Synthesis and pharmacological evaluation of chlorin derivatives for photodynamic therapy of cholangiocarcinoma

Article abstract of DOI:10.1016/j.ejmech.2020.112049

Photodynamic therapy (PDT) has been developed as a promising therapeutic method in cancer treatment. The discovery of effective photosensitizer, which is the key factor of PDT, is highly desired. This paper reports the synthesis of novel chlorin derivativ

Full text of DOI:10.1016/j.ejmech.2020.112049

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Synthesis and pharmacological evaluation of chlorin derivatives for photodynamic
therapy of cholangiocarcinoma
Ying-Hua Gao, Man-Yi Li, Faiza Sajjad, Jin-Hai Wang, Faiza Meharban, Malaz
Abdelazeem Gadoora, Yi-Jia Yan, Tebello Nyokong, Zhi-Long Chen
PII:
S0223-5234(20)30016-7
DOI:
https://doi.org/10.1016/j.ejmech.2020.112049
EJMECH 112049
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 October 2019
Revised Date: 17 December 2019
Accepted Date: 7 January 2020
Please cite this article as: Y.-H. Gao, M.-Y. Li, F. Sajjad, J.-H. Wang, F. Meharban, M.A. Gadoora, Y.-
J. Yan, T. Nyokong, Z.-L. Chen, Synthesis and pharmacological evaluation of chlorin derivatives for
photodynamic therapy of cholangiocarcinoma, European Journal of Medicinal Chemistry (2020), doi:
https://doi.org/10.1016/j.ejmech.2020.112049.
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Synthesis and pharmacological evaluation of chlorin derivatives for
photodynamic therapy of cholangiocarcinoma
1
1
1
1
1
Ying-Hua Gao , Man-Yi Li , Faiza Sajjad , Jin-Hai Wang , Faiza Meharban , Malaz
1
1
2
1
Abdelazeem Gadoora , Yi-Jia Yan , Tebello Nyokong *, Zhi-Long Chen *
1
Department of pharmaceutical Science & Technology, College of Chemistry and
Biology, Donghua University, Shanghai 201620, China
2
Department Chemistry, Rhodes University, P. O Box, Grahamstown, South Africa.
*
Corresponding author: Zhi-Long Chen, E-mail: zlchen1967@qq.com; Tebello
Nyokong, E-mail: t.nyokong@ru.ac.za.
Abstract
Photodynamic therapy (PDT) has been developed as a promising therapeutic method
in cancer treatment. The discovery of effective photosensitizer, which is the key
factor of PDT, is highly desired. This paper reports the synthesis of novel chlorin
derivatives,
chlorin
5,10,15,20-tetraphenyl-[2:3]-[(methoxycarbonyl,
carboxy)methano]
I
and
5,10,15,20-tetraphenyl-[2:3]-
{
[methoxycarbonyl,(2-hydroxyethyl)amide]methano}chlorin II. Their structures were
1
13
characterized with UV-vis, HNMR,
CNMR and HRMS spectroscopies.
Photophysical and photochemical experiments results showed that compound I and II
had an absorption maximum around 650 nm, with molar extinction coefficients of 1 ×
4
-1.
-1
1
0 M cm . They had strong fluorescence emission in 650 - 660 nm upon excitation
with 419 - 422 nm light. ESR showed that singlet oxygen was produced upon
irradiation of compounds with 650 nm light in the presence of molecular oxygen. The
photo-bleaching test indicated that the structure of compounds was stable. These new
compounds exhibit excellent anti-tumor effects and lower toxicity compared to
m-THPC in vitro and in vivo. Compound I and II had high tumor selectivity, which
could induced tumor cells shrinkage and necrosis under 650 nm laser irradiation.
Flow cytometry revealed that the compounds might mediate PDT effect at late
apoptotic phase. These results make these compound I and II promising candidates
for future study in photo-diagnosis and photodynamic therapy of cholangiocarcinoma.
Keywords: Photosensitizer, Photodynamic therapy, Chlorin derivatives, Tumor,
Cholangiocarcinoma
1
. Introduction
Cholangiocarcinoma (CCA) is the common malignant tumor worldwide [1, 2].
Due to latent symptoms, most CCA patients are diagnosed at a late stage and lost the
opportunities to accept radical surgery. Therefore, the survival prognosis of CCA is
generally poor with extremely high mortality rate [3]. Photodynamic therapy (PDT) is

a new local treatment technique for head and neck neoplasms, gastrointestinal
neoplasms, bladder neoplasms, actinic keratosis, pointed condyloma, age-related
macular degeneration and other diseases in clinics [4-7]. In recent years, PDT has
become an important therapeutic option for palliative treatment of
cholangiocarcinoma. Most photosensitizers have several absorption wavelengths, and
the relatively long wavelength of PSs is selectively used in the treatment of deep
seated tumors because the wavelength of light is positively correlated with the depth
of tissue penetration [8]. After irradiation with light at a specific wavelength, the
photosensitizer can produce reactive oxygen species (ROS) which ultimately leads to
cell death through apoptosis or necrosis [9]. Research has shown that one of the
drawbacks of PDT is weak light penetration capability [10]. Therefore, the
development of photosensitizer with absorption in the near-infrared region is very
important to PDT.
With relative strong absorption in the visible light region, high yield of ROS
generation and good cellular uptake efficiency, chlorin derivatives have the potential
to serve as ideal photosensitizers of PDT [11]. Among the second-generation
photosensitizers, meta-tetra(hydroxyphenyl)chlorin (m-THPC) possess efficacy with
two orders of magnitude higher than the first-generation photosensitizer porphyrin
derivatives HpD [12,13], and with low toxicity recognized in clinics [14,15].
Here, two chlorin derivatives, 5,10,15,20-tetraphenyl-[2:3]-[(methoxycarbonyl,
carboxy)methano]
[methoxycarbonyl,(2-hydroxyethyl) amide]methano}chlorin II (Scheme 1), were
synthesized and the photodynamic effects were investigated.
chlorin
I
and
5,10,15,20-tetraphenyl-[2:3]-
{
2
2
3
. Materials and methods
.1. Chemicals and reagents
-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), dimethyl
sulfoxide (DMSO), N,N-dimethylformamide (DMF), tetrahydrofuran (THF),
Polyoxyethylene castor oil, phosphate-buffered saline (PBS),
,2,6,6-tetramethylpiperidine (TEMP), alcohol, paraffin and Annexin V-FITC /
2
propidium iodide (PI) Apoptosis Detection Kit were purchased from Sigma (St. Louis,
MO, USA). RPMI-1640 medium, fetal serum (FBS), L-glutamine, penicillin, and
streptomycin were acquired from Hyclone (Logan, UT, USA). Other reagents were
purchased from Sinopharm Chemical Reagent Co., Ltd and used without further
purification. All solutions were freshly prepared. All the chemicals or reagents were
of analytical grade.
2
5
.2. Synthesis
,10,15,20-tetraphenyl-[2:3]-[bis(methoxycarbonyl)methano] chlorin (3)
A solution of K CO (620 mg, 4.5 mmol) and dimethyl malonate (0.77mL, 6.8 mmol)
2
3
in DMSO (20 mL) was stirred at 60 °C for 1 h under nitrogen. Compound 2 (800 mg,
.1 mmol) was added, and the solution was stirred at 100 °C for 3 h. The mixture was
1
cooled, diluted with dichlormethane (400 mL) and washed with water (6 × 200 mL).
The organic phase was dried over anhydrous Na SO , filtered, and evaporated to
2
4

dryness. The crude chlorin residue was purified by silica gel chromatography eluted
with ethyl acetate/ petroleum ether (1 : 20) to give purple powder product 3 ( 360 mg,
1
4
5.9%). HNMR (400 MHz, CDCl ) δ ppm: 8.72 (d, J = 4.9 Hz, 2H), 8.64 (d, J = 7.5
3
Hz, 2H), 8.54 (s, 2H), 8.49 (d, J = 5.0 Hz, 2H), 8.16 (dd, J = 14.2, 6.9 Hz, 4H), 7.89 -
.67 (m, 14H), 4.85 (s, 2H), 3.89 (s, 3H), 2.40 (s, 3H), -2.07 (s, 2H).
7
+
MS(MALDI-TOF) m/z: 745.3[M + H] .
5
,10,15,20-tetraphenyl-[2:3]-[(methoxycarbonyl, carboxy)methano]chlorin (I)
A solution of compound 3 (149 mg, 0.2 mmol) and 3 mol/L NaOH (6 mL) in THF (8
mL) was heated to reflux for 5 h under nitrogen. The solvent was removed in vacuo.
After addition of water (50 mL) and neutralization by 3 mol/L HCl (6 mL), the
residue was extracted by dichloromethane (100 mL). The organic phase was washed
with saturated brine (3 × 50 mL), dried over anhydrous Na SO , filtered, and
2
4
evaporated to dryness. The crude chlorin residue was purified by silica gel
chromatography eluted with 1 : 20 methanol / dichloromethane to give the purple
1
solid product I (89 mg, 60.9%). HNMR (400 MHz, DMSO-d ) δ: 8.70 (d, J = 4.9 Hz,
6
2
H), 8.53 (d, J = 7.5 Hz, 2H), 8.45 (d, J = 4.9 Hz, 2H), 8.41 (s, 2H), 8.12 (dd, J = 17.8,
6
2
.6 Hz, 4H), 7.90 (d, J = 5.9 Hz, 2H), 7.83 - 7.72 (m, 12H), 5.76 (s, 1H), 4.59 (s, 1H),
1
3
.34 (s, 3H), -2.19 (s, 2H). CNMR (100 MHz, DMSO-d ) δ: 206.98, 170.48, 165.30,
6
1
1
2
59.12, 153.02, 141.98, 141.51, 139.77, 135.53, 134.10, 133.95, 133.28, 132.79,
28.90, 128.54, 128.47, 128.28, 128.18, 127.49, 125.47, 122.88, 114.77, 51.90, 44.78,
+
2.55, 14.40. H RMS (DART Positive) m/z: calcd for C H N O [M+H] , 731.2653;
4
8
34
4
4
found, 731.2654.
5
,10,15,20-tetraphenyl-[2:3]-{[methoxycarbonyl,(2-hydroxyethyl)amide]methano}
chlorin (II )
A solution of compound 3 (372 mg, 0.5 mmol) and ethanolamine (10 mL) in THF (20
mL) was heated to reflux for 13 h under nitrogen. The solvent was removed in vacuo,
and the residue was diluted with dichloromethane (100 mL), washed with water (3 ×
5
0 mL), saturated brine (3 × 50 mL). The organic phase was dried over anhydrous
Na SO , filtered, and evaporated to dryness. The crude chlorin was purified by silica
2
4
gel chromatography eluted with 1 : 20 methanol / dichloromethane to give purple
1
solid product II (139 mg, 36%). HNMR (400 MHz, CDCl ) δ: 8.72 (d, J = 4.9 Hz,
3
2
2
2
H), 8.54 (s, 2H), 8.51 - 8.46 (m, 4H), 8.16 (dd, J = 13.2, 6.9 Hz, 4H), 7.89 - 7.85 (m,
H), 7.79 - 7.68 (m, 12H), 7.24 (t, J = 5.6 Hz, 1H), 4.86 (s, 2H), 3.81 (t, J = 5.0 Hz,
1
3
H), 3.56 (q, J = 5.3 Hz, 2H), 2.16 (s, 3H), -1.99 (s, 2H). CNMR (100 MHz, CDCl )
3
δ: 168.38, 167.55, 159.05, 153.29, 142.26, 142.01, 140.15, 135.90, 133.99, 133.82,
1
6
32.77, 132.68, 128.11, 127.79, 127.70, 127.58, 126.68, 124.75, 122.64, 114.59,
2.24, 51.58, 43.05, 36.64, 22.65, 14.03. HRMS (DART Positive) m/z: calcd for
+
C H N O [M+H] , 774.3075; found, 774.3076.
50
39
5
4
2
.3. Ultraviolet-visible and fluorescence spectroscopy
Compounds were dissolved in different solvents (DMSO, DMF) and emulsion
Polyoxyethylene castor oil + PBS). The drug delivery system were prepared as
(

follows: The compounds were dissolved in THF and mixed with polyoxyethylene
castor oil. THF was removed under vacuum and the resulted solution was diluted with
PBS. The Ultraviolet-visible absorption spectra were recorded by Ultraviolet-visible
Spectrophotometer (Model V-530, Japan) with 1 nm resolution. Fluorescence spectra
were carried out by Fluorescence Spectrometer (FluoroMax-4, France) in the range
5
50 - 800 nm with 420 nm excitation wavelength. Fluorescence quantum yields (Φ )
f
+
were measured using rhodamine 6G chloride cation (R6G ) in H O as a standard (Φ
2
f
=
0.9) [16]. The Φ was calculated with the following formula:
f
Φ , I and A were the the sample of fluorescence quantum yields, integrated
f
f
f
fluorescence intensity, absorbance, respectively. Φ , I and A were the the reference of
s
s
s
fluorescence quantum yields, integrated fluorescence intensity, absorbance,
respectively. I , was the fluorescence emission spectrum curve of the substance. λ ,
f
λ
1
λ2 were the fluorescence emission initiation and termination wavelengths of the
integrated fluorescence intensity, respectively. All the measurements were carried out
at room temperature in quartz cuvettes with a path length of 1 cm.
2
.4. ESR measurements
ESR measurements were performed with a Micro-ESR spectrometer (Bruker). TEMP
1
was used to obtain paramagnetic TEMPO via generated O . TEMP and PSs were
2
dissolved in DMF, respectively. The 20 µL TEMP (0.2 M) and 20 µL compound were
mixed in capillary tubes, which were irradiated every 1 min in the presence of oxygen
using a 5 mW Nd: YAG laser (650 nm) as the light source. The irradiation area of the
sample was 10 mm in diameter. ESR assays were all carried out at room temperature
operating at 9.65 GHz of the microwave frequency, 15 mW of microwave power,
3
400 - 3500 G of scanning field, 0.8 G of modulation amplitude, 15.5 S of the scan
time, and 32 scan number.
2
.5. Photo-bleaching assay
The photo-bleaching characteristics of compounds in DMSO were tested. The
absorption spectra were measured when the solution of the compound at final
concentrations of 10 µM was added to 1 cm quartz cuvettes. The solutions were then
irradiated with a semiconductor laser unit (PDT - 650 nm) with the output power and
2
discontinuous irradiation time fixed to 5 mW/cm and 90 min. After irradiation, the
absorbance was measured again. The extent of photo-bleaching was calculated
according to the equation: percent (%) = A / A 100, where A and A represent the
t
0 ×
0
t
absorbance before and after irradiation and t is the irradiation time (10 - 90 min).
2
.6. Cell culture and in vitro cytotoxicity assay

The in vitro anticancer activities of the PSs against cholangiocarcinoma cell line
QBC-939 were evaluated. The QBC-939 cells were obtained from ATCC and grown
in RIPM-1640 medium with 10% fetal serum, L-glutamine, penicillin, and
streptomycin at 37 °C in 5 % CO . The cells at logarithmic growth stage were
2
4
collected and seeded in 96-well plates at a density of 5 × 10 cells per well (five
replicates) in complete medium. After overnight incubation at 37 °C, the compounds
at different concentrations were added and then the cells were incubated for 24 h in
the dark. The original medium was removed and the cells were irradiated with 650 nm
2
light (20 mW/cm ) at different doses (1 J, 2 J, 4 J) respectively. The fresh medium
was added and the cells were incubated for another 24 h. Cell survival was assessed
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye
reduction assay [17]. The cell survival rate was calculated with the following formula:
cell viability (%) = (ODexpriment - ODblank) / (ODcontrol - ODblank) × 100%. The IC50
values were obtained by the SPSS program.
2
.7. Cellular uptake and subcellular localization
4
QBC-939 cells were cultured and grown in 12-well plates at 5 × 10 cells/well. 24 h
later, the medium was replaced with fresh medium containing 4 µM compound and
incubated for an additional 24 h. The cellular uptake of the compound was determined
by a fluorescence spectrophotometer according to the method previously reported
[
18].
To determine the subcellular localization of the compounds, the dye probes including
Hoechst 33342, Mito-Tracker Green, and Lyso-Tracker Blue, were used to stain
nucleus, label mitochondria, and lysosome, respectively. After digested with 2.5 %
trypsin and washed three times with PBS, the cells were collected, implanted into a 35
mm confocal plate and treated with 4 µM compound for 4 h in the dark. Following
three times washes with PBS, the cells were fixed with 4 % paraformaldehyde in PBS
for 30 min, and then incubated with 5 µg / mL Hoechst 33342 for 20 min, 0.2 µM
Mito-Tracker Green for 30 min, 5 µM Lyso-Tracker Blue for 30 min, respectively.
The dye probes were then removed by washing three times with PBS, and the
examination was performed under a confocal microscope.
2
.8. Apoptosis assay
Annexin V-FITC / propidium iodide (PI) Apoptosis Detection Kit was applied to
determine the model of cell death induced by PDT in QBC-939 [19]. Four groups,
namely, control group, light group, drug group, and drug + light group were set. The
cells were incubated with 2 µM compound for 2 h and 24 h, respectively. After
washed three times with PBS and added with fresh medium, the cells were irradiated
2
with 650 nm laser light (2 J, 20 mW /cm ). The cells were then collected, resuspended
in 500 µL binding buffer and stained with 5 µL Annexin V and 5 µL PI at room
temperature in the dark for 15 min. Apoptosis or necrosis was detected with
fluorescence activated cell sorting (FACS).
2
.9. Determination of plasma concentration

The mice were sacrificed at different times after a tail vein intravenous injection of
compounds (1 mg/ kg). Blood samples were collected into the polypropylene
microfuge tubes containing heparin sodium. Plasma was separated by centrifugation
at 12000 rpm for 10 min at 4 °C, and 300 µL of which was mixed with 2.7 mL DMSO.
The fluorescence intensity of the solution was measured with the fluorometric
method.
2
.10. In vivo imaging
The bio-distribution of compounds was investigated in nude mice xenograft tumor
7
model which was established by subcutaneous injection of 1 × 10 QBC-939 cells in
2
00 µL PBS into the right forelimb of five-week-old male Balb/C nu/nu mice.
Compound (1 mg/kg) was injected via tail vein when the tumors reached 8 - 10 mm in
diameter after implantation. The whole body fluorescence images were obtained from
an IVIS spectrum small animal imaging system (PerkinElmer, USA) at 0 min, 2 h, 8 h,
2
4
4 h after injection, respectively. The excitation and emission wavelength were set at
20 nm and 650 nm respectively. Images were analyzed with Living Image 4.5.1
software (PerkinElmer, USA).
2
.11. Skin phototoxicity
ICR mice (12 males and 12 females; 20 - 22 g) were assigned randomly into four
groups (n = 6): control group (no treatment), I treatment group (1 mg/kg), II
treatment group (1 mg/kg), and m-THPC treatment group (1 mg/kg). Osram simulated
solar light was used as solar light source (wavelength range 250 - 800 nm). The hair
of each mouse was removed from the back. After injection of compound (i. v.), the
2
mice were irradiated under light intensity of 10 mW/cm for 10 min. 3 mice from
each group were sacrificed 1d after light irradiation. The tissues were made into a
round shape (diameter 8 mm) and weighed. The auricular, exposed skin and trial
samples were also obtained from the mice, fixed in 4% paraformaldehyde and
embedded in paraffin wax. They were then sectioned, and stained with HE for
microscopic examination.
2
.12. In vivo anti-tumor study
BALB/c nu/nu mice (half males and half females, 18 - 22 g) were feed in a sterile
environment with 12 h light/dark cycle under constant temperature (25 - 26 °C) and
humidity (50 - 60 %). The animals were inoculated with QBC-939 cells suspension
6
with PBS (1 × 10 cells/mouse) in the right flank region by subcutaneous injection.
3
When the tumor volumes reached approximately 130 - 150 mm , the mice were
randomly divided into four groups with six animals separately: the untreated group,
2
the light group (120 J/cm ), the dark group (1 mg/kg), and the PDT group (1 mg/kg +
2
1
20 J/cm ). Tumor growth was evaluated by measuring tumor size via calipers daily,
and the inhibition effect was determined according to the tumor growth curve.
.13. Histology examination
2

After 1d post-PDT, tumor tissue and organs including heart, liver, spleen, lung, and
kidney in the control group and PDT treatment groups were separated and fixed with
1
0 % neutral buffered formalin and embedded in paraffin. The sliced tissues were
stained with hematoxylin-eosin (H&E) and examined under a microscope. The
TUNEL assay was carried out to detect cell apoptosis. The tumor-bearing mice were
anesthetized and killed 1 day after the irradiation, and the tumor was removed and
stored in 10 % neutral buffered formalin at room temperature. The paraffin-embedded
tissues were cut into 6-µm thick sections. The sections were deparaffinized with
xylene, rehydrated with graded ethanol series and treated with proteinase K in PBS.
After washed three times with PBS, the sections were incubated at 37 °C for 60 min
in the presence of TdT enzyme with a one-step TUNEL apoptosis assay kit. Then all
the sections were washed again with PBS and mounted with the fluorescence
anti-quenching agent. The observation was performed under a fluorescence
microscope.
2
.14. Statistical analysis
Graphics were done by origin pro 8.0 (Graph Software, USA). Statistical significance
was determined by unpaired two-tailed t-tests or two-way analysis of variance. * P
<0.05 was considered statistically significant.
3
3
. Results and discussions
.1. Synthesis and characterization
The synthesis of compound I involved an seven-steps process, as shown in Scheme 1.
meso-tetraphenylporphin 1, which was obtained after cyclization of pyrrole with
benzaldehyde and oxidation with air in refluxing proprionic acid with yield about
2
5%, was reacted with cupric acetate and then nitrated with cupric nitrate followed by
demetalization with H SO and CF COOH to give
-nitro-5,10,15,20-tetraphenylporphyrin (compound 2) with 85.8% yield as the
2
4
3
2
literature [20]. Compound 2 was reacted with dimethyl malonate in the presence of
K CO in anhydrous DMSO through Michael addition reaction to give compound 3,
2
3
which was then reacted with NaOH (3M) to hydrolize one ester group without the
hydrolization of both ester groups to get the product I. Compound 3 in anhydrous
THF was stirred with ethanolamine under reflux to afford the product II (Scheme 1). I
1
13
and II were purified with column chromatography and characterized by H NMR, C
NMR and HRMS (supporting information).

COOMe
COOMe
COOMe
COOH
Ph
NO2
Ph
Ph
NH
N
N
NH
N
N
NH
N
N
d
e
Ph
Ph
Ph
Ph
Ph
Ph
HN
HN
HN
Ph
Ph
Ph
2
3
a, b, c
f
COOMe
Ph
Ph
CONHCH CH OH
2
2
NH
N
N
NH
N
Ph
Ph
Ph
Ph
HN
N
HN
Ph
Ph
1
Scheme 1: Reagents and reaction conditions: (a) Cu(OAc) ·H O, CH Cl , CH COOH,
2
2
2
2
3
RT, 15 min, 94.1%; (b) Cu(NO ) ·3H O, Ac O, CH COOH, CH Cl , reflux 4 h, 96%;
3
2
2
2
3
2
2
(
c) H SO , TFA, 30 min. 95%; (d) K CO , CH (COOCH ) , DMSO, 4 h, 45.9%; (e)
2 4 2 3 2 3 2
3
M NaOH, THF, reflux 5 h, 60.9%.(f) NH CH CH OH, THF, reflux 13 h, 36%.
2 2 2
3
.2. Photophysical characteristics
The ideal photosensitizer should have long wavelength (650 - 800 nm) absorption in
order to treat deep-buried carcinoma efficiently [21]. Researchers found that chlorin
derivatives possess absorption in the near-infrared region. The absorption spectra of
the investigated compound I and II showed similar absorption bands in different
solvents (Fig. 1a, 1d). As seen, the absorption spectra of compound I exhibited a
strong absorption for the Q-band (at 656 nm) and soret band at 420 nm. In addition,
weak absorption bands were recorded at 520, 550, 597 nm. Compound II showed
similar spectral features with compound I.
Fluorescence is an important tool in photo-imaging and photo-diagnoses in clinics.
The pathological tissue could be discerned from normal tissue by comparing their
fluorescence intensity after excitation of photosensitizer by laser [22, 23]. The
fluorescence spectra of the compounds tested at concentration of 1 µM in different
solvents displayed that I and II had strong fluorescence emission in 650 - 660 nm
upon excitation with 420 nm light (Fig. 1b, 1e). In the emulsion of Polyoxyethylene
castor oil + PBS, the fluorescence intensity of I was markedly higher than II (Fig. 1c,
1
f). In addition, compound I and II had fluorescence quantum yield (Φ ) value of 0.15
f
and 0.04 (Table 1).

Fig. 1. (a, d) UV-vis absorption spectra of compound I and II (10 µM) in different solvents
DMSO, DMF) and Polyoxyethylene castor oil + PBS. (b, e) The fluorescence emission spectra of
compound I and II. (c, f) The matrix of excitation and emission spectra of compound I and II in
Polyoxyethylene castor oil + PBS.
Table 1 Absorption, emission spectral data of compound I and II in different solvents
Q band λmax, (nm)/Logɛ
Polyoxyethy
EmissionλEm, (nm)
Polyoxyeth
Φf
Polyoxyethy
lene castor
oil + PBS
0.15
compound
lene castor
oil + PBS
655/4.20
650/3.34
DMSO
DMF
ylene castor DMSO
oil + PBS
DMF
I
650/4.02 656/3.02
654/4.00 652/4.03
654
655
652
650
656
652
II
0.04
3
.3 ESR spectral measurements of singlet oxygen
1
As a major cytotoxic agent, singlet oxygen ( O ) is one of the important factors
2
producing a series of physiological changes and cytotoxicity in PDT [24 - 26].
1
Generation of O from PS irradiated in the presence of O in DMF was investigated
2
2
by ESR measurement, employing 2, 2, 6, 6-tetramethylpiperidine (TEMP) as a spin
trap [27]. TEMP itself has no ESR signal. However, as a stable substance, TEMPO
1
was produced from the reaction of TEMP with O and could be detected (Fig. 2a)
2
[
28]. The characteristic ESR spectrum of TEMPO consists of three equally intense
lines, whose intensities could be increased by changing the experimental conditions
29]. No obvious TEMP signals were showed in DMF at room temperature. The
[
intensities of these signals enhanced progressively when photo-irradiation time
increased to 1, 2, 3, 4, 5, 6 and 7 min, with no change in line-shape, respectively (Fig.
2
c, 2e, and S3a). ESR parameters of compounds I, II and m-THPC were determined,
and the hyperfine splitting constant (a ) and g-value were found to be 15.40 G and
N

2
.0048, and 15.22G and 2.0055, and 15.31G and 2.0039, respectively. Under the same
concentration and light condition, the rates of formation TEMPO was expressed in
Fig 2b. The values of compound I (k =8.44) and compound II (k =1.32) were higher
1
1
than m-THPC (k =0.77). Furthermore, it was apparent that the line-width of the signal
3
at 1 min is broader than that at 7min in Fig. 2d, 2f. This change was caused by
decreasing O concentration with irradiation time. The line-width of TEMPO
2
increased with the concentration of oxygen, as the collision of TEMPO with oxygen
could shorten its electron relaxation time [30, 31].
In addition, the formation of singlet oxygen also depended on the PS concentration.
As the concentration increased, the intensity of the ESR signal was enhanced under
the fixed exposure time. The results also showed that the signal intensity of
compound I and II was higher than m-THPC (Fig. 3 and S3), which indicated that the
quantity of singlet oxygen formed was dependent on the light intensity and
concentration dose.

1
Fig. 2. (a) The reaction of TEMP with O to produce TEMPO. (b) Rates of formation of
2
TEMPO. (c) ESR spectra as a function of time of compound I. (d) Compound I zoomed. (e) ESR
spectra as a function of time of compound II. (f) Compound II zoomed.
Fig. 3. Concentration-dependent of singlet oxygen formation of I and II photosensitized with 650
nm laser irradiation on 3 min; sample containing 0.2 M TEMP spin trap and different concentration
compound (0.1, 0.25, 0.5, 1 mM) in DMF, respectively.
3
.4 Photo-bleaching characteristics
The photo-bleaching rate plays an important role in PDT, by monitoring the
photobleaching could provide a quantifiable measure of the singlet oxygen production.
However, the photobleaching process remains unclear, as it involves complicated
photophysical and photochemical reactions that produce various photoproducts [32,
3
3]. Experiments about photo-bleaching properties of compounds I and II were
performed. As seen in Fig. 4, with the extension of irradiation time, the OD values of
maximum absorption wavelength had no significant change, which indicated that the
structure of compound I and II were stable.

Fig. 4. Photo-bleaching of compound I and II.
3
.5 Uptake and anti-tumor activity in vitro
Time-dependent uptake of compounds was shown in Fig. 5a. The concentration of
compound I and II rose dramatically within 4 h and reached a plateau within 24 h. So
the incubation time of drug chose for the MTT experiment was 24 hours.
The IC50 value is a measure of the laser-induced cytotoxic effectiveness of the
compound in relation to its dark cytotoxicity [34]. In Fig. 5, compound I and II
showed the less dark cytotoxicity than m-THPC. As summarized in Table 2,
compound I estimated the lower IC value at different laser dose (1 J, 2 J) vis II and
5
0
m-THPC, and photo-cytotoxicity was positively correlated with laser dose and drug
dose (Fig. 5c, 5d). Therefore, compound I had a relative distinct anti-cancer effect.

Fig. 5. (a) The QBC-939 cells uptake of compound I and II. (b, c, d) The cell viability of
QBC-939 cells treated by compounds m-THPC, I, II at the concentrations of 1 - 8 µM
2
with different light doses (Dark, 1, 2, 4 J/cm ) in MTT assay, respectively.
Table 2 The TC and IC values of compounds to QBC-939 cells
50
50
TC [µM]
IC [µM]
5
0
50
Compound
2
2
2
Dark
>8
1 J/cm
1.91
2 J/cm
4 J/cm
0.78
I
II
1.36
1.92
1.46
>8
3.41
1.17
m-THPC
6.08
2.96
0.63
3
.6 Cellular localization
The subcellular localization of the PS was another important factor since it determines
the site of primary photo damages and the type of cellular response to the therapy
[
35-37]. To understand the perinuclear localization of compounds, a dual staining
experiment was carried out. QBC-939 cells were incubated for 4 h with compound I
and II, followed by the treatment of the cells with the blue nucleic acid stain Hoechst
3
3342, green mitochondrial dye, and blue lysosome probe, respectively. Confocal
laser scanning microscope (CLSM) confirmed that the subcellular localization of
compound was exclusive to the cytoplasm while no nuclear localization was detected.

The red fluorescence of compounds I and II overlapped with Mito-Tracker Green,
Lyso-Tracker Blue, which indicated that the compounds almost accumulated in
subcellular organelles such as mitochondria, lysosome (Fig. 6). The absence of
nuclear localization suggested that this class of compounds would not induce direct
nuclear DNA damage but mediate their actions at extranuclear targets.
Fig. 6. Identification of subcellular localization in QBC-939 cells co-stained with Hoechst33342,
Mito-Tracker, and Lyso-Tracker. Scale bar: 20 µm.
3
.7 FACS analysis
Many excited PSs generate ROS that leads to induction of apoptosis in malignant
cells [38, 39]. The localization of photosensitizers to mitochondria and lysosome
benefits to induce cell apoptosis [40]. In this experiment, QBC-939 cells treated with
compound-PDT were incubated for 2 h and 24 h and stained with the V-FITC/PI
apoptosis assay kit. As shown in Fig. 7, no obvious apoptotic cells were detected in
the control group, light group and compound group. At 2 h post-PDT, the proportion
of apoptotic cells in I - PDT group and II - PDT group were 37.93% and 7%,
respectively. After 24 h incubation, the proportion of apoptotic cells increased to
9
0.11% and 64.7%, respectively. Besides, the cells apoptotic rate for I was markedly
higher than that of II. These results suggesting that the compounds might mediate
PDT effect at late apoptotic phase.

Fig. 7. Cell apoptosis necrotic by PDT in QBC-939 cells. The cells were loaded with compounds I
2
and II and irradiated with laser (2 J/cm ), then incubated for 2 h, 24 h. Cells were stained with
annexin V-fluorescein isothiocyanate (FITC) propidium iodide (PI) and analyzed by FACS.
3
.8 Determination of plasma concentration
The rate of drug scavenging in blood determines the minimum effective time for PDT
41]. Drug levels in the blood after single-dose intravenous administration were
[
shown in Fig. 8a. The plasma concentration in mice reached the maximum value at 1
min after administration, followed by a decrease with time increase. The fluorescence
intensity of drug in plasma decreased to half of the initial value
during the period of time from 1 h to 2 h after administration, then the decline slowed
down but a certain plasma concentration was still maintained within 24 h. The
fluorescence intensity of compound I in plasma was markedly higher than II.
3
.9 The fluorescence imaging in vivo
The target tissue can be distinguished from surrounding tissues by monitoring the
difference in fluorescence intensity, thus real-time fluorescence might assist to locate
cancer. Meanwhile, high tumor selectivity can greatly improve the therapeutic effect
[
42, 43]. The fluorescence imaging was performed by injecting 1mg / kg compound
into the mice via the tail vein. As shown in Fig. 8b, compounds I (left) and II (right)
distributed in various organ tissues throughout the body after administration. After 8 h,
the compounds were selectively retained in tumor tissue. Fluorescence in the tumor
could be observed during 24 h.

Fig. 8. (a) The plasma concentration of compound I and II. (b) In vivo distributions of compound
I (left) and II (right) in the tumor-bearing mice after intravenous injection with dosage 1 mg/kg.
Distribution of compounds in mice at a different post-injection time (0 h, 2 h, 8 h, 24 h).
3
.10. Skin phototoxic effects
The ICR mice model was selected and administered with therapeutic dose. ICR mice
2
were exposed to irradiation with simulated sunlight (10 mW / cm ) at 1 day after
intravenous injection of 1 mg / kg compounds of I, II and m-THPC. No obvious
changes in the surface of mice were inspected visually after I and II were
administered, while obvious erythema and swelling in the ears and skin in the
m-THPC group were shown. Compared with negative control, there was no
significant difference in the weight of tissues including ear, neck skin, the skin of
backs and tail in I, II groups after light irradiation, whereas the weight of tissues in
the m-THPC group increased significantly (Table 3). A representative H&E stained
micrographs of mice tissues after light irradiation were shown in Fig. 9. No
pathological changes were found in I, II groups in contrast to m-THPC group where
dermal inflammation was observed. These results indicated that skin phototoxic
effects of compound I, II were significantly lower than m-THPC.
Table 3. The weight of ears and back skin in ICR mice model after exposure to light irradiation
2
(
10 mW / cm ) at 1 day after intravenous injection of 1 mg/kg compounds
Weight (mg)
Group
ear
neck skin
36.5 ± 2.4
37.9 ± 2.2
skin of the backs
34.3 ± 2.1
tail
control
12.4 ± 1.5
12.8 ± 1.2
115.6 ± 5.6
116.5 ± 4.3
I
35.1 ± 1.8

II
13.2 ± 1.6
38.1 ± 1.7
35.3± 1.6
114.9 ± 3.9
m-THPC
15.6 ± 1.3*
42.7 ± 1.8*
39.2 ± 1.9*
120.4 ± 4.7*
*P<0.05 compared to the control group.
Fig. 9. Representative HE micrographs. (a) The auricles of the mice (left ear). (b) The exposed
neck skin. (c) The skin of the backs of mice. (d) The tail of the mice. Scale bar: 50 µm.
3
.11 PDT efficacy in the cholangiocarcinoma tumor model
To investigate the photodynamic anti-tumor efficacy of compounds on BALB/c nude
mice bearing QBC-939 tumor, the mice were randomly divided into 8 groups (Fig.
1
1
0a). It was shown that the tumor weight of the I-PDT treatment group was about
0 % of the control group and the II -PDT treatment group was about 17% while the
m-THPC-PDT treatment group was about 35%. However, the groups treated with 1
mg/kg I, II and m-THPC without irradiation exhibited no significant difference
compared to the negative control group (Fig. 10b). The tumor growth curve in Figure
1
0c revealed that PDT group could distinctively inhibit the tumor growth, especially
compound I. As seen in Figure 10d, HE staining showed significant tissue damage
and morphological changes in tumor tissue after PDT while no obvious changes in
mice organs (heart, liver, spleen, lung, and kidney).

Fig. 10. (a) Schematic image of treatment. (b) Tumor weight after 13d treatment. (c) Tumor
volume at different time points after treatment. (d) The changes of major organs in tumor mice
under treatment. Scale bar: 100µm. *P< 0.05.

3
.12 TUNEL staining
TUNEL staining was performed to confirm the effect of compound-PDT in QBC-939
cells. Under the fluorescence microscope, the apoptotic cells exhibited green
fluorescence in the nucleus after excitation with blue light. The number of tunnel
positives cells was exceeded in PDT treated groups than the control group, and more
apoptotic cells were observed in the I group than those in II and m-THPC groups (Fig.
11a, Fig. 11b).

Fig. 11. (a) Representative histopathological images of PDT treated tumors by fluorescent
microscopy (×200). (b) The apoptosis rate of groups (control, m-THPC-PDT, I-PDT, II -PDT).
*P<0.05.
4
. Conclusions
In summary, two chlorin derivatives, 5,10,15,20-tetraphenyl-[2:3]-[(methoxycarbonyl,
carboxy)methano] chlorin (I) and
,10,15,20-tetraphenyl-[2:3]-{[methoxycarbonyl,(2-hydroxyethyl)amide]methano}chl
5
orin (II ) were synthesized and characterized. The new PSs expressed high absorption
in the photo-therapeutic window (650nm), and strong fluorescence intensity. ESR
1
showed that O was produced under irradiation of compounds with 650 nm light and
2
the generation was enhanced with the increase of the PS concentration or light dose.
Furthermore, the two compounds exhibited more obvious anti-tumor activities against
QBC-939 cells in vitro and on Balb/c nude mice bearing QBC-939 tumor in vivo than
m-THPC. High tumor selectivity was indicated from the fluorescence of PSs being
activated in vivo. Subcellular assay showed that they could be located in mitochondria
and lysosome, and cell necrosis and apoptosis could be caused after PDT in flow
cytometry. Among them, I showed distinctive photodynamic anti-tumor effects, lower
dark-toxicity and weaker skin photo-toxicity, which suggested that compound I might
be appropriate to be developed as a potential anticancer agent.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No.
2
1
1977016), Foundation of Shanghai Science and Technology Committee (No.
7430741800, 17430711900, 18430713000, 18430731600；19410711000).
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1
2
) Two chlorin derivatives with high tumor selectivity were synthesized.
) The photophysical properties, photochemical properties and anti-tumor activities in vitro and in
vivo were investigated.
) The resultes demonstrated that compound I and II induced tumor cells apoptosis and necrosis
under 650 nm laser irradiation.
) The two compounds exhibited lower skin photo-toxicity than positive reference m-THPC in
vivo.
3
4

Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐
The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: