Photodynamic effects of isosteric water-soluble phthalocyanines on human nasopharynx KB carcinoma cells

Article abstract of DOI:10.1016/j.ejmech.2010.06.002

The photodynamic activity of water-soluble cationic zinc(II) phthalocyanines using human nasopharynx carcinoma (KB cells) was investigated. A sulfur-linked cationic dye, named: 2,9(10),16(17),23(24)-tetrakis[(2- trimethylammonium)ethylsulfanyl]phthalocyan

Full text of DOI:10.1016/j.ejmech.2010.06.002

European Journal of Medicinal Chemistry 45 (2010) 4129e4139
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Photodynamic effects of isosteric water-soluble phthalocyanines on human
nasopharynx KB carcinoma cells
,
,
,
Julieta Marino a 1, María C. García Vior b 1, Lelia E. Dicelio c, Leonor P. Roguin a, Josefina Awruch b
*
a Instituto de Química y Fisicoquímica Biológicas (UBA-CONICET), Facultad de Farmacia y Bioquímica, Junín 956, 1113 Buenos Aires, Argentina
b Departamento de Química Orgánica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina
c INQUIMAE, Departamento de Química Inorgánica, Analítica y Química Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
Ciudad Universitaria, Pabellón II, 1428 Buenos Aires, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
The photodynamic activity of water-soluble cationic zinc(II) phthalocyanines using human nasopharynx
carcinoma (KB cells) was investigated. A sulfur-linked cationic dye, named: 2,9(10),16(17),23(24)-tetrakis
[(2-trimethylammonium)ethylsulfanyl]phthalocyaninatozinc(II) tetraioidide (13) is the most active of
four sensitizer assays and shows a singlet oxygen quantum yield of 0.58 and a higher bathochromic shift
of 10 nm for the Q-band as compared with the oxygen-linked cationic aliphatic phthalocyanine: 2,9
(10),16(17),23(24)-tetrakis[(2-trimethylammonium)ethoxy]phthalocyaninatozinc(II) tetraioidide (11)
Received 13 January 2010
Received in revised form
7 May 2010
Accepted 2 June 2010
Available online 16 June 2010
and the best photo-stability in water in comparison with their tetra-a-substituted counterparts 1,8(11),15
Keywords:
(18),22(25)-tetrakis[(2-trimethylammonium)ethoxy]phthalocyaninatozinc(II) tetraioidide (12) and 1,8
(11),15(18),22(25)-tetrakis[(2-trimethylammonium)ethylsulfanyl]phthalocyaninatozinc(II) tetraioidide
(14). Phthalocyanine 13, partially localized in lysosomes, led to cell photoinactivation in a concentration-
and light dose-dependent manner. After photodynamic treatment, compound 13 induced an apoptotic
response e as indicated by morphological cell changes e an increase in the activity of caspase-3 and the
cleavage of poly-ADP-ribose-polymerase substrate (PARP).
Photodynamic therapy
Phthalocyanines
Singlet oxygen
Apoptosis
Lysosomes
KB cells
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
Zinc, aluminum, and silicon phthalocyanines that have been
found to be useful photosensitizers for PDT, are efficient generators
Photodynamic therapy (PDT) is emerging as
a
promising
of singlet oxygen, the cytotoxic species capable of destroying
malignant cells [10e14]. The silicon phthalocyanine Pc 4 is in
various stages of clinical evaluation for cutaneous and subcuta-
neous lesions from diverse solid tumor origins [15].
method for the treatment of a variety of oncological, dermatolog-
ical, cardiovascular and ophthalmic diseases [1e5]. PDT combines
the intravenous or topical administration of a photosensitizer
which preferentially localizes within the tumor, followed by
illumination of the target tissue thus resulting in the formation of
reactive oxygen species, believed to be responsible for the cascade
of cellular and molecular events that finally lead to selective tumor
destruction [1].
The uptake and efficacy of photosensitizers such as phthalocy-
anines, porphyrins, and core-modified porphyrins are directly
related to the number of hydrophilic groups [8,10] that the dye
carries on its structure. Thus, in the aluminum sulfonate phthalo-
cyanine series, those with two groups attached to the macrocycle
show greater uptake and phototoxicity than phthalocyanines with
three sulfonate groups which in turn show greater uptake and
efficacy than phthalocyanines with four sulfonate groups. In BALB/c
mice bearing EMT-6 tumors, aluminum sulfonate phthalocyanine
substituted with two sulfonate groups is 10 times more phototoxic
than aluminum phthalocyanine carrying four sulfonate groups [14].
Great efforts have been made to study the structureeactivity
relationship between the site [16,17] and kind of substitution [18]
and the photodynamic activity. However, to our knowledge,
studies directed to establish the structureeactivity relationship
between isosteric phthalocyanines are quite uncommon.
Phthalocyanines have been found to have applications as
phototoxic drugs for PDT [4,6e8]. In addition to their well-known
chemical stability, phthalocyanines possess characteristic absorp-
tion spectra [9], with a Soret band at approximately 350 nm and
a usually narrow but very strong Q-band around 675 nm, with
a molar absorption coefficient in the range of 105 Mꢀ1 cmꢀ1
.
* Corresponding author. Tel.: þ54 11 4964 8252; fax: þ54 11 4508 3645.
E-mail address: jawruch@ffyb.uba.ar (J. Awruch).
Both authors contributed equally to this work.
1
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.06.002

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J. Marino et al. / European Journal of Medicinal Chemistry 45 (2010) 4129e4139
We herein report the photobiological studies of four isosteric
With regard to the solubility of phthalocyanines 7e10 they are
soluble in almost all organic solvents, while cationic derivatives
11e14 are fully soluble in water and DMF.
water-soluble cationic zinc(II) phthalocyanines using human
nasopharynx KB carcinoma cells. The properties of these phthalo-
cyanines are compared and discussed in connection with their
photophysical and photochemical evaluation. Our results show the
efficiency of one of them: 2,9(10),16(17),23(24)-tetrakis[(2-trime-
thylammonium)ethylsulfanyl]phthalocyaninatozinc(II) tetraioidide
(13) for suitable PDT applications. To further elucidate the cellular
mechanism of action, we examined the intracellular localization for
the purpose of determining the primary photodamage site. In
addition, the prevailing mechanism of cell death after treatment
was evaluated.
2.2. Photophysical studies
The absorption spectra of zinc(II) phthalocyanines 11e14 in the
400e800 nm range are similar to those previously reported for
other analogs in homogeneous media [26,27] (Fig. 1). Phthalocya-
nines 11e14 are strongly aggregated in water; their absorption
spectra show an important dimer band blue-shifted even at low
concentrations as shown in Fig. 1. This situation is reverted when
DMF is employed as a solvent: the monomer band at c.a. 680 nm is
enhanced and the dimer absorption becomes negligible.
2. Results and discussion
The same behavior is evidenced by deviations of the Lam-
berteBeer law, in water (data not shown). The monomer spectra
are similar to that reported for different zinc(II) phthalocyanines.
On the other hand dimers are blue-shifted and different from the
corresponding spectra of other phthalocyanines of the same family
previously studied in our laboratory [26]. This agrees with the fact
that, while dimer spectra of metallated phthalocyanines are
dependent on the peripheral substituents and solvent polarity, that
of the monomer remains practically unchanged [28].
2.1. Chemistry
In order to evaluate the effect of substitution on the efficacy of
phthalocyanines, we synthesized cationic dyes 11e14. The
synthetic route is shown in Scheme 1; precursors 3e6 and phtha-
locyanines 7e14 were prepared by methods similar to those
described in the literature with minor modifications [19e23].
Taking into account that the above phthalocyanines are for
photophysical and photobiological studies, the synthesis of all
compounds was described under Experimental Section. Briefly,
phthalonitriles 3e6 were prepared by reaction of the correspond-
ing commercially available 4- and 3-nitrophthalonitrile with the
appropriate nucleophiles in the presence of potassium carbonate
[19,24,25] in dry N,N-dimethylformamide (DMF) that is more
effective than dimethylsulfoxide (DMSO) as the reaction solvent
because it resulted in improved yields of pure products.
Phthalocyanines 7e8 were readily prepared by cyclo-
tetramerization of phthalonitriles 3e4 employing 1,8-diazabicyclo
[5.4.0]undec-7-ene (DBU) and zinc acetate at 150 ꢁC whereas
phthalocyanines 9e10 were prepared by reacting 5e6 with DBU and
zinc acetate in butanol. Cationic phthalocyanines 11e14 were
obtained by treatment of 7e10 with methyl iodide in methylene
chloride. Intermediates 3e6 and dyes 7e14 were characterized by IR,
1H NMR and 13C NMR spectroscopy while EI-MS spectroscopy at
70 eV was performed for the characterization of 3e4, o-dinitriles
5e6 ESI-MS spectroscopy was determined with a Micromass Ultima
triple QUAD spectrometer since EI-MS at 70 eV or 40 eV only affor-
ded decomposition fragments. Besides, ESI-TOF mass spectroscopy
was employed for the characterization of phthalocyaninates 7e14.
Dyes were characterized by their photophysical properties; the
Q-band of substituted 1, 8(11), 15(18), 22(25) (
cyanines 8, 10, 12 and 14 lie at a longer wavelength (14e20 nm)
than 2,9(10),16(17),23(24) ( ) derivatives 7, 9, 11 and 13 (Table 1).
a) zinc(II) phthalo-
b
This red spectral shift is consistent with previous reports for
substituted zinc(II) phthalocyaninates with electron-releasing
groups [29,30] which can be reasonably explained by considering
the extent of the atomic orbital coefficients of the carbon atoms
derived from molecular orbital calculations. According to these
authors since the coefficient of the
that of carbon atoms in the HOMO, the destabilization extent of
this orbital by introducing electron-donating groups is larger when
said groups are linked to the -positions, a fact that makes the
a carbon atoms is larger than
b
a
HOMOeLUMO gap smaller and thereby results in the Q-band
shifting to a longer wavelength [30].
A bathochromic shift of 10 nm is observed for the Q-bands when
oxygen and sulfur are alternatively present. This effect was
observed for phthalocyanines 7e10 and cationic derivatives 11e14
respectively [31,32]. Such bathochromic shift into the therapeutic
window is highly promising for a second-generation photosensi-
tizer for biomedical purposes in photodynamic therapy. Cationic
R
N
N
N
N
R
R
NC
NC
NC
NC
N
Zn
N
N
N
R
NO
2
3-6
1,2
3, 4, 7, 8:
OCH CH N(CH )
R=
3 2
2
2
R
7-10
5, 6, 9, 10:
R=SCH CH N(CH )
3 2
2
2
+
-
OCH CH N(CH ) I
R=
11, 12:
2
2
3 3
+
-
13, 14:
SCH CH N(CH ) I
R=
2
2
3 3
11-14
Scheme 1. Synthetic route to phthalocyanines 11e14.

J. Marino et al. / European Journal of Medicinal Chemistry 45 (2010) 4129e4139
4131
dimethylaminosulfanyl derivatives 13 and 14 present higher molar
absorption coefficients ( ) in comparison with cationic dimethyl-
3
aminoethoxy analogs 11 and 12, this property improving skin light
penetration (Table 1).
The emission spectra were similar in all solvents studied indi-
cating that fluorescence can be attributed only to the monomer.
As previously reported [29,30] relative fluorescence quantum
yields (FF) are similar for compounds 7, 9, 11, 13 and present higher
FF values than those of compounds 8, 10, 12, 14.
The quantum yield of singlet oxygen production (FD) values
obtained for dyes 7e14 are listed in Table 1. Sample absorbances
were kept as low as possible to prevent aggregation yet enough to
obtain measurable values of quantum yield of singlet oxygen
production. FD in homogeneous solution are similar to those
previously published for lipophilic [26,33] as well as to cationic
dyes [27] corresponding to zinc(II) phthalocyanine monomers at
the concentration employed.
The photo-stability of 11e14 was analyzed inwater by measuring
the decrease in the Q-band over time irradiationwith redlight under
air. The time decay of the absorbance maxima of the Q-band for all
the compounds obeyed first-order kinetics as shown in Fig. 2.
The corresponding photodegradation constants k for phthalo-
cyanines 7e14 are listed in Table 1. Lesser values of k mean a higher
photo-oxidative stability. As shown in Table 1, tetra-
phthalocyanines 7, 9, 11 and 13 are more stable as compared with
their tetra- -substituted counterparts 8, 10, 12 and 14. Moreover,
b-substituted
a
the water solution of phthalocyanine 13 shows a lower value of k
thus indicating it is more stable than 14 at the different irradiation
times of our experiments and even in typical in vitro essays.
2.3. Photocytotoxicity studies
The effect of different concentrations of phthalocyanines 11e14
on KB cell survival was evaluated in the dark and upon exposure to
a light dose of 4.7 J cmꢀ2, 1.96 mW cmꢀ2 by employing the MTT
assay. No dark cytotoxicity was observed when cells were exposed
up to a 10 mM concentration of compounds 11, 12 and 13 (Fig. 3A).
On the contrary, when cells were treated with phthalocyanine 14,
cell viability diminished in a concentration-dependent manner, the
50% inhibition of cell proliferation (IC50
a concentration of 8 M (Fig. 3A). After irradiation, 13 and 14 were
found to be cytotoxic, and IC50 values from three independent
) being obtained at
m
experiments (mean ꢂ SE) were 2.7 ꢂ 0.6
m
M and 0.8 ꢂ 0.4
mM
respectively (Fig. 3B). No effect on cell proliferation was observed
after light exposure of KB cells previously incubated with phtha-
locyanines 11 and 12 (Fig. 3B). Since compound 14 diminished cell
growth even in the absence of light, it was not further studied. The
photocytotoxicity of 13 was also evaluated at different light doses.
As shown in Fig. 3C, the efficacy of this phthalocyanine at a 10
concentration was light dose-dependent, decreasing cell viability
by approximately 90% with a light dose of 4.7 J cmꢀ2
mM
.
It has been reported that cationic tetrasubstituted zinc(II)
phthalocyanines containing the dimethylaminoethoxy group
present photoredox activity on different malignant and non-
malignant cell lines such as XP 29MAmal, CX1, HeLa, S180 and
NO17 [34]; however, we have not detected, under our experimental
conditions, any anti-tumoral activity for phthalocyanines with
oxygen-linked cationic aliphatic chains (11 and 12).
In a recent manuscript [35] we stated that cultures of MCF-7c3
human breast cancer cells incubated with 2,3,9,10,16,17,23,24-
octakis[(N,N-dimethylamino)ethylsulfanyl]phthalocyaninatozinc
(II)-DMPC followed by a 27 J cmꢀ2 irradiation dose led to a 70%
Fig. 1. Absorption spectra of 11e14
in water.
1 ꢃ 10ꢀ6 M in DMF and
5 ꢃ 10ꢀ7
M
inactivation at 0.5 mM whereas a substantially greater phototoxic
effect was achieved at 1
100% cell death.
mM which resulted in approximately

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J. Marino et al. / European Journal of Medicinal Chemistry 45 (2010) 4129e4139
Table 1
Photophysical parameters obtained for phthalocyanines 7e10 in THF and 11e14 in DMF and water and photodegradation constants k for 7e10 in THF and 11e14 in water.
Pca
Solvent
Q-band
677
3
,
lmax Mꢀ1 cmꢀ1
FF
FD
k (10ꢀ3.minꢀ1
)
7
8
9
10
11
THF
THF
THF
THF
DMF
Water
DMF
Water
DMF
Water
DMF
Water
7.1 ꢃ 104
0.28 ꢂ 0.02
0.19 ꢂ 0.01
0.28 ꢂ 0.03
0.18 ꢂ 0.01
0.15 ꢂ 0.01
0.08 ꢂ 0.01
0.09 ꢂ 0.01
0.07 ꢂ 0.01
0.15 ꢂ 0.01
0.09 ꢂ 0.01
0.07 ꢂ 0.01
0.06 ꢂ 0.01
0.61 ꢂ 0.03
0.71 ꢂ 0.02
0.60 ꢂ 0.02
0.73 ꢂ 0.02
0.69 ꢂ 0.02
0.57 ꢂ 0.02
0.73 ꢂ 0.03
0.56 ꢂ 0.02
0.71 ꢂ 0.03
0.58 ꢂ 0.02
0.76 ꢂ 0.02
0.62 ꢂ 0.02
0.7
2.3
0.4
1.8
e
697
688
706
7.7 ꢃ 104
1.5 ꢃ 105
1.9 ꢃ 105
676
1.1 ꢃ 105
635, 674
696
657, 694
686
649, 684
700
653, 696
6.2 ꢃ 104 (674 nm)b
1.2 ꢃ 105
3.0
e
12
13
14
6.6 ꢃ 104 (694 nm)b
1.8 ꢃ 105
41
e
5.8 ꢃ 104 (684 nm)b
2.2 ꢃ 105
9.0
e
1.4 ꢃ 105 (696 nm)b
23
a
Experiments were carried out in concentrations ranging between 1 ꢃ 10ꢀ7 M and 6 ꢃ 10ꢀ6 M for 7e10 in THF and 11e14 in DMF and between 5 ꢃ 10ꢀ8 M and 5 ꢃ 10ꢀ7
M
for 11e14 in water.
b
3app at the monomer band.
2.4. Cellular uptake of phthalocyanine 13 in KB cells
The uptake of a 10 M solution of 13 was measured after incu-
The cellular uptake of phthalocyanines and related macrocycles
is known to depend on their hydrophobic character, overall charge
and charge distribution [36]. Thus, meso-tetraphenylporphyrin
derivatives bearing adjacent 5,15-di[4-(N-trimethylaminophenyl)-
15,20-diphenylporphyrin] (DADP-a) or opposite 5,10-di[4-(N-tri-
methylaminophenyl)-10,20-diphenylporphyrin] (DADP-o) cationic
N-(CH3)þ3 groups on two of the para-phenyl positions were exam-
ined for photodynamic properties as a function of charge distri-
bution. DADP-a selectively binds to mitochondria whereas DADP-o
which has a different charge distribution, showed lysosomal
affinity [37]. This property is shown by other sensitizers including
the chlorin NPe6 and the lutetium texaphyrin [38] since symmet-
rical cationic phthalocyanine with either Zn(II) or Si(IV) metal ions
localized preferentially within the cell lysosomes [36]. Fluorescence
images were also examined 3 h after irradiation. Under these
conditions, the punctuate pattern of the lysosomal probe was less
evident and no difference in mitochondrial labeling was detected
thus suggesting a possible lysosomal membrane permeabilization
(Figs. 5B and 6B). In addition, characteristic morphological changes
of apoptotic cells such as cell shrinkage and membrane blebbing,
were observed [39,40]. By using Hoechst 33258-staining, apoptotic
condensed and fragmented nuclei were also evident 3 h and 24 h
after irradiation of KB cells incubated with compound 13 (Fig. 7). A
nuclear localization of 13 was also detected, probably due to the
alteration of the nuclear membrane permeability (Figs. 5B and 6B).
m
bating KB cells for different time-periods in the dark. As shown in
Fig. 4A, a rapid significant uptake of 13 was observed after 30 min
exposure, reaching a plateau after 4 h. A cytosolic localization of 13
with a typical red fluorescence emission was observed by confocal
microscopy after 24 h incubation by exciting at 633 nm and
detecting the emission fluorescence at wavelengths >660 nm
(Fig. 4B). Cellular uptakes of compounds 11 and 12 were not
determined due to the absence of photocytotoxicity.
2.5. Intracellular localization of phthalocyanine 13
The subcellular localization of 13 was evaluated by confocal
microscopy after incubating KB cells for 24 h in the dark and
staining them with fluorescent dyes for specific organelles. As
shown in Fig. 5A, compound 13 e mainly found in the cytoplasmic
region e co-localized with the green fluorescence of the specific
lysosome probe. Thus, yellow vesicles were visualized from the
overlay of the red fluorescence from 13 and LysoTracker signals.
When MitoTracker Green was employed as
a mitochondria
fluorescent probe, it was found that compound 13 did not show
a mitochondrial localization, since no overlapping of red and green
signals was observed (Fig. 6A).
2.6. Evaluation of apoptosis mediated by phthalocyanine 13
3.0
It is well-known that the apoptosis is mediated by the activation
of aspartate specific cysteine proteases named caspases [39e44].
Activation of the initiator caspases-8 and -9 leads to the cleavage of
effector caspases such as caspase-3 which cleaves most cellular
substrates responsible for the typical morphological and
biochemical changes that occur during apoptosis. In order to
evaluate the induction of an apoptotic response mediated by
caspase activation, we measured the proteolytic activity of the
executioner caspase-3. As shown in Fig. 8, a slight increase in
caspase-3 activity was observed 1 h after light exposure of KB cells
preloaded with phthalocyanine 13. This activity reached a peak
after 3 h and remained high up to 24 h post-irradiation. No effect on
caspase activation was found in non-irradiated KB cells with or
without 13 or irradiated cells in the absence of 13. We also deter-
mined the ability of caspase-3 to hydrolyze PARP (Poly-ADP-
Ribose-Polymerase). PARP, a 113 kDa protein that binds and repairs
DNA strand breaks, is also a substrate for caspase-3 [45e47]. Thus,
a fragment of 89 kDa, unable to repair DNA damage originated
11
2.5
12
13
14
2.0
1.5
1.0
0.5
0.0
0
10
20
30
40
50
60
70
Time/min
Fig. 2. First-order plots for the photodegradation of phthalocyanines 11e14 in water.

J. Marino et al. / European Journal of Medicinal Chemistry 45 (2010) 4129e4139
4133
suggest that Pc 13 induced-apoptotic response mediated by
caspase-3 activation could be downstream a lysosomal event. Thus,
as previously reported,
a partial lysosomal membrane per-
meabilization and the consequent release of proteolytic enzymes
into cytosol lead to a lysosomal pathway of apoptosis which is
connected to mitochondrial dysfunction and caspase activation
[50e52].
3. Conclusions
The study herein investigates some biophysical aspects and the
in vitro photodynamic effects of a tetrasubstituted zinc(II) phtha-
locyanine replaced with sulfur-linked cationic aliphatic chains in
a human nasopharynx carcinoma cell line.
This water-soluble cationic phthalocyanine 13 was selected
from a panel of four analogs of oxygen- and sulfur-linked cationic
aliphatic isosteric compounds showing a FD of 0.58, a higher
bathochromic shift of 10 nm for the Q-band as compared with
oxygen-linked aliphatic phthalocyanine 11 and also the best photo-
stability in water as compared with their tetra-a-substituted
counterparts 12 and 14. The above shift into the therapeutic
window observed for 13 is an asset for biomedical applications in
photodynamic therapy. Besides, cationic dimethylaminosulfanyl
derivatives 13 and 14 present higher values of molar absorption
coefficients (3) in comparison with cationic dimethylaminoethoxy
analogs 11 and 12, this property improving skin light penetration.
Additionally, phthalocyanine 13 had no dark toxicity as compared
with its isomer 14 and showed an effective photodynamic activity
in KB cells which agrees with our previous finding about the
photodynamic efficiency of sulfur-linked zinc(II) phthalocyanines
[33]. This dye localized subcellularly within the lysosomes and
triggered after irradiation lysosomal-induced apoptosis leading to
a 90% cell death.
The above results have prompted us to consider performing in
vivo studies of 13.
4. Experimental
4.1. Materials and methods
4.1.1. Synthesis of photosensitizers
Melting points were determined on an Electrothermal 9100
capillary melting point apparatus. 1H NMR and 13C NMR were
recorded on a Bruker MSL 300 spectrometer. 1H NMR and 13C NMR
of phthalocyanines were recorded on a Bruker AM 500. Mass
spectra were obtained with a TRIO 2 (electronic ionization 70 eV)
spectrometer. Phthalocyanines ESI mass spectra were measured
with a Q-TOF Premier micromass/Waters Corporation spectrom-
eter. Electronic absorption spectra were determined with
a Shimadzu UV-3101 PC spectrophotometer. Fluorescence spectra
were monitored with a QuantaMaster Model QM-1 PTI spectro-
fluorometer. Infrared spectra were performed with a Perkin Elmer
Spectrum One FT-IR spectrometer.
Chromatography columns were prepared with TLC Kiesegel
(Merck), and Aluminum Oxide 90 standardized (Merck). N,N-
dimethylformamide was dried over 3 Ǻ molecular sieves during
72 h, then filtered and freshly distilled before utilization [53].
Diphenylisobenzofuran (DPBF), N,N-diethyl-4-nitrosoaniline, imi-
dazol and Methylene blue (MB) as well as all reagents were
provided by SigmaeAldrich. Tetra-t-butylphthalocyaninatozinc(II)
[26], 2,3,9,10,16,17,23,24-octakis(decyloxi)phthalocyaninatozinc(II)
(ZnPc) [33] and 2,3,9,10,16,17,23,24-octakis[(N,N-dimethylaminoe-
thylsulfanyl)]phthalocyaninatozinc(II) [31] were synthesized in our
laboratory.
Fig. 3. Effect of phthalocyanines 11e14 on KB cell viability. (A) Different concentrations
of 11 (C), 12 (o), 13 (-) and 14 ( ) were incubated with KB cells in the dark or (B)
7
were exposed to a light dose of 4.7 J cmꢀ2. (C) Cells preloaded with 10
mM of 13 (-)
were irradiated with various light doses. Control cells without 13 (,) were kept in the
dark. The MTT cytotoxicity assay was carried out 24 h after treatment as described
under Materials and methods. Results are expressed as the percentage of growth
obtained in the absence of phthalocyanines (control) and represent the mean ꢂ S.E. of
three different experiments.
during apoptosis, is obtained after proteolysis of PARP. Western blot
assays were performed to evaluate caspase-3-mediated PARP
cleavage (Fig. 9). A similar significant increase in cleaved PARP
(89 kDa fragment) was shown 3 h and 24 h after light exposure of
cells preloaded with 13. As controls, no effect on PARP cleavage was
observed in non-irradiated KB cells with or without 13 or irradiated
cells in the absence of 13. The intracellular localization of phtha-
locyanines or other compounds in lysosomes could contribute to
trigger an apoptotic cell death pathway [37,48,49]. Our results

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J. Marino et al. / European Journal of Medicinal Chemistry 45 (2010) 4129e4139
Fig. 4. Time-dependent uptake of phthalocyanine 13. (A) KB cells were incubated for different time-periods in the dark with a 10
mM concentration of 13. (B) Intracellular
localization of 13 was visualized by confocal microscopy after incubating KB cells for 24 h in the dark. The upper panel corresponds to phase contrast and lower panel to
fluorescence images.
4.1.2. 1,2-Dicyano-4-[2-(N,N-dimethylamino)ethoxy]benzene (3)
[19,34]
A mixture of 2-dimethylaminoethanol (0.30 mL, 2.983 mmol)
and K2CO3 (0.608 g, 4.4 mmol) in DMF (3 mL) was stirred under
argon at room temperature for 2 h and 4-nitrophthalonitrile (1)
(0.100 g, 0.58 mmol) was added and stirring continued for another
96 h. The reaction mixture was poured into water (50 mL) and
extracted with CH2Cl2 (3 ꢃ 30 mL) and the combined extracts were
washed with water (3 ꢃ 30 mL) and dried over Na2SO4. After
evaporation in vacuo, the residue was dissolved in a small volume
Fig. 5. Co-localization of 13 with lysosomes. KB cells incubated with 10 mM of 13 for 24 h were stained with LysoTracker Green DND-26 (75 nM, 30 min) and kept in the dark (A) or
exposed to a 4.7 J cmꢀ2 light dose and then incubated for an additional 3 h (B). Red fluorescence corresponds to 13 and green fluorescence represents the signal for LysoTracker
Green.

J. Marino et al. / European Journal of Medicinal Chemistry 45 (2010) 4129e4139
4135
Fig. 6. Intracellular localization of 13 and mitochondria staining. KB cells incubated with 10 mM of 13 for 24 h were stained with MitoTracker Green FM (100 nM, 45 min) and kept in
the dark (A) or exposed to a 4.7 J cmꢀ2 light dose and then incubated for an additional 3 h (B). Red fluorescence corresponds to 13 and green fluorescence represents the signal for
MitoTracker Green.
of CH2Cl2-MeOH (95:5) and filtered through a silicaegel column
packed and pre-washed with the same solvent. After evaporation of
the solvent, an oil was obtained. Yield: 0.110 g (89%). IR (KBr, cmꢀ1):
3065, 3030, 2967, 2235 (CN), 1599, 1562, 1494, 1473, 1310, 1293,
1257, 1182, 1104, 1033, 905, 858, 724, 642, 525, 462. 1H NMR
4.4 mmol) in DMF (3 mL) was reacted by applying the procedure
described for 3. An oil was obtained. Yield: 0.106 g (85%). IR (KBr,
cmꢀ1): 3000, 2229 (CN), 1662, 1583, 1473, 1345, 1289, 1062,
795,729. 1H NMR (300 MHz, CDCl3):
d (ppm) 2.67(s, 6H, CH3), 3.19
(t, 2H, J ¼ 5.13 Hz, CH2N), 4.51 (t, 2H, J ¼ 5.13 Hz, OCH2), 7.29 (d, 1H,
(300 MHz, CDCl3):
d
(ppm) 2.47(s, 6H, CH3), 2.93 (t, 2H, J ¼ 5.13 Hz,
J ¼ 8.72 Hz, Ar), 7.40 (d, 1H, J ¼ 7.7 Hz, Ar), 7.67 (t, 1H, J ¼ 7.95 Hz,
CH2N), 4.26 (t, 2H, J ¼ 5.13 Hz, OCH2), 7.24 (d, 1H, J ¼ 6.15 Hz, Ar),
Ar). 13C NMR:
d (ppm) 45.41, 57.92, 68.04, 101.68, 113.03, 116.31,
7.30 (s, 1H, Ar), 7.72 (d, 1H, J ¼ 8.72 Hz, Ar). 13C NMR:
d
(ppm) 45.35,
116.52, 120.90, 127.18, 134.11, 164.14. MS (EI, 70 eV): m/z (%) 214
(0.66) [Mþ ꢀ 1], 58 (100). Anal. Calcd. for C12H13N3O: C, 66.96; H,
6.09; N, 19.52. Found: C, 66.90; H, 6.07; N, 19.62.
58.02, 64.83, 106.82, 112.67, 115.30, 117.19, 119.78, 120.46, 135.13,
163.21. MS (EI, 70 eV): m/z (%) 215 (1.94) [Mþ], 216 (4.12) [Mþ þ 1],
58 (100). Anal. Calcd. for C12H13N3O: C, 66.96; H, 6.09; N, 19.52.
Found; C, 67.03; H, 6.06; N, 19.60.
4.1.4. 1,2-Dicyano-4-[2-(N,N-dimethylamino)ethylsulfanyl]benzene
(5) [20e22]
4.1.3. 1,2-Dicyano-3-[2-(N,N-dimethylamino)ethoxy]benzene
(4) [19]
A mixture of 3-nitrophthalonitrile (2) (0.100 g, 0.58 mmol),
2-dimethylaminoethanol (0.30 mL, 2.983 mmol), K2CO3 (0.608 g,
A mixture of 2-(dimethylamino)ethanethiol hydrochloride
(0.123 g, 0.867 mmol) and K2CO3 (0.608 g, 4.4 mmol) in DMF (3 mL)
was stirred under argon at room temperature for 2 h and 4-nitro-
phthalonitrile (1) (0.100 g, 0.58 mmol) was reacted by applying the
Fig. 7. Morphological changes visualization by Hoechst 33258-staining. KB cells incubated with 5
Non-irradiated cells (control) are shown in panel A. (Magnification 400ꢃ).
mM of 13 were stained 3 h (B) and 24 h (C) after irradiation with Hoechst 33258.

4136
J. Marino et al. / European Journal of Medicinal Chemistry 45 (2010) 4129e4139
1H, Ar), 7.64 (d, 1H, J ¼ 8.22 Hz, Ar). 13C NMR:
d (ppm) 30.71, 44.65,
58.30, 113.08, 114.72, 115.29, 115.38, 132.70, 136.46, 136.62, 141.83.
ESI-TOF MS: m/z (%) 232.2 (100)[Mþ þ 1], 233.2 (16.1) [Mþ þ 2].
Anal. Calcd. for C12H13N3S: C, 62.31; H, 5.66; N, 18.17. Found: C,
62.48; H, 5.69; N, 18.25.
4.1.5. 1,2-Dicyano-3-[(2-N,N-dimethylamino)ethylsulfanyl]benzene
(6) [21,22]
A
mixture of 2-(dimethylamino)ethanethiol hydrochloride
(0.123 g, 0.867 mmol) and K2CO3 (0.608 g, 4.4 mmol) in DMF (3 mL)
was reacted by applying the procedure described for 3. A white
solid was obtained that was recrystallized from ethanol. Yield:
0.074 g (55%); mp 94 ꢁC. IR (KBr, cmꢀ1):2969, 2943, 2862, 2824,
2792, 2777, 2229 (CN),1989,1812,1702,1566,1452,1421,1407,1377,
1303, 1289, 1269, 1253, 1229, 1196, 1156, 1100, 1067, 1042, 1011, 963,
903, 856, 797, 785, 727, 698, 621, 583, 552, 537, 499. 1H NMR (CDCl3,
300 MHz):
d
(ppm) 2.28(s, 6H, CH3),2.63 (t, 2H, J ¼ 7.18 Hz, CH2N),
3.17 (t, 2H, J ¼ 7.18 Hz, SCH2), 7.55 (dd, 1H, J ¼ 6.93, 2.31 Hz, Ar), 7.61
Fig. 8. Caspase-3 activation after irradiation. KB cells preloaded with 5 mM of 13 were
(s, 1H, Ar), 7.63 (d, 1H, J ¼ 6.93 Hz, Ar). 13C NMR:
d (ppm) 33.89,
irradiated with a light dose of 4.7 J cmꢀ2 and incubated for different time-periods. Cells
irradiated without 13 and incubated during the same periods were used as controls.
Since similar results were obtained, only the control value corresponding to time 0 is
shown. Caspase-3 activity was determined as indicated in Materials and methods.
44.54, 58.27, 113.20, 114.72, 115.33, 115.62, 123.65, 131.45, 132.10,
138.28. ESI-TOF MS: m/z (%) [M þ H]þ cald for: C12H13N3S 232.0902;
found: [M þ H]þ 232.0903. Anal. Calcd. for C12H13N3S: C, 62.31; H,
5.66; N, 18.17. Found: C, 62.39; H, 5.64; N, 18.28.
Enzymatic activity is expressed as the fluorescence per
mg of protein and represents
the mean values SE of three different experiments. Statistical significance in
ꢂ
comparison with the corresponding control is indicated by *p < 0.01 or **p < 0.0001.
4.1.6. 2,9(10),16(17),23(24)-Tetrakis[(2-dimethylamino)ethoxy]
phthalocyaninatozinc(II) (7) [23,34]
procedure described for 3. A solid residue was obtained that was
recrystallized from ethanol. Yield: 0.081 g (60%); mp 125e127 ꢁC. IR
(KBr, cmꢀ1): 2991, 2963, 2926, 2881, 2854, 2742, 2483, 2342, 2256,
2241(CN), 1981, 1842, 1793, 1736, 1610, 1534, 1357, 1298, 1263,
1179,1076, 924, 871, 801, 745, 600, 525, 489. 1H NMR (300 MHz,
A mixture of 3 (0.030 g, 0.139 mmol), anhydrous zinc acetate
(0.030 g, 0.137 mmol), and DBU (0.1 mL, 0.67 mmol) was stirred and
heated at 150 ꢁC under argon during 1 h. The mixture was cooled
down and 5 mL of CH2Cl2 was added, and centrifuged to eliminate
the excess of zinc acetate. The methylene chloride solution was
washed with water (3 ꢃ 5 mL) and evaporated in vacuo. The blue-
green solid was then applied to an aluminum oxide column packed
and pre-washed with CH2Cl2. After washing with CH2Cl2, the title
compound was eluted with CH2Cl2-methanol (98:2) and evapo-
rated. The dye was recrystallized from CH2Cl2ehexane. Yield
0.027 g (85%). IR (KBr, cmꢀ1): 2930, 1648, 1514, 1471, 1390, 1220,
CDCl3):
d
(ppm) 2.31(s, 6H, CH3), 2.65 (t, 2H, J ¼ 6.75 Hz, CH2N), 3.14
(t, 2H, J ¼ 6.75 Hz, SCH2), 7.51 (dd, 1H, J ¼ 8.22, 1.76 Hz, Ar), 7.58 (d,
1172, 1098, 832, 758. 1H NMR (500 MHz, CDCl3):
24H, CH3), 4.10 (m, 8H, CH2N), 4.65 (m, 8H, OCH2), 6.75e7.70 (m,
d(ppm) 3.35 (br s,
12H, Ar). 13C NMR:
d (ppm) 45.05, 57.85, 64.35, 74.50, 108.23, 116.37,
121.22, 124.51, 136.44, 142.13, 161.73. ESI-TOF MS: m/z [Mþ] calcd.
for C48H52N12O4Zn: 926.4094; found: [Mþ] 926.3523 and [Mþ þ 1]
927.3526.
4.1.7. 1,8(11),15(18),22(25)-Tetrakis[(2-dimethylamino)ethoxy]
phthalocyaninatozinc(II) (8) [22]
A mixture of 4 (0.040 g, 0.186 mmol), anhydrous zinc acetate
(0.040 g, 0.182 mmol), and DBU (0.1 mL, 0.67 mmol) was reacted by
applying the procedure described for 7. The dye was recrystallized
from CH2Cl2-hexane. Yield 0.031 g (73%). IR (KBr, cmꢀ1): 2925,1651,
1513, 1470, 1351, 1234, 1213, 1079, 831, 757. 1H NMR (500 MHz,
CDCl3):
d
(ppm) 2.34 (br s, 24H, CH3), 2.89 (m, 8H, CH2N), 3.82 (m,
(ppm) 45.52, 58.02,
8H, OCH2), 6.70e7.87 (m, 12H, Ar). 13C NMR:
d
65.26, 75.32, 81.73, 115.62, 121.41, 125.78, 130.65, 150.56, 159.84,
164.11, 176.70. ESI-TOF MS: m/z [Mþ] calcd. for C48H52N12O4Zn:
926.4094; found: [Mþ] 926.3655 and [Mþ þ 1] 927.3655.
4.1.8. 2,9(10),16(17),23(24)Tetrakis[(2-dimethylamino)
ethylsulfanyl]phthalocyaninatozinc(II) (9) [20e22]
A mixture of 5 (0.050 g, 0.216 mmol), anhydrous zinc acetate
(0.050 g, 0.225 mmol), and DBU (0.2 mL, 1.34 mmol) in anhydrous
butanol (4 mL) was stirred and heated at reflux temperature for 1 h
under argon. After evaporation in vacuo, the residue was treated
with methylene chloride (5 mL) and cooled during 24 h. After
centrifugation to eliminate the excess of zinc acetate, the solution
Fig. 9. PARP cleavage after PDT. KB cells with 5 mM of 13 were exposed to a light dose
of 4.7 J cmꢀ2, incubated for different time-periods and then submitted to Western blot
assays as described under Materials and methods (lanes 4e6). Non-irradiated cells
with or without 13 (lanes 1 and 2 respectively) or irradiated cells without 13 (lane 3)
were used as controls. Densitometric analyses show the relationship of the 89 kDa
fragment with regard to the total amount of PARP. Results are expressed as the
mean ꢂ SE of three different experiments (*p < 0.0001).

J. Marino et al. / European Journal of Medicinal Chemistry 45 (2010) 4129e4139
4137
was evaporated in vacuo leaving a blue solid, that was then applied
4.1.13. 1,8(11),15(18),22(25)-Tetrakis[(2-trimethylammonium)
to an aluminum oxide column packed and pre-washed with CH2Cl2.
After washing with CH2Cl2, the title compound was eluted with
CH2Cl2-methanol (98:2) and evaporated. The dye was recrystallized
from CH2Cl2-hexane. Yield 0.048 g (89%). IR (KBr, cmꢀ1): 2928,
1645, 1514, 1468, 1200, 1098, 1042, 958, 831, 758. 1H NMR
ethylsulfanyl]phthalocyaninatozinc(II) tetraioidide (14) [21,22]
This compound was prepared from 10 by applying the proce-
dure described for 13. Yield 0.043 g (91%). IR (KBr, cmꢀ1): 2929,
1620, 1474, 1385, 1315, 1107, 898, 742. 1H NMR (DMSO-d6,
500 MHz):
d (ppm) 2.41 (br s, 36H, CH3), 3.15 (m, 8H, CH2N), 4.17
(500 MHz, CDCl3):
d
(ppm) 1.61 (br s, 24H, CH3), 2.55 (m, 8H, CH2N),
(ppm) 30.88,
(m, 8H, SCH2), 7.13e8.02 (m, 12H, Ar). 13C NMR:
d (ppm) 53.80,
3.37 (m, 8H, SCH2), 6.80e7.80 (m, 12H, Ar). 13C NMR:
d
71.05, 121.98, 133.34, 151.08, 160.52, 172.03. ESI-TOF MS: m/z [Mþ]
44.91, 58.73, 121.05, 126.16, 128.59, 134.32, 139.73, 150.18, 165.25,
170.62. ESI-TOF MS: m/z [Mþ] calcd. for C48H52N12S4Zn: 990.6757;
found: [Mþ] 990.2611 and [Mþ þ 1] 991.2610.
calcd for C52H64N12S4I4Zn: 1558.9730; found [Mþ] 1558.9808.
4.2. Photophysical studies
4.1.9. 1,8(11),15(18),22(25)-Tetrakis[(2-dimethylamino)
ethylsulfanyl]phthalocyaninatozinc(II) (10) [21,22]
Absorption and emission spectra were recorded at different
concentrations employing a 10 ꢃ 10 mm quartz cuvette. All
experiments were performed at room temperature. Compounds
7e10 were measured in THF and 11e14 in DMF and water.
The emission spectra of 7, 9,11,13 were collected at an excitation
wavelength of 610 nm (Q-band) and recorded between 630 and
800 nm whereas 8, 10, 12, 14 were collected at an excitation
wavelength of 630 nm and recorded between 650 and 850 nm.
A mixture of 5 (0.050 g, 0.216 mmol), anhydrous zinc acetate
(0.050 g, 0.225 mmol), and DBU (0.2 mL, 1.34 mmol) in anhydrous
butanol (4 mL) was stirred and heated at reflux temperature for 2 h
under argon, following by the procedure described for 9. The dye
was recrystallized from CH2Cl2-hexane. Yield 0.038 g (70%). IR (KBr,
cmꢀ1): 2922, 2851,1622, 1459, 1385, 1315, 1261, 1107, 897, 798, 758,
742, 618. 1H NMR (500 MHz, CDCl3):
d
(ppm) 2.30(bs, 24H, CH3),
Fluorescence quantum yields
(FF) were determined by
2.65 (m, 8H, CH2N), 3.24 (m, 8H, SCH2), 7.10e7.83 (m, 12H, Ar). 13
C
comparison with those of tetra-t-butylphthalocyaninatozinc (II)
NMR:
d
(ppm) 31.62, 44.38, 58.71, 75.84, 122.59, 128.03, 133.13,
(
FF ¼ 0.30 in toluene) as a reference at lexc ¼ 610 nm for 7, 9, 11, 13
147.10, 159.23, 165.79, 172.15. ESI-TOF MS: m/z [Mþ] calcd. for
C48H52N12S4Zn: 990.2614; found [Mþ] 990.2596 and [M þ H]þ
991.2610.
and lexc ¼ 630 nm for 8, 10, 12, 14 [26].
Standard chemical monitor bleaching rates were used to calculate
the quantum yield of singlet oxygen generation rates [54]. For FD
studies in organic solvent, DPBF was used as a singlet oxygen chem-
ical quencher. To avoid chain reactions induced by DPBF in the
presence of singlet oxygen, the absorbance of DPBF was kept under
1.9. DPBF decay at 410 nm was monitored. For FD studies in aqueous
media, singlet oxygen monitor solutions composed of imidazol
4.1.10. 2,9(10),16(17),23(24)-Tetrakis[(2-trimethylammonium)
ethoxy]phthalocyaninatozinc(II) tetraioidide (11) [22,23,34]
Methyl iodide (3 mL, 48 mmol) was added to a solution of
phthalocyanine 7 (0.02 g, 0.013 mmol) in methylene chloride (5 mL)
and the solution was stirred for 48 h at 60 ꢁC. After cooling to room
temperature, the blue-green powder was centrifuged, dissolved in
methanol and precipitated with methylene chloride, and centri-
fuged again. Yield 0.031 g (95%). IR (KBr, cmꢀ1): 2922, 1608, 1489,
(8 mM) and N,N-diethyl-4-nitrosoaniline (40e50
mM), were air
saturated and irradiated. The bleaching of nitrosoaniline was fol-
lowed spectrophotometrically at 440 nm as a function of time.
Polychromatic irradiation was performed by using a projector lamp
(Philips 7748SEHJ, 24 Ve250 W) and a cut-off filter at 610 nm (Schott,
RG 610) and a water-filter were used to prevent ultraviolet and
infrared radiation. Samples 7e14 and references 2,3,9,10,16,17,23,24-
octakis[(N,N-dimethylaminoethylsulfanyl)]phthalocyaninatozinc(II)
1338, 1231, 1097, 968, 746. 1H NMR (DMSO-d6, 500 MHz):
3.38 (br s, 36H, CH3), 4.15 (m, 8H, CH2N), 4.69 (m, 8H, OCH2),
7.15e7.78 (m, 12H, Ar). 13C NMR:
(ppm) 54.91, 64.53, 66.15, 109.12,
d (ppm)
d
117.19, 124.61, 133.17, 149.76, 160.29, 163.18, 171.03. ESI-TOF MS: m/z
[Mþ] calcd for C52H64N12O4I4Zn: 1494.0647; found [Mþ] 1494.0723.
(
FD ¼ 0.69 in DMF) [31], ZnPc (FD ¼ 0.70 in THF) [33] and MB
(FD ¼ 0.56inwater) [55]were irradiated within the samewavelength
4.1.11. 1,8(11),15(18),22(25)-Tetrakis[(2-trimethylammonium)
ethoxy]phthalocyaninatozinc(II) tetraioidide (12) [22]
This compound was prepared from 8 by applying the procedure
described for 11. Yield 0.030 g (90%). IR (KBr, cmꢀ1): 2929, 1706,
1614, 1488, 1265, 1203, 1088, 979, 951,881, 805, 747. 1H NMR
interval l1el2, and FD was calculated according Amore et al. [56].
The photo-stability of phthalocyanines 11e14 was determined
by the decay of the Q-band intensity after exposure to red light [55]
The fluence rate was adjusted to 20 mW cmꢀ2. Measurements were
performed under air in water. Photodegradation rate constants k
were calculated as described elsewhere [57].
(DMSO-d6, 500 MHz):
d (ppm) 2.38 (br s, 36H, CH3), 3.02 (m, 8H,
CH2N), 3.99 (m, 8H, OCH2), 6.87e8.02 (m, 12H, Ar). 13C NMR:
d
(ppm) 54.90, 66.12, 109.25, 114.38, 116.30, 120.28, 126.63, 131.49,
4.3. Biological studies
150.05, 157.07, 165.29, 171.17, 176.72. ESI-TOF MS: m/z [Mþ] calcd for
C52H64N12O4I4Zn: 1494.0647; found [Mþ] 1494.0723.
4.3.1. Cells and culture conditions
Human nasopharynx carcinoma KB cells (ATCC CCL-17) were
maintained in Minimum Essential Medium (MEM, Gibco BRL)
containing 10% (v/v) fetal bovine serum (FBS, Gibco BRL), 2 mM
4.1.12. 2,9(10),16(17),23(24)-Tetrakis[(2-trimethylammonium)
ethylsulfanyl]phthalocyaninatozinc(II)tetraioidide (13) [20e22]
Methyl iodide (3 mL, 48 mmol) was added to a solution of
phthalocyanine 9 (0.03 g, 0.019 mmol) in methylene chloride
(5 mL) and the solution was stirred for 48 h at 60 ꢁC. After cooling to
room temperature, the blue-green powder was centrifuged, dis-
solved in methanol and precipitated with methylene chloride, and
centrifuged again. Yield 0.046 g (97%). IR (KBr, cmꢀ1): 2929, 1618,
L-glutamine, 50 U/mL penicillin, 50 mg/mL streptomycin, 1 mM
sodium pyruvate and 4 mM sodium bicarbonate, in a humidified
atmosphere of 5% CO2 at 37 ꢁC.
4.3.2. Dark cytotoxicity and photocytotoxicity
KB cells were plated at a density of 1 ꢃ 104 cells/well in 96-well
microplates and incubated overnight at 37 ꢁC until 70e80% of
confluence. Cationic phthalocyanines 11e14 were diluted in
1474, 1401, 1384, 1128, 605. 1H NMR (DMSO-d6, 500 MHz):
3.37 (br s, 36H, CH3), 4.21 (m, 8H, CH2N), 4.67 (m, 8H, SCH2),
d (ppm)
7.23e7.87 (m, 12H, Ar). 13C NMR:
d
(ppm) 53.80, 68.71, 70.12, 118.07,
a culture medium containing 4% FBS to give a 10 mM concentration.
2-Fold serial dilutions were then prepared and the cells were
incubated for 24 h. Compounds were then removed, a complete
131.50,140.43,157.03,160.29,171.18. ESI-TOF MS: m/z [Mþ] calcd for
C52H64N12S4I4Zn: 1558.9730; found [Mþ] 1558.9808.

4138
J. Marino et al. / European Journal of Medicinal Chemistry 45 (2010) 4129e4139
fresh culture medium was added and cells were exposed to a light
dose of 4.7 J cmꢀ2, 1.96 mW cmꢀ2 with a 150 W halogen lamp
equipped with a 10 mm water-filter to maintain cells cool and
attenuate IR radiation. In addition, a cut-off filter was employed to
bar wavelengths shorter than 630 nm. In parallel, non-irradiated
cells were used to study dark cytotoxicity. Following treatment,
cells were incubated for an additional 24 h period and cell viability
was determined by means of MTT reduction assay (Mosmann,
(per mg of protein) in relation to control. Non-irradiated cells
exposed or not to 13 and irradiated cells without 13 were treated as
described above and included as controls.
4.3.6. Western blot analysis
KB cells were incubated during 24 h with a 5 mM solution of 13,
then washed with PBS and exposed to a light dose of 4.7 J cmꢀ2 as
described above. After irradiation, cells were incubated for
different time-periods at 37 ꢁC and then, 1 ꢃ 106 cells were lysed
1983) [58]. Briefly, 100 mg of MTT tetrazolium salt were added and
incubated for 4 h at 37 ꢁC. Formazan crystals were solubilized in
0.01 M HCl in isopropyl alcohol and the absorbance (595 nm) was
measured in a Biotrack II Microplate Reader (Amersham Biosci-
ences). Different light doses were also employed to evaluate the
cytotoxic effect of 13. Irradiation of cells in the absence of 13 did not
modify cell viability (data not shown).
for 30 min at 4 ꢁC in a 10
mL lysis buffer (10% glycerol, 0.5% Triton X-
100, 1 mg/mL aprotinin, 1 mg/mL trysin inhibitor, 1 mg/mL leupeptin,
10 mM Na4P2O7, 10 mM NaF, 1 mM Na3VO4, 1 mM EDTA, 1 mM
PMSF, 150 mM NaCl, 50 mM Tris, pH 7.4). Clear cell lysate super-
natants were prepared by centrifugation and aliquots containing
100 mg of protein were resuspended in 0.063 M Tris/HCl, pH 6.8, 2%
SDS, 10% glycerol, 0.05% bromophenol blue, 5% 2-ME, submitted to
8% SDS-PAGE and then transferred to nitrocellulose membranes
(Amersham Biosciences, Piscataway, NY, U.S.A.) for 1 h at 100 V in
25 mM Tris, 195 mM glycine, 20% methanol, pH 8.2. After blocking
non-specific antibody binding sites with 10 mM Tris, 130 mM NaCl
and 0.05% Tween 20, pH 7.4, (TBS-T), containing 3% bovine ser-
albumin (BSA), membranes were incubated overnight at 4 ꢁC with
polyclonal antibody anti-PARP (Santa Cruz Biotechnology, CA, U.S.
A.) diluted in TBS-T, containing 1% BSA. Bound antibodies were
revealed with anti-rabbit IgG (horseradish peroxidase-conjugated
goat IgG from Santa Cruz Biotechnology, CA, U.S.A.) diluted in TBS-
T, 1% BSA. Immunoreactive proteins were visualized using the ECL
detection system (Amersham Biosciences, Piscataway, NY, U.S.A.)
according to the manufacturer’s instructions. For quantification of
band intensity, Western blots were scanned with a densitometer
(Gel Pro Analyzer 4.0). Non-irradiated cells exposed or not to 13
and irradiated cells without 13 were included as controls.
4.3.3. Time-dependent cellular uptake
KB cells (1 ꢃ 104 cells/well) were grown overnight at 37 ꢁC on
96-well microplates. A 10 mM solution of 13 in culture medium with
4% FBS was then exposed to cells from 30 min to 24 h in the dark.
After incubation, cells were washed with phosphate-buffer saline
(PBS) and solubilized in 100 mL/well of N,N-dimethylformamide.
Drug cellular uptake was measured by determining the fluores-
cence emission of 13 with an SLM-Aminco Bowman Series 2
Spectrofluorometer (Spectronic Instruments, Rochester, NY) at
680 nm excitation and 688 nm emission wavelengths. Results were
expressed as the emission relative to the total number of cells.
4.3.4. Intracellular localization
KB cells grown on coverslips were incubated with a 10
mM
solution of 13 for 24 h at 37 ꢁC in the dark. After removing the
compound with PBS, cells were stained with the following fluo-
rescent dyes for specific organelles diluted in the culture medium
without FBS: LysoTracker Green DND-26 (75 nM, 30 min) was used
to reveal lysosomes, and MitoTracker Green FM (100 nM, 45 min),
to visualize mitochondrias. Both organelle dyes were obtained from
Invitrogen. After washing with PBS, coverslips were fixed for 5 min
at room temperature with 4% paraformaldehyde and cells were
Acknowledgments
This work was supported by grants from the University of
Buenos Aires, the Consejo Nacional de Investigaciones Científicas y
Técnicas (CONICET) and the Agencia Nacional de Promoción Cien-
tífica y Tecnológica. We wish to thank the technical assistance as
regards chromatography of Ms. Juana Alcira Valdez. ESI-TOF mass
spectrometry assistance of Mr. Diego Cobice is also appreciated as
well as language supervision by Prof. Rex Davis.
then examined by fluorescence with
a confocal microscopy
Olympus FV 300. Phthalocyanine 13 was excited at 633 nm and its
emission was monitored at wavelengths >660 nm, and the
organelles (lysosomes and mitochondrias) were excited at 488 nm
and green fluorescence was detected at 510e530 nm. To examine
the localization of compound 13 after irradiation, cells treated as
described above were exposed to a light dose of 4.7 J cmꢀ2, and
then incubated for an additional 3 h previous to fixation.
References
[1] T.J. Dougherty, C.J. Gomer, B.W. Henderson, G. Jori, D. Kessel, M. Korbelik,
J. Moan, Q. Peng, J. Natl. Cancer Inst. 90 (1998) 889e905.
[2] L.B. Josefsen, R.W. Boyle, Br. J. Pharmacol. 154 (2008) 1e3 (and references therein).
[3] S.M. Fien, A.R. Oseroff, J. Natl. Compr. Canc. Netw. 5 (2007) 531e540 (and
references therein).
[4] R.K. Pandey, J. Porphyrins Phthalocyanines 4 (2000) 368e373.
[5] M.G.H. Vicente, Curr. Med. Chem. Anticancer Agents 1 (2001) 175e194.
[6] H. Ali, J.E. van Lier, Chem. Rev. 99 (1999) 2379e2450.
[7] I.J. MacDonald, T.J. Dougherty, J. Porphyrins Phthalocyanines 5 (2001) 105e129.
[8] M.R. Detty, S.L. Gibson, S.J. Wagner, J. Med. Chem. 47 (2004) 3897e3915.
[9] C.C. Leznoff, A.B.P. Lever (Eds.), Phthalocyanines: Properties and Applications,
vols. 1e4, VCH, New York, 1989, 1992, 1993, 1996.
4.3.5. Caspase activity assay
After incubating KB cells for 24 h with a 5 mM solution of 13, cells
were washed and then irradiated with a light dose of 4.7 J cmꢀ2 as
previously described. Cells were then incubated for different time-
periods at 37 ꢁC and 1 ꢃ 106 cells were lysed for 30 min at 4 ꢁC in
50
mL of lysis buffer (10 mM HEPES, pH 7.4, 50 mM NaCl, 2 mM
MgCl2, 5 mM EGTA, 1 mM PMSF, 2
m
g/mL leupeptin, 2 mg/mL
aprotinin) followed by three cycles of rapid freezing and thawing.
Cell lysates were centrifuged at 17,000g for 15 min and total protein
concentration was determined using Bradford reagent (Bio-Rad,
[10] B. Paquette, R.W. Boyle, H. Ali, A.H. MacLennan, T.G. Truscott, J.E. van Lier,
Photochem. Photobiol. 53 (1991) 323e327.
[11] P. Margaron, M.-J. Grégoire, V. Scasnárt, H. Ali, J.E. van Lier, Photochem.
Photobiol. 63 (1996) 217e223.
Hercules, CA, U.S.A.). Aliquots containing 100 mg of protein were
[12] K. Berg, J.C. Bommer, J. Moan, Photochem. Photobiol. 49 (1989) 587e594.
[13] V.C. Colussi, D.K. Feyes, J.W. Mulvihill, Y.-S. Li, M.E. Kenney, C.A. Elmets, N.
L. Oleinick, H. Mukhtar, Photochem. Photobiol. 69 (1999) 236e241.
[14] R.B. Boyle, B. Paquette, J.E. van Lier, Br. J. Cancer 65 (1992) 813e817.
[15] J.D. Miller, E.D. Baron, H. Scull, A. Hsia, J.C. Berlin, T. McCormick, V. Colussi, M.
E. Kenney, K.D. Cooper, N.L. Oleinick, Toxicol. Appl. Pharmacol. 224 (2007)
290e299.
[16] R.W. Boyle, C.C. Leznoff, J.E. van Lier, Br. J. Cancer 67 (1993) 1177e1181.
[17] M. Hu, N. Brasseur, S. Zeki Yildiz, J.E. van Lier, C.C. Leznoff, J. Med. Chem. 41
(1998) 1789e1802.
diluted in assay buffer (20 mM HEPES, 132 mM NaCl, 6 mM KCl,
1 mM MgSO4, 1.2 mM K2HPO4, pH 7.4), 20% glycerol, 5 mM DTT, and
incubated at 37 ꢁC for 2 h with 50
mM of the corresponding fluo-
rogenic substrate for caspase-3 (Ac-DEVD-AMC). Cleavage of the
substrate was monitored by AMC release in a SFM25 Konton
Fluorometer at 355 nm excitation and 460 nm emission wave-
lengths. Results were expressed as the change in fluorescence units

J. Marino et al. / European Journal of Medicinal Chemistry 45 (2010) 4129e4139
4139
[18] W. Liu, T.J. Jensen, F.R. Fronczek, R.P. Hammer, K.M. Smith, M.G.H. Vicente,
J. Med. Chem. 48 (2005) 1033e1041 (and references therein).
[19] W.O. Siegl, J. Heterocycl. Chem. 18 (1981) 1613e1618.
[20] D. Salih, G. Gulay, G. Ahmet, B. Ozer, J. Coord. Chem. 38 (1996) 287e293.
[21] M. Idowo, T. Nyokong, Polyhedron 28 (2009) 416e424.
[22] L. Zhang, J. Huang, L. Ren, M. Bai, L. Wu, B. Zhai, X. Zhou, Bioorg. Med. Chem.
16 (2008) 416e424.
[23] K. Kosuga, H. Hayashi, M. Handa, Chem. Lett. (1991) 1877e1880.
[24] T.M. Keller, T.R. Price, J.R. Griffith, Synthesis (1980) 613.
[25] D. Dei, G. Chiti, M.P. De Filippis, L. Fantetti, F. Giuliani, F. Giuntini, M.G. Soncin,
G. Jori, G. Roncucci, J. Porphyrins Phthalocyanines 10 (2006) 147e159.
[26] D.A. Fernández, J. Awruch, L.E. Dicelio, Photochem. Photobiol. 63 (1996)
784e792.
[27] D.A. Fernández, J. Awruch, L.E. Dicelio, J. Photochem. Photobiol. B, Biol. 41
(1997) 227e232.
[28] M.J. Stillman, T. Nyokong, Absorption and magnetic circular dichroism spec-
tral properties of phthalocyanines. Part 1: complexes of the dianion Pc (-2). in:
C.C. Leznoff, A.B.P. Lever (Eds.), Phthalocyanines: Properties and Applications.
VCH, New York, 1989, pp. 133e289.
[36] H. Li, T.H. Jensen, F.R. Fronczek, M.G.H. Vicente, J. Med. Chem. 51 (2008)
502e511 (and references therein).
[37] D. Kessel, R. Luguya, M.G.H. Vicente, Photochem. Photobiol. 78 (2003) 431e435.
[38] D. Kessel, Y. Luo, J. Porphyrins Phthalocyanines 5 (2001) 181e184.
[39] M.O. Hengartner, Nature 407 (2000) 770e776.
[40] H. Kaufmann, M.O. Hengartner, Trends Cell Biol. 11 (2001) 526e534.
[41] D.R. Green, J.C. Reed, Science 281 (1998) 1309e1312.
[42] N.A. Thornberry, Y. Lazebnik, Science 281 (1998) 1312e1316.
[43] M.G. Grutter, Curr. Opin. Struct. Biol. 10 (2000) 649e655.
[44] N.N. Danial, S.J. Korsmeyer, Cell 116 (2004) 205e219.
[45] A. Rodríguez-Hernández, G. Brea-Calvo, D.J. Fernández-Ayala, M. Cordero,
P. Navas, J.A. Sánchez-Alcáza, r, Apoptosis 11 (2006) 131e139.
[46] P.O. Hassa, S.S. Haenni, M. Elser, M.O. Hottiger, Microbiol. Mol. Biol. Rev. 70
(2006) 789e829.
[47] P.O. Hassa, M.O. Hottiger, Front. Biosci. 13 (2008) 3046e3082.
[48] D. Kessel, Y. Luo, P. Mathieu, J.J. Reiners Jr., Photochem. Photobiol. 71 (2000)
196e200.
[49] P. Boya, R.A. Gonzalez-Polo, D. Poncet, K. Andreau, H.L. Vieira, T. Roumier,
J.L. Perfettini, G. Kroemer, Oncogene 22 (2003) 3927e3936.
[50] M.E. Guicciardi, M. Leist, G.J. Gores, Oncogene 23 (2004) 2881e2890.
[51] C. Tardy, P. . Codogno, H. Autefage, T. Levade, N. Andrieu-Abadie, Biochim.
Biophys. Acta 1765 (2006) 101e125.
[29] N. Kobayashi, N. Sasaki, Y. Higashi, T. Osa, Inorg. Chem. 34 (1995) 1636e1637.
[30] N. Kobayashi, H. Ogata, N. Nonaka, E.A. Luk’yanets, Chem. Eur. J. 9 (2003)
5123e5134.
[31] C.A. Strassert, G.M. Bilmes, J. Awruch, L.E. Dicelio, Photochem. Photobiol. Sci. 7
(2008) 738e747.
[32] Z.A. Bayır, Dyes Pigm. 65 (2005) 235e242.
[33] M.E. Rodriguez, F. Morán, A. Bonansea, M. Monetti, D.A. Fernández, C.
A. Strassert, V. Rivarola, J. Awruch, L.E. Dicelio, Photochem. Photobiol. Sci. 2
(2003) 988e994.
[34] D. Wöhrle, N. Iskander, G. Graschew, H. Sinn, E.A. Friedrich, W. Maier-Borst,
J. Stern, P. Schlag, Photochem. Photobiol. 51 (1990) 351e356.
[35] N.B. Rumie Vittar, C.G. Prucca, C.A. Strassert, J. Awruch, V. Rivarola, Int.
J. Biochem. Cell Biol. 40 (2008) 2192e2205.
[52] P. Boya, G. Kroemer, Oncogene 27 (2008) 6434e6451.
[53] D.R. Burfield, R.H. Smithers, J. Org. Chem. 43 (1978) 3966e3968.
[54] M.G. Lagorio, L.E. Dicelio, E. San Roman, J. Photochem. Photobiol. A: Chem. 72
(1993) 153e161.
[55] F.Wilkinson,W.P.Helman, A.B.Ross, J.Phys. Chem. Ref.Data24(1995)663e1021.
[56] S. Amore, M.G. Lagorio, L.E. Dicelio, E. San Román, Prog. React. Kinet. Mech. 26
(2001) 159e177.
[57] G. Schnurpfeil, A.K. Sobbi, W. Spillger, H. Kliesch, D.J. Wöhrle, J. Porphyrins
Phthalocyanines 1 (1997) 159e167.
[58] T. Mosmann, J. Immunol. Methods 65 (1983) 55e63.