Comparison between porphin, chlorin and bacteriochlorin derivatives for photodynamic therapy: Synthesis, photophysical properties, and biological activity

Article abstract of DOI:10.1016/j.ejmech.2018.10.005

The development of novel photosensitizers is a challenging task for photodynamic therapy (PDT). Twelve novel photosensitizers (PSs), including porphins (P1-4), chlorins (C1-4) and bacteriochlorins (B1-4) were synthesized. The bacteriochlorins exhibited the longest absorption wavelength (λ_max = 736 nm), which is higher than that of porphins (λ_max = 630 nm) and chlorins (λ_max = 644 nm). In vitro photodynamic activities on Eca-109 human esophageal carcinoma cells were evaluated by standard assays and all PSs showed photodynamic activity. Among them, B2 displayed the highest phototoxicity and the lowest dark toxicity. In addition, B2 exhibited best photodynamic antitumor efficacy on BALB/c nude mice bearing Eca-109 cells tumor. Therefore, B2 is a powerful and promising antitumor photosensitizer for PDT.

Full text of DOI:10.1016/j.ejmech.2018.10.005

Accepted Manuscript
Comparison between porphin, chlorin and bacteriochlorin derivatives for
photodynamic therapy: Synthesis, photophysical properties, and biological activity
Wei Zhu, Ying-Hua Gao, Ping-Yong Liao, Dan-Ye Chen, Ning-Ning Sun, Phuong Anh
Nguyen Thi, Yi-Jia Yan, Xiao-Feng Wu, Zhi-Long Chen
PII:
S0223-5234(18)30867-5
10.1016/j.ejmech.2018.10.005
EJMECH 10791
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 September 2018
Revised Date: 28 September 2018
Accepted Date: 1 October 2018
Please cite this article as: W. Zhu, Y.-H. Gao, P.-Y. Liao, D.-Y. Chen, N.-N. Sun, P.A. Nguyen Thi, Y.-
J. Yan, X.-F. Wu, Z.-L. Chen, Comparison between porphin, chlorin and bacteriochlorin derivatives for
photodynamic therapy: Synthesis, photophysical properties, and biological activity, European Journal of
Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.ejmech.2018.10.005.
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Table of Contents (TOC) Graphic
Three series of porphins (P1-4), chlorins (C1-4) and bacteriochlorins (B1-4) were synthesized.
Among them, B2 is a powerful and promising antitumor PS for photodynamic therapy.
1

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Comparison between porphin, chlorin and
bacteriochlorin derivatives for photodynamic
therapy: synthesis, photophysical properties, and
biological activity
1
1
1
1
1
Wei Zhu , Ying-Hua Gao , Ping-Yong Liao , Dan-Ye Chen , Ning-Ning Sun , Phuong Anh
1
2,3
3
1
Nguyen Thi , Yi-Jia Yan , Xiao-Feng Wu * and Zhi-Long Chen *
1
Department of pharmaceutical Science & Technology, College of Chemistry and Biology,
Donghua University, Shanghai 201620, China
2
Ningbo Dongmi Pharmaceutical Co., Ltd, Ningbo 315899, China
3
Shanghai Xianhui Pharmaceutical Co., Ltd, Shanghai 200433, China
KEYWORDS：porphin, chlorin, bacteriochlorin, photodynamic therapy, antitumor
ABSTRACT: The development of novel photosensitizers is a challenging task for
photodynamic therapy (PDT). Twelve novel photosensitizers (PSs), including porphins (P1-4),
chlorins (C1-4) and bacteriochlorins (B1-4) were synthesized. The bacteriochlorins exhibited the
longest absorption wavelength (λmax= 736 nm), which is higher than that of porphins (λmax = 630
nm) and chlorins (λmax = 644 nm). In vitro photodynamic activities on Eca-109 human
1

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esophageal carcinoma cells were evaluated by standard assays and all PSs showed photodynamic
activity. Among them, B2 displayed the highest phototoxicity and the lowest dark toxicity. In
addition, B2 exhibited best photodynamic antitumor efficacy on BALB/c nude mice bearing Eca-
1
09 cells tumor. Therefore, B2 is a powerful and promising antitumor photosensitizer for PDT.
1
. Introduction
Cancer has been seriously threatening human health in contemporary society [1-3]. Diagnosis
and treatment of cancer have become the most pressing concern. PDT has emerged as an
alternative modality for specifically killing malignant tissues, especially for some localized and
superficial tumors [4-6]. The design and synthesis of novel photosensitizers (PSs) are central to
the development of efficient PDT modalities, particularly the reduction of the doses of drug and
photoirradiation by increasing photocytotoxicity, selective accumulation in the tumor, and rapid
clearance after treatment to reduce side effects such as prolonged skin sensitivity [7-8].
Commercial photofrin II is a porphyrin-based photosensitizer that has been commercially
produced and approved in several countries for the PDT [4, 9-11]. However, it still suffered from
several drawbacks such as chemical heterogeneity, poor tissue penetration due to its limited
maximum absorption wavelength, weak absorption at therapeutic wave length (630 nm), and
prolonged cutaneous photosensitivity caused by its slow elimination in normal tissue [12]. For
these reasons, several PSs possessing stronger and red-shifted absorption within the therapeutic
window have been investigated in PDT (Figure 1). Compared to porphins, chlorin-type PSs are
receiving considerable attention owing to their intense absorption in near infrared region (≥ 650
nm), which are relatively harmless and penetrate deeply in biological tissues [13-15]. Among
them, talaporfin [16-17] (mono-L-aspartyl chlorin e6), and temoporfin [18-19] (m-THPC) were
approved for PDT treatment. Bacteriochlorins are a class of tetrapyrrole macrocycles with two
2

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unsaturated pyrrolic rings. This ring structure occurs naturally in photosynthetic pigments
(bacteriochlorophylls a and b) from purple photosynthetic bacteria of the orders Rhodospirillales
and Rhizobiales [20]. Bacteriochlorin-type PSs received intensive attention due to their
advantage of maximum absorption in near infrared region (740 - 780 nm), enabling deep
penetration of light in animal tissues i.e. ∼8 mm [21-22]. Pd-bacteriopheophorbide, padeliporfin
and redaporfin have entered clinical trials [23-28]. However, many naturally occurring and
naturally derived bacteriochlorins were notoriously difficult to extract from a bacterial culture
with a low yield.
Banfi et al. had reported a series of diphenylporphins and one diphenylchlorin compound
without substituents [29]. Besides, di(3,5-dihydroxyphenyl)chlorin was reported by Patrice et al.
as an excellent PS [30]. The comparison between porphin, chlorin and bacteriochlorin with the
same substituents in the benzene ring has not been reported till now. In the present study, 12
novel PSs, including porphins (P1-4), chlorins (C1-4) and bacteriochlorins (B1-4) were
synthesized and characterized. Their photophysical properties, subcellular localization,
photodynamic activities in vitro and in vivo were reported herein.
Figure 1. The general structure of tetrapyrrole derivatives
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2
. Results and discussion
2
.1. Chemistry
A number of different procedures have been reported for the synthesis of 5,15-diarylporphins
31-32], mostly based on a (2 + 2)-type condensation in which two dipyrrolic derivatives
[
incorporating one type of carbon bridge are fused together, thereby forming the other type of
carbon bridge, the intermediate is then oxidized in situ to the final porphin [33-34]. The aromatic
aldehyde monomers (2a-c) were synthesized according to literature procedures [35-38].
Symmetric diarylporphins (3a-d) were synthesized via acid-catalyzed mixed condensation of
dipyrromethane with aromatic aldehydes, following the general procedure described by Lindsey
and co-workers [39]. The porphyrinogen intermediate was oxidized to porphin with 2,3-dichloro-
5
,6-dicyano-1,4-benzoquinone (DDQ). Then diarylporphins (3a-d) were treated with 2 N KOH,
and hydrolyzed to give the target compounds (P1-4). The overall yields of the products (43.4% -
3.1%) are excellent (Scheme 1).
5
a
Scheme 1 Synthesis of the porphins (P1-4)
R
R
OR1
OR2
CHO
CHO
N
HN
N
N
HN
N
a
b
c
NH
NH
HO
R1O
R
R
R1O
2
R O
R
R
1
1
a: R=-H
b: R=-OMe
2a: R=-H, R =-CH COOEt;
2b: R=-H, R =-CH CH CH COOEt;
1 2 2 2
3a: R=-H, R =-CH COOEt;
P1: R=-H, R =-CH COOH;
2 2
1
2
1
2
3b: R=-H, R1=-CH2CH2CH2COOEt;
3c: R=-OMe, R =-CH COOEt;
P2: R=-H, R2=-CH2CH2CH2COOH;
P3: R=-OMe, R =-CH COOH;
2
2
c: R=-OMe, R =-CH COOEt;
1 2
1
2
2
2
d: R=-OMe, R =-CH CH CH COOEt.
3d: R=-OMe, R =-CH CH CH COOEt.
P4: R=-OMe, R =-CH CH CH COOH.
2 2 2 2
1
2
2
2
1
2
2
2
a
2 3
Reagents and reaction conditions: (a) ethyl bromoacetate or ethyl 4-bromobutyrate, K CO , DMF, 100 ℃ , 9
h; (b) i) dipyrromethane, TFA, DCM, 12 h; ii) DDQ, Et N, 3 h; (c) 2 N KOH, MeOH/THF, reflux, 12 h.
3
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The synthetic route of the chlorins (C1-4) is shown in Scheme 2. According to the method of
Whitlock and co-workers [40], the diarylporphins were reduced with diimide. Briefly, the
compounds (3a-d) were treated with p-toluenesulfonyl hydrazide, and then p-chloranil was
added in portions until the absorption band at 734 nm disappeared. The chlorins (4a-d) were
treated with 2 N KOH, and hydrolyzed to give the target compounds (C1-4).
a
Scheme 2 Synthesis of the chlorins (C1-4)
a
Reagents and reaction conditions: (a) i) p-TsNHNH , K CO , pyridine, reflux, 8 h; ii) p-chloranil, ethyl
2
2
3
acetate, rt.; (b) 2 N KOH, THF/MeOH, reflux, 12 h.
Compared with the chlorins (C1-4), the synthesis of the bacteriochlorins (B1-4) was quite
difficult, requiring a long reaction time and a large amount of p-toluenesulfonyl hydrazine than
the similar method described for chlorin [40]. Due to the relative unstability of the
bacteriochlorins, their post-treatment need to be very careful and quick in the preparation process.
The yields of the compounds (5a-d) ranged from 18% to 35%. The bacteriochlorins (5a-d) were
treated with 2 N KOH, and hydrolyzed to give the target bacteriochlorins (B1-4) (Scheme 3).
The identities of porphins (P1-4), chlorins (C1-4), and bacteriochlorins (B1-4) were elucidated
1
13
by H NMR, C NMR and HR-MS (Figure S1-24).
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a
Scheme 3 Synthesis of the bacteriochlorins (B1-4)
a
Reagents and reaction conditions: (a) p-TsNHNH , pyridine, K CO , reflux 12 h. (b) 2 N KOH, THF/MeOH,
2
2
3
reflux, 18 h.
2
.2. UV-visible absorption and fluorescence spectra.
Due to the four conjugated pyrrole rings, porphins possess high rigidness and good
coplanarity, exhibiting strong UV-visible absorption and fluorescence at room temperature [41].
Schematic energy level diagram for highest occupied orbitals (HOMO) and lowest unoccupied
orbitals (LUMO) of parent porphin, chlorin and bacteriochlorin are illustrated in Figure 2.
Theoretical calculations of molecular orbitals for porphin, chlorin and bacteriochlorin show that
the LUMO energies are not very sensitive to the number of π electrons reduced from 22 to 20
and 18 respectively [42]. Compounds P2, C2 and B2 as examples, their UV-vis absorption
spectra were shown in Figure 3 at a concentration of 8 µM. The UV-vis absorption spectra of
P1-4, C1-4 and B1-4 were shown in concentrations ranging from 1 µM to 10 µM in
supplemental material (Figure S25). The fluorescence excitation and emission spectra of P1-4,
C1-4 and B1-4 were shown in Figure 4 and Table 1. Interestingly, the maximum absorption
wavelength of bacteriochlorin is about 736 nm which might be able to treat deeper tumors. The
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emission wavelength of bacteriochlorin is better than both porphin and chlorin, and evenly
centered at ~740 nm. This provides the possibilities of bacteriochlorins as near infrared (NIR)
fluorescence imaging agents.
Figure 2. The highest occupied orbitals (HOMO) and lowest unoccupied orbitals (LUMO) of porphin, chlorin
and bacteriochlorin
Figure 3. Absorption spectra of P2, C2 and B2 in DMSO at the concentration of 8 µM.
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Figure 4. Fluorescence excitation and emission spectra of P1-4, C1-4 and B1-4 in DMSO at the concentration
of 4 µM.
8

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a
Table 1. Photophysical data for compounds P1-4, C1-4 and B1-4 at T = 293 K.
b
Absorption λmax(nm)
Emission λmax
Stokes
shift
4
-1 .
-1
b
Compds
(
ε
×10 M cm )
(nm)
(
nm)
Soret
408
406
415
413
411
418
411
411
ε
(Soret)
Qy
ε
(Qy) Excitation Emission
P1
P2
P3
P4
C1
C2
C3
C4
B1
B2
B3
B4
37
630
630
633
634
644
645
644
644
736
0.375 408
633
632
644
639
649
649
650
651
738
740
738
738
3
2
11
5
5
4
6
2
2
2
2
2
41
0.20
0.72
0.40
4.20
3.40
4.15
4.05
5.20
406
415
413
411
418
411
412
373
14.22
23.22
18.35
13.97
19.97
12.20
349/373 5.85/7.15
349/373 12.97/58.54 736
11.02 373
352/373 7.47/6.92
352/373 3.52/3.7
736
736
6.02
3.72
373
373
a
b
Absorption, excitation and fluorescence emission wavelength, stokes shift. Solvent, DMSO.
2
.3. Singlet oxygen quantum yield.
As one of three elements of PDT, ROS are the key cytotoxic intermediates of PDT [43].
Therefore, the efficiency of the ROS generation upon irradiation is an important measure for the
cytotoxicity of PSs. The quantum yield of ROS generated by photosensitization of new PSs was
determined (Figure S26). The quantum yield of singlet oxygen value was determined by using 1,
3
- diphenylisobenzofuran (DPBF) as the scavenger. After the data were plotted as k= -ln
(
DPBF /DPBF )/t, straight lines were obtained for the sensitizers, and the slope for each
t
0
compound was obtained after fitting with a linear function. Table 2 lists singlet oxygen
9

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generation (ROS) rates. From holistic comparison, bacteriochlorin was significantly better than
chlorin and porphin. As shown in Table 2, the results indicate that P3 displayed the highest ROS
yield. Meanwhile, P2 and P4 were nearly similar and P1 was the worst in the porphin series.
Among chlorins (C1-4), the observed order is C2 > C4 > C3 > C1. Significantly, the ROS yield
of bacteriochlorins (B1-4) gave the order B2 > B4 > B3 >B1.
a
Table 2 Singlet oxygen generation (ROS) rates of compounds P1-4, C1-4 and B1-4
-2
-1
-2
-1
-2
-1
Compds
10 ×k[min ] Compds
10 ×k[min ] Compds
10 ×k[min ]
P1
0.829
1.928
2.406
1.681
C1
C2
C3
C4
4.056
B1
B2
B3
B4
30.85
P2
24.847
14.238
19.798
69.141
38.7
P3
P4
48.64
a
Solvent, DMF
2
.4. Cytotoxicity
Effective PS needs to possess high phototoxicity and low dark-toxicity [44]. To investigate
the potential of P1-4, C1-4 and B1-4 in PDT, their dark cytotoxicity and phototoxicity were
evaluated in esophageal cancer cells (Eca-109) exposed to various concentrations and light doses
(
Figure S27-28). IC50 value is an important index for the evaluation of antitumor activity of
drugs. The choice of light dose is based on our previous reports [45-47]. The IC values of their
5
0
−
2
dark-toxicity and photocytotoxicity irradiated by 16 J
almost no dark cytotoxicity observed against Eca-109 cells when exposed up to 10
the concentration was increased to 20 M, the survival fraction of Eca-109 cells is approximately
0%. The Nd: YAG laser ( = 635 nm, 650 nm and 730 nm) was respectively used to irradiate
these cells at a light dose of 16 J
ñcm were shown in Table 3. There was
µM. When
µ
8
λ
−
2
ñ
cm . As shown in Table 3, B2 showed the highest
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phototoxicity and the lowest dark toxicity among all the compounds toward Eca-109 cells.
Therefore, B2 was highly cytotoxic to malignant esophageal tumor cells.
a
b
c
d
Table 3 Dark-toxicity and photocytotoxicity of P1-4 , C1-4 and B1-4 in Eca-109 cells
Compds
P1
Dark-toxicity (IC ,
µM)
Phototoxicity (IC50,
µM)
5
0
74.63
9.41
8.19
12.50
10.14
3.31
2.65
P2
271.24
157.66
182.83
91.76
P3
P4
C1
C2
＞
300
111.29
300
162.26
500
186.75
300
C3
C4
B1
B2
B3
B4
3.14
2.83
2.62
2.43
3.09
2.47
＞
＞
＞
a
b
−2
c
−2
Without irradiation; The light dose was 16 J
ñ
cm
(λ
= 635 nm); The light dose was 16 J
ñ
cm
(λ = 650
d
−2
nm); The light dose was 16 Jñcm (λ =730 nm).
2
.5. Intracellular localization
The subcellular localization of the PS is of special significance, since it determines the site of
primary photodamages and the type of cellular response to the therapy. Cellular structures
containing PS would be preferentially damaged upon illumination [48-50]. The preferential sites
of subcellular localization of B2 were evaluated by laser confocal microscopy upon exposure of
Eca-109 cells to B2 (10 µM) for 4 h and staining with Mitro-tracker Green and Lyso-Blue for
mitochondria and lysosome, respectively. As shown in Figure 5, the merged stained images
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revealed the overlaps of B2 with Mitro-tracker Green (green signal) and Lyso Blue (blue signal),
suggesting that the PSs were mainly found in organelles of mitochondria and lysosome.
Figure 5. Subcellular location of B2 in Eca-109 cells at 10 µM for 4 h. Bright field; red fluorescence (B2);
mitochondria green fluorescence (Mitro-tracker Green) or lysosome (Lyso Blue) and merged images. Scale
bar: 20 µm.
2
.6. In vivo photosensitizing efficacy
The in vivo therapeutic efficacy of B2 was evaluated in Eca-109 tumor bearing BABL/c nude
mice. The tumor-bearing mice were injected with B2 (5 mg/kg) via the tail vein, and then
2
2
irradiated with 350 mW/cm lasers for 5 min (120 J/cm , λ = 730 nm) to the tumor area. The
mice in PDT group were treated with laser light at 24 h post-injection of the B2. The control
mice were not subjected to PS or light. After irradiation there was a hemorrhagic and necrotic
zone in the tumor sites of PDT group. With swelling, erythema subsided within 2 days. All the
tumors were measured in three dimensions every other day for 14 days. As shown in Figure 6,
the volume growth curves of tumors were provided. The volume of tumors in control, light and
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B2 group were larger than that in B2-PDT group at the same time. Therefore, the light group has
little influence on the growth of tumors, and there is no significant difference among the light
irradiation group, B2 group and the control group. After 14 days, the mean size of tumors only
3
3
reached 126 ± 25 mm in PDT group, compared to that of 785 ± 37 mm in the control group.
These results indicated that B2 could significantly inhibit the growth of the tumors.
Figure 6. In vivo photosensitizing efficacy of B2 against Eca-109 tumors (6 mice/group). (a). Images of tumor
bearing mice before and after treatment; (b). Tumor volume at different time after treatment. The data shown
are the means ± SD of three independent experiments. *P < 0.05 compared with control group.
3
. Conclusions
In summary, twelve novel PSs, including porphins (P1-4), chlorins (C1-4) and
bacteriochlorins (B1-4), were synthesized and characterized. The UV-vis absorption results are
consistent with the molecular orbital theory. The bacteriochlorins exhibited the longest
absorption wavelength (λmax = 736 nm), which is higher than that of porphins (λmax = 630 nm)
and chlorins (λmax = 644 nm). In comparison with porphins and chlorins, all bacteriochlorins
showed longer absorption and fluorescence emission wavelength. Meanwhile, all three series of
PSs can produce ROS and the bacteriochlorins also displayed higher ROS yields. All of the
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twelve novel compounds showed photodynamic activity. Among them, B2 showed the highest
phototoxicity and the lowest dark toxicity toward Eca-109 cells. Generally, our data support the
fact that B2 is a promising PS for PDT due to its long absorption and emission wavelength in the
phototherapeutic window (absorption λmax = 736 nm, emission λmax= 740 nm), high phototoxicity
and low dark-toxicity (phototoxicity IC = 2.43 µM, dark-toxicity IC ＞ 500 µM for Eca-109
50
5
0
cells) and excellent in vivo PDT antitumor efficacy.
4
. Experimental section
4
.1. Chemistry
All solvents and reagents were purchased from commercial suppliers and were used without
further purification unless otherwise stated. Melting points were obtained on a “Stuart” Bibby
1
13
apparatus and uncorrected. H-NMR and C-NMR spectra were recorded on a Bruker 400 MHz
spectrometer. Chemical shifts were reported as δ values relative to the internal standard TMS
(Me
4
Si). MALDI-TOF mass spectra were recorded on an AB SCIEX 4800 Plus MALDI
TM
TOF/TOF . HR-MS spectra were recorded on a Thermo Fisher Scientific LTQ FT Ultra Mass
Spectrometer. Column chromatography was performed on silica gel H (300 - 400 mesh). UV-vis
absorption spectra were recorded on an ultraviolet visible spectrophotometer (Jasco Model V-
5
30, Japan). Fluorescence spectra were measured on a Fluorescence Spectrophotometer
(
FluoroMax-4, France). Compounds 2a-d were synthesized according to previous reports.
4
.1.1 General procedure for the synthesis of 3a-d
To a solution of dipyrromethane (392 mg, 2.7 mmol) and compound 2 (2.7 mmol) in distilled
Cl (500 mL) was added dropwise trifluoroacetic acid (0.12 mL, 1.7 mmol) under nitrogen,
.
CH
2
2
and the mixture was stirred at room temperature for 12 h. 2,3-Dicyano-5,6-dichlorobenzoquinone
DDQ) (0.735 g, 3.24 mmol) and triethylamine (4 mL) were added, and then the mixture was
(
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continued for 1 h. The solvent was evaporated under reduced pressure and the resulting residue
was purified by silica gel chromatography to afford 3a-d as the purple solid.
5
,15-Di((3-ethoxycarbonylmethoxyl)phenyl)porphin (3a). Above described general method
was followed to afford the crude mixture which was purified by silica gel chromatography eluted
1
with CH Cl to give the purple solid 3a (527 mg, 58.6%). H NMR (400 MHz, CDCl ): δ ppm
2
2
3
1
7
0.33 (s, 2H), 9.40 (d, J = 3.5 Hz, 4H), 9.12 (d, 4H), 7.93 (d, J = 6.0 Hz, 2H), 7.83 (s, 2H), 7.76-
.70 (m, 2H), 7.40 (d, J = 8.1 Hz, 2H), 4.86 (s, 4H), 4.15 (q, J = 7.2 Hz, 4H), 1.28 (t, 6H), -3.16
(s, 2H).
5
,15-Di((3-ethoxycarbonylpropoxyl)phenyl)porphin (3b). Above described general method
was followed to afford the crude mixture which was purified by silica gel chromatography eluted
with dichloromethane/petroleum ether (v/v = 3:1) to give the purple solid 3b (512 mg, 52.5%).
1
H NMR (400 MHz, CDCl ): δ ppm 10.34 (s, 2H), 9.42 (d, J = 4.6 Hz, 4H), 9.17 (d, J = 4.6 Hz,
3
4
4
1
H), 7.91 (d, J = 7.4 Hz, 2H), 7.87 (s, 2H), 7.76-7.70 (m, 2H), 7.39 (dd, J = 8.3, 1.8 Hz, 2H),
.27 (t, J = 6.1 Hz, 4H), 4.20 (q, J = 7.1 Hz, 4H), 2.66 (t, J = 7.3 Hz, 4H), 2.31-2.24 (m, 4H),
.29 (t, J = 7.2 Hz, 6H), -3.08 (s, 2H).
5
,15-Di((3-carboxymethoxyl-4-methoxyl)phenyl)porphin (3c). Above described general
method was followed to afford the crude mixture which was purified by silica gel
chromatography eluted with dichloromethane/petroleum ether (v/v = 3:1) to give the purple solid
1
3
9
2
c (500 mg, 51.0%). H NMR (400 MHz, CDCl ): δ ppm 10.31 (s, 2H), 9.39 (d, J = 4.6 Hz, 4H),
3
.13 (d, J = 4.6 Hz, 4H), 7.89-7.83 (m, 2H), 7.78 (t, J = 2.3 Hz, 2H), 7.33 (dd, J = 8.2, 1.9 Hz,
H), 4.90 (s, 4H), 4.25 (q, J = 7.1 Hz, 4H), 4.21 (s, 6H), 1.18 (t, J = 7.1 Hz, 6H), -3.10 (s, 2H).
5
,15-Di((3-ethoxycarbonylpropoxyl-4-methoxyl)phenyl)porphin
(3d). Above described
general method was followed to afford the crude mixture which was purified by silica gel
15

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1
chromatography eluted with CH
2 2
Cl to give the purple solid 3d (485.8 mg, 46.1%). H NMR
(
400 MHz, CDCl ): δ ppm 10.30 (s, 2H), 9.39 (d, J = 4.6 Hz, 4H), 9.15 (d, J = 4.6 Hz, 4H), 7.90-
3
7
6
.83 (m, 2H), 7.81 (d, J = 8.1 Hz, 2H), 7.29 (d, J = 8.1 Hz, 2H), 4.24 (t, J = 6.3 Hz, 4H), 4.17 (s,
H), 4.10 (q, J = 7.2 Hz, 4H), 2.62 (t, J = 7.4 Hz, 4H), 2.27 (m, J = 6.9 Hz, 4H), 1.20 (t, J = 7.1
Hz, 6H), -3.07 (s, 2H).
.1.2 General procedure for the synthesis of P1-4
Porphin 3 (0.5 mmol) was dissolved in tetrahydrofuran/methanol (100 mL, v/v = 1/1)
4
.
followed by 2 N KOH (25 mL). The reaction mixture was stirred under reflux for 12 h under
nitrogen. After the reaction solution was cooled to room temperature, the solvent was evaporated
under reduced pressure. The residue was dissolved in water (30 mL) and the pH was adjusted to
3
-4 with 1 N HCl. The product (P1-4) was precipitated, collected by filtration and dried.
5
,15-Di((3-carboxymethoxyl)phenyl)porphin (P1). P1 was prepared by following the above
1
general procedure (276.4 mg, 90.6%). H NMR (400 MHz, DMSO-d ): δ ppm 10.57 (s, 2H),
6
9
4
1
.59 (d, J = 4.1 Hz, 4H), 9.09 (d, J = 3.4 Hz, 4H), 7.96-7.67 (m, 6H), 7.44 (d, J = 7.9 Hz, 2H),
1
3
6
.89 (s, 4H), -3.27 (s, 2H). C NMR (100 MHz, DMSO-d ): δ ppm 171.31, 157.40, 146.80,
45.21, 142.04, 133.02, 131.26, 128.59, 128.33, 121.52, 118.85, 114.92, 106.20, 65.99. HRMS
+
(
MALDI-TOF): m/z calcd for C H N O [M+H] , 611.1925; found, 611.1926.
3
6
27
4
6
5
,15-Di((3-carboxypropoxyl)phenyl)porphin (P2). P2 was prepared by following the above
1
general procedure (313.2 mg, 94.2%). H NMR (400 MHz, DMSO-d ): δ ppm 12.21 (s, 2H),
6
1
0.61 (s, 2H), 9.63 (d, 4H), 9.09 (d, 4H), 7.89-7.70 (m, 6H), 7.44 (s, 2H), 4.23 (t, 4H), 2.49 (t, J
1
3
=
8.4 Hz, 4H), 2.14-2.00 (m,4H), -3.28 (s, 2H). C NMR (100 MHz, DMSO-d ): δ ppm 174.68,
6
1
57.78, 146.84, 145.28, 142.29, 133.01, 131.27, 128.65, 128.02, 121.52, 118.92, 114.75, 106.20,
16

ACCEPTED MANUSCRIPT
+
6
35 4 6
7.45, 30.75, 24.89. HRMS (MALDI-TOF): m/z calcd for C40H N O [M+H] , 667.2551;
found, 667.2546.
5
,15-Di((3-carboxymethoxyl-4-methoxyl)phenyl)porphin (P3). P3 was prepared by following
1
the above general procedure (328.4 mg, 98.3%). H NMR (400 MHz, DMSO-d ): δ ppm 10.61
6
(s, 2H), 9.64 (d, J = 3.9 Hz, 4H), 9.13 (d, J = 3.7 Hz, 4H), 7.86-7.75 (m, 4H), 7.46 (d, J = 6.4 Hz,
1
3
2
1
6
6
H), 4.92 (s, 4H), 4.09 (s, 6H), -3.21 (s, 2H). C NMR (100 MHz, DMSO-d ): δ ppm 170.98,
6
49.42, 147.24, 146.34, 145.09, 132.97, 131.36, 128.95, 120.89, 120.64, 118.99, 111.65, 106.13,
+
5.61, 56.34. HRMS (MALDI-TOF): m/z calcd for C38
71.2139.
H
31
N
4
O
8
[M+H] , 671.2136; found,
5
,15-di((3-carboxypropoxyl-4-methoxyl)phenyl)porphin (P4). P4 was prepared by following
1
the above general procedure (354.4 mg, 97.7%). H NMR (400 MHz, DMSO-d ): δ ppm 12.14
6
(s, 2H), 10.60 (s, 2H), 9.63 (d, J = 4.7 Hz, 4H), 9.12 (d, J = 4.6 Hz, 4H), 7.98-7.86 (m, 2H), 7.77
(d, J = 7.9 Hz, 2H), 7.45 (d, J = 8.1 Hz, 2H), 4.17 (t, J = 6.1 Hz, 4H), 4.09 (s, 6H), 2.44 (t, J =
1
3
7
1
6
7
4
.4 Hz, 4H), 2.12-1.95 (m, 4H), -3.22 (s, 2H). C NMR (100 MHz, DMSO-d ): δ ppm 174.62,
6
49.61, 147.25, 147.02, 145.11, 133.33, 132.89, 131.36, 128.28, 120.52, 119.11, 111.36, 106.07,
+
8.14, 56.33, 30.62, 24.92. HRMS (MALDI-TOF): m/z calcd for C42
27.2762; found, 727.2763.
H
39
N
4
O
8
[M+H] ,
.1.3 General procedure for the synthesis of 4a-d
.
A solution of compound 3 (0.5 mmol) and K CO (828 mg, 6 mmol) dissolved in pyridine
2
3
(23 mL) was heated to reflux under nitrogen, and then 0.5 M p-toluenesulfonyl hydrazide in
pyridine (0.5 ml) was added every half hour in 16 batches over 8 h. The reaction mixture was
cooled to room temperature followed by the addition of ethyl acetate (100 mL) and distilled
water (50 mL) and stirred under reflux for 1 h. After cooling and stratification, the organic phase
17

ACCEPTED MANUSCRIPT
2 4
was washed with saturated brine (50 mL × 3), dried over anhydrous Na SO and filtered. The
filtrate was stirred at room temperature and chloranil (125 mg) was added in portions. The
reaction was stopped when the absorbance peak at 735 nm disappeared. The reaction mixture
was washed with 5% NaHSO solution (50 mL × 3), water (50 mL × 3), 2 N NaOH (10 mL × 3)
3
and saturated brine (50 mL × 3), dried over anhydrous Na SO , filtered, and evaporated under
2
4
reduced pressure. The residue was purified by column chromatography to afford 4a-d as dark
green solid.
5
,15-Di((3-ethoxycarbonylmethoxyl)phenyl)chlorin (4a). Above described general method
was followed to afford the crude mixture which was purified by silica gel chromatography eluted
1
with dichloromethane/petroleum ether (v/v = 1:1) to give the green solid 4a (121 mg, 36.2%). H
NMR (400 MHz, CDCl ): δ ppm 9.85 (s, 1H), 9.10 (d, J = 4.6 Hz, 1H), 8.99 (s, 1H), 8.97 (d, J =
3
4
.3 Hz, 1H), 8.87 (d, J = 4.7 Hz, 1H), 8.82 (d, J = 4.7 Hz, 1H), 8.67 (d, J = 4.2 Hz, 1H), 8.41 (d,
J = 4.6 Hz, 1H), 7.83 (d, J = 7.4 Hz, 1H), 7.74 (s, 1H), 7.65 (q, J = 8.4 Hz, 2H), 7.54 (d, J = 7.4
Hz, 1H), 7.49 (s, 1H), 7.35 (d, J = 8.3 Hz, 1H) , 7.27 (d, J = 8.3 Hz, 1H), 4.82 (d, J = 8.4 Hz,
4
1
H), 4.65 (q, J = 7.8 Hz, 2H), 4.37-4.27 (m, 6H), 1.29 (t, J = 7.1 Hz, 6H), -1.44 (s, 1H), -1.96 (s,
H).
5
,15-Di((3-ethoxycarbonylpropoxyl)phenyl)chlorin (4b). Above described general method
was followed to afford the crude mixture which was purified by silica gel chromatography eluted
1
with dichloromethane/petroleum ether (v/v = 3:1) to give the green solid 4b (200 mg, 55.2%). H
NMR (400 MHz, CDCl ): δ ppm 9.87 (s, 1H), 9.12 (d, J = 4.7 Hz, 1H), 9.02 (s, 1H), 8.99 (d, J =
3
4
.2 Hz, 1H), 8.89 (d, J = 4.7 Hz, 1H), 8.82 (d, J = 4.7 Hz, 1H), 8.72 (d, J = 3.2 Hz, 1H), 8.46 (d,
J = 4.6 Hz, 1H), 7.81 (d, J = 7.5 Hz, 1H), 7.76 (s, 1H), 7.66 (q, J = 7.7 Hz, 2H), 7.53-7.48 (m,
H), 7.32 (dd, J = 8.3, 2.4 Hz, 1H), 7.25 (dd, J = 8.4, 2.5 Hz, 1H), 4.69 (t, J = 7.7 Hz, 2H), 4.43-
2
18

ACCEPTED MANUSCRIPT
4
7
.36 (m, 2H), 4.25-4.17 (m, 8H), 2.64 (td, J = 7.3, 3.7 Hz, 4H), 2.28-2.22 (m, 4H), 1.30 (td, J =
.1, 1.5 Hz, 6H), -1.42 (s, 1H), -1.92 (s, 1H).
5
,15-Di((3-ethoxycarbonylmethoxyl-4-methoxyl)phenyl)chlorin
(4c). Above described
general method was followed to afford the crude mixture which was purified by silica gel
chromatography eluted with dichloromethane /petroleum ether (v/v = 6:1) to give the green solid
1
4
c (60.1 mg, 16.5%). H NMR (400 MHz, CDCl ): δ ppm 9.86 (s, 1H), 9.15 (s, 1H), 9.06-8.95
3
(
m, 2H), 8.91-8.79 (m, 2H), 8.69 (d, J = 4.2 Hz, 1H), 8.43 (d, J = 4.6 Hz, 1H), 7.84-7.74 (m,
H), 7.69 (s, 1H), 7.51 (m, 1H), 7.41 (s, 1H), 7.31-7.24 (m, 2H), 4.87 (d, J = 9.8 Hz, 4H), 4.69 (t,
J = 7.5 Hz, 2H), 4.37 (t, J = 6.6 Hz, 2H), 4.28-4.22 (m, 4H), 4.18 (d, J = 8.8 Hz, 6H), 1.21 (q, J =
.0 Hz, 6H), -1.43 (s, 1H), -1.92 (s, 1H).
,15-Di((3-ethoxycarbonylpropoxyl-4-methoxyl)phenyl)chlorin
1
7
5
(4d). Above described
general method was followed to afford the crude mixture which was purified by silica gel
1
chromatography eluted with CH Cl to give the green solid 4d (56.9 mg, 14.5%). H NMR (400
2
2
MHz, CDCl ): δ ppm 9.88 (s, 1H), 9.18 (d, J = 4.7 Hz, 1H), 9.09-8.99 (m, 2H), 8.93-8.81 (m,
3
2
7
H), 8.74 (d, J = 3.6 Hz, 1H), 8.48 (d, J = 4.6 Hz, 1H), 7.81-7.76 (m, 1H), 7.63-7.48 (m, 4H),
.32-7.27 (m, 1H), 4.74 (t, J = 7.8 Hz, 2H), 4.41-4.32 (m, 2H), 4.32-4.23 (m, 8H), 4.19 (d, J = 8
Hz, 6H), 2.64 (q, J = 7.3, 4H), 2.27-2.19 (m, 4H), 1.23 (t, J = 4 Hz, 6H), -1.41 (s, 1H), -1.88 (s,
1
H).
4
.1.4 General procedure for the synthesis of C1-4.
Compound 4 (0.5 mmol) was dissolved in tetrahydrofuran/methanol (100 mL, v/v = 1/1)
followed by 2 N KOH (25 mL). The reaction mixture was stirred under reflux for 12 h under
nitrogen. After the reaction solution was cooled to room temperature, the solvent was evaporated
19

ACCEPTED MANUSCRIPT
under reduced pressure. The residue was dissolved in water (30 mL) and the pH was adjusted to
3
-4 with 1 N HCl. The product (C1-4) was precipitated, filtered and dried.
5
,15-Di((3-carboxymethoxyl)phenyl)chlorin (C1). C1 was prepared by following the above
1
general procedure (280.4 mg, 91.6%). H NMR (400 MHz, DMSO-d ): δ ppm 9.87 (s, 1H), 9.13
6
(
d, J = 4.6 Hz, 1H), 9.02 (s, 1H), 8.99 (d, J = 4.3 Hz, 1H), 8.87 (d, J = 4.7 Hz, 1H), 8.82 (d, J =
.7 Hz, 1H), 8.69 (d, J = 4.2 Hz, 1H), 8.43 (d, J = 4.6 Hz, 1H), 7.86 (d, J = 7.4 Hz, 1H), 7.77 (d,
J = 2.5 Hz, 1H), 7.68 (q, J = 8.4 Hz, 2H), 7.57 (d, J = 7.4 Hz, 1H), 7.51 (d, J = 2.6 Hz, 1H), 7.37
dd, J = 8.3, 2.4 Hz, 1H), 7.30 (d, J = 2.5 Hz, 1H), 4.84 (d, J = 8.4 Hz, 4H), 4.68 (t, J = 7.9 Hz,
H), 4.35 (dqd, J = 10.7, 7.7, 7.1, 4.2 Hz, 6H), 1.32 (t, J = 7.1 Hz, 6H), -1.41 (s, 1H), -1.94 (s,
4
(
2
1
1
1
9
6
1
3
H). C NMR (100 MHz, DMSO-d ): δ ppm 171.19, 171.12, 168.11, 168.06, 158.08, 157.31,
6
52.58, 150.37, 142.95, 142.1, 140.53, 139.33, 135.38, 133.71, 133.60, 131.66, 129.53, 129.30,
28.63, 128.35, 127.56, 125.48, 125.31, 123.03, 120.63, 118.84, 114.76, 114.49, 111.67, 108.85,
+
7.63, 65.67, 65.61, 35.71, 35.32. HRMS (MALDI-TOF): m/z calcd for C H N O [M+H] ,
3
6
29
4
6
13.2082; found, 613.2079.
5
,15-Di((3-carboxypropoxyl)phenyl)chlorin (C2). C2 was prepared by following the above
1
general procedure (325.8 mg, 97.5%). H NMR (400 MHz, DMSO-d
6
): δ ppm 12.28 (s, 2H),
1
4
0.13 (s, 1H), 9.43 (dd, J = 4.8, 1.8 Hz, 1H), 9.26 (s, 1H), 9.13 (d, J = 4.2 Hz, 2H), 8.90 (d, J =
.7 Hz, 1H), 8.63 (d, J = 4.3 Hz, 1H), 8.46 (dd, J = 4.7, 1.7 Hz, 1H), 7.81-7.71 (m, 4H), 7.61 (d,
J = 2.3 Hz, 1H), 7.56 (d, J = 7.4 Hz, 1H), 7.46 (dt, J = 5.9, 3.0 Hz, 1H), 7.38 (dd, J = 8.4, 2.5 Hz,
1
2
1
1
H), 4.71 (t, J = 8.0 Hz, 2H), 4.47-4.23 (m, 6H), 2.61-2.57 (m, 2H), 2.53 (d, J = 7.2 Hz, 2H),
1
3
.18-2.10 (m, 4H), -1.46 (s, 1H), -2.00 (s, 1H). C NMR (100 MHz, DMSO-d ): δ ppm 174.64,
6
68.20, 168.11, 158.67, 157.79, 152.60, 150.40, 143.16, 142.28, 140.59, 139.42, 135.39, 133.76,
33.59,131.64, 129.66, 129.34, 128.66, 128.41, 127.15, 125.32, 125.23, 123.09, 120.71, 120.63,
20

ACCEPTED MANUSCRIPT
1
2
18.83, 114.60, 114.44, 111.80, 108.86, 97.68, 67.36, 67.27, 35.80, 35.44, 30.70, 30.67, 24.86,
+
4.83. HRMS (MALDI-TOF): m/z calcd for C H N O [M+H] , 669.2708; found, 669.2709.
4
0
37
4
6
5
,15-Di((3-carboxymethoxyl-4-methoxyl)phenyl)chlorin (C3). C3 was prepared by following
1
the above general procedure (324 mg, 96.4%). H NMR (400 MHz, DMSO-d ): δ ppm 10.03 (s,
6
1
1
4
H), 9.35 (s, 1H), 9.20 (s, 1H), 9.08-9.01 (m, 2H), 8.86 (d, J = 3.7 Hz, 1H), 8.59 (d, J = 3.9 Hz,
H), 8.41 (d, J = 3.7 Hz, 1H), 7.68 (s, 2H), 7.48 (d, J = 4.2 Hz, 2H), 7.40 (d, J = 24.2 Hz, 2H),
.81 (d, J = 19.8 Hz, 4H), 4.66 (t, J = 7.5 Hz, 2H), 4.37-4.28 (m, 2H), 4.05 (d, J = 14.6 Hz, 6H),
1
3
-
1.48 (s, 1H), -2.07 (s, 1H). C NMR (100 MHz, DMSO-d
6
): δ ppm 172.31, 163.78, 162.33,
1
1
1
53.61, 153.05, 148.83, 148.79, 146.09, 144.54, 143.78, 133.80, 133.40, 132.73, 132.10, 132.08,
30.94, 129.96, 125.59, 125.28, 125.15, 125.06, 121.28, 116.98, 114.05, 113.75, 113.53, 113.24,
13.13, 105.56, 101.44, 65.12, 59.91, 33.45, 31.80. HRMS (MALDI-TOF): m/z calcd for
+
C H N O [M+H] , 673.2293; found, 673.2301.
38
33
4
8
5
,15-Di((3-carboxypropoxyl-4-methoxyl)phenyl)chlorin (C4). C4 was prepared by following
1
the above general procedure (345.2 mg, 94.7%). H NMR (400 MHz, DMSO-d ): δ ppm 12.31
6
(s, 2H), 10.31 (s, 1H), 9.67 (s, 1H), 9.46 (s, 1H), 9.24 (d, J = 4.2 Hz, 2H), 9.01 (d, J = 4.7 Hz,
1
7
4
H), 8.73 (d, J = 4.3 Hz, 1H), 8.51 (d, J = 4.7 Hz, 1H), 7.61 (s, 2H), 7.56 (d, J = 2.4 Hz, 1H),
.46 (d, J = 6.4 Hz, 1H), 7.38 (m, 1H), 7.30 (m, 1H), 4.67 (t, J = 8.0 Hz, 2H), 4.52-4.31 (m, 6H),
1
3
.12 (d, J = 5.8 Hz, 6H), 2.64-2.58 (m, 4H), 2.23-2.16 (m, 4H), -1.53 (s, 1H), -2.07 (s, 1H). C
NMR (100 MHz, DMSO-d ): δ ppm 179.56, 163.78, 161.39, 153.67, 150.36, 148.56, 148.29,
6
1
1
5
46.39, 144.57, 142.88, 133.78, 133.45, 132.76, 132.10, 132.04, 130.98, 129.96, 125.57, 125.31,
25.12, 121.25, 116.92, 113.97, 113.51, 113.09, 113.01, 112.54, 105.56, 101.44, 67.91, 55.91,
41 4 8
5.85, 33.45, 31.80, 30.83, 24.61, 24.57. HRMS (MALDI-TOF): m/z calcd for C42H N O
+
[
M+H] , 729.8095; found, 729.8096.
21

ACCEPTED MANUSCRIPT
4
.1.5 General procedure for the synthesis of 5a-d
.
A solution of compound 3 (0.5 mmol) and K CO (828 mg, 6 mmol) dissolved in pyridine
2
3
(23 mL) was heated to reflux under nitrogen, and then 0.5 N p-toluenesulfonyl hydrazide in
pyridine (0.5 ml) was added every half hour in 24 batches over 12 h. The reaction mixture was
cooled to room temperature followed by the addtion of ethyl acetate (100 mL) and distilled water
(50 mL) and stirred under reflux for 1 h. After cooling and stratification, the organic phase was
washed with saturated brine (50 mL × 3), dried over anhydrous Na SO and filtered. The filtrate
2
4
was evaporated under reduced pressure. The residue was purified by column chromatography to
afford dark green solid 5a-d.
5
,15-Di((3-ethoxycarbonylmethoxyl)phenyl)bacteriochlorin (5a). Above described general
method was followed to afford the crude mixture which was was purified by silica gel
chromatography eluted with dichloromethane /petroleum ether (v/v = 6:1) to give dark green
1
solid 5a (95.2 mg, 28.4%). H NMR (400 MHz, CDCl ): δ ppm 8.79 (s, 2H), 8.57 (d, 2H), 8.19
3
(
d, J = 4.4 Hz, 2H), 7.64-7.59 (m, 2H), 7.52 (d, J = 7.5 Hz, 2H), 7.45 (d, J = 2.5 Hz, 2H), 7.24
dd, J = 8.4, 2.6 Hz, 2H), 4.80 (s, 4H), 4.55-4.41 (m, 4H), 4.31 (q, J = 7.2 Hz, 4H), 4.23-4.10 (m,
(
4
H), 1.32 (t, J = 7.1 Hz, 6H), -1.58 (s, 2H).
,15-Di((3-ethoxycarbonylpropoxyl)phenyl)bacteriochlorin (5b). Above described general
5
method was followed to afford the crude mixture which was purified by silica gel
chromatography eluted with dichloromethane /petroleum ether (v/v = 3:1) to give dark green
1
solid 5b (125.3 mg, 34.5%). H NMR (400 MHz, CDCl ): δ ppm 8.73 (s, 2H), 8.52 (d, 2H), 8.17
3
(d, 2H), 7.55 (dd, J = 8.3, 7.4 Hz, 2H), 7.44-7.38 (m, 4H), 7.16 (ddd, J = 8.3, 2.6, 1.0 Hz, 2H),
4
2
.72-3.96 (m, 16H), 2.57 (t, J = 7.3 Hz, 4H), 2.21-2.14 (m, 4H), 1.25 (t, J = 7.2 Hz, 6H), -1.60 (s,
H).
22

ACCEPTED MANUSCRIPT
5
,15-Di((3-ethoxycarbonylmethoxyl-4-methoxyl)phenyl)bacteriochlorin
(5c).
Above
described general method was followed to afford the crude mixture which was purified by silica
gel chromatography eluted with dichloromethane /petroleum ether (v/v = 6:1) to give dark green
1
solid 5c (96.8 mg, 26.5%). H NMR (400 MHz, CDCl ): δ ppm 8.78 (s, 2H), 8.56 (d, J = 4.2 Hz,
3
2
H), 8.20 (d, J = 4.2 Hz, 2H), 7.48 (q, J = 3.6 Hz, 2H), 7.36 (t, J = 2.5 Hz, 2H), 7.22 (d, J = 8.2
Hz, 2H), 4.83 (s, 4H), 4.49 (t, J = 8.3 Hz, 4H), 4.22 (dq, J = 13.4, 7.2 Hz, 8H), 4.14 (s, 6H), 1.22
t, J = 7.1 Hz, 6H), -1.57 (s, 2H).
,15-Di((3-ethoxycarbonylpropoxyl-4-methoxyl)phenyl)bacteriochlorin Above
(
5
(5d).
described general method was followed to afford the crude mixture which was purified by silica
1
gel chromatography eluted with CH Cl to give dark green solid 5d (72.5 mg, 18.4%). H NMR
2
2
(
400 MHz, CDCl ): δ ppm 8.78 (s, 2H), 8.56 (d, J = 4.5 Hz, 2H), 8.21 (d, 2H, J = 4.5 Hz), 7.44-
3
7
4
.39 (m, 4H),7.24 (d, J = 7.8 Hz, 2H), 7.19 (d, J = 8.1 Hz, 2H), 4.49 (t, J = 8.3 Hz, 4H), 4.23-
.09 (m, 18H), 2.60 (t, J = 7.3 Hz, 4H), 2.24 (p, J = 6.9 Hz, 4H), 1.22 (t, J = 7.1 Hz, 6H), -1.56
(
s, 2H).
.1.6 General procedure for the synthesis of B1-4
Compound 5 (0.5 mmol) was dissolved in tetrahydrofuran/methanol (100 mL, v/v = 1/1)
4
.
followed by 2 N KOH (25 mL). The reaction mixture was stirred under reflux for 18 h under
nitrogen. After the reaction solution was cooled to room temperature, the solvent was evaporated
under reduced pressure. The residue was dissolved in water (30 mL) and the pH was adjusted to
3
-4 with 1 N HCl. The product (B1-4) was precipitated, filtered and dried.
5
,15-Di((3-carboxymethoxyl)phenyl]bacteriochlorin (B1). B1 was prepared by following the
1
above general procedure (299.7 mg, 97.6%). H NMR (400 MHz, DMSO-d
6
): δ ppm 13.08 (s,
2
H), 8.91 (s, 2H), 8.74 (d, J = 4.5 Hz, 2H), 8.10 (d, J = 4.5 Hz, 2H), 7.64 (t, J = 7.8 Hz, 2H),
23

ACCEPTED MANUSCRIPT
7
1
1
6
6
.52-7.40 (m, 4H), 7.24 (d, J = 8.4 Hz, 2H), 4.85 (s, 4H), 4.45 (t, J = 8.4 Hz, 4H), 4.12 (qt, J =
1
3
6.9, 8.0 Hz, 4H), -1.65 (s, 2H). C NMR (100 MHz, DMSO-d ): δ ppm 170.81, 162.65, 162.15,
6
57.83, 143.29, 136.39, 135.12, 129.45, 125.35, 122.96, 121.81, 118.46, 114.38, 113.70, 100.03,
+
5.21, 34.97, 34.75. HRMS (MALDI-TOF): m/z calcd for C H N O [M] , 614.2160; found,
3
6
30
4
6
14.2163.
5
,15-Di((3-carboxypropoxyl)phenyl)bacteriochlorin (B2). B2 was prepared by following the
1
above general procedure (297 mg, 88.6%). H NMR (400 MHz, DMSO-d
6
): δ ppm 12.16 (s,
2
7
=
H), 8.90 (s, 2H), 8.73 (d, J = 4.5 Hz, 2H), 8.10 (d, J = 4.5 Hz, 2H), 7.62 (t, J = 7.8 Hz, 2H),
.42 (d, J = 9.2 Hz, 4H), 7.30-7.21 (m, 2H), 4.44 (t, J = 8.4 Hz, 4H), 4.24-4.02 (m, 8H), 2.44 (t, J
1
3
7.3 Hz, 4H), 2.02 (p, J = 6.9 Hz, 4H), -1.66 (s, 2H). C NMR (100MHz, DMSO-d ): δ ppm
6
1
1
74.64, 162.62, 162.14, 158.56, 143.41, 136.42, 135.11, 129.48, 124.90, 122.92, 121.83, 118.50,
14.25, 113.81, 99.99, 67.26, 34.96, 34.77, 30.69, 24.85. HRMS (MALDI-TOF): m/z calcd for
+
C H N O [M] , 670.2786; found, 670.2780.
40
38
4
6
5
,15-Di((3-carboxymethoxyl-4-methoxyl)phenyl)bacteriochlorin (B3). B3 was prepared by
1
following the above general procedure (312 mg, 92.6%). H NMR (400 MHz, DMSO-d
6
): δ ppm
1
2.02 (s, 2H), 8.90 (s, 2H), 8.72 (d, J = 3.9 Hz, 2H), 8.13 (d, J = 3.7 Hz, 4H), 7.49 (s, 2H), 7.36
(
(
d, J = 6.4 Hz, 2H), 7.28 (d, J = 8.2 Hz, 2H), 4.78 (s, 4H), 4.34-4.18 (m, 4H), 4.09 (s, 6H), -1.64
1
3
s, 2H). C NMR (100 MHz, DMSO-d ): δ ppm 175.31, 163.88, 162.38, 160.46, 153.68, 152.05,
6
1
1
48.01, 146.09, 142.75, 134.50, 133.56, 132.04, 128.58, 125.67, 125.32, 124.39, 117.92, 114.74,
13.48, 102.92, 61.82, 58.94, 33.45, 31.93. HRMS (MALDI-TOF): m/z calcd for C H N O
8
3
8
34
4
+
[
M] , 674.2371; found, 674.2370.
5
,15-Di((3-carboxypropoxyl-4-methoxyl)phenyl)bacteriochlorin (B4). B4 was prepared by
1
following the above general procedure (327.2 mg, 89.6%). H NMR (400 MHz, DMSO-d
6
): δ
24

ACCEPTED MANUSCRIPT
ppm 12.08 (s, 2H), 8.89 (s, 2H), 8.72 (d, J = 4.6 Hz, 2H), 8.13 (d, J = 4.6 Hz, 2H), 7.48 (s, 2H),
7
3
.37 (d, J = 7.7 Hz, 2H), 7.29 (d, J = 8.2 Hz, 2H), 4.44 (t, J = 8.4 Hz, 4H), 4.22-4.05 (m, 8H),
1
3
.99 (s, 6H), 2.42 (t, J = 7.4 Hz, 4H), 2.00 (q, J = 7.0 Hz, 4H), -1.65 (s, 2H). C NMR (100
MHz, DMSO-d ): δ ppm 180.36, 163.88, 162.37, 161.41, 154.61, 152.35, 148.29, 146.69,
6
1
5
7
4
4
42.75, 134.50, 133.53, 132.04, 128.38, 125.75, 124.35, 116.02, 113.69, 112.77, 102.92, 67.91,
+
5.91, 33.45, 32.90, 30.93, 24.87. HRMS (MALDI-TOF): m/z calcd for C H N O [M] ,
4
2
42
4
8
30.2997; found, 730.2996.
.2. Photophysical and photochemical measurements
.2.1. Absorption and emission spectra.
UV-visible absorption spectra were recorded on an ultraviolet visible spectrophotometer
Model V-530, Japan). Fluorescence spectra were measured on Fluorescence
Spectrophotometer (FluoroMax-4, France). Slits were kept narrow to 1 nm in excitation and 1 or
nm in emission. Right angle detection was used. All the measurements were carried out at
room temperature. P1-4, C1-4 and B1-4 were dissolved in DMSO to get different concentration
solutions (1-10 M).
.2.2. Singlet oxygen generation detection.
(
a
2
µ
4
The singlet oxygen ability of P1-4, C1-4 and B1-4 was monitored by chemical oxidation of
1
,3-diphenylisobenzofuran (DPBF) in the DMF solution. Typically, 3 mL portions of DMF
solution containing 20
µM DPBF and 0.5
µ
M PSs were placed in a sealed quartz cuvette and
2
respectively irradiated with 635 nm, 650 nm and 730 nm light at the 5 mW/cm laser intensity.
The natural logarithm values of absorption of DPBF at 410 nm were plotted against the
irradiation time and fit by a first-order linear least squares model to get the singlet oxygen
generation rate of the photosensitized process [51].
25

ACCEPTED MANUSCRIPT
4
4
.3. Cell studies.
.3.1. Cell lines and culture conditions
Human esophageal cancer cell line Eca-109 was obtained from the Type Culture Collection
of the Chinese Academy of Sciences. All cell culture related reagents were purchased from
Shanghai Ming Rong Bio-Science Technology Co., Ltd. Cells were cultured in normal RPMI-
1
640 culture medium with 10% fetal bovine serum (FBS), 50 units per mL penicillin and 50 mg
-1
o
2
mL streptomycin in 5% CO at 37 C.
4
.3.2. MTT cell viability assay
Eca-109 cells were cultured in RPMI-1640 medium with 10% (v/v) FBS, collected with
4
0
.25% (w/v) trypsin, and seeded in 96-well plates at 5
Õ
10 cells per well. The cells were
allowed to attach to the bottom of the wells for 24 hours prior to start the experiment. RPMI-
640 medium containing P1-4, C1-4 and B1-4 in different concentrations (range from 0 to 60
M) was administered to cells and allowed to uptake for 24 hours. Nonirradiated cells were used
1
µ
to investigate the dark cytotoxicity. RPMI-1640 medium containing PSs was removed and cells
were washed with fresh PBS before irradiation with different concentrations (range from 0 to 16
2
µ
M) and light doses (16 J/cm ) using an Nd : YAG laser at 635 nm, 650 nm and 730 nm. The
cell viability was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl- 2H-terazolium
bromide (MTT) colorimetric assay 24 hours after treatment. Irradiated cells were used to
investigate the phototoxicity.
4
.4. Intracellular localization.
The cells were plated on poly-L-lysine coated coverslips in 12-well plates at the density of 1
5
o
Õ
10 cells/mL. After 24 h, the cells were incubated in the dark at 37 C with 10 µM drug for 4
h, and then rinsed in the RPMI-1640 medium, followed by incubation with Mitro-tracker Green
26

ACCEPTED MANUSCRIPT
o
and Lyso-Blue for 20 min at 37 C in a 5% CO
2
incubator, respectively. After rinsed three times
o
with PBS, the coverslips were fixed with 4% (w/v) paraformaldehyde at 4 C for 30 min. The
cells were observed with confocal fluorescence microscopy (Carl Zeiss LSM 700, Jena,
Germany). Mitro-tracker Green, Lyso-Blue, and B2 were respectively excited at the wavelength
of 490, 373, 373 nm and the signals from different probes were acquired in a sequential scan
mode.
4
4
.5. In vivo experiments
.5.1. Tumor xenograft mice model.
Four-week-old female BALB/c nude mice were obtained from Shanghai SLAC Laboratory
6
Animal Company and housed in dedicated pathogen-free barrier facilities. The 5×10 Eca-109
cells were injected subcutaneously in 200
were approved by the Animal Care and Use Committee of Donghua University.
.5.2. In vivo photosensitizing efficacy.
When the tumor reached approximately 100 mm in size (about 14 days after inoculation), 24
µL PBS into the right forelimb. All animal protocols
4
3
tumor-bearing mice were divided into 4 groups: control, light only, B2 and B2-PDT (each group
contained 6 mice). PS was injected into the tail vein of mice in the B2 and B2-PDT groups at a
dose of 5 mg/kg in 0.2 mL solution via the lateral tail vein. Then the mice were restrained in
2
plastic plexiglass holders without anesthesia and treated with laser light (730 nm, 120 J/cm , 180
2
mW/cm ) except for the control and B2 group. The power was monitored during the entire
treatment. Post-PDT, the mice were observed daily for tumor regrowth or tumor cure. Visible
tumors were measured every two days using two orthogonal measurements L and W
2
(
perpendicular to L), and the volumes were calculated using the formula V = LW /2 and
recorded. After B2-PDT in 14 days, the tumor was weighed, and tumor inhibition rates were
27

ACCEPTED MANUSCRIPT
calculated. The statistical differences between groups with respect to the mean tumor volume
were evaluated by using SPSS 19.0 for windows software. The significance was accepted at P <
0
.05.
Notes
The authors declare no competing financial interest.
Acknowledgment
This work was supported by the Fundamental Research Fund for the Central Universities
(
No.17D110513), Foundation of Shanghai Science and Technology Committee
No.15431904100, 16ZR1400600, 17PJ1432800, 17431902600, 17430741800, 17430711900,
(
1
8430713000), and the Grant of Ningbo Science and Technology Bureau
(No.2017BS1CA01008).
Abbreviations used
PDT, photodynamic therapy; ROS, reactive oxygen species; PS, photosensitizer; DDQ, 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone; NMR, nuclear magnetic resonance; DPBF, 1,3-
2 2
diphenylisobenzofuran; CH Cl , dichloromethane; DMSO, dimethyl sulfoxide; DMF,
dimethylformamide; FBS, fetal bovine serum; PBS, phosphate buffered saline; MTT, 3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-terazolium bromide
Supporting Information
1
Supplementary data associated with this article can be found in the online version. H NMR,
1
3
C NMR, HR-MS, UV-visible absorption, ROS formation on photolysis, in vitro dark
cytotoxicity and phototoxicity (Fig. S1-28).
REFERENCES
28