Synthesis, spectral characterization, docking analysis, DNA binding/cleavage, antimicrobial and cytotoxic activity of new dimeric antipyrine-Schiff base metal complexes

Article abstract of DOI:10.14233/ajchem.2019.21680

In the present study, a new series of novel Schiff base ligand derived from (furan-2-carbaldehyde and 4-aminoantipyrine) and dimeric metal(II) complexes with the composition of [MLCl2]2, where M=Cu(II), Co(II), Ni(II) and Zn(II) have been synthesized and characterized by elemental analysis, magnetic susceptibility, molar conductivity measurements, FT-IR, UV-visible, ¹H NMR, ¹³C NMR, EPR, MS (ESI), XRD, SEM and EDX studies. The spectral data recommend that the dimeric metal complexes embrace octahedral geometry around the central metal ions. The DMF solutions of the dimeric metal complexes demonstrate the lower molar conductance values which might be due to the non-electrolytic nature of the complexes (5.67-13.24 ?^-1 mol^-1 cm^-2). The biological studies involved are DNA interaction (binding and damage) antimicrobial, anti-proliferative and molecular docking. DNA interaction of these complexes carried out with using calf thymus DNA (CT-DNA) by electronic absorption titration, viscometric measurement which revealed that the synthesized dimeric complexes interact with DNA through intercalative binding mode. A gel electro-phoresis assay testifies the ability of complexes to cleave supercoiled pUC19 DNA in the presence of hydrogen peroxide as an activator. The synthesized compounds were initiated from their biological perspective. The antimicrobial assay indicates that dimeric complexes are good antimicrobial agents. Besides, in vitro antiproliferative activity of dimeric complexes were investigated on MCF-7, Hep G2, HBL-100 cell lines using an MTT assay. In addition, the molecular docking study was accomplished to considerate the nature of binding of the synthesized compounds with protein and DNA.

Full text of DOI:10.14233/ajchem.2019.21680

Asian Journal of Chemistry; Vol. 31, No. 1 (2019), 199-212
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2019.21680
Synthesis, Spectral Characterization, Docking Analysis, DNA Binding/Cleavage,
Antimicrobial and Cytotoxic Activity of New Dimeric Antipyrine-Schiff Base Metal Complexes
1,*
2
3
4
ABDEL-NASSER M.A. ALAGHAZ , AMANI S. ALTURIQI , REDA A. AMMAR and MOHAMED E. ZAYED
1
2
3
4
Department of Chemistry, College of Science, Jazan University, Jazan, Saudi Arabia
Department of Chemistry, College of Science, Princess Nourah Bint Abdul Rahman University, Riyadh, Saudi Arabia
Department of Chemistry, College of Science, Al-Imam Mohammad Ibn Saud Islamic University, Riyadh, Saudi Arabia
Department of Botany and Microbiology, Faculty of Science, King Saud University, Riyadh 11541, Saudi Arabia
*
Corresponding author: E-mail: aalajhaz@hotmail.com
Received: 28 August 2018; Accepted: 24 September 2018;
Published online: 30 November 2018;
AJC-19183
In the present study, a new series of novel Schiff base ligand derived from (furan-2-carbaldehyde and 4-aminoantipyrine) and dimeric
metal(II) complexes with the composition of [MLCl , where M=Cu(II), Co(II), Ni(II) and Zn(II) have been synthesized and characterized
by elemental analysis, magnetic susceptibility, molar conductivity measurements, FT-IR, UV-visible, H NMR, C NMR, EPR, MS
ESI), XRD, SEM and EDX studies. The spectral data recommend that the dimeric metal complexes embrace octahedral geometry around
the central metal ions. The DMF solutions of the dimeric metal complexes demonstrate the lower molar conductance values which might
]
2 2
1
13
(
-1
-1
-2
be due to the non-electrolytic nature of the complexes (5.67-13.24 Ω mol cm ). The biological studies involved are DNA interaction
binding and damage) antimicrobial, anti-proliferative and molecular docking. DNA interaction of these complexes carried out with using
(
calf thymus DNA (CT-DNA) by electronic absorption titration, viscometric measurement which revealed that the synthesized dimeric
complexes interact with DNA through intercalative binding mode. A gel electro-phoresis assay testifies the ability of complexes to cleave
supercoiled pUC19 DNA in the presence of hydrogen peroxide as an activator. The synthesized compounds were initiated from
their biological perspective. The antimicrobial assay indicates that dimeric complexes are good antimicrobial agents. Besides, in
vitro antiproliferative activity of dimeric complexes were investigated on MCF-7, Hep G2, HBL-100 cell lines using an MTT assay.
In addition, the molecular docking study was accomplished to considerate the nature of binding of the synthesized compounds with
protein and DNA.
Keywords: Dimeric Schiff base complexes, Magnetic susceptibility, DNA binding, Antiproliferative activity, Molecular docking.
tion, drug design, catalysis and in the production of biological
probes and chemical sensors [3-6]. Stable complexes are formed
by the combination of Schiff bases and various transitions and
inner transition metals. Most of the metal complexes have
recently been tested by several bioinorganic chemists for deter-
miningand quantifying their anticancer properties [7-10]. DNA
damage is the major cause of cancer; strategies for controlling
DNA damage constitute the prime focus area for cancer related
research [11]. Many currently used chemotherapeutic agents
are reported to curb tumor growth by interfering with DNA
replication and transcription. In living cells, the primary function
of DNA is the storage and transport of genetic codes in a cell.
Furthermore DNA is a crucial target for drugs that are involved
INTRODUCTION
Schiff bases are considered an important class of ligands
because they exhibit synthetic flexibility in addition to being
selective regarding the central metal atom and sensitive to it.
In coordination chemistry, Schiff bases are known as privileged
ligands because they are prepared using simple condensation
reactions between aldehydes and primary amines [1,2]. The
azomethine (HC=N) group of a Schiff base ligand is primarily
responsible for coordination of metal ions through the lone
pair of electrons presents on N atom. Currently, Schiff bases
constitute an active area of research primarily because they have
numerous and diverse applications in optical material produc-
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200 Alaghaz et al.
Asian J. Chem.
in the intercellular control of cancer [12,13]. Different sequences
in the DNA are involved in various regulatory processes, such
as gene transcription, gene expression, carcinogenesis and
mutagenesis [14]. Interactions between metal complexes and
DNA may cause modifications in the DNA sequences, thereby
causing DNA damage. Zuber et al. [15] reported that the
interaction between small molecules and DNA can cause DNA
damage. In addition, it prevents cell division in cancer cells, which
results in cancer cell death [15].
obtained on a JEOL JMS-D300 mass spectrometer (scan range,
mass-to-charge (m/z) ratio 0-850) at Venture Business Labor-
atory, University of Toyama, Japan. The molar conductivity
-3
of complexes in DMF (10 M) solution was measured at room
temperature using deep vision 601 model digital conductivity
meter. The X-band of EPR spectrum was recorded at LNT (77 K)
and room temperature (300 K).Absorption spectra were recorded
using UV-visible spectrophotometer (Shimadzu model UV-1601)
at room temperature. The X-ray diffraction study of dimeric
Cu(II), Co(II), Ni(II) and Zn(II) complexes were recorded on
a Phillips PW 1130/00 diffractometer with scan axis-Gonio,
start position (2θ-10.004) end position (2θ-89.976) anode
material CuKαALPHA λ= 1.54060 Å and the generator settings
30mA, 40 KV. Surface morphology was studied by a field emis-
sion Scanning Electron Microscopy (SEM) (Model SUPRA
40) with a voltage of 30 KV. Energy Dispersive X-ray (EDX)
analysis was done by EDX (TESCAN) X-max version 4.1.17.D
/Mi 152. The TG, DTG and DTA of Co(II) complex is recorded
on NETZSCH STA 409 C/CD in nitrogen atmosphere at a
heating rate of 10 ºC/min.
Synthesis of Schiff base (L): Schiff base ligand (L) was
synthesized by modifying the procedure of Zhou et al. [28] by
dissolving 5 mmol of 4-aminoantipyrine in 20 mL of ethanol
and 5 mmol of furan-2-carbaldehyde in 20 mL of ethanol. The
mixture of two reactants was stirred for 5 h. The solvent was
then reduced to one-third of its volume and the resultant yellow
solid product was filtered and recrystallized from ethanol and
dried in vacuum at room temperature (Scheme-I).
4
-Aminoantipyrine and its derivatives have amyriad of
applications in catalysis, pharmacology and medicine [16-18].
Understanding the factors involved in drug development and
drug innovation is necessary for progress in health care as well
as future research and development in drug identification. New
types of chemotherapeutic agents containing Schiff bases have
generated tremendous interest among biochemists and some
aminopyridines are commonly administered intravenously for
the diagnosis of liver diseases in clinical practice [19-22]. To
probe their chemotherapeutic functions, applications and prop-
erties, several 4-aminoantipyrine based agents are routinely
administered intravenously for the diagnosis of liver diseases
at diagnostic centers [23]. Chromosensors are widely used for
environmental monitoring and in industrial and biological
processes; consequently, research efforts are now being directed
toward designing and developing sensitive yet simple 4-amino-
antipyrine based chromosensors. 4-Aminoantipyrine was used
as a key intermediate for the synthesis of heterocyclic comp-
ounds with biologically active moieties [24]. 4-Aminoanti-
pyrine administration led to a constant reduction in the contra-
cture of myometrium, whereas the cervix did not exhibitany
major effect [25]. Previous studies [26,27] have explored binding
reactions among 4-aminoantipyrine, human serum albumin
and DNA. 4-Aminoantipyrine derivatives are well-known
sensors in clinical practice. However, studies examining their
activity against cervical cancer are not currently available. In
this study, coordination behaviour of 4-aminoantipyrine ligand
was altered using condensation reactions involving aldehydes,
ketones, thiosemicarbazides and carbazides to obtain a flexible
ligand system. In this study, we reported the synthesis, charact-
erization, antimicrobial, molecular docking and cytotoxic studies
of novel transition metal complexes containing Schiff bases
derived from 4-aminoantipyrine and furan-2-carbaldehyde.
N
N
O
NH2
N
N
EtOH, 3 h
Reflux
+
N
O
O
O
O
Scheme-I: Synthesis of Schiff’s base ligand (L)
Synthesis of 4-(furan-2-ylmethyleneamino)-1,5-dimethyl-
2
-phenyl-1,2-dihydro-3H-pyrazol-3-one (L): Yield: 92 %.
m.f. C16
H
15
3 2
N O ; colour: yellow; Anal. cald. (found) %: C,
6
ν
8.31 (68.28), H, 5.37 (5.32), N, 14.94 (14.91). FT-IR (KBr,
-1
max, cm ): 1638 (-C=O), 1586 (-CH=N), 1027 (-C-O-C), ring
1
vibrations 1584, 1012, 923, 734, 727, 674, 607, 542, 444. H
NMR (DMSO-d ) (δ, ppm): 2.4 (s, 3H, C-CH ), 3.1 (s, 3H, N-
CH ), 7.8-6.8 (m, 8, ArH & hetero-H), 8.3 (s, 1H, -CH=N).
C NMR (DMSO-d ) (δ, ppm): 10.4 (s, 1C, C-CH ), 36.8 (s,
6
3
3
EXPERIMENTAL
13
6
3
In this research work, the chemicals were used asAnalaR
grade without further purification. However, the solvent is used
as a purified solvent by distillation method. The reagents are
furan-2-carbaldehyde and 4-aminoantipyine was procured
from Sigma Aldrich India. All other metal chloride salts were
collected from Merck products.
1C, N-CH ), 55.3-136.8 (m, 13, Ar-C & hetero-C), 145.7 (s, 1C,
-C-O-), 152.2 (s, 1C, C=O), 161.2 (s, 1C, -CH=N); ESI-MS:
281 (molecular ion peak).
Synthesis of dimeric metal(II) complexes:To synthesize
Schiff base metal complexes, Schiff base ligand (2 mmol) and
their corresponding metal chloride salt (2 mmol) were mixed
with 2:2 portion in hot ethanol (50 mL), then it was allowed to
stir with reflux for 8 h. The resultant product was washed with
ethanol, filtered and then recrystallized. The obtained solid
3
Metal contents were determined complexometrically by
standard EDTA titration and chloride content was tested gravi-
3
metrically usingAgNO .Vibration spectral data has been derived
by using FT-IR Shimadzu model IR-Affinity-1 spectrophoto-
product was dried over anhydrous CaCl under vacuum condi-
tion (Scheme-II).
2
1
13
meter using KBr discs. H NMR and C NMR spectral data of
ligand and their dimeric Zinc(II) complex were recorded by using
Bruker 400MHzAdvance III HD Nanobay NMR spectrometer
Synthesis of [Cu (µ-Cl) ((4-(furan-2-ylmethyleneamino-
2
2
1,5-dimethyl-2-phenyl-1,2-dihydro-3H-pyrazol-3-one) Cl ]
2
2
with DMSO-d
6
as a deuterated solvent. Mass spectra were
(1) [CuLCl ] : Yield: 78 %, m.w. 831.5, colour: brown.
2
2

Vol. 31, No. 1 (2019)
Studies of New Dimeric Antipyrine-Schiff Base Metal Complexes 201
2
.4 (s, 6, C-CH
hetero-H), 9.1 (s, 2H, -CH=N). C NMR (DMSO-d
10.5 (s, 2C, C-CH ), 36.4 (s, 2C, N-CH ), 55.2-136.3 (m, 26,
3
), 3.1 (s, 6, N-CH
3
), 7.8-6.8 (m, 16, ArH &
13
Cl
6
) (δ, ppm):
Cl
N
3
3
N
O
O
O
N
N
O
O
O
O
Cu
ArC & hetero-C), 149.8 (s, 4C, C-O-C), 154.3 (s, 2C, C=O),
Co
N
N
Cl
Cl
168.2 (s, 2C, CH=N), ring vibrations 1598, 1023, 855, 748,
N
N
Cl
Cl
+
6
88, 635, 576, 542, 448. ESI-MS: m/z 835 (M ).
N
O
Cu
Cl
N
N
Co
DNA binding experiments: In absorption spectral studies,
N
the intrinsic binding constant (K ) value of these complexes
b
Cl
are obtained by using the medium of 5 mM tris-HCl/50 mM
NaCl buffer (pH 7.2), DNA stock solution and fixed concentra-
tion solution of metal complexes in DMSO. These absorption
spectra were recorded on a Shimadzu model UV-1601 spectro-
photometer using cuvettes having 1 cm path length. The concen-
trated stock solution of metal complexes was diluting suitable
with corresponding buffer solution to required concentrations
for all experiments and the spectra of these complexes were
recorded before and after the addition of DNA [29]. The inter-
action of each complex with DNA was obtained from absorp-
tion date. These absorption spectral titrations were performed
by the incremental addition of nucleic acid to constant concen-
tration of the complex. This titration process was repeated until
there is no change in the absorption values, suggesting the
binding saturation.
Viscosity measurements:Viscosity measurements of all
the metal complexes were carried out by Ostwald viscometer,
at a constant temperature 30.0 ± 0.1 ºC by using thermostat
water-bath. Approximately 0.5mM sample of CT-DNA were
prepared by sonication in order to minimize the complexities
arising from CT-DNA flexibility [30]. Flow time was measured
for all the metal complexes in thrice using the digital stopwatch
and an average flow time was calculated at both pH. The data
(
1)
(
2)
Cl
Ni
Cl
N
N
N
O
O
O
N
O
O
Zn
N
N
Cl
Cl
N
N
Cl
Cl
N
Ni
O
N
N
O
Zn
O
N
Cl
3)
Cl
4)
(
(
Scheme-II: Proposed structures of dimeric Schiff base complexes
Anal. calcd. (found) % for C32
46.22); H, 3.65 (3.64); Cl, 16.95 (17.05); N, 10.08 (10.11);
Cu, 15.24 (15.28). Λ × 10 (DMF, Ω mol cm ) 13.24;
BM:1.87. UV-visible (EtOH): λmax, nm (ε, L mol cm ): 617
23843). FT-IR (KBr, νmax, cm ): 1667 (C=O), 1592 (-CH=N),
008 (-C-O-C), 537 (M-O), 427 (M-N), ring vibrations 1608,
018, 858, 745, 693, 632, 579, 531, 442. ESI-MS: m/z 831
H
30
N
6
O
4
Cl
4
Cu : C, 46.17
2
(
-
3
-1
-1
-2
m
-1
-1
-
1
(
1
1
+
(
M ).
Synthesis of [Co
,5-dimethyl-2-phenyl-1,2-dihydro-3H-pyrazol-3-one)
2) [CoLCl :Yield: 89.7 %, m.w. 822.3, colour: pink. Anal.
calcd. (found) % for C32 Cl Co : C, 46.74 (46.72); H,
.68 (3.66); Cl, 17.24 (17.21); N, 10.22 (10.24); Co, 14.33
2
(µ-Cl)
2
((4-(furan-2-ylmethyleneamino-
1
(
2
Cl
2
]
were calculated against η/η
complexes, where η is indicated as the viscosity of CT-DNA
with metal complexes and η is indicated as a viscosity of CT-
o
and the concentration of metal(II)
2 2
]
H
30
N
6
O
4
4
2
o
3
(
-3
-1
-1
-2
DNA without metal complexes. Viscosity values of complexes
were calculated after correcting the flow time of buffer alone
14.30). Λ
m
× 10 (DMF, Ω mol cm ): 10.32; BM: 5.18. UV-
-
1
-1
visible (EtOH): λmax, nm (ε, L mol cm ): 614 (24847). FT-IR
-1
(t
o
), η = (t − t
DNA damage studies:The extent of pUC19 DNA cleavage
in the presence of an activator H as an oxidizing agent was
0
)/t
0
.
(
KBr, νmax, cm ): 1645 (C=O),1590 (-CH=N), 1010 (-C-O-
C), 522 (M-O), 427 (M-N), ring vibrations 1602, 1014, 853,
+
O
2 2
7
42, 696, 634, 575, 534, 448. ESI-MS: m/z 822 (M ).
Synthesis of [Ni (µ-Cl) ((4-(furan-2-ylmethyleneamino-
,5-dimethyl-2-phenyl-1,2-dihydro-3H-pyrazol-3-one) Cl
:Yield: 89.8 %, m.w. 821.8, colour: green. Anal.
calcd. (found) % for C32 Cl Ni : C, 46.77 (46.72); H,
.68 (3.64); Cl, 17.25 (17.23); N, 10.23 (10.21); Ni, 14.28
monitored by using agarose gel electrophoresis [31]. In reaction
super coiled pUC19 plasmid DNA Form I (1 µg) in DMSO (1 %,
2
2
1
(
2
2
]
2
µL) was treated with metal complexes (250 µg) and oxidizing
2) [NiLCl
]
2 2
agent H (40 mmol, 5 µL). The samples were incubated at 1 h
2 2
O
H
30
N
6
O
4
4
2
at 310 K. A loading of buffer containing bromophenol blue
(0.25 %), glycerol (30 %) and xylenecyanol (0.25 %) was added
to a platform fixed with a comb to form slots. The electropho-
resis was performed at 50V for 1 h in tris-boric acid-EDTA buffer
using 1 % agarose gel containing 0.5 µg/mL ethidium bromide.
The effectiveness of the metal complexes was measured by deter-
mining the capacity of complexes converting the super coiled
form of DNA to circular form and finally linear form. It was
visualized by UV light to photograph the bands.
3
(
-
3
-1
-1
-2
14.25). Λ
m
× 10 (DMF, Ω mol cm ): 8.61; BM: 2.89. UV-
-
1
-1
visible (EtOH): λmax, nm (ε, L mol cm ): 624 (21843). FT-IR
-1
(
KBr, νmax, cm ): 1639 (C=O), 1598 (-CH=N), 1014 (-C-O-
C), 537 (M-O), 423 (M-N), ring vibrations 1604, 1021, 857,
+
7
46, 658, 639, 573, 535, 449. ESI-MS: m/z 821 (M ).
Synthesis of [Zn (µ-Cl) ((4-(furan-2-ylmethyleneamino-
,5-dimethyl-2-phenyl-1,2-dihydro-3H-pyrazol-3-one) Cl
:Yield: 88.3 %, m.w. 821.8, colour: pale yellow.
Anal. calcd. (found) % for C32 Cl Zn : C, 46.02 (46.00);
H, 3.62 (3.61); Cl, 16.98 (16.93); N, 10.06 (10.02); Zn, 15.66
2
2
1
(
2
2
]
2) [ZnLCl
2 2
]
Antimicrobial assay: Minimum inhibitory concentration
H
30
N
6
O
4
4
2
(MIC) is the very low concentration of an antimicrobial comp-
-3
-1
-1
-2
ound that will retard the visible growth of microorganisms at
310 K after overnight incubation. Minimum inhibitory concen-
trations are important in diagnostic laboratories to confirm the
resistance of microorganisms to antimicrobial agents and also
(
15.64). Λ
m
× 10 (DMF, Ω mol cm ): 5.67; µeff: diamagnetic.
-1
-1
UV-visible (EtOH): λmax, nm (ε, L mol cm ): 265 (35720). FT-
IR (KBr, νmax, cm ): 1633 (C=O), 1591 (CH=N), 1012 (-C-O-
C), 523 (M-O), 444 (M-N). H NMR (DMSO-d ) (δ, ppm):
-
1
1
6

202 Alaghaz et al.
Asian J. Chem.
to monitor the activity of new antimicrobial agents. The MIC
value of the ligand and its metal complexes were tested against
bacterial strains (Staphylococcus aureus, Bacillus subtilis,
Klebsiella pneumonia and Salmonella typhi) and fungi strains
ability to predict the binding-conformation of small molecule
ligands to the suitable target binding site. Characterisation of
the binding behaviour plays a significant role in the rational
design of drugs as well as to clarify the basic biochemical proc-
esses. Pavalue was given by the PASS online software.The enzyme
is responsible for inflammation of protein data bank. To establish
the optimized binding modes of synthesized ligand and its
complexes against COX-2 molecular docking studies were
accomplished using HEX 8.0 software. Hex 8.0 is a molecular
docking software and a bilateral molecular graphics progra-
mmes for studying the optimized docking [36] mode between
COX-2 and synthesized compound. The structure of ligand
and its metal complexes was transformed into PDB file format
using Pymole software [37]. The structure was optimized before
docking.
(
Aspergillus niger, Aspergillus flavus, Curvularia lunata and
Candida albicans) through a broth dilutions method. The results
of antimicrobial activity associated to the standard antibacterial
and antifungal strains as ampicillin and nystatin, respectively.
These test concentration of ligand and metal complexes were
made from 0.01 to 2.5 mg/ml in the sterile walls of microtiter
plates and using 50 µL of sterile nutrient broth. The MIC values
were determined by reading each well at 492 nm in an auto-
mated micro plate reader.
Cytotoxic activity (MTT assay):A pair of cancer cell lines
such as human breast adenocarcinoma (MCF-7), Human Liver
Cancer (Hep G2) and non-cancerous cell lines such as HBL-
RESULTS AND DISCUSSION
100, established from human breast milk were purchased from
National centre for cell science (NCCS) Pune, India. The cell
lines were grown in Dulbecco's modified Eagles medium (DMEM)
Four different dimeric metal(II) complexes were prepared
by using the Schiff base ligand and corresponding metal(II)
chloride in the ratio of 2:2 in an ethanol medium. In common
organic solvents, the ligand is soluble and its metal complexes
are freely soluble in DMSO and DMF, but they are insoluble
in many other common organic solvents. Both ligand and comp-
lexes are stable at room temperature. Analytical and spectral
data of these complexes provide the good result to predict the
geometry of the complexes and conductivity values of these
complexes indicate that they are electrolytic in nature.
(
Himedia, India) containing 10 % fetal bovine serum (FBS)
and 1% antibiotic-antimycotic solutions of all the compounds
were prepared in cell culture grade DMSO (Himedia). The effect
of treatment of compounds on cell viability was determined
using tetrazolium dye by 3-(4,5-dimethyl thiazol-2yl)-2,5-
diphenyl tetrazolium bromide (MTT) assay as reported earlier
[32]. The cytotoxic effect of dimeric complexes of Cu(II), Co(II),
Ni(II), Zn(II) against different cell lines was evaluated by MTT
assay. Briefly, the cells were seeded in a 96-well plate and kept
in CO for attachment and growth for 24 h and then the cells
were treated with various concentrations of complex dissolved
in DMSO and incubated for 24 h.After incubation, the culture
medium was removed and 15 mL of MTT was discarded and
DMSO (100 mL/well) was added to solubilize the purple form-
azan product. The experiment was carried out in triplicates and
the medium without complex served as control. The absorbance
was measured calorimetrically at 570 nm using an ELISA micro
plate reader. The percentage of cell viability was calculated
using the following formula:
FT-IR analysis: FT-IR spectra of compounds explore the
bonding nature of the organic compounds and functional group
determination. The free ligand affirms νC=O stretching fre-
-1
quency at 1638 cm [38], azomethine (HC=N) occurs at 1586
-1
-1
cm and (-C-O-C) furan ring at 1027 cm . These two peaks
are relocated to the region of 1667-1633, 598-1590 and 1014-
-1
1
008 cm , respectively in all the complexes owing to the
coordination of carbonyl and azomethine group of the ligand to
metal ion. This value is further supported by M-N characteristic
-1
peak at 444-423 and M-O band at 537-522 cm in all the metal
complexes [39].
Electronic spectral analysis: The electronic spectrum
of the Schiff base ligand L shows intense two bands in 272,
OD value of treated cells
IC =
×100
5
0
OD value of untreated cells (control)
*
*
2
93, 318 and 437 nm matched up to n → π and π → π transi-
A graph was plotted with the percentage of cell inhibition
versus concentration. From this graph IC50 (concentration of
compound to kill 50 % of cells) value was calculated.
in silico Biological activity of ligand:The priority of optim-
izing ADME-TOX properties of potential drug molecules is
now extensively identified [33]. in silico ADME-TOX prop-
erties of synthesized ligand were studied by usingVLS3D online
software. Huge number of compounds were barred in clinical
development because of awful pharmacokinetics properties
tions for the aromatic ring, CH=N and C=O groups, respectively.
However, these four absorption bands are also observed in the
electronic spectra of all synthesized complexes with slight
shifts to the higher region in wavelength. This shift may be
due to the alteration of molecular environment of Schiff base
ligand after the chelation with metal ions [40-42]. The electronic
spectra of the ligand and its dimeric Co(II) and Zn(II) complexes
are shown in Fig. 1.
1
13
1
H and C NMR spectra: In H NMR spectrum of the
[
34,35]. But along with the help of cheminformatics online
ligand displays the multiplet at 7.8-6.8 ppm which is associated
to the proton in the phenyl ring, and also it exhibits the peaks
software, it is possible to predictADME-TOX property through
reliable admetsar, Osiris calculator, SMOP, etc. In the present
study, absorption, distribution, metabolism, excretion and
toxicity properties of the synthesized ligand were calculated
by using admetsar.
at 2.4 (s, 3H), 3.1 (s, 3H) and 8.3 ppm (s, 1H) for C-CH
3
, N-
CH and CH=N, respectively. In complex CH=N group exhibits
3
a peak at 9.3 ppm, which indicates the coordination of
azomethine group during the complexation with zinc ion [43].
13
Molecular docking: Molecular docking is one of the most
powerful methods in structure-based drug design, due to its
C NMR spectrum of Schiff base ligand indicates the
carbon environment of phenyl group at 55.2-136.3 ppm. It

Vol. 31, No. 1 (2019)
Studies of New Dimeric Antipyrine-Schiff Base Metal Complexes 203
0
0
0
.3
.2
.1
0
(a)
3
000 G
2
00 G
ν
9.1 GHz
305
350
400
450
500
H Gauss
Wavelength (nm)
1
.0
.8
.6
.4
.2
0
0
0
0
0
.8
.6
.4
.2
0
Scan range
Fig. 2. EPR spectrum of Cu(II) complex in DMSO solution at LNT
0
0
0
0
(b)
It explained two g factors g|| and g
Hamiltonian spectral data of dimeric [CuLCl
in Table-1. From this spectral data, it is found that g|| (2.24) >
(2.03) > g (2.0024), which revealed that the one unpaired
⊥
, the calculated spin
]
2 2
are presented
g
⊥
e
5
00
600
700
800
electron is present in (dx2-y2) orbital and it is likely to be the
octahedral geometry as conferred in electronic spectroscopy
Wavelength (nm)
[
46]. The g|| value of dimeric Cu(II) complex can be used as a
measure of covalent behaviour of copper-ligand interaction.
If the value is higher than 2.4, copper-ligand bond is basically
ionic and the value less than the 2.4 is symptomatic of covalent
character. In the present ESR results, lesser value of g|| (2.24)
is correlated to 2.4 which is an excellent substantiation for the
covalent character of Cu-nitrogen interactions, as recommen-
ded by Kivelson and Neiman [47]. From the values of g factors,
the exchange coupling factor (G) can be expressed by the follo-
wing equation:
4
00
500
600
Wavelength (nm)
700
800
0.6
0.5
0.4
0.3
0.2
0.1
0
0
.35
.30
.25
.20
.15
.10
.05
0
0
0
0
0
0
0
(
c)
g − 2
|
|
G =
g⊥ − 2
5
00
550
600
650
700
750
800
Wavelength (nm)
It has been already investigated that if G > 4.0, the local
tetragonal axes are aligned parallel or slightly misaligned. If
G < 4.0, considerable exchange coupling might be present
and generate the considerable misalignment. In this study, the
4
00
500
600
Wavelength (nm)
700
800
observed G value of [CuLCl
2
] is 5.4 which explains that the
2
Fig. 1. Electronic spectra of (a) ligand, (b) [CoLCl
complexes
2
]
2
and (c) [ZnLCl
2
]
2
local tetragonal axes are aligned parallel or slightly misaligned
and exchange coupling could be worthless [48]. The observed
empirical ratio of g||/A|| value for the dimeric complex [CuLCl
is 162 which pointed out the octahedral geometry of dimeric
CuLCl with small distortion.
ESI-MS spectrum: Peaks conducted from mass spectra
2
]
2
also exhibits C-CH
0.4, 36.8, 161.2, 152. 2 and 145.7 ppm, respectively. In dimeric
ZnLCl complex, HC=N, C=O and C-O groups are shifted
to downfield due to coordination of azomethine and carbonyl
group containing donor atoms with zinc ion [44]. There is no
significant change in all other signals in the synthesized dimeric
3
, N-CH , HC=N, C=O and C-O signals at
3
1
[
[
]
2 2
]
2 2
of the ligand and its dimeric Cu(II), Co(II), Ni(II) and Zn(II)
complexes was attributed to the molecular ions m/z at 281,
+
+
8
31.5, 822.3, 821.8 M and 835.2 M , respectively. This fact
was matched to the suggested molecular formula for these syn-
thesized compounds i.e. ligand, [CuLCl , [CoLCl , [NiLCl
and [ZnLCl , where L = 4-(furan-2-ylmethyleneamino)-1,5-
[
ZnLCl
2
]
2
complex.
Electron spin resonance analysis: ESR spectroscopy has
]
2 2
2 2
]
]
2 2
been considered as the most admirable technique to scrutinize
the metal environment in the complexes [45]. It is used to analyze
the geometry and nature of bonding between copper ion and its
ligand, mainly in dimeric Cu(II) complex. In the present study,
]
2 2
dimethyl-2-phenyl-1,2-dihydro-3H-pyrazol-3-one. This
confirms the Schiff-base frame formation. Elemental analysis
values were appropriate with those values calculated from the
molecular formulae assigned to these complexes which are
further supported by mass studies.
ESR spectrum of dimeric [CuLCl
ded ESR spectrum of dimeric [CuLCl
2
]
2
was effectuated. The recor-
is represented in Fig. 2.
]
2 2

204 Alaghaz et al.
Asian J. Chem.
TABLE-1
THE SPIN HAMILTONIAN PARAMETERS OF Cu(II) COMPLEX IN DMSO SOLUTION AT LNT
-
4
-1
g-Tensor
A × 10 (cm )
Complex
Cu(II)
g||
G
⊥
giso
A||
A
⊥
Aiso
83
G
2.24
2.03
2.0024
134
69
5.4
XRD, EDX, SEM and TEM morphological studies: In
Fig. 3, XRD patterns of [CuLCl , [CoLCl , [NiLCl and
ZnLCl complexes are given. The indexing procedures were
performed using (CCP4, UK) CRYSFIRE program [49,50]
giving triclinic crystal system for [CuLCl having M(9) = 8,
F(6) = 7, [CoLCl monoclinic crystal system having M(9) =
, F(6) = 9, orthorhombic crystal system for [NiLCl having
M(6) = 15, F(6) = 5, and monoclinic crystal system for [CuLCl
and [ZnLCl
2
] they have been successfully integrated into the
2
2 2
]
2 2
]
2 2
]
sample structure. All positions contain predictable elements,
and different elements such as impurity were not detected.
[
]
2 2
Fig. 6a-c shows the TEM images of [CuLCl
2
]
2
, [CoLCl
2
]
2
,
2 2
]
[NiLCl and [ZnLCl complexes, respectively. The consistency
2
]
2
]
2 2
2 2
]
and resemblance in between the particle forms of synthesized
dimeric complexes, suggest that the structural phases have a
similar template. The particles diameter is found in the nano-
range as fellow: Cu(II), 57-228 nm; Co(II), 43-173 nm; Ni(II),
18-23 nm and Zn(II), 12-32 nm. Nanoparticle-size complexes
may act strong in different application areas between a
biological one. Complexation feature is also authenticated from
the SEM image of the respective dimeric complex [Cu(II),
Co(II), Ni(II), Zn(II)] has the different features with smooth
and rough surface regions. It is also further evidenced by the
brighter region, which indicates the presence of metallic species
in the samples and thus confirming the presence of dimeric
Cu(II), Co(II), Ni(II) and Zn(II) ions in the synthesized compound.
Thermal analysis: Thermal decomposition of dimeric
complexes (1-4), was studied in the temperature range 30-1200 ºC.
8
]
2 2
]
2 2
having M(6) = 7, F(6) = 11 as the superior solutions. Their cell
parameters are interpreted in Table-2.
The SEM images of [CuLCl
ZnLCl complexes are shown in Fig. 4a-d, respectively. The
micrograph of [CuL ]·2Cl complex shows peel shaped particles.
The [CoLCl and [ZnLCl complexes mention ice rock structure.
The [NiLCl and [ZnLCl complexes grain-structure of ice
2
]
2
, [CoLCl
2
]
2
, [NiLCl ]
2 2
and
[
]
2 2
2
2
]
2
]
2 2
2 2
]
]
2 2
was existent. The results by EDX analysis explained that copper,
cobalt, nickel, zinc and carbon, nitrogen and oxygen peaks,
which meant there were copper, cobalt, nickel, zinc and carbon,
nitrogen and oxygen deposited products as shown in Fig. 5a-d.
The EDX line traces indicate that [CuLCl
2
]
2
, [CoLCl
2
]
2
2 2
, [NiLCl ]
1
1
1
8000
6000
4000
3
2
2
1
1
0000
5000
0000
5000
0000
(a)
(b)
12000
1
0000
000
000
000
000
8
6
4
5
000
2
0
0
0
10
20
30
40
50
60
70
80
0
10
20
30
40
50
60
70
80
2θ
(°)
2θ
(°)
35000
30000
25000
20000
15000
10000
1
1
1
8000
6000
4000
(d)
(c)
12000
0000
000
000
000
000
1
8
6
4
2
0
5000
0
10
20
30
40
50
60
70
80
0
10
20
30
40
50
60
70
80
2θ
(°)
2θ (°)
Fig. 3. Powder XRD spectra of (a) [CuLCl
2
]
2
, (b) [CoLCl
2
]
2
, (c) [NiLCl
2
]
2
and (d) [ZnLCl
2
] complexes, respectively
2

Vol. 31, No. 1 (2019)
Studies of New Dimeric Antipyrine-Schiff Base Metal Complexes 205
TABLE-2
CRYSTALLOGRAPHIC DATA FOR THE SCHIFF BASE COMPLEXES [CuLCl ] , [CoLCl ] , [NiLCl ] AND [ZnLCl ]
2
2
2
2
2
2
2 2
Data
[CuLCl2]2
[CoLCl2]2
[NiLCl2]2
NiC H N O Cl
821.8
1.54056
Orthorhombic
[ZnLCl2]2
Empirical formula
Formula weight (g/mol)
Wavelength (Å)
Crystal system
CuC H N O Cl
CoC H N O Cl
ZnC H N O Cl
2
3
2
30
6
4
2
32 30
6
4
2
32 30
6
4
2
32 30
6
4
831.5
822.3
0.71073
Monoclinic
835.2
1.54056
Monoclinic
0.71073
Triclinic
P?
Space group
Pn
Pbca
Pn
a (Å)
b (Å)
c (Å)
α (°)
β (°)
γ (°)
5.8596(3)
9.6932(5)
12.8257(7)
116.649(6)
92.899(5)
102.230(5)
3325.7(4)
1.358
9.0086(7)
19.1090(13)
18.589(2)
90
101.46(3)
90
12.287 (5)
11.884 (5)
37.152 (5)
90
90
90
5854.3(4)
1.543
8.9981(5)
12.6314(8)
19.4346(10)
90
103.019(4)
90
3
Volume (Å )
854.36(4)
1.989
856.46(4)
1.993
–3
(
Calc.) density (g/cm )
2
θ range
1.71–28.27
–16 ≤ h ≥ 6
1.71–28.27
–6 ≤ h ≥ 7
–34 ≤ k ≤ 34
–12 ≤ l ≥ 9
4
10.25–67.27
0 ≤ h ≥ 15
0 ≤ k ≤ 15
0 ≤ l ≥ 47
8
12.56–68.00
–10 ≤ h ≥ 10
–12 ≤ k ≤ 12
–25 ≤ l ≥ 25
2
Limiting indices
–15 ≤ k ≥ 5
–15 ≤ l ≥ 5
Z
2
Rf
0.0000842
298
0.000847
298
0.0000323
298
0.0000907
298
Temperature (K)
Fig. 4. SEM images of (a) [CuLCl
2
]
2
, (b) [CoLCl
2
]
2
, (c) [NiLCl
2
]
2
and (d) [ZnLCl
2
2
] complexes

206 Alaghaz et al.
Asian J. Chem.
1.0
0.8
0.6
0.4
0.2
1.0
0.8
0.6
0.4
0.2
(a)
(b)
C
C
N
N
O
Cu
O
Co
Cl
Cl
Cl
Cl
Cu
7
Co
7
N
N
0
0
0
1
2
3
4
5
6
8
9
10
11
0
1
2
3
4
5
6
8
9
10
11
Energy (keV)
Energy (keV)
1
1
0
0
0
.5
2.5
(
d)
(
c)
.2
.9
.6
.3
0
2.0
1.5
1.0
0.5
0
Ni
Zn
O
C
Cl
Cl
N
Zn
Ni
N
Cu
1
O
C
0
2
3
4
5
6
7
8
9
10
11
0
1
2
3
4
5
6
7
8
9
10
11
Energy (keV)
Energy (keV)
Fig. 5. EDX images of (a) [CuLCl
2
]
2
, (b) [CoLCl
2
]
2
, (c) [NiLCl
2
]
2
and (d) [ZnLCl
2
] complexes, respectively
2
Fig. 6. TEM images of (a) [CuLCl
2
]
2
, (b) [CoLCl
2
]
2
, (c) [NiLCl
2
]
2
and (d) [ZnLCl
2
] complexes
2

Vol. 31, No. 1 (2019)
Studies of New Dimeric Antipyrine-Schiff Base Metal Complexes 207
All the thermogravimetric analysis curves for dimeric comp-
lexes (1-4) are shown in Fig. 7. No change in weight was observed
in the range 90-268 ºC, which indicated the absence of coord-
inated or uncoordinated water. During the experiment, all the
dimeric complexes first underwent a decomposition step and
exhibited a decrease of 7.18-14.76 % (7.24-14.89 %) in weight
in the range 265-270 ºC owing to the removal of chloride anions.
The complexes underwent a second decomposition step 79.32-
aureus (gram-positive bacterium), Bacillus subtilis (gram-positive
bacterium), Salmonella typhi and Escherichia coli (both gram-
negative bacterium) as well as those of fungi, such as Aspergillus
niger, Aspergillus flavus, Curvularia lunata and Candida albicans,
by using the broth micro dilution method.Ampicillin and nystatin
were used as standard drugs for antibacterial and antifungal activity
studies, respectively. The results of antimicrobial activity studies
with the Schiff base and its metal complexes are presented in
Table-4. The dimeric metal(II) complexes showed higher antimi-
crobial activities than did L. These observations can be explained
by Overtone's concept andTweedy's chelation theory [54].Accor-
ding to this concept, chelation considerably reduces the polarity
of metal ions because of partial donation of positive charges to
the donor groups and π-electron delocalization within the entire
chelating ring system.The reduction in polarity of the metal ion
increased the lipophilicity of complexes and enhanced the
interaction between the metal ion and lipid bilayer, thus facilitating
the diffusion of (chelated) metal ion through the cell membrane.
The minimum inhibitory concentration values of the complexes
indicated that the activity of the complexes against the selected
bacteria and fungi stains was higher than that of ligand because
of the presence of azomethine (HC=N) group in ligand and its
chelating effect on the central metal ion. The azomethine group
and its chelating effect improved the efficiency of antibacterial
agents, and the metal complexes exhibited either microbicidal
or microbiostatic effects by blocking the active sites of crucial
enzymes [55]. In addition, the main ligand containing (oxygen
and nitrogen) donor groups might reduce enzyme activity.
Because the enzymes need the groups (donor atoms) for their
activity, they are highly susceptible to deactivation by the metal
ion because of chelation.
8
1.59 % (79.38-81.62 %) in the temperature range 380-1100
ºC due to the loss of complete ligand. IR spectroscopy was
used to analyze and identify the final residue as metal oxide
corresponding to calculated value.
Kinetic studies: Graphical estimation methods, such as
Coats-Redfern [51], Horowitz-Metzger [52] and Piloyan-
Novikova [53] methods, were used to determine the kinetic
parameters (Table-3) such as thermal activation energy of decom-
*
*
position (E ), enthalpy (∆H ), entropy (∆S ) and Gibbs free
a
*
energy change of decomposition (∆G ) of the complexes.The
following conclusions were drawn from the results obtained:
•
The high values of the energy of activation, E of the
a
complexes indicated that the metal complexes were highly
stable owing to their covalent bond character.
•
The complexes that were analyzed exhibited positive
∆
G values, thus indicating that the free energy of final residue
was higher than that of initial compounds; hence, the complexes
underwent decomposition non-spontaneously. Furthermore,
the values of activation (∆G), increased significantly in the subse-
quent decomposition stages of a given complex.This observation
can be explained by the significant increase in the values of T∆S
from one step to the next, which predominated the values of ∆H.
•
The negative values of ∆S for the decomposition steps
indicated that all the studied dimeric complexes were more
ordered in their activated states than in their resting states.
Antimicrobial screening: Thein vitro antimicrobial activities
of synthesized Schiff base ligand (L) and its corresponding dimeric
metal(II) complexes were tested using cultures of Staphylococcus
in vitro Cytotoxicity assay: The in vitro antiproliferative
activity was assayed using two cancer cell lines, namely (MCF-7)
(human breast adenocarcinoma cancer cell line), Hep G2 (human
liver carcinoma cell line) and HBL-100, which is a non-canc-
erous cell line established from human breast milk), through
–
3
–
3
–3
9
8
7
6
5
4
3
2
1
0
-1
5.00×10
6
5
4
3
2
1
0
1
2.00×10
8
7
2.00×10
0
-
0
–3
–3
–3
–3
–2
–2
–2
0
2.00×10
–
3
6
5
4
-
-
-
-
-
2.00×10
–3
mg
mg/s
-4.00×10
-6.00×10
-8.00×10
1.00×10
1.20×10
5.00×10
mg
mg/s
–3
4.00×10
mg
–2
(a)
–
3
(c)
1.00×10
mg/s
6.00×10
3
2
1
(b)
-
-
–3
–2
8.00×10
1.50×10
–2
1.00×10
-1.40×10–
-1.60×10
–2
-1.80×10
–2
2
2.00×10
–
2
2
0
-1
-1.20×10
0
200
400 600
Temp. (°C)
800
1000
0
200
400
600
Temp. (°C)
800
1000 1200
0
200
400
600
Temp. (°C)
800
1000
1200
–2
–
2.50×10
-
-1.40×10
Fig. 7. TG/DTG of (a) Cu(II), (b) Co(II), and (c) Ni(II) complexes respectively up to 1000 °C
TABLE-3
KINETIC PARAMETERS OF Cu(II), Co(II) AND Ni(II) COMPLEXES
Decomposition
temp. (°C)
Parameters
Correlation
coefficient
Compound
Method
–
1
–1
*
–1
–1
*
–1
*
–1
E (kJ mol )
A (s )
∆S (J mol K ) ∆H (kJ mol )
∆G (kJ mol )
a
CR
HM
PN
CR
HM
PN
CR
HM
PN
38.9
42.3
41.7
68.8
68.3
69.1
66.6
67.2
66.8
162
163
162
180
182
179
169
170
171
–203
–207
–205
–238
–236
–237
–242
–244
–243
54.2
55.6
56.0
54.2
64.8
65.2
64.7
55.8
56.3
162
165
163
167
166
164
159
158
157
0.9924
0.9928
0.9929
0.9896
0.9899
0.9897
0.9887
0.9888
0.9885
[
[
CuLCl2]2
CoLCl2]2
142–372
193–347
187–374
[
NiLCl2]2

208 Alaghaz et al.
Asian J. Chem.
TABLE-4
MINIMUM INHIBITORY CONCENTRATION OF THE SYNTHESIZED FREE LIGAND
AND ITS METAL COMPLEXES AGAINST THE GROWTH OF BACTERIA AND FUNGI (?M)
4
4
Bacteria: MIC values (× 10 µM) SEM = ± 1.5
Fungi: MIC values (× 10 µM) SEM = ± 1.3
Compound
S. aureus
24.5
9.6
11.8
10.1
9.8
B. subtilis
25.8
92
10.8
11.6
10.4
3.2
S. typhi
27.5
10.4
11.4
9.7
9.4
4.2
–
E. coli
24.9
10.5
11.6
10.8
9.7
A. niger
26.8
11.3
13.5
12.7
11.9
–
A. flavus
25.3
11.3
13.9
12.8
12.5
–
C. lunata
25.8
12.4
11.6
13.9
11.2
–
C. albicans
25.7
12.7
12.7
13.5
11.6
–
Ligand
Cu(II)
Co(II)
Ni(II)
Zn(II)
Ampicillin
Nystatin
2.6
–
4.4
–
–
3.5
3.8
4.3
4.8
the MTT assay by using cisplatin as a standard (positive control).
The principle of MTT assay is that only viable cells can reduce
yellow MTT to form purple formazan products, whereas non-
viable cells cannot. Therefore, the metabolic activities of cells
were evaluated using their capability to cleave the tetrazolium
rings of yellow MTT and produce the purple formazan crystals.
The MTT assay results showed that the metal complexes hindered
the growth of cancer cells. The above-mentioned cell lines were
treated with the synthesized metal complexes at different
concentrations (0-100 mM) for 48 h. The results of cell inhibition
were expressed as IC50 values. These values are listed in Table-5
and cell viability depended on the concentration of dimeric
metal(II) complexes. The IC50 values decreased with an increase
in the concentration of dimeric metal(II) complexes, which
indicated that the cytotoxicity effect of synthesized dimeric
complexes depended on the dose and duration of exposure. All
the dimeric Cu(II), Co(II), Ni(II) and Zn(II) complexes showed
remarkable cytotoxicity toward the cancer cell lines, (MCF-7)
and HepG2. Among these complexes, copper complex caused
the highest growth hindrance in the MCF-7 and HepG2 under
the similar experimental conditions. The high cytotoxic effect
of copper complex may be associated with the size, charge (on
metal ion), ionic radius, primary ligand, steric and pharmaco-
kinetic factors, which collectively play a prominent role in the
potency of the drugs [56]. The IC50 values of complex in MCF-
for the blooming of modern drug design and findings as well
as to discover the exact binding site offered at the molecular target
DNA, mostly in a non-covalent interaction [57]. The probable
binding modes of the synthesized ligand and dimeric metal(II)
complexes were prearranged in HEX 8.0 software against
COX-2 (cyclooxygenase) receptor molecule which is based
on PASS biological activity prediction score. COX-2 enzyme
is an inflammation response, which was recovered from PDB
database (Protein Data Bank) (PDB ID:1CX2). It is universally
acknowledged that if the binding free energy is low, then the
binding affinity is more effective in between the receptor (DNA)
and "ligand" (Schiff base and its dimeric metal(II) complexes)
molecules, for the reasons, molecular docked model poses are
depicted in Fig. 8, which evidently explicates that Schiff base
ligand and its metal complexes with DNA through an intercal-
ation mode concerning outside edge stacking contact with the
oxygen atom of the phosphate backbone, it is clear that the
ligand and dimeric metal(II) complexes were to be positioned
suitably into the intercalation mode of targeted DNA. In addition,
resultant structures are stabilized by the vander Waal's inter-
action and hydrophobic contacts with DNA functional group
that delineate the stability of interaction [58]. The docking
scores of molecular docked ligand and the dimeric metal(II)
2+
2+
2+
2+
complexes (Cu , Co , Ni , Zn ) are foundto be (-253.4), (-289.7),
(- 289.5), ( -289.3), (-289.2), respectively. It is very enthralling
7
and HepG2 cell lines were 14 and 19 mM, respectively. In
to note that the binding energy of [CuL
that of other complexes and the order of binding energy is as
follows [CuLCl > [CoLCl > [NiLCl > [ZnLCl . These
data suggested that the dimeric metal(II) complexes have more
medicinal power and intended to reduce the inflammation or
swelling (due to heat and chemicals) than ligand.
2
]·2Cl is higher than
particular, in vitro antiproliferative activity testing of the synthe-
sized compounds against non-cancerous cell line HBL-100
showed that IC50 value was > 87 µM. This indicated that all the
dimeric complexes behave differently toward cancer cells and
non-cancerous cells and are less toxic to the HBL-100 cells than
to the cancerous cells. Thus, the IC50 values are inversely propor-
tional to the biological potential of the compound.
2 2
]
2 2
]
2
]
2
2 2
]
DNA Interaction studies
Electronic absorption titration: The UV-visible spectra
of [CuLCl
2
]
2
and [ZnLCl ] complexes in the presence or absence
2
2
TABLE-5
of CT-DNA are given in Figs. 9 and 10. DNA binding effects of
metal complexes possess powerful authentication for the growth
of efficient metal based chemotherapeutic drugs. The absorp-
tion spectral titration is a most credible instrument for observing
the metal-DNA interacting mechanism and complex formation.
The transition and inner transition metal complexes bind to
DNA through the covalent or non-covalent and electrostatic
binding interactions [59]. These binding propensities of
CYTOTOXICITY OF Cu(II), Co(II), Ni(II), Zn(II) COMPLEXES
AGAINST MCF-7, HepG2 AND HBL-100 HUMAN CELL LINES
IC values (mM)
50
Complexes
MCF-7
14 ± 0.9
28 ± 1.2
24 ± 0.7
20 ± 0.6
17 ± 1.2
HepG 2
19 ± 0.9
28 ± 1.2
22 ± 1.2
25 ± 0.7
21 ± 1.2
HBL-100
82 ± 0.9
95 ± 1.0
91 ± 0.5
87 ± 0.7
85 ± 1.1
Cu(II)
Co(II)
Ni(II)
Zn(II)
Cisplatin (standard)
[
CuLCl
2
]
2
, [CoLCl
2
]
2
, [NiLCl ]
2 2
and [ZnLCl
2
] complexes are
2
achieved by the diverse methods. One of the most important
method is electronic absorption titration. The deformation of
DNA structure is frequently correlated with the anticancer activity.
Molecular docking analysis: Molecular docking is a meti-
culous method to investigate the drug-nucleic acid interactions

Vol. 31, No. 1 (2019)
Studies of New Dimeric Antipyrine-Schiff Base Metal Complexes 209
Fig. 8. Binding model of synthesized compound with COX-2 receptor: (a) Schiff base ligand; (b) Cu(II), (c) Co(II), (d) Ni(II) and (e) Zn(II)
complexes
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0
0.6
.5
0.4
.3
0.2
0
0
0.1
0
265
285
305
325
345
265
285
305
325
345
Wavelength (nm)
Wavelength (nm)
Fig. 9. Electronic absorption spectra of [CuLCl
]
2 2
in 5 mM Tris-HCl/50
Fig. 10. Electronic absorption spectra of [ZnLCl ] in 5 mM Tris-HCl/50
2 2
mM NaCl buffer (pH = 7.2 at 298 K) in the presence of increasing
amount of CT-DNA
mM NaCl buffer (pH = 7.2 at 298 K) in the presence of increasing
amount of CT-DNA
Therefore, to conclude the anticancer mediator, DNA binding
study is considered as an essential one. In this binding study,
the above-mentioned metal complexes were recorded by using
tration of the complexes they were titrated with increasing
amount of CT-DNA (each addition of 30 µL). The increasing
amount of CT-DNA results from hypsochromic shift in the range
of 3-5 nm and significant hypochromicity [60]. The observed
hypochromism might to be accredited for the stacking interac-
tions between DNA and aromatic chromophore of dimeric comp-
lexes, which are consistent with all the dimeric metal(II) complexes
5
mM tris-HCl/50 mM NaCl buffer solution (pH = 7.2). The
absorption spectra of all the complexes indicated an intensive
absorption band in the region around 336-346 nm, in 5mM
tris-HCl/50 mM NaCl (pH 7.2) buffer solution.At a fixed concen-

210 Alaghaz et al.
Asian J. Chem.
binding to CT-DNA through the intercalative binding mode
and the absorbed hypochromicity (H %) of these synthesized
dimeric complexes exhibits ~ 11-17 % which clearly denotes
that all the dimeric metal(II) complexes strongly bind to DNA.
EB
1
2.4
2.0
1.6
1.2
4
3
b
The intrinsic binding constant values of K , were calculated
from the slope by intercept values obtained from the plots between
2
[
DNA]/(ε
b
− ε
f
) and [DNA] which are given in Table-6.
TABLE-6
ELECTRONIC ABSORPTION PARAMETERS
FOR THE INTERACTION OF DNA WITH Cu(II),
Co(II), Ni(II) AND Zn(II) COMPLEXES
a
H (%) = [(Afree – Abound)/Afree] × 100
4
λmax
K × 10
b
–1
a
1
2
3
4
5
6
Complexes
∆λ (nm)
H (%)
(
M )
Free
336
337
338
346
Bound
342
342
343
350
[Complex]/[DNA]
Cu(II)
Co(II)
Ni(II)
Zn(II)
5
4
4
3
16.9
14.6
11.8
11.2
9.7
7.4
5.7
6.3
Fig. 11. Effects of increasing amount of classical intercalator [EB] and
complexes on the relative viscosity of CT-DNA in 5 mM Tris-HCl/
50 mM NaCl buffer at room temperature, where (1) Cu(II), (2)
Co(II), (3) Ni(II) and (4) Zn(II) complexes
Viscosity measurements: The DNA-binding action of the
complex was determined using viscometric measurement. This
method is considered as the least ambiguous and most crucial
test for the DNA binding in solution. The viscosities were perfo-
rmed at room temperature. Partial and non-classical inter-
calative compounds can bend or kick the DNA, thus reducing
its effective length and minimizing the viscosity of the DNA.
This measurement provides the evidence to support the occur-
rence of intercalative binding. Furthermore, viscometry is the
useful tool for determining the changes in the length of DNA
and providing information about the binding mode of dimeric
metal(II) complexes with DNA. An increase in the viscosity
was observed owing to the intercalative binding in DNA because
the intercalation between the base pairs causes lengthening of
DNA polymer due to effective DNA base pairs separation.
Ethidium bromide is commonly used as a strong intercalating
agent in DNA-binding studies, because it can be easily accom-
modatedin between the DNA base pairs,which is expected to
lengthen the DNA double helix. The explicit viscosities of
Form II
Form III
Form I
Lane
I
II
III
IV
V
VI
Fig. 12. Gel electrophoresis method of cleavage showing pUC 19 DNA
treated with metal complexes: Lane 1: DNA control; Lane II: DNA
+
[
L + H
CoLCl
DNA + [ZnLCl
2
O
2
; Lane III: DNA + [CuLCl
+ H ; Lane V: DNA + [NiLCl
+ H
2
]
2
+ H
2
O
2
; Lane IV: DNA +
+ H and Lane VI:
2
]
2
2
O
2
2
]
2
2 2
O
2
]
2
2 2
O
migrated at a speed intermediate to those of Form I and Form II.
Unlike the ligand-induced cleavage of plasmid pUC19 DNA,
the cleavage of the plasmid induced by synthesized complexes
required activators such as hydrogen peroxide [Lanes (III-VI) in
Fig. 12]. Under the same conditions, a combination of ligand
1/3
DNA plotted against [complex]/[DNA] versus η/η
in Fig. 11, where η and η denote in the presence and absence
of the metal complexes. The value of viscosity was determined
from the observed flowing time η = (t − t )/t . Here t and t indi-
o
, are shown
o
and H
O did not cleave the pUC19 DNA. These results showed
2
2
that all the metal complexes efficiently cleaved the DNA from
Form I to Form III in the presence of activators such as hydrogen
peroxide. The activators generate hydroxyl free radicals that are
involved in the oxidation of deoxyribose moiety and hydrolytic
cleavage of sugar-phosphate backbone [62]. The copper complex
cleaved the pUC19 DNA more easily than the other complexes
at the same pH level. The high specificity of copper complex to
the pUC19 DNA may be attributable to its high binding efficiency
with DNA. This observation implies that DNA alters its
conformation due to the binding of dimeric metal(II) complexes.
This result indicates thatthe metal ions play a crucial role in the
cleavage of SC-DNA.
PASS-biological activity assess of ligand: PASS online
software (prediction of activity spectra for biological activity
ligand) was used to find out the biological activity of Schiff
base ligand. From biological data (Table-7), the synthesized
Schiff base ligand shows the biological activity. Ligand acquires
most powerful antineoplastic activity for lungs cancer, breast
o
o
o
cate the concentration of DNA with the metal complexes and
the metal complexes with respect to time. The relative viscosity
of DNA increased during the consecutive addition of comp-
lexes to a fixed amount of DNA solution. These data clearly
showed that the complexes could bind with DNA through the
intercalative binding mode [61].
DNA damage studies: DNA cleavage in the presence of
only L as well as in the presence of the complexes [CuLCl
2
]
2
,
[
CoLCl , [NiLCl and [ZnLCl was studied using gel electro-
2
]
2
2
]
2
2 2
]
phoresisand super coiled (SC) plasmid pUC19 DNA in tris-
acetate EDTA buffer (pH 7.2). Fig. 12 shows that when circular
plasmid DNA was subjected to gel electrophoresis, the intact
super coiled form of DNA (Form I) showed the highest mobility.
If a nick appeared on one strand, the SC-DNA assumed a
relatively slow-moving open circular form (Form II). When
both strands of DNA were cleaved, a linear form (Form III)

Vol. 31, No. 1 (2019)
Studies of New Dimeric Antipyrine-Schiff Base Metal Complexes 211
TABLE-7
BIOLOGICAL ACTIVITY ASSESSMENT OF SCHIFF BASE LIGAND (L) USING PASS ONLINE SOFTWARE
Pa
Pi
Activity
Anti-inflammatory
Analgesic
Pa
Pi
Activity
Antituberculosic
Thromboxane B2 antagonist
Anti-mycobacterial
DNA intercalator
Oxygen scavenger
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
.713
.686
.587
.555
.516
.511
.487
.464
.458
.417
.463
.448
.329
.336
.324
.282
.287
0.035
0.017
0.032
0.028
0.014
0.019
0.016
0.017
0.018
0.023
0.076
0.078
0.093
0.115
0.073
0.077
0.081
0.284
0.337
0.279
0.168
0.320
0.223
0.232
0.253
0.294
0.234
0.247
0.272
0.235
0.261
0.174
0.187
0.129
0.093
0.197
0.097
0.036
0.158
0.087
0.094
0.136
0.154
0.074
0.121
0.144
0.095
0.172
0.092
0.123
0.048
Antiviral (Picornavirus)
Leucopoiesis inhibitor
Antipyretic
Muscular dystrophy treatment
Rheumatoid arthritis treatment
Antineoplastic (colorectal cancer)
Antineoplastic (colon cancer)
Diabetic neuropathy treatment
Antineoplastic
Anthelmintic
Antiparasitic
Antiprotozoal (Coccidial)
Spermidine dehydrogenase inhibitor
Ferredoxin-nitrite reductase inhibitor
Steroid synthesis inhibitor
Anticonvulsant
Cell wall biosynthesis inhibitor
Cancer associated disorders treatment
Antineoplastic (renal cancer)
Eye irritation, inactive
Thioredoxin inhibitor
Cytochrome P450 stimulant
Nicotine dehydrogenase inhibitor
Lysostaphin inhibitor
Antineoplastic (breast cancer)
Cytostatic
Premenstrual syndrome treatment
Pa = Probability of active; Pi = Probability of inactive.
cancer, renal, colorectal, colon cancer and multiple myeloma.
It also possesses potent insulin inhibitor, anti-inflammatory,
DNA intercalator, kidney function stimulant, antibacterial,
antiviral, antiparasitic, anticonvulsant, antituberculosis,
rheumatoid arthritis and diabetic neuropathy treatment.
in silicoADME-TOX property:ADME-TOX (absorption,
distribution, metabolism, excretion and toxicity) properties of
the synthesized Schiff base ligand can be verified by VLS3D
ACKNOWLEDGEMENTS
This work was funded by the Deanship of Scientific Research
at Princess Nourah bint Abdulrahman University, through the
Research Groups Program Grant number (RGP-1438-00).
CONFLICT OF INTEREST
The authors declare that there is no conflict of interests
regarding the publication of this article.
(
Ligand-basedVirtual Screening a Database) software.As per
data derived from the online software ligand was easily
absorbed in human intestinal cells, where (Pa = 1) and taken
into the bloodstream. This implies that it may be useful for
the discovery of novel metal-based therapeutic drugs and
ligand also carry Pa equal to 0.9857 probabilities, so it moves
across the blood-brain barrier (BBB) quickly. This represents
it go across the central nervous system readily. It is a non-
substrate for CYP450 2C9 and CYP450 2D6 enzymes. It is
non-carcinogen and it is having ADME toxic value (Pa) of
REFERENCES
1
2
.
.
M. Nath and P.K. Saini, Dalton Trans., 40, 7077 (2011);
https://doi.org/10.1039/c0dt01426e.
P.G. Cozzi, Chem. Soc. Rev., 33, 410 (2004);
https://doi.org/10.1039/B307853C.
3. N.Ananthi, U. Balakrishnan, S.Velmathi, K.B. Manjunath and G. Umesh,
Opt. Photonics J., 2, 40 (2012);
https://doi.org/10.4236/opj.2012.21006.
4
5
6
.
.
.
R. Antony, S.T. David Manickam, P.V. Chandrasekar, K. Karuppasamy,
P. Kollu and S. Balakumar, RSC Adv., 4, 24820 (2014);
https://doi.org/10.1039/C4RA01960A.
M. Abu-Dief and I.M.A. Mohamed, Beni-Suef Univ. J. Basic Appl. Sci.,
4, 119 (2015);
https://doi.org/10.1016/j.bjbas.2015.05.004.
A. Kundu, P.S. Hariharan, K. Prabakaran and S.P.Anthony, Sens. Actuators
B Chem., 206, 524 (2015);
0
.8765 probabilities.
Conclusion
A new series of novel Schiff base ligand and its metal comp-
lexes of Cu(II), Co(II), Ni(II), and Zn(II) are synthesized and to
study their efficacy of DNA binding, DNA cleavage, antimicro-
bial and cytotoxic activity are also recorded. The synthesized
metal complexes are more efficient DNA binders, and the binding
properties of the metal complexes were examined by UV absor-
ption spectral studies and viscometric measurements. While
all the metal complexes interact with DNA, probably by the
intercalation mechanism. The DNA cleavage study was carried
out by the gel electrophoresis technique, and the result denotes
that copper and cobalt complexes are able to cleave the DNA
https://doi.org/10.1016/j.snb.2014.09.099.
7. H.K. Liu and P.J. Sadler, Acc. Chem. Res., 44, 349 (2011);
https://doi.org/10.1021/ar100140e.
8
.
U. Jungwirth, C.R. Kowol, B.K. Keppler, C.G. Hartinger, W. Berger
and P. Helffter, Antioxid. Redox Signal., 15, 1085 (2011);
https://doi.org/10.1089/ars.2010.3663.
9. S.L.H. Higgins, T.A.White, B.S.J. Winkel and K.J. Brewer, Inorg. Chem.,
0, 463 (2011);
https://doi.org/10.1021/ic100958r.
0. S. Kathiresan, S. Mugesh, J. Annaraj and M. Murugan, New J. Chem.,
5
1
1
1
through double strand helix in the presence of H
2
O activator.
2
4
1, 1267 (2017);
The antimicrobial activity of all the metal complexes shows
superior antibacterial and antifungal activity related to the free
ligand, Thus, this cumulative biological prowess of the metal
complexes from present study would be helpful in sensitive of
DNA interaction evinced by metal complexes and may lead to
establish novel metal-based therapeutic drugs.
https://doi.org/10.1039/C6NJ03501A.
1. N. Raman, M. Selvaganapathy and S. Radhakrishnan, Spectrochim.
Acta A Mol. Biomol. Spectrosc., 127, 185 (2014);
https://doi.org/10.1016/j.saa.2014.02.018.
2. M. Shamsi, S. Yadav and F. Arjmand, J. Photochem. Photobiol. B, 136,
1 (2014);
https://doi.org/10.1016/j.jphotobiol.2014.04.009.

212 Alaghaz et al.
Asian J. Chem.
1
3. P. Gurumoorthy, D. Mahendiran, D. Prabhu, C. Arulvasu and A.K.
Rahiman, RSC Adv., 4, 42855 (2014);
39. A.A. Abou-Hussein and W. Linert, Spectrochim. Acta A Mol. Biomol.
Spectrosc., 117, 763 (2014);
https://doi.org/10.1039/C4RA06941B.
https://doi.org/10.1016/j.saa.2013.06.078.
1
4. G. Dehghan, J.E.N. Dolatabadi,A. Jouyban, K.A. Zeynali, S.M.Ahmadi
and S. Kashaman, DNA Cell Biol., 30, 95 (2011);
40. E.M. Zayed,A.M.M. Hindy and G.G. Mohamed, Appl. Organomet. Chem.,
32, e3952 (2018);
https://doi.org/10.1089/dna.2010.1063.
https://doi.org/10.1002/aoc.3952.
1
1
5. G. Zuber, J.C. Quada and S.M. Hecht, J. Am. Chem. Soc., 120, 9368 (1998);
https://doi.org/10.1021/ja981937r.
6. T. Punniyamurthy, S.J.S. Kalra and J. Iqbal, Tetrahedron Lett., 36, 8497
41. M.M. Omar, G.G. Mohamed and A.A. Ibrahim, Spectrochim. Acta A
Mol. Biomol. Spectrosc., 73, 358 (2009);
https://doi.org/10.1016/j.saa.2009.02.043.
(
1995);
42. G.G. Mohamed, M.M. Omar and A.A. Ibrahim, Eur. J. Med. Chem.,
44, 4801 (2009);
https://doi.org/10.1016/0040-4039(95)01780-L.
7. R.K. Agarwal, L. Singh and D.K. Sharma, Bioinorg. Chem. Appl.,
Article ID 59509 (2006);
https://doi.org/10.1155/BCA/2006/59509.
8. S. Gopalakrishnan and J. Joseph, Mycobiology, 37, 141 (2009);
https://doi.org/10.4489/MYCO.2009.37.2.141.
1
https://doi.org/10.1016/j.ejmech.2009.07.028.
43. N. Raman, K. Pothiraj andT. Baskaran, J. Coord. Chem., 64, 3900 (2011);
https://doi.org/10.1080/00958972.2011.634005.
44. S. Srinivasan, P. Athappan and G. Rajagopal, Transition Met. Chem.,
26, 588 (2001);
1
1
9. C.A. Contreras-Celedón, J.A. Rincón-Medina, D. Mendoza-Rayo and
L. Chacón-García, Appl. Organomet. Chem., 29, 439 (2015);
https://doi.org/10.1002/aoc.3312.
0. V. Arun, N. Sridevi, P.P. Robinson, S. Manju and K.K.M. Yusuff, J.
Mol. Catal. A, 304, 191 (2009);
https://doi.org/10.1023/A:1011007429295.
45. C.X. Zhang and S.J. Lippard, Curr. Opin. Chem. Biol., 7, 481 (2003);
https://doi.org/10.1016/S1367-5931(03)00081-4.
46. R.F. Brissos, E. Torrents, F.M. dos Santos Mello, W. Carvalho-Pires,
E. de Paula Silveira-Lacerda, A.B. Caballero, A. Caubet, C. Massera,
O. Roubeau, S.J. Teat and P. Gamez, Metallomics, 6, 1853 (2014);
https://doi.org/10.1039/C4MT00152D.
2
2
https://doi.org/10.1016/j.molcata.2009.02.011.
1. C.A. Contreras-Celedón, D. Mendoza-Rayo, J.A. Rincón-Medina and
L. Chacón-García, Beilstein J. Org. Chem., 10, 2821 (2014);
https://doi.org/10.3762/bjoc.10.299.
47. D. Kivelson and R. Neiman, J. Chem. Phys., 35, 149 (1961);
https://doi.org/10.1063/1.1731880.
2
2
2. K. Satyanarayana and M.N.A. Rao, Eur. J. Med. Chem., 30, 641 (1995);
https://doi.org/10.1016/0223-5234(96)88280-8.
3. I.V. Shemarova, E.B. Maizel, I.V. Voznyi, N.P. Stepanova and A.E.
Khovanskikh, Pharm. Chem. J., 34, 530 (2000);
https://doi.org/10.1023/A:1010355129735.
4. N. Raman, S.J. Raja andA. Sakthivel, J. Coord. Chem., 62, 691 (2009);
https://doi.org/10.1080/00958970802326179.
5. R.Antony, S.T.D. Manickam, K. Karuppasamy, P.V. Chandrasekar, P. Kollu
and S. Balakumar, RSC Adv., 4, 42816 (2014);
https://doi.org/10.1039/C4RA08303B.
6. D. Kivelson and R. Neiman, J. Chem. Phys., 35, 149 (1961);
https://doi.org/10.1063/1.1731880.
48. P. Priya, S.V. Arunachalam, N. Sathya, C. Jayabalakrishnan and V.
Chinnusamy, Transition Met. Chem., 34, 437 (2009);
https://doi.org/10.1007/s11243-009-9214-z.
49. S. Sarkar, A. Mondal, M.S. El Fallah, J. Ribas, D. Chopra, H. Stoeckli-
Evans and K.K. Rajak, Polyhedron, 25, 25 (2006);
https://doi.org/10.1016/j.poly.2005.06.059.
50. M.S. El-Shahawi and A.F. Shoair, Spectrochim. Acta A Mol. Biomol.
Spectrosc., 60, 121 (2004);
https://doi.org/10.1016/S1386-1425(03)00191-4.
51. A.W. Coats and J.P. Redfern, Nature, 201, 68 (1964);
https://doi.org/10.1038/201068a0.
2
2
2
2
2
52. H.H. Horowitz and G. Metzger, Anal. Chem., 35, 1464 (1963);
https://doi.org/10.1021/ac60203a013.
53. G.O. Piloyan, I.D. Ryabchikov and O.S. Novikova, Nature, 212, 1229
(1966);
7. B.J. Hathaway and D.E. Billing, Coord. Chem. Rev., 5, 143 (1970);
https://doi.org/10.1016/S0010-8545(00)80135-6.
8. Y. Zhou, H. Zhou, J. Zhang, L. Zhang and J. Niu, Mol. Biomol. Spect.,
9
8, 14 (2012);
https://doi.org/10.1038/2121229a0.
https://doi.org/10.1016/j.saa.2012.08.025.
9. J.B. Chaires, N. Dattagupta and D.M. Crothers, Biochemistry, 21, 3933
54. H. Chen, J.A. Parkinson, R.E. Morris and P.J. Sadler, J. Am. Chem. Soc.,
125, 173 (2003);
2
(
1982);
https://doi.org/10.1021/ja027719m.
https://doi.org/10.1021/bi00260a005.
0. Y. Li, G. Zhang and M. Tao, J. Photochem. Photobiol. B, 138, 109 (2014);
https://doi.org/10.1016/j.jphotobiol.2014.05.011.
55. V.G. Vaidyanathan and B.U. Nair, Eur. J. Inorg. Chem., 9, 1840 (2004);
https://doi.org/10.1002/ejic.200300718.
56. F.A. Beckford, M. Shaloski Jr., G. Leblanc, J. Thessing, L.C. Lewis-
Alleyne, A.A. Holder, L. Li and N.P. Seeram, Dalton Trans., 10757
(2009);
3
3
1. A.K. Sadana,Y. Mirza, K.R. Aneja and O. Prakash, Eur. J. Med. Chem.,
3
8, 533 (2003);
https://doi.org/10.1016/S0223-5234(03)00061-8.
https://doi.org/10.1039/b915081a.
3
3
3
3
3
2. A. van Tonder, A.M. Joubert and A.D. Cromarty, BMC Res. Notes, 8, 47
57. V.L. Kinnula and J.D. Crapo, Free Radic. Biol. Med., 36, 718 (2004);
https://doi.org/10.1016/j.freeradbiomed.2003.12.010.
58. H.F. Abd El Halim and G.G. Mohamed, Appl. Organomet. Chem., 32,
e4176 (2017);
(
2015);
https://doi.org/10.1186/s13104-015-1000-8.
3. E.E. Oruç, S. Rollas, F. Kandemirli, N. Shvets and A.S. Dimoglo, J.
Med. Chem., 47, 6760 (2004);
https://doi.org/10.1021/jm0495632.
4. T. Sander, J. Freyss, M. von Korff, J.R. Reich and C. Rufener, J. Chem.
Inf. Model., 49, 232 (2009);
https://doi.org/10.1002/aoc.4176.
59. S.K. Mal, M. Mitra, G. Kaur, V.M. Manikandamathavan, M.S. Kiran,
A.R. Choudhury, B.U. Nair and R. Ghosh, RSC Adv., 4, 61337 (2014);
https://doi.org/10.1039/C4RA09448D.
60. W.D. Wilson, L. Ratmeyer, M. Zhao, L. Strekowski and D. Boykin,
Biochemistry, 32, 4098 (1993);
https://doi.org/10.1021/ci800305f.
5. M.D. Segall,A.P. Beresford, J.M.R. Gola, D. Hawksley and M.H. Tarbit,
Expert Opin. Drug Metab. Toxicol., 2, 325 (2006);
https://doi.org/10.1517/17425255.2.2.325.
https://doi.org/10.1021/bi00066a035.
61. V.A. Kawade, A.A. Kumbhar, A.S. Kumbhar, C. Näther, A. Erxleben,
U.B. Sonawane and R.R. Joshi, Dalton Trans., 40, 639 (2011);
https://doi.org/10.1039/C0DT01078B.
6. B. Mathew, J. Suresh and S. Anbazhagan, Biomed. Aging Pathol., 4,
3
27 (2014);
https://doi.org/10.1016/j.biomag.2014.08.002.
7. F. Liu and B. Fang, Chin. J. Biotechnol., 23, 133 (2007);
https://doi.org/10.1016/S1872-2075(07)60012-0.
8. J.R. Durig, J.J. Klaassen, B.S. Deodhar, T.K. Gounev, A.R. Conrad
and M.J. Tubergen, Spectrochim. Acta A, 87, 214 (2012);
https://doi.org/10.1016/j.saa.2011.11.041.
62. J. Liu, W. Zheng, S. Shi, C. Tan, J. Chen, K. Zheng and L. Ji, J. Inorg.
Biochem., 102, 193 (2008);
3
3
https://doi.org/10.1016/j.jinorgbio.2007.07.035.