Synthesis, structural characterization, DNA interaction, antibacterial and cytotoxicity studies of bivalent transition metal complexes of 6-aminobenzothiazole Schiff base

Article abstract of DOI:10.1016/j.inoche.2020.107767

Novel Schiff base (L) derived from 6-aminobenzothiazole and 4-methoxy salicylaldehyde and its bivalent Cu(II) (1), Ni(II) (2) and Co(II) (3) metal complexes were synthesized and thoroughly characterized by various analytical and spectral techniques. From the data a square planar geometry is assigned for all complexes. The DNA interaction studies of complexes with CT-DNA were carried out by UV–Vis absorption, fluorescence and viscosity measurements. These studies revealed that the complexes bind to CT-DNA by intercalative mode. DNA cleavage capacity of all the metal complexes was studied by agarose gel electrophoresis method. Further, the antibacterial studies of metal complexes were evaluated and it is observed that all complexes showed grater activity than free ligand. In addition, in-vitro cytotoxicity of these complexes on tumor cell lines HeLa, A549 and MCF-7 have been examined by MTT assay. The complexes showed greater cytotoxicity than the free Schiff base ligand.

Full text of DOI:10.1016/j.inoche.2020.107767

Journal Pre-proofs
Synthesis, structural characterization, DNA interaction, antibacterial and cyto-
toxicity studies of bivalent transition metal complexes of 6-aminobenzothia-
zole Schiff base
Narendrula Vamsikrishna, Sreenu Daravath, Nirmala Ganji, Nayeem Pasha,
Shivaraj
PII:
S1387-7003(19)31035-4
DOI:
https://doi.org/10.1016/j.inoche.2020.107767
INOCHE 107767
Reference:
To appear in:
Inorganic Chemistry Communications
Received Date:
Revised Date:
Accepted Date:
12 October 2019
2 January 2020
6 January 2020
Please cite this article as: N. Vamsikrishna, S. Daravath, N. Ganji, N. Pasha, Shivaraj, Synthesis, structural
characterization, DNA interaction, antibacterial and cytotoxicity studies of bivalent transition metal complexes of
6-aminobenzothiazole Schiff base, Inorganic Chemistry Communications (2020), doi: https://doi.org/10.1016/
j.inoche.2020.107767
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Synthesis, structural characterization, DNA interaction, antibacterial and cytotoxicity
studies of bivalent transition metal complexes of 6-aminobenzothiazole Schiff base
Narendrula Vamsikrishna, Sreenu Daravath, Nirmala Ganji, Nayeem Pasha and Shivaraj *
Department of Chemistry, Osmania University, Hyderabad, Telangana-500007, India.
Email: srajkavadi@gmail.com
Mobile No: +91 7842572128
Abstract
Novel Schiff base (L) derived from 6-aminobenzothiazole and 4-methoxy
salicylaldehyde and its bivalent Cu(II) (1), Ni(II) (2) and Co(II) (3) metal complexes were
synthesized and thoroughly characterized by various analytical and spectral techniques. From
the data a square planar geometry is assigned for all complexes. The DNA interaction studies
of complexes with CT-DNA were carried out by UV-Vis absorption, fluorescence and
viscosity measurements. These studies revealed that the complexes bind to CT-DNA by
intercalative mode. DNA cleavage capacity of all the metal complexes was studied by
agarose gel electrophoresis method. Further, the antibacterial studies of metal complexes
were evaluated and it is observed that all complexes showed grater activity than free ligand.
In addition, in-vitro cytotoxicity of these complexes on tumor cell lines HeLa, A549 and
MCF-7 have been examined by MTT assay. The complexes showed greater cytotoxicity than
the free Schiff base ligand.
Keywords. Schiff base; Binary complex; DNA interaction; Cytotoxicity; Antibacterial
activity.
1.
Introduction
The metal complexes have a wide range of medicinal applications both as diagnostic
agents and drugs for the treatment of the varied diseases, particularly used in chemotherapy
[1, 2]. Among all cisplatin has been extensively used as anti-metastatic drug for many cancers
since its discovery over 30 years ago, these drugs are very expensive and have serious toxic
efforts [3]. Therefore, need for the discovery of metallodrugs with less or no side effects and
capable of persuading apoptosis in the host. DNA controls many biological processes arising
in the cellular system and key target of several chemotherapeutic drugs. So, understanding
and mechanistic aspects of metal complexes binding with nucleic acid (DNA or tRNA) could
be helped to design new chemotherapeutic drugs. Generally DNA interactions are binding
and cleavage. DNA binding is classified as covalent and non-covalent (intercalation, groove
1

and electrostatic), whereas DNA cleavage is photolytic, hydrolytic and oxidative [4-7].
Schiff base is important and widespread ligand due to its synthetic flexibility, chelated
structure, moderate electron-donor and structural similarity with natural biomolecule. Among
all the Schiff bases, benzothiazole scaffolds have gained importance in medicinal chemistry
and pharmaceutical industries due to their broad range of biotic applications [8, 9]. Transition
metal complexes of Schiff bases act as artificial nuclease inhibitors as well as
chemotherapeutic agents. Cu(II) complexes show more anticancer activity than platinum
based drugs. Copper is a bio-relevant and bio-essential element. Numerous Cu(II) complexes
with biological activities such as anti-cancer and antimicrobial have appeared in the erstwhile
literature [10, 11]. Ni(II) complexes have wide applications in biological field and used as
catalysts in many organic transformations and some of those Ni(II) complexes also have
structural mimic with biological active centres [12]. Co(II) complexes used as potential
nuclease inhibitors due to their versatile biological properties. Thus, the development of a
highly efficient and easily accessible analytical method for detection of Co2+ in biological
and environmental applications is of considerable importance [13]. A thorough literature
survey has shown that not much work has been carried out on benzothiazole Schiff base
metal complexes. Previously synthesis, structural characterization and biological applications
of isoxazole, benzothiazole Schiff bases and their bivalent metal complexes have been
reported from our laboratory [14-16].
Considering the above observations herein we report the synthesis, characterization,
DNA binding, cleavage, cytotoxic and antibacterial studies of Cu(II), Ni(II) and Co(II)
complexes derived from novel benzothiazole Schiff base.
2. Synthesis of Schiff base and binary metal complexes
2.1 Synthesis of Schiff base (L)
Hot methanolic solution of 4-methoxy salicylaldehyde (10 mM) was added drop wise
to a hot methanolic solution of 6-Amino benzothiazole (10 mM). The resultant mixture was
refluxed for 2-4 h, the solid product obtained was filtered and dried under vacuum,
recrystallized from methanol. [BTEMMP] L;( C15H12N2OS) Anal. Calc (%): C, 63.36; H,
4.25; N, 9.85; S, 11.28. found: C, 63.33; H, 4.22; N, 9.82; S, 11.25. IR (KBr): (νO-H) 3433,
(νCH=N) 1622, (νC-O) 1163. UV-Vis (DMSO) max/nm (ε /M−1 cm−1): 257 (38910), 349
1
(28653). H-NMR (CDCl3) (δ): 13.58 (s, 1H); 8.95 (s, 1H); 8.57 (s, 1H); 8.15-8.08 (d, 1H);
7.77 (s, 1H); 7.45-7.43 (d, 1H); 7.29-7.24 (d, 1H); 6.53-6.64 (t, 2H); 3.84 (s, 3H). C13-NMR
(CDCl3) (δ): 164.19, 163.72, 162.16, 153.89, 151.82, 146.43, 134.97, 133.73, 124.10, 120.23,
2

113.84, 113.10, 107.36, 101.08, 55.5, shown in Figs. S1, S2. ESI-MS (m/z): Calc: 284.
Found: 285 [M+H]+ shown in Fig. S3. MP-195 oC.
2.2 Synthesis of binary metal complexes
To a hot methanolic solution of Schiff base ligand (L) (20 mM), copper(II) acetate
mono hydrate / nickel(II) acetate tetra hydrate / cobalt(II) acetate tetra hydrate (10 mM) was
added and the resulting mixture was being refluxed for 4-5 h and the reaction was maintained
in basic condition. The solid product obtained was separated, washed thoroughly with hot
methanol and dried in vacuum. The synthetic route of ligand and its complexes was shown in
Scheme 1.
2.2.1 [Cu(BTEMMP)2] (1); (C30H22N4O2S2Cu) Anal. Calc (%): C, 57.81; H, 3.52; N, 8.89;
S, 10.18. Cu, 13.24. Found: C, 57.79; H, 3.49; N, 8.86; S, 10.17. Cu,13.21. IR (KBr): ν(CH=N)
1610, ν(C-O) 1175, ν(M-O) 502, ν(M-N) 421. UV-Vis (DMSO) max/nm (ε/M−1 cm−1): 263
(38022), 301 (33112), 372 (26881), 556 (17985). eff (BM): 1.75. ESI-MS (m/z): Calc: 629.
Found: 630 [M+H]+ shown in Fig. S4. MP-290 oC.
2.2.2 [Ni(BTEMMP)2] (2); (C30H22N4O2S2Ni) Anal. Calc (%): C, 57.62; H, 3.35; N, 8.96;
S, 10.26; Ni, 9.39. Found: C, 57.59; H, 3.33; N, 8.94; S, 10.28; Ni, 9.37. IR (KBr): ν(CH=N)
1603, ν(C-O) 1170, ν(M-O) 518, ν(M-N) 433. UV-Vis (DMSO) max/nm (ε /M−1 cm−1): 263
(38022), 402 (24875), 587 (17035), 612 (16339). eff (BM): 0. ESI-MS (m/z): Calc: 624.
Found: 625 [M+H]+ shown in Fig. S5. MP-310 oC.
2.2.3 [Co(BTEMMP)2] (3); (C30H22N4O2S2Co) Anal. Calc (%): C, 57.60; H, 3.54; N, 8.96;
S, 10.25; Co, 9.42. Found: C, 57.63; H, 3.52; N, 8.98; S, 10.27; Co, 9.53. IR (KBr) ν(CH=N)
1604, ν(C-O) 1174, ν(M-O) 506, ν(M-N) 425. UV-Vis (DMSO) max/nm (ε /M−1 cm−1): 263
(38022), 361 (27700), 530 (18867). eff (BM): 2.14. ESI-MS (m/z) Calc: 625. Found: 648
[M+Na]+ shown in Fig. S6. MP-280 oC.
3

NH2
O
S
N
H
OH
methanol 2-4 h
reflux
70-80 0
C
OMe
OMe
HO
L
N
S
N
H
methanol 4-5 h
reflux
70-80 0
C
A
C
B
OMe
OMe
R
H
R
N
H
N
O
N
O
N
Co
Cu
O
O
H
R
H
MeO
OMe
R
MeO
3
1
R
H
N
O
N
Ni
O
H
R
MeO
2
A = Copper(II) acetate mono hydrate
B = Nickel(II) acetate tetra hydrate
C = Cobalt(II) acetate tetra hydrate
S
R =
N
Scheme 1. Synthesis of ligand and its metal complexes.
DNA binding studies
2.3
2.3.1. Electronic absorption study
Absorption titration experiments were carried out by varying the concentration of CT-
DNA while the metal complexes concentration was kept constant. Absorbance of CT-DNA in
tris-buffer solution gave 1.8-1.9 ratio at 260 and 280 nm [17, 18], conforming DNA
sufficiently free from protein. Using known molar extinction coefficient value (6600 M-1),
DNA concentration per nucleotide can be measured by spectrophotometrically. While
recording the absorption spectra equal increments of CT-DNA were added to both the
reference and compound solutions, to eliminate the absorbance of CT-DNA itself. The
compound solutions were incubated at 25 oC for 5 min before each absorption spectrum was
recorded.
4

2.3.2. Fluorescence quenching study
The competitive binding of complexes to EB bound DNA system has been
determined by fluorescence emission spectroscopy technique. First CT-DNA (125 µM) is
treated with EB (12.5µM), after that the quencher (0-60 µM) is added to that EB bound DNA
system. The emission intensity was decreased successively by adding quencher (complex).
The relative binding of complexes were determined by Stern-Volmer equation.
I0/I=1+KSV r
Where KSV is a linear Stern-Volmer constant and r is the concentration of quencher to that of
DNA, I and I0 are the fluorescence intensities in the presence and absence of complexes
respectively.
2.3.3. Viscosity study
Viscosity studies were carried out using an Ostwald viscometer maintained at a
constant temperature of 30±1 °C in a thermostatic water bath. Flow time is measured with a
digital stopwatch. Each sample was measured three times and an average flow time was
calculated. The plot made between (ɳ/ɳ0)1/3 versus [complex]/[DNA], where ɳ is the viscosity
of DNA in the presence of complex and ɳ0 is the viscosity of DNA alone. The relative
viscosity was calculated according to the relation ɳ = (t-t0)/t0, Where, t0 is the flow time for
the buffer and t is the observed flow time for DNA in the presence and absence of the
complex.
2.4. DNA cleavage
A mixture of Tris buffer, supercoiled pBR322 DNA (0.2 µg) and 20 µg of the
complexes (1-3) were incubated for 1 h at room temperature. The bromophenol blue dye
solution (0.2 µg) was added to the mixture proceeding to electrophoresis. The samples were
then analysed by 0.75% agarose gel and subjected to electrophoresis for 1 h at 70 mV. The
gel was blemished with EB and photographed under UV light.
2.5. Antibacterial assay
Ligand (L) and its complexes (1-3) were tested against gram positive and gram
negative bacterial strains by disc diffusion method. The nutrient agar media was used. The
standard and stock solutions of compounds were prepared by dissolving them in DMSO. The
paper disc, saturated in the required concentration of the compound solutions, was placed
upon the surface of the inoculated plates with tested microorganisms for 24 h at 30 °C. The
inhibition zones (mm) were compared with standard drugs.
5

2.6. Cytotoxic activity
Cytotoxicity of the Cu(II), Ni(II) and Co(II) complexes was carried out on HeLa
(cervical cancer cell), A549 (adenocarcinomic human alveolar basal epithelial cells ) and
MCF-7 (breast cancer cells) by using the MTT assay [19]. The HeLa, A549 and MCF-7 cells
were plated separately in 96-well plates at a density of 3×103 per well after counting on
Bright Line Haemocytometer. Complexes (1-3) of concentration ranging from 20 to 100 µM
dissolved in DMSO were seeded to the wells. After 48 h, the wells were treated with 0.4 mg/
mL of MTT and incubated at 37 °C for 2 h. The purple formazan crystals were gradually
extracted from test cultures, and 200 μL of DMSO was added. The plates were then shaken
for a while and the absorbance of the solution was dignified at a wavelength of 593 nm using
a Beckmann Coulter Elisa plate.
3. Results and discussion
The Schiff base and its metal complexes are coloured and stable at room temperature.
Schiff base is soluble in all organic solvents and complexes are insoluble in water, partially
soluble in methanol and chloroform, whereas soluble in DMF and DMSO. The analytical
data of all the synthesized complexes are in good agreement with calculated values for 1:2
stoichiometric ratios of metal to ligand.
3.1. IR spectra
Table 1 represents the important infrared spectral data of Schiff base and its metal
complexes (shown in Fig S7). The IR spectra of the Schiff base showed strong stretching
vibrations at 1622 and 1525 cm-1 due to azomethine ν(HC=N) and benzothiazole ring ν(HC=N)
stretching vibrations respectively. This azomethine stretching band was shifted to 1610, 1603
and 1604 cm-1 in 1, 2 and 3 complexes respectively, due to the nitrogen of azomethine group
is coordinated to the metal ion [20, 21] and there is no shifting in ν(HC=N) stretching frequency
of benzothiazole ring which emphasizes that azomethine nitrogen of benzothiazole ring is not
involved in coordination with metal ion. A broad band at 3433 cm-1 belongs to Schiff base
(L) hydroxyl group which is disappeared in all metal complexes indicating the coordination
of phenolic oxygen atom with metal ion by removal of hydrogen atom [22]. A sharp peak
observed at 1163 cm-1 is due to phenolic ν(C-O) stretching frequency of ligand. It is shifted to
the higher frequency region in complexes suggesting the phenolic hydroxyl group is involved
in coordination. The IR spectra of complexes showed new bands at 421-435 cm-1 and 502-
518 cm-1 due to ν(M-N) and ν(M-O), respectively [23, 24] which are absent in Schiff base.
Table 1. Some important IR absorption frequencies (cm-1) of Schiff base and its Cu(II),
Ni(II) and Co(II) complexes.
6

Compound
ν(O-H)
ν (CH=N)
ν(CH=N)
ν (C-O)
ν (M-O) ν(M-N)
BTEMMP (L)
3433
1622
1610
1603
1525
1524
1526
1163
1175
1170
-
-
[Cu(BTEMMP)2] (1)
[Ni(BTEMMP)2] (2)
502
518
421
433
-
-
[Co(BTEMMP)2] (3)
1604
1525
1174
506
435
3.2. Electronic spectra and magnetic moment
The synthesized compounds display UV-Visible absorption bands in DMSO solution at
room temperature in the region of 200-800 nm. The ligand showed two absorption bands at
257 nm and 349 nm due to π-π* transition of the aromatic benzenoid systems and n-π*
transition of azomethine groups, respectively (Fig. S8). All these bands are slightly shifted in
the corresponding complexes. The complexes have shown d-d bands in the visible region in the
range of 530-612 nm. The electronic spectra of Cu(II) complex display a band near 556 nm,
due to 2B1g-2Eg transitions [25]. Ni(II) complex exhibits two bands near 587 and 612 nm these
are assigned to 1A1g-1A2g and 1A1g-1B1g transitions [26]. While Co(II) complex exhibits a single
1
band at 530 nm it attributed to A1g-1B1g transition [27]. The magnetic measurement of Cu(II)
and Co(II) complexes showed 1.75 BM (1) and 2.14 BM (3) μeff values. Whereas Ni(II)
complex is diamagnetic [28]. According to these results, a square planar geometry is proposed
to all metal complexes.
3.3. Thermogravimetric analysis
Thermal stability of synthesized complexes 1-3 were determined by
thermogravimetric analysis (TGA) under nitrogen atmosphere in the temperature range 35-
1000 °C. The results indicate that all the complexes decompose in two stages. The first step
at 282-347 °C corresponding to the partial decomposition of starting ligand moiety and the
second step correspond to removal of total ligand moiety at the temperature range of 316-820
°C, above this temperature left out is metal oxide residue. It is observed that no weight loss at
temperature below 250 °C which indicates the absence of water molecules in both
coordination sphere and outer sphere of the compounds. The thermograms of complexes 1-3
are shown in Fig. 1.
7

Fig. 1. TGA curves of complexes 1, 2 and 3
3.4.
ESR spectra
The ESR spectra is useful to determine the coordination sphere around the Cu(II) ion
in complex. ESR Spectra of Cu(II) complex is recorded at liquid nitrogen temperature (77 K)
and shown in Fig. S9. It exhibit axial signal with two ‘g’ values which are higher than free
electron ‘g’ values [g║(2.12)>g(2.091)>ge(2.0023)] which provides an evidence for the
2
localization of the unpaired electron in dx – y2 orbital indicating square planar geometry [29].
The g║ <2.3 indicates the covalent nature of metal ligand bond. For this complex ‘G’ factor
[G= (g║-2.0023)/(g-2.0023)] value is 1.32 which suggests the considerable exchange
interactions between the Cu(II) centres in the complex [30, 31].
3.5.
DNA binding studies
3.5.1. Electronic absorption study
Electronic absorption titration studies are widely used to determine the binding
propensity of metal complexes with CT-DNA [32, 33]. In general absorbance of complex
decreased with successive addition of CT-DNA (hypochromism) and is also associated with
some red shift indicating an intercalative binding mode between CT-DNA and complex,
whereas increase in absorbance with increasing concentration of CT-DNA (hyperchromism)
showing some blue shift indicates a non-intercalative binding mode.
In present investigation, titration curves at 372 nm of complex 1, 374 nm of complex
2 and 350 nm of complex 3 show 15-20% hypochromism with slight (2-3 nm) red shift
8

shown in Fig. 2. These spectral results emphasized that the complexes bind with DNA by an
intercalative mode. These suggest that upon complexation the binding nature increases. The
strength of intercalative binding is depending upon the degree of the hypochromism [34]. The
propensity of binding strength of complexes measured quantitatively in terms of Kb values
calculated by the following equation.
[DNA]/(εa–εf) = [DNA]/ (εb–εf) +1/Kb(εb–εf).
Binding constant Kb is given by the ratio of the slope to intercept. Where Kb is the
intrinsic binding constant, [DNA] is the concentration of DNA, εa is apparent coefficient of
Aobsd/[complex], εb and εf correspond to the extinction coefficients of the fully bound and
free forms of the complex, respectively. The Kb values are found to be 3.34 ± 0.17 × 104 M-1
(1), 1.16 ± 0.14 × 104 M-1 (2) and 1.14 ± 0.19 × 104 M-1 (3) respectively. Results emphasized
that the copper complex have more binding affinity towards the DNA than the nickel and
cobalt complexes.
Fig. 2. UV-Vis absorption spectra of complexes 1-3, in Tris–HCl/NaCl buffer by addition of
CT-DNA. Arrow shows the hypochromic and bathochromic shifts upon increase of the DNA
9

concentration. Plots of [DNA]/(εa-εf) Vs [DNA] for the titration of DNA with metal
complexes.
3.5.2. Fluorescence quenching study
The intercalative binding mode between the CT-DNA and complexes 1-3 can be
substantiated by fluorescence titration study. EB shows low emission intensity in buffer
solution due to quenching of free EB by solvent molecules [35, 36], while in presence of
DNA, EB shows enhanced emission intensity due to intercalative binding. On addition of the
complexes 1-3 to EB bound DNA system the emission intensity at 591 nm was quenched by
25-28 % is due to the complexes probably compete with EB in EB bound DNA system [37]
(Fig. 3). The Ksv values are calculated from the slope of plot I0/I versus r [38] and Ksv values
are 9.11 ± 0.11 × 104 M-1 (1), 6.11 ± 0.12 × 103 M-1 (2) and 1.12 ± 0.14 × 103 M-1 (3)
respectively.
Fig. 3. Emission spectra of DNA-EB system of complexes 1-3. Arrows shows the emission
intensity changes upon increasing concentration of the complexes. Insert: I0/I versus r.
10

3.5.3. Viscosity studies
The binding mode between the complexes and CT-DNA was further confirmed by
viscosity measurements. Viscosity is least ambiguous test to evaluate the binding mode in
solution [39]. The classical interactions causes increasing length of CT-DNA, due to the
increase in separation of base pairs at sites of complex bind to DNA consequently length of
CT-DNA increases. The non-classical interactions such as groove and electrostatic binding
mode kink or bend the DNA helix resulting the decrease in the length of CT-DNA and
viscosity. In the present investigation with increasing the complexes concentration the
viscosity of CT-DNA is increased (Fig. 4). Among the three complexes, Cu(II) complex
showed high viscosity than Ni(II) and Co(II) complexes. The complexes compared with
groove binders [40] like mesalamine complex (GB) and it is observed that the complexes
bind to CT-DNA through an intercalative mode of binding.
Fig. 4. Viscosity of the EB, complexes 1-3 and GB.
3.6.
DNA cleavage activities
DNA cleavage activity of metal complexes 1-3 with super coiled pBR322 plasmid
DNA was carried out by agarose gel electrophoresis technique in oxidative and photolytic
methods [41]. When pBR322 DNA is subjected to electrophoresis, different forms of DNA
moves with different migration speed. If one strand is cleaved the super coiled DNA is
relaxed to generate a slower moving open circular form or form-II (OC-DNA). If both strands
are cleaved, a linear form (form-III) that migrates between Form I and Form II will be
observed [42].
11

In photolytic method in lane 3 (complex 1), lane 4 (complex 2) and lane 5 (complex
3) complexes cleaved supercoiled DNA into nicked form (Fig. 5). But no DNA cleavage was
observed in lane 1 (DNA control) and lane 2 (ligand L). In oxidative method no DNA
cleavage was observed in lane 1 (control), lane 2 (DNA+H2O2) and lane 3 (DNA+H2O2+L)
but in lane 4 (complex 1), lane 5 (complex 2) and lane 6 (complex 3) complexes efficiently
cleaved supercoiled DNA into nicked form (Fig.6). Further it is observed that the copper
complex more efficiently promoted conversion of the super coiled DNA into linear form.
Fig. 5. Photoactivated cleavage of supercoiled pBR322 DNA (0.2 µg/µL, 33.3 μM) by the
complexes at 37 °C in 5 mM Tris HCl/50 mM NaCl buffer (UV irradiation of wavelength
365 nm). Lane 1, DNA control; Lane 2, DNA + L (20 μM); Lane 3, DNA + complex 1 (20
μM); Lane 4, DNA + complex 2 (20 μM); Lane 5, DNA + complex 3 (20 μM).
Fig. 6. Oxidative cleavage of supercoiled pBR322 DNA (0.2 μg, 33.3 μM) at 37 °C in 5 mM
Tris HCl/50 mM NaCl buffer. Lane 1, DNA control; Lane 2, DNA + H2O2 (1mM); Lane 3,
DNA + H2O2 (1mM) + L; Lane 4, DNA + H2O2 (1mM) + complex 1 (20 μM); Lane 5, DNA
+ H2O2 (1mM) + complex 2 (20 μM); Lane 6, DNA + H2O2 (1mM) + complex 3 (20 μM).
3.7.
Antibacterial activity
Antibacterial activity of all the synthesized compounds were tested against Gram-
positive (Bacillus subtilis) and Gram-negative bacteria (Escherichia coli, Pseudomonas
putida, Klebsiella pneumonia.), shown in Fig. 7 and Table. 2. From the data it is observed
that antibacterial activity of metal complexes (1 mg/mL in DMSO) is higher than free ligand.
It is due to the change in structure upon coordination and chelation tends to make metal
complexes to act as potent bacteriostatic agents, thus inhibit the growth of the bacteria [43].
12

Chelation is one of the most important factor to the activity of metal complexes due to which
effective metal charge is decreased by partial distribution of positive charge with the donor
groups within the chelate ring system, this leads to increase in lipophilic nature of the central
metal ion and favoring its permeation through the lipid layer of the microorganism, thus
extinguishing them more destructively. Antibacterial activity results proved that the copper
complex showed more potent activity than nickel and cobalt complexes [44, 45].
Table 2. Inhibition zone (mm) of ligand and its metal complexes (1mg/mL)
Compound
Inhibition zone(mm)
Gram-negative bacteria Gram-positive bacteria
E.coli
11
P.putida K.pneumoniae
B.subtilis
BTEMMP (L)
10
21
18
21
29
13
19
17
19
25
12
[Cu(BTEMMP)2] (1)
[Ni(BTEMMP)2] (2)
[Co(BTEMMP)2] (3)
Amplicillin
23
22
22
30
21
21
20
28
Fig. 7. Antibacterial activity of Schiff base L and its metal complexes 1-3 with E.coli,
P.putida, K.pneumoniae (Gram Negative) and B.subtilis (Gram Positive) and amplicillin is
taken as a standard.
13

3.8.
Cytotoxicity
DNA is the primary intracellular target of many anticancer drugs, because the interaction
between the complexes and DNA can induce DNA damages in cancer cells, blocking the
division of aggressive growing cells leading cell death [46, 47]. Cytotoxicity of L and its
complexes 1, 2 and 3 has been investigated with following cancer cells such as HeLa
(cervical cancer cell), A549 (adenocarcinomic human alveolar basal epithelial cells) and
MCF-7 (breast cancer cells) by MTT assay. As concentration of compounds increases (20 to
100 µM) cell viability decreases shown in Figs. 8-10. Table 3 shows IC50 values of ligand and
its metal complexes (1-3) and cisplatin. The IC50 values suggest that the complex 1 showed
greater activity compared to the other metal complexes and the ligand. Further, it is observed
that these compounds are more active against HeLa cells than MCF-7 and A-549 cells. The
toxicity order of compounds found to be cisplatin > 1 > 2 > 3 > L. This momentous increase
in the cytotoxicity of compounds compared to ligand indicates that coordination strength of
the metal center in complexes has a noticeable effect on the anticancer activity. This may be
due to the charge of metal ion in complexes decreases which favors increasing the
permeability of complexes through the lipid layer of the cell membrane.
Fig. 8. Cytotoxicity of Ligand (L) and its metal complexes 1-3 on HeLa cell lines
14

Fig. 9. Cytotoxicity of Ligand (L) and its metal complexes 1-3 on A549 cell lines
Fig. 10. Cytotoxicity of Ligand (L) and its metal complexes 1-3 on MCF-7 cell lines.
Table 3. IC50 values of ligand (L) and its metal complexes (1-3) against HeLa, A549 and
MCF-7 cell lines
Compound
HeLa
A549
MCF-7
BTEMMP (L)
61 ± 0.17
32 ± 0.42
35 ± 0.23
37 ± 1.34
29 ± 0.54
62± 0.13
41 ± 0.16
44± 0.12
45 ± 0.34
30 ±0.42
67± 0.15
44 ± 0.17
48 ± 1.34
49 ± 0.65
28 ± 0.36
[Cu(BTEMMP)2](1)
[Ni(BTEMMP)2](2)
[Co(BTEMMP)2](3)
Cisplatin
4. Conclusion
In the present study, Cu(II), Ni(II) and Co(II) complexes have been synthesized from
novel Schiff base and thoroughly characterized by analytical and spectral techniques.
According to spectral and analytical data a square planar geometry is assigned to all
15

complexes. The binding studies of complexes with CT-DNA were investigated by UV-Vis
spectroscopy, fluorescence studies and viscosity measurements. Binding studies showed that
all complexes are involved in intercalative binding with CT-DNA. The DNA cleavage studies
of metal complexes revealed that these complexes effectively cleaved supercoiled pBR322
DNA both in the presence of UV light and H2O2. Further DNA cleavage activity of copper
complex showed comparatively higher activity than nickel and cobalt complexes. The
antibacterial data indicated that all complexes showed high antibacterial activity compared to
ligand. Cytotoxic activity results have shown that the complexes can act as antitumor agents.
Acknowledgements
Authors express heartfelt thanks to the Head, Department of Chemistry for providing
the necessary facilities, the Director, CFRD, Osmania University, Hyderabad. We are also
thankful to DST-SERB (SB/EMEQ-141/2014), DST-FIST, UGC-UPE (FAR)
(002/OU/UPE/FAR/CO/2013), OU-DST-PURSE-II(C-DST-PURSE-II/9/2017) and CSIR
(File No: 09/132(0864)/2017-EMR-I) and (Grant No.09/132(0799)/2012-EMR-I), New Delhi
for their financial assistance.
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GRAPHICAL ABSTRACT
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Highlights
 Ligand and its Cu(II), Ni(II) and Co(II) metal complexes have been synthesized.
 All the compounds were characterized by spectroscopic and analytical techniques.
 The DNA binding and cleavage studies have been studied.
 The compounds were screened for in-vitro antibacterial activity.
 The in-vitro cytotoxicity of the compounds was also investigated by MTT assay.
Declaration of interests
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
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