New porphyrin amino acid conjugates: Synthesis and photodynamic effect in human epithelial cells

Article abstract of DOI:10.1016/j.bmc.2010.06.030

The efficacy of new porphyrin amino acid conjugates as photosensitizers for photodynamic therapy (PDT) were assayed in vitro on tumoral (HeLa) and on non tumoral (HaCaT) human cell lines. The conjugates stable in liposomes are able to penetrate efficientl

Full text of DOI:10.1016/j.bmc.2010.06.030

Bioorganic & Medicinal Chemistry 18 (2010) 6170–6178
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New porphyrin amino acid conjugates: Synthesis and photodynamic effect
in human epithelial cells
V. Vaz Serra a, , A. Zamarrón b, , M. A. F. Faustino a, M. C. Iglesias-de la Cruz b, A. Blázquez b,
b
b,
*
J. M. M. Rodrigues a, M. G. P. M. S. Neves a, J. A. S. Cavaleiro a, , A. Juarranz , F. Sanz-Rodríguez
*
a QOPNA & Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
b Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, 28049 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The efficacy of new porphyrin amino acid conjugates as photosensitizers for photodynamic therapy (PDT)
were assayed in vitro on tumoral (HeLa) and on non tumoral (HaCaT) human cell lines. The conjugates
stable in liposomes are able to penetrate efficiently in the cytoplasm of cultured cancer and normal cells.
No dark cytotoxicity is observed at the same concentration used for PDT cell treatment and during long
incubation time (24 h). The cell survival after the PDT treatment with visible light is dependent upon light
exposure level and compound concentration. The tested compounds show higher photocytotoxicity in
tumoral HeLa cells than in no tumoral HaCaT cells. The results suggest that these amino acid porphyrin
conjugates are potential photosensitizers for PDT.
Received 8 April 2010
Revised 9 June 2010
Accepted 10 June 2010
Available online 17 June 2010
Keywords:
Porphyrins
Amino acids
Photodynamic therapy
HeLa cells
Ó 2010 Elsevier Ltd. All rights reserved.
HaCat cells
1. Introduction
PDT.8 The ideal molecule to be used as PS should exhibit a low level
of dark and systemic toxicity, good tumor selectivity avoiding
Photodynamic therapy (PDT) is an alternative treatment for
cancer and non-cancerous lesions. Malignant and abnormal cells
are destroyed by singlet oxygen and by reactive oxygen species
that are generated by a photosensitive drug (photosensitizer)
when light of appropriate wavelength is applied.1,2 PDT exhibits
several advantages over surgery, chemotherapy, and radiotherapy,
the standard modalities of tumor treatment. It is minimally inva-
sive, devoid of severe side effects, yields a good cosmetic result,
and often improves the patient’s quality of life.3
Porphyrins, due to their unique photophysical and photochem-
ical properties, have a special relevance in photodiagnosis (PD) and
in photodynamic therapy (PDT) of oncologycal and non oncology-
cal diseases. In the last decades, a large number of porphyrins and
porphyrin analogues (e.g., chlorins and phthalocyanines) have
been extensively exploited as photosensitizers (PS). Some com-
mercially available PS formulations that are currently in clinical
practice are based in this type of macrocycles.4–7 Porphyrin precur-
sors like 5-aminolevulinic acid (ALA) that generates endogenously
protoporphyrin IX, has been also widely used as a prodrug for
accumulation in the surrounding healthy tissues, and should be
rapidly eliminated from the organism to prevent prolonged photo-
sensitivity.7,9 With the aim to increase the biological effectiveness
of porphyrin as photosensitizers their conjugation to different mol-
ecules, like proteins,10–13 peptides,14–17 and sugars18 have been
investigated in recent years. In particular, some porphyrins cou-
pled with amino acids have also been reported to have interesting
characteristics for PDT. Some of these porphyrins are able to inter-
calate DNA sequences with high specificity19,20 and to enhance the
cellular uptake in tumors.11 They have good solubility in aqueous
solutions and also possible specific interactions with cell mem-
branes.21,22 Remarkable photocytotoxicity after visible light irradi-
ation is also reported for this kind of conjugates.23 Recent studies,
based on poly-S-lysine and meso-tetraarylporphyrins conjugates
reveal that they are efficient in the photoinactivation of antibiotic
resistant Gram-positive and Gram-negative bacteria. Other por-
phyrin analogues, like chlorin e6, coupled to poly-S-lysine are also
effective in the photoinactivation of these bacteria strains.25,26
Knowing that amino acid moieties improve the solubility in aque-
ous solutions and are able to stablish specific interactions with cell
membranes, porphyrins with amino acid moieties may be a good
candidates for PDT.
* Corresponding authors. Tel./fax: +34 914978243 (F.S.-R.); tel.: +351
234370717; fax: +351 234370084 (J.A.S.C.).
E-mail addresses: jcavaleiro@ua.pt (J.A.S. Cavaleiro), francisco.sanz@uam.es,
brig96@hotmail.com (F. Sanz-Rodríguez).
As part of a program related with the development of new com-
pounds with potentiality to be used in medicine24,27–29 we report
here the synthesis, structural and photophysical characterization
Both authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.030

V. V. Serra et al. / Bioorg. Med. Chem. 18 (2010) 6170–6178
6171
of a series of porphyrin derivatives conjugated with commercial
available amino acid residues. The photodynamic effect of the sta-
ble conjugates was evaluated on tumoral (HeLa) and no tumoral
(HaCaT) human cells in culture. The dark toxicity, phototoxicity,
intracellular localization of the PSs, and also the changes in
morphology of cells induced by the photodynamic treatment will
be presented and discussed.
to the amino acid moiety is summarized in Scheme 1. In our first
synthetic approach, the activation of the carboxylic function was
performed with thionyl chloride in dry pyridine, yielding the corre-
sponding porphyrinic acyl chloride. Due to the poor solubility of
the amino acid moiety, its direct addition to the porphyrinic acyl
chloride solution did not afforded the desired product. Further
addition of N-hydroxysuccinimide at 50 °C afforded the activated
ester 2 (Scheme 1). After the work up, porphyrin 2 was obtained
in 97% yield. The porphyrin amino acid conjugates were prepared
by reacting each amino acid with porphyrin 2, in the presence of
an excess of K2CO3 (Scheme 1). After stirring over night at 40 °C
in DMSO, washing with water, drying and rapid column chroma-
tography on silica gel, the new products were obtained from mod-
erate (51% for derivative 5) to excellent yields (>90% for porphyrins
3, 4, and 6). The structures of all the new compounds were con-
firmed by NMR spectroscopy (1H, 13C, COSY, HSQC, HMBC), ele-
mental analysis, MS and UV–vis techniques. An extensive study
on the characterization of these porphyrins by electrospray ioniza-
tion tandem mass spectrometry (ESI-MS/MS) was already reported
by our group.31
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of porphyrin amino acid conjugates
Based on the knowledge that porphyrins with meta-methoxy-
phenyl groups in meso positions show high cellular uptake28,29
we have chosen porphyrin 1 as the porphyrin moiety to be used
in the preparation of the porphyrin amino acid conjugates 3–6
(Scheme 1). The presence of the carboxyl group on one of the phe-
nyl substituents appears to us, an easy option to connect porphyrin
1 through an amide linkage to simple and commercially available
amino acids.
Porphyrin 1, with 3-methoxyphenyl and 4-carboxyphenyl meso-
substituents was synthesized by condensation of pyrrole with
3-methoxybenzaldehyde and 4-formylbenzoic acid (3:1) in a
refluxing mixture of acetic acid and nitrobenzene, as generally
described in the Rothemund modified procedure.24,30 This reaction
afforded porphyrin 1 after purification by silica gel chromatogra-
phy. The synthetic strategy used for the coupling of porphyrin 1
The 1H NMR spectra confirms that the obtained derivatives are
meso-substituted porphyrin derivatives, showing the resonances
of all the eight b-pyrrolic protons as multiplets (d 8.77–8.91). The
resonances of all the protons of the meso positions also appear at
characteristic chemical shifts. The para substitution of one of the
porphyrinic phenyl rings is also in accordance with the 1H NMR
assignments: (i) the resonances of the meta and ortho protons
appears as doublets at d 8.20–8.25 and d 8.28–8.32 (except for
Scheme 1. Reagents and conditions: (i) SOCl2, dry py, rt; (ii) N-hydroxysuccinimide, 50 °C; (iii) NH2CHR1CO2R2, DMSO, K2CO3, 40 °C.

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V. V. Serra et al. / Bioorg. Med. Chem. 18 (2010) 6170–6178
compound 5 where these resonances appears as a multiplet); (ii) it
can be distinguished the typical resonances of only three protons
of the para positions. The linkage of the amino acid residue through
an amide linkage is also confirmed by the resonance of the proton
of the amide group as a triplet at d 6.98 for compound 3. For
compounds 4, 5, and 6 the resonance of this proton appears as
duplets at d 7.44, 8.37, and 7.88, respectively. Along with the
several common resonance signals that reflect their common struc-
tural features, distinct resonance signs depending on the chemical
structure of the amino acid moiety can also be distinguished. In
particular, the resonance of the proton of the hydroxyl group for
compounds 4 and 5. The COSY spectrum of compound 4 allowed
us to identify that resonance to the multiplet at d 2.58–2.62 due to
the existence of only one correlation sign with the multiplet at d
4.22–4.25 assigned as the resonance of the CH2. For compound 5,
the HSQC spectrum allowed the unequivocal identification of the
signals of the aromatic carbons of the amino acid residue at d
130.3 for C-600 and C-1000 and at d 114.8 for C-700 and C-900. The HMBC
spectrum allowed us to identify the resonance of the proton of the
hydroxyl group at d 9.19 due to the long distance coupling (3J)
between this proton and C-700 and C-900.
2.1.3. Singlet oxygen production measurement
Today, it is well accepted that singlet oxygen plays a key role in
cell death induced by PDT. Considering the potential application of
the porphyrin amino acid conjugates 3–6 in PDT, we evaluated
their ability to generate singlet oxygen. The ability of these por-
phyrin derivatives to generate singlet oxygen, the basis of the pho-
toinactivation process, was qualitatively evaluated by monitoring
the photodecomposition of 1,3-diphenylisobenzofuran (DPBF).
The production of singlet oxygen was established qualitatively
using DPBF as a singlet oxygen quencher.33–35 Solutions of DPBF
(50 lM) and of the porphyrinic compounds (0.5 lM) in DMF were
irradiated, over a total period of 15 min, in glass cuvettes with
light at a fluence rate of 9.15 mW/cm2. The light beam was previ-
ous filtered using a cut-off filter (k <550 nm) in order to remove all
high-energy wavelengths. The photodecomposition of DPBF was
monitored by measuring the decreasing of the absorbance at
414 nm registered in 15 seconds intervals of irradiation and the
results are reported in Figure 2. The porphyrin 5,10,15,20-tetra-
phenylporphyrin (TPP) that is considered a good singlet oxygen
producer, was used as a reference.36 The DPBF photodegradation
is higher in the presence of all the photosensitizers and the decay
curves of all the compounds are quite comparable to TPP. On the
other hand, the DPBF decay rate is higher in the presence of the
porphyrin amino acid conjugate 4, denoting in this case a higher
efficiency to generate singlet oxygen under the same experimental
conditions than TPP.
The porphyrin 1 shows a typical ethio-type visible spectrum,
with an intense band centered at 419 nm (Soret band) and the four
Q bands at 514, 548, 587, and 645 nm. The insertion of the amino
acid moiety does not introduce significant changes in the UV–vis
spectrum of the new derivatives 3–6.
It is interesting to note that all the attempts to obtain the non
oxidized porphyrin–methionine conjugate were unsuccessful. The
FAB+ spectra of the derivative 6 clearly support the oxidized struc-
ture proposed, showing a molecular ion [M+H]+ at m/z 910.
2.2. Studies in vitro on HaCaT and HeLa cells
2.2.1. Cell viability
The potentiality of the porphyrin amino acid conjugates 3–6 as
photosensitizers was tested using HeLa human cervical cancer cell
line and HaCaT human normal skin keratinocytes cell line. The
cytotoxic activity of the conjugates was evaluated using the MTT
assay. Different concentrations of the porphyrin conjugates 3–6
(1.2 ꢀ 10ꢁ5–2.2 ꢀ 10ꢁ7 M) were administered to cell cultures in
liposomal solutions and the cells were incubated, in the dark, for
a period of 4 h. The cytotoxicity and the photocytotoxicity of com-
pound 5 were not determined because its incorporation into phos-
pholipids layers was not successful even after several attempts.
The survival rates obtained (Table 1) show that no significant dark
cytotoxicity is observed at concentrations up to 2.4 ꢀ 10ꢁ6 M.
For the photodynamic assays, the cell cultures were incubated
for a period of 4 h in the presence of non cytotoxic concentrations
of porphyrin conjugates and then submitted to different times of
irradiation with red light (5, 10, and 20 min) with a fluence rate
of 9.15 mW/cm2. The results obtained for the cell survival,
2.1.2. Photostability of the compounds
The rate of photodegradation exhibit by a compound under
light conditions is a very important parameter to assess when we
consider their potential application as photosensitizer. Porphyrin
derivatives can go through photobleaching when exposed to
UV–vis light and oxygen.32 A fast photobleaching would cause
the concentration of the drug to decrease, thus impairing the effec-
tiveness of the treatment. To induce photobleaching, aerated solu-
tions of compounds 3–6 in DMF, under magnetic stirring, were
irradiated with white light at a fluence rate of 9.15 mW/cm2. The
UV–vis spectra were recorded at different times of irradiation (0,
1, 2, 3, 4, 6, 8, 11, 15, and 20 min) and the absorbance intensity
of the Soret band was monitored and compared with the original
one. The UV–vis spectra of all target compounds did not show
any absorbance decay of the Soret and Q bands, during the total
irradiation period. As an example, the UV–vis spectra of compound
3 at two different irradiation times are shown in Figure 1. The re-
sults point out that all studied compounds are highly stable under
such conditions.
Figure 2. Comparative photooxidation of DPBF (50
photosensitizer (0.5 l
l
M) in DMF with or without
Figure 1. UV–vis spectra of compound 3 (DMF) after irradiation (white light,
M); white light (9.15 mW/cm2) filtered through a cut-off filter
9.15 mW/cm2) at 0 and 20 min (total light dose 11 J/cm2).
k <550 nm.

V. V. Serra et al. / Bioorg. Med. Chem. 18 (2010) 6170–6178
6173
Table 1
non tumoral HaCaT cells. So the conditions selected were:
(i) 2.5 ꢀ 10ꢁ7 M for compound 3 (cell survival of 50.96 1.3%);
(ii) 1.2 ꢀ 10ꢁ6 M for compound 4 (cell survival of 42.75 1.5%);
(iii) 3.0 ꢀ 10ꢁ7 M for compound 6 (cell survival of 53.02 8.4%)
and 10 min of red light irradiation. Figure 3 shows that LD50 condi-
tions for the non tumoral HaCaT cells, induced a 100% lethality in
the tumoral HeLa cells.
Percent survival of two cell lines, HeLa and HaCaT, incubated in the dark for a period
of 4 h at different concentrations of compounds 3, 4, and 6
Entry
Compound
Concentration (M)
Surviving fraction (% SD)
HaCaT
HeLa
1
3
2.2 ꢀ 10ꢁ7
6.1 ꢀ 10ꢁ7
1.2 ꢀ 10ꢁ6
2.4 ꢀ 10ꢁ6
1.2 ꢀ 10ꢁ5
2.2 ꢀ 10ꢁ7
6.1 ꢀ 10ꢁ7
1.2 ꢀ 10ꢁ6
2.4 ꢀ 10ꢁ6
1.2 ꢀ 10ꢁ5
2.2 ꢀ 10ꢁ7
6.1 ꢀ 10ꢁ7
1.2 ꢀ 10ꢁ6
2.4 ꢀ 10ꢁ6
1.2 ꢀ 10ꢁ5
94.98 5.21
97.96 6.26
102.19 8.01
109.91 5.0
24.30 2.47
101.49 1.2
104.72 4.49
102.65 2.98
97.54 12.0
96.98 11.54
99.20 6.70
101.50 6.60
103.40 4.90
101.30 12.70
16.90 3.35
107.31 2.73
110.38 3.37
106.97 6.64
104.89 8.81
60.42 3.62
102.58 1.36
100.28 1.26
102.78 0.09
91.48 0.23
67.33 8.51
93.26 9.66
92.61 5.04
95.69 4.81
93.50 9.18
16.93 3.17
Morphologically, HaCaT cells tend to form microcolonies with
well developed adhesion systems. At LD50 conditions the cells re-
tained the morphology quite similar to that control cells for all
the studied conjugates after 10 min of irradiation (Fig. 4A–D).
However, after 20 min of irradiation cells showed a partial retrac-
tion, loss of adhesion and cytoplasmic vacuolization (Fig. 4E–H).
These effects were more evident at the edge of the colonies than
in the center of them, because cells located at the edge of colonies
were more exposed to photosensitizers. PDT treatments induced
also morphological changes in HeLa cells, but substantially differ-
ent to those observed in HaCaT cells. Figure 4J, K shows some
morphological changes for compound 3 and 4 after 2 min of irradi-
ation. These alterations are more evident under LD50 conditions for
HeLa cells. (data not shown because after 10 min of PDT almost
100% of the cells were dead). LD50 of derivative 6 (Fig. 4L),
produced in HeLa cells a drastic effect such as cytoplasmic vacuo-
lization, followed by loss of adhesion and loss of basophilia, which
was more evident after 10 min of light irradiation.
2
3
4
6
Each point corresponds to the mean value SD from three different experiments.
summarized in Table 2, show that the cell photokilling is depen-
dent on both the photosensitizer concentration and the irradiation
time. A significant photocytotoxicity (survival fractions of about
20%) can be observed on non tumoral HaCaT cells when the photo-
sensitizers 4 and 6 were used at concentration 1.2 ꢀ 10ꢁ6 M and
after 20 min of irradiation. Furthermore, 100% lethality can be ob-
served for compound 3 under the same conditions (1.2 ꢀ 10ꢁ6 M,
20 min of irradiation). The phototoxic effect is less pronounced at
lower concentrations and shorter irradiation times (Table 2). For
example, a high HaCaT cell survival percentage of 82.69 0.01%
and 76.87 5.58% can be reached with compounds 3 and 6 at con-
centrations of 2.5 ꢀ 10ꢁ7 and 3.0 ꢀ 10ꢁ7 M, respectively, after
5 min of irradiation. Furthermore, no photocytotoxicity on HaCaT
cells is observed with compound 4 at 6.1 ꢀ 10ꢁ7 M and after
20 min of irradiation with red light. It is interesting to note that
compounds 3 and 4 at concentrations of 2.5 ꢀ 10ꢁ7 and 1.2 ꢀ 10
ꢁ6 M, respectively, show significant phototoxicity in HeLa cells
upon 5 min of irradiation; only surviving cells fractions of
3.74 3.40% (compound 3) and 8.00 3.90% (compound 4) are ob-
served. A less pronounced phototoxic effect (56.87 3.81) is de-
tected for compound 6 at 3.0 ꢀ 10ꢁ7 M and after 5 min. These
results, point out that compounds 3 and 4 provide a good photody-
namic effect in HeLa cells with minimal photocytotoxicity to non
tumoral cells at concentrations of 2.5 ꢀ 10ꢁ7 and 1.2 ꢀ 10ꢁ6 M.
To analyze the intracellular localization of the amino acids con-
jugates, the cells were incubated up to 24 h at the LD50 selected
concentration for HaCat in order to visualize the time-dependent
changes in intracellular localization of the PSs. For this purpose,
cells were incubated with compound
3
(2.5 ꢀ 10ꢁ7 M),
4
(1.2 ꢀ 10ꢁ6 M), and 6 (3.0 ꢀ 10ꢁ7 M) for 6 h and 24 h, respectively.
In HaCaT cells, all the compounds were concentrated mainly with-
in the cells and appeared as different size granules fitting the dis-
tribution pattern of lysosomes (Fig. 5).
In the same figure it can be observed that the PSs exhibit differ-
ent localization patterns in HeLa cells; conjugate is located at
lysosomes, and compounds 4 and 6 are located in the plasma
membrane at both studied incubation times (6 h and 24 h). We
did not see any localization patterns characteristic for mitochon-
dria (blue signal Fig. 5).
It is known that effective photodamage of PSs in both cell cul-
tures and tumors is related to the chemical structure, concentra-
tion, incubation time, vehicle, light dose, and site of subcellular
localization. Several cell components have been described as tar-
gets for the cytotoxic effects of ROS (reactive oxygen species)
including mitochondria, lysosomes, Golgi apparatus, plasma mem-
brane and nuclei. PSs localized in mitochondria and lysosomes are
highly effective for cell photodamage, triggering either apoptotic or
necrotic cell death responses.37
2.2.2. Subcellular localization and morphological studies
The subcellular localization experiments and morphological
studies were conduced under sublethal conditions (LD50) for the
Table 2
Percent survival of two cell lines, HeLa and HaCaT, at different times of irradiation with a fluence rate of 9.15 mW/cm2 and at different concentrations of
compounds 3, 4, and 6
Entry
Compound
Concentration (M)s
2.5 ꢀ 10ꢁ7
Irradiation time (min)
Surviving fraction (% SD)
HaCaT HeLa
3.74 3.4
1
2
3
3
4
6
5
10
20
5
10
20
5
82.69 0.01
50.96 1.3
22.11 1.3
ND
1.13 0.5
1.32 0.01
8.00 3.9
1.2 ꢀ 10ꢁ6
42.75 1.5
22.54 3.0
76.87 5.58
53.02 8.42
28.20 1.66
0
0
3.0 ꢀ 10ꢁ7
56.87 3.81
14.23 1.50
10.93 2.38
10
20
ND: not determined. Each point corresponds to the mean value SD from three different experiments.

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V. V. Serra et al. / Bioorg. Med. Chem. 18 (2010) 6170–6178
Further in vitro studies concerning the mechanism of cell death
produced by the photoactivation of these porphyrin amino acid
conjugates are presently in progress in our laboratory.
120
100
80
60
40
20
0
A
HaCaT
HeLa
3. Experimental
3.1. General
1H and 13C NMR spectra were recorded in a Bruker AMX 300
NMR spectrometer (at 300.13 and 75.47 MHz, respectively): CDCl3
was used as solvent and TMS as internal reference. The chemical
shifts are expressed in d (ppm) and the coupling constants (J) in
Hertz (Hz). Mass spectra were recorded on VG AutoSpec Q mass
spectrometer using CHCl3 as solvent and 3-nitrobenzyl alcohol
(NBA) as matrix. The UV–vis spectra were recorded on an UV-
2501 PC Shimadzu spectrophotometer using DMSO as solvent.
Melting points were measured on a Reichert Thermovar apparatus
fitted with a microscope and are uncorrected. Analytical TLC was
carried out on precoated sheets with silica gel (Merck, 60,
0.2 mm thick). Column chromatography was carried in silica gel
(Merck, 230–400 mesh). Pyridine was distilled from potassium
hydroxide, all other solvents and reagents were used without fur-
0
5
10
20
120
100
80
60
40
20
0
B
C
ther purification. Dipalmitoyl D-a-phosphatidylcholine from Sigma
was used in liposome preparation. The compounds purities were
confirmed by thin layer chromatography, 1H NMR and elemental
analysis.
0
5
10
20
120
100
80
60
40
20
0
3.1.1. 5-(4-Carboxyphenyl)-10,15,20-tris(3-methoxyphenyl)-
porphyrin (1)
4-Formylbenzoic acid (1.62 g, 10.8 mmol) and 3-methoxybenz-
aldehyde (2.63 mL, 21.6 mmol) were added to a refluxing mixture
of glacial acetic acid (200 mL) and nitrobenzene (150 mL). After
the dissolution of the 4-formylbenzoic acid, pyrrole (2.00 mL,
28.9 mmol) was added dropwise (ca. 4 min) to the mixture. The
reaction mixture was then refluxed for 1 h. The solvents were dis-
tilled under reduced pressure and the crude material was taken
into chloroform and directly chromatographed on a silica column
using chloroform as eluent. The first fraction was identified as
5,10,15,20-tetrakis (3-methoxyphenyl) porphyrin. The second frac-
tion, eluted with the same solvent, gave porphyrin 1 after evapora-
tion of the solvent and recrystallization from chloroform/light
petroleum. Yield: 8%. mp >300 °C. 1H NMR (300 MHz, CDCl3):
d = ꢁ2.80 (s, 2H, NH), 3.99 (s, 9H, 10,15,20-OCH3-Ph), 7.34 (dd,
J = 8.2 and 2.1 Hz, 3H, 10,15,20-H-p-Ph), 7.63–7.68 (m, 3H,
10,15,20-H-m-Ph), 7.79–7.83 (m, 6H, 10,15,20-H-o-Ph), 8.35 (d,
J = 7.6 Hz, 2H, 5-H-o-Ph), 8.51 (d, J = 7.6 Hz, 2H, 5-H-m-Ph), 8.80
and 8.92 (AB, J = 4.9 Hz, 4H, H-b), 8.90 (s, 4H, H-b). MS (FAB+)
0
5
10
15
20
irradiation time (min)
Figure 3. Photodynamic activity of porphyrin amino acid derivatives and effect of
LD50 treatments on HaCaT and HeLa cell lines. Cells were incubated for 4 h in the
presence of LD50 of (A) compound 3 (2.5 ꢀ 10ꢁ7 M), (B) compound 4 (1.2 ꢀ 10ꢁ6 M),
and (C) compound 6 (3.0 ꢀ 10ꢁ7 M) and subjected to red light irradiation for
variable times. Each points corresponds to the mean value SD from three different
experiments.
m/z: 749 [M+H]+. UV–vis (DMSO): kmax (log
e) = 419 (5.66), 514
(4.28), 548 (3.92), 587 (3.78), 645 (3.72) nm.
In our study, we have seen that all the analyzed compounds are
able to generate singlet oxygen, being compounds 4 and 6 the most
efficient ones. Although compound 3 is the less efficient single oxy-
gen producer, the fact that it is localized in lysosome membrane,
instead of plasma membrane like the others two, can be responsi-
ble by its higher photocytotoxicity in tumoral cells.
3.1.2. 5-[4-(Succinimide-N-oxycarbonyl)phenyl]-10,15,20-
tris(3-methoxyphenyl)porphyrin (2)
To a stirred solution of porphyrin 1 (31 mg, 42 lmol) in dry
pyridine was added SOCl2 (0.1 mL, 163 mmol). The reaction mixture
was maintained in the dark, protected from moisture with silica gel,
during 30 min at room temperature. Then, N-hydroxysuccinimide
In conclusion, four porphyrin amino acid conjugates 3–6 (gly-
cine, serine, tyrosine and methionine residues) have been synthe-
sized in good to excellent yields through an easy synthetic
approach. All of the compounds show high photostability and are
able to generate singlet oxygen. The conjugates 3, 4, and 6 show
significant photocytotoxic activity in HaCaT and HeLa cell lines.
Furthermore, the MTT cytotoxicity studies indicate that compound
3 had the highest efficacy at lower concentration. More, under LD50
conditions for non tumoral cells (HaCaT) compound 3 produced
100% lethality in tumoral cells (HeLa). These facts, associated with
the easy access to these porphyrin amino acid conjugates may pro-
vide valuable tools in PDT for the treatment of several diseases.
(74 mg, 643 lmol) was added and the temperature rised to 50 °C.
The reaction progress was monitored by TLC. When the reaction
was complete (3 h), the solvent was removed under reduced pres-
sure and the crude material was taken into chloroform. The mixture
was neutralized with aqueous sodium hydrogencarbonate solution
and extracted in chloroform. The organic layer was washed with
water (2 ꢀ 100 mL), dried over Na2SO4, and the solvent was
removed. The residue was purified by flash chromatography (silica
gel), using dichloromethane as eluent. Porphyrin 2 was crystallized

V. V. Serra et al. / Bioorg. Med. Chem. 18 (2010) 6170–6178
6175
Figure 4. Morphological changes induced by porphyrin amino acid derivatives on HaCaT (A–H) and HeLa (I–L) cell lines when incubated for 4 h in the presence of compound
3 (2.5 ꢀ 10ꢁ7 M), 4 (1.2 ꢀ 10ꢁ6 M), and 6 (3.0 ꢀ 10ꢁ7 M). HaCat cells: A–D: 10 min of irradiation, E–H: 20 min of irradiation. HeLa cells: J, K: 2 min of irradiation, I, L: 10 min of
irradiation. After light exposure, cells were fixed in cold methanol and stained with toluidine blue. Photographs are representative images of three different experiments for
each compound.
from dichloromethane/light petroleum. Yield: 34.4 mg (97%). mp
>300 °C. 1H NMR (300 MHz, CDCl3): d = ꢁ2.81 (s, 2H, NH), 3.02 (s,
4H, CH2), 3.99 (s, 9H, 10,15,20-OCH3-Ph), 7.34 (dd, J = 8.2 and
2.1 Hz, 3H, 10,15,20-H-p-Ph), 7.63–7.68 (m, 3H, 10,15,20-H-m-Ph),
7.78–7.82 (m, 6H, 10,15,20-H-o-Ph), 8.38 (d, J = 8.4 Hz, 2H, 5-H-o-
Ph), 8.55 (d, J = 8.4 Hz, 2H, 5-H-m-Ph), 8.76 (d, J = 4.8 Hz, 2H, H-b),
8.90 (s, 4H, H-b), 8.94 (d, 2H, J = 4.8 Hz, H-b). MS (FAB+) m/z: 846
sulfate. After the reaction work up, the solvent was removed under
pressure and each residue obtained was purified by flash chroma-
tography using an appropriate solvent.
3.2.1. Compound 3: MMA-Gly
[M+H]+. UV–vis (DMSO): kmax (log
e) = 419 (5.64), 514 (4.25), 548
(3.94), 587 (3.81), 645 (3.51) nm.
3.2. General procedure for the reaction of porphyrin 2 with
amino acids
Yield: 93%. mp = 270–272 °C. 1H NMR (300 MHz, CDCl3):
d = ꢁ2.81 (s, 2H, NH), 3.90 (s, 3H, H-500), 3.99 (s, 9H, OCH3-Ph),
4.44 (d, J = 4.9 Hz, 2H, H-300), 6.98 (t, J = 4.9 Hz, 1H, H-200), 7.34
(dd, J = 8.0 and 2.0 Hz, 4H, H-p-Ph), 7.64 (t, J = 8.0 Hz, 3H,
10,15,20-H-m-Ph), 7.78–7.82 (m, 6H, 10,15,20-H-o-Ph), 8.20 (d,
J = 8.1 Hz, 2H, 5-H-m-Ph), 8.31 (d, J = 8.1 Hz, 2H, 5-H-o-Ph), 8.77–
8.91 (m, 8H, H-b). EM (FAB)+ m/z: 819 [M+H]+, 704 [M+H]+:
The amino acid (4 equiv) was dissolved in DMSO (2 mL) and
then a large excess of K2CO3 and the activated ester 2 (1 equiv)
were added. The reaction was kept overnight at 40 °C, under stir-
ring, protected from light and from moisture with silica gel. Each
organic layer was neutralized with citric acid, washed with water,
extracted with dichloromethane, and dried over anhydrous sodium

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V. V. Serra et al. / Bioorg. Med. Chem. 18 (2010) 6170–6178
Figure 5. Intracellular localization of porphyrin amino acid derivatives in HaCaT and HeLa cells after incubation with each derivative for 6 h at concentration of 2.5 ꢀ 10ꢁ7
M
(3), 1.2 ꢀ 10ꢁ6 M (4), and 3.0 ꢀ 10ꢁ7 M (6) in complete medium. White arrows show the localization of the PSs those fluorescing in red and shown a lysosomal distribution.
Blue autofluorescence of mitochondria’s can be observed in both controls. Photographs concerning 24 h treatment are not shown due to similar pattern of PSs with 6 h
treatment.
C4H7NO3. UV–vis (DMSO): kmax (log
e
) = 414 (5.70), 514 (4.32), 549
500), 3.98 (s, 9H, 10,15,20-OCH3-Ph), 4.22–4.25 (m, 2H, H-1000),
5.07–5.10 (m, 1H, H-300), 7.32–7.34 (m, 3H, 10,15,20-H-p-Ph),
7.44 (d, J = 7.1 Hz, 1H, H-200), 7.64 (m, 3H, 10,15,20-H-m-Ph), 7.78
(s, 3H, 10,15,20-H-o-Ph), 7.80 (d, J = 7.1 Hz, 3H, 10,15,20-H-o-Ph),
8.25 (d, J = 8.0 Hz, 2H, 5-H-m-Ph), 8.32 (d, J = 8.0 Hz, 2H, 5-H-o-
Ph), 8.78, 8.89, and 8.91 (d, s, d, J = 4.9 Hz, 8H, H-b). 13C NMR
(75 MHz, CDCl3): d = 53.1 (C-500), 55.4 (C-300), 55.5 (10,15,20-
OCH3-Ph), 63.8 (C-1000), 113.6 (10,15,20-C-p-Ph), 118.4 (Porf-C-5),
120.1, 120.2, 120.4 (10,15,20-C-o-Ph), 125.6 (5-C-m-Ph), 127.5
and 127.6 (10,15,20-C-m-Ph and 10,15,20-C-o-Ph), 131.3 (C-b),
132.8, 134.7 (5-C-o-Ph), 143.3, 146.2 (C-10), 147.3, 157.9, 167.7
(C-100). EM (FAB)+ m/z: 850 [M+H]+, 703 [M+H]+: C5H9NO4. UV–vis
(3.90), 589 (3.79), 644 (3.61) nm. Anal. Calcd for C51H41N5O6ꢂH2O:
C, 73.10; H, 5.17; N, 8.36. Found: C, 73.20; H, 5.07; N, 8.52.
3.2.2. Compound 4: MMA-Ser
(DMSO): kmax (log e) = 414 (5.70), 514 (4.33), 549 (3.91), 589
Yield: 91%. mp = 270–272 °C. 1H NMR (300 MHz, CDCl3):
d = ꢁ2.81 (s, 2H, NH), 2.58–2.62 (m, 1H, Ser-OH), 3.93 (s, 3H, H-
(3.79), 644 (3.64) nm. Anal. Calcd for C52H43N5O7ꢂH2O: C, 71.96;
H, 5.23; N, 8.07. Found: C, 72.18; H, 5.23; N, 8.07.

V. V. Serra et al. / Bioorg. Med. Chem. 18 (2010) 6170–6178
6177
3.2.3. Compound 5: MMA-Tyr
fluence rate of 9.15 mW/cmꢁ2. During the irradiation period, the
solutions were stirred at room temperature. The generation of sin-
glet oxygen was followed by its reaction with DPBF. The breakdown
of DPBF was monitored by measuring the decreasing of the absor-
bance at 414 nm at irradiation intervals of 15 s. The photostability
of the photosensitizers was determined by irradiating 2 mL of
H
C
O
C
2' 3'
O
C
1''
2''
3''
1'
4'
OH
1'''
Porph
N
H
6' 5'
1 lM solutions of theporphyrins in DMF withwhite light at a fluence
CH
4''
2
rate of 9.15 mW/cmꢁ2. During such irradiation the solutions were
magnetically stirred and kept at room temperature. At fixed
intervals of time (0; 1; 2; 3; 4; 6; 8; 11; 15; 20 min), the concentra-
tion of the porphyrin was determined by visible absorption
spectrophotometry.
5''
6''
7''
10''
9''
8''
OH
3.4. Cell cultures
Yield: 51%. mp >300 °C. 1H NMR (300 MHz, CDCl3): d = ꢁ2.96 (s,
2H, Porf-NH), 3.07–3.18 (m, 2H, H-400), 3.92 (s, 6H, 10,20-OCH3-Ph),
3.95 (s, 3H, 15-OCH3-Ph), 4.55 (br s, 1H, H-300), 6.69 (d, J = 8.1 Hz,
2H, H-700 and H-900), 7.18 (d, J = 8.1 Hz, 2H, H-600 and H-1000), 7.40
(dd, J = 11.8 and 9.1 Hz, 3H, 10,15,20-H-p-Ph), 7.63–7.67 (m, 9H,
10,15,20-H-o-Ph and 10,15,20-H-m-Ph), 8.20 (d, J = 7.8 Hz, 2H, H-
30 and H-50), 8.28 (d, J = 7.8 Hz, 2H, H-20 and H-60), 8.37 (d,
J = 5.6 Hz, 1H, H-200), 8.83–8.87 (m, 8H, H-b), 9.19 (s, 1H, Tyr-OH).
13C NMR (75 MHz, CDCl3): d = 36.6 (C-400); 55.4 (10,15,20-OCH3-
Ph); 56.4 (C-300); 113.7 (10,15,20-C-p-Ph), 114.8 (C-700 and C-900);
119.1; 119.8; 119.9; 120.3 (10,15,20-C-o-Ph); 125.7 (C-30 and
C-50); 127.2 (10,15,20-C-o-Ph); 127.9 (10,15,20-C-m-Ph); 128.9
(C-500); 129.7 (C-800); 130.3 (C-600 and C-1000); 131.4 (C-b); 134.2
(C-20, C-60); 134.7 (C-5); 142.5; 143.8 (C-10); 155.5; 157.7; 165.5
(C@O). EM (FAB)+ m/z: 912 [M+H]+, 704 [M+H]+: C10H10NO4. UV–
The HeLa human cervical cancer cell line and HaCaT human
normal skin keratinocytes were stored frozen in liquid nitrogen.
The cells were grown as a monolayer employing Dulbecco’s Mod-
ified Eagle Medium (DMEM) containing 10% fetal calf serum
(FCS) and 50 units/mL penicillin and 50 lg/mL streptomycin (Sig-
ma). The cells were incubated at 37 °C in a humidified 5% CO2
atmosphere and the medium was changed daily. For the photocy-
totoxicity experiments, cells were plated on 24-well plates and for
fluorescence observation, cells were plated onto round coverslips
placed into wells.
3.5. Preparation of liposomes
Since these porphyrin conjugates are not soluble in PBS or
water, they were administered to cell cultures in liposomal solu-
tion. Each compound was dissolved in pyridine to a concentration
of 0.5 mg/mL and stored in the dark at 4 °C until use. The incorpo-
vis (DMSO): kmax (log e) = 418 (5.71), 514 (4.30), 549 (3.89), 588
(3.79), 644 (3.61) nm. Anal. Calcd for C57H45N5O7ꢂ3H2O: C, 70.87;
H, 5.32; N, 7.25. Found: C, 70.76; H, 4.91; N, 6.88.
ration of the sensitizers into liposomes of dipalmitoyl D-a-phos-
3.2.4. Compound 6: MMA-Met
phatidylcholine (Sigma, St Louis, MO, USA) was carried out by
the injection procedure as previously described.38 Liposomal sus-
pension of porphyrins amino acid conjugates were sterilized by fil-
tration through a Millipore filter (0.22 lM of pore size) and used
within 48 h after preparation. The concentration of the three
photosensitizers was estimated by the absorbance at the Soret
band using a Shimatzu UV 1601 spectrophotometer and diluted
with DMEM-1% FCS to obtain the desired concentrations.
3.6. Intracellular localization of each amino acid porphyrins
conjugates
The cells were seeded onto coverslips placed into 24-well plates
at densities of 3 ꢀ 104 (HaCaT and HeLa cells) and allow to grow for
24 h. After that, cells were incubated with the liposomal suspension
of porphyrins amino acid conjugates for variable times. The samples
were washed twice with PBS and then were mounted onto slides
with PBS. Samples were studied under fluorescence microscopy.
Yield: 90%. mp >300 °C. 1H NMR (300 MHz, CDCl3): ꢁ2.82 (s, 2H,
Porf-NH), 2.38–2.54, 2.84–2.92, 2.95–3.01, and 3.03–3.13 ppm (m,
4H, H-400 and H-500), 2.66 (s, 3H,H-600), 3.91 (s, 3H, H-2000), 3.99 (s, 3H,
10,15,20-OCH3-Ph), 5.07–5.09 (m, 1H, H-300), 7.34 (dd, J = 8.0 and
2.1 Hz, 3H, 10,15,20-H-p-Ph), 7.65 (t, J = 8.0 Hz, 3H, 10,15,20-H-
m-Ph), 7.78–7.82 (m, 6H, 10,15,20-H-o-Ph) 7.88 (d, J = 7.0 Hz, 1H,
H-200), 8.26–8.33 (m, 4H, H-20, H-30, H-50, H-60), 8.78–8.91 (m, 8H,
3.7. Photodynamic therapy treatment in vitro
H-b).UV–vis (DMSO): kmax (log e) = 419 (5.65), 513 (4.28), 548
(3.91), 588 (3.77), 645 (3.69) nm. EM (FAB)+ m/z: 910 [M+H]+,
703 [M+H]+: C6H13NO4S. HRMS (ESI) Calcd for C53H45N5O7S
[M+H]+: CH3: 895.3040, Found: 895.3026.
Cells seeded in 24-well plates were incubated with an appropri-
ate volume of each porphyrin amino acid conjugate, incorporated
into liposomes; 4 h after started the treatment, irradiations were
performed. After irradiations, the medium was replaced by fresh
complete medium without drugs and the cells were incubated in
dark for another 24 h and then tested their viability.
3.3. Singlet oxygen generation and photostability studies
Stock solution of each porphyrin derivative at 0.1 mM in DMF
and a stock solution of 1,3-diphenylisobenzofuran (DPBF) at
10 mM in DMF were prepared.
3.8. Light source
The reaction mixture of 50 lM of DPBF and 0.5 lM of a porphyrin
derivative in DMF in glass cells (2 mL) was irradiated with white
light filtered through a cut-off filter of wavelength <550 nm, at a
A LED array was constructed and used for irradiation. It is com-
posed of a matrix of 24 ꢀ 16 LEDs (Kingbright, model L53 SRCE),
which makes a total of 384 light sources with an emission peak

6178
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Acknowledgments
Thanks are due to Fundação para a Ciência e a Tecnologia (FCT,
Portugal) and POCI (FEDER) for funding the Organic Chemistry Re-
search Unit. V. Vaz Serra also thanks FCT for Ph.D. Grant (SFRH/BD/
28122/2006).
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