In vitro photodynamic activity of 5,15-bis(3-Hydroxyphenyl)porphyrin and its halogenated derivatives against cancer cells

Article abstract of DOI:10.1111/j.1751-1097.2009.00622.x

5,15-Diarylporphyrins (1-5) with hydroxyl groups and halogens as substituents were prepared by condensation between unsubstituted dipyrromethane and halogenated m-hydroxybenzaldehydes. Photophysical properties show that the nonhalogenated porphyrin 1 has higher fluorescence yield but lower singlet oxygen formation quantum yield than the halogenated derivatives due to the heavy atom effect. The in vitro activity of these derivatives was tested against WiDr colorectal adenocarcinoma and A375 melanoma cancer cells. All porphyrins present a much higher phototoxicity than Photofrin with IC ₅₀ values lower than the 50 nm level for WiDr cells and 25 nm level for A375 cancer cells. The most photoactive compound is the nonhalogenated porphyrin 1 which also presents the highest uptake. Halogenated derivatives present much lower uptakes than 1. However, their photoactivity is similar to compound 1 showing that their intrinsic photoactivity (ISP) is very high. Iodinated compound 4 presents the highest ISP. The greater ability of these porphyrins to destroy cancer cells could be related to their photophysical and photochemical properties.

Full text of DOI:10.1111/j.1751-1097.2009.00622.x

Photochemistry and Photobiology, 2010, 86: 206–212
In Vitro Photodynamic Activity of 5,15-bis(3-Hydroxyphenyl)porphyrin
and Its Halogenated Derivatives Against Cancer Cells
1
Armenio Serra , Marta Pineiro , Catarina Isabel Santos , Antonio Manuel d’A. Rocha Gonsalves*
1
1
1,2
´
´
,
Margarida Abrantes2,3, Mafalda Laranjo3 and Maria Filomena Botelho2,3
1
´
Chymiotechnon, Departamento de Quımica, Universidade de Coimbra, 3049-535, Coimbra, Portugal
2Centre of Investigation on Environment Genetics and Oncobiology (CIMAGO), Faculty of Medicine,
University of Coimbra, Coimbra, Portugal
3
´
´
Instituto de Biofısica ⁄ Biomatematica, IBILI, Faculdade de Medicina de Coimbra, 3000-354 Coimbra, Portugal
Received 17 June 2009, accepted 22 July 2009, DOI: 10.1111 ⁄ j.1751-1097.2009.00622.x
effect is not directly followed by an increase in photodynamic
activity (19). The structure of the PS and its interaction with
cell structures also have a major role in defining the best
properties for a PS.
ABSTRACT
5,15-Diarylporphyrins (1-5) with hydroxyl groups and halogens
as substituents were prepared by condensation between unsub-
stituted dipyrromethane and halogenated m-hydroxybenzalde-
hydes. Photophysical properties show that the nonhalogenated
porphyrin 1 has higher fluorescence yield but lower singlet
oxygen formation quantum yield than the halogenated deriva-
tives due to the heavy atom effect. The in vitro activity of these
derivatives was tested against WiDr colorectal adenocarcinoma
and A375 melanoma cancer cells. All porphyrins present a much
higher phototoxicity than Photofrinꢀ with IC50 values lower than
the 50 nM level for WiDr cells and 25 nM level for A375 cancer
cells. The most photoactive compound is the nonhalogenated
porphyrin 1 which also presents the highest uptake. Halogenated
derivatives present much lower uptakes than 1. However, their
photoactivity is similar to compound 1 showing that their
intrinsic photoactivity (ISP) is very high. Iodinated compound 4
presents the highest ISP. The greater ability of these porphyrins
to destroy cancer cells could be related to their photophysical
and photochemical properties.
Due to appropriate photophysical characteristics and affin-
ity for cancer cells, porphyrin derivatives are frequently used in
PDT studies (20). The easily prepared 5,10,15,20-tetraphenyl-
porphyrins (TPP) are very much studied porphyrin structures,
those having hydroxyl substituents in the phenyls being
particularly active. Among them, temoporfin has been the
most representative of this type of compounds (21). However,
the structures of such compounds are very different from the
natural porphyrins having unsubstituted methylenic positions.
This structural characteristic can be an obstacle for the
required elimination of the sensitizer by the body. A compro-
mise between photodynamic activity, simplicity of synthetic
methods and a more ‘‘natural’’ structure can be the prepara-
tion of 5,15-diarylporphyrins where only two groups at the
methylenic positions should be used to confer hydrophylicity
to the structure. Another advantage of these porphyrins is
their less voluminous structures and lower molecular weight,
increasing the possibility of having better characteristics for
passive diffusion across the blood–brain barrier (22). Examples
of the use of these kinds of porphyrins in PDT are described by
INTRODUCTION
Banfi et al. (23) and Bourre et al. (24). Also Senge et al.
´
prepared several derivatives, not mentioning biological studies
(25).
Photodynamic therapy (PDT) is one of the most selective
available treatments for oncological cancer diseases where the
destruction of cancerous tissues occurs only if light and a
photosensitizer (PS) reach the interior of cancer cells (1–3).
With these two requirements, the therapeutic species, singlet
oxygen, is locally generated and its effects confined to cancer
tissues with minimal side effects. Recently, some developments
were made regarding PS delivery (4–6), improvements on the
selectivity for cancer cells (7,8) and also new PSs have been
proposed (9–17).
One key characteristic of the PS is the singlet oxygen
quantum yield (FD) which is desirable to be as high as possible.
In recent work we showed that the presence of halogen atoms
as substituents on the PS causes an increase in FD (18) but this
We pursued our initial work (19) by exploiting halo-
genated porphyrins as PSs for PDT. The presence of
halogens in these structures can be an important factor for
the desired activity. Besides the effect on the photophysical
and photochemical properties of the porphyrin, it is possible
that the halogenated structures could interfere in the activity
of P-glycoprotein (P-gp). The P-gp is a membrane drug
transporter responsible for the molecular drug resistance
(MDR) of certain cancer types by promoting the elimination,
from the intracellular medium, of exogenous anti-cancer
drugs (26,27). Halogenated molecules (28–30) have the ability
to interfere, blocking the P-gp, inhibiting its action and
reversing the MDR. High anticancer activity due to the
presence of the halogen atoms was demonstrated in the case
of chlorine (31–33) and bromine (34).
*Corresponding author email: arg@qui.uc.pt (Anto
Gonsalves)
ꢁ 2009 The Authors. JournalCompilation. The AmericanSocietyof Photobiology 0031-8655/10
´
nio M. d’A. Rocha
206

Photochemistry and Photobiology, 2010, 86 207
Almost all colon cancers are primary carcinomas, which are
one of the most common cancers in the United States and
Western Europe. Melanoma is the most aggressive of the skin
cancers that, due to its localization, facilitates the irradiation
of the tumour, being a natural choice for treatment with PDT.
In this work we present a series of new 5,15-diarylporphyrins
substituted with halogen atoms and hydroxyl groups and the
study of their photophysical and photochemical properties as
well as their in vitro photodynamic activity against WiDr
adenocarcinoma cells and A375 melanoma cells. Some of our
compounds are very active against the two cancer cell lines and
seem to be promising PSs for PDT.
Synthesis of dipyrromethane 6. In a round-bottom flask pyrrole
(20 mL, 288 mmol), dichloromethane (20 mL), acetic acid (20 mL)
and trichloroacetic acid (0.3 g, 18 mmol) were added (37,38). The
solution was heated to reflux and a mixture of pyrrole (10 mL,
144 mmol) and paraformaldehyde (0.7 g, 23 mmol) in dichloro-
methane (20 mL) was added. The mixture was left in reflux for 2 h
giving a dark-green solution. After cooling, the organic phase was
carefully washed with a NaOH 10% solution, water and dried with
anhydrous sodium sulfate. The solution was concentrated in vacuo and
the excess of pyrrole removed by vacuum distillation. Dipyrrylmethane
6 was purified by flash chromatography (using dichloromethane with a
few drops of triethylamine as eluent). A white solid (0.977 g, 29%) was
obtained with mp: 73–74ꢂC (lit. 74ꢂC). (37); 1H NMR (CDCl3, 400
MH): d = 3.88 and 3.96 (s, 2H, methylene bridge-H), 6.03 (m, 2H, 3,3,
pyrrole-H), 6.14 (m, 2H, 4,4, pyrrole-H), 6.55 (m, 2H, 5,5, pyrrole-H),
7,83 (bs, 2H, NH); MS (EI): m ⁄ z 146 (100%, M+).
Synthesis of 5,15-bis(3-hydroxyphenyl)porphyrin 1. Porphyrin 1 was
prepared as described from the methoxy derivative (25); 1H NMR
(DMSOd6, 400 MH): d = 7.32 (m, 2H, ArH), 7.64–7.75 (m, 4H,
ArH), 9.14 (d, J = 4.6 Hz, 4H, pyrrole-H), 9.52 (d, J = 4.6 Hz, 4H,
pyrrole-H) 10.46 (s. 2H, methylene bridge-H); HRMS (FAB):
m ⁄ z calc. for C32H23N4O2 (M+H)+, 495.18155; found, 495.17969.
Synthesis of 5,15-bis(2-bromo-5-hydroxyphenyl)porphyrin 2. In a
1 L round-bottom flask with dichloromethane (650 mL), dipyrryl-
methane 6 (0.50 g, 3.4 mmol) and 2-bromo-5-hydroxybenzaldehyde
(8) (0.82 g, 4.1 mmol) were added. The solution was stirred at room
temperature for 20 min under nitrogen and then trifluoroacetic acid
(0.26 mL, 3.5 mmol) was added. The mixture was left overnight. The
mixture was neutralized with triethylamine and DDQ (1.17 g,
5.1 mmol) was added. After 2 h, the solution was evaporated to
dryness and the porphyrin purified by dry flash chromatography
(CH2Cl2 ⁄ AcOEt, 9 ⁄ 1). A violet solid was obtained (0.24 g, 11%). 1H
NMR (DMSOd6, 400 MH): d = 7.24 (2H, ArH), 7.70
(d, J = 3.03 Hz, 2H, ArH), 7.87 (d, J = 8.7 Hz, 2H, ArH), 8.95 (d,
J = 4.6 Hz, 4H, pyrrole-H), 9.38 (d, J = 4.6 Hz, 4H, pyrrole-H)
10.29 (s. 2H, methylene bridge-H). MS (ESI): m ⁄ z 653
MATERIALS AND METHODS
Chemical synthesis. Solvents were purified by standard methods before
use. Dichloromethane was dried over CaH2 and distilled. The
chloroform was neutralized with neutral active alumina. 9,10-Dimeth-
ylanthracene (DMA) was acquired from Aldrich and used without
further purification. Methylene Blue was used as received (Riedel-de
Haen). Pyrrole was distilled before used. For column chromatography,
silica gel 60-220-440mesh ⁄ 0.035–0.070 mm, from Fluka was used.
3-Hydroxybenzaldehyde from Aldrich was recrystallized from water.
2-Bromo-5-hydroxybenzaldehyde (8) (35) and 3-hydroxy-4-iodobenz-
aldehyde (9) (19) were prepared as described. Photofrinꢀ was kindly
offered by the Gastroenterology Service of the Hospitais da Univer-
sidade de Coimbra.
Absorption spectra were recorded with a Shimadzu UV-2100
spectrophotometer. Fluorescence spectra were measured with a Spex
Fluorolog 3 spectrophotometer. RMN spectra were recorded on a
400 MHz Bruker Advance III. Mass spectra of EI were obtained with
GC-MSD HP 6890 ⁄ 5973 and Finnigan Advantage for the MS
(electrospray ionization [ESI]) spectra. High resolution mass spectra
were recorded on a Bruker FTMS APEXIII instrument under ESI
(Vigo University).
[(M + 1)+].HRMS (FAB): m ⁄ z calc. for C32H21Br2N4O2 (M+H)+
653.0054; found, 652.99809.
Synthesis of 5,15-bis(4-iodo-3-hydroxyphenyl)porphyrin 3.
,
Synthesis of 2-iodo-3-hydroxybenzaldehyde 10. First, the 2-acetoxy-
mercury-3-hydroxybenzaldehyde (11) was prepared as follows (36). To
According to the procedure for porphyrin 2 with dipirrylmethane 6
(1.0 g, 6.8 mmol) and 4-iodo-3-hydroxybenzaldehyde (9) (2.0 g,
8.1 mmol) in dichloromethane (950 mL) and trifluoroacetic acid
(0.50 mL, 6.7 mmol) as catalyst. A solid was isolated (0.16 g, 3%) from
two consecutive flash chromatographies (CH2Cl2 ⁄ AcOEt, 3 ⁄ 1). 1H
NMR (DMSOd6, 400 MH): d = 6.88 (dd, J = 8.8, J = 3.0 Hz, 2H,
ArH), 7.00 (dd, J = 8.2, J = 1.8 Hz, 2H, ArH), 7.17 (d, J = 2.9 Hz,
2H, ArH), 7.27 (s, 2H, ArH), 7.76 (d, J = 8.8 Hz, 2H, ArH), 7.84 (d, 2H,
J = 8.2, ArH), 9.16–9.20 (m, 6H, pyrrole-H), 9.58–9.62 (m, 6H,
pyrroleH) 9.76–9.81 (m, 4H, pyrrole-H), 10.17 (s, 1H, methylene
bridge-H), 10.19 (s, 1H, methylene bridge-H), 10.26 (s, 2H, methylene
bridge-H); MS (ESI): m ⁄ z 747 [(M + 1)+].HRMS (FAB): m ⁄ z calc. for
C32H21I2N4O2 (M+H)+, 746.97484; found, 746.97258.
Synthesis of 5,15-bis(2-iodo-3-hydroxyphenyl)porphyrin 4. According
to the procedure for porphyrin 2 with dipyrrylmethane 6 (0.225 g,
1.54 mmol) and 2-iodo-3-hydroxybenzaldehyde (10) (0.382 g,
1.54 mmol) in dichloromethane (300 mL) and trifluoroacetic acid
(0.01 mL, 1.23 mmol) as catalyst. A solid was isolated (0.065 g, 6%)
from flash chromatography (CH2Cl2 ⁄ AcOEt, 9 ⁄ 1). 1H NMR
(DMSOd6, 400 MH): d = 7.48 (m, 2H, ArH), 7.65 (m, 2H, ArH), 7.83
(d, J = 8.5 Hz, 2H, ArH), 8.90 (d, J = 4.6 Hz, 4H, pyrrole-H), 9.37 (d,
J = 4.6 Hz, 4H, pyrrole-H), 10,31 (s. 2H, methylene bridge-H). MS
(ESI): m ⁄ z 747 [(M + 1)+].HRMS (FAB): m ⁄ z calc. for C32H21I2N4O2
(M+H)+, 746.97484; found, 746.97157.
Synthesis of 5,15-bis(2-chloro-5-hydroxyphenyl)porphyrin 5.
According to the procedure for porphyrin 2 with dipyrrylmethane 6
(0.88 g, 6 mmol) and 2-chloro-5-methoxybenzaldehyde (12) (0.84 g,
6 mmol) in dichloromethane (500 mL) and p-toluenesulfonic acid
(0.15 g, 0.8 mmol) as catalyst. A solid was isolated (0.13 g, 4%) from
flash chromatography (CH2Cl2) which was identified as porphyrin 13 by
mass spectrometry. MS (ESI): m ⁄ z 591 [(M + 1)+]. The demethylation
of 13 was carried out as follows. Porphyrin 13 (100 mg, 0.13 mmol) was
dissolved in dichloromethane (15 mL), the solution cooled to )25ꢂC and
1 mL of BBr3 solution was slowly added. The mixture was left overnight
at room temperature and was diluted with CH2Cl2 and treated with
a
mixture of 3-hydroxybenzaldehyde (5.0 g, 41 mmol) in
ethanol ⁄ water(25 ⁄ 25 mL),Hg(CH3COO)2(13.1 g,41 mmol)inethanol ⁄
water ⁄ acetic acid (50 ⁄ 49 ⁄ 1 mL) was added. The mixture was refluxed
for 6 h, concentrated in vacuum to 50 mL and kept overnight in a
refrigerator. Filtration gives a white solid (13.0 g, 83%) that can be
recrystallized from acetic acid. mp: >230ꢂC; 1H NMR (DMSOd6,
400 MHz): d = 10.05 (s, 1 H, CHO), 7.49 (d, J = 7.0 Hz, 1 H, ArH),
7.40 (t, J = 7.6 Hz, 1 H, ArH), 7.18 (d, J = 7.8 Hz, 1H, ArH), 1.96 (s,
1 H, CH3); 13C NMR (DMSOd6, 100 MHz): d = 194.7, 174.8, 161.2,
141.5, 130.3, 129.3, 126.2, 121.9, 23.2.
The aldehyde (11) (7.0 g, 18.3 mmol) is added in small amounts to a
solution of iodine (5 g, 19.6 mmol) and potassium iodide (5.0 g,
30 mmol) in 50 mL of water and the mixture stirred for 5 h. Aldehyde
10 formed as a light yellow solid (3.5 g, 77%), is filtered and
recrystallized from ethanol ⁄ water (15 ⁄ 85). mp: 150–152ꢂC (lit. 159–
160ꢂC) (36); 1H NMR (DMSOd6, 400 MHz): d = 10.04 (s, 1 H, CHO),
7,15(dd,J = 1.8, 7.0 Hz,1H,ArH),7.04(t, J = 7.3 Hz,1H,ArH),6.99
(dd, J = 1.8, 7.9 Hz, 1H, ArH), 5.90 (s, 1 H, OH); 13C NMR (DMSOd6,
100 MHz) d = 196.5, 157.1, 136.2, 129.1, 121.1, 120.6, 91.4; MS (EI):
m ⁄ z 248 (100%, M+).
Synthesis of 2-chloro-5-methoxybenzaldehyde 12. To a solution of
m-anisaldehyde (5 mL, 41 mmol) in methanol (50 mL), N-chlorosuc-
cinimide (6.0 g, 45 mmol) was added. The solution was stirred at 50ꢂC
overnight. The solvent was evaporated in vacuo, the residue treated
with hot petroleum ether (40–60ꢂC) and the solid filtered. The organic
fractions were collected, concentrated and chromatographed on a silica
gel column using dichloromethane ⁄ hexane (30 ⁄ 70) as eluent to give 12
as a crystalline white solid in the first fraction (1.1 g, 16%). The second
fraction is a mixture of compounds with some amount of 12. After
sublimation (40ꢂC, 60 mm Hg) it has mp 55ꢂC; 1H NMR (CDCl3,
400 MHz): d = 10.43 (s, 1 H, CHO), 7.41 (d, J = 3.0 Hz, 1 H, ArH),
7.34 (d, J = 8.8 Hz, 1 H, ArH), 7.09 (dd, J = 3.0, 8.8 Hz, 1H, ArH),
3.85 (s, 1 H, CH3); 13C NMR (CDCl3, 100 MHz) d = 189.8, 158.7,
132.9, 131.5, 129.8, 122.9, 111.9, 55.8; MS (EI): m ⁄ z 170 (100%, M+).

´
208 Armenio Serra et al.
methanol and triethylamine. The organic phase was carefully washed
with water, dried with sodium sulfate and evaporated in vacuum to give
67 mg (92%) of porphyrin 5. 1H NMR (DMSOd6, 400 MH): d = 7.24
(dd, J = 2.9, 8.7 Hz, 2H, ArH), 7.55–7.64 (m, 4H, ArH), 8.93 (d,
J = 4.6 Hz, 4H, pyrrole-H), 9.31 (d, J = 4.6 Hz, 4H, pyrrole-H), 10.21
(s. 2H, methylene bridge-H),10.26 (s. 2H, methylene bridge-H). MS
(ESI): m ⁄ z 563 [(M + 1)+]. HRMS (FAB): m ⁄ z calc. for
C32H21Cl2N4O2 (M+H)+, 563.10361; found, 563.10151.
1 X=Z=W=H
Z
X
Z
OH
HN
N
N
2 X=Br, Z=W=H
3 X=Z=H, W=I
4 X=W=H, Z=I
5 X=Cl, Z=W=H
W
W
NH
HO
X
Biological studies. Experimental details regarding photobleaching
of the sensitizer and cellular uptake measurements are described
elsewhere (19). The cell culture conditions are as follows. The human
colon carcinoma cell line WiDr and the human melanoma cell line
A375 were purchased from American Type Culture Collection. The
cell lines were cultured with Dulbecco’s modified Eagle medium (Sigma
D-5648; Sigma-Aldrich, Inc.) supplemented with 10% heat-inactivated
fetal bovine serum (Gibco 2010-04; Gibco Invitrogen Life Technolo-
gies), 1% penicillin-streptomycin (Gibco Invitrogen Life Technologies;
100 U mL)1 penicillin and 10 lg mL)1 streptomycin—Gibco 15140-
122) and 100 lM Sodium Piruvate (Gibco 1360; Gibco Invitrogen Life
Technologies) at 37ꢂC, in a humidified incubator with 95% air and 5%
CO2.
Scheme 1.
1
MeO
1.TFA
2.DDQ
3. BBr3/CH2Cl2
HO
7
OHC
8
Br
CHO
1.TFA
CH2Cl2/ACOH
TCA
+ (CH2O)n
2
Photodynamic treatment: For each experiment, cells were plated
in 48 multiwells (Corning Costar Corp) at a concentration of 40 000
cells mL)1 and kept in the incubator overnight, in order to allow the
attachment of the cells. The formulation of these sensitizers
N
N
N
H
2.DDQ
H
H
6
I
a a ternary mixture of
1 mg mL)1 solution in
HO
1.TFA
2.DDQ
consisted in
H2O:PEG400:EtOH (50 ⁄ 30 ⁄ 20, vol ⁄ vol ⁄ vol), the desired concentra-
tions being achieved by successive dilutions. The sensitizers were
administered in several concentrations (50 nM, 250 nM, 500 nM,
1 lM, 5 lM and 10 lM) and cells were incubated for 24 h. Cells
were washed with PBS and new drug-free medium was added. Each
plate was irradiated at a fluence rate of 7.5 mW cm)2 until a total of
10 J was reached. Cell viability was measured 24 h after the
photodynamic treatment.
9
CHO
3
Scheme 2.
The synthesis of PSs 4 and 5 requires the preparation of the
corresponding halogenated aldehydes. For the insertion of
iodine on the less accessible ortho position of the 3-hydroxy-
benzaldehyde we had to prepare the mercuric derivative 11
following an old procedure (36). Iodine substitution of the
mercury on 11 gives 10 with a moderate yield (36). NMR
analysis of 11 and 10 demonstrates the substitution at the less
accessible ortho position. Chlorination of 3-methoxybenzalde-
hyde with N-chlorosuccinimide gives a mixture of products
separable by silica chromatography. The first fraction is the
aldehyde with an NMR spectrum corresponding to 2-chloro-
5-methoxybenzaldehyde (12), even though the melting point of
the sample does not agree with the one reported from a
previously described procedure (Scheme 3) (40).
The reactions of dipyrromethane 6 with aldehydes 10 and 12
give PS 4 and the methoxy derivative 13, respectively.
Treatment of 13 with boron tribromide gives PS 5 (Scheme 4).
These 5,15-diarylporphyrins present the expected NMR
spectra, with a doublet for the eight b-protons and a singlet for
the two methylenic protons, except for the iodinated derivative
Measurement of cell viability: The sensitivity of the cell lines to the
sensitizers was analyzed using the MTT, (3-(4,5-Dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide, colorimetric assay (Sigma M2128;
Sigma-Aldrich, Inc.) to measure cell proliferation. Cytotoxicity was
expressed as the percentage of inhibition of cell proliferation correlated
with untreated cultures. This allows the calculation of the concentra-
tion that inhibits the culture cell proliferation in 50% (IC50). Each
experiment was performed in duplicate and repeated in three sets of
tests.
Briefly, cells were plated in 48 multiwells at a concentration of
40 000 cells mL)1 and kept in the incubator overnight. Solutions of
1 mg mL)1 of each sensitizer in a ternary mixture of H2O:PEG400:-
EtOH (50 ⁄ 30 ⁄ 20, vol ⁄ vol ⁄ vol), were administered in concentrations of
50 nM, 250 nM, 500 nM, 1 lM, 5 lM and 10 lM and cells were incubated
for 24 h. Cells were washed with PBS and a new drug-free medium was
added. Each plate was irradiated in a light box with an optical filter
(kcutoff < 560 nm) with a fluence rate of 7.5 mW cm)2 until a total of
10 J was reached. Cell viability was measured 24 h after the photo-
dynamic treatment using the MTT, (3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, colorimetric assay to measure cell
proliferation. The irradiation wavelength for the fluorimetric mea-
surement of the cellular uptake was 404 nm. The IC50 values were
obtained from the dose–response curves for WiDr human colon
adenocarcinoma cells and melanoma A375.
CHO
CHO
RESULTS AND DISCUSSION
CHO
I
I2/KI
HgOAc
OH
Hg(AcO)2
Porphyrin synthesis
OH
The structures of 5,15-diarylporphyrins used as PSs 1–5 are
shown in Scheme 1. The preparation of 1 to 5 requires the
synthesis of the unsubstituted dipyrromethane 6 (37–39) that
was prepared by the condensation of paraformaldehyde with
excess pyrrole catalyzed by trichloroacetic acid. PSs 1, 2 and 3
were prepared by the direct condensation between 6 and the
corresponding aldehydes 7, 8 (35) and 9 (19) using trifluoro-
acetic acid as catalyst followed by DDQ oxidation (Scheme 2).
OH
10
11
CHO
CHO
Cl
NCS/MeOH
OMe
OMe
12
7
Scheme 3.

Photochemistry and Photobiology, 2010, 86 209
CHO
absorption spectra and consequently the PSs present a short
Stoke shift. As a representative example, in Fig. 1 the
normalized Q-region of the UV–VIS spectrum and the
fluorescence spectrum of compound 5 are shown.
I
1.TsOH
2. DDQ
4
OH
MeO
10
+
Cl
The fluorescence quantum yields were measured using TPP
as reference following the standard procedure (41). The
fluorescence quantum yield of TPP in ethanol was measured
to be 0.09 using the quantum yield in toluene (0.10) as
reference (41) and the Parker equation (42) taking into account
the different refractive indexes of the solvents. The fluores-
cence quantum yields for the 5,15-diarylporphyrins in ethanol
are presented in Table 1. The fluorescence quantum yields are
lower when the halogen atoms are placed at the ortho position
of the phenyl group. Nonsubstituted porphyrin 1 presents the
highest fluorescence quantum yield. Among the halogenated
porphyrins the lowest value is observed for the iodinated
derivatives (ten times less) and the highest (two times less) for
the chlorine derivative, following the pattern expected from the
heavy atom effect already observed with tetraarylporphyrins
(18). In the case of the iodine atom, the influence on the
fluorescence quantum yield is very similar whether the substi-
tution is in the ortho or para position (compounds 4 and 3,
respectively). Singlet oxygen formation quantum yield, FD,
was measured by steady-state photolysis using DMA as target
and 5,10,15,20-tetrakis(2,6-dichlorophenyl)porphyrin as refer-
ence comparing the slopes of the semilogarithmic plots of the
decay of the absorption of the quencher at 380 nm with time
(ln(A0 ⁄ A) vs time) for the PSs compared with the correspond-
ing slope obtained for the reference. The singlet oxygen
formation quantum yields are presented in Table 1. The
influence of the halogen in the ortho position of the phenyl
ring is observed as an increase in the singlet oxygen quantum
yield, from 0.49 for compound 1 in the absence of halogen, to
0.64 with iodine in the ortho position, compound 4. The
presence of one chlorine atom at the ortho position, compound
5, increases the singlet oxygen quantum yield compared with
the unsubstituted porphyrin 1, no significant difference being
observed when this atom is replaced by bromine, as in
compound 2. The decrease in fluorescence quantum yield
observed in compounds 2 and 3 is not accompanied by a
significant increase in singlet oxygen formation, suggesting
that this type of substitution could be increasing both the
intersystem crossing between the first singlet excited state to
the first triplet state and also the intersystem crossing from this
state to the group state, favoring the formation of the triplet
N
N
H
H
CHO
NH
N
BBr3
6
1.TsOH
2. DDQ
Cl
5
HN
N
OMe
12
Cl
13
OMe
Scheme 4.
3. In this case we observed several singlets for the methylenic
protons and several doublets for the pyrrole-hydrogens; also
the substitution pattern of aromatic protons is much more
complicated, which means that noninterconverting isomers are
present.
Spectroscopic measurements and singlet oxygen production
The absorption maxima and absorption coefficients, the
fluorescence bands and the fluorescence quantum yield and
the singlet oxygen quantum yields of sensitizers 1–5 are
presented in Table 1.
The absorption spectra of porphyrins in ethanol show the
typical Soret (B(0,0) band) and Q-bands of these compounds.
Compared with the corresponding tetraarylporphyrins, the
absorption bands are shifted to the blue. The Qx(1–0) band of
5,15-diarylsubstituted porphyrins appears at ca 627 nm in-
stead of the 645 nm observed in the corresponding tetrasub-
stituted analogs. The insertion of chlorine or bromine at the
ortho position of the phenyl ring does not modify the position
of the UV–VIS bands; with the introduction of the iodine atom
at the same position, compound 4, there is a 2 nm blueshift of
the spectra that brings the absorption characteristics of these
compounds closer to the therapeutic window (600–800 nm)
and to the characteristics of the reference compound, Photo-
frin (kmax = 630 nm, e = 1.17 M cm)1) (1) accompanied by
)1
a decrease in the absorption coefficient. For concentrations
between 1 · 10)7 and 1 · 10)4 M no deviation of the Beer–
Lambert law was observed, indicating that the sensitizers are
not aggregated under the experimental conditions. The fluo-
rescence spectra present two bands at ca 630 and 695 nm,
accompanying the blueshift of the Qx(1–0) band of the
Table 1. Absorption and fluorescence data, fluorescence quantum yield and singlet oxygen formation quantum yield of photosensitizers (PS) 1–5.
Absorption, kmax (nm), e (M)1cm)1
)
Fluorescence, kmax (nm)
PS
1
B(0–0)
Qy(1–0)
Qx(0–0)
Qy(0–0)
Qx(1–0)
Q(0-0)
630
Q(0–1)
FF
FD
402
1.61 · 105
402
498
6.75 · 103
499
531
2.15 · 103
529
572
2.30 · 103
572
627
5.70 · 102
627
692
695
695
694
695
0.065
0.002
0.005
0.006
0.031
0.008
0.0007
0.001
0.002
0.006
0.49
0.56
0.53
0.64
0.55
0.03
0.07
0.04
0.05
0.04
2
630
3.12 · 105
403
1.13 · 103
494
2.70 · 103
524
3.57 · 103
566
5.72 · 102
616
3
628
4.12 · 105
404
1.42 · 104
500
3.7 · 103
531
4.85 · 103
575
1.0 · 103
629
4
630
1.45 · 105
404
6.82 · 103
496
1.67 · 103
530
2.15 · 103
567
2.53 · 102
627
5
632
9.28 · 104
5.09 · 103
2.33 · 103
2.16 · 103
1.57 · 103

´
210 Armenio Serra et al.
100
80
60
40
20
0
UV-Vis spectrum (Q bands)
Fluorescence spectrum
1,0
0,8
0,6
0,4
0,2
Photofrin
1
2
3
5
0,0
500
600
700
800
Controlo
50 nM
500 nM
250 nM
1 uM
5 uM
10 uM
λ (nm)
Concentration (M)
Figure 1. Q bands from the absorption spectrum and fluorescence
spectrum of compound 5 in ethanol.
Figure 3. Dose–response curves of photodynamic activity of photo-
sensitizers 1–5 and Photofrinꢀ against A375 melanoma cells.
state and also its nonradiative deactivation, decreasing the
photochemical capability to react with molecular oxygen.
To study the photobleaching process of the porphyrins we
used the same experimental conditions, namely, solvent,
concentration and light source, as those used in cytotoxicity
assays. The absorption of the solutions was measured after 6,
12, 18 and 24 h. After 24 h of irradiation, the analysis of the
absorption at the maxima of the Soret band (B(0–0)) shows
less than 10% degradation for all the compounds, indicating
that the degradation during the irradiation time could be
neglected.
carried out for each experimental set, in the absence of
sensitizer, reveals no cytotoxicity of the solvent mixture used to
administer the porphyrins. Similar experiments carried out in
the absence of light and with the porphyrin reveal no dark
cytotoxicity in the range of concentrations used. Photofrinꢀ,
the first PS approved by the FDA for use in PDT (1), was used
as a model compound to compare the cytotoxic activity of the
new PSs under the same experimental conditions. All our new
PSs have shown to be more cytotoxic than Photofrinꢀ, also
being more effective against A375 melanoma cells than against
WiDr cells. In this cell line the differences between the
photoactivity of porphyrins are more evident. The most active
PS is the unsubstituted porphyrin 1. Among the halogenated
porphyrins the chloroderivative 5 is more active than the
brominated one, 2, which is more active than iodinated
derivatives (3 and 4). Extrapolation of the IC50 values (the
concentration of porphyrin that provides 50% inhibition of
cancer cell growth) for this cell line shows that 1 and 2 are
about 10 to 15 times more active than Photofrinꢀ. For the
A375 melanoma cell line all porphyrins seem to be very efficient
PSs, being about 30 times more active than Photofrinꢀ.
Cytotoxicity assays and cellular uptake
The in vitro photodynamic activities of PSs 1–5 against WiDr
human colorectal adenocarcinoma cells and A375 melanoma
cells were investigated. Figures 2 and 3 present the cell viability
using different concentrations of the sensitizer solution in
cultures of WiDr and A375 cells, respectively. The control
100
The observed photoactivity of porphyrins in this kind of
experiments is dependent on the intrinsic photoactivity (ISP)
of the porphyrin (its capacity to produce singlet oxygen in cell
environment) and on its ability to penetrate to the interior of
cancer cells. Studies about the influence of porphyrin accu-
mulation inside cells on the activity have been contradictory
(43,44). To have a better understanding of the relation between
porphyrin structure and photoactivity we carried out the
analysis of the cellular uptake of the porphyrins in WiDr cells
(Fig. 4). The cellular uptake is presented as the relative
percentage of porphyrin present in cellular extracts relative
to the incubation dose. Experiments with different incubation
concentrations of porphyrins showed that the cellular uptake
was dose-dependent, the fluorescence intensity increasing with
the increasing concentration of porphyrin used in incubation
for all the sensitizers. A similar dose-dependent behavior was
observed with A375 cells. Fluorescence analysis of the cellular
extraction experiments with WiDr cells using 5 lM incubation
solutions of porphyrins 1–5 for 24 h estimates the intracellular
Photofrin
80
60
40
20
0
1
2
3
4
5
Controlo
50 nM
250 nM
500 nM
1 uM
5 uM
10 uM
Concentration (M)
Figure 2. Dose–response curves of photodynamic activity of photo-
sensitizers 1–5 and Photofrinꢀ against WiDr human colorectal
adenocarcinoma cells.

Photochemistry and Photobiology, 2010, 86 211
100
90
80
70
60
50
40
30
20
10
0
photodynamic effect against WiDr human adenocarcinoma
and A375 melanoma cancer cell lines analyzed. All porphyrins
are more active than Photofrinꢀ in both cell lines. Between cell
lines, porphyrin phototoxicity is higher for melanoma A375
than for colon WiDr cells. The most effective porphyrin is the
nonhalogenated porphyrin 1, followed closely by the porphy-
rin with a chlorine atom 5. The analysis of WiDr cancer cell
uptake by the different porphyrins showed very clear differ-
ences, namely that 1 has a much higher concentration inside
cells than the halogenated derivatives which can explain the
observed results. The balance between phototoxicity and
cellular uptake through the ISP led us to conclude that
incorporation of halogen atoms into the structure of these
porphyrins dramatically decreases the cellular uptake but
improves its capability to generate singlet oxygen, increasing
its intrinsic phototoxicity against cancer cells. In fact, with less
molecules of sensitizer inside the cell the photodynamic activity
is very similar, indicating that each molecule is much more
effective against cancer cells.
1
2
3
4
5
Photosensitizer
Figure 4. Cellular uptake of compounds 1–5 in WiDr human adeno-
carcinoma cells.
Acknowledgements—The authors thank Chymiotechnon, Ministerio
´
concentration of porphyrins to be 4.16
0.16 0.02, 0.023 0.002 and 1.01
0.38, 0.11
0.02 lM for com-
0.01,
da Economia ⁄ POE ⁄ Prime ⁄ Proj 3 ⁄ 293 ⁄ CLARO, Faculdade de
Medicina de Coimbra and CIMAGO for financial support and
Gastroenterology service of HUC for equipment facilities. Mass
spectra (MS[ESI]) studies were carried out by M. Alexandra Rocha
Gonsalves, Chymiotechnon and measurements of singlet oxygen
formation quantum yields by Sonia Ribeiro. We acknowledge the
Nuclear Magnetic Resonance Laboratory of the Coimbra Chemistry
Centre (http://www.nmrccc.uc.pt), Universidade de Coimbra, for
obtaining the NMR data.
pounds 1–5, respectively. Unsubstituted porphyrin 1 presents
the highest uptake by WiDr cancer cells, incorporation being
four times greater than for the chlorinated porphyrin 5 which
has similar photoactivity. Brominated and iodinated porphy-
rins (2–4) are much less incorporated by cancer cells.
The value of the cellular uptake indicates that the observed
phototoxicity of compound 1 is directly related to the high
concentration of this porphyrin inside the cells that are still
nontoxic in the absence of light. Halogenated porphyrins,
despite their lower uptake are unexpectedly photoactive. In
order to understand this behavior we quoted the IC50 ⁄ cellular
uptake quotient for compounds 1–5. This quotient indicates
the ISP of the compound as it indicates how photoactive a
single molecule is. In Fig. 5 we plot the values obtained for the
ISP of all the PSs 1–5. As can be observed, at the molecular
level, the most efficient PS is 4 and the less efficient is 1 which
has a correspondence with the highest and lowest values
obtained for the singlet oxygen formation quantum yield.
In summary, an ensemble of 5,15-diarylporphyrins with
different types of halogen substitution was prepared and its
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