Spectral, electrochemical, thermal, DNA binding ability, antioxidant and antibacterial studies of novel Ru(III) Schiff base complexes

Article abstract of DOI:10.1016/j.saa.2014.01.050

Four new air stable low spin Ru(III) complexes of the type [Ru(L ^1-4)(H₂O)₂]Cl have been synthesized, where L = dianion of the tetradentate Schiff base ligands namely N, N′bis(salicylaldehyde)4,5-dimethy-l,2-phenylendiammi

Full text of DOI:10.1016/j.saa.2014.01.050

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 404–415
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Spectral, electrochemical, thermal, DNA binding ability, antioxidant
and antibacterial studies of novel Ru(III) Schiff base complexes
a,c
Ayman A. Abdel Aziz a,b, , Hemmat A. Elbadawy
⇑
a Department of Chemistry, Faculty of Science, University of Tabuk, Tabuk 71421, Saudi Arabia
b Department of Chemistry, Faculty of Science, Ain Shams University, Cairo 11566, Egypt
c Department of Chemistry, Faculty of Science, Alexandria University, Alexandria 21641, Egypt
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
1.0
0.8
0.6
0.4
0.2
0.0
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Novel Ru(III)-NOON Schiff bases
complexes.
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
(1)
0
5
10
15
20
25
DNA
30
35
40
45
50
55
[
]
x
10-5
M
Spectral, electrochemical and
thermogravimetric studies.
Binding ability of Ru(III) complexes
with calf thymus DNA.
Antioxidant activity of the novel
complexes.
A
H
2O
Ru
DN
-
T
C
350
400
450
500
550
600
650
N
O
Wavelength (nm)
N
O
Cl
E
B
-
DN
400
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
A
(1)
Antibacterial screening.
350
300
250
200
150
100
50
H2
O
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5
(
I
I
I
)
[
R
u
c
o
m
p
l
e
x
]
0
500
550
600
650
700
Wavelength (nm)
a r t i c l e i n f o
a b s t r a c t
Four new air stable low spin Ru(III) complexes of the type [Ru(L1–4)(H2O)2]Cl have been synthesized,
where L = dianion of the tetradentate Schiff base ligands namely N,N0bis(salicylaldehyde)4,5-dimethy-
l,2-phenylendiammine (L1H2), N,N0bis(salicylaldehyde)4,5-dichloro 1,2-phenylendiammine (L2H2),
N,N0bis(o-vanillin)4,5-dimethy-1,2-phenylendiammine (L3H2) and N,N0bis(o-vanillin)4,5-dichloro-1,2-
phenylendiammine (L4H2). The complexes have been fully characterized by elemental analysis, infrared
spectroscopy, electronic spectroscopy, magnetic susceptibility and electron spin resonance spectroscopy.
Elemental analyses and spectroscopic data have been showed that, the stoichiometries of complexes
were 1:1 with an octahedral geometry for all the complexes. Thermal analysis measurements indicated
that the complexes have good thermal stability. The redox behavior of the complexes has been investi-
gated by the cyclic voltammetric technique. The interaction of these complexes with calf thymus DNA
(CT-DNA) was explored by different techniques which revealed that the complexes could bind to
CT-DNA through an intercalative mode. Furthermore, the antioxidant activity of the Ru(III) complexes
against superoxide and hydroxyl radicals was evaluated by using spectrophotometer methods in vitro.
The experiments on antioxidant activity show that the complexes were found to possess potent antiox-
idant activity. Additionally, as a potential application the antibacterial activity of the complexes was
assessed by testing their effect on the growth of various strains of bacteria.
Article history:
Received 13 October 2013
Received in revised form 21 December 2013
Accepted 8 January 2014
Available online 22 January 2014
Keywords:
Ru(III) complexes
Characterization
Cyclic voltammetry
CT-DNA binding
Antioxidant activity
Antibacterial activity
Ó 2014 Elsevier B.V. All rights reserved.
Introduction
Interactions between small molecules and DNA rank among the
primary action mechanism of anticancer activity and designing of
molecules that bind and cleave DNA have attracted extensive
⇑
Corresponding author at: University of Tabuk, Tabuk 71421, Saudi Arabia. Tel.:
+966 0595216274.
E-mail addresses: aabdelaziz@ut.edu.sa, aymanaziz31@gmail.com (A.A. Abdel
Aziz).
1386-1425/$ - see front matter Ó 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2014.01.050

A.A. Abdel Aziz, H.A. Elbadawy / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 404–415
405
attention [1–4]. Additionally, it has been demonstrated that free
radicals can damage proteins, lipids, and DNA of bio-tissues, lead-
ing to increased rates of cancer [5]. Fortunately, antioxidants can
prevent this damage, due to their free radical scavenging activity
[6]. Hence, it was very important to develop compounds with both
strong antioxidant and DNA-binding properties for effective cancer
therapy. Transition metal complexes of Schiff bases have been
widely exploited to develop synthetic binding and cleavage agents
for DNA [7–9].
Ruthenium’s properties are well suited towards pharmacologi-
cal oxidation states (II–IV) under physiologically relevant condi-
tions [10]. Also, the energy barrier for interconversion between
these oxidation states is relatively low, allowing for ready
oxidation state changes when inside the cell [11]. Furthermore,
ruthenium tends to form octahedral complexes, which gives the
chemist two more ligands to exploit compared with platinum(II)
complexes, which adopt a square planar geometry and can also
form strong chemical bonds with a range of different elements of
varying chemical ‘hardness’ and electronegativities, meaning that
ruthenium can bind to a range of biomolecules, not just DNA [12].
Ru(II) and Ru(III) complexes are presently an object of great
attention in the field of medicinal chemistry, as antitumor agents
with selective antimetastatic properties and low systemic toxicity.
Ruthenium compounds appear to penetrate reasonably well the
tumor cells and bind effectively to DNA [13,14].
The well-developed synthetic chemistry of ruthenium, particu-
larly with imine ligands provides for many approaches to innova-
tive new metallopharmaceuticals [15]. Advantages of utilizing
ruthenium imine complexes in drug development include; (i) reli-
able preparations of stable complexes with predictable structures,
(ii) the ability to tune ligand affinities, electron transfer, substitu-
tion rates, and reduction potentials, and (iii) an increasing knowl-
edge of the biological effects of ruthenium complexes.
However little work was focused on binding ability and antiox-
idant activity of ruthenium(III) Schiff bases complexes and as a
result of the continuing quest for new complexes of ruthenium,
in this work, we are reporting the synthesis, spectral and electro-
chemical characterization of a series of new Ru(III) complexes con-
taining tetradentate Schiff base ligands derived from condensation
reactions of 4,5-dimethyl-1,2-phenylendiammine and 4,5-di-
chloro-1,2-phenylendiammine with salicylaldehyde and o-vanillin.
Furthermore, the interaction of calf thymus DNA (CT-DNA) with
the novel Ru(III) complexes was investigated by UV–Vis. Spectro-
photometry, fluorescence quenching and viscosity measurements.
Furthermore, the antioxidant activity of the complexes was deter-
mined by superoxide and hydroxyl radical scavenging method
in vitro. In addition, the antibacterial activity of the reported com-
pounds was studied and the results were compared with standard
antibiotics.
complexes was estimated by using 1-nitroso-2-naphthol reagent
[16] by adopting spectrophotometric extraction technique [17].
The chloride content of the complexes was determined by photo-
metric method [18]. The FT-IR spectra of the samples in the
4000–400 cmꢂ1 region were obtained in KBr discs on a Unicam-
Mattson 1000 FT-IR. The molar conductivities of the complexes
(1 ꢃ 10ꢂ3 M) in dimethylformamide (DMF) solution were mea-
sured at room temperature by using Jenway 4010 conductivity me-
ter. Room temperature (298 K) magnetic susceptibilities were
measured using a Sherwood Scientific balance using Hg[Co(SCN)4]
as a calibrant. Diamagnetic corrections calculated from Pascal’s
constants [19] were used to obtain the molar paramagnetic sus-
ceptibilities. Electron spin resonance (ESR) measurements of solid
state Ru(III) complexes were recorded at room temperature
(298 K) and liquid nitrogen temperature (77 K) on Bruker EPR
spectrometer at 9.706 GHz (X-band), the microwave power was
(1.0 mW) with 4.0 G modulation amplitude, using 2,2-diph-
enylpyridylhydrazone (DPPH) as standard (g = 2.0037). Cyclic vol-
tammetric measurements were carried out using a Princeton EG
and GPARC model potentiostat using glassy carbon working elec-
trode and all the potentials were referred to Ag/AgCl. Thermogravi-
metric analyses (TGA) were carried out using a Shimadzu DT-50
thermal analyzer under nitrogen atmosphere with a heating rate
10 °C/min. The UV–Vis spectra were recorded on a Shimadzu UV
1800 spectrophotometer. Fluorescence spectra were recorded on
a Jenway 6270 fluorimeter at room temperature.
Syntheses
Microwave assisted solvent-free synthesis of the Schiff base ligands
(L1–4H2)
The ligands L1–4H2 were previously synthesized by Ref. [20].
However, microwave assisted solvent-free synthesis method is
used to enhance the yield and reduce the time. 0.1 mol of the dia-
mine derivative and 0.2 mol of aldehyde were mixed well in a
50 ml Pyrex beaker and the mixture was irradiated in a microwave
oven for one minute. The yellow product obtained was separated,
dried and recrystallized from ethanol and the purity of the ligands
was checked by TLC.
Synthesis of Ru(III) complexes (1–4)
All Ru(III) complexes were synthesized according to the general
procedure: a stoichiometric amount of RuCl3ꢁ3H2O (10 mmol) in
ethanol was added to a hot ethanol solution of the desired ligand
(10 mmol) and the reaction mixture was boiled under reflux with
stirring for 3 h. On cooling the desired complex was obtained as
powder. In some cases, complete precipitation was achieved by
the addition of diethyl ether to the cold reaction mixture. The sol-
vent was evaporated on a vacuum line. The residue was washed
several times with hot petroleum ether (60–80 °C) and recrystal-
lized from benzene/ethanol to give reddish-brown crystals. The
products were finally dried in vacuo over P2O5. Synthetic route of
[Ru(L1–4)(H2O)2]Cl complexes (1–4) is shown in Scheme 1.
Experimental
Materials
DNA-binding studies
4,5-Dimethyl-1,2-phenylendiammine, 4,5-dichloro-1,2-pheny-
lendiammine, o-vanillin, salicylaldehyde and RuCl3ꢁ3H2O were
supplied from Aldrich. Calf thymus DNA (CT-DNA) and ethidium
bromide (EB) were purchased from Sigma Chemicals Co. All
solvents used were of analytical reagent grade and used without
further purification.
All experimental involving CT-DNA were performed in HCl/NaCl
(5:50 mM) buffer solution (pH = 7.24). Tris–HCl was prepared
using deionized and sonicated triple distilled water and kept at
4 °C for 3 days. The absorption ratio of CT-DNA solutions A260
/
A280 was 1.8:1.9, indicating that the CT-DNA was sufficiently free
from protein [21]. The CT-DNA concentration was determined via
absorption spectroscopy using the molar absorption coefficient of
6600 Mꢂ1 cmꢂ1 (260 nm) [22]. Stock solutions of metal complexes
were prepared by dissolving them in dimethylformamide (DMF)
and suitably diluting them with the corresponding buffer to the
required concentrations for all experiments. The extent of DMF
Instruments
Carbon, hydrogen and nitrogen were determined using Perkin
Elmer 2400 CHN elemental analyzer. Ruthenium content of the

406
A.A. Abdel Aziz, H.A. Elbadawy / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 404–415
R
R
H2O
N
O
N
O
C2H5OH , Refulx
3 h
Cl
Ru
+
RuCl2.3H2O
N
N
HO
OH
H2O
R/
R/
R= CH3 and R/ = H
R= Cl and R/ = H
R= CH3 and R/ = OCH3
R= Cl and R/ = OCH3
H2L1
H2L2
H2L3
H2L4
Scheme 1. Synthetic route of [Ru(L1–4)(H2O)2]Cl complexes (1–4).
in the final concentration did not exceed 0.1% in the tested
solutions. At this concentration, DMF was not found to have any
effect on DNA conformation.
Viscosity measurements
Viscosity measurements were carried out using an Ostwald’s
capillary viscometer, immersed in a thermo stated water bath with
the temperature setting at 25 0.1 °C. CT-DNA samples with an
approximate average length of 200 base pairs were prepared by
sonication in order to minimize complexities arising from DNA
flexibility [26]. Titrations were performed for the complexes
Electronic absorption spectroscopy
Electronic absorption titration experiments were performed
with fixed Ru(III) complex concentration (10
increasing the concentration of CT-DNA (5.0–50.0
l
M), while gradually
M). When mea-
l
(0.0–5.0 lM), and each compound was introduced into a DNA solu-
suring the absorption spectra, an equal amount of CT-DNA was
added to both the complex solutions and the reference buffer solu-
tion to eliminate the absorbance of CT-DNA itself. The absorbance
values were recorded after each successive addition of CT-DNA
solution and equilibration for ꢄ5 min. Each sample was measured
three times and an average value was calculated. The intrinsic
binding constant Kb of Ru(III) complexes with CT-DNA was deter-
mined using the following equation [22]:
tion (0.30 mM) present in the viscometer. The flow times were
measured with a digital stopwatch. Each sample was measured
three times and an average flow time. The data were presented
1/3
as (
g
/
go
)
versus [Ru(III) complex]/[DNA] ratio of the concentra-
and go are the viscosity of
DNA in the presence and absence of the complex respectively
tion of the compound [27], where
g
[28,29]. The values of
equation:
g
and go were calculated by the following
½DNAꢅ
½DNAꢅ
1
g
¼ ðt ꢂ toÞ=to
ð3Þ
¼
þ
ð1Þ
ð
ea
ꢂ
ef
Þ
ð
eb
ꢂ
ef
Þ
Kbðeb
ꢂ
ef
Þ
where t is the observed flow time of DNA containing solution and, to
is the flow time of buffer alone. Relative viscosities for DNA were
calculated from the relation, g/go.
where [DNA] is the concentration of DNA in base pairs, ea is the
extinctions coefficient observed for the for the MLCT absorption
band at the given DNA concentration, ef is the extinction coefficient
of the free complex in solution and eb is the extinction coefficient of
the complex when fully bound to DNA. A plot of [DNA]/[eaꢂef] ver-
sus [DNA] gave a slope of 1/(eaꢂef) and Y intercept equal to (1/Kb)
(ebꢂef). The intrinsic binding constant (Kb) is given by the ratio of
the slope to the intercept.
Antioxidant activity
Superoxide radical scavenging activity
The superoxide radicals ðOꢂ2 ÅÞ were produced by the system
MET/VitB2/NBT [30]. The amount of OꢂÅ and suppression ratio for
2
Oꢂ2 Å can be calculated by measuring the absorbance at 560 nm, be-
Fluorescence spectroscopy
cause NBT can be reduced quantitatively to blue formazan by O2ꢂÅ
.
Further support for binding of Ru(III) complexes to CT-DNA was
studied through competitive emission quenching experiment.
Ethidium bromide (EB) is a common fluorescent probe for DNA
structure and has been employed in examinations of the mode
and process of metal complexes binding to DNA [23]. EB shows
weak reduced fluorescence intensity in buffer because of quench-
ing by solvent molecules, but its fluorescence is enhanced when
bound to DNA because of its intercalation into the helix and it is
quenched by the addition of another molecule that displaces EB
The solution of MET, VitB2 and NBT were prepared with 0.067 M
phosphate buffer (pH = 7.8) at the condition of avoiding light. The
tested Ru(III) complexes were dissolved in DMF. The reaction mix-
ture contained MET (0.01 mol Lꢂ1), NBT (4.6 ꢃ 10ꢂ5 mol Lꢂ1), VitB2
(3.3 ꢃ 10ꢂ6 mol Lꢂ1), phosphate buffer solution (0.067 mol Lꢂ1) and
a final concentration of Ru(III) complex from 0.03 to 1.5 lM. After
incubating at 30 °C for 10 min and illuminating with a fluorescent
lamp for 3 min, the absorbance (Ai) of the samples was measured
at 560 nm. The sample without the tested compound and avoiding
light was used as the control. The suppression ratio for Oꢂ2 Å was cal-
culated from the following expression:
from DNA [24]. A 2.0 mL solution of 4
(at saturating binding levels) was titrated by 2.5–25.0
l
M DNA and 0.32
lM EB
M the com-
l
plexes and ligand. Quenching data were analyzed according to the
Stern–Volmer equation which could be used to determine the fluo-
rescent quenching mechanism:
Io=I ¼ 1 þ Kq½RuðIIIÞ complexꢅ
Ao ꢂ Ai
Suppression ratio ð%Þ ¼
ꢃ 100
ð4Þ
Ao
ð2Þ
where Io and I are the fluorescence intensities in the absence and
presence of Ru(III) complex, respectively. Plots of Io/I versus [Ru(III)
complex] appear to be linear and Kq depends on temperature [25].
where Ai, the absorbance in the presence of the ligand or its
complexes; Ao, the absorbance in the absence of the ligand or its
complexes.

A.A. Abdel Aziz, H.A. Elbadawy / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 404–415
407
Hydroxyl radical scavenging activity
Results and discussion
The hydroxyl radical (HOÅ) in aqueous media was generated
through the Fenton reaction [31]. The solution of the tested com-
pounds was prepared with DMF. The reaction mixture contained
Characterization of the complexes
2.5 mL 0.15 M phosphate buffer (pH = 7.4), 0.5 mL 114
nin, 1.0 mL 945 M EDTA-Fe(II), 1 mL 3% H2O2 and 30
tested complex solution (the final concentration: Ci(i=1ꢂ9) = 0.3,
0.6, 0.9, 1.2, 1.5, 3.0, 6.0, 9.0. 12.0 and 15.00 M). The sample with-
out the tested compound was used as the control. The reaction
mixtures were incubated at 37 °C for 60 min in a water-bath.
Absorbances (Ai, Ao, Ac) at 520 nm were measured. The suppression
ratio for HOÅ was calculated from the following expression:
l
M safra-
The reactions of RuCl3ꢁ3H2O with tetradentate Schiff bases
ligands (L1–4H2) in a 1:1 M ratio in dry ethanol afforded new hexa-
coordinated low spin Ru(III) Schiff base complexes. Elemental anal-
yses and some physical properties of the reported ligands and their
Ru(III) complexes are listed in Table 1. The proposed molecular for-
mulae for all the complexes are in good agreement with the stoi-
chiometries concluded from their analytical data. The complexes
are stable in atmospheric conditions for extended periods and eas-
ily soluble in DMF and DMSO; slightly soluble in ethanol, methanol
and acetone; insoluble in benzene, water and diethyl ether. The
l
lL of the
l
Ai ꢂ Ao
Suppression ratio ð%Þ ¼
ꢃ 100
ð5Þ
Ao ꢂ Ac
molar conductance
(Km) values of the complexes in DMF
(1 ꢃ 10ꢂ3 M) at 25° fall in the range 99.79–107.21
X
ꢂ1 cm2 molꢂ1
where Ai, the absorbance in the presence of the tested compound;
Ao, the absorbance in the absence of the tested compound; Ac, the
absorbance in the absence of the tested compound, EDTA-Fe(II)
and H2O2. The antioxidant activity was expressed as the 50% inhib-
itory concentration (IC50). IC50 values were calculated from regres-
sion lines where: x was the tested compound concentration in mM
and y was percent inhibition of the tested compounds.
(Table 1), hence all complexes are considered as 1:1 electrolytes
in nature [34] and thus may be formulated as [Ru(L1–4)(H2O)2]Cl.
FT-IR spectra
The most important IR bands of the reported Schiff bases and
their Ru(III) complexes are listed in Table 2. On the basis of the very
similar spectra of the four complexes, it may be assumed that they
have the similar coordination structures. The IR spectra of Schiff
bases exhibited a broad band of medium intensity at 3389–
3444 cmꢂ1, strong band at 1608–1617 cmꢂ1, and a medium band
at 1274–1279 cmꢂ1, which were assigned to H-bonded AOH
Evaluation of antibacterial activity
In vitro antibacterial activity of ligands and their Ru(III)
complexes were assessed against a series of Gram positive (Bacillus
subtilis, Micrococcus luteus) and Gram negative (Pseudomonas aeru-
ginosa, Pseudomonas mendocina) using agar plate disc method
[32,33]. Stock solution was made by dissolving compound in
10 mL of DMSO. The media was made by dissolving nutrient agar
(15 g) in 1 L distilled water. The mixture was autoclaved for
15 min at 120 °C and then dispensed into sterilized petri dishes,
allowed to solidify and then used for inoculation. Target microor-
ganisms cultures were prepared separately in 15 ml of liquid nutri-
ent agar for activation. Inoculation was done with the help of
stretching
m(OH), azomethine m(C@N) group and phenolic oxygen
m(CAO) group vibrations respectively. In comparison with the
spectra of the Schiff bases, the
m(C@N) band exhibit downward
shift in the range 1605–1613 cmꢂ1 in the FT-IR spectra of the com-
plexes, which is in accordance with the coordination of the azome-
thine function to the metal ion for all the complexes [35].
Furthermore, on complexation, the medium band corresponding
micropipette with sterilized tips, 100
l
l of activated strain was
to phenolic oxygen m(CAO) is shifted to higher wave number in
placed onto the surface of agar plate, spread over the whole surface
and then two wells having diameter of 10 mm were dug in media.
Sterilized stock solutions were used for the application in the well
of inoculated agar plates. In this well of inoculated agar plates
the range 1299–1334 cmꢂ1 for all the complexes indicating that,
the ligands coordinate through their deprotonated form and
formation of metal–oxygen bonds. In addition, new bands were
observed in the region 503–525 cmꢂ1 and 455–480 cmꢂ1, which
were assigned to the formation of RuAO and RuAN bonds respec-
tively [36] which further supports the coordination of the azome-
thine nitrogen and the phenolic oxygen. Finally, the presence of
coordinated water was suggested by the very broad absorption
100 ll of solution was poured and incubated at 37 °C for 48 h.
Activity was determined by measuring the diameter of zone show-
ing complete inhibition and has been expressed in mm. All this
experiments were performed in triplicate.
Table 1
Yield %, molecular weight, microanalysis and conductivity data of the reported Schiff bases ligands (L1–4H2) and their Ru(III) complexes.
Compounds
Yield
(%)
M.Wt
Color
(Calc.) found (%)
Km (X )
ꢂ1 cm2 molꢂ1
C
H
N
Cl
–
Ru
–
L1H2 C22H20N2O2
88
77
89
73
94
82
92
74
344.41 Yellow
(76.72)
76.21
(5.85)
5.72
(8.13)
7.98
(5.44)
5.39
(7. 27)
7.25
(5.09)
5.00
(6.92)
6.78
(4.87)
4.77
(6.29)
6.11
(4.54)
4.45
–
[Ru(L1)(H2O)2]Cl (1) [C22H22N2O4Ru]Cl
L2H2 C20H14N2O2Cl2
514.95 Reddish (51.31)
(4.30)
4.21
(3.66)
3.60
(6.88)
6.79
(19.62)
19.59
–
101.45
–
brown
51.22
(62.35)
62.19
385.24 Yellow
(18.40)
18.38
(19.13)
19.00
–
[Ru(L2)(H2O)2]Cl (2) [C20H16N2O4Cl2Ru]Cl
L3H2 C24H24N2O4
555.78 Reddish (43.22)
(2.90)
2.83
(5.98)
5.77
(18.18)
18.02
–
105.70
–
brown
43.10
(71.27)
71.01
404.46 Orange
[Ru(L3)(H2O)2]Cl (3) [C24H26N2O6Ru]Cl
L4H2 C22H18N2O4Cl2
575.00 Reddish 50.13
4.55
(6.16)
6.00
(15.92)
15.75
(17.27)
12.057
(17.57)
17.41
–
107.21
–
brown
(49.99)
(59.34)
59.21
(4.48)
(4.07)
3.82
445.29 Yellow
[Ru(L4)(H2O)2]Cl (4) [C22H20N2O6Cl2Ru]Cl
615.84 Reddish (42.90)
brown 42.83
(3.27)
3.13
(16.41)
16.27
99.79

408
A.A. Abdel Aziz, H.A. Elbadawy / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 404–415
Table 2
The infrared (cmꢂ1) and UV–Vis. spectral data of the reported ligands (L1–4H2) and their Ru(III) complexes.
Compound
m
(OH)
m(C@N)
m
(CAO)
m(RuAO)
m(RuAN)
UV–Vis kmax (nm)
L1H2
3428(br.)
3381(br.)
3444(br.)
3398(br.)
3389(br.)
3324(br.)
3394(br.)
3373(br.)
1608(s)
1605(s)
1616(s)
1609(s)
1610(s)
1607(s)
1617(s)
1613(s)
1274(m)
1299(m)
1279(m)
1332(m)
1276(m)
1334(m)
1278(m)
1329(m)
–
–
216a, 297b
[Ru(L1)(H2O)2]Cl (1)
L2H2
511(w)
–
525(w)
–
507(w)
–
503w)
455(w)
–
480(w)
–
474(w)
–
461(w)
285a, 339b, 422c
214a, 295b
[Ru(L2)(H2O)2]Cl (2)
L3H2
284a, 338b, 419c
224a, 308b
[Ru(L3)(H2O)2]Cl (3)
281a, 334b, 414c
223a, 301b
L4H2
[Ru(L4)(H2O)2]Cl (4)
290a, 343b, 423c
ꢆs, strong; m, medium; w, weak; br., broad.
a
p
–
pꢆ
.
.
b
c
n–pꢆ
Charge transfer.
band in the region 3324–3398 cm–1 in the IR spectra of complexes
[37]. Thus, the FT-IR spectral data provide strong evidences for the
complexation of the tetradentate Schiff bases with ONNO se-
quence. Fig. S1 represents the FT-IR spectra of H2L3 ligand and its
Ru(III) complex.
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1
[Ru(L )(H O) ]Cl
2
2
2
[Ru(L )(H O) ]Cl
2
2
3
[Ru(L )(H O) ]Cl
2
2
4
[Ru(L )(H O) ]Cl
2
2
Electronic spectra
The electronic spectra were recorded in order to obtain infor-
mation about the geometry of the complexes. The electronic
spectra of the free ligands and the complexes were carried out in
DMF – buffer solution. The absorption region and the assignment
of the absorption bands of the ligands and complexes are listed
in Table 2. The electronic spectra of all the free ligands showed
two types of transitions, one appeared at the range 214–224 nm
200
250
300
350
400
450
500
550
600
which can be assigned to
p–
pꢆ transitions from the benzene ring
Wavelength (nm)
and the double bond of the azomethine group and the second
bands in the 295–308 nm region are due to n–pꢆ transition of
non-bonding electrons present on the nitrogen of the azomethine
group. These peaks exhibited bathochromic shift upon complex
formation, which supported the coordination of the ligands to
Ru(III) ion. The ground state of Ru(III) in an octahedral environ-
Fig. 1. UV–Vis. spectra of Ru(III) complexes.
two different ‘g’ values (gx = gy – gz) are an indicative of a tetrago-
nal distortion in these octahedral complexes [44]. In addition, the
nature and position of the lines in the spectra of these complexes
are similar to those of the other octahedral complexes [45]. The
EPR spectrum of [RuL1(H2O)2]Cl complex at room temperature
(RT) and at liquid nitrogen temperature (LNT) is depicted in
Fig. 2. The spectrum of at LNT show improved resolution with
the ‘g’ value and there is no much variation observed when com-
pared with that observed that of RT.
2
ment is T2g and the first excited doublet levels in the order of
increasing energy are A2g and T1g, which arise from t42geg1 config-
2
2
2
uration [38]. Hence, two bands corresponding to T2g
!
2A2g and
2T2g ! T1g are possible. Besides the
p–
pꢆ and n–pꢆ transitions,
2
the UV–Vis. spectra of the reported Ru(III) complexes show a third
intense absorption band in the region 414–423 nm, which can be
assigned to charge transfer (CT) transitions [39], which were ab-
sent in the spectra of the respective free ligands. In a d5 system,
especially in Ru(III) which has relatively high oxidizing properties,
Cyclic voltammetry study
the charge transfer bands of the type L y ? T2g are prominent in
The electrochemical studies of the Ru(III) complexes were car-
ried out in acetonitrile solution, in the range +2.0 to ꢂ2.0 V using
glassy carbon electrode as working electrode, Ag/AgCl as reference
electrode and tetrabutyl ammonium chloride (0.1 M) as supporting
electrolyte. The solution was deareated with a continuous flow of
nitrogen gas for 15 min. before scanning. A respective voltammo-
gram of the complex [Ru(H2O)2(L4)]Cl has been depicted in Fig. 3
and the data are given in Table 3. All the complexes are electroac-
tive only with respect to metal center. The complexes (10ꢂ3 M)
gave only quasi reversible cyclic voltammetric response due to
Ru(III)–Ru(II) couple in the range of E1/2 = ꢂ0.882 to ꢂ0.776 V, with
p
the low energy region, which obscures the weaker bands due to
d–d transitions [40]. Similar observations have been made for
other Ru(III) octahedral complexes [41] and in most Ru(III) Schiff
base chelates [42]. Electronic spectra of Ru(III) complexes are de-
picted in Fig. 1.
Magnetic moment and EPR spectra
The room temperature leff values per ruthenium ion for the re-
ported complexes were in 1.68–1.73 BM range (Table 3). The val-
ues obtained lie in the BM range corresponding to one unpaired
electron [43], which corresponds to the +3 state of ruthenium
and in consistent with non-interacting low spin t52g (S = 1/2) config-
uration. The EPR spectra of Ru(III) complexes provide information
of importance in studying the Ru(III) ion environment. The EPR
spectra of all the complexes were recorded at room temperature
and liquid nitrogen temperature. The ‘g’ values are listed in Table 3.
All Ru(III) complexes exhibited EPR spectra with gx = gy – gz. The
peak to peak separation (
to slow electron transfer and adsorption of the complexes onto
the electrode surface [46]. The E12 and Ep values are in good
DEp) of 0.195–0.240 V. This is attributed
D
agreement with those recently reported for other similar Ru(III)
Schiff base complexes [47,48]. The E12 (reduction) values of the
complexes containing one phenyl ring in the aldehyde part of the
Schiff base ligands range from 0.52 to 0.63 V [49]. When these
values are compared with that of new complexes, it has been

A.A. Abdel Aziz, H.A. Elbadawy / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 404–415
409
Table 3
EPRa, magnetic moment and electrochemicalb data of the reported Ru(III) complexes.
b
c
Complex
EPR parameter
leff (BM)
Epa(V)
Epc(V)
D
Ep
E1/2
(V)
(V)
a
gx
gy
gz
(gav.)
[Ru(L1)(H2O)2]Cl (1)
[Ru(L3)(H2O)2]Cl (2)
[Ru(L2)(H2O)2]Cl (3)
[Ru(L4)(H2O)2]Cl (4)
1.58
1.63
1.64
1.61
1.58
1.63
1.64
1.62
1.78
1.72
1.93
1.81
1.64
1.66
1.74
1.68
1.70
1.72
1.68
1.73
ꢂ0.785
ꢂ0.723
ꢂ0.761
ꢂ0.668
ꢂ0.980
ꢂ0.963
ꢂ0.983
ꢂ0.885
0.195
0.240
0.222
0.217
ꢂ0.882
ꢂ0.843
ꢂ0.872
ꢂ0.776
h
i
1
2
a
gav
¼
13 g2x
þ
13 g2y
þ
13 g2z
.
b
c
Supporting electrolyte [NBu4]ClO4 (0.1 M); scan rate, 100 mV/S; reference electrode Ag/AgCl;
D
Ep = EpaꢂEpc
.
E1/2 = 1/2 (Epa + Epc).
Table 4
Thermogravimetric characteristics of Ru(III) complexes.
EPR of [RuL1(H2O)2]Cl at LNT
EPR of [RuL1(H2O)2]Cl at RT
Complex Temp. Mass
Assignment
2 H2O
(27.11) 27.00 1/2 Cl2 + C8H8
(40.05) 40.00 2 C7H5N
Residue
RuO2
range (°C) loss (%)
(Calc.) found
[Ru(L1)(H2O)2]Cl (1) 136–245
296–336
(6.99) 6.97
420–499
[Ru(L2)(H2O)2]Cl (2) 149–256
308–336
(6.48) 6.27
(32.46) 32.37 1/2 Cl2 + C6H2Cl2
(37.10) 36.98 2 C7H5N
2 H2O
RuO2
426–488
[Ru(L3)(H2O)2]Cl (3) 182–261
324–401
(6.26) 6.19
(24.28) 24.14 1/2 Cl2 + C8H8
(46.31) 45.99 2 C8H7NO
2 H2O
RuO2
435–506
[Ru(L4)(H2O)2]Cl (4) 187–276
333–475
(5.85) 5.72
(29.29) 29.11 1/2 Cl2 + C6H2Cl2
(43.24) 43.10 2 C8H7NO
2 H2O
RuO2
2000 2250 2500 2750 3000 3250 3500 3750 4000
Magnetic Field (G)
520–598
Fig. 2. EPR spectra of [RuL1(H2O)2]Cl complex at RT (298 K) and at LNT (77 K).
observed that the addition of one phenyl ring in the ligand causes
positive shift in the E12 (reduction) values. This can be explained by
the fact that the additional phenyl ring of electron withdrawing
nature decreases the electron density around the metal center [50].
Thermogravimetric analysis
The thermal behavior of complexes under investigation was as-
sessed using thermogravimetric analysis (TGA). The samples were
analyzed in a platinum pan under N2 and the temperature was lin-
early increased with a heating rate 10 °C/min over a temperature
range 20–800 °C. The reported Ru(III) complexes were found to
be air stable and have higher thermal stability. The TG data for
the synthesized complexes are summarized in Table 4. The TG plot
of the four Ru(III) complexes showed similar patterns with three
resolved and well-defined decomposition steps. As an example
the TG plot of [Ru(L2)(H2O)2]Cl exhibited a first decomposition step
in the temperature range 149–256 °C with a net weight loss of
6.27% (calc. 6.48%) which has been consistent with the elimination
of two H2O molecule. The second decomposition step occurred in
the temperature range 308–336 °C with a net weight loss of
32.37.00% (calc. 32.46%). This decomposition step has been as-
signed to the elimination of 1/2 Cl2 and C6H2Cl2 organic moiety. Fi-
nally the third step occurs at the temperature range 426–488 °C
with a net weight loss 36.98% (calc. 37.10%) which was consistent
with the loss of two (C7H5N) moieties to give finally RuO2 residue
with a net weight of 26.03% (calc. 25.84%). TG plot of [RuL2
(H2O)2]Cl. complexes is depicted in Fig. 4 and its thermal decompo-
sition steps of is shown in Scheme 2.
Fig. 3. Cyclic voltammogram of complex [Ru (L4)(H2O)2]Cl.
change of the decomposition (D
Gꢆ) were evaluated graphically by
employing the Coats–Redfern equation [51]. It is obvious from
the data listed in Table 5 that all the complexes have negative
entropy values indicating that activated complexes have more
ordered systems than reactants.
DNA-binding studies
Previous reports [52] have suggested that ruthenium complexes
can interact with DNA through three non-covalent modes such as
electrostatic binding, groove binding, or intercalation. Among
these interactions, intercalative binding mode is one of the most
important DNA binding modes, which firstly proposed by Lerman
[53]. Intercalation usually occurs when the complexes insert their
Kinetic studies
The kinetic parameters such as activation energy (
py of activation (
Hꢆ), entropy of activation ( Sꢆ) and free energy
D
Eꢆ), enthal-
D
D

410
A.A. Abdel Aziz, H.A. Elbadawy / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 404–415
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0.050
0.025
0.000
-0.025
-0.050
-0.075
-0.100
-0.125
-0.150
Electronic absorption studies
Weight Loss -0.2839 mg
- 6.27 %
The binding modes of complexes to DNA are characterized clas-
sically through electronic absorption titrations method [54]. In the
intercalative binding mode, the pꢆ orbital of the intercalated ligand
Weight Loss -1.4660 mg
- 32.37 %
213.6
can couple with the
ing the
pꢆ transition energy and resulting in the bathochro-
mism. On the other hand, the coupling orbital is partially filled
p orbital of the DNA base pairs, thus, decreas-
p
?
p
by electrons, thus, decreasing the transition possibilities and con-
comitantly resulting in the hypochromism [55]. Generally, the
hypochromism and/or significant bathochromism in the absorp-
tion spectra arise from the strong stacking interaction between
the aromatic chromophore of the ligand and DNA base pairs and
the extent of hypochromism and bathochromism commonly con-
sistent with the strength of the intercalative interaction [56].
The UV–Vis titrations of Ru(III) complexes (1–4) with CT-DNA
were done in DMF-Tris buffer medium in the wave length range
of 200–800 nm. It was obviously that, the absorption band ap-
peared at 414–423 nm, which was obviously charge transfer in ori-
gin showed significant hypochromism with a red shift of 13 nm,
9 nm, 6 nm and 4 nm for complexes (1–4) respectively, suggesting
that the complexes used in this study showed strong binding to
DNA in an intercalative mode. The electronic absorption spectra
of all the complexes in the absence and presence of CT-DNA, using
471.2
Weight Loss -1.6748 mg
- 36.98 %
329.2
300
0
100
200
400
500
600
700
800
Temp. (oC)
Fig. 4. TG plot of [Ru(L2) (H2O)2]Cl complex.
149-256 oC
-(2H2O) (6.27 %)
[Ru(C20H12N2O2Cl2)(H2O)2]Cl
555.78
[Ru(C20H12N2O2Cl2)]Cl
519.75
a constant concentration of the complex (10 lM) are shown in
Fig. 5. The extent of hypochromism was 49.10% for complex 1,
42.10% for complex 2, 37.35% for complex 3, 31.6% for complex 4.
In order to compare quantitatively the binding strength of the
complexes was calculated using Eq. (1). The Kb values were
8.723 ꢃ 104 Mꢂ1 for complex 1, 6.354 ꢃ 104 Mꢂ1 for complex 2,
4.567 ꢃ 104, for complex 3, and 4.421 ꢃ 104 Mꢂ1 for complex 4
respectively, revealing that 1 > 2 > 3 > 4 in binding to CT-DNA.
This order can be explained as follows, by the methoxy in the
backbone of Schiff bases 3 and 4 severe steric constraints near
the core of Ru(III) when the complex intercalates into the DNA base
pairs. The methoxy groups may come into close proximity of base
pairs at the intercalation sites, which prevent the complexes 3 and
4 from intercalating effectively, compared to the complexes 1 and
2.
426-488 oC
[Ru(C14H10N2O2)]
339.31
RuO2
133.06
(26.03 %)
- (2C7H5N) (36.98 %)
Scheme 2. Thermal decomposition steps of [Ru(L2)(H2O)2]Cl complex.
Competitive studies with EB
planar aromatic ligand between DNA base pairs. Changes in the
structure of intercalative ligand could be used to attain diverse
DNA binding mode of ruthenium complexes, which would result
in the changes in the DNA-binding behavior, photophysical
properties, excited state reactivity and biological activities of the
complexes.
Further proof for the binding of Ru(III) complexes to DNA were
given by carrying out studying steady-state competitive binding
experiments using complexes (1–4) as quenchers were undertaken
to get further proof for the binding of the complexes to DNA. The
fluorescence measurement for Ru(III) complexes showed that no
Table 5
Thermodynamic parameters for the thermal degradation of Ru(III) complexes.
Complex
E* (kJ molꢂ1
)
A (sꢂ1
)
D
H* (kJ molꢂ1
)
D
S* (J molꢂ1 Kꢂ1
)
D )
G* (kJ molꢂ1
*
Ts
[Ru(L1)(H2O)2]Cl (1)
201
302
455
43.65
72.07
156.9
3.07 ꢃ 109
5.44 ꢃ 1011
7.64 ꢃ 107
55.15
76.86
121.70
ꢂ30.25
ꢂ72.26
67.62
86.82
143.9
ꢂ145.60
[Ru(L2)(H2O)2]Cl (2)
[Ru(L3)(H2O)2]Cl (3)
[Ru(L4)(H2O)2]Cl (4)
213.6
329.2
471.2
68.14
162.6
186.74
6.73 ꢃ 105
1.97 ꢃ 1011
9.04 ꢃ 106
58.38
186.70
215.90
ꢂ65.16
ꢂ165.90
ꢂ205.60
ꢂ56.35
ꢂ117.60
ꢂ147.3
74.47
185.70
225.7
212
315
488
77.62
175.00
222.9
1.89 ꢃ 107
4.63 ꢃ 1013
5.73 ꢃ 1010
3.05 ꢃ 109
5.27 ꢃ 1013
6.09 ꢃ 106
75.56
173.70
203.60
97.45
98.56
193.70
234
399
564
73.15
173.9
203.5
73.06
86.75
116.00
ꢂ63.98
ꢂ132.6
58.59
101.2
162.50
ꢂ193.200
*
Ts: the derivative peak temperature.

A.A. Abdel Aziz, H.A. Elbadawy / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 404–415
411
1.0
0.8
0.6
0.4
0.2
0.0
1.0
4.5
4.0
4.0
(1)
(2)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0.8
0.6
0.4
0.2
0.0
0
5 10 15 20 25 30 35 40 45 50 55
0
5 10 15 20 25 30 35 40 45 50 55
[DNA] x 10-5
M
[DNA] x 10-5
M
350
400
450
500
550
600
650
350
400
450
500
550
600
650
Wavelength (nm)
Wavelength (nm)
1.0
0.8
0.6
0.4
0.2
0.0
0.6
0.5
0.4
0.3
0.2
0.1
0.0
2.5
2.0
1.5
1.0
0.5
1.75
1.50
1.25
1.00
0.75
0.50
0.25
(3)
(4)
0.0
0
0.00
0
5
10 1520 25 30 35 40 45 50 55
[DNA] x 10-5 M
5
10 15 20 25 30 35 40 45 50 55
[DNA] x 10-5
M
350
400
450
500
550
600
650
350
400
450
500
550
600
650
Wavelength (nm)
Wavelength (nm)
Fig. 5. Absorption spectra of Ru(III) complexes (1–4) (10
arrow indicates the changes in absorbance with respect to an increase of DNA concentration (inset: plot of [DNA] versus [DNA]/(eaꢂef)).
lM) in the absence and (ꢁ ꢁ ꢁꢁ ꢁ ꢁ) and presence (—) of CT-DNA 5,10, 15, 20, 25, 30, 35, 40, 45 and 50 lM of CT-DNA. An
emission band either with or without CT-DNA at ambient temper-
ature. The fluorescence quenching spectra of DNA-bound EB at
602 nm by variable complex concentrations are shown in shown
in Fig. 6. Upon the addition of complexes, a significant decrease
in the fluorescence intensity of the EB-DNA system occurred,
which gave an indication of the binding of the Ru(III) complexes
to DNA. These results indicate that Ru(III) complex could partially
displace EB from the DNA-EB system, as often observed in interca-
lative complex-DNA modes.
The fluorescence quenching of EB bound to DNA by the com-
plexes (1–4) is plotted against [Ru(III) complex]/[DNA] values
and it was found to be in agreement with the Stern–Volmer equa-
tion (Fig. 6 insets). From the slope Io/I versus [Ru(III) complex]/
[DNA]-values plot using Eq. (2), The Stern–Volmer constant (Kq)
was calculated. The (Kq) value give an indication about the degree
of interaction the Ru(III) complexes to CT-DNA. The obtained Kq
values corresponding to Ru(III) complexes (1–4) were found to
be 6.583 ꢃ 103 Mꢂ1, 5.868 ꢃ 103 Mꢂ1, 4.235 ꢃ 103 and 3.174 ꢃ
103 Mꢂ1 respectively. These values indicated that, complex 1
showed highest binding ability with CT-DNA.
complexes and DNA. Hydrodynamic measurements, i.e. viscosity
and sedimentation that are sensitive to length changes are regarded
as the least ambiguous and most critical tests to a binding model in
solution in the absence of crystallographic structural data [57]. Vis-
cosity measurements are proved to be least ambiguous to support a
complex-DNA binding model, as these measurements are very
much sensitive to length change [58]. When a small molecule inter-
calate between the DNA base pairs, it unwinds the DNA helix and
hence increases lengthen it, resulting in significant increase in the
viscosity of DNA solution. However, a partial and/or non-classical
intercalation of ligand may bend (or kink) the DNA helix, resulting
in the decrease of its effective length and, concomitantly its viscos-
ity [59]. The binding of the complexes with CT-DNA was further
elucidated by measuring the relative specific viscosity of DNA after
the addition of varying concentration of complexes. The effect of
Ru(III) complexes (1–4) on the viscosity of rod-like CT-DNA at
25 0.1 °C is shown in Fig. 7. Viscosity experimental results clearly
showed that the relative viscosity of CT-DNA increases steadily on
addition of increasing concentration of the complexes (1–4).The in-
creased degree of viscosity, which may depend on its affinity to
DNA follows the order of 1 > 2 > 3 > 4. This observation can be ex-
plained on the fact that, classical intercalation model demands that
the DNA helix must lengthen as base pairs are separated to accom-
modate the binding complexes, leading to the increase of DNA
viscosity, as for the behaviors of the known DNA intercalators.
Viscosity studies
Optical or photophysical investigations are necessary but not
sufficient to establish the mode of binding between metal

412
A.A. Abdel Aziz, H.A. Elbadawy / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 404–415
Antioxidant activity
1.25
As a result of the fact that, Ru(III) complexes exhibited good
DNA binding affinity, it is considered worthwhile to investigate
their antioxidant activity. Reactive oxygen species (ROS), such as
superoxide anion ðOꢂ2 ÅÞ and hydroxyl radical (HOꢀ), are generated
by all aerobiccells during normal oxygen metabolism, and cumula-
tive information obtained has proved that the oxidation induced by
ROS is involved in the pathogenesis of various diseases through
direct effects on DNA directly and by acting as antitumor promoter
[60–62].
1.20
1.15
1.10
1.05
1.00
[RuL1(H2O)2]Cl
[RuL2(H2O)2]Cl
[RuL3(H2O)2]Cl
[RuL4(H2O)2]Cl
The inhibitory effects on the superoxide radical are increased
for greater concentrations of Ru(III) complexes, ranging from 0.03
to 1.5
effect of the four complexes is comparable at the concentration of
1.5 M, with a measured value of approximately 90%. These results
indicate that the four complexes will show almost the same activ-
ities when the concentration of complexes is as high as 1.5 M and
the differences can only be seen in low concentrations (<1.5 M).
lM as shown in Fig. 8a. It is noteworthy that the scavenging
0
3
6
9
12
15
18
[Complex]/[DNA] x 10-3
l
Fig. 7. Effect of increasing amounts of Ru(III) complexes on the relative viscosity at
25 ( 0.1) °C of calf thymus DNA (0.30 mM).
l
l
However, in our tested concentration, activities of four complexes
are also in the order of 1 > 2 > 3 > 4, with the IC50 value of 0.079,
0.140, 0.194 and 0.238 lM).
in Tables 6 and 7 provide evidence of the reliability in our experi-
mental conditions using the Fenton system to generate hydroxyl
radicals.
The scavenging abilities of the four Ru(III) complexes against
hydroxyl radicals were tested as a function of concentration, rang-
Compared to inhibitory effects on the hydroxyl radical (Fig. 9),
the four Ru(III) complexes exhibit greater activity (lower IC50 va-
lue) on the superoxide radical, which may be due to the higher
activity of the hydroxyl radical than the superoxide radical. Consid-
ering the mononuclear structure of the four complexes, differences
in the ligand structure are likely to induce variations in antioxidant
activities.
ing from 0.3 to 15
inhibitory effects of complexes on the hydroxyl radical are related
to concentration and saturate at a concentration of 9 M. Obvi-
lM, shown in Fig. 8b. It can be seen that the
l
ously, the scavenging activities of four Ru(III) complexes follow
the order of 1 (IC50 = 2.42) > 2 (IC50 = 4.28) > 3 (IC50 = 9.00) > 4
(IC50 = 42.00). The relatively small standard deviations (SD) listed
400
400
2.0
2.2
2.0
1.8
(1)
(2)
1.8
1.6
350
350
1.6
1.4
1.2
1.4
1.2
300
300
1.0
1.0
0.8
0.8
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5
[Ru(III) complex]
[Ru(III) complex]
250
250
200
150
100
50
200
150
100
50
0
0
500
500
550
600
650
700
550
600
650
700
Wavelength (nm)
Wavelength (nm)
400
400
1.8
1.6
1.4
1.2
1.0
0.8
1.6
1.4
1.2
1.0
0.8
(4)
(3)
350
300
250
200
150
100
50
350
300
250
200
150
100
50
0.0
2.5
5.0
7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5
[Ru(III) complex]
0.0
2.5
5.0
7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5
[Ru(III) complex]
0
0
500
500
550
600
650
700
550
600
650
700
Wavelength (nm)
Wavelength (nm)
Fig. 6. Emission spectra of DNA-EB system in the absence (ꢁ ꢁ ꢁꢁ ꢁ ꢁ) and presence (—) of 2.5, 5.0, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 and 25
lM Ru(III) complexes (1–4). An arrow
indicates the changes in emission intensity upon increasing the complex concentrations. (Inset: Stern–Volmer plot of the fluorescence titration data).

A.A. Abdel Aziz, H.A. Elbadawy / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 404–415
413
100
80
60
40
20
0
100
(a)
(b)
80
60
40
20
0
1
[Ru(L )(H O) ]Cl
1
2
2
[Ru(L )(H O) ]Cl
2
2
2
[Ru(L )(H O) ]Cl
2
2
2
[Ru(L )(H O) ]Cl
2
2
3
[Ru(L )(H O) ]Cl
3
2
2
[Ru(L )(H O) ]Cl
2
2
4
[Ru(L )(H O) ]Cl
4
2
2
[Ru(L )(H O) ]Cl
2
2
0
2
4
6
8
10
12
14
16
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Concentration (μM)
Concentration (μM)
Fig. 8. The scavenging effect of Ru(III) complexes on superoxide radical (a) and hydroxyl radical (b).
Table 6
The scavenging activities of Ru(III) complexes (1–4) against superoxide radical.
Complex
Average inhibition for O2ꢂÅ at different concentration (
l
M)
Equation
aIC50
(
lM)
R2
0.03
0.06
0.09
0.12
0.15
0.3
0.6
0.9
1.2
1.5
1
2
3
4
17.5
19.2
15.0
10.3
47.4
28.6
20.0
16.0
50.5
39.8
28.7
25.3
64.4
49.7
42.0
34.5
75.8
61.2
55.3
46.9
77.6
65.9
58.1
51.0
86.4
78.0
72.6
68.8
90.6
83.6
80.0
78.9
91.6
88.3
87.6
85.6
91.6
91.6
90.0
91.4
y = 39.02x + 39.028
y = 42.895x + 86.638
y = 46.434x + 83.018
y = 49.506x + 80.901
0.079
0.140
0.194
0.238
0.878
0.973
0.972
0.984
a
IC50 values were calculated from regression lines where: x was the log of the tested complexes concentration and y was the average inhibition of the tested complexes.
When the average inhibition of the tested complexes was 50%, the tested complex concentration was IC50
.
Table 7
The scavenging activities of Ru(III) complexes (1–4) against hydroxyl radical.
Complex
Average inhibition for OHÅ at different concentration (
l
M)
Equation
IC50
(lM)
R2
0.3
0.6
0.9
1.2
1.5
3.0
6.0
9.0
12
15
1
2
3
4
9.3
3.0
2.1
0.8
14.4
7.8
4.0
23.7
11.3
9.1
26.8
17.4
13.0
7.8
35.0
20.9
16.2
10.0
53.6
42.6
29.5
20.8
72.2
58.3
43.0
35.0
89.7
70.4
55.5
34.6
90.7
74.8
57.3
36.7
89.7
75.6
58.0
35.8
y = 55.11x + 28.858
y = 49.39x + 18.798
y = 38.12x + 13.630
y = 25.22x + 9.0087
2.42
4.28
9.00
42.0
0.975
0.971
0.968
0.950
1.7
6.7
0.25
0.20
0.15
0.10
0.05
0.00
50
40
30
20
10
0
(b)
(a)
1
2
3
4
1
2
3
4
Compounds
Ru(III) Complexes
Fig. 9. Radical scavenging activity of Ru(III) complexes.
Antibacterial screening
done by the agar plate disc method on the following strains i.e.,
Gram positive B. subtilis, M. luteus and Gram negative P. aeruginosa,
P. mendocina using different concentration of ligands and their
Determination of in vitro antibacterial activity of the reported
Schiff bases and their Ru(III) complexes are given in Table 8 and
their graphical representation in Fig. 10. Antibacterial studies were
complexes (50 and 100
lg/mL). Streptomycin was used as a stan-
dard drug for antibacterial activity. It may be concluded from the

414
A.A. Abdel Aziz, H.A. Elbadawy / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 404–415
Table 8
In vitro antibacterial activity of the Schiff bases ligands (L1–4H2) and their Ru(III) complexes.
Compound
Diameter of inhibition zone (mm)
(Gram +ve)
(Gram ꢂve)
R. subtilis
M. Luteus
P. aeruginosa
P. mendocina
50 (
lg/mL)
100 (lg/mL)
50 (l
g/mL)
100 (lg/mL)
50 (lg/mL)
100 (
lg/mL)
50 (lg/mL)
100 (lg/mL)
L1H2
9
5
10
7
17
13
14
19
22
10
7
11
9
18
15
20
17
25
9
6
11
7
17
13
19
15
20
11
8
8
5
9
7
10
8
11
17
15
19
16
25
9
5
10
6
15
14
18
15
25
11
9
L2H2
L3H2
13
10
20
17
22
19
23
14
10
18
16
21
18
27
L4H2
6
(1)
(2)
(3)
(4)
14
13
16
18
23
Streptomycin
30
25
20
15
10
5
30
25
20
15
10
5
(b)
(a)
B. subtilis
M. Luteus
P. aeruginosai
P. mendocine
B. subtilis
M. Luteus
P. aeruginosa
P. mendocina
0
0
1
2
3
4
1
2
3
4
L H L H L H L H (1) (2)
(3)
(4) Streptomycin
L H L H L H L H (1) (2)
(3)
(4) Streptomycin
2
2
2
2
2
2
2
2
Compounds
Compounds
Fig. 10. Zone of inhibitions of reported compounds and antibiotic ((a) 50 lg/mL and (b) 100 lg/mL) against gram (+) and gram (ꢂ) bacteria strains.
antibacterial screening data that: (i) The antibacterial activity of
ligands and their complexes was due to the presence of toxophor-
ically important imine groups where the mode of action of these
compounds may involve the formation of hydrogen bond through
azomethine group with the active center of cell constituents,
thereby resulting in interference with normal cell process. (ii) A
marked enhancement of in vitro biocidal studies of the ligands
was exhibited on coordination with Ru(III) ion against all microor-
ganisms strains under tested identical experimental conditions.
The increase in antibacterial activity may be explained on basis
of fact that on chelation the polarity of Ru(III) ion is reduced due
to overlap of ligand orbital and sharing of positive charge of
ruthenium ion with donor groups. Further it increases delocaliza-
tion of chelate ring and increases the lipophilicity of complexes.
This increased lipophilicity enhances penetration of complexes
there by disturbing the respiration process of cell and blocking
the synthesis of proteins, which further restricts growth of organ-
isms. (iii) It is evident from the data that the complexes were more
toxic towards Gram (+) strains as compared to Gram (ꢂ) strains
which may be attributed to the fact that the cell wall of Gram
(ꢂ) strains have more antigenic properties due to the presence of
an outer lipid membrane of lipopolysaccharides. (iv) Some com-
pounds have activity close to standard drug. It was clear from
the data that compounds with higher concentration were propor-
tionately more potent as compared to same compound with higher
concentration.
Conclusions
Four novel complexes of Ru(III)-tetradentate Schiff base ligands
have been synthesized and structurally characterized. Based on
elemental analysis, molar conductivity, UV–Vis, magnetic, EPR,
FT-IR spectral data and TG analysis, mononuclear octahedral
complexes of the general formula [RuL1–4(H2O)2]Cl are proposed,
where L = dianion of the tetradentate Schiff base ligand. The
CT-DNA binding abilities of the complexes have been studied.
The results suggest that all the complexes bind to CT-DNA by an
intercalative mode with different degrees. These studies form an
important rationale for drug design and warrant further in vivo
experiments and pharmacological assays. Additionally, Ru(III)
complexes also exhibited excellent antioxidant (Oꢂ2 Å and HOꢀ radi-
cal scavengers) and antibacterial activities. Therefore, the informa-
tion obtained from the present work would help in developing new
potent antioxidants and therapeutic drugs to cure certain valuable
diseases.
Acknowledgement
The authors would like to acknowledge financial support for
this work, from the Deanship of Scientific Research (DSR),
University of Tabuk, Tabuk, Saudi Arabia, under Grant No. (S141/
1434).

A.A. Abdel Aziz, H.A. Elbadawy / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 404–415
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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.2014.01.050.
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