Synthesis, spectral characterization, thermal, anticancer and antimicrobial studies of bidentate azo dye metal complexes

Article abstract of DOI:10.1007/s10973-015-5172-1

As a part of systematic investigation of biologically active compound, m-phenylene diamine, new azo dye ligand was synthesized by diazotization of m-phenylene diamine and coupled with coupling compound, p-methoxy benzaldehyde. A new series of Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) complexes derived from this azo dye ligand (L) were synthesized. The structures of the ligand and metal complexes were confirmed by elemental analysis, spectroscopic studies (IR, UV–Vis, ¹H NMR, mass spectrometry, electronic spectra, magnetic susceptibility and ESR), conductivity measurements, thermogravimetric analyses (TG-DTG) and X-ray powder diffraction. The molar conductance measurements of the complexes in DMF determine electrolytic nature of the complexes. On the basis of elemental and thermal analyses, magnetic moment, electronic and ESR spectral studies, an octahedral geometry was assigned for metal complexes. XRD data reflect that azo dye ligand and its Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II) and Zn(II) complexes are amorphous while Cd(II) complex is crystalline. Also, the newly synthesized azo dye ligand, in comparison with metal complexes, is screened for its antimicrobial and anticancer activity against breast cancer cell line (MCF7). The results showed that Mn(II), Ni(II) and Zn(II) metal complexes have higher anti-breast cancer activity than free ligand.

Full text of DOI:10.1007/s10973-015-5172-1

J Therm Anal Calorim
DOI 10.1007/s10973-015-5172-1
Synthesis, spectral characterization, thermal, anticancer
and antimicrobial studies of bidentate azo dye metal complexes
1
1
1
Walaa H. Mahmoud
•
M. M. Omar
•
Fatma N. Sayed
Received: 26 August 2015 / Accepted: 15 November 2015
Ó Akad e´ miai Kiad o´ , Budapest, Hungary 2015
Abstract As a part of systematic investigation of bio-
logically active compound, m-phenylene diamine, new azo
dye ligand was synthesized by diazotization of m-pheny-
lene diamine and coupled with coupling compound, p-
methoxy benzaldehyde. A new series of Cr(III), Mn(II),
Fe(III), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) complexes
derived from this azo dye ligand (L) were synthesized. The
structures of the ligand and metal complexes were con-
firmed by elemental analysis, spectroscopic studies (IR,
and Zn(II) metal complexes have higher anti-breast cancer
activity than free ligand.
Keywords p-Methoxybenzaldehyde Á Spectroscopy Á
Kinetics studies Á Antimicrobial activity Á Anticancer
activity
Introduction
1
UV–Vis, H NMR, mass spectrometry, electronic spectra,
magnetic susceptibility and ESR), conductivity measure-
ments, thermogravimetric analyses (TG-DTG) and X-ray
powder diffraction. The molar conductance measurements
of the complexes in DMF determine electrolytic nature of
the complexes. On the basis of elemental and thermal
analyses, magnetic moment, electronic and ESR spectral
studies, an octahedral geometry was assigned for metal
complexes. XRD data reflect that azo dye ligand and its
Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II) and Zn(II)
complexes are amorphous while Cd(II) complex is crys-
talline. Also, the newly synthesized azo dye ligand, in
comparison with metal complexes, is screened for its
antimicrobial and anticancer activity against breast cancer
cell line (MCF7). The results showed that Mn(II), Ni(II)
In the last few years, chemists have taken a growing
interest in the synthesis and physico-chemical studies of
first row transition metal complexes with a number of azo
dye ligands. Metal complexes of azo dye ligand have
played a central role in the development of coordination
chemistry [1]. Azo compounds are a very important class
of chemical compounds receiving attention in scientific
research. They are highly colored and they were studied
widely because of their excellent thermal and optical
properties in applications such as optical recording med-
ium, toner, ink-jet printing and oil-soluble lightfast dyes
[2]. Azo compounds are known to be involved in a number
of biological reactions, such as inhibition of DNA, RNA,
and protein synthesis, nitrogen fixation, and carcinogenesis
[
3]. Azo compounds were the subject of many spectral
studies for purpose of identification or structure elucida-
tion, as they are capable of forming metal chelates with
transition metal ions [4]. Metal-azo complexes are cur-
rently being involved as recording layers in a number of
high-density optical storage systems such as blue-ray discs
(
BDs) and high-density DVDs (HD-DVDs). For this pur-
&
Walaa H. Mahmoud
dr.walaa@yahoo.com
0
0
pose, new azo dye ligands derived from (3,3 -((1Z,1Z )-
pyridine-2,6-diylbis(diazene-2,1-diyl))bis(4-methoxyben-
zaldehyde)) was prepared using the recommended meth-
ods. The composition and stability of the complexes of the
1
Chemistry Department, Faculty of Science, Cairo University,
Giza 12613, Egypt
123

W. H. Mahmoud et al.
newly synthesized azo dye ligand with Cr(III), Mn(II),
Fe(III), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) were
and shaked carefully till complete dissolution. The med-
ium was then sterilized by filtration in a Millipore bac-
terial filter (0.22 lm). The prepared medium was kept in
a refrigerator (4 °C) and checked at regular intervals for
contamination. Before use, the medium was warmed at
37 °C in a water bath and supplemented with penicillin/
streptomycin and FBS. Sodium bicarbonate (Sigma
Chemical Co., St. Louis, Mo, USA) was used for the
preparation of RPMI-1640 medium. 0.05 % isotonictry-
pan blue solution (Sigma Chemical Co., St. Louis, Mo,
USA) was prepared in normal saline and was used for
viability counting. 10 % fetal bovine serum(FBS) (heat
1
investigated using elemental analyses, IR, H NMR, ESR,
UV–Vis, XRD, conductivity, mass spectra and magnetic
susceptibility measurements at room temperature, thermal
analyses. The azo dye ligand and its metal complexes were
screened for their antimicrobial and anticancer activity.
Experimental
Materials and reagents
-
1
inactivated at 56 °C for 30 min), 100 units mL peni-
-
1
All chemicals used in this study were of pure grade and of
highest purity available and used without further purifica-
tion. The chemicals used included m-phenylene diamine
cillin and 2 mg mL streptomycin were supplied from
Sigma Chemical Co., St. Louis, Mo, USA, and were used
for the supplementation of RPMI-1640 medium prior to
use. 0.025 % (w/v) trypsin was used for the harvesting of
cells, 1 % (v/v) acetic acid was used for dissolving the
unbound SRB dye, and 0.4 % sulforhodamine-B (SRB)
dissolved in 1 % acetic acid was used as a protein dye.
Stock solution of trichloro acetic acid (TCA, 50 %) was
prepared and stored. All of them were supplied from
Sigma. Fifty microliters of the stock solution was added
to 200 ll RPMI-1640 medium/well to yield a final con-
centration of 10 % used for protein precipitation. 100 %
isopropanol and 70 % ethanol were used. Tris base
10 mM (pH 10.5) was used for SRB dye solubilization.
121.1 g of tris base was dissolved in 1000 mL of distilled
water and pH was adjusted by HCl (2 M).
(
Sigma), p-methoxy-benzaldehyde (Aldrich), CrCl Á6H O
3
2
(
Sigma), MnCl Á2H O (Sigma), NiCl Á6H O (BDH),
2
2
2
2
FeCl Á6H O (Prolabo), CoCl Á6H O (Aldrich), CuCl Á2H O
3
2
2
2
2
2
(
Merck), ZnCl (Strem Chemicals) and CdCl (Aldrich).
2 2
Organic solvents were spectroscopic pure from BDH inclu-
ded ethanol and dimethyl formamide. Hydrochloric acid,
sodium nitrite and sodium acetate (A.R.) were used.
Human tumor cell line (breast cell line MCF7) was
obtained frozen in liquid nitrogen (-180 °C) from the
American Type Culture Collection and was maintained in
the National Cancer Institute, Cairo, Egypt, by serial sub-
culturing.
Solutions
Measurements
-
3
Stock solutions of metal complexes of 1 9 10 M were
prepared by dissolving the accurately weighed amount of
the complexes in DMF for measuring conductivity. Solu-
tions of the azo dye ligand and metal complexes ranging
Microanalyses of carbon, hydrogen and nitrogen were
carried out at the Microanalytical Center, Cairo University,
Egypt, using CHNS-932 (LECO) Vario Elemental Ana-
lyzer. Analyses of the metals followed the dissolution of
-
4
-5
from 1 9 10 to 1 9 10 M were prepared by accurate
dilution of the previous prepared stock solutions to mea-
sure UV–Vis spectra.
the solid complexes in concentrated HNO , neutralizing the
3
diluted aqueous solutions with ammonia and titrating the
1
metal solutions with EDTA. H NMR spectra, as a solution
Solutions for anticancer activity studies
in DMSO-d6, were recorded on a 300 MHz Varian-Oxford
Mercury at room temperature using TMS as an internal
standard. Electron spin resonance spectra were also
recorded on JES-FE2XG ESR spectrophotometer at
Microanalytical Center, Tanta University. The X-ray
powder diffraction analyses were carried out using Philips
Analytical X-ray BV, diffractometer-type PW 1840.
Radiation was provided by copper target (Cu anode
2000 W) high-intensity X-ray tube operated at 40 kV and
25 mA. Divergence and the receiving slits were 1 and 0.2,
respectively. Mass spectra were recorded by the EI tech-
-
A fresh stock solution of 1 9 10 M of azo dye ligand
3
-
0.00402 g L ) was prepared in the appropriate volume
1
(
of DMF. Dimethyl sulfoxide (DMSO) used in cryop-
reservation of cells and RPMI-1640 medium were sup-
plied from Sigma. The medium was used for culturing
and maintenance of the human tumor cell line. The
medium was supplied in a powder form. It was prepared
as follows: 10.4 g medium was weighed, mixed with 2 g
sodium bicarbonate, completed to 1 L with distilled water
1
23

Synthesis, spectral characterization, thermal, anticancer and antimicrobial studies of…
nique at 70 eV using MS-5988 GS-MS Hewlett–Packard
instrument at the Microanalytical Center, National Center
for Research, Egypt. FTIR spectra were recorded on a
spectrophotometrically at 564 nm with an ELIZA micro-
plate reader (Meter tech. R 960, USA).
-
1
Perkin-Elmer 1650 spectrometer (4000–400 cm ) in KBr
pellets. The electronic spectra were recorded in DMSO at
Synthesis of azo dye ligand
-
p-CH OPhCHO
3
-
-
HCl
NaNO2
- CH COONa
3
- 1h stirring
N
N
N
N
NH2
N
H N
Ice bath (0 °C)
2
N
Ice bath (0 °C)
O
Cl–
+N
N+
Cl–
O
O
O
room temperature on Shimadzu UV–Visible mini-1240
spectrophotometer. The molar magnetic susceptibility was
measured on powdered samples using the Faraday method.
The diamagnetic corrections were made by Pascal’s con-
In 250-mL quickfit round bottom flask, m-phenylene dia-
mine (3.00 g, 27.78 mmol) was dissolved in ethanol
(50 mL). The solution was cooled with stirring in an ice
bath to 0 °C until it becomes clear, then HCl solution
(4 mL) was added. Solution of sodium nitrite (3.80 g,
55.60 mmol) in water (20 mL) was added dropwise during
10 min and the reaction mixture was further stirred for
20 min in an ice bath at 0–5 °C. The solution was added
dropwise to p-methoxybenzaldehyde coupling component
(7.56 g, 55.60 mmol) in ethanol (30 mL) and aqueous
solution of sodium acetate (4 g) as catalyst, with stirring in
an ice bath for a further 1 h. The product was collected by
filtration and washed with water and dried under vacuum at
room temperature. The physical and analytical data of the
isolated azo dye ligand are listed in Table 1. The com-
pound has high melting point, and it is found to be air
stable [5].
stant, and Hg[Co(SCN) ] was used as a calibrant. Molar
4
-
3
conductivities of 10 M solutions of the solid complexes
in DMF were measured using Jenway 4010 conductivity
meter. The thermogravimetric analyses (TG and DTG) of
the solid complexes were carried using a Shimadzu TG-
5
0H thermal analyzer in a dynamic nitrogen atmosphere
-
1
(
flow rate 20 mL min
-
)
with
a heating rate of
1
1
0 °C min . The percentage mass loss was measured
from the ambient temperature up to 1000 °C. Highly sin-
tered a-Al O was used as a reference. Diffused reflectance
2
3
spectra analyses were carried out at the Microanalytical
Center, Cairo University, Egypt. The antimicrobial activi-
ties were carried out at the Microanalytical Center, Cairo
University, Egypt. The anticancer activity was performed
at the National Cancer Institute, Cancer Biology Depart-
ment, Pharmacology Department, Cairo University. The
optical density (O.D.) of each well was measured
Synthesis of metal complexes
N
N
N
N
reflux 2–3 h
+
MCl yH O
x. 2
ML complex
H CO
3
OCH3
CHO
OHC
(
0.4g, 9.95 × 10–4 mmol
in DMF)
123

W. H. Mahmoud et al.
Table 1 Analytical and physical data of azo dye ligand and its metal complexes
-
1
-1
2
Compound
molecular formula)
Color/%yield
M.p./°C
% Found/Calcd.
l
eff/BM
K
m
/X mol cm
(
C
H
N
Cl
–
M
–
L
Reddish brown
(85)
98
[300
[300
206
65.68
4.65
13.54
(13.93)
9.58
–
–
(
[
(
[
(
[
(
[
(
[
(
[
(
[
(
[
(
C
22
H
18
N
4
O
4
)
(65.67)
44.20
(4.48)
3.86
Cr(L)(H
2
O)Cl]Cl
CrN
O) ]Cl
NiN
O)Cl]Cl
FeN
O) ]Cl
CoN
2
ÁH
2
O
Black
(83)
18.06
8.54
3.80
118
108
99
C
22
H22Cl
3
4
O
6
)
(44.26)
46.90
(3.69)
4.21
(9.39)
9.77
(17.85)
12.42
(8.72)
9.35
Mn(L)(H
2
2
2
Black
(87)
5.42
C
22
H22Cl
2
4
O
6
)
(46.81)
43.50
(3.90)
4.02
(9.93)
9.50
(12.59)
18.02
(9.75)
9.62
Fe(L)(H
2
2
ÁH
2
O
Black
(78)
5.29
C
22
H22Cl
3
4
O
6
)
(43.96)
46.10
(3.66)
4.21
(9.33)
9.97
(17.74)
12.63
(9.33)
10.45
(10.34)
11.24
(11.04)
11.39
(11.09)
11.86
(12.08)
19.44
(19.20)
Co(L)(H
2
2
2
Black
(90)
230
5.30
126
38
C
22
H22Cl
2
4
O
6
)
(46.48)
49.48
(3.88)
3.59
(9.86)
10.15
(10.53)
10.05
(9.78)
9.98
(12.51)
12.99
Ni(L)Cl
2
]
Black
(89)
[300
280
3.27
C
22
H18Cl
2
NiN
4
O
4
)
(49.65)
46.04
(3.39)
4.04
(13.35)
12.64
Cu(L)(H
2
O)Cl]ClÁH
2
O
Black
(85)
1.79
71
C
22
H22Cl
2
CuN
4
O
6
)
(46.11)
49.50
(3.84)
3.05
(12.40)
13.33
Zn(L)Cl
2
]ÁH
2
O
Black
(81)
225
Diam.
Diam.
18
C
22
H20Cl
2
ZnN
4
O
6
)
(49.07)
44.83
(3.34)
3.25
(10.41)
9.12
(13.20)
11.99
Cd(L)Cl
2
]
Black
(86)
162
8
C
22
H18Cl
2
CdN
4
O
4
)
(45.10)
(3.07)
(9.57)
(12.13)
Azo dye ligand was dissolved in DMF (0.4 g, 9.95 9
of fungi (Candida albicans) by diffusion agar technique,
then, transferred to a saturated disk with a tested solution in
the center of Petri dish (agar plates). All the compounds
were placed at four equidistant places at a distance of 2 cm
from the center in the inoculated Petriplates. DMSO served
as control. Finally, all these Petri dishes were incubated at
25 °C for 48 h where clear or inhibition zones were detected
around each disk. Control flask of the experiment was
designed to perform under the same condition described
previously for each microorganism but with dimethyl for-
mamide solution only and by subtracting the diameter of
inhibition zone resulting with dimethyl formamide from that
obtained in each case, so antibacterial activity could be
calculated [6, 7]. Amikacin and ketoconazole were used as
reference compounds for antibacterial and antifungal activ-
ities, respectively. All experiments were performed as trip-
licate, and data plotted were the mean value.
-
4
1
0
mmol) and ethanolic metal salts with the same molar
ratio of 1:1 (M: azo dye) (0.265 g Cr(III), 0.161 g Mn(II),
.269 g Fe(III), 0.237 g Co(II), 0.237 g Ni(II), 0.134 g
0
Cu(II), 0.136 g Zn(II) and 0.183 Cd(II)) were added to the
azo dye ligand solution. The mixture was heated under
reflux for 2–3 h with stirring. The precipitates were filtered
off, washed with ethanol followed by diethyl ether and
dried in a vacuum desiccator over anhydrous CaCl2.
Spectrophotometric studies
-
The absorption spectra were recorded for 1 9 10 and
4
-
5
9 10 M solutions of the azo dye free ligand and of its
5
metal complexes dissolved in DMF. The spectra were
scanned within the wavelength range from 200 to 700 nm.
Pharmacology
Anticancer activity
Antimicrobial activity
Potential cytotoxicity of the complexes was tested using the
method of Skehan and Storeng [8], cells were plated in
96-multiwell plate (104 cells/well) for 24 h before treatment
with the complexes to allow attachment of cell to the wall of
the plate. Different concentrations of the complexes under
A filter paper disk (5 mm) was transferred into 250 mL
flasks containing 20 mL of working volume of tested solu-
-1
tion (100 mg mL ). All flasks were autoclaved for 20 min
at 121 °C. LB agar media surfaces were inoculated with four
investigated bacteria (Gram-positive bacteria: Bacillus sub-
tilis and Staphylococcus aureus, Gram-negative bacteria:
Neisseria gonorrhoeae and Escherichia coli) and one strain
-1
investigation (0, 5, 12.5, 25, 50 and 100 lg mL ) were
added to the cell monolayer triplicate wells and were pre-
pared for each individual dose. The monolayer cells were
incubated with the complexes for 48 h at 37 °C and in 5 %
1
23

Synthesis, spectral characterization, thermal, anticancer and antimicrobial studies of…
CO atmosphere. After 48 h, cells were fixed, washed and
2
stained with SRB stain. Excess stain was washed with acetic
acid and attached stain was recovered with tris–EDTA
buffer. The optical density (O.D.) of each well was mea-
sured spectrophotometrically at 564 nm with an ELIZA
microplate reader, and the mean background absorbance
was automatically subtracted and mean values of each drug
concentration was calculated. The relation between surviv-
ing fraction and drug concentration is plotted to get the
survival curve of breast tumor cell line for each complex.
Calculation
The percentage of cell survival was calculated as follows:
Survival fraction ¼ O:D:ðtreated cellsÞ=O:D:ðcontrol cellsÞ:
The IC50 values (the concentrations of the azo dye
ligand or its complexes required to produce 50 % inhibi-
tion of cell growth) were calculated. The experiment was
repeated three times for MCF7 cell line.
Results and discussion
Characterization of azo dye ligand
Free ligand and its metal complexes are soluble in DMF
and DMSO solvents and have high and sharp melting
points which indicated their pure nature. The IR spectrum
of the free azo dye ligand was carried out in the range of
-
1
4
000–400 cm , and the most effective bands are listed in
-
Table 2. The small band observed at 1680 cm , sharp
1
-
1
-1
band at 1599 cm and medium band at 1164 cm can be
attributed to C=O stretching [9], N=N [10] and C–O
methoxy [11], respectively.
1
H NMR spectrum of free azo dye ligand (L) in DMSO-d6
exhibited the following signals: 7.05–8.59 ppm (m, 10H,
Ar–H) [12], 3.85 ppm (s, 6H, OCH ) [4] and 9.86 ppm (s,
3
2
H, CHO) [13]. These signals indicated that three different
types of protons are present in the azo dye ligand.
The mass spectrum of the azo dye ligand displays the
?
predominant molecular ion peak (M ) at m/z = 402 with
complete agreement with results of elemental analysis. The
?
base peak appeared at m/z = 135 due to [C H O ] ion.
8
7 2
The other fragments give the peaks at 76, 104, 132, 135,
07, 239, 267, 282, 338 and 370 a.m.u. with various
2
intensities (Scheme 1).
Characterization of metal complexes
Elemental analyses
The stoichiometry and formulation of the free azo dye
(
L) ligand (Fig. 1) and its metal complexes were confirmed
123

W. H. Mahmoud et al.
Scheme 1 Suggested mass
fragmentation pattern of azo dye
ligand (Route 1)
N
N
N
N
N
N
N
–
C H O
8
7
2
N
O
O
OCH3
OHC
CHO
OHC
–
1
m/z = 267 g mol
f. = 269, R.I. = 47 %)
–
1
m/z = 402 g mol
(
(
f. = 402, R.I. = 42 %)
–N2
–
C H O
8
7
2
N
N
H CO
3
–
C H
6 4
CHO
–
1
N
N
m/z = 239 g mol
f. = 238, R.I. = 56 %)
N
N
(
–1
N
N
m/z = 132 g mol
(
f. = 132, R.I. = 51 %)
OCH3
–
C H O
8 7
2
–
2N2
OHC
m/z = 163 g mol
N
N
–
1
–N2
(
f. = 165, R.I. = 42 %)
–
N2
–
1
m/z = 104 g mol
f. = 104, R.I. = 44 %)
–1
m/z = 76 g mol
f. = 76, R.I. = 46 %)
(
(
CHO
H CO
3
–
1
m/z = 135 g mol
(
f. = 135, R.I. = 100 %)
base peak
by their elemental analysis (Table 1). The metal/ligand
ratio was found to be 1:1 in all complexes, which have
been arrived by estimating the carbon, hydrogen, nitrogen,
chloride and metal contents of the complexes. The ele-
mental analyses of the ligand and its complexes reveal
good agreement with the proposed structures. The ligand
and its metal complexes have high melting points, and they
are found to be air stable. The azo dye ligand is soluble in
common organic solvents, and all the complexes are freely
soluble in DMF and DMSO but insoluble in methanol,
ethanol and water.
[ML(H
x = 2, y = 1 and Cu(II); x = 1, y = 1.
[ML(H O) ]Cl where M = Mn(II), Co(II); x = 2.
[MLCl O where M = Ni(II), Zn(II) and Cd(II);
2
O)Cl]Cl
x
ÁyH
2
O where M = Cr(III), Fe(III);
2
2
x
]ÁyH
2
2
y = 0 for Ni(II), Cd(II); y = 1 for Zn(II).
IR spectral studies
The important infrared spectral bands and their tentative
assignments are discussed here. The IR spectra of the free
ligand (L) and its metal complexes were carried out in the
Molar conductance measurements
-
1
range of 4000–400 cm and the most effective bands are
listed in Table 2.
Molar conductivity measurements (Table 1) showed that
Cr(III), Mn(II), Fe(III) and Co(II) complexes are elec-
trolytes of the type 1:2, while Cu(II) complex is 1:1 elec-
trolytic type (Table 1). The data showed that Ni(II), Zn(II)
and Cd(II) chelates are non-electrolytes [14, 15]. These
results lead to general formulae:
On comparing the IR spectra of free azo dye ligand
(L) and its metal complexes, the bands at 1599 and
-
1
1164 cm in the ligand due to stretching t(N=N) and
t(C–O methoxy) groups, respectively, were shifted on
complexation and appeared at 1600–1606 and
1
23

Synthesis, spectral characterization, thermal, anticancer and antimicrobial studies of…
fragments and support the proposed structures (Fig. 2.).
Some of the fragments observed in the mass spectra of the
azo dye ligand (L) and its Co(II) and Cd(II) complexes are
shown in fragmentation Schemes 1–5.
N
N
N
N
O
O
UV–Vis absorption studies
O
O
UV–Vis spectra of azo dyes are generally affected by their
chemical structures such as chromophores, substituted
groups, number of azo groups, metal ions, pH values,
solvents. [20–24]. In order to explore and compare the
differences between free ligand and its metal complexes,
UV–Vis spectra of the azo dye ligand, Mn(II) and Co(II)
Fig. 1 Structure of the newly synthesized azo dye ligand
-
166–1173 cm , respectively, indicating the involvement
1
1
of nitrogen of azo group in chelation and coordination of
oxygen of methoxy group to metal ions [9–11]. Also,
1
aldehydic carbonyl group showed band at 1680 cm in
-
-5
-1
complexes in their 5.0 9 10 mol L DMF solutions
-
1
the free azo dye ligand which shifted to 1640–1660 cm
and Cr(III), Fe(III), Ni(II), Cu(II), Zn(II) and Cd(II) com-
-1
-
4
in the metal complexes. This explained as binding of the
azo group (which has withdrawing nature) to the metal ions
via its lone pair of electrons will lead to decrease in the
electron withdrawing effect of aldehydic group and hence
affect the position of aldehydic band in the metal com-
plexes of concentration 1.0 9 10
mol L
are deter-
mined. The results showed only one peak observed at
283 nm in the free azo dye ligand which assigned to the
moderate energy p–p* transition of the aromatic rings. This
peak showed shifting in the metal complexes as it appears
at 273–280 nm [2, 25].
-
1
plexes (1640–1660 cm ) [16].
In the far IR spectra of all the complexes, the non-ligand
-
1
bands observed at 521–570 and 420–442 cm
region
Electronic spectra and magnetic moment
measurements
assigned to t(M–O) [9, 16] and m(M–N) stretching vibra-
tions, respectively [10, 17].
The coordinating water in the Cr(III), Mn(II), Fe(III), Co(II)
and Cu(II) complexes is characterized by the appearance of
Cr(III) binary chelate with octahedral symmetry showed three
-1
spin allowed bands at 19,250, 20,110 and 21,350 cm . These
4
-1
4
m(M–O) at 517–524 cm . Also the stretching vibrations at
bands may be assigned to
4
A
2g(F) ?
T2g(F),
-
1
4
4
8
31–835 and 920–950 cm assigned to m(M / OH ), sup-
A2g(F) ? T1g(F) and A2g(F) ? 4T1g(P) transitions indi-
2
ported for the participation of water in coordination [18, 19].
cating the octahedral geometry of the complex. The magnetic
moment at room temperature is 3.80 B.M. which corresponds to
the expected value for octahedral Cr(III) complexes [26, 27].
Mn(II) complex showed a broadband centered at
1
H NMR spectral studies
-
1
6
4
16,500 cm
which corresponds to A1g ? T (4G)
1g
1
The HNMR spectra of the azo dye ligand and its Zn(II)
transition, and its magnetic moment value is found to be
5.42 B.M. This indicates an octahedral environment around
the Mn(II) ion [26].
and Cd(II) complexes are recorded in (DMSO-d ) solu-
6
tion using tetramethylsilane (TMS) as internal standard.
1
HNMR spectrum of the ligand revealed the following
The diffused reflectance spectrum of the Fe(III) chelate
-
displays band at 26,080 cm , which may be assigned to
1
signals at d = 3.85 (s, 6H, OCH ) [4], 7.05–8.59 (m,
3
6
5
1
0H, Ar–H) [12] and 9.86 ppm (s, 2H, CHO) [13], while
the A ? T (G) transition in octahedral geometry of
1
g
2g
1
the H NMR spectra of the Zn(II) and Cd(II) complexes
the complex [21, 24]. The two bands at 18,390 and
5
-
1
6
revealed the following signals at d = 3.73–3.86 (m, 6H,
OCH ), 7.05–7.94 (m, 10H, Ar–H) and 9.80–9.86 ppm
16,400 cm are the result of splitting of A ? T
1
g
1g
3
transition. The observed magnetic moment value of Fe(III)
(
s, 2H, CHO). It is found that the signal observed at
.33 ppm for Zn(II) complex is assigned to hydrated
complex is found to be 5.29 B.M. indicating octahedral
2
3
3
geometry with d sp hybridization in Fe(III) complex
[26, 28].
H O protons. The shift in the methoxy protons indicated
2
that oxygen atom of OCH groups is coordinated to the
3
Co(II) complex exhibits three bands at 8970, 13,500 and
-
1
4
4
Zn(II) and Cd(II) ions.
19,760 cm
4
which corresponds to T (F) ? T (F),
1
g
2g
4 4 4
T1g (F) ? A (F) and T (F) ? T (P), respectively,
2g
1g
1g
Mass spectral studies
and the observed magnetic moment value (5.20 B.M.)
indicates that it has an octahedral environment.
Mass spectral data of free azo dye ligand and its Co(II) and
Cd(II) complexes were consistent with the molecular ion
The nickel(II) complex showed four bands at 10,140,
-
16,125, 24,344 and 29,890 cm which are assigned to
1
123

W. H. Mahmoud et al.
Fig. 2 Mass spectra of (a) azo
dye ligand (L), (b) L–Cd
complex, (c) L–Co complex
(
a)
100
90
8
7
6
0
0
0
5
4
3
2
1
0
0
0
0
0
50
70
90
110
130
150
170
190
210
230
m/z
250
270
290
310
330
350
370
390
(
b)
100
9
9
8
8
7
7
6
6
5
5
4
4
3
3
5
0
5
0
5
0
5
0
5
0
5
0
5
0
2
2
5
0
Cd complex
(585 g/mol)
1
1
5
0
5
0
L (402 g/mol)
5
0
100
150
200
250
300
350
400
450
500
550
m/z
(
c)
100
9
5
9
8
8
0
5
0
7
7
6
6
5
5
4
4
3
3
2
2
1
1
5
0
5
0
5
0
5
0
5
0
5
0
5
0
5
0
Co complex
(568 g/mol)
L (402 g/mol)
5
0
100
150
200
250
300
m/z
350
400
450
500
550
1
23

Synthesis, spectral characterization, thermal, anticancer and antimicrobial studies of…
Scheme 2 Suggested mass
fragmentation pattern of azo dye
ligand (Route 2)
N
N
N
N
N
N
N
N
–CH3O
O
O
OCH3
CHO
OHC
CHO
OHC
–
1
–
1
m/z = 371 g mol
f. = 371, R.I. = 52 %)
m/z = 402 g mol
(
(
f. = 402, R.I. = 42 %)
–
CH O
3
N
N
N
–2CO, H2
N
N
N
N
N
–
1
CHO
m/z = 282 g mol
f. = 282, R.I. = 50 %)
OHC
–
1
m/z = 338 g mol
f. = 336, R.I. = 48 %)
(
(
–
C H
6 3
N
N
N
N
–
1
m/z = 207 g mol
f. = 208, R.I. = 50 %)
(
–
C H
6 3
–2N2
N
N
N
N
–
1
–
1
m/z = 76 g mol
f. = 76, R.I. = 46 %)
m/z = 132 g mol
f. = 132, R.I. = 51 %)
(
(
4
4
4
4
4
4
T (F) ? T (F), T (F) ? A (F), T (F) ? T
Cu(II) complex showed three bands at 12,122, 21,800
-
1
g
2g
1g
2g
1g
1g
1
2
(
P) and L ? M charge transfer, respectively. The calcu-
and 23,224 cm
2
corresponding to
2
B1g ? A ,
2
1g
2
lated magnetic moment (3.27 BM) confirms the octahedral
geometry [28].
B1g ? B and B1g ? E transitions in octahedral
2
2g
g
geometry [26, 29]. The magnetic moment of this complex
123

W. H. Mahmoud et al.
Scheme 3 Suggested mass
fragmentation pattern of Co(II)
complex (Route 1)
N
N
N
N
N
N
N
OH2
OH2
N
–Cl2
O
O
O
O
Co
Co
·Cl2
O
O
O
O
OH2
m/z = 497 g mol
(f. = 494, R.I. = 7.5 %)
OH2
m/z = 568 g mol
f. = 567, R.I. = 4.8 %)
–1
–
1
(
–H2
Co(OH)2
–
2H O
2
–
O
–
2
N
N
N
N
N
N
N
N
O
O
Co
O
O
O
O
–
1
m/z = 370 g mol
–1
m/z = 461 g mol
(
f. = 368, R.I. = 8 %)
(
f. = 463, R.I. = 4.1 %)
–
2C H O
8 10
–2N2
–
1
N
N
m/z = 76 g mol
f. = 77, R.I. = 77 %)
N
(
N
–
1
m/z = 132 g mol
(
f. = 135, R.I. = 32 %)
is 1.79 B.M. which fall within the range normally observed
for octahedral Cu(II) complex [29].
consistent with slightly distorted structure and showed poor
in-plane p bonding.
Zn(II) and Cd(II) complexes are diamagnetic and octa-
hedral geometry was proposed according to the proposed
formulae.
Powder X-ray diffraction spectroscopy
ESR studies
X-ray powder diffraction pattern in the 0° \ 2h \ 60° of
the free azo dye ligand and its metal complexes were
carried out in order to give an insight about the lattice
dynamics of these compounds. The X-ray powder diffrac-
tion obtained reflects a shadow on the fact that each solid
represents a definite compound of a definite structure
which is not contaminated with starting materials. Such
facts suggest that the L and its Cr(III), Mn(II), Fe(III),
Co(II), Ni(II), Cu(II) and Zn(II) complexes are amorphous
while Cd(II) complex is crystalline [30].
The ESR spectrum of the Cu(II) complex was recorded in
DMSO at 300 and 77 K, and its parameters are given in
(
(
Fig. 3). The observed order for Cu(II) complex (g||
3.22) [ g\ (2.073) indicated that the complex exerts an
octahedral geometry [29–31]. The observed value of G for
the Cu(II) complex (G = 1.337) implies that the exchange
coupling is not present and misalignment is appreciable.
The trend g [ g [ ge (2.0023) showed that the unpaired
|
|
\
electron is localized in the dx2-y2 orbital of the Cu(II) ion
in complex [5, 29, 32]. The giso (2.04) value less than 2.3
indicated the covalent character of the metal ligand bond
The average crystallite size (n) can be calculated from
the XRD pattern according to Debye–Scherrer equation
[33, 34]
2
and the a value (1.87) suggestive of in-plane covalency.
The calculated value (g /A ) 114 cm for the complex is
Kk
-
1
n ¼ b
:
ð1Þ
||
||
cos h
=2
1
1
23

Synthesis, spectral characterization, thermal, anticancer and antimicrobial studies of…
Scheme 4 Suggested mass
fragmentation pattern of Co(II)
complex (Route 2)
N
N
N
N
OH2
N
N
–2H2O
N
N
O
O
–CoCl2
H3CO
Co
OCH3
·Cl2
O
O
CHO
OHC
–
1
m/z = 402 g mol
(f. = 404, R.I. = 2.9 %)
OH2
–
1
m/z = 568 g mol
(
f. = 567, R.I. = 4.8 %)
–C8H7O2N2
–C8H7O2
N
N
N
N
N
N
OCH3
H3CO
OHC
–
1
m/z = 239 g mol
f. = 239, R.I. = 6 %)
CHO
(
–
1
m/z = 267 g mol
f. = 267, R.I. = 2.5 %)
(
–C8H7O2N2
–
C8H7O2
N
N
N
–2N2
–
1
m/z = 76 g mol
f. = 77, R.I. = 77 %)
N
(
–
1
m/z = 132 g mol
f. = 135, R.I. = 32 %)
(
of loss of masses (Fig. 4). Steps of decomposition of azo
dye ligand and metal complexes are shown in Table 3. For
the azo dye ligand, there are four stages of mass loss. The
first degradation stage takes place in the range of
The equation uses the reference peak width at angle (h),
where k is wavelength of X-ray radiation (1.541874 A), K
˚
is constant taken as 0.95 for organic compounds [21] and
b1/2 is the width at half maximum of the reference
diffraction peak measured in radians. The dislocation
density, d, is the number of dislocation lines per unit area
of the crystal. The value of d is related to the average
particle diameter (n) by the relation [35, 36]:
3
0–185 °C and corresponds to the loss of C H O mole-
2
4 2
cule, and represents a mass loss of 14.34 % and its cal-
culated value was 14.93 % with Ts at 144 °C. The second
degradation stage takes place in the range of 185–330 °C
which corresponding to the loss of C H O molecule and
2
4 2
representing a mass loss of 15.39 % and its calculated
value was 14.93 % with Ts at 283 °C. Finally, the third and
fourth degradation stages take place in the range of
1
d ¼
:
ð2Þ
2
n
The value of n is calculated and found to be 0.146 nm
for Cd(III) complex. The value of d is 46.76 nm for
Cd(II) complex.
3
30–800 °C with Ts at 368 and 597 °C that corresponding
-
2
to the loss of C H N molecule, representing a mass loss
1
8 10 4
of 70.27 % and its calculated value was 70.15 %.
The TG curves of the complexes are taken as a proof for
the existing of water molecules as well as the anions to be
in the coordination sphere. Decomposition of [Cr(L)(H2-
Thermal analysis studies
The TG curves for the azo dye ligand and its metal com-
plexes showed the temperature intervals and the percentage
O)Cl]Cl
2
ÁH
O complex occurs in three steps. The first and
2
second steps occur within the temperature range of
123

W. H. Mahmoud et al.
Scheme 5 Suggested mass
fragmentation pattern of Cd(II)
complex
N
N
N
N
N
N
N
Cl
Cd
N
O
O
O
O
–Cl2
Cd
O
O
O
O
Cl
–
1
m/z = 585 g mol
f. = 586, R.I. = 3 %)
–1
m/z = 514 g mol
(
–CdCl2
(f. = 517, R.I. = 2 %)
N
N
N
N
OCH3
H CO
3
OHC
CHO
–
1
jm/z = 402 g mol
f. = 402, R.I. = 2.9 %)
(
–
2C H O
8 7 2
–
C H ON
8 6 2
N
N
–
1
m/z = 87 g mol
f. = 88, R.I. = 4 %)
N
N
N N
(
–
1
m/z = 132 g mol
f. = 135, R.I. = 67 %)
(
–2N2
–
1
m/z = 76 g mol
f. = 77, R.I. = 100 %)
(
3
0–270 °C with maximum temperatures 60 and 244 °C due
decomposition occurs within the temperature range from
30 to 200 °C, with a maximum temperature at 113 °C, and
corresponds to the loss of one water molecule of hydration
and chlorine molecule with mass loss 15.50 %
(calcd. = 14.82 %). The second step of decomposition
occurs in the range of 200–315 °C, with one maxima at
240 °C, and corresponds to the elimination of coordinated
water molecule and chlorine gas with a mass loss of 7.90 %
(calcd. = 8.90 %). The last three steps of decompositions
occur within the temperature range of 315–800 °C, with
three maxima at 377, 552 and 779 °C and are simultane-
ously decomposed to ‘Fe O ? 15C with intermediate
to loss of two water and chlorine molecules. According to
the literature, the azo bonds in the azo metal complexes
breakdown when the temperature is higher than 260 °C
[
37–41]. As in the third step, the mass loss stage
(
270–800 °C) is due to the decomposition of a part of the
ligand (C H ClN O2.5). The remaining final product is
1
2
18
4
‘
Cr O contaminated with carbon atoms.
2 3
The TG curve of [Mn(L)(H O) ]Cl complex showed
2
2
2
three steps of mass loss. The first step at 30–95 °C with
maximum temperature 63 °C can be attributed to loss of
chlorine gas. The second and third steps at 96–800 °C with
maximum temperatures 172 and 227 °C can be attributed
to loss of the remaining ligand (C H ClN O ). The
2
3
formation of very unstable products which were not iden-
tified. They are accompanied by a mass loss of 32.50 %
(calcd. = 32.97 %) and corresponding to the loss of
C H N O molecule.
1
7
22
4 5
thermal decomposition of [Fe(L)(H O)Cl]Cl ÁH O com-
2
2
2
plex proceeds via five degradation steps. The first step of
7
18 4 3
1
23

Synthesis, spectral characterization, thermal, anticancer and antimicrobial studies of…
0
0
.2
.1
0
DATA
OFFSET
0
1000
2000
3000
4000
5000
6000
–
–
–
–
–
0.1
0.2
0.3
0.4
0.5
Fig. 3 ESR of L–Cu complex
The [Co(L)(H O) ]Cl complex is thermally decom-
2
contaminated with carbon atoms as final product from
which the metal content was found to be in good agreement
with the data obtained from complexometric analyses.
For [Zn(L)Cl ].H O complex, the TG curve showed
2
2
posed in two successive decomposition steps. The first
estimated mass loss of 16.48 % (calcd. = 15.67 %) within
the temperature range of 30–285 °C with maximum tem-
perature of 45 °C may be attributed to the loss of water and
chlorine molecules. The final mass loss is due to the
decomposition of the rest of the ligand molecule
2
2
mass loss in the temperature range of 30–290 °C (found
% = 16.26, calcd. % = 16.01) associated with two DTG
peaks at 47 and 192 °C. This step can be assigned to loss of
chlorine gas and water molecule in two steps. On further
heating, the DTG registered peaks at 311, 406 and 777 °C
within the temperature range of 290–800 °C that may
correspond to loss of remaining ligand fragment
(C H N O ) leaving ZnO contaminated with carbon as a
(
C H N O ) leaving cobalt oxide residue with contami-
21 20 4 4
nated carbon atom with total mass loss 84.66 %
calcd. = 84.68 %). The DTA curve of [Ni(L)Cl ] com-
(
2
plex showed endothermic peak in the temperature range
0–140 °C which is due to mass loss of 13.50 % (calcu-
lated mass loss: 13.35 %) which corresponds to the elim-
ination of chlorine gas. The second and third steps of the
thermal decomposition, which occur in the range
3
1
4 18 4 3
residue. This step had mass loss of 52.45 %
(calcd. = 52.16 %). The TG curve of [Cd(L)Cl ] complex
2
showed six decomposition steps. The first stage composed
1
40–800 °C, which are also endothermic process as shown
from the DTA curve, can assigned to the loss of
C H N O molecule (observed 70.84 %;
of two steps (40–275 °C) and showed the removal of
chlorine
gas
(found
mass
loss = 11.81 %;
calcd. = 12.14 %). The other four steps (275–840 °C)
2
1 18 4 3
calcd. = 70.30 %) leaving NiO contaminated with carbon
as residue.
represent the loss of remaining ligand (C H N O )
2
2 18 4 3
(found mass loss = 66.0 %; calcd. = 65.98 %) with CdO
The TG curve of [Cu(L)(H O)Cl]Cl.H O complex
2
residue.
2
indicated endothermic decompositions in four steps. The
first step within the temperature range 40–135 °C with
maximum temperature of 124 °C may be assigned to loss
of hydrated water molecule with mass loss 3.59 %
Structural interpretation
From all the previous data, the newly synthesized azo dye
ligand behaves as tetradentate ligand and coordinated to
metal ions through two nitrogens of azo groups (N=N) and
(
calcd. = 3.14 %). The second and third decomposition
steps occur at 135–380 °C with two maxima at 260 and
30 °C and include elimination of chlorine gas and water
3
two oxygens of methoxy groups (OCH ). The structure of
3
molecules with mass loss 15.96 % (calcd. = 15.54 %).
The fourth decomposition step occurs within temperature
range of 380–800 °C with maximum temperature 769 °C.
This step accompanied by complete decomposition of the
ligand (C H N O ) as well as formation of copper oxide
the newly synthesized ligand and its metal complexes and
coordination sites with metal ions can be predicted using
1
different techniques as elemental analysis, IR, HNMR,
mass spectrometry, electronic spectra, magnetic suscepti-
bility, ESR, conductivity measurements, thermogravimetric
8
18 4 3
123

W. H. Mahmoud et al.
(
a)
DTGA/
mg min
–
1
TG/%
–
–
0.00
0.05
1
00.00
3
68.16 °C
2
83.44 °C
1
44.32 °C
5
0.00
5
96.51 °C
–0.10
0
.00
0
.00
200.00
400.00
600.00
800.00
Temp/°C
DTGA/
mg min
–
1
(
b)
×
10–2
TG/%
0.50
1
00.00
–1.50
7
79.20 °C –3.50
2
44.23 °C
5
0.00
59.64 °C
–
–
5.50
7.50
4
54.81 °C
0
.00
0
.00
200.00
400.00
Temp/°C
600.00
800.00
(
c)
DTGA/
mg min
–
1
TG/%
0
–
–
.02
1
00.00
4
6.99 °C
310.49 °C
1
91.95 °C
0.08
0.18
7
76.60 °C
5
0.00
0
.00
4
06.13 °C
0
.00
200.00
400.00
600.00
800.00
Temp/°C
Fig. 4 Curves of a Azo dye ligand, b L–Cr(III) and c L–Zn(II) complexes
1
23

Synthesis, spectral characterization, thermal, anticancer and antimicrobial studies of…
Table 3 Thermoanalytical results (TG and DTG) of azo dye ligand and its metal complexes
Complex
TG range/°C DTGmax/°C
n* Mass loss/
found/
calcd
Total mass loss/ Assignment
% found/
calcd
Residues
%
L
30–185
85–330
31–800
30–270
70–800
30–95
144
1
1
2
2
1
1
2
1
1
3
1
1
1
2
1
2
1
2
3
2
4
14.34 (14.93)
15.39 (14.93)
70.27 (70.15)
17.65 (17.93)
Loss of C
Loss of C
2
H
4
O
2
2
–
1
3
283
H
2 4
O
368,597
60, 244
455
100 (100)
Loss of C18
H
10 4
N
[
[
[
Cr(L)(H O)Cl]Cl
2
2
ÁH
2
O
Loss of Cl
2
and 2H
2
O
‘Cr
2 3
O ? 10C
2
49.51 (49.20) 67.20 (67.14)
7.09 (6.29)
Loss of C12
H
18ClN
4
O
3
Mn(L)(H
2
O)
2
]Cl
2
63
Loss of ‘Cl
2
MnO ? 5C
9
5–800
172, 227
113
69.86 (70.48) 77.03 (76.77)
15.50 (14.82)
Loss of C17
Loss of H
Loss of ‘Cl
Loss of C
Loss of H
Loss of C21
Loss of Cl
Loss of C21
Loss of H
Loss of Cl
H
22ClN
4
O
5
Fe(L)(H O)Cl]Cl
2
2
ÁH
2
O
30–200
00–315
15–800
30–285
85–800
30–140
40–800
40–135
35–380
80–800
30–290
90-800
40–275
75–840
2
O and Cl
2
2 3
‘Fe O ? 15C
2
3
240
7.90 (8.90)
2
2
and H O
377,552,779
45
32.50 (32.97) 56.11 (56.70)
16.48 (15.67)
7 18 4 3
H N O
[
[
[
Co(L)(H O)
2
2
]Cl
2
2
O and Cl
2
CoO ? C
NiO ? C
2
1
661
68.15 (69.0)
13.50 (13.35)
84.66 (84.68)
H
20
N
4
O
4
Ni(L)Cl
2
]
78
2
196, 255
124
70.84 (70.30) 84.43 (83.65)
3.59 (3.14)
H
18
N
4
O
3
Cu(L)(H
2
O)Cl]ClÁH
2
O
2
O
CuO ? 14C
1
3
260, 330
769
15.96 (15.54)
2
and H
2
O
36.71 (38.08) 56.42 (56.77)
16.26 (16.01)
Loss of C
8 18 4 3
H N O
[
[
Zn(L)Cl
2
]ÁH
2
O
47, 192
311,406,777
115, 209
289, 370,
Loss of H
2
O and Cl
2
ZnO ? 8C
2
2
52.45 (52.16) 68.91 (68.17)
11.81 (12.14)
Loss of C14
Loss of Cl
Loss of C22
H
18
N
4
O
3
Cd(L)Cl
2
]
2
CdO
66.0 (65.98) 77.81 (78.12)
H
18
N
4
O
3
5
32, 686
n* number of decomposition steps
analyses (TG-DTG) and X-ray powder diffraction. The
proposed structures are given in Fig. 5.
Cd(II) complex has the highest antibacterial activity
against Staphylococcus aureus (Gram positive), Neisseria
gonorrhoeae (Gram negative) and Candida albicans (fun-
gus). The bacterial growth inhibitory capacity of the ligand
and its complexes follow the order:
Biological activities
The antimicrobial activities of the free azo dye ligand and
its metal complexes are given in Table 4 which also con-
tains standard antibiotic, amikacin for antibacterial activity
and ketoconazole as antifungal agent. The data listed in this
table showed that the azo dye ligand and [Mn(L)(H O) ]-
Zn(II) [ Cd(II) [ Co(II) [ Ni(II) [ Cu(II) [ Cr(III) [
amikacin [ L = Mn(II) = Fe(III)
Subtilis).
(for
Bacillus
Cd(II) [ Zn(II) [ Co(II) [ Ni(II) [ Fe(III) = Cu(II) =
amikacin [ L = Cr(III) = Mn(II) (for Staphylococcus
aureus)
2
2
Cl2 complex have no activities against all the tested
microorganisms (bacteria or fungi), while the other metal
complexes showed remarkable antimicrobial activities. The
Zn(II) [Cd(II) [Co(II) [Ni(II) [Cr(III) = Fe(III) = Cu
(II)[ amikacin [ L = Mn(II) (for E. coli)
-
1
inhibition zone diameter (mm mg sample) of bacterial
and fungal growth inhibition for the compounds and rela-
tion between atomic mass of metal ion and inhibition zone
diameter are summarized in their graphical representations
as shown in Fig. 6. On comparing the results of antimi-
crobial activity of the azo dye ligand and its metal com-
plexes, it was found that Zn(II) complex showed the
highest antibacterial activity against Bacillus Subtilis
Cd(II)[ Zn(II)[ Co(II)[ Ni(II) [ Cr(III) = Fe(III) =
Cu(II)[ amikacin[ L = Mn(II)
Gonorrhoeae).
(for
Neisseria
Higher activity of metal complex was probably due to
greater lipophilic nature of the complex. It increased
activity of the metal complex and can be explained on the
basis of Overtone’s concept and Tweedy’s chelation theory
[12, 42, 43]. According to Overtone’s concept of cell
(
Gram positive) and Escherichia coli (Gram negative), and
123

W. H. Mahmoud et al.
Fig. 5 The structure of the
complexes
N
N
N
N
OH2
N
N
N
Cl
O
N
O
O
M
·Cl2
M
O
O
O
O
OH2
Cl
M = Mn(II) and Co(II).
M = Ni(II), Zn(II) and Cd(II).
N
N
N
N
OH2
O
O
M
·
Cl ·yH O
x 2
O
O
Cl
M = Cr(III) and Fe(III); x = 2; y = 1.
Cu(II); x = y = 1.
Table 4 Biological activity of azo dye ligand and its metal complexes
-
1
Sample
Inhibition zone diameter/mm mg sample
Gram positive
Gram negative
Fungus
Bacillus Subtilis
Staphylococcus aureus
Escherichia coli
Neisseria gonorrhoeae
Candida albicans
Control: DMSO
L
0
0
0
0
0
0
0
0
0
0
[
[
[
[
[
[
[
[
Cr(L)(H
Mn(L)(H
Fe(L)(H O)Cl]Cl
Co(L)(H O) ]Cl
2
O)Cl]Cl
2
ÁH
2
O
O
9
0
9
9
0
2
O) ]Cl
2
2
0
0
0
0
0
2
2
ÁH
2
0
9
9
9
0
2
2
2
15
13
10
25
20
6
15
13
9
14
13
9
20
12
9
12
15
0
Ni(L)Cl
2
]
Cu(L)(H
2
O)Cl]ClÁH
2
O
Zn(L)Cl
2
]ÁH
2
O
20
30
9
17
15
7
22
26
6
0
Cd(L)Cl
2
]
23
–
Amikacin
Ketoconazole
–
–
–
–
9
permeability, the lipid membrane that surrounds the cell
favors the passage of only lipid-soluble materials due to
which liposolubility was considered to be an important
factor that controls the antimicrobial activity. On chelation,
the polarity of the metal ion will be reduced to a greater
extent due to the overlap of the ligand orbital and partial
sharing of positive charge of metal ion with donor groups
[44, 45]. Further, it increases the delocalization of the p
electrons over the whole chelate ring and enhances the
lipophilicity of the complex. This increased lipophilicity
enhanced the penetration of the complexes into lipid
membrane and thus blocks the metal binding sites on
enzymes of microorganisms [46]. These metal complexes
also disturb the respiration process of the cell and thus
1
23

Synthesis, spectral characterization, thermal, anticancer and antimicrobial studies of…
block the synthesis of proteins, which restricts further
growth of the organism [47].
(
a)
3
5
0
5
0
5
0
5
0
Bacillus Subtilis
Escherichia coli
3
2
2
1
1
Anticancer activity evaluation
Neisseria gonorrhoeae
Staphylococcus aureus
Candida albicans(fungus)
To study the cytotoxicity of the newly synthesized azo
dye free ligand and its metal complexes, the MCF7 [8]
cell line was chosen, as it is a breast cancer cell line.
Figure 7 illustrated cell viability in relation to increasing
concentrations of compounds. The values of concentra-
tion at which half of the maximal effect is observed (IC )
5
0
with the numerical data summarized in Table 5. Cr(III)
and Cu(II) complexes are inactive as they showed inhi-
bition ratio less than 70 %, while the free azo dye ligand
and other metal complexes have anti-breast cancer
activity. It is observed that the ligand has the lowest
-
1
activity as its IC50 = 26.8 lg mL while Zn(II) complex
-
1
of the lowest IC50 (12.0 lg mL ) showed the highest
anti-breast cancer activity. These results provided a per-
fect example of how changes in the chelation molecular
structure could lead to profound differences in anticancer
activity [48–50].
(
b)
3
3
2
2
1
1
5
Bacillus Subtilis
Escherichia coli
0
5
0
5
0
5
0
Neisseria gonorrhoeae
Staphylococcus aureus
Candida albicans(fungus)
C=0
C=5
C=12.5
C=25
C=50
1.2
1
0
0
0
0
.8
.6
.4
.2
0
52
55
56
59
59
63.5
65
112
Atomic mass/g mol–1
Fig. 6 a Biological activity of L and its complexes, b relationship
between inhibition zone diameter and atomic mass of metals
Fig. 7 Anticancer activity of L and its complexes
Table 5 Anti-breast cancer activity of azo dye ligand and its metal complexes
Complex Surviving fraction/MCF7
-
1
IC50/lg mL
-1
Conc./lg mL
.0
0
5.0
12.5
25.0
50.0
L
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.77
0.74
0.99
0.84
0.97
0.66
1.00
0.69
0.68
0.94
0.66
0.72
0.53
1.00
0.51
0.45
0.84
0.67
0.46
0.69
0.76
0.44
0.35
0.88
0.68
0.39
0.88
0.74
26.8
22.1
–
[
[
[
[
[
[
Mn(L)(H
Fe(L)(H O)Cl]Cl
Co(L)(H O) ]Cl
2 2 2
O) ]Cl
2
2
ÁH O
2
2
2
2
–
Ni(L)Cl
2
]
23.0
12.0
–
Zn(L)Cl
2
]ÁH
2
O
2
Cd(L)Cl ]
123

W. H. Mahmoud et al.
5
6
. Mahapatra BB, Mishra RR, Sarangi AK. Synthesis, characteri-
zation, XRD, molecular modelling and potential antibacterial
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. Sakiyan I, Logoglu E, Arslan S, Sari N. Antimicrobial activities
of N-(2-hydroxy-1-naphthalidene)-aminoacid(glycine, alanine,
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manganese(III) complexes. Biometals. 2004;17:115–20.
Conclusions
The newly synthesized azo dye ligand and its Cr(III),
Mn(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II)
complexes are prepared and their structures are confirmed
by elemental analysis, spectroscopic studies (IR, UV–Vis,
1
H NMR, mass spectrometry, electronic spectra, magnetic
susceptibility and ESR), conductivity measurements, ther-
mogravimetric analyses (TG-DTG) and further confirmed
by X-ray powder diffraction. Infrared spectra discussed the
chelation mode through two oxygen atoms of two methoxy
groups and two nitrogen atoms of two azo groups as the
ligand is tetradentate ligand. Molar conductance in DMF
indicates that all complexes are electrolytes except Ni(II),
Zn(II) and Cd(II) complexes are non-electrolytes. The
proposed structural formulas of the above complexes are:
7. Devi J, Batra N. Synthesis, characterization and antimicrobial
activities of mixed ligand transition metal complexes with isatin
monohydrazone Schiff base ligands and heterocyclic nitrogen
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. Skehan P, Storeng R. New colorimetric cytotoxicity assay for
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. Alaghaz AMA, Ammar YA, Bayoumi HA, Aldhlmani SA.
Synthesis, spectral characterization, thermal analysis, molecular
9
2 2
modeling and antimicrobial activity of new potentially N O
azo-dye Schiff base complexes. J Mol Struct. 2014;1074:
359–75.
1
1
1
0. Abo-Aly MM, Salem AM, Sayed MA, Abdel Aziz AA. Spec-
troscopic and structural studies of the Schiff base 3-methoxy-N-
salicylidene-o-amino phenol complexes with some transition
metal ions and their antibacterial, antifungal activities. Spec-
trochim Acta A. 2015;136:993–1000.
1. Chandra S, Gautama S, Rajor HK, Bhatia R. Syntheses, spec-
troscopic characterization, thermal study, molecular modeling,
and biological evaluation of novel Schiff’s base benzyl bis(5-
amino-1,3,4-thiadiazole-2-thiol) with Ni(II), and Cu(II) metal
complexes. Spectrochim Acta A. 2015;137:749–60.
[
ML(H O)Cl]Cl ÁyH O where M = Cr(III), Fe(III);
2
x
2
x = 2, y = 1 and M = Cu(II); x = 1, y = 1.
[ML(H O) ]Cl where M = Mn(II) and Co(II).
2 2 2
[
MLCl ]ÁyH O where M = Ni(II), Zn(II) and Cd(II);
2
2
y = 0 for Ni(II), Cd(II); y = 1 for Zn(II).
The magnetic and solid reflectance measurements con-
firm octahedral geometry of the complexes, and XRD
results showed that the free ligand and metal complexes
have amorphous nature except Cd(II) complex is crys-
talline. ESR spectra of solid Cu(II) complex at room
temperature showed axial type (dx2-y2) with covalent bond
character in an octahedral environment. The antimicrobial
test refer that all metal complexes except Mn(II) recorded a
significant antibacterial efficiency rather than the free azo
ligand, but only Co(II), Ni(II) and Cd(II) complexes have
antifungal activities upon the lipophilicity behaviors.
Ligand and some metal complexes showed inhibitory
activity against breast carcinoma (MCF7 cell line), espe-
cially Zn(II) complex showed the highest anticancer
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1
1
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