New Ni(II), Pd(II) and Pt(II) complexes coordinated to azo pyrazolone ligand with a potent anti-tumor activity: Synthesis, characterization, DFT and DNA cleavage studies

Article abstract of DOI:10.1002/aoc.4104

The absolute necessity to fight some class of tumor is perceived as serious health concerns, so the discovery and development of effective anticancer agents are urgently needed. (E)-4-((2-hydroxyphenyl)diazenyl)-3-phenyl-1H-pyrazol-5(4H)-one, HL, and its Ni(II), Pd(II) and Pt(II) complexes were synthesized and the biological activity was evaluated for antitumor, antioxidant and antimicrobial activity as well as DNA cleavage. Their structures were assigned depending on the elemental analysis, conductivity, magnetic moment, spectral measurements (IR, ¹HNMR, mass and UV–Vis) and thermal analysis. 3D molecular modeling using DFT method confirmed that the geometrical structures agree well with the suggested experimental ones. The antitumor activity was evaluated against four different cell lines using MTT assay. The ligand HL showed a potent cytotoxic activity compared to 5-fluorouracil as a reference drug. For metal complexes, the order of activity was: Pd(II)?>?Ni(II)?>?Pt(II). A remarkable antioxidant activity for the ligand HL was recorded. It was higher than that of the metal complexes. Results of antimicrobial experiments revealed that all compounds were moderate to highly active against selected bacterial strains but inactive as antifungal except Pd(II) which showed a moderate antifungal activity. Gel electrophoresis showed insignificant nucleases activity for the ligand or its metal complexes even in the presence of H₂O₂ providing protection of DNA from damage. The antitumor activity of our compounds may be not due to DNA cleavage but may be referred to a mechanism similar to that of 5-fluorouracil which interfere with DNA replication. The present work suggests the use of this ligand in the design and development of new anticancer drugs.

Full text of DOI:10.1002/aoc.4104

Received: 3 July 2017
DOI: 10.1002/aoc.4104
Revised: 18 August 2017
Accepted: 24 August 2017
F U L L P A P E R
New Ni(II), Pd(II) and Pt(II) complexes coordinated to azo
pyrazolone ligand with a potent anti‐tumor activity:
Synthesis, characterization, DFT and DNA cleavage studies
Eman A. Bakr1
| Gad B. Al‐Hefnawy1 | Mohamed K. Awad1 | Hossam H. Abd‐Elatty2 |
Mohamed S. Youssef3
1 Chemistry Department, Faculty of
The absolute necessity to fight some class of tumor is perceived as serious health
concerns, so the discovery and development of effective anticancer agents are
urgently needed. (E)‐4‐((2‐hydroxyphenyl)diazenyl)‐3‐phenyl‐1H‐pyrazol‐
5(4H)‐one, HL, and its Ni(II), Pd(II) and Pt(II) complexes were synthesized
and the biological activity was evaluated for antitumor, antioxidant and antimi-
crobial activity as well as DNA cleavage. Their structures were assigned depend-
ing on the elemental analysis, conductivity, magnetic moment, spectral
measurements (IR, 1HNMR, mass and UV–Vis) and thermal analysis. 3D molec-
ular modeling using DFT method confirmed that the geometrical structures
agree well with the suggested experimental ones. The antitumor activity was
evaluated against four different cell lines using MTT assay. The ligand HL
showed a potent cytotoxic activity compared to 5‐fluorouracil as a reference drug.
For metal complexes, the order of activity was: Pd(II) > Ni(II) > Pt(II). A remark-
able antioxidant activity for the ligand HL was recorded. It was higher than that
of the metal complexes. Results of antimicrobial experiments revealed that all
compounds were moderate to highly active against selected bacterial strains
but inactive as antifungal except Pd(II) which showed a moderate antifungal
activity. Gel electrophoresis showed insignificant nucleases activity for the
ligand or its metal complexes even in the presence of H2O2 providing protection
of DNA from damage. The antitumor activity of our compounds may be not due
to DNA cleavage but may be referred to a mechanism similar to that of 5‐fluoro-
uracil which interfere with DNA replication. The present work suggests the use
of this ligand in the design and development of new anticancer drugs.
Science, Tanta University, Tanta, Egypt
2 Department of Heavy metals, The
Ministry of Health Laboratories, Tanta,
Egypt
3 Botany Department, Faculty of Science,
Kafrelsheikh University, 33516
Kafrelsheikh, Egypt
Correspondence
Eman A. Bakr, Chemistry Department,
Faculty of Science, Tanta University,
Tanta, Egypt.
Email: emanbakr75@yahoo.com
KEYWORDS
antimicrobial, antioxidant, antitumor, azo‐pyrazolone dyes, DFT studies, DNA cleavage, metal
complexes
1 | INTRODUCTION
keto (C = O) group. Pyrazolone derivatives are notable
structural patterns due to the easy preparation and rich
Pyrazolone is one of the important heterocyclic com-
pounds having five‐membered ring and an additional
biological activity. It occurs in many drugs and synthetic
products. The compounds having pyrazolone moiety are
Appl Organometal Chem. 2017;e4104.
wileyonlinelibrary.com/journal/aoc
Copyright © 2017 John Wiley & Sons, Ltd.
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https://doi.org/10.1002/aoc.4104

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BAKR ET AL.
found to be remarkable anti‐tubercular, anti‐inflamma-
tory, antibacterial, antifungal and antitumor activity.[1]
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 considered an important class of azo dyes. The
azo‐derivatives of pyrazolone compounds have attracted
attention because of their potential importance in medic-
inal chemistry as antimicrobial and therapeutic agents,
chemically pure grade BDH or Merck. Deoxyribonucleic
acid sodium salt, pure, M.W. 50,000 to 100,000 Dalton
purchased from ACROS Organics was used for DNA
cleavage studies. The elemental microanalysis of the syn-
thesized compounds was carried out in the Micro analyt-
ical center, Cairo University using Perkin‐Elmer 2400 C.
H.N. Elemental Analyzer. The metal content of the syn-
thesized complexes was determined by atomic absorption
spectroscopy using PerkinElmer atomic absorption spec-
trophotometer PinAAcle 900 Ministry of Health laborato-
ries, Tanta, Egypt, after the whole decomposition in
concentrated HNO3 several times. The molar conductance
measurements were performed at room temperature
using Hanna 8733 conductivity meter. The Infrared spec-
tra within the range 4000–200 cm−1 were recorded on a
Perkin‐Elmer 1430 IR spectrophotometer as KBr discs.
The 1H NMR spectra were recorded on a Bruker AC spec-
trometer (400 MHz) at 25 °C in d6‐DMSO with TMS as an
internal standard. The electron impact mass spectra were
recorded using a Finnigan MAT8222 spectrometer at
70 eV in Micro analytical center, Cairo University. The
Nujol mull absorption spectra were obtained using
Shimadzu UV–Vis 240 spectrophotometer. The measure-
ments of magnetic susceptibility of the complexes were
performed at room temperature according to the Gouy's
technique using magnetic susceptibility balance (Johnson
Matthey) 436 Devon Park Drive, Cairo University. The
thermal analysis (TGA and DTG) of the solid complexes
were carried out in the range 30–800 °C using the
Schimadzu TG‐50 thermogravimetric analyzer under a
nitrogen atmosphere with a heating rate 20 °C/min. Anti-
microbial activity, antitumor and antioxidant activity
were carried out in Faculty of Pharmacy, El‐Mansoura
University, Microbiology Department.
[2]
in analytical chemistry as complexing agents having
an excellent ability to form chelates with a number of
metal cations[3] for the determination of these metal ions
spectrophotometrically[4], and in industrial chemistry as
dyestuffs.[5] Metal complexes of these compounds have
shown biological activity in comparison to without
metals.[6]
Recently, the computational chemistry has received
much attention as a result research into the theoretical
modeling of drug design, has become the main subject of
research work. Density functional theory (DFT) has been
a meteor in theoretical molecular modeling. By using
DFT method, it is possible to calculate many molecular
properties more accurate than the traditionally correlated
ab initio methods, at a more favourable computational
cost.[7] A literature survey showed that DFT has a major
precision in replicate the values suggested experimentally
for the geometrical structure.[8]
Pyrazolone nucleus has been reported as an interest-
ing template for combinatorial and medicinal chemis-
try.[9] The biological activity of azo pyrazolones has
been assigned to its scavenging activity against the harm-
ful species of reactive oxygen and nitrogen.[10] Cancer
has long been one of the most serious diseases menacing
human health. Subsequently, discovering new com-
pounds with potent anticancer activity is of ultimate
important.
Herein, we have synthesized and characterized an
arylazo dye containing the heterocyclic pyrazolone
nucleus; Namely (E)‐4‐((2‐hydroxyphenyl)diazenyl)‐3‐
phenyl‐1H‐pyrazol‐5(4H)‐one, HL, and their Ni(II),
Pd(II) and Pt(II) metal complexes. These compounds
are screened for their biological activity to be used as
anticancer, antioxidant and antimicrobial agents. As well
as their nuclease activity toward DNA has been
evaluated.
2.2 | Synthetic methods
2.2.1 | Synthesis of the ligand
The investigated ligand, HL (Figure 1), was synthesized
according to the following procedures for azo dye
preparation.[11] o‐Amino phenol (0.01 mol, 1.09 g) was dis-
solved in concentrated HCl (10 mL) and H2O (10 ml),
cooled to 5 °C and treated with a cold aqueous solution of
NaNO2 (0.01 mol, 0.69 g). The diazotized amine was added
gradually to an ice‐cold solution of 3‐phenyl‐5‐pyrazolone
2 | EXPERIMENTAL SECTION
2.1 | Materials and methods
The used compounds in the current investigation are of
high purity grade ready‐made from Aldrich, Sigma or
BDH. The solvents utilized for the spectral study are
FIGURE 1 Structure of the ligand HL

BAKR ET AL.
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(0.01 mol, 1.6 g) in 10% aqueous NaOH (50 ml). After the
addition was completed, the reaction mixture was left to
stand in a cold chest for 12 h. The product was filtered
off, washed with H2O and recrystallized from ethanol as
a faint brown crystal of HL. Yield: (84%). m.p. 219 °C. Ana-
lytical data for HL (C15H12N4O2; 280.28 g·mol−1), Calcd.
(%): C, 64.28; H, 4.32; N, 19.99. Found: C, 64.22; H, 4.44;
N, 19.87. EI‐mass spectra m/z “molecular ion peak” = 280.
IR (cm−1, KBr phase): 3193 (m) ν NH (ring), 3050 (m) ν CH
(ring), 1647 (m) ν C=O (ring), 1537 (sh) ν C=N (ring), 1496
δ: 6.3–8.4 (m, 9H, Ar‐H), 10.7 (s,1H, CH pyrazolone),
12.05 (s, 1H, NH in pyrazolone).
2.3 | Modeling studies
An attempt to get a preferable premeditation into the
molecular structure of the investigated compounds, full‐
unconstrained geometrical optimizations of the investi-
gated ligand and its metal complexes were performed at
a gradient corrected DFT using Becke's three ‐parameter
hybrid method and Lee–Yang–Parr correlation functional
(B3LYP)[12] combined with 6‐31G and LANL2DZ basis
sets[13] using Gaussian 03[14] in the gas phase. The studied
compounds were characterized with no imaginary fre-
quency in their potential energy surface. Geometric and
electronic structures of the molecules were calculated by
the optimization of their bond lengths, bond angles.
1
(m) ν N=N, 1240 (m) ν C‐O (PhOH). H NMR (400 MHz,
DMSO‐d6, δ, ppm): δ: 7.0–8.1 (m, 9H, Ar‐H), 10.7 (s, 1H,
CH pyrazolone), 12.05 (s, 1H, NH in pyrazolone), 14.01 (s
1H, ArOH).
2.2.2 | Synthesis of metal complexes
The following general procedures were used. A hot
ethanolic solution (30 ml) of HL (0.001 mol, 0.280 g)
was added to a hot ethanolic solution (30 ml) of
NiCl2.6H2O (0.001 mol, 0.2378 g), a hot aqueous
ethanolic solution 80% (30 ml) of K2PdCl4 (0.001 mol,
0.3264 g) or K2PtCl4 (0.001 mol, 0.415 g). The reaction
mixtures were mixed together and heated under reflux
on water bath for 12 hours with occasional stirring.
The reaction mixture was then concentrated by rotary
evaporation under reduced pressure until the onset of
precipitation of the product, which was separated and
washed successively with H2O, warm ethanol and
diethyl ether followed by drying in vacuum over anhy-
drous calcium chloride.
Ni complex (1): Yield: 46%. m.p. 266 °C Λm
(/Ω−1 cm2 mol−1) = 8. Anal. Calcd for 1 (C30H22N8NiO4;
617.24 g mol−1) (%): C, 58.38; H, 3.59; N, 18.15; Ni, 9.51.
Found: C, 58.11; H, 3.45; N, 17.96; Ni, 9.87. EI‐mass spec-
tra m/z “molecular ion peak” = 617. IR (cm−1, KBr
phase): 3190 (sh), 3061 (m), 1649 (m), 1530 (sh), 1474
2.4 | Biological activity studies
2.4.1 | In vitro anti‐cancer activity
Anticancer activity screening of the investigated com-
pounds was measured in vitro utilizing four different
human cancer cell lines namely; hepatocellular carci-
noma (HePG‐2), colorectal carcinoma (HCT‐116), Human
prostate carcinoma (PC‐3), and mammary gland breast
carcinoma (MCF‐7) provided by US National Cancer
Institute according to reported standard procedure using
the MTT assay described by Skehan et al.[15] The used
reagents are RPMI‐1640 medium, MTT, DMSO and 5‐
fluorouracil (Sigma co., St. Louis, MO, USA), Fetal Bovine
serum (GIBCO, Egypt). 5‐Fluorouracil was used as a stan-
dard anticancer drug for comparison. This colorimetric
assay is based on the transformation of the yellow tetrazo-
lium bromide (MTT) by mitochondrial succinate dehydro-
genase in viable cells to a purple formazan derivative. The
cells were cultured with 10% fetal bovine serum in RPMI‐
1640 medium. Antibiotics added were 100 units/ml peni-
cillin and 100 μg/ml streptomycin at 37 °C in a 5% CO2
incubator. The cells were seeded for 48 h under 5% CO2
at 37 °C in a 96‐well plate at a density of 1.0x104 cells/
well.[16] Then, the cells were treated with different con-
centration of compounds and incubated for 24 hrs. After
24 hrs of drug treatment, 20 μL of MTT solution at
5 mg/mL was added and incubated again for 4 hrs. The
volume of 100 μL of dimethyl sulfoxide (DMSO) was
added to each well to dissolve the purple formazan
formed. The colorimetric assay was measured and
recorded at the absorbance of 570 nm using a plate reader
(EXL 800, USA). The relative cell viability in percentage
was calculated as (A570 of treated samples/A570 of the
untreated sample) X 100. Values of IC50 were calculated
(m)), 1267 (m),604(w) ν Ni‐O, 427(m) ν Ni‐N
Pd complex (2): Yield: 64%. m.p. over 300 °C Λm
19. Anal. Calcd for 2
.
(/Ω−1 cm2 mol−1
)
=
(C15H13ClN4O3Pd; 439.16 g mol−1) (%): C, 41.02; H, 2.98;
N, 12.76; Pd, 24.23. Found: C, 40.92; H,2.91; N, 12.69; Pd,
24. 52. EI‐mass spectra m/z ‘molecular ion peak’ = 439.
IR (cm−1, KBr phase): 3195 (sh), 3061 (m), 1569 (m), 1537
(m), 1470 (m)), 1259 (m),610(w) ν Pd‐O, 452(m) ν Pd‐N
Pt complex (3): Yield: 54%. m.p. over 300 °C Λm
14. Anal. Calcd for
.
(/Ω−1 cm2 mol−1
)
=
3
(C15H15ClN4O4Pt; 545.84 g mol−1) (%): C, 33.01; H,
2.77; N, 10.26; Pt, 35.74. Found: C, 33.28; H, 2.61; N,
10.19; Pt, 36.18. EI‐mass spectra m/z ‘molecular ion
peak’ = 545. IR (cm−1, KBr phase): 3190 (m), 3071 (m),
1646 (m), 1539 (m), 1473 (m), 1259 (m),611(m) ν
,
Pt‐O
1
480(m) ν Pt‐N. H NMR (400 MHz, DMSO‐d6, δ, ppm):

4 of 18
BAKR ET AL.
from Boltzmann sigmoidal concentration‐response curve
using the nonlinear regression fitting models (Graph
Pad, Prism Version 5).
runs using methanol/phosphate buffer (1:1) instead of
tested compound.
2.4.3 | Methodology for preliminary test
against microorganisms
2.4.2 | Anti‐oxidant activity screening
assay
All selected strains were obtained from Faculty of Science,
Botany Department, Tanta University and maintained in
temperature freezer at ‐80 °C. Strains of human patho-
genic microorganisms used in this study were five differ-
ent types of bacteria; Escherichia coli (E. coli),
Staphylococcus aureus (S. aureus), Bacillus subtilis (B.
subtilis), Salmonella typhi (S. typhi) and Proteus SPP
(Pro. spp.) and two types of fungi; Candida albicans (C.
albicans) and Aspergillus niger (A. niger). The antimicro-
bial activities of the compounds under investigation were
determined versus the tested organisms by standard
methods using Cut plug method [19]. The assay was seeded
with 5x105 cfu/mL of bacteria and 4x105 cfu/ml of fungi
and filled with (0.01 g) powder samples for the tested bac-
teria and fungi, after solidification, they incubated at
37 °C for 3 days, after which the inhibition zones diame-
ters of the (mm) were measured.
Erythrocyte hemolysis
Erythrocytes were isolated from plasma then washed
three times with ten volumes of 0.15 M NaCl. To obtain
a constantly packed cell preparation, during the last
washing, the erythrocytes were centrifuged for 10 min at
2500 rpm. Erythrocyte hemolysis was mediated by peroxyl
radicals in this assay system.[17] A 10% suspension of
erythrocytes in pH 7.4 phosphate‐buffered saline (PBS)
was added to the same volume of 200 mM 2,20‐azobis(2‐
amidinopropane)dihydrochloride (AAPH) solution (in
PBS) containing samples to be tested at different concen-
trations. While the reaction mixture was being incubated
at 37 °C for 1 h, it is shaken gently. The reaction mixture
was then removed, diluted with 8 volumes of PBS and
finally centrifuged at 2500 rpm for 10 min. The absor-
bance A of the supernatant was measured at 540 nm. By
the same way, the reaction mixture was treated with 8 vol-
umes of distilled water till complete hemolysis, centri-
fuged then the absorbance B of the supernatant obtained
was measured at 540 nm. The percentage hemolysis was
calculated by equation (1‐ A/B) x 100%. The data were
expressed as mean standard deviation. L‐Ascorbic was
used as a positive control.
2.4.4 | MIC determination against test
microorganisms
Half‐fold serial dilutions were made of the compounds
under investigation in order to prepare concentrations of
2, 5, 10 and 20 mg/ml in distilled water, zero concentra-
tion was considered as a negative control. A previously
prepared pure spore suspension of each test microorgan-
ism (0.5 ml of about 106 cells/mL) was mixed with
9.5 ml of each concentration in sterile test tubes, incu-
bated for 3 days at 27 °C for fungi and 24 hours at 32 °C
for bacteria, then optical density of growth was measured
by spectrophotometer (Optima SP‐300, Tanta, Egypt) at
620 nm for each incubated mixture, and MIC was
recorded as Shadomy et al.[20]
ABTS method
For each of the investigated compounds, 2 ml of ABTS
solution (60 mM) was added to 3 M MnO2 solution
(25 mg/ml) all are prepared in phosphate buffer (pH 7,
0.1 M). The mixture was shaken, centrifuged, filtered,
and the absorbance (AControl) of the resulting green‐blue
solution (ABTS radical solution) was adjusted at ca. Abs.
= 0.5, λ = l734 nm. Then, 50 ml of (2 mM) solution of
the test compound in pure grade methanol/phosphate
buffer (1:1) was added. The absorbance (ATest) was mea-
sured and the reduction in color intensity was expressed
as % inhibition. The % inhibition for each compound
was calculated from the following equation:[18]
2.5 | DNA cleavage study
The DNA was dissolved in sterilized milli‐polar water
(MPW) and its concentration and purity were determined
by nanodrop ND‐1000 spectrophotometer. The 260/280
absorption ratio was 1.75 which indicates purity of DNA
and it is free from protein.
AControl − ATest
%Inhibition ¼
x 100:
AControl
2.5.1 | Agarose gel electrophoresis
Ascorbic acid (vitamin C) was used as a standard anti-
oxidant (positive control). Without ABTS blank sample
was prepared using methanol/phosphate buffer (1:1)
instead of a tested sample. A sample of negative control
The DNA cleavage activity of ligand and its metal com-
plexes were monitored by agarose gel electrophoresis on
Deoxyribonucleic Acid Sodium Salt, ACROS Organics™.

BAKR ET AL.
5 of 18
The reaction mixture was prepared by adding 100 μM of
ligand or its metal complex to 0.5 μg DNA to yield a final
volume of 500 μL in 1.5 ml Eppendorf tubes then were
mixed by inversion 3 times. The above reactions were
repeated but with the addition of 50 mM H2O2 after
10 minutes of incubating the DNA with HL or its metal
complexes. Then all reactions were incubated for 2 h at
room temperature. DNA (dissolved in sterilized MPW)
and DNA incubated with 50 μl of DMF were used as con-
trols. 10 μl of each reaction products were mixed with 2 μl
of 6X DNA Loading buffer containing 10 mM Tris–HCl
(pH 7.6), 0.03% bromophenol blue, 0.03% xylene cyanol
FF, 60% glycerol and 60 mM EDTA. 10 μl of mixed sam-
ples were loaded in 1% agarose gel containing 2 μL
ethidium bromide (10 mg/ml). In addition, 5 μl of Thermo
Scientific™ GeneRuler™ 100 BP DNA Ladder were loaded
for sizing the resolved DNA bands and judging the quality
of electrophoresis. The ladder is composed of ten chroma-
tography‐purified individual DNA fragments (in base
pairs): 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100.
The electrophoresis was carried out for 1 h at 50 V in
Tris–Borate–EDTA buffer (45 mM Tris, 45 mM H3BO3,
1 mM EDTA pH 8.3). After electrophoresis, the gel was
photographed under UV light.
groups in the moiety of the ligand HL and its metal com-
plexes, together with their assignments, were listed in
(Table S2). Three strong absorption peaks in the spectrum
of the ligand were observed at 1647, 1496 and 1240 cm−1
due to ν(C = O), ν(N = N) and ν(C–O) of the phenolic
group, respectively. Comparing these frequencies of the
ligand with that of the metal complexes, the following
results were found. Both of ν(N = N) and ν(C–O) of the
phenolic group bands were shifted appreciably, to lower
wave numbers by 22–26 for ν(N = N) and to higher wave
numbers 19–27 cm−1 for ν(C–O) compared to the values
for free HL. These shifts confirmed the coordination of
an azo–N and phenolic–O atom to the metal ions. How-
ever, the stretching band of carbonyl‐O of 5‐ pyrazolone
moiety showed no significant wavenumber shifts in com-
plexes 1 and 3 indicating non‐participation of the car-
bonyl‐O atom in binding to Ni(II) and Pt(II), whereas;
appreciable downward wavenumber shift (78 cm−1) in
complex 2 confirming that oxygen atom of pyrazolone
moiety was coordinated to Pd(II) ion. According to IR
data, the HL behaved as monobasic bidentate ligand
bonded to Ni(II) and Pt(II) ions but as monobasic
tridentate ligand bonded to Pd(II) ion. A broad hydroxyl
(−OH) peaks were observed within the region 3243–
3359 cm−1. The low frequency and the broadening of
these bands suggested that this ligand has a strong hydro-
gen bonding (O‐H· · ·N) in the solid state[22] (Scheme 1).
The IR spectra of the complexes also showed a broad band
around 3200 cm−1, assignable to the two (N–H) modes
overlapped with the stretching modes of water molecules.
The complexes showed bands in the 604–611 and 537–
565 cm−1 range assigned to ν(M–O) which could account
for the presence of different stretching modes of M–O
bonds of phenolic–O, water–O or carbonyl–O. Bands in
3 | RESULTS AND DISCUSSION
3.1 | Elemental analysis and molar
conductance
The results of elemental analysis of the synthesized ligand
and its metal complexes along with their physical proper-
ties were collected in Table S1. The metal complexes are
non‐hygroscopic, partially soluble in most common
organic solvents but entirely soluble in DMF and DMSO.
The analytical data revealed 1:2 (metal:ligand) stoichiom-
etry for Ni(II) but 1:1 (metal:ligand) stoichiometry for
Pd(II) and Pt(II). Based on these results, the correspond-
ing probable constitutional formulae of the metal com-
plexes were suggested as given in the same table. The
low molar conductance values measured in DMF solution
fall in the (ΛM = 8–19 Ω−1 cm2 mol−1) range, indicating
the non‐electrolytic nature of these complexes.[21]
−1
[23]
the 427–480 cm
range were assigned as ν(M–N).
3.2.2 | 1HNMR spectra of azo ligand HL
and Pt(II) complex (3)
Further support for the results obtained from the IR was
gained by considering the changes in the NMR spectra of
1
the ligand and its diamagnetic Pt(II) complex. The H
NMR spectra of the investigated ligand HL was recorded
in DMSO‐d6 as a solvent and tetramethylsilane as an
3.2 | Structural investigation of the
prepared compounds
O
O
H
HN
N
N
N
3.2.1 | Infrared spectra
N
N
O
N
H
O
N
H
In order to study the mode of binding between the ligand
and the metal ions in complexes, the free ligand IR spec-
trum was compared with that of the corresponding metal
complexes (Figure S1). The main IR bands of the active
Hydrazo form
II
Azo form
I
SCHEME 1 Tautomeric forms of the phenolic moiety of the azo‐
dye

6 of 18
BAKR ET AL.
1
internal standard. D2O was then added to check for
exchangeable protons (Figure 2). The H NMR spectrum
of 10.7 ppm.[25] The H NMR spectrum also confirmed
that the ligand exists in the azo keto form in solution
which is in agreement with IR spectra which revealed
the appearance of strong ν(C = O) band of the
pyrazolone ring suggesting that the pyrazolone side of
the azo‐dye is in the keto form, not enol form.[26] The
multiplet at 7.0–8.1 ppm was due to the aryl protons of
the two benzene rings. The 1H NMR spectrum
(Figure 2) of the diamagnetic Pt(II) complex exhibited
all the peaks of the ligand except the enolic OH peak
at δ 14.01 ppm. This indicates the coordination through
enolic oxygen where the metal has replaced the H
1
of azo ligand HL showed a singlet due to the hydrogen
bonded OH proton at 14.01 ppm. It is well known that
hydrogen bonded OH proton resonance appears at a
lower field than that of NH proton resonance.[24] There-
fore, it may be concluded that the ligand existed in the
1
azo form I in solutions (Scheme 1). The H NMR spec-
trum of the investigated ligand exhibited a sharp singlet
at 12.05 ppm which was assigned to the NH proton of
the pyrazolone ring. The proton at 4 position of the
pyrazolone ring appeared also as a singlet in the region
FIGURE 2 1H NMR spectra of HL and its Pt(II) complex

BAKR ET AL.
7 of 18
proton leading to the formation of a five‐membered che-
late. In the aromatic region, the differences between the
ligand and the complex were observed to be significant
due to the changing electronic nature of the
structures.[27]
N
HO
O
N
N
NH
m/z =174 (11%)
m/z =108 (Base peak)
HO
N
N
3.2.3 | EI mass spectra of HL and their
metal chelates
m/z =77 (90%)
m/z =280 (90%)
Molecular ion peak
N
N
H
O
-N2
The EI mass spectra of all metal chelates showed
molecular ion peak which confirmed the proposed for-
mula of the suggested compounds under investigation
(Figure S2). The mass spectrum of HL showed the M
peak at m/z 280 which is in agreement with the molecu-
lar formula C15H12N4O2. The peak at 252 pertained to
(M–N2)+ which is very characteristic of an azo group.
The base peak at m/z 174 indicated the fragment
[M–(C6H5N2]+. The other peaks of importance are
shown at m/z 160 [M‐(C9H7NO)], m/z 93 [M‐C6H5O)]
which also support the azo structure; where N = N cleav-
age occurs. Other prominent peaks were marked in the
spectrum. The EI mass spectra of metal complexes were
found to have very conspicuous M+ (m/z 617) peak
which affirmed the stoichiometry ML2 1:2 for Ni(II)
complex, M+ (m/z 439) peak for Pd(II) complex and M+
(m/z 545) peak for Pt(II) complex that confirmed the stoi-
chiometry ML 1:1 in both cases. The peak at (m/z 280)
which appeared in all spectra of metal complexes is that
of the ligand. Other prominent peaks were 421 (M–H2O)
in the case of Pd(II) complex and 509 [M–(2H2O)] in the
case of Pt(II) complex which further support the structure
of these complexes. The data of EI mass spectra of the
investigated metal complexes 1, 2 and 3 agree well with
their structural formula [Ni (L)2], [PdLCl](H2O) and
[PtLCl(H2O)](H2O), respectively. The fragmentation pat-
terns proposed for the mass spectra of the ligand HL
and Pt(II) complex ‘as a representative example’ in accor-
dance with the m/z of ions produced under electron
impact are shown in Schemes (2and 3).
HO
O
+
N
NH
m/z =160 (11%)
m/z =93 (11%)
-2H+
+H+
H2O
H2
O
N
N
m/z =77 (90%)
m/z =64 (90%)
SCHEME 2 Cleavage pathway of HL
band observed at wavelength 447 nm arises from a tran-
sition involving electron migration along the entire con-
jugate system of the ligand, i.e. it comprised a charge
transfer (C.T.) from the hydroxyphenyl ring to the car-
bonyl group by resonance and from hetero‐ring by induc-
tion.[28] In the UV–vis spectra of the investigated metal
complexes (Figure 3), the former band assigned to π–π*
transitions was unaffected by a complexation, whereas
the band assigned to n–π* transitions underwent a red
shift indicating that the azo nitrogen was involved in
the coordination with metal ions. The latter band could
be considered mainly due to metal–ligand charge‐trans-
fer transitions. In the case of Ni(II) complex LMCT band
has undergone much broadening extending to around550
nm. The bands of very low intensity are observed at 688,
720 and 767 nm which may be ascribed to d–d transition
within the nickel metal ion probably indicating a tetra-
hedral geometry. The magnetic moment of a regular tet-
rahedral Ni(II) should be approximately between 3.5 and
3.2 B.M.at room temperature, but it is appreciably
reduced if a distortion occurs.[29] The observed magnetic
moment value was found to be 2.86 B.M. which is a little
lower than specified limits for a regular tetrahedral one,
which means some distortion.[30] The distortion pro-
duced in a tetrahedral geometry may be attributed to a
steric hindrance produced by bulky two molecules of
ligand. In the case of Pd(II) and Pt(II) complexes, the
LMCT band appeared in the free ligand was shifted to
a longer wavelength 505 and 495 nm, respectively. This
band could be considered as a composite of charge trans-
fer transitions and some d → d transitions; the tails of
which obscured the weaker d‐d transitions. The magnetic
3.2.4 | Electronic spectra and magnetic
moments measurements
The room temperature magnetic moments, band posi-
tions (Nujol mull) and the proposed geometry of the
investigated compounds were given in Table S3. The
electronic spectrum of the free HL in Nujol mull
(Figure 3) displayed three characteristic absorption
bands. The first band at 245 nm was assigned to π–π*
transitions within both the pyrazolone and the phenyl
rings. While the second band at 314 nm was ascribed
to n–π* transition of the carbonyl (C = O) and azo
(N = N) groups within the system. The broad absorption

8 of 18
BAKR ET AL.
-H2O
-H2O
N
N
N
N
.H2O
O
O
N
N
O
Pt
Pt
Pt
N
Cl
N
Cl
OH2
N
Cl
OH2
N
H
O
N
H
O
N
H
O
m/z =509 (74.5%)
m/z =545 (61%)
Molecular ion peak
m/z =527 (63%)
+H+
-HCl
N
N
O
Pt
N
N
H
O
m/z =473 (55.5%)
PtO
+
N
N
N
N
O
m/zH=263 (61%)
m/z =211 (61%)
SCHEME 3 Cleavage pathway of Pt(II) complex
3.2.5 | Thermogravimetric and kinetic
parameters
The TG and DTG curves of Ni(II) 1, Pd(II) 2 and Pt(II) 3
complexes were recorded at an ambient temperature up
to 800°C under N2 gas flow and a heating rate suitably
controlled at 20 °C/min. The stages of decomposition,
temperature range, DTG peak temperature, decomposi-
tion product loss as well as found and the calculated
mass loss percentages were shown in Table S4. The TG
and DTG curves including the three representative com-
plexes were shown in Figure 4. The TG curve of [Ni
(L)2], 1, showed two major thermal processes. The first
lying in the region 265–377 °C with DTG peak at
280 °C, has a mass loss of 39.20 (Calcd.38.91%) corre-
sponding to decomposition of a part of the organic moi-
ety of the chelate 2(C6H4N2O) moles per mole of
[Ni(L)2]. The second thermal process was in the region
377–659 °C corresponding to the decomposition of the
rest of organic moiety of the chelate with DTG peak at
451 °C. There was no further mass loss beyond the latter
temperature ranges and the thermogram showed a pla-
teau corresponding to the formation of stable NiO as
FIGURE 3 Nujol mull electronic spectra of HL and its Ni(II) 1,
Pd(II) 2 and Pt(II) 3 complexes
moment values of Pd+2 and Pt+2 complexes indicated
that these complexes are diamagnetic as expected for a
square planar d8 system. These obscured d‐d bands may
1
1
1
1
be assigned to A1g → A2g(ν1) and A1g → B1g(ν2) tran-
sitions, respectively.[31]

BAKR ET AL.
9 of 18
FIGURE 4 TG and DTG curves of Ni(II) 1, Pd(II) 2 and Pt(II) 3 complexes of HL
revealed by the mass percentage of the end product. Both
thermograms of [PdLCl]H2O, 2, and [PtLCl(H2O)]H2O,
3, showed three decomposition steps. The thermogram
of [PdLCl]H2O, 2, showed a mass loss of 3.92 (Calcd.4.
1%) in the temperature range 30–145 °C with DTG peak
at 66.5 °C, corresponding to volatilization of one water
molecule of lattice type. In the 145–402 °C range with a
DTG peak at 355 °C, a mass loss of 15 (Calcd.14.45%)

10 of 18
BAKR ET AL.
was observed. This mass loss was consistent with elimi-
nation of N2 plus Cl atom. On further heating, the TG
curve showed the decomposition of the organic moiety
of the chelate till a constant mass is obtained where the
metal oxide residue (PdO) was formed as the final prod-
uct. Inspection of the TG curve for 1:1 Pt(II) complex
revealed an initial mass loss occurring at a temperature
less than 120 °C which was attributed to the loss of water
of hydration during the drying of the complex. At higher
temperature from 120–333 °C with a DTG peak at
176 °C, loss of one molecule coordinated water and elim-
ination of a Cl atom occurred. A third decomposition
step, in the 333–680 °C range, brought about a mass loss
of 27.47 (Calcd 28.44%) corresponding to decomposition
of organic ligand moiety. The presence of carbon residue
as an end product with PtO at 800 °C indicate insuffi-
cient decomposition of the organic moiety which may
need higher temperature till 1000 °C or more.
Depending on the previous results from the elemental
analysis, conductivity, magnetic moment spectral studies
and the finally thermal studies the following structural
formulas Scheme 4 were proposed for the complexes
under study.
FIGURE 5 Optimized structure of HL and its Ni(II) 1, Pd(II) 2
and Pt(II) 3 complexes
3.3 | Computational studies
3D molecular modeling of the proposed structure of the
investigated ligand and their metal complexes was per-
formed using DFT method. The correct geometrical struc-
tures of the metal complexes were doubtless confirmed
from the modification of the molecular coordinates to
obtain reasonable low energy molecular geometries.
Quantum chemical calculations showed a tetrahedral
geometry for Ni(II) complex and a square planar geome-
try for both Pd(II) and Pt(II) complexes (Figure 5). Selec-
tive measurements of bond lengths, bond angles were
given in Tables 1, 2. By discussion of the optimized molec-
ular geometry from the two tables, the following remarks
are concluded:
1. The optimized C15‐N14 bond length of the investi-
gated ligand was elongated upon coordination with
Ni(II), Pd(II) and Pt(II) by about 0.05, 0.005 and
0.096 Å, respectively. This is referred to the formation
of the M‐N14 bond, which let the C15‐N14 becomes
weaker.
N
N
O
N
H
N
O
Ni
N
N
O
O
N
H
N
1
2. Upon complexation, the C15‐C20 bond length for HL
was shortened in the case of Ni(II), Pd(II) and Pt(II)
complexes by about 0.015, 0.074 and 0.054 Å, respec-
tively. This confirms that the coordination takes place
via N14 atom of azo group.
N
N
.H2O
O
Pd
Cl
N
O
N
H
2
3. Also, the coordination through the N14 atom of the
azo group produced a shortening in C15‐C16 bond
lengths, in the case of Ni(II) and Pt(II) complexes by
0.019 and 0.002 Å, respectively. But in the case of
Pd(II), the bond was elongated by 0.013 Å which
may be due to the participation of carbonyl group of
pyrazolone moiety in complexation.
N
N
.H2O
O
Pt
N
OH2
Cl
N
H
O
3
SCHEME 4 Proposed structures of HL metal complexes

BAKR ET AL.
11 of 18
TABLE 1 Selected bond lengths (a°) of HL compared to their metal complexes
HL
1
2
3
C4‐C3
1.524
1.554
1.215
1.373
1.460
1.262
1.40
C3‐C4
1.529
1.505
1.257
1.469
1.472
1.282
1.450
1.396
1.407
1.401
1.461
1.828
1.818
C24‐C25
C23‐C24
C23‐O27
C23‐N22
C24‐N28
N28‐N29
N29‐C30
C30‐C35
C30‐C31
C31‐C32
C31‐O36
N29‐Ni15
O36‐Ni15
‐‐‐‐
1.523
1.513
1.258
1.342
1.465
1.272
1.442
1.398
1.406
1.396
1.466
1.823
1.818
C3‐C4
1.532
1.542
1.267
1.355
1.485
1.299
1.405
1.337
1.438
1.427
1.423
2.068
2.016
2.353
C3‐C4
1.541
1.565
1.238
1.391
1.466
1.288
1.496
1.357
1.423
1.413
1.356
1.80
C4‐C5
C5‐O6
C5‐N1
C4‐N13
N13‐N14
N14‐C15
C15‐C20
C15‐C16
C16‐C17
C16‐O21
‐‐‐‐
C2‐C3
C2‐C3
C2‐C3
C2‐O6
C2‐N1
C3‐N13
N13‐N14
N14‐C16
C16‐C21
C16‐C17
C17‐C18
C17‐O37
N14‐Ni15
O37‐Ni15
‐‐‐‐
C2‐O6
C2‐O6
C2−N1
C2‐N1
C3‐N13
N13‐N14
N14‐C16
C16‐C21
C16‐C17
C17‐C18
C17‐O22
N14‐Pd15
O22‐Pd15
Pd15‐Cl23
‐‐‐‐
C3‐N13
N13‐N14
N14‐C16
C16‐C21
C16‐C17
C17‐C18
C17‐O22
N14‐Pt15
O22‐Pt15
Pt15‐Cl23
Pt15‐O24
‐‐‐‐‐
1.411
1.425
1.403
1.344
‐‐‐‐
1.963
2.381
1.949
‐‐‐‐
‐‐‐‐
‐‐‐‐
‐‐‐‐
‐‐‐‐
‐‐‐‐
‐‐‐‐
O6‐Pd15
2.104
TABLE 2 Selected bond angles (°) of HL compared to their metal complexes
HL
1
2
3
C3C4N13
113.342
109.830
111.911
103.645
128.572
127.782
C4C3N13
111.438
110.448
110.386
107.768
126.101
126.129
C25C24N28 113.084
C23C24N28 112.886
C24C25N26 109.691
C23C24N22 107.019
C24C23O27 126.499
N22C23O27 126.477
C24N28N29 123.075
N28N29C30 124.129
N29C30C31 113.342
N29C30C35 124.725
C30C31O36 118.235
C32C31O36 122.490
C4C3N13
114.629
118.285
111.595
105.373
128.431
126.194
C4C3N13
C2C3N13
C3C4N5
C2C3N1
C3C2O6
N1C2O6
C3N13N14
120.701
118.754
110.554
103.138
128.613
128.114
122.164
C5C4N13
C4C3N2
C4C5N1
C4C5O6
N1C5O6
C2C3N13
C3C4N5
C2C3N1
C3C2O6
N1C2O6
C2C3N13
C3C4N5
C2C3N1
C3C2O6
N1C2O6
C4N13N14 113.123
N13N14C15 116.903
N14C15C16 125.278
N14C15C20 115.279
C15C16O21 122.603
C17C16O21 118.418
C3N13N14 124.403
N13N14C16 124.579
N14C16C17 113.194
N14C16C21 125.269
C16C17O37 117.784
C18C17O37 122.530
C3N13N14 114.929
N13N14C16 119.313
N14C16C17 113.802
N14C16C21 124.601
C16C17O22 120.852
C18C17O22 110.839
N13N14C16 108.836
N14C16C17 108.868
N14C16C21 130.201
C16C17O22 118.945
C18C17O22 121.251
‐‐‐‐
‐‐‐‐
‐‐‐‐
‐‐‐‐
‐‐‐‐
‐‐‐‐
N14Ni15O37 93.816
N29Ni15O36 94.492
N14Pd15O22 82.542
N14
Pt15O22
86.212
N29Ni15O37 111.713
N14Ni15O36 110.461
O22Pd15Cl23 93.911
‐‐‐‐
O22Pt15Cl23 85.594
‐‐‐‐
‐‐‐‐
‐‐‐‐
‐‐‐‐
‐‐‐‐
‐‐‐‐
‐‐‐‐
‐‐‐‐
N14Pt15O24 102.549
Cl23Pt15O24 85.640
‐‐‐‐‐‐‐‐‐‐
‐‐‐‐
O6Pd15N14 92.151
O6Pd15Cl23 91.251
‐‐‐‐‐‐‐‐
4. C16‐O21 bond length in the case of investigated ligand
was largely elongated upon coordination with Ni(II),
Pd(II) and Pt(II) by about 0.117, 0.079, 0.012 Å,
respectively. This weakens the bond character which
facilitates the chelation with the metal ion and leads
to the formation of the M‐O21 bond.

12 of 18
BAKR ET AL.
5. The other bond lengths within the ligand HL were
somewhat affected due to the coordination to the
metal ion.
hybridization for Ni(II) complex, whereas, a square
planar geometry predicts dsp2 hybridization for Pd(II)
and Pt(II), respectively.
6. The bond angle N14C15C20 was increased upon com-
plex formation by 9.99, 9.32 and 14.92° for Ni(II),
Pd(II) and Pt(II), respectively and was also increased
by 11.28, 1.806 and 9.04° for the bond angle
C4N13N14. But the bond angle C15C16O21 was
decreased by 4.82, 1.75 and 3.66° upon complexation
with Ni(II), Pd(II) and Pt(II), respectively. Also, the
bond angles N13N14C15 and C17C16O21 were strongly
affected upon complexation, which was reduced or
increased due to the complex formation.
3.3.1 | Frontier molecular orbitals and
chemical reactivity
The highest occupied molecular orbital (HOMO) and the
lowest unoccupied molecular orbital (LUMO) are the
most important orbitals in a molecule. The conjugated
molecules are characterized by a small (HOMO–LUMO)
separation. The Eigen values of HOMO and LUMO and
their energy gap between HOMO‐LUMO reflect the bio-
logical activity of the molecule. As the frontier orbital
gap is small, the molecule is more polarizable which
7. The calculated bond angles around the central metal
ion for the investigated complexes lied in the range
of the reported tetrahedral geometry predicting sp3
FIGURE 6 3D Plots frontier orbital of HL and its Ni(II) 1, Pd(II) 2 and Pt(II) 3 complexes

BAKR ET AL.
13 of 18
means a high chemical reactivity as well as a low
kinetic stability.[13,14] The charge distribution of the
HOMO level in case of ligand HL was highly central-
ized on the lone pair of N atoms of the azo group,
and the π‐ charge of the phenyl moiety. Furthermore,
the charge distribution of the LUMO level was mostly
delocalized over the azo group and phenyl moieties of
the ligand molecules with π* antibonding character as
can be seen in Figure 6. Accordingly, there are two
transitions, n‐ π* and π‐ π*, for the ligand which agrees
well with the experimental observations. Also, this con-
firms the presence of an electron donation from the
ligand toward the metal atom.
orbital of Ni metal (Figure 6 (1)). Also, the charge distri-
bution of the LUMO level was delocalized over the azo
group and phenyl moiety with antibonding character
and dz2 orbital of the Ni atom. This means that the calcu-
lated FMO shows different types of electronic transitions
for the investigated complexes, which is in a good agree-
ment with the experimental ones. Also, this confirms that
there is a σ‐donation from the ligand to the d‐orbital of
the metal and a back donation from the d‐orbital of the
metal ion to the π* antibonding of the ligand, both
increase the stability of the formation of the complex.
The same is for Pd (II) and Pt (II) complexes (Figure 6
(2, 3)). The calculations showed that the theoretical geo-
metrical structure of the investigated ligand HL and its
metal complexes agree well with the suggested experi-
mental geometry.
For Ni(II) complex, the calculations showed that the
charge density of the HOMO level was centralized on
the lone pair of N atom, phenolic moiety and the dxz
FIGURE 7 Survival curve of the tumour cell line for 5‐fluorouracil, HL and their metal complexes

14 of 18
BAKR ET AL.
cell lines with IC50 ranging from 10.73 to 35.62 μg/ml.
This behavior indicates that all complexes exhibited anti-
tumor activity from strong to moderate activity against
the cell lines. The order of activity is found to be
Pd(II) > Ni(II) > Pt(II).
3.4 | Biological evaluation
3.4.1 | In vitro cytotoxic activities (MTT‐
reduction assay)
In vitro cytotoxicity tests were conducted utilizing the
synthesized ligand and their Ni(II), Pd(II), Pt(II) com-
plexes against four different human tumour cell lines
HePG‐2, HCT‐116, PC3 and MCF‐7 using a colorimetric
assay (MTT assay). This method measures the mitochon-
drial dehydrogenase activity as an indication of the via-
bility of cells. The cytotoxicity results were described by
growth inhibitory concentration value (IC50), which
means the concentration of the compounds required for
production a 50% inhibition of cell growth after incuba-
tion for 72 h, parallel to untreated controls. The IC50
values were calculated from the survival curve of the
tumour cell line which was plotted between concentra-
tion and % cell viability. A wide range of different con-
centrations of each test compound (1.56, 3.125, 6.25,
12.5, 25, 50 and 100 μg/ml) were established (Figure 7).
The results of IC50 concentrations of the synthesized
compounds parallel to the vastly utilized anticancer
drug, 5‐fluorouracil as standard were presented in
Table 3. Each data point was an average of three inde-
3.4.2 | Anti‐oxidant activity
Antioxidants can provide a safeguard to living organisms
from damage coming from controlled producing of reac-
tive oxygen species and subsequent lipid peroxidation,
protein damage and DNA strand breaking and thus anti-
oxidants reduce the risk for chronic diseases including
cancer and heart diseases.[33] The antioxidant assay of
HL and its metal complexes 1–3 were evaluated by two
in vitro methods in order to compare the results erythro-
cyte hemolysis (Table 4) and ABTS method (Table 5).
The ligand HL manifested a potent anti‐oxidative activity
in the two methods. The marked increase in antioxidant
activity of free ligand compared to the metal complexes
could be attributed to the increased charge transfer of
the condensed ring system, which increases its capacity
to stabilize unpaired electrons of the azo system and
thereby, to scavenge free radicals. This was in agreement
with the reported results for the synthesized 4‐benzoyl‐
3‐methyl‐1‐phenyl‐2‐pyrazolin‐5‐one phenylhydrazone
Bmpp‐Ph with metal ion coordination.[34] The order of
pendent experiments and expressed as the value
SD.
Observing the results, we found that the ligand HL
showed potential cytotoxic activity higher than the metal
complexes Ni(II), Pd(II) and Pt(II) indicating that metal
complexation to the ligand clearly gave less effective
compounds against the tumour cell lines. The ligand
HL showed a better cytotoxic activity than 5‐fluorouracil
as reference drug (IC50 5.69, 6.80 μg/ml) against the
HePG2 and PC3 cell lines respectively. Hence, it can be
suggested that such compound could be used in the
designing and improvement of new anticancer drugs.
Such result is in accordance with the study of Wang
et al.,[9] they evaluated the cytotoxic activity of 13
pyrazolone derivatives against four tumour cell lines.
On the other hand, the three complexes covered a broad
range of cytotoxic activity towards the different tumour
TABLE 4 Anti‐oxidant assays by erythrocyte hemolysis (A/B x 100)
Compound
H2O (B)
Absorbance of samples
0.885
% Inhibition
‐‐‐‐
Vit–V
0.040
0.061
0.096
0.084
0.109
4.5%
6.9%
HL
1
10.8%
9.5%
2
3
12.3%
TABLE 3 In vitro cytotoxic activity of synthesized compounds towards the selected human tumour cell lines
In vitro cytotoxicity IC50 (μg/ml)
Compounds
HePG2
7.9 0.28
HCT‐116
PC3
8.3 0.25
MCF‐7
5‐Fu
5.2 0.14
5.5 0.21
HL
1
5.69 0.5
15.31 1.4
10.73 1.0
28.30 2.2
9.70 0.8
16.93 1.7
14.65 1.3
20.59 2.0
6.80 0.4
17.30 1.5
11.45 0.9
9.32 0.7
27.47 1.6
12.96 1.1
35.62 1.9
2
3
20.40 1.8
[32]
• IC50 (μg/mL): 1–10 (very strong). 11–20 (strong). 21–50 (moderate). 51–100 (weak) and above 100 (non‐cytotoxic)
5‐FU = 5‐Fluorouracil

BAKR ET AL.
15 of 18
TABLE 5 Anti‐oxidant assays by ABTS method [(Abscontrol
Abstest) /Abscontrol x 100]
‐
ligand. The difference in the efficiency of the studied
complexes versus the tested organisms may be due to
the impermeability of the microbes' cells or the differ-
ences in ribosome of the cells of microbes or might be
due to the anti‐proliferative activity reported for
pyrazolone derivative. Here, the complexes are active
against both types of the bacteria G+ and G−, which
may indicate broad‐spectrum activities.[35]
Minimum inhibitory concentration (MIC) is consid-
ered as the ‘gold standard’. It was used for determination
of the susceptibility of organisms to antimicrobials and
hence to judge the performance of all other methods of
susceptibility testing. The MICs of the investigated com-
pounds were determined using the five selected patho-
genic bacteria mentioned above. The concentrations
used for determining MICs of are 2, 5, 10, 20 mg/ml. It
can be seen from Table 7 that most of the microorganisms
were vulnerable against our ligand HL and its metal
complexes, giving MIC values varying from 2 mg/ml to
20 mg/ml. Pt (II) complex showed the highest MIC activ-
ity, the growth of the microorganisms was inhibited at
Compound
Absorbance of samples
0.488
% Inhibition
0%
Control of ABTS
Ascorbic‐acid
0.052
0.078
0.204
0.180
0.365
89.3%
84.0%
58.2%
63.1%
25.2%
HL
1
2
3
antioxidant activity for the metal complexes was found to
be Pd(II) > Ni(II) > Pt(II).
3.4.3 | In vitro antimicrobial activity and
minimum inhibitory concentration (MIC)
The screened antimicrobial activity of the synthesized
compounds against the selected microorganisms was
measured in vitro utilizing Cut plug method.[19] The
ligand and its metal complexes exhibited a considerable
degree of antimicrobial activity expressed by inhibition
zones ranged from 11–17 mm as represented in Table 6.
On the basis of the measured inhibition zones
diameters', it was found that all the tested compounds
were moderate to highly active against the selected
pathogenic bacterial strains. On the other hand, no anti‐
fungal activity was detected for all compounds except
for palladium complex 2 which indicate the broad bio
activity of Pd(II). The reason of this behavior may need
further specialist studies to can determine the mechanism
of the reaction between the Pd complex and fungus like
TEM macrograph. The metal complexes showed a consid-
erably higher antimicrobial activity than that of the free
TABLE 7 MIC values of the ligand and its complexes
Compound
Microorganisms
(G+) bacteria
HL
1
2
3
B. Subtilis
S. Aureus
(G−) bacteria
E. Coli
20
20
10
10
2
5
2
2
20
20
20
10
5
10
5
2
2
2
S. Typhi.
Pro. Spp.
10
2
TABLE 6 Antimicrobial activities of the investigated ligand and its metal complexes expressed by inhibition zone diameter (mm)
Organism
(G+) bacteria
(G−) bacteria
Fungi
Compound
HL
B. subtilis
S. aureus
E. coli
S. Typhi
Pro. Spp.
C.Albicans
A. niger
10.7 0.6
13.1 0.1
12.8 0.8
12.1 0.2
12.2 0.3
‐‐
‐‐
1
15.3 0.6
16.1 0.2
14.2 0.3
10.7 0.6
17.1 0.1
14.9 0.9
14.3 0.6
13.1 0.1
16.1 0.2
15.1 0.2
14.2 0.3
12.8 0.8
15.5 0.5
16.1 0.2
16.7 0.6
12.1 0.2
15.3 0.6
16.6 0.5
17.2 0.3
12.2 0.3
‐‐
‐‐
2
8.2 0.2
10.1 0.1
3
Sta
‐‐
‐‐
‐‐
‐‐
Less active (1–5 mm)
Moderately active (6–14 mm)
Highly active (15–30 mm)[35]
aSlandered drug for gram‐positive bacteria is Miphinicol.
For gram‐negative bacteria is Ciprofloxacin and for fungi is Flucoral.

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BAKR ET AL.
concentrations as low as 2 mg/ml. The Ligand showed the
lowest degree of MIC activity with a value of 20 mg/ml on
all the organisms. Such results indicate that the metal
complexes displayed lower toxicity than the free ligand.
This behavior may be assigned to low lipophilicity of the
metal complexes than free ligand and/or their disability
to chelate further metals, which are substantial for the
microorganisms' metabolism.[35] The order of antimicro-
bial activity for the metal complexes was found to be ≈
Pd(II) > Ni(II) > Pt(II) in accordance to that in case of
antitumor and antioxidant activity.
in the presence of metal ions. Role of H2O2 has been
fully explained by K. Midorikawa et al.[36] In the absence
of H2O2, one sharp band without tail or smear with a
molecular weight of about 100 BP (Figure 8) was detected
for all treatments. This sharp band indicates the integrity
of resolved DNA, and the investigated HL or its metal
complexes are completely inactive to cleave DNA in
absence of H2O2. On the other hand, when H2O2 is
involved in the reaction mixture, no significant nuclease
activity was detected for the ligand or its metal com-
plexes in comparison to H2O2 (Figure 9). The above
results agree with the antioxidant activity recorded for
ligand and complexes which provide protection for
DNA from damage. As scavengers of hydroxyl and super-
oxide radicals were seen to reduce the extent of DNA
cleavage.[37] Although other studies reported DNA cleav-
age activity for some metals coordinated to ligand con-
taining pyrazolones moiety.[38] Further studies are
undergoing to clarify the binding to DNA as well as to
disclaim the nuclease activity of HL ligand and its
complexes.
Gel electrophoresis experiments were performed using
Deoxyribonucleic acid Sodium Salt, ACROS Organics™
with DMF, ligand and its metal complexes. When DNA
was subjected to electrophoresis, no significant nucleases
activity was detected for the ligand or its metal complexes
(Figure 8 and 9). This result supported by the recorded
antioxidant activity for the ligand and its complexes pro-
vides protection for DNA from damage during treatment
of tumour cell. As it is evident from the literature survey,
the metals coordinated to other ligands have a DNA dam-
age action but its complexation to the investigated ligand
reduced its nucleases activity even in the presence of the
oxidizing agent (H2O2). Based on the above results, the
anti‐tumor activity recorded for the synthesized structures
cannot be explained on the basis of DNA cleavage. And
the anti‐ proliferative behavior for these compounds
may be due to interference with DNA replication or other
metabolic activity. So, we can say that antitumor activity
of our compounds is not due to DNA cleavage but may
be referred to the mechanisms similar to that of the 5‐
fluorouracil drug.[39,40] Sites of binding between DNA
and ligand or its metal complexes must be studied. The
mechanism for antimicrobial and antitumor activity
needs more interpretational studies. In our future plan,
DNA binding and docking may help in the interpretation
of this mechanism.
3.5 | DNA cleavage studies
The effect of both HL and its metal complexes on Deoxy-
ribonucleic Acid Sodium Salt (DNA), ACROS Organics™
in absence and presence of H2O2 have been investigated
by agarose gel electrophoresis (Figures 8 and 9). H2O2 is
considered as strong oxidizing agent and the solvent used
is DMF. However, purified DMF did not induce DNA
damage even in the presence of metal ions. Addition of
purified DMF enhanced DNA damage induced by H2O2
FIGURE 8 Gel electrophoresis diagram showing DNA (0.5 μg)
incubated with HL and its metal complexes (100 μM): Lane 1,
DNA control; lane 2, DNA+ DMF; lane 3, DNA + HL (100 μ M);
lane 4, DNA + [Ni(L)2]; lane 5, DNA + [PdLCl](H2O); lane 6,
DNA + [PtLCl(H2O)](H2O)
FIGURE 9 Gel electrophoresis diagram showing cleavage of
DNA (0.5 μg) by ligand and the synthesized complexes (100 μM)
in presence of H2O2: Lane 1, DNA control; lane 2, DNA+
DMF + 50 mM H2O2; lane 3, DNA + HL (100 M) + 50 mM H2O2;
lane 4, DNA + [Ni(L)2] + 50 mM H2O2; lane 5, DNA + [PdLCl]
(H2O) + 50 mM H2O2; lane 6, DNA + [PtLCl(H2O)]
(H2O) + 50 mM H2O2
4 | CONCLUSION
Synthesis and characterization of the aryl azo
pyrazolone ligand HL and its Ni(II), Pd(II) and Pt(II)

BAKR ET AL.
17 of 18
[14] M. J. Frisch et al., Gaussian 03, revision Inc., PA, Pittsburgh
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complexes have been discussed. The results revealed a
1:2 (metal: ligand) stoichiometry for Ni(II) but a 1:1
for Pd(II) and Pt(II) which has been confirmed
theoretically by 3D molecular modeling using DFT
method. The compounds are biologically screened as,
anticancer against four different human tumour cell
lines, antioxidant and antimicrobial agents. The ligand
HL shows a potential cytotoxic activity higher than the
metal complexes against the four tested tumour cell
lines and higher than the reference drug 5‐fluorouracil
against the HePG2 and PC3 cell lines respectively. Also,
the free ligand HL shows much higher antioxidant
activity than the metal complexes. The order of antioxi-
dant activity for the metal complexes is found to be
Pd(II) > Ni(II) > Pt(II). On contrast, the complexes show
higher antibacterial activity than the free ligand. Pt (II)
complex shows the highest MIC activity 2 mg/ml,
whereas the ligand shows the lowest degree of MIC activ-
ity with a value of 20 mg/ml. DNA cleavage study pro-
vides an interpretation for the biological activity
depending on protection for DNA from damage in the
same time of treatment.
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How to cite this article: Bakr EA, Al‐Hefnawy
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Ni(II), Pd(II) and Pt(II) complexes coordinated to
azo pyrazolone ligand with a potent anti‐tumor
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