New anthracene based Schiff base ligands appended Cu(II) complexes: Theoretical study, DNA binding and cleavage activities

Article abstract of DOI:10.1002/aoc.4128

New anthracene based Schiff base ligands L¹ and H(L²), their Cu(II) complexes [Cu(L¹)Cl₂] (1) and [Cu(L²)Cl] (2), (where L¹?=?N¹,N²-bis(anthracene-9-methylene)benzene-

Full text of DOI:10.1002/aoc.4128

Received: 14 July 2017
DOI: 10.1002/aoc.4128
Revised: 11 September 2017
Accepted: 11 September 2017
F U L L P A P E R
New anthracene based Schiff base ligands appended Cu(II)
complexes: Theoretical study, DNA binding and cleavage
activities
Ammavasai Gubendran1,2 | Gujuluva Gangatharan Vinoth Kumar3 |
Mookkandi Palsamy Kesavan3 | Gurusamy Rajagopal4 | Periakaruppan Athappan1 |
Jegathalaprathaban Rajesh1,3
1 Department of Inorganic Chemistry,
New anthracene based Schiff base ligands L1 and H(L2), their Cu(II) complexes
School of Chemistry, Madurai Kamaraj
[Cu(L1)Cl2] (1) and [Cu(L2)Cl] (2), (where L1 = N1,N2‐bis(anthracene‐9‐methy-
University, Madurai 625 021, Tamilnadu,
India
lene)benzene‐1,2‐diamine, L2 = (2Z,4E)‐4‐(2‐(anthracen‐9‐ylmethyleneamino)
2 Department of Chemistry, Saraswathi
phenylimino)pent‐2‐en‐2‐ol) have been prepared and characterized by elemen-
Narayanan College, Madurai 625 022,
Tamilnadu, India
tal analysis, NMR, FAB‐mass, EPR, FT‐IR, UV–Vis and cyclic voltammetry.
The electronic structures and geometrical parameters of complexes 1 and 2 were
analyzed by the theoretical B3LYP/DFT method. The interaction of these com-
plexes 1 and 2 with CT‐DNA has been explored by using absorption, cyclic
voltammetric and CD spectral studies. From the electronic absorption spectral
studies, it was found that the DNA binding constants of complexes 1 and 2 are
8.7 × 103 and 7.0 × 104 M−1, respectively. From electrochemical studies, the ratio
of DNA binding constants K+/K2+ for 2 has been estimated to be >1. The high
binding constant values, K+/K2+ ratios more than unity and positive shift of
voltammetric E1/2 value on titration with DNA for complex 2 suggest that they
bind more avidly with DNA than complex 1. The inability to affect the conforma-
tional changes of DNA in the CD spectrum is the definite evidences of electro-
static binding by the complex 1. It can be assumed that it is the bulky
anthracene unit which sterically inhibits these complexes 1 and 2 from interca-
lation and thereby remains in the groove or electrostatic. The complex 2 hardly
cleaves supercoiled pUC18 plasmid DNA in the presence of hydrogen peroxide.
The results suggest that complex 2 bind to DNA through minor groove binding.
3 Chemistry Research Centre, Mohamed
Sathak Engineering College, Kilakarai
623 806, Tamilnadu, India
4 PG and Research Department of
Chemistry, Chikkanna Government Arts
College, Tiruppur 641 602, Tamilnadu,
India
Correspondence
Dr. Jegathlaprathaban Rajesh, Associate
Professor, Chemistry Research Centre,
Mohamed Sathak Engineering College,
Killakarai ‐ 623 806, Ramanthapuram
(District), Tamilnadu, India.
Email: mkuraji@gmail.com
Funding information
DST‐SERB, Grant/Award Number: SB/
FT/CS‐130/2012
KEYWORDS
characterization, Cu(II) complexes, DNA binding, DNA cleavage, Schiff base ligands, theoretical
studies
1 | INTRODUCTION
The interaction of transition metal complexes with DNA
has been extensively studied in order to develop novel
probes of DNA structure, DNA mediated electron
transfer reactions and to find the potential biological
and pharmaceutical activity. Their activity depends on
the mode and affinity of the binding with DNA.[1–5] In
Appl Organometal Chem. 2017;e4128.
wileyonlinelibrary.com/journal/aoc
Copyright © 2017 John Wiley & Sons, Ltd.
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https://doi.org/10.1002/aoc.4128

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GUBENDRAN ET AL.
human blood plasma, the presence of transition metals
reveals their importance in the living organisms as accu-
mulated storage and transport. Transition metals play a
key role in biological systems such as cell division, respi-
ration, nitrogen fixation and photosynthesis.[6] Among
the variety of ligand system employed in transition metal
complexes, Schiff bases are an important class having
many applications and many complexes of different
Schiff bases have been reported by a number of
authors.[7–12] Chelating Schiff base ligands containing O
and N donor atoms show broad biological activity and
are of special interest because of the variety of ways in
which they are bound to metal ions.[13] Some of the
Schiff base metal chelates shown the minor changes in
the structure of the ligands containing hard soft donor
atoms, markedly affects the activity of the compounds.[14]
Schiff base complexes have received much attention as
biomimics model compounds.[15] [Fe(salen)] complex is
a model for natural iron protein, and hemerythrin has
the ability to bind molecular oxygen reversibly.[16] Schiff
base complexes incorporating two different metal ions
are of special interest, as they are similar to those found
2 | EXPERIMENTAL METHODS
2.1 | Materials
Anthracene‐9‐carbaldehyde, 1,2‐diaminobenzene and
acetylacetone were purchased from Alfa Aesar and used
as received. Copper(II) chloride (CuCl2. 2H2O), calf thy-
mus DNA and pUC18 plasmid DNA were obtained from
Sigma Aldrich. All reagents and solvents were purchased
from Merk and Loba chemie (India) and used without
further purification.
2.2 | Physical measurements
UV–Vis spectra of solutions were recorded using
Shimadzu spectrometer 2500 PC series. The FTIR
spectra were recorded from KBr pellets in the range
400–4000 cm−1 on a Perkin‐Elmer spectrometer. NMR
spectra were obtained on a Bruker 300 MHz spectrome-
ter. A mass analytical procedure was performed using a
system consisting of a LC10ADVP pump and a single
quadruple mass spectrometer with an electron spray
ionization (ESI) source (LCMS‐2010) (Shimadzu, Kyoto,
Japan). Electron paramagnetic resonance (EPR) spectra
were obtained using a Varian E‐112 EPR spectrometer.
CV measurements were carried out on Bio‐Analytical
System (BAS) model CV‐50 W electrochemical analyser.
in living organisms, like enzymes and proteins.[17]
A
large number of Schiff bases based metal complexes have
been studied for their interesting and vital properties
such as significant DNA binding ability, anticancer
activity, catalytic activity in the hydrogenation of olefins,
photochromic properties and so on.[18–23] Synthesis of
new Schiff bases and their metal complexes are still
pursued in many recent investigations.[24–26] Reddy et al.
has synthesized ternary Cu(II) complexes of the type
[Cu(L’)(L”)](ClO4), where L‘is N N‐donor phenanthroline
base and L’ is an ancillary Schiff base ligand.[27] Schiff‐
base complexes have a wide variety of structures, coordi-
nating to metal in either mono‐, bi‐ and tri‐dentate
modes, depending upon the aldehyde and amines.
Unsymmetrical Schiff‐base ligand complexes have been
suggested as useful biological models in understanding
irregular binding of peptides and also as catalysts in some
chemical processes.[11]
This paper deals with the preparation of new anthra-
cene derived Schiff base ligands L1 and H(L2) and their
Cu(II) complexes [Cu(L1)Cl2] (1), [Cu(L2)Cl] (2) and
their characterizations using different elemental, spectro-
scopic and analytical tools. In addition to that, the struc-
tural parameters of complexes 1 and 2 were investigated
by B3LYP/DFT studies. The DNA binding properties of
complexes 1 and 2 have been studied with a view to
evaluating their pharmaceutical activities. We believed
that the complexes of 1 and 2 having anthracene deriva-
tive ligands, which possess planar aromatic moiety, are
expected to bind strongly with DNA and to show prom-
ising chemotherapeutic activity.
2.3 | Synthetic protocols
2.3.1 | N1‐(anthracene‐9‐methylene)ben-
zene‐1,2‐diamine (L)
An ethanolic solution of anthracene‐9‐carbaldehyde
(0.206 g, 1 mmol was added dropwise to a solution of 1,
2‐diaminobenzene (0.108 g, 1 mmol dissolved in 20 ml
of ethanol under constant stirring and the stirring was
continued at room temperature for 6 h. After that, on slow
evaporation of the solvent at room temperature, white
shining precipitate was obtained. It was filtered, washed
with petroleum‐ether and recrystallized from ethanol.
Yield: 88%.
2.3.2 | N1,N2‐bis(anthracene‐9‐methylene)
benzene‐1,2‐diamine (L1)
An ethanolic solution of anthracene‐9‐carbaldehyde
(0.412 g, 2 mmol) was added drop wise into a ethanolic
solution of 1, 2‐diaminobenzene (0.108 g, 1 mmol) with
continuous stirring at room temperature. The resulting
solution was refluxed for 3 h and the solvent was evapo-
rated. The residue that formed was removed by filtration

GUBENDRAN ET AL.
3 of 11
and dried. The crude product was then recrystallized from
ethanol. Yield: 82%.
(DFT) analysis were executed by the hybrid exchange‐
correlation function with 6‐31G (d,p) and B3LYP/
LANL2DZ basis set using Gaussain 09 program.[28] Ini-
tially, the optimized geometries of the ligands (L1 and
H(L2)), complex 1 and 2 were obtained by the DFT‐
B3LYP program.
2.3.3 | (2Z, 4E)‐4‐(2‐(anthracen‐9‐
ylmethyleneamino)phenylimino)pent‐2‐
en‐2‐ol (H(L2))
An ethanolic solution of acetylacetone (0.103 ΜL, 1 mmol)
was slowly added to the ligand solution L (0.296 g, 1 mmol
in 25 ml ethanol) under continuous stirring. The resulting
mixtures was refluxed for 5 h and evaporated. The residue
that formed was removed by filtration and the resulting
solid was then recrystallized from ethanol. Yield: 70%
The synthetic schemes for ligands L, L1 and H(L2) are
given in Scheme 1.
2.5 | DNA binding experiments
The DNA binding experiments were carried out using
UV–visible absorption studies, cyclic voltammetry and
circular dichroism studies.
2.6 | DNA cleavage study
The cleavage of pUC18 DNA by complexes 1 and 2, in the
absence and presence of activating agents H2O2 was dem-
onstrated using agarose gel electrophoresis in 10%
DMSO–5 mM Tris–HCl–50 mM NaCl buffer at pH 7.2.
2.3.4 | [Cu(L1)Cl2] (1)
The ligand L1 (0.484 g, 1 mmol) was dissolved in 60 mL of
absolute ethanol. An aqueous solution of 1 mmol of the
CuCl2.2H2O was added dropwise to the ligand solution
with continuous stirring and refluxed for 5 h. The precip-
itate formed was filtered, washed with diethyl ether and
recrystallized from ethanol.
3 | RESULTS AND DISCUSSION
Initially, complexes 1 and 2 were characterized by ele-
mental analysis (Table 1). The elemental analyses result
of the complexes 1 and 2 are in good agreement with their
molecular formulas and the proposed chemical structure
of the complexes 1 and 2 are depicted in Figure 1.
2.3.5 | [Cu(L2)Cl] (2)
The complex 2 was prepared using ligand H(L2)
(0.378 g, 1 mmol) by adopting the same synthetic proce-
dure as given for complex 1.
3.1 | 1H and 13C–NMR spectra
The 1H and 13C–NMR spectra of ligand L1 were
recorded in CDCl3, though L and H(L2) were recorded
in DMSO‐d6. The 1H–NMR in addition to 13C–NMR
spectrum of L and L1 are shown in Figure S1 and S2,
respectively. In the 1H–NMR spectra, a sharp singlet
2.4 | Theoretical B3LYP/DFT studies
To get the electronic structure of the ground state com-
plexes 1 and 2, we carried out Density Functional Theory
SCHEME 1 Schematic representation
for the synthesis of ligands L, L1 and H(L2)

4 of 11
GUBENDRAN ET AL.
TABLE 1 Elemental analysis and electronic spectral data of complexes 1 and 2
Found (Calcd)
λmax (nm)
Complex
1
C
H
N
IL
LMCT
d‐d
69.76 (69.85)
3.93 (3.91)
4.44 (4.53)
255
377
915
2
66.15 (66.22)
4.41(4.49)
5.85 (5.94)
256
380
614
3.3 | EPR spectral analysis
EPR spectra of the complexes 1 and 2 were recorded in
DMSO solvent at liquid nitrogen temperature as well as
at room temperature. At room temperature complexes 1
and 2 exhibit single derivative peak with the g values of
2.131 and 2.163, correspondingly. But, at liquid nitrogen
temperature (77 K) the EPR spectra of the Cu(II) complex
1 exhibit a set of four well‐resolved peaks in the low field
region and a weaker signal in the high field region, corre-
sponding to g║and g⊥ respectively (Figure S4). The same
trend was observed in complex 2 (Figure S5).
FIGURE 1 The proposed chemical structure of the complexes 1
and 2
peak observed at δ, 8.99 (L) and 8.63 (L1) ppm is due
to = CH (azomethine) proton. The signal due to ‐OH pro-
ton of H(L2) is observed at δ, 12.25 ppm and methyl pro-
tons appear in the range δ, 1.92–2.24 ppm. The aromatic
protons are clustered at δ, (7.29–8.99 ppm). A broad signal
at δ, 6.3 in the spectrum of L may be assigned to ‐NH2 pro-
tons. The 13C–NMR spectral data of ligands confirm the
The g║and g⊥ values are 2.245, 2.019 for 1 and 2.318,
2.015 for 2, respectively. The trend observed as g║ > g⊥
> 2.02, for the complexes 1 and 2 is typical of a Cu(II)
ion (d9) in axial symmetry with the unpaired electron
present in the dx ‐y orbital.[29] It has been reported that
g║values are close to 2.4 for the complexes containing
copper–oxygen bonds and close to 2.3 for complexes con-
taining copper–nitrogen bonds.[30] The complex 2 has the
value of g║= 2.318 in conformity with the presence of
both Cu–O and Cu–N bonds. The quotient (g║/ A║),
which is empirically treated as a measure of the distortion
from planarity, has been found to be 186 and 164 cm−1 for
the complexes 1 and 2, respectively indicating distortion
from a square planar structure to a deformed tetrahedral
structure.[31]
2
2
1
results obtained from the H–NMR. The azomethine car-
bon atom (=CH) is observed at 149.78 ppm for L,
149.04 ppm for L1 and 156.02 ppm for H(L2). Aromatic
carbons are observed in the range δ, 113.46–156.02,
121.33–130.19 and 127.10–146.01 ppm for ligands L, L1
and H(L2) respectively. The 13C–NMR spectrum of
H(L2) exhibits –CH3, −CH = C‐CH3, and = C‐OH carbon
resonance signals respectively at δ, 19.86–24.20, 95.26 and
176.16 ppm, correspondingly. As a result, the NMR spec-
tral study well support with chemical structure of the
ligands L, L1 and H(L2).
3.4 | FT‐IR spectra
FT‐IR spectra of free ligands (L, L1 and H(L2)), complexes
1 and 2 were recorded using KBr disc and their character-
istic bands are summarized in Table 2. The IR spectra of
ligand L1 and complex 1 are shown in Figure S6. All the
3.2 | Mass spectra
The FAB‐mass spectra of the complexes 1 and 2 show
molecular ion peak consistent with the expected molecu-
lar weight and fragmentation pattern in accordance with
the molecular formula. The FAB‐mass spectrum of com-
plex 1 shows molecular ion peak at m/z = 620 correspond-
ing to its molecular weight. The other prominent peak at
m/z = 485 may be due to L1. The complex 2 (Figure S3)
shows a molecular ion peak at m/z = 496 corresponding
to its molecular weight. The other important peaks at
m/z = 441 is due to loss of ‐Cl and at m/z = 378 corre-
sponds to H(L2). Thus, mass spectral analysis corrobo-
rates well with the proposed chemical structure of the
complexes 1 and 2.
TABLE 2 FT‐IR spectral assignments of ligands (L, L1, H(L2)),
complexes 1 and 2 in cm−1
Compound
L
υ(O‐H)
υ(C = N)
υ(M‐N)
υ(M‐O)
‐
1618
‐
‐
L1
H(L2)
‐
1621
1624
1606
1606
‐
‐
3460
‐
‐
1
2
‐
‐
417
415
‐
560

GUBENDRAN ET AL.
5 of 11
ligands show a strong band in the range, 1624–1618 cm−1
due to azomethine (−C = N) stretching. These bands are
shifted to lower frequencies, 1610–1589 cm−1 in the com-
plexes, indicating coordination of the Schiff bases through
the azomethine nitrogen.[32] The enolic υ(OH) band of the
ligand H(L2) observed at 3460 cm−1 disappeared upon
complex formation with Cu(II), indicating the coordina-
tion of oxygen after deprotonation to the metal atoms.
For complex 1, the stretching vibration of υ(M‐Cl) is
observed below 350 cm−1 and not observed beyond
400 cm−1. The coordination of the ligands L1 and H(L2)
to the metal through azomethine nitrogen and oxygen
atom, after deprotonation, is further confirmed by the
appearance of new bands around 431–417 and
560–545 cm−1 regions corresponding to υ(M‐N) and
υ(M‐O) vibrational modes respectively.[33] The IR spectral
data are in good agreement with the proposed structure
for the complexes 1 and 2. The broad absorption around
3480 cm−1 is due to water molecules present in the
sample.
TABLE 3 Voltammetric behavior* of complexes 1 and 2 in DMSO
Epc
Epa
(V)
ΔEp
(V)
ipa/
ipc
Complex (V)
ipc (A)
ipa (A)
1
2
0.012 0.143 0.131 1.15 × 10−5 6.12 × 10−6 0.532
0.011 0.169 0.158 1.19 × 10−5 6.23 × 10−6 0.575
*Measured vs Ag/AgCl with TBAP as supporting electrolyte at 100 mVs−1
.
non‐equivalent current intensity at the cathodic and
anodic peaks (ipa/ipc = 0.532–0.575). The peaks for the
CuII/I couple for the complexes 1 and 2 were observed in
the potential range, + 0.400 V to – 0.400 V. The difference
in peak potentials, ΔEp = 0.131 (1) and 0.158 (2) V,
exceeds the Nernstian requirement (0.059 V) suggesting
that the complexes are quasi‐reversible for electrochemi-
cal redox reactions.
3.7 | The ground state structures by
theoretical DFT calculation
3.5 | Electronic absorption spectra
Density functional theory (DFT) calculations were used
to investigate the mode of complex formation; electronic
structures as well as geometrical parameters of com-
plexes 1 and 2, as their crystal structure have not been
obtained. The structure of complexes 1 and 2 in the gas
phase was determined to be Cu(II) centered complexes
with distorted tetrahedral geometry and the optimized
structures of complexes 1 and 2 are depicted in
Figure 2. These structures of complexes 1 and 2 are well
correlated with the results of spectroscopic and analytical
investigations.
The electronic spectral data of complexes 1 and 2 asso-
ciated structural assignments are given in Table 1. The
UV‐ Vis spectra of these complexes 1 and 2 in DMSO
exhibit a sharp absorption at 255 (1) & 256 (2) nm
and a broad absorption at 377 (1) & 380 (2) nm owing
to intra‐ligand and ligand to metal charge transfer
transitions, (LMCT) respectively (Figure S7). The very
broad band's obtained in the region at 915 (1) & 614
(2) nm may be attributed to the d–d transition of Cu(II)
ions.[34,35]
Broad and low energy bands are obtained for Cu(II)
complexes 1 and 2 at 915 and 885 nm, respectively. It
has already been reported that a lower energy transition
can be observed between 730 and 900 nm, corresponding
to the d–d transition.[36] This value is of particular impor-
tance since it was highly dependent on the geometry of
the molecule. It is known that the transition from a
square planar structure to a deformed tetrahedral struc-
ture leads to a red shift of absorption in the electronic
spectrum.[37] Thus, the complexes 1 and 2, are not per-
fectly square planar and the broad as well as low energy
bands obtained at longer wavelengths are attributed to
the deformed tetrahedral structure.
Further, the Frontier molecular orbital structures of
ligands (L1 and H(L2)), complex 1 and 2 are displayed in
Figure 3 and 4, respectively. In L1, HOMO is spread over
the whole π‐moiety and LUMO on the anthracene unit.
After complexation with Cu(II) (complex 1) HOMO of
L1 is localized on the part of the π‐moiety and more
spread in metal part and the same behavior is observed
as L1‐LUMO with little contribution of metal ion. Simi-
larly for H(L2), HOMO and LUMO spread over the whole
π‐moiety. Then, HOMO is spread on imine and metal
parts and LUMO is spread over the whole molecular
frame structure after complex 2 formations. Moreover,
the calculated HOMO‐LUMO energy gaps of L1 and
H(L2) are 2.82 eV and 2.97 eV, respectively. After com-
plexation with Cu2+, the HOMO‐LUMO energy gaps of
complex 1 and 2 are found to be 1.76 eV and 2.33 eV.
These results obviously indicate that the complexation of
ligands L1 and H(L2) with Cu2+, resulting in the disrup-
tion of the internal charge transfer and that resulted in
the changes in electronic properties.
3.6 | Electrochemical behavior
Electrochemical data for the complexes 1 and 2 are sum-
marized in Table 3 and their typical cyclic voltammetric
responses are shown in Figure S8. Complexes 1 and 2
exhibit a quasi‐reversible behavior as indicated by the

6 of 11
GUBENDRAN ET AL.
the intensity of the LMCT band at (380 nm) markedly
decreased in presence of DNA, 23–34% hypochromism,
with a red shift up to 4 nm. The binding constant of com-
plex 2 has been found to be 7.0 × 104 M−1 and this values
are higher than that obtained for complex 1 (8.7 × 103 M
−1). The intrinsic binding constant values obtained for 1
and 2 are lower than that of potential intercalator like
ethidium bromide (Kb = 7.0 × 107 M
ble to those observed for [Cu(phen)2]+ and
[Ru(phen)3]2+ [39]
)
−1 [38], but compara-
.
From the absorption titration results, it could be seen
that the spectral changes for the complexes 1 and 2 are
not uniform. The spectral characteristic of low
hypochromism, paltry red shift and low Kb values
observed for the complex 1 suggest that there is no likeli-
hood of intercalative binding to DNA due to two bulky
anthracene units which strongly prevent stacking the aro-
matic chromophore between the base pairs of the DNA.
The high Kb values and pronounced hypochromism for
complex 2 are possibly due to the presence of only one
anthracene moiety that facilitates groove binding with
the DNA with the base pairs.[40] Taking into account the
extent of hypochromism and binding constant values the
possibility of intercalative binding of DNA can be ruled
out and only groove binding may occur for complexes 1
and 2.
3.8.2 | Electrochemical studies
Electrochemical investigation of drug–DNA interactions
can provide a useful complement to other methods and
yield information about the mechanism of interaction
and the conformation of adduct.[41] The electrochemical
behavior of complexes 1 and 2, and their interaction with
CT‐DNA were carried out by CV. The cyclic voltammetric
data for complexes 1 and 2 in the absence and presence of
DNA at room temperature in Tris–HCl buffer (pH = 7.1)
is shown in Table 5.
FIGURE 2 Gas phase B3LYP/DFT optimized structure of
complexes 1 and 2
3.8 | DNA binding and cleavage studies
3.8.1 | Absorption spectroscopic titration
The cyclic voltammogram of complex 1 in the absence
of DNA reveals reduction of CuII to the CuI form at a
cathodic peak potential (Epc) of −0.010 V versus Ag/AgCl.
In the absence of DNA, the separation of the anodic and
cathodic peak potentials, ΔEp = 0.093 V for 1 indicates a
quasi‐reversible redox process. The formal potential, E1/2
(or voltammetric E1/2), taken as the average of Epc and
Epa is 0.037 V in the absence of DNA. The presence of
DNA in the solution at the same concentration of complex
causes a considerable decrease in the voltammetric cur-
rent. In addition, both the peak potentials, (Epc and Epa)
and E1/2 have shifted to more negative potential/less pos-
itive as shown in Figure 6.
Absorption titration experiments were performed by
maintaining the Cu(II) complexes 1 and 2 concentration
as constant at 20 μM while varying the concentration of
CT‐DNA in between 0–100 μM. Table 4 summarizes the
spectral changes when the complexes 1 and 2 are titrated
with DNA. LMCT bands (377 nm (1) – 380 nm (2)) are
monitored for absorption spectroscopic titration. Addition
of increasing amounts of DNA results in hypochromism
and a moderate bathochromic shift (2–4 nm) of the peak
at the LMCT band. The absorption spectra of complexes
1 and 2 in the presence of increasing amounts of DNA
are shown in Figure 5.
For complex 1, LMCT absorption (377 nm) shifts up to
only 2 nm with 14–18% hypochromism. As for complex 2
The cyclic voltammogram of complex 2 in the absence
of DNA features a quasi‐reversible redox wave with

GUBENDRAN ET AL.
7 of 11
FIGURE 3 Frontier molecular orbitals
of L1 and complex 1 (from left to right)
FIGURE 4 Frontier molecular orbitals
of H(L2) and complex 2 (from left to right)
TABLE 4 Absorption spectral titration data of the complexes 1 and 2 with DNA in Tris–HCl buffer solution
LMCT (λmax/nm)
Red
shift Δ
λ
Binding
constant
Hypochromism
(%)
Complex
1
Free
Bound
(nm)
Kb (M−1
)
8.7 × 103
377
379
14–18
23–34
2
4
2
380
384
7.0 × 104

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GUBENDRAN ET AL.
FIGURE 6 Cyclic voltammograms of 1 (a) and 2 (b) in the
absence and in presence of DNA in Tris–HCl buffer pH = 7.1.
[complex] = 100 μM. [R = 0, 4 & 8]
FIGURE 5 Absorption spectra of complex 1 (a) and 2 (b) in the
absence and presence of increasing amounts of DNA (0–240 μM)
in Tris–HCl buffer (pH = 7.1). Arrow mark indicates the absorbance
change upon increasing DNA concentration. [complex] = 20 μM
and voltammetric E1/2 at the same scan rate. After the
addition of DNA, E1/2 is shifted to −0.034 V, ΔEp is
0.080 V and ipa/ipc value is increased to 0.526.
TABLE 5 Voltammetric behavior of complexes 1 and 2 towards
DNA in Tris–HCl buffer solution
The drop in the voltammetric current in the presence
of DNA in complexes 1 and 2 can be attributed to the dif-
fusion of the Cu(II) complex bound to the large, slowly
diffusing DNA molecule. In the cyclic voltammograms
of 1 and 2, at different concentrations of DNA, the peak
currents decreased with increasing concentrations of
DNA while both the Epc and E’0 shifted to more positive
or less negative potentials. The phenomena of the shift of
E’0 and the decrease of peak current implied forming a
new association of DNA with complex. Based on the shift
of formal potentials in the cyclic voltammograms, the
interaction mode of compounds with DNA can be
inferred.[42] Therefore, in the light of Bard's report,[43]
complex 2 may interact with DNA by groove binding
mode in contrast to 1 whose interaction mode is electro-
static due to negative shift of E’0 in these cases. For com-
plex 1, K2+ is higher than K+ while the reverse is observed
for the complex 2. This suggests that the B form of DNA
tends to stabilize the Cu(II) over the Cu(I) state of the
complex 1 obviously by electrostatic interaction and
Epc
Epa ΔEp ipa/ E1/2
(V) (V) ipc (V)
ΔE1/2 K+/
\(V) K2+
Complex R (V)
1
0
4
8
0
4
8
−0.010 0.083 0.093 0.972 0.037
−0.044 0.077 0.121 1.129 0.017 −0.020 0.46
−0.102 0.051 0.153 1.057 −0.026 −0.063 0.12
−0.035 0.083 0.118 0.400 0.006
2
−0.077 0.074 0.151 0.526 0.017 −0.029 2.10
−0.114 0.051 0.165 0.983 −0.034 −0.080 7.43
Epc = −0.035 V at a scan rate of 100 mVs−1 and the mea-
sured half wave potential E1/2 for the Cu(II)/(I) couple is
0.006 V. The ratio of anodic to cathodic peak current is
0.4 which closely resembles that of the criteria of quasi‐
reversibility. Upon addition of increasing amounts of
DNA the cyclic voltammogram of the complex 2 experi-
enced a less negative shift in cathodic peak potential
and more positive shift in both the anodic peak potential

GUBENDRAN ET AL.
9 of 11
thereby indicating that the species Cu(II) interacts with
DNA to a greater extent than Cu(I). In short, the obvious
shift of peak potentials and the ratio of binding constant
values suggest a weak association of the complex 1 (K+/
K2+ < 1) with DNA than complex 2 (K+/K2+ > 1).
positive band along with a minor red shift (Figure 7) of
the band maxima is characteristic of groove binding that
stabilizes right‐handed B form of DNA.[45] The CD spec-
tral studies suggest that while the DNA binding of com-
plex 1 involves electrostatic mode, complex 2 wraps the
groove of the DNA. These conclusions are also in accor-
dance with those from electronic spectral and cyclic
voltammetric studies.
3.8.3 | Circular dichroism study
The results of CD studies for 1 and 2 are presented in
Figure 7. Pure DNA produced characteristic bands with
a positive band at 278 nm and a negative band at
244 nm. These bands were modified when DNA was
allowed to interact with complexes 1 and 2.
In presence of complex 1, the intensity of the negative
ellipticity band decreases almost similar to that for
the positive ellipticity band. This suggests that the DNA‐
binding of the complexes do not affect the conformational
changes of DNA. Furthermore, with increasing concen-
tration of complex 1, it proved difficult to detect any obvi-
ous perturbation in the CD spectrum; illustrating the
inability of complexes to affect the conformational hetero-
geneity of DNA anymore.[44]
3.8.4 | DNA cleavage study by gel
electrophoresis
Based on the above investigation, Complex 2 has potent
DNA binding ability than complex 1 and it needs to probe
the DNA cleavage characteristics of complex 2 for fulfill-
ing the basic requirements as an anticancer agent.
Figure 8 illustrates the gel electrophoretic separations
showing the cleavage of plasmid pUC18 DNA induced by
the complex 2 under aerobic conditions. With the increase
of complex concentration, no amount of the circular
supercoiled DNA is converted into nicked DNA via single
strand cleavage (lane 4). It reveals that pUC18 DNA
induced by the complex in the presence of high concen-
tration of H2O2 results in the insignificantly small conver-
sion of Form I to Form II (lane 6). H2O2 alone is incapable
of cleaving plasmid DNA (lane 3). It can be seen that
neither the complex 2 alone nor incubation with H2O2
without the complex causes any strand scission (lanes 2
and 3). These experiments demonstrate that both the
copper(II) complex and high amount of H2O2 are required
In presence of complex 2 an increase in the molar
ellipticity values of both the positive and negative elliptic-
ity bands of the DNA was observed, consistent with a sin-
gle binding mode. The enhancement of the intensity of
FIGURE 8 Agarose gel electrophoresis diagram showing the
cleavage of SC pUC18 DNA (500 ng) by complex 2 in Tris–HCl/
NaCl buffer (50 mM, pH = 7.2) lane 1, DNA control; lane 2,
Lane1 + 50 μM complex; lane 3, Lane1 + 100 μM H2O2; lane 4, lane
1 + 100 μM complex; lane 5, lane 1 + 100 μM complex +100 μM
H2O2; Lane6, lane 1+ 100 μM complex +200 μM H2O2
FIGURE 7 CD spectra of CT‐ DNA in the absence (i) and
presence (ii) of complex 1 (a) and complex 2 (b); [DNA] = 200 μM,
[complex] = 100 μM, (1/R = 0.5)

10 of 11
GUBENDRAN ET AL.
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to cleave plasmid DNA even to very small extent. The
inability of the complex 2 to cleave plasmid pUC18 DNA
can be attributed to the ratio K+/K2+, obtained from CV
studies, which is less than unity. To have an observable
nuclease activity the ratio K+/K2+ should be >1.[46,47]
The cleavage mechanism may involve a hydroxyl radical
oxidative mechanism.
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In summary, new anthracene based Schiff base ligands L1
and H(L2) and their derived Cu(II) complexes 1 and 2
have been synthesised and well characterized by using
various spectroscopic and analytical methods. The molec-
ular structures of the Cu(II) complexes 1 and 2 were
ascertained by a theoretical method (B3LYP/DFT analy-
sis). In vitro DNA binding studies have been performed
using various biophysical techniques viz., absorption,
cyclic voltammetry, and circular dichroism techniques,
to predict their binding mode as well as binding strength;
the results revealed higher binding affinity of complex 2
than complex 1, via groove mode of binding. For the rea-
son that, complex 1 has a bulkier in size than complex 2,
which make the barrier to bind with DNA via groove
mode of interaction. However, complex 1 showed signifi-
cant binding ability through electrostatic interaction with
DNA helix. We also analyzed the pUC18 DNA cleavage
capability of complex 2, which gives oxidative pathways
chase cleavage pattern. These results fortify our idea of
minor groove binding nature of complex 2 with DNA. In
conclusion, we believe that these Cu(II) complexes are
alternatives as better anticancer agents and additional
studies are needed on complex 2, to recommend as a
cancer drug in preclinical study.
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ACKNOWLEDGEMENTS
108.
[21] P. Kavitha, M. Rama Chary, B. V. V. A. Singavarapu, K. Laxma
This work was supported by the DST‐SERB (Grant. No:
SB/FT/CS‐130/2012). We express our gratitude to
Madurai Kamaraj University and Mohamed Sathak
Engineering College, Kilakarai for their lab facilities and
their instrumentation facilities.
Reddy, J. Saudi, Chem. Soc. 2016, 20, 69.
[22] A. Gubendran, M. P. Kesavan, S. Ayyanaar, J. Dhaveethu Raja,
P. R. Athappan, J. Rajesh, Appl. Organomet. Chem. 2017, e3708.
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ORCID
[24] A. Golcu, M. Tumer, H. Demicell, R. C. Wheatley, Inorg. Chim.
Acta 2005, 358, 1785.
Jegathalaprathaban Rajesh
http://orcid.org/0000-0001-
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