Antioxidant, DNA interaction, molecular docking and cytotoxicity studies of aminoethylpiperazine-containing macrocyclic binuclear copper(II) complexes

Article abstract of DOI:10.1002/aoc.3669

A series of new macrocyclic binuclear copper(II) complexes of the type [Cu₂L^1–5(ClO₄)](ClO₄) (1–5) were synthesized by template condensation between precursor compounds 2,6-bis(4-aminoethylpiperazin-1-ylmethyl)-

Full text of DOI:10.1002/aoc.3669

Received: 29 July 2016
DOI 10.1002/aoc.3669
Revised: 16 September 2016
Accepted: 27 September 2016
F U L L PA P E R
Antioxidant, DNA interaction, molecular docking and cytotoxicity
studies of aminoethylpiperazine‐containing macrocyclic binuclear
copper(II) complexes
C. Karthick1 | K. Karthikeyan2 | Purna Sai Korrapati2 | A. Kalilur Rahiman1
1 Post‐Graduate and Research Department of
Chemistry, The New College (Autonomous),
A series of new macrocyclic binuclear copper(II) complexes of the type
[Cu2L1–5(ClO4)](ClO4) (1–5) were synthesized by template condensation between
Chennai 600 014, India
2 Biomaterials Division, CSIR‐Central Leather
precursor compounds 2,6‐bis(4‐aminoethylpiperazin‐1‐ylmethyl)‐4‐substituted phe-
nols and 2,6‐diformyl‐4‐substituted phenols. The synthesized precursors and com-
plexes were characterized using regular physicochemical techniques. The rate
constant values obtained for the hydrolysis of 4‐nitrophenylphosphate were in the
Research Institute, Chennai 600 020, India
Correspondence
A. Kalilur Rahiman, Post‐Graduate and Research
Department of Chemistry, The New College
(Autonomous), Chennai – 600 014, India.
Email: akrahmanjkr@gmail.com;
akrahmanjkr@thenewcollege.in
range 1.83 × 10−2–4.19 × 10−2 min−1. Antioxidant studies against 2,2′‐diphenyl‐1‐
picrylhydrazyl revealed the antioxidant potency of the synthesized complexes. Binding
studies of the complexes with calf thymus DNA were conducted using electronic,
viscometric and voltammetric techniques, and the obtained results suggested a
non‐covalent groove mode of binding. The oxidative cleavage of pBR322 DNA in
the presence of co‐reactant H2O2 and radical scavengers showed single strand scission
and involvement of H2O2 radical in the cleavage process. Molecular docking studies
were performed to insert complexes into the crystal structures of 1BNA and VEGFR
kinase at active sites to determine the possible binding mode and predominant binding
interactions. In vitro cytotoxicity of the complexes was tested against human epider-
moid carcinoma cells (A431) by MTT assay, which revealed the effective anticancer
activity of the complexes. Live cell and fluorescent imaging of A431 cells showed that
the complexes induce cell death through apoptosis.
Funding information
UGC‐MRP, Grant/Award Number: 39‐797/2010
(SR).
KEYWORDS
antioxidant, apoptosis, copper(II) complexes, DNA interaction, molecular docking
1
|
INTRODUCTION
Discovery of drugs for cancer therapy is one of the most
rapidly developing areas of pharmaceutical research. Cancer
DNA is the primary intracellular agent in nucleic acid chem-
istry. The interaction of metal‐based drugs with DNA is an
essential issue in life sciences, and studies of DNA–metal
complex interactions play a critical role in the synthesis of
possible effective chemotherapeutic agents. The mode of
action of metal‐based anticancer drugs can be explained by
their ability to bind with DNA, which subsequently affects
DNA replication and thereby inhibiting the growth of tumour
cells.[1] Metal‐based small molecules able to cleave DNA
under physiological conditions are also called artificial nucle-
ases, which gives a deep insight to genomic research, foot-
print and therapeutic agents.[2–4]
involves malignant tumours where the growth and differenti-
ation of cells are out of control.[5,6] Compounds like cisplatin,
carboplatin, oxaliplatin, nedaplatin and bleomycin alone or in
combination with other drugs are widely used in chemother-
apy against testicular and ovarian carcinomas, head and neck
cancers, bladder tumours and osteosarcoma.[7–10] Despite
their clinical success, they possess some drawbacks. For
instance, cisplatin exhibits serious side effects like
nephrotoxicity, ototoxicity, neurotoxicity, nausea and
vomiting.[11–14] In addition to that, the efficacies of such
drugs are reduced by acquired and inherent resistance of can-
cer cells.[15,16] This state of condition has stimulated various
Appl Organometal Chem 2016; 1–15
wileyonlinelibrary.com/journal/aoc
Copyright © 2016 John Wiley & Sons, Ltd.
1

2
KARTHICK ET AL.
research groups to develop more effective, less toxic, site‐
specific novel metal‐based small molecules without undesir-
able side effects.[17–21]
tetramethyammonium
hydroxide,
2,2′‐diphenyl‐1‐
picrylhydrazyl (DPPH) and Cu(ClO4)2⋅6H2O were purchased
from Sigma Aldrich Ltd, USA. Tetra(n‐butyl)ammonium per-
chlorate (TBAP) used as supporting electrolyte in the electro-
chemical measurements was purchased from Fluka and
recrystallized from hot methanol. Calf thymus (CT) DNA
and supercoiled pBR322 DNA were obtained from Merck
Genei (India). The precursor compounds 2,6‐bis(4‐
aminoethylpiperazin‐1‐ylmethyl)‐4‐substituted phenols (PC‐
I–PC‐III) were synthesized by Mannich reaction. The polyaza
macrocyclic binuclear complexes (1–5) were synthesized by
treating the precursor compounds, Cu(ClO4)2⋅6H2O and 2,6‐
diformyl‐4‐substituted phenols, in a ratio of 1:2:1, using the
template method via Schiff base condensation (Scheme 1).
Micro‐analysis (C, H and N) was performed using a Carlo
Erba model 1106 elemental analyser. Infrared (IR) spectra
were recorded with an ABB Instruments MB‐3000 spectro-
Macrocyclic complexes with symmetric lateral chains
providing two phenolic oxygen atoms were first introduced
by Pilkington Robson.[22] Thereafter, polyaza macrocyclic
complexes were widely studied for their spectro‐chemical,
redox, optical, magnetic and biological importance.[23] Spe-
cial attention is given by many research groups to mimicking
the activities of metalloenzymes using transition metal com-
plexes as models, because many naturally occurring
metalloenzymes have two metal ions in close proximity.
The rational design and application of versatile artificial
bimetallic cores as models for mimicking activities of such
metalloenzymes and as bimetallic catalysts for hydrolysis of
phosphodiesters is an active area of research for bioinorganic
chemists. Copper is a known bio‐relevant essential trace ele-
ment associated with processes involving oxygen in biologi-
cal systems. The binuclear copper core is observed in
metalloproteins like hemocyanin, tyrosinase and laccase.[24]
The significant role of copper ions in metabolic pathways
can be well understood from mitochondrial respiration, free
radical scavenging and iron absorption activities. Biologi-
cally accessible redox potential and greater nucleobase affin-
ity of copper(II) complexes are primary factors behind their
potent artificial nuclease activity in both oxidative and
hydrolytic routes.[25] Generally, effective antioxidant active
molecules have been found to exhibit better anticancer, anti‐
cardiovascular and anti‐inflammatory properties.[26,27]
Plenty of reports are available in the literature for under-
standing the structural, magnetic, redox, catalytic and biolog-
ical properties of binuclear copper(II) complexes.[28–31] DNA
interaction and phosphatase‐like activity of some polyaza
macrocyclic copper(II) complexes containing N‐donor hetero-
cyclic bases as side arms have been reported.[32] Our group has
recently reported antimicrobial, DNA interaction and antican-
cer activities of some pendant‐armed binuclear macrocyclic
metal(II) complexes.[33] To the best of our knowledge, there
are no reports pertaining to DNA interaction and cytotoxicity
of macrocycles possessing the N‐(2‐aminoethyl)piperazine
moiety, which prompted us to design and develop new macro-
cyclic binuclear copper(II) complexes containing N‐(2‐
aminoethyl)piperazine units. The biological efficacy was stud-
ied in terms of catalytic, antioxidant, DNA interaction and
cytotoxic activities. Molecular docking analysis was also per-
formed with DNA and VEGFR protein kinase receptors.
photometer using KBr discs in the range 400–4000 cm−1
.
NMR spectral data were collected with a Varian‐VNMRS‐
400 in CDCl3 solution with tetramethylsilane as an internal
standard at ambient temperature. Electronic spectra of com-
plexes were recorded using a PerkinElmer Lambda‐45 spec-
trophotometer, in the range 200–1100 nm. Electron impact
(EI) mass spectra of precursors were recorded using a JEOL
GCMATE II GC‐MS, and electrospray ionization (ESI) mass
spectra with a Q‐Tof mass spectrometer. Cyclic voltammo-
grams were recorded with an electrochemical workstation
(602D, CH Instruments) in nitrogen atmosphere using TBAP
as supporting electrolyte. X‐band electron paramagnetic reso-
nance (EPR) spectra were recorded with a Varian EPR‐E 112
spectrometer using DPPH as reference. Variable‐temperature
magnetic susceptibilities were recorded with a vibrating sam-
ple magnetometer (model 7410, Lake Shore Cryotronics Inc.).
2.2 | Synthesis of 2,6‐Diformyl‐4‐substituted Phenols
2,6‐Diformyl‐4‐methylphenol was synthesized according to a
reported procedure.[34] 2,6‐Diformyl‐4‐tert‐butylphenol and
2,6‐diformyl‐4‐bromophenol were synthesized by adapting
a modified procedure reported in the literature.[35]
2
| EXPERIMENTAL
2.1 | Materials and methods
4‐Methylphenol, 4‐bromophenol and 4‐tert‐butylphenol
were purchased from SD Fine Chemicals Ltd, India.
SCHEME
1
Synthetic route for macrocyclic binuclear copper(II)
N‐(2‐Aminoethyl)piperazine,
4‐nitrophenylphosphate,
complexes (1–5).

KARTHICK ET AL.
3
2.2.1
|
2,6‐Diformyl‐4‐tert‐butylphenol
with constant stirring. This mixture was refluxed for 24 h
and the solvent volume was reduced to one‐third. The
resulting product was extracted successively with 10%
Na2CO3 (30 ml), CHCl3 (2 × 30 ml) and CH2Cl2 (30 ml).
The combined organic phases were collected and dried with
anhydrous Na2SO4. The oily compound obtained was washed
with diethyl ether and dried in vacuo.
Hexamethylenetetramine (9.60 g, 68.5 mmol) and parafor-
maldehyde (10.26 g, 342 mmol) were ground together and
this mixture was slowly added to 4‐tert‐butylphenol (5.14 g,
34.2 mmol) dissolved in 50 ml of glacial acetic acid in nitro-
gen atmosphere with stirring. After stirring continuously for
24 h, the mixture was refluxed for another 24 h during which
the colour of the reaction mixture changed from yellow to
orange red. The content was then poured in to 4 M HCl
(200 ml), and extracted with CH2Cl2 (2 × 150 ml). The com-
bined organic extracts were washed with 4 M HCl (2 × 200
ml), water (200 ml) and saturated NaCl (200 ml), then dried
with Na2SO4 and subsequently allowed to evaporate. A yel-
low residue was obtained and the product was purified using
a short column (silica gel, CH2Cl2). Pale yellow crystalline
needles were obtained, which were recrystallized from
cyclohexane.
Yield: 4.30 g (61%); colour: yellow; m.p. 105°C
(lit. 102–103°C). Anal. Calcd for C12H14O3 (203.24) (%):
C, 69.88; H, 6.84. Found (%): C, 69.86; H, 6.96.
Selected IR data (KBr disc, ν, cm−1): 3400 ν(OH), 1676
ν(HC=O). 1H NMR (CDCl3, 400 MHz, δ, ppm): 11.34
(s, 1H, OH), 10.28 (s, 2H–CHO), 8.0 (s, 2H, Ar–H), 1.35
(s, 9H, −C(CH3)3). 13C NMR (CDCl3, 100.50 MHz, δ,
ppm): 192.4 (−CHO), 162.0 (C–OH), 143.2 (Ar–C), 34.3
(−C(CH3)3).
2.3.1
| 2,6‐Bis(4‐aminoethylpiperazin‐1‐ylmethyl)‐4‐
methylphenol (PC‐I)
Yield: 4.1 g (55.3%); colour: pale yellow. Anal. Calcd for
C21H38N6O (390.56) (%): C, 64.58; H, 9.81; N, 21.52. Found
(%): C, 64.54; H, 9.84; N, 21.53. Selected IR data (KBr, ν,
cm−1): 3489 ν(OH), 2956 ν(NꢀH), 1374 ν(ArꢀO). EI‐MS
1
(m/z): 391.68 [M+]. H NMR (CDCl3, 400 MHz, δ, ppm):
11.38 (s, H, OH), 7.45 (s, 2H, Ar–H), 5.45 (br s, 2H, NH),
3.78–3.74 (m, 8H, CH2), 2.91–2.42 (m, 16H, CH2), 2.29 (s,
3H, CH3). 13C NMR (CDCl3, 100.50 MHz, δ, ppm): 159.3
(C–OH), 138.7–127.4 (Ar–C), 59.4–54.6 (4CH2), 46.0
(2CH2) and 20.2 (CH3).
2.3.2
| 2,6‐Bis(4‐aminoethylpiperazin‐1‐ylmethyl)‐4‐tert‐
butylphenol (PC‐II)
Yield: 4.75 g (57.8%); colour: yellow. Anal. Calcd for
C24H44N6O (432.64) (%): C, 66.63; H, 10.25; N, 19.42.
Found (%): C, 66.56; H, 10.31; N, 19.48. Selected IR data
(KBr, ν, cm−1): 3386 ν(OH), 2922 ν(NꢀH), 1369 ν(ArꢀO).
1
2.2.2
|
2,6‐Diformyl‐4‐bromophenol
EI‐MS: (m/z) 431.06 [M+]. H NMR (CDCl3, 400 MHz, δ,
ppm): 10.67 (s, H, OH), 7.42 (s, 2H, Ar–H), 5.53 (br s, 2H,
NH), 3.70–3.54 (m, 8H, CH2), 2.85–2.26 (m, 16H, CH2),
2.18 (s, 9H, 3CH3). 13C NMR (CDCl3, 100.50 MHz, δ,
ppm): 163.4 (C–OH), 135.6–128.4 (Ar–C), 60.9–52.3
(4CH2), 44.6 (2CH2).
Hexamethylenetetramine (11.17 g, 79.6 mmol) and parafor-
maldehyde (11.9 g, 373 mmol) were ground together and this
mixture was slowly added to 4‐bromophenol (6.45 g, 37.3
mmol) dissolved in 50 ml of glacial acetic acid with constant
stirring. The mixture was allowed to stir continuously for 72
h under nitrogen atmosphere during which the colour of the
reaction mixture became pale brown. Then, the mixture was
refluxed for about 3 h until the colour changed to orange
red. The mixture was cooled at 0°C, and then poured into
ice‐cooled 4 M HCl (300 ml) and refrigerated overnight.
The resulting product was extracted with CH2Cl2 (2 × 75 ml).
The yellow crystalline product obtained was dried at 70°C
and recrystallized from C2H5OH–H2O (1:1).
2.3.3
| 2,6‐Bis(4‐aminoethylpiperazin‐1‐ylmethyl)‐4‐
bromophenol (PC‐III)
Yield: 5.05 g (58.4%); colour: pale red. Anal. Calcd for
C20H35N6O (455.43) (%): C, 52.74; H, 7.75; N, 18.45. Found
(%): C, 52.72; H, 7.78; N, 18.46. Selected IR data (KBr, ν,
cm−1): 3398 ν(OH), 2937 ν(NꢀH), 1379 ν(ArꢀO). EI‐MS:
1
(m/z) 454.81 [M+]. H NMR (CDCl3, 400 MHz, δ, ppm):
10.23 (s, H, OH), 7.23 (s, 2H, Ar–H), 5.58 (br s, 2H, NH),
3.85–3.69 (m, 8H, CH2), 2.96–2.15 (m, 16H, CH2). 13C
NMR (CDCl3, 100.50 MHz, δ, ppm): 160.6 (C–OH),
137.6–129.4 (Ar–C), 61.6–52.2 (4CH2), 45.0 (2CH2).
Yield: 4.12 g (48%); colour: brownish yellow; m.p.
138°C (lit. 136°C). Anal. Calcd for C8H5O3Br (229.03) (%):
C, 41.95; H, 2.2. Found (%): C, 41.92; H, 2.28. Selected IR
data (KBr disc, ν, cm−1): 3439 ν(OH), 1680 ν(HC¼O).
1H NMR (CDCl3, 400 MHz, δ, ppm): 11.54 (s, 1H, OH),
10.19 (s, 2H, −CHO), 8.06 (s, 2H, Ar–H). 13C NMR (CDCl3,
100.50 MHz, δ, ppm): 191.4 (−CHO), 162.3 (C–OH), 139.7
(Ar–C).
2.4 | Template synthesis of Macrocyclic Binuclear
Copper(II) Complexes (1–5)
An ethanolic solution (20 ml) of Cu(ClO4)2⋅6H2O (0.67 g,
1.8 mmol) was added to 2,6‐diformyl‐4‐substituted phenols
(1.8 mmol) dissolved in ethanol (20 ml) followed by addition
of an ethanolic solution (20 ml) of precursor (PC‐I, 0.70 g;
PC‐II, 0.78 g; or PC‐III, 0.82 g; 1.8 mmol) and (C2H5)3N
(0.13 ml, 0.9 mmol), which was refluxed for 2 h. Then,
2.3 | Synthesis of precursor compounds (PC‐I–PC‐III)
N‐(2‐Aminoethyl)piperazine (4.91 g, 38 mmol) was added to
a well‐ground mixture of 4‐substituted phenols (19 mmol)
and paraformaldehyde (1.15 g, 38 mmol) in ethanol (60 ml)

4
KARTHICK ET AL.
another portion of Cu(ClO4)2⋅6H2O (0.67 g, 1.8 mmol)
followed by (C2H5)3N (0.13 ml, 0.9 mmol) were added, and
the mixture was refluxed for 24 h. The solvent volume was
reduced, and the product was washed with diethyl ether
followed by isopropyl alcohol for several times. Microcrys-
tals were obtained and dried in vacuo.
Caution! Compounds containing perchlorate are poten-
tiallyexplosive.Therefore, onlyasmallamountofthematerials
should be used at a time and handled with appropriate care.
[Cu2L1(ClO4)(H2O)](ClO4) (1). Yield: 2.22 g (71.6%);
colour: green. Anal. Calcd for C30H42N6O11Cl2Cu2
(860.69) (%): C, 41.86; H, 4.92; N, 9.76. Found (%): C,
41.83; H, 4.97; N, 9.79. Selected IR data (KBr, ν, cm−1):
3364 ν(ꢀOH), 1630 ν(C¼N), 1538 ν(bridging phenoxide),
1362 ν(ArꢀO), 1100 and 632 ν(ClO4−), 813 ν(rocking
OH)water, 474 ν(CuꢀO), 448 ν(CuꢀN). ΛM (Ω−1 cm2 mol−1,
in DMF): 70. UV–visible (λmax, nm (ε, M−1 cm−1) in DMF):
390 (21 000), 656 (102). ESI‐MS in CH3CN (m/z): 744
[L1 + 2Cu + ClO4 + H]+, 644 [L1 + 2Cu + H]+, 585
[L1 + Cu + 2H]+. μeff: 1.93 BM at 298 K.
(967.64) (%): C, 39.72; H, 4.69; N, 8.68. Found (%): C,
39.69; H, 4.75; N, 8.71. Selected IR data (KBr, ν, cm−1):
3374 ν(ꢀOH), 1620 ν(C¼N), 1534 ν(bridging phenoxide),
1358 ν(ArꢀO), 1100 and 628 ν(ClO4−), 818 ν(rocking
OH)water, 472 ν(CuꢀO), 450 ν(CuꢀN). ΛM (Ω−1 cm2 mol−1,
in DMF): 73. UV–visible (λmax, nm (ε, M−1 cm−1) in DMF):
366 (18 567), 645 (119). ESI‐MS in CH3CN (m/z): 851
[L5 + 2Cu + ClO4 + 2H]+, 689 [L5 + Cu + 2H]+. μeff:
1.87 BM at 298 K.
2.5 | Biological assays
Experiments for the kinetic studies of phosphate hydrolysis,
DPPH radical scavenging assay and cell proliferation assay
including MTT and apoptosis studies were carried out
according to literature methods[36–39] as given in the
supporting information.
2.6 | DNA binding experiments
A solution of CT DNA in Tris–HCl/NaCl buffer (5 mM Tris–
HCl/50 mM NaCl, pH = 7.2) gave a ratio of UV absorbance
at 260 and 280 nm of about 1.89:1, indicating that the DNA
was sufficiently free from proteins.[40] Stock solutions of CT
DNA were prepared in Tris–HCl/NaCl buffer and stored at
4°C for less than 4 days. The DNA concentration per nucle-
otide was determined by absorption spectroscopy using a
molar absorption coefficient of 6600 M−1 cm−1 at 260 nm.[41]
Stock solutions of complexes were prepared using 5% DMF
in Tris–HCl/NaCl buffer and diluting suitably with the
corresponding buffer to the required concentration for the
experiments.
[Cu2L2(ClO4)(H2O)](ClO4) (2). Yield: 2.54 g (74.7%);
colour: green. Anal. Calcd for C36H54N6O11Cl2Cu2
(944.85) (%): C, 45.76; H, 5.76; N, 8.89. Found (%): C,
45.72; H, 5.81; N, 8.92. Selected IR data (KBr, ν, cm−1):
3372 ν(ꢀOH), 1620 ν(C¼N), 1530 ν(bridging phenoxide),
1355 ν(ArꢀO), 1095 and 628 ν(ClO4−), 815 ν(rocking
OH)water, 470 ν(CuꢀO), 446 ν(CuꢀN). ΛM (Ω−1 cm2 mol−1,
in DMF): 68. UV–visible (λmax, nm (ε, M−1 cm−1) in DMF):
367 (17 452), 652 (107). ESI‐MS in CH3CN (m/z): 827
[L2 + 2Cu + ClO4 + 2H]+, 727 [L2 + 2Cu + H]+, 660
[L2 + Cu + 2H]+. μeff: 1.86 BM at 298 K.
[Cu2L3(ClO4)(H2O)](ClO4) (3). Yield: 2.53 g (70.9%);
colour: brown. Anal. Calcd for C28H36N6O11Br2Cl2Cu2
(990.43) (%): C, 33.96; H, 3.66; N, 8.48. Found (%): C,
33.94; H, 3.70; N, 8.51. Selected IR data (KBr, ν, cm−1):
3372 ν(ꢀOH), 1628 ν(C¼N), 1525 ν(bridging phenoxide),
1365 ν(ArꢀO), 1105 and 630 ν(ClO4−), 814 ν(rocking
2.6.1
| Absorption spectral titration
The most effective method for examining the binding mode
and strength of metal complexes with DNA is electronic
absorption spectroscopy. For absorption titrations, the
experiments were carried out using a fixed concentration
for complex (100 μM) to which increments of nucleic acid
(0–500 μM) were added. Complex–DNA solutions were
allowed to incubate for 30 min at room temperature before
measurements were taken. While measuring the absorption
spectra, equal amounts of DNA were added to both com-
plex solution and the reference solution to eliminate the
absorption of DNA itself. Titration curves were con-
structed from the fractional change in the absorption
intensity as a function of DNA concentration.[42,43] The
intrinsic binding constant Kb can be obtained using the
following equation:
OH)water, 470 ν(CuꢀO), 444 ν(CuꢀN). ΛM (Ω−1 cm2 mol−1
,
in DMF): 71. UV–visible (λmax, nm (ε, M−1 cm−1) in
DMF): 360 (17 460), 640 (126). ESI‐MS in CH3CN (m/z):
873 [L3 + 2Cu + ClO4 + 2H]+, 773 [L3 + 2Cu + 2H]+, 709
[L3 + Cu + 2H]+. μeff: 1.83 BM at 298 K.
[Cu2L4(ClO4)(H2O)](ClO4) (4). Yield: 2.27 g (68.1%);
colour: green. Anal. Calcd for C29H39N6O11BrCl2Cu2
(925.56) (%): C, 37.63; H, 4.25; N, 9.08. Found (%): C,
37.61; H, 4.32; N, 9.12. Selected IR data (KBr, ν, cm−1):
3371 ν(ꢀOH), 1633 ν(C¼N), 1540 ν(bridging phenoxide),
1363 ν(ArꢀO), 1105 and 630 ν(ClO4−), 817 ν(rocking
OH)water, 472 ν(CuꢀO), 450 ν(CuꢀN). ΛM (Ω−1 cm2 mol−1,
in DMF): 76. UV–visible (λmax, nm (ε, M−1 cm−1) in DMF):
368 (18 968), 648 (112). ESI‐MS in CH3CN (m/z): 808 [L4
+ 2Cu + ClO4 + 2H]+, 644 [L4 + Cu + 2H]+. μeff: 1.89 BM
at 298 K.
½DNAꢁ ½DNAꢁ
1
¼
þ
εa−εf
εb−εf
Kbðεb−εfÞ
[Cu2L5(ClO4)(H2O)](ClO4) (5). Yield: 2.31 g (66.3%);
colour: pale brown. Anal. Calcd for C32H45N6O11BrCl2Cu2
where [DNA] is the DNA concentration (per nucleotide)
in M, εa the absorption coefficient observed at a given

KARTHICK ET AL.
5
DNA concentration, εf the absorption coefficient of com-
plex in the absence of DNA, εb the absorption coefficient
of complex when fully bound to DNA and Kb the intrinsic
binding constant in M−1. Each set of data were fitted to
the above equation, and a plot of [DNA]/(εa − εf) versus
[DNA] gave a slope and y‐intercept that are equal to
1/(εb − εf) and 1/Kb(εb − εf), respectively. Kb was
obtained from the ratio of the slope to the intercept.
1BNA) was downloaded from the protein data bank. Visual-
ization of the docked pose was conducted using the PyMOL
molecular graphics program.[49]
2.8 | Molecular docking analysis with VEGFR2 Kinase
Inhibitor
Vascular endothelial growth factor receptor (VEGFR) kinase
was retrieved from the RCSB Protein Data Bank (PDB ID:
3VO3), and docking calculations were performed using
Schrödinger software. The protein was prepared using the
Protein Preparation Wizard, pre‐processed and hetero‐state
for co‐crystallized ligand was generated using Epik; proton-
ation state and optimization of hydrogen bonding of the pro-
tein side chains were assigned using protassign, energy
minimized (impref minimization) using OPLS2001 force
field. Receptor grid was prepared with default parameters
and without any constraints. Ligand docking was performed
using OPLS2001 force field. In order to predict the binding
affinity and preeminent docked structures, the combined
ligand docking and energy‐grid scores were ranked using E
model and Glide scores. Flexible docking was performed
using the Extra Precision (XP) feature of GLIDE 5.0 module
implemented in the Schrödinger software. Visualization and
analysis of protein–ligand complexes were performed using
PyMol v. 0.99 software (DeLano Scientific LLC, CA, USA).
2.6.2
| Viscosity (hydrodynamic) measurements
Viscometric titrations were carried out using an Ostwald
micro‐viscometer (2 ml capacity) immersed in a water bath
maintained at 25 0.2°C. CT DNA (100 μM) and complexes
(0–100 μM) were prepared using Tris–HCl/NaCl buffer (pH =
7.2). Solutions were mixed well by purging nitrogen gas
through the viscometer. The flow time was measured
using a digital stopwatch, and the experiment was repeated
in triplicate to get concurrent values. Data were presented
as (η/η0)1/3 versus binding ratio (1/R) [Complex]/[DNA],
where η and η0 are the specific viscosity of DNA in the
presence and absence of the complex, respectively.[44,45]
The values of η and η0 were calculated from the relation
η = (t − tb)/tb, where tb is the flow time of buffer alone
and t the observed flow time for DNA in the absence
and presence of complex.[46] Relative viscosities for DNA
were obtained from the ratio η/η0.
2.9 | Nuclease activity
2.6.3
| Electrochemical titration
The nature of the interaction between nucleic acid and redox
active molecules can additionally be inferred using the elec-
trochemical technique.[47] For electrochemical titration, a
three‐electrode system was employed consisting of a glassy
carbon electrode as working electrode, with Ag/AgCl as ref-
erence electrode and Pt electrode as auxiliary electrode in
DMF/Tris–HCl medium. Solutions of complex and DNA
were prepared by using 5% DMF in Tris–HCl/NaCl and
Tris–HCl/NaCl, respectively. The concentration of complex
was fixed as 50 μM and for DNA as 100 μM. Solutions were
deaerated by purging with nitrogen gas for 10 min prior to
measurements.
The agarose gel electrophoresis technique was used to moni-
tor the cleavage ability of the synthesized macrocyclic com-
plexes on pBR322 DNA (33.3 μM) in Tris–HCl/NaCl
buffer. In each experiment, 4 μl of plasmid DNA was treated
with 6 μl of metal complexes of concentration 100 μM along
with H2O2 (2 μl, 40 μM) and samples were incubated for
1.5 h at 37°C. A loading buffer containing 0.25%
bromophenol blue, 0.25% xylene cyanol and 30% glycerol
(3 μl) was added and the electrophoresis of DNA was per-
formed on agarose gel (0.8%) stained with ethidium bro-
mide (1 μg ml−1). The gels were run at 50 V for 1 h in
buffer (40 mM Tris base, 20 mM acetic acid, 1 mM
EDTA, pH = 8.3). The resulting bands were visualized
by UV light and photographed. In addition, the participa-
tion of reactive oxygen species was also studied in the
presence of H2O2 as external agent and adding standard
radical quenchers DMSO, NaN3 and KI for hydroxyl, sin-
glet oxygen and H2O2 radicals, respectively.
2.7 | Docking studies with DNA
Rigid molecular docking study of complexes was performed
using HEX 6.9 software, which is an interactive molecular
graphics program for calculating and displaying feasible
docking modes of a pair of protein and DNA molecule.[48]
The by default parameters were used for the docking calcula-
tion with correlation type shape only, FFT mode at 3D level,
grid dimension of 6 with receptor range 180 and ligand range
180 with twist range 360 and distance range 40. Structures of
the complexes were sketched using CHEMSKETCH and
converted into pdb format from mol format using online
molecular format converter Open Babel. The crystal structure
of the B‐DNA dodecamer d(CGCGAATTCGCG)2 (PDB ID:
3
| RESULTS AND DISCUSSION
The synthesized macrocyclic binuclear copper(II) com-
plexes were characterized using regular physicochemical
techniques. The elemental analysis, electronic spectral
and electrochemical data evidenced the formation of com-
plexes each with a binuclear core and the same was also

6
KARTHICK ET AL.
confirmed using ESI‐MS. Bio‐relevant activities of the
synthesized complexes were determined in terms of cata-
lytic, antioxidant, DNA interaction, molecular docking
and cytotoxic activities. Several attempts to grow single
crystals of the synthesized complexes with various sol-
vents and conditions were unsuccessful and only micro‐
crystals were obtained.
TABLE 1 Electronic, magnetic and electrochemical data for complexes 1–5
Reduction at
cathode
d–d band λ
Complex max (nm)
μeff
(BM)
ΛM (Ω−1
E1
(V)
E2
(V)
pc
pc
g
cm2 mol−1
)
1
2
3
4
5
656
652
640
648
645
2.226
2.224
2.210
2.214
2.216
1.93
1.86
1.83
1.89
1.87
70
68
71
76
73
−0.74
−0.71
−0.58
−0.70
−0.65
−1.76
−1.66
−1.60
−1.64
−1.63
3.1 | Spectral features
A comparison of IR spectral data of precursor compounds
and synthesized complexes provides valid information on
coordination mode of donor atoms with the metal centres
(Fig. S1). The precursors show a broad band around 3400
cm−1 due to the OꢀH stretching of phenol. All the complexes
show a broad band centred around 3375 cm−1 due to the
OꢀH stretching vibrations of water molecules. The broad-
ness may be due to intramolecular hydrogen bonding. The
appearance of a band at ca 815 cm−1 implies the rocking
mode of water, which clearly ratifies the coordination of
water molecule to metal centre.[50] Further, complex forma-
tion is strongly evidenced by the disappearance of carbonyl
(ca 1680 cm−1) and NꢀH of primary amine (2922–2956
cm−1) stretching frequencies of the precursors with the
appearance of a sharp band in the region 1632–1666 cm−1
corresponding to ν(C¼N), as a consequence of Schiff base
condensation. All the complexes show a strong stretching
band corresponding to perchlorate at 1100 cm−1 (antisym-
metric stretching) which is split, and a sharp band around
625 cm−1 (antisymmetric bending). In addition, the peak
observed around 930 cm−1 due to coordinated perchlorate
ion[51] clearly indicates the presence of coordinated perchlo-
rate anion in the complexes. The shift in frequency of
ν(ArꢀO) from precursors (1369–1379 cm−1) to complexes
(1355–1365 cm−1) by ca 15 cm−1 indicates the coordination
of metal ion with phenolic oxygen via deprotonation. The
new peak observed for complexes in the region 1530–1544
cm−1 is assigned to phenoxide bridging with metal ions.[52]
The non‐ligand bands of complexes in the regions 515–532
and 444–452 cm−1 are attributed to ν(CuꢀO) and ν(CuꢀN),
respectively. The absence of ν(HC¼O) and ν(NꢀH) and a
shift in ν(ArꢀO) of the precursors, and the appearance of
prominent bands due to ν(C¼N), ν(CuꢀO) and ν(CuꢀN)
for the complexes become strong evidence for the formation
of the complexes.
of the complexes were recorded in positive ion mode, which
also provides additional confirmation for the formation of
complexes (Fig. 1). The spectra of complexes show molecu-
lar ion peak corresponding to the entire complex excluding
one uncoordinated perchlorate anion, and some prominent
peaks correspond to the various fragments of complexes.
The proposed molecular formulae of the complexes corrobo-
rate well with ESI‐MS data of the complexes.
3.2 | Electrochemical studies
The molar conductance (ΛM) for complexes 1–5 was mea-
sured in DMF medium and the values are found to be
68–76 Ω−1 cm2 mol−1 corresponding to a uni‐univalent elec-
trolytic nature of the complexes.[54] These results indicate the
presence of an uncoordinated perchlorate anion in the
complexes.
Cyclic voltammetry was carried out for complexes 1–5 in
DMF using TBAP as supporting electrolyte in nitrogen atmo-
sphere (Fig. S3) and the reduction potential values are given
in Table 1. Complexes containing phenoxide bridge usually
undergo reduction at the cathodic region due to hard nature
and negative influence of phenoxide ion.[55] Generally, fac-
tors like degree and distribution of unsaturation, axial liga-
tion, nature of chelation and nature of substituent at the
para position of phenoxide group have control over the elec-
trochemical properties of metal complexes. The electrode
process is irreversible because no anodic peak in the cathodic
direction is obtained even when changing the scan rates. The
The electronic spectra of complexes were recorded at
room temperature in DMF. The absorption spectra of all com-
plexes exhibit three peaks with salient features (Fig. S2). The
intense peak observed below 300 nm is assigned to intra‐
ligand charge transfer transition (π → π*). The medium inten-
sity broad band in the region 372–400 nm is ascribed to
ligand‐to‐metal charge transfer transition. The spectra show
the d–d transition band in the range 640–656 nm with very
low absorptivity (Table 1), which is characteristic of Cu2+
ion in hexa‐coordination environment.[53] The ESI‐MS data
FIGURE 1 ESI mass spectrum of complex 1

KARTHICK ET AL.
7
first reduction potential (E1pc) values are obtained in the
range between −0.69 and −0.74 V and the second reduction
potential (E2pc) values are between −1.60 and −1.72 V. Here,
each reduction wave corresponds to one‐electron transfer, and
the reduction process can be assigned as follows:
gradually decreases to 0.118 cm3 K M−1 at 20 K. In this
region, the nature of the curve is uniform and indicates the
temperature dependence of the magnetic property. Intermo-
lecular antiferromagnetic coupling, intramolecular magnetic
coupling between two metal ions and orbital momentum con-
tribution are the usual factors responsible for temperature
dependence of magnetic moment in binuclear complexes.[59]
The two metal ions bridging through phenoxide ion give rise
to antiferromagnetic coupling, which leads to the unusual
magnetic behaviour of the complexes. The modified
Bleany–Bowers equation[60] is used to evaluate the singlet–
triplet energy separation (−2J) by fitting the magnetic sus-
ceptibility values:
CuIICuII→ CuIICuI→ CuICuI
The reduction potential value of 3 shifts more towards a
positive potential and this is because of the more negative
inductive effect (an electron displacement effect) exerted by
the electronegative bromine atom at the para position to the
phenoxide ion. The electron‐withdrawing bromine atom
decreases the electron density around the complex, which
favours easy reduction at less negative potential. The
observed behaviour is similar to that of previously reported
macrocyclic copper(II) complexes containing phenoxide
bridge and an electronegative atom in the para position to
the phenoxide ion.[56] Studies were also carried out to deter-
mine whether the redox behaviour of the complexes is due
to metal ion or ligand reactions[57] by recording the cyclic
voltammograms for precursor compounds, with the precur-
sors showing one irreversible peak in the cathodic region,
which may be due to an electron transfer of phenolic oxygen.
The voltammograms obtained for the complexes are entirely
different from those of the precursors, which suggests that
the complexes undergo reduction at negative potentials only
under the influence of the Cu(II) centre.
ꢀ
ꢁꢂ
ꢀ
ꢁꢃ
−1
Ng2β2
3KT
−2J
KT
0:45P
T
χM
¼
3 þ exp
ð1−PÞ þ
þ Nα
where χM is the paramagnetic susceptibility per metal
atom, N is the Avogadro number, g is the average gyromag-
netic ratio, Nα is the temperature‐independent fraction usually
assumed to be 120 × 10−6 cm3 M−1 for Cu(II) compounds, β is
the Bohr magnetron, K is the Boltzmann constant and P is the
percentage of monomeric impurities. The observed exchange
integral (−2J) value is 187 cm−1. The exchange interactions
are primarily controlled by Cu–O–Cu bridge angle. In addi-
tion, electronegativity of the bridging atom, degree of distor-
tion from planar geometry and dihedral angle between two
copper planes also influence the exchange interactions.[61]
3.3 | Magnetochemistry
3.4 | Phosphate hydrolysis ability
The solid‐state EPR spectra of the complexes were recorded
at room temperature with a field strength of 3200 G and fre-
quency of 9.414 GHz at 298 K (Fig. S5). The g values are
summarized in Table 1, and the obtained values are a measure
for the presence of magnetic interaction between copper
ions.[58] The room temperature magnetic moment (μeff)
values for the complexes lie between 1.83 and 1.93 BM, indi-
cating the presence of an antiferromagnetic interaction
between two copper(II) ions.
The magnetic exchange interaction was further supported
by temperature‐dependent magnetic susceptibility studies. A
variable temperature magnetometer with an applied magnetic
field of 0.2 T in the temperature range 20–300 K was
employed to measure magnetic susceptibility data. Plots of
χM and χMT versus T for complex 1 are depicted in Fig. S6.
A χM value of 0.24 × 10−3 cm3 M−1 at 300 K gradually
changes on lowering the temperature from 300 to 50 K.
Below 50 K, a steep increase in χM is observed and the value
reaches a maximum at 23 × 10−3 cm3 M−1 at 20 K. The
obtained χMT curve is entirely different from that of the χM
plot. In the temperature range between 300 and 150 K, no
significant change in the χMT plot is observed. Below 150
K, the value increases steeply towards a maximum at around
82 K with a χMT value of 0.136 cm3 K M−1, and then
The kinetic behaviour of the macrocyclic binuclear copper(II)
complexes was investigated spectrophotometrically in the
hydrolysis of 4‐nitrophenylphosphate at 420 nm for 45 min
at regular 5 min intervals, and an increase in absorption was
observed due to the formation of 4‐nitrophenolate anion
(Fig. S4). The obtained rate constant (k) value for the com-
plexes lies in the range 1.83 × 10−2–4.19 × 10−2 min−1
(Table 2). The coordination environment and electrochemical
properties are important factors in determining the kinetic
behaviour of the complexes. The para substituent of the
ligand influences the 4‐nitrophenylphosphate hydrolysis pro-
cess. The complexes with electron‐withdrawing group show
higher activity than complexes containing electron‐releasing
group,[62] whereas those with both electron‐withdrawing
and electron‐releasing substituents show moderate kinetic
activity.
3.5 | In Vitro radical scavenging assay
The evaluation of DPPH radical scavenging activity is a stan-
dard assay in antioxidant studies, and offers a rapid technique
for screening the radical scavenging properties of specific
compounds or extracts.[63] In general, many bio‐redox pro-
cesses generate free radicals, which may induce oxidative

8
KARTHICK ET AL.
TABLE 2 Initial rate constant values for hydrolysis of 4‐
nitrophenylphosphate, and antioxidant and intrinsic binding constant data for
complexes 1–5
mechanism of the radical scavenging activity of the com-
plexes under study remains unclear, these results would be
helpful in developing potent radical scavengers.
K
b (× 103 M−1
)
IC50 for
Compound
k (×10−2 min−1
)
DPPH (μM)
CV
UV–visible
3.6 | DNA binding activity
1
2.76
1.83
4.19
—
103.2 0.6
094.6 0.8
112.5 1.1
—
2.94
3.96
1.32
1.85
2.54
—
3.10
4.17
1.49
1.92
2.68
—
Covalent and/or non‐covalent interactions are usually
observed when transition metal complexes bind with DNA.
In covalent binding, nitrogen base of DNA such as guanine
N7 replaces the labile ligand of a complex, whereas in non‐
covalent binding, the double‐stranded DNA can bind in three
possible ways: (i) electrostatic interaction with the negatively
charged sugar–phosphate backbone, which is along the exter-
nal DNA double helix without any selectivity, (ii) intercala-
tion between the stacked base pairs of native DNA and (iii)
interactions with two grooves of the DNA double helix.[65]
2
3
4
5
—
—
Vitamin C
—
132.7 1.5
damage in various components of the body, including lipids,
proteins and DNA, and have been implicated in aging and a
number of life‐limiting chronic diseases like cancer, hyper-
tension, cardiac infarction, atherosclerosis, rheumatism, cata-
racts, etc.[64] Efforts to counteract the damage caused by
these species are gaining acceptance as a basis for novel ther-
apeutic approaches, and the field of preventive medicine is
experiencing an upsurge of interest in useful antioxidants.
The stable free radical DPPH shows a strong absorption
band at 517 nm due to the presence of an odd electron, and
the absorption strength of this band decreases stoichiometri-
cally when the odd electron becomes paired off by accepting
an electron or hydrogen radical. Hence, the more rapidly the
absorbance decreases the more potent is the antioxidant activ-
ity. The scavenging activity of the complexes was compared
with that of the well‐known natural antioxidant vitamin C
(ascorbic acid). The complexes showing better scavenging
activity could be due to coordination of metal after complex-
ation, increasing its capacity to stabilize the unpaired electron,
and thereby to arrest free radicals. The antioxidant potency
(IC50) follows the order 2 > 1 > 3 > vitamin C (Table 2).
The effect of scavenging property (Fig. 2) varies by changing
the substituent in the ligand, i.e. the complexes containing
electron‐releasing substituent show higher activity than those
with electron‐withdrawing substituent. Although the
3.6.1
| Electronic absorption titration
The binding mode of metal complexes with CT DNA is usu-
ally characterized using the electronic absorption technique
by following the changes in absorbance and shift in wave-
length upon varying the concentration of DNA.
Hyperchromism and hypochromism are the two spectral fea-
tures of DNA concerning its double helix structure.[66] When
a complex is bound to DNA through groove binding,
hyperchromism is observed, whereas a complex bound to
DNA via intercalation mode exhibits hypochromism.
The absorption band intensity of complexes increases on
increasing the DNA concentration, which indicates strong
binding of the complexes to CT DNA. The observed
hyperchromic effect with a slight red shift (1–3 nm) strongly
indicates the interaction of complexes to DNA through
groove mode of binding. The hydrogen binding sites avail-
able in the structure of DNA might be a reason for such bind-
ing. The absorption spectral plots for complexes 2 and 4 with
varying concentration of CT DNA are shown in Fig. 3. The
binding propensities of the complexes are usually expressed
with the intrinsic binding constant (Kb) value (Table 2), and
the binding affinity (103 M−1) of the complexes follows the
order 2 (4.17) > 1 (3.10) > 5 (2.68) > 4 (1.92) > 3 (1.49).
The higher binding strength of complex 2 is attributed to
the hydrophobic interaction of tert‐butyl group with the
hydrophobic DNA surface, which leads to the enhancement
of DNA binding affinity.[67] The lower binding affinity of
complex 3 may be due to a higher density created by bromine
substituents. The presence of more nitrogen atoms in the
complexes plays a vital role for the secondary interaction of
complexes with DNA. The Kb values obtained for the com-
plexes are comparable with those for some other reported
polyaza macrocyclic copper(II) complexes.[68]
3.6.2
| Viscosity (hydrodynamic) measurement
In the absence of a crystal structure for a complex, one of the
best methods to ascertain the mode of interaction between the
complex and DNA is viscometric titration.[69] In intercalation
FIGURE 2 DPPH radical scavenging activity of complexes 1–3

KARTHICK ET AL.
9
3.6.3
| Electrochemical titration
Knowledge of DNA interaction of the reduced and/or the
oxidized form of a metal can also be complemented by
studying the electrochemical behaviour of metal complexes
with DNA.[42] For studying the binding mode of complexes
with DNA using cyclic voltammetry, the redox potential
was measured for a fixed concentration of the complex with
increases in the concentration of CT DNA. Usually, a posi-
tive shift in potential is observed for intercalation mode of
binding, whereas a negative shift is observed for electro-
static interaction. Both intercalation and electrostatic interac-
tion can be inferred when there is simultaneous existence of
more than one potential. On increasing the concentration of
DNA for a fixed concentration of complex, a decrease in
cathodic peak current is observed without any new redox
peaks (Fig. 4). The decrease in current intensity may be
due to the diffusion of an equilibrium mixture of free and
DNA‐bound complex on the electrode surface, suggesting
the occurrence of an interaction between each complex
and DNA.
The sequential drop in peak current of complexes 1–5 on
the addition of varying concentrations of DNA, ranging from
25 to 100 μM, can be used to quantify the binding constant
using the following equation:[71,72]
ꢀ
ꢁ
ꢀ
ꢁ
1
I
IO
log
¼ logKb þ log A −IA
½DNAꢁ
where Kb is the binding constant and IO and IA are the
peak currents of the complex in the absence and presence
of DNA, respectively.
FIGURE 3 Absorption spectra of complexes (a) 2 and (b) 4 (100 μM) in
Tris–HCl/NaCl (pH = 7.2) buffer upon addition of CT DNA (0–250 μM).
Arrow shows that absorbance increases with increasing DNA concentration.
Insets: plots of [DNA]/(εa − εf) versus [DNA] for absorption titration of
DNA with complexes
The binding constants, Kb = 1.32 × 103–3.96 × 103 M−1
,
for the interaction of the complexes with DNA were
obtained from the intercept of plots of log(1/DNA) versus
log(IA/IO − IA) (Fig. 5). The higher Kb of complex 2 than
the others is presumably due to the additional hydrophobic
interactions of the bulky butyl groups with nitrogenous
bases of DNA.[73] The obtained results imply that the bind-
ing of the complexes to DNA is through a non‐covalent
mode of interaction. The binding ability of complexes fol-
lows the order 2 > 1 > 5 > 4 > 3. The Kb values are
found to be comparable with the values obtained from
electronic titration. The results of absorption spectral titra-
tion, viscosity measurement and electrochemical titration
indicate an identical order for the degree of interaction of
the complexes with CT DNA, and it is strongly inferred
that a non‐covalent groove mode of binding is operative
in the present case.
mode, the relative viscosity of DNA solution increases due to
an increase in overall DNA contour length similar to the clas-
sic intercalator ethidium bromide. In contrast, in partial or
non‐intercalative binding, the DNA helix could bend or kink
and as a consequence the relative viscosity of DNA solution
decreases.[70] In the case of electrostatic or groove binding,
there will be little or no effect on the relative viscosity of
DNA solution.
The relative specific viscosity (η/η0) was determined, and
plotted against 1/R = 0.1–1, where R = [DNA]/[complex]
(Fig. S7). From the obtained data, only a slight increase in
the relative viscosity is observed, which is a positive sign of
groove binding mode. When the concentration of complexes
increases, the relative viscosity of DNA also increases. This
behaviour is similar to that of distamycin (D), a classic
minor‐groove binder. The binding ability of the complexes fol-
lows the order D > 2 > 1 > 5 > 4 > 3. Increase in viscosity of
DNA refers to an increase in length of DNA and, among the
complexes, the affinity of binding is most for 2 and least for 3.
3.7 | Docking studies with DNA
Molecular docking is an attractive technique for elucidating
the interaction mode and binding affinity between a drug
and various biological molecules at a supramolecular level.
In our experiment, molecular docking studies of complexes

10
KARTHICK ET AL.
FIGURE 4 Cyclic voltammograms of complexes (a) 1 and (b) 2 in the
absence (solid curve) and presence (dashed curve) of CT DNA
FIGURE 6 Molecular docking of complexes (a–d) 1–4 with the DNA
duplex of sequence d(CGCGAATTCGCG)2
FIGURE 5 Plots of log(IA/IO − IA) versus log(1/[DNA]) used to calculate
the binding constants of complexes 1–5 with DNA
the DNA groove. It is observed that the complexes interact
through outside edge and A–T regions suggesting the groove
nature of binding to DNA, and stabilized by van der Waals
interaction and hydrophobic contacts with DNA functional
groups, which define the stability of the groove[74] as shown
in Fig. 6.
1–5 with DNA duplex of sequence d(CGCGAATTCGCG)2
dodecamer (PDB ID: 1BNA) were performed in order to pre-
liminarily predict the chosen binding site along with the pre-
ferred orientation of sterically acceptable complexes inside

KARTHICK ET AL.
11
According to the docking results (Table 3), the binding
Gly1011, Asn1162, Gln1185 and Cys862. The selection of
best docked pose has been chosen based on empirical and
force‐field‐based terms. The results reveal that complexes 4
and 5 are half‐surrounded within sub‐domain IIA hydropho-
bic cavity, and is in close proximity to hydrophobic residues
such as Lys195, Trp827, Arg835, Leu896, His895, Lys920,
Arg863 and Glu828 of sub‐domain IIA of BSA, suggesting
the existence of hydrophobic interaction between them
(Fig. 7). The lower the relative binding energy, the more
potent is the binding affinity between BSA and target mole-
cules. The more negative relative binding energy of complex
5 indicates its stronger binding ability to BSA than complex
4, because it contains a large hydrophobic cavity in sub‐
domain IIA, which plays a key role in metabolism and trans-
portation of BSA. The results obtained from molecular
docking studies indicate that the interaction between com-
plexes and BSA is dominated by hydrophobic forces and
π–π stacking interactions, which is consistent with our
binding titration techniques. The observed features are in
good agreement with the results obtained from DNA binding
titration techniques.
affinity of the complexes with DNA indicates that van der
Waal interactions are dominant over electrostatic interactions.
Since the conformation of complexes had not changed in the
minimum energy docked poses, the total internal energy
remains zero. The molecular docking results show that com-
plexes 1–5 bind efficiently with the DNA receptor and
exhibit free energy of binding (FEB) values from −2.61 to
−10.34 kcal mol−1. Thus, the observed more negative FEB
values suggest an effective binding affinity, which correlates
well with the experimental DNA binding results. The more
negative relative binding energy of complex 2 indicates its
higher DNA binding affinity than the other complexes,
because 2 shows strong hydrophobic interactions with right‐
handed DNA.[75] Thus, we can conclude that there is a
mutual complement between spectroscopic techniques and
molecular docking modelling, which substantiates our exper-
imental results, and provides further evidence of intercalative
binding.
3.8 | Molecular Modelling with VEGFR2 Kinase
Inhibitor
3.9 | DNA Cleavage Activity
VEGF is a mitogen for vascular endothelial cells derived
from arteries, veins and lymphatics. VEGF also elicits a pro-
nounced angiogenic response in a wide variety of in vivo
models.[76] VEGFR2 appears to be the most important recep-
tor in VEGF‐induced mitogenesis and permeability.[77] Clin-
ically successful small molecules in the treatment of tumours,
like axitinib, pazopanib, sorafenib and sunitinib, possess the
therapeutic receptor VEGFR2.[78–81] Therefore, understand-
ing VEGF/VEGFR2 signalling in therapeutics becomes inev-
itable. In order to study the binding site selection, complexes
4 and 5 were docked into the three‐dimensional structure
(PDB ID: 3VO3) of BSA using AutoDock, and the docking
results are listed in Table 3. The predicted active sites consist
of amino acids such as Phe1018, Gln1165, Lys1038,
Ser1037, Leu896, Lys920, His895, Gly893, His856,
Asn900, Leu902, Asp857, Ile856, Arg863, Phe854,
Arg835, Phe829, Glu828, Trp827, Lys826, Thr864,
The chemistry of DNA cleavage associated with redox‐
active/photo‐activated complexes is a useful tool in the
characterization of DNA recognition of transition metal com-
plexes.[82] The cleavage activity of complexes can be studied
in two different ways: the cleavage of DNA in the absence
and presence of co‐reactant. Different DNA cleavage ability
of the complexes may be due to the different binding affinity
of the complexes to the DNA. In gel electrophoresis, the seg-
regation of DNA molecules is based on the relative rate of
movement under the influence of an electric field. DNA is
a negatively charged species and when it is placed in an elec-
tric field it will migrate towards the anode, and the extent of
migration depends on the strength of the electric field, nature
of buffer, gel density and DNA size. DNA cleavage is moni-
tored by relaxation of the supercoiled circular form of DNA
into the nicked circular and linear forms. When circular
TABLE 3 Molecular docking parameters for complexes 1–5
Final intermolecular energy
Estimated free
energy of binding
(kcal mol−1
)
Unbound system
energy (= (2))
vdW + H bond +
Complex dissolving energy
Electrostatic
energy
Final total internal
Torsional free
((1) + (2) + (3) −
Total (1)
energy (kcal mol−1) (2)
energy (kcal mol−1) (3)
(kcal mol−1) (4)
(4)) = (kcal mol−1
)
1BNA
1
−5.43
−5.75
−2.61
−4.45
−4.67
−5.11
−4.58
−1.32
−4.21
−4.89
−10.54
−10.33
−3.93
−8.66
−9.56
−0.48
+0.00
+0.00
+0.00
+0.00
+0.55
+0.00
+0.00
+0.00
+0.00
−0.48
+0.00
+0.00
+0.00
+0.00
−9.99
−10.33
−3.93
−8.66
−9.56
2
3
4
5
VEGFR2 kinase
4
5
−3.12
−3.44
−4.26
−4.37
−7.38
−7.81
−0.31
+0.82
+0.00
−0.31
−6.56
−7.81
+0.00
+0.00

12
KARTHICK ET AL.
The interaction of complexes with plasmid DNA is explained
as follows. The central metal ion may bind with phosphodiester
bond of the nucleic acid through the coordinate linkage and/or
electrostatic interaction. As a consequence, a phosphorus atom
of DNA is activated followed by nucleophilic attack of metal
ion (Lewis acid) via charge neutralization, and finally one of
the PꢀO ester bonds of DNA is cleaved. Sometimes the water
molecules associated with the complexes may lead to direct
hydrolysis of diester bonds by attacking the phosphorus atom,
resulting in a trigonal bipyramidal phosphorus intermediate,
and finally one of the phosphodiester bonds of DNA is cleaved.
The cleaving ability of complexes on DNA may be due to the
influence of substituents.
The degradation of DNA was also examined in the presence
of co‐reactant H2O2 (oxidative pathway). Generally, in the pres-
ence of co‐reactant, it is believed that various oxygen‐based
intermediates in the form of non‐diffusible metal–peroxo species
or diffusible radicals like hydroxyl, hydroperoxyl, hydrogen per-
oxide, singlet oxygen etc. are generated. Thus, cleavage can
occur by direct oxidation of deoxyribose ring, which involves
non‐diffusible species or by damage of deoxyribose ring (C3′)
of nucleic acid involving diffusible reactive oxygen species.[84]
In general, the nuclease activity of metal‐based molecules acting
as chemotherapeutics depends on co‐reactants for initiating the
DNA cleavage process.
In the present case, enhanced cleavage activity is
observed in the presence of co‐reactant. To gain information
on the involvement of reactive oxygen species, radical scaven-
gers were included in the cleavage reactions (Fig. 8). Lanes
2–10 represent the cleavage activity of complexes in the pres-
ence of co‐reactant H2O2 and radical scavengers DMSO
(hydroxyl radical), NaN3 (singlet oxygen) and KI (H2O2).[85]
For complexes 1, 3 and 4 (lanes 4, 7 and 10), the cleavage
activity is completely inhibited in the presence of KI suggest-
ing H2O2 might be the diffusible reactive oxygen species for
DNA cleavage. In addition, complex 4 (lane 9) shows inhibi-
tion of cleavage in the presence of DMSO and in this case
hydroxyl radical might also be a reactive oxygen species. Pro-
nounced cleavage activity is observed in the presence of co‐
reactant when compared to the activity in the absence of H2O2.
3.10 | Cytotoxicity
3.10.1
| MTT assay
FIGURE 7 Docking poses of complexes (a, b) 4 and (c, d) 5 with VEGFR2
kinase receptor
The cytotoxicity of complexes 1, 3 and 4 was examined
against human epidermoid carcinoma cell line (A431), by
maintaining the cells in varying concentrations of complexes
plasmid DNA is run on a horizontal gel, the fastest migration
will be observed for the supercoiled form (SC, Form I). Nicked
circular form (NC, Form II) will be produced as a consequence
of relaxation of supercoiled form, when a single strand of
DNA alone cleaved. On the other hand, a linear form (LC,
Form III) that migrates in between Form I and Form II will
be generated when both strands of DNA are cleaved.[83]
Hydrolytic cleavage pathway of complexes 1–5 demon-
strates that the cleaving ability is dose dependent (Fig. S8).
FIGURE 8 Oxidative cleavage of pBR322 DNA by complexes 1, 3 and 4
(100 μM) in Tris–HCl/NaCl buffer (pH = 7.2) with H2O2 and incubated for
90 min at 37°C. Lane 1: DNA control; lanes 2, 5, 8: DNA + 1/3/4 + NaN3;
lanes 3, 6, 9: DNA + 1/3/4 + DMSO; lanes 4, 7, 10: DNA + 1/3/4 + KI

KARTHICK ET AL.
13
for 24 h, using MTT reduction assay. This assay gives an
index of cell viability by detecting the reduction of yellow tet-
razolium salt to purple blue formazan, developed as a result
of a metabolic change, which is mitochondrial enzyme activ-
ity of succinate dehydrogenase in living cells. The inhibitory
concentration required to reduce the size of a cell population
by 50% (IC50 value) of complexes 1, 3 and 4 is 150.06,
442.02 and 281.47 μg ml−1, respectively. Results of MTT
assay (Fig. S9) clearly indicate that the complexes inhibit
the growth of epithelial cancer cells significantly in a dose‐
dependent manner. Complex 1 shows a significant decrease
in the viability of carcinoma cells in comparison with
FIGURE 9 Morphology of (a) control and A431 cell line treated with
complexes (b) 1, (c) 3 and (d) 4
FIGURE 10 Propidium iodide stained morphology of (a) control and A431
cell line treated with complexes (b) 1, (c) 3 and (d) 4

14
KARTHICK ET AL.
ACKNOWLEDGEMENTS
complexes 3 and 4. Beyond 125 μM concentration, it has a
significant anticancer activity, and at a concentration of 250
μM the cell viability becomes as low as 4%. For complex 4,
a considerable activity is observed at a concentration of 125
μM and the activity progresses in a linear fashion. From the
results, it may be concluded that complex 1 has more potent
anticancer activity against A431 cell line than complexes 3
and 4.
A.K.R. gratefully acknowledges the financial assistance from
the University Grants Commission (UGC), New Delhi,
through a Major Research Project Grant (F. no. 39‐797/
2010 (SR)). C.K. thanks UGC, New Delhi, for providing
financial assistance in the form of a fellowship.
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oxidant, DNA interaction, molecular docking and
cytotoxicity studies of aminoethylpiperazine‐contain-
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