Synthesis, spectroscopic characterization and in vitro studies of new heteroleptic copper (II) complexes derived from 2-hydroxy napthaldehyde Schiff's bases and N, N donor ligands: Antimicrobial, DNA binding and cytotoxic investigations

Article abstract of DOI:10.1016/j.ica.2015.04.033

A series of eight new copper (II) complexes of types [Cu(HL¹)(B)] (4a-4d) and [Cu(HL²)(B)] (4e-4h), where HL¹ is N-(2-hydroxy-1-naphthalidene)-2-aminophenol, HL² is N-(2-hydroxy-1-naphthylidene)-2-mercaptoaniline and B is N, N donor ligands viz bipyridyl (bpy), 1,10-phenanthroline (phen), dipyridoquinoxaline (dpq) and dipyridophenazine (dppz) have been synthesized and characterized by elemental analysis as well as by spectroscopic techniques (IR, UV, ESI mass and EPR spectra). The DNA-binding properties of these copper complexes (4a-4h) have been investigated by electronic absorption, viscosity measurements and docking analyses. The results obtained indicated that the complexes get bonded with DNA via an intercalation binding mode with an intrinsic binding constant, K_b (1.116 ± 0.21 to 7.227 ± 0.21) × 10⁴ M^-1. The antimicrobial activity assays with the Schiff's bases and their heteroleptic copper (II) complexes of 4a and 4b exhibited profound activity against gram positive bacteria, gram negative bacteria and fungi. Further, complexes 4d and 4h displayed significant cytotoxicity when examined in vitro on a panel of cancerous cell line - human liver cancer cell line - HepG-2 cells (IC₅₀ = 40.82 and 29.74 μg/ml).

Full text of DOI:10.1016/j.ica.2015.04.033

Accepted Manuscript
Synthesis, spectroscopic characterization and in-vitro studies of new heterolep-
tic copper (II) complexes derived from 2-hydroxy napthaldehyde Schiff’s bases
and N, N donor ligands: Antimicrobial, DNA binding and cytotoxic investiga-
tions
Tummalapalli Kiran, Vuppalapati Giri Prasanth, M.M. Balamurali, C.S. Vasavi,
Punnagai Munusami, Kulathu Iyer Sathiyanarayanan, Madhvesh Pathak
PII:
S0020-1693(15)00221-2
http://dx.doi.org/10.1016/j.ica.2015.04.033
ICA 16526
DOI:
Reference:
Inorganica Chimica Acta
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 December 2014
20 April 2015
27 April 2015
Please cite this article as: T. Kiran, V.G. Prasanth, M.M. Balamurali, C.S. Vasavi, P. Munusami, K.I.
Sathiyanarayanan, M. Pathak, Synthesis, spectroscopic characterization and in-vitro studies of new heteroleptic
copper (II) complexes derived from 2-hydroxy napthaldehyde Schiff’s bases and N, N donor ligands: Antimicrobial,
DNA binding and cytotoxic investigations, Inorganica Chimica Acta (2015), doi: http://dx.doi.org/10.1016/j.ica.
2015.04.033
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Synthesis, spectroscopic characterization and in-vitro studies of new
heteroleptic copper (II) complexes derived from 2-hydroxy napthaldehyde
Schiff’s bases and N, N donor ligands: Antimicrobial, DNA binding and
cytotoxic investigations
Tummalapalli Kirana, Vuppalapati Giri Prasantha, Balamurali M M a,C.S.Vasavib, Punnagai
Munusamib, Kulathu Iyer Sathiyanarayanan†a, Madhvesh Pathak*a
a Chemistry Division, School of Advanced Sciences, VIT University, Vellore, Tamilnadu 632014, India
b Bioinformatics Division, School of Bio Sciences and Technology, VIT University, Vellore, Tamil Nadu 632 014,
India.
* Corresponding author. Tel.: +91-416-2202553; E-mail: madhveshpathak@vit.ac.in (M. Pathak)
† Co-corresponding author. Tel.: +91-416-2202081. E-mail: sathiya_kuna@hotmail.com (K. I. Sathiyanarayanan)
ABSTRACT
A series of eight new copper (II) complexes of types [Cu(HL1)(B)] (4a-4d) and
[Cu(HL2)(B)] (4e-4h), where HL1 is N-(2-hydroxy-1-naphthalidene)-2-aminophenol, HL2 is N-
(2-hydroxy-1-naphthylidene)-2-mercaptoaniline and B is N, N donor ligands viz bipyridyl (bpy),
1,10- phenanthroline (phen), dipyridoquinoxaline (dpq) and dipyridophenazine (dppz) have been
synthesized and characterized by elemental analysis as well as by spectroscopic techniques (IR,
UV, ESI mass and EPR spectra). The DNA-binding properties of these copper complexes (4a-
4h) have been investigated by electronic absorption, viscosity measurements and docking
analyses. The results obtained indicated that the complexes get bonded with DNA via an
intercalation binding mode with an intrinsic binding constant, Kb (1.116 0.21 to 7.227 0.21)
×104 M-1. The antimicrobial activity assays with the Schiff’s bases and their heteroleptic copper
(II) complexes of 4a and 4b exhibited profound activity against gram positive bacteria, gram
negative bacteria and fungi. Further, complexes 4d and 4h displayed significant cytotoxicity
when examined in-vitro on a panel of cancerous cell line - human liver cancer cell line - HepG-2
cells (IC50= 40.82 and 29.74 µg/ml).
Key words: Copper (II) complexes, DNA binding, DNA cleavage, Molecular docking Studies,
Antimicrobial and Cytotoxicity.
1

1. Introduction
The role of transition metal complexes in biological and medical sciences has been well
established during the past few decades. The emergence of metallo-therapeutics has been made
possible through the recent achievements in bioinorganic chemistry. Though the discovery of
cis-platin and other platinum containing drugs resulted in coining the terminology of elemental
medicines, it exhibits high toxicity to normal cells which leads to undesirable side-effects.
Moreover, majority of these medicines are inactive against many cancer cell lines and metastasis
(secondary) cancers [1]. In order to overcome these challenges, considerable attempts have been
made to substitute the cis-platin with suitable metal (copper, zinc, nickel and vanadium) based
drugs. Among the transition metals, copper (II) is the third most bio-essential ion due to its labile
nature towards coordinating the ligands [2] proceeding iron and zinc. It also plays a pivotal role
in chemotherapy as it accumulates in tumor cells for its selective permeability through cancer
cell membranes [3]. At present, DNA-binding properties of copper (II) complexes with small
molecules have been in focus for researchers [4] round the world for two reasons. (i) DNA has
been the most preferred target for most of the biological activities serving anti-cancer and anti-
viral therapies [5], (ii) the differential binding mode between transition metal complexes and
DNA have been the scaffold for bioinorganic medicinal chemistry. Especially, copper (II)
complexes with Schiff’s base ligands have been extensively studied because of their strong
interactions with DNA via surface associations or intercalation [6] and DNA cleavage abilities
through hydrolytic or oxidative mechanisms [7]. Due to appreciable chelating and stable
complex forming behavior (in their neutral or deprotonated forms), Schiff’s base ligands have
been at focus for many years [8]. According to various reports, a wide range of biological
activities of Schiff’s base-copper complexes such as antibacterial [9, 10], antifungal [11, 12],
antioxidant [13] and anticancer [14, 15] have made them to exhibit themselves as efficient
biological agents. In recent years, heteroleptic derivatives of copper with Schiff’s base and
ancillary ligands have expedited the idea of potential antimicrobial agents compared to mono-
ligand system [16]. In view of their DNA binding behavior, ancillary ligands such as bipyridine,
phenanthroline, dipyridoquinoxaline and dipyridophenazine provide planarity to metal
complexes, thereby strengthening their binding ability to DNA. Planarity also adds to the
intercalation behavior of the complexes and in turn increases the binding potential. Hence,
2

complexes with N, N donor phenanthroline have drawn much attention as they exhibit high
nucleolytic efficiency [17].
Thus, encouraged by the above-said advancements and in addition to the availability of a
few analogous complexes in the literature [18, 19] as well as to fill some gaps left, herein we
report our newly synthesized heteroleptic complexes of copper (II) derived from tri-dentate
Schiff’s bases and other ancillary ligands together with their characterization by employing
techniques IR, UV-Visible, ESI-mass and EPR spectroscopy. The bioactivity of these complexes
was assayed on gram positive, gram negative and fungal strains. Further, intercalation of the
copper (II) complexes with the DNA was also studied to get additional features of their binding
modes. In addition, cytotoxicity of the complexes was also examined in-vitro on a panel of
cancerous cell lines - HepG2 to elaborate their potential activity of concerns.
2. Experimental
2.1 Materials
All the chemicals were purchased from Sigma Aldrich and SDFCL and used without
further purification. Thin layer chromatography (TLC) was performed on preparative plates of
silica gel. Visualization was carried out with UV illuminated chamber. Melting points were
obtained using microprocessor digital melting point apparatus and were corrected with standard
benzoic acid. IR spectra were recorded on Shimadzu IR affinity-1 in the range of 4000-400 cm-1
using KBr pellets. UV-Vis spectra were obtained from Jasco V670. The NMR spectra were
recorded on a Bruker Avance III- 400 MHz spectrometer with Trimethylsilane (TMS) as internal
standard. Elemental analyses of the metal complexes were recorded on Elementar Vario EL III
instrument. The molecular weights of the metal complexes were obtained from Electron Spray
Ionization (ESI) mass spectroscopy (Agilent-1100, PE SCI EX API 3000). The viability of cells
was assessed by MTT assay [20] using HepG2 cell lines.
2.2 Synthesis of Schiff bases
To a methanolic solution of 2-hydroxy-napthaldehyde 1 (1.72g, 10 mmol), 2-amino
phenol 2a (1.09g, 10 mmol) / 2-amino thiophenol 2b (0.91mL, 10 mmol) and catalytic amount of
glacial acetic acid were added and the reaction mixture was allowed to stir at room temperature
for 4h. The completion of the reaction was monitored by TLC. Afterwards, the reaction mixture
3

was filtered and washed with cold methanol. Products 3a and 3b were recrystallized with hot
methanol (Scheme 1).
Scheme 1: Synthesis of Schiff’s base ligands (HL1 and HL2).
HL1 Yield: 82%, yellow solid, m.p. = 249-251°C. IR (KBr cm−1): 3446 br (O–H), 1622 (C= N).
UV–vis [(λmax(nm), ε (L mol−1 cm−1)]: 472 (26702), 449 (26852), 326 (15392), 262 (27360). 1H
NMR (DMSO-d6, 400 MHz): δ (ppm): 6.80-6.77 (d, J=9.6Hz, 1H), 7.01-6.93 (m, 2H), 7.13-7.09
(p, J=15.6, 6.4 Hz, 1H), 7.28-7.25 (t, J=14.4Hz, 1H), 7.50-7.46 (p, J=15.6, 7.6Hz, 1H), 7.69-7.67
(d, J=8Hz, 1H), 7.82-7.79 (d, J=12Hz, 1H), 7.94-7.92 (t, J=8 Hz, 1H), 8.39-8.37 (d, J=8Hz, 1H),
13
9.50-9.48 (d, J=9.6 Hz, 1H), 10.38 (s, OH), 15.72-15.69 (d, J=9.6Hz, OH). C NMR (DMSO-
d6, 100MHz): δ (ppm): 107.63, 115.90 , 117.51, 119.67, 119.80, 123.02, 125.08, 125.79, 126.72,
128.09, 128.47, 128.96, 133.88, 137.94, 148.36, 149.33, 177.68.
HL2 Yield: 78%, yellow solid, m.p. = 142-144 °C. IR (KBr cm−1): 2551 w (SH), 1618 (C= N).
1
UV–vis [(λmax(nm), ε (L mol−1 cm−1)]: 393 (16700), 326 (13072), 262 (24160). H NMR
(DMSO-d6, 400MHz): δ (ppm): 7.18-7.15 (d, J=9.2Hz, 1H), 7.31-7.27 (t, J=15.2Hz, 1H), 7.44-
7.38 (dd, J=16, 8Hz, 3H), 7.64-7.58 (dd, J=16.8, 8Hz, 2H), 7.82-7.80 (d, J=7.6, 1H), 7.90-7.88
(d, J=8Hz, 1H), 8.05-8.03 (d, J=9.2Hz, 1H), 8.6 2-8.60 (d, J=8.8 Hz, 1H), 9.76 (s, SH), 15.04 (s,
OH). 13C NMR (DMSO-d6, 100MHz): δ (ppm): 109.51, 119.21, 120.09, 120.90, 123.83, 127.28,
127.51, 127.96, 128.15, 128.86, 129.01, 129.42, 132.69, 136.31, 145.27, 158.81, 165.0.
2.3 General procedure for the Synthesis of Complexes
Synthetic route for these new copper (II) complexes was employed as reported [21]. A
methanolic (10 ml) solution of copper acetate monohydrate (0.2 g, 1.0 mmol) along with N, N
donor ligands (1.0 mmol) was stirred continuously for 30 mins followed by the addition of
4

methanolic solution of Schiff’s base 3a-3b (0.263 g, 1.0 mmol) and the stirring was further
continued for next 1h. A brown solid started settling in the reaction mixture. This was filtered
and washed with cold methanol followed by diethyl ether and was then dried under vacuum
(Scheme 2).
Scheme 2: Synthesis of heteroleptic copper (II) complexes (4a-4h).
[Cu (HL1) (bpy)] (4a) Yield: 80%, Colour: brown solid, m.p: 243-245 °C, Anal Calcd. For
C27H19CuN3O2: C, 67.42; H, 3.98; N, 8.74; O, 6.65. Found: C, 67.65; H, 3.86; N, 8.68; O,
6.76.IR (KBr cm−1): 3041 w (CHAr), 1579 m (C=N), 1394 w (C-N), 567 w (Cu-N), 426 w (Cu-
O) cm−1 (br, broad; s, strong; m, medium; w, weak). UV–vis [(λmax (nm), ε (L mol−1 cm−1)]: 472
(11115), 443 (13300), 296 (25975), 243 (29660). ESI-MS: 481.0 (M+1), 482.2 (M+2).
Kb=1.116× 104 M¯1.
[Cu (HL1) (phen)] (4b) Yield: 78%, brown solid, m.p. = 269-271 °C. Anal Calcd. For
C29H19CuN3O2: C, 68.91; H, 3.79; N, 8.32; O, 6.34. Found: C, 68.83; H, 3.67; N, 8.25; O, 6.46.
IR (KBr cm−1): 3014 w (CHAr), 1581 m (C=N), 1404 w (C-N), 565 w (Cu-N), 428 w (Cu-O)
cm−1. UV–vis [(λmax (nm), ε (L mol−1 cm−1)]: 445 (17460), 270 (81294), 237 (53400). ESI-MS:
505.3 (M+1), 506.2 (M+2). Kb=1.271× 104 M¯1.
[Cu (HL1) (dpq)] (4c) Yield: 75%, brown solid, m.p. = 248-250 °C. Anal Calcd. For
C31H19CuN5O2: C, 66.84; H, 3.44; N, 12.57; O, 5.74. Found: C, 66.93; H, 3.31; N, 12.63; O,
5.81. IR (KBr cm−1): 3041 w (CHAr), 1579 m (C=N), 1388 s (C-N), 565 w (Cu-N), 428 w (Cu-O)
5

cm−1. UV–vis [(λmax (nm), ε (L mol−1 cm−1)]: 460 (14985), 260 (39340). ESI-MS: 557.3 (M+1).
Kb=2.108× 104 M¯1.
[Cu (HL1) (dppz)] (4d) Yield: 70%, brown solid, m.p. = 272-274 °C. Anal Calcd. For
C35H21CuN5O2: C, 69.24; H, 3.49; N, 11.54; O, 5.27. Found: C, 69.07; H, 3.37; N, 11.43; O,
5.34. IR (KBr cm−1): 3053 w (CHAr), 1581 m (C=N), 1392 w (C-N), 563 w (Cu-N), 430 w (Cu-
O) cm−1. UV–vis [(λmax (nm), ε (L mol−1 cm−1)]: 470 (11295), 276 (22835). ESI-MS: 607.4
(M+1), 608.3(M+2). Kb=5.343× 104 M¯1.
[Cu (HL2) (bpy)] (4e) Yield: 82%, brown solid, m.p. = 236-238 °C. Anal Calcd. For
C27H19CuN3OS: C, 65.24; H, 3.85; N, 8.45; O, 3.22; S, 6.45. Found: C, 65.32; H, 3.76; N, 8.37;
O, 3.31; S, 6.34. IR (KBr cm−1): 3053 w (CHAr), 1537 m (C=N), 1365 w (C-N), 530 w (Cu-N),
418 w (Cu-O) cm−1. UV–vis [(λmax (nm), ε (L mol−1 cm−1)]: 426 (10440), 240 (21590). ESI-MS:
497.4 (M+1). Kb=1.485× 104 M¯1.
[Cu (HL2) (phen)] (4f) Yield: 79%, brown solid, m.p. = 245-247 °C. Anal Calcd. For
C29H19CuN3OS: C, 66.84; H, 3.68; N, 8.06; O, 3.07; S, 6.15. Found: C, 66.91; H, 3.59; N, 7.96;
O, 3.16; S, 6.21. IR (KBr cm−1): 3051 w (CHAr), 1535 w, 1365 w, 534 w (Cu-N), 422 w (Cu-O)
cm−1. UV–vis [(λmax (nm), ε (L mol−1 cm−1)]: 472 (11115), 443 (13300), 296 (25975), 243
(29660). ESI-MS: 521.3 (M+1). Kb=1.558× 104 M¯1.
[Cu (HL2) (dpq)] (4g) Yield: 76%, brown solid, m.p. = 221-223 °C. Anal Calcd. For
C31H19CuN5OS: C, 64.97; H, 3.34; N, 12.22; O, 2.79; S, 5.59. Found: C, 65.06; H, 3.23; N,
12.10; O, 2.86; S, 5.43. IR (KBr cm−1): 3053 w (CHAr), 1537 m, 1367 m, 563 w (Cu-N), 418 w
(Cu-O) cm−1. UV–vis [(λmax (nm), ε (L mol−1 cm−1)]: 447 (4725), 376 (8840), 360 (9180), 274
(35715). ESI-MS: 573.4 (M+1), 574.4 (M+2). Kb=4.277× 104 M¯1.
[Cu (HL2) (dppz)] (4h) Yield: 73%, brown solid, m.p. = 229-231 °C. Anal Calcd. For
C35H21CuN5OS: C, 67.46; H, 3.40; N, 11.24; O, 2.57; S, 5.15. Found: C, 67.39; H, 3.29; N,
11.15; O, 2.69; S, 5.23. IR (KBr cm−1): 3051 w (CHAr), 1537 w, 1361 w, 561 w (Cu-N), 418 w
(Cu-O) cm−1. UV–vis [(λmax (nm), ε (L mol−1 cm−1)]: 435 (2915), 272 (13720), 244 (15470). ESI-
MS: 623.1 (M+1), 624.1(M+2). Kb=7.227× 104 M¯1.
6

2.4 DNA Binding and chemical nuclease activity Studies
DNA binding property of the synthesized metal complexes were carried out by UV
absorption spectroscopy using calf thymus DNA in 5 mM of Tris-HCl buffer (pH 7.2). 260:280
absorbance ratio of ca. 1.9:1 indicated the absence of any protein impurities in the DNA sample
[22]. The concentration of DNA was determined using molar absorption co-efficient (6600 M-
1cm-1) at 260 nm [23]. Stock solution of the metal complexes was prepared by dissolving in 5
mM Tris-HCl, pH 7.2 and 10% DMSO (Dimethyl Sulfoxide) and further dilutions were done in
the same buffer without DMSO. The effect on the electronic absorption spectrum of various
metal complexes was monitored by titrating against different concentrations of CT-DNA (2 – 26
µM). For the above titration, buffer containing an equivalent amount of CT-DNA was used as
blank. The binding strength of the synthesized complexes with CT-DNA were compared and
calculated by using the equation
[DNA]/ (εa- εf) = [DNA]/ (εb- εf) +1/ Kb (εb- εf)
Equation 1
where [DNA], is the concentration of the DNA, the apparent absorption coefficients εa, εf and εb
correspond to Aobsd / [complex], extinction coefficient for complex in free form and extinction
coefficient for complex in bounded form, respectively.
The DNA binding was probed with the help of viscosity measurements using Schott
Gerate AVS 310 Automated Viscometer at 37 ( 0.1) °C, in a water bath. The concentration of
CT-DNA was maintained at 100 µM in nucleotide pairs (NP) and the flow time of the same was
calculated in the absence and in the presence of complexes (20-140 µM) with the help of a
digital stop watch. All the experiments were carried out in triplicates and the average flow time
was calculated for both the CT-DNA and complex bound CT-DNA. Data was presented as
(η/η0)1/3 vs [complex]/ [DNA] where η is the viscosity of DNA bound metal complex and η0 is
the viscosity of DNA alone.
The DNA cleaving efficiency of metal complexes (4a-4h) was determined by agarose gel
electrophoresis. The experiment was performed with super coiled pBR322 DNA (500 ng) mixed
with complexes (5% DMF in 10 mM Tris buffer pH 7.2, containing 50 mM NaCl,) and H2O2 as
oxidizing agent. The samples were incubated in the dark for 1h at 37 °C, followed by the
addition of loading dye (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol, 10mM
7

of EDTA). The samples were loaded and analyzed by 1% slab gel containing 1µg/ml of ethidium
bromide in TBE (Tris-boric acid ethylenediaminetetraacedicacid) buffer. The gel was viewed on
an illuminator and photographed for analyses. The chemical nuclease activity was measured by
determining the ability of the complexes to convert the super coiled (SC) DNA to the nicked
circular (NC) form.
2.5 Molecular docking studies
Docking calculations were performed with Auto Dock 4.2. Grid potential maps between
the DNA and various atom types were calculated using Auto Grid program. A stochastic
Lamarckian genetic algorithm method was used for docking calculations with default
parameters. Auto Dock uses AMBER force field to evaluate conformations during docking
calculations. The binding energy was evaluated using the following scoring function.
∆ꢀ= ∆ꢀꢁꢂꢃ +ꢄ∆ꢀꢅꢆꢇꢈꢂ + ∆ꢀꢉꢊꢉꢋ +ꢄ∆ꢀꢌꢇꢍ + ∆ꢀꢂꢉꢎꢇꢊꢁ
First three terms are van der Waal’s, hydrogen bonding, and electrostatics, respectively.
The term ∆ꢀꢌꢇꢍis for rotation and translation and ∆ꢀꢂꢉꢎꢇꢊꢁis for desolvation upon binding and the
hydrophobic effect.
This study will aid in understanding the binding as well as the orientation of metal
complexes with DNA [24, 25]. The X-ray structure of DNA (Pdb id: 355D) obtained from
Brookhaven Protein Data Bank was considered for docking studies [26]. The crystal structure
was refined by removing the water molecules, spermine molecule and magnesium ion. Hydrogen
atoms and Gasteiger-Marsili charges [27] were added using AutoDockTools-1.5.6 [28]. The 3D
coordinates of the synthesized compounds 4a-4h was constructed and optimized using molecular
mechanics.
2.6 Cyclic voltammetry
In order to determine the redox behavior of metal complexes cyclic voltammetry (CV)
was performed using a CHI 660C electrochemical work station (TX, USA) with three electrode
cell consisting of a glassy carbon electrode as a working electrode, silver/silver nitrate as a
reference electrode and platinum wire as a counter electrode. Electrochemical measurements
8

were performed in acetonitrile solution with 0.1 M tetra butyl ammonium hexa fluorophosphate
at room temperature (25 2°C).
2.7 Biological Studies
2.7.1 Antimicrobial Assay
Antimicrobial activities of the target complexes and ligands were evaluated by Agar well
diffusion method. The anti-bacterial study was carried out against various gram positive
(Bacillus Subtilis, Staphylococcus Aureus), gram negative (Pseudomonas aeruginosa, Klebsilla
pneumoniae) and fungal (Candida albicans, Penicillium chrysogenum) strains. Mueller Hinton
Agar and potato dextrose agar were used as media for anti-bacterial and anti-fungal studies
respectively. Inoculum suspension was streaked uniformly by using glass spreader and allowed
to dry at room temperature for 3-5 minutes under sterile conditions. Agar-borer of 8 mm
diameter was preheated and pressed on the inoculated agar plate in order to make 5 wells per
plate. To each well, 100 µg of each complex or ligand solution in a volume of 100 µL was added
with the help of micropipette. After addition, the plates were incubated at 37°C for 24 h.
Ampicillin and ketoconazole were used as a positive control respectively for bacterial and fungal
strains and neat DMSO solvent was used as a negative control. The experiments were done in
duplicates and also supported with docking analyses.
2.7.2 Evaluation of cytotoxic activity cell culture
The human liver carcinoma cell line, HepG-2 was obtained from the American Type
Culture Collection (ATCC HB-8065), USA. The cells were cultured in a sterile T75 flask
containing DMEM medium supplemented with fetal bovine serum (10% v/v) at 37 ºC in an
incubator with humidified atmosphere of 95% air and 5% CO2.
The compound was dissolved in 5 mM Tris-HCl buffer, pH 7.2 containing 10% DMSO.
The final DMSO concentration was maintained below 0.5% for cell survival. The HepG-2 cells
were plated separately in 96 well plates at a concentration of 1x105 cells/well. After 24 h, the
cells were washed twice with 100 µl of serum-free medium and starved for an hour at 37°C.
After starvation, the cells were treated with different concentrations of complexes (10-500
µg/ml) for 24 h. At the end of the treatment period, the medium was aspirated and serum free
9

medium containing MTT (0.5 mg/ml) was added and incubated for 4 h at 37 ºC in a CO2
incubator. The MTT containing medium was then discarded and the cells were washed with PBS
(200 µl). Only the live cells can take in MTT and convert it in to formazan crystals. The cells
were then lysed by adding DMSO which further dissolved formazan and imparted purple blue
colour to the buffer solution. This conversion was monitored by change in absorbance at 579 nm
in a microtitre plate reader (Biorad 680). Cytotoxicity was determined using Microsoft excel.
The percentage of cell viability was calculated using the formula
%viability = {OD of the experiment samples/OD of the control} × 100
3. Results and discussion
3.1 Spectral characterization of Copper (II) complexes and ligands
The FT-IR spectra of the ligands HL1 and HL2exhibit a broader absorption band at 3446
cm-1 and a weak stretching frequency at 2551 cm-1 due to free –OH and –SH groups,
respectively. Interestingly, in HL2 the appearance of weak stretching peaks at 3637 cm-1 is an
indication of intra-molecular H-bonding between –OH and azine nitrogen. Similarly, the FT-IR
spectra of the copper (II) complexes (4a-4d) have been compared with that of free ligand (HL1)
to locate their coordination sites for proper chelation. The shift in the absorption band from 1622
cm-1 (C=N) of free ligand (HL1) to 1606 cm-1 for the complexes reveals the formation of the co-
ordination bond between copper (II) ion and nitrogen atom of azine group. On the other hand,
the disappearance of –OH band of free ligand (HL1) from 3400 cm-1 and the appearance of a
peak at 428 cm-1 (Cu-O) reveals the successful formation of complexes. Similarly, the IR spectra
of the free ligand (HL2) and the complexes (4e-4h) were closely compared and corresponding
observations made.
UV-visible spectra of these new copper (II) complexes were recorded in the range 200-
800 nm. Intra ligand transitions (π-π*, n-π*) were observed at 230 nm and 335 nm as sharp peaks
in complex 4a due to the ligand. Ancillary ligand e.g. bipyridyl showed characteristic intra
ligand transition at 283 nm. Complexation of copper (II) ion to bipyridyl led to a blue shift in UV
region to 274 nm in 4a. The weaker d-d transitions were observed as a broad peak in visible
region from 400-800 nm. The broad peak due to ligand metal charge transfer (LMCT) was
10

noticed in the range of 402-488 nm of complexes 4a-4h. The relevant λmax values of the
remaining complexes, along-with HL1, HL2 and ancillary ligands, are tabulated in Table 1.
Table 1 The UV–vis absorption bands (λmax) of the ligands and its metal (II) complexes.
π-π*, n-π* Intra ligand
Compound
LMCT (nm)
Transition (nm)
225, 262, 326, 472
221, 262, 326, 393, 472
283
HL1
HL2
Bpy
Phen
DPQ
DPPZ
4a
-
-
-
265
-
231, 261, 342
269, 363, 381
230, 274, 335
231, 226, 335
230, 261, 335
227, 271, 338
226, 260, 332
225, 261, 341
231, 261, 341
231, 271, 344
-
-
457
488
402
422
458
425
458
427
4b
4c
4d
4e
4f
4g
4h
ESI mass spectroscopic data of the complexes 4a-4h confirm the complex formation and
their corresponding data have been summarized in Table 2. The mass spectrum of copper (II)
derivative 4a with theoretical molecular weight 480.08 reveals the molecular ion peak M+1 at
481.2 and M+2 ion peak at 482.2 which confirms its existence in mono nuclear state (Figure 1).
Similarly, both M+1 and M+2 molecular ion peaks were observed for the complexes 4b, 4d, 4g
and 4h as illustrated in Table 2 (Fig. S9-15 in supplementary information). However, the
11

complexes 4c, 4e and 4f were found to exist only in M+1 ion form. The ESI mass spectral data
of all the eight new heteroleptic copper (II) complexes are in good agreement with their proposed
monomeric structure.
Table 2 The ESI mass spectral data of copper(II) complexes.
Observed Molecular Weight
Theoretical Molecular
Weight
S. No.
Complex
M+1
M+2
1
2
3
4
5
6
7
8
480.08
504.08
556.08
606.10
496.05
520.05
572.06
622.08
481.0
505.3
557.3
607.4
497.4
521.3
573.4
623.1
482.2
506.2
-
608.3
-
4a
4b
4c
4d
4e
4f
-
574.4
624.1
4g
4h
12

Fig. 1. ESI mass spectra of Complex 4a.
The X-band EPR spectra (Fig. S16-23. in supplementary information) of copper (II)
complexes were recorded in the solid state at room temperature (298K) by employing micro
wave frequency of about 9.4 GHz. The results indicate that all the complexes show broad
2
component system with dx -y2 ground state of mononuclear nature of copper(II) complex [29].
The calculated g values are in the range of 2.070 - 2.071 for all the complexes 4a – 4h which is
similar to the expected values for penta coordinated geometry of copper(II) complexes [30].
3.2 Binding constants reveal the affinity for Calf Thymus-DNA by the complexes
UV spectral titration and docking studies were performed to determine the binding mode
of the complexes 4a-4h with calf-thymus DNA. UV absorption spectrum of complexes 4a-4h at
20 µM concentration, in the absence and presence of CT-DNA, is shown in the (Fig. 2). Binding
13

of metal complex to CT-DNA induces a shift in the metal-ligand centered band of the UV
spectrum. Absorption bands of the complexes were affected with increasing concentrations of
DNA (2-26 µM). The hypochromism observed in the spectral band could be due to the
intercalation of metal complexes with the DNA. The value of Kb was calculated from the plot of
[DNA] against [DNA]/ (εa - εf) (Equation 1). The binding constants Kb of the complexes were
determined to be (1.116 0.21 to 7.227 0.21) ×104 M-1. These values of binding constants
suggest that complex 4h has stronger binding affinity for calf thymus DNA than other
complexes.
Fig. 2. Absorption spectra of complex 4a upon addition of CT-DNA, Inset: plots of [DNA]/ (εa-
εf) vs. [DNA].
In order to determine the DNA binding mode of the synthesized metal complexes,
viscosity studies were carried out in the absence and in the presence of CT-DNA and at different
concentration values of the complexes. The plot of relative specific viscosity (η/η0)1/3 vs
([complex]/ [DNA]) displayed a moderate change in the viscosity for complexes 4d and 4h
indicating an intercalation mode of DNA binding (Fig.3). The observation of minor changes for
the other complexes (4a-4c, 4e-4g) indicates the possibility of groove binding or partial
intercalation of the complexes with the DNA. To summarize, bpy, phen and dpq containing
14

complexes exhibits groove binding while dppz complexes show partial DNA intercalative
binding. The order of binding as revealed from the plot is depicted as 4d, 4h > 4c, 4g >4b, 4f
>4a, 4e.
Fig. 3. Effect of complexes (4a-4h) on the viscosity of CT-DNA. Relative specific viscosity
versus [complex]/ [DNA].
Docking calculations were performed by considering both major groove and minor
groove as a binding site for the metal complexes. It was observed that the compounds prefer to
bind to the major groove of double stranded DNA than the minor groove. This is due to the steric
clashes between the copper atom and the backbone atoms of DNA strand. The binding (∆GBE)
and intermolecular energies (∆Gintermol) of the compounds 4a-4h are given Table 3. The best
docked conformation of 4d and 4h obtained by docking calculations is shown in Fig.4. The
docked compounds 4a-4h showed binding energy in the range of −9.0 to −12.0 kcal/mol. The
compounds 4d and 4h exhibit high binding energies of −12.0 and −11.7 kcal/mol, respectively.
This could be due to strong contribution of intermolecular van der Waals, hydrogen bonding, and
desolvation energies. The high binding affinity of complexes 4d and 4h are in agreement with
the experimental results.
15

Table 3 The binding (∆GBE) and intermolecular energies (∆Gintermol) of the compounds 4a-4h
with DNA are given below. All the energies are reported in kcal/mol.
∆Gintermol
Compounds
∆GBE
∆Gvdw_hb_desol
−10.15
−9.77
∆Gelec
−0.05
0.02
−10.20
−9.75
4a
4b
4c
4d
4e
4f
−10.81
−12.03
−9.45
−10.75
−11.99
−9.49
−0.06
−0.04
0.04
−9.99
−9.96
−0.03
−0.07
−0.02
−10.71
−11.68
−10.64
−11.66
4g
4h
(a)
(b)
Fig. 4. Docked conformation of the compounds (a) 4d and (b) 4h with crystal structure of DNA
(Pdb id: 355D) is shown. The compounds are represented in ball and stick model. The maroon
dashed line represents the hydrogen bonds between the metal complex and DNA.
16

The ability of the complexes (4a -4h) to cleave the pBR322 DNA has been tested. In all
the cases, potent cleavage activity was observed in the presence of hydrogen peroxide (H2O2).
The efficiency was determined from band intensities in the agarose gel and by comparing with
the control sample [31, 32]. Complexes 4d and 4h exhibit high cleavage efficiency compared to
other complexes. Such complexes pave way for the development of efficient drugs that can
target nucleic acids. Gel electrophoresis study clearly indicates that complexes show potent
cleavage activity in the presence of hydrogen peroxide (H2O2) (Fig.5a and 5b).
Fig. 5a and 5b. Gel electrophoresis diagram showing the chemical nuclease activity of the
complexes in the presence of H2O2.5a: (4a-4d),Lane 1: DNA; lane 2: DNA + H2O2; lane 3: DNA
+ 4a+ H2O2; lane 4: DNA + 4b+ H2O2; lane 5: DNA + 4c+ H2O2; lane 6: DNA + 4d+ H2O2,
5b:(4e-4h), Lane 1: DNA; lane 2: DNA + H2O2; lane 3: DNA +4e+ H2O2; lane 4: DNA + 4f+
H2O2; lane 5: DNA + 4g+ H2O2; lane 6: DNA + 4h+ H2O2.
Nuclease activity of the synthesized copper complexes was checked in pBR322 plasmid
DNA. Complexes 4d and 4h exhibited its ability to cleave the DNA as revealed by agarose gel
electrophoresis (Fig.5c). The complexes could exhibit nuclease activity only in the presence of
hydrogen peroxide, indicating that the cleavage is induced either by hydroxyl or singlet oxygen
radicals [33-35]. In order to identify the responsible radical, cleavage experiment was carried out
in the presence of hydroxyl radical scavenger (DMSO) as well as by applying singlet oxygen
radical scavenger (NaN3) too. As appeared in Fig.5c, the activity was inhibited in the presence of
DMSO, revealing that the active radical involved in the cleavage activity is hydroxyl radical. It
was evident that the presence of azide did not have any effect in the cleavage process of DNA.
The possible mechanism behind this activity could be the oxidation of copper complexes in the
presence of H2O2 that could further be involved in the process of DNA cleavage.
17

Fig. 5c. Agarose gel electrophoresis depicting the nuclease activity of copper complexes
4d (top) and 4h (bottom) in pBR322 plasmid DNA. Lane 1: DNA alone, lane 2: DNA in the
presence of copper complex, lane 3: DNA in the presence of copper complex and H2O2 without
any radical scavengers, lane 4: DNA in the presence of copper complex and H2O2 with DMSO as
hydroxyl radical scavenger and lane 5: DNA in the presence of copper complex and H2O2 with
sodium azide as singlet oxygen radical scavenger.
3.3 Redox behaviour of complexes
Cyclic voltammetry (CV) studies of complexes 4a-4h were carried out with a glassy
carbon electrode at a scan rate of 0.05V/s in dimethyl formamide (DMF) solution containing 0.1
M tetra butyl ammonium hexa fluorophosphate as a supporting electrolyte. The results obtained
through CV confirmed the complexes are redox active and showed a quasireversible CV
response at 0.247 and -0.064 V vs. Ag/AgNO3 for 4f complex (Fig.6). The redox peaks can be
assigned to CuII /CuI couple and the redox potential of the complexes was accessible for
nucleobases. [36, 37]
18

Fig. 6. Cyclic voltammogram of complex 4f in DMF (0.1 M nBu4N PF6) on a GC working
electrode. Scan rate: 0.05 V s-1.
3.4 Complexes inhibit microbial growth – Antimicrobial activity
The parent ligands and the metal complexes were tested for inhibiting the growth of
Gram positive (Bacillus Subtilis and Staphylococcus Aureus), Gram negative bacterial
(Pseudomonas aeruginosa and Klebsilla Pneumoniae) and fungal (Candida albicans and
Penicillium chrysogenum) strains. The anti-bacterial and anti-fungal results are represented in
Table 4. Comparing the activities of the ligands and complexes with standard drugs, it was found
that all the complexes exhibit better inhibition against B. subtilis and the complexes 4a, 4b and
4c show better inhibition only for K. pneumoniae. In case of anti-fungal activity, complexes 4a
and 4d show good activity against C. albicans and compound 4a exhibits good activity against
P. chrysogenum (Fig. 7 and 8). The enhanced growth inhibition activity of the synthesized metal
complexes when compared to their ligands might be due to their chelating ability. The redox
behavior of the copper ion in the complexes might also add to the higher anti-microbial activity.
The results from these studies could pave the way for the development of next generation
antibiotics for therapeutic use.
19

Fig. 7. Comparison of antibacterial activity for ligands (HL1 and HL2) and their metal (II)
Complexes 4a-4h.
Fig. 8. Comparison of antifungal activity of ligands (HL1 and HL2) and their metal (II)
Complexes 4a-4h.
20

Table 4 Zone of inhibition (%) of antibacterial and antifungal activity for ligands and their metal
(II) complexes [zone of inhibition (mm)].
Zone of inhibition (mm)
Compound
Bacteria
Fungi
B.subtilis
S.aureus
P.aeruginosa
K.pneumoniae
C.albicans
P.chysogenum
20
11
13
14
12
7
19
13
11
09
7
4a
4b
24
15
15
16
13
14
16
13
11
25
NT
-
15
9
21
17
11
13
9
11
9
4c
9
10
12
10
9
8
4d
11
8
9
6
4e
11
-
8
4f
-
-
7
4g
-
-
-
8
6
4h
HL1
HL2
Amp
Ketoc
DMSO
14
10
29
NT
-
12
13
28
NT
-
11
14
26
NT
-
9
8
11
NT
15
-
7
NT
12
-
Amp-Ampicilin, Ketoc-Ketoconazole, NT-Not Tested
21

3.5 Complexes influence the cell viability - Cytotoxic activity
The viability of HepG-2 cells was tested after 24h by MTT assay. Different
concentrations of dppz ligand containing 4d and 4h, complexes were used as representatives
since they are reported to be efficient DNA cleavers [38]. The results of both the complexes,
summarized in (Fig.9), show a dose-dependent activity towards the HepG-2 cell line. The results
are illustrated as percentage viability and represented as mean standard deviation (n= 2). It can
be concluded that 4d and 4h display promising cytotoxic activity towards HepG-2 cells with IC50
values of 40.82µg/ml and 29.74µg/ml, respectively. Among the two, 4h showed better
cytotoxicity compared to 4d.
120
4d
100
4h
80
60
40
20
0
0
10
50
100
200
500
Concentration in µg/ml
Fig. 9. Comparison of cytotoxicity (MTT assay) for complex 4d and 4h against human liver
carcinoma cell line (HepG-2).
4. Conclusion
On the basis of the above discussion for characterization of heteroleptic copper (II)
complexes incorporated with Schiff’s base and ancillary ligands in appropriate stoichiometry,
mononuclear molecules were found with penta-coordination around copper (II) ion. The free
ligands and their copper (II) complexes were screened for antimicrobial, cytotoxic and DNA
absorption studies. Out of eight synthesized molecules, 4d and 4h showed synergistic DNA
binding propensity when compared with other derivatives of the series. The molecular docking
studies are in agreement with the experimental observations. Further, all these complexes
exhibited good antimicrobial activity due to synergetic effect of copper (II) ion. In addition, the
22

in-vitro cytotoxic assay carried out on HepG-2 cell lines with complexes 4d and 4h exhibited
pronounced activity. Eventually, it is concluded that the outcome of the present study would
contribute to the chemistry of Cu (II) complexes and their biological activities.
Acknowledgement
The authors wish to thank VIT University, Vellore, India for providing necessary
infrastructure facilities and financial support. We extend our hearty thankfulness to Prof. A.
Senthil Kumar, SAS, VIT Vellore for carrying out cyclic voltammetry of the samples. One of the
authors (T.K.) is grateful to VIT University for awarding research associate-ship.
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24

Eight novel metallacyclic derivatives of copper (II) have been synthesized and structurally
characterized as a pent-coordination around central metal. Significant biological applications e.g.
DNA binding, DNA cleavage, antimicrobial, molecular docking and cytotoxicity of these
complexes were found remarkably well.
25

26

ꢀ Synthesis of new metallo organic derivatives of copper (II) in ambient conditions.
ꢀ Characterization of monomeric copper (II) complexes by spectroscopic techniques.
ꢀ All eight complexes have been tested for DNA binding, observations were encouraging.
ꢀ An interesting variation in antimicrobial activity has been noticed in these complexes.
ꢀ Cytotoxic studies only for two complexes have been carried out with fruitful results.
27