Novel bioactive azo-azomethine based Cu (II), Co (II) and Ni(II) complexes, structural determination and biological activity

Article abstract of DOI:10.1016/j.molstruc.2019.04.022

A novel bioactive azo-azomithine tridentate ligand N'-(E)-{2-hydroxy-3-methoxy-5-[(E)phenyldiazenyl]phenyl}methylidene]pyridine-3 carbohydrazide (HL) and its Cu(II), Co(II) and Ni(II) complexes were synthesized and their structure determined by elemental

Full text of DOI:10.1016/j.molstruc.2019.04.022

Accepted Manuscript
Novel bioactive azo-azomethine based Cu (II), Co (II) and Ni(II) complexes, structural
determination and biological activity
N. Venugopal, G. Krishnamurthy, H.S. Bhojyanaik, M. Giridhar
PII:
S0022-2860(19)30417-X
DOI:
https://doi.org/10.1016/j.molstruc.2019.04.022
MOLSTR 26394
Reference:
To appear in: Journal of Molecular Structure
Received Date: 18 January 2019
Revised Date: 8 March 2019
Accepted Date: 4 April 2019
Please cite this article as: N. Venugopal, G. Krishnamurthy, H.S. Bhojyanaik, M. Giridhar, Novel
bioactive azo-azomethine based Cu (II), Co (II) and Ni(II) complexes, structural determination
and biological activity, Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/
j.molstruc.2019.04.022.
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“Novel bioactive azo-azomethine based Cu (II), Co (II) and Ni(II)
complexes, structural determination and biological activity”
Venugopal N a, Krishnamurthy G a*, Bhojyanaik H. S. b* Giridhar M c.
aDepartment of chemistry, Sahyadri Science College, Kuvempu University, Shivamogga-
577201, Karnataka, India.
bDepartment of Industrial chemistry, Kuvempu University, Jnana Sahyadri, Shankaraghatta,
Shivamogga-577451, Karnataka, India.
cGovernment First Grade College (GFGCH), Hassan-573201, Karnataka, India.
*Email: gkmnaiksahydri@gmail.com
Abstract
A novel bioactive azo-azomithine tridentate ligand N'-[(E)-{2-hydroxy-3-methoxy-5-
[(E)phenyldiazenyl]phenyl}methylidene]pyridine-3 carbohydrazide (HL) and its Cu(II),
Co(II) and Ni(II) complexes were synthesized and their structure determined by elemental
1
analysis, spectroscopic studies (FTIR, H NMR, 13C NMR, LC-MS, UV-vis, ESR, and
powder XRD) and magnetic susceptibility measurements. The molar conductivity
measurement proved that all the complexes are non-electrolytic in nature. On the basis of
physicochemical and spectroscopic data, the octahedral geometry is assigned for all the metal
complexes. The thermal decomposition curve indicated the existence of lattice water
molecule. The binding studies of all the synthesized complexes against CT-DNA suggest that
the intercalation binding mode. A DNA nuclease activity exhibited that all complexes
cleaved pBR322 DNA in an efficient manner. Consequently, the cytotoxic activity of the
compounds was screened against A-549 (lung carcinoma), MDA-MB 231 (breast carcinoma)
and normal HEK293 (human embryonic kidney cells) cell lines using MTT assay.
Keywords: Metal chelates; P‒XRD; TGA-DTG; DNA interaction; Cytotoxicity.

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Introduction
Over the last few decades, remarkable step have been taken in designing of transition
metal chelates of azomethines, which are utilized in an extensive range of biological and
clinical applications along with remedial drug against cancer cells [1]. Cancer is becoming a
leading cause of premature lethal death in most of the developed as well as developing
countries [2]. To examine the clinical application of transition metal based tumour drugs one
important point is minimization the undesirable side effect of the drug [3]. Cisplatin,
carboplatin, and oxaliplatin (with many severe side effects) were major tumour drugs for
some decades thus developing novel metal–based complexes with less-toxicity and target
specific in an active area of research[4].The deoxyribonucleic acid (DNA) plays an essential
role in the replication of cells and storage of genetic information[5]. DNA is the primary
intercellular target for many tumour drugs in clinical use and important to understand the
binding of metal ion to DNA through both covalent and non-covalent interactions [6]. Three
known non-covalent binding modes of small molecule to DNA are intercalative binding,
groove binding and external electrostatic binding [7]. Among these, intercalative binding
mode is most important and coherent to the anticancer activity the metal ion [8].
The pyridine-anchored hydrazone moiety have recognised significant interest due to the
presence of donar atom like nitrogen and oxygen has achieved dazzling coordination capacity
and diverse pharmacological activities [9-11]. In addition, the phenolic aldehyde compounds
like vanillin are well known and perform a class of molecule which show a comprehensive
biological property [12, 13]. An added privilege of using 2-hydroxy-3-methoxy-5-[(Z)-
phenyldiazenyl] benzaldehyde over vanillin is the position of hydroxyl group, this group is
present at ortho position can readily deprotonate and coordinate with metal centre [14].
Hence, an imense number of Cu (II), Co (II) and Ni (II) complexes of azo-dye Schiff bases
have been extensively studied due to their diverse antifungal, antibacterial, anti-tuberculosis,
anticonvulsant and anticancer activities. Thus, the design of new chemotherapeutic drug is
now engaging the consideration of medicinal chemists which reduce the toxicity [15-17].
Furthermore, the interaction of compounds with double helix DNA has been widely studied,
due to the site of specific binding and many significant applications in cancer therapy. These
coordination compounds were also important in developing probes for nucleic acid structures
and cancer drugs [18, 19].
This paper describes, the synthesis of novel bioactive Cu (II), Co (II) and Ni (II) complexes
of the azo linked Schiff base N'-[(E)-{2-hydroxy-3-methoxy-5-[(E)phenyldiazenyl]
phenyl}methylidene]pyridine-3 carbohydrazide (HL) and characterized by various analytical

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and spectroscopic techniques. The electronic and thermal techniques were used to deduce the
geometry of the metal complexes. DNA-binding behaviour of metal complex was
investigated via electronic absorption spectroscopy. The DNA cleavage activity of metal
complexes performed using gel electrophoresis technique using supercoiled pBR322 DNA.
Additionally, in-vitro anticancer activity of synthesized complexes was assessed by MTT
assays, against cancer cell lines A-549, MDA-MB 231 and normal HEK293 cells.
2. Experimental
2.1. Reagents and Physical Measurements
All the reagents needed for the present work were commercially available, analytical
grade and obtained from Sigma Aldrich, Merck and Hi Media Ltd., utilized without further
purification. Microanalytical data (Carbon, Hydrogen and Nitrogen) was carried out on Vario
EL.CHNOS elemental analyser. The electronic spectra of the compounds were recorded on
shimadzu model 1650 UV- visible double beam spectrometer in the range of 200‒800 nm in
DMF solution (10-3M). FTIR spectra recorded using KBr discs on a Bruker alpha-T FTIR
1
Spectrophotometer in the range of 4000-400cm-1. H and 13C NMR spectra were obtained
using a Bruker DPX200 instrument (400 MHz) in DMSO-d6 as a solvent consisting TMS as
the internal reference compound. The mass spectral analysis was done on LC-MS: water
AQUITY-2777C mass spectrometer. The conductivity measurement of the metal complexes
was carried out in (10-3M) DMF solution using an ELICO-CM82 conductivity bridge. The
EPR spectrum of copper complex was recorded on JES - FA200 ESR spectrometer with solid
state X-band at room temperature. The magnetic moment values of the complexes were
calculated at room temperature on a Gouy balance model 7550 applying Hg [Co (NCS)4] as a
calibrant. Thermal analysis was recorded using SII Exstar TG/DTA 6300 instrument in the
o
o
temperature range 30 to 800 C with a heating rate of 10 C min. The powder X- ray
diffraction studies of the complexes were obtained on Bruker AXS D8 prior instrument.
2.2. Synthesis of of Azo Schiff base ligand (HL)
Azo-linked Schiff base ligand (HL) was prepared in two steps. In the first step,
diazonium solution was prepared by dissolving (2mmol) of aniline in 20 mL of distilled water
o
and (4mL) of concentrated hydrochloric acid. The solution was cooled to 0-5 C in an ice
bath and a cold aqueous solution of sodium nitrite (2 mmol) in 3mL of concentrated sulphuric
acid was added dropwise with continuous stirring. The obtained diazonium salt solution was
o
stirred and cooled to 0-5 C in an ice bath for about 1h and then poured drop wise to the

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chilled solution of 2-hydroxy-4 methoxybenzaldehyde (2mmol) in alkaline media. The
resulting mixture was stirred for about 2 h at the same temperature and the pH was
maintained around 7.0-8. Coupling to the o-vanillin has occurred in basic media at the para
position.
In the second step, 1:1 condenstion reaction occured between (2 mmol) 2-hydroxy -3
methoxy -5-(phenyldiazenyl) benzaldehyde and (2 mmol) of isonicotine hydrazide which
were dissolved in hot ethanolic solution in the presence of 2-3 drops of glacial acetic acid as
a catalyst. The reaction mixture was refluxed and continuously stirred for about 3-4h. After
cooling, the azo-azomethine a dye was collected by filtration, washed with ethanol and dried
in vacuum desiccator over CaCl2. The purity of the compounds was achieved by the
recrystallization in ethanol and the schematic depiction of the synthesis of ligand displayed in
Scheme 1.

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NH2
O
O
Diazotisation,0 -5oC
N
OH
N
+
Coupling
OH
OCH3
OCH3
O
Ethanol, reflux
3-4h
NH2
NH
N
OCH3
OH
N
N
N
N
NH
O
Azo linked schiff base HL1
M= Cu2+, Co2+
Ni2+
NH
OCH3
O
N
N
N
n H2O
N
Cu
O
N
O
N
CH3O
O
NH
Where M=Cu (II) n=3, Co (II) and Ni(II) n=2.

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2.2.1 N'-[(E)-{2-hydroxy-3-methoxy-5-[(E)-phenyldiazenyl]phenyl}methylidene]pyridine-3
carbohydrazide (HL).
Yield: (77.0 %), Colour: orange, melting Point: 254–256°C. Anal. Calc. For [C20H17N5O3]
C, 57.42; H, 3.76; N, 10.65. Found C, 57.44; H, 3.78; N, 10.62. IR data on KBr pellet ν(cm-1)
:3246 ν(‒OH), 3058 ν(N‒H), 1602 ν(CH=N),1661 ν(C=O), 1444 ν(‒N=N‒).1H NMR (400
MHz, DMSO-d6, δ ppm): 12.40 (s, 1H, OH), 11.38 (s, 1H, NH); 8.85 (s, 1H, N=CH), 8.84
(d, 2H, J=10 Hz, pyridine ꢀ-H), 8.00–7.53 (m, 9H, Ar-H), 3.97 (s, 3H, OCH3). 13CNMR (100
MHz, DMSO-d6, δ ppm): 161.98, 152.4, 150.88, 149.36, 147.55, 131.38, 129.91, 122.71,
119.77, 118.70, 104.79, and 56.49. LC-MS: m/z 376 [M+1]. UV–Vis (λmax, nm): 246, 410.
2. 3 Synthesis of Cu (II), Co (II) and Ni (II) complexes
To the hot ethanolic solution of azo-azomethine ligand (2mmol) and appropriate
metal chlorides Cu(II), Co(II) and Ni(II) in ethanol (1mmol) was added drop wise with
constant stirring. The resulting suspension was refluxed on a steam bath for about 3-4 h. On
cooling, the colored solid product was filtered and washed thoroughly with hot water and
ethanol and dried in a vacuum over anhydrous CaCl2.
2.4 Biological studies
2.4.1 DNA interaction studies
The binding interaction between the metal ions and CT-DNA was investigated using
UV-vis spectroscopy. The required amount of DNA solution was prepared by dissolving in
buffer medium (50 mM NaCl, 5 mM Tris-HCl and pH 7.2) UV absorbance value at 260 and
280 nm gave a ratio of about 1.8–1.9, revealing that DNA is clear from protein
contamination. The concentration of CT-DNA per nucleotide was determined from its
1
1
absorbance by employing a molar extinction coefficient value of 260 nm (Ɛ = 6600 Mꢀ cmꢀ )
[20]. The metal complexes were dissolved in DMSO solvent because of low solubility in
buffer solution. Absorption titration studies were carried with a fixed concentration of the
complexes (20 mM), by varying the concentration of DNA (20-350 mM). An equal volume
of CT-DNA was added to both complex solution and reference solution in order to remove
the absorbance of CT-DNA itself. The intrinsic binding constant (Kb) values of the
complexes with DNA were measured by the following equation.
[DNA]/(Ԑa– Ԑf) = [DNA]/(Ԑb – Ԑf) + 1/Kb (Ԑb – Ԑf)
1
Where [DNA] is the concentration of CT-DNA in base pairs, Ԑa is apparent
coefficient of Aobsd/[complex], Ԑf and Ԑb correspond to the extinction coefficients of the free

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and fully bound forms of the complex, respectively. A plot of [DNA] / (Ԑa– Ԑf) versus [DNA]
gave a slope of 1/(Ԑb – Ԑf) and Y-intercept equal to 1/ Kb(Ԑb– Ԑf), Kb is the ratio of slope to
intercept.
2.4.2 DNA cleavage activity
The interaction of newly synthesized metal complexes with DNA was carried out by
using gel-electrophoretic technique [21]. The experiment involves pBR322 DNA (2 ꢀL, 2
ꢀg), 100 ꢀg metal complexes, 50 mM Tris-HCl buffer, 50 mM NaCl were mixed and pH 7.2
was mantained. The contents were allowed to proceed with incubation for 2 h at 37 °C. After
incubation, 5 ꢀL of loading buffer (bromophenol blue 0.25%, Xylene cyanol 0.25% and
glycerol 30%) and samples were poured into the well along with standard DNA marker on
3
1% agarose gel for 1 h at 60 mV. The gel was further blemished with 1ꢀg cmꢀ ethidium
bromide solution for 10 min. The bands were observed under ultraviolet light and picture was
taken.
2.4.3 Cell culture
The cell lines A-549, MDA-MB 231 and normal HEK293 were procured from NCCS
Pune. The cells were cultivated in monolayer in RPMI 1640 medium, added with 10% fetal
bovine serum (FBS) 2 mM L-glutamine, and 1% penicillin. Cells were preserved in 5% CO2
at 37 °C in 95% relative humidity and clear from mycoplasma contamination.
2.4.4 Cell viability assay
The in vitro-anticancer activity was performed using MTT assay. Cells were grown in
96 well tissue culture plates at a concentration of (1×104 cells/well) and overnight incubation
at 37 °C in 5% CO2 under a moistened air atmosphere to permit the growth and attachment.
Then the cells were treated with various concentration of complexes dissolved in DMSO (1-
100ꢀg mL-1) and incubated for 24h. After incubation, the culture is detached and 20 ꢀl MTT
solution was added to each well. The plates were further incubated in darkness at the
temperature of 37°C for about 4 h in CO2 atmosphere. The following media is discarded in
order to solubilise the purple colored formazan product by adding 200ꢀl of DMSO is poured
to each well. The absorbance of the wells was measured using the ELISA reader at 570 nm
[22, 23]. The % of cell inhibition expressed as
Inhibition (%) = 1 ‒ 100 × (OD toxicant) / (OD ‒ve control).

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IC50 values were evaluated using the nonlinear regression program from origin.
3. Result and Discussion
The newly synthesized azo-azomethine dye ligand (HL) and their metal complexes were
found to be tinted solid, stable at room temperature, soluble in DMF and DMSO. The
observed molar conductivity measurement proved that all the metal complexes are non-
electrolytic in nature [24]. Melting points of the prepared complexes were found to be above
300 o C. From the analytical data, it is evident that 1:2 stoichiometric ratio of metal to ligand
with general a formula [M (L) 2] nH2O for all the complexes. The elemental analysis data are
in good accordance with the calculated values. Physical measurement and analytical data of
the ligands and its metal complexes are depicted in Table 1.
<Insert Table 1>
3. 1 1H and 13C NMR spectrum of ligand
1
13
The H and C NMR spectrum of the ligand (HL) (Fig. S1 and S2) was obtained in
DMSO-d6 at room temperature using TMS as an internal standard. The 1H NMR spectrum of
azo azo-mithine ligand displayed a signal at 12.40 ppm as singlet which is attributed to the
hydroxyl group attached to vanillin ring. The NH proton attached to isonicotine hydrazide
next to carbonyl group appeared as a singlet at 11.38 ppm. The signal appeared at 8.85 ppm
as a singlet is ascribed to azomethine proton (‒CH=N). Another doublet found at 8.84 ppm is
considered as the signal of the two protons at the alpha position of pyridine. Other aromatic
ring protons of the ligands were resonated as multiplets in the range 8.00–7.53 ppm. Another
singlet appeared at 3.97 ppm corresponds to the methoxy proton attached to vanillin ring [25-
27].
The 13C NMR spectrum of the ligand showed a signal at δ 161.98 ppm due to carbon
of carbonyl group. The peak at δ 152.4 ppm due to azomethine carbon. All the other aromatic
carbon shifts were observed in the range of δ 104.7–150.8 ppm. The peak at δ 55.9 ppm is
assigned to the carbon atom of methoxy group [28].
3. 2 Mass spectra
The mass spectra of the ligand and its metal complexes were recorded and obtained
molecular ion peak confirms the proposed formulae. The mass spectrum of ligand (HL)
shows (Fig. S3) a well defined molecular ion peak at m/z 377 which was coincident with

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(M+1) proposed molecular formula weight of azo-azomethine ligand. The molecular ion
peaks of the Cu(II), Co(II) and Ni(II) complexes were appeared at m/z of 848, 844 and 845
respectively, which are equivalent to the stoichiometric ratio of 1:2 (M:L) type. Further, the
observed molecular mass in all the spectra of the prepared complexes are in consistency with
their proposed molecular structures.
3.3. Infrared spectra
The Infrared spectra of ligand (HL) and their metal complexes were recorded as KBr
pellets in the region 4000-400 cm-1 and the data indicate the binding modes of the ligand to
metal ion as presented in Table 2. The ligand exhibited a sharp band at 1602 cm-1 is due to ʋ
(C=N) stretching vibration. On complexation, this band shifted to a lower frequency in the
range of 1545-1563 cm-1 by about 29-55cm-1in all the complexes indicating the involvement
of azomethine nitrogen atom in coordination with the metal ion [29]. The appearance of a
strong absorption band at 1661 cm-1 in spectrum of ligand assigned to ʋ(C=O) vibration
which is shifted to lower frequency by 54 to 63 cm-1 in complexes and observed in the range
1598-1607cm-1 respectively, suggesting that the involvement of carbonyl group in bonding
with the metal ions [30]. The characteristic broad band at 3058 cm-1 in the uncoordinated
ligand is assignable to ʋ(NH) stretching vibration, which remains more or less at the same
position in the spectra of all the complexes, indicating their non-involvement of amide group
on complexation [31]. The IR spectra of free ligand showed a absorption band at 3225 cm-1 is
ascribed to ʋ(-OH) group which disappeared in their spectra of metal complexes, indicating
that the complex formation has taken place by deprotonation of phenolic oxygen atom.[32]
The appearance of medium intensity band at 1444 cm-1 in the free ligand was assigned to
stretching frequency of azo group (‒N=N‒). In all the complexes, this band lies at the same
position confirming the non-participation of azo group in complex formation [33].
Furthermore, the existence of two new non-ligand bands in the region 495–460 cm-1 and
622– 555 cm-1 has been ascribed to M–N and M–O bands, respectively [34]. In conclusion,
the IR data confirms a tridentate nature of the ligand.
<Insert Table 2>
3.4. Electronic spectral studies
The electronic absorption spectra of all the compounds were recorded using DMF
solution in the range 200-800 nm at room temperature. The spectral data of the uncoordinated
ligand exhibited two recognizable absorption bands. The absorption band appeared at 287 nm

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(34,843cm-1) may be ascribed to the ꢁ → ꢁ* transition of the aromatic rings, while the other
band appeared at 349 nm (28,653cm-1) may be assigned to n→ꢁ* electronic transition due to
the azo-methine group respectively. Further, it is recognized that the absorptions bands were
shifted to longer wavelength in all the metal complexes suggesting that ligands coordinate to
metal ions [35].
Electronic spectrum of Cu(II) complexes exhibited two intra ligand charge transfer
bands in the range of 24,875, 30,769 along with d–d transition band at 17,064 cm-1 due to the
2Eg →2T2g transition
. The position of these bands suggests that the Cu(II) complex has a
distorted octahedral geometry [36]. This is further favoured by its magnetic moment value of
1.82 B.M. The electronic spectrum of Co(II) complex exhibit three bands at 14,598, 16393
4
4
4
4
4
and 23,529cm-1 which are attributed to T1g → T2g(F), T1g→ A2g(F) and T1g→4Tg(P)
transition, respectively. The position of these bands and their magnetic moment value of 4.21
B.M, suggests that the Co(II) complex has a characteristic of octahedral geometry [37]. The
electronic spectrum of Ni (II) complexes exhibit three bands at 16,103, 24,630 and 32,258
1
1
cm-1 attributed to A1g ꢀ B1g 1A1g ꢀ 1A2g, and charge transfer transition. The positions of
,
these bands and its magnetic moment value of 3.28 B. M, indicate an octahedral geometry
[38].
3. 5 Electron spin resonance (ESR)
The X-band ESR spectrum of the Cu(II) complex of ligand HL was measured in the
solid state at room temperature and is displayed in Fig. 1. The investigation of g value of
copper complex provided the g tensor value g|| =2.1438, g⊥=2.0380 and G=4.04. It is known
that if g|| value is greater than 2.3, then the M‒L bond is virtually anionic character and the
value is less than 2.3 is an indication of covalent nature [39]. In present case the observed g||
value of the copper complex is less than 2.3 indicating that appreciable covalency in
metal‒ligand bond. The trend in observed g values of the Cu(II) complex is g|| (2.1438) > g⊥
(2.0380) > 2.0023, indicating that the unpaired electron in the ground state of Cu(II) is
predominantly in dx2-y2 and the complex is axially symmetric. Thus, an octahedral structure is
deduced for the complex [40]. G = (g|| -2) / (g⊥-2) which determines the exchange interaction
between the copper centres in the polycrystalline compound have been evaluated. According
to Hathaway and Billing expression, if the G value is less than 4, the exchange interaction
between the copper centers is noticed whereas if its value is greater than 4 the exchange
interaction is negligible [41]. The calculated G value for the present Cu(II) complex was

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4.042 indicating that there is an absence of exchange interaction between the copper ions.
The observed gav is 2.053. The ESR spectral parameters are given in Table 3.
<Insert Table 3>
<Insert Figure 1>
3.7. Thermal analysis
The thermal properties of Cu(II), Co(II) and Ni(II) complexes were carried out to
understand the thermal stability and molecular structure. The tentative thermal decomposition
of metal complex with respect to temperature and formation of the respective metal oxides
are displayed in Table 4. The Thermal behaviour of the complexes were done from room
temperature to 800 °C at a heating rate of 10 °C/min in a N2 atmosphere is shown in Fig. 2.
The thermogram of [Cu(C40H38N10O9)] complex (1a) showed three decomposition stages, the
first stage of decomposition occur in the temperature range 30-78°C is due to the elimination
of three lattice water molecules with an approximate loss of 6.06% (calcd 6.23%). The
second stage of decomposition occurs below the temperature 250 °C which corresponds to
the loss of C4O2 molecules with a mass loss 9.18% (calcd 9.30%). The third stage of
decomposition occurs within the temperature range 250-370 °C and corresponds to the loss of
C10H10N2 molecules with a mass loss 17.96% (calcd 18.26%). Further the complex
underwent fourth stage of decomposition due to complete decomposition of organic portion
C24H28N8O3 of complex in the temperature range 370-697°C with weight loss of 54.72%
(calcd 55.02%). The weight of the residue corresponds to the one mole of cupric oxide and
two moles of carbon.
The thermogram of [Co(C40H36N10O8)] (1b) showed three decomposition stages, the first
stage of decomposition occured within the temperature range at 26-75 °C is due to the loss of
two water molecules with a weight loss of 4.26% (calcd 4.35%). The second stage of
decomposition occurs within the temperature range 75-315 °C and corresponds to the loss of
C12H10N2O2 molecules with a mass loss 24.29% (calcd 24.48%). Further third stage of
decomposition occur in the temperature range 315-495 °C due to the loss of organic moiety
of the complex C28H24N8O3 with the weight loss 55.86% (calcd 56.46%). The final weight of
the residue corresponds to the one mole of cobalt oxide.
Similarly, the thermogram of [Ni(C40H36N10O8)] (1c) showed four decomposition stages, in
the temperature range 28-65 °C, 65-175 °C, 175-378 °C and 378-510 °C respectively. The

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weight loss 4.25% (calcd 4.46%), % 15.56 (calcd 15.71%), 12.64% (calcd 13.03%) and
57.10% (calcd 58.29%) are tentatively ascribed to the removal of 2H2O, C8H4O2, C4H2N2O2
and C28H28N8O3 fragments. Finally, one mole nickel oxide as residue was observed.
<Insert Table 4>
<Insert Figure 3>
3.8. Powder XRD studies
The powder X-ray diffraction patterns of the newly synthesized metal complexes
were recorded because these complexes are soluble in polar organic solvents, like DMSO and
DMF. Therefore single crystal could not be obtained. In order to understand the degree of
crystallinity of the synthesized metal chelates, the X-ray diffractogram of Cu(II), Co(II) and
Ni(II) complexes were obtained in the range of 10-80 (2θ) at a wavelength of 1.54 Å. All the
metal complexes were displayed a certain amount of well defined sharp crystalline peaks
proved inherent crystalline nature of complexes Fig. 3.
Powder X-ray diffraction pattern for Cu(II) complex showed 8 reflections in the range
of 9-40 ° (2θ). The inter-planar spacing (d values) of the reflections were calculated using
Bragg’s equation nλ= 2dsinθ (where λ=1.5406 Å). The observed and calculated inter-planar
spacing (d) simultaneously, with relative intensities with respect to most intense peak are
consistent and are depicted in Table 5. Further, the unit cell parameters were evaluated for
cubic symmetry by all the important peaks, and h2 + k2+ l2 values were ascertained. The
observed inter-planer d-spacing values have been compared with the calculated ones and it
was found to be in good agreement. The h2 + k2 + l2 values for Cu(II) complex are 1, 2, 3, 4,
6, 10 and 15. It was noticed that the existence of forbidden number 15 suggests that the
Cu(II) complex may reside to hexagonal or tetragonal systems. The calculated lattice
parameter for Cu (II) complex is found to be a = b = c = 8.05.
Similar calculations were carried out for Co(II) and Ni(II) complexes and all the
obtained major intense peaks have been indexed and observed values of inter-planar d-
spacing have been compared with the calculated ones. The h2 + k2 + l2 values for Co(II)
complex are 1,1, 2, 2, 3, 4, 6, 7, 8, 10, 14 and for Ni(II) complex are 1, 1, 2, 3, 4, 6, 8, 10, 15.
The presence of forbidden numbers like 7 for Co(II) complex and 15 for Ni (II) complex
indicates both the complexes may belong to hexagonal or tetragonal systems. The lattice
parameter for the Co(II) and Ni(II) complexes were found to be a = b = c = 7.86 and 8.108
respectively.

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<Insert Table 5 >
<Insert Figure 3>
3.8. Scanning electron microscopy (SEM)
The surface morphology of the azo-azomethine ligand and its complexes were
examined by scanning electron microscopy (SEM). Fig. 4 illustrates the SEM photographs of
the studied compounds. The broken rock like structure was observed in ligand (HL).The ice
block like structure with layered morphology has observed in Cu (II) complex. Particles are
embede in otton-like a filamentary matrix has observed in Co(II) complex and Ni(II)
C
complex seemed as non uniform grain like structure, respectively. SEM images disclosed that
the surface morphology of metal complexes vary from ligand and other because of the
complexation and change of metal ion.
<Insert Figure 4>
4. Biological activities
4.1. DNA binding experiments
Electronic absorption spectroscopy is universally engaged technique to examine the
strength and mode of binding of the metal complexes with DNA which generally involves the
changes in absorbance and wavelength. The absorption spectra of Cu (II), Co (II) and Ni (II)
complexes recorded in the absence and presence of CT-DNA are depicted in Fig.5 The
absorption titration were carried using a fixed metal complexes concentration to which
increment of CT-DNA stock solutions was added in different ratio ranging from 25 mL to
350 mL. While calculating the absorption spectra of complexes, an equal amount of DNA
was added to both the compound solution and the reference solution to eliminate the
absorbance of DNA itself. The binding nature of metal ions to DNA was determined from the
changes in absorbance spectrum of every complex upon supplement of CT-DNA. After the
interaction of compounds with CT-DNA base pairs, the π* orbital of the intercalated ligand on
the complexes can couple with π orbitals of the DNA base pairs of DNA which leads to
decrease in π-π* transition energies. On the other hand, as the coupling π* orbital are partially
filled by electrons, decreases the transition probabilities resulting in hypochromism [42].
Upon gradually increasing the amount of CT-DNA to the complexes leads to considerable
hypochromic effect without varying the absorption intensities. The hypochromicity, feature
of intercalation is frequently assigned to the interaction between the planar aromatic

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chromospheres of molecule and base pair of DNA [43]. These results revealed that the
complex can bind to DNA via an intercalation mode of the double helix DNA. The
percentage of hypochromism of the complexes were found to be 24 ‒37% with red shift of 1-
4 nm.
In order to determine significantly the binding potency of these complexes, the intrinsic
binding constant (Kb) of the metal complexes (1a–1c) with DNA was determined by equation
(1). The Kb values are found to be 5.6 × 105M−1, 4.48 × 105 M−1 and 3.79 × 105 M-1,
respectively, revealing greater binding tendency of complex 1a as compared to 1b and 1c.
From the above result it is clear that, the Cu (II) complex show strong binding affinity to
DNA than Co (II) and Ni (II) complexes. The binding efficiency of metal chelates toward
DNA depends on the coordination number and nature of metal atom in the complex.
<Insert Figure 5>
4.2.DNA cleavage studies
The investigation on the cleaving affinity of metal complexes to DNA is very fascinating
because it can furnish to understanding the toxicity mechanism of them and to develop novel
artificial nuclease. The DNA cleavage study of the synthesized metal complexes was
performed by agarose gel electrophoretic technique using pBR322 plasmid DNA as a
substrate. When circular plasmid DNA is subjected to electrophoresis, the relatively fastest
journey will be obtained for the supercoiled form (Form I). If one strand slashed, the
supercoil will unwind to produce a slowest moving nicked form (Form II). If both strands are
cleaved, a linear form (Form III) will be produced and migrates in between Form I and Form
II [44]. The plasmid pBR322 plasmid DNA (100 ng) were mixed with a fixed concentration
of metal complexes. M – is standard DNA molecular weight marker, Lane 1 applies to the
untreated pBR322 plasmid DNA (control DNA) which was initially found to be mixture of
mainly of super coiled form I and a small amount of singly-nicked form II [45] as shown in
Fig.6. The plasmid DNA interaction with the complexes leads to one strands break of the
DNA, and the circular form (II) is observed. Lane 2 (1a) has partially cleaved both the form I
and form II DNA, lane 3 and 4 (1b & 1c) were effectively cleaved supercoiled DNA (form I)
to nicked DNA (form II). Thus, the complexes show DNA nuclease activity in the absence of
any external agent. Moreover, the quantity of helical unwinding caused by the complex
bound to supercoiled offers proof for the intercalation mode of interaction between the
compounds and DNA [46]. Thus, we can draw a conclusion that the newly synthesized metal

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complexes under the present study are good inhibitor for the growth of the pathogenic
microorganism by cleaving the plasmid DNA.
<Insert Figure 6>
4.3.In-vitro cytotoxic activity by MTT assay
The cytotoxic activity of the synthesized azo-azomithine ligand and its metal
complexes on the proliferation of human lung cancer cells (A-549), breast cancer cells
(MDA-MB 231) and normal embryonic kidney cells (HEK293) were examined by MTT
assay. Cisplatin was taken as a reference drug and the cells were treated with various
concentrations of metal complexes (1, 2, 5, 10, 25, 50 and 100 ꢀg mL-1) for 24 h. The dose
dependent half-inhibitory effects of ligand and its complexes on cell viability of different
human cancer cell lines showed in Fig. 7-9. From the figures, it is clear that on increment in
the concentrations of compounds exhibited diminish in cell viability deducing enhanced
cytotoxicity. The IC50 values obtained for the compounds against the screened cell lines are
offered in Table 5, and were compared with cisplatin. All the complexes exhibited strong
potent cytotoxicity against A-549 and MDMB-231 cancer cells, IC50 value ranged from 4.759
-
41.25 and 5.836–58.51 ꢀg/ ml. While all the metal complexes are less toxic toward the
normal cell as it evident from the higher IC50 value 25.17–79.52 ꢀg/ml, respectively.
Evidently, the azo-azomethine ligand exhibited less inhibition on the cancer cells, but the
complexes showed good cytotoxic activities relative to cisplatin. The higher activity of metal
complex may be due to the increase in their lipophibility on chelation. Moreover, the activity
of the metal complex depends on the nature of central metal ion present in the metal
complexes. In the present work, the Cu (II) complex showed good activity when compared to
the remaining complexes against all the screened cell lines.
<Insert Table 6>
<Insert Figure 7, 8 and 9 >
3. Conclusion
The azo azo-methine ligand HL and their Cu(II), Co(II) and Ni(II) complexes were
synthesized and structurally characterized by various analytical and spectroscopic techniques.
The analytical data reveal that the metal complexes have 1:2 metal to ligand ratio. The molar
conductance data indicate that the complexes are non-electrolytic in nature. The electronic
spectra and magnetic moment measurement indicated that all the complexes having an

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octahedral geometry. The synthesized metal complexes showed different surface
morphological structure as appeared in SEM and powder XRD, TGA spectra of metal
complexes showed the existence of lattice water molecule and gave good result in thermal
stability. Moreover, the interaction of metal complexes with CT-DNA have been effectively
investigated by electronic absorption spectroscopy, the result revealed that the metal
complexes bind to CT-DNA through an intercalative mode. The DNA cleavage activity
revealed that all the metal complexes effectively cleaved supercoiled pBR322 DNA without
any external agent. Moreover, the in-vitro cytotoxicity of ligand and its metal complexes
were tested on A-549, MDA-MB 231 and HEK293 cell lines. All compounds exhibit good
potent cytotoxic agents that might become potent anticancer agent in clinical research.
Acknowledgment
We express our sincere thanks to the Chairman, Department of Industrial Chemistry,
and Kuvempu University for providing laboratory facilities. Authors are also grateful to IISC
Banglore, MIT Manipal, STIC Cochin and SAIF IIT Bombay for providing spectral data. We
are thankful to the SDM Centre for Research in Ayurveda and Allied Sciences, Udupi for
providing anticancer activity and Biogenic Hubli for providing DNA cleavage studies.
References
1. C. Rajarajeswari, R. Loganathan, M. Palaniandavar, E. Suresh, A. Riyasdeen, M. A.
Akbarsha, Dalton Trans 42 (2013) 8347-8363.
2. Cancer Facts & Figures 2015, Atlanta, American Cancer Society, (2015).
3. T. C. Johnstone, K. Suntharalingam, S. J. Lippard, Chem. Rev.116 (2016) 3436-3486.
4. S. Banerjee, A.R. Chakravarty, Acc. Chem. Res. 48 (2015) 2075–2083.
5. B. Rosenberg, L.V. Camp, T. Krigas, Nature 205 (1965) 698–699.
6. G. Barone, A. Terenzi, A. Lauria, A. M. Almerico, J. M. Leal, N. Busto, B. García, C.
Chem. Rev 257 (2013) 2848-2862.
7. VM. Manikandamathavan, T. Weyhermüller, RP. Parameswari, M. Sathishkumar, V.
Subramanian, Nair BU. Dalton Trans. 43 (2014) 13018-13031.
8. R. Palchaudhri, P.J. Hergenrother. Curr. Opin. Biotechnol. 18 (2007) 497–503.
9. H. Abd El-Wahab, M Abd El-Fattah, A.H Ahmed, A.A Elhenawy, N.A Alian, Journal
of Organometallic Chemistry (2015), doi: 10.1016/j.jorganchem.2015.05.024.
10. Yunpeng Du, Wei Chen, Xiang Fu, Hang Deng, JunGang Deng. RSC Adv. 6 (2016)
109718-109725.

ACCEPTED MANUSCRIPT
11. G.L. Backes, D.M. Neumann, B.S. Jursic. Bioorg. Med.Chem. 22 (2014) 4629-4636.
12. R.N. Singh, Amit Kumar, R.K. Tiwari, Poonam Rawat, Divya Verma, Vikas Baboo.
J. Mol. Struct. 1016 (2012) 97-108.
13. Nives Galic, Zvjezdana Cimerman, Vladislav Tomisic. Spectrochim. Acta A 71
(2008) 1274–1280.
14. Prateek Tyagi, Monika Tyagi, Swati Agrawal, Sulekh Chandra, Himanshu Ojha,
Mallika Pathak. Spectrochim. Acta A 171 (2017) 246–257.
15. L.H. Abdel-Rahman, A.M. Abu-Dief, R.M. El-Khatib, S.M. Abdel-Fatah, J.
Photochem. Photobiol. B 162 (2016) 298–308.
16. S. Budagumpi, N. V. Kulkarni, G. S. Kurdekar, M. P. Sathisha, V. K. Revankar,
European J. Med. Chem., 45 (2010) 455-462.
17. S. Daravath, M.P. Kumar, A. Rambabu, N. Vamsikrishna, G. Nirmala, Shivaraj. J.
Mol. Struct. 1144 (2017) 147-158.
18. P.R. Reddy, A. Shilpa, N. Raju, P. Raghavaiah, J. Inorg. Biochem. 105 (2011) 1603.
19. Hamid Khanmohammadi, Malihe Erfantalab, Spectrochim. Acta Part A 86 (2012) 39–
43.
20. B. Sreekanth, G. Krishnamurthy, H. S. Bhojya Naik, T. K.Vishnuvardhan,
Nucleosides, Nucleotides and Nucleic Acids. 31 (2012) 1-13.
21. S. Dhar, D. Senapati, P.K. Das, P. Chattopadhyay, M. Nethaji, A.R. Chakravarty, J.
Am. Chem. Soc. 125 (2003) 12118–12124.
22. T.R. Mosmann, J. Immunol. Methods 65 (1983) 55-63.
23. S. K. N, G. Krishnamurthy, Y. D. Bodke, V. H. Malojirao, R. N. TR, S. Kandagalla
and P. B T, New J. Chem., 2018, DOI: 10.1039/C8NJ03057J
.
24. W.J. Geary, Coord. Chem. Rev. 7 (1971) 81.
25. Abdel-Nasser M.A. Alaghaz , Mohamed E. Zayed, Suliman A. Alharbi, Reda A.A.
Ammar, Ahmed Elhenawy. J. Mol. Struct. 1084 (2015) 352–367.
26. Gokhan Yeginer, Mehmet Gulcan, Sema Isik, Gulsiye Ozturk Urut, Sadin Ozdemir
Mukerrem Kurtoglu. J Fluoresc 27 (2017) 2239–2251.
27. Ganga naik Krishnamurthy and Narashimaiah Shashikala. J. Serb. Chem. Soc. 74 (10)
1085–1096 (2009).
28. Munire Sarigul, Aysel Sari, Muhammet Kose, Vickie McKee, Mahfuz Elmastas,
Ibrahim Demirtas,Mukerrem Kurtoglu. Inorganica Chimica Acta 444 (2016) 166–
175.

ACCEPTED MANUSCRIPT
29. A.Z. El-Sonbati, M.A. Diab, A.A. El-Bindary, M.I. Abou-Dobara, H.A. Seyam
Journal of Molecular Liquids 218 (2016) 434–456.
30. A.M.A. Alaghaz, B.A. El-Sayed, A.A. El-Henawy, R.A.A. Ammar, Mol. Struct. 1035
(2013) 83–93.
31. Sirin Bitmez , Koray Sayin, Baris Avar, Muhammet Kose, Ahmet Kayraldız,
Mukerrem Kurtoglu. J. Mol. Struct. 1076 (2014) 213–226
32. Nakamoto K (1997) Infrared and Raman spectra of inorganic and coordination
compounds. Wiley, New York.
33. S.M. Pradeepa, H.S. Bhojya Naik, B. Vinay Kumar, K. Indira Priyadarsini, Atanu
Barik, M.C. Prabhakara. Spectrochim. Acta Part A 141 (2015) 34–42.
34. E. Ispir, Dyes Pigments 82 (2009) 13–19.
35. Lever ABP (1984) Inorganic electronic spectroscopy. Elsevier, Amsterdam
36. T. Manjuraj, G. Krishnamurthy, Yadav D. Bodke, H.S. Bhojya Naik, H.S. Anil
Kumar. J. Mol. Struct. 1171 (2018) 481-487.
37. D. N Sathyanarayana, electronic absorption spectroscopy and related technique
University Press (India), Hyderabad 2001.
38. P.P. Dholakiya, M.N. Patel, Syn. React. Inorg. Met. Org. Nano-Met. Chem. 32 (2002)
819–829.
39. Mohamed Gaber, Yusif S. El-Sayed, Kamal El-Baradie, M. Rowaida, Fahmy. J. Mol.
Struct. 1032 (2013) 185–194.
40. A.M.F. Benial, V. Ramakrishnan, R. Murugesan, Spectrochim. Acta Part A. 56
(2000) 2775–2781.
41. B. J. Hathaway and D. E. Billing. Coordin. Chem. Rev, 5 (1970) 143-207
42. Sreenu Daravath, Marri Pradeep Kumar, Aveli Rambabu, Narendrula Vamsikrishna,
Nirmala Ganji, Shivaraj. J. Mol. Struct. 1144 (2017) 147-158.
43. P.R. Reddy, S. Rajeshwar, B. Satyanarayana, J. Photochem. Photobiol. B 160 (2016)
217–224.
44. Nirmala Ganji, Vijay Kumar Chityala, Pradeep Kumar Marri, Rambabu Aveli,
Vamsikrishna Narendrula, Sreenu Daravath, Shivaraj, J. Photochem. Photobiol. B.
175 (2017) 132–140.
45. N. Divya Hegde, S. Ganesh Naik, Ramesh Vadavi, V. Shyam Kumar, A. Delicia
Barretto, B. Kalagouda Gudasi. Inorganica Chimica Acta 461 (2017) 301–315.
46. D.J. Che, G. Li, X.L. Yao, Y. Zhu, D.P. Zhou, J. Chem. Soc., Dalton Trans. (1999)
2683–2687.

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Manuscript Id: Our reference: MOLSTR 26394
Article reference: MOLSTR_MOLSTRUC-D-19-00214
: “Novel bioactive azo-azomethine based Cu (II), Co (II) and Ni(II)
complexes, structural determination and biological activity”
Title
As requested by the respective editorial team, the following corrections have been made and i
request you to incorporate the changes given below. The corrections made were highlighted
by the red colour font.
Table 4: Thermal decomposition of metal complexes.
Complex
Stage Decomposition
temp (0C)
Probable
assignment
Loss of mass
in (%)
Residual
species
Obsd Cald
6.06 6.23
9.18 9.30
Cu[C40H38N10O9]
1st
2nd
3rd
4th
30–78 °C
79–250 °C
251–370 °C
371-697 °C
3H2O
C4O2
C10H10N2 17.96 18.26
C24H28N8O3 54.72 55.02
CuO+2corbon
CoO
2H2O
C12H10O2N2
C28H24N8O3
1st
2nd
3rd
26–75 °C
75–315 °C
315–495°C
4.26 4.35
24.29 24.48
55.88 56.46
Co[C40H36N10O8]
Ni[C40H36N10O8]
2H2O
C8H4N2
C4H2N2O2
C28H28N8O3
1st
28-65 °C
65-175 °C
175-378 °C
378-510 °C
4.25 4.46
15.56 15.71
13.46 13.03
57.10 58.29
2nd
3rd
4th
NiO
Table 5: Powder X-ray data of Cu (II) complex.
Peak
2θ
θ
sinθ
sin2θ
1000 sin2θ
h2+k2+l2
hkl
1 0 0
1 1 0
1 1 1
2 0 0
2 1 1
3 1 0
-
d
a in
Å
8.052
obs
calc
1
2
3
4
5
6
7
10.98
17.05
18.28
5.49
8.50
9.14
0.0956
0.1478
0.1588
0.1949
0.229
0.0091
0.0218
0.0252
0.0379
0.0524
0.0899
0.1389
9.153
21.86
25.23
37.99
52.49
89.97
138.93
1(1)
8.0575 8.387
5.2117 5.199 8.061
4.8507 4.834 8.099
3.9521 3.922 8.089
3.3637 3.352 8.057
2.5762 2.564 8.053
2.0706 1.988 8.048
2.3882(2)
2.7564(3)
4.1505(4)
5.7347(6)
9.8295(10)
15.178(15)
22.48 11.24
26.49
34.91
43.77
13.24
17.45
21.88
0.229
0.372

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Fig. 1 ESR spectrum of complex 1a

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Fig. 2 TG curves of metal complexes (1a-1c)

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2000
1500
1000
Co
500
3720
Ni
2790
1860
930
3080
2310
1540
770
Cu
0
20
40
60
80
2θ
(degree)
Fig. 3 Powder X-ray diffraction pattern of metal complexes (1a-1c)

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Fig.4 SEM images of ligands and its metal complexes

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Fig.5 Electronic spectra of the complexes in Tris-HCl buffer upon addition of CT-DNA.
Arrow shows the absorption intensities decrease in complex (1a-1c).

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Fig. 6 Cleavage of supercoiled pBR322 DNA (2 µL, 2µg) at 37 0C in 5 mM Tris HCl/5 mM
NaCl buffer by the metal complexes, M:. Marker, Lane 1: DNA control (pBR322), lane 2:
DNA+1a complex, lane 3: DNA+1b complex, lane 4: DNA+1c complex.
Fig. 7 Cytotoxicity effect of ligand and its complexes (1a-2c) on A549 cells assessed by
MTT assay

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Fig. 8 Cytotoxicity effect of ligand and its complexes on MDA-MB-231 cells assessed by
MTT assay
Fig. 9 Cytotoxicity effect of ligand and its complexes on normal Hek-293 cells assessed by
MTT assay.

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Highlights
ꢀ Synthesis and characterization of novel azo-azomethine ligand and its metal
complexes.
ꢀ The ESR spectra of Cu (II) complexes confirm an octahedral geometry.
ꢀ
ꢀ
Metal complexes act as good DNA intercalating agents and also cleave DNA efficiently.
All metal complexes have showed good cytotoxic activity towards tested cell lines.