Modulating In Vitro Photodynamic Activities of Copper(II) Complexes

Article abstract of DOI:10.1002/ejic.201800081

The photodynamic activities of selected copper(II) complexes [Cu(L¹)B]ClO₄ (1,2) and [Cu(L²)B]ClO₄ (3,4), where L¹ = 2-[(pyridin-2-yl)methyleneamino]phenol, L² = 2-[(pyridin-2-yl)methylenea

Full text of DOI:10.1002/ejic.201800081

A Journal of
Accepted Article
Title: Modulating In Vitro Photodynamic Activities of Copper(II)
Complexes
Authors: Dulal Musib, Md Kausar Raza, Somashree Kundu, and
Mithun Roy
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To be cited as: Eur. J. Inorg. Chem. 10.1002/ejic.201800081
Link to VoR: http://dx.doi.org/10.1002/ejic.201800081

European Journal of Inorganic Chemistry
10.1002/ejic.201800081
FULL PAPER
Modulating In Vitro Photodynamic Activities of Copper(II)
Complexes
Dulal Musib , Md Kausar Raza , Somashree Kundu and Mithun Roy*[a]
[
a]
[b]
[c]
3
Abstract: Photo-dynamic activity of selected copper(II) complexes
2
that require simultaneous presence of molecular oxygen ( O ),
non-toxic red light and a photo-sensitizer.
1
2
1
8-10
[
Cu(L )B]ClO
4
(1-2) [Cu(L )B]ClO
4
(3-4), where L = 2-((pyridin-2-
Photo-activated
2
yl)methyleneamino)phenol,
L =
2-((pyridin-2-
photosensitizers quickly relaxed into the low-lying triplet excited
state through intersystem crossing (ISC), followed by transfer of
energy to the triplet oxygen into cytotoxic singlet oxygen.
Photofrin , oligomeric mixture of porphyrins derivatives, was used
yl)methyleneamino)benzenethiol and
B
are 1H-imidazo[4,5-
3
11
f][1,10]phenanthroline (L ) and 1-(pyren-2-yl)-1H-imidazo[4,5-
4
®
f][1,10]phenanthroline (L ) were studied in detail in SAR-based
approach to explore the role of S-coordination to Cu(II) in PDT to
HeLa cells. Although all the complexes were toxic in dark (IC50 ~ 10
µM), we observed enormous enhancement (~10 fold) in cytotoxicity
as first-generation PDT drugs along with other porphyrins or
pthalocyanines.12 Although severe hepatotoxicity and prolonged
®
skin photo-sensitivity associated with Photofrin and other side
-
2
of the complexes in visible light (400-700 nm, 10 Jcm ) resulting in
IC50 ~ 1.0 µM. Intracellular ROS generated in HeLa cells during photo-
effects related to the tumor non-specificity, PDT has lost clinical
1
3
acceleration. Photo-activated transition metal complexes, often
associated with ligand exchange, intramolecular redox reaction or
generation of various ROS through energy transfer to molecular
oxygen on photo-activation, are of particular interest in
activation of the complexes was probed by DCFH-DA assay. Degree
1
of photo-cytotoxicity of the complexes was related to the
O
2
generation. S-coordinated complexes exhibiting enhanced photo-
cytotoxicity was due to the presence of low-lying, long-lived triplet
14-17
developingmetal-based photo-chemotherapeutic agents.
Low
1
3
excited state and hence increased ability to generate O
2
from O
2
in
energy UV-visible absorption (>600) nm by the complexes is one
of the essential criteria for photo-chemotherapeutic applications
considering greater tissue penetration of longer wavelength light.
Recently complexes of bio-compatible first row transition metals
with low energy charge transfer (CT) or d-d bands are extensively
type-II photo-process. We observed dual photo-sensitization of S-
coordination and pyrenyl moiety for the complex 4 leading to
remarkable PDT effect in HeLa cells with IC50 ~ 0.3 µM and the nature
of cell death was apoptotic in nature. Overall SAR-based approach in
modulating the in vitro photodynamic activity of the copper(II) is of
paramount importance is designing copper-based next generation
PDT agents.
1
8-20
explored for visible light-induced cytotoxicity.
Copper being
one of the non-toxic, bio-essential element, photo-
chemotherapeutic applications of copper-based complexes
would be an interesting and attractive option. Several copper(II)
1
complexes are already explored for singlet oxygen ( O
2
) mediated
21-22
photobiological investigations.
However, toxicity of the
Introduction
copper(II) complexes in dark itself have limited their applications
as the ideal photosensitizers in photodynamic therapy (PDT).
Therefore, it become important to explore copper(II) complexes
for further PDT applications by modulating the photo-chemistry of
the copper(II) complexes. In our present studies in probed the
degree of in vitro photodynamic activity of the copper(II)
complexes in SAR-based approach to explore the role of Sulphur.
Here in we report the synthesis, analytical characterization,
photo-physical studies and theoretical evaluation of singlet
oxygen generation on photo-activation, cellular localization,
photo-cytotoxicity studies in HeLa cells, photo-activated ROS
generation of several new ternary copper(II) complexes
Transition metal complexes with wide range of coordination
number and geometry, accessible redox states, tunable
thermodynamic and kinetic of ligand-exchange, isomerization,
facile electron transfer reactions have emerged as attractive class
1
-5
of compounds for chemotherapeutic applications.
Systemic
side effects in chemotherapy due to high dose requirement and
tumor non-specificity caused a serious concern leading to search
for cost-effective, more tolerable and tumor specific treatment
6
,7
modality for cancer.
Photodynamic Therapy (PDT) has
emerged as a tumor specific, non-invasive treatment modality
1
2
1
[
2
Cu(L )B]ClO
4
(1-2) [Cu(L )B]ClO
4
(3-4), where L = 2-((pyridin-
2
-yl)methyleneamino)phenol,
L
=
2-((pyridin-2-
yl)methyleneamino)benzenethiol and B are 1H-imidazo[4,5-
[
a]
Department of Chemistry, National Institute of Technology Manipur
Langol, Imphal West, Pin: 795004 (INDIA)
3
f][1,10]phenanthroline (L ) and 1-(pyren-2-yl)-1H-imidazo[4,5-
E-mail: mithunroy@nitmanipur.ac.in
4
f][1,10]phenanthroline (L ) to explore the role of sulphur and
[
[
b] Department of Inorganic and Physical Chemistry
oxygen coordination to Cu(II) in photocytotoxicity (Scheme 1).
Nanomolar level visible light induced cytotoxicity of the copper(II)
complexes with theoretical and spectroscopic exploration to
probe the role of sulphur is unprecedented and remarkable in
chemistry of metal-based PDT.
Indian Institute of Science, Bangalore
CV Raman Avenue, Bangalore-560012 (INDIA)
UGC-DAE Consortium for Scientific Research, Kolkata Centre,
III/LB-8 Bidhannagar, Kolkata 700098, (INDIA)
c]
Supporting information for this article is given via a link at the end of
the document.
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European Journal of Inorganic Chemistry
10.1002/ejic.201800081
FULL PAPER
infrared spectra along with the characteristic broad NHstr at
-
-
1
1
~
2990-3020 cm and C=Nstr at ~1590-1610 cm . In addition,
-
1
Cu-Sstr, Cu-Nstr at 570, 510 cm respectively were also identified
2
4
in the solid-phase IR spectra of the complexes (Figure S5).
Electronic spectra of the ligands and complexes (1-4) were
recorded in H O/DMF (10% DMF v/v) (Figure1, Table 1, Table S1,
2
Figure S6). Complexes (1-4) were characterized with broad and
weak d-d bands in the range of 670-750 nm in addition to an
intense band near 480 nm for the complexes 1-2 and 550 nm for
the complex 3-4 assignable to LMCT transitions. Highly intense
ligand-centered bands assignable to -* or n-* transitions
were observed in the UV region of the electronic spectra of the
complexes. Optimized structure, HOMO, and LUMO
stereographs determined by DFT calculation were presented in
Figure S7. Detail of orbital contribution, energetics and other
related parameters obtained from TD-DFT calculations were
presented in the supplementary section (Figure S8, Table S2).
Low energy d-d bands >700 nm for the complexes 2 and 4 along
with highly intense MLCT/LMCT bands prompted us to utilize
such transitions for photodynamic application in present studies.
Normalized emission spectra ligands and the complexes (1-4)
Scheme 1. Schematic representation of the complexes (1-4).
2
was recorded in H O/DMF (10% DMF v/v) at room temperature
(
4
Figure S9). Although the ligands were highly emissive in 430-
70 nm on UV-A light excitation, paramagnetic complexes display
typically poor luminescence in 430-460 nm under the same
condition with luminescence quantum yield (, 0.01-0.06).
Pyrenyl complexes having comparatively higher luminescence
3
attributable to the IL emission of the pyrene core (Table 1). This
assignment was supported by the spin density calculation of T
1
state of the complex (Figure 5). Reduced fluorescence intensity
of the complexes could be due to the paramagnetic quenching
effect of copper(II) center. However, we explored the blue
luminescence of the complexes to probe the cellular uptake.
Figure 1. UV-visible spectrum of the complexes 1 - 4 in 10% v/v DMF-H
room temperature with the inset showing metal centered d-d bands.
2
O at
Solubility and stability
Complexes as the perchlorate salt were readily soluble in 10%
Results and Discussion
(
v/v) aqueous DMF along with other solvents like MeCN, MeOH,
DMSO. Complexes were less soluble in water. Stability of the
complexes was determined by recording UV-visible spectral
traces in different time interval till 72 hrs. Complexes were stable
in aqueous-DMF (10% v/v) medium up to 72 hrs in the dark.
Synthesis, characterization and general aspects
Complexes (1-4) were prepared from methanolic solution of
1
2
Cu(NO
3
)
2
.3H
2
O on slow addition of N,N,O (L ) or N,N,S (L ) donor
3
ligands followed by the addition of N,N-diimine-based ligands (L ,
Triplet excited state and luminescence life-time
4
L ). Complexes were isolated or precepted as perchlorate salt
with ~60% yield and the complexes were dried in vacuo over
We probed the existence of low-lying triplet excited state of the
complexes. Triplet-triplet annihilation (TTA) upconversion
requires triplet photo-sensitizer for absorbing of the excitation
energy and triplet acceptor for the upconverted emission. The
energy is transferred from the photosensitizer to the acceptor by
triplet-triplet energy transfer (TTET) process followed by the
singlet excited state of the acceptor is produced and the emission
intensity of the acceptor increased. Typical increase emission
spectrum of perylene in MeCN was observed when perylene
solution was exposed to the visible light (400-700 nm) in the
presence of the complexes (3, 4) (Figure 2a, Figure S10). We
assumed that when the complexes were photo-activated, those
4
P O10 after washing. Elemental analysis revealed that the
complexes were obtained in analytically pure form and later used
for characterization and further studies. Complexes were
characterized analytically and spectroscopically (Table S1).
+
Single molecular ion characterized by [M-ClO
4
]
or [M-H-
+
+
ClO ]Na /K along with other fragments were identified in Q-TOF
ESI mass spectra of all the complexes recorded in acetonitrile
4
(
Figure S1-S4). Molar conductance values of all the complexes in
aqueous DMF (10% DMF v/v) were typically ranges in 110-125
mS indicating 1:1 electrolyte corresponding to the complex salt
2
3
[
M]ClO
4
where M is the complex cation. All the complexes
display characteristic sharp ClO
4
stretching at ~1090 cm-1 in the
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European Journal of Inorganic Chemistry
10.1002/ejic.201800081
FULL PAPER
quickly relaxed into the low-lying triplet excited state which was
moiety have sufficiently longer luminescence life-time due to the
rich photo-physical properties of pyrene.
2
5
quenched by perylene resulting in emissive singlet perylene.
Emission spectra recorded for perylene in the presence of the
complexes in dark was considered as negative control. Relative
intensity of the complexes indicated the extent of inter-system
crossing in the photo-activated complexes. Higher emission
intensity of triplet perylene in the presence of 4 indicated effective
inter-system crossing into triplet excited state for complex 4.
Table 1. Selected photo-physical data for the complexes (1–4).
Complexes
1
2
3
4
-

cm
ex/nm(,Lmol
482(18000),
670sh (150)
427
518(3350),
695sh (75)
451
525(270),
740sh (470)
445
476(15000),
725sh(220)
460
1
-1 [a]
)
[
b]

Ф
em / nm
[
c]
0.011
0.219
6.21
0.047
0.303
12.13
0.017
0.273
7.56
0.059
0.381
16.45
1
[d]
Ф( O
2
)
Figure 3. Plot of the relative change of the absorbance of DPBF at 417 nm
[e]
τ
L
/RT(ns)
(
A/A )) with time of visible light exposure treated with the complexes (1-4),
0
showing a linear decrease of the DPBF absorbance indicating photo-induced
generation of singlet oxygen in type II photo-process.
[a]10% v/vH
O-DMF at 20 °C; [b]10% v/vH
O-DMF at 20 °C; [c]fluorescence
, 0.27 in ethanol)
2
2
quantum yields of the complexes in MeOH and anthracene (Φ
F
[
d]
as standard; singlet oxygen quantum yield of the complexes with DPBF and
Rose Bengal as standard in DMSO at 20 °C; [ Luminescence lifetime of the
complexes in MeOH at RT[ex(laser), 375 nm].
e]
1
Singlet oxygen ( O
2
) generation
Later we probed the generation of singlet oxygen on photo-
-2
activation (Visible light, 400-700 nm, 10 Jcm ) of the complexes
by using diphenylisobenzofuran (DPBF), Rose Bengal as a
2
6
standard (Figure 3, Figure S12, Figure S13). UV-visible spectral
titration was carried out to record the change in absorbance (A417
of DPBF (50 µM) at max = 417 nm in aqueous DMSO against
)
-2
visible-light exposure (400-700 nm, 10 J cm ) time in the
presence of complexes (50 µM). Gradual decrease in A417 of
DPBF indicated photo-induced production of singlet oxygen from
complexes. Several control experiments with DPBF alone and
DPBF in the presence of the complexes in dark indicated negative
1
response. Measure of singlet oxygen ( O
2
) by the complexes was
indicated by the extent of decrease in absorbance (A417) of DPBF
on photo-activation. Relative singlet oxygen generation by the
photo-activated complexes was graphically represented by
Figure 2. (a) Upconversion emission spectra of mixture of the photo-activated
complexes (3, 4) and acceptor (Perylene) recorded in MeCN indicating the
presence of triplet excited state in the complexes; [complex] = 50 µM, [Perylene]
plotting the extent of decrease of absorbance of DPBF (A/A
0
)
=
250 µM; Inset shows blue fluorescence images (i) perylene + visible light, (ii)
against photo-exposure time (t/min), where
A
was the
was the
perylene + 4 in dark, (iii) perylene + 4+ visible light, ex = 360 nm; (b) Luminiscence
decay of the complexes 3 and 4 in MeOH in RT [ex(laser), 375 nm].
absorbance of DPBF at particular time while A
0
absorbance of DPBF at t = 0 min. We observed almost linear
decrease of absorbance of DPBF with exposure time indicating
photo-induced generation of 1O
from 3
O via type II photo-
2 2
We have determined the steady state luminescence life-time of
the ligands and the complexes (1-4) at ambient temperature in
MeOH (Figure 2b, Figure S11). First order luminescence decay
of the ligands and the complexes gave the life-time in nano
second scale (Table 1). Although luminiscence ligands are
deactivated through radiative decay with typical life-time of 5-8
ns under the ambient condition. Complexes, however, exhibited
longer luminescence life-time in the range 6.21-16.45 ns. Longer
luminescence life-time of the complexes could be speculated as
the presence of low-lying triplet excited state in the complexes,
although we lack other, enough evidences in favor of the
presence of triplet excited state. Complexes 2 and 4 with pyrene
process. The plot qualitatively explained higher amount of singlet
oxygen generation from the S-coordinated complexes. Complex
4
having pyrenyl moiety generated highest amount of singlet
oxygen. Subsequently we quantified the singlet oxygen
generation by the photo-activated complex in terms of quantum
yield () that was determined with reference to Rose Bengal in
DMSO at room temperature (Table 1, Figure S13).22 The singlet
oxygen quantum yield was determined to be in the range 0.2-0.4
for the complexes (1-4). Complex 4 generated singlet oxygen
1
(
2
O ) to the significant amount, enough to induce PDT effect to
the cancer cells.
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European Journal of Inorganic Chemistry
10.1002/ejic.201800081
FULL PAPER
Figure 4. Proposed type-II photo-process and energetics in the complexes (1-4) on photo-activation.
1
TD-DFT calculations on O
2
generation
presence of relatively low-lying, long-lived triplet excited states
for the complexes, those are rapidly quenched by triplet oxygen
(3O
) and generated singlet oxygen ( O
1
) by energy transfer
and T
Time-dependent density functional theory (TD-DFT) calculations
on the excited state of Cu(II) complexes (1-4) were carried out
using unrestricted B3LYP density functional theory. Basis sets
were employed by using 6-31G(d,p) for H, C, N, S, O atoms and
2
2
process. Hence, low energy gap (0.17 eV) between D
1
1
facilitating faster intersystem crossing, sufficiently long-lived
3
triplet excited state (T
to generate O
1
) (, 27 ns), efficient quenching of T
1
by O
2
2
7
1
LANL2DZ for Cu atom in the gas phase. Initially we searched
for the lowest energy excited triplet and doublet states using their
corresponding roots followed by the determination of vertical
excitation energies and oscillator strength. Further we examined
the presence of lowest excited triplet state in all the complexes by
carrying out self-consistent, unrestricted B3LYP calculations both
at the ground-state geometry as well as geometrically optimized
triplet state. The vertical excitation energies of lowest doublet and
triplet states of the complex 2 were 2.06 eV, 1.53 eV and 1.67 eV,
2
with high quantum yield (, 0.4) made complex
4 for potential candidate in photodynamic therapy.
Table 2. Selected low-lying excited states, Calculated Energies (E), and
Dominant Orbital Excitation from TD-DFT Calculations for all Cu(II)
complexes low-laying doublet and triplet state Calculated at
TDDFT/B3LYP/6-31G(d,p)/LanL2DZ level.
State
Orbitalexcitation
Character
-E[a] (a.u)
1
.45 eV for the complex 4. The nature of the low-lying excited
1
2
3
4
T
1
HOMO2(β)-LUMO(β)(97%)
HOMO(β)-LUMO5(β)(28%)
HOMO6(β)-LUMO(β)(25%)
HOMO5(β )-LUMO(β) (96%)
HOMO5(β)-LUMO2(β) (40%)
HOMO(β)-LUMO3(β) (40%)
HOMO2(β)-LUMO(β) (31%)
HOMO1(β)-LUMO2(β)(46%)
MLCT
1550.179
1550.142
2177.225
2177.209
1486.443
1486.417
2112.120
2112.114
states was determined to be typically MLCT and ILCT type.
Corresponding HOMO had significantly metallic character while
LUMO was localized in the ligands. TD-DFT approach to derive
vertical excitation energies was compared with the experimental
D
1
ILCT
T
1
ILCT/MLCT
MLCT
D
1
T
1
ILCT/MLCT
ILCT/MLCT
ILCT
spectroscopic data. Doublet excited (D
absorption of visible light (D ) efficiently populated further
the triplet excited state (T ) via strong inter-system crossing.
1
) state, populated upon
D
1
0
→D
1
T
1
1
D1
LMCT/ILCT
Energy difference between low lying doublet excited and triplet
[a]The energy value corresponds to the absolute energy of the respective
states.
excited states of complexes (1-4) were 0.99, 0.41, 0.69 and
0
.174 eV (Table 2, Figure 4). Consequently, we expected better
and faster inter-system crossing for the pyrenyl complexes (2 and
4
). This is even more pronounced in S-coordinaed complexes.
Long-lived triplet excited state in complex 4 was efficiently
quenched by molecular oxygen in generating singlet oxygen.
Spin density calculation in optimized triplet excited state of the
complexes (1-4) revealed the localization of spin density in
pyrenyl moiety for complexes 2 and 4 indicating typically IL state
of the triplet excited state (T
time for the complex 4 (Figure 5).
1
) and probing relatively longer life-
25
Based on the results obtained on photo-physical studies and TD-
DFT calculations for the photo-activated complexes, we assumed
a type II photo-process leading to the generation of singlet
Figure 5. Isosurfaces of the spin density of the complexes (1-4) for optimized
1
3
oxygen ( O
2
) from
O
2
by energy transfer. We observed the
T
1
excited-state geometry (isovalue
±
0.0004)
calculated at TD-
DFT/B3LYP/6-31G(d,p)/LanL2DZ level in gas phase with Gaussian 09W.
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European Journal of Inorganic Chemistry
10.1002/ejic.201800081
FULL PAPER
Cytotoxicity and Photo-dynamic effect
The cytotoxicity of the complexes (1-4) along with the ligands was
studied by MTT assay in HeLa (human cervical cancer) cells
(
Table 3, Figure 8, Figure S14) and the IC50 values for the
complexes 1-4 obtained by non-linear regression analysis of the
dose-response curve are listed in Table 3. Cells were initially
incubated with the complexes in dose dependent manner at 37C
for 4 h in the dark followed by photo-irradiation with visible light
-2
(
400-700 nm, 10 Jcm ) for 1 h. Proper dark control was
maintained under similar experimental condition the photo-
irradiation. We observed toxicity of the complexes in HeLa cells
with the IC50 values of ~10 µM and their cytotoxicity could be
related to their ability to alter GSH levels and changes in the
mitochondrial membrane potential or GSH could readily reduce
Cu(II) to Cu(I) and generated toxic ROS from molecular oxygen.
The dark toxicity of the copper(II) complexes were of no exception
3
4
Figure 6. Cellular incorporation of L , L and complexes 2 and 4 in HeLa cells.
3
4
FACS analysis of cellular uptake of L , L and complexes 2 and 4 into HeLa
cells in 4hwith colour code Black, HeLa cells only; light blue, HeLa cells + L ;
green, HeLa cells + L ; red, HeLa cells + 2; blue, HeLa cells + 4
3
4
2
4
from other copper(II)-based cytotoxic compounds. Ligands or
the copper(II) salt were not cytotoxic to HeLa cell at all.
Table 3. Cytotoxicity data (IC50/M) of the complexes in dark and on
visible light exposure
Complexes
HeLa cells
IC50 / µM
Dark[a]
Light[b]
1
2
3
4
9.2(±0.7)
9.5(±0.6)
8.7(±0.4)
12.6(±1.0)
2.5(±0.6)
1.0(±0.5)
1.0(±0.3)
0.3(±0.1)
Figure 7. Cellular incorporation of complexes 2 and 4 in HeLa cells. Confocal
images of the HeLa cells treated with complexes showing intracellular
localization of the complexes (a) Bright Field images of HeLa cells, (b) HeLa cells
treated with propidium iodide (PI), (c) HeLa cells treated with complex 4, (d)
Merged. Panels (e) – (f) are the confocal image of HeLa cells while treated with
PI and complex 2.
Cu(NO
3
)
2
.3H
2
O
>100
>100
Cellular incorporation and uptake
63.4 (±3.8)
56.2 (±4.2)
L3
L4
ND
24
ND
We explored the blue luminescence of the complexes (2 and 4)
to study the cellular localization. Incubation time for in vitro studies
before light irradiation was standardized by cellular incorporation
assay. Cellular uptake of the complexes 2 and 4 was studied by
FACS analysis in HeLa cells (Figure 6). The experimental result
indicated almost complete drug incorporation of the complexes in
cisplatin
>
41
4.3 (±0.2)
Photofrin®
[a]IC50 values correspond to 24
correspond to 4 h incubation in the dark followed by photoexposure to visible
light (400-700 nm,10 J cm- ).ND: Not determined due to insolubility
h
incubation in dark. [b]IC50 values
2
4h of incubation. However, luminescent ligands were not
incorporated in the cells. Confocal microscopy for the complexes
confirm primarily cytosolic localization of the complexes in HeLa
cells on merging the fluorescence of the complexes and
propidium iodide (PI) (nucleus targeting dye) after 4h of
incubation (Figure 7).
However, important finding in this work is the enhancement (5-10
fold) in cytotoxicity to HeLa cells when exposed to the visible light
in the presence of the complexes (1-4) (Table 3, Figure 8). We
followed SAR-based approach in photocytotoxicity of the
complexes and S-coordinated complexes (3-4) were found to be
more photo-responsive in comparison to the O-coordinated
copper(II) complexes (1-2). We observed even nanomolar level
cytotoxicity for the complex 4 with IC50 value 0.3 µM in visible light
and this is a remarkable enhancement (~40 fold) in comparison
to the cytotoxicity in dark (IC50, 12.6 µM). Another aspect of the
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European Journal of Inorganic Chemistry
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FULL PAPER
study is the higher photo-cytotoxicity of the pyrenyl (2 and 4)
compared to the non-pyrenyl complexes (1 and 3).
A greater shift signified a higher amount of oxidized DCF and thus
greater intracellular ROS generation. We observed similar shift
fluorescence band intensity of DCFH-DA in HeLa cells treated
with the complexes in dark indicating generation of ROS. Further
shift in flurescence intensity of DCF in HeLa cells treated with
photo-activated copper(II) complexes indicated enhanced
generation of intracellular ROS with highest shift for the complex
4
as it was already probed to generate high amount of singlet
oxygen (ROS) when photo-activated and hence proved to the
most photo-cytotoxic relative to the other complexes.
Apoptosis
Fluorescence microscopy was performed to explore the nature of
cell death by observing the change in nuclear morphology of the
cancer cells treated with complex 4 (5.0 µM) in the presence of
DNA targeting fluorescent dye like DAPI (Figure10). Treatment of
the HeLa cells with the complex 4 for 4 h in the dark and visible
light exposure (1 h) exhibited characteristic changes in the
nuclear morphology like shrinkage in cell volume and membrane
blabbingin addition to the condensed or convoluted nuclei of DAPI
stained HeLa hallmarking the apoptosis in HeLa cells.
Figure 8. Cell viability (MTT assay) plots showing the cytotoxicity of the
copper(II) complexes (1 - 4) in Human Cervical Carcinoma (HeLa) cells in the
dark (black symbols) and in the presence of visible light (red symbols, 400-700
-
2
nm, 10 J cm ). The number inside the plot indicate the complexes.
Figure 10. DAPI nuclear staining of untreated and complex 4 (5 µM) treated
HeLa cells permeabilized with 0.1% Triton X-100 and fixed with
paraformaldehyde (4%) in dark as well as 1 h after photoexposure (400-700 nm,
1
0Jcm- ) to identify nuclear morphology: (a) untreated cells, in dark (b) treated
2
with 4, in dark (c) treated with 4, in visible light.
Figure 9. Shift in band positions of complexes 2 and 4 in HeLa cells in light
-
2
(
400–700 nm, 10 J cm ) with colour code: purple, cells only; green, cells +
DCFDA; magenta, cells + DCFDA+ 2 in dark; yellow, cells + DCFDA + 4 in dark;
red, cells + DCFDA + 2 in visible light; blue, cells + DCFDA + 4 in visible light
Conclusions
(
400–700 nm).
Overall, our present study investigated the role of S-coordination
to copper(II) in modulating the photodynamic effect of the
copper(II) complexes to HeLa cells. We observed remarkable
enhancement in cytotoxicity of the complex 4 in HeLa cells when
ROS generation in vitro
The intracellular ROS generation by the complexes on photo-
activation was probed by using non-polar cell permeable 2’,7’-
dichlorofluorescein diacetate (DCFH-DA). The dye after
hydrolysis by the intracellular esterases was accumulated inside
the cells. The hydrolysed dye on oxidation by intracellular ROS or
ROS generated in situ, is transformed into 2’,7’-
dichlorofluorescein (DCF) which is highly fluorescent emitting
green light. Intracellular ROS generation using DCFH-DA was
studied by flow cytometric analysis (FACS) on HeLa cells (Figure
-2
exposed to visible light (400-700 nm, 10Jcm ). Photo-cytotoxicity
of the copper(II) complexes were related to the photo-activated
1
2
generation of singlet oxygen ( O ). Several photo-physical
studies like quenching of DPBF, determination of singlet oxygen
quantum yield, luminescence life-time measurements and TD-
DFT calculations signified the type II photo-process for the
copper(II) complexes in generating singlet oxygen as the key
cytotoxic species leading to apoptosis in HeLa cells. Presence of
low-lying and long-lived triplet excited state in complex 4 facilitate
effective intersystem crossing into the triplet excited state which
efficiently transfer its energy to triplet oxygen into cytotoxic singlet
9). Shift in fluorescence intensity compared to cells alone or only
DCFH-DA gave us the measure of intensity of intracellular ROS.
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European Journal of Inorganic Chemistry
10.1002/ejic.201800081
FULL PAPER
oxygen with significantly high 1O
quantum yield leading to
2
-1
525 (2735) 740sh (466) (sh, shoulder). 
10 at 298 K.
M 2
(Sm mol ) in 10% v/v DMF-H O:
2
1
remarkable photo-cytotoxicity of the complex 4. SAR-based
approach in modulating the photo-cytotoxicity of the copper(II)
complexes is pivotal in developing next generation copper(II)-
based PDT agents.
6 4
Anal. Calc. for C41H25ClCuN O S (4): Calculated C,61.81; H, 3.16; N, 10.55.
-
1
Found: C, 60.25; H, 3.07; N, 10.59; FT-IR, cm : 3375br (H
2
O), 3012br (NH),
-
1594s (C=N), 1304s, 1021s (ClO
4
), 854m, 765m, 576m (Cu-S), 503m
+
(
Cu-N). ESI-MS in MeCN: m/z696.1157 [M-(ClO4)] . UV-visible in 10% v/v
3
-1
DMF-H
2
O [max, nm (, dm mol-1 cm )]: 476 (14998), 726 sh (216), (sh,
2
-1
Experimental Section
M 2
shoulder).  (Sm mol ) in 10% v/v DMF-H O: 120 at 298 K.
Materials and measurements
CAUTION! The perchlorate salts could be potentially explosive. Therefore,
only small quantity of the sample was handled to avoid any possible
explosion
The reagents and all chemicals were obtained from Sigma-Aldrich (USA),
SD-Fine chemicals (India), HIMEDIA and used as received without any
further purification. DCFH-DA, diphenylisobenzofuran (DBPF), 4',6-
diamidino-2-phenylindole
aminobenzenethiol,
(DAPI),
pyridin-2-carboxaldehyde,
1,10-phenanthroline,
2-
Acknowledgements
2-Aminophenol,
formaldehyde and pyrene-1-carbaxyldehyde were procured from Sigma
Acknowledgements Text. We thank the Science and Engineering
Research Board, Government of India, New Delhi for financial
(
USA). A reported synthetic procedure was used to synthesize 1,10-
1
phenanthroline-5,6-dione, 2-(Pyridin-2-ylmethyleneamino)-phenol (L ), 2-
{
f][1,10]phenanthroline and
f][1,10]phenanthroline (L ) with minor modification.
[(Pyridin-2-yl)methyl]amino}benzenethiol
(L2),
1H-imidazo[4,5-
support [ECR-2016-000839/CS] and NIT Manipur for providing
infrastructure. We sincerely thank Prof. Akhil R. Chakravarty, IISc
Bangalore for providing the facility of cellular studies. We thank
Prof. Abhijit Saha, UGC-DAE Consortium for Scientific Research,
Kolkata for measuring luminescent decay for the complexes.
(L3)
1-(pyren-2-yl)-1H-imidazo[4,5-
4
28-30
Detail of synthesis
of ligands, analytical and photo-physical measurements, cell viability assay,
ROS assay, nuclear staining can be found in supplementary section.
Synthesis
Keywords: Ternary copper(II) complexes•Modulation of in vitro
PDT effect•Role of sulphur•Singlet Oxygen•Photo-physical
studies and TD-DFT
General procedure for synthesis of the complexes
Complexes 1-4 were prepared by a general procedure in which 0.2 g (1
mmol) Cu(NO
3
)
2.3H
2
O in 10 mL of MeOH was mixed with Schiff-Base
1
2
ligands (L , 0.24g, 1.2 mmol; L , 0.26g, 1.2 mmol) pre-dissolved in 10 mL
MeOH followed by the slow addition of NN-diimine heterocyclic base (1H-
imidazo[4,5-f][1,10]phenalthroline (0.18g, 0.8 mmol); 2-(pyrene-1-yl)-1H-
imidazo[4,5-f][1,10]phenalthroline (0.34g, 0.8 mmol) with constant stirring
at room temperature for 30 min. The reaction mixture was stirred for 1 h,
and the product was isolated as a light green solid in approximate 70%yield
on addition of a methanolic solution of NaClO4 (1.0 mmol,0.12 g). The solid
was isolated in analytically pure form, washed with cold methanol, and
[1]
T. C. Johnstone, K. Suntharalingam,S. J. Lippard, Chem. Rev. 2016, 116,
436.
3
[2]
[3]
[4]
[5]
D. M. Cheff, M. D. Hall, J. Med. Chem. 2017, 60, 4517.
K. D. Mjos, C. Orvig, Chem. Rev. 2014, 114, 4540.
C. S. Allardyce, P. J. Dyson, Dalton Trans. 2016, 45, 3201.
A. Mukherjee, P. J. Sadler, Wiley Encyclopedia of Chemical Biology,
2009, John Wiley & Sons, Inc, doi: 10.1002/9780470048672.wecb333
[6]
[7]
[8]
https://www.cancer.org/treatment/treatments-and-side-effects.html
S. S. Lucky, K. C. Soo, Y. Zhang, Chem. Rev. 2015, 115, 1990.
(a) G. Mion, C. Mari, T. Da Ros, R. Rubbiani, G. Gasser,
T.Gianferrara,Chemistry Select 2017, 2, 190. (b) Bonnett, Photodynamic
Action, Chemical Aspects of Photodynamic Therapy, vol. 1, Gordon &
Breach, London, U.K, 2000, 57.
4
finally dried in vacuo over P O10.
6 5
Anal. Calc. for C25H17ClCuN O (1): Calculated C, 51.73; H, 2.95; N, 14.48.
Found: C, 51.05; H, 2.89; N, 14.52; FT-IR (KBr phase; br, broad; vs, very
-
1
strong; s, strong; m, medium; w, weak), cm : 3212br (H
2
O), 3010br (NH),
-
1
5
1
599s (C=N), 1310s, 1037s (ClO
4
) ,1010w, 832m, 759m, 542m (Cu-S),
09m (Cu-N). ESI-MS in MeCN: m/z 480.0760 [M-(ClO4)] . UV-visible in
[9]
(a) D.E.Dolmans, D. Fukumura R.K. Jain, Nat. Rev. Cancer 2003, 3, 380.
(b) T. J. Dougherty, C. J. Gomer, B. W. Henderson, G. Jori, D. Kessel, M.
Korbelik, J. Moan, Q. Peng, J. Natl. Cancer Inst. 1998, 90, 889.
+
3
-1
-1
0% DMF-H
2
O [max, nm (, dm mol cm )]: 482 (18014), 670sh (153),
O: 112 at 298 K.
M
2
-1
(
Sm mol ) in 10% v/v DMF-H
2
[10] (a) J.P. Celli, B.Q. Spring, I. Rizvi, C.L. Evans, K.S. Samkoe, S. Verma,
P.W. Pogue, T. Hasan, Chem. Rev. 2010, 110, 2795. (b) J. D. Knoll, C.
Turro, Coord. Chem. Rev. 2014,17.
6 5
Anal. Calc. for C41H25ClCuN O (2): Calculated C, 63.08; H, 3,23; N, 10.77.
-
1
[11] R. Lincoln,L. Kohler,S. Monro, H.Yin,M.Stephenson,R. Zong, A.Chouai,C.
Dorsey,R. Hennigar,R.P. Thummel, S. A. McFarland, J. Am. Chem. Soc.
2013, 135, 17161.
Found: C, 62.88; H, 3.11; N, 10.68; FT-IR, cm : 3363br (H
2
O), 2995br (NH),
-
1
(
583s (C=N), 1304s, 1427m ,1010s (ClO
4
), 770m, 556m (Cu-S), 515m
Cu-N). ESI-MS in MeCN: m/z 680.1386 [M-(ClO4)] . UV-visible in 10% v/v
+
3
-1
-1
(Sm2
[12]
P. Ung,M. Clerc, H. Huang,K. Qiu,H.Chao,M.Seitz, B. Boyd,B.
DMF-H
2
O [max, nm (, dm mol cm )]: 518(3349), 695sh (73). 
O: 125 at 298 K.
M
-1
Graham,G.Gasser, Inorg. Chem. 2017, 56, 7960.
mol ) in 10% v/v DMF-H
2
[
13] (a) M.J. Ochsner, Photochem. Photobiol. B, 1996, 32, 3. (b) The data was
obtained from FDA (Food and Drug
Administration),http://www.accessdata.fda.gov/drugsatfdadocs/label/20
8/020451s019lbl.pdf.
Anal. Calc. For C25
H
17ClCuN
6
O
4
S (3): Calculated C, 50.34; H, 2.87; N,
-
1
14.09. Found: C, 50.11; H, 2.78; N, 14.12; FT-IR, cm : 3260br (H
2
O),
0
-
3020br (NH), 1596s (C=N), 1321s,1106m ,1010m(ClO
4
), 832m, 757m,
[
14] (a) T. C. Johnstone, K. Suntharalingam, S. J. Lippard, Chem. Rev. 2016,
116, 3436. (b) Md. K. Raza, S. Gautam, A. Garai, K. Mitra, P. Kondaiah,
A. R Chakravarty,Inorg. Chem., 2017, 56, 11019.
+
562m (Cu-S), 448m (Cu-N). ESI-MS in MeCN: m/z 496.0531 [M-(ClO4)] .
3
-1
-1
UV-visible in 10% v/v DMF-H O [max, nm (, dm mol cm )]: 416 (8686),
2
This article is protected by copyright. All rights reserved.

European Journal of Inorganic Chemistry
10.1002/ejic.201800081
FULL PAPER
[
15] (a) I. Romero-Caneló and P. J. Sadler, Inorg. Chem. 2013, 52, 12276. (b)
N.J. Farrer,L. Salassa,P. J. Sadler, Dalton Trans. 2009, 10690. (c) Md. K.
Raza, K. Mitra, A. Shettar, U. Basu, P. Kondaiah, A. R. Chakravarty,
Dalton Trans. 2016, 45,13234.
2560. (c) J. Zhao, S. Ji, H. Guo, RSC Adv. 2011,1, 937. (d) W. Wu, J.
Zhao, H. Guo, J. Sun, S. Ji,Z. Wang, Chem. Eur. J. 2012, 18, 1961.
(a) J. Sun, F. Zhong, X. Yi and J. Zhao, Inorg. Chem. 2013,52, 6299;(b)
W. Wu, J. Zhao, H. Guo, J. Sun, S. Ji and Z. Wang, Chem. Eur. J. 2012,18,
1961.
[26]
[16]
M. Frezza,S. Hindo,D. Chen,A. Davenport,S. Schmitt,D. Tomco, Q. P.
Dou, Curr. Pharm. Des. 2010, 16, 1813.
[27] (a) M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb,
J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson,
H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino,
G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J.
Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T.
Vreven, J. A. Montgomery Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J.
Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J.
Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J.
Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J.B. Cross,
V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O.
Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin,
K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J.
Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V.
Ortiz, J. Cioslowski, and D. J. Fox,Gaussian 09, 2009, Rev. A.02.
Gaussian, Inc., Wallingford; D. D. Roy, A. K. Todd, and M. M. John,
Gauss View 5.0.8., 2009,Gaussian, Inc., Wallingford; A. D. Becke, J.
Chem. Phy., 1993, 98, 5648. (b) GaussView 5.0 (Gaussian Inc.,
Wallingford, CT, USA), 2009.
[
17] U. Schatzschneider, Eur. J. Inorg. Chem. 2010, 1451.
[18]
U. Basu, I. Khan, A. Hussain, P. Kondaiah, A.R. Chakravarty, Angew.
Chem. Int. 2012, 51, 2658.
[19] (a) S. Banerjee, A.R. Chakravarty, Acc. Chem. Res., 2015, 48, 2075; (b)
A.R. Chakravarty and M. Roy, in: K.D. Karlin (Ed.), Prog. Inorg. Chem.,
2012,57, 119.
[
20] S. B. Chanu, S. Banerjee, M. Roy, Eur. J. Med. Chem., 2017, 125, 816.
21] (a) N. Mukherjee, S. Poddar, S. Banerjee, S. Majumdar, D. Nandi, Eur. J.
Med. Chem. 2016, 122, 497. (b) S. Dhar, D. Senapati, P. K. Das, P.
Chattopadhyay, M. Nethaji, A. R. Chakravarty, J. Am. Chem. Soc. 2003,
[
1
25, 12118. (c) S. Banerjee, A. Dixit, K. S. Maheswaramma, B. Maity, S.
Mukherjee, A. Kumar, A. Karande, A. R. Chakravarty, J. Chem. Sci. 2016,
28, 165.
(a) R. Lin, C. Chiu, C. Hsu, Y. Lai, P.Venkatesan, P. Huang, P. Lai,
C,Lin, Chem.Eur.J.2017,10.1002/chem.201705640. (b) A.
1
[22]
Bhattacharyya, A. Dixit, S. Banerjee, B. Roy, A. Kumar, A. A. Karande, A.
R. Chakravarty, RSC Adv. 2016, 6, 104474.
[
[
[
23] (a)T. K. Goswami, S. Gadadhar, M. Roy, M. Nethaji, A. A. Karande, A. R.
Chakravarty, Organometallics, 2012, 31, 3010. (b) B.S. Mendiguchia, I.
Aiello, A. Crispini, Dalton Trans., 2015,44, 9321.
[28] M. Steiner and A. Duerkop, Analyst, 2011, 136, 148.
[29]
(a) X. Li, J. Zhang, S. Suo, B. Han, US Patent, 2013, US20130070722
A1, 21 Mar 2013.(b) C. Kobelt, C. Burschka, R. Bertermann,C.F. Guerra,
F. M. Bickelhaupt, R. Tacke, Dalton Trans.2012, 41, 2148.
24] K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination
Compounds: Part A: Theory and Applications in Inorganic Chemistry, 6th
Ed., 2008, John Wiley & Sons, Inc. (ISBN: 9780471743392).
[30]
(a)X. Qi, R. Lin, Q. Chen, J. Lin, J. Zhang, X. Chen, Chem. Sci.2011, 2,
2214; (b) M. Mariappan, B. G. Maiya, Eur. J. Inorg. Chem.2005, 11, 2164.
25] (a) J. Sun, F. Zhong, X. Yi,J. Zhao, Inorg. Chem.2013, 52 ,6299. (b) T.
N. Singh-Rachford, F. N. Castellano, Coord. Chem Rev. 2010, 254, 21,
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European Journal of Inorganic Chemistry
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Entry for the Table of Contents
FULL PAPER
Dulal Musib, Md Kausar Raza,
Somashree Kundu, Mithun Roy*
Page No. – Page No.
Title: Modulating in vitro photodynamic
activities of copper(II) complexes
1
Role of sulphur was probed for singlet oxygen ( O ) mediated in vitro photo-dynamic
2
activity of copper(II) complexes by modulating the photo-chemistry. Ramarkable
photo-cytotoxicity of the copper(II) complexes is of paramount importance in
desigining metal-based PDT agent.
Copper(II)-based photo-chemotherapeutics
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