Photocytotoxic ternary copper(II) complexes of histamine Schiff base and pyridyl ligands

Article abstract of DOI:10.1007/s12039-015-1017-5

Ternary copper(II) complexes of salicylaldehyde-histamine Schiff base (HL) and pyridyl ligands, viz. [Cu(bpy)(L)](ClO₄) (1) and [Cu(dppz)(L)](ClO₄) (2), where bpy is 2,2 ′ ^{prime } -bipyridine (in 1) and dppz is dipyrido[3,2-a:2 ′ ^

Full text of DOI:10.1007/s12039-015-1017-5

c
J. Chem. Sci. ꢀ Indian Academy of Sciences.
DOI 10.1007/s12039-015-1017-5
Photocytotoxic ternary copper(II) complexes of histamine Schiff base
and pyridyl ligands
SAMYA BANERJEEa, AKANKSHA DIXITb, K SESHA MAHESWARAMMAa,
BASUDEV MAITYa, SANJOY MUKHERJEEa, ARUN KUMARa, ANJALI A KARANDEb,∗
and AKHIL R CHAKRAVARTYa,∗
aDepartment of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560 012, India
bDepartment of Biochemistry, Indian Institute of Science, Bangalore 560 012, India
e-mail: anjali@biochem.iisc.ernet.in; arc@ipc.iisc.ernet.in
MS received 19 October 2015; accepted 21 November 2015
Abstract. Ternary copper(II) complexes of salicylaldehyde-histamine Schiff base (HL) and pyridyl ligands,
viz. [Cu(bpy)(L)](ClO4) (1) and [Cu(dppz)(L)](ClO4) (2), where bpy is 2,2ꢁ-bipyridine (in 1) and dppz is
dipyrido[3,2-a:2ꢁ,3ꢁ-c]phenazine (in 2), were synthesized, characterized and their DNA binding, photo-activated
DNA cleavage activity and photocytotoxicity studied. The 1:1 electrolytic one-electron paramagnetic com-
plexes showed a d-d band near 670 nm in aqueous DMF (1:1 v/v). The crystal structure of complex 1 showed
the metal in CuN4O distorted square-pyramidal geometry. Complex 2 intercalatively binds to calf-thymus (ct)
DNA with a binding constant (Kb) of ∼105 M−1. It exhibited moderate chemical nuclease activity but excellent
DNA photocleavage activity in red light of 647 nm forming •OH radicals. It showed remarkable photocytotox-
icity in human cervical cancer cells (HeLa) giving IC50 of 1.6 μM in visible light (400-700 nm) with low dark
toxicity. The photo-induced cell death is via generation of oxidative stress by reactive oxygen species.
Keywords. Copper; dipyridophenazine; medicinal chemistry; photocytotoxicity; apoptosis.
1. Introduction
efficacy and to overcome the intrinsic resistance of the
platinum based drugs by photo-activating platinum(IV)
prodrugs specifically within the target cancer cells, thus
not damaging the unexposed normal cells.1,3,20,21 A new
model in PDT is reported for an azopyridine manganese
complex [Mn(azpy)Br(CO)3] showing release of cyto-
toxic carbon monoxide (CO) on exposure to low energy
visible light leading to cellular apoptosis.22 Dunbar,
Turro and co-workers have reported various polypyridyl
ruthenium(II) complexes having photoactive terpyri-
dine and biologically active 5CNU moieties (5CNU,
5-cyanouracil) that are photocytotoxic upon visible
light irradiation.23,24 We have recently reported several
metal-based photo-chemotherapeutic agents which
show efficient DNA photocleavage and photocytotoxic
activity in various cancer cells in visible or near-IR
red light.25–30 In addition, dichloro-oxovanadium(IV)
complexes are shown to form photo-induced ds-DNA
crosslinks resulting apoptotic cell death.31
Photoactive metal complexes with their tunable coordi-
nation geometry, versatile redox and spectral properties
are of current interest as the non-porphyrinic metal-based
photochemotherapeutic agents for cancer treatment.1–7
Such metal complexes can be selectively activated by low
energy visible light to display cytotoxicity in the cancer
cells, while remaining non-cytotoxic in the dark. They
act as potential alternatives to the FDA approved photo-
R
ꢀ 8–12
dynamic therapy (PDT) drug Photofrin
.
PDT
as a promising therapeutic methodology has received
considerable attention due to its non-invasive nature,
reduced side effects, and for its tumor selective mode of
action leaving the normal cells unaffected.13–16 The or-
ganic PDT agents on photoactivation in red light gene-
rally form cytotoxic singlet oxygen (1O2) as the reactive
oxygen species via type-II pathway.14,15 The porphyrin
dyes are known to lead skin irritation and hepatotoxicity
due to formation of bilirubin on oxidative degradation.14,16
In contrast, redox active 3d-metal complexes could show
visible light induced photocytotoxicity by an alter-
nate photo-redox pathway forming hydroxyl radicals as
the reactive oxygen species (ROS).17–19 Sadler et al.
have recently developed a new strategy to increase the
The present work stems from our interest to design
new copper(II) complexes that are capable of cleaving
DNA in near IR red light and show visible light induced
photocytotoxicity with low dark toxicity. Copper is
a bioessential element and copper(II) complexes are
reported as anticancer agents and extensively used as
synthetic nucleases.2,32 Further, copper(II) complexes
∗For correspondence

Samya Banerjee et al.
showing a low energy metal centered d-d band within Aldrich, USA) and used as such without any further
purification. Solvents used were purified by standard
procedures.35 Calf thymus (ct) DNA, Dulbecco’s phos-
phate buffered saline (DPBS), ethidium bromide (EB),
Hoechst 33258, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT), 2ꢁ,7ꢁ-dichlorofluore-
sceindiacetate (DCFDA), agarose (molecular biology
grade), distamycin, catalase, superoxide dismutase
(SOD), 2,2,6,6-tetramethyl-4-piperidone (TEMP), pro-
pidium iodide (PI), Dulbecco’s modified eagle medium
(DMEM) and fetal bovine serum (FBS) were pur-
chased from Sigma (USA). Tris-(hydroxymethyl)ami-
nomethane-HCl (Tris-HCl) buffer was prepared using
deionized and sonicated triple distilled water using a
quartz water distillation setup. The N, N,-donor hetero-
cyclic base, viz. dipyrido[3, 2-a:2ꢁ, 3ꢁ-c]phenazine (dppz),
was synthesized following the literature procedures.36
Tetrabutylammonium perchlorate (TBAP) was prepared
from tetrabutylammonium bromide and perchloric acid.
The elemental analyses were carried out using a
Thermo Finnigan Flash EA 1112 CHNS analyser. The
infrared (IR) and electronic spectra were recorded using
Bruker Alpha and Perkin-Elmer Spectrum 650 spectro-
photometers, respectively, at 25◦C. Molar conductivity
measurements were made using a Control Dynamics
(India) conductivity meter. Electrospray ionization (ESI)
mass spectral measurements were done using Agilent
6538 Ultra high definition (UHD) accurate Mass-Q-
the PDT spectral window of 600-800 nm are suitable
for PDT applications with their excellent DNA photo-
cleavage activity in near IR light. Herein, we present the
synthesis, characterization, DNA binding, DNA pho-
tocleavage activity and visible light-induced photocy-
totoxicity of two new ternary copper(II) complexes,
viz., [Cu(bpy)(L)](ClO4) (1) and [Cu(dppz)(L)](ClO4)
(2), where bpy is 2,2ꢁ-bipyridine (in 1), dppz is
dipyrido[3,2-a:2ꢁ, 3ꢁ-c]phenazine (in 3) and L is the
monoanionic form of salicylaldehyde-histamine Schiff
base (figure 1). Complex 1 is structurally characterized
by X-ray crystallography. The planar phenazine moiety
in 2 is expected to show dual activity with its intercala-
tive DNA-binding and excellent photosensitizing abil-
ities. We have used the salicylaldehyde Schiff base of
histamine considering its inherent anti-tumorigenesis
activity.33,34 The planar polypyridyl ligands are well
known for their intercalative DNA binding, photoac-
tivity and preferential nuclear uptake.8,9,19,25 Signifi-
cant results of this study include remarkable DNA-
photocleavage activity in red light involving hydroxyl
radicals (·OH) and visible light induced photocytotoxi-
city of [Cu(dppz)(L)](ClO4) (2) in HeLa cells with low
cytotoxicity in the dark. The cell death is found to be via
apoptotic pathway involving photo-induced generation
of intracellular reactive oxygen species (ROS).
1
TOF (LC-HRMS) model mass spectrometer. H NMR
spectra were recorded at room temperature on a
Bruker 400 MHz NMR spectrometer. Flow cytometric
analysis was performed using FACS Calibur (Becton
Dickinson (BD) cell analyzer) at FL1 channel (595 nm).
2. Experimental
2.1 Materials and Methods
All the chemicals and reagents were purchased from
various commercial sources (SD Fine Chemicals, India;
2.2 Synthesis of the complexes
The complexes were prepared by following a general
synthetic procedure. A mixture of histamine dihydro-
chloride (0.184 g, 1 mmol) was initially reacted with tri-
ethylamine (0.202 g, 2 mmol) under stirring in MeOH
(15 mL) for 15 min and then salicylaldehyde (0.122 g,
1 mmol) was added slowly. The resulting mixture was
heated to reflux for 1h with continuous stirring. The
color of the solution changed to bright yellow indicat-
ing formation of salicylaldehyde-histamine Schiff base.
In a separate flask, copper(II) nitrate (0.24 g, 1 mmol)
was dissolved in 15 mL aqueous MeOH (1:4 v/v). A
methanol solution (10 mL) of 2,2ꢁ bypyridyl (0.156 g,
1 mmol) for complex 1 or dppz (0.282 g, 1 mmol) for
complex 2 was added slowly into the copper(II) nitrate
solution. The resulting blue colored solution was stirred
for 1h at room temperature. The solution was cooled
Figure 1. Schematic diagram of the copper(II) complexes
1 and 2 and the ligands used.

Photocytotoxic ternary copper(II)...
to 0◦C using ice bath and the Schiff base solution
the molecule was obtained using ORTEP.39 CCDC-
1428741 contains the crystallographic data for this arti-
cle. The crystallographic parameters are listed in table 1.
was added slowly with continued stirring for 1 h. The
solution turned to green color due to the formation of
ternary complexes. Sodium perchlorate was added to
this solution under stirring for another 15 min. The com-
plex was precipitated out as a green solid which was
collected by filtration, washed with cold methanol and
diethyl ether and finally dried in vacuum over P4O10.
2.4 Theoretical study
The energy optimised structures of the complexes 1
and 2 were obtained by density functional theory
(DFT) calculation.40 The hybrid U3LYP functional and
LANL2DZ basis set were used in all calculations as
incorporated in Gaussian 09 package.41 Visualizations
of the optimized structures and the MOs were done
using Gauss view 5.0. Frequency test was performed to
ascertain the stationary points.
2.2a [Cu(bpy)(L)](ClO4)(1): [Yield: 78%] Analysis:
Calculated for C22H20ClCuN5O5: C, 49.54; H, 3.78; N,
13.13%. Found: C, 49.83; H, 3.86; N, 13.10%. ESI-MS
inMeCN:m/z 433.0978[M–ClO4]+. IR / cm−1:3360 m,
1626 vs, 1532 m, 1444 m, 1354 s, 1091 vs, 763s, 625 w
(vs, very strong; s, strong; m, medium; w, weak; br,
broad). UV-visible in 50% aqueous DMF [λmax/nm
(ε/ M−1 cm−1)]: 673 (65), 361 (3400), 273 (15700).μeff
at 298K: 1.84 μB. Conductivity in DMF (ꢀM): 72 S
cm2M−1.
2.5 DNA binding experiments
The experiments were done using calf thymus (ct) DNA
in Tris-HCl/NaCl buffer (5 mM Tris-HCl, 5 mM NaCl,
pH 7.2) using DMF solution of the complexes at room
temperature following the reported procedures.8,9,17,19
The intrinsic equilibrium binding constant (Kb) of 1
and 2 to ct-DNA were obtained by McGhee-von Hippel
2.2b [Cu(dppz)(L)](ClO4)(2): [Yield: 72%] Analysis:
Calculated for C30H22ClCuN7O5: C, 54.63; H, 3.36; N,
14.87%. Found: C, 54.37; H, 3.47; N, 14.95%. ESI-
MS in MeCN: m/z 559.1211 [M–ClO4]+. IR / cm−1:
3425 m, 2918 br, 1621 vs, 1537 w, 1458 s, 1350 w,
1095 vs, 759 w, 615 w. UV-visible in 50% aqueous
DMF [λmax/nm (ε/ M−1 cm−1)]: 658 (130), 377 (13000),
360 (14000), 272 (58000). μeff at 298K: 1.81 μB. Con-
ductivity in DMF (ꢀM): 78 S cm2M−1.
Table 1. Selected crystallographic data for complex 1.
Formula
C22H20ClCuN5O5
533.42
Fw (g M−1
)
Crystal system
Space group (no.)
a (Å)
Monoclinic
P21/c
11.6846(11)
17.9514(16)
11.0165(10)
90.0
98.215(2)
2287.1(4)
4
293(2)
1.595
0.71073
1.117
2.3 X-ray Crystallography
b (Å)
c (Å)
The crystal structure of [Cu(bpy)(L)](ClO4)(1) was
obtained by the single-crystal X-ray diffraction method.
The green crystals were isolated from an acetonitrile
solution on slow evaporation of the solvent. Crystal
mounting was done on a glass fiber with epoxy cement.
All geometric and intensity data were collected at room
temperature using an automated Bruker SMART APEX
CCD diffractometer equipped with a fine-focus 1.75
kW sealed tube Mo-Kα X-ray source (λ = 0.71073 Å)
α = γ (◦ )
β (◦ )
V (Å3)
Z
T (K)
ρcalc (g cm−3
)
λ (Å)(Mo Kα)
μ (mm−1
F(000)
)
1092
65193
5272
3302
311/ 0
1.074
0.0566
0.1303
Reflections collected
with increasing ω (width of 0.3◦ per frame) at a scan Unique reflections
Reflections with I>2σ(I)
speed of 5 sec per frame. Intensity data, collected using
ω-2θ scan mode, were corrected for Lorentz-polari-
zation effects and absorption.37 Structure solution was
done by the combination of Patterson and Fourier tech-
niques and refined by full-matrix least-squares method
using the SHELX programs.38 Hydrogen atoms of the
complex were placed in their calculated positions and
refined using a riding model. The non-hydrogen atoms
parameters/restraints
Goodness-of-fit on F2
R(Fo)a [I >2σ(I)]
wR (Fo)b[I >2σ(I)]
R [all data] (wR [all data])
0.1079 (0.1598)
0.568, −0.426
Largest difference in peak and hole [e. Å−3
]
aR = ꢁ ||Fo| − |Fc||/ꢁ|Fo|. wR = {ꢁ[w(Fo2 − F2c)2]/
b
ꢁ[w(Fo)2]}1/2; w = [σ2(Fo)2 + (AP)2 + BP]−1; where P =
were refined anisotropically. The perspective view of (Fo2 + 2Fc2)/3. CCDC number: 1428741.

Samya Banerjee et al.
(MvH) method using the expression of Bard et al. Meter (Model 407A). The solution path length used for
by monitoring the change of the absorption intensity illumination in the glass vial was ∼5 mm. Mechanis-
of the spectral band with increasing concentration of tic investigations were done using various reactive oxy-
ct-DNA.42,43 DNA thermal denaturation experiments gen species (ROS) quenchers/scavengers to ascertain
were carried out by monitoring the absorbance of ct- formation and nature of the ROS.
DNA (190 μM) at 260 nm by varying the temperature
from 40 to 90◦C, both in the presence and absence of
2.7 Cytotoxicity from MTT assay
the complexes (20 μM) using a Perkin-Elmer Lambda
650 UV/Vis spectrometer with a temperature controller
The cytotoxicity of the complexes was studied using
at an increase rate of 0.5◦C per min of the solu-
MTT assay in light and dark conditions in human
tion. The viscosity titrations were done using a Schott
cervical carcinoma (HeLa) cells following reported
Gerate AVS 310 automated viscometer attached with
procedures.17,19 The MTT assay method is based on the
a constant temperature bath at 37( 0.1)◦C following
ability of mitochondrial dehydrogenases of viable cells
the experimental procedure reported earlier.8,9,17,19 The
to cleave the tetrazolium rings of MTT, forming dark
purple membrane impermeable crystals of formazan
that can be estimated from the spectral measurements
concentration of ct-DNA stock solution was 150 μM
(NP) in 5 mM Tris-HCl buffer. The complex was added
gradually so that the concentration of the complexes
in DMSO at 540 nm.46 Photo-irradiation was done with
varied from 0 to 120 μM. The apparent DNA bind-
a broad band visible light (400−700 nm, 10 J cm−2)
ing constants were obtained from fluorescence spectral
using Luzchem Photoreactor (Model LZC-1, Ontario,
measurements. Ethidium bromide (EB), a known DNA
Canada, Sylvania fluorescent white tubes with a fluence
intercalator, did not show any emission in the Tris-HCl
rate of 2.4 mW cm−2 to provide a total dose of 10 J
buffer due to solvent quenching. In presence of duplex
cm−2). The absorbance was measured at 540 nm using a
DNA EB showed red emission at 601 nm on excitation
Molecular Devices Spectra Max M5 plate reader. Cyto-
at 546 nm due to intercalation. Hence, the fluorescence
toxicity of the complexes was measured as the per-
intensity of EB bound to ct-DNA was monitored upon
centage ratio of the absorbance of the treated cells to
addition of the complex. On increasing the complex
the untreated controls. The cytotoxicity was expressed
concentration the fluorescence intensity of EB showed
in terms of the IC50 values which were determined by
a gradual decrease in intensity due to its displacement
nonlinear regression analysis using GraphPad Prism 5.
by the complex.44,45 The apparent DNA binding con-
stants (Kapp) of the complexes were obtained using the
2.8 DCFDA assay for ROS generation
equation: Kapp × [complex]50 = KEB × [EB], where
[complex]50 is the concentration of the complex at 50%
reduction of the fluorescence intensity, KEB = 1.0 × 107
DCFDA assay was carried out to detect any genera-
tion of intracellular ROS.47 Cell permeable DCFDA
M
−1 and [EB] = 2 μM.
on oxidation by cellular ROS generates green fluores-
cent 2ꢁ, 7ꢁ-dichlorofluorescein (DCF) with an emission
maxima around 525 nm.48 To detect intracellular ROS
generation, HeLa cells were incubated with 2 μM of
complex 2 for 4 h followed by photo-exposure to visible
light (400-700 nm) for 1 h in PBS (50 mM phosphate
buffer, pH, 7.2 containing 150 mM NaCl). After har-
vesting the cells by trypsinization, a single cell suspen-
sion of 1×106 cells mL−1 was prepared. The cells were
then treated with 10 μM DCFDA in dark for 15 min at
room temperature. The distribution of the HeLa cells,
stained by DCFDA, was determined by FACS analysis.
2.6 DNA Cleavage Experiments
The DNA cleavage activity of the complexes was stud-
ied in presence of external reducing agents to ascer-
tain the chemical nuclease activity and in presence of
light in Tris-HCl buffer medium using SC pUC19 DNA
(0.2 μg, 30 μM, 2686 base pairs) by agarose gel elec-
trophoresis in 50 mM Tris-HCl buffer of pH 7.2 and
50 mM NaCl containing 10% DMF following reported
procedures.8,9,17,19 The chemical nuclease activity was
studied using glutathione in its reduced form (1 mM).
The photo-induced DNA cleavage experiments were
carried out in the red light of 647 nm (50 mW)
using a continuous-wave (CW) Ar-Kr laser (laser beam
2.9 Annexin-V FITC and Propidium Iodide (PI) Assay
diameter = 1.8 mm, beam divergence = 0.70 mrad, To investigate the pathway of cell death, HeLa cells (4 ×
Spectra Physics Water-Cooled Mixed-Gas Ion Laser 105 cells mL−1) were incubated with complex 2 (2 μM)
R
ꢀ
Stabilite 2018-RM). The power of the laser beam in 10% DMEM for 4 h, followed by irradiation by vis-
was measured using Spectra Physics CW Laser Power ible light of 400-700 nm for 1 h. The cells were then

Photocytotoxic ternary copper(II)...
cultured for 12 h, harvested and washed with chilled region. The complexes showed their n → π∗ transition
PBS at 4◦C. The cells were then re-suspended in due to the presence of histamine moiety at ∼360 nm.
100 μL Annexin-V binding buffer (100 mM HEPES/ The dppz complex 2 showed an additional n → π∗ tran-
NaOH, pH 7.4 containing 140 mM NaCl and 2.5 mM sition at 377 nm due to the phenazine moiety (figure
CaCl2), stained with Annexin-V FITC and PI, and 2).19,25 The redox active complexes showed only reduc-
incubated for 15 min in dark. A 400 μL of binding tive cyclic voltammetric responses involving the metal
buffer was added to the cells after incubation, and flow centre and the polypyridyl bases in DMF-0.1 M TBAP.
cytometry was done for analysis.49
A quasi-reversible reduction peak observed near 0.0 V
for 1 and at –0.1 V for 2 is due to Cu(II)-Cu(I) couple.
Complex 2 also showed dppz based ligand reduction
peaks at –1.16 and –1.69 V vs. SCE.
3. Results and Discussion
3.1 Synthesis and characterization
3.2 Crystal structure
The ternary copper(II) complexes [Cu(bpy)(L)](ClO4)
(1) and [Cu(dppz)(L)](ClO4) (2), where bpy is
2,2ꢁ-bipyridine (in 1), dppz is dipyrido[3,2-a:2ꢁ,3ꢁ-
c]phenazine (in 2) and L is monoanion of a Schiff base
derived from salicylaldehyde and histamine, were pre-
pared in good yield from the reaction of the Schiff base
ligand with copper(II) nitrate and the pyridyl base in
methanol. The complexes were isolated as their per-
chlorate salt and were characterized from the analyti-
cal and physicochemical data that are given in table 2.
The schematic views of the complexes and the ligands
are shown in figure 1. The major peak observed in
the ESI-MS spectra was assigned to the [M–ClO4]+ in
acetonitrile. The complexes are 1:1 electrolyte giving
molar conductance of ∼75 S m2 M−1 in DMF at 25◦C.
They are one-electron paramagnetic with effective mag-
netic moment values of ∼1.81 μB. The IR peaks at
∼1090 cm−1 and ∼1620 cm−1 are assigned to the per-
chlorate anion and the coordinated C=N moiety.50 The
electronic absorption spectra of the complexes in 50%
aqueous DMF showed the d-d band at ∼660 nm.
The ligand centered absorption bands were in the UV
Complex 1 was structurally characterized by single
crystal X-ray diffraction. It crystallizes in P 21/c space
group of the monoclinic crystal system with four
molecules per unit cell. The molecular structure of
Figure 2. The electronic absorption spectra of the com-
plexes 1(· · · ) and 2 (—) in 50% aqueous DMF. The inset
shows the Cu(II) centered d–d bands of the complexes in the
near-IR region.
Table 2. Selected physicochemical and ct-DNA binding data of the complexes
1 and 2.
1
2
IRa (cm−1) [U(C=N)]
Electronicb: λ(nm) (ε / M−1 cm−1
μceff (μB)
1626
673 (65)
1.84
1621
658 (130)
1.81
¯
)
ꢀdM (S cm2M−1
)
72
–0.05
78
–0.1
Efe (V)
Kbf(M−1
)
1.5( 0.2) × 104
1.3( 0.3) × 105
ꢂTmg(◦C)
1.4
5.1
Kahpp(M−1
)
5.0( 0.5) × 105
1.02( 0.2) × 107
b
c
aIn KBr phase. Visible electronic spectral band in 50% aqueous DMF. Magnetic
moment of solid sample at 298 K. dꢀM, molar conductance in DMF at 25◦C. eCathodic
peak potential in DMF having 0.1 M TBAP as the supporting electrolyte at a scan rate
of 50 mVs−1. fEquilibrium ct-DNA binding constant determined from the UV-Visible
absorption titration. Change in the ct-DNA melting temperature. Apparent ct-DNA
binding constants determined from the EB displacement assay.
g
h

Samya Banerjee et al.
complex 1 showed a discrete mononuclear penta- and the bpy showing tridentate and bidentate mode of
coordinated axially elongated square-pyramidal CuN4O
coordination geometry (τ = 0.13) with the Schiff base
binding to the copper(II) centre. An ORTEP view of
the cationic complex is shown in figure 3 and selected
bond distances and angles are given in table 3. The
equatorial Cu-O bond length is 1.903(3) Å. The Cu-N
bond distances in the basal plane are in the range
of 1.957(3) to 2.015(3) Å. The axial Cu(1)-N(1)
bond distance of 2.227(4) Å is significantly longer
in this distorted square-pyramidal geometry. The bpy
ligand shows axial-equatorial bidentate chelating mode
of binding to the copper(II) centre. That leaves the tri-
dentate Schiff base binding to the metal in a meridional
fashion in the basal plane.
3.3 Theoretical study
Figure 3. An ORTEP view of the cationic complex of 1
with atom labeling scheme for the metal and heteroatoms
showing 50% probability thermal ellipsoids. The hydrogen
atoms are not shown for clarity.
Computational studies were performed to rationalize
the photophysical properties of the complexes. The
energy optimized structures of the complexes showed
that the copper(II) centre is coordinated to the chelat-
ing N,N-donor bpy (in 1) or dppz (in 2), O,N,N-
donor HL giving a distorted square pyramidal CuIIN4O
coordination geometry as observed in the crystal struc-
ture of complex 1 (figure 4). The bonding parameters
are similar to those obtained from the crystal structure.
The FMOs (frontier molecular orbitals) of the com-
plexes showed active participation of the metal centre
and the Schiff base moiety in forming the HOMOs and
contribution of the bpy or dppz ligand in forming the
LUMOs (figure 4).
Table 3. Selected bond distances (Å) and angles (◦) for
the bpy complex 1 with e.s.d.s in the parentheses.
Cu(1)–O(1)
Cu(1)–N(1)
Cu(1)–N(2)
Cu(1)–N(3)
Cu(1)–N(4)
1.903(3) O(1)–Cu(1)–N(4) 150.06(15)
2.227(4) N(1)-Cu(1)-N(2) 76.79(14)
2.015(3) N(1)-Cu(1)-N(3) 97.72(14)
1.957(3) N(1)-Cu(1)-N(4) 98.42(14)
1.996(3) N(2)–Cu(1)–N(3) 173.84(14)
O(1)-Cu(1)-N(1) 109.49(14) N(2)–Cu(1)–N(4) 90.53(13)
O(1)–Cu(1)–N(2) 85.65(14) N(3)–Cu(1)–N(4) 93.07(14)
O(1)–Cu(1)–N(3) 93.68(14)
Figure 4. The energy optimized structure of complex 2 along with the FMOs
[color codes: C black, N green, O blue, Cu red, and H white].

Photocytotoxic ternary copper(II)...
denaturation studies in phosphate buffer (pH = 7.2)
3.4 DNA binding properties
showed stabilizing interaction of the complexes with
duplex ct-DNA (figure 5(b)). The change in DNA
melting temperature with respect to free duplex ct-
DNA (ꢂTm) for the complexes 1 and 2 were 1.4 and
5.1◦C, respectively, while it is 10.6◦C for ethidium bro-
mide (EB).9 The DNA melting data indicate partial
intercalative mode of binding of the dppz complex 2.
A similar order is also observed from the viscosity
titration experiments. Hoechst dye as a groove binder
and EB as a DNA intercalator were used as standards.
The plots of (η/η0)1/3 vs. [complex]/[DNA] ratio show
partial intercalative binding mode of the dppz com-
plex 2 to ct-DNA, whereas complex 1 showed primarily
groove binding propensity (figure 6(a)). The apparent
DNA binding constants (Kapp) of the complexes were
determined using EB displacement assay in Tris-HCl
The equilibrium binding constant (Kb) of the complexes
to ct-DNA were determined by UV-visible absorption
titration method. The changes in the absorption spec-
tra of the complexes were monitored on addition of
the ct-DNA solution. The complexes showed signif-
icant hypochromicity at ∼270 nm upon addition of
DNA. The spectral changes for complex 2 are shown in
figure 5(a). The Kb value of the dppz complex is
1.3( 0.3) × 105 M−1 whereas it is 1.5( 0.2) × 104M−1
for the bpy complex 1. The dppz complex shows higher
DNA binding strength than its bpy analogue due to
presence of planar aromatic phenazine moiety. This is
in accordance with the reported results showing high
DNA binding strengths for the complexes having lig-
ands with extended planarity.9,17,19,25 DNA thermal
Figure 5. (a) Spectral traces showing the effect of gradual addition of ct-
DNA (210 μM NP) to the 20 μM solution of complex 2 in Tris-HCl buffer
medium. Inset shows the plot of ꢂεaf/ꢂεbf vs. [ct-DNA] for the calculation
of binding constant. (b) Thermal denaturation plots for 190 μM ct-DNA alone
and in the presence of complexes 1 and 2 (20 μM) in 5mM phosphate buffer at
pH = 7.2.
Figure 6. (a) Effect of increasing the concentration of the complexes 1(ꢀ), 2 (ꢁ), ethidium
bromide (EB,ꢂ) and Hoechst 33258 (•) on the relative viscosities of 150 μM ct-DNA in 5
mM Tris-HCl buffer at 37.0( 0.1)◦C. (b) The spectral traces showing the decrease of emis-
sion intensity of ct-DNA bound EB with the increasing concentration of the complex 2 in 5
mM Tris-HCl buffer medium. The inset shows effect of addition of increasing concentration
of the complexes 1 (ꢂ) and 2 (ꢁ) to an EB bound ct-DNA solution in a 5 mM Tris-HCl buffer
(pH, 7.2) at 25◦C.

Samya Banerjee et al.
buffer. EB is non-fluorescent in Tris-HCl due to solvent
quenching, but in presence of DNA it emits strong red
fluorescence with emission maxima at 601 nm due to
intercalation of EB between the DNA base pairs.44,45
Addition of the complex solution into the EB bounded
DNA showed displacement of EB from the ct-DNA
with a reduction of its emission intensity. This emis-
sion band was used as a probe in determining the com-
petitive DNA binding affinity of the complexes. The
effect of addition of complex 2 into the DNA-bound EB
is shown in figure 6(b). The Kapp values for the com-
Figure 7. Gel electrophoresis diagram showing the red
light-induced (647 nm) DNA cleavage activity of the com-
plexes 1 and 2 using SC pUC19 DNA (0.2 μg, 30 μM) for
a photo-exposure time of 1 h: lane-1, DNA control; lane-2,
plexes 1 and 2 are 5.0( 0.5) × 105M−1and 1.02( 0.2) DNA + 1 (20 μM) in the dark; lane-3, DNA + 1 (20 μM);
× 107M−1, respectively. The dppz complex 2 having
the planar aromatic moiety exhibits the higher DNA
binding affinity compared to its bpy analogue.
lane-4, DNA +2 (7.5μM); lane-5, DNA + 2 (7.5μM) in the
dark.
3.5 DNA cleavage activity
The chemical nuclease activity of the complexes was
studied in the dark in presence of glutathione (GSH) in
its reduced form (1 mM) as a reducing agent due to the
presence of near zero volt (vs. SCE) Cu(II)-Cu(I) redox
couple. The bpy complex 1 did not show any activity
whereas a 7.5 μM of the dppz complex 2 showed ∼57%
cleavage of the supercoiled (SC) pUC19 DNA into its
nicked circular (NC) form in the presence of GSH. This
could be due to higher DNA binding affinity of the
dppz complex. The mechanistic aspects of the chemi-
cal nuclease activity of 2 were studied in presence of
various ROS quenchers and scavengers. The hydroxyl
radical scavengers like DMSO, KI and catalase inhib-
·
ited the nuclease activity indicating formation of OH
radical as the reactive oxygen species responsible for
the nuclease activity. The singlet oxygen quenchers like
sodium azide and TEMP did not inhibit any cleavage
activity thus ruling out formation of singlet oxygen as
the ROS. The photo-induced DNA cleavage activity of
the complexes was studied using near-IR red light of
647 nm. The choice of this wavelength was based on
the d-d absorption band at ∼660 nm. The complexes
Figure 8. Bar diagram showing the photo-cleavage of
pUC19 DNA (0.2 μg, 30 μM) by complex 2 (7.5 μM) in
the presence of different additives in near-IR light of 647 nm
for an exposure time of 1 h (D2O, 16 μL; NaN3, 0.5 mM;
TEMP, 0.5 mM; DMSO, 4 μL; KI, 0.5 mM; catalase, 4 units;
SOD, 4 units). The + sign indicates treatment of the specified
additive with complex 2 and SC DNA.
were incubated with SC pUC19 DNA (0.2 μg, 30 μM) complex did not show any apparent DNA photocleav-
for 1 h prior to photo-irradiation. The DNA photocleav- age activity in an argon atmosphere thus indicating pos-
age data show that only complex 2 is active in red light sible involvement of ROS. Addition of singlet oxygen
and this could be due to the presence of a photoac- quenchers, viz., NaN3 and TEMP had no apparent
tive phenazine moiety. Complex 2 showed ∼92% cleav- effect on the DNA cleavage activity of the complex.
age of DNA at a concentration of 7.5 μM (figure 7). This ruled out formation of singlet oxygen as the ROS.
The observation of red light induced DNA cleavage is Hydroxyl radical scavengers (catalase, DMSO and KI)
of importance towards developing copper-based photo- showed significant reduction in the DNA photocleavage
chemotherapeutic agents considering the greater tissue activity in red light. Generation of superoxide radical
penetration power of red light.
as intermediate was evidenced from the inhibitory role
The mechanistic aspects of the pUC19 DNA pho- of SOD to the DNA photo-cleavage. The results sug-
tocleavage activity of 2 were studied to explore any gested the formation of both hydroxyl and superoxide
involvement and nature of the ROS (figure 8). The radicals as the ROS in a photo-redox pathway which

Photocytotoxic ternary copper(II)...
is known for the copper(II)–dipyridoquinoxaline (dpq) cytotoxicity on light irradiation or in the dark (IC50
complexes.51,52 To determine the DNA minor or major 100 μM).
>
grove binding preference of complex 2, cleavage exper-
iments were carried out in presence of distamycin as
the DNA minor-groove binder and methyl green as the
DNA major-groove binder. DNA photocleavage by 2
was found to be significantly inhibited in the presence
of methyl green (25 μM), whereas no such reduction in
cleavage activity was observed in the presence of dis-
tamycin (50 μM) indicating DNA major groove binding
preference of the dppz complex 2.
3.7 DCFDA Assay for ROS
We examined any generation of intracellular ROS by
complex 2 on visible light (400-700 nm) exposure in
HeLa cells using DCFDA assay. DCFDA, a cell per-
meable fluorogenic probe, on oxidation by intracellular
ROS generates green fluorescent DCF with an emis-
sion maxima at 525 nm which can be examined by flow
cytometry.47,48 The cells treated with the dppz complex
2 in the dark did not show any significant generation of
DCF. However, cells treated with the complex on expo-
sure to visible light (400-700 nm) showed a significant
increase in the intensity of the intracellular emission
indicating generation of fluorescent DCF via the ROS
induced oxidation of non-fluorescent DCFDA (figure
10). The assay data suggest generation of ROS only on
visible light irradiation with insignificant quantity in the
dark.
3.6 Photocytotoxicity from MTT Assay
The cytotoxicity of the complexes 1 and 2 was studied
in human cervical cancer (HeLa) cells by MTT assay
both in the dark and upon photo-irradiation with visible
light (400-700 nm). A dose-dependent anti-proliferative
activity of the complexes was observed. Photo-exposure
with visible light resulted in an enhanced cytotoxicity
of complex 2 due to the presence of photoactive phe-
nazine moiety. The dppz complex 2 showed ∼10 fold
increase in cytotoxicity in visible light (400-700 nm)
when compared to that in the dark thus giving moderate
PDT effect (figure 9). Importantly, complex 1 which
lacks a photoactive moiety failed to induce any photo-
triggered enhancement of cytotoxicity giving IC50
values of 10.3 0.8 μM in the dark and 6.8 0.4 μM
on light irradiation indicating no apparent PDT effect.
The IC50 values of 2 were found to be 1.2 0.5 μM
with visible light irradiation, while it is 12.8 0.7 μM
in the dark. The dppz ligand gave an IC50 value of
60.1 2.3 μM in visible light under similar experimen-
tal condition. This ligand did not show any apparent
cytotoxicity (IC50 >100 μM) in HeLa cells in the dark.
The Schiff base ligand (HL) also did not show any
3.8 Apoptosis from Annexin V-FITC/PI assay
This assay which provides a convenient way to detect
apoptosis was performed in HeLa cells with the dppz
complex 2. The early stages of apoptosis are marked
by the flipping of phosphatidylserine from the inner
surface of the plasma membrane to the outer surface.
This can be readily detected by a fluorophore con-
jugated annexin V protein which is known to bind
the surface exposed phosphatidylserine.49 The assay is
based on dual staining by green fluorescent annexin V-
FITC (fluorescein isothiocyanate) and propidium iodide
(PI, a red fluorescent DNA binding dye). The cells
Figure 10. The DCFDA assay to detect generation of any
ROS in the HeLa cells on exposure to light (400-700 nm).
Colour code: black, cells only; red, cells + DCFDA; blue,
Figure 9. Photocytotoxicity of complex 2 in HeLa cells on cells + DCFDA + 2 (in dark); green, cells + DCFDA +
4 h incubation in the dark (D) followed by exposure to visible complex 2 (in light). Generation of ROS is marked by the
light (L) of 400-700 nm (10 J cm−2) for 1 h.
shift in the fluorescence intensity.

Samya Banerjee et al.
excellent photocleaver of DNA in red light of 647 nm
by generating hydroxyl radicals as the ROS via a photo-
redox pathway involving the redox active metal center.
Unlike other copper(II) complexes of phenanthroline
bases that are known to show undesirable high chemical
nuclease activity, the dppz copper(II) complex with
moderate chemical nuclease activity is suitable for PDT
applications. Complex 2 displays significant photocy-
totoxicity in HeLa cells upon visible light irradiation,
while remaining less-toxic under darkness. The cell
death upon photo-exposure is via apoptotic pathway
most likely by generation of intracellular ROS. Consid-
ering the bio-essential nature of copper, the presence
of a low-energy d-d band within the PDT spectral win-
dow and the biocompatibility of the ligands used, the
results are of significant importance towards developing
copper-based PDT agents.
Supplementary Information (SI)
Figure 11. FACS analysis of Annexin-V FITC and PI
stained HeLa cells undergoing apoptosis induced by com-
plex 2 under darkness and upon visible light (400-700 nm)
exposure.
All additional information pertaining to the charac-
terization of the complexes using ESI-MS technique
(Figures S1, S2), IR spectra (Figures S3, S4), cyclic
voltammograms (Figure S5), unit cell packing diagram
of complex 1 (Figure S6), energy optimised structure of
1 along with FMO (Figure S7), EB displacement assay
plot for complex 1 (Figure S8), Gel diagram (Figure
S9), MTT assay data of complex 1 (Figure S10) and
coordinates for the energy minimized structures (Tables
S1, S2) are given as supporting information, available
at www.ias.ac.in/chemsci.
undergoing early apoptosis are stained only by annexin
V-FITC, while the late apoptotic cells are dual stained
by both the dyes having a compromised plasma mem-
brane which allows PI entry. The necrotic cell popula-
tion is stained only by PI. To study whether complex
2 could induce visible light-mediated apoptotic cell
death, we stained the HeLa cells that were pretreated
with 2 μM of the complex in the dark as well as in light
with Annexin V-FITC and PI. The results indicated
that complex 2 induced significant apoptosis only after
visible light (400-700) irradiation (figure 11). About
23% population of the cells were in an early apoptotic
stage and ∼13% of the cells were in a late apoptotic
stage. Cell population in the necrotic mode was found
to be negligible. In the dark, the complex did not induce
any significant cell death at this concentration. The data
suggest an overall apoptotic nature of the photo-induced
cell death.
Acknowledgements
We thank the Department of Science and Technology
(DST), Government of India, for financial support
(SR/S5/MBD-02/2007). ARC thanks the DST for J.C.
Bose national fellowship and the Alexander von Hum-
boldt Foundation, Germany, for donation of an electro-
chemical system. S.B. thanks Indian Institute of Science
(IISc) for a postdoctoral fellowship. A.D. thanks the
University Grants Commission (UGC), New Delhi, for
research fellowship. K.S.M. thanks Indian Academy of
Sciences for a summer teacher fellowship. The use of
the FACS facility of the Department of Biotechnology,
Government of India, is gratefully acknowledged.
4. Conclusions
Ternary pyridyl copper(II) complexes of a Schiff base
derived from salicylaldehyde and histamine is shown to
display red light induced plasmid pUC19 DNA cleav-
age activity and visible light assisted photocytotoxicity
in HeLa cancer cells. The complexes with CuN4O
square pyramidal coordination geometry showed inter-
calative ct-DNA binding. The dppz complex 2 is an
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