Synthesis, DNA/protein binding, molecular docking, DNA cleavage and in vitro anticancer activity of nickel(ii) bis(thiosemicarbazone) complexes

Article abstract of DOI:10.1039/c5ra04498g

A series of N-substituted isatin thiosemicarbazone ligands (L1-L5) and their nickel(ii) complexes [Ni(L)₂] (1-5) were synthesized and characterized by elemental analyses and UV-Visible, FT-IR, ¹H & ¹³C NMR, and mass spectr

Full text of DOI:10.1039/c5ra04498g

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Synthesis, DNA/protein binding, molecular docking, DNA cleavage and in vitro anticancer
activity of nickel(II) bis(thiosemicarbazone) complexes
a
a
b
c
Jebiti Haribabu, Kumaramangalam Jeyalakshmi, Yuvaraj Arun, Nattamai S. P. Bhuvanesh,
b
a
Paramasivan Thirumalai Perumal, and Ramasamy Karvembu *
Abstract
A series of Nꢀsubstituted isatin thiosemicarbazone ligands (L1ꢀL5) and their nickel(II)
complexes [Ni(L) ] (1ꢀ5) were synthesized and characterized by elemental analyses and UVꢀ
2
1
13
Visible, FTꢀIR, H & C NMR, and mass spectroscopic techniques. The molecular structure of
ligands (L1 and L2) and complex 1 was confirmed by single crystal Xꢀray crystallography. The
single crystal Xꢀray structure of 1 showed distorted octahedral geometry. The interaction of calf
thymus (CT) DNA and bovine serum albumin (BSA) with nickel(II) complexes was explored
using absorption and emission spectral methods. DNA cleavage study showed that the
complexes cleaved DNA without any external agents. The alterations in the secondary structure
of protein by the nickel(II) complexes (1ꢀ5) were confirmed by synchronous and three
dimensional fluorescence spectroscopic studies. The interaction of the complexes with
DNA/protein has also been supported by molecular docking studies. An in vitro cytotoxicity
study of the complexes found significant activity against human breast (MCF7) and lung (A549)
cancer cell lines, with the best results for complexes 4 and 2 respectively, where IC50 value is
less than 0.1 µM concentration.
_________________________________________________________________________
a
Department of Chemistry, National Institute of Technology, Tiruchirappalli ꢀ 620015, India
Eꢀmail address: kar@nitt.edu (R. Karvembu); Fax: +91 431 2500133; Tel: +91 431 2503631
b
Organic Chemistry Division, CSIRꢀCentral Leather Research Institute, Chennai ꢀ 600020, India
c
Department of Chemistry, Texas A & M University, College Station, TX 77842, USA
Electronic supplementary information (ESI) available: Crystallographic data for the structures
reported in this paper have been deposited with the Cambridge Crystallographic Data Centre
(CCDC) as supplementary publication numbers (CCDC 1052678, CCDC 1052679 and CCDC
1052682, for L1, L2, and 1 respectively). Copies of the data can be obtained free of charge from

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the CCDC (12 Union Road, Cambridge CB2 1EZ, UK; Tel.: + 44ꢀ1223ꢀ336408; Fax: + 44ꢀ1223ꢀ
36003; eꢀmail: deposit@ccdc.cam.ac.uk ; Web site http://www.ccdc.cam.ac.uk).
3
1
. Introduction
Transition metal complexes have been extensively studied as anticancer drugs because of
1
ꢀ9
their diverse spectral, chemical, and electrochemical properties. It is well known that DNA is
an important cellular receptor. The interaction of metal complexes with DNA has recently gained
much attention because it indicates that the complexes may have potential biological activity and
10
their activity depends on the mode and affinity of the binding with DNA. Platinum based drugs
1
1
have been in clinical use for cancer treatment for more than 30 years. The landmark discovery
of the antitumoral properties of cisplatin by Rosenberg in 1965 heralded a new area of anticancer
1
2
research based on metallopharmaceuticals. Although the mechanism by which cisplatin
selectively kills cells is not entirely understood, it is generally believed that the therapeutic
1
3
effects arise from covalent binding of the drug to DNA. The efficacy of cisplatin, however, is
reduced by increasing tumor resistance and high toxicity. These limitations have aroused interest
toward the design and evaluation of transition metal complexes other than platinum based
1
4
derivatives for therapeutic use. Similarly, protein was also established as one of the main
15
molecular targets in the action of anticancer agents. The interaction between protein and drugs
provides useful information on the structural features that determine the therapeutic effectiveness
1
6,17
of drugs and also to study the pharmacological response of drugs.
Therefore interaction of
the proteins with the metal complexes become important in the search of new drug molecules.
Thiosemicarbazones are a class of Schiff base compounds that contain a thiourea moiety.
Thiosemicarbazones (TSCs) are versatile ligands and efficient metal chelators and due to which
its coordination chemistry has been developed especially for the first row transition metals. A
number of reasons have been offered as responsible for their versatility in coordination, such as
intramolecular hydrogen bonding, bulkier coligand, steric crowding on the azomethine carbon
atom, and π−π stacking interactions. Thiosemicarbazones usually bind to a metal ion, either in
the neutral thione form (a) or in the anionic thiolate form (b), as bidentate N, S donor ligands
1
8,19
forming fiveꢀmembered chelate rings.
However, incorporation of a third donor site (D) into

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these thiosemicarbazone ligands, linked to the carbonylic carbon via one or two intervening
1
8, 20ꢀ21
atoms, normally results in D, N, S tricoordination (c) (Fig. 1).
Fig. 1 Different coordination modes of thiosemicarbazone.
Thiosemicarbazones have attracted considerable attention by chemists and biologists
because of their wide range of pharmacological effects such as antibacterial, antiviral, antifungal
2
2
and particularly, antitumor and antileukemic activities. Thiosemicarbone (Triapine) has already
evaluated in several clinical phase I and II trials as anticancer drug. Unfortunately it has ended
with severe side effects. Therefore the search for new TSCs metal complexes with
pharmaceutical importance is being dealt with. In recent years, several nickel based complexes
have been investigated for potential antitumor activity. Nickel is present in the active sites of
several important classes of metalloproteinase, such as 2ꢀmercaptoethanolꢀinhibited urease and
2
3,24
Ni/Fe hydrogenases.
Complementing its natural biochemical functions, complexes of nickel
also display pharmacological potential. Nickel(II) complexes with pyrazolate ligands display
2
5
cytotoxicity towards HLꢀ60, NALMꢀ6 (leukemia cells) and WMꢀ 115 (melanoma cells). Nickel
complexes containing 2ꢀphenylquinolineꢀ4ꢀcarboxylic acid hydrazide ligand showed significant
cytotoxicity against MFC cell lines and also bind DNA via a groove binding mode; further, these
2
6
complexes can cleave pBR322 DNA. In the present work, we report the synthesis and
characterization of nickel(II) complexes containing istain based thiosemicarbazones. The
interaction of the nickel(II) complexes with CTꢀDNA and BSA was studied using
spectrophotometric methods. We have also tested the cytotoxicity of the nickel(II) complexes
against MCF7 and A549 cancer cell lines. Interestingly, it was found that Nꢀalkylation of the
ligand affects the DNA / protein binding ability and cytotoxicity of the complexes.

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2
. Results and discussion
2.1. Synthesis and characterization
Isatin thiosemicarbazone ligands (L1ꢀL5), were prepared by condensation of substituted
isatin with 4ꢀmethyl thiosemicarbazide in the presence of glacial acetic acid (Scheme 1). The
corresponding nickel(II) complexes were synthesized using [NiCl (PPh ) ] as the metal precursor
2
3 2
(Scheme 2). All the ligands and their nickel(II) complexes were characterized by elemental
analyses and various spectroscopic techniques. The molecular structure of L1, L2 and 1 was
confirmed by single crystal Xꢀray diffraction study.
Scheme 1 Synthetic scheme of isatin thiosemicarbazone ligands.
Scheme 2 Synthetic scheme of nickel(II) complexes.

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2.2. Spectroscopy
The electronic spectra of the ligands showed two bands around 264ꢀ265 and 364ꢀ365 nm,
which correspond to π−π* and nꢀπ∗ transitions respectively. The spectra of the complexes
exhibited three bands. Two bands appeared around 265ꢀ293 and 364ꢀ388 nm correspond to
2
7
intraligand transitions. The band in the region 440ꢀ453 nm is attributed to dꢀd transition. In the
FTꢀIR spectra of the ligands, two bands were observed for N−H groups in the range of 3469ꢀ
ꢀ1
ꢀ1
3
231 cm . The C=N stretching frequency of the ligands was observed at 1561ꢀ1552 cm . The
ꢀ1
ꢀ1
C=O and C=S stretching frequencies appeared around 1689ꢀ1682 cm and 1285ꢀ1271 cm
respectively. On complexation there is a decrease in carbonyl, thiocarbonyl and azomethine
stretching frequencies, which suggested that thiosemicarbazones coordinate to nickel(II) through
2
8
sulfur, carbonyl oxygen and the azomethinic nitrogen atoms. The observed magnetic moment
8
29
lies within 1.95–2.17 BM, which is attributable to the octahedral nickel(II) center (d , S = 1).
1
On examination of the H NMR spectra of the ligands (L1ꢀL5), H−N−C=S protons resonate in
the regions 12.39ꢀ12.78 and 7.57ꢀ9.26 ppm. The signal of amide N−H (L1 and L5) proton
13
appeared at 11.29ꢀ11.10 ppm. C NMR spectra of the ligands showed resonances due to thiolato
carbon (C=S), carbonyl carbon (C=O) and imine carbon (C=N) in the regions 178.0ꢀ179.1,
161.1ꢀ163 and 141.6ꢀ143.9 ppm respectively. Chemical shift of all other aromatic and aliphatic
2
7
protons / carbons was observed in the expected regions.
2.3. Single crystal X-ray crystallography
Thermal ellipsoid plots of ligands (L1 and L2) and complex 1 with the atomic labelling
schemes are shown in the Figs. 2ꢀ4. Crystal data and selected inter atomic bond lengths and
angles are given in the Tables 1ꢀ4. Yellow crystals of ligands suitable for Xꢀray diffraction study
were obtained by slow evaporation of its ethanol solution. The fiveꢀ and sixꢀmembered rings of
the isatin in L1 and L2 are virtually planar. An intraꢀmolecular hydrogen bonding between the
N3 proton and O1 is present in ligands L1 [N(3)−H(3)…O(1) = 2.721 Å] and L2
[
N3−H(3)…O(1) = 2.738 Å], creating a sixꢀmembered ring and favoring the Zꢀisomer with
respect to the imine C=N bond. This hydrogen bond renders the isatin and TSC
3
0
(
N2−N3−C9−S1−N4) moieties essentially coplanar. The crystal structure of L1 showed the
existence of an intraꢀmolecular hydrogen bond between sulfur and water molecule
O(1W)−H(1WA)…S(1) = 3.337 Å].
[

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Complex 1 crystallized in monoclinic C 1 2/c 1 space group with two molecules of the
ligand per nickel. The nickel(II) complex adopts a distorted octahedral geometry with binding of
the ligands as monobasic tridentate (ONS) donors. Two molecules of dimethyl formamide were
found in the asymmetric unit. The S−Ni1 bond is longer than the O1−Ni1, and N2−Ni1 bond
lengths (2.367>2.212>2.016 Å), which are in the expected range for thiosemicarbazone
27
complexes. The S1−C9 bond length (1.709 Å) in 1 is higher than that of L1 (1.690 Å). There is
an increase in the C−O and C−N bond lengths (involved in coordination) in 1 compared to L1.
The torsional angles in 1 are 3.66 (Ni−S1−C9−N3), ꢀ176.70 (Ni−S1−C9−N4), 0.68
(
Ni−N2−N3−C9), 176.68 (Ni−N2−C2−C1), 177.22 (Ni−O1−C1−N1) and 3.49°
Ni−O1−C1−C2).
(
2.4. DNA binding studies
2.4.1. Electronic absorption titration
The DNA binding experiments were carried out in TrisꢀHCl buffer (pH 7.2). The
nickel(II) complexes (1, 3ꢀ5) showed absorption bands at 273ꢀ285 and 356ꢀ369 nm, which were
assigned to intraligand transitions. But in complex 2 there are three absorption bands at 284, 365
and 458 nm. Upon the incremental addition of CT DNA to the complexes, the intensity of
absorption decreases resulting in hypochromism (ꢁε, 18ꢀ37%) with a small red shift.
Intercalative mode of binding due to the strong stacking interaction between an aromatic
chromophore and the base pairs of DNA usually results in hypochromism along with or without
3
1
a small red or blue shift. The extent of shift and hypochromism are normally found to correlate
with the intercalative binding strength. The magnitude of hypochromism is in the order of
4>2>3>1>5, which reflects the DNA binding affinities of the complexes. The absorption spectra
of the complexes (1ꢀ5) in the presence and absence of CT DNA are shown in Figs. 5 and S1. The
binding constant of the complexes with CT DNA (K ) was obtained from the ratio of slope to
b
3
2
intercept by ploting [DNA]/(ε ꢀε ) versus [DNA] according to the equation [DNA]/(ε ꢀε ) =
a
f
a
f
[
DNA]/(ε ꢀε ) + 1/K (ε ꢀε ) where [DNA] is the concentration of DNA in base pairs, ε is the
b f b b f a
apparent extinction coefficient value found by calculating A(observed)/[complex], ε is the
f
extinction coefficient for the free compound, and ε is the extinction coefficient for the
b
compound in the fully bound form. Each set of data, when fitted into the above equation, gave a
straight line with a slope of 1/(ε − ε ) and an yꢀintercept of 1/K (ε − ε ) (Fig. 7). The magnitudes
b
f
b
b
f

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of intrinsic binding constants (K ) are given in Table 5. The observed values of K (1.40ꢀ2.90 ×
b
b
4
ꢀ1
33
1
0 M ) revealed that the nickel(II) complexes bind to DNA via intercalative mode.
Complexes 4 and 2 showed better DNA binding affinity compared to the other complexes. The
enhanced binding of 4 and 2 may be due the presence of benzyl and allyl groups respectively.
2.4.2. Fluorescence spectroscopic studies
Fluorescence property has not been observed for the complexes at room temperature in
solution or in the presence of CT DNA. So the binding of the complexes with DNA could not be
directly predicted through the emission spectroscopy. Hence, competitive binding study was
34ꢀ36
done to understand the mode of DNA interaction with the complexes.
EB) emits intense fluorescence in the presence of CT DNA because of strong intercalation of
the planar EB phenanthridine ring between adjacent base pairs in the double helix; therefore, EB
Ethidium bromide
(
3
7
has been considered as a typical indicator of intercalation. If another molecule which can bind
to DNA more strongly than EB was added, the molecule will replace the bound EB and there
was a quenching in the DNA induced EB emission. The extent of quenching of CT DNAꢀEB
reflects the extent of interaction with the added molecule. On adding nickel(II) complexes (0ꢀ50
µM) to CT DNAꢀEB, the quenching in the emission of DNA bound EB takes place (Figs. 6 and
3
8
o
S2). Fluorescence quenching is explained by the SternꢀVolmer equation F /F = 1 + K [Q]
q
ο
where F and F are the fluorescence intensities in the absence and presence of complex
respectively, K is a linear SternꢀVolmer quenching constant, and [Q] is the concentration of
q
o
complex. The slope of the plot of F /F versus [Q] gave K (Fig. 8). The apparent DNA binding
q
constant (K ) values were calculated by using the equation KEB [EB] = Kapp [complex] where
app
[
complex] is the complex concentration at 50% reduction in the fluorescence intensity of EB,
7
ꢀ1
K = 1.0 × 10 M and [EB] = 5 µM. The quenching constant (K and K ) values are listed in
EB
q
app
Table 5.
2.4.3. CD spectral studies
Circular dichroic spectral technique gave us useful information on how the conformation
of DNA was influenced by the binding of the metal complex to DNA. The observed CD
spectrum of CT DNA consists of a positive band at 277 nm due to base stacking and a negative
band at 245 nm due to helicity, which is characteristic of DNA in the rightꢀhanded B form.
While groove binding and electrostatic interaction of small molecules with DNA show little or

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no perturbation on the base stacking and helicity bands, intercalation changes the intensity of
both the bands. In the present case, the intensity of both the negative and positive bands changes
significantly (shifting to zero levels) (Figs. 9 and S3). This suggests that the binding of DNA
with the complexes (1ꢀ5) induces certain conformational changes, such as the conversion from a
3
9
more Bꢀlike to a more Cꢀlike structure within the DNA molecule. These changes are indicative
4
0
of a nonꢀgroove mode of binding of the complexes and offer support to its intercalation mode.
2
.5. DNA cleavage
To explore the DNA cleavage ability of complexes (1ꢀ5), supercoiled (SC) pUC19 DNA
40 ꢂM in base pairs) was incubated at 37 °C with the complexes (150 µM) in a 5% DMF/5 mM
(
TrisꢀHCl/50 mM NaCl buffer at pH 7.2 for 3 h. Complexes (1ꢀ5) cleave SC (Form I) DNA into
nicked circular (NC) (Form II) DNA without any external agents (Fig. 10), and the DNA
cleavage efficiency follows the order 2 (99.8%) > 1 (99.2%) > 5 (96.5%) > 4 (14.1%) > 3
(6.6%). The study reveals that 2 cleaves DNA more efficiently than the other complexes,
because of the strong partial intercalation of the allyl group.
2.6. BSA interaction studies
2.6.1. Absorbance and fluorescence studies
Figs. 11 and S4 show the fluorescence emission spectra of BSA after the addition of the
complexes (1ꢀ5). When increasing amount of complex solution was added to a fixed quantity of
BSA, there observed a decrease in the fluorescence intensity of BSA at 345 nm, upto 78.1, 89.0,
87.7, 88.3 and 79.4% with bathochromic shift of 15, 13, 12, 15 and 14 nm for complexes 1ꢀ5
respectively. The observed hypochromicity has revealed that the complexes interact
4
1
hydrophobically with the BSA protein.
UVꢀVisible absorption titration of BSA with the complexes (1ꢀ5) was done to predict the
type of quenching process. Addition of the complex to BSA lead to an increase in BSA
absorption intensity without affecting the position of absorption band. This indicates that the type
4
2
of interaction between nickel(II) complexes and BSA was mainly a static quenching process.
The representative absorption titration spectrum is shown in Fig. 12. The fluorescence quenching
o
o
is described by the SternꢀVolmer relation F /F = 1 + K [Q] where F and F demonstrate the
q
fluorescence intensities in the absence and presence of quencher, respectively. K is a linear
q
SternꢀVolmer quenching constant, and [Q] is the quencher concentration. The quenching

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o
constant (K ) can be calculated using the plot of log (F /F) versus log [Q] (Fig. 13). When small
q
molecules bind independently to a set of equivalent site, on a macromolecule, the equilibrium
43,44
o
between free and bound molecules is represented by the Scatchard equation
log[(F ꢀF)/F] =
log K + n log[Q] where K is the binding constant of the complex with BSA and n is the number
b
b
o
of binding sites. From the plot of log[(F ꢀF)/F] versus log[Q] (Fig.14), the number of binding
sites (n) and the binding constant (K ) values have been obtained. The quenching constant (K ),
b
q
binding constant (K ) and number of binding sites (n) for the interaction of the nickel(II)
b
complexes with BSA are shown in Table 6. In all the complexes, only one binding site is
available to interact with BSA. Results showed that complexes 2, 4 and 1 interact strongly with
BSA compared to 3 and 5.
2.6.2. Characteristics of synchronous fluorescence spectra
To obtain reasonable information on the molecular microenvironment, particularly in the
vicinity of the fluorophore functional groups, the structural changes occurring to BSA upon the
4
5
addition of the new complexes were studied through synchronous fluorescence spectra. It is
well known that the fluorescence of BSA is normally due to the presence of tyrosine, tryptophan
and phenylalanine residues and hence spectroscopic methods are usually applied to study the
conformation of serum protein. The difference between the excitation and emission wavelength
4
6
(
ꢁλ) reflects the nature of the chromophore. The large ꢁλ value, such as 60 nm, is
characteristic of tryptophan residue and a small ꢁλ value, such as 15 nm, is characteristic of
tyrosine. The synchronous fluorescence spectra of BSA with various concentrations of nickel(II)
complexes (1-5) were recorded at ꢁλ = 15 nm and ꢁλ = 60 nm. On addition of the complexes,
the fluorescence intensity of tryptophan residue at 340 nm decreased in the magnitude of 73.5,
8
6.2, 81.7, 84.0 and 73.9% for complexes 1-5 respectively (Figs. 15 and S5). Similarly, there
was also decrease in the intensity of tyrosine residue at 300 nm. The magnitude of decrease was
5.4, 89.4, 83.4, 88.1 and 77.7% for complexes 1ꢀ5 respectively (Figs. 16 and S6). The
7
synchronous fluorescence spectral studies clearly suggested that the fluorescence intensities of
both the tryptophan and tyrosine were affected with increasing concentration of the complexes.
The results indicate that the interaction of complexes with BSA affects the conformation of both
4
7
tryptophan and tyrosine microꢀregions.

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2.6.3. 3D fluorescence spectra
Application of threeꢀdimensional fluorescence technique is seen in recent years for the
4
8
investigation of conformational changes of proteins interacting with drugs. The excitation
wavelength, emission wavelength and fluorescence intensity can be used as axes in the
fluorescence emission spectra. The maximum emission wavelength and fluorescence intensity of
the residues showed a close relation to the polarity of their microenvironment. The three
dimensional fluorescence spectra and the corresponding contour diagrams for BSA, BSA–2, and
BSA–4 are shown in Fig. 23 and for BSA–1, BSA–3 and BSA–5 are shown in Fig. S9. Two
typical fluorescence peaks 1 and 2 were observed. Among these two, peak 2 mainly revealed the
spectral characteristics of tryptophan and tyrosine residues. The reason is that, when protein is
excited at 280 nm, it mainly reveals the intrinsic fluorescence of tryptophan and tyrosine
residues. The excitation wavelength of peak 1 was noticed at 240 nm which was mainly
attributed to the n−π∗ transition of protein’s characteristic polypeptide backbone structure,
4
9
C=O. The results showed that the threeꢀdimensional fluorescence contour map of protein and
protein-(1-5) was clearly different. From Figs. 17 and S7, it is clear that the fluorescence
intensities of peak 2 decreased significantly in the presence of complexes (1ꢀ5) indicating the
quenching of fluorescence of BSA by the complexes. The results shown in Table 7 indicated the
50
conformational changes of BSA upon interaction with the complexes.
2.7. Molecular docking
2.7.1. Molecular docking studies with DNA
Computational docking is extremely useful tool to gain an understanding of synthesized
compounds and biological drug target interactions which is very important in drug discovery. In
our study, the synthesized nickel(II) complexes were subjected to molecular docking studies
using the AutoDock Tools (ADT) version 1.5.6 and AutoDock version 4.2.5.1 docking
programs, which are interactive molecular graphics programs, to understand the drug–DNA
interactions to investigate the potential binding mode and energy.
Docked ligand conformation was analyzed in terms of energy, hydrogen bonding, and
hydrophobic interaction between synthesized nickel complexes and BꢀDNA (PDB ID: 1BNA).
Detailed analyses of the complexꢀDNA interactions were carried out, and final coordinates of the
complex and receptor were saved. From the docking scores, the free energy of binding (FEB) of

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the compound was calculated and details were shown in (Table 8).
In silico molecular docking experiment reveals that the docked complexes fit into the
DNA comfortably involving van der Waals interaction, hydrophobic and hydrogen bonding
contacts with DNA functional groups, resulted in the binding energy between ꢀ7.26 to ꢀ9.05
kcal/mol. Binding interactions of all the nickel(II) complexes with the BꢀDNA receptor are
shown in the Figs. 18 and S8.
Nickel(II) complexes (1ꢀ5) show the binding energy value ꢀ8.71, ꢀ7.26, ꢀ7.34, ꢀ7.77 and ꢀ
9.05 kcal/mol with four, two, two, one and six hydrogen bonds respectively, with the docked
DNA receptor. Among all the complexes docked, 5 shows the highest binding energy value ꢀ9.05
kcal/mol with six hydrogen bonds with the docked DNA receptor. In complex 5, one of the
methyl attached N−H interacts with the two oxygens of phosphate of DCꢀ21 and forms two
hydrogen bonds with the bond length of 2.2 and 2.4 Å. Also, another methyl attached N−H
interacts with the phosphate of DAꢀ6 and forms a hydrogen bond with the bond length of 1.9 Å.
Furthermore, one of the oxindole N−H interacts with the oxygen of sugar moiety of DTꢀ7 and
nitrogen of base moiety of DAꢀ6 and forms two hydrogen bonds with the bond length of 2.4 and
2.4 Å respectively. Another oxindole N−H interacts with the C=O of base moiety of DTꢀ21 and
forms a hydrogen bond with the bond length of 2.0 Å.
2.7.2. Molecular docking studies with human DNA topoisomerase I
All the nickel(II) complexes were also subjected to molecular docking studies with
human DNA topoisomerase I using the AutoDock Tools (ADT) version 1.5.6 and AutoDock
version 4.2.5.1. The Xꢀray crystallographic structure of the humanꢀDNAꢀTopoisomerase I
complex (PDB ID: 1SC7) was retrieved from Protein Data Bank in which TopoꢀI is bound to the
oligonucleotide sequence 50ꢀAAAAAGACTTsXꢀGAAAATTTTTꢀ30 where ‘s’ is 50ꢀbridging
phosphorothiolate of the cleaved strand and ‘X’ represents any of the four bases A, G, C or T.
The SH of G11 on the scissile strand was changed to OH and phosphoester bond of G12 in 1SC7
5
1
was rebuilt. The calculated free energy of binding (FEB) of the nickel(II) complexes with
DNAꢀTopoisomerase I complex is shown in Table 9.
The molecular docking result revealed that all the complexes approach towards the DNA
cleavage site in the DNAꢀTopoisomerase I and forming a stable complex through nonꢀcovalent
interactions like hydrogen bonding, hydrophobic and van der Waals interactions, results in the

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binding energy between ꢀ6.93 and ꢀ9.86 kcal/mol, subsequently leading to inhibitory effect on
DNAꢀTopoisomerase I. Binding interactions of the complexes with the DNAꢀTopoisomerase I
receptor are shown in Figs. 19 and S9.
2.7.3. Molecular docking studies with human protein
Molecular docking technique is an attractive scaffold to understand the ligandꢀprotein
interactions which can substantiate our experimental results. The GOLD scores of the individual
complexess corresponding to different poses have been tabulated and the highest score for each
of the complexes has shown in Table 10. Complex 4 has the highest score followed by 2
predicting the formation of stable complex between protein (3V03) and complexes. Complex 1
could also be considered favourable based on their scores and number of poses generated.
However 1 and 5 could generate only three possible poses unlike the others generating 10 poses
each. Binding interaction of the complexes with the RCSB protein is shown in Figs. 20 and
S10.
After docking, the docked complexes corresponding to the highest scores for each
complex were imported as.pdb files into PYMOL software for evaluating the hydrogen bonds
…
formed. It is found that many N−H O bonds are formed between the complexes and the protein
(Table 11). The main residues involved in the interactions are Arg 215, Val 290 and Asp 448. It
is found that complex 1 formed the maximum number of hydrogen bonds. The complexes 2, 3
and 5 also involved in hydrogen bond interaction with Asp 448 and Arg 215. But complex 4 with
highest GOLD score shows no hydrogen bond formation.
2.8. Cytotoxicity
All the nickel(II) complexes have been tested for their in vitro cytotoxicity against human
breast (MCF7) and human lung (A549) cancer cell lines using sulphorhodamine B (SRB) assay.
Results were expressed as IC50 values and were compared with adriamycin (positive control)
[
IC50 < 0.1 µM for MCF7 and A549 under identical conditions] (Table 12). Figs. 21, 22, 23 and
4 show the cytotoxicity of the complexes (1-5) after 24 h incubation on MCF7 and A549
2
cancer cell lines, respectively. Complexes 4 and 2 showed excellent cytotoxicity (< 0.1 µM)
against MCF7 and A549 cancer cell lines respectively. All the other complexes showed
significant activity. The calculated IC50 values were in the range of 14.9 to 48.9 µM for the
other complexes (1ꢀ3, 5) against MCF7 cell lines. The complexes (1, 3ꢀ5) showed the IC50 value

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in the range of 5.7 to 29.3 µM against A549 cell lines. Higher cytotoxicity of 4 and 2 is in line
with their DNA and protein binding abilities.
3
. Conclusion
Five nickel(II) complexes with Nꢀalkylated isatin based thiosemicarbazone ligands have
been synthesized and characterized. The coordination geometry around nickel(II) is described as
distorted octahedral. The DNA binding affinity of the complexes was revealed using
spectrophotometric methods. The results supported the interaction of the complexes with CT
DNA through intercalation. Complexes 2 and 1 have better DNA cleaving ability compared to
the other complexes. According to the results of the fluorescence quenching experiments of
BSA, we conclude that the complexes have binding affinity in static mode. In addition, the
synchronous and 3D fluorescence spectral investigations revealed that the complexes induced a
small change in the secondary structure of the protein at the studied conditions. The molecular
docking studies also confirmed the interaction of the complexes with DNA and protein. In vitro
cytotoxicity results showed that the complexes 4 and 2 exhibit remarkable cytotoxicity against
MCF7 and A549 cancer cell lines respectively. All the other complexes showed significant
activity which is comparable with the adriamycin drug
4
. Experimental
4.1. Materials and methods
All the chemicals were purchased from Sigma Aldrich / Merck and used as received.
Solvents were purified according to the standard procedures. The melting points were
determined on Lab India instrument and are uncorrected. The elemental analyses were performed
using a Vario EL−III CHNS analyzer. Magnetic susceptibilities were measured using Sherwood
scientific auto magnetic susceptibility balance. FTꢀIR spectra were obtained as KBr pellets using
a NicoletꢀiS5 spectrophotometer. UVꢀVisible spectra were recorded using a Shimadzuꢀ2600
spectrophotometer. Emission spectra were measured on a Jasco Vꢀ630 spectrophotometer using
5
% DMF in buffer as the solvent. NMR spectra were recorded in DMSOꢀd or CDCl by using
6 3
TMS as an internal standard on a Bruker 400 MHz spectrometer.

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4.2. Synthesis of isatin thiosemicarbazones (L1- L5)
The isatin based thiosemicarbazone ligands were synthesized using the standard
5
2
procedure. 4ꢀMethyl thiosemicarbazide (1 mmol) and substituted isatin (1 mmol) were
suspended in ethanol (20 mL) containing a few drops of glacial acetic acid. The mixture was
refluxed for 3 h and cooled to room temperature. The yellow product formed was filtered off,
washed with cold ethanol and ether, and dried in vacuo. It was recrystallized from ethanol to get
crystals of the title compounds.
4.2.1. (Z)-N-methyl-2-(2-oxoindolin-3-ylidene)hydrazinecarbothioamide (L1)
4
ꢀMethyl thiosemicarbazide (0.105 g, 0.001 mole) and isatin (0.147 g, 0.001 mole) were
o
used. Yield: 91%. Yellow. m.p.: 151 C. Anal. Calc. for C H N OS (%): C, 51.27; H, 4.30; N,
1
0
10
4
2
3.91; S, 13.69. Found: C, 51.35; H, 4.25; N, 23.67; S, 13.61. UV−Vis (CH OH): λmax, nm (ε,
3
3
ꢀ1
ꢀ1
ꢀ1
dm mol cm ) 265 (21300), 364 (16600). FTꢀIR (KBr): ʋ, cm 3469, 3420, 3236 (N−H), 1689
1
(
C=O), 1557 (C=N), 1274 (C=S). H NMR (400 MHz, DMSOꢀd ): δ, ppm 12.59 (s, 1H), 11.10
6
(s, 1H), 9.09ꢀ9.08 (d, J = 4.0 Hz, 1H), 7.62ꢀ7.60 (d, J = 8.0 Hz, 1H), 7.29ꢀ7.26 (t, J = 8.0 Hz,
1H), 7.04ꢀ7.00 (t, J = 8.0 Hz, 1H), 6.90ꢀ6.88 (d, J = 8.0 Hz, 1H), 3.09ꢀ3.08 (d, J = 4.0 Hz, 3H).
1
3
C NMR (100 MHz, DMSOꢀd ): δ, ppm 178.2 (C=S), 163.0 (C=O), 142.5 (C=N), 131.8, 131.3,
6
+
1
22.6, 121.1, 111.4 (aromatic C), 31.6 (aliphatic C). ESIꢀMS m/z = 234.38 [M] .
4.2.2. (Z)-2-(1-allyl-2-oxoindolin-3-ylidene)-N-methylhydrazinecarbothioamide (L2)
4
ꢀMethyl thiosemicarbazide (0.105 g, 0.001 mole) and allyl isatin (0.187 g, 0.001 mole)
o
were used. Yield: 89%. Yellow. m.p.: 163 C. Anal. Calc. for C H N OS (%): C, 56.91; H,
1
3
14
4
5
.14; N, 20.42; S, 11.69. Found: C, 56.84; H, 5.01; N, 20.33; S, 11.61. UV−Vis (CH OH): λmax,
3
3
ꢀ1
ꢀ1
ꢀ1
nm (ε, dm mol cm ) 265 (35800), 365 (62000). FTꢀIR (KBr): ʋ, cm 3465, 3232 (N–H), 1682
1
(
C=O), 1555 (C=N), 1285 (C=S). H NMR (400 MHz, CDCl ): δ, ppm 12.78 (s, 1H), 7.82 (s,
3
1H), 7.59ꢀ7.57 (d, J = 8.0 Hz, 1H), 7.37ꢀ7.33 (m, 1H), 7.12ꢀ7.08 (t, J = 8.0 Hz, 1H), 6.89ꢀ6.87
(d, J = 8.0 Hz, 1H), 5.89ꢀ5.80 (m, 1H), 5.28ꢀ5.27 (d, J = 4.0 Hz, 1H), 5.248ꢀ5240 (t, J = 1.6 Hz,
13
1
H), 4.39ꢀ4.37 (d, J = 8.0 Hz, 1H), 3.30ꢀ3.29 (d, J = 4.0 Hz, 3H). C NMR (100 MHz, CDCl ):
3
δ, ppm 179.1 (C=S), 161.1 (C=O), 143.2 (C=N), 132.1, 131.1, 123.2, 120.6, 118.8, 117.4, 110.0
+
(
aromatic C), 42.1, 31.4 (aliphatic C). ESIꢀMS m/z = 297.11 [M+Na] .

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4.2.3. (Z)-N-methyl-2-(1-methyl-2-oxoindolin-3-ylidene)hydrazinecarbothioamide (L3)
4
ꢀMethyl thiosemicarbazide (0.105 g, 0.001 mole) and methyl isatin (0.161 g, 0.001
o
mole) were used. Yield: 90%. Yellow. m.p.: 157 C. Anal. Calc. C H N OS (%): C, 53.21; H,
1
1
12
4
4
.87; N, 22.56; S, 12.91. Found: C, 53.11; H, 4.76; N, 22.65; S, 12.82. UV−Vis (CH OH): λmax,
3
3
ꢀ1
ꢀ1
ꢀ1
nm (ε, dm mol cm ) 265 (6200), 364 (35800). FTꢀIR (KBr): ʋ, cm 3458, 3227 (N–H), 1680
1
(
C=O), 1552 (C=N), 1285 (C=S). H NMR (400 MHz, DMSOꢀd ): δ, ppm 12.51 (s, 1H), 9.27ꢀ
6
9.26 (d, J = 4.0 Hz, 1H), 7.66ꢀ7.64 (d, J = 8.0 Hz, 1H), 7.45ꢀ7.41 (t, J = 8.0 Hz, 1H), 7.17ꢀ7.10
1
3
(
m, 2H), 3.20 (s, 3H), 3.09ꢀ3.08 (d, J = 4.0 Hz, 3H). C NMR (100 MHz, DMSOꢀd ): δ, ppm
6
1
3
4
78.0 (C=S), 161.2 (C=O), 143.9 (C=N), 131.5, 131.2, 123.3, 120.8, 119.6, 110.2 (aromatic C),
+
1.8, 26.1 (aliphatic C). ESIꢀMS m/z = 249.25 [M+H] .
.2.4. (Z)-2-(1-benzyl-2-oxoindolin-3-ylidene)-N-methylhydrazinecarbothioamide (L4)
4
ꢀMethyl thiosemicarbazide (0.105 g, 0.001 mole) and benzyl isatin (0.237 g, 0.001
o
mole) were used. Yield: 87%. Yellow. m.p.: 177 C. Anal. Calc. C H N OS (%): C, 62.94; H,
17
16
4
4
.97; N, 17.27; S, 9.88. Found: C, 62.84; H, 4.90; N, 17.40; S, 9.81. UV−Vis (CH OH): λmax,
3
3
ꢀ1
ꢀ1
ꢀ1
nm (ε, dm mol cm ) 264 (17300), 364 (24800). FTꢀIR (KBr): ʋ, cm 3463, 3231 (N–H), 1689
1
(
C=O), 1561 (C=N), 1271 (C=S). H NMR (400 MHz, DMSOꢀd ): δ, ppm 12.54 (s, 1H), 9.34ꢀ
6
9.33 (d, J = 4.0 Hz, 1H), 7.71ꢀ7.69 (d, J = 8.0 Hz , 1H), 7.37ꢀ7.26 (m, 6H), 7.17ꢀ7.13 (t, J = 8.0
1
3
Hz, 1H), 7.04ꢀ7.02 (d, J = 8.0 Hz, 1H), 4.90 (s. 2H), 3.11ꢀ3.10 (d, J = 4.0 Hz, 3H). C NMR
100 MHz, DMSOꢀd ): δ, ppm 178.1 (C=S), 161.2 (C=O), 142.8 (C=N), 136.1, 131.4, 130.9,
(
6
1
29.1, 128.1, 127.9, 123.4, 121.0, 119.9, 110.8 (aromatic C), 43.0, 31.8 (aliphatic C). ESIꢀMS
m/z = 347.21 [M+Na]+.
.2.5. (Z)-2-(5-bromo-1-methyl-2-oxoindolin-3-ylidene)-N-methylhydrazinecarbothioamide
L5)
4
(
4
ꢀMethyl thiosemicarbazide (0.105 g, 0.001 mole) and 5ꢀbromo isatin (0.226 g, 0.001
o
mole) were used. Yield: 93%. Yellow. m.p.: 159 C. Anal. Calc. C H BrN OS (%): C, 38.35; H,
1
0
9
4
2
.90; N, 17.89; S, 10.24. Found: C, 38.29; H, 2.81; N, 17.83; S, 10.12. UV−Vis (CH OH): λmax,
3
3
ꢀ1
ꢀ1
ꢀ1
nm (ε, dm mol cm ) 265 (22100), 364 (26600). FTꢀIR (KBr): ʋ, cm 3468, 3427, 3233 (N–H),
1
1
1
6
683 (C=O), 1559 (C=N), 1281 (C=S). H NMR (400 MHz, DMSOꢀd ): δ, ppm 12.39 (s, 1H),
6
1.29 (s, 1H), 9.35ꢀ9.26 (d, J = 4.0 Hz, 1H), 7.80 (s, 1H), 7.48ꢀ7.47 (d, J = 4.0 Hz, 1H), 6.88ꢀ
1
3
.86 (d, J = 8.0 Hz), 3.10ꢀ3.09 (d, J = 4.0 Hz, 3H). C NMR (100 MHz, DMSOꢀd ): δ, ppm
6

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178.0 (C=S), 162.6 (C=O), 141.6 (C=N), 133.5, 130.6, 123.5, 122.7, 114.6, 113.4 (aromatic C),
+
3
1.7 (aliphatic C). ESIꢀMS m/z = 313.64 [M] .
4.3. Synthesis of nickel(II) complexes (1-5)
The methanolic solution of [NiCl (PPh ) ] (1 mmol) was added into an appropriate
2
3 2
substituted isatin thiosemicarbazone ligands (1 mmol) in methanol and a few drops of triethyl
amine was added. The reaction mixture was stirred for 5 h under reflux, and then the precipitate
2
7
formed was filtered and washed with methanol. The suitable crystals for Xꢀray diffraction were
grown from CHCl /DMF mixture (1:1).
3
4.3.1. Bis((Z)-N-methyl-2-(2-oxoindolin-3-ylidene)thiosemicarbazonato)nickel(II) (1)
[
NiCl (PPh ) ] (0.654 g, 0.001 mole) and L1 (0.234 g, 0.001 mole) were used. Yield:
2
3 2
89%. Light brown solid. m.p.: 254 °C. Anal. Calcd. For C H N NiO S : C, 45.73; H, 3.45; N,
20 18 8 2 2
21.33; S, 12.21. Found: C, 45.99; H, 3.23; N, 21.02; S, 12.00. UV−Vis (DMF): λmax, nm (ε,
3
ꢀ1
ꢀ1
ꢀ1
dm mol cm ) 268 (24100), 376 (20800), 446 (6900). FTꢀIR (KBr): ʋ, cm 3464, 3426 (N–H),
+
1
640 (C=O), 1519 (C=N), 1237 (C= S). ESIꢀMS m/z = 525.04 [M] . µ = 2.09 BM.
4.3.2. Bis((Z)-2-(1-allyl-2-oxoindolin-3-ylidene)thiosemicarbazonato)nickel(II) (2)
[
NiCl (PPh ) ] (0.654 g, 0.001 mole) and L2 (0.274 g, 0.001 mole) were used. Yield:
2 3 2
85%. Light brown solid. m.p.: 270 °C. Anal. Calcd. For C H N NiO S : C, 51.59; H, 4.33; N,
26 26 8 2 2
18.51; S, 10.59. Found: C, 51.21; H, 4.02; N, 18.11; S, 10.06. UV−Vis (DMF): λmax, nm (ε,
3
ꢀ1
ꢀ1
ꢀ1
dm mol cm ) 269 (14600), 376 (10400), 447 (6600). FTꢀIR (KBR): ʋ, cm 3461 (N–H), 1647
+
(
C=O), 1522 (C=N), 1251 (C= S). ESIꢀMS m/z = 605.10 [M] . µ = 1.98 BM.
4.3.3. Bis((Z)-N-methyl-2-(1-methyl-2-oxoindolin-3-ylidene)thiosemicarbazonato)nickel(II)
(3)
[
NiCl (PPh ) ] (0.654 g, 0.001 mole) and L3 (0.248 g, 0.001 mole) were used. Yield:
2 3 2
8
1
2
3%. Brown. m.p.: 261 °C; Anal. Calcd. for C H N NiO S : C, 47.76; H, 4.01; N, 20.25; S,
22 22 8 2 2
3
ꢀ1
ꢀ1
1.59 Found: C, 46.91; H, 4.18; N, 20.00; S, 11.37. UV−Vis (DMF): λmax, nm (ε, dm mol cm )
ꢀ1
67 (26100), 382 (8800), 448 (6500). FTꢀIR (KBr): ʋ, cm 3453 (N–H), 1636 (C=O), 1525
+
(
C=N), 1220 (C= S). ESIꢀMS m/z = 553.07 [M] . µ = 1.95 B.M.
4
.3.4. Bis((Z)-2-(1-benzyl-2-oxoindolin-3-ylidene)thiosemicarbazonato)nickel(II) (4)
NiCl (PPh ) ] (0.654 g, 0.001 mole) and L4 (0.324 g, 0.001 mole) were used. Yield:
[
2
3 2
8
7%. Light brown solid. m.p.: 293 °C. Anal. Calcd. for C H N NiO S : C, 57.88; H, 4.29; N,
34 30 8 2 2

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15.88; S, 9.09. Found: C, 57.01; H, 4.51; N, 15.32; S, 8.71. UV−Vis (DMF): λmax, nm (ε,
3
ꢀ1
ꢀ1
ꢀ1
dm mol cm ) 267 (22500), 388 (15900), 440 (5900). FTꢀIR (KBr): ʋ, cm 3463 (N–H), 1644
+
(
C=O), 1524 (C=N), 1227 (C= S). ESIꢀMS m/z = 705.13 [M] . µ = 2.13 BM.
4.3.5. Bis((Z)-2-(5-bromo-1-methyl-2-oxoindolin-3-ylidene)thiosemicarbazonato)nickel(II)
(5)
[
NiCl (PPh ) ] (0.654 g, 0.001 mole) and L5 (0.403 g, 0.001 mole) were used. Yield:
2 3 2
7
9%; Light brown solid. m.p.: 272 °C. Anal. Calcd. for C H Br N NiO S : C, 35.17; H, 2.36;
20 16 2 8 2 2
N, 16.41; S, 9.39 Found: C, 34.83; H, 2.59; N, 16.12; S, 9.54. UV−Vis (DMF): λmax, nm (ε,
3
ꢀ1
ꢀ1
ꢀ1
dm mol cm ) 293 (26500), 380 (10500), 453 (6600). FTꢀIR (KBr): ʋ, cm 3470, 3423 (N–H),
+
1
642 (C=O), 1533 (C=N), 1223 (C= S). ESIꢀMS m/z = 683.09 [M] . µ = 2.17 BM.
4.4. Single crystal X-ray diffraction studies
A Bruker APEX2 Xꢀray (threeꢀcircle) diffractometer was employed for crystal screening,
unit cell determination, and data collection. The Xꢀray radiation employed was generated from a
Mo sealed Xꢀray tube (Kα = 0.70173 Å with a potential of 40 kV, 40 mA) fitted with a graphite
monochromator in the parallel mode (175 mm collimator with 0.5 mm pinholes). Sixty data
0
frames were taken at widths of 0.5 . These reflections were used in the autoꢀindexing procedure
to determine the unit cell. A suitable cell was found and refined by nonlinear least squares and
Bravais lattice procedures. The unit cell was verified by examination of the h k l overlays on
several frames of data by comparing with both the orientation matrices. No superꢀcell or
erroneous reflections were observed. After careful examination of the unit cell, a standard data
collection procedure was initiated using omega scans. Integrated intensity information for each
5
3
reflection was obtained by reduction of the data frames with the program APEX2. The
integration method employed a three dimensional profiling algorithm and all data were corrected
for Lorentz and polarization factors, as well as for crystal decay effects. Finally, the data were
5
4
merged and scaled to produce a suitable data set. The absorption correction program SADABS
was employed to correct the data for absorption effects. Systematic reflection conditions and
statistical tests of the data suggested the space group. Solution was obtained readily using
5
5
SHELXTL (XS). Hydrogen atoms were placed in idealized positions and were set riding on the
respective parent atoms. All nonꢀhydrogen atoms were refined with anisotropic thermal
parameters. The structure was refined (weighted least squares refinement on F2) to
5
6
55
convergence. Olex2 was employed for the final data presentation and structure plots.

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4.5. DNA binding studies
The interaction of metal complexes with CT DNA was carried out in Tris HCl/NaCl
buffer (pH 7.2). The bulk solution of CT DNA was prepared by diluting the CT DNA using Tris
HCl/NaCl buffer followed by stirring at 4 °C for three days, and kept at 4 °C for not more than a
week. The stock solution of CT DNA gave a ratio of UV absorbance at 260 and 280 nm
(A260/A280) of 1.89, indicating that the DNA was sufficiently free of proteins. The bulk DNA
solution was further diluted to 10 folds to show maximum absorbance at 260 nm. The absorption
ꢀ1
ꢀ1
57
coefficient of CT DNA was 6600 cm M per nucleotide. Ni(II) complexes of required
concentration were prepared by dissolving the calculated amount of the complexes in 5%
DMF/Tris HCl/NaCl. Complex solution of concentration 15 µM was taken in cuvette and CT
DNA of equivalent concentration (5ꢀ50 µM) was added each time and the significant absorbance
change was noted.
The competitive binding of each complex with EB has been investigated by fluorescence
spectroscopic technique in order to examine whether the complex can displace EB from its CT
DNAꢀEB complex. Ethidium bromide solution was prepared using Tris HCl/NaCl buffer (pH
7.2). The test solution was added in aliquots of 5 µM concentration to DNAꢀEB and the change
in fluorescence intensities at 596 nm (450 nm excitation) was noted down.
Circular dichroic spectra were recorded with a Jꢀ715 spectropolarimeter (Jasco) at 25 °C
with a 0.1 cm path length cuvette. The spectra were recorded for 200 ꢂM of DNA in the absence
58
and presence of 40 ꢂM of the nickel(II) complexes (1ꢀ5) in the region 220–300 nm.
4.6. DNA cleavage studies
A mixture of Tris buffer (5 mM TrisꢀHCl/50 mM NaCl buffer, pH 7.2), pUC19 plasmid
DNA (150 ꢂg/mL) and complexes (1ꢀ5) were incubated for 3 h at 37 °C. A dye solution (0.05%
bromophenol blue and 5% glycerol) was added to the reaction mixture prior to electrophoresis.
The samples were then analyzed by 1.5% agarose gel electrophoresis [TrisꢀHCl/Boric
−
1
acid/EDTA (TBE) buffer, pH 8.0] for 2 h at 60 mV. The gel was stained with 0.5 ꢂg mL
ethidium bromide, visualized by UV light and photographed. The extent of cleavage of pUC19
was determined by measuring the intensity of the bands using AlphaImager HP instrument
.

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4.7. Protein binding studies
The binding of nickel(II) complexes (1ꢀ5) with BSA was studied using fluorescence
spectra recorded at a fixed excitation wavelength corresponding to BSA at 280 nm and
monitoring the emission at 335 nm. The excitation and emission slit widths and scan rates were
constantly maintained 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
solutions of each test compound were prepared by dissolving them in DMF–phosphate buffer
(5:95) and diluted with phosphate buffer to get required concentrations. 2.5 ml of BSA solution
−
6
was titrated by successive additions of a 10 M stock solution of the complexes using a
micropipette. For synchronous fluorescence spectra measurements, the same concentration of
BSA and the complexes were used and the spectra were measured at two different ꢁλ (difference
between the excitation and emission wavelengths of BSA) values of 15 and 60 nm.
In three dimensional fluorescence studies, the emission wavelength was recorded
between 240 and 440 nm, and the excitation wavelength was recorded from 200 to 360 nm at 10
nm increments. The other scanning parameters were just the same as the fluorescence quenching
59
experiments.
4.8. Molecular docking studies of DNA
The Xꢀray crystal structure of BꢀDNA (PDB ID: 1BNA) dodecamer
d(CGCGAATTCGCG) and human DNA TopoꢀI complex (PDB ID: 1SC7) was obtained from
2
6
0
the Protein Data Bank. 2D structure of nickel(II) complexes was drawn using ChemDraw Ultra
2.0 (ChemOffice 2010). Chem3D Ultra 12.0 was used to convert 2D structure into 3D and the
1
energy was minimized using semiꢀempirical AM1 method. Molecular docking studies have been
done using the AutoDock Tools (ADT) version 1.5.6 and AutoDock version 4.2.5.1 docking
6
8
programs. The energy calculations were made using genetic algorithms. The outputs were
exported to PyMol for visual inspection of the binding modes and for possible polar and
hydrophobic interactions of the complexes with DNA.

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4.9. Molecular docking studies of protein
GOLD (Genetic Optimization for Ligand Docking) is a genetic algorithm for docking
flexible ligands into protein binding sites. GOLD enables to make confident binding mode
predictions and achieve high database enrichments. Gold reliably identifies the correct binding
mode for a large range of test set cases and has been shown to perform favorably against other
docking tools in numerous independent studies.
6
1
The structure of the protein was obtained from RCSB Protein Data Bank , (with PDB ID
3V03) and the structure of the protein was minimized by running 1000 steps of steepest descent
:
algorithm using the UCSF Chimera software. All water molecules were deleted while
minimizing. The minimized protein was then imported into the Hermes Visualizer, the graphical
interface of GOLD. The structure of the ligands was drawn and 3D optimized using chemsketch
software. The target protein was kept rigid and the ligands being docked were kept flexible.
4.10. Cytotoxic studies using SRB assay
The sulphorhodamine B (SRB) assay represent an appropriate and sensitive assay to
measure the drug induced cytotoxicity and cell proliferation for large scale drug screening
6
0
applications. The cell lines were grown in RPMI 1640 medium containing 10% fetal bovine
serum and 2 mM Lꢀglutamine. For screening experiment, cells were inoculated into 96 well
microtiter plates in 90 µL at plating densities depending on the doubling time of individual cell
lines. After cell inoculation, the microtiter plates were incubated at 37 °C, 5 % CO , 95 % air and
2
100 % relative humidity for 24 h prior to addition of experimental drugs.
After 24 h, one plate of each cell line was fixed in situ with trichloroacetic acid (TCA), to
represent a measurement of the cell population for each cell line at the time of drug addition
Tz). Experimental drugs were solubilized in appropriate solvent at 400ꢀfold the desired final
(
maximum test concentration and stored frozen prior to use. At the time of drug addition, an
aliquot of frozen concentrate was thawed and diluted to 10 times the desired final maximum test
ꢀ3
concentration with complete medium containing test article at a concentration of 10 . Additional
three, 10ꢀfold serial dilutions were made to provide a total of four drug concentrations plus
control. Aliquots of 10 µL of these different drug dilutions were added to the appropriate microꢀ
titer wells already containing 90 µL of medium, resulting in the required final drug
concentrations.
After compound addition, plates were incubated at standard conditions for 48 h and assay

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was terminated by the addition of cold TCA. Cells were fixed in situ by the gentle addition of 50
µL of cold 30 % (w/v) TCA (final concentration, 10 % TCA) and incubated for 60 minutes at 4
°
C. The supernatant was discarded and the plates were washed five times with tap water and air
dried. Sulforhodamine B (SRB) solution (50 µL) at 0.4 % (w/v) in 1 % acetic acid was added to
each of the wells, and plates were incubated for 20 minutes at room temperature. After staining,
unbound dye was recovered and the residual dye was removed by washing five times with 1 %
acetic acid. The plates were air dried. Bound stain was subsequently eluted with 10 mM trizma
base, and the absorbance was read on an Elisa plate reader at a wavelength of 540 nm with 690
nm reference wavelength. Percentage growth inhibition was calculated as:
[
[
(Ti−Tz)/(C−Tz)] × 100 for concentrations for which Ti>/=Tz (Ti−Tz) positive or zero
(Ti−Tz)/Tz] × 100 for concentrations for which Ti<Tz. (Ti−Tz) negative
where time zero (Tz), control growth (C), and test growth in the presence of drug at the four
concentration levels (Ti). IC50 values can be derived from curve fitting methods with statistical
analysis software.
Acknowledgements
J. H. thanks the University Grants Commission for the fellowship (F1ꢀ17.1/2012ꢀ
13/RGNFꢀ2012ꢀ13ꢀSTꢀANDꢀ18716). We thank anti cancer drug screening facility (ACDSF) at
ACTREC, Tata Memorial Centre, Navi Mumbai for cytotoxicity studies and CLRI, Chennai for
providing CD facility.

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Fig. 2 Thermal ellipsoid plot of L1.
Fig. 3 Thermal ellipsoid plot of L2.

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Fig. 4 Thermal ellipsoid plot of 1 (Symmetry related atoms are not labelled for clarity).
Fig. 5 Absorption spectra of complexes (2 and 4) in TrisꢀHCl buffer upon addition of CT DNA.
−5
[
Complex] = 1.5 × 10 M, [DNA] = 0ꢀ50 µM. Arrow shows that the absorption intensities
decrease upon increasing DNA concentration.

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Fig. 6 Fluorescence quenching curves of EB bound to DNA in the presence of 2 and 4. [DNA] =
µM, [EB] = 5 µM and [complex] = 0ꢀ50 µM.
5
Fig. 7 Plots of [DNA]/(ε − ε ) versus [DNA] for the titration of the complexes with CT DNA.
a
f

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Fig. 8 SternꢀVolmer plots of fluorescence titrations of the complexes with CT DNA.
Fig. 9 CD spectra of CT DNA (200 ꢂM) in the absence and presence of 40 ꢂM of complex 2 and
4
.