Synthesis, photophysical properties and biological evaluation of β-alkylaminoporphyrin for photodynamic therapy

Article abstract of DOI:10.1016/j.bmc.2016.09.060

A series of β-alkylaminoporphyrins conjugated with different amines at β position (D1–D3) or with electron-donating and electron-withdrawing substituents at phenyl position (D4–D6) were synthesized. Their photophysical and photochemical properties, intracellular localization, photocytotoxicities in vitro and vivo were also investigated. All target compounds exhibited no cytotoxicities in the dark and excellent photocytotoxicities against HeLa cells. Among them, D6 showed the highest phototoxicity and the lowest dark toxicity, which was more phototoxic than Hematoporphyrin monomethyl ether (HMME). In addition, D6 exhibited best photodynamic antitumor efficacy on BALB/c nude mice bearing HeLa tumor. Therefore, D6 is a powerful and promising antitumor photosensitizer for photodynamic therapy.

Full text of DOI:10.1016/j.bmc.2016.09.060

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, photophysical properties and biological evaluation
of b-alkylaminoporphyrin for photodynamic therapy
Ping-Yong Liao a, Xin-Rong Wang a, Ying-Hua Gao a, Xiang-Hua Zhang b, Li-Jun Zhang a, Chun-Hong Song a,
Dan-Ping Zhang a, Yi-Jia Yan c, Zhi-Long Chen a,
⇑
a Department of Pharmaceutical Science & Technology, College of Chemistry and Biology, Donghua University, Shanghai 201620, China
b Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200433, China
c Ningbo Dongmi Pharmaceutical Co., Ltd, Ningbo 315899, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of b-alkylaminoporphyrins conjugated with different amines at b position (D1–D3) or with elec-
tron-donating and electron-withdrawing substituents at phenyl position (D4–D6) were synthesized.
Their photophysical and photochemical properties, intracellular localization, photocytotoxicities
in vitro and vivo were also investigated. All target compounds exhibited no cytotoxicities in the dark
and excellent photocytotoxicities against HeLa cells. Among them, D6 showed the highest phototoxicity
and the lowest dark toxicity, which was more phototoxic than Hematoporphyrin monomethyl ether
(HMME). In addition, D6 exhibited best photodynamic antitumor efficacy on BALB/c nude mice bearing
HeLa tumor. Therefore, D6 is a powerful and promising antitumor photosensitizer for photodynamic
therapy.
Received 6 August 2016
Revised 22 September 2016
Accepted 24 September 2016
Available online xxxx
Keywords:
Tetraphenylporphyrin
Photosensitizer
Photodynamic therapy
Antitumor
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
efficiency,10 (4) high photocytotoxicity and non-toxicity in the
dark, (5) selective accumulation and rapid clearance.11,12
Photodynamic therapy (PDT) is an attractive therapeutic proce-
dure utilizing a photosensitizer (PS) activated by light to generate
highly reactive oxygen species (ROS).1,2 Compared with traditional
cancer therapies such as surgery, chemotherapy, PDT damages the
tumor cells selectively without destructing to surrounding normal
tissues.3 There are two mechanisms that cause destruction of
tumor cells in the process of oxidation of biomolecules and leading
to formation of ROS: excited-state electron transfer to oxygen sub-
strates (type I) to generate radicals, peroxides, superoxide radicals
anions or excited-state energy transfer to molecular O2 (type II) to
produce singlet oxygen.4 Typical PSs undergo type II mechanism.5
PDT also has the advantages of repeated dose tolerance and high
specificity, which are achieved through the precise application of
light.6 The synthesis of PS with desired physical, chemical and
biological properties is considered to be an important bottleneck
in PDT.7 An ideal PS should have the following characteristics:
(1) high purity and extraordinary chemical stability,8 (2) strong
light absorption at long wavelength (photo therapeutic window
600–800 nm) for deeper penetration into biological tissues,3,9 (3)
high molar absorption coefficient and singlet oxygen generation
Porphyrin-based PS hematoporphyrin derivatives (HPD, Photo-
frin) was discovered by Lipson and Baldes in 1960s and is one of
first generation PS to receive regulatory approval for the treatment
of various tumors in more than 40 countries throughout the
world.13,14 But it is a complex derivative and its selectivity and
photocytotoxicity is insufficient for tumor therapy.15 Compared
with HPD, Hematoporphyrin monomethyl ether (HMME) has a
known structure which has been used in clinic in China. Experi-
mental studies and clinical trials demonstrated that HMME has a
strong photodynamic effect with lower toxicity.16,17 Nowadays,
various meso-substituents have been incorporated into porphyrin
molecule. The design and synthesis of novel PSs are central to
the development of efficient PDT modalities, particularly the
reduction of the doses of drug and photo irradiation by increasing
photocytotoxicity, selective accumulation in the tumor, and rapid
clearance after treatment to induce side effects such as prolonged
skin sensitivity.18
As the limited solubility of porphyrin and aggregation in
commonly used solvents affects their optical properties,19 many
methods have been proposed to overcome these limitations. One
method relies on inducing steric isolation of the porphyrins’ core
through their substitution with bulky groups.20 Other method
which relies on the introduction to appropriate functional groups,
⇑
Corresponding author. Tel./fax: +86 21 67792654.
E-mail address: zlchen1967@qq.com (Z.-L. Chen).
http://dx.doi.org/10.1016/j.bmc.2016.09.060
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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P.-Y. Liao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
such as amide, carboxylic, or sulfonic group.21 Synthesis of
b-heteroatom-substituted porphyrin often utilize metal-catalyzed
carbon-heteroatom formation. Many researchers have established
a powerful new strategy for synthesis of heteroatom-substituted
porphyrin from catalytic reactions of halogenated porphyrins with
amines, amides and alcohols.22 Another strategy for preparation of
b-heteroatom-substituted porphyrin is palladium-catalyzed
amination reactions of 2-amino-porphyrin nickel(II) with bro-
mobenzene derivatives.23 2-Nitroporphyrins or 2-nitroporphyrins
copper have been extensively used as starting materials for the
synthesis of other porphyrin derivatives by direct nucleophilic sub-
stitution of the 2-nitro group by alkoxides,24 1,2-dicarbonyl com-
pounds.25 In this work, b-alkylaminoporphyrins were synthesized
by nucleophilic substitution of 2-nitroporphyrin copper derivatives
with amine derivatives and then demetalization. Their photophys-
ical properties, subcellular localization, photodynamic activities
in vitro and vivo are reported herein.
Briefly, pyrrole and benzaldehyde derivatives (1a–d) were
added to propanoic acid. After refluxing, the solution was cooled
to room temperature and filtered to give purple solid compounds
2a–d. 2a–d were reacted with cupric acetate monohydrate in
dichloromethane to afford the corresponding 5,10,15,20-
tetraphenylporphyrin copper(II) (3a–d) in about 98% yield.26 Reac-
tion of the metalloporphyrins 3a–d and cupric nitrate trihydrate in
refluxing dichloromethane gave 2-nitro-5,10,15,20-tetraphenyl-
porphyrin copper(II) (4a–d) in about 96% yield. Compounds 5a–f
were prepared by the nucleophilic substitution of 2-nitroporphyrin
copper derivatives (4a–d) with different amines under reflux at
nitrogen atmosphere. Demetalization of 5a–f with H2SO4 and
CF3COOH gave target compounds D1–D6. The combination of 1H
NMR, 13C NMR and HR-MS was used to elucidate the identities of
D1–D6. All spectra are provided in Supplementary information
(Figs. S1–S18).
2.2. UV–visible absorption and fluorescence spectra
2. Results and discussion
The UV–vis spectra of the porphyrins were highly sensitive to
the aggregation characteristics, which can be used to indicate the
aggregation status. The aggregation behaviors in the presence of
D1–D6 in DMSO were investigated through the analysis of the con-
centration dependence of their UV–vis spectra in concentrations
2.1. Synthesis and characterization
An useful synthesis method of b-alkylaminoporphyrins with a
variety of substituents was developed and outlined in Scheme 1.
R1
R1
R1
N
N
N
N
NH
N
N
c
a
b
R1
R1
Cu
R1
R1
HN
CHO
1a-d
1a:
R1 = H
1b: R1 = Cl
1c: R1 = Me
1d: R1 = OMe
R1
3a-d
R1
2a-d
R1
R1
R1
R2
R2
NO2
NH
N
N
N
N
N
N
N
N
e
d
R1
R1
R1
Cu
R1
R1
Cu
R1
N
HN
N
R1
R1
R1
5a-f
D1-D6
4a-d
N
N
N
O
N
N
N
O
D1: R1 = H, R2 =
D2: R1 = H, R2 =
D3: R1 = H, R2 =
5a: R1 = H, R2 =
5b:
R1 = H, R2 =
5c: R1 = H, R2 =
5d: R1 = Cl, R2 =
5e: R1 = Me, R2 =
5f: R1= OMe, R2 =
N
N
N
D4:
N
N
N
R1 = Cl, R2 =
D5: R1 = Me, R2 =
D6:
R1 = OMe, R2 =
Scheme 1. Reagents and reaction conditions: (a) pyrrole, propanoic acid, reflux 30 min; (b) Cu(OAc)2ÁH2O, CH2Cl2, CH3COOH, RT, 15 min; (c) Cu(NO3)2Á3H2O, Ac2O, CH3COOH,
CH2Cl2, reflux 4 h; (d) amine, K2CO3, reflux; (e) H2SO4, TFA, 30 min.
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P.-Y. Liao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
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ranging from 1
l
M to 10
l
M. As shown in Figure 1 and Table 1, the
The fluorescence excitation and emission spectra of D1–D6 were
determined. As shown in Figure 2, the shapes of their excitation
spectra were similar to those of the absorption spectra.
spectra of D1–D6 had no new bands appearing because the aggre-
gated species and normalized spectra at all concentrations could be
superimposed without a change in the shape of the soret band. The
intensity of the absorption of the soret band is directly propor-
tional to concentration, obeying the Lambert–Beer law, which sug-
gested that aggregation of these compounds in DMSO was
negligible. All of the synthesized PSs showed a bathochromic shift
of their long wavelength absorption bands which could be
explained by stereo–hindrance effect derived from the connection
of amino groups at b position, because pyrrolidine group is smaller
than morpholine group or piperidine group. D6 possessed the lar-
gest molar absorption coefficient at a long wavelength of 668 nm.
2.3. Singlet oxygen quantum yield
Reactive oxygen species (ROS) are the key cytotoxic intermedi-
ates of photodynamic therapy. Therefore, the efficiency of the ROS
generation upon photo irradiation is an important measure of the
cytotoxicity of photosensitizers. The quantum yield of singlet oxy-
gen (UD) values generated by photosensitization of new PSs were
determined (Fig. S19 in Supplementary information). After the data
were plotted as ln[DPBF0]/[DPBFt] versus irradiation time t,
Figure 1. Absorption spectra of compounds D1–D6 at various concentrations in DMSO.
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P.-Y. Liao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 1
The absorption and emission properties of compounds D1–D6
Compd
Absorption kmax (nm) (
e
 104 MÀ1 cmÀ1
)
Emission kmax (nm)
Excitation Emission
Stokes shift (nm)
Soret
Q band
D1
D2
D3
D4
D5
D6
417 (16.08)
412 (14.53)
412 (8.32)
412 (14.75)
414 (16.52)
417 (13.43)
522 (1.37)
528 (1.30)
524 (0.57)
521 (1.45)
531 (1.64)
535 (1.24)
596 (0.69)
601 (0.44)
596 (0.27)
594 (0.73)
602 (0.58)
603 (0.68)
650 (0.56)
664 (0.25)
650 (0.16)
648 (0.67)
666 (0.34)
668 (0.54)
417
412
412
412
414
417
663
665
669
665
671
683
13
1
19
17
4
15
Figure 2. Fluorescence excitation and emission spectra of compounds D1–D6 in DMSO at the concentration of 4 lM.
Table 2
straight lines were obtained for the sensitizers, and the slope for
each compound was obtained after fitting with a linear function
(Fig. 3). Table 2 lists the quantum yield of singlet oxygen (UD) val-
ues, D1–D6 showed better UD values than HMME, and D6 was sig-
nificantly better than HMME.
Ratios of singlet oxygen yields and singlet oxygen values (UD
)
Compd
10À2 Â k [minÀ1
]
UD
D1
D2
D3
D4
D5
D6
2.071
3.646
1.573
1.721
4.566
4.67
0.25
0.43
0.19
0.20
0.54
0.56
0.18
2.4. Cytotoxicity on HeLa cells
To investigate the potential of D1–D6 in PDT, the toxicities of
the PSs toward HeLa cells with or without irradiation were inves-
tigated and compared with HMME (as shown in Fig. S20). All syn-
thesized PSs showed no cytotoxicity in the dark. IC50 value is an
important index for the evaluation of antitumor activity of drugs.
HMME
1.556
As shown in Table 3, after exposure to light (16 J/cm2), D2 exhib-
ited highest phototoxicity among D1–D3. Because pyrrolidine
group is smaller than morpholine or piperidine group. For the phe-
nyl substituted derivatives D4–D6, D6 showed better phototoxicity
than D2. It could be explained that methoxyl group, as an electron-
donating group and polar group, could promote the uptake of drug.
Among D1–D6, D6 showed the highest phototoxicity toward HeLa
cells, which was more than 2 folds higher than HMME. These
results suggest that the substituted types of phenyl and b position
influenced significantly on the phototoxicity and D6 is an effective
photosensitizer in vitro.
Table 3
Phototoxicity for D1–D6 and HMME toward HeLa cells
Compd
Dark toxicity (IC50
l
M)
Phototoxicitya (IC50
lM)
D1
D2
D3
D4
D5
D6
HMME
93.4***
171.6***
109.9***
81.6***
5.06***
4.56***
5.56***
6.43***
5.33***
4.38***
9.46
280.0***
386.9***
344.7
Figure 3. First-order plots for the photodecomposition of DPBF photosensitized by
compounds D1–D6 and HMME (monitoring the maximum absorption of DPBF at
410 nm every minute).
a
Visible light k = 650 nm, light dose = 16 J/cm2.
P <0.001 represents a significant difference relative to the control group.
***
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P.-Y. Liao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
2.5. Intracellular localization
The intracellular localization of D6 in HeLa cells was investi-
gated by confocal microscopy after incubating HeLa cells for 12 h
and staining them with fluorescent dye (Hoechst 33342) for cell
nucleus. As shown in Figure 4, D6 was mainly found in cytoplasm
and nuclear membranes corresponding to the red fluorescent and
nucleus colocalized with the blue fluorescence of the nucleus
probe.
2.6. In vivo PDT efficacy
The in vivo therapeutic efficacy of PDT using D1–D6 was evalu-
ated in HeLa tumor bearing BABL/c nude mice. By comparing the
tumor weight in different groups and defining group without any
treatment as a control, the inhibition rates of tumor growth could
be calculated. Inhibition rates of compounds D1–D6 against HeLa
were shown in Figure 5a, the light irradiation only has little influ-
ence on the growth of tumors, and there is no significant difference
between the light group and the control group. Because of its best
comprehensive performance, D6 was selected as a typical repre-
sentative. As shown in Figure 5b, the volume growth curves of
tumors were provided. The volume of tumors in control, light
and D6 group were larger than that in D6–PDT group at the same
time. The light group has little influence on the growth of tumors,
and there is no significant difference among the light irradiation
group, D6 group and the control group. The tumor volume
increased about 10 folds for 14 days in control group. D6–PDT
decreased the tumor volume at the 5th day post treatment, and
the tumor growth was slower than that in control group. These
results demonstrate that compound D6 is effective to tumor in
PDT in vivo.
3. Conclusions
In summary, a series of b-alkylaminoporphyrins were devel-
oped. Their synthesis, photophysical properties, in vitro and vivo
antitumor activities were reported. They were prepared via nucle-
ophilic substitution of 2-nitroporphyrin copper derivatives with
amine derivatives and then demetalization. All target compounds
exhibited no cytotoxicity in the dark and excellent photocytotoxi-
city against HeLa cells. Among them, D6 showed the highest
phototoxicity and the lowest dark toxicity, which was more
phototoxic than HMME. In addition, D6 also exhibited better
in vivo photodynamic antitumor efficacy on BALB/c nude mice
bearing HeLa tumor. So D6 is a powerful and promising antitumor
PS for photodynamic therapy.
Figure 5. (a) Inhibition rates of compounds D1–D6 against HeLa tumor. (b) Tumor
volume of D6 at different time points post-PDT. The date shown are the means SD
of three independent experiments.
Meanwhile, it was found that the substituted types of phenyl
and b position of porphyrin ring influenced significantly on the
phototoxicity. D2 with a pyrrolidine group linked at b position
showed better phototoxicity than others which linked with a
morpholine group (D1) or piperidine group (D3). Different
phototoxicities could be observed when electron-donating and
Figure 4. Intracellular location of D6 in HeLa cells. Hela cells were incubation with 5
corresponds to D6, blue fluorescence represents the signal for Hoechst 33342.
lM D6 for 4 h in dark, and then stained with Hoechst 33342. Red fluorescence
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P.-Y. Liao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
electron-withdrawing substituents were present at phenyl
(D2, D4–D6). Compounds with electron-donating substituent or
electron-withdrawing substituent has effects on inhibition rate
and the effect is required further study.
ether (1:8) to give the desired product 5e (44.6 mg, 24.2%).
Mp >300 °C. MS (MALDI-TOF): m/z 801.3 [M+H]+.
4.2.1.6. 2-Pyrrolidine-5,10,15,20-tetra(4-methoxylphenyl)por-
phyrin copper(II) (5f).
The crude porphyrin was purified by
silica gel chromatography eluted with dichloromethane/petroleum
ether (1:2) to give the desired product 5f (73.5 mg, 37%).
Mp >300 °C. MS (MALDI-TOF): m/z 865.3 [M+H]+.
4. Experimental section
4.1. Materials and Instruments
4.2.2. General procedure for the synthesis of D1–D6
All solvents and reagents were obtained from commercial sup-
pliers and used without further purification unless otherwise sta-
ted. Melting points were obtained on a ‘stuart’ Bibby apparatus
and were uncorrected. 1H NMR and 13C NMR spectra were
recorded on a Bruker 400 MHz spectrometer. Chemical shifts were
reported as d values relative to the internal standard tetramethyl-
silane. MADLI-TOF mass spectra were recorded on an AB SCIEX
4800 Plus MALDI TOF/TOFTM. HR-MS spectra were recorded on a
Thermo Fisher Scientific LTQ FT Ultra Mass Spectrometer. Column
chromatography was performed using silica gel H (300–400 mesh).
UV–vis absorption spectra were recorded on an ultraviolet visible
spectrophotometer (Model V-530, Japan). Fluorescence spectra
To compound 5 (1 mmol) in a round-bottomed flask, CF3COOH
(8 mL) was added. Concentrated H2SO4 (2 mL) was then added
dropwise to this stirred mixture until the porphyrin was dissolved.
The reaction continued with stirring at room temperature for
30 min, after which the mixture was poured into ice water
(100 mL). Then the pH was adjusted to 7 by adding aqueous
NaHCO3. The water phase was extracted with dichloromethane
(3 Â 50 mL). The combined organic layer was washed with water
(3 Â 100 mL), saturated brine (3 Â 100 mL), dried over anhydrous
Na2SO4, and concentrated in vacuo to give purple solid of com-
pound D.
were measured on
oroMax-4, France).
a Fluorescence Spectrophotometer (Flu-
4.2.2.1. 2-Morpholine-5,10,15,20-tetraphenylporphyrin (D1). D1
was prepared by following the above general procedure. Yield:
96%. Mp >300 °C. 1H NMR (400 MHz, CDCl3): d ppm -2.60 (s, 2H),
3.14–3.36 (m, 8H), 7.73–7.78 (m, 12H), 8.18 (s, 1H), 8.22–8.28
(m, 8H), 8.64–8.79 (m, 6H). 13C NMR (100 MHz, CDCl3): d ppm
142.69, 142.32, 141.88, 141.61, 135.50, 134.69, 134.63, 134.37,
127.72, 127.68, 127.49, 126.89, 126.77, 126.67, 120.86, 119.97,
118.64, 118.34, 118.18, 66.46, 54.27. HRMS (DART Positive): m/z
calcd for C48H38N5O [M+H]+, 700.3071; found, 700.3071.
4.2. Synthetic methods
4.2.1. General procedure for the synthesis of 5a–f
The compounds 4a–d were synthesized according to literature
procedure.26–28
A solution of 4 (0.23 mmol) and K2CO3 (360 mg, 2.56 mmol) in
dry amine (12 mL) was stirred at reflux for about 10 h under nitro-
gen until no more starting material was detected by TLC. The mix-
ture was cooled, diluted with dichloromethane (150 mL), washed
with water (3 Â 100 mL) and saturated brine (3 Â 100 mL), dried
over anhydrous Na2SO4 and concentrated. The crude porphyrin
was purified by silica gel chromatography to afford the desired
product 5.
4.2.2.2. 2-Pyrrolidine-5,10,15,20-tetraphenylporphyrin (D2).D2
was prepared by following the above general procedure. Yield:
96%. Mp >300 °C. 1H NMR (400 MHz, CDCl3): d ppm 8.82–8.71
(m, 3H), 8.69–8.58 (m, 3H), 8.34 (dd, J = 7.8, 1.6 Hz, 2H), 8.29–
8.15 (m, 6H), 7.91 (s, 1H), 7.79–7.64 (m, 12H), 3.08–2.95 (m, 4H),
1.66–1.61 (m, 4H), -2.39 (s, 2H). 13C NMR (100 MHz, CDCl3): d
ppm 143.03, 142.50, 142.00, 141.32, 136.08, 134.82, 134.68,
134.29, 127.65, 127.54, 127.48, 127.40, 127.10, 126.78, 126.67,
121.21, 119.30, 118.97, 117.06, 112.50, 53.26, 25.04. HRMS (DART
4.2.1.1. 2-Morpholine-5,10,15,20-tetraphenylporphyrin copper
(II) (5a).
The crude porphyrin was purified by silica gel chro-
matography eluted with dichloromethane/petroleum ether (1:1) to
give the dark green product 5a (87.4 mg, 50%). Mp >300 °C. MS
(MALDI-TOF): m/z 761.2 [M+H]+.
Positive): m/z calcd for
C
48H38N5 [M+H]+, 684.3122; found,
684.3110.
4.2.2.3. 2-Piperidine-5,10,15,20-tetraphenylporphyrin (D3).
D3 was prepared by following the above general procedure. Yield:
96%. Mp >300 °C. 1H NMR (400 MHz, CDCl3): d ppm 8.76 (d,
J = 7.6 Hz, 3H), 8.68 (dd, J = 9.4, 4.8 Hz, 2H), 8.59 (d, J = 4.8 Hz,
1H), 8.33–8.25 (m, 2H), 8.24–8.18 (m, 6H), 8.09 (s, 1H), 7.78–
7.69 (m, 12H), 3.14 (m, 4H), 1.88 (m, 4H), 1.08 (m, 2H), -2.59 (s,
2H). 13C NMR (100 MHz, CDCl3) d ppm 142.92, 142.47, 142.00,
141.45, 135.73, 134.73, 134.65, 134.38, 127.67, 127.61, 127.55,
127.12, 126.88, 126.77, 126.73, 126.65, 120.84, 119.66, 118.92,
117.84, 117.14, 55.43, 29.75, 25.38. HRMS (DART Positive): m/z
calcd for C49H40N5 [M+H]+, 698.3278; found, 698.3270.
4.2.1.2. 2-Pyrrolidine-5,10,15,20-tetraphenylporphyrin copper
(II) (5b).
The crude porphyrin was purified by silica gel chro-
matography eluted with dichloromethane/petroleum ether (1:3) to
give the dark green product 5b (65.2 mg, 38%). Mp >300 °C. MS
(MALDI-TOF): m/z 745.2 [M+H]+.
4.2.1.3. 2-Piperidine-5,10,15,20-tetraphenylporphyrin copper(II)
(5c).
The crude porphyrin was purified by silica gel chro-
matography eluted with dichloromethane/petroleum ether (1:3)
to give the desired product 5c (61.2 mg, 35%). Mp >300 °C. MS
(MALDI-TOF): m/z 759.1 [M+H]+.
4.2.2.4.
phyrin (D4).
2-Pyrrolidine-5,10,15,20-tetra(4-chlorinephenyl)por-
D4 was prepared by following the above general
4.2.1.4.
phyrin copper(II) (5d).
silica gel chromatography eluted with dichloromethane/petroleum
ether (1:8) to give the desired product 5d (64.9 mg, 32%).
Mp >300 °C. MS (MALDI-TOF): m/z 881.1 [M+H]+.
2-Pyrrolidine-5,10,15,20-tetra(4-chlorinephenyl)por-
The crude porphyrin was purified by
procedure. Yield: 88%. Mp >300 °C. 1H NMR (400 MHz, CDCl3): d
ppm 8.75–8.73 (m, 3H), 8.68–8.64 (m, 3H), 8.28 (d, J = 8.0 Hz,
2H), 8.22–8.08 (m, 6H), 7.90 (s, 1H), 7.83–7.69 (m, 8H), 3.07 (t,
J = 4 Hz, 4H), 1.76–1.70 (m, 4H), -2.45 (s, 2H). 13C NMR (100 MHz,
CDCl3): d ppm 141.17, 140.65, 140.17, 139.55, 137.04, 135.75,
135.59, 135.27, 134.33, 134.21, 134.05, 134.02, 127.37, 127.13,
127.11, 127.01, 120.03, 118.02, 117.87, 115.99, 112.70, 53.46,
4.2.1.5.
phyrin copper(II) (5e).
silica gel chromatography eluted with dichloromethane/petroleum
2-Pyrrolidine-5,10,15,20-tetra(4-methylphenyl)por-
The crude porphyrin was purified by
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P.-Y. Liao et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
24.99. HRMS (DART Positive): m/z calcd for C48H34N5Cl4 [M+H]+,
4.4.2. MTT cell viability assay
820.1545; found, 820.1563.
HeLa cells were cultured in DMEM medium with 10% (v/v) FBS,
collected with 0.25% (w/v) trypsin, and seeded in 96-well plates at
2 Â 104 cells per well. The cells were allowed to attach to the bot-
tom of the wells for 24 h prior to start the experiment. The tested
compounds were diluted to different final concentrations, and
allowed to uptake for 24 h in the dark. DMEM medium containing
photosensitizer was removed and cells were washed twice with
fresh PBS before irradiation with different light doses (0, 1, 2, 4,
6, 8, 16 J/cm2) using an Nd: YAG laser at 650 nm. The cell viability
was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazolium bromide (MTT) colorimetric assay 24 h after treat-
ment. In parallel, non-irradiated cells were used to investigate
the dark cytotoxicity. The percentage cell survival was
calculated by normalization with respect to the value for no drug
treatment.
4.2.2.5.
2-Pyrrolidine-5,10,15,20-tetra(4-methylphenyl)por-
phyrin (D5).
D5 was prepared by following the above general
procedure. Yield: 86%. Mp >300 °C. 1H NMR (400 MHz, CDCl3): d
ppm 8.77–8.73 (m, 3H), 8.69–8.65 (m, 3H), 8.21 (d, J = 7.6 Hz,
2H), 8.12 (dd, J = 14.2, 7.5 Hz, 6H), 7.93 (s, 1H), 7.57–7.51 (m,
8H), 3.03 (t, J = 8 Hz, 4H), 2.72–2.65 (m, 12H), 1.65 (t, J = 6.0 Hz,
4H), -2.37 (s, 2H). 13C NMR (100 MHz, CDCl3): d ppm 140.13,
139.67, 139.15, 138.49, 137.24, 137.09, 136.97, 136.00, 134.82,
134.62, 134.22, 127.79, 127.49, 127.38,121.12, 119.20, 118.83,
116.96, 112.39, 53.19, 25.02, 21.57, 21.54, 21.52, 21.48. HRMS
(DART Positive): m/z calcd for C52H46N5 [M+H]+, 740.3748; found,
740.3730.
4.2.2.6. 2-Pyrrolidine-5,10,15,20-tetra(4-methoxylphenyl)por-
phyrin (D6).
D6 was prepared by following the above general
4.4.3. Intracellular localization
procedure. Yield: 98%. Mp >300 °C. 1H NMR (400 MHz, CDCl3): d
ppm 8.79 (t, J = 4.1 Hz, 2H), 8.75 (d, J = 4.7 Hz, 1H), 8.70 (d,
J = 4.8 Hz, 1H), 8.64 (q, J = 4.8 Hz, 2H), 8.27 (d, J = 8.2 Hz, 2H),
8.17 (dd, J = 17.3, 8.1 Hz, 6H), 7.90 (s, 1H), 7.33–7.28 (m, 8H),
4.16–4.07 (m, 12H), 3.09–3.02 (m, 4H), 1.70 (q, J = 3.3 Hz, 4H),
À2.34 (s, 2H). 13C NMR (100 MHz, CDCl3): d ppm 159.39, 159.28,
159.12, 137.22, 136.00, 135.71, 135.51, 135.29, 135.08, 134.50,
134.01, 120.86, 118.85, 116.52, 118.40, 112.69, 112.29, 112.24,
112.20, 111.96, 55.63, 55.59, 55.57, 55.56, 53.13, 25.14. HRMS
(DART Positive): m/z calcd for C52H46N5O4 [M+H]+, 804.3544;
found, 804.3536.
The cells were plated on poly-L-lysine coated coverslips in
12-well plates at the density of 1 Â 105 cells/mL. After 24 h, the
cells were incubated in the dark at 37 °C with 5 lM drug for 4 h,
then rinsed in the DMEM medium and incubated with Hoechst
33342 (1 g/mL) for 10 min at 37 °C. After rinsing three times with
l
PBS, the coverslips were fixed with 4% (w/v) paraformaldehyde at
4 °C for 30 min. The cells were observed by a confocal fluorescence
microscopy (Carl Zeiss LSM 700, Jena, Germany). Drug was excited
at 417 nm and monitored through a 600–700 nm band-pass filter,
and Hoechst 33342 was excited at 350 nm and blue fluorescence
was detected through a 450–500 nm band-pass filter.
4.3. Photophysical and photochemical measurements
4.5. In vivo experiments
4.3.1. Absorption and emission spectra
4.5.1. Tumor xenograft mice model
UV–visible absorption spectra were recorded on an ultraviolet
visible spectrophotometer (Model V-530, Japan). Fluorescence
spectra were measured on a Fluorescence Spectrophotometer (Flu-
oroMax-4, France). Slits were kept narrow to 1 nm in excitation
and 1 or 2 nm in emission. Right angle detection was used. All
the measurements were carried out at room temperature. D1–D6
were dissolved in DMSO to get different concentration solutions
Four-week-old female BALB/c nude mice were obtained from
the Shanghai SLAC Laboratory Animal Company and housed in
dedicated pathogen-free barrier facilities. And 5 Â 106 HeLa cells
were injected subcutaneously in 200 lL PBS into right forelimb.
All animal protocols were approved by the Animal Care and Use
Committee of Donghua University.
(1, 2, 4, 6, 8, 10 lM).
4.5.2. In vivo PDT efficacy
When the tumor reached approximately 100 mm3 in size (about
14 days after inoculation), the tumor-bearing mice were divided
into 4 groups: control, light, PS and PS-PDT (each group contained
6 mice). PS was injected into the tail vein of mice in the PS and PS-
PDT groups at a dose of 5 mg/kg. Then the mice were restrained in
plastic plexiglass holders without anesthesia and treated with laser
light (650 nm, 100 J/cm2, 180 mW/cm2) except for the control and
PS 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 (perpendicular to L), and
the volumes were calculated using the formula V = LW2/2 and
recorded. After PS-PDT at 14 days, the tumor weights were
weighed, and tumor inhibition rates were calculated.
4.3.2. Singlet oxygen generation detection
The singlet oxygen ability of D1–D6 and HMME were moni-
tored by chemical oxidation of 1,3-diphenylisobenzofuran (DPBF)
in the DMF solution. Typically, a 3 mL portion of DMF solution con-
taining 20 lM DPBF and 0.5 lM photosensitizer was placed in a
sealed quartz cuvette and irradiated with 650 nm light at the laser
intensity of 5 mW/cm2. 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.29 The rate constant
was converted into 1O2 quantum yield by comparison with the rate
constant for DPBF photo-oxidation sensitized by methylene blue.
4.4. In vitro experiments
4.6. Statistical analysis
4.4.1. Cell lines and culture conditions
HeLa cells were 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 Dulbecco’s Modified Eagle’s Medium
(DMEM) with 10% fetal bovine serum (FBS, BioChrom, Cambridge,
UK) in 5% CO2 at 37 °C in a humidified incubator.
All experiments were performed in triplicate and the data were
expressed as mean plus and minus the standard error of the mean.
Analysis of variance (ANOVA) and Student’s t-test were used to
determine the statistically significant difference among different
groups when appropriate.
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Acknowledgements
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This work was supported by National Natural Science Founda-
tion of China (Nos. 21372042, 81301878, 21402236), Foundation
of Shanghai Government (Nos. 15XD1523400, 14140903500,
15431904100, 14ZR1439900, 14ZR1439800, 15ZR1439900,
16ZR1400600, 15411960400) and Foundation of Songjiang
Government (Nos. 14SJGGYY08, 15SJGG45).
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A. Supplementary data
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