Nano-synthesis, characterization, modeling and molecular docking analysis of Mn (II), Co (II), Cr (III) and Cu (II) complexes with azo pyrazolone ligand as new favorable antimicrobial and antitumor agents

Article abstract of DOI:10.1002/aoc.4606

Novel nanosized Mn (II), Co (II), Cr (III) and Cu (II) complexes were synthesized with 2-((5-oxo-1,3-diphenyl-4,5-dihydro-1H-pyrazol-4-yl)diazenyl) benzoic acid, HL applying precipitation method. Their structures were characterized based on the elemental and thermal analyses, spectra (FT-IR, UV–Vis, MS, ESR and XRD), conductivity and magnetic moment measurements. IR spectra offered that HL behaves as monobasic tri-dentate ligand towards Mn (II), Cr (III) and Cu (II) and monobasic bi-dentate towards Co (II). The XRD results unambiguously confirmed the crystalline nature and nano-sized particles of Cu (II) complex while HL and other complexes exhibited amorphous phases. The magnetic moment data, UV–Vis and ESR spectra supported the formation of octahedral geometries for Mn (II), and Cr (III) complexes, whereas Co (II), and Cu (II) complexes showed tetrahedral arrangement. The activation parameters for the thermal degradation stages were theoretically calculated using TGA curves. The obtained data showed the inspected complexes as favorable antimicrobial drug candidates. The studied compounds were screened out for their antitumor and antimicrobial activities. The inspected compounds exhibited a reasonable antibacterial activity and weak antitumor efficacy. The in vitro results were confirmed using the in silico molecular docking analysis (docking server) applying x-ray crystallographic structures of the proteins (4?m01, 3?t88, 1zap & 4ynt) from PDB (Protein Data Bank). HL and probably its complexes displayed adequate binding with the receptors of 4?m01, 3?t88, 1zap, and 4ynt microorganisms. The obtained data show the inspected complexes as favorable antimicrobial drug candidates.

Full text of DOI:10.1002/aoc.4606

Received: 18 May 2018
DOI: 10.1002/aoc.4606
Revised: 19 August 2018
Accepted: 25 August 2018
F U L L P A P E R
Nano‐synthesis, characterization, modeling and molecular
docking analysis of Mn (II), Co (II), Cr (III) and Cu (II)
complexes with azo pyrazolone ligand as new favorable
antimicrobial and antitumor agents
1
1,2
3,4
Mohamed Gaber
| Abdalla M. Khedr
| Mohammed A. Mansour
|
1
Mohsen Elsharkawy
1
Chemistry Department, Faculty of
Science, Tanta University, Tanta 31527,
Egypt
Novel nanosized Mn (II), Co (II), Cr (III) and Cu (II) complexes were synthe-
sized with 2‐((5‐oxo‐1,3‐diphenyl‐4,5‐dihydro‐1H‐pyrazol‐4‐yl)diazenyl) benzoic
acid, HL applying precipitation method. Their structures were characterized
based on the elemental and thermal analyses, spectra (FT‐IR, UV–Vis, MS,
ESR and XRD), conductivity and magnetic moment measurements. IR spectra
offered that HL behaves as monobasic tri‐dentate ligand towards Mn (II), Cr
2
Department of Chemistry, Faculty of
Applied Science, Umm Al–Qura
University, Makkah, Saudi Arabia
3
Biochemistry Division, Chemistry
Department, Faculty of Science, Tanta
University, Tanta 31527, Egypt
(III) and Cu (II) and monobasic bi‐dentate towards Co (II). The XRD results
4
Institute of Cancer Sciences, University
unambiguously confirmed the crystalline nature and nano‐sized particles of
Cu (II) complex while HL and other complexes exhibited amorphous phases.
The magnetic moment data, UV–Vis and ESR spectra supported the formation
of octahedral geometries for Mn (II), and Cr (III) complexes, whereas Co (II),
and Cu (II) complexes showed tetrahedral arrangement. The activation param-
eters for the thermal degradation stages were theoretically calculated using
TGA curves. The obtained data showed the inspected complexes as favorable
antimicrobial drug candidates. The studied compounds were screened out for
their antitumor and antimicrobial activities. The inspected compounds exhib-
ited a reasonable antibacterial activity and weak antitumor efficacy. The
in vitro results were confirmed using the in silico molecular docking analysis
of Glasgow, Garscube Estate, Switchback
Road, Glasgow G61 1QH, UK
Correspondence
Abdalla M. Khedr, Department of
Chemistry, Faculty of Applied Science,
Umm Al–Qura University, Makkah, Saudi
Arabia.
Email: abkhedr2010@yahoo.com
(docking server) applying x‐ray crystallographic structures of the proteins
(4 m01, 3 t88, 1zap & 4ynt) from PDB (Protein Data Bank). HL and probably
its complexes displayed adequate binding with the receptors of 4 m01, 3 t88,
zap, and 4ynt microorganisms. The obtained data show the inspected
1
complexes as favorable antimicrobial drug candidates.
KEYWORDS
antimicrobial, antitumor, modeling, molecular docking, nanosized metal complexes
Appl Organometal Chem. 2018;e4606.
wileyonlinelibrary.com/journal/aoc
© 2018 John Wiley & Sons, Ltd.
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https://doi.org/10.1002/aoc.4606

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GABER ET AL.
1
| INTRODUCTION
(kcal/mol), total intermolecular energy (kcal/mol), and
electrostatic Energy (kcal/mol) and were determined.
Also, the totally interaction profile (hydrogen bonds &
hydrophobic interaction), and hydrogen bonding interac-
tions (HB plot) were investigated.
Pyrazolones are defined as oxo five‐membered heterocy-
cle derivatives comprising two neighboring nitrogen
atoms. They contain two double bonds inside the moiety;
imparting an aromatic character to these molecules.
Pyrazolones are still economically important precursors
for both pharmaceuticals and dyes. Pyrazolone deriva-
tives have numerous pharmacological applications
like analgesic, antipyretic, anti‐inflammatory, anti-
[1]
2 | EXPERIMENTAL
[2]
[3]
1,3‐Diphenyl‐5‐pyrazolone, anthranilic acid, triethylamine,
[4]
[5]
[6]
ethanol, diethylether, DMSO, DMF, MnCl
2
•4H O,
2
bacterial, antifungal,
anti‐infective,
antioxidant,
CoCl •6H O, CrCl •6H O and CuCl •2H O were of high
2
2
3
2
2
2
anticonvulsant, anti‐depressant, anti‐hyperglycemic and
anti‐proliferative agents. Besides, they have the ability to
extend noteworthy anticancer influences by inhibiting
diverse types of enzymes that act serious roles in cell
purity and was applied as purchased.
2
.1 | Synthesis of azo Pyrazolone ligand
[7]
division.
4‐Aminoantipyrine is one of the superior
(HL)
celebrated pyrazolone derivatives which is applied for
the protection towards oxidative stress and has a prophy-
lactic influence on various diseases involving cancer.
Due to the reactivity of position‐4 in the pyrazolone ring,
it undergoes a coupling reaction with a number of aryl
diazonium chlorides forming 4‐arylazo derivatives, which
are considered as an important class of azo dyes. The
azo‐derivatives of pyrazolone compounds have enticed
great interest because of their prospect prominence in
medicinal chemistry as antimicrobial agents.
more, antipyrine derivatives and their complexes with
different metal ions can serve as antiparasitic agents
and antitumor substances.
complexes in which the azo group is included in bonding
are derived from azo compounds comprising donor func-
o‐Amino benzoic acid (0.01 mol, 1.37 g) was dissolved in
[
8,9]
concentrated HCl (10 ml) and H O (10 ml), cooled to 5 °C
2
and treated with a cold aqueous solution of NaNO2
(
0.01 mol, 0.69 g). The diazotized amine was added grad-
ually to an ice‐cold solution of 1,3‐diphenyl‐5‐pyrazolone
0.01 mol, 2.25 g) in 10% aqueous NaOH (50 ml). After
(
completing the addition process, the reaction mixture
was left to stand in a cold chest for 12 hr. The obtained
[10]
precipitate was filtered off, washed with H O and recrys-
Further-
2
tallized using ethanol as an orange crystal of HL
(
Figure 1). The purity of ligand was examined applying
melting point constancy and thin layer chromatographic
TLC) tools.
[11,12]
Moreover, azo dye metal
(
tions such as COOH, OH, SH, NH , in a congenial posi-
2
2
.2 | Synthesis of the Nano‐sized metal
[
13]
tion so as to form five or six membered chelates.
In
chelates
view of the obove facts and the importance of such azo
[12,13]
dye bearing NO donor atoms,
coordination character of azo dye ligand derived from
‐aminoantipyrine towards Mn (II), Co (II), Cr (III) and
we reported here the
The following recommended general procedures were
used. A hot ethanolic solution (30 ml) of HL (0.001 mol,
0.384 g) was mixed with 1 mM of the metals salts
[MnCl •4H2O (0.1979 g), CoCl •6H O (0.2379 g),
4
Cu (II) ions. The structural formulae of the complexes
are investigated using analytical and spectral techniques.
The molecular modeling was performed for the ligand
and all complexes to assert their structural formula.
Moreover, the antimicrobial efficiencies of the inspected
compounds were examined towards different types of
bacteria and fungi. Also, their antitumor activities were
investigated against the human hepatocellular carcinoma
cells HEPG2. Molecular docking analysis was achieved in
silico to examine the ligand for its inhibitory performance
on the receptor of 1zap (secreted aspartic protease from
Candida albicans), 4ynt (Aspergillus flavus FAD glucose
dehydrogenase), 3 t88 (Escherichia coli), and 4 m01
2
2
2
CrCl Cl •6H O (0.2664 g), CuCl •2H O (0.1705 g), dis-
3
2
2
2
2
solved in ethanol (40 ml) in the presence of six drops of
triethylamine as a basic medium. The reaction mixture
was mixed together and heated under reflux on water
bath for 12 hours with occasional stirring. Both the pres-
ence of triethylamine as basic medium and contentious
(
Staphylococcus aureus adhesion protein). Different
parameters like free energy of inhibition constant, Ki
μM), vdW + Hbond + desolv energy (kcal/mol), binding
FIGURE 1 Molecular structure of 2‐((5‐oxo‐1,3‐diphenyl‐4,5‐
dihydro‐1H‐pyrazol‐4‐yl)diazenyl) benzoic acid (HL)
(

GABER ET AL.
3 of 10
stirring enhance the formation of nano‐sized particles.
The volume of the reaction mixture was reduced by evap-
oration of solvent until precipitation of the complex,
which was filtered off and washed consequently with
were computed using MOPAC2009 and then added to
the organic ligand atoms. Rotatable bonds were specified
in the docking process, and non‐polar hydrogen atoms
were merged. The calculations were executed to validate
the binding of the ligand with the receptor of 1zap
(secreted aspartic protease from Candida albicans), 4ynt
H O, warm ethanol and diethyl ether followed by drying
2
in vacuum (over anhydrous calcium chloride).
(
(
Aspergillus flavus FAD glucose dehydrogenase), 3 t88
Escherichia coli), and 4 m01 (Staphylococcus aureus
2
.3 | Instrumentations, measurements
adhesion protein). AutoDock tools were applied to
append the solvation parameters, essential hydrogen
atoms, and Kollman united atom type charges. In order
to calculate the electrostatic terms, and the van der
Waals, auto Dock parameter distance‐ and set‐ dependent
dielectric functions were applied, respectively. The Solis
& Wets local search method and The Lamarckian genetic
algorithm (LGA) were applied in order to implement the
docking simulations.
and methods
Perkin‐Elmer 2400 CHN Elemental Analyzer was applied
to determine C, H and N contents. The metal content
was investigated by the aid of inductively coupled plasma
(ICP) technique after decomposition of the metal com-
plexes using nitric acid. The molar conductance of metal
−
3
−1
complexes were determined in DMF (10 mol L ) by
the aid of a conductivity meter JENWAY model 4070
Conductance Bridge at room temperature. Infrared
spectra were achieved as KBr discs using a Jasco FT‐IR–
3
| RESULTS AND DISCUSSION
.1 | Nature and stoichiometry of the
−
1
4
100 (Tsukuba, Japan) within 4000–200 cm range. Stan-
dard electron impact mass spectra (E.I) of the free ligand
and complexes 1–4 were performed by the aid of a
Finnigan MAT 8222 Spectrometer at 70 eV. UV–Vis spec-
tra were taken in freshly prepared DMF solution from
3
formed complexes
The physical and analytical characteristics of the scruti-
nized ligand and complexes 1─4 are collected in Tables 1.
The chemical composition and stoichiometry of the pre-
pared metal complexes were confirmed by the results of
elemental analysis. The gained data showed satisfactory
coincidence within the proposed molecular formulae.
These data supported formation of complexes exhibit 1:1
(M:L) stoichiometry. Complexes 1─4 were found to be
stable in air (for a long time), insoluble in diversified
organic solvents but soluble to great extent in DMSO
and DMF. The molar conductance values (Table 1) are
2
00 to 800 nm with T80 + UV–Vis spectrometer. Magnetic
moments of the complexes were determined applying a
Sherwood scientific magnetic susceptibility balance. EPR
spectrum of Cu (II) complex (4) as powder was performed
using a Jeol JES‐RE1X EPR spectrometer working in the
X‐band, 9.435 GHz (at Alexandria University). XRD
patterns of the inspected materials were obtained using a
X‐ray diffractometer (GNR, APD2000PRO, Italy) at
Central Laboratory, Tanta University, Egypt. Whole
−
1
patterns were measured at 0.03 ° min scanning rate,
applying Cu/Kα radiation with a graphite monochroma-
−
1
2
−1
found to be within the range 8.50–14.00 Ω cm mol .
These values indicated the non‐electrolytic character of
1
tor. TGA (thermal gravimetric analysis) of complexes 1–4
were investigated (under nitrogen as atmosphere) using a
Shimadzu TG‐50 thermal analyzer from ambient tempera-
ture up to 800 C (with 10 C/min heating rate). Antifungal
and antimicrobial activities of all compounds under
interest were examined at Micro‐Analytical Center, Cairo
University applying a modified Kirby‐Bauer disc diffusion
[
18]
the complexes 1─4.
The metal complexes which con-
tain chloride ion were also tested qualitatively with silver
nitrate and no precipitation was detected which can be
taken as a proof that the complexes are non‐electrolytes.
ο
ο
1
3
.2 | FT‐IR and H NMR spectra and
[14,15]
method.
Evaluation of the antitumor activities of the
bonding modes
investigated compounds against human liver Carcinoma
cell lines (HEPG2) was carried out at the Regional Center
for Microbiology and Biotechnology (Al‐Azhar University)
The inspection of IR spectrum of the parent ligand HL
compared with that of complexes 1─4 (Figure 1S)
denoted certain characteristics differences as presented in
Table 2. The infrared spectra of the metal chelates were
closely similar among themselves. In HL spectrum, the
[15–17]
using the well‐known recommended method.
2.4 | Molecular docking using docking
−
1
band appeared at 1520 cm
corresponding to ν
N=N
server
−
1
shifted to higher frequency by 15–40 cm in complexes
1─4 spectra supporting its coordination to Mn (II), Co
Docking Server software was applied to perform the
molecular Docking calculations. The empirical charges
[
19]
(II), Cr (III) and Cu (II) ions.
The sharp carboxylic vsym

4
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GABER ET AL.
TABLE 1 Microanalysis, physical characteristics, and molar conductivity data of HL and complexes 1–4
Microanalysis; Found (Calc.) %
Comp.
No.
Molecular formula
(Empirical formulae)
M. Wt. Found
(Calc.)
Colour
(Λ
m
)
%C
%H
%N
%M
Ligand
HL
(384.55)
(384.39)
Orange
(−‐‐)
69.14
(68.74)
4.95
(4.20)
14.52
(14.58)
__
(−‐‐)
(C
22
H
16
N
4
O
3
)
1
2
3
4
[MnLCl (H
23ClMnN
2
O)
2
]•2H
2
O
(545.11)
(545.83)
D. yellow
(8.50)
48.36
(48.41)
4.36
(4.25)
10.49
(10.26)
9.98
(10.07)
(C
22
H
4
O
7
)
[CoLCl (H O)]•C
23ClCoN
[CrLCl (H O)]•2H
CrN
[CuLCl]•H
17ClCuN
2
2
H
5
OH
(541.11)
(541.85)
D. brown
(10.00)
52.97
(53.20)
4.76
(4.28)
10.52
(10.34)
10.00
(10.88)
(C
24
H
4
O
5
)
2
2
2
O
(560.01)
(560.33)
D. Orange
(14.00)
46.78
(47.16)
5.88
(3.78)
9.55
(10.00)
9.74
(9.28)
(C
22
H21Cl
2
4
O
6
)
2
O
(500.74)
(500.39)
Green
(11.00)
52.49
(52.81)
3.12
(3.42)
11.30
(11.20)
12.55
(12.70)
(C
22
H
4
O
4
)
−
1
TABLE 2 Important IR spectral bands (cm ) and their assignment for the ligand HL and its metal complexes
IR spectra
Compounds
Ν
N=N
ν
C=O (ring)
ν
C=N (ring)
ν
sym(COO‐)
ν
M‐O
ν
M‐N
HL
1
1520
1560
1556
1553
1535
1665
1656
1658
1652
1647
1493
1487
1489
1489
1497
1783
1734
1712
1727
1727
‐‐
‐‐
603
612
609
612
504
433
491
492
2
3
4
−
−1
(
COO ) of the free ligand HL appeared at 1783 cm
which elucidates the assignment of NH proton of the
pyrazolone ring. The proton at 4 position of the
pyrazolone ring appears as a singlet at 7.67 ppm inside
the multiplet region. The multiplet at 7.26–8.11 ppm is
due to the aryl protons of the two benzene rings.
showed obvious shifts to lower frequencies in the metal
−
1
complexes, and appeared within 1712–1734 cm range.
This behavior confirmed that the ligand coordinates to
the metal ions through the O‐atom of the carboxylate
group. The stretching band of carbonyl group ν (C=O)
of 5‐pyrazolone moiety did not show significant wave-
number shift in complex 2 indicating non‐participation
of the carbonyl‐O atom in binding to Co (II), whereas;
appreciable downward wavenumber shifts 9–18 cm in
complexes 1, 3, and 4 confirming that oxygen atom of
pyrazolone moiety was coordinated to Mn (II), Cr (III)
and Cu (II) ions. According to these IR data, HL behaves
as monobasic tri‐dentate ligand towards Mn (II), Ni (II)
and Cu (II) and monobasic bi‐dentate towards Co (II).
In the spectra complexes 1─4, the broad band around
429–3440 cm are attributed to the presence of coordi-
nated water. Appearance of non‐ligand bands ranging
between 603–612 cm and 433–504 cm in the com-
plexes are characteristic of M‐O and M‐N bands,
respectively.
The H NMR spectrum of the investigated ligand HL
Figure 2S) showed two singlets at 14.91 and 12.06 ppm;
each one is integrated for one proton. NH Proton of the
pyrazolone ring appeared in the same position as HL
3.3 | Inspection of mass spectra
Mass spectra were applied to underline the constitutions
and pureness of the synthesized ligand and its chelates
(Figure 3S). The free ligand fragmentation showed the
molecular ion peak at 384.55, which is coincide with its
theoretical value (384.39). Mass spectra of [MnLCl
(H O) ]•2H O, [CoLCl (H O)]•C H OH, [CrLCl (H O)]
−
1
2
2
2
2
2
5
2
2
•2H O, [CuLCl]•H O presented precise molecular ion
2
2
peaks (calc.) at 545.11 (545.83), 541.11 (541.85), 560.01
−
1
3
(560.33), 500.74 (500.39), respectively, corresponding to
+
the parent ion [ML] . Also, appearance of various peaks
−
1
−1
due to assorted fragments trough sequential degradation
of the base compound is excellent evidence supporting
the proposed molecular structure [15]. Ideal evidence
backing the suggested structures of the complexes result
from the decomposition of complexes 1─4 via elimination
of HL, which confer rise to the occurrence of a molecular
ion peak assignable to the free ligand. This is triumphant
[15]
1
(

GABER ET AL.
5 of 10
[22]
character for metal complexes involving various types of
suggested by Coats‐Redfern (Figures 5S‐8S).
The
ligands (ML) that decompose via incision of the ligand–
metal bond during the ionization process.
thermo‐kinetic activation parameters of thermal decompo-
sition steps of complexes 1─4 are presented in Tables 1S
and prove the following points;
[20]
3
.4 | Thermo‐gravimetric analytical
a‐ ΔH* exhibited positive values denoting the endother-
mic nature of all thermal decomposition processes.
b‐ ΔS* have positive values for complexes 1─4, reveal-
ing that the activated complex is less ordered
compared with the reactants and/or the reactions
studies
The TGA curves of complexes 1─4 were on performed
under N2 gas flow from ambient temperature up to
8
00 °C at a heating rate of 10 °C/min (Figures 4S). Apply-
[
23]
are very quick.
ing TGA curves, the mass loss was determined for the
various thermal degradation stages in comparison with
those theoretically computed for the suggested formula
depending on the results of micro‐analyses. In generality,
TGA denoted the formation of metal oxide as a residue
from which the percentage of metal (%) is calculated
and found to be in favorable conformity with that gained
c‐ Increasing of ΔG* values in appropriately or signifi-
cant way for the subsequently decomposition steps
as a result of increasing TΔS* values on going from
one step to another which disregard values of ΔH*.
This reverberates that the rate of abstraction of the
posterior parts of ligand will be lower than that of
[
24]
[21]
the forerunner.
This can be bemoaned to the
from analytical measurements.
The acquired data
structural rigidity of the vestige complex after libra-
tion of one or more ligand parts, in comparison with
the precedent complex, which needs more energy for
its rearrangement before sustaining any further
compositional alteration.
showed that complexes 1─4 decomposed in four steps
for complex 1 and three steps for complexes 2, 3 and 4.
The decomposition steps, theoretical calculated, and
found mass losses, temperature ranges, as well as final
products observed in each thermal decomposition process
are presented in Table 3.
3.5 | Thermo‐kinetic parameters
3.6 | ESR Spectrum of cu (II) complex
The order of reaction (n) mechanism of thermal degrada-
tion process, frequency factor (A), andactivation energy
The powder X‐band ESR spectra of Cu (II) complex (4) at
room temperature displayed a broadened characteristic
without hyperfine splitting due to the dipolar interaction
from the ESR spectrum of a set of magnetic parameters.
(
E), for various stages in thermal decomposition com-
plexes 1─4 were investigated using integral method
TABLE 3 Thermogravimetric analysis (TGA) of complexes 1─4
Mass loss %
Molecular formula
(Empirical formulae)
Temp. range
(°C)
No
Found
Calc.
Assignment
1
[MnLCl (H
2
O)
2
]•2H
2
O
)
31–93
93–225
6.68
6.79
28.03
45.50
6.59
6.59
28.48
45.31
Loss of hydrated 2H
Removal of coordinated 2H
Elimination of Cl, CO , C H .
6 5
Decomposition of the organic ligand and
formation of MnO as a residual product.
2
O molecules.
O molecules.
(C
22
H23ClMnN
4
O
7
2
2
3
25–346
46–390
2
2
3
4
[CoLCl (H
2
O)]•C
2
H
5
OH
31–85
85–326
8.50
29.50
46.00
8.48
29.62
45.83
Evaporation of C
2
H
5
OH molecule.
, C
(C
24
H23ClCoN
4
O
5
)
Removal of H O, Cl, CO
2
2
5 3
H .
326–376
Decomposition of the organic ligand and
formation of CoO + C as a final product.
[CrLCl
2
(H
2
O)]•2H
CrN
2
O
45–115
115–287
7.22
16.23
54.29
6.42
15.88
55.53
Removal of hydrated 2H
Loss of coordinated H O + 2Cl molecules.
Decomposition of the organic ligand and
formation of Cr + 6C as final product.
2
O molecule.
(C
22
H21Cl
2
4
O
6
)
2
287–470
[CuLCl]•H
2
O
40–140
140–301
3.40
6.22
71.46
3.59
7.09
71.02
2
Loss of hydrated H O molecule.
(C
22
H17ClCuN
4
O
4
)
Loss of coordinated Cl molecules.
Decomposition of the organic ligand and
formation of CuO + C as a residue.
301–465

6
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GABER ET AL.
Two anisotropic signals was appeared in ESR spectrum of
Cu (II) complex 4 (Figure 9S). The shape of ESR spectrum
supports tetrahedral geometry around Cu (II) center. Cu
(
II) complex 4 showed g ‐value (2.1222) with a positive
eff
mutation from the free electron value (2.0023) which is
assigned to the significant covalent character in the
bonding between the ligand and Cu (II) ion. Hence, the
metal ligand bonding in these complexes exhibit ulti-
[25]
mately covalent character.
3
.7 | UV–vis spectra and magnetic
moment studies
The electronic absorption spectra of ligand and complexes
1
─4 were achieved applying in Nujol mull technique. The
−
1
spectrum of HL displayed band at 24752 cm correspond-
ing to charge transfer transition within the ligand
molecule. This band exhibited red shift in the range 422–
FIGURE 2 XRD spectral patterns of HL and complexes 1─4
−
1
1
168 cm
in complexes 1─4 spectra supporting the
[
26]
participation of C=O and N=N in complex formation.
patterns revealed that Cu (II) complex 4 exhibit nano‐
crystalline phase, whereas HL and complexes 1─4
showed amorphous nature. This behavior may suppos-
The calculated magnetic moment value for Mn (II)
complex 1 was found to be 6.10 B.M. The spectrum of the
Mn (II) complex displayed a weak bands at 17391 and
edly be assigned to the incorporation of the H O mole-
2
−
1
6
4
2
3255 cm corresponding to the A g → Eg(4D) and
cules. A comparative analysis between the patterns of
ligand and those of metal complexes refers to the absence
of smearing or contamination with starting reactants. The
value of average particle size of Cu (II) complex 4 was
calculated applying the Deby‐Scherrer equation, and
found to be 5.3627 nm.
1
6
4
[27]
A g → T g(4D) transitions, respectively. These results
1
1
supported the high spin octahedral geometry around the
[
28]
Mn (II) ion and considerable covalent character.
Co
(
II) complex 2 presented two bands at 17513 and
−
1
4
4
2
8326 cm which can be attributed to A → T (F),
2 1
4
4
A → T (P) transitions, respectively, in a tetrahedral
The occurrence of the novel compounds in such scru-
pulous modernistic nanometer scale ordinarily attract
formidable attention due to their outstanding functional
properties and a wide range of interesting technological
applications, including microelectronics, optics, catalysis,
2
1
[29]
arrangement.
Complex 2 exhibited a moment value
equals 3.80 BM confirming its paramagnetic character.
Cr (III) complex 3 showed two bands at 18726, and
−
1
4
4
a
4
4 b
2
7322 cm can be assigned to B → E , and B → E
1g g 1g g
[
33]
transitions, respectively, reinforcing the lifting of the
degeneracy of the orbital triplet (in octahedral symmetry)
in the order of increasing energy and assuming D
chemical and bio‐sensors.
4
h
3.9 | Molecular modeling studies
[30]
symmetry.
It has 5.40 BM magnetic moment values in
agreement with the suggested geometry. Cu (II) complex
The model geometric structures of HL and complexes
1─4 have been achieved by optimization of their bond
lengths, dihedral angles and bond angles, using the
−
1
2
2
4
showed a band at 18552 cm attributed to T → E
2
transition supporting tetrahedral geometry around the
[34]
central Cu (II) ion. The subnormal μeff value of Cu (II)
hyper chem. 8.03 molecular modeling program.
The
[
31]
complex (1.10 BM) announces the Cu‐Cu interaction.
structures were optimized (Figures 3–7) with minimum
energies acquired applying the quantum chemical calcu-
lations. Selected bond lengths and bond angles of signifi-
cant importance are presented in Tables 2S–6S. As shown
presented in Figure 3, the studied ligand has a planner
form. The bond lengths between atoms in coordination
center are C(3)‐O(13) = 1.192, C(4)‐N(14) = 1.318,
N(14)‐N(15) = 1.338, N(15)‐C(21) = 1.452, C(27)‐
O(28) = 1.366 and C(27)‐O(29) = 1.208 Å. Upon chela-
tion, the bond lengths of free ligand HL are altered to
some extent, essentially for the atoms in direct interaction
3.8 | XRD studies
Meaningful structural data concerning microcrystalline
nature of HL and complexes 1─4 can be obtained from
[32]
inspecting their XRD.
The diffraction pattern of all
compounds were achieved within 2θ (scattering angle)
from 10 to 90°. XRD pattern of free ligand HL are fully
different compared with those of each metal complex
(Figure 2), supporting complex formation. The XRD

GABER ET AL.
7 of 10
with the metal ions. Otherwise, the bond lengths of
the rest atoms in HL are slightly or not altered upon
chelation with the metal ions under interest. The
optimization revealed that Mn (II) complex 1 has an
octahedral geometry. The bond lengths of central metal
atom with the chelating atom are Mn(31)‐N(20) = 1.786,
O(29)‐Mn(31) = 1.888, O(22)‐Mn(31) = 1.884, Mn(31)‐
O(35) = 2.375 and Mn(31)‐O(32) = 1.893 Å. Also, The
bond lengths of Cr (II) ion with the chelating atoms
are Cr(30)‐N(15) = 1.968, O(13)‐Cr(30) = 1.937, O(29)‐
FIGURE 3 Optimized molecular structure of 2‐((5‐oxo‐1,3‐
diphenyl‐4,5‐dihydro‐1H‐pyrazol‐4‐ yl)diazenyl) benzoic acid (HL)
Cr(30)
=
1.847, Cr(30)‐Cl(31)
=
2.260, Cr(30)‐
O(33) = 1.968 Å. The bond angles around Mn and Cr
atoms (Tables 3S and 5S) are quite near to an octahedral
[
35]
3 2
geometry,
indicting sp d hybridization. Co (II) and
Cu (II) complexes are found to attain distorted tetrahe-
dral structures where the hydroxyl oxygen atom and
N‐atom are coordinated to the metal center. In Cu (II)
complex 4, the bond lengths of Cu atom and chelating
atoms are Cu(30)‐N(15) = 1.902, O(13)‐Cu(30) = 1.849,
O(29)‐Cu(30) = and Cu(30)‐Cl(31) = 2.160 Å. The bond
angles of ligand are changed upon chelation with Cu
FIGURE 4 Optimized molecular structure of Mn (II)‐complex (1)
(
II) and Co (II) ions. The largest changes occur for those
bonding the N‐atom and hydroxyl oxygen supporting
their co‐ordination to the metal centers (Tables 4S and
6S). As example, the optimization bond angles around
Co‐atom are C(31)‐N(15)‐Co(30) = 115.66, N(15)‐Co(30)‐
Cl(31) = 110.79, Co(30)‐O(32)‐H(33) = 109.47 and O(29)
Co(30)‐O(32) = 110.80, which confirms the distorted
tetrahedral geometry of the complex 2. The computed
bond angles around Cu atom are C(3)‐O(13)‐
Cu(30) = 116.02, O(13)‐Cu(30)‐N(15) = 100.12, N(15)‐
FIGURE 5 Optimized molecular structure of Co (II)‐complex (2)
ο
Cu(30)‐O(29) = 97.38 and C(21)‐N(15)‐Cu(30) = 125.17 .
From the change between minimum and maximum bond
angles around Co (II) and Cu (II) ions distorted tetrahe-
[
36]
dral geometries are proposed.
The evanescence of
O‐H bond and appearance of novel M‐O bond supported
the participation of carboxylic hydroxyl group in chelate
[
37]
formation.
As a result of electronegativity difference
between the oxygen and nitrogen atoms, the M‐O bond
[
38,39]
length differs than that of M‐O bond.
FIGURE 6 Optimized molecular structure of Cr (III)‐complex (3)
3
.10 | In‐vitro antifungal and antibacterial
assays
The in‐vitro antimicrobial efficiencies of HL and the
inspected complexes 1─4 were examined against E. coli,
P. vulgaris, S. aureus, B. subtillis, A. fumigatus and C.
albicans stratify the modified Kirby–Bauer disc diffusion
[
14,15]
technique.
Table 7S. The free ligand under interest and complexes
─4 did not display any anti‐fungal activities against
The obtained results are displayed in
1
the examined organisms. The free organic ligand and com-
FIGURE 7 Optimized molecular structure of Cu (II)‐complex (4)
plexes 1─4 showed moderate to high efficiencies across

8
of 10
GABER ET AL.
E. coli and P. vulgaris. Furthermore, the HL and complexes
and 3 exhibited moderate activities against S. aureus and
B. subtillis. This character may be clearfield depending on
3.12 | Molecular docking analysis
2
The inhibition constant, Ki (μM), free energy of binding,
total estimated energy of vdW + Hbond + desolv
[40]
the concepts of chelation theory. This theory states that
a diminution in the metal polarizability could reinforce
the lipophilicity of the metal complexes. This causes a
slump of the permeability of the cells, resulting in inter-
vention with normal processes of the cells. Hence, the
complexation leads to make the azo compounds act as
more potent and vigorous antimicrobial agents, inhibiting
the growth of bacteria more and more compared with than
(
kcal/mol), total intermolecular energy parameters and
electrostatic energy were computed to evaluate the favor-
able binding of HL to the proteins. Table 4 presented the
complete profile of all mentioned parameters of HL for its
interaction with receptor of 4 m01 (Staphylococcus aureus
adhesion protein), 3 t88 (Escherichia coli), 1zap (secreted
aspartic protease from Candida albicans), and 4ynt
(Aspergillus flavus FAD glucose dehydrogenase). One of
the most favorable bindings of the ligand was its binding
with 4ynt and 3 t88 and proteins with evaluated free
energy of binding −6.91 and − 6.98 kcal/mol, respec-
tively. Also, the ligand showed the inhibition constant
(ki) of 7.59 and 8.56 μM of 3 t88 and 4ynt, respectively.
Figure 10S displays the binding of the azo pyrazolone
ligand (HL) to the protein 3 t88. A 2D plot was generated
where hydrogen bonds, non‐ligand bond, and ligand
bond, in addition to their lengths were mentioned
[41]
the parent organic ligand.
So, it is concluded that the
chelation process significantly affects the efficacy of mate-
rials that are potent towards microbial strains.
3.11 | Antitumor activity evaluation
Novel anticancer agents are in need to combat the
[
42]
acquired drug resistance often seen in cancer patients.
The anticancer performance of HL and complexes 1─4
was detected in vitro against human hepatocellular carci-
noma cells (HEPG2) utilizing Doxorubicin (Dox) as a ref-
erence drug (IC50 = 4.73). Also, the untreated cells were
applied as a reference control. Each data point was deter-
mined as the average of three separated experiments and
specified as mean ± SD. Growth inhibition of 50% (IC )
(
Figure 10S). A HB plot has been generated to mention
interactions with various amino acids of the protein
Figure 8). Similarly, Figures 11S–16S showed the binding
pattern of the azo pyrazolone ligand (HL) with 1zap
secreted aspartic protease from Candida albicans), 4ynt
(
(
50
is determined as the compound concentrations, which
leads to a 50% lowering in cell proliferation pending the
[43]
tested compound incubation.
From the gained results
it is shown that the growth inhibition of the tested cells
is strongly affected by the character of metal ion and
increases in the order; Mn (II) complex < Cr (III) com-
plex < Co (II) complex < Cu (II) complex < HL. This
denotes that the type of metal ion play a significant role
[
44]
in determining the anticancer efficiency.
to Shier.
1
According
the compounds displaying IC50 values within
0.00–25.00 μg/ml range are considered weak anticancer
agents, compounds exhibit IC50 value between 5.00–
0.00 μg/ml are moderate, whereas those below 5.00 μg/ml
are strong anticancer agents. HL and complexes 1, 2, 3 and
exhibited a weak activity towards human liver Carci-
noma cell line (HEPG2) and showed IC50 values of 15.3,
74, 52.4, 102 and 23.5 μg/ml, respectively.
[45]
1
4
FIGURE 8 HB plot of interaction between azo pyrazolone ligand
(HL) and receptor of Escherichia coli (3 t88)
1
TABLE 4 Binding energies for the interactions between azo pyrazolone ligand (HL) and 3t88, 4m01, 1zap and 4ynt
Est. Free Energy of Est. Inhibition vdW + Hbond + desolv Electrostatic
Total Intermolec.
Interaction
Binding (kcal/mol) Constant, Ki (μM) Energy (kcal/mol)
Energy (kcal/mol) Energy (kcal/mol)
Ligand‐3 t88
−6.98
7.59
394.25
49.61
8.56
−8.94
−5.53
−7.63
−8.07
−0.02
+0.06
−0.04
−0.01
−8.96
−5.47
−7.67
−8.08
Ligand‐4 m01 −4.64
Ligand‐1zap
Ligand‐4ynt
−5.87
−6.91

GABER ET AL.
9 of 10
(
4
Aspergillus flavus FAD glucose dehydrogenase), and
m01 (Staphylococcus aureus adhesion protein).
[9] P. Santos, A. Antunes, J. Noronha, E. Fernandes, A. J. S. C. Vieira,
Eur. J. Med. Chem. 2010, 45, 2258.
Colletively, the in silico molecular docking analysis
confirm the in vitro findings of the anti‐microbial activity
of azo pyrazolone ligand (HL) and possibly its complexes.
Hence, our findings present these complexes as novel
nano‐synthesized azopyrazolone complexes with antimi-
crobial activities.
[10] A. A. S. Al‐Hamdani, W. Al‐Zoubi, Spectrochim. Acta A 2015,
137, 75.
[11] T. Aysha, A. Lycka, S. Lunák Jr., O. Machalický, M. Elsedik,
R. Hrdina, Dyes Pigm. 2013, 98, 547.
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14] A. C. Scott, Laboratory Control of Antimicrobial therapy, in
Practical Medical Microbiology, 13th ed. (Eds: J. G. Collee,
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Livingestone, Edinburgh 1981.
4
| CONCLUSION
Four novel nano‐size Mn (II), Co (II), Cr (III) and Cu (II)]
complexes with 2‐((5‐oxo‐1,3‐diphenyl‐4,5‐dihydro‐1H‐
pyrazol‐4‐yl)diazenyl) benzoic acid (HL) were synthe-
sized. The complexes were investigated using spectral
[
[
15] M. Gaber, A. M. Khedr, M. Elsharkawy, Appl. Organomet.
Chem. 2017, 31. https://doi.org/10.1002/aoc.3885
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FT‐IR, UV–Vis, ESR, MS), thermal and elemental analy-
[17] A. P. Wilson, in Cytotoxicity and viability assays in animal cell
culture: A practical Approach, 3rd ed. (Ed: J. R. W. Masters),
Oxford University Press, Oxford 2000.
ses. The XRD confirmed the nano‐sized nature of Cu (II)
complex. Mn (II) and Cr (III) complexes have octahedral
arrangement while Co (II) and Cu (II) complexes have
distorted tetrahedral configuration. Molecular modeling
was performed by the molecular mechanic calculation
using the hyper chem. 8.03 molecular modeling program
to give the most stable geometry. The mechanism, E, n,
ΔH*, ΔS*, A, and G* values for thermal decomposition
of the inspected complexes were determined from TGA
curves applying Coats‐Redfern method. Azo pyrazolone
ligand (HL) and complexes 1─4 displayed a weak anti-
cancer activity and a reasonable anti‐microbial activity
as confirmed by the in vitro and in silico studies.
[
18] D. C. Onwudiwe, A. C. Ekennia, E. Hosten, J. Coord. Chem.
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10.1002/aoc.4606