Synthesis and photodynamic properties of adamantylethoxy Zn(II) phthalocyanine derivatives in different media and in human red blood cells

Article abstract of DOI:10.1016/j.ejmech.2012.02.005

Novel unsymmetrically substituted Zn(II) phthalocyanine bearing an adamantylethoxy group (AZnPc) was synthesized by the ring expansion reaction of boron(III) subphthalocyanine chloride with an appropriated phthalonitrile derivative (APc). Also, APc was us

Full text of DOI:10.1016/j.ejmech.2012.02.005

European Journal of Medicinal Chemistry 50 (2012) 280e287
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and photodynamic properties of adamantylethoxy Zn(II)
phthalocyanine derivatives in different media and in human red blood cells
*
A. Laura Ochoa, Tomas C. Tempesti, Mariana B. Spesia, M. Elisa Milanesio, Edgardo N. Durantini
Departamento de Química, Facultad de Ciencias Exactas Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Agencia Postal Nro. 3,
X5804BYA Río Cuarto, Córdoba, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel unsymmetrically substituted Zn(II) phthalocyanine bearing an adamantylethoxy group (AZnPc)
was synthesized by the ring expansion reaction of boron(III) subphthalocyanine chloride with an
appropriated phthalonitrile derivative (APc). Also, APc was used to obtain a new Zn(II) phthalocyanine
bearing four adamantylethoxy groups (A4ZnPc) by cyclotetramerization reaction. The spectroscopic and
photodynamic properties of these photosensitizers were compared with those of a Zn(II) phthalocyanine
substituted by four methoxy groups (M4ZnPc) in different media. Similar results were obtained in N,N-
dimethylformamide. However, a higher photodynamic activity was found for AZnPc in a biomimetic
system formed by reverse micelles. This behavior was also observed in the presence of human red blood
(HRB) cells, which were used as an in vitro cellular model. Thus, AZnPc was the most effective photo-
sensitizer to produce HRB cells hemolysis. The photodynamic effect produced a decrease in the HRB cells
osmotic stability leading to the release of hemoglobin. Studies of photodynamic action mechanism
showed that photohemolysis of HRB cells was protected in the presence of azide ion, while the addition
of mannitol produced a negligible effect on the cellular photodamage, indicating the intermediacy of
O2(1Dg). Therefore, the presence of an adamantyl unit in the phthalocyanine macrocycle represents an
interesting molecular architecture for potential phototherapeutic agents.
Received 7 October 2011
Received in revised form
2 February 2012
Accepted 3 February 2012
Available online 10 February 2012
Keywords:
Synthesis
Zinc(II) phthalocyanine
Adamantane
Photodynamic properties
Human red blood
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
covalently linked to the macrocycle. Different strategies have been
employed for the preparation of these A3B-symmetry derivatives.
Phthalocyanine derivatives have attracted
a
considerable
However, the most efficient selective approach involves ring
expansion reaction of subphthalocyanines using a substituted
phthalonitrile [4e7].
amount of attention as second-generation of phototherapeutic
agents for the treatment of malignant tumors by photodynamic
therapy (PDT) [1,2]. These photosensitizers generally have larger
extinction coefficients in the therapeutically convenient
650e800 nm range and appropriated photochemical properties [3].
To date, a great variety of symmetrically substituted phthalocya-
nines have been extensively studied. The intrinsic symmetry of the
phthalocyanines sometimes represents a limitation for many
purposes. In contrast, unsymmetrically substituted phthalocya-
nines offer a possibility of improving selectivity since groups with
different properties may be attached to the ring. Thus, the chance of
designing and synthesizing unsymmetrical compounds with
substituents located at specific positions may facilitate enhanced
applications of phthalocyanines [4]. The design of A3B-phthalocy-
anines bearing one different (B) and three identical (A) isoindole
subunits can be used to incorporate a biological active structure
Adequate photosensitizers for PDT should have specific chem-
ical and biological properties [8]. Two of the photochemical
requisites are a high absorption coefficient in the visible region of
the spectrum and a long lifetime of triplet excited state to produce
efficiently reactive oxygen species (ROS). In this approach visible
light is used to active a photosensitizer, which can react with
molecules from its direct environment by electron or hydrogen
transfer, leading to the production of radicals (type I reaction), or it
can transfer its energy to oxygen, generating the highly reactive
singlet molecular oxygen, O2(1Dg) (type II reaction). Both mecha-
nisms can occur simultaneously and the ratio between the two
processes is influenced by the sensitizer, substrate and the nature of
the microenvironment [9].
In biological media, cell membranes seem to be important
targets for many antineoplastic photosensitizer agents. In this
sense, mammalian erythrocytes serve as convenient model cells for
exploration of physiological and molecular mechanisms involved in
* Corresponding author. Tel.: þ54 358 4676157; fax: þ54 358 4676233.
E-mail address: edurantini@exa.unrc.edu.ar (E.N. Durantini).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.005

A.L. Ochoa et al. / European Journal of Medicinal Chemistry 50 (2012) 280e287
281
PDT [10]. Red blood cells have been often used for in vitro PDT
studies [11e16]. Though erythrocytes are not ideal cells to evaluate
or quantitatively compare photosensitizers, they are useful to
elucidate the cellular and molecular principles of PDT [17].
In this paper, novel Zn(II) phthalocyanine derivatives
substituted by one (AZnPc) or four (A4ZnPc) adamantylethoxy
groups were synthesized. In previous studies, Zn(II) phthalocya-
nines substituted with four adamantane moieties were synthesized
from the corresponding phthalonitriles. However, in vitro photo-
dynamic activity using adamantyl phthalocyanines has not yet
been reported [18,19]. Adamantane is a highly lipophilic compound,
which has been used to modify the biological availability of several
molecules. Among the major biological activities displayed by
adamantane derivatives, the antiviral, antibacterial, antifungal,
anti-inflammatory are the most important ones [20,21]. The value
of the adamantyl group in drug design has been recognized most
recently in agents to treat iron overload disease, malaria and type 2
diabetes [22]. Also, in the present work, the spectroscopic and
photodynamic characteristics of AZnPc and A4ZnPc were studied in
a homogeneous medium and in reverse micelles. The results ob-
tained for these new adamantylethoxy phthalocyanines were
compared with those of Zn(II) 2,9,16,23-tetrakis(methoxy)phtha-
locyanine (M4ZnPc), which is an active photosensitizer for potential
use in PDT [23e25]. Photodynamic action was then evaluated
in vitro using human red blood (HRB) cells under different condi-
tions to obtain information about the effect produced by these
phthalocyanines.
0.58 mmol) in 5 mL of DMSO was stirred for 20 min at room
temperature under argon atmosphere. Then, the mixture was
treated with water (20 mL) and extracted with dichloromethane
(two portions of 20 mL each). The solvent was removed under
reduced pressure. The product was purified by flash chromatog-
raphy column (silica gel, dichloromethane/n-heptane 30%) yielding
113 mg (72%) of the pure phthalonitrile APn. TLC (silica gel) Rf
(dichloromethane/n-heptane 50%) ¼ 0.30. 1HNMR (CDCl3, TMS)
d
[ppm] 1.48 (t, 2H, CH2CH2O, J ¼ 6.0 Hz), 1.52 (br, s, 6H, CH2), 1.68
(br, s, 6H, CH2), 1.98 (bs, s, 3H, bridgehead, CH), 4.10 (t, 2H, CH2O,
J ¼ 6.0 Hz), 7.17 (dd, 1H, J ¼ 2.6 Hz, 9.0 Hz), 7.25 (d, 1H), 7.69 (d, 1H,
J ¼ 9.0 Hz). EI-MS [m/z] 306 (Mþ) (306.1732 calculated for
C20H22N2O). FT-IR (KBr) cmꢀ1 2916, 2848, 2232 (CN), 1596, 1450,
1344, 1325, 1289, 1255, 1087, 1011, 873, 835. Anal. calcd. C 78.40, H
7.24, N 9.14, found C 78.34, H 7.29, N 9.09.
2.2.2. Zn(II) 2-[2-(1-adamantyl)ethoxy]phthalocyanine (AZnPc)
A solution of APn (13 mg, 0.042 mmol) and 1,8-diazabicyclo
[5.4.0]undec-7-ene (DBU) (8 mL, 0.08 mmol) in DMSO (2 mL) was
heated to 130 ꢁC under an argon atmosphere. Then, a suspension of
boron(III) subphthalocyanine chloride (SubPc, 43 mg, 0.10 mmol)
and Zn(II) acetate dihydrate (30 mg, 0.14 mmol) in DMSO/1-
chloronaphthalene (2 mL, 2:1) was added drop-wise to the
heated mixture over a period of 1 h. The reaction was kept at
130e140 ꢁC for 24 h. The reaction mixture was cooled to room
temperature and precipitated with cool water (25 mL). The solid
was separated by centrifugation and the product was purified by
flash chromatography column (silica gel, dichloromethane/n-
heptane 10%) yielding 10 mg (15%) of the pure AZnPc. TLC (silica
gel) Rf (dichloromethane/n-heptane 10%) ¼ 0.10. 1HNMR (CDCl3,
2. Materials and methods
2.1. General
TMS) d [ppm] 1.50e1.80 (m, 14H, CH2CH2O and adamantanyl CH2),
1.98 (bs, s, 3H, bridgehead, CH), 4.25 (t, 2H, CH2O, J ¼ 6.0 Hz),
7.30e8.00 (m, 15H). ESI-MS [m/z] 755.2225 (M þ H)þ (754.2147
calculated for C44H34N8OZn). FT-IR (KBr) cmꢀ1 2922, 2850, 1602,
1454, 1331, 1279, 1089, 1057, 879, 750. Anal. calcd. C 69.89, H 4.53, N
14.82, found C 69.97, H 4.46, N 14.89. Absorption spectrum lmax
(DMF) [nm] (log ε) 343 (4.95), 604 (4.53), 668 (5.29).
Absorption and fluorescence spectra were recorded on a Shi-
madzu UV-2401PC spectrometer (Shimadzu Corporation, Tokyo,
Japan) and on a Spex FluoroMax spectrofluorometer (Horiba Jobin
Yvon Inc, Edison, NJ, USA), respectively. Proton nuclear magnetic
resonance spectra were recorded on an FT-NMR Bruker Advance 200
(Rheinstetten, Germany) at 200 MHz and on an FT-NMR Bruker
Avance DPX400 spectrometer at 400 MHz. Mass Spectra were taken
with a Bruker micrO-TOF-QII (Bruker Daltonics, MA, USA) equipment
with an ESI source operated in positive/negative mode, using
nitrogen as nebulizing and drying gas and sodium formiate (10 mM)
as internal calibrant. IR spectra were recorded on a Bruker Tensor 27
FT-IR (Ettlingen, Germany). The visible light source used to irradiate
erythrocytes was a Novamat 130 AF (Braun PhotoTechnik, Nürnberg,
Germany) slide projector equipped with a 150 W lamp. The light was
filtered through a 2.5 cm glass cuvette filled with water to absorb
heat. A wavelength range between 350 and 800 nm was selected by
optical filters. The light intensity at the treatment site was 300 W/m2
(Radiometer Laser Mate-Q, Coherent, Santa Clara, CA, USA).
2.2.3. Zn(II) 2,9(10),16(17),23(24)-tetrakis[2-(1-adamantyl)ethoxy]
phthalocyanine (A4ZnPc)
A solution of APn (20 mg, 0.065 mmol) and Zn(II) acetate
dihydrate (11 mg, 0.05 mmol) in 2 mL of n-pentanol was stirred for
10 min under argon atmosphere. Then, DBU (10 mL, 0.10 mmol) was
added and the mixture was refluxed for 15 h. The reaction mixture
was cooled at room temperature and precipitated with methanol
(25 mL). The solid was filtered and washed with water to yield 6 mg
(27%) of A4ZnPc. TLC (silica gel) Rf (dichloromethane/n-heptane
10%) ¼ 0.78. 1HNMR (CDCl3, TMS)
d [ppm] 1.50e1.80 (m, 56H,
CH2CH2O and adamantyl CH2), 1.99 (bs, s, 12H, bridgehead, CH),
4.10e4.20 (m, 8H, CH2O), 7.30e8.00 (m, 12H). ESI-MS [m/z]
1323.5909 (M þ Cl)ꢀ (negative mode) (1288.6220 calculated for
C80H88N8O4Zn). FT-IR (KBr) cmꢀ1 2920, 2848, 1605, 1447, 1329,
1277, 1093, 1049, 876, 747. Anal. calcd. C 74.43, H 6.87, N 8.68, found
C 74.38, H 6.94, N 8.62. Absorption spectrum lmax (DMF) [nm] 352
(4.98), 612 (4.59), 680 (5.31).
All the chemicals from Aldrich (Milwaukee, WI, USA) were used
without further purification. Silica gel thin-layer chromatography
(TLC) plates 250 microns from Analtech (Newark, DE, USA) were
used. Solvents (GR grade) from Merck (Darmstadt, Germany) were
distilled. Ultrapure water was obtained from a Labconco (Kansas,
MO, USA) equipment model 90901-01.
2.3. Spectroscopic studies
2.2. Synthesis of phthalocyanines
Absorption and fluorescence spectra were recorded at
25.0 ꢂ 0.5 ꢁC using 1 cm path length quartz cells. The fluorescence
Zn(II) 2,9,16,23-tetrakis(methoxy)phthalocyanine (M4ZnPc) was
synthesized as previously described [23].
quantum yield
(FF) of phthalocyanines were calculated by
comparison of the area below the corrected emission spectrum in
N,N-dimethylformamide (DMF) with that of Zn(II) phthalocyanine
(ZnPc) as a reference (FF ¼ 0.28) [26]. Sample and reference
absorbances were matched at the excitation wavelength (610 nm)
2.2.1. 4-[2-(1-Adamantyl)ethoxy]phthalonitrile (APn)
A mixture of 4-nitrophthalonitrile (90 mg, 0.52 mmol), 2-(1-
adamantyl)ethanol (97 mg, 0.54 mmol) and t-BuOK (65 mg,

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A.L. Ochoa et al. / European Journal of Medicinal Chemistry 50 (2012) 280e287
and the areas of the emission spectra were integrated in the range
together with 5 mM phthalocyanine. Then, the samples were
630e800 nm.
treated as described above.
2.8. Osmotic fragility evaluation
2.4. Steady state photolysis
Suspensions of HRB cells were treated with 5 mM photosensi-
Solutions of 9,10-dimethylanthracene (DMA, 35
mM) and
tizer as described above and irradiated for 3 min with visible light.
After that 100 L of HRB cells suspension were diluted in NaCl
photosensitizer in DMF were irradiated in 1 cm path length quartz
cells (2 mL) with monochromatic light at lirr ¼ 670 nm (sensitizer
absorbance 0.2), from a 75 W high-pressure Xe lamp through a high
intensity grating monochromator (Photon Technology Instrument,
Birmingham, NJ, USA). The light fluence rate was determined as
15 W/m2. The kinetics of DMA photooxidation were studied by
following the decrease of the absorbance (A) at lmax ¼ 378 nm. The
observed rate constants (kobs) were obtained by a linear least-
squares fit of the semilogarithmic plot of Ln A0/A vs. time. Photo-
oxidation of DMA was used to determine O2(1Dg) production by the
photosensitizers. ZnPc (FD ¼ 0.56) was used as a reference in DMF
[27]. Measurements of the sample and reference under the same
conditions afforded FD for phthalocyanines by direct comparison of
the slopes in the linear region of the plots. All the experiment were
performed at 25.0 ꢂ 0.5 ꢁC. The pooled standard deviation of the
kinetic data, using different prepared samples, was less than 10%.
m
solution (2 mL) of various concentrations (0.2e0.9% w/v) buffered
with 5 mM sodium phosphate (pH 7.4) [16]. Then, the suspensions
were incubated for 24 h at room temperature, centrifuged and the
percentage of osmotically lysed cells was estimated [15].
In all studies using HRB cells, control experiments were carried
out without illumination in the absence and presence of photo-
sensitizer and irradiating the HRB cells without photosensitizer.
Each experiment was repeated separately three times. Variation
between groups was evaluated using the Student t-test, with
a confidence level of 95% (p < 0.05) considered statistically
significant.
3. Results and discussion
3.1. Synthesis of phthalocyanines derivatives
2.5. Studies in reverse micelles
Two new Zn(II) phthalocyanines substituted by adamantyl units
were synthesized from phthalonitrile by a two-step procedure.
First, a phthalonitrile derivative (APn) bearing an adamantyl group
was prepared by a nucleophilic ipso-nitro substitution reaction of
4-nitrophthalonitrile with 2-(1-adamantyl)ethanol in DMSO
(Scheme 1). The reaction was carried out in the presence of t-BuOK
at room temperature. This basic medium was used to activate the
nucleophilic capacity of 2-(1-adamantyl)ethanol. The product APn
was isolated by flash chromatography with 72% yield. This phtha-
lonitrile derivative was employed to synthesize the AZnPc by ring
expansion of SubPc (Scheme 1). The reaction was performed in the
presence of DBU and Zn(II) acetate in DMSO/1-chloronaphthalene.
After heating the mixture for 24 h, the AZnPc phthalocyanine was
purified by flash chromatography with a 15% yield. Under these
conditions, only one phthalocyanine was obtained facilitating the
purification process. Therefore, this approach has advantages over
statistical condensation of two differently substituted phthaloni-
triles. The latter procedure can results in a complex mixture of
products, which difficult the purification process.
On the other hand, the cyclotetramerization of APn with Zn(II)
acetate in the presence of organic base DBU was performed in n-
pentanol (Scheme 1). After reflux for 15 h, the reaction results in the
formation of the corresponding A4ZnPc, which was precipitated
with methanol, filtered and washed with water to obtain 27% yield.
This method of synthesis produced a mixture of four regioisomers
with an adamantyl group at the 2- or 3-positions of each benzene
ring in the A4ZnPc molecule.
Studies in reverse micelles were performed using a stock solu-
tion of (AOT) 0.1 M, which was prepared by weighing and dilution
in n-heptane. The addition of water to the corresponding solution
was performed using a calibrated microsyringe. The amount of
water present in the system was expressed as the molar ratio
between water and the AOT present in the reverse micelle
(W0 ¼ [H2O]/[AOT]). In all experiments, W0 ¼ 10 was used. Pho-
tosensitized decomposition of DMA was made as explained above
in homogenic solution. The values of FD were calculated using ZnPc
(FD ¼ 0.56) as reference [28].
2.6. Studies in HRB cells
HRB cells were obtained from healthy donors. After extraction,
the whole blood was taken into anticoagulant EDTA, centrifuged
(3000 rpm for 5 min) to remove plasma and the leukocyte layer,
washed three times with phosphate buffer saline (PBS) solution
(137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4 and 8 mM Na2HPO4)
and centrifuged again. Erythrocytes were re-suspended in saline
solution (0.9% NaCl) to get a concentration of w106 cells/mL and
maintained at 4 ꢁC until use [29].
2.7. Photohemolysis assays
The HRB cell suspensions (w106 cells/mL) were placed in glass
flasks (2 mL) and dark incubated with different concentrations
Due to the lipophilicity of adamantane, the incorporation of the
adamantyl moiety in phthalocyanine macrocycle results in
compounds with relatively higher lipophilicity, which in turn can
modify the biological availability of these photosensitizers. In
AZnPc and A4ZnPc structures, the adamantyl center is isolated from
the phthalocyanine macrocycle by a two carbon aliphatic chain.
This spacer provides a higher mobility of the adamantyl moiety,
which could facilitate the interaction with the membrane.
(1e10
mM) of phthalocyanine at room temperature for 30 min.
Phthalocyanine was added from a 0.5 mM stock solution in DMF.
After incubation HRB cells suspensions were irradiated with visible
light as described above. Photosensitized hemolysis was carried out
at room temperature. After irradiation, the samples were kept 24 h
at room temperature in dark. Then, the tubes were centrifuged and
100 mL of supernatants were diluted in 2 mL of distilled water. The
hemoglobin content was determined by measuring the absorbance
at 413 nm. Results are expressed as percentage of hemolysis taking
100% as the absorbance obtained from a sample lysed in distilled
water [11]. Sodium azide (50 mM) or mannitol (50 mM) were added
to HRB cell suspensions from 2.5 M stock solutions in water and the
cells were incubated for 30 min at room temperature in dark
3.2. Spectroscopic studies
Absorption spectra of AZnPc and A4ZnPc were studied in
different solvents (Fig. 1). In DMF, both phthalocyanines showed an
intense Q-bands in the region of w670 nm, which are characteristic

A.L. Ochoa et al. / European Journal of Medicinal Chemistry 50 (2012) 280e287
283
Scheme 1. Synthesis of phthalocyanine derivatives AZnPc and A4ZnPc.
of Zn(II) phthalocyanine derivatives dissolved as monomeric
molecules [30,31]. Varying the solvent polarity, a small sol-
vatochromic effect is observed on the location of the absorption Q-
bands. These typical Q-bands were also observed for AZnPc in the
other organic solvents studied (Fig. 1A). The spectrum of AZnPc in
DMSO (Fig. 1A) was as intensive as in DMF at the same concen-
tration. The introduction of four adamantyl substituents in A4ZnPc
was accompanied by an increase in the lipophilic character of the
phthalocyanine macrocycle. Thus, A4ZnPc was better solubilized as
monomer in less polar solvents, for example toluene (Fig. 1B).
However, A4ZnPc was very poorly solubilized as monomer in more
polar solvents, such as DMSO and methanol, as shown by the
broadening and the low intensity of the Q-bands (Fig. 1B). Many
phthalocyanines have tendency to aggregate in polar solvents
especially when the macrocycle is substituted by lipophilic groups
[32]. On the other hand, both new adamantyl phthalocyanines are
hydrophobic in nature and they were not soluble in pure water.
In Table 1, the spectroscopic properties of AZnPc and A4ZnPc Q-
band were compared with that of M4ZnPc in DMF. The shape and
the Q-band absorption maximum of AZnPc closely match the
spectrum of ZnPc (668 nm) [30]. The Q-bands of A4ZnPc and
M4ZnPc present a w10 nm bathochromic shift respect to ZnPc due
to the auxochromic effect of the four ether groups.
The steady-state fluorescence emission spectra of AZnPc and
A4ZnPc were performed in DMF. As shown in Fig. 2, the spectra
showed two bands in the red spectral region, which are charac-
teristic for similar Zn(II) phthalocyanines [3]. As expected from the
absorption data, the emission maxima of A4ZnPc and M4ZnPc are
bathochromically shifted with respect to that of AZnPc. By
comparison with ZnPc as a reference, the values of fluorescence
quantum yields (FF) were calculated in DMF. The results of FF are
summarized in Table 1 and these values are according with previ-
ously reported for similar Zn(II) phthalocyanines in solution and
A
0.5
0.4
0.3
0.2
0.1
0.0
DMF
DMSO
DCM
toluene
methanol
500
600
700
800
Wavelength (nm)
B
0.6
0.5
0.4
0.3
0.2
0.1
0.0
DMF
DMSO
DCM
toluene
methanol
Table 1
Spectroscopic characteristics of phthalocyanines and fluorescence quantum yield
(FF) in DMF.
500
600
700
800
Phthalocyanine lmax (Abs)a lmax (Emission) ε (Lmolꢀ1 cmꢀ1
)
FF
Wavelength (nm)
AZnPc
A4ZnP
M4ZnP
668
680
676
672
688
678
1.96 ꢃ 105
2.08 ꢃ 105
2.01 ꢃ 105
0.17 ꢂ 0.02
0.25 ꢂ 0.02
0.26 ꢂ 0.02
Fig. 1. Absorption spectra of (A) AZnPc and (B) A4ZnPc in different solvents: N,N-
dimethylformamide (DMF), dimethylsulfoxide (DMSO), dichloromethane (DCM),
toluene and methanol; [phthalocyanine] ¼ 2
a
mM.
Q-band.

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A.L. Ochoa et al. / European Journal of Medicinal Chemistry 50 (2012) 280e287
1.0
0.8
0.6
0.4
0.2
0.0
A
0.6
AZnPc
A4ZnPc
0.5
M4ZnPc
0.4
0.3
0.2
0.1
0.0
650
700
750
800
0
50 100 150 200 250 300
Time (s)
Wavelength (nm)
Fig. 2. Normalized fluorescence emission spectra of AZnPc (solid line), A4ZnPc (dashed
line) and M4ZnPc (dotted line) in DMF; lexc ¼ 610 nm.
B
0.5
0.4
0.3
0.2
0.1
0.0
AZnPc
A4ZnPc
they are appropriated values for quantification of phthalocyanine
by fluorescence emission techniques [23].
M4ZnPc
3.3. Photodynamic properties
Photooxidation of 9,10-dimethylanthracene (DMA) sensitized
by AZnPc and A4ZnPc phthalocyanines was studied in DMF under
aerobic condition (Fig. 3A). Also, the DMA reaction was evaluated in
the presence of M4ZnPc. The values of the observed rate constant
(kobs) were calculated from first-order kinetic plots of the DMA
absorption at 378 nm vs. time (Table 2). In this medium, these
phthalocyanines photosensitized the decomposition of DMA with
similar rates.
0
500
1000
Time (s)
1500
2000
Taking into account that DMA quenches O2(1Dg) exclusively by
chemical reaction, this substrate can be used as a method to eval-
uate the ability of the sensitizers to produce O2(1Dg) in solution
[33]. The quantum yield of O2(1Dg) production (FD) were calculated
comparing the kobs for the corresponding phthalocyanine with that
for the reference (ZnPc, kobs ¼ (1.67 ꢂ 0.05) ꢃ 10ꢀ3 sꢀ1). Very close
values of FD were obtained for these phthalocyanines indicating
that they are photosensitizers higher efficient to produce O2(1Dg) in
DMF (Table 2). However, the values of FD can significantly change in
a different medium, diminishing mainly when the sensitizer is
partially aggregated [32]. Also, the photophysics of the phthalocy-
anines established in solution can be significantly modified in the
biological microenvironment where the sensitizer is localized. This
limits to predict the photodynamic efficiencies of phthalocyanines
in biological systems on the basic of photophysical investigations in
homogeneous medium.
Also, photooxidation of DMA sensitized by phthalocyanines
under aerobic conditions was performed in n-heptane/AOT (0.1 M)/
water (W0 ¼ 10). Microheterogeneous systems such as reverse
micelles are frequently used as an interesting model to mimic the
water pockets that are found in various bioaggregates [34,35]. In
reverse micelles, compounds with different polarities can be
located in a variety of microenvironments namely the organic
surrounded solvent, the water pool or at the micellar interface.
Because DMA is a non-polar compound, it is assumed that this
substrate is mainly solubilized in the organic pseudophase (n-
heptane) of the micellar system [34]. In this biomimetic microen-
vironment, the substrate reacts with the O2(1Dg) photosensitized by
phthalocyanine. From the plots in Fig. 3B, the values of the kobs
Fig. 3. First-order plots for the photooxidation of DMA photosensitized by AZnPc (:),
A4ZnPc and M4ZnPc (-) in (A) DMF and (B) n-heptane/AOT (0.1 M)/water
(W0 ¼ 10). Values represent mean ꢂ standard deviation of three separate experiments.
(
)
;
were calculated for DMA reaction in micellar system. As can be
observed in Table 2, the value of kobs for AZnPc is higher than those
for A4ZnPc and M4ZnPc, indicating that AZnPc was the better
photosensitizer to decompose DMA in reverse micelles of AOT.
Using ZnPc as a reference, the FD values were calculated for these
phthalocyanines (Table 2). As can be observed, AZnPc was the most
efficient photosensitizer to generate O2(1Dg
) in this micro-
heterogeneous medium. The reason for the monomeric state of
these lipophilic phthalocyanines is given by hydrophobic interac-
tions of the photosensitizer with the micelles. In micellar system,
phthalocyanines can be located in different microenvironments
depending on their structure. In the case of two strong hydrophobic
phthalocyanines (AZnPc and A4ZnPc) the different behavior
observed can be due to the asymmetry of AZnPc, which could
promote a better location in the micellar interface. The photody-
namic activity induced by A4ZnPc was very similar to that found for
M4ZnPc. Possibly; these symmetrically substituted phthalocya-
nines are located in the same micellar microenvironment. Thus,
photodynamic activity induced by these phthalocyanines in
micelles differs from that found in DMF, which is a good solvent for
all photosensitizers.
As can be observed in Table 2, the reaction rates of DMA sensi-
tized by these phthalocyanines in DMF are faster than in the AOT

A.L. Ochoa et al. / European Journal of Medicinal Chemistry 50 (2012) 280e287
285
Table 2
Kinetic parameters (kobs) for the photooxidation reaction of DMA and quantum yield of O2(1Dg) production (FD) of phthalocyanines in different media.
a
a
b
b
Phthalocyanine
kDobMsA (sꢀ1
)
FD
kDobMsA (sꢀ1
)
FD
AZnPc
A4ZnP
M4ZnP
(2.16 ꢂ 0.08) ꢃ 10ꢀ3
(2.16 ꢂ 0.08) ꢃ 10ꢀ3
(2.06 ꢂ 0.07) ꢃ 10ꢀ3
0.72 ꢂ 0.05
0.72 ꢂ 0.05
0.69 ꢂ 0.05
(2.20 ꢂ 0.08) ꢃ 10ꢀ4
(1.59 ꢂ 0.07) ꢃ 10ꢀ4
(1.63 ꢂ 0.07) ꢃ 10ꢀ4
0.75 ꢂ 0.05
0.55 ꢂ 0.05
0.56 ꢂ 0.05
a
in DMF.
b
in n-heptane/AOT (0.1 M)/water (W0 ¼ 10).
micellar system. Similar behavior was previously observed in AOT
reverse micelles using porphyrins as sensitizers [36]. In the AOT
micellar system, O2(1Dg) is partitioned between the internal and
external pseudophases with a partition constant of 0.11 [37].
Consequently, the photooxidation rate of DMA was diminished in
the organic pseudophase.
the dye binding to the cells and, as a consequence, the cytotoxic
efficiency [40]. Therefore, the existence of monomers in biological
surroundings is important because it allows obtaining a high
photosensitizer activity leading to cell damage [41].
Photolytic cellular activities produced by different concentra-
tions of AZnPc (1e10
mM) are shown in Fig. 4B. An increase in the
amount of AZnPc from 1 to 5
mM was accompanied by an
enhancement in the HRB photodamage. After a short irradiation
period of 5 min, the hemolytic effect increased from 13% to 75% for
3.4. Photohemolysis of HRB cells
cells treated with 1 and 5 mM, respectively. Under the last condition,
complete hemolysis was reached after 30 min of irradiation. Also,
In vitro the photodynamic activity of AZnPc, A4ZnPc and M4ZnPc
was evaluated by photohemolytic effect on HRB cells. In accordance
with cell-culture assays, where cell death is monitored by
membrane disruption, hemolysis of red blood cells can be regarded
as a mode of measuring death of erythrocytes [10,38]. Hemoglobin
absorption spectrum shows an intense Soret band at 413 nm and
two Q-bands at 500e600 nm [39] Thus, spectroscopic measure-
ments of hemoglobin released are much easier and faster to carry
out than cell staining and counting. In this way, phthalocyanines
are appropriated photosensitizers in presence of blood because
they absorb in the phototherapeutic window.
A
100
80
60
40
20
0
Suspensions of HRB cells (w106 cells/mL) in saline solution were
treated with different concentration of phthalocyanine (1e10 mM)
for 30 min at room temperature in dark. In these experiments,
the photosensitizers were added from a stock solution (0.5 mM) in
DMF because this solvent is soluble in PBS and it can dissolve both
phthalocyanines as monomers. Also, control assays using the
highest volume of DMF added to the cells (40 mL in 2 mL of cell
suspension) showed negligible hemolytic effect on HRB cells. After
24 h without irradiation negligible hemolysis was found indicating
no dark toxicity induced by these phthalocyanines. On the other
hand, light alone at the highest fluence rate used, did not induce
photohemolysis either. Therefore, the hemolysis obtained after
irradiation of the erythrocytes cells treated with the phthalocya-
nine was due to the photosensitization effect of the agent produced
by visible light.
0
10
20
30
Irradiation time (min)
B
100
80
60
40
20
0
The consequent cellular photohemolytic activities produced by
each phthalocyanine were compared using 5
mM and different
irradiation periods. As can be observed in Fig. 4A, the photo-
hemolysis after HRB irradiation with visible light was dependent
upon the phthalocyanine structure and light exposure level. The
photohemolysis produced by AZnPc was always higher than
M4ZnPc. After 5 min of irradiation, AZnPc induced a w75% of
cellular lysis, while the phototoxic activity of M4ZnPc was w38%. In
contrast, this was not the behavior found for the A4ZnPc, which
only induced a w5% of hemolysis after 30 min irradiation. One of
the problems that affect the photosensitizing ability of the phtha-
locyanines is the aggregation tendency due to the large
p conjugate
systems [32]. The aggregates present an efficient nonradiative
energy relaxation pathway, diminishing the triplet-state pop-
ulation and the O2(1Dg) quantum yield. In the present study, the
decrease in the photohemolytic activity induced by A4ZnPc with
respect to AZnPc can be due to its higher lipophilic character as
result of the four symmetrically distributed adamantyl groups
around the macrocycle. It is known that the nature of chemical
compounds and their hydrophilic/lipophilic nature strongly affects
0
10
20
30
Irradiation time (min)
Fig. 4. Photohemolysis of HRB cells (w106 cells/mL) treated with (A) 5
mM AZnPc (:),
A4ZnPc ( ) and M4ZnPc (-) and (B) 1 mM (-), 5 mM (C) and 10 mM (:) AZnPc for
;
30 min in dark and irradiated with visible light for different times (300 W/m2) in PBS
0.9% w/v NaCl solution. HRB cells control untreated with photosensitizer and irradiated
(B). Values represent mean ꢂ standard deviation of three separate experiments.

286
A.L. Ochoa et al. / European Journal of Medicinal Chemistry 50 (2012) 280e287
a phthalocyanine concentration of 10 mM did not produce signifi-
cant difference in the HRB photohemolysis in comparison with
M of sensitizer. This behavior may be due to saturation in the
5
m
amount of intracellular photosensitizer located in the photody-
namic action sites. Thus, a further increase in the amount of AZnPc
did not produce a greater effect on the cellular damage.
On the other hand, the membrane fragility after photodynamic
treatment was compared between AZnPc and M4ZnPc. Osmotic
fragility curves of erythrocytes treated with 5 mM phthalocyanine
and irradiated for 3 min with visible light are shown in Fig. 5. This
irradiation time was selected to produce a partial damage in the
HRB cells. The results indicate that the irradiation of HRB cells in the
presence of the photosensitizer produced an increase in the HRB
cells osmotic fragility. Thus, the concentration of NaCl corre-
sponding to 50% osmotic hemolysis of native erythrocytes is about
0.43% w/v NaCl, whereas that corresponding to cells treated with
AZnPc and M4ZnPc were 0.70% and 0.64% w/v, respectively. This
tendency was previously observed for ZnPc irradiated with various
light power [16]. Therefore, these phthalocyanines induced
a membrane photodamage, which is evidenced by a diminished in
the osmotic resistance. Membrane fluidization may be due to the
formation of membrane defects in the HRB cells caused by photo-
chemical oxidation, which plays a significant role in the formation
of hemolytic holes [16]. Such impairments of membrane integrity
decreased HRB cells osmotic stability.
Fig. 6. Photograph showing morphology of HRB cells (A) control untreated with
photosensitizer and irradiated for 3 min; (B) control treated with 5 M AZnPc in dark
and (C) treated with 5
M AZnPc irradiated for 3 min with visible light (300 W/m2);
100ꢃ microscope objective.
m
m
The morphology of erythrocytes was observed by microscopy
after incubation with 5 mM AZnPc and irradiated for 3 min. Repre-
In order to elucidate the photodynamic mechanism involved in
the photosensitized damage of the HRB membrane, the effect of
two suppressors, sodium azide and mannitol, were investigated.
Sodium azide is a know quencher of singlet oxygen O2(1Dg) pre-
venting type II photoprocess [43]. Thus, HRB cells were incubated
with 50 mM azide ion and this concentration was not hemolytic in
the dark or under irradiation without phthalocyanine (results not
sentative results are shown in Fig. 6. Microscopic observations
showed that the HRB cells changed their normal biconcave discoid
shape after photodynamic treatment. Erythrocytes lost their normal
profile and presented a spiny configuration with blebs in their
surfaces. This effect was present in all studied samples with partial
photohemolysis. These results provided evidence about membrane
deformation and an increase in the volume of cells indicating
a higher fragility after the photodynamic action. Similar deformation
of membrane has been previously found in oxidative damage of
human erythrocytes [42]. Photosensitized injure of HRB cells
produces a significant increase of membrane fluidity due to the
formation of defects in HRB cell membrane. This effect decreases
erythrocytes osmotic stability and releases the hemoglobin.
shown). In presence of 5 mM phthalocyanine and light, the addition
of azide ion produced a reduction in the photohemolysis of HRB
cells (Fig. 7, line 3). After 5 min of irradiation, the hemolysis
percentage of erythrocytes was reduced from 75% to 50% for AZnPc,
80
60
40
20
0
100
80
60
40
20
0
1
2
3
4
1
2
3
4
0.2
0.4
0.6
0.8
1.0
AZnPc
M ZnPc
[NaCl] (w/v)
4
Fig. 5. Osmotic fragility of HRB cells (w106 cells/mL) incubated with 5
mM of AZnPc
Fig. 7. Influence of azide ion and mannitol on the photohemolysis of HRB cells
(w106 cells/mL) treated with 5
M of AZnPc and M4ZnPc for 30 min in dark in 0.9% w/v
NaCl solution; 1) cells control in dark; 2) cells irradiated for 5 min; 3) cells containing
50 mM sodium azide and irradiated for 5 min; 4) cells containing 50 mM mannitol and
irradiated for 5 min; visible light (300 W/m2). Values represent mean ꢂ standard
deviation of three separate experiments.
(:) and M4ZnPc (-) for 30 min in dark and irradiated 3 min with visible light (300 W/
m2). After irradiation the HRB cells were immediately washed and incubated 30 min in
different buffered NaCl solutions. HRB cells control untreated and irradiated (B) and
treated with photosensitizer in dark (C). Values represent mean ꢂ standard deviation
of three separate experiments.
m

A.L. Ochoa et al. / European Journal of Medicinal Chemistry 50 (2012) 280e287
287
while the hemolysis was reduced from 38% to 12% for M4ZnPc.
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Acknowledgments
Authors thank Consejo Nacional de Investigaciones Científicas y
Técnicas (CONICET) of Argentina, SECYT Universidad Nacional de
Río Cuarto, MINCyT Córdoba and Agencia Nacional de Promoción
Científica
y Tecnológica (ANPCYT) of Argentina for financial
support. M.B.S., T.C.T., M.E.M. and E.N.D. are Scientific Members of
CONICET. A.L.O. thanks to CONICET for a doctoral fellowship.
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