Synthetic porphyrins bearing β-propionate chains as photosensitizers for photodynamic therapy

Article abstract of DOI:10.1142/S1088424610002227

Porphyrins with different numbers of β-propionate chains mimicking natural porphyrins were prepared via the 2+2 MacDonald type approach. Photodynamic activity against WiDr colon adenocarcinoma cells showed that activity is related to the number of β-propi

Full text of DOI:10.1142/S1088424610002227

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2010; 14: 438–445
DOI: 10.1142/S1088424610002227
Published at http://www.worldscinet.com/jpp/
Synthetic porphyrins bearing β-propionate chains as
photosensitizers for photodynamic therapy
Nelson Pereiraa, Arménio C. Serraa, Marta Pineiroa, António M. d’A. Rocha
Gonsalves*a,c◊, Margarida Abrantesb,c, Mafalda Laranjob and Filomena Botelhob,c
a Chymiotechnon, Departamento de Química, Universidade de Coimbra, 3004-535 Coimbra, Portugal
b Instituto de Biofísica/Biomatemática, IBILI, Faculdade de Medicina de Coimbra, 3000-354 Coimbra, Portugal
c Centro de Investigação em Ambiente, Genética e Oncobiologia, (CIMAGO), Faculdade de Medicina de Coimbra,
3000-354 Coimbra, Portugal
Received 22 July 2009
Accepted 20 October 2009
ABSTRACT: Porphyrins with different numbers of β-propionate chains mimicking natural porphyrins
were prepared via the 2+2 MacDonald type approach. Photodynamic activity against WiDr colon
adenocarcinoma cells showed that activity is related to the number of β-propionate chains, with the
derivatives with two carboxylic groups showing higher activity.
KEYWORDS: photodynamic therapy, colon adenocarcinoma, β-propionate porphyrins, anti-cancer,
WiDr cancer cells.
treatment [8]. We decided to make use of our synthetic
capacities to prepare porphyrinic photosensitizers hav-
ing β-propionic side-chains to exploit the effect of the
presence of halogen on the photodynamic activity previ-
ously demonstrated for meso-tetraphenyl porphyrins [9].
In this work, we describe the preparation of porphyrins
INTRODUCTION
Natural porphyrins, their chemically modified deriva-
tives or purely synthetic structures are the source of
photosensitizers for PDT [1]. Among the photosensitiz-
ers approved for use in PDT, some are based on natu-
rally occurring porphyrins or their chemically modified
incorporating halogenated meso-phenyl groups and also
derivatives: polyhematoporphyrin ether/ester, mono-L-
propionic acid chains at β-positions, synthetic structures
aspartylchlorin e6, benzoporphyrin derivative monoacid
more similar to natural porphyrins than those previously
ring A, Tookad® and Photofrin® [2–4]. Typically, they
have free meso-positions and alkyl acid chains at the
β-positions. Studies with synthetic porphyrin sensitizers
have concentrated on the more easily available meso-aryl
studied. The photodynamic activity of these new porphy-
rins was tested against WiDr adenocarcinoma cells.
substituted β-free structures. Consistent results obtained
with cell cultures, animals and membrane models pres-
ent strong evidence that the extracellular-intracellular
pH gradient in the tumor cells might play an important
role in the accumulation and selectivity of sensitizers that
possess carboxylic chains [5–7]. Hydrophilic and hydro-
phobic characteristics of the sensitizer affect the cellular
uptake and localization inside cells and can play a deci-
sive role on the results obtained in the photodynamic
EXPERIMENTAL
General
Solvents were purified by standard methods before
use. Dichloromethane was dried over CaH2 and distilled.
Chloroform was filtered through neutral active alumina.
9,10-dimethylanthracene (DMA) was obtained from
Aldrich and used without further purification. Methylene
Blue was used as received (Riedel-de Haën). Pyrrole was
distilled before used. For column chromatography, silica
gel 60–220 mesh/0.035–0.070 mm, from Fluka was used.
Photofrin was kindly offered by the Gastroenterology
ž
SPP full member in good standing
*Correspondence to: António M. d’A. Rocha Gonsalves, email:
arg@qui.uc.pt, tel: +351 239-854-479, fax: +351 239-826-068
Copyright © 2010 World Scientific Publishing Company

SYNTHETIC PORPHYRINS BEARING β-PROPIONATE CHAINS AS PHOTOSENSITIZERS
439
Service of the Hospitais da Universidade de Coimbra.
for 15 min. Trifluoroacetic acid (50 µl, 0.65 mmol) was
added and the mixture was stirred under N2 for 15 min at
room temperature. A NaOH solution (25 mL, 0.1 M) was
carefully added and the mixture was extracted two times
with ethyl acetate. The extracts were washed with water,
dried over anhydrous sodium sulfate, and the solvent
evaporated. The excess of pyrrole was removed under
reduced pressure with slight heating to yield a dark oily
residue. This was purified by column chromatography
(silica gel; eluent: CH2Cl2) to give, after evaporation of
the solvent in vacuo, the desired product as a light brown
Absorption spectra were recorded with a Shimadzu
UV-2100 spectrophotometer. Fluorescence spectra were
measured with a Spex Fluorolog 3 spectrophotometer.
NMR spectra were recorded on a Bruker Avance III
400 MHz spectrometer. Mass spectra of EI were obtained
with GC-MSD HP 6890/5973 and Finnigan Advantage
for the MS (ESI) spectra. High resolution mass spectra
were recorded on a Bruker FTMS APEXIII instrument
under electrospray ionization (ESI) (Vigo University).
1
oil that solidified upon standing (1.19 g, 73% yield). H
Porphyrin synthesis
NMR (400 MHz; CDCl3; Me4Si): δH, ppm 7.89 (2H, br
s, 2 × NH), 7.42 (2H, d, J = 8.2, PhBr), 7.07 (2H, d, J =
8.2, PhBr), 6.68 (2H, s), 6.15 (2H, d, J = 2.8), 5.88 (2H,
s), 5.41 (1H, s).
4-acetyl-5-oxo-hexanoate (1). Obtained as described
1
[10] as a colorless oil (50.2 g, 55% yield). H NMR
(400 MHz; CDCl3; Me4Si; enol-ketone tautomers): δH,
ppm ketone: 8.55 (1H, t, J = 6.9, COCH), 3.68 (3H, s,
CO2CH3), 2.32 (2H, t, J = 7.3, CH2CH2CO2), 2.21 (6H, s,
2 × CH3), 2.13–2.15 (2H, m, CH2CH2CO2), enol: 3.69
(3H, s, CO2CH3), 2.59–2.62 (2H, m, CH2CH2CO2),
2.39–2.43 (2H, m, CH2CH2CO2), 2.17 (6H, s, 2 × CH3).
GC-MS (EI): m/z 186, calcd. for [M]+ 186.
5-(2-bromophenyl)dipyrrylmethane (7b). The
dipyrrylmethane 7b was prepared according to the pro-
cedure described above for 7a using 2-bromobenzalde-
1
hyde (94% yield). H NMR (400 MHz; CDCl3; Me4Si):
δH, ppm 7.97 (2H, br s, 2 × NH), 7.56 (1H, d, J = 7.6,
PhBr), 7.23 (1H, d, J = 7.2, PhBr), 7.09–7.12 (2H, m,
PhBr), 6.70 (2H, s), 6.15 (2H, d, J = 2.8), 5.89 (2H, s),
5.88 (1H, s).
Benzyl 4-(2-methoxycarbonylethyl)-3,5-dimethyl-
pyrrole-2-carboxylate (3). Obtained as described [10]
1
as colorless needles (44.2 g, 52% yield). H NMR
5-phenyldipyrrylmethane (7c). The dipyrrylmethane
7c was prepared according to the procedure described
(400 MHz; CDCl3; Me4Si): δH, ppm 8.55 (1H, br s, NH),
7.32–7.46 (5H, m, Ph), 5.28 (2H, s, CH2Ph), 3.66 (3H, s,
CO2CH3), 2.70 (2H, t, J = 7.7, CH2CH2CO2), 2.42 (2H,
t, J = 7.7, CH2CH2CO2), 2.29 (3H, s, CH3), 2.20 (3H, s,
CH3).
1
above for 7a using benzaldehyde (54% yield). H NMR
(400 MHz; CDCl3; Me4Si): δH, ppm 7.88 (2H, br s, 2 ×
NH), 7.17–7.32 (5H, m, Ph), 6.67 (2H, s), 6.14 (2H, d,
J = 6.1), 5.90 (2H, s), 5.45 (1H, s).
Benzyl
5-acetoxymethyl-4-(2-methoxycarbonyl-
5-(4-bromophenyl)-1,9-diformyldipyrrylmethane
(8a) [14]. The Vilsmeier reagent used in the formyla-
tion was prepared by adding POCl3 (3.0 mL, 32 mmol)
dropwise under N2 to N,N-dimethylformamide (20 mL)
at 0 °C. Dipyrrylmethane 7a (1.35 g, 4.5 mmol) was
dissolved in N,N-dimethylformamide (15 mL) and the
solution was cooled to 0 °C. To this stirred solution was
added the Vilsmeier reagent dropwise (7 mL, 2.4 equiv.)
and the mixture was stirred for 1.5 h at 0 °C under N2.
Saturated aqueous sodium acetate (50 mL) was carefully
added and the mixture was stirred for a further 4 h at room
temperature. The solution was extracted three times with
ethyl acetate and the extracts were washed with brine and
water, dried over anhydrous sodium sulfate and the sol-
vent evaporated in vacuo to yield a brown oil. This was
purified by column chromatography (silica gel; eluent:
CH2Cl2/10% ethyl acetate) to give, after evaporation of
the solvent in vacuo, the desired product as a brown solid
(0.60 g, 47% yield). 1H NMR (400 MHz; CDCl3; Me4Si):
δH, ppm 10.20 (2H, br s, 2 × NH), 9.27 (2H, s, 2 × CHO),
7.48 (2H, d, J = 8.3, PhBr), 7.14 (2H, d, J = 8.3, PhBr),
6.88 (2H, s), 6.05 (2H, s), 5.52 (1H, s); GC-MS (EI): m/z
358. Calcd. for [M+] 358.
ethyl)-3-methylpyrrole-2-carboxylate (4). Obtained as
described [11] (4.7 g, 88% yield). H NMR (400 MHz;
1
CDCl3; Me4Si): δH, ppm 9.07 (1H, br s, NH), 7.34–7.40
(5H, m, Ph), 5.30 (2H, s, CH2Ph), 5.05 (2H, s, CH2OAc),
3.65 (3H, s, CO2CH3), 2.78 (2H, t, J = 7.7, CH2CH2CO2),
2.46 (2H, t, J = 7.7, CH2CH2CO2), 2.29 (3H, s, CH3), 2.06
(3H, s, COCH3).
Dibenzyl 3,7-bis(2-methoxycarbonylethyl)-2,8-di-
methyldipyrrylmethane-1,9-dicarboxylate (5). Obtained
as described [12] as a solid (1.82 g, 74% yield). 1H NMR
(400 MHz; CDCl3; Me4Si): δH, ppm 9.05 (2H, br s, 2 ×
NH), 7.30–7.39 (10H, m, Ph), 5.25 (4H, s, CH2Ph), 3.97
(2H, s,), 3.57 (6H, s, 2 × CO2CH3), 2.76 (4H, t, J = 6.9,
2 × CH2CH2CO2), 2.51 (4H, t, J = 6.9, 2 × CH2CH2CO2),
2.28 (6H, s, 2 × CH3).
3,7-bis(2-methoxycarbonylethyl)-2,8-dimethyl-
dipyrrylmethane-1,9-dicarboxylic acid (6) [12].
Obtained from hydrogenolysis of the dipyrrylmethane 5
1
[12] (1.12 g, 87% yield). H NMR (400 MHz; CDCl3/
(CD3)2SO; Me4Si): δH, ppm 10.49 (2H, br s, 2 × NH), 3.87
(2H, s,), 3.67 (6H, s, 2 × CO2CH3), 2.76 (4H, t, J = 7.7,
2 × CH2CH2CO2), 2.40 (4H, t, J = 7.7, 2 × CH2CH2CO2),
2.25 (6H, s, 2 × CH3).
5-(4-bromophenyl)dipyrrylmethane (7a) [13]. A
mixture of 4-bromobenzaldehyde (1.0 g, 5.40 mmol)
and pyrrole (10 mL, 0.15 mol) was bubbled with dry N2
5-(2-bromophenyl)-1,9-diformyldipyrrylmethane
(8b). The dipyrrylmethane 8b was prepared according
to the procedure described above for 8a (18% yield).
1H NMR (400 MHz; CDCl3; Me4Si): δH, ppm 9.82 (2H,
Copyright © 2010 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2010; 14: 439–445

440
N. PEREIRA ET AL.
br s, 2 × NH), 9.30 (2H, s, 2 × CHO), 7.62 (1H, d, J =
7.9, PhBr), 7.28–7.32 (1H, m, PhBr), 7.17–7.21 (2H, m,
PhBr), 6.88 (2H, s), 6.05 (1H, s) 6.04 (2H, s). GC-MS
(EI): m/z 356, calcd. for [M]+ 356.
CH2CH2CO2), -3.40 (2H, br s, 2 × NH). MS (ESI): m/z
667.27 [M + H]+. Anal. calcd. for C36H33BrN4O4·1/2H2O:
C, 64.10; H, 5.08; N, 8.31. Found C, 63.89; H, 4.98; N,
8.35. UV-vis (CH2Cl2): λmax, nm (ε %) 404 (100), 499
(10), 530 (4), 569 (5), 622 (2).
1,9-diformyl-5-phenyldipyrrylmethane (8c). The
dipyrrylmethane 8c was prepared according to the pro-
5-(2-bromophenyl)-13,17-bis(2-methoxycarbonyl-
ethyl)-12,18-dimethyl-porphyrin (11b). Porphyrin 11b
was prepared by reacting dipyrrylmethane dicarboxylic
acid 6 (1.1 mmol) with diformyldipyrrylmethane 8b
(1.3 mmol) by following the procedure used for porphy-
1
cedure described above for 8a (46% yield). H NMR
(400 MHz; CDCl3; Me4Si): δH, ppm 10.07 (2H, br s, 2 ×
NH), 9.25 (2H, s, 2 × CHO), 7.23–7.66 (5H, m, Ph), 6.87
(2H, s), 6.08 (2H, s), 5.57 (1H, s). GC-MS (EI): m/z 278,
calcd. for [M]+ 278.
1
rin 9 (0.044 g, 6% yield). H NMR (400 MHz; CDCl3;
5,15-di(4-bromophenyl)-3,7,13,17-tetra(2-methoxy-
Me4Si): δH, ppm 10.18 (2H, s, meso), 10.09 (1H, s,
meso), 9.32 (2H, d, J = 4.6, beta), 8.85 (2H, d, J = 4.6,
beta), 8.19 (1H, dd, J3 = 6.9, J4 = 2.2, PhBr), 8.04 (1H, dd,
J3 = 7.3, J4 = 1.8, PhBr), 7.66–7.75 (2H, m, PhBr), 4.43
(4H, t, J = 7.6, 2 × CH2CH2CO2), 3.65 (12H, s, 4 × CH3),
3.31 (4H, t, J = 7.7, 2 × CH2CH2CO2), -3.40 (2H, br s,
2 × NH). MS (ESI): m/z 667.33 [M + H]+. Anal. calcd. for
C36H33BrN4O4·3H2O: C, 60.09; H, 5.46; N, 7.79. Found
C, 60.79; H, 5.01; N, 7.31. UV-vis (CH2Cl2): λmax, nm
(ε %) 403 (100), 499 (9), 533 (5), 569 (6), 622 (4).
carbonylethyl)-2,8,12,18-tetramethylporphyrin
(9)
[15]. A mixture of 4-bromobenzaldehyde (0. 320 g, 1.7
mmol) and dipyrrylmethane dicarboxylic acid 6 (0.500
g, 1.2 mmol) in dichloromethane (90 mL) was bubbled
with dry N2 for 15 min. A solution of p-toluenesulfonic
acid (1 g) in methanol (17 mL) was added to the mix-
ture and stirred under N2 for 24 h at room temperature.
A solution of zinc acetate (1 g) in methanol (15 mL) was
added to the mixture, bubbled with air and stirred for a
further 24 h under air at room temperature. The solution
was washed with water and saturated aqueous sodium
bicarbonate, dried over anhydrous sodium sulfate and the
solvent evaporated in vacuo. The residue was chromato-
graphed over silica gel (eluent: dichloromethane/10%
ethyl acetate). To the fractions with the zinc porphyri-
nate dissolved in dichloromethane (30 mL) was added
trifluoroacetic acid (1 mL) and the mixture stirred for
30 min. The solution was washed with water and satu-
rated aqueous sodium bicarbonate, dried over anhydrous
sodium sulfate and the solvent evaporated. The resulting
Zn-free porphyrin was chromatographed over a short
alumina column eluted with dichloromethane and the
solvent evaporated. The porphyrin was recrystallized
from dichloromethane/methanol, isolated and dried in
vacuo (0.24 g, 40% yield.). 1H NMR (400 MHz; CDCl3;
Me4Si): δH, ppm 10.29 (2H, s, meso), 7.94 (4H, d, J =
8.5, PhBr), 7.90 (4H, d, J = 8.0, PhBr), 4.36 (8H, t, J =
7.8, 4 × CH2CH2CO2), 3.66 (12H, s, 4 × CO2CH3), 3.16
(8H, t, J = 7.8, 4 × CH2CH2CO2), 2.54 (12H, s, 4 × CH3),
-2.47 (2H, br s, 2 × NH). MS (ESI): m/z 1021.27 [M +
H]+. Anal. calcd. for C52H52Br2N4O8·6H2O: C, 55.32; H,
5.71; N, 4.96. Found: C, 54.75; H, 4.07; N, 3.91. UV-vis
(CH2Cl2): λmax, nm (ε %) 408 (100), 505 (15), 538 (8),
573 (8), 624 (5).
13,17-bis(2-methoxycarbonylethyl)-12,18-dimethyl-
5-phenylporphyrin (11c). Porphyrin 11c was prepared by
reacting dipyrrylmethane dicarboxylic acid 6 (1.1 mmol)
with diformyldipyrrylmethane 8c (1.3 mmol) by follow-
ing the procedure used for porphyrin 9 (0.018 g, 28%
1
yield). H NMR (400 MHz; CDCl3; Me4Si): δH, ppm
10.18 (2H, s, meso), 10.05 (1H, s, meso), 9.32 (2H, d,
J = 4.6, beta), 9.02 (2H, d, J = 4.6, beta), 8.24–8.27
(2H, m, Ph), 7.78–7.80 (3H, m, Ph), 4.42 (4H, t, J = 7.7,
2 × CH2CH2CO2), 3.66 (6H, s, 2 × CO2CH3), 3.65 (6H,
s, 2 × CH3), 3.31 (4H, t, J = 7.7, 2 × CH2CH2CO2), -3.39
(2H, br s, NH). MS (ESI): m/z 587.40 [M + H]+. Anal.
calcd. for C36H34N4O4: C, 73.70; H, 5.84; N, 9.55. Found
C, 73.23; H, 5.87; N 9.53. UV-vis (CH2Cl2): λmax, nm
(ε %) 401 (100), 498 (10), 530 (4), 569 (5), 621 (2).
5,15-di(4-bromophenyl)-3,7,13,17-tetra(2-hy-
droxycarbonylethyl)-2,8,12,18-tetramethylporphyrin
(10). To porphyrin 9 (50 mg) dissolved in tetrahydrofuran
(30 mL) a solution of potassium hydroxide (250 mg) in
water (1 mL) was added and the mixture was stirred over-
night under N2 at room temperature. The potassium salt
of the porphyrin precipitated, was filtered off, washed
with tetrahydrofuran and dissolved in water. The car-
boxylic acid porphyrin was precipitated by neutralization
of the aqueous solution with acetic acid. The porphyrin
was collected by filtration, washed well with water and
dried in vacuo (0.036 g, 76% yield). 1H NMR (400 MHz;
CDCl3/TFA; Me4Si): δH, ppm 10.55 (2H, s, meso), 8.07
(8H, mL, Ph), 4.06 (8H, t, J = 6.2, 4 × CH2CH2CO2),
2.90 (8H, t, J = 6.2, 4 × CH2CH2CO2), 2.31 (12H, s,
4 × Me(β)), -2.13 (4H, s, 2 × NH). MS (ESI): m/z 965.27
[M + H]+. HRMS (FAB): m/z calcd. for C48H45Br2N4O8
[M + H]+, 963.15987; found 963.16013; ∆ = -0.26 mmu
[M + H]+.
5-(4-bromophenyl)-13,17-bis(2-methoxycarbonyl-
ethyl)-12,18-dimethyl-porphyrin (11a). Porphyrin 11a
was prepared by reacting dipyrrylmethane dicarboxylic
acid 6 (1.1 mmol) with diformyldipyrrylmethane 8a (1.3
mmol) by following the procedure used for porphyrin 9
(0.081 g, 11% yield). 1H NMR (400 MHz; CDCl3; Me4Si):
δH, ppm 10.66 (1H, s, meso), 10.59 (2H, s, meso), 9.33
(2H, d, J = 4.7, beta), 8.86 (2H, d, J = 4.7, beta), 8.33
(2H, d, J = 7.3, PhBr), 8.12 (2H, d, J = 8.2, PhBr), 4.41
(4H, t, J = 7.4, 2 × CH2CH2CO2), 3.65 (6H, s, 2 × CO2
CH3), 3.58 (6H, s, 2 × CH3), 3.20 (4H, t, J = 7.4, 2 ×
5-(4-bromophenyl)-13,17-bis(2-hydroxycarbonyl-
ethyl)-12,18-dimethyl-porphyrin (12a). Porphyrin 12a
Copyright © 2010 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2010; 14: 440–445

SYNTHETIC PORPHYRINS BEARING β-PROPIONATE CHAINS AS PHOTOSENSITIZERS
441
was prepared by hydrolysis of porphyrin 11a using the
procedure followed for porphyrin 10 (90% yield). H
ternary mixture of H2O:PEG400:EtOH (50/30/20, v/v/v),
the desired concentrations being achieved by successive
dilutions. The sensitizers were administered in several
concentrations and cells were incubated for 24 hours.
Cells were washed with PBS and new drug-free medium
was added. Each plate was irradiated with a fluence rate
of 7.5 mW/cm2 until a total of 10 J was reached. Cell
viability was measured 24 hours after the photodynamic
treatment. Dark controls were made as described above
except that no irradiation were made.
1
NMR (400 MHz; CDCl3/TFA; Me4Si): δH, ppm 10.80
(1H, s, meso), 10.75 (2H, s, meso), 9.46 (2H, d, J = 4.2,
beta), 8.98 (2H, d, J = 4.2, beta), 8.34 (2H, d, J = 7.8,
PhBr), 8.19 (2H, d, J = 7.7, PhBr), 4.46 (4H, t, J = 5.8,
2 × CH2CH2CO2), 3.69 (6H, s, 2 × CH3), 3.27 (4H, t, J =
5.8, 2 × CH2CH2CO2). MS (ESI): m/z 639.33 [M + H]+.
HRMS (FAB): m/z calcd. for C34H30BrN4O4 [M + H]+,
637.14449; found 637.14530; ∆ = -0.81 mmu [M + H]+.
5-(2-bromophenyl)-13,17-bis(2-hydroxycarbonyl-
ethyl)-12,18-dimethyl-porphyrin (12b). Porphyrin 12b
was prepared by hydrolysis of porphyrin 11b using the
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-Aldrich, Inc; Sigma M2128) to measure cell pro-
liferation. Cytotoxicity was expressed as the percentage
of inhibition of cell proliferation correlated with irradi-
ated cells with only the ternary solvent mixture. This
allows the calculation of the concentration that inhibits
the culture cell proliferation in 50% (IC50). Each experi-
ment was performed in duplicate or triplicate and repeated
in three sets of tests.
1
procedure followed for porphyrin 10 (87% yield). H
NMR (400 MHz; CDCl3/TFA; Me4Si): δH, ppm 10.86
(1H, s, meso), 10.71 (2H, s, meso), 9.49 (2H, d, J =
4.7, beta), 8.96 (2H, d, J = 4.7, beta), 8.19–8.24 (2H,
m, Ph), 7.89–7.93 (2H, m, Ph), 4.49 (4H, t, J = 6.7, 2
× CH2CH2CO2), 3.68 (6H, s, 2 × CH3), 3.22 (4H, t, J =
6.7, 2 × CH2CH2CO2). MS (ESI): m/z 639.33 [M + H]+.
HRMS (FAB): m/z calcd. for C34H30BrN4O4 [M + H]+,
637.14449; found 637.14274; ∆ = +1.75 mmu [M + H]+.
13,17-bis(2-hydroxycarbonylethyl)-12,18-dimethyl-
5-phenylporphyrin (12c). Porphyrin 12c was prepared
by hydrolysis of porphyrin 11c using the procedure fol-
lowed for porphyrin 10 (73% yield). 1H NMR (400 MHz;
CDCl3/TFA; Me4Si): δH, ppm 10.90 (1H, s, meso), 10.71
(2H, s, meso), 9.42 (2H, d, J = 4.6, beta), 8.98 (2H, d,
J = 4.6, beta), 8.47 (2H, s, Ph), 8.03 (3H, s, Ph), 4.43
(4H, t, J = 5.8, 2 × CH2CH2CO2), 3.68 (6H, s, 2 × CH3),
3.33 (4H, t, J = 5.8, 2 × CH2CH2CO2), -2.00 (2H, br s,
NH). MS (ESI): m/z 559.53 [M + H]+. UV-vis (CH3OH):
Cellular uptake
Cells, 1 × 105, were incubated with solutions of the
sensitizer of 5 µM, 1 µM and 0.5 µM concentrations for
24 hours, released by trypsinization and washed with cold
PBS. To ensure full disaggregation and cell rupture, 5 mL
of methanol was added and the resulting suspension left
in the dark for 24 hours. The fluorescence intensity of the
supernatant was determined by fluorescence spectroscopy
with a SPEX fluoromax 322-2 spectrophotometer using
410 nm excitation wavelength. The intracellular concen-
tration was determined using a calibration curve obtained
from the fluorescence intensity in methanol solutions for
each sensitizer.
λ
max, nm (ε %) 398 (100), 497 (12), 527 (6), 567 (7), 618
(4). HRMS (FAB): m/z calcd. for C34H31N4O4 [M + H]+,
559.23398; found 559.23219; ∆ = +1.79 mmu [M + H]+.
Cell culture conditions
The human colon carcinoma cell line WiDR was pur-
chased from American Type Culture Collection. The
cell lines were cultured with Dulbecco’s Modified Eagle
medium (Sigma-Aldrich, Inc; Sigma D-5648) supple-
mented with 10% heat-inactivated fetal bovine serum
(Gibco Invitrogen Life Technologies; Gibco 2010-04),
1% Penicillin-Streptomycin (Gibco Invitrogen Life
Technologies; 100 U/mL penicillin and 10 µg/mL strep-
tomycin – Gibco 15140-122) and 100 µM Sodium Piru-
vate (Gibco Invitrogen Life Technologies; Gibco 1360)
at 37 °C, in a humidified incubator with 95% air and
5% CO2.
RESULTS AND DISCUSSION
Synthesis of porphyrins
The synthesis of porphyrins having four β-propionic
acid chains 10 and two β-propionic acid chains 12a–c is
outlined in Scheme 1.
The synthesis starts with the preparation of the pro-
pionate substituted pyrrolic precursors. For the prepara-
tion of the methyl 4-acetyl-5-oxohexanoate (1) we used
a Michael addition of acetylacetone to methyl acrylate,
catalyzed by sodium ethoxide [10]. The oxime 2 was pre-
pared from the addition of a solution of sodium nitrite
to benzyl acetoacetate. The tetrasubstituted pyrrole 3
was synthesized from the reaction of dione 1 with ben-
zyl oxyiminoacetate (2) in the presence of zinc dust and
sodium acetate. Reduction of the compound 2 to the
Photodynamic treatment
For each experiment, cells were plated in 48 multi-
wells (Corning Costar Corp), in a concentration of 40,000
cells/mL and kept in the incubator overnight, in order to
allow the attachment of the cells. The formulation of
these sensitizers consisted of a 1 mg/mL solution in a
Copyright © 2010 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2010; 14: 441–445

442
N. PEREIRA ET AL.
NaNO2
AcOH
T<10ºC
Na/EtOH
80ºC
O
O
O
O
O
O
O O
55%
OBn
+
OBn
+
NOH
1
MeO2C
CO2Me
2
Zn/AcOH
65ºC
52%
CHO
R1
CO2Me
Pb(CH3CH2)4
88%
CO2Me
OAc
+
N
H
R2
BnO2C
N
BnO2C
N
TFA
H
H
HCl/MeOH
3
70ºC
74%
4
CO2Me
MeO2C
7a R1=H, R2=Br; 73%
7b R1=Br, R2=H; 94%
7c R1=H, R2=H; 54%
N
R1
N
H
H
HN
NH
CO2Bn
CO2Me
BnO2C
R2
5
H2/Pd
87%
POCl3
DMF
MeO2C
+
CHO
OHC
HN
NH
N
R1
N
H
H
CO2H
HO2C
1) TsOH
8a R1=H, R2=Br; 47%
8b R1=Br, R2=H; 18%
8c R1=H, R2=H; 46%
6
1) TsOH
CHO
CH2Cl2; N2
2) Zn(AcO)/
MeOH; air
3)TFA
CH2Cl2; N2
2) Zn(AcO)/
MeOH; air
3)TFA
R2
Br
R2
40%
R1
CO2Me
MeO2C
NH
N
N
11a R1=H, R2=Br; 11%
11b R1=Br, R2=H; 6%
11c R1=H, R2=H; 28%
NH
N
N
HN
Br
Br
HN
MeO2C
CO2Me
9
MeO2C
CO2Me
CO2H
KOH
THF
KOH
THF
76%
R2
HO2C
R1
12a R1=H, R2=Br; 90%
12b R1=Br, R2=H; 87%
12c R1=H, R2=H; 73%
NH
NH N
HN
N
Br
Br
N
N
HN
HO2C
CO2H
HO2C
CO2H
10
Scheme 1. Synthetic routes to porphyrins 10 and 12a–c
respective amine, followed by the condensation in situ
with 1 gave the intended pyrrole 3 in 52% [10]. Pyrrole
3 was α-acetoxylated with lead tetraacetate originating 4
[11] and underwent a self-condensation process in acid
medium to give the dipyrromethane 5 in 77% yield [12].
Hydrogenolysis of the benzyl group with 10% palladium
on charcoal at room temperature and 1 atm of hydrogen
originated the diacid dipyrrylmethane 6. Condensation
of the dipyrrylmethane 6 with 4-bromobenzaldehyde in
the presence of p-toluenesulfonic acid using zinc acetate
Copyright © 2010 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2010; 14: 442–445

SYNTHETIC PORPHYRINS BEARING β-PROPIONATE CHAINS AS PHOTOSENSITIZERS
443
template originated the tetrasubstituted propionate por-
phyrin which was demetalated with TFA and hydrolyzed
to give the tetraacid derivative 10 [15, 16].
For the synthesis of porphyrins 12a–c a different
approach was required. Substituted dipyrrylmethanes
7a–c were obtained by reaction of the substituted benzal-
dehyde with an excess of pyrrole and a catalytic amount
of trifluoroacetic acid [13]. These dipyrrylmethanes were
diformylated to dipyrrylmethanes 8a–c by treatment with
Vilsmeyer reagent (POCl3/dimethylformamide) [14].
Condensation of the dipyrrylmethane 6 with dipyrryl-
methanes 8a–c catalyzed by p-toluenesulfonic acid in the
presence of zinc acetate [15] gaves, after demetalation,
porphyrins 11a–c. In order to increase water solubility of
the products for the photodynamic studies, the ester func-
tion was hydrolysed with KOH to the carboxyl deriva-
tives porphyrins 12(a,b,c).
Fig. 1. Absorption, fluorescence and fluorescence excitation
normalized spectra of sensitizer 12c
Evaluation of the photodynamic activity of porphyrins
A complete spectroscopic characterization of sensitiz-
ers 10 and 12a–c by UV-vis spectroscopy and fluores-
cence was carried out (Table 1).
The activity of the synthesized porphyrins as pho-
tosensitizers in photodynamic therapy was evaluated
against WiDr colon adenocarcinoma cells. For the pho-
todynamic experiments, cancer cells were plated in 48
multiwell plates (Corning Costar Corp), in a concentra-
tion of 40,000 cells/mL (well) and kept in the incubator
overnight, in order to allow the attachment of the cells.
The sensitizers 10, 12a–c were administered to cells
dissolved in a ternary mixture of H2O/PEG400/ethanol
(50:30:20, v/v/v) in several concentrations and incubated
for 24 h. In the control experiments cells were incubated
only with the ternary solvent mixture. Subsequently,
cells were washed with PBS and new drug-free medium
was added. Each plate was then irradiated with a fluence
rate of 7.5 mW/cm2 until a total of 10 J of energy was
reached. Cell viability was evaluated after 24 h using the
MTT test. Figure 2 shows the concentration-dependent
cell survival curves for 10, 12a–c and for Photofrin® as
reference compound.
All sensitizers present absorption spectra of the phyllo
type [17]. The absorption coefficients were calculated
using the Beer-Lambert law from methanolic solutions
with concentrations between 10-5-10-7 M. No aggregation
was observed under these conditions. The Q(0,0) band
around 625 nm (into the therapeutic window) has char-
acteristic absorption coefficients ca. 103 M-1.cm-1, values
very similar to Photofrin® (λ = 630 nm, ε = 1170 M-1.
cm-1) [2]. The fluorescence spectra of the new sensitizers
are very similar to the Photofrin spectrum [18], show-
ing two bands of similar relative intensity at ca. 625 and
700 nm corresponding to Q(0-0) and Q(0-1) emissions
respectively. The last fluorescence band is much more
intense than the corresponding band in Photofrin® or
meso-phenylporphyrins, indicating a high contribution
of the emission to the first vibrational level. The fluo-
rescence excitation spectra at the maxima of the fluores-
cence emission bands fit well with the absorption spectra
of each sensitizer. In Fig. 1, the normalized spectra of
absorption, fluorescence and fluorescence excitation of
porphyrin 12c are shown.
The photodynamic activity of this series of porphy-
rins is relatively lower than that observed for our tetra-
hydroxyphenyl porphyrins for the same cancer cell type
under the same conditions [9]. Clearly, from Fig. 2, the
Table 1. Absorption and fluorescence data of porphyrins 10 and 12a–c in methanolic solutions
Compound
Absorption λmax, nm (ε, M-1.cm-1)
Fluorescence λmax, nm
B(0-0)
Qy(1-0)
Qx(0-0)
Qy(0-0)
Qx(1-0)
Q(0-0)
630
Q(0-1)
694
10
406.5
2.85 × 105
399.0
507.4
2.16 × 104
498.8
541.0
8.76 × 103
529.0
575.0
1.05 × 104
568.0
629.0
3.57 × 103
627.2
12a
12b
12c
627
627
627
688
691
688
1.68 × 105
1.05 × 104
7.80 × 103
8.40 × 103
1.26 × 103
400.5
497.3
535.6
569.8
623.2
8.23 × 104
399.2
5.37 × 103
498.4
3.33 × 103
528.8
2.71 × 103
620.5
8.27 × 102
627.2
9.50 × 104
7.19 × 103
2.58 × 103
3.57 × 103
1.26 × 103
Copyright © 2010 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2010; 14: 443–445

444
N. PEREIRA ET AL.
between hydrophilic and hydrophobic regions. The por-
phyrin with two propionate chains may correspond to
the best balance of these two factors and can also have a
better distribution inside cells for photodynamic action
[8, 20].
In summary, a series of porphyrins with propionate
chains at the β-positions have been synthesized and their
in vitro photocytotoxicity evaluated on human colon
tumor WiDr cells. Photosensitizer 10 with four propi-
onate chains showed lower cellular uptake and much
lower activity than photosensitizer 12c with only two
propionic chains. Photodynamic activity of these sensi-
tizers depends on the number of propionic acid chains
and the kind of substitution. Brominated porphyrins 12a
and 12b and Photofrin® demonstrated similar photocy-
totoxicities. Non-halogenated porphyrin 12c showed the
highest photocytotoxicity on cells.
Fig. 2. Cell viability (%) for WiDr human colon adenocarci-
noma cells using photosensitizers 10, 12a–c and Photofrin®
porphyrins with two propionate groups are more active
than the porphyrin with four propionate groups (10).
Photofrin®, which is a mixture of oligomers of hemato-
porphyrin (two propionate chains) appears to behave like
monomeric porphyrins with two propionate groups. Sen-
sitizers 12a–c and Photofrin® present high photodynamic
activity for concentrations above 1 µM, with cellular
viabilities below 15%; dipropionate porphyrins present
higher activity than Photofrin®. The photodynamic activ-
ity order is 12c > 12a > 12b > Photofrin® > 10 with IC50
(sensitizer dose (µM) necessary to observe a 50% cel-
lular viability) of 0.14, 0.27, 0.34 and 0.46, respectively.
Among dipropionate porphyrins, non-halogenated por-
phyrin 12c is the most active sensitizer.
Acknowledgements
The authors thank Chymiotechnon, Ministério da
Economia/POE/Prime/Proj 3/293/CLARO, and Gastro-
enterology service of HUC for equipment facilities. Mass
spectra studies were carried out by Alexandra Rocha
Gonsalves-Chymiotechnon. NMR data was obtained
at the Nuclear Magnetic Resonance Laboratory of the
Coimbra Chemistry Centre (www.nmrccc.uc.pt), Univer-
sidade de Coimbra, supported in part by grant REEQ/481/
QUI/2006 from FCT, POCI-2010 and FEDER, Portugal.
REFERENCES
1. Dolmans DE, Fukumura D and Jain RK. Nature
Rev. Cancer 2003; 3: 380.
2. Serra A, Pineiro M, Pereira N, Gonsalves AMd’AR,
Laranjo M, Abrantes M and Botelho MF. Oncol.
Rev. 2008; 2: 235.
3. Nyman EA and Hynninen PA. J. Photochem.
Photobiol. B 2004; 73: 1.
4. Detty MR, Gibson SL and Wagner SJ. J. Med.
Chem. 2004; 47: 3897.
5. Bonneau S, Maman N and Brault D. Biochim.
Biophys. Acta 2004; 1661: 87.
6. Das K, Jain B, Dube A and Gupta PK. Chem. Phys.
Lett. 2005; 401: 185.
7. Mojzisova H, Bonneau S, Vever-Bizet C and Brault
D. Biochim. Biophys. Acta 2007; 1768: 366.
8. Boyle RW and Dolphin D. Photochem. Photobiol.
1996; 64: 469.
9. Serra A, Pineiro M, Pereira N, Gonsalves AMd’AR,
Laranjo M, Abrantes M, Santos AC and Botelho
MF. J. Photochem. Photobiol. B 2008; 92: 61.
10. Bullock E, Johnson AW, Markham E and Shaw KB.
J. Chem. Soc. 1958; 1430.
Activity of these compounds is related to the ability
to enter the cells and the presence of carboxylic acid
groups may have a decisive role [19]. Cellular uptake
was carried out for porphyrins 10 with four carboxylic
groups and 12c with two carboxylic groups. For the cel-
lular uptake measurements, 1 × 105 cells were incubated
with solutions of the sensitizer of 5 µM concentration for
24 hours, released by trypsinization and washed with cold
PBS. To ensure full disaggregation and cell rupture,
5 mL of methanol were added. The intracellular concen-
tration was determined from the fluorescence intensity
(λex = λmax of the Soret band ca. 400 nm) directly measured
from these solutions using a calibration curve obtained
from the fluorescence intensity in methanol for concen-
trations between 10-6 and 10-8 M. The results showed that
the cellular uptake of sensitizer 10 (0.06 0.02 µM) is
three times lower than the cellular uptake of sensitizer
12c (0.18 0.02 µM). Also, a report with meso-phenyl-
carboxyl groups showed the same behavior [19]. The
higher hydrophilicity (logP = -1.9) of porphyrin 10 rela-
tive to the value obtained for 12c (logP = 1.8) can explain
the more difficult penetration in cells and the less favor-
able results for porphyrin 10. The photodynamic activity
of photosensitizers of different structures has been related
to the amphilicity of the molecule, which is a balance
11. Cavaleiro JAS, Gonsalves AMd’AR, Kenner GW
and Smith KM. J. Chem. Soc., Perkin Trans. 1 1973;
2471.
Copyright © 2010 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2010; 14: 444–445

SYNTHETIC PORPHYRINS BEARING β-PROPIONATE CHAINS AS PHOTOSENSITIZERS
445
12. Cavaleiro JAS, Gonsalves AMd’AR, Kenner GW
16. Arsenault GP, Bullock E and MacDonald SF. J. Am.
Chem. Soc. 1960; 82: 4384.
17. Bonnett R. Chemical Aspects of Phtotodynamic
Therapy, Gordon and Breach Science Publishers:
2000; pp 149–156.
18. Finlay JC, Mitra S, Patterson MS and Foster TH.
Phys. Med. Biol. 2004; 49: 4837.
19. YouY, Gibson SL, Hilf Russell, Davies SR, Oseroff
AR, Roy I, Ohulchanskyy TY, Bergey EJ and Detty
MR. J. Med. Chem. 2003; 46: 3734.
and Smith KM. J. Chem. Soc., Perkin Trans. 1 1974;
1771.
13. Littler BJ, Miller MA, Hung CH, Wagner RW,
O’Shea DF, Boyle PD and Lindsey JS. J. Org.
Chem. 1999; 64: 1391.
14. Bruckner C, Posakony JJ, Johnson CK, Boyle RW,
James BR and Dolphin D. J. Porphyrins Phthalo-
cyanines 1998; 2: 455.
15. Pandey SK, Gryshuk AL, Graham A, Ohkubo
K, Fukuzumi S, Dobhal MP, Zheng G, Ou RZZ,
Kadish KM, Oseroff A, Ramaprasad S and Pandey
RK. Tetrahedron 2003; 59: 10059.
20. Osterloh J and Vicente MGH. J. Porphyrins
Phthalocyanines 2002; 6: 305.
Copyright © 2010 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2010; 14: 445–445

Copyright of the works in this Journal is vested with World Scientific Publishing. The
article is allowed for individual use only and may not be copied, further disseminated, or
hosted on any other third party website or repository without the copyright holder’s
written permission.