Heteroscorpionate-based heteroleptic copper(II) complexes: Antioxidant, molecular docking and in vitro cytotoxicity studies

Article abstract of DOI:10.1002/aoc.3809

Three new heteroscorpionate ligands, (2-hydroxyphenyl)bis(imidazol-1-yl)methane (HL¹), (4-diethylamino-2-hydroxyphenyl)bis(imidazol-1-yl)methane (HL²) and (5-bromo-2-hydroxyphenyl)bis(imidazol-1-yl)methane (HL³), and their heteroleptic copper(II) complexes of the type [Cu(L^1–3)diimine]ClO₄ (1–6; where diimine =2,2′-bipyridyl or 1,10-phenanthroline) have been synthesized and characterized using spectroscopic methods. The molecular structure of ligand HL¹ was determined by single-crystal X-ray diffraction. UV–visible, electron paramagnetic resonance and theoretical studies suggest a distorted square pyramidal geometry around copper(II) ion. Analyses of highest occupied and lowest unoccupied molecular orbitals have been used to explain the charge transfer taking place within the complexes. The antioxidant activities of the heteroscorpionate ligands and their heteroleptic copper(II) complexes were determined using ABTS, DPPH and H₂O₂ free radical scavenging assays with respect to standard antioxidant ascorbic acid. In molecular docking studies, the complexes showed π–π, hydrogen bonding, van der Waals and electrostatic interactions with fibroblast growth factor receptor kinase. In vitro cytotoxicity activities of ligands and copper(II) complexes were examined on human breast adenocarcinoma (MCF-7), cervical (HeLa) and lung (A549) cancer cell lines and normal human dermal fibroblast cell line using MTT assay. Complex 4 exhibited higher anticancer activity than the other complexes against all three cancer lines, being more potent than the standard drug cisplatin.

Full text of DOI:10.1002/aoc.3809

Received: 26 December 2016
DOI: 10.1002/aoc.3809
Revised: 20 February 2017
Accepted: 24 February 2017
F U L L PA P E R
Heteroscorpionate‐based heteroleptic copper(II) complexes:
Antioxidant, molecular docking and in vitro cytotoxicity studies
1,2
1
3
3
1
S. Jayakumar | D. Mahendiran | V. Viswanathan | D. Velmurugan | A. Kalilur Rahiman
1
Post‐Graduate and Research Department of
Three new heteroscorpionate ligands, (2‐hydroxyphenyl)bis(imidazol‐1‐yl)methane
Chemistry, The New College (Autonomous),
Chennai 600 014, India
1
2
(
HL ), (4‐diethylamino‐2‐hydroxyphenyl)bis(imidazol‐1‐yl)methane (HL ) and (5‐
2
Department of Chemistry, Misrimal
3
bromo‐2‐hydroxyphenyl)bis(imidazol‐1‐yl)methane (HL ), and their heteroleptic
copper(II) complexes of the type [Cu(L )diimine]ClO4 (1–6; where diimine
=2,2′‐bipyridyl or 1,10‐phenanthroline) have been synthesized and characterized
Navajee Munoth Jain Engineering College,
Thoraipakkam, Chennai 600 097, India
1
–3
3
CAS in Crystallography and Biophysics,
1
University of Madras, Guindy Campus,
Chennai 600 025, India
using spectroscopic methods. The molecular structure of ligand HL was deter-
mined by single‐crystal X‐ray diffraction. UV–visible, electron paramagnetic reso-
nance and theoretical studies suggest a distorted square pyramidal geometry
around copper(II) ion. Analyses of highest occupied and lowest unoccupied
molecular orbitals have been used to explain the charge transfer taking place within
the complexes. The antioxidant activities of the heteroscorpionate ligands and their
Correspondence
A. Kalilur Rahiman, Post‐Graduate and
Research Department of Chemistry, The
New College (Autonomous), Chennai 600
0
14, India.
Email: akrahmanjkr@gmail.com;
akrahmanjkr@thenewcollege.in
heteroleptic copper(II) complexes were determined using ABTS, DPPH and H O2
2
free radical scavenging assays with respect to standard antioxidant ascorbic acid.
In molecular docking studies, the complexes showed π–π, hydrogen bonding, van
der Waals and electrostatic interactions with fibroblast growth factor receptor
kinase. In vitro cytotoxicity activities of ligands and copper(II) complexes were
examined on human breast adenocarcinoma (MCF‐7), cervical (HeLa) and lung
(A549) cancer cell lines and normal human dermal fibroblast cell line using MTT
assay. Complex 4 exhibited higher anticancer activity than the other complexes
against all three cancer lines, being more potent than the standard drug cisplatin.
KEYWORDS
antioxidant activity, cytotoxicity, heteroleptic complexes, heteroscorpionate ligands, molecular docking
1
| INTRODUCTION
can be obtained by the replacement of pyrazolyl in
homoscorpionates
with
triazolyl,
imidazolyl
and
Tris(pyrazolyl)borate (homoscorpionate) ligands are one of
the most widely exploited classes of ligands in inorganic
chemistry, first reported by Trofimenko, and modifications
of these ligands can be made by replacement of the boron
bridging atom by other elements such as carbon, silicon or
methimazolyl moieties or by changing the substituents on
the heterocyclic ring, and they exhibit a wide range of cata-
[
7–11]
lytic and biological applications.
This has led to a series
of mixed heteroscorpionate ligand systems that incorporate
histidine‐like nitrogens and one or more alkoxy, carboxylate,
thiolate, thioether or phenolate donor atoms to bind a
metal ion.
The discovery of cisplatin followed by other platinum‐
based antitumour drugs such as carboplatin, oxaliplatin and
[
1–6]
phosphorus.
These homoscorpionate ligand systems are
completely symmetric with all‐nitrogen donor atoms and
many metalloprotein active sites do not have such
monofunctional binding sites. The heteroscorpionate ligands
Appl Organometal Chem. 2017;e3809.
wileyonlinelibrary.com/journal/aoc
Copyright © 2017 John Wiley & Sons, Ltd.
1 of 14
https://doi.org/10.1002/aoc.3809

2
of 14
JAYAKUMAR ET AL.
nedaplatin was important for their use as chemotherapeutic
agents. However, severe side effects, toxicity and increasing
drug resistance of these drugs have limited their clinical
2 | EXPERIMENTAL
2.1 | Materials
Copper(II) perchlorate hexahydrate and other commercially avail-
able reagents 1,1′‐carbonyldiimidazole, 2‐hydroxybenzaldehyde,
[
12,13]
applications.
In order to overcome these challenges,
considerable attempts are being made to substitute the plati-
num‐based drugs with suitable alternatives, and numerous
metal‐based antitumour drugs entered clinical trials, and have
4‐(diethylamino)‐2‐hydroxybenzaldehyde and 5‐bromo‐2‐
hydroxybenzaldehyde were purchased from Aldrich and used
without further purification. Cobalt(II) chloride hexahydrate,
byp and phen were commercial products from SD Fine and
AVRA chemicals (India). Solvents were dried and purified before
being used according to standard procedures and stored over
molecular sieves. Tetra(n‐butyl)ammonium perchlorate (TBAP)
used as supporting electrolyte in the electrochemical measure-
ments was purchased from Fluka and recrystallized from hot
methanol. (Caution! Perchlorate salts and TBAP are potentially
explosive; hence care should be taken in handling these
compounds.)
[14–19]
been screened for their anticancer activities.
Among
these, copper complexes have been investigated as most
[
20,21]
promising substitutes for the platinum‐based drugs.
Copper is an essential micronutrient for most living organ-
isms, which plays an important role either in biological sys-
tems or as unique chemotherapeutic agents. The redox
property of copper ion is important for several biological pro-
cesses such as metabolism, free radical scavenging, mito-
chondrial respiration and iron absorption, where it acts as a
[
22]
catalytic co‐factor.
Copper(II) complexes exhibit various
tunable coordination geometries in the redox state, which
have better applications in the treatment of various chronic
diseases and also act as antimicrobial, antioxidant and
2.2 | Physical methods
Elemental analyses were performed with a Carlo Erba model
106 elemental analyser. Fourier transform infrared (IR)
spectra were recorded using KBr pellets in the range
[
23–26]
antitumour agents.
In this regard, the nitroimidazole‐
1
and glucosamine‐conjugated heteroscorpionate copper(II)
complexes have been investigated as effective chemothera-
1
4
00–4000 cm− with a Bruker ALPHA FT‐IR spectropho-
[
27]
peutic agents against tested human tumour cell lines.
tometer. Electronic spectra of complexes were recorded in
HPLC‐grade DMF with an Agilent‐8453 spectrophotometer
in the range 200–800 nm at ambient temperature. H NMR
spectral data of ligands were collected using a VNMRS‐400
spectrometer in deuterated dimethylsulfoxide (DMSO‐d6)
Interestingly, the heteroleptic copper(II) complexes derived
from heteroscorpionate ligands containing N,O donor ligands
with different heterocyclic N,N′ diimines as co‐ligands have
been reported as effective anticancer, DNA binding and
1
[
28]
cleavage agents.
solution with tetramethylsilane as an internal standard at
In view of all these observations, herein we report
ambient temperature. Electrospray ionization (ESI) mass
spectra were recorded with a Q‐Tof mass spectrometer using
acetonitrile as the carrier solvent. Redox properties of com-
plexes were determined using a CHI 602D (CH Instruments
Co., USA) electrochemical analyser. The measurements were
carried out with a three‐electrode cell in which a glassy car-
bon, saturated Ag/AgCl and platinum wire were used as the
working, reference and auxiliary electrodes, respectively.
TBAP was used as supporting electrolyte for the electro-
chemical work and nitrogen gas was purged prior to all the
measurements. X‐band electron paramagnetic resonance
the synthesis of heteroscorpionate‐based heteroleptic
copper(II) complexes of the type [Cu(L )diimine]ClO4,
containing three biologically active heteroscorpionate
1–3
core ligands, (2‐hydroxyphenyl)bis(imidazol‐1‐yl)methane
1
(
HL ), (4‐diethylamino‐2‐hydroxyphenyl)bis(imidazol‐1‐yl)
2
methane
(HL )
imidazol‐1‐yl)methane (HL ), and diimine (2,2′‐bipyridyl
byp) or 1,10‐phenanthroline (phen)) as co‐ligand. Geometry
and
(5‐bromo‐2‐hydroxyphenyl)bis
3
(
(
optimization and highest occupied molecular orbital
HOMO)–lowest unoccupied molecular orbital (LUMO)
analysis were realized using density functional theory
DFT) calculations. The antioxidant activity was evaluated
(
(EPR) spectra were recorded at room temperature with a
(
Varian EPR‐E‐112 spectrometer using DPPH as the field
marker. Room temperature magnetic moment was measured
with a PAR (model 155) vibrating sample magnetometer.
using ABTS (2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sul-
fonic acid)), DPPH (2,2′‐diphenyl‐1‐picrylhydrazyl) and
H O free radical scavenging assays. Further, in vitro cyto-
2
2
toxicity was tested against human breast adenocarcinoma
(
MCF‐7), cervical (HeLa) and lung (A549) cancer cell lines
2.3 | General procedure for synthesis of
and normal human dermal fibroblast (NHDF) cell line using
MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium
bromide) assay. A molecular docking study was performed
with fibroblast growth factor receptor (FGFR) using the
AutoDock Tools (ADT) version 1.5.6 and AutoDock version
Heteroscorpionate ligands
2
.3.1 | (2‐Hydroxyphenyl)bis(imidazol‐1‐yl)
1
methane (HL )
A mixture of 1,1′‐carbonyldiimidazole (4.38 g, 27 mmol), 2‐
hydroxybenzaldehyde (2.87 ml, 27 mmol) and CoCl ⋅6H O
4
.2.5.1 docking programs.
2
2

JAYAKUMAR ET AL.
3 of 14
(
0.20 g, 0.84 mmol) was heated to 100 °C with vigorous stir-
2.4 | General procedure for synthesis of
Heteroscorpionate‐based Heteroleptic
copper(II) complexes (1–6)
ring for 6 h. The reaction mixture was then allowed to cool
slowly to room temperature. The obtained brown melt was
then dissolved in methanol (100 ml), and washed with deion-
ized water (3 × 30 ml). The organic phase was dried over
MgSO , filtered and volatiles removed under reduced pres-
sure yielding a brown solid. The crude product was dried
and recrystallized from hot methanol to give HL as brown
crystals.
Yield 0.18 g (77%); m.p. 218 °C. Anal. Calcd for
C H N O (240.26) (%): C, 64.99; H, 5.03; N, 23.32. Found
To a methanolic solution of Cu(ClO ) ⋅6H O (1 mmol),
4
2
2
1–3
appropriate heteroscorpionate ligand (HL , 1 mmol) was
added dropwise with constant stirring for 30 min at room
temperature followed by the dropwise addition of the metha-
nolic solution (10 ml) of heterocyclic base (byp/phen,
4
1
1
mmol). Then, the reaction mixture was stirred for 2 h and
the resulting precipitate was filtered, washed with methanol
and diethyl ether, and finally evaporated under vacuum.
1
3 12 4
(%): C, 64.92; H, 4.99; N, 24.32. Selected IR data (KBr, ν,
−
1
1
cm ): 3216 ν(O─H), 1648 ν(C═N), 1372 ν(Ar─O). H
NMR (DMSO‐d , 400 MHz, δ, ppm): 6.75 (d, 4H, imidazole
protons), 6.84 (t, 1H, ArH), 6.92 (d, 1H, ArH), 7.24 (t, 2H,
ArH), 8.15 (s, 2H, imidazole protons), 10.25 (br s, 1H,
1
[
Cu(L )byp]ClO (1)
4
6
Yield 0.39 g (86%). Anal. Calcd for C H N O ClCu
23 19 6 5
(558.43) (%): C, 49.46; H, 3.43; N, 15.05. Found (%): C,
−
1
−
1
−1
49.42; H, 3.40; N, 15.04. Selected IR data (KBr, ν, cm ):
604 ν(C═N, imidazole), 1576 ν(C═N, byp), 1347
Ar─OH). UV–visible (λmax, nm (ε, M cm ) in DMF):
64 (22 186), 358 (4241).
1
2
−
ν(Ar─O), 1095 and 621 (uncoordinated ν(ClO )).
4
UV–visible (λmax, nm (ε, M− cm ) in DMF): 269 (21 268),
1
−1
1
+
2
.3.2 | (4‐Diethylamino‐2‐hydroxyphenyl)
359 (2864), 656 (91). ESI‐MS (m/z): [Cu(L )(byp)] (459).
2
−
1 2
−1
bis(imidazol‐1‐yl)methane (HL )
Conductance (Λ , Ω cm mol ) in DMF: 73. EPR at
M
1
298 K: g|| = 2.176, g = 2.053. μeff = 1.82 BM at 298 K.
⊥
The procedure for the synthesis was similar to that of HL
except that 4‐(diethylamino)‐2‐hydroxybenzaldehyde was
used in place of 2‐hydroxybenzaldehyde to give HL as a
purple solid.
1
2
[Cu(L )phen]ClO (2)
4
Yield 0.40 g (83%). Anal. Calcd for C H N O ClCu
582.45) (%): C, 51.55; H, 3.29; N, 14.43.Found (%): C,
1.51; H, 3.26; N, 14.42. Selected IR data (KBr, ν, cm ):
609 ν(C═N, imidazole), 1521 ν(C═N, phen), 1348
ν(Ar─O) 1090 and 624 (uncoordinated ν(ClO )).
UV–visible (λmax, nm (ε, M cm ) in DMF): 274 (24
64), 337 (2923), 648 (94). ESI‐MS (m/z): [Cu(L )(phen)]
483). Conductance (Λ , Ω cm mol ) in DMF: 84. EPR
2
5 19 6 5
(
5
1
Yield 0.25 g (79%); m.p. 202 °C. Anal. Calcd for
C H N O (311.38) (%): C, 65.57; H, 6.80; N, 22.49. Found
−
1
1
7 21 5
(
%): C, 65.51; H, 6.74; N, 22.48. Selected IR data (KBr, ν,
−
−
1
4
cm ): 3221 ν(O─H), 2970 ν(N─C H ), 1632 ν(C═N),
1
2
5
−
1
−1
1
376 ν(Ar─O). H NMR (DMSO‐d , 400 MHz, δ, ppm):
6
1
+
5
(
1
.10–1.14 (t, 6H, ─N(CH CH ), 3.40–3.42 (m, 4H,
2
3
−
1
2
−1
M
─
N(CH CH ), 6.05 (s, 1H, ArH), 6.34 (d, 4H, imidazole
2 3
at 298 K: g = 2.178, g = 2.063. μ = 1.78 BM at 298 K.
||
⊥
eff
proton), 6.65 (d, 1H, ArH), 7.42 (d, 1H, ArH), 8.15 (s, 2H,
imidazole protons), 9.62 (br s, 1H, Ar─OH). UV–visible
(
2
λmax, nm (ε, M− cm ) in DMF): 278 (17 128), 361 (3144).
1
−1
[
Cu(L )byp]ClO (3)
4
Yield 0.42 g (80%). Anal. Calcd for C H N O ClCu
27 28 7 5
(
5
2
629.55) (%): C, 51.51; H, 4.48; N, 15.57. Found (%): C,
2
.3.3 | (5‐Bromo‐2‐hydroxyphenyl)
−1
1.46; H, 4.45; N, 15.57. Selected IR data (KBr, ν, cm ):
974 ν(N─C H ), 1603 ν(C═N, imidazole), 1536 ν(C═N,
3
bis(imidazol‐1‐yl)methane (HL )
2
5
1
byp), 1352 ν(Ar─O), 1092 and 620 (uncoordinated
ν(ClO )). UV–visible (λmax, nm (ε, M cm ) in DMF):
2
(
The procedure for the synthesis was similar to that of HL
except that 5‐bromo‐2‐hydroxybenzaldehyde was used in
place of 2‐hydroxybenzaldehyde to give HL as a yellow
solid.
Yield 0.24 g (75%); m.p. 198 °C. Anal. Calcd for
C H N BrO (319.16) (%): C, 48.92; H, 3.47; N, 17.55.
−
−1
−1
4
2
3
79 (22 635), 328 (2715), 641 (98). ESI‐MS (m/z): [Cu(L )
+
−1
2
−1
byp)] (530). Conductance (Λ , Ω cm mol ) in DMF:
M
9
2. EPR at 298 K: g = 2.183, g = 2.048. μ = 1.80 BM
||
⊥
eff
at 298 K.
1
3 11 4
Found (%): C, 48.87; H, 3.44; N, 17.54. Selected IR data
−1
2
(
KBr, ν, cm ): 3218 ν(O─H), 1642 ν(C═N), 1383
[Cu(L )phen]ClO
4
(4)
1
ν(Ar─O). H NMR (DMSO‐d , 400 MHz, δ, ppm): 6.75 (d,
Yield 0.43 g (78%). Anal. Calcd for C H N O ClCu
29 28 7 5
(653.57) (%): C, 53.29; H, 4.32; N, 15.00. Found (%): C,
53.24; H, 4.28; N, 14.99. Selected IR data (KBr, ν, cm ):
2979 ν(N─C H ), 1611 ν(C═N, imidazole), 1570 ν(C═N,
phen), 1365 ν(Ar─O), 1093 and 621 (uncoordinated
6
1
H, ArH), 6.86 (d, 1H, ArH), 7.33 (d, 4H, imidazole pro-
tons), 7.95 (s, 1H, ArH), 8.28 (s, 2H, imidazole protons),
0.72 (br s, 1H, Ar─OH). UV–visible (λmax, nm (ε, M
−
1
1
2
5
−
1
−1
cm ) in DMF): 259 (19 621), 354 (2624).

4
of 14
JAYAKUMAR ET AL.
ν(ClO )). UV–visible (λ , nm (ε, M−1 cm ) in DMF):
−
−1
ORTEP‐PLATON program. The hydrogen atoms were
placed in calculated positions with C─H = 0.93 to 0.98 Å
and O─H = 0.82 Å by using a riding model, but not refined
and with fixed isotropic displacement parameters:
4
max
2
[
71 (19 265), 332 (2241), 634 (102). ESI‐MS (m/z):
2 + 2
−1
−1
Cu(L )(phen)] (554). Conductance (Λ , Ω cm mol )
M
in DMF: 97. EPR at 298 K: g = 2.123, g = 2.056. μ = 1.81
||
⊥
eff
BM at 298 K.
U (H)
iso
=
1.5Ueq(C) for methyl groups and
U (H) = 1.2Ueq(C) for C aromatic. The methyl groups were
allowed to rotate but not to tip.
iso
3
[
Cu(L )byp]ClO (5)
4
Yield 0.43 g (81%). Anal. Calcd for C H N O BrClCu
2
3 18 6 5
(
4
1
637.33) (%): C, 43.34; H, 2.85; N, 13.18. Found (%): C,
3.30; H, 2.82; N, 13.17. Selected IR data (KBr, ν, cm ):
−1
2.6 | Computational details
604 ν(C═N, imidazole), 1572 ν(C═N, byp), 1349
[
29]
−
The Gaussian 03 software package
was used for
ν(Ar─O), 1098 and 623 (uncoordinated ν(ClO )).
UV–visible (λmax, nm (ε, M cm ) in DMF): 268 (20
9
(
4
−1
−1
theoretical calculations. The quantum chemical calculations
were performed by applying the DFT method with Becke‐
3
+
43), 334 (3142), 645 (96). ESI‐MS (m/z): [Cu(L )(byp)]
−
1
2
−1
3‐Lee‐Yang‐Parr (B3LYP) supplemented with the standard
538). Conductance (Λ , Ω cm mol ) in DMF: 94. EPR
M
[30]
LANL2DZ and GEN basis set.
The optimized geometry
at 298 K: g = 2.092, g = 2.020. μ = 1.79 BM at 298 K.
||
⊥
eff
corresponding to the minimum on the potential energy
surface was obtained by solving self‐consistent field equation
iteratively.
3
[
Cu(L )phen]ClO (6)
4
Yield 0.46 g (81%). Anal. Calcd for C H N O BrClCu
2
5 18 6 5
(
4
1
661.35) (%): C, 45.40; H, 2.74; N, 12.71. Found (%): C,
5.36; H, 2.72; N, 12.70. Selected IR data (KBr, ν, cm ):
−1
2.7 | In Vitro antioxidant Activity
603 ν(C═N, imidazole), 1574 ν(C═N, phen), 1372
−
ν(Ar─O), 1092 and 621 (uncoordinated ν(ClO )).
UV–visible (λmax, nm (ε, M cm ) in DMF): 262 (19
2
(
4
In vitro antioxidant activity of both the heteroscorpionate
ligands and their heteroleptic copper(II) complexes was
evaluated via their ability to scavenge ABTS, DPPH and
H O radicals and comparing the results with standard anti-
−1
−1
3
+
63), 321 (2432), 628 (108). ESI‐MS (m/z): [Cu(L )(phen)]
−
1
2
−1
562). Conductance (Λ , Ω cm mol ) in DMF: 87. EPR
M
2
2
[
31,32]
at 298 K: g = 2.171, g = 2.074. μ = 1.81 BM at 298 K.
||
⊥
eff
oxidant ascorbic acid.
The percentage inhibition was
calculated from the following formula:
2
.5 | Single‐crystal X‐ray diffraction studies
Acontrol−Asample
X‐ray diffraction intensity data were collected at room
temperature (293 K) using a Bruker SMART APEX single‐
crystal X‐ray diffractometer equipped with graphite
monochromatized Mo Kα (λ = 0.71073 Å) radiation
Radial scavenging ð%Þ ¼
×100
Acontrol
and CCD detector.
A
crystal of dimensions of
0.18 × 0.14 × 0.12 mm was mounted on a glass fibre using
2.7.1 | ABTS scavenging assay
cyanoacrylate adhesive. The unit cell parameters were deter-
mined for 36 frames measured (0.5° phi‐scan) from three
different crystallographic zones using the method of differ-
ence vectors. The intensity data were collected with an aver-
age four‐fold redundancy per reflection and optimum
resolution (0.75 Å). The intensity data collection, frame inte-
gration, Lorentz and polarization corrections and decay cor-
rection were carried out using SAINT (version 7.06a)
software. An empirical absorption correction (multi‐scan)
was performed using the SADABS program. The crystal
structure was solved by direct methods using SHELXS‐97
and refined by full‐matrix least‐squares using SHELXL‐
ABTS (54.8 mg, 2 mM) was mixed with potassium persulfate
0.3 ml, 17 mM) in distilled water (50 ml) to produce ABTS
(
radical cation. The reaction mixture was left to stand at room
temperature overnight in the dark before usage. To 0.2 ml of
various concentrations of samples/standard, freshly distilled
DMSO (1.0 ml) and 0.16 ml of ABTS solution were added
to make a final volume of 1.36 ml. After 20 min, absorbance
was measured spectrophotometrically at 734 nm.
2.7.2 | DPPH scavenging assay
2014. Molecular geometry was calculated using PARST.
The assay was carried out in a 96‐well microtitre plate. To
200 μl of DPPH solution, 10 μl of each of the samples or
standard solution was added separately in wells of the micro-
titre plate. The plates were incubated at 37 °C for 30 min and
the absorbance of each solution was measured at 517 nm.
All non‐hydrogen atoms were refined using anisotropic ther-
mal parameters. The hydrogen atoms were included in the
structure factor calculation at idealized positions using a
riding model, but not refined. Images were created with the

JAYAKUMAR ET AL.
5 of 14
2
.7.3 | H O scavenging assay
3 | RESULTS AND DISCUSSION
2
2
A solution of H O (20 mM) was prepared in phosphate buff-
2
2
The heteroscorpionate ligands (HL1–3) were obtained in
good yield and their heteroleptic copper(II) complexes
ered saline (pH = 7.4). Various concentrations of 1 ml of
samples or standard were added to 2 ml of H O solution in
2
2
(
1–6) were synthesized from the reaction between equimo-
phosphate buffered saline. After 10 min, the absorbance
was measured at 230 nm.
lar amount of heteroscorpionate ligands and heterocyclic
base (byp or phen) as co‐ligand in the presence of
copper(II) perchlorate in methanol (Scheme 1). All the
complexes were obtained in good yield as air‐stable solids,
2
.8 | Molecular docking studies
which are readily soluble in CH Cl , CH CN, DMF and
2 2 3
DMSO. The stoichiometry of the synthesized complexes
was determined based on elemental analysis and spectral
techniques.
Molecular docking with FGFR was carried out by following
the procedure reported in our earlier publication.
[33]
2
2
.9 | In Vitro cytotoxicity studies
.9.1 | Cell lines and cell culture conditions
1
3
.1 | Crystal structure of HL
1
Heteroscorpionate ligand HL crystallizes in monoclinic
crystal system with C2/c space group and the measured unit
cell dimensions are a = 16.1890(17), b = 7.7234(8),
c = 18.8760(2) Å, and α = 90°, β = 92.446(7)°, γ = 90°
and Z = 8. The three‐dimensional molecular structure of this
ligand was determined using X‐ray crystallography with
SHELXS‐97 and later refined by SHELXL‐2014 to a final
R factor of 0.0413. The crystal data and structure refinement
parameters are given in Table 1. Selected bond lengths and
bond angles are given in Table 2. The details of hydrogen
bonding and selected torsion angles are given in Tables 3
and S1, respectively.
MCF‐7, HeLa, A549 and NHDF cell lines were obtained
from the National Centre for Cell Science (NCCS), Pune,
India, and cultured in Dulbecco's modified Eagle's medium.
The medium was supplemented with 10% foetal bovine
−
1
−1
serum, 100 μg ml penicillin, 20 μg ml streptomycin,
1
2
0 μg ml− kanamycin acid sulfate and 7.5% sodium bicar-
bonate solution. The cell lines were maintained at 37 °C in
a 5% CO incubator and the medium was changed frequently.
2
MTT (a yellow tetrazole) was obtained from Himedia, India.
1
The molecular structure of ligand HL is shown in
2
.9.2 | MTT assay
Figure 1. The two imidazole and one phenyl rings are in an
almost planar conformation in the structure. The imidazole
rings 1 and 2 make a dihedral angle of 72.91(9)° from the
mean plane. The mean plane of the phenyl ring makes a
dihedral angle of 82.75(8)° and 84.97(8)° with the
imidazole rings 1 and 2, respectively. Oxygen atom O1
In vitro cytotoxicity of both the heteroscorpionate ligands
and their heteroleptic copper(II) complexes against three can-
cerous cell lines (MCF‐7, HeLa and A549) and NHDF cell
line was determined using MTT assay by following the pro-
[33]
cedure reported in our earlier publication.
SCHEME 1 Schematic route for
synthesis of heteroscorpionate ligands
1–3
(
HL ) and their heteroleptic copper(II)
complexes (1–6)

6
of 14
JAYAKUMAR ET AL.
1
TABLE 1 Crystal data and structure refinement for ligand HL
TABLE 2 Selected bond lengths (Å) and bond angles (°) for ligand
HL
1
(CCDC 1475335)
Empirical formula
C
13
H
12
N
4
O
Bond length (Å)
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
240.27
296(2)
0.71073
N1–C1
1.3418(16)
1.3675(17)
1.4737(14)
1.4501(16)
1.5113(16)
1.3587(16)
1.3573(17)
1.3708(18)
1.3886(18)
1.4002(17)
N3–C1
N3–C2
N4–C7
N4–C6
C5–C6
C3–C2
C5–C6
C9–C10
C11–C10
C8–C9
1.3122(17)
1.3561(19)
1.304(2)
N1–C3
N1–C4
Monoclinic
C4–N2
1.368(2)
C2/c
C4–C8
1.345(2)
O1–C13
1.3464(19)
1.345(2)
16.1890(17)
7.7234(8)
18.876(2)
90
N2–C7
b (Å)
N2–C5
1.380(2)
c (Å)
C13–C12
C13–C8
1.377(2)
α (°)
1.3863(17)
β (°)
92.446(7)
90
Bond angle (°)
C1–N1–C3
C1–N1–C4
C3–N1–C4
N2–C4–N1
N2–C4–C8
N1–C4–C8
C7–N2–C5
C7–N2–C4
C5–N2–C4
O1–C13–C12
γ (°)
106.41(11)
128.93(11)
124.61(10)
109.85(9)
C1–N3–C2
C3–C2–N3
C5–C6–N4
C6–C5–N2
N3–C1–N1
O1–C13–C8
C10–C9–C8
C9–C8–C13
C13–C8–C4
C11–C12–C13
105.46(11)
110.15(13)
110.86(15)
105.77(14)
111.72(12)
117.46(11)
120.70(13)
119.44(12)
118.05(11)
120.03(13)
3
Volume (Å )
2357.9(4)
8
Z
Calculated density (Mg m−
Absorption coefficient (mm−
3
)
1
1.354
)
0.091
110.44(10)
111.62(10)
106.43(12)
126.73(12)
126.83(11)
123.11(12)
F(000)
1008
Crystal size (mm)
0.18 × 0.14 × 0.12
θ range for data collection (°)
2.16–28.40
Limiting indices
−21 ≤ h ≤ 21, −10 ≤ k ≤ 10,
25 ≤ l ≤ 24
−
Reflections collected/unique
Completeness to theta (%)
Absorption correction
10 709/2877 [R(int) = 0.0301]
97.30
1
TABLE 3 Hydrogen bonding parameters for ligand HL
Semi‐empirical from equivalents
0.9892 and 0.9838
Max. and min. transmission
Refinement method
d(D–H)
d(H∙∙∙A)
d(D∙∙∙A) ∠(DHA)
a
2
D–H∙∙∙A (Å)
(Å)
(Å)
(Å)
(°)
Full‐matrix least‐squares on F
O1–H1∙∙∙N3 (i)
C9–H9∙∙∙N1
0.82
0.93
0.93
1.87
2.53
2.58
2.6848(5)
2.8576(17)
3.5139(17)
172
101
133
Data/restraints/parameters
2877/0/165
1.014
2
Goodness‐of‐fit on F
C3–H3∙∙∙Cg1
Final R indices [I > 2sigma(I)]
R indices (all data)
R
R
1
1
= 0.0413, wR
2
2
= 0.1068
= 0.1156
(
ii)
C5–H5∙∙∙Cg2
iii)
= 0.0542, wR
0.93
2.73
3.6687(16)
154
Extinction coefficient
Largest diff. peak and hole (e Å− ) 0.296 and −0.160
0.0020(5)
(
3
a
Symmetry codes: (i) − ½ + x, −½ + y, z; (ii) − x, 1 − y, 1 − z; (iii) − x, y, ½ − z.
attached to the phenyl ring lies in a plane evidenced from the
torsion angle values of [C9–C8–C13–O1] = −179.9° and
generates a C(8) chain running along [100] plane and its view
down the b‐axis is shown in Figure 2. The two molecules are
also held together by C3─H3∙∙∙Cg1 and C5─H5∙∙∙Cg2 inter-
actions with the hydroxyl phenyl ring (symmetry code of
Cg1 = −x, 1 − y, 1 − z and Cg2 = −x, y, ½ − z). These
two C─H∙∙∙π interactions generate a chain running along
the c‐axis and its view down the b‐axis is shown in Figure
S1. In the crystal, the molecules are stacked in layers held
together by π–π interactions, with a distance of Cg3 and
[C11–C12–C13–O1] = −179.6°. In this crystal, the packing
is stabilized by intermolecular O─H∙∙∙N and intramolecular
C─H∙∙∙N types of hydrogen bonds. In addition, the crystal
packing is also stabilized by C─H∙∙∙π interaction. An intra-
molecular C9─H9∙∙∙N1 hydrogen bond in the crystal gener-
ates an S(5) ring motif as shown in Figure 1. The
intermolecular interaction of O1─H1∙∙∙N3 hydrogen bond

JAYAKUMAR ET AL.
7 of 14
3.2 | Spectral characterization
IR spectra of the heteroscorpionate ligands (HL1–3) exhibit a
broad band in the region 3216–3221 cm− assigned to the
phenolic ν(O─H) stretching vibration (Fig. S3). The com-
plexes fail to display this ν(O─H) stretching band, which
implies the absence of phenolic group due to deprotonation
upon complexation (Figs S4 and S5). Further, the ν(C═N)
1
−
1
stretching frequency (1632–1648 cm ) of imidazole moiety
of heteroscorpionate ligands and the ν(C═N) ring stretching
−
1
frequency of diimines (1578–1586 cm ) are shifted towards
−1
lower frequencies (1603–1611 and 1521–1574 cm , respec-
tively), indicating the coordination of nitrogen atoms of both
heteroscorpionate ligands and diimines with the copper ion.
The free ligands exhibit a band in the region 1372–1383 cm
−
1
due to ν(Ar─O), which is shifted to lower frequency
−
1
(1347–1365 cm ) upon complexation, suggesting coordina-
tion via phenoxide atom. The presence of perchlorate anion
in the complexes is observed at ca 1100 cm− (ν antisym-
1
1
FIGURE 1 ORTEP diagram of ligand HL , showing the atom
labelling and displacement ellipsoids drawn at 50% probability level.
The C─H∙∙∙N contact is shown as a thin dashed line
3
metric stretching) which is slightly broadened but not split,
and the band at ca 625 cm− (ν antisymmetric bending).
1
4
−1
The absence of band at ca 930 cm
(ν2 symmetric
Cg4 of 3.4953(9) Å, between the centroids of two adjacent
stretching) due to coordinated perchlorate ion clearly indi-
imidazole rings (symmetry code of Cg3 is x, y, z; Cg4 is
cates the presence of uncoordinated perchlorate anion in the
complexes.
[
34,35]
½
− x, ½ − y, 1 − z) as shown in Fig. S2.
1
FIGURE 2 Crystal packing diagram of ligand HL

8
of 14
JAYAKUMAR ET AL.
The newly synthesized heteroscorpionate ligands were
The X‐band EPR spectra of copper(II) complexes 1–6
were recorded in solid state under a magnetic field strength
of 3200 G (frequency of 9.414 GHz) at 298 K (Fig. S10).
The trend g > g > g (2.0023) indicates the presence of an
1
characterized using H NMR spectra (Figs S6 and S7).
2
Ligand HL shows triplet in the region 1.10–1.14 ppm and
multiplet in the region 3.40–3.42 ppm due to methyl and
||
⊥
2
methylene protons, respectively, of (─N─C H ) substituent
unpaired electron in the d
x2−y2
molecular orbital ( B state)
2
5
1g
36]
[
attached to aromatic ring. The aromatic and imidazole
protons in the ligands show a signal in the region
of the Cu(II) ion, and square pyramidal geometry.
The
observed room temperature magnetic moment (μ ) values
eff
6
9
.05–8.28 ppm. All the ligands show a signal at
.62–10.72 ppm attributed to phenolic (─OH) proton.
The ESI mass spectra of all the complexes were studied in
of one electron paramagnetic copper(II) complexes were
obtained in the region 1.78–1.82 BM, which is slightly
higher than the spin only value (1.73 BM), indicating a dis-
crete monomeric nature of the complexes.
positive mode, which confirm the formation of heteroleptic
copper(II) complexes (Figures 3 and S8). The mass spectra
of the complexes show molecular ion peaks corresponding
to the molecular weight of the entire complex excluding the
counter ion. The base peak observed corresponds to the
molecular weight of the ligands (HL ). The peak intensities
are in accordance with the abundance of the ions. The mass
spectral data provide additional confirmation for the forma-
tion of heteroleptic complexes, which agrees well with the
proposed molecular formulae.
3.3 | DFT Calculations
3.3.1 | Geometry optimization
1–3
DFT calculations give suitable information about structural
description, in the absence of crystal data, in addition to the
energy minimized conformation, and a tool of growing
importance for the structural investigation of coordination
and organometallic compounds. In this regard, the complexes
1–6 were optimized at B3LYP/LANL2DZ and B3LYP/GEN
levels in gas phase and the optimized ground‐state geometry
structures are shown in Figure 4. The calculated structural
parameters (bond lengths and bond angles) of the complexes
are given in Table S2. The calculated values of bond angles
obtained using B3LYP/LANL2DZ level are more appropriate
than those obtained using B3LYP/GEN level. Therefore,
DFT/B3LYP/LANL2DZ level was preferred to B3LYP/
GEN level for determining the molecular structure. The
five‐coordinated copper(II) complexes coordinate through
1–3
The electronic spectra of ligands HL
and complexes
1–6 were recorded in the region 200–800 nm (Fig. S9). The
electronic spectra of ligands exhibit intense absorption bands
in the UV region 264–361 nm attributed to intra‐ligand transi-
tions (π–π* and n–π*). The complexes exhibit three bands in
the regions 262–279, 321–359 and 628–656 nm. The first
band is assigned to intra‐ligand charge transfer transition
(
π–π*) and the second band is due to ligand‐to‐metal charge
transfer transition (n–π*) whereas the third band with low
intensity is assigned to d–d transition, which suggest distorted
square pyramidal geometry around the copper(II) ion.
[
36]
FIGURE 3 ESI mass spectrum of heteroleptic copper(II) complex 5

JAYAKUMAR ET AL.
9 of 14
predicted by calculating τ values using the following equa-
[
39]
tion:
β−α
τ ¼
6
0
where α and β are the equatorial and axial bond angles, respec-
tively. Generally, when the τ value is close to 0, then the geom-
etry is square pyramidal; when the τ value is close to 1, then
the geometry is trigonal bipyramidal. If the τ value is between
0
and 0.5, the molecular geometry is distorted trigonal bipyra-
midal. For complexes 1–6, α (N(2)–Cu–N(6)) values are
3.8°, 94.12°, 93.69°, 92.99°, 92.63° and 93.41°, respectively,
and β (N(7)–Cu–(O1)) values are 89.89°, 90.17°, 88.25°,
0.32°, 89.89° and 89.12°, respectively. The observed results
9
9
suggest square pyramidal geometry for the synthesized
heteroleptic complexes as their τ values are equal to 0.
3.3.2 | Frontier molecular orbitals
Frontier molecular orbital values are used to exemplify the
chemical reactivity and kinetic stability of molecules, which
also play critical role in the study of electrical, optical and
[
40,41]
chemical properties.
The HOMO–LUMO energy gap
has also been used to confirm the bioactivity from intramo-
[
42]
lecular charge transfer.
Interestingly, the HOMO–LUMO
energy gaps are essential for evaluating the reduction and
oxidation potentials of molecules. Information about these
orbitals may be important for drug metabolism, because most
drugs are metabolized by oxidation and reduction reactions.
Moreover, molecules having high EHOMO are good electron
donors while those having low ELUMO are good electron
acceptors. The orbital energy level analysis for complexes
1–6 shows that EHOMO values are −4.29, −4.12, −3.92,
−
−
4.42, −4.01 and −3.86 eV while ELUMO values are −1.21,
1.18, −2.14, −3.12, −1.86 and −1.01 eV, respectively
(Figure 5). The ΔE values of complexes 1–6 are 3.08, 2.94,
1
.78, 1.30, 2.15 and 2.85 eV, respectively. The low values
of energy gap may be due to the groups that enter into
conjugation, and the resulting small energy gap values of
complexes 3 and 4 support the easy charge transfer in these
complexes, which influences the biological activity of the
molecules.
FIGURE 4 Optimized structures of the heteroleptic copper(II)
complexes 3 (a) and 4 (b)
3.4 | Electrochemistry
one phenolic ─OH, two imidazole nitrogens (N1 and N2) and
two nitrogen atoms of diimine (byp or phen). The observed
bond length values are comparable with previously reported
The molar conductivity values of the synthesized complexes
1–6 in DMF at 25 °C are in the range 73–97 Ω− cm mol
1
2
−1
indicating the uni‐univalent electrolyte nature of the
[37,38]
[43]
experimental values.
The B3LYP method in combina-
complexes.
tion with the LANL2DZ basis gives an excellent estimation
of Cu─N and Cu─O bond distances: Cu─N1, Cu─N2,
Cu─N3, Cu─N4 and Cu─O. Molecular geometries can be
The electrochemical properties of complexes are of
interest, which depend on a number of factors such as
chelate ring/size, axial ligation, degree and distribution of

1
0 of 14
JAYAKUMAR ET AL.
[
44]
unsaturation and substitution pattern in the chelate ring.
The electrochemical properties of complexes 1–6 were stud-
ied using cyclic voltammetry in the potential range from 0
to −1.0 V versus Ag/AgCl using DMF containing 0.1 M
TBAP as supporting electrolyte under similar conditions
(Figure 6). All mononuclear copper(II) complexes show a
well‐defined irreversible one‐electron reduction wave in the
cathodic region (E ) between −0.62 and −0.67 V. The
pc
reduction process is assigned as follows:
CuðIIÞ→CuðIÞ
3.5 | Antioxidant Activity
1–3
The antioxidant activities of free ligands HL
and their
complexes 1–6 were determined against ABTS, DPPH and
H O radicals at concentrations varying from 2.5 to
2
2
1
00 μM with respect to the standard antioxidant ascorbic acid
(Figure 7). The IC50 values for the inhibitory effects of the
ligands and their complexes on the tested radicals are given in
Table 4. It is observed that the inhibitory effects of the tested
compounds against all radicals are increased with increasing
concentration of the compounds. All the complexes exhibit
higher scavenging activity when compared with their corre-
sponding ligands due to the chelation with the copper ion.
In general, the antioxidant activity of ligands and complexes
in all concentrations against the tested free radicals ABTS,
DPPH and H O with respect to standard antioxidant ascor-
2
2
bic acid was found to decrease in the order 4 > ascorbic acid
2
1
3
>
3 > 2 > 1 > 6 > 5 > HL > HL > HL . Among the tested
complexes, complex 4 shows higher free radical scavenging
activity against all the tested radicals. Interestingly, complex
4
also shows higher antioxidant activity than the standard
antioxidant ascorbic acid. Lower IC50 values observed in
FIGURE 5 Frontier molecular orbitals of the heteroleptic copper(II)
complexes 3 (a) and 4 (b)
FIGURE 6 Reduction cyclic voltammograms of heteroleptic
copper(II) complexes (1–6)

JAYAKUMAR ET AL.
11 of 14
of mouse genetics targeted on FGF/FGFR and the analysis
of FGFR mutations involved in human skeletal disorders
revealed essential molecular mechanisms by which
[
46]
FGF/FGFR signalling controls bone formation.
For this
reason, we were interested in studying the interaction of
the synthesized complexes 1–6 with FGFR kinase receptor
(Figure 8). It can be seen that the complexes mostly bind
with hydrophobic pocket of the FGFR kinase receptor.
The observed free binding energy values are listed in Table
S3. All the complexes strongly interact with FGFR via π–π,
hydrogen bonding, electrostatic and van der Waals interac-
tions. Complex 3 is stabilized by one hydrogen bonding
interaction formed between hydrogen atom of LYS 129
and phenolic oxygen atom (bond length: H∙∙∙O = 3.2 Å).
Besides, the binding model with FGFR kinase receptor is
enhanced by electrostatic interaction formed between this
complex and residues GLU 12, GLY 13, THR 14, ASP
2 2
FIGURE 7 Trends in the inhibition of ABTS, DPPH and H O
radicals by the heteroscorpionate ligands (HL ) and their heteroleptic
1–3
copper(II) complexes (1–6)
1
−3
TABLE 4 Radical scavenging activity of ligands HL
complexes 1–6
and their
1
27, LYS 129 and GLU 162, and van der Waals interac-
tions formed between the complex and the residues TYR
5, GLN 131, ASN 132, ASP 145, VAL 163, VAL 164,
IC50 (μM)
1
Compound
ABTS
DPPH
2
H O
2
THR 165 and ARG 169. Complex 4 shows one π–π inter-
action formed between the imidazole ring and LYS 129
1
HL
31.07 ± 0.21
29.11 ± 0.09
33.18 ± 0.35
19.16 ± 0.11
18.02 ± 0.25
17.99 ± 0.21
14.08 ± 0.17
22.01 ± 0.06
21.87 ± 0.20
17.90 ± 0.10
27.56 ± 0.19
26.45 ± 0.39
29.52 ± 0.21
22.67 ± 0.17
14.23 ± 0.51
13.19 ± 0.09
10.23 ± 0.07
19.18 ± 0.61
18.09 ± 0.14
12.42 ± 0.15
29.98 ± 0.36
27.51 ± 0.26
31.72 ± 0.41
26.37 ± 0.54
17.03 ± 0.41
18.01 ± 0.11
13.11 ± 0.17
22.81 ± 0.21
19.64 ± 0.28
16.97 ± 0.08
(bond length: 3.6 Å). Further, the binding model is
2
HL
enhanced by electrostatic interaction formed between this
complex and residues GLU 12, GLY 13, THR 14, LYS
129, GLN 131, ASN 132, ASP 145 and GLU 162, and
van der Waals interactions formed between the complex
and the residues TYR 15, LYS 33, LEU 134, ALA 144
and VAL 164.
The observed data indicate FGFR kinase receptor as a
potent inhibitor for all the complexes, because all the com-
plexes are retained tightly by the binding pocket of FGFR.
Comparing the binding affinity of the complexes, the binding
of complex 4 with FGFR is stronger than that of the other
complexes. The lower the relative binding energy, the more
potent is the binding affinity between FGFR and target
molecules. The obtained results from molecular docking
studies indicate that the interaction between complexes and
FGFR is dominated by electrostatic, van der Walls, hydrogen
bonding and π–π interactions.
3
HL
1
2
3
4
5
6
Ascorbic acid
antioxidant assays demonstrate that these complexes have a
strong potential as drugs for eliminating radicals. The effect
of radical scavenging activity of the complexes
varies on changing the substituent of the ligand and the rela-
tive π‐acidity of co‐ligand (byp/phen) in the complexes.
Further, the results obtained confirm that the heteroleptic
copper(II) complex containing ─N(C H ) substituent and
2
5 2
phen as co‐ligand is more effective in arresting the formation
of free radicals.
3.7 | In Vitro cytotoxicity
In vitro cytotoxicity of the synthesized ligands HL1–3 and
their complexes 1–6 was evaluated for three cancer cell lines
(
MCF‐7, HeLa and A549) and NHDF cell line using MTT
3
.6 | Targeting of FGFR
assay with cisplatin as standard (positive control). The meta-
bolic activity of the cells was assessed by their ability to
cleave the tetrazolium rings of the pale yellow MTT to pro-
duce a dark blue water‐insoluble formazan crystal: only live
cells reduce yellow MTT to blue formazan products but not
dead cells. In the present cytotoxicity studies, five different
concentrations (5, 10, 20, 50 and 100 μM) of compounds
FGFRs are a family of heparin‐binding growth factors,
which play a critical role in the control of cell proliferation,
cell differentiation, cellular processes and development. It
has also been show that FGFR‐activating mutations and
over‐expression are closely associated with the development
[45]
and progression of tumours in humans.
The development

1
2 of 14
JAYAKUMAR ET AL.
FIGURE 8 Molecular docking poses of the heteroleptic copper(II) complexes 3 (a) and 4 (b) with FGFR kinase receptor (PDB ID: 3C4F)
were tested for 48 h. The cell viability is dose‐dependent,
which decreases gradually with increasing concentration of
the compounds. A dose–response graph (Figure 9).was
plotted and the IC50 values are given Table 5. Further,
both ligands and complexes are active against the tested
cancer cell lines. The cytotoxicity of both ligands and
complexes on the NHDF cells is much lower than that on
the three tumorigenic cell lines. Interestingly, complex 4
1−3
TABLE 5 In vitro cytotoxicity for ligands HL and their complexes
–6
1
IC50 (μM)
MCF‐7
Compound
HeLa
A549
NHDF
1
HL
38.17 ± 0.32 28.12 ± 0.24 33.74 ± 0.23 >100
36.19 ± 0.42 27.15 ± 0.31 31.23 ± 0.04 >100
41.28 ± 0.05 29.64 ± 0.63 35.98 ± 0.14 >100
23.64 ± 0.01 18.14 ± 0.10 20.82 ± 0.61 >100
23.42 ± 0.05 16.31 ± 0.12 19.23 ± 0.05 >100
19.00 ± 0.07 15.93 ± 0.76 16.45 ± 0.11 >100
2
HL
3
HL
1
2
3
4
13.63 ± 0.14
9.50 ± 0.18 11.87 ± 0.05 >100
5
28.17 ± 0.12 22.88 ± 0.11 25.93 ± 0.67 >100
26.76 ± 0.87 17.65 ± 0.08 25.03 ± 0.48 >100
FIGURE 9 In vitro cytotoxicity of heteroscorpionate ligands (HL –3
and their heteroleptic copper(II) complexes (1–6) against three cancer
and one normal cell lines
1
)
6
Cisplatin
15.87 ± 0.09 12.93 ± 0.22 13.01 ± 0.16
94.6

JAYAKUMAR ET AL.
13 of 14
shows the highest anticancer activity against all three cancer
lines, and is more potent than cisplatin (positive control).
The potency of the complexes to kill cancer cells follows
the order 4 > 3 > 2 > 1 > 6 > 5, which is based on
observed IC50 values. A possible explanation for the
cytotoxicity properties of the synthesized copper(II)
complexes could be arrived at by changing the substituent
of the ligand and the relative π‐acidity of co‐ligand
are also grateful to Sophisticated EPR Facility, Chemical
Physics Laboratory, CSIR‐CLRI, Chennai, for EPR spectral
facility.
REFERENCES
[1] S. Trofimenko, J. Am. Chem. Soc. 1967, 89, 3170.
[2] S. Trofimenko, J. Am. Chem. Soc. 1967, 89, 6288.
(
byp/phen) in the complexes. The complexes containing
[
3] S. Trofimenko, Scorpionates: The Coordination Chemistry of
Polypyrazolylborate Ligands, Imperial College Press, London
1999.
electron‐releasing substituent (─N(C H ) ) with extended
2
5 2
planar structure, which induces the π–π* conjugation,
show higher cytotoxicity than the complexes containing
electron‐withdrawing group (─Br). The copper complexes
with phen ligand are more reactive than similar ones with
[
[
4] S. Trofimenko, J. Am. Chem. Soc. 1970, 92, 5118.
5] T. Psilakis, J. C. Jeffery, J. A. McCleverty, M. D. Ward, J. Chem.
Soc. Dalton. Trans. 1997, 1645.
[
47]
byp ligand.
[
[
6] E. E. Pullen, D. Rabinovich, C. D. Incarvito, T. E. Concolino,
A. L. Rheingold, Inorg. Chem. 2000, 39, 1561.
7] C. Pettinari, C. Santini, in Comprehensive Coordination Chemistry
II: From Biology to Nanotechnology, (Eds: J. A. McCleverty,
T. J. Meyer), Elsevier, Oxford 2004 159.
4
| CONCLUSIONS
Six heteroleptic mononuclear copper(II) complexes of the
[
8] A. Otero, J. Fernandez‐Baeza, A. Antinolo, J. Tejeda, A.
Lara‐Sanchez, Dalton Trans. 2004, 1499.
1–3
type [Cu(L )diimine]ClO (1–6) containing bis(imidazol‐
4
1‐yl)methane‐based heteroscorpionate ligands and diimines
[
9] M. Pellei, C. Santini, G. G. Lobbia, F. Cantalamessa, C. Nasuti,
M. D. Prinzio, R. Gabbianelli, G. Falcioni, Appl. Organometal.
Chem. 2005, 19, 583.
as co‐ligands have been synthesized and characterized using
spectroscopic methods. The spectral and DFT results
suggested distorted square pyramidal geometry for all the
complexes in which the five coordination positions were
occupied by tridentate heteroscorpionate ligand and diimine.
The observed small energy gap values of complexes 3 and 4
support their higher biological activity. The heteroleptic
copper(II) complex 4 exhibited higher scavenging activity
than the standard ascorbic acid and other complexes against
all the tested radicals. All the complexes showed π–π, hydro-
gen bonding, van der Waals and electrostatic interactions
with FGFR kinase. Further, the cytotoxicity of the complexes
on three different cancer lines cell lines (MCF‐7, HeLa and
A549) was examined. Interestingly, complex 4 showed
higher cytotoxicity against all three cancer lines than the
widely used drug cisplatin and the other complexes with
respect to their IC50 values. The potent efficacy of complex
[
[
10] C. Marzano, M. Pellei, D. Colavito, S. Alidori, G. G. Lobbia,
V. Gandin, F. Tisato, C. Santini, J. Med. Chem. 2006, 49, 7317.
11] S. Alidori, G. G. Lobbia, G. Papini, M. Pellei, M. Porchia,
F. Refosco, F. Tisato, S. L. Jason, C. Santini, J. Biol. Inorg. Chem.
2
008, 13, 307.
[
[
12] S. H. V. Rijt, P. J. Sadler, Drug Discovery Today 2009, 14, 1089.
13] S. Iglesias, N. Alvarez, M. H. Torre, E. Kremer, J. Ellena, R. R.
Ribeiro, R. P. Barroso, A. J. Costa‐Filho, M. G. Kramer, G.
Facchin, J. Inorg. Biochem. 2014, 139, 117.
[
14] L. Ronconi, P. J. Sadler, Coord. Chem. Rev. 2007, 251, 1633.
15] G. N. Kaluderovic, R. Paschke, Curr. Med. Chem. 2011, 18, 4738.
[
[16] Z. F. Chen, Y. F. Shi, Y. C. Liu, X. Hong, B. Geng, Y. Peng,
H. Liang, Inorg. Chem. 2012, 51, 1998.
[17] A. L. Noffke, A. Habtemariam, A. M. Pizarro, P. J. Sadler, Chem.
Commun. 2012, 48, 5219.
4
─
may be due to the strong influence of the substituent
N(C H ) and the extended aromaticity of phen unit. It is
2
5 2
[18] S. Sathiyaraj, K. Sampath, R. J. Butcher, R. Pallepogu,
concluded that the outcome of the present investigation
would contribute to the chemistry of heteroscorpionate‐based
heteroleptic copper(II) complexes and their biological activi-
ties and also the present studies reveal that complexes 3 and 4
may be used as chemotherapeutic agents and hold much
promise in the field of drug discovery.
C. Jayabalakrishnan, Eur. J. Med. Chem. 2013, 64, 81.
[
[
19] N. P. Barry, P. J. Sadler, Chem. Commun. 2013, 49, 5106.
20] J. D. Ranford, P. J. Sadler, D. A. Tocher, J. Chem. Soc. Dalton
Trans. 1993, 3393.
[
21] M. Magnarin, A. Bergamo, M. E. Carotenuto, S. Zorzet, G. Sava,
Anticancer Res. 2000, 20, 2939.
[
[
22] J. R. J. Sorenson, Prog. Med. Chem. 1989, 26, 437.
ACKNOWLEDGEMENTS
23] M. L. P. D. Santos, A. F. Alario, A. S. A. Mangrich, A. M. C.
Ferreira, J. Inorg. Biochem. 1998, 71, 71.
The authors are grateful to Sophisticated Analytical Instru-
ment Facility (SAIF), Indian Institute of Technology Madras
[
24] A. Robert, B. Meunier, Chem. – Eur. J. 1998, 7, 1287.
(IIT M), Chennai, for ESI mass spectral analysis. The authors
[25] R. S. Kumar, S. Arunachalam, Polyhedron 2007, 26, 3255.

1
4 of 14
JAYAKUMAR ET AL.
[26] C. D. Fan, H. Su, J. Zhao, B. X. Zhao, S. L. Zhang, J. Y. Miao, Eur.
J. Med. Chem. 2010, 45, 1438.
[36] S. Dhar, P. A. N. Reddy, M. Nethaji, S. Mahadevan, M. K. Saha,
A. R. Chakravarty, Inorg. Chem. 2002, 41, 3469.
[
37] T. C. Higgs, C. J. Carrano, Inorg. Chem. 1997, 36, 291.
[
27] M. Pellei, G. Papini, A. Trasattia, M. Giorgetti, D. Tonelli, M.
Minicucci, C. Marzano, V. Gandin, G. Aquilanti, A. Dolmellad,
C. Santinia, Dalton Trans. 2011, 40, 9877.
[38] P. Gurumoorthy, J. Ravichandran, A. K. Rahiman, J. Chem. Sci.
2014, 126, 783.
[28] C. Santini, M. Pellei, V. Gandin, M. Porchia, F. Tisato,
[39] A. W. Addison, T. N. Rao, J. Reedijk, J. V. Rijn, G. C. Verschoor,
C. Marzano, Chem. Rev. 2014, 114, 815.
J. Chem. Soc. Dalton Trans. 1984, 1349.
[
40] M. Govindarajan, S. Periandy, K. Carthigayen, Spectrochim. Acta
[
29] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A.
Robb, J. R. Cheeseman, J. A. Montgomery Jr., T. Vreven, K. N.
Kudin, J. C. Burant, J. M. Millam, S. S. Iyengar, J. Tomasi,
V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G. A.
Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda,
J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao,
H. Nakai, M. Klene, X. Li, J. E. Knox, H. P. Hratchian, J. B. Cross,
V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann,
O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski,
P. Y. Ayala, K. Morokuma, A. Voth, P. Salvador, J. J. Dannenberg,
V. G. Zakrzewski, S. Dapprich, A. D. Daniels, M. C. Strain,
O. Farkas, D. K. Malick, A. D. Rabuck, K. Raghavachari, J. B.
Foresman, J. V. Ortiz, Q. Cui, A. G. Baboul, S. Clifford,
J. Cioslowski, B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz,
I. Komaromi, R. L. Martin, D. J. Fox, T. Keith, M. A. Al‐Laham,
C. Y. Peng, A. Nanayakkara, M. Challacombe, P. M. W. Gill, B.
Johnson, W. Chen, M. W. Wong, C. Gonzalez, J. A. Pople,
Gaussian 03, Revision D.01, Gaussian Inc., Pittsburgh, PA 2005.
A 2012, 97, 411.
[41] I. Fleming, Frontier Orbitals and Organic Chemical Reactions,
John Wiley, New York 1976.
[
42] D. Mahendiran, R. S. Kumar, V. Viswanathan, D. Velmurugan,
A. K. Rahiman, Dalton Trans. 2016, 46, 7794.
[
43] W. J. Geary, Coord. Chem. Rev. 1971, 7, 81.
[44] E. K. Barefield, G. M. Freeman, D. G. V. Derveer, Inorg. Chem.
1986, 25, 552.
[
45] I. A. N. Brooks, E. Kilgour, P. D. Smith, Clin. Cancer Res. 2012,
1
8, 1855.
[
[
46] S. Sperber, E. Spector, Mol. Pathol. Clin. Pract. 2016, 177.
47] P. P. Silva, W. Guerra, J. N. Silveira, A. M. D. C. Ferreira, T.
Bortolotto, F. L. Fischer, H. Terenzi, A. Neves, E. C. Pereira‐Maia,
Inorg. Chem. 2011, 50, 6414.
SUPPORTING INFORMATION
[
[
30] A. D. Becke, J. Chem. Phys. 1993, 98, 5648.
Additional Supporting Information may be found online in
the supporting information tab for this article.
31] R. Re, M. Pellegrini, A. Proteggente, A. Pannala, M. Yang, E. C.
Rice, Free Radical Biol. Med. 1999, 26, 1231.
[
[
[
[
32] N. T. T. Huong, K. Malsumato, R. Kasai, K. Yamasaki, H.
Watanabeth, Biol. Pharm. Bull. 1998, 21, 978.
How to cite this article: Jayakumar S, Mahendiran
D, Viswanathan V, Velmurugan D, Kalilur Rahiman
A. Heteroscorpionate‐based heteroleptic copper(II)
complexes: Antioxidant, molecular docking and
in vitro cytotoxicity studies. Appl Organometal Chem.
33] S. Jayakumar, D. Mahendiren, T. Srinivasan, G. Mohanraj, A. K.
Rahiman, J. Photochem. Photobiol. B 2016, 155, 66.
34] K. Nakamoto, Infrared and Raman Spectra of Inorganic and
Coordination Compounds, Wiley‐Interscience, New York 1978.
2
017;e3809. https://doi.org/10.1002/aoc.3809
35] P. Arthi, A. Haleel, P. Srinivasan, D. Prabhu, C. Arulvasu, A. K.
Rahiman, Spectrochim. Acta A 2014, 129, 400.