Synthesis and photodynamic activity of zinc(II) phthalocyanine derivatives bearing methoxy and trifluoromethylbenzyloxy substituents in homogeneous and biological media

Article abstract of DOI:10.1016/j.bmc.2004.10.003

Two zinc(II) phthalocyanines bearing either four methoxy (ZnPc 3) or trifluoromethylbenzyloxy (ZnPc 4) substituents were synthesized and their photodynamic activity compared in both homogeneous medium containing photooxidizable substrate and in vitro on Hep-2 human larynx-carcinoma cell line. Although both sensitizers present similar behavior in organic solvent, a higher cell photoinactivation was found for ZnPc 3. Two zinc(II) phthalocyanines bearing either four methoxy (ZnPc 3) or trifluoromethylbenzyloxy (ZnPc 4) substituents have been synthesized by a two-step procedure starting from 4-nitrophthalonitrile. Absorption and fluorescence spectroscopic studies were analyzed in different media. These compounds are essentially non-aggregated in the organic solvent. Fluorescence quantum yields (φ_F) of 0.26 for ZnPc 3 and 0.25 for ZnPc 4 were calculated in tetrahydrofuran (THF). The photodynamic activity of these compounds was compared in both THF containing photooxidizable substrates and in vitro on Hep-2 human larynx-carcinoma cell line. The production of singlet molecular oxygen, O₂( ¹Δ_g), was determined using 9,10-dimethylanthracene yielding values of ～0.56 for both sensitizers. Under these conditions, the addition of β-carotene (Car) suppresses the O₂( ¹Δ_g)-mediated photooxidation. In biological medium, no dark cytotoxicity was found for cells incubated with 0.1 μM of phthalocyanines 3 and 4 for 24 h. However, under similar conditions 0.5 μM of ZnPc 4 was toxic (70% cell survival). The uptake into Hep-2 cells was evaluated using 0.1 μM of sensitizer, reaching values of ～0.05 nmol/10⁶ cells after 3 h of incubation at 37°C. The cell survival after irradiation of the cultures with visible light was dependent upon both light exposure level and intracellular sensitizer concentration. A higher photocytotoxic effect was found for ZnPc 3 with respect to 4 (32%/70% cell survival after 15 min of irradiation). Also, these studies were performed treating the cells with 0.5 μM of ZnPc 3. In this case, an increase in the uptake (～0.28 nmol/10 ⁶ cells) was observed, which is accompanied by a higher photocytotoxic activity (20% cell survival). These results show that even though both sensitizer present similar photophysical properties in homogeneous medium, the photodynamic behavior in cellular media can significantly be changed.

Full text of DOI:10.1016/j.bmc.2004.10.003

Bioorganic & Medicinal Chemistry 13 (2005) 39–46
Synthesis and photodynamic activity of zinc(II) phthalocyanine
derivatives bearing methoxy and trifluoromethylbenzyloxy
substituents in homogeneous and biological media
b
E. Ines Yslas, Viviana Rivarola and Edgardo N. Durantini
b
a,
*
´
a
´
´
´
Departamento de Quımica, Universidad Nacional de Rıo Cuarto, Agencia Postal Nro 3, X580BYA Rıo Cuarto, Argentina
´
b
´ ´
Departamento de Biologıa Molecular, Universidad Nacional de Rıo Cuarto, Agencia Postal Nro 3, X580BYA Rıo Cuarto, Argentina
Received 10 September 2004; revised 1 October 2004; accepted 4 October 2004
Available online 28 October 2004
Abstract—Two zinc(II) phthalocyanines bearing either four methoxy (ZnPc 3) or trifluoromethylbenzyloxy (ZnPc 4) substituents
have been synthesized by a two-step procedure starting from 4-nitrophthalonitrile. Absorption and fluorescence spectroscopic stud-
ies were analyzed in different media. These compounds are essentially non-aggregated in the organic solvent. Fluorescence quantum
yields (/F) of 0.26 for ZnPc 3 and 0.25 for ZnPc 4 were calculated in tetrahydrofuran (THF). The photodynamic activity of these
compounds was compared in both THF containing photooxidizable substrates and in vitro on Hep-2 human larynx-carcinoma cell
line. The production of singlet molecular oxygen, O2(1Dg), was determined using 9,10-dimethylanthracene yielding values of ꢀ0.56
for both sensitizers. Under these conditions, the addition of b-carotene (Car) suppresses the O2(1Dg)-mediated photooxidation. In
biological medium, no dark cytotoxicity was found for cells incubated with 0.1lM of phthalocyanines 3 and 4 for 24h. However,
under similar conditions 0.5lM of ZnPc 4 was toxic (70% cell survival). The uptake into Hep-2 cells was evaluated using 0.1lM of
sensitizer, reaching values of ꢀ0.05nmol/106 cells after 3h of incubation at 37ꢀC. The cell survival after irradiation of the cultures
with visible light was dependent upon both light exposure level and intracellular sensitizer concentration. A higher photocytotoxic
effect was found for ZnPc 3 with respect to 4 (32%/70% cell survival after 15min of irradiation). Also, these studies were performed
treating the cells with 0.5lM of ZnPc 3. In this case, an increase in the uptake (ꢀ0.28nmol/106 cells) was observed, which is accom-
panied by a higher photocytotoxic activity (20% cell survival). These results show that even though both sensitizer present similar
photophysical properties in homogeneous medium, the photodynamic behavior in cellular media can significantly be changed.
ꢁ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
the main species responsible for cell inactivation.1 Both
reactions can occur simultaneously and the ratio be-
tween two processes depends on the sensitizer, substrate
and the nature of the medium.1
One of more recent and promising applications of ph-
thalocyanine in medicine is in the detection and cure
of tumors.1,2 Photodynamic therapy (PDT) is an early
new technique for treating cancer.3,4 After administra-
tion of a photosensitizer, which is selectively retained
by tumor cells, the subsequent irradiation with visible
light in the presence of oxygen specifically inactivates
neoplastic cells.5,6 Basically two types of reactions can
occur after photoactivation of the photosensitizer. One
involves the generation of free radicals (type I photo-
chemical reaction) and in the other, the production of
singlet molecular oxygen, O2(1Dg), (type II) which is
Phthalocyanines derivatives exhibit a high absorption
coefficient in the visible region of the spectrum, mainly
in the phototherapeutic window (600–800nm) and a
long lifetime of triplet excited state to produce efficie-
ntly O2(1Dg).1,2 Liposomal zinc(II) phthalocyanine
(ZnPc) has proved very useful for photokilling of HeLa
cells and tumor localization in vivo and PDT of murine
tumors.7,8 Also, the photobiological properties of vari-
ous phthalocyanine derivatives indicate that they can
be very promising photosensitizers for clinical applica-
tion of PDT.2,6
Keywords: Phthalocyanines; Photosensitizer; Singlet oxygen; Photo-
inactivation; Photodynamic therapy.
*
In previous studies, we have investigated the photody-
namic activity of porphyrin derivatives substituted by
Corresponding author. Tel.: +54 0358 4676157; fax: +54 0358
4676233; e-mail: edurantini@exa.unrc.edu.ar
0968-0896/$ - see front matter ꢁ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.10.003

40
E. I. Yslas et al. / Bioorg. Med. Chem. 13 (2005) 39–46
methoxy groups in different biomimetic and biological
media.9–12 The presence of methoxy groups appears to
be beneficial from the standpoint of tumor localiz-
ation.13,14 Also, a novel meso-2,4,6-trimethoxyphenyl
porphyrin covalently linked to a trifluoromethylphenyl
derivative was evaluated as interesting photosensi-
tizer.15,16 An attractive photobiological feature is its
ability to inactivate cultured tumor cells with high effi-
ciency by apoptotic or necrotic modes depending on
the light dose.16,17 The influence of the trifluoromethyl
group in biologically active molecules is often associated
with the increased lipophilicity that this substituent im-
parts.18 Fluorinated porphyrin derivatives have been
synthesized for application in diagnosis and therapy of
cancer.19,20 Likewise, fluorinated zinc phthalocyanines
offer some advantages over non-fluorinated derivatives
as photosensitizer.21 Also, studies using zinc hexadeca-
fluorophthalocyanine in mice indicate a favorable
tumor/muscle ratio.22
RO
OR
N
Zn
N
DBU
CN
CN
N
N
N
N
N
N
Zn(H3CCOO)2
1-pentanol
RO
RO
OR
3 R: CH3
4 R: CH2
CF3
Scheme 2. Synthesis of phthalocyanine derivatives.
2.2. Spectroscopic studies
The absorption spectra of ZnPcs 3 and 4 show the typ-
ical Soret and Q-bands bands characteristic of zinc(II)
phthalocyanine (ZnPc).25 Figure 1A compares the
1.0
0.8
0.6
0.4
0.2
0.0
A
Taking into account these considerations, in this paper
we report the synthesis and the photodynamic activity
of two zinc(II) phthalocyanine derivatives substi-
tuted by either four methoxy (ZnPc 3) or four tri-
fluoromethylbenzyloxy (ZnPc 4) groups. In particular,
the presence of four –CF3 groups was used to obtain
a highly lipophilic sensitizer.18 The photosensitizing
properties of these compounds were compared in
homogeneous medium bearing photooxidizable
substrates and in Hep-2 human larynx-carcinoma cell
line. The results contribute to understand the photo-
dynamic process induced by these agents in PDT
treatments.
400
500
600
700
wavelength (nm)
1.0
0.8
0.6
0.4
0.2
0.0
B
2. Results and discussion
2.1. Synthesis
Zinc(II) phthalocyanines (ZnPcs) 3 and 4 were synthe-
sized by a two-step procedure.23,24 First, dinitriles 1
and 2 were prepared by a nucleophilic ipso-nitro substi-
tution reaction of 4-nitrophthalonitrile with the corre-
spondent alcohol in the presence of K2CO3 (Scheme
1). Dinitriles 1 and 2 were isolated by flash chromatog-
raphy with 68% and 60% yield, respectively. The cyclo-
tetramerization of dinitriles 1 and 2 with zinc(II) acetate
in the presence of organic base 1,8-diazabicy-
clo[5.4.0]undec-7-ene (DBU) was performed in n-penta-
nol. After reflux for 10h, the reaction results in the
formation of the corresponding ZnPcs 3 and 4 as mix-
tures of constitutional isomers, which were purified by
re-crystallization to yield 38% and 41%, respectively
Scheme 2.
650
700
750
800
wavelength (nm)
1.0
0.8
0.6
0.4
0.2
0.0
C
300
400
500
600
700
wavelength (nm)
CN
CN
CN
ROH
1 R: CH3
2 R: CH2
Figure 1. (A) Absorption spectra; (B) fluorescence emission spectra
(kexc = 608nm) and (C) fluorescence excitation spectra (kem = 740nm)
of ZnPc 3 (solid line), ZnPc 4 (dashed line) and ZnPc (dotted line) in
THF.
K2CO3
DMF
CF3
O2N
CN
RO
Scheme 1. Synthesis of nitrile derivatives.

E. I. Yslas et al. / Bioorg. Med. Chem. 13 (2005) 39–46
41
Table 1. Absorption and fluorescence emission data for phthalocya-
nines 3 and 4 in different solvents
found between the absorption and excitation spectra,
indicating that sensitizers 3 and 4 are essentially nonag-
gregated in these solutions.
Solvent
Absorption kmax
(nm)
Emission kmax
(nm)b
2.3. Photosensitized decomposition of substrates and
singlet oxygen production
ZnPc 3
Toluene
THF
352
347
358
339
611
608
614
627
678
675
682
682
687
685
693
690
758
753
766
764
Pyridine
FCSa
The aerobic irradiation with monochromatic light of
photosensitizer 3 and 4 in THF was performed in the
presence of 9,10-dimethylanthracene (DMA). Figure
2A shows typical semilogarithmic plots describing the
progress of the reaction for DMA. From these plots
the values of the observed rate constant (kobsDMA) were
obtained, irradiating the system with light of 675nm
(Table 2). From the slopes of Figure 2A the quantum
yield of O2(1Dg) production (UD) were calculated com-
paring the slope for the phthalocyanines with the corre-
sponding slope obtained for the reference, ZnPc.28 The
results show a similar value of UD ꢀ 0.56 for both
phthalocyanines and it is a quite reasonable value for
ZnPc derivatives in this solvent.25,28
ZnPc 4
Toluene
THF
358
349
357
345
612
607
615
628
680
673
684
683
688
685
693
687
759
755
764
758
Pyridine
FCSa
a 10% vol/vol in PBS.
b kexc = 608nm.
ZnPcs spectra recorded in THF. Values of
e = 2.15 · 105 Mꢁ1 sꢁ1 at 675 and 2.20 · 105 Mꢁ1 sꢁ1 at
673 were calculated for 3 and 4, respectively. The Q-
band presents a bathochromic shift by ꢀ10nm when
compared with that of ZnPc. In addition, the wave-
length absorption maxima of these ZnPcs were studied
in different media (Table 1). A sharp absorption band
was obtained indicating that there is no aggregation of
these ZnPcs in the organic solvents. A small solvato-
chromic effect in pure solvents is observed on the loca-
tion of the absorption band showing a slight red-shift
upon solubilization in pyridine.25 These ZnPcs are not
soluble in water as monomers, the absorption spectrum
shows a unique broad band at ꢀ630nm (not shown).
Many phthalocyanines have tendency to aggregate in
aqueous solutions especially of high ionic strength.1
The addition of an excess of protein into such solutions
usually breaks aggregates to monomers followed by the
binding of the monomer.26 In our cases, when fetal calf
serum (FCS) is added to a fixed amount of PcZns
(1nmol) in saline PBS solution produces a narrowing
and enhancement in intensity of Q-bands (Table 1).
Under these conditions, lipophilic phthalocyanines are
preferentially bound to the hydrophobic environment
of the lipoproteins in the serum.27
To evaluate the mediation of O2(1Dg) in the photooxid-
ation of DMA sensitized by ZnPcs, the reaction was
studied in the presence of b-carotene (Car, 12lM).
Under this condition, Car can quench O2(1Dg) through
energy transfer or chemical reaction.29–32 From the sem-
ilogarithmic plots (Fig. 2B) the values of the observed
rate constant in the presence (kobsDMA + Car) of Car
were calculated. The results are gathered in Table 2.
0.4
A
0.3
0.2
0.1
0.0
0
200
400
600
800
irradiation time (s)
The steady-state fluorescence emission spectra of these
ZnPcs present two maxima, as shown in Figure 1B
and Table 1. As can be observed, the kem values are
not very different in these media and no changes in the
fluorescence profile were observed, showing that the
same species were responsible for the fluorescence emis-
sion under these conditions. The fluorescence quantum
yields (/F) of the ZnPcs were calculated by steady state
B
0.12
0.08
0.04
0.00
comparative method using ZnPc as
a reference
(/F = 0.28 in THF). Values of /F = 0.26 0.01 and
0.25 0.01 were obtained for 3 and 4, respectively.
These results are consistent with those reported for sim-
ilar ZnPcs.1 The fluorescence of these sensitizers are
appropriate for detection and quantification of the agent
located in biological media.12
0
400
800
1200
1600
irradiation time (s)
Figure 2. First-order plots for the photooxidation of (A) DMA
(30lM) and (B) DMA (30lM) in the presence of Car (12lM) by ZnPc
3 (j), ZnPc 4 (d) and ZnPc (m), in THF, kirr = 675nm. Values
represent mean standard deviation.
Also, fluorescence excitation spectra of ZnPcs 3 and 4
were recorded in the different media. In Figure 1C they
are compared in THF. In all cases, a close relationship is

42
E. I. Yslas et al. / Bioorg. Med. Chem. 13 (2005) 39–46
Table 2. Kinetic parameters for the photooxidation reaction of DMA
without b-carotene and in the presence of b-carotene (12lM) in THF
0.4
0.3
0.2
0.1
0.0
A
DMA
DMA + Car
a
gq
Phthalocyanine kobs
(sꢁ1
)
kobs
(sꢁ1 b
)
ZnPc
(4.92 0.05) · 10ꢁ4 (8.1 0.1) · 10ꢁ5
(4.95 0.07) · 10ꢁ4 (7.9 0.1) · 10ꢁ5
(4.94 0.06) · 10ꢁ4 (8.3 0.1) · 10ꢁ5
0.84
0.84
0.83
3
4
a gq = (1ꢁkobsDMA + Car/kobsDMA).
As can be observed the reaction of DMA was quenched
with an efficiency (gq = 1ꢁkobsDMA + Car/kobs
DMA
)
0
5
10
15
20
25
of ꢀ0.84 for both phthalocyanines as sensitizers.
incubation time (h)
On the other hand, the Stern–Volmer equation
=
B
control
3
4
(kobsDMA/kobs
1 + KSV [Car], KSV = s0kq
DMA + Car
100
where kq represents the quenching constant of Car and
s0 the excited state lifetime of O2(1Dg) in the absence
of Car) was used to analyze the O2(1Dg) mediation
in this precess.12 Taking into account the value
of KSV = 3.9 0.2 · 105 Mꢁ1 previously reported in
THF, a ratio of kobsDMA + Car/kobsDMA = 0.17 was cal-
culated for [Car] = 12lM.33 This result is in agreement
with that found for gq sensitized by ZnPcs 3 and 4, indi-
cating that O2(1Dg) is the main species responsible for
DMA photooxidation in THF.
80
60
40
20
0
5
0
15
irradiation time (min)
2.4. Studies in vitro on Hep-2 cells
Figure 3. (A) Uptake of ZnPc 3 (j) 0.1lM, ZnPc 3 (h) 0.5lM and
ZnPc 4 (d) 0.1lM into Hep-2 cells as a function of incubation time;
(B) inactivation of Hep-2 cells treated with phthalocyanine and
exposed to different irradiation times with visible light; [phthalocya-
nine] = 0.1lM; incubation time 24h. Values represent mean stand-
ard deviation of three separate experiments.
The uptake and photodynamic activity of ZnPcs 3 and 4
were compared in vitro using Hep-2 human larynx-
carcinoma cell line. Both sensitizers are not soluble in
PBS, therefore they were added to the cell cultures
from
a
liposomal solution of L,D-a-dipalmitoyl
phosphatidylethanolamine containing 20% moles of
cholesterol.
value of uptake is lower than those previously found for
porphyrin substituted by methoxy groups (ꢀ2nmol/106
cells) in Hep-2 cells.11 However, in the last case the cells
were incubated with 1lM of porphyrin.
Cell toxicity induced by the photosensitizers was first
analyzed in the dark. The Hep-2 cellular cultures were
treated with 0.1lM of ZnPcs 3 and 4 at 37ꢀC in the
dark. Under these experimental conditions, sensitizers
3 and 4 were not significant toxic after 24h of incub-
ation. Also, no toxicity was found using 0.5lM of ZnPc
3 but 4 produced a decrease in cell survival of ꢀ30%
after 24h of dark incubation.
After the treatment of Hep-2 cells with 0.1lM of the
ZnPcs 3 or 4 for 24h, the cultures were irradiated with
visible light. The irradiation system used in these studies
is described in Experimental section. The corresponding
cell survival values for each photosensitizer are shown in
Figure 3B. No significant lethality was found for cell
cultures not treated with the sensitizer and irradiated;
indicating that the cell mortality obtained after irradia-
tion of the cultures treated with the phthalocyanine is
due to the photosensitization effect of the agent pro-
duced by visible light.
Therefore, the uptake of ZnPcs 3 and 4 into Hep-2 cells
was evaluated at different incubation times using
0.1lM. In each case, the sensitizer intracellular concen-
tration was determined by fluorescence analysis (see
Experimental section) and the results are summarized
in Figure 3A. As can be seen, both phthalocyanines
are rapidly incorporated in the Hep-2 cells in the initial
time (<3h) and the uptake tends to a saturation value at
incubation times P3h. These values were estimated as
ꢀ0.05nmol/106 cells for ZnPc 3 and ꢀ0.06nmol/106 cells
for ZnPc 4. Also, ZnPc 3 was studied using 0.5lM. Un-
der this condition, the profile is similar to that found at
lower concentration (0.1lM), reaching a saturation va-
lue of ꢀ0.28nmol/106 cells. As can be noted, the amount
of ZnPc 3 incorporated is approximately proportional
to the concentration of sensitizer used to incubate the
cells at least in the range of concentrations studied. This
The cell survival after irradiation of the cells with visible
light was dependent upon light exposure level. As shown
in Figure 3B, the cell survival fraction was 0.32 for cells
treated with ZnPc 3 and 0.70 for ZnPc 4 after 15min of
irradiation. Taking into account the light intensity at the
treatment site, the light exposure levels required to inac-
tivate 50% of cell population (D50) were estimated.
Value of D50 = 28 and 67J/cm2 were found for sensitizer
3 and 4, respectively. Thus, even though these phthalocy-
anines present similar uptake by Hep-2 cells, a higher

E. I. Yslas et al. / Bioorg. Med. Chem. 13 (2005) 39–46
43
photodynamic efficiency is obtained using ZnPc 3 than 4
under the same conditions. Also, these experiments were
performed using 0.5lM of ZnPc 3. Under these condi-
tions, an increase in the cell photoinactivation was ob-
tained. The cell survivals were 62% and 20% after 5
and 15min of irradiation, respectively. From these
results, a value of D50 = 19J/cm2 was estimated for ZnPc
3. A similar photokilling behavior was previously
observed for HeLa cells treated with different concen-
tration of ZnPc.7 On the other hand, a small photo-
cytotoxicity was found for HeLa cells treated with
0.1lM of zinc hexadecafluorophthalocyanine for 2h
and irradiated with 31J/cm2.21 The low efficiency of this
hydrophobic compound was apparently influenced by
the dissociation degree of the sensitizer and the phthalo-
cyanine localization site in the cells. Also, sensitizer 3 is
more effective than 5,10,15,20-tetrakis(4-methoxyphen-
yl)porphyrin (D50 = 45J/cm2) and its metal complex
with Zn(II) (D50 = 30J/cm2) in Hep-2 cells.11
ceived. Solvents (GR grade) from Merck were distilled.
Ultrapure water was obtained from Labonco equipment
model 90901-01.
3.2. Synthesis
4-Methoxyphthalonitrile 1. To a mixture of 4-nitriph-
thalonitrile (400mg, 2.31mmol) and methanol (1mL,
38.25mmol) in DMF (15mL) was added anhydrous
potassium carbonate (1.2g, 8.68mmol). The mixture
was heated for 3h at 70ꢀC. Then, the mixture was par-
titioned between dichloromethane (50mL) and water
(50mL). The aqueous layer was separated and extracted
with dichloromethane (3 · 50mL). The solvents of com-
bined organic layers were evaporated under reduced
pressure. The residue was subjected to flash column
chromatography (silica gel) using dichloromethane
as eluent to give 244mg (68%) of pure dinitrile 1.
Rf (dichloromethane) = 0.51. MS [m/z] 158 (M+)
(158.0481 calculated for C9H6N2O1). Anal. Calcd C
68.35, H 3.82, N 17.71; found C 68.28, H 3.84, N 17.75.
In conclusion, two zinc(II) phthalocyanine derivatives
have been synthesized bearing either four methoxy
(ZnPc 3) or trifluoromethylbenzyloxy (ZnPc 4) groups
by a two-step procedure starting from 4-nitrophthalo-
nitrile. Both phthalocyanines show similar spectroscopic
properties and production of O2(1Dg) in organic solvent.
In biological medium, the uptake of these ZnPcs into
Hep-2 cells reaches similar values (ꢀ0.05nmol/106 cells)
after 3h of incubation with 0.1lM of sensitizer at 37ꢀC.
However, the photocytotoxic effect was considerably
higher for ZnPc 3 than ZnPc 4. The low photokilling
efficiency of highly lipophilic sensitizer 4 could be due
to the formation of aggregate in the cellular microenvi-
ronment where the agent is localized. Also, a different
intracellular distribution could be affecting the photody-
namic activity of ZnPc 4. This comparative study indi-
cates that the behavior observed in homogeneous
solution is not always the case in cellular systems, where
the biological microenvironment of the phthalocyanines
can induce significant changes in the photophysics of the
sensitizers.34,35 Further in vitro studies concerning the
mechanism of cell death produced by the photoactiva-
tion of ZnPcs 3 and 4 are presently in progress in our
laboratory.
4-(40-Trifluoromethylbezyloxy)phthalonitrile 2. The
reaction was performed as described above for
compound 1, using 4-nitrophthalonitrile (433mg,
2.50mmol), and 4-(trifluoromethyl)benzyl alcohol
(500mg, 2.84mmol) to give 450mg (60%) of pure dinit-
rile 2. Rf (dichloromethane) = 0.60. MS [m/z] 302
(M+) (302.0668 calculated for C16H9F3N2O1). Anal.
Calcd C 63.58, H 3.00, N 9.27; found C 63.62, H 3.05,
N 9.23.
Zinc(II) 2,9,16,23-tetrakis(methoxy)phthalocyanine 3. A
mixture of dinitrile 1 (90mg, 0.57mmol) and zinc acetate
dihydrate (47mg, 0.21mmol) in n-pentanol (5mL) was
heated at 90ꢀC. Then, DBU, (86lL, 0.57mmol) was
added and the mixture was stirred for 10h at reflux.
The volatiles were removed under reduced pressure
and the solid obtained was dissolved in a minimum
amount of THF. Then methanol and water were added
to induce precipitation. The solid was filtered and
washed sequentially with water (15mL), methanol
(15mL), and n-hexane (25mL). The solid was redis-
solved in THF and the precipitation process repeated
one more time. The product was dried under vacuum
to yield 38mg (38%) of pure ZnPc 3. Rf (ethyl acetate/
n-hexane 1:1) = 0.20. 1H NMR (300.08MHz, CDCl3,
TMS) d [ppm] 3.7 (br, 12H, –OCH3); 7.3–8.0 (m,
12H). MS [m/z] 696 (M+) (696.1212 calculated for
C36H24N8O4Zn). Anal. Calcd C 61.95, H 3.47, N
16.05; found C 61.87, H 3.54, N 16.14.
3. Experimental
3.1. General
UV–vis and fluorescence spectra were recorded on a
Shimadzu UV-2401PC spectrometer and on a Spex
FluoroMax fluorometer, respectively. Proton nuclear
magnetic resonance (1H NMR) spectra were recorded
on a Bruker ARX 300 multinuclear spectrometer at
300MHz. Mass spectra were taken with a ZAB-SEQ
Micromass equipment. Uniplate Silica gel GHLF 250
microns thin layer chromatography plates from Anal-
tech (Newark, DE, USA) was used. All the chemicals
from Aldrich (Milwaukee, WI, USA) were used without
further purification. Sodium dodecyl sulfate (SDS) from
Merck (Darmstadt, Germany) and b-carotene (Car)
from Sigma (St. Louis, MO, USA) were used as re-
Zinc(II)
2,9,16,23-tetrakis(4-trifluoromethylbenzyl-
oxy)phthalocyanine 4. According to the above proce-
dure described for ZnPc 3, using dinitrile 2 (100mg,
0.33mmol) and zinc acetate dihydrate (30mg,
0.14mmol) to yield 43mg (41%) of pure ZnPc 4. Rf
(ethyl acetate/n-hexane 1:1) = 0.53. 1H NMR
(300.08MHz, CDCl3, TMS) d [ppm] 5.2 (br, 8 H, –O–
CH2–Ph) 7.3–8.0 (m, 28H). MS [m/z] 1272 (M+)
(1272.1959 calculated for C64H36F12N8O4Zn). Anal.
Calcd C 60.32, H 2.85, N 8.79; found C 60.25, H 2.78,
N 8.83.

44
E. I. Yslas et al. / Bioorg. Med. Chem. 13 (2005) 39–46
3.3. Spectroscopic studies
confluent cell layers. Then, an appropriate amount of
the phthalocyanine incorporated into liposomes was
added to a stock of medium bearing 4% fetal calf serum
(FCS) to yield the desired final concentration of treat-
ment. An aliquot (2mL) of this medium was added to
the culture dishes. The cells were treated with the sensi-
tizer for 24h in the dark. Afterwards, the medium con-
taining the photosensitizer was discarded. Cells were
washed three times with medium and kept in 2mL of
it containing 7% of FCS. The dishes were exposed for
different time intervals to visible light. The light source
used was a Novamat 130 AF slide projector equipped
with a 150W lamp. The light was filtered through a
3cm glass cuvette filled with water to absorb heat. A
wavelength range between 350 and 800nm was selected
by optical filters. The light intensity at the treatment site
was 45mW/cm2 (k = 670nm) (Radiometer Laser Mate-
Q, Coherent).39 After each irradiation time, the viability
of the cells was estimated 24h after treatments by 3-[4,5-
dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide
(MTT) spectrophotometric method.40 Also, the viability
was established by microscopy with trypan blue exclu-
sion test using a Neubauer chamber counter.12 In both
cases, similar results were obtained. The same procedure
was carried out without irradiation for determining dark
toxicity. The uptake was determined adding 1.0mL of
6% SDS in PBS solution to 1mL of cellular suspension.
The mixture was incubated further for 24h in the dark
and room temperature, sonicated for 15min and centri-
fuged at 9000rpm for 30min. Then, 1mL of THF was
added to the supernatant. The concentration of sensi-
tizer in the supernatant was measured by spectrofluori-
metry (kexc = 672nm, kem = 682nm). The fluorescence
values obtained from each sample were referred to the
total number of cells contained in the suspension. The
concentration of the phthalocyanine in this sample was
estimated by comparison with a calibration curve ob-
tained with standard solutions of the sensitizer in the
same medium (2% SDS in water/THF 2:1, [sensi-
tizer] ꢀ 0.05–0.5lM). Five culture dishes were used for
each incubation time. Every experiment was compared
with a culture control without photosensitizer.
Absorption spectra were recorded at 25.0 0.5ꢀC using
1cm path length cells. The fluorescence quantum yield
(/F) of phthalocyanines were calculated by comparison
of the area below the corrected emission spectrum in
tetrahydrofuran (THF) with that of zinc phthalocyanine
(ZnPc) as
k
a
fluorescence standard, exciting at
ex = 608nm.36 A value of /F = 0.28 for ZnPc in THF
was calculated by the comparison with the fluorescence
spectrum in pyridine using /F = 0.30 and taking into
account the refractive index of the solvents.36,37
3.4. Steady state photolysis
Solutions of 9,10-dimethylanthracene (DMA, 30lM)
and photosensitizer (k = 675nm, absorbance 0.3) in
THF (2mL) were irradiated in quartz cuvettes with
monochromatic light at k = 675nm from a 75W high-
pressure Xe lamp through a high intensity grating mono-
chromator (Photon Technology Instrument). The light
intensity was determined as 1.5mW/cm2 (Radiometer
Laser Mate-Q, Coherent). The kinetics of DMA photo-
oxidation were studied by following the decrease of the
absorbance (A) at kmax = 378nm. The observed rate
constants (kobs) were obtained by a linear least-squares
fit of the semilogarithmic plot of Ln A0/A. Photooxida-
tion of DMA was used to determine singlet molecular
oxygen, O2(1Dg), production by the photosensitizers.15,28
ZnPc was used as the standard (UD = 0.56).38 Measure-
ments of the sample and reference under the same con-
ditions afforded UD for ZnPcs 3 and 4 by direct
comparison of the slopes in the linear region of the
plots. The studies in the presence of b-carotene (Car,
12lM) were performed using the same condition
described above. The pooled standard deviation of the
kinetic data, using different prepared samples, was less
than 5%.
3.5. Cell photosensitization studies and quantification
The incorporation of the phthalocyanines into the
phospholipid bilayer of the D,L-a-dipalmitoyl phospha-
tidylethanolamine was carried out according to the pre-
viously described procedure.12 Phthalocyanine (1lMol),
phospholipid (33lMol), and cholesterol (7lMol) were
dissolved in ethanol–THF binary mixture (400lL, 1:1
vol/vol). The solution was injected into 5mL of phos-
phate-buffered saline (PBS) solution at 80ꢀC. The injec-
tion was performed at a speed of 50lL/min with
magnetic stirring and the final volume was reduced to
5mL by evaporation of organic solvent. The phthalo-
cyanine concentration (ꢀ0.2mM) in the liposomal solu-
tion was determined by absorption spectroscopy
diluting 10lL of the sample in 2mL of THF.
Acknowledgements
Authors are grateful to Consejo Nacional de Investigac-
´
iones Cientıficas y Tecnicas (CONICET) of Argentina
and Fundacion Antorchas for financial support.
´
´
E.N.D. is Scientific Members of CONICET.
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