Photocytotoxic Copper(II) Complexes with Schiff-Base Scaffolds for Photodynamic Therapy

Article abstract of DOI:10.1002/chem.201705640

Photodynamic therapy (PDT) is a promising and minimally invasive method for the treatment of superficial diseases, and photosensitizers with high phototoxicity indices (defined as (IC₅₀^dark)/(IC₅₀^irradiation)) a

Full text of DOI:10.1002/chem.201705640

A Journal of
Accepted Article
Title: Photocytotoxic Copper (II) Complexes with Schiff Base Scaffolds
for Photodynamic Therapy
Authors: Rong-Kai Lin, Chin-I Chiu, Cho-Han Hsu, Ying-Ju Lai,
Parthiban Venkatesan, Po-Han Huang, Ping-Shan Lai, and
Chu-Chieh Lin
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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to this Accepted Article as a result of editing. Readers should obtain
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to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201705640
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Photocytotoxic Copper (II) Complexes with Schiff Base Scaffolds
for Photodynamic Therapy
[
a]
[a]
[a]
[a]
[a]
Rong-Kai Lin, Chin-I Chiu, Cho-Han Hsu, Ying-Ju Lai, Parthiban Venkatesan, Po-Han Huang,
[
a]
[a]
[a]
Ping-Shan Lai* and Chu-Chieh Lin*
Abstract: Photodynamic therapy (PDT) is a promising and minimally
invasive method for the treatment of superficial diseases, and
photosensitizers with high phototoxicity indexes (defined as
based ligand complexes have exhibited high reactivity toward
DNA cleavage and apoptosis.
[
20-24]
In a similar fashion, metal complexes with Schiff base scaffolds
have been effectively applied as antibacterial chemotherapeutic
Dark
Irridation
(IC50
)/(IC50
)) are essential for the development of ideal
[
25-26]
photosensitizing properties for this technology. In this study, a series
moieties in medicine.
Cu(II)-containing Schiff base
R
of photocytotoxic copper(II) complexes [Cu( QYMP)(dppn)] (QYMP
complexes might have a greater chemotherapeutic effect via
=
NNO-tridentate
Schiff
base
derivatives,
dppn
=
tuning the electronic properties of the metal center or the ligands
[
27-29]
benzo[i]dipyrido[3,2-a;2´,3´-c]-phenazine) were reported, with their
respective structural confirmations by mass spectrometry and FTIR
by means of coordination.
Furthermore, copper complexes
have been widely utilized in chemical nucleases as oxidizing or
spectroscopy. X-ray crystallography revealed that the CuN
4
O core of
) complex (3) has a distorted square
pyramidal geometry. From our results, the phototoxicity index of
reducing co-reactants to induce the hydrolysis or oxidative
cumy
[30-31]
the [Cu(
QYMP)(dppn)](ClO
4
cleavage of DNA.
For instance, recently, a square-planar
Cu(II) complex chelated by an NNO-tridentate Schiff base (with
phenolic and pyridyl ring binding sites) was reported to efficiently
catalytically cleave DNA without the presence of a reductant co-
factor. Deliberately, the DNA cleavage activity of this type of
complex allows for a rational approach to develop Cu(II)
3
-OMe
[
Cu(
squamous cell carcinoma (SCC15) and 296 in basal cell carcinoma
BCC) cell lines, involving the formation of reactive oxygen species,
4
QYMP)(dppn)](ClO ) (4) was shown to reach 329 in human
(
cell apoptosis and caspase-3 activation and, hence, indicating the
high potential of complex 4 as a photosensitizer candidate in PDT.
Thus, copper complexes bearing suitable Schiff base ligands with a
dppn co-ligand can be considered for the design of efficient metal-
based anticancer agents for PDT.
[
32-37]
complexes with potential as anticancer drugs.
In this report, copper complexes comprising an NNO-tridentate
Schiff base ligand with benzo[i]dipyrido[3,2-a;2´,3´-c]-
a
phenazine (dppn) moiety were studied for in vitro
photocytotoxicity in photodynamic therapy and apoptosis. The
R
synthesized copper complexes [Cu( QYMP)(dppn)] (QYMP =
NNO-tridentate Schiff base) (1-6) were well characterized by
spectral and cyclic voltammetric studies. Furthermore, the
crystal structure of complex 3 was demonstrated by single-
crystal X-ray analysis. Notably, the [Cu( QYMP)(dppn)]
complexes demonstrated DNA cleavage ability and cell viability
and induced reactive oxygen species (ROS) generation in cell
Introduction
Due to the lower side effects of photodynamic therapy (PDT) as
compared to chemo- and radiotherapies, PDT has been
effectively applied in the development of therapeutic cancer
R
[
1-4]
treatment.
For this purpose, several photosensitizers, such as
apoptosis. Ultimately, the photodynamic properties of the
Photofrin, 5-aminolevulinic acid (ALA), Foscan and Verteporn,
have been clinically approved for the treatment of cancers and
skin diseases with light irradiation.
selectivity and efficacy against cancer in PDT treatments,
transition metal complexes have been considered.
R
[
Cu( QYMP)(dppn)]
complexes
were
monitored
and
demonstrated by flow cytometry and confocal microscopy
imaging investigations.
[
5-6]
Recently, to improve the
[
7-10]
Among
the essential transition metals, copper is the third most abundant
crucial transition metal ion in human tissue and plays important
biological roles in the active site of cytochrome c oxidase and as
Results and Discussion
Synthesis and General Considerations
[
11-14]
a catalyst in oxidation-reduction reactions in many proteins.
The synthesis of the NNO-tridentate Schiff base ligands
Furthermore, compared to platinum and ruthenium complexes,
copper complexes have demonstrated good antitumor activity
R
(
QYMP-H) (R = H; 3,5-di-tert-butyl; 3,5-di-cumyl; 3-OMe; 4-
OMe; 5-OMe) were prepared by reacting 8-aminoquinoline with
the respective substituted salicylaldehyde derivatives (Scheme
[
15-19]
with fewer side effects.
Moreover, a variety of pyridine-
S1, see the Supporting Information). The designed heteroleptic
R
copper(II) complexes, [Cu( QYMP)(dppn)](ClO
4
)
(1-6), were
[a]
Ping-Shan Lai and Chu-Chieh Lin,
Department of Chemistry,
National Chung Hsing University,
synthesized from [Cu(OAc)
2
‧H
2
O] with a bipyridine-based ligand
R
(dppn), followed by the addition of QYMP-H and NaClO ,
4
No. 250, Kuo-Kuang Road, Taichung 402, Taiwan,
Tel: +886-4-22840411 ext. 428. Fax: +886-4-22862547
E-mail: pslai@email.nchu.edu.tw (Ping-Shan Lai),
Tel:+886-4-22840411 ext. 718 Fax:+886-4-22862547
E-mail: cchlin@mail.nchu.edu.tw (Chu-Chieh Lin)
Supporting information for this article is given via a link at the end of
the document.
providing good yields, as presented in Chart 1. Copper(II)
complexes 1-6 were characterized by IR spectroscopy and
electrospray ionization (ESI) mass spectrometry (see the
Supporting Information).
IR spectra are additional supporting information to confirm the
functional groups of copper complexes (1-6). The free ligands
-
1
stretching bands of C=N is appeared at 1639-1602 cm and
-
4
copper complexation of ClO
ion corroborated from the
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-
1
appearance of characteristic stretching band at 1080 cm . In
addition, EPR spectroscopic studies of these complexes
revealed a quartet splitting due to the coupling of the copper
Table 1. Selected Physicochemical data for complexes 1-6
nuclei (I = 3/2) with a g value of ~2.00 in DMF solution at 77 K.
b
f p
E / V (ΔE / mV)
9
B
Furthermore, the magnetic moment values of ~1.8 μ suggest d
-
1
IR/cm
ν(C=N)]
λ
max/nm
Cu(II)-
Cu(I)
Cu(I)-
Cu(0)
c
paramagnetic copper(II) centers in these copper(II) complexes,
as summarized Table 1. The UV-vis spectra of copper
complexes 1-6 exhibited a broad and weak copper centered d-d
transition band between 500-700 nm (Figure 1 and Figure S7,
Supporting Information). Noticeably, complexes 2 and 3 have
steric effects to enhance their stability. By contrast, the electron-
donating groups in copper(II) complexes 4-6 might enhance
ligand-to-metal charge transfer (LMCT) from the phenolate π
orbitals to the dπ* orbitals. Next, the electrochemical properties
via cyclic voltammetric measurements of copper complexes 1-6
revealed the redox activities, displaying a quasi-reversible cyclic
voltammetric response of the Cu(II)-Cu(I) couple near 0.14 V (vs
complex
4
-1
-1
a
μ
eff /μ
B
[
(ε/10 M cm )
3
15(7.62),
-
1
1637
1639
0.18
0.17
1.77
1.78
415(1.93)
0.52
3
15(8.39),
-
2
3
414(1.60),
4
0
.67
63(1.25)
15(8.25),
3
-
1604
415(1.60),
66(1.36)
0.13
1.78
0.68
-
4
314(6.66),
4
4
5
1604
1606
0.11
0.12
1.77
1.77
14(1.50)
SCE) in 0.1
M tetrabutylammonium hexafluorophosphate
[
42-43]
(
TBAP) in DMF
(Table 1, Figure 1).
3
4
14(7.96),
15(2.84)
-
0.06
313(6.92),
6
1602
415(1.50),
67(1.25)
0.14
-
1.78
4
a
b
In DMF−H
2
O (v/v). Cu(II)−Cu(I) and Cu(I)−Cu(0) couple in DMF−0.1 M TBAP,
= (Epa−Epc), where Epa and Epc are the anodic and
E
f
= 0.5(Epa+Epc), ΔE
p
−1 c
cathodic peak potentials. Scan rate = 100 mV s . Magnetic moment at 77 K.
X-ray Crystallography.
The crystal structure of [Cu(
cumy
4
QYMP)(dppn)](ClO ) (3) was
obtained from single-crystal X-ray diffraction analysis. Single
crystals of complex 3 were grown in a 1:2 CH Cl /hexane (v/v)
mixture via slow evaporation of the CH Cl solution, obtaining
R
2
2
Chart 1. Structures of the copper(II) complexes [Cu( QYMP)(dppn)](ClO
4
) (1-
2
2
6).
the data shown in Table 2. A summary of selected bond lengths
and angles for 3 is listed in Table S1 (see the Supporting
Information). The crystal structure of complex 3 corresponded to
the monoclinic space group C
center. The Cu atom is bound to the NNO-tridentate
2
/c with a penta-coordinated Cu(II)
cumy
QYMP
Schiff base and NN-bidentate dppn co-ligand. Furthermore, the
molecular structure of complex 3 revealed the geometry around
the Cu center, which is a distorted square pyramid (= 0.27), as
shown by the ORTEP diagram in Figure 2. The Schiff base
binds to copper through the phenolate oxygen atoms and a
nitrogen atom and, hence, validated the tridentate N,N,O-donor
binding mode. The bond distances are Cu-O (1) = 1.877 (2) Å,
Cu-N(1) = 1.949 (3) Å, Cu-N(2) = 2.012 (3) Å, Cu-N(3) = 2.027
(3) Å and Cu-N(4) = 2.223 (3) Å.
2
Figure 1. Electronic spectra of complex 4 in DMF-H O (v/v). The arrows show
the wavelength used for the DNA photocleavage study. (Inset) Cyclic
voltammetric response of complex 4 in 0.1 M TBAP in DMF (scan rate, 100
-1
mV s ).
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Figure 2. ORTEP diagram of cationic complex 3, showing 50% probability
thermal ellipsoids and the atom-numbering scheme for the metal and
heteroatoms. C(8)-C(15) and C(17)-C(24) of the cumyl groups and all the H
atoms are omitted for clarity.
the lowest unoccupied molecular orbitals (LUMOs) of complexes
1–6 are 0.034 to 0.534 eV (Table 3). Indeed, the HOMOs are
primarily localized on the phenanthroline moiety of each
complex, whereas the LUMOs are vacant orbitals mainly
localized on the NNO-tridentate Schiff base ligand moiety.
Hence, charge transfer upon photoexcitation from the
phenanthroline moiety to the coordinated NNO-tridentate Schiff
base ligand at the Cu(II) center is expected.
Table
2.
Selected
Crystallographic
Data
for
Complexes
cumy
u(
QYMP)(dppn)](ClO
4
) (3).
3
Empirical formula
Formula weight
Temperature
56 6 5
C H43ClCuN O
978.95
110(2) K
0.71073 Å
Monoclinic
C2/c
Wavelength
Crystal system
Space group
Unit cell dimensions
a = 34.669(4) Å
 = 90º
b = 13.0804(8) Å
c = 26.435(3) Å
=127.214(17) º
= 90º
3
Volume
95469(16) Å
Z
8
3
Density (calculated)
Absorption coefficient
F(000)
1.362 Mg/m
-
1
0.571 mm
4056
Theta ranges for data collection
Index ranges
2.82 to 29.28°
-39<=h<=47, -11<=k<=17, -33<=l<=30
10960
Reflections collected
Figure 3. Optimized structures, HOMO and LUMO of the Cu(II) complexes 1–
6
Independent reflections
Completeness to theta = 26.00°
Absorption correction
10960 [R(int) = 0.0000]
99.80%
Semi-empirical from equivalents
Table 3. Molecular orbital (HOMO-LUMO) of the complexes 1-6 obtained from
2
DFT calculations.
Refinement method
Full-matrix least-squares on F
HOMO-LUMO energy
complexes
Energy (eV)
HOMO
Energy
gap (eV)
Data / restraints / parameters
10960 / 36 / 592
2
LUMO
Goodness-of-fit on F
0.815
1
2
3
4
5
6
-10.5365
-9.7669
-10.0018
-9.5756
-9.9463
-10.0018
-9.9463
-9.8668
-0.5347
-0.1913
-0.0346
-0.3069
-0.1670
-0.3755
Final R indices [I>2sigma(I)]
R indices (all data)
R1= 0.0652, wR2 = 0.1310
R1 = 0.1790, wR2 = 0.1465
-
3
Largest diff. peak and hole
0.971 and -0.663 e.Å
-9.9117
-10.3087
-10.1133
-10.2423
Computational Methods
Quantum chemical calculations based on density functional
theory (DFT) were performed using the Gaussian 09 program.
The optimized structures of complexes 1-6 were obtained using
DFT with the hybrid-generalized gradient approximation (HGGA)
functional B3LYP. The LanL2DZ basis set was used for
complexes 1-6, as shown in Figure 3. The energy differences
between the highest occupied molecular orbitals (HOMOs) and
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(
A) complex 1, (B) complex 2, (C) complex 3, (D) complex 4, (E) complex 5,
DNA Binding and Cleavage Studies.
and (F) complex 6.
DNA binding plays an essential role in DNA cleavage. Many
metal complexes inhibit DNA replication and subsequently
decrease RNA synthesis via binding to nuclear DNA. However,
the DNA binding abilities of the complexes using EB bound to
CT-DNA in Tris-HCl/NaCl (50 mM, pH, 7.2) buffer were
determined by fluorescence-quenching experiments. During
these investigations, no fluorescence emission of EB at 601 nm
Cytotoxicity Study.
The cytotoxicity, photocytoxicity and the phototoxicity index (PI,
using an MTT assay) of copper complexes 1-6 were investigated
in HeLa (human cervical cancer), SCC15 and BCC cancer cells,
as shown in Table 5. Initially, in the dark, complexes 1-6 were
incubated with different cell lines for 20 h using a half maximal
(excitation at 546 nm) was observed with respect to increasing
Dark
inhibitory concentration (IC50 ). In addition, the photocytoxicity
the copper complex concentration with EB. By contrast, EB with
CT-DNA showed enhanced emission intensity due to DNA
binding. Due to the displacement of the complexes by EB,
further addition of the copper(II) complexes may lead to a
considerable decrease of the fluorescence emission intensity.
Furthermore, from the emission-based calculations, the
apparent binding constant values (Kb) of copper complexes 1-6
of these complexes were evaluated with different cell lines upon
further irradiation under blue light (430 nm) and then incubated
for 18 h in the dark. According to the above experiments, in the
HeLa, SCC15 and BCC cells, the cytotoxicity order in the dark
Dark
(
IC50 ) was 3 > 6 > 4 > 1 > 5 > 2, 6 > 4 > 1 > 3 > 5 > 2 and 1 >
4
> 6 > 3 > 2 > 5, respectively.
6
-1
were between 1.24 ~ 2.28×10 M (Figure S8 and Table S2,
Supporting Information). DNA endonucleolytic cleavage
responses, which are activated by metal ions near the DNA helix
that locally generate oxygen or hydroxide radicals to induce
efficient DNA cleavage reactions, are of considerable interest.
To investigate photoinduced DNA cleavage, the activity of
copper complexes 1-6 were studied by gel electrophoresis using
SC (supercoiled) pUC19 DNA (30 μM, 0.2 μg) in Tris-HCl/NaCl
(50 mM, pH, 7.2) buffer (Figure 4). Initially, in the dark,
complexes 1-6 (20 µM) appeared to be inactive. However, under
irradiation with 430 nm light, each complex led to photoinduced
DNA cleavage, as shown in Figure 5. Complexes 2 and 3
exhibited 40% NC DNA formation from SC DNA. However,
complexes 4-6 resulted in the formation of 13% SC DNA, and
complex 1 showed no apparent DNA photocleavage. As shown
Figure 5. Bar diagram showing the photoinduced DNA cleavage activities of
complexes 1-6 using SC pUC19 DNA (0.2 μg) over an exposure time of 1 h
in Table 4, the activity order for photoinduced DNA cleavage
t-buty
with the complexes was 2 (Cu-
QYMP-dppn) ≈ 3 (Cu-
(
color code: black, in the dark; blue, under 430 nm light).
cumy
4-OMe
3-OMe
QYMP-dppn) > 5 (Cu-
QYMP-dppn) > 4 (Cu-
QYMP-dppn) > 1 (Cu- QYMP-dppn]).
5-OMe
H
QYMP)dppn) ≈ 6 (Cu-
These results demonstrate that complexes 2 and 3 possess
Table 4. Selected DNA (SC pUC19, 0.2 μg) Cleavage Data for Complexes 1-6
in light of 430nm.
good photoinduced DNA cleavage activities.
Reaction
conditions
% NC form
λ= 430 nm
a
SC
98
93
60
60
87
85
87
NC
2
DNA control
DNA + 1
DNA + 2
DNA + 3
DNA + 4
DNA + 5
DNA + 6
7
40
40
13
15
13
Figure 4. Gel electrophoresis diagrams showing the photoinduced DNA
cleavage activities of complexes 1-6 using SC pUC19 DNA (0.2 μg) in 50 mM
Tris-HCl buffer: lane 1, DNA in the dark; lane 2, DNA + complex in the dark;
lane 3, DNA under 430 nm light; lane 4, DNA + complex under 430 nm light.
a
In 50 mM Tris-HCl buffer. λ, 430 nm. Photoexposure time (t) = 1 h.
Concentration of complexes 1-6 used for 430 nm experiments is 20 μM. SC
and NC are supercoiled and nicked circular forms of DNA.
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Table 5. Cytotoxicity of Compounds 1-6 Against Human Tumor Cells (IC50 ± SD, μM).
HeLa
SCC15
BCC
Dark
a
Irr
b
c
Dark
a
Irr
b
c
Dark
a
Irr
b
c
compound
IC50 / μM
IC50 / μM
PI
IC50 / μM
IC50 / μM
PI
IC50 / μM
IC50 / μM
PI
1
2
3
4
5
6
6.26±0.55
3.25±0.64
10.20±2.20
7.25±1.25
4.43±0.97
7.47±1.58
8.95±0.2
>20
0.06±0.01
0.11±0.01
0.16±0.04
0.07±0.02
0.18±0.01
0.17±0.02
1.29±0.04
10.0±3.0
104
30
64
104
25
44
7
11.96±2.81
3.28±0.57
9.21±1.59
23.00±1.40
7.81±0.77
23.70±2.90
0.05±0.01
0.03±0.01
0.04±0.01
0.07±0.01
0.06±0.01
0.08±0.01
239
109
230
329
130
296
32.60±2.20
7.71±0.55
14.00±1.00
20.70±2.80
6.79±1.02
17.60±1.30
0.26±0.03
0.06±0.01
0.07±0.01
0.07±0.01
0.05±0.01
0.06±0.01
125
129
200
296
136
293
d
[Cu(Fc-Trp)(dppz)]
e
8-MOP
>2
>1
f
ALA
>50
45.5±3.2
g
h
cisplatin
11.5±2.9
22.2±5.7
na
a
b
IC50 values correspond to 24 h incubation in dark. IC50 values correspond to 2 h incubation in the dark followed by photoexposure to 430 nm for 10 min, then
c
Dark
Irr 55 d
45 e
incubated for 24 hrs. PI = Phototoxicity index; PI = (IC50 )/(IC50 ).
treatment followed by irradiation at 365 nm (0.793 J/cm ) and 24 h incubation. 4 h treatment followed by 15 min irradiation at 420 nm (6.95 J/cm ) and 44 h
incubation. 4 h treatment followed by 15 min irradiation at 420 nm (6.95 J/cm ) and 44 h incubation. Not applicable.
4 h treatment followed by 60 min irradiation at 400-700 nm and 20 h incubation. 1 h
2
4
4
f
2
5
6 g
2
h
Irr
By contrast, after irradiation, the IC50 value of complex 1 (60
nM) in the HeLa cells demonstrated the lowest photocytoxicity.
Complex
2 (30 nM) in the SCC15 cells showed high
photocytoxicity. However, complex 5 in the BCC cells exhibited
Irr
higher photocytoxicity (IC50 value of 50 nM) than the other
complexes. Moreover, the photocytotoxicity of 4 was slightly
Dark
lower than the other complexes. In the dark, the IC50
= 7.25
μM in the HeLa cells, 23.0 μM in the SCC15 cells and 20.70 μM
in the BCC cells, while 4 exhibited enhanced cytotoxicity upon
exposure to blue light (430 nm) with an IC50 value of 70 nM in
Figure 6. Cell viability plots showing the cytotoxicity of dppn complex 4 in the
dark (black symbols) and in the presence of blue light (430 nm, 10 min): (A)
cytotoxicity of complex 4 in the SCC15 cells; (B) cytotoxicity of complex 4 in
the BCC cells.
Irr
these human tumor cells, as shown in Figure 6. In this context,
the PI value of 4 is 300-fold greater than those of the other
complexes and only 6 showed a similar result with the BCC cell
line. Next, complexes 2 and 3 revealed better DNA cleavage
abilities than the other complexes, as shown in Figure 5;
however, insufficient cell death results were observed in the cell
studies. We speculate that the internalization pathway and
intracellular distribution/location of these complexes are different
and, therefore, cause different amounts of cell death.
min. A considerable change in the nuclear morphology, i.e.,
chromatin aggregation or nuclear condensation, was observed,
as shown in Figure 7A. Furthermore, ROS have a central role in
photodynamic cytotoxicity. The generation of ROS is induced by
[46-48]
apoptosis and necrosis to directly kill tumor cells.
Following
the absorption of light (photons), the photosensitizer (PS) was
transformed from its ground state into a relatively long-lived
Irr
Impressively, both the IC50 value (0.07 ± 0.02 μM) and the PI of
the copper(II) complexes 4 showed considerable improvements
as compared to chemotherapeutic cisplatin (22.2 ± 5.7 μM),
other copper complexes (1.29 ± 0.04 μM), clinically established
PDT agents ALA (45.5 ± 3.2 μM) and 8-MOP (10.0 ± 3.0 μM)
electronically excited state (triplet state) via a short-lived excited
[49]
singlet state.
Upon returning to the ground state, a H atom
(electron) may be transferred to the substrate to form radicals.
Impressively, the data showed that complexes 1-6 regulated the
effects of PDT by type I and type II reactions (Figure S9,
Supporting Information). These radicals interact with oxygen to
produce ROS (type I reaction). Alternatively, the triplet can
transfer its energy directly to oxygen atoms to form singlet
(Table 5). ROS generation and the mechanism of cell death of 4
with irradiation are further investigated.
Changes in the Nuclear Morphology of the Cells
Changes in the nuclear morphology of BCC cells induced by
complex 4 were investigated using DAPI nuclear staining.
Compared to the control cell line, the BCC cells were treated
with 4 for 2 h and then irradiated with blue light (430 nm) for 10
1
[50-51]
oxygen species ( O
2
) (type II reaction).
Thereafter, both
type I and type II reactions simultaneously occur, and the ratio
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Chemistry - A European Journal
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between these processes depends on the type of sensitizer
used.
the integrity of the plasma membrane, the highly condensed
chromatin in dead cells with apoptotic nuclei emitted a bright-
pink fluorescence. However, the necrotic cells were swollen and
had irregular bright-pink fluorescent chromatin. The merged
[
52-53]
Here, the formation of cellular ROS after PDT with the complex
was determined using a H DCFDA probe. In addition, the SOSG
reagent (Invitrogen), which is highly selective for O
2
image of complex 4 upon irradiation showed bright-pink
1
2
, was used
fluorescent chromatin that was highly condensed even in a
shrunken cell. These results clearly demonstrate that cell death
was induced by apoptosis, resulting from the BCC cell treatment
of complex 4 via PDT.
to determine the singlet oxygen population after PDT with a
complex. Moreover, this method demonstrated ROS generation
by complex 4, as shown by irradiating the BCC cells after
2
staining the cells with the ROS probe H DCFDA (Figure 7B).
The phototoxicity of 4 was observed upon irradiation with an
increasing generation of intracellular ROS concentrations.
Hence, complex 4 is a strong photosensitizer, and upon
irradiation, complex 4 mediates its biological effects via oxygen-
independent reactions (type I). The generation of ROS strongly
induces cell death in the presence of complex 4 via PDT.
Caspase-Independent Apoptosis.
Next, to determine the apoptotic pathway in the PDT with
complex 4 after treatment of the SCC15 and BCC cells, flow
cytometry using the BD Pharmingen Caspase-3 Assay Kit was
performed. As shown in Figure 8 and Figure S11, complex 4
incubated with caspase-3 was observed in the dark and
irradiated by blue light. Complex 4 exhibited the highest increase
in caspase-3 activity in the BCC cell line with a 100-fold higher
activity than the untreated cells. In a similar fashion, the highest
caspase-3 activity exhibited an 80-fold increase in the SCC15
cells upon photoexposure to blue light (430 nm). Programmed
cell death could be induced through different pathways. A major
pathway could be mediated by caspase-3, the promoter of
apoptosis. These results confirm that complex 4 is capable of
inducing apoptosis in the SCC15 and BCC cells by activation of
caspase-3.
Figure 7. Induction of apoptosis in BCC cells treated with complex 4 during
PDT. (A) Analysis of apoptosis by nuclear condensation. DAPI staining shows
the typical apoptotic morphological changes after the different treatments. (B)
Accumulation of ROS in the cells. The cells were treated differently, followed
by incubation with DCFH-DA for 30 min. (C) Cell death revealed by Hoechst
33342/propidium iodide staining for 1 h after different treatments. Upon PDT,
complex 4 mainly induced apoptosis. Dark: in the dark, Light: irradiated with
blue light (430 nm) for 10 min, 4 (Dark): cells were incubated with 4 for 2 h,
and 4 (Light): cells were incubated with 4 for 2 h and then irradiated with blue
light (430 nm) for 10 min. Fluorescence images were acquired by fluorescence
microscopy (Olympus IX71). Scale bar: 100 μm.
Induction of Apoptosis in the BCC Cells.
Apoptosis is the process of programmed cell death. Defective
apoptosis has been identified in disease states, such as cancer.
In the present study, the double staining assay with Hoechst
Figure 8. Flow cytometric analysis of complex 4 in the SCC15 and BCC cells.
Bar diagram shows the caspase-3 activation as a fold increase compared to
the dark. Values are the means ± the S.D. from at least three independent
experiments. The asterisks represent significantly different values from the
control (P < 0.001).
3
3342 and propidium iodide was utilized to determine the
morphological changes in the cell nuclei by fluorescence
microscopy. The cell nuclei of BCC cells treated with compound
4
after PDT showed considerable morphological alterations
Conclusion
compared to those of the control. As observed in Figure 7C, the
untreated (dark) or control (light and 4-dark) cells exhibited less
bright-blue fluorescence as well as an absence of red
fluorescence. Furthermore, the cells treated with complex 4
upon irradiation (4-light) exhibited typical features of apoptosis.
The chromatin of the apoptotic nuclei showed highly condensed
and homogeneous fluorescence with bright-blue spherical beads
In summary, novel NNO-tridentate Schiff base copper(II)
complexes 1-6 were synthesized and characterized, and their
biological potentials in PDT were studied. FTIR and EPR studies
indicated that copper was coordinated to the dppn ligand via the
two nitrogen atoms and the NNO-tridentate Schiff base ligand.
Moreover, DNA cleavage did not occur in the dark but occurred
under blue light irradiation for all the metal complexes. Indeed,
complexes 2 and 3 exhibited higher DNA cleavage activities
[
54]
(typical detection of early apoptosis).
Cells emit pink
fluorescence during late apoptosis or necrosis. Upon the loss of
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Chemistry - A European Journal
10.1002/chem.201705640
FULL PAPER
compared to the other metal complexes after irradiation.
Importantly, the cytotoxicities and photocytotoxicities of all the
metal complexes (1-6) were studied in different cell lines. For
117.15, 113.18, 107.68, 101.58, 55.40. Anal. Calcd. (Found) for
17 14 2
C H N O2: C, 73.37 (72.20); H, 5.07 (4.85); N, 10.07 (10.12) %. Mp:
-
1
+
1
04 °C. IR (KBr, cm ): 1619 ν(C=N). ESI-MS in DMSO: m/z 278.2 (M ).
complex
4 in the dark and under irradiation, reasonable
5
-
Preparation of (E)-4-methoxy-2-[(quinolin-8-ylimino)methyl]phenol
(
cytotoxicity and photocytotoxicity activities were observed.
Furthermore, complex 4-treated cells exhibited nuclei
morphological changes and cell apoptosis via caspase-3
activation. This study provides copper (II) complexes containing
dppn moiety as DNA binder and potential tridentate Schiff base
ligand NNO-donor to give stability to the structure with the metal
in (II) oxidation state and minimize the dark cellular toxicity for
photosensitizer design. Further optimization of intracellular
localization and delivery of metal complexes photosensitizers
can be carried out by incorporation of penetrating and targeting
moieties for topical PDT applications.
OMe
QYMP-H). A mixture of 2-hydroxy-5-methoxybenzaldehyde (1.055 g,
.93 mmol) and 8-aminoquinoline (1.00 g, 6.93 mmol) was refluxed in
EtOH (10 mL) for 12 h. The resulting reaction mixture was cooled to
room temperature, and an orange precipitate was obtained. After filtration
and evaporation, the orange powder was recrystallized from hexane.
, ppm): δ 13.50(1H, s, N=CH), 8.95
(1H, dd, J = 1.2, 4 Hz, QuH), 8.84(1H, s, OH), 8.13(1H, dd, J = 0.8, 8.4
Hz, QuH), 7.67(1H, d, J = 8 Hz, QuH), 7.54-7.41(3H, m, QuH), 7.40(1H, s,
).
, ppm): δ 164.12, 156.29, 151.90, 150.34, 145.27,
42.16, 135.86, 129.02, 126.43, 126.11, 121.61, 120.71, 118.97, 118.73,
18.28, 114.96, 55.79. Anal. Calcd. (Found) for C17 2: C, 73.37
6
1
Yield: 1.08 g (56%). H NMR (CDCl
3
ArH), 7.00(1H, d, J = 0.8 Hz, ArH), 6.91(1H, s, ArH), 3.78 (3H, s, OCH
3
13
C NMR (CDCl
3
1
1
14 2
H N O
-
1
(
1
72.48); H, 5.07 (5.10); N, 10.07 (10.02) %. Mp: 118 °C. IR (KBr, cm ):
Experimental Section
+
619 ν(C=N). ESI-MS in DMSO: m/z 278.9 (M ).
R
Materials and Instruments
General procedures for the preparation of [Cu( QYMP)(dppn)](ClO
R=H, t-butyl, cumyl, 3-OMe, 4-OMe and 5-OMe ). mixture of
Cu(OAc) ·H O (0.100 g, 0.50 mmol) and dppn (0.55 mmol) in methanol
4
)
(
A
2
,2,6,6-Tetramethyl-4-piperidone (TEMP), ethidium bromide (EB), 3-(4,5-
2
2
R
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2′,7′-
dichlorofluoresceindiacetate (DCFDA), singlet oxygen sensor green
reagent (SOSG), 4’,6-diamidino-2-phenylindole (DAPI), Hoechst 33342,
propidium iodide, and the FITC Active Caspase-3 Apoptosis Kit were
purchased from Fine Chemicals and Sigma-Aldrich. All the solvents used
were of HPLC grade; anhydrous solvents were obtained by standard
distillation drying procedures. Supercoiled (SC) pUC19 DNA (cesium
chloride purified) was purchased from Bangalore Genie. Calf-thymus
DNA (CT-DNA) was purchased from Sigma-Aldrich (USA). Tris-
(MeOH, 20 mL) was stirred for 0.5 h, and then, QYMP-H (0.55 mmol)
was added. The above mixture was stirred at 25 °C for 3 h, followed by
the addition of NaClO (0.067 g, 0.55 mmol), yielding a grass-green
4
precipitate. The solid was filtered and washed with MeOH (1 mL) three
times and dried under vacuum. Complexes 2-4 and 6 were further
recrystallized from saturated dichloromethane solution.
H
[
Cu( QYMP)(dppn)](ClO
4
) (1). Yield: 0.31 g (81%). Mp: >300 °C. Anal.
Calc. for [C38H23ClCuN O ·MeOH]: C, 60.47; H, 3.51; N, 10.85. Found: C,
4
0.08; H, 3.09; N, 10.98%. ESI-MS in DMSO: m/z 642 [M-ClO ] . IR (KBr,
6 5
(
hydroxymethyl)aminomethane-HCl (Tris-HCl) buffer was prepared using
+
6
deionized and sonicated triple-distilled water.
-1
cm ): 1637 (C=N).
1
13
The H and C NMR spectra were recorded on a Varian Mercury 400
MHz NMR spectrometer. Microanalyses were performed using
Heraeus CHN-O-RAPID instrument. Electron paramagnetic resonance
EPR) spectroscopy was performed on a Bruker EMX-10: CW EPR
instrument at -78 °C using CH Cl as a solvent. Electronic absorption
spectra were recorded by a JASCO U-530 UV/Vis spectrometer at 25 °C
using CH Cl as a solvent. Fluorescent images were captured on a Leica
t-buty
[
Cu(
QYMP)(dppn)](ClO
4
) (2). Yield: 0.32 g (71%). Mp: 232 °C. Anal.
Cl ]: C, 62.24; H, 4.49; N, 9.37. Found:
a
Calc. for [C46
H
39ClCuN ·0.5CH
6
O
5
2
2
+
C, 61.92; H, 5.05; N, 9.36%. ESI-MS in DMSO: m/z 754 [M-ClO
IR (KBr, cm ): 1639 (C=N).
4 3
-2CH ] .
(
-1
2
2
cumy
[
Cu(
QYMP)(dppn)](ClO
43ClCuN
Found: C, 65.53; H, 4.36; N, 8.02%. ESI-MS in DMSO: m/z 878 [M-ClO
4
) (3). Yield: 0.43 g (86%). Mp: 220-222 °C.
2
2
Anal. Calc. for [C66
H
6
O
5
·0.5CH Cl ]: C, 66.44; H, 4.34; N, 8.23.
2
2
TCS-SP5-X AOBS confocal fluorescence microscope.
4
-
+
-1
2
3
CH ] . IR (KBr, cm ): 1604(C=N).
Synthesis: The tridentate-NNO Schiff base ligands (E)-2-[(quinolin-8-
H
ylimino)methyl]phenol ( QYMP-H), (E)-2,4-di-tert-butyl-6-[(quinolin-8-
3-OMe
[
Cu(
Calc. for [C39
Found: C, 57.54; H, 3.32; N, 9.98%. IR. ESI-MS in DMSO: m/z 671.9
QYMP)(dppn)](ClO
4
) (4). Yield: 0.28 g (73%). Mp: 236 °C. Anal.
t-buty
ylimino)methyl]phenol (
[
[
QYMP-H), (E)-2,4-bis(2-phenylpropan-2-yl)-6-
H
25ClCuN ·0.5CH
6
O
6
2
Cl ]: C, 58.20; H, 3.22; N, 10.31.
2
cumy
(quinolin-8-ylimino)methyl]phenol (
QYMP-H) and (E)-2-methoxy-6-
3
-OMe
(quinolin-8-ylimino)methyl]phenol (
QYMP-H) were synthesized as
+.
-1
[
M] IR (KBr, cm ): 1604 (C=N).
[
38]
per the reported literature.
4
-OMe
[
Cu(
QYMP)(dppn)](ClO
4
) (5). Yield: 0.23 g (58%). Mp: >300 °C.
·MeOH]: C, 59.70; H, 3.63; N, 10.44.
4
-
Preparation of (E)-5-methoxy-2-[(quinolin-8-ylimino)methyl]phenol
(
Anal. Calc. for [C39
Found: C, 59.99; H, 3.25; N, 10.80%.ESI-MS in DMSO: m/z 672.2 [M]
IR (KBr, cm ): 1606 (C=N).
H
25ClCuN O
6 6
OMe
QYMP-H). A mixture of 8-aminoquinoline (1.00 g, 6.93 mmol), 2-
hydroxy-4-methoxybenzaldehyde (1.055 g, 6.93 mmol), and
benzenesulfonic acid (0.1 mL) was refluxed in ethanol (EtOH, 10 mL) for
+.
-1
1
2 h. The resulting reaction mixture was cooled to room temperature,
5-OMe
[
0
Cu(
4 2 2
QYMP)(dppn)](ClO ) (6). A mixture of Cu(OAc) ·H O (0.100 g,
and a yellow precipitate was obtained. After filtration and evaporation, the
yellow powder was recrystallized from hexane. Yield: 1.08 g (56%).
NMR (CDCl , ppm): δ 14.81 (1H, s, N=CH), 9.00 (1H, dd, J = 2.0, 4.4 Hz,
QuH), 8.73 (1H, s, OH), 8.18 (1H, dd, J = 2.2, 8.2 Hz, QuH), 7.69 (1H, dd,
J = 1.6, 8.0 Hz, QuH), 7.58-7.44 (3H, m, QuH), 7.25 (1H, d, J = 2.8 Hz,
ArH), 6.47 (1H, d, J = 2.4 Hz, ArH), 6.42 (1H, dd, J = 2.4, 8.8 Hz, ArH),
3
1
.50 mmol) and dppn (0.55 mmol) in MeOH (20 mL) was stirred for 0.5 h,
1
H
5-OMe
and then,
was stirred at 25 °C for 6 h, followed by the addition of NaClO
.50 mmol), yielding a brown precipitate. The brown solid was filtered
and washed with MeOH (1 mL) three times and dried under vacuum.
QYMP-H (0.139 g, 0.50 mmol) was added. The mixture
3
4
(0.061 g,
0
Yield:
0.29
g
(76%).
Mp:
214
°C.
Anal.
Calc.
for
13
.85 (3H, s, OCH
3 3
). C NMR (CDCl , ppm): δ 168.91, 165.11, 160.31,
[
C
39
H25ClCuN
6
O
6
·CH
2
Cl ]: C, 56.02; H, 3.17; N, 9.80. Found: C, 56.73; H,
2
50.29, 143.27, 141.73, 135.89, 133.75, 129.06, 126.51, 125.36, 121.78,
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Chemistry - A European Journal
10.1002/chem.201705640
FULL PAPER
+
-1
4
3
.55; N, 9.37%. ESI-MS in DMSO: m/z 672.2 [M] . IR (KBr, cm ): 1602
Approximately, 5 × 10 cells of human squamous cell carcinoma
(
C=N).
(SCC15) and basal cell carcinoma (BCC) were placed in 96 well culture
plates in DMEM/F12 medium containing 10% fetal bovine serum (FBS)
2
and, after 48 h of incubation at 37 °C in a CO incubator, concentrations
of the Cu(II) complexes dissolved in <1% DMSO. After the incubation
period, 125 μL of MTT was added to each well and incubated for an
additional 4 h. Then, the culture medium was discarded and 200 μL of
DMSO was added to dissolve the purple formazan crystals. The
absorbance at 570 nm was noted using a PerkinElmer 1420 Multilabel
Counter. The cytotoxicities of the complexes were measured as the
percentage ratio of the absorbance of the treated cells to the untreated
controls. Ultimately, the IC50 values were determined by nonlinear
regression analysis using GraphPad Prism analysis software.
X-ray Crystallography. Suitable crystals of complex 3 were mounted
onto a glass fiber using perfluoropolyether oil and cooled rapidly in a
stream of cold nitrogen gas using an Oxford Cryosystems Cryostream
unit. Diffraction data were collected at 110 K using an Oxford Gemini S
diffractometer. Intensity data were collected in 1350 frames with an
increasing w (width of 0.5° per frame). The absorption correction was
based on the symmetry-equivalent reflections using the SADABS
program. Then, the space group was determined from the Laue
symmetry and systematic absence and confirmed through the structure
determination. The structure was solved by direct methods using the
SHELXTL package. All non-H atoms were located from successive
Fourier maps, and the H atoms were refined using a riding model.
Anisotropic thermal parameters were used for all non-H atoms, and fixed
isotropic parameters were used for the H atoms.
Measurement of the Intracellular ROS Accumulation. The intracellular
generation of ROS was measured by performing a dichloro-dihydro-
fluorescein diacetate (DCFH-DA) assay. After deacetylation in the cells,
DCFH-DA is converted by intracellular radicals to a fluorescent product,
2
’,7’-dichlorofluorescein (DCF), which is intracellularly retained. BCC
cells were cultured in 6 cm for 48 h and then serum-starved for 24 h.
After subsequent treatment with the Cu(II) complexes for 2 h, the cells
were exposed to 430 nm for 10 min, washed with phosphate-buffered
DNA Binding Methods. The apparent binding constant (Kapp) values of
complexes 1-6 were obtained by monitoring their respective fluorescence
emission spectra changes in a mixed solution of EB (13 μM) and CT-
DNA (10 μM) in Tris-HCl/NaCl buffer (pH, 7.2). In addition, the
fluorescence intensities of EB at 600 nm (546 nm excitation) were
examined with an increasing concentration of the ternary complex.
Finally, the Kapp values were calculated from the following equation.
2
saline (PBS), and incubated with 10 μM H DCF-DA (Sigma-Aldrich) at
3
7 °C for 30 min. The cells were then washed with PBS (1 mL), and the
fluorescence images of the cells were photographed with an Olympus
IX71.
Fluorescence Microscopy of the BCC Cells with Copper Complex 4.
KEB [EB] = Kapp [complex]
DAPI staining is used in nucleus staining for cell death detection. After
7
−1
staining with DAPI, cell death was detected by a fluorescence
where KEB (1 × 10
M ) is the binding constant of EB toward CT-DNA,
microscope. The apoptotic cells were identified by highly condensed
nuclei. By contrast, the cells in the dark or the cells treated with the
complexes but unexposed to light showed intact cellular nuclei
morphologies. Hoechst 33342 and propidium iodide staining assays for
apoptosis based on fluorescence detection at the condensed state of the
[
EB] is the concentration of EB, and [complex] is the concentration of the
complex when the EB fluorescence is reduced to half of the initial
intensity.
[39-40]
DNA Cleavage Experiments. The cleavage of (SC) pUC19 DNA by the
copper(II) complexes was investigated using an agarose gel
electrophoresis method. Reactions were performed both in the dark and
under illuminated conditions using a 430 nm blue light (3.5 mW). An
Eppendorf tube was used for the respective UV experiments in a dark
room at 25 °C using (SC) pUC19 DNA (1 μL, 0.5 μg) in 50 mM Tris-HCl
buffer (pH 7.2) containing NaCl (50 mM) and the copper complex (1.8 μL
in dimethylformamide (DMF)) with constant concentrations. In the above
case, the concentration of the complexes in DMF corresponded to the
quantity after dilution with a 1.8 μL stock solution to 18 μL as the final
volume using Tris-HCl buffer. Following light exposure, each sample was
incubated at 37 °C for an hour and then analyzed for photocleavage
products using gel electrophoresis. Thereafter, the extent of DNA
cleavage was measured from the intensities of the bands using the
UVITEC Gel Documentation System. Corrections were made for the low
level of the nicked circular (NC) form present in the original SC pUC19
DNA sample and for the low affinity of the SYBR safe binding to the SC
compared to the NC and linear forms of DNA. The concentrations of the
complexes and reagents were related to the final sample quantity (18 μL;
after dilution with Tris-HCl buffer). Here, the observed error during the
measurement of the band intensities ranged between 4 and 7%.
[41]
chromatin in apoptotic cells were conducted. Briefly, approximately 5 ×
5
1
0
cells were allowed to adhere overnight on culture dishes. At that
juncture, the cells were treated with or without copper complex 4 at 70
nM for 2 h in the dark, followed by irradiation at 430 nm for 10 min. After
2
hours, the cells were washed with cold PBS. A Hoechst 33342 solution
(
10 μg/mL) was added to the cells. After incubation with Hoechst 33342
for 10 minutes, the cells were stained with propidium iodide (2.5 μg/mL).
The samples were maintained in the dark for 15 minutes. Thereafter, the
cells were washed with PBS and observed by an inverted fluorescence
microscope (Olympus IX71). The cells were measured at the 360 nm
excitation and 460 nm emission of the Hoechst 33342 dye and observed
with a fluorescence microscope using the 488 nm excitation and >575
nm emission of propidium iodide under magnification.
Assessment of the Caspase-3 Assay. Caspases are key mediators of
cell apoptosis. A caspase-3 activity assay was performed using the FITC
Active Caspase-3 Apoptosis Kit (BD Pharmingen). These probes
covalently and irreversibly bind at the active site of the active caspase
heterodimer, emitting a green fluorescent signal detected at the FL-1
channel. Briefly, the cancer cells were treated with 70 nM copper
complex 4 for 2 h in the dark, followed by irradiation at 430 nm for 10 min.
The assay was performed based on the manufacturer’s protocol. The
cells were analyzed using BD Accuri C6 flow cytometry and the BD
CFlow software.
Cell Cytotoxicity Assay. The cytotoxicities of the Cu(II) complexes were
measured
using
a
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazolium bromide (MTT) assay based on the ability of mitochondrial
dehydrogenases in the viable cells to cleave the tetrazolium rings of MTT
to form dark-blue membrane-impermeable crystals of formazan, which
can be quantified at 570 nm on detergent solubilization. The quantity of
the formazan product provided a measure of the number of viable cells.
Acknowledgements
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Chemistry - A European Journal
10.1002/chem.201705640
FULL PAPER
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