In-situ nickel(II) complexes of 3-(dimethylamino)-1-propylamine based Schiff base ligands: Structural, electrochemical, biomolecular interaction and antimicrobial properties

Article abstract of DOI:10.1016/j.ica.2018.07.018

Five new di-Schiff base nickel(II) complexes (1–5) of salen type ligands were synthesized in-situ by condensation of 3-(dimethylamino)-1-propylamine with 2-hydroxybenzaldehyde, 2-hydroxy-5-methyl benzaldehyde, 2-hydroxy-5-bromo benzaldehyde, 2-hydroxy-5-nitrobenzaldehyde and 2-hydroxy-1-naphthaldehyde to obtain L¹–L⁵, respectively and complexed with nickel chloride. Single crystal X-ray diffraction studies of complexes 1 and 5 showed a distorted octahedral and distorted square-planar geometry, respectively around nickel atoms. The cyclic voltammetry of complexes 1–5 showed redox peaks near cathodic and anodic regions assignable to the Ni²⁺/Ni⁺ and Ni²⁺/Ni³⁺ redox couples, respectively. The binding studies of complexes with calf thymus DNA (ctDNA) showed an intercalation mode of binding. The nuclease activity of the complexes with pBR322 plasmid DNA showed efficient oxidative cleavage by the formation of singlet oxygen species in presence of H₂O₂. All nickel(II) complexes were found to have greater zone inhibition diameter when analyzed for antimicrobial property against four bacterial species and two human pathogenic fungal species.

Full text of DOI:10.1016/j.ica.2018.07.018

Accepted Manuscript
Research paper
In-situ nickel(II) complexes of 3-(dimethylamino)-1-propylamine based Schiff
base ligands: Structural, electrochemical, biomolecular interaction and antimi-
crobial properties
Arumugam Jayamani, Soundarajan Nagasubramanian, Vijayan Thamilarasan,
Stephen O. Ojwach, Gopalakrishnan Gopu, Nallathambi Sengottuvelan
PII:
S0020-1693(18)30847-8
https://doi.org/10.1016/j.ica.2018.07.018
ICA 18364
DOI:
Reference:
Inorganica Chimica Acta
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Received Date:
Revised Date:
Accepted Date:
1 June 2018
10 July 2018
10 July 2018
Please cite this article as: A. Jayamani, S. Nagasubramanian, V. Thamilarasan, S.O. Ojwach, G. Gopu, N.
Sengottuvelan, In-situ nickel(II) complexes of 3-(dimethylamino)-1-propylamine based Schiff base ligands:
Structural, electrochemical, biomolecular interaction and antimicrobial properties, Inorganica Chimica Acta (2018),
doi: https://doi.org/10.1016/j.ica.2018.07.018
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In-situ nickel(II) complexes of 3-(dimethylamino)-1-propylamine based Schiff base
ligands: Structural, electrochemical, biomolecular interaction and antimicrobial
properties
Arumugam Jayamania,b, Soundarajan Nagasubramaniana, Vijayan Thamilarasana,c, Stephen
O. Ojwachb, Gopalakrishnan Gopua, Nallathambi Sengottuvelana*
aDepartment of Industrial Chemistry, School of Chemical Sciences, Alagappa University,
Karaikudi – 630003, Tamilnadu, India
bDepartment of Chemistry School of Chemistry and Physics, University of KwaZulu-Natal,
Private Bag X01, Scottsville, Pietermaritzburg 3209, South Africa
cDepartment of Chemistry and Nano Science, Ewha Womans University, Seoul-120 750,
Republic of Korea.
Abstract
Five new di-Schiff base nickel(II) complexes (1–5) of salen type ligands were
synthesized in-situ by condensation of 3-(dimethylamino)-1-propylamine with 2-
hydroxybenzaldehyde, 2-hydroxy-5-methyl benzaldehyde, 2-hydroxy-5-bromo
benzaldehyde, 2-hydroxy-5-nitrobenzaldehyde and 2-hydroxy-1-naphthaldehyde to
obtain L1-L5, respectively and complexed with nickel chloride. Single crystal X-ray
diffraction studies of complexes 1 and 5 showed a distorted octahedral and distorted
square-planar geometry, respectively around nickel atoms. The cyclic voltammetry of
complexes 1–5 showed redox peaks near cathodic and anodic regions assignable to the
Ni2+/Ni+ and Ni2+/Ni3+ redox couples, respectively. The binding studies of complexes
with calf thymus DNA (ctDNA) showed an intercalation mode of binding. The
nuclease activity of the complexes with pBR322 plasmid DNA showed efficient
oxidative cleavage by the formation of singlet oxygen species in presence of H2O2. All
nickel(II) complexes were found to have greater zone inhibition diameter when

analyzed for antimicrobial property against four bacterial species and two human
pathogenic fungal species.
Keywords: Nickel(II) complexes; Single crystal, Oxidative cleavage, Antimicrobial
activity
1. Introduction
The salen type di-Schiff base complexes of transition metal ions showed a continuous
interest and were extensively studied over past few decades due to their stability, chelating
properties, fascinating structural features, efficient electrochemical and magnetic properties,
and biological applications as metalloenzymes [1–3]. They play a vital role in the field of
bioinorganic chemistry as they can easily form stable complexes [4,5]. The transition metal
complexes of Schiff bases derived from aromatic compounds containing nitrogen and oxygen
atoms are of current interest as simple structural models of biological systems [6–9]. Among
the transition metal ions, the study of nickel compounds is of great interest in various aspects
of chemistry [10,11]. In general, the reaction of salen or related ligands with Ni(II) yields
complexes in which the deprotonated, di-negative Schiff base acts as a tetradentate chelating
ligand [12–14]. However, in some cases, it has been found that the oxygen atoms of the
Schiff base are not deprotonated in case of having bulky substituents [15]. In this work, an
attempt was made to study the steric and electronic effect of ligand substituent on the
geometry and biomolecular interaction of the Ni(II) complexes.
In the present work, nickel(II) complexes [Ni(L1)2] (1), [Ni(L2)2] (2), [Ni(L3)2] (3),
[Ni(L4)2] (4) and [Ni(L5)2]Cl2 (5) were synthesized with Schiff base ligands prepared by in-
situ condensation of 3-(dimethylamino)-1-propylamine with different aldehydes. Some of
these compounds and similar complexes were previously reported by Sacconi et al,
synthesized from bis(salicylaldehyde) nickel(II) complexes studies and studied their
four/five/six coordination [16-18]. The DNA binding and cleavage ability of all the nickel(II)

complexes were evaluated using calf thymus DNA (ctDNA) and plasmid pBR322 DNA,
respectively. The antimicrobial properties of the complexes were evaluated against four
bacteria and two pathogenic fungi.
2. Experimental
2.1. Materials
All the chemicals used were of analytical grade and were used as received without
any further purification. The amine, 3-(dimethylamino)-1-propylamine and aldehydes 2-
hydroxybenzaldehyde,
2-hydroxy-5-methyl
benzaldehyde,
2-hydroxy-5-bromo
benzaldehyde, 2-hydroxy-5-nitrobenzaldehyde, 2-hydroxy-1-naphthaldehyde were obtained
from Alfa Aesar, (India). The calf thymus and plasmid DNA’s were purchased from SRL
(India), Tris–HCl, Tris–base and NaCl were purchased from Merck (India).
2.2. Instrumentation
The UV-Visible spectra were recorded on
a
Shimadzu UV-3101PC
spectrophotometer. FT-IR spectra were recorded in the 4000– 400 cm-1 regions using KBr
pellets on a Bruker EQUINOX 55 spectrometer. The elemental composition of the complexes
was evaluated on an Elementar Vario MACRO cube elemental analyzer. The electron spray
ionization mass spectra (positive mode) were recorded on an Aligent 6520 Q-T mass
spectrometer (Central Drug Research Institute, Lucknow, India). The fluorescence quenching
experiment of DNA-bound to EB for the synthesized complexes were recorded on an Elico
SL-174 spectrofluorometer. The redox potential of the complexes was measured on a
Biologic CHI604D electrochemical analyzer using a three-electrode cell under nitrogen
atmosphere where a glassy carbon, Ag/AgCl and a platinum wire were used as working
electrode, a reference electrode and the auxiliary electrode, respectively. The complexes (1
mM) in dimethylformamide with 0.1 M supporting electrolyte, tetra(n-butyl) ammonium

perchlorate were used for cyclic voltammetry studies. All the solvents were purified
according to standard procedures.
2.3. Synthesis of in-situ nickel (II) complexes
2.3.1. [Ni(L1)2](1): To a solution of NiCl2.6H2O (0.24 g, 1 mM) in methanol (25 mL), 2-
hydroxybenzaldehyde (0.24 g, 2 mM) in methanol was added slowly with constant stirring,
then 3-(dimethylamino)-1-propylamine (0.20 g, 2 mM), was added to the reaction mixture
and the content was stirred at room temperature for three hours followed by reflux for about
two hours. The green coloured precipitate formed was dried and recrystallized in acetonitrile
to obtain green crystals after a few days. Yield: 0.51 g, 56 %. m.p.: 201 °C (dec). Anal. data
for C24H34N4O2Ni: Calc. (%): C, 61.43; H, 7.25; N, 11.94; Ni, 12.51; Found (%): C, 61.37;
H, 7.24; N, 11.86; Ni, 12.43; FT–IR (ν, cm−1) (KBr Disc): 1636(ν C=N), 565(ν Ni-O), 492(ν
Ni-N); UV-Vis [λmax/nm (εmax/ mol-1 cm-1)]: 272 (60910), 310 (33460), 376 (56060), 538
(15), 745 (10); Molar conductivity (m/Scm2mol-1) in acetonitrile 15.
Other nickel(II) complexes 1-5 were synthesized by a procedure similar to that of complex 1.
2.3.2. [Ni(L2)2] (2): 2-hydroxy-5-methyl benzaldehyde (0.27 g, 2 mM), NiCl2.6H2O (0.24 g,
1 mM), 3-(dimethylamino)-1-propylamine (0.20 g, 2 mM). The greenish yellow coloured
precipitate obtained was filtered and dried. Yield: 0.56 g, 60 %; m.p.: 188 °C (dec). Anal.
data for C26H38N4O2Ni Calc. (%): C, 62.79; H, 7.65; N, 11.27; Ni, 11.80; Found (%): C,
62.73; H, 7.74; N, 11.14; Ni, 11.67; FT–IR (ν, cm−1) (KBr Disc): 1629(ν C=N), 556(ν Ni-O),
468(ν Ni-N); UV-Vis [λmax/nm (εmax/ mol-1 cm-1)]: 273(6685), 388(6023), 653(19), 695(18);
Molar conductivity (mScm-1mol-1) in acetonitrile 16.
2.3.3. [Ni(L3)2] (3): 2-hydroxy-5-bromo benzaldehyde (0.42 g, 2 mM), NiCl2.6H2O (0.24 g, 1
mM), 3-(dimethylamino)-1-propylamine (0.20 g, 2 mM). The green coloured precipitate
obtained was filtered and dried. Yield: 0.64 g, 60 %; m.p.: 199 °C (dec). Anal. data for
C24H32Br2N4O2Ni Calc. (%): C, 45.97; H, 5.14; N, 8.94; Ni, 9.36; Found (%): C, 45.9; H,

5.18; N, 8.81; Ni, 9.25; FT–IR (ν, cm−1) (KBr Disc): 1625(ν C=N), 570(ν Ni-O), 490(ν Ni-
N); UV-Vis [λmax/nm (εmax/ mol-1 cm-1)]: 275 (6665), 387 (6275), 635 (10), 699 (10); Molar
conductivity (mScm-1mol-1) in acetonitrile 12.
2.3.4. [Ni(L4)2] (4): 2-hydroxy-5-nitrobenzaldehyde (0.33 g, 2 mM), NiCl2.6H2O (0.24 g, 1
mM), 3-(dimethylamino)-1-propylamine (0.20 g, 2 mM). The green coloured precipitate
obtained was filtered and dried. Yield: 0.65 g, 65 %; m.p.: 183 °C (dec.) Anal. data for
C24H32N6O6Ni Calc. (%): C, 51.54; H, 5.72; N, 15.03; Ni, 10.50; Found (%): C, 51.48; H,
5.64; N, 14.92; Ni, 10.30; FT–IR (ν, cm−1) (KBr Disc): 1625(ν C=N), 568(ν Ni-O), 464(ν Ni-
N); UV-Vis [λmax/nm (εmax/ mol-1 cm-1)]: 288 (7920), 404 (58180), 585 (19), 740(3); Molar
conductivity (mScm-1mol-1) in acetonitrile 14; ESI-MS (CH3CN) m/z (%): 558.7(6)
[Ni(L4)2+H]+, 369.2(38), 300.9(76), 251.8(32), 148.6(65), 113.7(100)
2.3.5. [Ni(L5)2]Cl2 (5): 2-hydroxy-1-naphthaldehyde (0.34 g, 2 mM), NiCl2.6H2O (0.24 g, 1
mM), 3-(dimethylamino)-1-propylamine (0.20 g, 2 mM). The green coloured precipitate
formed was dried and recrystallized in acetonitrile to obtain green crystals by slow
evaporation after few days. Yield: 0.61g, 60 %; m.p.: 191 °C (dec); Anal. data for
C32H40N4O2NiCl2 Calc. (%): C, 59.84; H, 6.28; N, 8.72; Ni, 9.14; Found (%): C, 59.40; H,
6.41; N, 8.61; Ni, 9.16; FT–IR (ν, cm−1) (KBr Disc): 3336(ν O-H), 2980(s), 1621(ν C=N),
568(ν Ni-O), 496(ν Ni-N); UV-Vis [λmax/nm (εmax/ mol-1 cm-1)]: 277 (24030), 305 (16150),
404 (11806), 635 (10); Molar conductivity (mScm-1mol-1) in acetonitrile 122.
2.4. Single crystal XRD
The in-situ Schiff base nickel complexes 1 and 5 were obtained as green crystals
suitable for X-ray diffraction studies by recrystallization using acetonitrile solvent. The X-ray
diffraction analysis of the complexes was carried out on Bruker SMART APEX-II CCD
diffractometer, using a single crystal with different dimensions with graphite
monochromatized Mo-Kα radiation (λ = 0.71073 Å). Data reduction and corrections for

absorption and decomposition were achieved using the SAINT/X-PREP software [19]. The
structure was solved using the direct methods and successive Fourier difference synthesis
thermal parameters for all non-hydrogen atoms (SHELXL-97) and all nonhydrogen atoms
were refined anisotropically by full-matrix least-square procedures. Hydrogen atoms were
added theoretically and refined with riding model position parameters and fixed isotropic
thermal parameters.
2.5. DNA binding and cleavage studies
The ctDNA was used in binding experiments due to greater similarity with that of
mammalian DNA, also at high molecular weights, the impurity due to the presence of protein
can be easily identified. The UV absorption spectral method was used to study the binding
ability of synthesized nickel(II) complexes with ctDNA. The stock solution of ctDNA was
prepared in 5 mM Tris–HCl/ 20 mM NaCl buffer (pH = 7.2), stored at 4 °C and used within 4
days. The solution of ctDNA gave a ratio of UV absorbance at 260 nm and 280 nm, A260/
A280, of 1.8-1.9, indicating that the ctDNA was sufficiently free of proteins. Absorption
titration experiments were performed by varying the DNA concentration while keeping the
complex concentration as constant. To compare quantitatively, the intrinsic binding constant
Kb of complexes with ctDNA were determined according to the following equation, [DNA]
/(εa-εf) = [DNA] /(εb-εf) + 1/Kb(εa-εf), where, [DNA] is the concentration of DNA in base
pairs, the apparent absorption coefficients εa, εf, and εb correspond to Aobsd/[complex], the
extinction coefficient for the free complexes and the extinction coefficient for the complexes
in the fully bound form, respectively. Double distilled water was used to prepare all buffer
solutions.
The complexes did not show luminescence behaviour at room temperature in any of
the organic solvents examined and in the presence of ctDNA. Hence, the fluorescence
binding studies were performed using ethidium bromide (EB) as a reference to determine the

relative DNA binding properties of complexes with ctDNA in 5 mM Tris–HCl/ 5 mM NaCl
buffer, pH 7.2. Fluorescence intensities of EB at 596 nm with an excitation wavelength of
515 nm were measured at different complex concentrations.
The DNA cleavage studies were performed by gel electrophoresis experiment using
pBR322 plasmid DNA (which show clear DNA fragmentation pattern when compared to
ctDNA). The cleavage activity was evaluated by monitoring the conversion of supercoiled
plasmid DNA (Sc – form I) to nicked circular DNA (Nick- form II) and linear DNA (Lin –
form III). Each reaction mixture was prepared by adding 2 μL (200 ng) of supercoiled DNA,
2 μL of 500 mM Tris – HCl/500 mM NaCl buffer (pH = 7.4), 4 μL of hydrogen peroxide and
6 μL of the complex dissolved in DMF. The final reaction volume was 20 μL, the final buffer
concentration was 50 mM and the final concentration of complex varied between 100 and
200 μM. Samples were typically incubated for 1h at 37 ºC and after incubation, 5 μL of DNA
loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol in water) were
added to each tube. The sample was then loaded onto a 0.8% agarose gel in TBE buffer (89
mM Tris-borate, 1.0 mM EDTA pH 8.4) containing ethidium bromide (0.5 μg/mL). Negative
and positive controls were loaded on each gel electrophoresis and the experiment was carried
out for 1.30 h at 50 V. The reaction was also carried out in the presence of KI (40 mM)
(hydrogen peroxide scavenger), DMSO (40 mM) (hydroxy radical scavenger) and NaN3 (40
mM) (singlet oxygen quencher).
2.6. Molecular docking
The molecular docking studies for all complexes were performed in-silico by using
HEX 8.0 software [20]. The coordinates of nickel(II) complexes 1 and 5 were taken from
their respective crystal structure as a CIF file and converted to the PDB format using Mercury
3.3 software (http://www.ccdc.cam.ac.uk/). The structures of complexes without single
crystal data were sketched by CHEMSKETCH (http://www.acdlabs.com) and converted to

PDB format from MOL format by Mercury 3.3 software. The crystal structure of the B-DNA
dodecamer d(CGCGAATTCGCG)2 (PDB ID: 1BNA) was downloaded from the protein data
bank (http://www.rcsb.org./pdb). All calculations were carried out on an Intel pentium4, 2.4
GHz based machine running MS Windows XP SP2 as the operating system. The docked pose
of
complex
and
DNA
was
visualized
using
CHIMERA
software
(www.cgl.ucsf.edu/chimera).
2.7. Antimicrobial screening
Antimicrobial activity of nickel(II) complexes 1 – 5 were tested against the bacterial
species (Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Pseudomonas
aeruginosa) cultured on nutrient agar medium and against the fungal species (Aspergillus
niger and Candida albicans) cultured on potato dextrose agar medium by the well-diffusion
method. The medium preparation was detailed in the supplementary article. For the
investigation of the antimicrobial activity, all the complexes were dissolved in
dimethylsulfoxide (DMSO) to a final concentration of 100 μg/mL. The sample was filled into
o
the wells of agar plates directly, incubated at 37 C for 24 h for bacteria and 38 h for fungi.
The diameter of the inhibition zone around each well was measured after the incubation
period and studies were performed in duplicate. The solvent was also examined as a control
to study the effect of DMSO (solvent) on the growth of microorganisms, which showed no
inhibition.
3. Results and discussion
3.1. Synthesis and characterization
The synthesis of the nickel(II) complexes was done using a common procedure, by
reaction of a stoichiometric amount of nickel chloride salts with the respective ligands in
methanol as given in scheme 1. The analytical data obtained for complexes 1–5 were
consistent with the formation of di-Schiff's base nickel(II) complexes. The obtained

complexes were insoluble in water, methanol, ethanol, and chloroform but soluble in
acetonitrile, dimethylformamide (DMF) and DMSO. The lower molar conductivity values of
complexes 1–4 showed the neutral non-electrolyte behaviour, and complex 5 with higher
conductivity showed 1:2 electrolyte behaviour.
3.2. Crystal structure
Crystal data and details of the structure determination of complexes 1 and 5 were
presented in Table 1, selected bond lengths and bond angles were listed in Table 2. Suitable
crystals for the single crystal X-ray diffraction were grown by slow evaporation of complex
dissolved in acetonitrile solvent. The ORTEP views shown in Figures 1 and 2 suggested the
complex 1 with slightly distorted octahedral coordination geometry and complex 5 with
distorted square-planar geometry. The X-ray structure of the complex 1 showed the
hexacoordinated nickel(II) ion with two N, N, O donors of two Schiff’s base ligand
molecules in distorted octahedral geometry [18]. In the crystal structure of 1, it was noticed
that the basal plane is formed by the two N atoms of the azomethine group and the two O
atoms of the phenolic hydroxyl group of salicylaldehyde moiety, where the nickel atom was
in the equatorial plane of the octahedron. The axial positions of the distorted octahedron were
observed to be occupied by the nitrogen atoms containing dimethyl groups in the Schiff's
base ligand. The two 3-(dimethylamino)-1-propylamine ligands joined with nickel atom and
form the chair conformation. The larger axial Ni–N1 and Ni–N3 distances of 2.302 Å, and
2.330 Å, respectively were observed for the complex 1. The equatorial Ni–N2 and Ni–N4
bonds of the complex were observed to be (2.050 Å and 2.055 Å) greater than the equatorial
Ni–O1 and Ni–O2 bonds (2.014(15) Å and 2.010(15)) [21].
The trans angles O1–Ni–O2, N1–Ni–N3 and N2–Ni–N4 is found to be 179.23(6),
179.82(7) and 179.77(8)º, respectively; and the cis angles N2–Ni–O2 and N4–Ni–O1 is
present at 88.33(7) and 87.67(7)º, respectively. The trans and cis angles of the complex

deviate from 180º and 90º shows the presence of slight distortion in the octahedral structure
of nickel(II) complex. The packing diagram of complex 1 (Figure S1) showed the
intermolecular hydrogen bonding contacts formed at the para position to the carbon attached
to azomethine nitrogen moiety. The hydrogen bonding also occurs between the two methyl
groups of the adjacent molecule in crystal packing.
The crystal structure of complex 5 showed the four-coordinated nickel(II) ions with
two N, O donors of two molecules of Schiff’s base ligand in distorted square-planar
geometry. The trans two O atoms coordinated to nickel atom with Ni1–O2 bond lengths of
1.830 Å and the two azomethine N atoms coordinated to nickel atom with Ni–N bond lengths
of 1.897 Å. The cis angle N–Ni–O in the asymmetric unit in a plane with ligand molecule
was 91.26° and out of a plane with ligand molecule was 91.26°; the trans angles N–Ni–O was
found to be 180° between azomethine nitrogen one ligand and oxygen of another ligand
molecule.
The one-half of the Schiff’s base ligand molecule containing the 3-(dimethylamino)-
1-propylamine unit and another half of the Schiff’s base ligand with naphthaldehyde unit
were observed above and below the N2O2N2O2 plane, both incline to the plane at an angle
of 120.6° and 96.92°, respectively. The position of the ligand molecule causes a change in the
cis angles of the coordination sphere and causes a slight distortion in the square planar
geometry of the complex. The packing diagram of complex 5 (Figure S2) showed the
intermolecular hydrogen bonding in the complex and weak intramolecular hydrogen
interactions at H10B···H14 at 2.358(7) Å. The hydrogen bonding also observed between the
two methyl groups of the adjacent molecule in crystal packing. The presence of interchain π–
π stacking interactions extends the structure into an interesting 3D supramolecular array and
stabilizes the packing of the complex in the crystal lattice.
3.3. Physico-chemical characterizations

The IR spectra of complexes 1–5 showed a sharp band in the region of 1619-1636 cm-
1 attributed to ν(C=N) stretching vibration [22], which confirms the presence of Schiff’s base
in the complex. All complexes have bands in the region of 3070–3095 cm-1 and 2876–2895
cm-1, which can be assigned to C–H stretching vibrations. Further evidence of coordination of
ligands with the metal ions was revealed by the band at 456–622 cm-1 assigned to the metal-
oxygen (M–O) vibration in all complexes [23].
The absorption spectral data obtained experimentally for all the nickel(II) complexes
in DMF solution show peaks near 272 - 288 nm due to π → π* transition of Schiff’s base
ligands. In the UV region of complexes 1–5, broad/slightly intense peaks or a shoulder was
observed in the region of 310–419 nm which could be assigned for the ligand to metal charge
transfer transitions [24]. In the visible region of nickel(II) complexes 1–4, the absorption
bands near 538–653 nm and 695–745 nm can be attributed to 3T2g ← 3A2g and 3T1g(F) ← 3A2g
transitions [25], which were consistent with the distorted octahedral geometry. The visible
spectrum of complex 5 displayed the absorption bands below 670 nm characteristic of the
spin allowed d–d transitions which have a low-spin d8 nickel(II) ion in a square-planar
environment. Thus, the absorption peak at 635 of nickel(II) complex 5 can be assigned to
1A1g→1A2g [26], which strongly favours a square-planar geometry around the central metal
ion.
The electron spray ionization (ESI) mass spectrum of nickel(II) complexes 2 and 4
(Figures S3&S4) showed their corresponding molecular ion peak at m/z 497.0 and m/z
558.7(6) assignable to [Ni(L2)2+H]+ and [Ni(L4)2+H]+, respectively. The base peaks at m/z
220.9(100) for complex 2 and at m/z 113.7 (100) for complex 4 were attributed to
[C13H20N2O]+ and [C6H14N2]+ fragments. The ESI mass spectral data of the Schiff base
nickel(II) complexes are in good agreement with the proposed structure of complexes.
3.4. Electrochemical studies

The molar conductivity of the freshly prepared complex in acetonitrile solution was
observed to be 12-28 mScm-1mol-1 indicating that the complexes 1-4 were neutral. The
electrochemical behaviour of the nickel(II) complexes was studied in DMF solution using
cyclic voltammetry in the potential range of +1.80 to -1.80 V containing 10-1 M tetra(n-
butyl)ammonium perchlorate. The voltammetric data of nickel(II) complexes (scan rate 50
mV s-1) were summarized in Table 3 and displayed in Figure 3. Controlled potential
electrolysis performed at a potential 100 mV showed more negative than the reduction wave
indicates the consumption of one electron per molecule.
The nickel(II) complexes showed a metal-centred quasi-reversible cyclic
voltammetric response in the cathodic region as given in Table 3 due to the Ni(II)/Ni(I)
couple. The ratio of cathodic to anodic peak height was less than one. However, the peak
current increases with the increase of the square root of scan rates. From these data, it can be
deduced that the redox couple was related to a quasi-reversible one-electron transfer process
controlled by diffusion. The reduction potential of nickel(II) complexes were in the order 4 <
5 < 3 < 2 < 1, suggesting that the electron donating/withdrawing groups influences on the
electron density at the metal center, that tries to stabilize nickel(I) ions leading to easy
reduction and shifts to a less negative potential [27, 28]. One quasi-reversible redox wave
was observed at an anodic region which can be assigned to Ni(II)/Ni(III) [29].
3.5. DNA binding studies
3.5.1. Absorption spectroscopy
Absorption experiments were performed in UV-Visible spectroscopy by titrating the
fixed concentrations of the complexes (40 μM) with an increase in the concentration of
o
ctDNA (10 mM) at 25 C. The absorbance of DNA itself was eliminated by adding equal
amounts of DNA to both the complex solution and the reference solution. The intrinsic
binding constant of complexes with ctDNA was determined by monitoring the absorption

intensity near 260 - 350 nm. Upon addition of increasing amount of ctDNA to complexes 1–
5, a significant “hypochromic” shift (8-39%) in the intra-ligand bands were observed near
260-350 nm accompanied by a redshift of 2–3 nm, indicative of stabilization of the DNA
helix, which suggest that the complexes bind to DNA by intercalation.
The “hypochromic effect” may be due to the presence of planar aromatic
chromophore that facilitates a strong binding interaction of the complexes with ctDNA
thereby, providing an opportunity for the complex to bind with the ctDNA via partial
insertion of the aromatic moiety in between the stacking base pair [30]. The
spectrophotometric titrations of the complex 5 were shown in Figure 4 and other complexes
1–4 are shown in Figure S5. In plots of [DNA]/(εb–εf) vs [DNA], Kb is given by the ratio of
the slope to the intercept. The Kb values obtained for nickel(II) complexes 1–5 were 1.50 ×
104, 3.74 × 104, 4.02 × 104, 8.56 × 104, and 1.35 × 105 M-1, respectively, following the order
5 > 4 > 3 > 2 > 1. The complex containing 2-hnap was having higher binding affinity than
that of complexes, this may be due to the better coplanarity and square planar geometry of the
complex [31,32]. The binding constant Kb for the nickel(II) complexes 1–5 are in the range
103-105 M-1 were in good agreement with values of similar DNA intercalators [33, 34].
3.5.2. Fluorescence spectroscopy
The fluorescence spectroscopy technique is an effective method to study metal
complex interaction with DNA. The addition of the complex to DNA pretreated with EB
causes an appreciable reduction in the fluorescence intensity, indicating that complexes 1–5
compete with EB to bind with DNA. The reduction of the emission intensity gives a measure
of the DNA-binding propensity of the complexes and stacking interaction between adjacent
DNA base pairs [35]. The intensity of the emission band near 590 nm of the EB–DNA
system decreased (up to 90% of the initial fluorescence intensity for 1, up to 87% for 2, up to
60% for 3, up to 79% for 4 and up to 55% for 5) upon addition of each complex 1–5 at

diverse r values (Figure S6). The emission spectra of EB bound to DNA in the absence and
presence of complex 5 was shown in Figure 5 (complexes 1–4 are shown in Figure S7).
According to the classical Stern–Volmer equation, I0/I = 1 + Ksv [Q], I0 and I are the
fluorescence intensities in absence and presence of quencher, respectively; Ksv is a linear
Stern–Volmer quenching constant; [Q] is the concentration of the quencher.
The fluorescence quenching curve of DNA-bound EB by complexes 1–5 illustrated
that the quenching of EB bound to DNA by complexes 1–5 was in good agreement with the
linear Stern–Volmer equation. In the linear fit plot of I0/I versus [complex]/[DNA], Ksv is
given by the ratio of the slope to the intercept. The Ksv values for nickel(II) complexes 1–5
are 1.30 × 104 M-1, 1.50 × 104 M-1, 4.02 × 104 M-1, 8.56 × 104 M-1 and 1.37 × 105 M-1,
respectively. The hydrophobic property of the rigid complex facilitates the DNA binding [36,
37]. The binding constant value obtained for the complexes were like that of other complexes
with Schiff’s base ligand, which reveals that the synthesized complexes have a good binding
propensity. The Ksv values imply that all the complexes can strongly interact with DNA and
are protected by DNA efficiently since the hydrophobic environment inside the DNA helix
reduce the accessibility of solvent water molecules to the complex and the complex’s
mobility is restricted at the binding site [38].
3.6. Molecular docking studies
The complexes 1−5 were successively docked within the DNA duplex of sequence
d(CGCGAATTCGCG)2 dodecamer (PDB ID: 1BNA) in order to predict the chosen binding
site along with the preferred orientation of the ligand inside the DNA. The minimum energy
docked poses (Figure S8) revealed that complexes fitted into the curved contour of the
targeted DNA in the minor groove and is situated within G–C/ A–T region, and slightly
bends the DNA in such a way that a part planar aromatic rings make favorable stacking
interactions between DNA base pairs leading to van der Waals interaction and hydrophobic

contacts with DNA functional groups that define the groove [39]. The resulting relative
binding energies of docked metal complexes 1–5 with DNA were found to be –251.20, –
279.51, –285.37, –270.21 and –337.85 Kcal/mol, respectively, which was also in accordance
with in-vitro DNA binding studies, that the nickel complexes with 2-hydroxy-1-
naphthaldehyde is more prominent DNA binder.
3.7. DNA cleavage studies
The DNA cleavage activities of nickel(II) complexes (1–5) have been studied by
using supercoiled pBR322 plasmid DNA as a substrate in a medium of 50 mM Tris–HCl/50
mM NaCl buffer (pH = 7.4) in the presence of hydrogen peroxide (oxidizing agent). The
control experiments with the ligand or metal salts or DNA alone do not reveal any significant
cleavage. All the complexes do not show cleavage in absence of oxidizing agent even at a
higher concentration.
The gel electrophoresis separations of plasmid pBR322 DNA by the nickel(II)
complexes 1–5 in the presence of H2O2 was shown in Figure 6. The supercoiled form was
converted to nicked circular form about 50% at 100 μM concentration of the complexes 1–5
(Figure 6, lanes 3–7 respectively) in the presence of H2O2. As the concentration of complexes
increased to 200 μM, Form I was maximum converted to Form II and no linear form was
observed (Figure 6, lanes 8–12). At 200 μM concentration of complex 5, multiple scissions
occur in a cleavage showing efficient cleavage (Figure 6, lane 12) compared to other
nickel(II) complexes. The maximum cleavage was observed for complex 5 containing
Schiff’s base ligand of 2-hnap than that of other complexes. It shows that the presence of
more aromatic, more planar ligand and geometry of the complex influence on the cleavage
process [40 –42].
To establish the DNA cleavage mechanism of nickel(II) complexes, the cleavage of
DNA was also further investigated in the presence and absence of scavengers DMSO, KI

(hydroxyl radical scavengers) and NaN3 (singlet oxygen quencher) for oxidative species. The
additives have no effect on DNA cleavage activity in control experiments (Figure 7a, lanes 3
and 9; Figure 7b, lane 3) which indicates their non-involvement in DNA cleavage activity.
The result illustrated in Figure 7 shows no inhibition of cleavage process in the presence of
DMSO (Figure 7a, lanes 4-8) and significant inhibition in the cleavage process in presence of
KI (Figure 7a, lanes 10-14) indicating that hydroxyl radical was not involved in the cleavage
process but hydrogen peroxide influence on the cleavage process. The addition of sodium
azide (singlet oxygen quencher) inhibits the cleavage process (Figure 7b, lanes 4-8)
indicating that 1O2 was the activated oxygen intermediate and responsible for the cleavage of
Ni(II) complexes. The significant increase in the DNA cleavage activity of nickel(II)
complexes in the presence of H2O2 and the inhibition of activity in the presence of DMSO,
KI and NaN3 suggest that the cleavage was preferentially proceeded by the formation of
singlet oxygen species [43].
3.8. Antimicrobial activity
The inhibition efficiencies of the complexes 1–5 were tested against two gram-
negative (Escherichia coli and Pseudomonas aeruginosa) and two gram-positive bacteria
(Staphylococcus aureus and Bacillus subtilis) and two fungal species (Aspergillus niger and
Candida albicans) (Figures S9-11). The experimental result shown in Table 4 indicates that
all the complexes exhibit higher inhibition efficiency, which can be explained based on
chelate formation [40, 41]. The chelation reduces the polarity of ligand due to the overlap of
the ligand orbital and partial sharing of the positive charge of the metal ion with donor
groups. Further, it increases the delocalization of π-electrons over the whole chelate ring and
enhances the lipophilic nature of the complexes. This increased lipophilicity enhances the
transportation of the complexes into the lipid membrane and restricts further multiplicity of
the microorganisms. By comparing the bacterial species, the complexes exhibit higher

inhibition efficiency for gram-positive bacteria than gram-negative bacteria. In fungal
species, the complexes show greater activity for C. albicans than that for A. niger species.
The complex containing 2-hydroxy-1-naphthaldehyde appended Schiff base ligand
has higher efficiency than other complexes with sal, msal, bsal and nsal as a substituent. This
may be due to the presence of additional phenyl group and higher planarity in the complex
containing a ligand with 2-hnap. The obtained results of complexes have also been compared
with those of the standard drugs, amikacin and ketoconazole for bacteria and fungi,
respectively. The complexes exhibited similar activities compared with the respective
standard drugs.
4. Conclusions
The nickel(II) complexes were synthesized in-situ by using Schiff base ligands and
characterized by physiochemical methods. The single crystal analyses, molar conductivity
values and absorption studies confirm the distorted octahedral and distorted square-planar
geometry of complexes 1 and 5, respectively. The complexes showed efficient DNA binding
following the order of 5 > 4 > 3 > 2 > 1. The gel electrophoresis results for the complexes
with plasmid DNA showed an oxidative (O2-dependent pathway) cleavage in the presence of
hydrogen peroxide by the hydroxy radical mechanism. All complexes showed a larger zone
of inhibition for Gram-positive bacteria than that of Gram-negative bacteria. The 2-hydroxy-
1-naphthaldehyde based Schiff base Ni(II) complex showed better results than other
complexes due to its better electronic/steric property and square planar geometry. Thus, the
above results evidenced that the steric factor influences the structural geometry, electronic
properties, and biomolecular activity of the complex.
Supplementary material
Crystallographic data in CIF format for compounds 1 and 5 have been deposited at
the Cambridge Crystallographic Data Centre, CCDC No. 1010002 and 949770. Copies of

CIFs are available free of charge from The Director, CCDC, 12 Union Road, Cambridge,
CB2 1EZ, UK (fax: -/44-1223-336-033; email: deposit@ccdc.cam.ac.uk or
http://www.ccdc.cam. ac.uk). The mass spectra of complexes 2 and 4; absorption,
fluorescence spectral studies of complexes 1–4 are given in the supplementary material.
Acknowledgements
The authors thank the STIC, Cochin University of Science and Technology, Cochin
for single crystal XRD and SAIF, Central Drug Research Institute (CDRI), Lucknow for
analyzing the mass spectra.
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Table 1. Crystallographic data and structure refinement parameters for complexes 1 and 5
1
5
.
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system, space group
a (Å)
C24H34N4NiO2
496.26
C32H38N4NiO2.Cl2 2H2O
569.36
296(2)
0.71073
293(2)
0.71073
Monoclinic, P21/c Monoclinic, P21/c
10.2312(6)
15.3327(9)
15.3774(8)
90
12.5721(4)
10.8367(4)
12.2393(4)
90
B(Å)
c (Å)
α (°)
β (°)
107.0350(10)
90
95.522(2)
90
γ (°)
Volume (Å3)
2306.4(2)
4, 1.351
0.869
1659.74(10)
2, 1.353
0.787
Z, calculated density (mg m-3)
Absorption coefficient (mm-1)
F(000)
1000
712
Crystal size (mm)
Theta range for data collection (°)
Limiting indices, h,k,l
0.30 × 0.25 × 0.20 0.35 × 0.30× 0.25
1.92 to 25.00
–12 ≤ h ≤ 12,
–18 ≤ k ≤ 18,
–17≤ l ≤ 18
31264/ 4057
0.0303
1.63 to 28.32
–16 ≤ h ≤ 15,
–14 ≤ k ≤ 14,
–16≤ l ≤ 11
12672 / 4063
0.0248
Reflections collected/ unique
Rint
Data / restraints / parameters
Goodness-of-fit on F2
Final R indices [I > 2σ (I)]
4057 /0 / 280
1.137
4063 / 3 / 207
0.704
R1 = 0.0310, wR2 R1 = 0.0423, wR2 =
= 0.0759 0.1391
R1 = 0.0454, wR2 R1 = 0.0626, wR2 =
R indices (all data)
= 0.0887
Largest difference peak and hole / e Å-3 0.724 and –0.196
0.1768
0.729 and –0.331

Table 2．Selected bond lengths (Å) and bond angles (°) for 1 and 5
1
5
Ni(1) -N(1)
Ni(1) -N(2)
Ni(1) -N(3)
Ni(1) -N(4)
Ni(1) -O(1)
Ni(1) -O(2)
2.302(19) Ni(1)-O(2)#1
2.050(17) Ni(1)-O(2)
2.329(18) Ni(1)-N(2)
2.055(18) Ni(1)-N(2)#1
1.8302(14)
1.8302(14)
1.8971(18)
1.8971(18)
180.00(10)
88.74(7)
2.014(15) O(2)#1-Ni(1)-O(2)
2.010(15) O(2)#1-Ni(1)-N(2)
179.2(6) O(2)-Ni(1)-N(2)
O(2)-Ni(1)-O(1)
O(2)-Ni(1)-N(2)
O(1)-Ni(1)-N(4)
N(2)-Ni(1)-N(4)
O(2)-Ni(1)-N(3)
N(1)-Ni(1)-N(3)
91.26(7)
88.3(7)
O(2)#1-Ni(1)-N(2)#1 91.26(7)
87.67(7) O(2)-Ni(1)-N(2)#1
179.76(8) N(2)-Ni(1)-N(2)#1
87.55(7)
88.74(7)
179.999(1)
179.82(7)

Table 3. Electrochemical data of nickel (II) complexes
Complexes
At cathodic region
At anodic region
Epc
(V)
Epa
(V)
E1/2
ΔE
Epa
Epc
(V)
E1/2
(V)
ΔE
(V) (mV) (V)
(mV)
-0.81 -0.58 -0.70 230
-0.84 -0.61 -0.73 230
-0.79 -0.62 -0.71 170
-0.69 -0.51 -0.60 180
-0.84 -0.67 -0.76 170
+1.06 +0.88 +0.97 180
+1.16 +1.06 +1.11 100
+1.51 +1.34 +1.42 170
+1.14 +1.04 +1.09 100
+1.20 +1.03 +0.98 170
1
2
3
4
5

Table 4. Antimicrobial activities of salen type di-Schiff base nickel (II) complexes 1–5
Zone inhibition diameter (mm)
Complex
Bacteria
Fungi
A. niger C. albicans
Gram-negative
Gram-positive
E. coli P. aeruginosa B. subtilis S. aureus
15
14
12
14
16
18
NT
9
15
15
13
15
15
18
NT
17
16
12
13
14
18
NT
10
12
13
14
14
NT
19
19
12
13
14
16
NT
19
1
2
3
4
5
15
14
13
15
17
NT
Amikacin
Ketokonazole

Figure captions
Figure 1. 30% probability ORTEP diagram of complex 1. Hydrogen atoms are omitted for
clarity.
Figure 2. 30% probability ORTEP diagram of complex 5. Hydrogen atoms are omitted for
clarity.
Figure 3. Cyclic voltammograms of di-Schiff base nickel(II) complexes 1–5 for (a)
Reduction process & (b) Oxidation process
Figure 4. Absorption spectra of nickel(II) complex 5 (10–5 M) in 5 mM Tris–HCl/20 mM
NaCl buffer at pH 7.2 in the absence and presence of increasing amounts of ctDNA Inset:
plot of [DNA]/[εa- εf] vs. [DNA].
Figure 5. Emission spectra of EB bound to ctDNA in the presence of complex 5. The inset
shows the plot of I0/I vs [DNA]/[complex].
Figure 6. Cleavage of SC pBR322 DNA (0.2µg, 33.3 µM) by Ni(II) complexes 1–5(100µM,
200µM) in the presence of the H2O2 (100 µM) in 50 mMTris–HCl/50mM NaCl buffer (pH
7.2). Lane 1, DNA control; lane 2, DNA + H2O2; lanes 3–7, DNA + H2O2 + 1- 5 (100µM),
respectively; lanes 8–12, DNA + H2O2 + 1-5 (200µM), respectively.
Figure 7. Cleavage of SC pBR322 DNA (0.2µg, 33.3 µM) by Ni(II) complexes 1–5 (100µM)
in the presence of H2O2 (100 µM), hydroxyl radical scavengers (DMSO–40 mM, KI–40 mM)
and singlet oxygen scavenger (NaN3–40 mM) in 50 mMTris–HCl/50mM NaCl buffer (pH
7.2). (a) Lane 1, DNA control; lane 2, DNA + H2O2; lane 3, DNA + H2O2 + DMSO; lanes 4–
8, DNA + H2O2 + DMSO + 1- 5 (100µM), respectively; lane 9, DNA + H2O2 + KI; lanes 10-
14, DNA + H2O2 + KI + 1- 5 (100µM), respectively. (b) Lane 1, DNA control; lane 2, DNA
+ H2O2; lane 3, DNA + H2O2 + NaN3; lanes 4–8, DNA + H2O2 + NaN3 + 1- 5 (100µM),
respectively.

Highlights
 di-Schiff base nickel(II) complexes prepared by insitu condensation & complexation
 Complexes presented good binding propensity to ctDNA
 Complex with bulky substituent showed square planar geometry and greater DNA
interaction
 The nickel(II) complexes showed efficient cleavage of plasmid DNA