Synthesis and in vitro photodynamic activities of water-soluble fluorinated tetrapyridylporphyrins as tumor photosensitizers

Article abstract of DOI:10.1016/j.bmcl.2007.02.083

A series of water-soluble fluorinated cationic porphyrins were designed, synthesized, and characterized. In vitro photocytotoxicity of these compounds was evaluated by MTT assay on HeLa cells. Their photocytotoxicity was dependent on the positions of the cations and the fluorines in the pyridine ring, and 5,10,15,20-tetrakis-(N-methyl-2-fluoro-pyridin-3-yl)-porphyrin (8) showed the most potent photo-induced cytotoxicity without photobleaching. PDT-induced ROS inside HeLa cells was measured with flow cytometry using ROS-sensitive fluorometric probe, 2,7-dichlorofluororescin (DCF), which revealed high correlations of ROS with cellular cytotoxicity. FACS analysis shows that PDT with porphyrin 8 induced apoptosis in HeLa cells. In summary, efficient generation of ROS, biological effectiveness, and good photostability of porphyrin 8 indicate its potential application in photodynamic therapy (PDT) in the near future.

Full text of DOI:10.1016/j.bmcl.2007.02.083

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2789–2794
Synthesis and in vitro photodynamic activities of water-soluble
fluorinated tetrapyridylporphyrins as tumor photosensitizers
Yoon-Joo Ko,a Kyung-Jin Yun,b Min-Seok Kang,b Jongmin Park,a
a,
a,
*
Kyung-Tae Lee,b,* Seung Bum Park and Jung-Hyu Shin
*
aDepartment of Chemistry, Seoul National University, Seoul 151-747, South Korea
bCollege of Pharmacy, Kyung-Hee University, Dongdaemun-ku, Seoul 130-701, South Korea
Received 4 September 2006; revised 5 February 2007; accepted 26 February 2007
Available online 12 March 2007
Abstract—A series of water-soluble fluorinated cationic porphyrins were designed, synthesized, and characterized. In vitro photo-
cytotoxicity of these compounds was evaluated by MTT assay on HeLa cells. Their photocytotoxicity was dependent on the posi-
tions of the cations and the fluorines in the pyridine ring, and 5,10,15,20-tetrakis-(N-methyl-2-fluoro-pyridin-3-yl)-porphyrin (8)
showed the most potent photo-induced cytotoxicity without photobleaching. PDT-induced ROS inside HeLa cells was measured
with flow cytometry using ROS-sensitive fluorometric probe, 2,7-dichlorofluororescin (DCF), which revealed high correlations of
ROS with cellular cytotoxicity. FACS analysis shows that PDT with porphyrin 8 induced apoptosis in HeLa cells. In summary,
efficient generation of ROS, biological effectiveness, and good photostability of porphyrin 8 indicate its potential application in pho-
todynamic therapy (PDT) in the near future.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Photodynamic therapy (PDT) is a cancer treatment
leading to the selective destruction of malignancies by
visible light in the presence of a photosensitizer and oxy-
gen.1 Upon the irradiation of visible light with appropri-
ate wavelength, the photosensitizer can drive molecular
oxygen into excited triplet state, transferring energy into
ground state molecular oxygen to produce singlet molec-
ular oxygen.2 Activated singlet oxygen, or reactive oxy-
gen species (ROS) in general, plays an important role in
cytotoxic effects on tumor tissues. PDT can be applied
as an effective cancer treatment due to enhanced perme-
ability and retention (EPR) effect in tumors in compar-
ison with normal tissues and is easily controlled by
limiting the area of light irradiation.3 Therefore, diver-
gence in selective distribution to tumors and efficiency
in visible light energy transfer to an intermediate are
important properties of photosensitizers.
Porphyrin is a heterocyclic natural ligand of heme
groups in hemoglobin or electron carriers such as cyto-
chromes. Porphyrin-based macrocycles are one of the
most frequently used photosensitizers and have been in
the center of interest for the development of more sensi-
tive analogues,4 due to its excellent properties, especially
selective association with membranes of malignant cells
in vitro or in vivo studies. They also have low toxicity
since they are easily cleared from tissues and blood flu-
ids.5 Furthermore, porphyrin-based compounds are effi-
cient generators of reactive oxygen species (ROS) by the
absorption of photons in visible region.6a In addition,
porphyrin-based compounds showed diverse patterns
of intracellular localization, predominantly at lysosomes
and mitochondria, based on their structures, lipophilic-
ity, and charges.6b However, a number of challenges are
ahead of us to develop efficient porphyrin-based photo-
sensitizers; longer retention time in tumor tissue, better
water-solubility, and high quantum yield of reactive
oxygen species. To address this issues, cationic porphy-
rins gained attentions in scientific community due to
their advantages as a photosensitizer, such as improved
water-solubility and selectivity to malignant tumor
cells.7 They also intercalate with DNA and inhibit the
production of telomerase associated with the prolifera-
tion and immortality of cancerous cells.8 In this report,
Keywords: Fluorinated tetrapyridylporphyrins; Photodynamic therapy
Reactive
(PDT); Photosensitizer;
Photocytotoxicity.
oxygen
species
(ROS);
*
Corresponding authors. Tel.: +82 2 880 6655; fax: +82 2 889 1568
(J.-H.S.); e-mail addresses: ktlee@khu.ac.kr; sbpark@snu.ac.kr;
junghyu@snu.ac.kr
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.02.083

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Y.-J. Ko et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2789–2794
In this paper, we reported the synthesis, photophysical
+
N
N
properties, and photostability of fluorinated cationic
porphyrin derivatives. The results of photocytotoxicity
studies as well as cellular location and efficiency of reac-
tive oxygen species (ROS) generation in HeLa cells
in vivo are also presented.
Y
X
Y
X
X
Y
Y
X
X
Y
Y
NH
N
NH
N
+
N
N
N
+
N
N
HN
N
HN
X
The porphyrins were prepared through a typical reac-
tion between equimolar amounts of fluoropyridine carb-
aldehyde and pyrrole in refluxing propionic acid.11
Porphyrins 112 and 213 were prepared from pyridine-4-
carbaldehyde and 3-fluoropyridine-4-carbaldehyde,
respectively. The synthesis of 3 was achieved in five steps
(Scheme 1). After formylation of 3,5-dibromopyridine at
the 4-position, the formyl group was converted into a
1,3-dioxolane group via a conventional protection pro-
tocol. The NFSi-mediated difluorination and subse-
quent deprotection of the 1,3-dioxolane group to an
aldehyde yielded compound 9.14 The typical reaction
of aldehyde 9 with pyrrole in propionic acid generated
an 8% yield of porphyrin 3 purified by silica gel flash col-
umn chromatography (MeOH/CHCl3, 1:40). Porphyrin
4 was successfully obtained from highly volatile 2-flu-
oropyridine-3-carbaldehyde15 at a 6% yield which was
purified by silica gel flash column chromatography
(MeOH/CHCl3, 1:40). Methylation of porphyrins (1–4)
with trimethyloxonium tetrafluoro-borate afforded an
excellent yield (>90%) of the water-soluble cationic por-
phyrins (5–8). Porphyrins 3 and 4 and their salts, 7 and
8, are novel compounds. These compounds were charac-
terized by conventional spectroscopic techniques
(NMR, HRMS).16 19F NMR signals of the fluorinated
porphyrins 5–8 showed a single peak at around d
150 ppm in DMSO-d6.
-
4BF4
X
Y
X
Y
N
N
+
5: X=H,Y=H
6: X=H,Y=F
7: X=F,Y=F
1: X=H,Y=H
2: X=H,Y=F
3: X=F,Y=F
+
N
N
F
F
F
F
NH
N
N
N
+
NH
N
N
N
+
N
HN
N
HN
F
F
-
4BF4
F
F
N
N
+
8
4
Figure 1. Porphyrin analogues.
we replaced some of the hydrogen atoms on cationic
porphyrin system with fluorine to obtain high quantum
yields and effective energy absorption. Generally, the
fluorinated porphyrins have greater triplet quantum
yields,9 and their fluorine substitution is known to play
a key role in anti-tumor activity.10 For systematic com-
parison of the structure–activity relationship, non-fluo-
rinated cationic porphyrin 5 and three fluorinated
cationic porphyrin analogues (6–8) were synthesized in
our laboratory. The structures of these compounds are
shown in Figure 1.
The fluorescence emission and UV–visible spectral
data of these cationic porphyrins in water are listed in
Table 1. The fluorescence maxima show two bands
(ꢀ660 and 720 nm) upon the excitation by visible light
O
O
CHO
Br
Br
1) HCO2CH3
F
F
1) NFSi
Br
Br
2) 12N HCl
2) OH(CH)2OH
N
N
N
9
pyrrole
propionic acid
+
N
N
F
F
F
F
F
F
F
F
F
F
F
NH
N
N
NH
N
(CH3)3OBF4
+
N
N
N
N
+
N
HN
HN
F
-
F
F
F
F
4BF4
N
N
+
3
7
Scheme 1.

Y.-J. Ko et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2789–2794
Table 1. Fluorescence emission and UV–vis data of porphyrin derivatives
2791
Compound
kem (nm)
kmax (nm) (e · 10À4, MÀ1 cmÀ1
Visible bands
550 (0.6)
)
Soret band
5
6
7
8
660
658
665
662
720
717
721
726
422 (17.2)
424 (21.1)
420 (9.7)
424 (28.3)
515 (1.5)
513 (2.0)
513 (1.3)
516 (1.9)
589 (0.6)
582 (1.2)
587 (0.6)
593 (0.6)
647 (0.3)
—
563 (1.2)
—
552 (0.6)
659 (0.3)
648 (0.2)
with absorption maxima wavelength in Q bands. Their
UV–visible spectra were consisting of the intense Soret
band around 420 nm and weaker Q bands in the 510–
660 nm intervals with very little difference in extinction
coefficients. Based on our observation, fluorinated sub-
stituents on cationic porphyrins do not significantly
influence their fluorescence emission maxima (626–
630 nm) as well as UV–vis absorption maxima (Fig. 2).
growth inhibition by photosensitizers using MTT assay.
Figure 2 shows the concentration-dependent survival
curves for porphyrins 5–8. Most of the survival rates
ranged up to 90% in the absence of light under the wide
range of concentration, which revealed the minimal or
moderate cytotoxicity in darkness (Fig. 3).
To evaluate the biological functions of our novel com-
pounds, we initiated an in vitro cytotoxicity assay with
photodynamic activity of porphyrins (5–8) on HeLa
cells.16 Cells were pre-incubated with various concentra-
tions of porphyrins (5–8) for 1 h followed by irradiation
with fluorescent lamp (530–620 nm) at a fluence rate
of 1.6 J/cm2 (0.44 mW/cm2 for 1 h) without washing.
Then, HeLa cells were incubated for 48 h at 37 ꢁC. The
cell survival fraction was measured for photo-induced
a
Figure 3. Photocytotoxic effect of porphyrin derivatives on HeLa cells
in the absence of light and the irradiation of visible light for 1 h.
b
Figure 4. Porphyrins induce the generation of ROS. HeLa cells were
incubated in the presence or absence of 30 lg/mL porphyrins for 2 h.
DCF fluorescence was detected by flow cytometry after 2 h of
porphyrin treatment.
Figure 2. (a) UV–vis absorbance spectra of cationic porphyrin
derivatives (5–8); (b) Emission spectra of cationic porphyrin deriva-
tives (5–8).

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Y.-J. Ko et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2789–2794
As shown in Figure 3, porphyrins 7 and 8 exhibit sig-
nificantly higher photocytotoxic effects on HeLa cells.
IC50 values are 0.87 lM for porphyrin 7 and 1.1 lM
for porphyrin 8, while IC50 values for porphyrin 5
and 6 are 3 lM and 40 lM, respectively. Particularly,
porphyrin 8 showed a remarkable difference in photo-
sensitizing activity from porphyrin 6, which led us to
the conclusion that the position of positive charges
on pyridyl moieties exerts a strong influence on the
photosensitizing activity. Among 7 and 8, although
both porphyrins give a significantly high photocyto-
toxicity, porphyrin 8 turned out to be a more effective
PDT photosensitizer based on the studies with HeLa
cells.
Figure 5. Intracellular localization images of porphyrin 8 captured by Axiovert200 (Zeiss, Germany). HeLa cells were cultured in the presence of
30 lM porphyrin 8 for 24 h. (Left) Phase contrast image; (Right) Fluorescence image.
a
4.6 μM
9.2 μM
0 μM
18.4 μM
27.6 μM
36.8 μM
40
30
20
10
0
b
*
*
*
*
*
0 (Con)
4.6
9.2
18.4
27.6
36.8
Concentration (μM)
Figure 6. Sub-G1 cell population in 8-treated HeLa cells stained with propidium iodide (PI) was determined by FACS flow cytometry. (a) data from
a typical experiment; (b) histogram from FACS analysis of cells incubated under control conditions in the presence of various concentrations of 8.
The values are means S.D. of three independent experiments and analyzed by using Student’s t-test. (*p < 0.001 when compared to the control
(0 lM)).

Y.-J. Ko et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2789–2794
2793
The cytotoxicity of photosensitizer is caused by reac-
Acknowledgments
tive oxygen species (ROS), generated upon the irradi-
ation. ROS has been demonstrated to be an early
signal mediating apoptosis.17 To investigate whether
intracellular ROS is involved in cationic porphyrin-
mediated cell death, we next measured the level of
ROS within the cells using a ROS-sensitive fluoromet-
ric probe, 2,7-dichlorofluororescin (DCF), that detects
a wide range of ROS by flow cytometric analysis.18 As
shown in Figure 4, the basal level of DCF-sensitive
ROS in HeLa cells was not readily detectable,
however significant generation of ROS was observed
upon the treatment of 30 lg/mL porphyrins followed
by visible light irradiation. The PDT-induced ROS
generation was highly correlated with the cytotoxic
data of porphyrins 5–8 in HeLa cells. Especially,
porphyrins 7 and 8 showed significant enhancement
of ROS inside HeLa cells, which is consistent with
the data of cytotoxicity assay. In addition, cellular up-
take image of porphyrin 8 indicates that porphyrin 8
is localized to the cytoplasm (possibly to the mito-
chondria) as previously reported (Fig. 5).19 Therefore,
we can conclude that ROS generation in the
cytoplasm by PDT-sensitizers is the key element for
cellular cytotoxicity.
Support of this work from the Brain Korea 21 program
is greatly appreciated. S.B. Park is supported by Korea
Science and Engineering Foundation (KOSEF), and
Molecular and Cellular BioDiscovery Research Pro-
gram from the Ministry of Science and Technology
(MOST).
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.02.083.
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We also performed a photobleaching experiment with
cationic porphyrins under the same irradiation condi-
tions used for photocytotoxicity in the HeLa cells.
However, we observed minimal decay of emission inten-
sity at kem = 662 nm even with extensive irradiation (up
to 500-fold light dosage of PDT cytotoxicity study) of
visible light at 516 nm (see Supporting information). It
is noteworthy that porphyrins 8 and 7 showed high
photostability, which is the desired property of a PDT-
sensitizer.
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peak on DNA content frequency histograms revealed
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characterized. The potentials of fluorinated cationic por-
phyrin derivatives as PDT-sensitizers were evaluated by
in vitro photocytotoxicity measurement using MTT as-
say on HeLa cells. Notably, porphyrin, 7 and 8 show
significantly higher photocytotoxicity, presumably due
to the efficient generation of reactive oxygen species
(ROS) in the cytoplasm and their photostability. FACS
analysis also showed that PDT with porphyrin 8 in-
duced apoptosis in HeLa cells. Further developments
and improvements using water-soluble fluorinated por-
phyrins are in progress and more extensive biological
studies are on going.
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