Inorganica Chimica Acta 482 (2018) 160–169

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Inorganica Chimica Acta

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Research paper

Pyridazine-based heteroleptic copper(II) complexes as potent anticancer T

drugs by inducing apoptosis and S-phase arrest in breast cancer cell
Ummer Muhammed Rafia, Dharmasivam Mahendirana, Venkat Gayathri Devib, Mukesh Dobleb,
⁎
Aziz Kalilur Rahimana,
a
Post-Graduate and Research Department of Chemistry, The New College (Autonomous), Chennai 600 014, India
b
Bioengineering and Drug Design Laboratory, Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036, India

A R T I C LE I N FO A B S T R A C T

Keywords: A new series of heteroleptic copper(II) complexes of the type [Cu(L1−3)(diimine)](ClO4) (1–6) have been syn-
DFT analysis thesized using three pyridazine-based ligands (3-chloro-6-(salicylidenehydrazinyl)pyridazine (HL1), 3-chloro-6-
Docking studies (4-diethylaminosalicylidenehydrazinyl)pyridazine (HL2) and 3-chloro-6-(5-bromosalicylidenehydrazinyl)pyr-
Anticancer activity idazine (HL3), and diimine (2,2′-bipyridine (bpy) or 1,10-phenanthroline (phen)) as co-ligands. The ligands and
Apoptosis
their copper(II) complexes have been characterized by elemental analyses and spectroscopic methods. The
Cell cycle arrest
copper(II) complexes display ligand-field band in the region 641–661 nm suggesting square pyramidal geometry.
The optimized structures of the complexes and their molecular orbital calculations obtained by the density
functional theory (DFT) also showed five coordinated distorted square pyramidal geometry around the copper
(II) ion. The cyclic voltammetric analyses of copper(II) complexes exhibit one-electron irreversible reduction
wave (Epc = −0.596 to −0.641 V) in the cathodic potential region. Anti-proliferative activity of the complexes
against breast cancer MDA-MB-231 cell line was evaluated by MTT cell proliferation assay, and the clonogenic
assay revealed improved cytotoxicity for the complexes with potency higher than the standard drug cisplatin.
Since the complexes 3 and 4 with diethylamino substituent displayed higher anti-proliferative activity than the
other complexes, these complexes were chosen for apoptosis and cell cycle analysis. The apoptosis induction was
analyzed by AO/EB staining, and the flow cytometry showed the inhibition of cell growth at the S-phase of the
cell cycle. Additionally, the interaction of copper(II) complexes with FGFR kinase receptor have been studied by
in silico molecular docking studies.

1. Introduction imidazo[1,2-b]pyridazine scaffold was found to act as tumor necrosis

Diazines are six-membered aromatics with two nitrogen atoms,
which belong to the most important N-containing heterocycles.
According to the relative position from the nitrogen atoms, three dif-
ferent structures can be distinguished viz., pyridazine (1,2-diazine) [1],
pyrimidine (1,3-diazine) [2] and pyrazine (1,4-diazine) [3]. Among
these, pyridazine is a highly π-deficient aromatic compound and the
most basic with pKa = 2.3, which favors protonation, hydrogen bond
formation and chelation through nitrogen atoms of the pyridazine ring.
The pyridazine core has also been found in a large range of biologically
active structures such as herbicides [4] and pharmaceuticals [5,6]. The
metal(II) complexes derived from pyridazine-based ligands exhibited
anticancer activity against non-small-cell lung (A549), breast (MCF-7/
MDA-MB-231), cervical (HeLa), hepatoma (HepG-2) and osteosarcoma
(MG-63) cancer cell lines [7–9]. A series of compounds derived from
factor (TNF-α) production inhibitor [10], and diarylpyridazine scaffold
functionalized with different sulfenamide and sulfonamide acted as
selective cannabinoid receptor (CB1R) antagonists [11]. Pyridazine-
bridged complexes also exhibit photo-physical properties favorable for
luminescent bio-imaging applications [12].
Cancer remains the second leading cause of human deaths after
cardiovascular diseases [13]. The design and synthesis of new metal-
based anticancer agents with a better biological activity, better se-
lectivity, lower toxicity and different mechanism of action to solve the
unresolved clinical problems of cisplatin analogous drugs have been the
focus of bioinorganic and medicinal chemists [14,15]. Many metal
complexes have been extensively investigated as they act as potent
growth inhibitors of human cancer cells by in vitro and in vivo experi-
ments [16]. More efforts are being made to synthesize Ni(II), Cu(II), Zn
(II) and Ru(II) complexes to act as better anticancer drugs, which could

⁎
Corresponding author.
E-mail address: akrahmanjkr@thenewcollege.in (A.K. Rahiman).

https://doi.org/10.1016/j.ica.2018.06.007
Received 5 March 2018; Received in revised form 1 June 2018; Accepted 4 June 2018
0020-1693/ © 2018 Elsevier B.V. All rights reserved.
U.M. Rafi et al.
overcome the limited activity of cisplatin [17,18]. Among the metal(II)
complexes, copper(II) complexes exhibit remarkable anticancer activity
with lower toxicity than the platinum compounds, and act as potential
reagents for DNA cleavage both oxidatively [19] and hydrolytically
[20]. Copper(II) complexes derived from 1-(6-chloropyridazin-3-yl)-5-
(2-hydroxyphenyl)-3-methyl-1H-pyrazole-4-carboxylic acid methyl
ester, which exhibited cytotoxicity activity against leukemia HL-60 and
NALM-6 cell lines [21], and pyridazolato-bridged copper(II) complexes,
which exhibited anti-proliferative and apoptosis inducing activity
against estrogen independent breast BT-20 and androgen independent
prostate PC-3 cancer cells have been reported [22].
Heteroleptic complexes play an important role in biological pro-
cesses, which are evident from the activation of enzymes by metal ions
[23]. 2,2′-Bipyridine and 1,10 phenanthroline as co-ligands act as po-
tential antitumor agents [24] and if these chelating agents are masked
by copper(II) ions they can exhibit even better anticancer activity.
Sigman et al. [25] have first reported the DNA cleavage ability of
copper(II) complexes of 1,10 phenanthroline. Since then heteroleptic
copper(II) complexes containing diimines as co-ligands have been ex-
plored extensively for their strong interactions with DNA and cyto-
toxicity and antiviral activities [26–28].
Human breast cancer cell line (MDA-MB-231) belongs to triple-ne-
gative breast cancer (TNBC), which lacks estrogen receptor (ER), pro-
gesterone receptor (PR) expression and human epidermal growth factor
receptor-2 (HER2). Among the 1.38 million new cases per year, more
than 15% are designated to be TNBC, which has no efficient treatment.
Dealing with the scarcity of well-defined molecular targets is still a
challenge as its prognosis remains bleak [29]. Since, breast cancer
therapy does not provide any effective drugs on TNBC, it is desirable to
develop novel cytotoxic drugs for the treatment of TNBC. Hence, our
strategy was to synthesize heteroleptic copper(II) complexes using three
pyridazine-based Schiff base tridentate ligands and diimines (2,2′-bi-
pyridine (bpy) and 1,10 phenanthroline (phen)) as co-ligands, and to
assess their in vitro anticancer activity against one human breast cancer
(MDA-MB-231) and one myoblast normal (L6) cell lines by MTT assay.
The apoptotic study was carried out using AO/EB staining method and
the cell cycle arrest was performed by flow cytometry.
2. Experimental section
2.1. Materials and instrumentation
2,2′-Bipyridine, 1,10-phenanthroline, 3,6-dichloropyridazine, sali-
cylaldehyde, 5-bromosalicylaldehye and 4-(diethylamino)salicylalde-
hyde were purchased from Aldrich and used without further purifica-
tion. Copper(II) carbonate was purchased from Fischer Scientific-
Qualigens, India. Copper(II) perchlorate hexahydrate was prepared by
the reaction of 70% perchloric acid with copper(II) carbonate. Solvents
used in the synthesis were dried and purified before being used ac-
cording to standard procedure [30]. The ligands 3-chloro-6-(salicyli-
denehydrazinyl)pyridazine (HL1), 3-chloro-6-(4-diethylaminosalicyli-
denehydrazinyl)pyridazine (HL2) and 3-chloro-6-(5-
bromosalicylidenehydrazinyl)pyridazine (HL3) were prepared ac-
cording to the procedure reported in our earlier publication [7].
The melting points were obtained on an Electro thermal capillary
apparatus and are uncorrected. Elemental analyses for C, H and N were
obtained on a Carlo Erba model 1106 elemental analyzer. IR spectra
were recorded on a Perkin-Elmer 297 spectrophotometer in the range
4000–400 cm−1. 1H and 13C NMR spectra were recorded on an Avans
Bruker 400 MHz spectrometer using DMSO-d6 as solvent. ESI mass
spectra were recorded on a Q-Tof mass spectrometer. Electronic spectra
were recorded on a Perkin-Elmer Lambda 25 version 1.27 spectro-
photometer using DMSO as solvent. EPR spectra (X-Band) were re-
corded on a Varian EPR-E 112 spectrometer using 2,2′-diphenyl-1-pi-
crylhydrazyl (DPPH) as the reference at 25 °C. The room temperature
magnetic moment of the complexes have been recorded on a PAR
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Inorganica Chimica Acta 482 (2018) 160–169
(model-155) vibrating sample magnetometer. Elico digital conductivity
bridge model CM-88 was used to measure molar conductance using a
freshly prepared DMF solution of the complexes. Cyclic voltammograms
were obtained on CHI 602D (CH Instruments Co., UA) electrochemical
analyzer under oxygen free conditions, equipped with a three-electrode
cell consisting of a platinum wire counter electrode, glassy carbon
working electrode and Ag/AgCl reference electrode (saturated KCl so-
lution). A DMF solution of TBAP (0.1 M) as the supporting electrolyte
and a ferrocene/ferrocenium (Fc/Fc+) couple was used as an internal
standard. The reported potentials were measured relative to the Ag/
Ag+ reference electrode and E1/2 of the Fc/Fc+ couple. The con-
centration of complex solutions were taken around 1.0 × 10−3 M and
the scan rate was 100 mVs−1.
Caution! Metal perchlorate salts with organic ligands should be
handled with care as they can cause explosion.
2.2. Synthesis of pyridazine-based heteroleptic copper(II) complexes
The heteroleptic copper(II) complexes of pyridazine-based ligands
(HL1−3) and diimines (2,2′-bipyridine or 1,10-phenanthroline) were
prepared by following the same procedure as given below: A metha-
nolic solution (15 mL) of Cu(ClO4)2·6H2O (0.37 g, 1.0 mmol) was added
to the appropriate ligand (HL1−3, 1.0 mmol) in 1:1 methanol/DMF
(20 mL) and an equimolar amount of triethylamine with constant stir-
ring for 30 min followed by diimine (2,2′-bipyridine (0.16 g, 1 mmol) or
1,10-phenanthroline (0.23 g, 1 mmol)) in methanol (15 mL). The stir-
ring was continued for 1 h and refluxed on a water bath for additional
2 h. The content was filtered while hot, and the filtrate was allowed to
stand at room temperature for few days. The solid complexes obtained
were recrystallized using hot methanol.
[Cu(L1)(bpy)](ClO4) (1)
Yield: 0.41 g (72%); Color: Greenish brown. Anal. Calc. for
C21H16N6O5Cl2Cu, (FW: 566.84): C, 44.50; H, 2.85; N, 14.83; Found: C,
44.46; H, 2.82; N, 14.82%. Selected IR data (cm−1): 3117 ʋ(eNH),
1603 ʋ(eC]N), 1251 ʋ(AreO), 1568 ʋ(eN]N), 1073 & 621 ʋ(ClO4−,
uncoordinated). UV-Vis (DMSO) λmax (nm) (ε (M−1cm−1)): 287
(13,650), 349 (4041), 485 (1372), 645 (6 3 5). ESI-MS (m/z): 466.04
([Cu(L1)(bpy)]+; 100%). Conductance (ΛM, Ω−1 cm2 mol−1) in DMF:
68. g|| = 2.20, g⊥ = 2.09. μeff = 1.84 B.M.
[Cu(L1)(phen)](ClO4) (2)
Yield: 0.44 g (75%); Color: Greenish brown. Anal. Calc. for
C23H16N6O5Cl2Cu, (FW: 590.86): C, 46.75; H, 2.73; N, 14.22; Found: C,
46.71; H, 2.71; N, 14.20%. Selected IR data (cm−1): 3064 ʋ(eNH),
1606 ʋ(eC]N), 1243 ʋ(AreO), 1541 ʋ(eN]N), 1083 & 624 ʋ(ClO4−,
uncoordinated). UV-Vis (DMSO) λmax (nm) (ε (M−1cm−1)): 266
(14,089), 367 (4070), 456 (1319), 661 (7 8 0). ESI-MS (m/z): 490.04
([Cu(L1)(phen)]+; 100%). Conductance (ΛM, Ω−1 cm2 mol−1) in DMF:
81. g|| = 2.22, g⊥ = 2.09. μeff = 1.81 B.M.
[Cu(L2)(bpy)](ClO4) (3)
Yield: 0.45 g (71%); Color: Greenish brown. Anal. Calc. for
C25H25N7O5Cl2Cu, (FW: 637.96): C, 47.07; H, 3.95; N, 15.37; Found: C,
47.03; H, 3.92; N, 15.36%. Selected IR data (cm−1): 3121 ʋ(eNH),
1612 ʋ(eC]N), 1245 ʋ(AreO), 1577 ʋ(eN]N), 1090 & 623 ʋ(ClO4−,
uncoordinated). UV-Vis (DMSO) λmax (nm) (ε (M−1cm−1)): 273
(13,500), 355 (5450), 410 (1215), 642 (5 9 9). ESI-MS (m/z): 537.11
([Cu(L2)(bpy)]+; 100%). Conductance (ΛM, Ω−1 cm2 mol−1) in DMF:
73. g|| = 2.22, g⊥ = 2.09. μeff = 1.80 B.M.
[Cu(L2)(phen)](ClO4) (4)
U.M. Rafi et al.
Yield: 0.49 g (74%); Color: Greenish brown. Anal. Calc. for
C27H25N7O5Cl2Cu, (FW: 661.68): C, 48.99; H, 3.81; N, 14.81; Found: C,
48.94; H, 3.78; N, 14.79%. Selected IR data (cm−1): 3105 ʋ(eNH),
1612 ʋ(eC]N), 1248 ʋ(AreO), 1575 ʋ(eN]N), 1072 & 621 ʋ(ClO4−,
uncoordinated). UV-Vis (DMSO) λmax (nm) (ε (M−1cm−1)): 268
(15,110), 388 (5540), 468 (1315), 658 (6 5 2). ESI-MS (m/z): 561.11
([Cu(L2)(phen)]+; 100%). Conductance (ΛM, Ω−1 cm2 mol−1) in DMF:
77. g|| = 2.25, g⊥ = 2.10. μeff = 1.88 B.M.
[Cu(L3)(bpy)](ClO4) (5)
Yield: 0.48 g (74%); Color: Pale green. Anal. Calc. for
C21H15N6O5BrCl2Cu, (FW: 645.74): C, 39.06; H, 2.34; N, 13.02; Found:
C, 39.03; H, 2.33; N, 13.01%. Selected IR data (cm−1): 3103 ʋ(eNH),
1614 ʋ(eC]N), 1244 ʋ(AreO), 1590 ʋ(eN]N), 1068 & 622 ʋ(ClO4−,
uncoordinated). UV–Vis (DMSO) λmax (nm) (ε (M−1cm−1)): 277
(13,410), 386 (4505), 450 (1105), 657 (7 3 8). ESI-MS (m/z): 545.97
([Cu(L3)(bpy)]+; 100%). Conductance (ΛM, Ω−1 cm2 mol−1) in DMF:
62. g|| = 2.20, g⊥ = 2.09. μeff = 1.79 B.M.
[Cu(L3)(phen)](ClO4) (6)
Yield: 0.51 g (76%); Color: Pale green. Anal. Calc. for
C23H15N6O5BrCl2Cu, (FW: 669.76): C, 41.25; H, 2.26; N, 12.55; Found:
C, 41.21; H, 2.24; N, 12.54%. Selected IR data (cm−1): 3155 ʋ(eNH),
1617 ʋ(eC]N), 1258 ʋ(AreO), 1589 ʋ(eN]N), 1078 & 620 ʋ(ClO4−,
uncoordinated). UV–Vis (DMSO) λmax (nm) (ε (M−1cm−1)): 270
(13,720), 397 (3988), 453 (1265), 641 (6 6 2). ESI-MS (m/z): 569.95
([Cu(L3)(phen)]+; 100%). Conductance (ΛM, Ω−1 cm2 mol−1) in DMF:
59. g|| = 2.20, g⊥ = 2.08. μeff = 1.83 B.M.
2.3. Computational studies
Theoretical and quantum chemical calculations of the heteroleptic
copper(II) complexes (1–6) have been carried out using the density
functional theory (DFT) incorporating the Becke’s three parameter ex-
change (B3) with Lee, Yang and Parr (LYP) correlation functional
supplemented with the internally stored 6-31G(d) and LANL2DZ basis
set [31] employing the Gaussian 03 software package [32].
2.4. Cell culture
Human breast cancer MDA-MB-231 and normal rat myoblast L6 cell
lines were purchased from National Center for Cell Science (NCCS),
Pune, India, and cultured in Dulbecco's Modified Eagle's Medium
(DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco, Life
Technologies Corporation, Grand Island, NY, USA) and 1% anti-
biotic–antimycotic solution, in a humidified atmosphere of 5% CO2 at
37 °C. Stock solutions of all the complexes were prepared in cell culture
grade dimethyl sulfoxide (DMSO).
2.5. In vitro anticancer activity
The capacity of synthesized heteroleptic copper(II) complexes to
interfere with the growth of the cells was determined by using a tet-
razolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) as reported earlier [7,33]. Cell clonogenic assay,
apoptosis using AO/EB fluorescent double staining method and the cell
cycle distribution by flow cytometry for complexes 3 and 4 were per-
formed by employing the procedure as reported in the literature
[7,34,35].
2.6. Protein FGFR kinase interaction by molecular docking method
Molecular docking studies were performed on AutoDock Tools
(ADT) version 1.5.6 and AutoDock version 4.2.5.1 docking programmes
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Inorganica Chimica Acta 482 (2018) 160–169
Scheme 1. Synthesis of heteroleptic copper(II) complexes of pyridazine-based
ligands and diimines (1–6).
to identify the binding site of copper(II) complexes 3 and 4 by em-
ploying the procedure as reported by us previously [7]. The available
crystal structure of FGFR (PDB ID: 3C4F) was obtained from the Protein
Data Bank (http://www.rcsb.org./pdb).
3. Results and discussion
Three pyridazine-based ligands (HL1−3) were synthesized by the
condensation reaction between aromatic aldehydes (salicylaldehyde, 4-
(diethylamino)salicylaldehyde or 5-bromosalicylaldehyde) and 3-
chloro-6-hydrazinopyridazine in ethanol. The heteroleptic copper(II)
complexes (1–6) were synthesized by reacting equimolar quantities of
copper(II) perchlorate hexahydrate with the ligands (HL1−3) and the
corresponding diimine in the presence of triethyamine in methanol
(Scheme 1). The complexes are soluble in DMSO and DMF. The pro-
posed structure of the complexes is well in agreement with the ele-
mental and spectral analyses.
3.1. Spectral characterization
IR spectra was used to determine the mode of coordination of the
ligand with the copper(II) ion (Table S1) and significant differences
were analyzed by comparing the spectra of the complexes with the li-
gands. The ʋ(C]N) stretching vibrations of azomethine group
(1622–1631 cm−1), ʋ(AreO) stretching vibration of phenolic group
(1282–1292 cm−1) and ʋ(N]N) stretching vibration of the un-
symmetrical pyridazine ring (1526–1535 cm−1) observed for the li-
gands (HL1−3) appeared with significant shifts for all the complexes at
1603–1617, 1243–1258 and 1541–1590 cm−1, respectively [36,37].
These shift suggests the participation of azomethine nitrogen, phenolic
oxygen and pyridazine nitrogen of the ligands in coordination with the
copper(II) ion. The characteristic ring stretching frequency ʋ(C]N) of
free bipy/phen (1421–1443 cm−1) is shifted to higher frequencies
(1494–1514 cm−1) upon complexation, indicating the coordination of
the heterocyclic nitrogen atoms to the copper(II) ion [38]. All the
complexes exhibit typical stretching vibrations at ∼1100 cm−1 (ʋ3-
antisymmetric stretching) and ∼625 cm−1 (ʋ4-antisymmetric bending)
attributable to the perchlorate anion. The absence of band around
930 cm−1 (ʋ2-symmetric stretching) due to coordinated perchlorate
indicates the presence of uncoordinated anions in the complexes [39].
This was further supported by the conductivity measurements in which
the observed molar conductivity values (59–81 Ω−1 cm2 mol−1) at
25 °C in DMF solution (10−3 M) implies the 1:1 electrolytic nature of
the complexes [40]. Thus, both IR spectra and conductivity measure-
ments authenticate the presence of uncoordinated perchlorate ion in
U.M. Rafi et al. Inorganica Chimica Acta 482 (2018) 160–169

the complexes. The weak bands at 538–568 and 467–486 cm−1 were
assigned to ʋ(MeO) and ʋ(MeN) stretching frequencies, respectively
[41]. These features are consistent with the proposed structures of the
complexes in which the copper(II) ion is coordinated to monoanionic
tridentate ligand through a nitrogen of the azomethine group, one of
the nitrogen of the pyridazine ring, an oxygen of the phenolic group,
and two nitrogen atoms of the diimine ring.
1
H NMR spectra of ligands (HL1−3) show a signal at 8.23–8.43 ppm
for the imine group, which confirms the formation of ligands (Figs.
S1–S3). The peak at 11.27–11.73 ppm indicates the presence of phe-
nolic hydroxyl group and the peaks corresponding to the aromatic
protons are observed in the region 6.12–7.87 ppm. 13C NMR spectra of
the ligands (Figs. S1–S3) showed characteristic signals at
137.67–143.89 ppm for the azomethine carbon (C]N). The spectra are
further characterized by the disappearance of signal at 190 ppm due to
carbonyl carbon.
The ESI mass spectra provides a vital clue for elucidating the
structure of complexes. All the complexes exhibit molecular ion peaks
corresponding to the mass of entire complex excluding perchlorate
counter ion. The ESI mass spectra for the complexes 5 and 6 are shown
Fig. 2. EPR spectrum of the heteroleptic copper(II) complex 5.
in Fig. 1, which shows the molecular ion peaks at m/z = 545.97 and
569.95 consistent with the proposed molecular formulae [Cu(L3)
(bpy)]+ and [Cu(L3)(phen)]+, respectively. The obtained results

Fig. 1. ESI-mass spectra of the heteroleptic copper(II) complexes 5 (a) and 6 (b).

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Table 1
EPR data of the heteroleptic copper(II) complexes (1–6).
Complexes g|| g⊥ a
gav G μeff

1 2.20 2.09 2.13 2.22 1.84

2 2.22 2.09 2.13 2.37 1.81
3 2.22 2.09 2.14 2.48 1.80
4 2.25 2.10 2.16 2.52 1.88
5 2.20 2.09 2.12 2.35 1.79
6 2.20 2.08 2.12 2.49 1.83

a
gav = (g|| + 2 g⊥)/3.

suggest that the complexes are mononuclear in nature. Thus, the data
obtained from ESI mass spectra of the complexes are in good agreement
with the proposed empirical molecular formulae.
In order to obtain further structural information, the electronic
spectra of the ligands (HL1−3) and copper(II) complexes (1–6) were
recorded in DMSO in the range 200–900 nm (Fig. S4), and the data are
presented in Table S2. The absorption spectra of the ligands (HL1−3)
are characterized by bands at 301–371 nm. The copper(II) complexes
show two intra-ligand transitions, at 266–287 nm and 349–397 nm, Fig. 3. Reduction voltammograms of the heteroleptic copper(II) complexes
while the ligand to metal charge transfer (LMCT) band was observed at (1–6).
410–485 nm. A weak d–d band at 641–661 nm for the complexes can be
assigned to 2B1g → 2B2g (ν1) (dx2-y2 → dyz) transition consistent with
negative reduction potential observed for complexes of ligands HL3
that of the square pyramidal geometry around copper(II) ion [38].
when compared to other complexes of ligands HL1&2 is due to presence
Electron paramagnetic resonance (EPR) spectroscopy provides va-
of the electron withdrawing substituent in the phenyl ring, which de-
luable information about the local environment of the paramagnetic
creases the electron density around the complex and favors easy re-
metal ions, their distribution and metal-metal interactions of transition
duction [40].
metal complexes. It is a powerful tool to infer details about the structure
of complexes formed by paramagnetic metal ions. EPR spectra of the
polycrystalline samples of copper(II) complexes (1–6) were recorded at 3.3. Theoretical calculations
room temperature (Fig. 2) and their data are tabulated in Table 1. The
calculated g||, g⊥ and gav for the complexes (1–6) are in the range 3.3.1. Geometry optimization
2.20–2.25, 2.08–2.10 and 2.12–2.16, respectively. The observed trend DFT calculations are a tool of growing importance for the structural
g|| > g⊥ > 2.0023 support the presence of unpaired electron in dx2–y2 investigation of coordination and organometallic compounds by pro-
ground state and distorted square pyramidal structure for these com- viding tremendous information about structural description, in the
plexes [42]. The g|| value, a moderately sensitive function is used for absence of crystal data, in addition to the energy minimized con-
the indication of covalency. The g|| values for the complexes are less formation. Hence, all the copper(II) complexes (1–6) were optimized at
than 2.3, indicate significant increase in covalency of the metal-ligand B3LYP/LANL2DZ and B3LYP/GEN levels in gas phase. The optimized
(MeL) bonds [43]. The axial symmetry parameter G, which measures ground state geometry structures are deduced (Fig. 4) and the calcu-
the exchange interaction between the metal centers in a polycrystalline lated structural parameters (bond lengths and bond angles) of the
solid, was calculated using the equation: complexes are summarized in Table S4. The five coordinated copper(II)
complexes coordinate through one phenolic oxygen, one azomethine
G= (g‖−2.0023)/(g⊥−2.0023) nitrogen, one pyridazine nitrogen, and two nitrogen atoms of diimine
The G values for the complexes (1–6) are in the range of 2.22–2.52, (2,2′-bipyridyl (bpy) or 1,10-phenanthroline (phen)) co-ligands. The
which is less than 4, indicating dx2–y2 as ground state and having the observed bond length values of Cu–O (2.0481–2.0892 Å), CueN (azo-
methine N1: 1.9622–1.9841 Å), CueN (pyridazine N2:
presence of exchange interaction for the solid complexes [44].
The magnetic moment (μeff) values of copper(II) complexes (1–6) at
room temperature were found in the range 1.79−1.88 B.M. indicates
the paramagnetic nature of these complexes. The values are slightly
higher than the spin only moment 1.73 BM due to mixing-in of some
orbital angular momentum from the closely lying excited states via spin-
orbit coupling, which indicates the monomeric nature of these com-
plexes [45].

3.2. Electrochemical measurements

The electrochemical properties of complexes have gained our at-

tention because the redox properties provides information for its fur-
ther application in electrochemistry and catalytic reactions. The re-
duction properties of the copper(II) complexes (1–6) were studied in
DMF using TBAP as the supporting electrolyte by cyclic voltammetry in
the range 0 to −1.0 V (Fig. 3) and the data are summarized in Table S3.
All the copper(II) complexes display a well-defined irreversible one-
electron reduction wave in the cathodic region (Epc) between −0.596 Fig. 4. The optimized geometric structures of the heteroleptic copper(II) com-
and −0.641 V, which can be assigned to CuII/CuI couple. The less plexes 3 (a) and 4 (b).

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2.0162–2.0921 Å), CueN (diimine N3: 1.9800–1.9916 Å) and (diimine
N4: 1.9609–1.9809 Å) for the complexes (1–6) are consistent with the
reported values [39,46,47]. The B3LYP method in combination with the
LANL2DZ basis gives an excellent estimation of CueN and CueO bond
distances for CueN1, CueN2, CueN3, CueN4 and CueO. Molecular
geometries can be predicted by calculating τ values using the following
equation [48]:
β−α
τ=
60
where α and β are the equatorial and axial bond angles, respectively.
When the τ value is close to 0, then the geometry is square pyramidal,
and when the τ value is close to 1, then the geometry is trigonal bi-
pyramidal. If τ value is between 0 and 0.5, the molecular geometry is
distorted trigonal bipyramidal. For the complexes (1–6) α (N(1)eCueN
(3)) values are 101.72, 100.87, 102.59, 105.35, 101.01 and 102.12 Å,
and β (N(2)eCueO) values are 102.41, 101.93, 100.28, 106.10, 102.98
and 103.07 Å, respectively. The observed results indicate square pyr-
amidal geometry for the complexes 1–6 as their τ values are equal to 0.
3.3.2. HOMO-LUMO analysis
The study of frontier molecular orbitals (FMOs) are used to under-
stand the electrical, optical and chemical properties of the compounds
including chemical reactivity and kinetic stability [49,50]. The FMOs,
namely, highest occupied molecular orbital (HOMO), represents the
ability to donate an electron (π donor) and the lowest unoccupied
molecular orbital (LUMO) is an electron acceptor (π acceptor), which
represents the ability to obtain an electron. Molecules with small
frontier orbital gap is more polarizable, having high chemical reactivity
and low kinetic stability are termed as soft molecule [49]. The energy of
the HOMO is directly related to the ionization potential while LUMO
energy is directly related to the electron affinity. Recently, the HOMO-
LUMO energy gap has been used to confirm the bioactivity from in-
tramolecular charge transfer [51]. The HOMO-LUMO energy gaps are
essential for evaluation of reduction and oxidation potentials of the
molecule. These orbitals provide information, which may be important
Fig. 5. Frontier molecular orbitals of the heteroleptic copper(II) complexes 3 (a) and 4 (b).
165
Inorganica Chimica Acta 482 (2018) 160–169
Table 2
Half maximal inhibitory concentrations (IC50) of the heteroleptic copper(II)
complexes (1–6) using MTT assay.
Complexes *IC50 (μM)
MDA-MB-231 L6
1 20.57 ± 1.0 9.14 ± 0.84
2 8.27 ± 0.56 17.48 ± 0.09
3 5.84 ± 0.82 8.58 ± 1.12
4 5.69 ± 0.41 11.36 ± 0.68
5 6.95 ± 0.96 9.57 ± 0.12
6 6.26 ± 1.1 7.80 ± 0.19
Cisplatin 6.19 ± 0.45 8.08 ± 0.32
*Results are presented as mean IC50 values, calculated from three independent
experiments.
for drug metabolism, as most of the drugs are metabolized by oxidation
and reduction reactions [52]. The orbital energy level analysis for
complexes 1–6 showed that EHOMO values are −3.26, −3.86, −4.36,
−5.09, −2.18 and −2.02 eV, while ELUMO values are −1.42, −2.18,
−3.01, −3.86, −4.42 and −4.09 eV, respectively (Fig. 5). The ΔE
values of complexes 1–6 are 1.84, 1.68, 1.35, 1.23, 2.24 and 2.07 eV,
respectively. The resulting small energy gap value between HOMO and
LUMO of complexes 3 and 4 support easy charge transfer in these
complexes, which influences the biological activity of the molecule and
the low energy gap value is also due to the groups that enter into
conjugation.
3.4. In vitro anticancer activity
3.4.1. Cell proliferation assay
In vitro cytotoxicity of the heteroleptic copper(II) complexes (1–6)
against human breast cancer (MDA-MB-231) and normal rat myoblast
(L6) cell lines were screened using MTT assay with DMSO as a positive
control. The cells were treated with increasing concentration of
U.M. Rafi et al. Inorganica Chimica Acta 482 (2018) 160–169

complexes (3.9, 7.8, 15.6, 31.25, 62.5, 125, 250 and 500 μM) for 24 h.
After 24 h, MTT was added and incubated for 4 h [7]. The IC50 values
towards the tested cell lines were obtained by non-linear fitting of log
dose-response curve using the GraphPad Prism software (Table 2; Fig.
S5). IC50 value represents the concentration required to achieve the
50% of cell viability when compared to control. Complexes with lower
IC50 value are more potent than those with higher IC50 value. One of the
key requirements for a chemical entity to act as an anticancer agent is
to selectively kill cancer cells without causing excessive damage to
normal cells [53]. The complexes exhibited sufficient toxicity toward
the cancer cells and may be useful in selectively targeting cancer cells
than normal cells as they have less cytotoxicity effects on normal cells
than the cancer cell line. The complexes 1 and 2 exhibited relatively
high IC50 values against cancer cells. Though, the complex 6 exhibit low
IC50 value against cancer and normal cells, there is no considerable
difference in their IC50 values. The complexes 3, 4 and 5 are found to be
more active against cancer cells than the other complexes in terms of
lower IC50 values with considerable differences in IC50 values between
normal and cancer cells. This indicates that these complexes are rela-
tively less toxic to normal cell line (L6) than against the tested cancer
cell line (MDA-MB-231), thereby, exhibiting more selectivity to MDA-
MB-231 cell line when compared to other synthesized complexes. The
copper(II) complexes 3 and 4 are very similar in their structure as both
have an electron releasing diethylamino group. The proliferation-in-
hibitory activity of complexes 3 and 4 on the human breast cancer cell
line (MDA-MB-231) is higher than that of cisplatin and other complexes
derived from pyridazine-based ligands [7]. The higher cytotoxicity
observed for the complexes than the other reported pyridazine-based
complexes may be due to increase in hydrophobic character in these
complexes because of the inclusion of heterocyclic bases [54]. Based on
these results, the complexes 3 and 4 were chosen to carry out clono-
genic assay, apoptosis evaluation and cell cycle analysis.

3.4.2. Clonogenic assay

Clonogenic assay (colony formation assay) is an in vitro cell survival
assay based on the ability of a single cell to grow into a colony. The
colony is defined to consist of at least 50 cells. Cell clonogenic assay can
be used to determine and confirm the cytotoxicity effect of complexes
on cancer cells. The ability of copper(II) complexes 3 and 4 to inhibit
the colony formation of MDA-MB-231 cells was done by treating the
cells with IC50 concentration of these complexes for 24 h, and the co-
lonies were counted and photographed (Fig. S6). It can be observed that
both the complexes resulted in decrease in number and size of colonies
when compared with untreated controls. Thus, the complexes 3 and 4
causes cancer cells to loose their reproductive integrity and inhibit the
growth and proliferation of MDA-MB-231 cells, which seems to be
consistent with the result obtained from the MTT assay.

3.4.3. Apoptotic evaluation by AO/EB fluorescent staining method

All multicellular organisms use apoptosis during development,
homeostasis, defense, metamorphosis, terminal differentiation, immune
response and cellular response to growth factors and hormones [55].
Apoptotic cells have the distinct morphological features such as cell
shrinkage, chromatin condensation, membrane blebbing and formation
of apoptotic bodies, and shows no inflammatory response when com-
pared to normal cells [55]. Most tumour cells retain their sensitivity to
some apoptotic stimuli from chemotherapeutic agents, and in this
context, the apoptosis-inducing ability of drugs seems to be a primary
factor in determining their efficacy. Necrotic and apoptotic cells can be
distinguished on the basis of overall cell morphology and cell mem-
brane integrity using fluorescence microscopy. Dual AO/EB fluorescent
staining, visualized under a fluorescent microscope has been used to
identify apoptosis-associated changes in MDA-MB-231 cells induced by
the copper(II) complexes 3 and 4 at IC50 concentration for 24 h. The
treatment did not cause necrosis of cells. Live cells and apoptotic cells
can be easily distinguished by the percentage uptake of AO/EB. AO
Fig. 6. Morphological observation of apoptosis on MDA-MB-231 cells with
0.1% DMSO (control) (a), and the heteroleptic copper(II) complexes 3 (b) and 4
(c).
permeates all live cells, and hence appeared green. EB is taken up only
when the cells have lost their cytoplasmic membrane integrity and
hence appear red. It has been observed that live cells appear to have
normal nuclei staining, which has green chromatin with organized
structures while apoptotic cells contain condensed or fragmented or-
ange chromatin, and necrotic cells have normal nuclei staining with
orange chromatin. Control sample showed green nuclei with organized
structure indicating live cells whereas treated samples showed frag-
mented chromatin (Fig. 6). The morphological evaluation confirmed

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Fig. 7. Cell cycle analysis on MDA-MB-231 cells showing the distribution of
cells in sub G0, G0/G1, S and G2/M phases after treatment with 0.1% DMSO
(control) (a), and the heteroleptic copper(II) complexes 3 (b) and 4 (c). (Data is
represented as average of four independent experiments).
apoptosis induction as the number of reddish-orange and red cells were
observed with the loss of membrane integrity indicating the occurrence
of late apoptosis and secondary necrosis, which is a natural con-
sequence of a complete apoptotic cycle. The percentage of apoptotic
and necrotic cells were calculated. AO/EB staining data indicated that
the percentage of apoptosis was almost 90% for both the complexes 3
and 4. The data shows that both the complexes exhibit cytotoxicity
activity in MDA-MB-231 cells via apoptotic pathway. Thus, the com-
plexes 3 and 4 has higher apoptosis inducing ability, which determines
the efficacy for being an anticancer drug. Additionally, the
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Inorganica Chimica Acta 482 (2018) 160–169
morphological changes observed for complex 4 suggest that apoptotic
cell death of MDA-MB-231 cells are more efficient compared to complex
3, which may be due to the extended planar surface and hydrophobicity
of 1,10-phenanthroline co-ligand that facilitates the permeability of the
complex across the cell membrane.
3.4.4. Cell cycle arrest by flow cytometry
The cell cycle profile of copper(II) complexes 3 and 4 at IC50 con-
centrations for 24 h was performed by flow cytometry using propidium
iodide staining, and the changes in cell size distribution is shown in
Fig. 7. The populations in the G1, G2/M and S phase can be quantified if
the fluorescence is measured in the FL2 red channel, which can be re-
lated to the number of DNA copies present in the single cell suspension.
The MDA-MB-231 cells treated with complexes 3 and 4 showed an in-
crease in the proportion of cells in S phase and sub G0 phase, and this
increase was accompanied by a decrease in the proportion of cells in the
G0/G1 and G2/M phases compared to untreated cells after treatment. A
slight escalation in the percentage of the cells in S phase for complex 3
was observed from 20.0% to 26.3% whereas that with complex 4 was to
22.5%. So the complexes 3 and 4 induces apoptosis in cells by causing
DNA damage, which causes arrrest of cells in S phase. The increse in
population of sub G0 phase indicates the hypodiploid cells that have
fragmented DNA and thus late apoptotic cells [56]. Cisplatin and ox-
aplatin are known to induce cell cycle arrest at the S phase, which
signifies inhibition of DNA synthesis [57,58].
3.5. Molecular docking with FGFR
Fibroblast growth factors (FGFs) regulate prenatal and postnatal
bone formation through activation of FGF receptors (FGFRs) and
downstream signaling events. The development of mouse genetics tar-
geted on FGF/FGFR and the analysis of FGFR mutations involved in
human skeletal disorders revealed essential molecular mechanisms by
which FGF/FGFR signaling controls bone formation [59]. FGFRs induce
intracellular signaling networks that tightly regulate key biological
processes including proliferation, angiogenesis and migration, upon
binding with the ligands. Therapeutic strategies targeting FGFs and
FGFRs in human cancer are therefore currently under investigation as
drug targets in breast cancer patients [60]. Hence, for this reason, the
interaction of synthesized copper(II) complexes (1–6) with FGFR kinase
receptor have been studied by in silico molecular docking studies.
The energy-minimized docked images (Figs. 8 & S7) illustrate that
the complexes (1–6) most likely binds to the hydrophobic pocket and
the molecular docking parameters are listed in Table 3. It can be ob-
served that all the complexes potentially interact with FGFR via π–π,
π–σ, hydrogen bonding, electrostatic and van der Waals interactions.
The docking analysis of complexes 3 and 4 alone are discussed here, as
they show higher cytotoxicity activity than the other complexes. The
complex 3 shows π–π interaction between the bipyridyl ring and
His1026 (bond length: 3.6 Å), the bipyridyl ring and Lys868 (bond
length: 3.5 Å) and the bipyridyl ring and His1026 (bond length: 3.7 Å).
This complex also interacts with binding site around Asp1046 through
pyridazine ring especially by π–σ interaction (bond length: 3.4 Å), as
well as stabilized by hydrogen bonding interaction with oxygen atom of
Cys1045 and hydrogen atom of pyridazine ring (bond length:
O⋯H = 3.2 Å). The binding model was also enhanced by electrostatic
interaction formed between complex 3 and residues Glu885, Val899,
His1026, Ile1044, Cys1045 and Asp1046, and van der Waals interaction
formed between the complex 3 and residues Val848, Lys868, Ile888,
Leu889, Ile892, Val898, Val914, Val916, Leu1019 and Phe1047. The
complex 4 shows three π–π interactions, one between the phenan-
throline ring and His1026 (bond length: 3.7 Å), second between the
pyridazine ring and Arg1027 (bond length: 3.6 Å), and third between
the pyridazine ring and His1026 (bond length: 3.6 Å). Complex 4 was
stabilized by two hydrogen bonding interactions, first one was formed
between oxygen atom of Asp1046 and hydrogen atom of pyridazine
U.M. Rafi et al. Inorganica Chimica Acta 482 (2018) 160–169

Fig. 8. Docking poses of the heteroleptic copper(II) complex 3 located within the hydrophobic pocket of FGFR (a), and the interaction mode between 3 and FGFR (b).
Docking poses of the copper(II) complex 4 located within the hydrophobic pocket of FGFR (c), and the interaction mode between 4 and FGFR (d).

Table 3
Molecular docking parameters (kcal mol−1) of the heteroleptic copper(II) complexes (1–6) with FGFR kinase receptor.
Complexes vdW + H bond + dissolving energy Electrostatic energy Total internal Torsional free energy Unbound system’s Binding free energy
(ΔGvdW+hb+desolv) (ΔGelec) energy (ΔGtotal) (ΔGtor) energy (ΔGunb) (ΔGbinding)a

1 −8.08 −0.11 −0.23 +2.11 −0.15 −6.45

2 −7.69 −0.09 −0.32 +1.74 −0.23 −6.59
3 −8.24 −0.67 −0.63 +1.13 −0.09 −8.32
4 −8.89 −0.99 −0.94 +1.07 −0.11 −9.86
5 −6.86 −0.77 −0.12 +0.79 −0.05 −7.01
6 −7.93 +0.13 −0.34 +0.86 −0.08 −7.36

a
ΔGbinding = ΔGvdW+hb+desolv + ΔGelec + ΔGtotal + ΔGtor + ΔGunb.

ring (bond length: O∙∙∙H = 3.1 Å), and second one was formed between
oxygen atom of Asp1046 and hydrogen atom of pyridazine ring (bond
length: O⋯H = 3.3 Å). Besides, the binding model was enhanced by
electrostatic interaction formed between complex 4 and residues
Cys817, Lys868, Glu885 and Asp1046, and van der Waals interaction
formed between the complex 4 and residues Val848, Ile888, Ile892,
Leu889, Val889, Val898, Val914, Val916, Leu1019, Ile1044, Cys045
and Phe1047.
The obtained results indicate FGFR kinase receptor to be a potent
inhibitor for all the complexes (1–6), which were retained tightly by the
binding pocket of FGFR. While comparing the binding affinity of the
complexes, the complex 4 binds to FGFR stronger than the other
complexes. The lower the relative binding energy, the more potent the
binding affinity is between the FGFR and target molecules. The more
negative relative binding energy of complex 4 indicate its stronger
binding affinity to the BSA than the other complexes, which play a key
role in metabolism and transportation of BSA. The obtained results from
molecular docking studies indicate that the interaction between com-
plexes and FGFR was dominated by π–π, π–σ, hydrogen bonding,
electrostatic and van der Waals interactions.
4. Conclusion
Six heteroleptic copper(II) complexes of tridentate pyridazine-based
ligands and neutral co-ligands (2,2′-bipyridine (bpy) or 1,10-phenan-
throline (phen)) have been synthesized and characterized. Electronic
spectral data, magnetic moment measurements and theoretical analysis
indicate the square pyramidal geometry for copper(II) complexes. The
optimized parameters reflects that the B3LYP/LANL2DZ method is
preferable to B3LYP/Gen (LANL2DZ for Cu and 6-31G(d) for the re-
maining elements), and also the DFT studies revealed the obtained
smaller energy gap, which supports the bio-efficacy of the complexes.
The cytotoxicity of all the complexes against MDA-MB-231 cancer cell
line was evaluated and compared with L6 myoblast normal cells by
MTT assay. The complexes 3 and 4 with diethylamino substituent have
been found to exhibit higher in vitro cytotoxicity towards MDA-MB-231
cell line, than the other complexes. The cell cycle analysis indicates that
these complexes inhibited cell growth at S phase of the cell cycle. The
calculated higher negative free energy values of the complexes 3 and 4
suggested strong binding to FGFR than the other complexes, which can
assist in the design and development of new FGFR inhibitors. These
results suggest that the complexes 3 and 4 can be introduced as an

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effective metal-based anticancer drugs and further experiments in re-
levance to potential in vivo anticancer activity of these complexes can
be considered in future.
Acknowledgments
The authors acknowledge Department of Chemistry, Indian Institute
of Technology Madras (IIT-M), Chennai 600 036, for recording ESI mass
spectra, and Central Leather Research Institute (CLRI), Chennai 600
020, for EPR analysis.
Disclosure statement
No conflict of interest was reported by the authors.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.ica.2018.06.007.
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