Synthesis, characterization and in vitro anticancer, DNA binding and cleavage studies of Mn (II), Co (II), Ni (II) and Cu (II) complexes of Schiff base ligand 3-(2-(1-(1H-benzimidazol-2-yl)ethylidene)hydrazinyl)quinoxalin-2(1H)-one and crystal structure of the ligand

Article abstract of DOI:10.1002/aoc.4526

A Schiff base ligand, 3-(2-(1-(1H-benzimidazol-2-yl)ethylidene)hydrazinyl)quinoxalin-2(1H)-one (BZHQO), has been synthesized by the condensation of 3-hydrazinylquinoxalin-2(1H)-one and 1-(1H-benzoimidazol-2-yl) ethanone and characterized using spectral and single-crystal X-ray analyses. Mn (II), Co (II), Ni (II) and Cu (II) complexes of the BZHQO ligand have been synthesized and characterized. The interactions of the ligand and its metal complexes with calf thymus DNA have been investigated using absorption spectroscopy and the intrinsic binding constant has been evaluated. Agarose gel electrophoresis has been performed to study the abilities of the ligand and its metal complexes to cleave supercoiled pBR322 DNA into nicked circular form. In vitro anticancer activities of the Cu (II) and Ni (II) complexes have been investigated by MTT assay, using the cell lines HeLa, B16-F10, SKOV3 and MCF7. The Cu (II) complex has been found to be active against HeLa, B16-F10 and MCF7, while the Ni (II) complex has been found to be active against MCF-7.

Full text of DOI:10.1002/aoc.4526

Received: 11 April 2018
DOI: 10.1002/aoc.4526
Revised: 29 June 2018
Accepted: 3 July 2018
F U L L P A P E R
Synthesis, characterization and in vitro anticancer, DNA
binding and cleavage studies of Mn (II), Co (II), Ni (II) and
Cu (II) complexes of Schiff base ligand 3‐(2‐(1‐(1H‐
benzimidazol‐2‐yl)ethylidene)hydrazinyl)quinoxalin‐2(1H)‐
one and crystal structure of the ligand
Panaganti Sukanya | Chittireddy Venkata Ramana Reddy
Department of Chemistry, Jawaharlal
A Schiff base ligand, 3‐(2‐(1‐(1H‐benzimidazol‐2‐yl)ethylidene)hydrazinyl)
Nehru Technological University
Hyderabad, Hyderabad, India
quinoxalin‐2(1H)‐one (BZHQO), has been synthesized by the condensation of
3
‐hydrazinylquinoxalin‐2(1H)‐one and 1‐(1H‐benzoimidazol‐2‐yl) ethanone
Correspondence
Chittireddy Venkata Ramana Reddy,
Department of Chemistry, Jawaharlal
Nehru Technological University
Hyderabad, Hyderabad 500085, India.
Email: vrr9@yahoo.com
and characterized using spectral and single‐crystal X‐ray analyses. Mn (II),
Co (II), Ni (II) and Cu (II) complexes of the BZHQO ligand have been synthe-
sized and characterized. The interactions of the ligand and its metal complexes
with calf thymus DNA have been investigated using absorption spectroscopy
and the intrinsic binding constant has been evaluated. Agarose gel electropho-
resis has been performed to study the abilities of the ligand and its metal com-
plexes to cleave supercoiled pBR322 DNA into nicked circular form. In vitro
anticancer activities of the Cu (II) and Ni (II) complexes have been investigated
by MTT assay, using the cell lines HeLa, B16‐F10, SKOV3 and MCF7. The Cu
(II) complex has been found to be active against HeLa, B16‐F10 and MCF7,
while the Ni (II) complex has been found to be active against MCF‐7.
KEYWORDS
anticancer activity, complex, crystal structure, DNA binding, DNA cleavage, Schiff base
1
| INTRODUCTION
Several derivatives of quinoxaline were reported to
exhibit a wide range of pharmacological activities such
[
10]
[11]
Transition metal complexes of multidentate heterocyclic
ligands containing nitrogen donor sites have gained
prominence due to their versatile structural features, var-
ied ligational behaviour, beneficial biological activities
and potential applications in the fields of medicine, mate-
rials research and catalysis.
larly those containing sp hybrid nitrogens as part of the
aromatic system of the ligands, find significant applica-
as antimicrobial,
anti‐inflammatory,
and antidepressant
antidia-
activities,
[
12]
[1,13]
[14]
betic,
antitumour
in addition to their use in catalysis and organic synthe-
[
15,16]
sis.
Schiff base ligands derived from quinoxaline
are extensively employed in the synthesis of metal com-
[1,2]
Metal complexes, particu-
plexes with a variety of geometries and varied analytical,
2
[17–21]
industrial and pharmaceutical applications.
The
design of Schiff bases of 3‐hydrazinylquinoxalin‐2(1H)‐
one has been of interest due to the predictable coordina-
tion properties of the ligands and their prominent phar-
[3–5]
tions as drugs.
erocyclic moieties act as efficient ligands and form
chelating complexes with transition metal ions.
Schiff bases containing nitrogen het-
[
6–9]
[22–26]
macological activities.
Appl Organometal Chem. 2018;e4526.
wileyonlinelibrary.com/journal/aoc
© 2018 John Wiley & Sons, Ltd.
1 of 11
https://doi.org/10.1002/aoc.4526

2
of 11
SUKANYA AND VENKATA RAMANA REDDY
Benzimidazole is also an important constituent in sev-
as calibrant. Diamagnetic corrections were computed
using Pascal's constants. DNA cleavage experiments were
performed using a Biotech electrophoresis system and the
bands were visualized and photographed with a Biotech
Transilluminator.
eral natural and synthetic compounds. It is an integral
part of the structure of vitamin B12 and its broad spec-
[
27]
trum of biological activities such as antioxidant,
anti-
and
[28]
[29]
[30,31]
histamine,
anti‐allergic,
DNA binding
[
32]
anticancer
activities make it an important scaffold in
the development of several therapeutic agents.
2
.2 | DNA Binding Studies
Considering all these observations, we report here
the synthesis and characterization of Mn (II), Co (II),
Ni (II) and Cu (II) complexes of the tridentate
Schiff base 3‐(2‐(1‐(1H‐benzimidazol‐2‐yl)ethylidene)
The interactions of the metal complexes with CT‐DNA
were investigated using electronic absorption spectro-
scopic titrations. CT‐DNA was dissolved in KH PO and
2
4
hydrazinyl)quinoxalin‐2(1H)‐one
(BZHQO)
derived
K HPO4 buffer and its concentration was determined
2
[
33]
from 3‐hydrazinylquinoxalin‐2(1H)‐one and 1‐(1H‐
benzoimidazol‐2‐yl)ethanone. We also report the in vitro
anticancer activities and DNA binding and cleavage abil-
ities of the ligand and its complexes.
from the absorption intensity
at 260 nm and εvalue
−
1
−1
of 6600 M cm . Stock solutions were kept at 4 °C
and used within four days. Dilute solutions of the com-
pounds in DMSO were used. The titrations were carried
out by keeping the metal complex concentration constant
and varying the CT‐DNA concentration. The solutions
after each addition of CT‐DNA were incubated for about
2
2
| EXPERIMENTAL
10 min and the electronic absorption spectra were
.1 | Materials and Methods
recorded. Each sample solution was scanned from 200
to 800 nm. The mode of binding was studied by monitor-
ing the spectral changes, and the quantitative binding
strength of the complexes was evaluated in terms of the
All the chemicals used were of reagent grade, and used
without any further purification. Calf thymus DNA (CT‐
DNA) was procured from Sigma‐Aldrich and used as
received. Plasmid pBR322 DNA was obtained from SRL.
Cell lines HeLa, B16‐F10, SKOV3, MCF7 and CHO‐KI
for in vitro anticancer activity studies were procured from
the American Type Culture Collection (ATCC, Bethesda,
MD, USA).
intrinsic binding constant (K ) using the following
b
[
34]
equation:
½
DNAꢀ ½DNAꢀ
1
¼
þ
εa−εf
εb−εf
Kbðεb−εf Þ
Infrared (IR) spectra were recorded using a Shimadzu
FTIR‐5300 spectrometer with KBr pellets from 250 to
−
1
4
000 cm . Electronic absorption spectra were recorded
where [DNA] is the concentration of DNA. The apparent
in dimethylsulfoxide (DMSO) from 200 to 1200 nm with
a Shimadzu UV‐3600 spectrophotometer. H NMR spec-
extinction coefficient, ε , is calculated as Aobserved/[com-
a
1
plex]. Parameters ε and ε are the extinction coefficients
b
f
tra were recorded with a Bruker 400 MHz spectrometer
using DMSO‐d6 solvent. Electron paramagnetic reso-
nance (EPR) spectra at room temperature were obtained
using a Bruker Xenon EMX‐ER073 spectrometer. Powder
X‐ray diffractograms were recorded with a Bruker
SMART D8 Advance X‐ray diffractometer using Cu K
αradiation (λ= 1.5406 Å) at 40 kV and 30 mA. Mass spec-
tra were recorded using a Shimadzu mass spectrometer.
Elemental analysis was carried out using a FLASH Ea
of fully bound and free complex, respectively. K is found
b
as the ratio of slope to intercept of a plot of [DNA]/(ε − ε )
a
f
versus [DNA].
2.3 | DNA Cleavage Studies
The ability of the ligand and its metal complexes to cleave
supercoiled pBR322 DNA was studied using agarose gel
[
35]
electrophoresis.
The pBR322 DNA dissolved in 5 mM
1
112 series CHNS analyser. Thermogravimetric analysis
Tris–HCl/50 mM NaCl buffer was treated with the ligand
and its metal complexes. The mixture was incubated for
2 h at 37 °C. A loading buffer containing 1%
bromophenol blue and 40% sucrose (1 μl) was added to
the incubated solution and loaded onto a 0.8% (w/v) aga-
(TGA) and differential thermal analysis (DTA) were car-
ried out using a TG/DTA EXTAR 6300 in the temperature
range 25–800 °C under nitrogen atmosphere with a
heating rate of 10 °C min . Electrical conductance
values were measured using 10 M solutions of the com-
plexes in DMSO with an Elico conductivity bridge and
dip‐type cell calibrated with KCl solution. Magnetic sus-
ceptibilities were measured with a Faraday balance
model 7550 at room temperature using Hg [Co (NCS)4]
−
1
−
3
−1
rose gel containing ethidium bromide (1 μg ml ). Con-
trol solution using DNA alone was also loaded. The gel
was run in Tris acetate EDTA buffer (40 mM Tris base,
20 mM acetic acid, 1 mM EDTA), at a constant voltage
of 60 V for 2 h until the bromophenol blue travelled

SUKANYA AND VENKATA RAMANA REDDY
3 of 11
through 70% of the gel. The bands were photographed
using a UV Transilluminator.
2.5.2 | Synthesis of 3‐
hydrazinylquinoxalin‐2‐(1H)‐one
A
mixture
of
quinoxaline‐2,3‐(1H,4H)‐dione
(
(
16.2 g,100 mmol), hydrazine hydrate (50 ml) and water
2
.4 | Cytotoxicity
[
38]
50 ml) was refluxed (Scheme 1).
The reaction was
Cytotoxic activity was evaluated for the Cu (II) and Ni (II)
complexes against four different cancer cell lines by
microculture tetrazolium assay using 3‐(4,5‐dimethyl-
thiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT).
The cell lines, human cervix adenocarcinoma (HeLa),
mouse skin melanoma (B16‐F10; ATCC CRL‐6475™),
human ovarian cancer (SKOV3; ATCC HTB‐77™),
human breast adenocarcinoma (MCF7; ATCC HTB‐
monitored by TLC. The reaction mixture was cooled after
h. The yellow solid obtained was collected by filtration,
2
washed with water and recrystallized from n‐butanol.
Yield: 86%; m.p. >350°C.
2.5.3 | Synthesis of 1‐(1H‐benzo [d]
imidazol‐2‐yl)ethanol
2
2™) and normal mammalian cell line Chinese hamster
o‐Phenylenediamine (10.8 g, 100 mmol) was dissolved in
2
ovary cells (CHO‐K1; ATCC CCL‐61™), were procured
from ATCC and maintained in Dulbecco's modified
Eagle's medium supplemented with 10% foetal bovine
5 ml of 4 N HCl. To this solution, 50 ml of water and lac-
tic acid (10.4 ml, 150 mmol) were added and the mixture
was refluxed for about 3 h (Scheme 2). The reaction was
monitored by TLC using 9:1 benzene and methanol. After
completion of the reaction, the reaction mass was
neutralized with ammonia. The solid obtained was fil-
tered, washed repeatedly with water and dried. The
−
1
serum, 100 U ml penicillin, 2 mM L‐glutamine and
−
1
1
00 μg ml streptomycin at 37 °C in a 5% CO incubator.
2
The cells were seeded in a 96‐well culture plate and
allowed to attach properly. Various concentrations of
solutions of the metal complexes in DMSO, ranging from
[39]
colourless product was recrystallized from hot water.
M.p. 179 °C.
1
2
to 50 μM, were added in triplicate and incubated for
4 h. The cells were then incubated with MTT
−
1
(0.5 mg ml ) for 3 h. DMSO (100 μl) was added to each
well to dissolve the insoluble formazan crystals. Finally
the absorbance of the plates was measured using a Syn-
ergy H1 multi‐mode plate reader (USA). Mitomycin‐C
for normal cells and doxorubicin for cancer cells were
2
.5.4 | Synthesis of 1‐(1H‐benzo [d]
imidazol‐2‐yl)ethanone
1‐(1H‐Benzo [d]imidazol‐2‐yl) ethanol (16.2 g, 100 mmol)
[36]
was dissolved in 5% sulfuric acid. A solution of potassium
dichromate in sulfuric acid was added slowly in an ice
bath with constant stirring. The reaction was monitored
by TLC using 9:1 benzene and methanol. After comple-
tion of the reaction, the reaction mass was neutralized
used as positive controls for comparison.
2
2
.5 | Synthesis of Ligand
.5.1 | Synthesis of quinoxaline‐2,3‐
and the solid obtained was filtered, washed repeatedly
with water and dried
(1H,4H)‐dione
[40]
(Scheme 2).
o‐Phenylenediamine (10.8 g,100 mmol) and oxalic acid
12.6 g,100 mmol) were dissolved in 50 ml of 4 N HCl and
heated in a water bath for 20 min (Scheme 1). A crystal-
line solid was formed. The reaction mixture was cooled and
the product was filtered, repeatedly washed with water to
remove traces of HCl and dried. M.p. >350°C.
(
2
.5.5 | Synthesis of BZHQO
[37]
3‐Hydrazinylquinoxalin‐2‐(1H)‐one (3.52 g, 20 mmol)
was dissolved in hot methanol, and 1‐(1H‐
benzoimidazol‐2‐yl) ethanone (3.2 g, 20 mmol) and a
NH2NH2.H2O
H
N
H
N
HO
HO
O
O
H O
O
NH2
+
NH2
O
O
4N HCl
Heat
2
C
C
NH2
SCHEME 1 Synthesis of 3‐
hydrazinylquinoxalin‐2‐(1H)‐one
N
H
Heat
N
N
H
4
N HCl
NH2
NH2
O OH
OH
K Cr O
N
2
2
7
N
O
C
CH3
+
HO
C
C
H
H
C
H
SCHEME 2 Synthesis of 1‐(1H‐benzo
Reflux (3 h)
N
H
CH3 dil.H SO /Ice
N
H
2
4
[
d]imidazol‐2‐yl)ethanone

4
of 11
SUKANYA AND VENKATA RAMANA REDDY
1
catalytic amount of acetic acid were added to it and
refluxed for 5 h. The reaction was monitored by TLC
using 9:1 chloroform and methanol. The reaction mixture
was cooled after 5 h. The yellow solid obtained was col-
lected by filtration and washed with methanol and n‐hex-
ane (Scheme 3). The product was recrystallized from
dimethylformamide (DMF). Single crystals suitable for
X‐ray diffraction were collected by slow diffusion of water
into a saturated solution of the compound in DMF. Yield:
quinoxaline ring, 1010 ν(N─ N) (Figure 2). The
H
NMR spectrum of BZHQO (Figure 3) shows the ─ NH
protons of quinoxaline ring and benzimidazole ring reso-
[
41]
nate at 12.8 and 10.61 ppm, respectively.
The ─ NH
proton of hydrazone fragment is observed at 11.8 ppm.
The aromatic protons (8H) and methyl protons (3H) have
been identified as a multiplet in the region 7.06–7.7 ppm.
The UV–visible spectrum of BZHQO displays peaks at
272, 311 and 326 nm corresponding to π →π* transitions.
The peaks at 384 and 402 nm are due to n →π* transi-
tions associated with the carbonyl and azomethine
groups.
7
9%; m.p. 292 °C.
2
.6 | Synthesis of Complexes
The structure of BZHQO confirmed from spectral and
analytical data is shown in Figure 4.
To a hot methanolic solution of metal chloride in a two‐
necked round‐bottom flask, a hot methanolic suspension
of the ligand was added in small increments. A clear solu-
tion was obtained after each addition. The pH of each
reaction mixture was adjusted to about 8 using alcoholic
ammonia. The reaction mixture was then refluxed in a
water bath for about 4 h. The coloured solids obtained
were filtered in hot condition, washed successively with
methanol and n‐hexane and dried.
3
.1.2 | Crystal structure of BZHQO ⋅H O
2
Single‐crystal X‐ray crystallographic data corroborate the
structure of BZHQO. Analysis of the crystal structure
indicates the presence of one solvent (water) molecule.
3
3
| RESULTS AND DISCUSSION
.1 | Characterization of Schiff Base
Ligand
3.1.1 | Spectral data
Elemental analysis indicates the formation of the Schiff
base corresponding to the formula C H N O. The m/z
17
14 6
+
peak at 319 [M + 1] in the LC–MS spectrum, as shown
in Figure 1, is consistent with the molecular formula
C H N O.
17
14 6
−
1
IR spectral data (KBr, cm ): 3344ν(N─ H), 1680
ν(C═ O), 1617 ν(N═ C) of free azomethine group, 1579
ν(C═ N) of benzimidazole ring, 1478 ν(C═ N) of
FIGURE 2 IR spectrum of BZHQO
SCHEME 3 Synthesis of BZHQO
FIGURE 1 Mass spectrum of BZHQO

SUKANYA AND VENKATA RAMANA REDDY
5 of 11
1
FIGURE 3 H NMR spectrum of BZHQO
CCDC 1829210 contains the supplementary crystallo-
graphic data for this paper. These data can be obtained
free of charge via http://www.ccdc.cam.ac.uk/conts/
retrieving.html, or from the Cambridge Crystallographic
Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK;
fax: (+44) 1223 336 033; e‐mail: deposit@ccdc.cam.ac.uk.
H
N
O
N
N
CH3
NH
N
N
H
C
FIGURE 4 Structure of BZHQO
3.2 | Characterization of Metal Complexes
An ORTEP view of BZHQO ⋅H O is shown in Figure 5.
of BZHQO
2
The compound crystallizes in a monoclinic system with
3.2.1 | Elemental analysis
the space group P2 /c, with four molecules of BZHQO
1
and four molecules of H O. The crystal structure is stabi-
lized by hydrogen bonding network between water and
Elemental analysis data reveal the metal to ligand ratio to
be 1:2 in all the complexes. The results of analysis of the
complexes and their formulae are presented in Table 4.
2
BZHQO molecules involving N nitrogen of hydrazone
4
moiety, ring nitrogens of quinoxaline and benzimidazole
rings and hydrogens of the solvent. A perspective view
of the two‐dimensional network through solvent mole-
3
.2.2 | Molar conductance
cules in BZHQO ⋅H O along the b‐axis is shown in
The molar conductance values of the Mn (II), Co (II), Ni
(II) and Cu (II) complexes, measured in 10 M solutions
of the complexes in DMSO, are 124, 122, 136 and 127
2
−
3
Figure 6. Crystallographic data and structure refinement
parameters are presented in Table 1. Selected bond
lengths and angles are presented in Table 2. Details of
hydrogen bonding are presented in Table 3.
−
1
2
−1
Ω
cm mol , respectively, indicating that all the com-
plexes are 1:2 electrolytes suggesting the presence of two
ionizable chloride ions.
presence of chloride ions in the ionization sphere.
[
42]
Volhard test confirmed the
[43]
3.2.3 | IR spectra
The characteristic azomethine peak of medium intensity
−1
observed at 1617 cm in the spectrum of the ligand experi-
−1
ences a downward shift by 22–26 cm and appears as a
shoulder in the spectra of all the complexes indicating the
participation of nitrogen of free ─ N═ CH─ group in the
−1
coordination with the metal ions. The peak at 1579 cm in
the spectrum of the ligand assigned to benzimidazole ring
C═ N also shifted to lower wavenumbers in the spectra of
FIGURE 5 ORTEP view of BZHQO. Thermal ellipsoids are
represented at the 30% probability level

6
of 11
SUKANYA AND VENKATA RAMANA REDDY
FIGURE 6 Thw‐dimensional network
diagram of BZHQO along the b‐axis
all the complexes, confirming the participation of tertiary
weight loss (9.67%) up to 130 °C corresponds to the loss
[34]
[46]
nitrogen of the benzimidazole ring in complexation. The
of two chloride ions
from the ionization sphere (calcd
−1
medium intensity peak at 1478 cm in the spectrum of the
ligand attributed to ν(C═ N) of the quinoxaline ring experi-
enced a negative shift in the spectra of the complexes suggest-
ing the involvement of quinoxaline ring nitrogen in
9.37%). Further heating results in gradual loss of ligand.
TGA–DTA data of the Co (II) and Cu (II) complexes also
exclude the presence of lattice as well as coordinated
water. The complexes decompose gradually on increasing
the temperature. An endotherm at 85 °C in DTA data of
the Ni (II) complex indicates the presence of lattice water,
which is in accordance with the broad peak correspond-
ing to the presence of water in the IR spectrum of the
complex. TGA indicates the gradual decomposition of
the complex upon increasing the temperature. The first
stage decomposition between 33 and 128 °C (11.4%) cor-
responds to the loss of 1 mol of lattice water and 2 mol
of lattice chloride ions (calcd 11.34%) per mole of the
complex. The second stage weight loss observed between
−1
coordination. A broad peak at 3392 cm in the spectrum of
[
44]
the Ni (II) complex suggests the presence of lattice water
−1
in the complex. The band observed at 935 cm for the com-
plex is attributed to bending vibration of water. The new
[45]
bands of medium intensity in the low‐frequency region
in the spectra of the complexes reveal the presence of M─
N bonds. The important IR spectral frequencies along with
their assignments are summarized in Table 5. (IR spectra of
the complexes are reported in supporting information S1–S4.)
128 and 470 °C (40.954%) may be attributed to the loss of
3
.2.4 | Mass spectra
one ligand moiety (calcd 40.58%). Further loss in weight
is equivalent to the decomposition of the second ligand.
The weight of residue (9.89%) corresponds to metal oxide
indicating the composition of the complex to be 1:2
Mass spectra of the complexes were recorded and com-
pared with that of the ligand to evaluate the stoichiome-
tries of the complexes. The molecular ion peaks
observed at 763.5476, 766.6921 and 785.5864 in high‐reso-
lution mass spectra of the Mn (II), Co (II) and Ni (II)
complexes correspond to the respective molecular formu-
lae assigned. The LC–MS spectrum of the Cu (II) complex
shows a molecular ion peak at 772. The CHN analysis
results and mass spectral data (Table 4) are in agreement
with the values calculated from the molecular formulae
assigned to the complex. (Mass spectra of the complexes
are reported in supporting information S5–S8.)
(
metal to ligand). (Thermograms of the complexes are
shown in supporting information S9–S12.)
3.2.6 | Magnetic data
The μeff value of the Mn (II) complex, 6.08 BM, is within the
5
range of the magnetic moment for d electronic configura-
tion. The Co (II) complex has a magnetic moment of 4.72
BM, which is in agreement with the values reported for
[
47]
the presence of three unpaired electrons. For the Ni (II)
complex, the μvalue obtained, 3.12 BM, is consistent with
3.2.5 | TGA and DTA
8
[48]
the d electronic configuration.
The observed magnetic
TGA–DTA data of the Mn (II) complex suggest the
absence of lattice and coordinated water. The first stage
moment value of the Cu (II) complex, 1.86 BM, is due to
the presence of one unpaired electron in the metal ion.
[49]

SUKANYA AND VENKATA RAMANA REDDY
7 of 11
[
50]
TABLE 1 Crystal data and structure refinement for BZHQO
ligand‐to‐metal charge transfer.
The band at 499 nm
in the spectrum of the Ni (II) complex is in accordance
Identification code
shelx
3
3
with the A2g (F) → T (P) transition for six‐coordinated
1g
Empirical formula
Formula weight
Temperature
C
17
H
16
N
6
O
2
[50]
octahedral Ni (II) complex.
The electronic spectrum of
336.36
298(2) K
1.54184 Å
the Cu (II) complex shows a band at 491 nm correspond-
2
2
ing to B1g → E transition, indicating the complex to
1g
[
19]
have distorted octahedral geometry.
(Electronic
Wavelength
absorption spectra of the ligand and its complexes are
shown in supporting information S13.)
Crystal system
Space group
Monoclinic
P21/c
Unit cell dimensions
a = 5.2594(2) Å, α= 90°
b = 13.2099(5) Å, β = 92.265(3)°
c = 23.1884(8) Å, γ = 90°
3.2.8 | EPR spectra
The EPR spectrum of the Mn (II) complex recorded at
room temperature is broad with no hyperfine splitting.
The g value calculated is 2.1. The broadening of the spec-
tral line indicates large spin–lattice interactions. The EPR
spectrum of the Cu (II) complex is isotropic and the spec-
tral parameters g (2.29) >g (2.14) >2.0023 indicate
3
Volume
1609.78(10) Å
Z
4
−
3
Density (calculated)
Absorption coefficient
F (000)
1.388 mg m
−
1
0.791 mm
704
|
|
⊥
[
50]
tetragonal geometry for the complex.
The axial sym-
3
Crystal size
0.30 × 0.28 × 0.20 mm
metry parameter G (2.04) indicates that considerable
exchange interactions in the solid complex are possible.
The μvalue (1.86 BM) is also in agreement with the para-
magnetic nature of the Cu (II) complex. (EPR spectra of
the Mn (II) and Cu (II) complexes are shown in
supporting information S14 and S15.)
θrange for data collection 3.852° to 71.682°
Index ranges
−6 ≤h ≤5, −14 ≤k ≤15, −28 ≤l ≤ 24
Reflections collected
Independent reflections
Completeness to θ=
5921
3088 [R (int) = 0.0158]
99.8%
Based on the spectral and analytical data, the possible
structures for the complexes are shown in Figure 7.
6
7.684°
Absorption correction
Semi‐empirical from equivalents
Max. and min.
Transmission
1.00000 and 0.35855
3.3 | Biological Activity of Metal
Complexes
2
Refinement method
Full‐matrix least‐squares on F
Data/restraints/parameters 3088/4/240
3
.3.1 | DNA binding investigated using
2
Goodness‐of‐fit on F
Final R indices [I > 2σ(I)]
R indices (all data)
1.078
electronic absorption titrations
R
1
1
= 0.0420, wR
2
2
= 0.1228
= 0.1284
The DNA binding properties of the metal complexes were
quantified by employing electronic absorption spectros-
copy. The ligand and its metal complexes were screened
against CT‐DNA and the spectral changes were moni-
tored, which reflect the changes in DNA structure during
interaction with the metal complexes. In general, for
complexes with aromatic moieties that bind to DNA
through intercalation, hypochromism results due to the
stacking interactions between the base pairs of DNA
and aromatic chromophores of the complexes, while
damage of the double helix of DNA results in
R
= 0.0473, wR
Extinction coefficient
0.0119(8)
−
3
Largest diff. Peak and hole 0.200 and − 0.223 e Å
3.2.7 | Electronic spectra
In the spectra of the metal complexes, the bathochromic
shift of the peak due to n →π* transitions associated with
the azomethine group of the ligand indicates the partici-
pation of azomethine nitrogen in coordination. The elec-
tronic absorption spectrum of the Mn (II) complex
exhibits a peak at 409 nm with a shoulder at 468 nm cor-
[
21,51]
hyperchromism.
The binding curves are illustrated in Figures 8–12.
Initial spectrum corresponds to the complex in the
absence of CT‐DNA. BZHQO and its Mn (II), Co (II), Ni
(II) and Cu (II) complexes show a considerable decrease
in intensity of the bands at 384, 395, 327, 491 and
312 nm, respectively, with the corresponding intrinsic
6
4
responding to A1 → A transitions, suggesting an octa-
g
1g
hedral geometry. The Co (II) complex shows absorption
bands at 495 and 531 nm assignable to T1 (F) → T
4
4
g
1g
(
P) transitions, characteristic of octahedral Co (II) com-
[19]
7
5
5
plex.
A shoulder observed at 466 nm is assigned to
binding constants of 1.44 × 10 , 1.21 × 10 , 6.58 × 10 ,

8
of 11
SUKANYA AND VENKATA RAMANA REDDY
2
TABLE 2 Bond lengths and angles for BZHQO ⋅H O
Bond
Bond length (Å)
Angle
Bond angle (°)
Angle
Bond angle (°)
O(1)–C(1)
N(1)–C(1)
N(1)–C(7)
N(2)–C(2)
N(2)–C(8)
N(3)–N(4)
N(3)–C(2)
N(4)–C(9)
N(5)–C(11)
N(5)–C(12)
N(6)–C(11)
N(6)–C(13)
C(1)–C(2)
C(8)–C(3)
C(8)–C(7)
C(7)–C(6)
C(6)–C(5)
C(5)–C(4)
C(4)–C(3)
C(9)–C(11)
C(9)–C(10)
C(12)–C(17)
1.2385(19)
1.342(2)
1.386(2)
1.291(2)
1.385(2)
1.3604(17)
1.361(2)
1.281(2)
1.360(2)
1.3761(19)
1.3188(19)
1.391(2)
1.4822(19)
1.395(2)
1.404(2)
1.392(2)
1.376(3)
1.394(3)
1.374(3)
1.468(2)
1.490(2)
1.395(2)
C(12)–C(13)
1.400(2)
1.400(2)
C(6)–C(7)–C(8)
121.09(15)
119.41(16)
120.13(17)
120.50(17)
120.67(16)
114.47(13)
126.62(13)
118.91(13)
113.59(13)
124.53(14)
121.87(13)
132.44(15)
105.46(13)
122.05(14)
109.93(13)
129.92(16)
120.12(15)
117.62(17)
121.71(16)
122.03(17)
116.46(17)
C(13)–C(14)
C(5)–C(6)–C(7)
C(14)–C(15)
1.372(3)
C(6)–C(5)–C(4)
C(15)–C(16)
1.390(3)
C(3)–C(4)–C(5)
C(16)–C(17)
1.379(2)
C(4)–C(3)–C(8)
C(1)–N(1)–C(7)
C(2)–N(2)–C(8)
N(4)–N(3)–C(2)
C(9)–N(4)–N(3)
C(11)–N(5)–C(12)
C(11)–N(6)–C(13)
O(1)–C(1)–N(1)
O(1)–C(1)–C(2)
N(1)–C(1)–C(2)
N(2)–C(2)–N(3)
N(2)–C(2)–C(1)
N(3)–C(2)–C(1)
N(2)–C(8)–C(3)
N(2)–C(8)–C(7)
C(3)–C(8)–C(7)
N(1)–C(7)–C(6)
N(1)–C(7)–C(8)
123.52(12)
117.71(12)
119.47(12)
117.44(13)
106.69(12)
104.32(13)
124.18(13)
121.46(14)
114.34(13)
122.58(12)
124.87(13)
112.54(13)
119.99(14)
121.82(14)
118.19(14)
121.18(14)
117.71(13)
N(4)–C(9)–C(11)
N(4)–C(9)–C(10)
C(11)–C(9)–C(10)
N(6)–C(11)–N(5)
N(6)–C(11)–C(9)
N(5)–C(11)–C(9)
N(5)–C(12)–C(17)
N(5)–C(12)–C(13)
C(17)–C(12)–C(13)
N(6)–C(13)–C(12)
N(6)–C(13)–C(14)
C(12)–C(13)–C(14)
C(15)–C(14)–C(13)
C(14)–C(15)–C(16)
C(17)–C(16)–C(15)
C(16)–C(17)–C(12)
TABLE 3 Hydrogen bonds with H⋅⋅⋅A <r (A) + 3.000 Å and ∠DHA >110°
D–H
d (D–H)
d (H⋅⋅⋅A)
∠DHA
d (D⋅⋅⋅A)
A
N1–H1
0.860
0.860
0.865
0.851
0.851
1.944
2.003
2.181
2.125
2.569
172.74
169.88
174.73
169.14
120.42
2.799
2.854
3.044
2.966
3.089
O1 (−x, −y + 2, −z)
N5–H5
O2
O2–H2A
O2–H2B
O2–H2B
N6 (−x + 2, y – ½, −z + ½)
N2
N4
TABLE 4 Mass and CHN analysis data of complexes of BZHQO
Calcd (found) (%)
Carbon
Molecular formula of complex
Molecular weight
Hydrogen
Nitrogen
C
34
C
34
C
34
C
34
H
28
H
28
H
28
H
28
N
N
N
N
12
12
12
12
O
O
O
O
2
2
2
2
MnCl
2
763.54
766.69
785.58
772
53.55 (53.42)
53.27 (53.18)
52.06 (52.18)
52.95 (52.85)
3.70 (3.65)
3.68 (3.61)
3.85 (3.46)
3.65 (3.59)
22.04 (22.16)
21.92 (21.88)
21.43 (21.65)
21.79 (21.65)
CoCl
2
2 2
NiCl ⋅H O
CuCl
2

SUKANYA AND VENKATA RAMANA REDDY
9 of 11
−
1
TABLE 5 Significant IR frequencies of BZHQO and its complexes (cm
)
Ligand/complex
ν(C═ N)free
ν(C═ N)ring benzimidazole
ν(C═ N)ring quinoxaline
ν(M─ N)
BZHQO
1617
1593
1591
1593
1595
1579
1541
1556
1550
1541
1478
1460
1458
1460
1469
—
[
[
[
[
Mn (BZHQO)
2
]Cl
]Cl
]Cl
2
604, 477, 359
665, 584, 505
609, 437, 376
613, 433, 356
Co (BZHQO)
2
2
Ni (BZHQO)
2
2
⋅H
2
O
Cu (BZHQO)
2
]Cl
2
NH
NH
O
O
N
N
NH
N
NH
N
HN
CH3
NH
HN
CH3
N
N
C
C
M
Ni
2
2
Cl .H O
Cl2
C
C
H3C
N
H3C
N
N
N
HN
HN
NH
N
N
O
O
HN
HN
M=Mn(II), Co(II), Cu(II)
(a)
(b)
FIGURE 7 Structures of complexes of BZHQO
2 2
FIGURE 10 Absorption spectra of [Co (BZHQO) ]Cl in the
presence and absence of DNA
FIGURE 8 Absorption spectra of BZHQO in the presence and
absence of DNA
2 2 2
FIGURE 11 Absorption spectra of [Ni (BZHQO) ]Cl ⋅H O in the
presence and absence of DNA
FIGURE 9 Absorption spectra of [Mn (BZHQO)
2
]Cl
2
in the
2 2
FIGURE 12 Absorption spectra of [Cu (BZHQO) ]Cl in the
presence and absence of DNA
presence and absence of DNA

10 of 11
SUKANYA AND VENKATA RAMANA REDDY
6
6
−1
1
.18 × 10 and 1.94 × 10 M . The binding constants
4 | CONCLUSIONS
indicate that all the molecules have strong affinity to bind
with DNA, while the hypochromism observed suggests
that the molecules interact with DNA through intercala-
tion mode.
The Schiff base ligand BZHQO was synthesized by the
condensation of 3‐hydrazinylquinoxalin‐2‐(1H)‐one and
1‐(1H‐benzoimidazol‐2‐yl) ethanone and characterized
using spectral and single‐crystal X‐ray analyses. The Mn
(
II), Co (II), Ni (II) and Cu (II) complexes of BZHQO
were synthesized and characterized using various
spectroanalytical methods. The ligand behaves as a neu-
tral tridentate N,N,N‐donor, coordinating with the metal
ions through free azomethine, benzimidazole and
quinoxaline ring nitrogens. All the complexes obtained
are mononuclear 1:2 electrolytes and have octahedral
geometry. The ligand and its complexes were screened
against CT‐DNA using electronic absorption spectros-
3
.3.2 | DNA cleavage
The extent of cleavage of supercoiled pBR322 DNA to
nicked circular form was studied using agarose gel elec-
trophoresis. When plasmid DNA is subjected to electro-
phoresis, the fastest migration will be observed for the
supercoiled form (Form I). If one strand is cleaved, the
supercoils will relax to produce a relatively slow‐moving
open circular form (Form II). If both strands are cleaved,
a linear form (Form III) that moves at intermediate rate
copy. The intrinsic binding constant (K ) values calcu-
b
lated and hypochromism observed indicate that all the
molecules have strong affinity to bind with DNA by inter-
calation mode. Agarose gel electrophoresis revealed that
the ligand and its complexes cleaved supercoiled
pBR322 DNA efficiently into nicked form. Cytotoxic
activity studies indicated that the Cu (II) complex was
active against HeLa, B16‐F10 and MCF7 cells, while the
Ni (II) complex was active against MCF7 cells.
[20]
will be generated.
The control experiment using
DNA alone shows no significant cleavage even for longer
time of exposure. The ligand and metal complexes are
able to cleave the supercoiled DNA and convert it to open
circular form (Form II). However, Form III is not
observed in any of these cases (Figure 13).
3.3.3 | Anticancer activity
ACKNOWLEDGMENTS
In vitro anticancer activity studies of the Ni (II) and Cu
The authors thank the UGC networking resource centre,
University of Hyderabad, India for providing spectral and
single‐crystal XRD facilities and Vasavi College of Engi-
neering, Hyderabad, India for its support.
(
II) complexes of BZHQO were investigated using MTT
[36,52]
assay,
with cell lines HeLa, B16‐F10, SKOV3,
MCF7 and CHO‐K1. The results are expressed as IC50
values that represent the concentration of complex
required for the inhibition of the growth of 50% of cells
in vitro. IC50 values of the Cu (II) complex are
ORCID
4
0.73 ± 0.39, 20.81 ± 2.89, 19.88 ± 1.94 and 26.86 ± 2.73
Chittireddy Venkata Ramana Reddy
http://orcid.org/
μM, respectively, for HeLa, B16‐F10, MCF7 and CHO‐
K1, while the complex is inactive towards SKOV3. The
Ni (II) complex was only active against MCF7 cells with
an IC50 value of 21.30 ± 2.84 μM. The Ni (II) complex
did not show any effect on the CHO‐K1 cell line.
0000-0003-3374-2791
REFERENCES
[1] L. W. Deady, A. J. Kaye, G. J. Finlay, B. C. Baguley, W. A.
Denny, J. Med. Chem. 1997, 40, 2040.
[
2] J. A. Castillo‐Garit, M. C. Vega, M. Rolón, Y. Marrero‐Ponce, A.
Gómez‐Barrio, J. A. Escario, A. A. Bello, A. Montero, F.
Torrens, F. Pérez‐Giménez, V. J. Arán, C. Abad, Eur. J. Med.
Chem. 2011, 46, 3324.
[
[
[
3] K. Skalsky, D. Yahav, A. Lador, N. Eliakim‐Raz, L. Leibovici,
M. Paul, Clin. Microbiol. Infect. 2013, 19, 370.
4] Y. Takano, F. Shiga, J. Asano, W. Hori, K. Fukuchi, T. Anraku,
T. Uno, Bioorg. Med. Chem. 2006, 14, 776.
5] A. Gazit, H. App, G. McMahon, J. Chen, A. Levitzki, F. D.
Bohmer, J. Med. Chem. 1996, 39, 2170.
FIGURE 13 DNA cleavage patterns. Lane 1: DNA control; lane
2
: DNA + BZHQO; lanes 3–6: DNA + Mn (II), Co (II), Ni (II) and
Cu (II) complexes, respectively
[6] B. Tamami, S. Ghasemi, J. Org. Chem. 2015, 794, 311.

SUKANYA AND VENKATA RAMANA REDDY
11 of 11
[
7] V. Kuchtanin, L. Kleščíková, M. Šoral, R. Fischer, Z.
Růžičková, E. Rakovský, J. Moncoľ, P. Segľa, Polyhedron
[33] M. E. Reichman, S. A. Rice, C. A. Thomas, P. Doty, J. Am.
Chem. Soc. 1954, 76, 3047.
2
016, 117, 90.
[34] F. Arjmand, S. Parveen, M. Afjal, M. Shahid, J. Photochem.
[
8] N. Beyazit, D. Çakmak, C. Demetgül, Tetrahedron 2017, 73, 2774.
Photobiol. B 2012, 114, 15.
[
9] M. Jafari, M. Salehi, M. Kubicki, A. Arab, A. Khaleghian, Inorg.
Chim. Acta 2017, 462, 329.
[35] N. Raman, R. S. Johnson, J. Serb. Chem. Soc. 2007, 72983.
[36] V. S. Bollu, S. K. Nethi, D. Rama Krishna, N. R. Soma Shiva, S.
[
[
10] N. Padmaja, M. Ravinder, P. Shilpa, S. Srihari, J. Pharm. Chem.
011, 5, 31.
Misra, C. R. Patra, Nanotoxicology 2016, 10, 413.
2
[
[
[
[
37] M. A. Phillips, J. Chem. Soc. 1928, 0, 2393.
11] R. A. Smits, H. D. Lim, A. Hanzer, O. P. Zuiderveld, E. Guaita,
38] G. W. H. Cheeseman, M. Rafiq, J. Chem. Soc (C) 1971, 453.
39] S. Babu, S. Selvakumar, Int. J. Biol. Pharm. Res. 2012, 3, 275.
M. Adami, G. Coruzzi, R. Leurs, I. J. P. de Esch, J. Med. Chem.
2
008, 51, 2457.
40] N. Maheswaran, M. F. Saleshier, M. Mahalakshmi, N.
[
[
12] P. Nobia, M. Vieites, M. H. Torre, A. J. Costa‐Filho, H.
Cerecetto, M. Gonzalez, M. L. Lavaggi, Y. Adochi, H. Sakurai,
D. Gambino, J. Inorg. Biochem. 2006, 100, 281.
Parthiban, V. Sureshkannan, K. Ananda Reddy, Int. J. Chem.
2012, 10, 43.
[
41] M. Ayaz, Z. Xu, C. Hulme, Tetrahedron Lett. 2014, 55, 3406.
13] C. Urquiola, D. Gambino, M. Cabrera, M. L. Lavaggi, H.
Cerecetto, M. Gonzalez, A. Lopez de Cerian, A. Monge, A. J.
Costa‐Filho, M. H. Torre, J. Inorg. Biochem. 2008, 102, 119.
[
42] S. J. Swami, A. Dharma Reddy, K. Bhaskar, Indian J. Chem.
Sect. A. 2001, 40A, 1166.
[
43] J. Mendham, R. C. Denney, J. D. Barnes, M. J. K. Thomas,
Vogel's Textbook of Quantitative Chemical Analysis, 6th ed.,
Pearson Education 2008.
[
[
[
[
[
14] M. R. Suryawanshi, V. M. Kulkarni, K. R. Mahadik, S. H.
Bhosale, Arch. Appl. Sci. Res. 2011, 3, 380.
15] S. Adewuyi, Gangli, S. Zhang, W. Wang, P. Hao, W. Sun, N.
[
[
44] M. Sebastian, V. Arun, P. P. Robinson, P. Leeju, D. Vargese, G.
Tang, J. Yi, J. Org. Chem. 2007, 692, 3532.
Varsha, K. K. M. Yusuff, J. Coord. Chem. 2010, 63, 307.
16] M. Sebatian, V. Arun, P. P. Robinson, A. A. Varghese, R. Abra-
45] P. A. Praveen, R. Ramesh Babu, K. Jothivenkatachalam, K.
ham, E. Suresh, K. K. M. Yusuff, Polyhedron 2010, 29,3014.
Ramamurthi, Spectrochim. Acta A 2015, 150, 280.
17] S. Mayadevi, P. G. Prasad, K. K. M. Yusuff, Synth. React. Inorg.
Met.‐Org. Chem. 2003, 33, 481.
[46] P. Kavitha, K. Laxma Reddy, Arabian J. Chem. 2016, 9, 640.
[47] M. Sunitha, P. Jogi, Ushaiah, C. Gyanakumari, J. Chem. Pharm.
Res. 2012, 4, 1553.
18] N. V. Kulkarni, N. H. Bevinahalli, K. Vidyanand, R. Dhanraj,
J. Coord. Chem. 2010, 63, 1785.
[
48] M. Ur Rehman, M. Arif, M. I. M. Farooq, Am. J. Chem. 2014, 4, 10.
[
[
[
19] C. J. Dhanaraj, J. Johnson, Spectrochim. Acta A 2014, 118, 624.
20] J. D. Chellaian, J. Johnson, Spectrochim. Acta A 2014, 127, 396.
21] C. J. Dhanaraj, J. Johnson, J. Therm. Anal. Calorim. 2017, 127,
[
49] M. Bala, K. K. Mishra, S. Kumar, L. K. Mishra, Am. Int. J. Res.
Formal Appl. Nat. Sci. 2013, 3, 23.
[
[
[
50] A. Majid, A. Adam, A. Alhadhrami, H. A. Saad, M. S. Refat,
1
845.
22] P. V. A. Lakshmi, T. Satyanarayana, P. S. Reddy, Chin. J. Chem.
012, 30, 935.
23] A. Lakshmi, S. Reddy, J. Raju, Bull. Chem. Soc. Ethiop. 2008, 22,
85.
24] S. Rani, J. Raju, A. Lakshmi, Indian J. Chem. Sect. A 1999, 38A,
85.
Appl. Organomet. Chem. 2018, 32, 3950.
[
[
[
51] P. K. Sasmal, A. K. Patra, A. R. Chakravarty, J. Inorg. Biochem.
2
2008, 102, 1463.
52] V. Novohradsky, L. Zerzankova, J. Stepankova, A. Kisova, H.
Kostrhunova, Z. Liu, P. J. Sadler, J. Kasparkovab, V. Brabec,
Metallomics 2014, 6, 1491.
3
3
[
[
25] A. Lakshmi, S. J. Raju, Polish J. Chem. 2009, 83, 1555.
SUPPORTING INFORMATION
26] P. V. Ananthalakshmi, D. Sandhyarani, V. Jayatyagaraju, Asian
J. Chem. 1995, 7, 296.
Additional supporting information may be found online in
the Supporting Information section at the end of the article.
[
27] B. Can‐Eke, M. O. Puskullu, E. Buyukbingol, M. Iscan, Chem.‐
Biol. Interact. 1998, 113, 65.
[
28] S. Grassman, B. Sadek, X. Ligneau, S. Elz, C. R. Ganellin, J. M.
Arrang, J. C. Schwartz, H. Stark, W. Schunack, Eur. J. Pharm.
Sci. 2002, 12, 367.
How to cite this article: Sukanya P, Venkata
Ramana Reddy C. Synthesis, characterization and
in vitro anticancer, DNA binding and cleavage
studies of Mn (II), Co (II), Ni (II) and Cu (II)
complexes of Schiff base ligand 3‐(2‐(1‐(1H‐
benzimidazol‐2‐yl)ethylidene)hydrazinyl)
quinoxalin‐2(1H)‐one and crystal structure of the
ligand. Appl Organometal Chem. 2018;e4526.
https://doi.org/10.1002/aoc.4526
[
29] C. Beaulieu, Z. Wang, D. Denis, G. Greig, S. Lamontagne, G.
O'Neill, D. Slipetz, J. Wang, Bioorg. Med. Chem. Lett. 2004, 14, 3195.
[
30] F. Arjmand, B. Mohani, S. Ahmad, Eur. J. Med. Chem. 2005, 40,
1
103.
[
[
31] F. Arjmand, M. Chauhan, Helv. Chim. Acta 2005, 88, 2413.
32] A. Shah, E. Nosheen, R. Qureshi, M. M. Yasinzai, S. K.
Lunsford, D. D. Dionysiou, Z. Rehman, M. Siddiq, A. Badshah,
S. Ali, Int. J. Org. Chem. 2011, 1, 183.