Cobalt(II), Nickel(II) and Copper(II) complexes of a tetradentate Schiff base as photosensitizers: Quantum yield of 1O2 generation and its promising role in anti-tumor activity

Article abstract of DOI:10.1016/j.saa.2012.09.062

In the present investigation, a Schiff base N′¹, N′³-bis[(E)-(5-bromo-2-hydroxyphenyl)methylidene]benzene-1, 3-dicarbohydrazide and its metal complexes have been synthesized and characterized. The DNA-binding studies were performed using absorption spectroscopy, emission spectra, viscosity measurements and thermal denatuaration studies. The experimental evidence indicated that, the Co(II), Ni(II) and Cu(II) complexes interact with calf thymus DNA through intercalation with an intrinsic binding constant K_b of 2.6 × 10⁴ M ^-1, 5.7 × 10⁴ M^-1 and 4.5 × 10 ⁴ M^-1, respectively and they exhibited potent photodamage abilities on pUC19 DNA, through singlet oxygen generation with quantum yields of 0.32, 0.27 and 0.30 respectively. The cytotoxic activity of the complexes resulted that they act as a potent photosensitizers for photochemical reactions.

Full text of DOI:10.1016/j.saa.2012.09.062

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 101 (2013) 132–139
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Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
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Cobalt(II), Nickel(II) and Copper(II) complexes of a tetradentate Schiff base
1
as photosensitizers: Quantum yield of O2 generation and its promising role
in anti-tumor activity
a
b
b
S.M. Pradeepa a, H.S. Bhojya Naik a, , B. Vinay Kumar , K. Indira Priyadarsini , Atanu Barik ,
⇑
T.R. Ravikumar Naik c
a Department of Studies and Research in Industrial Chemistry, Kuvempu University, Shankaraghatta 577 451, Shimoga, India
b Radiation Chemistry Section, Radiation and Photochemistry Division, Bhabha Atomic Research Centre, Mumbai 400 085, India
c Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, India
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
"
"
"
"
"
We have synthesized biologically
active M(II) (where M@Co, Ni, Cu)
complexes.
The DNA-binding modes of M(II)
complexes were studied by different
techniques.
The DNA-photocleavage abilities of
M(II) complexes were studied on
pUC 19 DNA.
The quantum yields of 1O2
generation of M(II) complexes were
determined.
The cytotoxicity of M(II) complexes
were performed on A549 lung cancer
cell lines.
a r t i c l e i n f o
a b s t r a c t
1
In the present investigation, a Schiff base N0 ,N03-bis[(E)-(5-bromo-2-hydroxyphenyl)methylidene]ben-
zene-1,3-dicarbohydrazide and its metal complexes have been synthesized and characterized. The
DNA-binding studies were performed using absorption spectroscopy, emission spectra, viscosity mea-
surements and thermal denatuaration studies. The experimental evidence indicated that, the Co(II), Ni(II)
and Cu(II) complexes interact with calf thymus DNA through intercalation with an intrinsic binding con-
stant Kb of 2.6 ꢀ 104 Mꢁ1, 5.7 ꢀ 104 Mꢁ1 and 4.5 ꢀ 104 Mꢁ1, respectively and they exhibited potent photo-
damage abilities on pUC19 DNA, through singlet oxygen generation with quantum yields of 0.32, 0.27
and 0.30 respectively. The cytotoxic activity of the complexes resulted that they act as a potent photosen-
sitizers for photochemical reactions.
Article history:
Received 3 July 2012
Received in revised form 7 September 2012
Accepted 20 September 2012
Available online 28 September 2012
Keywords:
Schiff base
Photosensitizers
DNA binding
DNA photocleavage
Singlet oxygen
Ó 2012 Elsevier B.V. All rights reserved.
Introduction
tosensitizer compound (PS) by radiation of appropriate wavelength
has been applied in PDT to treat diseases characterized by uncon-
Photosensitizers (PSs) are a group of compounds which performs
an vital role in photodynamic therapy (PDT). The activation of a pho-
trolled cell growth, such as cancer [1]. A huge number of photosen-
sitizers together with their generating mechanisms of singlet
oxygen have been discussed in detail [2–4]. Among these studies,
the types of photosensitizers generating singlet oxygen frequently
concentrate on the organic molecules, such as organic dyes, porphy-
⇑
Corresponding author. Fax: +91 8282 257302.
E-mail address: hsb_naik@rediffmail.com (H.S. Bhojya Naik).
1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2012.09.062

S.M. Pradeepa et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 101 (2013) 132–139
133
rins, phthalocyanines, and some macrocyclic systems etc. However,
many of the studied PSs are unstable against light, which alters their
capability to absorb light during illumination, resulting in a de-
creased efficiency of production of excited molecules [5].
flux for 5–6 h. The resulting solution was concentrated to half of
its initial volume and then cooled to room temperature. The yellow
precipitate was separated by filtration, washed with EtOH and
dried in a vacuum desiccator. The yield was 76% and mp = 180–
182 °C. Anal. (%) Calc. for [C22H16Br2N4O4]: C, 47.17; H, 2.88; N,
10.00. Found: C, 47.19; H, 2.87; N, 10.02. LC–MS (m/z) 559
[C22H16 Br2N4O4AH]+. IR (KBr,cmꢁ1): 3217 (AOH), 1658 (C@O),
1557 (C@N), 3058 (NAH), 1270 (NAN); 1H NMR (DMSO-d6,
400 MHz, d ppm): 11.20 (s, OAH, 2H), 12.30 (s, NAH, 2H), 9.54 (s,
Success of photofrin as an efficient photosensitizer agent has
drawn the concentration of many bioinorganic chemists towards
developing other active non-porphyrin transition metal complexes
with superior efficiency [6–10]. Under the irradiation of suitable
light, these metal complexes will be excited and, further, produce
reactive oxygen species in the presence of molecular oxygen, and
display potential utility on PDT. Recently, many of transition metal
complexes have been found to be more efficient as the singlet oxy-
gen producers than the well studied organic systems. For example,
some complexes of ruthenium(II) and platinum(II) can be the pro-
ficient photosensitizers to induce singlet oxygen [11,12]. The inter-
action of Schiff base metal complexes with DNA has been widely
studied in the past decades. Due to the site specific binding prop-
erties and many fold applications in cancer therapy, these coordi-
nation compounds were suitable candidates as DNA secondary
structure probes, photo cleavers, and antitumor drugs [13–15]. In
addition to this, several metal complexes of Schiff bases derived
from salicylaldehyde and amino acid [16–22] and reduced salicy-
lidene amino acid [23–25] were reported and some of them have
been confirmed to be efficient DNA cleavers [26–28] and as novel
tumor chemotherapeutic and tumor radio imaging agents [29].
In view of the diversified roles of Schiff base metal complexes,
ACH@N), 6.8–8.65 (m, Ar-10H). UV–Visible in DMF [kmax/nm (e/
Mꢁ1 cmꢁ1)]: 360 (798), 310 (7522), 298 (7800).
Synthesis of Co(II), Ni(II) and Cu(II) complexes
A ethanolic solution of ligand (0.01 mol) and respective metal
chloride salt (0.01 mol) was refluxed for 2–3 h under nitrogen.
The complex was precipitated by adding distilled water. The com-
plex separated was filtered, washed with water, then with hot
alcohol and finally dried in vacuum desiccator over P2O5 (yield
60–70%).
Co(II) complex
Yield: 70%; M.P.: > 300 °C; Anal. (%) Calc. for [C22H18Br2CoN4O6]:
C, 40.46; H, 2.78; N, 8.58. Found: C, 40.44; H, 2.80; N, 8.55; LC–MS
(m/z) 652 [C22H18Br2CoN4O6AH]+. IR (KBr, cmꢁ1): 3402 (OH lattice
H2O), 1658 (C@O), 1608 (C@N), 540 (MAO), 425 (MAN); UV–Visi-
1
3
we synthesized a Schiff base ligand N0 ,N0 -bis[(E)-(5-bromo-2-
hydroxyphenyl)methylidene]benzene-1,3-dicarbohydrazide (L1)
and its Co(II), Ni(II) and Cu(II) complexes and the structures were
elucidated by physico-chemical methods. Their DNA binding,
photosensitizing ability and antitumor activity were also
described.
ble in DMF [kmax/nm (e
/Mꢁ1 cmꢁ1)]: 440 (2540), 334 (5614), 302
(7450), 288 (8350).
Ni(II) complex
Yield:68%; M.P.: > 300 °C; Anal. (%) Calc. for [C22H18Br2NiN4O6]:
C, 40.47; H, 2.78; N, 8.58. Found: C, 40.45; H, 2.79; N, 8.57; LC–MS
(m/z) 651 [C22H18Br2NiN4O6AH]+. IR (KBr, cmꢁ1): 3425 (OH lattice
H2O), 1658 (C@O), 1609 (C@N), 545 (MAO), 437 (MAN); UV–Visi-
Experimental
ble in DMF [kmax/nm (e
/Mꢁ1 cmꢁ1)]: 430 (2690), 335 (4630), 302
(6266).
Materials
Cu(II) complex
All reagents and solvents required were of AR grade, purchased
commercially. All the solvents were purified by distillation and
CoCl2ꢂ6H2O, NiCl2ꢂ6H2O, CuCl2ꢂ2H2O, and Tris–HCl were purchased
from Merck (India), calf thymus (ds)DNA and super coiled (SC)
pUC19DNA were purchased from Bangalore Genie (India), Agarose
(molecular biology grade) and ethidium bromide were purchased
from Himedia. Tris–HCl buffer solution used for binding and cleav-
age studies was prepared using deionised double distilled water.
Yield: 65%; M.P.: > 300 °C; Anal. (%) Calc. for [C22H14Br2CuN4-
O4]: C, 42.50; H, 2.27; N, 9.01. Found: C, 42.48; H, 2.25; N, 8.98;
LC–MS (m/z) 620 [C22H14Br2CuN4O4AH]+. IR (KBr,cmꢁ1): 1658
(C@O), 1614 (C@N), 548 (MAO), 440 (MAN); UV–Visible in DMF
[kmax/nm (e
/Mꢁ1 cmꢁ1)]: 407 (6032), 336 (6560), 321 (6520), 303
(6380).
DNA-binding experiments
Physical measurements
Electronic absorption spectroscopy has been widely employed
to determine the binding characteristics of metal complexes with
DNA. The DNA concentration per nucleotide was determined by
absorption spectroscopy using the molar absorption coefficient
(6600 Mꢁ1 cmꢁ1) at 260 nm [30]. Relative binding of the complex
to CT DNA was studied by fluorescence spectrometry using ethi-
dium bromide (EB) bound CT DNA solution in Tris–HCl/NaCl buffer
(pH 7.2) at room temperature [31].
Viscosity measurements were carried out using a semi micro
dilution capillary viscometer at room temperature. Flow time
was measured with a digital stopwatch, and each sample was mea-
sured three times, and an average flow time was calculated. Data
The melting points are determined by open capillary methods
and are uncorrected. The UV–Visible spectra are recorded on a Shi-
madzu model impact 1650 UV–Visible double beam spectrometer.
The FT-IR spectra are recorded on a Shimadzu model impact 8400S
FT-IR spectrometer (KBr pellets, 3 cmꢁ1 resolution), 1H, 13C NMR
spectra on a Bruker 400 MHz and mass spectra are recorded on
LCMS Shimadzu, Japan 800 MHz spectrometer. Elemental analyses
are done on Vario EL.CHNOS elemental analyser. Viscosity mea-
surements are studied by semi micro dilution capillary viscometer
(Viscomatic Fica MgW) with a thermostated bath D40S.
were presented as (g/go) vs [complex]/[DNA], where g is the vis-
Synthesis of Schiff base ligand (L1)
cosity of DNA in the presence of the complex and g0 is that of
DNA alone [32,33].
A solution of benzene-1,3-dicarbohydrazide (0.01 mol) in anhy-
drous EtOH (50 cm3) were added drop wise to the ethanolic solu-
tion (50 cm3) of 5-bromo-2-hydroxybenzaldehyde (0.02 mol) in
the presence of acetic acid and the mixture was heated under re-
Thermal denaturation experiments were carried out with a
Shimadzu Model UV-160A spectrophotometer coupled to
a
temperature controller (Model TCC-240A) by monitoring the
absorption at 260 nm of CT-DNA at various temperatures [34,35].

134
S.M. Pradeepa et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 101 (2013) 132–139
5% CO2 atmosphere at 37 °C, respectively. For cell viability assay,
h
γ
the cancer cells were plated at a density of 104 cells/ well in 96-
well plates and incubated for 24 h. The medium was removed,
and complex solutions were added into a new medium without
O2+Photosensitizer
O
O
O
FBS to the required final concentrations between 5
200 M. The untreated A549 cells were used as a control.
After 72 h of incubation, 10 l of a stock MTT solution was
added to give a final concentration of 0.5 mg/ml and incubated
for a further 4 h. Then, the medium was replaced with 100 l of
lM and
Scheme 1. The reaction of DPBF with singlet oxygen.
l
l
DNA cleavage experiments
l
pure dimethyl sulfoxide and the absorbance of the dark blue for-
mazan was measured with an ELISA plate reader at 550 nm. Cell
viability = (Asample/Acontrol) ꢀ 100%. After 72 h of incubation with
For the gel electrophoresis experiments, supercoiled pUC19
DNA was treated with Co(II), Ni(II) and Cu(II) complexes in tris buf-
fer (50 mM tris–acetate, 18 mM NaCl, pH 7.2), and the solution was
irradiated at room temperature with a UV lamp (365 nm, 10 W)
after being incubated at 37 °C for 1 h. Electrophoresis was carried
out at 50 V for 2 h in tris–borate EDTA (TBE) buffer. Bands were
visualized by UV light and photographed to determine the extent
of DNA cleavage from the intensities of the bands using UVITEC
Gel Documentation System [36,37].
complexes, 30
with 30
ll of washed cell suspension was mixed thoroughly
ll of trypan blue solution (0.4%) and allowed to stand for
3 min at room temperature. The total number of cells and the num-
ber of blue-stained cells (dead cells) were counted using a micro-
scope [39].
Results and discussion
Quantum yield of 1O2 generation
1
3
One pot synthesis of the Schiff base ligand N0 ,N0 -bis[(E)-(5-
bromo-2-hydroxyphenyl)methylidene]benzene-1,3-dicarbohyd-
razide (L1) (Scheme 2) and its metal(II) complexes were prepared
by direct reaction of metal chlorides and the ligand in 1:1 M ratio
(metal to ligand), using methanol as the reaction medium. The
complexes are stable in both air and light. They are soluble in
DMF, DMSO and buffer solution. The conductivity measurement
in DMF at 10ꢁ3 M concentration gave a Ko value in the range of
65–70 ohmꢁ1 molꢁ1 cmꢁ1 at 300 k, suggesting that the complexes
are non-electrolytic in nature [40].
The reaction of 1O2 with 1,3-diphenylisobenzofuran (DPBF),
(Scheme 1) was adopted to evaluate the quantum yield of 1O2 gen-
eration by Co(II), Ni(II) and Cu(II) complexes. A sequence of 2 mL of
air-saturated DMSO solutions containing DPBF (20 lM) and com-
plexes, of which the absorbance at 440, 430 and 407 nm originat-
ing from the absorption of Co(II), Ni(II) and Cu(II) complexes was
adjusted to the same (OD440 nm, OD430 nm and OD407 nm = 0.25),
were separately charged into an opened 1 cm path quartz cuvette
and illuminated with light of 440 nm, 430 nm and 407 nm (ob-
tained from a Shimadzu model impact 1650 UV–Visible double
beam spectrophotometer, 2.0 nm of slit width). The consumptions
of DPBF were followed by monitoring its loss of absorbance at
417 nm (k of irradiation = 440, 430 and 407 nm) at different irradi-
ation time. [Ru(bpy)3]2+ was used as standard, whose 1O2 genera-
tion quantum yield was determined to be 0.81 in air saturated
methanol [38].
Spectroscopic studies
It is well established in the IR spectral studies that the Schiff
base having o-hydroxy group either on aldehyde or on amine
residue can form intramolecular hydrogen bond. This has direct
impact on the t(OH) vibration, and shifts to the lower frequency
with broadening. The extent of shift depends on the strength of
hydrogen bonding observed [41]. The observed bands at
3217 cmꢁ1, a strong band at 1557, and 1658 cmꢁ1 in the IR
spectrum of the Schiff base are assigned to H-bonded AOH
Antitumor activity
Human lung cancer cell line A549 was cultured in Dulbecco’s
modified Eagle’s medium (Thermo Scientific HyClone, Logan,
USA) supplemented with 10% (v/v) fetal bovine serum (FBS) in
stretching,
t(C@N) of azomethine and carbonyl t(C@O) vibrations,
respectively. An intense band at 3058 cmꢁ1 is due to the
O
HO
Acetic acid
Ethanol
2
O
O
+
NH
NH
NH2
H2N
NH
N
NH
O
O
Br
N
Br
Br
OH
HO
(L1)
M= Co2+, Ni2+
Cu2+
O
O
O
O
O
O
NH
N
NH
NH
N
NH
X=H2O
X
N
N
M
M
Br
Br
O
Br
Br
O
X
Scheme 2. Synthesis of Co(II), Ni(II) and Cu(II) complexes.

S.M. Pradeepa et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 101 (2013) 132–139
135
ANHA vibrations of the hydrazine group, the band at 1270 cmꢁ1 is
assigned to hydrazinic (NAN) of the free ligand.
t
For the Co(II), Ni(II) and Cu(II) complexes, the following changes
were observed; the high intense band due to phenolic AOH ap-
peared in the region at 3217 cmꢁ1 in the Schiff base was disap-
peared in the complexes. These observations support the
formation of MAO bonds via deprotonation. So, the H-bonded
AOH groups have been replaced by the metal ion. The presence
of broad stretching vibrations in the 3402–3452 cmꢁ1 region can
be attributed to coordinated or lattice water molecules in these
complexes. The medium intense band at 1608 cmꢁ1 due to
t
(C@N) indicates that the C@N of the ligand coordinates to the me-
tal through nitrogen. The unaltered position of the (C@O) (car-
bonyl) confirms non-involvement in coordination. The (MAO)
and (MAN) bands have been assigned in the region 540–
550 cmꢁ1 and 425–450 cmꢁ1, respectively.
t
t
t
The electronic spectra of all the compounds were recorded in
DMF solution in the range 200–800 nm. The electronic spectra of
the free dihydrazone ligand exhibit characteristic bands at
298 nm (
360 nm (
298–310 nm are assigned to intraligand
e e
max, 7800 Mꢁ1 cmꢁ1), 310 nm ( max, 7522 Mꢁ1 cmꢁ1) and
e
Fig. 1. Absorption spectral traces of [Co(L1)(H2O)2] complex in Tris–HCl buffer
max, 798 Mꢁ1 cmꢁ1). The bands present in the region
p⁄ transition while
(0.01 M, pH 7.2) upon the addition of CT-DNA = 0.5
30 m; 40 m; shows the absorbance changing upon an increase of DNA concen-
tration. Arrow shows the absorbance changing upon the increase of DNA concen-
tration. The inner plot of [DNA]/(ea f) vs [DNA] for the titration of DNA with Co(II).
lm, = 10 lm, drug, 20 lm;
l
l
p
–
the band at 360 nm is assigned to the n–p⁄ transition which is
characteristic of azomethine (C@N) function of the Schiff base.
The electronic spectra for the complexes show two to four bands
in the 300–450 nm region. The ligand bands at 298, 310 and
360 nm show red shift on complexation. The bands appearing in
the region 302–340 nm, in the complexes are attributed to intrali-
–e
gand p–
p⁄ transition. On the other hand, the appearance of a new
bands with high molar extinction coefficient in the region 440–
407 nm for Co(II), Ni(II) and Cu(II) complexes, may be assigned
for n–p⁄ and ligand to metal charge transfer transitions. The red
shift of the ligand bands provides good evidence for chelation by
ligand L1 to the metal center. The magnitude of shift of ligand
bands on complexation indicates strong bonding between the li-
gand and the metal center [42,43].
1H NMR spectra
The 1H NMR spectra of the Schiff base ligand L1 was recorded in
DMSO-d6. The down field shift of the OH proton in the ligand which
resonates at 11.2 ppm in its 1H NMR spectrum indicates that the
OH proton in ligand is probably involved in the formation of strong
intramolecular hydrogen bonding.
The 1H NMR spectrum of the ligand L1 exhibits NH proton at
12.3 ppm, aromatic ring protons at 6.8–8.65 ppm (each as a dou-
blet). The 1H NMR spectra of the complexes cannot be obtained
due to interference in their paramagnetic properties.
Fig. 2. Absorption spectral traces of [Ni(L1)(H2O)2] complex in Tris–HCl buffer
(0.01 M, pH 7.2) upon the addition of CT-DNA = 0.5 m, = 10 m, drug, 20 m;
30 m; 40 m; shows the absorbance changing upon an increase of DNA concen-
tration. Arrow shows the absorbance changing upon the increase of DNA concen-
tration. The inner plot of [DNA]/(ea f) vs [DNA] for the titration of DNA with Ni(II).
l
l
l
l
l
ꢁ
e
DNA binding properties of the complexes
bathochromism of about 2 nm (Fig. 1). Similarly, Ni(II) and Cu(II)
complexes exhibits an evident hypochromism of about 23.3% and
25.1% at 430 and 407 nm, respectively, without any apparent bath-
ochromic shift (Figs. 2 and 3).
The interactions of Co(II), Ni(II) and Cu(II) complexes in the ab-
sence and presence of increasing amount CT-DNA (at a constant
concentration of complexes) are given in Figs. 1–3, respectively.
The electronic absorption spectroscopy is one of the most com-
mon method to study the DNA-binding properties of M(II) com-
plexes, since there are strong MLCT (metal-to-ligand), LMCT
(ligand-to-metal) and IL (intraligand) charge transfer features were
observed in the absorption spectra of these complexes. In general,
Hypochromism is suggested to occur due to an intercalative mode
of binding involving a tough stacking interaction between an
aromatic chromophore and the base pairs of DNA [44–46]. Upon
addition of CT-DNA, the observed absorption band of Co(II)
complex at 288 nm exhibited hypochromism of about 20.8%, with-
out showing any changes in the wavelength, while the LMCT band
at 440 nm exhibited hypochromism of about 23.0%, with a slight
The spectral changes are characterized by an isosbestic point
maintained throughout the set of experiments. Small changes in
kmax and hypochromicities have been observed in all the metal
complexes upon interaction with CT-DNA. The isosbestic points
was observed at 364 nm for Co(II) complex (Fig. 1), 356 nm for
Ni(II) complex (Fig. 2), 451 and 341 nm for Cu(II) complex
(Fig. 3), respectively. The appearance of strong hypochromism in
the absorption spectra of the complexes is attributable to the inter-
action between the electronic states of the binding chromophore
and that of CT-DNA. The intrinsic binding constant (Kb) for the
association of Co(II), Ni(II) and Cu(II) complexes with CT-DNA
(insets of resp. Fig.) were found to be 2.6 ꢀ 104 Mꢁ1
,

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S.M. Pradeepa et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 101 (2013) 132–139
0.8
0.7
0.6
0.5
0.4
0.3
0.2
Complex 1
Complex 2
Complex 3
1
2
3
4
5
6
7
8
9
[Complex]/[DNA]
Fig. 4. Effects of increasing amount of Co(II) 1, Ni(II) 2 and Cu(II) 3 complexes on
the relative viscosity of CT-DNA at 25 0.1 °C.
Fig. 3. Absorption spectral traces of [Cu(L1)] complex in Tris–HCl buffer (0.01 M,
Fluorescence studies
pH 7.2) upon the addition of CT-DNA = 0.5
40 m; shows the absorbance changing upon an increase of DNA concentration.
Arrow shows the absorbance changing upon the increase of DNA concentration. The
inner plot of [DNA]/(ea f) vs [DNA] for the titration of DNA with Cu(II).
lm, = 10 lm, drug, 20 lm; 30 lm;
l
The interactions of Co(II), Ni(II) and Cu(II) complexes with CT-
DNA were also monitored via steady-state fluorescence emission
spectra, and the results are shown in Fig. 5. It reveals that, upon
the addition of CT-DNA, the emission strength of the complexes
exhibits obvious reduction [49,50].
The addition of each of the complexes to DNA, causes obvious
reduction in emission intensity, and the quenching constants K of
all the complexes were calculated according to the classical
Stern–Volmer equation [51]:
ꢁe
5.7 ꢀ 104 Mꢁ1, 4.5 ꢀ 104 Mꢁ1, respectively which are comparable
to that observed for typical classical intercalators [44–46], it sug-
gests a mode of intercalative binding that involves a stacking inter-
action between the complexes and the base pairs of DNA.
Viscosity measurements
I0=I ¼ 1 þ Kr
Optical photophysical techniques are widely used to study the
binding of ligands and their complexes to DNA, but do not give suf-
ficient information to determine a binding model. Therefore, vis-
cosity measurements were taken to further clarify the interaction
between metal complexes and DNA. Hydrodynamic measurements
that are sensitive to the length change of DNA (i.e., viscosity and
sedimentation) are regarded as the least ambiguous and the most
critical tests of a binding model in solution in the absence of crys-
tallographic structural data [47,48]. For the ligand and complexes,
as the amount of compound is increased, the viscosity of DNA in-
where I0 and I are the fluorescence intensities in the absence and
presence of the complex, and r is the ratio [M]/[DNA]. K is a linear
Stern–Volmer quenching constant. The fluorescence quenching
curves of complexes are shown in Fig. 6. The quenching plots
illustrate that the binding of DNA by the complexes is in good
agreement with the linear Stern–Volmer equation. In the linear fit
plot of I0/I vs [complex], the K values calculated for Co(II), Ni(II)
and Cu(II) complexes are 3.4, 3.6 and 4.1, respectively. The above re-
sults showed that all the complexes could replace EB from the
DNA–EB system, and a complex-DNA system was formed. The
reduced emission of the DNA–EB system was caused by EB being
expelled from the hydrophobic environment into the water solution
[49,50]. Hence, the fluorescence studies indicated that Co(II), Ni(II)
and Cu(II) complexes binds to DNA by intercalation.
creases steadily. The values of (g/g0) were plotted against [com-
pound]/[DNA] (Fig. 4). In classical intercalation, the DNA helix
lengthens as base pairs are separated to accommodate the ligand,
leading to increased DNA viscosity, whereas a partial, non-classical
ligand intercalation causes a bend (or kink) in the DNA helix, so
reducing its effective length and thereby its viscosity [47,48].
Fig. 5. The emission spectra for Co(II), Ni(II) and Cu(II) metal complexes in the presence and absence of CT-DNA.

S.M. Pradeepa et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 101 (2013) 132–139
137
0.6
0.5
0.4
0.3
0.2
Complex 1
Complex 2
Complex 3
Fig. 8. Photo-induced DNA cleavage by the M(II) complexes. The complexes were
irradiated with UV light at 365 nm, Lane; 1: control DNA (with out complexes),
Lane; 2: 20
Ni(II), Lane; 6: 20
l
M Co(II), Lane; 3: 40
l
M Co(II), Lane; 4: 20
M Cu(II).
lM Ni(II), Lane; 5: 40 lM
lM Cu(II), Lane; 7: 40
l
3
4
5
6
[M]/[DNA]
Fig. 6. The fluorescence quenching curves of Co(II) 1, Ni(II) 2 and Cu(II) 3 complexes
in the presence and absence of CT-DNA.
Thermal denaturation studies
The thermal behavior of DNA is a measure of the stability of
DNA double helix temperature. An interaction between DNA and
complexes were indicated by the increase in the thermal melting
temperature (Tm) as shown in Fig. 7. Thermal denaturation exper-
iments also revealed the intercalation of these complexes with
DNA. The CT-DNA alone melt at 61 1 °C (10 mmol NaCl, 1 mmol
phosphate) in the absence of any added complex. The rT values
of DNA were also increased by 4 1 °C for these complexes. The in-
crease Tm and rT of DNA could be interpreted in terms of the sta-
bilization that results from the intercalation of these metal
complexes with DNA. The observations made during the absorp-
tion titration, viscosity measurements and thermal denaturation
experiments are reminiscent of those reported earlier for various
metallointercalators, thus suggesting that the complexes bound
to DNA by intercalations [52–55].
Fig. 9. DNA cleavage by the M(II) complexes. Lane; 1: control DNA (with out
complexes), Lane; 1: control DNA (with out complexes), Lane; 2: 60 M Co(II), Lane;
3: 80 M Co(II), Lane; 4: 60 M Ni(II), Lane; 5: 80 M Ni(II), Lane; 6: 60 M Cu(II),
Lane; 7: 80 M Cu(II).
l
l
l
l
l
l
Fig. 10. Light-induced DNA cleavage by the M(II) complexes. Supercoiled DNA runs
at position I (SC), nicked DNA at position II (NC) and Linear DNA at position III (LC).
DNA photocleavage studies
Lane; 1: control DNA (with out complexes), Lane; 2: 100
Co(II), Lane; 4: 100 M Ni(II), Lane; 5: 120 M Ni(II), Lane; 6: 100
7: 120 M Cu(II).
l
M Co(II), Lane; 3: 120
lM
l
l
lM Cu(II), Lane;
The ability of the complexes to mediate DNA cleavage was as-
sayed using agarose gel electrophoresis at physiological pH and
temperature. When supercoiled circular pUC 19 DNA is subjected
to electrophoresis, relatively fast migration will be observed for
the intact supercoiled form (Form I). If scission occurs on one
strand, the supercoiled form will relax to generate a slower-
moving open nicked form (Form II), and if both strands are cleaved,
l
a linear form (Form III) that migrates between Form I and Form II
will be observed. Figs. 8–10 show the gel electrophoretic separa-
tions of pUC 19 DNA induced by Co(II), Ni(II) and Cu(II) complexes.
All three complexes promote photocleavage of DNA under physio-
logical conditions (pH 7.2, 37 °C), but with similar cleavage activi-
ties. As shown in Figs. 8–10 the DNA cleavage activities of the
complexes are obviously concentration-dependent. With the in-
crease of complex concentration, the supercoiled DNA decreases
and nicked circular DNA gradually increases. It was found that
Co(II), Ni(II) and Cu(II) complexes were the most potent nuclease
mimic in terms of molecular structure. Chemical environment
and their geometric structures may also affect the nucleolytic effi-
ciency of these complexes. The different DNA-cleavage efficiency of
the complexes may be considered due to the different binding
affinity to DNA [36,37,56].
12
10
8
DNA
Complex 1
Complex 2
6
4
2
0
Complex 3
Quantum yield of 1O2 generation
20
40
60
80
100
120
Temparature
Numerous organic compounds can react with reactive oxygen
species such as 1O2, leading to the changes of absorbance and/or
fluorescence intensity, and consequently can be utilized to
Fig. 7. Melting curves of CT-DNA in the presence and absence of Co(II) 1, Ni(II) 2
and Cu(II) 3 complexes.

138
S.M. Pradeepa et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 101 (2013) 132–139
quantitatively evaluate the 1O2 generation quantum yields of
photosensitizers by simply monitoring the fluorescence or UV–Vis-
ible spectrum [57]. 1,3-Diphenylisobenzofuran (DPBF, Scheme 1)
with a reported b value of about 10ꢁ4 is presently one of the most
reactive with 1O2. The consumptions of DPBF were followed by
monitoring its loss of absorbance at 417 nm and be articulated as
a function of photosensitizer’s 1O2 generation quantum yield
Anti-tumor study on A549 (lung carcinoma) cell
The cytotoxicity of Co(II), Ni(II) and Cu(II) complexes to human
lung tumor cells of A549 was measured by MTT reduction assay
[60]. The metabolic activity of the cells was assessed by their
ability to cleave the tetrazolium rings of the pale yellow MTT
and form a dark blue water-insoluble formazan crystal. Fig. 12
shows the results of the cell viability experiments intended at
determining the cytotoxicity of Co(II), Ni(II) and Cu(II) complexes.
Fig. 12a showed dose dependent, dark cytotoxicity of all the three
complexes to human lung carcinoma cells of A549 and Fig. 12b
showed significant dose dependent, photocytotoxicity to human
lung carcinoma cells of A549.
(
UD) according to the literature method [58,59]. Using [Ru(bpy)3]2+
as standard (UDs = 0.81), the UD of Co(II), Ni(II) and Cu(II) com-
plexes in DMSO were determined to be 0.32, 0.27 and 0.30 respec-
tively (Fig. 11), implying their prospective application in 1O2-
involving processes, such as photocleavage of DNA.
In the present investigation, we supplementary employed try-
pan blue exclusion assay to compute the cytotoxicity of Co(II),
Ni(II) and Cu(II) complexes to human lung tumor cells of A549. Via-
ble cells characterized by a structurally vital cell membrane do not
uptake trypan blue dye. In contrast, dead cells (necrotic or late
apoptotic cells) characterized by the failure of integrity of their
membrane are stained blue by the dye. At all concentration, in
the absence of irradiation the Co(II), Ni(II) and Cu(II) complexes
moderately reduced the viable A549 cells after staining with try-
pan blue at 72 h (effects on cell proliferation), which was slightly
more at higher concentrations (50–100 lM) (Fig. 12a). On the
other hand, under similar experimental conditions but with irradi-
ation, the complexes show enhanced cytotoxicity and reduced
maximum number of viable A549 cells at both lower and higher
concentrations (Fig. 12b). These results further sustaining the con-
clusion reached by MTT reduction assay that these complexes
show considerable dose dependent cytotoxicity to A549 cells in
presence of light irradiation through singlet oxygen generation.
Fig. 11. The DPBF consumption percentage as a function of irradiation time in the
air-equilibrated DMSO solution of Co(II) (d), Ni(II) (J) and Cu(II) (j). [Ru(bpy)3]2+
(I) as standard (UDs = 0.81) in air equilibrated CH3OH.
Conclusion
In summary, three novel M(II) complexes of Schiff base ligand
1
3
N0 ,N0 -bis[(E)-(5-bromo-2-hydroxyphenyl)methylidene]benzene-
1,3-dicarbohydrazide have been synthesized and structurally char-
acterized. The DNA binding abilities resulted that the complexes
bind to CT-DNA by an intercalative mode. When irradiated by
UV–Visible light (at 365 nm) the complexes are found to be effi-
cient photocleaving agents of DNA. The mechanism studies reveal
that singlet oxygen (1O2) play an important role in the DNA
photocleavage. Hence, these complexes may be useful as potential
PDT agents in photochemical therapy.
Acknowledgment
Authors are thankful to Board of Research in Nuclear Sciences,
Mumbai, INDIA for supported the work through a Major Research
project (No.2009/34/44/BRNS/3227).
References
[1] E.D. Sternberg, D. Dolphin, C. Brückner, Tetrahedron 54 (1998) 4151–4202.
[2] J. Ma, J. Chen, M. Idowu, T. Nyokong, J. Phys. Chem. B 112 (2008) 4465–4469.
[3] L. Shi, B. Hernandez, M. Selke, J. Am. Chem. Soc. 128 (2006) 6278–6279.
[4] K. Ishii, M. Shiine, Y. Shimizu, S. Hoshino, H. Abe, K. Sogawa, N. Kobayashi, J.
Phys. Chem. B 112 (2008) 3138–3143.
[5] I.J. Macdonald, T.J. Dougherty, J. Porphyrins Phthalocyanines. 5 (2001) 105–
129.
[6] Y. Jung, S.J. Lippard, Chem. Rev. 107 (2007) 1387–1407.
[7] X. Wang, Z. Guo, Dalton Trans. (2008) 1521–1532.
[8] M.H. Garcia, T.S. Morais, P. Florindo, M.F.M. Piedade, V. Moreno, C. Ciudad, V.
Noe, J. Inorg. Biochem. 103 (2009) 354–361.
[9] C.-H. Wu, D.-H. Wu, X. Liu, G. Guoyiqibayi, D.-D. Guo, G. Lv, X.-M. Wang, H. Yan,
H. Jiang, Z.-H. Lu, Inorg. Chem. 48 (2009) 2352–2354.
[10] E. Kim, P.T. Rye, J.M. Essigmann, R.G. Croy, J. Inorg. Biochem. 103 (2009) 256–
261.
Fig. 12. (a) Cytotoxicity of Co(II), Ni(II) and Cu(II) complexes to A549 cells in the
absence of irradiation. (b) Cytotoxicity of Co(II), Ni(II) and Cu(II) complexes to A549
cells in the presence of irradiation.

S.M. Pradeepa et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 101 (2013) 132–139
139
[11] R. Gao, D.G. Ho, B. Hernandez, M. Selke, D. Murphy, P.I. Djurovich, M.E.
Thompson, J. Am. Chem. Soc. 124 (2002) 14828–14829.
[34] R. Augamuthu, V. Rajendiran, P. UmaMaheshwari, R. Balamurugan, C.A. Kilner,
M.A. Halcrow, M. Palaniandavar, J. Inorg. Biochem. 99 (2005) 1717–1732.
[35] Z. Hang, S.L. Chun, H.B. Xian, Y. Ming, J. Inorg. Biochem. 99 (2005) 1119–
1125.
[36] C.N. Sudhamani, H.S. Bhojya Naik, T.R. Ravikumar Naik, M.C. Prabhakara,
Spectrochim. Acta A. 72 (2009) 643–647.
[37] M.C. Prabhakara, H.S. Bhojya Naik, V. Krishna, H.M. Kumaraswamy, Nucleos.
Nucleot. Nucl. 26 (2007) 459–471.
[38] A.A. Abdel-Shafi, P.D. Beer, R.J. Mortimer, F. Wilkinson, J. Phys. Chem. A 104
(2000) 192–202.
[12] M. Selke, W.L. Karney, S.I. Khan, C.S. Foote, Inorg. Chem. 34 (1995) 5715–5720.
[13] S. Chan, W.T. Wong, Coord. Chem. Rev. 138 (1995) 219–296.
[14] G. Pratviel, J. Bernadou, B. Meunier, Adv. Inorg. Chem. 45 (1998) 251–312.
[15] F. Liang, P. Wang, X. Zhou, T. Li, Z.Y. Li, H.K. Lin, D.Z. Gao, C.Y. Zheng, C.T. Wu,
Bioorg. Med. Chem. Lett. 14 (2004) 1901–1917.
[16] C. Gruning, H. Schmidt, D. Rehder, Inorg. Chem. Commun. 2 (1999) 57–59.
[17] I. Cavaco, J. Costa Pessoa, M.T.L. Duarte, R.T. Henriques, P.M. Matias, R.D.
Gillard, J. Chem. Soc. Dalton Trans. (1996) 1989–1996.
[18] I. Cavaco, J. Costa Pessoa, M.T.L. Duarte, R.D. Gillard, P. Matias, Chem. Commun.
11 (1996) 1365–1366.
[39] P. Zhang, J. Chen, Y. Liang, Acta Biochim. Biophys. Sin. 42 (2010) 440–449.
[40] W.J. Geary, Coord. Chem. Rev. 7 (1971) 81–122.
[19] J. Costa Pessoa, I. Cavaco, I. Correia, M.T. Duarte, R.D. Gillard, R.T. Henriques, F.J.
Higes, C. Madeira, I. Tomaz, Inorg. Chim. Acta. 293 (1999) 1–11.
[20] J. Costa Pessoa, M.J. Calhorda, V. Felix, S. Gama, I. Correia, M.T. Duarte, S.
Marcao, M.F.M. Piedade, I. Tomaz, J. Inorg. Biochem. 86 (2001) 188.
[21] J. Costa Pessoa, M.T. Duarte, R.D. Gillard, C. Madeira, P.M. Matias, I. Tomaz, J.
Chem. Soc., Dalton Trans. (1998) 4015–4020.
[41] B. Turkkan, B. Sarıbog, N. Sarıbog, Transit. Met. Chem. 36 (2011) 679–684.
[42] B. Ulkuseven, T.B. Demirci, M. Sahin, Rev. Inorg. Chem. 26 (4) (2006) 367–378.
[43] M. Sahin, T.B. Demirci, G.P. Soylu, B. Ulkuseven, Inorg. Chim. Acta 362 (2009)
2407–2412.
[44] B.Y. Wu, L.H. Gao, Z.M. Duan, K.Z. Wang, J. Inorg. Biochem. 99 (2005) 1685–
1691.
[22] R. Karmakar, C.R. Choudhury, S. Mitra, L. Dahlenburg, Struct. Chem. 16 (2005)
611–616.
[23] L.L. Koh, J.O. Ranford, W. Robinson, J.O. Svensson, A.L.C. Tan, D. Wu, Inorg.
Chem. 35 (22) (1996) 6466–6472.
[24] C.T. Yang, B. Moubaraki, K.S. Murray, J.D. Ranford, J.J. Vittal, Inorg. Chem. 40
(2001) 5934–5941.
[25] C.T. Yang, B. Moubaraki, K.S. Murray, J.J. Vittal, J. Chem. Soc., Dalton Trans.
(2003) 880–889.
[45] E.C. Long, J.K. Barton, Acc. Chem. Res. 23 (1990) 271–273.
[46] J.M. Kelly, A.B. Tossi, D.J. McConnell, C. Oh, Uigin, Nucleic Acids Res. 13 (1985)
6017–6034.
[47] S. Sathyanarayana, J.C. Dabrowiak, J.B. Chaires, J. Biochem. 31 (1992) 9319–
9324.
[48] S. Sathyanarayana, J.C. Dabrowiak, J.B. Chaires, J. Biochem. 32 (1993) 2573–
2584.
[49] C.V. Kumar, J.K. Barton, N.J. Turro, J. Am. Chem. Soc. 107 (1985) 5518–5523.
[50] J.K. Barton, J.M. Goldberg, C.V. Kumer, N.J. Turro, J. Am. Chem. Soc. 108 (1986)
2801.
[26] P.A.N. Reddy, M. Nethaji, A.R. Chakravarthy, Eur. J. Inorg. Chem. 7 (2004) 1440–
1446.
[27] R. Pradhan, A.M. Thomas, A. Mukherjee, S. Dhar, M. Nethaji, A.R. Chakravarthy,
Indian J. Chem. Sect. A. 44A (2005) 18–26.
[28] P.K. Sasmal, A.S. Patra, M. Nethaji, A.R. Chakravarthy, Inorg. Chem. 46 (26)
(2007) 11112–11121.
[51] J.R. Lakowicz, G. Webber, Biochemistry 12 (1973) 4161–4170.
[52] M. Sethuraman, P. Mallayan, Inorg. Chem. 37 (1998) 693–700.
[53] G.A. Neyhart, N. Grover, S.R. Smith, W.A. Kalsbeck, T.A. Fairley, M. Cory, H.H.
Thorp, J. Am. Chem. Soc. 115 (1993) 4423–4428.
[29] M.-Z. Wang, Z.-X. Meng, B.-L. Liu, G.-L. Cai, C.-L. Zhang, X.-Y. Wang, Inorg.
Chem. Commun. 8 (4) (2005) 368–371.
[30] B. Vinay Kumar, H.S. Bhojya Naik, D. Girija, N. Sharath, S.M. Pradeepa, J.
Macromol. Sci. Part A Pure Appl. Chem. 49 (2012) 139–148.
[31] M.F. Reichmann, S.A. Rice, C.A. Thomas, P. Doty, J. Am. Chem. Soc. 76 (1954)
3047.
[54] R.M. Hartshorn, J.K. Barton, J. Am. Chem. Soc. 114 (1992) 5919–5925.
[55] K.A. Meadows, F. Liu, J. Sou, B.P. McMillin, Inorg. Chem. 32 (1993) 2919–2923.
[56] M.C. Prabhakara, B. Basavaraju, H.S. Bhojya Naik, Bioinorg. Chem. Appl. 2007
(2007) 1–7.
[57] H.Y. Ding, X.S. Wang, L.Q. Song, J.R. Chen, J.H. Yu, C. Li, B.W. Zhang, J.
Photochem. Photobiol. A Chem. 177 (2006) 286–294.
[32] J.B. Chaires, N. Dattagupta, D.M. Crothers, Biochemistry 21 (1982) 3933–3940.
[33] B. Vinay Kumar, H.S. Bhojya Naik, D. Girija, N. Sharath, S.M. Pradeepa, H. Joy
Hoskeri, M.C. Prabhakara, Spectrochim. Acta A. 94 (2012) 192–199.
[58] R.H. Young, K. Wehrly, R.L. Martin, J. Am. Chem. Soc. 93 (1971) 5774–5779.
[59] I.E. Kochevar, R.W. Redmond, Methods Enzymol. 319 (1992) 20–28.
[60] T. Mosmann, J. Immunol. Methods 65 (1983) 55–63.