Fabrication, spectroscopic characterization, calf thymus DNA binding investigation, antioxidant and anticancer activities of some antibiotic azomethine Cu(II), Pd(II), Zn(II) and Cr(III) complexes

Article abstract of DOI:10.1002/aoc.4943

This study was conducted to prepare novel azomethine chelates of Cu(II), Pd(II), Zn(II) and Cr(III) with tridentate dianionic azomethine OVAP ligand 2-[(2-hydroxyphenylimino)methyl]-6-methoxyphenol. The prepared compounds were characterized using elementa

Full text of DOI:10.1002/aoc.4943

Received: 27 January 2019
DOI: 10.1002/aoc.4943
Revised: 19 March 2019
Accepted: 19 March 2019
F U L L P A P E R
Fabrication, spectroscopic characterization, calf thymus
DNA binding investigation, antioxidant and anticancer
activities of some antibiotic azomethine Cu(II), Pd(II),
Zn(II) and Cr(III) complexes
1
1
2
Ahmed M. Abu‐Dief
| Hussein M. El‐Sagher | Mohamed R. Shehata
1
Chemistry Department, Faculty of
This study was conducted to prepare novel azomethine chelates of Cu(II),
Pd(II), Zn(II) and Cr(III) with tridentate dianionic azomethine OVAP ligand
Science, Sohag University, 82524, Egypt
2
Chemistry Department, Faculty of
Science, Cairo University, Giza, Egypt
2
‐[(2‐hydroxyphenylimino)methyl]‐6‐methoxyphenol. The prepared com-
pounds were characterized using elemental analyses and spectral, conductivity,
magnetic and thermal measurements. The spectroscopic data suggest that the
parent azomethine ligand binds to the investigated metal ions through both
deprotonated phenol oxygen and azomethine nitrogen atoms, and adopts
distorted octahedral geometry in the case of Cr(III) and Cu(II) ions while tetra-
hedral and square planar geometries for Zn(II) and Pd(II) ions, respectively. In
order to confirm the molecular geometry of the investigated azomethine chela-
tor and its complexes, theoretical density functional theory calculations were
employed. Correlation between experimental observations and theoretical cal-
culations of geometry optimization results are in a good agreement. Absorption
titration was used to explore the interaction of the investigated azomethine
metal chelates with calf thymus DNA, and the binding constant as well as
Gibbs free energy were evaluated. Viscosity measurements and gel electropho-
resis studies suggest intercalative and replacement binding modes of the
azomethine metal chelates with calf thymus DNA. Additionally, the antimicro-
bial activity of the complexes was screened against some pathogenic bacteria
and fungi. This biological study shows that the complexes exhibit a marked
inhibitory effect compared to the corresponding ligand and standard drugs.
Furthermore, the effect of the novel compounds as antioxidants was deter-
mined by reduction of 1,1‐diphenyl‐2‐picrylhydrazyl and compared with that
of vitamin C. Finally, in vitro cell proliferation via MTT assay was investigated
against colon carcinoma cells (HCT‐116), hepatic cellular carcinoma cells
Correspondence
Ahmed M. Abu‐Dief, Chemistry
Department, Faculty of Science, Sohag
University, 82524, Egypt.
Email: ahmed_benzoic@yahoo.com
(HepG‐2(and breast carcinoma cells (MCF‐7) to calculate the cytotoxicity of
the prepared compounds. Cell proliferation is inhibited for all compounds
and in a dose‐dependent manner in the sequence of OVAPPd > OVAPCu >
OVAPZn > OVAPCr > OVAP azomethine ligand.
Appl Organometal Chem. 2019;e4943.
wileyonlinelibrary.com/journal/aoc
© 2019 John Wiley & Sons, Ltd.
1 of 23
https://doi.org/10.1002/aoc.4943

2
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ABU‐DIEF ET AL.
KEYWORDS
anticancer, antioxidant, azomethine complexes, calf thymus DNA interaction, DFT calculations, In
vitro antimicrobial activity
1
| INTRODUCTION
chromium(III) nitrate (Cr(NO ) ⋅6H O), zinc(II) nitrate
3
3
2
(
Zn(NO ) ⋅4H O), copper chloride (CuCl ⋅4H O) and pal-
3
2
2
2
2
There has been a considerable increase in the use of
metal chelates for cancer treatment after the accidental
discovery of the biological activity of platinum complex
ladium acetate (Pd(OAC) ) were obtained from Sigma‐
Aldrich.
2
Ethanol, N,N′‐dimethylformamide (DMF), dimethyl-
sulfoxide (DMSO) and HCl were used without distillation.
The important reagents for DNA interaction studies,
CT‐DNA, tris(hydroxymethyl)aminomethane (Tris) and
EDTA, were obtained from Sigma‐Aldrich. CT‐DNA was
dissolved in Tris–HCl buffer (pH 7.2). Tris–HCl buffer
solution was prepared using deionized water and was
used to control the pH of reaction systems.
[1]
cisplatin in 1965 by Rosenburg. The organic molecules
are designed to combine biological functional groups
with a suitable metal ion to mimic active sites of
[2]
metalloproteins. In recent years, many investigations
have been concerned with metal–drug adducts showing
promising biological activity and are of great interest in
biology
and
chemistry.
Metals
like
zinc(II),
chromium(III) and copper(II) have great affinity for coor-
dination because of their small size and high nuclear
charge. The development of metal–drug adducts as che-
motherapeutic agents basically depends on the ability of
Agarose was purchased from Fischer Biotech (GE
Heath Care). Tracking dye (0.20% bromophenol blue,
35% sucrose, 200 mM EDTA and0.20% xylene cyanole)
and Ready‐load 100 BP DNA ladder were used as the
native size DNA and purchased from Bio Labs.
The chemical used for antioxidant activity study, 2,2‐
diphenyl‐1‐ picrylhydrazyl (DPPH), was purchased from
Merck.
To evaluate the antimicrobial activity, bacterial and
fungal strains (Micrococcus luteus (+ve), Serratia
marcescence (−ve), Escherichia coli, Pseudomonas aureus,
Aspergillus flavus, Geotrichum candidum and Fusarium
oxysporum) were used. These strains were cultured in
nutrient agar and Muller‐Hinton medium. Ofloxacin and
fluconazole were used as standard drugs for comparison.
[3–12]
the metallodrug to provoke DNA cleavage.
It is
worth mentioning that metal chelate pharmacological
efficacy mainly depends on the nature of metal ions and
ligands. The literature suggests that ligands with different
[13–21]
metal ions possess different biological properties.
Our aim was to undertake a systematic study of
the preparation, spectral characterization, DNA binding
and biological activities of new transition metal
complexes involving the OVAP azomethine ligand 2‐[(2‐
hydroxyphenylimino)methyl]‐6‐methoxyphenol.
The
structure of the studied complexes was elucidated using
elemental analyses, Fourier transform infrared (FT‐IR),
1
13
H NMR, C NMR and UV–visible spectroscopies, mag-
netic moment and molar conductance measurements
and thermal analysis and confirmed by density functional
theory (DFT) calculations. Moreover, the antimicrobial,
antioxidant and cytotoxic effects of the investigated com-
pounds were screened. Furthermore, electronic absorp-
tion, viscosity and gel electrophoresis measurements
were used to study the ability of the investigated com-
plexes to bind to calf thymus DNA (CT‐DNA).
2.2 | Instrumentation and methods
NMR spectra of the prepared azomethine ligand was
measured in DMSO‐d and recorded at the Central Labo-
6
ratory, Department of Chemistry, Faculty of Science,
Sohag University at 298 K with a FT‐NMR spectrometer
1
(Bruker ARX400) at 400.1 MHz ( H) and 100.6 MHz
13
( C). Elemental analyses (C, H, N) were performed with
a PerkinElmer 2408 CHN analyser at the Micro‐analytical
Center, Cairo University. Room temperature FT‐IR spec-
tra of the prepared azomethine ligand and its complexes
were recorded as KBr pellets with a Shimadzu model
8101 FT‐IR spectrophotometer in the range 400–
2
2
| EXPERIMENTAL
.1 | Reagents
−
1
4
000 cm at the Department of Chemistry, Faculty of
All the chemicals and solvents used in this study were of
reagent grade and used as received without additional
purification processes. Organic compounds 3‐methoxthy-
salicyaldehyde and 2‐aminophenol and metal salts
Science, Sohag University. Electronic spectra were
obtained with a Jasco P‐530 UV–visible spectrophotome-
ter in DMF at 298 K. Magnetic susceptibilities of the
investigated metal azomethine chelates were measured

ABU‐DIEF ET AL.
3 of 23
1
3
utilizing the modified Gouy method at 298 K using a
magnetic susceptibility balance (Johnson Matthey). An
Elico digital pH meter (model LI‐127) equipped with a
CL‐51B combined electrode was used for pH measure-
ments and calibrated against standard buffers (pH 4.02
and 9.18) before measurements. Thermogravimetric anal-
ysis (TGA) was conducted using a Shimadzu 60H
(3H, CH3 of methoxy group), 3.84 (s, 2H, H O).
C
2
NMR (δ, ppm) of OVAPZn complex in DMSO‐d : 162,
152.5, 151, 149, 135, 128.5, 124, 120, 119, 118, 117, 116,
6
115.5, 56.4.
2.5 | Determination of stoichiometry
−
1
analyser under air at a heating rate of 10°C min from
ambient temperature to 750°C at the Department of
Chemistry, Faculty of Science, Thermal Analysis Unit,
Assiut University, Egypt. DFT calculations were carried
out to investigate the equilibrium geometry of the ligand
Job's method (continuous variation method)[22–26] was
used to determine the stoichiometry of the investigated
complexes. A mixture of metal salt and ligand solution
was mixed, stirred and left to equilibrate, and the absor-
bance measured at λmax up to 2 h. The absorbance of each
solution was then plotted against the mole fraction of
metal ion or mole fraction of ligand.
[2]
and complexes using the Gaussian 09 program at the
B3LYP/LANL2DZ level of theory for the ligand and
complexes.
2
.6 | Kinetic parameters for TGA of the
2
.3 | Synthesis of OVAP azomethine
prepared complexes
ligand
The decomposition and thermal dehydration of the metal
chelates were studied kinetically using the integral
method applying the Coats–Redfern method. Moreover,
the thermodynamic activation parameters of degradation
steps of dehydrated complexes, namely energy of activa-
tion (E), frequency factor (A), enthalpy of activation
The OVAP azomethine ligand was synthesized by a con-
ventional method according to the following procedure.
A
hot ethanolic solution (10 ml) of 3‐
methoxthysalicyaldehyde (10 mmol) was added dropwise
to another hot ethanolic solution (10 ml) of 2‐
aminophenol (10 mmol). After completion of addition, a
yellow‐coloured solid formed on vigorous stirring of the
solution. The reaction mixture was refluxed in a water
bath for 1 h and then allowed to cool. The completion
(ΔH), entropy of activation (ΔS) and free energy change
of the decomposition (ΔG), were evaluated from TGA
[6–
data graphically utilizing the Coats–Redfern relation
10,27–30]
in the following form:
of the reaction was monitored by TLC.
1
ꢀ
ꢁ
H NMR (δ, ppm), in DMSO‐d :14.01 (1H, OH of phe-
6
logðW∞=ðW∞ − WÞÞ
log
nyl ring), 9.72(s, 1H, OH of methoxy phenyl ring), 8.96 (s,
2
T
ꢀ
ꢂ
ꢃꢁ
1
H, CH═N), 7.38–6.98(m, 7H, aromatic), 2.50 (3H, CH of
E*
3
AR
φE*
2RT
E*
13
¼
log
1 −
−
(1)
methoxy group). C NMR (δ, ppm), in DMSO‐d : 162,
6
2:303RT
1
52, 151, 149, 135, 128, 124, 120, 119, 118, 117, 116, 115,
5
6.
where W∞ is the mass loss at the completion of the
decomposition reaction, W is the cluster loss up to tem-
perature T, R is the general gas constant and ϕ is the
2
.4 | Synthesis of OVAP azomethine
*
complexes
heating rate. Since 1 − 2RT/E ≈ 1, a plot of the left‐hand
side of equation (1) against 1/T would give a straight line.
E was calculated from the slope and the Arrhenius con-
*
To a hot stirred solution of OVAP azomethine ligand
(10 mmol) in ethanol (20 ml) was added a solution of
stant, A, was determined from the graph intercept. The
metal(II) salt (10 mmol) made in warm ethanol (10 ml)
and the resultant mixture was refluxed for 2 h. The solid
formed during refluxing or upon cooling was collected by
suction filtration. Thorough washing with hot ethanol
followed by ether or recrystallization from aqueous etha-
nol gave purified products.
other kinetic parameters, namely the entropy of activa-
*
*
tion (ΔS ), enthalpy of activation (ΔH ) and free energy
*
change of activation (ΔG ), were calculated using the fol-
lowing equations:
Ah
*
ΔS ¼ 2:303R log
(2)
(3)
(4)
K T
B
1
H NMR (δ, ppm) of OVAPPd complex in DMSO‐d6:
8
.87 (s, 1H, CH═N), 7.99–6.61(m, 7H, aromatic), 2.51
*
*
ΔH ¼ E − RT
(
3H, CH of methoxy group), 3.79 (s, 2H, H O).
H NMR (δ, ppm) of OVAPZn complex in DMSO‐d6:
.96 (s, 1H, CH═N), 7.39–6.8 (m, 7H, aromatic), 2.51
3
2
1
*
*
*
8
ΔG ¼ ΔH − TΔS

4
of 23
ABU‐DIEF ET AL.
where K and h are the Boltzmann and Planck constants,
respectively.
η0 is that of DNA alone. The values of viscosity were cal-
culated from the flow time of DNA‐containing solutions
B
(t) with subtraction of that of only buffer solution
(
5 mM Tris–HCl/50 mM NaCl) (t ): η = (t − t ).
The cleavage efficiency of prepared azomethine metal
0
0
2
.7 | Investigation of DNA interaction
with azomethine metal chealates
complexes with calf thymus DNA was examined by the
gel electrophoresis method. Hot Tris–acetate–EDTA
All DNA binding experiments were done in 10 mM Tris–
HCl buffer (pH = 7.4) containing 50 mM NaCl. A solu-
tion of CT‐DNA gave a ratio of UV absorbance at 245
and 262 nm of greater than 1.8, indicating that the DNA
(
TAE) buffer (50 ml) (4.84 g of Tris base, pH = 8.0,
0.5 M EDTA) was used to dissolve agarose (500 mg) then
heated until boiling for a few minutes. When the gel
reached approximately 55°C, it was poured into a gas cas-
sette fitted with comb. After that, the gel was left to solid-
ify slowly by cooling to room temperature and then the
comb was removed carefully. Then gel in a solid form
was placed in the electrophoresis chamber including
TAE buffer. The prepared metal chelate samples were
subjected to electrophoresis on 2% agarose gel prepared
in TBE buffer (45 mM boric acid, 45 mM Tris and
[15–17,31,32]
was adequately free from protein.
absorption spectroscopic experiments were executed at
5°C, maintaining the concentration of each metal che-
Molecular
2
late constant (10.0 μM) while altering the concentration
of DNA from 0 to 15.0 μM. After each addition of DNA
to the metal complex, the resulting solution was allowed
to equilibrate at 25°C for 10 min, after which the change
in absorbance of the ligand‐to‐metal charge transfer band
was recorded. While measuring the absorption spectra,
an appropriate amount of CT‐DNA in Tris–HCl buffer
solution (pH = 7.4) was added to both metal chelate solu-
tion and the blank reference to eliminate the absorption
of CT‐DNA itself. The intrinsic binding constant (Kb)
was determined by way of plotting [DNA]/(ε − ε ) versus
1
mM EDTA, pH 7.1). Then 20 μl of each of the incubated
metal chelates and 20 μl of DNA were used. The resulting
mixture was incubated for 25 min at 37°C. After that, it
was loaded with tracking dye (0.30% bromophenol blue)
on the prepared gel. The electric current was turned off
at the end of migration of DNA. Ethidium bromide solu-
a
f
−1
[
29,32,33]
tion in water (0.5 μg ml ) was used to stain the gel for
[
DNA] according to the following expression:
40 min at room temperature and later the gel was visual-
ized under UV light utilizing a transilluminator. The illu-
minated gel was photographed using a Panasonic digital
camera.
½
DNAꢀ
½DNAꢀ
ε − ε
1
À
Á ¼ À
Á þ Â
À
Á
(5)
ε − ε
K ε − ε ꢀ
a
f
b
f
b
b
f
where [DNA] is the concentration of DNA in base pairs
and ε , ε and ε are the apparent, free and completely
bound metal chelate eradication coefficients, respectively.
a
f
b
2.8 | Antimicrobial assessment
Specifically, ε was assessed from the calibration curve of
the separated metal chelate; confirmation of Beer's law.
εa was assessed as the proportion among the determined
f
The antimicrobial activity of the free ligand and its corre-
sponding metal complexes were primarily screened
against pathogenic bacteria and fungi that are common
contaminants of the environment in Sohag, Egypt, and
some of which are involved in human and animal dis-
eases, such as Aspergillus flavus, Geotrichum candidum
and Fusarium oxysporum, or frequently reported from
contaminated soil, water and food substances, e.g. Serratia
marcescence, Escherichia coli and Micrococcus luteus. In
vitro biological activities of OVAP azomethine ligand and
its metal chelates in DMSO medium were investigated
for their microbial inhibition using standard agar as the
absorbance and the M(II) chelate concentration, Aobs
metal chelate). The information became reasonable for
the above equation with a slope equivalent to 1/(ε − ε )
/
(
b
f
and y‐intercept equivalent to 1/(K (ε − ε )); Kb was
b
b
f
obtained from the ratio of slope to intercept. The standard
Gibbs free energy for DNA binding was assessed from the
following expression:
≠
b
ΔG ¼ −RT ln Kb
(6)
[
29,32–34]
Hydrodynamic measurements (viscosity) were executed
utilizing an Ostwald viscometer at room temperature
using 20 μM CT‐DNA, complex concentrations of 0 to
medium using the a well diffusion method.
Wells
were set up in cooled agar plates (1 cm). An amount of
10 mg of each compound was dissolved in 3 ml of DMSO
and 100 μl was loaded in a well. The activity or sensitivity
was observed after 24–48 h incubation at 37°C. The mini-
mum inhibitory concentration (MIC) ranges were deter-
mined. Zone of inhibition of the investigated metal
complexes was compared with that of available standard
1
00 μM and measuring the flow times with a digital
timer. Each sample was measured three times for accu-
racy, and an average flow time was calculated. Data were
1/3
presented as (η/η0) versus [complex]/[DNA], where η is
the viscosity of DNA in the presence of metal chelate and

ABU‐DIEF ET AL.
5 of 23
[
33,35]
drugs.
The MIC is the smallest concentration of com-
2.10.1 | Cytotoxicity by MTT assay
pound that inhibits the growth of microorganisms.
The viability of cancer cells against the synthesized com-
pounds was examined using MTT assay as previously
[
36,37]
described.
Colon carcinoma cells (HCT‐116), hepatic
2
.9 | Antioxidant assay (DPPH free radical
cellular carcinoma cells (HepG‐2) and breast carcinoma
cells (MCF‐7) were seeded in 96‐well plates at a density
of 5 × 10 cells per well. After 24 h they were exposed
scavenging activity)
3
In vitro antioxidant activity of the newly synthesized
OVAP azomethine ligand and its metal complexes was
evaluated using a modified stable DPPH radical scaveng-
to different concentrations of compounds (ranging
−
1
between 0 and 10 μg ml ) for 48 h. After the treatment,
the medium containing the compounds was removed and
[32]
ing method.
The stable DPPH radical scavenging
−
1
1
0 μl of MTT (5 mg ml in phosphate‐buffered saline)
method is used to determine antioxidant activities of the
prepared compounds in a relatively short time compared
with other methods. The change of absorption, easily
observed as a colour change from purple to yellow, of
the radical when deactivated by antioxidant is a reliable
test in the DPPH radical scavenging model. Methanol–
DMSO (4:1) stock solutions of the investigated com-
pounds were prepared then diluted to final concentra-
tions of 10, 25, 50, 100 and 150 M. A (1 ml, 0.3 mmol)
methanolic solution of DPPH was added to 3.0 ml of
the synthesized compounds as well as standard com-
pound (ascorbic acid). Each tube was kept in the dark
for 30 min and the absorbance was measured in triplicate
at 517 nm using methanol as a blank. The positive control
was vitamin C. The reduction in the absorbance of DPPH
was calculated relative to the measured absorbance of the
control. The DPPH radical scavenging percentage of
activity was calculated using the following equation:
was added to each well and incubated at 37°C for 3 h.
Then, 100 μl of DMSO was added to dissolve the purple
crystals. The optical density values of wells were mea-
sured at 570 nm. Each concentration was tested three
times and five replica wells were used for controls and
5‐fluorouracil was used as standard drug. With the con-
trol wells representing 100% viability, the absorption
values of each well were expressed as the percentage cell
viability. C50 values were calculated from the dose–
response curves using Origin software, representing the
concentration of test compound to inhibit cell viability
by 50%. Every concentration for each compound was
tested in triplicate and mean values ± SD were recorded.
3 | RESULTS AND DISCUSSION
3.1 | Identification of prepared
compounds
A − A
C
S
DPPH scavenging activity ð%Þ ¼
(7)
The obtained solid complexes are coloured and quite sta-
ble in air and moisture (Scheme 1). The complexes were
found to have ligand‐to‐metal ratio of 1:1. Table 1 presents
all the analytical and physical data of the OVAP
azomethine ligand and its metal chelates. Good agreement
between the elemental analyses with the chemical formu-
lae of the proposed structures of the prepared compounds
was observed. FT‐IR, H NMR, C NMR and mass spectra
and microanalyses were used to elucidate the structure of
OVAP azomethine ligand and its metal chelates formed as
well as the coordination mode of the ligand.
AC
where A is the absorbance of vitamin C (standard) and
A is the absorbance of sample under investigation. The
effective concentration of sample required to scavenge
DPPH radicals by 50% (IC50 value) was obtained by lin-
ear regression analysis of dose–response curve plotting
between percent inhibition and concentration.
C
S
1
13
2
.10 | Cell culture and experimental
conditions
3.1.1 | IR spectra and mode of bonding for
prepared OVAP azomethine ligand and its
complexes
Cancer cell lines were cultured in RPMI 1640 medium in
culture flasks, supplemented with 10% foetal bovine
serum and 100 μg ml penicillin and streptomycin. Can-
−
1
In order to authenticate the formation of ligand and
metal complexes, the FT‐IR spectra of OVAP azomethine
ligand and its metal chelates are discussed (Figures S1
and S2; and Table 1). A comparative study of their FT‐
IR spectra indicates that certain peaks are of interest.
cer cells were incubated at 37°C in a 5% CO ‐supple-
2
mented atmosphere for at least 24 h before the
appropriate treatments and used to determine the cell
viability against the synthesized compounds.

6
of 23
ABU‐DIEF ET AL.
SCHEME 1 Synthetic strategy for preparation of the investigated complexes
Thus, only important peaks that are shifted or have newly
coordination sphere. The characteristic frequencies of
the coordinating nitrate group in the Cr(III) complex
−
1
appeared are discussed. The band at 1628 cm in the FT‐
IR spectrum at of OVAP ligand corresponds to ν(HC═N)
group. In the FT‐IR spectra of the metal complexes, it was
observed that the band undergoes a blue shift to lower
−
1
shows three non‐degenerate modes at 1430 cm
of
of
−
1
−1
ν(NO2) , 1384 cm
of ν(NO2)sy and 828 cm
asy
46]
[
ν(NO).
Based on these spectral observations, we con-
−
1
values by 16–27 cm , which implies the formation of a
bond between nitrogen of azomethine group and metal
ion and a reduction of the double bond character of
clude that OVAP azomethine ligand coordinates in a bi‐
negative tridentate manner around the Cr(III), Zn(II),
Cu(II) and Pd(II) metal ion centres.
[38–41]
carbon–nitrogen bond.
Supporting that, new bands
due to ν(M─N) bond appeared in the range 442–
−
1
1
13
4
94 cm . The broad band corresponding to ν (─OH)
3.1.2 | H NMR, C NMR and mass spec-
tra of prepared OVAP azomethine ligand
and its diamagnetic complexes
−
1
stretching vibrations, which appeared at 3376 cm for
the ligand, disappeared after complex formation, pointing
to the deprotonation of phenolic OH during complex for-
[42,43]
1
mation.
This is further supported by the appearance
To investigate the encapsulation behaviour, H NMR and
CNMR studies were used for demonstrating the interac-
tion of the host molecule and guest molecule between OV
and AP. The H NMR and C NMR results are displayed
in Figure S2 (supporting information) and detailed in
section 2.
13
of new bands due to metal–oxygen bond formation in the
−
1 [44]
region 529–562 cm .
3
Broad bands in the region 3378–
−
1
1
13
423 cm point to the presence of coordinated water
molecules in the complexes. This is further supported by
the presence of bands corresponding to ν (─OH) rocking
−
1 [45]
1
and wagging mode of vibrations at 737–742 cm .
In
The H NMR spectrum of OVAP azomethine ligand
the spectrum of the Cr(III) complex, new peaks appeared
as a result of binding of nitrate group inside the
(Figure 1) demonstrated singlet signals at 14.01 and
9.72 ppm which were attributed to proton hydroxyl group

ABU‐DIEF ET AL.
7 of 23
of phenyl ring and methoxy of phenyl ring, respectively.
Also it shows a singlet signal at 8.96 ppm, which was
assigned to the azomethine group. Also, it displays multi-
plet signals at 7.38–6.98 ppm for seven aromatic CH for
the two rings in the ligand. Also, it displays a singlet sig-
nal at 2.50 ppm due to 3H protons of methoxy group. The
13
C NMR spectrum of OVAP ligand (Figure S3) displayed
a signal at 162.00 ppm which probably corresponded to
azomethine carbon. The signals in the range 152–
115 ppm were assigned to phenyl carbons as demon-
strated in Figure S3. Furthermore it displays singlet signal
at 56 ppm due to aliphatic carbon of methoxy group.
1
The HNMR spectrum of OVAPPd complex clearly
demonstrates the presence of the framework protons of
the OVAP molecule. It shows a singlet signal at
8
.37 ppm which assigned to the proton of azomethine
group. Moreover, it shows multiplet signals at 7.99–
.61 ppm for aromatic 7‐CH protons (Figure S4 and sec-
tion 2). Additionally, it displays a singlet signal at
.51 ppm due to 3H protons of methoxy group. Further-
more, the OVAPPd complex shows a singlet signal at
6
2
3
.79 ppm attributed to the two protons of H O molecule.
The H NMR spectrum of the OVAPZn complex clearly
2
1
demonstrates the presence of the framework protons of
the OVAP molecule. It shows a singlet signal at
8.96 ppm which was assigned to the proton of azomethine
group. Moreover, it shows multiplet signals at 7.39–
6
2
.8 ppm for aromatic 7‐CH protons (Figure S5 and section
). Also, it displays a singlet signal at 2.51 ppm due to 3H
protons of methoxy group. Furthermore, the OVAPZn
complex shows a singlet signal at 3.84 ppm, attributed to
the two protons of H O molecule. Thus the formation of
2
OVAP ligand and its complexes with the proposed chemi-
13
cal structures was established. The C NMR spectrum of
OVAPZn complex exhibits resonances with slight chemi-
cal shifts (Figure S6 and section 2).
The ESI mass spectrum of ligand OVAP, for further
structural information, is given in Figure S7. The spec-
trum shows the molecular ion peak at m/z 244 and the
+
isotopic peak at m/z 244 (M ) due to the presence of
1
3
15
C and N isotopes. The base peak at m/z 228 is due to
+
(
C H NO ) ion. Another intense peak at m/z 120 is
14
12
2
+
due to (C H O ) ion. The intensity of these peaks
7
4 2
reflects the stability and abundance of the ions.
3
.1.3 | Electronic spectra, conductivity
measurements and magnetic moments for
OVAP azomethine ligand and its metal
chelates
Electronic spectra of the investigated complexes were
measured in DMF as solvent. The highest energy band is

8
of 23
ABU‐DIEF ET AL.
1
FIGURE 1 H NMR spectrum of OVAP ligand
assigned to π–π* transition while the shoulder may be
assigned to an intramolecular charge transfer. OVAP
azomethine ligand exhibits absorption bands in the UV–
visible region at 288 and 350 nm which are assigned to
n → π* transition originating from the azomethine func-
tion of the ligand which is shifted on complexation. The
absorption bands of complexes at λmax = 318–490 nm are
assigned to charge transfer transitions. These transitions
probably occur from the p‐orbitals of the azomethine to
the d‐orbitals of the metal ions. The appearance of a low‐
intensity broad band lying in the region 430–520 nm in
the electronic spectra of the complexes that is absent in
the spectrum of the free ligand is further evidence of the
complexation process. This band could be mainly ascribed
to the d → d transition in the structure of the investigated
1
0
metal chelates. Since zinc ion has a d configuration, dia-
magnetic, the observed absorption spectra of the Zn(II)
complex could be assigned in terms of the absence of any
d–d electronic transitions (cf. Figure 2).
FIGURE 2 Molecular electronic spectra of of OVAP azomethine
ligand and its complexes in DMF (10 M) at 298 K
−
3
Orbital contribution is the reason for the variation of
spin‐only value that varies with the nature of coordination
and consequent delocalization. Diamagnetic behaviour
with zero μeff value of OVAPPd and OVAPZn complexes
suggests that they have square planar and tetrahedral
The molar conductance (Λ ) values for the prepared
M
2
−1
−1
complexes are found in the range 6.5–11.6 cm Ω mol ,
indicating a non‐electrolytic nature and there is no coun-
ter ion present outside the coordination spheres of the
complexes. So, we conclude that all the prepared com-
[
48]
geometry, respectively.
The magnetic moment value
[38,43,47]
plexes are neutral in nature.
observed for the OVAPCr complex is 3.78 BM, and is close
3+
3
The magnetic moment for complexes is generally diag-
nostic of the coordination geometry about the metal ion.
to theoretical spin‐only values for Cr (3d system) of
[
20]
octahedral geometry.
Magnetic susceptibility value of

ABU‐DIEF ET AL.
9 of 23
the OVAPCu complex is 1.87 BM, which suggests a
The thermogram of OVAPCr shows five decomposi-
tion steps within the range 36–618°C. Within the range
36–115°C, a decomposition step corresponds to the loss
of half a hydrated water molecule with a mass loss of
2.27% (calcd 2.25%). A second step of decomposition
within the range 117–295°C corresponds to the loss of
nitrate and two coordinated water molecules with a mass
loss of 24.22% (calcd 24.25%). The third step up to 330°C
with mass loss of 7.76% (calcd 7.75%) indicates the
removal of the methoxy group part of the azomethine
[49]
distorted octahedral geometry for the Cu(II) ion.
The Nujol mull spectrum of the OVAPCr complex
−
1
showed two bands at 18 781 and 27 415 cm which
4
4
a
4
4 b
can be assigned to B1 → E and B g → E g transi-
g
g
1
tions, respectively, reinforcing the lifting of the degener-
[
20]
acy of the orbital triplet (in octahedral symmetry).
−
1
The band at 28 822 cm in the spectrum of the OVAPPd
complex is assignable to a combination of, for example,
d , d )–b (dx –y ), i.e. A → E and metal‐to‐ligand
2
2
1
1
(
yz zx
1g
1g
g
charge transfer indicating a square planar geome-
ligand (OCH ). The fourth step in the range 332–505°C,
3
[48,50,51]
2+
try.
The electronic spectrum of the Cu complex
with an estimated mass loss of 20.71% (calcd 20.75), cor-
responds to loss of part of the azomethine ligand
(C H O ) followed by removal of the remaining part of
−
1
2
2
displayed a band at 17 790 cm assigned to E → T
g
2g
transition indicative of octahedral geometry around the
6
3 0.5
[
49,52]
Cu(II) ion.
The electronic spectrum of the OVAPZn
the ligand (C H N) up to 618°C leaving Cr O as a resi-
7 5 2 3
10
complex, as d system, exhibited a non‐ligand band at
1 217 cm , corresponding to ligand‐to‐metal charge
due. The TGA data (Table 2) show good agreement with
the suggested stoichiometry abstracted from findings of
elemental analysis.
−
1
2
transfer transition. The clear shifts in the Schiff base
[53,54]
bands are indicative of complex formation.
3.1.5 | Kinetic and thermodynamic studies
Table 2 presents the thermodynamic activation parame-
ters from the Coats–Redfern graphical evaluation
method, namely the activation energy (ΔE*), entropy
3
.1.4 | TGA of prepared azomethine
complexes
(
(
ΔS*) and Gibbs free energy change of decomposition
ΔG*), of the Cr(III), Zn(II), Cu(II) and Pd(II) complexes.
The metal complexes show similar decomposition pat-
terns as evident from their TGA curves (Figure S8).
The characteristic thermogram of OVAPCu shows five
distinct mass losses up to 640°C. Dehydration of one
water molecule in the first step up to 110°C is followed
by removal of three coordinated water molecules up to
Form the obtained data, the following observations can
be summarized:
*
i. The ΔS values for the complexes were found to be
negative. This implies that the activated complex is
more ordered than starting materials and/or the
1
87°C. During further heating, a complete decomposition
[
55,56]
of the OVAPCu azomethine complex in three steps
occurs with mass losses of about 8.22% (calcd 8.23%),
reactions are slow.
*
ii. The negative values of ΔH means that the decompo-
2
4.20% (calcd 24.15%) and 27.31% (calcd 27.35%) in the
sition processes are exothermic.
iii. The positive values of ΔG for the investigated com-
*
temperature ranges 189–301, 303–379 and 381–640°C,
respectively, leaving a residue of CuO.
plexes increases significantly for the subsequent deg-
radation steps due to increasing values of TΔS from
*
The thermogram of OVAPPd shows loss in weight
within the range 36–112°C, which is due to removal of
the hydrated water molecule (found 4.57%, calcd 4.67%)
followed by loss of one coordinated water molecule
within the range114–191°C. Also, it shows three mass
losses within the range 193–730°C, which is due to
removal of ligand moiety leaving PdO as a final residue.
The characteristic thermogram of OVAPZn shows five
distinct mass losses up to 697°C. In the first step up to
one step to another which override the values of
*
ΔH . This increase indicates that the rate of removal
of the subsequent azomethine ligand will be slower
than that of the preceding ligand.
[
57]
f
TABLE 2 Formation constant (K ), stability constant (pK) and
*
Gibbs free energy (ΔG ) values of the synthesized azomethine
complexes at 298 K
*
Type of
ΔG (kJ
1
10°C, it dehydrates two water molecules followed by
−1
Complex
complex
K
f
Log K
f
mol )
removal of one coordinated water molecule up to 185°C.
During further heating, the OVAPZn complex undergoes
complete decomposition in three steps and shows mass
losses of about 8.62% (calcd 8.59%), 25.19% (calcd
4
4
4
4
OVAPPd
OVAPCr
OVAPCu
OVAPZn
1:1
1:1
1:1
1:1
2.62 × 10
4.15 × 10
1.28 × 10
3.85 × 10
4.41
4.61
4.11
4.58
−25.20
−26.33
−23.42
−26.15
2
5.23%) and 28.63% (28.56%) up to 697°C of the ligand
moiety leaving ZnO as a residue.

10 of 23
ABU‐DIEF ET AL.
3
.1.6 | Stoichiometry determination for
This indicates that the formation of the complex greatly
stabilizes the OVAP aazomethine ligand. Consequently,
the optimum pH range for different applications of the
complexes is from pH = 5 to 11. Correlation between
the data obtained from elemental analyses, molar con-
ductance and magnetic measurements, infrared and elec-
tronic spectra and the suggested composition of the
complexes was identified.
prepared complexes utilizing spectropho-
tometric method
Based on the methods which were utilized and the exper-
imental data, the stoichiometry of the prepared metal
chelates is 1:1. The curves of the continuous variation
method (Figure 3) give a maximum absorbance at mole
fraction Xligand = 0.5–0.6, showing the complexation of
metal ions to OVAP azomethine ligand in a molar ratio
of 1:1. Moreover, the data resulting from utilizing the
molar ratio method support the same metal ion to ligand
3
.2 | Molecular DFT calculation for OVAP
azomethine ligand and its complexes
[22–26]
ratio of the prepared complexes (Figure S9).
3.2.1 | Molecular DFT calculation of
OVAP azomethine ligand
3
.1.7 | Formation and stability constants
Figure 5 shows the optimized structures of OVAP as the
lowest energy configurations. The natural charges
obtained from natural bond orbital (NBO) analysis show
that the more negative active sites are in the order O1
for investigated complexes
Spectrophotometric measurements utilizing the continu-
ous variation method were used to determine the forma-
tion constants (K ) of the resultant azomethine complexes
which formed in solution (Table 3). The estimated Kf
values show the high stability of the complexes. The order
(−0.731) > O3 (−0.729) > O2 (−0.601) > N1 (−0.545).
f
The metal ions prefer tridentate coordination to O1, O3
and N1 forming five‐ and six‐membered rings.
of decreasing values of K is as follows: OVAPCr >
f
OVAPZn > OVAPPd > OVAPCu. Moreover, the values
3
.2.2 | Molecular DFT calculation of
of the stability constant (pK) and Gibbs free energy
OVAP ligand and [Cu(OVAP)(H O) ]
complex
≠
2
3
(
ΔG ) of the complexes were calculated. The observed
negative values of Gibbs free energy give an indication
of the spontaneous complexation reaction. The diagram
for stability of the investigated metal chelates at different
pH values (Figure 4) shows typical dissociation curves
and a high pH stability range (5–11) of the metal chelates.
Figure
6 shows the optimized structures of the
[Cu(OVAP)(H O) ] complex as the lowest energy config-
2
3
urations. The copper atom is six‐coordinate in a distorted
octahedral geometry with O4 and O5 of water molecules
being in axial positions. The dihedral angles of atoms in
the three perpendicular planes O1, N1, O3 and O6
(−1.023°), N1, O4, O6 and O5 (3.661°) and O1, O5, O3
and O4 (−4.364°) indicate that these atoms are almost
in one plane. The bond angles in the octahedral geometry
range from 84.89° to 95.04° (Table 4). The distances
between donor atoms O1⋅⋅⋅N1 and O1⋅⋅⋅O3 involved in
coordination are largely decreased upon complex forma-
tion from 4.024 and 6.616 Å (in free ligand) to 2.912
and 3.914 Å (in the complex), respectively (Table 4).
The natural charges computed from the NBO analysis
on the coordinated atoms are Cu (+0.992), O1 (−0.771),
N1 (−0.578), O3 (−0.724), O4 (−0.941), O5 (−0.933) and
O6 (−0.934) (Figure 6).
3
.2.3 | Molecular DFT calculation of
[
Cr(OVAP)NO (H O) ] complex
FIGURE 3 Continuous variation plots for the prepared OVAPCr,
OVAPV, OVAPZn and OVAPCu complexes in aqueous–ethanolic
medium at [OVAPCr] = [OVAPPd] = [OVAPZn] = [OVAPCu] =
3
2
2
Figure
7 shows the optimized structures of the
−
3
1
0
M and 298 K
[Cr(OVAP)NO (H O) ] complex as the lowest energy
3 2 2

ABU‐DIEF ET AL.
11 of 23
TABLE 3 Thermal decomposition steps, mass loss, proposed lost segments, final residue and thermo‐kinetic activation parameters of each
decomposition step for the prepared complexes
*
Mass loss (%)
E
*
*
*
Decomp.
temp.
(°C)
(kJ
mol
)
ΔH
ΔG
ΔS
(J mol
Proposed
segment
(kJ mol
(kJ mol
−
1
−1
−1
−1
−1 −1
Complex
Found
(Calcd)
A (s
)
)
)
K
)
OVAPCu
35–110
4.75
14.36
8.22
24.20
27.31
(4.78)
(14.33)
(8.23)
(24.15)
(27.35)
H
3H
CH
2
O
28.50
25.40
30.10
49.80
0.082
0.022
0.074
0.49
27.90
27.20
26.46
25.70
24.30
46.20
66.20
91.20
116.70
162.10
−253.93
−260.04
−264.12
−266.86
−270.21
1
1
3
3
12–187
89–301
03–379
81–640
2
O
3
O
C
C
6
7
H
H
3
5
O
N
Residue
> 640
21.09
(21.11)
CuO
—
—
—
OVAPPd
36–112
14–191
93–340
42–527
29–730
4.57
4.63
8.12
23.76
26.89
(4.69)
)4.69)
)8.08)
)23.72)
)26.84)
H
H
CH
2
2
O
O
24.80
24.10
23.20
21.80
20.20
44.40
65.30
96.50
143.20
198.40
−265.10
−271.09
−275.74
−279.81
−282.91
1
1
3
5
3
O
C
C
6
7
H
H
3
5
O
N
Residue
>730
31.96
(31.90)
PdO
2H
H O
2
CH
—
—
—
OVAPZn
35–110
12–185
87–346
48–549
51–697
9.99
4.97
8.62
25.19
28.63
(9.98)
(4.99)
(8.59)
(25.23)
(28.56)
2
O
29.50
28.90
27.90
26.40
24.90
48.10
67.50
98.80
147.90
195.10
−254.90
−260.78
−265.69
−270.02
−272.74
1
1
3
5
3
O
C
C
6
7
H
H
3
5
O
N
Residue
35–110
22.57
(22.56)
ZnO
0.5 H
2H O + NO
CH
—
—
—
OVAPCr
36–115
17–295
97–330
32–505
07–618
2.27
24.22
7.76
20.71
25.74
(2.25)
(24.50)
(7.75)
(20.75)
(25.75)
2
O
49.20
48.10
47.20
46.30
45.10
67.20
99.10
125.90
152.30
189.10
−239.41
−247.81
−251.29
−253.69
−256.15
1
2
3
5
2
3
3
O
C
C
6
7
H
H
3
5
O
0.5
N
Residue
> 618
18.97
(19.00)
2 3
0.5Cr O
configurations. The chromium atom is six‐coordinate in a
distorted octahedral geometry with O7 and O8 of water
molecules in axial positions. The dihedral angles of atoms
in the three perpendicular planes N1, O7, O4 and O8
(
0.378°), O3, O8, O1 and O7 (13.12°) and O1, N1, O3
and O4 (−9.251°) indicate that these atoms are almost
in one plane. The deviation may be attributed to hydro-
gen bonding and the restriction caused by the tridentate
ligand. The bond angles in the octahedral geometry range
from 78.96° to 97.54° (Table 5). The distances between
donor atoms O1⋅⋅⋅N1 and O1⋅⋅⋅O3 involved in coordina-
tion are largely decreased upon complex formation from
4.024 and 6.616 Å (in free ligand) to 2.713 and 3.661 Å
(
in the complex), respectively (Table 5). Also, the bond
angle of C7–N1–C8 is smaller in the complex (122.8°)
compared to in the free ligand (130.1°) due to complex
formation. The natural charges computed from the NBO
analysis on the coordinated atoms are Cr (+0.714), O1
(−0.543), N1 (−0.451), O3 (−0.610), O4 (−0.467), O7
FIGURE 4 Dissociation curves of the prepared OVAPCr,
OVAPPd, OVAPZn and OVAPCu complexes in DMF
(−0.865) and O8 (−0.876) (Figure 7).

12 of 23
ABU‐DIEF ET AL.
FIGURE 5 Optimized structure of OVAP, vector of dipole moment, and natural charges on active centres of OVAP by density function
B3LYP/LANL2DZ
3
.2.4 | Molecular DFT calculation of
[
Pd(OVAP)H O] complex
2
TABLE 4 Important optimized bond lengths (Å) and bond angles
(
2 3
°) of OVAP and [Cu (OVAP)(H O) ] complex
Figure
8 shows the optimized structures of the
Bond
length(Å) Type of
Complex
[
Pd(OVAP)H O] complex as the lowest energy configura-
2
Bond length(Å)
Type of
bond
tions. The palladium atom is four‐coordinate in a
distorted square planar configuration with atoms O1,
N1, O3 and O4 almost in one plane deviating by
bond
OVAP Complex
Cu–N1
Cu–O1
Cu–O3
Cu–O4
2.073
1.960
1.956
2.251
Cu–O5
Cu–O6
—
—
2.237
2.135
−
0.380°. The bond angles in the square planar geometry
range from 83.50° to 96.56° (Table 6). The distances
between donor atoms O1⋅⋅⋅N1 and O1⋅⋅⋅O3 involved in
coordination are largely decreased upon complex forma-
tion from 4.024 and 6.616 Å (in free ligand) to 2.958
and 4.002 Å (in the complex), respectively (Table 6). Also,
the bond angle of C7–N1–C8 is smaller in the complex
O1⋅⋅⋅N1
O1⋅⋅⋅O3
4.024 2.912
6.616 3.914
Angle (°)
Type of
angle
Angle (°)
Complex
Type of angle Complex
N1–Cu–O1 92.43
N1–Cu–O3 84.89
O1–Cu–O6 87.63
O3–Cu–O6 95.04
O4–Cu–N1 91.76
O4–Cu–O1 86.25
O4–Cu–O3 91.79
O4–Cu–O6 87.75
O5–Cu–N1 92.18
O5–Cu–O1
O5–Cu–O3
O5–Cu–O6
N1–Cu–O6
O1–Cu–O3
O4–Cu–O5
88.70
93.43
88.32
179.5
176.6
173.7
(
126.0°) compared to in the free ligand (130.1°) due to
complex formation. The natural charges computed from
the NBO analysis on the coordinated atoms are Pd
(
(
+0.715), O1 (−0.665), N1 (−0.483), O3 (−0.681) and O4
−0.929) (Figure 8).
a
a
a
O1–N1–O3–O6 −1.023
N1–O4–O6–O5 3.661
O1–O5–O3–O4 −4.364
3
[
.2.5 | Molecular DFT calculation of
Zn(OVAP)H O] complex
2
a
Figure
9 shows the optimized structures of the
Dihedral angle.
[
Zn(OVAP)H O] complex as the lowest energy
2
2 3
FIGURE 6 Optimized structure, vector of dipole moment, and natural charges on active centres of [Cu(OVAP)(H O) ] using B3LYP/
LANL2DZ. H atoms are omitted on C atoms for clarity

ABU‐DIEF ET AL.
13 of 23
FIGURE 7 Optimized structure, vector of dipole moment, and natural charges on active centres of [Cr(OVAP)NO
3
(H
2
O)
2
] complex by
density function B3LYP/LANL2DZ. H atoms are omitted on C atoms for clarity
TABLE 5 Important optimized bond lengths (Å) and bond angles
distances between donor atoms O1⋅⋅⋅ N1 and O1⋅⋅⋅O3
involved in coordination are largely decreased upon com-
plex formation from 4.024 and 6.616 Å (in free ligand) to
2.958 and 4.002 Å (in the complex), respectively (Table 7).
Also, the bond angle of C7–N1–C8 is smaller in the com-
plex (126.4°) compared to in the free ligand (130.1°) due
to complex formation. The natural charges computed
from the NBO analysis on the coordinated atoms are Zn
(+0.715), O1 (−0.665), N1 (−0.483), O3 (−0.681) and O4
(
3 2 2
°) of OVAP and [Cr(OVAP)NO (H O) ] complex
Bond
length(Å) Type of
Complex
Bond length(Å)
OVAP Complex
Type of
bond
bond
Cr–N1
Cr–O1
Cr–O3
Cr–O4
Cr–O7
Cr–O8
2.052
1.828
1.886
2.043
2.122
1.969
O8–H14
O8–H15
—
—
1.027
0.970
(−0.929) (Figure 9).
Figure 10 shows the natural charges (obtained using
O1⋅⋅⋅N1
O1⋅⋅⋅O3
4.024 2.713
6.616 3.661
Angle (°)
NBO calculation) on the atoms of the ligand, which indi-
cate large electron density (more negative) of the coordi-
nated atoms N1, O1and O3. Figure 10 shows the
molecular electrostatic potential surfaces, which describe
the positive (blue colour) and the negative (red colour;
bound loosely or excess electrons) charged electrostatic
potential in the molecule. The computed total energy,
the highest occupied molecular orbital (HOMO) energies,
the lowest unoccupied molecular orbital (LUMO) ener-
gies and the dipole moment for the ligand and complexes
were calculated (Figure 11 and Table 8). The more nega-
tive values of total energy of the complexes than those of
free ligand indicate that the complexes are more stable
than the free ligand. Also, the energy gaps
Type of
angle
Angle (°)
Complex
Type of angle Complex
N1–Cr–O1 88.56
O1–Cr–O4 94.94
N1–Cr–O3 81.88
O3–Cr–O4 93.99
O7–Cr–N1 98.72
O7–Cr–O1 84.71
O7–Cr–O3 80.26
O7–Cr–O4 78.96
O8–Cr–N1 89.89
O8–Cr–O1
O8–Cr–O3
O8–Cr–O4
N1–Cr–O4
O1–Cr–O3
O7–Cr–O8
N1–O7–O4–O8
99.17
97.54
92.22
175.6
160.7
170.7
a
a
0.378
a
O3–O8–O1–O7 −13.12
O1–N1–O3–O4 −9.251
(Eg = ELUMO − EHOMO) are smaller in the case of the
a
Dihedral angle.
complexes than in the ligand due to chelation of ligand
to metal ions (Table 8). The lowering of E in complexes
g
compared to ligand explains the charge transfer interac-
tions upon complex formation.
configurations. The zinc atom is four‐coordinate in geom-
etry between distorted square planar and distorted tetra-
hedral configurations with atoms O1, N1, O3 and O4
deviating by 18.62° from the plane. The bond angles in
the square planar geometry range from 78.16° to 106.7°
3.3 | Complex–DNA binding
3.3.1 | Absorption titration analysis
(Table 7). The diagonal angle N1–Zn–O4 (154.2°) largely
deviates from 180°, while O1–Zn–O3 (173.1°) is restricted
by the tridentate ligand and is close to 180° (Table 7). The
The absorption titration technique is very useful, promi-
nent and reliable for studying the binding mode of small

14 of 23
ABU‐DIEF ET AL.
FIGURE 8 Optimized structure, vector of dipole moment, and natural charges on active centres of [Pd(OVAP)H
2
O] complex by density
function B3LYP/LANL2DZ. H atoms are omitted on C atoms for clarity
TABLE 6 Important optimized bond lengths (Å) and bond angles
binding constant of the synthesized compounds to CT‐
DNA, we monitored the absorption maxima with gradual
addition of DNA. For noncovalent interaction, i.e.
intercalation, hypochromism with or without a red shift
(
2
°) of OVAP and [Pd(OVAP)H O] complex
Bond
length(Å) Type of
Bond length(Å)
Type of
bond
[
6–9]
Complex
bond
OVAP Complex
takes place;
while hyperchromism takes place for
electrostatic binding. The observation of this study reflects
the intercalative binding mode of the compounds to CT‐
DNA. Binding constants reflect the compound–DNA
complex stability, while negative values of free energy
indicate the spontaneity of compound–DNA binding.
The electronic absorption spectra of the metal com-
plexes in the absence and presence of DNA are shown in
Figure 12 and Figure S10. Table 9 presents spectral
parameters for the interaction of DNA with the studied
complexes. Upon gradual addition of DNA, the absorp-
tion intensities of ligand‐to‐metal charge transfer band
gradually increased. As the DNA concentration is
increased, a decrease of absorbance for each investigated
complex was observed. Representative spectra illustrate
this hypochromicity observed for the interaction of the
investigated complexes with CT‐DNA. With increasing
amounts of DNA, the absorption bands of each investi-
gated complex were affected. The determined intrinsic
binding constants for the investigated complexes are in
Pd–N1
Pd–O1
Pd–O3
1.971
1.991
2.011
Pd–O4
—
2.110
O1⋅⋅⋅N1
O1⋅⋅⋅O3
4.024 2.958
6.616 4.002
Type of
Angle
Angle (°)
Complex
Type of
Angle
Angle (°)
Complex
N1–Pd–O1 96.56
N1–Pd–O3 84.48
O1–Pd–O4 83.50
O3–Pd–O4 95.46
N1–Pd–O3
O1–Pd–O3
179.9
178.9
a
O1–N1–O3–O4 −0.380
a
Dihedral angle.
molecules to a biopolymer like DNA. In this study, the
binding mode of compounds to CT‐DNA was investi-
gated. Absorption spectra are very sensitive to changes
in structure of compounds, and the structural
change affects the absorption maxima. To determine the
2
FIGURE 9 Optimized structure, vector of dipole moment, and natural charges on active centres of [Zn(OVAP)H O] complex by density
function B3LYP/LANL2DZ. H atoms are omitted on C atoms for clarity

ABU‐DIEF ET AL.
15 of 23
TABLE 7 Important optimized bond lengths (Å) and bond angles
3
.3.2 | Hydrodynamic methods for inves-
(
2
°) of OVAP and [Zn(OVAP)H O] complex
tigation of DNA interaction with
complexes
Bond
length(Å) Type of
Bond length(Å)
Type of
bond
Complex
bond
OVAP Complex
Optical spectroscopic tools provide significant, but not
sufficient, evidence to support the binding mode for
DNA interaction with the studied azomethine complexes.
It is known that intercalation and groove binding affect
the length of DNA, and hence the viscosity of DNA
increases; while electrostatic binding does not affect the
length of DNA and hence its viscosity remains con-
Zn–N1
Zn–O1
Zn–O3
Zn–O4
2.068
1.914
1.981
2.061
O1⋅⋅⋅N1
O1⋅⋅⋅O3
4.024
6.616
2.898
3.889
Type of
angle
Angle (°)
Complex
Angle (°)
Type of angle Complex
[
29,32,58]
stant.
Accordingly, to clarify the mode of interac-
tion of the complexes with DNA, successive addition of
complex to a fixed concentration of DNA was carried
out. Upon addition of the prepared complexes, the rela-
tive viscosity of DNA increased as well as the respective
complexes (Figure 13). This observation is explained on
the basis that insertion of complex compound into the
base pairs of DNA increases the length of the DNA chain
and consequently increases the separation of base pairs
which leads to the occurrence of intercalation of com-
N1–Zn–O1
N1–Zn–O3
93.33
83.54
N1–Zn–O4
O1–Zn–O3
154.2
173.1
O1–Zn–O4 106.7
O3–Zn–O4 78.16
a
O1–N1–O3–O4 18.62
a
Dihedral angle.
[
29,32]
pounds into DNA.
3.3.3 | Proposed mechanism of interaction
with DNA
In our investigation, correlation between spectroscopic
characteristics and hydrodynamic measurements of the
interaction between the investigated complexes and
DNA can suggest different binding modes. Most likely,
DNA interacts through electrostatically or hydro-
phobically with the complexes. Since the OVAPCr com-
plex gains positive charge as a result of replacement of
[
29]
nitrate ligand by H O molecules in solution,
an elec-
2
trostatic interaction of the OVAPCr complex ion with
negatively charged backbone in phosphate group at the
periphery of the double helix of CT‐DNA easily takes
place. Also due to the removal of nitrate ligand from
the complex in solution, the investigated complexes will
have a flat part in the middle. Therefore, possible interac-
tion of for example the OVAPCr complex with DNA
could be as follows (Scheme 2):
FIGURE 10 Molecular electrostatic potential surface of ligand
OVAP) and complexes ([Cu(OVAP)(H O) ], [Cr(OVAP)
NO (H O) ], [Pd(OVAP)H O] and [Zn(OVAP)H O]) using B3LYP/
LANL2DZ
(
2
3
3
2
2
2
2
•
•
First, electrostatic interaction of coordination sphere
with base pairs of DNA or interaction of Cr(III) com-
plex with base backbone of DNA.
Second, the flat part of the complex is inserted
between the base pairs and consequently there is coor-
3+
the following order OVAPCr > OVAPZn > OVAPPd >
OVAPCu. The free energy values of the prepared com-
plexes are negative. This indicates the spontaneity of
complex–DNA interaction.
dination of Cr with base pairs of DNA.
For the OVAPPd complex, the possible interaction
with DNA can occur through coordination of Pd(II) with

16 of 23
ABU‐DIEF ET AL.
FIGURE 11 HOMO and LUMO charge density maps of ligand (OVAP) and complexes using B3LYP/LANL2DZ
TABLE 8 Calculated total energies, HOMO, LUMO, energy gap and dipole moments of OVAP azomethine ligand and its complexes at
B3LYP/LANL2DZ level
a
b
c
d
E
HOMO
LUMO
E
g
Dipole moment (D)
OVAP
−821.590
−1245.897
−1432.862
−1023.632
−962.521
−5.6790
−4.7928
−5.9949
−5.2948
−5.1245
−1.9273
−1.7270
−3.0546
−1.9665
−1.8277
3.7517
3.0658
2.4903
3.3283
3.2968
7.6786
6.2332
10.262
3.6576
2.9618
[Cu(OVAP)(H
[Cr(OVAP)NO
[Pd(OVAP)H O]
[Zn(OVAP)H O]
2
O)
3
]
3
(H
2 2
O) ]
2
2
a
Total energy (a.u.).
b
c
HOMO: highest occupied molecular orbital (eV).
LUMO: lowest unoccupied molecular orbital (eV).
d
Energy gap = ELUMO − EHOMO (eV).

ABU‐DIEF ET AL.
17 of 23
base pairs of DNA through replacement binding mode
and insertion of aromatic ring of OVAP ligand between
base pairs of DNA (Scheme 3).
3.3.4 | DNA interaction via gel
electrophoresis
The gel electrophoresis technique was used to investigate
the binding of the investigated complexes with DNA. The
results are shown in Figure 14. The technique involves
comparing bands with different band widths and bright-
ness to the control. The diversity in DNA cleavage effi-
ciency of the investigated complexes was attributed to
their difference in binding ability to DNA. It was clearly
concluded that when complexes cleaved DNA, patho-
genic growth was arrested through destruction of the
genome of the organism.
FIGURE 12 Absorption spectra of OVAPCr complex in 0.01 M
Tris buffer (pH 7.5, 298 K) upon addition of CT‐DNA in absence
−
3
(
lower) and presence of CT‐DNA (top) at [OVAPCr] = 10 M and
CT‐DNA] = 0–30 μM
[
TABLE 9 Spectral parameters for interaction of prepared azomethine complexes with DNA
λ
max free
λ
max bound
Type of
chromism
Binding
constant (× 10 )
ΔG
(kJ mol )
a
5 b
−1
Complex
(nm)
(nm)
nΔ
Chromism (%)
OVAPPd
239
237
317
410
2
1
19
3.59
31.75
41.25
Hypo
2.38
−29.99
3
4
18
29
OVAPZn
OVAPCu
240
239
252
270
281
352
1
0
0
9
12
5.29
5.15
5.15
7.08
8.72
Hypo
3.20
1.30
−30.71
2
2
2
3
52
70
90
64
239
239
252
270
289
362
386
399
431
0
0
1
0
−1
0
12.15
11.76
11.83
11.52
19.88
18.90
13.33
12.50
Hypo
−28.53
252
271
289
361
386
399
432
0
1
OVAPCr
240
240
252
270
287
361
386
398
431
480
0
0
0
1
0
−12.23
−12.41
−12.50
−12.08
−16.39
−14.75
−14.63
−13.43
2.56
Hyper
6.82
−32.54
252
270
288
361
386
398
430
498
0
0
−1
−19
a
Chromism (%) = (Absfree − Absbound)/Absfree
.
b
−1
3
b
Binding constant K in mol dm .

18 of 23
ABU‐DIEF ET AL.
3.4 | Antimicrobial activities
Many pathogens acquire resistance to antibiotics; there-
fore, a necessity for new antibiotics has arisen. Out of
the five prepared compounds, the OVAPPd complex
displayed broad‐spectrum antimicrobial activity against
all tested bacterial strains with MIC values of 4.25–
−
1
6
.00 μg l (Tables 10 and 11; Figure 15). The investi-
gated azomethine metal chelates reveal significantly
enhanced antimicrobial activity against microbial strains
in comparison to the free ligand (Tables 10 and 11;
Figure 15). Hydrogen bond formation with azomethine
group by the active sites is the proposed pathway of
action of the metal chelates resulting in interference with
cell wall synthesis. This hydrogen bond formation dam-
ages the cytoplasmic membrane and cell permeability
may also occur resulting in the death of the cell.
Certainly steric and pharmacokinetic factors also play a
FIGURE 13 Effect of increasing amount of synthesized
complexes on relative viscosities of CT‐DNA at [DNA] = 0.5 mM
at 298 K
SCHEME 2 Suggested mechanism for interaction of OVAPPd with DNA via (a) intercalation binding mode

ABU‐DIEF ET AL.
19 of 23
SCHEME 3 Suggested mechanism for interaction of OVAPPd with DNA via replacement binding mode
decisive role in deciding the potency of an antimicrobial
agent. Thus, the antimicrobial property of metal com-
Activity indexðAÞ
inhibition zone of complexðmmÞ
plexes cannot be ascribed to chelation alone but it is an
intricate blend of coordinating sites, nature of the metal
ion, nature of the ligand, geometry of the metal chelate,
and concentration, hydrophilicity, lipophilicity and the
presence of co‐ligands. The data reported in Table 10
show that the target azomethine OVAP ligand and
its complexes are more efficient and active against
Gram‐positive than Gram‐negative bacteria. This could
be rationalized in terms of Gram‐positive bacteria
possessing a thick cell wall incorporating many layers
of teichoic acids and peptidoglycan. On the other hand,
Gram‐negative bacteria possess a relatively thin cell wall
of a few layers of peptidoglycan surrounded by a second
lipid membrane incorporating lipoproteins and lipopoly-
saccharides. The variance in the structure of cell wall
led to differences in antibacterial activity. The activities
of the tested complexes were confirmed by calculating
the potency index (Table S2) according to the following
¼
× 100
inhibition zone of standard drugðmmÞ
On comparison of our obtained data with those of the
literature, the activity of the palladium complex is higher
[
59,60]
than those of other complexes.
The determined
properties of the synthesized compounds are of interest
in terms of extending antibacterial and antifungal arsenal
of existing drugs.
3
.5 | Antioxidant activities
Free radical oxidative processes play a significant patho-
logical role in causing many human diseases together
[
61]
with aging. In vitro antioxidant activities of the newly
synthesized OVAP azomethine ligand and its metal che-
lates were evaluated usingthe DPPH free radical scav-
enging method due to its ease and convenience.
Sample solutions of different concentrations (10, 25,
[6–10]
relation:

20 of 23
ABU‐DIEF ET AL.
FIGURE 14 Interaction of investigated complexes with CT‐DNA
studied by agarose gel electrophoresis. Lane 1: CT‐DNA + OVAPPd;
lane 2: CT‐DNA + OVAPZn; lane 3: CT‐DNA + OVAPCu; lane 4:
CT‐DNA + OVAPCr; lane 5: OVAPPd complex; lane 6: OVAPCu
complex; lane 7: OVAPZn complex; lane 8: OVAPCr complex
−
1
5
0, 100 and 150 μg ml ) were prepared to evaluate the
antioxidant activity. A solution of ascorbic acid was
used as a standard for comparison. From the results
obtained, it is clear that the metal chelates exhibit better
antioxidant activity than the ligand. The results indicate
that the antioxidant activity increases with increasing
concentration for all complexes under study. In terms
of IC50, the data show that the OVAPCu complex has
potent antioxidant activity with the lowest IC50 value
−
1
of 22 μg ml , while the OVAPCr complex exhibited
−
1
least antioxidant activity with IC50 of 75 μg ml , as
shown in Figure 16.
3.6 | Cytotoxicity studies
The anticancer activities of OVAP azomethine ligand and
its metal chelates against HCT‐116, HepG‐2 and MCF‐7
cells were screened using MTT assay. The results were
analysed in terms of cell viability curves and expressed
as IC50 values as shown in Figure 17 and Table S3. The

ABU‐DIEF ET AL.
21 of 23
TABLE 11 Minimum inhibition zone for antimicrobial assay of prepared Schiff base and its complexes
Bacteria
Fungi
Compound
M. luteus
E. coli
S. marcescence
A. flavus
G. candidum
F. oxysporum
OVAP
10.00
6.75
4.25
4.50
5.50
10.70
7.50
6.00
6.50
7.25
9.00
5.75
4.75
5.00
5.50
10.75
6.75
5.00
5.50
6.25
9.00
6.00
4.50
5.00
5.25
9.50
6.50
4.75
5.25
5.75
OVAPCr
OVAPPd
OVAPCu
OVAPZn
FIGURE 15 Histogram showing the
comparative antibacterial activities of
compounds against M. luteus (+ve) at 15
and 30 μg/ml
FIGURE 16 Trends in inhibition of
DPPH radical by OVAP azomethine ligand
and its complexes
FIGURE 17 IC50 values of the ligand
and its complexes against human colon
carcinoma cells (HCT‐116), hepatic
cellular carcinoma cells (HepG‐2) and
breast carcinoma cells (MCF‐7)

22 of 23
ABU‐DIEF ET AL.
maximal inhibition concentrations (IC ) given in Table
ORCID
50
S3 show that the cytotoxicity efficiencies of the com-
pounds under investigation follow the order: OVAPPd >
OVAPCu > OVAPZn > OVAPCr > OVAP ligand. From
the results it is evident that the OVAPPd complex exhib-
ited higher in vitro cytotoxicity against all selected cell
lines when compared to the ligand as well as to the stan-
dard drug, vinblastine. In contrast, the OVAPCr complex
showed lower anticancer activity when compared to the
standard drug. It is generally thought that the cytotoxicity
of metal chelates depends on their ability to bind DNA
and thus damage its structure, which is followed by
inhibition of replication and transcription processes and
Ahmed M. Abu‐Dief
https://orcid.org/0000-0003-3771-
9011
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