Complex formation, thermal behavior and stability competition between Cu(II) ion and Cu0 nanoparticles with some new azo dyes. Antioxidant and in vitro cytotoxic activity

Article abstract of DOI:10.1016/j.saa.2013.01.039

Four triazole and thiadiazole-based azo chromophores namely [(E)-4-((1H-1,2,4-triazol-3-yl)diazenyl)benzene-1,3-diol.(HL¹), (E)-4-((5-(methylthio)-1H-1,2,4-triazol-3-yl)diazenyl)benzene-1,3-diol.(HL ²), (E)-4-((1,3,4-thiadiazol-2-yl)

Full text of DOI:10.1016/j.saa.2013.01.039

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 359–370
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Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Complex formation, thermal behavior and stability competition between Cu(II)
ion and Cu0 nanoparticles with some new azo dyes. Antioxidant and in vitro
cytotoxic activity
1
2
⇑
M. Gaber , Y.S. El-Sayed , K.Y. El-Baradie , R.M. Fahmy
Chemistry Department, Faculty of Science, Tanta University, 31527 Tanta, Egypt
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
"
"
"
Cu(II) complexes of four triazole and
thiadiazole-based azo chromophore
were synthesized and characterized.
The mode of interaction between the
synthesized azo ligands and copper
nanoparticles was studied.
The antitumor and antioxidant
activities of the synthesized azo
ligands and their Cu(II) azo
complexes have been evaluated.
a r t i c l e i n f o
a b s t r a c t
Article history:
Four triazole and thiadiazole-based azo chromophores namely [(E)-4-((1H-1,2,4-triazol-3-yl)diaze-
nyl)benzene-1,3-diol.(HL1), (E)-4-((5-(methylthio)-1H-1,2,4-triazol-3-yl)diazenyl)benzene-1,3-diol.(HL2),
(E)-4-((1,3,4-thiadiazol-2-yl)diazenyl)benzene-1,3-diol.(HL3) and (E)-4-((5-mercapto-1,3,4-thiadiazol-2-
yl)diazenyl)benzene-1,3-diol.(HL4)] were synthesized and characterized by elemental analyses, IR,
UV–Vis as well as mass spectroscopy. Cu(II) complexes of the investigated azo dyes have been synthe-
sized and characterized by elemental analyses, IR, electronic and ESR spectra, magnetic susceptibility
and thermogravimetric analyses. The bond lengths and bond angles have been calculated to confirm
the geometry of the ligands and their Cu(II) complexes. The mode of interaction of the azodyes to copper
nanoparticles was described as coordination mode of charged dye molecules on the colloidal Cu0 surface
through anchoring AOHꢀ group. The apparent association constants of the colloidal copper nanoparticles
azodye complexes in solution were evaluated using the spectral method and compared with the forma-
tion constant of the Cu(II) azo complexes. The antitumor and antioxidant activities of the synthesized azo
dyes and their Cu(II) azo complexes have been evaluated.
Received 25 October 2012
Received in revised form 25 December 2012
Accepted 11 January 2013
Available online 31 January 2013
Keywords:
Azo
Complexes
Copper nanoparticles
Antitumor
Antioxidant
Ó 2013 Elsevier B.V. All rights reserved.
Introduction
a great deal of interest in the synthesis and characterization of azo
compounds and their metal complexes [7–13].
Metal complexes of azo compounds have remained an attrac-
tive area of research for coordination and structural chemists.
The importance of the heterocyclic azo dyes may stem from its bio-
logical activity and analytical applications [1–6]. Recent years have
Metal complexes of 1,2,4-triazole derivatives are biologically
active having nuclease like activity [14], anti-proliferative [15],
antibacterial and antifungal activity [16–19], antitumor [20] and
anticancer [21]. Also, the chemistry of 1,3,4-thiadiazole derivatives
received much attention because of their significant biological
activities [22] and anticancer activity [23].
In the present work, we synthesized new four azo dyes com-
pounds (HL1–HL4). The structures of these azo compounds were
elucidated by elemental analysis, IR, UV–Vis and mass spectra.
The coordination behavior of the investigated azo dyes towards
⇑
Corresponding author. Tel./fax: +20 40 3350804.
E-mail address: abuelazm@yahoo.com (M. Gaber).
Prepared Basic Science, Deanery of Academic Services, Taibah University, Al-
1
Maddinah, Saudi Arabia.
2
Faculty of Science and Arts at Al-Rass, Qassim, Saudi Arabia.
1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.01.039

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M. Gaber et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 359–370
Cu(II) ion was studied via the elemental analysis, IR, electronic, ESR
spectra, magnetic moment and molar conductance measurements.
In addition, the thermal behavior of Cu(II) complexes was studied.
The kinetic and thermodynamic parameters for the decomposition
steps have been calculated. The mode of interaction between the
synthesized azo-compounds and copper nanoparticles in solution
was studied. The biological activities (antitumor and antioxidant)
of the azo-compounds and their Cu(II) complexes were evaluated.
desiccator over anhydrous CaCl2. The analytical and physical data
of the synthesized azo compounds were presented in Table 1.
Preparation of Cu(II) azo complexes
The solid Cu(II) azo complexes were prepared in molar ratio 1:1
by dropwise addition of 50 mL hot ethanolic Cu(II) acetate solution
(1 mmol) to the azo-compounds (1 mmol) in 50 mL of hot ethanol,
whereupon suspensions of the Cu(II) azo complexes resulted. The
reaction mixture was then heated at ꢁ50 °C under refluxed for
4–5 h. TLC plates were used to detect the reaction finishing. The
precipitated solids were collected by filtration, washed several
times with bidistilled water and then dried in desiccator over
anhydrous CaCl2. The analytical and physical data of the synthe-
sized complexes were presented in Table 1.
Experimental
Reagents
All the reagents and solvents were of analytical grade quality.
3-Amino-1H-1,2,4-triazole, 3-amino-5-methylmercapto-1H-1,2,
4-triazole, 2-amino-1,3,4-thiadiazole, 2-amino-5-mercapto-1,3,
4-thiadiazole, cetyltrimethyl ammonium bromide (CTAB) and
1,3-dihydroxybenzene were purchased from Sigma–Aldrich Co.
and were used without further purification.
Synthesis of copper nanoparticles
Cu nanoparticles were synthesized as previously described [20]
by reduction of Cu+2 to Cu0. 10 ml of 0.003 M Cu(NO3)2ꢂ3H2O pre-
pared in isopropanol (IPA) solution was added dropwise to 10 ml of
0.09 M of cetyltrimethylammonium bromide/isopropanol (CTAB/
IPA) solution. The reaction mixture was stirred vigorously giving
a violet colloid absorbing at 560 nm. The appearance of a violet col-
or indicated the presence of copper nanoparticles [25]. CTAB was
used as catalyst for the reduction of Cu2+ with IPA and as stabilizer
to protect Cu nanoparticles from oxidation.
Physical measurements
Elemental analyses of the azo ligands and their Cu(II) azo com-
plexes were performed with the aid of Perkin–Elmer model 2400
automated analyzer. Infrared spectra for both azo ligands and their
Cu(II) azo complexes were recorded on Perkin Elmer 1430 Infrared
Spectrophotometer using KBr discs in the range 200–4000 cmꢀ1
.
Molecular modeling
Electronic absorption spectra of azo ligands and their Cu(II) azo
complexes were recorded in the range 200–700 nm on a Shimadzu
Recording UV–Vis spectrophotometer model 240 A with the aid of
1 cm quartz cuvettes. The electronic absorption spectra of the solid
Cu(II) azo complexes were recorded using Nujol mull technique
[24].
An attempt to gain a better insight on the molecular structure of
the ligands and their complexes, geometry optimization and
conformational analysis has been performed by the use of
MM + force-field as implemented in hyperchem 8.0 [26]. Semi
empirical methodPM3 is then used for optimizing the full geome-
try of the system using Polak–Ribiere (conjugate gradient) algo-
rithm and Unrestricted Hartee–Fock (UHF) is employed keeping
RMS gradient of 0.01 kcal/mol.
The mass spectra of azo compounds were recorded using Shi-
madzu Qp-2010 plus. Magnetic susceptibilities of the prepared so-
lid Cu(II) azo complexes were measured at room temperature at
25 °C on Sherwood Scientific Magnetic Susceptibility Balance using
Hg[Co(SCN)4] as calibrant. The ESR spectra of powdered samples of
Cu(II) azo complexes were recorded at room temperature with the
aid of JEOL JES-FE2XG Spectrometer equipped with an E101 micro
wave bridge. The magnetic field was calibrated with diphenyl pic-
rylhydrazide (DPPH). The conductance measurements for the pre-
pared solid Cu(II) azo complexes were recorded with the aid of
Hana model 1331 conductometer. The thermal analysis (TGA–
DrTGA) was performed using Shimadzu TG-50 thermal analyzer
up to 800 °C at a heating rate 10 °C minꢀ1 in an atmosphere of
N2. The morphology of the prepared copper nanoparticles was
investigated using JEOL-JEM-100SX electron microscope. The parti-
cle size and size distributions were obtained by image analyses.
The antitumor activity of investigated azo compounds (HL1–HL4)
and their Cu(II) azo complexes were evaluated against Ehrlish Asci-
tes Carcinoma cells. Also, the antioxidant assay for some synthe-
sized azo ligand and their Cu(II) azo complexes was performed.
Results and discussion
Study of the azo ligands (HL1–HL4)
U.V–Vis spectra
The electronic absorption spectra of 5 ꢃ 10ꢀ5 M azo ligands
(HL1–HL4) in methanol showed three main absorption bands. The
first band appeared around 210 nm assigned to the moderate en-
ergy
sented the (1La ? 1A) state. The second band observed in the
range 250–310 nm attributed to low energy
pꢄ electronic transi-
p–
pꢄ electronic transition within the phenyl moiety repre-
p–
tion of the heterocyclic moiety and phenyl ring corresponded to
the (1Lb ? 1A) state. The third band appeared within the range
380–470 nm derived from n to pꢄ electronic transition involving
the whole electron system and charge transfer interaction within
the molecule.
Synthesis of the azo ligands (HL1–HL4) and their Cu(II) azo complexes
Solvent effect on UV–Vis spectra
The electronic absorption spectra of 10ꢀ5 M azo ligands
(HL1–HL4) under investigation were also recorded in Ethanol,
Di-methylformamide, Acetonitrile, Methylene chloride and
n-Heptane.
Synthesis of azo compounds
Azo dyes (HL1–HL4) Fig. 1, were synthesized by coupling the
diazonium salt of 3-Amino-1H-1,2,4-triazole, 3-amino-5-methyl-
mercapto-1H-1,2,4-triazole, 2-amino-1,3,4-thiadiazole, 2-amino-
5-mercapto-1,3,4-thiadiazole with 1,3-dihydroxybenzene at ꢀ5
to 0 °C. The precipitated solids were filtered off, washed several
times with bidistilled water, purified by further recrystallization
from hot ethanol to give the pure azo ligands and finally dried in
The plot of charge transfer energy (ECT) against the solvent
parameters such as dielectric constant (D) given by Suppan [27],
refractive index relation F(n) of Bayliss and Mac Rae, Dimroth
and Reichardit’s ET(30), Kosower’s Z, Kamlet and Taft’s pꢄ solvato-
chromic scales,
a scale of acidity and b scale of basicity, gave non

M. Gaber et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 359–370
361
H
H
N
O
N
N
N
N
OH
H
(E)-4-((1H-1,2,4-triazol-3-yl)diazenyl)benzene-1,3-diol.(HL1).
H3CS
H
N
O
N
N
N
N
OH
H
(E)-4-((5-(methylthio)-1H-1,2,4-triazol-3-yl)diazenyl)benzene-1,3-diol.(HL2).
H
N
O
N
S
N
N
OH
H
(E)-4-((1,3,4-thiadiazol-2-yl)diazenyl)benzene-1,3-diol.(HL3).
H
N
O
N
S
N
N
OH
HS
4
(E)-4-((5-mercapto-1,3,4-thiadiazol-2-yl)diazenyl)benzene-1,3-diol.(HL ).
Fig. 1. Structure of ligands (HL1–HL4).
Table 1
Elemental analysis and physical properties of the prepared azo ligands (HL1–HL4) and their Cu(II) azo complexes.
Ligand/complex
Formula/(mol.wt)
Color
m.p. °C
Content% (calcd.) found
%C
%H
%N
%Cu
–
(17.5)17.31
–
(14.89)14.8
–
(14.8)14.64
–
(16.0) 15.0
HL1
C8H7N5O2/(205)
Yellow
Reddish violet
Orange
Reddish violet
Greenish yellow
Reddish violet
Reddish orange
Reddish violet
200
>300
212
>300
237
>300
131
(46.87) 46.8
(33.11) 33.28
(43.07) 43.00
(30.90) 30.79
(43.28) 43.11
(27.9) 27.78
(37.83) 37.70
(30.2) 30.11
(3.44) 3.41
(3.6) 3.64
(3.60) 3.51
(4.0) 4.1
(2.70) 2.60
(3.3) 3.21
(2.38) 2.25
(3.5) 3.35
(34.16) 34.1
(19.3) 19.22
(27.90) 27.77
(16.4) 16.37
(25.24) 24.98
(13.0) 12.73
(22.06) 21.97
(14.1) 13.91
[CuHL1(H2O)]ꢂOAcꢂH2O/362.79
HL2
C9H9N5O2S/(251)
[CuHL2(H2O)]ꢂOAcꢂ2H2O/426.9
HL3
C8H6N4O2S/(222)
[CuHL3(2H2O)]ꢂOAcꢂH2O/397.9
HL4
C8H6N4O2S2/(254)
[CuHL4(2H2O)]ꢂOAcꢂH2O/429.9
>300
linear relationships. This indicated that each solvent parameter
had its effective contribution to the CT band which depends on
the nature of the solute. Accordingly, it can be concluded that
the change of the CT band position with solvent was the net result
of the influence of all these parameters.
Moreover, the contribution of each solvent parameter in con-
trolling the ECT was calculated. This contribution was calculated
for each of Kamlet–Abboud–Taft’s three solvent parameters and
Katritzky’s three solvent parameters. Tables (S1, S2),
1. The energy of charge transfer band (ECT) for all ligands was sol-
vent dependent.
2. It was clear that, the Kamlet Taft parameters equation has the
higher correlation coefficient (0.804–0.994) than Katritzky
parameters equation (0.576–0.953) and so, Kamlet Taft equa-
tion can be used as indication for the solvatochromic properties
for azo ligands (HL1–HL4).
3. The value of contribution of each solvent parameter measured
the magnitude of the effect on the energy of charge transfer
band. It was found that hetero cyclic moiety in ligands
From the data one can notice that:

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M. Gaber et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 359–370
(HL1–HL4) is responsible for the band shift which appeared from
the difference of the contribution results which was {pꢄ,b} for
Kamlet–Abboud–Taft’s equation and {F(e),F(n)}for Katritzky’s
Table 2
Spectrophotometric data, stability constants and analytical data (Molar absorptivity
), specific absorptivity (a), Sandell sensitivity (S), Standard deviation (S.D.),
correlation coefficient (C.C.) and obeyence of Beer’s law for Cu(II) with (HL1–HL4).
(e
equation.
Data
Complex 1 Complex 2 Complex 3 Complex 4
kmax (nm)
Beer’s range (ppm)
Ringbom range (ppm) 0.281–
0.58
CC
510
0–3.175
540
460
550
Mass spectra
0–3.81
0.212–
0.58
0.99439
0.01851
8.7 ꢃ 103
0.14
0.007
1:1
1:1
1:1
3.67
5003.8
5
5
0–3.175
0.173–
0.50
0.99788
0.01253
8 ꢃ 103
0.126
0.008
1:1
1:1
1:1
4.69
6394.4
5
0–6.35
0.344–
0.80
0.995
0.00691
1.9 ꢃ 103
0.03
0.033
1:1
1:1
1:1
5.32
7253.4
5
5
The mass spectra of the ligands displayed molecular ions which
confirmed their molecular weights (Scheme S1–S4). The mass
spectra of ligands showed the molecular ion peak [M+] at m/z =
205(80%) with a great abundance equivalent to its molecular mass
attributed to C8H7N5O2 formula for HL1; at m/z = 251(53%) related
to C9H9O2N5S formula for HL2; at m/z = 222(1%) corresponded to
the mass of C8H6N4O2S formula for HL3 and at m/z = 254(11%)
corresponded to C8H6N4S2O2 formula with moderate abundant
for HL4
0.99383
0.00971
5.6 ꢃ 103
0.088
0.011
1:1
1:1
1:1
5.18
7062.5
5
S.D.
e
(l molꢀ1 cmꢀ1
)
a (ml gꢀ1 cmꢀ1
)
S (
l
g cmꢀ2
)
MRM
CVM
CM
Log bn
ꢀ
D
Gꢄ
IR spectral studies
Y ꢃ 10ꢀ5
X ꢃ 10ꢀ5
5
5.1
4752
46E + 8
The IR spectra of the azo compounds (Table S3) showed the
characteristic bands due to stretching frequency of the OH group
in the region 3329–3427 cmꢀ1. The broadening of this peak indi-
cated that these compounds have strong intramolecular hydrogen
bonding OAH . . . N between the hydroxyl group and the azo nitro-
gen [28]. A strong band within the region 1247–1297 cmꢀ1 could
K (bulk-copper)
K (nano-copper)
12765
85E + 8
10566
53E + 8
3915
20E + 8
Where: MRM: mole ratio method. CVM: continuous variation method. CM: con-
ductometric method. bn: stability constant of complex. X: concentration of prepared
sample (mol Lꢀ1). ‘‘EDTA-titration’’. Y: concentration of sample (mol Lꢀ1) calculated
experimentally. ‘‘EDTA-titration’’.
D
G: Gibbs free energy change (Cal molꢀ1). (K):
be attributed to
m(CAO) vibration of the CAOAH group [29]. In
Formation constant of Cu(II) azo complexes. (Kapp): Apparent association constant
addition, IR spectra of the azo compounds showed an absorption
band in the region 1434–1495 cmꢀ1, which assignable to the
stretching vibration of (N@N) group. The data obtained from spec-
tral measurements and elemental analyses, confirmed the pro-
posed formula of the investigated azo compounds. The IR
spectrum of (HL4) showed a band at 2633 cmꢀ1 assigned to
of Cu0 azo complexes.
for the titration process of the Cu(II) ion with EDTA as a standard
solution using (HL1–HL4) as indicators. The results indicated that
the Cu(II) ion was successfully determined up to the range listed
in Table 2. The results obtained indicated that the applied ligands
can be used as indicators for the spectrophotometric titration of
the Cu(II) with EDTA.
m
(SAH) vibration mode indicating the existence of thiol form. Also,
the band appeared at 661 cmꢀ1 is attributed to
m(CASCH3) while
the CASAC stretching vibration appeared at 629 and 633 cmꢀ1
for ligands HL3 and HL4, respectively.
Stability constant measurement
The addition of different concentration of Cu(II) acetate to azo
ligands (HL1–HL4) were studied, where a new absorption band ap-
peared at 510, 540, 460 and 550 nm related to the complex forma-
tion of Cu(II) with HL1, HL2, HL3 and HL4, respectively. The
formation constant of the complexes was estimated from Benesi
and Hildebrand equation [34]
Study of Cu(II) azo complexes (1–4) in solution
UV–Vis spectra
The evaluation of the optimum conditions for complexation of
Cu(II) ion with azo compounds (HL1–HL4) included a careful inves-
tigation of all factors involved in the procedure. The optimum stoi-
chiometry of the colored Cu(II) azo complexes was determined
using mole ratio method (MRM) [30], Fig. S1 and continuous vari-
ation method (CVM) [31], Fig. S2 as well as conductometric titra-
tion method (CM). All the previous methods were agree with the
stoichiometry of 1:1 (M:L) complexes. The stability constants of
Cu(II) azo complexes were determined by the aid of the data ob-
tained from (MRM) and (CVM) applying the Harvey and Manning
equation [32], Table 2. The conductometric method (CM) showed
that the conductance increased with increasing the addition of li-
gand. This indicated that the reaction between Cu(II) ion and azo
occurred via the formation of a covalent linkage with oxygen of
the phenolic OH group, i.e. the conductance increases due to the
liberation of H+ ion.
½Loꢅ
1
ꢀ
1
¼
þ
eML
A ꢀ Ao eML
eL
ð
ꢀ
eLÞK½Moꢅ
where, Ao and A are absorbance of the free azo ligand solutions and
the corresponding Cu(II) azo complexes, respectively, at a given
The limits of Cu(II) ion concentration at which the MAL system
obeyed Beer’s law, molar absorbativity, Sandell sensitivity, Stan-
dard deviation and Correlation coefficient were listed in Table 2.
These values confirmed the possible application of the present
spectrophotometric method for the determination of Cu(II) ion.
Ringbom [33] concentration range was also determined and the re-
sults were listed in Table 2. The obeyance to Beer’s law and Ring-
bom plots of Cu(II) complexes were shown in Figs. (S3, S4).
The spectrophotometric titration of Cu(II) ion with EDTA using
the azo compounds (HL1–HL4) were carried out. The titration
curves shown in Fig. 2. exhibited the presence of two intersected
straight lines. The point of intersection indicated the end point
Fig. 2. Spectrophotometric titration of Cu(II) with EDTA using azoligands (HL1–
HL4)as indicators.

M. Gaber et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 359–370
363
Azo ꢀ ligand þ Cu0nanoparticles ꢀ Azo ꢀ ligand ꢀ ꢀ ꢀ ꢀ
ꢀ Cu0nanoparticle
wavelength; eL and eML are the molar extinction coefficients of the
azo ligands and their Cu(II) azo complexes, respectively; [Lo] is
the initial concentration of azo ligands and finally [Mo] is the Cu(II)
concentration. Formation constant (K) values, correlation coefficient
and standard deviation for the complexes of azo ligands (HL1–HL4)
with Cu(II) ion were calculated from the plots of [Lo]/(A–Ao) versus
1/[Mo], Fig. (S5) and values were listed in Table 2. The results ob-
tained revealed that the Cu(II) complexes of the triazole azocom-
pounds were more stable than those of thiadiazole ones due to
the formation of two rings giving stability larger than one ring,
which give a great evidence to the proposal structure of the pre-
pared complexes.
ð1Þ
h
i
Azo ꢀ ligand . . . Cu0nanoparticles
h
i
Kapp
¼
½Azo ꢀ ligandꢅ ꢂ Cu0nanoparticles
The change in intensity of the absorption peak due to the forma-
tion of the surface complex was utilized to obtain Kapp according to
Benesi and Hildebrand.11
Aobs ¼ ð1 ꢀ
aÞCoeazoꢀligand
þ
aCoecomplex
ð2Þ
where Aobs is the observed absorbance of the solution containing
Coordination with colloidal copper nanoparticles
different concentrations of colloidal Cu0 nanoparticles,
is the de-
gree of association between azo ligand and Cu0 nanoparticles,
azo-ligand and complex are the molar extinction coefficients at the de-
a
The interaction between azo ligands (HL1–HL4) and colloidal Copper
Nanoparticles
e
e
fined wavelength of azo ligand and the formed complex, respec-
tively. In water, Eq. (2) can be expressed as Eq. (3), where Ao and
Ac are the absorbance of azo ligand and the complex, respectively,
with a concentration of Co:
The interaction between azo ligands (HL1–HL3) and copper
nanoparticles may occurred through the free anchoring (AOH)
group as a result of contribution of the other group in intermolec-
ular hydrogen bond with nitrogen atom of azo group besides the
great steric hindrance with hetero ring. Furthermore, for (HL4)
the interaction occured through the thiol and hydroxyl groups,
Scheme 1.
Aobs ¼ ð1 ꢀ
a
ÞAo þ
a
Ac
ð3Þ
At relatively high Cu0 nanoparticles concentrations,
a
can be equa-
ted to (Kapp[Cu0 nanoparticles])/(1 + Kapp[Cu0 nanoparticles]). In this
case, Eq. (3) can be changed to Eq. (4):
Absorption characteristics of azo ACu nanoparticles
Fig. 3. showed the absorption spectrum of azo ligand (HL1) in
the absence and presence of different concentrations of colloidal
Cu0 nanoparticles. As the the concentration of colloidal Cu0 nano-
particles increased, the absorption of azo ligand decreased regu-
larly with appearance of new peak due to the formation of
surface complex to a limits in which the Cu0 surface was saturated
with ligand molecules and up this concentration there was no ob-
served increasing in absorption spectra (Fig. S6) for TEM image for
the complex formation [35].
1
1
1
¼
þ
ð4Þ
KappðAc ꢀ AoÞ½Cu0nanoparticlesꢅ
Aobs ꢀ Ao Ac ꢀ Ao
The enhancement of absorbance was due to absorption of the
surface complex, i.e. donor–acceptor behavior [36,37] based on
the good linear relationship between 1/(Aobs–Ao) versus reciprocal
concentration of colloidal Cu0 nanoparticles with a slope equal to
1/Kapp(Ac–Ao) and an intercept equal to 1/(Ac–Ao) Fig. 4. The corre-
lation coefficient, standard deviation and values of the apparent
association constant (Kapp) were determine from this plot. Further,
the reason for the highest apparent association constant of Cu0
nanoparticles compared to that bulk copper may be due to the lar-
ger surface area of the Cu0 nanoparticles.
The apparent association constant (Kapp) for the complex be-
tween azo ligand and colloidal Cu0 nanoparticles can be given by
the equation:
R2
Studies of the solid complexes (C1–C4)
H
N
O
H
N
N
N
The azo compounds reacted with Cu(II) acetateꢂ4H2O in 1:1 mol
ratio leading to the formation of complexes having the general
formula [CuHL1ꢀ4(xH2O)]ꢂOAcꢂyH2O, Table 1. The isolated solid
N
Cu0
O
H
H
O
H
H
(a)
Free ligand
1.0
[Cu0] = [4.23E-11]
[Cu0] = [8.46E-11]
[Cu0] = [1.27E-10]
H
N
N
S
N
[Cu0] = [1.69E-10]
N
0.8
[Cu0] = [2.12E-10]
[Cu0] = [2.54E-10]
[Cu0] = [3.38E-10]
Cu0
O
R1
[Cu0] = [4.23E-10]
0.6
0.4
0.2
0.0
H
H
(b)
H
N
O
H
H
H
N
S
N
N
Cu0
O
Cu0
S
200
300
400
500
600
700
(c)
Wavelength (nm)
Scheme 1. The mode of interaction between (a) HL1, HL2. (b) HL3 and (c) HL4 with
Fig. 3. Showed the absorption spectrum of azo ligand (HL1) in the absence and
the surface of colloidal Cu0.
presence of different concentrations of colloidal Cu0 nanoparticles.

364
M. Gaber et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 359–370
complexes were stable and insoluble in common organic solvents
but soluble in DMF and DMSO. The solid Cu(II) azo complexes were
subjected to elemental analyses, spectral, conductance, magnetic
measurements as well as thermal analysis. The elemental analyses
were in agreement with the formula suggested in Table 1 and
Scheme 2.
From the above arguments together with the elemental analy-
ses, it was concluded that the azo compounds (HL1,HL2) behave
as mono-negative tridentate ligands with ONN donor sites while
azo compounds (HL3,HL4) behave as mono-negative bidentate li-
gands with ON donor sites.
Electronic spectra and magnetic measurements
Conductivity measurement
The electronic absorption spectra of Cu(II) azo complexes (C1
and C2) exhibited two bands at (14925,18667) and
(15152,17857) cmꢀ1 attributed to 2B1g ? 2A1g and 2B1g ? 2E1g
transitions which favored square planar geometry around Cu(II)
ion [39]. The electronic spectra of Cu(II) azo complexes (C3 and
C4) exhibited characteristic band at 16949 and 16129 cmꢀ1 corre-
sponding to the tetrahedral geometry. Cu(II) complexes (C1–C4)
have normal magnetic moments values (1.75–1.87 BM) corre-
sponding to the mono-nuclear Cu(II) complexes [8].
The molar conductance values of the isolated complexes mea-
sured in DMF solution (10ꢀ3 M) at room temperature are 80, 100,
90 and 110
X
ꢀ1 molꢀ1 cm2 for Cu(II) complexes C1, C2, C3 and C4,
respectively, indicating the ionic nature of the complexes [38].
Also, the addition of FeCl3 solution to the solution of complexes
gave the reddish brown coloration confirming the non coordina-
tion of acetate anion, i.e. acetate anion was outside the coordina-
tion sphere.
IR spectra
ESR spectra
The infrared spectra of the solid Cu(II) azo complexes displayed
interesting changes that may give a reasonable idea about the
structure of the metal complexes, (Table S3)
The ESR spectra of the powdered Cu(II) azo complexes (C1–C4)
were recorded at room temperature (25 °C) and the g-values are
listed in Table 3. The ESR spectra of complexes were characteristic
of a monomeric configuration and having an axial symmetry type
of dx2–y2 ground state which is most common for Cu(II) complexes.
The ESR spectra of Cu(II) azo complexes exhibited two g-values.
The g-values of all complexes have a positive contribution from the
value of the free electron (2.0023) due to the measurable covalent
character in the bonding between the azo ligands and Cu(II) ion.
The values of g11 < 2.3 indicating that the Cu(II)-azo compounds
bonds have the covalent character [40]. Also, the g values are re-
lated by the expression G = (g11 ꢀ 2)/(g\ ꢀ 2). If the value of
G > 4, the exchange interaction is negligible whereas if G < 4, a con-
siderable exchange interaction is indicated in the solid complexes.
The G values of Cu(II) azo complexes were higher than 4 indicated
that there was no interaction between Cu centers. The g-values of
Cu(II) azo complexes with a 2B1g ground state (g11 > g\) may be ex-
pressed [41] by:
ꢆ All complexes showed a broad band in the range 3330–
3420 cmꢀ1 assigned to
t(OH) of the coordination water mole-
cules associated with the complexes.
ꢆ The (N@N) of the free azo compounds exhibited a shift to
t
lower values on complex formation indicating the coordination
of the azo nitrogen to the Cu(II) ion, which was supported by
the appearance of a new band within the range 444–486 cmꢀ1
due to MAN bond formation.
ꢆ For complexes C1 and C2, the
t(C@N) group of the triazole ring
was shifted to lower wavenumbers indicating the participation
of azomethine nitrogen atom on complex formation. For Cu(II)
complexes C3 and C4,
t(C@N) remained unchanged indicating
that the (C@N) group of the thiadiazole ring was not involved
in the coordination.
ꢆ The phenolic (CAO) stretching vibration of the free azo com-
pounds was shifted to higher values by 25–85 cmꢀ1 indicating
the involvement of the phenolic oxygen in complex formation.
ꢆ The appearance of a new band in the spectra of Cu(II) complexes
within the range 532–583 cmꢀ1 which may be assigned to MAO
bond.
K211 ¼ ðg11 ꢀ 2:0023Þ
D
2=8k
k2? ¼ ðg? ꢀ 2:0023Þ
k2 ¼ ½k211 þ 2k2?ꢅ=3
D
1=2k
where k211 and k2? were the parallel and perpendicular components,
respectively, of the orbital reduction factor k, k was the spin–orbit
coupling constant (ꢀ828 cmꢀ1) for free copper, D1 and D2 were
the electronic transition 2B1g ? 2E1g and 2B1g ? 2B2g respectively,
ꢆ The appearance of new bands at 3420, 1610, 940 and 630 cmꢀ1
due to t(OH), d(H2O), qrock (H2O) and qwagg (H2O), respectively.
corresponding to coordination water.
the calculated values of k211, k2 indicated that k11 < k\ which
\
2
0.0020
was a good evidence for the assumed B1g ground state. The lower
values of k than unity were indicative of their covalent nature
which in agreement with the conclusion obtained from the values
of g11.Significant information about the nature of bonding in Cu
complex can be derived from the relative magnitudes of k11 and
L4
0.0015
)
k\ In case of pure
plies considerable in-plane
bonding, k11 > k\ For the Cu(II) complexes under investigation,
k11 < k\, implying a greater contribution from in-plane bonding
bonding [42].
r
-bonding, k11ꢁk\ꢁ0.77, wheresas k11 < k\ im-
0
p
bonding while for out-of-plane
p
0.0010
1/(-A
L3
p
than from out-of-plane
p bonding in metal ligand p
0.0005
0.0000
L2
L1
Thermal analysis
Thermogravimetric analyses. The TGA as well as DrTGA data of the
thermal decomposition of the Cu(II) azo complexes (C1–C4) were
listed in Table 4. As an example, the TG curve of C1 is shown in
Fig. S7. The correlation between the different decomposition steps
of the complexes with the corresponding weight losses discussed
in term of the proposal formula of the complexes as follows:
0.00E+000 5.00E+009 1.00E+010 1.50E+010 2.00E+010 2.50E+010
1/[M0 ]
Fig. 4. Linear dependence of 1/(A–Ao) on the reciprocal concentration of colloidal-
copper nanoparticles according to Benesi and Hildebrand equation.

M. Gaber et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 359–370
365
Complex C1 (R2 =H; n=1)
Complex C2 ( R2 = SCH3 )
Complex C3 ( R1 = H; n=1)
Complex C4 ( R1 = SH; n=1)
Scheme 2. Structure of complexes (C1–C4).
Table 3
Spin hamiltonian and bonding parameters of Cu(II) azo complexes (C1–C4).
of this step is 23.8 kJ/mol. The second degradation step within
the temperature range 133–221 °C with an estimated mass loss
of 4.98% (calcd. 4.84%) was assigned to the loss of two mole-
cules of coordination water. The activation energy of this step
is 49.25 kJ/mol. The third degradation step within the tempera-
ture range 221–335 °C with an estimated mass loss of 17.26%
(calcd. 17.29%) corresponded to the loss of acetate molecule.
The activation energy of this step is 49.34 kJ/mol. The fourth
degradation step within the temperature range 335–461 °C
with an estimated mass loss of 51.2% (calcd. 51.27%) was
assigned to the loss of N2 gas, hetero ring and C6H4O with the
formation of CuO as a final product. The activation energy of
this step is 98.7 kJ/mol.
ꢆ C4 was thermally decomposed within the temperature range
25–800 °C at successive four degradation steps. The first dehy-
dration step within the temperature range 38–111 °C with an
estimated mass loss of 4.37% (calcd. 4.71%) corresponded to
the loss of one molecule of lattice water. The activation energy
of this step is 52.6 kJ/mol. The second degradation step within
the temperature range 111–187 °C with an estimated mass loss
of 4.58% (calcd. 4.61%) corresponded to the loss of two mole-
cules of coordination water. The activation energy of this step
is 32.4 kJ/mol. The third degradation step within the tempera-
ture range 187–380 °C with an estimated mass loss of 31.25%
(calcd. 31.26%) corresponded to the loss of hetero ring. The acti-
vation energy of this step is 39.4 kJ/mol. The fourth degradation
step within the temperature range 380–667 °C with an esti-
mated mass loss of 49.85% (calcd. 49.85%) was assigned to the
loss of N2 gas, acetate and C6H4O with the formation of CuO
as a final product. The activation energy of this step is 30.2 kJ/
mol
State
C1
C2
C4
g11
g\
gav
G
2.244
2.061
2.124
4
2.110
2.026
2.054
4.23
2.40
2.06
2.173
6.66
–
k11
k\
0.813
0.8897
0.80
0.8897
–
ꢆ C1 was thermally decomposed within the temperature range
25–800 °C at successive four degradation steps. The first step
within the temperature range 39–92 °C with an estimated mass
loss of 4.96% (calcd. 5.03%) corresponded to the loss of one mol-
ecule of lattice water. The activation energy of this step is
58.5 kJ/mol. The second degradation step within the tempera-
ture range 92–141 °C with an estimated mass loss of 5.22%
(calcd. 5.17%) can be assigned to the loss of one molecule of
coordination water molecule. The activation energy of this step
is 74.6 kJ/mol. The third degradation step within the tempera-
ture range 141–444 °C with an estimated mass loss of 20.52%
(calcd. 20.49%) corresponded to the loss of hetero ring. The acti-
vation energy of this step is 9.3 kJ/mol. The fourth degradation
step within the temperature range 444–664 °C with an esti-
mated mass loss of 47% (calcd. 48%) can be assigned to the loss
of N2 gas, acetate and C6H4O with the formation of CuO as a final
product. The activation energy of this step is 147.5 kJ/mol
ꢆ C2 was thermally decomposed within the temperature range
25–800 °C at successive four degradation steps. The first degra-
dation step within the temperature range 53–148 °C with an
estimated mass loss of 8.44% (calcd. 8.43%) corresponded to
the loss of two molecules of lattice water. The activation energy
of this step is 27.8 kJ/mol. The second degradation step within
the temperature range 148–217 °C with an estimated mass loss
of 4.61% (calcd. 4.41%) was assigned to the loss of two mole-
cules of coordination water. The activation energy of this step
is 109.1 kJ/mol. The third degradation step within the tempera-
ture range 217–342 °C with an estimated mass loss of 30.62%
(calcd. 30.55%) corresponded to the loss of hetero ring. The acti-
vation energy of this step was 52.8 kJ/mol. The fourth degrada-
tion step within the temperature range 342–474 °C with an
estimated mass loss of 50.66% (calcd. 50.76%) was assigned to
the loss of of N2 gas, acetate and C6H4O with the formation of
CuO as a final product. The activation energy of this step is
562.6 kJ/mol.
The thermal analysis of Cu(II) complexes confirmed the compo-
sition of the complexes and allow the number and the nature of the
water molecules determination. Complex C4 loss coordination
water at temperature lower than complex C3. Also, complex C1 loss
coordination water at lower temperature than complex C2. The fact
that the activation energy of the second step of decomposition is
greater than that of the first step may be due to the less steric
strain occurring at that point [43].
Kinetic and thermodynamic studies. The kinetic parameters of acti-
vation energy (E), reaction order (n) and Arrhenius pre-exponential
factor (A) were calculated from the integral method proposed by
Coats–Redfern [44] and the approximation method proposed by
Horowitz–Metzger [45], Fig. 5. The data listed in Table 5 showed
that the values obtained by approximation method were different
from those calculated by integral method due to the different
mathematical treatment of the obtained data [46].
ꢆ C3 was thermally decomposed within the temperature range
25–800 °C at successive four degradation steps. The first degra-
dation step within the temperature range 50–133 °C with an
estimated mass loss of 4.74% (calcd. 4.73%) corresponded to
the loss of one molecule of lattice water. The activation energy
The selection of the mechanism which best describes the ther-
mal decomposition of the desired complexes was deduced using

366
M. Gaber et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 359–370
Table 4
Thermo analytical results (TGA and DrTGA) of Cu(II) azo complexes (C1–C4).
Complex
(TGA)
Step
Temperature (°C)
Wt.%loss (calcd.)/found
Assignment
[CuHL1(H2O)]ꢂOAcꢂH2O
1st
39.89–92.04
92.04–140.62
140.62–444
444–663.7
(4.96)/5.03
(5.22)/5.17
(20.52)/20.49
(47)/47.4
Loss of lattice water
Loss of coordination water
Loss of hetero ring
2nd
3rd
4th
Loss of N2, C6H4O and acetate
[CuHL2(H2O)]ꢂOAcꢂ2H2O
[CuHL3(2H2O)]ꢂOAcꢂH2O
[CuHL4(2H2O)]ꢂOAcꢂH2O
1st
53.23–147.9
(8.44)/8.43
(4.61)/4.41
(30.62)/30.55
(50.6)/50.7
Loss of two molecules of lattice water
Loss of coordination water
Loss of hetero ring
2nd
3rd
4th
147.9–217.11
217.11–342.44
342.44–473.5
Loss of N2, C6H4O and acetate
1st
49.6–132.5
132.5–220.9
220.9–334.9
334.9–460.6
(4.74)/4.73
(4.98)/4.84
(17.16)/17.29
(52.1)/52.17
Loss of lattice water
Loss of two molecules of coordinated water
Loss of acetate
2nd
3rd
4th
Loss of N2, C6H4O and hetero ring
1st
38.3–110.5
(4.37)/4.71
(4.58)/4.61
(31.25)/31.26
(50.66)/50.66
Loss of lattice water
Loss of two molecules of coordinated water
Loss of hetero ring
2nd
3rd
4th
110.5–186.6
186.61–379.63
379.63–667.2
Loss of N2, C6H4O and acetate
served peak temperature. The thermodynamic parameters correspond-
ing to each chosen function form were listed in Table 6.
According to the data obtained, the following remarks can be
pointed out:
the method proposed by the integral method (Coats–Redfern)
according to equation:
ln½gð
a
Þ=T2ꢅ ¼ lnðAR=QEÞ ꢀ E=RT
g(
a
) suggested by Satava [47] were used to enunciate the mech-
ꢆ The high values of the energy of activation of the complexes
anism of thermal decomposition in each stage see Table (S4):
where Q is the heating rate, is the fraction decomposed, A is
Arrhenius pre-exponential factor, R is the universal gas constant,
(D
Eꢄ) reflect the high stability of the investigating complexes
a
due to their covalent character [48]. The positive sign of
D
Gꢄ
for the Cu(II) azo complexes under investigation revealed that
the free energy of the final residue is higher than that of the ini-
tial compound and all the decomposition steps are non-sponta-
neous processes.
T is the absolute temperature, g(a) is the kinetic model and E is
the activation energy.
The quantity ln[g(
The correlation coefficients for nine forms were calculated and
the form of g( ) for which the correlation has a maximum value
was chosen as the mechanism of reaction.The thermodynamic
parameters of activation
Hꢄ, Sꢄ and Gꢄ were estimated accord-
a
)/T2] was plotted as a function of (1000/T).
a
The values of
D
Gꢄ increased for the subsequent decomposition
Gꢄ of a given
stages of a given complex. Increasing the values of
D
D
D
D
complex reflected that the rate of removal of the subsequent ligand
was lower than that of the precedent ligand, this is due to increas-
ing to the previous methods with the aid of the following
expressions:-
ing in the values of T
D
Sꢄ from one step to another which overrides
D
D
D
Hꢄ ¼ E ꢀ RT
the values of
D
Hꢄ [49].
Sꢄ ¼ RlnðhA=KBTÞ
Gꢄ ¼
D
Hꢄ ꢀ T
D
Sꢄ
ꢆ The negative values of
complex than reactant and/or the reaction was slow [50].
ꢆ The positive values of
Hꢄ means that the decomposition pro-
cesses were endothermic.
D
Sꢄ indicated a more order activated
where
D
Hꢄ is the activation enthalpy (kJ molꢀ1),
D
Sꢄ is the activation
D
entropy (Jmolꢀ1 kꢀ1),
D
Gꢄ is the Gibbs activation free energy (kJ molꢀ1),
h is the Plank constant, KB is the Boltzmann constant and T is the ob-
(a)
(b)
-1
-2
-3
-4
-5
-6
step1
step2
step3
-15.0
step4
step1
step2
step3
step4
-15.5
-16.0
-16.5
-17.0
-17.5
Y
1.0
1.5
2.0
2.5
3.0
-60
-40
-20
0
20
40
60
-1
θ
1000/T K
Fig. 5. (a) Plotting Y = log(ꢀlog(1 ꢀ
a
)/T2) versus 1000/T (kꢀ1) for n = 1 and Y = log(1ꢀ(1 ꢀ
a
)
1ꢀn/T2(1 ꢀ n2)) versus 1000/T (kꢀ1) for n – 1 for the different decomposition steps
of complex C1 using Coats–Redfern equation. (b) Plotting Y = log(ꢀlog(1 ꢀ
a
)/T2) versus h for n = 1 and Y = log(1ꢀ(1 ꢀ
a
)
1ꢀn/T2(1 ꢀ n2)) versus h for n – 1 for the different
decomposition steps of complex C1 using Horowitz–Metzger equation.

M. Gaber et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 359–370
367
Table 5
120
Kinetic parameters of activation for complexes (C1–C4) using Coats–Redfern (CR) and
Horowitz–Metzger (HM) methods.
100
80
60
40
20
0
ED 100
Complex
C1
Step
1st
Method
n
r
E
A
ED 50
ED 25
CR
HM
CR
HM
CR
HM
CR
1
2
1
2
1
2
1
2
0.9840
0.9967
0.9905
0.9905
0.9922
0.9972
0.9922
0.9927
58.49
64.44
74.59
81.69
9.25
974.9571
46529943
43.721
1882421
5.24E + 09
0.552203
102.4779
1.25E + 10
2nd
3rd
4th
21.14
147.49
170.08
HM
C2
C3
C4
1st
CR
HM
CR
HM
CR
HM
CR
0
0
1
2
1
2
0
0
0.9923
0.9953
0.9995
0.9890
0.9874
0.9889
0.9896
0.9898
27.78
33.36
109.14
119.20
52.85
67.68
562.61
573.92
11116286
803.5078
0.80649
3.18E + 11
1501931
84353.55
1.28E-34
3.24E + 46
Fig. 6. In vitro cytoxcity of azo ligands (HL1–HL4) and their Cu(II) azo complexes
(C1–C4) on EAC compared to 5-fluorouracil.
2nd
3rd
4th
HM
1st
CR
HM
CR
HM
CR
HM
CR
0
0
1
2
0
0
1
2
0.99981
0.98718
0.9963
0.9953
0.9938
0.9963
0.9929
0.9911
23.48
29.16
49.25
57.65
49.34
58.57
98.70
113.76
71666998
89.41566
1620272
22172.16
10753305
2855.612
2373.113
1.25E + 08
120
100
80
60
40
20
0
2nd
3rd
4th
HM
1st
CR
HM
CR
HM
CR
HM
CR
1
3
1
2
1
2
1
0.5
0.9973
0.9970
0.9965
0.9970
0.9886
0.9829
0.9925
0.9893
52.56
58.94
32.42
40.87
39.41
50.68
30.24
39.94
5830.452
7137804
33583537
607.998
31620512
933.7278
5.67E + 08
9.470626
2nd
3rd
4th
Control of
L-
acid
HL2
HL4
C2
C4
ABTS Ascorbic
Fig. 7. Antioxidant assay for azo ligand (HL2,HL4) and their Cu(II) azocomplexes.
HM
Molecular modeling
2. There is a variation in CAO bond lengths on complexation. It
becomes slightly longer (for complexes C1 and C2) or shorter
(for complexes C3 and C4) as the coordination takes place via
o-atom of CAO group that is formed on deprotonation of CAOH
leading to the formation of CuAO bond [52].
3. The bond length of CuAO increases in the order C1 > C2 > C3 > C4
while CuAN increases in the order C1 > C2 > C4 > C3.
4. The bond angles around the N@N group were reduced upon
coordination.
The molecular structure along with atom numbering of the li-
gands and complexes are shown in structures (1–8). Data analysis
in Tables S5–S12 (Supplementary material) for bond lengths and
bond angles of the ligands and their complexes revealed the fol-
lowing remarks:
1. The N@N bond length is elongated due to the formation of
CuAN bond. The same trend was observed for the C@N bond
length of the hetero ring [51].
Table 6
Integral methods and their corresponding thermodynamic parameters for Cu(II) azo complexes (C1–C4).
No.
C1
Complex
Step
g(
a
)
r
Thermodynamic parameters
Eꢄ Hꢄ
D
D
A
ꢀ
D
Sꢄ
D
Gꢄ
[CuHL1(H2O)].OAc.H2O
1st
[1ꢀ(1 ꢀ
[1ꢀ(1 ꢀ
[1ꢀ(1 ꢀ
[1ꢀ(1 ꢀ
a2
[1ꢀ(1 ꢀ
[1ꢀ(1 ꢀ
a2
a
a
a
a
)
)
)
)
]
]
]
]
0.98542
0.99118
0.99683
0.99236
122.40
155.01
28.06
119.64
151.82
22.81
1.52E + 09
1.97E + 12
1.06E-07
4.8E + 12
0.070
0.012
0.385
0.010
142.82
156.28
266.02
303.18
1/3
1/3
1/3
1/3
2
2
2
2
2nd
3rd
4th
301.68
295.24
C2
C3
C4
[CuHL2(H2O)].OAc.2H2O
[CuHL3(2H2O)].OAc.H2O
[CuHL4(2H2O)].OAc.H2O
1st
0.99366
0.99951
0.99190
0.98976
60.47
225.38
46.99
57.56
221.55
42.29
4.46
3.25E + 16
4.4E-5
0.234
0.068
0.334
0.436
139.54
252.63
230.85
2023.36
1/3
1/3
2
2
2nd
3rd
4th
a
a
)
)
]
]
1135.55
1130.38
1.35E + 88
1st
a2
0.99984
0.99679
0.99468
0.98907
52.96
105.78
107.88
192.84
49.96
101.91
103.28
187.53
0.09
0.267
0.199
0.211
0.093
146.30
194.69
220.167
246.73
1/3
1/3
2
2
2nd
3rd
4th
[1ꢀ(1 ꢀ
a
a
)
)
]
369.40
106.12
1.91E + 08
a2
[1ꢀ(1 ꢀ
]
1/3
1/3
1/3
1/3
2
2
2
2
1st
[1ꢀ(1 ꢀ
[1ꢀ(1 ꢀ
[1ꢀ(1 ꢀ
[1ꢀ(1 ꢀ
a
a
a
a
)
)
)
)
]
]
]
]
0.99760
0.99713
0.98892
0.99313
110.57
71.58
80.03
66.89
107.70
68.03
75.06
60.01
27108733
0.16
0.08
0.104
0.263
0.272
0.322
143.46
180.60
237.56
326.55
2nd
3rd
4th
0.0003
Where
D D D D
Eꢄ, Hꢄ and Gꢄ in kJ molꢀ1 while Sꢄ in J molꢀ1 Kꢀ1 and A in Sꢀ.

368
M. Gaber et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 359–370
5. The bond angle in Cu(II) complexes C1 and C2 lie in the range
reported for square planar geometry while for complexes C3
and C4 the bond angle are quite near to the tetrahedral
geometry.
2,2-azino-bis(3-ethyl benzthiazoline-6-sulfonic acid) (ABTS) as
stable free radical to assess antioxidant potential of the investi-
gating compounds.
L-ascorbic acid is a well known antioxidant, a reducing agent, a
powerful electron donor is known to play a vital role in a number
of chemical, biological and physiological reactions in the human
body and used as stander antioxidant (positive control). Blank
sample was run without ABTS and using instead of tested com-
pounds while negative control was run with ABTS and MeOH/
phosphate buffer (1:1) only [53].
Some azo ligands and their copper complexes were
subjected to anti-oxidant assay by ABTS method and
experimental data were tabulated in Table (S14) and illustrated
in Fig. 7. From the data obtained experimentally, we can derive
these points:
Biological activities
In vitro antitumor activity
Antitumor activity of the investigated azo ligands (HL1–HL4)
and their Cu(II) azo complexes (C1–C4) were evaluated against Ehr-
lish Ascites Carcinoma (EAC) derived from ascetic fluid of the fe-
male Swiss albino mice (purchased from National Cancer
Institute, Cairo, Egypt) after 2 weeks of their injection with
1 ꢃ 106 EAC. Different concentrations of the tested compounds dis-
solved in DMSO were prepared (100, 50 and 25 ll/ml from 1 mg/
ml in DMSO) then 0.1 ml of each concentration was added to the
suspension of tumor cells which was then cultured in round bot-
tom 96-well plate for 2 h at 37 °C in humidified atmosphere with
5% CO2. The same volume of DMSO was added to somewells as
control. 5-fluorouracil was used as a standard cytotoxic agent.
The viability of the cells was determined under microscopial exam-
ination using hemocytometer after staining with trypan blue. In
this assy, blue cells absorb the dye and appear blue while the viable
cells appear unstained. The data are summarized in Table (S13) and
illustrated in Fig. 6.
(a) All complexes subjected to antioxidant assay were less
potent active antioxidant agents compared to corresponding
azo ligands, the reason may be related to the consumption of
free electrons in coordination.
(b) As increasing number of sulphur atoms in compounds under
study, their reactivity increased which was shown from
results that HL4 was the highest active ones than and HL2
which may be related to the high electronegativity of nitro-
gen atom than sulphur one.
By comparing the cytotoxicity of the investigated compounds, it
was found that
Conclusion
(a) All Cu(II) azo complexes expressed less cytotoxic than their
free azo ligands which may be a result of coordination and
(b) The highest cytotoxic effects of HL4 at its lowest does (ED25)
as shown in Table S13 could be attributed to its high of sul-
pher atom number as well as to its high anti-oxidant effects
as shown in Fig. 7 and Table S14.
The structure assignment of Cu(II) complexes under investiga-
tion was carried out using the elemental analysis, molar conductiv-
ity, spectroscopic data (IR, UV–Vis, ESR), magnetic measurements
and thermal analysis (TG). The IR spectra showed that the triazole
azodyes behave as mono basic tridentate ligands with ONN donor
sites while the thiadiazole azodyes behave as mono basic bidentate
ligands with ON donor sites. A square planar structure was pro-
posed for Cu(II) complexes C1 and C2 and a tetrahedral structure
for Cu(II) complexes C3 and C4. The reaction between the azodyes
and Cu(II) ion were also studied spectrophotometrically in solu-
tion. The stoichiometry and stability constants were evaluated.
The analytical data indicated that the investigated azodyes can
be used as an indicator for spectrophotometric determination of
Cu(II) ion. The calculated bond lengths and bond angles confirmed
the structures of Cu(II) complexes. The interaction between the
synthesized azo ligands and copper nanoparticles was studied.
The spectral data showed the formation of a surface complex be-
tween the azodye and colloidal Cu nanoparticles through anchor-
ing AOHꢀ group. The antitumor and antioxidant activities of the
synthesized azo ligands and their Cu(II) azo complexes have been
evaluated.
ABTS antioxidant activity screening
The antioxidant activity assay employed here is one of the sev-
eral assays that depend on measuring the consumption of stable
free radicals. The methodology assumes that the consumption of
the stable free radical (Xꢂ) will be determined by reactions as
follows:
Xꢂ þ YH ! XH þ Yꢂ
The rate and/or the extent of the process measured in terms
of the decrease in Xꢂ concentration would be related to the abil-
ity of the added compounds to trap free radicals. The absorbance
was measured at a specific wavelength and the reduction in col-
or intensity of the free radical solution due to scavenging of the
free radical by the antioxidant was expressed as inhibition per-
centage. The assay employs the radical action derived from
Structure 1: Molecular model of HL1
Structure 2: Molecular model of HL2

M. Gaber et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 107 (2013) 359–370
369
Structure 3: Molecular model of HL3
Structure 4: Molecular model of HL4
Structure 5: Molecular model of Cu (II) complex C1
Structure 6: Molecular model of Cu (II) complex C2
Structure 7: Molecular model of Cu (II) complex C3
Structure 8: Molecular model of Cu (II) complex C4
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Appendix A. Supplementary material
[17] Z. Chohan, S. Sumrra, M. Toussoufi, T. Hadda, Eur. J. Med. Chem. 45 (2010)
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[18] A.M. Khedr, M. Gaber, E.H. Abd El-ZaherChin, J. Chem. 29 (2011) 2421–2427.
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.saa.2013.01.039.
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