Photophysical properties and photodynamic anti-tumor activity of corrole-coumarin dyads

Article abstract of DOI:10.1142/S1088424618500724

A new non-conjugated corrole-coumarin dyad and its gallium complex has been synthesized. Photophysical properties of the dyads were tested in two solvents, exhibiting strong solvent effect on the absorption and fluorescence spectra. Absorption spectra of

Full text of DOI:10.1142/S1088424618500724

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2018; 22: 1–13
DOI: 10.1142/S1088424618500724
Published at http://www.worldscinet.com/jpp/
Photophysical properties and photodynamic anti-tumor
activity of corrole-coumarin dyads
a
a
a
b
b
Fan Cheng , Hua-Hua Wang , Atif Ali , Jaipal Kandhadi , Hui Wang* ,
a
a◊
Xiang-Li Wang and Hai-Yang Liu*
a
Department of Chemistry, Key Laboratory of Functional Molecular Engineering of Guangdong Province,
South China University of Technology, Guangzhou, Guangdong 510641, China
State Key Laboratory of Optoelectronics Materials and Technologies, Sun-Yat Sen University, Guangzhou,
b
Guangdong 510275, China
Dedicated to Professor Naisheng Chen on the occasion of his 80th birthday
Received 28 March 2018
Accepted 24 April 2018
ABSTRACT: A new non-conjugated corrole-coumarin dyad and its gallium complex has been
synthesized. Photophysical properties of the dyads were tested in two solvents, exhibiting strong solvent
effect on the absorption and fluorescence spectra.Absorption spectra of the dyads are a linear combination
of the spectra of their corresponding monomers, demonstrating a negligible electronic communication
between coumarin and corrole moiety. However, fluorescence emission of coumarin entity in all dyads
were quenched significantly as compared to pristine coumarin; this was attributed to intramolecular
energy transfer from coumarin to the corrole. Photodynamic anti-tumor tests revealed that gallium
corrole-coumarin dyads (2-Ga) exhibited good PDT activity towards SiHa cells. After PDT treatment,
2
-Ga could induce apoptosis in SiHa cells, which was associated to cell S phase arrest, collapse of the
mitochondrial membrane potential and increase of the intracellular ROS level.
KEYWORDS: corrole, coumarin, gallium, energy transfer, PDT.
Coumarin-tetrapyrrolic macrocycle conjugates have
attracted much interest in the past years [10]. They have
potential uses in organic light-emitting diodes (OLEDs)
11, 12], light harvesting [13, 14], fluorescent probes
15, 16] and anti-cancer drugs [17]. Although many
coumarin-porphyrin conjugates have been reported
18–21], studies of coumarin–corrole conjugates are
scarce. In 2010, Gryko and co-workers reported the
first synthesis of corrole-coumarin dyads [13]. They
had synthesized a series of conjugated corrole-coumarin
dyads using direct condensation of formyl-coumarins
and dipyrromethanes [22, 23]. Interestingly, gallium
corrole-coumarin dyads could be used as fluorescent
probes for anionic or cationic ions [16]. Partly inspired
by the lack of literature reporting on corrole-coumarin,
and to explore its potential uses in light harvesting and
photodynamic anti-cancer therapy, we here wish to
report the preparation of a new coumarin–corrole dyad
and its gallium complex in which the coumarin unit is
INTRODUCTION
Corrole is a tetrapyrrolic macrocycle closely related to
porphyrin with a direct pyrrole−pyrrole linkage. It shows
a relatively higher fluorescence quantum yield and more
intense absorption of red light than its corresponding
porphyrin [1]. Corroles have potential applications in
the field of photochemical sensors [2, 3], and biomedical
devices [4, 5]. Coumarin, also known as 2H-chromen-2-
one, is a kind of flavonoid compound of secondary plant
metabolites which displays relatively high fluorescent
quantum yield. Coumarin derivatives are highly desirable
for numerous applications in diverse fields including
laser dyes [6], chemical sensor [7], medicinal chemistry
[
[
[
[
8] and light harvesting [9].
◊SPP full member in good standing
*
Correspondence to: Hai-Yang Liu, tel.: +86 020-22236805,
fax: +86 020-22236805, email: chhyliu@scut.edu.cn.
Copyright © 2018 World Scientific Publishing Company

2
F. CHENG ET AL.
Scheme 1. Cor-Cou dyads and the reference corrole examined in this study
linked to the para-position of 10-phenyl group of the
corrole (Scheme 1). The compounds were characterized
1
19
by H NMR, F NMR, HR-MS, IR and UV-vis spectral
Figs S1−S17). Their photophysical properties, in vitro
(
photodynamic anti-cancer activity was also investigated.
RESULTS AND DISCUSSION
Photophysical properties
The UV-vis spectra of the dyads and their reference
corroles have been measured in two solvents. As an
example, the absorption spectra of free-base dyad 2,
corrole 1 and coumarin in DMF are shown in Fig. 1. The
absorption spectrum of the dyad is a linear combina-
tion of the spectrum of the corresponding monomers,
demonstrating that coumarin was attached with the
corrole successfully. Spectral data including maximum
absorption wavelength and molar extinction coefficients
(e) are summarized in Table 1. All the dyads showed an
intense Soret band located at 418 nm to 429 nm, and
Q bands with a lower intensity located at 540–640 nm.
These bands characterize the compounds derived from
corrole group, while another absorption band around
3
24 nm corresponds to the coumarin moiety. Interestingly,
the Soret band of the free-base dyad was split in DMF
Fig. 1), but a normal Soret band around 429 nm was
(
observed in toluene (Fig. S17). This phenomenon is
probably caused by deprotonation of the macrocycle in
polar solvents or hydrogen bonding with an internal N–H
group inducing solvent dependent changes [24]. From
the above observations, we believe the free-base dyad is
more solvent dependent, including band shift (Table 1)
and Soret band splitting.
The fluorescence spectral studies for dyads along
with their monomers were carried out in toluene and
DMF (Fig. 2). Spectral data including emission maxima
and quantum yields are summarized in Table 1. As an
example, the fluorescence emission spectra were recorded
at a 420 nm excitation wavelength in toluene, and the
Fig. 1. Absorption spectra of 5 mM complex 1, 2 and coumarin
in DMF
around 641 nm while for 2-Ga was observed at 618 nm,
this fluorescence shift in line with the corresponding
Q-band absorption shifts. In addition, an obvious blue
shift and fluorescence quenching were observed when
the fluorescence spectrum of the dyads was compared
with reference corroles (Fig. 2). This behavior suggests
that intermolecular interactions are present.
Fluorescence quantum yield measurements were
performed with excitation at 535 nm, and the absorbance
Cor-Cou dyad
2 displayed an intense and broad band
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PHOTOPHYSICAL PROPERTIES AND PHOTODYNAMIC ANTI-TUMOR ACTIVITY OF CORROLE-COUMARIN DYADS
3
Fig. 2. Fluorescence spectra (l = 420 nm) of 5 mM complex 1, 1-Ga, 2 and 2-Ga in DMF and toluene
ex
Fig. 3. Comparison of the normalized absorption and fluorescence spectra in DMF at 298K. (a) The absorption spectra of corrole 1
and gallium corrole 1-Ga are in red and blue respectively and fluorescence spectra of coumarin (l = 320 nm). Note that the folded
ex
conformation drawn for the dyads is only to make them fit within the graphs. (b) Normalized fluorescence spectra of 5 mM coumarin
and dyads in DMF (lex = 320 nm)
of samples at the excitation wavelength was less than 0.1.
The fluorescence quantum yield of the gallium dyad was
higher than for the free-base dyad. This phenomenon can
be explained in part by ring distortion variations [25]. As
showninTable1,thequantumyieldsofthedyadsarelower
than the reference corroles in same solution, suggesting
that probably an intramolecular energy transfer or charge
transfer occurred. The quenched emission spectra of
and absorption spectra of the dyad was also evident
(Fig. 3a). The fluorescence quenching demonstrates that
an intramolecular fluorescence resonance energy transfer
(FRET) occurred from the singlet state of coumarin to
corrole.
The fluorescence lifetime of the complexes were
measured in two solvents at the excitation wavelength
in 405 nm (Table 1). The fluorescence lifetime of the
complexes were smaller in DMF as compared to toluene.
dyad (l = 320 nm) was compared to that for individual
ex
constituent coumarins (between 330–550 nm), and the
quenching efficiency was in the range of 96–97%. In
addition, an emission band corresponding to the corrole
moiety around 600–750 nm was observed (Fig. 3b).
A large area overlay of fluorescence spectra of coumarin
Furthermore, the fluorescence lifetime of dyad
2 (3.96 ns)
was slightly larger than for the reference corrole
1
(3.85 ns). This may be due to weak or negligible electronic
interactions from corrole to coumarin in the ground states
of the dyads.
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4
F. CHENG ET AL.
Table 1. Photophysical properties of the examined corroles
3
-1
.
-1
Compound
Absorption [nm] (e [10 M cm ])
Emission
fF
t [ns] FD
[
nm]
Soret Q band
Cou
toluene 324
DMF 324
toluene 325 (24.3) 429 (29.3)
DMF 323 (23.4) 424 (50.0), 439 (44.3)
-Ga toluene 324 (14.9) 422 (58.4)
—
—
—
—
—
—
—
—
—
—
0.08
—
—
—
—
393
656
641
613
618
662
644
615
621
0.08 [26] 0.19
2
561 (6.8) 748 (3.0)
—
3.96
546 (7.2) 582 (8.6) 628 (14.7)
0.14
0.38
0.46
0.12
0.18
0.61
0.58
3.70 0.96
2.69
2.21 0.28
3.85
3.65 0.83
2.52
2.17 0.68
2
1
576 (7.5) 600 (9.6)
576 (6.3) 606 (12.8)
—
—
—
DMF
315 (8.8) 422 (101.1)
1
toluene
DMF
—
—
—
—
418 (109.8)
563 (15.8) 619 (9.4) 639 (8.3)
—
424 (139.1), 439 (118.0) 549 (6.4) 589 (14.1) 631 (41.3)
-Ga toluene
422 (212.5)
422 (204.3)
576 (20.5) 603 (28.6)
578 (17.2) 608 (29.1)
—
—
—
DMF
e-molar absorption coefficient; stokes shift; F -fluorescence quantum yield; t [ns]-fluorescence lifetime; F -singlet oxygen quantum
F
D
yield.
dyads could promote the singlet oxygen generation.
Generation of singlet oxygen is an important step in
photodynamic therapy; therefore, the results support that
Cor-Cou dyads could be used as potential photosensitizers
in photodynamic therapy of cancer.
Cyclic voltammetry
The electrochemical properties of dyad 2 and its
gallium complex 2-Ga, as well as the reference compounds
1
and 1-Ga were further examined by cyclic voltammetry
in DMF at room temperature. The redox data vs. the
ferrocene/ferrocenium couple (Fc/Fc+) are collected in
Table 2. The coordinating metal cation Ga(III) does not
show a redox behavior: all the redox processes occur only
on the corrole ring and the coumarin. For corrole 1, two
Fig. 4. Plot of change in absorbance of DPBF at 418 nm vs.
irradiation time in the presence of 1, 1-Ga, 2 and 2-Ga in DMF
reversible reduction peaks and one oxidation peak were
observed. The reversible wave at 0.47V is due to the redox
peak of the internal standard (Fc/Fc+). Other reduction
peaks at -0.72 and -1.73 V can be assigned to the reduction
of the corrole ring while the oxidation peak (0.21 V)
belongs to the oxidation of corrole ring [28]. Compared
with corrole, the dyad showed two similar reduction peaks
(-1.10 V, -0.65) and an oxidation peak (0.30 V) attributed
to the corrole-based reductions and oxidation respectively.
Another reduction peak at -1.83 V was assigned to
reduction of coumarin. The reduction potentials of the
dyad are shifted to a more positive state, indicating that
the Cor-Cou dyad is easier to reduce and harder to oxidize
compared to its corresponding monomers.
Singlet oxygen quantum yield
Singlet oxygen generation was determined in DMF
using an indirect method by DPBF as singlet oxygen
scavenger. A red LED lamp (650 nm) was used as a light
source, and absorbance of DPBF at 418 nm decreased in
the presence of each complex with increasing illumination
time (0–90 s) as shown in Fig. S18. From the slope of the
line, we can compare the relative generation of singlet
oxygen: the data in Fig. 4 showed that the singlet oxygen
of free-base dyad 2 was higher than reference corrole 1,
while the singlet oxygen generated by 2 is greater than for
In case of gallium dyad, the first reduction peak (-1.53)
should be assigned to reduction of the corrole ring, and
the second reversible reduction peak (-1.84V) should be
assigned to coumarin-based reductions. No oxidation
peak could be observed in the cyclic voltammogram
2-Ga and singlet oxygen generated by 1 is greater than
for 1-Ga, which is consistent with previous reports [27].
Overall, the results showed the possibility of singlet oxy-
gen generation by Cor-Cou dyads, and corrole-coumarin
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PHOTOPHYSICAL PROPERTIES AND PHOTODYNAMIC ANTI-TUMOR ACTIVITY OF CORROLE-COUMARIN DYADS
5
Table 2. Redox potentials of porphyrins (V vs. Ag/AgNO ; Pt electrode as a working
3
electrode; TBAP 0.1 M, DMF)
Ered3 /V
Ered2 /V
Ered1 /V
Fc/Fc+
Eox1 /V
1.23
Coumarin
-1.92
—
—
0.47 (0.09)
a
1
—
—
-1.73 (0.15)
-1.47 (0.15)
-1.10
-0.72 (0.11)
—
0.47 (0.08)
0.53 (0.09)
0.49 (0.07)
0.21 (0.06)
—
1
2
-Ga
-1.83
-0.65
0.30
2
-Ga
-1.84
-1.53
—
0.49 (0.09)
—
a
Reversible redox. DE = E –E in brackets.
pa
pc
Calculated distribution patterns of the free-base
dyad are shown in Fig. 5, where it can be seen
that the frontier molecular orbitals HOMO,
HOMO-1, LUMO and LUMO+1 orbital of the
complex are all localized on the corrole dye but
not on the substituent. However, HOMO-2 is
localized on the coumarin molecule, including
the meso linker group, as well as the LUMO+2
orbital (data not shown). Similar results were
obtained for the gallium complexes (Fig. S19).
The TD-DFT calculated spectra of the dyads
in the two solvents are presented in Fig. 6. With
toluene as the solvent, the calculated Q band
was located at 626 nm, which corresponds to the
HOMO → LOMO (90%). The intensive Soret
band composed of two transitions with absorption
maxima at 439 nm and 393 nm, represents
the HOMO-1 → LUMO (40%), HOMO →
LUMO+1 (55%) and HOMO-1 → LUMO+1
(45%) and HOMO-2 → LUMO (17%) transfers.
Another less intensive band at 300–400 nm
with absorption maxima at 344 nm, represents
HOMO → LUMO+3 (74%). Most of these
transitions take place on the corrole part of
the molecule, with only HOMO-2 → LUMO
participation of the coumarin part. This suggests
a weak charge-transfer (CT) character from the
corrole to the coumarin part. Similar results
were observed in DMF: the calculated Q band
is formed by some transitions with absorption
maxima at 668 nm, which corresponds to
HOMO → LUMO (96%). Two intensive Soret
bands were observed at 472 nm, representing
Fig. 5. DFT optimized geometry of dyads and frontier molecular orbitals
of complex 1, 2 optimized at the B3LYP/6-31G (d,p) level
of the gallium dyad. A similar phenomenon was also
the HOMO → LUMO+1 (60%), HOMO-1 → LUMO
38%) and at 413 nm, corresponding to HOMO-1
LUMO+1 (66%) HOMO-2 → LUMO (14%).
Another band at 318 nm corresponds to HOMO-2 →
LUMO+2 (83%) and HOMO-3 → LUMO+2 (13%). The
calculated UV-vis spectra of the free-base dyads showed
an obvious shift in two solvents, consistent with the
experimental results. Furthermore, TD-DFT calculated
spectra of the gallium dyads are presented in Fig. S20,
observed for monomer gallium corrole 1-Ga
.
(
→
Theoretical studies
Detail of the DFT calculations shown in the
experimental section. Calculated optimized geometry
shows that the non-conjugated arm does not influence
the structure of corrole molecule, and the gallium corrole
appears more planar than free-base corrole (Fig. S19).
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6
F. CHENG ET AL.
Fig. 6. Computed TD-DFT spectra of dyad 2 with inclusion of DMF or toluene as solvent
Table 3. The IC50 (μM) values of corrole and dyads towards Bel-7402, A549 and SiHa cell lines
Com
BEL-7402
A549
SiHa
dark
light
dark
light
dark
light
2
>100
>100
>100
>100
>100
>100
>100
>100
>100
2
1
-Ga
1
35.4 ± 1.4
1.1 ± 0.4
0.2 ± 0.01
75.4 ± 1.1
0.7 ± 0.1
0.2 ± 0.04
15.8 ± 1.2
3.5 ± 0.8
0.1 ± 0.01
29.1 ± 2.3
8.3 ± 1.1
>100
>100
-Ga
1.6 ± 0.1
11.4 ± 0.1
-2
Results represent means ± SD of three independent experiments. Light dose = 22 J·cm .
proving the weak charge-transfer (CT) character from the
gallium corrole to the coumarin.
also observed the poor solubility of the free-base dyad
compared with the free-base corrole, which may hinder
the compound’s ability to enter into cells smoothly.
Gallium corroles can increase the cytotoxicity and
showed brighter red fluoresence compared with free-
base corroles in our study. The reference gallium corrole
showed stronger cytotoxicity in both dark and light
compared with the corrole. The target Ga(III)corrole-
coumarin complex showed low cellular toxicity in the
dark, with an IC50 value over 100 mM, and the IC50
exhibited significant reduction after exposure to red
light (650 nm), especially towards SiHa cells, where
the phototoxicity increased more than 6 times compared
with dark toxicity. Despite the fact that the dyads did not
show cytotoxicity as expected, this data still provides
a case that gallium complexes may be good candidates
for antitumor therapy. Next, SiHa cells were taken as
an example to investigate the antitumor mechanism of
gallium corrole-coumarin dyads.
Cytotoxicity assay
The light-induced antitumor activities of the
dyads were investigated in vitro by the MTT method,
using three tumor cell cultures for study. A red LED
lamp (650 nm) was used as a light source, and light
wavelengths used were within the range of effective
PDT wavelengths (600–800 nm); the results are listed
in Table 3. Reference corrole 1 showed lower toxicity
in the dark; after exposure to the light, the half-maximal
inhibitory concentration (IC ) value even dropped
5
0
to 0.7 mM towards A549 cells. However, the target
compound (2) showed no apparent toxicity even at a
high concentration of 100 mM in either dark or light. The
above research indicates that the corrole-coumarin dyad
2
enhanced the singlet oxygen quantum yield compared
to the reference corrole. Unfortunately this enhancement
effect did not work in antiproliferative activities of tumor
cells. This may be explained by optimizing the structure
of DFT calculation. The increased p–p stacking leads to
self-aggregation of either corrole or coumarin, and we
Intracellular location
It is well known that intracellular uptake is an important
property of a photosensitizer. DAPI was used as the
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PHOTOPHYSICAL PROPERTIES AND PHOTODYNAMIC ANTI-TUMOR ACTIVITY OF CORROLE-COUMARIN DYADS
7
Fig. 7. Cellular uptake of SiHa cells with exposure to 30 mM 2-Ga (up red channel) for 24 h at 200× magnification
Fig. 8. (a) Assay of SiHa cells mitochondrial membrane potential with JC-1 as fluorescence probe staining method. (b) Intracellular
ROS was detected in SiHa cells. All were treated with red light (650 nm) for 2 h and incubated for 24 h at 200× magnification
nuclear staining dye to evaluate the intracellular location
and thereby to further investigate the mechanism of PDT
action. SiHa cells were treated with 30 mM dyad 2-Ga
for 24 h, Fig. 7 shows that the bright red fluorescence of
sincedecreasedMMPhasbeenimplicatedasanearlyevent
in apoptotic cells. Dye JC-1 shows red fluorescence in its
aggregated form and exists as a green-emissive monomer
after loss of the mitochondrial membrane potential.
Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a
mitochondrial electron transport chain inhibitor, was used
as a positive control. Figure 8a shows that the SiHa cells
lost the MMP completely after treatment with CCCP. SiHa
cells exposed to gallium dyads and then treated with a red
light (650 nm) for 2 h showed reduced red fluorescence
intensity with a concomitant increase in the green signal.
Furthermore, there was an obvious increase of green
fluorescence as the concentration of 2-Ga increased from
15 to 30 mM.All of the above suggest that 2-Ga can reduce
the MMP of SiHa cells during the apoptosis process.
2
-Ga can be uptaken by SiHa cells without the help of an
internalizing protein. The merged spectrum shows that
the red fluorescence signals from 2-Ga accumulated not
only in the cytoplasm but also in the nucleus of the SiHa
cells after incubation for 24 h.
Effects on mitochondrial dysfunction
and ROS generation
The cell-permeant dye, JC-1, was used to monitor any
loss of mitochondrial membrane potential (MMP, Ym),
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8
F. CHENG ET AL.
Mitochondria are an important intracellular source of
reactive oxygen species (ROS) [29]. To further explore
the mechanism of the examined dyads for anti-cancer
cells, we also explored intracellular ROS levels of SiHa
cells after exposure to 2-Ga, using an oxidation-sensitive
fluorescent probe DCFH-DA, to measure the ability of
2-Ga to generate ROS. As shown in Fig. 8b, the green
fluorescence which reflects the level of intracellular ROS,
was difficult to observe in the control group. In contrast,
for SiHa cells exposed to gallium dyads and then treated
with LED light (650 nm) for 2 h and incubated for 24 h, the
intensity of the green fluorescence was brighter than that
of the control group. Furthermore, the green fluorescence
became progressively brighter when the concentration
of gallium dyad was increased. Altogether, according to
the observed loss of mitochondrial potential, dyad 2-Ga
was able to increase ROS levels and eventually triggered
apoptotic cell death.
Fig. 9. Comet assay of EB-stained control and 15 μM of
complexes 2-Ga treated SiHa cancer cells. Red well-formed
comets were observed
SiHa cells by flow cytometry. As shown in Fig. 10, the
complex led to an accumulation of cells in the S phase,
from 18.10% to 45.64%, with a concomitant decrease
of cells in the G1 and G2/M phase cells. Furthermore,
the apoptotic cells increased from 0.24% to 4.89% after
exposure to 2-Ga. These results imply that dyad 2-Ga
induces cell cycle arrest at the S phase, probably through
induction of apoptosis.
Comet assay
DNA fragmentation is a hallmark of apoptosis, mitotic
catastrophe or both [30]. Single cell gel electrophoresis
(comet assay) in an agarose gel matrix was used to study
DNA fragmentation. As shown in Fig. 9, in the control,
SiHa cells failed to show a comet-like appearance.
Treatment of SiHa cells with 15 mM of complex showed
statistically significant and well-formed comets, with the
length of the comet tail representing the extent of DNA
damage. These results clearly indicate that 2-Ga indeed
induced DNA fragmentation, which is further evidence
of apoptosis.
EXPERIMENTAL
Materials and methods
All reagents were purchased from commercial sources
and used without further purification unless otherwise
mentioned. UV-vis absorption spectra were measured
on a Hitachi 3900H spectrophotometer in 1 cm optical
path length quartz cells at room temperature. Fluoresce
emission spectra were measured using a Hitachi F-4500
fluorescence spectrophotometer. Intracellular location
Cell-cycle assay
In the following study, the 2-Ga complex was
examined for its effect on cell cycle progression of
Fig. 10. Cyclic progression of SiHa cells treated with 15 μM 2-Ga. Cells were harvested, fixed with 70% ethanol, and stained with
propidium iodide (PI). The cellular DNA content was then determined by flow cytometry analysis
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PHOTOPHYSICAL PROPERTIES AND PHOTODYNAMIC ANTI-TUMOR ACTIVITY OF CORROLE-COUMARIN DYADS
9
was visualized using an Axio Observer Z1 fluorescence
microscope. HR-MS spectra were recorded on an
Esquire HCT PLUS mass spectrometer. H NMR spectra
Singlet oxygen quantum yield
For the singlet oxygen generation experiment, an
aerated solution of 1,3-diphenylisobenzofuran (DPBF)
1
were recorded on a 400 MHz spectrometer in CDCl or
3
(
20 mM) and photosensitizer (0.5 mM) in DMF (3 mL)
DMSO-d solution.
6
was irradiated at 650 nm under a LED lamp (650 nm) at
All biological experiments were prepared using
Ultrapure MilliQ water. A red LED lamp (650 nm) was
used as the light source. The distance between the light
source and the surface of the sample was kept constant
1
2
5°C for 60 s intervals. Reaction of DPBF with O was
2
monitored by the decreasing intensity of the absorption
band at 418 nm over time.
(
10 cm) in all experiments. LED irradiance was measured
Theoretical calculations
using a TES-1330A luxmeter (TES, Taiwan) at the plate
surface. The irradiance dose used for the present work
is 3 mw·cm
irradiation for 2 h. Cell lines of human hepatocellular
carcinomas (Bel-7402), human lung cancer (A549) and
human cervical cancer (SiHa) were purchased from the
American Type Culture Collection. Cancer cells were
cultured in RPMI 1640, with the supplement of 10% fetal
bovine serum (FBS; Gibco, US).
Full geometry optimization computations of the
dyads porphyrin-coumarin dyads were carried out with
the DFT-B3LYP method using the 6-31G (d,p) basis set
and for the Ga atom, the lanl2DZ basis set. Frequency
analysis confirmed that the obtained geometries are
genuine global minimum structures. All calculations
were performed with the Gaussian G09 package [32].
-2
-2
, and the light reached to 22 J·cm after
Cyclic voltammetry
Fluorescence quantum yield
All cyclic voltammograms (CV) were performed in
DMF solution containing 0.1 M tetrabutylammonium
perchlorate (TBAP) and complexes (1 mM) under
nitrogen atmosphere at ambient temperature. The scan
rate was 100 mV/s. A three-electrode system consisting
of a glassy carbon working electrode, a platinum wire
Luminescence quantum yields (F ) were measured in
F
dilute N,N-Dimethylformamide (DMF) solutions with an
absorbance below 0.1 by using Equation 1 where A (l)
is the absorbance at the excitation wavelength (l), n is
the refractive index, and I is the integrated luminescence
intensity. Subscripts “r” and “s” stand for reference and
sample, respectively.
counter electrode and a saturated Ag/AgNO electrode
3
as the reference electrode was employed. The Ag/
AgNO electrode contained 1 M TBAP in DMF. Half-
wave potentials (E1/2) for reversible or quasi-reversible
redox processes were calculated as E1/2 = (Epa + Epc)/2,
3
2
ΦFs
Ar (λ)
Is
ns
=
×
×
(1)
2
ΦFs
As (λ) Ir
nr
where Epa and Epc represent the anodic and cathodic peak
potentials, respectively. The E1/2 value for the ferrocene
couple under these conditions was 0.47 V.
5
,10,15,20-Tetraphenylporphyrin (TPP) in aerated
toluene was used as a standard with F = 0.11 [31].
F
Cytotoxicity assays
Fluorescence lifetime
3
An MTT assay was carried out using 5 × 10 cells
Fluorescence lifetimes of porphyrin and dyads were
measured by time-correlated single-photon counting
using a FLSP920 Combined Fluorescence Lifetime and
Steady State Spectrometer (Edinburgh Instruments) with
a pulse diode laser EPL 405 (Edinburgh Instruments)
with spectral full-width half maximum of 405 ± 10 nm
and pulse width 58.8 ps at a 10 MHz repetition rate.
Fluorescence decays of porphyrin and dyads were
observed at 650 nm. Highly dilute colloidal silica (Ludox)
in water as a scattered solution was used to acquire the
instrument response function (IRF). Data acquisition
took place until a maximum count in a single channel
reached at least 10 . All experiments were performed at
room temperature for air-equilibrated solutions using
quartz cuvettes.
Fluorescence lifetimes of coumarin were measured
with a laser excitation wavelength at 310 nm, and
fluorescence decays were observed at 410 nm.
in 100 μl of medium seeded on 96-well plates and
grown overnight at 37°C in a 5% CO incubator. Serial
2
dilutions of the complex ranging from 0.2–100 mM in
DMSO were added to the monolayer. The plates were
incubated at normal condition for 48 h. The cultures
-
1
were assayed after the addition of 20 ml of 5 mg·mL
MTT and incubation for 4 h at 37°C. Finally, the
MTT-containing medium was aspirated, and the buffer
(100 mL) containing DMSO (50%) and sodium dodecyl
sulfate (20%) was added to solubilize the MTT formazan.
The optical density of each well was measured with a
microplate spectrophotometer at a wavelength of 490 nm.
The IC50 values were determined by plotting the
percentage of cell viability vs. concentration on a
logarithmic graph and reading off the concentration
at which 50% of cells remained viable relative to the
control. Each experiment was repeated at least three
times to obtain the mean values.
4
Copyright © 2018 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2018; 22: 9–13

1
0
F. CHENG ET AL.
Intracellular location
The experimental group was incubated with 15 mM of
complex treated with red light 2 h and then incubated for
A549 cells were placed in 24-well micro assay culture
2
4 h at 37°C. The cells were harvested by a trypsinization
4
plates (4 × 10 cells per well) and grown overnight at
process at 24 h. A total of 100 mL of 0.5% normal agarose
in PBS was dropped gently onto a fully frosted microslide,
covered immediately with a coverslip, and then placed
at 4°C for 10 min. The coverslip was removed after
the gel had set. 50 mL of the cell suspension (200 cells/
μL) was mixed with 50 mL of 1% low-melting agarose
preserved at 37°C. A total of 100 mL of this mixture
was applied quickly on top of the gel, coated over the
microslide, covered immediately with a coverslip, and
then placed at 4°C for 10 min. The coverslip was again
removed after the gel had set. A third coating of 50 mL
of 0.5% low melting agarose was placed on the gel and
allowed to set at 4°C for 15 min. After solidification of
the agarose, the coverslips were removed, and the slides
were immersed in an ice-cold lysis solution (2.5 M NaCl,
100 mM EDTA, 10 mM Tris, 90 mM sodium sarcosinate,
NaOH, pH 10, 1% Triton X-100 and 10% DMSO) and
placed in a refrigerator at 4°C for 2 h. All of the above
operations were performed under low light conditions to
avoid additional DNA damage. The slides, after removal
from the lysis solution, were placed horizontally in an
electrophoresis chamber. The reservoirs were filled with
an electrophoresis buffer (300 mM NaOH, 1.2 mM
EDTA) until the slides were immersed in it, and the DNA
was allowed to unwind for 30 min in an electrophoresis
solution. Then electrophoresis was carried out at 25 V
and 300 mA for 20 min. After electrophoresis, the slides
were removed, washed three times in a neutralization
buffer (400 mM Tris, HCl, pH 7.5). Nuclear DNA was
stained with 20 mL of EtBr (20 mg/mL) in the dark for
3
7°C in a 5% CO incubator. Then the test compounds
2
were added to the wells. The plates were incubated at
7°C in a 5% CO incubator for 24 h. Upon completion
3
2
of the incubation, the wells were washed three times
with PBS. After removing the culture medium, the cells
were stained with DAPI and imaged by fluorescence
microscopy.
Mitochondrial membrane potential assay
5
SiHa cells in a growth medium (2 × 10 cells/well)
were seeded on a sterilized coverslip in six-well plates
and grown overnight at 37°C in a 5% CO incubator.
2
2
-Ga was added to the wells and incubated at 37°C in
5% CO for 4 h, then cells were exposed to the red light
2
for 2 h. After incubation for 18 h, the cells were incubated
for 20 min with 1 mg/mL of 5,5′,6,6′-tetrachloro-
1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) in
culture medium at 37°C in the dark. The cell pellets were
suspended in PBS and then imaged under a fluorescence
microscope.
Reactive oxygen species (ROS) level detection
SiHa cells were seeded into six-well plates at a density
5
of 2 × 10 cells per well and incubated for 24 h at 37°C
in 5% CO . The medium was removed and replaced with
2
medium containing 2-Ga.After incubation at 37°C in 5%
CO for 4 h, the cells were irradiated for 2 h. Followed
2
by incubation for 18 h, the medium was removed.
The fluorescent dye 2′,7′-dichlorodihydrofluorescein
diacetate (DCHF-DA, 10 mM) was added to the medium
and the cells were imaged by fluorescence microscopy.
2
0 min. The slides were washed in chilled distilled water
for 10 min to neutralize the excess alkali, air-dried and
scored for comets by fluorescence microscopy.
Cell cycle arrest studies
Synthetic procedures
SiHa cells were seeded into six-well plates at a
6
The reference corrole
groups were synthesized according to the general method
1 and 1-Ga with mono-hydroxyl
density of 1 × 10 cells per well and incubated for 24 h.
The cells were cultured in RPMI 1640 supplemented
with 10% of FBS and incubated at 37°C and 5% CO2.
The medium was removed and replaced with medium
containing complex 2-Ga (15 mM). Then the cells were
treated with red light for 2 h and incubated for 24 h at
[
33, 34].
1
1
. H NMR (400 MHz, CDCl , Me Si): d 9.11 (d, J =
3
4
4
.3 Hz, 2H, pyrrole–H), 8.71 (s, 4H, pyrrole–H), 8.57 (d,
J = 4.2 Hz, 2H, pyrrole–H), 8.02 (d, J = 8.3 Hz, 2H, Ph),
1
9
7
.14 (d, J = 8.0 Hz, 2H, Ph). F NMR (376 MHz, CDCl )
3
3
7°C. After incubation, the cell layer was trypsinized
and washed with cold PBS and fixed with 70% ethanol.
0 mL of RNAse (0.2 mg/mL) and 20 mL of propidium
d -137.86 (dd, J = 23.6, 7.9 Hz), -152.90 (t, J = 20.9 Hz),
-1
-161.80 (dt, J = 23.1, 7.6 Hz). IR (KBr) nmax, cm : 3328–
2
3
1
508 (N–H, O–H), 1649, 1606, 1521, 1494, 1434, 1282,
iodide (0.02 mg/mL) were added to the cell suspensions
and the mixtures were incubated at 37°C for 30 min. The
samples were then analyzed with a FACSCalibur flow
cytometer.
176, 1159, 1058, 982, 928, 847, 795, 758.
1
1
-Ga. H NMR (400 MHz, DMSO, Me Si): d 9.77 (s,
4
1
H, –OH), 9.30 (s, 2H, pyrrole–H), 9.11–8.64 (m, 6H,
pyrrole-H), 8.54 (d, J = 6.8 Hz, 2H, pyridine–H), 7.96 (d,
J = 7.0 Hz, 2H, Ph), 7.77 (t, J = 6.7 Hz, 1H, pyridine–H),
Comet assay
7
.36 (d, J = 4.5 Hz, 2H, pyridine–H), 7.19 (d, J = 7.2 Hz,
19
DNA damage was investigated by means of a comet
assay. The control SiHa cells were incubated for 24 h.
2H, Ph). F NMR (376 MHz, DMSO) d -139.06 (d, J =
25.9 Hz), -155.48 (t, J = 22.2 Hz), -163.29 (dd, J = 34.5,
Copyright © 2018 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2018; 22: 10–13

PHOTOPHYSICAL PROPERTIES AND PHOTODYNAMIC ANTI-TUMOR ACTIVITY OF CORROLE-COUMARIN DYADS
11
-
1
1
1
7
3.9 Hz). IR (KBr) nmax, cm : 3436 (O–H), 1612, 1519,
492, 1332, 1290, 1234, 1168, 1060–1035 850, 793, 760,
04–692.
silica gel chromatography (100–200 mesh) with DCM
to provide the crude product. The crude product was
purified by silica gel chromatography (300–400 mesh)
with DCM/hexane (1:1) used as the eluent, and
2 was
Synthesis of (4-(2-coumarin-7-oxy)ethoxy)
benzaldehyde
obtained as a dark violet solid after recrystallization from
DCM and hexane (yield 29%).
+
2
. MS (HR-MS): m/z 911.1716 (calcd for [M]
The aldehyde was synthesized as shown in Scheme 1.
First, 4-hydroxybenzaldehyde (5.0 g, 40.9 mmol) was
reacted with 1,2-dibromoethane (11.5 g, 61.4 mmol),
dissolved in dry DMF (20 mL), then K CO was added
1
9
11.1711); H NMR (400 MHz, DMSO, Me Si): d 9.21
s, 2H, pyrrole–H), 8.94 (s, 2H, pyrrole–H), 8.72 (s, 2H,
4
(
pyrrole–H), 8.61 (s, 2H, pyrrole–H), 8.04 (d, J = 12.5 Hz,
2
3
3
2
8
H, Ph, cou–H), 7.71 (d, J = 7.6 Hz, 1H, cou–H), 7.43 (s,
H, cou–H), 7.16 (d, J = 24.0 Hz, 2H, Ph), 6.34 (d, J =
and the mixture was stirred under room temperature
for 24 h. The progress of the reaction was monitored by
thin-layer chromatography (TLC). After the reaction, the
product was washed several times with dichloromethane
and saturated salt water, and the organic phase was
collected. The crude product was purified by silica gel
chromatography (100–200 mesh) with DCM/hexane (1:2)
used as the eluent, and 4-(2-bromoethoxy)benzaldehyde
was obtained as a pale yellow solid (yield 76%). Second,
1
9
.8 Hz, 1H, cou–H), 4.64 (s, 4H, –CH –CH ). F NMR
2
2
(
(
376 MHz, DMSO) d -139.59 (d, J = 22.7 Hz), -154.82
t, J = 21.4 Hz), -162.88 (t, J = 21.2 Hz). IR (KBr) nmax,
-1
cm : 3437 (N–H), 2927–2850 (aliphatic–CH), 1739
(
(
C=O), 1650, 1608, 1521, 1495, 1426, 1283, 1248–1231
C–O), 1178, 1159, 1124 (C–O), 1059, 984, 930, 837,
7
97, 758.
4
-(2-bromoethoxy)benzaldehyde (5.0 g, 21.8 mmol) was
Synthesis of gallium dyad 2-Ga
The corrole-coumarin dyad (30.0 mg, 0.033 mmol)
reacted with 7-hydroxycoumarin (7.1 g, 43.7 mmol),
K CO was added dissolved in dry DMF, reflux reacted
2
2
3
for 24 h, then the product was washed several times with
dichloromethane and water, and the organic phase was
collected. The crude product was purified by silica gel
chromatography (100–200 mesh) with DCM/hexane
and Gallium(III) chloride (58 mg, 0.33 mmol) were
dissolved in pyridine (10 mL), reflux stirred under argon
atmosphere for 2 h. After the reaction was complete, the
mixture was cooled to room temperature, the solvent
was evaporated and the residue was dissolved in DCM,
filtered, dried, and evaporated. The crude product was
purified by silica gel chromatography (200–300 mesh)
with DCM/pyridine (100:1) used as the eluent. A red
fraction was collected. The solvent was evaporated under
vacuum and 2-Ga was obtained as a purple-red solid after
recrystallization from DCM and hexane in an 81% yield.
2-Ga. MS (HR-MS): m/z 999.0549 (calcd for [M –
(
1:2) used as the eluent. (4-(2-coumarin-7-oxy)ethoxy)
benzaldehyde was obtained as a pale yellow solid (yield
1%).
-(2-Bromoethoxy)benzaldehyde. HNMR(400MHz,
CDCl , Me Si): d 9.85 (s, 1H, –CHO), 7.80 (d, J = 8.6 Hz,
6
1
4
3
4
2
1
H, Ph), 6.97 (d, J = 8.5 Hz, 2H, Ph), 4.35 (dd, J = 21.7,
5.6 Hz, 2H, –CH ), 3.63 (t, J = 6.1 Hz, 2H, –CH ).
2
2
(
4-(2-Coumarin-7-oxy)ethoxy)benzaldehyde. MS
+
1
(
HR-MS): m/z 333.0740 (calcd for [M + Na] 333.0733);
pyridine + Na] 999.0551); H NMR (400 MHz, DMSO,
1
H NMR (400 MHz, CDCl , Me Si): d 9.90 (s, 1H,
Me Si): d 9.31 (d, J = 3.6 Hz, 2H, pyrrole–H), 8.99 (s,
3
4
4
–
1
CHO), 7.86 (d, J = 8.2 Hz, 2H, Ph), 7.64 (d, J = 9.5 Hz,
H, cou–H), 7.39 (d, J = 8.3 Hz, 1H, cou–H), 7.06 (d,
2H, pyrrole–H), 8.90 (s, 2H, pyrrole–H), 8.72 (d, J =
4.4 Hz, 2H, pyrrole–H), 8.56 (s, 2H, pyridine–H), 8.07
(dd, J = 19.5, 8.6 Hz, 3H, Ph), 7.86–7.65 (m, 2H, cou–H,
pyridine–H), 7.56–7.29 (m, 4H, cou–H, pyridine–H),
7.20 (s, 1H, Ph), 7.13 (d, J = 8.8 Hz, 1H, cou–H), 6.34
(d, J = 9.6 Hz, 1H, cou–H), 4.65 (s, 4H, –CH –CH ).
J = 8.3 Hz, 2H, Ph), 6.89 (d, J = 10.2 Hz, 2H, cou–H),
6
–
1
1
1
.27 (d, J = 9.5 Hz, 1H, cou–H), 4.43 (d, J = 4.2 Hz, 4H,
13
CH –CH ). C NMR (101 MHz, CDCl ) d 190.83 (s),
2
2
3
63.52 (s), 161.74 (s), 161.13 (s), 155.97 (s), 143.40 (s),
32.15 (s), 130.61 (s), 129.04 (s), 115.04 (s), 113.69 (s),
13.12 (d, J = 4.6 Hz), 101.77 (s), 66.94 (s), 66.61 (s).
2
2
1
9
F NMR (376 MHz, DMSO) d -139.07 (dd, J = 25.7,
6.0 Hz), -155.45 (t, J = 21.9 Hz), -163.00 – -163.71 (m).
-1
IR (KBr) nmax, cm : 3448 (O–H), 2929–2858 (aliphatic–
CH), 1735 (C=O), 1610, 1519, 1492, 1332, 1290, 1230,
Synthesis of dyad 2
-(pentafluorophenyl) dipyrromethane (5F-DPM) was
prepared according to previously published methods [35].
Synthesis of : 5-(pentafluorophenyl) dipyrrylmethane
0.25 g, 0.8 mmol) and (4-(2-coumarin-7-oxy)ethoxy)
1
122 (C–O), 1031, 986, 957, 837, 795, 762, 692.
5
2
CONCLUSIONS
(
benzaldehyde (0.13 g, 0.4 mmol) were dissolved
in dichloromethane (DCM, 10 mL), mixed in 6 mL
THF, stirred for 5 h at room temperature. Then 12 μL
trimethylamine, 40 mL DCM and 0.18 g (0.8 mmol)
DDQ were added and the mixture was stirred at room
temperature for 1 h. DDQ was removed through the
In summary, we have synthesized a new corrole-
coumarin dyad and its gallium complex. Intramolecular
energy transfer from the coumarin to the corrole moiety
was observed after excitation of coumarin, due to the
spectral overlap between the emission spectrum of
the coumarin subunit and the absorption spectrum
Copyright © 2018 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2018; 22: 11–13

1
2
F. CHENG ET AL.
of the corrole moiety. In vitro experiments showed
that corrole-coumarin dyads exhibited significant
photodynamic anticancer activity toward all tested tumor
cell lines. Further mechanistic studies using the SiHa
cell line revealed that the absorbed 2-Ga was extensively
distributed in the cytoplasm and the nuclei. After PDT
treatment, 2-Ga induced tumor cell S phase arrest, ROS
level increase and mitochondrial membrane potential
destruction, which finally led to the apoptosis of SiHa
cells.
12. Concellón A, Marcos M, Romero P, Serrano JL,
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Acknowledgments
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0: 3361–3370.
This work was supported by the National Natural
Science Foundation of China (Nos. 21671068, 81400023),
the National Basic Research Program (973 Program)
of China under Grant 2013CB922403 and the Open
Fund of State Key Laboratory of Optoelectronic Materi-
als and Technologies (Sun Yat-Sen University) (No.
OEMT-2015-KF-05).
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Supporting information
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Figures S1–S20 are given in the supplementary
material. This material is available free of charge via the
Internet at http://www.worldscinet.com/jpp/jpp.shtml.
2
2. Tasior M, Voloshchuk R, Poronik YM, Rowicki T
and Gryko DT. J. Porphyrins Phthalocyanines
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