Photoactivated DNA cleavage and anticancer activity of pyrenyl-terpyridine lanthanide complexes

Article abstract of DOI:10.1016/j.ejmech.2012.02.011

Lanthanide(III) complexes [Ln(R-tpy)(acac)(NO₃)₂] (Ln = La(III) in 1, 2; Gd(III) in 4, 5) and [Ln(py-tpy)(sacac)(NO₃) ₂] (Ln = La(III), 3; Gd(III), 6), where R-tpy is 4′-phenyl-2, 2′:6′,2″-terpyridine (ph-tpy in 1, 4), 4′-(1-pyrenyl)-2, 2′:6′,2″-terpyridine (py-tpy in 2, 3, 5 and 6), acac is acetylacetonate and sacac is 4-hydroxy-6-{4-[(β-d-glucopyranoside)oxy] phenyl}hex-3,5-dien-2-onate, were prepared to study their DNA photocleavage activity and photocytotoxicity. Complexes [La(ph-tpy)(acac)(EtOH)(NO ₃)₂] (1a) and [Gd(ph-tpy)(acac)(NO₃) ₂] (4) were characterized by X-ray crystallography. The 1:1 electrolytic complexes bind to calf thymus DNA. The py-tpy complexes cleave pUC19 DNA and exhibit remarkable photocytotoxicity in HeLa cells in UV-A light of 365 nm with apoptotic cell death (IC₅₀: ～40 nM in light, >200 μM in dark). Confocal microscopy using HeLa cells reveal primarily cytosolic localization of the complexes.

Full text of DOI:10.1016/j.ejmech.2012.02.011

European Journal of Medicinal Chemistry 50 (2012) 319e331
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Photoactivated DNA cleavage and anticancer activity of pyrenyl-terpyridine
lanthanide complexes
,
Akhtar Hussain a, Sudarshan Gadadhar b, Tridib K. Goswami a, Anjali A. Karande b
**
,
,
Akhil R. Chakravarty a
*
a Department of Inorganic and Physical Chemistry, Indian Institute of Science, Sir C.V. Raman Avenue, Bangalore 560012, India
b Department of Biochemistry, Indian Institute of Science, Sir C.V. Raman Avenue, Bangalore 560012, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Lanthanide(III) complexes [Ln(R-tpy)(acac)(NO3)2] (Ln ¼ La(III) in 1, 2; Gd(III) in 4, 5) and [Ln(py
etpy)(sacac)(NO3)2] (Ln ¼ La(III), 3; Gd(III), 6), where R-tpy is 40-phenyl-2,20:60,200-terpyridine (phetpy
in 1, 4), 40-(1-pyrenyl)-2,20:60,200-terpyridine (pyetpy in 2, 3, 5 and 6), acac is acetylacetonate and
Received 16 November 2011
Received in revised form
1 February 2012
Accepted 3 February 2012
Available online 10 February 2012
sacac is 4-hydroxy-6-{4-[(b-D-glucopyranoside)oxy]phenyl}hex-3,5-dien-2-onate, were prepared to
study their DNA photocleavage activity and photocytotoxicity. Complexes [La(ph-tpy)(acac)(E-
tOH)(NO3)2] (1a) and [Gd(phetpy)(acac)(NO3)2] (4) were characterized by X-ray crystallography. The 1:1
electrolytic complexes bind to calf thymus DNA. The pyetpy complexes cleave pUC19 DNA and exhibit
Keywords:
remarkable photocytotoxicity in HeLa cells in UV-A light of 365 nm with apoptotic cell death (IC50
w40 nM in light, >200
localization of the complexes.
:
Medicinal chemistry
Lanthanide complexes
Pyrenyl-terpyridine photosensitizer
Crystal structure
m
M in dark). Confocal microscopy using HeLa cells reveal primarily cytosolic
Ó 2012 Elsevier Masson SAS. All rights reserved.
Photocytotoxicity
Cellular imaging
1. Introduction
PDT agents are primarily porphyrin and phthalocyanine bases. The
mode of action of the organic PDT agents is to generate cytotoxic
3
Metal-based photoactivated chemotherapeutic agents are of
current interests for their potential utility in the photodynamic
therapy (PDT) of cancer [1e4]. PDT has emerged as a new modality
of cancer cure due to its non-invasive nature and for its selectivity
in which the photo-irradiated cancer cells are selectively damaged
leaving the unexposed normal cells intact [5e8]. The currently used
singlet oxygen (1O2) upon photoactivation involving the
(p
-p
*)
state of the photosensitizer and molecular oxygen (3O2) [9e11]. The
major drawbacks of such bases include skin sensitivity and hepa-
totoxicity which leads to jaundice [12,13]. Besides, the potency of an
organic PDT agent depends largely on the high quantum yield of
singlet oxygen generation which is often difficult to achieve thus
making them unsuitable for phototherapeutic applications [11].
These problems could be circumvented using metal-based PDT
agents which could be suitably designed utilizing tunable coordi-
nation geometries, wide spectral and redox properties thus
providing alternate type-I and/or photo-redox pathways to cause
cellular photodamage in addition to the generation of singlet
oxygen species in a type-II process. The 4d and 5d metal-based
anticancer agents, viz., platinum(IV), ruthenium(II) and rhodiu-
m(II) complexes, are reported to display photocytotoxicity in
a variety of cancer cells [14e19].
Abbreviations: AO, acridine orange; CCDC, Cambridge Crystallographic Data
Centre; ct-DNA, calf thymus DNA; DABCO, 1,4-diazabicyclo[2.2.2]octan; DMEM,
Dulbecco’s Modified Eagle’s Medium; DMF, dimethylformamide; DMSO, dimethyl
sulfoxide; DNA, deoxyribonucleic acid; DOTA, 1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetate; dppz, dipyrido[3,2-a:20,30-c]phenazine; DTPA, diethylene-
triaminepentaacetate; EB, ethidium bromide; EDTA, ethylenediaminetetraacetate;
FBS, foetal bovine serum; GLUTs, glucose transporters; ISC, intersystem crossing;
MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide;
MvH,
McGhee-von Hippel; NC, nicked circular; PBS, phosphate buffered saline; PDT,
photodynamic therapy; PI, propidium iodide; RME, receptor-mediated endocytosis;
SC, supercoiled; SOD, superoxide dismutase; TEMP, 2,2,6,6-tetramethyl-4-
piperidone.
Our work on the 3d metal-based complexes has shown that
iron(III) and oxovanadium(IV) complexes are remarkably photo-
cytotoxic in various cancer cells in visible light [20e24]. While
transition metal-based PDT agents are reported from various
research groups, the lanthanide-based PDT agents are virtually
* Corresponding author. Tel.: þ91 80 22932533.
** Corresponding author. Tel.: þ91 80 22932306.
E-mail addresses: anjali@biochem.iisc.ernet.in (A.A. Karande), arc@
ipc.iisc.ernet.in (A.R. Chakravarty).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.011

320
A. Hussain et al. / European Journal of Medicinal Chemistry 50 (2012) 319e331
unknown except few complexes containing macrocyclic organic
dyes [25e27]. For example, a lutetium(III) texaphyrin (LUTRINÒ)
complex is an effective PDT agent in near-IR light of 732 nm [25].
The lanthanide complexes are being used as luminescent tags for
bio-analytical applications [28e30]. Gadolinium(III) complexes,
viz., [Gd(DTPA)(H2O)]2ꢀ (MagnevistÔ) and [Gd(DOTA)(H2O)]ꢀ
(DotaremÔ), are used as magnetic resonance imaging (MRI)
contrast agents [31e33]. The success of the Gd-based MRI agents
provides strong impetus to design new lanthanide complexes and
explore their photochemotherapeutic potentials. The present work
stems from our continued interest to enrich the chemistry of
photocytotoxic Ln(III) complexes using their high coordination
number and oxophilicity [34,35].
We have chosen terpyridine bases as photoactive organic
ligands to achieve high photocytotoxicity in cancer cells. The
Ln(III) complexes are expected to be non-toxic in dark owing to
their redox stability thus making them suitable for cellular
applications in the presence of reducing thiols, viz., glutathione.
We have recently reported the La(III) and Gd(III) complexes
[Ln(dppz)2(NO3)3] and [Ln(dppz)(acac)3] of dipyridophenazine
(dppz) as DNA photocleaving agents showing photocytotoxicity in
HeLa cells on irradiation with UV-A light of 365 nm while
remaining essentially non-toxic in dark [34,35]. We have now
designed and synthesized a series of lanthanide complexes having
pyrene-appended terpyridine and acetlyacetonate as ligands. The
choice of pyrenyl terpyridine (pyetpy) as a ligand is based on the
fact that tpy with a pendant pyrenyl moiety could serve as
Scheme 1. Schematic drawing of the La(III) and Gd(III) complexes 1e6 and the ligands
used.
Significant results of this study include remarkable photocytotoxicity
(PDTeffect) of the pyrenyl terpyridine complexes in HeLa cells in UV-
A light, while being less toxic in the dark. The pyetpy complexes
showed cytosolic localization as evidenced from the confocal fluo-
rescence imaging studies.
2. Chemistry
The substituted terpyridine ligand (phetpy and pyetpy) was
prepared by a reaction of the corresponding aldehyde with 2-
acetylpyridine in the presence of NaOH and subsequent conden-
sation of the intermediate product with ammonium acetate in
a
photosensitizer-cum-DNA binder. The photoactive pyrenyl
moiety, also being a fluorophore, could be used for confocal fluo-
rescence microscopy to study the localization of the complex
within the cancer cell and for its cytotoxicity on photoactivation.
We have also prepared the corresponding phenyl analogues
(phetpy complexes) as controls to understand the role of the
refluxing ethanol [39,40]. The glucose appended
(Hsacac) was prepared in five synthetic steps. -Glucose was acyl-
ated to get -glucose pentaacetate which was subsequently
reacted with Br2/red P to afford the highly reactive -aceto-
bromoglucose. The -acetobromoglucose was reacted with para-
b-diketone ligand
D
b-D
a
pyrenyl moiety in the cellular damage. The O,O-donor b-diketo-
a
nate ligand is chosen for strong complexing nature of the oxophilic
lanthanide ions resulting in the formation of a stable complex. We
have also used a carbohydrate-appended acetlyacetonate ligand to
increase the aqueous solubility of the complexes and to augment
their cancer cell targeting potential. There is an upregulation of
glycolysis and a decrease in the oxidative phosphorylation in
cancer cells compared to the normal cells which results in ineffi-
ciency in the metabolism of cancer cells. Consequently, the glucose
requirements of cancer cells are significantly higher than the
normal cells for their uncontrolled growth and proliferation
resulting in the overexpression of certain proteins known as
GLUTs which form a class of transmembrane proteins mediating
the transport of glucose to the cells [36,37]. We have designed the
present complexes based on our dual strategy of combining
imaging with therapy. In addition, the presence of the nitrate
anions in the complexes would improve their aqueous solubility.
The use of tridentate terpyridine, as compared to the bidentate
phenanthroline bases, has led to higher coordination number of
the complexes thus decreasing the possibilty of any hydrolytic
DNA damage since lanthanides are known to display significant
hydrolytic DNA cleavage activity [38].
hydroxy benzaldehyde in the presence of NaOH using
tetrabutylammonium bromide as the phase transfer catalyst to
obtain the corresponding
reacted with 2,4-pentatedione in presence of boric anhydride using
dry DMF as a solvent to get the acetyl protected -diketone ligand
[41]. Finally, the deprotection of the acetyl group by standard
method afforded the desired glucose appended -diketone ligand
b-glycoside which was subsequently
b
b
(Hsacac) in moderate yield. Lanthanide(III) complexes 1e6 were
prepared by a general method in two synthetic steps. Reaction of
a CH2Cl2 solution of phetpy or pyetpy with an ethanol solution of
the corresponding metal nitrate afforded the precursor complex
[Ln(phetpy)(NO3)3] or [Ln(pyetpy)(NO3)3] in good yield. The
precursor complex upon reaction with Hacac or Hsacac in the
presence of triethylamine gave the desired complex in high yield
(Scheme 1).
3. Pharmacology
The lanthanide(III) complexes 1e6 were evaluated for their DNA
binding, DNA cleavage, and cytotoxic activities. Binding studies for
the complexes with ct-DNA were done using spectroscopic
methods such as electronic absorption titration and DNA melting
and hydrodynamic method, viz., solution viscosity measurement of
complex bound ct-DNA. The DNA cleavage activity of the complexes
was studied by determining the ability of each complex to convert
the supercoiled (SC) form of DNA to its nicked circular (NC) form.
Photocytotoxicity of the complexes was assessed against HeLa cells
by MTT assay [42]. The mechanistic aspects of cell death was
studied by EB/AO dual staining method using fluorescence
microscopy of the HeLa cells in the presence or absence of the
Herein, we report the synthesis, characterization, DNA binding,
DNA cleavage activity and photocytotoxicity of the lanthanide(III)
complexes[Ln(R-tpy)(acac)(NO3)2](Ln¼ La(III),1and2;Gd(III), 4 and
5) and [Ln(pyetpy)(sacac)(NO3)2] (Ln ¼ La(III), 3; Gd(III), 6), where
R-tpy is an N,N,N-donor ligand, viz., 40-phenyl-2,20:60,20’-terpyridine
(phetpy in 1, 4), 40-(1-pyrenyl)-2,20:60,20’-terpyridine (pyetpy in 2,
3, 5 and 6), acac is acetylacetonate and sacac is 4-hydroxy-6-{4-[(
b-
D-glucopyranoside)oxy]phenyl}hex-3,5-dien-2-onate (Scheme 1).
Complexes 1 as [La(pyetpy)(sacac)(EtOH)(NO3)2] (1a) and 4 were
structurally characterized by single crystal X-ray diffraction method.

A. Hussain et al. / European Journal of Medicinal Chemistry 50 (2012) 319e331
321
complexes in UV-A light and in dark. The cellular localization
studies were carried out on HeLa cells by confocal fluoresence
microscopy using the blue light emitting fluorescent pyrenyl
complexes of La(III).
4. Results and discussion
4.1. Synthesis and general aspects
Reaction of a dichloromethane solution of the phetpy or pyetpy
ligand with an ethanol solution of the corresponding metal nitrate
afforded the precursor complex [Ln(phetpy)(NO3)3] or
[Ln(pyetpy)(NO3)3]. The desired complexes 1e6 were prepared by
reacting the corresponding precursor complex with 3 equivalents
of Hacac or Hsacac in the presence of three equivalents of trie-
thylamine as a base (Scheme 1). The complexes were characterized
from the analytical and mass spectral data. Selected physico-
chemical data are given in Table 1. The molar conductivity value of
w105 S cm2 Mꢀ1 suggests 1:1 electrolytic nature of the complexes
in aqueous DMF (1:5 v/v). The nitrate ligand is likely to dissociate
from the complex in a solution phase to form 1:1 electrolyte. This is
further supported from the ESI-MS spectral data in aqueous
methanol or aqueous acetonitrile showing the presence of
a prominent [M-(NO3)]D peak corresponding to the loss of one
nitrate anion. The complexes are thus formulated as [Ln(R-tpy)(a-
cac/sacac)(NO3)2] in the solid state and as [Ln(R-tpy)(acac/sacac)(-
NO3)](NO3) in an aqueous phase (R ¼ ph or py). The IR spectra of
the complexes in the solid state showed three strong bands around
Fig. 1. Absorption spectra of 1e3 in DMF (solid curves). Fluorescence emission spectra
(dotted curves) of 1e3 in 25% aqueous DMSO (excitation wavelengths are: 285 nm for
1 and 350 nm for 2 and 3).
displayed fluorescence spectral band around 350 nm when excited
at 285 nm with respective quantum yield value of 0.04 and 0.02.
The pyetpy complexes in aqueous DMSO (1:4 v/v) exhibited
intense emission band at w420 nm when excited at 350 nm with
fluorescence quantum yields in the range of 0.14e0.22 (Fig. 1). The
phetpy and pyetpy ligands showed similar emission spectral
properties (Table 1) [45]. The emission property of the pyetpy
complexes allowed us to study the cellular internalization of
these complexes by confocal fluorescence microscopy. The py-tpy
complexes were tested for their susceptibility towards phot-
bleaching under UV-A light illumination by monitoring the
decrease (if any) in the emission intensity in the presence of dis-
solved oxygen [46]. The emission intensities were recorded at an
1585, 1515 and 1380 cmꢀ1 corresponding to the C]O, C]C (
diketonate) and NOꢀ3 stretching vibrations, respectively, indicating
b-
bidentate coordination of the b
-diketonate and NOꢀ3 ligands [43].
The 1H-NMR spectra of the diamagnetic La(III) complexes (1e3)
showed characteristic spectral features of the metal-bound poly-
pyridyl ligand and
b-diketonates. The large J value of 15 Hz for the
protons at the 5,6-positions of the coordinated
b
-diketonate sacac
ligand indicates the presence of a conjugated C]C double bond
[44]. Room temperature solution magnetic susceptibility
measurements of the paramagnetic Gd(III) complexes (4e6) in
DMSO-d6 gave magnetic moment value of w8.0 mB suggesting the
presence of seven unpaired electrons. The electronic absorption
spectra of the complexes in DMF displayed an intense ligand cen-
interval of 5 min (upto 60 min) after irradiation of a 10 mM solution
of the complex in aqueous DMSO (1:4 v/v) with UV-A light of
365 nm (100 W lamp). No significant decrease in the fluorescence
intensity of the solution was observed during the time-course
studied. This observation suggests the fact that the pyetpy
complexes do not suffer any photobleaching under intense UV-A
light illumination.
tred
showed an additional band centred at 350 nm assignable to the low
lying intraligand * transitions [40]. The broad band centred at
p / p* transition at w278 nm (Fig. 1). The pyetpy complexes
p
/ p
4.2. Crystal structure
350 nm, observed due to sacac ligand in complexes 3 and 6,
obscures the absorption features arising from the pyetpy ligand
[41]. The phetpy complexes 1 and 4 in aqueous DMSO (1:4 v/v)
Complexes 1 as [La(phetpy)(acac)(EtOH)(NO3)2] (1a) and 4
were structurally characterized by single crystal X-ray diffraction
ꢀ
method. Both the crystals belonged to the triclinic space group P ı.
The ORTEP views of the complexes are shown in Fig. 2. The
complexes are discrete mononuclear species with the La(III) centre
in a ten-coordinate LaO7N3 and Gd(III) centre in a nine coordinate
GdO6N3 coordination geometry. The terpyridine ligand displays
a tridentate mode of binding. The monoanionic acetylacetonate
and two nitrate ligands bind in a bidentate chelating fashion. The
coordination of a solvent ethanol molecule is observed in 1a. The
pyridine rings of the terpyridine moiety of phetpy ligand in
complex 1a are out of plane possibly due to coordination of the
solvent ethanol molecule resulting in a cis disposition of two
bidentate nitrate anions in the coordination sphere. In contrast, the
pyridine rings of the phetpy ligand in complex 4 lie in a plane and
a trans-disposition of two bidentate nitrate anions is observed. The
OH group of the coordinated ethanol molecule in 1a is hydrogen-
bonded to the non-bound oxygen atom of one of the nitrate
anions of another complex. In addition, the phenyl ring of the
Table 1
Selected physicochemical data and DNA binding parameters for the Ln(III)
complexes 1e6.
Complex IR (cmꢀ1
)
meff
lf (nm)
Ffb
Mc Kb (Mꢀ1) [s]d
D
Tm
a
e
[
]
(ꢁC)
C]O NOꢀ3
1
2
3
4
5
6
1595 1378
1580 1389
1581 1389
350 [0.04] 101 (4.6 ꢂ 0.5) x 104 [0.2] 1.5
420 [0.22]
98 (2.9 ꢂ 0.3) x 106 [0.5] 4.5
420 [0.18] 110 (9.8 ꢂ 0.4) x 105 [0.4] 4.4
1590 1380 8.01 350 [0.02] 105 (5.0 ꢂ 0.3) x 104 [0.3] 1.7
1581 1389 7.98 420 [0.15] 103 (3.2 ꢂ 0.2) x 106 [0.6] 4.8
1584 1390 8.05 420 [0.12] 108 (9.5 ꢂ 0.5) x 105 [0.5] 4.6
a
meff in mB using DMSO-d6 solutions of the Gd(III) complexes at 25 ꢁC.
Emission spectral band in H2O-DMSO (1:4 v/v). The quantum yield values of the
b
phetpy and pyetpy ligands in H2O-DMSO (1:4 v/v) are 0.06 and 0.34, respectively.
c
LM, molar conductivity in S cm2 Mꢀ1 in 20% aqueous DMF at 25 ꢁC.
Kb, the calf thymus DNA binding constant [s, binding site size].
Change in the calf thymus DNA melting temperature.
d
e

322
A. Hussain et al. / European Journal of Medicinal Chemistry 50 (2012) 319e331
Fig. 3. Absorption spectral traces of complex 3 in 5 mM TriseHCl buffer (pH 7.2) on
increasing the quantity of calf thymus DNA. The inset shows the least-squares fit of
vs. [DNA] for [La(phetpy)(acac)(NO3)2] (1, :), [La(pyetpy)(acac)(NO3)2] (2,
D
3 /
af
D 3
bf
C) and [La(pyetpy)(sacac)(NO3)2] (3, -).
phetpy analogues beacuse of the presence of a planar pendant
pyrenyl moiety in the pyetpy ligand. This is expected since metal
complexes of pyetpy are known to form
supramolecular arrays supporting the fact that they can strongly
-stack in between the DNA base pairs [48]. The fitted parameter s
p-assembled metallo-
p
of the MvH equation gives an estimate of the number of DNA bases
associated with the complex and its value of <1.0 is typically
interpreted as arising from the aggregation of hydrophobic mole-
cules on the surface of DNA. The greater value of s for the pyetpy
complexes than the phetpy analogues also indicates better DNA
binding strength of the pyetpy complexes [49].
Fig. 2. ORTEP views of the complexes 1a (a) and 4 (b) showing 50% probability thermal
ellipsoids and the atom numbering scheme for the metal and hetero atoms. The
hydrogen atoms are not shown for clarity.
phetpy ligand makes an angle with the middle pyridine ring of the
terpyridine moiety due to steric encumbrance among the aromatic
hydrogen atoms giving a dihedral angle of 8.41ꢁ in complex 1a and
26.39ꢁ in complex 2. The LaeO and LaeN distances in complex 1a
are in the range of 2.398(2) to 2.722(3) Å and 2.711(3) to 2.731(3) Å,
respectively. The GdeO and GdeN distances in complex 2 are in the
range of 2.295(2) to 2.529(2) Å and 2.533(2)e2.576(2) Å, respec-
tively. The LneO bond distances involving the acac ligand are
shorter than those involving the nitrate ligands. The significantly
long La(1)eO(6) and La(1)eO(8) bond lengths in 1a that are trans to
the phetpy ligand could result in the dissociation of the nitrate
ligand in a polar solvent. The coordination behaviour observed in
the present lanthanide complexes is similar to the previously re-
ported structures for analogous lanthanide complexes [47].
4.3.2. DNA melting studies
Thermal DNA denaturation experiments were performed to
obtain insights into the binding of the complexes 1ꢀ6 to ct-DNA
(Fig. 4(a)). A positive shift in the DNA melting temperature (DTm)
was observed upon addition of the complexes to the ct-DNA. The
increase in the ct-DNA melting temperature suggests an increase in
the stability of the double helix upon addition of the complex. The
D
Tm value of w2 ꢁC indicate primarily groove binding nature of the
phetpy complexes to ct-DNA and a partial intercalative mode of
binding of the pyetpy complexes to ct-DNA giving a large positive
D
Tm value of w5 ꢁC [50].
4.3.3. Viscosity measurements
Viscosity measurements were carried out to examine the effect
of the complexes on the relative specific viscosity of the solution
4.3. DNA binding properties
containing ct-DNA (Fig. 4(b)). Since the relative specific viscosity (h/
h0) of a solution containing DNA gives a measure of the increase in
contour length of DNA associated with the separation of base pairs
caused by intercalation, a classical intercalator, viz., ethidium
bromide would result in a significant increase in the viscosity of the
4.3.1. Electronic absorption spectroscopy
DNA being one of the most important targets of many anticancer
agents, designing and developing metal complexes that avidly bind
to DNA are of paramount importance. The electronic absorption
titrations were carried out to monitor the binding interactions of
the complexes 1e6 with ct-DNA (Fig. 3). The intrinsic equilibrium
ct-DNA binding constants (Kb) which give a measure of the strength
of binding of a complex to ct-DNA are given in Table 1. The Kb values
DNA solution (where
h and h0 are the respective specific viscosities
of a DNA solution in the presence and absence of the complexes). In
contrast, a partial and/or non-intercalating molecule could result in
less pronounced effect on the viscosity [51]. The groove binder
Hoechst 33258 was used as a reference compound that showed no
apparent change in the solution viscosity of ct-DNA. The results
indicate a two step binding process in which the Ln(III) complexes
in the range of 104e106
M
ꢀ1 follow the order: 6, 5, 3, 2 > 1, 4. The
pyetpy complexes have significantly higher Kb values than their

A. Hussain et al. / European Journal of Medicinal Chemistry 50 (2012) 319e331
323
1/3
Fig. 4. (a) Derivative plots of dA260/dT vs. T for the thermal denaturation of 180
mM calf thymus DNA alone and on addition of the complexes 4e6. (b) Plots of (
h/
h0)
vs.
[compound]/[DNA] showing the effect of increasing the concentration of the complexes [Gd(ph-tpy)(acac)(NO3)2] (4, A), [Gd(pyetpy)(acac)(NO3)2] (5, -), [Gd(pyetpy)(sa-
cac)(NO3)2] (6, ), ethidium bromide (EB, ,) and Hoechst 33258 (B) on the relative viscosities of CT-DNA at 37.0 (ꢂ0.1) ꢁC in 5 mM TriseHCl buffer (pH 7.2) containing 2.5e20%
;
DMF and 180 mM calf thymus DNA.
possibly first interact with the ct-DNA surface followed by groove
the complexes was studied using a minor groove binder
binding and/or partial intercalation [52].
distamycin-A and a major groove binder methyl green. Distamycin-
A and methyl green showed w15% cleavage of SC DNA in UV-A
light. Addition of complex 2 to the distamycin-A bound SC DNA
did not inhibit the DNA cleavage activity. Addition of methyl green
to complex 2, however, inhibited the DNA photocleavage activity
suggesting DNA major groove binding mode of the pyetpy
complexes (Fig. 5) [53]. We also studied the DNA photocleavage
activity of the complexes in natural light. The phetpy complexes 1
and 4 did not show any significant cleavage activity when treated
4.4. Photoinduced DNA cleavage activity
The photo-induced DNA cleavage activity of the complexes 1ꢀ6
was studied using supercoiled (SC) pUC19 DNA (30 mM, 0.2 mg) in
TriseHCl/NaCl (50 mM, pH, 7.2) buffer by irradiating the samples
with a low power monochromatic UV-A light of 365 nm (6 W
power). Selected DNA photocleavage data are given in Table 2. The
phetpy complexes 1 and 4 showed only moderate DNA cleavage
activity in UV-A light. The pyetpy complexes with greater DNA
binding strength and photosensitizing ability showed efficient
photo-induced DNA cleavage activity. The pyetpy complexes
showed essentially complete cleavage of SC DNA to its NC form at
with DNA and exposed to natural light. A 10 mM solution of the
pyetpy complexes 2, 3, 5 and 6 showed 40e45% cleavage of SC DNA
when irradiated with natural light for 1 h (lanes 18 and 19 in Fig. 5).
The moderate photcleavage activity shown by the pyetpy
complexes could be a result of photsensitization of the pyetpy
moiety by high energy region of the solar spectrum. Therefore,
the pyetpy complexes are much less cleavage active in natural light
compared to that in UV-A light.
a complex concentration of 1.0 mM for an exposure time of 1 h
(Fig. 5, lanes 8, 9, 11, 12). Control experiment performed with only
SC DNA did not show any apparent cleavage of DNA in UV-A light.
The ligand pyetpy alone showed w15% cleavage of SC DNA. The
complexes were not cleavage active in dark thus ruling out any
possibility of hydrolytic DNA damage (Fig. 5, lane 5). The effect of
La(III) vs. Gd(III) metal ion is not apparent from the DNA photo-
cleavage data. The complexes having pyetpy ligand are more active
than the phetpy complexes. Binding of py-tpy to the Ln(III) thus
remarkably enhances its DNA photocleavage activity compared to
the ligand or the metal salt alone. The DNA groove binding nature of
4.5. Mechanistic studies
The mechanistic aspects of the UV-A light-induced DNA
cleavage activity of the pyetpy complexes were studied using
different additives (Fig. 6). The DNA photocleavage reactions
involving molecular oxygen could proceed via two major pathways,
viz., the type-II process forming singlet oxygen (1O2) or a photo-
redox pathway forming reactive hydroxyl radicals (ꢃOH). Addition
of singlet oxygen quenchers, viz., sodium azide, TEMP or DABCO to
SC DNA showed partial inhibition of the DNA photocleavage
activity. Hydroxyl radical scavengers, viz., DMSO or catalase also
showed partial inhibition in the DNA cleavage activity. The results
suggest the formation of both singlet oxygen and hydroxyl radicals
as the reactive oxygen species (ROS) responsible for the photo-
damage of DNA. The singlet oxygen formation was also evidenced
from the enhancement of the DNA cleavage activity in D2O due to
longer lifetime of 1O2 in this medium [54]. While the photoactive
pyrenyl moiety could generate the singlet oxygen species in a type-
II process in UV-A light, the formation of hydroxyl radicals could
take place from electron transfer to the oxygen molecule from the
photo-excited pyetpy ligand generating radical cation [55]. The
photocleavage of DNA at low complex concentration is a significant
result in the chemistry of metal-based photochemotherapeutic
agents. The presence of heavy lanthanide ion seems to facilitate the
Table 2
Selected DNA cleavage dataa for the complexes 1e6 in UV-A light of 365 nm.
Reaction condition
[Complex] (
mM)
t (h)b
%NC
DNA control
DNA þ 2 (in dark)
DNA þ pyetpy
DNA þ 1
e
1
e
1
1
1
1
1
1
1
2
10
15
25
88
81
22
86
78
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
DNA þ 2
DNA þ 3
DNA þ 4
DNA þ 5
DNA þ 6
a
SC and NC are supercoiled and nicked circular forms of pUC19 DNA (0.2
m
M)
g,
30
m
M). The DNA cleavage data for the ligands (1.0 M) and metal salts (1.0 m
m
giving %NC for 1 h photoexposure time are: phetpy, 7%; Hsacac, 5%; Hacac, 5%;
La(NO3)3, 5% and Gd(NO3)3, 7%.
b
Exposure time, t.

324
A. Hussain et al. / European Journal of Medicinal Chemistry 50 (2012) 319e331
Fig. 5. Gel electrophoresis diagram showing the cleavage of SC pUC19 DNA (0.2
ligands alone (1.0 M) and the groove binding agents in 50 mM TriseHCl/NaCl buffer (pH, 7.2) containing 5% DMF on irradiation with UV-A light (lanes 1e16) of 365 nm (6 W) or
natural light (lanes 17e19) for 1 h except lanes 5 and 6 that were in dark: lane 1, DNA control; lane 2, DNA þ phetpy; lane 3, DNA þ pyetpy; lane 4, DNA þ Hsacac; lane 5, DNA þ 1
(in dark lane 6, DNA þ 6 (in dark); lane 7, DNA þ 1; lane 8, DNA þ 2; lane 9, DNA þ 3; lane 10, DNA þ 4; lane 11, DNA þ 5; lane 12, DNA þ 6; lane 13, DNA þ distamycin-A (50 M);
lane 14, DNA þ distamycin-A (50 M) þ 2; lane 15, DNA þ methyl green (200 M); lane 16, DNA þ methyl green (200 M) þ 2; lane 17, DNA control; lane 18, DNA þ 2; lane 19,
DNA þ 5. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
mg, 30 mM) by the complexes (1.0 mM of 1e6 in UV-A light and 10 mM of 2, 5 in natural light), the
m
m
m
m
m
intersystem crossing (ISC) for efficient generation of singlet oxygen
and hydroxyl radicals [25,26].
carbohydrate appended acac and the acac ligand upon 4 h incu-
bation of the complexes in dark followed by photoexposure. To
obtain a deeper insight into the cellular uptake behaviour of the
complexes, we carried out time-dependent photocytotoxicity
studies using complex 2 having no pendant glucose moiety and
complex 3 having a pendant glucose moiety. A 1.0 h incubation of
the complexes 2 and 3 in dark followed by photoexposure gave IC50
values of 80(ꢂ10) and 40(ꢂ10) nM, respectively. The IC50 values for
2 h incubation of the complexes in the dark followed by photo-
exposure were 50(ꢂ10) nM for 2 and 30(ꢂ10) nM for 3. Therefore,
a distinct difference in the IC50 values for 2 and 3 is seen at 2 h
incubation. Complex 3 bearing a pendant glucose moiety was found
to be more cytotoxic compared to the glucose-free complex 2. The
photocytotoxicity of the complexes was found to be very similar for
an incubation time of 4 h prior to photoexposure. The results
suggest two different cellular uptake pathways for the complexes.
The acac complexes being more lipophilic could have a passive
4.6. Cell viability assay
MTT assay was carried out to evaluate the photocytotoxic
potential of the complexes 1ꢀ6 upon photoexcitation on human
cervical cancer HeLa cells (Fig. 7). The phetpy complexes upon
prior incubation for 4 h in the dark and subsequent photoexposure
to UV-A light of 365 nm for 15 min showed moderate decrease in
the cell viability giving IC50 values of 38(ꢂ4)
m
M for 1 and 25(ꢂ2)
m
M for 4 in light and >200 M for both in dark (Fig. 7(a)). The
m
pyetpy complexes 2, 3, 5 and 6 showed a dose-dependent decrease
in cell viability with IC50 values of 40(ꢂ10), 30(ꢂ10), 50(ꢂ10) and
30(ꢂ10) nM, respectively (Fig. 7(b)). The cells unexposed to light
exhibited an IC50 value of >200
mM for the complexes. The
complexes are thus non-toxic in dark but become highly cytotoxic
upon photo-irradiation. The pyrenyl ligand is less toxic compared to
its complexes giving an IC50 value of 70(ꢂ10) nM in light while
being non-toxic in dark. This observation suggests a more facile
intersystem crossing (ISC) mediated by the heavy lanthanides
resulting in the generation of singlet oxygen compared to the
pyetpy ligand alone [25,26]. No significant difference in the IC50
values in light is observed between the pyrenyl complexes bearing
Fig. 7. Cell viability plots showing the photocytotoxicity of the complexes 1 and 4 in
panel (a) and 2, 3, 5 and 6 in panel (b) In HeLa cells on 4 h incubation in dark followed
by exposure to UV-A light of 365 nm (0.55 J cmꢀ2) for 15 min, as determined from the
MTT assay. The non-linear fitted curves are shown by circles (B) for complex 1 and
squares (,) for complex 4 in panel (a) and the fitted-curves are shown by circles (B)
Fig. 6. Bar diagram showing the photocleavage of SC pUC19 DNA (0.2 mg, 30 mM) by
[Gd(pyetpy)(acac)(NO3)2] (5) (light shade) and [Gd(pyetpy)(sacac)(NO3)2] (6) (dark
shade) in the presence of various additives in TriseHCl buffer containing 10% DMF. The
complex concentration and exposure time are 1.0
additive concentrations/quantities are: sodium azide, 0.5 mM; KI, 0.5 mM; TEMP,
0.5 mM; DABCO, 0.5 mM; D2O, 16 L; DMSO, 4 L; catalase, 4 units and SOD, 4 units.
mM and 1 h, respectively. The
for complex 2, squares (,) for complex 3, triangles (
(>) for complex 6 in panel (b).
6) for complex 5 and diamonds
m
m

A. Hussain et al. / European Journal of Medicinal Chemistry 50 (2012) 319e331
325
diffusion uptake process. The complexes bearing the carbohydrate
moiety could have a receptor-mediated endocytic pathway. With
reduced lipophilicity of sacac due to the presence of the carbohy-
drate functionality, the overexpressed glucose transporters (GLUTs)
in HeLa cells could be responsible for the endocytosis of the
complexes.
We earlier reported the photocytotoxicity of the lanthanide
complexes of dipyridophenazine base, viz., [Ln(dppz)2(NO3)3] and
[Ln(dppz)(acac)3] in HeLa cells (Table 3) [34,35]. The MTTassay data
show that the pyetpy complexes are more photocytotoxic than
their phetpy or dppz analogues, possibly due to greater photo-
sensitizing ability of the pyetpy ligand. Cisplatin is known to be
and the nuclei condensed significantly. The nuclei of the cells
treated in dark and the untreated control cells remained intact and
stained evenly with AO but not with EB. This difference in staining
is because AO is cell-permeable whereas EB is not. In viable cells
having an intact plasma membrane, AO crosses the cell membrane
and stains the DNA whereas EB is excluded. The cells undergoing
apoptosis on treatment with the complex in the presence of light
lose membrane integrity during the late stages of apoptosis, cells
become permeable to EB and stain orange [57].
4.8. Cellular uptake
toxic at an IC50 value of 7.5
bation [34]. Incubation of the HeLa cells for 4 h with cisplatin in
dark and subsequent photoexposure to UV-A light is known to
m
M in dark in HeLa cells on 24 h incu-
The cellular uptake of an anticancer drug is of importance for its
effectiveness against tumours [58]. We explored the localization of
the complexes inside the HeLa cancer cells from confocal imaging
study which was carried out using fluorescent pyetpy complexes 2
and 3 (Fig. 9). Complex 3 having a pendant glucose moiety was used
for the confocal study to see the effect of the carbohydrate moiety
on the cellular uptake behaviour since GLUT receptors are known to
overexpress in a majority of cancer cells and cancer cell lines. There
are reports on the overexpression GLUTs, particularly GLUT1, in
rapidly growing HeLa cells [59,60]. Confocal studies were carried
exhibit IC50 values of 71.3 mM in dark and 68.7 mM in UV-A light
(
l
¼ 365 nm) with no apparent PDT effect as expected [34]. Pho-
tofrinÒ with an IC50 value of 4.3(ꢂ0.2)
m
M in 633 nm light (5 J cmꢀ2
power) and >41
mM in dark in HeLa cells is the FDA approved
PDT drug [56]. Complex trans-[Pt(N3)2(OH)2(NH3)(py)] is known to
have an IC50 value of 6.1(ꢂ0.5) M in UV-A light of 365 nm and
>244.3 in dark in HaCaT cancer cells [15]. Lanthanide
m
mM
complexes of the py-tpy ligand are thus remarkably photocytotoxic
in UV-A light at nanomolar concentrations while remaining
essentially non-toxic in dark. The lanthanide complexes are thus
potent PDT agents in blue light.
out using 10
mM solution of the complexes with propidium iodide
(PI) as a nuclear staining agent which also served as a reference. The
confocal images were taken after 15 min, 30 min, 1 h and 4 h of
incubation of the complexes to see their time-dependent uptake
and localization inside the cells. The images taken after 15 min
showed little uptake of both the complexes as seen from the
intracellular fluorescence intensity seen in the cells in panels (a),
(b) and (c) in Figs. 9 and 10. Images taken after 30 min showed no
significant enhancement in the fluorescence intensities for both the
complexes (panels (d), (e) and (f) in Figs. 9 and 10). Interestingly,
images of the cells treated with the glucose-bearing complex 3
collected after 1 h showed remarkable enhancement in intensity
having diffused cytosolic distribution with punctate staining of the
cells around the perinuclear region (panels (g), (h) and (i) in Fig.10).
The images were found to be of much less intensity for glucose-free
complex 2 (panels (g), (h) and (i) in Fig. 9). However, the confocal
images of the cells taken after 4 h showed similar intensity pattern
for both the complexes (panels (j), (k) and (l) in Figs. 9 and 10). The
intracellular localization of complex 2 did not show any granular
appearance, but largely a diffused staining of the whole cytoplasm.
The nucleus gave little or no blue emission, indicative of negligible
nuclear uptake in both the cases. These observations suggest two
possible cellular uptake pathways for the glucose-free complex 2
taken up by a diffusion process and the glucose-bearing complex 3
internalized by receptor-mediated endocytosis (RME). The obser-
vation of similar punctate staining of cells treated with the metal
complex bearing a pendant glucose moiety has been reported in
the literature [60]. No change in the nuclear morphology was seen
in the confocal images of the cells indicating the fact that the
complexes remain harmless in the dark but show potent PDT effect
upon photoexposure. The results are indeed interesting considering
that the present complexes could serve the dual purpose of
detecting the tumour and damaging the DNA upon photoactivation
leading to apoptosis. The lanthanide-based MRI agents are only
useful for tumour detection but not for its selective damage.
4.7. Photodynamic effect on nuclear morphology
Since the pyetpy complexes showed remarkable photo-
cytotoxicity in UV-A light, we performed fluorescent ethidium
bromide-acridine orange (EB/AO) dual staining of the HeLa cells
treated with complexes 2 and 3 to understand the mechanism of
cell death by monitoring the changes in the nuclear morphology
upon PDT (Fig. 8). The cells treated with the complexes in the dark
did not show any significant nuclear morphological change. But
a significant number of cells treated with the complexes and
subsequently photo-irradiated with UV-A light of 365 nm showed
apoptotic nuclear morphology. Cell membrane blebbing was seen
Table 3
A
comparison of the IC50 values of the complexes 1e6 with other relevant
compounds in HeLa cell.
Compound
IC50 in lighta
IC50 in darkb
1
2
3
4
5
6
38(ꢂ4)
m
M
>200
>200
>200
>200
>200
>200
11.6 mM
>100
>100
m
m
m
m
m
m
M
M
M
M
M
M
40(ꢂ10) nM
30(ꢂ10) nM
25(ꢂ2)
mM
50(ꢂ10) nM
40(ꢂ10) nM
dppzc
0.41
0.57
m
m
M
M
[Gd(dppz)2(NO3)3]c
[Gd(dppz)(acac)3]d
PhotofrinÒe
Cisplatin
m
m
M
M
M
0.53(ꢂ0.03)
m
m
M
M
4.28(ꢂ0.20)f
>41
m
68.7g
m
M
7.5h
m
M
a
IC50 values correspond to 4 h incubation in dark followed by photoexposure to
UV-A light of 365 nm (0.55 J cmꢀ2). The respective IC50 values for the phetpy,
pyetpy and Hsacac ligands are 47(ꢂ3)
m
M, 80(ꢂ10) nM and >200
mM in UV-A light
and >200 M in dark.
m
b
5. Conclusions
IC50 values correspond to 24 h incubation in dark.
The IC50 values are taken from Ref.[35].
The IC50 values are taken from Ref.[36].
The IC50 values are from Ref.[68].
c
d
e
f
The photo-induced DNA cleavage activity and photocytotoxicity
of six lanthanide complexes having tridentate N,N,N-donor ter-
pyridine and acetylacetonate ligands are presented. The La(III) and
Gd(III) complexes show LnO6(7)N3 core in the solid state and
LnO4N3 core in a solution phase. The pyetpy complexes with
Visible light source of 630 nm.
g
IC50 value of cisplatin in dark for 4 h incubation is 71.3 mM (from Ref.[35]). The
value is 68.7
m
M after exposure to light.
h
IC50 value on incubation for 24 h in dark. The data from Ref.[35].

326
A. Hussain et al. / European Journal of Medicinal Chemistry 50 (2012) 319e331
Fig. 8. Ethidium bromide/acridine orange staining of HeLa cells treated with complex 2 (50 nM) and complex 3 (50 nM) to identify nuclear morphology. Panels (a) and (d)
correspond to the cells treated with respective complex 2 and 3 in dark, panels (b) and (e) correspond to the cells treated with respective complex 2 and 3 and irradiated with UV-A
light of 365 nm (0.55 J cmꢀ2) showing significant apoptosis of the cells and panels (c) and (f) correspond to the cells in panels (b) and (e) respectively visualized at 63ꢄ magnification
showing the membrane blebbing of the apoptotic cells. The scale bar corresponds to 10
to the web version of this article.)
mm. (For interpretation of the references to colour in this figure legend, the reader is referred
a planar pyrenyl moiety show intercalative DNA binding propen-
sity. The pyetpy complexes with the photoactive pyrenyl moiety
display efficient UV-A light-induced DNA cleavage activity at low
complex concentration via mechanistic pathways that involve
formation of both singlet oxygen and hydroxyl radicals. The DNA
without further purifications. Solvents were purified by standard
procedures [61]. Supercoiled (SC) pUC19 DNA (caesium chloride
purified) was purchased from Bangalore Genie (India). Tris-
(hydroxymethyl)aminomethane-HCl (TriseHCl) buffer (pH ¼ 7.2)
was prepared using deionized and sonicated triple distilled
water. Calf thymus DNA, agarose (molecular biology grade),
distamycin-A, methyl green, catalase, SOD, TEMP, DABCO, EB, PI and
MTT were purchased from Sigma (U.S.A.). The terpyridine deriva-
tives, viz., 40-phenyl-2,20:60,200-terpyridine (phetpy) and 40-(1-
pyrenyl)-2,20:60,200-terpyridine (pyetpy) were prepared following
published procedures [39,40]. The carbohydrate-appended ligand,
photocleavage observed at only 1.0 mM complex concentration is
significant towards using lanthanide-based complexes as synthetic
photonucleases. The complexes do not show any apparent DNA
hydrolytic cleavage activity thus making them suitable for potential
photoactivated chemotherapeutic applications. The pyetpy
complexes display remarkable photocytotoxic effect in HeLa cells
giving IC50 values in the nanomolar range and a time-dependent
photocytotoxicity is observed for the complexes. The EB/AO dual
staining of the HeLa cells treated with complexes 2 and 3 shows
apoptosis as the major pathway of the cell death. The confocal
fluorescence microscopy of HeLa cells using the pyetpy complexes
2 and 3 reveals localization of the complex primarily in the cytosol
of the HeLa cells. The uptake of the pyetpy complexes 3 and 6
having an appended glucose moiety seems to be endocytosis
mediated by overexpressed GLUTs in the HeLa cells. Interestingly,
the complexes are non-toxic in dark but become highly cytotoxic on
photoactivation. With significant PDT effect at nanomolar complex
concentration range, the present pyetpy lanthanide complexes
could serve dual purpose of detecting the tumour while remaining
dormant inside the cell in dark till subjected to photoactivation
causing apoptotic cellular death selectively at the photo-exposed
cells leaving unexposed healthy cells unaffected. In contrast, the
lanthanide-based MRI agents are useful for only tumour detection
but not for tumour damage. The present results are indeed
encouraging and are likely to presage designing and developing
new lanthanide complexes as potent photochemotherapeutic and
tumour imaging agents.
4-hydroxy-6-{4-[(b-D-glucopyranoside)oxy]phenyl}hex-3,5-dien-
2-one (Hsacac), was prepared according to a literature method [41].
6.2. Instrumentation
The elemental analyses were done using a Thermo Finnigan
Flash EA 1112 CHNS analyzer. The infrared spectra were recorded
on a Bruker ALPHA FT-IR spectrometer. Electronic spectra were
recorded on a PerkinElmer Lambda 650 UVvis spectrometer.
Molar conductivity measurements were performed using
a Control Dynamics (India) conductivity metre. Room temperature
magnetic susceptibilities of the DMSO-d6 solutions of the Gd(III)
complexes containing 1% TMS (v/v) as the internal reference were
obtained by a solution NMR method with a Bruker AMX-400 NMR
spectrometer [62,63]. The magnetic moments were calculated by
Evans method using the equation: meff ¼ 0.0618(
DfT/fM), where Df
is the observed shift in frequency of the TMS signal, T is the
temperature (K), f is the operating frequency (MHz) of the NMR
spectrometer, and M is the molarity of the complex in the solu-
tion. Electrospray ionization mass spectral (ESI-MS) measure-
ments were carried out using Bruker Daltonics make Esquire 300
Plus ESI Model instrument. 1H-NMR spectra of the diamagnetic
La(III) complexes were recorded at room temperature on a Bruker
400 MHz NMR spectrometer. Room temperature fluorescence
quantum yield measurements were done using a PerkinElmer LS
55 fluorescence spectrometer using pure anthracene as a standard
[64]. Photobleaching experiments were carried out using a Perki-
nElmer LS 55 fluorescence spectrometer by monitoring the
6. Experimental protocols
6.1. Materials
All reagents and chemicals were purchased from commercial
sources (S.D. Fine Chemicals, India; SigmaeAldrich, U.S.A.) and used

A. Hussain et al. / European Journal of Medicinal Chemistry 50 (2012) 319e331
327
Fig. 9. A time-course collection of confocal microscopic images of the HeLa cells treated with [La(pyetpy)(acac)(NO3)2](2, 10
m
M) and propidium iodide (PI). Panels (a), (d), (g) and
(j) correspond to the blue emission of complex 2 upon excitation at 365 nm and the respective images were taken after 15 min, 30 min, 1 h and 4 h. Panels (b), (e), (h) and (k)
correspond to the red emission of PI. Panels (c), (f), (i) and (l) are the merged images of the first two panels. The scale bar corresponds to 10
to colour in this figure legend, the reader is referred to the web version of this article.)
mm. (For interpretation of the references
change in the emission intensity of the pyetpy complexes after
irradiation of a 10 M solution of the complex in aqueous DMSO
carried out on Leica DM IL microscope with integrated Leica
DFC400 camera and IL50 image software. Confocal microscopy
was done using confocal scanning electron microscope (Leica, TCS
SP5 DM6000).
m
(1:4 v/v) with UV-A light of 365 nm (UV lamp, 100 W). The room
temperature emission spectra were recorded at an interval of
5 min post-irradiation upto a total duration of 60 min. The test
solutions were continuously stirred during the experiment to
ensure homogeneous bleaching (if any). The emission signals due
to the pyetpy chromophore were recorded (excitation wave-
length, 350 nm). Fluorescence microscopic investigations were
6.3. Synthesis of the complexes 1e6
The complexes were prepared by following a general synthetic
method. An ethanol solution (15 ml) of Ln(NO3)3.6H2O (0.43 g for

328
A. Hussain et al. / European Journal of Medicinal Chemistry 50 (2012) 319e331
Fig. 10. A time-course collection of confocal microscopic images of the HeLa cells treated with [La(pyetpy)(sacac)(NO3)2](3, 10
mM) and propidium iodide (PI). Panels (a), (d), (g) and
(j) correspond to the blue emission of complex 3 upon excitation at 365 nm and the respective images were taken after 15 min, 30 min, 1 h and 4 h. Panels (b), (e), (h) and (k)
correspond to the red emission of PI. Panels (c), (f), (i) and (l) are the merged images of the first two panels. The scale bar corresponds to 10
to colour in this figure legend, the reader is referred to the web version of this article.)
mm. (For interpretation of the references
La(III) and 0.45 g for Gd(III), 1.0 mmol) was added dropwise to
a dichloromethane solution (15 ml) of the terpyridine ligand
(phetpy, 0.31 g; pyetpy, 0.44 g; 1.0 mmol) with stirring for 1 h at
25 ꢁC. The reaction mixture was cooled to 0 ꢁC to afford a crystalline
solid of [Ln(phetpy)(NO3)3] or [Ln(pyetpy)(NO3)3] as the precursor
complex which was filtered, washed with ice-cold ethanol followed
by diethyl ether and finally dried in vacuum and used directly in the
next step.
To an ethanol suspension (15 ml) of the precursor complex
[Ln(phtpy)(NO3)3] (0.32 g, La(III); 0.33 g, Gd(III), 0.5 mmol) or
[Ln(pyetpy)(NO3)3] (0.38 g, La(III); 0.39 g, Gd(III), 0.5 mmol) was
added the
b-diketone ligand (0.15 g, Hacac; 0.55 g, Hsacac,
1.5 mmol) dissolved in methanol (15 ml) and the pH of the reaction
mixture was adjusted to 7.5 using triethylamine. After 1h of stirring
at room temperature, a clear solution was obtained which was
further stirred for 2 h and filtered. Slow evaporation of the solution

A. Hussain et al. / European Journal of Medicinal Chemistry 50 (2012) 319e331
329
gave a crystalline solid of the desired product which was isolated
and washed with cold methanol followed by diethyl ether and
finally dried in vacuum over P4O10 (Yield: w70%). The character-
ization data for the complexes are given below.
w, 628 m, 590 w, 560 w, 540 w, 521 w, 482 w, 460 w, 421 w.
UVevisible in DMF [lmax, nm (
, Mꢀ1 cmꢀ1)]: 350 (48,060), 331
3
(36,930), 316 (21,200), 278 (70,020), 265 (62,060). meff ¼ 7.98 mB at
298 K. Molar conductance (LM) in 1:5 aqueous DMF at 298 K:
103 S cm2 Mꢀ1
.
6.3.1. [La(phetpy)(acac)(NO3)2] (1)
Anal. calc. for C26H22N5O8La: C, 46.51; H, 3.30; N, 10.43. Found:
C, 46.65; H, 3.50; N, 10.38%. ESIeMS in 20% aqueous MeOH (m/z):
609.10 [M-(NOꢀ3 )]þ. FTeIR, cmꢀ1: 3065 w, 1595 vs, 1572 m, 1512 s,
1450 s, 1415 s, 1378 s, 1351 m, 1290 s, 1263 m, 1165 m, 1074 w, 1028
m, 1010 s, 922 w, 880 w, 840 m, 815 w, 790 m, 764 s, 732 m, 691 m,
658 w, 635 m, 617 m, 533 w, 497 w, 410 w (vs, very strong; s, strong;
6.3.6. [Gd(pyetpy)(sacac)(NO3)2] (6)
Anal. calc. for C49H40N5O14Gd: C, 55.49; H, 3.73; N, 6.48. Found:
C, 55.69; H, 3.65; N, 6.62%. ESIeMS in 10% aqueous MeOH (m/z):
1018.33 [M-(NOꢀ3 )]þ. IR data, cmꢀ1: 3312 s, 3045 w, 1584 vs, 1565 s,
1508 s, 1470 m, 1390 s, 1231 s, 1175 m, 1067 m, 1040 m, 1012 m, 989
m, 896 w, 840 s, 790 m, 783 w, 760 w, 744 m, 722 w, 678 w, 660 w,
620 w, 590 w, 557 m, 537 w, 476 w, 456 w, 420 w. UVevisible in DMF
m, medium; w, weak). UVevisible in DMF [lmax, nm (
3
, Mꢀ1 cmꢀ1)]:
310sh (15,670), 278 (38,680) (sh, shoulder). 1H NMR in DMSO-d6 (
d,
[
lmax, nm (
3
, Mꢀ1 cmꢀ1)]: 350 (57,600), 280 (53,800). meff ¼ 8.05 mB
ppm): 8.80 (d, 2H, J ¼ 4.5 Hz), 8.72 (s, 2H), 8.68 (d, 2H, J ¼ 8.2 Hz),
8.15 (d, 2H, J ¼ 8.0 Hz), 7.92 (d, 2H, J ¼ 7.5 Hz), 7.50e7.65 (m, 5H),
5.40 (s, 1H), 1.80 (s, 6H) (s, singlet; d, doublet; m, multiplet). Molar
at 298 K. Molar conductance (LM) in 1:5 aqueous DMF at 298 K:
95 S cm2 Mꢀ1
.
conductance (LM) in 1:5 aqueous DMF at 298 K : 101 S cm2 Mꢀ1
.
6.4. Solubility and stability
6.3.2. [La(pyetpy)(acac)(NO3)2] (2)
All the complexes showed good solubility in DMF and DMSO and
moderate solubility in methanol, ethanol and acetonitrile.
Complexes 3 and 6 were moderately soluble in water but showed
poor solubility in chlorinated solvents due to the presence of
a carbohydrate moiety. The complexes were found to be stable in
the monocationic form due to dissociation of the nitrate anion and
the solution stability of the cationic species was ascertained from
the ESI-MS as well as from the molar conductivity data.
Anal. calc. for C36H26N5O8La: C, 54.35; H, 3.29; N, 8.80. Found: C,
54.50; H, 3.33; N, 8.75%. ESIeMS in 10% aqueous MeOH (m/z):
733.50 [M-(NOꢀ3 )]þ. FTeIR, cmꢀ1: 3050 w, 1580 s, 1560 m,1515 m,
1460 s, 1435 s, 1410 s, 1389 m, 1293 s, 1187 w, 1086 w, 1041 w, 1005
m, 893 w, 840 s, 780 m, 737 m, 720 m, 683 m, 658 w, 625 m, 580 w,
560 w, 507 w, 475 w, 462 w, 415 w. UVevisible in DMF [lmax, nm (
ꢀ1 cmꢀ1)]: 350 (45,790), 331 (34,320), 315 (19,940), 278 (78,250),
268 (64,830). 1H NMR in DMSO-d6
, ppm): 8.98 (s, 2H), 8.77 (d,
3 ,
M
(
d
2H, J ¼ 7.7 Hz), 8.58 (d, 2H, J ¼ 4.6 Hz), 8.50e8.25 (m, 8H), 8.11 (t,1H,
6.5. X-ray crystallographic procedure
J ¼ 7.9 Hz), 7.95 (m, 2H), 7.70 (m, 2H), 5.5 (s, 1H), 1.78 (s, 6H). Molar
conductance (LM) in 1:5 aqueous DMF at 298 K: 108 S cm2 Mꢀ1
.
The
crystal
structures
of
the
complexes
1
as
[La(phetpy)(acac)(EtOH)(NO3)2] (1a) and 4 were obtained by single
crystal X-ray diffraction method. Single crystals of 1a were grown
by slow evaporation of a solution of the complex in EtOH-MeOH
mixture (1:1 v/v). The single crystals of 4 were obtained by
vapour diffusion of diethyl ether into a methanol solution of the
complex. Crystal mounting was done on glass fibre 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-
6.3.3. [La(pyetpy)(sacac)(NO3)2] (3)
Anal. calc. for C49H40N5O14La: C, 55.43; H, 3.80; N, 6.60. Found:
C, 55.50; H, 3.92; N, 6.67%. ESIeMS in 10% aqueous MeOH (m/z):
999.11 [M-(NOꢀ3 )]þ. FTeIR, cmꢀ1: 3312s, 3060 w, 1581 vs, 1566 s,
1510 s, 1465 m, 1389 s, 1233 s, 1176 m, 1070 m, 1040 m, 1018 m, 989
m, 893 w, 843 s, 790 w, 780 w, 755 w, 740 w, 720 w, 681w, 660 m,
623 w, 588 w, 558w, 540 w, 517 w, 474 w, 458 w, 426 w. UVevisible
in DMF [lmax, nm (
NMR in DMSO-d6
3
, Mꢀ1 cmꢀ1)]: 350 (57,730), 278 (45,280). 1H
, ppm): 9.01 (s, 2H, J ¼ 8.5 Hz), 8.80 (d, 2H,
(
d
Ka X-ray source (
frame) at a scan speed of 8 s per frame for complex 1a and 5 s per
frame for complex 4. Intensity data, collected using -2 scan
l
¼ 0.71073 Å) with increasing
u
(width of 0.3ꢁ per
J ¼ 7.8 Hz), 8.61 (d, 2H, J ¼ 4.4 Hz), 8.48e8.20 (m, 8H), 7.90 (t, 1H,
J ¼ 7.9 Hz), 7.72 (m, 2H), 7.65 (d, 1H, J ¼ 15.1 Hz), 7.50 (m, 2H),
7.40e7.28 (m, 4H), 6.52 (d, 1H, J ¼ 15.1 Hz), 5.72 (s, 1H), 4.96 (d, 1H,
J ¼ 7.8 Hz), 4.81(m, 1H, J ¼ 7.1, 6.8 Hz), 4.72 (m, 1H), 4.58 (m, 1H),
4.35 (m, 1H), 4.26 (m, 1H), 3.85 (m, 1H), 2.15 (s, 3H). Molar
u
q
mode, were corrected for Lorentzepolarization effects and for
absorption [65]. The structures were solved by a combination of
Patterson and Fourier techniques and refined by full-matrix least-
squares method using SHELX system of programs [66]. All
hydrogen atoms belonging to the complexes were refined using
a riding model. All non-hydrogen atoms were refined anisotropi-
cally. Perspective views of the molecules were obtained by ORTEP
[67]. The CCDC numbers for the complexes 1a and 4 are 844349 and
844350, respectively.
conductance (LM) in 1:5 aqueous DMF at 298 K: 110 S cm2 Mꢀ1
.
6.3.4. [Gd(phetpy)(acac)(NO3)2] (4)
Anal. calc. for C26H22N5O8Gd: C, 45.28; H, 3.21; N, 10.15. Found:
C, 45.40; H, 3.33; N, 10.25%. ESIeMS in 20% aqueous MeOH (m/z):
628.15 [M-(NOꢀ3 )]þ. FTeIR, cmꢀ1: 3040 w, 1590 vs, 1572 w, 1520 s,
1460 vs, 1410 m, 1380 s, 1282 s, 1262 m, 1160 m, 1076 w, 1030 m,
1006 s, 925 m, 894 w, 850 w, 820 w, 795 m, 765 s, 735 m, 695 m, 660
w, 635 w, 620 m, 533 w, 498 w, 422 w. UVevisible in DMF [lmax, nm
Crystal data for 1a: C28H27LaN5O9, M ¼ 716.46, triclinic, space
ꢀ
group Pı, a ¼ 8.3959(3); b ¼ 10.1193(3); c ¼ 18.0790(6) Å,
a
¼ 95.588(2),
b
¼ 100.109(2),
g
¼ 101.898(2)ꢁ, V ¼ 1465.38(8) Å3,
(
3
, Mꢀ1 cmꢀ1)]: 309sh (15,830), 280 (39,050). meff ¼ 8.01 mB at 298 K.
Z ¼ 2, Dc ¼ 1.522 Mg mꢀ3, T ¼ 293(2) K, 30.53 ꢅ
q
ꢅ 2.08ꢁ,
Molar conductance (LM) in 1:5 aqueous DMF at 298 K:
m
¼ 1.511 cmꢀ1, F(000) ¼ 668, GOF ¼ 0.997, R1 ¼ 0.0469,
105 S cm2 Mꢀ1
.
wR2 ¼ 0.0958 for 8730 reflections with I > 2
s(I) and 388
parameters [R1 (F2) ¼ 0.074 (all data)]. Weighting scheme:
6.3.5. [Gd(pyetpy)(acac)(NO3)2] (5)
w ¼ [
s
2(Fo)2 þ (0.0412 P)2 þ 0.0000 P]ꢀ1, where P ¼ (Fo2 þ 2F2c)/3.
Anal. calc. for C36H26N5O8Gd: C, 53.13; H, 3.22; N, 8.60. Found: C,
53.31; H, 3.34; N, 8.68%. ESIeMS in 10% aqueous MeOH (m/z):
752.05 [M-(NOꢀ3 )]þ. IR data, cmꢀ1: 3065 w, 1581 s, 1560 s, 1505 s,
1470 m, 1430 m, 1415 m, 1389 s, 1280 m, 1183 m, 1072 s, 1040 m,
1015 m, 893 w, 841 s, 795 m, 773 w, 757 w, 742 m, 716 m, 683 w, 661
Crystal data for 4: C26H22GdN5O8, M ¼ 689.74, triclinic, space
ꢀ
group Pı, a ¼ 9.4899(6); b ¼ 10.8580(8); c ¼ 14.1264(10) Å,
a
¼ 77.276(4),
b
¼ 81.896(4),
g
¼ 69.773(4)ꢁ, V ¼ 1328.95(16) Å3,
Z ¼ 2, Dc ¼ 1.724 Mg mꢀ3, T ¼ 293(2) K, 30.46 ꢅ
q
ꢅ 1.48ꢁ,
m
¼ 2.554 cmꢀ1, F(000) ¼ 682, GOF ¼ 1.039, R1 ¼ 0.035,

330
A. Hussain et al. / European Journal of Medicinal Chemistry 50 (2012) 319e331
wR2 ¼ 0.075 for 7843 reflections with I > 2
s
(I) and 361
(1.0
50 mM NaCl and the complex (2.0
The concentration of the complexes in DMF or the additives in
buffer corresponded to the quantity in 2.0 L stock solution after
dilution to the 20 L final volume using TriseHCl buffer. The solu-
m
L, 30
m
M) in 50 mM TriseHCl buffer (pH 7.2) containing
parameters [R1 (F2) ¼ 0.059 (all data)]. Weighting scheme:
mL) with varying concentrations.
w ¼ [
s
2(Fo)2 þ (0.0313P)2 þ 0.0000P]ꢀ1, where P ¼ (Fo2 þ 2F2c)/3.
m
6.6. Pharmacological evaluations
m
tion path length in the sample vial was w5 mm. After light expo-
sure, each sample was incubated for 1.0 h at 37 ꢁC and analyzed for
the photo-cleaved products using gel electrophoresis. Experiments
to study the groove binding selectivity of the complexes were
6.6.1. DNA binding studies
6.6.1.1. Electronic absorption spectroscopy. DNA binding studies
were carried out in TriseHCl/NaCl buffer (5 mM TriseHCl, 5 mM
NaCl, pH 7.2) using DMF solutions of the complexes 1e6. Calf
carried out in the presence of distamycin (50
mM) and methyl green
thymus DNA (ca. 350
m
M NP) in this buffer medium gave a ratio of
(200 M) as the DNA minor and major groove binder, respectively
m
ca. 1.9:1 of the UV absorbance at 260 and 280 nm indicating the
DNA apparently free from protein impurities. The DNA concen-
tration was estimated from its absorption intensity at 260 nm with
[71,72]. The SC DNA was pre-treated with distamycin or methyl
green and then treated with the complex as described above.
Mechanistic studies were done using different additives (NaN3,
a known molar extinction coefficient (
3
) value of 6600 Mꢀ1 cmꢀ1
0.5 mM; TEMP, 0.5 mM; DABCO, 0.5 mM; DMSO, 4 mL; KI, 0.5 mM;
[68]. Absorption titration experiments were made by varying the
concentration of the DNA while keeping the complex concentra-
tion constant. Due correction was made for the absorbance of DNA
itself. Each spectrum was recorded after equilibration of the
sample for 5 min. The intrinsic equilibrium binding constant (Kb)
and the fitting parameter (s) of the complexes 1e6 to DNA were
obtained by McGhee-von Hippel (MvH) method using the
expression of Bard et al. by monitoring the change of the
absorption intensity of the spectral bands with increasing
concentration of calf thymus DNA by regression analysis using
catalase, 4 units; SOD, 4 units) prior to the addition of the lantha-
nide complex. For the D2O experiment, this solvent was used for
dilution of the sample to 20 mL sample volume. The samples after
incubation in a dark room were added to the loading buffer con-
taining 0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol
(3.0
containing 1.0
m
L) and the solution was finally loaded on 1% agarose gel
m
g mlꢀ1 ethidium bromide (EB). Electrophoresis was
done in a dark room for 2.0 h at 60 V in TAE (Tris-acetate EDTA)
buffer. Bands were visualized in UV light and photographed. The
extent of SC DNA cleavage was estimated from the intensities of the
bands using UVITEC Gel Documentation System. Due corrections
were made for the low level of nicked circular (NC) form of DNA
present in the original SC DNA sample and for low affinity of EB
binding to SC compared to NC form of DNA [73]. The observed error
in measuring the gel band intensities was in the range of 3e5%.
equation: (
3
ꢀ
3
f)/(
3
ꢀ
3
f) ¼ (b ꢀ (b2 ꢀ 2Kb2Ct[DNA]t/s)1/2)/2KbCt,
a
b
where b ¼ 1 þ KbCt þ Kb[DNA]t/2s and
3
a
is the extinction coeffi-
cient observed for the absorption band at a given DNA concen-
tration,
3
f
is the extinction coefficient of the complex free in
solution, b is the extinction coefficient of the complex when fully
3
bound to DNA, Kb is the equilibrium binding constant, Ct is the
total metal complex concentration, [DNA]t is the DNA concentra-
tion in nucleotides and s is the fitting parameter for the MvH
equation giving an estimate of the binding site size in base pairs
[69,70]. The non-linear least-squares analyses were done using
Origin Lab, version 8.1.
6.6.3. Cell culture
HeLa (human cervical carcinoma) cells were maintained in
DMEM supplemented with 10% FBS, 100 IU mlꢀ1 of penicillin,
100 m
g mlꢀ1 of streptomycin and 2 mM of Glutamax at 37 ꢁC in
a humidified incubator at 5% CO2. The adherent cultures were
grown as monolayer and were passaged once in 4e5 days by
treatment with 0.25% Trypsin-EDTA.
6.6.1.2. DNA melting experiments. DNA melting experiments were
carried out by monitoring the absorbance of the ct-DNA (180 mM) at
260 nm at various temperatures, both in the absence and presence
of the complexes. Measurements were carried out using a Perki-
nElmer Lambda 650 UV/VIS spectrometer with a PTP-1þ1 Peltier
temperature controller at an increase rate of 0.5 ꢁC per min.
6.6.4. Photocytotoxicity experiments
Photocytotoxicity of the complexes 1e6 was studied using MTT
assay which is based on the ability of mitochondrial dehydroge-
nases of viable cells to cleave the tetrazolium rings of MTT forming
dark purple membrane impermeable crystals of formazan that
could be measured at 540 nm after solubilization in DMSO [42].
Approximately, 104 HeLa cells in a 96-well culture plate in DMEM
containing 10% FBS were incubated for 24 h at 37 ꢁC in a CO2
incubator, followed by addition of the complex solution in
a medium containing 1% DMSO. After 4 h of incubation in dark, the
medium was replaced with phosphate buffered saline (PBS) and the
culture plate was exposed to UV-A light of 365 nm (6 W). PBS was
replaced with fresh medium, and cells were further incubated for
6.6.1.3. Viscosity experiments. Viscometric titrations were per-
formed with a Schott Gerate AVS 310 Automated Viscometer
thermostated at 37 ꢁC in a constant temperature bath. The
concentration of ct-DNA was 150 mM in NP (nucleotide pair) and the
flow times were measured using an automated timer. Each sample
was measured 3 times and an average flow time was calculated.
Plots of (h/h0) h is the
1/3 vs. [compound]/[DNA] were made, where
viscosity of the DNA solution in the presence of the compound and
h0 is that of the DNA solution alone. Viscosity values were calcu-
lated from the observed flow time of the DNA-containing solutions
24 h in the dark. Finally, a 25
each well and again incubated for an additional 3 h. After dis-
carding the culture medium, 200 L of DMSO was added to dissolve
m
L of 4 mg mlꢀ1 of MTT was added to
(t) corrected for that of the buffer alone (t0),
corrections were made for the viscosity of DMF solvent present in
the aqueous buffer solution of DNA.
h
¼ (t ꢀ t0)/t0. Due
m
the formazan crystals formed, and the absorbance at 540 nm was
measured using a BIORAD ELISA plate reader. The cytotoxicity was
measured from the absorbance ratio of the treated cells and
untreated controls. The IC50 values were determined by non-linear
regression analysis using GraphPad Prism software.
6.6.2. DNA cleavage experiments
The cleavage of supercoiled (SC) pUC19 DNA (30 mM, 0.2 mg,
2686 base-pairs) was studied by agarose gel electrophoresis. For
photo-induced DNA cleavage studies, the reactions were carried
out under illuminated conditions using UV-A light of 365 nm (6 W,
Model LF-206.LS of Bangalore Genei). Eppendorf vials were used for
photocleavage experiments in a dark room at 25 ꢁC using SC DNA
6.6.5. Nuclear staining experiments
HeLa cells cultured on cover-slips were photo-irradiated with
a UV-A light of 365 nm (6 W) following 4 h of incubation in the dark
in the presence of 50 nM of the complexes 2 and 3. The cells were

A. Hussain et al. / European Journal of Medicinal Chemistry 50 (2012) 319e331
331
[23] P.K. Sasmal, S. Saha, R. Majumdar, R.R. Dighe, A.R. Chakravarty, Chem. Com-
mun. (2009) 1703e1705.
[24] P.K. Sasmal, A.K. Patra, M. Nethaji, A.R. Chakravarty, Inorg. Chem. 46 (2007)
11112e11121.
[25] J.L. Sessler, R.A. Miller, Biochem. Pharmacol. 59 (2000) 733e939.
[26] W.-H. Wei, Z. Wang, T. Mizuno, C. Cortez, L. Fu, M. Sirisawad, L. Naumovski,
D. Magda, J.L. Sessler, Dalton Trans. (2006) 1934e1942.
[27] J.C. Frias, G. Bobba, M.J. Cann, C.J. Hutchison, D. Parker, Org. Biomol. Chem. 1
(2003) 905e907.
then allowed to recover for 1 h, washed three times with PBS and
stained with an EB/AO mixture (1:1, 10 M) for 15 min and
m
observed at 20ꢄ magnification with a fluorescence microscope
[20].
6.6.6. Confocal microscopy
HeLa cells (4 x 104 cells/mm2) plated on cover slips, were
[28] E.J. New, D. Parker, D.G. Smith, J.W. Walton, Curr. Opin. Chem. Biol. 14 (2010)
238e246.
incubated with 10 mM of the complexes 2 and 3 for different time
intervals of 15 min, 30 min, 1 h and 4 h in dark, fixed with 4%
paraformaldehyde for 10 min at 25 ꢁC and washed with PBS. This
was followed by incubation with propidium iodide (PI) staining
solution for 1.0 h at 42 ꢁC. The cells were washed free of excess PI
and mounted. Images were acquired using the confocal scanning
microscope (Leica, TCS SP5 DM6000) and analysed using the LAS AF
Lite software.
[29] E.J. New, D. Parker, Org. Biomol. Chem. 7 (2009) 851e855.
[30] J.C.G. Bunzli, Chem. Rev. 110 (2010) 2729e2755.
[31] A. Datta, K.N. Raymond, Acc. Chem. Res. 42 (2009) 938e947.
[32] P. Caravan, Acc. Chem. Res. 42 (2009) 851e862.
[33] M. Bottrill, L. Kwok, N.J. Long, Chem. Soc. Rev. 35 (2006) 557e571.
[34] A. Hussain, D. Lahiri, M.S.A. Begum, S. Saha, R. Majumdar, R.R. Dighe,
A.R. Chakravarty, Inorg. Chem. 49 (2010) 4036e4045.
[35] A. Hussain, S. Saha, R. Majumdar, R.R. Dighe, A.R. Chakravarty, Ind. J. Chem.
Sec. A 50A (2011) 519e530.
[36] R.A. Gatenby, R.J. Gillies, Nat. Rev. Cancer 4 (2004) 891e899.
[37] M. Hanif, S.M. Meier, W. Kandioller, A. Bytzek, M. Hejl, C.G. Hartinger,
A.A. Nazarov, V.B. Arion, M.A. Jakupec, P.J. Dyson, B.K. Keppler, J. Inorg. Bio-
chem. 105 (2011) 224e231.
[38] S.J. Franklin, Curr. Opin. Chem. Biol. 5 (2001) 201e208.
[39] S.A. Moya, R. Pastene, H.L. Bozec, P.J. Baricelli, A.J. Pardey, J. Gimeno, Inorg.
Chim. Acta 312 (2001) 7e14.
Acknowledgements
We thank the Department of Science and Technology (DST),
Government of India, for financial support (SR/S5/MBD-02/2007).
We are thankful to DST for a CCD diffractometer facility and the
Department of Biotechnology for the imaging facility. A.R.C. thanks
the DST for J. C. Bose national fellowship. A.H., S.G. and T.K.G. thank
the Council of Scientific and Industrial Research (CSIR), New Delhi,
for research fellowships. The authors thank IRIS, IISc and P.
Janardhan for assistance with the confocal fluorescence microscopy
experiments.
[40] W. Leslie, R.A. Poole, P.R. Murray, L.J. Yellowlees, A. Beeby, J.A.G. Williams,
Polyhedron 23 (2004) 2769e2777.
[41] B. Arezzini, M. Ferrali, E. Ferrari, C. Frassineti, S. Lazzari, G. Marverti,
F. Spagnolo, M. Saladini, Eur. J. Med. Chem. 43 (2008) 2549e2556.
[42] T. Mosmann, J. Immunol. Methods 65 (1983) 55e63.
[43] K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination
Compounds, third ed. John Wiley & Sons, New York, 1978.
[44] K. Mohri, Y. Watanabe, Y. Yoshida, M. Satoh, K. Isobe, N. Sugimoto, Y. Tsuda,
Chem. Pharm. Bull. 51 (2003) 1268e1272.
[45] S. Roy, S. Saha, R. Majumdar, R.R. Dighe, E.D. Jemmis, A.R. Chakravarty, Dalton
Trans. 40 (2011) 1233e1242.
[46] (a) D.J. Toffoli, L. Gomes, N.D. Vieira Jr., L.C. Courrol, J. Opt. A: Pure Appl. Opt.
10 (2008) 104026 (8pp);
Appendix. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2012.02.011.
(b) J.R. Lakowicz, Principles of Fluorescence Spectroscopy, third ed. Springer,
New York, 2006.
[47] Y. Fukuda, A. Nakao, K. Hayashi, J. Chem. Soc. Dalton Trans. (2002) 527e533.
[48] H. Krass, E.A. Plummer, J.M. Haider, P.R. Barker, N.W. Alcock, Z. Pikramenou,
M.J. Hannon, D.G. Kurth, Angew. Chem. Int. Ed. 40 (2001) 3862e3865.
[49] A.M. Angeles-Boza, P.M. Bradley, P.K.-L. Fu, S.E. Wicke, J. Bacsa, K.R. Dunbar,
C. Turro, Inorg. Chem. 43 (2004) 8510e8519.
[50] Y. An, S.-D. Liu, S.-Y. Deng, L.-N. Ji, Z.-W. Mao, J. Inorg. Biochem. 100 (2006)
1586e1593.
[51] J.M. Veal, R.L. Rill, Biochemistry 30 (1991) 1132e1340.
[52] P.A. Pellegrini, J.R. Aldrich-Wright, Dalton Trans. (2003) 176e183.
[53] K.E. Erkkila, D.T. Odom, J.K. Barton, Chem. Rev. 99 (1999) 2777e2795.
[54] A.U. Khan, J. Phys. Chem. 80 (1976) 2219e2227.
References
[1] N.J. Farrer, L. Salassa, P.J. Sadler, Dalton Trans. (2009) 10690e10701.
[2] H.T. Chifotides, K.R. Dunbar, Acc. Chem. Res. 38 (2005) 146e156.
[3] (a) N.L. Fry, P.K. Mascharak, Acc. Chem. Res. 44 (2011) 289e298;
(b) A.D. Ostrowski, P.C. Ford, Dalton Trans. (2009) 10660e10669.
[4] U. Schatzschneider, Eur. J. Inorg. Chem. (2010) 1451e1467.
[5] R. Bonnett, Chemical Aspects of Photodynamic Therapy, Gordon & Breach,
London, U.K, 2000.
[55] M. Tanaka, K. Ohkubo, S. Fukuzumi, J. Phys. Chem.
A 110 (2006)
[6] M. Ethirajan, Y. Chen, P. Joshi, R.K. Pandey, Chem. Soc. Rev. 40 (2011)
340e362.
11214e11218.
[56] E. Delaey, F. Van Laar, D. De Vos, A. Kamuhabwa, P. Jacobs, P. De Witte,
J. Photochem. Photobiol. B. 55 (2000) 27e36.
[57] (a) J. Portugal, BMC Pharmacol. 9 (2009) 11;
[7] M.R. Detty, S.L. Gibson, S.J. Wagner, J. Med. Chem. 47 (2004) 3897e3915.
[8] A.P. Castano, P. Mroz, M.R. Hamblin, Nat. Rev. Cancer 6 (2006) 535e545.
[9] M.C. DeRosa, R.J. Crutchley, Coord. Chem. Rev. 233ꢀ234 (2002) 351e371.
(b) N.J. Wheate, C.R. Brodie, J.G. Collins, S. Kemp, J.R. Aldrich-Wright, Mini Rev.
Med. Chem. 7 (2007) 627e648.
[10] I.J. MacDonald, T.J. Dougherty, J. Porphyrins Phthalocyanines
5 (2001)
105e129.
[58] (a) T. Storr, K.H. Thompson, C. Orvig, Chem. Soc. Rev. 35 (2006) 534e544;
(b) K.H. Thompson, C. Orvig, Dalton Trans. (2006) 761e764.
[59] R.A. Madina, G.I. Owen, Biol. Res. 35 (2002) 9e26.
[60] M.-W. Louie, H.-W. Liu, M.H.-C. Lam, Y.-W. Lam, K.K.-W. Lo, Chem. Eur. J. 17
(2011) 8304e8308.
[61] D.D. Perrin, W.L.F. Armarego, D.R. Perrin, Purification of Laboratory Chemicals,
Pergamon Press, Oxford, 1980.
[62] D.F. Evans, J. Chem. Soc. (1959) 2003e2005.
[11] K. Szacilowski, W. Macyk, A. Drzewiecka-Matuszek, M. Brindell, G. Stochel,
Chem. Rev. 105 (2005) 2647e2694.
[12] S.I. Moriwaki, J. Misawa, Y. Yoshinari, I. Yamada, M. Takigawa, Y. Tokura,
Photodermatol. Photoimmunol. Photomed. 17 (2001) 241e243.
[13] M. Ochsner, J. Photochem, Photobiol. B. Biol. 32 (1996) 3e9.
ꢁ
[14] F.S. Mackay, J.A. Woods, P. Heringová, J. Kaspárková, A.M. Pizarro,
S.A. Moggach, S. Parsons, V. Brabec, P.J. Sadler, Proc. Natl. Acad. Sci. U.S.A. 104
(2007) 20743e20748.
[63] D.F. Evans, T.A. James, J. Chem. Soc. Dalton Trans. (1979) 723e726.
[64] A.T.R. Williams, S.A. Winfield, J.N. Miller, Analyst 108 (1983) 1067e1071.
[65] N. Walker, D. Stuart, Acta Crystallogr. Sect. A 39 (1983) 158e166.
[66] G.M. Sheldrick, Acta Cryst. A64 (2008) 112e122.
[67] (a) C.K. Johnson, ORTEP-III, Report ORNLe5138, Oak Ridge National Labora-
tory, Oak Ridge, TN, 1976;
(b) L.J. Farrugia, J. Appl. Cryst. 30 (1997) 565e566.
[68] M.E. Reichmann, S.A. Rice, C.A. Thomas, P. Doty, J. Am. Chem. Soc. 76 (1954)
3047e3053.
[15] A.M. Angeles-Boza, H.T. Chifotides, J.D. Aguirre, A. Chouai, P.K.-L. Fu,
K.R. Dunbar, C. Turro, J. Med. Chem. 49 (2006) 6841e6847.
[16] M.J. Rose, N.L. Fry, R. Marlow, L. Hinck, P.K. Mascharak, J. Am. Chem. Soc. 130
(2008) 8834e8846.
[17] (a) S. Swavey, K.J. Brewer, Inorg. Chem. 41 (2002) 6196e6198;
(b) A.A. Holder, S. Swavey, K.J. Brewer, Inorg. Chem. 43 (2004) 303e308.
[18] (a) D.A. Lutterman, P.K.-L. Fu, C. Turro, J. Am. Chem. Soc. 128 (2006) 738e739;
(b) D. Loganathan, H. Morrison, Curr. Opin. Drug Discov. Devel. 8 (2005)
478e486.
[69] J.D. McGhee, P.H. von Hippel, J. Mol. Biol. 86 (1974) 469e489.
[70] M.T. Carter, M. Rodriguez, A.J. Bard, J. Am. Chem. Soc. 111 (1989)
8901e8911.
[71] M.L. Kopka, C. Yoon, D. Goodsell, Proc. Natl. Acad. Sci. U.S.A. 82 (1985)
1376e1380.
[72] S.K. Kim, B. Norden, FEBS Lett. 315 (1993) 61e64.
[73] J. Bernadou, G. Pratviel, F. Bennis, M. Girardet, B. Meunier, Biochemistry 28
(1989) 7268e7275.
[19] N.J. Farrer, J.A. Woods, L. Salassa, Y. Zhao, K.S. Robinson, G. Clarkson,
F.S. Mackay, P.J. Sadler, Angew. Chem. Int. Ed. 49 (2010) 8905e8908.
[20] S. Saha, D. Mallick, R. Majumdar, M. Roy, R.R. Dighe, E.D. Jemmis,
A.R. Chakravarty, Inorg. Chem. 50 (2011) 2975e2987.
[21] S. Saha, R. Majumdar, M. Roy, R.R. Dighe, A.R. Chakravarty, Inorg. Chem. 48
(2009) 2652e2663.
[22] B. Banik, P.K. Sasmal, S. Roy, R. Majumdar, R.R. Dighe, A.R. Chakravarty, Eur. J.
Inorg. Chem. 9 (2011) 1425e1435.