Inorganica Chimica Acta 363 (2010) 2752–2761

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Inorganica Chimica Acta

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A new blue luminescent dichlorido-bridged dinuclear copper(II) complex with

DNA binding and cytotoxic activities: Synthesis, structure and DFT studies
Soma Mukherjee a,*, Chandrama Basu a, Santanu Chowdhury a, Asoke P. Chattopadhyay b, Atanu Ghorai c,
Utpal Ghosh c, Helen Stoeckli-Evans d
a
Department of Environmental Science, University of Kalyani, Kalyani, Nadia 741 235, West Bengal, India
b
Department of Chemistry, University of Kalyani, Kalyani, Nadia 741 235, West Bengal, India
c
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, Nadia 741 235, West Bengal, India
d
Institute of Physics, University de Neuchatel, CH 2009, Neuchatel, Switzerland

a r t i c l e i n f o a b s t r a c t

Article history: A blue luminescent dichlorido-bridged dinuclear copper(II) (S = 1/2) complex, [CuII2(HL)2(l-Cl)2]2H2O,
Received 30 January 2010 1a was synthesized with the 1:1 reaction of the acyclic tridentate salicylaldehyde 2-pyridyl hydrazone
Received in revised form 7 April 2010 ligand, HL, 1. The complex 1a displays multiple bands in the visible region (400–470 nm). The association
Accepted 14 April 2010
constant (Kass, UV–Vis) was found to be 1.186  104 for 1a at 298 K. The copper(II)–copper(III) oxidation
Available online 20 April 2010
potential lies near 0.32 V versus Ag/AgCl electrode. On excitation at 390 nm, the ligand 1 strongly emits at
Dedicated to Prof. Animesh Chakravorty 444 nm due to an intraligand 1(p–p*) transition. Upon complexation with copper(II) the emission peak is
slightly red shifted (kex 390 nm, kem 450 nm, F/F0 0.81) with little quenching. Molecular structure of 1a
Keywords: (CuCu 3.523 Å) has been determined by single crystal X-ray diffraction studies. DFT and TDDFT calcu-
Luminescence lations strongly support the spectral behavior of the ligand and the complex. The complex 1a exhibits a
Dinuclear copper(II) complex strong interaction towards DNA as revealed from the Kb (intrinsic binding constant) 2.05  104 M1 and
Structure Ksv (Stern–Volmer quenching constant) 2.47 values. The complex exhibits cytotoxic effect and the LD50
DFT and TDDFT value for HeLa cells was calculated as 5.44 lM at which the cell cycle was arrested at G2/M phase.
DNA binding Ó 2010 Elsevier B.V. All rights reserved.
Cytotoxicity

1. Introduction [4,17–20]. The ligand displays intramolecular azo-imine tautomer-

In the course of development of supramolecular chemistry,
there has been a growing interest in the rational design of dinucle-
ar assemblies that consists of spacers and/or bridging ligands link-
ing two redox active metal termini that permits electron flow
along the molecular backbone [1–9]. Among inorganic anions,
halides are particularly interesting since they are capable of bind-
ing metal centers in a unidentate or bridging mode and exhibit
interesting electronic, magnetic, redox, photochemical and struc-
tural properties as well as biological activities [10–16]. The above
objective has been realized in a rational manner via design, synthe-
sis and characterization of a new blue luminescent (kem 450 nm)
redox active dinuclear copper(II) complex (S = 1/2) with
[Cu2(l-Cl)2] core unit. The binding pattern of the complexation
equilibrium has been examined via binding constant determina-
tion and thermodynamic parameter calculations. DFT and TDDFT
calculations were performed to describe the nature of orbitals in-
volved in the spectral transitions of the ligand and the complex
* Corresponding author. Tel.: +91 (0)33 2582 8378, +91 (0)33 2582 8750x291,
292; fax: +91 33 2582 8282.
E-mail address: sommukh445@yahoo.co.in (S. Mukherjee).
ism [21–23] supported further by DFT studies. In recent years cop-
per(II) complexes have received much attention due to their
numerous biological activities [24,25]. Moreover, considerable
interest has been placed to the interaction of copper(II) complexes
with DNA [5,26–29] due to their significance in the development of
new therapeutic agents in medicine. Herein, we have investigated
the DNA binding activity of the complex 1a by UV absorption spec-
troscopy, fluorescence spectroscopy and cyclic voltammetric stud-
ies. Moreover, cytotoxic activity of 1a for HeLa cells has been
observed by fluorescence microscopy and flow cytometric analysis.
2. Experimental
2.1. Materials
All starting materials and solvents were purchased from Sigma–
Aldrich Chemical Company and were used without further purifi-
cation unless otherwise stated. Ethidium bromide and DNA
(pUC19 and CT-DNA) were purchased from Bangalore GeNei.
Hoechst dye, propidium iodide and RNase A were purchased from
Sigma Chemicals (USA). Culture media MEM and serum was

0020-1693/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2010.04.026
 S. Mukherjee et al. / Inorganica Chimica Acta 363 (2010) 2752–2761 2753

purchased from HiMedia, India. Other molecular biology grade fine Table 1
chemicals were procured locally. Crystallographic data for [CuII2(HL)2(l-Cl)2]2H2O, 1a.

Compound 1a
2.2. Physical measurements Empirical formula C24H20Cl2Cu2N6O22H2O
Formula mass 658.47
T (K) 173
A Perkin–Elmer 2400 C Elemental Analyzer was used to col- Wavelength (Å) 0.71073
lect microanalytical data (C, H, N). Sartorius CP64 balance was Crystal system monoclinic
used for weighing purpose. FTIR data were collected with the Space group C2/c (No. 15)
help of FTIR Shimadzu 8400 Spectrophotometer. UV–Vis spectra a (Å) 16.2959(18)
b (Å) 7.419(1)
of the ligand and the complex were recorded on Shimadzu UV-
c (Å) 20.541(2)
1700 spectrophotometer and corrected for background due to a (°) 90.00
solvent absorption. Fluorescence spectra were recorded on Per- b (°) 97.000(8)
kin–Elmer LS 50B Luminescence Spectrometer. For binding con- c (°) 90.00
stant measurements solutions were prepared at a fixed V (Å3) 2464.9(5)
Z 4
concentration of HL (1.0  105 M) and at a concentration of me- Dcalc (g cm3) 1.775
tal ions ranging from 1.0 to 10.0  106 M at room temperature. l (mm1) 1.989
Room temperature magnetic susceptibility was measured with F(0 0 0) 1336
the model 155 PAR vibrating sample magnetometer fitted with Crystal dimension (mm) 0.40  0.40  0.45
h Range (°) 2.52, 26.07
a Walker scientific L75FBAL magnet. Electrochemical measure-
Limiting indices 19 < h < 19, 6 < k < 8,
ments were performed under nitrogen atmosphere on a Versas- 24 < l < 23
tat II PAR Electrochemistry system using a platinum electrode. Reflections collected/unique 4828/2020
NH4PF6 was used as a supporting electrolyte and the potentials Refinement method Full-matrix least squares on |F2|
are referenced to the Ag/AgCl electrode. HPLC grade solvents Data/restraints/parameters 2020/3/181
Goodness-of-fit (GOF) on F2 1.005
were used for spectroscopic and electrochemical studies. Axio- R1a, wR2b [I > 2r(I)] 0.0556, 0.1424
skop 2 plus (Carl Zeiss) fluorescence microscope was used for R1a, wR2b (all data) 0.0612, 0.1452
imaging with AXIOVISION 3.1 software and FACS Calibur™ (BD Bio- Largest differences in peak and hole 0.68, 0.92
science, USA) fluorescence-activated cell sorter was used for flow (e Å3)
cytometry. a P P
R1 = [ ||Fo|  |Fc||]/ |Fo| (based on F).
P P
b
wR2 = [[ w(|F 2o  F 2c |)2]/[ w(F 2o )2]]1/2 (based on F2).
2.3. X-ray structure determination

Single crystals of [CuII2(HL)2(l-Cl)2]2H2O, 1a, were grown by

slow evaporation from methanol. A rod shaped crystal was
mounted on a glass fiber. Intensity data were collected at
173 K by the x-scan method over the 2h range 2.52–26.07° on
a Stoe Mark II-Image Plate Diffraction System [30] equipped
with a two-circle goniometer and using Mo Ka graphite mono-
chromated radiation (k = 0.71073 Å). Structure solution and
refinement were done by direct methods using SHELXS-97 and
SHELXL-97 programs [31–33]. The structure was successfully
solved in space group C2/c (No. 15). Of the 4828 independent
reflections, 2020 satisfying I > 2r(I) were used for structure solu-
tion. The NH H-atoms were located from Fourier difference maps
and refined isotropically. The water H atoms were located from
Fourier difference maps and refined with distance restraints.
The remainders of the H-atoms were included in calculated posi-
tions and treated as riding atoms using SHELXL default parameters.
All non-hydrogen atoms were refined anisotropically using
weighted full-matrix least squares on F2. The molecular structure
and crystallographic numbering scheme are illustrated in the
PLATON drawing [34]. Details concerning crystal data and refine-
ment are given in Table 1.
2.4. Syntheses of the ligand, 1 and the complex, 1a
The ligand salicylaldehyde-2-pyridyl hydrazone, HL, 1, was syn-
thesized following the literature method [23,35].
2.4.1. [CuII2(HL)2(l-Cl)2]2H2O, 1a
To a stirred methanolic solution of CuCl22H2O (0.03 g,
0.2 mmol) was added a solution of HL, 1, (0.04 g, 0.2 mmol) in
methanol (5 ml) and the resulting green solution was stirred for
0.5 h. The solvent was evaporated by rotary evaporator and on
recrystallization from methanol, deep green crystals of
[CuII2(HL)2(l-Cl)2]2H2O, 1a were obtained. Yield. 84%, 0.11 g. Anal.
Calc. for C24H20Cl2Cu2N6O22H2O: C, 43.76; H, 3.65; N, 12.76.
Found: C, 43.74; H, 3.62; N, 12.74%. FTIR (KBr pellets, cm1):
3598, 3477, 3417, 3172, 2931, 1656, 1627, 1602, 1577, 1546,
1481, 1456, 1425, 1375, 1348, 1284, 1255, 1203, 1149, 1110,
1093, 1070, 1016, 960, 931, 862, 757, 626, 513, 474, 418. UV–Vis
(CH3OH), kabs (nm) (e, M1 cm1) for 1a: 650(300), 450(1800),
400(12 700), 329(12 400), 290(16 500), 273(16 200), 247(22 600).
UV–Vis (CH3OH), kabs (nm) (e, M1 cm1) for 1: 335.6(19 600),
309(14 000), 238(13 000).
2.5. DFT calculations
DFT calculations were carried out on the gas phase structure of
the ligand HL, 1 and the single crystal X-ray structure of
[CuII2(HL)2(l-Cl)2]2H2O, 1a. The GAMESS-US (Version 2008, April
11 (R1) [36] and GAUSSIAN-03W (Revision C.01) [37] packages were
used for the calculations. The valence-only 6-31G(d) basis set
[38–40] and B3LYP functional [41–43] were used as earlier [44].
Electronic transitions were checked using TDDFT method in both
the packages. The MO pictures and molecular structures were
obtained with MASK [45,46] software.
2.6. DNA binding experiments
The DNA binding experiments were performed at 25.0 ± 0.2 °C.
The CT-DNA concentration per nucleotide was determined by elec-
tronic absorption spectroscopy using the known molar extinction
coefficient value of 6600 M1 cm1 at 260 nm [47]. The UV–Vis
titration of the complex, 1a, was performed in a buffer (50 mM
NaCl–50 mM Tris–HCl buffer, pH 7.2) medium using a fixed
complex concentration to which increments of the CT-DNA stock
solutions (0.0–4.0  105 M) were added. The resulting solutions
were incubated for 10 min before the absorption spectra were
recorded.
2754 S. Mukherjee et al. / Inorganica Chimica Acta 363 (2010) 2752–2761

For fluorescence quenching experiments initially the CT-DNA
was pretreated with ethidium bromide (EB) for 0.5 h and the
complex, 1a, was added to this mixture. On excitation at 490 nm,
emission peak was observed between 500 and 700 nm.
The electrochemical titration experiments were performed by
keeping the concentration of the complex constant while varying
the CT-DNA concentration using Tris buffer (pH 7.2) at room
temperature.
2.7. Cytotoxicity studies
3. Results and discussion
3.1. Synthesis and characterization
The ligand HL, 1 binds in a tridentate manner utilizing imine,
phenolic and pyridyl functions as potential donor sites. The 1:1
reaction of HL with CuCl22H2O in methanol at room temperature
afforded blue luminescent (kem 450 nm) [CuII2(HL)2(l-Cl)2]2H2O,
1a in excellent yields. The complex is highly soluble in methanol.
The room temperature (298 K) magnetic moment of the complex
is 1.82 lb (S = 1/2) as expected for d9 system.

2.7.1. Cell culture 3.2. Spectroscopic studies

Human cervical cancer cell line HeLa was obtained from Na-
tional Centre for Cell Sciences, Pune, India. HeLa cells were grown All the spectroscopic measurements were carried out in de-
in MEM supplemented with 10% bovine serum (complete medium) gassed methanol at room temperature. The bands appearing in
at 37 °C in humidified atmosphere containing 5% CO2. the UV region (200–395 nm) are characteristics of the ligand, HL,
1. In the complex these ligand centered bands are accompanied
by multiple bands extending into the visible (400–470 nm) region.
2.7.2. Cell viability and morphology
On excitation at 390 nm, the ligand 1, strongly emits in the blue re-
After inoculation in a fresh medium HeLa cells were incubated
gion (444 nm) due to intraligand 1(p–p*) transition [51]. In the case
for 18 h and then treated with different concentrations of the com-
of [CuII2(HL)2(l-Cl)2]2H2O, 1a, the emission peak is slightly red
plex 1a (0–10 lM) for 17 h. The cells were harvested, washed with
shifted with a little quenching in emission intensity (kex 390 nm,
phosphate buffered saline (PBS) twice. Then the cells were incu-
kem 450 nm, F/F0 0.81). Once excited, both the ligand 1 and the
bated with 0.1% trypan blue for 5 min at room temperature and
complex, 1a undergo auto increment in emission intensity at room
counted by hemocytometer under light microscope. Each experi-
temperature without the influence of any external reagent as re-
ment was repeated 4 times and the mean of % viable cells at each
ported earlier [23]. This may probably due to the photo induced
dose was compared with the mean of untreated control using one
azo-imine tautomerism exhibited by 1 and 1a (Scheme 1, vide su-
way ANOVA with a post-hoc test viz. Dunnett’s test [48].
pra) [21,22]. The rate of increment is highest in 1, but is restricted
For cell morphology study, cells were grown over cover slips
in 1a, possibly due to the structural rigidity of dinuclear copper(II)
placed inside the plate for overnight. After 17 h treatment with dif-
complex.
ferent concentrations of 1a (0–10 lM), the cover slips were
washed with phosphate buffered saline (PBS) twice and placed
3.3. Thermodynamics of binding
over clean microscopic slides. The photographs were taken under
normal light microscope.
The association constant (Kass) of the complex [CuII2(HL1)2(l-
Cl)2]2H2O, 1a can be estimated spectrophotometrically (Fig. 1)
2.7.3. Cell cycle analysis by flow cytometry according to the Eq. (1) [52,53] where X represents the absorption
After 17 h treatment, the cells were trypsinized, washed twice intensity, CH and CG are the corresponding concentrations of the li-
with cold phosphate buffered saline (PBS), fixed with 70% ethanol gand and the metal ion. The Kass value was found to be 1.186  104
in PBS for 5 h at 4 °C, and then stained with a solution containing at 298 K.
10 lg/ml propidium iodide (PI), 100 lg/ml of DNase-free RNase A X ¼ X o þ ðX lim  X o Þ=2C o fC H þ C G þ 1=K ass
(Sigma) and 0.1% (v/v) Triton X-100 in the dark for 30 min at room
temperature before flow cytometric analysis. The intracellular DNA  ½ðC H þ C G þ 1=K ass Þ2  4C H C G 1=2 g ð1Þ
was then labeled with PI and the PI fluorescence of individual nu-
clei was determined by a FACS Calibur (BD Bioscience, USA) fluo-
rescence-activated cell sorter [49,50] at 488 nm excitation.
20,000 cells were taken for each set and fluorescence data were
plotted using CELLQUEST software (Becton Dickinson). Using this
raw data, the percentages of cells in each phase of the cell cycle
was obtained using MODFIT LT software (Becton Dickinson). Three
independent experiments were performed. The p-values were cal-
culated using one way ANOVA with a post-hoc test viz. Dunnett’s
test.

2.7.4. Cell staining by the complex 1a phosphate buffered saline (PBS)

HeLa cells were inoculated on cover slip placed inside the plate
in complete MEM medium. After overnight incubation in CO2 incu-
bator, the cover slip was taken out, washed with PBS twice and
then incubated in presence of 2 lM of the complex 1a in PBS. After
25 min incubation in dark at room temperature the cover slip was
taken out and placed upside down over a glass slide, so that cells
were in touch with the glass slide. The slide was observed under
fluorescence microscope with UV excitation and photographed at
0 and 30 min exposure of time keeping the slide under fluores-
cence microscope. Scheme 1.
 S. Mukherjee et al. / Inorganica Chimica Acta 363 (2010) 2752–2761 2755

Fig. 3. Crystal packing diagram of [CuII2(HL)2(l-Cl)2]2H2O, 1a viewed along the c-

axis. The hydrogen atoms are not shown for clarity.
Fig. 1. Absorbance spectra of HL, 1 (1.0  105 M) upon addition of CuCl22H2O
(1.0  106 to 1.0  105 M) in degassed methanol (pH 7.0) at 298 K.

The temperature dependence of binding constant was studied

between 293 and 308 K. The values of the thermodynamic param-
eters for the binding were obtained by variable temperature UV–
Vis titration in degassed methanol at 298 nm. The standard Gibb’s
free energy change, DG0 = 5591.2 cal/M, the standard enthalpy
change, DH0 = 557.7 cal/M, and the standard entropy change,
DS0 = 19.61 cal/M/° are calculated using Van’t Hoff’s Equation. It
is seen that the binding process is favored by the negative DH0
and positive DS0.

Fig. 4. Intermolecular H-bonding in [CuII2(HL)2(l-Cl)2]2H2O, 1a forming one

3.4. X-ray structure of [CuII2(HL)2(l-Cl)2]2H2O, 1a
dimensional hydrogen bonded polymer.

The molecular structure of the complex [CuII2(HL)2(l-Cl)2]

2H2O, 1a is shown in Fig. 2. Crystal packing diagrams illustrating
Table 2
different hydrogen bonding are shown in Figs. 3 and 4. Selected
Selected bond lengths (Å) and angles (°) for [CuII2(HL)2(l-Cl)2]2H2O, 1a.
bond lengths and angles are listed in Table 2. Details of the hydro-
gen bonding are given in Table 3. The molecular structure of 1a Complex, 1a
clearly shows that it is a centrosymmetric dichlorido-bridged dinu- Bond lengths
clear copper(II) complex, with a [Cu2(l-Cl)2] core unit (Fig. 2). Each Cu(1)–Cl(1) 2.2616(12) Cu(1)–Cl(1i) 2.9284(12)
Cu(1)–N(1) 1.994(4) Cu(1)–O(1) 1.900(4)
copper atom lies in a square pyramidal N2OCl2 coordination envi-
Cu(1)–N(3) 1.955(3)
ronment and is connected to the other by means of two chloride
Bond angles
groups bridging in an end-to-end fashion. The Tor value for this
Cl(1)–Cu(1)–O(1) 91.06(10) O(1)–Cu(1)–N(3) 91.94(14)
complex is 0.034, which indeed indicates that it can be considered Cl(1)–Cu(1)–N(1) 95.51(11) N(1)–Cu(1)–N(3) 81.09(15)
Cl(1)–Cu(1)–N(3) 173.93(12) Cl(1i)–Cu(1)–N(1) 86.17(11)
Cl(1)–Cu(1)–Cl(1i) 95.53(4) Cl(1i)–Cu(1)–N(3) 89.28(11)
O(1)–Cu(1)–N(1) 171.87(14) Cl(1i)–Cu(1)–O(1) 97.98(10)

as having an almost perfect square–pyramidal geometry (Tor = 0

Fig. 2. Molecular structure of the complex [CuII2(HL)2(l-Cl)2]2H2O, 1a with
thermal ellipsoids drawn at the 50% probability level. Water molecules have been
omitted for clarity.
for square pyramidal and Tor = 1 for trigonal bipyramidal) [54].
The ligand binds to the metal in a tridentate manner utilizing phe-
nolato-O, pyridyl-N and imine-N as potential donor sites, Cu(1)–
O(1) 1.900(4) Å, Cu(1)–N(1) 1.994(4) Å, Cu(1)–N(3) 1.955(3) Å.
The penta-coordination of the copper atoms is completed by two
chloride atoms Cu(1)–Cl(1) 2.2616(12) Å, Cu(1)–Cl(1i) 2.9284
(12) Å [Symmetry operation: (a) = x, y, z + 1].
The Cu(1)Cu(1i) distance is ca. 3.523 Å. Atoms N1, N3, Cl1 and
O1 define the basal plane, with atom Cl1i occupying the axial posi-
tion. The angles at the metal center between cis-positioned donor
pairs span the range 81.09(15)–97.98(10)° and those between
trans-positioned pairs are 171.87(14)–173.93(12)°. The water mol-
ecules and chloride ions occur as discrete entities in the lattice and
2756 S. Mukherjee et al. / Inorganica Chimica Acta 363 (2010) 2752–2761

Table 3 in Table 4. The HOMO and LUMO values, in lower and upper rows
Hydrogen bonding (Å) and angles (°) for [CuII2(HL)2(l-Cl)2]2H2O, 1a. with a gap in between, are underlined.
D–HA D–H HA DA D–HA
O(1W)–H(1WA)Cl(1) 0.89(2) 2.81(5) 3.404(4) 126(4)
O(1W)–H(1WA)O(1) 0.89(2) 2.02(3) 2.867(4) 160(6)
O(1W)–H(1WB)Cl(1) 0.88(5) 2.62(5) 3.396(3) 148(5)
N(2)–H(2N)O(1W) 0.8800 1.9400 2.789(5) 161.00
C(1)–H(1)Cl(1) 0.9500 2.7100 3.279(5) 119.00
C(4)–H(4)Cl(1) 0.9500 2.6600 3.608(4) 177.00

Symmetry operations: (a) = 1/2  x, 3/2  y, z; (b) = 1/2  x, 1/2 + y, 1/2  z; (c) = 1/
2  x, 1/2  y, z; (d) = x, 1  y, 1/2 + z, (e) = 1/2 + x, 1/2 + y, z; (f) = 1/2 + x, 1/2  y,
1/2 + z; g) = x, y, 1/2  z; (h) = x, 1  y, z; (i) = x, y, 1/2 + z; (j) = 1/2 + x,
1/2 + y, z; (k) = x, 1  y, 1/2 + z; (l) = 1/2  x, 1/2 + y, 1/2  z; (m) = 1/2 + x, 1/2  y,
1/2 + z; (n) = 1  x, y, 1/2  z; (o) = x, y, 1/2  z; (p) = 1/2 + x, 1/2  y, 1/2 + z;
(q) = 1/2  x, 1/2 + y, 1/2  z; (s) = x, y, 1z; (t) = 1/2 + x, 1/2  y, 1/2 + z;
(u) = x, y, 1/2 + z.

participate in intermolecular hydrogen bonding O(1 W)–
H(1 WA)Cl(1) 3.404(4) Å, O(1 W)–H(1 WA)O(1) 2.867(4) Å,
O(1 W)–H(1 WB)Cl(1) 3.396(3) Å, N(2)–H(2)O(1W) 2.789(5) Å,
C(1)–H(1)Cl(1) 3.279(5) Å, C(4)–H(4)Cl(1) 3.608(4) Å (Table 3)
leading to one dimensional hydrogen bonded polymeric structure
(Fig. 4).
3.5. Electronic structure calculations and correlation with spectral
transitions
To gain detailed insight into the azo-imine tautomerism exhibited
by the ligand HL, 1 as well as the complex, 1a, the possible transi-
tion state structures were determined using the CHEM-3D ultra soft-
ware and are depicted below. DFT calculations were carried out in
the gas phase on each of the four structures A ? D, with 6-31G(d)
basis and B3LYP functional. The four structures have two possible
orientations of the OH attached to the phenyl ring, i.e. trans and
cis with respect to the HC@N–NH-moiety. The MO levels and the di-
pole moments (in Debye units) of all the eight structures are given
It is important to note that these are all gas phase calculations and
solvent effects of all these forms may add (or subtract) considerably
to the fairly low differences between A, B and D forms. It is known
that polar solvents would tend to stabilize the azo form more than
the imine form [55], thereby possibly bringing down the energies of
the final azo forms lower than the starting imine forms, or at least at
par with them. To estimate the effect of solvation, we carried out
Self-Consistent Reaction Field (SCRF) calculations [56] under the
DFT formalism with the same basis set and functional, but for meth-
anol as solvent (dielectric constant = 32.63, cavity radius = 5.9 Å).
No appreciable difference was observed in the physical structures
of the cis and trans forms optimized in solvent from the gas phase,
nor in the electronic structures or energies. Consequently the low-
est few transitions were, essentially, unchanged. Also noteworthy
is the greater stability of the intermediates possibly arising out of
the cis form and the final product obtained from it, although the
starting cis configuration is a little higher in energy (by 2.3 kcal).
As solvent effects could not be computed for all the intermediates
and final structures, we cannot pinpoint the detailed mechanism
which may be responsible. However, the observation remains valid
that the azo-imine tautomerism is definitely possible here, probably
having a modest energy barrier.
The dipole moment values reported in Table 4 give an estimate
of the charge separation in the ground state of the structure con-
cerned. The magnitudes increase from the imine form to the azo
form as expected. But the variations among the cis and trans forms
of the intermediates B and C are curious. The radical C form (of cis/
trans) has higher lD among all the structures studied. DFT and
TDDFT calculations were also carried out on the gas phase struc-
ture of the ligand, HL, 1 and the complex, [CuII2(HL)2(l-Cl)2]2H2O,
1a. These structures were optimized at the DFT level with 6-31G(d)
basis and B3LYP functional. The highest occupied (HOMO) and the
lowest unoccupied (LUMO) molecular orbitals are abbreviated as H
and L, respectively, and other orbitals are referred accordingly.

Table 4
MO levels (eV) of the possible conformations of the ligand, HL, 1.

Optimized ligand A B C D
Trans Cis Trans Cis Trans Cis Trans Cis Trans Cis
0.370 0.693
0.083 0.474 0.158 0.204 0.506 0.693
0.526 0.811 0.376 0.650 0.557 0.909 0.592 0.797 0.898 1.122
L 1.124 1.328 1.242 1.471 0.801 1.276 1.583 1.587 1.358 1.958
H 5.190 5.432 5.216 5.459 5.801 5.453 5.083 5.301 5.989 5.982
6.213 6.197 6.323 6.304 5.923 6.137 5.434 5.535 5.992 6.378
6.835 6.951 6.836 6.940 6.694 7.105 6.753 6.760 6.641 6.629
7.112 7.363 6.875 7.114 6.885 7.220 6.997 7.139 7.290 7.494
7.419 7.691 7.375 7.713 7.333 7.638 7.150 7.369 7.595 7.793
lD 3.0358 2.3238 3.5693 1.2898 2.2252 2.9459 6.3752 4.2936 4.9984 3.9997
 S. Mukherjee et al. / Inorganica Chimica Acta 363 (2010) 2752–2761
Fig. 5. Some MO pictures (HOMO, HOMO1, LUMO, LUMO+1) of 1 and 1a.
Fig. 5 shows pictures of few of the frontier MOs of the ligand, 1 and
the complex, 1a.
The electronic structure of the optimized geometry of the ligand
(in the trans form) is as follows. This shows a HOMO at 5.190 eV,
consisting of a p MO over the whole molecule, and a LUMO at
1.124 eV, mainly over the pyridine and the chain, with little on
the phenyl ring. The L+1 is a p MO mainly on the phenyl ring much
less on the pyridine. H1 at 6.213 eV is a p MO delocalized over
the whole molecule, but the density is a little more on the phenyl
ring. H2 at 6.835 eV is a r MO of the molecule, with more elec-
trons on the chain. The later describes lone pair type orbitals on the
nitrogen. H3 at 7.112 eV is a p MO over the whole molecule.
H4 at the 7.419 eV, is another r MO on pyridine and the middle
chain only.
The electronic structure of the optimized geometry of the cis
form of the ligand is similar to the trans form. Both the HOMO at
5.432 eV and the LUMO at 1.328 eV are molecular p MOs.
H1 at 6.197 eV is also a p MO, with weight a little more on ben-
zene, as in the trans form. H2 at 6.951 eV is a r MO with more
density on the chain. H3 at 7.363 eV is a p MO. H4 at
7.691 eV is again a r MO as in the trans form, over the pyridine
and the middle chain only.
For the complex [CuII2(HL)2(l-Cl)2]2H2O, 1a, HOMO
(3.798 eV) and LUMO (3.080 eV) are both mainly Cu dr type
orbitals, bonding with nearest neighbors of Cu. H1 and H2 are
two p orbitals, lying close together (5.033 and 5.123 eV), cen-
tered on phenyl ring, with little contribution from the C–N–N chain
between phenyl ring and pyridine rings. H3 (5.787 eV) is a p
bond between Cu and Cl, with more density on Cl. This is followed
by another close lying pair of p MOs, H4 and H5 (5.812 and
5.877 eV), which are distributed over the ligand. H6, H7 and
H8 (6.199, 6.211 and 6.318 eV) all describe the dp–pp type
bonding between Cu and Cl. H9 and H10 (6.694 eV and
6.699 eV) are two very close orbitals both of which describe r
bonds between Cu (d) and its near neighbors, including, Cl. H11
(6.818 eV) is a p MO, mainly on the ligand, with no contribution
from Cl and O. H12 (6.944 eV) is of the same nature. H13 to
H16 (7.122, 7.147, 7.169 and 7.236 eV) consist of Cu dp
orbitals interacting with near neighbors, with little contribution
of Cl. The last two have some density on phenyl ring and O, but
very little on pyridine. H17 (7.698 eV) shows strong Cu–Cu
bond which includes Cls and is of dp–pp in nature. Because of
the much reduced HOMO–LUMO gap in the complex, 1a, vis-a-vis
the ligand HL, 1, we expect a greater number of higher wavelength
(lower energy) bands in 1a, as compared to 1.
2757
The TDDFT results and the experimental data are quite reveal-
ing. For the ligand HL, 1, in the trans form, the calculated transi-
tions are (p–p*) type. The most intense transition (H ? L) is
observed at 332.87 nm, the other transitions are at 280.36 and
235.74 nm, respectively. In case of the optimized cis form, most in-
tense transition (H ? L) is observed at 332.72 nm, the other transi-
tions are at 291.39 and 226.85 nm, respectively. The calculated
values are quite close to the observed kmax values at 335.6, 309
and 238 nm. We have also computed the lowest few electronic
transitions by TDDFT method and it was found that the nature of
the frontier orbitals do not change very much between A, B and
D forms, and the transitions are similar. The C form has a couple
of MOs mostly involving lone pairs on the chain nitrogens and
transitions P400 nm were observed.
For the complex, 1a the major low-lying transitions are at
656.83, 622.68, 560.61, 433.63, 390.59 and 383.32 nm. All these in-
volve transitions from H3, H5, H8, H9 or H11 to L (for the
first three transitions), and from H1, H2, H3 and H4 to L+1
and L+2 (for the latter three transitions), implying a ML ? ML
(mainly L ? M) CT type of transition for 1a. These values compare
well with experimental kmax values at 650, 450, 400, 329, 290, 273
and 247 nm.
3.6. DNA binding studies
3.6.1. Absorption spectral studies of CT-DNA with 1a
Binding of metal complexes with DNA generally results in hyp-
ochromism and hyperchromism due to a strong interaction be-
tween an aromatic chromophore and the base pairs of DNA [57].
To evaluate the binding capacity of the complex, 1a, towards CT-
DNA, UV–Vis titration was performed in buffer (50 Mm Tris–HCl–
NaCl pH 7.2) medium at room temperature. As shown in Fig. 6,
with the addition of CT-DNA (0.0–4.0  105 M) to the solution
of 1a, (1.0  105 M), hyperchromism with blue shift [5] was ob-
served suggesting that there exists a strong interaction between
1a and DNA.
In order to further investigate the intensity of interactions be-
tween 1a and CT-DNA, we have calculated the intrinsic binding
constant, Kb, from the plot of [DNA]/(ea  ef) versus [DNA] using
the following Eq. (2), [26,27,58]
½DNA=ðea  ef Þ ¼ ½DNA=ðeb  ef Þ þ 1=K b ðeb  ef Þ ð2Þ
Fig. 6. Variation of UV–Vis absorption spectra for CT-DNA (4.0 x 105 M) in
presence of 1a (1.0  105 M) in a buffer (Tris–HCl–NaCl, pH 7.2) medium at room
temperature. Inset: plot of [DNA]/(ea  ef) vs. [DNA].
2758 S. Mukherjee et al. / Inorganica Chimica Acta 363 (2010) 2752–2761

3.6.2. Ethidium bromide induced emission spectral studies

Fig. 7. Variations of emission spectra of EB with CT-DNA (4.0  105 M) on addition
of the complex 1a. Inset: plot of I0/I vs. r.
where [DNA] is the concentration of DNA in base pairs, the molar
absorption coefficients ea, eb and ef represent the apparent absorp-
tion coefficient for the complex and the extinction coefficient for
the complex in the fully bound form and the extinction coefficient
for the free dinuclear complex, respectively. The Kb is obtained by
the ratio of the slope to the intercept and is found to be
2.05  104 M1 for 1a (Fig. 6).
Competitive ethidium bromide (EB) binding study was carried
out to understand the mode of DNA interaction of 1a. The molecu-
lar fluorophore EB emits intense fluorescence at 600 nm in the
presence of CT-DNA due to its strong intercalation between the
adjacent DNA base pairs. Addition of a second molecule, which
binds to DNA more strongly than EB, would quench the DNA-in-
duced EB emission [59,60]. The extent of quenching of the fluores-
cence of EB bound to DNA would reflect the extent of DNA binding
of the second molecule. Here, upon addition of the complex, 1a, in
Tris–HCl–NaCl buffer (pH 7.2) to CT-DNA (4.0  105 M) pre-
treated with EB the emission intensity decreases (Fig. 7) almost
up to 50%.
The quenching of the EB bound to DNA by the complex is in
agreement with the linear Stern–Volmer equation, Eq. (3) [60],
I0 =I ¼ 1 þ K sv r ð3Þ
where, I0 and I represent the fluorescence intensities in the absence
and presence of the complex, respectively, r is the concentration ra-
tio of the complex, 1a to DNA. Ksv is the linear Stern–Volmer
quenching constant. The Ksv value is obtained from the slope of I0/
I versus r linear plot and is found to be 2.47 which indicates a strong
interaction of 1a with CT-DNA [61].

Fig. 8. Flow cytometric profiles of cell cycle distribution after analysis with MODFIT LT software. DNA content (PI) staining is shown in the X-axis (FL2-A) in the all figures.
Figures (A), (B), (C), (D), (E) and (F) represent control (untreated), 2, 4, 6, 8 and 10 lM of 1a, respectively.
 S. Mukherjee et al. / Inorganica Chimica Acta 363 (2010) 2752–2761 2759

Table 5
Cell cycle distribution in HeLa cells by FACS. Cells were treated with different concentrations of 1a (0–10 lM). The percentages of cell cycle distribution were evaluated by MODFIT LT
software using PI staining. Values represent the means ± SD of three independent experiments.

Dose (lM) G0/G1 S G2/M

0 64.92 ± 0.3 24.73 ± 0.19 10.33 ± 0.31
2 64.28 ± 0.72 (p = 0.918) 25.37 ± 0.99 (p = 0.991) 10.34 ± 0.52 (p = 1.000)
4 49.63 ± 0.89 (p = 0.000) 34.00 ± 2.91 (p = 0.000) 16.36 ± 2.36 (p = 0.019)
6 51.46 ± 0.55 (p = 0.000) 5.88 ± 2.65 (p = 0.000) 42.65 ± 2.28 (p = 0.000)
8 39.67 ± 1.49 (p = 0.000) 0.23 ± 0.39 (p = 0.000) 60.09 ± 1.66 (p = 0.000)
10 36.13 ± 1.76 (p = 0.000) 1.91 ± 2.03 (p = 0.000) 61.94 ± 3.70 (p = 0.000)

3.6.3. Cyclic voltammetric studies 3.7.2. Cell cycle analysis

Cyclic voltammetry also provides useful information regarding
the binding of metal complexes with DNA [62]. In the absence of
CT-DNA, the complex 1a exhibits a quasireversible cyclic response
near 0.32 V (DEp 0.17 V) versus Ag/AgCl corresponding to the
Cu(II,II)/Cu(II,III) oxidation. While in the presence of CT-DNA the
formal potential as well as the voltammetric peak currents de-
creased, apparently which indicate that there exists interaction be-
tween the complex 1a and CT-DNA [63]. The peak-to-peak
separation becomes larger with DEp value of 0.23 V and E1/2 value
of 0.30 V versus Ag/AgCl electrode.
3.7. Cytotoxicity studies
3.7.1. Cell viability and morphology
The percentage of viable HeLa cells was decreased dose-depen-
dently with increase of concentration of 1a. The LD50 value was cal-
culated as 5.44 lM. Morphology of HeLa cells was observed to be
changed with increase in concentration of 1a. Healthy HeLa cells
generally look like elongated, attached and in irregular shape but
when treated with 1a (10 lM) the cells get damaged and looks like
almost round, wrinkled and short.
The cell cycle distribution in HeLa cells treated with 1a was ana-
lyzed using MODFIT LT software and pictures for a typical experiment
were shown in Fig. 8, where A, B, C, D, E and F represent control
(untreated) and cells treated with 2, 4, 6, 8 and 10 lM of 1a,
respectively. The percentage of cell population in different phases
were obtained using MODFIT LT software and the mean % cell popula-
tion with standard deviation at each phase obtained from three
independent experiments were shown in Table 5. The p-values
were calculated at each dose with respect to untreated control
and shown within bracket as shown in Table 5.
There was a dose-dependent decrease of G0/G1 cell population
and dose-dependent increase of G2/M population compared with
the control. Such decrease and increase in the respective cases
was not significant at 2 lM but significant at 4 lM and its higher
doses. Importantly, the S-phase cell population increased signifi-
cantly (p = 0.000) at 4 lM but at higher doses (6–10 lM) decreased
significantly (p = 0.000) with respect to control. This result indicated
that the complex 1a arrested the cells in G2/M phase at and above
6 lM concentration so that no more cells were coming from G2/M
to G0/G1 and subsequently S-phase. That’s why population in
S-phase and G2/M phase decreased significantly at and above

Fig. 9. (A) and (C) represent HeLa cells stained with 2 lM of 1a at 0 and 30 min exposure and photographed under fluorescence microscope in automatic mode. (B) and (D)
images taken under light microscope with the same field corresponding to (A) and (C), respectively. (E) represents HeLa cells stained with Hoechst dye under fluorescence
microscope.
2760 S. Mukherjee et al. / Inorganica Chimica Acta 363 (2010) 2752–2761
6 lM concentration. This result corroborate with the LD50 value
which is 5.44 lM obtained from cell viability assay.
3.7.3. Cell staining by 1a in PBS
Interestingly the complex 1a exhibits cell staining ability which
is relatively scarce among the first transition metal complexes [64].
The complex 1a can enter into the cells upon incubation in dark at
the sub-lethal dose of 2 lM and the cells were visualized under
fluorescence microscope with UV excitation. The staining ability
of 1a increases gradually with the exposure time (0–30 min) of
UV light under fluorescence microscope. The reason may probably
due to the auto increment phenomenon of 1a upon UV excitation
(vide infra). Typical pictures at 0 and 30 min exposure were shown
in Fig. 9A and C. The same slides were observed under light micro-
scope and shown in Fig. 9B and D, respectively. A typical picture of
HeLa cells stained with Hoechst dye, commonly used in cell biol-
ogy is shown as control (Fig. 9E) for comparison.
4. Conclusion
A new blue luminescent dinuclear copper(II) complex
[CuII2(HL)2(l-Cl)2]2H2O, 1a has been successfully designed and
synthesized with the ligand HL, 1. The complex 1a exhibits quasi-
reversible one-electron oxidation in aqueous medium. DFT and
TDDFT studies indicate the spectral transition of the ligand and
the complex. The molecular structure of 1a shows that it is a cen-
trosymmetric dichlorido-bridged copper(II) complex with a
[Cu2(l-Cl)2] core unit. The water molecule present in the crystal
lattice undergoes strong intermolecular hydrogen bonding to form
a one dimensional polymeric structure. The absorption, emission
characteristics and cyclic voltammetric studies indicate the inter-
action of 1a with DNA. The complex shows cytotoxic effect with
HeLa cells and the cell cycle was arrested at the G2/M phase. Be-
sides, the complex has the unique property of cell staining at
sub-lethal dose. Further studies in this field are in progress.
Acknowledgements
Financial support received from UGC, New Delhi, CSIR, New
Delhi and DST-FIST, New Delhi are gratefully acknowledged. We
are grateful to the University of Kalyani for providing infrastructur-
al facilities.
Appendix A. Supplementary material
CCDC 712057 contains the supplementary crystallographic data
for [CuII2(HL)2(l-Cl)2]2H2O. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif. Detailed information re-
lated to Cyclic Voltammetry, DFT and TDDFT studies, Cell viability
and morphology are given in the supplementary section. Supple-
mentary data associated with this article can be found, in the on-
line version, at doi:10.1016/j.ica.2010.04.026.
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