitrogen and the oxygen atom [N(2)...O(5) (2.863 A˚)] of the water which came through the
solvent of crystallization (Fig. S10). The presence of four intermolecular hydrogen bondings
in complex 3 creates a 2D network (Table S1). First two hydrogen bonding comes through
interaction N(2) and N(5) nitrogen atoms of the coordinated thiosemicarbazone moieties and
with the hydrogen atoms of water molecule which is coming through solvent of
crystallisation (Fig. S11). The third intermolecular hydrogen bonding is between hydrogen

atom of N(3) terminal nitrogen atom and oxygen atom of methanol which is used as solvent
for crystallisation and the fourth hydrogen bonding is between water and methanol moieties
of crystallisation solvents. All the above four intermolecular hydrogen bonding led give 2D
layer structure to the complex 3.
Fig. 1. ORTEP diagram of [Ni2(Msal-tsc)2(µ-dppm)] (1)

Fig. 2. ORTEP diagram of [Ni2(Msal-etsc)2(µ-dppm)] (3)

Table 1. Crystallographic data of complexes 1 and 3
Identification code
[Ni2(Msal-tsc)2(ꢀ-dppm)](1) [Ni2(Msal-etsc)2(ꢀ-dppm)](3)
Empirical formula
Formula weight
C43H40N6Ni2O4P2S2
984.32
C47H48N6Ni2O4P2S2
1072.46
Temperature
293(2) K
293(2) K
Wavelength
Crystal system
Space group
1.54184 Å
Monoclinic
P21/C
1.54184 Å
Monoclinic
P21/C
Unit cell dimensions
a
B
C
11.3353(9) Å
14.0555(10) Å
28.3292(2) Å
90°
10.55971(13) Å
15.68622(13) Å
31.4025(3) Å
90°
α
β
94.3615(7)°
90°
94.1646(9)°
90°
γ
Volume
Z
4500.44 Å3
4
5187.84Å3
4
Density
Absorption coefficient,
F(000)
1.453 Mg/m3
3.020 mm-1
2040
1.373 Mg/m3
2.678 mm-1
2240
Crystal size
Crystal shape
θ range for data collection
Limiting indices
0.16 × 0.16 × 0.07 mm
Block
0.16 × 0.16 × 0.12 mm
Block
4.421 to 73.635°
-13 ≤ h ≤ 13, -16 ≤ k ≤ 17,
-34 ≤ l ≤ -35
20206
8894 (R(int) = 0.0339)
66.97° 99.62 %
multi-scan
4.182 to 73.606°
-12 ≤ h ≤ 12, -19 ≤ k ≤ 19,
-39 ≤ l ≤ 38
23092
10291(R(int) = 0.0270)
66.97° 99.89 %
multi-scan
Reflections collected
Independent reflections
Completeness to θ
Absorption correction
Refinement method
Full-matrix least-squares on Full-matrix least-squares on
F2
F2
Data / restraints / parameters 8894 / 0 / 567
10291/ 0/631
1.138
R1 = 0.0508, wR2 = 0.1383
R1 = 0.0542, wR2 = 0.1410
Goodness-of-fit on F2
Final R indices [I>2σ(I)]
R indices (all data)
1.024
R1 = 0.0331, wR2= 0.0875
R1 = 0.0366, wR2 = 0.0910

Table 2: Selected bond lengths(Å) and bond angles(°) of the complexes (1) and (3)
BOND LENGTHS
Ni(1)-S(1)
Ni(1)-P(1)
Ni(1)-O(1)
Ni(1)-N(1)
Ni(2)-S(2)
Ni(2)-P(2)
Ni(2)-O(3)
Ni(2)-N(4)
1
3
2.1370(7)
2.1974(5)
1.838(2)
1.884(2)
2.1410(6)
2.1968(6)
1.850(1)
1.885(2)
2.139(1)
2.2075(8)
1.852(2)
1.885(3)
2.1254(9)
2.1254(9)
1.862(2)
1.881(3)
BOND ANGLES
S(1)-Ni(1)-P(1)
S(1)-Ni(1)-O(1)
S(1)-Ni(1)-N(1)
P(1)-Ni(1)-O(1)
P(1)-Ni(1)-N(1)
O(1)-Ni(1)-N(1)
S(2)-Ni(2)-P(2)
S(2)-Ni(2)-O(3)
S(2)-Ni(2)-N(4)
P(2)-Ni(2)-O(3)
P(2)-Ni(2)-N(4)
O(3)-Ni(2)-N(4)
96.27(2)
174.66(5)
87.68(5)
81.01(5)
174.99(5)
94.79(7)
94.99(2)
176.54(5)
87.32(5)
82.59(5)
175.61(6)
94.94(7)
94.58(4)
174.48(8)
86.88(9)
83.42(7)
176.95(9)
94.9(1)
91.58(3)
177.52(7)
87.01(8)
86.21(7)
173.32(8)
95.1(1)
2.4 DNA binding studies
The interaction between DNA and metal complexes is of paramount importance in
understanding the mechanism of any drug action. Thus, the mode and propensity for binding
of the new nickel(II) complexes 1-4 to CT DNA were studied with the help of electronic
absorption and fluorescence quenching techniques. The stability of the complexes in DMSO,
1% aqueous DMSO and tris-HCl buffer have been carried out by using UV-Vis
spectrophotometer. The results showed that all the complexes are stable in these solutions
allowing us to proceed further for DNA binding studies. The absorption spectra of the
complexes (1–4) at constant concentration (10 µM) in the presence of different
concentrations of CT-DNA (5–50 µM) are given in Fig. 3.
The absorption spectra of complex 1 consist of three resolved bands centered at 284 nm
(intraligand transition), 373 and 425 nm (charge transfer). As the DNA concentration is
increased, hypochromism (A=0.3099–0.1563) with a red shift of 2 nm (up to 286 nm) was
observed in the intraligand band. The CT band at 373 and 425 nm showed modest
hypochromism (A=0.1685–0.0912 and A=0.0990-0.0550 respectively) with negligible shifts

in the wavelength. The binding behavior of complexes 2, 3 and 4 is also quite similar.
Complex 2 exhibited hypochromism in the intraligand band at 265 nm (A=0.7609–0.2771)
with 1 nm red shift and hypochromism at 367 nm with 1 nm red shift (LMCT) (A=0.4323–
0.1371). Complex 3 exhibited hypochromism in the intraligand band at 304 nm (A=0.6458–
0.3818) with 1 nm red shift and hypochromism at 369 nm (LMCT) (A=0.2842–0.1433). The
spectra of complex 4 consist of two resolved bands centered at 262 nm and 308 nm
(intraligand transition). As the DNA concentration is increased, hypochromism (A=0.2876–
0.1748) with a red shift of 1 nm was observed in the intraligand band. The another IL band at
308 nm (A=0.3250–0.2016) showed hypochromism with negligible shifts in the wavelength.
The observed hypochromic effect with a red shift suggested that the new complexes may bind
to CT-DNA by intercalation. Further, the binding mode has been evidented through
competitive binding studies using Ethidium Bromide (EB)
The intrinsic binding constant Kb is a useful tool to monitor the magnitude of the
strength with the compounds which bind to CT-DNA (Table 3). It can be determined by
monitoring the changes in the absorbance in the intraligand band at the corresponding ꢅmax
with increasing concentration of DNA and is given by the ratio of slope to the ‘y’ intercept in
plots of [DNA]/(ꢆa-ꢆf) versus [DNA] ( Fig. 4) [43].
To further confirm the interaction between complexes 1–4 and CT-DNA, competitive
binding studies using Ethidium Bromide (EB) were conducted. The emission intensity of EB
is used as a structural probe as EB shows reduced emission intensity in buffer solution due to
either solvent quenching or photoelectron transfer mechanism[47] and enhancement in the
emission intensity when it bound intercalatively to DNA. While adding the complexes 1–4 to
DNA pretreated with EB, the DNA-induced emission intensity at 604 nm was decreased
(Fig. S12). This indicates the replacement of EB by complexes from the EB–DNA system.
Such a characteristic change is often observed in intercalative DNA interactions. The
quenching extents of complexes 1-4 were evaluated qualitatively by employing Stern–
Volmer equation (1).
Iꢇ/I = Ksv[Q] + 1
(1)
Where Io is the emission intensity in the absence of compound, I is the emission
intensity in the presence of compound, Ksv is the quenching constant, and [Q] is the
concentration of the compound. The Stern–Volmer quenching constant KSV, obtained as the
slope of Iꢇ/I versus the concentration of the quencher (Table 3) (Fig.S13). A decrease in the

emission intensity suggests stronger binding affinity of the complexes to CT-DNA [43,48].
Furthermore, the observed quenching constants and binding constants of the new complexes
suggested an intercalative binding mode the complexes to DNA.
0.4
1
1.0
2
0.8
0.3
0.6
0.2
0.4
0.1
0.2
0.0
0.0
300
400
500
300
400
500
WAVELENGTH(nm)
WAVELENGTH(nm)
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.35
4
3
0.30
0.25
0.20
0.15
0.10
0.05
0.00
300
400
500
300
400
500
WAVELENGTH(nm)
WAVELENGTH(nm)
Fig. 3. Changes in the electronic absorption spectra of complexes 1-4 (10 µM) with
increasing concentrations (5-50 µM) of CT-DNA (tris HCl buffer, pH 7.2).

1
2
3
4
0.0024
0.0022
0.0020
0.0018
0.0016
0.0014
0.0012
0.0010
0.0008
0.0006
0.0004
0.0002
0.0000
0
10
20
30
40
50
[DNA]
Fig. 4. plot of [DNA]/(ꢈa-ꢈf) versus [DNA]
Table 3 :The Kb and Ksv values for the interactions of complexes (1–4) with CT-DNA
Complex
Kb/M-1
KSV/M-1
1
2
3
4
2.765 0.120×105
1.079 0.050×105
4.311 0.056×105
3.384 0.140×105
5.99 0.029×103
6.79 0.021×103
8.67 0.034×103
6.85 0.026×103
2.5 Protein binding studies
2.5.1 Quenching mechanism of BSA by complexes
Binding of Schiff base metal complexes with the most abundant carrier proteins
(serum albumins) have also been an area of interest as such drug–protein binding greatly
influences absorption, drug transport, storage, metabolism and excretion properties of typical
drugs in vertebrates [49]. Since serum albumins are well known to bind with small aromatics,
the possible binding interactions of the nickel(II) thiosemicarbzone complexes (1-4) with
BSA have been investigated by absorption/emission-titration experiments at room
temperature. Prior to the interaction fluorescence studies, the stability of complexes (1-4) was
studied in DMSO, 1% aqueous DMSO and in phosphate buffer using UV-Vis
spectrophotometer. All the complexes were found to be stable in these solutions for, at least,
24 h, allowing us to carry on with the studies with albumin. In the absorption spectra of BSA,

the addition of 10 µM concentrations of the four complexes (1-4) (Fig. S14) showed
enhanced absorption intensity of BSA with a blue shift of complex–BSA spectrum (from 265
to 261 nm). The formation of non-fluorescence ground-state complex induced the change in
absorption spectrum of fluorophore and possible quenching mechanism of BSA by
complexes (1-4) was found as static quenching [50].
2.5.2. Fluorescence quenching studies of BSA
A solution of BSA (10 µM) was titrated with various concentrations of the complexes
(0–100 µM). Fluorescence spectra were recorded in the range of 290–500 nm upon excitation
at 280 nm. The changes observed on the emission spectra of BSA by the addition of
increasing amounts of the complexes (1-4) which are shown in Fig. 5.
Upon the addition of complexes 1–4 to BSA, a significant decrease in the
fluorescence intensity was observed at 346 nm with hypochromism of 8.98 %, 10.33 %,
13.95 % and 3.04 % respectively accompanied by 1-4 nm blue shifts. The observed
quenching may be attributed to the possible changes in secondary structure of protein and this
indicating the binding of complex to BSA [22]. According to Stern−Volmer quenching
equation, eq 1. The KSV value was found to be 8.75 0.021 x 103 M-1, 9.23 0.016 x 103
M-1,1.367 0.036 x 104 M-1 and 4.09 0.009 x 103 M-1 corresponding to complexes 1, 2, 3
and 4 respectively. The observed linearity in the plots (Fig. 6; Table 4) indicates the ability
of the complexes to quench the emission intensity of BSA. From KSV values, the complex 3
exhibited better protein-binding. For the static quenching interaction, if it is assumed that
there are similar and independent binding sites in the biomolecule, the binding constant (Kb)
and the number of binding sites (n) can be determined according to the method [51] using the
Scatchard equation (2).
log [(Fꢇ−F)/F]= log Kb+ n log [Q] (2)
where, in the present case, Kb is the binding constant for the complex–protein interaction and
‘n’ is the number of binding sites per albumin molecule, which can be determined by the
slope and the intercept of the double logarithm regression curve of log [(Fꢇ−F)/F] versus
log[Q] (Fig. 7; Table 4).

400
300
200
100
0
2
300
200
100
0
1
300
350
400
450
300
350
400
450
WAVELENGTH(nm)
WAVELENGTH(nm)
350
300
250
200
150
100
50
3
400
4
200
0
300
350
400
450
500
300
350
400
WAVELENGTH(nm)
450
500
WAVELENGTH(nm)
Fig. 5. The emission spectra of BSA (10 µM; ꢅexc= 280 nm; ꢅemi= 346 nm) in the presence
of increasing amounts of complexes 1-4 (0–100 µM). The arrow shows the emission
intensity changes upon increasing complex concentration.

Complex 1
Complex 2
Complex 3
Complex 4
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0
20
40
60
80
100
[Q]
Fig. 6. Stern−Volmer plot of the fluorescence titration of the complexes (1-4) (10-100 µM)
with BSA (10 µM).
Complex 1
Complex 2
0.4
0.2
Complex 3
Complex 4
0.0
-0.2
-0.4
-0.6
-0.8
-1.0
-1.2
-1.4
-1.6
-5.0
-4.8
-4.6
-4.4
-4.2
-4.0
log [Q]
Fig. 7. Scatchard plot of the fluorescence titration of the complexes (1-4) (10-100 µM) with
BSA(10 µM).

Table 4: Quenching constant (Ksv), binding constant (Kbin) and number of binding sites (n)
for the interactions of complexes (1–4) with BSA
Complex
KSV/M-1
Kbin/M-1
‘n’
1
2
3
4
8.75 0.021×103
9.23 0.016×103
13.67 0.036×103
4.09 0.009×103
5.637 0.020×103
4.747 0.011×103
7.584 0.017×103
7.097 0.018×103
0.9499
0.9262
0.9422
1.0574
2.5.3. Synchronous fluorescence spectroscopic studies of BSA:
Synchronous fluorescence spectral study involves simultaneous scanning of the
excitation and emission monochromators while maintaining a constant wavelength interval
between them. It was used to obtain information about the molecular environment in the
vicinity of the fluorophore moieties of BSA [52]. Synchronous fluorescence spectra show
tyrosine residues of BSA only at the wavelength interval ꢉꢅ of 15 nm whereas tryptophan
residues of BSA at ꢉꢅ of 60 nm. While the amount of complexes (1-4) added to BSA (10
µM) is increased, there is a decrease in the fluorescence intensity of the complexes 1 and 4
and increase in the fluorescence intensity of the complexes 2 and 3 corresponding to tyrosine
residue with a red shift (1-3 nm) (Fig. S15) was noted. In addition, significant decrease in
fluorescence intensity of tryptophan residues together with a small blue shift (1 nm) in the
emission wavelength was also observed for the complexes (Fig. S16). These experimental
results indicate that the metal complexes affect the microenvironment of both tyrosine and
tryptophan residues during the binding process and synchronous measurements confirmed the
effective binding of all the complexes with BSA.
2.6 Antioxidant capacity
Since the experiments conducted so far revealed that the complexes exhibit significant
DNA and protein binding affinity, it is considered worthwhile to study the antioxidant
activity of these compounds. Free radicals play an important role in the inflammatory
process. Thus, compounds with possible antioxidant properties could play a crucial role
against inflammation and lead to potentially effective drugs. Antioxidants that exhibit radical
scavenging activity are receiving increased attention since they present interesting anticancer,
anti-ageing and anti-inflammatory activities. Therefore, compounds with antioxidant
properties may offer protection against rheumatoid arthritis and inflammation.45 The radical
scavenging activities of our compounds along with standards, such as ascorbic acid and

butylated hydroxytoluene (BHT) in a cell free system, have been examined with reference to
−
DPPH radicals (DPPH˙) and superoxide anion radicals (O2 ˙) and their reducing power and
total antioxidant activity were examined.The power of the compounds ability to scavenge
various radicals was found to be slightly higher than that of the standard antioxidants
(Ascorbic acid and BHT).
The free radical scavenging activity of NiCl2.6H2O, ligands, 1,1ꢁ-
bis(diphenylphosphino)methane and new Ni(II) complexes were tested by its ability to bleach
the stable radical DPPH. This assay provided information on the reactivity of the compound
with a stable free radical. Because of the odd electron, DPPH shows a strong absorption band
at 517 nm in the visible spectrum [53]. As this electron becomes paired off in the presence of
a free radical scavenger, the absorption vanishes, and the resulting decolonization is
stoichiometric with respect to the number of electrons taken up. The compounds represent
moderate to significant reducing ability of DPPH radical when compared to the reference
compound ascorbic acid. The new complexes exhibited a significant DPPH radical
scavenging effect greater than that of well established vitamin C, a reference drug established
elsewhere [45,54]. The free radical scavenging activity of the compounds increased with an
increase in the concentration of the compounds and it is measured interms of IC50values (Fig.
8 and 10; Table S2). Our results were compared with the literature data, the new binuclear
Ni(II) complexes showed moderate scavenging activity.[45,55,56]
DPPH RADICAL SCAVENGING ASSAY
90
80
70
60
50
40
30
20
STD
H2L1
H2L2
H2L3
H2L4
NiCl2.6H20
dppm
Complex 1
Complex 2
Complex 3
Complex 4
20
40
60
80
100
CONCENTRATION (µg/ml)

Fig. 8. DPPH scavenging activity of ligands, 1,1ꢁ-bis(diphenylphosphino)methane (dppm),
NiCl2.6H2O and new Ni(II) complexes. Error bars represent the standard deviation of the
mean (n=3).
.-
Superoxide (O2 ) is highly reactive compound among the reactive oxygen species
(ROS). These species can disintegrate cell membranes and damage protein and DNA
structures and are largely responsible for many diseases, such as cancer, liver injury and
cardiovascular complications[36]. The ability of Nickel chloride, ligands, 1,1ꢁ-
bis(diphenylphosphino)methane and new Ni(II) complexes were appreciable for the
superoxide radicals when compared to the standards (Table S2; Fig 9-10). It can be seen that
.-
the inhibitory effects of the tested compounds on O2 is concentration related and the
suppression ratio increases with the increasing sample concentration in the range of the tested
concentration. As seen from the results, the compounds exhibited significant activity[57].
STD
SUPEROXIDE RADICAL SCAVENGING ASSAY
H2L1
H2L2
75
H2L3
70
H2L4
NiCl2.6H2O
dppm
Complex 1
Complex 2
Complex 3
Complex 4
65
60
55
50
45
40
35
30
25
20
20
40
60
80
100
CONCENRATION (µg/ml)
Fig. 9. Superoxide scavenging activity of ligands, bis(diphenylphosphino)methane (dppm),
NiCl2.6H2O and new Ni(II) complexes. Error bars represent the standard deviation of the
mean (n=3).

DPPH
SUPEROXIDE
100
90
80
70
60
50
40
30
20
10
0
STD H2L1 H2L2 H2L3 H2L4 Ni DPPM
1
2
3
4
--
Fig. 10. Antioxidant activity (IC50 values) of ligands, bis(diphenylphosphino)methane
(DPPM), NiCl2.6H2O and new Ni(II) complexes . Error bars represent the standard deviation
of the mean (n=3).
Fig.
11
shows
the
reducing
power
of
NiCl2.6H2O,
ligands,
bis(diphenylphosphino)methane and new Ni(II) complexes as a function of their
concentration. In this assay, the yellow color of the test solution changed to various shades of
green and blue depending upon the reducing power of each compound. The presence of
reducers (i.e., antioxidants) causes the reaction of the Fe3+/ferricyanide complex to the
ferrous form the Perl’s Prussion blue after the addition of trichloroacetic acid and ferric
chloride, that can be monitored at 700 nm. The reducing power of the standard BHT at
various concentrations was higher than that of test compounds. The reducing power of test
compounds solutions in DMSO increased with increase in concentration and showed
absorbances nearer to those of the standard. All the metal complexes showed much better
activity than the ligands, bis(diphenylphosphino)methane and Ni salt (Fig.11). Thus the
coupling of metal ions with the ligands is the important features for increased reducing power

of these compounds[58]. The complex 3 has the highest antioxidant activity when compared
to the other complexes as it showed the reducing power of 0.71 0.003/ 100µg ml-1 .
STD
H2L1
0.8
REDUCTIVE ABILITY ASSAY
H2L2
H2L3
H2L4
0.7
NiCl2.6H2O
0.6
0.5
0.4
0.3
0.2
0.1
0.0
dppm
Complex 1
Complex 2
Complex 3
Complex 4
20
40
60
80
100
CONCENTRATION (µg/ml)
Fig. 11. Reductive ability of ligands, bis(diphenylphosphino)methane (dppm), NiCl2.6H2O
and new Ni(II) complexes. Error bars represent the standard deviation of the mean (n=3).
Among the complexes, total antioxidant capacity was found to be high in complex 3
and low in bis(diphenylphosphino)methane. (Table 5) Moreover, from the results obtained
for the four Ni(II) complexes, complex 3 showed the better activity may be due to the ethyl
substitution in the terminal nitrogen of the ligand. From the above results, it can be concluded
that the scavenging effects of the nickel salt, bis(diphenylphosphino)methane and free ligands
is significantly less when compared to their corresponding Ni(II) complexes, which is mainly
due to the chelation of the organic ligand with the Ni(II) ion[55,56].

Table 5: Estimation of Total antioxidant capacity of ligands, bis(diphenylphosphino)methane
(dppm), NiCl2.6H2O and new Ni(II) complexes
Compounds
H2L1
µg Ascorbic acid equivalents/ml
9.84 0.04
H2L2
10.95 0.06
9.52 0.05
12.27 0.07
H2L3
H2L4
NiCl2.6H2O
dppm
13.66 0.11
7.87 0.04
Complex 1
Complex 2
Complex 3
Complex 4
10.57 0.04
25.33 0.05
35.08 0.03
16.14 0.06
2.7 Cytotoxic studies
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay
The positive results obtained from the previous biological studies namely, DNA
binding, BSA binding and antioxidative studies of compounds encouraged us to test their
cytotoxicity against human lung cancer cell line (A549). The tumour inhibiting capacity of
the compounds against A549 cells (a model cell line for lung cancer) by the MTT assay, that
is based on the mitochondrial reduction of the tetrazolium salt by actively growing cells to
produce blue insoluble formazan crystals, was undertaken. Compounds were dissolved in
DMSO and blank samples containing same volume of DMSO are taken as controls to identify
the activity of solvent in this cytotoxicity experiment. Cisplatin was used as a positive control
to assess the cytotoxicity of the test compounds. The results were analyzed by means of cell
viability expressed as IC50 values are shown in Table 6. Appropriate Ligands, 1,1ꢁ-
bis(diphenylphosphino)methane and NiCl2.6H2O used in the synthesis of the complexes were
also tested for cytotoxicity. Though the ligands and metal precursor showed moderate
inhibiting activity against lung cancer cell line (A549), the new complexes exhibited
significant activity against A549. The complexes exhibited good cytotoxic effects on lung
cancer cells with lower IC50 values indicating their efficiency in killing the cancer cells even
at low concentrations which confirmed that the chelation of the ligand with the Ni(II) ion is
the only responsible factor for the observed cytotoxic properties of the new complexes. The
Ni(II) complexes which possess better cytotoxic activities than ligand may be attributed to
the extended planar structure induced by the ꢊ/ꢊ* conjugation resulting from the chelating of
the metal ion with ligand.Some promising cytotoxic effects were observed for new binuclear

Ni(II) complexes toward the A549 cell line as reported in Table 6 (Fig.S17). These results are
much better than those previously reported for other mononuclear Ni(II) complexes [43]. The
complex 3 showed higher cytotoxic effect followed by the complexes 4 and 2. It is interesting
to note that the complexes 2, 3 and 4 exhibited significant activity than cisplatin. The highest
activity was noted for the complex 3 and the least activity was observed with the complex 1.
In addition, though the ligand coordinated as bibasic tridentate to Ni(II) in all the complexes,
the inhibitory activity of 3 against cancer cell is higher, which may be due to the electron
donating ethyl substitution in the terminal nitrogen of the ligand.
Table 6: The IC50 values for the human lung cancer cell line A549 after an incubation period
of 48 hours with the ligands, bis(diphenylphosphino)methane (dppm), NiCl2.6H2O and new
Ni(II) complexes
Complexes
IC50value(µM)
A549
H2L1
H2L2
34 0.32
29 0.26
27 0.23
30 0.28
31 0.35
43 0.52
26 0.28
23 0.20
12 0.14
20 0.19
25 0.23
H2L3
H2L4
NiCl2.6H2O
dppm
Complex 1
Complex 2
Complex 3
Complex 4
Cisplatin
3. Conclusion
New binuclear nickel(II) thiosemicarbazone complexes have been synthesized and
characterized by various spectro and analytical techniques. The true nature of coordinating
ligand with the central metal atom was established by X-ray crystallographic studies. From
the study, it is inferred that in complexes 1 and 3 the ligands coordinated as tridentate bibasic
nature. In order to study the potential binding ability of the new complexes, CT-DNA and
BSA protein were taken as models. The complexes bind to CT-DNA through intercalation
mode and with BSA, the quenching was found as static. Among the four complexes,
complex 3 exhibited better binding affinity with BSA. All the complexes exhibited

significant antioxidant activity. The cytotoxic studies showed that the complexes 2, 3 and 4
exhibited good cytotoxic activity against A549 cell line and complex 1 showed IC50
cytotoxicity coefficients similar to those of cisplatin. The IC50 value of the complexes in
A549 cell line was relatively low as compared with the standard cisplatin , ligands, 1,1ꢁ-
bis(diphenylphosphino)methane, NiCl2.6H2O. Complex 3 exhibited relatively high cytotoxic
activity against lung cancer A549 cells with low IC50 value(IC50 =12 µM) than the standard
cisplatin. In general, complex 3 showed bettter biological activity in all the experiments. The
substitution of an ethyl group at terminal nitrogen of the coordinated ligand of complex 3
may be responsible for its observed good biological activity over the other three complexes.
4. Experimental
4.1. Complex synthesis and characterization
1,1ꢁ bis(diphenylphosphino)methane and NiCl2.6H2O were purchased from Sigma
Aldrich Ltd. The ligands [H2L]1-4 were synthesized according to the standard literature
procedure [41]. All the reagents used were Analar grade, were purified and dried according
to the standard procedure [59]. CT-DNA, EB and BSA were obtained from Himedia. Infrared
spectra were measured as KBr pellets on a JASCO FT-IR 4100 instrument between 400–
4000 cm−1. Elemental analyses of carbon, hydrogen, nitrogen, and sulfur were determined by
using Vario EL III CHNS at the Department of Chemistry, Bharathiar University,
Coimbatore, India. Melting points were measured in a Lab India apparatus. The electronic
spectra of the complexes have been recorded in DMSO using a JASCO V-630
Spectrophotometer in the 800–200 nm range. Emission spectra were recorded by using
JASCO FP 6600 Spectrofluorometer. 1H NMR spectra were recorded in DMSO and CDCl3 at
room temperature with a Bruker 400 MHz instrument, chemical shift relative to
tetramethylsilane. Single crystal data collections and corrections for the new Ni(II)
complexes (1 and 3) were done at 293 K with a CCD kappa Diffractometer using graphite
monochromated MoKꢋ (l = 1.54184 A˚) radiation [60]. The structural solutions were done by
using SHELXS-97 [61] and refined by full matrix least square on F2 using SHELXL-97 [62].
Preparation of new nickel(II) complexes
4.1.1. Preparation of [Ni2(Msal-tsc)2(µ-dppm)] (1)

3-Methoxysalicylaldehydethiosemicarbazone (0.095 g; 0.420 mmol) was dissolved in
dichloromethane (25 cm3) and this was added to an ethanolic solution of NiCl2.6H2O (0.100
g; 0.420 mmol). The mixture was stirred during a clear reddish brown solution was formed.
To this 1,1ꢁ-bis(diphenylphosphino)methane (0.080 g, 0.210 mmol) was added immediately.
After 24 hours stirring, the reaction mixture was filtered and concentrated, whereby a reddish
brown solid was obtained. It was washed with petroleum ether (60–80°C) and recrystallized
from toluene and methanol to yield reddish brown crystals suitable for X-ray crystallography.
Yield: 53% . M.p. 272 °C. Anal. Calcd for C43H40N6O4S2Ni2P2: C, 54.48; H, 4.25; N, 8.86; S,
6.76. Found: C, 54.42; H, 4.20; N, 8.79; S, 6.71%. FT-IR (cm−1) in KBr: 1606 (ꢂC=N), 1305
(ꢂC–O), 737 (ꢂC–S); UV-Vis (DMSO), ꢅmax: 263 (93720) nm (dm3mol−1cm−1) (intra-ligand
transition); 321(82831) nm (dm3mol−1cm−1) (LMCT s ꢃ d); 1H NMR (CDCl3, ꢄ ppm, J Hz):
ꢄ 7.835-7.857(d, (J= 8.8), CH=N), ꢄ 6.559- 6.578 (d, (J= 7.6), C4-H), ꢄ 6.419- 6.458 (t,
(JH4H5= 7.6), (JH5H6= 8), C5-H), ꢄ 6.677- 6.697 (d, (J= 8), C6-H), ꢄ 4.57 (s, –NH2), ꢄ 3.61
(s,-OCH3), ꢄ 4.108- 4.182 (t, (–CH2–)), ꢄ 7.184- 7.276 & ꢄ 7.912-7.953(m, dppm protons).
The very similar method was followed to synthesize other complexes.
4.1.2. Preparation of [Ni2(Msal-mtsc)2(µ-dppm)] (2)
The complex 2 was prepared by the procedure as used for (1) with
3-methoxysalicylaldehyde-4(N)-methylthiosemicarbazone [H2-Msal-mtsc](0.100 g; 0.420
mmol),
NiCl2.6H2O
(0.100
g;
0.420
mmol)
and
1,1ꢁ-bis(diphenylphosphino)methane (0.080 g, 0.210 mmol).Yield: 51% . M.p: 208 °C. Anal.
Calcd for C45H44N6O4S2Ni2P2: C, 55.37; H, 4.54; N, 8.61; S, 6.57. Found: C, 55.33; H, 4.51;
N, 8.58; S, 6.52%.
FT-IR (cm−1)
in KBr: 1594 (ꢂC=N), 1310
(ꢂC–O), 731 (ꢂC–S); UV-Vis (DMSO), ꢅmax: 265 (79916) nm (dm3mol−1cm−1) (intra-ligand
transition); 313(48072) & 365 (37518) nm (dm3mol−1cm−1) (LMCT s ꢃꢌꢍꢎꢏꢌꢐꢑꢒꢌꢓꢑꢔꢕꢖꢑꢎ nm
(dm3mol−1cm−1)
forbidden
(dꢃꢍꢎꢌ
ꢗꢘꢙꢚꢛꢜꢗꢜꢝꢚꢏꢌ
1H
NMR
(CDCl3, ꢄ ppm, J Hz): ꢄ 6.539- 6.562 (dd, (J=1.6, 7.6), C4-H), ꢄ 6.400- 6.438 (t, (JH4H5=
7.6), (JH5H6= 8), C5-H), ꢄ 6.671- 6.695 (dd, (J= 1.6, 8), C6-H), ꢄ 4.529 – 4.542 (d, (J= 5.2),
terminal –NH), ꢄ 3.599 (s, –OCH3), ꢄ 2.913- 2.926 (d (J=5.2), –CH3), ꢄ 4.092-4.166 (t, (–
CH2–)), ꢄ 7.173- 7.277 & ꢄ 7.911-7.966(m, dppm protons and –CH=N).
4.1.3. Preparation of [Ni2(Msal-etsc)2(µ-dppm)] (3)

The complex 3 was prepared by the procedure as used for (1) with
3-methoxysalicylaldehyde-4(N)-ethylthiosemicarbazone [H2-Msal-etsc] (0.106 g; 0.420
mmol), NiCl2.6H2O (0.100 g; 0.420 mmol) and 1,1ꢁ-bis(diphenylphosphino)methane (0.080
g, 0.210 mmol).Yield: 57%. M.p: 173 °C. Anal. Calcd for C47H48N6O4S2Ni2P2: C, 56.22; H,
4.81; N, 8.37; S, 6.38. Found: C, 56.18; H, 4.78; N, 8.33; S, 6.34%. FT-IR (cm−1) in KBr:
1611 (ꢂC=N), 1326 (ꢂC–O), 738 (ꢂC–S); UV-Vis (DMSO), ꢅmax: 266 (89502) nm
(dm3mol−1cm−1) (intra-ligand transition); 318 (61779) nm (dm3mol−1cm−1) (LMCT s ꢃꢌꢍꢎꢏꢌ
1
418 (18500) nm (dm3mol−1cm−1) forbidden (dꢃd) transition; H NMR (CDCl3, ꢄ ppm, J
Hz): ꢄ 6.540- 6.559 (d, (J= 7.6), C4-H), ꢄ 6.396- 6.435 (t, (JH4H5= 7.6), (JH5H6= 8), C5-H),
ꢄ 6.670- 6.690 (d, (J= 8), C6-H), ꢄ 4.450 (s, terminal –NH), ꢄ 3.599 (s, –OCH3), ꢄ 3.286-
3.335 (m, –CH2), ꢄ 1.143-1.179 (t, (J= 7.2), –CH3), ꢄ 4.095-4.169 (t, (–CH2–)), ꢄ 7.171-
7.261 & ꢄ 7.892-7.969 (m, dppm protons and –CH=N protons).
4.1.4. Preparation of [Ni2(Msal-ptsc)2(µ-dppm)] (4)
The complex 4 was prepared by the procedure as used for (1) with
3-methoxysalicylaldehyde-4(N)-phenylthiosemicarbazone [H2-Msal-ptsc] (0.106 g; 0.420
mmol), NiCl2.6H2O (0.100 g; 0.420 mmol) and 1,1ꢁ-bis(diphenylphosphino)methane (0.080
g, 0.210 mmol).Yield: 63% . M.p: 226 °C. Anal. Calcd for C55H48N6O4S2Ni2P2: C, 60.05; H,
4.40; N, 7.64; S, 5.83. Found: C, 60.01; H, 4.35; N, 7.59; S, 5.78 %. FT-IR (cm−1) in KBr:
1592 (ꢂC=N), 1315 (ꢂC–O), 732 (ꢂC–S); UV-Vis (DMSO), ꢅmax: 268 (51668) nm
(dm3mol−1cm−1) (intra-ligand transition); 327 (34652) &371 (25668) nm (dm3mol−1cm−1)
3
−1
−1
1
ꢃd)
(LMCT s ꢃ d); 429 (13729) nm (dm mol cm ) forbidden (d
transition; H NMR
(CDCl3, ꢄ ppm, J Hz): ꢄ 8.122- 8.144 (d, (J=8.8), CH=N), ꢄ 6.589- 6.605 (d, (J= 6.4), C4-H),
ꢄ 6.449- 6.488 (t, (JH4H5= 7.6), (JH5H6= 8), C5-H), ꢄ 6.743- 6.763 (d, (J= 8), C6-H), ꢄ
7.495-7.518 (d, (J= 9.2), C2’-H & C6’-H), ꢄ 7.941-7.986 (t, (J (H6’H5’) =8.4, J(H2’H3’)
=9.6), C3’-H & C5’-H), ꢄ 6.944-6.995 (m, ( C4’-H), ꢄ 5.29 (s, terminal –NH), ꢄ 3.631 (s, –
OCH3), ꢄ 4.159-4.232 (t, (–CH2–)), ꢄ 7.192-7.473 (m, dppm protons).
4.2. DNA binding study
All of the experiments involving the binding of the compounds with CT DNA were
carried out in deionised water with tris(hydroxymethyl)-aminomethane (Tris, 5 mM) and
sodium chloride (50 mM) and adjusted to pH 7.2 with hydrochloric acid at room temperature.
The concentration of CT-DNA was determined by absorption spectroscopy, using the molar
absorption coefficient (6600 M-1cm-1) at 260 nm. A stock solution of CT-DNA was stored at

277 K and used after no more than 4 days. The complexes were dissolved in minimum
amount of DMSO and diluted with deionized water to the required concentrations prior to
use (1% DMSO (v/v) in the final solution). The absorption titrations of the Ni(II) complexes
were done using a fixed concentrations of the the Ni(II) complexes (10µM) with varying
concentration of CT-DNA (5-50µM). While measuring the absorption spectra, an equal
concentration solution of CT-DNA was added to both the nickel complex solution and the
reference solution to eliminate the absorbance of CT-DNA itself. Control experiments with
DMSO were performed and no changes in the spectra of CT-DNA were observed. The
magnitude of the binding strength of the compounds with CT-DNA can be estimated through
the binding constant Kb, which can be obtained by monitoring the changes in the absorbance
of the corresponding ꢅmax with increasing concentrations of CT-DNA and is given by the (eqn
(3)) [63,64].
[DNA]/[ꢈa-ꢈf]) = [DNA]/[ꢈb-ꢈf]+1/Kb[ꢈb-ꢈf]
(3)
The absorption coefficients ꢈa , ꢈf and ꢈb correspond to Aobsd / [DNA], the extinction
coefficient for the free complex and the extinction coefficient for the complex in the fully
bound form respectively. The slope and the intercept of the linear fit of the plot of
[DNA]/[ꢈa −ꢈf] versus [DNA] give 1/[ꢈa−ꢈf] and 1/Kb[ꢈb−ꢈf], respectively. The intrinsic
binding constant Kb can be obtained from the ratio of the slope to the intercept [63].
Luminescence titration in the presence of ethidium bromide (EB)
The competitive interactions of each of the complexes 1–4 with EB have been
investigated by fluorescence spectroscopy in order to inspect whether the compound can
displace EB from its DNA–EB complex. EB fluorescence displacement experiments were
conducted by adding the solution of the complexes to the Tris-HCl buffer of a DNA–EB
mixture. Before measurements, the mixture was shaken up and recorded. The fluorescence
spectra of DNA bound EB were obtained in the excitation (ꢅex) and emission (ꢅem)
wavelengths of 515 and 602 nm, respectively. Luminescence titration quenching experiments
were conducted by keeping the constant concentration of DNA in buffer-EB (10 µM) with
the various concentrations (10-100 µM) of Ni(II) complex solutions. Control experiments
with DMSO were performed and no changes in the spectra of DNA-EB were observed. The
Stern–Volmer constant KSV is used to evaluate the quenching efficiency of each complex.
4.3. Bovine serum albumin binding study

Binding of the test commplexes (1-4) with bovine serum albumin (BSA) was studied
using fluorescence spectra recorded with an excitation at 280 nm and corresponding emission
at 346 nm assignable to that of free bovine serum albumin (BSA). The excitation and
emission slit widths and scan rates were maintained constant for all the experiments. Stock
solution of BSA was prepared in 50 mM phosphate buffer (pH=7.2) and stored in the dark at
4°C for further use. Concentrated stock solution of complexess were prepared by dissolving
the compounds in DMSO and diluted suitably with deionised water to required
concentrations for all the experiments (1% DMSO (v/v) in the final solution). Titrations were
manually done by using micropipette for the addition of compounds. Control experiments
with DMSO were performed and no changes in the spectra of DNA-EB were observed. For
synchronous fluorescence spectra also, the same concentration of BSA and compounds were
used and the spectra were measured at two different ꢉꢅ (difference between the excitation
and emission wavelengths of BSA) values such as 15 and 60 nm [43].
4.4 Evaluation of antioxidant activity
4.4.1 DPPH (1,1-diphenyl-2-picryl-hydrazyl) radical scavenging assay
The potential antioxidant activity of the compounds were evaluated by DPPH radical-
scavenging assay by using the method of Szabo [65]. DPPH free radicals are used for rapid
analysis of antioxidants.While scavenging the free radicals, the antioxidants donate hydrogen
and form a stable DPPH-H molecule. Briefly, the various concentrations of the complexes
(20-100 µg/ml) in 1 ml of 10% DMSO were added to 5 ml of DPPH (0.1 mM in methanol)
and were mixed rapidly. Radical scavenging capacity was measured at 10 min intervals using
spectrophotometer by monitoring the decrease in absorbance at 517 nm. The IC50 values of
DPPH decolonization of the complexes were calculated. Ascorbic acid was used as a positive
control.
4.4.2 Reductive ability
Total reducing power was determined as described by Oyaizu [66]. The various
concentrations(20-100 µg/ml) of the complexes in 1 ml of 10% DMSO were mixed with 2.5
ml of phosphate buffer (0.2 mol/l, pH 6.6) and 2.5 ml of potassium ferricyanide (1%). The
mixture was incubated at 50 ºC for 20 min. To this mixture 2.5 mL of 10% trichloro-acetic
acid was added and the mixture was centrifuged at 3000 rpm for 20 min. The upper layer (2.5
mL) was mixed with 2.5 mL of deionised water and 0.5 mL of 0.1% ferric chloride and the

absorbance was measured at 700 nm using a spectrophotometer. Increased absorbance of the
reaction mixture indicated increased reducing power.
4.4.3 Superoxide anion scavenging activity
Measurement of superoxide anion scavenging activity of the new nickel(II)
complexes were done based on the method described by Liu [67]. Super oxide radicals were
generated in PMS-NADH systems by oxidation of NADH and assayed by the reduction of
nitroblue tetrazolium (NBT). 3 ml of sample solutions at different concentrations (20-100
µg/ml) were mixed with 1 ml of NBT (156 µM) and 1 ml of NADH (468 µM). The reaction
was initiated by adding 0.1 ml of phenazine metho sulphate (PMS) solution (60 µM) to the
mixture. The reaction was incubated at 25 ºC for 5 min, and the absorbance at 560 nm was
measured against blank. Decreased absorbance of the reaction mixture indicates increased
super oxide anion scavenging activity. Butylated hydroxy toluene (BHT) was used as the
standard.
4.4.4 Estimation of Total antioxidant capacity
Total antioxidant was determined by phosphomolybdenum method [68] followed by
Samples and standard (1ml) was mixed with 2 ml of reagent solution [ammonium molybdate
(4 mM), sodium phosphate (28 mM) and sulphuric acid (0.6 M)]. All the reaction mixtures
were incubated at 95 ºC for 90 min. The absorbance was measured at 695 nm. Total
antioxidant activity was expressed as the number of equivalent of ascorbic acid (µg/ml AA).
For the above four assays, all the tests were run in triplicate and various
concentrations of the complexes were used to fix a concentration at which complexes showed
in and around 50 % of activity. In addition, the percentage of activity was calculated using
the formula, % of activity=[(A0 -AC)/A0] ×100. A0 and AC are the absorbance in the absence
and presence of the tested complex respectively. The IC50 (half maximal inhibitory
concentration) is a measure of the effectiveness of the substance in inhibiting a specific
biological or biochemical function. The 50 % of activity (IC50) can be calculated using the
percentage of activity results. All experimental results were expressed as the mean and ( )
standard deviation (SD) of triplicate determinations.
4.5 Cytotoxic studies
Cell-culture

Human lung adenocarcinoma cells, A549 were obtained from NCCS, Pune, India. Cells were
grown in DMEM and 10% FBS (v/v), containing 100 units/ml penicillin, 30 mg/ml
streptomycin and 20mg/ml gentamicin in a CO2incubator with 5% CO2.
Cell proliferation (MTT) assay
Effect of the complexes on the viability of human lung cancer cells (A549) was
assayed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
[69]. The cells were seeded at a density of 10,000 cells per well, in 200 µl DMEM medium
and were allowed to attach overnight in a CO2 incubator. Then flick off the media and the
complexes (1-4) dissolved in 10% DMSO and diluted in cell culture media were added to the
cells at a final concentration of 1, 10, 25 and 50 µM. Triplicate was maintained and the
medium containing without the compounds were served as control. After 48 h, the wells were
treated with 20µl MTT (5 mg/ml PBS) and incubated at 37 °C for 4 h. The purple formazon
crystals formed were dissolved in 200µl DMSO and read at 570 nm in a micro plate reader.
The mean absorbance for each drug dose was expressed as a percentage of the untreated
control well absorbance and was plotted vs. drug concentration. IC50 values represent the
drug concentrations that reduced the mean absorbance at 570 nm to 50% of those in the
untreated control wells and a graph was plotted with the percentage of cell inhibition versus
concentration. From this, the IC50 value was calculated.
% inhibition = [mean OD of untreated cells(control)/mean OD of treated cells (control)]×100
ACKNOWLEDGEMENT
Department of Science and Technology (SR/FT/CS-32-/2011 dated 04.05.2012)
and Council of Science and Industrial Research (01(2553)/12/EMR-II dated 03.04.2012)
New Delhi, India are greatly acknowledged for their financial support. The author G.K.
greatly acknowledged DST, New Delhi, India for INSPIRE fellowship (IF140225 dated
23.01.2014). The author A.S. greatly acknowledged UGC, New Delhi, India for UGC-BSR
fellowship (F.25-1/2013-14(BSR)/7-26/2007(BSR)dated 30.05.2014).
Supporting information
Crystallographic data for the complexes 1 and 3 have been deposited at the
Cambridge Crystallographic centre as supplementary publication (CCDC No.1443219 and